PMID- 8610743 TI - Comparison of anterior colporrhaphy and retropubic urethropexy for patients with genuine stress urinary incontinence. AB - OBJECTIVE: Our purpose was to compared the efficacy of anterior colporrhaphy and retropubic urethropexy performed for genuine stress urinary incontinence. STUDY DESIGN: A retrospective analysis was performed on women who underwent either anterior colporrhaphy or retropubic urethropexy for genuine stress urinary incontinence. Patients were identified by a computer-assisted search, and these women were contacted by telephone. The interview was used to assess current continence status. Variables reviewed included demographic data, medications, hormonal status, current smoking history, significant medical and surgical history, and time to recurrence of incontinence. Operative procedure, prior or concomitant hysterectomy, history of previous incontinence procedures, concomitant surgery for repair of other pelvic floor defects, experience level of the primary surgeon, and duration of postoperative catheterization were also documented. RESULTS: Seventy-six women who had undergone surgery for genuine stress incontinence during a 4-year period were identified and evaluated by telephone interview. Fifty-six had undergone anterior colporrhaphy and 20 retropubic urethropexy. Both groups of patients were comparable in age, social status, race, parity, and weight. The duration of follow-up (mean +/- SD) was 66.6 +/- 14.2 months (range 48 to 96 months). Concurrent surgery to repair other pelvic floor defects was more common in patients undergoing anterior colporrhaphy than in patients undergoing retropubic urethropexy (p < 0.05). Of the 56 patients treated with anterior colporrhaphy, 26 (46%) were continent at the time of interview versus 15 of 20 (75%) treated with retropubic urethropexy (p < 0.05). Times to recurrence for anterior colporrhaphy and retropubic urethropexy were not significantly different. History of previous incontinence procedures, concomitant hysterectomy, previous hysterectomy, duration of postoperative catheterization, obesity, chronic lung disease, and smoking were not correlated with success for either procedure. Experience of the primary surgeon did have a significant effect on success, with attending staff having a better cure rate than resident surgeons (p < 0.05). CONCLUSION: Retropubic urethropexy was significantly more effective than anterior colporrhaphy for long-term cure of genuine stress urinary incontinence. We believe these conclusions should be tempered because of the complex nature of genuine stress incontinence. Patients having anterior colporrhaphy may represent a high-risk group because nearly all of them had associated pelvic floor defects. Experience of the surgeon seems to enhance the likelihood of success and may reflect subtle modifications of technique. PMID- 8610744 TI - Wound strength in abdominal incisions: a comparison of two continuous mass closure techniques in rats. PMID- 8610745 TI - Effects of sacrocolposuspension on the lower urinary tract. PMID- 8610746 TI - Sacrocolpopexy and the anterior compartment: support and function. PMID- 8610747 TI - Modified Le Fort partial colpocleisis with Kelly urethral plication and posterior colpoperineoplasty in the medically compromised elderly: a comparison with vaginal hysterectomy, anterior colporrhaphy, and posterior colpoperineoplasty. PMID- 8610748 TI - Periurethral and paravaginal anatomy: an endovaginal magnetic resonance imaging study. PMID- 8610749 TI - Granular cell tumors of the vulva. PMID- 8610750 TI - Cherney versus midline vertical incision for myomectomy or hysterectomy of a significantly enlarged uterus. PMID- 8610751 TI - The Mersilene mesh suburethral sling: a clinical and urodynamic evaluation. PMID- 8610752 TI - Minilaparotomy for the ambulatory management of ovarian cysts. PMID- 8610753 TI - A comparison of the relative risk of vessel injury with conical versus pyramidal laparoscopic trocars in a rabbit model. AB - OBJECTIVE: Our purpose was to compare the relative risk of vessel injury after use of a 5 mm conical-tipped trocar, a 5 mm pyramidal-tipped trocar, and a 10 mm pyramidal-tipped trocar in a rabbit vessel model. STUDY DESIGN: Plastic templates were placed in front of and behind 108 mesenteric vessels in 11 anesthetized New Zealand White rabbits. Laparoscopic trocars were inserted through the templates and mesentery. The incidence of vessel injury was determined at distances from the vessels ranging from 0 to 5 mm. RESULTS: The 5 mm conical trocar resulted in a vessel injury rate of 88% at 0 mm from the vessel but 0% at 1 or 2 mm. The 5 mm pyramidal trocar resulted in 100%, 88%, and 62% injury rates of 0, 1, and 2 mm from the vessels, respectively. The 10 mm pyramidal trocar resulted in a 100% injury rate at 0, 1, 2, or 3 mm from the vessels and 80% and 40% at 4 mm and 5mm, respectively. CONCLUSION: The relative risk of vessel injury is significantly increased by the use of pyramidal-tipped trocars when compared with conical tipped trocars, especially if larger diameter trocars are used. PMID- 8610754 TI - Headaches in women, including women who are pregnant. AB - There are new concepts in headache definition and pathophysiologic characteristics, but both classification and management remain largely the responsibility of the principal care physician. Women are particularly vulnerable for some types of headache. When a woman needs medical help she usually turns to their personal physician, who can be expected to diagnose and successfully treat most headaches. PMID- 8610755 TI - Associations between adverse perinatal outcome and serially obtained second- and third-trimester maternal serum alpha-fetoprotein measurements. AB - OBJECTIVE: Our purpose was to determine whether third-trimester maternal serum alpha-fetoprotein predicts adverse perinatal outcome and whether use of both second- and third-trimester maternal serum alpha-fetoprotein enhances the positive predictive value for various abnormal outcomes. STUDY DESIGN: A cohort study with obstetric outcome assessed by chart analysis after delivery was performed at Regional Medical Center (Memphis, Tennessee), a hospital staffed by university-based physicians serving a large urban population with many indigent patients. A total of 650 women attending prenatal clinics in the above setting with a singleton pregnancy without a neural tube defect, contributing a maternal blood samples in both the second and third trimesters, and delivered in the above hospital participated. Various pregnancy outcomes were correlated with maternal serum alpha-fetoprotein levels in the second and third trimesters and in both. RESULTS: In the third trimester no significant associations were found between maternal serum alpha-fetoprotein elevations and pregnancy complications. In the second trimester elevation ( > or = 2.0 multiples of the median) were, by contrast, significantly associated with preterm premature rupture of the membranes, preterm birth, and low birth weight. No association was found with certain other complications. When second-trimester data were grouped according to the types of complications occurring in individual women, only preterm premature rupture of the membrane proved statistically significant. CONCLUSIONS: Second trimester but not third-trimester maternal serum alpha-fetoprotein is significantly elevated with preterm premature rupture of the membranes, preterm birth, and low birth weight; in this cohort study no association was found with preeclampsia, oligohydramnios, or polyhydramnios. PMID- 8610756 TI - The origin of alpha-fetoprotein in first-trimester anembryonic pregnancies. AB - OBJECTIVE: Our purpose was to evaluate the origin of alpha-fetoprotein in the maternal circulation and coelomic fluid of pregnancies with an empty gestational sac on first-trimester ultrasonographic examination. STUDY DESIGN: The alpha fetoprotein level and the affinity of alpha-fetoprotein for concanavalin A Sepharose was measured between 8 and 11 weeks of gestation in the maternal serum and coelomic fluid of nine pregnancies complicated by an empty gestational sac and of 27 normal pregrancies. RESULTS: The maternal serum alpha-fetoprotein level in patients with an empty gestational sac was high in seven cases and normal in two cases. In these cases the median level was significantly (p < 0.01) higher in the serum and lower in the coelomic fluid compared with normal pregnancies. In eight cases of the nine pregnancies with an empty sac, > 50% of alpha-fetoprotein molecules in the coelomic fluid were of the concanavalin A nonreactive fraction, whereas in one case the coelomic fluid sample contained < 5% of this fraction. A similar distribution was found in the corresponding serum samples. CONCLUSION: Normal or high maternal serum AFP levels and alpha-fetoprotein molecules predominantly of yolk sac origin in the coelomic fluid of pregnancies with an empty gestational sac on ultrasonography provide further evidence that the most likely explanation for this feature is the early death of the embryo with persistence of the placental tissue. PMID- 8610757 TI - Prenatal and perinatal influences on long-term psychomotor development in offspring of diabetic mothers. AB - OBJECTIVE: Our purpose was to assess to what extent disturbances in antepartum maternal metabolism and perinatal complications and morbidities contribute to poorer psychomotor development in offspring of diabetic mothers. STUDY DESIGN: One hundred ninety-six pregnant women and their singleton offspring participated in this prospective cohort-analytic study. Ninety-five women had pregestational diabetes mellitus, and 101 women had gestational diabetes mellitus. Serial estimates of circulating maternal fuels were obtained throughout each index pregnancy along with detailed records of the perinatal course and outcome. Offspring were administered the psychomotor development index of the Bayley Scales of Infant Development at age 2 years and the Bruininks-Oseretsky Test Of Motor Proficiency at ages 6, 8, and 9 years. Tests were performed blinded to the mother's antepartum metabolic status, and perinatal history, and the child's previous test scores. Partial correlations and analyses of covariance were used to control for other influences and confounds, such as family socioeconomic status, racial or ethnic origin, patient group (i.e., pregestational or gestational diabetes mellitus), and sex of child. RESULTS: Children's average score on the Bruininks-Oseretsky test at ages 6 to 9 years correlated significantly with maternal second (p < 0.02) and third trimester (p < 0.001) beta-hydroxybutyrate. There was also a borderline association between the children's scores on the psychomotor development index at age 2 years and maternal third-trimester beta-hydroxybutyrate levels (p = 0.06). No other correlations approached significance. CONCLUSIONS: Intrauterine metabolic experiences continue to influence the neurodevelopmental course in offspring of diabetic mothers. Prevailing practices in diabetes management and obstetric and neonatal care appear to effectively mitigate the potential long-term effects of most perinatal complications and morbidities. Management and obstetric and neonatal care appear to effectively miltigate the potential long-term effects of most perinatal complications and morbidities. PMID- 8610758 TI - Follow-up of children born with an umbilical arterial blood pH < 7. AB - OBJECTIVE: We performed neurodevelopmental assessment in children born with an umbilical artery pH < 7. STUDY DESIGN: All infants born with an umbilical artery pH < 7 from a 19-month period were retrieved from the obstetric database. Obstetric, neonatal, and pediatric records were reviewed. At an age of 1 to 3 years, children were visited at home for semi-structured questioning of the mother and a Denver Developmental Screening Test of the child. RESULTS: During the study period 1614 umbilical artery pH measurements were entered in the database. Thirty (1.9%) were < 7. From this group 23 infants were admitted to the neonatal intensive care unit, and 8 of them required intubation. Twenty-eight children survived the neonatal period. Three children experienced an episode of mild hypertonia. One child had a mild motor developmental delay. CONCLUSION: Babies born with an umbilical artery pH < 7 are at greater risk to experience considerable short-term morbidity. Those who leave the neonatal intensive care unit without major problems have good outcomes, and pessimism in counseling their parents in unwarranted. PMID- 8610759 TI - Ultrasonographic assessment of fetal growth: comparison between human and ovine fetus. AB - OBJECTIVE: Our purpose was to evaluate the rate of ovine fetal growth for several body parameters by serial ultrasonographic measurements and to compare them with analogous data in the human fetus. STUDY DESIGN: Forty-three ewes with singleton gestations were studied. Four parameters were measured: biparietal diameter, abdominal circumference, femur length, and tibia length. Ultrasonographic examinations were performed weekly from 50 to 138 days of gestation (term 147 days). Quadratic regression analysis was used to describe each data set. RESULTS: The biparietal diameter showed a significant deceleration of its growth rate. The abdominal circumference showed a linear growth pattern. Both femur and tibia revealed a significant acceleration of the growth rate. CONCLUSION: The ovine fetal growth pattern is different from that observed in the human fetus, in which all four parameters show deceleration of the growth rate in late gestation. In comparison to the ovine, the human fetus reaches similar abdominal circumference and femur length values at term, but in a gestational period that is twice as long. In sharp contrast to abdominal circumference and femur length growth, the biparietal diameter has a similar growth rate in both species. Thus the human fetus has a slower rate of somatic growth and its greater biparietal diameter at term results from the longer gestational period. PMID- 8610760 TI - Intrauterine ultrasonographic assessments of embryonic development. AB - OBJECTIVE: Our purpose was to describe embryonal anatomic structures by use of intrauterine ultrasonography with a 20 MHz flexible catheter-based, high resolution, real-time miniature transducer. STUDY DESIGN: Thirty-four women about to undergo therapeutic abortion from 7 to 9.9 weeks' gestation were studied with specially developed catheter-based, high-resolution, real-time miniature (2.4 mm outer diameter) ultrasonography transducer (20 MHz). A percentage of anatomic structures visualized at each gestational age is presented. RESULTS: The number and the clarity of structures increased from 7 to 8 weeks of gestation; however, the image quality was degraded because of the increasing fetal size at 9 weeks. At 8 weeks secondary brain vesicles, spine, midgut herniation, liver, upper and lower limb buds, and sacral tail were visualized in all fetuses. The four-chamber view was first identified at 8 weeks, as were fingers or toes. The stomach was first noted at 9 weeks. The umbilical cord cyst was visualized in 8% of embryos at 7 weeks' gestation and in 29% of embryos at 8 weeks. One cystic hygroma was diagnosed at 8 weeks 5 days. CONCLUSION: Intrauterine ultrasonography provides information on the visualization of anatomic structures of the embryo. In this limited series one embryonic malformation was demonstrated, and thus there is a potential for its use in the detection of malformations. These results suggest that intrauterine ultrasonography has the potential to be a supplement to transvaginal ultrasonography during the first trimester in high-risk pregnancies. PMID- 8610761 TI - Cardiac and venous blood flow in fetuses of insulin-dependent diabetic mothers: evidence of abnormal hemodynamics in early gestation. AB - OBJECTIVE: Our purpose was to determine whether in early gestation cardiac and venous blood flow patterns of fetuses of insulin-dependent diabetic mothers differ from those of normal fetuses. STUDY DESIGN: Serial recordings were obtained at 12, 16, and 20 weeks of gestation in 11 normal fetuses, 16 fetuses of insulin-dependent diabetic mothers with first-trimester glycosylated hemoglobin levels < or = 8.5% (group 1), and 11 fetuses of insulin-dependent diabetic mothers with first-trimester glycosylated hemoglobin levels > 8.5% (group 2). Velocity waveforms at the level of atrioventricular valves, inferior vena cava, and umbilical vein were recorded by means of color and pulsed Doppler equipment by either transvaginal or transabdominal approaches. The following variables were measured: ratio between the peak velocities during early passive ventricular filling and active atrial filling at the level of atrioventricular valves, percent reverse flow during atrial contraction in inferior vena cava, and pulsations in umbilical vein. RESULTS: In all the fetuses the ratios between early and active ventricular filling increased linearly with advancing gestation, whereas the percent reverse flow in the inferior vena cava decreased linearly. However, fetuses of diabetic mothers showed significant differences in the slope of the functions describing the development with gestation of these index values, resulting in lower values of the ratios between early and active ventricular filling at the level of both ventricular valves and higher values of percent reverse flow in inferior vena cava. These differences were more evident in group 2 fetuses of diabetic mothers, and statistically significant differences were found in the slope values compared with group 1 fetuses. In normal fetuses umbilical vein pulsations were present only in two fetuses at 12 weeks of gestation (18.18%) and were never evidenced later in gestation. A significantly higher incidence of pulsations was found at 12 weeks in fetuses of diabetic mothers (group 1, 56.25%; group 2, 81.81%) and pulsations were present until 16 weeks (group 1, 37.5%; group 2, 45.47%). CONCLUSIONS: An impaired development of cardiac and venous blood flow patterns occurs in fetuses in insulin-dependent diabetic mothers. These abnormalities are more evident in pregnancies with poorer glycemic control but still occur in the presence of stricter metabolic control. PMID- 8610762 TI - Human placental monoamine transporters as targets for amphetamines. AB - OBJECTIVES: The use of amphetamine and its derivatives during pregnancy is known to have adverse effects on the outcome of pregnancy. These effects are at least partly a result of impairment of placental function caused by these abusable drugs. We hypothesized that the two monoamine transporters, namely, the serotonin transporter and the norepinephrine transporter, that are expressed in the human placenta are direct targets for these drugs. STUDY DESIGN: The interaction of amphetamine and methamphetamine with human placental serotonin and norepinephrine transporters was examined. Activity of the serotonin transporter was assessed by serotonin uptake in both maternal-facing brush border membrane vesicles isolated from normal term human placentas and in JAR choriocarcinoma cells. Activity of the norepinephrine transporter was assessed by dopamine uptake and nisoxetine binding in placental brush border membrane vesicles. RESULTS: Amphetamine and methamphetamine are potent inhibitors of the serotonin and norepinephrine transporters expressed in the human placenta. The inhibitory potency of amphetamine is greater than that of methamphetamine. In each case, the S(+)diastereoisomer is more potent than the corresponding R(-)diastereoisomer. The sensitivity of the norepinephrine transporter to inhibition by these drugs is at least two orders of magnitude greater than that of the serotonin transporter. At concentrations known to occur in the plasma of users, these drugs cause a marked inhibition of the norepinephrine transporter. CONCLUSIONS: The results show that the norepinephrine transporter and, to a lesser extent, the serotonin transporter are cellular targets in the human placenta for the abusable drugs amphetamine and methamphetamine. PMID- 8610763 TI - Accuracy of prenatal diagnosis of renal agenesis with color flow imaging in severe second-trimester oligohydramnios. AB - OBJECTIVE: Our purpose was to examine the potential of color flow imaging to assess the presence of renal arteries in second-trimester pregnancies complicated by severe oligohydramnios. STUDY DESIGN: Thirty-three consecutive second trimester pregnancies referred with severe oligohydramnios were prospectively studied with high-resolution color Doppler ultrasonography to establish the presence or absence of renal arteries. Prenatal findings were correlated with the presence or absence of fetal kidneys at postmortem or postnatal examination. RESULTS: Neither renal artery was visualized in eight fetuses; postmortem examination confirmed bilateral renal agenesis in seven and unilateral renal agenesis with a contralateral atrophic multicystic kidney in the other. Only one renal artery was seen in three; postmortem examination demonstrated unilateral renal agenesis in two fetuses and bilateral multicystic dysplastic kidneys in the other. Postmortem or postnatal evaluation confirmed the presence of both kidneys in all 22 fetuses in which both renal arteries were identified prenatally. CONCLUSIONS: Color Doppler ultrasonography is useful in the prenatal evaluation of fetuses with severe second-trimester oligohydramnios to demonstrate the presence or absence of renal arteries. This technique should be added to the armamentarium of prenatal tests to evaluate second-trimester fetuses with severe oligohydramnios. PMID- 8610764 TI - Contractile proteins in human fetoplacental vessels. AB - OBJECTIVE: Our purpose was to compare the protein isoform composition of the contractile apparatus at different levels of the fetoplacental vessel musculature at term. STUDY DESIGN: Umbilical, chorionic, and stem villi vessel protein extracts were run on one- and two-dimensional gel electrophoresis; previously characterized human myometrium proteins were used as the smooth muscle proteins of reference. RESULTS: Fetoplacental vessel musculature exhibited a high actin/myosin ratio. The presence, in varying quantities, of myosin heavy chain and actin isoforms of smooth muscle type in the different vessels reflected their degree of differentiation. The presence of nonmuscle protein isoforms, particularly in stem villi vessels, indicated a certain degree of immaturity. CONCLUSIONS: The presence of smooth muscle contractile protein isoforms indicates that fetoplacental vessel musculature is highly differentiated. Regional modulation of fetoplacental blood flow could be, in part, the result of local differences in contractile apparatus protein composition. PMID- 8610766 TI - A randomized trial of open versus closed vaginal vault in the prevention of postoperative morbidity after abdominal hysterectomy. AB - OBJECTIVE: Our purpose was to evaluate the effects of two surgical techniques, closed vaginal vault with two layers of continous 3-0 polyglactin suture versus open vaginal vault with a locking 3-0 polyglactin suture, after abdominal hysterectomy. STUDY DESIGN: A prospective, randomized trial was performed. During the hospital stay and 4 to 8 weeks after the operation patients were observed for evidence of morbidity. RESULTS: Of the 273 evaluable subjects, 141 had the vaginal vault left open whereas 132 were closed. Ten (7.1%) and eight patients (6.1%) had infections at the operative site (pelvis or abdominal wound) (p = 0.92). A urinary tract infection was diagnosed in three (2.1%) and in four subjects (3.0%) (p = 0.46). A pelvic hematoma developed in two patients of each group (p = 0.66). Vault granulations were recorded in 11% and 12% of subjects (p = 0.97). CONCLUSIONS: This study failed to show some benefit in favor of either of the two surgical policies. A careful surgical technique and antibiotic prophylaxis seem to remain the most important factors in the prevention of postoperative morbidity. PMID- 8610765 TI - Absence of relaxation to lactate in human placental vessels of pregnancies with severe preeclampsia. AB - OBJECTIVE: Our objective was to determine whether the observed relaxation to lactate and other agents in placental vessels of normal pregnancies is altered in severe preeclampsia. STUDY DESIGN: Isolated placental arteries and veins from women with severe preeclampsia and uncomplicated term pregnancies were precontracted with prostaglandin F2 alpha under 5% oxygen and 5% carbon dioxide with the balance nitrogen (Po2 35 to 38 torr) and then exposed to lactate (1 to 10 mmol/L, pH 7.4, n = 8 to 15), arachidonic acid (0.01 to 10 mumol/L, n = 6 to 13), nitroglycerin (1 nmol to 1 mumol/L, n = 4 to 12), or forskolin (0.01 to 10 mumol/L, n = 6 to 9). The response to lactate was also examined in placental vessels from appropriate-for-gestational-age preterm deliveries (n = 8) for comparison with a similar group with severe preeclampsia (n = 8). The t test and analysis of variance statistics were used. RESULTS: Relaxation to lactate was markedly inhibited in both placental arteries and veins of women with severe preeclampsia compared with vessels from uncomplicated term or preterm pregnancies. Responses to the other relaxing agents were not altered in the severely preeclamptic vessels. CONCLUSIONS: In severe preeclampsia absence of lactate-induced dilatation of placental vessels may contribute to the fetal complications associated with impaired blood flow and vasospasm. PMID- 8610767 TI - Single-use versus reusable laparoscopic surgical instruments: a comparative cost analysis. AB - OBJECTIVE: Our purpose was to determine and compare the total annual costs for reusable and single-use laparoscopic instruments. STUDY DESIGN: Records were kept over a 12-month period of all laparoscopic operations (performed only with reusable instrumentation), the surgical instruments used, depreciation costs, and all associated expenses (repairs, maintenance, replacements, cleaning, sterilization, wages). The total cost was then calculated and compared with the total cost (purchase price plus disposal fees) that single-use instruments would have caused for the same operations. RESULTS: The total cost for single-use instruments would have been more than seven times that for reusable instruments. CONCLUSION: We have decided to continue using reusable instruments for most of our laparoscopic operations. However, single-use instruments are used in situations in which they present a definite advantage. A proper balance is thus sought between the importance of cost factors, on the one hand, and the recognition of the need for the best instrument available for certain procedures, on the other. PMID- 8610769 TI - Platelet-derived growth factor promotes endometrial epithelial cell proliferation. AB - OBJECTIVE: Our purpose was to determine the effects of platelet-derived growth factor on the proliferation of endometrial epithelial cells. Platelet-derived growth factor and its receptors have been identified in the endometrium, and platelet-derived growth factor is a mitogen for endometrial stromal cells. Released from macrophages and platelets at sites of ectopic endometrial growth, platelet-derived growth factor could promote the progression of endometriosis and endometrial cancer. STUDY DESIGN: Endometrial epithelial cell lines were developed from proliferative-phase endometria from two patients without endometrial lesions. Cell lines were confirmed to be epithelial. Proliferation assays were conducted on both lines with recombinant human platelet-derived growth factor-AA, AB, and BB. Assays were also performed at different doses, times, and cell densities with platelet-derived growth factor. RESULTS: All isoforms of platelet-derived growth factor were potent mitogens for both endometrial epithelial cell lines. The greatest proliferative responses were achieved at 10 ng/ml and at 24 hours. Responses decreased significantly in confluent cultures. CONCLUSIONS: These results suggest that endometrial epithelial cells have functional platelet-derived growth factor-alpha receptors that signal cell replication. The greater activity of platelet-derived growth factor in subconfluent cultures may indicate that receptor numbers or affinity are up-regulated when cell-cell contact is disrupted. These data support a role for platelet-derived growth factor in normal endometrial proliferation and in pathologic proliferation such as endometriosis and endometrial cancer. PMID- 8610768 TI - Hormone replacement therapy reduces the reactivity of monocytes and platelets in whole blood--a beneficial effect on atherogenesis and thrombus formation? AB - OBJECTIVE: Our purpose was to investigate the effects of hormone replacement therapy on the reactivity of monocytes and platelets in whole blood, measured by tissue factor activity, tumor necrosis factor-alpha, and thromboxane B2. STUDY DESIGN: Thirty-two women were randomized into either transdermal or oral combined hormone replacement therapy and underwent blood sampling before and after 3 and 12 months of treatment. The tissue factor activity in monocytes was measured both in unstimulated whole blood and after a weak lipopolysaccharide stimulation. Tumor necrosis factor-alpha and thromboxane B2 formation in plasma were measured after a weak lipopolysaccharide stimulation of whole blood. RESULTS: After 12 months of hormone replacement therapy there were significant reductions of tissue factor activity in both unstimulated and lipopolysaccharide-stimulated monocytes (p < 0.001) and significant reductions in the formation of tumor necrosis factor alpha (p < 0.03) and thromboxane B2 (p < 0.02). There were no differences in these parameters between the transdermal and the oral groups. No changes were observed after 3 months of therapy. CONCLUSION: Twelve months of hormone replacement therapy reduces cellular activation of blood monocytes and platelets; these changes may account for some of the beneficial effects in reducing the risk of cardiovascular disease. PMID- 8610770 TI - Photosensitization of the endometrium with topical 5-aminolevulinic acid. AB - Photosensitization of the endometrium was attempted in vitro and in vivo by intrauterine administration of 5-aminolevulinic acid, which is converted to the photosensitizer protoporphyrin IX. Protoporphyrin IX was found in both functional and basal layers of the endometrial glands at concentrations nine and 10 times higher than myometrium in in vitro and in vivo experiments, respectively. Selective endometrial photosensitization is possible with topical 5 aminolevulinic acid, but it might not be distributed sufficiently evenly for use as a sensitizing agent in photodynamic ablation. PMID- 8610771 TI - Multivariate survival analysis of clinicopathologic features in surgical stage I endometrioid carcinoma including analysis of HER-2/neu expression. AB - OBJECTIVE: We previously described vascular invasion-associated changes, defined as the presence of vascular invasion or perivascular lymphocytic infiltrates, as key prognostic indicators in stage I endometrioid carcinoma. The current study was undertaken to examine the prognostic value of HER-2/neu expression in relation to other factors, including vascular invasion-associated changes, in surgical stage I endometrioid carcinoma. STUDY DESIGN: Seventy-one patients with surgical stage I endometrioid carcinoma treated by hysterectomy and followed up were randomly chosen for retrospective analysis of prognostic indicators including standard clincopathologic features, deoxyribonucleic acid ploidy, and HER-2/neu expression. The latter was examined by an objective computerized quantitative immunohistochemical system. RESULTS: By univariate analysis many factors were found to correlate with outcome, including age, tumor grade, depth of invasion, ploidy, HER-2/neu expression, and vascular invasion-associated changes. By multivariate analysis only vascular invasion-associated changes, aneuploidy, and HER-2/neu overexpression were found to independently correlate with survival. Stratification of patients on the basis of these three features revealed survival rates of 100%, 92%, and 60% when none, one, and two or three features were present, respectively. CONCLUSION: This study suggests that HER 2/neu expression correlated with outcome independent of other factors in endometrial carcinoma and may aid in estimating prognosis. The prognostic value of HER-2/neu overexpression independent of vascular invasion suggests that this factor may operate by increasing the ability of tumor cells to grow at a distal site once vascular invasion occurs. PMID- 8610772 TI - Effects of Taxol on choriocarcinoma cells. AB - OBJECTIVE: Taxol (Bristol-Myers Squibb) (paclitaxel) has been shown to be a potent inhibitor of cell growth for a variety of tumors. We were interested in the antiproliferative efficacy and biologic properties of this novel antineoplastic agent in choriocarcinoma cells. STUDY DESIGN: Human choriocarcinoma cell lines JAR and BeWo were cultured as monolayers and treated with Taxol. RESULTS: Proliferation of JAR and BeWo cells was inhibited by Taxol in a dose-related manner and 1 to 3 nmol/L was sufficient to achieve 50% growth reduction. This effect was accompanied by a marked induction of human chorionic gonadotropin secretion. The effect on human chorionic gonadotropin secretion was dependent on intact protein biosynthesis but not mediated by augmented messenger ribonucleic acid expression. In these choriocarcinoma cells Taxol promoted differentiation as shown by an increase in syncytiotrophoblastic-like cells. Combination of Taxol with either etoposide or methotrexate resulted in antagonistic growth inhibition. CONCLUSION: Taxol is a highly effective antineoplastic agent in choriocarcinoma cells, and clinical trials in refractory disease would therefore be warranted. However, substances other than etoposide or methotrexate should be evaluated for combined treatment. In addition to growth inhibition, differentiation is also induced by Taxol, as shown by increased human chorionic gonadotropin secretion and changed morphologic features. PMID- 8610773 TI - A computer-controlled, continuously circulating, hot irrigating system for endometrial ablation. AB - OBJECTIVE: This study evaluated the efficacy of a computer-controlled thermal catheter system to ablate the endometrium by use of 5 to 15 ml of hot saline solution. STUDY DESIGN: Five in vivo sheep uteri and 32 freshly excised human uteri were studied by inserting a self-contained heater catheter equipped with thermocouples and circulating hot saline solution for 15 minutes within the uteri at various temperature settings ranging from 60 degrees C to 80 degrees C. The uteri were grossly examined and microscopically evaluated by hematoxylin and eosin and reduced nicotinamide adenine dinucleotide-diaphorase stains for degree of acute destruction, depth of thermal injury, and predicted viability. RESULTS: All 80 degrees C sheep treatments resulted in complete loss of viability at mucosal, submucosal, and myometrial levels. In human uteri temperatures > or = 80 degrees C produced grossly recognizable thermal damage to the endometrium. Full thickness irreversible damage was seen in the endometrium. Loss of viability was also demonstrated 1 to 3 mm deep in the superficial myometrium. Intrauterine pressure was demonstrated to range from 20 to 40 mm Hg, with a mean of 26 mm Hg. No leakage of fluid from the oviducts was observed. CONCLUSION: The data presented here demonstrate that a simple technique coupled with a sophisticated monitoring control system could successfully ablate the endometrium without the vagaries associated with individual operator skill levels. PMID- 8610774 TI - The effect of estrogen replacement therapy with or without progestogen on the fibrinolytic system and coagulation inhibitors in postmenopausal status. AB - OBJECTIVE: The aim of this study was to analyze several fibrinolytic components and coagulation inhibitors in postmenopausal women and had to evaluate the effect of hormone replacement therapy. STUDY DESIGN: Several hemostatic parameters were evaluated in 75 postmenopausal women before and after 3 to 4 and 12 months of hormone therapy. RESULTS: An increase in plasma fibrinolytic activity primarily related to a significant increase in tissue-type plasminogen activator and a decrease in plasminogen activator inhibitor type 1 was observed in women receiving hormone replacement therapy. A significant decrease in protein S and lipoprotein(a) was detected under therapy. No modifications in tissue-type plasminogen activator/plasminogen activator inhibitor-1 and activated protein C/alpha 1-antitrypsin complexes, urokinase activity, plasminogen, and antithrombin III were detected. CONCLUSIONS: The increase in fibrinolytic activity and the decrease in lipoprotein(a) levels observed in women receiving hormone replacement therapy could help decrease the risk of coronary disease associated with the postmenopausal state. PMID- 8610775 TI - Efficacy of outpatient induction with low-dose intravaginal prostaglandin E2: a randomized, double-blind, placebo-controlled trial. AB - OBJECTIVE: Our purpose was to determine whether a protocol for outpatient induction is safe and effective for initiating labor. STUDY DESIGN: A randomized, double-blind, placebo-controlled trial was performed with 100 low-risk patients having well-dated pregnancies. Women with a Bishop score < or = 6 at 38 to 40 weeks' gestation were administered either 2 mg of intravaginal prostaglandin E2 gel or placebo for 5 consecutive days as outpatients while undergoing fetal monitoring. RESULTS: The median interval from randomization to delivery was 4 days in the prostaglandin E2 group (range 0 to 28 days) versus 10 days in the placebo group (range 0 to 26 days, p = 0.002). Twenty-seven of 50 patients (54%) in the prostaglandin E2 group were admitted for labor during the dosing interval compared with 10 placebo-treated patients (20%, p = 0.001). The mean gestational age at delivery was significantly reduced in the treatment group (39.9 +/- 1.0 weeks vs 40.5 +/- 0.99 weeks, p = 0.003) as was the incidence of postdates pregnancy (40% vs 66%, p = 0.016). Hyperstimulation was observed in one prostaglandin E2-treated patient, but no intervention was required. CONCLUSIONS: Outpatient low-dose prostaglandin E2 gel administration is effective for initiating labor in patients with an unfavorable cervix and appears safe if performed with adequate monitoring. PMID- 8610776 TI - Randomized comparison of the effects of endocervical and vaginal prostaglandin E2 gel in women with various degrees of cervical ripeness. Dutch Collaborative Prostaglandin Trialists' Group. AB - OBJECTIVE: The trail was conducted to obtain an unbiased comparison of the relative merits of endocervical and vaginal prostaglandin E2 gel in a weighted case mix of parous and nulliparous women with favorable and unfavorable cervical features. STUDY DESIGN: Multicenter randomized trial with 285 participants, (three exclusions) was performed with sealed envelopes stratified for parity and Bishop score. RESULTS: Outcomes of labor and delivery were clearly related to the cervical score at trial entry, especially in nulliparous women. Endocervical prostaglandin E2 had a more marked effect on cervical ripeness than did vaginal prostaglandin E2, but this did not result in any differences in more substantive outcomes. Frequencies of delivery within 12 (50%) and 24 hours (77.7%), cesarean section (7.3%), instrumental vaginal delivery (11.7%), and poor infant outcomes were similar with both preparations. CONCLUSION: Because differences in effectiveness between endocervical and vaginal prostaglandin E2 in triacetin gel are marginal, preferences of women and clinicians can determine the choice between them. PMID- 8610777 TI - Monitored outpatient management of mild gestational hypertension remote from term in teenage pregnancies. AB - OBJECTIVE: Our purpose was to compare maternal and perinatal outcomes of teenage and adult pregnancies with mild gestational hypertension remote from term managed with an outpatient program. STUDY DESIGN: A matched cohort design was used. Maternal and perinatal outcomes of 60 teenage pregnancies with mild gestational hypertension remote from term were compared with 120 adult controls 20 to 42 years old. The groups were matched for race, gestational age, and proteinuria status at enrollment. All were monitored on an outpatient basis with four times daily automated blood pressure measurement and daily assessment of weight, proteinuria, and fetal movement. RESULTS: The mean gestational age at enrollment was 33.5 +/- 2.6 weeks for both groups (range 27 to 36 weeks). Only 60% of teenagers had a high school degree or equivalent compared with 76% of adults (p = 0.024). The teenagers were more likely than the adults to be of single marital status (75% vs 13%, p = 0.015). The mean gestational age at delivery (37.0 +/- 2.0 vs 37.0 +/- 2.2 weeks), mean pregnancy prolongation (23.5 +/- 19.0 vs 24.5 +/ 17.4 days), and mean birth weights (2915 +/- 669 vs 2879 +/- 678 gm) were not statistically different between the teenagers and adults (all p > 0.05). There were no stillbirths, neonatal deaths, or cases of eclampsia in either group. CONCLUSIONS: In spite of a study population characterized by limited education, single marital status, and young age at enrollment, monitored outpatient management of mild gestational hypertension remote from term in teenage pregnancies is associated with maternal and perinatal outcomes similar to those observed in adults. PMID- 8610778 TI - Heparin levels to guide thromboembolism prophylaxis during pregnancy. AB - OBJECTIVE: Our purpose was to determine the dose of heparin required in pregnant women to achieve the same heparin levels as standard doses of 5000 units given subcutaneously every 12 hours in the nonpregnant population. STUDY DESIGN: Fourteen pregnant women placed on heparin prophylaxis for a history of thromboembolism had blood drawn for 64 anti-Xa level determinations in the second and third trimesters. Heparin doses were adjusted in an attempt to achieve a midinterval or peak level of 0.05 to 0.25 U/ml, which corresponds to the range seen in nonpregnant patients given standard doses of 5000 units subcutaneously every 12 hours. RESULTS: A standard heparin dose of 5000 units given subcutaneously every 12 hours was inadequate to achieve the desired range in this pregnant population. In five of nine second-trimester pregnancies 7500 units given subcutaneously every 12 hours was inadequate to attain this range. In six of 13 third-trimester pregnancies, > 10,000 units subcutaneously every 12 hours was needed. CONCLUSIONS: Heparin requirements may increase and are highly variable in patients during pregnancy. Until appropriate clinical outcomes trials can determine optimal dosing, measuring anti-Xa activity may be useful to guide therapy. PMID- 8610779 TI - Low-dose versus high-dose oxytocin augmentation of labor--a randomized trial. AB - OBJECTIVE: Our purpose was to compare the efficacy and safety of low-dose versus high-dose oxytocin regimens in the augmentation of labor. STUDY DESIGN: Three hundred ten term pregnancies requiring augmentation of labor underwent randomization to receive either a low-dose or high-dose oxytocin augmentation regimen. Maternal demographics, labor-delivery data, and neonatal outcome were compared. RESULTS: The high-dose oxytocin group had a significantly lower cesarean section rate, regardless of parity (10.4% vs 25.7%, p < 0.001), with no differences in maternal complications and neonatal outcomes. The time needed to correct the labor abnormality was also significantly decreased (1.24 +/- 1.4 hours vs 3.12 +/- 1.6 hours, p < 0.001) in the high-dose group. CONCLUSIONS: The use of high-dose oxytocin regimen benefits both nulliparous and multiparous women requiring labor augmentation by significantly lowering both the time necessary to correct the labor abnormality and the need for cesarean section. PMID- 8610780 TI - Naturally occurring insulin autoantibodies in neonates of normal pregnancies and their relationship to insulinemia and birth weight. AB - OBJECTIVE: The objectives of this study were to determine whether insulin autoantibodies are present in umbilical cord blood from normal pregnancies, determine whether cord blood insulin autoantibody levels correlate with respective maternal levels at delivery, determine whether cord blood insulin autoantibody levels are related to cord blood or maternal insulin levels, and to determine what relationship neonatal birth weight has with either cord blood insulin autoantibody and insulin levels or maternal insulin autoantibody and insulin levels. STUDY DESIGN: Paired umbilical cord and maternal serum samples were taken from 70 normal subjects at delivery. Measurements of serum insulin autoantibody (competitive charcoal radiobinding assay) and insulin (radioimmune inhibition assay) levels were performed. Multiple linear regression analysis and paired t tests were used for data analyses. RESULTS: Neonatal insulin autoantibody levels (120 nU/ml) were more than two times higher than maternal levels (49 nU/ml) (p < 0.001). No correlation was observed between neonatal and maternal insulin autoantibody levels (r = 0.14, p = 0.25). A positive correlation of both neonatal and maternal insulin with birth weight was observed (r = 0.28, p < 0.02; and r = 0.36, p < 0.01, respectively). CONCLUSIONS: These results suggest that the insulin autoantibody levels in fetal cord blood are not related to maternal levels in normal uncomplicated pregnancies. In addition, insulin levels in both maternal and neonatal circulations were positively correlated with increased birth weight in the normal pregnancies studied. PMID- 8610781 TI - The coelomic cavity--a reservoir for metals. AB - OBJECTIVE: The concentrations of metals in fluids surrounding the first-trimester fetus were measured. STUDY DESIGN: Atomic absorption spectrometry was used to measure concentrations of metals in matched samples of amniotic and extraembryonic coelomic fluids in 17 women between 9 and 12 weeks of pregnancy. RESULTS: Concentrations of calcium, magnesium, iron, copper, and manganese (but not zinc, cadmium, or lead) were significantly higher in coelomic than in amniotic fluid. There was no significant difference between levels of iron, manganese, and lead in controls and amniotic fluid or between concentrations of manganese, cadmium, and lead in controls and coelomic fluid. There was no relationship between the concentrations of each metal in amniotic and coelomic fluid. CONCLUSION: The extraembryonic coelom is an important site of concentration of metals in early pregnancy. This might represent a store of metals essential for normal embryonic and fetal development or constitute a defense mechanism against environmental conditions adverse to the fetus. PMID- 8610782 TI - Jelly beans as an alternative to a cola beverage containing fifty grams of glucose. AB - OBJECTIVE: Our purpose was to test the diagnostic value and patient tolerance of jelly beans as an alternative to a 50 gm glucose solution. STUDY DESIGN: Pregnant women between 26 to 30 weeks of gestation confirmed by early ultrasonography were recruited to participate in the study. Each participant was given a cola beverage containing 50 gm of glucose. The plasma glucose level was determined 1 hour later. Within 2 weeks of the 50 gm glucose test, each patient ate 18 jelly beans and had her plasma glucose levels tested after 1 hour. Finally, within 2 weeks of the jelly bean test a 100 gm, 3-hour glucose tolerance test was performed on each subject. The results of the 3-hour test were used to define the presence or absence of gestational diabetes and carbohydrate intolerance by the criteria of The American College of Obstetricians and Gynecologists. Patient tolerance was rated by responses to questions regarding side effects. RESULTS: One hundred fifty-seven women completed the study. The mean maternal age, gravidity, parity, and number of abortions were 26.06 years, 2.66, 0.96, and 0.69. By use of a 140 mg/dl threshold, the sensitivity, specificity, and positive predictive value of the cola beverage was 46%, 81%, and 18%. These values at a 120 mg/dl threshold for jelly beans were 54%, 81%, and 20%, respectively. The patient tolerance was greater for the jelly beans compared with the 50 gm cola beverage. CONCLUSION: Jelly beans may serve as an alternative to a cola beverage containing 50 gm of glucose. PMID- 8610783 TI - Sterility, loss of the fertilized ovum, fecundity and childbirth and female and newborn diseases...(Observations diverses sur la sterilite, perte de fruict, foecondite accouchements et maladies des femmes et enfants nouveaux naiz...). PMID- 8610784 TI - MOMs (multiples of the median) may know best: there are insufficient data to suggest that DADs (discriminant aneuploidy detection) are superior. PMID- 8610785 TI - Are DADs (discriminant aneuploidy detection) as good as MOMs (multiples of the median)? PMID- 8610786 TI - Platelet response to vasopressin in preeclampsia. PMID- 8610787 TI - The expert witness. PMID- 8610788 TI - Clarification of source. PMID- 8610789 TI - Are more male infants affected by preterm premature rupture of the membranes? PMID- 8610790 TI - Delayed functional loss in glaucoma. LII Edward Jackson Memorial Lecture. AB - PURPOSE: This study is a systematic exploration of why some patients with glaucoma continue to lose visual field long after therapeutic normalization of their increased intraocular pressures. METHODS: Three cases of glaucoma are described that had increased intraocular pressures and good initial visual fields. RESULTS: The following four hypotheses are offered to explain delayed functional loss in these patients: (1) A process independent of intraocular pressure is killing ganglion cells. (2) Unmeasured increases of pressure are killing ganglion cells. (3) The ganglion cells have a genetically determined hypersensitivity to intraocular pressure. (4) The ganglion cells have been rendered hypersensitive to intraocular pressure by irreversible damaging effects of previously increased intraocular pressures CONCLUSION: The current state of knowledge does not permit the elimination of any of the four hypotheses. An additional hypothesis is that the final stage of ganglion cell death is mediated by apoptosis. If so, a potential new treatment for glaucoma would be to inhibit the apoptotic pathway. PMID- 8610791 TI - Retinal nerve fiber layer assessment by scanning laser polarimetry and standardized photography. AB - PURPOSE: To determine whether, in a clinical setting, scanning laser polarimetry and retinal nerve fiber layer photography provide equivalent information on the retinal nerve fiber layer. METHODS: We prospectively studied 60 patients with glaucoma or ocular hypertension and 24 healthy subjects. With scanning laser polarimetry, an estimate of the cross section of the retinal nerve fiber layer was obtained. By using a photographic reference set, we quantified photographs of the retinal nerve fiber layer. Visual fields were used to relate the results of both methods to functional damage. RESULTS: The scanning laser polarimetry measurements yielded reproducible cross-section values (coefficient of variation, 6.6%). Comparison of cross-section values and photograph scores gave Pearson correlation coefficients smaller than r = .4 (P < .01), improving to a maximum of r = .53 after compensation for offset. When cross-section values were compared to the mean deviation of the visual field, the Spearman correlation coefficients varied from an r of -.34 to -.53 (P < .01). Correction for offset resulted in an r of -.54 to -.65. When photograph scores and mean deviation of the visual field were compared, the Spearman correlation coefficients varied from an r of -.65 to .71 (P < .01). CONCLUSIONS: Because r was maximal at .53, the information on the retinal nerve fiber layer obtained with scanning laser polarimetry and photography seems not equivalent. This result could not have been because of lack of reproducibility. Although the results suggested possible offset in scanning laser polarimetry, other methodologic differences must be considered to explain the differences between the two techniques. PMID- 8610792 TI - Comparison between tomographic scanning evaluation and photographic measurement of the neuroretinal rim. AB - PURPOSE: To compare laser tomographic scanning evaluation with photographic measurement of size and shape of the neuroretinal rim. METHODS: For 25 normal eyes and 32 glaucomatous eyes, the optic disks were examined with the Heidelberg Retina Tomograph (software version 1.11) for confocal laser tomographic scanning evaluation, and color stereoscopic optic disk photographs for planimetric measurements. Area and width of the neuroretinal rim were determined as percentages of the disk area and diameter, respectively. RESULTS: For the normal and glaucomatous eyes, the tomographic results compared with the photographic measurements disclosed significantly larger values for the relative width and relative area of the neuroretinal rim. The differences between both methods were most marked in the nasal part of the optic disk and least marked in the temporal disk region. The relative differences increased significantly (P < .05) with increasing degree of glaucomatous optic nerve damage. CONCLUSIONS: In normal and glaucomatous eyes, the Heidelberg Retina Tomograph determines the neuroretinal rim, expressed as percentage of optic disk measurements, to be significantly larger than when the rim is evaluated on optic disk photographs. Because parts of the central retinal vessel trunk are defined as neuroretinal rim in the algorithm of the Heidelberg Retina Tomograph, the differences between Heidelberg Retina Tomograph measurements and photographic determinations of the rim are largest in the nasal disk region and smallest in the temporal disk area. The neuroretinal rim shape and neuroretinal rim area differ appreciably between the two methods. These differences increase with increasing degree of glaucomatous optic nerve damage. PMID- 8610793 TI - Color Doppler imaging in patients with asymmetric glaucoma and unilateral visual field loss. AB - PURPOSE: To determine whether lower blood velocities and high resistive index in the retrobulbar arteries are primary or secondary to glaucomatous damage in patients with open-angle glaucoma. METHODS: Color Doppler imaging was performed in 32 glaucomatous patients with unilateral visual field loss and in 31 control subjects. Peak systolic velocity and end diastolic velocity were measured, and resistive index was calculated in the central retinal artery and short posterior ciliary arteries. RESULTS: In patients with glaucoma, both the more affected and the contralateral eyes with normal visual fields had significantly lower peak systolic velocity and end diastolic velocity in their central retinal artery and short posterior ciliary arteries than did the control subjects of similar age (P < or = .03). The resistive index of the central retinal artery of both eyes of patients with glaucoma was also significantly higher than in the control subjects (P = .001). When considering the 16 patients who had the greatest visual field asymmetry, the more affected eyes had lower peak systolic velocity and end diastolic velocity in the central retinal artery than the contralateral eyes did (P = .02). CONCLUSIONS: Even eyes with normal visual fields in patients with asymmetric disease had decreased blood velocities in their retrobulbar vessels, suggesting that these circulatory changes probably precede detectable damage. Furthermore, the finding of lower central retinal artery blood velocities in the more affected eye of asymmetric patients suggests that low blood velocities may be one of the lateralizing factors in those patients and that they have a possible role in the pathogenesis of the disease. PMID- 8610794 TI - Neural networks to identify glaucoma with structural and functional measurements. AB - PURPOSE: Neural networks can recognize patterns and classify complex variables. We assessed the ability of neural networks to discriminate between normal and glaucomatous eyes by using structural and functional measurements. METHODS: Several neural network algorithms were tested with a database of 185 eyes of patients with early glaucomatous visual field loss (average mean defect, 4.5 dB) and 54 eyes of age-matched normal control subjects. The information used included automated visual field indices (mean defect, corrected loss variance, and short term fluctuation) and structural data (cup/disk ratio, rim area, cup volume, and nerve fiber layer height) from computerized image analysis. RESULTS: A back propagation network with two intermediate layers assigned an estimated probability of being glaucomatous to each eye and correctly identified 88% of all eyes with 90% sensitivity and 84% specificity. The same neural network trained with only structural data correctly identified 80% of the eyes with 87% sensitivity and 56% specificity, and when trained with functional data only, it correctly identified 84% of the eyes with 84% sensitivity and 86% specificity. CONCLUSION: Analysis of several optic nerve and visual field variables by neural networks can help identify early glaucomatous damage and assign an estimated probability that early damage is present in individual patients. PMID- 8610795 TI - Timolol hemihydrate vs timolol maleate to treat ocular hypertension and open angle glaucoma. AB - PURPOSE: We compared the therapeutic efficacy and safety of timolol hemihydrate to timolol maleate in patients with ocular hypertension and chronic open-angle glaucoma. METHODS: We conducted this three-month study as a multicentered, masked, parallel group comparison. Both the 0.25% and 0.5% concentrations were evaluated against similar concentrations of timolol maleate. Dosing was twice daily. An open-label, nine-month study followed the masked portion of the protocol, in which all patients received either 0.25% or 0.5% timolol hemihydrate. A total of 371 patients were included in both the 0.25% and 0.5% studies. RESULTS: We found statistically similar intraocular pressures with both the 0.25% (18.3 and 18.6 mm Hg for the hemihydrate and maleate groups, respectively) and 0.5% (19.9 and 19.5 mm Hg for the hemihydrate and maleate groups, respectively) concentrations of timolol hemihydrate and timolol maleate after three months of masked treatment. Likewise, peak intraocular effect at two hours after taking the medication was statistically similar between medicines at both concentrations. Likewise, both ocular and systemic safety were similar between the maleate and hemihydrate preparations at both concentrations. In the nine-month open-label protocol, therapeutic efficacy (19.9 and 19.1 mm Hg for the 0.25% and 0.5% concentrations, respectively) and safety of timolol hemihydrate were similar to effect and safety of the three-month protocol. CONCLUSIONS: This study suggests that timolol hemihydrate had an ocular hypotensive efficacy and safety profile statistically equivalent to that of timolol maleate for up to three months of therapy. Timolol hemihydrate showed efficacy and safety similar to that observed within the first three months, for up to one year of therapy. PMID- 8610796 TI - Late onset of corneal scar after excimer laser photorefractive keratectomy. AB - PURPOSE: We studied the occurrence of late scarring after photorefractive keratectomy and its response to topical corticosteroids and debridement during the course of follow-up of 950 eyes that had photorefractive keratectomy with excimer laser. METHODS: Five eyes of four patients developed localized corneal scars, decreased visual acuity, and increased myopia after five to 33 months of good visual acuity, with trace haze. In two eyes, scars were removed by debridement alone. In these two eyes, recurrent scars were treated by debridement, followed by aggressive treatment with topical corticosteroids. Two other eyes were treated with topical corticosteroids alone. The fifth eye, which developed a scar after debridement to correct a subjective visual distortion after photorefractive keratectomy, was treated with debridement followed by aggressive topical corticosteroids. RESULTS: Treatment with topical corticosteroids alone in two eyes improved uncorrected visual acuity slightly and decreased myopia, although the scars remained unchanged. Debridement without aggressive topical corticosteroid use resulted in rapid return of the scars and a decrease in visual acuity. Subsequent debridement after aggressive topical corticosteroid treatment resulted in resolution of scars and no recurrence after discontinuation of corticosteroids in one case. In another case, the scar recurred eight months after discontinuation of topical corticosteroids. In Case 4, the scar has not recurred as the topical corticosteroid dosage has been reduced. CONCLUSIONS: Patients who undergo photorefractive keratectomy should be counseled concerning the risk of late scarring, reexamined frequently after photorefractive keratectomy, and treated with topical corticosteroids after corneal trauma. Long-term treatment with topical corticosteroids may be required to prevent the recurrence of scars after debridement. PMID- 8610797 TI - Corneal pannus associated with contact lens wear. AB - PURPOSE: To determine whether contact lens, related corneal pannus regressed when eyes were refitted with rigid gas-permeable contact lenses and to investigate the role of hypoxia as the putative origin of this complication. METHODS: Twenty three eyes of 14 patients with corneal pannus were identified retrospectively from a referral practice population and were separated into two subgroups. One subgroup accepted refitting with rigid gas-permeable contact lenses or discontinued contact lens wear, and the other elected to continue wearing hydrogel or polymethylmethacrylate contact lenses. The response of pannus was observed by slit-lamp biomicroscopy and photographs and was compared between the two subgroups. One eye of each of six patients identified with bilateral corneal pannus secondary to daily wear of hydrogel contact lenses was selected at random. Duplicates of the hydrogel contact lenses worn by these eyes were obtained, and their peripheral oxygen transmissibility (Dk/Lp) values were compared with those of a control group of hydrogel contact lenses identical to those worn by another group of patients, matched by age, gender, and laterality to the study group, but who did not have corneal vascularization. RESULTS: Vascularization regressed in 14 eyes of nine patients refitted with rigid gas-permeable contact lenses and also in both eyes of one patient who discontinued contact lens wear. All seven eyes of five patients who declined refitting showed persistence or increase of pannus. The mean Dk/Lp of the hydrogel contact lenses for six eyes with pannus was 3.8 X 10(-9) cm ml O2/s ml mm Hg; mean Dk/Lp was 8.6 X 10(-9) cm ml O2/s ml mm Hg for the lens of the control eyes. This difference was found to be statistically significant (P = .028). CONCLUSION: Peripheral, contact lens induced hypoxia is a risk factor for corneal pannus, and conversion from hydrogel or polymethylmethacrylate contact lenses to daily wear rigid gas-permeable contact lenses in these eyes successfully reversed this complication. PMID- 8610798 TI - Contrast sensitivity and other vision tests in the optic neuritis treatment trial. AB - PURPOSE: To determine the intercorrelation, prevalence of abnormality, and incremental detection value of vision tests in optic neuritis. METHODS: We calculated the linear correlation of paired vision tests and prevalence of abnormal test values from baseline and six-month measurements of Snellen visual acuity, Pelli-Robson contrast sensitivity, Humphrey Field Analyzer mean deviation, and Farnsworth-Munsell 100-hue color vision in 438 patients entered in the Optic Neuritis Treatment Trial from 1988 to 1991. The incremental detection value of nonvisual acuity tests was defined as their frequency of abnormality when visual acuity was 20/20 or better. RESULTS: All four vision-test results were highly intercorrelated at baseline and at six months. At baseline, contrast sensitivity had the highest prevalence of abnormality, but all vision tests were so often abnormal that differences were not clinically relevant. At six months, when visual recovery had occurred, contrast sensitivity was most often abnormal (2.2 X visual acuity; 1.8 X mean deviation; 1.5 X Farnsworth-Munsell 100-hue color vision test); when contrast sensitivity, mean deviation, or Farnsworth Munsell 100-hue color vision was normal, visual acuity was 20/25 or better in 98% of patients. CONCLUSIONS: The high intercorrelation of four vision tests suggests that optic neuritis affects a broad range of visual functions. Among non-visual acuity tests, Pelli-Robson contrast sensitivity proved to be a particularly practical and sensitive indicator of visual dysfunction in optic neuritis. PMID- 8610799 TI - Recombinant hirudin for prevention of experimental postoperative intraocular fibrin. AB - PURPOSE: To determine the efficacy of a specific antithrombin agent (recombinant desulphatohirudin variant 1 [Revasc, Ciba-Geigy, Ltd., Basel, Switzerland]) administered in the infusion fluid to prevent early postoperative fibrin formation in a rabbit lensectomy and vitrectomy model. METHODS: Standard fragmatome lensectomies and core vitrectomies were performed prospectively in a masked fashion on ten control eyes with lactated Ringer's infusion and on ten eyes treated with 10 microgram of recombinant hirudin/ml in the infusate. The amounts of fibrin and hemorrhage were graded in a masked fashion by using slit lamp examination and indirect ophthalmoscopy on postoperative days 1 through 5 and on day 7. RESULTS: The difference in the mean grade of fibrin formed on the first postoperative day in the eyes treated with recombinant hirudin (mean, 0.9) in relation to the mean grade of fibrin in the control eyes (mean, 3.5) was statistically significant (P = .004). This difference was also significant on the second postoperative day (P = .01). None of the treated eyes developed intraoperative or postoperative hemorrhage. CONCLUSIONS: Recombinant desulphatohirudin variant 1 is an effective inhibitor of postoperative fibrin formation in a rabbit model and is not associated with an increased risk of intraoperative or postoperative bleeding at the tested dose. This drug may be a useful adjunct in vitreous surgery for both proliferative vitreoretinopathy and the complications of proliferative diabetic retinopathy. PMID- 8610800 TI - p53 gene and cell cycling in uveal melanoma. AB - PURPOSE: To determine whether alterations of p53, a tumor suppressor gene, were present in uveal melanoma, and to characterize further the nature of those changes. METHODS: Immunohistochemical analysis with a monoclonal antibody was used to determine whether alterations of p53 were present in 35 enucleated archival uveal melanomas. Further characterization was done by comparing the p53 gene and cell cycling status by using bromodeoxyuridine staining. The alterations in p53 were characterized using polymerase chain reaction single-strand conformational polymorphism analysis and sequencing. RESULTS: Four of 35 uveal melanomas showed low levels (0.5% to 5.0%) of positive immunostaining for altered p53 in tumor cell nuclei using monoclonal antibody DO-7. These four tumors had the three highest and the 14th highest bromodeoxyuridine labeling indices, ranging from 1.3% to 7.0%. Polymerase chain reaction single-strand conformational polymorphism analysis of p53 exons 5 to 8 was performed on three p53-positive and six p53-negative tumors, and no altered motility shifts were detected. Sequencing of one of the positive staining specimens confirmed no mutations in exons 5 through 8 in the p53 gene. Double immunohistochemical labeling for both bromodeoxyuridine and p53 in one tumor showed that most of p53-positive cells were in S phase. CONCLUSIONS: Mutation of p53 is an uncommon event in uveal melanomas. Nuclear accumulation of p53 protein was found in three of the four tumors with the highest levels of cell cycling. PMID- 8610801 TI - Centenary of the Japanese Ophthalmological Society and the American Academy of Ophthalmology. PMID- 8610802 TI - Corneal scarring after photorefractive keratectomy in a penetrating keratoplasty. AB - PURPOSE: To report severe scarring in a corneal graft after excimer laser photorefractive keratectomy. METHODS: A 35-year-old man underwent photorefractive keratectomy twice for severe compound myopic astigmatism and anisometropia after penetrating keratoplasty. RESULTS: Corneal opacity corresponded to areas of irregular epithelial thickness, focal absence of the basement membrane, loss of Bowman's layer, and stromal scarring in the ablation zone. CONCLUSION: There may be an increased risk of severe corneal stromal scarring from photorefractive keratectomy in eyes that have had previous penetrating keratoplasty. PMID- 8610803 TI - Recurrent corneal ulcerations associated with smokeable methamphetamine abuse. AB - PURPOSE: We studied a case of chronic, recurrent, bilateral, corneal ulcerations associated with smokeable methamphetamine abuse, commonly known as "ice," in an otherwise healthy 31-year-old woman. METHODS: Every few months the patient had recurrent corneal ulcerations. Each time, she was hospitalized and treated successfully with topical antibiotics. RESULTS: Even though she had undergone numerous formal attempts at drug rehabilitation, she continued to have relapses, and ulceration recurred only during periods of smokeable methamphetamine abuse. CONCLUSION: Illicit use of smokeable methamphetamine may result in corneal ulceration. PMID- 8610804 TI - Stress fractures after folding an acrylic intraocular lens. AB - PURPOSE: To manage the complication of stress fractures in foldable acrylic intraocular lenses. METHODS: During an implantation of a foldable acrylic intraocular lens (AcrySof, Alcon Laboratories, Fort Worth, Texas), the optic was noted to have developed paracentral stress fractures along the fold approximately 50% deep. RESULTS: The lens was explanted and replaced without difficulty. CONCLUSIONS: The observed complication can be avoided by recognizing that it might occur, wetting the lens before slowly folding it, and inspecting it carefully before insertion. PMID- 8610805 TI - Condensation of silicone oil on the posterior surface of a silicone intraocular lens during vitrectomy. AB - PURPOSE: We experienced a complication caused by the condensation of silicone oil on the surface of a silicone intraocular lens during pars plana vitrectomy. METHODS: A 74-year-old woman with proliferative vitreoretinopathy underwent a pars plana vitrectomy in her left eye, which contained silicone oil and a silicone intraocular lens. RESULTS: Intraoperatively, condensation of silicone oil on the posterior surface of a silicone intraocular lens caused a loss of visibility during fluid/gas exchange. The silicone oil droplets could not be removed. CONCLUSION: Surgeons should avoid direct contact of silicone oil with silicone intraocular lens. PMID- 8610806 TI - Postoperative hypopyon after intravitreal bovine thrombin for macular hole surgery. AB - PURPOSE: We used intravitreal autologous fibrinogen with bovine thrombin for the surgical closure of macular holes in 60 cases. METHODS: Pars plana vitrectomy with separation of the posterior cortical vitreous was performed after air/fluid exchange. One to two drops each of autologous fibrinogen and bovine thrombin (20 to 80 U) were instilled in each patient. RESULTS: Five (8%) of 60 patients developed a hypopyon without unusual pain on the first postoperative day. Inflammation responded to frequent topical corticosteroids within 48 to 72 hours. CONCLUSION: Postvitrectomy hypopyon after the use of bovine thrombin may represent an immune reaction that must be differentiated from endophthalmitis. We recommend careful observation and frequent topical corticosteroids. PMID- 8610807 TI - Choroidal hemorrhage associated with systemic tissue plasminogen activator. AB - PURPOSE: To determine the cause of spontaneous choroidal hemorrhage in a 67-year old man after a myocardial infarction and administration of tissue plasminogen activator. METHODS: The patient underwent ocular examination. RESULTS: The patient retained excellent visual acuity and the choroidal hemorrhage resolved completely within two months. CONCLUSION: The administration of tissue plasminogen activator was responsible for the large extent of hemorrhage and should be considered in the differential diagnosis of hemorrhagic choroidal detachment. PMID- 8610808 TI - Uveal effusion syndrome associated with primary pulmonary hypertension and vomiting. AB - PURPOSE: We treated a 69-year-old woman with bilateral uveal effusions associated with primary pulmonary hypertension, congestive heart failure, and vomiting. METHODS: The patient underwent ophthalmoscopic, echocardiographic, and fluorescein angiographic examinations. RESULTS: Increased venous pressure caused by congestive heart failure and vomiting was implicated as the cause of bilateral uveal effusions. The uveal effusions responded well to systemic furosemide. CONCLUSION: Primary pulmonary hypertension with right-sided congestive heart failure and vomiting can lead to the development of uveal effusions. PMID- 8610809 TI - Abduction defect associated with aberrant regeneration of the oculomotor nerve after intracranial aneurysm. AB - PURPOSE: To determine the cause of delayed-onset ipsilateral abduction defect associated with aberrant regeneration of the oculomotor nerve. METHODS: Isolated oculomotor palsy was noted after successful basilar artery aneurysm surgery in a 35-year-old patient. Several months later, aberrant regeneration of the oculomotor nerve and an ipsilateral abduction defect were first detected. RESULTS: Ocular electromyography demonstrated failure of relaxation of the ipsilateral medial rectus muscle on attempted abduction, suggesting cocontraction of horizontal recti muscles as the origin of the abduction defect. CONCLUSION: A late-onset ipsilateral abduction defect caused by failure of relaxation of the medial rectus muscle may be associated with basilar aneurysm. PMID- 8610810 TI - Visual function after laser hyperthermia and chemotherapy for macular retinoblastoma. AB - PURPOSE: To determine whether treatment of macular retinoblastoma with chemotherapy and laser hyperthermia can lead to satisfactory visual acuity. METHODS: A child with unilateral macular retinoblastoma was treated with chemotherapy and laser hyperthermia. Visual function was assessed one year after treatment. RESULTS: At age 25 months, the patient had normal visual behavior and a normal spatial-sweep visual-evoked potential. CONCLUSION: Treatment of macular retinoblastoma with chemotherapy and laser hyperthermia may enable recovery of satisfactory visual acuity. PMID- 8610811 TI - Cerebellar astrocytoma manifesting as isolated, comitant esotropia in childhood. AB - PURPOSE: We encountered a 4 1/2-year-old girl with gradual onset of intermittent, comitant esotropia in the absence of diplopia and other neurologic findings. METHODS: Because of the patient's relatively advanced age and lack of response to hyperopic correction for accommodative esotropia, computed tomography of the head was performed. RESULTS: A large cerebellar astrocytoma was identified and successfully resected. After strabismus surgery, fusion was reestablished. CONCLUSIONS: The onset of comitant esotropia in an older child may indicate an underlying neurologic disorder. PMID- 8610812 TI - Simultaneous silicone intubation through the osteotomy and the nasolacrimal duct during dacryocystorhinostomy. AB - PURPOSE: To determine whether simultaneous silicone intubation of the osteotomy and the nasolacrimal duct is advantageous in some dacryocystorhinostomy procedures. METHODS: Silicone tubing was placed through both the osteotomy and the nasolacrimal duct in two patients who underwent dacryocystorhinostomy for dacryolithiasis. RESULTS: Postoperatively, signs and symptoms of nasolacrimal obstruction were relieved, and lacrimal irrigation confirmed anatomic patency of the tear drainage canals in both cases. CONCLUSIONS: Simultaneous intubation of the osteotomy and the nasolacrimal duct prevents prolapse of the silicone stent and may decrease the frequency of postoperative scarring. PMID- 8610813 TI - Unilateral open-angle glaucoma secondary to idiopathic dilated episcleral veins. AB - PURPOSE: To determine the cause of unilateral dilated tortuous episcleral vessels in a 34-year-old patient. METHODS: The patient underwent slit-lamp examination, visual field testing, tonographic measurement, orbital ultrasound examination, orbital color Doppler blood flow measurement, dye-enhanced computed topographic scan, and selective carotid angiography. RESULTS: Disk cupping and early scotoma were present. Gonioscopy showed Schlemm's canal engorged with blood. The only pathologic findings were an increased tonographic resistance to outflow and increased arterial and venous episcleral flow on color-coded Doppler sonography. CONCLUSION: The origin of this episcleral vessel abnormality is still unknown. PMID- 8610814 TI - Prevent Blindness America Visual Field Screening Study. PMID- 8610815 TI - Impact of recent economic changes in psychiatry on academic psychiatry programs. PMID- 8610816 TI - Images in neuroscience. Progressive dysarthria: structural and brain correlations. PMID- 8610817 TI - Newer antidepressants and the cytochrome P450 system. AB - OBJECTIVE: This review evaluates the in vitro and in vivo evidence for inhibition of cytochrome P450 enzymes by the newer antidepressants and provides clinical recommendations for avoiding and managing drug interactions. METHOD: The international literature on the cytochrome P450 system and related drug interactions from 1966 to 1995 was reviewed. In vitro studies, pharmacokinetic trials in human subjects, and case reports were assessed. RESULTS: The newer antidepressants each inhibit a different cluster of cytochrome P450 enzymes, which are of relevance to the potential for drug interactions. Cytochrome P450 1A2 is inhibited by fluvoxamine and is implicated in drug interactions with theophylline, clozapine, and others. Fluoxetine, norfluoxetine, sertraline, and paroxetine are potent in vitro inhibitors of cytochrome P450 2D6 and are capable of causing marked elevations in plasma desipramine and nortriptyline concentrations. Fluoxetine, sertraline, and fluvoxamine are believed to inhibit cytochrome P450 2C because of observed interactions with phenytoin, diazepam, and other drugs metabolized by these enzymes. Cytochrome P450 3A4 metabolizes terfenadine, astemizole, carbamazepine, alprazolam, triazolam, and other benzodiazepines. Plasma concentrations of these drugs have increased when they are administered with fluvoxamine, nefazodone, fluoxetine, and sertraline. CONCLUSIONS: The majority of the newer antidepressants are associated with a risk for clinically significant drug interactions. A rapidly growing body of literature provides evidence for a distinct profile of cytochrome P450 inhibition and drug interaction risks by individual antidepressants. These findings underscore the need for definitive in vivo interaction studies of plasma from phenotyped patients treated with clinically effective antidepressant doses of medication, for direct comparative clinical studies, and for studies assessing the utility of phenotyping in clinical practice. PMID- 8610818 TI - What are the functional consequences of neurocognitive deficits in schizophrenia? AB - OBJECTIVE: It has been well established that schizophrenic patients have neurocognitive deficits, but it is not known how these deficits influence the daily lives of patients. The goal of this review was to determine which, if any, neurocognitive deficits restrict the functioning of schizophrenic patients in the outside world. METHOD: The author reviewed studies that have evaluated neurocognitive measures as predictors and correlates of functional outcome for schizophrenic patients. The review included 1) studies that have prospectively evaluated specific aspects of neurocognition and community (e.g., social and vocational) functioning (six studies), 2) all known studies of neurocognitive correlates of social problem solving (five studies), and 3) all known studies of neurocognitive correlates and predictors of psychosocial skill acquisition (six studies). RESULTS: Despite wide variation among studies in the selection of neurocognitive measures, some consistencies emerged. The most consistent finding was that verbal memory was associated with all types of functional outcome. Vigilance was related to social problem solving and skill acquisition. Card sorting predicted community functioning but not social problem solving. Negative symptoms were associated with social problem solving but not skill acquisition. Notably, psychotic symptoms were not significantly associated with outcome measures in any of the studies reviewed. CONCLUSIONS: Verbal memory and vigilance appear to be necessary for adequate functional outcome. Deficiencies in these areas may prevent patients from attaining optimal adaptation and hence act as "neurocognitive rate-limiting factors." On the basis of this review of the literature, a series of hypotheses are offered for follow-up studies. PMID- 8610819 TI - Impact of visit copayments on outpatient mental health utilization by members of a health maintenance organization. AB - OBJECTIVE: The authors examined the impact of increasing cost sharing on use of outpatient mental health services. METHOD: A quasi-experimental design was used to study outpatient utilization by members of a health maintenance organization (HMO) who were subject to increasing copayments for mental health visits (state government employees and dependents). Their outpatient mental health utilization was compared with that of similar HMO members who were not subject to cost sharing (federal government employees and dependents). Analyses compared both likelihood of any service use and number of visits per year among service users. RESULTS: Institution of $20/visit copayments was associated with a 16% decrease in likelihood of service use but no change in visit rate among service users. A subsequent copayment increase to $30/visit resulted in no significant change in likelihood of use but was associated with a 9% decrease in visits per year among those using services. The impact of the first copayment change on likelihood of using services did not vary according to level of clinical need (as measured by prior service use and psychotropic drug use). CONCLUSIONS: In this staff-model HMO, modest visit copayments significantly reduced initial access to mental health treatment and had a smaller effect on treatment intensity. Copayments restricted access regardless of clinical need. Designers of mental health benefits must consider the impact of copayments on those with the greatest need for treatment. PMID- 8610820 TI - Controlling inpatient psychiatric utilization through managed care. AB - OBJECTIVE: There is little current understanding of how managed care strategies affect hospital inpatient psychiatric care for mentally ill patients. This study examined one prominent form of managed care, utilization management, which reviews requests for psychiatric care and authorizes provision of care deemed appropriate and clinically necessary. METHOD: The authors analyzed data on 2,265 utilization management reviews conducted during 1989-1992 for patients insured by a single large commercial insurance company. Three utilization management procedures were examined: preadmission review, continued-stay review, and case management. The performance indicators analyzed included percent of admission requests granted, number of days requested and approved, and number of treatment extensions granted. RESULTS: Utilization management initially approved inpatient psychiatric treatment for nearly all (98.8%) of the patients but authorized, on average, only one-third of the days requested (6.9 versus 19.0). On average, 23.5 (total) days of care were requested and 16.8 days were approved. Care for patients with alcohol or drug dependence diagnoses was more restricted than was care for other patients. CONCLUSIONS: These data suggest that managed care does restrict inpatient psychiatric care, primarily by managing length of stay. The fact that almost all patients were approved for the same initial length of stay implies adherence to strict treatment protocols that do not distinguish among different clinical or patient factors. There is a need for careful study of the effects of managed care on outcomes and quality of psychiatric care. PMID- 8610821 TI - Blunted change in cerebral glucose utilization after haloperidol treatment in schizophrenic patients with prominent negative symptoms. AB - OBJECTIVE: The purpose of this report was to determine 1) the effects of chronic haloperidol treatment on cerebral metabolism in schizophrenic patients, 2) the relation between negative symptoms and haloperidol-induced regional changes in cerebral glucose utilization, and 3) the relation between metabolic change and clinical antipsychotic effect. METHOD: Cerebral glucose utilization, as determined by position emission tomography (PET), was studied in 18 male schizophrenic subjects before and after chronic treatment with haloperidol at a standardized plasma level. RESULTS: Overall, haloperidol caused a widespread decrease in absolute cerebral glucose metabolism. The cerebral metabolic response to haloperidol was blunted in patients with high pretreatment negative symptom scores. CONCLUSIONS: Taken together with the results from a previously reported PET study of the effects of an acute amphetamine challenge (in which 14 of the current subjects participated), these data suggest that the negative symptom complex is associated with diminished cerebral response to change in dopaminergic activity. This deficit cannot be solely accounted for by structural differences. PMID- 8610822 TI - Temporal lobe morphology in childhood-onset schizophrenia. AB - OBJECTIVE: Neurodevelopmental models of schizophrenia imply that a more severe early brain lesion may produce earlier onset of psychotic symptoms. The medial temporal lobes have been proposed as possible locations for such a lesion. The authors tested this hypothesis in a group of children and adolescents with childhood-onset schizophrenia who had severe, chronic symptoms and who were refractory to treatment with typical neuroleptics. METHOD: Anatomic brain magnetic resonance imaging scans were acquired with a 1.5-T scanner for 21 patients (mean age=14.6 years, SD=2.1) who had onset of schizophrenia by age 12 (mean age at onset=10.2, SD=1.5) and 41 normal children. Volumes of the temporal lobe, superior temporal gyrus, amygdala, and hippocampus were measured by manually outlining these structures on contiguous 2-mm thick coronal slices. RESULTS: Patients with childhood-onset schizophrenia had significantly smaller cerebral volumes. With no adjustment for brain volume, no diagnostic differences were observed for any temporal lobe structure. Unexpectedly, with adjustment for total cerebral volume, larger volumes of the superior temporal gyrus and its posterior segment and a trend toward larger temporal lobe volume emerged for the patients with schizophrenia. These patients lacked the normal (right-greater-than left) hippocampal asymmetry. CONCLUSIONS: These findings do not indicate a more severe medial temporal lobe lesion as the basis of very early onset schizophrenia. PMID- 8610823 TI - Eye movements in spectrum personality disorders: comparison of community subjects and relatives of schizophrenic patients. AB - OBJECTIVE: The purpose of the current study was to test the specificity of an association between eye tracking abnormality and schizophrenia spectrum personality symptoms in the family members of schizophrenic patients. The studies of biological markers for genetic vulnerability to schizophrenia, which test an association between a biological measure and schizophrenia spectrum personality symptoms, are constrained, since these personality symptoms may lack the specificity for a schizophrenic phenotype. An association between a behavioral measure and these personality symptoms in general can easily be false (i.e., not related to schizophrenic vulnerability). In contrast, a strong deviant finding in the relatives of schizophrenic patients with spectrum personality symptoms, in the presence of a relatively normal finding in spectrum subjects without a known history of schizophrenia, makes the biobehavioral measure an interesting candidate for such investigations. METHOD: Seventy-five subjects recruited from the community who did not have a family history of psychosis completed the study (24 of the 75 had significant schizophrenia spectrum personality symptoms). Thirty-two first-degree relatives of schizophrenic patients (13 with spectrum symptoms) completed the study. Subjects were 18-45 years old and had no DSM-III-R axis I diagnosis. RESULTS: Qualitative smooth pursuit eye movement score was significantly worse in relatives with the spectrum symptoms than in spectrum subjects without a family history of schizophrenia and the nonspectrum relatives. Schizotypal and schizoid symptoms explained a significant amount of the variance in the eye tracking measure in the relatives (31% and 20%, respectively) but not in the community subjects (less than 2%). Relatives of schizophrenic patients with and without the spectrum symptoms had significantly longer antisaccade latency, in spite of comparable latency for visually guided saccades, than the community subjects. CONCLUSIONS: Smooth pursuit abnormality in subjects with schizophrenia spectrum personality disorders is specifically associated with a family history of schizophrenia. PMID- 8610824 TI - Chronic PTSD in Vietnam combat veterans: course of illness and substance abuse. AB - OBJECTIVE: The purpose of this study was to measure the longitudinal course of specific symptoms of posttraumatic stress disorder (PTSD) and related symptoms of alcohol and substance abuse and the effects of alcohol and substances on the symptoms of PTSD. METHOD: A structured interview for the assessment of PTSD and alcohol and substance abuse, as well as other factors such as life stressors and treatment, was administered to 61 Vietnam combat veterans with PTSD. RESULTS: Onset of symptoms typically occurred at the time of exposure to combat trauma in Vietnam and increased rapidly during the first few years after the war. Symptoms plateaued within a few years after the war, following which the disorder became chronic and unremitting. Hyperarousal symptoms such as feeling on guard and feeling easily startled developed first, followed by avoidant symptoms and finally by symptoms from the intrusive cluster. The onset of alcohol and substance abuse typically was associated with the onset of symptoms of PTSD, and the increase in use paralleled the increase of symptoms. Patients reported a tendency for alcohol, marijuana, heroin, and benzodiazepines to make PTSD symptoms better, while cocaine made symptoms in the hyperarousal category worse. There was no relationship between treatment interventions and the natural course of PTSD. CONCLUSIONS: These findings suggest that symptoms of PTSD begin soon after exposure to trauma, that hyperarousal symptoms are the first symptoms to occur, that the natural course of alcohol and substance abuse parallels that of PTSD, and that specific substances have specific effects on PTSD symptoms. PMID- 8610825 TI - Relationship of childhood anxiety to adult panic disorder: correlates and influence on course. AB - OBJECTIVE: This study investigated the correlates of a childhood history of anxiety disorders in adult patients participating in a longitudinal study of panic disorder. The authors hypothesized that a history of anxiety during childhood would be associated with higher rates of comorbid anxiety and depressive disorders, greater likelihood of anxiety disorders in family members, and greater chronicity, as reflected by decreased time spent in remission. METHOD: The presence of a childhood history of anxiety disorders was assessed by structured interview, and its association with comorbid anxiety and depressive disorders, family history, and select anxiety severity variables was examined in a replication sample of 94 patients. The influence of childhood anxiety on the prospectively ascertained course of disorder was assessed in a full group of 194 patients. RESULTS: Over half (54%) of the patients experienced anxiety disorders during childhood. These patients experienced higher rates of comorbid anxiety and depression, family history of anxiety, and increased levels of agoraphobia, panic frequency, and global severity of illness at baseline evaluation. Childhood anxiety disorders were not independently associated with the number of months in remission or the severity of illness over time, although a modest effect for this variable was evident when degree of avoidance and anxiety sensitivity at baseline were statistically controlled. CONCLUSIONS: Adult panic patients with a history of anxiety disorders in childhood have elevated rates of comorbid anxiety and depressive disorders and a tendency toward increased avoidance, but there was not strong evidence that these patients respond differently to treatment over time. PMID- 8610826 TI - Self-regard: a new measure. AB - OBJECTIVE: The status of patients and research subjects is usually considered in terms of self-reported symptoms. Measures seldom include disturbances in a conscious sense of the self. An additional brief measure of the sense of current self-regard is desirable, since a conscious lapse in an integrated self-concept may occur under stressful circumstances. The authors constructed and tested such a measure. METHOD: Clinical interviews had indicated five common experiences that occurred more frequently as complaints during stress-induced regressions in the sense of the self as a functioning mind-body agency. An anchored five-item scale, the Self-Regard Questionnaire, was constructed and tested with 79 subjects who were in the midst of grief from the death of a spouse. Data analyses included checks on the internal coherence of questionnaire scale scores and their association with symptom, personality, and social desirability measures. RESULTS: The five-item Self-Regard Questionnaire was completed quickly, in less than a minute, and led to internally consistent and unique data. Low levels of overall self-regard were correlated with higher levels of distress and predicted prolonged distress. CONCLUSIONS: These results suggest that the questionnaire is a useful, quick, and easy-to-score self-report tool for assessing, and reassessing over time, current experiences of the self. The five questions may also be useful to clinicians who evaluate patients in contexts other than research. PMID- 8610827 TI - Bias and bulimia nervosa: how typical are clinic cases? AB - OBJECTIVE: Since patients being treated for bulimia nervosa constitute only a minority of persons with the disorder, the cases seen in clinics may be subject to sampling bias. The aim of this study was to investigate sampling bias as it affects secondary referrals for bulimia nervosa. METHOD: The personal and family characteristics of a consecutive series of 60 women with secondary referrals for bulimia nervosa (clinic subjects) were compare with those of 83 subjects with bulimia who were recruited directly from the community. Most of the data were collected by interview. RESULTS: The demographic characteristics of the two groups were similar. The clinic subjects had a more severe eating disorder and much greater impairment of social functioning. There was no difference between the groups in duration of the eating disorder or level of general psychiatric disturbance. The community subjects were heavier and had stronger family histories of obesity. CONCLUSIONS: There is sampling bias among secondary referrals for bulimia nervosa. The relative absence of persons prone to obesity among secondary subjects is important, since there is evidence that vulnerability to obesity is a poor prognostic feature as well as being a risk factor for the development of bulimia nervosa. The greater social impairment among the clinic subjects is suggestive of greater personality disturbance in this group. Caution is warranted when generalizing from clinic cases to the disorder as a whole. PMID- 8610828 TI - Retrospective study of 2,200 involuntary psychiatric admissions and readmissions. AB - OBJECTIVE: The authors describe demographic data, the distribution of diagnoses, and comorbid psychoactive substance use in a large sample of patients involuntarily admitted to a psychiatric hospital from multiple crisis centers and explore the relative roles these variables may play in service utilization and admission rates. METHOD: Data on demographic characteristics and comorbid psychoactive substance use in 2,200 consecutive involuntary hospital admissions of 1,755 psychiatric patients were gathered. Pertinent demographic and comorbidity data at first admission for the 1,755 patients, 314 of whom were admitted more than once, were analyzed; then the data for the 1,441 single admission patients and the data at first admission for the 314 patients who had multiple admissions were compared. Finally, the diagnostic distribution and comorbid psychoactive substance use in all 2,200 admissions were investigated, with attention to a subgroup of 88 high-risk patients (those with three or more admissions) who represented a total of 307 admissions. RESULTS: Specific demographic characteristics were represented in the patient group at a high level of statistical significance. The diagnosis of schizophrenia was significantly overrepresented. Schizophrenia and psychosis not elsewhere classified clustered in the subgroup with a high risk of readmission. CONCLUSIONS: The results suggest a specific profile for the patient with heightened risk of hospital admission: a young, unmarried, African American male who has schizophrenia without comorbid substance abuse. An effect size data analysis identified marital status and a diagnosis of schizophrenia as the variables associated with the greatest likelihood of admission. Unexpectedly, the impact of comorbid psychoactive substance use was relatively modest and showed a uniform distribution among diagnostic groups. PMID- 8610829 TI - Four-year follow-up of the influence of psychological comorbidity on medical rehospitalization. AB - OBJECTIVE: This study tested the hypothesis that psychiatric comorbidity measured in medical and surgical general hospital inpatients predicts increased readmissions and days spent rehospitalized at the same hospital up to 4 years after discharge. METHOD: A convenience sample of 273 medical/surgical inpatients aged 18 years and older were given psychological tests during their third to fifth hospital days on medical and surgical units from June 1, 1985, through June 30, 1986. The main outcome measure was the number of medical/surgical readmissions and number of days rehospitalized during a 4-year follow-up at the same institution. RESULTS: Compared to the rest of the study group, the cognitively impaired patients (according to the Mini-Mental State examination) averaged twice as many rehospitalizations and three times as many days rehospitalized at 6-month follow-up and twice as many days rehospitalized at 2 year follow-up. Compared to the rest of the group, the patients who were depressed or who had high interpersonal sensitivity scores at the index admission spent twice as many days rehospitalized during the 4-year follow-up, while the patients with high hostility scores had almost twice as many readmissions. These results remained statistically significant after separate partialing out of the effects of severity of functional impairment, age, cognitive impairment, and number of admissions or days spent hospitalized before the index admission. CONCLUSIONS: Psychiatric comorbidity, previously well documented as contributing to increased length of stay in the general hospital, is associated with increased hospital utilization for at least 4 years after discharge. PMID- 8610830 TI - Psychiatric complications in a patient with complex partial seizures. AB - This case highlights the complexities of evaluating and treating psychiatric symptoms that are concurrent with a seizure disorder. Interictal and postictal psychoses, affective disorders, personality changes, and cognitive deficits are common problems that require modified psychiatric treatments. PMID- 8610831 TI - Change in thyroid hormone levels following response to cognitive therapy for major depression. AB - OBJECTIVE: Various somatic antidepressant treatments of depression are associated with substantial but limited decreases in T4 levels. This study examined changes in patients' thyroid hormone levels during a period of cognitive behavior therapy. METHOD: Thyroid hormone levels of 30 patients with major depressive disorder were measured before and after they received 20 weekly sessions of cognitive behavior therapy. RESULTS: There were significant decreases in measures of T4 in the 17 responders to cognitive behavior therapy and increases in the 13 nonresponders. CONCLUSIONS: Cognitive behavior therapy has an effect on the thyroid axis similar to that of somatic antidepressant treatments. PMID- 8610832 TI - Fluvoxamine alone in the treatment of delusional depression. AB - OBJECTIVE: The aim of this study was to evaluate the efficacy of fluvoxamine in the treatment of delusional depression. METHOD: Fifty-nine inpatients who met the DSM-III-R criteria for major depression with psychotic features were treated with fluvoxamine for 6 weeks. Patients were assessed at baseline and weekly thereafter with the Hamilton Depression Rating Scale and the Dimensions of Delusional Experience rating scale. RESULTS: Of the 57 subjects completed the trial, 84.2% (N=48) recovered. The index episodes of the patients who did not respond to fluvoxamine were of significantly longer duration than those of the responders. CONCLUSIONS: Fluvoxamine has a response rate similar to that of the currently most efficacious treatments for delusional depression, including antidepressants plus antipsychotics and ECT. PMID- 8610833 TI - Different side effect profiles of risperidone and clozapine in 20 outpatients with schizophrenia or schizoaffective disorder: a pilot study. AB - OBJECTIVE: The purpose of this study was to compare the side effect +profiles of clozapine and risperidone. METHOD: The subjects were 20 outpatients with schizophrenia or schizoaffective disorder who were clinically stable on a regimen of clozapine at the time of screening. They underwent a randomized-order crossover comparison of 6 weeks of risperidone treatment and 6 weeks of clozapine treatment. Clinical and neurocognitive variables were assessed by raters blind to medication status, and severity of side effects was determined from patients' self-reports. RESULTS: Side effect measures, but not clinical ratings, were significantly different after 6 weeks of treatment with the two drugs. Patients required more benztropine for motor effects and complained of more insomnia with risperidone and more sedation with clozapine. Body weight was higher at the end of clozapine treatment than at the end of risperidone treatment. CONCLUSIONS: In this exploratory study, the side effect profiles of clozapine and risperidone were consistent with the different pharmacodynamic profiles of the two drugs. PMID- 8610834 TI - Prospective study of increased platelet membrane fluidity as a risk factor for Alzheimer's disease: results at 5 years. AB - OBJECTIVE: The primary goal of this study was to evaluate increased platelet membrane fluidity as a putative risk factor for Alzheimer's disease. METHOD: This report describes the initial results of a prospective, longitudinal study of 330 initially asymptomatic, first-degree relatives of probands with Alzheimer's disease. RESULTS: Five incident cases of Alzheimer's disease were detected during the first 1,582 subject-years of the follow-up period. The age-specific incidence of Alzheimer's disease was several-fold higher than corresponding figures that were obtained in two prospective community studies. Most important, both age and increased platelet membrane fluidity made significant independent contributions to the risk of developing Alzheimer's disease. CONCLUSIONS: These results validate age and a family history of Alzheimer's disease as risk factors for this disorder and provide the first prospective evidence of increased platelet membrane fluidity as a biological risk factor for Alzheimer's disease. PMID- 8610835 TI - Negative symptoms in Alzheimer's disease. AB - OBJECTIVE: This study was undertaken to demonstrate that negative symptoms are prominent in patients with Alzheimer's disease and are distinct from depression. METHOD: patients with Alzheimer's disease (N=24) and a group of cognitively intact older adults (N=26) were compared in terms of scores on the Scale for the Assessment of Negative Symptoms in Alzheimer's Disease. RESULTS: Negative symptoms were more severe in patients with Alzheimer's disease than in the healthy elderly comparison subjects. Among the patients with Alzheimer's disease, negative symptoms were correlated with dementia severity. CONCLUSIONS: Patients with Alzheimer's disease display little interest in self-care, work and household tasks, social and family activities, and emotional needs of others, despite an absence of depressive symptoms, positive symptoms, comorbid systemic illnesses, or medication exposure. Such negative symptoms may contribute to functional disability and thus complicate management of such patients. PMID- 8610836 TI - Eliminating (almost) treatment dropout of substance abusing or dependent delinquents through home-based multisystemic therapy. AB - OBJECTIVE: This study examined the effects of an innovative treatment model that was designed to reduce treatment dropout among substance abusing or dependent juvenile offenders. METHOD: One hundred eighteen delinquents who met diagnostic criteria for substance abuse or dependence were randomly assigned to receive either home-based multisystemic therapy (N=58) or treatment that was provided by the usual community services (N=60). RESULTS: In the multisystemic therapy condition, 98% (N=57) of the families completed a full course of treatment, which lasted an average of 130 days. In contrast, 78% (N=47) of the families assigned to treatment through the usual community services received no mental health or substance abuse treatment in the 5 months after referral. CONCLUSIONS: The serious and long-standing problem of high dropout rates in the substance abuse field can be greatly attenuated by services that increase accessibility and place greater responsibility for engagement on service providers. PMID- 8610837 TI - Physician caseloads at public mental hospitals. AB - OBJECTIVE: The purpose of the study was to gather baseline data on physician caseloads, particularly psychiatric caseloads, at public mental hospitals. METHOD: A 26-item survey was sent to medical directors of public mental hospitals in the United States and its territories. Questions focused on hospital demographics, programs, and medical staffing. Survey data were analyzed and descriptive statistics were determined. RESULTS: Of 295 surveys mailed, 195 (66%) usable surveys were returned. The hospitals had a mean bed capacity of 347 (SD=301, range=10-1,926), a mean current patient population of 321 (SD=277, range=7-1,815), and 950 mean annual admissions (SD=891, range=3-5,100). Acute care was the most common treatment program (81%), followed by long-term care (71%). A wide range of psychiatric caseloads by type of program existed, with approximately equal mean and median amounts. CONCLUSIONS: The caseloads reported were, overall, reasonable and expected by the type of treatment program. The range of caseloads, however, included extremely high outliers that raise concerns about the quality of care delivered. PMID- 8610838 TI - Fear of thinness. PMID- 8610839 TI - Risperidone treatment of a psychotic adolescent. PMID- 8610840 TI - Bupropion treatment of depression to assist smoking cessation. PMID- 8610841 TI - New-onset auditory hallucinations after right temporal lobectomy. PMID- 8610842 TI - Prolonged bleeding time in a patient treated with sertraline. PMID- 8610843 TI - Porphyria cutanea tarda associated with carbamazepine treatment. PMID- 8610844 TI - Improvement of clozapine-induced sialorrhea after septoplasty. PMID- 8610845 TI - Treatment of clozapine-induced parotid gland swelling. PMID- 8610846 TI - Clozapine treatment of a schizophrenic patient with polydipsia and hyponatremia. PMID- 8610847 TI - Do antidepressants promote tumor growth? PMID- 8610848 TI - Poststroke depression and fluoxetine. PMID- 8610849 TI - Evaluating CBF velocity changes with transcranial Doppler ultrasound. PMID- 8610850 TI - Aging and neuroleptic-induced acute dystonia. PMID- 8610851 TI - Psychopathology in women with HIV disease and drug abuse. PMID- 8610852 TI - Fluoxetine and extrapyramidal side effects. PMID- 8610853 TI - Alternative diagnostic procedures. PMID- 8610854 TI - Genetic anticipation. PMID- 8610855 TI - Etiology of ADHD: nature or nurture? PMID- 8610856 TI - Lidocaine plasma concentrations in pediatric patients after providing airway topical anesthesia from a calibrated device. AB - The aim of this prospective study was to evaluate plasma lidocaine concentrations in infants and children after laryngeal spray using a calibrated device. Twenty one patients aged 3 to 24 mo requiring laryngoscopy or bronchoscopy were included in the study. Anesthesia was induced via a mask with halothane up to 2% in 100% O2. Lidocaine was administered using a 5% lidocaine spray. For patients weighing less than 10 kg, one spray (8 mg of lidocaine) was administered. For those weighing from 10 to 20 kg, two sprays (16 mg) were given. The dose of lidocaine administered ranged between 0.9 and 2.6 mg/kg. Maximum plasma lidocaine concentration (Cmax) was 1.05 +/- 0.55 micrograms/mL (mean +/- SD; range 0.24 2.29 micrograms/mL). With this procedure, we demonstrated the safety of administering lidocaine to children by laryngeal spraying using a 5% sprayer. PMID- 8610857 TI - Auto-positive end-expiratory pressure during one-lung ventilation using a double lumen endobronchial tube. AB - The present study was undertaken to investigate the possible relationships between the magnitude of autopositive end-expiratory pressure (auto-PEEP) and measured PaO2 during one-lung ventilation (OLV). Forty-one adults received OLV anesthesia using a tidal volume of 8 mL/kg and a respiratory rate of 12 breaths/min. Auto-PEEP was quantified using an end-expiratory port occlusion method. During two-lung ventilation (2LV), auto-PEEP was observed in 18 of 41 patients and ranged from 0.5 to 2.5 cm H2O. During OLV, auto-PEEP was observed in 34 of 41 patients and ranged from 0.5 to 10 cm H2O. The mean (+/- SD) value of auto-PEEP was significantly higher during OLV than during 2LV (3.2 +/- 3.3 cm H2O versus 0.5 +/- 0.7 cm H2O, P < 0.0001). Auto-PEEP during OLV correlated inversely with preoperative forced expiratory volume in 1 s/forced vital capacity (y = 12.5 - 0.13x, r = -.05, P < 0.005). During OLV, there was no significant correlation between auto-PEEP and measured PaO2. These findings confirm that many patients do not exhale completely to functional residual capacity during OLV. PMID- 8610858 TI - The use of a circumferential cathode improves amplitude of intraoperative electrical transcranial myogenic motor evoked responses. AB - Measurement of motor evoked responses to transcranial electrical stimulation (tc MER) is a technique for intraoperative monitoring of motor pathways. Since most anesthetics significantly reduce motoneuronal excitability, optimal stimulation paradigms should be sought. We compared the efficiency of stimulus delivery using two different configurations of the cathode component of the stimulating electrode pair (circumferential: Fz, F3, F4, A1, and A2 versus a single cathode at Fz). The anode was positioned at Cz with both cathode configurations. Fourteen neurologically normal patients undergoing spinal surgery were anesthetized with sufentanil-N2O-ketamine. Partial neuromuscular blockade (single twitch height 25%) was maintained with vecuronium. Compound action potentials to transcranial stimulation with both cathode configurations were recorded from the tibialis anterior muscle. All recordings were completed before spinal manipulation. The median amplitude response using the Fz cathode configuration was 256 microV (10th 90th percentiles: 50-641 microV). With the circumferential cathode configuration, tc-MER amplitude increased to 281 (87-1479) microV (P < 0.01). There was no significant difference in onset latency between electrode configurations. The observed tc-MER amplitude augmentation with the use of a circumferential cathode might allow tc-MER monitoring in those patients who do not have sufficiently reproducible responses when a single cathode is used. A possible explanation is that the circumferential cathode alters the direction of the electrical currents in the cortex, resulting in more efficient depolarization of cortical motor neurons. PMID- 8610859 TI - The effects of propofol on the 40-Hz auditory steady-state response and on the electroencephalogram in humans. AB - The auditory steady-state response (ASSR) is a nearly sinusoidal electrical response of the brain to auditory stimuli delivered at fast rates. The amplitude of the response is largest for stimulus rates near 40/s, hence the label 40-Hz ASSR. We have studied the effects of propofol (1.5 mg/kg) on the 40-Hz ASSR in 14 patients. The spectral edge frequency (SEF) and median frequency (MDF) of the electroencephalogram were recorded for comparison. The study was limited to 6 min after the injection of propofol. Recordings were obtained every minute. Consciousness, defined as responsiveness to verbal command, was assessed in all patients within 1 min. Nine patients, regained consciousness before the end of the study. Propofol caused disappearance of the 40-Hz ASSR for 2 min. Th 40-Hz ASSR reappeared afterward, reaching, at the end of the study period, an amplitude of about 65% of baseline. Recovery of the 40-Hz ASSR occurred whether or not consciousness was regained during the study, but the amplitude tended to be larger in patients who had regained consciousness compared with patients still unconscious during the same recording. The SEF was reduced by 24% within 2 min and recovered more quickly than the 40-Hz ASSR, reaching 91% of baseline within 4 min. The SEF tended to be higher in patients who had regained consciousness. The MDF was reduced by 27% within 2 min, and exceeded baseline values by 25% during recovery. The MDF was not higher in patients who had regained consciousness. We conclude that propofol transiently abolishes the 40-Hz ASSR. Recovery of the 40 Hz ASSR occurs whether or not consciousness is regained, but the 40-Hz ASSR tends to be larger after the return of consciousness. An association between higher amplitude 40-Hz ASSR and the return of consciousness could not be conclusively established, perhaps because of low signal-to-noise ratio in three patients. The 40-Hz ASSR did not offer any clear advantage over the SEF in predicting the return of consciousness. PMID- 8610860 TI - Hemodynamic effects of intravenous isoproterenol versus epinephrine in the chronic maternal-fetal sheep preparation. AB - Isoproterenol 5 micrograms may be an effective marker of accidental intravascular injection in women in labor; however, before isoproterenol can be incorporated in routinely used epidural test doses, the safety and usefulness should be determined in an animal model. This study was designed to examine the hemodynamic effects of isoproterenol in comparison with epinephrine in the pregnant ewe. Five doses of isoproterenol were tested and compared with two doses of epinephrine in a randomized cross-over fashion. After administration of isoproterenol there was a small decrease of uterine blood flow (UBF) and maternal mean artery pressure (MMAP), which both almost immediately returned to baseline. When epinephrine was used a more pronounced and more prolonged decrease of UBF occurred. Increasing doses of isoproterenol resulted in dose-dependent increases in maternal heart rate (MHR), while with epinephrine this was not the case. A significant increase in the cardiac output was seen after isoproterenol. Neither isoproterenol nor epinephrine affected fetal heart rate (FHR), fetal mean arterial pressure (FMAP), amniotic fluid pressure (Amn-pr), blood gases, or acid base status in the mother and the fetus. Provided that neurotoxic effects are absent, isoproterenol might be a better alternative than epinephrine as a test dose for possible intravenous placement of an epidural catheter in pregnant women. PMID- 8610861 TI - Effect of cesarean delivery on perioperative regulation of the beta-adrenergic receptor system of human lymphocytes. AB - We studied the perioperative regulation of the beta-adrenergic receptor (beta AR) system in lymphocytes obtained before and after surgery from 12 patients undergoing cesarean delivery with spinal anesthesia. Receptor number (Bmax) and binding affinity (KD) were determined by Scatchard analysis of [125I]iodopindolol saturation binding curves. Receptor function was assessed by measuring cyclic adenosine 3',5'-monophosphate (cAMP) production in the unstimulated state and in response to stimulation by isoproterenol, forskolin, and prostaglandin E1. Basal cAMP production increased 48% postoperatively (P < 0.05), while stimulated cAMP production and Bmax and KD were not significantly changed after surgery. The response to surgery of the beta AR system on these patients differed from that of patients undergoing cardiothoracic and abdominal surgery in whom we previously found postoperative down-regulation and desensitization of the beta AR system. It is possible that this difference in response is due to amelioration of the stress response to surgery by regional anesthesia, and/or alterations in beta AR status by pregnancy. We conclude that pregnancy and regional anesthesia, but not changes in lymphocyte subset distribution, contributed to the lack of effect of surgery on the beta AR system in the patients studied. PMID- 8610862 TI - Bolus metoclopramide does not enhance morphine analgesia after cesarean section. AB - Intravenous metoclopramide potentiates the analgesic effects of opioids in postoperative patients. We speculate that increased spinal concentrations of acetylcholine from metoclopramide-induced acetylcholinesterase inhibition is the mechanism responsible for enhanced morphine analgesia from metoclopramide. Sixty patients undergoing elective cesarean section with subarachnoid anesthesia were randomized to receive either 20 mg metoclopramide or saline intravenously 30-60 min prior to subarachnoid injection. Prior to subarachnoid injection of local anesthetic, 2 mL of cerebrospinal fluid (CSF) was aspirated for cholinesterase activity measurement. Visual analog scale (VAS) scores for pain were obtained prior to drug administration, at first patient request for analgesia, and at discharge from the postanesthesia care unit. Total morphine use was recorded in the recovery room and for 24 h postoperatively. There were no significant differences in VAS scores, morphine use, or CSF cholinesterase activity between groups. CSF cholinesterase activity was similar to values in nonpregnant patients demonstrated previously. This study failed to confirm the morphine-enhancing action of 20 mg intravenous metoclopramide in postoperative patients. Furthermore, this dose of metoclopramide does not inhibit CSF acetylcholinesterase. PMID- 8610863 TI - Postarthroscopic meniscus repair analgesia with intraarticular ketorolac or morphine. AB - Both ketorolac, a nonsteroidal antiinflammatory drug, and morphine, an opioid agonist, provide enhanced patient analgesia after arthroscopic knee surgery when administered via the intraarticular route. This study was designed to determine whether ketorolac or morphine results in better patient analgesia and whether their combination would provide superior analgesia to either drug alone. Patients undergoing arthroscopic knee meniscus repair under local anesthesia with sedation were evaluated. Each of the study groups evaluated received ketorolac (either via the parenteral or intraarticular route) and morphine (via either the parenteral or intraarticular route). This study revealed a significant benefit from the individual intraarticular administration of both morphine and ketorolac. The combination of these drugs did not result in decreased postoperative pain or need for postoperative analgesics, and it did not result in an increased analgesic duration. We conclude that the use of either intraarticular ketorolac or intraarticular morphine improves the comfort in patients undergoing arthroscopic meniscus repair and that their combination offers no advantage over either drug alone. PMID- 8610864 TI - Dural puncture with a 26-gauge spinal needle affects spread of epidural anesthesia. AB - Combined spinal and epidural anesthesia may increase the risk of epidurally administered drugs spreading into the subarachnoid space through the dural hole. We studied the effect of dural puncture with a 26-gauge needle on the spread of analgesia induced by epidural injection of local anesthetics. Forty patients were randomly assigned to control and dural puncture groups. In the dural puncture group, the dura was punctured with a 26-gauge Whitacre spinal needle at L2-3 but no drug was injected. In both groups, an 18-gauge epidural catheter was inserted 4 cm cephalad into the epidural space at L2-3 and 15 mL of 2% mepivacaine without epinephrine was injected. Analgesia was assessed by pinprick at 5, 10, 15, and 20 min after injection and at the end of surgery. The caudal spread of analgesia was significantly greater in the dural puncture group than in the control group 15 and 20 min after injection (P < 0.01), but the cranial spread of analgesia was not different between the two groups. We conclude that dural puncture (without drugs) using a 26-gauge Whitacre spinal needle before epidural injection increases caudal spread of analgesia induced by epidural local anesthetics. PMID- 8610865 TI - A multidimensional comparison of morphine and hydromorphone patient-controlled analgesia. AB - Although patient-controlled analgesia (PCA) pumps have been in use for more than a decade, the optimal PCA analgesic has yet to be identified. Many drugs are used; however, morphine remains the "gold standard" of opioid analgesics worldwide. The present study evaluated morphine and hydromorphone (Dilaudid) PCA with respect to analgesic efficacy, side effects, mood, and cognitive function. Sixty-one opioid naive patients undergoing lower abdominal surgery participated in the double-blind protocol. Verbal rating scores, use of medication, and side effects for the two medications were recorded. Cognitive functioning was assessed by computation of Digit Symbol and Trails Making B Tests. Self-reported affective state (mood) was measured by Profile of Mood States (POMS) inventory. Both medications provided adequate analgesia without a difference in side effects. Cognitive performance was poorer in the hydromorphone group (P < 0.05). Patients receiving hydromorphone reported less anger/hostility (P < 0.01) and generally better mood elevations on the other subscales than those receiving morphine. A similar incidence of side effects and dose medication can be anticipated with morphine and hydromorphone. When considering cognitive effects, morphine had less adverse consequences, while hydromorphone appeared to result in improved mood. We conclude that hydromorphone may provide a suitable alternative to morphine. PMID- 8610866 TI - The effect of thoracic epidural anesthesia on hypoxic pulmonary vasoconstriction in dogs: an analysis of the pressure-flow curve. AB - The aim of the present study was to examine whether hypoxic pulmonary vasoconstriction (HPV) is preserved during one-lung ventilation combined with thoracic epidural anesthesia (TEA) in dogs. Using a separately ventilated left lower lobe (LLL) in situ, the pressure-flow (P-Q) curve was obtained. The HPV response was assessed by the shift of the P-Q curve, changes in blood flow diversion rate (FDR) and decrease in PaO2 during hypoxic gas ventilation of LLL. In the control group (n = 7), the shift of P-Q curve, changes in FDR, and decrease in PaO2 remained constant during four consecutive hypoxic stimulations. In the TEA group (n = 6), the P-Q curve shifted to the left during hyperoxia, but the magnitude of the shift during hypoxia was unchanged. FDR and decrease in PaO2 were significantly reduced compared with baseline values (P < 0.05 with analysis of variance). TEA reduced heart rate, cardiac output, mean arterial pressure, mean pulmonary arterial pressure, and mixed venous oxygen tension. Our results suggest that TEA did not affect the primary pulmonary vascular tone at baseline or during lobar hypoxia, but enhanced the diversion of blood flow and arterial blood oxygenation during lobar hypoxia. This enhanced HPV response probably reflects hemodynamic changes, such as decreased cardiac tension, due to sympathetic nerve activity blockade by TEA. PMID- 8610867 TI - Epidural test dose: isoproterenol is a reliable marker for intravascular injection in anesthetized adults. AB - Epidural test doses containing more than 15 micrograms epinephrine are reliable for the detection of intravascular injection based on the conventional systolic blood pressure (SBP) criterion (positive if > or = 15 mm Hg increase) but not on the heart rate (HR) criterion (positive if > or = 20 bpm increase) in adult patients anesthetized with isoflurane. The present study was designed to test whether isoproterenol could be used as a reliable marker. Thirty adult patients were randomly assigned to one of two groups, each of which was anesthetized with 1% end-tidal isoflurane and nitrous oxide after endotracheal intubation. Isoproterenol group (n = 15) was given 3 mL of 1.5% lidocaine with 3 micrograms isoproterenol intravenously (i.v.) to simulate an i.v. administered test dose. The saline group (n = 15) was identical to the isoproterenol group but received 3 mL of normal saline. HR and arterial blood pressure were measured at 20-s intervals for 4 min after i.v. injection. Mean HR in the isoproterenol group was significantly higher than in the saline group from 40 to 240 s after i.v. test dose with a mean maximum HR increase of 32 +/- 7 bpm (mean +/- SD) occurring at 68 +/- 19 s. All 15 patients in the isoproterenol group developed HR increases > or = 20 bpm (sensitivity 100%). Since HR was essentially unchanged in the saline group, specificity and positive and negative predictive values were all 100%. On the other hand, 12 of 15 patients in the isoproterenol group and none in the saline group exhibited SBP increases > or = 15 mm Hg, resulting in 80% sensitivity and 83% negative predictive value. In the isoproterenol group, however, transient systolic hypotension ( < 80% of the preinjection value) occurred in five patients without untoward clinical sequelae. These results indicate that, based on the peak HR response, the epidural test dose containing 3 micrograms isoproterenol is a reliable marker for intravascular injection in adult patients during isoflurane anesthesia. PMID- 8610868 TI - The role of hyaluronidase on lidocaine and bupivacaine pharmacokinetics after peribulbar blockade. AB - Orbital regional anesthesia is the only circumstance where hyaluronidase is routinely added to local anesthetics to accelerate the onset of the block. The aim of this study was to compare the pharmacokinetics of lidocaine and bupivacaine with or without hyaluronidase for peribulbar blockade. Twenty-one patients scheduled for cataract surgery with lens implantation were included in this prospective randomized study. Peribulbar blocks were achieved with plain bupivacaine 0.5% (5.5 mL), lidocaine 2% (5.5 mL), and hyaluronidase (100 IU = 2 mL) (n = 10) ir sterile water (2 mL) (n = 11). Plasma bupivacaine and lidocaine concentrations were measured by high-performance liquid chromatography at regular intervals from the end of the local anesthetic injection until the 360th minute. Maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were obtained for all the patients except one who needed a supplementary injection and was excluded from the study. The time to onset and duration of the analgesia and akinesia were monitored at the times of sampling. Motor blockade was incomplete in two patients in each group without affecting surgery. The Tmax and absorption half-life (t1/2a) of lidocaine and bupivacaine were not different within each group (P > 0.05). The Tmax of lidocaine was shorter in the presence of hyaluronidase (17.1 +/- 2.6 min vs 32.7 +/- 6.0 min) as well as the Tmax of bupivacaine (16.8 +/- 3.0 min vs 26.5 +/- 4.4 min). The Cmax of lidocaine and bupivacaine were not modified by the addition of hyaluronidase. The clearance, terminal half-life, and volume of distribution were not different between groups. The absorption of lidocaine and bupivacaine from the peribulbar space are hastened by the addition of hyaluronidase. The Tmax of lidocaine is not different from that of bupivacaine within each group suggesting that the absorption of local anesthetics is minimally influenced by the liposolubility of the drugs. Moreover, hyaluronidase influences the absorption kinetics of both lidocaine and bupivacaine in the same manner. PMID- 8610869 TI - Video as a patient teaching tool: does it add to the preoperative anesthetic visit? PMID- 8610870 TI - Epidural abscess--early magnetic resonance imaging detection and conservative therapy. PMID- 8610871 TI - Epidural hematoma associated with enoxaparin. PMID- 8610872 TI - Intrathecal hyperbaric 0.5% tetracaine as a possible cause of transient neurologic toxicity. PMID- 8610873 TI - Delayed bronchial stenosis after blunt chest trauma. PMID- 8610874 TI - Hypotension resistant to therapy with alpha receptor agonists complicating cardiopulmonary bypass: lithium as a potential cause. PMID- 8610875 TI - Intraoperative ventricular tachycardia responsive to adenosine. PMID- 8610876 TI - Right ventricular perforation by a pulmonary artery catheter during coronary artery bypass surgery. PMID- 8610877 TI - The use of small-dose intravenous nitroglycerin in a case of uterine inversion. PMID- 8610878 TI - Recurrence of herpes simplex virus blepharitis after cesarean section and epidural morphine. PMID- 8610879 TI - Refractory idiopathic erythromelalgia. PMID- 8610880 TI - Failure of intraoperative liquid crystal temperature monitoring. PMID- 8610881 TI - Slow awakening after electroconvulsive therapy due to unrecognized attempted suicide. PMID- 8610882 TI - Anaphylactic reaction to latex in health care workers. PMID- 8610883 TI - Preventing desaturation during single-lung anesthesia. PMID- 8610884 TI - Raising the operating table causing a sudden anesthesia system obstruction. PMID- 8610885 TI - A safer approach to retrograde-guided fiberoptic intubation. PMID- 8610886 TI - Crystalloids, colloids, and spinal anesthesia. PMID- 8610887 TI - Transesophageal atrial pacing in patients with permanent pacemakers. PMID- 8610888 TI - Paraplegia in a patient who by chance missed the insertion of an epidural catheter. PMID- 8610889 TI - Optimal loading dose of milrinone for weaning from cardiopulmonary bypass. PMID- 8610890 TI - Lidocaine and no carrier fluid for the prevention of pain due to injection of propofol. PMID- 8610891 TI - Invalid determination of renal blood flow during hypothermic cardiopulmonary bypass. PMID- 8610892 TI - Mallampati grade and laryngeal mask placement. PMID- 8610893 TI - Additional test to confirm placement of pediatric lines. PMID- 8610894 TI - The arterial to end-tidal carbon dioxide difference in neurosurgical patients during craniotomy. PMID- 8610895 TI - Does mannitol save the kidney? PMID- 8610896 TI - Effects of hyperosmotic mannitol infusion on hemodynamics of dog kidney. AB - This study evaluated the effect of systemic infusion of hypertonic mannitol on renal hemodynamics (aortic pressure [P]-renal blood flow [RBF] relationship, glomerular filtration rate [GFR], and effective renal plasma flow [ERPF]) during 50% reduction of left kidney blood flow. Conditioned mongrel dogs anesthetized with halothane were hydrated by continuous infusion of lactated Ringer's solution containing creatinine to measure GFR and p-aminohippurate (PAH), to measure ERPF. The left kidney was exposed and two hydraulic occluders were placed, one around the aorta just above the renal arteries and the other around the left renal artery. Experimental design consisted of measuring P near the left renal artery, RBF by electromagnetic flowmeter, and ERPF and GFR by clearance methods in both kidneys in response to stepwise reduction in the aortic pressure by aortic occlusion before and after 50% reduction in the left kidney blood flow. The P-RBF relationship, GFR, and ERPF thus obtained were compared with those obtained during systemic intravenous infusion of 20% mannitol for a period of 1 h. We found that 1) a transient increase occurred in RBF with step reduction of P from 80 to 60 mm Hg under control conditions; 2) reducing the RBF by 50% changed the shape of the P-RBF relationship from a convex to the P axis to a linear form with a marked shift toward the P axis; 3) infusion of mannitol, during reduced RBF, caused a significant shift of the P-RBF curve toward the RBF axis and returned the linear P-RBF relationship toward normal, but had no effect on altered yield pressure; and 4) infusion of hypertonic mannitol had slightly increased GFR and ERPF in the right (unconstricted) kidney. However, hypertonic mannitol significantly increased GFR and ERPF values in the left (constricted) kidney suggesting a beneficial effect of mannitol on ischemic kidney. The results are consistent with the hypothesis that infusion of hypertonic mannitol to ischemic kidney increases RBF, presumably by decreasing the intrarenal vascular resistance. We speculate that this compensatory response may be mediated either 1) by stimulating the release of a vasodilator substance (e.g., prostaglandins), or 2) by washing out interstitial sodium, thereby reducing the sensitivity of the renal vasculature to ischemia-induced stimulation of renin-angiotensin system. PMID- 8610897 TI - The effects of sevoflurane and isoflurane anesthesia on renal tubular function in patients with moderately impaired renal function. AB - Increasing evidence indicates that sevoflurane anesthesia does not impair renal function in healthy patients despite higher concentrations of plasma inorganic fluoride. However, whether sevoflurane further affects renal tubular function in patients with impaired renal function is not known. We compared the effect of sevoflurane anesthesia with that of isoflurane anesthesia on renal tubular function in patients with moderately impaired renal function. Fourteen patients with creatinine clearance between 10 and 55 mL/min were anesthetized with either sevoflurane or isoflurane using a semiclosed circuit system. Plasma inorganic fluoride concentrations and urine N-acetyl-beta-D-glucosaminidase (NAG), gamma glutamyltranspeptidase (gamma-GTP), and beta 2-microglobulin (beta 2MG) excretions were measured up to post-anesthetic day 14. Although both the peak plasma inorganic fluoride concentrations and the areas under the curve of plasma inorganic fluoride concentration versus time were significantly greater in the sevoflurane group than in the isoflurane group, urine NAG, gamma-GTP, and beta 2MG excretions per day did not differ between the two groups. These results indicate that sevoflurane and isoflurane may have similar effects on the renal tubules in patients with moderately impaired renal function. PMID- 8610898 TI - Spectral analysis of arterial pressure variability during induction of propofol anesthesia. AB - We studied the effect of continuous infusion of propofol on spectral components in systemic arterial pressure (SAP) signals in 35 consenting patients undergoing abdominal surgery. Anesthesia was induced with intravenous bolus administration of propofol (2.0 mg/kg), followed by infusion at either 5 mg.kg-1.h-1 (Group 1, n = 18) or 10 mg.kg-1.h-1 (Group 2, n = 17). Tracheal intubation was facilitated by administration of vecuronium (0.1 mg/kg). The SAP signal was subjected to off line spectral analysis to obtain changes in power of the very low frequency (VLF; 0.00-0.08 Hz), low frequency (LF; 0.08-0.15 Hz), high frequency (0.15-0.25 Hz); and very high frequency (VHF; 0.80-1.60 Hz) components. Venous blood for the measurement of plasma concentration of propofol was collected at 5 min before bolus injection of propofol; at 5, 10, and 15 min after infusion of propofol; and immediately after endotracheal intubation. Infusion of propofol significantly decreased the total power of SAP spectrum in both groups, especially the VLF, LF, and VHF components at all intervals except postintubation. Immediately after tracheal intubation, patients in Group 1 showed a significant increase in mean arterial pressure when compared with those in Group 2 (118 +/- 5 mm Hg vs 102 +/- 5 mm Hg, P < 0.05). Similar change was also seen in the VLF component (7.4 +/- 0.7 mm Hg2 vs 4.4 +/- 0.5 mm Hg2, P < 0.05). After tracheal intubation, patients in Group 1 showed 15.7-, 3.3-, and 4.4-fold increase in the VLF, LF, and VHF components, respectively. There were 14.1-, 2.8-, and 2.8-fold increases in the respective components of the SAP signal in Group 2. At all intervals, the spectral components of SAP, however, did not correlate well with the plasma concentration of propofol in either group. These results suggest that spectral analysis of SAP signals may provide an alternative for assessing autonomic activities, such as the sympathetic response, to tracheal intubation during propofol anesthesia. PMID- 8610899 TI - The effect of a cyclodextrin vehicle on the cardiovascular profile of propofol in rats. AB - We studied the aqueous solution of propofol dissolved in hydroxypropyl-beta cyclodextrin (HP beta CD) 20% to determine whether the cardiovascular profile differed from that measured for propofol prepared in Intralipid 10% (Diprivan). Conscious male rats were given an intravenous bolus of propofol, 5.0 mg/kg, the minimum dose that induces a loss of righting. Immediately severe bradycardia occurred which was the result of a combination of sinus arrest and atrioventricular block; a significant decrease of blood pressure resulted. A bolus of HP beta CD produced no significant changes in heart rate rhythm. The severe bradycardia produced by propofol in HP beta CD was blocked by both atropine and bilateral cervical vagotomy. Therefore, the effects of propofol in HP beta CD are cholinergic and neurally mediated. These results are consistent with the hypothesis that propofol reduces sympathetic tone prior to reduction in vagal tone, and thereby produces a period of time during which vagal tone is dominant. PMID- 8610900 TI - Rapid core-to-peripheral tissue heat transfer during cutaneous cooling. AB - Perioperative thermal manipulations are usually directed at the skin surface because methods of directly warming the core are invasive or ineffective. However, inadequate heat flow between peripheral and core compartments will decrease the rate at which core temperature changes. We therefore determined whether core hypothermia is delayed after initiation of surface cooling. Six volunteers were anesthetized with propofol and midazolam, and maintained under three layers of passive insulation for 2.5-4 h. Subsequently, the skin surface was cooled using forced air, 1000 L/min, at 10 degrees C. Isoflurane was added as necessary to maintain arteriovenous shunt vasodilation. Overall heat balance was determined from the difference between cutaneous heat loss (thermal flux transducers) and metabolic heat production (oxygen consumption). Average arm and leg (peripheral) tissue temperatures were determined from 19 intramuscular needle thermocouples, 10 skin temperatures, and "deep" foot temperature. Overall body heat content decreased approximately 234 kcal during 2.5 h of active cooling. Core temperature, which was nearly constant before active cooling, decreased approximately 1.3 degrees C/h. There was no delay between initiation of active cooling and the decrease in core temperature. Furthermore, peripheral (arm and leg) and core (trunk and head) tissue heat contents decreased at virtually the same rates: approximately 50 kcal/h and approximately 47 kcal/h, respectively. These data indicate that there is little restriction of heat flow between peripheral and core tissues in vasodilated, anesthetized subjects. PMID- 8610901 TI - The effects of transnasal butorphanol on mood and psychomotor functioning in healthy volunteers. AB - Transnasal butorphanol is effective in relieving migraine and postoperative pain. The extent to which this drug preparation impacts on cognitive and psychomotor performance, as well as mood, has not been examined. Accordingly, the cognitive and psychomotor, subjective, and physiological effects of two clinically relevant doses of transnasal butorphanol (1 and 2 mg) were compared to that of placebo, and a common analgesic drug combination given for pain relief in ambulatory settings, 600 mg of acetaminophen and 60 mg of codeine, in healthy volunteers (n = 10). The larger transnasal butorphanol dose impaired psychomotor performance for up to 2 h, and produced subjective effects for up to 3 h. The smaller dose had no psychomotor-impairing effects, but had subjective effects (including increased ratings of "sleepy"). All three active drug conditions including miosis. These laboratory results suggest that patients should use caution when using the 1-mg dose of transnasal butorphanol, and should curtail certain activities if they administer the 2-mg dose of transnasal butorphanol for analgesia. PMID- 8610902 TI - Identification of cytochrome P450 3A1/2 as the major P450 isoform responsible for the metabolism of fentanyl by rat liver microsomes. AB - The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism. Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl.min-1.mg-1 microsomal protein, and the apparent Km values were 32 and 47 microM, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes. PMID- 8610903 TI - The effect of sevoflurane and isoflurane on the neuromuscular block produced by vecuronium continuous infusion. AB - Volatile anesthetics enhance the action of neuromuscular blockade to various degrees, although the influence of sevoflurane on the neuromuscular block has not yet been characterized. The purpose of this investigation is to determine the vecuronium infusion rate requirement under sevoflurane anesthesia and to compare it to that of isoflurane anesthesia. Twenty patients scheduled for otorhinolaryngologic surgery were randomly assigned to receive either sevoflurane (SEV) or isoflurane (ISO) at 1 minimum alveolar anesthetic concentration (MAC) (1.7% and 1.2%, respectively) in combination with 67% nitrous oxide. Anesthesia was induced with 5 mg/kg thiopental and muscle relaxation was obtained by a bolus of vecuronium infusion to achieve 90% muscle relaxation. The plasma concentrations of vecuronium (CVEC) and 3-desacetylvecuronium (CDES) at steady state were measured with a gas chromatographic assay. There was no difference between SEV and ISO in the following variables: the vecuronium infusion rate requirements to achieve 90% muscle relaxation (0.42 +/- 0.11 [SEV] vs 0.40 +/- 0.10 [ISO] microgram-kg-1.min-1), CVEC (144.4 +/- 38.1 [SEV] vs 149.7 +/- 69.2 [ISO] ng/mL), CDES (57.2 +/- 20.3 [SEV] vs 65.3 +/- 26.1 [ISO], ng/mL), and plasma vecuronium clearance (2.85 +/- 0.86 [SEV] vs 3.19 +/- 1.24 [ISO] mL.kg 1.min-1). This study indicates that SEV at 1 MAC requires a vecuronium infusion rate similar to that of ISO at 1 MAC to achieve 90% muscle relaxation. PMID- 8610904 TI - Lipoteichoic acid from Staphylococcus aureus depresses contractile function of human arteries in vitro due to the induction of nitric oxide synthase. AB - The aim of this study was to clarify the role of Gram-positive organisms in the genesis of sepsis. In the present study, we investigated the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus on contractions elicited by norepinephrine (NE) in rings cut from human gastroepiploic arteries. LTA diminished the contractile response to NE. This attenuation began after several hours of exposure, whether or not endothelium was present. The cyclic guanosine monophosphate content of LTA-treated rings was higher than that of control rings, whether there was a functional endothelium. These LTA-mediated responses were reduced significantly by inhibitors of nitric oxide (NO) synthase and guanylate cyclase. All of this indicates that the main underlying cause of the vascular hyporeactivity to NE was a massive generation of No. In addition, cycloheximide, an inhibitor of inducible NO synthase, prevented the attenuation of NE-induced contractions caused by LTA. Thus, our results offer strong supporting evidence that the important factor in the genesis by Gram positive organisms of a diminished contractile response to pressor drugs is their induction of inducible NO synthase in smooth muscle. PMID- 8610905 TI - The in vitro effects of phosphodiesterase inhibitors on the human internal mammary artery. AB - The internal mammary artery (IMA) is the preferred conduit for myocardial revascularization, but it changes diameter in response to injury or thromboxane release to decrease myocardial blood supply. Papaverine, a phosphodiesterase (PDE) inhibitor, is injected in the IMA bed during surgery to prevent spasm. We evaluated the ability of papaverine and cyclic adenosine monophosphate PDE Type III (cAMP-PDE) inhibitors (amrinone, enoximone, and milrinone) in vitro to reverse the constriction of human IMA rings, induced by a thromboxane A2 analog, U46619, and evaluated amrinone's ability to modify the constricting effect of norepinephrine (NE). All cAMP-PDE inhibitors produced complete relaxation of U46619-induced contractions. The contractions necessary to produce 50% relaxation (EC50) were within therapeutic ranges. The vasodilatory potency of amrinone was greater after NE than after U46619 (EC50, 1.9 +/- 0.5 vs 4.3 +/- 2.2 x 10(-5)M; mean +/- SD; P < 0.05). Response to constriction after a submaximal dose of NE was attenuated to 38% (P < 0.001) from that observed in the control rings by a pretreatment with amrinone. These results suggest that cAMP-PDE inhibitors have the potential utility to reverse IMA spasm, and represent a potential therapeutic modality for IMA spasm after myocardial revascularization. PMID- 8610906 TI - Perioperative distribution of pulmonary vascular resistance in patients undergoing coronary artery surgery. AB - This study was undertaken to measure distribution of pulmonary vascular resistance (PVR) perioperatively in patients undergoing coronary artery bypass grafting (CABG) and to examine the effects of cardiopulmonary bypass (CPB) on pulmonary capillary pressure (Pc) relative to wedge pressure (Pw). Pulmonary artery catheters were placed before anesthetic induction in 18 patients scheduled for elective CABG and systemic hemodynamic variables were measured. Pulmonary artery pressure was recorded during balloon inflation and stored for off-line determination of Pc. Data were collected prior to induction (baseline), as well as after induction and intubation, skin incision, sternotomy, protamine administration, and chest closure. At each data point, downstream (capillary plus venous segments) resistance (Rds) contributed approximately 60% of total PVR and did not change significantly during the operation. PVR decreased (P < 0.05) after CPB and protamine administration, primarily due to a decrease in the absolute magnitude of the upstream (arterial) resistance. Administration of large-dose opioid anesthesia had no significant effect (P > 0.05) on total PVR or on segmental distribution of vascular resistance. At all data points, Pc was significantly larger than Pw (P < 0.05). This study demonstrates that perioperative measurement of Pc is feasible, that during CABG under these conditions, relative contribution of arterial and venous resistances remain relatively unchanged, that Pc is always larger than Pw, and that the administration of large-dose opioid anesthesia has a minimal effect on pulmonary vascular hemodynamics. PMID- 8610907 TI - Jugular venous bulb oxyhemoglobin saturation during cardiac surgery: accuracy and reliability using a continuous monitor. AB - Previous studies have demonstrated the feasibility of continuously monitoring jugular venous oxygen saturation (SjO2) with a fiberoptic catheter during hypothermic cardiopulmonary bypass (CPB). In the present study, with patients maintained at either moderate (28 degrees C) or mild (32-34 degrees C) hypothermia during CPB, SjO2 values obtained from a fiberoptic catheter were compared to intermittent samples analyzed by a co-oximeter. Twenty patients scheduled for elective coronary artery or valvular surgery had a 5.5 Fr Opticath catheter inserted into the left internal jugular bulb after induction of general anesthesia. The catheter was calibrated in vitro and in vivo according to the manufacturer's specifications. Catheter and co-oximetry SjO2 values obtained at four time points--1) pre-CPB, 2) target CPB temperature, 3) mid-rewarming, and 4) post-CPB--were compared using linear regression, Bland-Altman analysis, and Shrout-Fleiss interclass correlation coefficient analysis. These statistical methods revealed poor correlation between the catheter and co-oximetry SjO2 values: r = 0.44 by linear regression and 0.32 by interclass correlation coefficient analysis, and was unacceptably discrepant by Bland-Altman analysis. Oxyhemoglobin saturation values obtained continuously from a jugular venous bulb fiberoptic catheter during CPB may not accurately reflect true oxyhemoglobin saturation, and caution is warranted when interpreting SjO2 values obtained from a fiberoptic catheter during CPB. PMID- 8610908 TI - Steady-state myogenic response of rat coronary microvessels is preserved by isoflurane but not by halothane. AB - The myogenic response of vascular smooth muscle produces vasomotion in response to changes in vessel transmural pressure. While this is an important determinant of coronary blood distribution, the effect of volatile anesthetics on the response has not been previously investigated. In this study, we examined the effect of isoflurane and halothane on this myogenic response. Coronary resistance arteries were isolated from Wistar rats. As the intraluminal pressure was increased from 10 to 120 mm Hg in the presence of either isoflurane (1%, 2%, and 3%), halothane (1% and 2%), or no volatile agent (control), the vessel intraluminal diameter was monitored using a video detection system. Passive changes in vessel diameter were measured after exposure to papaverine 100 microM. Additionally, the myogenic responses of endothelium-intact and endothelium denuded vessels were compared. Endothelium-intact control vessels demonstrated myogenic constriction above 80 mm Hg of intraluminal pressure. This response was not affected by endothelial denudation. The response was preserved by isoflurane 1%, 2% or 3% but abolished by halothane 1% or 2%. We conclude that, in rat coronary resistance arteries, myogenic constriction can be demonstrated above 80 mm Hg of intraluminal pressure and is endothelium independent. This response is preserved by isoflurane but abolished by halothane. These findings may have implications for the effect of the anesthetics on coronary blood flow distribution. PMID- 8610909 TI - Interaction of heart rate and hypothermia on global myocardial contraction of the isolated rabbit heart. AB - We studied the effects of mild hypothermia on cardiac contractility in isolated rabbit hearts perfused with Krebs-Henseleit solution according to the technique of Langendorff. Isovolumetric left ventricular pressure (LVP) was measured with a fluid-filled balloon. Hearts were paced after induction of atrioventricular block. At low heart rates ( < 30 bpm) mild hypothermia (cooling to 30 degrees C) induced a 32% increase in LVp (146.5 +/- 10 mm Hg at 30 degrees C vs 110.7 +/- 13 mm Hg at 37 degrees C) but this positive inotropic response was progressively lost by increasing heart rate. At pacing rates > or = 90 bpm, lower systolic LVP, higher diastolic LVP, and lower positive and negative LV dP/dt were obtained in hypothermic (93 +/- 12 mm Hg, 55 +/- 18 mm Hg, 584 +/- 137 mm Hg/s, and 323 +/- 57 mm Hg/s at 210 bpm, respectively) compared to normothermic hearts (123 +/- 4 mm Hg, 10 +/- 4 mm Hg, 1705 +/- 145.5 mm Hg/s, and 1155 +/- 78 mm Hg/s at 210 bpm, respectively.) The duration of mechanical diastole was reduced or suppressed in these hearts. Exposure to the beta-adrenoreceptor agonist, isoproterenol, improved this diastolic dysfunction during hypothermia and pacing at high rates, suggesting that the sarcoplasmic reticulum Ca2+ uptake might be involved. Our data are also consistent with an increase in myofilament Ca2+ sensitivity that is opposed by isoproterenol during hypothermia. PMID- 8610911 TI - Tracheal extubation of children in the operating room after atrial septal defect repair as part of a clinical practice guideline. AB - Early tracheal extubation in the operating room after atrial septal defect (ASD) surgery was recommended as part of a clinical practice guideline (CPG) established in the Cardiovascular Program at the Children's Hospital, Boston, MA. This retrospective review was undertaken to determine whether this practice was efficient without compromising patient care. The charts and hospital charges for 102 patients undergoing secundum ASD or sinus venosus defect surgery between March 1992 and July 1994 were reviewed; 36 patients (Group I) had surgery prior to introduction of the CPG, and 66 patients were managed according to the CPG. Of the latter, 25 patients (Group II) were tracheally extubated in the operating room (OR) and 41 patients (Group III) were extubated in the cardiac intensive care unit (CICU). Patients in all three groups were similar with respect to height, weight, and surgical conditions including cardiopulmonary bypass time, lowest esophageal temperature, hematocrit, total OR time, and the time from completion of bypass to leaving the OR. Patients in Group II received significantly less fentanyl during anesthesia, were more likely to have a respiratory acidosis on admission to the CICU, and had an increased frequency of vomiting in the CICU. There was no difference in duration of CICU stay among groups. The length of hospital stay was reduced in Groups II and III after introduction of the CPGs, but was not influenced by tracheal extubation in the OR. There was no difference among groups in the hospital charges for OR, anesthesia and CICU time. However, when the combined hospital charges for services provided both in the OR and CICU were included, patients in Group II were charged significantly less, and this primarily reflects the absence of postoperative mechanical ventilation charges. Tracheal extubation in the OR after ASD surgery in children can result in lower patient charges without significantly compromising patient care. PMID- 8610910 TI - Sigma receptor activation does not mediate fentanyl-induced attenuation of muscarinic coronary contraction. AB - Our overall goal was to investigate the mechanism by which fentanyl attenuates acetylcholine-induced contraction in porcine coronary artery. We tested the hypothesis that fentanyl attenuates muscarinic coronary contraction via sigma receptor activation. Left coronary artery vascular rings were isolated from porcine hearts and were suspended in organ chambers for isometric tension recording. In untreated coronary vascular rings, acetylcholine administration resulted in dose-dependent contraction. Fentanyl attenuated acetylcholine-induced contraction. The sigma ligands--(+)-pentazocine, (+)-cyclazocine, haloperidol, and 1,3-di-o-tolylguanidine--also inhibited acetylcholine-induced contraction. In contrast, the selective sigma ligand, (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine failed to have an inhibitory effect on acetylcholine-induced contraction. Moreover, metaphit (1-[1(3 isothiocyanatophenyl)cyclohexyl]piperidine), which causes irreversible acylation of sigma receptors, only inhibited acetylcholine-induced contraction when it was present in the organ chamber. We also assessed the effects of inhibiting various points in the signal transduction pathway distal to naloxone-sensitive opioid receptor activation on acetylcholine-induced contraction. Selective (glybenclamide) and nonselective (tetraethylammonium) K(+)-channel inhibition, guanosine triphosphate-binding protein inactivation (pertussis toxin), and Type 1 and Type 2 dopamine receptor inhibition all failed to alter the attenuating effect of fentanyl on acetylcholine-induced contraction. Thus, neither sigma or opioid receptor activation is a prerequisite for fentanyl-induced inhibition of muscarinic coronary contraction. PMID- 8610912 TI - Comparison of rocuronium and mivacurium to succinylcholine during outpatient laparoscopic surgery. AB - Tracheal intubating conditions and neuromuscular effects of succinylcholine, rocuronium, and mivacurium were studied in 100 healthy women undergoing outpatient laparoscopic surgery. After a standardized fentanyl-thiopental induction, tracheal intubation was facilitated with succinylcholine 1 mg/kg in Groups I (n = 23) and II (n = 25), rocuronium 0.6 mg/kg in Group III (n = 27), or mivacurium 0.2 mg/kg in Group IV (n = 25). If clinically indicated, bolus doses of rocuronium 5-10 mg (Groups I and III) or mivacurium 2-4 mg (Groups II and IV) were administered during the maintenance period. Anesthesia was maintained with desflurane and nitrous oxide 60% in oxygen. At the end of the surgery, residual neuromuscular block was reversed with edrophonium 0.5 mg/kg and atropine 10 micrograms/kg, if needed. The neuromuscular function was assessed using electromyography with a train-of-four mode of stimulation every 10 s at the wrist. Intubating conditions 90 s after succinylcholine and rocuronium were significantly better than after mivacurium. The onset time (from the end of injection until 95% suppression of the first twitch [T1]) for succinylcholine (63 +/- 21 s and 62 +/- 17 s in Groups I and II, respectively) were significantly shorter than for rocuronium (158 +/- 76 s) or mivacurium (210 +/- 93 s). Moreover, the onset times for rocuronium were significantly shorter than mivacurium. The recovery times (of T1 to 25% of the control value) were significantly shorter with succinylcholine and mivacurium than rocuronium. Significantly fewer patients needed reversal of residual neuromuscular blockade after mivacurium compared to rocuronium. One patient in Group I and six patients in Group IV displayed erythema on the upper body. Postoperative myalgia were experienced by 16% of the patients in Groups I and II compared to none in Groups III and IV. There was on difference in the incidence of postoperative nausea and vomiting among the four groups. In conclusion, rocuronium appears to be an acceptable alternative to succinylcholine for tracheal intubation. However, rocuronium's longer duration of action increases the need for reversal drugs. When rapid tracheal intubation is unnecessary, mivacurium is also an acceptable alternative to succinylcholine and is associated with a more rapid spontaneous recovery than rocuronium. PMID- 8610913 TI - Spontaneous versus edrophonium-induced recovery from paralysis with mivacurium. AB - This study compared spontaneous with edrophonium-induced recovery of neuromuscular transmission (NMT) after mivacurium infusion. During nitrous oxide narcotic-propofol anesthesia, the electromyogram (EMG) of the adductor pollicis (AP) was recorded and the movement of the first toe in response to stimulation of the posterior tibial nerve was noted. Mivacurium infusion was titrated to produce posttetanic count of 1-5 at the toe and absence of NMT at the AP. Thirty children were assigned to three groups on the basis of age. Edrophonium, 1 mg/kg, with atropine 10 micrograms/kg, was given after the mivacurium infusion when NMT of the AP was 1% or 10% of baseline. In the third group, spontaneous recovery was observed. Edrophonium given when NMT was 11% +/- 1% SEM produced the most rapid recovery, 7.5 +/- 0.6 min to a train-of-four (TOF) ratio (T4/T1) of 0.9 and the shortest interval from T4/T1 of 0.4-0.9, when residual block was likely to be underestimated, 4.8 +/- 0.6 min. Edrophonium given when block was greater produced recovery of the T4/T1 to 0.4 in 2.8 +/- 0.7 min, but the time from then to T4/T1 = 0.9 was 7.9 +/- 1.1 min, as long as during spontaneous recovery. Spontaneous recovery to T4/T1 = 0.9 occurred 12.9 +/- 0.7 min after the first measurable AP EMG. There was no significant relationship between duration of infusion, which ranged from 16 to 135 min, and time to appearance of AP EMG after the infusion, which averaged 3.1 +/- 0.5 min. We recommend that administration of edrophonium to induce reversal of mivacurium be delayed until two responses to a TOF stimuli are observed because this will produce the most rapid recovery and decrease the interval in which residual block may be underestimated. PMID- 8610914 TI - Effective maintenance treatment of reflux esophagitis with low-dose lansoprazole. A randomized, double-blind, placebo-controlled trial. AB - OBJECTIVE: To compare the efficacy of two doses of lansoprazole with that of placebo in preventing recurrence of erosive esophagitis in a 12-month period. DESIGN: Randomized, double-blind, parallel, placebo-controlled trial. SETTING: 25 U.S. medical centers. PATIENTS: 173 patients with documented healing of erosive esophagitis after 8 weeks of acid-suppressing therapy. INTERVENTION: Lansoprazole, 15 mg or 30 mg, or placebo once daily for as long as 12 months. MEASUREMENTS: Endoscopy and symptom evaluation after 1, 2, 3, 6, 9, and 12 months of treatment. Endoscopy was also done whenever symptoms suggested erosive changes. RESULTS: Lansoprazole was significantly superior to placebo in maintaining healing and preventing recurrence of symptoms. By month 1, 45% of placebo recipients remained healed compared with more than 90% of patients in either lansoprazole group. By month 12, only 24% of placebo recipients remained healed compared with 79% of patients receiving 15 mg of lansoprazole and 90% of patients receiving 30 mg of lansoprazole. During the same period, 35% of placebo recipients remained asymptomatic compared with 72% of recipients of 15 mg of lansoprazole and 67% of recipients of 30 mg of lansoprazole. The 15-mg and 30-mg lansoprazole doses did not differ significantly in maintaining healing and controlling symptoms. Follow-up after recurrence of erosion indicated that during the 12 months, 35% of placebo recipients and 2% of lansoprazole recipients had three or more recurrences. CONCLUSION: Lansoprazole effectively maintains healing of erosive esophagitis. The 15-mg and 30-mg lansoprazole doses did not differ significantly for use as maintenance treatment. PMID- 8610915 TI - Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Hepatitis Interventional Therapy Group. AB - OBJECTIVE: To 1) verify the validity of a new line probe assay for hepatitis C virus (HCV) genotyping and 2) determine the distribution of HCV genotypes and the association between HCV genotype and clinical variables in patients with chronic hepatitis C seen in tertiary referral centers in the United States. DESIGN: Retrospective cross-sectional analysis. PATIENTS: 438 patients with chronic hepatitis C from 10 tertiary referral centers. MEASUREMENTS: The validity of the line probe assay was first verified against a panel of serum specimens that had previously been characterized by six different HCV genotyping methods. Specimens from all 438 patients were then genotyped using this line probe assay. The associations between HCV genotype and clinical variables were examined using analysis of variance. Pairwise testing was used when the F test showed a statistically significant difference. Nonparametric alternatives were used for variables for which normality could not be assumed. RESULTS: The line probe assay was quick and reproducible, and it showed good concordance with other tests. In our sample, the proportions of patients with HCV types 1, 2, 3, and 4 were 71.5%, 13.5%, 5.5%, and 1.1%, respectively. Subtypes 1a and 1b were seen in approximately equal proportions of patients with HCV type 1. Mixed infection was detected in 3.7% of specimens, and 4.8% of specimens either had negative results on polymerase chain reaction or could not be typed. A higher proportion of patients with HCV type 1 than of patients with HCV-type 1 had acquired HCV through transfusion of blood products (50% compared with 25%; P < 0.001). Patients with HCV type 1 also had a longer estimated duration of infection compared with patients with HCV type 3 (P = 0.004) and type 4 (P = 0.049). Disease activity did not differ among patients infected with HCV types 1, 2, or 3. Levels of viremia were similar in patients with HCV types 1, 2, or 3, but patients with HCV type 4 had a lower level of viremia than did patients with HCV type 1 (P = 0.047). CONCLUSIONS: The line probe assay can be used in patients with chronic HCV infection in the United States. In patients with chronic hepatitis C referred to tertiary centers in the United States, type 1 is the most common HCV genotype. Disease activity and viremia levels do not differ among patients chronically infected with HCV types 1, 2, or 3. PMID- 8610916 TI - Association of the auscultatory gap with vascular disease in hypertensive patients. AB - OBJECTIVE: To assess the relation of the auscultatory gap during blood pressure measurement to cardiovascular structure and function. DESIGN: Cross-sectional study. SETTING: A hypertension center in a university hospital. PATIENTS: 168 persons with hypertension who were otherwise healthy and were not receiving medication. MEASUREMENTS: Wideband external pulse recordings and ultrasonographic examination of the left ventricle and extracranial carotid arteries. Vascular stiffness was evaluated using simultaneous carotid pressure waveforms obtained by applanation tonometry of the contralateral carotid artery. RESULTS: Classic auscultatory gaps were present in 21% of patients and were associated with older age (mean age SD, 64 11 years for patients with gaps and 55 13 years for patients without gaps; P < 0.001), female sex (67% of patients with gaps and 44% of patients without gaps were female; P < 0.05), and increased arterial stiffness (arterial stiffness index, 8.5 4.6 in patients with gaps and 5.8 3.2 in patients without gaps; P < 0.005). The prevalence of atherosclerotic plaques was increased more than twofold among patients with gaps compared with patients without gaps (50% compared with 22%; p < 0.002). Patients with and without auscultatory gaps had similar blood pressures, left ventricular structure and function, serum cholesterol levels, and smoking history. Logistic regression analysis indicated that only female sex (P < 0.02), arterial stiffness (P < 0.002), and atherosclerotic plaque (P < 0.02) were independently associated with the presence of an auscultatory gap. CONCLUSIONS: This study provides strong evidence that auscultatory gaps are related to carotid atherosclerosis and to increased arterial stiffness in hypertensive patients, independent of age. Although these observations need to be confirmed prospectively, they suggest that auscultatory gaps may have prognostic relevance. PMID- 8610917 TI - Implementing antibiotic practice guidelines through computer-assisted decision support: clinical and financial outcomes. AB - OBJECTIVE: To determine the clinical and financial outcomes of antibiotic practice guidelines implemented through computer-assisted decision support. DESIGN: Descriptive epidemiologic study and financial analysis. SETTING: 520-bed community teaching hospital in Salt Lake City, Utah. PATIENTS: All 162 196 patients discharged from LDS Hospital between 1 January 1988 and 31 December 1994. INTERVENTION: An antibiotic management program that used local clinician derived consensus guidelines embedded in computer-assisted decision support programs. Prescribing guidelines were developed for inpatient prophylactic, empiric, and therapeutic uses of antibiotics. MEASUREMENTS: Measures of antibiotic use included timing of preoperative antibiotic administration and duration of postoperative antibiotic use. Clinical outcomes included rates of adverse drug events, patterns of antimicrobial resistance, mortality, and length of hospital stay. Financial and use outcomes were expressed as yearly expenditures for antibiotics and defined daily doses per 100 occupied bed-days. RESULTS: During the 7-year study period, 63 759 hospitalized patients (39.3%) received antibiotics. The proportion of the hospitalized patients who received antibiotics increased each year, from 31.8% in 1988 to 53.1% in 1994. Use of broad-spectrum antibiotics increased from 24% of all antibiotic use in 1988 to 47% in 1994. The annual Medicare case-mix index increased from 1.7481 in 1988 to 2.0520 in 1993. Total acquisition costs of antibiotics (adjusted for inflation) decreased from 24.8% ($987,547) of the pharmacy drug expenditure budget in 1988 to 12.9% ($612,500) in 1994. Antibiotic costs per treated patient (adjusted for inflation) decreased from $122.66 per patient in 1988 to $51.90 per patient in 1994. Analysis using a standardized method (defined daily doses) to compare antibiotic use showed that antibiotic use decreased by 22.8% overall. Measures of antibiotic use and clinical outcomes improved during the study period. The percentage of patients having surgery who received appropriately timed preoperative antibiotics increased from 40% in 1988 to 99.1% in 1994. The average number of antibiotic doses administered for surgical prophylaxis was reduced from 19 doses in the base year to 5.3 doses in 1994. Antibiotic-associated adverse drug events decreased by 30%. During the study, antimicrobial resistance patterns were stable, and length of stay remained the same. Mortality rates decreased from 3.65% in 1988 to 2.65% in 1994 (P < 0.001). CONCLUSIONS: Computer-assisted decision support programs that use local clinician-derived practice guidelines can improve antibiotic use, reduce associated costs, and stabilize the emergence of antibiotic-resistant pathogens. PMID- 8610918 TI - Successful treatment of Behcet disease with pentoxifylline. PMID- 8610919 TI - Successful treatment of relapse of acute promyelocytic leukemia with a new synthetic retinoid, Am80. PMID- 8610920 TI - Breast cancer in black women. AB - PURPOSE: To review the current knowledge about breast cancer in black women- including epidemiology, risk factors, screening practices, pathology, clinical manifestations, treatment, and outcome--with emphasis on issues that might explain why the survival rate in this population of women is lower than that in white women. DATA SOURCES: The MEDLINE database from 1966 to 1995 and the bibliographies of all related articles. STUDY SELECTION: Review articles and clinical studies related to all aspects of breast cancer in black women. DATA SYNTHESIS: The incidence of breast cancer is lower in black women (95.8 cases per 100 000 women) than in white women (112.7 cases per 100 000 women). Differences in reproductive factors may partially explain the lower risk for breast cancer among black women in the United States. Breast tumors in black women are consistently diagnosed at a more advanced stage of disease: Forty-two percent of black women present with cancer confined to the breast compared with 53% of white women. In addition, the cancers of black women tend to be more poorly differentiated and are less likely to be estrogen receptor positive. Treatment of breast cancer in black women appears to be similar to that in white women, but little is known about systemic therapy choices and efficacy. Overall, despite their lower risk for breast cancer, black women have a mortality rate from breast cancer similar to that of white women because they have a lower 5-year disease specific survival rate (64% in black women compared with 80% in white women). CONCLUSIONS: The discrepancy in survival rate between black and white women exists because black women have tumors that are more advanced at the time of diagnosis, because tumor biology in black women is different from that in white women (in particular, black women have a higher frequency of poorly differentiated tumors and a lower frequency of hormone receptor-positive tumors), and because of confounding comorbid conditions and socioeconomic factors. Current efforts to improve survival rates in black women with breast cancer should focus on community education, screening efforts, and early detection. As more information is gained about breast cancer treatment in black women, this may also be an important area for intervention. PMID- 8610921 TI - The effect of pharmaceutical benefits managers: is it being evaluated? AB - Over the last decade, the number of pharmaceutical benefits managers has increased, and their influence has expanded rapidly. These managers now provide prescription drug coverage to more than 100 million Americans. The effect of pharmaceutical benefits managers on health care delivery remains unclear. We review the development of these organizations, their current role in the delivery of pharmaceutical therapies to patients, and their relationship with pharmaceutical manufacturers. We discuss potential advantages and disadvantages of pharmaceutical benefits manager practices and suggest ways in which these organizations can be made more accountable to the employer groups that hire them. PMID- 8610922 TI - Cholesterol reduction: weighing the benefits and risks. AB - The National Cholesterol Education Program recommends reducing total and low density lipoprotein cholesterol levels to decrease the risk for coronary heart disease. The available evidence clearly indicates that higher cholesterol levels increase the risk for coronary heart disease and that cholesterol reduction results in corresponding decreases in risk. In contrast, existing data do not strongly support the idea that cholesterol reduction causes increases in any specific nonvascular cause of death. The outcomes of ongoing, large-scale trials will enable existing guidelines to be refined. However, current recommendations, which encourage nonpharmacologic interventions for about 30% of U.S. adults and cholesterol-reducing drugs for about 7% of U.S. adults, seem both justified and warranted. PMID- 8610923 TI - Cultural competence: essential measurements of quality for managed care organizations. PMID- 8610925 TI - Annual meeting: perspectives. PMID- 8610924 TI - Lipid formulations for amphotericin B: does the emperor need new clothes? PMID- 8610926 TI - Misoprostol and gastrointestinal complications in patients taking nonsteroidal anti-inflammatory drugs for rheumatoid arthritis. PMID- 8610927 TI - Misoprostol and gastrointestinal complications in patients taking nonsteroidal anti-inflammatory drugs for rheumatoid arthritis. PMID- 8610928 TI - Misoprostol and gastrointestinal complications in patients taking nonsteroidal anti-inflammatory drugs for rheumatoid arthritis. PMID- 8610929 TI - Misoprostol and gastrointestinal complications in patients taking nonsteroidal anti-inflammatory drugs for rheumatoid arthritis. PMID- 8610930 TI - Giant cell arteritis. PMID- 8610931 TI - Esophageal ulcers in AIDS. PMID- 8610932 TI - Esophageal ulcers in AIDS. PMID- 8610933 TI - Ventilator circuit changes. PMID- 8610934 TI - Ventilator circuit changes. PMID- 8610935 TI - Blood donors with antibody to hepatitis C virus. PMID- 8610936 TI - Blood donors with antibody to hepatitis C virus. PMID- 8610937 TI - Hepatic injury caused by acarbose. PMID- 8610938 TI - Hemolysis after autologous transfusion. PMID- 8610939 TI - Residency overwork. PMID- 8610940 TI - Residency overwork. PMID- 8610941 TI - Residency overwork. PMID- 8610942 TI - Residency overwork. PMID- 8610943 TI - Residency overwork. PMID- 8610944 TI - Residency overwork. PMID- 8610945 TI - Residency overwork. PMID- 8610946 TI - Delayed tuberculin reactivity in persons of Indochinese origin: implications for preventive therapy. AB - OBJECTIVES: To 1) study a variant delayed reaction to tuberculin testing as a way to enhance screening for tuberculosis among high-risk persons and 2) correlate the delayed reaction with lymphocyte blastogenesis. DESIGN: Cross-sectional study. SETTING: 2 public health department clinics in North Carolina. PARTICIPANTS: 121 adults who had recently emigrated from Vietnam to North Carolina and who were ethnic Vietnamese and ethnic Dega, a minority population group from the central highlands region of Vietnam. MEASUREMENTS: Medical history, physical examination, laboratory evaluation, and standard purified protein derivative (PPD) testing (Mantoux method). Skin test results were read at 72 hours and again at 6 days. Variant reactivity was defined as induration of less than 10 mm at 72 hours that, when reassessed at 6 days, had increased in size to 10 mm or greater. Persons with negative (n=54) and variant (n=32) PPD results also had booster testing at 10 to 12 weeks. Serum samples were obtained from 57 participants for lymphocyte blastogenesis studies. RESULTS: 26% of participants had variant tuberculin reactivity. Variant reactivity was strongly associated with booster positivity: Sixty-five percent of persons with variant PPD results had booster positivity compared with 16% of persons with negative PPD results (P<0.001). The lymphocyte blastogenesis response of persons with variant PPD results was between the response of persons with negative PPD results and that of persons with positive PPD results. CONCLUSION: Variant reactivity in this high-risk group was a predictor of booster positivity. Together with the blastogenic response pattern, this association strongly suggests that variant reactivity has a high positive predictive value for tuberculous infection. Clinicians should incorporate these findings into their approach for choosing candidates for preventive therapy. PMID- 8610947 TI - Azithromycin compared with amoxicillin in the treatment of erythema migrans. A double-blind, randomized, controlled trial. AB - OBJECTIVE: To determine whether azithromycin or amoxicillin is more efficacious for the treatment of erythema migrans skin lesions, which are characteristic of Lyme disease. DESIGN: Randomized, double-blind, double-dummy, multicenter study. Acute manifestations and sequelae were assessed using a standardized format. Baseline clinical characteristics and response were correlated with serologic results. Patients were followed for 180 days. SETTING: 12 outpatient centers in eight states. PATIENTS: 246 adult patients with erythema migrans lesions at least 5 cm in diameter were enrolled and were stratified by the presence of flu-like symptoms (such as fever, chills, headache, malaise, fatigue, arthralgias, and myalgias) before randomization. INTERVENTION: Oral treatment with either amoxicillin, 500 mg three times daily for 20 days, or azithromycin, 500 mg once daily for 7 days. Patients who received azithromycin also received a dummy placebo so that the dosing schedules were identical. RESULTS: Of 217 evaluable patients, those treated with amoxicillin were significantly more likely than those treated with azithromycin to achieve complete resolution of disease at day 20, the end of therapy (88% compared with 76%; P=0.024). More azithromycin recipients (16%) than amoxicillin recipients (4%) had relapse (P=0.005). A partial response at day 20 was highly predictive of relapse (27% of partial responders had relapse compared with 6% of complete responders; P<0.001). For patients treated with azithromycin, development of an antibody response increased the possibility of achieving a complete response (81% of seropositive patients achieved a complete response compared with 60% of seronegative patients; P=0.043). Patients with multiple erythema migrans lesions were more likely than patients with single erythema migrans lesions (P<0.001) to have a positive antibody titer at baseline (63% compared with 17% for IgM; 39% compared with 16% for IgG). Fifty-seven percent of patients who had relapse were seronegative at the time of relapse. CONCLUSIONS: A 20-day course of amoxicillin was found to be an effective regimen for erythema migrans. Most patients were seronegative for Borrelia burgdorferi at the time of presentation with erythema migrans (65%) and at the time of relapse (57%). PMID- 8610949 TI - Polymerase chain reaction for the diagnosis of HIV infection in adults. A meta analysis with recommendations for clinical practice and study design. AB - PURPOSE: To do a meta-analysis of studies that have evaluated the sensitivity and specificity of polymerase chain reaction (PCR) assay for the diagnosis of human immunodeficiency virus (HIV) infection in adults. Evaluating the performance of PCR is difficult because in certain clinical situations, the sensitivity or specificity of PCR may exceed those of the current reference standard tests (enzyme immunoassay followed by confirmatory Western blot analysis). Therefore, an additional goal was to develop recommendations for 1) the design of future evaluative studies of PCR and 2) the use of PCR in persons with suspected HIV infection. DATA SOURCES: Studies published between 1988 and 1994 that were identified in a search of 17 computer databases, including MEDLINE, and abstracts identified from conference proceedings. STUDY SELECTION: Studies were included if DNA amplification by PCR was done on peripheral blood mononuclear cells from adults. Ninety-six studies met the inclusion criteria. DATA EXTRACTION: Data were extracted independently by two reviewers. Study design was assessed independently by two investigators blinded to study results. RESULTS: Reported sensitivities for PCR range from 10% to 100%, and specificities range from 40% to 100%. A summary receiver-operating characteristic curve based on all 96 studies has a maximum joint sensitivity and specificity (upper left point on the curve, where sensitivity equals specificity) of 97.0% to 98.1%. If the threshold value that defines a positive PCR result is chosen so that sensitivity is higher than 98.1%, specificity will decrease to less than 98.1%. Conversely, if the threshold value that defines a positive PCR result is chosen so that specificity is greater than 98.1%, sensitivity will decrease to less than 98.1%. If sensitivity and specificity are chosen to be equal, the corresponding false-positive rate is 1.9% to 3.0%. At the maximum joint sensitivity and specificity, the positive predictive value of PCR ranges from 34% to 85% as the prevalence of HIV increases from 1.0% to 10%. We identified seven areas in which study design could be modified to 1) reduce susceptibility to bias in estimates of the sensitivity and specificity of PCR and 2) to increase the generalizability of the study results. These modifications will also help to overcome methodologic problems created by the lack of a reference standard test. CONCLUSIONS: The PCR assay is not sufficiently accurate to be used for the diagnosis of HIV infection without confirmation. Use of PCR for the diagnosis of HIV in adults should be limited to situations in which antibody tests are known to be insufficient. Future studies of PCR performance should be sufficiently large and should use adequate reference standard tests and standardized methods for the performance of PCR. Specimens should be evaluated by persons blinded to clinical status and to the results of other diagnostic tests for HIV infection. PMID- 8610948 TI - Comparison of three regimens for treatment of mild to moderate Pneumocystis carinii pneumonia in patients with AIDS. A double-blind, randomized, trial of oral trimethoprim-sulfamethoxazole, dapsone-trimethoprim, and clindamycin primaquine. ACTG 108 Study Group. AB - OBJECTIVE: To compare the tolerability and efficacy of three oral regimens for the treatment of patients with the acquired immunodeficiency syndrome (AIDS) and Pneumocystis carinii pneumonia. DESIGN: A randomized, double-blind study. SETTING: 24 U.S. academic medical centers. PATIENTS: 181 patients with morphologically confirmed P. carinii pneumonia and alveolar-arterial oxygen differences (PAO2-PaO2) of 45 mm Hg or less. INTERVENTION: Patients were randomly assigned to receive trimethoprim-sulfamethoxazole, dapsone-trimethoprim, or clindamycin-primaquine for 21 days. Patients with a PAO2-PaO2 of 35 to 45 mm Hg at study entry also received prednisone. MEASUREMENTS: Serial clinical and laboratory evaluations for therapeutic response and toxicity. Therapeutic failure at day 21 was defined by any of the following: increase in PAO2-PaO2 of greater than 20 mm Hg; no remission of baseline signs and symptoms; and change in antipneumocystis therapy for reasons other than toxicity, intubation, or death. Dose-limiting toxicity was defined as discontinuation of therapy by the primary physician because of one or more adverse reactions. RESULTS: No statistically significant differences were seen among treatment groups in the proportions of patients who had dose-limiting toxicity (P=0.2), therapeutic failure (P>0.2), or a complete course of therapy (P>0.2). Survival during therapy or for 2 months thereafter did not differ among the three groups (P>.02). However, elevation of serum aminotransferase levels to more than five times the baseline levels was more frequent in the trimethoprim-sulfamethoxazole group (P=0.003), and one or more serious hematologic toxicities (neutropenia, anemia, thrombocytopenia, or methemoglobinemia) occurred more frequently in the clindamycin-primaquine group (P=0.01). CONCLUSIONS: The rates of dose-limiting toxicity, therapeutic failure, and survival did not differ among patients with AIDS who were receiving oral trimethoprim-sulfamethoxazole, dapsone-trimethoprim, or clindamycin-primaquine for mild to moderate P. carinii pneumonia. However, the limited sample size prevents the unequivocal demonstration of the equality of these three regimens. Differences in expected categories of toxicities associated with each regimen should guide the clinician in choosing first-line therapy, particularly for patients with baseline hepatic insufficiency or myelosuppression. PMID- 8610950 TI - Necessity of routine chest roentgenography after thoracentesis. AB - OBJECTIVE: To determine the necessity of posteroanterior chest roentgenography for the identification of pneumothorax and other complications after thoracentesis. DESIGN: Prospective cohort study. SETTING: Tertiary care teaching hospital. PATIENTS: 67 men and 43 women (mean age +/- SD, 62.4 +/- 13.2 years). Exclusion criteria included age younger than 18 years, concurrent pleural biopsy, ultrasound guidance, and use of mechanical ventilation. MEASUREMENTS: 174 thoracenteses done between March 1991 and June 1993. RESULTS: 2 hemothoraces (1.2%) occurred, and 8 patients had a total of 9 pneumothoraces (5.2%). The roentgenograms obtained immediately after the procedures identified 8 pneumothoraces; the other pneumothorax was seen incidentally on a delayed roentgenogram obtained 3 days later. Pneumothorax was suspected in 5 of the 8 cases, and tube thoracostomy was done in 4 of these 5 cases. Patients with unsuspected pneumothorax identified on the roentgenogram obtained immediately after the procedure did not receive treatment for their pneumothoraces. Univariate analysis showed that the variables that correlated significantly with pneumothorax were aspiration of air during the procedure (relative risk ratio, 12.3; 95% CI, 3.7 to 41.4), number of passes with the thoracentesis needle (relative risk ratio, 6.1; CI, 1.6 to 23.3), history of thoracic radiation therapy (relative risk ratio, 10.5; CI, 2.5 to 44.4), and operator suspicion of pneumothorax (relative risk ratio, 25.9; CI, 8.6 to 78.5). CONCLUSION: Among hospitalized patients with pleural effusions, we identified subgroup of patients in whom the risk for pneumothorax is low enough (approximately 1%) with sufficiently minimal clinical consequences to justify the avoidance of about 60% of chest roentgenograms obtained after thoracentesis. These patients are clinically stable, have not previously received chest irradiation, had only one pass at thoracentesis attempted without the aspiration of any air, and give no other indication of pneumothorax. PMID- 8610951 TI - Aerosolized prostacyclin and iloprost in severe pulmonary hypertension. AB - OBJECTIVE: To compare the effects of aerosolization of prostacyclin and its stable analog iloprost with those of nasal oxygen, inhaled nitric oxide, and intravenous prostacyclin on hemodynamics and gas exchange in patients with severe pulmonary hypertension. DESIGN: Open uncontrolled trial. SETTING: Justus-Liebig University, Giessen Germany. PATIENTS: 4 patients with primary pulmonary hypertension and 2 patients with severe pulmonary hypertension associated with calcinosis, the Raynaud phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (the CREST syndrome). All were classified as New York Heart Association class III or class IV. INTERVENTION: Short-term applications of O2, inhaled nitric oxide, intravenous prostacyclin, aerosolized prostacyclin, and aerosolized iloprost during repeated catheter investigation of the right side of the heart within a 1-month period. One patient had long-term therapy with inhaled iloprost. RESULTS: Aerosolized prostacyclin decreased pulmonary artery pressure in 6 patients from (mean +/- SE) 62.3 +/- 4.1 mm Hg to 50.8 +/- 5.5 mm Hg and reduced pulmonary vascular resistance from 1721 +/- 253 dyne/s cm-5 to 1019 +/- 203 dyne/s cm-5, and it increased cardiac output from 2.75 +/- 0.21 L/min to 4.11 +/- 0.54 L/min, mixed venous oxygen saturation from 51.1% +/- 3/4% to 66.3% +/- 4.1% and arterial oxygen saturation from 90.6% +/- 2.7% to 93.8% +/- 23% (P<0.05 for all changes). Mean systemic arterial pressure was only slightly affected. The responses lasted for 10 to 30 minutes after inhalation was terminated. Aerosolized iloprost had an identical efficacy profile but was associated with a longer duration of the pulmonary vasodilatory effect (60 min to 120 min). In comparison, intravenous prostacyclin reduced pulmonary vascular resistance with corresponding efficacy but produced a more pronounced decline in systemic artery pressure and no clinically significant decrease in pulmonary artery pressure. Nitric oxide and O2 were less potent pulmonary vasodilators in these patients. In one patient, 1 year of therapy with aerosolized iloprost (100 microgram/d in six aerosol doses) resulted in sustained efficacy of the inhaled vasodilator regimen and clinical improvement. CONCLUSION: Aerosolization of prostacyclin or its stable analog iloprost causes selective pulmonary vasodilatation, increases cardiac output, and improves venous and arterial oxygenation in patients with severe pulmonary hypertension. Thus, it may offer a new strategy for treatment of this disease. PMID- 8610952 TI - Dietary calcium and blood pressure: a meta-analysis of randomized clinical trials. AB - PURPOSE: To assess the effect of dietary calcium supplementation on blood pressure. DATA SOURCES: Published reports of trials studying the effect of dietary calcium supplementation on blood pressure were identified by a search of previous reviews, a MEDLINE search, a manual review of journal articles, and a review of abstracts from scientific meetings. STUDY SELECTION: Randomized clinical trials in which dietary calcium intake varied by intervention group were selected. Multifactorial trials were not included. DATA SYNTHESIS: Data from 28 active treatment arms or strata from 22 randomized clinical trials were pooled using a weighted average method, with weights proportional to the inverse of the variance of the treatment effect. The total sample comprised 1231 persons. Because trials of both normotensive and hypertensive persons were included, subgroup analyses could be done. Pooled estimates of the effect of calcium supplementation on blood pressure were -0.18 mm Hg for diastolic blood pressure (95% CI, -0.75 to 0.40 mm Hg) and -0.89 mm Hg for systolic blood pressure (CI, 1.74 to -0.05 mm Hg). Pooled estimates for systolic blood pressure were -0.53 mm Hg (CI, -1.56 to 0.49 mm Hg) for trials of normotensive persons and -1.68 mm Hg (CI, -3.18 to -0.18 mm Hg) for trials of hypertensive persons. Diastolic blood pressure was not significantly affected in either subgroup. CONCLUSION: The pooled estimate shows a statistically significant decrease of systolic blood pressure with calcium supplementation, both for hypertensive persons and for the overall sample. However, the effect is too small to support the use of calcium supplementation for preventing or treating hypertension. PMID- 8610953 TI - From outcomes research to disease management: a guide for the perplexed. AB - Outcomes research is a rapidly evolving field that incorporates epidemiology, health services research, health economics, and psychometrics. Measurement of clinical and other outcomes has become increasingly important to the stakeholders in a rapidly changing health care environment. The desire to improve outcomes and control costs has stimulated greater interest in cost-effectiveness studies, which determine how well effective therapies work in the usual practice setting and how much they cost. The application of outcomes principles to the practices of health care providers has resulted in efforts to implement disease management programs. Unlike traditional programs carried out by physicians, these new efforts are based on systematic population-based approaches to identifying persons at risk, intervening with specific programs of care, and measuring clinical and other outcomes. The new efforts depend heavily on modern information systems. PMID- 8610954 TI - Disease management: new wine in new bottles? AB - Cost pressures are driving the reorganization of the health care delivery system in the United States, causing health care delivery organizations to become larger and more diversified. These new health care delivery entities are arriving at the same time as a new approach to clinical care: commercial population-based medicine. This approach is sharing an increasing amount of space with the traditional physician-patient-based approach and offers strategies for managing a population's risk, demand, diseases, and outcomes. Although commercial population based medicine strategies such as disease management may use many of the same tools as traditional public health approaches, they focus on the relatively short term health needs of defined populations because of the underlying economic incentives facing payers. Population-based-medicine promises better use of resources and a systems approach to health care delivery, but it also presents the pitfalls of subversion by the needs of commercial companies, unanticipated adverse effects, and wide application without adequate preparation. PMID- 8610955 TI - Platelet glycoprotein IIb/IIIa receptor inhibitors in coronary artery disease. PMID- 8610956 TI - Voluntary purchasing pools: a market model for improving access, quality, and cost in health care. American College of Physicians. AB - States and small businesses have been rapidly establishing voluntary health care purchasing pools during the past few years. Purchasing pools can decrease health care costs, improve access for some small businesses and individual persons, allow greater choice among health care plans, and provide continuity of care. Purchasing pools also help to even the balance of power in the health care marketplace, which has come increasingly under the control of huge proprietary managed care corporations. This position paper of the American College of Physicians discusses how a system of well-designed voluntary purchasing pools can help protect the integrity of health care in the emerging managed care marketplace. PMID- 8610957 TI - Diagnosing ehrlichiosis. PMID- 8610958 TI - Hepatotoxicity and rash associated with zidovudine and zalcitabine chemoprophylaxis. PMID- 8610959 TI - Tamoxifen-induced steatohepatitis. PMID- 8610960 TI - Physician-controlled utilization management. PMID- 8610961 TI - Oral tolerance: mechanisms and applications. Introduction. PMID- 8610962 TI - Oral Tolerance: Mechanisms and Applications. Proceedings of a conference. New York City, New York, March 30-April 2, 1995. PMID- 8610963 TI - History of oral tolerance and mucosal immunity. AB - Mucosal immunity depends on antigen stimulation in specialized lymphoepithelial structures such as the Peyer's patches. Although these inductive compartments were discovered more than 300 years ago, their functional role has become clear only over the last few decades. Research on homing of primed lymphoid cells to the intestinal mucosa began with animal experimentation in the 1960s and 1970s and has recently been brought to the molecular level. The major effector substance of mucosal immunity is secretory IgA (SIgA). The first evidence for its local antibody activity was obtained in humans in 1922, but its unique properties were not defined until the mid-1960s. Several models were subsequently proposed for selective external transport of IgA involving the secretory component (SC). In the early 1970s SC was suggested to act as a transmembrane polymeric Ig receptor common for dimeric IgA and pentameric IgM; this transport mechanism has now been confirmed by detailed studies at the level of cellular/molecular biology. Although SIgA antibodies performing immune exclusion are the main goal for exploitation of the mucosal immune system by oral vaccination, little is known about the precise mechanisms for induction of mucosal immunity against soluble proteins and chemicals. A peripheral immunosuppressive effect of oral immunization with such substances was apparently exploited by ancient people, and "oral tolerance" has since 1910 been subjected to numerous feeding experiments in rodents. The basis for the whole phenomenon appears to be intact epithelial barrier. Mucosal induction of suppression may in the future be exploited not only to modulate autoimmune diseases through the gut but also to prevent the development of IgE-mediated allergy and other untoward immune reactions by way of the respiratory tract. PMID- 8610964 TI - Induction of anergy in Th1 lymphocytes by oral tolerance. Importance of antigen dosage and frequency of feeding. AB - Oral tolerance, a biologically relevant pathway for inducing peripheral tolerance in T lymphocytes, occurs by two distinct mechanisms. Multiple low doses of antigen induce regulatory T lymphocytes that secrete immunosuppressive cytokines, whereas feeding a single high dose of antigen induces anergy of antigen-specific Th1 lymphocytes (diminished IgG2a, IL-2, and IFNgamma) with intact Th2 responses (IgG1 and IL-4). Anergy was demonstrated by the ability to reverse tolerant state after culturing tolerant cells in rIL-2. Reversal of the tolerant state was established in vitro by increase in frequency of IL-2 secreting cells, and in vivo by specific IgG2a production in irradiated mice adoptively transferred with cells cultured in rIL-2. Inasmuch as the induction of anergy was inhibited by the presence of antibodies specific for the tolerizing antigen, it appears that the oral induction of anergy might depend on the systemic dissemination of antigen (or its fragments) absorbed from the gut. It is suggested that tolerance is insured by the fact that this absorbed antigen is presented to Th1 lymphocytes in draining lymph nodes in the absence of inflammatory and costimulatory molecules. PMID- 8610965 TI - Dose-dependent activation and deletion of antigen-specific T cells following oral tolerance. PMID- 8610966 TI - Inactivation of Th1 and Th2 cells by feeding ovalbumin. AB - Several different mechanisms have been implicated in oral tolerance to protein antigens, depending on the nature and dose of antigen used and the species under study. Here, we have investigated the basis of unresponsiveness in a well established model of oral tolerance in mice fed 25 mg ovalbumin (OVA). Our results show that CD8+ T-cell activity is suppressed by feeding OVA and that these cells are not required for the induction of tolerance. CD4+ T cells are essential for tolerance to occur, but both Th1 and Th2 cell-dependent functions are tolerized equally in OVA-fed mice. Peripheral lymph node cells from tolerized mice rapidly undergo apoptosis when cultured in vitro but produce substantial amounts of transforming growth factor beta (TGFbeta) in response to OVA. The appearance of tolerance in vivo is preceded by a transient phase of T-cell priming, and we propose that this model of oral tolerance reflects partial activation of T cells by fed antigen, leading to selective production of TGFbeta and consequent inactivation of all effector T cells. These findings indicate that the active suppression and clonal anergy identified previously in mice with oral tolerance may not be mutually exclusive phenomena. PMID- 8610967 TI - The role of chemokines in oral tolerance. Abrogation of nonresponsiveness by treatment with antimonocyte chemotactic protein-1. AB - Peripheral antigen-specific tolerance can be induced by feeding protein antigens. The mechanism has been described as either clonal anergy/deletion or induction of antigen-specific regulatory cells that produce transforming growth factor (TGF) beta, depending on the dose of antigen fed. Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, can be prevented by feeding myelin basic protein (MBP) or proteolipid protein (PLP). We decided to address the role of chemokines in the induction of oral tolerance. We have used a model antigen system of feeding a high dose of human gamma globulin (HGG) to mice that have been subsequently immunized with HGG emulsified in CFA. The result was decreased recall proliferative, delayed-type hypersensitivity (DTH) and Th1 cytokine responses. By contrast, Th2 cytokine responses were enhanced. Interestingly, macrophage inflammatory protein (MIP)-1alpha production was decreased, whereas monocyte chemotactic protein (MCP)-1 production was enhanced. Induction of oral tolerance was prevented by the administration of anti-MCP-1 to mice fed HGG. These results show that chemokines play an important role in the induction of oral tolerance. PMID- 8610968 TI - Effect of oral beta interferon on subsequent immune responsiveness. AB - Oral administration of myelin antigens reduces the incidence and severity of EAE in rat and mouse models and decreases the frequency of MBP-reactive cells and the frequency of attacks in some patients with multiple sclerosis. Low-dose oral tolerance has been shown to be mediated by Th2-type regulatory cells that secrete TGFbeta and IL-4/IL-10. Adjuvants and cytokines may modulate oral tolerance. The addition of betaIFN to the experimental therapy regimen, either orally or by intraperitoneal injection, has been shown to enhance the suppressive effects of oral myelin antigens when either are fed the suboptimal dosing regimen to suppress EAE. The current studies were conducted to elucidate the mechanism of the observed in vivo synergy of betaIFN and antigen feeding. Analysis of the in vitro proliferative response and cytokine production by lymphocytes from fed animals in response to specific antigen in culture shows that the synergistic effect may be related to both independent suppression of the immune response by oral betaIFN and enhanced production of TGFbeta and IL-4/IL-10. There was an unexpected increase in the production of gammaIFN by lymphocytes in vitro after three doses of oral betaIFN in vivo. These observations have important implications for the use of cytokines to modulate oral tolerance. PMID- 8610970 TI - Effects of oral antigen in T-cell receptor transgenic mice. PMID- 8610969 TI - Oral-antigen delivery by way of a multiple emulsion system enhances oral tolerance. PMID- 8610971 TI - Molecular mechanisms securing "unresponsiveness" in lamina propria T lymphocytes. PMID- 8610972 TI - Cholera toxin B subunit as transmucosal carrier-delivery and immunomodulating system for induction of antiinfectious and antipathological immunity. PMID- 8610973 TI - Induction of tolerance in humans: effectiveness of oral and nasal immunization routes. PMID- 8610974 TI - Active immunity or tolerance to foods in patients with celiac disease or inflammatory bowel disease. PMID- 8610975 TI - Oral tolerance in experimental autoimmune encephalomyelitis. AB - In work performed by a number of laboratories, it has become quite clear that the oral administration of autoantigens exerts a profoundly suppressive effect on the development and long-term clinical course of autoimmune disease. Specific peptide sequences derived from the autoantigens are similarly suppressive. An interesting sidelight to emerge from specificity studies is that oral administration of a self-protein or peptide sequence (i.e., rat MBP peptide administered to a rat) is markedly less tolerogenic than oral administration of a non-self or even closely related sequence (guinea pig MBP peptide administered to a rat). The dose of oral antigen is now known to play a critical role in determination of the mechanism of oral tolerance, with low doses of antigen causing active suppression with concomitant release of TGFbeta1. Studies outlined here suggest that oral administration of higher antigen doses (e.g., 20 mg MBP to rats or mice) results in deletion of specific antigen-reactive T lymphocytes. This conclusion stems from the fact that injections of IL-2 could not reverse high-dose tolerance while reversing low-dose oral tolerance. Moreover, feeding MBP to MBP-TCR transgenic mice caused trafficking of transgenic cells to the intestine followed by a profound depletion of transgene-positive cells and reduction in proliferative function in all peripheral lymphoid organs. Oral tolerance has proven to be of therapeutic benefit in other animal models of autoimmune disease as well, including uveitis, collagen-induced arthritis, adjuvant arthritis, thyroiditis, myasthenia gravis, and diabetes. Initial human trials in multiple sclerosis, rheumatoid arthritis, and uveitis show promising results. PMID- 8610976 TI - Mucosal tolerance in a murine model of experimental autoimmune encephalomyelitis. AB - Attempts to induce oral tolerance in a murine model of EAE with either the dominant T-cell epitope or whole protein have failed. These results may, in part, be due to the extraordinarily low affinity for class II MHC displayed by the dominant T-cell epitope. This belief is supported by experiments using a high affinity analogue of the peptide that was capable of inducing tolerance at a high dose. By contrast, peptide inhalation has proven an effective route for induction of mucosal tolerance in this model. Most importantly, the inhalation of a single peptide could inhibit disease induced by the complex mixture of antigens found in whole myelin. Peptide inhalation was effective both before and after disease induction, and there was a positive correlation between affinity of class II binding and tolerogenicity of a panel of analogues of the N-terminal peptide of myelin basic protein. PMID- 8610977 TI - Three-year open protocol continuation study of oral tolerization with myelin antigens in multiple sclerosis and design of a phase III pivotal trial. PMID- 8610978 TI - Antigen-specific TGF-beta1 secretion with bovine myelin oral tolerization in multiple sclerosis. AB - Multiple sclerosis is a presumed autoimmune disease, associated with inflammation in the CNS white matter, mediated by autoreactive T cells. We previously reported that oral myelin tolerization of relapsing-remitting MS patients resulted in fewer attacks, as compared to a placebo-fed group. Here, we examined whether oral tolerization with bovine myelin resulted in altered autoreactive T-cell populations or altered T-cell fraction. We generated 4,620 T-cell lines from 34 relapsing-remitting MS patients (17 were fed bovine myelin daily), and each line was examined for proliferation to MBP, PLP, and TT and for secretion of IL-4, IFN gamma, and TGF-beta1. The frequency of TGF-beta1-secreting T-cell lines after MBP and PLP stimulation in fed patients was greater than that of nonfed patients. These experiments demonstrate that oral tolerization with autoantigen results in altered cytokine secretion in a human autoimmune disease with the generation of TGF-beta1-secreting T cells that may regulate the inflammatory response at the site of the demyelinating lesions in multiple sclerosis. These data provide the first evidence of antigen-specific modification of cytokine secretion in a human autoimmune disease. PMID- 8610979 TI - Oral tolerance in myasthenia gravis. AB - Because of the antibody-mediated pathogenesis of MG, it is of particular interest to understand the effects of oral administration of the autoantigen AChR on the disease process. It is now clear that feeding AChR prior to immunization can prevent clinical manifestation of EAMG. It initially primed, then inhibited, antibody responses to foreign (Torpedo) AChR and self (rat) AChR, with a delayed onset. Cellular responses to AChR, evaluated by lymphocyte proliferation and IL-2 production, were markedly inhibited. The effects were dependent on the dose and purity of the fed antigen. Tolerance to an orally administered unrelated antigen, OVA, was more prompt in development and more profound, illustrating the influence of the nature of the antigen on tolerance. The tolerance induced was antigen specific. Oral administration of AChR after immunization resulted in inhibition of the clinical manifestation of EAMG, concomitant with a paradoxical enhancement of the AChR-antibody responses. Both the clinical benefit and the antibody response appear to be dependent on the feeding protocol. These findings suggest that a molecule with less immunogenic potential than native AChR may be required for safe and effective oral treatment of ongoing disease. PMID- 8610980 TI - Mucosal tolerance to experimental autoimmune myasthenia gravis is associated with down-regulation of AChR-specific IFN-gamma-expressing Th1-like cells and up regulation of TGF-beta mRNA in mononuclear cells. AB - Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG. PMID- 8610981 TI - Antigen trafficking in the intestine. PMID- 8610982 TI - Antigen-presenting function of the mouse CD1 molecule. AB - CD1 molecules are distantly related to major histocompatibility complex (MHC) encoded class I molecules, and they are coexpressed with beta2 microglobulin (beta2m). In the mouse, CD1 is expressed by intestinal epithelial cells and also by some cells in spleen and lymph node. We have shown that surface expression of mouse CD1 (mCD1) is not dependent upon a functional transporter associated with antigen processing (TAP). This, and other data, suggest that mCD1 may acquire peptides in an intracellular compartment other than the endoplasmic reticulum, where classical class I molecules bind peptide. mCD1 molecules also are distinct from classical class I molecules with regard to the types of peptides that they bind. We have demonstrated that mCD1 molecules preferentially bind peptides much longer than the 8-9 amino acids typical of the peptides that bind to classical class I molecules. The sequence motif for mCD1 peptide binding is characterized by the presence of bulky and hydrophobic amino acid side chains. We have generated mCD1-restricted and peptide-specific T-cell lines, thereby demonstrating the immunologic relevance of peptide binding to mCD1. The reactive T cells are TCR alphabeta+ and CD8+, a phenotype typical of many lymphocytes in both lymph node and intestinal mucosae. We speculate that mCD1 molecules may be capable of sampling peptides from the gut lumen and presenting them to mucosal T lymphocytes. In this way, they may function in the maintenance of normal mucosal homeostasis, and perhaps also in the induction of systemic tolerance to antigens delivered by the oral route. In summary, CD1 molecules are a novel category of antigen-presenting molecules that have features in common with class I molecules, features in common with class II, and properties distinct from either subset of antigen-presenting molecules. Further studies of the antigen-presenting function of these molecules are certain to yield new insight into immune regulation and perhaps also into the mechanism of oral tolerance. PMID- 8610983 TI - Arthritis: animal models of oral tolerance. PMID- 8610984 TI - Evidence that type II collagen feeding can induce a durable therapeutic response in some patients with rheumatoid arthritis. PMID- 8610985 TI - Cytokine-dependent modulation of oral tolerance in a murine model of autoimmune uveitis. AB - In summary, our data suggest that oral tolerance in the mouse EAU model may occur by anergy/deletion or by suppression, depending on the feeding regimen. Tolerance involving putative regulatory cells appears to require the ability to produce both IL-4 and IL-10, whereas induction of tolerance involving anergy may not require the presence of Il-4 and IL-10. We propose that regulatory cells induced by three feedings of IRBP can be selectively enhanced through the use of cytokines. From the point of view of clinical therapy, it would be worthwhile to explore postimmunization feeding regimens involving administration of IL-4 and IL 10. PMID- 8610986 TI - Intraocular inflammatory disease (uveitis) and the use of oral tolerance: a status report. AB - Intraocular inflammatory disease, or uveitis, is a disorder that mostly affects children and young adults. It is the cause of about 10% of the severe visual handicap in the United States. Many of the severe, sight-threatening uveitic conditions are thought to be driven by putative autoimmune mechanisms, often with high-dose oral prednisone use as treatment, along with cytotoxic agents, antimetabolites, and cyclosporine adjunctively. The feeding of the uveitogenic retinal S-Ag to rats immunized with the same antigen resulted in clinical protection. A pilot study in which two patients, one with pars planitis and the other with Behcet's disease, were fed with the retinal S-Ag resulted in these patients' immunosuppressive medication being decreased and/or stopped. The trial also provided us with information concerning dosage and expected immune responses. A randomized, masked study looking at the effect of feeding retinal antigens to uveitis patients is ongoing. PMID- 8610987 TI - Mechanisms of oral tolerance by MHC peptides. AB - Recent evidence indicates that MHC peptides play an important role in T-cell recognition of alloantigen. We studied the tolerogenicity of orally administered synthetic MHC allopeptides in the rat model. Initially, we demonstrated that oral administration of synthetic class II MHC allopeptides significantly inhibited the DTH response to the peptides as well as to donor-derived cells. The tolerogenic effect was antigen specific and was induced by immunogenic, but not by nonimmunogenic, allopeptides. Immunohistological studies of DTH skin lesions showed that oral tolerance is associated with a state of "immune deviation" to a predominance of Th2 cell function in the lesions. We recently extended the above observations and examined the tolerogenic effect of orally administered synthetic MHC allopeptides on MLR and CTL generation. We found that oral administration of the class II allopeptides effected significant reduction of MLR proliferation and CTL generation, which was antigen specific. In addition, similar to the DTH results when we compared the tolerogenicity of the immunogenic versus the nonimmunogenic peptides, MLR and CTL suppression was significantly higher with the immunogenic peptides. The gut immune system play an important role in oral tolerance by MHC peptides. Initial experiments showed that intestinal epithelial cells pulsed in vitro with immunogenic MHC allopeptides, or in vivo by oral administration of immunogenic peptides, were capable of presenting these peptides to primed T cells in vitro. Whether such presentation by intestinal epithelial cells or other gut antigen-presenting cells leads to preferential activation of Th2 regulatory cells, which ultimately suppress Th1 alloimmune responses, remains to be determined. PMID- 8610988 TI - Oral tolerance to insulin and the insulin B-chain: cell lines and cytokine patterns. PMID- 8610989 TI - Local and systemic immune responses in SJL/J mice during prolonged oral myelin basic protein administration. PMID- 8610990 TI - The role of the thymus in intestinal intraepithelial T-cell development. PMID- 8610991 TI - Active suppression of diabetes after oral administration of insulin is determined by antigen dosage. AB - We have previously demonstrated that feeding six-week-old female mice with 20 units of human insulin every 2 - 3 days for 15 or 30 days induced an active mechanism of suppression through the generation of regulatory T cells that reduced the number of successful diabetic transfers in irradiated NOD recipients. In the present study, we analyzed the effects of antigen dosage and the critical period of cell injection to obtain protection. The effects of the dose of insulin feeding were therefore compared during cotransfer experiments of 5 x 10(6) T cells from diabetic mice and 5 x 10(6) T cells from the spleen of mice receiving 10 units, 20 units, or 40 units of insulin or saline every 2 - 3 days for 15 days. Only T lymphocytes from mice fed with 20 units conferred active cellular protection during adoptive transfer with a significant delay in diabetes onset (p = 0.002). No significant difference was noticed during histological analysis of pancreatic glands, indicating tha insulitis was not prevented. However, mice receiving T lymphocytes from the 20 units of insulin-fed animals had a milder form of inflammation, with a significantly lower percentage of severely infiltrated islets. Injecting regulatory T cells 7 days and 14 days after iv injection of diabetogenic T cells did not modify the incidence curves of diabetes in the recipients, suggesting that cellular interactions and delay in cell trafficking were determinants. These results may have important clinical implications in humans. In conclusion, this study indicates the importance but also the limits of antigen therapy in type I diabetes. Antigen dosage is a critical element for active suppression. Such analysis is important to perform in humans before the initiation of a large-scale prevention trial in prediabetic individuals. PMID- 8610992 TI - Induction of transplantation tolerance by feeding or portal vein injection pretreatment of recipient with donor cells. PMID- 8610993 TI - Intranasal administration of insulin peptide B: 9-23 protects NOD mice from diabetes. PMID- 8610994 TI - Breaking of oral tolerance by an encapsulated antigen. PMID- 8610995 TI - Effects of cyclosporin A on the induction of oral tolerance. PMID- 8610996 TI - Rheumatoid arthritis and the drop in tolerance to foods: elimination diets and the reestablishment of tolerance by low-dose diluted food. PMID- 8610997 TI - Oral tolerance in autoimmune encephalomyelitis. In vivo reversal of anergy. PMID- 8610998 TI - Protein B: an important human IgA-binding reagent. AB - Protein B had a much higher affinity for human IgA than Jacalin, increasing the sensitivity and specificity of the measurement of total human IgA. Protein B, used as a capturing agent, greatly enhanced the measurement of antigen-specific IgA as compared to alpha chain-specific antibodies. PMID- 8610999 TI - Multiple emulsions oral vaccine vehicles for inducing immunity or tolerance. PMID- 8611000 TI - In vivo administration of IL-4 induces TGF-beta-producing cells and protects animals from experimental autoimmune encephalomyelitis. PMID- 8611001 TI - Oral tolerance in experimental autoimmune encephalomyelitis: specificity of peptide-induced oral tolerance. PMID- 8611002 TI - Mucosal tolerance induced by flour dust. PMID- 8611003 TI - Pilot study of oral tolerance to keyhole limpet hemocyanin in humans: down regulation of KLH-reactive precursor-cell frequency. PMID- 8611004 TI - The protective role of enteral IgA supplementation in neonatal gut-origin sepsis. PMID- 8611005 TI - Immune deviation during the induction of tolerance by way of nasal installation: nasal installation itself can induce Th-2 responses and exacerbation of disease. PMID- 8611006 TI - Oral tolerance in myelin basic protein TCR transgenic mice. PMID- 8611007 TI - The effects of oral myelin basic protein and dexamethasone treatment on experimental autoimmune encephalomyelitis. PMID- 8611008 TI - Oral insulin does not prevent insulin-dependent diabetes mellitus in BB rats. PMID- 8611009 TI - Mucosal tolerance to aflatoxin B1. PMID- 8611010 TI - Tolerance to an arthritogenic T-cell epitope of HSP65 and the regulation of experimental arthritis. PMID- 8611011 TI - IL-4 is not involved in the early MLN T-cell response to antigen given orally with cholera toxin, but those cells can express IL-4R. PMID- 8611012 TI - Development of immune response to orally administered cow milk protein in young children. PMID- 8611013 TI - Intestinal intraepithelial lymphocyte responses to glucocorticoid signaling. PMID- 8611014 TI - Regulation of chemokine gene expression by contact hypersensitivity and by oral tolerance. AB - In mice with hapten-induced CH, T cells of the CD4+ and CD8+ phenotypes activated the gene for JE, whereas CD8+ T cells alone caused activation of the gene for IP 10. In animals tolerized by feeding either TNCB or OX, hapten-induced expression of IP-10 but not JE mRNA was lost. The down-regulation of IP-10 gene activation was adoptively transferred from tolerized mice to naive mice by CD4+ splenic T cells. These findings reflect the differential roles of individual T-cell subsets in both enhancing and diminishing chemokine gene expression in contact hypersensitivity reactions. PMID- 8611015 TI - The role of dendritic cells in antigen processing in the Peyer's patch. PMID- 8611016 TI - Role of gamma delta T cells in the regulation of mucosal IgA response and oral tolerance. AB - In this short review, we first described experiments that show that prolonged oral immunization with a protein vaccine, such as DT, induced systemic unresponsiveness in the presence of antigen-specific mucosal IgA responses. Mucosal T cells, such as IEL, may play an important role for the maintenance of antigen-specific IgA responses because these T cells are able to respond to stimulation signals provided by antigen even when T-cell unresponsiveness was induced in systemic tissue, such as spleen of mice orally tolerized with the protein DT. Inasmuch as IEL contain a high frequency of gamma delta T cells, it was logical to postulate that mucosal gamma delta T cells are essential regulatory T cells for the induction of IgA responses in oral tolerance. To this end, our previous studies showed that adoptive transfer of mucosal gamma delta T cells from IEL of mice orally tolerized with SRBC to the recipient mice with systemic unresponsiveness to the same antigen resulted in the abrogation of unresponsiveness to Ig synthesis, including those of IgA isotype. In this regard, when the mucosal immune system of TCR-delta-/- and their control mice was examined, lower numbers of IgA antibody-producing cells were noted in TCR-delta-/ mice in comparison to control background mice. Further, the level of IgA in fecal extracts was also low in TCR-delta-/- mice. These findings suggested that loss of gamma delta T cells in down-regulation of IgA B-cell responses. PMID- 8611017 TI - The role of Th1 and Th2 cells for mucosal IgA responses. AB - We have used cytokine-knockout mice to help determine the precise requirements for CD4+ Th cell regulation of IgA responses. In these studies, we have used two different oral delivery systems to induce mucosal and systemic antibody responses to the vaccine TT. In normal mice, oral administration of TT with CT as adjuvant induces Th2 cells and cytokines, which give rise to mucosal IgA and serum IgG1, IgA, and IgE responses. On the other hand, oral immunization with rSalmonella expressing Tox C results in Th1-type responses as well as Th2 cell-derived IL-10 and macrophage-derived IL-6, which correlate with mucosal IgA and serum IgG2a antibody responses. Two major conclusions can be drawn from our studies with these two regimens in normal, IFN-gamma-/-, and IL-4-/- mice. First, oral administration of rSalmonella, which elicits classical Th1-type responses also induces significant mucosal IgA responses when given to mice with defective Th1- (IFN-gamma-/-) or Th2- (IL-4-/-) cytokine pathways. Interestingly, we detect Th2 type cells producing IL-10 and macrophage-secreting IL-6 in both normal and cytokine-deficient mice, and we postulate that these two cytokines are of most importance for murine IgA responses. Second, oral administration of TT plus CT as adjuvant induces classical Th2-type responses in both normal and IFN-gamma-/- mice. Further, lack of IL-4 results in failure to induce mucosal IgA responses. Thus, the IL-4 pathway is necessary for the CT adjuvant effect for mucosal IgA responses after oral immunization with a protein vaccine. PMID- 8611018 TI - Studying immunological tolerance by physically monitoring antigen-specific T cells in vivo. PMID- 8611019 TI - Parenteral and oral administration of tolerogens: protein-IgG conjugates. PMID- 8611020 TI - Neonatal oral tolerance. PMID- 8611021 TI - In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface. AB - To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to be 5' end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly2 in the myristoylation signal with Ala entirely inhibited the incorporation of [3H] myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro translation/transcription system. PMID- 8611022 TI - Uptake of iron by isolated rat hepatocytes from a hydrophilic impermeant ferric chelate, Fe(III)-DTPA. AB - We studied uptake of iron from Fe(III)-diethylenetriamine pentaacetate (DTPA) in isolated rat hepatocytes. This uptake is specific with an affinity of 600 nM and shows an optimum pH of 6. The specificity is indicated by inhibition by ferric citrate and diferric transferrin. Iron uptake from Fe(III)-DTPA is completely inhibited by trypsinization of the cell surface, by strong impermeant ferric chelators (DTPA, apo-transferrin, polymer-conjugated desferrioxamine), both hexacyanoferrates, copper and zinc, and partly by dipyridyl, manganese, cobalt, N ethylmaleimide, and citrate. The lysosomotropic agent chloroquin inhibits weakly; proton pump inhibitors are without effect. Ascorbate and Tiron both effectively stimulate the uptake and also mobilize iron from DTPA in vitro. Approximately 75% of the freshly acquired intracellular iron is found in ferritin even after uptake at lowered temperature (16 degrees C). We conclude that a rate-limiting mobilization of iron from the DTPA chelate by a cell-surface activity is required before iron can actually enter the cell. This can be enhanced by mediators of iron release, but does not seem to require reduction of iron. The use of DTPA as chelator offers the possibility of studying this putative activity because the Fe(III)-DTPA chelate is stable in the presence of transferrin or desferroxamine, in contrast to ferric citrate or Fe(NTA)2. PMID- 8611023 TI - Binding of myristoylated alanine-rich protein kinase C substrate to phosphoinositides attenuates the phosphorylation by protein kinase C. AB - The myristoylated aline-rich protein kinase C substrate (MARCKS) is a peripheral membrane protein that undergoes phosphorylation-dependent translocation between membrane and cytosol. MARCKS binds to acidic phospholipids with high affinity (Kd less than 0.5 microM) but binds poorly to neutral phospholipids. Although interaction of MARCKS with acidic phospholipids lacks specificity when determined by binding assay, these phospholipids exert distinctive effects on the phosphorylation of this protein by protein kinase C (PKC). Preincubation of MARCKS with phosphatidylserine (PS) or phosphatidylglycerol enhanced the phosphorylation; whereas with phosphatidic acid, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, or phosphatidylinositol-4,5-biphosphate inhibited the phosphorylation of this substrate by PKC. Phosphoinositide inhibition of MARCKS phosphorylation was apparently directed at the substrate rather than at the kinase as the phosphorylation of two other phospholipid binding PKC substrates, neuromodulin and neurogranin, exhibited different responses from those of MARCKS. Furthermore, the inhibition of phosphoinositides on MARCKS phosphorylation was seen with PKC isozymes alpha, beta, gamma, and delta and with the catalytic fragment of PKC, protein kinase M. A 25-amino-acid synthetic peptide corresponding to the phosphorylation site domain (PSD) of MARCKS, but not to the myristoylated N-terminal peptide, competed equally effectively with MARCKS in binding to either PS- or PI-containing vesicles, suggesting that both phospholipids bind to the PSD of MARCKS. Binding of PI to MARCKS inhibited PKC phosphorylation of all three phosphorylation sites. These results suggest that phosphoinositides and PS bind at different residues within the MARCKS PSD, so that the resulting phospholipid/MARCKS complexes are differentially phosphorylated by PKC. PMID- 8611024 TI - Measurement of metabolic fluxes through pyruvate kinase, phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, and pyruvate carboxylate in hepatocytes of different acinar origin. AB - Isolated hepatocytes were prepared from the periportal and perivenous regions of the liver of 18-h-starved rats. These showed characteristics enzyme patterns and an enhanced rate of ureagenesis in the periportal cells; however, total cellular ATP content was unchanged in the two cell types. Measurements of pyruvate kinase flux showed no significant difference in the overall rate in the two cell types; however, the flux through phosphoenolpyruvate (PEP) carboxykinase was significantly higher in the periportal cells, such that the percentage of PEP being metabolized by pyruvate kinase was enhanced in the perivenous cells. The increase in partitioning of PEP through pyruvate kinase could account for only a small percentage of the difference in gluconeogenic flux in the two cell types, suggesting that the rate of provision of PEP was the principal limiting factor for glucose synthesis. The flux through pyruvate dehydrogenase showed no significant metabolic zonation, whereas pyruvate carboxylase flux was enhanced in the periportal zone. The partitioning of pyruvate between pyruvate carboxylase and pyruvate dehydrogenase was increase 2.8-fold in the periportal cells compared to that in the perivenous cells and it is suggested that this, together with possible alterations in phosphoenolpyruvate carboxykinase, is primarily responsible for the different gluconeogenic rates in the two zones of the liver. PMID- 8611025 TI - Determination of rate constants of the reactions of thiols with superoxide radical by electron paramagnetic resonance: critical remarks on spectrophotometric approaches. AB - Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s 1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols. PMID- 8611026 TI - Upregulation of the Drosophila 1731 retrotransposon long-terminal repeat by UV-B irradiation requires a short sequence in the U3 region. AB - A 1731 is a Drosophila melanogaster retrotransposon, the transcripts of which decrease in Drosophila cells after treatment by the 20-hydroxyecdysone (20-OH), the steroid-molting hormone of insects. In order to analyze the regulation of the long terminal repeat (LTR) directed transcription by UV-B, S2 Drosophila cells were transfected with various chimeric constructs carrying the LTR of 1731 linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and then subjected to UV-B irradiation. The results demonstrated that the 1731 LTR is activated by UV-B irradiation in a dose- and time-dependent manner. Using constructions expressing the reporter gene under the control of either the entire or deleted LTRs of 1731, we established that a sequence located in the U3 region was required for the retrotransposon to respond to UV-B. The cis-acting element is identical to the binding sequence of the dorsal transcription factors. In addition, the S2 Drosophila cell produced extracellular factor(s) in response to UV-B irradiation which activated the 1731 LTR in nonirradiated cells. This factor(s) was detected when responding cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures. PMID- 8611027 TI - Superoxide production during reduction of molecular oxygen by assimilatory nitrate reductase. AB - Assimilatory NADH:nitrate reductase catalyzes the transfer of reducing equivalents from NADH to molecular oxygen. Initial rate studies performed under conditions of optimal pH (8.0) and constant ionic strength (mu = 0.2) revealed that the maximal rate of activity with molecular oxygen was 0.5% (0.44 mumol NADH consumed/min/nmol heme) with a Km for O2 of 586 microM. NADH:molecular oxygen reductase activity exhibited a pH optimum of 9.2, was inhibited by cyanide, and was unaffected by changes in ionic strength or the presence of phosphate ions. Spectroscopic studies indicated NADH:molecular oxygen reductase activity resulted in the production of the superoxide radical, detected as the formation of adrenochrome from epinephrine and by the formation of adrenochrome from epinephrine and by the reduction of nitroblue tetrazolium, both of which could be inhibited by the addition of superoxide dismutase and were unaffected by the addition of catalase. Direct observation of superoxide production using spin trapping in combination with EPR spectroscopy resulted in the detection of the spin adduct 5.5-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH). The formation of this spin adduct was abolished either in the absence of nitrate reductase, NADH, or DMPO or the the addition of superoxide dismutase or nitrate and was greatly reduced by the presence of cyanide. Inclusion of catalase or ethanol had no effect on the formation of the spin adduct. These results indicate that nitrate reductase can utilize molecular oxygen as an electron acceptor and that the product, O2.(-), is primarily generated via the Mopterin center. PMID- 8611028 TI - Lovastatin inhibits the stimulation of mitogen-activated protein kinase by insulin in HIRcB fibroblasts. AB - Lovastatin, a cholesterol biosynthesis inhibitor, has recently been shown to inhibit mitogenesis and tumor growth. We have investigated the effects of lovastatin on the activation of MAP kinase by insulin using as a model HIRcB cells, a rat fibroblast cell line that overexpresses the human insulin receptor. Treatment with lovastatin (1-30 microM) for 24 h decreased the level of activation of MAP kinase by insulin by as much as 60%. Immunoblotting experiments using a specific anti-MAP kinase monoclonal antibody demonstrated that the amount of MAP kinase protein in the cells was not altered by lovastatin treatment. Likewise, lovastatin had no apparent effects on the expression of the insulin receptor. Treatment with lovastatin (20 microM) reduced the percentage of farnesylated Ras by 50%. Immunoprecipitation of tyrosine phosphorylated proteins from HIRcB cell lysates followed by immunodetection of MAP kinase using specific antibodies demonstrated a reduced level of insulin-induced tyrosine phosphorylation levels of MAP kinase in lovastatin-treated cells. Furthermore, immunodetection of the beta-subunit of the insulin receptor in anti phosphotyrosine immunoprecipitates revealed that treatment with lovastatin reduced the tyrosine phosphorylation levels of the receptor. Lysates obtained from cells treated with increasing concentrations of lovastatin demonstrated a dose-dependent inhibition of the insulin-induced tyrosine phosphorylation of the receptor. Treatment with mevalonic acid prevented the effects of lovastatin demonstrating that the effects of the drug are a consequence of its inhibitory effects on the synthesis of steroids. It is concluded that, in addition to inhibition of Ras farnesylation, lovastatin reduces receptor tyrosine phosphorylation levels which also contributes to the blockade of MAPK activation by the insulin receptor. PMID- 8611029 TI - Glucose 6-phosphate and mannose 6-phosphate are equally and more actively hydrolyzed by glucose 6-phosphatase during hysteretic transition within intact microsomal membrane than after detergent treatment. AB - We have studied the rapid kinetics of glucose-6-phosphatase (Glc6Pase) toward glucose 6-phosphate (Glc6P) and mannose 6-phosphate (Man6P) in intact and detergent-treated microsomes, using a radiometric assay based on the use of [U( )14C]hexose 6-phosphates. We show that a hysteretic transition of Glc6Pase from a rapid hydrolytic form to slower kinetic form within the intact membrane occurs for both substrates with the same relaxation time. During the hysteretic transition, preceding the steady-state rate of hydrolysis, Glc6Pase is able to hydrolyze both Glc6P and Man6P at very similar rates. Only Glc6P is significantly hydrolyzed at steady state. Moreover, the initial rates of hydrolysis of both Glc6P and Man6P in intact microsomes are higher than the respective rates of hydrolysis after detergent treatment of the membrane at high substrate concentrations (10 and 20 microM), while these rates are not different at lower substrate concentrations. These data show that the marked specificity of Glc6Pase at steady state in the membrane is acquired owing to a hysteretic transition induced by the hydrolytic phenomenon, independently of the nature of the prior phosphate donor. The role of the membrane in this phenomenon is crucial, since the transition does not occur in its absence. PMID- 8611030 TI - Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA. AB - To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs. Similar observations were made in P19 cells expressing a chimeric beta-APP-751 mRNA where endogenous beta APP-751 mRNA levels also were decreased. Also, suppression of beta-APP-770 and 751 mRNAs was observed in human kidney cells expressing exogenous beta-APP-695 mRNA. PMID- 8611031 TI - Hemoglobin induced apolipoprotein B crosslinking in low-density lipoprotein peroxidation. AB - Oxidative modification of human low-density lipoprotein (LDL) is thought to play a major role in the development of atherosclerosis. Free hemin, hemoglobin, myoglobin, and horseradish peroxidase (HRP) were reported in different studies as promoters of LDL lipid oxidation. Based on our previous finding that hemin induced oxidative crosslinking of the LDL protein, apolipoprotein B (apo B) (Y. I. Miller and N. Shaklai (1994) Biochem. Mol. Biol. Int. 34, 1121-1129), we compared the ability of free hemin and the above hemoproteins to induce peroxidation modification of apo B using SDS-PAGE. The levels of the final products of lipid peroxidation were determined as thiobarbituric acid-reactive substances. Hemoglobin and myoglobin were found to be as active as free hemin and all these were much more active than the classic peroxidase HRP. Moreover, the products of oxidized apo B differed: hemoglobin, myoglobin, and hemin induced mostly covalent aggregates, while HRP caused fragmentation of apo B. Hemoglobin reactivity was expressed at low H2O2 concentrations even in the absence of molecular oxygen. Desferal, along with other antioxidants, inhibited the hemoglobin-induced LDL oxidation independently of its iron-chelating property. The high peroxidative reactivity of hemoglobin is explained by its ability (unlike HRP) to transfer the oxidative equivalents from the heme active site, through the globin, to LDL. The apo B radicals thus formed are terminated, yielding intermolecular crosslinked protein. It is suggested that small amounts of the highly reactive hemoglobin in plasma, suffice to trigger LDL protein oxidation (along with its lipid oxidation), thereby inflict the atherosclerosis precondition. PMID- 8611032 TI - Oxidation of 1,2,4,5-tetramethoxybenzene by lignin peroxidase of Phanerochaete chrysosporium. AB - We have reinvestigated the lignin peroxidase-catalyzed oxidation of 1,2,4,5 tetramethoxybenzene (TMB) by using presteady-state and steady-state kinetic methods. Our presteady-state kinetic results show that the reaction of compound I with TMB obeyed second order kinetics with a rate constant of 1.1 x 10(7) M-1s-1. The reaction of compound II with TMB exhibits a hyperbolic concentration dependence with a Kd of 16 microM and K = 24 s-1. The stoichiometry of TMB oxidation during steady state is two TMB cation radicals formed per H2O2 consumed. These results clearly show that TMB is a good substrate for both compounds I and II of lignin peroxidase. PMID- 8611033 TI - Endotoxin treatment increases lung mitochondrial scavenging of extramitochondrial superoxide in hyperoxia-exposed rats. AB - Mitochondria scavenge extramitochondrial superoxide anion via a respiration dependent (i.e., non-enzymatic) mechanism. This study reports that hyperoxia exposure ( > 95 % O2 for 48h) reduced (P < 0.05) lung mitochondrial respiration dependent superoxide scavenging by 56% compared to lung mitochondria from untreated control rats. In comparison, endotoxin treatment (5 micrograms insufflated intratracheally 24 h earlier) increased (P < 0.05) lung mitochondrial respiration-dependent superoxide scavenging by 57% compared to mitochondria from untreated control rats. Further, lung mitochondria from rats given endotoxin 24 h prior to hyperoxia exposure had more than twice the respiration-dependent superoxide scavenging capacity as compared to mitochondria from untreated rats exposed to hyperoxia (P < 0.05). In contrast, endotoxin treatment did not increase (P < 0.05) lung mitochondrial enzymatic (i.e., superoxide dismutase) scavenging activity when corrected for mitochondrial protein content in either hyperoxia-exposed or air-exposed rats. Therefore, hyperoxia exposure decreased, whereas endotoxin treatment increased, respiration-dependent lung mitochondrial scavenging of extramitochondrial superoxide. This recently identified cellular antioxidant defense appears to be an early target in hyperoxia but its induction provides an important component of endotoxin-induced tolerance to hyperoxic lung damage. PMID- 8611034 TI - Coupling between [arginine8]-vasopressin-activated increases in protein tyrosine phosphorylation and cellular calcium in A7r5 aortic smooth muscle cells. AB - Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca2+]i and protein tyrosine phosphorylation induced by 20 nM [arginine 8]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells. In fura-2-loaded cells, AVP induced a rapid (0.5-2 min) transient increase in [Ca2+]i that was followed by a smaller sustained increase in [Ca2+]i. In 66% of the cells, the transient response involved both influx of extracellular Ca2+ and release of intracellular Ca2+: influx accounted for 6% of the response, and release accounted for 40%. However, in 34% of the cells, the relative contribution of influx and release during the transient could not be assessed. In all cells, the smaller sustained response was entirely dependent on extracellular Ca2+. Genistein (148 microM) always blocked the transient and sustained components of the Ca2+ response showing that both influx and release were genistein-sensitive. Isobestic fluorescence analysis, in medium containing 0.5 mM Mn2+ in place of Ca2+, showed that the influx pathway was selective because it did not conduct Mn2+. It also confirmed that Ca2+ release was blocked by genistein. In contrast, 105 microM lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 microM), did not alter the transient or sustained components of the Ca2+ response. No AVP-induced increases in tyrosine phosphorylation were detected unless special procedures were used. When cells were preincubated in 10 mM vanadate, a tyrosine phosphatase inhibitor, AVP induced a transient increase in tyrosine phosphorylation (5-60 s). The time course for AVP-induced phosphorylation was similar to that for increase in [Ca2+]i. Vanadate alone increased tyrosine phosphorylation and induced a slow small increase in [Ca2+]i that was dependent on extracellular Ca2+. Genistein blocked tyrosine phosphorylation induced by AVP and vanadate, and it blocked the increase in [Ca2+]i induced by vanadate alone. In contrast, lavendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylation induced by vanadate alone and precluded assessment of AVP-induced tyrosine phosphorylation in the presence of vanadate. Lavendustin produced time-dependent enhancement of vanadate induced increase in [Ca2+]i. These results underscored the need for measuring cellular changes in protein tyrosine phosphorylation to assess potential functions of tyrosine kinase activity. Under conditions where changes in phosphorylation could be measured, the results suggested that AVP-activated increases in tyrosine phosphorylation may be coupled to AVP-activated mechanisms that regulate influx of extracellular Ca2+ and release of intracellular Ca2+. PMID- 8611035 TI - Regulation of peroxisome proliferator-activated receptor-alpha mRNA in rat liver. AB - Chemical-induced peroxisome proliferation in rodent liver is postulated to occur via activation of members of the steroid hormone receptor superfamily, the peroxisome proliferation-activated receptors (PPARs). In the present study, the expression of the predominant liver subtype PPAR alpha was examined and compared to that of acyl-CoA oxidase (ACO), a marker for peroxisome proliferation and a prototype for genes regulated via PPARs. Despite the induction of both mRNA species in vivo by the peroxisome proliferator perfluorodecanoic acid (PFDA), dose response and time course indicate PPAR alpha and ACO are not controlled similarly. Messenger RNA levels for ACO increased rapidly in rat liver and declined over the subsequent 7 days following PFDA administration, while PPAR alpha mRNA increased slower and remained elevated over this period. In addition, PPAR alpha mRNA accumulation in PFDA-treated rats appears to be due primarily to hypophagia as pair feeding and complete caloric restriction result in a large increase in the concentration of this messenger RNA. Nuclear run-on experiments in vivo suggest that, unlike ACO, PFDA as well as caloric restriction results in accumulation of PPAR alpha mRNA which cannot be explained solely by transcriptional activation. These data indicated that PPAR alpha mRNA accumulation has a very small peroxisome proliferator-dependent component and that other factors may be involved. A rat hepatoma cell line was examined to determine the direct effect on peroxisome proliferators on PPAR alpha mRNA. PPAR alpha and ACO mRNA levels were increased rapidly in the rat hepatoma cell line FaO after treatment with PFDA or the prototypical peroxisome proliferator Wy 14,643. In this cell line, PPAR alpha mRNA levels are not affected by glucagon or insulin and in addition to peroxisome proliferators are induced in this cell line by oleic acid and dexamethasone. The latter treatment had the greatest effect on PPAR alpha mRNA accumulation while having a minimal effect on ACO mRNA. Treatment of FaO cells with actinomycin D prior to Wy 14,643 abolished ACO and PPAR alpha mRNA accumulation, demonstrating that there must be a transcriptional component of the peroxisome proliferator response. Therefore, although PPAR alpha is responsive to peroxisome proliferators and direct effects are observed in cell cultures, mRNA accumulation in vivo is predominantly posttranscriptional, and endogenous regulators such as glucocorticoids may play critical roles in the tissue- and developmentally specific expression of this steroid hormone receptor. PMID- 8611036 TI - Effect of interferon on protein translation during growth stages of 3T3 cells. AB - Interferons (IFNs) elicit a spectrum of biological responses from target cells, including inhibition of proliferation in several types of cells in vivo and in culture. The mechanism of action of IFN is complex and not fully understood. Previous evidence has indicated that part of the antiproliferative effect of IFN is due to modulation of protein translation. Here we report that there is a transient autocrine production of beta-interferon during specific periods of growth of mouse 3T3-F442A and 3T3-C2 cells. Treatment of preconfluent mouse 3T3 C2 cells with interferon reduced protein synthesis in these cells. This reduction began after 3 h of interferon treatment and was correlated with the appearance of phosphorylated double-stranded RNA dependent eIF-2 alpha kinase (PKR) measured in vitro. This inhibition of protein synthesis was associated with diminished exchange of GTP for GDP in the eLF-2.GDP complex. This diminished guanine nucleotide exchange activity was due to the inhibition of eukaryotic initiation factor eIF-2B, the factor required for the dissociation of GDP from eIF-2, and the formation of the functional eIF-2.GTP complex. The autocrine effect of IFN resulted in elevated PKR activity, increased phosphorylation of eIF-2 alpha, and diminished eIF-2B activity. These results suggest that interferon regulates the initiation of protein synthesis by a mechanism involving PKR, eIF-2 alpha phosphorylation, and eIF-2B activity. Since 3T3-F442A cells produce and secrete interferon in a transient fashion during growth, this regulatory mechanism may be significant in the normal growth and differentiation of these cells. PMID- 8611037 TI - cDNA-directed expression of two allelic variants of cytochrome P450 2C11 using COS1 and SF21 insect cells. AB - Cytochromes P450 (CYP) are ubiquitous enzymes which metabolize multiple endogenous and exogenous substrates. CYP2C11 is the most abundant among the P450s present in untreated rat liver (approximately 50% of total P450) and is known for metabolizing testosterone mainly in positions 2 alpha and 16 alpha. In Gunn rats, the specific enzymatic activities of CYP2C11 are decreased by 90%, although the protein is present at levels similar to that of the Wistar reference strain. In order to explain this difference, we cloned and expressed CYP2C11 Wistar and Gunn cDNAs in two heterologous systems. Three mutations were identified in Gunn CYP2C11 form: Val4 --> Ala in the transmembrane region, Asn116 --> Ser in the substrate recognition site SRS-1, and Phe187 --> Leu in the (E --> F) interhelical region when compared to the spatial structure of CYP101. Sf21 insect cells displayed high yields of expressed CYP2C11 proteins (up to 2 nmol CYP2C11/mg microsomal proteins) which are more than 60-fold those observed in COS1 cells and 4-fold those present in hepatic microsomes. Testosterone hydroxylating activities from CYP2C11 Gunn expressed in insect cells were decreased by 40% when compared to CYP2C11 Wistar, reflecting with a lesser extent the decrease we have previously reported in liver microsomes. The high level of active protein obtained with the baculovirus/insect cell expression system will be useful to analyze activities of site-directed mutants of CYP2C11 in order to elucidate the respective influence of the identified mutations on the enzymatic activities. PMID- 8611038 TI - [Has the clinical trials in Japan been accepted by the society?]. AB - The difference between clinical trial and clinical practice of cancer treatment were discussed. The problems of clinical trials in Japan was clarified. The conditions how clinical can be accepted by the society were discussed. PMID- 8611039 TI - [The importance of scientific clinical trials]. AB - Well-designed clinical trials have been the only ways to answer many questions as to the "uncertainty" of cancer treatment. However, general physicians and mass media have not been willing to accept clinical trials because they were experiments on patients. In order to break through the present bottleneck it is very important to recognize the clinical significance of clinical trials by physicians themselves and to establish a system for reviewing and managing protocol study and for collecting clinical data. It is also important to obtain honest informed consent from patients tested in clinical trials. To accumulate the scientific evidences from clinical trials was the best way to achieve significant advances in cancer therapy. PMID- 8611040 TI - [Current status and problems in clinical trial for new anticancer drug in Japan]. AB - Results of clinical trials for 28 anti cancer drugs in 20 pharmaceutical companies were analyzed to describe current status and problems in clinical trial for new anticancer drug in Japan. Phase I studies have been carried out in one to 19 institutes (median; 12), and in the period of 4 to 35 or more months (median; 13 months). Median number of patients entered into phase II studies was 2.9 per institute. Out of 21 drugs which finished the clinical trial, 4 were approved, 6 were made an application for approval, and 11 were discontinued the clinical development. Following problems are arisen in clinical trial for anticancer drug in Japan. Decision network for clinical development of new drug should be organized. Development of derivatives should be prudently done because it is difficult to overcome the parent product in efficacy. Phase I institutes for new anticancer drug evaluation should be designated by authority. Decision of sample size, patient selection, quality control, and informed consent are critical problems in clinical trials for new drug. PMID- 8611041 TI - [Getting informed consent in clinical trials on Japanese cancer patients]. AB - According to the survey in the spring, 1995 of the Minister of Health and Welfare, only 20% of recently died patients had been told the truth, having cancer, in Japan. This suggest that most of clinical trials on cancer in Japan involved patients without real Informed consent (IC) from patients themselves. Recent effort of the Japanese Clinical Oncology Group are breaking through this situation. In the first part, principles of IC in cancer patients and IC in trials were explained. Then the results of a questionnaire are shown. From my personal experience of over 700 patients told the truth, all that doctors should tell to patients in western countries could be told without causing troubles even in Japan. The results of the questionnaire answered by 388 patients of various cancers treated by the author, showed clearly the feasibility of telling the truth to Japanese cancer patients. This has long been regarded impossible or unacceptable. Only 2% of them regretted to be told the truth, only 7% did not desire to know the prognosis, more than 90% wanted to be told that they have recurrence when they would have recurrence. Even when they would be incurable, 66% of them required to be told so and only 16% did not. In such difficult situation, 36% would insist anticancer treatment, while 41% would prefer best supportive care. No one but patients themselves can make this choice. PMID- 8611042 TI - [Problems in conducting clinical trials for cancer patients]. AB - There are many problems and difficulties in conducting clinical trials for cancer patients. Firstly, the public does not understand well the concept of clinical trials. They often can't distinguish clinical trials for comparing two treatment methods from ones for developing new chemotherapeutic agents. Secondly, clinical trials per se can hardly fulfill the necessary conditions. At least four conditions are necessary to allow clinical trials for cancer patients: (1) the possibility of improved survival by the new methods; (2) A necessity to conduct the clinical trials; (3) the possibility of conducting clinical trials; and (4) the permissibility of conducting said trials. However, none of these conditions are fulfilled. The reasons why clinical trials are not allowed in the field of cancer treatment are discussed. PMID- 8611043 TI - [The guideline for clinical trials proposed by Japan Society for Cancer Therapy (JSCT)]. AB - The purpose of the guideline is to improve the quality of clinical trials (especially in phase III) which are performed by the members of Japan Society for Cancer Therapy. It was summarized the following 5 points: 1) clinical studies using new anti cancer drugs after its permission by Ministry of Health of Welfare, 2) not only evaluation of the effect of new drugs, but also evaluation of multidisciply therapy of specific organ cancer, 3) the dose and administration method of the drug, which was admitted by ministry of Health & Welfare may be deviated by researcher, 4) true endpoint the therapy is patients' survival and 5) verification of hypothesis is requested to perform well planned randomized controlled trials. Much more, the problems of its clinical application, eg. qualification and authorization of coordinator, evaluation criteria (each organ specific evaluation criteria made by JSCT must be used in the clinical studies), evaluation of QOL, were referred. PMID- 8611044 TI - [Current status of phase I and phase II trials--experience in Habikino from 1988 to 1994]. AB - Introduction of a new agent in the treatment for cancer will require usually three phases of investigation. A new drug will be tested in a small series of patients for toxicity and feasibility; the main objective of a phase I trial is to determine the maximum tolerated dose with a specific type of administration. Demonstration of activity is the major goal of phase II trials, which will place some confidence interval on the efficacy of the new agent. Information obtained from phase I and II studies should be the basis for phase III trials. Based upon our experience with the clinical development of CPT-11, this paper reviews the two factors which influence the quality of the phase I and II trials. Firstly, adequately designed and prepared plans for every clinical trial are essential minimum requirement. Another issue which has been frequently overlooked has been variation from the planned protocol treatment. Such variations can be due to the intolerance of the patient to toxicity as well as the individual physician's poor perceptions and the physician's intolerance of toxicity. PMID- 8611045 TI - [Quality control in clinical trials]. AB - Quality control (QC) in clinical trials means the procedures which insure protection of human subjects from research risk, reliability of the data, and thereby assures internal consistency. This has been developed since 1970s in the US, by establishing various regulations which are now called GCP. From the viewpoint of total QC, it should be emphasized that rigorous review of protocol by the Institutional Review Board and obtaining Informed Consent are prerequisites for insuring the quality of the given trial at high scientific level. When pursuing a clinical trial, first of all, facilities of the institutions and the ability of investigators must be of high quality. For this reason, at each institution previous data related to trials should be thoroughly reviewed and analyzed prior to developing a protocol. Educational courses in QC in clinical practice are invaluable. QC of diagnosis means, for example, central pathology review and standardization of diagnostic procedures and process. Secondly, at each institution, data managers collect the data and submit them to the central office at the indicated time. In order to evolve clinical trial, continuous education for data managers and expansion of their job are encouraged. Thirdly, at the statistical center independent from the research group office, subject-specific data managers, the biostatistical staff, must check submitted forms for completeness, consistency and accuracy. Finally, at the data analysis, quality evaluation of the research should also be carried out. Throughout the trial, monitoring and audit are particularly important to assure quality. The sponsor has the responsibility of monitoring the trial and make rigorous onsite visits, and the individual study group also have a monitoring program, while the FDA and the NCI audit by themselves. The purpose of audit is not only to assure data reliability but also to check out patient compliance to drug, education as to regulations and rules of clinical trials and the analysis of violations so as to provide suggestions to improve medical care. PMID- 8611046 TI - [Quality control of surgery in multicenter study--interinstitutional and individual differences]. AB - In a prospective randomized controlled trial (PRCT) of adjuvant chemotherapy for gastric cancer patients participated by 6 cancer center hospitals, inter institutional and inter-individual differences about several factors related to the surgery and remote survivals were analyzed. From 1988 to 1992, 1,049 evaluable patients were enrolled and stratified to 6 groups according to the grade of the serosal invasion and curability, and analyzed statistically. RESULTS: 1) There were no inter-institutional differences about postoperative survival rate, but there were slight differences about patient's background factors and entry policy of each institution. 2) There were no inter institutional differences about several factors related to the surgery such as operative time, bleeding volume during operation, and the length of the administration periods. 3) There were no inter-individual differences about the length of the administration periods, but there were a little differences about operative time and the volume of hemorrhage. When the PRCT of adjuvant chemotherapy of which the end point is survival would be designed, no inter institutional differences about remote survival and surgical operation in each institution should be confirmed before starting the trial. PMID- 8611048 TI - [Design of a new antitumor nucleoside CNDAC, against solid tumors]. AB - The design, antitumor activity in vitro as well as in vivo, and mechanism of CNDAC have been described. CNDAC had potent antitumor effects against various solid tumors in vitro as well as in vivo. CNDAC was phosphorylated by deoxycytidine kinase, followed by certain nucleotide kinases to afford its 5' triphosphate (CNDACTP), which was a potent inhibitor of DNA polymerase alpha. Using a chain-extension method with Vent (exo-) DNA polymerase and a short primer/template system, we found that CNDACTP was incorporated into the primer. After further chain-extension reaction of the primer containing CNDAC at the 3' terminus, chain elongation was not observed. Therefore, CNDACTP appeared to act as a chain-terminator. Analyses of the structure of the 3'terminus in the primer revealed the presence of ddCNC together with CNDAC and CNDC. The existence of ddCNC in the 3'-end of the primer would be due to the self-strand-break by the nucleotide incorporated next to CNDAC. PMID- 8611047 TI - [Novel targets for cancer chemotherapy]. AB - Data resulting from new insights into targets for cancer chemotherapy have been stored after the educational symposium entitled "Novel Targets for Cancer Therapy" was held at the 27th Annual Meeting of the American Society of Clinical Oncology in 1991. Novel targets for cancer chemotherapy, which have been found or assessed to be useful after the meeting, are summarized. The contents are as follows: 1) inhibitors of signal transduction pathway, 2) inducers of apoptosis and/or differentiation, 3) agents acting on DNA directly and indirectly, 4) inhibitors of telomerase, 5) agents acting on cytoplasmic microorgans, 6) inhibitors of cytoplasmic metabolism, 7) modulators of multidrug resistance, and 8) unknown or complex targets of drug action. Targets described in each passage may be applicable to the development of new anticancer agents. Agents acting on the targets have been optionally chosen and listed. The significance of important targets and agents in cancer chemotherapy is described in more detail. PMID- 8611049 TI - [Current problems in development of new anticancer agents for new molecular targets]. AB - In this report, we present some current problems in the development of new anticancer agents for new molecular targets in the development process: basic research, developing research and clinical trials. Also new proposals are made to solve these problems from our corporate point of view. It will be necessary to work out new rational methods to evaluate toxicity and efficacy considering the mechanisms and pharmacokinetics of drugs to develop new anticancer agents. Changes in clinical evaluation criteria for antitumor agents, from tumor size to survival time an improvement of QOL, will mean increased costs and a longer time for clinical trials. Given these conditions, it will be necessary for companies to co-operate with public research institutes in order to develop new anticancer agents, and to revise the guidelines and systems of clinical trials. PMID- 8611050 TI - [Combination phase I trials]. AB - Most successes in the treatment of advanced cancers have required the use of combinations of cytotoxic agents. Therefore, the development of combinations of new active compounds is one of the key challenges of clinical oncology. Reasons for the use of combinations include: 1) biochemical synergism, 2) lack of cross resistance, and 3) higher achievable dose-intensity by exploiting nonoverlapping dose-limiting toxicities. The first step in clinical development of combinations is to find doses of the drugs that are tolerated when administered together. This is a purpose of a phase I study. However, there are some differences in phase I trials between single agents and combinations. For combinations of drugs, the maximum-tolerated does (MTD) of both active agents is already known. There are almost countless possibilities as to which drugs to combine and in what proportions. A method for selecting drugs and doses for a combination regimen from among many possibilities was discussed in this symposium. PMID- 8611051 TI - [Informed consent in phase I trial]. AB - Informed consent in phase I trials involves very difficult problems related to experimental factors of the phase I trial, a toxicity study conducted in human cancer patients. However, this is the first clinical step in new drug development, and patients must participate in phase I only when there is full disclosure of correct trial information. Disclosure should include the purpose of the trial, the procedure, and the risk/benefits related to the trial. Informing the patient of the cancer diagnosis and the extent of disease is also necessary before entry into a phase I trial. Although phase I trials are conducted with the precious contribution of patients, it is important that informed consent should not become a type of legal contract. Informed consent in phase I trials is an essential process, just like a roundtable discussion of patient and physician before fighting an incurable cancer. PMID- 8611052 TI - [Development of new anti-cancer agents in Europe]. AB - CPT-11 was synthesized 12 years after its mother compound, camptothecin, had been dropped from development by NCI (USA). A new drug application with CPT-11 was filed in 1991 after phase I and phase II clinical studies, and the marketing approval was authorized in 1994 with indications of non-small cell and small cell lung cancers, cervical cancers, and ovarian cancers. Phase I studies in France were initiated in 1990 one year after late phase II studies were started in Japan. A new drug application was filed in 1994 after phase II studies and marketing approval was authorized by the French government in May, 1995 with indication of advanced colon cancers. Clinical development of new anticancer agents in France was discussed. PMID- 8611053 TI - [Early clinical trials in ECTG. Early Clinical Trial Group]. AB - Methodology to conduct phase I trials in the Early Clinical Trial Group (ECTG) in Europe was reviewed and compared with current status of the trials in Japan. Single dose acute toxicity studies and repeated dose toxicity studies were required in order to enter phase I trials in Europe, while in Japan those results and various other toxicity studies such as mutagenecity, carcinogenecity and reproductive toxicity were required. Since significant numbers of new anticancer drugs are dropped in the early stage of clinical investigations, it is recommended that unnecessary toxicological studies be avoided. Repeated administration and dose escalations in the same patients are allowed during phase I trials in ECTG, partly in consideration of the patient's desire to obtain antitumor effects. Dose intensity and quality of life are considered in order to decide the optimal dose and schedule in phase II trials. PMID- 8611054 TI - [Contribution of Japan to develop new anticancer agents]. AB - Many drugs developed in Japan have been started their clinical trials in USA and European countries. The clinical trials in Japan will be requested the strict standard in accordance with the introduction of ICH-GCP. It is strongly requested for the medical student to be educated on the importance and methodology of clinical trials at university. PMID- 8611055 TI - Alcohol research: at the cutting edge. PMID- 8611056 TI - An 8-year follow-up of 450 sons of alcoholic and control subjects. AB - BACKGROUND: Between 1978 and 1988, 453 sons (age range, 18-29 years) of alcoholic and control subject were evaluated for their level of reaction (LR) to alcohol. This article presents the results of the 8.2-year follow-up of 450 of these men. The three goals were (1) to attempt to replicate results of the follow-up of the first 223 subjects, (2) to evaluate the potential impact of the quantity and frequency of drinking at the time of the original study on the relationship between LR and alcoholic outcome (ALC), and, most importantly, (3) to test if the relationship between family history (FH) and ALC might be mediated by LR in a subset of the sample. METHODS: Face-to-face structured follow-up interviews were carried out with the subjects and separately with an additional informant, and blood samples, as well as urine specimens, were obtained for determination of state markers of heavy drinking and drug toxicology screens. RESULTS: First, the rate of development of DSM-III-R abuse and dependence on alcohol was 14.1% and 28.6%, respectively, for family history positive (FHP) subjects, compared with 6.6% and 10.8%, respectively, for family history negative (FHN) men. Second, neither consideration of the quantity nor the frequency of drinking at the time of the original study, nor their combination, effectively diminished the relationships between LR and ALC. Third, among men who drank and demonstrated the 15% highest and lowest scores of LR at about the age of 20 years (ie, 30% of the relevant population), the correlation between FH and ALC was greatly reduced when LR was considered, but the correlation between LR and ALC was not greatly diminished when the impact of FH was evaluated. CONCLUSIONS: In this sample of moderately functional white men, the development of alcoholism occurred in relationship to an FH of alcoholism, but alcohol abuse or dependence was unrelated to prior psychiatric disorders. For this group, LR at the age of 20 years was associated with future alcoholism in a manner that was independent of the drinking practices at the time of the original study. At least among those men with clearly high and low LR scores, these data are consistent with the conclusion that LR might be a mediator of the alcoholism risk. PMID- 8611057 TI - Early-onset alcoholics have lower cerebrospinal fluid 5-hydroxyindoleacetic acid levels than late-onset alcoholics. AB - BACKGROUND: We investigated the interrelationships of age at onset of excessive alcohol consumption, family history of alcoholism, psychiatric comorbidity, and cerebrospinal fluid monoamine metabolite concentrations in abstinent, treatment seeking alcoholics. METHODS: We studied 131 recently abstinent alcoholics. Supervised abstinence was maintained on a research ward at the National Institutes of Health Clinical Center for a minimum of 3 weeks. All alcoholics received a low-monoamine diet for a minimum of 3 days before lumbar puncture. Lumbar punctures were performed in the morning after an overnight fast. Monamine metabolites and tryptophan in cerebrospinal fluid were quantified with liquid chromatography by means of electrochemical detection. Psychiatric diagnoses were established from blind-rated Schedule for Affective Disorders and Schizophrenia Lifetime version interviews administered by a research social worker. Severity and age at onset of excessive alcohol consumption were documented with a structured lifetime drinking history questionnaire and with selected alcoholism screening questionnaires (CAGE and Michigan Alcoholism Screening Test). Family history of alcoholism was obtained from the probands. RESULTS: A majority of the treatment-seeking, primarily white male alcoholics had a lifetime history of psychiatric disorders other than alcoholism. None fulfilled criteria for antisocial personality disorder. Early-onset alcoholics (onset of excessive consumption before 25 years of age) had a more severe course of alcoholism and lower mean cerebrospinal fluid 5-hydroxyindoleacetic acid concentration than late onset alcoholics. Patients who reported both parents to be alcoholics had particularly low mean cerebrospinal fluid 5-hydroxyindoleacetic acid, homovanillic acid, and tryptophan concentrations. CONCLUSION: Among treatment seeking alcoholics, early age at onset is generally associated with a more severe course of alcoholism and lower cerebrospinal fluid 5-hydroxyindoleacetic acid concentration. PMID- 8611058 TI - Six-month follow-up of naltrexone and psychotherapy for alcohol dependence. AB - BACKGROUND: The goal of this study was to examine the persistence of naltrexone's effects on drinking outcomes among alcoholics following discontinuation of treatment and to determine whether coping skills therapy improves long-term outcomes compared with supportive therapy. METHODS: Eighty of 97 alcohol dependent subjects randomized to receive naltrexone or placebo and either coping skills therapy or supportive therapy for 12 weeks were assessed at a 6-month off treatment follow-up. RESULTS: Subjects who received naltrexone were less likely to drink heavily or to meet criteria for alcohol abuse or dependence than subjects who received placebo. The effect of naltrexone therapy on abstinence rates persisted only through the first month of follow-up. Coping skills therapy was associated with decreased levels of drinking among subjects who received placebo. Psychotherapy condition, however, did not predict alcohol diagnosis at follow-up. CONCLUSIONS: Some but not all of the benefits resulting from short term naltrexone treatment persist after discontinuation of treatment. The findings suggest that continued treatment with naltrexone may be beneficial for some patients. PMID- 8611059 TI - The obsessive compulsive drinking scale: A new method of assessing outcome in alcoholism treatment studies. AB - BACKGROUND: the 14-item Obsessive Compulsive Drinking Scale (OCDS) is a quick and reliable self-rating instrument that provides a total and two subscale scores that measure some cognitive aspects of alcohol "craving". This study validated further its utility as an alcoholism severity and treatment outcome instrument. METHODS: Alcoholism severity and analogue craving scales were administered at baseline, and the OCDS was given at baseline and weekly to 41 alcohol-dependent individuals who participated in a 12-week pharmacologic and cognitive-behavioral treatment trial. Repeated-measures analysis of variance was used to examine group differences in the OCDS scores of those individuals who remained abstinent or drank during the trial. RESULTS: At baseline, the OCDS was correlated with the alcohol composite score of the addiction severity index (r=.48), the alcohol dependence scale (r=.42), the analogue craving measures (range r=.40 to .57), and prestudy alcohol consumption (r=.60). Most importantly the OCDS total and subscale scores were significantly different between individuals who had relapse drinking, who had "slip" drinking, and who remained abstinent, with relapsers showing the highest scores. CONCLUSIONS: The OCDS scores appear to be sensitive to alcoholism severity and change during abstinence and relapse drinking. Since the shared variance with analogue craving measures is only about 20% to 30%, it appears to be measuring a largely independent dimension of alcohol dependence. Its ease of use (5 minutes per self-rating), reliability, validity, and analytic capabilities support its utility as a tool to measure severity and improvement during alcoholism treatment trials. PMID- 8611060 TI - Imipramine treatment of alcoholics with primary depression: A placebo-controlled clinical trial. AB - BACKGROUND: Depressive disorders are commonly comorbid with alcoholism, particularly in treatment-seeking samples. If antidepressant treatment were safe and improved the treatment outcome in the subset of actively drinking alcoholics with depression, this would be of clinical importance. METHODS: We conducted a randomized, 12-week placebo-controlled trial of imipramine hydrochloride combined with weekly relapse prevention psychotherapy. The subjects were 69 actively drinking alcoholic outpatients with current depressive disorders. The first onset of depression was either antecedent to the abuse of alcohol or occurred during prolonged periods of sobriety. Depression and drinking outcomes at 12 weeks, as well as their relationship, were measured. RESULTS: Imipramine treatment was safe and associated with improvement in depression in both adequately treated and intention-to-treat samples. While there was no overall effect on drinking outcome, patients whose mood improved showed decreased alcohol consumption that was more marked in those treated with imipramine. CONCLUSIONS: Imipramine treatment is effective for primary depression among actively drinking alcoholic outpatients, and may improve alcoholic outcome for those whose depression responds to treatment. PMID- 8611061 TI - A long-term follow-up of male alcohol abuse. AB - BACKGROUND: This study attempted to determine the course of male alcohol abuse from the age of 40 years to 60 or 70 years, to estimate the duration of abstinence required for stable remission and to study the hypothesis of progression of symptoms in chronic alcohol abuse. METHODS: The subjects were 268 former Harvard University (Cambridge, Mass) undergraduates (college sample) and 456 nondelinquent inner-city adolescents (core city sample) who had been repeatedly studied in multidisciplinary fashion since 1940. Since 47 years of age, these men have been followed up biennially by questionnaire and every 5 years by physical examination. At some point during their lives, 55 (21%) of the college and 150 (33%) of the core city men met DSM-III criteria for alcohol abuse. The college cohort has been followed until the age of 70 years, the core city cohort until age 60 years. The dependent variables were mortality and alcohol abuse status every 5 years. RESULTS: By 60 years of age, 18% of the college alcohol abusers had died, 11% were abstinent, 11% were controlled drinkers, and 59% were known to be still abusing alcohol. By 60 years of age, 28% of the core city alcohol abusers had died, 30% were abstinent, 11% were controlled drinkers, and only 28% were known to be still abusing alcohol. CONCLUSIONS: In three respects the two socially divergent samples resembled each other. After abstinence had been maintained for 5 years, relapse was rare. In contrast, return to controlled drinking without eventual relapse was unlikely. Alcohol abuse could continue for decades without remission or progression of symptoms. The samples differed in that the core city men began to abuse alcohol when younger and, although they were more likely than the college men to become alcohol dependent, the core city men were twice as likely to achieve stable abstinence. PMID- 8611062 TI - Enhanced sensitivity of pituitary beta-endorphin to ethanol in subjects at high risk of alcoholism. AB - BACKGROUND: Previous studies have demonstrated that a moderate dose of ethanol induced a significant increase in the plasma beta-endorphin content of subjects from families with a history of alcoholism (high risk (HR)), but not subjects from families without a history of alcoholism (low risk (LR)). The objective of this study was to examine the response of the pituitary beta-endorphin and adrenal cortisol systems to various concentrations of ethanol in male and female subjects at high and low risk of the future development of alcoholism. METHODS: All subjects participated in four experimental sessions. In each session the subjects were given a drink containing one of the following doses of ethanol: 0, 0.25, 0.50, and 0.75 g of ethanol per kilogram of body weight (for a 60- to 70-kg individual). Blood samples were taken at 0 minutes and at 15, 45, 120, and 180 minutes after the drink for estimation of the blood alcohol, plasma beta endorphin, and plasma cortisol levels. RESULTS: The concentration of alcohol in the blood at various intervals after the drink was similar among the subjects, regardless of the risk group. Ethanol increased the plasma level of beta endorphin-related peptides of the HR but not of the LR subjects in a dose dependent manner. All subjects showed a small decrease in plasma cortisol level with time, but ethanol ingestion did not significantly alter the plasma cortisol levels. CONCLUSION: This study indicates that the pituitary beta-endorphin system, but not the adrenal cortisol system, of the HR subjects shows an enhanced sensitivity to ethanol, which may be an important factor in controlling ethanol consumption. PMID- 8611063 TI - The electroencephalogram after alcohol administration in high-risk men and the development of alcohol use disorders 10 years later. AB - BACKGROUND: In 1979 through 1980, electroencephalographic (EEG) responses to an alcohol challenge in 19 year-old sons of alcoholics as well as in sons of nonalcoholic control subjects were examined. The familial risk status of the subjects and greater EEG sensitivity to alcohol were hypothesized to predict the development of alcoholism 10 years later. METHODS: In 1990 through 1992, diagnostic interviews were completed to ascertain alcohol and other substance use disorders in these subjects and to update their family history. RESULTS: Updated family history of alcoholism predicted the development of substance dependence. Density of alcoholic relatives (the number of alcoholic relatives divided by the number of known relatives) was positively related to the severity of alcohol use disorders in the probands. Contrary to expectation, a greater EEG response at age 19 years was not related to the later development of alcohol dependence. Instead, the opposite was observed: a smaller EEG alpha frequency response to alcohol at age 19 years was related to the development of alcohol dependence and high quantity and frequency of alcohol consumption 10 years later. CONCLUSIONS: Lower EEG response to a small dose of alcohol may be associated with the later development of alcohol dependence. This result is based on a small number of subjects and should be interpreted with caution. Although this result is opposite to our 1980 hypothesis, it is consistent with much of the recent literature. PMID- 8611064 TI - The effects of alcohol consumption on laboratory-induced panic and state anxiety. AB - BACKGROUND: This study tested whether alcohol consumption reduces anxiety and panic associated with a panic-challenge procedure. METHODS: Subjects with panic disorder were randomly assigned to consume either a moderate dose of alcohol or a nonalcoholic placebo. All subjects were told that they were drinking alcohol to control beverage expectancies. Following the beverage administration, subjects underwent a panic challenge (35% carbon dioxide) and a series of anxiety symptom assessments. RESULTS: Subjects who consumed alcohol reported significantly less state anxiety both before and after the challenge. In response to the challenge, subjects who consumed alcohol experienced significantly fewer panic attacks when applying liberal panic criteria; however, this effect only approached significance when applying conservative panic criteria. CONCLUSIONS: These findings suggest that alcohol acts acutely to reduce both panic and the anxiety surrounding panic, and they lend support to the view that drinking behavior among those with panic disorder is reinforced by this effect. We suggest that this process may contribute to the high rate at which alcohol-use disorders co-occur with panic disorder. PMID- 8611066 TI - Surgeries. PMID- 8611065 TI - William Halsted, his family and 'queer business methods'. AB - The life and times of William Stewart Halsted have become a blend of fact and sometimes fiction. Lost in this hagiographic haze are certain true aspects of his upbringing, family life, and professional activities. Whether Halsted remains as monumental a figure in the evolution of American surgery as he is presently perceived, remains a master of historical inquiry. For instance, the important consideration of Halsted's independent wealth and its impact on his ability to accept a "full-time" faculty position at The Johns Hopkins Hospital is a question of interest. Newly available information shows that Halsted's father, William Mills Halsted, Jr, was involved in numerous financial irregularities centered around the family's business, Halsted, Haines & Co. Among the father's alleged misconduct was the apparent embezzlement and fraudulent assignment of company funds. Included in these abuses was, at the time of the firm's bankruptcy, the providing of "preference loans" to William Stewart Halsted, which became the basis for the surgeon's later affluence. PMID- 8611067 TI - Bedside percutaneous tracheostomy with bronchoscopic guidance in critically ill patients. AB - BACKGROUND: Bedside percutaneous dilational tracheostomy, a relatively new method of tracheal cannulation, provides safe and ready access to the trachea to relieve airway obstruction and tracheopulmonary secretions. The dilational technique has undergone various modifications during the past decade. Complications of this procedure are primarily related to the lack of direct visualization during tracheostomy tube placement and to poor patient selection. OBJECTIVE: To report the utility of percutaneous dilational tracheostomy with bronchoscopic guidance in 162 critically ill patients. MAIN OUTCOME MEASURES: Mortality rates and complications associated with this technique. RESULTS: Twenty-five patients (15.4%) died while hospitalized. No deaths were related to tracheostomy. There were four (2.5%) major complications: one pneumothorax and three posterior tracheal tears, which healed spontaneously. There were five (3.1%) minor complications: one posterior mucosal disruption, one minor bleeding episode, and three minor episodes of cellulitis. One hundred thirty-seven patients (84.6%) were discharged. Twenty-nine patients (21.2%) were available for follow-up and were experiencing no significant problems or complications following the procedure. Compared with standard open tracheostomy, charges were reduced by $1628.20 per patient ($263,768.40 total savings). CONCLUSIONS: Bedside percutaneous tracheostomy with bronchoscopic guidance is safe and cost-effective. Complications compare favorably with that of open tracheostomy. Major complications should be avoided with continuous bronchoscopic observation during the procedure. PMID- 8611068 TI - Paramedic vs private transportation of trauma patients. Effect on outcome. AB - BACKGROUND: Prehospital emergency medical services (EMS) play a major role in any trauma system. However, there is very little information regarding the role of prehospital emergency care in trauma. To investigate this issue, we compared the outcome of severely injured patients transported by paramedics (EMS group) with the outcome of those transported by friends, relatives, bystanders, or police (non-EMS group). DESIGN: We compared 4856 EMS patients with 926 non-EMS patients. General linear model analysis was performed to test the hypothesis that hospital mortality is the same in EMS and non-EMS cases, controlling for the following confounding factors, which are not affected by mode of transportation: age, gender, mechanism of injury, cause of injury, Injury Severity Score (ISS), and severe head injury. Crude, specific, and adjusted mortality rates and relative risks were also derived for the EMS and non-EMS groups. SETTING: Large, urban, academic level I trauma center. PATIENTS: All patients meeting the criteria for major trauma. RESULTS: The two groups were similar with regard to mechanism of injury and the need for surgery or intensive care unit admission. The crude mortality rate was 9.3% in the EMS group and 4.0% in the non-EMS group (relative risk, 2.32; P < .001). After adjustment for ISS, the relative risk was 1.60 (P = .002). Subgroup analysis showed that among patients with ISS greater than 15, those in the EMS group had a mortality rate twice that of those in the non-EMS group (28.8% vs 14.1%). After controlling for confounding factors, the adjusted mortality among patients with ISS greater than 15 was 28.2% for the EMS group and 17.9% for the non-EMS group (P < .001). CONCLUSIONS: Patients with severe trauma transported by private means in this setting have better survival than those transported via the EMS system. Large prospective studies are needed to identify the factors responsible for this difference. PMID- 8611069 TI - Reappraisal of the systematic management of complicated hepatolithiasis with bilateral intrahepatic biliary strictures. AB - OBJECTIVE: To compare the results, limitations, and complications of the surgical treatment of bilateral hepatolithiasis and intrahepatic biliary strictures with left hepatectomy and without left hepatectomy. DESIGN: Case-controlled study. SETTING: Referral center. PATIENTS: During a 12-year period, 103 patients with bilateral hepatolithiasis and intrahepatic biliary strictures underwent surgical treatment. Group A (n = 73) received left hepatic resection (lateral segmentectomy or lobectomy) and postoperative biliary dilatation with residual stone extraction. Group B (n = 30) underwent the same procedures except for left hepatectomy. INTERVENTIONS: Left lateral segmentectomy or left lobectomy, choledocholithotomy, postoperative cholangioscopic treatments (electrohydraulic lithotripsy, other lithotripsy, lithotomy, balloon dilatation, etc. via T tube or precutaneous transhepatic route). MAIN OUTCOME MEASURES: Days of hospitalization, incidence of major and minor complications, mortality rates, and the rates of residual stones and stone recurrence were compared. RESULTS: Group A and B had similarly low postoperative 1-month mortality rates of 5.5% and 6.7%, respectively. The main cause of death in both groups was uncontrollable septicemia. The main major complications in group A were intra-abdominal abscess and upper gastrointestinal bleeding; the major complication in group B was massive hemobilia. Group B had a significantly higher overall rate of complications (53.3% vs 23.3%, P < .01) and a longer hospital stay than group A (median, 72 days vs 28 days, P < .03). When complications were classified as major or minor, only minor complications showed a significant difference (30% vs 13.7%, P = .05). After using biliary stricture dilatation and stone extraction, the rate of residual stones in the right lobe was similar in both groups, but patients in group B had a significantly higher rate of residual stones (12.5% vs 0%, P < .02) and stone recurrence in the left lobe (19% vs 0%, P < .003) than those in group A. CONCLUSIONS: Partial resection of the left lobe in cases of bilateral hepatolithiasis and biliary strictures can effectively simplify problems in the treatment of bilateral hepatolithiasis and intrahepatic biliary strictures. In addition, not only were surgical complications not increased, but a decrease in complications from postoperative manipulations for stone clearance was noted in our series. PMID- 8611070 TI - Pyogenic liver abscess. An audit of experience over the past decade. AB - OBJECTIVES: To audit our experience in managing patients with pyogenic liver abscesses since 1984 and to identify any risk factor associated with hospital mortality. DESIGN: Retrospective review. SETTING: A tertiary referral center. PATIENTS: Eighty-three patients with pyogenic liver abscesses were studied to determine demographic characteristics; clinical features, laboratory, imaging, and microbiologic findings; methods of treatment; and final outcome. The median follow-up period was 9.8 months. INTERVENTION: All patients were treated with intravenous antibiotic drugs. Fifty-three patients were to image-guided percutaneous aspiration of the abscess. A percutaneous drainage catheter was inserted after aspiration in 27 patients. Laparotomy was performed in 27 patients; seven of them underwent an elective operation. MAIN OUTCOME MEASURE: Hospital mortality, defined as death within the same hospital admission for management of liver abscess. RESULTS: Biliary tract disease was the most frequently identifiable cause. The right lobe abscess was more frequently cryptogenic, while the left lobe abscess was more frequently related to intrahepatic stones (P < .001). The overall hospital mortality rate was 18% (15/83). On univariate analysis, female gender, rupture on presentation, emergency laparotomy, management without aspiration or catheter drainage, presence of malignancy, hyperglycemia, hyperbilirubinemia, elevated prothrombin time, and elevated activated partial thromboplastin time were significantly associated with hospital mortality. On multivariate logistic regression analysis, presence of malignancy, hyperbilirubinemia, and elevated activated partial thromboplastin time were found to be independent risk factors. CONCLUSIONS: Pyogenic liver abscess is still a disease with significant mortality. Early diagnosis and prompt treatment are necessary to further improve our results of management. PMID- 8611071 TI - Improvement in the organ donation rate at a large urban trauma center. AB - OBJECTIVE: To implement and then determine the efficacy of a "hospital development" (HD) plan designed to increase organ donation rates at an urban trauma center. DESIGN/SETTING: Retrospective reviews of all deaths at an urban, level I trauma center for 1991 to 1994. SUBJECTS: Potential organ donors were identified by standardized criteria, and the reasons why potential donors did not become actual organ donors ("nonproductive donors") were categorized. Actual donors were defined as individuals in whom one transplantable organ was recovered. Results also were expressed as percentages of potential donors for each year. Changes in actual donor numbers and in nonproductive donor categories were compared for the "pre-HD" (1991-1992) and "post-HD" (1993-1994) periods. INTERVENTION: The HD plan had six components: identification of key contact individuals, development and modification of relevant hospital policies, improvement in procurement agency visibility in hospital units, education of hospital staff regarding organ donation, institution of early on-site donor evaluations, and provision of feedback to hospital staff about the disposition of potential organ donors. RESULTS: Institution of the HD plan was associated with a highly significantly increase in actual donors for the post-HD period as compared with the pre-HD period (P < .001), and pre-HD and post-HD donor rates were 26.1% and 49.5%, respectively. This increase was due primarily to a marked improvement in hospital staff identification and referral of potential donors (P < .001). CONCLUSIONS: A coordinated plan incorporating continuing staff education, organ donation policy refinement, and increased visibility and availability of organ procurement agency personnel can substantially increase organ donation at an urban trauma center. PMID- 8611072 TI - Correcting prolonged bleeding during renal transplantation with estrogen or plasma. AB - OBJECTIVE: To determine the efficacy and relative effectiveness of conjugated entrogens (CE) and fresh-frozen plasma (FFP) in normalizing prolonged preoperative bleeding times during renal transplantation. DESIGN: Prospective, randomized trial. SETTING: A university regional referral center for transplantation. PATIENTS: Patients scheduled for renal transplantation with preoperative bleeding times greater than 10 minutes (normal, < 7 minutes) following informed consent were asked to participate in the randomized protocol. Those with bleeding times of 8 to 9.5 minutes were asked, following informed consent, to be a control group receiving neither CE nor FFP. INTERVENTIONS: Following induction of anesthesia and drawing of baseline laboratory tests, patients were administered randomly, using a table of random numbers, either 50 mg of CE or 2 U of FFP. MAIN OUTCOME MEASURES: Bleeding time measurements and other laboratory tests were repeated at the end of surgery as well as at 24 and 48 hours postoperatively. RESULTS: Treatment with CE and FFP decreased the patients' bleeding times from 16.68 +/- 0.8 (SEM) and 17.13 +/- 0.85 minutes to 7.67 +/- 0.79 (P < .001) and 10.50 +/- 1.27 minutes (P < .001), respectively, by the end of surgery. At 24 and 48 hours postoperatively, the CE group had bleeding times of 9.77 +/- 0.99 and 9.81 +/- 1.24 minutes (P < .001 for both), respectively, whereas the FFP group bleeding times were 12.76 +/- 1.57 (P = .003) and 12.14 +/- 1.56 minutes (P = .001), respectively. There were no statistical differences for the control group compared with baseline either at the end of surgery or at 24 hours. CONCLUSIONS: Although both CE and FFP significantly decreased prolonged preoperative bleeding times during renal transplantation, CE might be preferred because of lower risk and cost, as well as a longer duration of action. PMID- 8611073 TI - Gastrointestinal 'crosses'. A new shade from an old palette. AB - BACKGROUND: We present our experience with an as yet undescribed type of intentionally ingested metallic foreign body that was specially designed to arrest in its passage and cause perforation of the gastrointestinal tract. The two halves of a standard paper clip are tied crosswise with a rubber band, forming an elastic "cross." With its branches squeezed to lie parallel, the cross is wrapped into a small strip of paper and ingested. After being released from its wrapper, the branches of the cross spring back to their original position and cause perforation of the bowel wall. DESIGN: Case series. SETTING: University hospital, Bulgaria. PATIENTS: Five male prisoners from the same jail undergoing laparotomy for foreign body removal during 1 week in January 1994. INTERVENTIONS: Laparotomy, removal of the foreign bodies through incisions of the bowel wall, immediate restoration of the gastrointestinal tract continuity, and peritoneal lavage and drainage. OUTCOME MEASURES: Morbidity and mortality. RESULTS: Two of the patients were initially observed but subsequently underwent surgery for perforations of the gastrointestinal tract. Three of the patients underwent immediate operative treatment. There were 20 total crosses ingested; all foreign objects impacted and perforated the stomach (50%), first duodenal portion (25%), and fourth duodenal portion (20%). There was no morbidity or mortality among the five patients treated. CONCLUSIONS: Because ingested crosses that have been released from their wrappers never move distally, the only definitive solution is to operate early. PMID- 8611074 TI - Racial factors cannot explain superior Japanese outcomes in stomach cancer. AB - OBJECTIVE: To compare the stage-stratified survival of Japanese patients treated in Honolulu according to Western techniques with that of Japanese patients treated in Tokyo according to Japanese techniques, thus eliminating race as a potentially confounding variable. DESIGN AND PATIENTS: Of 312 Honolulu Japanese patients surviving Western-type gastric resection for neoplasm between 1974 and 1985, 279 were identified with invasive gastric adenocarcinoma unassociated with any second malignancy. This Honolulu cohort, treated by Western methods, was retrospectively compared with a similar, previously described cohort of 3176 Tokyo Japanese patients treated according to Japanese methods. MAIN OUTCOME MEASURES: American Joint Committee on Cancer/Union Internationale Contre le Cancer criteria for stage-stratified survival. RESULTS: Despite non-TNM prognostic factors favoring higher survival for the Honolulu Japanese patients, for every TNM stage, we observed higher survival for the Tokyo Japanese patients who were treated according to Japanese techniques. For stage I disease, the survival rates were 86% vs 96%, respectively (P < .001); for state II, 69% vas 77% (P = .15); for stage III, 21% vs 49% (P < .001); and for stage IV, 4% vs 14% (P < .001). CONCLUSIONS: Because all patients in this study are Japanese, race related factors or the "different-disease" hypothesis cannot explain these results. Lymphadenectomy-related stage-migration and/or differing therapeutic efficacy seem more likely explanations. PMID- 8611075 TI - Microbial translocation in neonates and infants receiving long-term parenteral nutrition. AB - OBJECTIVE: To explore whether episodes of endogenous septicemias due to microbial translocation are clinically relevant in neonates and infants who are receiving long-term parenteral nutrition (PN). DESIGN: Prospective observational cohort study of 2 years. SETTING: Neonates and infants who underwent surgical procedures and required PN because of gastrointestinal abnormalities. MEASUREMENTS: Surveillance cultures of the oropharynx and gut were obtained at the first of PN and thereafter twice each week. These cultures were processed for all microorganisms, except for coagulase-negative staphylococci, in a semiquantitative manner to detect overgrowth. A blood sample was taken for culture from both the central venous line and peripheral vein on clinical indication only. Microbial translocation was diagnosed when the microorganisms that were isolated from the blood sample were also carried in the throat and/or rectum within the 2 weeks preceding the episode of septicemia. MAIN RESULTS: Of 94 infants, 10 (11%) experienced 24 episodes of septicemia (ie, 7.3 septicemic episodes per 1000 days of PN). Six infants experienced 15 episodes of microbial translocation due to enteric microorganisms, including Escherichia coli, Klebsiella, Candida species, and enterococci. Microbial translocation occurred after a median of 58 days of PN (range, 32 to 286 days). The enteric organisms that caused septicemia were always present in the throat and/or rectum and in high concentrations ( > 10(5) colony-forming units per gram [ie, overgrowth]) in 60% of the translocation episodes. All but one episode occurred in infants with an abnormal serum bilirubin level ( > 17 mumol/L [0.99 mg/dl]). CONCLUSIONS: In neonates and infants who are receiving PN, septicemia may be a gut-related phenomenon. PMID- 8611076 TI - Prognosis and treatment of peritonitis. Do we need new scoring systems? AB - OBJECTIVE: To assess the clinical significance of present scoring systems for prognosis and treatment in patients with secondary bacterial peritonitis and to define risk factors for patient survival and outcome not included in the scores. A secondary objective was to review our therapeutic regimens and the need for reoperation with regard to outcome. DESIGN: Prospective observational study. SETTING: University hospital, secondary referral center. PATIENTS: From 1992 to 1995, 92 patients with secondary peritonitis were examined at the University Surgical Clinic, Vienna, Austria. the populations as a whole consisted of 56 men and 36 women with an average age of 56 +/- 19 years. Forty-four percent of patients had postoperative peritonitis. OUTCOME MEASURES: Mortality, multiple organ system failure (MOSF), relaparotomy. RESULTS: The mortality rate in patients with an APACHE II (Adult Physiology and Chronic Health Evaluation) score of less than 15 was 4.8%, while mortality rose to 46.7% in those with a score of 15 or higher (P = .001). The average total mortality rate was 18.5%. The prognosis for patients without organ failure or with failure of one organ system was excellent (mortality rate, 0%); quadruple organ failure, however, had a mortality rate of 90%. Initial thrombocytopenia ( < 60 x 10(9)/L), four-quadrant peritonitis, and diabetes mellitus were associated with significantly higher mortality. Leukopenia (white blood cells, < 6 x 10(9)/L) and inappropriate antibiotic therapy as determined by the antibiogram were mildly significant for higher mortality. The need for relaparotomy resulted in substantially higher mortality (P < .001). The impossibility of definitive operative resolution of the intra-abdominal pathologic findings at initial operation had no significant effect on mortality, possibly because planned reoperations were always carried out in those cases. For patients with definitive resolution at initial operation, it was possible to reduce the traditionally high mortality rate associated with relaparotomy on demand by making the decision for reexploration promptly, within the first 48 hours. Nevertheless, the 52.4% mortality rate observed in those cases was still much higher than the 33% found in patients who were not free of disease after the initial operation. CONCLUSION: The prognosis in peritonitis is decisively influenced by the health status of the patient at the beginning of treatment and by any concomitant risk factors. As a result, a fairly accurate prediction of the outcome of the disease can initially be made on the basis of the APACHE II score and the MOSF score according to Goris. However, the certainty that severely ill patients with high scores often die has little clinical relevance, since it does not provide any therapeutic alternatives to the attending physician. The decision to perform a relaparotomy must be made as soon as possible, at least before MOSF emerges. Already existing MOSF will lead to the "point of no return." PMID- 8611077 TI - Small carcinomas of the thyroid. A long-term follow-up of 867 patients. AB - OBJECTIVE: To determine the adequate extent of surgery for small carcinomas of the thyroid. DESIGN: Retrospective cohort study of 867 consecutive patients with small carcinomas of the thyroid (lesions < 10 mm in diameter) who were operated on at the Noguchi Thyroid Clinic, Oita, Japan, between 1965 and 1987. Mean follow up was 12.8 years. SETTINGS: A center for treatment of thyroid disease, where about 1400 thyroid operations are performed per year. PATIENTS: Thyroidectomy was performed in patients with a preoperative diagnosis of Graves' disease, Graves' disease with nodules, solitary thyroid nodules, multinodular goiters, cysts, chronic thyroiditis and small carcinomas of the thyroid, in 394, 22, 136, 193, 18, 28, and 76 patients, respectively. RESULTS: Operations were conservative. Three patients who had adenomatous nodular goiters underwent total thyroidectomy. Modified radical neck dissection was performed in 66 patients. Of these 66 patients, 30 had grossly noticeable nodal metastases and 17 had microscopic metastases. Another 50 patients underwent selective lymph node excision, and 28 patients had nodal metastases. Recurrence from remnants of thyroid was seen in five patients. They were treated by surgery. Recurrence in lymph nodes was observed in five patients, and four of them were successfully treated. Recurrence in bone was observed in two patients; one with recurrence in the femur was successfully treated. Two patients died with recurrent cancer. CONCLUSIONS: Small carcinomas of the thyroid can be fatal. Total thyroidectomy is unnecessary. Modified radical neck dissection is unnecessary unless gross nodal metastases are present. Long-term follow-up is mandatory. A PMID- 8611078 TI - Impact of different classes antimicrobial agents on plasma endotoxin activity. AB - OBJECTIVE: To investigate the influence of different classes and doses of antibiotics on endotoxin release in gram-negative infection in a rat model of intra- abdominal infection. DESIGN: Immediately after intraperitoneal inoculation of Escherichia coli (5 x 10(7) colony-forming units/kg), anesthetized Wistar rats were treated with a single intravenous dose of an antimicrobial agent: cefotaxime (40 mg/kg), ciprofloxacin (3 mg/kg or 6 mg/kg), imipenem (7 mg/kg or 14 mg/kg), or gentamicin (5 mg/kg). An untreated control group received 0.9% sodium chloride instead of antibiotic. Plasma endotoxin activity, blood bacteria count, and mean arterial pressure were monitored at 60-minute intervals for 5 hours. At the end of the experiment, lavage was performed to determine the bacteria count in the peritoneal cavity. RESULTS: In the untreated group, the blood bacteria count increased rapidly. Five hours after therapy, the plasma endotoxin activity in the cefotaxime group was higher by a factor of 3.6 than in the untreated group. Compared with the cefotaxime group, endotoxin activity was approximately 26% lower in the ciprofloxacin (3 mg/kg) group, 35% lower in the imipenem groups, and 38% lower in the gentamicin group. The lowest endotoxin levels were in the high dose ciprofloxacin group. Bacteria counts in the peritoneal cavity were lowest in the gentamicin and high-dose ciprofloxacin groups. Except in the high-dose ciprofloxacin group, the endotoxin increase in the therapy groups was associated with a significant (P < .05) decrease in mean arterial pressure. CONCLUSIONS: In the early phase of therapy, antibiotic-induced endotoxin release is influenced by the mode of action of the agent class. This is not the sole influence in every class. With quinolones, this effect is also influenced considerably by dosage, ie, by pharmacodynamics. PMID- 8611079 TI - Mononuclear cell line THP-1 internalizes bactericidal/permeability-increasing protein by a non-receptor-mediated mechanism consistent with pinocytosis. AB - BACKGROUND: Bactericidal/permeability-increasing protein (BPI) binds lipopolysaccharide and neutralizes its toxic effects in vitro and in endotoxemic animals. Our recent work identified physiologically significant interactions between BPI, lipopolysaccharide, and mononuclear cells. OBJECTIVE: To determine whether the interaction between BPI and mononuclear cells is receptor mediated. DESIGN: Labeled BPI was incubated with THP-1 cells in the presence of up to 100 fold excess of unlabeled BPI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were performed to evaluate competitive binding and total uptake of BPI. Crosslinking was performed to determine whether BPI binds to a single protein entity. Acid washing experiments and flow cytometric analysis were performed to determine whether BPI remains on the cellular surface. Finally, flow cytometry analysis was used to determine whether BPI incubation with THP-1 cells affects the surface expression of the lipopolysaccharide-binding protein-lipopolysaccharide receptor CD14. RESULTS: Labeled BPI uptake was not inhibited by the presence of 100-fold excess of unlabeled BPI at 37 degrees C or 4 degrees C in the presence of azide. Uptake was not saturable under either condition with incubation concentrations up to 10 microgram/mL. Cross-linking did not show BPI bound to a single entity. Acid washing and flow cytometry experiments disclosed rapid internalization of BPI. Finally, BPI uptake by THP-1 cells had no effect on the surface expression of CD14. CONCLUSIONS: Bactericidal/permeability-increasing protein is rapidly internalized by mononuclear cells in a nonspecific fashion not saturable at very high doses, which is consistent with pinocytosis. This process may represent a disposal mechanism for lipopolysaccharide in closed-space infections and may be partially responsible for the rapid clearance of BPI from the peripheral circulation. PMID- 8611080 TI - Esophageal carcinoma and coexisting hepatocellular carcinoma resected simultaneously. AB - We have experience with two cases in which esophageal carcinoma and coexisting hepatocellular carcinoma were resected simultaneously. One patient had advanced esophageal carcinoma located in the thoracic esophagus and solitary hepatoma in the posterior segment of the liver with normal liver function. The other patient had superficial esophageal carcinoma in the thoracic esophagus and solitary hepatoma in the posterior segment of the liver with impaired liver function. Subtotal resection of the esophagus and posterior segmentectomy of the liver were performed simultaneously in both patients. In the patient with impaired liver function, postoperative management of respiration and bleeding was difficult, and intensive care was needed. Mediastinal lymph node resection was modified. Postoperative course was considered to have a close relationship to liver function. Thus, close evaluation of liver function is important to decide suitable treatment of patients with primary hepatocellular carcinoma and coexisting malignant neoplasms. With close evaluation of liver function and intensive postoperative care, simultaneous resection of esophageal carcinoma and hepatocellular carcinoma is not impossible or difficult. PMID- 8611081 TI - Operative injury to the hepatic artery. Consequences of a biliary-enteric anastomosis and principles for rational management. AB - Although the interruption of the hepatic arterial flow usually is well tolerated, this is not always the case, and it is important to predict in which circumstances complications are likely to occur. The main determinants that should guide the surgeon confronted with this problem are (1) whether the portal circulation is normal, (2) whether structures carrying collateral blood supply have been interrupted, and (3) whether some form of biliary reconstruction is needed. We present our experience with three patients in whom the hepatic artery was damaged at operation as examples of how this injury can be dealt with in practice and discuss the measures to prevent or treat the complications that developed. PMID- 8611082 TI - A false-positive localization of a parathyroid adenoma with technetium Tc 99m sestamibi scan secondary to a thyroid follicular carcinoma. AB - Using technetium Tc 99m sestamibi for localization of a parathyroid adenoma in primary hyperparathyroidism, we describe the first reported case of increased uptake due to a thyroid follicular carcinoma. Prior false-positive results have been reported exclusively with follicular adenomas. PMID- 8611083 TI - Increased tumor establishment and growth after laparotomy vs laparoscopy. PMID- 8611084 TI - Systemic leakage and side effects of tumor necrosis factor alpha administration via isolated limb perfusion can be manipulated by flow rate adjustment. PMID- 8611085 TI - A plea for uniform reporting of patient outcome in chronic pancreatitis. PMID- 8611086 TI - The evolution of a contemporary academic health care system. AB - The story I wish to tell today is of the emergence, over the last two centuries, of an integrated academic health care delivery system from the foundations of this country's fourth medical school, New Hampshire's first major hospital, and a multidisciplinary practice group, unique in its time. I will trace the covergence of each of these into a system that we offer as an answer to the contemporary puzzle of how to provide the best available care at value and still support the academic missions of education and research. We believe this to be the current delemma of American medicine, and while our answer may not be either successful or universally applicable, we are committed to the attempt. As background, let me provide some perhaps familiar details of the growth of these Hanover [NH] and Lebanon [NH] institutions. PMID- 8611087 TI - Severe pancreatitis. Determinants of mortality in a tertiary referral center. AB - OBJECTIVE: To determine factors associated with mortality in patients with severe pancreatitis. DESIGN: Retrospective review. SETTING: University tertiary referral center intensive care unit (ICU). PATIENTS: Thirty patients admitted to the ICU with the primary diagnosis of pancreatitis from 1986 to 1995. MAIN OUTCOME MEASURE: Survival vs nonsurvival. RESULTS: Twenty-seven patients were transferred from another institution. At the time of ICU admission, subsequent death was not associated with the following: systolic blood pressure, pulse rate, hemoglobin level, leukocyte count, platelet count, or serum calcium concentration. The patients who died during the study were older at admission (age [mean+/-SD] of those who lived, 47+/-17 years; age of those who died, 64+/-8 years; P=.01) and their serum creatinine concentrations were higher (creatinine concentrations [mean +/-SD] of those who lived, 150+/-90 micromol/L [1.7+/-1.0 mg/dL]; creatinine concentrations of those who died, 410+/-250 micromol/L [4.6+/-2.8 mg/dL]; P=.001). Clinical events not associated with mortality included respiratory failure, insulin use, positive blood cultures, positive pancreatic cultures, and abdominal surgery for pancreatitis and infected pancreatic necrosis. Death was associated with the use of inotropic and/or vasopressor support (P=.05) and renal failure (creatinine, >170 micromol/L[>2.0 mg/dL]) at any time during the ICU stay (P=.01). Patients with renal failure were no older than the patients without, but were admitted later after the onset of pancreatitis (mean+/-SD, 5.9+/-7.2 days vs 1.5+/-1.1 days; P=.03; median, 2 days vs 1 days). CONCLUSIONS: After hospital transfer to a teritiary referral center, only older age, use of inotropic and/or vasopressor support, and evidence of renal malfunction are associated with death. Prompt recognition of severe pancreatitis, especially in older patients, aggressive hemodynamic management, and/or earlier transfer to a tertiary care center may diminish the incidence of renal failure and mortality in severe pancreatitis. PMID- 8611088 TI - Long-term results of pylorus-preserving pancreatoduodenectomy for chronic pancreatitis. AB - OBJECTIVE: To assess the long-term outcome of patients following pylorus preserving pancreatoduodenectomy (PPPD) for chronic pancreatitis. DESIGN: Retrospective study with mean follow-up of 63 months (range, 1 month to 13.7 years). SETTING: Tertiary referral hospital. PATIENTS: Records of all patients who underwent PPPD for chronic pancreatitis at Lahey Clinic were reviewed. All patients who were alive were contacted by telephone. In cases where patients had died, information was gathered from family members and hospital records. RESULTS: Forty-five patients underwent PPPD for disabling chronic pancreatitis. The mean preoperative duration of pain was 50 months, with 32 patients (70%) requiring daily narcotics. In one patient resection of the portal vein was required. One patient died within 30 days of the operation. Forty-one patients (92%) had improvement of pain at 5 years. The mean pain score (on a scale of 0 to 10) was 9.2 preoperatively and 1.5, 0.8, 1.1, and 1.1 at 6 months, 1 year, 2 years, and 5 years, respectively. Thirty-three patients (74%) had a postoperative weight gain to an average of 92% of their pre-illness weight. New-onset diabetes occurred in six patients (14%) by 6 months and in 21 patients (46%) by 5 years. Hypoglycemia was the cause of death in one patient who underwent total pancreatectomy. Four patients died of causes unrelated to PPPD. Marginal ulcers occurred in five patients (10%). Nine patients required late operations. CONCLUSIONS: In selected patients, resection of the head of the pancreas achieves long-term pain improvement in over 90% of cases. The early development of diabetes mellitus is infrequent, but over longer follow-up periods it reaches prevalence rates similar to those described in patients who have not undergone resection. Weight gain in this group was superior to that previously reported for our patients who underwent "standard Whipple" operation for chronic pancreatitis. PMID- 8611089 TI - Computed tomography in blunt hepatic trauma. AB - BACKGROUND: Nonoperative management of blunt hepatic injury in hemodynamically stable trauma patients is now common. Recently, it has been proposed that the finding of hepatic periportal tracking (PPT) of blood on the initial computed tomographic (CT) scan is a sensitive marker of significant hepatic and subhepatic injury that might militate against nonoperative management. While CT scan is useful in diagnosing the injury, the utility of follow-up CT scans has not been elucidated. DESIGN: Retrospective chart review. SETTING: Regional trauma center. PATIENTS: The records of 58 hemodynamically stable patients with blunt hepatic trauma were reviewed and the following data recorded: age, outcome, Injury Severity Score (ISS), operative intervention, and complications. Computed tomographic scans were taken on admission and reviewed for the presence of PPT. The timing and radiographic appearance of follow-up CT scans was also recorded. RESULTS: Seventeen patients (29%) had radiographic evidence of PPT while 41 patients (71%) did not. Age, ISS, injury grade, overall success rate of nonoperative management, and incidence of complications were not statistically significant between the two groups. In no instance did a routine follow-up CT scan affect subsequent management of the patient. CONCLUSIONS: The finding of PPT on the admission CT scan is not clinically significant and does not preclude nonoperative management of patients with blunt hepatic injury. Furthermore, routine follow-up CT scans are not indicated, as treatment is not influenced by their results. Rather, follow-up CT scans should be obtained as dictated by the patient's clinical course. Extrapolation of these findings to all patients with blunt hepatic trauma in the United States would result in considerable savings of health care dollars, without negatively affecting patient care. PMID- 8611090 TI - Management of complicated appendicitis. A rational approach based on clinical course. AB - OBJECTIVE: To better define the appropriate management of children with complicated appendicitis, using an outcome approach based on clinical parameters. DESIGN: Retrospective study. SETTING: A 500-bed tertiary care university-based hospital. PATIENTS: Fifty-six consecutively admitted children (age <19 years) with a diagnosis of complicated appendicitis (gangrenous or perforated) confirmed at laparotomy. INTERVENTION: All children were managed postoperatively using an institutionally established protocol requiring hospitalization and broad-spectrum intravenous antibiotics until three criteria were met permitting discharge: (1) resolution of fever for 24 hours; (2) normalization of white blood cell count; and (3) normal results of clinical examination. MAIN OUTCOME MEASURES: Length of stay, costs, and infectious complications. RESULTS: Overall, infectious complications occurred in only two patients (3.5%). No complications occurred in any patient who met the criteria for discharge. The average length of stay for all patients was 5.1+/-3.0 days (range, 3 to 18 days). Using this approach instead of current standards reported in the literature resulted in an estimated savings of over $4000 per patient and $224000 for the entire cohort. CONCLUSIONS: Postoperative management of complicated appendicitis can be safely based on a defined clinical algorithm that should replace empirical therapy as the "gold standard." PMID- 8611091 TI - Percutaneous dilatational tracheostomy. A safe, cost-effective bedside procedure. AB - OBJECTIVE: To evaluate the safety and cost-effectiveness of percutaneous dilatational tracheostomy performed in the intensive care unit. DESIGN: Retrospective review of 65 patients with cost-effectiveness analysis. SETTING: University-affiliated tertiary care teaching hospital with a 34-bed combined medical-surgical intensive care unit. PATIENTS: All patients who underwent percutaneous dilatational tracheostomy under the supervision of a single general surgeon during a 19-month period. Cost analysis was based on comparison with standard operative tracheostomies performed during the same period. RESULTS: Percutaneous dilatational tracheostomy was completed in all patients in whom it was attempted, regardless of airway anatomy, body habitus, and ventilator settings. The mean duration of the procedure performed in the intensive care unit was 13.6 minutes (95% confidence interval, 11.7 to 15.5 minutes). Intraoperative complications occurred in 14 patients (22%), most of which were minor technical difficulties, and none resulted in serious morbidity. Postoperative complications occurred in six patients (9%), including one death secondary to premature decannulation, three bleeding complications, one episode of subcutaneous emphysema, and one air leak. Two long-term airway complications after percutaneous dilatational tracheostomy were documented during a mean 7.5-month follow-up of 28 patients. Mean patient charges for the procedure performed in the intensive care unit by a surgeon, nurse, and respiratory therapist were $997 (95% confidence interval, $975 to $1018) compared with $2642 (95% confidence interval, $2513 to $2772) for standard tracheostomy (P<.001). This represented a savings of $1645 (95% confidence interval, $1492 to $1798) per tracheostomy. CONCLUSIONS: Percutaneous dilatational tracheostomy is a safe, rapid, cost-effective alternative to standard open tracheostomy. It can be performed at the bedside, obviating the need to transport critically ill patients from their optimal intensive care unit environment. PMID- 8611092 TI - Complete intraoperative small-bowel endoscopy in the evaluation of occult gastrointestinal bleeding using the sonde enteroscope. AB - OBJECTIVE: To review our experience with intraoperative small-bowel Sonde enteroscopy in evaluating occult bleeding in the small intestine. DESIGN: Retrospective study with 100% follow-up. SETTING: University-affiliated, tertiary care teaching hospital. PATIENTS: Sixteen consecutive patients referred with occult gastrointestinal bleeding in whom esophagogastro-duodenoscopy , push enteroscopy, and colonoscopy had failed to identify the source of bleeding. Fourteen of the 16 patients had required one or more transfusions. MAIN OUTCOME MEASURE: Completeness of visualization, diagnostic accuracy, and complications of the procedure and follow-up for recurrent bleeding. RESULTS: In all 16 patients, intraoperative Sonde enteroscopy allowed visualization of the entire small bowel. In 14 of the 16, it revealed the cause of bleeding, which was ileal angiodysplasia in three patients, ileal ulcers in six patients, neoplasia in two patients, and ileal ulcers caused by Crohn's disease, small-intestinal enteropathy and varices caused by portal hypertension, and radiation stricture in one patient each. Two patients had normal small bowel mucosa. The patients with mucosal disease underwent small-bowel resection or oversewing of bleeding sites. Two surgical complications occurred: prolonged postoperative ileus (one patient) and small-bowel obstruction that resolved without surgery (one patient). Two of the patients with angiodysplasia had recurrent bleeding postoperatively. CONCLUSIONS: Intraoperative Sonde enteroscopy is safe and effective in localizing small-intestinal bleeding sites, providing complete visualization of the small bowel mucosa without enterotomy while avoiding the trauma that can be caused by push endoscopy. It is the diagnostic assessment of choice in selected patients with occult gastrointestinal bleeding of presumed small-bowel origin. PMID- 8611093 TI - Multidisciplinary approach to pseudoaneurysms complicating pancreatic pseudocysts. Impact of pretreatment diagnosis. AB - OBJECTIVE: To determine the effectiveness of thin-section, dynamic-contrast computed tomography and angiography in detecting the presence of pancreatic pseudoaneurysms. DESIGN: This case series consisted of 57 patients who were being examined for endoscopic drainage of pancreatic pseudocysts. SETTING: All patients were examined in a tertiary care, teaching hospital. PATIENTS: Fifty-seven consecutive patients were examined for 2 years. Follow-up ranged from 6 months to 2 years. INTERVENTIONS: All patients underwent thin-section, high-speed, dynamic contrast computed tomography. Those patients with findings that were consistent with the presence of a pseudoaneurysm underwent angiography. Embolization was attempted if a pseudoaneurysm was present. Endoscopic retrograde cholangiopancreatography was used to determine pancreatic ductal anatomy before operation. MAIN OUTCOME MEASURE: No undetected pseudoaneurysm has complicated this series of endoscopically drained pseudocysts. RESULTS: Five patients had findings that were consistent with a pancreatic pseudoaneurysm on computed tomography. Angiographic findings confirmed a pseudoaneurysm in four patients, and angiographic embolization was successful in three. Four patients underwent resection, while one was treated with embolization and endoscopic stenting of a compressed pancreatic duct. There were no mortalities. CONCLUSIONS: Before endoscopic drainage of a pancreatic pseudocyst, a thin-section, high-speed, dynamic-contrast computed tomographic scan is essential. If there are findings consistent with the development of a pseudoaneurysm, angiography must be performed. This allows delineation of the arterial anatomy, as well as the option of performing angiographic embolization. While patients with pseudoaneurysms in the body and tail of the pancreas underwent resection, angiographic embolization alone was an acceptable alternative when the lesion was located in the head of the pancreas. PMID- 8611094 TI - Hepatitis C viral infection in liver transplantation. AB - OBJECTIVE: To study the outcomes of patients who underwent liver transplantation for the primary diagnosis of chronic active hepatitis secondary to hepatitis C virus (HCV). DESIGN AND SETTING: Retrospective review within a university medical center. PATIENTS: Seventy-four adult recipients who received 78 orthotopic liver allografts for the primary diagnosis of chronic active hepatitis secondary to HCV between January 1990 and December 1994. Sixty-seven patients (91%) survived more than 2 months and were analyzed further for recurrent HCV infection. MAIN OUTCOME MEASURE: Recurrence of HCV infection, hepatitis, or cirrhosis and survival rates for patients who were undergoing orthotopic liver transplantation for chronic active hepatitis secondary to HCV. RESULTS: Actuarial survival rates for the entire group were 79.3%, 70.9%, and 64.5% at 1,2, and 3 years, respectively. Four patients (5% underwent retransplantation with an actuarial survival rate of 14.3% at 1 year (P<.05). Thirty-eight patients (57%) had evidence of posttransplant HCV infection, 31 patients (46%) showed histologic evidence of viral hepatitis, and 11 patients (16%) experienced portal fibrosis or cirrhosis. Seven (33%) of the deaths and all retransplantations were secondary to recurrent HCV infection. There were no significant differences in age, sex, United Network of Organ Sharing status, associated diagnoses, intraoperative packed red blood cell requirements, OKT3 use, or 1-, 2-, and 3-year survival rates in the recurrent vs nonrecurrent HCV infection groups. A higher incidence of posttransplant cirrhosis was observed in patients who were treated with tacrolimus (FK 506) (31.8% vs 8.9%, P<.05). Twenty-one patients (70%) received interferon alfa antiviral therapy with a significant benefit in the liver function test results during therapy (P<.01). CONCLUSIONS: Despite recurrence of HCV infection in most patients after transplantation, survival following primary orthotopic liver transplantation for chronic active hepatitis secondary to HCV infection remains favorable, and these patients should continue to be candidates for liver transplantation. In contrast, survival following retransplantation for HCV infection is poor and should be reconsidered. There is an apparent association between the intensity of immunosuppression and recurrent HCV infection and cirrhosis that warrants continued evaluation. Interferon therapy appears to afford benefit to patients in whom recurrent HCV hepatitis develops after transplantation. PMID- 8611095 TI - Multimodality treatment of hepatocellular carcinoma in a hepatobiliary specialty center. AB - OBJECTIVES: To review the experience of the treatment of hepatocellular carcinoma by a single multimodality team during a 6-year period, including all patients who were referred for possible surgical intervention, to evaluate prognostic factors at presentation, and to determine the results of the different modalities of treatment that were used. DESIGN: Retrospective study of 154 patients who were referred to our Hepatobiliary Surgical Unit with the diagnosis of hepatocellular carcinoma from January 1988 through August 1995. SETTING: Tertiary care center. RESULTS: Methods of treatment included surgical resection (n=49), transplantation (n=22), hepatic artery chemoembolization (n=30), systemic chemotherapy (n=25), and no treatment (n=22). Predictive prognostic factors included coexisting cirrhosis, symptoms at presentation, and abnormal liver function test results. Unfavorable tumor characteristics were size (diameter, >5 cm) and multicentricity. For patients who underwent surgical exploration, advanced staging according to the manual of the American Joint Committee on Cancer, vascular invasion, and a margin of less than 1 cm in the group for patients who underwent resection impacted negatively on the prognosis. The median survival (42.4 months) for the group of patients who underwent resection was significantly higher than that for the groups of patients who did not undergo resection. Chemoembolization was associated with significantly better survival results than was systemic chemotherapy. CONCLUSIONS: Hepatic resection offers the best chance at cure for patients with hepatocellular carcinoma. The high association between hepatocellular carcinoma and cirrhotic liver disease makes surgical resection, even in favorable tumor types, a difficult task based on low hepatic reserve whose tumors are considered unresectable can be considered for chemoembolization. Liver transplantation should be reserved for selected patients with cirrhotic liver disease who have tumors (diameter, <5 cm) in the contest of neoadjuvant protocols. PMID- 8611096 TI - The new era in breast cancer. Invasion, size, and nodal involvement dramatically decreasing as a result of mammographic screening. AB - OBJECTIVE: To describe the magnitude of changes and opportunities that may arise for simplified surgical procedures for women with breast cancer because of the decreasing size and lymph node involvement in invasive breast cancer and earlier presentation of noninvasive and invasive breast cancer. DESIGN AND MAIN OUTCOME ASSESSMENT: Cases (N=1001) of breast cancer from a tertiary and a community hospital between 1989 and 1993 were analyzed for invasion, size, nodal status, and change over time. RESULTS: Ductal carcinoma in situ constituted 14% and 18% of the cancers at the two hospitals. At the tertiary and community hospitals, the mean maximum diameters were 2.1 and 2.0 cm, respectively, and the median maximum diameters were 1.5 and 1.7 cm, respectively, for invasive breast cancer. Twenty nine percent and 28%, respectively, were 1 cm or less in diameter. Axillary nodal metastases occurred in only 31% of the invasive cancers (tertiary hospital); only 10% had more than three nodal metastases. In the T1a and T1b cases, nodal metastases occurred in only 10% and 43% of the positive nodes were solitary; only 16% had more than three nodal metastases. The proportion of ductal carcinoma in situ, T1a and T1b, and node-negative cases increased significantly over time. CONCLUSIONS: Within the next decade, the proportion of all breast cancers that are ductal carcinoma in situ will approach 33%, and invasive cancers will approach 1 cm in median maximum diameter. Therapy simplification will be logical because of very small size, low risk of recurrence after breast conservation, and excellent prognosis, and might include increased breast conservation, avoidance of axillary nodal dissection, and omission of radiation therapy to conserve breasts. Adjuvant therapy will be based on the prognostic features of the primary cancer and findings from careful histologic examination of the sentinel lymph nodes. PMID- 8611097 TI - Selective nonoperative management of liver and spleen injuries in neurologically impaired adult patients. AB - BACKGROUND: Nonoperative management of blunt liver and spleen injuries in hemodynamically stable, neurologically intact patients has become an accepted treatment in recent years. OBJECTIVE: To determine the morbidity and mortality in neurologically impaired adult patients who had sustained blunt liver or spleen injuries and who had been managed nonoperatively in a monitored setting, owing to the preponderance of blunt trauma and associated head injuries in Vermont. DESIGN: Case-control study. SETTING: Regional level I trauma center in northern Vermont. PATIENTS: One hundred eighty-seven consecutive patients with documented blunt splenic or hepatic trauma who were admitted to a regional rural trauma referral center in Vermont during an 8-year period, beginning in January 1987, were studied. Hemodynamically stable patients underwent diagnostic imaging studies and were classified by mental status as either normal or altered. Patients who required operative intervention were excluded. MAIN OUTCOME MEASURES: Morbidity and mortality rates for each group were recorded and compared to determine if statistically significant differences between the two groups existed. RESULTS: The groups were similar in age, systolic blood pressure, and hematocrit at admission. The group of patients with an altered mental status were more severely injured and had a longer hospital stay. Intensive care unit stays were not significantly different. Transfusion requirements for both groups were minimal; however, the group of patients with an altered mental status received more blood transfusions compared with the group of patients with a normal mental status. There was no significant difference in morbidity and mortality between the two groups. There were no failures of nonoperative management, no complications, and no missed visceral injuries in the group of patients with an altered mental status. Patients older than 50 years had higher morbidity and mortality. CONCLUSIONS: Nonoperative management in patients with an altered mental status can be done safely in a monitored setting. This challenges the current criteria of excluding neurologically impaired patients with liver or spleen trauma from nonoperative management. PMID- 8611098 TI - The effect of peripheral vascular disease on long-term mortality after coronary artery bypass surgery. Northern New England Cardiovascular Disease Study Group. AB - OBJECTIVE: To examine the effect of peripheral vascular disease (PVD) on long term mortality after successful myocardial revascularization. METHODS: We performed a regional cohort study of 2871 consecutive patients discharged alive after coronary artery bypass graft surgery at five tertiary care centers in Maine, New Hampshire, and Vermont between 1987 and 1989. Data reflecting patient characteristics, heart disease severity, and comorbidity were collected prospectively; the presence of clinical and subclinical indicators of PVD was determined by medical record review; and vital status was determined using the National Death Index (mean follow-up, 4.4 years). RESULTS: Five-year mortality following coronary artery bypass graft surgery was substantially higher in the 755 patients with indicators of PVD (20%; 95% confidence interval [CI], 17% to 23%) than in the 2116 patients without PVD (8%, 95% CI, 7 to 9; P<.001). The crude hazard ratio of long-term mortality associated with PVD was 2.77 (95% CI, 2.19 to 3.50; P<.001). After adjusting for their higher comorbidity scores, more advanced cardiac disease, and age, mortality rates in patients with PVD remained twice as high as those in patients without PVD (adjusted hazard ratio, 2.01; 95% CI, 1.57 to 2.58; P<.001). Long-term mortality was increased in patients with any of the indicators of PVD. Patients with multilevel PVD had especially high late mortality rates (adjusted hazard ratio, 2.46; 95% CI, 1.64 to 3.68; P<.001). CONCLUSIONS: Even after successful myocardial revascularization, patients with PVD remain at substantially increased risk for long-term mortality. The presence of clinical or subclinical PVD is important when predicting both short- and long term outcomes in patients considering coronary artery bypass graft surgery. PMID- 8611099 TI - Patient selection for hepatic resection of colorectal metastases. AB - OBJECTIVE: To determine the major factors governing patient outcome after hepatic resection of metastatic colorectal cancer and to formulate criteria for optimal resection. PATIENTS: We reviewed records of 74 patients (44 men, 30 women) who underwent resection of colorectal liver metastases. MAIN OUTCOME MEASURES: Sex, age, primary tumor location; Dukes tumor stage; disease-free interval after primary resection (synchronous vs metachronous); location, number, size, and distribution of liver metastases; operative complications; and mortality. RESULTS: The primary tumor location was rectosigmoid in 46 patients and the colon in the others. The tumor stage was Dukes A in one patient, Dukes B in 16, Dukes C in 31, and Dukes D (synchronous metastases) in 26. The disease-free interval was less than 12 months in 38 patients and 12 months or more in 36. The location of the metastases was the right lobe in 42 patients, left lobe in 22, and bilateral in seven. The cancer was unilobar in 55 patients and bilobar in 18. Surgical resection included wedge resection in 23 patients, segmentectomy in 30, lobectomy in seven, and trisegmentectomy in 12. The number of lesions resected was one in 50 patients, two or three in 18, and four or more in five. Nine patients had repeated liver resections because of recurrence. There were five postoperative deaths within 60 days (7%), four of which occurred after extended resection and were complicated by delayed liver failure and multisystem failure. An additional death occurred at 65 days after an apparently uneventful initial convalescence. Overall median survival was 35 months; actuarial 5- and 10-year survival rates were 24% and 12% respectively. There were significant relationships with survival (P<.05) for the number of metastases (three or fewer vs four or more), bilobar vs unilobar metastases, and extent of liver resection (wedge and segmental vs lobectomy and trisegmentectomy). A multiple logistic regression model (multivariate analysis) showed a significant correlation with survival (P<.05) for distribution of metastases (bilobar vs unilobar) and extent of resection (wedge and segmental vs lobectomy and trisegmentectomy). CONCLUSION: Patient selection for hepatic resection of colorectal cancer metastases based on standard clinical and tumor outcome variables should be expected to achieve long-term survival with low morbidity and mortality in bilobar disease or extended resection should generally be avoided, especially in medically compromised patients. PMID- 8611100 TI - Emerging and reemerging microbial threats. Nosocomial fungal infections. AB - The incidence of nosocomial fungal infections had been increasing steadily for the past 25 years. Although they were once believed to be of little clinical consequence, there is now compelling evidence that fungal agents represent a bona fide microbial threat with substantial morbidity and high mortality. Reporting on a series of 30447 nosocomial fungal infections that occurred in the decade from 1980 to 1990, Beck-Sague and Jarvis noted increases in incidence from 90% to 175%. Infection rates rose from 2.0 per 1000 discharges to as high as 6.6 infections per 1000 discharges. Fungal infections increased at all major anatomic sites, including surgical wounds, lung, urinary tract, and bloodstream. Candida species accounted for 78.3% of nosocomial fungal infections, while torulopsis species and aspergillus species accounted for 7.3% and 1.3%, respectively. PMID- 8611101 TI - Antibiotic-resistant enterococci and the changing face of surgical infections. AB - BACKGROUND: Enterococci have not been thought to play an important role in intra abdominal infections because of their relatively low virulence. However, this notion is changing because of the recent emergence of these microbes as significant nosocomial pathogens. OBJECTIVES: To review the mechanisms of antibiotic resistance of enterococci and to discuss the significance of multidrug resistant enterococci in surgical infections. DATA SOURCES: Medical and basic science literature relating to enterococci. DATA SYNTHESIS: In addition to having intrinsic resistance to a number of antibiotics, enterococci have the ability to acquire resistant genes through the exchange of plasmids or transposons from other bacterial species. Moreover, enterococci have been shown to transmit these genes to other bacterial species in turn. The extensive resistance of these microorganisms has led to their emergence as significant nosocomial pathogens, ranking second only to Escherichia coli in the number of pathogenic isolates recovered from patients in intensive care units. There has also been a marked increase in vancomycin-resistant enterococcal infections in surgical patients in the last 5 years. Some studies associate the prior use of vancomycin or third generation cephalosporins with the emergence of these strains. Overall, enterococcal infections are associated with increased morbidity and mortality. CONCLUSIONS: In view of the marked resistance of enterococci to antibiotics and their ability to disseminate resistance genes, these microbes have become important pathogens. Enterococci pose a threat to surgical patients, often causing significant therapeutic dilemmas. PMID- 8611102 TI - Josiah Nott (1804-1873) PMID- 8611103 TI - [45th general meeting of the Japanese Society of Allergology. Tokyo, Japan. October 25-27, 1995. Abstracts]. PMID- 8611104 TI - George Adlington Syme Oration. The International Year of Tolerance: its challenge to Australia. PMID- 8611105 TI - The role of cellular adhesion molecules in surgery. AB - The purpose of this review is to detail the roles played by the cellular adhesion molecules (CAM) in inflammatory and immunological reactions relevant to surgery. The interactions between leucocytes and endothelial cells which are mediated by CAM are central to the development of ischaemia/reperfusion injury (IRI) as occurs when blood flow is restored after an ischaemic period; for example, following revascularization of replanted digits and microvascular tissue transfers, angioplasty and tourniquet procedures. Cellular adhesion molecules are also important in wound healing and other inflammatory processes. In addition, the immunological response to organ allograft transplantation is mediated by cellular interactions mediated by CAM. This review details the functions and regulation of the various CAM involved in inflammation and allograft rejection and summarizes the results of previous surgical studies in which various techniques have been used to block the interactions mediated by CAM in an attempt to improve surgical outcomes. PMID- 8611106 TI - Patterns of injury from major trauma in Victoria. AB - Basic demographic and injury data were collected on all major trauma patients (ISS > 15) presenting to 25 Victorian hospitals over a 1 year period (March 1992 February 1993). A total of 1076 patients were identified with an Injury Severity Score (ISS) > 15. Of these, 957 resulted from blunt trauma, 68 from penetrating trauma and 51 from burns. Most serious blunt injury was transport-related (n = 652) but falls made up a significant proportion (n = 206). The pattern of injury in blunt trauma demonstrated in this study showed a preponderance of serious head, thoracic and limb injuries with less frequent occurrences of abdominal, spine and facial injuries. In major penetrating trauma, serious injuries of the thorax and abdomen were more frequent. Head injury is the most common cause of morbidity in major trauma patients. Motor vehicle accidents caused the majority of head injuries but, proportionately, head injury was more common in pedal cycle, pedestrian, motorcycle injuries and falls. The low frequency of major abdominal trauma has important implications for surgical training and resource allocation. In Victoria, various injury prevention interventions have been introduced such as compulsory wearing of bicycle helmets, a safer home environment and behavioral modifications through advertising. Injury prevention strategies must continue to target the populations at risk and assess the impact of interventions by accurate injury surveillance. PMID- 8611108 TI - A review of intestinal injury from blunt abdominal trauma. AB - Eighty-five patients who suffered blunt trauma to the small intestine and/or to the colon were treated at either the Royal Brisbane Hospital (RBH), Brisbane, Queensland or the Flinders Medical Centre (FMC), Adelaide, South Australia, between 1980 and 1991. Data were collected by retrospective review of case notes from the medical records departments of both hospitals and analysed with respect to the cause, the anatomical distribution, the diagnostic methods and the mortality of these injuries. There were 129 intestinal injuries (44 colonic and 85 small bowel). Five (5.9%) deaths were recorded. Seventy-two patients (84.7%) were injured in vehicular accidents. Fifty-three patients (62.4%) underwent laparotomy based on clinical findings alone. Diagnostic peritoneal lavage (DPL) was used in 24 cases and was positive in 22 (91.7%). The most common small bowel injury was ?blowout' perforation on the antimesenteric border of the bowel (55.5%). The most common colonic injury was a serosal tear/bruise (62.2%). PMID- 8611107 TI - Evaluation of the prevalence of drug and alcohol abuse in motor vehicle trauma in south western Sydney. AB - This study estimated prospectively the prevalence of high drug and alcohol levels in road trauma cases who met the criteria for activation of the Liverpool Hospital's trauma team. Urine analysis of road trauma victims between October 1992 and October 1993 was undertaken for drug and alcohol estimation. A total of 164 drivers were studied. A urine alcohol concentration (UAC) exceeding 0.08 g/dL was detected in 27 drivers (16.5%). Cannabinoids were detected in the urine of 25 drivers (15.2%), in 17 the concentrations exceeded 400 ng/mL. In one instance amphetamine, cocaine and heroin were detected in the same injured driver. Combined use of alcohol with some other drugs was detected in only four drivers. Alcohol and cannabinoid levels were prevalent in the urine of injured drivers in this study, particularly in young males who remain over-represented in the group of injured drivers. In the population surveyed other drugs were rarely detected. The role of cannabinoids in road trauma and the use of cannabinoids in young male drivers will however need to be monitored more extensively. PMID- 8611109 TI - Review of liver trauma management in Tasmania: an analysis of risk factors for mortality and morbidity. AB - A review of liver trauma treated by the major trauma care facilities of Tasmania in the 5 year period between 1989 and 1993 is presented. The aim of this retrospective review was to provide an audit of the management of liver trauma in the island of Tasmania and to analyse the risk factors contributing to mortality and major morbidity. Thirty-seven patients were treated with a median Injury Severity Score (ISS) of 14 (range 9-34). The overall mortality rate of this series was 5.8%. Age, mechanism of injury (blunt or penetrating), delay prior to hospital presentation and modality of treatment (operative or non-operative) were not significant risk factors for mortality and morbidity; however, transfusion requirement of over 10 units of blood (P < 0.005), ISS score of over 20 (P < 0.0005), haemodynamic instability at presentation (P < 0.05) and a Hepatic Injury Score (HIS) grade of 3 or more (P < 0.05) were statistically significant risk factors. PMID- 8611110 TI - Bone allograft banking in South Australia. AB - The South Australian Bone Bank had expanded to meet an increased demand for allograft bone. During a 5 year period from 1988 to 1992, 2361 allografts were harvested from 2146 living donors and 30 cadaveric donors. The allografts were screened by contemporary banking techniques which include a social history, donor serum tests for HIV-1, HIV-2, hepatitis B and C, syphilis serology, graft microbiology and histology. Grafts were irradiated with 25 kGy. The majority of grafts were used for arthroplasty or spinal surgery and 99 were used for tumour reconstruction. Of the donated grafts 336 were rejected by the bank. One donor was HIV-positive and two had false positive screens. There were seven donors with positive serology for hepatitis B, eight for hepatitis C and nine for syphilis. Twenty-seven grafts had positive cultures. Bone transplantation is the most frequent non-haematogenous allograft in South Australia and probably nationally. The low incidence of infectious viral disease in the donor population combined with an aggressive discard policy has ensured relative safety of the grafts. The frequency of graft rejection was similar to other bone banks but the incidence of HIV was lower. PMID- 8611111 TI - Island flaps including the Bezier type in the treatment of malignant melanoma. AB - Despite the trend in current surgical practice in the treatment on melanoma to produce smaller excisional defects, any technique which can introduce a surgical closure that does not require split skin grafting must be of benefit. This paper introduces and illustrates a range of island flap techniques that employ no skin grafting for the treatment of malignant melanoma defects. The new cutaneous island flap described, termed the Bezier or the French Curve, employs a double V Y appositional closure method, thus giving a more refined reconstructive result that fits into the line of the body curves aesthetically. The design of the Bezier flap is almost identical in size and shape to the excisional defect, with a facial or muscular base for vascular support. Appropriate guidelines that determine the design and application of this island flap technique are listed. They are illustrated both diagrammatically and clinically. Other flaps illustrated include fasciocutaneous island flaps and myocutaneous island flaps that use a single V-Y flap appositional closure technique. All these flaps were designed with special reference to the dermatomes, which act as an aid memoire upon which the flaps are marked. PMID- 8611113 TI - End-to-end anastomosis at the duodenojejunal flexure: is it safe? AB - Resection of the proximal jejunum in the duodenojejunal junction area is described in four patients, in diverse clinical settings. The conventional approach would have been to perform a gastrojejunostomy because of the possibility of ischaemia at and around the duodenojejunal flexure. A uniform surgical approach was followed for mobilization, resection and end-to-end duodenojejunal anastomosis and the outcome was successful in all cases. PMID- 8611112 TI - High dose loperamide suppositories: a novel approach for improving clinical function after restorative proctocolectomy. AB - The effect of loperamide suppositories on patients following restorative proctocolectomy was studied by means of a randomized, double-blind, crossover trial comparing active suppositories (20 mg b.d. x 1 week) with placebo. Ten patients (8 male, 2 female; 7 J pouch, 3 W pouch; 8 ulcerative colitis, 2 familial adenomatous polyposis) were studied 3-60 months (median, 31.5) after ileostomy closure. Ages ranged from 24 to 63 years (medium, 41.5). All patients kept a diary of their bowel habits and eight underwent a standardized test of pouch compliance. Urgency volume (UV) and maximum tolerable volumes (MTV) and the volume at onset of large isolated pouch contractions (LIC) were recorded. Statistical analysis was by the Wilcoxon test for paired data. Mean daily stool frequency during the placebo phase ranged from 3.7 to 7.8. It was reduced during the active phase in only seven patients (P > 0.1) but was reduced in all six patients whose placebo phase stool frequency was five or more. Urgency volume was increased by use of active suppositories in six of the eight patients tested (P > 0.01). There was no consistent effect on MTV. Large isolated pouch contractions were not seen in either test in one patient. In all of the remaining seven patients LIC were recorded after the placebo phase. After the active phase LIC first appeared at higher volumes in three but were not seen at all in four patients. High dose loperamide suppositories suppress pouch contractions and tend to lower stool frequency especially when high initially. They represent a novel therapeutic approach to high stool frequency in pouch patients. PMID- 8611114 TI - The beginning of antiseptic surgery in Australia. PMID- 8611115 TI - Muskets, musket balls and the wounds they made. AB - Gunshot wounds are a common occurrence today in both military and civilian situations. This paper discusses the development of the weapons, their capabilities, the type of wounds produced and their management in earlier times. A brief comparison is made with the wounds produced by modern firearms. It may be concluded that the earlier wounds were simpler due to the lower velocity of the projectile and therefore a more conservative approach to treatment was acceptable. PMID- 8611116 TI - Locally advanced breast cancer. PMID- 8611117 TI - Locally advanced breast cancer. PMID- 8611118 TI - Surgical presentation of hepatobiliary disease due to Salmonella typhi. PMID- 8611119 TI - Dermatofibrosarcoma protuberans in a renal transplant patient. AB - Dermatofibrosarcoma protuberans in an unusual soft tissue tumour with a propensity for local recurrence and rarely metastatic spread. Given its indolent course it may be mistaken for atypical scarring or a keloid growth. Early diagnosis and prompt wide local resection of this entity are required in order to prevent a local recurrence. We report a patient who developed locally invasive dermatofibrosarcoma protuberans 4 years after a successful renal transplantation. Although other forms of sarcoma have been reported as a complication of organ transplantation and immunosuppression, this pathology has, so far, not been reported in a transplant recipient. PMID- 8611120 TI - Obturation of the small bowel. AB - Obturation of the small bowel is an uncommon but important cause of small bowel obstruction. There are a great number of substances that may impact at the narrow portions of the small bowel. More than one object may be present in the alimentary tract and should be actively sought. Certain groups of patients are more likely to suffer from the condition. We present two cases of small bowel obstruction due to obturation and review the relevant literature. PMID- 8611121 TI - Arteriovenous fistulae complicating orthopaedic procedures. AB - Arterial injuries in general and arteriovenous fistulae in particular are uncommon complications of orthopaedic procedures. We report the case of a 75 year old patient who suffered an arteriovenous fistula following bone graft insertion for an un-united tibial fracture. Aetiology diagnosis and management of these potentially serious injuries are reviewed. PMID- 8611122 TI - A pulsatile gluteal mass due to sciatic artery aneurysm. AB - A persistent sciatic artery is a rare congenital anomaly where the internal iliac artery and the original axial artery of the embryo continue to provide the major blood supply to the lower limb after birth. Its predisposition to atherosclerosis, aneurysmal degeneration of its gluteal segment and association with other congenital anomalies are important for its management. We report a patient who highlights these aspects and we provide a brief review of this unusual condition. PMID- 8611123 TI - Laparoscopic surgery: an ethical dilemma. PMID- 8611124 TI - Treatment principles in advanced colorectal cancer. AB - BACKGROUND: Colorectal cancer is an important disease in the Australian community. Whilst there has been much discussion about the appropriate management of local disease, particularly with respect to the issue of adjuvant therapy, there has been relatively little local discussion of treatment options in metastatic colorectal cancer. METHODS: The critical principles underlying treatment in this setting are briefly outlined in the present article, indicating wherever possible what options could be considered standard, and based on the available literature, which approaches must still be considered experimental at this time. RESULTS: Overall, the results of treatment in this group of patients remain poor. CONCLUSION: Further clinical trials are required to address the issues affecting patients with metastatic colorectal cancer. PMID- 8611125 TI - Intensive care unit admissions following laparoscopic surgery: what lessons can be learned? AB - BACKGROUND: Laparoscopic surgery has been widely embraced, often without adequate data concerning the range and incidence of complications. In the present series, our experience of complications requiring Intensive Care Unit (ICU) admission following laparoscopic surgery is described. METHODS: The records of patients requiring ICU admission at John Hunter Hospital (JHH) following laparoscopic surgery over a 39 month period were retrospectively reviewed by an independent multidisciplinary panel. RESULTS: Twenty-three ICU admissions were identified. Twenty-one followed general surgical laparoscopic procedures and two followed gynaecological laparoscopies. Ten cases were operated on initially at JHH and 13 were transferred from other hospitals. During the study period, 2444 laparoscopic surgical cases were performed at JHH; 725 general surgical procedures (1.37% admitted to ICU) and 1719 gynaecological procedures (no ICU admissions). Twelve cases suffered surgical complications (including five gastrointestinal tract perforations and three biliary tract injuries) and 11 cases were admitted for non surgical problems. In 75% of surgical complications there was delay in diagnosis of more than 24 h. The duration of ICU stay for surgical complications (16.4 days) was significantly longer than for the non-surgical group (3.9 days). CONCLUSION: There was a greater likelihood of ICU admission following general surgical rather than gynaecological laparoscopy. Fifty-two per cent of the admissions were for surgical complications. Surgical complications are characterized by delay in diagnosis and longer ICU admission periods. Strategies to prevent some of these complications are discussed. PMID- 8611126 TI - Pancreatic stents in the management of chronic pancreatitis. AB - BACKGROUND: Elevated pancreatic duct pressure is a potential source of pain in patients with chronic pancreatitis. Endoscopic pancreatic duct stenting is a minimally invasive way of reducing this pressure and may be a useful adjunct to surgery in these patients. METHODS: We prospectively reviewed a series of nine symptomatic patients with obstructive chronic pancreatitis and relative contraindications to open surgery, who were managed by attempted endoscopic placement of a pancreatic stent. RESULTS: Stents were successfully inserted endoscopically into the main or accessory duct in six patients and into a pseudocyst, transduodenally, in one patient. Of the two unsuccessful insertions, one proceeded to longitudinal pancreato-jejunostomy and in the other a stent was inserted at distal pancreatic cyst-jejunostomy. Median follow up was 21 months (range 14-43). In all eight cases with stent insertion there was rapid pain resolution, pain scores falling from 9/10 (8-10) to 2 (1-5) after 2 days (1-7). Associated symptoms of weight loss, nausea and vomiting settled in all eight cases. In one patient with a persistent pancreatic fistula, the fistula resolved. In the three with pseudocysts, the cysts resolved on computed tomography (CT) (one recurred). Five patients subsequently proceeded to stent removal after 6 months (5-23). In three of these, the stent was removed endoscopically, and replaced endoscopically in two cases, with pain resolution. Two patients underwent transduodenal pancreatic duct septectomy (one had stent change prior) and one proceeded to pseudocyst-gastrostomy, with pain resolution. the remaining three patients with stents in situ remain symptom-free. No patient suffered acute pancreatitis. CONCLUSIONS: In selected patients with obstructive chronic pancreatitis, insertion of a pancreatic stent is a safe procedure, which can lead to rapid symptomatic control over the intermediate period. A significant proportion will need further intervention. PMID- 8611127 TI - Prolonged survival follows resection of oesophageal SCC downstaged by prior chemoradiotherapy. AB - BACKGROUND: The poor survival rate of surgically treated patients with oesophageal cancer has not improved substantially over the last 25 years, but combined modality therapy has shown early promising results. METHODS: A prospective study was undertaken to determine the effect of pre-operative synchronous chemoradiotherapy followed by oesophagectomy in 53 patients with squamous cell carcinoma (SCC) of the oesophagus. The patient group was unselected, other than by fitness for surgery. RESULTS: In 25% of patients, complete pathological regression of the tumour was achieved. All but one of the patients in this subgroup had T2 tumours on pre-operative clinical staging and two had evidence of lymph node involvement, but postoperative pathological examination revealed that pre-operative chemoradiotherapy had downstaged their disease to T0N0. There was no hospital mortality in this subgroup and the actuarial 7 year survival was 69%. CONCLUSIONS: For squamous oesophageal tumours deep to the submucosa this is an extremely good survival. For the present, this form of therapy for SCC of the oesophagus appears capable of achieving results comparable to, or better than, those reported for 3-field lymphadenectomy. PMID- 8611128 TI - The morbidity of defunctioning stomata. AB - BACKGROUND: The choice of a defunctioning stoma in restorative resection of rectal carcinoma is unclear. Traditionally, the loop colostomy has been preferred. METHODS: All patients with either a defunctioning loop ileostomy or colostomy treated by a single surgeon (MS) were studied. The morbidity of stoma construction and closure, as well as problems experienced in the intervening period, were entered on a database. RESULTS: No significant difference in the morbidity of closure was noted when loop ileostomy was compared to loop colostomy. Although a trend favouring loop ileostomy was noted when interval morbidity was examined, this difference was not statistically significant. CONCLUSIONS: Loop colostomy and ileostomy are both effective in defunctioning the distal colon following a restorative resection for rectal carcinoma. There is some morbidity associated with both but when a stoma is constructed, loop ileostomy is preferable. The loop ileostomy is generally easier to manage and is not associated with a greater rate of complications (in its construction and closure) than the loop colostomy. PMID- 8611129 TI - Long-term experience with sclerotherapy for treatment of epididymal cyst and hydrocele. AB - BACKGROUND: Symptomatic testicular hydrocele and cyst of the epididymis may be treated with either operation or sclerotherapy. METHODS: The current report presents the experience of a 9 year prospective study using sodium tetradecyl sulphate (STD) sclerotherapy for the treatment of symptomatic hydrocele and/or epididymal cyst. RESULTS: A total of 102 lesions were treated during the study period, with an initial success rate of 76% which improved to 94% with multiple treatments. The overall median follow up during the study was 30 months (range 2 100). CONCLUSIONS: Sclerotherapy offers a cost-effective outpatient method for the treatment of symptomatic scrotal cysts. PMID- 8611130 TI - Non-tuberculous mycobacterial lymphadenitis. AB - BACKGROUND: Non-tuberculous mycobacterial lymphadenitis has been recognized since medieval times by an array of names. The condition is familiar to paediatricians and paediatric surgeons but it often is not recognized in its early stages. A paediatric surgeon's experience of the condition in Newcastle over 30 years is reviewed. METHODS: A personal series of children with non-tuberculous mycobacterial lymphadenitis treated in Newcastle from 1966 to 1994 is reviewed. Clinical diagnosis was supported by multiple Mantoux skin testing in most patients using human purified protein derivative (PPD) and avian antigens. All were treated surgically with histological confirmation of the diagnosis. A total of 89 patients were encountered. Twenty-two were seen in hospital practice between 1966 and 1976 and have already been reported. The current paper presents the results of analysis of the clinical features, diagnosis and surgical treatment of the remaining 67 patients seen in paediatric surgical practice between 1976 and 1994. RESULTS: There was equal sex distribution. Ages ranged from 1 to 10 years, with none under 1 year, and 82% of the patients were in the pre-school age group. Cervical lymph nodes were involved in all, the majority being jugulo-digastric or submandibular. Surgical excision by limited dissection of lymph nodes was performed in 55 patients with one recurrence, and by excision and curettage in eight patients with two recurrences. Simple bacterial wound infection occurred in four patients and two had prolonged postoperative suppuration as a result of mycobacterial wound infection. Paresis of the mandibular or cervical branch of the facial nerve occurred in 50% of patients where the nerve was at risk, but the majority of these recovered although it took over 6 months in some children. Culture for mycobacterial organisms was positive in only 29 patients. CONCLUSIONS: The diagnosis of non-tuberculous mycobacterial lymphadenitis is clinical and its early recognition requires an awareness of the condition. It can be confirmed by multiple Mantoux testing or fine needle aspiration biopsy. The treatment is local excision of the affected lymph nodes. Histological examination and mycobacterial culture should be performed. PMID- 8611131 TI - Auger injuries in the Wimmera region 1987-95. AB - BACKGROUND: Eighteen cases of auger injuries in the Wimmera region of Victoria were treated over 8 years. METHODS: The records of auger related injuries presenting to Wimmera Base Hospital from March 1987 to March 1995 were reviewed. RESULTS: Five of these were severe injuries. Sixteen were male farmers. Their fingers were most commonly injured by being caught in the auger flight. CONCLUSIONS: Augers have numerous mechanical features which make them one of the most potentially dangerous pieces of farm equipment. This, combined with human error and fatigue, results in significant but preventable morbidity in a hardworking population. PMID- 8611132 TI - Carotid artery surgery in the octogenarian. AB - BACKGROUND: The value of carotid surgery is largely dependent on the safety of the procedure. With changes in population life expectancy, increasing numbers of elderly patients are being considered for surgery. METHODS: In the present paper, the results of 113 patients (octogenarians: > 80 years of age) who underwent carotid endarterectomy in the 17 years prior to 1994 are reported. This group composed 6.2% of the 1818 patients treated in the period, 665 (36.6%) operations were performed on patients 50-65 years inclusive and 1040 (57.2%) on patients aged 66-80 years inclusive. RESULTS: The overall peri-operative stroke rate was 2.5% and the postoperative mortality was 1.9% with no statistical difference apparent between the age groups despite hemispheric strokes being the most common indication for operation in the octogenarian group (29.5%) and the least common indication in the youngest age group (16.2%, P <0.01). Long-term follow up (2-7 years) of octogenarian patients undergoing carotid surgery suggested maintenance of pre-operative levels of living independence. CONCLUSIONS: The results of the study indicated that octogenarian patients should not be denied carotid endarterectomy on the basis of age alone and that results comparable to those of younger patients can be anticipated. PMID- 8611133 TI - Gastrin receptor antagonist CI-988 inhibits growth of human colon cancer in vivo and in vitro. AB - BACKGROUND: Whilst gastrin has been found to be trophic for some colorectal cancer cell lines, and gastrin receptor antagonists are able to block this phenomenon, their potency has been modest. METHODS: The effect of a new, potent and selective CCK B receptor antagonist, CI-988 on the growth of LoVo, a human colon cancer cell line both in vitro and in vivo was instigated. RESULTS: Basal growth of LoVo in vitro was inhibited by up to 58.93 +/- 7.30% with concentrations of CI-988 as low as 1 X 10(-11) mol/L whereas the addition of gastrin (G17) at 0.5 nmol/L had no effect. LoVo was also grown in vivo for 10 days in nude mice subsequently treated with CI-988 at 10 mg/kg per day orally for 20 days. CI-988 inhibited the growth of xenografts by 53%. CONCLUSION: This was the first study in cancer with this potent gastrin receptor antagonist, CI-988. The results suggest that CI-988 may be of use in inhibiting the growth of colorectal cancer. PMID- 8611134 TI - Surgeon-superintendents on convict ships. AB - Surgeon-superintendents on the convict ships transporting convicted men, women and youths to Australia played a key role in the evolution of medical standards in Australia. The British Transportation Acts of 1717 and 1825 added the punishment of exile and banishment to the prevailing penology of the era, that of retribution and deterrence. The surgeon-superintendents formed a bulwark during the sea voyages (between 88 and 258 days), protecting the convicts against the potential abuses of the time. Between 1787 and 1868, some 160 000 convicts were transported to the open air gaols at Sydney, Norfolk Island, Van Diemen's Land (Hobart, Macquarie Harbour, Maria Island and Port Arthur), Moreton Bay, Melville Island and Fremantle. Seventeen convict ships left England for Australia in 1823 alone. The surgeon-superintendent's role on the high seas evolved over this time from one of amateur casualness with a primary responsibility to the system rather than to individual convicts, to that of a highly efficient, courageous professionalism. It became a new specialty discipline in its own right. Mortality on the convict transportation voyages fell from one in three convicts embarked in 1790 to zero mortality in the convicts transported on the Sultana in 1859. The key role of the surgeon-superintendent, in the context of preventive medicine, is developed in the present paper. Historical nodes in the evolution of the new discipline of prison doctor were the 1814 Report of Redfern (himself a former convict), and the evolution from the 1820s of doctors who became the pioneers of the specialty discipline of the Prison Medical Service in Australia. The experiences and influences of surgeon-superintendents on convict transportation vessels formed the catalyst for the Passenger Act (UK) of 1855 which, for the remaining decades of the 19th century, regulated the lives of millions of immigrants to Australia and New Zealand. PMID- 8611135 TI - Gastropericardial fistula. PMID- 8611136 TI - Giant cell arteritis of the breast. PMID- 8611137 TI - Cervical osteophytes: an unusual cause of dysphagia. PMID- 8611138 TI - Fatigue fracture of the medial malleolus in a junior roller skater. PMID- 8611139 TI - Emphysematous cholecystitis. PMID- 8611140 TI - Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2. AB - By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development. PMID- 8611141 TI - The role of Stat and C/EBP transcription factors in the synergistic activation of rat serine protease inhibitor-3 gene by interleukin-6 and dexamethasone. AB - The rat serine proteinase inhibitor 3 gene is activated by interleukin 6 (IL-6) and glucocorticoids in hepatic cells. We report here that a 147 bp promoter is sufficient for both IL-6 stimulation and glucocorticoid enhancement of IL-6 induced transcription. Within this region we identified two functional elements binding transcription factors from the C/EBP (CCAAT/enhancer binding proteins) and Stat (signal transducers and activators of transcription) families. Mutations introduced into the Stat binding site resulted in a loss of responsiveness, showing that this element is indispensable for activation. In contrast, the promoter containing the mutated C/EBP binding site was still responsive to IL-6 and glucocorticoids; however, the magnitude of the induction was decreased by 50%. The Stat binding element is an enhancer capable of conferring both responsiveness to IL-6 and partial enhancement of glucocorticoids on to a heterologous promoter. In response to IL-6 this element rapidly binds acute-phase response factor (APRF/Stat3) and, later, the protein(s) that require ongoing protein synthesis and is recognized by anti-Stat3 antibodies. In addition, long term treatment with IL-6 results in sustained phosphorylation of APRF /Stat3. PMID- 8611142 TI - Identification and characterization of a 44 kDa protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization. AB - Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041-8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA-protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDa RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail. PMID- 8611143 TI - The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes. AB - We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31 8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC alpha and PKC-beta in plasma membranes of rat adipocytes. PMID- 8611146 TI - Chemical inhibition of myristoylation of the G-protein Gi1 alpha by 2 hydroxymyristate does not interfere with its palmitoylation or membrane association. Evidence that palmitoylation, but not myristoylation, regulates membrane attachment. AB - The alpha-subunit of the G-protein Gi1 alpha is normally dually acylated at its N terminus with the saturated fatty acids myristate and palmitate. Inhibition of protein myristoylation by treatment with 2-hydroxymyristate prevented neither the incorporation of [3H]palmitate nor the membrane association of this protein when expressed in the COS cells. Construction of a mutant of Gi1 alpha in which serine 6 was replaced by aspartic acid prevented both myristoylation and palmitoylation, and the expressed protein was found primarily in the cytoplasmic fraction. These data indicate the myristoylation is not an absolute requirement for palmitoylation of Gi1 alpha and that palmitoylation, but not myristoylation, plays a key role in membrane association of this G-protein alpha-subunit. PMID- 8611145 TI - Peptidoglycan structure of Enterococcus faecium expressing vancomycin resistance of the VanB type. AB - Resistance to glycopeptide antibiotics in enterococci is due to the synthesis of UDP-MurNAc-tetrapeptide-D-lactate (where Mur is muramic acid) replacing the normal UDP-MurNAc-pentapeptide precursor. The peptidoglycan structures of an inducible VanB-type glycopeptide-resistant Enterococcus faecium, D366, and its constitutively resistant derivative, MT9, were determined. Using HPLC, 17 muropeptides were identified and were present regardless of whether resistance was expressed or not. The structures of 15 muropeptides were determined using MS and amino acid analysis. The cross-bridge between D-alanine and L-lysine consisted of one asparagine. No monomer pentapeptide or tetrapeptide-D-lactate could be identified. These results obtained with D366 (non-induced) and MT9 indicate that, in the absence of vancomycin, the cell wall synthetic machinery of E. faecium can process the lactate-containing precursor as efficiently as the normal pentapeptide. In contrast, the presence of subinhibitory inducing concentrations of vancomycin interfered with the synthesis of oligomers. PMID- 8611147 TI - Influence of experimental errors on the determination of flux control coefficients from transient metabolite concentrations. AB - The influence of experimental errors on the determination of flux control coefficients from transient metabolite concentrations with the method proposed by Delgado and Liao [(1992) Biochem. J. 282, 919-927] has been investigated by using Monte Carlo simulations. The method requires least-squares fitting of the transient metabolite concentrations. Three different fitting methods have been evaluated. Simulated metabolite concentrations of a fictive metabolic pathway were scattered randomly, emulating experimental errors, before performing the fits. This was repeated a large number of times; the mean values and standard deviations of the resulting control coefficients are reported. The results show that the proposed method for determining control coefficients is too sensitive to experimental errors to be practicable, with theoretically justified fitting methods. This is in particular due to the high degree of correlation between the concentrations. An alternative ad hoc fitting method produced biased mean values of the estimates of the control coefficients, but with remarkably low standard deviations. PMID- 8611144 TI - The role of cellular hydration in the regulation of cell function. PMID- 8611148 TI - Phospholipid and cation activation of chimaeric choline/ethanolamine phosphotransferases. AB - The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ethanolaminephosphotransferase, respectively. Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids. A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/Ept1p enzymes to gain insight into their mechanism(s) of activation. Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, and Mn2+ and Co2+. This allowed the use of chimaeric enzymes to determine the mechanism of cation activation. Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves. Cpt1p and Ept1p also displayed distinct phospholipid activation profiles. Phospholipid activation required a phosphate and/or glycero phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle. Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation. Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S. cerevisiae. Hence CPT displays product activation. Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition. PMID- 8611149 TI - Identification of a transcription factor that binds to the S box of the I-A beta gene of the major histocompatibility complex. AB - Class II genes of the MHC show a striking homology upstream of the transcription start site that is composed of three conserved sequences (S, X and Y boxes, each separated by 15-20 bp). The presence of the S-box sequence in the mouse MHC class II gene I-A Beta was examined for its influence on the expression of this gene. Deletion or mutation of the S box decreased the induction of chloramphenicol acetyltransferase (CAT) activity in B lymphocytes by 32%. In macrophages, deletion or mutation of the S box abolished interferon-gamma (IFN-gamma) inducibility of CAT activity. Using a gel-retardation assay, we have identified a nuclear factor whose binding site overlaps the 7-mer conserved sequence of the S box. This factor is present in lymphocytes, macrophages, mastocytes and fibroblasts. Surprisingly, binding of this nuclear factor to DNA was induced by IFN-gamma in bone-marrow-derived macrophages, but not in macrophage-like cell lines. The binding site for this factor was defined by DNase I footprinting and partially purified by using an affinity column containing double-stranded oligonucleotides containing a sequence of the S box. A prominent protein of 43 kDa was found that bound specifically to the S-box sequence. PMID- 8611150 TI - Expression of laminin gamma 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter. AB - Laminin gamma 1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin gamma 1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5' flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that A M(r) 60,000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures. PMID- 8611151 TI - Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus. AB - Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed. PMID- 8611152 TI - Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis. AB - A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with MRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 degrees C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus. PMID- 8611153 TI - Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa. AB - A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells. The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex. The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex. No complex can be reformed in the absence of CSI or CSII. Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared. More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase. The results support the idea of a 'metabolon' in this organism, with the composition of the CS component varying during the growth cycle. PMID- 8611154 TI - Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions. AB - The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells. PMID- 8611155 TI - Low-density lipoprotein is the major carrier of lipid hydroperoxides in plasma. Relevance to determination of total plasma lipid hydroperoxide concentrations. AB - High-density lipoprotein (HDL) has been proposed as the principal carrier of hydroperoxides in plasma, based upon data gathered with an HPLC-chemiluminescence technique. To test this hypothesis we have measured total lipid hydroperoxides in native plasma using the ferrous oxidation in Xylenol Orange (FOX) assay and then fractionated plasma into very-low-density lipoprotein (LDL) and HDL fractions. Hydroperoxides were found to accumulate principally (more than 65%) in LDL, as judged by hydroperoxide content per amount of protein or cholesterol, or expressed as a proportion of total hydroperoxide in plasma. Plasma was also incubated at 37 degrees C in the presence and absence of 2,2'-azo-bis-(2 amidinopropane) hydrochloride (AAPH), an azo-initiator of lipid peroxidation. The majority of hydroperoxides generated in plasma were recovered in the LDL fraction. Furthermore, when isolated lipoproteins were subject to oxidation initiated by AAPH, very-low-density lipoprotein and LDL showed the greatest propensity for hydroperoxide accumulation, whereas HDL seemed relatively resistant. Estimates for plasma and LDL peroxidation based upon techniques which measure total lipid hydroperoxides suggest that levels of hydroperoxides in plasma and LDL are far higher than that those estimates generated by ostensibly more selective techniques. Higher levels of hydroperoxides in LDL than those reported by HPLC-chemiluminescence also seem in greater accordance with other available data concerning LDL oxidation. PMID- 8611156 TI - Early proteolytic cleavage with loss of a C-terminal fragment underlies altered processing of the beta-galactosidase precursor in galactosialidosis. AB - Processing of human beta-galactosidase (beta-GAL) was studied in permanently transfected Chinese hamster ovary (CHO) cells and compared with that in normal cells and in cells from subjects with GM1-gangliosidosis, galactosialidosis and I cell disease. Biosynthesis of beta-GAL in CHO cells results in the synthesis of an 88 kDa glycosylated and phosphorylated monomer precursor which is enzymically active and is secreted into the medium. Post-translational processing begins at the C-terminal end of the protein and gives rise to structurally related 67 and 64 kDa mature forms. These are subsequently degraded to give several inactive products of which a 50 kDa and a 18 kDa species are prominent. In normal fibroblasts only the 84 kDa precursor is readily detected inside cells, while the 88 kDa precursor is the only form secreted from cells in the presence of ammonium chloride. Processing of the precursor in normal fibroblasts results in the appearance of both the 67 and 64 kDa mature forms, which are also degraded to give 50 and 18 kDa products, as in transfected CHO cells. As affected controls, GM1-gangliosidosis cells showed a general loss of all forms of the enzyme, while in I-cell fibroblasts only the 84 kDa precursor and an 18 kDa degradation form were prominent. In galactosialidosis fibroblasts, taken from two different subjects, processing of beta-GAL was characterized by the respective appearance of intermediate 80 and 72 kDa enzymically inactive polypeptides, at levels lower than the normal amounts of the 67 and 64 kDa mature forms and higher than the normal amounts of degradation products, one of which is of 45 kDa and arises by endoproteolytic cleavage of the 80 kDa polypeptide. Incubation for up to 72 h in medium containing leupeptin, a potent inhibitor of thiol-dependent proteases, resulted in a significantly increased level of beta-GAL activity to near normal levels in fibroblasts from one galactosialidosis subject. Concordant with this, the abundance of the 84 kDa precursor was increased and the levels of the 80 kDa, 45 kDa and 18 kDa digestion products were diminished. However, in fibroblasts from the second galactosialidosis subject, the amount of the abnormal 72 kDa polypeptide was not influenced by leupeptin treatment. Leupeptin treatment did not increase enzymic activity levels in normal, GM1-gangliosidosis or I-cell disease fibroblasts, despite the fact that the production of the 50 kDa and 18 kDa degradation products was blocked in the presence of leupeptin. We concluded that in galactosialidosis the leupeptin-inhibitable proteolytic cleavage of a small fragment causes a conformational change of the precursor that precludes its further normal processing and results in its enzymic deficiency. This early abnormal trimming of beta-GAL is ascribable to a deficiency in the functional protective protein, the function of which is absolutely essential to render beta GAL cryptic from at least two distinct and separate proteolytic attacks that together remove at least 12 kDa from the C-terminal end of the enzyme. PMID- 8611157 TI - Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts. AB - In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts results from alternative RNA splicing (designated ET-AR delta 4 and ET-AR delta 3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-AR delta 4 and ET-AR delta 3,4 as well as ET AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression. PMID- 8611158 TI - Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides. AB - Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine(biotin-x-cadaveri ne; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 muM CA2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 muM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting. PMID- 8611159 TI - Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin. AB - Recent studies have indicated that regulatory mechanisms underlying the oxygen dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific. PMID- 8611160 TI - Ca2+ entry modulates oscillation frequency by triggering Ca2+ release. AB - As in many cells, the frequency of agonist-induced cytosolic Ca2+ concentration ([Ca2+]1) oscillations in exocrine avian nasal gland cells is dependent on the rate of Ca2+ entry. Experiments reveal that the initiation of each oscillatory spike is independent of the relative fullness of the stores and, furthermore, the oscillating pool is normally fully refilled by the end of each [Ca2+]1 spike. Therefore, contrary to current models, the interspike interval (which essentially sets the frequency) does not reflect the time taken to recharge the oscillating stores. Instead, the data show that it is the previously demonstrated role that Ca2+ entry plays in triggering the repetitive release of Ca2+ from the oscillating stores, rather than the recharging of those stores, that provides the basis for the observed effects of Ca2+ entry rate on oscillation frequency. PMID- 8611161 TI - Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl CoA hydrolase). AB - A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses omicron nitrophenyl acetate and alpha-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688-29696; Yan, Yang, and Parkinson (1995) Arch. Biochem. Biophys. 317, 222-234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE. PMID- 8611162 TI - Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A: immunoreactivity analyses. AB - By immunoreactivity analysis using monoclonal antibodies, we showed that the C terminal domain [R415-631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ("tail" portion of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1-417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 through the same binding sites as does proMMP-2, and also that the high order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of pro-MMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase-TIMP-2 complex or the gelatinase B(MMP-9)-TIMP-2 complex was able to form a ternary complex with proMMP 2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9--TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9. PMID- 8611163 TI - Surfactant-associated protein A is important for maintaining surfactant large aggregate forms during surface-area cycling. AB - Alveolar surfactant can be separated into two major subfractions, the large surfactant aggregates (LAs) and the small surfactant aggregates (SAs). The surface-active LAs are the metabolic precursors of the inactive SAs. This conversion of LAs into SAs can be studied in vitro using a technique called surface-area cycling. We have utilized this technique to examine the effect of trypsin on aggregate conversion. Our results show that trypsin increases the conversion of LAs into SAs in a concentration- and time-dependent manner. Immunoblot analysis revealed that surfactant-associated Protein A (SP-A) was the main target of trypsin. To examine further the role of SP-A in aggregate conversion, we tested the effect of Ca2+ and mannan on this process. The absence of Ca2+ (l mM EDTA) and the presence of mannan both increased the formation of SAs. Electron microscopy revealed that highly organized multilamellar and tubular myelin structures were present in samples that converted slowly to SAs. We concluded that SP-A is important for maintaining LA forms during surface-area cycling by stabilizing tubular myelin and multilamellar structures. PMID- 8611164 TI - Mechanism of indole-3-acetic acid oxidation by plant peroxidases: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases. AB - Indole-3-acetic acid (IAA) is a powerful plant growth regulator. The oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA. Horseradish peroxidase isoenzyme C and an anionic tobacco peroxidase isolated from transgenic Nicotiana sylvestris have been used in experiments in vitro designed to determine the mechanism of IAA oxidation. In particular, the initial reduction of ferric to ferrous enzyme, a key step in previously proposed mechanisms, has been investigated by rapid-scan stopped-flow spectrophotometry under strictly anaerobic conditions and at defined oxygen concentrations. The data provide the first evidence for a ternary complex comprising peroxidase, IAA and oxygen that is kinetically competent both at the initiation stage and during the catalytic cycle of IAA oxidation. A general scheme describing the oxidative cycles of both anionic and cationic peroxidases is proposed that includes native ferric enzyme and compound II as kinetically competent intermediates. For anionic peroxidases, addition of hydrogen peroxide switches on the oxidative cycle thereby promoting IAA oxidation. 2-Methyl-IAA is not a substrate of the oxidase reaction, suggesting a specific interaction between plant peroxidases and IAA. PMID- 8611165 TI - Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+. AB - Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from 1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0 -C14:0 saturated fatty acids as for arachidonate. At 35 microM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are constant with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu. PMID- 8611166 TI - Kinetics and thermodynamics of the binding of riboflavin, riboflavin 5'-phosphate and riboflavin 3',5'-bisphosphate by apoflavodoxins. AB - The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with riboflavin 5'-phosphate (FMN), riboflavin 3',5'-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5-8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes with FMN and riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized/semiquinone and semiquinone/hydroquinone couples of the riboflavin and FMN complexes were used with K delta values for the complexes with the oxidized flavins to calculate values for K delta for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the redox potential of the semiquinone/hydroquinone couple. PMID- 8611167 TI - Phosphorylation of c-Jun stimulated in primary cultured rat liver parenchymal cells by a coplanar polychlorinated biphenyl. AB - Phosphorylation of c-Jun was stimulated in primary cultured rat liver parenchymal cells by treatment with a coplanar polychlorinated biphenyl congener, 3,3'4,4'5 pentachlorobiphenyl (PenCB), as well as by epidermal growth factor, but was not stimulated by the non-coplanar form. However, the amount of c-Jun mRNA did not increase with PenCB treatment. PenCB may activate a signal-transducing pathway consisting of protein kinases. PMID- 8611168 TI - Inhibition of endothelin- and phorbol ester-stimulated tyrosine kinase activity by corticotrophin in the rat adrenal zona glomerulosa. AB - 1. The experiments described in this study were carried out to investigate the role of tyrosine kinase in the acute adrenal response to peptide hormone stimulation, and to determine whether the activity of this kinase may be subject to regulation by other intracellular signalling mechanisms in the adrenal zona glomerulosa. 2. Previous studies from this laboratory have shown that angiotensin II stimulates tyrosine kinase activity in the rat adrenal cortex. This study has shown, for the first time, that endothelin-1 also stimulates tyrosine kinase activity in this tissue. 3. Using the specific inhibitor of protein kinase C (PKC) activity, Ro 31-8220, we have shown that stimulation of tyrosine kinase activity, in response to endothelin-1, angiotensin II or the phorbol ester phorbol 12-myristate 13-acetate, is at least partly dependent on increased PKC activity. 4. The data presented also provide further evidence of cross-talk between signalling systems in the adrenal cortex. Corticotrophin and its intracellular second messenger, cyclic AMP, significantly attenuate the increment in tyrosine kinase activity seen in response to each of the effectors used. 5. The results of this study provide important new evidence for the regulation of protein kinases by other intracellular second messenger systems. PMID- 8611169 TI - Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression. AB - Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 micrograms/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83-136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63 65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats. PMID- 8611170 TI - Cellular activation of mesangial gelatinase A by cytochalasin D is accompanied by enhanced mRNA expression of both gelatinase A and its membrane-associated gelatinase A activator (MT-MMP). AB - Activation of gelatinase A represents a crucial regulatory step in the control of its enzymic activity. Rat kidney mesangial cells secrete predominantly latent gelatinase A that can be activated following treatment with cytochalasin D. In the present paper we provide new evidence, using reverse transcription-PCR, that treatment of rat mesangial cells with cytochalasin D enhances the steady-state level of mRNA of the membrane-type matrix metalloproteinase (MT-MMP), as well as of gelatinase A, with no change in the level of tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA. Since the TIMP-2 protein level is reduced in conditioned medium from cytochalasin D-treated cells, the results of the present study are consistent with a model in which the action of cytochalasin D is to cause extracellular gelatinase A and TIMP-2 to be sequestered at the plasma membrane, forming a heterotrimeric complex with MT-MMP. In this manner, TIMP-2 may assume a bifunctional role causing: (i) inhibition of gelatinase A in the extracellular compartment; and (ii) guiding gelatinase A to activation through a membrane association with MT-MMP. PMID- 8611171 TI - Determination of the three-dimensional structure of hordothionin-alpha by nuclear magnetic resonance. AB - The high-resolution three-dimensional solution structure of the plant toxin hordothionin-alpha obtained from korean barley was determined by using two dimensional NMR techniques combined with distance geometry and restrained molecular dynamics. Experimentally derived restraints including 292 interproton distances from nuclear Overhauser effect measurements, 16 hydrogen bond restraints together with four disulphide bridge restraints were used as input to calculations of distance geometry and restrained molecular dynamics. Also included in the calculations were 36 phi and 17 chi 1 torsion angles obtained from 33JHN alpha and 3J alpha beta coupling constants in double quantum filtered COSY and primitive exclusive COSY experiments, respectively. The overall protein fold is similar to crambin and purothionin-alpha 1. Two alpha-helices running in opposite directions are found on the basis of 3JHN alpha and 3J alpha beta and deuterium exchange rates for backbone NH protons, and encompass residues 7-18 and 22-28. These two helices are connected by a turn and form a 'helix-turn-helix' motif. A short stretch of an anti-parallel beta-sheet exists between residues 1-4 and 31-34. the two protein termini of hordothionin-alpha are 'well-anchored'; the N-terminus of the protein is immobilized by this short beta-sheet whereas the C terminus is 'pasted' to the carbonyl group of Cys-4 by a very stable hydrogen bond. The average root-mean-square differences for the backbone and heavy atoms after the restrained molecular dynamics calculations are 0.62 and 1.16 A respectively. These numbers represent a significant improvement over the corresponding values for the previous NMR structures of other thionins. The distance violation from the experimental interproton distances for the final structures is 0.14 for all atoms. PMID- 8611172 TI - Forms of lipoprotein lipase in rat tissues: in adipose tissue the proportion of inactive lipase increases on fasting. AB - Previous studies have shown that the ratio of lipoprotein lipase (LPL) catalytic activity to LPL mass in tissues differs in different conditions, but it is not clear whether this occurs by a change in the catalytic efficiency of the LPL molecules, or because of a shift in the relation between active and inactive forms of the enzyme. To explore this, we have measured LPL activity and mass in detergent extracts of rat tissues. LPL specific activity was high and similar in heart, skeletal muscle, lung and brain. The liver had significantly lower specific activity, which is in accord with previous findings that the liver takes up and catabolizes LPL. The specific activity was also low in adipose tissue from fasted rats. When tissue extracts were applied to columns of heparin-agarose and eluted by a gradient of NaCl, a peak of active LPL was eluted at 1.0 M NaCl, but there was also a peak of inactive LPL protein, which was eluted at 0.6 M NaCl. In adipose tissue, LPL activity decreased by 70-80% during an overnight fast, whereas LPL mass decreased by only 20-40%. The mass ratio between inactive and active LPL, as separated by heparin-agarose chromatography, increased from 0.5 to over 2 during the fast. In hearts there was no significant difference between fed and fasted rats in total LPL activity, LPL mass or in the distribution between inactive and active forms. The results indicate that the relation between inactive (probably monomeric) and active (dimeric) forms of LPL is a target for post-translational regulation in adipose tissue. PMID- 8611174 TI - Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells. AB - The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2(+)-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+. PMID- 8611173 TI - Integrin alpha 4 cysteines 278 and 717 modulate VLA-4 ligand binding and also contribute to alpha 4/180 formation. AB - Here we describe experiments in which we mutated four of the six integrin alpha 4 subunit cysteine residues that are not present in most other integrin alpha subunits that lack an I domain. In four different types of ligand binding assay we found that optimal integrin alpha 4 beta 1 and/or to CS1 peptide required the presence of both alpha 4 Cys 278 and Cys 717. In addition, optimal ligand binding required divalent cations and reduced cysteines, as evidenced by EDTA and N ethylmaleimide inhibition results. In a control experiment, an alpha 4 mutation that completely eliminated the alpha 4 80/70 proteolytic cleavage site had no effect on ligand binding. Notably, although Cys 278 an Cys 717 mutations markedly altered ligand binding, they had no adverse effect on cell adhesion. Thus, compared with cell adhesion, ligand binding is a distinct and apparently more stringent test of VLA-4 integrin-ligand interactions. In addition, we have established that the formation of the previously described alpha 4/180 [Parker, Pujades, Brenner and Hemler (1993) J. Biol. Chem. 268, 7028-2035] also requires Cys 278 and Cys 717, divalent cations and reduced cysteines. thus alpha 4/180 appears to be more functionally relevant than alpha 4/150. PMID- 8611175 TI - Hormonal regulation of concentrative nucleoside transport in liver parenchymal cells. AB - Na(+)-dependent uridine uptake is stimulated in isolated rat liver parenchymal cells by glucagon. This effect is transient, reaches maximum levels of stimulation 10 min after hormone addition, and is dose-dependent. Glucagon action can be mimicked by agents that are able to hyperpolarize the plasma membrane (e.g. monensin) and by dibutyryl cyclic AMP. The effects triggered by glucagon, monensin and dibutyryl cyclic AMP are not additive, suggesting a common mechanism of action. 8-(4-Chloro-phenylthio)adenosine 3':5'-cyclic monophosphate (PCT), a cyclic AMP analogue but also a nucleoside analogue, markedly stimulates Na(+) dependent uridine uptake in an additive manner to that triggered by monensin, similarly to the effect described for nitrobenzylthioinosine. Considering the roles reported for nucleosides in liver metabolism, the use of PCT as a cyclic AMP analogue should be precluded. Insulin is also about to up-regulate Na(+) dependent uridine uptake by a mechanism which involves a stable induction of this transport activity at the plasma-membrane level. This is consistent with a mechanism involving synthesis and insertion of more carriers into the plasma membrane. It is concluded that the recently characterized hepatic concentrative nucleoside transporter is under short-term hormonal regulation by glucagon, through mechanisms which involve membrane hyperpolarization, and under long-term control by insulin. This is the first report showing hormonal modulation of the hepatic concentrative nucleoside transporter. PMID- 8611176 TI - Effect of channelling on the concentration of bulk-phase intermediates as cytosolic proteins become more concentrated. AB - This paper shows that metabolic channelling can provide a mechanism for decreasing the concentration of metabolites in the cytoplasm when cytosolic proteins become more concentrated. A dynamic complex catalysing the direct transfer of an intermediate is compared with the analogous pathway lacking a channel (an "ideal" pathway). In an ideal pathway a proportional increase in protein content does not result in a change in the steady-state concentration of the bulk-phase intermediate, whereas in a channelling pathway the bulk-phase intermediate either decreases or increases depending on the elemental rate constants within the enzyme mechanisms. When the concentration of the enzymes are equal, the pool size decreases with increasing protein concentration if the elemental step depleting the bulk-phase intermediate exerts more control on its concentration than the step supplying the intermediate. Results are illustrated numerically, and a simplified dynamic channel is analysed in which the concentration of the enzyme-enzyme forms. For such a "hit-and-run" channel it is shown that, when the product-releasing step of the enzyme located upstream is close to equilibrium, the pool size decreases as the concentrations of the enzymes increase in proportion, regardless of the rate, equilibrium constants and concentration ratios of the two sequential enzymes. PMID- 8611177 TI - Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis. AB - High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40 50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin. PMID- 8611178 TI - Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage. AB - The content of the C-terminal region of aggrecan was investigated in samples of articular cartilage from individuals ranging in age from newborn to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by proteolytic cleavage, but the age related changes in its abundance in human cartilage have not been described previously. The analysis was performed by immunosorbant assay using an antiserum (JD5) against recombinant amino acid residues of human aggrecan, on crude extracts of cartilage without further purification of aggrecan. The results showed that the content of the C-terminal region decreased with age relative to the G1 domain content (correlation coefficient = 0.463). This represented a 92% fall in the content of this region of the molecule from newborn to 65 years of age. furthermore, when the G1 content of the cartilage extracts was corrected to only include the G1 attached to aggrecan and to exclude the G1 fragments which accumulate as a by-product of normal aggrecan turnover (free G1), the age-related decrease in the C-terminal region remained very pronounced. Analysis by composite agarose/PAGE showed that the number of subpopulations of aggrecan resolved increased from one in newborn to three in adult cartilage. All of these reacted with an antiserum to the human G1 domain, but only the slowest migrating species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (200 kDa) proteins reactive with JD5, but no reactive fragments of lower electrophoretic mobility were detected. In contrast, when probed with the antiserum to the human G1 domain, the immunoblots showed protein species corresponding to the free G1 and G1-G2 fragments, which were present at high concentrations in adult cartilage. The results suggest that the loss of the C-terminal region is not directly part of the process of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slower. Young cartilage with the fastest turnover contains least molecules lacking the C terminal region, whereas in old tissue with slow turnover few molecules retain this region. An increase in the cleavage of this region with age may also contribute to this change. The content of the C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan. PMID- 8611179 TI - Comparative analysis of the QUTR transcription repressor protein and the three C terminal domains of the pentafunctional AROM enzyme. AB - The AROM protein is a pentadomain protein catalysing steps two to six in the prechorismate section of the shikimate pathway in microbial eukaryotes. On the basis of amino acid sequence alignments and the properties of mutants unable to utilize quinic acid as a carbon source, the AROM protein has been proposed to be homologous throughout its length with the proteins regulating transcription of the genes necessary for quinate catabolism. The QUTR transcription repressor protein has been proposed to be homologous with the three C-terminal domains of the AROM protein and one-fifth of the penultimate N-terminal domain. We report here the results of experiments designed to overproduce the QUTR and AROM proteins and their constituent domains in Escherichia coli, the purpose being to facilitate domain purification and (in the case of AROM), complementation of E. coli aro- mutations in order to probe the degree to which individual domains are stable and functional. The 3-dehydroquinate dehydratase domain of the AROM protein and the 3-dehydroquinate dehydratase-like domain of the QUTR spectroscopy and fluorescence emission spectroscopy. The CD spectra were found to be virtually superimposable. The fluorescence emission spectra of both domains had the signal from the tryptophan residues almost completely quenched, giving a tyrosine dominated spectrum for both the AROM- and QUTR-derived domains. This unexpected observation was demonstrated to be due to a highly unusual environment provided by the tertiary structure, as addition of the denaturant guanidine hydrochloride gave a typical tryptophan-dominated spectrum for both domains. The spectroscopy experiments had the potential to refute the biologically-based proposal for a common origin for the AROM and QUTR proteins; however, the combined biophysical data are consistent with the hypothesis. We have previously reported that the AROM dehydroquinate synthase and 3-dehydroquinate dehydratase are stable and functional as individual domains, but that the 5-enol-pyruvylshikimate-3 phosphate synthase is only active as part of the complete AROM protein or as a bi domain fragment with dehydroquinate synthase. Here we report that the aromA gene (encoding the AROM protein) of Aspergillus nidulans contains a 53 nt intron in the extreme C-terminus of the shikimate dehydrogenase domain. This finding accounts for the previously reported observation that the AROM protein was unable to complement aroE- (lacking shikimate dehydrogenase) mutations in E. coli. When the intron is removed the correctly translated AROM protein is able to complement the E. coli aroE- mutation. An AROM-derived shikimate dehydrogenase domain is, however, non-functional, but function is restored in a bi-domain protein with e dehydroquinate dehydratase. This interaction is not entirely specific, as substitution of the 3-dehydroquinate dehydratase domain with the glutathione S transferase protein partially restores enzyme activity. Similarly an AROM-derived shikimate kinase domain is non-functional, but is functional as part of the complete AROM protein, or as a bi-domain protein with 3-dehydroquinate dehydratase. PMID- 8611180 TI - Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors. AB - A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2'. The specificity constants (k(cat)/Km) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P' residues for cruzipain specificity. PMID- 8611182 TI - Structural studies on a lipoarabinogalactan of Crithidia fasciculata. AB - The monosaccharide D-arabinopyranose has only been found in glycoconjugates of the trypanasomatid parasites Leishmania major, Endotrypanum schaudinni and Crithidia fasciculata. The donor molecule for the relevant arabinosyltransferases is known to be GDP-alpha-D-Arap in L. major and C. fasciculata, and the latter organism is being used to study the biosynthesis of GDP-alpha-D-Arap. In this study, we describe the structure of the terminal product of arabinose metabolism in C. fasciculata, namely lipoarabinogalactan. This molecule was purified by hydrophobic-interaction chromatography and studied by a variety of techniques, including gas chromatography-mass spectrometry, electrospray mass spectrometry and chemical and enzymic digestions. These data show that lipoarabinogalactan contains a previously described D-arabino-D-galactan polysaccharide component covalently attached to a glycosylphosphatidylinositol type of membrane anchor that is similar to, but not identical with, that found in the lipophosphoglycans of the Leishmania. PMID- 8611181 TI - Intracellular pH governs the subcellular distribution of hexokinase in a glioma cell line. AB - Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pH1) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pH1 variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pH1 changes were verified microspectrofluorimetrically by using the pH1-sensitive probe SNARF-1-AM (seminaphtho-rhodafluor-1-acetoxymethyl ester). Imposition of an acidic pH1 for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pH1, was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pH1 and increased by basic pH1. Conversely, the glucose 6-phosphate level was increased by acidic pH1 and decreased by basic pH1. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pH1 was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pH1, and regulates hexokinase activity. PMID- 8611183 TI - Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells. AB - Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium, salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB --> methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the beta-hydroxylase enzyme system itself. PMID- 8611185 TI - Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells. AB - Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lacK (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24 +/- 0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21 +/- 0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09 +/- 0.31 and 1.53 +/- 0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538 +/- 82 nmol/24 per mg of DNA; CHOm+: 616 +/- 88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80 +/- 28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080 +/- 320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by [1-(13)C]glycine dilution was lower in CHOm- (1200 +/- 200 nmol/24 h per mg of DNA) than CHOm+ (10200 +/- 1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800 +/- 1200 mu mol/24 h per mg of DNA) than CHOm+ (6600 +/- 1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion. PMID- 8611184 TI - Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes. AB - In this study the possible role of Na+ influx, arachidonate mediators and alpha subunit phosphorylation in the stimulatory response of hepatic Na+/K(+)-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4( +/- 1.3) x 10(-10) M. Analysis of the K(+)-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent kappa 0.5. Unlike monensin, glucagon stimulation of Na+/K(+)-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K(+)-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo oxygenase, lipoxygenase, cytochrome p-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb uptake, hepatocytes were metabolically radiolabelled with [32P]P(i), Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that comigrates with Na+/K(+)-ATPase alpha-subunit immunoreactivity in two-dimensional gel electrophoresis. The alpha-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K(+)-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]P(i), the immunoprecipitated alpha subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197 +/- 21% (n = 6). Similar results were observed with a rabbit anti-peptide serum ('anti-LEAVE' serum) prepared against an amino acid sequence in the alpha-subunit. The EC50 for glucagon-stimulated phosphorylation of the alpha-subunit (approximately 1 x 10(-10) M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K(+)-ATPase mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na(+)-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the alpha-subunit. PMID- 8611186 TI - Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique. AB - 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11 beta-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11 beta-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11 beta-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11 beta-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11 beta-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer M(r) approximately 40,000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11 beta-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity. PMID- 8611187 TI - Organochlorine compounds in relation to breast cancer, endometrial cancer, and endometriosis: an assessment of the biological and epidemiological evidence. AB - There is an increasing public and scientific concern that certain chlorinated compounds, recognized as environmental pollutants, may cause estrogen-related neoplastic disease in humans. The main hypothesis has been that certain organochlorines, through their estrogenic actions, might cause breast cancer. From experimental studies, both in vitro and in vivo, there is evidence that certain organochlorine compounds may cause estrogenic effects, whereas others may cause antiestrogenic effects. In limited studies, some of these compounds in high doses have also been shown to increase and reduce the frequency of estrogen related tumors in animals. The epidemiological findings regarding the association between organochlorines and breast cancer are inconclusive. However, the largest and best designed study has been interpreted as negative with respect to DDT and polychlorinated biphenyls (PCB) in relation to breast cancer. Associations between organochlorine exposure and endometrial cancer or endometriosis have even more limited empirical basis. The hypothesis that human exposure to environmental levels or organochlorines would favor an estrogenic overactivity leading to an increase in estrogen-dependent formation of mammary or endometrial tumors is not supported by the existing in vitro, animal and epidemiological evidence. It can, however, not be conclusively rejected on the basis of available data. PMID- 8611188 TI - The pathology of the endocrine pancreas in type 1 (insulin-dependent) diabetes mellitus. AB - Type 1 diabetes is an organ-specific autoimmune disease in which the insulin secreting B cell is destroyed. Both genetic factors (linked to class II MHC genes) and environmental agents (viruses, diet) appear to be involved in the aetiology. Study of autopsy pancreases of children who die at presentation of their disease has proved elucidating. In such pancreases islets before, during and after B cell destruction, are all visible. The earliest defined immunological event in the disease process appears to be expression of interferon-alpha by insulin-containing B cells. Secretion of this cytokine is associated with hyperexpression of class I MHC by all the endocrine cells within insulin containing islets. Another immunological phenomenon which is unique to type I diabetes is the presence of aberrant class II MHC molecule expression by B cells. This may induce autoimmunity by allowing antigen presentation of B cell specific antigens. If the onset of the disease process is marked by interferon-alpha expression by B cells then a search for the presence of a continuing viral infection in these cells may prove profitable, although no viruses have been found in them to date. PMID- 8611189 TI - Epidemiological observations on Streptococcus pyogenes serotype T1 in Uppsala county, Sweden 1989-1995. AB - During the period 1989-1995, the T-type distribution pattern of Streptococcus pyogenes pharyngeal isolates was compared with the annual incidence of culture verified S. pyogenes bacteremias in Uppsala county, Sweden. An exceptional increase in the number of S. pyogenes bacteremias was seen in 1994. During the study period, the annual number of blood culture isolates was shown to correlate with the proportion of S. pyogenes serotype T1 among pharyngeal isolates. PMID- 8611190 TI - Microbicidal mechanisms of liver macrophages in experimental visceral leishmaniasis. AB - To study the differential microbicidal potentials of liver macrophages, the oxygen-dependent and oxygen-independent pathways in Kupffer cells and immigrant macrophages of Leishmania donovani-infected BALB/c mice were investigated. Hydrogen peroxide assay was performed using horse radish peroxidase-dependent oxidation of phenol red to quantitate the reactive oxygen species produced. To examine the oxygen-independent pathway, the enzymes N-acetyl-beta-glucosaminidase (NAG) and beta-glucuronidase (beta G) were investigated after exposure of cells to lipopolysaccharide. Hydrogen peroxide release by Kupffer cells was significantly decreased only at 21 days postinfection, whereas hydrogen peroxide release by immigrant macrophages was significantly increased on all postinfection days with a maximum at 21 days postinfection. The pattern of release of NAG and beta G was similar in both cell populations with a peak at 21 days postinfection. The present study therefore suggests that Kupffer cells and immigrant macrophages adopt different pathways to cope with this infection. PMID- 8611191 TI - Induction of nitric oxide and nitric oxide synthase mRNA by silica and lipopolysaccharide in PMA-primed THP-1 cells. AB - Nitric oxide (NO), a nitrogen-free radical, plays an important role in mediating inflammatory reaction and cytotoxicity of tissue. To determine whether NO was involved in silica-induced pulmonary tissue damage, we studied the effects of silica on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression by THP-1 cells, a monocyte-like cell line with properties of the pulmonary alveolar macrophage. Experimental results showed that silica elicited a marked stimulation of nitric oxide production in a time-dependent manner by THP-1 cells in vitro following the priming of these cells with the phorbol ester PMA. Both nitric oxide synthase inhibitor N-monomethyl-L-arginine (NMMA) and xanthine oxidase inhibitor allopurinol can partially suppress silica-induced NO production in PMA-primed THP-1 cells. Northern blot analysis indicated that, after 2 h of silica exposure, PMA-primed THP-1 cells began to express iNOS mRNA, which reached peak expression at 8 h. Endotoxin treatment of these cells produced a similar effect. These results indicated that silica is a potent inducer of NO production in macrophages and its ability to induce tissue damage may partially be attributed to its ability to initiate excessive production of nitric oxide from macrophages. PMID- 8611193 TI - Atrophy of the aortic media in giant cell arteritis. AB - Aortic tissue from seven patients with giant cell arteritis (GCA) was investigated using light microscopy and immunocytochemistry. Four surgical cases and three autopsy cases were included. All the specimens displayed a severe reduction in the size and number of media smooth muscle cells immunopositive for alpha-smooth muscle actin (alpha-SMA). A subtotal loss of alpha-SMA-positive cells was seen in non-inflamed media tissue, continuing gradually towards multiple calcified acellular lesions totally devoid of alpha-SMA immunoreactivity. There was a slight to moderate granulomatous inflammatory reaction in the tissue surrounding part of the acellular lesions. Foreign body giant cell reaction and elastin degradation were found at the ends of the acellular calcified areas. The present findings indicate that the atrophy and the loss of alpha-SMA-positive cells in the aortic media in GCA is primary, and that the granulomatous reaction is secondary and directed against atrophic calcified media tissue. PMID- 8611192 TI - The association of vascular proliferation with HPV status and epithelial PCNA positivity in cervical intraepithelial lesions. AB - In this study we investigated the number of blood vessels and vascular proliferation in subepithelial areas of 80 cervical condylomas and cervical intraepithelial neoplasias (CIN). The number of blood vessels was determined by counting factor VIIIRAg-positive vascular channels in areas beneath the epithelial lesions. Vascular proliferation was evaluated by determining the number of proliferating cell nuclear antigen (PCNA)-positive endothelial cells in the subepithelial connective tissues. The results were compared with the expression of human papillomavirus (HPV) DNA subgroups (6/11 (low-risk) and 16/18/31/33/35 (high-risk) of the lesions, as determined by dot-blot and in situ hybridization, and with epithelial cell proliferation as determined by immunohistochemistry for PCNA. Also p53 immunohistochemistry of the lesions was performed. Even though CIN II-III lesions on average contained more factor VIIIRAg-positive blood vessels compared to condylomas and CIN I lesions, no significant association was found between their number and the degree of dysplasia. However, moderate or strong PCNA staining in vascular endothelial cells was seen significantly more often in CIN II-III lesions than in condylomas and CIN I lesions (p = 0.008): 34/80 (45%) cases contained detectable HPV DNA as determined by dot-blot or in situ hybridization. There was no correlation between the presence or absence of HPV DNA and the number of PCNA-positive endothelial cells. Nine cases showed p53-positive cell nuclei and in three cases there was more than 1% positive nuclei in the lesion. No association was found between the vascularity or the number of PCNA-positive endothelial cells and the p53 immunoreactivity. The increased proliferative activity of endothelial cells in CIN II-III lesions suggests that they are angiogenically more active than condylomas and CIN I lesions. This activity does not, however, depend on the HPV or p53 status. This is the first report in which endothelial cell PCNA positivity was used as a marker for vascular proliferation. PMID- 8611194 TI - Determination of gamma/delta and other T-lymphocyte subsets in bronchoalveolar lavage fluid and peripheral blood from patients with sarcoidosis and idiopathic fibrosis of the lung. AB - We measured five different lymphocyte subpopulations, including gamma/delta-T cells, in peripheral blood and bronchoalveolar lavage (BAL) fluid from 48 consecutive patients undergoing diagnostic BAL (patients with sarcoidosis (n = 19), patients with idiopathic pulmonary fibrosis (IPF; n = 11) and patients with other diseases of the lung). Quantitative analysis of CD3+, CD4+, CD8+, CD16/56+ cells and gamma/delta-T cells was done by flow cytometry. The proportion of CD3+ and CD4+ cells in the peripheral blood of patients with sarcoidosis and IPF was significantly diminished, while CD8+ lymphocytes and NK cells were significantly elevated compared to healthy controls. There was no significant difference for the gamma/delta-T cell subpopulation between patients with sarcoidosis, IPF and control group. The proportion of CD3+ and CD4+ cells in BAL fluid was significantly elevated in sarcoidosis compared to IPF, while CD8+ cells were significantly diminished. Natural killer cells, defined as CD16/56+ CD3- cells, showed comparable low numbers in sarcoidosis and IPF. For gamma/delta-T cells no significant difference was found between patients with sarcoidosis and IPF. PMID- 8611195 TI - Bacteriocins in Neisseria meningitidis. Screening of systemic patient strains and pharyngeal isolates from healthy carriers. AB - Systemic meningococcal isolates and meningococci from healthy pharyngeal carriers in Norway were screened for production of growth antagonistic substances. Seven (4.9%) of a total of 142 systemic strains and 3 (2.1%) of 140 carrier isolates spontaneously released diffusible growth antagonistic substances. Properties shown by these substances complied with the criteria used in the definition of a bacteriocin. A cluster of producers among systemic strains registered during the first half of 1975 in North Norway (13.5% of the isolates) was observed, coinciding with the peak in incidence of meningococcal disease of the Norwegian epidemic starting in that region. Among more recent isolates, producers occurred at approximately the same rate in systemic strains (2.5%) as in carrier isolates. The meningocin-producing isolates detected were either of serogroup A and generally sulfonamide-resistant, or serogroup B and sulfonamide-sensitive. The group A strains isolated from disease cases in North Norway during the first half of 1975 were mostly sulfonamide-resistant. Except for the producers, all these strains revealed distinctly higher sensitivity to meningocin than did serogroup B sulfonamide-resistant strains, which became predominant among meningococci causing disease in Norway from that time on. PMID- 8611196 TI - Interleukin-1 alpha, interleukin 6 and tumor necrosis factor alpha increase the synthesis and expression of the functional alternative and terminal complement pathways by human umbilical vein endothelial cells in vitro. AB - The proinflammatory cytokines interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) modulate the synthesis of complement factors B and C3 by endothelial cells (EC), and are considered to play an important role in the development of sepsis. By using agarose beads activating the alternative pathway of complement, we wanted to study the net effect of these cytokines on EC synthesis of the alternative and terminal pathways, measured by binding of anti-C3c and anti-TCC (terminal complement complex) antibodies to beads kept with the EC. Addition of IL-1 alpha and TNF alpha at concentrations of 50 and 100 U/ml resulted in a significant increase in binding of these antibodies to co-incubated beads, most pronounced for anti-C3c. IL-6 from 50-200 U/ml resulted in a stronger (two to fourfold) binding for both antibodies compared to experiments with IL-1 alpha and TNF. However, increased concentrations of IL-1 alpha (200 U/ml) and IL-6 (400 U/ml) resulted in a strong reduction in binding of anti-C3c and anti-TCC antibodies to the co-cultured beads. This study indicates that proinflammatory cytokines upregulate the synthesis by EC of the functional alternative and terminal pathways of complement. PMID- 8611198 TI - In vitro activity of six macrolides, clindamycin and tetracycline on Streptococcus pneumoniae with different penicillin susceptibilities. AB - A collection of 99 clinical isolates of Streptococcus pneumoniae, chosen due to their different susceptibilities to penicillin, were investigated with respect to their susceptibility to the macrolides azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, and to clindamycin and tetracycline by the agar dilution method. We found complete cross resistance among the macrolides. The pneumococci were either susceptible, MIC < or = 0.5 micrograms/ml, or resistant, MIC > or = 16 micrograms/ml, to the tested macrolides, giving a bimodal distribution. In addition, complete cross resistance was observed between clindamycin and macrolides. Pneumococci resistant to macrolides were also resistant to tetracycline, and 26% of the macrolide susceptible strains were tetracycline resistant. PMID- 8611197 TI - Distribution of liver haemosiderin iron in 187 patients with various types of hepatic diseases. AB - The purpose of this study was to evaluate the distribution of haemosiderin iron in various regions of the liver (central, intermediary and peripheral hepatocytes, Kupffer cells, portal macrophages and bile duct epithelial cells) in 174 patients with different hepatic diseases (alcoholic cirrhosis (n = 49), alcoholic steatosis (n = 60), non-alcoholic cirrhosis (n = 16), acute hepatitis (n = 20), clinically overt untreated hereditary haemochromatosis (n = 3), miscellaneous disorders (n = 26)), and in 13 subjects with a normal liver biopsy. Furthermore, the relationship between liver haemosiderin iron, biochemical iron status markers and biochemical liver tests was investigated. In haemochromatosis iron was consistently present in all examined regions of the liver, and in 43% of patients with alcoholic liver disease haemosiderin was present in at least one region of the liver lobule. In 65% of patients with acute hepatitis, haemosiderin was present in macrophages and Kupffer cells. In other hepatic diseases and in normal livers, haemosiderin was rarely seen. Regression analyses showed a correlation between iron status markers in most patients, except in those with high serum aspartate aminotransferase levels. In conclusion, haemosiderin iron is distributed in a typical pattern in haemochromatosis, alcoholic liver disease and acute hepatitis. Both histochemical liver iron and serum ferritin are of value as indirect markers of liver iron stores in patients with moderate hepatocellular damage. PMID- 8611199 TI - Paraganglioma of the cauda equina. A case report and review of the literature. AB - A 59-year-old man presented with clinical evidence of a primary tumor of the cauda equina region. It was well circumscribed and was completely removed by neurosurgery. Routine staining showed that it had structural similarities to an ependymoma, but immunohistochemistry with antisera to synaptophysin, NSE, chromogranin-A and PGP 9.5 proved it to be a neuroendocrine tumor, i.e. a paraganglioma. We propose the use of endocrine markers in cases with tumors of the cauda equina to differentiate a paraganglioma from an ependymoma. Paragangliomas appear to have a better clinical outcome than ependymomas. Recurrence after surgery for a paraganglioma in the cauda equina region, especially if it is encapsulated, is rarely encountered. PMID- 8611200 TI - Issue dedicated to the memory of Professor Salvatore Ugo D'Arca, 1916-1992. PMID- 8611201 TI - [Prof. Salvatore Ugo D'Arca in the recollections of his students and colleagues]. PMID- 8611202 TI - [The scientific publications of Prof. Salvatore Ugo D'Arca]. PMID- 8611203 TI - [The history of the Code for Public Hygiene of Agostino Bertani]. PMID- 8611204 TI - [The history of the Code for Public Hygiene of Agostino Bertani]. PMID- 8611205 TI - [The birth of experimental hygiene and the founding of the Istituto di Igiene of the University of Rome]. PMID- 8611206 TI - [Avicenna, a forerunner of hygiene and preventive medicine]. PMID- 8611208 TI - [The health district in primary health care and the community health officer]. PMID- 8611207 TI - [A reflection on the past to give an explanation to the present]. PMID- 8611209 TI - [Epidemiology in the health services]. PMID- 8611210 TI - [The health care problems of planning services in a sample of persons with AIDS in Naples]. PMID- 8611211 TI - [Quality nursing: where are we headed? The importance of the development of nursing standards for a quality assurance management system]. PMID- 8611212 TI - [The accessibility and the index of coverage of a pediatric consultation service. A comparison between 2 USSLs (local health screening units) in the Milan area]. PMID- 8611214 TI - [Home health care within direct social interventions for the protection of the elderly, the disabled and patients with chronic diseases]. PMID- 8611213 TI - [Health care and hospitalization for the elderly]. PMID- 8611215 TI - [Informatics in the hospital]. PMID- 8611216 TI - [The peripheral offices of naval and aviation health administration]. PMID- 8611217 TI - [The social and health problems involving migration]. PMID- 8611218 TI - [General mortality as a health indicator: the validity of the indicator and its use in community diagnosis]. PMID- 8611219 TI - [The evaluation of the PYLL (potential years of life lost) with relation to Lucca and Pisa provinces in the biennium 1989-1990)]. PMID- 8611220 TI - A century of infant mortality in Italy: the years 1870-1990. PMID- 8611221 TI - [The cardiovascular risk of essential arterial hypertension: general concepts]. PMID- 8611222 TI - [Essential arterial hypertension: risk factors, pathogenetic mechanisms, possibilities for control]. PMID- 8611223 TI - [The mortality trend for respiratory system tumors in 1979-1990 in Italy]. PMID- 8611224 TI - [Breast carcinoma: its descriptive epidemiology and risk factors]. PMID- 8611226 TI - [The Mediterranean and zoonoses]. PMID- 8611225 TI - Accumulation levels of organochlorine pesticides in human adipose tissue: a new sample survey in Abruzzo. PMID- 8611227 TI - [Control over the possibilities for the transmission of microbial agents from one patient to another via tonometers]. PMID- 8611228 TI - [The prevalence of anemia in women of reproductive age belonging to rural Somali communities]. PMID- 8611229 TI - [Mortality from hypokinetic disease and institutionalization in patients over 65 in Italy]. PMID- 8611230 TI - [The evaluation of the nasopharyngeal carriage of pathogenic staphylococci, enterotoxic or not, in food handlers in the province of Pisa for the purpose of issuing a health card]. PMID- 8611231 TI - [Epidemiological aspects of acute respiratory infections in childhood: hospital discharge data from Modena (USL no. 16) and the Emilia Romagna region]. PMID- 8611232 TI - [An epidemiological analysis of epidemic strains of Salmonella typhimurium: a comparison between gene amplification with "arbitrary" primers and rRNA-DNA hybridization]. PMID- 8611233 TI - [Epidemiological studies in 2 episodes of food poisoning by Salmonella enterica serovar enteritidis and the phenotypic and genotypic characterization of the epidemic strains]. PMID- 8611234 TI - [The evaluation of the currency of the knowledge on viral hepatitis in a group of primary physicians of Rome]. PMID- 8611235 TI - [Hepatitis B and C in institutionalized mental patients]. PMID- 8611236 TI - [The prevalence of anti-HCV antibodies in the sera from dialysis patients collected in 1980]. PMID- 8611237 TI - [A serological study of the spread of hepatitis C infection in 14-year-old residents of the Lazio region]. PMID- 8611239 TI - [Parasitic worm infestations of food origin: a serious environmentally related public health problem]. PMID- 8611238 TI - [The sexual transmission of the acquired immunodeficiency virus: the reasons for greater male-to-female than female-to-male transmission efficiency]. PMID- 8611240 TI - Epidemiological characterization of human and environmental Pseudomonas aeruginosa strains: use of plasmid pattern analysis in addition to other typing methods. PMID- 8611241 TI - [The relationship between Streptococcus mutans in the saliva and the indices of carious pathology]. PMID- 8611242 TI - [Poliomyelitis: vaccination is still important]. PMID- 8611243 TI - [The direct prophylaxis of hepatitis A]. PMID- 8611244 TI - Correlates of HIV infection knowledge in samples of Italian young population. PMID- 8611245 TI - [Use of the condom for the prevention of AIDS: a review of 10 studies from Europe and the United States]. PMID- 8611246 TI - [The level of information on AIDS among the university and secondary school students of Camerino]. PMID- 8611247 TI - [The policy of drug liberalization in the Netherlands]. PMID- 8611248 TI - [Health education and smoking: a controlled study of an intervention carried out among the junior high school students in a local health screening unit]. PMID- 8611249 TI - [The prevention of dental caries: a preliminary study for an intervention aimed at health education]. PMID- 8611250 TI - [The carioprotective effect of milk]. PMID- 8611251 TI - [The nutritional status of the pediatric population in Albania]. PMID- 8611252 TI - [The validity of a Mediterranean-type food model]. PMID- 8611253 TI - [The intake of dithiocarbamate fungicides with a total hypercaloric diet prepared in a large Roman hospital (1991-1992)]. PMID- 8611254 TI - [Atmospheric contamination by aromatic polycyclic hydrocarbons in an urban area]. PMID- 8611255 TI - [Atmospheric pollution: acid depositions]. PMID- 8611256 TI - [A simulated microclimate analysis of Fanger's indices]. PMID- 8611257 TI - [Indoor pollution: the microbiological findings in some work environments]. PMID- 8611259 TI - [The Fiuggi water: health and hygiene aspects and a legislative update]. PMID- 8611258 TI - [Anesthetic gas pollution in operating rooms: the health and hygiene and management problems]. PMID- 8611260 TI - [Pharmaco-toxicological and analytical chemical aspects of the new regulations on mineral waters]. PMID- 8611261 TI - [Bacterial regrowth in water-supply networks and the health and hygiene aspects]. PMID- 8611262 TI - [The feasibility of fluoridation of the drinking water in the Rome metropolitan area]. PMID- 8611263 TI - [Research on the microbiological and chemical quality of the water from the river Idice with special reference to metals, volatile organic halogens and pesticides]. PMID- 8611264 TI - [The search for nontuberculous mycobacteria in bottled mineral waters found in commerce]. PMID- 8611265 TI - [Evaluation of the quality of waters destined for human consumption]. PMID- 8611266 TI - [Volatile organic halogen compounds in the drinking water of the city of Rome]. PMID- 8611267 TI - [Aeromonas spp. as an indicator of the trophic state of bodies of water]. PMID- 8611268 TI - Scanning electron microscopy analysis of bacterial colonization in crustaceans. PMID- 8611269 TI - Poliovirus detection in environmental samples by cell cultures and hybridization test. PMID- 8611270 TI - Environmental impact of sewage sludge: possibility and limits for agricultural use. PMID- 8611272 TI - [Innovations and the outlook in industrial hygiene and safety]. PMID- 8611271 TI - [Comparison between the efficacy of oxygen and atmospheric air in sewage water purification installations]. PMID- 8611273 TI - A survey of the medical consequences of working with video display terminals (VDT). PMID- 8611274 TI - [The intelligent building, well-being and the computerization of libraries]. PMID- 8611275 TI - [Technological innovations and civil protection: the minimal security requirements in buildings]. PMID- 8611276 TI - [New regulations in the matter of the safety of gas installations for domestic use: what is new?]. PMID- 8611277 TI - Left ventricular diastolic function in young adults: the Coronary Artery Risk Development in Young Adults Study. AB - Doppler transmitral flow velocities have been used to assess left ventricular diastolic function. Associations of transmitral velocities with specific physiologic variables and cardiovascular risk factors have not been reported previously in a large population-based study of young adults. We performed Doppler analysis of left ventricular inflow in 3492 black and white men and women (aged 23 to 35 years) in the year-5 examination of the Coronary Artery Risk Development in Young Adults (CARDIA) Study. First third filling fraction, peak flow velocity in early diastole (PFVE), peak flow velocity in late diastole (PFVA), and the PFVA/PFVE ratio were measured. Women had higher PFVE and PFVA than had men (PFVE: 0.81 +/- 0.13 m/sec versus 0.76 +/- 0.13 m/sec; PFVA: 0.47 +/ 0.11 m/sec versus 0.43 +/- 0.10 m/sec; both p < 0.001). Gender-specific multiple regression analyses showed that age, heart rate, systolic blood pressure, left ventricular percent fractional shortening, and body weight were independently and positively related to PFVA (all p < 0.001) in men and women. Age, heart rate, and forced expiratory lung capacity in 1 second were inversely related to PFVE and first third filling fraction (both p < 0.01). Left ventricular percent fractional shortening was positively related to PFVE and first third filling fraction (p < 0.001). Age, heart rate, and body weight were positively correlated with the PFVA/PFVE ratio (all p < 0.001). Height had weak negative associations with PFVA and PFVE in women only. These results suggest that, in young adults, Doppler measures of left ventricular diastolic filling are related to age, sex, body weight, blood pressure, heart rate, left ventricular systolic function, and lung function. PMID- 8611278 TI - Comparison of M-mode and two-dimensional echocardiographic algorithms used to estimate left ventricular mass: the Coronary Artery Risk Development in Young Adults Study. AB - Left ventricular (LV) mass as measured from M-mode echocardiography has been shown to be an important predictor of subsequent cardiovascular morbidity and death. Investigators have debated the advantages of LV mass calculations derived from M-mode versus various two-dimensional (2D) echocardiographic algorithms. The purpose of this study was to compare measurements of LV mass made from M-mode and 2D echocardiographic formulas in 325 healthy young adults of the Coronary Artery Risk Development in Young Adults cohort. M-mode LV mass was calculated according to a necropsy-validated formula, whereas 2D LV mass was calculated according to two established algorithms (i.e., the biplane Simpson and truncated ellipsoid methods). LV mass derived from M-mode echocardiography was 162.7 +/- 52 gm (mean +/- SD). Mean (+/- SD) LV mass derived from 2D echocardiographic measurements were as follows: with the biplane Simpson method (four-chamber view), 164.2 +/- 42 gm; with the biplane Simpson method (two-chamber view), 159.8 +/- 44 gm; with the truncated ellipsoid method (four-chamber view), 139.8 +/- 37 gm; and with the truncated ellipsoid method (two-chamber view), 143.1 +/- 38 gm. Correlations between M-mode and 2D methods ranged from 0.75 to 0.81 (p < 0.0001 for each comparison), and correlations between 2D methods were all greater than 0.90. This study has demonstrated that measurements of LV mass calculated from M-mode and 2D formulas correlate well with each other. Nonetheless, LV mass calculated from the truncated ellipsoid formula averages approximately 20 gm less than that calculated from the 2D biplane Simpson or M-mode echocardiographic formulas. These systematic differences in calculated values for LV mass must be taken into account when choosing an LV mass algorithm for use in cross-sectional and serial studies. PMID- 8611279 TI - Echocardiographic reference values for aortic root size: the Framingham Heart Study. AB - The objectives of this study were to develop sex-, age-, and body size-specific nomograms and partition values for upper and lower limits of M-mode echocardiographic aortic root measurements derived from a large population-based cohort. The study sample consisted of 1433 male and 1816 female participants in the Framingham Heart Study and Framingham Offspring Study who were normotensive and free of clinically apparent heart disease at the baseline examination. Aortic root measurements were obtained by M-mode echocardiography by a leading-edge to leading-edge technique. The relations of age and measures of body size with aortic root dimensions were evaluated with sex-specific correlations and multiple stepwise linear regression analyses. Age was the most important determinant of aortic root size in both men and women in the multivariable regression models. Models with age and body surface area yielded R2 values of 0.214 in men and 0.222 in women. Models with age and height yielded lower R2 values of 0.136 in men and 0.181 in women. Thus aortic root dimensions vary widely with the age, sex, and body size of individuals. Sex-specific reference nomograms of aortic root dimensions in relation to age and body size (body surface area or height) are presented to facilitate the detection of abnormalities of aortic root size. PMID- 8611280 TI - Left atrial mechanical function in the healthy elderly: new insights from a combined assessment of changes in atrial volume and transmitral flow velocity. AB - To assess left atrial mechanical function in the elderly, 35 old (age > 70 years) and 18 sex-matched young (age < 50 years) healthy subjects were studied. Transmitral flow velocities were recorded with pulsed Doppler echocardiography. Left atrial volumes were measured echocardiographically at mitral valve opening (maximal) and closure (minimal) and at onset of atrial systole (P wave of the electrocardiogram) according to the biplane area-length method. Left atrial passive emptying was assessed with the passive emptying volume (maximal-volume at onset of atrial systole) and fraction (passive emptying volume/maximal). Left atrial active emptying was assessed with the active emptying volume (volume at onset of atrial systole-minimal) and fraction (active emptying volume/volume at onset of atrial systole) and with left atrial ejection force = 0.5.blood density.volume at onset of atrial systole.active emptying fraction.(A velocity)2/A integral. Left atrial volumes were greater in old compared with young subjects (maximal: 31 +/- 10 cm3/m2 vs 24 +/- 8 cm3/m2, p = 0.02; at onset of atrial systole: 23 +/- 8 cm3/m2 vs 15 +/- 5 cm3/m2, p = 0.0002; minimal: 13 +/ 5 cm3/m2 vs 9 +/- 4 cm3/m2, p = 0.001). Passive emptying volume and fraction were lower (7.8 +/- 1.7 cm3/m2 vs 9.2 +/- 3.2 cm3/m2 [p = 0.04] and 26.4% +/- 9.8% vs 37.9% +/- 11.2% [p = 0.003], respectively), whereas atrial ejection force and active emptying volume were greater in old compared with young subjects (6.8 +/- 3.3 kdynes/m2 vs 4.2 +/- 2.8 kdynes/m2 [p = 0.007] and 9.2 +/- 4.1 cm3/m2 vs 5.7 +/- 2.9 cm3/m2 [p = 0.002], respectively). The active emptying fraction was similar in the two groups (39.7% +/- 11% vs 38.4% +/- 13%; difference not significant). Thus advanced age is associated with depressed left atrial passive emptying function and increased left atrial volume. Left atrial dilation contributes to an increase in atrial ejection force and the amount of blood ejected during left atrial systole and may represent an important compensatory mechanism in this age population. PMID- 8611281 TI - Evaluation of a semiautomatic contour detection approach in sequences of short axis two-dimensional echocardiographic images. AB - Quantitative analysis of echocardiographic sequences has been limited by time consuming and strenuous manual tracing approaches. To circumvent these disadvantages, we have developed the EchoCardiographic Measurement System (ECHO CMS). ECHO-CMS employs the robust, model-based Minimum Cost Contour Tracking technique for semiautomatic detection of left ventricular (LV) endocardial contours in sequences of consecutive echocardiographic images. An evaluation study was carried out on 20 short-axis patient studies (10 transesophageal and 10 transthoracic studies), each consisting of 16 consecutive images covering approximately one cardiac cycle. The LV endocardial contours in the 320 images were analyzed both by manual tracing and semiautomatically. In addition, interobserver and intraobserver variabilities were determined for both techniques in two patients (32 images). Manual editing was required in only 57 (18%) of all 320 contours detected. Average processing time per patient for manual tracing was 25 minutes (of which 18 1/2 minutes was for drawing and corrections) and for semiautomatic tracing it was only 5 1/2 minutes (of which just 1 1/2 minutes was for corrections). Regression analysis showed excellent correspondence between manual and semiautomatic tracing: semiautomatic = 1.01 manual + 5.58%; r = 0.989; standard error of the estimate = 11.9% (n = 320 contours). Interobserver and intraobserver variabilities were smaller for semiautomatic than for manual tracing, although not in all cases statistically significant. In conclusion, semiautomatic LV short-axis endocardial contour detection by ECHO-CMS provides contours that are highly similar to those drawn by an expert; it is five to 10 times faster than manual tracing and reduces intraobserver and interobserver variabilities. This demonstrates that ECHO-CMS is a useful tool for clinical echocardiographic research studies. PMID- 8611282 TI - The clinical utility of automatic boundary detection for the determination of left ventricular volume: a comparison with conventional off-line echocardiographic quantification. AB - The aim of this study was to compare measurements of echocardiographic volume with an on-line automatic boundary detection imaging system with those of a conventional off-line method for routine clinical studies. Automatic boundary detection imaging shows promise as a rapid, on-line method for quantitating left ventricular volumes by echocardiography. However, there is little information about the role of automatic boundary detection for routine clinical studies. Ninety-seven patients with a variety of clinical diseases who were referred for clinical transthoracic echocardiographic evaluation were studied in apical four chamber and two-chamber imaging planes. End-diastolic volume, end-systolic volume, and ejection fraction obtained with automatic boundary detection images were compared with those of conventional off-line analysis. Segmental endocardial definition and border tracking were evaluated on all automatic boundary detection images. Left ventricular end-diastolic volumes obtained by automatic boundary detection correlated well but were systematically under-estimated compared with off-line analysis for the apical two-chamber (r = 0.83; underestimation = 42 +/- 33 ml; p < 0.05) and four-chamber views (r = 0.83; underestimation = 43 +/- 31 ml; p < 0.05). Left ventricular end-systolic volumes also correlated well but were underestimated by automatic boundary detection for the apical two-chamber (r = 0.83; underestimation = 14 +/- 26 ml; p < 0.05) and four-chamber views (r = 0.83; underestimation = 18 +/- 24 ml; p < 0.05). Ejection fraction was not predicted accurately for the entire study population (n = 97). However, for patients with complete endocardial definition (n = 32), automatic boundary detection accurately predicted ejection fraction with no systematic error compared with manually traced images for both the apical two-chamber (r = 0.86; p < 0.05) and four-chamber (r = 0.82; p < 0.05) views. Segmental analysis of endocardial tracking revealed significantly better tracking of the septal and lateral walls compared with other regions (p < 0.05). End-diastolic and end systolic volumes determined by automatic boundary detection correlate well but underestimate volume compared with conventional off-line analysis. However, ejection fraction compares favorably for the two methods when there is complete endocardial definition. PMID- 8611283 TI - The variation of integrated backscatter in human hearts in differing ultrasonic transthoracic views. AB - It has been shown previously that in normal subjects the interventricular septum imaged in the long-axis view (LAX) and the left ventricular posterior wall imaged in both the LAX and the short-axis view (SAX) exhibit cyclic variation of integrated backscatter (IB) throughout the cardiac cycle, with maximum values occurring at end diastole (ED) and minimum at end systole (ES). The ability to demonstrate this cyclic variation within these myocardial regions in only two ultrasonic views has limited the potential clinical utility of an IB imaging system. To determine whether clinically useful information on the variation of IB is available from different myocardial regions in different ultrasonic views, we measured ED to ES variation of IB from the parasternal and apical views in normal subjects with a radiofrequency acquisition technique. Two independent clinical observers analyzed ED to ES variation of IB from 14 normal volunteers (mean age 32 +/- 6 years; range 21 to 45 years) in reconstructed two-dimensional ultrasonic images obtained from the parasternal LAX and SAX and apical two-chamber (2C) and four-chamber (4C) views. ED to ES variation of IB was measured from manually traced regions of interest (ROI) within the myocardium. These ROIs were chosen interactively and were located within the midposterior wall and the midanteroseptum in LAX views; within the midposterior wall, midanteroseptum, midseptum, and midlateral wall in SAX views; within the midseptum and the midlateral wall in 4C views; and within the midinferior wall and the midanterior wall 2C views. In all analyzed ROIs within the parasternal and apical views, ED to ES variation of IB was found. We have shown that the maximum magnitude of IB was at ES within the midseptum and in 10 out of 14 volunteers in the midanteroseptum measured from SAX views, the midanterior wall from 2C views, and the midlateral wall from 4C views. The rest of the ROIs analyzed exhibited the maximum value of IB cyclic variation at ED. We have confirmed that the ED to ES variation of IB is present not only when measured from the two standard parasternal views but also from the two apical views in all analyzed myocardial walls, and the minimum of this cyclic variation was not always coincident with ES nor the maximum with ED. PMID- 8611284 TI - Intravenous Albunex during transesophageal echocardiography: quantitative assessment by videodensitometry and integrated backscatter analysis from unprocessed radiofrequency signals. AB - This study investigates the comparative sensitivity of video and radiofrequency imaging to detect changes of the myocardial acoustic properties after intravenous Albunex. Thirty-six patients received Albunex, 0.08 and 0.22 ml/kg intravenously, during transesophageal imaging of the ventricular short axis. Analysis of video images was performed in all patients and of radiofrequency data in 20 patients. Although myocardial videointensity remained unchanged, 57% of the myocardial backscatter plots demonstrated significant contrast enhancement. The study demonstrates that intravenous Albunex is capable of myocardial contrast enhancement and proves the diagnostic superiority of radiofrequency compared with video imaging. Ultrasonic radiofrequency imaging may provide a technical basis for future noninvasive assessment of myocardial perfusion. PMID- 8611285 TI - Safety and efficacy of computerized closed-loop delivery of arbutamine: a new pharmacologic myocardial stress modality for the assessment of coronary artery disease. The European Arbutamine Study Group. AB - This study evaluated the efficacy of computer-controlled closed-loop delivery of a new synthetic catecholamine, arbutamine, when given to induce myocardial ischemia detected by electrocardiography and echocardiography with a high (10 beats/min/min) and low (6 beats/min/min) rate of increase in heart rate (heart rate slope) in 70 patients with coronary artery disease. The electrocardiographic sensitivity for the detection of myocardial ischemia was 52% for the low slope and 51% for the high slope. The corresponding figures for echocardiographic sensitivity were 83% and 79% for the low and high slopes, respectively. There were no significant differences in changes from baseline to maximum in pharmacodynamic variables, although the mean times to reach maximum heart rate and systolic blood pressure were 1.4 minutes shorter (p = 0.001) and 3.7 minutes shorter (p < 0.05), respectively, for the high-slope regimen. The duration of the infusion was shorter (p < 0.001) for the high slope. In this study, closed-loop arbutamine administration was effective and safe in the detection of coronary artery disease for both heart rate slope regimens. PMID- 8611286 TI - Evaluation of the extent and timing of the coronary hyperemic response to dipyridamole: a study with transesophageal echocardiography and positron emission tomography with oxygen 15 water. AB - Coronary flow reserve may be measured with Doppler-derived coronary blood-flow velocity or scintigraphic assessment of myocardial perfusion. The purpose of this study was to compare coronary flow velocity and perfusion measures of flow reserve with respect to their extent and time course. Coronary flow velocity reserve in the proximal left anterior descending coronary artery measured by pulsed-wave Doppler at transesophageal echocardiography, with measures of perfusion reserve obtained in the corresponding territory, were measured by a standard O15 water technique at positron emission tomography. Eighteen male volunteers underwent both tests on different days in random order, with dipyridamole stress (0.56 mg/kg). After correction of resting flow and perfusion measurements to a standard cardiac workload (to compensate for heterogeneity in hemodynamics between the two studies), coronary flow reserve was calculated as the ratio between dipyridamoleand corrected resting flow. The uncorrected perfusion reserve measured by positron emission tomography was 3.7 +/- 1.2, compared with a corrected perfusion reserve of 2.3 +/- 0.7. This correlated with a corrected flow velocity reserve of 2.9 +/- 1.0 at transesophageal echocardiography (R = 0.92; p < 0.001). The mean difference between these results was 0.58 +/- 0.41; discrepant results occurred at higher flows, in the presence of discordant blood pressure responses to stress, and because of intersubject variations in the timing of the peak coronary flow response, which were detected by continuous monitoring at transesophageal echocardiography. Measurement of coronary flow reserve at transesophageal echocardiography correlates well with measurements at positron emission tomography, and discrepancies are minimized if measurements are taken at the same time after dipyridamole. PMID- 8611287 TI - Measurement of aortic flow velocity during transesophageal echocardiography in the transgastric five-chamber view. AB - Continuous-wave Doppler echocardiography of aortic flow velocity has a variety of clinical and research applications. Recently, continuous-wave Doppler echocardiography has been added to transesophageal echocardiographic systems. However, alignment of the Doppler beam with aortic flow is not possible with standard single and biplane views. A modified transesophageal echocardiographic view; the transgastric five-chamber (TG5C) view, allows for measurement of aortic flow velocity but its feasibility and accuracy in an unselected consecutive population have not yet been described. The feasibility of obtaining the TG5C view and measuring aortic flow velocity was assessed in 58 consecutive transesophageal echocardiographic investigations. The TG5C view was obtained in 97% and adequate Doppler flow velocity signals were obtained in 91% of patients. The accuracy of measurements was assessed in 24 patients in whom flow signals from both the TG5C and standard transthoracic views could be obtained. The correlation between TG5C and transthoracic views was excellent, with r values of 0.968 and 0.952 for peak aortic flow velocity and mean aortic flow velocity, respectively. Accurate aortic flow velocity measurements can be obtained in most patients during transesophageal echocardiography with the TG5C view. This view has great utility in a variety of situations in which adequate transthoracic imaging is not possible, especially the operating room and intensive care unit. PMID- 8611288 TI - The effect of technical factors on the quality of pulmonary venous flow from the transverse and longitudinal imaging planes with transesophageal echocardiography. AB - Right and left upper pulmonary venous flow is usually assessed with monoplane transesophageal echocardiography (TEE) in the transverse imaging plane. Pulmonary venous flow in the transverse imaging plane may be relatively difficult to record because of the larger angle between the pulmonary vein and the transducer beam. To compare the quality of echocardiographically derived Doppler flows of the right and left upper pulmonary veins between the longitudinal and transverse imaging planes with TEE, we performed pulsed-wave Doppler TEE of both upper pulmonary veins in transverse and longitudinal imaging planes in 36 patients with various diseases. We also recorded a quality index for each flow profile and the angle between the transducer beam and the pulmonary vein. The quality index of the left pulmonary venous flow assessed with the longitudinal and transverse imaging planes was similar in 35 (95%) of 36 patients, whereas the longitudinal imaging plane was superior to the transverse plane in one patient (3%). In contrast, the quality index of the right pulmonary venous flow assessed with the longitudinal and transverse imaging planes was similar in only 19 (53%) of 36 patients, whereas in 17 patients (47%) the longitudinal imaging plane was superior to the transverse imaging plane. The quality index had a significant effect on the Doppler flow recordings; suboptimal-quality flow recordings significantly underestimated the pulmonary venous diastolic flow integrals. The left atrium was larger in those patients with unobtainable flows than in those patients with exclusively obtainable flows (p < 0.001). The angle between the sample volume and the right pulmonary vein was larger in the transverse imaging plane than in the longitudinal plane (p < 0.001). In conclusion, the longitudinal imaging plane is generally superior to the transverse imaging plane for assessing right pulmonary venous flow and is recommended for performing a comprehensive assessment of pulmonary venous flow. The ability to obtain quality images and accurate assessment of flow may be related to the size of the left atrium and angle of the pulmonary vein. PMID- 8611289 TI - Evaluation of pulmonary vein stenosis by transesophageal echocardiography. AB - Pulmonary vein stenosis was diagnosed by transesophageal echocardiography in five patients who underwent the study for different clinical indications. Stenosis was encountered in the right upper pulmonary vein in two patients, the right lower pulmonary vein in two patients, and at the confluence of the left pulmonary veins in one patient. In only one patient was the diagnosis suspected on transthoracic echocardiography. Contralateral normal veins from the same patient served as the control. Vessel diameter and peak flow velocity were measured and compared. The diameter of the stenosed veins ranged from 0.3 to 0.8 cm (mean 0.4 +/- 0.09 cm [SEM]), whereas for normal veins the diameter was 0.9 to 1.2 cm (mean 1.0 +/- 0.05 cm [SEM]; p < 0.001). Peak flow velocity in the stenosed veins ranged from 1.1 to 1.6 m/sec (mean 1.4 +/- 0.1 m/sec [SEM]), whereas in normal veins peak flow velocity ranged from 0.4 to 0.7 m/sec (mean 0.6 +/- 0.04 m/sec [SEM]; p < 0.001). There was a strong negative correlation between vessel diameter and peak flow velocity (R = 0.89; p < 0.001). Peak flow velocity of 0.8 m/sec appears to provide the best separation between normal and stenosed pulmonary veins. We conclude that pulmonary vein stenosis is associated with increased flow velocity and turbulence and deformity of the flow signal. Transesophageal echocardiography is a powerful tool in the study of pulmonary vein stenosis. PMID- 8611290 TI - Validation of a new Doppler-echocardiographic method for quantifying mitral regurgitation. AB - The noninvasive determination of severity of mitral regurgitation (MR) is often of major clinical importance. We performed simultaneous Doppler echocardiography and left ventricular angiography in 24 patients with MR, comparing a new Doppler echocardiographic technique with angiographic criteria for severity of MR. Angiographic severity was measured on a 1+ to 4+ scale, determined by the degree of opacification of the left atrium during left ventricular systole. The echocardiographic examination consisted of color flow imaging and continuous-wave Doppler echocardiography across the mitral valve. The product of the mitral regurgitant color flow jet area and the mitral regurgitant time-velocity integral was obtained, yielding a Doppler-derived regurgitant volume across the mitral valve. Good correlations were demonstrated between the severity of angiographic MR and the Doppler-derived regurgitant volume (r = 0.831; p < 0.005) and between the angiographic severity of MR and the Doppler-determined mitral regurgitant jet diameter (r = 0.833; p < 0.005). We conclude that a new Doppler echocardiographic method for quantitating the severity of MR correlates well with qualitative angiographic grading. Further study is needed to compare this technique with other quantitative Doppler indexes of severity of MR. PMID- 8611291 TI - Estimation of volume flow rate by surface integration of velocity vectors from color Doppler images. AB - A new Doppler echocardiographically based method has been developed to quantify volume flow rate by surface integration of velocity vectors (SIVV). Electrocardiographic-gated color Doppler images acquired in two orthogonal planes were used to estimate volume flow rate through a bowl-shaped surface at a given time and distance from the probe. To provide in vitro validation, the method was tested in a hydraulic model representing a pulsatile flow system with a restrictive orifice. Accurate estimates of stroke volume (+/- 10%) were obtained in a window between 1.2 and 1.6 cm proximal to the orifice, just before the region of prestenotic acceleration. By use of the Bernoulli's equation, the estimated flows were used to generate pressure gradient waveforms across the orifice, which agreed well with the measured flows. To demonstrate in vivo applicability, the SIVV method was applied retrospectively to the determination of stroke volume and subaortic flow from the apical three-chamber and five chamber views in two patients. Stroke volume estimates along the left ventricular outflow tract showed a characteristic similar to that in the in vitro study and agreed well with those obtained by the Fick oxygen method. The region where accurate measurements can be obtained is affected by instrumental factors including Nyquist velocity limit, wall motion filter cutoff, and color flow sector angle. The SIVV principle should be useful for quantitative assessment of the severity of valvular abnormalities and noninvasive measurement of pulsatile volume flows in general. PMID- 8611293 TI - Detection of partial anomalous right pulmonary venous return with an intact atrial septum by transesophageal echocardiography. AB - Partial anomalous pulmonary venous drainage with intact atrial septum is a rare congenital anomaly. We report a case of a 63-year-old woman who recently had dyspnea on minimal exertion and was found to have pulmonary hypertension, right atrial enlargement, and right ventricular enlargement by two-dimensional transthoracic echocardiography. Transesophageal echocardiography demonstrated anomalous venous drainage of the right lung into the superior vena cava with an intact interatrial septum. This diagnosis was confirmed by angiography and the patient underwent successful repair. This case illustrates the importance of locating all four pulmonary veins in patients with pulmonary hypertension or suspected intracardiac shunt in addition to scanning the atrial septum. PMID- 8611292 TI - Three-dimensional flow images by reconstruction from two-dimensional vector velocity maps. AB - A method for constructing three-dimensional images of flow is described. The technique involves the acquisition of numerous closely spaced planes, each comprised of a map of the two-dimensional velocities measured in that plane. Each such vector velocity map is formed by tracking the motion of small regions of ultrasonic speckle between two ultrasonic acquisitions separated by a short time interval. In contrast to current Doppler velocity methods, this technique measures both the axial and lateral components of flow and is not subject to aliasing. The resulting series of two-dimensional vector velocity maps is then combined into a three-dimensional data set, which can be manipulated with appropriate software to yield quantitative three-dimensional displays of the flow within the interrogated volume. In this article we present such images obtained from measurements of in vitro laminar flow in a vessel, as well as a free jet phantom. The results allow comprehensive visualization of the three-dimensional flow characteristics, indicating promise for more complete and quantitative clinical assessment of blood flow. PMID- 8611294 TI - Intracardiac echocardiography-guided biopsy of intracardiac masses. AB - A 69-year-old man diagnosed with lung cancer had a transesophageal echocardiogram performed because of suspicion of intramyocardial tumor. The transesophageal echocardiogram confirmed the presence of both a right and left atrial mass. The lung cancer was believed to be potentially resectable if this mass did not represent tumor; therefore, biopsy of the intracardiac mass was requested. Intracardiac ultrasound was used to guide the biopsy procedure. Using intracardiac ultrasound guidance, a successful biopsy was performed that revealed the presence of tumor cells. PMID- 8611295 TI - Intracardiac extension of a lung tumor causing left ventricular inflow obstruction. AB - A patient with the diagnosis of small cell lung cancer had syncopal episodes. The tumor was found to invade the right upper pulmonary vein with extension into the left atrium. The mass was protruding across the mitral valve producing ball-valve blockade. In this report the echocardiographic signs of a primary lung tumor with intraatrial extension causing left ventricular inflow tract obstruction are described. PMID- 8611296 TI - Cardiac sarcoidosis masquerading as a metastatic tumor: the role of transthoracic and transesophageal echocardiography. AB - We present an unusual case of biopsy-proven myocardial sarcoidosis in which the transthoracic and transesophageal echocardiographic findings suggested metastatic tumor involvement of the myocardium and pericardium. The pathologic, clinical, and echocardiographic features of cardiac sarcoidosis are reviewed, with emphasis on the role of echocardiography. PMID- 8611297 TI - Transesophageal echocardiographic diagnosis of right atrial thrombi associated with the antiphospholipid syndrome. AB - Thromboembolic disorders are a hallmark of the antiphospholipid antibody syndrome. We describe a patient with IgM antiphospholipid antibodies associated with pulmonary emboli and in situ thrombosis within an otherwise normal right atrium. Echocardiography, particularly the transesophageal study, proved invaluable in providing a diagnosis and guiding our patient's evaluation and treatment. PMID- 8611299 TI - Failure of transesophageal echocardiography to visualize a large mitral prosthesis vegetation detected solely by transthoracic echocardiography. AB - Transesophageal echocardiography yields a highly accurate diagnosis in the detection of valvular vegetations. We describe a case in which omniplane transesophageal echocardiography failed to demonstrate a large vegetation protruding from a Starr-Edwards mitral prosthesis, toward the left ventricular outflow tract. The vegetation could be detected solely by transthoracic echocardiography. PMID- 8611298 TI - Systolic aortic regurgitation: a hemodynamic challenge for the clinician. AB - Aortic regurgitation is considered a diastolic phenomenon. We describe a case in which premature beats resulted in a left ventricular systolic pressure that was lower than the aortic pressure, and thus aortic regurgitation continued throughout the systole of the premature beat. This sequence of events was clearly demonstrated by Doppler echocardiography. PMID- 8611300 TI - Disseminated coccidioidomycosis with rapid progression to effusive-constrictive pericarditis. AB - This case reports coccidiomycosis presenting as pericarditis with tamponade rapidly progressing to effusive-constrictive pericarditis and death over 72 hours. Coccidiomycosis pericarditis is a rapidly progressing disease requiring early and complete pericardiectomy to avoid the hemodynamics of constriction. We illustrate the use of echocardiography in this case and demonstrate the histopathology. We review the literature and discuss therapy and management. Coccidiomycosis is often clinically unsuspected and unrecognized by the health care worker unfamiliar with the disease process. PMID- 8611301 TI - Cor triatriatum dexter in an adult diagnosed by transesophageal echocardiography: a case report. AB - Cor triatriatum dexter is a rare congenital heart malformation in which a persistent right sinus venosus valve divides the right atrium into two chambers. Before echocardiography, this anomaly has been rarely diagnosed before surgery or death. This is a case of cor triatriatum dexter in an adult with lifelong exertional cyanosis and dyspnea. A definitive diagnosis of cor triatriatum dexter with associated heart defects was best made by transesophageal echocardiography at 47 years of age. Subsequent surgical intervention confirmed all of the echocardiographic findings and successful correction of the defects was performed. PMID- 8611302 TI - Aneurysm of the membranous ventricular septum and accessory valvular tissue causing obstruction of the pulmonary outflow tract in corrected transposition. AB - Aneurysm of the membranous portion of the ventricular septum and accessory valvular tissue are two infrequent congenital lesions. This case illustrates a rare association of aneurysm of the membranous septum and accessory valvular tissue originating from the mitral valve, causing right-sided ventricular outflow tract obstruction in a patient with corrected transposition. Multiplane transesophageal echocardiography allowed for determination of the dual nature of the obstruction. PMID- 8611303 TI - Identification of a mobile intracardiac rheumatoid nodule mimicking an atrial myxoma. AB - Cardiovascular involvement occurs in 30% to 50% of patients with rheumatoid arthritis and typically presents as nonspecific inflammation or fibrosis of cardiac structures or as nodules embedded in the various cardiac tissues. This case report describes a unique pedunculated, mobile left atrial rheumatoid nodule prolapsing through the mitral valve plane and mimicking an atrial myxoma. A brief discussion of intracardiac rheumatoid nodules and their potential significance follows. PMID- 8611304 TI - Transesophageal echo probe compression of an aberrant right subclavian artery. AB - Transesophageal echocardiography (TEE) is frequently used in the operating room to assess surgical repairs in children with congenital heart disease. Complications from the procedure are unusual, but the potential for TEE probe compression of normal and abnormal posterior vascular structures has been recognized. We report a case of TEE probe compression of an aberrant right subclavian artery in a patient undergoing repair of an atrioventricular septal defect. PMID- 8611305 TI - The position and function of the mandibular hinge axis. AB - It is widely believed that the mandibular hinge axis is anatomically located through the condylar head or its superior surface. This study, using markers placed over the average hinge axis point prior to orthopantograms, an analysis of lateral skull cephalometric radiographs and skull extrapolation, shows that the hinge axis is remote from the condylar head and in the region of the neck of the condyle. This site is consistent with the anatomical consideration of the function of the temporo-mandibular ligament. PMID- 8611306 TI - The endalveolar crest: anatomic and prosthodontic considerations of an overlooked mandibular landmark. AB - Surgical and prosthodontic treatment of edentulous patients have confirmed the presence of a seldom-described crest of bone situated above and distal to the mylohyoid ridge of the mandible. This study aimed to establish the frequency of occurrence and range of expression of the feature, which it is suggested should be called the endalveolar crest in humans and other primates. Evidence of the crest was noted in all specimens examined although the expression varied. Dissection of fresh and preserved cadavers indicated that the crest provided attachment for part of the superior constrictor muscle and that it may also afford some protection to the lingual nerve in the retromolar region. PMID- 8611307 TI - Wide diameter implants: indications, considerations and preliminary results over a two-year period. AB - Wide diameter implants may be a useful alternative to conventional implant rehabilitation. Despite the fact that most major implant manufacturers are supplying wide diameter implants, very few articles actually address the indications, benefits and results of the use of wide diameter implants. Benefits and indications include increased implant surface area, "back-up" implant for when the initial implant lacks stability, immediate replacement of non-integrated or fractured implants, immediate placement of an implant after tooth extraction, anatomical considerations, increased prosthetic stability and biomechanical factors. Over a two year period, 268 wide diameter implants were placed in 196 patients. Preliminary results are presented in this paper and indicate an overall success rate, up to two years after placement, of approximately 96 per cent. All failures occurred before stage two surgery, due to non-integration of the implant. PMID- 8611308 TI - The captive screw retained sectional denture. AB - Sectional dentures, where retention is gained, not by flexible metal arms engaging "desirable" undercuts, have occasionally been described in the dental literature. The captive screw retained sectional denture described here is an effective replacement for one or two missing teeth in selected patients. It consists of separate labial and lingual sections, which, when assembled, provide an aesthetic appearance, functional occlusion and excellent retention, with a minimum of abutment tooth preparation and at low cost. The two sections are connected by a captive screw, which allows the sections to be separated for denture insertion, but which is tightened to join the two sections. Portions of each section then engage tooth undercuts to provide retention. The screw is loosened and tightened by means of a torque-limiting screwdriver. These dentures have a high level of patient acceptability, and do not appear to promote dental caries or gingival irritation. PMID- 8611309 TI - A history of dental polymers. AB - The disadvantages of the use of vulcanite as a denture base material prompted the development of a wide range of thermoplastic polymers as alternatives to vulcanite. Heat-cured acrylic resin materials are the most widely used polymeric denture base materials at present, but research into further improvements in denture base materials continues. PMID- 8611311 TI - Bruxism: aetiology, clinical signs and symptoms. AB - The aetiology of bruxism and therefore its management is poorly understood by dentists and their focus on a local dental cause has lead to much unnecessary irreversible dental treatment, with little impact on the incidence of bruxism. Clinical and neurophysiological evidence suggests that there is a strong link between bruxism and tooth wear in man and its counterpart in animals. In animals, keeping teeth sharp has importance for food retrieval and defense. In man, although this is no longer necessary, remnants of this mechanism remain as an inherited predisposition. PMID- 8611310 TI - Resin luting cements for full coverage restorations. AB - Adhesive resin luting cements bond to enamel, dentine, porcelain and some metal alloys. The use of adhesive resins, in the restoration of teeth with short, tapered clinical crowns or other unfavourable geometric configurations, may be particularly advantageous because an approach less destructive than traditional crown preparation may be possible. This paper reviews the composition, adhesive, physical, and biological properties, and handling characteristics of Panavia and 4-META containing resins (C & B Metabond and Super-Bond C & B). It is concluded that clinical trials are needed to determine the structural longevity and clinical efficacy of these adhesive systems. PMID- 8611312 TI - Effect of bone density of the head on total body DEXA measurements in 100 healthy Swedish women. AB - PURPOSE: The aims of this study were to examine the bone areal density of the head and how it varied in relation to the density of the rest of the skeleton, and with age, and body mass index (BMI). Our intention was to study the feasibility of excluding the head from the rest of the body, a method which might improve the fracture prediction power of bone mineral measurements. MATERIAL AND METHODS: Bone mineral per area (BMA) and bone mineral content (BMC) (g) were determined in 100 consecutive female volunteers, aged 17 to 78 years, with total and partial body measurements. RESULTS: BMC of the head was found to be 20.2 +/- 2.2% of that for the total body. The BMA of the head was 2.38 +/- 0.21 times higher than that of the rest of the body. The correlation between the BMA of the head and the rest of body was significant (r = 0.73). The average change in z score (referred to the same age group in our material) was 0.20 when the head was excluded from total body BMA. The BMA of a) total body, b) total body, head excluded, and c) head decreased with age. The BMA of the head was correlated to BMI in the older age groups (p < 0.01). The relative statistical uncertainty for repeated measurement of head BMA was 1.8%. CONCLUSION: The change of the bone density of the head with age and BMI, in comparison to that of the rest of the skeleton, suggests that when the head is excluded from total body BMA better predictive value for fracture risk is obtained. PMID- 8611313 TI - Dynamic range control processing of digital chest images. A clinical evaluation. AB - PURPOSE: The clinical usefulness of an advanced image-processing system called "dynamic range control processing" was investigated, with which selected parts of the dynamic range of digital chest images could be controlled. MATERIAL AND METHODS: A comparative study of 3 different post-processed formats of storage phosphor (SR) images was performed in 35 patients with abnormalities in the chest. The 3 formats were SR images with standard mode (SR-standard), SR images with strong edge-enhancement (SR-enhanced), and dynamic range controlled SR images (SR-controlled). RESULTS: For lung abnormalities, there was no difference among the 3 SR image formats. For normal mediastinal structures and lung abnormalities covered by the heart or diaphragm, SR-controlled and SR-enhanced images were significantly superior to SR-standard images, while no difference was found between SR-controlled and SR-enhanced images except for the trachea and bony structures. CONCLUSION: Dynamic range control processing appears to be a useful method for displaying SR chest images with an extremely wide dynamic range. PMID- 8611315 TI - Professional confidentiality is a major right to which our patients are entitled. PMID- 8611314 TI - Conditioned taste aversion in rats following intrathecal administration of contrast media. AB - PURPOSE: The occurrence of side-effects such as visceral malaise after intrathecal administration of the non-ionic radiography contrast media iomeprol, iopamidol, and iotrolan was assessed in rats by the conditioned tasted aversion procedure. METHODS: Reduced preference towards a saccharose solution compared with normal water following intraventricular administration of a contrast medium was used as a measure of the aversive response. RESULTS: At a dose of 100 mg I/kg none of the tested contrast media induced aversion. At 200 and 300 mg I/kg, both iopamidol and iomeprol induced significant aversive responses with respect to control, although the response of the iomeprol group appeared milder than that of the iopamidol group at a dose of 200 mg I/kg. Iotrolan could be tested only at the lowest dose since the high doses caused excessive mortality. CONCLUSION: Intrathecally administered iomeprol appeared to be well tolerated in rats at doses higher than those suggested for clinical use. PMID- 8611316 TI - Ultrasound at scintigraphic "intermediate probability of pulmonary embolism". AB - For diagnosis of pulmonary embolism (PE), ventilation/perfusion lung scintigraphy is routinely used; approximately one-third of the patients will have the diagnosis "intermediate probability of PE" (inconclusive). In this group only about 33% are found to have pulmonary emboli if examined with pulmonary angiography. To evaluate the diagnostic, therapeutic, and economic consequences of ultrasound of the legs as a complementary diagnostic investigation to "intermediate probability", 72 consecutive patients were investigated with bilateral ultrasound of the proximal deep veins of the legs and pulmonary angiography in a prospective study. Ten patients had PE, of whom 7 had deep venous thrombosis, and 62 had no PE, of whom 2 had deep venous thrombosis. The negative predictive value of ultrasound was 0.95. In view of the importance of adequate treatment and rational use of public health care expenditure, complementary diagnostics should be performed, and ultrasound is an adequate complementary investigation. PMID- 8611317 TI - Myocardial cell death in reperfused and nonreperfused myocardial infarctions. MR imaging with dysprosioum-DTPA-BMA in the pig. AB - PURPOSE: To investigate whether Dy-DTPA-BMA-enhanced MR imagining would permit identification of myocardial cell death, myocardial infarction was induced in 12 domestic pigs. MATERIAL AND METHODS: In 6 pigs with irreversible cell damage, Dy DTPA-BMA (1.0 mmol/kg b.w.) was administered i.v. 70 min after coronary occlusion. In 6 other pigs, the infarctions were reperfused 80 min after the occlusion, followed by injection of Dy-DTPA-BMA after 30 min of reperfusion. In 4 additional pigs, the hearts were reperfused after 2 min of occlusion. All 16 pigs were sacrificed 10 min after the injection of Dy-DTPA-BMA. The hearts were excised and imaged with MR. RESULTS: Reversibly injured myocardium could not be distinguished from adjacent nonischaemic myocardium after the administration of Dy-DTPA-BMA. Reperfused, infarcted myocardium demonstrated a high signal intensity in the proton-density- and T2-weighted sequences, despite a 5-fold higher Dy concentration compared with both nonreperfused infarcted and nonischaemic myocardium. CONCLUSION: This lack of susceptibility effect in infarcted myocardium, due to a homogeneous distribution of Dy, indicates the usefulness of Dy as a marker of tissue viability. PMID- 8611318 TI - Ischemic heart disease a major health problem in the West. PMID- 8611319 TI - Double-contrast MR imaging of reperfused porcine myocardial infarction. An experimental study using Gd-DTAA and Dy-DTPA-BMA. AB - PURPOSE: Myocardial infarctions were induced in 12 pigs to investigate whether a double-contrast method, combining a positive and a negative MR contrast agent, could improve the visualization of reperfused myocardial infarctions. MATERIAL AND METHODS: All 12 pigs were subjected to 80 min of occlusion followed by reperfusion. In the double-contrast group (6 pigs), Gd-DTPA-BMA (0.3 mmol/kg b.w.) and Dy-DTPA-BMA (1.0 mmol/kg b.w.) were administered i.v. after 30 min of reperfusion. In the remaining 6 pigs, a single injection of Gd-DTPA-BMA (0.3 mmol/kg b.w.) was given after 30 min of reperfusion. All pigs were sacrificed 10 min post-contrast injection, corresponding to a reperfusion time of 40 min. The hearts were excised and imaged with MR. The concentrations of GD and Dy were measured in infarcted and nonischaemic myocardium using ICP-AES. RESULTS AND CONCLUSION: Contrast media concentrations were more than 4-fold higher in infarcted compared with nonischaemic myocardium. The infarctions were best shown on T1-weighted images, and there were no differences between the double and single contrast groups. In the T2-weighted images, the infarctions were significantly better visualized in the double-contrast group, due to a Dy-induced signal intensity loss in nonischaemic myocardium. PMID- 8611320 TI - MR angiography of abdominal and peripheral arteries. Techniques and clinical applications. AB - This review article deals with MR angiography (MRA) of abdominal and peripheral arteries. Pulsatile flow, respiratory motion and peristalsis impose difficulties in imaging the vascular structures in the abdomen and the lower extremities. Development of new techniques, such as segmentation of the data acquisition, using specific acquisition windows in relation to a cardiac trigger, magnetization preparation of the tissue and phase-encoding re-ordering or sorting, have reduced the artifacts associated with abdominal and peripheral MRA. Clinical MR investigations of the arteries branching from the abdominal aorta such as the renal and mesenteric arteries and arteries in the lower extremities have revealed that severe stenoses or occlusions can be diagnosed accurately while the grading of less severe stenosis is more difficult. The phase-contrast method has been used to quantify blood flow and study the hemodynamics in abdominal and peripheral vessels. Quantitative flow information can be used to diagnose vascular disease and provides important physiological information. More prospective clinical studies, in which recently developed MRA techniques are compared with conventional angiography, are necessary before conclusive decisions can be made as to whether MRA may replace these methods. PMID- 8611321 TI - Long-term results of percutaneous transluminal angioplasty in renovascular hypertension. AB - PURPOSE: The long-term effect of percutaneous transluminal renal angioplasty (PTRA) was assessed in 50 patients with renovascular hypertension. MATERIAL AND METHODS: Thirty-eight of the patients had atherosclerotic disease and 12 patients fibromuscular dysplasia (FMD). Dilatation with a balloon catheter were performed at 6 to 8 atm for 15 to 30 s. RESULTS: PTRA was technically successful in 46 (92%) patients. Three of the failures underwent surgical revascularization. There was no mortality connected with PTRA. Minor complications occurred in 6 (12%) patients, and surgical intervention was required in 1. In 4 patients with restenosis, repeated PTRA was performed in 2, and surgery in the other 2 patients. Bilateral disease occurred in 12 patients, and 3 had sequential bilateral PTRA. In 9 patients with atherosclerotic ostial stenosis, PTRA was technically successful in 8 (89%). Thirty-eight patients were re-examined with a mean follow-up of 4 years. At follow-up, 5 (45%) of the patients with FMD were classified as cured, 6 (55%) as improved, and none as failed. In the 27 patients with atherosclerotic disease, 23 (85%) had long-term benefit, 3 (11%) were cured, 20 (74%) were improved, and 4 (15%) were failures. In the 8 patients with ostial atherosclerotic lesions and successful PTRA, there was a 75% long-term benefit. Two patients died during follow-up, both from myocardial infarction. CONCLUSION: The result suggest that PTRA is effective in long-term management of renovascular hypertension, not only in patients with fibromuscular stenosis, but also in patients with atherosclerotic disease, even when ostial lesions are present. PMID- 8611322 TI - Duplex US of the external carotid artery. AB - The ratio between the systolic peak velocities of the internal and common carotid arteries (vpICA/vpCCA), vpICA and grey-scale imaging measurement are generally used to evaluate internal carotid stenosis against known flow criteria in order to differentiate non-significant from significant stenosis. The same criteria are also used for evaluating the external carotid artery (ECA). Our data on 707 normal or stenotic ECA nevertheless showed that the systolic peak velocity of the normal ECA (vpECA) and its ratio to the systolic velocity of the CCA (vpECA/vpCCA) are higher than vpICA and vpICA/vpCCA. vpECA/vpCCA is about 2 in > 0-49% ECA stenosis. Only in severe stenosis are the peak velocities almost comparable. The ratio between the peak end diastolic velocities (edvECA/edvCCA) and edvECA proved to be unreliable, as did grey-scale imaging measurement of the external carotid stenosis. In addition, ipsi-lateral internal carotid stenosis greatly affects the non-stenotic external carotid flow values, and probably has the same effect on the flow values of a stenotic external artery. Thus, external carotid flow values must be considered carefully. PMID- 8611324 TI - Carotid-cavernous sinus fistula and aneurysmal rupture associated with fibromuscular dysplasia. A case report. AB - A patient with carotid-cavernous sinus fistula associated with fibromuscular dysplasia, who died from rupture of an associated splenic aneurysm despite successful treatment of the CCF, is reported. When multivessel involvement with aneurysmal dilatation is observed in patients with fibromuscular dysplasia, the possibility of aneurysm ruptures in any of the arteries should be considered. PMID- 8611323 TI - Traumatic bilateral carotid-cavernous fistulas treated with detachable balloon. A case report. AB - A 29-year-old male developed ptosis and progressive pulsating protrusion of the right eye, accompanied by conjunctival injection and tinnitus following a bicycle accident. MR angiography revealed dilation of both superior ophthalmic veins and facial veins. Right internal carotid angiography demonstrated right carotid cavernous fistula (CCF) at the C3 portion of the right internal carotid artery with abnormal venous drainage. After right carotid balloon occlusion test had been performed, a detachable balloon was introduced into the right CCF while preserving the lumen of the right internal carotid artery. A left CCF, which was detected after closure of the right CCF, was also closed with a detachable balloon. Follow-up carotid angiographies showed complete closure of both CCFs and no abnormal venous drainage. After 1 year no abnormal physical manifestations, or abnormal neurologic signs or symptoms were present. PMID- 8611325 TI - Chemoembolization of head and neck cancer with carboplatine microcapsules. AB - Fourteen patients with malignant tumor in the head and neck region were treated with infusion of carboplatine microcapsules (CBDCA-mc) via percutaneous super selective catheterization. A microcatheter was advanced into a feeding artery using a coaxial catheter system. Eleven patients had over 30% reduction of the tumor size on CT within 1 month after embolization. Twelve patients had an increased amount of low attenuation tissue in the tumor on CT after embolization, suggesting increased necrosis in the tumor. No definite hematologic toxicity was found. A majority of patients complained of moderate pain in the embolized region immediately after embolization, easily relieved by i.v. analgesics. Chemoembolization using CBDCA-mc may be an effective therapeutic modality in advanced cases of head and neck cancer. PMID- 8611326 TI - Size and asymmetry of the planum temporale. A new three-dimensional method for analysis of the supratemporal plane using MR imaging and computer-aided graphics. AB - The planum temporale of the supratemporal plane is important for language function and shows left-right asymmetry in most brains. To estimate the size and allow side comparison of the planum temporale, we developed a new technique for 3 D MR analysis of the supratemporal plane using a personal computer and computer aided graphics. The temporal lobes of 5 human cadavers were imaged by MR in the sagittal plane, at a slice thickness of 2 mm. The images of the supratemporal plane were entered into a personal computer using the original software to determine the positions of anatomic landmarks and the size of the planum temporale. The data were then transferred to a supercomputer to reconstruct the 3 D surface image of the supratemporal plane. Computer images of the supratemporal plane agreed with macroscopic observations. The positions of anatomic landmarks and the size of the planum temporale also agreed with macroscopic measurements. Thus, the present technique provides valuable anatomic data on the supratemporal plane which should be useful for further clarification of the anatomic basis of language function. PMID- 8611327 TI - A new liposomal contrast medium for CT of the liver. An imaging study in a rabbit tumour model. AB - A new type of liposomal liver-specific contrast medium was studied in an experimental tumour model. Rabbits were inoculated with VX2-carcinoma directly in the liver of laparotomy. CT studies were carried out 14 days after inoculation. The liver-specific contrast medium consisted of a suspension of liposomes in a 100 mg I/ml iodixanol solution, with equal amounts of encapsulated and nonencapsulated iodixanol. It was administered at a dose of 200 mg I/kg. The contrast of normal liver tissue to tumorous tissue was significantly increased by contrast medium administration, the increase being largest 10 min after injection. PMID- 8611328 TI - Small hepatocellular carcinoma. Detection with US, CT, MR imaging, DSA, and Lipiodol-CT. AB - Twenty-two patients with 37 small (3 cm or less) nodular lesions of hepatocellular carcinoma (HCC) were examined with ultrasonography (US), CT, MR imaging, digital subtraction angiography (DSA), and CT following intraarterial injection of Lipiodol (Lipiodol-CT). All patients subsequently underwent surgery, and the gold standard was provided by intraoperative US. The detection rate was 70% for US, 65% for CT, 62% for MR imaging, 73% for DSA, and 86% for Lipiodol-CT. A significant difference (p < 0.05) was observed between the detection rate of Lipiodol-CT and the detection rates of all the other imaging modalities. The difference was even more manifest (p < 0.02) when only lesions smaller than or equal to 1 cm were considered. It is concluded that Lipiodol-CT is the single most sensitive examination to detect small nodules of HCC. It should therefore be considered a mandatory step in the preoperative evaluation of patients with HCC considered to be surgical candidates after noninvasive imaging studies. PMID- 8611329 TI - Percutaneous pancreatography under ultrasonographic guidance. AB - PURPOSE: The aim of the study was to evaluate percutaneous pancreatography as an alternative method for pancreatic duct visualisation in patients with pancreatic disease. MATERIAL AND METHODS: In 21 patients with pancreatic disease and previously unsuccessful ERCP, puncture of the pancreatic duct was carried out under ultrasonographic guidance with an 0.7-mm Chiba needle, and contrast injection was made under fluoroscopic control in the pancreatic duct. RESULTS: The procedure was successful in 18 patients (86%). In 10 patients, chronic pancreatitis was found, and in 8 patients, pancreatic carcinoma. CONCLUSION: Percutaneous pancreatography is a good alternative method for visualisation of the pancreatic duct in patients with pancreatic disease and previously unsuccessful ERCP. PMID- 8611330 TI - MR velocity mapping measurement of renal artery blood flow in patients with impaired kidney function. AB - Renal blood flow (RBF) was measured in 9 patients with chronic impaired kidney function using MR velocity mapping and compared to PAH clearance and 99mTc-DTPA scintigraphy. An image plane suitable for flow measurement perpendicular to the renal arteries was chosen from 2-dimensional MR angiography. MR velocity mapping was performed in both renal arteries using an ECG-triggered gradient echo pulse sequence previously validated in normal volunteers. Effective renal plasma flow was calculated from the clearance rate of PAH during constant infusion and the split of renal function was evaluated by 99mTc-DTPA scintigraphy. A reduction of RBF was found, and there was a significant correlation between PAH clearance multiplied by 1/(1-hematocrit) and RBF determined by MR velocity mapping. Furthermore, a significant correlation between the distribution of renal function and the percent distribution of RBF was found. PMID- 8611331 TI - Percutaneous lumbar discectomy. Technique and clinical result. AB - Lumbar disc herniation was treated by percutaneous discectomy using a new instrument for automatic aspiration and cutting of disc material. The inclusion criteria were limited to patients with pure disc herniation without stenosis or any other additional factors. Only contained hernias with a maximum size of 50% of the thecal sac were included. Three procedures out of 45 were technical failures. At 1-year follow-up 69% of the patients were satisfied. No complications were seen. The result was not influenced by the amount of disc material removed, age, duration of symptoms or the size of the disc hernia. Reduced size of disc hernia was found in 13 out of 14 satisfied patients followed by CT. All unsatisfied were conventionally operated on. The percutaneous treated patients had 1 day of hospitalisation and on average 11 weeks of sick-leave compared to an average of 6 days and 16 weeks following conventional discectomy. PMID- 8611332 TI - MR diagnosis of retropatellar chondral lesions under compression. A comparison with histological findings. AB - PURPOSE: The aim of the study was to improve the chondromalacia patellae (CMP) diagnosis by MR imaging under defined compression of the retropatellar cartilage, using a specially designed knee compressor. The results were compared with histological findings to obtain an MR classification of CMP. METHOD: MR imaging was performed in in vitro studies of 25 knees from cadavers to investigate the effects of compression on the retropatellar articular cartilage. The results were verified by subsequent histological evaluation. RESULTS: There was a significant difference in cartilage thickness reduction and signal intensity behaviour under compression according to the stage of CMP. CONCLUSION: Based on the decrease in cartilage thickness, signal intensity behaviour under compression, and cartilage morphology, the studies permitted an MR classification of CMP into stages I-IV in line with the histological findings. Healthy cartilage was clearly distinguished, a finding which may optimize CMP diagnosis. PMID- 8611333 TI - Posttraumatic spinal osteolysis in ankylosing spondylitis as part of pseudoarthrosis. A case report. AB - A case of ankylosing spondylitis with pronounced osteolysis of the 12th thoracic vertebral body as part of posttraumatic pseudoarthrosis is described. The appearance of posttraumatic osteolysis simulates a malignant lesion, and it is important to consider the diagnosis in cases of osteolytic lesions of unknown origin. PMID- 8611334 TI - Renal tubular handling of potassium in children with insulin-dependent diabetes mellitus. AB - To clarify the mechanism by which renal potassium (K) excretion is reduced in children with insulin-dependent diabetes mellitus, we studied two groups of patients: (A) at diagnosis and (B) after at least 1 year of follow-up. Group A (15 children) was studied twice: on the day of admission and after 1 month of insulin therapy. On admission, urinary K excretion, fractional K excretion, and transtubular K concentration gradient (TTKG) were significantly decreased, but became normal after extended insulin therapy. TTKG was inversely correlated with blood glucose (P < 0.001) and hemoglobin A1c (HbA1c, P < 0.001). Group B (73 children with a mean follow-up of 54 +/- 36 months) was subdivided according to the TTKG: 30 patients had a low TTKG < 4.0 (median 3.2) and 43 patients had a normal TTKG > or = 4.0 (median 5.2). Patients had a low TTKG and those with a normal TTKG had an identical duration of follow-up and similar values for plasma renin activity, aldosterone concentration, calciuria, magnesiuria, albumin excretion rate, and creatinine clearance. However, those with a low TTKG had significantly higher blood HbA1c levels, urine volume, and glucosuria. Logistic regression analysis showed that the only independent variables predicting a low TTKG were blood HbA1c and glucosuria (P < 0.001). These data confirm that a reduced renal K excretion is a characteristic feature of diabetic children; this is reversible with appropriate insulin therapy, largely depends on the metabolic control of the disease, and, specifically, on the degree of hyperglycemia and/or glucosuria. PMID- 8611337 TI - Pediatric renal transplantation into the abnormal urinary tract. AB - Many children with end-stage renal disease have significant urinary tract problems other than irreversible loss of native kidney function. These significant other urinary tract problems, if not corrected prior to transplantation, may significantly increase recipient mortality, graft loss, and patient morbidity. These other urinary tract problems may cause hydroureteronephrosis in the transplanted kidney, lead to an increased incidence of graft rejection, be the source of sepsis after subsequent immunosuppression, and cause hypertension. In addition, pre-existing urinary diversion, large interabdominal masses, or previous cancer require specific pre-transplant management plans. Potential pediatric transplant recipients with other significant urinary tract problems can be classified according to three parameters; anatomical extent, pathology, and pathophysiology of the significant other problems. Particular attention must be paid to pre-existing lower tract problems. Strategy must be worked out pre transplant as to how the lower urinary tract is going to store, hold, and empty urine. The means for assessing the potential recipients and strategies and techniques for correcting pre-existing problems have been summarized in this article. PMID- 8611336 TI - The renin-angiotensin system in transgenic rats. AB - Transgenic animals are used to study the function, regulation and in vivo expression of genes. The effects of the genes of the renin-angiotensin system on blood pressure regulation and hypertension were invested in transgenic rats. The role of the renin-angiotensin system in the development of the cardiovascular hypertrophy or hypertensive renal damage was analysed, as well as its interaction with other hormonal systems, i.e., adrenal steroids. The development of a transgenic rat strain carrying the mouse REN-2 gene has provided a new model of hypertension with systolic blood pressure values of 200 mmHg. This model is characterised by low active plasma renin, hyperproreninaemia and high expression of renin in the adrenal gland and other external tissues. Transgenic rats with the human components of the renin-angiotensin system expressed the human renin and angiotensinogen proteins which interacted species-specifically in transgenic rats. These transgenic models demonstrate the feasibility of studying the function of candidate hypertension genes in transgenic animals. In the future, further refinements in transgene construction, mutation, and modification can be tested in such transgenic animal models. PMID- 8611335 TI - The hepatorenal syndrome. AB - Hepatorenal syndrome may occur in any form of severe liver disease. It appears less common in children than adults, but still carries a poor prognosis. There are several factors involved in its aetiology, including a decreased renal perfusion pressure, activation of the renal sympathetic nervous system and increased synthesis of several vasoactive mediators, which may modulate glomerular filtration by acting as both renal vasoconstrictors and dynamic regulators of the glomerular capillary ultrafiltration coefficient, through their action on mesangial cells. This review will discuss the pathophysiology of the hepatorenal syndrome and some of the principles of management of patients with renal failure and severe liver disease. The role of renal support and liver transplantation will also be covered. PMID- 8611338 TI - Clinical quiz. Osteopetrosis with carbonic anhydrase II deficiency. PMID- 8611339 TI - Syndrome of inappropriate secretion of antidiuretic hormone in tuberculous meningitis. PMID- 8611340 TI - Central venous line blood sampling for coagulation tests in hemodialysis patients. PMID- 8611343 TI - Should a cystogram be carried out on every baby diagnosed as having a dilated renal pelvis, either unilateral or bilateral, before or after birth? PMID- 8611342 TI - Pulseless disease with renal amyloidosis. PMID- 8611341 TI - Chronic renal failure in south eastern Anatolia. PMID- 8611344 TI - Nitrogen mustard therapy and nephrotic syndrome. PMID- 8611345 TI - Nitrogen mustard therapy of pediatric nephrotic syndrome. PMID- 8611346 TI - Nephrotic syndrome, was found to be positive for hepatitis B surface antigen. PMID- 8611347 TI - Collagen distribution in human membranous glomerulonephritis. AB - In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (alpha 1/alpha 2, alpha 3, alpha 4, alpha 5, alpha 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the alpha 3, alpha 4, and alpha 5 chains of type IV collagen. In contrast, the alpha 1/alpha 2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the alpha 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type IV collagen play an important role in the thickening of the GBM in human MGN. PMID- 8611348 TI - Tuberous sclerosis has several angiolipomata in both kidneys? PMID- 8611349 TI - Ultrasound findings in juvenile nephronophthisis. AB - The ultrasound finding of renal medullary cysts associated with increased echogenicity has been suggested to be diagnostic of juvenile nephronophthisis. The lack of cysts in several of our patients with juvenile nephronophthisis lead us to review the ultrasound findings at presentation in our patient population. Of 11 children with the diagnosis of juvenile nephronophthisis, 10 demonstrated increased echogenicity with loss of corticomedullary differentiation on initial ultrasound. Only 2 children had a single cyst each. On follow-up ultrasound, 2, 4.5, and 7 years later, 3 patients developed visible renal cysts. We conclude that at presentation the ultrasound finding consistent with the diagnosis of juvenile nephronophthisis is most often that of hyperechogenic kidneys without cysts; namely the lack of cysts does not rule out the diagnosis of juvenile nephronophthisis. PMID- 8611350 TI - 125Iodine-iothalamate clearance in children. A simple method to measure glomerular filtration. AB - Glomerular filtration rate (GFR) is the most widely used test to evaluate renal function. Several clearance markers have been used to measure GFR in adults. In children, however, a simple and reliable method to measure GFR is not available. Renal 125iodine (I)-iothalamate clearance, after a single subcutaneous injection, is a simple and accurate test to measure GFR in adults. The validity of unlabelled iothalamate, as a marker for measurement of GFR in children, was reported recently. Unfortunately, the unlabelled iothalamate assay is arduous. We report our experience with a single subcutaneous injection of 125I-iothalamate to measure GFR in normal children and those with renal disease. A weight-adjusted dosing regimen was adopted. This regimen resulted in sufficient above-background radioactivity in both blood and urine for reproducible measurement of GFR. Intra test variability for GFR was not affected by the degree of renal insufficiency. The test was well tolerated with only 2 patients developing mild headache during the procedure. Our study showed that renal clearance of 125I-iothalamate is reproducible, simple, and practical in healthy children and those with mild and advanced renal disease. PMID- 8611351 TI - Renal collecting duct carcinoma in an 8-year-old child. AB - A fatal collecting duct carcinoma, presenting with pleural metastases, arose from the right kidney in an 8-year-old child. A distal nephron origin of the tumor is supported by positive tumor staining with Ulex europaeus and Arachis hypogaea, and a lack of staining with Tetragonolobus lotus. The ultrastructural features of short stubby microvilli, smooth basal cell membranes, and lateral membrane infoldings also support a distal nephron origin (inner most inner medullary collecting duct). This rare childhood renal neoplasm behaved similarly to that reported in adults with metastatic disease at presentation and a short fatal clinical course. PMID- 8611352 TI - Sodium or chloride deficiency lowers muscle intracellular pH in growing rats. AB - Sodium deficiency and chloride deficiency are associated with a contracted extracellular (ECF) volume and impaired growth in young children and growing rats. In cell culture, lowering sodium in the medium reduces growth factor stimulated Na+/H+ exchange activity, intracellular pH (pHi), and DNA synthesis. We studied the effect of chronic sodium deficiency and chloride deficiency upon growth, extracellular acid base status, and muscle pHi in young rats. We fed growing rats for 21 days either a control diet, or one deficient in sodium (0.005%), chloride (0.005%), or calories. Muscle pHi was measured using 31phosphorus nuclear magnetic resonance spectroscopy. Rats fed either the sodium deficient or chloride-deficient diet developed ECF volume contraction and hyponatremia; growth in length and weight was impaired. Muscle pHi was decreased (pHi = 7.074 +/- 0.006, 7.078 +/- 0.006 vs. control 7.100 +/- 0.002; P < 0.02). In calorie-restricted rats, growth was impaired but pHi was not affected (pHi 7.103 +/- 0.008). Metabolic alkalosis developed in the chloride-deficient group; acid base status was not affected in the sodium-deficient group. Despite differences in ECF acid base status, both groups had a low muscle pHi. We speculate that the low muscle pHi was a result of the ECF volume contraction and hyponatremia; low muscle pHi may contribute to retarded cell growth. PMID- 8611353 TI - Sensorineural hearing loss in patients reaching chronic renal failure in childhood. AB - The incidence of sensorineural hearing loss (SNHL) was investigated in 68 patients who reached chronic renal failure (CRF) in childhood with the aim of identifying possible risk factors. Tests were carried out by means of pure-tone and impedance audiometry. SNHL was found in 29% of patients on conservative treatment, 28% of patients on hemodialysis, and 47% after renal transplantation. Differences among groups were not significant. A significant correlation was found with the administration of ototoxic drugs (aminoglycosides and furosemide). We hypothesize that SNHL may be reduced in patients with CRF or on renal replacement therapy by strictly monitoring ototoxic therapy. PMID- 8611354 TI - Mortality trends in pediatric patients with chronic renal failure. AB - Mortality trends were analyzed in 441 children and adolescents with chronic renal failure (CRF) observed over a 24-year period before and after institution of renal replacement therapy (RRT). A total of 93 patients died. Overall mortality rate (MR) per 100 patient years decreased from 6.6 in 1969-1978 to 2.5 in 1979 1988 and increased slightly to 2.9 in 1989-1992. The fall involved all four modes of treatment: conservative, hemodialysis (HD), continuous peritoneal dialysis (CPD), and transplantation (TX). From 1979-1988 to 1989-1992 MR on conservative and on dialysis treatment changed only slightly and was similar on HD and CPD. An alarming rise in MR was noted after TX in 1989-1992, mainly due to malignant tumors. In 44 patients who died on conservative treatment, the reasons for non acceptance for RRT were analyzed: in 22 multi-morbidity was the main reason, usually because of a congenital neurological disorder. Some patients died from advanced uremia or unexpected events after the decision to institute RRT. Our experience demonstrates a persistent mortality in pediatric patients with CRF, which in recent years is primarily ascribed to congenital multi-morbid conditions which make RRT unfeasible, infections on dialysis treatment, and malignancies after TX. PMID- 8611355 TI - Intracellular amino acid concentrations in children with chronic renal insufficiency. AB - We studied amino acid concentrations in granulocytes and plasma of 24 children with chronic renal failure and 15 healthy children. Granulocytes were isolated from 10 ml of blood using a dextran-Ficoll-Hypaque procedure. Intracellular levels of leucine, isoleucine, methionine, phenylalanine, lysine, histidine, tyrosine, and arginine were significantly lower in children with chronic renal failure than healthy children. There were no significant differences in intracellular and plasma amino acid concentrations between children with chronic renal failure on a well-balanced protein-restricted diet and children with chronic renal failure with a normal protein intake. PMID- 8611357 TI - Bacteremia in a pediatric hemodialysis unit secondary to Enterococcus fecalis. AB - Bacteremia is often a serious and recurring problem in children with hemodialysis catheters. We report an outbreak of Enterococcus bacteremia in a pediatric hemodialysis unit occurring from June 1992 to June 1993. During this period, 18 episodes of bacteremia occurred in eight children; 11 infections were polymicrobial. Enterococcus fecalis was associated with 13 infections in five patients (8 polymicrobial). Other pathogens included Enterobacter cloacae (5 infections), Staphylococcus (3), Staphylococcus epidermidis (2), and Klebsiella pneumoniae (2). All Enterococcus infections occurred in patients with dual-lumen subclavian venous catheters. Skin and catheter sites were culture negative, except in one patient. Rectal swabs were positive for Enterococcus in five patients. Enterococcus was not isolated from any source within the unit. Serotypes of all Enterococcus isolates were different, except for 2 isolates in the same patient. Starting in June 1993, catheters were flushed after dialysis with vancomycin or ampicillin. Since initiating this procedure, further episodes of Enterococcus bacteremia have not occurred. A questionnaire sent to other pediatric hemodialysis units failed to identify Enterococcus among 26 cases of bacteremia. IN CONCLUSION: (1) Enterococcus is an unusual pathogen for hemodialysis-related bacteremia in children; (2) patients with dialysis catheters were predisposed to this infection; (3) a common source for Enterococcus could not be identified by either culture or by serotyping; (4) flushing catheters with antibiotics after dialysis was effective prevention. PMID- 8611356 TI - Renal failure in the surviving monochorionic twin after death of the co-twin in utero. AB - Renal failure in the newborn infant is mainly determined by vascular causes. In this report we describe a patient with a particular vascular cause of renal failure. The patient was the product of a twin pregnancy in which the twin partner died in utero. In retrospect, the twins appeared to be monozygotic. As the pregnancy was studied carefully prenatally by ultrasound, we were able to observe the development of this condition, characterized by oliguria, oligohydramnios, and lung hypoplasia: the oligohydramnios sequence. After organ development had been normal initially, renal function was lost and the oligohydramnios sequence developed in the survivor after the co-twin had died in utero. PMID- 8611359 TI - Diabetes as a complication of tacrolimus (FK506) in pediatric renal transplant patients. AB - Three cases of insulin-requiring diabetes mellitus associated with tacrolimus (FK506) therapy in pediatric renal transplant patients are presented. New-onset diabetes mellitus has been reported with tacrolimus therapy post liver and kidney transplants in up to 12% of adult patients, but is thought to be rare in pediatrics. Although insulin requirement with tacrolimus therapy has been occasionally reported in adolescent patients post liver transplant, only a single case in a pediatric kidney transplant recipient has been previously documented. These cases illustrate the significant diabetogenic effect of tacrolimus in pediatric renal transplant patients. As the use of tacrolimus becomes more prevalent in pediatric kidney transplantation, pediatric nephrologists should be aware of this potential complication. PMID- 8611358 TI - Haemodynamic effects of arteriovenous and venovenous haemofiltration in piglets. AB - The aim of this study was to compare the early haemodynamic effects of continuous arteriovenous haemofiltration (CAVH) with those of continuous venovenous haemofiltration (CVVH) in normal and endotoxic piglets, within the framework of a two-period cross-over trial. Sixteen domestic piglets (weight 6-18 kg) underwent 1 h of CAVH followed by 1 h of CVVH or 1 h of CVVH followed by 1 h of CAVH. Six were pre-treated with a graded endotoxin infusion to simulate clinical sepsis. The main measurements included: heart rate; mean arterial (MAP), pulmonary artery, central venous and pulmonary artery occlusion pressures; thermodilution cardiac output; and calculated systemic (SVRI) and pulmonary vascular resistance indexes. Each measurement was performed immediately before and 30 min after commencement of each technique of filtration. Commencement of haemofiltration in normal piglets caused minimal haemodynamic effects. In endotoxic piglets, commencement of filtration, whether CAVH or CVVH, caused a haemodynamic change which was significantly more pronounced in the first filter (SVRI -39%, MAP -32%) than the second filter (SVRI +22%, MAP +0.9%) (SVRI, P=0.01, first filter vs. second) (MAP, P=0.009 first filter vs. second). In conclusion, there were no significant differences between the early haemodynamic effects of CAVH and CVVH in normal or endotoxic piglets. The haemodynamic effects of either technique may become more significant in the presence of sepsis. PMID- 8611360 TI - Long-term immunosuppressive treatment of a child with Takayasu's arteritis and high IgE immunoglobulins. AB - A 7-year-old child presented with a severe form of Takayasu's arteritis, with two consecutive episodes involving the right testis and then the left kidney 6 months later. The renal artery obstruction was accompanied by severe hypertension. An aortography showed a complete obstruction of the left renal artery and a narrowing of the right subclavian artery. Plasma renin activity was high. Serum immunoglobulins were within the normal range, except for an increase in IgE (880 mu/l). Despite 4 months', treatment with antihypertensive drugs, prednisone, cyclophosphamide, and anticoagulant, the blood pressure never returned to normal and the left renal function remained completely absent. A nephrectomy was performed which immediately normalized plasma renin activity and blood pressure. The child was subsequently treated with alternate-day prednisone for 3 months, alternating with 3 months of cyclophosphamide or, later, azathioprine. Persantine (dipyridamole) and acetylsalicylic acid were administered continuously. The right radial pulse returned to normal within 2 years. An 8-year follow-up failed to detect any new episode of arteritis. The right kidney showed signs of compensatory hypertrophy. Finally, a recent arteriography demonstrated not only a normal right renal artery blood flow but almost total disappearance of the right subclavian artery obstruction. However, the IgE remained abnormally high (2,023 micrograms/l). PMID- 8611361 TI - Membranous nephropathy, interstitial nephritis, and Fanconi syndrome-- glomerular antigen. AB - We characterized the glomerular antigen of membranous nephropathy (MN) in a child with the triad of MN, proximal renal tubular basement membrane autoantibody (TBMAb)-associated interstitial nephritis (ITN), and Fanconi syndrome. Granular staining was demonstrated for human gp600 in the vicinity of immune deposits of MN along glomerular capillary loops, using a monospecific polyclonal antibody to human gp600 by indirect immunofluorescence. However, no staining was observed in the MN deposits for receptor-associated protein. Membranous nephropathy preceded the development of TBMAbs, ITN, and Fanconi syndrome by 1 year, showing that the MN lesion does not result from the initial immunological injury to the tubulointerstitium, as postulated earlier. We confirmed the reactivity of TBMAbs with the recently described rabbit 58-kilodalton (kDa) tubular basement membrane antigen (TBMAg). However, this is the first report to show reactivity of these antibodies with the human 58-kDa protein. Also, we found that TBMAg is comprised of a single protein band of 58 kDa, unlike the previously described combination of two protein band (58 kDa and 175 kDa). In this patient, following prednisone treatment, the TBMAbs became undetectable, and the nephrotic syndrome and Fanconi syndrome resolved, thus suggesting a causal role of TBMAbs in the pathogenesis of Fanconi syndrome. We postulate that in this rare disorder, renal lesions result from an autoimmune response to the 58-kDa TBMAg and possibly to gp600, and that the predisposition to autoimmunity is genetically linked to the HLA B7 serotype. PMID- 8611362 TI - Hemolytic uremic syndrome in families-an Argentinian experience. AB - Six hundred and thirty-one patients with hemolytic uremic syndrome (HUS) were treated from 1960 to 1992; 19 (3%) were familial cases, of which 9 were classified as concomitant (including twins), 6 as non-concomitant, and 4 as recurrent. In the recurrent group there were 15 HUS episodes, 10 being concomitant in 2 patients. Prodromal diarrhea was present only in concomitant and non-concomitant cases. Patients with recurrences were sisters from a single family. Concomitant and non-concomitant cases had clinical features, course, and age similar to typical endemoepidemic forms of HUS, in which an association with verocytotoxin-producing Escherichia coli has been reported. There may be a genetic determinant in concomitant cases; these occurred outside the season during which endemoepidemic forms are typically detected. In patients with recurrent disease a genetic factor which may lead to the development of the disease when triggered by viral infections is likely. PMID- 8611363 TI - Nephrotic syndrome in a mother and her infant: relationship with cytomegalovirus infection. AB - This case report describes infantile nephrotic syndrome (NS) in a baby girl with a clinically severe cytomegalovirus (CMV) infection. Culture of the baby's urine was positive for CMV and IgM anti-CMV antibodies were detected. After an unsuccessful course of corticosteroids, gancyclovir treatment was started and a remission of cutaneous, pulmonary, and renal symptoms was achieved. As the mother also developed NS at the end of pregnancy, a common etiology could be postulated, although there were no signs of recent CMV infection in the mother, only anti-CMV IgG. The relationship between CMV infection and glomerular disease is still unclear; NS may represent another manifestation of CMV disease. PMID- 8611364 TI - Membranous nephropathy following perinatal transmission of hepatitis B virus infection--long-term follow-up study. AB - In children with hepatitis B-associated membranous glomerulonephritis, the time of onset of the infection and the duration of the carrier state before diagnosis of the renal disease are always unknown. Moreover, follow-up is usually short. We report the unique observation of a French girl who was infected with hepatitis B virus by her mother who had acute hepatitis during the immediate postpartum period; the girl developed proteinuria at 6 years of age. The onset of the infection in the perinatal period, the mild liver abnormalities, and the absence of nephrotic syndrome did not justify any treatment. Spontaneous seroconversion to anti-HBe antibody positive occurred at 12 years of age. Proteinuria gradually diminished and was absent at 18 years. However, HBs antigenemia persists. PMID- 8611365 TI - "Neonatal variant" of Bartter syndrome presenting with acidosis. AB - An infant is reported with the "neonatal variant" of Bartter syndrome, presenting at 5 weeks of age with metabolic acidosis associated with a life-threatening water and electrolyte depletion. Alkalosis was first shown after 2 weeks of vigorous fluid, sodium, and potassium substitution. We suggest that the extreme fluid and electrolyte losses associated with the "neonatal form" of Bartter syndrome could lead to acidosis more often than previously suspected, and may cause underdiagnosis of a possibly fatal condition. PMID- 8611366 TI - Cyclosporine-induced transient renal hypoperfusion in adolescent transplant recipients. AB - We investigated the acute hemodynamic effect of a single oral dose of cyclosporine A (CsA) given as part of the immunosuppressive schedule in six adolescents with renal transplants. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were determined by continuous infusion of inulin and amino hippuric acid for 12 h. A fall in both GFR and RPF was observed 4-6 h after peak plasma CsA levels. No significant correlation was found with CsA dosage or any pharmacokinetic parameters. This study demonstrates that CsA also has a vasoconstrictory effect in adolescent recipients; this could be one of the causes of its nephrotoxicity. PMID- 8611367 TI - Haemophilus influenzae type b immunization in infants on peritoneal dialysis. Pediatric Peritoneal Dialysis Study Consortium. AB - As part of a multi-center collaborative study, we measured antibody levels to Haemophilus influenzae type b (Hib) in ten chronic peritoneal dialysis (CPD) patients, aged 39 months or less, who were immunized while on CPD. Nine of the ten developed protective antibody levels to Hib. Four patients had serial measurements of antibody and all maintained protective levels, although the levels did decrease in two patients. Thus most, but not all, infants immunized with Hib vaccine while on CPD develop protective antibody levels. The factors responsible for vaccine failure are not clear. Whether patients maintain protective antibody over time needs to be determined. PMID- 8611368 TI - Use of prostaglandin I2 in three small children at high risk of early renal graft thrombosis. AB - We report the use of prostaglandin I.2. (PGI2) in three small children weighing less than 15 kg at high risk of graft thrombosis after cadaveric renal transplantation complicated by acute tubular necrosis. PGI2 was started at a dose of 5 ng/kg per min within the first 6 h after transplantation, and was continued for 12-15 days. Before and during PGI2 infusion, color-coded and pulsed Doppler sonography was performed. We found immediate restoration of diastolic flow, consistent with a decrease in vascular resistance. During the subsequent days, the sonographically assessed flow pattern and clinical graft function improved gradually. None of the three consecutively treated children developed graft thrombosis or lost his graft; no clinically relevant bleeding or adverse hemodynamic or pulmonary effects were seen. PMID- 8611370 TI - Metabolism of the broad-spectrum neuropeptide growth factor antagonist: [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P. AB - Broad-spectrum neuropeptide growth factor antagonists, such as [D-Arg1, D-Phe5, D Trp7,9, Leu11]substance P (antagonist D) and [Arg6, D-Trp7,9, NmePhe8]substance P(6-11) (antagonist G), are currently being investigated as possible anti-tumour agents. These compounds are hoped to be effective against neuropeptide-driven cancers such as small-cell lung cancer. Antagonist D possesses a broader antagonistic spectrum than antagonist G and hence may be of greater therapeutic use. The in vitro metabolism of antagonist D has been characterised and the structures of two major metabolites have been elucidated by amino acid analysis and mass spectrometry. Metabolism was confined to the C-terminus where serine carboxypeptidase action produced [deamidated]-antagonist D (metabolite 1) and [des-Leu11]-antagonist D (metabolite 2) as the major metabolites. Biological characterisation of the metabolites demonstrated that these relatively minor changes in structure resulted in a loss of antagonist activity. These results provide some of the first structure-activity information on the factors that determine which neuropeptides these compounds inhibit and on the relative potency of that inhibition. PMID- 8611369 TI - Hemostatic complications in renal disorders of the young. AB - This review focuses on the hemorrhagic and thrombotic complications sometimes associated with the most common renal disorders in children. A Medline search of the literature was conducted from 1966 to January 1995, using combinations of key words appropriate for each disorder. Additional references were located through the bibliographies of the publications and recent journals were searched independently. The most common renal disorders with hemostatic complications in children were: renal vein thrombosis (268 children in 80 publications), hemolytic uremic syndrome (473 children in 29 publications), nephrotic syndrome (4,158 children in 51 publications), renal transplantation (3,976 children in 14 publications), glomerulonephritis (20 publications), end-stage renal disease, and dialysis (22 publications). The age distribution, clinical presentation, etiology, diagnosis, treatment, and outcome of the affected children were analyzed for each disorder. Children with inherited pre-thrombotic disorders usually do not present during childhood unless there is a secondary risk factor. Similarly, most children with renal disease do not develop thromboembolic complications. Therefore, when a child with a renal disorder develops a thromboembolic event, evaluation for an inherited pre-thrombotic disorder should be seriously considered. Guidelines for the use of heparin and warfarin in these children (both therapeutically and prophylactically) are given. At this time, the risk/benefit of thrombolytic therapy in children is not known and a general recommendation for thrombolytic therapy cannot be made. PMID- 8611371 TI - The role of apoptosis in bispecific antibody-mediated T-cell cytotoxicity. AB - In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2 negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2 positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 51Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 51Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAb-mediated cell killing. PMID- 8611372 TI - A clinicopathological study on overexpression of cyclin D1 and of p53 in a series of 248 patients with operable breast cancer. AB - Overexpression of cyclin D1 is frequently found in various types of human tumours and results from clonal rearrangement and/or amplification involving chromosomal region 11q13. In order to evaluate the pathological relevance of cyclin D1 overexpression in human breast cancer, we generated a polyclonal antiserum against the carboxy-terminal part of the cyclin D1 protein. After affinity purification, the antiserum specifically detected overexpression of cyclin D1 in formalin-fixed, paraffin-embedded tumour material also. The intensity of the nuclear stainings was, in general, proportional to the degree of cyclin D1 amplification. We did not encounter significant variability of staining within individual tumours with overexpression of cyclin D1. Overexpression of cyclin D1 appeared to be associated with oestrogen receptor-positive breast tumours, but not with any other clinicopathological parameter tested. Overexpression of cyclin D1 was not prognostic value for recurrence of survival in a consecutive series of 248 operable breast cancer patients (stage I and II). Overexpression of p53 was also not of prognostic significance in this series, but was associated with undifferentiated histology and oestrogen receptor-negative breast tumours, as has been reported previously by others. A high proportion of breast tumours with a low grade of malignancy in this series of operable breast cancer patients may explain discrepancies concerning the prognostic value of amplification and of overexpression of cyclin D1. PMID- 8611373 TI - Variation of hormonal receptor, pS2, c-erbB-2 and GSTpi contents in breast carcinomas under tamoxifen: a study of 74 cases. AB - Seventy-four post menopausal patients with primary non-metastatic invasive ductal carcinomas of the breast were first treated with tamoxifen alone (30 mg p.o. daily) for 5 months. To study changes induced by tamoxifen, core biopsies before treatment and surgical specimens after hormonal therapy were assayed by immunohistochemistry for oestrogen (ER) and progesterone receptors (PR), pS2, GSTpi and c-erbB2. After tamoxifen, ER and PR significantly decreased in 60 and 44 cases respectively, whereas 11 and 19 cases showed no variation and 2 and 11 cases showed an increase (P<10(-4)). GSTpi and pS2 showed a significant increase in 43 and 41 cases, a decrease in 2 and 21 cases and no variation in 29 and 12 cases (P<10(-4) and P=0.04 respectively). c-erbB-2 showed no significant variation under tamoxifen, increased in only three cases and decreased in 13 cases. No relation was found between these variations and efficiency of hormone therapy. Our results allow a better knowledge of protein expression modifications occurring in breast cancer cells under tamoxifen therapy. They are also more consistent with clone selection rather than with phenotype modification. PMID- 8611374 TI - Phase I and pharmacological study of the new topoisomerase I inhibitor GI147211, using a daily x 5 intravenous administration. AB - Topoisomerase I inhibitors are interesting anti-cancer agents with a novel mechanism of action. We performed a phase I study with intravenous GI147211, a new semisynthetic camptothecin analogue, using a daily x 5 schedule administered every 3 weeks, to evaluate the side-effects and pharmacokinetics of the agent. Patients with a histologically confirmed diagnosis of a solid tumour refractory to standard froms of therapy were eligible for the study. GI147211 was given as a 30 min intravenous infusion daily for 5 consecutive days, repeated every 3 weeks. In subsequent patient cohorts the dose was escalated from 0.3 to 1.5 mg m-2 day 1. Pharmacokinetics analysis was performed on days 1 and 4 of the first course using a validated high-performance liquid chromatographic assay and non compartmental methods. A total of 19 patients were entered into the study, one patient was not evaluable for toxicity because only one drug administration was given. Eighteen patients received a total of 67 courses through four dose levels. The dose-limiting toxicities were neutropenia and thrombocytopenia at the dose of 1.5 mg m-2 day-1. Nadirs occurred on day 15 and day 15 respectively. Other toxicities were mild and infrequent and included nausea/vomiting, headache and alopecia. The maximal tolerated dose was 1.2 mg m-2 day-1. One partial response was observed in a patient with colorectal cancer. The total plasma clearance was 999+/-184 ml min-1 (range 640-1329). The volume of distribution was 190+/-461 m-2 and the terminal half-life was 3.7+/-1.2 h. The AUC increased linearly with the administered dose. A steep and significant sigmoid relationship was established between the AUC and the percent decrease of ANC. GI147211 is a new topoisomerase I inhibitor that induced dose-limiting neutropenia and thrombocytopenia in this phase I study. The recommended dose for phase II studies with this schedule is 1.2 mg m-2 x 5 every 3 weeks. PMID- 8611375 TI - Variations in the management and survival of women under 50 years with breast cancer in the South East Thames region. AB - A retrospective, population-based study was undertaken to determine variations in the management of women aged less than 50 years with primary breast cancer in different hospital settings and the influence of these variations on survival. A total of 1757 women who were resident in the South East Thames Health Region aged less than 50 years at the time of diagnosis of breast cancer and who presented during a 5 year period (January 1984 to December 1988) were recorded by the Thames Cancer Registry. The hospitals at which primary surgery was undertaken were categorised as teaching or non-teaching hospitals. The non-teaching hospitals were grouped according to the mean number of patients treated annually during the study period (< or = 2, 3-9, > or = 10 each year). The following factors were compared between these groups: age, extent of disease, tumour morphology, extent of primary surgery (mastectomy vs less than mastectomy), use of axillary surgery (any vs none) and use of systemic adjuvant therapy. Survival rates for the different groups were compared. Registration rates did not differ significantly between health districts. A total of 1485 (85%) women underwent surgery in over 90 different hospitals. In 1324 (86%) of these cases the surgery was undertaken in a total of 42 NHS hospitals within SE Thames Health Region or in seven teaching hospitals in adjacent regions. Mastectomy rates decreased from 52% in 1984 to 28% in 1988 (P<0.0001), but were consistently higher in teaching hospitals (P=0.01). The use of any form of axillary surgery decreased from 49% to 36% over the 5 year period (P=0.003), with significantly lower rates of axillary surgery being performed in non-teaching hospitals (P<0.0001). The proportion of cases recorded as having non-specific morphology was higher in nonteaching than in teaching hospitals (P<0.0001). On multivariate analysis survival was significantly (P<0.001) influenced by stage and tumour histology. Among patients who underwent surgery, the type of hospital in which this was undertaken did not appear to influence survival significantly. This analysis of routine cancer registry data indicates that patients were widely dispersed in a large number of different hospitals and that there were marked variations in practice according to the type of hospital to which patients presented. The treatments provided were frequently at variance with those recommended at a consensus conference held during the study period, particularly in relation to the use of axillary surgery and adjuvant systemic therapy. The way in which services are currently provided may hamper the delivery of appropriate management and comprehensive support. These data thus have implications for the purchasing and provision of services for this common condition. PMID- 8611376 TI - Assesssment of the effect of pretreatment with neoadjuvant therapy on primary breast cancer. AB - Patients with invasive cancer of the breast (T1-4, N0-2, M0) were assigned to pretreatment based on oestrogen receptor (ER) status; patients with ER-negative tumours received chemotherapy [mitozantrone, methotrexate and mitomycin C (MMM)] for 3 months, patients with ER-positive tumours underwent endocrine therapy [luteinising hormone releasing hormone (LHRH) agonist goserelin (leuprolide premenopausal) or 4-hydroxyandrostenedione (formestane-post-menopausal)] for 3 months. Of the first 100 patients assessed at 3 months, 47 with ER-positive tumours had a 40.4% response (premenopausal 53.8%; post-menopausal 35%) and 53 with ER-negative tumours had a 60% response (premenopausal 57%; post-menopausal 63%). Patients with early breast cancer (T1/T2) had a complete clinical resolution in 41% (16/39) of cases after MMM and in 20% (7/35) of cases following endocrine therapy compared with 14% (2/14) advanced tumours (T3/T4) following MMM and (0/12) following endocrine therapy. However, in those patients achieving a complete clinical response, subsequent appropriate surgery showed that 16 of 19 patients (84%) had evidence of residual viable tumour on histological examination. PMID- 8611377 TI - Glutathione-S-transferase activity and isoenzyme levels measured by two methods in ovarian cancer, and their value as markers of disease outcome. AB - A study has been carried out to investigate the cellular distribution and levels of glutathione-S-transferase isoenzymes (GST), acidic (pi), basic (alpha) and neutral (mu), in ovarian tumour biopsies, and to measure GST activity in the same tumour specimens. Two methods of assessing isoenzyme levels (immunohistochemistry and Western blot) were compared. Well-known important clinicopathological features were correlated with response to treatment, overall survival and progression-free survival for each of 97 patients from whom biopsies had been obtained. The glutathione-S-transferase isoenzyme levels were also correlated with overall and progression-free survival, and with the important clinicopathological features. As expected, there was a significant correlation between FIGO stage, histological grade of tumour, amount of residual disease after staging laparotomy, response to chemotherapy, and both overall and progression-free survival. Glutathione-S-transferase isoenzyme levels (acidic, basic and neutral) measured by Western blot were not found to be significantly correlated with any of the clinicopathological parameters tested. Using the immunohistochemistry method of detection there was a correlation between the GST acidic isoenzyme level and the amount of residual disease remaining after initial debulking surgery (higher levels were detected in the group with no residual disease, P=0.034), and also between the GST acidic isoenzyme level and the type of chemotherapy regimen used. Higher levels of the acidic isoenzyme were present in tumour biopsies taken from the patient group who had received a combination regimen (cyclophosphamide, carboplatin, ifosfamide and doxorubicin). The neutral and basic GST isoenzyme levels were not significantly correlated with any of the clinicopathological parameters. None of the GST isoenzyme levels were significantly correlated with response to treatment, overall survival or progression-free survival (using either method of detection). Similarly, glutathione transferase activity showed no significant correlation with prognosis or survival. PMID- 8611379 TI - Pharmacokinetically guided dosing of carboplatin and etoposide during peritoneal dialysis and haemodialysis. AB - Two patients with relapsed Wilms' tumour and renal failure requiring dialysis were given carboplatin and etoposide by pharmacokinetically guided dosing. The target area under the drug plasma concentration vs time curve (AUC) was 6 mg ml-1 min for carboplatin and 18 and 21 mg ml-1 min for etoposide. On course 1 measured AUCs of carboplatin and etoposide were 6 and 20 mg ml-1 min for patient 1 and 6 and 21 mg ml-1 min for patient 2 respectively. Peritoneal dialysis did not remove carboplatin or etoposide from the plasma, however carboplatin but not etoposide was cleared by haemodialysis. Therapy with carboplatin and etoposide is possible in children and adults with renal failure who require dialysis, but in this situation pharmacokinetic monitoring is essential. PMID- 8611378 TI - Intraoperative detection of somatostatin-receptor-positive neuroendocrine tumours using indium-111-labelled DTPA-D-Phe1-octreotide. AB - After injection of 111In-labelled DTPA-D-Phe1-octreotide, intraoperative tumour localisation was performed using a scintillation detector in 23 patients with neuroendocrine tumours. Count rates from suspect tumour lesions and adjacent normal tissue were expressed as a ratio before (Rin situ) and after (Rex vivo) excision. 111In activity concentration ratios of tumour tissue to blood (T/B) were determined in a gamma counter. In patients with midgut carcinoids, (all scintigraphy positive), false Rin situ recordings were found in 4/29 macroscopically identified tumours. T/B ratios were all high (27-650). In patients with medullary thyroid carcinomas (eight out of ten scintigraphy positive), misleading Rin situ results were found in 4/37 macroscopically identified tumours. T/B ratios were lower (3-39) than those seen in midgut carcinoids. Two out of four patients with endocrine pancreatic tumours had positive scintigraphy, reliable intraoperative measurements and very high T/B ratios (910-1500). One patient with a gastric carcinoid had correct measurements in situ and ex vivo with high T/B ratios (71-210). In situ measurements added little information to preoperative scintigraphy and surgical findings using the present detection system. Rex vivo measurements were more reliable. The very high T/B ratios seen in midgut carcinoids and some endocrine pancreatic tumours would be favourable for future radiation therapy via somatostatin receptors. PMID- 8611380 TI - Implicit rationing criteria in non-small-cell lung cancer treatment. AB - Data collected from lung cancer patients attending the Victoria Clinic of the British Columbia Cancer Agency are used to investigate how resources are rationed in the treatment of non-small-cell lung cancer (NSCLC). An ordered logit model is estimated to analyse empirically the relationship between treatment selection and: tumour stage, size and differentiation; the Feinstein index; Karnofsky performance status (KPS); and the patient's age, gender and marital and smoking status. Implicit rationing is found to occur with respect to all of these factors except the Feinstein index, gender and marital status. With respect to age, KPS and smoker status the main empirical results are: (a) an increase in age from 50 to 85 reduces the expected treatment expenditure by 50-70%, depending on the patient's KPS and smoker status; (b) patients with a KPS less than 80 and of 80, receive 30-46% and 75-85%, respectively, of the expected treatment expenditure for patients with a KPS of 90 or 100, depending on age and smoker status; (c) the expected treatment expenditure for active smokers is about 71-86% of the expenditure for non- or former smokers depending on age and KPS. PMID- 8611381 TI - Recombinant human interleukin 6 in metastatic renal cell cancer: a phase II trial. AB - A phase II trial investigating the anti-tumour effects of recombinant human interleukin 6 (rhIL-6) in patients with metastatic renal cell cancer was carried out. RhIL-6 (150 microgram) was administered as a daily subcutaneous injection for 42 consecutive days on an outpatient basis. Forty-nine patients were studied, 12 with and 37 without previous immunotherapy. Forty patients were evaluable for response. A partial remission was noted in two patients, stable disease in 17 and progressive disease in 21. Toxicity was moderate and reversible and consisted mainly of fever, flu-like symptoms, nausea, weight loss and hepatotoxicity. Anaemia, leucocytosis and thrombocytosis and induction of acute phase protein synthesis were noted in most patients. In 15% of the patients anti-IL-6 antibodies developed, and were neutralising in only one patient. Baseline plasma IL-6 concentrations did not correlate with tumour behaviour before or after rhIL 6 treatment. In conclusion, rhIL-6 can be safely administered on an outpatient basis for prolonged period of time and has moderate, reversible toxicity. Its administration induces IL-6-antibody production in only a minority of patients. Antitmour effects of rhIL-6 in metastatic renal cancer are limited. PMID- 8611383 TI - Clinicopathological characteristics of gastric cancer in the elderly. AB - The clinicopathological features of 380 elderly patients 70 years of age or older with gastric cancer were reviewed retrospectively from hospital records between 1969 and 1993. They were then compared with 1134 middle-aged patients between 40 and 69 years. The elderly constituted 18.4% of all gastric cancer patients 20 years ago but now comprise 24.4% of all patients in the most recent decade, despite the overall decrease in the rate of gastric cancer. The distinguishing histological features of gastric cancer in the elderly were an intestinal type of cancer, expansive tumour growth and synchronous multiplicity of the lesions. Elderly patients had a similar rate of tumour extension but had poorer survival as compared with the middle-aged patients. Post-operative death within 30 days after surgery was also higher in the elderly than in the middle-aged patients. PMID- 8611382 TI - Experience with poorly myelosuppressive chemotherapy schedules for advanced myeloma. The Cooperative Group of Study and Treatment of Multiple Myeloma. AB - In a multicentre study, 83 patients with advanced and previously uniformly treated multiple myeloma (MM) were randomised between cyclophosphamide (600 mg m 2) and epirubicin (70 mg m-2), administered every 3 weeks for three courses and both associated with prednisone and interferon-alpha2b. Both regimens were administered on an outpatient basis and had low haematological toxicity. Clinical results were similar. Overall response rate (43%) and median response and survival (5.9 and 14.1 months respectively) compare well with those obtained with more aggressive chemotherapy schedules. PMID- 8611384 TI - Tallimustine in advanced previously untreated colorectal cancer, a phase II study. AB - Tallimustine is a novel benzoyl mustard derivative from distamycin A with a unique mode of action. It is a DNA minor groove binder and produces highly sequence-specific alkylations. Previous studies have shown significant anti tumour effects in animal models. We performed a phase II study in previously untreated patients with advanced colorectal cancer, using a schedule of i.v. bolus infusions of 900 microgram m-2 once every 4 weeks. Seventeen patients were enrolled, and no responses were documented in 14 evaluable patients. Toxicity mainly consisted a highly selective neutropenia, which warrants further investigation of this agent in combination with myeloid growth factors. PMID- 8611385 TI - Trends in survival from cancers of the oral cavity and pharynx in Scotland: a clue as to why the disease is becoming more common? AB - Data were examined to determine trends in survival from cancers of the oral cavity and pharynx in Scotland between 1968 and 1987, and to analyse survival rates and the previously noted increases in the incidence of such cancers according to the level of social deprivation. Incidence data on oral cavity and pharyngeal cancer and survival rates following diagnosis were obtained from the Information and Statistics Division of the Common Services Agency for the National Health Service in Scotland, covering the period 1968-92. It was found that survival rates for cancers of the tongue, mouth and pharynx diagnosed among persons less than 65 years of age decreased between 1968-72 and 1983-87. Five year relative survival rates fell from 47% to 39% over this period, while the equivalent rates among persons older than 65 years have shown a modest improvement from 34% to 38%. When considered by level of social deprivation, survival is lower among persons from the most deprived areas, and it is among such persons that the recent increases in occurrence of cancers of the oral cavity and pharynx have primarily occurred. The poorer survival among those from more socially deprived areas, and the evidence that the largest increase in incidence has occurred in such areas may to some extent explain the non favourable trends in mortality. More importantly it emphasises the potential benefits of targeting such a population for oral health information. An educational campaign should include both information on the risk factors for developing oral cancer, and also the importance of seeking an early professional consultation in the case of symptoms. PMID- 8611386 TI - Frequent gene deletions in potentially malignant oral lesions. AB - Some oral cancers are preceded by premalignant lesions which include leucoplakia and erythroplakia. At present there are no reliable markers to identify lesions that may progress to malignancy. We have analysed 30 potentially malignant oral lesions for deletions at chromosomal regions that harbour tumour-suppressor genes for oral cancer. A total of 16 of 30 cases (53%) showed loss of heterozygosity (LOH) or allele imbalance at TP53, DCC, 3p21.3-22.1 or 3p12.1-13. These genetic alterations were detected in dysplastic lesions but not in histologically normal mucosa and may be early events in the carcinogenic process. A total of 64% of dysplastic lesions that recurred during the study showed LOH or allele imbalance in the initial biopsy and the number of genetic abnormalities increased in the tumours that developed. This type of molecular profiling may help to identify patients with lesions that may recur or acquire additional genetic events and progress to malignancy. PMID- 8611387 TI - A case-control study of lactation and cancer of the breast. AB - We have examined the relation of lactation, by total duration, with breast cancer risk among pre- and post-menopausal women. In a hospital-based case-control study conducted in Athens (1989-91), involving 820 patients with confirmed breast cancer and 795 orthopaedic patient controls and 753 hospital visitor controls, logistic regression was used to analyse the data controlling for demographic, nutritional and reproductive factors, including parity and age at any birth. Among post-menopausal women, there was no association between breastfeeding and breast cancer risk, but among premenopausal women those who has breastfed for > or = 24 months had an odds ratio of 0.50 (95% confidence interval 0.23-1.41). A reduction of the odds ration was also evident among premenopausal women who had breastfed between 12 and 23 months (odds ratio 0.70; 95% confidence interval 0.34 1.60). In conjunction with several other recent reports these results support the hypothesis that breastfeeding of prolonged duration may reduce the risk of breast cancer among premenopausal women but not among post-menopausal women. The biology underlying this different effect remains unknown, and the practical implication of the finding is a marginal importance. PMID- 8611388 TI - Helicobacter pylori gastritis and serum pepsinogen levels in a healthy population: development of a biomarker strategy for gastric atrophy in high risk groups. AB - This study aimed to estimate the prevalence and type of chronic gastritis in an asymptomatic working population and to determine whether a combination of serum pepsinogen levels and Helicobacter pylori serology could be used to identify a subgroup with atrophic gastritis at elevated risk of gastric carcinoma. A 10% subsample of 544 male volunteer factory workers aged 18-63 years and participating in a larger study underwent endoscopy and biopsy. Of these men, 29 were seropositive for Helicobacter pylori; all but three (89.7%) had chronic gastritis. Serum pepsinogen A levels increased with progression from a corpus predominant pattern of gastritis through pangastritis to an antral predominant pattern. Nine subjects had corpus atrophy, which was in most cases accompanied by fasting hypochlorhydria and hypergastrinaemia. A combination of pepsinogen A below 80 ng ml-1 and Helicobaceter pylori seropositivity detected corpus atrophy with sensitivity 88.9% and specificity 92.3%. A second screening stage, using a pepsinogen A/C ratio of below 2.5 as a cut-off, resulted in a reduction in numbers requiring further investigation but with some loss of sensitivity (77.8%). Application of this two-stage screening programme to the original sample of 544 workers would have resulted in 11 (2.2%) men being selected for follow-up, excluding 25 (5.1%) false negatives. Our results suggest that a combination of serum pepsinogen levels and Helicobacter pylori serology could be useful as a biomarker strategy for detection of individuals at increased risk of gastric carcinoma and for non-invasive investigation of the natural history of Helicobacter pylori gastritis. PMID- 8611389 TI - Risk factors for intellectual and educational sequelae of cranial irradiation in childhood acute lymphoblastic leukaemia. AB - Long-term cognitive and educational sequelae have been inconsistently reported in children who received cranial irradiation (CRT) to prevent central nervous system (CNS) disease in acute lymphoblastic leukaemia (ALL). This study investigates a large and representative sample of survivors of ALL and compares them with non irradiated survivors of cancer and healthy control children to determine the effect of CRT on cognitive and educational ability. Three groups of children were studied: Group 1 (n=100) survivors of ALL treated with chemotherapy and CRT, group 2 (n=50) children with a variety of malignancies treated with chemotherapy alone, group 3(n=100) healthy children. Cognitive and educational abilities of these groups were evaluated using standardised psychometric techniques. Significant differences in cognitive and educational abilities were found between the children in group 1 (chemotherapy + CRT) and the two control groups, with the children receiving CRT performing less well in a range of tests. Greatest differences were detected for tasks dependent on language function including verbal IQ, reading and spelling. Within group 1 a younger age at treatment (less than 5 years) and a higher dose of CRT (24 Gy vs 18 Gy) were predictive of poor long-term outcome for cognitive and education ability. In contrast, children who received chemotherapy alone, with or without intrathecal methotrexate, performed similarly to healthy controls. No gender differences were detected for these measures. PMID- 8611390 TI - Epidemiological evidence of cervical intraepithelial neoplasia without the presence of human papillomavirus. AB - The aim of this paper was to provide epidemiological evidence to support the notion that cervical intraepithelial neoplasia (CIN) without human papillomavirus (HPV) is a true entity. If a diagnosis of HPV-negative cervical neoplasia is erroneous, one would not expect there to be any differences in risk factors between HPV-positive and HPV-negative patients. Patients at a gynaecological outpatient clinic of a university hospital [a total of 265 consecutive women with dyskaryotic cervical smears who were subsequently diagnosed with CIN I (n=37), CIN II (n=48) or CIN III (n=180)] completed a structured questionnaire regarding smoking habits and sexual history. Analysis of an endocervical swab for Chlamydia trachomatis, analysis of a cervical scrape for HPV, and morphological examination of cervical biopsy specimens were also performed. HPV was found in 205 (77.4%) out of the 265 women. Univariate analysis showed that current age (P=0.02), current smoking behaviour (P=0.002) and the number of sexual partners (P=0.02) were significantly associated with the presence of HPV. Age at first sexual intercourse, a past history of venereal disease or genital warts, and current infection with Chlamydia trachomatis were not associated with the presence of HPV. Using multivariate logistic regression analysis, the number of sexual partners and current smoking behaviour showed an independent significant association with HPV. HPV-negative and HPV-positive CIN patients differ with respect to the risk factors for HPV. These findings suggest that HPV-negative CIN is a separate true entity. PMID- 8611391 TI - The South Australian Breast X-Ray Service: results from a statewide mammographic screening programme. AB - The South Australian Breast X-Ray Service is a centralised breast cancer screening programme in the State of South Australia. In its first 5 years of operation nearly 100 000 screens were performed. This study reports the clinical performance of the programme and compares it with other published series. Women aged 40 years and over were screened with two-view mammography every 2 years. Radiologists double-read the screening films and multidisciplinary teams assessed the recalled women at a single centre. In the prevalent round 76 106 women were screened, and subsequently 21 506 of them were rescreened. The recall rate for further investigation was 4.9% in the prevalent round and 2.4% in the incident rounds. The cancer detection rate per 1000 women was 7.0 in the prevalent screening round and 3.4 in the incident rounds. Forty-two per cent of invasive carcinomas measured < or = 10 mm in the prevalent screening round and the median tumour size was 12 mm. The benign to malignant biopsy ratio was 1:1.4 in the prevalent round and 1:2.8 in the incident rounds. In the prevalent round 77% of invasive tumours were lymph node negative and this proportion increased to 86% in the incident rounds. PMID- 8611392 TI - Rising incidence of pancreatic carcinoma in middle-aged and older women-time trends 1961-90 in the city of Malmo, Sweden. AB - The city of Malmo (population 230 000), situated in the south of Sweden, is in an area which has the highest incidence of pancreatic cancer in the country. The present study was designed to assess time trends of the incidence of pancreatic cancer 1961-90. The 1314 incident cases, 651 men and 663 women, were retrieved from the Regional Tumour Register and the National Cause-of-Death Register. In 75% of cases diagnosis was based on autopsy. Twenty per cent of the these cases were first found at autopsy, being undiagnosed. The average age-standardised incidence was 20.4 per 10(5) person-years for men and 13.7 for women. The incidence was higher for men than for women in all age groups above 44 years. No change in incidence over time was observed for men. In older and middle-aged women there was however a statistically significant increase. The average relative change in women above age 64 was 1.7% per year after age adjustment and in women aged 55-64 years 2.6% per year. We have found no results indicating that this increasing incidence could be caused by detection bias as a result of changing autopsy rates during the study period and hence conclude that the observed increase is explained by a growing number of women being exposed to factors with a potential tumour-promoting or -initiating effect. PMID- 8611393 TI - Cancer in the offspring of survivors of childhood leukaemia and non-Hodgkin lymphomas. PMID- 8611394 TI - A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells. AB - A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility. PMID- 8611395 TI - Dipyridamole increases VP16 growth inhibition, accumulation and retention in parental and multidrug-resistant CHO cells. AB - Dipyridamole (DP) has been shown to reverse multidrug resistance (MDR) via interactions with P-glycoprotein (P-gp). The effect of DP on VP16 growth inhibition was investigated in parental (CHO-K1) and MDR (CHO-Adr(r)) Chinese hamster ovary cells. CHO-Adr(r) cells were 18-fold resistant to VP16 and intracellular accumulation was 28% less than in CHO-K1 cells. DP reduced the resistance of CHO-Adr(r) to VP16 by a factor of 2-3 and caused a similar potentiation of VP16 growth inhibition in the parental cells. A time-dependent increase in intracellular VP16 accumulation, which was similar in both cell lines, was caused by DP. The intracellular retention of VP16 was increased 2- to 3-fold by DP in both cell lines. The magnitude of the effect of DP on all three parameters measured was similar (2- to 4-fold), suggesting that the increased growth inhibition was related to increased intracellular exposure to VP16 owing to the inhibition of the efflux of VP16 by DP. However, since the effect of DP was similar in both parental and P-gp-overexpressing cells it is unlikely that the potentiation of VP16 by DP is mediated via an interaction with P-gp. PMID- 8611397 TI - Distribution and activity of doxorubicin combined with SDZ PSC 833 in mice with P388 and P388/DOX leukaemia. AB - SDZ PSC 833 (PSC 833) is a non-immunosuppressive analogue of cyclosporin A and is a potent modifier of P-glycoprotein (P-gp)-mediated multidrug resistance. The present study was undertaken to evaluate whether doxorubicin (DOX) pharmacokinetic and anti-tumour activity on P388- and P388/DOX-resistant leukaemia was modified by PSC 833 pretreatment. P388- or P388/DOX-bearing mice were given PSC 833 intraperitoneally 30 min before an intravenous injection of DOX. The levels of DOX were determined by a high-performance liquid chromatography method in leukaemic cells and in normal tissues (heart, lung, liver, small intestine, kidney and spleen). In all tissues, DOX concentrations were significantly increased in mice pretreated with PSC 833. The difference was greatest in P-gp-overexpressing P388/DOX cells, the DOX area under the curve being approximately seven times greater after PSC 833 and DOX than after DOX alone. In P388 cells the difference was approximately 2.5 times, as in the majority of normal tissues. As expected DOX levels in P388 cells were higher than in P388/DOX cells in mice treated with DOX alone, whereas after PSC 833 and DOX the levels of DOX were similar in the two leukaemic lines. In spite of this PSC 833 was unable to reverse the resistance to DOX of P388/DOX leukaemia in vivo, suggesting that mechanisms other than P-gp expression are responsible for resistance. PMID- 8611398 TI - Ranitidine and cimetidine differ in their in vitro and in vivo effects on human colonic cancer growth. AB - Histamine has recently been shown to be a growth factor for some gastric and colorectal cancer cells. Previous studies have shown that cimetidine blocks in vitro and in vivo histamine-stimulated growth and cAMP release from the human colonic cancer cell line, C170. In this study, ranitidine, another H2 receptor antagonist, did not affect either basal or histamine-stimulated in vitro proliferation of C170, and failed to prevent cAMP release in vitro. Ranitidine did not inhibit in vivo growth of C170 at a dose of 1, 10, 25, 50 or 100 mg/kg, in contrast to 50 mg/kg/day cimetidine, which produced 39.3% inhibition of tumour volume (p<0.01) after 23 days' treatment. Ranitidine did not inhibit in vivo histamine-stimulated growth of C170 cells . LIM2412, another colonic cancer cell line, was significantly stimulated by both cimetidine and ranitidine in vivo. Ranitidine had no effect on in vitro cell proliferation. PMID- 8611396 TI - 3-methyladenine-DNA-glycosylase and O6-alkyl guanine-DNA-alkyltransferase activities and sensitivity to alkylating agents in human cancer cell lines. AB - The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3 meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes. PMID- 8611399 TI - Human liver cancer cells and endothelial cells incorporate iodised oil. AB - Iodised oil (lipiodol) administered via the hepatic artery localises selectively in primary liver cell cancers (hepatocellular carcinomas or HCCs) for prolonged periods and has been used as a vehicle for cytotoxic agents. Despite clinical use, the mechanism of lipiodol retention by tumours has remained unclear, embolisation of oil droplets in the tumour vasculature being the prevailing hypothesis. We have investigated the role of tumour and endothelial cells in lipiodol retention. Human liver tumour (Hep G2) cells and human umbilical vein endothelial cells in culture were exposed to lipiodol. Light microscopy using selective silver impregnation stains and transmission electron microscopy revealed lipiodol incorporation by both cell types, probably by pinocytosis. This was not associated with cellular injury in terms of cell lysis, cell replication or radio-labelled leucine uptake. Histological analysis of 24 HCCs either surgically resected or discovered incidentally at liver transplantation (with prior arterial injection of lipiodol) revealed vesicles of lipiodol in the cytoplasm of tumour cells and endothelial cells lining tumour vessels. Thus, lipiodol is likely to deliver cytotoxic agents directly into tumour cells and endothelial cells, both in vitro and in vivo. This may also apply to other lipids and to other human tumours. These findings have significant therapeutic implications. PMID- 8611400 TI - PET imaging of primary mediastinal tumours. AB - Mediastinal masses include a wide variety of tumours and remain an interesting diagnostic challenge for radiologist. We performed positron emission tomography (PET) studies of primary mediastinal tumours in order to predict the malignancy of these tumours preoperatively. Twenty-two patients with primary mediastinal tumours were studied with PET using 2-deoxy-2-[18F]fluoro-D-glucose (FDG). The histological findings of surgical pathology or biopsy, or mediastinoscopy were compared with those of computerised tomography (CT) and PET. PET images were evaluated semiquantitatively using the differential uptake ratio (DUR). Increased FDG uptake was observed in nine of ten patients with malignant tumours, including thymic carcinomas, lymphomas, invasive thymomas and a case of sarcoidosis. A moderate level of FDG uptake was found in a myeloma, non-invasive thymomas, and a schwannoma, whereas a low uptake was observed in a teratoma and various benign cysts. The mean FDG uptake of malignant tumours was significantly higher than that of benign tumours. Both thymic cancer and invasive thymoma showed a high FDG uptake. CT examination resulted in three false-negative and two false-positive cases when used in predicting tumour invasion, while PET was associated with a false-positive and a false-negative case. In conclusion, the use of FDG with PET is clinically helpful in evaluating the malignant nature of primary mediastinal tumours. Our results also suggest that a high FDG uptake reflects the invasiveness of malignant nature of thymic tumours. PMID- 8611401 TI - Multiplicity of fibronectin-binding alpha V integrin receptors in colorectal cancer. AB - Current data from in vitro and in vivo animal models indicate that fibronectin binding integrin receptors expressed by colon cancer cells may regulate tumour growth. While individual members of the beta 1 subfamily of integrins have now been clearly identified in colorectal cancer, little information exists with respect to the alpha V subfamily. In the present study we show that alpha V can associate with multiple and different beta subunits capable of binding fibronectin in this tumour type. This is likely to have functional implications for growth and spread of colorectal cancer. PMID- 8611402 TI - Distribution of lymphoid nodules, aberrant crypt foci and tumours in the colon of carcinogen-treated rats. AB - Sprague-Dawley rats were given eight weekly subcutaneous injections of 1,2 dimethylhydrazine (DMH) or of vehicle then were sacrificed at 1, 5 or 24 weeks after the last injection of DMH. The locations of pre-existing aggregates of lymphoid nodules (ALNs), the location and multiplicity (size) of aberrant crypt foci (ACF), and the locations of tumours in the colon were determined. A trimodal distribution of pre-existing ALNs along the length of the colon was significantly correlated with the timodal distribution of DMH-induced adenocarcinomas (ACs). A unimodal peak in ACF of all sizes occurred between the sites of two distal ALNs. Thus, the distribution of ACF at 1 or 5 weeks did not correlate with distribution of AC found at 24 weeks. Of the 2640 ACF observed at 1 or at 5 weeks, none were found in the proximal 25% of the colon where ACs eventually occurred. It was concluded that: (1) ALNs play a promotional role in AC formation; (2) the ACs which form in the proximal quarter of the colon seldom if ever form via an ACF precursor; and (3) the location, the number and the size of ACF observed early after DMH exposure did not correlate with the location or predict the incidence of ACs which eventually formed in the colon. PMID- 8611403 TI - A study on the levels of calmodulin and DNA in human lung cancer cells. AB - In order to study the role of calmodulin (CaM) in the proliferation of lung cancer cells, the CaM level of the specimens of 40 cases of primary lung cancers and the DNA content of the specimens of 35 cases of primary lung cancers were determined with phosphodiesterase assay and flow cytometry respectively. It was found that the CaM level of lung cancers was significantly higher than that of host lungs, benign lung diseases and normal lungs (p<0.001) and that it was significantly correlated with the histopathological grading and TNM staging of the lung cancers. It was also found that the cellular DNA content of lung cancers, like the CaM level, was also significantly higher than that of benign lung diseases and normal lungs (p<0.001). There was a significant positive correlation between the cellular DNA content and tissue CaM level in lung cancers (r=0.885). It is believed that CaM plays an important role in the proliferation of lung cancer cells through the mechanism of the promotion of an uncontrolled synthesis of DNA in the cells. Consequently, it is inferred that CaM antagonists may be tried as a chemotherapeutic agent for lung cancer. PMID- 8611404 TI - Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin. AB - The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis. PMID- 8611405 TI - Expression of tropomyosin isoforms in benign and malignant human breast lesions. AB - High molecular weight tropomyosins (tms) are commonly down-regulated in fibroblasts transformed by oncogenes. Previous studies have also demonstrated that specific tm isoforms are down-regulated in human breast carcinoma cell lines. We examined tropomyosin isoforms in cells prepared from non-cancerous breast lesions and primary human breast carcinomas. The average level of expression of all three high molecular weight tm isoforms (tm 1-3) in carcinomas was generally found to be less than 25% of that observed in non-cancerous breast lesions. Interestingly, the expression of tm 1 was found to be 1.7-fold higher in primary tumours with metastatic spread to axillary lymph nodes compared with primary tumours with no evidence of metastasis (p<0.05). Similarly, tm 1 expression was higher in two 12V-H-ras transformed fibroblast cell lines capable of experimental metastasis compared with three weakly metastatic cell lines. We conclude from these studies that expression of high molecular weight tm isoforms is low in primary breast carcinomas, and that metastatic tumours express relatively high levels of tm 1. PMID- 8611406 TI - P53 expression, DNA ploidy and S-phase cell fraction in operable locally advanced non-small-cell lung cancer. AB - The identification of biomarkers to complement pathological stage for a more accurate prognosis and help clinicians decide on treatment is still an open problem for patients with lung cancer. Expression of P53 protein was detected by an immunohistochemical approach using the monoclonal antibody PAb1801 on paraffin embedded sections of tumours obtained surgically from 102 stage II - IIIa patients with non-small-cell lung cancer (52 squamous cell carcinomas, 50 adenocarcinomas). [3H]Thymidine labelling index, an indicator of the S-phase cell fraction, was evaluated on histological sections of [3H]thymidine-labelled tumour samples. DNA ploidy was defined by flow cytometric analysis on frozen tumour tissue. The biomarkers, histology and pathological stage were analysed in relation to relapse-free survival in univariate and multivariate analyses. Stage and interaction between [3H]thymidine labelling index and histology provided significant prognostic information for the overall series. [3H]thymidine labelling index was an independent prognostic indicator of 3 year relapse-free survival in patients with adenocarcinoma. The results indicate the importance of cell proliferation to complement prognostic information provided by pathological stage in patients with stage II-IIIa adenocarcinomas. PMID- 8611407 TI - Measurement of extracellular fluid carboplatin kinetics in melanoma metastases with microdialysis. AB - Clinical anti-tumour efficacy of anti-cancer drugs is a function of dose intensity, i.e. the concentration--time profile in tumour tissue. Hence, information on drug concentration profiles in tumours is of critical importance but appropriate methods for measurement are lacking. The aim of the present study was to obtain, by microdialysis sampling, concentration--time profiles in a solid tumour (melanoma) of a model anti-cancer drug, carboplatin, and thereby to assess the scope of microdialysis for tumour pharmacokinetic studies in man. Six patients with cutaneous melanoma metastases at the extremities or body trunk, scheduled to receive carboplatin (400 mg m-2 i.v.) were studied. Carboplatin concentrations were monitored in serum, intratumoral and subcutaneous tissue. Calibration of the microdialysis probes was carried out in vitro and in vivo with use of the retrodialysis method. Complete carboplatin concentration vs time profiles in tumour and subcutaneous tissue were obtained. Major pharmacokinetic parameters (maximum concentration, time to maximum concentration, area under the curve, elimination half-life) were calculated for tissues and tumour/serum concentration ratios for carboplatin were derived. Mean free concentrations of carboplatin in cutaneous melanoma metastases reached only about 50-60% of total serum levels; maximal intratumoral concentrations were 7.6 (+/-2.0; s.e.m.) microgram/ml, mean concentrations in subcutaneous tissue were similar to those in tumour. The present study demonstrates that microdialysis is a novel tool for measuring drug concentrations in solid tumours in humans in vivo and appears to be a valuable addition for pharmacokinetic/pharmacodynamic studies in oncology. PMID- 8611408 TI - Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice. AB - In this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4h) or systemically (200 mg/kg,i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVB irradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 +/- 20 nm and fluorescence was detected through a filter that passes light of 670 +/- 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and a 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved. PMID- 8611410 TI - Cancer in electrical workers: an analysis of cancer registrations in England, 1981-87. AB - Associations between work in the electrical and electronic industry and cancer incidence were assessed using data for 371 890 cancers registered in England between 1981 and 1987, of which 7981 were in electrical workers. Proportional registration ratios (PRRs) were calculated, both with and without the commonest cancers, with adjustment for age, social class, cancer registry of origin and sex. Of four cancers previously linked with work in the electrical and electronic industry (leukaemia, brain, breast and melanoma), only two were significantly raised: leukaemia (PRR=124, 95% CI=109-142, based on 217 cases) and malignant brain cancer (PRR=118, 95% CI=103-136, based on 204 cases). A significantly increased risk was also observed for pleural cancer (PRR=201, 95% CI=167-241, based on 115 cases). The histology of almost 90% of pleural cancers was coded as mesothelioma, confirming the previously observed association between pleural cancer and exposure to asbestos in electrical workers. The extent to which workplace exposures to extremely low frequency electromagnetic fields explains the excesses seen here for leukaemia and brain cancer requires further study. PMID- 8611409 TI - Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma. AB - Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms. PMID- 8611412 TI - Have increases in solar ultraviolet exposure contributed to the rise in incidence of non-Hodgkin's lymphoma? AB - The incidence of non-Hodgkin's lymphoma (NHL) has increased substantially in many countries over recent decades. The aetiology of this cancer is poorly understood, and this rise is largely unexplained. The incidence of NHL is known to increase markedly following immune suppression. In the light of evidence that exposure to ultraviolet radiation (UVR) may cause systemic immune suppression, part of the recent increase in NHL incidence may reflect population-based increases in UVR exposure. That such exposure increases have occurred is inferred from the widespread increases in skin cancer incidence in fair-skinned populations, especially malignant melanoma (MM), over recent decades. Epidemiological evidence presented here in support of the proposed UVR-NHL relationship includes the following: in Caucasian populations there is a moderate positive correlation between ambient UVR level, by latitude, and NHL incidence; there is also a positive correlation between time trends in MM incidence and NHL; there is some evidence that migration across latitude gradients induces concordant shifts in risks of NHL and MM. Data from two historical cancer patient registers show that, in individuals, these two cancers concurred a little more often than expected. These findings support recent suggestions that UVR-induced impairment of immune functioning contributes to the aetiology of NHL. PMID- 8611411 TI - Comparison between lentigo maligna melanoma and other histogenetic types of malignant melanoma of the head and neck. Scottish Melanoma Group. AB - A study of 953 invasive cutaneous malignant melanomas of the head and neck was performed to determine differences between lentigo maligna melanoma and other histogenetic types with regard to patients and sites affected; prognosis was analysed in 595 of these cases. The cases studied comprised all head and neck melanomas registered with the Scottish Melanoma Group between 1979 and 1992, apart from the 3% of cases that were unclassifiable or rare histogenetic types. The histogenetic types of melanoma were 498 (52%) lentigo maligna melanoma (LMM), 237 (25%) superficial spreading melanoma (SSM) and 218 (23%) nodular melanoma (NM). All types increased in incidence throughout the study period. Patients with LMM (mean age 73 years) and NM (mean 68 years) were significantly older than those with SSM (mean 57 years). There were significant anatomical subsite differences related to sex of patients and histogenetic type of melanoma; melanomas on the face were more frequent in females and 90% of LMM occurred at this site, whereas melanomas on the scalp, neck and ears were more frequent in men. Kaplan-Meier estimates of the probability of survival were produced for the 595 of these 953 patients with 5 year follow-up details. In this group of patients the prognostic significance of tumour thickness, Clark level of invasion, ulceration, histogenetic type of melanoma and number of mitoses were studied using stepwise variable selection of procedures. Each of these possible prognostic factors attained individual significance but the tumour thickness was the dominant risk factor in the proportional hazards analysis. When patients were divided into four sex/ulceration subgroups (male/ulcerated, female/ulcerated, male/non-ulcerated, female/non-ulcerated) and analysed by proportional hazards analysis, no variable other than the tumour thickness had any further prognostic effect. Histogenetic type did not remain an independent prognostic variable at this stage. Despite sex and subsite differences, the prognosis for invasive lentigo maligna melanoma does not differ from that for other histogenetic types after controlling for tumour thickness. PMID- 8611413 TI - Recent increase in the incidence of non-Hodgkin's lymphoma among young men and women in Denmark. AB - Time-related trends in the incidence of non-Hodgkin's lymphoma (NHL) in Denmark were analysed for the period 1943-89. A total of 13 822 patients (7565 men and 6257 women) were included in the study. In men, world-standardised incidence rates per 100 000 population increased from 2.5 in 1943-47 to 9.3 in 1988-89. In women, a similar increase was seen, i.e. from 1.9 in 1943-47 to 6.5 per 100 000 population in 1988-89. For all birth cohorts, the male-to-female incidence ratio was highest among young subjects and fell significantly after the age of 29 years. Trends in age-specific incidence were analysed separately for two periods, i.e. 1943-77 and 1978-89, reflecting an early, pre-AIDS period and a later period possibly influenced by AIDS. In both periods, the incidence of NHL increased in all age groups. However, in recent years a noticeable increase in incidence averaging 8% annually was observed in men and women aged 40-49 years. A number of factors including changes in the perception of NHL and in the diagnostic methods available are considered insufficient to explain the observed increase. The remarkable and parallel time trends observed in young men and women in recent years indicate that factors other than AIDS must be considered. PMID- 8611414 TI - Breast cancer mortality rates are levelling off or beginning to decline in many western countries: analysis of time trends, age-cohort and age-period models of breast cancer mortality in 20 countries. AB - Age-standardised mortality rates for breast cancer were examined for 20 countries in Europe, North America, Australia and New Zealand from 1950 to 1992 and age birth cohort and age-period of death models were fitted to the data. Breast cancer mortality rates generally increased in the earlier decades, but more recently rates have levelled off or begun to decline in most countries. Only in 4 of the 20 countries studied, Belgium, Hungary, Poland and Spain, was there no evidence of a decline or leveling off or mortality in recent birth cohorts or in recent years. In the other countries the decline in mortality appeared to be in part due to birth cohort effects and in part due to period effects. The birth cohort effects were suggestive of a decline in breast cancer rates among women born after about 1920 and were evident in many countries especially Canada, The Netherlands, The United Kingdom and the United States. The decline in mortality in women born after 1920 appeared to be in part related to a reduction in childlessness and a reduction in age at first birth in those generations. As well as the birth cohort effects, there was some evidence of a recent overall decline in mortality rates in several countries, e.g. Austria, FRG, Greece and the UK, and this may be due to an increase in survival resulting from improved management and treatment of women with breast cancer. PMID- 8611415 TI - Breast cancer incidence subsequent to surgical reduction of the female breast. AB - The incidence of breast cancer among 1240 women who were treated surgically for breast hypertrophy in Copenhagen, Denmark between 1943 and 1971 was determined and compared with age- and calendar period-specific rates for the Danish female population. A total of 32 cases of breast cancer had developed by the end of 1990; the expected number was 52.55, yielding a relative risk (RR) of 0.61 [95% confidence interval (CI) 0.42-0.86]. The greatest reduction in risk was observed for women who had 600 g or more of breast tissue removed (RR=0.30; 95% CI 0.10 0.69). This suggests that the number of potential foci is important for cancer development in the female breast. In the group of women who were operated on before the age of 20, four cases of breast cancer developed, compared with 2.23 expected cases, to give an RR of 1.79, suggesting that the aetiology of their breast hypertrophy may be different from that for the rest of the group. PMID- 8611416 TI - Familial testicular cancer in Norway and southern Sweden. AB - Information about occurrence of testicular cancer (TC) in relatives of TC patients has been collected using questionnaires from 797 out of 922 consecutive Norwegian and 178 out of 237 Swedish patients with TC seen at the Norwegian Radium Hospital and the University Hospital Lund in Sweden during 1981-91. Fifty one Norwegian and five Swedish patients had a relative with confirmed TC. Thus, 51/922 (5.5%) of the Norwegian and 5/237 (2.1%) of the Swedish patients treated during the time interval investigated were considered to have familial TC. Thirty two of the patients had an affected first-degree relative. Expected numbers of cancers in the relatives were computed from data in the Norwegian and Swedish Cancer Registries. Standardised incidence ratios (SIRs) were taken as observed numbers of TC/expected numbers of TC in the relatives. The SIR for brothers was 10.2 (95% confidence interval 6.22-15.77). SIR for fathers was 4.3 (1.6-9.3) and for sons 5.7 (0.7-23.2). The point estimate for the risk to brothers in the Norwegian part of the sample to develop TC by the age of 60 was 4.1% (95% CI 1.7 6.6%). This study indicates that genetic factors may be of greater importance in TC than previously assumed. Patients with familial testicular cancer had bilateral tumours more often than sporadic cases (9.8% bilaterality in familial vs 2.8% in sporadic cases, P=0.02). For patients with seminoma age of onset was lower in familial than in sporadic cases (32.9 vs 37.6 years, P=0.06). In father son pairs, there was a statistically significant earlier age of diagnosis in the generation of sons (28.8 years vs 44.9 years, P=0.04). The prevalence of undescended testis (UDT) did not seem to be higher in familial than in sporadic TC (8.2% in familial TC and 13.3% in sporadic cases). This may indicate that different factors are of importance for the development of familial TC and UDT. PMID- 8611417 TI - Risk of cancer in relatives of testicular cancer patients. AB - The incidence of cancer at sites other than the testis has been investigated in the families of 797 Norwegian and 178 Swedish patients diagnosed with testicular cancer during 1981-91. In the families of the Norwegian patients, the total number of cancers in the relatives was significantly lower than the expected number derived from national incidence rates [observed number of cancers 250, expected number of cancers 281.92, standardised incidence ratio (SIR) 0.89, 95% confidence interval (CI) 0.78-1.00]. This finding can be accounted for almost entirely by the finding of fewer than expected prostate and gastrointestinal cancers in the parents of cases. The other common cancers were found at slightly lower than or near the expected levels in the relatives. In the Swedish cohort, which accounts for less than 20% of cases, the observed number of cancers was very close to the expected number. Fourteen fathers of cases had prostate cancer compared with 27.57 prostate cancers expected, giving a SIR of 0.51 (P=0.006). Mothers had more lung cancers (ten cases observed, SIR=2.11, P=0.04) and cancers of the endometrium than expected (13 cases observed, SIR=1.73, P=0.09). These findings may be interpreted as support for theories proposing hormonal dysfunction as causing testicular cancer. Fifty-four gastrointestinal cancers were observed in the parents compared with 68.48 expected (SIR=0.78, P=0.082). Furthermore, testicular cancer was not found to be associated with the known dominantly inherited cancer syndromes [Familial breast (-ovarian) cancer, hereditary no-polyposis colon cancer]. However, one patient belonged to a Li Fraumeni family, raising the possibility that testicular cancer may be an infrequent component of this rare cancer syndrome. This study supports the hypothesis that families of testicular cancer patients are not prone to cancer. PMID- 8611418 TI - Estimating the efficacy of screening by auditing smear histories of women with and without cervical cancer. The National Co-ordinating Network for Cervical Screening Working Group. AB - The screening histories of all 348 women with invasive cervical cancer diagnosed in 1992 in 24 self-selected district health authorities and health boards in England, Wales and Scotland were compared with those of 677 age- and residency matched controls. The controls were randomly selected from the family health services authority (FHSA) register. Screening histories, comprising the dates and results of all smears taken before the date of diagnosis of the patient's cancer, were determined from the FHSA computer and laboratory records. We estimate that the number of cases of cervical cancer in participating districts in 1992 would have been 57% (95% confidence interval 28-86%) greater if there had been no previous screening. In women under the age of 70 it would have been approximately 75% (31-115%) greater. Extrapolation of the results from this pilot suggests that screening prevented between 1100 and 3900 cases of invasive cervical cancer in the UK in 1992. Women with stage 1B cancer or worse were more likely to have no record of previous screening than controls: 47% of these women under the age of 70 had been adequately screened according to current (5 yearly screening) guidelines, compared with 75% of matched controls. Thirteen per cent of all patients under age 70 had screening histories indicative of inadequate follow-up of smears requiring colposcopy. The proportion of microinvasive cases with screening predating diagnosis was similar to the proportion of controls. There was a strong correlation between stage and age: 56% of cancers in women under 35 were microinvasive compared with just 9% in women 65 years or over. The 'relative protection' following a negative smear was greatest in the first 12 months and fell off towards the end of the fifth year. These data suggest that full adherence to current guidelines could perhaps have prevented another 1250 cases, but additional steps would have been required to prevent some of the 2300 remaining cases in women under the age of 70. PMID- 8611419 TI - Childhood leukaemia in Europe after Chernobyl: 5 year follow-up. AB - The European Childhood Leukaemia - Lymphoma Incidence Study (ECLIS) is designed to address concerns about a possible increase in the risk of cancer in Europe following the nuclear accident in Chernobyle in 1986. This paper reports results of surveillance of childhood leukaemia in cancer registry populations from 1980 up to the end of 1991. There was a slight increase in the incidence of childhood leukaemia in Europe during this period, but the overall geographical pattern of change bears no relation to estimated exposure to radiation resulting from the accident. We conclude that at this stage of follow-up any changes in incidence consequent upon the Chernobyl accident remain undetectable against the usual background rates. Our results are consistent with current estimates of the leukaemogenic risk of radiation exposure, which, outside the immediate vicinity of the accident, was small. PMID- 8611420 TI - Characterisation of antimitotic products from marine organisms that disorganise the microtubule network: ecteinascidin 743, isohomohalichondrin-B and LL-15. AB - The effect of selected marine compounds with anti-tumoral activity on the cell microtubule network was tested by immunofluorescence analyses, or by other in vitro analyses involving competition with colchicine or with GTP for tubulin binding and tubulin polymerisation, studies that were carried out in parallel with other microtubule poisons used as controls. Three compounds were found to disorganise the microtubule network: isohomohalichondrin B, LL-15 and ecsteinascidin 743. The first two compounds prevent microtubule assembly and GTP binding to tubulin. Ecteinascidin 743 disorganises the microtubule network but it does not seem to interact directly with tubulin. PMID- 8611421 TI - Thymidine phosphorylase/platelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma. AB - It is known that angiogenesis plays an important role in the growth and metastasis of solid tumours. Several angiogenic factors have been identified and platelet-derived endothelial cell growth factor (PD-ECGF) is thought to be one such factor. Recently, it was reported that thymidine phosphorylase (dThdPase) is identical to PD-ECGF. Using immunohistochemical staining with an anti-dThdPase antibody, we investigated the correlation between dThdPase expression and the microvessel density in 120 gastric carcinomas. The microvessel density, determined by immunostaining for factor VIII-related antigen, was significantly higher in dThdPase-positive tumours than in dThdPase-negative tumors. There was a significant correlation between dThdPase expression and the increment of microvessel density. Moreover, regarding distant organ metastasis, the frequency of hepatic metastasis was significantly higher (P < 0.01) in patients with dThdPase-positive tumours than in those with dThdPase-negative tumors. In summary, it was suggested that dThdPase expression is closely associated with the promotion of angiogenesis and hepatic metastasis in gastric carcinoma. PMID- 8611422 TI - Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas. AB - Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl 2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form. PMID- 8611423 TI - High frequency of p53 gene mutations in primary breast cancers in Japanese women, a low-incidence population. AB - The pattern of acquired mutations in the p53 tumour-suppressor gene is potentially useful for determining factors contributing to carcinogenesis in diverse populations differing in incidence and/or mortality from the disease. We previously reported differences in mutational patterns of the p53 gene in primary breast cancers from Midwest US Caucasian, African-American and Austrian women. Herein, we report 16 mutations in 27 primary breast cancers from Japanese women from Hirosaki, a population with a low incidence of breast cancer. The frequency of 59.3% of p53 mutations is the highest reported in breast cancers from a particular ethnic group thus far. A relatively high number of mutations (7/16) were heterozygous in at least some tumour cell clusters. Intergroup comparisons of the mutational pattern between this population and several other US, European and Japanese populations do not show any statistically significant differences. There were recurrent mutations at two sites, codon 273 (R --> H; three mutations), a common hotspot of mutations in breast and other cancers, and codon 183 (S --> Stop; two mutations), a very rare location for p53 mutations. These mutations were shown to be independent and presumably not in the germ line. The highest frequency of p53 mutations raises the possibility that p53 mutagenesis is a predominant factor for breast cancer development in this low-risk Japanese group, whereas in other cohorts different mechanisms are likely to account for the higher proportion of breast cancer. Further studies are needed to confirm the present observations. PMID- 8611424 TI - The relationship between smoking exposure and p53 overexpression in colorectal cancer. AB - Although epidemiological studies of the relationship between cigarette smoking and colorectal cancer risk have been equivocal, a positive association is consistently found for colorectal adenoma development. We performed an epidemiological study to determine whether p53 protein overexpression, in tumours obtained at the time of resection, is associated with cigarette exposure in colorectal cancer. A total of 163 colorectal cancer cases and 326 healthy controls responded to a standardised questionnaire on colorectal cancer risk factors including detailed information on their history of cigarette smoking. All patients' tumours were analysed immunohistochemically for p53 overexpression using an avidin-biotin immunoperoxidase procedure and polyclonal anti-p53 antibody CM1. Comparison of colorectal cases with controls revealed an elevated risk for ex-smokers (OR = 1.34, 95% CI 0.85-2.12) and current smokers (OR = 1.13, 95% CI 0.63-2.02) when compared with non-smokers. No dose-response relationship was found for total pack-years of smoking (trend test: P = 0.19). However, a trend for total pack-years of smoking was found when p53-positive cases were compared with p53-negative cases suggesting aetiological, heterogeneity (trend test: P = 0.06). Estimating the individual relative risk of developing a p53 positive tumour relative to controls showed no associations for smoking status or total pack-years of smoking. However, when p53-negative cases were compared with controls, an elevated risk was found for ex-smokers (OR = 1.84, 95% CI 1.00-3.37) and current years of smoking (trend test: P = 0.03). Colorectal tumours developing through p53-positive dependent pathways were not associated with smoking exposure. A significant increase in risk was observed for the p53 negative independent pathway with smoking. p53 overexpression appears to be associated with smoking exposure in colorectal cancer. PMID- 8611425 TI - Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas. AB - Biopsies from 61 sporadic metastatic malignant melanomas and five melanoma cell lines were examined for homozygous deletions and mutations in the CDKN2 gene (p16). As the p16 protein is involved in a cell cycle regulatory pathway consisting of at least pRb, cdk4 and cyclin D1, the tumours were also screened for amplifications of the last two genes. Moreover, the transcript levels of the genes were determined and the results compared with the immunohistochemically assessed expression of pRb. Altogether, homozygous deletions of CDKN2 were found in seven tumours (11%) and two of five cell lines, whereas a mutation was detected in only one biopsy, indicating that in sporadic melanomas the former mechanism is predominant for inactivating this gene. Notably, in total 59% of the metastatic lesions lacked detectable expression of p16 mRNA, whereas all the biopsies were found to express pRb. In accordance with the postulated negative feedback loop between p16 and pRb, one melanoma cell line showed overexpression of CDKN2 mRNA together with very low levels of the Rb protein. Amplification of the other two genes may not be important in the tumorigenesis of melanomas, as only one CDK4 and no CCND1 amplification was observed. However, highly elevated CDK4 mRNA levels, compared with that seen in a panel of normal tissues, were observed in 76% of the tumours, accompanied in 71% of the cases by high expression of the CCND1 cyclin activator. Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas. PMID- 8611427 TI - Characterisation of circulating chromogranin A in human cancer patients. AB - The structure of circulating chromogranin A (CgA) of phaeochromocytoma patients was characterised and compared with that of CgA extracted from tumours. Size exclusion chromatography experiments provided evidence that CgA is present in the blood of different patients, as well as in tumour extracts, as multiple forms having different hydrodynamic sizes of 600 kDa (CgA-I), 100 kDa (CgA-II) and 55 kDA (CgA-III). The amount of each CgA form as a proportion of the total antigenic material was different in different patients. Western blot analysis of chromatographic fractions indicated that these forms are made up by polypeptides of similar molecular weight (about 60-70 kDa). All CgA forms express the epitopes recognised by two monoclonal antibodies (A11 and B4E11), directed against residues 68-70 and 81-90 of human CgA. However, their relative immunoreactivity was markedly different. No evidence for the presence of multimeric complexes in the CgA-I fraction was obtained by various immunological and biochemical methods. These results suggest that circulating CgA in phaeochromocytoma patients consists of at least three forms that appear to be made up by polypeptides with similar molecular weight and different hydrodynamic properties and immunoreactivity. We hypothesise that different conformations and shapes contribute to the heterogeneity of circulating CgA. PMID- 8611426 TI - Streptococcal glycoprotein-induced tumour cell growth inhibition involves the modulation of a pertussis toxin-sensitive G protein. AB - We studied the mechanism of anti-tumour action of sulphydryl glycoprotein (SAGP) purified from an extract of Streptococcus pyogenes in vitro. SAGP rapidly inhibited the incorporation of nucleic acid precursors into murine fibrosarcoma (Meth A) cells before it inhibited the cell growth. SAGP-induced cell growth inhibition was diminished by incubating the cells with pertussis toxin (IAP), whereas the SAGP activity was augmented by incubating the cells with cholera toxin (CTX). Meth A cells exposed to SAGP underwent an increase in labelling of the alpha-subunit of an inhibitory guanine nucleotide-binding (Gi) protein in a subsequent IAP-catalysed [32P]ADP ribosylation of the cell membrane fraction. Gi alpha labelling was not increased either in the membrane from the Meth A cells exposed to heat-inactivated SAGP or in the membrane from L929 cells exposed to SAGP, in which growth was also unaffected. By contrast, SAGP caused no alteration in labelling the alpha-subunit of stimulatory guanine nucleotide-binding (Gs) protein in a subsequent CTX-catalysed ADP ribosylation of membrane fractions of Meth A and L929 cells. The amount of intracellular cAMP was decreased slightly in Meth A cells incubated with SAGP. Although the precise roles of Gs protein and adenylate cyclase in the cell growth inhibition induced by SAGP are not clear, these findings suggested that the modulation of Gi protein is involved in such SAGP-induced cellular events as the inhibition of nucleic acid synthesis and cell growth inhibition. PMID- 8611428 TI - Response of Photofrin-sensitised mesothelioma xenografts to photodynamic therapy with 514 nm light. AB - We have studied the response of human mesothelioma xenografts in nude mice to Photofrin-sensitised photodynamic therapy with 514 nm light. Delays in tumour regrowth following four different 514 nm irradiation regimens were compared with results obtained with the more commonly used 630 nm light. One of these 514 nm regimens, which consisted of 1 h of irradiation at an incident fluence rate of 20 mW cm-2 and a second hour at a fluence rate of 28 mW cm-2, produced tumour volume doubling times that were statistically indistinguishable from results that were observed when tumours were irradiated for 2 h with 630 nm light at an incident fluence rate of 50 mW cm-2. The three other 514 nm light protocols tested were found to be less effective than the 630 nm regimen. The 514 nm treatment protocols were devised on the basis of attempts to equate the photodynamic dose and the dose rate at these two wavelengths, with photodynamic dose defined as the number of photons absorbed by the sensitiser. Photosensitiser extinction coefficients, photon energies and tissue optical properties were considered in these attempts. Our results indicate that, under certain conditions, photodynamic therapy performed with 514 nm light can provide tumour control that is similar to that achieved with 630 nm, with potential for diminished normal tissue damage. PMID- 8611429 TI - A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model. AB - An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed. PMID- 8611430 TI - Enhancement of photodynamic therapy by mitomycin C: a preclinical and clinical study. AB - Photodynamic therapy (PDT) using Photofrin was used in combination with a hypoxic toxin (mitomycin C, MMC) to treat four patients with recurrent skin metastasis of a mammary carcinoma. In preclinical experiments an additive effect was found for the combination of MMC and PDT for treating subcutaneous RIF1 tumours in mice. When interstitial PDT was combined with a low dose of MMC (administered 15 min before illumination), the Photofrin dose or light dose could be reduced by a factor of 2 in order to obtain equivalent cure rate or growth delay. In the clinical pilot study, a low dose of Photofrin (0.75 mg kg-1) was used for PDT alone (superficial illumination) or combined with low-dose MMC (5 mg m-2). Different tumour areas were illuminated with or without a preceding infusion of MMC. Both tumour response and skin photosensitivity were scored. After 8-12 weeks of treatment, tumour cure could be achieved by administering light doses > or = 150 J cm-2 for PDT alone and similar effects were obtained when light doses of 75 87.5 J cm-2 were given after infusion with MMC. In all cases necrotic tissue of both tumour and surrounding skin was observed, which lasted for a mean of 5 months (range 2-20 months). Skin phototoxicity, tested by using a standardised illumination of skin patches on the back, lasted maximally 3 weeks. Three main conclusions could be drawn from these studies: (1) The enhanced effects of the combination of PDT and MMC observed in mouse tumours can be extrapolated to patients with mammary skin metastasis. (2) The combination of PDT and hypoxic toxins facilitates treatment by permitting lower doses of photosensitiser to be used (thereby reducing skin phototoxicity) or lower light doses (thereby reducing illumination times and allowing the possibility to treat larger tumour areas). (3) Restoration of skin after PDT in previously treated tumour areas (chemotherapy, radiation therapy and surgery) is very low. PMID- 8611431 TI - Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro. AB - Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic. PMID- 8611432 TI - Increasing hepatic arterial flow to hypovascular hepatic tumours using degradable starch microspheres. AB - The effect of degradable starch microspheres (DSM) on the intrahepatic distribution of a low molecular weight marker, 99Tcm-labelled methylene diphosphonate (MDP), was studied in rats with hypovascular HSN liver tumours. MDP was injected regionally, via the hepatic artery, alone or co-administered with DSM, with or without subsequent occlusion of either the hepatic artery or the portal vein. Tumour vascularity was measured with 57Co-labelled microspheres. Co injection with DSM immediately significantly increased hepatic retention of marker in both tumour (T) (median 22.40 (range 16.82-39.58)% injected dose) and normal liver (N) (9.08 (4.85-12.59) %ID) the greater effect seen in T (P < 0.01). After DSM degradation, very little MDP remained in N (0.61 (0.28-1.40) %ID) but there was significant retention in T (10.01 (6.73-20.28) %ID, P < 0.01). Clamping the hepatic artery had minimal effect on the retention of MDP when administered alone. Regional injection of 16.5 microM 57Co microspheres resulted in a N:T ratio of 2.25:1. Concomitant injection of the 40 microM DSM was 57Co microspheres reversed this ratio to 1:2. The results indicate that DSM selectively enhances the retention of MDP to a hypovascular hepatic tumour, not by causing intra tumour stasis, but by directing a greater arterial flow to hypovascular areas in the liver. PMID- 8611433 TI - Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine. AB - Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco's modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate. PMID- 8611435 TI - Pharmacokinetics and pharmacodynamics of carboplatin administered in a high-dose combination regimen with thiotepa, cyclophosphamide and peripheral stem cell support. AB - The aim of this pharmacokinetic/pharmacodynamic study was to define the relationships of the carboplatin exposure with the toxicity in patients treated with high dose carboplatin (400 mg m-2 day-1), cyclophosphamide (1500 mg m-2 day 1) and thiotepa (120 mg m-2 day-1) for four consecutive days, followed by peripheral stem cell transplantation. Exposure to carboplatin was studied in 200 treatment days by measuring the area under the carboplatin plasma ultrafiltrate (pUF) concentration vs time curve (AUC). The AUC was obtained by using a previously validated limited sampling model. A total of 31 patients was studied who received one, two or three courses of this high-dose chemotherapy regimen. The unbound, plasma ultrafiltrate carboplatin was almost completely cleared from the body before each next treatment day in a course; the day-to-day AUC variation was 3.3%. The mean cumulative AUC over 4 days was 19.6 (range 14.1-27.2) mg ml-1 min-1. In 97 treatment days the carboplatin dose was calculated using the Calvert formula with the creatinine clearance as the measure for the glomerular filtration rate (GFR). For these courses, the inter-patient variability in pharmacokinetics was significantly reduced from 21% to 15% (P = 0.007) in comparison with the schemes where it was given as a fixed dose of 400 mg m-2. There were no relationships found between toxicity and the AUC of carboplatin, which may be due to the influence of overlapping toxicities of cyclophosphamide and thiotepa. However, the ototoxicity was strongly related to the cumulative carboplatin AUC. This toxicity was dose limiting for carboplatin in this schedule. It appeared that the carboplatin pharmacokinetics in these regimens were similar to those reported at conventional dosages. To reduce the inter patient variation, the carboplatin dose can be calculated using the Calvert formula with the creatinine clearance as the measure for the GFR. PMID- 8611434 TI - Expression of collagenase (MMP2), stromelysin (MMP3) and tissue inhibitor of the metalloproteinases (TIMP1) in pancreatic and ampullary disease. AB - It is now recognised that epithelial-stromal interactions are important in a wide range of disease processes including neoplasia and inflammation. Metalloproteinases are central to matrix degradation and remodelling, which are key events in tumour invasion and metastasis and may also be involved in tissue changes occurring in chronic inflammation. Immunohistochemistry was performed on sections from 50 patients with pancreatic cancer (n = 27), ampullary cancer (n = 12), low bile duct cancer (n = 3), neuroendocrine tumours (n = 3) and chronic pancreatitis (n = 5), using antibodies raised against collagenase (MMP2), stromelysin (MMP3) and tissue inhibitor of metalloproteinase (TIMP1) and developed using the avidin-biotin complex method. Abundance of MMP2, MMP3 and TIMP1 was greater in pancreatic and ampullary cancer than any other pathology and immunoreactivity in the malignant epithelial cells in pancreatic and ampullary cancer was greater than in the stromal tissues (in pancreatic cancer: MMP2 100% vs 37%, MMP3 93% vs 15%, TIMP1 93% vs 4%, P < 0.0001). There were strong correlations between the immunoreactivity of the two antibodies for MMP2 (P < 0.0001), between MMP2 and TIMP1 (P < 0.0001) and between MMP3 and TIMP1 (P < 0.0001). The immunoreactivity for TIMP1 in pancreatic and ampullary cancers with lymph node metastases was significantly less compared with those cases without lymph node metastases (P < 0.02) and there was an association between increased immunoreactivity for MMP2 and the degree of tumour differentiation (P < 0.01). The results implicate MMP2, MMP3 and TIMP1 in the invasive phenotype of pancreatic and ampullary cancer. PMID- 8611436 TI - Isoelectric focusing of alphafetoprotein in patients with hepatocellular carcinoma--frequency of specific banding patterns at non-diagnostic serum levels. AB - Serum levels of alphafetoprotein are raised in 60-80% of patients with hepatocellular carcinoma. Although widely used as a serum marker, frequent false positive results in patients with benign liver disease, result in poor specificity. This occurs particularly when levels of alphafetoprotein fall between 50-500 ng ml-1, the so-called 'grey area'. Recent reports suggest that isoelectric focusing of alphafetoprotein demonstrates certain bands that are more specific for hepatocellular carcinoma. Our aim was to determine whether the apparent specificity of this new approach is gained at the expense of decreased sensitivity. Sera from 110 patients with a 'non-diagnostic' serum alphafetoprotein level (50-500 ng ml-1) were examined by isoelectric focusing and quantified by densitometric scanning. Ten patients with chronic liver disease and a raised serum alphafetoprotein level (50-500 ng ml-1), but with no evidence of hepatocellular carcinoma, were also studied. Isoelectric focusing revealed characteristic hepatocellular carcinoma bands (bands +II and +III) in 96% patients overall, and 100% of those with levels of total alphafetoprotein greater than 100 ng ml-1. No such bands were seen among ten subjects with cirrhosis but without hepatocellular carcinoma. Bands that are characteristic of hepatocellular carcinoma (bands +II or +III) are seen in the great majority of hepatocellular carcinoma patients; their absence makes a diagnosis of hepatocellular carcinoma extremely unlikely. PMID- 8611437 TI - Efficacy and safety of Stealth liposomal doxorubicin in AIDS-related Kaposi's sarcoma. The International SL-DOX Study Group. AB - The utility of current chemotherapeutic regimens in the treatment of AIDS-related Kaposi's sarcoma (AIDS-KS) is often compromised by both limited efficacy and substantial toxicity. Pegylated (Stealth) liposomal doxorubicin hydrochloride (SL DOX) has been demonstrated specifically to deliver high concentrations of doxorubicin to Kaposi's sarcoma (KS) lesions. This phase II study was performed to evaluate the efficacy and safety of SL-DOX in the treatment of moderate to severe AIDS-KS. Patients were treated biweekly with 10, 20, or 40 mg m-2 SL-DOX. Tumour response was assessed according to AIDS Clinical Trials Groups (ACTG) criteria before each cycle. Best response was determined for 238 patients and was achieved after a mean of 2.3 cycles (range 1-20). Fifteen patients (6.3%) had a complete response to SL-DOX, 177 (74.4%) had a partial response, 44 (18.5%) had stable disease and two (0.8%) had disease progression. SL-DOX was well tolerated: ten patients discontinued therapy because of adverse events, in four cases because of neutropenia. Grade 3 or 4 neutropenia occurred after 281 of 2023 cycles (13.9%) but involved 137 of 240 patients (57.1%) for whom data were available. SL-DOX has substantial activity in AIDS-KS. Best response is typically seen after fewer than three cycles of chemotherapy and in some cases may be prolonged. The most important adverse event is neutropenia, which occurs after a minority of cycles but which may occur in over half of all patients. PMID- 8611438 TI - Nasopharyngeal carcinoma: an EBV-associated tumour not significantly influenced by HIV-induced immunosuppression. The AIDS/Cancer Working Group. AB - We used a link between cancer (859,398 reports) and AIDS (50,050 reports) registries in the United States to study whether nasopharyngeal carcinoma (NPC) was increased in the population with AIDS. There was no indication of a significantly increased risk up to or after the AIDS diagnosis, which argues against progressively failing immunity being important in the development of this malignancy. PMID- 8611439 TI - Pernicious anaemia and cancer risk in Denmark. AB - A cohort of 5072 patients with pernicious anaemia was identified in the Danish Hospital Discharge Register from 1977 to 1989 and, through linkage to the Danish Cancer Registry, the occurrence of cancer in the cohort was determined up to 1991. Observed numbers of cancer cases during 1-15 years of follow-up were compared with expected numbers based on national incidence rates. Besides the well-established increased risk for stomach cancer, the analysis also revealed a 2-fold increase in the relative risk for cancer of the buccal cavity and pharynx among pernicious anaemia patients in accordance with previous studies; previously reported elevated risks for other digestive tract cancers were not confirmed. There was a non-significantly increased risk for lymphatic and haematological malignancy but the risk tended to disappear after 5 years of follow-up, indicating a possible selection bias. Decreased risks for cervical cancer and non melanoma skin cancer were also seen. PMID- 8611440 TI - Thrombocytopenia in the antiphospholipid syndrome. PMID- 8611442 TI - Bone marrow assessment in B-cell chronic lymphocytic leukaemia: aspirate or biopsy? A comparative study in 258 patients. AB - To evaluate the relative merits and prognostic value of bone marrow aspirate and bone marrow biopsy in the assessment of bone marrow infiltration in B-cell chronic lymphocytic leukaemia (CLL), two observers independently reviewed the percentage of lymphocytes in bone marrow aspirate (lymphocytic infiltration, LI) and the bone marrow histological pattern (BMP) in 258 patients. The inter observer reproducibility and agreement was higher for BMP than for LI. BMP was an independent prognostic factor for survival in the whole series, whereas LI only had independent predictive value in stage A patients. In the entire series, disease progression was predicted by either BMP and LI, whereas in stage A patients only by BMP. Regarding low-risk CLL (smouldering CLL or A"1 substage), BMP led to a more reproducible identification of these clinical forms than LI. In conclusion, although both BMP and LI are of value to estimate bone marrow infiltration in CLL and to predict the outcome of the disease, BMP is more reliable and reproducible than LI. PMID- 8611441 TI - Serum p53 accumulation and altered antibody responses to Epstein-Barr virus proteins precede diagnosis of haemopoietic malignancies of lymphoid origin. AB - We analysed antibodies to Epstein-Barr virus nuclear antigens (EBNAs 1, 2, 5 and 6) and the presence of serum p53 in 100 individuals, 37 of whom has developed a haemopoietic malignancy during a 12-year follow-up of 39,000 Finnish adults. Serum p53 was detectable in six of the 63 (10%) matched controls and in 13/31 (42%) patients who developed a malignancy of lymphoid origin approximately 7 years after serum withdrawal. Six patients who developed a malignancy of myeloid origin were negative for p53. The presence of p53 alone was associated with a highly significant increased risk of lymphoid malignancies (relative risk (RR)p53 = 6.7, 95% confidence limits (CL) 1.9, 24) whereas high levels of antibody to EBNA2 seemed to be inversely related to the risk (RREBNA2 = 0.1, CL 0.0, 1.1). Among lymphoid malignancies, a combination of serum p53 and high EBNA1 antibody levels gave a greater than expected risk (RRp53 and EBNA1 = 14, CL 1.4, 130; RRexpected = 4.4), whereas interaction with high levels of EBNA5 antibody gave an expected risk (RRp53 and EBNA5 = 19, CL 1.7, 220; RRexpected = 17). Thus detectable levels of p53 appear early in the development of lymphoid malignancies, and high EBNA1 antibody levels, and accumulated p53 may both be synergistic risk indicators for lymphoid malignancies, whereas high EBNA5 antibody levels and accumulation of p53 seem to raise the RR independently of each other. PMID- 8611443 TI - Glycophorin A mutations and risk of secondary leukaemia in patients treated for childhood acute lymphoblastic leukaemia. AB - Survivors of child acute lymphoblastic leukaemia (ALL) have a higher than expected risk of developing secondary acute myeloid leukaemia (AML). The glycophorin A (GPA) mutation assay measures the frequency of variant NO and NN erythrocytes in MN heterozygotes. A raised variant frequency (Vf) has been shown in patients treated with chemotherapy known to be at risk of secondary leukaemia. ALL patients were investigated for increased Vf using the GPA assay. Vfs at diagnosis were not significantly different from controls (NO Vf P = 0.193; NN Vf P = 0.790). During treatment Vfs increased significantly (No Vf P = 0.001; NN Vf P = 0.001). NO Vf returned to control values (P = 0.169) within 5 years from diagnosis but NN Vf remained significantly raised (P = 0.014). Three study patients developed secondary AML. At diagnosis of AML all three had significantly increased Vf. The first had a significantly raised Vf at routine follow-up 19 years following diagnosis of ALL then developed AML 3.5 years later. The second had a significantly raised NN Vf at diagnosis of ALL indicating possibly prior exposure to a mutagen or defective DNA repair involving erythroid stem cells. We conclude that a raised Vf detected by the GPA assay can act as a marker for the development of secondary induced leukaemia and can be used to screen individuals at a known high risk of this complication. PMID- 8611444 TI - CD20 and CD40 mediated mitogenic responses in B-lineage acute lymphoblastic leukaemia. AB - Activation of CD20, a cross-membrane ion channel, induces cell cycle progression from G0 to G1 in B lymphocytes. Subsequent activation of CD40, a membrane receptor of the nerve growth factor receptor superfamily, transits the B cells to the S phase. CD40 may also act synergistically in combination with IL-4 (B lymphocytes) or IL-3/IL-7 (B-cell precursors). We investigated the proliferative responses of B-lineage acute lymphoblastic leukaemia (ALL) cells to CD20/CD40 activation. In 18/56 ALL cases, CD20 activation resulted in significant increases in DNA synthesis. Similar, although more moderate, effects were seen of activation of CD40 in 10/44 cases. Responses to CD20 or CD40 activation were independent of co-stimulation with IL-3, IL-4 or IL-7, and various cocktails of the different growth stimuli did not act synergistically. PMID- 8611445 TI - Effects of recombinant human thrombopoietin on megakaryocyte colony formation and megakaryocyte ploidy by human CD34+ cells in a serum-free system. AB - In serum-free cultures of human CD34 cells, recombinant human thrombopoietin (TPO) induced megakaryocyte colony formation a dose-dependent fashion that was further enhanced by the presence of interleukin-3 (IL-3) and stem cell factor (SCF), but not by IL-6, IL-11 or erythropoietin. TPO gave rise to much smaller colonies and at an earlier time than IL_3, indicating that TPO affects predominantly more mature megakaryocytic progenitors. In liquid cultures. TPO increased the percentage and the absolute number of > or = 8N megakaryocytes, but it did not shift their modal ploidy from 2N. TPO-induced endomitosis was totally inhibited by the presence of or previous exposure of cells to, IL-3 and /or SCF. The mechanism by which TPO overcomes in vivo the negative effects of IL-3 and SCF on megakaryocyte ploidy remains unknown. PMID- 8611447 TI - Association of acquired Pelger-Huet anomaly with taxoid therapy. AB - We describe the occurrence of acquired Pelger-Huet anomaly (APHA) in 23 patients treated with paclitaxel (13) or docetaxel (10). A consistent peak of Pelger-Huet cells (PHC) within a range of 3-9 d after treatment with taxoids was noted. The APHA generally disappeared by day 21 after treatment. Peak PHC values for the first course were significantly different in paclitaxel versus docetaxel versus control groups (P < 0.0001) with the maximum PHC counts being significantly higher for docetaxel compared with paclitaxel (P < 0.001) and for paclitaxel compared with controls (P = 0.007). We conclude that taxoid therapy produces transient APHA which peaks between days 3 and 9 and is more pronounced with docetaxel than with paclitaxel. PMID- 8611446 TI - A phase II trial of partially incompatible bone marrow transplantation for high risk acute lymphoblastic leukaemia in children: prevention of graft rejection with anti-LFA-1 and anti-CD2 antibodies. Societe Francaise de Greffe de Moelle Osseuse. AB - Bone marrow transplantation (BMT) from matched sibling donors has been useful for the treatment of acute lymphoblastic leukaemia in children with a poor prognosis but is not available to more than two-thirds of patients who do not have a matched allogeneic donor. This study was undertaken to assess one strategy of marrow graft rejection prevention when alternative marrow sources such as HLA phenoidentical unrelated volunteers and HLA-partially incompatible relatives were used. Results have been compared with two matched groups of children with the same risks factors and disease status who underwent HLA-genoidentical or autologous BMT. The conditioning regimen was the same for the three groups of patients; in the study group anti-LFA-1 and anti-CD2 monoclonal antibodies combined with T-cell depletion of the marrow was added to prevent graft rejection and graft-versus-host disease. Nineteen patients were included and followed for a median of 25 months (14 months to 3 years). Bone marrow engraftment occurred in 83% of the evaluable patients. Post-transplantation infectious diseases were the most frequent causes of death in the study group, occurring in 31% of patients. No fatal infections occurred in the two control groups. Post-transplantation relapse of leukaemia occurred in 26% of study group's patients, in 58% of autologous BMT control group's patients and 5% of HLA-genoidentical allogeneic group's patients. The event-free survival was 83% in the HLA-genoidentical control group, and 30% and 24% in the study group and in the autologous control group, respectively. In conclusion, a high rate of engraftment was achieved by the use of anti-LFA-1 and anti CD2 antibodies. Occurrence of a long-lasting immunodeficiency, however, led to a high incidence of lethal infections and relapses. Combined approaches are therefore to be investigated accelerating immune reconstitution after transplantations of T-depleted HLA partially incompatible marrow. PMID- 8611448 TI - Peripheral blood progenitor cell collections in multiple myeloma: predictors and management of inadequate collections. AB - Thirty-seven patients with previously treated multiple myeloma (MM) underwent peripheral blood progenitor cell (PBPC) collection following high-dose cyclophosphamide and GM-CSF or sequential IL-3 and GM-CSF. Patients with an inadequate collection were considered for a second or third collection. 25 patients underwent subsequent autotransplant. The only variable predictive of CFU GM yield was the extent of prior melphalan therapy. All repeat collections were unsuccessful and patients infused with an autograft obtained from multiple sets of collections had a high incidence of delayed engraftment. We conclude that melphalan should be avoided or PBPC collection performed early in the disease course in patients who are potential transplant candidates. PMID- 8611449 TI - Sustained trilineage recovery and disappearance of abnormal chromosome clone in a patient with myelodysplastic syndrome following combination therapy with cytokines (granulocyte colony-stimulating factor and erythropoietin) and high dose methylprednisolone. AB - We report a case of hypoplastic myelodyplastic syndrome (MDS) (refractory anemia (RA)) in which sustained trilineage haematological response and persistent disappearance of an abnormal chromosome clone were achieved after treatment with combination therapy of cytokines (granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo)) and methylprednisolone (mPSL) pulse dose. The patient's haematological recovery was rapid and maintained even after cessation of the therapy. In addition, the predominant chromosome clone 13q- in bone marrow cells disappeared in the fourth week. The patient's improved bone marrow haemopoiesis and disappearance of the abnormal chromosome has continued to the present, 13 months after treatment. The occurrence of both trilineage response and abnormal chromosome disappearance in MDS patients treated with cytokine(s) or steroids is rare. Combination therapy might therefore be advantageous in MDS. PMID- 8611450 TI - Humanized CD52 monoclonal antibody Campath-1H as first-line treatment in chronic lymphocytic leukaemia. AB - The humanized CD52 monoclonal antibody Campath-1H was used as first-line therapy in nine patients with progressive chronic lymphocytic leukaemia (CLL). Intravenous (n = 5) or subcutaneous (n = 4) injections (up to 30 mg/inj.) were given three times a week for a maximum of 18 weeks. Three patients achieved a complete remission (CR) and five patients a partial remission (PR) (response rate 89%). CLL cells were cleared from blood in all patients and from the bone marrow in seven patients. The response duration time was 8 + -24+ months. Adverse events were mild except for one patient who developed CMV pneumonitis. All patients developed lymphocytopenia (B and T cells) but other haematological toxicities were negligible. Campath-1H is a highly effective and well-tolerated agent in patients with previously untreated CLL and further studies are warranted. PMID- 8611451 TI - Serum erythropoietin in chronic lymphocytic leukaemia. AB - Anaemia is a frequent complication of advanced chronic lymphocytic leukaemia (CLL) and several cytokines known to inhibit erythropoietin (Epo) formation are produced by CLL B cells. Therefore we measured serum Epo levels in 47 CLL patients to determine whether Epo was a significant factor in the development of their anaemia. Epo levels were increased compared to normal individuals and this elevation appeared adequate for the degree of anaemia. The slope of the regression of Epo versus haemoglobin (Hb) was similar to that of a reference group. Serum transferrin receptor (sTfR) levels were also appropriately elevated for the degree of anaemia and correlated with serum Epo. Advanced stage was not associated with reduction of Epo production but diminished erythropoietic activity was observed in several patients. The results indicate that anaemia in CLL is not characterized by inadequate Epo production. PMID- 8611452 TI - T-cell form of chronic lymphocytic leukaemia: a reaffirmation of its existence. AB - Early in the 1980s three categories of T-cell chronic lymphocytic leukaemia were recognized: CD4+ CD8- knobby type, CD4- CD8+ azurophilic type and CD4+ CD8- adult T-cell leukaemia (ATL) type. Both azurophilic and ATL types were later shown to be distinctive disorders, whereas the knobby type has been largely neglected and even considered non-existent by some authors. In this report we describe two patients with leukaemia of CD3+ CD4+ CD8- post-thymic T lymphocytes presenting with marked lymphocytosis, generalized lymphadenopathy and hepatosplenomegaly. We believe that CLL of the post-thymic T-lymphocytes is a distinct entity, and merits a separate designation from other T-cell leukaemias. PMID- 8611453 TI - Microsatellite instability in follicle centre cell lymphoma. AB - Fluorescent polymerase chain reaction (PCR) was used to assay 12 microsatellite markers (APC x 2, DCC, P53 x 2, RB1, NM23, WT1, D6S260, D6S262, D6S281 and TNFa) to look for evidence of microsatellite instability in 40 cases of follicle centre cell lymphoma (FCC). Evidence of novel alleles seen in the tumour tissue but not the normal uninvolved tissue was seen in seven cases (17%). In only two of these cases (5%) was more than one locus involved but in these cases multiple affected loci were seen (4/12 and 7/12 respectively). The detection of microsatellite instability indicates a DNA repair defect such as that which would be predicted to occur in cells with mutated mismatch repair genes, a novel finding in FCC lymphoma. PMID- 8611454 TI - Detection of donor cell derived acute myelogenous leukaemia in a patient transplanted for chronic myelogenous leukaemia using fluorescence in situ hybridization. AB - The recurrence of leukaemia following allogeneic bone marrow transplantation appears to develop rarely in donor cells. However, the standard method for assigning the origin of recurrence, metaphase analysis, can be unreliable. We have applied the technique of fluorescence in situ hybridization (FISH) directly on archival Wright stained bone marrow slides obtained from a patient who developed acute myelogenous leukaemia (AML) following allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukaemia (CML). Using a chromosome specific DNA probe we linked a chromosomal aberration, previously detected by conventional metaphase analysis, directly to morphologically identifiable blast cells. In this way we were able to assess cell-lineage involvement of the secondary leukaemia and assign a donor origin. PMID- 8611455 TI - Discordant clinical presentation and outcome in infant twins sharing a common clonal leukaemia. AB - We report the different presentation features and clinical outcome between two identical infant twins with acute lymphoblastic leukaemia with a shared clonal disease and MLL gene rearrangement. One twin relapsed and died, but the other is in complete remission > 4 years after diagnosis. These data, and similar observations on other twin infants with leukaemia, suggest that despite a common clonal in utero, post-natally these leukaemias can evolve independently, at different rates, in the twinned individuals, and that the usually fatal leukaemia associated with t(4;11) MLL gene rearrangement can be effectively treated when the leukaemic burden is small. PMID- 8611457 TI - Fibrinogen Lincoln: a new truncated alpha chain variant with delayed clotting. AB - A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non-reducing SDS PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single-stranded conformational polymorphism) analysis of exon 5 of the A alpha gene, which encodes the C terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758-4770), resulting in a frame-shift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (-476His-Cys-Leu-Ala-Stop). The presence of a circulating truncated A alpha chain was confirmed when SDS-PAGE gels were probed with an alpha chain specific antisera; which showed that the variant A alpha chain comigrated with gamma chains. The truncation results in a variant A alpha chain with a deletion of 131 amino acids (480-610), and four new amino acids at the C-terminal. PMID- 8611458 TI - Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells. AB - We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AMI) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34+CD33- cells expressed P gp strongly, CD34+CD33- cells moderately, and CD34-CD33+ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34-CD33+ but not CD34+CD33- at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations. PMID- 8611456 TI - Evidence for an active fibrinolytic system in normal human bone marrow. AB - Normal human bone marrow from patients undergoing heart surgery was analysed quantitatively for components of the fibrinolytic system, using functional and immunological assays. Marrow was found to contain considerable fibrinolytic activity, reflecting high levels of t-PA (tissue-type plasminogen activator). The t-PA was in an active form, despite the presence of the inhibitors PAI-1 and PAI 2. Plasminogen and alpha2-antiplasmin (alpha2-AP) were also present in marrow. The balance of proteases and inhibitors differed dramatically from that observed in plasma, with higher levels of t-PA, PAI-1 and PAI-2, and lower levels of u-PA (urokinase), plasminogen, alpha2-AP and t-PA-PAI-1 complex in bone marrow, and resulted in favourable conditions for fibrinolysis. The presence of plasmin alpha2-AP complex at concentrations of the same order of magnitude as total plasminogen and alpha2-AP demonstrated that active generation of plasmin was indeed occurring. A role for the active fibrinolytic system in normal human bone marrow may be the removal of unnecessary fibrin deposits formed in the cavities of the marrow, in order to maintain flow through this tissue. PMID- 8611459 TI - Beta 2-glycoprotein I in thrombosis: evidence for a role as a natural anticoagulant. AB - Although the physiological role of beta2-glycoprotein (B2GPI) is unknown, in vitro evidence indicates that B2GPI may be a natural anticoagulant. In this study we have examined whether fluctuations of plasma B2GPI occur in in vivo coagulation. Serial measurements of B2GPI and other anticoagulant proteins were performed in 51 patients with thrombotic (group 1: six patients with disseminated intravascular coagulation (DIC), group 2: venous (n = 4) or arterial (n = 170 thrombosis) and non-thrombotic disease (group 3: 24 patients undergoing elective surgery). Reductions in plasma B2GPI levels were seen in most patients which were roughly proportional to the severity of their illness. Particularly striking reductions of B2GPI, protein C (PC) and antithrombin III (AT-III) (mean +/- 95% CI: 42.7 +/- 8.6%, 42.1 +/- 14.8%, 39.1 +/- 28.4% respectively) were seen in group 1. The reductions in plasma B2GPI were significantly greater in group 1 than in the other groups. Dilutional factors explain most of the reductions in B2GPI, PC and AT-III in groups 2 and 3, but contribute little to group 1. In conclusion, although B2GPI behaves as a 'negative acute phase reactant', the magnitude of reduction of plasma B2GPI levels, accompanied by reductions in other anticoagulant proteins in patients with DIC, suggests specific consumption of B2GPI in in vivo coagulation. This study provides further evidence that B2GPI is an anticoagulant of physiological importance. PMID- 8611460 TI - Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo. AB - The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Hautchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear-stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post-infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel-Palade Body (WPB) content observed by EM in previously reported studies using this model. PMID- 8611461 TI - Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation. AB - Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3 fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125I-labelled Fg, 70-255 nm with Bmax = 10000-25000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly by plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research. PMID- 8611462 TI - Treatment of homozygous protein C deficiency with subcutaneous protein C concentrate. AB - Subcutaneous protein C concentrate (Immuno, Vienna) was used to treat a child with homozygous protein C deficiency who was formerly treated with intravenous protein C concentrate. After 3000 units subcutaneous protein C concentrate (250 iu/kg), protective protein C levels were maintained for 48 h after infusion, with peak levels at 12 h. Subcutaneous protein C concentrate is given every third day and is well tolerated by the patient. No thrombotic events have occurred. We conclude that subcutaneous administration of protein C concentrate is a valuable therapeutic option in the long-term management of homozygous protein C deficiency and avoids the potential hazards of long-term central venous lines. PMID- 8611463 TI - A case of cryofibrinogenaemia responsive to stanozolol. AB - Cryofibrinogen is a plasma protein complex whose presence in the peripheral blood is generally asymptomatic, but may sometimes cause multiple thromboembolism in the skin, lung and myocardium. The pathological manifestations associated with cryofibrinogenaemia have been treated with plasmapheresis and fibrinolytic drugs such as streptokinase, streptodornase and/or urokinase. Good results have recently been reported with stanozolol. This prompted us to treat a patient suffering from cryofibrinogenaemia with this androgenic hormone. The patient was a 66-year-old woman with rapidly evolving leg ulcers. Stanozolol was orally administered at 4 mg b.i.d. for 5 months and then gradually reduced. Plasma cryofibrinogen disappeared after 45 d from the start of therapy and cutaneous ulcers healed in 5 months. PMID- 8611464 TI - Implication of CD44 in adhesion-mediated overproduction of TGF-beta and IL-1 in monocytes from patients with bone marrow fibrosis. AB - Myelofibrosis (MF) is characterized by bone marrow (BM) fibrosis and excessive deposits of extracellular matrix (ECM) proteins which include fibronectin (FN), collagen type I and hyaluronic acid (HA). We have previously reported that adhesion to polystyrene over-activates MF monocytes. We now confirm their activation by increased CD25 expression and tyrosine phosphorylation. We hypothesize that ECM protein-adhesion molecule interactions induce overproduction of fibrogenic cytokines in MF monocytes leading to BM fibrosis. In this study we found that FN, collagen type I and HA induce 2-3-fold more TGF-beta and 6-9 fold more interleukin (IL)-1 in MF monocytes than normal controls (NC). Since CD44 can function as the natural ligand for these proteins, its role was studied. We found that CD44 mediated most of the TGF-beta and IL-1 produced. Immunoprecipitation of CD44 revealed three proteins at approximately 11 kD in MF monocytes and one in NC. Our results indicated that adhesion is important in overproduction of TGF beta and IL-1, and that their production is at least partly mediated by adhesion molecule-ECM protein interactions. These results implicate at least one adhesion molecule, CD44, in the pathophysiology of the BM fibrosis. PMID- 8611465 TI - Clinical and laboratory factors associated with platelet transfusion refractoriness: a case-control study. AB - In recent years clinical factors have largely surpassed alloimmunization as the predominant cause of platelet refractoriness. This makes it necessary to properly identify and weigh the non-immune factors that have a major impact of refractoriness. A case-control study is suitable for such an analysis, and to our knowledge has not previously been performed to assess this issue. Fifty-two refractory patients were compared with 52 control patients who were transfused at the same time. Only one transfusion event was analysed per patient. Clinical and laboratory data were recorded at the time of selected transfusion, and their association with refractoriness was investigated by the contingency table method and the Cox stepwise logistic regression. There were 16 (31%) patients with HLA antibodies in the index group and only one in the control group. The corrected count increment in the group of patients refractory due to HLA antibodies was significantly lower than that in non-alloimmunized refractory patients [median (range): 48.5 (-3560, 4614) and 4058 (-4417, 6886), respectively; U = 493, P < 0.0001]. In the multivariate analysis, factors associated with refractoriness were the presence of HLA antibodies (odds ratio (OR) 50.7; 95% CI 5.5-463); fever (odds ratio 7.2; 95% CI 2.5-21) and BMT because of chronic myeloid leukaemia (odds ratio 7.3; 95% CI 1.8-30). The latter two were the only factors that remained independently associated with refractoriness after excluding alloimmunized patients and their controls. We conclude that HLA antibodies are strongly associated with platelet transfusion refractoriness, but account for less than a third of these patients. Fever and BMT because of chronic myeloid leukaemia were the only non-immune factors independently associated with refractoriness. PMID- 8611466 TI - Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays. AB - Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte-derived inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) may contribute to a larger number of unexplained non-antibody-mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. we have monitored the plasma of PCs prepared by the platelet-rich plasma method (PRP), the buffy-coat method or by apheresis for IL-6, IL-1, transforming growth factor-beta (TGF-beta), TNF and interferon gamma (IFN gamma). Biologically active IL-6 increased in stored PRP-PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL-8, as detected by immunoassay, were evident in PRP-PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL-1 with only minimal biological activity were present in some PRP-PCs by day 5/6. No significant increase in the levels of IL-8, IL-6 or IL-1 were seen in buffy-coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL-8 content, but not in IL-6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGF beta were present, but there was no evidence of any biologically active or immunoreactive TNF alpha. Pre-storage filtration of PRP PCs for depletion of leucocytes prevented the increase in IL-8 and IL-6 levels of these PCs. Our results show that leucocyte reduction by buffy-coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines. PMID- 8611467 TI - NA-phenotype-dependent differences in neutrophil Fc gamma RIIIb expression cause differences in plasma levels of soluble Fc gamma RIII. AB - Soluble Fc gamma RIII in plasma is primarily derived from neutrophils and is a measure of the total body neutrophil mass. We have developed a new, sensitive 'sandwich' ELISA to measure soluble Fc gamma RIII in plasma and released Fc gamma RIII in cell supernatants. Both sFc gamma RIIIa, derived from NK cells and sFc gamma RIIIb, derived from neutrophils are detected in the assay. However, plasma analysis of Fc gamma RIIIB gene-deficient donors suggested that sFc gamma RIIIa contributes only marginally to the total amount measured in healthy individuals. Furthermore, we observed that plasma of homozygous NA1-positive donors contained lower amounts of sFc gamma RIII than plasma of homozygous NA2-positive donors. Heterozygous donors were found to have intermediate levels of sFc gamma RIII in their plasma. Hemizygous Fc gamma RIIIB gene-deficient donors were found to have half the amount of sFc gamma RIII in their plasma compared to donors with two Fc gamma RIIIB alleles. These NA phenotype-dependent differences in plasma sFc gamma RIII could not be contributed to either an assay artefact or NA-dependent differences in shedding of Fc gamma RIIIb upon neutrophil activation. Calibration curves constructed with plasma of homozygous donors did nor reveal NA-dependent differences in antibody affinity. Measurement of released Fc gamma RIIIb in supernatants of neutrophils stimulated with PMA, and inhibition of this signal with human IgG revealed no NA-dependent differences. However, NA-dependent differences in neutrophil Fc gamma RIIIb expression were present, comparable to the differences found in plasma levels of sFc gamma RIII. Differences in the amounts of released Fc gamma RIII in supernatants of NA-typed apoptotic neutrophils were similar to initial differences in Fc gamma RIIIb expression, again being lower in NA1-positive than in heterozygous and NA2-positive donors. In conclusion, NA-dependent differences in plasma levels of soluble Fc gamma RIII seem to be caused by differences in expression of the receptor on the neutrophil membrane. PMID- 8611468 TI - Trisomy 3 in marginal zone B-cell lymphoma: a study based on cytogenetic analysis and fluorescence in situ hybridization. AB - Trisomy 3 represents the most frequent and consistent chromosomal abnormality characterizing the recently defined entity marginal zone B-cell lymphoma (MZBCL). By cytogenetic analysis and/or fluorescence in situ hybridization (FISH) on interphase nuclei we found in increased copy number of chromosome 3 in 22/36 (61%) successfully analysed cases, including 8/12 cases with extranodal MZBCL, 8/13 cases with nodal MZBCL, and 6/11 patients with splenic MZBCL. Sensitivity of interphase cytogenetics was somewhat higher than that of conventional cytogenetic investigation. Structural chromosomal changes involving at least one chromosome 3 were seen in 11/20 cases with an increased copy number of chromosome 3: +de(3)(p13) was demonstrated in three cases, and was the sole chromosomal abnormality in one of them; +i(3)(q10) was seen in two other patients; and rearrangements involving various breakpoints on the long arm of chromosome 3 were found in the remaining cases. FISH on metaphase spreads confirmed these structural abnormalities and additionally showed two unexpected translocations involving chromosome 3. We conclude that: (1) trisomy 3 occurs in a high proportion of extranodal, nodal and splenic MZBCL; (2) FISH on interphase nuclei is an additional and sensitive tool in detecting an increased copy number of chromosome 3 in MZBCL; (3) additional structural abnormalities involving the long arm of chromosome 3 are frequent but non-recurrent and are perhaps secondary changes; and (4) abnormalities such as +del(3)(pl3) and +i(3)(q10) suggest that genes located on the long arm of chromosome 3 are of particular importance in the pathogenesis of MZBCL. PMID- 8611469 TI - Bone marrow transplantation and severe hypophosphataemia. PMID- 8611470 TI - Detection of monoclonality in bone marrow plasma cells by flow cytometry: limitations for minimal residual disease detection. PMID- 8611472 TI - Cytoskeletal behaviour in spectrin and in band 3 deficient spherocytic red cells: evidence for differentiated splenic conditioning role. AB - Based on quantitative analysis of red cell membrane proteins, hereditary spherocytosis (HS) can be divided into two main groups including isolated or ankyrin combined spectrin deficiency and band 3 reduction. Protein methyl esterification catalysed by protein carboxyl methyl-transferase (PCMT type II; EC 2.1.1.77) is a post-biosynthetic modification which is involved in the metabolism of damaged membrane proteins. We utilized the evaluation of erythrocyte membrane protein methyl esterification as a marker of cytoskeletal disarray in seven HS subjects with spectrin reduction and in seven patients with HS due to band 3 deficiency. Our results support the notion that band 3 deficient erythrocytes are not affected by an extensive cytoskeletal derangement. On the contrary, we found a remarkable increase of membrane methylation in the unsplenectomized, spectrin deficient. HS patients, suggesting a striking membrane skeleton disarray. This phenomenon was not observed in the spectrin-deficient red cells of splenectomized patients. Therefore in spectrin deficient erythrocytes the induction of cytoskeletal damage, specifically recognized by PCMT type II, could be one of the splenic steps producing conditioned spherocytes. PMID- 8611471 TI - Macrophages are the major target cell for HIV infection in long-term marrow culture and demonstrate dual susceptibility to lymphocytotropic and monocytotropic strains of HIV-1. AB - Haematological abnormalities are often seen in patients infected with HIV. A number of mechanisms are thought to contribute to this bone marrow suppression, including impaired stromal function and direct infection of progenitor cells. Evidence suggests that both bone marrow progenitor cells and perhaps stromal cells are open to infection by HIV, which raises the possibility that bone marrow stromal cells may serve as a reservoir for HIV. This study investigated the cellular targets and kinetics of in vitro infection of stroma in long-term bone marrow culture (LTBMC) using both mono- and lymphocytotropic strains of HIV-1. p24 ELISA and reverse transcriptase (RT) assay demonstrated that stroma could be infected with HIV and release infectious virions. The target cells for infection were shown to be macrophages by immunohistochemistry (APAAP), dual immunofluorescence staining (using CD68 and p24) and electron microscopy. The data show that it was possible to infect stroma in LTBMC with HIV and that such infection was productive. The main target for infection was bone marrow macrophages. In contrast to peripheral blood derived macrophages, these cells were susceptible to both lymphocytotropic and monocytotropic strains of HIV-1. The data suggests that these bone marrow macrophages may act as a reservoir for HIV, Infection of bone marrow macrophages may affect haemopoiesis either by transmission of HIV infection to developing progenitor cells through direct cell to-cell contact or by altering the ability of the stroma to support normal development. PMID- 8611473 TI - Correction of aplastic anaemia complicating paroxysmal nocturnal haemoglobinuria: absence of eradication of the PNH clone and dependence of response on cyclosporin A administration. AB - Paroxysmal nocturnal haemoglobinuria (PNH) is defined as a somatic mutation of a clonal population of stem cells. Consequently, when aplastic anaemia (AA) occurs in patients with a history of PNH, allogeneic bone marrow transplantation is considered as the only effective treatment. The impact of immunosuppressive therapy has not been reported in this situation. We present observations of three PNH patients who developed AA and were effectively treated with cyclosporin A (CSA). Because of lack of improvement with other treatments, CSA alone was given at a dose of 5-10mg/kg/d. Complete response (CR) was obtained in two patients after 6 and 24 months respectively. A partial response (PR) was observed in the third patient after 12 months. Transient elevated LDH and haemosiderinuria persisted in all cases. DAF and MIRL deficiency were still documented in the two patients in CR. Two patients (one CR, one PR) ceased CSA therapy after 12 months and relapsed within 3-6 months. CSA was reinitiated and led to platelet recovery in one patient after 6 months. The persistence of the abnormal PNH clone is coherent with the hypothesis that CSA does not act directly on the PNH clone but probably acts through regulation of the inhibitory effects of immunocompetent cells on haemopoiesis. These observations suggest that patients suffering from severe AA complicated PNH should not be excluded from immunosuppressive therapy. PMID- 8611474 TI - Plasma soluble interleukin-2 receptor level in patients with primary myelodysplastic syndromes: a relationship with disease subtype and clinical outcome. AB - To assess the hypothesis that the plasma soluble interleukin-2 receptor (sIL-2R) level may have predictive value for morbidity/mortality in patients with myelodysplastic syndromes (MDS), we determined in plasma sIL-2R level of 80 MDS patients and examined their subsequent clinical course. Compared with low-risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts) patients and normal subjects, the plasma sIL-2R level was significantly elevated in high-risk MDS (three other MDS subtypes and acute leukaemia following MDS) patients (high-risk MDS versus low-risk MDS, P < 0.01; high-risk MDS versus normal subjects, P < 0.01). 14/40 low-risk MDS patients developed at least one of the following during the follow-up period: erythrocyte transfusion dependence, infections requiring hospitalization, disease progression or MDS-related death. The plasma sIL-2R level was higher in these eventful subjects than in event-free low-risk subjects (P < 0.0001), and all of 10 low-risk subjects with a plasma sIL-2R level > 540 U/ml experience at least one event. By logistic regression analysis of various parameters in these 40 low-risk subjects, the plasma sIL-2R level was identified as the strongest independent parameter for predicting eventful subjects (P < 0.0047). The plasma sIL-2R level did not show a predictive value in high-risk MDS. This study revealed that the plasma sIL-2R level is significantly elevated in high-risk MDS and suggested that the plasma sIL-2R level is a valuable predictive factors for the clinical outcome in low-risk MDS. PMID- 8611475 TI - Demonstration of developing myelodysplasia/acute myeloid leukaemia in haematologically normal patients after high-dose chemotherapy and autologous bone marrow transplantation using X-chromosome inactivation patterns. AB - Autologous bone marrow or peripheral blood stem cell transplantation may carry an increased risk of secondary myelodysplasia (MDS) and acute myeloid leukaemia (AML), which are already recognized as complications of conventional treatment for lymphoid malignancies. In order to ascertain whether it is possible to detect the evolution of such a clone at an early stage in its development we have studied X-chromosome inactivation patterns (XCIPs) in three informative females who developed abnormal myelopoiesis after high-dose chemotherapy and ABMT. In one patient transplanted for relapsed Hodgkin's disease a leukaemic clone comprising approximately 50% of the patient's myeloid cells was detectable by comparison of peripheral blood granulocyte and T-cell XCIPs when the full blood count and morphology were normal. She presented with AML 7 months later. In two patients transplanted for AML, XCIP analysis was complicated by constitutively skewed Lyonization patterns, nevertheless a progressive alteration could be demonstrated by serial analyses. In one patient a difference was detectable 28 months before presentation with MDS. In the other patient, despite evident mild pancytopenia and alterations in her XCIPs over the past 4 years, she has developed no definitive myelodysplastic features and oligoclonality due to stem cell failure cannot be excluded. These studies show that XCIPs can be used to predict development of MDS/AML in some patients, but the technique is limited by technical variability and frequent constitutional skewing in the haemopoietic system. PMID- 8611476 TI - Unrelated donor marrow transplantation for myelodysplasia (MDS) and MDS-related acute myeloid leukaemia. AB - Allogeneic marrow transplantation using related marrow donors for myelodysplasia (MDS) and acute myeloid leukaemia (AML) arising from MDS results in 35-56% actuarial disease-free survival. Because the use of unrelated donors has not been well-characterized, we report on the outcome of 52 patients with MDS or MDS related AML consecutively treated between 1987 and 1993 with unrelated donor marrow transplantation. The median age was 33 (range 1-53) years. 33 patients received chemotherapy and total body irradiation and the remainder busulfan and cyclophosphamide. The donors were phenotypically identical at the HLA-A, B and Dw/DRB1 loci in 34 cases and mismatched for one HLA locus in 17 cases and two loci in one case. Marrow was non-T-cell depleted and methotrexate with cyclosporine or FK506 was used for postgrafting immunosuppression. The 2-year disease-free survival, relapse, and non-relapse mortality rates were 38%, 28% and 48%, respectively. One patient who relapsed survives disease-free after withdrawal of immunosuppressive therapy. 16/19 survivors have a performance status of 90-100%. Patients with MDS in transformation or with AML had a significantly higher risk of relapse than patients with less advanced disease (P = 0.0014). Increased non-relapse mortality was significantly associated with higher age, longer disease duration before transplant, lower neutrophil count on admission and, unexpectedly, being seronegative for cytomegalovirus. We conclude that the outcome with transplantation using unrelated donors is similar to reported results using related donors and that a meaningful proportion of eligible patients with an otherwise incurable disease may be cured with this treatment. However, mortality from the transplant procedure is high and future studies should focus on reducing toxicity. PMID- 8611477 TI - The effect of the GM-CSF/IL-3 fusion protein PIXY321 on bone marrow and circulating haemopoietic cells of previously untreated patients with cancer. AB - This is a phase I/II study of the GM-CSF/IL-3 fusion protein (PIXY321. Patients were treated with PIXY321 at a daily subcutaneous dose of 500, 750 and 1000 micrograms/m2 for 14 d. Side-effects were mild and consisted mainly of injection site reactions and constitutional symptoms. A biphasic modest increase of white blood count (2-5-fold) and platelets (1-1.5 fold) was seen, accompanied by an increased bone marrow cellularity and an increase in circulating progenitors. Colony-forming cells in the blood rose to a median of 184 granulocyte/macrophage colony forming cells (GM-CFC)/ml, eight Mix-CFC/ml, 250 burst forming units erythroid (BFU-E)/ml and 140 CFU-mega-karyocytes/ml, corresponding to a 10-, 2.5, 8- and 30-fold increase respectively. When seeded for long-term culture on irradiated bone marrow stroma, the mobilized cells were not able to sustain haemopoiesis in vitro to the same degree as bone marrow. Taken together these results indicate that PIXY321 has a biological effect in humans more similar to that of IL-3 than to that of GM-CSF. PMID- 8611478 TI - Establishment of a myeloid leukaemia cell line (Kasumi-4) with t(9;22;11)(q34;q11;q13), inv(3)(q21q26) and the EVI1 gene activation from a patient with chronic myelogenous leukaemia in blast crisis. AB - A novel human leukaemia cell line (Kasumi-4) was established from the peripheral blood of a 6-year-old girl suffering from chronic myelogenous leukaemia (CML) in blast crisis. The Kasumi-4 cells had the following characteristic features: undifferentiated blasts which were positive from CD34, CD33 and CD13 surface markers, but negative for myeloperoxidase platelet peroxidase, CD36, CD41 and CD42; chromosome abnormalities of t(9;22;11) (q34;q11;q13), inv(3)(q21q26); and elevated expression of EVI1 gene which is located at chromosome band 3q26. Megakaryocytic maturation was not observed in the liquid culture following the addition of TPA, IL3, IL-6 or GM-CSF, b2-a2 type of BCR-ABL chimaeric messenger RNA was detected by RT-PCR analysis. This the first leukaemia cell line with a three-way translocation containing the the Ph chromosome and the second cell line with an inv(3)(q21q26). This cell line appears to be useful for studying the mechanisms of leukaemogenesis involving these chromosomal abnormalities and related oncogenes. PMID- 8611479 TI - Molecular characterization of the 7q deletion in myeloid disorders. AB - Deletion of the long arm of chromosome 7 is a common karyotypic finding in myeloid disorders and in particular is found in association with secondary leukaemias. We have used restriction fragment length polymorphisms and gene dosage experiments to assess the loss or retention of sequences localized to chromosome 7q in five patients with clonal myeloid disorders and a 7q deletion. The deletion was interstitial in all cases with retention of the anonymous marker pS194 located at 7q36-qter. Three out of five cases also retained the more proximal gene T-cell receptor beta (TCR beta) located at 7q35. The proximal breakpoints of all five cases were localized to 7q22 by cytogenetic analysis. In two cases the proximal breakpoint lay between the genes for elastin (ELN) and collagen type 1 alpha (COL1A2) and in three cases distal to this region between the genes for erythropoietin (EPO) and acetylcholinesterase (ACHE). The genes of ACHE, plasminogen activator inhibitor 1 (PLANH1), CCAAT displacement protein (CUTL1) and Met proto-oncogene (MET) were deleted in all cases. Molecular analysis of the 7q deletion in myeloid leukaemias demonstrates heterogeneity of the breakpoints, supporting a recessive mechanism of tumourigenesis. PMID- 8611481 TI - Treatment of newly-diagnosed acute myelogenous leukaemia in patients aged 80 years and above. AB - In order to assess outcome following treatment of acute myeloid leukaemia (AML) in patients aged 80 years and above, we have studied 33 patients aged > or = 80 years treated between 1980 and 1994; 29 of these received treatment. The median age was 82 years (range 80-89). Three patients received daunorubicin ( > or = 60 mg/m2 daily x 3) alone or with low-dose ara-C, two patients received '3+7' with post treatment GM-CSF; 24 patients had higher doses of ara-C, generally with anthracyclines or fludarabine, and in nine cases with G or GM-CSF. The median survival of the treated patients was 3-4 weeks and only two were alive after 1 year (at 66 and 79 weeks). Complete remission (CR) occurred in 9/29 (31%). Only one of the nine remains alive in remission, at 76 weeks after the date of CR, whereas the other eight died in remission or had disease recurrence at a median of 11 weeks (range 5-37 weeks) after CR. The median survival of the four untreated patients was 10 weeks (range 3-38). Patients aged > or = 80 had, on average, worse outcomes than those observed in patients aged 70-79. Our results confirm that currently available chemotherapy is generally not indicated in patients aged 80 or over with AML. PMID- 8611480 TI - Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications. AB - The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56+ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16+ cells did not differ significantly from that of normal BM samples. The number of CD56+CD3- lymphocytes increased significantly within high-risk patients (869 +/- 671) as compared to intermediate (388 +/- 212) and low-risk patients (274 +/- 199) (P = 0.04). Moreover, patients with high values of CD56+CD3- lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high beta 2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56+CD3-lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57+CD8- cells had a better outcome. PMID- 8611482 TI - Somatostatin receptor scintigraphy in the initial staging of Hodgkin's disease. AB - Somatostatin receptor (SS-R) scintigraphy has been successfully used in the visualization of a variety of neuroendocrine tumours. In vitro studies have shown that SS-Rs are present in human malignant lymphomas. We conducted a prospective study in 56 consecutive untreated patients with histologically proven Hodgkin's disease (HD) and compared the results of SS-R scintigraphy with physical and radiological examinations as initial evaluation. SS-R scintigraphy was positive in 55/56 (98%) patients at sites of documented disease. In 20 patients SS-R scintigraphy disclosed lymphoma localizations not revealed following procedures of conventional staging. As a result in 12 patients (21%) SS-R scintigraphy produced a change of stage and in seven patients (13%) the additional information obtained from SS-R scintigraphy led to a change of treatment. SS-R scintigraphy failed to visualize sites of HD in four patients, mainly in the abdominal area. In three patients a false-positive result was obtained. These data show that SS-R scintigraphy provides an imaging technique that appears to visualize tumours in most patients with HD and may be clinically useful in the management of these patients. PMID- 8611483 TI - Clinical management of ectodermal dysplasia. AB - Children with ectodermal dysplasia may have hypodontia or anodontia and are often treated dentally with conventional adult appearing prosthesis which are focused only on the oral manifestations of the syndrome. This case report describes the management of an ectodermal dysplasia patient to provide improved aesthetics, function, and emotional development. PMID- 8611484 TI - Treatment of facial asymmetry with a functional appliance: a case report. AB - A five-year-old girl, who had a facial asymmetry from trauma to the condyle, was treated by a hybrid functional appliance. At the end of 1 year 5 months of treatment, symmetry was gained and condylar process showed a significant development. PMID- 8611485 TI - Orthodontic goniometry: a control technique after P. Planas transversal expansion method: neuro-occlusal rehabilitation: Part 3. AB - Orthodontic goniometry applied to expansion cases treated by P. Planas method (neuro-occlusal rehabilitation) demonstrates considerable bimaxillary transverse expansion (crown and apices). Maximal expansion of 10 mm was noted for mandibular apices with a mean of 5.8 mm. Maximal expansion of 11.5 mm was noted for maxillary apices with a mean of 8.11 mm. Results were stable, some being controlled more than eight years out of retention. PMID- 8611486 TI - Shear strength of ceramic brackets bonded to etched or unetched enamel. AB - The purpose of this study was to compare the shear bond strengths and enamel surface morphology after debonding a polycrystalline ceramic bracket (Transcend 2000) bonded with a light-cured resin cement (Transbond) without enamel etching or by etching for 15 seconds with 10% or 37% phosphoric acid and 10% maleic acid. Forty extracted noncarious human premolars were used. The buccal enamel surfaces were used and the teeth randomly divided in to four groups of 10 teeth each: Group 1: No enamel etching; Group 2: Enamel etching for 15 seconds with 10% phosphoric acid; Group 3: Enamel etching for 15 seconds with 37% phosphoric acid; and Group 4: Enamel etching for 15 seconds with 10% maleic acid. The brackets were bonded to the etched enamel surfaces according to manufacturers' instructions except the etching time variations. All specimens were stored in distilled water for 24 hours and then thermocycled for 300 cycles between 5 degrees C and 55 degrees C. The specimens were mounted in dental stone and placed in the Instron at a crosshead speed of 0.5 mm/min using a knife-edged blade. Immediately after debonding, the enamel surface and bracket-enamel interface were evaluated visually and with a stereomicroscope. Representative samples were then examined with the SEM. ANOVA and Student-Newman-Keuls tests were performed. The results (in MPa) were: Group 1:11.83 (+3.9); Group 2: 28.80 (+12.6); Group 3: 26.25 (+5.3); Group 4: 18.06 (+6.9). Groups 2 and 3 were statistically significantly different (p<0.0001) from Groups 1 and 4. Groups 2 vs. 3 or 1 vs. 4 were not statistically different. Debonding occurred mainly at the bracket-resin interface in all groups, except Group 2 which displayed two samples with enamel cohesive failures and two fracturing the bracket. The SEM evaluation revealed that after debonding, the group etched with the 37% phosphoric acid gel had the roughest enamel surface and was the only group to present enamel fractures. Bracket bonding with unetched enamel and enamel etched with 10% phosphoric acid gel should be clinically investigated using the products tested. PMID- 8611487 TI - Resin adhesion to the primary enamel: influence of light-irradiation times. AB - The purpose of this study was to investigate the influence of the light irradiation times to the bonding agent and the composite resin on resin adhesion to the primary enamel. One hundred bovine mandibular ground primary incisors were used. Materials used in this study were 40% phosphoric acid gel, Photo Bond and Photo Clearfil A (Kuraray Co., Kurashiki, Japan). The etching time was 30 seconds. The light-irradiation times for the bonding agent were 10, 20 and 30 seconds and the light-irradiation times for the composite resin were 20, 40 and 60 seconds. Specimens were divided into the non-thermal cycled groups and the thermal cycled groups. Shear bond strengths were tested. After the shear bond strength test. the test surfaces of the enamel and the resin specimens were observed using the SEM. The optimum light-irradiation time on the bonding agent was 10 seconds. Both in the non-thermal cycled group and the thermal cycled group, the light irradiation time on the composite resin which showed the significantly higher bond strength was 40 seconds. Higher bond strength was not always obtained with the longer light-irradiation time on the bonding agent and the composite resin. PMID- 8611488 TI - Hyoid bone and atlas vertebra in established mouth breathers: a cephalometric study. AB - The position of hyoid bone and atlas vertebra in 29 established mouth breathers (17 boys and 12 girls) in the age group of 10-14 years were cephalometrically evaluated and compared with 23 nose breathers (11 boys and 12 girls). The children of both the groups were selected on the basis of history and clinical examination. The comparisons were made using univariate analysis for male and female groups separately as well as combined. It was observed that mouth breathers do maintain an extended head posture, which was evident from a decrease in distance between the occiput and dorsal arch of atlas vertebra. However the results of the present study did not reveal any distinct characteristics of hyoid bone and atlas vertebra that can be used to predict or associate the craniofacial pattern of mouth breathers. PMID- 8611489 TI - Ultrasonographic observation of the circumoral musculature: an in-vivo study. AB - The aim of the present in-vivo study was to determine whether circumoral musculature could be imaged using an ultrasound with a high frequency probe and to record the changes, if any, in the thickness and shape of the images of the lips occurring amongst normal and different malocclusion states during the relaxed and contracted states. Thirty children with varying classes and types of malocclusion in the age group of 9-12 years were included in this investigation. A real time ultrasound system with a 10 MHz sector scan transducer was used to do the imaging. The results indicated that the circumoral region could be imaged ultrasonographically and that significant changes occurred in the thickness of this muscle in both relaxed and contracted states as well as in varying malocclusion states. Significant increase in muscle thickness was aslo noted in these subjects who were given muscle exercises. Long term observations utilizing this technique in following growth and age related changes will provide a wealth of information for clinical practice in future. PMID- 8611490 TI - Inverted impaction of second premolar: two case reports. AB - This report documents two patients with inversion and impaction of the second premolar tooth. Family and personal histories of both patients were unremarkable with no abnormalities in general growth and development nor any history of trauma. Patient 1 was a 10-year-old female with inversion and impaction of the mandibular right second premolar. Fifteen months previously, pre-operative radiographic examination prior to extraction of the mandibular right second primary molar revealed a normal direction and eruptive pattern of the tooth in question. However, following extraction of the mandibular right second primary molar, the developing succedaneous premolar was seen to be inverted. This was suggestive of inversion resulting from an iatrogenic force during tooth extraction. The second patient was a 9-year-old male with inversion and impaction of the maxillary right second premolar. Radiographic examination 3 months prior to the initial visit at our hospital showed an existing inversion of this tooth. This pointed to a developmental abnormality in the location of the original tooth bud. PMID- 8611491 TI - Preparation of feeding obturators for infants with cleft lip and palate. AB - Clefts of the lip and palate are the commonest congenital craniofacial malformations in children. Sucking is impaired in infants born with complete clefts of the lip and palate. Feeding obturators improve feeding thereby contributing to weight gain and a thriving state of health, a prerequisite for surgical repair of the defects. A pediatric dentist may be required to fabricate the obturator. A method for constructing the appliance is presented. The severity of the clefts varies so much that stock trays are not always useful for the impression of the infant's maxillary arch. A preliminary impression is taken by introducing a thermoplastic impression material with the index and middle fingers as the tray. A model is produced from which a custom tray is constructed. The final maxillary impression is taken using an irreversible hydrocolloid with the child in an upright position. An obturator is constructed on the stone model by sprinkling soft autopolymerizing acrylic resin on the palate extending well into the mucobuccal fold area. The cured appliance is trimmed and polished prior to insertion. PMID- 8611492 TI - Congenital hypodontia: a pedigree and dermatoglyphic study. AB - The dermatoglyphs and the pedigrees of 11 male and 10 female, a total of 21 patients with congenital hypodontia (CH) were investigated. The dermatoglyphics of the patients were compared with those of 250 male and 250 female control cases. There were more arches on the finger-tips of the patients with absence of the lateral incisors, absence of the second premolars and a total patients with CH. The patients with absence of the lateral incisors had more palmar ll and plantar lll loops and p triradii. The patients with absence of the second premolars had more H and H loops, P triradii and plantar ll loops. A total patients with CH had more palmar ll and plantar lll, IV loops and p and z triradii. The pedigrees of the patients with both absence of the lateral incisors and the second premolars showed that the inheritance patterns of both conditions would seem autosomal recessive. PMID- 8611493 TI - Fluoridated drinking water and maturation of permanent teeth at age 12. AB - Dental age was evaluated in 88 children aged 12 years + 6 months in both areas of Boston with fluoridated drinking water and nonfluoridated areas surrounding Athens, Greece. Fluoridation of drinking water in the Boston areas was 1.0 ppm, a level considered 'optimal' in the USA. Girls from the fluoridated Boston area were shown in this study to have a significantly (p<0.05) delayed dental age when compared to their chronological age according to the tables of Nolla. Boys from the Boston area and boys and girls from the Athens area showed no significant difference when comparing dental age to chronological age. PMID- 8611494 TI - Assembly of light-harvesting chlorophyll a/b complex in vitro. Time-resolved fluorescence measurements. AB - The assembly kinetics of a pigment-binding membrane protein, the light- harvesting chlorophyll a/b complex of green plants, have been determined in vitro. Time-resolved fluorescence spectroscopy, with millisecond time resolution, has been used to monitor changes in both protein and chlorophyll (Chl) fluorescence, as well as in energy transfer from Chl b to Chl a, during complex assembly. Three reaction steps could be resolved after rapid, stopped-flow, mixing of the apoprotein (light-harvesting Chl a/b protein, LHCP) and pigments, solubilized in sodium dodecyl sulfate (SDS) and octyl glucoside (OG), respectively. A fast step in the range of 10 ms was detected regardless of whether the reaction mixture contained pigments or proteins, or both, and is interpreted as being connected with the formation of mixed SDS and OG detergent micelles. Two further steps were resolved: one with a time constant of about 1 min and another, slow step with a time constant of several minutes. Both of these were dependent on the presence of protein, Chls, and xanthophylls. Most, if not all, of the energy transfer from Chl b to Chl a was established during the slow step, indicating the the juxtaposition of these pigments, either by a structural rearrangement of the complex of by additional pigment binding, is the final stage in LHCII assembly in vitro. PMID- 8611495 TI - Solvation energies of amino acid side chains and backbone in a family of host guest pentapeptides. AB - Octanol-to-water solvation free energies of acetyl amino amides (Ac-X-amides) [Fauchere, J.L., & Pliska, V. (1983) Eur. J. Med. Chem. --Chim. Ther. 18,369] form the basis for computational comparisons of protein stabilities by means of the atomic solvation parameter formalism of Eisenberg and McLachlan [(1986) Nature 319, 199]. In order to explore this approach for more complex systems, we have determined by octanol-to-water partitioning the solvation energies of (1) the guest (X) side chains in the host-guest pentapeptides AcWL-X-LL, (2) the carboxy terminus of the pentapeptides, and (3) the peptide bonds of the homologous series of peptides AcWLm (m = 1-6). Solvation parameters were derived from the solvation energies using estimates of the solvent-accessible surface areas (ASA) obtained from hard-sphere Monte Carlo simulations. The measurements lead to a side chain solvation-energy scale for the pentapeptides and suggest the need for modifying the Asp, Glu, and Cys values of the "Fauchere-Pliska" solvation-energy scale fro the Ac-X-amides. We find that the unfavorable solvation energy of nonpolar residues can be calculated accurately by a solvation parameter of 22.8 +/- 0.8 cal/mol/A2, which agrees satisfactorily with the AC-X amide data and thereby validates the Monte Carlo ASA results. Unlike the Ac-X amide data, the apparent solvation energies of the uncharged polar residues are also largely unfavorable. This unexpected finding probably results, primarily, from differences in conformation and hydrogen bonding in octanol and buffer but may also be due to the additional flaking peptide bonds of the pentapeptides. The atomic solvation parameter (ASP) for the peptide bond is comparable to the ASP of the charged carboxy terminus which is an order of magnitude larger than the ASP of the uncharged polar side chains of the Ac-X-amides. The very large peptide bond ASP, -96 +/- 6 cal/mol/A2, profoundly affects the results of computational comparisons of protein stability which use ASPs derived from octanol-water partitioning data. PMID- 8611496 TI - Partitioning roles of side chains in affinity, orientation, and catalysis with structures for mutant complexes: asparagine-229 in thymidylate synthase. AB - Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS.dCMP shows sCMP 4-NH2 excluded from the space between Asn-229 and His-199 by the hydrogen bonding and steric properties and Asn-229. Consequently, 6-C of dCMP is over 4 A from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180 degrees to and orientation the could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Asn-229 is replaced by a cysteine, the Cys-229 S gamma rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D.dCMP, Asp-229 forms hydrogen bonds to 3-N and 40NH2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or 4-NH2) have been proposed to stabilize reaction intermediates. If their absence in TS N229D.dUMP persists in the ternary complex, it could explain the 10(4)-fold decrease in kcat/Km for dUMP. PMID- 8611497 TI - Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4 epimerase from Escherichia coli: implications for the catalytic mechanism. AB - UDP-galactose 4-epimerase is one of three enzymes in the metabolic pathway that converts galactose into glucose1-phosphate. Specifically this enzyme catalyzes the interconversion of UDP-galactose and UDP-glucose. The molecular structure of the NADH/UDP-glucose abortive complex of the enzyme from Escherichia coli has been determined by X-ray diffraction analysis to a nominal resolution of 1.8 A and refined to an R-factor of 18.2% for all measurement X-ray data. The nicotinamide ring of the dinucleotide adopts the syn conformation in relationship to the ribose. Both the NADH and UDP-glucose are in the proper orientation for a B-side specific transfer from C4 of the sugar to C4 of the dinucleotide. Those residues implicated in glucose binding include Ser 124, tyr 149, Asn 179, Asn199, Arg 231, and Tyr 299. An amino acid sequence alignment of various prokaryotic and eukaryotic epimerases reveals a high degree of conservation with respect to those residues involved in both NADH and substrate binding. The nonstereospecificity displayed by epimerase was originally thought to occur through a simple rotation about the bond between the glycosyl C1 oxygen of the 4-ketose intermediate and the beta-phosphorous of the UDP moiety, thereby allowing the opposite side of the sugar to face the NADH. The present structure reveals that additional rotations about the phosphate backbone of UDP are necessary. Furthermore, the abortive complex model described here suggests that Ser 124 and Tyr 149 are likely to play important roles in the catalytic mechanism of the enzyme. PMID- 8611498 TI - Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR. AB - The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein. PMID- 8611499 TI - Conformational preferences of a chimeric peptide HIV-1 immunogen from the C4-V3 domains of gp120 envelope protein of HIV-1 CAN0A based on solution NMR: comparison to a related immunogenic peptide from HIV-1 RF. AB - A critical problem to overcome on HIV vaccine design is the variability among HIV strains. One strategy to solve this problem is the construction of multicomponent immunogens reflective of common HIV motifs. Currently, it is not known if these motifs should be based primarily on amino acid sequence or higher-order structure of the viral proteins of a combination of the two. In this paper, we report NMR derived solution conformations for a sympathetic peptide taken from the C4 and V3 domains of HIV-1 CAN0A gp120 envelope protein. This peptide, designated T1 SP10CAN0(A), is compared to a recently reported C4-V3 peptide. T1-SP10RF(A) from the HIV-1 RF strain [de Lorimier et al. (1994) Biochemistry 33, 2055-2062], in terms of conformational features and immune responses in mice [Haynes et al. (1995) AIDS Res. Hum. Retroviruses 11, 211-221]. The T1 segment of 16 amino acids from the gp120 C4 domain is identical in both peptides and exhibits nascent helical character. The SP10 region, taken from the gp120 V3 loop, differs from that of T1-SP10RF(A) in both sequence and conformations. A reverse turn is observed at the conserved GPGX sequence. The rest of the Sp10 domain is extended with the exception of the last three residues which show evidence for a helical arrangement. Modeling of the turn region of the T1-SP10CAN0(A) peptide shows exposure of a continuous apolar stretch of side chains similar to that reported in the crystal structure of a V3 peptide from HIV-1 MN complexed with a monoclonal antibody [Rini et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6325 6329]. this hydrophobic patch is interrupted by a charged Lys residue in the T1 SP10RF(A) peptide. This observation suggests that the HIV-1 CAN0A and HIV-1 RF C4 V3 peptides can induce widely different anti-HIV antibodies. consistent with immunogenic results. PMID- 8611500 TI - Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line. AB - Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2 3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT 3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates. PMID- 8611501 TI - Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy. AB - The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs. PMID- 8611502 TI - Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in vivo. AB - The formation of isoaspartyl sites during aging of rat tubulin in vitro and in vivo has been studied. When incubated in vitro at pH 7.4, 37 degrees C, purified rat brain tubulin accumulated isoaspartyl sites at a rate > or = 2.4 isoaspartyl sites per 100 tubulin subunits (50 kDa) per day for 30 days. Isoaspartate levels were estimated by the transfer of radiolabeled methyl groups from S-adenosyl-L [methyl-3H]-methionine in a reaction catalyzed by protein-L-isoaspartyl methyltransferase. isoaspartate formation occurred in parallel with, but was not dependent upon, extensive cross-linking of tubulin via formation of intermolecular disulfide bonds. When rat PC12 cells were incubated for 24 or 72 h in the presence of adenosine dialdehyde, a potent methyltransferase inhibitor, a substantial and consistent increase in the isoaspartate content of tubulin was observed. This suggests that tubulin constantly undergoes isoaspartate formation in vivo, but that the levels are normally kept low by methylation-dependent repair. These findings support the hypothesis that protein-isoaspartyl methyltransferase plays a key role in countering spontaneous damage reactions to proteins associated with cell aging. These results also suggest that tubulin is an important target for protein-isoaspartyl methyltransferase in vivo. PMID- 8611504 TI - The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior. AB - A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function. Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments. Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile. Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding. The global nature of the flexibility is demonstrated by 1H NMR studies. Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons. Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone. This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states. The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling. PMID- 8611503 TI - Role of diacylglycerol and apolipophorin-III in regulation of physiochemical properties of the lipophorin surface: metabolic implications. AB - Manduca Sexta adults insects have two defined lipophorin species of densities 1.09 g/mL, [high-density lipophorin (HDLp)] and 1.02 g/mL [low-density lipophorin (LDLp)], respectively, and a continuous broad range of lipophorin particles of intermediate size and density, intermediate-density lipophorin (IDLp). The transformation of HDLp into IDLp and LDLp is the result of the progressive loading of HDLp with diacylglycerol (DG) and an exchangeable apolipoprotein, apolipophorin-III (apoLp-III). In this paper, we describe the physiochemical changes which occur in the lipophorin surface as a result of the transformation of HDLp into LDLp. (1) The increase in apoLp-III content, from 0 to 16 molecules per particle, is accompanied by a gradual increase in the zeta-potential which, at pH 8.6 ranges from /1.02 mV for lipophorins without apoLp-III to -7.76 mV for lipophorins containing 16 molecules of apoLp-III. (2) As judged by the changes in the partition constant for trimethylammonium diphenylhexatriene and oleic acid, an average 2-fold increase in the size of the lipophorin lipid surface takes place when HDLp is loaded with Dg and transformed into LDLp. (3) These data, as well as the results obtained by end point lipolysis with a triacylglycerol (TG) lipase, indicated that the accessible DG content increases 4-7 times when HDLp is converted in LDLp. (4) Fluorescence polarization of the cationic and anionic lipid probes, trimethylammonium diphenylhexatriene and cis-parinaric acid, embedded in eight different subspecies of lipophorin, containing from 12 to 50% DG, showed a small decrease in the surface lipid order when going from HDLp (25% DG) to LDLp (50% DG). (5) Porcine pancreatic phospholipase A2 was used as a probe of the lipoprotein surface. As the DG content of the lipoprotein increased, a higher enzyme activity against the lipoprotein-phospholipids was observed, with a maximum activity 5-fold higher against LDLp than against HDLp. Overall, the changes observed as the lipoprotein particles are loaded with DG and apoLp-III provide a link between the structure and properties of the lipophorin surface and the physiological roles of HDLp and LDLp particles. PMID- 8611505 TI - Characterization of detergent-solubilized beef liver mitochondrial NAD+ glycohydrolase and its truncated hydrosoluble form. AB - Membrane-bound beef liver mitochondrial NAD+ glycohydrolase (NADase) was partially purified after its solubilization by either detergent or crude pancreatic lipase, steapsin. Solubilization by steapsin yielded a homogeneous water-soluble enzyme. A fluorescence assay was developed that allowed visualization of NADase activity directly within the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular masses of the detergent- and steapsin-solubilized forms were estimated to be about 30,000 and 28,000, respectively. The small part that was cleaved by steapsin represents presumably the membrane anchor of the mitochondrial NADase, as its removal converted the enzyme from a highly hydrophobic to a hydrosoluble protein. The fluorescence staining for activity was also successfully applied to other NADases. Kinetic analyses of the two forms of solubilized mitochondrial NADase revealed that the catalytic properties were unaffected after the steapsin treatment. Neither the binding affinity of the substrate analog 1, N6-etheno-NAD+ nor the inhibition by nicotinamide differed significantly between these two forms of the enzyme. Moreover, the dependence of the enzyme activity on temperature, pH, or ionic strength was also similar for both preparations. However, activity of the detergent-solubilized but not of the truncated steapsin-solubilized enzyme was strongly dependent on the presence of bivalent metal ions such as ZN2+. These results suggest that the membrane part of the mitochondrial NAD+ glycohydrolase is not required for catalysis. It appears, however, to be of importance for the regulation of the enzyme. PMID- 8611506 TI - Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli. AB - The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D galactopyranoside quenches the fluorescence of 2-(4' maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule. PMID- 8611508 TI - Phospholipase D regulation by a physical interaction with the actin-binding protein gelsolin. AB - Increases in intracellular phosphatidic acid levels caused by receptor- mediated activation of phospholipase D (PLD) have been implicated in many signal transduction pathways leading to cellular activation. PLD is known to be regulated by several means, including tyrosine kinase activity, increases in Ca2+, receptor-coupled G proteins, small GTP binding proteins, ceramide metabolisms, and protein kinase C. We have investigated a additional regulatory effect on PLD activity involving nucleoside triphosphates (NTPs). A NTP binding protein copurifies with LPD activity from rabbit brains using a GTP-agarose affinity column, and this protein stimulates PLD activity only in the absence of NPTs. The NTP effect is reversible and labile, and the binding protein is separable from the PLD activity by heparin-agarose chromatography. We identified this protein as the actin- binding protein gelsolin by amino acid sequencing following peptide mapping. This finding was verified by the co immunoprecipitation of gelsolin and PLD activity as well as by the reconstitution of gelsolin- dependent nucleotide sensitive PLD activity by the addition of purified gelsolin-free PLD. Our data indicate that actin rearrangements and PLD signaling are coordinately regulated through the physical association between PLD and gelsolin and that this interaction may also serve to amplify both PLD signaling and actin reorganization. PMID- 8611507 TI - Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase-1 inhibitor. AB - The cDNA encoding human brain protein phosphatase inhibitor-1 (I-1) was expressed in Escherichia coli. Following PKA phosphorylation at a threonine, recombinant human I-1 was indistinguishable from rabbit skeletal muscle I-1 as a potent and specific inhibitor of the type-1 protein serine/threonine phosphatase (PP1). N Terminal phosphopeptides of I-1 that retained the selectivity of intact human I-1 highlighted a functional domain that mediates PP1 inhibition. Substituting alanine in place of threonine-36 eliminated I-1 phosphorylation by PKA and its phosphatase inhibitor activity. An acidic residue was substituted in place of the phosphoacceptor to produce I-1(T35D), a constitutive phosphate inhibitor. I 1(T35D) was an equally effective inhibitor of PP1 and the type-2 phosphatase, PP2A. However, CNbr digestion of I-1(T35D) yielded an N-terminal peptide that showed 100-fold increased specificity as a PP1 inhibitor. This provided new insight into a unique conformation of the phosphorylated I-1 that accounts for selective inhibition of PP1 activity. Truncation of an active I-1 phosphopeptide identified an N-terminal sequence that was reduced in addition to threonine-35 phosphorylation to inhibit PP1 activity. Biosensor studies demonstrated that PP1 bound to both Phosphorylated and dephosphorylated I-1 and suggested that distinct elements of I-1 structure accounted for PP1 binding and inhibition. Our data point to multiple interactions between the I-1 functional domain. and the PP1 catalytic subunit that define this phosphoprotein as a physiological regulator of the type-1 protein phosphatase. PMID- 8611509 TI - Polymorphism of F-actin assembly. 1. A quantitative phase diagram of F-actin. AB - We have made the first quantitative phase diagram of actin filament (F-actin) assembly represented by the concentration of F-actin and the chi parameter which characterizes solvent-solute interaction energy. We manipulated the chi value of F-actin by adding a high molecular weight poly- (ethylene glycol) with average molecular weight 6000 (PEG 6K). The preferential exclusion of PEG 6K from the region adjacent to F-actin increases the chi value of F-actin. We qualified the PEG 6K-induced increase of the chi value through analysis of the PEG-induced solubility change of protein. The phase diagram shows that F-actin changes its assembly structure from isotropic disordered distribution to anisotropic ordered phase of a lyotropic liquid crystalline with an increase in the concentration and to concentrated anisotropic ordered phase of a crystalline-like bundle with a small increase in chi respectively, in the physiological concentration range. The formation of the crystalline- like bundle suggests that some specific force may act between F-actin. The present results demonstrate that F-actin can take various assembly structures as observed in cytoplasm by itself, indicating that the versatility of F-actin assembly in cytoplasm may be based on the thermodynamic properties of F-actin as a rod-like molecule. PMID- 8611510 TI - Polymorphism of F-actin assembly. 2. Effects of barbed end capping on F-actin assembly. AB - In the accompanying paper [Suzuki, A., Yamazaki, M., & Ito, T. (1996) Biochemistry 35, 5238-5244], we presented a quantitative phase diagram of actin filament ( (F-actin) described with the F-actin concentration and delta chi value which characterizes the affinity of F-actin with solvent. The phase diagram shows that F-actin changes its assembly structure from an isotropic disordered distribution to a dilute ordered assembly of a lyotropic crystalline with an increase in the concentration an to a concentrated ordered assembly of a crystalline-like bundle with an increase in the delta chi value (i.e., with a decrease in the affinity with the solvent), respectively, in the physiological concentration range. We report here that capping the barbed end of F-actin significantly affects the phase diagram. The F-actin capped by gelsolin (capped F actin) decreased the delta chi value required for the formation of the concentrated ordered assembly. The time taken for the decrease in the delta chi value to reach a stationary state after the barbed end capping was proportional to the filament length (approximately 1 h/microm length). the electron microscopic morphology of the concentrated ordered assembly of the capped F-actin was a wide and loose bundle, which was distinctly different from the crystalline like bundle of the uncapped F-actin. Fragmin from the acellular slime mould, which has similar functions to gelsolin, showed the same effects. These results suggest that the barbed end capping of F-actin gradually changes the nature of the whole filament so as to make the interaction with the solvent more unstable, and the F-actin loses the ability to make a crystalline-like bundle. PMID- 8611511 TI - Expression of the mouse mastocytoma glucosaminyl N-deacetylase/ N sulfotransferase in human kidney 293 cells results in increased N-sulfation of heparan sulfate. AB - The biosynthesis of heparin and heparan sulfate involves a series of polymer modification reactions that is initiated by N-deacetylation and subsequent N sulfation of N-acetylglucosamine residues. These reactions are catalysed by a combined N-deacetylase/N-sulfotransferase. Proteins expressing both activities have previously been purified from mouse mastocytoma, which generates heparin, and from rat liver, which produces heparan sulfate. In the present study, the mouse mastocytoma enzyme has been expressed in the human kidney cell line, 293, to investigate whether it could promote modification of the endogenous heparan sulfate precursor polysaccharide into a heparan-like molecule. The N-deacetylase activity of the stably transfected cell clones as approximately 8-fold higher, on a cell-protein basis, than that of control cells, while the N-sulfotransferase activity was increased approximately 2.5 fold. The amounts of glycosaminoglycans synthesized were the same in control and transfected cells, measured as incorporation of [3H]-glucosamine, whereas 35S-labeled glycosaminoglycans were approximately 50% increased in transfected cells, with an increased relative content of heparin sulfate. Structural analysis demonstrated the the glucosamine units of the "heparan sulfate" from transfected cells were almost exclusively N sulfated, as expected for heparin, whereas more than half of the glucosamine units of the control polysaccharide remained N-acetylated. Notably, the increased N-sulfation was not accompanied by increased O-sulfation, not by C-5 epimerization of D-glucuronic to L-iduronic acid units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process. PMID- 8611512 TI - Self-association of spectrin's repeating segments. AB - We have examined the self-association behavior in solution of one of the repeating conformational segments of Drosophila spectrin, D-alpha-14, as well as of the two-segment unit, D-alpha-14,15. In both polypeptides, sedimentation equilibrium and nondenaturing gel electrophoresis detect a reversible, moderate affinity (K2 approximately equal to 10(4) M-1) dimerization reaction. Equilibration between monomer and dimer is kinetically limited near 5 degrees C, but occurs at a measurable rate at temperatures > or = 20 degrees C. The temperature dependence for equilibration is consistent with the requirement for extensive disruption of helix-helix packing as the reaction proceeds in either direction. Hydrodynamic studies by means of sedimentation velocity confirm that in solution the C helix in the monomer of D-alpha_14 is folded back to interact with the A and B helices, and that the form of monomeric subunit observed in the crystal structure, in which the A and B helices are continuous, does not persist in the monomer in solution. Both the dimer of D-alpha-14 and the monomer of D alpha-14,15 appear to be twice the length of the D-alpha-14 monomer, while the frictional ration of the D-alpha-14,15 dimer is consistent with four end-to-end triple alpha-helical domains. PMID- 8611513 TI - Functional and sequence characterization of coagulation factor IX/factor X binding protein from the venom of Echis carinatus leucogaster. AB - A new coagulation factor IX/factor X-binding protein (IX/X-bp) from Echis carinatus leucogaster venom has been purified and designated ECLV IX/X-bp. ECLV IX/X-bp binds factor IX and X in a Ca(2+)-dependent manner and is devoid of thrombin-inhibitory and platelet-aggregating activities. The apparent dissociation constants (Kd) for binding of ECLV IX/X-bp to factor IX and factor X are 6.6 and 125 nM, respectively. Upon the addition of Mg2+, the required Ca2+ concentration for optimal binding of ECLV IX/X-bp to factor IX and factor X was prominently reduced. Mg2+ also increases the affinity of factor X for the venom protein. Direct binding of IX/X-bp to factor IX and X could also be detected by far-Western blotting, and results of the experiment ruled out the lectin-like mechanism of ECLV IX/X-bp. The complete amino acid sequence and the disulfide pattern of ECLV IX/X-bp was deduced by enzymatic hydrolysis and automated sequencing of the S-pyridylethylated protein. The venom protein is a heterodimer with one subunit of 131 amino acid residues and another of 125 residues. Both subunits are homologous to each other and to other snake venom proteins of the C type lectin superfamily. PMID- 8611515 TI - The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis. AB - The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AAtase in which Tyr-225 is converted to Phe (Y225F) was investigated. The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm. The primary C alpha 2H-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AAtase at pH 8.9 and 7.5 on kcat/KMAsp are approximately 2, and the KIEs on kcat are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C alpha 2H-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values. The solvent deuterium KIEs (SKIEs) on kcat for reactions with L-Asp catalyzed by wild type AATase and Y225F at their pH/pD maxima approximately 2, and the SKIE on kcat/kMAsp is increased from 1.3 to 2.3 by the mutation. The C4' (S)-2H pyridoxamine 5'-phosphate KIE values on reactions of alpha-ketoacids with both enzymes are near unity. The viscosity effects on kcat/KMAsp and kcat for wild type AAtase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited. The viscosity effects on kcat/KMAsp and kcat for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited. A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AAtase. C alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F- catalyzed reaction. PMID- 8611514 TI - A highly salt-dependent enthalpy change for Escherichia coli SSB protein-nucleic acid binding due to ion-protein interactions. AB - We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to 34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = 144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions. PMID- 8611516 TI - Sarcosine oxidase contains a novel covalently bound FMN. AB - Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric protein containing three different enzymes: noncovalent FAD, noncovalent NAD+, and covalently bound flavin which is released as 8 alpha-(N3-histidyl)riboflavin upon complete hydrolysis of the protein. The following results show that the covalent flavin is not at the FAD level, as previously proposed, but it is rather as 8 alpha-(N3- histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treated with phosphodiesterase or subjected to mild acid hydrolysis. The enzyme contains a total of 5 mol of phosphate. Only one phosphate is covalently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD+. The 31P NMR spectrum of native enzyme exhibits resonances due to a single phosphate monoester an two pyrophosphates. Only a resonance due to phosphate monoester is observed after removal of the noncovalent cofactors and proteolytic digestion of the protein moiety. The 8 alpha-(N3 histidyl)FMN found in corynebacterial sarcosine oxidase represents a novel type of covalent flavin. Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomonas sp. show that these heterotetrameric enzymes also contain covalently bound FMN plus noncovalently bound FAD and NAD+, similar to corynebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxidases (from Bacillus sp. and an unidentified microorganism) were found to contain only covalently bound FAD. PMID- 8611517 TI - Dimerization of the human cytomegalovirus protease: kinetic and biochemical characterization of the catalytic homodimer. AB - The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication. PMID- 8611518 TI - Ribozyme-mediated cleavage of a substrate analogue containing an internucleotide bridging 5'-phosphorothioate: evidence for the single-metal model. AB - An oligonucleotide substrate containing a 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanisms of the hammerhead ribozyme and to probe the catalytic role of the metal cofactor(s). Specifically, we tested the hypothesis that a second metal interacts with the 5'-leaving group to facilitate the cleavage event. To this end, we have examined the ribozyme-mediated cleavage activity of the phosphorothioate substrate at pH 7.5 with a series of divalent metal in both the presence and absence of the polycation spermine. The cleavage products are found to be the same as for the native sequence under a variety of reaction conditions. The influence of divalent metal ion concentration, temperature, and pH on the cleavage rate also has been examined for both the oxo linkage and the thio analogue. Spermine (but not spermidine or NaCl) is shown to support efficient cleavage of the thio analogue in the absence [5 mM ethylenediaminetetraacetic acid (EDTA)] of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effect of 3.6, but a similar effect is not observed with the thio analogue. The pseudo-first-order rate constants for cleavage of the thio analogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 x 10(-3) min-1, respectively. The native oxo linkage is cleaved at essentially the same rate as the thio analogue (35 and 97 x 10(-3) min-1 for Mg2+ and Mn2+, respectively). The absence of an appreciable thio effect and the lack of a preference for either Mg2+ or Mn2+ provides compelling evidence that the metal cofactor does not interact with the 5'-thioanion (or oxyanion) leaving group in the transition state. These rate comparisons additionally reveal the the departure of the 5'-leaving group is not the rate-limiting step of the cleavage reaction catalyzed by the hammerhead ribozyme. PMID- 8611519 TI - Inhibitors of Escherichia coli RNA polymerase specific for the single-stranded DNA of transcription intermediates. Tetrahedral cuprous chelates of 1,10 phenanthrolines. AB - Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives. The cleavage sites are observed in the open, initiating, and elongating complexes. Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription. Although these cuprous chelates of neocuproine bind at multiple sites within three distinct enzyme intermediates, the highest affinity site is within the elongation complex. The I50 of 5 microM for the 2:1 5-phenylneocuproine cuprous complex ((5 phi NC)2Cu+) in runoff transcription therefore primarily reflects its intermediate. The neocuproine cuprous chelates are novel transcription inhibitors because they bind to single stranded DNA sites generated during the course of catalysis by RNA polymerase. PMID- 8611520 TI - SH2 domains mediate the sequential phosphorylation of HS1 protein by p72syk and Src-related protein tyrosine kinases. AB - The protein tyrosine kinase p72syk readily phosphorylates hematopoietic linkage cell-specific protein p50/HS1 with high stoichiometry (up to 4 mol of Pi/mol of protein) and favorable kinetic constants (Km 77 nM, kcat 0.37 s-1), at sites that display the motif that is specifically recognized by the HS2 domains of Src tyrosine kinases. Such a phosphorylation converts p50/HS1 into a good substrate for c-Fgr, which in contrast is nearly inactive on nonphosphorylated p50/HS1. A phosphopeptide reproducing one of the main p50/HS1 site affected by p72syk, but neither its dephosphorylated derivative nor other phosphopeptides with different structure, blocks the secondary phosphorylation of phospho(p50/HS1) by c-Fgr but not its primary phosphorylation by p72syk. It also prevents the coimmunoprecipitation of phospho(HS1) with c-Fgr by anti-(c-Fgr) antibodies. In contrast the HS1[393-402] phosphopeptide is ineffective on the kinase activity of c-Fgr when tested with peptide substrates, showing that inhibition of p50/HS1 phosphorylation is not exerted at the catalytic site of c-Fgr. The sequential phosphorylation of p50/HS1 as well as its specific blockage by the HS1 phosphopeptide is also observable if c-Fgr is replaced by two other Src-related kinases, namely, Lyn and Fyn, as secondary phosphorylating agents. None of these Src-related kinases, however, can carry out the phosphorylation of p50/HS1 at the sites affected by p72syk, even after prolonged incubation. Our data suggest that sequential phosphorylation might represent a general mechanism by which p72syk and other Syk-related kinases generate substrates for Src-related protein tyrosine kinases. They also show that sequential phosphorylation (requiring the concerted action of a primary and a secondary kinases) cannot be surrogated by "processive" phosphorylation where a single kinase catalyzes both the primary and secondary phosphorylation, although both these modes of multiple phosphorylation are based on interactions between SH2 domains of the kinases and phosphotyrosyl sites of the substrate. PMID- 8611521 TI - Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli. AB - Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix VI and the flanking hydrophilic loops (residues 164- 211) was replaced individually with Cys. Of the 48 mutants, 43 accumulate lactose at highly significant rates to > 80% of the steady state observed with C-less permease. Three mutants (Phe185--> Cys, Ala187--> Cys, and Phe208--> Cys) exhibit lower but significant levels of accumulation (30-60% of C-less). Cys replacement for Ala177 or Leu184 results in low transport activity (ca. 20%) in the C-less background but much higher activity (60-70%) in the wild type. Immunoblot analysis reveals that all of the mutants are inserted into the membrane at concentrations comparable to that of C less perrmease. The transport activity of the great majority of the mutants is unaffected by treatment with N-ethylmaleimide (NEM). Relatively modest but significant inactivation (ca. 50%) is observed with mutants Phe170--> Cys, Gly173 -> Cys, and Ala187--> Cys, and these positions cluster on the same face of the helix VI. Moreover, the two positions where single Cys replacements result in low activity (Ala177 and Lcu184) are on the same face of helix VI. The results demonstrate the following. (i) Permease function is not disrupted by replacement of most residues with Cys, but function is disrupted when some of the residues are further altered by addition of the NeM moiety. (ii) The latter residues lie on a stripe down one face of an alpha-helix, and within the same stripe are residues where Cys substitution itself leads ti inhibition of function. PMID- 8611522 TI - Incorporation of a non-nucleotide bridge into hairpin oligonucleotides capable of high-affinity binding to the Rev protein of HIV-1. AB - A bridge containing a rigid trans-stilbene group, -P(O)(O-)O(CH2)3NHC(O)- C6H4 CH=CHC6H4C(O)NH(CH2)3OP(O)(O-)-, has been incorporated into several oligonucleotide sequences based on the minimal Rev Binding Element (RBE) of HIV 1. This bridge was found to be effective as a UUCG tetraloop in stabilizing short RNA duplex structures containing mismatched bases and bulged out nucleotide residues and to be more effective than either a TTTT loop or a triethyleneglycol linker in stabilizing similar DNA structures. Evaluation of stilbene-containing RNA RBE sequences of varying length for their ability to bind the Rev protein of HIV-1 showed that a 22-nucleotide stilbenedicarboxamide conjugate bound Rev almost as well as a 94-base fragment of the Rev Responsive Element (RRE). A DNA hairpin mimetic with the same sequence was incapable of Rev binding. Taken together, these experiments serve as an example for how in vitro selection and chemical modification can be combined to generate high-affinity mimetics of nucleic acid sequence and structure. PMID- 8611524 TI - Aluminum fluoride inhibition of nitrogenase: stabilization of a nucleotide.Fe protein.MoFe-protein complex. AB - Coupling of ATP hydrolysis to electron transfer in nitrogenase has properties similar to nucleotide-dependent switch proteins. Aluminum fluoride, a powerful inhibitor of some switch proteins, is a progressive, slowly reversible (t1/2 for reversal > 21 h) inhibitor of nitrogenase that requires both component proteins (Fe-protein and MoFe-protein) and nucleotide (either ATP or ADP). The pseudo first-order inhibition is dependent on the aluminum fluoride species, AlF4, and is linear with [Al] concentration (nonsaturating) at a pH optimum near 7.1-7.3. The inhibitor appears to react with the transient complex of the two component proteins and nucleotide. Although ADP can support the AlF inhibition, the rate of inhibition is more than 30-fold greater with ATP, which suggests the reactive conformation more closely resembles ATP hydrolysis. Conditions that increase enzymic turnover (protein concentration and component ratio) also increase the rate of inhibition, while ionic strength which slows enzymic activity spares the inhibition. The inhibited protein was isolated by gel filtration chromatography and found to be an AlF4-ADP-Fe-protein.MoFe-protein complex with the ratio of 2:1 that is consistent with two active sites per MoFe-protein alpha 2 beta 2 tetramer. Hence, inhibition by AlF4 is the stabilization of a complex that no longer hydrolyzes ATP or reduces substrates. We propose that AlF-ADP is tightly bound only in Fe-protein conformations obtained in the complex with MoFe-protein. Ligands (including Arg-46) at the base of a flexible flap on the Fe-protein could immobilize MoFe-protein--Fe-protein interface, thereby preventing dissociation of the complex. PMID- 8611523 TI - Metal ion interaction with urease and UreD-urease apoproteins. AB - Klebsiella aerogenes urease in a Ni-containing enzyme (two Ni per alpha beta gamma unit) that is purified as an apoprotein from cells grown in Ni-free medium. Partial activation of urease and UreD-urease apoproteins is achieved in vitro by incubation in the presence of Ni(II) and CO2, whereas incubation of these proteins with Ni alone leads to the formation of inactive species [Park, I.-S., & Hausinger, R. P. (1995) Science 267, 1156-1158]. Here we determined the kinetics of these inhibitory reactions and demonstrated the presence of two Ni ions per alpha beta gamma unit in the inactive proteins. Although metal-substituted urease has never been purified from Ni-deprived cell, several other metal ions were shown to bind to the urease apoproteins. Divalent Zn, C, Co, and Mn all inhibited Ni- and Co2-promoted urease activation at concentrations below that of Ni, whereas Mg and Ca ions did not inhibit this process. Ni-inhibited species recovered their ability to be partially activated after EDTA treatment. In contrast, samples that were exposed to Co or Cu ions were irreversibly inactivated, and EDTA treatment of Zn- or Mn-inhibited samples led to reduced levels of activation competence. Mn-substituted urease, generated from urease apoprotein samples in a Mn- and Co2-dependent manner, was shown to be active, whereas other metal-substituted forms if urease lacked activity. The Mn-protein possessed only 2% of the activity of Ni-activated apoprotein [ approximately 8.0 vs approximately 400 mumol min-1 (mg protein)-1], but its KM value was only moderately altered from that of the native enzyme (3.86 +/- 0.15 mM vs 0.2 mM). Unlike the Ni-containing enzyme, Mn-urease was inhibited by EDTA. Given the evidence that urease apoprotein binds numerous metal ions, we speculate on possible roles for the UreD, UreF, and UreG accessory proteins in urease activation. PMID- 8611525 TI - On the dynamics and conformation of the HA2 domain of the influenza virus hemagglutinin. AB - To investigate the dynamics and conformation of the membrane-interacting HA2 domain of the hemagglutinin protein of influenza virus, the peripheral part of the HA2 domain (aa 1-127) was expressed in Escherichia coli. Four consecutive single-cysteine mutants, F63C, H64C, Q65C, and I66C, were generated using site directed mutagenesis. This region is proposed to undergo a conformational change from a loop to a helical coiled-coil when going from the native to the fusion active state [Bullough et al. (1994) Nature (London) 371, 37-43]. In the trimeric coiled-coil geometry positions 63 and 66 belong to the core so that cysteines from individual monomers are spatially close. On the other hand, positions 64 and 65 face the aqueous phase so that cyteines from monomers are spatially remote. The mutants were studied with cysteine-cysteine cross-linking and the spin labeling electron paramagnetic resonance (EPR) in both the membrane-bound state and in the detergent-solubilized state. Extensive intramolecular cysteine cysteine cross-linking was observed not only for F63C and I66C but also for H64C. Rates of cross-linking were comparable for these three mutants at physiological temperatures. These results are inconsistent with what is expected for a well defined coiled-coil and suggest that the region containing the mutation sites is flexible. However, a characteristic cross-linking pattern consistent with a well defined coiled-coil developed at very low temperatures. Line shapes of EPR spectra also indicate that this region is dynamic at ambient temperatures. Such flexibility perhaps arises from an equilibrium between a coiled-coil and a random coil conformation. No significant changes of the EPR spectra were observed upon lowering the pH to fusogenic conditions, suggesting that this flexible structure is the stable conformation at both neutral and low pH. The dynamic flexibility of this region may have important implications for the mechanism of HA-induced membrane fusion; for example it may be required for the apposition of the viral and endosomal membranes. PMID- 8611526 TI - Hierarchical modeling of phenolic ligand binding to 2Zn--insulin hexamers. AB - Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)-insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)-insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The binding isotherms were modeled and thermodynamic parameters were quantified using a novel, flexible algorithm that permitted the development of a hierarchical series of physical models. With the insulin hexamer represented as a dimer of trimers, the modeling demonstrated that ligand binding is highly cooperative in nature, both intra- and inter-trimer. The isotropic inter-trimer cooperativity was dominant and negative in every system studied, with initial binding constants typically an order of magnitude greater for the binding of ligands to the first trimer relative to the second. The inter-trimer cooperatively estimated from the modeling of solution calorimetry data is consistent with a T6 <--> T3R3 <--> R6 equilibrium first proposed from crystallographic investigations. Intra-trimer cooperatively was present only in the enthalpy coefficient space, not in the equilibrium coefficient space, and therefore, less of a factor. The order of binding affinity for the ligands studied in resorcinol >> phenol > or = m-cresol as determined from their overall free energies of binding to the 2Zn(II)-insulin hexamer ( 26.6, -23.4, and -23.4 kcal/mol, respectively) and their intrinsic binding constants (8780, 5040, and 3370 L/mol, respectively) at 14 degrees C. The temperature dependence of phenol binding to 2Zn(II)-insulin hexamer was modeled. Increasing temperature decreased the magnitude of both the intrinsic binding constant and the inter-trimer was cooperatively. The second phase of the ITC binding profile was also found to be highly temperature dependent. At lower temperatures the second phase is endothermic but gradually decreases with increasing temperature and subsequently becomes exothermic. This effect is attributed to loss of water from the hydration shell of the insulin hexamer with increasing temperature and consequently reduces the entropic contributions to the T <--> R transition in the phenol/2Zn(II)-insulin hexamer system. PMID- 8611527 TI - Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean. AB - Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution. PMID- 8611528 TI - Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica. AB - The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation. PMID- 8611529 TI - Ligand geometry of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase from rotational-echo double-resonance NMR. AB - The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog. PMID- 8611530 TI - X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. AB - The structure of the vanadate-trapped ADP complex of a truncated head of Dictyostelium myosin II consisting of residues Asp 2-Asn 762 has been determined by molecular replacement at 1.9 A resolution and refined to a crystallographic R factor of 19.4%. The crystals belong to the orthorhombic space group C2221 where a = 84.50 A, b = 145.4 A, and c = 152.8 A. The conformation of the protein is similar to that of MgADP.AlF4.SlDc [Fisher, A.J., et al. (1995) Biochemistry 34, 8960-8972]. The nucleotide binding site contains a complex between MgADP and vanadate where MgADP exhibits a very similar conformation to that seen in previous complexes. The vanadate ion adopts a trigonal bipyramidal coordination. The three equatorial oxygen ligands are fairly short, average 1.7 A, relative to a single bond distance of approximately 1.8 A and are coordinated to the magnesium ion, N zeta of Lys 185, and five other protein ligands. The apical coordination to the vanadate ion is filled by a terminal oxygen on the beta phosphate of ADP and a water molecule at bond distances of 2.1 and 2.3 A, respectively. The long length of the apical bonds suggests that the bond order is considerably less than unity. This structure confirms the earlier suggestion that vanadate is a model for the transition state of ATP hydrolysis and thus provides insight into those factors that are responsible for catalysis. In particular, it shows that the protein ligands and water structure surrounding the gamma phosphate pocket are oriented to stabilize a water molecule in an appropriate position for in-line nucleophilic attack on the gamma-phosphorus of ATP. This structure reveals also an orientation of the COOH-terminal region beyond Thr 688 which is very different from that observed in either MgADP.BeFx.SlDc or chicken skeletal myosin subfragment 1. This is consistent with the COOH-terminal region of the molecule playing an important role in the transduction of chemical energy of hydrolysis of ATP into mechanical movement. PMID- 8611531 TI - Identification of the ATP transporter of rat liver rough endoplasmic reticulum via photoaffinity labeling and partial purification. AB - In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4 azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax of 1 nmol min-1 (mg of protein)-1. The transport of ATP was specifically inhibited by AB-ATP and 4, 4'-diisothiocyanatostilbene-2', 2' disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with Ki value of 0.19 microM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS- PAGE analysis of RER vesicles photolabeled with [gamma-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56 kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-32P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER. PMID- 8611532 TI - Probing the function of Asp128 in the lower molecular weight protein-tyrosine phosphatase-catalyzed reaction. A pre-steady-state and steady-state kinetic investigation. AB - The role of Asp128 in the catalytic mechanism of the low Mr protein-tyrosine phosphatase (PTPase) from the fission yeast Schizosaccharomyces pombe has been investigated by a combination of site-directed mutagenesis and pre-steady-state and steady-state kinetic analysis. The corresponding aspartic acid in the bovine enzyme is located on a loop adjacent to the phosphate-binding loop and forms a hydrogen bond with the oxygen atom of the bound sulfate or phosphate that is structurally homologous to the ester oxygen in substrates [Su et al. (1994) Nature 370, 575-578; Zhang, M., et al. (1994) Biochemistry 33, 11097-11105]. Asp128 has been replaced by a Glu, an Asn, and an Ala. The kcat for the hydrolysis of p-nitrophenyl phosphate (pNPP) decreases by factors of 6.7, 400, and 650 for the mutants D128E, D128N, and D128A, respectively. Compared to the wild type, the binding affinity for phosphate is decreased 2 and 4.3-fold, respectively, for the D128A and D128N mutants, whereas no change in affinity is observed for the D128E mutant. An evaluation of the burst kinetics demonstrates that Asp128 plays a role in both the phosphoenzyme intermediate formation (k2) and breakdown (k3). Thus, substitution at Asp128 by a Glu, an Asn, or an Ala reduces k2 by 17, 7480, and 11900 and reduces k3 by 6.2, 380, and 40. The greater effect on k2 and k3 is consistent with a dissociative transition-state for the low M(r)PTPase-catalyzed reaction. Results from the rapid kinetics, partition experiments, and leaving group dependence experiments suggest that for the wild type and D128E mutant, the rate-limiting step is k3, whereas k2 has become rate limiting for the D128N mutant. With the exception of pNPP, k2 may also be rate limiting for D128A. Taken together, these results are consistent with Asp128 or Glu128 acting as a general acid to donate a proton to the phenolate leaving group in the phosphorylation step, and the carboxylate side chain plays a role as a general base to activate a nucleophilic water molecule in the dephosphorylation step. The presence of the general acid ensures productive partitioning toward phosphoenzyme formation. In the absence of the general acid, the nature of the transition-state for the phosphorylation step is sensitive to the pKa of the attacking active site thiol group and changes with the structure of the leaving group. PMID- 8611533 TI - Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase. AB - The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3 phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes. PMID- 8611534 TI - Purification and characterization of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans. AB - Xanthine dehydrogenase (XDH) is induced in Comamonas acidovorans cells incubated in a limited medium with hypoxanthine as the only carbon and nitrogen source. The enzyme has been purified to homogeneity using standard techniques and characterized. It contains two subunits with M(r) values of 90 and 60 kDa. Gel filtration studies show the enzyme to have an alpha 2 beta 2 native structure. No precursor form of the enzyme is observed on Western blot analysis of cell extracts obtained at various stages of enzyme induction. Metal analysis of the purified enzyme shows 1.1 Mo, 4.0 Fe, and 3.6 phosphorus atoms per alpha beta protomer. Cofactor analysis shows the enzyme to contain a single molybdopterin mononucleotide and one FAD per alpha beta protomer. Electron spin resonance and circular dichroism spectral studies of the oxidized and reduced forms of the enzyme suggest the Fe centers to be two nonidentical [2Fe-2S] clusters. Electron spin resonance signals due to Mo(V) and neutral FAD radical are also observed in the reduced form of the enzyme. Purified enzyme preparations ranged from 70% to 100% functionality. The enzyme is irreversibly inactivated by CN- and is inhibited on incubation with allopurinol. With xanthine and NAD+ as substrates the enzyme has a specific activity of 50 units/mg, a kcat value of 120 s-1, an activity/flavin ratio of 1930, and respective Km values of 66 and 160 mM. Using 8 D-xanthine as substrate, a DV value of 1.8 is found with no change in Km. Thus, the Km and KD values of the enzyme for xanthine are equal. These data show Comamonas XDH to exhibit structural properties similar to bovine milk xanthine oxidase/dehydrogenase and to chicken liver xanthine dehydrogenase. Although the bacterial enzyme exhibits a 6-7-fold greater turnover rate than bovine or avian enzymes, the catalytic efficiencies (as measured by V/K) are similar for all three enzymes. PMID- 8611535 TI - Isotope partitioning with Ascaris suum phosphofructokinase is consistent with an ordered kinetic mechanism. AB - Isotope partitioning and initial velocity studies have been used to study the kinetic mechanism of Ascaris suum phosphofructokinase (PFK) at pH 8.0 for the native enzyme (nPFK), and at pH 6.8 for a form of enzyme desensitized (dPFK) to hysteresis in the reaction time course, to ATP allosteric inhibition, and to F6P homotropic cooperativity. Complete trapping (P*max approximately equal to 100%) of the E:MgATP* complex as fructose (1-32P)-1, 6-bisophosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism [Rao, G.S.J., Harris, B.G., & Cook, P.F. (1987) J.Biol. Chem. 262, 14074-14079] with MgATP binding before fructose 6-phosphate (F6P). K'F6P values for trapping of MgATP of 0.54 +/- 0.09 mM for nPFK and 0.85 +/- 0.15 mM for dPFK were obtained. Saturating amounts of the heterotropic activator fructose 2, 6 bisphosphate (F26P2) gives no change in the trapping parameters for nPFK with a P*max of 100% and a K'F6P of 0.40 +/- 0.06 mM. For dPFK, however, F26P2 causes a decrease in both parameters, giving a P*max of 54% and a K'F6P of 0.26 +/- 0.07 mM. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initial velocity studies confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the E:[14C]F6P complex could be detected, indicating either that the E:[14C]F6P complex does not form in a significant amount under the conditions used or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. The data are consistent with a predominantly ordered mechanism with MgATP binding prior to F6P. The minor pathway with MgATP dissociating from the E:F6P:MgATP ternary complex becomes apparent for the dPFK in the presence of F26P2. PMID- 8611536 TI - Identification of Asp 549 as the catalytic nucleophile of glycogen-debranching enzyme via trapping of the glycosyl-enzyme intermediate. AB - Glycogen-debranching enzyme catalyzes the removal of branching from glycogen via a two-step process involving first the transfer of a maltotriosyl unit from the branch to the main chain and second the hydrolysis of the residual alpha-(1,6) linked glucose moiety. Since the transfer occurs with retention of anomeric configuration, a mechanism involving a maltotriosyl-enzyme species is presumed. 4 Deoxy-alpha-maltotriosyl fluoride functions as an incompetent substrate for this transferase activity since a glycosyl-enzyme species in formed, as witnessed by a "burst" of fluoride release, but turned over only very slowly unless a suitable acceptor such as maltotriose is added, at which point 4-deoxymaltohexaose is released. Peptic proteolysis of this trapped enzyme generated a mixture of peptides which was separated by reverse phase high-performance liquid chromatography, and the glycosylated peptide was located by use of tandem mass spectrometry in the neutral loss mode. Subsequent tandem mass spectrometric experiments on this peptide identified it as one surrounding Asp 549. This amino acid is completely conserved in all alpha-glucanotransferases and alpha glucosidases belonging to this sequence -related family and is hereby identified as the catalytic nucleophile. PMID- 8611537 TI - Evidence for association of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from pancreatic islets and HIT insulinoma cells with a phosphofructokinase like protein. AB - Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet beta-cells, and ATP has attracted interest as a messenger of glucose metabolism within beta-cells. Glucose-induced insulin secretion from islets and HIT insulinoma cells is accompanied by activation of an ATP stimulatable Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme, the catalytic activity of which resides in a 40 kDa protein. An analogous PLA2 enzyme in myocardium was recently found to consist of a complex of a 40 kDa catalytic protein with a tetramer of an isoform of the glycolytic enzyme phosphofructokinase (PFK). Association of the PFK isoform with the myocardial PLA2 catalytic protein was found to confer ATP sensitivity onto the enzyme complex. Here we demonstrate that the majority of HIT cell and islet ASCI-PLA2 catalytic activity elutes from a gel filtration column in a region corresponding to 400 kDa, suggesting that the 40 kDa beta-cell ASCI-PLA2 catalytic protein exists as part of a larger molecular mass complex. Islet and HIT cell ASCI-PLA2 activities were immunoprecipitated by antibodies directed against PFK, and the immunoprecipitates contained 40 and 85 kDa proteins which correspond to the molecular masses of the PLA2 catalytic protein and of a PFK monomer, respectively. Islet and HIT cell ASCI-PLA2 activities were selectively and reversibly adsorbed to affinity matrices containing immobilized PFK but not to similar matrices containing immobilized transferrin or bovine serum albumin. Addition of free PFK prevented binding of HIT cell ASCI-PLA2 activity to immobilized PFK matrices and promoted desorption of activity previously bound to such matrices. These results suggest that beta-cell ASCI-PLA2, like the myocardial enzyme, exists as a complex comprised of a catalytic protein and a PFK like protein and raise the possibility that the ASCI-PLA2 complex may represent a component of the beta-cell glucose sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved in glucose-induced insulin secretion. PMID- 8611538 TI - Fragmin, a microfilament regulatory protein from Physarum polycephalum, is phosphorylated by casein kinase II-type enzymes. AB - Fragmin is a 42 kDa regulatory protein involved in actin microfilament organization in Physarum polycephalum. We show that fragmin is a target of casein kinase II (CK II) enzymes isolated from evolutionarily divergent species. In Physarum microplasmodia, two such kinases were identified. A serine residue located in the sequence Gly-Gly-Ser-Asp-Leu-Glu constitutes the phosphorylation site and was identified by phosphopeptide sequencing, mass spectometry analysis, and inhibition studies with a synthetic peptide corresponding to this site. Interestingly, the actin-fragmin dimer (A--F) as well as the actin2-fragmin trimer (A2--F) are equally efficient targets, and phosphorylation had no effect on the actin-binding properties of fragmin. Actin-fragmin isolated from microplasmodia revealed a minor acidic fragmin isoform, suggesting that fragmin is phosphorylated in vivo. The actin-fragmin complex is also phosphorylated on the actin subunit by an endogenous actin-fragmin kinase [Gettemans, J., De Ville, Y., Vandekerckhove, J., & Waelkens, E. (1992) EMBO J. 11, 3185-3191]. We show that the two phosphorylation events act independently of each other. PMID- 8611539 TI - High affinity inositol 1,3,4,5-tetrakisphosphate receptor from rat liver nuclei: purification, characterization, and amino-terminal sequence. AB - Inositol 1,3,4,5-tetrakisphosphate (InsP4) mediates nuclear calcium signalling [Koppler P., Matter, N., Malviya A.N. (1993) J. Biol. Chem. 268, 26248-26252], and a distinct high affinity InsP4 binding site is identified with rat liver nuclei [Koppler, P., Mersel, M., & Malviya, A.N. (1994) Biochemistry 33, 14707 14713] as compared with other rat liver membrane fractions. A novel InsP4 receptor protein derived from rat liver nuclei has been purified to apparent homogeneity employing preparative isoelectric focusing, electrophoretic mobility, nondenaturating polyacrylamide gel electrophoresis, and electroelution. Isoelectric focusing indicated an isoelectric pH around 4.3 +/- 0.2 which was further confirmed by bidimensional electrophoresis. The high affinity nuclear InsP4 receptor was identified as a 74 kDa protein both on the SDS-PAGE and on the bidimensional electrophoresis. Partial microsequence analysis showed that the N terminal end of nuclear InsP4 receptor consists of amino acids: PNHKNEIAGNFS. The 74 kDa nuclear InsP4 receptor protein is a distinct protein from the other InsP4 receptors purified from other sources and documented in the literature. PMID- 8611540 TI - FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O. AB - Fourier transform infrared (FTIR) spectroscopy was employed to examine the thermal denaturation of the Fe(III), Fe(II), and Fe(II)-CO forms of cytochrome c peroxidase and horseradish peroxidase in phosphate buffer at pD 7.0. The amide I' regions of the deconvolved spectra are consistent with predominantly alpha helical secondary structure around room temperature, but the alpha-helical absorption of the two peroxidases differs significantly; bands assigned to alpha helical components occur at 1659 and 1649 cm-1 in horseradish peroxidase and at 1652 and 1637 cm-1 in cytochrome c peroxidase. The thermal denaturation mechanisms of the peroxidases also vary. All three forms of cytochrome c peroxidase retain their secondary structure up to 50 degrees C, when bands characteristic of aggregation (1616 and 1684 cm-1) appear in the amide I' region, and above 55 degrees C rapid loss of secondary structure is accompanied by enhanced aggregation. In horseradish peroxidase, on the other hand, the Fe(III) and Fe(II) states exhibit dissimilar denaturation mechanisms. Slow, gradual alteration of secondary structure is observed for Fe(III) horseradish peroxidase on heating, and polypeptide unfolding appears to be complete around 90 degrees C, without aggregation. In Fe(II) and Fe(II)-CO horseradish peroxidase, aggregation bands appear at approximately 55 degrees C, signaling the onset of denaturation. Frequency shifts in the v(CO) bands above room temperature reveal the conformational changes in the heme cavity precede global conformational changes in cytochrome c peroxidase but not in horseradish peroxidase. The reduction in amide II intensities, due to peptide H-D exchange on heating the peroxidases in D2O, indicates the formation above room temperature of partially unfolded states with increased solvent accessibility but intact secondary structures. PMID- 8611541 TI - Interstrand complex formation of purine oligonucleotides and their nonionic analogs: the model system of d(AG)8 and its complement, d(CT)8. AB - We have investigated the role of purines in interstrand complex formation with regard to substitution of the negatively-charged, phosphodiester backbone by a nonionic, internucleoside linkage. Using the purine oligomer, d(AG)8, its methylphosphonate analog, d(AG)8, and the complementary pyrimidine oligomer, d(CT)8, as a model system, the stoichiometry, conformation, and stability of complexes formed at pH 8 were studied by spectroscopic and electrophoretic methods. When there is only one oligomer species in solution, d(AG)8 behaves as a single-stranded molecule. In contrast, the d(AG)8 oligomer readily forms an intermolecular self-complex, particularly in the presence of magnesium ion. Using either purine oligomer, duplexes can form with the d(CT)8 strand which differ in terms of their conformation and in the dependence of their thermal stability on sodium and magnesium ions. All studies show that a stable triplex forms with a 1:2 d(CT)8:d(AG)8 stoichiometry which does not require high concentrations of sodium or magnesium ions. Triplex formation between the d(CT)8 strand and two d(AG)8 strands was not observed. Native gel electrophoresis suggests that a 1:1:1 d(CT)8:d(AG)8:d(AG)8 complex may be formed. In regard to triplex formation, the advantage of the methylphosphonate backbone on the purine strand is clearly demonstrated. PMID- 8611542 TI - Thermodynamic analysis of small ligand binding to the Escherichia coli repressor of biotin biosynthesis. AB - BirA is the transcriptional repressor of biotin biosynthesis and a biotin holoenzyme synthetase. It catalyzes synthesis of biotinyl-5'-AMP from the substrates biotin and ATP. The adenylate is the activated intermediate in the biotin transfer reaction as well as the positive allosteric effector for site specific DNA binding. The affinity of BirA for the adenylate is considerably greater than its affinity for biotin, and both binding reactions are coupled to changes in the conformation of the protein. The temperature dependencies of the two binding interactions have been determined using kinetic techniques. Van't Hoff analysis of the equilibrium dissociation constants derived from the kinetic data indicate that while the two binding processes are characterized by large negative enthalpies, the entropic contributions are small for both. Binding enthalpies have also been determined by isothermal titration calorimetry. Consistent with the results of the van't Hoff analyses, the calorimetric enthalpies are large and negative. The greater precision of the calorimetric measurements allowed more accurate estimation of the entropic contributions to the binding processes, which are of opposite sign for the two ligands. In addition, the heat capacity changes associated with the two binding reactions are small. The measured thermodynamic parameters for binding of biotin and bio-5'-AMP to BirA have been utilized to dissect out structural contributions to the binding energetics. Results of these calculations indicate equivalent contributions of burial of polar and apolar surface area to both binding processes. The total loss of solvent accessible surface area is, however, greater for biotin binding. The analysis indicates furthermore that although both binding reactions are coupled to losses in configurational entropy, the magnitude of the conformational change is significantly larger for biotin binding. PMID- 8611543 TI - The Fc segment of IgE influences the kinetics of dissociation of a symmetrical bivalent ligand from cyclic dimeric complexes. AB - As part of a systematic effort to determine the features of immunoglobulin E receptor (IgE-Fc epsilon RI) aggregation that are critical for cellular activation, we used fluorescence to examine the dissociation of a soluble bivalent ligand, N, N'-bis[[epsilon-[(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl] L-cystine ((DCT)2-cys), from soluble bivalent IgE and its bivalent F(ab')2 and monovalent Fab' fragments. Cross-linking of Fab' fragments by (DCT)2-cys is limited to linear dimers, and we find that (DCT)2-cys dissociation from Fab' occurs with a single kinetic coefficient [(4.2 +/- 0.6) x 10-3 s-1] that corresponds to the lower of the two kinetic coefficients observed with the bivalent IgE [(4.7 +/- 0.7) x 10-2 s-1 and (4.4 +/- 0.3) x 10-3 s-1]. Similarly, the lower value is obtained for dissociation of (DCT)2-cys that is monovalently bound to IgE after incubation with a large excess of the ligand. (DCT)2-cys can bind to bivalent F(ab')2 fragments and form a variety of linear and cyclic aggregates, similarly to IgE, but, unlike IgE, we find that dissociation occurs with a single kinetic coefficient similar to that observed for Fab'. We find that IgE and its (Fab')2 fragments form highly stable cyclic dimer rings with two (DCT)2-cys. We demonstrate that the kinetic coefficients are independent of enhanced fluorescence quenching observed for bound sites in cyclic dimers. Together, the results show that the rate constant for breaking a linear cross link formed by (DCT)2-cys is the same as that for dissociation of the monovalently bound (DCT)2-cys. Further, they show that opening of a bond in a dimer ring for the F(ab')2 fragment occurs with approximately the same dissociation rate constant as opening a bond in a linear cross-link. This rate constant is about three times smaller than that observed with IgE, suggesting that steric strain is caused by apposed Fc segments in cyclic IgE dimers. Such structural interference may affect the functional consequences of IgE-Fc epsilon RI aggregation on the cell surface. PMID- 8611544 TI - High-level expression and characterization of a purified 142-residue polypeptide of the prion protein. AB - The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt. PMID- 8611545 TI - Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. AB - The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (INC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (IC) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of INC) consistent with a strong decrease in the population of INC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of INC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of IC, the subsequent partially folded intermediate, INC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha helices. PMID- 8611546 TI - Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped flow double-mixing technique. AB - Often protein folding reactions show complex kinetics, because multiple unfolded species are present, which refold simultaneously. After conformational unfolding, these species are formed by the slow cis/trans equilibrations at Xaa-Pro peptide bonds. To dissect the roles of individual prolines for unfolding and refolding, we used ribonuclease T1, a protein with two cis prolyl peptide bonds, preceding Pro39 and Pro55, and two variants with substitutions at these positions. A stopped-flow double-mixing technique was employed (i) to measure the rates of the individual prolyl isomerizations in the unfolded proteins and (ii) to study the refolding of transient species that are not well populated at equilibrium. In particular, the elusive species with correct prolyl isomers could be produced by short unfolding pulses, and its refolding kinetics could be measured. The two isomerizations in unfolded ribonuclease T1 could be assigned to Pro39 and Pro55, because they occurred with almost identical rates in the wild-type protein, in the single-cis proline variants, and in tetrapeptide-4-nitroanilides, which contained prolines in the same sequential context at Pro39 and Pro55 or ribonuclease T1. The direct refolding reaction of the unfolded molecules with correct prolyl isomers shows a time constant of 180 ms (at 25 degrees C, pH 4.6). This reaction is almost unaffected by the proline substitutions. It depends nonlinearly on temperature with a maximum near 25 degrees C, which suggest that the activated state for this reaction resembles the native rather than the unfolded state in heat capacity. The formation of a transient intermediate with incorrect prolyl isomers could be studied as well. Surprisingly, this reaction is only about 5-fold slower than direct folding, and it is also accompanied by a strong decrease in the apparent heat capacity. PMID- 8611547 TI - Infrared spectroscopy of the cyanide complex of iron (II) myoglobin and comparison with complexes of microperoxidase and hemoglobin. AB - The cyanide complex of FeIIMb prepared and maintained at temperatures below 0 degrees C is sufficiently stable to permit spectroscopic characterization and allow comparison with free HCN and other ferric and ferrous CN complexes. The visible absorption spectrum of FeIIMb-CN has a split alpha band maxima at 571 and 563 nm, suggesting distortion in the x-y plane of the porphyrin. FeIIMb-CN, like the CO complex, was found to be optically active by circular dichroism. The C-N stretching frequencies for the CN-ferrous complexes are very sensitive to parameters within the heme pocket. The values are as follows: FEIIMb at pH 8, 2057 cm-1 with a shoulder appearing at 2078 cm-1 at pH 5.6; FeIIMp, 2034 cm-1. In contrast, the frequencies for C-N stretch differ little among ferric heme complexes, ranging from 2123 to 2125 cm-1 for myoglobin, hemoglobin, and microperoxidase. These values compare with free HCN (2094 cm-1) or CN- (2080 cm 1). Quantum chemical modeling of the neutral iron-porphyrin complex with imidazole and cyanide and of its anion was used to explain the effects of the cyanide coordination and of iron reduction on the C-N stretching frequencies. The lower nu C-N for FeIIMb-CN relative to the ferric complex is attributed to the appearance of additional electron density on all the anti-bonding CN orbitals. The extra electron density was also used to explain that the band width of C-N stretching mode was greater in the ferrous complexes than in the ferric complex. Finally, the calculation shows that sigma donation weakens the Fe-C bond, in qualitative agreement with the spontaneous dissociation of CN- from FeIIMb at -5 degrees C. The sensitivity of CN complexes of ferrous heme proteins to the heme pocket environment and the ability to correlate spectroscopic parameters with calculated electron density suggest that infrared spectroscopy of the CN ligand is an appropriate tool to study ferrous heme proteins. PMID- 8611548 TI - Effect of the tyrosyl radical on the reduction and structure of the Escherichia coli ribonucleotide reductase protein R2 diferric site as probed by EPR on the mixed-valent state. AB - It was recently shown by EPR that high yields of a sterically constrained mixed valent species may be formed in radical free protein metR2 of Escherichia coli ribonucleotide reductase by gamma-irradiation at 77 K [Davydov, R., Kuprin, S., Graslund, A., & Ehrenberg, A. (1994) J. Am. Chem. Soc. 116, 11120]. This species, with S = 1/2, essentially retains the ligand geometry of the original diferric center and should be a sensitive probe for structural changes in the diferric centers. Here we apply this probe and demonstrate that there is a structural difference between the diferric iron center of the complete site of protein R2, with a tyrosyl radical, and that of metR2, without radical. The EPR spectrum of the mixed-valent species of metR2 shows pure axial symmetry, while complete sites show rhombic distortion and a shifted high-field turning point. Differences also remain in the EPR of the first S = 9/2 species obtained by annealing at 165 K, but disappear after relaxation at 200 K. In addition, the diferric center of a complete site is not reduced radiolytically until the associated tyrosyl radical has been reduced, indicating that an electron first reaching the iron center may be transferred to the radical. This route of electron transfer and the influence of the radical on the structure of the iron center are likely to have functional roles for the formation of the proposed substrate radical and regulation of redox processes within the enzyme. The sensitivity of the structure of the iron site to the structure of the Tyr-122 site is also demonstrated by the strong influence the mutation Y122F has on the EPR spectra of the corresponding mixed-valent species. PMID- 8611549 TI - Is the function of the cdc2 kinase subunit proteins tuned by their propensities to oligomerize? Conformational states in solution of the cdc2 kinase partners p13suc1 and p9cksphy. AB - The cdc2 kinase subunit (cks) proteins play an essential function in the control of mitosis through their molecular complexes with the cdc2 kinase. In this work, we characterize the conformational state(s) in solution of the cks proteins p13suc1 from Schizosaccharomyces pombe and p9cksphy from Physarum polycephalum. Monomers of p13suc1 and p9cksphy were found to be markedly nonglobular, presumably with a long, nonfolded C-terminal moiety. This was in contrast to the previously published structure of p13suc1, derived from crystallographic studies on a zinc-promoted p13suc1 dimer, in which the individual p13suc1 subunits had a globular conformation. This disparity was resolved when we found that the globular p13suc1 fold undergoes a conformational transition into nonglobular monomers upon dissociation of the dimers following chelation of the zinc ions by ethylenediaminetetraacetic acid (EDTA). We also found that p13suc1, but not p9cksphy, forms stable dimers in the absence of metal ions. The topology of these EDTA-insensitive dimers likely resembles that of the human p9ckshs2 protein, characterized by beta 4 strand exchange from each nonglobular monomer. PMID- 8611550 TI - Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single stranded DNA. AB - Replication protein A (RPA) is multisubunit single-stranded DNA-binding protein required for multiple processes in DNA metabolism including DNA replication, DNA repair, and recombination. Human RPA is a stable complex of three subunits of 70, 32, and 14 kDa (RPA70, RPA32, and RPA14, respectively). We examined the structure of both wild-type and mutant forms of human RPA by mapping sites sensitive to proteolytic cleavage. For all three subunits, only a subset of the possible protease cleavage sites was sensitive to digestion. RPA70 was cleaved into multiple fragments of defined lengths. RPA32 was cleaved rapidly to a approximately 28-kDa polypeptide containing the C-terminus that was partially resistant to further digestion. RPA14 was refractory to digestion under the conditions used in these studies. The digestion properties of a complex of RPA32 and RPA14 were similar to those of the native heterotrimeric RPA complex, indicating that the structure of these subunits is similar in both complexes. Epitopes recognized by monoclonal antibodies to RPA70 were mapped, and this information was used to determine the position of individual cleavage events. These studies suggest that RPA70 is composed of at least two structural domains: an approximately 18-kDa N-terminal domain and a approximately 52-kDa C-terminal domain. The N-terminus of RPA70 was not required for single-stranded DNA-binding activity. Multiple changes in the digestion pattern were observed when RPA bound single-stranded DNA: degradation of the approximately 52-kDa domain of RPA70 was inhibited while proteolysis of RPA32 was stimulated. These data indicate that RPA undergoes a conformational change upon binding to single-stranded DNA. PMID- 8611551 TI - Mutational analysis suggests the same design for editing activities of two tRNA synthetases. AB - Although the structural basis for amino acid activation by class I tRNA synthetases is known, that for their editing activities has remained elusive. Two class I tRNA synthetases discriminate closely similar amino acids by RNA independent and RNA-dependent mechanisms. In the absence of tRNA, isoleucyl-tRNA synthetase misactivates valine, while valyl-tRNA synthetase misactivates threonine. Both enzymes improve amino acid discrimination by tRNA-dependent hydrolytic editing reactions. Recent mutational analysis of an isoleucyl-tRNA synthetase showed that discrimination of valine from isoleucine by amino acid activation was functionally independent of discrimination by editing. In this work, we used mutational analysis to test whether the two types of amino acid discrimination were functionally independent in valyl-tRNA synthetase. We obtained four mutations in the valine enzyme which severely affected amino acid activation. The two most defective enzymes reduced kcat/Km for activation of valine by more than 4 orders of magnitude and were essentially inactive for aminoacylation. These two defective enzymes were tested and found to be unaltered in catalysis of rapid and selective removal of threonine misacylated onto valine tRNA. On the basis of these data, and in spite of there being few residues conserved between the two proteins in a region believed important for editing, we propose that the valine and isoleucine enzymes share a global design which functionally separates amino acid editing from amino acid activation. PMID- 8611552 TI - Chimeras of yeast and chicken calmodulin demonstrate differences in activation mechanisms of target enzymes. AB - Various chimeric proteins were constructed from yeast (Saccharomyces cerevisiae) and chicken calmodulin (CaM), and regions essential for target activation and responsible for the specific features of the yeast CaM were identified. The chimeric CaMs were designed so that each Ca2+ binding site of the yeast CaM was replaced in series from the C-terminus. Resulting CaM proteins showed Ca2+ binding properties inherent to the original Ca2+ binding site. Cooperative Ca2+ binding and a suitable rearrangement of the two EF-hand sites in each half molecular domain were shown to be important for high-affinity interaction with CaM-dependent cyclic nucleotide phosphodiesterase (PDE). Residues in chicken CaM sequences 129-148 and 88-128, respectively, were required for low values of Kact (the concentration of CaM required for the half-maximal activation) in the activation of PDE and myosin light chain kinase (skMLCK and smMLCK). The difference in the structural requirements indicated different manners of the interaction. While PDE was activated to similar levels by different chimeras, the maximum activity (Vmax) given by chicken CaMs was not achieved by any chimeric CaMs in MLCKs. Residues in chicken CaM sequences 1-50 and 88-129, in addition to Ca2+ binding to the fourth site, were important for high values of Vmax of skMLCK. On the other hand, Met51 and residues in chicken CaM sequence 88-129 were critical for the high Vmax of smMLCK. These residues may work to form the active structure of the catalytic site of each MLCK, while simple binding of CaM seems sufficient to expose the active site of PDE. PMID- 8611553 TI - Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide. AB - We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny. PMID- 8611554 TI - Oxidation of 9-alkylanthracenes by cytochrome P450 2B1, horseradish peroxidase, and iron tetraphenylporphine/iodosylbenzene systems: anaerobic and aerobic mechanisms. AB - Variously substituted alkylanthracenes were studied as models for polycyclic hydrocarbon oxidations. 9-Methylanthracene was oxidized to 9 (hydroxymethyl)anthracene, 10-methyl-10-hydroxy-9-anthrone, and anthraquinone in several systems, including (i) NADPH- and O2-fortified rat liver microsomes, (ii) cytochrome P450 (P450) 2B1 Supported by either iodosylbenzene (PhIO) or a mixture of NADPH-P450 reductase, NADPH, and O2, (iii) horseradish peroxidase and either H2O2 or ethyl hydroperoxide, and (iv) a mixture of iron tetraphenylporphine (FeTPP) and PhIO (in anhydrous CH2Cl2/MeOH). The microsomal system also formed dihydrodiols from 9-methyl- and 9-ethylanthracenes. The formation of the three oxidized products by the P450/NADPH/O2 system was dependent upon O2 label from 18O2 was incorporated into the products, and no label from H2(18O) was incorporated. No label from 18O2 was incorporated into the three products in the FeTPP/PhIO system. In the horseradish peroxidase/H2O2 system, the formation of the three products was decreased when O2 was omitted, and label from both H2(18)O and 18O2 was incorporated into all three products. The results are interpreted in terms of three mechanisms. One is used by the FeTPP and P450 systems, with all oxygen transfers coming from an FeO entity. The other two pathways are utilized by horseradish peroxidase and begin with formation of a radical cation, which can undergo reactions either with H2O or with O2 to form the products detected here. The involvement of a 9-methylanthracene radical cation in the P450 and FeTPP pathways is a possibility, but rapid rearrangement and oxygen rebound must be invoked. Comparisons of products from various 9-alkylanthracenes do not provide evidence that one-electron oxidation is an integral part of the epoxidation process with these compounds. The significance of the lack of trapping of radical (by H2(18O) in the P450 reactions to DNA adduct formation is considered. PMID- 8611555 TI - Influence of cholesterol on the association of plasma proteins with liposomes. AB - The in vivo association of blood proteins with large unilamellar liposomes composed of saturated phosphatidylcholines was analyzed to determine the effect of membrane fluidity and hydrocarbon chain length on liposome-plasma protein interactions and liposome clearance. Liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidoylphosphatidylcholine (DAPC) were administered via the lateral tail vein of CD-1 mice and were subsequently isolated from the blood at 2 min postinjection. The protein binding ability (PB, grams of protein bound per mole total lipid) of the liposomes was quantified and related to their circulation half-lives. Liposomes composed of long-chain saturated phospholipids that exist in the gel (frozen) state at 39 degrees C (DPPC,DSPC and DAPC) bound large quantities of blood proteins, in excess of 48 g of protein per mole total lipid, and were found to be rapidly cleared from the circulation. The incorporation of cholesterol into DSPC liposomes resulted in significantly decreased PB values and enhanced circulation lifetimes for this lipid system. This cholesterol effect plateaued at 30 mol % cholesterol, corresponding to the loss of the gel-liquid crystalline phase transition, and resulted in PB values of 23-28 grams of protein per mole of total lipid. The types of blood proteins binding to DSPC liposomes were not significantly altered by the inclusion of cholesterol. This is the first demonstration of rapid clearance of neutral large unilamellar liposomes having high levels of bound protein. PMID- 8611556 TI - Sequence evidence for strong conservation of the photoactive yellow proteins from the halophilic phototrophic bacteria Chromatium salexigens and Rhodospirillum salexigens. AB - The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria. They have photochemical activity remarkably similar to that of the bacteria rhodopsins. In contrast to rhodopsins, however, the PYPs are small water-soluble proteins. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three dimensional structure of the prototypic protein from Ectothiorhodospira halophila. Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical. All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment. This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment. It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein". R. salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge. Therefore it may also be classified as a "halophilic protein". PMID- 8611557 TI - Co-crystallization and characterization of the photosynthetic reaction center cytochrome c2 complex from Rhodobacter sphaeroides. AB - The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex. PMID- 8611558 TI - Mutational analysis of the endothelin type A receptor (ETA): interactions and model of selective ETA antagonist BMS-182874 with putative ETA receptor binding cavity. AB - Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist-ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383-12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxlic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors an, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS 182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS 182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ETA receptor complex formation. PMID- 8611559 TI - Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli. AB - UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP glucose through a mechanism involving the transient reduction of NAD+. Here we describe the X-ray structures for epimerase complexed with NADH/UDP, and NAD+/UDP, refined to 1.8 and 2.0 angstrom, respectively. The alpha-carbon positions for the two forms of the enzyme are superimposed with a root-mean square deviation of 0.36 A. Overall, the models for the reduced and oxidized proteins are very similar except for the positions of several side chains including Phe 178 and Phe 218. The most striking difference between the oxidized and reduced enzymes is the conformation of the nicotinamide ring of the dinucleotide. In the reduced protein, the nicotinamide ring adopts the anti conformation while in the oxidized enzyme the syn conformation is observed. There are also significant structural differences in UDP binding between the oxidized and reduced forms of the protein which most likely explain the observation that uridine nucleotides bind more tightly to epimerase/NADH than to epimerase/NAD+. Both van der Waals and electrostatic interactions between epimerase and NAD+ are extensive with 35 contacts below 3.2 angstrom as would be expected for enzyme that binds the dinucleotide irreversibly. This is in sharp contrast to the patterns typically observed for the NAD+-dependent dehydrogenases which bind nucleotides in a reversible fashion. While it has been postulated that the active site of epimerase must contain a base, the only potential candidates within approximately 5 A of both the NAD+ and the UDP are Asp 31, Asp 58, and ASP 295. These amino acid residues, however, are intimately involved in nucleotide binding and most likely do not play a role in the actual catalytic mechanism. Thus it may be speculated that an amino acid residue, other than glutamate, aspartate, or histidine, may be functioning as the active site base. PMID- 8611560 TI - Crystal structures of the recombinant kringle 1 domain of human plasminogen in complexes with the ligands epsilon-aminocaproic acid and trans-4 (aminomethyl)cyclohexane-1-carboxylic Acid. AB - The X-ray crystal structures of the complexes of the recombinant kringle 1 domain of human plasminogen (Klpg) with the ligands epsilon-aminocaproic acid (EACA) and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), which are representative of a class of in vivo antifibrinolytic agents, have been determined at 2.1 angstroms resolution. Each Klpg/ligand unit cell contained a dimer of the complexes, and some small differences were noted in the kringle/ligand interatomic distances within the monomeric components of the dimers. The structures obtained allowed predictions to be made of the amino acid residues of Klpg that are likely important to ligand binding. In the crystal structure, the anionic center of Klpg responsible for coordinating the amino group of the ligands is composed of both Asp54 and Asp56, and the cationic center that stabilizes binding of the carboxylate moiety of the ligands is Arg70, with a possible contribution from Arg34. A hydrogen bond between the carboxylate of the ligand to the hydroxyl group of Tyr63 also appears to contribute to the kringle/ligand binding energies. The methylene groups of the ligand are stablized in the binding pocket by van der Waals contacts with side-chain atoms of Trp61 and Tyr71. These conclusions are in general agreement with site-directed mutagenesis results that implicate many of the same amino acid residues in the binding process, thus showing that the crystal and solution structures are in basic accord with each other. Further comparisons of the X-ray crystal structures of the Klpg/ligand complexes with each other and with apo-Klpg show that while small differences in Klpg side-chain geometries may exist in the three structures, the binding pocket can be considered to be preformed in the apokringle and not substantially altered by the nature of the omega-amino acid ligand that is inserted into the site. From the similar geometries of the binding of EACA and AMCHA, it appears that the kon is an important component to the tighter binding of AMCHA to Klpg, as compared to EACA. Ordered solvation effects of the bound AMCHA may contribute to its longer lifetime on Klpg, thereby retarding koff, both effects thus accounting for the higher binding energy of AMCHA as compared to EACA. PMID- 8611561 TI - Conformation of an RNA molecule that models the P4/P6 junction for group I introns. AB - We present a three-dimensional structure of a 34-nucleotide RNA molecule determined by NMR spectroscopy. The molecule was designed to form a junction between two double-helical stems whose sequence was based on the P4/P6 domain from group I introns. There are 5' and 3' single-strand overhangs at the junctions of the stems. Contrary to our expectations, we found that the 3' end of the molecule is placed in the minor and not the major groove of the P4 helix. As a result of tertiary contacts and stacking interactions from nucleotides in the 3' end, the junction helices are rotated in a left-handed fashion and do not stack coaxially. This conformation is highly dependent on the presence of single stranded nucleotides at the 3' overhang. When the 3' end is removed, the molecule assumes a radically different structure with 5' end in the minor groove of the P6 helix and overall right-handed rotation between the stems. Only one nucleotide at the 3' end is sufficient to change the geometry of the junction. PMID- 8611562 TI - Structure and function of S-adenosylmethionine synthetase: crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 28 angstroms resolution. AB - S-Adenosylmethionine synthetase (MAT,ATP:L-methionine S-adenosltransferase, EC 2.5.1.6) plays a central metabolic role in all organisms. MAT catalyzes the two step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi), and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. The first crystal structure of MAT from Escherichia coli has recently been determined [Takusagawa et al. (1995) J. Biol. Chem. 271, 136-147]. In order to elucidate the active site and possible catalytic reaction mechanism, the MAT structures in the crystals grown with the substrate ATP (and BrATP) and the product PPi have been determined (space group P6(2)22; unit cell a = b = 128.9 Angstroms, c= 139.8 Angstroms, resolution limit 2.8 Angstroms; R O.19; Rfree 0.26). The enzyme consists of four identical subunits; two subunits form a spherical dimer, and pairs of these tightly bound dimers form a tetrameric enzyme. Each dimer has two active sites which are located between the subunits. Each subunit consists of three domains related to each other by a pseudo 3-fold symmetry. The crystal structures showed that the ATP molecules were hydrolyzed to ADP and Pi by the enzyme. Those products were found at the active site along with the essential metal ions (K+ and Mg2+). This rather unexpected finding was first confirmed by the structure of the complex with PPi and later by an HPLC analysis. The enzyme hydrolyzed ATP to ADP and Pi in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and Pi interacts extensively with amino acid residues from the two subunits of the enzyme, whereas the adenine and ribose moieties have little interaction with the enzyme. The enzyme structure is little changed upon binding ADP. All amino acid residues involved in the active site are found to be conserved in the 14 reported sequences of MAT from a wide range of organisms. Thus the structure determined in this study can be utilized as a model for other members of the MAT family. On the basis of the crystal structures, the catalytic reaction mechanisms of AdoMet formation and hydrolysis of tripolyphosphate are proposed. PMID- 8611563 TI - Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. AB - The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues. PMID- 8611564 TI - Poly(ethylene glycol)-lipid conjugates promote bilayer formation in mixtures of non-bilayer-forming lipids. AB - The influence of poly(ethylene glycol)-lipid conjugates on phospholipid polymorphism has been examined using 31P-NMR and freeze--fracture electron microscopy. An equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cholesterol adopts the hexagonal (HII) phase when hydrated under physiological conditions but can be stabilized in a bilayer conformation when a variety of PEG lipid conjugates are included in the lipid mixture. These PEG conjugates produced an increase in the bilayer to hexagonal (HII) phase transition temperature and a broadening of the temperature range over which both phases coexisted. Further, the fraction of phospholipid adopting the bilayer phase increased with increasing mole fraction of PEG-lipid such that at 20 mole % DOPE--PEG2000 no HII phase phospholipid was observed up to a least 60 degrees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (while maintaining the PEG--lipid molar ratio constant) increased the proportion of lipid in the bilayer phase. In contrast, varying the acyl chains of the PE anchor had no effect on polymorphic behavior. PEG--lipid conjugates in which ceramide provides the hydrophobic anchor also promoted bilayer formation in DOPE:cholesterol mixtures but at somewhat higher molar ratios compared to the corresponding PEG--PE species. The slightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosphorus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG--PE mixtures experienced isotropic motional averaging with this proportion being sensitive to both temperature and PEG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG--ceramide was used in place of PEG- PE. Consistent with the 31P-NMR spectra, freeze-fracture electron microscopy showed the presence of small vesicles (diameter <200 nm) and lipidic particles in DOPE:cholesterol mixtures containing PEG--PE. We conclude that the effects of PEG -lipid conjugates on DOPE:cholesterol mixtures are 2-fold. First, the complementary "inverted cone" shape of the conjugate helps to accommodate the "cone-shaped" lipids, DOPE and cholesterol, in the bilayer phase. Second, the steric hindrance caused by the PEG group inhibits close apposition of bilayers, which is a prerequisite for the bilayer to HII phase transition. PMID- 8611565 TI - Poly(ethylene glycol)--lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine. AB - The effect of poly(ethylene glycol)--lipid (PEG--lipid) conjugates on liposomal fusion was investigated. Incorporation of PEG--lipids into large unilamellar vesicles (LUVs) composed of equimolar phosphatidylethanolamine (PE) and phosphatidylserine (PS) inhibited calcium-induced fusion. The degree of inhibition increased with increasing molar ratio of the PEG conjugate and with increasing size of the PEG moiety. Inhibition appeared to result from the steric barrier on the surface of the liposomes which opposed apposition of bilayers and interbilayer contact. In the presence of a large excess of neutral acceptor liposomes, however, fusogenic activity was restored. The rate of fusion under these conditions depended on the initial molar ratio of the PEG conjugate in the PE:PS vesicles and the length and degree of saturation of the acyl chains which composed the lipid anchor. These results are consistent with spontaneous transfer of the PEG--lipid from PE:PS LUVs to the neutral lipid sink reducing the steric barrier and allowing fusion of the PE:PS LUVs. The primary determinant of the rate of fusion was the rate of transfer of the PEG--lipid, indicating that liposomal fusion could be programmed by incorporation of appropriate PEG--lipid conjugates. Interestingly, increasing the size of the PEG group did not appear to affect the rate of fusion. The implications of these results with respect to the design of fusogenic liposomal drug delivery systems are discussed. PMID- 8611566 TI - Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin. AB - In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein. PMID- 8611567 TI - Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release. AB - The seven types (A--G) of botulinum neurotoxin (BoNT) are Zn2+ -dependent endoproteases that potently block neurosecretion. Syntaxin is presently thought to be the sole substrate for BoNT/C1, and synaptosomal-associated protein of Mr = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+ -regulated exocytosis of dense core granules from adreno chromaffin cells were examined together with its underlying molecular action. Intact chromaffin cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin cleavage by Western blotting. A good correlation was obtained between degradation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secretion. However, blotting with antibodies against a C-terminal peptide of SNAP-25 revealed the additional disappearance of immunoreactivity, with the same toxin concentration dependency as syntaxin breakdown. Notably, the cleaved SNAP-25 product was similar in size to that produced by BoNT/A; however, contamination of BoNT/C1 by serotypes A or E was eliminated. Therefore, it is concluded that syntaxin 1A/B and SNAP-25 are cleaved in intact cells poisoned with only C1. Notably, C1 treatment of chromaffin cells abolished Ca2+ -evoked secretion following digitonin permeabilization, compared with partial inhibition by BoNT/A, suggesting the importance of syntaxin for catecholamine release. Unexpectedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even though it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in intact cells, possibly due to the existence therein of a unique conformation and/or the participation of accessory factors. PMID- 8611568 TI - Mechanism of hydroperoxide reduction by mangano-prostaglandin endoperoxide synthase. AB - Reaction of manganese-reconstituted prostaglandin endoperoxide synthase (Mn-PGHS) with 15-hydroperoxyeicosatetraenoic acid (15-HPETE) generates two products in nearly equal amounts: 15-hydroxyeicosatetraenoic acid (15-HETE) and 15 ketoeicosatetraenoic acid (15-KETE) [Kulmacz et al. (1994) Biochemistry 33, 5428 5439]. Their proposed mechanism to explain 15-KETE formation, namely oxidation of 15-HETE by the peroxidase activity of MnPGHS, was tested and found not to occur. Instead, 15-KETE may arise by one-electron reduction of 15-HPETE followed by oxidation of an intermediate alkoxyl radical. The mechanism of hydroperoxide reduction by Mn-PGHS was investigated using 10-hydroperoxyoctadeca-8,12-dienoic acid (10-OOH-18:2), a diagnostic probe of hydroperoxide reduction pathways. Reaction of Mn-PGHS with 10-OOH-18:2 generated the two-electron reduced product, 10-hydroxyoctadeca-8,12-dienoic acid (10-OH-18:2), as well as the one-electron reduction products, 10-oxooctadeca-8,12 dienoic acid (10-oxo-18:2) and 10-oxodec 8-enoic acid (10-oxo-10:1) in relative yields of 82, 10, and 7%, respectively. The identity of the one-electron reduction products was confirmed by electrospray ionization mass spectrometry. The detection of 10-oxo-10:1 provides strong evidence for the production of an alkoxyl radical during 10-OOH-18:2 reduction by Mn-PGHS. Like 15-HPETE, reaction of Mn-PGHS with 13-hydroperoxyoctadeca-8,12 dienoic acid (13-OOH-18:2) generated two products in equal amounts: 13 hydroxyoctadeca-8,12-dienoic acid (13-OH-18:2) and the keto fatty acid 13 oxooctadeca-8,12-dienoic acid (13-oxo-18:2). Comparison of the three hydroperoxides demonstrates that 15-HPETE is a much better substrate for Mn-PGHS than 10-OOH-18:2 or 13-OOH-18:2 with 10-fold greater turnovers. The results show that Mn-PGHS catalyzes both one- and two-electron hydroperoxide reduction and that the pathway of alkoxyl radical decomposition is influenced by the protein component of Mn-PGHS and the structure of the alkoxyl radical intermediate. PMID- 8611569 TI - Formation of vesicles by the action of acyl-CoA:1-acyllsophosphatidylcholine acyltransferase from rat liver microsomes: optimal solubilization conditions and analysis of lipid composition and enzyme activity. AB - The enzyme acyl coenzyme A:1-acyllysophosphatidylcholine acyltransferase (acyl CoA:lysoPC acyltransferase) can be isolated in newly formed phosphatidylcholine (PC) vesicles by solubilization of rat liver microsomes with the two substrates lysoPC and acyl-CoA. In this study, we sought to optimized the conditions for the formation of PC vesicles and analyzed the lipid composition and enzyme activity of the newly formed vesicles. Analysis of PC vesicles formed by incubation of the microsomal preparation with 1-(C16:0)lysoPC and C18:1CoA, C18:2CoA, or C20:4CoA showed that the optimal protein:lysoPC ratio was 1:5 (by weight) and the optimal lysoPC:acyl-CoA ratio was 1:1 (molar amounts). PC formation increased with incubation time; after 20 h of incubation at 37 degrees C, approximately 75% of the lysoPC was converted to PC in the incubation mixture. The phospholipid molecular species composition of the vesicles reflected almost exclusively the substrates used; the vesicles contained approximately 33% of the total acyl CoA:lysoPC acyltransferase activity from the microsomes and demonstrated a single protein band with a molecular mass of 21 kDa by gel electrophoresis. The procedure selected for the enzyme specific for lysoPC acylation, as enzyme activity toward lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), and lysophosphatidylinositol (lysoPI), was very low. In addition, the utilization of different acyl-CoA substrates for acylation of lysoPC was different from that in microsomes. These results show that an enzyme specific for the formation of PC from lysoPC can be isolated in PC vesicles with a designed phospholipid molecular species composition and that the lipid environment plays an important role in the regulation of the enzyme's affinity for its substrates. PMID- 8611570 TI - A 50 KDa protein modulates guanine nucleotide binding of transglutaminase II. AB - Regulation of cellular response is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins (GTPases) and their associated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, G alpha h (transglutaminase II), consistently copurified with an approximately 50 kDa protein (G Beta h) which is dissociated from G alpha h upon activation with GTP gamma S or AlF4-. Present immunological and biochemical studies on the regulation of the GTPase cycle of G alpha h, which involves the alpha 1-adrenoceptor and 50 KDa G beta h, reveal that the 50 kDa protein is indeed a G alpha h-associated protein and down regulates functions of G alpha h. Thus, polyclonal antibody against G Beta h coimmunoprecipitates GDP-bound G alpha h but not the GDP-AlF4- bound form. The GTP gamma S binding and GTPase activity of G alpha h are inhibited in a G beta h concentration dependent manner. Supporting this notion, G beta h accelerated GTP gamma S release from G alpha h and changes the affinity of G alpha h from GTP to GDP. Moreover, the ternary complex preparation exhibits TGase activity that is inhibited in the presence of the alpha 1-agonist and GTP. The GTP gamma S binding by the ternary complex, consisting of the alpha 1 agonist, the receptor, and Gh, is also inhibited by G beta h. The inhibition of GTP gamma S binding with the ternary complex requires a > or = 2.7-fold higher concentration of G beta h than the G alpha h alone, indicating that the receptor enhances the affinity of G alpha h for GTP. In addition, G beta h copurifies with an alpha 1-agonist, adrenoceptor, and G alpha h ternary complex, showing that the complex is a heterotetramer. Our data also suggest that G beta h does not directly interact with alpha 1-adrenoceptor. These findings clearly demonstrate that G alpha h associates with a novel protein which modulates the affinity of G alpha h for guanine nucleotides and that the GDP-bound Gh is the ground state for the counterpart activator, the alpha 1-adrenoceptor, in this signaling system. PMID- 8611571 TI - Affinity Labeling of the active site of rabbit muscle adenylosuccinate lyase by 2 [(4-bromo-2.3-dioxobutyl)thio] adenosine 5'-monophosphate. AB - Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 muM 2 -[(4-bromo 2.3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, ph 7.0 and 10 degrees C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration 2 BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (K1=8.5 microM. 5.2 microM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB [14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5'-AMP and by 0.1 mM 5'-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5'-AMP alone, 5 mM fumarate alone, or 0.1 mM 5'-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase. PMID- 8611572 TI - Sedimentation analyses of the salt- and divalent metal ion-induced oligomerization of nucleolar protein B23. AB - Protein B23 is a major nonribosomal nucleolar protein and putative ribosome assembly factor that has been demonstrated to form oligomers. Sedimentation velocity and equilibrium analyses were used to examine the oligomerization properties of recombinant proteins B23.1 and B23.2. Under low ionic strength conditions protein B23.1 was predominantly a 2.1S monomer with small amounts of a 7.1S oligomer. At NaCl concentrations of 0.1 M and above the protein was almost exclusively the 7.1S oligomer. The oligomer remained the predominant species in NaCl concentrations as high as 1 M, suggesting that oligomers are not stabilized by electrostatic interactions. Low concentrations of divalent metal ions (0.1 - 1mM Ca2+ or Mg2+) also promoted oligomerization. Reducing agents had no effect on oligomerization, indicating that disulfide bridges are not important in oligomer formation. Protein B23.2, the carboxyl-terminal truncated isoform, had sedimentation characteristics similar to that of protein B23.1, suggesting that the carboxyl-terminal end of protein B23.1 is not essential for oligomerization. Protein B23.1 was previously shown to bind nucleic acids [Wang, D., Baumann, A., Szebeni, A., & Olson, M. O. J.(1995) J. Biol. Chem. 269, 30994-30998]. The effect of protein B23.1 oligomerization on its interaction with a 230 base pair DNA fragment was examined by sedimentation analyses. Under conditions where significant amounts of monomer were present, protein B23.1 was capable of binding DNA, whereas conditions that strongly favored oligomerization caused a nearly complete abolition of DNA binding activity. These studies suggest that protein B23 exists in an equilibrium between monomer and oligomer and that the quaternary structure of the protein may regulate its DNA binding properties. PMID- 8611573 TI - Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions. AB - The DNA binding domain (residues 1--65) of the yeast transcriptional activator GAL4 is only partially folded. While residues 10-41, the DNA recognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In order to describe the mobility of the protein, we have characterized the frequency spectrum of the motions of N-H bond vectors of GAL4(1-65) using a reduced spectral density mapping approach (an approximation of the full spectral density mapping technique) [Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308-332; Peng. J. W., & Wagner, G. (1992b) Biochemistry 31, 8571-8586]. 15N spin-lattice relaxation [Rn(Nz)], spin-spin relaxation [Rn(Nx,y)], cross-relaxation [RN(Hz-->Nz)], two-spin order [RNH(2HzNz)], and antiphase [RNH(2HzNx,y)] rates were determined for 52 of the 65 backbone amide groups at 10 degrees C and ph 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of RN(Nz),RN(Nx,y),RN(Hz-->Nz), and RNH(2HzNz) gave excellent agreement with those calculated using all six sets. The reduced method has the added advantage that the errant behavior seen at high field values is circumvented. A linear correlation was found between J(omega N) and J(0) with a limited and clearly defined range of J(0) values which defines the range of rates for internal motions in GAL4(1-65). It appears that all residues experience a combination of two movements: one of the overall tumbling (correlation time, 8.65 ns) and the other of fast internal fluctuations of the structure. The respective weights of these contributions vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(omega N) versus J(0) indicates the amount of angular averaging relative to the overall motion of the protein. A spectral density function for internal motions can be described. PMID- 8611574 TI - Vibrational dephasing of long- and short-lived primary donor excited states in mutant reaction centers of Rhodobacter sphaeroides. AB - Femtosecond spectroscopy was used to study vibrational dynamics in the first singlet excited state (P*) of the primary donor of bacterial reaction centers (RC)in which primary electron transfer dynamics have been altered by single amino acid modifications. We studied intracytoplasmic RC-only membranes containing Rhodobacter sphaeroides wild-type RCs and RCs bearing mutations in the vicinity of P, where Tyr M210 was modified to His, Leu, and Trp and where Phe L181 was modified to Tyr. These mutations do not change the frequencies of the main low frequency activated modes, which is consistent with a description in which these modes involve extended regions of the protein. Electron transfer in FL181Y, YM210H, and wild-type RCs at 10 K occurs in approximately 1 ps or less, and damping of the coherences occurs simultaneously with the decay of the P* excited state. These results, and a comparison with YM210L RCs, show that in wild-type RCs the damping is primarily determined by the depletion of P* and not by vibrational dephasing induced by interactions with the bath or nonharmonic coupling. In the YM210L and W mutants, electron transfer occurs on a time scale of hundreds of picoseconds at 10 K. Analysis of the longer-lasting vibrational dynamics in these mutants sets a new lower limit for the intrinsic vibrational dephasing time of 1.2 ps for some modes, but of approximately 2 ps for most activated modes. PMID- 8611575 TI - A comparative thermodynamic analysis by laser-flash absorption spectroscopy of photosystem I reduction by plastocyanin and cytochrome c6 in Anabaena PCC 7119, Synechocystis PCC 6803 and Spinach. AB - A comparative thermodynamic analysis of photosystem I (PSI) reduction by plastocyanin (Pc) and cytochrome c6 (Cyt) has been carried out by laser-flash absorption spectroscopy in the cyanobacteria Anabaena PCC 7119 and Synechocystis PCC 6803 as well as in spinach. These three organisms have been reported to exhibit different reaction mechanisms [Hervas, M., Navarro, J. A.. Diaz, A., Bottin, H., & De la Rosa, M. A. (1995) Biochemistry, 34, 11321-11326]. Whereas the activation free energy for the overall reaction is mainly enthalpic in nature, long-range electrostatic interactions appear to be attractive in Anabaena, but repulsive in Synechocystis and spinach. The net interaction between PSI and its two donor proteins in Anabaena is similarly affected by ionic strength (the rate constant decreases with increasing salt concentration), but the activation parameters delta H+/+ delta S+/+ show different dependencies on ionic strength. A compensation effect between entropy and enthalpy at varying ionic strength is found in all these Pc/PSI and Cyt/PSI systems, except with Cyt and PSI from Anabaena. Such a compensation effect is proposed to be mainly due to stabilization of the intermediate electrostatic complex by hydrophobic forces. The electron transfer step seems to be well optimized in the Anabaena Cyt/PSI couple, which exhibits a temperature-independent fast kinetic phase and, therefore, a low activation energy barrier. Short-distance forces appear to have gained relevancy in the reaction mechanism of PSI reduction by Cyt and Pc throughout evolution, whereas long-range interactions are prevalent in less evolved organisms. PMID- 8611576 TI - Function of the [2FE-2S] cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor. AB - Ferrochelatase (E.C. 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway, catalyzing the insertion of ferrous iron into protoporphyrin. In mammals the enzyme contains a labile [2Fe-2S] center. Although this cluster is absent in all prokaryotic, plant, and yeast ferrochelatases, its destruction or elimination from the mammalian enzyme results in loss of enzyme activity. In the current study we present data which clearly demonstrate that mammalian ferrochelatase is strongly inhibited by nitric oxide and that this effect is mediated via destruction of the [2Fe-2S] cluster. Carbon monoxide has no inhibitory effect, and yeast ferrochelatase, which lacks the [2Fe-2S] cluster, is not affected by NO (or CO). EPR and UV-visible absorption of purified recombinant human ferrochelatase provides evidence that NO is targeting the [2Fe-2S] center. UV visible absorption spectroscopy of both human and murine recombinant ferrochelatase incubated with NO or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), indicate a rapid loss of the visible absorption spectrum of the [2Fe-2S] cluster. EPR studies of the resulting samples reveal the characteristic axial S = 1/2 resonance, g perpendicular = 2.033, and g parallel = 2.014 of a cysteinyl coordinated monomeric iron-dinitrosyl cluster degradation product. Parallel spectroscopic studies of spinach ferredoxin, which also contains a [2Fe-2S] cluster, gave no indication of NO-induced cluster degradation under the same experimental conditions. Exposure of DMSO-induced murine erythroleukemia cells exposed to SNAP results in an initial decrease in heme production, suggesting that in vivo the cluster is rapidly destroyed. The potential physiological relevance of these data to the anemias that are found in individuals with chronic infections is discussed. PMID- 8611577 TI - Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles. AB - The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6 pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ2. The Kmin (kcat/Km) for CoQ1 is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of kmin is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kroger, A., & Klingenberg, M. (1973) Eur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool. PMID- 8611578 TI - Characterization of electrostatic and nonelectrostatic components of protein- membrane binding interactions. AB - A general method was developed to determine the thermodynamic parameters for the interaction of protein with membranes. Protein intrinsic tryptophan fluorescence was quenched by titration with large unilamellar vesicles containing 9,10 dibrominated distearoylphosphatidylcholine (Br4-DSPC) or a small amount of trinitrophenylphosphatidylethanolamine (TNP-PE), Binding was modeled as a bimolecular reaction of free protein with a unit of "n" lipid molecules and a dissociation constant, Kd. The contribution of residual fluorescence and light scattering could be eliminated by using the second derivative of the titration function as the basis for calculations. For the binding of C-terminal channel domain polypeptides(178-190 residues) of the colicin El ion channel, n=50-60 and Kd=2-3 nM at pH 4, ionic strength, I=0.12 M, and anionic lipid content = 40% (surface potential, psi o =-30 mV), conditions for which the protein has high activity. Values of n = 95 and 210 for the binding of a C-terminal 293-residue colicin fragment and the 522 residue intact colicin E1 molecule scale qualitatively according to the increase in molecular size. General methods are presented to distinguish the electrostatic (delta G el) and nonelectrostatic (delta G nel) components of the total delta G for binding. Using Br4DSPC as the quencher, the binding of the channel polypeptide, P178, was characterized by delta G approximately -9.8 kcal/mol, and delta G el approximately -7.0 kcal/mol, and delta G el= -2.8 kcal/mol (psi o = -30 mV). Using TNP-PE as the quencher, similar values of delta G approximately -9.3 to -9.9 kcal/mol were determined, a somewhat smaller value for delta G nel approximately -5.0 kcal/mol, and a correspondingly larger value for deltaGnel approximately -4.9 kcal/mol. The existence of a delta G nel component of this magnitude may distinguish proteins that have the potential to insert into the membrane. PMID- 8611579 TI - Formation of electrostatic interactions on the protein-folding pathway. AB - We describe a novel method of obtaining information about the structures of transient conformations on the folding pathway from their ionization equilibria: the H+ -titration behavior of a protein residue is determined in detail by its environment. We follow the consolidation of electrostatic interactions in the folding process by comparing the acid-titration behavior of four conformations on the folding pathway of barnase: the denatured state (D); the folding intermediate (I); the major transition state(+); and the native state (N) in the scheme D <==>I<==>(+)<==)N. The results show that strong electrostatic interactions are present in the major transition state: some of its carboxylate groups display the highly anomalous pKA values of <2 that are found in N. However, the network of ionic surface interactions is not formed in (+), and the overall protection of titrating residues is weakened. The results are consistent with the transition state being an expanded form of the native state, with a weakened but poorly hydrated core and a loosened periphery. The surface residues in such an expanded conformation are, on average, farther apart than are those in the center of the molecule. The results concerning the folding intermediate are less clear cut. We show that the interpretation of kinetic data relating to folding intermediates depends critically on assumptions about their equilibrium with other denatured states. We have, however, characterized the pH and ionic strength dependence of an apparent stability of I, using the deviation from two-state folding behavior, which can be used to investigate electrostatic properties of folding intermediates from a variety of mechanisms. In general, the data imply that I is somewhat similar to (+). Apparently odd titration properties of I are investigated further in the accompanying paper [Oliveberg, M., & Fersht, A. (1996) Biochemistry 35, 2738-2749]. The approach in this study may be of particular use in testing theoretical results since the relationship between H+ titration properties and protein structure can be treated by classical electrostatics. PMID- 8611580 TI - Thermodynamics of transient conformations in the folding pathway of barnase: reorganization of the folding intermediate at low pH. AB - New classes of small proteins have recently been found that refold rapidly with two-state kinetics from a substantially unfolded conformation ("U") and without the accumulation of a folding intermediate. Barnase, on the other hand, is representative of a class of proteins that display multistate kinetics and refold from a partly structured conformation, a folding intermediate (I). The accumulation of I on the folding pathway of barnase is highly dependent on the experimental conditions: a transition from multistate to two-state folding behavior can be induced simply by changing the reaction conditions away from physiological, i.e., elevated temperatures, high concentration of denaturant, or low pH. We argue that the change in folding behavior results from the denatured state changing under different conditions. The denatured state seems compact and partly structured at conditions that favor folding but is disorganized at denaturing conditions. At physiological pH and temperature, the denatured state (Dphys) is the folding intermediate because it is the most stable of the denatured conformation, i.e., Dphys is identical to I. At high temperature or [urea], however, Dphys becomes destabilized relative to less structured denatured states ("U"). Kinetics under these extreme conditions is two-state because the refolding reaction is from "U" to the native state with no significant accumulation of Dphys (identical to I) which is here a high-energy intermediate. The two-state behavior at low pH results from a different cause. The acid denatured state of barnase (Dacid) is not as unfolded as "U" but energetically similar to Dphys (identical to I). It appears that protonation of Dphys has only marginal effects on its stability, so that the protonated form of Dphys constitutes the acid-denatured state at equilibrium. The energetic similarity between Dphys and Dacid gives rise to two-state kinetics at low pH, although the refolding is from a compact denatured state throughout the pH range. Protonation of Dphys to give Dacid causes the structure to become more disorganized and hydrated. The heat capacity of Dphys (identical to I) at pH 6.3 is in between that of "U" and the native protein. We suggest that protonation of folding intermediates disrupts their structural integrity and allows isoenergetic reorganizations that increase the solvation of charged residues. Such protonated and reorganized folding intermediates may then constitute the molten globules, which are compact denatured states that are sometimes observed at equilibrium at low pH and high ionic strength. Under all experimental conditions, the heat capacity of the major transition state is close to that of the native protein. This, together with its titration properties, shows that the transition state is an expanded form of the native state with a weakened but poorly hydrated hydrophobic core, and with disrupted surface regions. PMID- 8611581 TI - Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer. AB - The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1. PMID- 8611582 TI - Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S. AB - Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S. PMID- 8611583 TI - Interdomain interactions between the hydrophilic domains of the mannitol transporter of Escherichia coli in the unphosphorylated and phosphorylated states. AB - Interdomain interactions in the mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli play a key role in the mechanism of mannitol transport across the membrane [Boer et al. (1995) Biochemistry 34, 3239-3247; Loikema et al. (1991) Biochemistry 30, 6716-6721]. In this study, we focus on the interaction between the hydrophilic A and B domains and try to determine those as a function of the phosphorylation state of the enzyme. To this end, unfolding studies on the subcloned domains IIAmtl and IIBmtl, as well as on the binary combination IIBAmtl, were performed, both in the unphosphorylated and in the phosphorylated states, using GuHCl and heat as the denaturant. It is shown that IIAmtl and IIBmtl, as well as P-IIAmtl and P-IIBmtl, unfold according to a two-state mechanism but that IIBAmtl and P2 IIBAmtl do not exhibit such behavior. Two transitions are observed instead, indicating a lack of strong positive cooperative interactions. DSC studies of the unphosphorylated proteins showed a destabilization of the B domain in IIBAmtl with respect to the free IIBmtl as indicated by a lowereing of the melting temperature and a lower enthalpy of unfolding. Furthermore, it is shown that phosphorylation has a destablilizing effect on both IIAmtl and IIBAmtl but not on IIBmtl. Possible explanations for this behavior and the biological relevance of the destabilizing forces in IIBAmtl are discussed. PMID- 8611584 TI - Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase. AB - In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced under conditions of oxidative stress, we have examined wheat germ CaM for the presence of highly reactive sites that correlate with the loss of function. Using reversed-phase HPLC and FAB mass spectrometry after proteolytic digestion, we have identified the sites of modification by hydrogen peroxide. We find that one of the vicinal methionines (i.e., Met146 or Met147) near the C-terminus of CaM is selectively oxidized. The ability of CaM to bind and to activate the plasma membrane (PM)-Ca-ATPase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is a little change in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory sequence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the amino-terminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain correlates with the loss of CaM-dependent activation of the PM-Ca-ATPase. The presence of oxidatively modified CaM prevents native CaM from activating the PM Ca-ATPase, indicating that the oxidatively modified CaM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivity of CaM to the oxidation of one of the C-terminal vicinal methionines permits CAM to serve a regulatory role in modulating cellular metabolism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in terms of the enhanced solvent accessibility of these vicinal methionines. PMID- 8611585 TI - Evaluation of isofagomine and its derivatives as potent glycosidase inhibitors. AB - A pseudo-aza-monosaccharide and several pseudo-aza-disaccharide compounds were constructed based on replacement of the anomeric carbon with a nitrogen and the ring oxygen with a carbon. The inhibition constants of these compounds toward five different glycosidases, alpha-glucosidase, beta-glucosidase, isomaltase, alpha-mannosidase, and glucoamylase, were obtained. Isofagomine, the pseudo-aza monosaccharide, shows a broad spectrum of strong inhibition against glycosidases. It is the most potent inhibitor of beta-glucosidase from sweet almonds reported to date and also a strong inhibitor of glucoamylase, isomaltase, and alpha glucosidase. Isofagomine inhibits beta-glucosidase, glucoamylase, and isomaltase more strongly than 1-deoxynojirimycin where the ring oxygen has been replaced with a nitrogen. The alpha-1,6- linked pseudo-disaccharide showed very strong inhibition toward glucoamylase, being nearly as potent an inhibitor as acarbose. Pseudo-disaccharides in which the anomeric nitrogen was methylated to favor formation of either the alpha or beta substrate linkage generally had weakened inhibition for the glycosidases studied most likely due to steric interference with the various active sites. These results indicate that the presence of a basic group at the anomeric center is important for carbohydrase inhibition. The presence of a charged carboxylate group near the anomeric carbon which interacts with the basic nitrogen is suggested for these enzymes, particularly for beta glucosidase. The presence of a second alpha-linked glucosyl residue is also critical for strong inhibition of glucoamylase. PMID- 8611586 TI - Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi. AB - Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA. PMID- 8611587 TI - EPR spectroscopic characterization of neuronal NO synthase. AB - Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exits as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 HZ at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin spin coupling. The flavin radical, probably the semiquinone FMNH., was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D=5.2cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron. PMID- 8611588 TI - Nucleotide and amino acid sequences for cytochrome caa3-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c. AB - A pseudo-sigmoidal cytochrome c-dependence curve of oxidase activity was observed with cytochrome oxidase from the Bacillus stearothermophilus strain K1041, while the other thermophilic Bacillus PS3 which has been extensively studied possessed normal Michaelis-Menten type kinetics. The genes coding for four subunits of cytochrome caa3-type oxidase and for heme O synthase were isolated from a genomic DNA library of K1041 by using a PS3 DNA fragment containing the highly-conserved region of the largest subunit as a probe, and sequenced. Most residues in subunits I (COI/caaB product), III (COIII/caaC product), and IV (COIV/caaD product) of K1041 were highly conserved when compared with those of PS3. However, the sequence of K1041 subunit II (COII/caaA product) was distinctly different from that of the PS3 subunit II. These Bacillus COIIs have an additional sequence for cytochrome c after the CuA binding protein portion with two transmembrane segments which is homologous to the mitochondrial counterpart, and represents the site of electron ingress. Several charged residues in the vicinity of cytochrome c moiety are replaced by oppositely charged residues. It is likely that these amino acid replacements in subunit II are the cause of the abnormal sigmoidal saturation curve for extrinsic cytochromes c of the K1041 enzyme. PMID- 8611589 TI - The membrane-bound cytochrome cy of Rhodobacter capsulatus can serve as an electron donor to the photosynthetic reaction of Rhodobacter sphaeroides. AB - Rhodobacter capsulatus has two different pathways for reduction of the photo oxidized reaction center, one using water-soluble cytochrome c2, the other via membrane-associated cytochrome cy. Rhodobacter sphaeroides differs in that it lacks a cytochrome cy homologue capable of functioning in photosynthetic electron transfer; cytochrome c2 is thus the sole electron carrier, and is required for photosynthetic (Ps+) growth. Genetic evidence indicates that cytochrome cy of R. capsulatus can complement a Ps- cytochrome-c2-deficient mutant of R sphaeroides (Jenny, F.E. and Daldal, F (1993). EMBO J. 12, 1283-1292). Here, we show that it transfers electrons from cytochrome bc1 complex to the reaction center in R. sphaeroides, albeit at a lower rate than that catalyzed by the endogenous cytochrome c2. When cytochrome cy is expressed in R. sphaeroides in the presence of cytochrome c2, there is an increase in the amount of photo-oxidizable c-type cytochrome. In the absence of cytochrome c2, electron transfer via cytochrome cy shows significantly different kinetics for reaction center reduction and cytochrome c oxidation. These findings further establish that cytochrome cy, the electron carrier permitting soluble cytochrome c2-independent photosynthetic growth in R. capsulatus, can function in a similar capacity in R. sphaeroides. PMID- 8611590 TI - NADPH-specific quinone reductase is induced by 2-methylene-4-butyrolactone in Escherichia coli. AB - 2-Methylene-4-butyrolactone (MBL), an inducer of NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.699.2) in animal cells, was found to induce NADPH-specific quinone reductase about 25-fold in Escherichia coli. MBL induced NADPH-quinone reductases with relative mobilities (Rm) of 0.70, 0.76 and 0.91 on polyacrylamide gel electrophoresis (PAGE). These three enzymes were found to be charge isomers with the same molecular size of 42 kDA. Two NADPH-quinone reductases (A and B) were purified to single proteins both with an apparent mass of 21 kDa on SDS PAGE. Enzyme A corresponded to the activity of the band at Rm 0.76 with a minor active band at Rm 0.70, and enzyme B to the activity of band Rm 0.91. Both enzymes reacted exclusively with NADPH and were most active toward quinone derivatives and ferricyanide with the optimum pH at 7.0. The reaction followed a ping-pong mechanism with Km values for NADPH and menadione of 10.5 microM and 6 microM, respectively. The sequences of 20 amino acids at the N-terminal of enzymes A and B were identical, and furthermore coincided with that of the E. coli modulator of drug activity (mda66) submitted under the accession number U18656. PMID- 8611591 TI - Different cardiac myosin isoforms exhibit equal force-generating ability in vitro. AB - We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin. PMID- 8611592 TI - Isolation of the vma-6 gene encoding a 41 kDa subunit of the Neurospora crassa vacuolar ATPase, and an adjoining gene encoding a ribosome-associated protein. AB - The vma-6 gene, encoding a membrane-associated subunit of the vacuolar H+-ATPase from Neurospora crassa, was cloned and sequenced. The gene contains three small introns and encodes a protein of 41 005 Da. When compared with homologous polypeptides from other species, the N. crassa protein contains a unique glycine rich region. Three conserved cysteine residues, previously unrecognized, have been identified. An unrelated gene encoding a protein of 31 701 Da was found 2.1 kb downstream of vma-6. The second appears to encode the N. crassa homolog of a ribosome-associated protein identified previously in several plant and mammalian cells, and was named rap-1. PMID- 8611593 TI - The coxD gene for heme O synthase in Synechocystis. AB - The cyanobacterial coxD gene for heme O synthase was cloned from Synechocystis sp. PCC 6803 and its nucleotide sequence was determined. The deduced amino-acid sequence of the gene was homologous to the amino-acid sequences of bacterial heme O synthesis. In contrast to the genes for heme O synthases in other prokaryotes, which are clustered together with genes for the structural subunit(s) of cytochrome oxidase, the coxD gene is not linked to such genes on the chromosome of Synechocystis. PMID- 8611594 TI - NADPH oxidase of neutrophils. PMID- 8611595 TI - Genistein inhibition of fast Na+ current in uterine leiomyosarcoma cells is independent of tyrosine kinase inhibition. AB - Possible regulation of fast Na+ channels by tyrosine kinase was examined in human uterine smooth muscle cell line, using whole-cell voltage clamp (at a holding potential of - 90 mV). Bath application of genistein, an inhibitor tyrosine kinase, decreased the fast Na+ current (INa(f)) dose-dependently. The maximal inhibition of INa(f) was 98%, and the concentration for half-maximal inhibition (IC50) was 9 microM. The effect of genistein was rapidly reversible. Daidzein, an inactive analog of genistein, had a similar inhibitory effect on INa(f). These results suggest that the fast Na+ channels in uterine sarcoma cells may be directly blocked by genistein and daidzein, i.e., their effect may be independent of tyrosine kinase inhibition. PMID- 8611596 TI - Transport mechanisms of nucleosides and the derivative, 6-mercaptopurine riboside across rate intestinal brush-border membranes. AB - Na+ -driven nucleoside transport processes across rat intestinal brush-border membrane vesicles were investigated. 6-Mercaptopurine riboside (6-MPR), an analogue of purine-nucleoside such as adenosine and inosine, was recognized by its purine- and pyrimidine-nucleosides transport system, but their nucleobases did not entirely inhibit the 6-MPR transport. The analysis according to the Hill equation of the curve of Na+ activation of 6-MPR uptake was consistent with the notion of a Na+/6-MPR coupling stoichiometry of 1:1. The expressed transport activities of adenosine, uridine, and 6-MPR were Na+ -dependent and saturable, and their affinity constants (Km value) obtained by Eadie-Hofstee analysis were approx. 20, 15 and 100 microM. Moreover, the uptake of radiolabeled adenosine and uridine was trans-stimulated by 6-MPR inside vesicles in the absence of an inwardly directed Na+ gradient. On the other hand, uridine did not exhibit any inhibitory effects on the uptake of adenosine despite the fact that adenosine was a potent inhibitor of uridine uptake by intestinal brush-border membrane vesicles. These differences in the inhibition may be explained by the multiplicity of the nucleoside transport systems. PMID- 8611597 TI - Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli. AB - Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu 325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 - > Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325. PMID- 8611598 TI - Identification of a sodium-bicarbonate symport in human platelets. AB - Intracellular pH (pH(i)) was measured in human platelets using fluorescent probes. Basal pH(i) was higher in HC(O3-)- buffered solutions (7(7.33 +/- 0.01) than in nominally HCO3- free, Hepes buffered solutions (7.16 +/- 0.01, P < 0.05). Addition of EIPA caused to fall in Hepes, but did not inhibit the increase of pH(i) when platelets maintained in Hepes were transferred to a CO2/HCO3- buffer. After an intracellular acidosis induced by an NH4Cl prepulse, the initial velocity of recovery (d(pH)/dt(i), in pH units/min) was 3.32 +/- 0.69 in Hepes buffered solution and 2.85 +/- 0.88 in HCO3- media. Taking into account the differences in buffer capacity, the efflux of acid equivalents after 1.2 min was twice as much in the presence of bicarbonate. The addition of 30 mumol/1 EIPA effectively blocked acid efflux (d(pH)/dt(i) = 0.08 +/ 0.04) in a nominally HCO3- free solution, whereas the recovery was reduced but not abolished (d(pH)/dt(i) = 0.37 +/- 0.10, P < 0.05) in the presence of bicarbonate. The stilbene derivative SITS further inhibited the EIPA-resistant pH(i) recovery. Removal of external Na+ inhibited the HCO(3-)-dependent recovery whereas depletion of internal Cl-, did not suppress it. Depolarization of the membrane had no effect on this recovery. The results suggest the contribution of an electroneutral Na+/HCO3- cotransport in the recovery of pH(i) following an acid load. Both the Na+/H+ antiport and the HCO(3-)-dependent mechanism contribute approx. 50% each to the total acid equivalent efflux during the recovery from a pH(i) 6.46 +/- 0.14 to the basal pH(i) in human platelets. PMID- 8611599 TI - Interaction of dipyridamole with lipids in mixed Langmuir monolayers. AB - Dipyridamole (DIP), a well known coronary vasodilator and coactivator of anti tumor activity of a number of drugs, forms stable Langmuir monolayers with the zwitterionic lipid dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) at an air/aqueous solution interface. The drug binds to the lipid molecules and change their packing density in the monolayer in the process of compression, the effect depending on the drug location in the monolayer, protonation of the drug and also on the charge state of the lipid. The incorporation of dipyridamole (DIP) into neutral DPPC monolayers causes them to be more expanded at low DIP concentrations but more condensed at high concentrations, resembling the effect of cholesterol. Maximum expansion occurs for a DIP concentration of 2 mol%. For slightly charged DPPG monolayers spread on ultra pure water, the monolayers become increasingly more expanded with increasing DIP concentrations. For the negatively charged DPPG monolayers spread on buffer solutions, the incorporation of DIP has similar effects to that observed for DPPC monolayers. This is probably due to the interaction between the charged DPPG molecules and the protonated DIP molecules. Also, introduction of protonated DIP brings an increase in surface potential of DPPG monolayers because the negative contribution from the double layer is decreased. The results indicated that DIP molecules are located deeper in the hydrophobic region of DPPC monolayers, whereas in DPPG ones they appear to be located very close to the polar head region. Due to the electrostatic interaction of protonated DIP with the charges on the polar heads of lipids it is inclined with respect to the plane of the monolayer. PMID- 8611600 TI - A cation non-selective channel induced by extracellular ATP in macrophages and phagocytic cells of the thymic reticulum. AB - Extracellular ATP4- can bind to P2Z purinergic receptors including depolarization and cytoplasmic membrane permeabilization to small molecular weight solutes in macrophages, thymocytes, mast cells, phagocytic cells of the thymic reticulum and other cell types. An ATP(4-)-induced cation current has been described in whole cell records of some of these cells but it is currently not clear whether these currents and the phenomenon of membrane permeabilization are a consequence of only one type of P2Z-associated channel/pore or two different phenomena triggered by one or more receptors. Here we use the outside-out patch-clamp technique to describe a single channel associated with this cation current in two murine phagocytic cells: intraperitoneal macrophages and phagocytic cells of the thymic reticulum. Multi channel currents could be readily observed in 77% of the outside out patches of macrophages. Single channels of 7.8 pS could usually be resolved only in tail currents. Reversal potential measurements and ion replacement experiments indicated a lack of cation selectivity, similarly to what has already been described for the ATP(4-)-induced whole-cell inward current. No large conductance channels that could explain the permeabilization to small molecular weight studies solutes was observed under our experimental conditions. A single channel of approx. 5 pS was also observed in phagocytic cells of the thymic reticulum under similar conditions. We conclude that the channel here described is the main carrier of cation current usually associated with the binding of ATP4 to P2Z receptors in whole-cell and outside-out patch-clamp experiments. PMID- 8611601 TI - Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium. AB - Human platelets were studied by patch clamp recordings from inside-out membranes; there were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20 degrees C in symmetrical 150 mM NaCl, spontaneous channel openings were rarely observed at negative potentials, whereas depolarised potentials (+ 60 to + 100 mV) elicited sustained channel activity in 38% of patches. The single channel conductance was 53 +A- 1 pS at + 80 mV (outward current), decreasing to 20 +/- 2 pS at -80 mV (inward current). Ion substitution experiments indicated that this channel conducts Cl- and not Na+. Furthermore, 5 nitro-2-(3-phenylpropylamino)benzoate (100 microM), a recognized inhibitor of anion channels, induced a reversible 'flickery' channel block. We estimate that each platelet possesses < or = 30 such channels. Kinetic analysis suggested at least two open channel states (tau = 0.8 +/- 0.2 ms, tau = 22 +/- 14 ms, n = 4) and two closed states (tau = 0.8 +/- 0.2 ms, tau = 12 +/- 0.6 ms, n = 4). Increasing [Ca2+]i to 10 microM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca2+]i (300 nM-10 microM) increased the number of anion channels activated by subsequent depolarisation. This study represents the first recordings of ionic currents in excised, inside-out membrane patches from human platelets, and provides further evidence for the existence of chloride channels in these cells. PMID- 8611602 TI - Binding and uptake of liposomes containing a poly(ethylene glycol) derivative of cholesterol (stealth liposomes) by the macrophage cell line J774: influence of PEG content and its molecular weight. AB - The binding and intake of liposomes containing a different molar content and chain length of a PEG-Chol derivative had been studied in cultured macrophage cell line J774. The decrease in binding and endocytosis of the liposomes containing PEG-Chol is dependent on (i) the PEG chain length, (ii) the molar content of the surfactant, (iii) the liposome concentration in the external medium. The best results in reducing the uptake of liposomes were obtained by a PEG-Chol liposome suspension with a high molar content (25%) which presents a non negligible amount of free PEG-Chol. Moreover, we could show an increase by 2 for binding and by about 5 for endocytosis of filtrated-liposomes containing 25 mol% of 8800PEG-Chol, in the absence of free PEG-Chol in the suspension. Binding and intake of control liposomes was also inhibited in the presence of free PEG-Chol. Fluid phase endocytosis of SRh was inhibited up to 45% of control in the presence of liposomes containing PEG-Chol or free PEG-Chol. Based on the comparison of 4400PEG-Chol with the most commonly used PEG derivative 5000PEG-PE, PEG-Chol is more powerful in terms of reducing their binding and endocytosis by J774 cells. Inhibition of the fluid phase endocytic process is attributed to the binding of PEG-Chol to the cells' plasma membrane inducing a decrease in surface hydrophobicity of the cells, resulting in a marked decrease in the extent of phagocytic ingestion. PMID- 8611603 TI - Is control of distribution of liposomes between tumors and bone marrow possible? AB - The objective of this study is to clarify to what extent the accumulation of liposomes from the blood into the tumor and bone marrow can be controlled by liposome size and membrane fluidity. Liposomes with different diameters (50-400 nm) and different membrane fluidity were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol (Ch) and dicetylphosphate in various molar ratios. These liposomes were injected intravenously into rats bearing Yoshida sarcoma, and the ratios of the accumulation of liposomes in the tumor to those in the bone marrow, liver and spleen were compared. The tumor-to-bone marrow accumulation ratio increased with the decrease in liposome size from 400 to 50 nm. This ratio was greater than those for the liver and spleen at all sizes. Although tumor-to-liver accumulation ratios of 50- and 100-nm HEPC-containing liposomes were higher than those of EPC containing liposomes, no obvious difference in tumor-to-bone marrow or tumor-to spleen accumulation ratios was found between these liposomes. Tumor-to-bone marrow accumulation ratio of HEPC-containing liposomes increased remarkably with the decrease in Ch content from 40 to 30 or 20 mol% compared with ratios for the liver and spleen. Interestingly, the tumor uptake clearance of liposomes of the same size was constant regardless of their membrane fluidity. These findings show that the increases in these accumulation ratios are due to their decreased uptake clearance by the bone marrow. Furthermore, the uptake of 50-nm HEPC-containing liposomes by the bone marrow was specifically inhibited by preinjection of other liposomes, but not when they were exposed in advance to in vivo components. These observations suggest the involvement of in vivo component(s) in the uptake of these liposomes by the bone marrow. We conclude that small HEPC-liposomes with low Ch content show their significantly decreased uptake by the bone marrow due to their decreased recognition by this tissue. PMID- 8611604 TI - Effect of cholesterol on the tight insertion of cytochrome b5 into large unilamellar vesicles. AB - When cytochrome b5 is added to large unilamellar vesicles (LUVs) of 1-palmitoyl-2 oleoylphosphatidylcholine (POPC), it binds predominantly in a 'loose,' or transferable form. Prolonged incubation of 30 degrees C leads to insertion in the physiological 'tight,' nontransferable form, with a halftime for the loose --> tight conversion of approx. 9 days. In this study, the effect of cholesterol on the rate of tight insertion was determined. Tight binding was assayed by depleting the LUVs of loose cytochrome b5 with an excess of SUV acceptors and then separating the liposome populations by gel-filtration or velocity sedimentation. Incorporation of cholesterol into the LUVs was found to markedly increase the rate of tight insertion, even though cholesterol decreases the equilibrium binding constant and saturation level of protein binding. The effect is not a continuously increasing function of cholesterol content, but attains a maximum at 20-25% mol%, where the rate enhancement is approx. 10-fold over baseline. At higher cholesterol levels, the rate decreases, returning to baseline at 40 mol% cholesterol. These observations are highly unusual in that cholesterol generally decreases the membrane binding affinity and the permeability of solutes, and does so as a monotonic function of cholesterol concentration (above the liquid-crystalline phase transition of the phospholipids). It is suggested that tight insertion is enhanced by lipid-protein packing mismatches and by bilayer fluidity; the former increases monotonically with increasing cholesterol whereas the latter decreases monotonically. At 20-25 mol% cholesterol the optimum balance of these physical properties is obtained for tight insertion. PMID- 8611605 TI - Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. AB - Cationic liposomes mediate efficient transfection of mammalian cells, but the manner in which cells internalize and process cationic liposome-DNA complexes has not been well characterized. We exposed several cell types, including human and murine erythroleukemia cells. African green monkey kidney cells (CV-1), isolated rat alveolar type II cells and alveolar macrophages to DNA-cationic liposome complexes containing N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and Dioleylphosphatidylethanolamine (DOPE). The morphology of liposome-cell interactions was assessed by electron microscopy. Liposome preparations were complexed to colloidal gold particles or to both plasmid DNA and gold particles. Cells treated with DOTMA liposome-DNA complexes demonstrated endocytosis of the liposome-DNA complexes in coated pits, which were seen in early endosomes, late endosomes, and lysosomes. In isolated alveolar type II cells, the gold-labelled DOTMA lipid apparently mixed with the contents of lamellar bodies. In most cells, gold particles were dispersed throughout the cytoplasmic matrix. In a small proportion of CV-1 and U937 cells, a membrane system resembling the endoplasmic reticulum developed within the nucleus. This novel structure was also present in nuclei after they were isolated from CV-1 cells and then mixed with DOTMA containing liposomes. Membranes which form after exposure to DOTMA-containing liposomes were 10 nm in thickness as compared to the approx. 8 nm thickness of endogenous cellular membranes. Based on these morphologic observations, we propose that the main route of entry of cationic liposomes into cells is by endocytosis. In some instances, the endosomal compartment releases its cationic liposome-DNA contents into the cytoplasmic matrix. Occasionally, liposomes may enter the nucleus by fusion with the nuclear envelope, creating vesicular and reticular intranuclear membranes. It is not clear at present which, if any of these morphological observations correlates with transfection mediated by cationic liposomes. PMID- 8611606 TI - Serum independent liposome uptake by mouse liver. AB - The rate of liposome clearance from blood by the reticuloendothelial system (RES), primarily the Kupffer cells of the liver, depends largely on liposome composition. Inclusion of phosphatidylserine or dicetyl phosphate into liposomes with a simple composition of phosphatidylcholine and cholesterol increases liposome clearance, while inclusion of GM1 or amphipathic poly(ethylene glycol) decreases the rate of liposome clearance. To understand the underlying mechanism by which liposome clearance is regulated by the RES, we have developed a simple liver perfusion system. Using mouse liver as a model, we demonstrated that hepatic uptake of neutral or negatively charged liposomes does not involve serum components. Liver uptake of liposomes is directly related to the surface characteristics of liposomes. Liposomes with a neutral composition of phosphatidylcholine and cholesterol exhibit relatively low liver uptake. Inclusion of PS or DCP into these liposome dramatically enhances liposome uptake by the perfused liver. Conversely, inclusion of GM1 or PEG derivatives into liposomes greatly reduces the liposome uptake by the mouse liver. In contrast to the neutral or negatively charged liposomes, serum enhances the liver uptake of positively charged liposomes. Such serum effect on liver uptake of the positively charged liposomes is likely due to liposome aggregations caused by serum proteins. Inhibition of the liver uptake for PS-containing liposomes using liposomes with different compositions suggests that liver uptake of liposomes may involve different receptors. PMID- 8611607 TI - Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor mediated gene transfer into hepatic cells by asialofetuin-labeled liposomes. AB - Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2 fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner. PMID- 8611609 TI - Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition. AB - Electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9- > 12- > 5- > 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8 degrees C and the cooperative unit to 1/5 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase. PMID- 8611608 TI - Receptor-mediated endocytosis of poly(acrylic acid)-conjugated liposomes by macrophages. AB - The uptake characteristics of negatively-charged liposomes made by conjugation of poly(acrylic acid) (PAA) were studied with respect to cultured RAW macrophages. The PAA-conjugated liposomes were internalized and digested in an acidic compartment at a much faster rate than the unmodified phosphatidylcholine (PC) liposomes. After incubation for 18 h, an over 5-fold increase in the uptake of PC liposomes was obtained by PAA conjugation. Subsequently, part of the aqueous phase of the internalized liposomes was exocytosed. Recognition of PAA by the macrophages seems to be responsible for the enhanced uptake of PAA-conjugated liposomes. Cross-competition experiments showed that PAA-conjugated liposomes inhibited the uptake of acetylated-low density lipoprotein (acetyl-LDL) by the macrophages and vice versa. The uptake of PAA-conjugated liposomes was also inhibited by dextran sulfate and maleylated-bovine serum albumin (maleyl-BSA), which are also known to bind to scavenger receptors. Poly(C) and BSA, which are not ligands for the scavenger receptor, competed poorly with the uptake of PAA conjugated liposomes. Enhanced uptake of PAA-conjugated liposomes by CHO cells with low scavenger receptor expression was not observed. Unexpectedly, LDL, which is not a ligand for scavenger receptor, also partially inhibited the uptake of PAA-conjugated liposomes. The interaction of PAA-conjugated liposomes with macrophages is complex, and the endocytosis of PAA-conjugated liposomes most likely involves multiple receptors and/or pathways. The data obtained suggest that the high affinity binding of PAA-conjugated liposomes to macrophages may be due to recognition of the negative charges of PAA by cell surface receptors, including the scavenger receptor. PMID- 8611610 TI - Kinetics of membrane micellization by the hydrophobic polyelectrolyte poly(2 ethylacrylic acid). AB - Rates of pH-dependent micellization of multilamellar vesicles by the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) (PEAA) have been measured turbidometrically. This polymer shows a strong ph-dependence in its affinity for phospholipid membranes, binding in increasing amounts as pH is lowered and ultimately solubilizing membranes to form mixed micelles (Tirrell, Takigawa and Seki (1985) Ann. N.Y. Acad. Sci. 446, 237). The rate of solubilization of dipalmitoylphosphatidylcholine (DPPC) vesicle suspensions by PEAA increases approximately linearly with reductions in pH below a threshold at pH 6.55. Interestingly, negatively-charged dipalmitoylphosphatidylglycerol membranes showed qualitatively similar behavior in the presence of PEAA, and incorporation of 10% or 20% dipalmitoylphosphatidic acid in DPPC membranes did not affect solubilization rates, demonstrating that membrane charge is not an important factor in determining micellization kinetics. Micellization of DPPC and dimyristoylphosphatidylcholine membranes occurs most rapidly at their respective gel-liquid crystalline transition temperatures (Tm); the rate enhancement is correlated with a peak in the temperature-dependent binding of a fluorescently modified PEAA in slightly alkaline solutions in which no micellization is observed. The lateral compressibility of the membrane, which has a similar peak at Tm, is proposed to be an important determinant of the rate and extent of polymer adsorption, and consequently of the rate of micellization. PMID- 8611611 TI - Contribution to the biophysics of the lethal effects of electric field on microorganisms. AB - The proposed model assumes that the criteria leading to the lethal breakdown of microorganisms suspended in a continuous medium depend on two parameters: (a) the applied electric field must exceed the critical field of membrane to create holes and (b) the Joule energy (deposited in the membrane) must exceed the minimum value beyond which the cell can not recover. The first parameter initiates (reversible) breakdown and the second one, the completion of the (irreversible) electrical breakdown leading to death of the cell. The number of cells surviving the electric field treatment is related to statistical distribution of cell size. Comparison between theory and the experimental results of Kinosita and Tsong (1977); Hulsheger et al. (1980, 1981, 1983); Rosemberg and Korenstein (1990) and others is given. PMID- 8611612 TI - Cyclodextrins: a new tool for the controlled lipid depletion of thylakoid membranes. AB - Cyclodextrins (CDs) have been used in controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono-- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with alpha-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DM-beta-CD), were as follows. (1)Acyl lipids were removed from thylakoid membranes by both CDs (DM-beta-CD being more efficient than alpha-CD; the extent of removal depended on both CD and chlorophyll concentrations. (2) alpha-CD presented a higher selectivity towards lip classes than did DM-beta-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) alpha-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-beta-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-beta-CD > alpha-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0 degrees than at 20 degrees C. (7) The presence of MgCl(2) reduced the removal of PG and SQDG but not affect galactolipid depletion levels. (8) Staple lipid depletion levels in thylakoid membranes were reached after 5-10 min of CD treatment at 0 degrees C. (9) Of the four CDs tested, only three (alpha-CD, beta-CD, and DM-beta-CD) promoted lipid depletion whereas one (hydroxypropyl-beta CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationship in photosynthetic membranes. PMID- 8611613 TI - Effect of X31 influenza virus fusion on phosphatidylserine asymmetry in erythrocytes. AB - Influenza virus fusion is mediated by its fusion protein, hemagglutinin (HA). HA undergoes a low pH dependent conformational change that results in insertion into the cell membrane bilayer, formation of a fusion pore, and merging of membrane lipids and establishment of cytoplasmic continuity. Erythrocytes, which can serve as targets of influenza virus fusion, display an asymmetric transbilayer arrangement of their phospholipids. The effect of influenza virus fusion on erythrocyte phosphatidylserine asymmetry was determined. Influenza virus were bound to erythrocytes containing the fluorescent membrane probe NBD-PS in the inner leaflet. Induction of fusion by exposure to a low pH environment resulted in movement of PS to the outer leaflet of the cell as well as hemolysis. Insertion of the fusion protein into erythrocytes and subsequent fusion can be distinguished from hemolysis by examining the interaction of a soluble form of HA (BHA) with cells and by monitoring viral fusion at low temperatures. No hemolysis was observed under either condition. BHA binding and insertion into cells did not affect the asymmetry of PS. Incubation of influenza virus fusion at pH 5, 0 degrees C resulted in complete fusion but no outward movement of PS was observed. These findings suggest the viral fusion pore does not involve a rearrangement of the transbilayer phospholipid organization of the target membrane. PMID- 8611614 TI - Cloning and sequence analysis of human butyrophilin reveals a potential receptor function. AB - Human butyrophilin was cloned and sequenced from a human breast cDNA library. The derived amino acid sequence shows 84% sequence identity and identical domain arrangements with the previously reported bovine sequence. Sequence analysis reveals an immunoglobulin constant (IgC) domain that was not previously identified in the bovine sequence. The extracellular domain composition of butyrophilin suggests a cell surface receptor function. PMID- 8611615 TI - Regulation of NHE1 expression in L6 muscle cells. AB - We examined regulation of expression of the NHE1 promoter on rat L6 cells. Transient transfection of these cells showed that there are two regions critical for basal expression in this cell type. One is from bp -155 to -171 and second more proximal region is between bp -92 and -125. When cells were induced to differentiate by serum withdrawal, mRNA levels rose 2-3-fold. To investigate the mechanisms of this phenomenon a series of stable transfectants were made of the NHE1 promoter in L6 cells. Muscle differentiation caused a significant stimulation of transcriptional activity in the stable cells containing the more distal regions of the promoter. The results show that basal expression of the NHE1 promoter is mediated largely by two proximal regions of the gene. However, during the process of differentiation more distal regions of the gene are involved in elevation of the level of expression. PMID- 8611616 TI - Corrigendum to "Cloning of cDNA for rat eosinophil major basic protein" [Biochim. Biophys. Acta 1264 (1995) 261-264]. PMID- 8611618 TI - Structure of a cluster of mouse histone genes. AB - The structure of a 25 kilobase region of mouse DNA containing 6 functional histone genes and an H2a pseudogene has been determined. The sequences and levels of expression of the H3 and H2b gene as well as the sequence of the H2a pseudogene have been determined. PMID- 8611617 TI - Cloning and sequencing of a human cDNA encoding a putative transcription factor containing a bromodomain. AB - A 2985 bp cDNA was isolated from a Lambda Zap Express library and sequenced. The cDNA appeared to represent a previously unknown gene that encodes and acidic 757 amino acid protein containing a bromodomain, several potential sites for phosphorylation by casein kinase-II and small proline-rich segments. The results suggest that the encoded protein might be a novel transcription factor. PMID- 8611619 TI - Cloning and expression of an Entamoeba histolytica NAPD+(-)dependent alcohol dehydrogenase gene. AB - In this paper we cloned, sequenced and expressed a novel Entamoeba histolytica alcohol dehydrogenase gene (Ehadh3). Ehadh3 has a predicted 383 amino acids open reading frame, encoding for a 42.3 kDa protein. The deduced amino acid sequence showed 24 to 26% identity to other type III alcohol dehydrogenases found in prokaryotic and lower eukaryotic organisms, but not in mammalia. There are at least two Ehadh3 gene copies in the genome, but only a 1.2 kb transcript was detected. The EhADH3 fusion protein showed a NADP+(-)dependent ADH activity. Ehadh3 may be a good target for the developing of anti-E. histolytica drugs, without producing damage to the human. PMID- 8611621 TI - The rat FGF-5 mRNA variant generated by alternative splicing encodes a novel truncated form of FGF-5. AB - We isolated rat FGF-5 cDNA and its variant from the embryo. The FGF-5 cDNA encodes a protein of 266 amino acid residues. The variant cDNA encodes a protein of 121 amino acid residues, the N-terminal 117-amino acid sequence of which is identical to that of FGF-5. Thus the variant cDNA encodes a novel truncated form of FGF-5. The rat FGF-5 gene consists of three exons (exons 1-3). The variant cDNA lacks the nucleotide sequence of exon 2, indicating that the variant mRNA is generated by alternative splicing. The variant mRNA as well as the FGF-5 mRNA was detected in the embryo and adult brain. This is the first description of the mRNA for a truncated form of FGF within the FGF family. PMID- 8611620 TI - Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39. AB - A human cDNA encoding a human homologue of the rat phosphoribosylpyrophosphate synthetase-associated protein of 39 kDa was isolated. The deduced protein contains 356 amino acids and has calculated molecular mass of 38561. The amino acid sequence is 98% identical to that of the rat. The corresponding mRNA is present in all human tissues examined. PMID- 8611622 TI - Promoter region of the rat phosphoribosylpyrophosphate synthetase-associated protein 39. AB - The 5' region of the 39-kDa rat phosphoribosylpyrophosphate synthetase-associated protein (PAP39) gene was isolated and sequenced. The promoter region of the rat PAP39 is GC-rich and contains potential binding sites for regulatory factors. Its promoter activity was demonstrated by transfection of the promoter region in fusion with the chloramphenicol acetyltransferase gene into rat pheochromocytoma PC12 cell. PMID- 8611623 TI - The Ca2+(-)binding proteins parvalbumin and oncomodulin and their genes: new structural and functional findings. PMID- 8611624 TI - The HIR protein family: isolation and characterization of a complete murine cDNA. AB - A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome. This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast. The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family. The two cDNAs are highly conserved within the coding regions, but also in the entire 5' untranslated region and in a strikingly long stretch of nucleotides in the 3' untranslated region. PMID- 8611626 TI - A third human tissue transglutaminase homologue as a result of alternative gene transcripts. AB - A 2.4 kilobase (kb) cDNA encoding a new form of human tissue transglutaminase homologue (TGH2) was isolated from retinoic acid-induced human erythroleukemia cell (HEL) library. Full-length cDNA analysis gives an open reading frame coding for a polypeptide of 349 amino acid residues with a molecular mass of 38,700 Da. This variant differs from the previously reported homologue TGH in that it is 199 amino acids shorter and has an alternative, 63 amino acid COOH-terminal peptide. The 3'-untranslated region of the cDNA also differs from the previously reported sequences for both TGH and human tissue transglutaminase. The region coding for the first 286 amino acids of TGH2, which contains the active site is identical to TGH. Immunoprecipitation of the in vitro translation product from a synthetic TGH2 mRNA and immunoprecipitation of total protein of human heart, liver, kidney and cultured erythroleukemia HEL cell, revealed a protein with a molecular mass of 37,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the cDNA sequence for the previously known tissue transglutaminases with genomic DNA and the TGH2 cDNA described here indicate that the sequence divergence points correlate with known intron-exon boundaries. The smaller RNA species encode for truncated proteins with novel carboxyl termini. The TGH cDNA and the TGH2 cDNA both produce transcripts which start with the regular coding sequence for TGase and then fail to splice at specific donor sites, resulting in the use of an alternative exon that contains a stop codon. PMID- 8611625 TI - In vitro and in vivo gene transfer to pulmonary cells mediated by cationic liposomes. AB - Cationic liposomes have been proposed as alternative to adenovirus in the treatment of cystic fibrosis lung disease. Therefore, we have investigated the efficiency of two lipid mixtures in mediating gene transfer in in vitro and in vivo models. The cationic lipid DOTMA (N-(1-(2,3(dioleyloxy)propyl)-n,n,n trimethylammoniumchloride++ +) and DOGS (dioctadecylamidoglycylspermine) were used in combination with the neutral lipid DOPE (dioleoylphosphatidylethanolamine). The relative transfection efficiencies of the two cationic liposomes were tested using the bacterial beta-galactosidase (lacZ) and the firefly luciferase genes. Gene expression was detected in both cell limes and primary culture of rhesus monkey airway epithelium after transfection with plasmid DNA complexed with DOGS/DOPE or DOTMA/DOPE. Transfection efficiency of both types of lipids was higher in the mouse fibroblast 3T3 cell line as compared to human carcinoma A549 cells and primary epithelial cultures. Administration of DNA-liposome complexes via intratracheal instillation resulted in expression of the lacZ and luciferase marker gene in the mouse airways. In vivo transfection mediated by both types of liposomes were proven to be far less efficient than adenovirus treatment. PMID- 8611628 TI - Isolation of a cDNA clone, TRX encoding a human T-cell lymphotrophic virus type-I Tax1 binding protein. AB - Tax1 is essential for human T-cell lymphotropic virus type I (HTLV-I) virus replication and transformation. We have identified and characterized a Tax1 binding protein, TRX, by cDNA screening of a Jurkat T-cell cDNA library. TRX mRNA is ubiquitously expressed in human tissues tested and cell lines analyzed. PMID- 8611627 TI - Characterization of the human AMPD3 gene reveals that 5' exon useage is subject to transcriptional control by three tandem promoters and alternative splicing. AB - Previous work has identified multiple human AMPD3 transcripts proposed to differ by mutually exclusive alternative splicing of three exons located at, or near, the 5' end of the gene. In this study, we perform a more comprehensive evaluation of human AMPD3 gene expression. Combined Northern blot and RNase protection analyses show that alternative mRNAs are widely expressed in human tissues and cells, but at variable relative abundances. Sequencing of human genomic clones, together with human-mouse somatic cell hybrid analysis, demonstrates that the entire gene is comprised of seventeen exons spanning approx. 60 kilobases on the short arm of chromosome 11 in the region p13-pter. Together, RT-PCR and additional RNase protection analyses establish that exons 1a, 1b, and 1c are 5' terminal sequences in alternative transcripts. Transient transfection experiments show fusion constructs containing proximal flanking and 5' untranslated sequence from each of these exons are able to direct expression of a reporter luciferase gene in mammalian cell lines. These combined results reveal that AMPD3 gene expression is subject to transcriptional control by three tandem promoters. Differential regulation of the exon 1b promoter in skeletal myocytes, as compared to retinal pigment epithelial cells, is proposed to be mediated by skeletal muscle-specific basic helix-loop-helix protein/E-box interactions. Finally, an internal splice acceptor site in exon 1c is shown to be used alternatively to retain the 3' portion of this exon in mature AMPD3 transcripts initiating upstream in exon 1b. PMID- 8611629 TI - Dielectric studies of intermolecular interactions in native DNA. AB - The concentration dependence of the high frequency dielectric dispersion exhibited by solutions of DNA and the sensitivity of this to the ionic strength of the solution are described. Dielectric data obtained are consistent with a model involving the fluctuation of counterions along short segments of the DNA chain. The concentration and ion strength dependencies are discussed in terms of intermolecular interactions which are considered responsible for the existence of structure in DNA solutions. PMID- 8611630 TI - Bovine alpha 1-microglobulin/bikunin. Isolation and characterization of liver cDNA and urinary alpha 1-microglobulin. AB - cDNA coding for alpha 1-microglobulin, an immunoregulatory plasmaprotein, was isolated from bovine liver. The sequence of a total of 1258 nucleotides revealed an open reading frame of 352 amino acids. This included alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasmaprotein inter-alpha inhibitor, 147 amino acids. The two proteins were connected by a basic tetrapeptide, R-A-R-R, which conforms to the consensus sequence recognized by endoproteolytic cleavage enzymes. The deduced amino acid sequence showed a high degree of identity with alpha 1-microglobulin and bikunin sequences from other species, and the alpha 1-microglobulin part displayed sequence motifs typical for members of the lipocalin protein superfamily. A single alpha 1 microglobulin/bikunin mRNA with a size of around 1300 nt was found in bovine liver. The mature alpha 1-microglobulin protein was isolated from bovine urine, and partly characterized. It was found to be a globular molecule with an apparent molecular weight of 23,300, containing one N-linked and at least on O-linked oligosaccharide, one intra-chain disulfide bridge and an electrophoretic heterogeniety with a pI-value of 4.1-5.2. PMID- 8611631 TI - Tumor necrosis factor-alpha induces the 85-kDa cytosolic phospholipase A2 gene expression in human bronchial epithelial cells. AB - Phospholipase A2 (PLA2) activity has been suggested to mediate some of the tumor necrosis factor (TNF) induced cellular responses including cytotoxicity. We evaluated the induction of both the 85-kDa cytosolic phospholipase A2 (cPLA2) and non-pancreatic group II PLA2 gene expression by TNF-alpha in a human bronchial epithelial cell line (BEAS 2B cell). TNF-alpha (20 ng/ml) induced a significantly increased release of prelabeled [3H]arachidonic acid (AA) following 4-24 h incubation. Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the TNF-alpha-treated cells. In vitro activity assay revealed that TNF-alpha increased the dithiothreitol (DTT)-resistant PLA2 activity which was blocked by the cPLA2 inhibitor AACOCF3. Treatment with TNF-alpha for 4-24 h increased the cPLA2 protein and mRNA levels which were blocked by the broad inhibitor of protein kinases staurosporine, the protein kinase C (PKC) inhibitor calphostin C, and to a lesser extent the calcium/calmodulin-dependent protein kinase inhibitor W-7. Reverse transcription and polymerase chain reaction amplification of the group II PLA2 mRNA showed that it is expressed in human lung but not in the bronchial epithelial cell line. TNF-alpha failed to induce the expression of group II PLA2 in the BEAS 2B cells. These results demonstrate that the cPLA2 gene expression is up-regulated by TNF-alpha and this effect may contribute to the TNF-alpha stimulated AA release in airway epithelial cells. PMID- 8611632 TI - Cell adhesion to substratum and activation of tyrosine kinases are essentially required for G1/S phase transition in BALB/c 3T3 fibroblasts. AB - Cell adhesion to substratum and activation of tyrosine kinases are essential for the progression of cell cycle through G1 phase in mammalian cells. The kinetic studies of mouse BALB/c 3T3 fibroblasts showed that serum was no longer required for the progression of G1/S phase transition. In contrast, cell adhesion was essentially required in late G1 phase, especially at the period of G1/S transition. Among the kinase inhibitors used to elucidate the signal transduction caused by cell adhesion, tyrosine kinase inhibitors, genistein and herbimycin A, blocked the G1/S transition most effectively when cells were exposed to the inhibitors at the period of G1/S transition. Cell adhesion was not critically required for cells to undergo DNA synthesis once they had passed the G1/S boundary, and the effects of tyrosine kinase inhibitors on the progression of S phase were also not critical. The expressions of histone H2B and dihydrofolate reductase (DHFR) genes (S phase specific genes) and also the transcription factor E2F-1 gene (an activator of DHFR gene) were suppressed when cells were cultured without adhesion or exposed to the tyrosine kinase inhibitors. These results suggest that cell adhesion to substratum plays an important role in the G1/S phase transition of mouse BALB/c 3T3 fibroblasts through the activation of tyrosine kinases other than growth factor receptor-tyrosine kinases. PMID- 8611633 TI - Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator. AB - The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytes and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12 myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering the RA is now being tested as an anti-cancer agent and proposed for prevention studies. PMID- 8611634 TI - Cell-specific regulation of the stably expressed serotonin 5-HT1A receptor and altered ganglioside synthesis. AB - Neurotransmission is dependent on the presence of neuronal receptors at the synapses, and important cell surface molecules such as gangliosides are pivotal in the maintenance of synaptic contacts. To study the interrelationship between these two classes of molecules, we achieved stable expression of the hippocampus- and CNS-localized serotonin 1A receptor (5-HT1A-R) in three 5-HT1A-R-deficient neuronal cell lines and also the control, non-neural CHO cells. A strong passage dependence of 5-HT1A-R expression, as measured by mRNA levels as well as membrane binding to the selective agonist [3H]8-OH-DPAT, was observed only in the HN2 (hippocampal) and NCB-20 (CNS) cells which are derived from tissues of natural occurrence of the 5-HT1A-R. A paradigm of stress was obtained by carrying out continuous culture of cells without feeding. During this time a dramatic increase in 5-HT1A-R mRNA and [3H]8-OH-DPAT binding was observed only in the neuronal cells after confluence and during decreased cell viability (days 10/11). This was not due to differentiation, since deliberate serum deprivation and differentiation of cells did not result in any dramatic increase in 5-HT1A-R expression. Analysis of ganglioside synthesis by pulse labeling of the transfected cells produced striking results. In the dorsal root of the ganglion (DRG) derived F-11 cells which show low but significant levels of complex gangliosides before transfection, the mere presence of the serotonin 1A receptor resulted in a dramatic increase in synthesis of gangliosides comigrating with GM2, GD1a, GD1b, and GT1b (20-fold by densitometry). In contrast, there was only a 2-fold increase in the overall content of complex gangliosides in the presence of the 5-HT1A-R. In the NCB-20 cells which contain only GD1a but no GD1b or GT1b before transfection, a decrease in GD1a synthesis was observed following transfection. Also agonist (8-OH-DPAT) binding to the serotonin 1A receptor in NCB-20 cells produced a 3-fold increase in synthesis of a ganglioside comigrating with GM3. Thus, our neuroblastoma transfectants help demonstrate stress-induced regulation of the 5-HT1A-R, which in turn exerts a strong and cell type-specific control over such essential cell-surface determinants like gangliosides. PMID- 8611636 TI - Hormonal modulation of c-fos expression in isolated hepatocytes. Effects of angiotensin II and phorbol myristate acetate on transcription and mRNA degradation. AB - It has been shown that angiotensin II and PMA increase the expression of proto oncogenes (c-fos, c-myc and c-mos) in liver cells. In this study the effects of angiotensin II and PMA on c-fos transcription and mRNA stability were investigated. Using nuclear run-off transcription assays, it was observed that PMA and angiotensin II induced a rapid increase in c-fos transcription. The transcription rate of the GAPDH gene did not change, indicating that the effects were not general on gene transcription. The ability of these agents to modulate proto-oncogene mRNA stability was tested by measuring c-fos mRNA half-life. It was observed that c-fos mRNA half-life was relatively short (approximately 14-18 min) and that angiotensin II and PMA markedly stabilized mRNA, increasing its half-life (approximately 4-fold and approximately 2-fold, respectively). The protein synthesis inhibitor cycloheximide increased mRNA stability to a much greater extent. Our results clearly demonstrate that angiotensin II and PMA increased c-fos mRNA accumulation in liver cells through two actions: induction of c-fos gene transcription and increase in mRNA stability. PMID- 8611635 TI - Suppression of Ca2+ oscillations in glucagon-producing alpha 2-cells by insulin/glucose and amino acids. AB - The cytoplasmic Ca2+ concentration ([Ca2+]i) was continuously monitored in single glucagon-producing alpha 2-cells isolated from the mouse pancreas and later identified by immunostaining. Up to 60% of the alpha 2-cells exhibited spontaneous [Ca2+]i oscillations (frequency 0.1-0.3/min) in a medium containing 3 mM glucose. In originating from a basal level of 60-100 nM, reaching peak values of 300-400 nM and promptly disappearing after blocking voltage-dependent Ca2+ channels with methoxyverapamil, the oscillations resembled those in insulin releasing beta-cells stimulated by glucose. The oscillatory activity was suppressed when combining elevation of glucose to 20 mM with the addition of 2 2000 ng/ml insulin. Whereas 10 mM of L-arginine or l-glycine transformed the oscillations into sustained elevation of [Ca2+]i, there was no response to 1 mM tolbutamide or 0.1-1 mM gamma-aminobutyric acid. The observations that alpha 2 cells differ from islet cells secreting insulin and somatostatin in responding to adrenaline with mobilisation of intracellular calcium can be used for their rapid identification. It is suggested that the oscillations reflect periodic entry of Ca2+ due to variations of the membrane potential. PMID- 8611637 TI - Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP. AB - T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al. (1990)) Nature 364, 719-723). In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomic Jurkat cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA induced, but also TCR-mediated formation of Ras.GTP. Furthermore, down-regulation of PMA-sensitive PKC severely impaired the activation of Ras in response to TCR stimulation. Tyrosine-phosphorylation and translocation to the particulate fraction of phospholipase C-gamma 1 (PLC-gamma 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-gamma 1 by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interleukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gs alpha, is shown to inhibit the activation of IL-2 promoter. TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-gamma 1 , and PKC. An activated Gs alpha mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects. PMID- 8611638 TI - Apyrases (ATP diphosphohydrolases, EC 3.6.1.5): function and relationship to ATPases. PMID- 8611639 TI - Collisions and encounters in simulations of receptor/GTP-binding protein interactions via simple diffusion. AB - In two intact cell systems in which GTP-binding protein (G) activity is initiated by the presence of agonist-bound receptors (R), it has been demonstrated that the rate of G activation is influenced by the rate of turnover of agonist occupancy among the receptor population. G activity is reduced when a low concentration of agonist-occupied receptors comprised by low fractional occupancy of a large receptor population is replaced by the presence of the same concentration of 100% occupied receptors. This effect has been proposed to be due to a time interval of interaction between R and G (an encounter) that is long compared to the time of a single collection between R and G and long compared to the lifetime of an agonist receptor complex. In a recent simulation study of R-G interaction via diffusion, the effect of agonist occupancy turnover was observed but it was assumed that long encounters were not operative. In this study, encounter intervals in simulations of R-G interaction by simple diffusion were measured in order to address that difference. The results demonstrate that relatively long encounters comprised of multiple, separate collisions are an inherent part of R-G interaction as modelled by diffusion. The implications for further implementation of simulation studies of R-G interaction are discussed. PMID- 8611640 TI - Molecular characterisation of the genomic locus of the mouse MRP8 gene. AB - MRP8 is an inflammatory marker protein specifically expressed throughout the myeloid cell lineage in mouse and humans. Here the nucleotide sequence and the genomic structure of the mouse MRP8 gene (MM-MRP8) is presented. A strong homology between the mouse and human MRP8 promoters reflects the highly specific expression pattern of both genes and suggests that a conserved transcriptional machinery regulates these genes. PMID- 8611641 TI - Diallyl disulfide inhibits the proliferation of human tumor cells in culture. AB - Diallyl disulfide (DADS), an oil-soluble organosulfur compound in processed garlic, was more effective in inhibiting the in vitro growth of human tumor cell lines: HCT-15 (colon), A549 (lung), and SK MEL-2 (skin) than isomolar quantities of the water-soluble compound S-allyl cysteine (SAC). Addition of DADS (100 microM) was cytostatic to all three cell lines. The importance of the allyl and the disulfide groups were revealed by the lack of a comparable depression in the growth of HCT-15 cells exposed to its saturated analogue, dipropyl disulfide (DPDS). Treatment with DADS also resulted in a dose-dependent increase in intracellular free calcium in cells. A dose-dependent decrease in the activity of calcium-dependent ATPase enzyme occurred in HCT-15 cells exposed to increasing quantities of DADS. A correlation (r = -0.975) was found between the intracellular free calcium levels and the Ca-ATPase activity in DADS-treated cells. These studies document that DADS, a constituent of garlic oil, is an effective inhibitor of the growth of human neoplastic cells. Alterations in calcium hemostasis are likely involved in the growth inhibition/cytotoxicity caused by DADS. PMID- 8611642 TI - Protection against oxidative injury and permeability alteration in cultured alveolar epithelium by transferrin-catalase conjugate. AB - The successful prevention of hydrogen peroxide-induced alveolar permeability alterations and cell injury by transferrin-catalase conjugate is described in this study. Permeability alterations and cell injury were induced in cultured alveolar epithelial monolayers by hydrogen peroxide. Transepithelial transport of a permeability marker, [14C] mannitol, and cellular nuclear fluorescence of a membrane integrity indicator, propidium iodide, were used to quantitate epithelial permeability and damage respectively. Hydrogen peroxide (0.1 - 10 mM) induced a dose-dependent increase in both alveolar permeability and cellular damage; however, the oxidant effect on monolayer permeability did not require prior cell damage. Electron spin resonance measurements using the spin trap 5,5 dimethyl-l-pyrroline-N-oxide indicated the formation of hydroxyl radicals in hydrogen peroxide-treated cells. Chelation of the cellular pool of iron by deferoxamine inhibited radical formation and helped protect the cells from oxidative changes. Prior treatment of the cells with catalase (0.1 U-10 U/ml) had minimal protective effects on cell injury and permeability alterations. In contrast, transferrin-catalase conjugate, at the same concentration range, exhibited much improved protective effects on the cells in response to oxidant stress. This enhanced protection was found to correlate well with an increase in cellular uptake of the enzyme conjugate via the transferrin receptor endocytosis pathway. Effective protection by the enzyme conjugate was shown to require both the antioxidant enzyme moiety and the cognate moiety for the cell surface receptor. These findings indicate the potential therapeutic merit of transferrin catalase conjugate for the treatment of pathological processes in the lung, whenever oxidative stress is involved. PMID- 8611643 TI - Identification and synthetic pathway of sialyl-Lewis(x)-containing neolacto series gangliosides in lens tissues. 2. Enzymatic synthesis of sialyl-Lewis(x) gangliosides in monkey and rat lenses. AB - In Japanese monkey lenses, 3H-labeled fucose and N-acetylneuraminic acid were enzymatically transferred to neolactotetraosylceramide (nLc4) and III 3 FucnLc4, respectively, suggesting the presence of a synthetic pathway of IV3 NeuAcIII3 FucnLc4 via III3 FucnLc4 in monkey lenses. Six rat strains, Wistar, Sprague Dawley and pigmented strains, contained sialyl-Lewis(x) gangliosides in non cataractous lenses in a strain-specific manner. Glycosyltransferase assay revealed that the transfer of 3H-labeled fucose to nLc4 occurred in all the strains, but that the transfer of 3H-labeled N-acetylneuraminic acid to III3 FucnLc4 was strain-specific. These results suggested that sialyl-Lewis(x) gangliosides were generally synthesized from neolactotetraosylceramide via Lewis(x) glycolipid (III3 FucnLc4) in lens tissues, differing from other tissues. Combining our results, we propose two synthetic pathways of sialyl-Le(x)- containing neolacto-series gangliosides and A-pathway ganglio-series gangliosides in human senile cataractous lens: one to sialyl-Lewis(x) gangliosides from nLc4 via Lewis(x) glycolipid, and the other to GD1a from GM3, via GM2 and GM1. PMID- 8611644 TI - Reduced carnitine palmitoyl transferase activity and altered acyl-trafficking in red blood cells from hemodialysis patients. AB - We measured carnitine palmitoyl transferase activity, free carnitine, and long chain acyl carnitine levels in erythrocytes from 15 uremic patients and 25 controls. Carnitine palmitoyl transferase levels in patients were significantly lower than in controls. The levels of free carnitine and long chain acyl carnitines as well as the long chain acyl carnitine/free carnitine ratio were significantly higher in patients than in controls. Our results suggest that hemodialysis causes alteration in the acyl-trafficking in red blood cells membrane. PMID- 8611645 TI - Conformations of synthetic beta peptides in solid state and in aqueous solution: relation to toxicity in PC12 cells. AB - The secondary structures of peptides beta 25-35 (the active toxic fragment) and beta 35-25 (reverse sequence and non-toxic fragment), as well as of the amidated beta (25-35)-NH2 peptide were investigated in aqueous solution and in the solid state by means of Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy. The conformations of the beta 25-35 and beta 35-25 in solid state were identical and contained mostly beta-sheet structures. In solid state the amidated beta (25-35)-NH2 peptide also contained mostly beta-sheet structures. Freshly prepared aqueous solutions of the beta 25-32 (0.5 - 3.8 mM) contained a mixture of beta-sheet and random coil structures. Within 30-60 min incubation at 37 degrees C in water or in phosphate-buffered saline solution (PBS), beta 25-35 was almost fully converted to a beta-sheet structure. Decreasing the temperature from 37 degrees C to 20 degrees C decreased the rate of conversion from random coil to beta-sheet structures, 1-2 h being required for complete conversion. In contrast beta 35-25 in water or in PBS buffer had mostly a random coil structure and remained so for 6 days. The amidated beta(25-35)-NH2 peptide in water (2.7 mM) was also mostly random coil. However, when this peptide (2-2.7 mM) was dissolved in PBS (pH 7.4) or in 140 mM NaCl, a gel was formed and its conformation was mostly beta-sheet. Decreasing the concentration of beta (25 35)-NH2 peptide in 140 mM NaCl aqueous solution from 2 mM to 1 mM or below favored the conversion from beta-sheet structures to random coil structures. The beta 25-35 was toxic to PC12 cells while beta 35-25 was not. The amidated peptide beta (25-35)-NH2 was at least 500-fold less toxic than beta 25-35. Structural differences between these beta peptides in aqueous solutions may explain the difference in their respective toxicities. PMID- 8611646 TI - Altered calcium homeostasis: a possible mechanisms of aluminium-induced neurotoxicity. AB - The effect of aluminium, A1(3+) (10 mg/kg body weight/day i.p.) for a period of 4 weeks was examined on the calcium homeostatic mechanisms in rat central nervous system. Incubation of synaptosomes prepared from rat brain, with aluminium in vitro had a detrimental effect on the activity of Ca2+ ATPase which could be reversed completely on exogenous addition of desferrioxamine (10 microM) and partially with glutathione (1 mM). In vivo administration also revealed a similar observation. A marked increase in the levels of intracellular calcium was observed after aluminium treatment. Concomitant to the increased levels of intracellular calcium, there was an increase in the levels of lipid peroxidation and a consequent decrease in fluidity of synaptic plasma membranes. In addition, aluminium also had an inhibitory effect on the depolarization-induced calcium uptake which was found to be of a competitive type. The biological activity of calcium regulatory proteins calmodulin and protein kinase C was considerably affected by aluminium. The results suggest that aluminium exerts its toxic effects by modification of the intracellular calcium messenger system with detrimental consequences on neuronal functioning. PMID- 8611647 TI - Derangement in aerobic and anaerobic energy metabolism in skeletal muscle of critically ill and recovering rats. AB - As part of our research into the mechanisms of protein wasting and muscle weakness during critical illness, we here investigate various aspects of energy metabolism. Intraperitoneal injection of zymosan in rats leads to an acute phase of critical illness followed by a prolonged recovery phase. Previously we observed low activities of mitochondrial enzymes, reduced protein synthesis rates and low concentrations of glutamine in skeletal muscle of zymosan-treated rats. In the present study we investigated (1) whether decreases in high energy phosphates are present in skeletal muscle of these rats and (2) whether an impairment in the glycolytic pathway or the tricarboxylic acid cycle leads to these decreases. Concentrations of creatine phosphate and ATP were decreased in zymosan-treated rats to approx. 85% of pair-fed control values respectively on day 2 and on days 4 and 6 after treatment. Concentrations of tricarboxylic acid (TCA) cycle intermediates were decreased to 80% on day 6 after zymosan treatment. Lactate/pyruvate ratio and concentrations of lactate and glycogen were normal at all sampling times. We conclude that no major changes in concentrations of high energy phosphates and in concentrations of intermediates of TCA cycle, glycolysis and glycogenolysis were present. This indicated that, although the maximal oxidative capacity (mitochondrial content) is decreased, no derangement in energy metabolism seems to be present in skeletal muscle of critically ill and recovering rats. PMID- 8611648 TI - Cotranscription of two RNA coding for the cell adhesion regulator and its variant in Reh leukemia cells. AB - Human genomic DNA analysis reveals the existence of polymorphisms at the cell molecular adhesion regulator (CMAR) locus. In order to choose between the two possible open frames deduced from the variant sequence, we have sequence both the human 5' non-coding region and the mouse CMAR variant DNA. We found that both mRNA species coexist in human cells. PMID- 8611649 TI - Alterations in erythrocyte membrane fluidity and fatty acid composition in glycogen storage disease. AB - Liver glycogen storage diseases (GSD) are disorders associated with severe dyslipidaemia which can induce cell membrane alterations. Reduced erythrocyte membrane fluidity has been associated with ischaemic cardiovascular disease. Our study has been designed to investigate membrane erythrocyte fluidity, and to determine its lipid composition and peroxidation parameters. Membrane erythrocyte fluidity has been studied by electron spin resonance (ESR) with two fatty acid nitroxide probes (5NS and 16NS). Twenty-five GSD cases aged 1-27 years and 15 controls aged 1-28 years were included. The erythrocyte membrane of GSD patients appeared less fluid with the two probes (P < 0.001). The membrane fatty acid pattern explained this reduced fluidity. Patients showed a relative saturated fatty acid (SFA) increase and polyunsaturated fatty acid (PUFA) decrease which induced lower PUFA/SFA ratio than in controls. We have provided evidence that the PUFA decrease was independent of the oxidative process. These findings should be taken into account for the management of the dietary treatment of GSD patients. PMID- 8611651 TI - Arginine to tryptophan substitution in the active site of a human lactate dehydrogenase variant--LDHB GUA1: postulated effects on subunit structure and catalysis. AB - A variant of lactate dehydrogenase (LDHB GUA1) was previously identified among the Guaymi Indians of Panama and Costa Rica. The LDHB GUA1 variant is enzymatically inactive; however, the variant subunits alter the electrophoretic mobility of the tetramers that include active LDHA and LDHB subunits. The kinetic properties of the tetrameric enzyme, comprised of the inactive B plus active A subunits, are similar to properties of the heterotetramers with active B subunits, except for the reduced specific activity. We have determined that a single C.G to T.A transition changes an Arg to a Trp at amino acid residue 106. This substitution explains the increase in net negative charge observed by protein electrophoresis. This Arg 106 residue is absolutely conserved throughout evolution. Published high-resolution crystal structures of LDH reveal that this residue is within the hinge of a loop that closes over the active site of the subunit upon binding of substrate and cofactor and also has a direct role in catalysis. Computer modeling of the variant enzyme suggests that replacement of this Arg residue with a Trp does not induce significant change in the structure of the active site. However, this substitution would result in disruption of enzyme activity through the inability of the uncharged tryptophan side-chain to polarize the substrate carbonyl bond. This would explain the loss of the catalytic function with maintenance of normal kinetic properties in the heterotetramers containing the variant subunits. The ability to maintain normal, tissue-specific kinetic properties could explain the absence of clinical manifestations in the homozygous LDHB GUA1 individuals. PMID- 8611650 TI - Ageing is associated in females with a decline in the content and activity on the b-c1 complex in skeletal muscle mitochondria. AB - The activity of cytochrome-c oxidase [E.C. 1.9.3.1] and b-c1 complex [E.C. 1.10.2.2] and the content of cytochromes b, c + c1 and a + a3 in human skeletal muscle mitochondria from orthopaedic patients (108 women and 68 males), of age ranging between 10 and 50 years, have been analyzed. The activity of cytochrome c oxidase declines with age both in females and males. The activity of b-c1 complex, which in young females is significantly higher than in young males, declines sharply in females, but not in males, with ageing. These results reveal that the content of active b-c1 complex in muscle mitochondria is specifically controlled by female sex hormones. PMID- 8611652 TI - Non-rhizomelic and rhizomelic chondrodysplasia punctata within a single complementation group. AB - Several patients have been described recently who suffer from a non-rhizomelic type of chondrodysplasia punctata (CDP), but who show all the biochemical abnormalities characteristic of the rhizomelic form of chondrodysplasia punctata (RCDP), a peroxisomal disorder. We have used protease protection experiments and microinjection of reporter-protein-encoding expression plasmids to show that peroxisomal thiolase fails to be imported into peroxisomes in cells from non rhizomelic CDP patients, as has already been found in cells from classical RCDP patients. Furthermore, complementation analysis after somatic cell fusion indicates that the non-rhizomelic CDP patients are impaired in the same gene as classical RCDP patients. We conclude that defects in a single gene can give rise to both clinical phenotypes. PMID- 8611654 TI - Five isoenzymes of protein kinase C are expressed in normal and STZ-diabetic rat hepatocytes: effect of phorbol 12-myristate 13-acetate. AB - Using isoenzyme-specific antisera, five Protein Kinase Cs (PKCs) were detected in cytosol and membrane hepatocytes from normal rats: PKC alpha (80 kDa), PKC beta II (40, 50, 55, 85 kDa), PKC delta (74, 76 kDa), PKC epsilon (95 kDa), PKC zeta (65, 70 kDa). STZ-diabetes induced a lower expression of the five PKCs, a higher localization in the cytosol, a preferential expression of PKC delta as the 76 kDa phosphorylated species and a decreased kinase activity towards Histone III-S. A 1 microM phorbol 12-myristate 13-acetate (PMA) incubation induced similar translocation to the membrane of PKCs alpha, native 85 kDa beta II and epsilon. The 74 kDa PKC delta was switched to the 76 kDa species, the normal form in STZ diabetic cells. The truncated PKC beta II and PKC epsilon were unchanged. PMID- 8611653 TI - Evidence for McKusick's hypothesis of deficient collagen cross-linking in patients with homocystinuria. AB - Osteoporosis occurs commonly in homocystinuria. The underlying pathobiochemical mechanism remains unclear; disturbed cross-linking of collagen has been suggested but this hypothesis has not been fully tested, nor have studies on collagen synthesis been performed. We therefore used recently available noninvasive tests for collagen synthesis and cross-linking to examine 10 patients with homocystinuria. Synthesis of collagen type I and type III was not different from age-matched healthy controls as reflected by comparable plasma levels of carboxyterminal propeptide of type I procollagen (PICP) and of plasma levels of N terminal propeptide of procollagen type III (PIIINP). Collagen type I cross-links expressed by serum carboxyterminal telopeptide of collagen type I (ICTP) were 1.14 +/- 0.24 micrograms/l in the patient group versus 3.29 +/- 0.32 micrograms/l in the control group. This significant reduction of cross-links in the group with homocystinuria did not correlate with serum homocysteine or homocysteic acid concentrations. Our data clearly indicate that the disturbed cross-linking hypothesis still holds and that the bone manifestations of homocystinuria are not due to deficient collagen synthesis. PMID- 8611655 TI - Isolation of up- and down-regulated cDNAs associated with hepatocellular carcinoma by a subtraction-enhanced display technique. AB - Identification of gene products exclusively or abundantly expressed in hepatocellular carcinoma (HCC) and in normal liver may yield novel tumor markers. We have isolated 36 up- and down-regulated cDNAs from diethylnitrosamine-induced rat hepatocellular carcinoma and normal liver tissue by using the subtraction enhanced display technique. Nucleotide sequence analysis revealed that the majority of 20 subtraction-enriched cDNA fragments were well-characterized oncogenes and tumor-associated genes, like c-myc, alpha-prothymosin, p21, glutathione-S transferase (G-ST) and alpha 1-acid glycoprotein (AGP). As demonstrated by Northern blot detection, all of them were preferentially expressed either in HCC or in normal liver (2- to 7-fold). As paradigm, G-ST and AGP were shown to be exclusively overexpressed in tumor nodules by in situ hybridization. In addition, 14 of the remaining 16 novel genes were analysed on Northern blot, 10 of which were differentially expressed in HCC. PMID- 8611656 TI - Glycation-induced inactivation of malate dehydrogenase protection by aspirin and a lens molecular chaperone, alpha-crystallin. AB - Non-enzymic glycosylation (glycation) of structural proteins has been widely studied as a possible mechanism in the long-term complications of diabetes. Here we show that glycation inactivates malate dehydrogenase. Aspirin affords some protection against the glycation, but alpha-crystallin, a lens protein which appears to act as a molecular chaperone in other systems, is much more effective. For example, 5 mM glucose completely inactivates malate dehydrogenase in four days, and 5 micrograms alpha-crystallin/ml provides complete protection against this inactivation. Fructose, a superior glycating agent, inactivates the enzyme in 24 hours but even so the same low concentration of alpha-crystallin is able to protect 80% of the activity. Other proteins provide no protection at the same concentration. The inactivation of malate dehydrogenase and other enzymes by glycation could play a role in diabetic complications, and molecular chaperones like alpha-crystallin could serve to protect them. PMID- 8611657 TI - Human eosinophils lack human leukocyte elastase. AB - Recently, human leukocyte elastase has been detected in human eosinophils. Reinvestigating these findings, 2.5 pg active human leukocyte elastase (E.C. 3.4.21.37) were found per neutrophil isolated from peripheral blood, whereas the elastase activity of eosinophil preparations was linearly correlated with the content of contaminating neutrophils. Also spontaneous or stimulated release of active elastase was absent in eosinophils. By immunohistochemistry no elastase immunoreactivity could be demonstrated in human eosinophils. Therefore, we conclude that human eosinophils do not contain considerable amounts of human leukocyte elastase. PMID- 8611658 TI - The alpha / beta and alpha 2 / alpha 1-globin mRNA ratios in different forms of alpha-thalassemia. AB - The present study provides information about the alpha / beta and alpha 2 / alpha 1-mRNA ratios in reticulocytes of normal adults and individuals with different alpha-globin gene deficiencies; it found its origin in analytical data of blood samples from a Laotian couple and their newborn baby. The father carried the 4.2 kb deletion on one chromosome and a TAA --> CAA mutation at the terminating codon of the alpha 2 gene (Hb Constant Spring or CS) on the other chromosome. The mother had the 3.7 kb deletion on one chromosome and a TA A --> TAT mutation at the terminating codon of the alpha 2-globin gene (Hb Pakse) of the second chromosome. The baby was a compound heterozygote for the two termination codon mutations. The mRNA data for this family were compared to those for persons with several well-defined alpha-globin gene deficiencies. The results confirm the importance of the alpha 2 alpha 1-mRNA for the synthesis of alpha chains in alpha thalassemia-2 homozygotes (-alpha/-alpha) and in patients with Hb H disease due to the deletion of three alpha-globin genes (-alpha/--). Furthermore, the MRNA production of the alpha 1-globin gene on the chromosome with the alpha CS mutation (alpha CS alpha) is only one-half of that by the alpha 2 alpha 1-globin gene of a chromosome with a 3.7 or 4.2 kb deletion, explaining the greater severity of, and higher Hb H level in Hb H patients with the alpha CS alpha condition (alpha CS alpha/--) as compared to those with the three gene deletion ( alpha/--). The methodology could be useful as a preliminary screening for the presence of point mutations leading to the functional loss of a single alpha globin gene, provided common deletional alleles have been excluded. PMID- 8611659 TI - Increase of lipid peroxidation in rat liver microsomes by dehydroepiandrosterone feeding. AB - Oral administration of the adrenal steroid dehydroepiandrosterone (DHEA), a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in microsomes isolated from rat liver and kidney cortex, but not from brain. The increase of liver microsomal lipid peroxidation was greater in male than in female rats. the effect of DHEA on lipid peroxidation became discernible after feeding steroid-containing diet (0.6%) to male and female rats for 2 and 3 days and reached maximal levels at 1 and 2 weeks, respectively. The increase of microsomal lipid peroxidation reached a plateau stimulation at 0.05% in the diet. The addition of DHEA in the concentration range 0.1-100 microM to microsomes isolated from control rats had no effect on lipid peroxidation. Furthermore, a significant increase of the endogenous concentration of thiobarbituric acid reactive substances was found in microsomes after DHEA-administration at 0.05% in the diet. These results provide in vivo evidence that DHEA can cause lipid peroxidation in rat liver. Administration of DHEA at 0.6% in the diet for 7 consecutive days also significantly enhanced NADH- and ascorbate-dependent lipid peroxidation in liver microsomes. The DHEA-stimulated rat liver microsomal lipid peroxidation was completely inhibited by EDTA but not by superoxide dismutase, catalase or mannitol applied as OH-radical scavenger. The findings indicate that membrane lipid peroxidation is an early effect of DHEA, and that this process may be involved in the steroid-induced carcinogenesis in rats. PMID- 8611660 TI - Subunit specific monoclonal antibodies show different steady-state levels of various cytochrome-c oxidase subunits in chronic progressive external ophthalmoplegia. AB - Monoclonal antibodies recognizing the mitochondrially encoded subunits I and II, and the nuclear-encoded subunits IV, Va, Vb and VIc of human cytochrome-c oxidase were generated. These antibodies are highly specific and allow the assessment of subunit steady-state levels in crude cell extracts and tissue sections. In the experimental human cell line 143B206, which is devoid of mitochondrial DNA, immunovisualization with the antibodies revealed that the nuclear-encoded subunits IV and Va were present in amounts close to that of the parental cell line despite the absence of the mitochondrially encoded subunits. In contrast, the nuclear-encoded subunits Vb and VIc were severely reduced in cell line 143B206, suggesting that unassembled nuclear-encoded subunits are degraded at different rates. In skeletal muscle sections of a patient with chronic progressive external ophthalmoplegia known to harbor the 'common deletion' in a subpopulation of her mitochondrial DNA, most cytochrome-c oxidase activity negative fibers had greatly reduced levels of subunits I, II, Va, Vb and VIc of cytochrome-c oxidase. The steady-state level of subunit IV, however, was less affected. This was particularly evident in cytochrome-c oxidase activity negative fibers with accumulated mitochondria ('ragged-red' fibers) where immunodetection with anti-subunit IV resulted in intense staining. The data presented in this paper demonstrate that the battery of monoclonal antibodies can be employed for diagnostic purposes to analyze steady-state levels of mitochondrially and nuclear encoded subunits of cytochrome-c oxidase. PMID- 8611661 TI - Abnormalities in hepatic fatty-acid metabolism in a surfactant/influenza B virus mouse model for acute encephalopathy. AB - Abnormalities in fatty-acid metabolism are believed to play a role in nonspecific acute encephalopathy (AE) with hepatomegaly, although the specific nature of these abnormalities and their temporal relationship to the pathology are not well defined. We have examined hepatic fatty-acid beta-oxidation and metabolism in a mouse model for AE in which neonatal mice were exposed dermally to nontoxic doses of the industrial surfactant, Toximul MP8 (Tox), daily from days 1 to 12 after birth, and then infected with a sublethal dose (LD30) of mouse-adapted human influenza B (Lee) virus (FluB). The number of deaths in the group treated with Tox + FluB were significantly higher than those in the group infected with virus alone. Under optimal in vitro assay conditions, beta-oxidation of [1-14C]palmitic acid was approximately 15% higher in liver homogenates from mice painted with Tox for 12 days (P < 0.02); catabolism of [1-14C]octanoic acid to 14C-labelled water soluble products (14C-WSP) and 14CO2 was unaltered by Tox. Infecting Tox-free mice with FluB inhibited beta-oxidation of both [1-14C]palmitate and [1 14C]octanoate by 20-30% (P < 0.001). On days 18-19, when most Tox + FluB dependent deaths occurred, the inhibition of oxidation was increased to approximately 50% in mice given the combined treatment. Treatment of the mice with Tox/FluB also altered the pattern of incorporation of fatty acids into complex lipids. Hepatic levels of thiobarbituric acid reactive substance (TBARS), a marker for lipid peroxides, were approximately 15% higher in Tox-painted than in control mice (P < 0.01); FluB alone had no effect. In Tox + FluB-treated animals, TBARS levels were > 2-fold higher than in any other experimental group (P < 0.001). These studies demonstrated that nasally-administered FluB has profound effects on hepatic fatty-acid metabolism, particularly beta-oxidation. Exacerbation of this and related effects by exposing young animals to xenobiotic surfactants could be the basis of surfactant-mediated potentiation of virus induced mortality. PMID- 8611662 TI - Effects of HMG-CoA reductase inhibitors on growth and differentiation of cultured rat skeletal muscle cells. AB - HMG-CoA reductase inhibitors have been associated with skeletal muscle myopathy, ranging from asymptomatic elevations of serum creatine kinase (CK) activity to rhabdomyolysis. In this study, we assessed the effects of addition of different concentrations of simvastatin and pravastatin on growth and differentiation of cultured primary rat skeletal muscle cells. Protein concentrations, CK activity and percentage CK-MM, which is a parameter for maturation, were determined. Effects were generally stronger if inhibitors were added to both growth and differentiation medium rather than only to differentiation medium. Addition of 25 microM pravastatin caused only a decrease of CK activity. Addition of 1-5 microM simvastatin resulted in a decrease of protein concentration, CK activity and percentage CK-MM, whereas 25 microM simvastatin resulted in cell death. Addition of mevalonic acid or cholesterol could not prevent the effects of 1 microM simvastatin. In addition, 1 microM simvastatin did not influence the cholesterol and phospholipid content of the cells. Superfusion of cultured cells with simvastatin concentrations of 10 microM and higher caused a transient increase of the cytoplasmic calcium concentration followed by an apparent second rise and cell puncture. The results indicate that HMG-CoA reductase inhibitors may affect skeletal muscle cell regeneration in vivo by a direct toxic effect on growth and differentiation. PMID- 8611663 TI - Changes of ionized magnesium and free fatty acids in serum after acute myocardial infarction. AB - The most feared early complications after an acute myocardial infarction are ventricular arrhythmias. These may be initiated by changed concentrations of catecholamines and electrolytes. The present study shows a reduction of total serum magnesium after acute myocardial infarction which is normalized within a few days. Further, it could be shown that a more significant decrease of ionized Mg2+ (iMg2+) takes place at the day of acute myocardial infarction in the total group of myocardial infarction patients (n = 36). A closer investigation reveals that iMg2+ was considerably decreased in one third of the patients, whereas two thirds showed minor changes of iMg2+ in both directions. The pronounced decrease of iMg2+ in the first sub-group can be explained by the time course of free fatty acids in serum. On the day of the myocardial infarction free fatty acids in serum were increased. This is probably caused by beta-adrenergic-induced lipolysis due to catecholamines released by the stressful situation of an acute myocardial infarction. The increased free fatty acids in serum bind Mg2+, thus reducing iMg2+. As long as a beneficial effect of a general Mg infusion in all acute myocardial infarction patients is controversial, iMg2+ should be measured and Mg infusion therapy should be applied only in patients with low iMg2+. PMID- 8611664 TI - Neopterin and beta 2-microglobulin as serum markers in a placebo-controlled anti HIV therapy trial. AB - The purpose of this study was to evaluate the use of the biologic immune activation markers neopterin and beta 2-microglobulin in monitoring human immunodeficiency virus (HIV)-positive patients without acquired immunodeficiency syndrome (AIDS) treated with isoprinosine and placebo. Serum samples obtained at the commencement of study and samples obtained after 24 weeks were available from 277 HIV-positive patients in the Scandinavian multicentre isoprinosine trial. After 24 weeks' treatment, the concentrations of beta 2-microglobulin and neopterin had increased both in the isoprinosine group and the placebo group. However, in the isoprinosine group the relative increase within beta 2 microglobulin was significantly smaller. Within neopterin, the increase from baseline level was small and not significantly different from the change in the placebo group. The beta 2-microglobulin data might reflect a suppressive effect of isoprinosine on the HIV-induced activation of the cellular immune system. Because of the minor changes, there is no real evidence of neopterin and beta 2 microglobulin being valuable as surrogate markers in monitoring therapy effects of isoprinosine. PMID- 8611665 TI - Estimation of the epidermal growth factor receptor by the hydroxyapatite method in human breast cancer. AB - Results of epidermal growth factor (EGF) receptor in human breast tumours show large variations, mainly due to the lack of standardization of the assays. Our EGF receptor values are higher than those reported previously which may be due to the use of the hydroxyapatite to separate bound and non-bound ligand in a radioligand assay. We found EGF receptors in 58% (103/178) of the tumours (EGF receptor levels: 3 to 625 fmol/mg of membrane protein, mean = 33.3, median = 17.4), with a median Kd of 0.642 nmol/l. There was an inverse correlation between EGF receptors and estrogen receptors (r = -0.215, p = 0.00002, Kendall correlation). There was a difference between EGF receptor content in grade II (mean = 16.9) and grade III tumours (mean = 59.3) (p = 0.027), but not between histopathological types and lymph node status. The relevance of EGF receptor largely depends on the reliability of its determination. The standardized EORTC methodology is a reproducible alternative which will expand EGF receptor determination and permit comparability of data between laboratories. PMID- 8611666 TI - Autoregulation of actin synthesis by physiological alterations of the G-actin level in hepatocytes. AB - Hypotonic treatment of cultured rat hepatocytes significantly decreased the monomeric G-actin level by 18% after 120 min while the level of filamentous F actin remained essentially unchanged. Simultaneously the level of cellular actin mRNA was increased by 53%. Incubation of hepatocytes for 120 min with the F-actin stabilizing toxin phalloidin from Amanita phalloides led to a decrease of G-actin by 70% and an increase of F-actin by 55%. Although the toxin dependent decrease of G-actin was much more pronounced than the decrease after hypotonic treatment, the increase of actin mRNA was similar under both conditions. Simultaneous treatment with hypotonic medium did not result in a further decrease of the G actin level. On the other hand, the G-actin elevating C2 toxin from Clostridium botulinum completely blocked the effects of osmotic stress on G-actin and actin mRNA content. The results demonstrate that already an essentially physiological decrease of G-actin without alterations of F-actin results in a substantial enhancement of the actin mRNA level, indicating the physiological significance of this autoregulation. PMID- 8611667 TI - Urinary N-acetyl-beta-D-glucosaminidase determination in newborns and children: methods and diagnostic applications. AB - More recently numerous papers have been written on the successful paediatric applications of some well-established urinary N-acetyl-beta-D-glucosaminidase assays by several authors. Regarding these developments we tried to collect and summarize all the available literature data published in this field thus far and, in addition, to encourage and promote the wider utilization of these effective methods for the diagnosis of very different primary and secondary renal damages. Besides the chemical and biochemical background of the assays the pathological conditions and medical treatments which may lead to tubulopathy and, therefore, to a significantly elevated urinary enzyme excretion are also discussed. PMID- 8611668 TI - Study of the stability of creatine kinase in control materials. AB - We examined the stability of twelve control materials from different suppliers containing creatine kinase. The stability was assessed storing preparations at 4 degrees C, 27 degrees C and 37 degrees C and periodically measuring creatine kinase catalytic concentration. The activity was significantly greater when the lyophilized material was reconstituted with water at 4 degrees C as compared to 27 degrees C and 37 degrees C. All preparations tested showed good stability at 4 degrees C over a 72 hour period. We considered the suitability of tested control materials for quality-control purposes. PMID- 8611669 TI - Use of azure-D2 for the measurement of uric acid in serum. AB - It has been suggested that achieving a chromogenic endpoint with an absorbance read at 600 nm or greater will reduce the degree of spectral interference in many colorimetric methods. We have examined a uricase/peroxidase-based system utilising a novel oxygen acceptor (azure-D2) as chromogen which produces a chromophor with an absorbance which can be measured at 600 nm (Synermed). Results (median, range, mumol/l) obtained on patient sera (n = 113) using the Synermed method (297; 38-847) were lower than those obtained using a 293 nm uricase method (Du Pont Ltd., 312; 62-874) (p < 0.001, Synermed = -16.709 + 1.0065 Du Pont). Within- and between-batch CV's were < 3% in all cases. Results obtained in one external quality assessment scheme (WEQAS) were significantly lower (p < 0.001) than the method group mean (Synermed = -17.298 + 1.0056 WEQAS) but in a second scheme (NEQAS) results did not differ significantly (p > 0.05) from the method group mean (Synermed = -29.315 + 1.0570 NEQAS). Bilirubin had a negative effect (p < 0.0001; 300 mumol/l producing a 23 mumol/l reduction in uric acid) and haemoglobin had a small positive effect (p < 0.05; 5 g/l increasing uric acid by 8 mumol/l) on the assay. Lipaemia did not interfere (p > 0.05) but both ascorbic acid (100 mumol/l reducing uric acid by 68 mumol/l) and N-acetylcysteine (3 mmol/l reducing uric acid by 95 mumol/l) had significant negative effects (p < 0.0001 in both cases). Uraemic serum had no effect on the assay (p > 0.05) but serum storage for 72 hours at room temperature resulted in a significant (p < 0.0005) increase in measured uric acid. The Synermed method is a precise and accurate assay for serum uric acid. However, although generally showing low levels of spectral interference, chemical interferences in the assay from antioxidant components of serum may be problematic. This paper shows that the use of longer wavelengths of detection can reduce the significance of common spectral interferences. PMID- 8611672 TI - Application of IUPAC-IFCC recommendations on quantities and units to WHO biological reference materials for diagnostic use. International Union of Pure and Applied Chemistry (IUPAC) and International Federation of Clinical Chemistry (IFCC). AB - This document deals with the nature of WHO biological reference materials, their development for the control of therapeutic substances and recommendations to improve their application in diagnosis. The nature of international units specified by WHO biological reference materials is contrasted with that of SI units, and the method for assigning values in international units to such reference materials is described. The document recommends the use of SI units (mole) with existing and proposed WHO biological reference materials whenever the elementary entity of the stated component can be recognized. It also recommends that the description of quantities having no recognized kind-of-quantity with a definable dimension should be clearly distinguished by the term "arbitrary" and include a reference to the procedure and calibrator used. The WHO is urged to involve appropriate non-governmental organizations in advising on the need for, and the suitability of international reference materials. PMID- 8611670 TI - Comparative study of four extraction procedures for urokinase type plasminogen activator and plasminogen activator inhibitor-1 in breast cancer tissues. AB - The urokinase type plasminogen activator (u-PA) and the plasminogen activator inhibitor-1 (PAI-1) are among the best second-generation prognostic tissue factors in breast cancer. However, different extraction procedures and assay kits are used in different laboratories. A total of 79 breast tumour tissues stored in liquid nitrogen were analysed in this study. We compared u-PA and PAI-1 levels determined with the American Diagnostics (AD) kit after various extraction procedures. The median cytosolic extraction yield in the presence of 0.4 mol/l KCl, calculated relative to extraction in the presence of 10 ml/l Triton X100 when adapted to standard laboratory working hours (incubation for 2 h instead of 12 h) was 74.4% for u-PA and 85.8% for PAI-1. In addition, the correlations were acceptable. Cytosolic extracts prepared with KCl could permit optimal u-PA and PAI-1 assays while also enabling hormone receptors to be determined with the same specimens. Further studies with clinical data are now necessary to determine the prognostic relevance of this extraction procedure. PMID- 8611671 TI - Thyroid testing using the Cobas Core immunoassay system. A multicentre study. AB - The random access immunoanalyzer Cobas Core and the Cobas Core Thyroid Assays were assessed as to their clinical usefulness in the analysis of different thyroid diseases. Four centres participated in this study measuring the following five thyroid tests per sample on the instrument: thyrotropin, free thyroxine, thyroxine, triiodothyronine and free triiodothyronine. The assessment was based on studies of precision and clinical samples. Within- and between-series precisions showed a mean CV over all assays of 4.3 and 6.1%, respectively. Comparison of the test results with clinical data demonstrated that the Cobas Core results are in accordance with diagnosed thyroid diseases. A good discrimination between normal and disease status and between untreated and treated status was found. Furthermore, 370 sera measured with Technicon Immuno-1 TSH and FT4 assays were compared to the respective Cobas Core EIAs). A good correlation between the assays was demonstrated. PMID- 8611673 TI - Properties and units in the clinical laboratory sciences. I. Syntax and semantic rules (recommendation 1995). International Union of Pure and Applied Chemistry (IUPAC) and International Federation of Clinical Chemistry (IFCC). PMID- 8611674 TI - International Society on Thrombosis and Haemostasis (ISTH), and International Union of Pure and Applied Chemistry (IUPAC), Clinical Properties and units in the clinical laboratory sciences. V. Properties and units in thrombosis and haemostasis (recommendation 1995). International Society on Thrombosis and Haemostasis (ISTH) and International Union of Pure and Applied Chemistry (IUPAC),and International Federation of Clinical Chemistry (IFCC). PMID- 8611675 TI - Inherited thrombophilia: pathogenesis, clinical syndromes, and management. PMID- 8611676 TI - Single-chain urokinase-type plasminogen activator bound to its receptor is relatively resistant to plasminogen activator inhibitor type 1. AB - Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule to its cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affinity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of scuPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor bound tcuPA. Binding of PaI-1 to suPAR/scuPA complexes is totally reversible and can be overcome by increasing the concentration of plasminogen, suggesting a competitive mechanism of inhibition (Ki = 18 nmol/L). Binding of scuPA to suPAR also retards its cleavage by plasmin. These results indicates that binding of single-chain urokinase to its receptor promotes its activity, retards its inhibition, and protects it from conversion to a two-chain form of the enzyme, a step that may precede its inactivation and clearance from cell surfaces. These results are consistent with a physiologic role for receptor-bound single-chain urokinase as a cellular plasminogen activator. PMID- 8611677 TI - CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein. AB - CD34 is expressed on the surface of hematopoietic stem/progenitor cells, stromal cells, and on the surface of high-endothelial venules (HEV). CD34 binds L selectin, an adhesion molecule important for leukocyte rolling on venules and lymphocyte homing to peripheral lymph nodes (PLN). We generated CD34-deficient mutant animals through the use of homologous recombination. Wild-type and mutant animals showed no differences in lymphocyte binding to PLN HEV, in leukocyte rolling on venules or homing to PLN, in neutrophil extravasation into peritoneum in response to inflammatory stimulus, nor in delayed type hypersensitivity. Anti L-selectin monoclonal antibody (MEL-14) also inhibited these immune responses similarly in both CD34-deficient and wild-type mice. However, eosinophil accumulation in the lung after inhalation of a model allergen, ovalbumin, is several-fold lower in mutant mice. We found no abnormalities in hematopoiesis in adult mice and interactions between mutant progenitor cells and a stromal cell line in vitro were normal. No differences existed in the recovery of progenitor cells after 5-fluorouracil treatment, nor in the mobilization of progenitor cells after granulocyte colony-stimulating factor treatment compared with wild-type animals. Surprisingly, although CD34 was not expressed in these mice, a portion of its 90-kD band crossreactive with MECA79 remained after Western blot. Thus, we have identified an additional molecule(s) that might be involved in leukocyte trafficking. These results indicate that CD34 plays an important role in eosinophil trafficking into the lung. PMID- 8611678 TI - Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38- cells and maintains progenitor cells in vitro. AB - Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with interleukin-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( < or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (35 to 60 days) and extended long-term culture initiating cell (ELTC-IC) period ( > 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P < .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P < .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro. PMID- 8611679 TI - Factor V Quebec revisited. AB - Factor V Quebec has been described as a bleeding disorder that exhibits an autosomal dominant inheritance pattern and presents severe bleeding after trauma. Two members of a fourth-generation (IV.13 and IV.15) Canadian family have been studied in detail and are the subject of this report. Their clinical presentations and histories have been described previously (Tracy et al: J Clin Invest 74:1221, 1984). Persistent abnormalities include mild thrombocytopenia and defective platelet factor V. Plasma factor V is present at near normal concentration and is fully functional. Thus, the bleeding diathesis appears to reflect the absence of platelet factor V activity. The recent report (Hayward et al: Blood 84:110a, 1994 [suppl, abstr]) of multimerin deficiency in these individuals led us to reevaluate these patients. Western blot analyses of platelet lysates developed with a variety of monoclonal antibodies show that the alpha-granule proteins, fibrinogen, von Willebrand factor, factor V and osteonectin are decreased in concentration and significantly degraded in the platelets of these patients. Thrombospondin, while not degraded, is substantially decreased. In contrast, platelet factor 4 and beta-thromboglobulin do not appear to be affected. These observations suggest that the alpha-granules are correctly assembled but the contents are subsequently subjected to proteolytic degradation. The results indicate that factor V Quebec disorder is probably associated with a generalized defect that leads to degradation of most proteins of the alpha granules. PMID- 8611680 TI - Molecular definition of a narrow interval at 7q22.1 associated with myelodysplasia. AB - Chromosome 7 translocations, deletions, or monosomy are associated with myelodysplasia (MDS) and acute myeloid leukemia both in children and adults. These chromosomal anomalies represent one of the most common cytogenetic abnormalities associated with these diseases and usually herald a poor prognosis. In this study two cosmid DNA probes that mapped to 7q22.1 and were known to be separated by approximately 500 kb were identified to flank the proximal inversion breakpoint in a patient carrying a constitutional inversion (7q22.1-34) associated with MDS. A yeast artificial chromosome (YAC) clone that encompassed the two cosmids was identified and shown to span the breakpoint. Fluorescence in situ hybridization was then used to analyze six additional patients with myelodysplasia and chromosomal rearrangements of the 7q22 region (three patients had translocations and three carried deletions). The breakpoint in one of the patients was found to be contained within the same YAC clone that spanned the inversion breakpoint. Moreover, this same interval was determined to be absent in all three patients with chromosomal deletions. These results suggest that this segment of DNA on chromosome 7q22.1 may contain specific gene(s) that have a significant role in myeloid malignancies. PMID- 8611681 TI - Specific human cellular immunity to bcr-abl oncogene-derived peptides. AB - Chronic myelogenous leukemia (CML) cells are characterized by a t(9;22) translocation, which can encode one of two chimeric P210 bcr-abl fusion proteins, comprising products of either the b2a2 or the b3a2 exon junction. The junctional sequences represent potentially immunogenic tumor-specific antigens. Despite their intracellular location, the fusion proteins might be recognized immunologically by T lymphocytes if peptides, derived from these unique sequences, are capable of presentation by the major histocompatibility complex molecules. We previously found that four peptides, 9 to 11 amino acids long, spanning the b3a2 CML breakpoint bind with high or intermediate affinity to purified HLA class I molecules A3, A11, B8, or both A3 and A11. We tested the ability of these peptides to elicit specific class I restricted cytotoxic T lymphocytes (CTLs) in vitro in HLA-matched healthy donors. In addition, a longer b3a2 CML-breakpoint-derived peptide, 25 aminoacids in length (b3a2-25), was studied for its ability to induce peptide-specific, class II-mediated, T-cell proliferation. In four of four HLA-A3 donors tested, CML-A3/A11-peptide specific CTLs were induced that killed an allogeneic HLA-A3-matched peptide pulsed leukemia cell line. In two of three HLA-A3 donors, the CML-A3/A11 peptide was able to induce killing of autologous and allogeneic HLA-matched peptide-pulsed peripheral blood mononuclear cells (PBMC). CML-A3 peptide induced peptide specific CTLs in one of the four HLA A3 donors tested. No killing was observed in two HLA-B8 and two HLA-A11 donors. PBMC from seven donors were also tested for anti b3a2-25 peptide proliferation in a thymidine incorporation assay. Specific proliferation was detected in three donors, all of the HLA-DR11 haplotype. These data represent the first evidence of a cytolytic human immune response against CML bcr-abl oncogene-derived peptides and provide a rationale for developing peptide-based vaccines for this disease. PMID- 8611682 TI - FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo. AB - Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo. PMID- 8611683 TI - Glycosylphosphatidylinositol-anchor-deficient mice: implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria. AB - Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis. Abnormal hematopoietic cells from patients with PNH are deficient in glycosylphosphatidylinositol (GPI) anchored proteins and clonally dominate various hematopoietic lineages in the bone marrow and the peripheral blood. Analysis of many patients with PNH has showed that somatic mutation in the X-linked gene PIG-A is responsible for the GPI-anchor deficiency in PNH. The PIG-A mutation must also be relevant to the clonal dominance of GPI-anchor deficient (GPI-) blood cells because two or more PIG-A mutant clones become dominant in many patients. However, whether the PIG-A mutation alone is sufficient for clonal dominance is not known. To address this question, we generated chimeric mice using Pig-a (the murine homologue of PIG-A) disrupted embryonic stem (ES) cells, in which the animals are chimeric with respect to the surface expression of GPI-anchored proteins. The chimerism of hematopoietic and nonhematopoietic tissues in such mice was always low, suggesting that the higher contribution of Pig-a disrupted GPI- cells had a lethal effect on the chimera. GPI- cells appeared in the peripheral blood of some of the chimeric mice. However, the percentage of GPI- erythrocytes did not increase for 10 months after birth, implying that the Pig-a mutation alone does not immediately cause the clonal dominance of GPI- blood cells; another pathologic or physiologic change(s) in the hematopoietic environments or in the clone itself may be necessary. PMID- 8611684 TI - A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. AB - Thrombocytopenia is a complication of cancer treatment that can limit dose intensity. Interleukin-11 (IL-11) is a growth factor that increases platelet production. We conducted a multicenter, randomized, placebo-controlled trial of recombinant human IL-11 (rhIL-11) in 93 patients with cancer who had already been transfused platelets for severe thrombocytopenia resulting from chemotherapy. The patients had received platelet transfusions for nadir platelet counts of < or = 20,000/microL during the chemotherapy cycle immediately preceding study entry. Chemotherapy was continued during the study without dose reduction. Patients were randomized to receive placebo or rhIL-11 at 50 or 25 micrograms/kg subcutaneously once daily for 14 to 21 days beginning 1 day after chemotherapy. Eight of 27 (30%) evaluable patients treated with rhIL-11 at a dose of 50 micrograms/kg did not require platelet transfusions versus 1 of 27 (4%) patients who received placebo (P < .05). Five of 23 (18%) patients treated with rhIL-11 at 25 micrograms/kg avoided platelet transfusions (P = .23). Side effects were fatigue and cardiovascular symptoms, including a low incidence of atrial arrhythmias and syncope. There were no differences among treatment groups in the incidence of neutropenic fever, days of hospitalization, or number of red blood cell transfusions. This study shows that rhIL-11 treatment of a dose of 50 micrograms/kg significantly increases the likelihood that patients who have already been transfused platelets for severe chemotherapy-induced thrombocytopenia will not require platelet transfusions during a subsequent chemotherapy cycle. PMID- 8611685 TI - A phase I trial of recombinant human interleukin-11 (neumega rhIL-11 growth factor) in women with breast cancer receiving chemotherapy. AB - We performed a phase I trial of recombinant human interleukin-11 (rhIL-11) in women with breast cancer. Cohorts of three to five women were accrued to five dosage levels of rhIL-11 (10, 25, 50, 75, and 100 micrograms/kg/d). rhIL-11 alone was administered by a daily subcutaneous injection for 14 days during a 28-day prechemotherapy "cycle 0." Patients (pts) subsequently received up to four 28-day cycles of cyclophosphamide (1,500 mg/m2) and doxorubicin (60 mg/m2) chemotherapy followed by rhIL-11 at their assigned dose (days 3 through 14). Sixteen pts (13 stage IV, 3 stage IIIB) were accrued to this study. Median age was 53 years and median Eastern Cooperative Oncology Group Performance Status was 0. A grade 3 neurologic event was seen in 1 pt at 100 micrograms/kg. Because of the degree of grade 2 constitutional symptoms (myalgias/arthralgias and fatigue) at 75 micrograms/kg, dose escalation was stopped and 75 micrograms/kg was the maximally tolerated dose. No other grade 3 or 4 adverse events related to rhIL-11 were seen. The administration of rhIL-11 was not associated with fever. Reversible grade 2 fatigue and myalgias/arthralgias were seen in all pts at 75 micrograms/kg. Weight gain of 3% to 5% associated with edema was seen at doses > 10 micrograms/kg but a capillary leak syndrome was not seen. rhIL-11 alone was associated with a mean 76%, 93%, 108%, and 185% increase in platelet counts at doses of 10, 25, 50, and 75 micrograms/kg, respectively. No significant changes in leukocytes were seen. A mean 19% decrease in hematocrit was observed. Acute phase proteins increased with treatment at all doses. Compared with patients at the 10 micrograms/kg dose, patients receiving doses > or = 25 micrograms/kg experienced less thrombocytopenia in the first two cycles of chemotherapy. We conclude that rhIL-11 has thrombopoietic activity at all doses studied, is well tolerated at doses of 10, 25, and 50 micrograms/kg, and at doses > or = 25 micrograms/kg has the potential to reduce chemotherapy-induced thrombocytopenia in this model. PMID- 8611686 TI - Second malignancies after treatment for laparotomy staged IA-IIIB Hodgkin's disease: long-term analysis of risk factors and outcome. AB - The survival of patients with Hodgkin's disease has dramatically improved over the past 30 years because of advances in treatment. However, concern for the risk of long-term complications has resulted in a number of trials to evaluate reduction of therapy. The consequences of these trials on recurrence, development of long-term complications, and survival remain unknown. One major consequence of successful treatment of Hodgkin's disease is the development of second malignant neoplasms. We sought to determine the factors most important for development of second tumors in pathologically staged and treated Hodgkin's disease patients followed for long intervals to provide background information for future clinical trials and guidelines for routine patient follow-up. Between April 1969 and December 1988, 794 patients with laparotomy staged (PS) IA-IIIB Hodgkin's disease were treated with radiation therapy (RT) alone or combined radiation therapy and chemotherapy (CT). There were 8,500 person-years of follow-up (average of 10.7 person-years per patient). Age and gender-specific incidence rates were multiplied by corresponding person-years of observation to obtain expected numbers of events. Observed to expected results were calculated by type of treatment, age at treatment, sex, and time after Hodgkin's disease. Absolute (excess) risk was expressed as number of excess cases per 10,000 person-years. Seventy-two patients have developed a second malignant neoplasm. Eight patients developed acute leukemia, 10 had non-Hodgkin's lymphoma (NHL), and 53 patients developed solid tumors at a median time of 5 years, 7.25 years, and 12.2 years, respectively, after Hodgkin's disease. One patient developed multiple myeloma 16.5 years after Hodgkin's disease. The relative risk (RR) of developing a second malignancy was 5.6. The absolute excess risk per 10,000 person-years (AR) of developing a second malignancy was 69.6 (7.0% excess risk per person per decade of follow-up). The highest RR occurred for the development of leukemia (RR = 66.2), however because of the low expected risk, the AR was only 9.3. The RR of solid tumors after Hodgkin's disease was lower (4.7); however, the AR was greater (49) than for acute leukemia. Among the solid tumors, breast, gastrointestinal, lung, and soft tissue cancers had the highest absolute excess risks. The risk for developing breast cancer after Hodgkin's disease was greatest in women who were under the age of 25 at treatment. The most significant risk factor for the development of both leukemia and solid tumors was the combined use of radiation therapy and chemotherapy. The RR following RT alone was 4.1 (AR = 51.1); for RT + CT (initially or at relapse) the RR was 9.75 (P < 0.05, nonoverlapping confidence limits, AR = 123.9). Survival following development of a second malignancy was poor in patients with leukemia, gastrointestinal tumors, lung cancer, and sarcoma. Survival from other malignancies including NHL and breast cancer was more encouraging. Second malignant neoplasms are a major cause of late morbidity and mortality following treatment for Hodgkin's disease. The most significant risk factor for the development of second tumors is the extent of treatment for Hodgkin's disease. Recommendations are presented for both prevention and early detection of these tumors. PMID- 8611687 TI - Malignant neoplasms following bone marrow transplantation. AB - We undertook an analysis of 2,150 recipients of bone marrow transplant (BMT) at the University of Minnesota to determine the incidence of post-BMT malignant neoplasms (MNs). Fifty-one patients developed 53 MNs, compared with 4.3 expected from general population rates (standardized incidence ratio [SIR], 11.6, 95% confidence interval [CI], 8.2-14.5). These included 22 occurrences of B-cell lymphoproliferative disorder (BLPD), 17 solid nonhematopoietic tumors, 10 myelodysplastic syndromes (MDS), 1 acute myelogenous leukemia (AML), 2 non Hodgkin's lymphoma (NHL), and 1 Hodgkin's disease (HD). The estimated actuarial incidence of any post-BMT malignancy was 9.9% +/- 2.3% at 13 years posttransplant. The cumulative probability of BLPD plateaued at 1.6% +/- 0.3% by 4 years from transplant and factors independently associated with increased risk included in vitro T-cell depletion of marrow (relative risk (RR) = 11.9, P < .001), HLA mismatch (RR = 8.9, P < .001), use of antithymocyte globulin (ATG) for graft versus host disease (GVHD) prophylaxis (RR = 5.9, P < .001) or in the preparative regimen (RR = 3.1, P = .03) and primary immunodeficiency (RR = 2.5, P = .06). The cumulative probability of developing solid malignancy was 5.6% +/- 2.2% at 13 years from BMT. Malignant melanomas were the most common (SIR, 10.3, 95% CI 1.9 to 25.4). The actuarial incidence of MDS/AML plateaued at 2.1% +/- 0.8% at 9 years and was seen most often in older patients receiving autologous peripheral blood stem cells for HD or NHL. These data document that BMT recipients are at an increased risk of later malignancy, which may add significant morbidity and mortality to the transplant process. Methods for screening and identification of individuals at increased risk need to be addressed in future studies. PMID- 8611688 TI - Radioimmunotherapy of relapsed B-cell lymphoma with yttrium 90 anti-idiotype monoclonal antibodies. AB - Tumor-specific anti-idiotype (anti-Id) monoclonal antibodies (MoAbs) to B-cell lymphomas have been administered to patients, resulting in significant clinical responses. However, clinical responses have been limited by the emergence of Id negative lymphoma. To overcome the problem of tumor heterogeneity, we conducted a pilot evaluation of the safety and effectiveness of yttrium 90 (90Y)-labeled anti Id and shared Id (sId) MoAbs in non-Hodgkin's B-cell lymphoma. Nine patients with relapsed B-cell lymphoma in whom tumor was successfully targeted with 111In labeled anti-Id MoAb were treated with 90Y-labeled anti-Id MoAb. A total of 19 courses (one to four per patient) were administered using 1,000 to 2,320 mg unlabeled clearing MoAb and 10 to 54 mCi 90Y MoAb per patient. Two of nine patients had a complete response, one a partial response, three stable disease, and three disease progression. Time to progression varied from 1 to 12 months. Toxicities were predominately hematologic, and only one patient developed infection and required transfusion. At progression, three of five assessable patients had Id-positive lymphoma and two had Id-negative lymphoma. Human antimouse antibodies (HAMA) did not develop in the patients after treatment. 90Y anti-Id MoAbs demonstrated excellent in vivo stability, produced significantly tumor regression in three of nine patients, exhibited acceptable toxicities, and elicited no HAMA formation. Further investigation of repetitive, low-dose 90Y anti-Id and MoAb therapy is warranted; however, the advantages of a pan B MoAb may prove the latter to be the agent of choice for the radio immunotherapy of B cell lymphoma. PMID- 8611689 TI - Alterations in tretinoin pharmacokinetics following administration of liposomal all-trans retinoic acid. AB - We administered liposome-encapsulated all-trans retinoic acid (L-ATRA) to 48 patients with refractory hematologic malignancies using an every-other-day schedule for 28 days and doses of 15 to 175 mg/m2. In 19 patients, pharmacology studies were conducted after the first (day 1) and seventh (day 15) doses. In contrast to the decline in tretinoin concentration seen within 3 to 4 days of administration of daily oral ATRA, there were no differences between the area under the curve (AUC) of tretinoin concentration versus time on day 1 and day 15 (P = .98, Wilcoxon signed-rank test). Peak day 1 concentrations after 15 mg/m2 were higher than those reported after 45 mg/m2 oral ATRA. Six patients with relapsed acute promyelocytic leukemia (APL) were treated. Three, each in first relapse and at least year from the last exposure to oral ATRA, achieved a complete response (CR). Disease recurred in two (one at 3 months despite maintenance L-ATRA and similarity in tretinoin AUC on days 1 and 85, and the other at 5 months, 2 months after discontinuation of L-ATRA) and the third was transplanted 1 month into CR. The three nonresponders were in at least a second relapse and failed to respond to oral ATRA before or immediately after receiving L-ATRA. Severe toxicity developed in three of eight patients treated at 175 mg/m2 (joint pains in two, skin in one). The maximum tolerated dose (MTD) was determined to be 140 mg/m2, at which dose grade 2 toxicity (primarily headache and skin) occurred in eight of eight patients, but grade 3 to 4 toxicity in none. Compared with oral ATRA, L-ATRA apparently results in greater exposure to tretinoin and for a longer time. PMID- 8611690 TI - Signaling pathways activated in a unique mast cell line where interleukin-3 supports survival and stem cell factor is required for a proliferative response. AB - Stem cell factor (SCF) and interleukin-3 (IL-3) both act on several target hematopoietic populations, including mast cells. We have isolated a unique murine mast cell line, B6M, that is phenotypically similar to immature mast cells. For B6M cells, IL-3 is a survival factor and alone does not stimulate proliferation. SCF can stimulate proliferation of B6M cells, and together IL-3 and SCF synergize to stimulate optimal proliferation and long-term growth. A sustained induction of c-myc is observed only in the presence of SCF (with or without IL-3). In B6M cells, both IL-3 and SCF stimulate phosphorylation of Shc and activation of the Ras, Raf-1, MAPK pathway. Interestingly, IL-3 plus SCF synergistically activate MAPK. IL-3, but not SCF, leads to activation of Jak 2 and Stat 5 and induces pim 1 expression. From these data, we suggest that the induction of pim-1 and c-myc is independently regulated. Furthermore, IL-3-stimulated activation of the Jak 2/Stat 5 pathway, induction of pim-1, and activation of the Ras/MAPK pathway are insufficient to mediate proliferation of B6M cells. The unusual IL-3 response of B6M cells provides a useful model to dissect signals required for IL-3-stimulated survival and proliferation. PMID- 8611691 TI - The role of MAP kinase kinase in interleukin-3 stimulation of proliferation. AB - Expression of a dominant interfering mutant of MAP kinase kinase (MAPKK) inhibits interleukin-3 (IL-3) activation of MAP kinase in the murine bone marrow-derived cell line BAF3. This results in an increase in the level of IL-3 required to stimulate cell proliferation and suppress apoptosis. When apoptosis is constitutively inhibited by coexpression of bcl-2, the dominant interfering MAPKK inhibits IL-3 driven cell cycle progression. Thus, MAPKK function is necessary for optimal IL-3 inhibition of apoptosis and optimal IL-3 stimulation of entry into S phase. Expression of a constitutively activated mutant of MAPKK does not replace IL-3, but renders cells able to proliferate in a density-dependent manner. Cell contact is required to allow cell proliferation; such contact can be supplied by cells without activated MAPKK. PMID- 8611692 TI - CD98: a type II transmembrane glycoprotein expressed from the beginning of primitive and definitive hematopoiesis may play a critical role in the development of hematopoietic cells. AB - In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells. PMID- 8611693 TI - JAK2 is associated with the c-kit proto-oncogene product and is phosphorylated in response to stem cell factor. AB - Stem cell factor (SCF) is a hematopoietic growth factor that interacts with the receptor tyrosine kinase, c-kit. We have found that SCF-stimulates rapid and transient tyrosine phosphorylation of JAK2 in human and murine cell lines, as well as in normal human progenitor cells. JAK2 and c-kit were associated in unstimulated cells with further recruitment of JAK2 to the c-kit receptor complex after SCF stimulation. Treatment of cells with JAK2 antisense oligonucleotides resulted in a 46% decrease in SCF-induced proliferation. These data demonstrate that SCF induces tyrosine phosphorylation of JAK2 and suggest that JAK2 is a component of the SCF signal transduction pathway. PMID- 8611695 TI - Macrophage-stimulating protein activates STK receptor tyrosine kinase on osteoclasts and facilitates bone resorption by osteoclast-like cells. AB - Recently we cloned a novel receptor tyrosine kinase, STK. STK belongs to the hepatocyte growth factor receptor family and was identified as the receptor for macrophage-stimulating protein (MSP). STK is expressed on a restricted, macrophage population such as peritoneal macrophages, but not on mononuclear phagocytes of peripheral blood, bone marrow, or alveoli. Using an anti-STK monoclonal antibody, we observed STK expression on multinuclear osteoclast-like cells (OCLs) formed by murine bone marrow cultures in the presence of 1,25 dihydroxyvitamin D3, and interleukin-3. The OCLs expressed both the calcitonin receptor and STK. We also detected STK expression in bone-derived mouse osteoclasts. The addition of MSP to OCLs induced rapid morphologic changes such as cytoplasmic contraction and formation of ruffled border. In addition, MSP caused rapid redistribution of src to the borders of cytoplasm. These phenomena were associated with enhanced bone resorption. MSP caused a threefold increase in pit formation compared with control OCLs. These findings suggest that by involving src kinase, the MSP/STK signal transduction pathway induces rapid cytoskeletal reorganization in osteoclasts and facilitates bone resorption by osteoclasts. PMID- 8611694 TI - Adjacent, cooperative elements form a strong, constitutive enhancer in the human granulocyte-macrophage colony-stimulating factor gene. AB - Both copies of a repeated sequence CATT(A/T), located between bp -53 and -39 in the upstream region of the human GM-CSF gene, are required for mitogen-inducible promoter activity in T lymphocytes. However, the proteins that recognize this region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, and are responsible for its transcriptional regulatory activity, have not been clearly identified. Using transient transfection assays, we demonstrate that a 19 bp oligonucleotide containing the CATT(A/T) repeats has strong constitutive enhancer activity in both T cell and non-T-cell lines, even though GM-CSF is not normally constitutively expressed by these cells. A 12-bp oligonucleotide, containing only the sequence CATTAATCATTT, lacks enhancer activity indicating that the nucleotides surrounding these sequences are critical for this enhancer activity. The sequence TTTCCT, which can bind members of the ets family of transcription factors, is located just 3' of these CATT(A/T) repeats, and mutagenesis of the CCT sequence abolishes (1) the constitutive (and mitogen inducible) enhancer activity of the 19-bp GM-CSF sequences, (2) the responsiveness to transactivation by ets-1, and (3) the ability to specifically bind ets-1 and elf-1 in electrophoretic mobility shift assays (EMSA). We demonstrate that although T cells contain nuclear proteins capable of independently recognizing the ets binding site and the CATT(A/T) repeats in EMSAs, both of these regulatory elements are required for enhancer function. The strong constitutive activity of this 19-bp region suggests that negative regulation of the GM-CSF promoter is critical for the restricted expression pattern of GM-CSF mRNA. PMID- 8611696 TI - Constitutive expression of GATA-1 interferes with the cell-cycle regulation. AB - GATA-1, mainly expressed during erythroid differentiation, has been shown to regulate the genes specifically expressed in the late stages of erythropoiesis and to protect erythroid cells from apoptosis, suggesting that it might interfere with the cell cycle. By expressing the retrovirally transduced human GATA-1 cDNA in NIH3T3 fibroblasts, we have shown that GATA-1 alone was unable to transactivate its erythroid-specific target genes in these nonerythroid cells. However, GATA-1 expression had a dramatic effect on the proliferation of these fibroblasts. The cloning efficiency of the GATA-1-expressing fibroblasts was maintained but their S phase was greatly elongated and their G1 and G2/M phases were reduced, impairing substantially their proliferation. When cultured at low serum concentrations for 48 hours, GATA-1-expressing fibroblasts failed to accumulate in the G0/G1 phases but did not become serum independent. GATA-1 expressing fibroblasts expressed D1, A, and B1 cyclin mRNAs under conditions of serum starvation or at confluence, whereas these cyclin mRNAs were downregulated in the parental NIH3T3 cells cultured under the same conditions. Moreover, these effects of GATA-1 expression on proliferation were not limited to NIH3T3 cells, since different clones of hGATA-1 virus-infected FDCP-1 cells, a murine interleukin-3-dependent hematopoietic cell line, had a slower growth rate than control cells. Based on these data, we hypothesize that GATA-1 plays a role in the regulation of the cell cycle during terminal erythroid differentiation. PMID- 8611697 TI - Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: partial reconstitution of cells from patients with X linked chronic granulomatous disease by gp91-phox expression. AB - The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase. PMID- 8611698 TI - Severe perinatal thrombosis in double and triple heterozygous offspring of a family segregating two independent protein S mutations and a protein C mutation. AB - Molecular genetic and phenotypic analyses were performed in a highly unusual case of combined protein S and protein C deficiency manifesting in a family in which a child had died perinatally from renal vein thrombosis. Antenatal diagnosis in a second pregnancy was initially performed by indirect restriction fragment length polymorphism (RFLP) tracking using a neutral dimorphism within the PROS gene and served to exclude severe protein S deficiency. Am umbilical vein blood sample at 22 weeks gestation showed isolated protein C deficiency. This pregnancy proceeded to a full-term delivery without thrombotic complications. Molecular genetic analysis of the PROC and PROS gene segregating in the family then yielded one PROC gene lesion in the father and two PROS gene lesions, one in each parent. These lesions were shown to segregate with the respective deficiency states through the family pedigree. Analysis of DNA from paraffin-embedded liver tissue taken from the deceased child showed the presence of both PROS mutations, as well as the PROC mutation. Genotypic analysis of the second child showed a PROC mutation, but neither PROS mutation consistent with its possession of normal protein S levels and a low/borderline protein C level. Antenatal diagnosis was then performed in a third pregnancy by direct mutation detection. However, although the fetus carried only the paternal PROS and PROC gene lesions, the child developed renal thrombosis in utero. It may be that a further genetic lesion at a third locus still remains to be defined. Alternatively, the intrauterine development of thrombosis in this infant could have been caused, at least in part by a transplacental thrombotic stimulus arising in the protein S deficient maternal circulation. This analysis may, therefore, serve as a warning against extrapolating too readily from genotype to phenotype in families with a complex thrombotic disorder. PMID- 8611699 TI - Studies of the activation of factor VII bound to tissue factor. AB - Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I-factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed. PMID- 8611700 TI - Regulation of type I plasminogen activator inhibitor by fibrin degradation products in rat lung fibroblasts. AB - Persistent fibrin deposition in tissues characterizes the early pathology of many types of injury. In an animal model of bleomycin-induced lung fibrosis, increased expression of type 1 plasminogen activator inhibitor (PAI-1) is associated with accumulation of fibrin in fibroproliferative lesions. Plasmin proteolysis of cross-linked fibrin generates fibrin degradation products (FDPs) with multiple biological activities in several cell types. We reasoned that fibrin fragments may also regulate fibroblast-mediated fibrinolysis. In this study, we describe induction of PAI-1 mRNA, protein, and activity by soluble FDPs and fibrinogen in rat lung fibroblast monolayers. FDPs are more potent than fibrinogen, inducing a concentration-dependent, maximal 3.7 (+/- 0.9)-fold increase in PAI-1 mRNA as measured by northern blotting and a 9.0 (+/- 1.3)-fold induction of PAI-1 antigen levels. Active PAI-1 is demonstrated in fibrinogen- and FDP-stimulated conditioned media. Further characterization of this response shows that PAI-1 expression is induced by the DD/D fragments, but not by immunopurified fragment E. Experiments using Actinomycin D and puromycin indicate that the induction appears to be transcriptionally regulated and is not dependent on new protein synthesis. FDP induction of PAI-1 suggests a matrix-cell feedback process in which a fibrin fragment modulates expression of an important regulator of fibrinolysis. PMID- 8611701 TI - Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo. AB - The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2 c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131 1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets. PMID- 8611702 TI - Monocyte procoagulant activity induced by in vivo administration of the OKT3 monoclonal antibody. AB - The first injection of OKT3 in kidney transplant recipients activates the common pathway of coagulation. This may result in early thrombosis of graft vessels. To this day, the cells involved in this phenomenon have not been identified. The aim of this study was to investigate whether circulating monocytes participated in this OKT3-induced coagulopathy. The procoagulant activity (PCA) of circulating monocytes rose from (mean +/- SEM) 0.15 +/- 0.02 mU/mL to 0.40 +/- 0.05 mU/mL at 3 hours (P = .002) and 0.56 +/- 0.21 at 5 hours (P = .045) after the initial OKT3 injection. These monocytes displayed increased tissue factor expression at the same moments (mean flourescence intensity: 14 +/- 2 before OKT3 injection versus 54 +/- 14 at 3 hours, P = .008 and 34 +/- 7 at 5 hours, P = .01). Tissue factor mRNA was detected in blood by reverse transcriptase-polymerase chain reaction as early as 2 hours after OKT3 administration. The circulating monocytes also displayed a steady increase in membrane expression upregulation of ICAM-1, CD29, CD11b, and CD11c. In vitro experiments showed that OKT3 as well as 2 mitogenic, humanized anti-CD3 antibodies potently induced monocytic PCA whereas the 4 nonmitogenic anti-CD3 antibodies tested were over 1,000-fold less potent than OKT3. We conclude that (1) OKT3 induces in vivo tissue factor gene upregulation and membrane expression resulting in increased PCA of circulating monocytes; and (2) nonmitogenic anti-CD3 antibodies seem devoid of significant procoagulant properties. PMID- 8611704 TI - Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. AB - Src-family nonreceptor protein tyrosine kinases (NRPTK) are associated with cell surface receptors in large detergent-resistant complexes: in epithelial cells, yes is selectively located in vesicle structures containing caveolin ("caveolae"). These formations are typically also endowed with glycophosphatidylinositol (GPI)-anchored proteins. In the present study, we observed lck, lyn, src, hck, CD4, CD45, G proteins, and CD55 (decay-accelerating factor) expression in the buoyant low-density Triton-insoluble (LDTI) fraction of selected leukemic cell lines and granulocytes. We provide a detailed analysis of the two most highly expressed NRPTK, p53/p56lyn and p56lck, which are involved in the transduction of signals for proliferation and differentiation of monocytes/B lymphocytes and T lymphocytes, respectively. We show that lyn is selectively recovered in LDTI complexes isolated from human leukemic cell lines (promyelocytic [HL-60], erythroid [K562] and B-lymphoid [697]) and from normal human granulocytes, and that lck is recovered from LDTI fractions of leukemic T- and B-lymphoid cell lines (CEM, 697). In LDTI fractions of leukemic cells, lck and lyn are enriched 100-fold as compared with the total cell lysates. Analysis of these fractions by electron microscopy shows the presence of 70- to 200-nm vesicles: lyn and lck are homogenously distributed in the vesicles, as revealed by an immunogold labeling procedure. These novel results propose a role for these vesicles in signal transduction mechanisms of normal and neoplastic hematopoietic cells. In support of this hypothesis, we further observed that molecules participating in B- and T-cell receptor activation cofractionate in the LDTI fractions, CD45/lyn (B cells) and CD45/lck/CD4 (T cells). PMID- 8611703 TI - Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow. AB - Changes in intracellular calcium concentration [Ca2+]i of fura-2-loaded human platelet during its adhesion to a fibrinogen-coated surface were studied, using a flow chamber mounted on an epifluorescence microscope equipped with digital-ratio imaging. Adherent platelets were individually mapped under a scanning electron microscope to establish the possible correlation between adhesion-associated shape alterations and [Ca2+]i changes. We found that 1) there was no immediate [Ca2+]i elevation on platelet adhesion; 2) [Ca2+]i changes varied drastically platelets with a lag time ranging 10 to 200 s, averaging about 1 minute; 3) the pattern of [Ca2+]i changes varied drastically among individual adherent platelets; 4) the degree of [Ca2+]i elevation appeared to correlate with the extent of morphology change, with the vast majority ( > 90%) of spread platelets showed detectable [Ca2+]i changes; 5) neither morphological nor [Ca2+]i changes correlated with the lag time; 6) platelets treated with dimethyl-BAPTA (15 mumol/L) underwent normal shape change without [Ca2+]i elevation; 7) cytochalasin D (10 mumol/L) inhibited both shape change and [Ca2+]i elevation; 8) colchicine (1 mmol/L) was ineffective in both regards. We conclude that although platelet adhesion-associated shape changes may be accompanied with heterogeneous [Ca2+]i changes that are microfilament-dependent, [Ca2+]i changes do not happen immediately after platelet-surface contact and they are not required for adherent platelets to undergo postcontact morphological changes. PMID- 8611705 TI - Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia. AB - Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire. PMID- 8611706 TI - HLA class II as potential target antigen on malignant B cells for therapy with bispecific antibodies in combination with granulocyte colony-stimulating factor. AB - We have investigated the capacity of polymorphonuclear phagocytes (PMN) to lyse malignant B-cell lines using antibodies and antibody derivates to a range of different B-cell antigens. PMN were found to mediate lysis of all tested B-cell lines in the presence of HLA class II antibodies L227, L243, F3.3, and CR3/43. Target cell lysis was significantly enhanced when PMN isolated during granulocyte colony-stimulating factor (G-CSF) treatment were compared with PMN from healthy donors. Only G-CSF primed PMN, expressing Fc gamma RI (CD64), lysed B cells in the presence of monoclonal antibody (MoAb) 1D10 or Lym-1 to HLA class II related epitopes. Remarkably, PMN were consistently unable to kill malignant B cells with antibodies to the B-cell related antigens CD19, CD20, CD21, CD37, and CD38. These target antigen restriction was not observed with mononuclear effector cells, which mediated cytotoxicity with antibodies to HLA class II, but also with mouse/human chimeric constructs to CD19, CD37, and CD38. Blocking studies with Fc gamma RI antibodies and reverse antibody-dependent cellular cytotoxicity (ADCC) experiments against Fc gamma R antibody expressing hybridoma targets confirmed the pivotal role of Fc gamma RI in enhanced killing by G-CSF primed neutrophils. Bispecific antibodies (BsAb) with one specificity for Fc gamma RI, and another for a tumor associated antigen, offer an interesting approach to improve effector cell recruitment for immunotherapy. In our studies, very effective lysis was observed with G-CSF primed PMN and an [HLA class II x Fc gamma RI] BsAb. The therapeutic implications of these findings and the possible use of BsAb in combination with G-CSF are discussed. PMID- 8611707 TI - Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma. AB - To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies. PMID- 8611708 TI - A new method of "in-cell reverse transcriptase-polymerase chain reaction" for the detection of BCR/ABL transcript in chronic myeloid leukemia patients. AB - Methods of detecting minimal residual disease (MRD) in chronic myeloid leukemia (CML) include chromosome analysis, Southern blotting, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) techniques. We report a novel method to detect intracellular messenger RNA (mRNA) by combining the techniques of reverse transcription (RT) and PCR performed directly inside the cells, without extraction of the nucleic acid. We applied this method, which we call "in cell RT-PCR", to detect hybrid BCR/ABL transcript within single cells. After cellular permeabilization and fixation of single cells in suspension, the neoplastic mRNA was reverse transcribed into cDNA, and the cDNA was amplified by PCR with fluorescent primers, specific for bcr/abl. Flow cytometry was used to detect cells positive for the amplified DNA within the cell cytoplasm. After transferring the amplified cells onto slides by cytospin, the positive cells for BCR/ABL cDNA were observed by fluorescent microscopy. The technique was capable of detecting low abundancy signals and distinguishing different levels of gene expression. The amplification products were found in the cells and supernatants. The distribution was critically affected by the protease digestion condition. The specificity of amplification was confirmed by a nested RT-PCR of BCR/ABL performed on extracted mRNA from the same sample, and by reamplification of supernatants. We have used the technique to study 10 Ph+ CML patients and three normal subjects as controls. Four patients were 100% Ph+ at diagnosis time and RT PCR+ at cytogenetic and molecular analysis, respectively. In-cell RT-PCR showed that the residual non-neoplastic cells could be observed in all cases. In two patients undergoing interferon-alpha (IFN-alpha) therapy and in four bone-marrow transplanted patients, the in-cell RT-PCR was used to compare the level of Ph+ positivity detected by cytogenetic analysis with the number of cells expressing BCR/ABL transcript. In this manner, we could estimate the MRD. Our preliminary application of the technique suggests that it is capable of accurately identifying cells transcribing bcr/abl, and that it may have significant clinical applications in the detection of MRD. PMID- 8611709 TI - The consistent association between Epstein-Barr virus and Hodgkin's disease in children in Kenya. AB - Recent studies have suggested that Epstein-Barr virus (EBV) may play a role in the etiology of Hodgkin's disease (HD). In a previous study, we used a latent membrane protein 1 (LMP1)-specific antibodies to examine archival material from 74 British children with HD and found 50% of cases to be positive. It is known that there are geographic and ethnic variations in the incidence of HD. We have investigated LMP1 status in formalin-fixed, paraffin wax-embedded lymph nodes with HD involvement from 53 children and 48 adults from Kenya using immunohistochemical staining. We also developed sensitive and specific in vitro gene amplification protocols for examining the EBV strain type in such material using several combinations of primers derived from the EBNA 2 and EBNA 3 coding regions. LMP1 positivity was present in 100% of the pediatric cases (two lymphocyte-predominant, 25 nodular sclerosis, 16 mixed cellularity, 5 lymphocyte depletion, and 5 unclassified) and in 66% of the adult cases (two of three lymphocyte-predominant, 26 of 39 nodular, sclerosis, two of two mixed cellularity, and two of four lymphocyte depletion). Tests to type the EBV strain were undertaken in 25 EBV-positive pediatric cases. A combination of type specific polymerase chain reactions for EBNA 2 and EBNA 3C genes indicated that seven patients had type 1, eight had type 2, and 10 had dual infections with both types. Five cases with dual infections were further investigated using a sensitive in situ hybridization for the EBV-encoded, small nuclear nonpolyadenylated RNAs (EBERs). EBER transcripts were detected in Reed-Sternberg and Hodgkin cells and in occasional infiltrating lymphocytes. These observations indicate that in Kenya EBV is consistently associated with pediatric cases of HD, and that biopsies from a number of such cases appear to carry both type 1 and type 2 viral sequences. PMID- 8611710 TI - Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL. AB - The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells. PMID- 8611711 TI - Analysis of major histocompatibility complex class I expression on Reed-Sternberg cells in relation to the cytotoxic T-cell response in Epstein-Barr virus-positive and -negative Hodgkin's disease. AB - To get insight into the failure of the immune system to eradicate Epstein-Barr virus (EBV) harboring Hodgkin and Reed-Sternberg cells (H-RS cells), expressing the latent membrane protein 1 (LMP1), we analyzed major histocompatibility complex (MHC) class I expression on H-RS cells in relation to the presence of activated cytotoxic cells, i.e., granzyme-B-expressing lymphocytes. H-RS cells in EBV+ cases of Hodgkin's disease (HD) were found to express significantly higher levels of MCH class I heavy- and light-chain molecules compared with EBV- HD cases. When low levels of MHc class I expression were found (mainly in EBV- cases), these were not associated with low levels of the transporter protein associated with antigen presentation 1 (TAP-1). The relatively high levels of MHC class I expression in H-RS cells in EBV+ HD cases were accompanied by significantly higher numbers of activated cytotoxic T lymphocytes (CTLs) as shown by the presence of increased numbers of CD8 and granzyme B+ lymphocytes. However, these cells were only sporadically detected in the close vicinity of the H-RS cells. These data suggest that mechanisms other than downregulation of MHC class I or TAP-1 expression on H-RS cells are involved in the failure of the immune system to eradicate EBV harboring H-RS cells. Probably, the function of activated CTLs is locally inhibited by the H-RS cells or by reactive cells in the vicinity of the H-RS cells. PMID- 8611712 TI - Interleukin-9 stimulates the proliferation of human myeloid leukemic cells. AB - Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh) IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction of prevention of apoptosis by IL-9. Il-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU-L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9-supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (i.e., SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P < .05) and induced an increase of G1- and S-phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle. PMID- 8611713 TI - Occurrence of the t(2;5)(p23;q35) in non-Hodgkin's lymphoma. AB - Primary CD30(Ki-1)-positive anaplastic large-cell lymphoma (ALCL) is considered by some to be a distinct clinicopathologic entity associated with the t(2;5) (p23;q35). However, the specificity of t(2;5) for ALCL has not been carefully studied. Therefore, we performed a detailed analysis of all cases of ALCL with abnormal cytogenetics results in the Nebraska Lymphoma Study Group registry, as well as all other cases of non-Hodgkin's lymphoma with t(2;5) in the registry. We found the t(2;5) in only five of 10 cases of ALCL, four of whom were young patients. However, we also found the t(2;5) in 11 other cases of nonanaplastic lymphoma, including eight children with typical peripheral T-cell lymphomas of various types. The t(2;5) was also found in three older adults with B-cell lymphomas of various types. Thus, the t(2;5) was not specific for CD30+ ALCL. However, t(2;5) may define a clinicopathologic entity in children and young adults characterized by variable morphologies with a T-cell or indeterminate phenotype, CD30-positivity, nodal disease with frequent extranodal involvement, advanced stage, and an excellent response to therapy, including bone marrow transplantation for relapsed disease. The clinical relevance of the t(2;5) in older patients requires further study. PMID- 8611714 TI - Restricted immunoglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia. AB - Between 10% and 25% of chronic lymphocytic leukemia (CLL) patients have episodes of autoimmune hemolytic anemia (AIHA) during the course of their disease. The anti-erythrocyte autoantibodies in most cases are polyclonal and express a different heavy chain isotype than the malignant clone, indicating that they are secreted by normal autoreactive B lymphocytes. To further investigate the pathogenesis of the AIHA in CLL, we analyzed the lg heavy (H) chain variable region genes expressed by leukemic cells from CLL patients with and without AIHA. Two VH genes were preferentially expressed by the leukemic cells in the CLL cases with AIHA and were present in 9 of the 12 investigated cases. The 51p1/DP-10 gene was expressed in 5 of these cases and was absent in the control group of 12 consecutive CLL cases without AIHA, whereas the DP-50 gene was present in 4 CLL AIHA cases and only once in the control CLL group. A strikingly similar H-chain CDR3 region that contained a single reading frame of the DXP4 DH gene segment, and N-encoded proline at the DH/JH boundary, and a tyrosine-rich region encoded by the JH6 gene segment was observed in four CLL-AIHA cases. The preferential expression of two VH gene segments and a particular CDR3 region by the leukemic cells of patients with AIHA suggests that the antibodies produced by the CLL cells are directly involved in the pathogenesis of the hemolytic anemia. PMID- 8611716 TI - Interferon-alpha restores normal beta 1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia. AB - Chronic myelogenous leukemia (CML) progenitors show decreased adhesion to stroma and fibronectin (FN) through beta 1 integrin receptors. We have previously shown that interferon-alpha (IFN-alpha) restores beta 1 integrin-mediated adhesion of CML progenitors to stroma. Because beta1 integrins transmit proliferation inhibitory signals from the microenvironment to normal hematopoietic progenitors, we hypothesized that decreased integrin-mediated adhesion of CML progenitors contributes to their continuous proliferation when in contact with stroma and that IFN-alpha treatment, by restoring integrin-mediated adhesion, also restores integrin-mediated microenvironmental inhibition of CML progenitor proliferation. We show here that, in contrast to normal colony-forming cells (CFC), the percentage of malignant CML CFC in S-phase was not significantly reduced following coculture with stromal layers. However, IFN-alpha treatment resulted in a significant reduction in the proliferation of CML CFC on coculture with stroma. This effect was not because of a direct antiproliferative effect of IFN-alpha on CML CFC because the proliferation of IFN-alpha treated CML CFC kept in suspension culture was not reduced. We examined the role of restored signaling through beta 1 integrin receptors in IFN-alpha induced inhibition of CML progenitors in two sets of experiment. In the first set of experiments, we demonstrated that proliferation of IFN-alpha-treated CML CFC, but not untreated CML CFC, was significantly reduced following coculture with 33/66-kD and 75-kD FN fragments, recognized by alpha 4 beta 1 and alpha 5 beta 1 integrins respectively. In a second set of experiments, we demonstrate that direct stimulation of integrin receptors by crosslinking with blocking antibodies to alpha 4, alpha 5, and beta 1 integrins and secondary goat antimouse antibodies resulted in significant reduction in proliferation of normal and IFN-alpha treated CML progenitors but not untreated CML CFC. These studies indicate that CML hematopoietic progenitors are unresponsive to beta 1-integrin mediated proliferation inhibition and that IFN-alpha not only restores beta 1 integrin-mediated adhesion but also beta1 mediated microenvironmental inhibition of CML progenitor proliferation. These observations may explain, at least in part, the therapeutic efficacy of IFN-alpha in CML. PMID- 8611715 TI - Inhibition of angiogenesis by interleukin-12 is mediated by the interferon inducible protein 10. AB - Interleukin 12 (IL-12), a multifunctional cytokine produced by macrophages and B cell lines, induces interferon-gamma (IFN-gamma) production, stimulates growth of both T and natural killer cells, promotes Th1-type helper T-cell responses, and inhibits neovascularization. Because the human interferon-inducible protein 10 (IP-10) can also inhibit neovascularization, we tested whether IP-10, induced by IL-12 through the intermediate IFN-gamma, might be a mediator of IL-12 angiogenesis inhibition. We report here that murine IL-12 profoundly inhibited basic fibroblast growth factor (bFGF)-induced Matrigel neovascularization in vivo, and that this effect of IL-12 was neutralized by systemic administration of antibodies to either murine IFN-gamma or IP-10. Murine IL-12 induced murine IP-10 expression in mouse splenocytes, and human IFN-gamma induced human IP-10 expression in purified human endothelial cells, suggesting that IL-12 can induce IP-10 expression in certain cells. These results document the important role of IP-10 as a mediator of angiogenesis inhibition by IL-12, and raise the possibility that IP-10 may also contribute to the antitumor effect of IL-12. PMID- 8611717 TI - Cure of multidrug-resistant human B-cell lymphoma xenografts by combinations of anti-B4-blocked ricin and chemotherapeutic drugs. AB - The CD-19-directed immunotoxin anti-B4-blocked ricin (anti-B4-bR) is currently in clinical trials for the treatment of B-cell malignancies. To explore the potential of using anti-B4-bR with chemotherapy protocols we tested the in vivo efficacy of the immunotoxin in combination with two multi-drug chemotherapeutic regimens in severe combined immunodeficient (SCID) mice bearing disseminated tumors of the multidrug-resistant human B-cell lymphoma Namalwa/mdr-1. In cytotoxicity studies in vitro, combinations of the immunotoxin with cisplatin produced supra-additive killing effects on both Namalwa and Namalwa/mdr-1 cells, whereas anti-B4-bR combined with 4-hydroperoxy-cyclophosphamide caused additive killing of both cell lines. In vivo cyclophosphamide, cisplatin, vincristine, doxorubicin, and etoposide as single agents, were effective in prolonging the survival of SCID mice burdened with the Namalwa tumor, whereas only cyclophosphamide and cisplatin were effective on Namalwa/mdr-1 tumors. Treatment of Namalwa/mdr-1-bearing mice with anti-B4-bR alone or with the drug combination CHOE (consisting of cyclophosphamide, vincristine, doxorubicin, and etoposide) alone increased the lifespan of the tumor-burdened mice by 58% and 73%, respectively. However, treatment with five daily bolus intravenous injections of anti-B4-bR followed by CHOE increased the lifespan by 173%, and 20% of the mice were cured. The drug combination CCE (cyclophosphamide, cisplatin, and etoposide) alone could increase the lifespan of the Namalwa/mdr-1 tumor-burdened mice by 129% compared with untreated controls. Combination therapy with anti-B4-bR and CCE produced long-term cures in 50% of the tumor-burdened mice. These results suggest that anti-B4-bR in combination with current multidrug regimens may constitute a highly efficacious modality for the treatment of drug-resistant B cell malignancies. PMID- 8611718 TI - Routine bone marrow exam during first remission of acute myeloid leukemia. AB - To detect relapse acute myeloid leukemia (AML) treatment protocols have called for bone marrow exams every 2 to 4 months in remission. To investigate the effect of replacing this policy with one calling for marrows only when blood count is abnormal (platelets < 100,000, neutrophils < 1,000, circulating blasts > 0%, unrelated to prior chemotherapy), we reviewed the records of all 444 patients with AML whose disease recurred ( > or = 5% marrow blasts unrelated to prior chemotherapy) for the first time between 1980 and 1995. The 375 patients with adequate follow-up were classified as (1) simultaneous--blood count abnormal when relapse noted without a normal marrow intervening between first abnormal count and relapse, 289 patients (77% of 375), (2) marrow first--blood count normal when relapse noted, 60 patients (16%), or (3) blood first--a normal marrow intervened between first abnormal blood count and relapse, 26 patients (7%). Interval between marrow exams and blood counts did not differ in the three groups (a 25 patient sample of the 289 patient simultaneous group was analyzed as representative of this group) with marrows done at a median of once monthly and blood counts at a median of once weekly from complete remission (CR) date to relapse date. The three groups also had similar first CR duration, and pretreatment cytogenetics. CR rates following salvage chemotherapy were 32% to 33% in the simultaneous and marrow first groups and 17% in the blood first group. We conclude that routine marrow exams for morphology are not needed in the great majority of AML patients in first CR. PMID- 8611719 TI - Human herpesvirus-8 DNA sequences in human immunodeficiency virus-negative angioimmunoblastic lymphadenopathy and benign lymphadenopathy with giant germinal center hyperplasia and increased vascularity. AB - Human herpesvirus-8 (HHV-8) DNA sequences have been reported to be strictly associated not only with various forms of Kaposi's sarcoma but also with an unusual subgroup of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphomas. A possible relation of this putative virus also with multicentric Castleman's disease (MCD) has been recently suggested. We used polymerase chain reaction to look for the presence of HHV-8 sequences in a well characterized series of benign, atypical, and malignant lymphoid tissues from 45 Hodgkin's disease and 43 non-Hodgkin's lymphoma (NHL) cases, as well as from 5 MCD, 15 angioimmunoblastic lymphadenopathy (AILD), and 23 benign lymphadenopathy cases. Among the 38 AIDS-related lymphoid lesions, only 1 NHL and 1 persistent generalized lymphadenopathy (PGL) case were positive. Furthermore, among the 92 non-AIDS-related lymphoproliferative disorders, HHV-8 sequences were detected in 3 classic AILD cases and in 4 reactive lymphadenopathies. Six of 9 HHV-8 positive lymphoid lesions (1 NHL, 1 PGL, 1 AILD, and 3 reactive lymphadenopathy cases) were also positive for Epstein-Barr viral sequences. The four human immunodeficiency virus (HIV) negative lymphadenopathies positive for HHV-8 sequences showed an almost identical histology, characterized by a predominantly follicular lesion, with giant germinal center hyperplasia, and increased vascularity, resembling HIV-related lymphadenopathy and MCD. Our results, while providing the first evidence of the presence of HHV-8 sequences in AILD cases, suggest a possible association of these herpes viral sequences with a distinct histologic type of non-neoplastic lymphadenopathy, not associated with other common herpes infections. PMID- 8611720 TI - Subcellular localization of transforming growth factor-alpha in human eosinophil granulocytes. AB - Eosinophils are involved in the inflammatory response seen in allergy and helminthic infestations. Eosinophils synthesize transforming growth factor-alpha (TGF-alpha), which may play a role in the development of the characteristic fibrosis seen in longstanding high eosinophilia. Using immunoelectron microscopic techniques, eosinophils from peripheral blood of healthy individuals and from one patient with high eosinophilia showed presence TGF-alpha in matrix of the specific crystalloid-containing granules. In cryosections, TGF-alpha was also visualized in a vesicular compartment of the cytoplasm. In double-labeling experiments, the TGF-alpha of this latter compartment did not colocalize with CD63, a marker for lysosomes, nor with albumin of secretory vesicles. In extracts from eosinophils, obtained from healthy donors, immunoreactive TGF-alpha could be detected by enzyme-linked immunosorbent assay-technique. In addition, sera from two patients with high eosinophilia showed TGF-alpha concentrations of 1.5 ng/mL and 164 pg/mL, respectively, whereas TGF-alpha could not be detected in serum from healthy controls. In conclusion, TGF-alpha is present in the specific granules, and in an additional vesicular compartment of the cytoplasm of eosinophils. PMID- 8611722 TI - Total cell content of CR3 (CD11b/CD18) and LFA-1 (CD11a/CD18) in neonatal neutrophils: relationship to gestational age. AB - Neonatal neutrophils (PMN) exhibit a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b/CD18), a critical adhesion molecule, on chemoattractant-activated PMN. We recently documented that deficient surface expression of CR3 on stimulated neonatal PMN is due principally to a deficiency in total cell content of CR3. In the current studies, we tested the hypothesis that total cell CR3 content of PMN is even more profoundly deficient in premature infants and that PMN CR3 content is directly related to gestational age. A sandwich enzyme-linked immunosorbent assay for CR3 showed that PMN lysates from term neonates ( > or = 37 weeks gestation) contain about 60% of adult PMN levels of CR3, whereas PMN from premature infants (range of 27 to 36 weeks gestation) contained a mean of about 30%, ranging from 10% to 48% (P < .001 for term [n = 6] v premature [n = 11] by unpaired t-test). When the relationship between total cell CR3 and gestational age (n = 15) was analyzed, the correlation coefficient was .94 by linear regression, and the Spearman rank correlation was significant with P < .001. PMN content of LFA-1 (CD11a/CD18) was similarly measured for 14 neonates. Term neonates were equivalent to adults in LFA-1 content of their PMN (99.4% +/- 3.2% of paired adult values, n = 6), whereas prematures (28 to 36 weeks gestation) were deficient, overall (69.1% +/- 10.4%, n = 8, P = .035). Below 35 weeks gestation, LFA-1 values ranged from 26% to 65% of paired adult control values, but no infant of > or = 35 weeks gestation had PMN LFA-1 content that was less than 85% of its adult control. We concluded that CR3 total cell content is more profoundly deficient in premature than in term neonates, that at birth there is a direct relationship between PMN CR3 content and gestational age, and that LFA-1 is deficient only in prematures less than 35 weeks of gestational age. Below 30 weeks gestation, CR3 content of PMN approached that seen in genetic deficiency of the CD18 family of leukocyte integrins, or type 1 leukocyte adhesion deficiency, underscoring the severity of this host impairment in very early preterm neonates. PMID- 8611721 TI - Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide. AB - The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell associated and released CP-10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection. PMID- 8611723 TI - Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation. AB - Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5' end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma. PMID- 8611724 TI - Human red blood cell Wright antigens: a genetic and evolutionary perspective on glycophorin A-band 3 interaction. AB - The Wright (Wra/Wrb) blood group polymorphism is defined by an allelic change (Lys658Glu) in the band 3 protein; nevertheless, the Wrb antigen apparently requires glycophorin A (GPA) for surface presentation. To gain insight into the structural basis for this protein-protein interaction and delineate its relationship with Wrb antigen expression, we investigated GPA and band 3 sequence polymorphisms occurring in rare humans and nonhuman primates. The lack of GPA or amino acid residues 59 through 71 of GPA results in the absence of Wrb from human red blood cells (RBCs) exhibiting the MkMk, En(a-), or MiV phenotype. However, the SAT homozygous cells carried a Glu658 form of band 3 and a hybrid glycophorin with the entire GPA extramembrane domain from residues 1 through 71, yet expressed no Wrb antigen. This finding suggests that formation of the Wrb antigenic structure is dependent on protein folding and that the transmembrane junction of GPA is important in maintaining the required conformation. Comparative analyses of GPA and band 3 homologues led to the identification in the interacting regions of conserved and dispensable amino acid residues that correlated with the Wrb positive or negative status on nonhuman primates. In particular, the chimpanzee RBCs cells expressed Wrb and the Glu658 form of band 3, which is identical to humans, but their GPA contained the Gly rather than Arg residue at position 61. Taken together, the results suggest that (1) Arg61 of GPA and the proposed Arg61-Glu658 charge pair are not crucial for Wrb antigen exhibition and (2) the role of GPA for interaction with band 3, including Glu658, probably involves a number of amino acid residues located in the alpha-helical region and transmembrane junction. PMID- 8611725 TI - Catalysis of soluble hemoglobin oxidation by free iron on sickle red cell membranes. AB - Abnormal deposition of hemichrome on the inner aspect of the sickle red cell membrane promotes premature cell demise. The steps proximate to hemichrome formation in these cells are poorly understood. To test the hypothesis that the pathologic deposits of free ferric iron located on the inner aspect of sickle cell membranes would be redox active and promote oxidation of soluble oxyhemoglobin, we incubated native versus iron-stripped sickle or normal ghost membranes with oxyhemoglobin S. We found that sickle membranes exerted an exaggerated effect on methemoglobin formation in solution, an effect completely accounted for by their abnormal content of free iron. This ability of sickle membranes to promote hemoglobin oxidation was not diminished by catalase or by presence of a high-affinity, iron-inactivating chelator that is unable to remove membrane iron. Examination of those membranes likewise revealed that their free iron content promoted deposition of additional heme-protein. These results establish that the potential redox couple formed by membrane-associated ferric iron and cytoplasmic oxyhemoglobin is promotive of hemoglobin oxidation and deposition of hemichrome on the membrane. This predicts that removal of pathologic membrane iron might help prevent the detrimental formation of methemoglobin and hemichrome in vivo, insofar as this is accelerated by transition metal. PMID- 8611726 TI - Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean. AB - Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization. PMID- 8611727 TI - The effect of lymphocytotoxic antibody reactivity on the results of single antigen mismatched platelet transfusions to alloimmunized patients. AB - Despite selection strategies that attempt to maximize the platelet donor pool, significant numbers of alloimmunized patients have few if any available donors. Although the number of potential donors increases when one antigen mismatched platelet transfusions (OAMPT) are considered, transfusions from such donors are often cited to fail to produce satisfactory platelet count increments. The presence of lymphocytotoxic antibody (LCTAB) correlates well with responsiveness to random donor platelet transfusions and serves as a good serologic screen for the diagnosis of alloimmunization. We therefore reviewed the results of OAMPT to alloimmunized patients and assessed the relationship between LCTAB levels in the recipient and posttransfusion platelet count increments. We noted an unexpectedly high percentage of good responses in our patient population: 73% of all OAMPT to recipients with LCTAB < 60% reactive, resulted in successful increments. In recipients with LCTAB > or = 60%, 58% of all transfusions were still successful. Despite a statistically significant inverse relationship between the level of LCTAB and the response of OAMPT to alloimmunized patients, 58% to 73% of recipients will have a satisfactory platelet recovery posttransfusion. These data support extending donor searches for alloimmunized patients to include any single mismatch particularly if a recipient's LCTAB has lower reactivity. PMID- 8611728 TI - Quantitation of mafosfamide-resistant pre-colony-forming units in allogeneic bone marrow transplantation: relationship with rate of engraftment and evidence for long-lasting reduction in stem cell numbers. AB - Current assays of human committed-stem cells are of limited value in predicting the rate of engraftment or in assessing the integrity of the stem cell pool after allogeneic bone marrow (BM) transplantation (BMT). We have used a limiting dilution assay of mafosfamide-resistant progenitors (pre-colony-forming units [CFU]), which are ancestral to committed progenitors such as CFU-granulocyte macrophage (GM) to analyze the kinetics of myeloid engraftment after BMT and to assess the size of the stem cell pool at intervals up to 66 months thereafter. In 24 patients transplanted for chronic myeloid leukemia in chronic phase (eight with matched unrelated donors and 16 with sibling donors), the rate of neutrophil engraftment correlated strongly with the number of pre-CFU transfused per kilogram recipient body weight (r = .7, P < .005) but not with CFU-GM per kilogram or nucleated cells per kilogram. In 25 patients studied 6 to 66 months after allogeneic BMT, the mean number of pre-CFU in the marrow was 3.1/10(5) mononuclear cells (MNC) (median, 3.47; range, 0.4 to 23.3), compared with 24.7/10(5) MNC (median, 27.3; range, 4.2 to 180) in 25 normal subjects. CFU-GM were also reduced in these patients, but with considerable overlap into the normal range (mean +/- SD: 54 +/- 45.6 per 10(5) MNC; normal, 129 +/- 61.6). Low pre-CFU but not low CFU-GM levels were associated with reduced peripheral blood white blood cell counts in post-BMT patients. Pre-CFU and CFU-GM levels were not related to the interval posttransplant and remained low for up to 66 months. We conclude that the pre-CFU assay measures a population of stem/progenitor cells that are important in the kinetics of engraftment after allogeneic BMT. Our data suggest that pre-CFU levels may remain low for some years after BMT in humans. PMID- 8611729 TI - Detection of empty HLA class II molecules on cord blood B cells. AB - Fetal mononuclear cells are increasingly used in transplantation of hematopoietic cells due to a reportedly lower incidence of graft-versus-host disease. Previous studies of immune responses of fetal lymphocytes have indicated a general hyporesponsiveness in response to polyclonal stimulation. Fetal B lymphocytes display many features typical of the resting state such as a low level of HLA class II expression, but a large proportion of cells also carry the activation associated CD23 antigen. We show here that despite a low cell surface level of all three HLA class II isotypes on fetal B cells, their allogeneic capacity, measured as the ability to elicit a mixed lymphocyte reaction, is similar to that of adult B cells. Allogeneic stimulation is believed to be peptide-dependent. Surprisingly, the majority of the HLA class II molecules on cord blood B cells appeared to be devoid of stably bound peptide as detected by the binding of a recombinant and soluble invariant chain, as well as by the absence of sodium dodecyl sulfate (SDS) stable alpha beta heterodimers in whole cell lysates. Immunoblot experiments showed that HLA class II molecules of fetal B cells were predominantly present in high molecular weight aggregates in stark contrast to B cells of adult origin. However, a sensitive cell surface labeling technique followed by immunoprecipitation enabled us to detect an SDS-stable 120-kD molecule on fetal B cells. We propose that the 120-kD molecules could correspond to HLA class II doubledimers or superdimers. We hypothesize that the 120-kD HLA class II molecule functions as the antigen-presenting molecule in the mixed lymphocyte reaction of fetal B cells, as it is the major species detected on the surface. Secondly, we suggest that a high level of empty HLA class II molecules may be indicative of a particular stage in B-cell ontogeny. PMID- 8611730 TI - Newborn blood can engraft adult mice without inducing graft-versus-host disease across non H-2 antigens. AB - Cells derived from human cord blood were recently used instead of bone marrow (BM) for transplantation. However, several questions concerning the potential of these cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce a graft-versus-host disease (GVHD) remain unanswered. Here we used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non H-2 antigens. Few mature T cells were found in NBB as in adult BM and both contain around 10% to 20% SCA-1+ pluripotent progenitor cells. Yet numerous immature CD4+CD8+ TCR alpha/beta low Thy-1high T cells were present in NBB. In contrast, adult peripheral blood (PB) exhibits a mature T-cell phenotype and contains few progenitor cells. The injection of blood pooled from one to three newborn mice resulted in the engraftment of 71% to 86% of F1 recipients, which survived more than 100 days without clinical signs of GVHD. The injection of BM leads to 100% survival whereas the injection of adult PB resulted in rapid mortality, both without signs of GVHD. In NBB-engrafted F1 surviving mice, T-cell reconstitution preceded B-cell reconstitution, and the degree of donor cell chimerism increased progressively with time. Additionally, 2 to 4 months after transplantation, T cells from these mice were tolerant to host non H-2 antigens but kept reactivity against third-party Ags. Host specific tolerance in NBB-engrafted F1 mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells were able to reconstitute the lymphohematopoietic system of lethally irradiated adult mice without inducing GVHD against incompatible non H-2 antigens. Thus, this experimental model in vivo may be relevant to the human cord blood transplantation. PMID- 8611731 TI - Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts. AB - We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion > 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( > 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( < 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells. PMID- 8611732 TI - Natural killer (NK)-cell function and antileukemic activity of a large population of CD3+/CD8+ T cells expressing NK receptors for major histocompatibility complex class I after "three-loci" HLA-incompatible bone marrow transplantation. AB - We have shown that addition of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to the marrow inoculum allows engraftment of T cell depleted, "three loci" HLA-incompatible marrow transplants for acute leukemia. The event-free survival of patients at high risk for potential of this transplant. Tumor-cell lysis by natural killer (NK) cells is regulated by inhibitory receptors for specific HLA class I alleles. Here, we report the postgrafting emergence of a large, donor-type CD3+/CD8+ T-cell receptor (TcR) alpha beta+ cell population, barely detectable in normal subjects, that expresses 58 kD, "p58," NK receptors for HLA-C locus alleles. Analysis of > 900 clones revealed that 40% to 80% of these T cells exhibit NK-like function, i.e., they lysed class I- targets and were functionally blocked by class I alleles on target cells. Monoclonal antibody-mediated blocking of class I recognition by these cells induced lysis of HLA-protected, autologous targets. The class I-mediated inhibitory signaling through the NK receptors also blocked TcR/CD3-triggered cytotoxicity of these cells, indicating that their antigen-specific responses may be impaired. However, the NK-like function of these cells allows them to discriminate normal cells, protected from lysis, from leukemic cells that were lysed and may be targets for a graft-versus-leukemia effect. PMID- 8611733 TI - In vivo inhibition of cytokine responsiveness and graft-versus-host disease mortality by rapamycin leads to a clinical-pathological syndrome discrete from that observed with cyclosporin A. AB - Rapamycin (RAPA) has been shown to be a highly effective means of reducing the lethality of graft-versus-host disease (GVHD) in B10.BR recipients of allogeneic C57BL/6 donor cells. RAPA-treated mice had no clinical (e.g., weight loss, diarrhea, lethargy) or histologic evidence of classical acute or chronic GVHD but did develop a clinical-pathological syndrome consisting of ulcerative dermatitis, bile duct proliferation, and a nondestructive peribronchiolar pulmonary infiltration. Because RAPA was found to interfere with the deletion of self reactive T cells, we wondered whether the RAPA-induced syndrome was related to failed negative selection or altered alloreactivity. We now show that the RAPA induced syndrome is due to effects on mature, donor-derived alloreactive T cells. By titering the number of T cells infused we were able to vary the syndrome incidence. In contrast to the syndrome seen after cyclosporin A (CsA) administration, the RAPA syndrome did not require an intact thymus and the disease could not be adoptively transferred. The addition of CsA (which blocks T cell cytokine production) to RAPA (which blocks T-cell cytokine response) prevented the generation of this syndrome, suggesting that the tissue manifestations seen in RAPA only treated recipients were caused by cytokine production and release. RAPA also caused this alloimmune syndrome in recipients of minor histocompatibility antigen disparate donor cells, showing that the RAPA effects were not restricted to a single donor-recipient strain combination or to instances in which the donor and recipient were fully major histocompatibility complex disparate. We conclude that RAPA is a highly effective means of preventing murine acute GVHD, and that when combined with CsA, warrants consideration for human investigations. PMID- 8611734 TI - The first clinical use of donor leukocyte infusions for the treatment of leukemia relapsing after allogeneic bone marrow transplantation. PMID- 8611735 TI - Allogeneic cell therapy: the treatment of choice for all hematologic malignancies relapsing post BMT. PMID- 8611737 TI - Utility of fingernail DNA for evaluation of chimerism after bone marrow transplantation and for diagnostic testing for transfusion-associated graft versus-host disease. PMID- 8611736 TI - Identification of the lineage of mature neutrophils and delineation of lipid body formation and apoptosis therein. PMID- 8611738 TI - Serum interleukin-8 (IL-8) and IL-6 concentrations in patients with hematologic malignancies. PMID- 8611739 TI - Stem cell factor serum levels may not be clinically relevant. PMID- 8611740 TI - How do antisense oligodeoxynucleotides inhibit the growth of chronic myelogenous leukemia cells? PMID- 8611742 TI - Effects of interleukin-4 administration on endocrine function and lipid profile of patients with malignant diseases. PMID- 8611741 TI - Long-term follow-up of 12 pediatric patients with primary myelodysplastic syndrome treated with HLA-identical sibling donor bone marrow transplantation. PMID- 8611743 TI - Effects of sesamin and alpha-tocopherol, individually or in combination, on the polyunsaturated fatty acid metabolism, chemical mediator production, and immunoglobulin levels in Sprague-Dawley rats. AB - Feeding sesamin and alpha-tocopherol in combination, both at the 0.5% dietary level, to Sprague-Dawley rats for 3 weeks resulted in a trend toward decreasing the proportion of 20:4n-6 and 22:5n-6 and increasing that of 18:2n-6 in phosphatidylcholine from various tissues, suggesting interference with the metabolism of linoleic acid. This dietary manipulation significantly reduced the production of leukotriene C4 in the lung, the splenic production of leukotriene B4, and reduction of the plasma histamine level. Simultaneous administration of sesamin and alpha-tocopherol significantly increased the production of IgA, IgG, and IgM by mesenteric lymph node lymphocytes, while the IgE level tended to be reduced. These effects were not necessarily apparent by feeding these compounds separately. Thus, sesamin and alpha-tocopherol in combination would be effective for regulating the eicosanoid production and modifying the immune function. PMID- 8611744 TI - Purification and properties of a novel enzyme, maltooligosyl trehalose synthase, from Arthrobacter sp. Q36. AB - Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (alpha-maltooligosyl alpha-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The Km of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9 mM, 8.7 mM, 1.4 mM, and 0.9 mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40 degrees C, and was stable from pH 6.0 to 9.5 and up to 40 degrees C. The enzyme activity was inhibited by Hg2+ and Cu2+. PMID- 8611745 TI - Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp. Q36. AB - A novel enzyme, maltooligosyl trehalose trehalohydrolase from Arthrobacter sp. Q36 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE Toyopearl 650S, and Toyopearl HW-55S. The enzyme specifically catalyzed the hydrolysis of the alpha-1,4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. The Km of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose was 5.5 mM, 4.6 mM, 7.0 mM, and 4.2 mM, respectively. The enzyme had a molecular mass of 62,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was threonine. The enzyme showed the highest activity at pH 6.5 and 45 degrees C, and was stable from pH 5.0 to 10.0 and up to 45 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, and Zn2+. PMID- 8611746 TI - Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. AB - The keratinase produced by Bacillus licheniformis PWD-1 was induced by feather powder. Maximal enzyme production could be achieved by culturing in a medium containing 1% hammer-milled feather powder (100 mesh) at 45 degrees C for 30 h. Maximal growth of PWD-1 was achieved at 50 degrees C, and maximal enzyme induction was at 45 degrees C. The molecular mass and isoelectric point of this enzyme were 31.4 kDa and 8.5, respectively. This enzyme was stable from pH 5 to 12. The optimal reaction pHs for feather powder and casein were 8.5 and 10.5 to 11.5, respectively. The optimal reaction temperature was 50 degrees C to 55 degrees C. The relative activity of this enzyme toward casein, feather powder, keratin, elastin, and collagen was 100:52:41:18:7, and 100:56:32:3 for Suc-AAPL pNA, Suc-AAPF-pNA, Suc-AAPM-pNA, and Suc-AAVA-pNA (Suc, succinyl; pNA, p nitrophenylanilide). PMID- 8611747 TI - Characteristic expression of three amylase-encoding genes, agdA, amyB, and glaA in Aspergillus oryzae transformants containing multiple copies of the agdA gene. AB - In Aspergillus oryzae wild-type strains, the expression of the agdA gene encoding alpha-glucosidase (AGL) is induced by maltose at the transcriptional level in a similar manner to the amyB gene encoding Takaamylase A (TAA) and the glaA gene encoding glucoamylase (GLA). In A. oryzae transformants containing multiple copies of the agdA gene, a high-level of AGL activity was observed. This was accompanied by a significant reduction in TAA and GLA activities. Moreover, transformants with the highest AGL activity showed the lowest degree of TAA and GLA activities. Northern blot analyses showed that the transcriptional levels of amyB and glaA in the AGL-overproducing transformant were drastically reduced when large amounts of agdA mRNA were detected in maltose-grown mycelia. In addition, the glucose concentration of the maltose-containing medium that was used to grow the AGL-overproducing transformant for RNA extraction was higher than that of the control transformant. These results suggest that the reduced expression of the amyB and glaA genes in the AGL-overproducing transformant was due to either titration of a common regulatory protein(s) involved in maltose induction or carbon catabolite repression. PMID- 8611749 TI - High cell density cultivation and high recombinant protein production of Escherichia coli strain expressing uricase. AB - Uricase from Cellulomonas flavigena SK-4 is an industrially useful enzyme for commercial formulations of hair coloring. The uricase production by recombinant Escherichia coli strain with a high cell density cultivation technique was described. Of three kinds of media, synthetic media with the feeding of a high concentration of glucose solution were suitable for high cell density cultivation. As for feeding, both biomass concentration and uricase productivity were increased by about two (61.2 g dry cell weight (DCW)/liter) and three times (1037 U/ml broth), respectively, in 24 h by continuous supply. In the case of feeding by a DO-stat method, however, cell concentration was comparable to continuous glucose supply but uricase activity was reduced. By supplying pure oxygen to compensate for oxygen limitation during cultivation, the highest values of 77.4g DCW/liter and 1113 U/ml broth of the uricase activity were achieved with the total cultivation time of 15 h. PMID- 8611748 TI - Effects of freezing on the proteolysis of beef during storage at 4 degrees C. AB - The effects of freezing on the proteolysis of beef during storage at 4 degrees C after being thawed was investigated. A sarcoplasmic 32-kDa protein in frozen as well as unfrozen beef decreased rapidly during storage at 4 degrees C, and a more than 100-kDa protein appeared in both beef samples. And the increment of peptides in the frozen beef during the storage at 4 degrees C was larger than that in the unfrozen beef, suggesting that the proteolysis was faster during the storage of the former than the latter. However, its increment in the frozen beef for 10 weeks during the storage at 4 degrees C became smaller than that of the one frozen for less than 5 weeks. To discover an indicator for evaluation of the conditioning of frozen and unfrozen beef, peptides produced during the storage of beef at 4 degrees C were surveyed. A peptide, APPPPAEVPEVHEEV, was detected and seemed to be available as an indicator in the conditioning of beef. PMID- 8611750 TI - Interaction of sesamin and eicosapentaenoic acid against delta 5 desaturation and n-6/n-3 ratio of essential fatty acids in rat. AB - Sesamin is a specific inhibitor of delta 5 desaturation, the conversion from dihomo-gamma-linolenic acid (20:3, n-6) to arachidonic acid (AA, 20:4, n-6). Previously, we reported that sesamin inhibited delta 5 desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20:4 (n-3) to eicosapentaenoic acid (EPA, 20:5, n-3). In this study, we investigated the interaction of sesamin and EPA on delta 5 desaturation of both series and the n 6/n-3 fatty acids ratio by measuring actural fatty acid contents in vivo. Rats were fed three types of dietary oils; 1) linoleic acid (LA, 18:2, n-6): linolenic acid (LLA, 18:3, n-3) = 3:1, n-6/n-3 ratio of 3:1 (LA group), 2) LA:LLA = 1:3, n 6/n-3 ratio of 1:3 (LLA group), 3) LA:LLA:EPA = 1:0.5:3, n-6/n-3 ratio of 1:3.5 (EPA group) with or without sesamin (0.5% w/w) for 4 weeks. In all groups, sesamin administration increased the content of dihomo-gamma-linolenic acid (20:3, n-6) in the liver and decreased the delta 5 desaturation index of n-6 fatty acid, the ratio of 20:4/20:3 (n-6). On the contrary, the delta 5 desaturation index of n-3 fatty acid, the ratio of 20:5 + 22:5 + 22:6/20:4 (n-3), was increased by the administration of sesamin. These results suggest that sesamin inhibits the delta 5 desaturation of n-6 fatty acid, but not that of n-3 fatty acid in rat livers. Sesamin administration decreased incorporation of EPA (n-3) and simultaneously increased the AA (n-6) content in the liver. The n-6/n-3 ratio in the liver was increased by administering sesamin under n-3 rich conditions, i.e., the LLA and EPA groups. PMID- 8611751 TI - Alpha s1-casein-specific CD8+ T cell clone that inhibits its own interferon-gamma production by producing interleukin-10. AB - A CD8+ T cell clone specific to alpha s1-casein, one of the major allergens in milk, is shown to inhibit its own production of interferon (IFN)-gamma by producing interleukin (IL)-10. Anti-IL-10 antibodies enhanced the production of IFN-gamma induced by the antigen plus antigen-presenting cells from 12 h onward after initiating the culture. This enhancing effect was observed only when the cells were stimulated in the presence of the antigen-presenting cells. Neither IL 2 nor IL-4 abrogated this enhancing effect. This reveals a new regulating mechanism for IFN-gamma production from CD8+ T cells. PMID- 8611752 TI - A new enzymatic method for the measurement of creatinine involving a novel ATP dependent enzyme, N-methylhydantoin amidohydrolase. AB - A new enzymatic method for the measurement of serum and urine creatinine is described. The method is based on a novel microbial creatinine degradation pathway via N-methylhydantoin [Shimizu et al., Clin. Chim. Acta, 185, 241-252 (1989)]. By using two novel enzymes, N-methylhydantoin amidohydrolase and N carbamoylsarcosine amidohydrolase, as key enzymes, coupled with a colorimetric procedure for hydrogen peroxide detection, the creatinine level can be measured. The results obtained for human serum and urine show good correlation with those obtained by a standard chemical method based on the Jaffe reaction. The new method is simple and specific, and shows excellent sensitivity and reliability. PMID- 8611753 TI - Pseudomonas fluorescens KKL101, a benzoic acid degrader in a mixed culture that degrades biphenyl and polychlorinated biphenyls. AB - A mixed culture we had isolated, which degrades biphenyl/polychlorinated biphenyls, is composed of two strains, Pseudomonas fluorescens KKL101 and Pseudomonas sp. strain KKS102. KKS102 produces benzoic acid as a dead-end metabolite in the degradation of biphenyl. In this study we showed that KKL101 grew on benzoic acid as a sole source of carbon. This indicated a role of KKS102 in the growth of KKL101 and KKL101 for the growth of KKS102 in the mixed culture. PMID- 8611754 TI - Effects of degraded schizophyllans on regeneration of protoplast cells of Saccharomyces cerevisiae. AB - Schizophyllan was heated at 100 degrees C in 85% dimethyl sulfoxide (DMSO) containing 0.01 M H2SO4 for various times, and fractionated by gel-permeation chromatography. Molecular weights (M(r)) of the depolymerized products thus obtained were measured in water and DMSO by GPC-LALLS to estimate their conformations in water. The products with triple helical structure stimulated regeneration of Saccharomyces cerevisiae protoplast cells, while those of single chain conformation were totally inactive. PMID- 8611755 TI - Six-membered cyclic phosphonate GABA antagonists, 2,5-disubstituted 1,3,2 dioxaphosphorinanes. AB - The trans isomer of 2-(4-bromophenyl)-5-tert-butyl-2-thiono-1,3,2 dioxaphosphorinane competitively inhibited the specific binding of 35S-tert butylbicyclophosphorothionate to rat brain membranes with an IC50 value of 0.52 microM, and showed insecticidal activity against houseflies with an LD50 value of 2.4 micrograms/fly. This compound and its analogues acted as noncompetitive GABAA receptor antagonists (NGRAs), and phosphorus-containing cyclohexane skeletons may prove useful for the design of novel NGRAs. PMID- 8611756 TI - Oxidation of acetophenone by Aspergillus species and their possible contribution to Katsuobushi flavor. AB - Acetophenone was converted to phenol by A. glaucus MA0200. Production of phenol, which has a pungent flavor, seemed to give a contrary effect on the creation of flavor of molded Katsuobushi. Production of phenol is the process of degradation of acetophenone, also with a pungent flavor. It would play a role in the decreasing of the pungent flavor of Katsuobushi. PMID- 8611757 TI - Tocopherol levels in the plasma lipoproteins from Japanese eel Anguilla japonica. AB - The alpha- and gamma-tocopherol levels in the plasma of Japanese eel (Anguilla japonica) were 53.9 micrograms and 1.3 micrograms/ml of plasma, respectively. The alpha-tocopherol in the plasma was mainly distributed as very-low-density lipoprotein and low-density lipoprotein (LDL), while LDL and high-density lipoprotein constituted most of the gamma-tocopherol. A highly positive coefficient of correlation was observed between the alpha-tocopherol and lipoprotein contents in Japanese eel plasma. PMID- 8611758 TI - Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill). AB - The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule. PMID- 8611759 TI - Purification and some properties of alpha-galactosidase from Penicillium purpurogenum. AB - alpha-Galactosidase was purified by ion-exchange chromatographies on DEAE cellulose and SE-cellulose columns from the culture filtrate of Penicillium purpurogenum No. 618. The final preparation was judged homogeneous by SDS-PAGE and its molecular mass and isoelectric point were estimated to be 67 kDa and 4.1, respectively. The N-terminal amino acid sequence of the enzyme was analyzed and aligned with those of other alpha-galactosidases. In addition, the enzyme acted on the stubbed alpha-galactosyl residue connected to the beta-1,4-manno oligosaccharide chain, indicating that this specificity was quite different from that of Mortierella vinacea alpha-galactosidase. PMID- 8611760 TI - Blasticidin S deaminase gene (BSD): a new selection marker gene for transformation of Arabidopsis thaliana and Nicotiana tabacum. AB - Arabidopsis thaliana and Nicotiana tabacum were transformed to blasticidin S (BS) resistance with BSD (the BS deaminase gene from Aspergillus terreus) using the Agrobacterium-mediated transformation method. Expression of BSD allowed direct selection of transformants by the fungicide, and both kinds of transgenic plants showed high level of resistance phenotype at 100 ppm of BS sprayed on the leaves. Using Botrytis cinerea, the causal fungus of gray mold disease, it was exemplified that application of BS could control the disease in transgenic tobacco with negligible phytotoxicity. PMID- 8611761 TI - Transcriptional regulation of tyrosine phenol-lyase gene of Erwinia herbicola AJ2985. AB - Induction and repression of tyrosine phenol-lyase (TPL; EC 4.1.99.2) of Erwinia herbicola AJ2985 were examined on the transcriptional level and it was shown that transcription of tpl was increased by the addition of tyrosine and decreased by the addition of glucose in the medium. The 5' flanking region of its gene, tpl, was analyzed and its transcriptional start point was determined. A presumed -35, 10 promoter region, TyrR box, and operator-like region were also found in this region. PMID- 8611763 TI - BbrPI, an extracellular proteinase inhibitor of Bacillus brevis, protects cells from the attack of exogenous proteinase. AB - BbrPI, an extracellular serine proteinase inhibitors of B. brevis HPD31, is activated by proteolytic processing of an inactive precursor. To discover the physiological role of BbrPI, its deficient mutants were genetically constructed. Although the BbrPI deficient mutant had a higher extracellular proteinase activity than the parent strain, it grew normally. The BbrPI deficient mutant showed higher sensitivity to added trypsin than that of the parent. These observations suggest that the BbrPI may play a role in cellular protection from the attack of exogenous proteinases. PMID- 8611762 TI - Potentiation of the antihypertensive activity of orally administered ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidylcholine. AB - Ovokinin, a vasorelaxing octapeptide derived from ovalbumin, significantly lowered the systolic blood pressure of spontaneously hypertensive rats (SHR) when orally administered as an emulsion in 30% egg yolk at a dose of 25 mg/kg, this effect being larger than that of the peptide administered as a solution at a dose of 100 mg/kg. Egg phospholipid, especially phosphatidylcholine, showed essentially the same effect as egg yolk. However, egg neutral lipid was ineffective. Soybean phospholipid was less effective than egg phospholipid in potentiating the antihypertensive activity of ovokinin. PMID- 8611764 TI - Formation of 1:1 complex of the cytokine receptor homologous region of granulocyte colony-stimulating factor receptor with ligand. AB - The cytokine receptor homologous (CRH) region of the murine granulocyte colony stimulating factor (G-CSF) receptor was secreted using a Escherichia coli maltose binding protein (MBP) fusion system. The CRH region was prepared from the periplasmic fraction by G-CSF affinity column chromatography and restriction protease factor Xa digestion, and was purified to homogeneity. The purified CRH region specifically bound G-CSF, with an apparent dissociation constant (kd) of about 1.5 x 10(-9) M. A 1:1 CRH.G-CSF complex was established by gel-filtration high pressure liquid chromatography (HPLC). However, a 2:1 stoichiometric complex was not established, as in the case of the growth hormone (GH) receptor [Recent Prog. Hormone Res., 48, 233-275 (1993)]. PMID- 8611765 TI - A toluene-tolerant mutant of Pseudomonas aeruginosa lacking the outer membrane protein F. AB - The outer membrane protein profiles of a toluene-tolerant mutant, Pseudomonas aeruginosa, strain PAK103, were compared with those of its parent strain PAO1161. Protein F (OprF), the most abundant outer membrane protein in the parental strain PAO1161, was missing in the toluene-tolerant strain PAK103. The absence of OprF may lead to the loss of toluene diffusion across in the outer membrane of the mutant cells. PMID- 8611766 TI - Small bowel transplantation. PMID- 8611767 TI - Housing reform: getting tough on poor people. PMID- 8611768 TI - Near patient testing in primary care. PMID- 8611769 TI - Impaired glucose tolerance. PMID- 8611770 TI - Chronic coronary artery disease: drugs, angioplasty, or surgery? PMID- 8611772 TI - Tobacco executive goes public over company lies. PMID- 8611771 TI - Renal services in UK are underfunded, says report. PMID- 8611773 TI - Australia targets men's health. PMID- 8611774 TI - Religion should not put a child's health at risk. PMID- 8611775 TI - uS has oversupply of doctors. PMID- 8611776 TI - Environment and genetic link found. PMID- 8611777 TI - Fat substitute gets FDA approval. PMID- 8611778 TI - Irish transfusion service accused of criminal negligence. PMID- 8611779 TI - Saudi Arabia curbs tranquilliser prescribing. PMID- 8611780 TI - Health repercussions of the Harman case. PMID- 8611781 TI - Neglected aspects of false positive findings of mammography in breast cancer screening: analysis of false positive cases from the Stockholm trial. AB - OBJECTIVES: To examine the implications of false positive results of mammography in terms of the time lag from screening and complete mammography to the point when women with false positive results are declared free of cancer; the extra examinations, biopsies, and check ups required; and the cost of these extra procedures. DESIGN: Review of women with false positive results from the Stockholm mammography screening trial. SETTING: Department of Oncology, South Hospital, Stockholm. SUBJECTS: 352 and 150 women with false positive results of mammography from the first and second screening rounds of the Stockholm trial. MAIN OUTCOME MEASURES: Extra examinations and investigations required and the cost of these procedures. RESULTS: The 352 women from the first screening round made 1112 visits to the physician and had 397 fine needle aspiration biopsies, 187 mammograms, and 90 surgical biopsies before being declared free of cancer. After six months 64% of the women (219/342) were declared cancer free. The 150 women in the second round made 427 visits to the physician and had 145 fine needle aspiration biopsies, 70 mammograms, and 28 surgical biopsies, and after six months 73% (107/147) were declared cancer free. The follow up costs of the false positive screening results were Kr2.54m (250,000 pounds) in the first round and Kr0.85m (84,000 pounds) in the second round. Women under 50 accounted for about 41% of these costs. CONCLUSIONS: The examinations and investigation carried out after false positive mammography --especially in women under 50--and the cost of these procedures are a neglected but substantial problem. PMID- 8611784 TI - Seasonal variation in deep vein thrombosis. PMID- 8611782 TI - Asymptomatic gonorrhoea and chlamydial infection in rural Tanzanian men. AB - OBJECTIVE: To measure the prevalence of urethritis due to Neisseria gonorrhoeae and Chlamydial infection trachomatis in rural Tanzanian men DESIGN: About 500 men aged 15-54 years were selected from each of 12 rural communities by random cluster sampling; interviewed concerning past or present symptoms of sexually transmitted diseases; and asked to provide a first catch urine specimen, which was tested for pyuria with a leucocyte esterase dipstick test. Subjects with symptoms or with a positive result on testing were examined, and urethral swabs were taken for detection of N gonorrhoeae by gram stain and of C trachomatis by antigen detection immunoassay. SETTING: Mwanza region, north western Tanzania. SUBJECTS: 5876 men aged 15-54 years. MAIN OUTCOME MEASURES: Prevalence of urethral symptoms, observed urethral discharge, pyuria, urethritis ( > 4 pus cells per high power field on urethral smear), N gonorrhoeae infection (intracellular gram negative diplococci), and C trachomatis infection (IDEIA antigen detection assay). RESULTS: 1618 (28%) subjects reported ever having a urethral discharge. Current discharge was reported by 149 (2.5%) and observed on examination in 207 (3.5%). Gonorrhoea was found in 128 subjects (2.2%) and chlamydial infection in 39 (0.7%). Only 24 of 158 infected subjects complained of urethral discharge at the time of interview (15%). CONCLUSION: Infection with N gonorrhoeae and C trachomatis is commonly asymptomatic among men in this rural African population. This has important implications for the design of control programmes for sexually transmitted disease. PMID- 8611783 TI - Randomised trial comparing hysterectomy with endometrial ablation for dysfunctional uterine bleeding: psychiatric and psychosocial aspects. AB - OBJECTIVE: To compare in psychiatric and psychosocial terms the outcome of hysterectomy and endometrial ablation for the treatment of dysfunctional uterine bleeding. DESIGN: Prospective randomised controlled trial. SETTING--Obstetrics and gynaecology department of a large teaching hospital. SUBJECTS: 204 women with dysfunctional bleeding for whom hysterectomy would have been the preferred treatment were recruited over 24 months and randomly allocated to hysterectomy (99 women) or to hysteroscopic surgery (transcervical resection (52 women) or laser ablation (53 women). MAIN OUTCOME MEASURES: Mental state, martial relationship, psychosocial and sexual adjustment in assessments conducted before the operation and one month, six months, and 12 months later. RESULTS: Both treatments significantly reduced the anxiety and depression present before the operation, and there were no differences in mental health between the groups at 12 months. Hysterectomy did not lead to postoperative psychiatric illness. Sexual interest after the operation did not vary with treatment. Overall, 46 out of 185 (25%) women reported a loss sexual interest and 50 out of 185 (27%) reported increased sexual interest. Marital relationships were unaffected by surgery. Personality and duration of dysfunctional uterine bleeding played no significant part in determining outcome. CONCLUSIONS: Hysteroscopic surgery and hysterectomy have a similar effect on psychiatric and psychosocial outcomes. There is no evidence that hysterectomy leads to postoperative psychiatric illness. PMID- 8611785 TI - Concerns about AIDS in general practice. PMID- 8611786 TI - Methods for managing the increased workload in anticoagulant clinics. PMID- 8611788 TI - Effects on asylum seekers of ill treatment in Zaire. AB - To describe the health effects of the political system in Zaire on asylum seekers seen at the Medical Foundation for the Care of Victims of Torture a retrospective study was performed of the records of 92 asylum seekers from Zaire who were seen for medical reports at the medical foundation in 1993 and 1994. Eighty one had been imprisoned; the others had been severely treated at home by the security services. Sixty six had been detained for up to one year. Prison conditions were invariably unsanitary, and food of poor quality when provided. All had been beaten on arrest, and all but two had been beaten repeatedly in prison. Nearly all the women and some of the men described sexual abuse. Almost all left prison through bribery or because a guard had a similar background. Seventy two asylum seekers had scarring, consider to be consistent with the history, and 70 were considered to have suffered persistent psychological damage. Asylum seekers from Zaire will have health effects from experiences unimaginable to the ordinary Briton. An understanding of the background will help clinicians manage them. PMID- 8611789 TI - Acute laryngeal obstruction in rheumatoid arthritis. PMID- 8611787 TI - Deprivation and cause specific morbidity: evidence from the Somerset and Avon survey of health. AB - OBJECTIVE: To investigate the association between cause specific morbidity and deprivation in order to inform the debates on inequalities in health and health services resource allocation. DESIGN: Cross sectional postal questionnaire survey ascertaining self reported health status, with validation of a 20% sample through general practitioner and hospital records. SETTING: Inner city, urban, and rural areas of Avon and Somerset. SUBJECTS: Stratified random sample of 28,080 people aged 35 and over from 40 general practices. MAIN OUTCOME MEASURES: Age and sex standardised prevalence of various diseases; Townsend deprivation scores were assigned by linking postcodes to enumeration districts. Relative indices of inequality were calculated to estimate the magnitude of the association between socioeconomic position and morbidity. RESULTS: The response rate was 85.3%. The prevalence of most of the conditions rose with increasing material deprivation. The relative index of inequalilty, for both sexes combined, was greater than 1 for all conditions except diabetes. The conditions most strongly associated with deprivation were diabetic eye disease (relative index of inequality 3.21; 95% confidence interval 1.84 to 5.59), emphysema (2.72; 1.67 to 4.43) and bronchitis (2.27; 1.92 to 2.68). The relative index of inequality was significantly higher in women for asthma (P < 0.05) and in men for depression (P < 0.01). The mean reporting of prevalent conditions was 1.07 for the most deprived fifth of respondents and 0.77 in the most affluent fifth (P < 0.001). CONCLUSIONS: Material deprivation is strongly linked with many common diseases. NHS resource allocation should be modified to reflect such morbidity differentials. PMID- 8611790 TI - Department of Health shoots itself in the hip. Why the report of the Advisory Group on Osteoporosis undermines evidence based purchasing. PMID- 8611791 TI - Department of Health is fair to patients with osteoporosis. PMID- 8611792 TI - ABC of general surgery in children. Problems with the penis and prepuce. PMID- 8611793 TI - Who should be doing what in the international health: a confusion of mandates in the United Nations? AB - Since 1945 at least five United Nations organisations have become substantially involved in international health activities. This has led to considerable confusion among policy makers, scholars, and UN staff over distinct and appropriate mandates. Interviews with staff an a historical analysis have shown that while formal mandates have been complementary, effective mandates have led to an unclear delineation of activities. The process of translating formal into effective mandates have been influenced by the decentralised nature of the UN, lack of a master plan for its activities, the considerable growth in the policy agenda and the shift towards a multisectoral approach to health. The identification of each organisation's comparative advantage, at both the global and country levels, is one way of understanding what each organisation does best and perhaps should be doing. There is a need for improved mechanisms to define effective mandates, taking into account comparative advantages, if the mandates of UN organisations are appropriate to meet future challenges in international health. PMID- 8611794 TI - Laparoscopic versus open repair of inguinal hernia. Long-term recurrence may be lower with laparoscopic repair. PMID- 8611795 TI - Laparoscopic versus open repair of inguinal hernia. Paper should have put more emphasis on benefits of laparoscopic repair. PMID- 8611796 TI - Laparoscopic versus open repair of inguinal hernia. Surgeons in study were inexperienced at laparoscopic repair. PMID- 8611797 TI - Laparoscopic versus open repair of inguinal hernia. Hernia repair should be individualised to the patient. PMID- 8611799 TI - Provision of palliative care is suboptimal. PMID- 8611798 TI - Laparoscopic versus open repair of inguinal hernia. Lichtenstein technique should be used. PMID- 8611800 TI - Fc epsilon RI-beta polymorphisms unlikely to contribute substantially to genetic risk of allergic disease. PMID- 8611801 TI - Training of overseas qualified doctors in Britain. Training programmes in countries overseas should be supported. PMID- 8611802 TI - Training of overseas qualified doctors in Britain. View of overseas doctors training in obstetrics and gynaecology. PMID- 8611803 TI - Publicity about certain aspects of meningococcal disease is still needed. PMID- 8611804 TI - Breast feeding should be promoted as a public health measure. PMID- 8611805 TI - Neonatal screening in New Zealand. PMID- 8611806 TI - Treatment of toenail tinea infection. True cure seems unlikely. PMID- 8611807 TI - Surgical bleeding and calcium antagonists. PMID- 8611808 TI - Insulin dependent diabetes mellitus in twins. Differences between monozygotic and dizygotic twins may need to be taken into account. PMID- 8611809 TI - Career choices of trainees in general practice. PMID- 8611810 TI - Inquiry is needed into difficulties of developing academic careers. PMID- 8611811 TI - Use of predeposit autologous blood infusion varies greatly. PMID- 8611812 TI - Concerns about "lookback" exercise for hepatitis C. PMID- 8611813 TI - Hospital patients in Bosnia are nutritionally vulnerable. PMID- 8611814 TI - Moving house unlikely to pose substantial risk of childhood asthma. PMID- 8611815 TI - Somatic gene therapy. PMID- 8611816 TI - Health promotion in primary care. PMID- 8611817 TI - Treatment resistance in schizophrenia. PMID- 8611818 TI - Education for educating surgeons. PMID- 8611819 TI - Autistic spectrum disorders. PMID- 8611820 TI - South Africa poised for national health system. PMID- 8611821 TI - US rape case leaves ethical uproar. PMID- 8611822 TI - Medical litigation faces British revolution. PMID- 8611823 TI - Canadian women compensated for sterilisation. PMID- 8611824 TI - Israelis destroy Ethiopian blood donations. PMID- 8611825 TI - French AIDS group criticises treatment of immigrants. PMID- 8611826 TI - Australia is failing with childhood immunisation. PMID- 8611827 TI - Britain investigates Gulf war syndrome. PMID- 8611828 TI - GP fundholder reported to GMC. PMID- 8611829 TI - Disease in children infected with HIV in Abidjan, Cote d'Ivoire. AB - OBJECTIVE: To document the range of disease in African children infected with HIV. DESIGN: Necropsy results in consecutive children aged 1 month or more who were HIV positive and in children who were HIV negative for comparison; IgA western blots on serum samples from children under 2 years of age who were positive for HIV-1 to test the validity of routine HIV serology. SETTING: Largest hospital in Abidjan, Cote d'Ivoire. SUBJECTS: 78 children who were HIV positive and 77 children who were HIV negative on whom a necropsy was performed; their median ages at death were 18 and 21 months respectively. 36 HIV positive children and 29 HIV negative children were 1-14 months old; 42 HIV positive and 48 HIV negative children were > or = 15 months old. MAIN OUTCOME MEASURES: Cause of death and prevalence of diseases confirmed pathologically. RESULTS: Respiratory tract infections were more common in HIV positive than in HIV negative children (73 (94%) v 52 (68%); P < 0.05), and were aetiologically heterogeneous. Pneumocystis carinii pneumonia was found in 11 out of 36 (31%) HIV positive children aged < 15 months, but in no HIV negative children. Among older children measles was more common in HIV positive children (8/42 (19%) v 2/48 (4%); P < 0.06). Pyogenic meningitis was present in similar proportions of HIV positive and HIV negative children aged < 15 months (7/36 (19%) and 7/29 (24%)). In HIV positive children tuberculosis (1/78), lymphocytic interstitial pneumonitis (1/78), and HIV encephalitis (2/78) were rare. CONCLUSIONS: There is greater overlap between diseases associated with HIV infection and other common health problems in African children than there is in adults. Compared with adults, HIV positive children had a high prevalence of P carinii pneumonia and a low prevalence of tuberculosis. Measles, but not malaria, was associated with HIV infection. PMID- 8611830 TI - Single or multiple daily doses of aminoglycosides: a meta-analysis. AB - OBJECTIVE: To assess relative efficacy and toxicity of aminoglycosides given by single daily dose compared with multiple daily doses. DESIGN: Meta-analysis of 21 randomised trials identified through MEDLARS (1966 to January 1995). Data were overviewed with fixed effects and random effects models and with meta-regression analysis. SUBJECTS: Total of 3091 patients with bacterial infection, most without pre-existing renal disease. INTERVENTIONS: Patients were randomized to receive aminoglycosides once daily or multiple times daily with similar total daily dose. MAIN OUTCOME MEASURES: Clinical failure of treatment, nephrotoxicity, ototoxicity, and mortality. RESULTS: Single daily dose regimen produced a non significant decrease in risk of antibiotic failures (random effects risk ratio 0.83 (95% confidence interval 0.57 to 1.21)). Benefit of once daily dosing was greater when the percentage of pseudomonas isolates in a trial was larger. Once daily administration reduced risk of nephrotoxicity (fixed effects risk ratio 0.74 (0.54 to 1.00)). Similar trends were noted for patients with febrile neutropenia and for children. There was no significant difference in ototoxicity between the two dosing regimens, but the power of the pooled trials to detect a meaningful difference was low. There was no significant difference in mortality. CONCLUSIONS: Once daily administration of aminoglycosides in patients without pre existing renal impairment is as effective as multiple daily dosing, has a lower risk of nephrotoxicity, and no greater risk of ototoxicity. Given the additional convenience and reduced cost, once daily dosing should be the preferred mode of administration. PMID- 8611832 TI - Absence of dyspeptic symptoms as a test of Helicobacter pylori eradication. PMID- 8611831 TI - Compliance therapy in psychotic patients: randomised controlled trial. AB - OBJECTIVE: To determine whether compliance therapy, a cognitive-behavioural intervention, could improve compliance with treatment and hence social adjustment in acutely psychotic inpatients, and if so, whether the effect persisted six months later. DESIGN: Randomised controlled trial of compliance therapy and non specific counselling, each comprising 4-6 sessions lasting 10-60 minutes. SETTING: Acute psychiatric admissions ward serving an inner London catchment area. SUBJECTS: 47 patients with psychosis. MAIN OUTCOME MEASURES: Informant and observer reported measure of compliance; observer assessed global functioning after intervention and three and six months later; self-rated attitudes to drug treatment after the intervention and one month later; symptom scores after intervention and six months later. RESULTS: 25 patients received compliance therapy and showed significantly greater improvements in their attitudes to drug treatment and in their insight into illness and compliance with treatment compared with the control group. These gains persisted for six months. The intervention group was 5.2 times more likely than the control group to reach a criterion level of compliance (95% confidence interval 1.5 to 18.3). Global functioning showed a tendency to improve more in the intervention group after a delay (odds ratio 3.0 (0.8 to 11.5) to reach the criterion level at six months). Four subjects given compliance therapy and six in the control group were readmitted during follow up (odds ratio 2.0 (0.48 to 8.2)). CONCLUSIONS: Compliance therapy is a pragmatic method for improving compliance with drug treatment in psychotic inpatients and its gains persist for at least six months. Overall functioning may also be enhanced. PMID- 8611833 TI - Experience of medical senior house officers in preparing discharge summaries. PMID- 8611834 TI - Life expectancy in osteogenesis imperfecta. PMID- 8611835 TI - Future provision of out of hours primary medical care: a survey with two general practitioner research networks. AB - OBJECTIVE: To ascertain general practitioners' views about the future provision of out of hours primary medical care. DESIGN: Self completing postal questionnaire survey. SETTING: Wessex and north east England. SUBJECTS: 116 general practitioners in the Wessex Primary Care Research Network and 83 in the Northern Primary Care Research Network. MAIN OUTCOME MEASURES: Intention to reduce or opt out of on call; plans for changing out of hours arrangements; the three most important changes needed to out of hours care; willingness to try, and perceived strengths and limitations of, three alternative out of hours care models--primary care emergency centres, telephone triage services, and cooperatives. RESULTS: The overall response rate was 74% (Wessex research network 77% (89/116), northern research network 71% (59/83)). Eighty three per cent of respondents (123/148) were willing to try at least one service model, primary care emergency centres being the most popular option. Key considerations were the potential for a model to reduce time on call and workload, to maintain continuity of care, and to fit the practice context. Sixty one per cent (91/148) hoped to reduce time on call and 25% (37/148) hoped to opt out completely. CONCLUSIONS: General practitioners were keen to try alternative arrangements for out of hours care delivery, despite the lack of formal trials. The increased flexibility in funding brought about by the recent agreement between the General Medical Services Committee and the Department of Health is likely to lead to a proliferation of different schemes. Careful monitoring will be necessary, and formal trials of new service models are needed urgently. PMID- 8611837 TI - Ways of measuring rates of recurrent events. AB - Recurrent events are common in medical research, yet the best ways to measure their occurrence remain controversial. Moreover, the correct statistical techniques to compare the occurrence of such events across populations or treatment groups are not widely known. In both observational studies and randomised clinical trials one natural and intuitive measure of occurrence is the event rate, defined as the number of events (possibly including multiple events per person) divided by the total person-years of experience. This is often a more relevant and clinically interpretable measure of disease burden in a population than considering only the first event that occurs. Appropriate statistical tests to compare such event rates among treatment groups or populations require the recognition that some individuals may be especially likely to experience recurrent events. Straightforward approaches are available to account for this tendency in crude and stratified analyses. Recently developed regression models can appropriately examine the association of several variables with rates of recurrent events. PMID- 8611838 TI - ABC of general surgery in children. Lumps and swellings of the head and neck. PMID- 8611836 TI - A rational approach to the management of hepatitis C infection. PMID- 8611839 TI - Cerebral Whipple's disease. PMID- 8611840 TI - Avoiding premature coronary deaths in Asians in Britain. Cultural factors are important. PMID- 8611841 TI - Avoiding premature coronary deaths in Asians in Britain. Several key facts need to be considered. PMID- 8611842 TI - Avoiding premature coronary deaths in Asians in Britain. General practitioners should identify risks opportunistically. PMID- 8611843 TI - Avoiding premature coronary deaths in Asians in Britain. Benefit from controlling blood pressure was understated. PMID- 8611844 TI - Avoiding premature coronary deaths in Asians in Britain. Guidelines for pharmacological intervention are needed. PMID- 8611845 TI - Beta blockers have important role in pulmonary oedema due to mitral stenosis. PMID- 8611846 TI - Circumcision of children. PMID- 8611847 TI - Ratio of waist circumference to height may be better indicator of need for weight management. PMID- 8611848 TI - Female genital mutilation. PMID- 8611849 TI - Faecal incontinence in hospitals and residential and nursing homes for elderly people. PMID- 8611850 TI - Weightings for analysing general practices' prescribing. Pooling of data from practices was inappropriate. PMID- 8611851 TI - Early controlled clinical trials. PMID- 8611852 TI - Recent advances in obstetrics. Epidural analgesia in labour. PMID- 8611853 TI - Recent advances in obstetrics. Figures on screening for Down's syndrome are inaccurate. PMID- 8611854 TI - Recent advances in obstetrics. Testing for Down's syndrome carries too much stress. PMID- 8611855 TI - Recent advances in obstetrics. Transmission of HIV from mother to infant depends on many factors. PMID- 8611856 TI - Evidence-Based Medicine. Commentaries should be evidence based too. PMID- 8611857 TI - Management of blood loss in children of Jehovah's Witnesses. PMID- 8611858 TI - Dangers of cocaine and adrenaline paste. PMID- 8611859 TI - Few reprint requests come from less privileged countries. PMID- 8611860 TI - Life's unfair. PMID- 8611861 TI - Hormone replacement for men. PMID- 8611862 TI - The prone position in acute respiratory distress syndrome. PMID- 8611863 TI - Second primary cancers after childhood cancer. PMID- 8611865 TI - Promoting environmental health. PMID- 8611864 TI - Long term care of older people. PMID- 8611866 TI - Enteral nutrition after surgery. PMID- 8611868 TI - Rwanda's children struggle to face the future. PMID- 8611867 TI - Seascale cancer cluster not due to radiation. PMID- 8611869 TI - Young children want to look like supermodels. PMID- 8611870 TI - Europe needs gene patent laws. PMID- 8611871 TI - Too many doctors, and more on the way. PMID- 8611872 TI - Randomised trial of safety and efficacy of immediate postoperative enteral feeding in patients undergoing gastrointestinal resection. AB - OBJECTIVES: To assess whether immediate post-operative enteral feeding in patients who have undergone gastrointestinal resection is safe and effective. DESIGN: Randomised trial of immediate post-operative enteral feeding through a nasojejunal tube v conventional postoperative intravenous fluids until the reintroduction of normal diet. SETTING: Teaching hospitals in London. SUBJECTS: 30 patients under the care of the participating consultant surgeon who were undergoing elective laparotomies with a view to gastrointestinal resection for quiescent, chronic gastrointestinal disease. Two patients did not proceed to resection. MAIN OUTCOME MEASURES: Nutritional state, nutritional intake and nitrogen balance, gut mucosal permeability measured by lactulose-mannitol differential sugar absorption test, complications, and outcome. RESULTS: Successful immediate enteral feeding was established in all 14 patients with a mean (SD) daily intake of 6.78 (1.57)MJ (1622 (375) kcal before reintroduction of oral diet compared with 1.58 (0.14) MJ (377 (34) kcal) for those on intravenous fluids (P < 0.0001). Urinary nitrogen balance on the first postoperative day was negative in those on intravenous fluids but positive in all 14 enterally fed patients (mean (SD) - 13.2 (11.6) g v 5.3 (2.7) g; P < 0.005). There was no difference by day 5. There was no change in gut mucosal permeability in the enterally fed group but a significant increase from the test ratios seen before the operation in those on intravenous fluids (0.11(0.06) v 0.15 (0.12); P < 0.005). There were also fewer postoperative complications in the enterally fed group (P < 0.005). CONCLUSIONS: Immediate postoperative enteral feeding in patients undergoing intestinal resection seems to be safe, prevents an increase in gut mucosal permeability, and produces a positive nitrogen balance. PMID- 8611873 TI - Cohort study of predictive value of urinary albumin excretion for atherosclerotic vascular disease in patients with insulin dependent diabetes. AB - OBJECTIVE: To examine whether slightly elevated urinary albumin excretion precedes development of atherosclerotic vascular disease in patients with insulin dependent diabetes independently of conventional atherogenic risk factors and of diabetic nephropathy. DESIGN: Cohort study with 11 year follow up. SETTING: Diabetes centre in Denmark. SUBJECTS: 259 patients aged 19-51 with insulin dependent diabetes of 6-34 years' duration and without atherosclerotic vascular disease or diabetic nephropathy at baseline. MAIN OUTCOME MEASURES: Baseline variables: urinary albumin excretion, blood pressure, smoking habits, and serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor. END POINT: atherosclerotic vascular disease assessed by death certificates, mailed questionnaires, and hospital records. RESULTS: Thirty patients developed atherosclerotic vascular disease during follow up of 2457 person year. Elevated urinary albumin excretion was significantly predictive of atherosclerotic vascular disease (hazard ratio 1.06 (95% confidence interval 1.02 to 1.18) per 5 mg increase in 24 hour urinary albumin excretion, P = 0.002). Predictive effect was independent of age; sex; blood pressure; smoking; serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor; level of haemoglobin A(lc); insulin dose, duration of diabetes, and diabetic nephropathy (hazard ratio 1.04 (1.01 to 1.08) per 5 mg increase PMID- 8611874 TI - Sensitivity and specificity of QTc dispersion for identification of risk of cardiac death in patients with peripheral vascular disease. AB - OBJECTIVE: To determine whether QTc dispersion, which is easily obtained from a standard electrocardiogram, can predict those patients with peripheral vascular disease who will subsequently suffer a cardiac death, despite having no cardiac symptoms or signs. DESIGN: Patients with peripheral vascular disease were followed up for five years after they had had coronary angiography, radionuclide ventriculography, and their QTc dispersion calculated from their 12 lead electrocardiogram. SUBJECTS: 49 such patients were then divided into three groups: survivors (34), cardiac death (12), and non-cardiac death (3). MAIN OUTCOME MEASURE: Survival. RESULTS: The mean (SD; range) ejection fractions were similar in all three groups: survivors 45.9 (11.0; 27.0-52.0), cardiac death 44.0 (7.90; 28.5-59.0), and non-cardiac death 45.3 (4.55; 39.0-50.0). QTc dispersion was significantly prolonged in the cardiac death group compared with in the survivors (86.3(23.9; 41.0-139) v 56.5 (25.4; 25.0-164); P = 0.002). A QTc dispersion > or = 60 ms had a 92% sensitivity and 81% specificity in predicting cardiac death, QTc dispersion in patients with diffuse coronary artery disease was significantly (P < 0.05) greater than in those with no disease or disease affecting one, two, or three vessels. CONCLUSIONS: There is a strong link between QTc dispersion and cardiac death in patients with peripheral vascular disease. QTc dispersion may therefore be a cheap and non-invasive way of assessing the risk of cardiac death in patients with peripheral vascular disease. PMID- 8611875 TI - Twin births to mothers who are twins: a registry based study. AB - OBJECTIVES: To estimate the risk of having twin infants for mothers who are twins; to investigate the genetic influence on twinning. DESIGN: Retrospective study of multiple births in two nationwide registries. SETTING: Sweden. SUBJECTS: Multiple births among 31,586 deliveries between 1973 and 1991 to women who were twins. MAIN OUTCOME MEASURES: Numbers of monozygotic and dizygotic twin births expected and estimated. RESULTS: Women who are dizygotic twins have a moderately increased risk of having twins (relative risk 1.30, 95% confidence interval 1.14 to 1.49) which seems to be completely the result of dizygotic twinning. When a mother is a monozygotic twin, her risk of having twins of the same sex is significantly increased (1.47; 1.10 to 1.97). This is the result of an excess of monozygotic twins (39 pairs estimated, 18 expected). CONCLUSIONS: Women who are twins have an increased risk of giving birth to twins. Genetic components of monozygotic and dizygotic twinning seem to be independent. PMID- 8611876 TI - Haem iron intake in 12-36 month old children depleted in iron: case-control study. AB - OBJECTIVE: To compare the intakes of haem and non-haem iron in iron depleted and iron replete children. DESIGN: Case-control study. SETTING: Early Childhood Centres and a long day care centre in Sydney, Australia. SUBJECTS: Children aged 12-36 months depleted in iron and controls matched for age and sex. MEAN OUTCOME MEASURES: Iron status by using plasma ferritin concentration. A three day weighed dietary intake record completed by the parents. Risk factors for iron deficiency assessed by questionnaire. RESULTS: Fifty six iron depleted and 68 iron replete children participated. The average daily intake of haem iron was significantly lower in the iron depleted group (t = 2.392, P = 0.018); there was a tendency towards a lower average daily intake of non-haem iron (t = 1.724, P = 0.086) and vitamin C (t = 1.921, P = 0.057) for iron depleted children. Low intake of haem iron (< 0.71 mg/day) was significantly associated with iron depletion with an odds ratio fo 3.0 (P = 0.005). The proportion of iron depleted children who were given whole cows' milk before 12 months of age was almost double that of iron replete children; multivariate analysis showed that both haem iron intake and age of introduction of cows' milk were independently associated with iron depletion. CONCLUSIONS: The results of this study show that, in young children in developed countries, a lower haem iron intake is a major risk factor for iron depletion; the introduction of whole cows' milk before 12 months is further confirmed as a risk factor. Parental education on nutrition should now focus on these two aspects of nutrition for infants and young children. PMID- 8611877 TI - Crossover study of glyceryl trinitrate patches for controlling pain in women with severe dysmenorrhoea. PMID- 8611878 TI - Intake, output, and drop out in United Kingdom medical schools. PMID- 8611879 TI - Bronchospasm induced by isotretinoin. PMID- 8611880 TI - Pravastatin: interaction with oral anticoagulant? PMID- 8611881 TI - Ranitidine and aseptic meningitis. PMID- 8611882 TI - Waiting list management in general practice: a review of orthopaedic patients. AB - OBJECTIVE: To review all patients on a current general practice orthopaedic waiting list for outpatient appointments with regard to accuracy of the list, clinical priority, and need for further radiological investigation before hospital attendance. DESIGN: Record review by one general practitioner and a radiologist, and discussion with patients of management alternatives. SETTING: Six partner city centre urban fund-holding general practice, list size 8651 (29% low deprivation payment status). SUBJECTS: 116 adults on an orthopaedic waiting list. MAIN OUTCOME MEASURES: List accuracy (patient details and status on waiting list); clinical priority (severity of condition); further investigations (results of tests after radiological review). RESULTS: 32 patients (28%) were removed from the waiting list because of inaccuracies. 14 patients were considered to be high priority and referred to other hospitals by utilising waiting list initiative funds. Of these patients, five agreed to referral to another hospital (treatment completed on average within three months of rereferral), six did not wish to be rereferred, and two did not attend to discuss the offer and remained on the original waiting list. One prioritised patient had further radiological investigations, was reassured, and was taken off the waiting list. 10 patients had further investigations. These resulted in six patients being referred to other hospitals, three being taken off the waiting list, and one seeking private care. CONCLUSIONS: Systematic review of patients on an orthopaedic waiting list of one general practice, though time consuming, led to the identification of inaccuracies in the list and changes in management. Costs need further evaluation, but if the findings occur widely substantial benefits could be achieved for patients. PMID- 8611883 TI - Ophthalmology. PMID- 8611884 TI - Career destinations in 1994 of United Kingdom medical graduates of 1983: results of a questionnaire survey. AB - Cohort studies of doctors' career choices and career progression since the mid 1970s have shown important changes in the medical workforce, in specialist training, and in employment. Examples of these changes are the increasing proportion of women doctors and of doctors who wish to work part time, the emigration patterns of doctors, and the development of vocational training for general practice. Studies enable the effects of longer term changes to be assessed, and sometimes they inform current debate. PMID- 8611885 TI - Something in the air: survival after dramatic, unsuspected case of accidental carbon monoxide poisoning. PMID- 8611887 TI - ABC of urology. Subfertility and male sexual dysfunction. PMID- 8611886 TI - Grand Rounds--City Hospital, Nottingham. A complicated case of community acquired pneumonia. PMID- 8611888 TI - Contemplating a one child world. Fundamental rethinking is needed. PMID- 8611889 TI - Contemplating a one child world. Medical profession should give more decisive leadership. PMID- 8611890 TI - Contemplating a one child world. Medical students should be taught to appraise evidence on global health issues. PMID- 8611891 TI - Abortion without the woman's consent is unlikely to improve her depression. PMID- 8611892 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Counselors do not have to be genetic nurse specialists. PMID- 8611893 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Realistic cost must be established for genetic counselling in two step screening. PMID- 8611894 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Couple screening should be preferred for medical reasons. PMID- 8611895 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Authors should have used marginal analysis. PMID- 8611896 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Couple screening would be easier for many centres. PMID- 8611897 TI - Cost effectiveness of antenatal screening for cystic fibrosis. Study might be better described as a cost description of screening. PMID- 8611898 TI - Clinical trials and rare diseases. Statistical results should be expressed in different ways, depending on circumstances. PMID- 8611899 TI - Clinical trials and rare diseases. Trials of adequate size are possible with the right organisation. PMID- 8611900 TI - GMSC's advice on intrapartum care is unhelpful. PMID- 8611901 TI - Deaths in police custody are being analysed retrospectively. PMID- 8611902 TI - Role of police surgeons is changing. PMID- 8611903 TI - Drugs for atrial fibrillation. Measurement of the pulse deficit is unnecessary. PMID- 8611905 TI - Prescribing of quinine and drugs that induce cramp. Study did not consider several factors. PMID- 8611904 TI - Drugs for atrial fibrillation. Digoxin comes from Digitalis lanata. PMID- 8611906 TI - Techniques of vasovasostomy should be improved for men seeking fertility. PMID- 8611907 TI - Prescribing of quinine and drugs that induce cramp. GPs should not be deterred from prescribing quinine. PMID- 8611908 TI - BMA should look at inequalities in the NHS. PMID- 8611909 TI - Specialist breast and ovarian cancer clinics should be staffed by oncologist nurses. PMID- 8611910 TI - The 13 steps to community care. PMID- 8611912 TI - My brain stem stroke. PMID- 8611911 TI - Brain injury rehabilitation: jaw jaw not war war. PMID- 8611913 TI - Human resources. Rewards over the long haul. PMID- 8611914 TI - Finance. Teaching hospitals face credit crunch. PMID- 8611915 TI - The mouse that roared. AB - They don't call the onslaught of managed care a revolution for nothing: everything is different now. Especially communications technology. The key tasks of managed care, from identifying best practices to coordinating physicians, are impossible when relying on paper records alone. That's why more health systems are depending on automated medical records. Though it's still not too late to catch up to stay competitive, now could be your last chance. PMID- 8611916 TI - Ceo summit. At risk. PMID- 8611917 TI - Blessed be the board. PMID- 8611918 TI - Training. Back to school at Bradley U. PMID- 8611919 TI - Innovators. A doc who doubles as inventor. PMID- 8611920 TI - Integration. Taking care of business. PMID- 8611921 TI - Online. Dialing for patient data. PMID- 8611922 TI - Patrick Hays: CEO, Blue Cross and Blue Shield Association of America. Interview by Terese Hudson. PMID- 8611923 TI - Delivery channels. Call in for a consult. PMID- 8611924 TI - Quality patrol. Getting to the heart of chest pain. PMID- 8611925 TI - Governance. Strength in numbers. PMID- 8611926 TI - Marketing. A pitch for organ donations. PMID- 8611927 TI - Another nurse-case scenario. PMID- 8611928 TI - Hospital volunteerism in the '90s. PMID- 8611929 TI - [Is cervical screening justified in adolescents?]. PMID- 8611930 TI - [Physiopathologic anomalies in ovarian response]. PMID- 8611931 TI - [Tocolytic drugs]. PMID- 8611932 TI - [The condom. A little known product in 1995]. PMID- 8611933 TI - [Magnetic resonance imaging in gynecology]. PMID- 8611934 TI - [Epidemiology of ectopic pregnancy: incidence and risk factors]. AB - During the past two decades, the incidence of ectopic pregnancy (EP) has doubled or tripled in many parts of the world. In France, EP currently constitutes 2 % of livebirths and 1.6 % of all reported pregnancies. These rates seem presently stable. The main identified risk factors include pelvic inflammatory disease (PID)--in particular that due to Chlamydial infection, previous ectopic pregnancy and cigarette smoking. More than 50 % of EP cases are attributable to infectious factors and cigarette smoking, suggesting dramatic effects on EP rates of appropriate prevention programs. A third risk which could be prevented is induced conception cycles, especially by Clomiphene. Other risk factors are pelvic surgery, previous EP and maternal age (especially over 35 years). Further epidemiologic research is needed to identify new risk factors, to monitor incidence rates and to evaluate the effects of public health policies on EP occurrence. French EP registers will certainly contribute to this research. PMID- 8611935 TI - [Radical laparoscopic surgery of ectopic pregnancy: results from a continuous series of 383 interventions]. AB - From January 1983 to December 1993, 375 patients underwent laparoscopic salpingectomy. Six complications (1.5 %) were observed. Among 145 patients with a patent contralateral tube who desired pregnancy, 73 had an intrauterine pregnancy (50.3 %) and 22 had ectopic pregnancy (15.2 %). These results are discussed according to the contralateral tube status and gynecologic past history. PMID- 8611936 TI - [Hormonal replacement treatment: therapeutic protocols]. AB - Hormonal replacement therapy concerns an increasing number of women: therefore it would be beneficial to vary its doses and administration modes in order to ensure the treatment's efficiency and innocuity. Furthermore, this treatment has to take into account the fact that women often wish not to have withdraw bleedings. It also has to adapt to various clinical situations like fibroids, adenomyosis, endometriosis, hysterectomy and old age. The general situation and particular cases are discussed in this text. PMID- 8611937 TI - [Breast cancer during pregnancy. Epidemiology--diagnosis--prognosis]. AB - The association "breast cancer and pregnancy" is rare. The hypervascularisation of the pregnant breast, the role of hormones, the immunological factors have been incriminated. The rate of diagnosis is often too late because of the changes in the pregnant breast. Among the investigations, besides mammogram, the interest of ultrasound examination especially cyto-punction is underlined. Prognosis appears more severe because the delay in diagnosis and the young age of patients. However that is true for N+ patterns, and not for N- cases. We detail the experience of the Centre des Maladies du Sein de l'Hopital Saint-Louis which concerns 15 cases. PMID- 8611938 TI - [Influence of infertility etiology and follicular stimulation protocols on pregnancy outcomes by in vitro fertilization]. AB - During a retrospective study on 152 singles pregnancies obtained by in vitro fecondation at Tenon hospital, and followed in this department between January 1990 and December 1994, we have studied the influence of the IVF (tubal origin, masculine or idiopathic) and the type of stimulation (human menopausal gonadotrophin: hMG or follicle stimulating hormone: FSH) on the weight of the newborn and the pathologies that occurred during the pregnancy. No difference in the antecedents has been found in the different group of patients. This study shows a significative difference (p < 0.001) of the newborn's weight when the indication of IVF is tubal origin or masculine. Also, in all IVF indications, the weight is significantly (p < 0.01) higher after a follicular stimulation by FSH versus hMG. For the pregnancy pathologies, no significative difference has been noted, although arterial hypertension, fetal growth retardation and gestational diabetes appear to be more frequent in the group of women who had stimulation by hMG. PMID- 8611939 TI - [Comparison of the effects of fenticonazole and econazole on the aspartic proteinase secreted by Candida albicans]. AB - In this note, the effect of fenticonazole on secretory aspartic proteinase of the human opportunistic fungus Candida albicans is shown, in a comparison with econazole. Both antigenic and enzymatic assays demonstrate that fenticonazole, in contrast to econazole, greatly reduces the production of the virulence enzyme by stationary-phase C. albicans. This inhibitory effect was specific and was not mediated by the inhibition of fungal growth. These results confirm fenticonazole's unique property, already shown in previous studies. PMID- 8611940 TI - Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography. AB - The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA2 were used in this study. Protein mixture I was PLA2 obtained from Sigma which contained approximately 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from flesh bovine pancreatic tissue and contained > 100 proteins including the target protein, PLA2. After adsorbing Sigma PLA2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA2. Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels. PLA2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA2 from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%. PMID- 8611941 TI - High-performance liquid chromatographic analysis of retinal and retinol isomers. AB - Normal-phase chromatograms of retinal- and retinol isomers using various mobile phrases are presented, showing that in n-hexane--tert.-butyl methyl ether and also in n-heptane--tert.-butyl methyl ether the elution order for retinol is 13 cis-, 11-cis-, 9-cis- and all trans-retinol, whereas in n-hexane-1,4-dioxane 11 cis- elutes before 13-cis-retinol. Assignment of these main retinal and retinol peaks was performed using pure crystals of isomers, measurement of absorbance spectra and analysis of NMR spectra. The new mobile phase n-heptane-tert.-butyl methyl ether allows best baseline separation of the commonly occurring isomeric forms in reasonably short analysis time. In addition, eight di-cis- and tri-cis retinol isomers were tentatively identified. The chromatograms show that former identifications in the literature are inconsistent or wrong. PMID- 8611942 TI - Quantitative structure-activity relationships studies with micellar electrokinetic chromatography. Influence of surfactant type and mixed micelles on estimation of hydrophobicity and bioavailability. AB - Applications of micellar electrokinetic chromatography (MEKC) in quantitative structure-activity relationships (QSAR) were studied. First, quantitative structure-retention relationships (QSRR), which describe the correlation between logarithm of capacity factor (log k') in MEKC and logarithm of distribution coefficient between 1-octanol and water (log P(ow)), were investigated for 60 aromatic compounds and 9 corticosteroids using three different anionic surfactants [e.g., sodium dodecyl sulfate (SDS), sodium cholate (SC), and lithium perfluorooctane sulfonate (LiPFOS)], one cationic surfactant (C14TAB), and mixed anionic micellar systems. Linear solvation energy relationships (LSER) and solvatochromic parameters were used to shed light on the different log k' vs. log P(ow) relationships of the various surfactants. It was concluded that hydrogen bonding interactions have a great influence on retention behavior in MEKC and its relationships with hydrophobicity. Interestingly, bile salt surfactants (e.g., SC) and mixed bile salt micellar systems provide better correlations for log k' vs. log P(ow) than SDS and/or SDS with buffer additives (e.g., beta-cyclodextrin, urea, and acetonitrile). Using SC micelles, only one line was adequate to describe the relationship between retention in MEKC and hydrophobicity for a group of 60 aromatic compounds. The existence of higher correlation for the SC system was attributed to a similar hydrogen bonding pattern between SC micelles and 1-octanol. In the SDS system, however, three lines were recognized for the congeneric subgroups of compounds. This is due to the hydrogen bond door (HBD) characteristic of SDS micelles that selectively differentiate between the solutes with different hydrogen bond acceptor (HBA) strength, thus demonstrating that retention is not solely based on hydrophobicity. A similar result was observed for a C14TAB-MEKC system, however, the HBA characteristic of C14TAB selectively differentiates between the solutes with different HBD strength. In addition, quantitative retention-activity relationships in MEKC were also investigated for 9 corticosteroids. Two types of biological activities [small intestinal absorption in the rat (log A/NA) and protein binding to human serum albumin (log B/F) were examined in this work. High correlations were observed between bioactivity and log k' in MEKC using bile salt surfactants and mixed bile salt systems. PMID- 8611943 TI - Micellar electrokinetic capillary chromatography of fungal metabolites. Resolution optimized by experimental design. AB - Fungal metabolites were extracted from two isolates of Penicillium commune and two isolates of Aspergillus versicolor grown on yeast extract agar (YES) and Czapek yeast extract agar (CYA). Optimized conditions for analysing the extracts by high-performance capillary electrophoresis (HPCE) were obtained by experimental designs. The following nine factors were examined by a two-level fractional factorial design: concentration of the buffer ion PO4(3-) and B4O7(2 ), ionic strength of buffer, buffer pH, addition of sodium dodecyl sulphate (SDS) and sodium deoxycholate (SCD), addition of acetonitrile and methanol and voltage. Four factors significantly influenced the separation of peaks in all the fungal extracts. For optimization of the HPCE method a subsequent response-surface experiment with the important factors was made with an extract from an isolate of Aspergillus versicolor. Optimum separation conditions were obtained, which gave good resolution of the components in the extract. PMID- 8611944 TI - Characterization of ewe's milk by capillary zone electrophoresis. AB - The purpose of the present work was to develop a procedure able to separate and identify the major protein components of ewe's milk by capillary zone electrophoresis (CZE). Thirty-five individual milk samples of Massese breed were analyzed using a coated capillary. The analyses were performed at pH 3.0 at a temperature of 40 degrees C in the presence of 6 M urea. The purification of casein fractions was carried out by preparative fast protein liquid chromatography and the CZE results were confirmed by polyacrylamide agarose gel electrophoresis (PAAGE). The identification of whey proteins was also carried out by comparison with high-performance liquid chromatography data. The present study permitted the identification of the major components of ewe's milk by high resolution electropherograms and characteristic migration times (tM). it was also possible to detect the presence of genetic variants of beta-lactoglobulin. The tM of k-casein was determined after enzymatic action of chymosin by verifying the simultaneous formation of p-k-casein. In most of the samples a fast moving alpha s2-casein variant was identified by comparison with PAAGE results. Minor genetic differences were found in other casein fractions for this pool of samples. PMID- 8611945 TI - Rapid high-performance liquid chromatographic method for determination of beta carotene in milk. AB - An HPLC method for the determination of beta-carotene in milk samples is described. The method has many advantages over previous methods due to its rapidity, convenience and the small volumes of solvents required. Beta-Carotene was 100% recovered and the relative standard deviation of repeatability was 5.3%. The limit of quantitation was found to be about 30 ng/ml and the system was linear in the concentration range investigated, from 0.67 to 4 microgram/ml. PMID- 8611946 TI - Liquid chromatographic determination of okadaic acid and dinophysistoxin-1 in shellfish after derivatization with 9-chloromethylanthracene. AB - The reagent 9-chloromethylanthracene was evaluated for derivatization of the diarrhetic shellfish poisons, okadaic acid and dinophysistoxin-1 (DTX-1), to form fluorescent products separable by liquid chromatography. The toxins were reacted with the reagent in acetonitrile in the presence of tetramethylammonium hydroxide for 1 h at 90 degrees C. The products were purified by using two silica solid phase extraction cartridges before being determined by reversed-phase liquid chromatography with fluorescence detection. The results are comparable to those obtained using 9-anthryldiazomethane (ADAM) for okadaic acid and DTX-1 in mussel tissue. Detection limits were estimated to be about 70-100 ng/g hepatopancreas (equivalent to 12-20 ng/g whole tissue) for each toxin. PMID- 8611947 TI - Determination of lactic acid and pyruvic acid in serum and cerebrospinal fluid by ion-exclusion chromatography with a bulk acoustic wave detector. AB - A chromatographic method base don a combination of ion-exclusion chromatography separation and bulk acoustic wave series piezoelectric quartz crystal detector quantification for the determination of pyruvic acid and lactic acid in serum and cerebrospinal fluid (CSF) was developed. The separation was carried out using a Shim-pak SCR-102H ion-exclusion column with phosphoric acid solution as eluent. The method shows an acceptable detection limit and anti-interference ability. Serum and CSF from healthy individuals and patient were analysed successfully. PMID- 8611948 TI - Determination of epitestosterone and testosterone in urine by high-performance liquid chromatography. AB - This paper describes an alternative HPLC method for the determination of testosterone and epitestosterone, which is simple, rapid, selective, sensitive and reproducible. Samples were prepared using a previous enzymatic hydrolysis with liquid-liquid extraction. The determination was carried out on a Hypersil BDS-C18 reversed-phase column with gradient elution and UV absorbance detection (240 nm). The limits of quantification (signal-to-noise ratio = 6) were 20 ng/ml for testosterone and 30 ng/ml for epitestosterone. PMID- 8611949 TI - High-performance liquid chromatographic determination of urinary 2,5-hexanedione as mono-2,4-dinitrophenylhydrazone using ultraviolet detection. AB - The good correlation between exposure to n-hexane and 2,5-hexanedione urinary excretion confers on this diketone an important toxicological meaning. this paper proposes a reversed-phase HPLC method which includes, after acid hydrolysis, a derivatization step of 2,5-hexanedione with 2,4-dinitrophenylhydrazine at 70 degrees C for 20 min. The reaction conditions, such as temperature, reagent concentration and time, are optimized so as to allow the condensation of a single carbonyl group. A linear response was obtained in the 0.19-20.0 mg/l range with a detection limit of 0.03 mg/l, corresponding to a signal-to-noise ratio of 3. A phosphate buffer (pH 3.3)-acetonitrile mixture (50:50) as the eluent and UV detection at 334 nm were used. PMID- 8611950 TI - Supercritical fluid extraction of drugs in drug addict hair. AB - Opiates were extracted from sixteen hair samples of drug addicts using a supercritical fluid extraction method with supercritical carbon dioxide and a modifier solution of methanol-triethylamine-water (2:2:1, v/v). the concentrations, as determined by GC-MS, ranged from 1.22 to 21.73 (mean 7.60 ng/mg), 0.17 to 1.54 (mean 0.69 ng/mg) and 0.15 to 14.009 ng/mg hair (mean 3.78 ng/mg) for codeine, morphine and 6-monoacetylmorphine, respectively. The reproducibility of the total procedure had a relative standard deviation of 13%, 17% and 14% for codeine, morphine and 6-monoacetylmorphine, respectively. But this method, concentrations of 0.3, 0.2 and 0.1 ng/mg hair for codeine, morphine and 6-monoacetylmorphine, respectively, could be detected. Relative extraction recoveries were 61%, 53% and 96% for codeine, morphine and 6-monoacetylmorphine, respectively. PMID- 8611951 TI - Determination of loratadine and pheniramine from human serum by gas chromatography-mass spectrometry. AB - In this work, a method for the determination of the antihistaminic drugs loratadine and pheniramine from human serum is presented. Serum samples are extracted under basic conditions with hexane-n-amyl alcohol (95:5, v/v), the analytes are reextracted into diluted hydrochloric acid and, after basification, are once again extracted into the organic phase. The samples are measured by GC MS. The limits o detection of the assay are 0.5 ng/ml for loratadine and 2 ng/ml for pheniramine. The R.S.D.s in the day-to-day precision test for loratadine are 7.0% at 20 ng/ml and 12.4% at 2 ng/ml. for pheniramine, the R.S.D. are 6.4% at 300 ng/ml and 10.2% at 20 ng/ml. PMID- 8611952 TI - Gas chromatography with surface ionization detection: a highly sensitive method for determining underivatized codeine and dihydrocodeine in body fluids. AB - Underivatized codeine and dihydrocodeine in human plasma and urine have been determined with a high degree of accuracy by capillary gas chromatography (GC) with surface ionization detection (SID). The drugs were extracted with the aid of Sep-Pak C18 cartridges. Recovery of both drugs was > or = 90%. The calibration curves obtained with dimemorfan as an internal standard showed linearity in the range 4.5-72.3 and 3.0-75.5 ng/ml of plasma for codeine and dihydrocodeine, respectively. The detection limit was about 100 pg on column (2.5 ng/ml sample). Codeine was determined quantitatively in plasma and urine obtained from a volunteer who had received 10 mg codeine phosphate orally 3 h before the sampling: the levels were found to be 14.1 and 142 ng/ml, respectively. The present GC-SID method has been compared carefully with GC-NPD (nitrogen phosphorus detection) using the same extracts; the sensitivity of GC-SID was more than ten times greater than that of GC-NPD, with background noise correspondingly lower. PMID- 8611953 TI - Determination of chloramphenicol in muscle, liver, kidney and urine of pigs by means of immunoaffinity chromatography and gas chromatography with electron capture detection. AB - A rapid and specific clean-up procedure based on immunoaffinity chromatography (IAC) with polyclonal antibodies for the gas chromatographic determination with electron-capture detection of chloramphenicol in pig muscle tissue, organs and urine is described. A commercially available IAC material was used for the analysis. A decrease in the capacity of the column after being used more than 100 times was observed. Mean recoveries were 69, 54, 62 and 95% for spiked pig muscle tissue, liver, kidney and urine, respectively. The limit of detection was 0.2 micrograms/kg for muscle tissue, 2.0 micrograms/kg for liver and kidney and 0.4 micrograms/kg for urine. PMID- 8611954 TI - Determination of cyclophosphamide metabolites by gas chromatography and thermionic specific detection. Interindividual differences in hepatic biotransformation of cyclophosphamide in man in vitro. AB - Sensitive methods for the determination of the cyclophosphamide metabolites nornitrogen mustard, 4-ketocyclophosphamide and carboxyphosphamide are presented. After liquid-liquid extraction and derivatization, the metabolites are determined by gas chromatography and thermionic specific detection. The methods were used to study the in vitro biotransformation of cyclophosphamide with S-9 liver fractions of human donors. The results show large interindividual differences in the formation of nornitrogen mustard and carboxyphosphamide. 4-Ketocyclophosphamide was not detected. PMID- 8611955 TI - Rapid quantitative determination of a collagenase inhibitor and its major metabolite by on-line liquid chromatography with ionspray tandem mass spectrometric detection. AB - A rapid, sensitive and specific analytical method has been developed and validated to quantify the collagenase inhibitor N2-(2(5) [(hydroxycarbamoyl)methyl)-4-methylvaleryl]-N1, 3-dimethyl-L-valin-amide (I) and its major metabolite (II) from plasma and urine. This method employs an automated solid-phase extraction procedure to isolate the analytes and the internal standard from the biological matrix. Reconstituted extracts were analyzed by HPLC ionspray MS-MS. Chromatography was performed on a 4.6 mm I.D. reversed-phase guard column. The retention times of the analytes and the internal standard were approximately 1.3 min. The assay has a limit of quantification of 5 ng/ml plasma and a limit of detection of 1 ng/ml, based on 100-micro l plasma aliquots. No sample-drying step was required. The standard curves were linear form 5 to 5000 ng/ml using weighted linear regression analysis (1/y2). The intra- and inter assay precision were better than + or - 10% with intra- and inter-assay accuracies between 95 and 105%. this new HPLC-MS-MS assay procedure for I and II in plasma and urine has proven to be specific, sensitive, accurate and robust, and is being used routinely for the analysis of I in pre-clinical and clinical trial samples. Up to 200 unknowns may be analyzed each 24 h per analyst. PMID- 8611956 TI - Selective extraction of quercetrin in vegetable drugs and urine by off-line coupling of boronic acid affinity chromatography and high-performance liquid chromatography. AB - Quercetrin, quercetin and chlorogenic acid were measured in urine or in drugs by combination of boronic acid affinity chromatography and HPLC. Simple reversed phase HPLC with UV detection was used to determine quercetrin in five different Solidago virgaurea drugs. For determination of quercetrin in human urine immobilized boronic acid was applied for sample pretreatment. this procedure leads to a determination limit of 0.01 micrograms/ml with a recovery rate of 95.3%. The first results using this method for quercetrin pharmacokinetics are presented. PMID- 8611957 TI - Determination of ceftiofur and its desfuroylceftiofur-related metabolites in swine tissues by high-performance liquid chromatography. AB - An HPLC method was developed and validated for the determination of ceftiofur related metabolites that have the potential to be microbiologically active in swine muscle, kidney, liver and fat. Its performance was evaluated against incurred-residue swine tissues. This method is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their conjugate sulfur containing moiety using dithioerythritol to yield desfuroylceftiofur, and further stabilization to desfuroylceftiofur acetamide. The limit of quantitation was 0.1 micrograms ceftiofur equivalents/g tissue. The assay is specific for ceftiofur-related metabolites when evaluated against commercially available antibiotics for swine. PMID- 8611958 TI - High-performance liquid chromatographic determination of thiopentone enantiomers in sheep plasma. AB - An HPLC method was developed to determine the plasma concentrations of R(+)- and S(-)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma sample were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm x 4 mm I.D. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k' values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(-)-pentobarbitone, R(+)-thiopentone, S( )-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S( )-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 micrograms/ml were, respectively, 4.8, 4.4 and 3.5% for R(+) thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(-)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentone and 8.8, 7.4 and 9.6% for S(-)-thiopentone. Linearity over the standard range, 0.01-40 micrograms/ml, was shown by correlation coefficients > 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone enantiomers. PMID- 8611959 TI - High-performance liquid chromatographic method for the analysis of imipramine metabolism in vitro by liver and brain microsomes. AB - A new sensitive method for resolution and quantitation metabolites of in vitro imipramine metabolism has been developed for use in liver and brain microsomes. Separation of metabolites was done using a Supelcosil PCN column with a mobile phase of acetonitrile-methanol-potassium phosphate dibasic (40:35:25, v/v/v), pH 7. Resolution is achieved for 2- and 10-hydroxyimipramine, and desipramine. Varying levels of these metabolites formed during in vitro incubations of rat liver and brain microsomes following treatments. PMID- 8611960 TI - Sensitive high-performance liquid chromatographic method for the determination of N4-hexadecyl- and N4-octadecyl-1-beta-D-arabinofuranosylcytosine in plasma and erythrocytes. AB - N4-Hexadecyl- and N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NHAC, NOAC) are two new cytostatic derivatives of cytosine arabinoside (ara-C) with improved cytostatic activity and stability against deamination. A high-performance liquid chromatography (HPLC) method was developed for the specific determination of NHAC and NOAC in plasma and erythrocytes, after solid-phase extraction using UV detection at 275 mm. Because of the strong binding of the drugs to proteins and membranes, the samples have to be pretreated with urea (plasma) or butanol and ultrasonication (erythrocytes). The calibration curves are linear for both drugs (r > 0.999) in the concentration ranges 20-2100 micrograms/l for plasma and 40 4200 micrograms/l for erythrocytes, respectively. The within-day and between-day precision studies showed a good reproducibility, with coefficients of variation below 8.5%. The recoveries of the lipophilic ara-C derivatives are greater than 66%. The method described can be applied to pharmacokinetic studies with NHAC and NOAC. PMID- 8611962 TI - High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum. AB - AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 3331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4'-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C18 analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50-2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml. PMID- 8611961 TI - High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions. AB - A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb. PMID- 8611963 TI - Solid-phase extraction of arginine vasotocin and isotocin in fish samples and subsequent gradient reversed-phase high-performance liquid chromatographic separation. AB - Gradient high-performance liquid chromatography (HPLC) preceded by a solid-phase extraction (SPE) step is described for determining arginine vasotocin and isotocin, the neurohypophysial nonapeptides, in fish plasma samples. The combination of these two methods significantly improves the separation and increases the sensitivity of the assay. The proposed assay may be a useful alternative for analysis of similar nonapeptides in plasma without the use of radioisotopes, while taking into consideration a difference in detection sensitivity. PMID- 8611964 TI - Determination of the beta-blocker carteolol in human plasma by a sensitive gas chromatographic-negative-ion chemical ionization high-resolution mass spectrometric method. AB - A specific and sensitive gas chromatographic-high-resolution mass spectrometric method for the determination of 5-(3-tert.-butylamino-2-hydroxy)propoxy-3,4 dihydrocarbostyril (carteolol), which is a beta-blocker giving depression of intraocular pressure, was developed to elucidate the pharmacokinetics of its ophthalmic application. Carteolol has been determined by high-performance liquid chromatography but with less satisfactory sensitivity. Carteolol was derivatized with pentafluorobenzoyl (PFB) amide followed by dimethylethylsilyl (DMES) ether, resulting in a high negative-ion current. The PFB-DMES derivative of carteolol was determined by the gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICI-MS) using selected-ion monitoring at low and high mass spectrometric resolution. the detection limit was less than 100 fg when the fragment ion was monitored at m/z 552.2067 in the NICI mode using methane as a reagent gas. The quantification limit of carteolol in human plasma with this method was less than 30 pg/ml. The proposed GC-MS method is considered to have sufficient specificity and sensitivity to study the pharmacokinetics of carteolol used as an ophthalmic solution. PMID- 8611965 TI - Solid-phase microextraction for the determination of the free concentration of valproic acid in human plasma by capillary gas chromatography. AB - The potential of solid-phase microextraction in the bioanalysis of drugs is demonstrated. The free concentration of valproic acid in human plasma was determined by equilibrium dialysis at room temperature. To the dialysate was added an internal standard and the pH was adjusted to 2.5. The polymethylsiloxane coated fused-silica fibre of the solid-phase microextraction device was inserted into the dialysate for 3 min. The sorbed analytes were then thermally desorbed at 210 degrees C in the split-splitless injection port of the gas chromatograph, separated on a Nukol capillary column and detected with a flame ionization detector. The method was shown to be highly reproducible with a detection limit of 1 microgram/ml of free valproic acid in human plasma. PMID- 8611966 TI - Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography. AB - A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 microliters of 6 M nitric acid, passed through a Sep-Pak C18 cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. the separations were carried out using a mu Bondapak c18 column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40 degrees C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r > 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 micrograms/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%. PMID- 8611967 TI - Column liquid chromatographic determination of clozapine and N-desmethylclozapine in human serum using solid-phase extraction. AB - A 0.5-ml aliquot of a serum sample, after the addition of a 50-microliters aliquot of a 5 microgram/ml solution of amoxapine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifuged. The supernatant is applied to a 1-ml BondElut C18 silica extraction column-conditioned with subsequent washings with 1 M HCL, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 15 microliters aliquot of the eluate is injected onto a 150 x 4.6 mm I.D. column packed with 5-microns C8 silica particles and eluted at ambient temperature with a mobile phase of 0.1% tetramethylammonium perchlorate-acetonitrile (73:27, v/v) adjusted to pH 4.2 with 10% perchloric acid. The peaks are detected with an absorbance detector at 245 nm. PMID- 8611968 TI - Determination of ofloxacin in human plasma using high-performance liquid chromatography and fluorescence detection. AB - A new method for the determination of ofloxacin in human plasma was developed. Plasma proteins were precipitated with acetonitrile, the supernatant concentrated and injected into a reversed-phase C18 column. Enoxacin was used as an internal standard. The fluorimetric detection was performed at 282 nm for excitation and 450 nm for emission. Limit of quantitation was 20 ng/ml and the calibration curve was linear up to 6900 ng/ml. PMID- 8611969 TI - High-performance liquid chromatographic determination of the silicon phthalocyanine Pc 4 in human blood. AB - A sensitive and reproducible method has been developed for the measurement of the silicon phthalocyanine Pc 4 in red blood cell concentrates (RBCC). The procedure involves extraction of the RBCC with acetonitrile, purification of the extracts with reversed-phase Sep-Pak C18 cartridges and determination of Pc 4 in the extracts by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. The detection limit with 1-ml RBCC samples is 2 ng. This method is applicable to monitoring Pc 4 during its use as a photosensitizer for the inactivation of viruses in RBCC prior to transfusion. It has the potential to be adapted for measuring Pc 4 in tissues during its use in photodynamic therapy of cancer. PMID- 8611970 TI - Is the right sided atrioventricular valve trileaflet? PMID- 8611971 TI - Is the morphologically right atrioventricular valve tricuspid? AB - BACKGROUND AND AIM OF STUDY: Classification of the components of the right atrioventricular valve by its anatomical configuration is problematic and needs clarification. METHODS: Fifty hearts were studied so as to analyze carefully the structure of the right atrioventricular valve and to define its component parts. Cross-sectional echocardiograms were reviewed to demonstrate the valve in closed position, and reconstructed in three-dimensional fashion. RESULTS: The majority of specimens (62%) had three readily identifiable leaflets, but some (30%) could be described as having two leaflets while (8%) had four leaflets. The cross sectional echocardiograms reviewed showed three definite lines of closure in all cases. CONCLUSIONS: The eccentric placement of the anterior papillary muscle causes the leaflets to close with three lines of apposition, and this makes the valve readily distinguishable from its mitral partner. When present, extra "scallops" in the structure of the right atrioventricular valve merely allow better coaption of the leaflets. So, although the term "cusp" is less than adequate as the descriptive basis for the structure of an atrioventricular valve, the right atrioventricular valve should still be considered to be tricuspid. PMID- 8611972 TI - Skirts, slits, scallops and semantics. PMID- 8611973 TI - Bicuspid aortic valves in hearts with other congenital heart disease. AB - The bicuspid aortic valve is the most frequent congenital cardiac malformation; it may be isolated or associated with other congenital heart disease. The present investigation consists of a study of bicuspid aortic valves in 1022 heart specimens belonging to the anatomical collection of the Institute of Pathological Anatomy of the University of Padua. A bicuspid aortic valve was observed in 95 specimens. It occurred as an isolated congenital cardiac defect in 28 cases, seven of which had spontaneous laceration of the aortic valve (aortic dissection). It was associated with other congenital cardiac malformations in 67 out of the remaining 994 specimens (6.7%), 41 of which (61.2%) showed obstruction of the aortic arch. The frequency of bicuspid aortic valve in specimens with complete transposition of great arteries was only 1%. Bicuspid aortic valve was particularly frequent in association with ventricular septal defect and was significantly more frequent in cases with (51.1%) than in cases without (20.5%) aortic arch obstruction (p < 0.001). There was no significant relationship between the occurrence of bicuspid aortic valves and left ventricular outflow tract obstructions or mitral valve malformations. The morphology of the pulmonary valve was also examined. Concurrence of a bicuspid aortic and pulmonary valve was detected in 11 specimens, five of these had trisomy-18. Our findings cast doubt on the assumption that altered fetal blood flow through the aortic valve may be the main factor producing the bicuspid condition. Indeed, they rather support the hypothesis that most bicuspid aortic valves are expressions of a developmental complex that affects the aortic arch and the wall of the ascending aorta as well as the aorta valve. PMID- 8611974 TI - Doppler echocardiography in normally functioning replacement heart valves: a literature review. AB - BACKGROUND AND AIM OF THE STUDY: The English literature on replacement heart valves up to the end of 1994 was reviewed to establish normal ranges for forward function and patterns of regurgitation. METHODS: A total of 48 papers were collected, but 16 were excluded leaving normative data for 14 different designs on a total of 1595 valves in the aortic and 1127 valves in the mitral position. Values for velocity, derived pressure drop, effective orifice area and pressure half-time were tabulated by annulus diameter for each class of valve. RESULTS: Comparison between valve designs was difficult as a result of differences in methodology. For the aortic position, differences in velocity and derived pressure drop within one valve type were similar to those between all valve designs. The homograft and Labcor-Santiago valves appeared the least obstructive although using flow-corrected effective orifice area, there were no material differences between the valve designs. For the mitral position, there was a narrow range in mean pressure drop of 2.0 mmHg to 7.8 mmHg. Little data for the tricuspid position were available. Patterns of normal transvalvar regurgitation varied widely between the valves, but obvious transvalvar regurgitation was a feature of most mechanical valves. CONCLUSION: Much work using standardized protocols and with appropriate hemodynamic formulae needs to be done to improve our knowledge of replacement valve function. PMID- 8611975 TI - Functional significance of a flexible mitral annuloplasty ring: a three dimensional echocardiographic assessment. PMID- 8611976 TI - Three-dimensional analysis of configuration and dynamics in patients with an annuloplasty ring by multiplane transesophageal echocardiography: comparison between flexible and rigid annuloplasty rings. AB - BACKGROUND AND AIM OF THE STUDY: Multiplane transesophageal echocardiography provides three-dimensional reconstruction of the mitral annulus from multiple cross-sectional views from a stable transducer position by rotating around a central axis. This study was designed to evaluate the effect of the type of annuloplasty ring on mitral annular configuration and dynamics using three dimensional reconstruction by multiplane transesophageal echocardiography. METHODS: Ten patients who underwent mitral valve repair for pure mitral regurgitation (five patients with flexible Duran ring, five patients with rigid Carpentier ring) and five normal subjects were studied with multiplane transesophageal echocardiography. Three-dimensional configuration of the mitral annulus was obtained from multiple cross-sectional views of multiplane transesophageal echocardiography. RESULTS: In normal subjects, the mitral annulus had a non-planar configuration and reduced its area in systole. In patients with a Duran ring, the mitral annulus had a non-planar configuration and reduced its area in systole. In patients with a Carpentier ring, the mitral annulus had a planar configuration and the mitral annular area did not change during the cardiac cycle. CONCLUSION: Three dimensional reconstruction of the mitral annulus using multiplane transesophageal echocardiography revealed that mitral annular configuration and dynamics are more physiologic in patients with a flexible Duran ring that with a rigid Carpentier ring. PMID- 8611977 TI - Volume-rendered three-dimensional dynamic anatomy of the mitral annulus using a transesophageal echocardiographic technique. AB - Mitral annulus anatomy and dynamics were evaluated in 12 subjects using a three dimensional transesophageal echocardiographic technique. The mitral annular area, diameters and distance from the left ventricular apex were measured in end diastole, mid-systole, end-systole and mid-diastole. The mitral annulus had its largest area in end-systole and the smallest area in end-diastole. The shape of the annulus changed during the cardiac cycle with the maximal change occurring in the diameters passing close to the middle of the mitral leaflets. In the vertical plane, the annulus had a shallow ski-slope shape, with the attachment of the anterior leaflet being farthest from the apex. In other words, the highest point of the annulus was situated anteromedially and was visualized in the long axis imaging plane. PMID- 8611978 TI - Seven-and-a-half years clinical experience with the CarboMedics prosthetic heart valve. AB - Seven and a half year clinical experience with the CarboMedics prosthetic heart valve is presented. A total of 287 valves were inserted in 277 patients. The first 132 patients were followed in a prospective, and the remaining 145 patients in a partly prospective and partly retrospective manner. The follow up was 98.9% complete with a total of 1,055 patient-years. Actuarial survival at 7.5 years was 74.0% +/- 3.5% overall; 76.0% +/- 4.3% for single aortic, 75.0% +/- 6.5% for single mitral and 76.0% +/- 11.4% for double valve replacements. The actuarial rates of freedom from complications were as follows: valve thrombosis 99.6% +/- 0.4%, embolism 96.0% +/- 1.7%, and anticoagulant-related bleeding 88.0% +/- 2.4%. There was no hemolysis, prosthetic valve dysfunction, or structural deterioration. The linearized rates per 100 patients-years were as follows: valve thrombosis 0.09 (mitral 0.30); embolism 0.75 (aortic 0.31, mitral 1.80); anticoagulant related bleeding 2.84; paravalvular leakage overall 0.19 (aortic 0.31); prosthetic valve endocarditis 0.19 (aortic 0.31). Over a 7.5-year time frame, the CarboMedics prosthetic heart valve has been highly reliable with a low incidence of valve related complications. PMID- 8611979 TI - Long term follow up of 292 patients after valve replacement with the Omnicarbon prosthetic valve. AB - In a prospective study, 292 consecutive patients received 336 Omnicarbon cardiac valves from September 1984 through September 1992 at the Montpellier University Hospital. There were 153 aortic (52%), 95 mitral (33%) and 44 double (15%) mitral and aortic replacements. Mean age was 58 years; 57% were male. Total follow up was 1,383 patient-years, with a maximum of nine and a mean of 4.87 years. Early mortality was 2.75% overall. Late mortality occurred at a rate of 1.9%/patient year. Nine-year probability of freedom from mortality (including early mortality) was 85.0% +/- 2.6% overall. There were no cases of structural failure. Thromboembolic events occurred in nine patients, producing a linearized rate of 0.7%/patient-year. Hemorrhage associated with anticoagulant therapy occurred at a rate of 0.8%/patient-year. Therefore, the combined rate of thromboembolic/hemorrhagic events was 1.5%/patient-year. Cumulative overall freedom from thromboembolism and hemorrhage was 91.5% +/- 1.9% at nine years; it was 86.2% +/- 4.3% after mitral and 95.1% +/- 2.0% after aortic valve replacement. Hemolytic anemia was not observed. Endocarditis occurred eight times (0.6%/patient-year), and there were seven cases of perivalvular lead (0.5%/patient-year). At the end of the follow up, 86% of the patients were in NYHA class I. It is concluded that clinical results over a nine-year period are excellent with the Omnicarbon prosthesis. PMID- 8611980 TI - Manufacturing characteristics associated with strut fracture in Bjork-Shiley 60 degrees Convexo-Concave heart valves. AB - Bjork-Shiley Convexo-Concave (CC) valves sometimes experience fracture of the outlet strut. Previously implicated valve characteristics that predict strut fracture include larger valve size, larger opening angle (70 degrees vs 60 degrees), remilling, weld date, and implant in the mitral position. While the associations between risk, size, and opening angle suggest that part of the elevated incidence of strut fracture might be due to the design of the Bjork Shiley valves, only a small fraction of implanted valves have experienced strut fracture. In consequence, previously unexamined variations in the manufacturing process have been suggested as possible factors affecting the failure risk of individual valves; materials, manufacturing steps, quality control, and specific workers have all been put forward as potential explanations for valve-to-valve variation in risk. We conducted a case-control study of CC60 degrees valves implanted in the USA and Canada and manufactured between January 1, 1979 and March 31, 1984. Cases included all verified strut fractures reported to the manufacturer from 1979 through January, 1992. up to 10 controls were selected for each case. Controls were matched to cases on implanting surgeon and were required to have been implanted and functioning at least as long as their respective case valves. We reviewed case and control manufacturing records. There were 150 cases and 1095 surgeon-matched controls. Large mitral valves were at greatest risk of strut fracture; 33mm mitral valves were estimated to be 23 times more likely to fracture than 21-25mm aortic valves. Valves welded in 1979 and 1980 were less likely to fracture than those welded in any other time period; however, no specific manufacturing procedures or personnel were uniquely associated with this time period. Valves with more flexible outlet struts, as determined by the hook deflection and load deflection tests during manufacture, appear to have been at higher risk than valves with more rigid outlet struts. There were three welders who had worked on a sufficient number of valves to allow separate estimation of the risk in the valves they welded. One welder's work was associated with about one-third the risk of valves worked on by the other two. Examination of receiver operating characteristic curves revealed, however, that welder identity added little to the discriminating information already available in the form of valve size and implant position. It is concluded that welder identity and strut flexibility appear to contribute to the risk of outlet strut fracture in Bjork Shiley CC60 degrees valves. Neither of these factors, however, is sufficient to account for much of the previously unexplained variation in risk. No other characteristic measurable in existing manufacturing records appears to predict risk of strut fracture in any useful way. PMID- 8611981 TI - Acoustic characterization of mechanical valve condition and loading. AB - Both closing dynamics and the mechanical condition of a Bjork-Shiley Convexo Concave (BSCC) valve are significant in assessing the risks of outlet strut fracture. Risk of fracture increases with the presence of a pre-existing fracture in one of the two strut legs and with magnitude and frequency of loading. Recent analyses of in vivo data collected in clinical studies, and in vitro data from a computer-controlled pulse duplicator, indicate that the condition of an outlet strut can be evaluated by non-invasive passive acoustic measurement. The technique utilizes heuristic methods to identify features in time and frequency in the closing sound of BSCC valves. Because of patient-to-patient and beat-to beat variability in the waveforms of closing sounds, the sound of beats are cross correlated to identify thirteen characteristic waveform groups that are independent of valve strut condition. The groups are used for subsequent acceptance of each closing event. For each group, a Mahalanobis distance technique is used to identify features in time and frequency that characterize the mechanical condition of the BSCC valve. A Volterra expansion is used to optimize the features. A similar approach, where strain gages supply the measured strut load, is used to identify features associated with valve closing load, and to predict outlet strut forces on a beat-for-beat basis in vitro and in sheep. The characterization is based on a set of acoustic recordings made on patients prior to explant of each valve. Analysis is made using blinded and leave-one-out methods, preventing overlap between the data used in training and that used in testing. Results have demonstrated a sensitivity and specificity to strut fracture of 100 percent on a group of 33 patients for whom gold standard data was available. Analysis of additional blinded data will be useful to further quantify the robustness of the detection method. The relative ease with which data can be collected, and the excellent results, indicate that the method may develop into a practical and effective screen for outlet strut condition. PMID- 8611982 TI - Use of larger sized aortic homograft conduits in right ventricular outflow tract reconstruction. AB - Between 1973 and 1993 sixty aortic homograft valved conduits in fifty-six patients were used to establish continuity between the right ventricle and the pulmonary artery in congenital heart disease. Age range was one day to 23.5 years (median 3.6 years) which included twenty-six patients less than one-year-old. Conduit size ranged from 11 to 23 mm (median 17.6 mm). there were nine hospital deaths and eight late deaths. The 45 survivors have been followed for a median of 8.6 years (range 6 months to 20 years). All patients have had serial echocardiographic assessments and 35 have had post repair cardiac catheterization. Almost all patients had mild-to-moderate degrees of homograft regurgitation. There were eleven with severe homograft regurgitation and two are being considered for reoperation. The follow up homograft gradient ranged from 0 to 64 mmHg (mean 24 mmHg). Freedom from reoperation for conduit obstruction was 98.2% at five years falling to 91% (C.L. 82%-100%) at 10 years. Of the 23 homografts inserted more than 10 years ago, only one (4.3%) has been replaced because it was causing important obstruction. None have been replaced for regurgitation. Our results indicate that larger sized aortic homografts used in reconstruction of the right ventricular outflow tract give satisfactory results and there is a low incidence of reoperation for replacement at medium term follow up. PMID- 8611983 TI - Partial mitral homograft: a new technique for mitral valve repair. AB - Partial mitral homograft was used in 22 patients with localized lesions contraindicating a conventional valve repair. The etiologies of valve disease were: calcified rheumatic stenosis (n=14) and acute bacterial endocarditis (n=8). One patient died three months after surgery from cancer. Another patient required reoperation for residual stenosis 14 months postoperatively. All other patients had an excellent functional result with 19 patients in sinus rhythm. In this series, partial homograft replacement of the mitral valve significantly extended the possibilities of reconstructive surgery. PMID- 8611984 TI - Is aprotinin safe when used in the context of profound hypothermia and circulatory arrest? A literary review. PMID- 8611985 TI - Aprotinin use with hypothermic circulatory arrest for aortic valve and thoracic aortic surgery: renal function and early survival. AB - BACKGROUND AND AIM OF STUDY: Aprotinin is widely used during high risk cardiac surgery to reduce blood loss. Concern has been expressed about the safety of aprotinin in association with hypothermic circulatory arrest for surgery of the thoracic aorta and aortic valve. METHODS: A consecutive series of 19 patients undergoing surgery of the ascending aorta and/or the aortic arch using hypothermic circulatory arrest (15 - 20 degrees C) in conjunction with the use of aprotinin were studied prospectively from January 1993 to October 1994. The indications for operation were aortic dissection (n = 15) (11 acute) or annuloaortic ectasia (n = 4); 11 were emergency procedures. Ten patients underwent aortic valve replacement as part of a composite aortic root replacement and in seven patients aortic valve resuspension was possible. RESULTS: Mean total chest tube drainage was 878 +/- 548 ml (range 300 - 2,000 ml) with a mean usage of homologous blood of 2,328 +/- 1,600 ml. All but one patient survived (mortality 5.3%). None of the survivors experienced any adverse cardiac or neurological events. Serum creatinine rose significantly from a mean of 102 +/- 17 micromol/L preoperatively, to a mean of 172 +/- 100 micromol/L postoperatively (p<0.05), however, none of the patients became anuric or required dialysis and all values returned to preoperative levels by six weeks after surgery. Median intensive care stay was two days (range 1 - 20 days) and the median postoperative hospital stay was 11 days (range 6 - 50 days). CONCLUSION: These data suggest that aprotinin in conjunction with hypothermic circulatory arrest for surgery of the thoracic aorta and aortic valve has no adverse effect on early survival. However, significant though transient postoperative renal dysfunction was commonly observed in our experience. PMID- 8611986 TI - Role of esophagogastroscopy in application and follow-up of high-dose-rate brachytherapy (HDRB) for treatment of esophageal carcinoma. AB - Flexible esophagogastroscopy (EG) and external beam radiotherapy (EBR) have become important means of diagnosing and treating both squamous and adenocarcinoma of the esophagus and gastroesophageal (GE) junction. Recently, new technology, termed high-dose-rate brachytherapy (HDRB), utilizing the placement of radioisotopes in the esophagus by endoscopic techniques has been introduced. This report describes the endoscopic application of the brachytherapy afterloading catheters and the additional role of EG in the posttreatment assessment of these patients. Twenty-four patients (21 esophageal, 3 GE junction) were treated using HDRB delivered by afterloading catheter techniques utilizing flexible EG. Radiation dosages ranged from 5 Gy (500 rads) to 8 Gy (800 rads) delivered to the tumor bed over an average of three applications. All patients were followed to assess swallowing ability, endoscopic evidence of tumor reduction, and complications resulting from intraluminal radiation therapy. Fifteen patients had reduction in intraluminal tumor based on endoscopic evaluation. Seven had partial or complete relief of dysphagia. Nine patients required gastrostomy tube placement for alimentation before or after therapy. Four patients had complications of perforation (1), fistula (1), or bleeding (2) after HDRB. Overall survival ranged from 2 to 27 months (mean = 8.9 months) after the first HDRB treatment. EG proved to be an efficient and safe technique for the introduction of intraluminal esophageal radiation therapy. PMID- 8611987 TI - Laparoscopic retrograde cholecystectomy (from fundus downward) facilitated by lifting the liver bed up to the diaphragm for inflammatory gallbladder. AB - In order to safely and reliably perform laparoscopic cholecystectomy in severe inflammatory cases (e.g., acute or chronic cholecystitis), we have designed a method of suturing the liver bed to the diaphragm, lifting it cephalad so as to maintain a good operative field. Initially, we dissect the gallbladder fundus, fully dissecting the neck of the gallbladder from the liver and finally dissecting the cystic duct (laparoscopic retrograde cholecystectomy facilitated by lifting the liver bed up to the diaphragm; Lap-RC). This method is different from laparoscopic standard cholecystectomy (Lap-SC), in which dissection of the cystic duct is done first. One hundred and twenty-nine consecutive laparoscopic cholecystectomies for various gallbladder diseases were carried out at Nerima General Hospital between August 1991 and June 1994. Fifteen cases of Lap-RC and six cases of Lap-SC in a severe inflammatory group were comparatively evaluated. Thirteen cases of Lap-RC and 92 cases of Lap-SC in a noninflammatory group were also comparatively evaluated. The rates of conversion to laparotomy were 0% in Lap-RC cases (0/15) and 33% in Lap-SC cases (3/9) in the severe inflammatory group. The incidences of major postoperative complications were 0% in Lap-RC cases (0/15) and 17% in Lap-SC cases (1/6) in the severe inflammatory group. In conclusion, Lap-RC showed satisfactory results in terms of both safety and reliability in patients with severe inflammatory disease. PMID- 8611988 TI - Assessing the safety of pediatric laparoscopic surgery. AB - We studied deviations from normal physiology in piglets (n = 10; average weight 5.75 kg) during carbon dioxide (CO2) pneumoperitoneum. Cardiopulmonary data were gathered during varying intraabdominal pressures (IAP = 8, 12, 15, 20 mm Hg), each sustained for 10 mins. Each animal was its own preinsufflation and exsufflation control. A rapid, significant rise in arterial CO2 pressure from preinsufflation (46.5 +/- 6.7 mm Hg) to insufflation at 20 mm Hg (72.9 +/- 15 mm Hg; p < 0.05) initiated further cardiac adjustments. Responses included a sustained increase in cardiac index (presufflation = 3.1 +/- 1.4; 20 mm Hg IAP = 3.6 +/- 1.2), increased heart rate (preinsufflation = 121 +/- 21; 20 mm Hg IAP = 150 +/- 28; p < 0.05), and left ventricular stroke work (20 mm IAP = 22.7 +/- 8.9; exsufflation 20 min = 15.3 +/- 9.4 g.m/m2; p < 0.05). There was a significant arterial-end CO2 tidal difference throughout insufflation, as great as 15 mm Hg (p < 0.05), suggesting increasing ventilation dead space. Core temperature decreased significantly from preinsufflation (35.3 +/- 1.3 degrees C) to 20 mm Hg IAP (33.6 +/- 1.5 degrees C, p < 0.05). We suggest the following guidelines based on the above data: (a) preoperative examination screening for cardiopulmonary abnormalities; (b) fluid replacement to normal hydration only; (c) cuffed endotracheal tubes for effective ventilation; (d) careful adjustment of minute ventilation to achieve normocapnia; (e) CO2 warming; (f) maximal insufflation pressure of 12 mm Hg; (g) postoperative care emphasizing respiratory and thermoregulation status. PMID- 8611989 TI - Laparoscopic microwave coagulonecrotic therapy for hepatocellular carcinoma. AB - The present study reports on the usefulness of laparoscopic microwave coagulonecrotic therapy as a new option in the treatment of hepatocellular carcinoma. Five patients with liver tumors associated with cirrhosis were treated from July 1993 to March 1994 with a microwave electrode (output 100 W, 3 to 4 cm long) devised for laparoscopic use. The tumors, all with diameters less than 3 cm and superficially located, were coagulated for a total radiation period of 20 to 30 min under laparoscopic, intraoperative ultrasonographic control. Postoperative complications were negligible, and laboratory values (glutamate-pyruvate transaminase, bilirubin, prothrombin time, platelet count) returned to preoperative levels within 7 days. Complete necrosis, including the surrounding liver tissue, was confirmed by a follow-up dynamic computed tomography scan during the follow-up period of 6 to 17 months (mean, 13 months). Laparoscopic microwave coagulonecrotic therapy can exert an effect on tumor equivalent to that obtained from a wedge resection but is noninvasive and may represent a new option for unresectable liver cancers. PMID- 8611990 TI - Totally preperitoneal laparoscopic approach combined with minianterior dissection in the treatment of indirect inguinal hernias. AB - Totally preperitoneal laparoscopic hernioplasty has become more popular recently and will possibly replace the transabdominal preperitoneal procedure. This procedure, however, is more demanding for the surgeon, especially in large indirect hernias. We describe an alternative technique derived from Darzi's anterior endoscopic approach. To date, it has been used in 15 patients, all with good success. The technique results in a shorter operative time and is easier for the surgeon. PMID- 8611991 TI - To do or not to do an endoscopic retrograde cholangiopancreatography in acute biliary pancreatitis? AB - In 16 patients with acute biliary pancreatitis, we performed endoscopic retrograde cholangiopancreatography (ERCP) 10 to 14 days after onset of the attack. All patients had mild pancreatitis. In 10 patients with normal-appearing common bile duct (CBD) on ultrasonography, ERCP was normal also. In two patients with dilated CBD on ultrasonography, stones were found in the CBD on ERCP, sphincterotomy was performed, and the stones were extracted endoscopically. We think there is no need for preoperative ERCP in patients with mild attacks of biliary pancreatitis if the CBD appears undilated on ultrasonography. PMID- 8611992 TI - The laparoscopic learning curve. AB - To characterize the learning curve for laparoscopic cholecystectomy, we compared the first 47 cases (group A), which were performed by two senior attending surgeons who assisted each other when the procedure was introduced into clinical practice (1990-1991), with the first 46 cases (group R) performed by two surgical chief residents who were assisted by members of the teaching faculty in 1992 1993. The patient groups were comparable in terms of age, sex, and anesthetic class, but pathologically proven acute cholecystitis was more common in group R (33% vs. 9%; p < 0.005). To analyze operative procedures and outcomes, we compared operative time, frequency of successful operative cholangiography (attempted in all cases), frequency of conversion to open cholecystectomy, major complication rate, and days of postoperative stay for all patients and for those without complications. Of these parameters, only operative time for nonacute cases differed significantly between the groups (144 min for group A vs. 114 min for group R; p < 0.05). Complications in group A included one ductal injury and one case of postoperative pancreatitis; group R had one ductal injury and two cases of postoperative bleeding. We conclude that (a) the learning curve has similar structure for senior surgeons and resident trainees; and (b) the resident learning curve is not hazardous when teaching assistants are trained in the procedure, which has implications for safe instruction and proctoring of residents and staff. PMID- 8611993 TI - Bipolar versus monopolar cautery scissors for laparoscopic cholecystectomy: a randomized, prospective study. AB - The most efficacious energy source for laparoscopic surgery is constantly being debated. Monopolar electrocautery has gained wide popularity over laser energy because of its lower cost and ease of use. Bipolar current has been extensively used by gynecologists for peritoneoscopic surgery after reports of injuries from monopolar current. The purpose of this study was to compare bipolar scissors (Evershears, Everest Medical) and monopolar scissors (Endoshears, United States Surgical Corporation). Eighty patients were prospectively randomized as to the type of scissors to be used for dissection of the gallbladder from the liver bed during laparoscopic cholecystectomy. The scissors were evaluated subjectively with regard to cutting, coagulation, char, and smoke. The two groups were similar in age, sex, weight, blood loss, and hospital stay. Cutting ability and charring were equal. Coagulation was superior in the monopolar group. Smoke was much less with bipolar energy. Overall satisfaction was similar. We concluded that bipolar scissors are a good, safe device for dissection of the gallbladder from the liver bed during laparoscopic cholecystectomy and compare favorably with monopolar scissors. PMID- 8611994 TI - Hand-assisted laparoscopic splenectomy. AB - A method of performing a laparoscopic splenectomy with the aide of intraabdominal manipulation is described. We believe that this is a versatile technique that compares quite favorably with a pure cannula approach. It is likely that this approach is safer because vascular control can readily be assured by the intraabdominal operator. It is also less costly because it is more rapid than a procedure done solely by cannula techniques. Moreover, it is reproducible by an experienced general surgeon. The results appear equal in terms of access morbidity and hospitalization time to those seen with a purely laparoscopic approach. Experience with 21 splenectomies is described and compared with 20 others performed by the traditional open approach. PMID- 8611995 TI - Laparoscopic colon resection: 60 cases. AB - The first 60 successfully completed laparoscopic colectomies in our series are reported. Patients used moderate amounts of narcotic postoperatively, tolerated oral intake early postoperatively (mean, 1.5 days), and returned to work 2.5 weeks postoperatively. Mean blood loss was 127 cc. Morbidity (11.6%) and mortality (1.6%) were acceptable. Length of stay, complications, and operating time all decreased with experience suggesting a steep learning curve. PMID- 8611996 TI - Total laparoscopic proctocolectomy and laparoscopy-assisted proctocolectomy for inflammatory bowel disease: operative technique and preliminary report. AB - We present the operative technique of laparoscopic proctocolectomy along with the clinical course of the first four patients to undergo this procedure. The operation was conducted through five 12-mm ports. In total laparoscopic proctocolectomy, the dissection began with the sigmoid, left colon, and rectum. The gastrocolic ligament was opened at the level of the midtransverse colon and dissected along with the transverse mesocolon toward the splenic flexure. The attachments and vessels of the right side of the gastrocolic ligament and the right colon were taken last. This sequence was followed because gradual mobilization of the colon displaced all structures in the middle of the abdomen and obscured vision. The specimen was extracted through the anus. In laparoscopy assisted proctocolectomy, it was necessary to incise only the white line of Toldt of the ascending and descending colon, mobilize the hepatic and splenic flexures, and ligate the vessels of the gastrocolic ligament. Then the vessels of the mesentery were ligated near the bowel wall through a 6.5-cm midline subumbilical incision from which the abdominal colon was also extracted. The rectum was then completely dissected and sectioned at 10-15 cm from the anus, everted, and resected at the dentate line. Mean operative time was 7 h, 18 min, and average blood loss was 493 ml. One patient had urinary retention. Return to liquid diet took a mean of 4 days. Average postoperative stay, which depended on full return of bladder function and teaching of stoma care, was 10 days. PMID- 8611997 TI - Minimally invasive colectomy in elderly patients. AB - The safety of minimally invasive colectomy (laparoscopic or laparoscopically assisted colectomy) has not been evaluated in the elderly patient. Therefore, a prospective study of the outcome of minimally invasive colectomy (MIC) in patients aged 65 years or older was undertaken. Between October 1991 and September 1993, 103 elderly patients underwent attempted MIC (right colectomy, 53; left colectomy, sigmoid colectomy, or anterior resection, 36; abdominoperineal resection, 12; and total proctocolectomy with ileostomy, 2); 81 procedures were successfully completed. Complications occurred in 23% of patients converted to laparotomy (including one death) and in 25% undergoing successful MIC (two deaths, p = ns). The average length of postoperative stay was 5.3 days in the MIC group and 8.1 days for patients converted to laparotomy (p < 0.001). These results compare favorably with published results of traditional colectomy for elderly patients. We conclude that MIC is safe in the elderly patient and that further studies are warranted. PMID- 8611998 TI - VATS resection of a mediastinal neurogenic dumbbell tumor. AB - Some posterior mediastinal tumors can be safely resected with video-assisted thoracic surgery (VATS), but the safety of resecting a dumbbell-shaped tumor has been questioned. This article reports the successful removal of a dumbbell-shaped cystic schwannoma at T4 using a posterior laminectomy and VATS, with the patient in the prone position. PMID- 8611999 TI - Laparoscopic assisted percutaneous endoscopic gastrostomy. AB - Percutaneous endoscopic gastrostomy (PEG) cannot be accomplished in some patients and should not be performed if a distinct indentation in the stomach is not seen with finger pressure on the abdominal wall. We describe a technique of laparoscopic assisted PEG as an alternative to evaluate the intraabdominal organs after failed PEG placement. A needle is placed percutaneously into the stomach under laparoscopic and gastroscopic control. A wire is placed through the needle, encircled with a snare, and the PEG completed. We have performed this technique in three patients without complication. This simple and safe procedure has become our technique of choice for gastrostomy tube placement in those patients where upper endoscopy is possible but a PEG alone cannot be performed safely. PMID- 8612000 TI - Laparoscopic and minilaparotomy proximal gastrectomy and esophagogastrostomy: technique and case report. AB - A laparoscopic and minilaparotomy proximal gastrectomy with esophagogastrostomy (end-to-side anastomosis) was performed on a 56-year-old woman with a leiomyoma located just below the esophagogastric junction. Gastroscopic examination revealed a leiomyoma (diameter of 2.5 cm) just below the esophagogastric junction. We considered a laparoscopic proximal gastrectomy safer than a laparoscopic partial gastrectomy because of the risk of postoperative anastomotic stenosis in this case. Therefore, the patient underwent laparoscopic minilaparotomy proximal gastrectomy with esophagogastrostomy. On postoperative day 1, she was able to walk. On postoperative day 4, she started on a clear liquid diet and was discharged on postoperative day 14. During her postoperative recovery, the patient experienced little pain and did not request narcotic analgesia. PMID- 8612001 TI - Laparoscopic and open appendectomies. PMID- 8612002 TI - Effects of microcystin-LR on actin and the actin-associated proteins alpha actinin and talin in hepatocytes. AB - Microcystin-LR (MCLR) is a commonly encountered blue-green algal hepatotoxin and a known inhibitor of cellular protein phosphatase types 1 and 2A. The toxin causes alterations in, and redistribution of, intermediate filaments, microtubules, and actin microfilaments (MFs) in affected cells. In this study, the effect of MCLR on the sequence of alterations in MFs and actin-associated proteins (AAPs) of isolated hepatocytes was examined in an effort to determine whether morphologic changes induced in MFs by microcystins are a result of prior dislocation of AAPs. We studied the effects of MCLR exposure on alpha-actinin and talin, two AAPs that play a role in the orientation of the MFs. Primary hepatocytes were incubated with 10 microns MCLR for 0-64 min. The distribution of actin, alpha-actinin, and talin were examined using fluorescence microscopy. MCLR induced similar changes in the distribution of actin and the AAPs. Actin filament redistribution was first observed after 12 min of MCLR exposure, and was characterized by detachment of MFs from the cell periphery, followed by condensation at distinct focal points and progressive collapse into the interior of affected cells. Changes in alpha-actinin and talin distribution were first observed after 20 min of toxin exposure. The AAPs appeared to detach from focal contacts on the cytoplasmic surface of the plasma membrane, condense into cytoplasmic aggregates, and ultimately collapse into a juxtanuclear bundle. The results of this study indicate that, in hepatocytes exposed to MCLR, the collapse of actin MFs occurs prior to the dislocation of alpha-actinin and talin. Changes in these actin associated proteins are not likely to account for the initial changes in actin MFs. PMID- 8612003 TI - Documented case of ciguatera on the Mexican Pacific coast. AB - Serranidae and Labridae fish caught in 1993 at Alijos Rocks, 300 miles off East Magdalena Bay, Southern Baja California, caused severe illness of a fishing boat crew. The described symptoms resembled those of ciguatera. The presence of ciguatera-like toxins was confirmed on extracts from these fish using the mouse bioassay procedure, showing activities between 220 to 390 mouse units (M.U.). The founding of ciguatoxin at Alijos Rocks 24 degrees 57' N, 115 degrees 45' W) extends to the northeast its geographical distribution in the Pacific and locates the outbreak near the continental coastline. PMID- 8612004 TI - Maitotoxin increases voltage independent chloride and sodium currents in GH4C1 rat pituitary cells. AB - Maitotoxin (MTX) is a 3,424 dalton polyether marine toxin that causes influx of calcium through type L voltage-dependent calcium channels (L-VDCC) in GH4C1 rat pituitary cells, presumably as the result of membrane depolarization. In this study we have investigated the ionic conductances responsible for MTX-induced depolarization under voltage clamp conditions using the perforated and ruptured patch methods. MTX-induced steady-state voltage independent currents of nearly 400 pS/pF within seconds of addition to the bath. Ion substitution experiments demonstrated these currents are consistent with the conductance of sodium and chloride, but not calcium, ions. MTX induction of the voltage-independent chloride conductance in GH4C1 cells occurred concurrently without modification of L-VDCC currents. Pretreatment with nimodipine eliminated voltage activation of L VDCC, and reduced by two thirds the voltage independent current. Analysis as a function of time of MTX exposure revealed that the first 60 sec of MTX-induced currents were not affected by nimodipine pretreatment, but subsequent additional currents were prevented. This indicates that the initial currents induced by MTX occur independently of L-VDCC mediated calcium entry, but full activation of these currents by MTX likely requires the involvement L-VDCC. Taken together this work identifies a voltage-independent sodium/chloride conductance as an initial action of MTX, one that may promote the sequence of ionic events leading to activation of L-VDCC and massive calcium entry. PMID- 8612005 TI - Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudo-nitzschia multiseries. AB - Axenic cultures of Pseudo-nitzschia multiseries (formerly Pseudonitzschia pungens f. multiseries) produce less domoic acid (DA) than the original bacteria containing cultures. Bacterial strains isolated from two nonaxenic P. multiseries clones were reintroduced individually into cultures of three axenic P. multiseries strains. The bacteria did not substantially affect division rates or cell yields. However, they did cause a 2- to 95-fold enhancement of DA production (per cell basis) relative to the axenic culture, depending on the P. multiseries and bacterial strain used. Bacteria isolated from a nontoxic Chaetoceros sp. culture also enhanced DA per cell (by 115-fold), showing that it is not necessary for the bacteria to be isolated from a toxic culture in order to enhance toxin production. There was no evidence of intracellular bacteria in disrupted P. multiseries cells obtained from axenic cultures. Our results demonstrate an important, but nonessential, role of extracellular bacterial in DA production. Characterization of the bacterial strains using morphology, substrate utilization, and restriction fragment length polymorphism (RFLP) analyses clearly showed that we had isolated different species of bacteria from the various nonaxenic cultures. We conclude that not one but several bacterial species enhance DA production by P. multiseries. PMID- 8612006 TI - Ochratoxin A levels in human milk and related food samples: an exposure assessment. AB - Ochratoxin A (OA) is a mycotoxin detected in a variety of food and feeds mostly from countries with temperate or continental climate, because the fungi that produce it, mainly Aspergillus ochraceus, Penicillium verrucosum, and Penicillium viridicatum, can grow under a great variety of climate conditions. The aim of this article was, firstly, to confirm the occurrence of OA in human milk in Italy. Then, a preliminary calculation of OA intake via human milk was made, from ingested food. For this investigation, food and milk samples were collected, continuously for a week, from 4 lactating mothers. The obtained results revealed a significant exposure of sucklings and mothers to OA levels higher than the tolerable daily intake as estimated from animal models. On the basis of these data, a major effort in planning surveillance and research programs to control OA contamination in food, feed, and biological fluids should be pursued. PMID- 8612007 TI - Natural co-occurrence of Fusarium toxins and aflatoxin B1 in corn for feed in north Vietnam. AB - Natural occurrence of Fusarium mycotoxins (trichothecenes and fumonisins) and aflatoxin B1 (AFB1) were surveyed in 32 corn samples, harvested in 1993 and randomly sampled in 1994 in several districts of Hanoi, Vietnam. Corn samples were first milled into fine powder, extracted with methanol-water (3:1) and the crude extracts obtained from the same samples were used for the simultaneous analysis of the trichothecenes such as nivalenol (NIV), deoxynivalenol (DON), and T-2 toxin (T-2) by gas chromatography/mass spectrometry (GC/NS); fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) by high-performance liquid chromatography (HPLC) with a flourescence detector; and AFB1 by and ELISA kit based on a monoclonal antibody. The data revealed that 14, 8, 4, 3, and 2 out of 15 corn kernel samples were positive for AFB1, FB1, FB2, FB3, and NIV with the average levels being 28, 1, 101, 276, 232, and 858 ppb, respectively, and neither DON nor T-2 were detected. As for the other 17 samples of corn powder, 13, 15, 12, 10, 4 and 2 were positive for AFB1, FB1, FB2, FB3, DON, and NIV with the average being 30, 780, 289, 176, 3, 170, and 1,365 ppb, respectively, and T-2 was not detected. Although their positive rates and levels fell in the ranges reported elsewhere, it was found for the first time that the Fusarium toxins (NIV, DON, and fumonisins) and an Aspergillus toxin (AFB1) were naturally co-contaminated in selected samples of corn produced in north Vietnam. PMID- 8612009 TI - [Etiology of isolated pruritus in dermatology consultations at Lome (Togo)]. AB - A prospective study was conducted to determine the etiologies of isolated pruritus among out-patients attending a hospital dermatology clinic in Lome. Two hundred and twenty patients (120 men and 100 women) suffering from isolated pruritus were included in the study. The most frequent etiologies were; digestive parasitosis (n = 50), onchocercosis (n = 45), drug allergy (n = 26), food allergy (n = 10), psychological pruritus (n = 25) and hepatitis (n = 13). The etiology was not determined for 29 cases. This study shows the large contribution of parasites to the pathogenesis of pruritus in tropical Africa. PMID- 8612008 TI - [Control of hepatitis B in French Polynesia with a program of systematic vaccination of newborns with the Genhevac B vaccine]. AB - In 1988, a 5-year vaccination program against hepatitis B was launched for all newborns in a pilot area, the Austral archipelago in French Polynesia. Genhevac B, a recombinant vaccine produced from mammalian cells was administered. Three different immunization schedules were used, none of them including additional specific immunoglobulin: i) four doses, one at each months (M) 0, 1, 2, and 12; ii) three doses one at each MO, M1 and M6; and iii) three doses one at each MO, M1 and M12. Each year during the 5 year period a serological survey was conducted. Of the 837 children who received at least one vaccine dose, 5 were HBsAg carriers. Seroprotection rates for anti-HBs and anti-PreS2 antibodies were 88% after one dose and 97% after two doses. After the third dose, seroprotection rates and geometric mean titers of anti-HBs antibodies were 95% and 217 mIU/ml for schedule (i) (three dose only); 92% and 389 mIU/ml for schedule (ii) and 93% and 344 mIU/ml for schedule (iii) respectively. After four doses (schedule i) the values were 100% and 1228 mIU/ml. Of the 18 newborns whose mothers were positive for both HBsAg and HBeAg, one was a HBsAg carrier. The estimated protective rate for prevention of perinatal transmission was 94%. This study suggests that in field conditions, systematic vaccination of newborns without using specific immunoglobulins can confer early protection. The schedule recommended for use in French Polynesia was three doses, at MO, M1 and M6-12 (between 6 and 12 months) with an additional booster dose at age 6 years, the last year of nursery school. Since April 1992, all children born in French Polynesia have been vaccinated according to this schedule. A catch-up program has been implemented for children aged 4 to 10 years old using a similar immunization schedule. PMID- 8612010 TI - [Generic drugs in developing countries]. AB - Drugs are generally considered as consumer goods which serve to protect, maintain and restore health. In recent years, the proportion of national expenditure devoted to health has increased in all developing countries. The use of generic drugs can reduce health costs, if part of a national drug action plan, and their quality is confirmed (quality control, distribution system, correct utilization). Nevertheless, a large proportion of the population in developing countries has no access to these drugs. This situation requires establishing financing schemes for drug supplies, with community involvement and participation of the population. However, such schemes can result in limited access and inequity. PMID- 8612011 TI - [The fluorosis problem in Senegal: data evaluation and presentation of a new method for defluoridating the water supply]. AB - Fluoride intoxication is a serious public health problem in Senegal. The concentration of fluoride ions in underground water in Senegal is higher than the acceptable standard (0.7 mg/l at 25 degrees C) varying between 5 and 15 mg/l according to the season. The hydrological pattern of fluoride ions in Senegalese water is presented. The high levels of fluoride ions are due to the presence of phosphate ores. The health problems associated with the ingestion of toxic doses (about 4 mg/l) are discussed. Epidemiological investigation shows that the intoxication in Senegal is serious. In addition to fluoride in drinking water, high levels are also found in food, particularly tea and vegetables. Nanofiltration is a low cost membrane-based process which can be used to eliminate fluoride from drinking waters. Also known as low pressure reverse osmosis, this process is selective for small molecules and ions. It can thus sterilize and partially demineralize water. Furthermore, it can require only small amounts of energy, such that it can be run from photoelectric cells. PMID- 8612012 TI - [Smoking among medical students in Tunisia: trends in behavior and attitudes]. AB - We report a cross sectional survey to analyze the effects of medical training on the smoking habits of Tunisian medical students, and their attitudes and knowledge about smoking. Two groups of medical students were studied. One group was 257 first year students at the Medical Faculties of Tunis and Sfax, in 1987, the other 211 final year students at the same Faculties in 1994 and who had been in the first year in 1987. A questionnaire bases on that of the WHO and International Union against Lung Disease for health professionals was administered. It was completed by 95% of the students. Fifty-four % were men and 46% women, and 70.2% lived in an urban area before attending university (table 1). Nonsmokers were defined as those who had never smoked. Exsmokers were those who had formerly smoked but no longer did so. Smokers were divided into those who smoked occasionally and those who smoked daily. The prevalence of smoking was higher among the final year students than the first year students. Combined daily and occasional smoking was 24.1% among first year students and 37.1% among final year students (table 2). The rates among men for daily smoking were 19.2% in the first year and 38.9% in the final year, whereas for women the corresponding rates were 1.8% and 2% (table 3). The prevalence of occasional smoking among men was 17.8% for the first year and 17.7% in the final year. Among women, this behavior increased from 5.5% to 16.8%. Men exsmokers increased from 6.2% to 16.8% and women from 4.6% to 13.4% from the first to the final year. The proportion of first year smokers who reported a serious attempt to stop was 64.8% and that of final year students was 50%. Protected personal health was the most common reason (table 4). In the final year, 94.1% of te students agreed strongly with the view that smoking is harmful to health (table 5). However, there was substantial underestimation of the contribution of tobacco to causing serious diseases including bladder cancer, coronary artery disease, peripheral vascular disease, emphysema and neonatal morality (table 6). The study evidences insufficient awareness of medical students about their responsibility for health education and prevention. There was little interest in preventive action for patients. Only 4.5% of the final year students felt that they were equipped to advise patients about smoking. Similarly, 65.7% would not advise patients to stop smoking if they had no smoking-related symptoms and did not raise the question themselves (table 7). There were major deficiences in knowledge of preventive measures. Only 45.5% of final year students considered that they had adequate knowledge to advise patients about smoking (table 8) and 72.5% thought that they should have received more specific training about counseling (table 9). This work shows that, like in Africa, Asia and Europe, Tunisian medical students have an unsatisfactory knowledge of tobacco and its effects. There were no substantial changes in the students' knowledge of, or attitude to, smoking between the first and final year of training. Simply stimulating the interest of these future doctors in the problem of smoking is insufficient. PMID- 8612013 TI - [Myth reconstruction and health representations in a Southern Congolese therapeutic sect]. PMID- 8612014 TI - [Present status of ringworm: from France to Africa]. PMID- 8612015 TI - [The investigational vaccine SPf66 against Plasmodium falciparum: myth or reality?]. PMID- 8612016 TI - [Attitudes to counseling related to HIV infection screening in Bobo-Dioulasso, Burkina Faso]. AB - Two approaches, a questionnaire administered to pregnant women and interviews independently with selected individuals in posts of responsibility, were used to assess (i) the perception of HIV screening among patients attending a Maternal and Child Health Clinic and (ii) the attitudes of health staff and local authorities. One hundred and thirty five pregnant women were individually interviewed in their native language at the Farakan Maternal and Child Health Clinic, Bobo Dioulasso. Ninety six percent accepted the principle of being screened for HIV, and 91% wanted to be given the result, 46% in the presence of a family member. The person most often chosen to share the announcement of the result was the regular partner. Three physicians, two midwives, a hospital director, a parish priest and a politician were interviewed. One midwife did not approve of offering screening tests in the absence of available treatment. Four of these individuals suggested that the presence of a member of the family during counselling would be valuable to promote a sense of partnership. Many reasons were given to explain the difficulties associated with announcing the results of tests. They included: the absence of treatment; the cost of the test; the lack of confidentiality; psychosocial consequences; the reaction, particularly fear, of the patient; difficulties associated with communication and with changing behavior. These observations contribute to a basis for analysis to develop a local and national consensus about HIV testing and counselling for pregnant woman. PMID- 8612017 TI - Peripheral bone mass measurements--is there any clinical value in rheumatoid arthritis? PMID- 8612018 TI - Undergraduate education in rheumatology. PMID- 8612019 TI - The role of endogenous serotonin in adjuvant-induced arthritis in the rat. AB - We have investigated the effects of serotonin depletion on the progress and severity of adjuvant-induced arthritis in the Piebald-Viral-Glaxo (PVG) strain of rat. Total body depletion of serotonin was achieved using p-chlorophenylalanine given i.p. Two paradigms were investigated. First we depleted serotonin at the time of injection of the adjuvant to determine whether serotonin was involved in the initial induction phase. Secondly, we depleted serotonin at the time of onset of the inflammation. Serotonin levels in the hypothalamic paraventricular nucleus (PVN) were reduced by > 95%. Depletion at the time of induction had no effect on the severity of the disease (determined by the increase (determined by the increase in hind paw volume) 14 days after injection of the adjuvant. In contrast, depletion at the time of onset of the disease resulted in a significant reduction in severity at day 14, suggesting a pro-inflammatory role for serotonin in this model. The decrease in corticotrophin-releasing factor (CRF) mRNA in the PVN associated with the development of adjuvant arthritis in PVG rat was reversed in the serotonin-depleted animals. Central serotonin could be one of the factors responsible for the reduced expression of CRF mRNA in response to adjuvant induced arthritis in this rat strain. These data suggest that serotonin antagonists may be efficacious in reducing the severity of acute inflammatory episodes. PMID- 8612020 TI - Changes in glycosylation of IgG during fasting in patients with rheumatoid arthritis. AB - Patients with rheumatoid arthritis (RA) have a higher proportion of agalactosyl IgG than healthy individuals. Glycosylation status was examined in 26 RA patients who fasted for 7-10 days and afterwards followed a vegetarian diet for 3.5 months. The decrease in the proportion of agalactosyl IgG correlated significantly with the clinical improvement after the fasting period, but not after the vegetarian diet period. Although the glycosylation status of IgG may have played a role in the improvement of disease during the fasting period, it did not seem to be associated with, and therefore responsible for, the clinical improvement observed after the vegetarian diet. PMID- 8612021 TI - Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid. AB - In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF 41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease. PMID- 8612022 TI - Similarly increased serum IgA1 and IgA2 subclass antibody levels against Klebsiella pneumoniae bacteria in ankylosing spondylitis patients with/without extra-articular features. AB - IgA1 and IgA2 subclass serum antibodies against whole Klebsiella pneumoniae bacteria were studied earlier in the sera of 98 patients with ankylosing spondylitis (AS) and in 100 healthy blood donors by enzyme immunoassay. In this study, the patients were divided into groups according to the clinical picture, i.e., the presence or absence of iritis and enthesitis. The previous findings of increased IgA1 and IgA2 subclass antibody levels against K. pneumoniae in AS patients when compared to the healthy controls were not specifically associated with any single AS patient group in the present study, but instead were similarly seen in all patient groups with/without extra-articular features. This is in line with the previous studies suggesting a role for K. pneumoniae in the pathogenesis of AS. PMID- 8612023 TI - Prevalence and clinical significance of antibodies to ribonucleoproteins in systemic lupus erythematosus in Malaysia. AB - One hundred and seventy patients with systemic lupus erythematosus (SLE) were studied for the prevalence of antibodies to the small RNA-associated proteins Ro/SSA, La/SSB, Sm, U1RNP and Sm. The relationship of these autoantibodies to different races, sexes and clinical manifestations of SLE was evaluated. Passive immunodiffusion was employed using human spleen extract as antigen source for Ro and rabbit thymus extract for La, Sm and U1RNP. We found the prevalence of antibodies to be as follows: anti-Ro/SSA, 36%; anti-La/SSB, 8%; anti-Sm, 15% ; anti-U1RNP, 21%. Except for a low prevalence of anti-La, the prevalence of these antibodies was similar to that in Western studies, The prevalence of anti-Ro/SSA is similar to that reported in the Western studies, but lower than that reported in the Oriental patients from Singapore and Hong Kong. Linkages of anti-Ro with anti-La antibodies were usual; however, although anti-Sm antibodies were usually associated with anti-U1RNP, they were more frequently associated with anti-Ro antibodies. The Malay patients had a high prevalence of anti U1RNP compared to other races. No gender difference was detected. Anti-Sm antibody was associated with serositis and anti-U1RNP antibodies with Raynaud's phenomenon. No association was found between the presence of skin renal or cerebral manifestations and any specific antibodies or combination of antibodies. PMID- 8612025 TI - The ten-year mortality in Behcet's syndrome. AB - We surveyed the 10-yr mortality among 152 Behcet's syndrome (BS) patients who had registered at a BS out-patient clinic and compared it to the expected mortality in the general population. Information on mortality was available in 79% of the study group, among whom six patients (all males) had died. The observed mortality of two patients in the 15-24 yr age bracket was significantly above that expected in the general population. BS is a cause of increased mortality in the young male patients PMID- 8612024 TI - Increased rate of lupus flare during pregnancy and the puerperium: a prospective study of 78 pregnancies. AB - The objective was to determine whether the frequency of flare in systemic lupus erythematosus (SLE), patients is increased during pregnancy and the puerperium. Seventy-eight pregnancies in 68 SLE patients attending the lupus pregnancy clinic, at St. Thomas' Hospital, during the last 5 yr were included. The pregnancy period and 8 weeks post-delivery were considered. This group was compared with a control group of 50 consecutive, non-pregnant, age-matched SLE patients attending our weekly lupus clinic. Additionally, 43 of the pregnant patients carried on attending the lupus clinic for the year after puerperium, and their course was compared with themselves during pregnancy. SLE activity was assessed using the Lupus Activity Index (LAI) score. An increase > or = 0.26 in the score was considered as a flare of the disease. Pregnancy and control groups were homogeneous for age, race, disease duration and distribution of autoantibodies. Sixty-five per cent of the patients flared during pregnancy and/or the puerperium and 42% flared in the control group (P = 0.015). The rates of flare per patients/month were 0.082 +/- 0.004 for the pregnancy group and 0.039 +/- 0.003 for the control group (P < 0.001). The 43 patients whose course was controlled after the puerperium flared more frequently during pregnancy that thereafter (McNemar test, P = 0.003). The rates of flare per patient/month were 0.093 +/- 0.006 during pregnancy and the puerperium, and 0.049 +/- 0.004 after the puerperium (P = 0.0015). Kidney and central nervous system involvement was not different between the pregnancy and control groups. In terms of frequency of flares, there was no difference in any of the groups between patients taking and not taking steroids. We conclude that SLE tends to flare during pregnancy. Flares are maximal during the second and third trimester and the puerperium. Flares are not more severe than in non-pregnant patients, and most of the flares can be managed conservatively. Prednisolone does not prevent flares. PMID- 8612026 TI - Wegener's granulomatosis--increased incidence or increased recognition? AB - We aimed to calculate the annual incidence of Wegener's granulomatosis (WG) in Norfolk (UK) and to compare the clinical spectrum of disease to that seen in tertiary centres in both the UK and USA. We also aimed to examine the seasonal onset of symptoms. This was a prospective study of all patients presenting with WG to a district hospital with a well-defined stable population (515,000) during the period 1988-1993. The annual incidence for WG in the adult population is 8.5/million (95% CI 5.2-12.9). These are the first incidence figures for a well defined population and are higher than previously published. The clinical spectrum of disease in Norfolk was similar to that seen in tertiary centres and there is supportive evidence for a seasonal variation in the onset of symptoms (highest in winter). PMID- 8612027 TI - Knee pain amongst the poor and affluent in Pakistan. AB - The frequency of joint symptoms was determined amongst 2022 affluent and 2210 poor adults in Karachi, Pakistan. Joint pain was significantly (P = 0.025) more common amongst the affluent (6.6%) compared with the poor (5%) and this was due to a significantly greater frequency of knee pain in the richer community (3% vs 1.8%; P = 0.008). The prevalence increased with age and was more common in females. Almost half were associated with varus deformity, suggesting the presence of associated OA in a high proportion. The overall frequency of knee pain seemed no greater than in series reported from the West. Compared with age- and sex-matched controls, body weight was significantly greater amongst those with knee pain, both amongst the affluent (P = 0.005) and the poor (P = 0.02). Control subjects were heavier in the affluent population, suggesting that the greater frequency of knee symptoms in this community was due to their relative obesity. Knee bending at prayer was most common amongst the affluent controls and may indicate that religious observance also contributed to the problem in the richer population. Squatting was a characteristic of the poor who had less knee pain than the affluent. Knee flexing could not therefore be confidently implicated. No relationship could be demonstrated between knee pain and joint laxity. PMID- 8612029 TI - Long-term sonographic follow-up of rheumatoid and psoriatic proliferative knee joint synovitis. AB - The potential role of sonography in evaluating the response to therapy of persistent knee joint synovitis (KJS) was assessed in a longitudinal study in pre and post-arthroscopic (AS) synovectomy in rheumatoid and psoriatic patients. At entry to the study ultrasound (US) detection of synovial proliferation was compared with arthroscopic visualization as the 'gold standard' reference. US joint effusion and synovial thickness measures and predominant patterns of synovial proliferation were recorded by comparing clinical and US indices before and at 2, 6 and 12 months after AS synovectomy, or after KJS relapse up to 24 months. A 12 month survival analysis of clinical and US outcomes of arthroscopic synovectomy was also performed. US detection of morphology and degree of synovial proliferation was correlated with AS macroscopic evaluation. After AS synovectomy, the clinical index and both US joint effusion and synovial thickness were significantly reduced, whereas US patterns of synovial proliferation did not show significant changes. US and clinical indices were significantly correlated in all follow-up measurements and US joint effusion was significantly increased in the relapsed compared with the non-relapsed KJS group. The probability at 12 months of reaching maximum improvement in US joint effusion and synovial thickness outcomes was 99 and 58%, respectively; that for clinical remission of KJS was 72%. Ultrasound evaluation has proven reliable and accurate by the arthroscopic gold standard in detecting changes of rheumatoid arthritis and psoriatic arthritis knee joint synovitis. The correlation of US with clinical findings in pre-and post synovectomy patients suggests that sonography can be used as an objective method in monitoring the response to therapy of inflammatory knee joint disease. PMID- 8612028 TI - A randomized double-blind controlled trial of sulphasalazine combined with pulses of methylprednisolone or placebo in the treatment of rheumatoid arthritis. AB - Thirty-eight patients with rheumatoid arthritis meeting American College of Rheumatism (ACR) criteria were entered in a randomized controlled trial (RCT) of 6 months to assess whether monthly treatment with i.v. methylprednisolone (MP) enhances or accelerates the efficacy of sulphasalazine (SSZ). All patients had failed at least one second-line agent and were randomized to receive SSZ (2g/day) and pulses of MP (5 mg/kg), or SSZ+ (2 g/day) and pulses of saline (SA). A single infusion of 2 h was carried out in both groups for a total of three times (0, 1 and 2 months). The two groups were comparable at baseline regarding their demographic and clinical characteristics. Disease activity was evaluated every 2 months by means of: (1) joint count; (2) morning stiffness; (3) grip strength; (4) visual analogue pain score; (5) health assessment questionnaire; and (6) erythrocyte sedimentation rate. All outcome measures improved significantly in both groups (P < 0.001). Evaluation at each follow-up visit showed no significant differences between the groups in any of the adverse effects attributable to SSZ therapy (one SA vs two MP). Adverse effects attributable to SA/MP therapy were rare and mild. We concluded that repeated pulses of MP during the first 3 months of treatment did not improve the efficacy of SSZ. Therefore, there is no justification for using MP in this way during the induction phase of SSZ therapy. PMID- 8612030 TI - Use of sequential DTPA clearance and high resolution computerized tomography in monitoring interstitial lung disease in dermatomyositis. AB - Interstitial lung disease complicating polymyositis-dermatomyositis has a grave prognosis. We report the case of a 50-yr-old woman with dermatomyositis and interstitial lung disease monitored by sequential high-resolution computerized tomography (HRCT) of lung and aerosol clearance times of the radionuclide 99Technetium-diethylene triamine pentacetate (DTPA). She was treated with oral cyclophosphamide and prednisolone with good outcome. Pulmonary response to therapy was followed with sequential HRCT and DTPA scanning. DTPA clearance a measure of lung inflammation, and HRCT paralleled clinical course during the treatment of interstitial lung disease. Sequential HRCT and DTPA were useful adjuncts in the initial assessment and monitoring of interstitial lung disease in association with dermatomyositis. PMID- 8612031 TI - The effect of 30 min cycle ergometry on ankylosing spondylitis. AB - The effect of 30 min cycle ergometry at approximately 100 W (mean 98.8 W; range 34-151 W) in 11 male patients who had no hip involvement were studied. In most patients, exercise produced immediate increases in spinal flexibility and bilateral cervical tilt, and a reduction in pain. However, these improvements steadily waned and all had disappeared by 3-5 h. Exercise induced marked changes in the numbers of circulating leucocytes and platelets, and in the distribution of lymphocytes subsets, similar to those previously reported to occur in individuals without the disease. In a majority of patients there were positive associations (Kendall's tau test) between Schober's index and the platelet count, and negative associations between Schober's index and the percentage of CD4 positive cells over a 5 h period on the exercise day, whereas there were negative associations between the pain score and the leucocyte and neutrophil counts over a comparable period on a control day without exercise. We conclude that exercising those regions of the body unaffected by disease can elicit short-term beneficial effects by a systemically mediated mechanism(s). PMID- 8612032 TI - A prospective controlled study of low back school in the general population. AB - There are no data on the efficacy of a back school in primary prevention of back pain in the general population or on the characteristics of the population who volunteers. After announcement in the local press, 494 healthy adults volunteered and paid for a back school course in Switzerland. A total of 371 controls were matched for sex, age, profession, nationality and back pain. A statistically significant decrease in numbers of doctor's visits was found by the participants during the following 6 months compared with the controls. However, there were no significant between-group difference in the four remaining parameters (presence and intensity of back pain, drug intake and sick leave). Three-quarters of participants changed their attitudes after the back school. Volunteering for a back pain prevention programme was associated with the presence of back pain problems. Reasons for volunteering are further discussed. Overall, the results of this study showed that a back school for the general population may not solve the problem of low back pain, but improves self-help in a subgroup of the population. PMID- 8612033 TI - An overview of disability. PMID- 8612034 TI - Silent myocardial infarction in Wegener's granulomatosis. AB - Histological cardiac abnormalities in Wegener's granulomatosis can frequently be demonstrated at post-mortem examination, but clinically significant cardiac involvement is rare. We describe a massive silent myocardial infarction leading to intractable heart failure and death in a young man with Wegener's granulomatosis, occurring at a time when other features of the disease were responding to aggressive immunosuppression. PMID- 8612036 TI - A case of chronic Escherichia coli osteomyelitis of the clavicle. PMID- 8612035 TI - The value of HLA-B27 typing in the diagnosis of early, oligosymptomatic spondylarthropathies. PMID- 8612037 TI - Pericardial effusion and giant cell arteritis. PMID- 8612038 TI - Data on flares among SLE patients. PMID- 8612039 TI - Thyroid autoimmunity in systemic lupus erythematosus: the clinical significance of a fluctuating course. PMID- 8612040 TI - Chemical mechanisms underlying the vasodilator and platelet anti-aggregating properties of S-nitroso-N-acetyl-DL-penicillamine and S-nitrosoglutathione. AB - The chemistries of S-nitroso-DL-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) in relation to their ability to relax vascular smooth muscle and prevent platelet aggregation have been investigated. Metal ion catalysis greatly accelerates the decomposition of SNAP, but has little effect on GSNO. Instead, NO release from GSNO is effected either by NO transfer to a free thiol (e.g. cysteine), or by enzymatic cleavage of the glutamyl-cystyl peptide bond. In both cases the resulting nitrosothiol (i.e. S-nitrosocysteine and S nitrosocystylglycine, respectively) is susceptible to metal ion catalysed NO release. We conclude that transnitrosation or enzymatic cleavage are obligatory steps in the mechanism of NO release from GSNO, whereas SNAP needs only the presence of metal ions to effect this process. The different modes of NO production may go some way towards explaining the different physiological effectiveness of these S-nitrosothiols as vasodilators and inhibitors of platelet aggregation. PMID- 8612041 TI - Synthesis and properties of oligonucleotides containing the mutagenic base O4 benzylthymidine. AB - The preparation of synthetic oligodeoxynucleotides containing O4-benzylthymidine (Tbn) is described. The use of standard and t-butylphenoxyacetyl amino protecting groups is compared. The thermal stabilities of duplexes containing Tbn paired with adenine and guanine have been measured. PMID- 8612042 TI - Inactivation of gamma-aminobutyric acid aminotransferase by L-3-chloroalanine hydroxamate. AB - The mechanism of inactivation of gamma-aminobutyric acid aminotransferase (GABA AT) by L-3-chloroalanine hydroxamate (1) was investigated. Inactivation of [3H]PLP-reconstituted GABA-AT with 1 followed by denaturation gave no PMP or enamine adduct to the PLP; however, a new unknown metabolite was observed which was identical to the metabolite formed upon inactivation of GABA-AT by L cycloserine. Time-dependent inactivation occurs, but the kinetics are second order; the rate of inactivation increases with time. After inactivation occurs the addition of fresh enzyme results in a faster rate of inactivation than prior to the initial inactivation. This indicates that the actual inactivator is generated from L-3-chloroalanine hydroxamate, and is not L-3-chloroalanine hydroxamate itself. Added gabaculine-inactivated enzyme to fresh enzyme does not increase the rate of inactivation, suggesting that the conversion of L-3 chloroalanine hydroxamate to the active form is not catalyzed by peripheral amino acid residues. L-3-Chloroalanine hydroxamate was shown to undergo buffer catalyzed cyclization to L-cycloserine, which is the actual inactivator of GABA AT. PMID- 8612043 TI - Synthesis and evaluation of oligodeoxynucleotides containing acyclic nucleosides: introduction of three novel analogues and a summary. AB - Novel flexible oligodeoxynucleotides containing (S)-1-(2,3 dihydroxypropyl)thymine or 2',3'-seco-thymidine nucleoside analogues were synthesized on an automated DNA-synthesizer. Oligodeoxynucleotides with one, two or three acyclic nucleosides incorporated in the middle or in the ends of 17-mers have been evaluated. 3'-End-modified oligomers were significantly stabilized towards 3'-exonucleolytic degradation compared to unmodified analogues and showed acceptable hybridization properties as measured by UV experiments. For oligodeoxynucleotide analogues containing the three novel acyclic monomers in the middle, a more pronounced reduction in duplex stability was observed. All oligodeoxynucleotides containing acyclic nucleoside analogues made so far are evaluated with respect to stability towards 3'-exonucleolytic degradation and hybridization properties. PMID- 8612044 TI - Bioisosterism in drug design: identification of and structure-activity relationships in a series of glycine anilide ACAT inhibitors. AB - To examine the effects of bioisosteric replacement on the biological activity of our previously disclosed disubstituted urea inhibitors of the enzyme acyl CoA:cholesterol acyltransferase (ACAT), we prepared a series of N'-substituted and N',N'-disubstituted glycine anilides. These compounds were tested for the ability to inhibit ACAT in vitro and lower plasma total cholesterol in cholesterol-fed rats given a single high-fat, high-cholesterol meal. ACAT inhibitory potency was greatest in compounds containing 2,6-diisopropyl substituents in the anilide portion with the glycine nitrogen substituted by a 1,1-diphenylmethyl moiety. Small improvements in potency in vitro were obtained by substitution of electron donating groups in the 2-, 3- or 5-positions of the aryl rings of the 1,1-diphenylmethyl moiety, but not by substitution in the 4 position. In vitro potency was maintained, but not improved by acylation of the glycine nitrogen. Through a QSAR analysis of in vitro ACAT inhibition for this set of compounds, an equation could be derived which accounted for 85% of the variance in the dataset. An optimal clogp of 6.65 was found, comparable to that found for other series of ACAT inhibitors. In general, compounds from this series displayed inhibitory potency against ACAT in vitro and hypocholesterolemic activity in the in vivo rat model of hypercholesterolemia comparable to that found with the ureas. PMID- 8612045 TI - Molecular associations of flavins with betacarbolines and related indoles. AB - The interactions of a set of structurally selected betacarbolines (BC), (9H pyrido[3,4-b]indoles) and indoles (IND) with two representative flavins (FN): riboflavin (RFN) and flavin mononucleotide (FMN) have been investigated by absorption and fluorescence spectroscopies. Spectral results provided evidence on the formation of 1:1 non-fluorescent molecular complexes, whose stability constants and other related thermodynamic parameters have been estimated from Stern-Volmer quenching analysis. The FMN complexes are somewhat more stable than the RFN complexes. The stabilities of the IND and BC complexes for a given FN follow approximately the order IND approximately tetrahydro BC < dehydro BC < fully aromatic BC. Protonation of the pyridinic nitrogen atom of BCs has a destabilizing effect, which is more pronounced for fully aromatic than for tetrahydro derivatives. The influence of structural factors on the stability of the complexes has been discussed and, aided by theoretical AM1 calculations, a qualitative model for the structure (stacking) and binding forces (cooperative localized charge transfer and dispersion forces) of the complexes has been proposed. PMID- 8612046 TI - Antiviral effect of phosphorothioate oligodeoxyribonucleotides complementary to human immunodeficiency virus. AB - Modifications of oligodeoxyribonucleotides include the replacement of the backbone phosphodiester groups with phosphorothioate (S-ODNs) groups and the substitution of phosphorothioate (SO-ODNs) groups at both the 3'- and 5'-ends. In assays for HIV, oligomers (S-ODNs) were more active at the micromolar range than were SO-ODNs of the same sequence. Furthermore, the abilities of antisense-, sense-, random-, and mismatched-oligomers, or homo-oligomers containing internucleotidic phosphorothioate linkages to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-, sense-, mismatched-, and homo-oligomers of the same length or with the same internucleotide modification. Five different target sites (gag, pol, rev, tat, and tar) within the HIV genes were also studied with regard to the inhibition of HIV replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of initiation sequences and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting viral replication was also investigated. Of particular interest was the S-ODNs-rev 15 mer, which possessed higher anti-HIV activity than the sense-, random-, mismatched-, and homo-20 mers. PMID- 8612047 TI - Application of chemical cytochrome P-450 model systems to studies on drug metabolism--VIII. Novel metabolism of carboxylic acids via oxidative decarboxylation. AB - The oxidative decarboxylation of carboxylic acids by the chemical cytochrome P 450 model and rat liver microsomal systems was investigated. In the chemical system using meso-tetrakis(pentafluorophenyl)porphyrin iron chloride [Fe(TPFPP)Cl] with iodosylbenzene (PhIO), alpha-arylcarboxylic acids and alpha,alpha,alpha-trisubstituted acetic acids are converted to the corresponding one-carbon-reduced alcohol (I) and carbonyl derivatives (II) via oxidative decarboxylation. These products were then used as standards to identify the metabolites in vivo and in vitro. Biliary excretion of Ia and IIa in bile duct cannulated rats after oral administration of ketoprofen amounted to 0.22 and 0.03% of the dose, respectively. In the case of indomethacin, Ib and IIb were detected as metabolites in the rat liver microsomal system, in yields of 2.8 and 0.29%, respectively. Further, the yields of Ib and IIb were decreased in the presence of SKF-525A. Thus, these metabolites were formed by cytochrome P-450 dependent reactions. Metabolites Ia, Ib, IIa and IIb had moderate to strong inhibitory activities on arachidonic acid-induced platelet aggregation and cyclooxygenase activity in vitro, comparable to those of the parent compounds. PMID- 8612048 TI - Diastereoselective Dieckmann condensation suitable for introduction of the duocarmycin A C6 center: development of a divergent strategy for the total synthesis of duocarmycins A and SA. AB - The development of a divergent approach to the introduction of the C-ring of the duocarmycin A and SA alkylation subunits is detailed and includes the development of a diastereoselective Dieckmann condensation suitable for introduction of the duocarmycin A C6 center with control of its relative and natural R absolute configuration. PMID- 8612049 TI - A new procedure for the labeling of peptides and amino acids. AB - Several stable, water soluble cyclohexa-2,4-dien-1-ones have been prepared and their photolytic coupling reactions in aqueous solution at 28 degrees C with various amino acids and dipeptides investigated. This procedure represents a new and high yielding method for the labeling of the terminal amino functionality of peptides and amino acids. PMID- 8612050 TI - Synthesis and alpha-adrenoreceptor blocking properties of phenoxybenzamine related (2-chloroethyl)-(2,3-dihydrobenzo[1,4]dioxin- 2-ylmethyl)-(2 phenoxyethyl) amines. AB - A series of beta-chloroethylamines, structural hybrids of WB 4101, a competitive alpha 1-adrenoreceptor antagonist, and phenoxybenzamine, an irreversible alpha adrenoreceptor antagonist, has been synthesized and tested in isolated rat vas deferens alpha-adrenoreceptors. Although, for all compounds, apparent blocking potency and alpha 1-selectivity are quite similar to those of phenoxybenzamine, affinity values calculated by taking into account the actual concentration of aziridinium ion in solution, reveal that compounds bearing a 1,4-benzodioxan-2 ylmethyl moiety, display a significantly higher potency for both alpha 1- and alpha 2-adrenoreceptors than compounds having a benzyl group. In addition, two of the compounds, having both methyl and methoxy groups in their structure, show a marked discontinuity in the alpha 1-adrenoreceptor concentration-inhibition curve, with a plateau in the range 30-100 nM. Stereochemical aspects are also shown to play an important role in the binding. The biological results suggest that the two irreversible antagonists may be able to discriminate between two alpha 1-adrenoreceptor subtypes, which are both involved in the noradrenaline induced contraction of the epididymal portion of rat vas deferens. PMID- 8612051 TI - Synthesis and elastase inhibitory activity of 6 alpha-chloro-2,2-dimethyl-3 alpha (pivaloyloxy)methylpenam sulfone, 6 alpha-chloro-2,2-dimethyl-3-exo methylenepenam sulfone, benzyl and methyl 6 alpha-substituted penicillanate sulfones. AB - The triflates and pivalates of 3 alpha-hydroxymethyl-6-substituted-2,2 dimethylpenam sulfones 3, 5; methyl and benzyl 6-substituted penicillanates 6-9 and 3-exo-methylene-6-substituted-2,2-dimethylpenam sulfone 4 were synthesized. These novel compounds were evaluated as elastase inhibitors using porcine pancreatic elastase. The effects that structural modifications of substituents on C-3 and C-6 in the penam nucleus have on elastase activity were examined and several similarities and distinctions were identified when compared to the reported penicillin esters and amides elastase inhibitors. PMID- 8612052 TI - A study of gunshot suicides in Northern Ireland from 1989 to 1993. AB - A study of 104 gunshot suicides, including six women, in Northern Ireland over a 5-year period. Forty-five suicides in the security forces are compared with 59 which took place in the civilian population. The former were commonly associated with marital problems and overwhelmingly occurred in young males under the age of 40, whereas the civilian deaths were predominantly associated with mental ill health, with a wider age range distribution. The security forces used rifled weapons in 44 cases, whereas civilians used shotguns in 46 cases. Twelve out of the 45 were witnessed, compared to one in the civilian population. The security forces favoured the head as site of entry in 40 cases compared to 35 in the civilian population. Alcohol consumption was involved in 23 of the security forces suicides and 18 civilian. Of the 6 women, one was in the security forces and 4 had a history of mental illness. PMID- 8612053 TI - Post firing visualisation of fingerprint on spent cartridge cases. AB - A considerable range of techniques is available to visualise latent fingerprints on smooth surfaces. Published methods and those developed or modified in the authors' laboratory have been evaluated for spent cartridge cases. The most suitable were found to be vacuum cyanoacrylate (with fluorescent staining) and selenious acid treatments. Both were used to investigate the post firing recovery of finger-marks on spent cartridge cases from the ammunition and weapon systems encountered in terrorist casework in Northern Ireland. Only certain revolvers and the SLR rifle yielded identifiable ridge detail. These results are borne out by casework experience. PMID- 8612054 TI - Acute poisoning with carbamate pesticides: the Cretan experience. AB - In Crete, in southern Greece, a number of fatal carbamate poisonings were investigated over a period of 2 years, from 1991 to 1993. Five cases are reported, involving the fatal ingestion of methomyl (Lannate), a cholinesterase inhibiting carbamate insecticide. Analysis of samples of blood plasma and serum showed more than 90% inhibition of cholinesterase. The blood methomyl concentrations had a mean value of 26.7 mg/l, and a range of 5.6-57.0 mg/l. These values are much higher than those previously reporter in similar cases (0.57-1.4 mg/l). Methomyl concentrations in organs and tissues were found to be significantly lower than those in blood and vitreous humour. PMID- 8612055 TI - The analysis of methomyl, a carbamate pesticide, in post-mortem samples. AB - Extraction procedures and analytical methods are described for the quantitation of methomyl, a carbamate insecticide, in autopsy tissue and fluid samples. The analytical results from case work demonstrate the rapid metabolism of methomyl in the body. PMID- 8612056 TI - The use of a digital imaging technique to aid bite mark analysis. AB - Criminal cases occasionally involve the analysis of bite mark injuries. This paper presents a method of digital image enhancement of an injury and an overlay production method to improve the comparison between the dentition of a suspect and the image of the injury. A comparison method entirely within the digital image software is also illustrated. The use of digitally-produced overlays appears to provide a more objective method of comparison. The use of this method for shoe print, tyre mark and other blunt instrument injury analysis is noted. PMID- 8612057 TI - Enhancement of fire scene investigations using accelerant detection canines. PMID- 8612058 TI - Alcoholism: the clinical/basic science interface. PMID- 8612059 TI - Neurobehavioral aspects of the pharmacotherapy of alcohol dependence. AB - A neurobehavioral basis for the pharmacologic treatment of alcoholism is beginning to emerge. Preclinical and clinical findings have provided valuable information on which to build bridges of understanding regarding the biological causes and treatment of alcoholism. Reinforcement and stress reduction are prominent in the initiation of alcohol use, while neuroadaptation to chronic alcohol exposure and Pavlovian conditioning of alcohol-like effects appear to be involved in the development of alcohol dependence. Impulsivity may play a crucial role in the rapidity with which alcohol dependence develops. This article presents a model that attempts to integrate these neurobehavioral phenomena with neurochemical systems. The pharmacological agents that have been studied for the treatment of alcoholism are reviewed in the context of this model. While medications that affect the serotonin system have been the most widely studied for the treatment of alcoholism, their clinical effects have been modest or inconsistent. Medications that affect dopaminergic neurotransmission have received less research attention, and their potential clinical utility may be limited by their side effect profile. The most efficacious agents for the treatment of alcoholism have been the opiate antagonists, including naltrexone. Naltrexone recently received approval from the U.S. Food and Drug Administration for relapse prevention in alcoholism. A number of recent clinical and animal studies suggest potential mechanisms of action for opiate antagonists in the treatment of alcoholism. Knowledge in this field is advancing rapidly. Developments in neurobiology, coupled with improvements in both animal models of alcohol self-administration and clinical trial methodology will surely further our understanding of the pathophysiology and pharmacotherapy of alcohol dependence. PMID- 8612061 TI - Molecular and cellular adaptations in signal transduction pathways following ethanol exposure. AB - The purpose of this review is to provide an overview of the acute actions of ethanol on signal transduction, as well as a selective consideration of some of the long-term adaptive changes in signal transduction pathways that may underlie clinical manifestations of ethanol dependence, tolerance, withdrawal, and addiction. The acute intoxicating effects of ethanol have been widely attributed to its ability to block voltage-gated Ca2+ and Na+ channels and N-methyl-D aspartate glutamate receptor cation channels, and to facilitate GABAA receptor Cl channels. Adaptive changes in these same proteins following chronic ethanol exposure may contribute to physical and psychological signs of ethanol dependence and withdrawal. Ethanol, as with other drugs of abuse, also acutely activates the mesolimbic dopamine pathway, an effect which likely accounts, at least in part, for ethanol's acute reinforcing properties. Studies directed at unraveling the biochemical and molecular basis of ethanol's acute and chronic actions may lead to the development of novel pharmacotherapeutics that mitigate aspects of acute ethanol intoxication and, more importantly, treat the effects of withdrawal and addiction (craving) associated with long-term ethanol abuse. PMID- 8612060 TI - Cellular and behavioral neurobiology of alcohol: receptor-mediated neuronal processes. AB - Ethanol can be viewed as a receptor modulator, selectively altering neurochemical processes in discrete regions of the CNS. The actions of ethanol at two inotropic receptor systems, gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) glutamate receptors, are highlighted in this review. In addition, evidence is presented and discussed concerning the possible effects of ethanol that arise from interactions between these two receptor systems, the interactions of ethanol with phosphorylation states of receptor proteins and with metabotropic receptor systems. PMID- 8612062 TI - Candidate genes in alcoholism. AB - Individual alleles identified by candidate gene analysis have been shown to profoundly influence certain complex behavioral syndromes including vulnerability to alcoholism. The alcohol and aldehyde dehydrogenase polymorphisms, ADH2(2) and ALDH2(2), respectively, are associated with lower vulnerability to alcoholism both in the Orient and also in North America among populations of Taiwanese and Koreans who have immigrated there. Protein structural variants have recently been identified for a series of genes involved in dopamine and serotonin function. Three dopamine receptors exhibit such structural variants, and these include the DRD2 dopamine receptor, which has three relatively rare amino acid substitutions. Structural polymorphisms were detected in five serotonin receptors (5HT1A, 5HT1Db, 5HT2A, 5HT2C, and 5HT7). Association studies between neurotransmitter gene variants and alcoholism are at an earlier stage than with ADH2(2)/ALDH2(2), but results are thus far negative (dopamine DRD4 receptor), equivocal (dopamine DRD2 receptor), or preliminary (tryptophan hydroxylase, 5HT2C and 5HT1Db). PMID- 8612063 TI - Pharmacogenetic models of alcoholism. AB - This article reviews recent efforts in developing laboratory animal models for the study of alcoholism and abnormal alcohol-seeking behavior. Through selective breeding, stable lines of rats that reliably exhibit high and low voluntary alcohol consumption have been raised. The high preference animals self-administer ethanol by free-choice drinking, and operantly for intragastric infusion, in amounts that produce intoxication. With chronic free-choice drinking, the preferring rats develop tolerance and physical dependence. Low to moderate concentrations (50-150 mg%) of ethanol are reinforcing to the preferring rat, as evidenced by intracranial self-administration studies. Compared with nonpreferring animals, they are less affected and develop tolerance more quickly to the sedative-hypnotic effects of ethanol. Neurochemical, -anatomical and pharmacological studies indicate innate differences between the alcohol preferring and -nonpreferring lines in the brain limbic structures. Depending on the animal model under study, a change in the main dopaminergic pathway and/or the serotonergic, opioid, and GABAergic systems that regulate this pathway may underlie the vulnerability to the abnormal alcohol-seeking behavior in these pharmacogenetic animal models of alcoholism. PMID- 8612064 TI - Membrane proteins in the USA. PMID- 8612065 TI - Parallel worlds. PMID- 8612066 TI - Ligands and electrons and haem proteins. PMID- 8612067 TI - Picture story. Not just another copycat. PMID- 8612068 TI - The muscle in kinesin. PMID- 8612069 TI - Solution structure of a peptide nucleic acid-DNA duplex. PMID- 8612070 TI - The catalytic mechanism of cytochrome P450 BM3 involves a 6 A movement of the bound substrate on reduction. PMID- 8612071 TI - Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor. AB - We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, Fc epsilon RI. The structure of the second domain of CD2 was used as a modelling template for the second alpha-chain domain of Fc epsilon RI, the C-C' loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C' region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C' peptide mimics act as competitive inhibitors of IgE binding. The cyclic L- and retro D peptides exhibited KDs of approximately 3 microM and 11 microM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response. PMID- 8612072 TI - Rapid, electrostatically assisted association of proteins. AB - The rapid association of barnase and its intracellular inhibitor barstar has been analysed from the effects of mutagenesis and electrostatic screening. A basal association rate constant of 10(5) M(-1) s(-1) is increased to over 5 x 10(9) M( 1) s(-1) by electrostatic forces. The association between the oppositely charged proteins proceeds through the rate-determining formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase association rate constants between molecules and its principles may be used for protein design. PMID- 8612073 TI - The kinetic folding pathway of the Tetrahymena ribozyme reveals possible similarities between RNA and protein folding. AB - We have probed the nature of the individual kinetic steps in the folding of the Tetrahymena ribozyme by studying the folding kinetics of mutant ribozymes. After rapid formation of the first structural subdomain, a slow step precedes stable formation of the second subdomain. The two central helices of the second subdomain form in an interdependent manner, and this structural subunit therefore also constitutes a kinetic folding unit. The slow folding step includes formation of tertiary interactions in a triple-helical scaffold that orients the two subdomains of the RNA. The rapid and early formation of short range secondary structure, the hierarchical formation of kinetic folding units corresponding to structural subdomains, and the formation of tertiary interactions between subdomains late during the folding process appear to be common features of the folding mechanism for both RNA and proteins. PMID- 8612074 TI - Packing interactions in the apomyglobin folding intermediate. AB - The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate has been studied by urea denaturation monitored by circular dichroism and fluorescence. Mutations disrupting native packing sites within the subdomain formed by the A, G and H helices destabilize the intermediate, in contrast to the conclusion drawn from earlier studies of pH induced unfolding. Based on these results, the intermediate is proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate. The results help to explain how the intermediate acquires its structure and stability. PMID- 8612075 TI - In vitro evolution of thermodynamically stable turns. AB - To determine the role of primary structure in specifying turns, random sequences (guests) were substituted for the native turn sequences in a series of proteins (hosts) of differing thermodynamic stabilities.The fraction of inserts that result in active proteins is measured as a function of the stability of the host and temperature. With a highly stable host, more than half of the inserts give functional proteins. However, a smaller fraction of sequences supports folding as the stability of the host decreases, and the temperature increases. The sequences of many of the selected inserts resemble the wild-type turn, and those that diverge match other established turn preferences. Thermodynamic measurements show that turn sequences selected under stringent conditions result in the most stable proteins. Thus, beta-turns appear to be under evolutionary pressure favouring thermodynamically stable structures. PMID- 8612076 TI - Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin. AB - Haloarcula marismortui is an archaebacterium that flourishes in the world's saltiest body of water, the Dead Sea. The cytosol of this organism is a supersaturated salt solution in which proteins are soluble and active. The crystal structure of a 2Fe-2S ferredoxin from H. marismortui determined at 1.9 A is similar to those of plant-type 2Fe-2S ferredoxins of known structure, with two important distinctions. The entire surface of the protein is coated with acidic residues except for the vicinity of the iron-sulphur cluster, and there is an insertion of two amphipathic helices near the N-terminus. These form a separate hyperacidic domain whose postulated function to provide extra surface carboxylates for solvation. These data and the fact that bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non halophilic protein crystal structure support the notion that haloadaptation involves better water binding capacity. PMID- 8612077 TI - Concerted movement of side chains in the haem vicinity observed on ligand binding in cytochrome c' from rhodobacter capsulatus. AB - We have determined the structure of n-butylisocyanide-bound Rhodobacter capsulatus cytochrome c'. This is the first example of a ligand-bound structure of a class IIa cytochrome c. Compared with the structure of native cytochrome c', there are significant conformational changes of amino acid residues in the haem vicinity, accompanied by a rearrangement of the hydrogen bonding pattern. The results suggest that rearrangements resulting from ligand binding could drive dimer dissociation in some species and also that the haem propionate may participate in proton transfer. PMID- 8612078 TI - Retrovirus envelope domain at 1.7 angstrom resolution. AB - We report the crystal structure of an extraviral segment of a retrovirus envelope protein, the Moloney murine leukemia virus (MoMuLV) transmembrane (TM) subunit. This segment, which comprises a region of the MoMuLV TM protein analogous to that contained within the X-ray crystal structure of low-pH converted influenza hemagglutinin, contains a trimeric coiled coil, with a hydrophobic cluster at its base and a strand that packs in an antiparallel orientation against the coiled coil. This structure gives the first high-resolution insight into the retrovirus surface and serves as a model for a wide range of viral fusion proteins; key residues in this structure are conserved among C- and D-type retroviruses and the filovirus ebola. PMID- 8612079 TI - The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution. AB - Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose 6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 A crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other. PMID- 8612080 TI - Suspected obstructive pyelocaliectasis: Doppler ultrasonography compared with diuretic renal scintigraphy in proven cases. AB - OBJECTIVE: To compare Doppler ultrasonography with diuretic renal scintigraphy in the differentiation of obstructive from nonobstructive pyelocaliectasis. PATIENTS AND METHOD: The authors reviewed the findings of Doppler ultrasonography and diuretic renal scintigraphy performed over a 3-year period for 27 pyelocaliectatic kidneys (17 obstructed and 10 unobstructed) in 20 individuals ranging in age from 19 to 88 years. The kidneys were classified as "obstructed" or "unobstructed" on the basis of the resistive index as calculated from Doppler ultrasonographic results and as "obstructed", "unobstructed" or "indeterminate" on the basis of the clearance half-time determined from scintigraphic findings. RESULTS: On the basis of scintigraphy, the obstruction status was indeterminate in 12 of the 27 kidneys. The data were analysed for sensitivity and specificity in two ways, first by classifying the kidneys with indeterminate obstruction status as "obstructed, " and then by excluding them from the analysis altogether. In both situations, the differences in sensitivity and specificity between Doppler ultrasonography and scintigraphy were not statistically significant (chi 2 test, p > 0.05). When the kidneys with indeterminate obstruction status were included in the obstructed category, the sensitivity and specificity of Doppler ultrasonography were 94% and 90% respectively and of scintigraphy 100% and 70% respectively; when the kidneys with indeterminate obstruction status were excluded, the sensitivity and specificity of Doppler ultrasonography were 83% and 89% respectively and of scintigraphy 100% and 78% respectively. CONCLUSIONS: These results suggest that Doppler ultrasonography is comparable to diuretic renal scintigraphy in the work-up of potential obstructive pyelocaliectasis. Because grey-scale ultrasonography is usually performed before scintigraphy, a Doppler examination could be added at this stage, which might reduce the time necessary to establish the diagnosis. PMID- 8612081 TI - Dermoid cyst of the ovary: computed tomography diagnosis of an unusual case. AB - Dermoid cyst is a common benign tumour of the ovary. Eighty percent of these tumours are discovered in premenopausal women. The authors report a case of dermoid cyst of the left ovary, which was diagnosed in a 78-year-old-woman on the basis of computed tomography (CT) findings. Radiologic findings consistent with ovarian dermoid cyst are discussed, with reference to the role of CT as a diagnostic tool. PMID- 8612082 TI - Infected third branchial cleft cyst: retropharyngeal extension to the superior mediastinum. AB - The presentation of an infected branchial cleft cyst extending into the retropharyngeal space is extremely rare. However, differentiation of this lesion from a simple retropharyngeal abscess is important for surgical planning. The authors describe a 44-year-old woman in whom an infected third branchial cleft cyst was initially misdiagnosed as an abscess and drained surgically. Repeat surgery was necessary when the patient's symptoms recurred several weeks later. PMID- 8612083 TI - Accuracy of direct signs of tears of the anterior cruciate ligament. AB - OBJECTIVE: To evaluate the accuracy of direct magnetic resonance imaging (MRI) signs of tears of the anterior cruciate ligament. PATIENTS AND METHODS: Over the period April 1991 to February 1994, 92 consecutive MRI studies of the knee were obtained for which arthroscopic data were also available. The MRI studies were retrospectively evaluated for course, continuity, signal intensity, morphologic features, contour and visualization of the anterior cruciate ligament. Arthroscopic findings were correlated with individual primary signs and the overall MRI diagnosis. RESULTS: Among the cases studied were 4 partial and 32 complete tears of the anterior cruciate ligament (as determined by arthroscopy). Because of the low number of partial tears, it was not possible to draw any meaningful conclusions about the MRI diagnosis of this type of tear. For complete tears, the criteria with the highest accuracy were abnormal course of the ligament (96.0%) and high signal intensity (89.3%). The overall diagnostic accuracy of MRI was 98.8%. CONCLUSIONS: The most accurate direct MRI finding in patients with a complete tear of the anterior cruciate ligament was abnormal course of the ligament, followed by abnormally high signal intensity. PMID- 8612084 TI - Congenital-infantile fibrosarcoma: magnetic resonance imaging findings. AB - Congenital-infantile, fibrosarcoma is a rare tumour presenting at birth or in the neonatal period. Few such tumours have been reported, and imaging details in particular are scant. The authors describe two neonates with congenital-infantile fibrosarcoma, the first case involving the right thigh and extending into the pelvis, and the second involving the calf and the ankle. In both cases magnetic resonance imaging (MRI) demonstrated well-demarcated, low-signal-intensity soft tissue masses with t1-weighting and inhomogeneous, high-signal-intensity masses with T2-weighting. MRI was superior to other imaging modalities in the assessment of soft-tissue involvement and proved especially useful in planning therapy and monitoring chemotherapeutic response. PMID- 8612085 TI - Neuroepithelial cyst of the posterior fossa: two case reports with radiologic pathologic correlation. AB - The clinical, radiologic and pathological findings in two cases of neuroepithelial cyst of the posterior fossa are described. The first patient was a 43-year-old woman who presented with clumsiness of the hands, gait disturbance and slurring of speech. Magnetic resonance imaging (MRI) revealed a large cystic structure between the cerebellum and the brain stem on the left side, accompanied by compression of the medulla oblongata and displacement of the forth ventricle to the right. The second patient was an 11-month-old boy who presented with progressive bulbar dysfunction. He had previously undergone surgery for myelomeningocele and hydrocephalus associated with Arnold-Chiari type 2 malformation. MRI revealed a large cystic mass isointense with cerebrospinal fluid between the cerebellum and the midbrain, compressing the fourth ventricle and displacing the brain stem to the right. The cases were pathologically similar, the cyst containing a single layer of cuboidal epithelium and rims of neuroglial cells. Immunohistochemical staining was consistent with neuroepithelial cyst. It is important to distinguish this type of cyst from a malignant mass to ensure appropriate treatment and determine the prognosis. In addition, even though neuroepithelial cysts of the posterior fossa are benign, they are often symptomatic because of their mass effect. The second case was unusual because the cyst developed over a period of 2 months, which shows that this type of cyst can develop rapidly. To the authors' knowledge, the association with Arnold-Chiari type 2 malformation has not previously been reported. PMID- 8612086 TI - [Multiple intracerebral granulocytic sarcomas in a patient with chronic myeloid leukemia]. AB - The authors describe a young man with known chronic myeloid leukemia who sought medical attention after a first episode of seizure. The hematologic investigation revealed hyperleukocytosis without evidence of blastic transformation in peripheral blood or bone marrow. Numerous intracerebral nodules, characterized pathologically as granulocytic sarcomas, were demonstrated by computed tomography and magnetic resonance imaging (MRI). The appearance of a granulocytic sarcoma in chronic myeloid leukemia is unusual, and location of the lesion in the central nervous system is rare. Follow-up MRI after treatment showed almost complete regression of all lesions. The radiologic features of this case are discussed with reference to others reported in the literature. PMID- 8612087 TI - Clinico-radiologic-pathologic conference. Abnormal findings on chest radiographs of an asymptomatic patient. PMID- 8612088 TI - Residents' corner. Answer to case of the month #36. Hepatic lipoma. PMID- 8612089 TI - Esophageal rupture after routine bowel preparation for barium enema study. PMID- 8612090 TI - Radiology at Kenyatta National Hospital, Nairobi, Kenya. PMID- 8612091 TI - Epidermal inclusion cysts in the male breast. AB - The authors present the second and third reported cases of an epidermal inclusion cyst presenting as a breast mass in a male. In each of the two patients the mass appeared on mammography as a well-defined, noncalcified mass, and breast cancer was suspected. Biopsy was required to establish the correct diagnosis. PMID- 8612092 TI - Nontraumatic subcutaneous emphysema in association with rectal carcinoma. AB - Nontraumatic subcutaneous emphysema is a rare complication of colorectal cancer. To the authors' knowledge, only eight cases have been reported to date. The initiating event was free colonic perforation in seven of the patients and contained colosubcutaneous fistula in the eighth. In nontraumatic subcutaneous emphysema typical fecal flora is cultured, whereas in nontraumatic gas gangrene, a different clinical entity that has a fulminant course, the bacteria are generally clostridial. The authors report a case of nontraumatic subcutaneous emphysema associated with rectal carcinoma and describe the associated computed tomography findings. PMID- 8612093 TI - Yolk sac number, size and morphologic features in monochorionic monoamniotic twin pregnancy. AB - OBJECTIVE: To determine the relation between three characteristics of the yolk sac (number, size and morphologic features) and outcome in monochorionic monoamniotic twin pregnancy. METHODS: The authors reviewed data for the four sets of monochorionic monoamniotic twins detected by first-trimester ultrasonography between January 1990 and June 1994 at their institution. Data analysed included yolk sac number, size and morphologic features, as well as the outcome of the pregnancy. RESULTS: In all four sets of twins, only one yolk sac was identified. In one case the yolk sac was irregular in contour, and in two it was abnormally large. Two of the four mothers delivered healthy twins at 34 weeks gestational age, one had conjoined twins ( and underwent elective termination of the pregnancy), and one had a twin ectopic pregnancy (and underwent salpingectomy). CONCLUSION: A single yolk sac in cases of monochorionic monoamniotic twins may be a normal finding. PMID- 8612094 TI - Age-related changes in G proteins in rat aorta. AB - Blood vessels from aged animals and humans have impaired relaxation to beta adrenergic stimulation. We hypothesized that a loss of stimulatory G protein (Gs) or an increase in inhibitory G proteins (Gi) could explain this impairment. Aortic membranes from Fischer 344 rats of 4 age groups (6 week to 24 month) were studied. G-protein levels were initially assessed using cholera and pertussis toxin labeling. There was a marked decline in cholera toxin labeling (which primarily labels Gs alpha) from 6 weeks to 6 months which persisted in 12-month and 24-month animals. Pertussis toxin labeling (which primarily labels Gi alpha) showed only a slight decline with age. Western blotting was performed using specific antibodies for the alpha subunit of Gs, Gi1&2, Gi3, and G beta. There was no significant change in Gs alpha, Gi alpha, or G beta protein levels with age. We conclude there is a loss of cholera toxin-catalyzed ADP ribosylation with age, which does not represent a loss of the stimulatory alpha subunit of G protein. These data suggest that the loss of cholera toxin labeling seen with age may be a marker for loss of Gs alpha function. PMID- 8612095 TI - Complexity of age-related change in skeletal muscle. AB - Age-related changes in skeletal muscle mass, fiber area, and contractile function were examined in pathogen-free rats at 6, 12, 28 and 36 mos of age. The intent of this study was to clarify age-related decline, particularly in contractile force, and to determine if the decline in contractile tension with age is due to alterations at the neuromuscular junction. A variable amount of age-associated reduction in muscle mass was noted for the soleus (18%), extensor digitorum longus (EDL-16%), plantaris (37%), and gastrocnemius (38%) muscles. The decline in fiber area for these four muscles was between 5 and 16% greater than the loss in muscle wet weight. A variable amount of change in peak contractile force between 6 and 36 mos was observed for the soleus (62%), EDL (48%), and plantaris (34%). For soleus and EDL, the decline in peak tetanic tension exceeded the decline in muscle mass and fiber area. Most of the declines for the animals used in this study did not become significant until after the age of 28 mo. The marked reduction in peak tetanic tension, fiber area, and muscle mass between 28 and 36 mos indicates an accelerated age-related decline in this time period. The reduced peak twitch and peak tetanic tension in the oldest animals was not due to likely age-related changes at the neuromuscular junction. Peak values for tetanic tension were similar, whether tension was elicited via direct muscle stimulation or through stimulation of the nerve. Results underscore the complexity of age related change and suggest that multiple mechanisms contribute to the decline of skeletal muscle. PMID- 8612096 TI - Effect of aging on the click-rate induced facilitation of acoustic reflex thresholds. AB - Acoustic reflex thresholds are known to improve with an increase in the click repetition rates from 50/sec to 300/sec. In the current study this improvement was used to evaluate auditory processing in older subjects. Acoustic reflex thresholds were obtained from 16 human adult ears within each of the following four groups: young male, young female (18-28 years), older male and older female (50-65 years). The probe tone frequency was 226 Hz and the intensity of the probe tone was 85 dB SPL (sound pressure level). Clicks were delivered ipsilaterally to each ear at repetition rates of 50, 100, 150, 200, and 300/sec. The mixed MANOVA revealed a significant effect for the repetition rate and a significant age and rate interaction. Rate integration in dB was computed by subtracting the highest acoustic reflex threshold from the lowest threshold of each ear. Statistical analyses revealed reduced rate integration in the older subjects, suggesting less efficient processing of faster stimuli within the acoustic reflex pathway. PMID- 8612097 TI - Aged murine T-lymphocytes are more resistant to oxidative damage due to the predominance of the cells possessing the memory phenotype. AB - Glutathione (GSH) is the most important cytosolic antioxidant. Since GSH levels are decreased with age, we hypothesized that T-lymphocytes from old mice would be more sensitive to oxidative stress. T-lymphocytes from young and old mice were exposed to hypoxanthine/xanthine oxidase, and lymphocyte viability, proliferation, GSH content, and calcium signaling were measured. Before exposure, proliferation of T-lymphocytes from young mice was greater than that of old; following exposure, the converse was true. This was in spite of the fact that old mice had lower total GSH levels and greater levels of glutathione disulfide. After oxidative challenge, intracellular calcium responses to anti-CD3 were decreased in naive T-lymphocytes from all mice, while memory lymphocytes were less affected. Higher proportions of memory lymphocytes in old mice resulted in their greater overall preservation of lymphocyte function following oxidative injury, contrary to expectations that lower lymphocyte GSH content with age would increase susceptibility to oxidative stress. PMID- 8612098 TI - Glycine enhances binding to the NMDA receptor complex in aged mice, but does not correct the aging change. AB - The purpose of this study was to determine whether glycine is effective in correcting the age-related change in the N-methyl-D-aspartate (NMDA) receptor complex. Autoradiographic analysis of [3H]MK801 and NMDA-displaceable [3H]glutamate binding was performed with 0, 10, 30 and 100 microM glycine on brain tissue from 3-, 10-, and 24-30-month-old C57B1 mice. Glycine, even at 10 microM, enhanced binding to both the NMDA and MK801 binding sites in all age groups. Glycine was not able to reduce the absolute difference in [3H]glutamate binding to NMDA sites between 3- and 24-month-olds. Low micromolar concentrations of glycine exacerbated the aging difference in [3H]MK801 binding between young and old mice, but 100 microM glycine produced smaller aging differences that were similar to those seen with no glycine. This study suggests that glycine may enhance NMDA receptor function in old animals, but it does not appear to correct the underlying problem with the NMDA receptor in aged animals. PMID- 8612099 TI - Effects of age on energy expenditure and substrate oxidation during experimental overfeeding in healthy men. AB - Relatively little is known about the influence of age on energy regulation during energy imbalance. We compared the effects of overfeeding on changes in energy expenditure, substrate oxidation, and energy deposition between young men (age 23.7 +/- 1.1 [SEM] years) and older men (age 70.0 +/- 7.0) of normal body weight who were leading unrestricted lives. Changes in total energy expenditure, resting energy expenditure (REE), the thermic effect of feeding (TEF), respiratory quotient (RQ), and body energy content were determined in response to overeating by 4.09 +/- 0.07 Megajoule (MJ)/day for 21 days in 16 healthy subjects consuming a typical diet. After excluding data from one young subject with unusual results and adjusting for individual differences in excess energy intake, there was a tendency towards a smaller increase in REE in older men compared to the young men (p = .07) which was accounted for by their lower fat-free mass (p = .016). There was also a significantly smaller increase in resting energy expenditure averaged over fasting and fed states (i.e, REE + TEF) with overfeeding in older men than in young men (p < .01). Combined, these smaller increases in energy expenditure with overfeeding in the older subjects averaged an estimated 365 kilojoule (kJ)/day (8.9% of the excess energy intake) (p < .02). There were also significant effects of age on fasting RQ (p < .001) and the change in RQ with overfeeding (p < .001), but no significant increase in energy expenditure for physical activity and thermoregulation with overfeeding in either age-group. These results are consistent with the suggestion that older individuals experience both a reduction in the ability to increase energy expenditure, and an alteration in the pattern of substrate utilization, in response to overfeeding. These changes may promote cumulative increases in body energy during normal cycles of positive energy balance unless compensated for by adaptive variations in energy intake. PMID- 8612101 TI - Moderate caloric restriction prevents the age-related decline in growth hormone receptor signal transduction. AB - A decline in plasma concentration of insulin-like growth factor-1 (IGF-1) has been hypothesized to contribute to a decrease in tissue protein synthesis and function in aging animals and man. In this study, the effects of aging and long term caloric restriction on growth hormone receptor signal transduction were assessed in hepatic tissue to determine whether alterations in tissue responsiveness to growth hormone contribute to the decline in IGF-1 gene expression. Liver slices from female C57/BL mice (10, 17, and 31 months) were prepared in media and stimulated with growth hormone (2 nM). An increase in growth hormone receptor binding was observed in 31-month ad libitum-fed animals (p < .01) compared to 10- or 17-month-old animals), and this effect was partially attenuated by moderate caloric restriction. However, growth hormone (2 nM) induced IGF-1 gene expression was significantly lower in old ad libitum-fed animals (p < .05 compared to 10-month-old ad libitum and 31-month-old caloric restricted animals). Further analysis revealed that growth hormone receptor and JAK2 kinase phosphorylation as well as mitogen-activated protein (MAP) kinase activity were significantly lower in old animals compared to the adult or middle age groups (p < .05). Old caloric-restricted animals demonstrated a significant increase in growth hormone receptor and JAK2 kinase phosphorylation and MAP kinase activity in response to growth hormone. The results demonstrate that growth hormone increases growth hormone receptor and JAK2 kinase phosphorylation as well as MAP kinase activity in liver. These responses decrease with age and are attenuated by moderate, long-term caloric restriction. PMID- 8612100 TI - Effects of age on energy expenditure and substrate oxidation during experimental underfeeding in healthy men. AB - Relatively little is known about the influence of age on energy regulation during energy imbalance. We compared the effects of underfeeding on changes in energy expenditure, substrate oxidation, and body energy loss between young men (age 22.0 +/- 0.9 [SEM] years) and older men (age 66.0 +/- 1.8) who were leading unrestricted lives. Changes in total energy expenditure (TEE), resting energy expenditure (REE), the thermic effect of feeding (TEF), respiratory quotient, and body energy loss were determined in response to undereating by 3.17 +/- 0.16 Megajoule (MJ)/day for 21 days in 19 healthy subjects consuming a typical diet. No significant effects of age were observed in changes in TEE, REE, TEF, or body energy loss in response to underfeeding. Thus, older men do not appear to have any impairment of energy conservation during negative energy balance compared to young men. This normal pattern of energy conservation during undereating together with the previously demonstrated reduction in energy dissipation during overeating can be predicted to promote body fat deposition in older men during the cycles of positive and negative energy balance that occur during normal life. This finding may help to explain the increased body fat mass in older individuals. PMID- 8612102 TI - Effect of age and food restriction on alkaline protease activity in rat liver. AB - The effect of age and food restriction on the hepatic alkaline protease activity of 100,000 x g supernatant has been investigated using 7-, 16-, and 26-month-old Fischer 344 rats. The proteasome, a major component of alkaline protease activity, is activated by sodium dodecyl sulfate (SDS) and this property was exploited to gain insight into the effects of age and food restriction on proteasome activity. Three alkaline protease activities, chymotrypsin-like (ChT L), trypsin-like (T-L), and peptidylglutamyl peptide hydrolyzing (PGPH) activities were measured. These activities are also commonly used as measurement of proteasomal activities. Basal ChT-L and PGPH activities were not markedly altered by either age or food restriction. The level of T-L activity did not change with age, but was decreased by food restriction. SDS-activated ChT-L activity increased 15% between 7 and 26 months of age and this increase was blocked by food restriction. SDS-activated PGPH activity decreased 40% and the decrease was ameliorated by food restriction. In conclusion, we have shown that the alteration of alkaline protease activities by age and food restriction is not uniform and that the changes observed are likely due to alterations of proteasomal activity. The lack of uniformity in these alterations indicates that any assessment of alkaline protease activity requires the measurement of more than one of the enzymatic activities. Lastly, the first evidence suggesting that age and food restriction can modulate proteasomal activity is presented. PMID- 8612103 TI - Postural sensitivity to visual flow in aging adults with and without balance problems. AB - BACKGROUND: This study tested balance behavior of young adults and aging adults with and without balance problems in response to visual flow from a moving visual surround. METHODS: Balance behavior was indexed by force plate measures of maximum anterior/posterior displacement of the center of foot pressure and horizontal shear forces. The sample included normal young adults (n = 13; mean age 23 years, +/- 7.5), normal aging adults (n = 13; mean age 76 years, +/- 6.5), and aging adults with balance problems not directly attributable to a diagnosable neurological disease or dysfunction (n = 13; mean age 79 years, +/- 5.8). RESULTS: The balance-affected aging group had statistically greater sway responses than the young group when the stimulus was unexpected (as in the first trial; p < .05). Some individuals in each group had large responses that were statistical outliers from the group median. The balance-affected group had significantly greater shear forces than the young group. CONCLUSIONS: Greater sway responses suggest over-reliance on visual cues for posture control in the balance-affected aging group, which may be related to underlying, borderline somatosensory deficits, as indicated by the patterns of subclinical indications for somatosensory impairments on neurological exams in this group. Visually sensitive postural control, however, may issue from several different underlying processes. Elevated shear forces during balance responses in the balance-affected group suggest a greater use of hip movements in addition to ankle movements for postural adjustments. PMID- 8612104 TI - Postural balance and its sensory-motor correlates in 75-year-old men and women: a cross-national comparative study. AB - BACKGROUND: There are no earlier cross-national comparative studies analyzing the functions of the posture control mechanisms and its sensory-motor correlates in elderly subjects. We investigated whether there are differences in balance between elderly subjects living in different geographical areas, and analyzed the sensory-motor associates of balance in men and women separately. METHOD: Using a force platform method, the functioning of the posture control system under three standardized conditions (normal standing, eyes open; normal standing, eyes closed; and tandem standing, eyes open) was studied among samples of 75-year-old residents in three Nordic localities, namely Glostrup in Denmark, Goteborg in Sweden, and Jyvaskyla in Finland. The associations of the variables describing performance in each test with other sensory and motor functions were studied using correlation analyses and multivariate regression models. RESULTS: Differences between the populations were observed in both tests with visual control, favoring the participants from Glostrup and Jyvaskyla compared with those from Goteborg. However, only minor differences between the subjects from different localities were observed in the test performed with the eyes closed. In all localities there was a primary sex difference in favor of the women which, however, mainly disappeared when body height was taken into the analyses as a covariate. A good performance in the balance tests (body height-adjusted values) was associated with good visual acuity, low vibrotactile thresholds, and high psychomotor speed. Also, isometric muscle strength, especially hand grip and body extension, was positively associated with good performance in the balance tests. Among the women, a poorer balance was observed in women with a smaller body mass. The results of the multivariate analyses showed that among the men, the most important predictors of good performance in the balance tests were low vibrotactile threshold on the foot, high isometric hand grip strength, and low body stature. Among the women, the most important predictors were low body stature, high body mass, high isometric body extension strength, and high psychomotor speed. However, only a small proportion of the variance in balance (about 13% in the men and 11% in the women) could be explained by the help of these factors. CONCLUSIONS: As the same procedure was applied to the analysis of postural balance, some differences between the populations living in different localities could be detected in some of the tests. The better performance of the women in the balance tests may partly be explained by anthropometric factors, especially differences in body height. There may also be differences in sensory motor associates of balance in elderly men and women. On the basis of the associations observed, it is difficult to explain the differences in balance between the sexes or subjects living in different localities. Within the sexes, only a small proportion (10-13%) of the variation in balance during normal standing with eyes open could be explained by the factors included in the study. PMID- 8612105 TI - The effect of exercise on gait patterns in older women: a randomized controlled trial. AB - BACKGROUND: This study was undertaken to determine (a) whether a program of regular exercise can improve gait patterns in older women, and (b) whether any such improvement in gait is mediated by increased lower limb muscle strength. METHOD: A 22-week randomized controlled trial of exercise was conducted as part of the Randwick Falls and Fractures Study in Sydney, Australia. Subjects were 160 women aged 60-83 years (Mean age 71.1, SD = 5.2) who were randomly recruited from the community. Exercise and control subjects were tested prior to and at the end of the trial. At initial testing, exercisers and controls performed similarly in the strength and gait parameters. They were well matched in terms of age and a number of health and life-style characteristics. RESULTS: At the end of the trial, the exercise subjects showed improved strength in five lower limb muscle groups, increased walking speed, cadence, stride length, and shorter stride times as indicated by both reduced swing and stance duration. There were no significant improvements in any of the strength or gait parameters in the controls. Within the exercise group, increased cadence was associated with improved ankle dorsiflexion strength, and increased stride length was associated with improved hip extension strength. Exercise subjects with initial slow walking speed showed greater changes in velocity, stride length, cadence, and stance duration than those with initial fast walking speed. CONCLUSION: These findings show that exercise can increase gait velocity and related parameters in older persons, and that part of this increase may be mediated by improved lower limb muscle strength. PMID- 8612106 TI - Effect of age on body water and resting metabolic rate. AB - BACKGROUND: We previously reported that differences in fat-free mass (FFM) estimated by isotope dilution of 18O-labeled water could not fully account for lower resting metabolic rates (RMR) in old men and women compared to RMR in young men. METHODS: Since age-related changes in the distribution of water between extracellular and intracellular spaces could lead to overestimation of FFM in the old, we reanalyzed our data using estimates for total body and intracellular water (TBW and ICW, respectively) derived from published equations and included data from adolescent boys and girls studied under similar conditions. RESULTS: In both sexes, the age-related reduction in RMR remained significant after adjustment for estimated body water compartments (p < .05). While adjusted RMR differed in boys and girls (p < .0001), it did not in old men and women (p = .15). CONCLUSION: We conclude that aging per se reduces RMR in lean tissue, a difference which cannot be fully explained by changes in body water or its distribution. Investigators should be cautious when selecting models and equations to estimate body water compartments. PMID- 8612107 TI - The impact of ibuprofen on the efficacy of antihypertensive treatment with hydrochlorothiazide in elderly persons. AB - BACKGROUND: Nonsteroidal antiinflammatory drugs (NSAIDs) may alter blood pressure through their inhibitory effects on prostaglandin biosynthesis. Such potential hypertensive effects of NSAIDs have not been adequately examined in the elderly, who are the largest group of NSAID users. METHODS: We performed a randomized, double-blind, two-period crossover trial of ibuprofen (1800 mg per day) vs placebo treatment in patients older than 60 years of age with hypertension controlled with hydrochlorothiazide. While continuing their usual thiazide dosage, subjects were randomized to a 4-week treatment period (ibuprofen or placebo) followed by a 2-week placebo wash-out period and a second 4-week treatment period with the alternative therapy. Supine and standing systolic and diastolic blood pressures were measured weekly. RESULTS: Of 25 randomized subjects, 22 completed the study protocol (mean age = 73 +/- 6.7 years). Supine systolic blood pressure and standing systolic blood pressure were increased significantly with ibuprofen treatment, compared with placebo. Mean supine systolic blood pressures were 143.8 +/- 21.0 and 139.6 +/- 15.9 mmHg on ibuprofen and placebo, respectively (p = .004). Mean standing systolic blood pressures were 148.1 +/- 19.9 and 143.4 +/- 17.9 mmHg on ibuprofen and placebo, respectively (p = .002). CONCLUSION: We conclude that 1800 mg per day of ibuprofen does induce a significant increase in systolic blood pressure in older hypertensive patients treated with hydrochlorothiazide. NSAID therapy may negatively impact the control of hypertension in elderly patients. PMID- 8612108 TI - Glomerular dysfunction in the aging Fischer 344 rat is associated with excessive growth and normal mesangial cell function. AB - BACKGROUND: Fischer 344 (F344) rats display focal and diffuse glomerulosclerosis with aging postulated to result from loss of normal mesangial cell intrinsic function, e.g., vasoactive hormone signaling, or preservation of normal responsiveness to extrinsic growth factors. METHODS: In 3-, 17-, and 24-month-old F344 male rats, glomerular structure, measured by PC-based morphometry, and function were compared. Immunoperoxidase staining of glomerular proliferating cell nuclear antigen (PCNA) detected cellular proliferation. Primary cultured mesangial cells from the 3 age groups were studied in parallel. Calcium (Ca2+) signaling, measured by Fura-2 fluorescence, contraction to vasopressin (AVP) 1 microM, measured by videomicroscopy, and proliferative response to platelet derived growth factor-beta beta (PDGF) were compared. RESULTS: Proteinuria was 13 +/- 4, 38 +/- 17, and 110 +/- 35 mg/24 hours at 3, 17, and 24 months, respectively (n = 5, mean +/- SE, p < .01, 3 vs 24 months), with no change in 24 hour creatinine clearances. Glomerular volumes (n = 200/group) for 3, 17, and 24 months, respectively, were .30 +/- .01, .60 +/- .02, .74 +/- 0.2 x 10(6) micron3 (p < .001, 3 months vs 17 months, and 17 vs 24 months). Glomerular basement membrane (GBM) widths and fractional mesangial volumes increased significantly with aging. Glomerular cell PCNA staining remained positive at 24 months. Cultured mesangial cell Ca2+ signaling and contraction to AVP were unchanged with aging. Proliferation to PDGF, which was partially inhibited with verapamil, was similar at 3 and 24 months. CONCLUSIONS: In the Fischer 344 rat, mesangial cell Ca2+ signaling, contraction, and proliferation responsiveness are unchanged with aging. Continued growth is associated with the glomerulosclerosis of aging. PMID- 8612109 TI - Longitudinal changes in smell identification. AB - BACKGROUND: Cross-sectional studies have demonstrated that olfactory function diminishes with increasing age, which may impact on the safety and quality of life of older persons. To date, however, there have been no published longitudinal studies on olfaction. The purpose of this study was to examine the influence of age and gender on smell identification over a 3-year period in a group of generally healthy men and women. METHODS: Males (n = 85) and females (n = 76) between the ages of 19 and 95 years were administered the University of Pennsylvania Smell Identification Test (SIT) over a 3-year span as part of the oral physiology component of the Baltimore Longitudinal Study of Aging. A linear mixed-effects regression model was used to determine how longitudinal changes in SIT scores differ with respect to gender, history of medical problems, and use of prescription medications. RESULTS: Over the 3-year period, SIT scores diminished progressively with increased age. Women and men in the eighth decade of life experienced a decline of greater than one SIT point per year. Females consistently performed better than males in smell identification. Similar results were obtained regardless of medical problems or medication usage. CONCLUSIONS: These results extend the conclusions of previous cross-sectional olfactory studies and indicate that smell identification deteriorates progressively with greater age. Furthermore, age-related declines in olfaction occur even in the absence of overt medical problems. PMID- 8612110 TI - Immunotherapy for childhood asthma: is there a rationale for its use? AB - OBJECTIVE: This paper reviews the literature regarding immunotherapy in the management of childhood asthma. Immunotherapy is a well established treatment of venom allergy and allergic rhinitis, however its use in asthma remains controversial. DATA SOURCES: We reviewed the pediatric literature from 1966 to 1994 and evaluated the existing studies for clinical efficacy of immunotherapy in childhood asthma. STUDY SELECTION: Only 12 purely pediatric studies existed over the time period that we reviewed. The studies used a variety of different antigens including house dust, house dust mite, grass, mold, cat, dog, and combinations of antigens. RESULTS: In reviewing the studies, we assessed study duration, number of subjects, whether it was blinded, placebo controlled or open labeled, the measures of clinical efficacy and the assessments of specific and nonspecific bronchial reactivity. The studies were very heterogeneous, and therefore direct comparison and extrapolation of conclusions was difficult. The majority of the studies demonstrated either an improvement in asthmatic symptoms or a decrease in bronchial reactivity to the specific antigen employed, or both. The minority of studies demonstrated no clinical efficacy. The most consistent evidence of benefit was suggested in those trials employing house dust mite therapy, while immunotherapy for grass and cats demonstrated some benefit but the number of studies employing these treatments was very small. There are no trials that provide convincing evidence that immunotherapy with dog and mold antigens is effective for childhood reactive airway disease. CONCLUSION: Asthma is a multifactorial disease with many triggers. In establishing a role for immunotherapy one must consider all the different aspects such as allergic triggers, environmental stresses, and viral infections. The literature is unclear as to when immunotherapy should be initiated for childhood asthma. While there are suggestions that immunotherapy should be considered for the child with mild or moderate asthma and dust mite sensitivity when pharmacotherapy is not efficacious, the immunomodulatory properties of immunotherapy may actually be more tailored for early intervention in asthma rather than for use once symptoms have occurred. More research is required in order to clarify whether immunotherapy should be recommended more often for the treatment of childhood asthma. PMID- 8612111 TI - Chronic cough in a child. PMID- 8612112 TI - Recurrent Wegener's granulomatosis: a case report and review. AB - BACKGROUND: Wegener's granulomatosis is a granulomatous systemic necrotizing vasculitis with prominent upper airway involvement. Complete remissions can be induced with aggressive management. Despite successful treatment, relapses and recurrences of active disease may occur. OBJECTIVE: We present a patient who presents with histologically confirmed active disease 17 years following successful therapy. METHODS: A case report of a 72-year-old man who initially presented at age 50 with necrotizing vasculitis of the upper and lower respiratory tract. Eighteen years following his initial therapy he presented with isolated retroorbital disease. Following re-institution of cytotoxic therapy he appears to be disease-free. RESULTS: Following re-treatment with cytoxan and prednisone, a second remission was induced. The patient is currently entirely well 1 year off therapy. CONCLUSION: Wegener's granulomatosis may recur many years following apparent successful and aggressive cytotoxic and anti inflammatory therapy. PMID- 8612113 TI - Hypersensitivity to banana in latex-allergic patients. Identification of two major banana allergens of 33 and 37 kD. AB - BACKGROUND: Allergy to banana often occurs in patients sensitized to latex. The spectrum of IgE-mediated responses to latex allergens is more and more documented but banana allergens and epitopes shared by these two allergens have not yet been characterized, even though the existence of cross-reacting IgE antibodies has been demonstrated. OBJECTIVE: The purpose of this study was to assess the relationship between banana hypersensitivity and latex allergy in a population of 19 latex-allergic patients and to identify allergenic components in banana and the common structures shared with latex. METHODS: The in vivo study was conducted in our outpatient department in patients with well-documented latex allergy. Clinical histories were evaluated and skin prick tests were performed with banana and latex extracts. IgE responses from 19 patients were investigated by means of CAP RAST assay and SDS PAGE immunoblotting. Epitopes shared by banana and latex were investigated by means of immunoblotting experiments. RESULTS: Eight of 16 patients (50%) reported symptoms after eating bananas and banana skin prick tests were positive in 5 of 14 patients (36%). Banana RAST results were positive in 12 of the 19 patients (63%). In immunoblot experiments, 17 of the 19 patients (89%) exhibited specific banana IgE antibodies and 16 allergenic components were identified with molecular weights ranging from 17 to 128 kD. Two were considered as major allergens: 33 kD was detectable in 15 of 19 sera (88%) and 37 kD in 13 of 19 sera (76%). Inhibition studies by preincubation of two individual sera with banana or latex extract demonstrated the complete disappearance of IgE binding on banana blotted allergens. CONCLUSION: This study confirms the "latex-fruit syndrome" already described by Blanco et al. Two major allergens were revealed in banana at 33 and 37 kD and the presence of more than ten common components with latex was observed. PMID- 8612114 TI - Selective effect of levocabastine on histamine receptor and histamine release from human leukocytes and guinea pig isolated tissue. AB - BACKGROUND: Levocabastine is a potent histamine H1 receptor antagonist used topically in the treatment of patients with allergic rhinitis. It has been suggested that antihistamines also have anti-inflammatory properties. OBJECTIVE: The present study was performed to investigate whether levocabastine, in addition to the anti-H1 receptor activity, has anti-inflammatory properties and thus is able to modulate the release of histamine and cytokines, such as interleukin 5 from human leukocytes and isolated tissues. METHODS: Leukocytes suspensions were prepared by dextran sedimentation of peripheral venous blood drawn from allergic and healthy volunteers. Leukocytes obtained from allergic volunteers were preincubated for 30 minutes with levocabastine (doses 10(-8) M to 10(-6) M) and thereafter incubated with allergen. Leukocytes obtained from healthy volunteers were incubated for zero to three hours with levocabastine (doses 10(-14) M to 10( 3) M). Histamine release was measured by an automated fluorometric method. Interleukin-5 release was measured by enzyme linked immunoassay. Contractile responses to histamine on guinea pig trachea and lung parenchyma as well as the release of histamine and interleukin-5 by the tissues were investigated in the absence or presence of levocabastine and/or the histamine H2 receptor antagonist cimetidine. RESULTS: Levocabastine did not influence allergen-induced histamine release from leukocytes obtained from allergic volunteers. High concentrations (10(-4)and 10(-3) M) of levocabastine, however, caused release of histamine from leukocytes obtained from healthy volunteers as well as guinea pig airway smooth muscle tissues. Pretreatment with levocabastine dose-dependently decreased the contractile response to histamine, showing an irreversible competitive mechanism. Interleukin 5 release from human leukocytes and by guinea pig airway smooth muscle was not detectable. CONCLUSIONS: These findings indicate that the H1 receptor blocker, levocabastine, has probably no anti-inflammatory properties, measured as histamine release, and that the histamine release from both human leukocytes and guinea pig trachea and lung parenchyma is significantly increased by the drug only at high concentrations. PMID- 8612115 TI - Effects of theophylline and ipratropium on cognition in elderly patients with chronic obstructive pulmonary disease. AB - OBJECTIVES: To determine whether chronic therapy with theophylline or ipratropium has an adverse effect on cognition and psychomotor skills in geriatric patients with chronic obstructive pulmonary disease. DESIGN: The study design was a randomized, repeated measures, double-blind, double-dummy, placebo-controlled comparison of theophylline and ipratropium treatments. SETTING: Ambulatory patients were tested at the Clinical Trials Center of the University of California, San Diego, Medical Center. PATIENTS: Ambulatory patients with chronic obstructive pulmonary disease aged 65 years or more with FEV1 less than 60% predicted, FEV1/FVC less than 70%, and post bronchodilator FEV1 less than 70%. INTERVENTIONS: Patients received either theophylline or ipratropium for 2 weeks, followed by a 1-week placebo control period, then a 2-week treatment period of the alternative drug therapy. A standard therapy of albuterol MDI, 2 puffs (180 microg) qid was given throughout the study. MEASUREMENTS: The main response level was an 11-part battery of psychometric tests. Tests were administered at the end of each treatment period and at the end of the washout period. Covariates were sequence of treatment, pulmonary function tests, age, and baseline psychometric test scores. RESULTS: There was no difference in performance scores on the cognitive tests among the three treatment periods. CONCLUSIONS: We were unable to detect a harmful effect of treatment with either theophylline or ipratropium on the performance of elderly patients with chronic obstructive pulmonary disease on a battery of psychometric tests, suggesting that significant cognitive impairment in the elderly is not commonly associated with treatment with either theophylline or ipratropium. PMID- 8612116 TI - Preimmunization and postimmunization pneumococcal antibody titers in children with recurrent infections. AB - BACKGROUND: Patients with recurrent infections and normal IgG levels may have an abnormal response to pneumococcal polysaccharides. The ability to develop antibodies against different pneumococcal polysaccharides develops gradually in the first years of life, but the sequence of development and the influence of preexisting antibody titers has not been defined. METHODS: Preimmunization and postimmunization IgG antibody titers against pneumococcal serotypes 3, 7F, 9N, and 14 were evaluated in a population of 100 1- to 18-year-old children referred to a pediatric allergy-immunology clinic because of recurrent respiratory infections. None of the patients had a known immunodeficiency syndrome; all had normal total IgG levels. Postimmunization antibody levels were obtained 4 to 6 weeks after immunization. Patients less than/=5 years of age who failed to develop antibody levels above 200 ng Ab N/mL against any serotype and older patients who failed to develop these levels against a second serotype in addition to serotype 3 were considered for IgG replacement therapy. RESULTS: Prior to immunization, 50% of 51 patients did not have protective antibody levels against any of the serotypes tested. Immunization induced a high response to serotype 3 in all age groups, but responses to serotypes 7F and 14 increased with age. Five of 78 patients (6.4%) failed to develop protective antibody levels against any serotype tested. Three of these patients had clinical criteria that justified the use of IgG replacement therapy; all improved. Three patients were re-immunized 1 to 2 years after the first immunization and all developed protective levels of antibodies against serotype 3 after the second immunization. CONCLUSION: We conclude that, although measurement of antibody levels against pneumococcal serotype 3 allows a good differentiation of patients who are able to develop anti polysaccharide antibodies from those who are not, further studies of the development of specific antibodies against other vaccine serotypes in normal populations of different ages are needed to define a normal response to pneumococcal polysaccharides. PMID- 8612118 TI - Management of adverse reactions to prophylactic trimethoprim-sulfamethoxazole in patients with human immunodeficiency virus infection. AB - BACKGROUND: In hospitals attended by patients with human immunodeficiency virus infection, adverse reactions are often observed to trimethoprim-sulfamethoxazole, particularly cutaneous reactions. Given the importance of this drug for prophylaxis we have attempted to establish a desensitization or tolerance protocol so that patients can continue the drug without repeated adverse reactions. METHODS: We studied 34 HIV patients with adverse cutaneous reactions to trimethoprim-sulfamethoxazole, slight to moderate in nature but not life threatening. Skin tests (prick and intradermal) were done in an attempt to rule out a mechanism of hypersensitivity. Subsequently, trimethoprim-sulfamethoxazole was administered orally in increasing doses beginning with trimethoprim, 0.2 mg, and sulfamethoxazole, 1 mg. The same dose was repeated after 12 hours and then doubled every 24 hours until the therapeutic dose was achieved. If adverse reactions appeared we maintained the last dose administered and administered antihistamines until the reactions cleared or improved. RESULTS: None of the patients had positive skin tests (immediate or delayed). Twenty- seven patients were satisfactorily desensitized. After a follow-up of 3 months, 25 patients were still incident-free on trimethoprim-sulfamethoxazole prophylaxis, and 19 returning for check-ups at 6 months could still tolerate the drug well. CONCLUSIONS: Our data indicate that patients with adverse reactions to trimethoprim-sulfamethoxazole can continue prophylactic treatment after oral desensitization. PMID- 8612117 TI - Motivation for participation in clinical trials of drugs for the treatment of asthma, seasonal allergic rhinitis, and perennial nonallergic rhinitis. AB - BACKGROUND: While previous studies of enrollment motivation have been conducted with either healthy subjects or subjects with certain other diseases, little is known about the motives of subjects with asthma or rhinitis symptoms who seek to enter clinical trials. OBJECTIVE: This study was conducted to assess the self reported role that altruism, healthcare receipt, and financial gain play in the motivation of subjects with symptoms of bronchial asthma, seasonal allergic rhinitis, and perennial nonallergic rhinitis who attempted to enroll in clinical trials. METHODS: Subjects with symptoms of asthma, allergic rhinitis, and perennial nonallergic rhinitis who sought to enroll in phase III clinical trials completed surveys from December 1991 to August 1992 (n = 295). The importance of altruistic and nonaltruistic motives was rated on numerical scales. RESULTS: Improved control of symptoms and learning more about the illness and medications for treatment were the most important nonaltruistic motives (P less than .05). Financial motives and second opinion were moderately important but less important than healthcare motives (P less than .05). This population as a whole agreed that the altruistic motives listed in the survey were reasons to enroll. CONCLUSION: Subjects with symptoms of asthma, allergic rhinitis, and perennial nonallergic rhinitis entered clinical trials for altruistic reasons and to receive healthcare treatment for their chronic illness including related health education. For the entire group, self-reported financial motives were less important than illness related healthcare. PMID- 8612119 TI - In vitro release of soluble CD23 by human lymphocytes in ragweed-sensitive versus nonatopic patients following stimulation with ragweed antigen E. AB - BACKGROUND: Soluble CD23 is the proteolytic cleavage product of the low affinity receptor (FcERII). Functions of CD23 and its soluble products may include upregulation of IgE production and stimulation of B lymphocyte growth. METHODS: Soluble CD23 was quantitated in supernatants of lymphocytes from nine ragweed sensitive and eight nonatopic subjects stimulated in vitro by antigen E (amb Al), crude ragweed extract, and pokeweed mitogen (PWM). RESULTS: Although PWM stimulation produced no significant difference between groups, sCD23 release was significantly elevated in the cells of nonatopic patients stimulated with antigen E and crude ragweed extract (P less than .05). CONCLUSIONS: This finding supports the concept of separate pathways of activation by antigen and mitogen for sCD23 release and suggests ragweed-sensitive and nonatopic patients have fundamental differences in the response of sCD23 release to ragweed antigen stimulation. PMID- 8612121 TI - Osteoporosis in steroid-dependent asthma. AB - BACKGROUND: Patients on prolonged corticosteroid therapy are at risk of developing osteoporosis. Some patients with severe asthma are difficult to wean off corticosteroids and are therefore at risk of developing bony complications due to steroids. OBJECTIVE: The purpose of this study was to examine the relationship of cumulative steroid dosage and duration of therapy with osteoporosis. METHODS: We obtained bone mineral density studies using dual photon absorptiometry, and radiographs of the lumbar spine of 16 steroid-dependent patients with asthma. Patients with conditions affecting bone metabolism were excluded. RESULTS: We studied 16 male steroid-dependent patients with asthma who received 4 to 41 grams equivalent dose of prednisone over a period of 1 to 15 years. The overall prevalence rate for abnormal age-matched bone mineral density was 50%. Abnormal bone mineral density was more commonly noted in the lumbar spine (38%) than in the femoral neck (19%). The lowest dose of corticosteroid associated with a decrease in bone mineral density was a cumulative steroid dose of 5.6 equivalent grams-prednisone. CONCLUSION: Prolonged corticosteroid therapy can cause significant osteoporosis among male patients with steroid-dependent asthma. Bone loss due to corticosteroid therapy occurs at different rates at different bony sites. PMID- 8612122 TI - Duration of inhibitory effect of terfenadine on histamine-induced skin wheals. AB - BACKGROUND: The duration of inhibitory effect of terfenadine on histamine-induced skin wheals has been reported differently by different workers. OBJECTIVE: The present study was carried out to study the duration of inhibitory effect of terfenadine on histamine-induced skin wheals. METHODS: In nine normal, healthy adult volunteers, baseline skin reactivity was established by performing intradermal skin tests with histamine and normal saline on three consecutive days. The effect of two regimens of terfenadine was evaluated: 1-day treatment with 60 mg bid and 1-week treatment with the same dosage. Skin tests were performed every day following the treatment until the wheal size had recovered to within 90% of the baseline value. RESULTS: The mean duration of inhibition with the 1-day treatment was 70.66 +/- 17.47 hours while that following the 1-week treatment was 78.66 +/- 20.00 hours (P less than .05). The inhibition was about 40% at 24 hours and 17% at 48 hours with 1-day treatment while it was 37% and 18%, respectively, with 1-week treatment. In individual subjects, the duration of inhibitory effect varied from 36 to 84 hours with 1-day treatment and 60 to 108 hours with 1-week treatment. CONCLUSION: The study showed that terfenadine has no significant cumulative antihistaminic effect on repeated dosing. It is suggested that terfenadine should be stopped at least three days before performing skin tests for type I hypersensitivity. PMID- 8612120 TI - Comparison of efficacy, safety, and skin test inhibition of cetirizine and astemizole. AB - BACKGROUND: Astemizole, an H1-histamine-receptor antagonist prescribed for seasonal allergic rhinitis, has a slow onset of action and a strong suppressive effect on the wheal and flare reaction, which interferes with skin testing results. The newer antihistamine cetirizine appears to have a rapid onset of action and a low potential to interfere with posttreatment skin testing results. OBJECTIVE: To compare the efficacy, safety, and skin test inhibition of astemizole and cetirizine in the treatment of seasonal allergic rhinitis. METHODS: In a double-blind, parallel-group study conducted at six sites during ragweed pollination season, 263 subjects were randomized to receive 10 mg of astemizole, 5 mg of cetirizine, or 10 mg of cetirizine daily for 2 weeks. The subjects rated seven allergic rhinitis symptoms daily, the subjects and investigators provided global assessments of the responses to the treatments, and the subjects rated their satisfaction with the treatments. Thirty-nine subjects at one study site underwent quantitative skin testing before and after treatment. RESULTS: As measured by reduction from baseline in total symptom severity score, which was the primary efficacy measure in the study, all three treatments significantly relieved the symptoms of allergic rhinitis (P less than .05). This finding was supported by the global ratings and the subject satisfaction ratings. There were no significant differences among the three treatments for reduction from baseline in total symptom severity score. The mean subject satisfaction score with 10 mg of cetirizine was significantly greater than that with astemizole (P less than .05). In the skin tests performed 3, 7, and 14 days after the end of antihistamine treatment, the subjects who had received the cetirizine doses had significantly greater mean sum of wheal and mean sum of erythema values than those who had received the astemizole dose (P less than .05). Sensitivity to ragweed pollen extract returned to 90% of baseline within three days of the end of cetirizine treatment. Both drugs were well tolerated and their adverse event profiles were similar. CONCLUSIONS: Astemizole and cetirizine are effective and well tolerated in alleviating the symptoms of ragweed-induced allergic rhinitis. Cetirizine inhibits skin test results to a much lesser extent than does astemizole. Physicians may wish to consider the potential for skin test inhibition when selecting an antihistamine for patients with allergic rhinitis. PMID- 8612123 TI - A fatality from idiopathic anaphylaxis. AB - BACKGROUND: Idiopathic anaphylaxis by definition is a form of anaphylaxis where no identifiable stimulus can be found. In our series of over 350 patients with idiopathic anaphylaxis there have been no reported deaths. OBJECTIVE: To report the clinical course of the first fatality in our series from idiopathic anaphylaxis. METHODS: A patient was evaluated for idiopathic anaphylaxis and started on our previously published protocol of corticosteroids, sympathomimetics, and antihistamines. RESULTS: Despite following our protocol- which has been effective in decreasing the number of emergency room visits, hospitalizations, and intensive care unit admissions--controlling the disease, and placing a high percentage of patients into remission, the patient with idiopathic anaphylaxis died unexpectedly after experiencing symptoms similar to previous episodes of idiopathic anaphylaxis. CONCLUSION: This is the first reported fatality from idiopathic anaphylaxis in our extensive series. PMID- 8612126 TI - Allergy injections at home? PMID- 8612127 TI - IL-15: a pleiotropic cytokine with diverse receptor/signaling pathways whose expression is controlled at multiple levels. PMID- 8612128 TI - MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. AB - The generation of mature CD4 T cells from CD4+CD8+ precursor thymocytes usually requires corecognition of class II MHC by a TCR and CD4, while the production of mature CD8 T cells requires corecognition of class I MHC by a TCR and CD8. To assess the role of the CD4 coreceptor in development and lineage commitment, we generated CD4-deficient mice expressing a transgenic class II-specific TCR. Surprisingly, in the absence of CD4 a large number of T cells mature, but these cells appear in the CD8 lineage. Thus, when CD4 is present, the majority of immature T cells with this class II-specific TCR choose the CD4 lineage but develop in the CD8 pathway when CD4 is absent. The results indicate that even for TCRs that are not dependent on coreceptor for MHC recognition, the coreceptor can influence the lineage choice. These findings are considered in terms of a quantitative signaling model for CD4/CD8 lineage commitment. PMID- 8612129 TI - In vivo cross-priming of MHC class I-restricted antigens requires the TAP transporter. AB - Recent in vitro evidence suggests two alternative mechanisms by which bone marrow derived APCs may process exogenous antigens for presentation to CTL in vivo, a phenomenon termed cross-priming. Although in vitro studies have suggested that both TAP-dependent and TAP-independent pathways exist, we have now demonstrated an absolute requirement for a functional TAP for cross-priming to occur in vivo. Bone marrow chimeras reconstituted with marrow from TAP-defective donors develop functional CD8+ CTL, but have APCs with disrupted TAP function. In such chimeras, in vivo priming of naive CTL was observed when antigen was targeted to the ER in a TAP-independent fashion, but cross-priming could not be demonstrated. These results support the TAP-dependent mechanism of cross-priming. PMID- 8612130 TI - Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. AB - Destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complexes to the cell surface. The cysteine protease cathepsin S is highly expressed in spleen, lymphocytes, monocytes, and other class II-positive cells, and is inducible with interferon-gamma. Specific inhibition of cathepsin S in B lymphoblastoid cells prevented complete proteolysis of li, resulting in accumulation of a class II associated 13 kDa li fragment in vivo. Consequently, the formation of SDS-stable complexes was markedly reduced. Purified cathepsin S, but not cathepsin B, H, or D, specifically digested li from alpha beta li trimers, generating alpha beta CLIP complexes capable of binding exogenously added peptide in vitro. Thus, cathepsin S is essential in B cells for effective li proteolysis necessary to render class II molecules competent for binding peptides. PMID- 8612131 TI - The specificity and orientation of a TCR to its peptide-MHC class II ligands. AB - A T cell-mediated immune response is mainly determined by the 3-5 aa residues that protrude upwards from a peptide bound to an MHC molecule. Alterations of these peptide residues can diminish, eliminate or radically alter the signal that the T cell receives through its T cell receptor (TCR). We have used peptide immunizations of normal mice and mice carrying alpha or beta chain TCR transgenes to identify three distinct peptide contact points. One, near the carboxyl terminus of the peptide, involves the beta chain CDR3 region; the second was centrally located and interacted with both the alpha and beta chain CDR3 loops; the third was near the amino terminus of the peptide, and affected V alpha gene usage, but not the structure of CDR3 of either TCR chain. Based on these results, we propose an orientation for the TCR of this cloned line and argue for its generality. PMID- 8612132 TI - Deletion of the Ig kappa light chain intronic enhancer/matrix attachment region impairs but does not abolish V kappa J kappa rearrangement. AB - Roles of the kappa intronic enhancer (iE kappa) and its associated matrix attachment region (MAR) during B cell development were examined using mutant embryonic stem (ES) cell lines in which the entire region on both chromosomes was replaced with either a recombined LoxP site (E kappa ND) or the PGK-neomycin resistance (PGK-neo(r)) gene (E kappa NI). B cells derived from E kappa ND ES cells had greatly impaired V kappa J kappa rearrangement, normal levels of kappa expression, and kappa:lambda ratios of 1:1 instead of the usual 10:1. Furthermore, lambda-producing hybridomas derived from E kappa ND cells displayed little kappa rearrangement. Thus, the MAR and iE kappa are quantitatively significant for kappa rearrangement but not necessary. In addition, little V kappa J kappa rearrangement could be detected in B cells derived from E kappa NI ES cells, demonstrating that an inserted PGK-neo(r) gene dominantly suppresses V kappa J kappa rearrangement. PMID- 8612133 TI - TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. AB - The death domain of tumor necrosis factor (TNF) receptor-1 (TNFR1) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD. Here, we show that TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. We also show that RIP is a serine threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process. Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis. However, expression of the death domain of RIP Induces apoptosis, but potently inhibits NF-kappa B activation by TNF. These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation. PMID- 8612134 TI - Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice. AB - NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response. PMID- 8612135 TI - Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. AB - Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria. CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS. The role of CD14 in bacterial-induced and LPS induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria. PMID- 8612136 TI - Post-traumatic epilepsy: mechanisms and prevention. AB - An effective method to disrupt the process of post-traumatic epilepsy must await scientific understanding of epileptogenesis. If hemorrhage-induced focal lipid peroxidation is operant, then a strategy designed to disrupt initiation reactions may prove successful. PMID- 8612137 TI - Analyses of the molecular basis of kindling development. AB - Kindling has become the most widely studied animal model of limbic epilepsy. Understanding the molecular basis of kindling development may provide novel therapeutic approaches to prevention of limbic epileptogenesis. I briefly describe the kindling model and outline the basis for my thinking that kindling represents a synaptic reorganization triggered by pathologic activity in the mature nervous system. The pathologic activity is postulated to evoke a cascade of gene expression driven, at least in part, by glutamate receptor activation. Evidence in support of this hypothesis is presented, as are future challenges that permit critical tests of these ideas. PMID- 8612138 TI - Lennox-Gastaut syndrome: a new vista. AB - Lennox-Gastaut syndrome (LGS) is regarded as a model of the epileptic syndrome because of its specific clinicoelectrical manifestation. However, a close investigation reveals that its outline is somewhat vague, having the borderland around it. Precise diagnosis in an individual case is not always easy. In this paper, the diagnostic criteria of LGS are described. According to these criteria, cases with LGS were subclassified into the typical and the atypical cases, and also cases in the borderland of LGS were reviewed. On the other hand, our prospective long-term follow-up study revealed that cortical mechanisms played an important role in the pathophysiology, clinical features and refractoriness of LGS. Secondary bilateral synchrony (SBS) is supposed to be a mode of expression of cortical mechanisms of LGS. A newly developed method with coherence and phase analysis demonstrated that the pathophysiology was based on SBS in 33% of the typical LGS cases. This finding is not only crucial for the choice of rational treatment including epilepsy surgery, such as callosotomy, but also contributes to a more refined subclassification of LGS. PMID- 8612140 TI - Single photon emission computed tomography studies of partial epilepsies. AB - At present, in the field of clinical epilepsy, it has been established that single proton emission computed tomography (SPECT) is useful for the regional determination of epileptic foci. In particular, it seems to be of great advantage to the field of clinical epilepsy that epileptic foci situation in the deep regions of the brain are detectable by the three-dimensional resolution power in SPECT, because such foci are not often represented on routine scalp electroencephalograph (EEG). In reference to our review published previously, recent findings in SPECT studies of partial epilepsies are surveyed here. PMID- 8612139 TI - Serial position emission tomography study in West syndrome. PMID- 8612141 TI - A new method of presurgical evaluation of partial epilepsy with 31P MRS/CSI. PMID- 8612142 TI - Epilepsy and genetics. PMID- 8612143 TI - Role of immediate early gene expression during establishment of epileptogenesis in the brain of EL mice. PMID- 8612144 TI - Clinical genetics of epilepsy. PMID- 8612145 TI - The alteration of AP-1 DNA-binding activity in seizure-prone EL mouse brain. PMID- 8612146 TI - Induction of c-fos and reduction of dynorphin in dentate granule cells of a rat model of epilepsy produced by systemic administration of kainic acid: an immunohistochemical study. PMID- 8612147 TI - Molecular changes of NMDA receptor subunit mRNA in amygdala-kindled rat. PMID- 8612148 TI - Regional increase in brain-derived neurotrophic factor and nerve growth factor, but not acidic and basic fibroblast growth factor, mRNA in kindling. PMID- 8612150 TI - Comparison of IMP-single photon emission computed tomography findings to Wechsler Intelligence Scale and Benton Visual Memory Scale. PMID- 8612151 TI - Rolandic discharges and somatosensory evoked potentials in benign childhood partial epilepsy: magnetoencephalographical study. PMID- 8612149 TI - Manual and digitizer measurements of amygdalohippocampal volume: reliability in comparison to computer-based measurement. PMID- 8612152 TI - Changes in water diffusion of rat limbic system during status epilepticus elicited by kainate. PMID- 8612153 TI - Identification of the epileptic focus by the dipole tracing method: single layered model vs Scalp-Skull-Brain model. PMID- 8612154 TI - Ictal electroencephalographic findings of spasms in West syndrome. PMID- 8612155 TI - Correlation between EEG activity and event-related potential (P300) in childhood partial epilepsy. PMID- 8612157 TI - Prolongation in RR-interval derived from brain activity at atypical absence seizures. PMID- 8612156 TI - Evaluation of semantic processing in patients with temporal lobe epilepsy using event-related potential. PMID- 8612158 TI - The treatment of West syndrome by child neurologists in Japan. PMID- 8612159 TI - Pharmacokinetics of slow-release preparations of sodium valproate. PMID- 8612160 TI - The effect of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, major metabolites of phenytoin, on the occurrence of chronic side effects: in vivo and in vitro studies. PMID- 8612161 TI - Changes in the lymphocyte subsets during high dose immunoglobulin therapy for intractable childhood epilepsy. PMID- 8612162 TI - Effect of vagal stimulation on intractable epilepsy. PMID- 8612164 TI - Immunohistochemical changes of perilesional GABA neurons in alumina cream-induced focal motor epilepsy. PMID- 8612163 TI - Surgical management of intractable epilepsy arising from central area. PMID- 8612165 TI - Clinical course of intractable epilepsy: a 15 year follow-up study. PMID- 8612167 TI - Clinico-electroencephalographic study in intractable epilepsy beginning at infancy. PMID- 8612166 TI - Recurrence of seizures in childhood epilepsy after discontinuation of antiepileptic drugs. PMID- 8612168 TI - Delayed myelination in West syndrome. PMID- 8612169 TI - Prevalence and annual incidence of psychosis in patients with epilepsy. PMID- 8612170 TI - The nature of the depressive symptoms in adult outpatients with epilepsy. PMID- 8612171 TI - Neuropsychological study of localization-related epilepsy in children. PMID- 8612172 TI - Low rate of depressive symptomatology in patient with idiopathic generalized epilepsy. PMID- 8612173 TI - In vivo microdialysis monitoring for extracellular glutamate and GABA in the ventral hippocampus of the awake rat during kainate-induced seizures. PMID- 8612174 TI - Increase of central nitric oxide during pentylenetetrazol-induced seizures in rats. PMID- 8612175 TI - Audiogenic seizure induces c-fos mRNA expression in the inferior colliculus and not in the hippocampus. PMID- 8612176 TI - Translational activity of endogenous and exogenous mRNA in a cell-free translation system from seizure-prone EL mouse brains. PMID- 8612177 TI - Neuropathological study on a newly developed epileptic rat mutant with limbic like seizures. PMID- 8612179 TI - Concurrent development of BECCT in a pair of monozygotic twins. PMID- 8612178 TI - A study of the association between Japanese juvenile myoclonic epilepsy patients and HLA class II antigens. PMID- 8612180 TI - Epileptic activity in the cat perirhinal cortex in response to amygdala kindling. PMID- 8612181 TI - Convulsive seizures induced by micro-injection of bicuculline methiodide into the interpeduncular nucleus in rats. PMID- 8612182 TI - A case of intractable epilepsy with mental deterioration: detection of measles virus genome in cerebrospinal fluid and peripheral mononuclear cells using reverse transcriptase-polymerase chain reaction. PMID- 8612183 TI - Seizures-in-series observed in symptomatic generalized epilepsy other than West syndrome. PMID- 8612184 TI - Seizures and psychiatric symptoms of epileptics with frontally localized foci. II. The localization of the EEG abnormalities and psychiatric symptoms. PMID- 8612186 TI - A case of epilepsia partialis continua associated with hemimegalencephaly. PMID- 8612185 TI - Difficulty in differential diagnosis of atypical absence seizures and complex partial seizures in childhood. PMID- 8612187 TI - Assessing quality of life for the measurement of outcome after epilepsy surgery. PMID- 8612188 TI - Effect of the medication management module evaluated using the role play test. AB - Training in social skills has been shown to have a strong, positive impact, according to behavioral measures, on social skills, self-rated assertiveness, and the hospital discharge rate. It is important to establish a system of assessing social skills because it is necessary for the effects of social skills training to be assessed in Japan. In Project 1, we devised a Japanese version of the role play test to quantify social skills using a standard method. We tested 30 patients attending the day hospital who were considered to need intensive rehabilitation. We found the role play test had high inter-rater and test-retest reliability, and had construct validity and criterion related validity. Thus, the role play test was thought to be a useful tool for assessing social skills. For Project 2, eight inpatients who were ready for discharge but who needed to improve their skills in self-managed medication participated in this study. The social skills of self-managed medication assessed using the role play test were significantly improved after the subjects participated in the Medication Management Module of the UCLA social and independent living skills program. Knowledge of self-managed medication also tended to improve after training. This study is preliminary and it should be confirmed that improved skills influenced by the medication management module decreases the relapse rates. PMID- 8612189 TI - Shortening of the hippocampal formation in first-episode schizophrenic patients. AB - Shortening of hippocampal formation (HF) in chronic schizophrenic patients have been demonstrated in our previous study. The purpose of the present study is to test if shortening of the HF occurs in schizophrenic patients suffering their initial psychotic episode. We performed contiguous, 1 mm thick, magnetic resonance imaging scans in 20 first-episode schizophrenic patients, 21 chronic schizophrenic patients, and 25 healthy subjects. Both groups of schizophrenic patients demonstrated significant shortening of the HF compared with normal controls (first-episode schizophrenia, 5.3%; chronic schizophrenia, 8.0%). However, the HF length was not significantly different between the first-episode and chronic schizophrenic patients. No significant correlation was seen between the HF length and the duration of illness in chronic schizophrenic patients. These results suggest that the HF shortening observed in schizophrenic patients may be genetic and/or developmental in origin. PMID- 8612190 TI - Prognosis of epilepsy withdrawn from antiepileptic drugs. AB - Antiepileptic drugs (AED) were discontinued in 55 epileptics who had been free from seizures treated with AED, in accordance with the following criteria and procedures. (i) A reduction in AED commences when patients have been free from seizures for at least 2 years and epileptic discharges have also disappeared in repeated electroencephalogram (EEG) recordings during that period. (ii) AED are gradually reduced if no relapse is seen in clinical seizures and epileptic discharges in EEG. (iii) As a rule at least 2 years are required as the interval from the onset of a reduction to the withdrawal of AED. Forty-three patients were followed up by a questionnaire and/or by telephone and the follow-up period from the withdrawal of AED to the survey ranged from 0.9 to 8.8 years; in 38 patients (88.4%) the period was longer than 2 years. No relapse of seizures was found in any of the 43 patients. The severity of epilepsy judged by the total number of frequency of seizures, the presence of neuropsychiatric complications, the combination of different types of seizures, and the duration of epilepsy from the seizure onset to the last seizure appeared not to be risk factors for the recurrence of seizure. Normal EEG was, however, considered to be an important prerequisite for a good prognosis. PMID- 8612191 TI - Epidemiology of inflammatory neurological and inflammatory neuromuscular diseases in Tottori Prefecture, Japan. AB - We investigated the incidence of the following conditions: inflammatory neurological and neuromuscular diseases, adult meningitis and adult encephalitis in Yonago City, and Guillain-Barre syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), polymyositis/dermatomyositis (PM/DM), periarteritis nodosa (PN) and HTLV-1 associated myelopathy (HAM) during the period 1988-1992 in Tottori Prefecture, Japan. The annual incidence per 100,000 population was as follows: meningitis, 4.38; encephalitis, 0.90; GBS, 1.14; PM/DM, 1.01; and PN, 0.32. The prevalence per 100,000 population CIDP, 0.81; PM/DM, 9.92; PN, 2.59; and HAM, 1.30. There were marked localization of HAM in western Tottori, and there was seasonal variation in the prevalence of meningitis, encephalitis and GBS. The mean age at onset of meningitis was lower than that for encephalitis. Comparison with reported data revealed interracial differences in the epidemiology of PM/DM and PN. PMID- 8612193 TI - Cold Spring Harbor Laboratory. PMID- 8612192 TI - Screening for mutations at codon 717 of the amyloid precursor protein gene in Alzheimer's disease. AB - Three kinds of missense mutation at codon 717 of amyloid precursor protein (APP) gene (Val --> Ile; Val --> Gly; Val --> Phe) were screened in 114 patients with familial and sporadic Alzheimer's disease (AD), using a rapid testing method for each Val --> Gly and Val --> Phe mutation and Goate's method for Val --> Ile mutation based on the polymerase chain reaction. Mutations were not found in the subjects, confirming earlier suggestions that these three mutations at codon 717 of APP gene account for only a small proportion of cases of not only familial AD but also sporadic AD. PMID- 8612194 TI - Glutamine repeats as polar zippers: their role in inherited neurodegenerative disease. PMID- 8612195 TI - Antigen-specific therapy for autoimmune disease: prospects for the prevention of insulin-dependent diabetes. PMID- 8612196 TI - Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis. AB - BACKGROUND: The histopathology of multiple sclerosis (MS) is characterized by a loss of myelin and oligodendrocytes, relative preservation of axons, and a modest inflammatory response. The reasons for this selective oligodendrocyte death and demyelination are unknown. MATERIALS AND METHODS: In light of the T lymphocyte and macrophage infiltrates in MS lesions and the numerous cytokines these cells secrete, the direct influence of cytokines on survival of cultured oligodendrocytes and sensory neurons was investigated. Expression of cytokines in vivo was determined by immunolabeling cryostat sections of snap-frozen tissue containing chronic active lesions from four different patients. The samples were also analyzed for the presence of apoptotic nuclei by in situ labeling of 3'-OH ends of degraded nuclear DNA. RESULTS: The results showed: (i) interferon-gamma (IFN gamma) to be a potent inducer of apoptosis among oligodendrocytes in vitro and that this effect can be reversed by leukemia inhibitory factor (LIF); (ii) IFN gamma has a minimal effect on the survival of cultured neurons; (iii) IFN gamma at the margins of active MS plaques but not in unaffected white matter; (iv) evidence for apoptosis of oligodendrocytes at the advancing margins of chronic active MS plaques. CONCLUSIONS: Injury to a substantial number of oligodendrocytes in MS is the results of programmed cell death rather than necrotic cell death mechanisms. We postulate that IFN gamma plays a role in the pathogenesis of MS by activating apoptosis in oligodendrocytes. PMID- 8612197 TI - Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations. AB - BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection. PMID- 8612198 TI - Compounds that target novel cellular components involved in HIV-1 transcription. AB - BACKGROUND: Therapeutic intervention designed to block expression of human immunodeficiency virus (HIV) at a cellular level may slow the clinical progression of HIV-1 disease. MATERIALS AND METHODS: Cellular models of latent (OM-10.1 and U1) and chronic (8E5) HIV infection were used to evaluate two benzothiophene derivatives, PD 121871 and PD 144795, for an ability to inhibit HIV activation and expression. RESULTS: The benzothiophene derivatives were effective at micromolar concentrations in preventing tumor necrosis factor alpha (TNF alpha)-induced HIV-1 expression in OM 10.1 and U1 cultures. These compounds inhibited the activation of HIV-1 transcription; however, this inhibition was selective in that another TNF alpha-induced response, the transcription of autocrine TNF alpha, was unaffected. Constitutive HIV-1 expression by chronically infected 8E5 cells was also significantly reduced when treated with these experimental compounds. In TNF alpha-treated OM-10.1 cultures, the inhibition of HIV-1 transcription by these compounds was not due to a block of nuclear factor kappa B induction. The benzothiophene derivatives also inhibited HIV-1 activation by phorbol ester treatment of OM-10.1 promyelocytes, although no inhibition of cellular differentiation toward a macrophage-like phenotype was observed. Furthermore, these experimental compounds induced a state of HIV-1 latency in cytokine-activated OM-10.1 cultures even when maintained under constant TNF alpha stimulation. The benzothiophene derivatives did not inhibit the activity of the HIV-1 trans-activator, Tat, when evaluated in transient transfection assays. CONCLUSIONS: The benzothiophene derivatives appear to inhibit a critical cellular component, distinct from nuclear factor-kappa B, involved in HIV transcription and may serve to identify new therapeutic targets to restrict HIV expression. PMID- 8612200 TI - Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland. AB - BACKGROUND: Macrophage migration inhibitory factor (MIF) was one of the first lymphokine activities to be discovered and was described almost 30 years ago to be a soluble factor(s) produced by activated T lymphocytes. In more recent studies, MIF has been "rediscovered" to be an abundant, pre-formed constituent of the anterior pituitary gland and the macrophage, and to be a critical component in the host response to septic shock. Pituitary-derived MIF enters the circulation after infectious or stressful stimuli and appears to act to counterregulate glucocorticoid suppression of cytokine production. MATERIALS AND METHODS: Immunoelectron microscopy utilizing a combination of anti-MIF and anti pituitary hormone-specific antibodies was used to study the ultrastructural localization of MIF within the anterior pituitary gland. Pituitaries were obtained from resting, unstimulated mice and from mice 16 hr after endotoxin administration. The release of MIF also was investigated in vitro by examining the effect of corticotropin-releasing hormone (CRH_ on the AtT-20, corticotrophic cell line. RESULTS: MIF localizes to granules present exclusively in ACTH and TSH secreting cells. Within each cell type, a subset of granules was found to contain both MIF and ACTH, or MIF and TSH. The pituitary content of MIF-containing granules decreased significantly after experimentally induced endotoxemia. In seven pituitaries examined 16 hr after LPS injection, the number of MIF-positive granules diminished by 38% in corticotrophic cells and by 48% in thyrotrophic cells when compared with controls (p < 0.05). CRH was observed to be a potent MIF secretagogue in vitro, inducing the release of MIF from corticotrophic cells at concentrations lower than that required for ACTH release. CONCLUSION: These data provide ultrastructural information that identify MIF to be a novel anterior pituitary hormone, support earlier studies showing a time-dependent release of pituitary MIF during endotoxemia, and suggest an important, systemic role for MIF in the stress response to infection and other stimuli. PMID- 8612201 TI - Detection of circulating tumor cells in colorectal cancer by immunobead-PCR is a sensitive prognostic marker for relapse of disease. AB - BACKGROUND: Recurrent and metastatic carcinoma of the colorectum remains a major problem, with survival at 5 years post curative resection still only about 50%. Moreover, up to 30% of patients who present with early stage disease also relapse and die within 5 years, suggesting the presence of micrometastatic disease at diagnosis. One route of metastatic spread is via the blood stream, hence the detection of tumor cells in blood is likely to provide an important predictive tool with respect to relapse of disease. We have developed a sensitive molecular technique to identify tumor cells in blood using mutations in codon 12 of the K ras gene as a marker. MATERIALS AND METHODS: Twenty-seven patients whose tumor carried a mutation in codon 12 of K-ras were studied for the presence of tumor cells in perioperative peripheral blood samples. Immunomagnetic beads, labeled with an epithelial-specific antibody, were used to harvest epithelial cells from blood. K-ras mutations were identified in this selected population using a polymerase chain reaction (PCR)-based analysis (immunobead-PCR). RESULTS: Circulating K-ras mutant cells were detected in 9 or 27 patients; seven of these nine patients have since died due to recurrent or metastatic disease. Mutant cells were not detected in 18 patients, and 16 or 18 have remained disease free (median follow-up: 16 months; range: 7-42 months). Kaplan-Meier analysis showed that detection of K-ras mutant cells in bloods was associated with significantly reduced disease-free survival (p = 0.0001). CONCLUSION: This study indicates that detection of circulating tumor cells perioperatively by immunobead-PCR provides a sensitive prognostic marker for recurrent and metastatic colorectal cancer. PMID- 8612199 TI - VH3-21 B cells escape from a state of tolerance in rheumatoid arthritis and secrete rheumatoid factor. AB - BACKGROUND: Rheumatoid factor (RF) is a characteristic but not pathognomic feature in patients with rheumatoid arthritis (RA). It is unknown whether the repertoire of immunoglobulin genes utilized by RF+ B cells of RA patients is unique and whether RF+ B cells in normal individuals are silenced or deleted. MATERIALS AND METHODS: Clonal B cell populations were established from the peripheral blood of normal donors (127 B cell clones), RA patients (113 RF- and 60 RF+ B cell clones) and patients with primary Sjogren's syndrome (82 RF- and 47 RF+ B cell clones) by coculturing with anti-CD3-stimulated T helper cell clones. The cross-reactivity pattern of antibodies secreted by the B cell clones was determined by ELISA on a panel of antigens. The molecular structure of the IgM heavy chains was characterized by VH family-specific RT-PCR and sequencing. VH elements which correlated with RF specificity were identified. The responsiveness of B cells expressing these VH elements to T helper cell signals was compared in normal individuals and RA patients. RESULTS: The majority of RF+ B cells were monospecific when specificity was tested on five antigens. RF+ B cells expressed a significantly different repertoire of VH gene segments than RF- B cells. In particular, the VH3 gene segment V3-21 was not detected in B cell clones from normals but was the most frequent VH element in RF+ B cell clones from RA patients. Most of the V3-21 sequences were in germline configuration. The correlation between RF specificity and V3-21 gene segment usage was maintained in patients with Sjogren's syndrome. V3-21 transcripts were present in peripheral blood B cells from normal individuals. VH3-21+ B cells from RA patients but not from normal donors were responsive to preactivated T helper cells. Stimulation with a bacterial superantigen could overcome the nonresponsiveness of V3-21+ B cells in normal donors and induce the secretion of RF. CONCLUSIONS: RF production is correlated with the usage of the V3-21 gene segment in two distinct RF+ diseases. In patients with these diseases, V3-21+ B cells secrete antibodies with RF activity in response to activated T helper cells. V3-21+ B cells remain in a state of nonresponsiveness in normal individuals that can be broken by superantigen stimulation. The germline configuration of VH3-21+ RF+ immunoglobulins in RA patients suggests that the loss of tolerance is not an antigen-driven process. PMID- 8612202 TI - Administration of neutralizing antibodies to interleukin-6 (IL-6) reduces experimental autoimmune encephalomyelitis and is associated with elevated levels of IL-6 bioactivity in central nervous system and circulation. AB - BACKGROUND: We previously demonstrated the local production of the pleiotropic cytokine interleukin-6 (IL-6) in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. MATERIALS AND METHODS: To assess the role of IL-6 in autoimmune CNS inflammation, we administered neutralizing antibodies to IL-6 in the EAE model. Their effect was examined at the clinical and histopathological level. Levels of administered antibody and IL-6 bioactivity were followed in serum and cerebrospinal fluid (CSF). RESULTS: Systemically administered antibodies penetrated into the fluid CSF in animals in which EAE was induced. Administration of anti-IL-6 reduced the development of actively induced as well as adoptively transferred EAE and was associated with increased levels of IL-6 activity in the CSF and to a lesser extent in the serum. Anti-IL-6 was still effective when given 1 day before the onset of disease signs in adoptively transferred EAE. The disease-reducing effect of anti-IL-6 was also reflected at the pathological level by the absence of inflammatory infiltrates in the CNS. CONCLUSIONS: Our study indicates that IL-6 plays an important role in autoimmune CNS inflammation. However, due to the complex nature of the in vivo interactions of administered antibodies, the disease-reducing effect of the anti-IL-6 antibodies could be caused by neutralization of IL-6 activity or by enhancement of IL-6 activity via induction of higher IL-6 levels in the CNS. PMID- 8612203 TI - Differences in the expression of heat-shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulin-dependent diabetes mellitus. AB - BACKGROUND: It has previously been observed that the insulin-producing cells of human pancreatic islets are more resistant to alloxan-, streptozotocin-, nitroprusside-, or cytokine-induced injury than those of mouse and rat islets. MATERIALS AND METHODS: Human pancreatic islets were obtained from heart-beating organ donors. The expression of the stress proteins heat shock protein 70 (hsp70) and heme oxygenase and the anti-apoptosis gene bcl-2 was determined in isolated rat, mouse, and human islets, either cultured in vitro or transplanted under the kidney capsule of nude mice, using immunoblot analysis. Rat and human islet sensitive hydrogen peroxide was assess by glucose oxidation measurements. Isolated islets were also analyzed for their catalase and superoxide dismutase activities, and the islet cell levels of reduced glutathione were determined in response to hydrogen peroxide and nitroprusside. Programmed cell death in human and rat islets in response to streptozotocin was evaluated using TUNEL staining. RESULTS: Cultured human islets expressed higher contents of hsp70 than mouse and rat islets at basal conditions. Also after 4 weeks under the kidney capsule of normoglycemic mice, the hsp70 levels were higher in human islets than in rat islets. The expression of another stress protein, heme oxygenase (HO), was strongly increased in cultured rat islets, but was not affected in human islets. Expression of the bcl-2 gene could not be detected in human islets. In spite of this, 0.5 mM streptozotocin induced apotosis in rat but not in human islet cells. Hydrogen peroxide (0.1 and 0.4 mM) decreased glucose oxidation rates in rat but not in human islets. The levels of reduced glutathione were moderately decreased in human and rat islet cells and sharply decreased in mouse islet cells in response to hydrogen peroxide. Moreover, the activities of catalase and superoxide dismutase (SOD) were markedly lower in mouse islets than in human islets. The activity of catalase was lower in rat islets than in human islets. CONCLUSION: Human islets differ clearly from mouse and rat islets in their increased expression of hsp70, catalase, and SOD, which may explain the increased resistance of human islets to beta cell toxins. PMID- 8612206 TI - Molecular medicine database. PMID- 8612204 TI - Local expansion of allergen-specific CD30+Th2-type gamma delta T cells in bronchial asthma. AB - BACKGROUND: T lymphocytes infiltrating airways during the allergic immune response play a fundamental role in recruiting other specialized cells, such as eosinophils, by secreting interleukin 5 (IL-5), and promoting local and systemic IgE synthesis by producing IL-4. Whether these presumed allergen-specific T cells are of mucosal or systemic origin is still a matter of conjecture. MATERIALS AND METHODS: Immunophenotype, IL-4 production, and in vitro proliferative response to specific or unrelated allergens were analyzed in the bronchoalveolar lavage (BAL) fluid lymphocyte suspensions obtained from untreated patients with allergic asthma. Healthy subjects and patients affected by pulmonary sarcoidosis, a granulomatous lung disease characterized by infiltrating Th1 CD4+ lymphocytes, served as controls. RESULTS: The proportions of gamma delta T lymphocytes, mostly CD4+ or CD4- (-)CD8-, was higher in asthmatic subjects than in controls (p < 0.05). Most BAL gamma delta CD4+ lymphocytes of asthmatic patients displayed the T cell receptor (TCR)-gamma delta V delta 1 chain. While CD30 antigen coexpression on the surface of BAL alpha beta(+) T lymphocytes was low (ranging from 5 to 12%), about half of pulmonary gamma delta T cells coexpressed it. These cells produced IL-4 and negligible amounts of interferon-gamma (IFN gamma), and proliferated in vitro in response to purified specific but not unrelated allergens. In contrast, control or sarcoidosis gamma delta T cells never displayed the CD30 surface molecule or produced significant quantities of IL-4. CONCLUSIONS: These findings not only confirm our previous hypothesis that the allergen-specific Th2-type lymphocytes found in the lungs of asthmatic patients are gamma delta T cells belonging to airway mucosal immunocytes, but also strongly support the notion that asthma is a local rather than a systemic disease. PMID- 8612207 TI - AIDS -- a treatable disease at last. PMID- 8612205 TI - Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE). AB - BACKGROUND: Heme oxygenase (HO; EC 1.14.99.3) catalyzes the conversion of heme to biliverdin, which is reduced enzymatically to bilirubin. Since bilirubin is a potent antioxidant and heme a pro-oxidant, HO may protect cells against oxidative damage. HO-1 is highly inducible by diverse chemical agents, resembling those evoking induction of phase 2 enzymes (i.e., Michael reaction acceptors, heavy metals, trivalent arsenicals, and sulfhydryl reagents). Phase 2 enzymes (glutathione transferases; NAD (P)H:quinone reductase; glucuronosyltransferases) are regulated by antioxidant-responsive elements (ARE), and their induction protects against chemical carcinogenesis. Is HO-1 regulated by chemical agents and enhancer elements similar to those controlling phase 2 enzymes? MATERIALS AND METHODS: Induction of HO-1 by phorbol ester and heavy metals is transcriptionally controlled through a 268-bp SX2 fragment, containing two phorbol ester-responsive (TRE) sites (TGAC/GT C/AA) which overlap ARE consensus sequences (TGACNNNGC). Therefore, mutations of the SX2 element designed to distinguish ARE from TRE were inserted into chloramphenicol acetyltransferase (CAT) reporter plasmids, and the response of the CAT activity of murine hepatoma cells stably transfected with these constructs was examined with a wide range of inducers of phase 2 enzymes. RESULTS: All compounds raised HO-1 mRNA and CAT expression constructs containing wild-type SX2. When the SX2 region was mutated to alter TRE consensus sequences without destroying the ARE consensus, full inducibility was preserved. Conversely, when the ARE consensus was disturbed, inducibility was abolished. CONCLUSION: Induction of heme oxygenase-1 is regulated by several chemically distinct classes of inducers (mostly electrophiles), which also induce phase 2 enzymes, and these inductions are mediated by similar AREs. These findings support the importance of HO-1 as a protector against oxidative damage and suggest that HO-1 induction is part of a more generalized protective cellular response that involves phase 2 enzymes. PMID- 8612209 TI - The true Western diet. PMID- 8612208 TI - Cyclin E -- a better prognostic marker for breast cancer than cyclin D? PMID- 8612210 TI - p53 and chemosensitivity. PMID- 8612211 TI - Edward Jenner 200 years on. PMID- 8612212 TI - Saquinavir: too early to market? PMID- 8612214 TI - Boboon graft fails, but patient thrives. PMID- 8612213 TI - Optimism invades HIV conference. PMID- 8612215 TI - From genes to mechanisms to therapies: lessons to be learned from neurological disorders. PMID- 8612216 TI - Hantaviruses are likely threat to NATO forces in Bosnia and Herzegovina and Croatia. PMID- 8612217 TI - Imaging brain tumors -- beyond three dimensions. PMID- 8612218 TI - A new role for a fat actor. PMID- 8612219 TI - Closing in on human immunodeficiency virus-1. PMID- 8612220 TI - Peptide-displaying phages for targeted gene delivery? PMID- 8612222 TI - Shock revelations about HLA-DR4 -- a shortcut to rheumatoid arthritis? PMID- 8612221 TI - Human herpesvirus 8 in lymphoma and Kaposi's sarcoma: now the virus can be propagated. PMID- 8612223 TI - Epigenetics and human disease. PMID- 8612224 TI - Dental caries diagnosis -- toward the 21st century. PMID- 8612225 TI - Cuts and scrapes? Plasmin heals! PMID- 8612226 TI - Impaired wound healing in mice with a disrupted plasminogen gene. AB - Activation of plasminogen (Plg) has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling events, including wound healing. However, there has been no definitive proof of involvement of Plg in such processes. We now report that healing of skin wounds is severely impaired in mice made deficient in Plg by targeted gene disruption. The results demonstrate that Plg is required for normal repair of skin wounds in mice and support the assumption that it also plays a central role in other disease processes involving extracellular matrix degradation, such as cancer invasion. PMID- 8612227 TI - Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. AB - Catalytically active thrombin, acting locally, is thought to mediate neointima formation after arterial injury. We constructed an adenovirus vector, AdHV-1.2, containing a complementary DNA for the thrombin inhibitor hirudin. AdHV-1.2 directed the synthesis and secretion of biologically active hirudin from vascular cells in vitro. In vivo gene transfer of hirudin into smooth muscle cells of injured rat carotid arteries resulted in peak secretion of at least 34+/-23 pg hirudin per vessel per 24 hours, and resulted in a significant (P<0.05) 35% reduction in neointima formation. Systemic partial thromboplastin times were not affected by local hirudin expression. These results support the hypothesis that local thrombin activity contributes to neointima formation after arterial injury and suggest that local delivery of a highly specific antithrombin may constitute an effective intervention for arterial proliferative disease. PMID- 8612228 TI - Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. AB - Ideal gene therapy vectors would be delivered intravenously to transfect only specific cells. Existing vectors only transfect cells in vivo in a manner determined by blood flow and the site of introduction. As a general and systematic approach for generating cell-targeting ligands for gene therapy vectors, we have used peptide-presenting phage libraries to select peptides that bind and enter several different cell types. Because of their small size, cell binding peptides such as these could be incorporated into biological or physical gene therapy vectors. In addition, peptide-presenting phage themselves may also be candidates for gene therapy vectors. PMID- 8612229 TI - HLA-DR4 and HLA-DR10 motifs that carry susceptibility to rheumatoid arthritis bind 70-kD heat shock proteins. AB - Most patients with rheumatoid arthritis express particular HLA-DR alleles. The DRbeta1 chains of these alleles share a highly homologous amino acid motif, in their third hypervariable (HV3) region, and this motif seems to help the development of rheumatoid arthritis via unknown mechanisms. In an attempt to identify a ligand of this motif, we screened bacterial proteins. HV3 peptides from HLA-DRB1 alleles containing a QKRAA or RRRAA motif bound the 70-kD heat shock protein (HSP) from Escherichia coli, dnaK. In lymphoblastoid cells homozygous for these same HLA-DRB1 alleles the constitutive 70-kD HSP, HSP73, that targets selected proteins to lysosomes coprecipitated with HLA-DR. Thus the QKRAA and RRRAA amino acid motifs of HLA-DR mediate binding of HLA-DR to HSP73. This property may influence the intracellular route, processing or peptide associations of the HLA-DRbeta1 chain in these two rheumatoid arthritis associated alleles. PMID- 8612230 TI - Somatic overgrowth associated with overexpression of insulin-like growth factor II. AB - Overexpression of the normally imprinted fetal insulin-like growth factor II (IGF2) has been implicated in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). We have detected constitutional relaxation of imprinting of IGF2 in four children with somatic overgrowth who do not show diagnostic features of BWS. Three children showed constitutional abnormalities of H19 methylation. All four children showed nephromegaly and two developed Wilms' tumors. Gene methylation is known to be associated with gene silencing, and three children showed constitutional abnormalities of H19 gene methylation. Disruption of H19 methylation, and concomitant relaxation of IGF2 imprinting, provides another mechanism that can increase IGF2 expression in children with overgrowth. The accumulated data on normal and pathologic IGF2 expression are now sufficient to define an entity, "IGF2 overgrowth disorder," of which BWS may be one extreme manifestation. These findings have broad implications for the characterization of idiopathic overgrowth. PMID- 8612231 TI - Fas ligand in human serum. AB - The Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis in Fas-bearing cells. The membrane-bound human FasL was found to be converted to a soluble form (sFasL) by the action of a matrix metalloproteinase like enzyme. Two neutralizing monoclonal anti-human FasL antibodies were identified, and an enzyme-linked immunosorbent assay (ELISA) for sFasL in human sera was established. Sera from healthy persons did not contain a detectable level of sFasL, whereas those from patients with large granular lymphocytic (LGL) leukemia and natural killer (NK) cell lymphoma did. These malignant cells constitutively expressed FasL, whereas peripheral NK cells from healthy persons expressed FasL only on activation. These results suggested that the systemic tissue damage seen in most patients with LGL leukemia and NK-type lymphoma is due to sFasL produced by these malignant cells. Neutralizing anti-FasL antibodies or matrix metalloproteinase inhibitors may be of use in modulating such tissue damage. PMID- 8612232 TI - Accurate, noninvasive diagnosis of human brain tumors by using proton magnetic resonance spectroscopy. AB - Although conventional proton magnetic resonance imaging has increased our ability to detect brain tumors, it has not enhanced to nearly the same degree our ability to diagnose tumor type. Proton magnetic resonance spectroscopy is a safe, noninvasive means of performing biochemical analysis in vivo. Using this technique, we characterized and classified tissue from normal brains, as well as tissue from the five most common types of adult supratentorial brain tumors. These six tissue types differed in their pattern across the six metabolites measured. 'Leaving-one-out' linear discriminant analyses based on these resonance profiles correctly classified 104 of 105 spectra, and, whereas conventional preoperative clinical diagnosis misclassified 20 of 91 tumors, the linear discriminant analysis approach missed only 1. Thus, we have found that a pattern recognition analysis of the biochemical information obtained from proton magnetic resonance spectroscopy can enable accurate, noninvasive diagnosis of the most prevalent types of supratentorial brain tumors. PMID- 8612233 TI - HMG-CoA reductase mediates the biological effects of retinoic acid on human neuroblastoma cells: lovastatin specifically targets P-glycoprotein-expressing cells. AB - The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, involved in de novo cholesterol synthesis and cell-cycle progression, was identified as a potential mediator of the growth inhibitory effects of retinoic acid on human neuroblastoma. Lovastatin, a nonreversible inhibitor of HMG-CoA reductase, induced extensive cytotoxicity that was restricted to drug-resistant P glycoprotein-expressing neuroblastoma cell lines. This response was potentiated by dibutyryl cyclic AMP but not retinoic acid. Patients with advanced-stage metastatic neuroblastoma often display an acquired chemoresistant phenotype, which may in part be mediated by P-glycoprotein. Our studies support the application or use of HMG-CoA reductase inhibitors as potential therapeutic agents in the treatment of these patients who are refractory to chemotherapy. PMID- 8612234 TI - Auxotrophic vaccines for tuberculosis. AB - Tuberculosis is responsible for the deaths of more people each year than any other single infectious disease, with greater than 7 million new cases and 2 million deaths annually. It remains the largest attributable cause of death in HIV-infected individuals, responsible for 32% of deaths of HIV-infected individuals in Africa. The only currently available vaccine for tuberculosis, bacille Calmette-Guerin (BCG) is the most widely used vaccine in the world, being administered to approximately 100 million children each year. Although untoward effects were not seen in several studies of HIV-seropositive children, the safety of live attenuated BCG vaccine in HIV-positive adults remains unknown and a matter of some concern. To obviate potential adverse affects of BCG vaccines in immunodeficient individuals, we have studied five auxotrophic strains of BCG produced by insertional mutagenesis for safety in administration to mice with severe combined immunodeficiency disease (SCID), and for protection in a susceptible strain of mice. The results indicate that viable BCG could no longer be detected in mice receiving the auxotrophs after 16-32 weeks, and that infected SCID mice survived for at least 230 days. In contrast, all SCID mice succumbed within eight weeks to conventional BCG vaccine. When susceptible BALB/c mice were immunized with auxotrophs and subsequently challenged with virulent Mycobacterium tuberculosis, several of the auxotrophs produced comparable protection against intravenous and intratracheal challenge with M. tuberculosis relative to conventional BCG. These results suggest that auxotrophic strains of BCG represent a potentially safe and useful vaccine against tuberculosis for populations at risk for HIV. PMID- 8612235 TI - Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. AB - Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response. PMID- 8612236 TI - Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture. AB - Kaposi's sarcoma (KS) is the leading neoplasm of AIDS patients, and HIV infection is known to be a major risk factor for its development. However, KS can occur in the absence of HIV infection and the risk of KS development varies widely even among HIV-infected patients, with homosexual men with AIDS being 20 times more likely to develop KS than AIDS-afflicted children or hemophiliacs. These and other data strongly suggest that a sexually transmitted agent or co-factor may be involved in KS pathogenesis. Recently, DNA sequences corresponding to the genome of a novel member of the herpesvirus family have been identified within AIDS-KS biopsies, and several reports indicate that these sequences are also present in all forms of HIV-negative KS. These and other findings suggest this new agent, referred to as KS-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), as a candidate for the putative etiologic cofactor. However, the role of this agent in KS remains hotly debated. Further progress in understanding its biology has been severely hampered by the lack of a cell culture system for virus growth. Here we report the development of a system for the lytic growth of this virus in a latently infected B cell line and present the first ultrastructural visualization of the virus. This system will facilitate the detailed study of the molecular biology of viral replication, the testing of antiviral drugs and the development of diagnostic tests for viral infection. PMID- 8612237 TI - Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. AB - At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions. PMID- 8612239 TI - Magnetic resonance imaging with laser-polarized noble gases. PMID- 8612238 TI - Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin. AB - Immunotoxin LMB-1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin. B3 recognizes a carbohydrate antigen (Le(Y)) present on many human solid tumors. LMB-1 has excellent antitumor activity in nude mice bearing Le(Y)-positive tumors. We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis. Three patients (two colon, one breast cancer) had minor responses. The maximum tolerated dose of LMB-1 is 75 microgram/kg given intravenously three times every other day. The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema. Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors. PMID- 8612240 TI - Madeleine's death. PMID- 8612241 TI - Madeleine's death. PMID- 8612242 TI - Madeleine's death. PMID- 8612243 TI - Madeleine's death. PMID- 8612244 TI - Madeleine's death. PMID- 8612245 TI - Does iron prevent effects of lead exposure? PMID- 8612246 TI - Need for university student health services growing. PMID- 8612247 TI - Physician-patient relationships: patients as friends and patients who harass. PMID- 8612248 TI - Physician-patient relationships: patients as friends and patients who harass. PMID- 8612249 TI - Informal clinical consulting via the Internet. PMID- 8612250 TI - The emerging role of the physician in genetic counselling and testing for heritable breast, ovarian and colon cancer. AB - As a genetic testing for susceptibility to breast, ovarian and colon cancer becomes more readily available, physicians are faced with an increasing demand for information about inherited cancer risk. Because advances in treatment have not kept pace with advances in predictive testing, the provision of genetic counselling and testing marks a departure from the traditional role of the physician. A systematic framework is needed within which the physician's emerging role in predictive testing for heritable cancer can be delineated. The development of such a framework will require collaboration among professionals in a range of scientific disciplines, as well as the suspension of traditional assumptions about the physicians role. PMID- 8612251 TI - Global physician budgets as common-property resources: some implications for physicians and medical associations. AB - Since 1990 payment for physician services in the fee-for-service sector has shifted from an open-ended system to fixed global budgets. This shift has created a new economic context for practising medicine in Canada. A global cap creates a conflict between physicians' individual economic self-interest and their collective interest in constraining total billings within the capped budget. These types of incentive problems occur in managing what are known in economics as "common-property resources." Analysts studying common-property resources have documented several management principles associated with successful, long-run use of such resources in the face of these conflicting incentives. These management principles include early defining the boundaries of the common-property resource, explicitly specifying rules for using the resource, developing collective decision-making arrangements and monitoring mechanisms, and creating low-cost conflict-resolution mechanisms. The authors argue that global physician budgets can usefully be viewed as common-property-resources. They describe some of the key management principles and note some implications for physicians and the provincial and territorial medical associations as they adapt to global budgets. PMID- 8612252 TI - Contact dermatitis associated with the use of Always sanitary napkins. AB - OBJECTIVE: To report a clinical association between vulvar irritation or contact dermatitis and the use of Always sanitary napkins. DESIGN: Case series. SETTING: A gynecology practice in Montreal. PATIENTS: Women presenting between September 1991 and September 1994 with itching or burning of areas that would be in contact with a sanitary napkin (mons pubis, external surfaces of the vulva and perineum) beginning at least 1 day after the use of the napkins was started and less than 5 days after the use was stopped. RESULTS: Twenty-eight women experienced vulvar itching and burning, often associated with eruptions resembling contact dermatitis, of the vulvar and perineal surfaces after using Always sanitary napkins. Twenty-six reported that symptoms disappeared after they stopped using that brand of sanitary napkin. Seven women who later used the same brand again reported a recurrence of the vulvar irritation. CONCLUSION: The findings of this case series reveal Always sanitary napkins as a potentially important cause of recurrent vulvitis. Physician awareness of the association will enable effective advice and relief for a large number of women suffering "chronic vaginitis". PMID- 8612253 TI - Do too many cooks spoil the broth? Multiple physician involvement in medical management of elderly patients and potentially inappropriate drug combinations. AB - OBJECTIVES: To determine (a) whether the risk of a potentially inappropriate drug combination (PIDC) increases with the number of physicians involved in the medical management of an elderly patient and (b) whether the risk of a PIDC is reduced if a patient has a single primary care physician or a single dispensing pharmacy, or both. DESIGN: Cross-sectional retrospective provincial database study. PARTICIPANTS: A regionally stratified random sample of 51,587 elderly medicare registrants in Quebec who (a) visited at least one physician in 1990, (b) were not living in a health care institution for the entire year and (c) had been dispensed at least one prescription for a cardiovascular drug, a psychotropic drug or a nonsteroidal anti-inflammatory drug (NSAID). OUTCOME MEASURES: Information on all physician visits and drugs dispensed during 1990. Physician claims were used to identify the number of physicians involved in a patient's management and whether the patient had one primary care physician. Prescription claims were used to identify the number of PIDCs, prescribing physicians and dispensing pharmacies. RESULTS: The prevalence of PIDCs ranged from 4.0% (among those in the NSAID group) to 20.3% (among those in the psychotropic drug group). Of the PIDCs identified, 17.6% to 25.8% resulted from contemporaneous prescribing by different physicians. The number of prescribing physicians was the most important risk factor for a PIDC in all drug groups (odds ratio [OR] 1.44 to 1.71). The presence of a single primary care physician lowered the risk for cardiovascular and psychotropic PIDCs (OR 0.70 and 0.79 respectively) but not for NSAID PIDCs (OR 0.94). The use of a single dispensing pharmacy lowered the risk of a PIDC in all drug groups (OR 0.68 to 0.79). CONCLUSION: The greater the number of physicians prescribing medications for an elderly patient, the greater is the risk that the patient will receive a PIDC. A single primary care physician and a single dispensing pharmacy may be "protective" factors in preventing PIDCs. PMID- 8612254 TI - Opportunity lost: a frontline view of reference-based pricing. AB - The introduction in October 1995 of reference-based pricing as a cost-saving measure for British Columbia's drug benefit program represented an opportunity for collaboration between frontline practitioners and the bureaucracy that supports some of their work. If well-established principles of continuing education, quality improvement and modern management had been followed, practitioners in the field could have focused their individual and collective talents effectively and constructively on the task of improving cost effectiveness in drug prescribing. Although the reference-based pricing program may well achieve its purpose of saving money, it is sad that it was not used to build bridges of common interest and mutual trust between two camps that are often in conflict. PMID- 8612255 TI - A closer look at reproductive technology and postmenopausal motherhood. AB - Although reproductive technologies have been aimed at young, infertile women, evidence suggests that postmenopausal women are also taking advantage of them. Dr. Eike-Henner Kluge asserts in an article in CMAJ (1994; 151; 353-355) that there are ethical reasons to deny older women access to these technologies. Kluge's comparison of postmenopausal women to prepubescent girls is fallacious. His assertion that older parents harm children by denying them a "normal" childhood is not supported by any empiric data. Kluge's distinction between medical intervention, in offering reproductive technologies to a woman in her reproductive years, and "improving on nature", by offering these technologies to postmenopausal a woman is spurious. Unless technologies that are expensive and minimally successful, such as in-vitro fertilization, are denied to everyone, there are no grounds for denying them to postmenopausal women. PMID- 8612256 TI - Sleep stages, memory and learning. AB - Learning and memory can be impaired by sleep loss during specific vulnerable "windows" for several days after new tasks have been learned. Different types of tasks are differentially vulnerable to the loss of different stages of sleep. Memory required to perform cognitive procedural tasks is affected by the loss of rapid-eye-movement (REM) sleep on the first night after learning occurs and again on the third night after learning. REM-sleep deprivation on the second night after learning does not produce memory deficits. Declarative memory, which is used for the recall of specific facts, is not similarly affected by REM-sleep loss. The learning of procedural motor tasks, including those required in many sports, is impaired by the loss of stage 2 sleep, which occurs primarily in the early hours of the morning. These findings have implications for the academic and athletic performance of students and for anyone whose work involves ongoing learning and demands high standards of performance. PMID- 8612257 TI - Guidelines for control of measles outbreaks in Canada (revised 1995). Advisory Committee on Epidemiology. PMID- 8612258 TI - Do you have any regrets about entering medicine? AB - Do today's students have any second thoughts about their decision to pursue a career in medicine? Gabriel Leung, who graduates in June from the University of Western Ontario, considers the problems and dilemmas that have confronted him and his classmates during their short careers and muses on the uncertain future today's physicians face. PMID- 8612259 TI - Frustrated residents worry as they look toward an uncertain future. AB - Frustrated, disheartened and angry that most provinces have introduced disincentive measures that target young physicians, today's residents are discouraged and uncertain about their future. Not only will the medical profession lose because of this situation, the residents say, but so will patients who are deprived of new ideas and physicians with the most current training. Nancy Robb examines the mood of the country's physicians-in-waiting. PMID- 8612260 TI - Mood still angry following bitter Saskatchewan strike. PMID- 8612261 TI - Conferences point to growing concern about possible links between breast cancer, environment. AB - Evidence is growing that there may be a connection between certain chemicals in the environment and the rising incidence of breast cancer in North America. Two recent Canadian conferences have been held to disseminate information and another is planned for 1996. "We have a situation that is similar to global warming, " Devra Lee Davis, founder of the US Breast Cancer Prevention Collaborative Research Group, warned people attending a conference in Niagara Falls, Ont. "Breast cancer continues to increase. The increase is greatest among older women who have fewer of the known risk factors. It makes sense to try to limit exposure to things that could be promoting the disease." PMID- 8612262 TI - Humane handling of end-of-life issues the aim of program at U of O medical schools. AB - The University of Ottawa medical school has launched a program to help students deal with end-of-life issues. One of its speakers is a cancer patient upset with a physician's handling of his case. Organizers hope to gauge the impact of the program through a study of its effectiveness. PMID- 8612263 TI - Notion of "virtual library" developing as medical schools cope with rising journal costs. AB - The skyrocketing cost of medical and scientific journals has caused most Canadian universities to cut their subscription lists by an average of 20% over the last 3 years. Researchers say this causes delays in getting pertinent articles and journals and is an impediment to their work, but on the positive side the continuing decline in library holdings is stimulating libraries and researchers alike to use alternative strategies when seeking information, including computer databases, e-mail and the Internet. PMID- 8612264 TI - Quebec considering universal drug insurance. PMID- 8612265 TI - Quality control in Mu DNA transposition. PMID- 8612266 TI - A molecular perspective on pollination in flowering plants. PMID- 8612267 TI - Self/nonself recognition in fungi: old mysteries and simple solutions. PMID- 8612268 TI - Active and inactive protein kinases: structural basis for regulation. PMID- 8612269 TI - The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. AB - Petunia embryos carrying the no apical meristem (nam) mutation fail to develop a shoot apical meristem. Occasional shoots on nam- seedlings bear flowers that develop ten instead of five primordia in the second whorl. Double mutants with the homeotic gene green petals show that nam acts independently of organ identify in whorl 2 and now also affects primordium number in whorl 3. The nam gene was isolated by transposon tagging. The encoded protein shares a conserved N-terminal domain with several other proteins of unknown function and thus represents a novel class of proteins. Strikingly, nam mRNA accumulates in cells at the boundaries of meristems and primordia. These data indicate a role for nam in determining positions of meristems and primordia. PMID- 8612270 TI - Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. AB - The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development. PMID- 8612271 TI - HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl. AB - Bending in plant tissues results from differential cell elongation. We have characterized Arabidopsis "hookless" mutants that are defective in differential growth in the hypocotyl. HOOKLESS1 was cloned and its predicted protein shows similarity to a diverse group of N-acetyltransferases. HOOKLESS1 mRNA is increased by treatment with ethylene and decreased in the ethylene-insensitive mutant ein2. High level expression of HOOKLESS1 mRNA results in constitutive hook curvature. The morphology of the hookless hypocotyl is phenocopied by inhibitors of auxin transport or by high levels of endogenous or exogenous auxin. Spatial patterns of expression of two immediate early auxin-responsive genes are altered in hookless1 mutants, suggesting that the ethylene response gene HOOKLESS1 controls differential cell growth by regulating auxin activity. PMID- 8612273 TI - SNARE-mediated retrograde traffic from the Golgi complex to the endoplasmic reticulum. AB - Operation of the secretory pathway in eukaryotic cells requires the selective docking and fusion of transport vesicles with the appropriate target organelle. This is mediated in part by integral membrane proteins termed v-SNAREs (on vesicles) and t-SNAREs (on the target membranes). We describe a novel yeast t SNARE that resides on the endoplasmic reticulum and mediates retrograde traffic from the Golgi complex. Mutation of this protein prevents both the HDEL receptor and a membrane protein bearing a dibasic retrieval signal from recycling to the endoplasmic reticulum. Forward traffic is also blocked, but only indirectly. Comparison with other yeast mutants indicates that Sec21p (gamma-COP) and Sec20p (an endoplasmic reticulum membrane protein) are also involved primarily, if not exclusively, in retrograde transport. PMID- 8612272 TI - LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. AB - In C. elegans, the anchor cell signal induces Pn.p cells to form the vulva by activating a conserved receptor tyrosine kinase pathway. lin-2 and lin-7 mutants exhibit a vulvaless phenotype similar to the phenotype observed when this signaling pathway is defective. We have found that LIN-7 is a cell junction associated protein that binds to the LET-23 receptor tyrosine kinase. LET-23 is also localized to the cell junctions, and both LIN-2 and LIN-7 are required for this localization. LET-23 overexpression rescues the lin-2 or lin-7 vulvaless phenotype, suggesting that increased receptor density can compensate for mislocalization. These results suggest that proper localization of LET-23 receptor to the Pn.p cell junctions is required for signaling activity. PMID- 8612274 TI - Sequence-specific alteration of the ribosome-membrane junction exposes nascent secretory proteins to the cytosol. AB - Tight docking of the ribosome at the translocation channel ensures that nascent secretory proteins are shielded from the cytoplasm during transfer into the endoplasmic reticulum. Discrete pause transfer sequences mediate the transient stopping of translocation in certain proteins. Here we show that during a translocational pause, the junction between the ribosome and translocation channel is opened, exposing the nascent chain to the cytosol. While transient, this opening is sufficient to demonstrate macromolecular interactions between the translocating chain and molecules added to the cytosol, such as antibodies and site-specific proteases. Moreover, this opening is accompanied by alterations in the proteins that neighbor the nascent chain. These results demonstrate that specific sequences within a translocating nascent chain can elicit dramatic and reversible structural changes in the translocation machinery. Thus, the translocon is dynamic and can be regulated. PMID- 8612275 TI - Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. AB - Despite an increased understanding of the cellular and molecular biology of the CFTR Cl- channel, it is not known how defective Cl- transport across airway epithelia causes chronic bacterial infections in cystic fibrosis (CF) airways. Here, we show that common CF pathogens were killed when added to the apical surface of normal airway epithelia. In contrast, these bacteria multiplied on CF epithelia. We found that bactericidal activity was present in airway surface fluid of both normal and CF epithelia. However, because bacterial killing required a low NaCl concentration and because CF surface fluid has a high NaCl concentration, CF epithelia failed to kill bacteria. This defect was corrected by reducing the NaCl concentration on CF epithelia. These data explain how the loss of CFTR Cl- channels may lead to lung disease and suggest new approaches to therapy. PMID- 8612276 TI - Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. AB - The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH module. The domain consists of a stable beta alpha alpha beta beta alpha fold. On the basis of our results, we suggest a potential surface for RNA binding centered on the loop between the first two helices. Substitution of a well-conserved hydrophobic residue located on the second helix destroys the KH fold; a mutation of this position in FMR1 leads to an aggravated fragile X phenotype. PMID- 8612277 TI - The 1.85 A structure of vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends. AB - VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap specific RNA 2'-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 A crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis data suggest two possible RNA-binding sites with novel underlying architecture, one of which forms a cleft spanning the region adjacent to the methyltransferase active site. This report provides a prototypic structure for an RNA methyltransferase, a protein that interacts with the mRNA 5' cap, and an intact poxvirus protein. PMID- 8612278 TI - The interwoven architecture of the Mu transposase couples DNA synapsis to catalysis. AB - Mu transposition occurs exclusively using a pair of recombination sites found at the ends of the phage genome. To address the mechanistic basis of this specificity, we have determined both where the individual subunits of the tetrameric transposase bind on the DNA and where they catalyze DNA joining. We demonstrate that subunits do not catalyze recombination at the site adjacent to where they are bound, but rather on the opposite end of the phage genome. Furthermore, subunits bound to two different sites contribute to catalysis of one reaction step. This interwoven subunit arrangement suggests a molecular explanation for the precision with which recombination occurs using a pair of DNA signals and provides an example of the way in which the architecture of a protein DNA complex can define the reaction products. PMID- 8612279 TI - Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. AB - Central to the Mu transpositional recombination are the two chemical steps; donor DNA cleavage and strand transfer. These reactions occur within the Mu transpososome that contains two Mu DNA end segments bound to a tetramer of MuA, the transposase. To investigate which MuA monomer catalyzes which chemical reaction, we made transpososomes containing wild-type and active site mutant MuA. By pre-loading the MuA variants onto Mu end DNA fragments of different length prior to transpososome assembly, we could track the catalysis by MuA bound to each Mu end segment. The donor DNA end that underwent the chemical reaction was identified. Both the donor DNA cleavage and strand transfer were catalyzed in trans by the MuA monomers bound to the partner Mu end. This arrangement explains why the transpososome assembly is a prerequisite for the chemical steps. PMID- 8612281 TI - Complement receptor type 3 mediates phagocytosis and killing of Listeria monocytogenes by a TNF-alpha- and IFN-gamma-stimulated macrophage precursor hybrid. AB - Previous work demonstrated that engagement of complement receptor type 3 (CR3) was required for inflammatory peritoneal macrophages to phagocytose and kill the facultative intracellular bacterium Listeria monocytogenes. The experiments described here tested the role of CR3 in phagocytosis and killing of Listeria by a clonal population of TNF-alpha/IFN-gamma-stimulated macrophage precursor hybrids. Stimulation with TNF-alpha and IFN-gamma increased CR3 expression 20 fold and induced a big increase in phagocytic activity. Phagocytosis and killing of Listeria by these cells were inhibited when bacteria were opsonized with complement-depleted serum or by incubation of the macrophages with anti-CR3 mAb. Furthermore, cytokine-stimulated macrophages could not kill Listeria opsonized with heat-inactivated anti-Listeria antiserum, indicating that macrophage receptors which mediate phagocytosis do not necessarily promote bactericidal activity. These data suggest that upregulation of CR3 and CR3-mediated phagocytosis are mechanisms by which TNF-alpha and IFN-gamma stimulate nonphagocytic, nonbactericidal macrophage precursors to kill intracellular bacterial pathogens. PMID- 8612280 TI - Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family. AB - The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7. PMID- 8612282 TI - CD53 antigen and epidermal growth factor induce similar changes in the pattern of phorbol ester binding in a B cell lymphoma. AB - The CD53 antigen is a prototype member of the transmembrane-4 superfamily which includes several tumor antigens. In this report we have studied the changes in the cellular binding of phorbol esters after stimulation with monoclonal antibody (mAb) MRC OX-44 (anti-CD53) and epidermal growth factor (EGF) using a fluorochrome-phorbol ester binding assay. Incubation of a rat B cell lymphoma cell line with this mAb or EGF induces the appearance of high- and low-affinity phorbol ester binding sites and changes the total number of binding sites. Four binding sites with different characteristics have been detected. The binding data suggest that two structurally different receptors, CD53 antigen and EGF receptor, induce a similar change in the functional protein kinase C expressed in the cell which might be implicated in the responses elicited after cell stimulation. PMID- 8612283 TI - Tetanus toxin-sensitive VAMP-related proteins are present in murine macrophages. AB - The light chain of tetanus neurotoxin (TeTx) is a zinc endopeptidase specific for VAMP/synaptobrevin (VAMP), a 120-amino-acid integral protein previously described in the small synaptic vesicles of neuronal cells. TeTx has been shown to be active also on nonneuronal cells. By SDS-PAGE and quantitative immunoblotting on proteins derived from murine macrophages (Mphi) exposed to TeTx, we have shown that: (1) VAMP-related proteins are also present in Mphi and (2) such proteins are sensitive to TeTx proteolytic cleavage. The demonstration that TeTx acts on VAMP-related proteins also in Mphi offers a new and useful tool for molecular studies on Mphi exocytosis. PMID- 8612284 TI - Dexamethasone and prostaglandin E2 modulate T-cell receptor signaling through a cAMP-independent mechanism. AB - One possible explanation for the link between stress and increased incidence of infection can be attributed to concomitant increases in levels of glucocorticoids (GS) and prostaglandin E2 (PGE2), both of which possess potent immunoregulatory activities. We have previously demonstrated that concentrations of PGE2 and the synthetic glucocorticoid, dexamethasone (DEX), which individually do not inhibit human T-cell responsiveness to anti-CD3 monoclonal antibody (mAb), act synergistically to inhibit IL-2 secretion and subsequent T-cell proliferation. In the present paper, we demonstrate that treatment of anti-CD3 mAb-stimulated T cells with low (10(-8) and 10(-9) M) concentrations of DEX and PGE2 results in the inhibition of steady-state levels of IL-2 mRNA. Initial studies to elucidate the biochemical mechanisms involved indicate that the inhibitory effects of DEX and PGE2 cannot be correlated with increased levels of intracellular cAMP or the induction of apoptosis. However, the data indicate that DEX and PGE2 when added together interrupt anti-CD3 mAb-induced tyrosine phosphorylation of substrate proteins. Furthermore, the synergistic effect of DEX and PGE2 is mimicked by agonists for the cAMP-independent EP3 subtype of the PGE2 receptor. These data suggest that DEX and PGE2 elicit cAMP-independent signaling pathways which interact to inhibit the T-cell receptor-linked signal transduction cascade in anti-CD3 mAb-stimulated T-cells. PMID- 8612285 TI - Effects of N(G)-Nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis, on IL-2-induced LAK cell generation in vivo and in vitro in healthy and tumor-bearing mice. AB - We had earlier shown that therapy with N(G)-nitro-L-arginine methyl ester (L NAME), an inhibitor of nitric oxide (NO) synthesis, had antitumor and antimetastatic effects in C3-L5 mammary adenocarcinoma-bearing mice. When combined with interleukin-2 (IL-2) therapy, L-NAME augmented antitumor effects of IL-2. In the present study, we tested whether the L-NAME effects were due, at least in part, to a potentiation of antitumor cytotoxicity of host effector cells. We examined the effects of L-NAME on IL-2-induced generation of antitumor cytotoxicity in vivo and in vitro in splenocytes of healthy and C3-L5 tumor bearing C3H/HeJ mice, using 51Cr release assay. IL-2 treatment, in vivo or in vitro, markedly stimulated splenocyte tumoricidal activity against NK-sensitive (YAC-1) and -resistant (C3-L5) targets, accompanied with an increase in NO production measured in the serum or culture medium. Addition of L-NAME to IL-2 therapy blocked IL-2-induced NO production in vivo and improved IL-2-induced splenocyte cytotoxicity as well as tumor regression. Addition of L-NAME in vitro also reduced IL-2-induced NO production in the medium and enhanced IL-2 induced cytotoxicity of splenocytes of healthy but not tumor-bearing mice. These results reveal that IL-2-induced increase in NO production in vivo causes a suppression of LAK cell activation, which can be overcome by NO inhibition with L-NAME therapy. These findings, combined with our observation that L-NAME can mitigate IL-2 -induced capillary leakage in healthy and tumor-bearing mice, suggest that L NAME could be a valuable adjunct to IL-2 therapy of cancer and infectious diseases. PMID- 8612287 TI - The specificity of adhesive interactions between rat lymphocytes and salivary gland epithelia. AB - Salivary immune responses depend on localization of immunocytes in salivary glands. We tested effects of anti-adhesion molecule antibodies and several ligand analogs on in vitro adherence of rat thoracic duct lymphocytes (TDL) to parotid and submandibular gland sections. While TDL adherence to both tissues was markedly decreased by anti-L-selectin mAbs, binding ability after removal of L selectin by chymotrypsin or PMA suggested that other adhesion systems were involved. Integrin involvement in parotid interactions was indicated by inhibitory effects of anti-HEBF(PP), LFA-1, ICAM-1, and alpha4 integrin antibodies as well as by the PMA-enhanced adherence. Anti-Thy-1 partially inhibited TDL binding to parotid gland, and anti-CD44 partially inhibited submandibular binding. The majority of salivary gland-bound TDL were sIg+ B cells. FACS analysis showed differences in parotid and submandibular endogenous lymphocyte adhesion molecule expression with greater percentages of L-selectin, HEBF(PP), alpha4 integrin, LFA-1, ICAM-1, CD44, and Thy-1-positive cells present in parotid gland. While precise roles of known or novel adhesion molecules in salivary gland lymphocyte retention are not clear, these data suggest that selectins (parotid, submandibular), integrins (parotid), Thy-1 (parotid), and CD 44 (submandibular), as well as other unidentified molecules, are involved. PMID- 8612286 TI - Conjugation between cloned human NK cells (H7.8) and K562/MOLT4 tumor cell systems: saturability, binding parameters, and population distribution of conjugates. AB - Effector-target conjugation between cloned NK(H7.8)-K562 and NK(H7.8)-MOLT4 tumor cells has been studied from binding isotherms. Nonlinear and linear regression methods were used to calculate the maximum effector and target conjugate frequencies as well as the dissociation constant of the conjugates formed. The results obtained show there is an enhancement of the effector-target saturability and effector-target affinity in comparison with the values previously observed for polyclonal NK effector cells. Population distributions revealed that different types of conjugates were formed as the effector-to-target ratio was changed in the NK(H7.8)-K562 and NK(H7.8)-MOLT4 tumor cells. In both cases conjugates where one effector cell was bound to several target cells and conjugates with one target cell bound to several effector cells were found. At all values of R the prevailing conjugates were those with one effector cell bound to one target cell. PMID- 8612288 TI - Nitric oxide and superoxide anion production decrease with age in resident and activated rat peritoneal macrophages. AB - Superoxide anion and nitric oxide production have been studied in resident and activated peritoneal macrophages of 3-, 12-, and 24-month-old rats. Some key enzymes involved in the metabolism of glucose were also studied in relation to aging. Production of O2 and NO was reduced in all cases in middle-aged (12 months) and old (24 months) animals. Malic enzyme and citrate synthase activity shows a progressive reduction with age. Hexoquinase, pyruvate quinase, and lactate dehydrogenase activities decrease sharply from 3 to 12 months with no significant change between 12 and 24 months. Taken as a whole, the results of enzyme activity suggest that aging may reduce the capacity for glucose utilization in macrophages. PMID- 8612289 TI - Specific tolerization of active cytolytic T lymphocyte responses in vivo with soluble peptides. AB - A promising approach toward preventing and treating autoimmune disease involves identifying the mediating antigen and then tolerizing the autoreactive T cells with the corresponding antigen. For success, this method will require the specific tolerization of active helper or CTL responses while maintaining the integrity of the immune system. In this report, we selectively eliminated an ongoing CTL response by administering soluble peptide. BALB/c mice were immunized with two H-2Kd-restricted immunodominant CTL epitopes derived from HIV and malaria together with a T helper epitope to elicit a strong CTL response. Beginning 3 days later, mice were injected 3 times at 3-day intervals with 500 micrograms of only one or both of these epitopes in PBS. Following these injections, only one of two active CTL responses was tolerized without affecting T helper cells. This tolerization state requires antigen for its maintenance, may be retolerized upon return, and is not due to active or antigen-driven bystander suppression. This study suggests that soluble peptides may be utilized to treat or prevent autoimmune diseases caused by autoreactive CTLs. PMID- 8612290 TI - Adoptive transfer of resistance to murine retrovirus-induced immune suppression. AB - Infection of certain strains of mice, such as C57BL/6 and C57BL/10 [B10], with LP BM5 murine leukemia virus (MuLV) rapidly causes a profound and lethal immune suppression. The H2d congenic strain of B10, B10.D2, is resistant to disease, but B10 x B10.D2 F1 mice are susceptible, indicating that disease sensitivity is dominant. To determine whether disease resistance could be adoptively transferred to a sensitive host, radiation chimeras (B10.D2 --> B10 x B10.D2 F1 and F1 --> F1) were challenged with LP-BM5 virus. Infected B10.D2 --> F1 chimeras showed no loss of immune function, whereas F1 --> F1 chimeras infected with LP-BM5 MuLV developed MAIDS and became completely immune suppressed. These results, coupled with previous studies, indicate resistance or sensitivity to disease is an inherent property of the hematopoietic system that can be transferred by bone marrow grafts. PMID- 8612291 TI - Cure of fibroblast monolayers from murine cytomegalovirus infection: phenotypic assessment of rat lymphoid cell population developed on fibroblast monolayers. AB - Rat and mouse fibroblast monolayers were infected with murine cytomegalovirus (MCMV) by 2 hr incubation to allow virus to penetrate the cells. Then lymph node cells (LNC) from rats infected with MCMV and from untreated rats were added together with human recombinant interleukin-2 (hrIL-2). Cytopathic plaques appeared within 2-3 days. In culture of fibroblasts only, 30-40 plaques per well progressed into confluent cytopathy within 6-8 days. In cultures with LNC syngeneic to fibroblasts, plaques appeared; however, the cytotoxic T lymphocyte population that developed and specific apoptotic fragmentation eliminated the cytomegalic cells in the plaques. The surrounding cells stretched to the area, the cytopathic plaques disappeared, and the monolayer resumed its uninfected texture. No plaque-forming units could be isolated from such cured cultures. In allogeneic combination there was no apoptotic target cell killing. However, in cultures stimulated by hrIL-2, plaque growth was arrested and the plaques remained rudimentary. Similarly, arrest of plaques was also obtained in cultures containing LNC from uninfected rats, but only if stimulated by hrIL-2. In mouse fibroblasts carrying the rat LNC, plaque growth was not arrested, and the culture developed into confluent cytopathy. Interferon (IFN)-gamma or -alpha,beta added 24 hr before and 2 hr after infection abolished plaque appearance or arrested growth. IFN-gamma appeared to be the most effective. Fluids harvested from cured cultures also protected from plaque development. Antibodies to IFN-gamma, but not to IFN-alpha,beta, neutralized this capacity in the culture fluids. It is concluded that IFN-gamma produced by the LNC played a major role in the cure of the fibroblast culture from MCMV infection. A mechanism of cell-mediated immunity operating in resolving virus infection is proposed. PMID- 8612292 TI - HIV-1 Tat protein and its inhibitor Ro 24-7429 inhibit lymphocyte proliferation and induce apoptosis in peripheral blood mononuclear cells from healthy donors. AB - To explore further the effects of the HIV-1 transactivator protein Tat on human lymphoid cell function we examined the effects of Tat on lymphocyte proliferation and programmed cell death (apoptosis). We found that the HIV-1 Tat protein induced apoptosis and inhibited lymphocyte proliferative responses in lymphocytes obtained from healthy HIV-1 seronegative donors. Surprisingly, the Tat inhibitor Ro 24-7429 also induced apoptosis and inhibited antigen-induced lymphocyte proliferation. In checkerboard experiments, each agent could antagonize the effects of the other in both assays. These data suggest that Tat and its inhibitor may interact with a host element critically important in the processes of lymphocyte proliferation and programmed cell death. The HIV-1 Tat protein may affect both lymphocyte survival and function in HIV-1 infection, thereby contributing to the immune deficiency of HIV-1 disease. PMID- 8612293 TI - Regulation of the insect immune response: the effect of hemolin on cellular immune mechanisms. AB - Hemolin is a bacteria-inducible protein of the immunoglobulin superfamily identified in the silk moth Hyalophora cecropia. The role of this protein, in hemocyte aggregation and phagocytosis, was studied in vitro. Hemocyte aggregation, stimulated by phorbol myristate acetate or lipopolysaccharide (LPS), was prevented by hemolin in a dose-dependent fashion, but hemolin did not disrupt aggregates once they had been formed. Furthermore, hemolin was able to stimulate phagocytic activity in both hemocytes and hemocytic mbn-2 cells and this activity was enhanced by LPS. The enhanced phagocytosis produced by a combination of hemolin and LPS was prevented by the protein kinase C (PKC) inhibitors staurosporine and H-7, and PKC activity in hemocyte crude extracts was enhanced by hemolin and LPS, with the highest activity observed in the presence of both. Hemolin affected tyrosine phosphorylation of hemocyte proteins, enhancing the phosphorylation of two proteins of 20 and 30 kDa and preventing tyrosine phosphorylation of two proteins of 35 and 40 kDa. These results suggest that hemolin is involved in the regulation of the cellular immune responses via a pathway that includes PKC activation and protein tyrosine phosphorylation. PMID- 8612295 TI - Dysregulation of temperature and liver cytokine gene expression in immunodeficient wasted mice. AB - Wasted mice bear the spontaneous autosomal recessive mutation wst/wst; this genotype is associated with weight loss beginning at 21 days of age, neurologic dysfunction, immunodeficiency at mucosal sites, and increased sensitivity to the killing effects of ionizing radiation. The pathology underlying the disease symptoms is unknown. Experiments reported here were designed to examine thermoregulation and liver expression of specific cytokines in wasted mice and in littermate and parental controls. Our experiments found that wasted mice begin to show a drop in body temperature at 21-23 days following birth, continuing until death at the age of 28 days. Concomitant with that, livers from wasted mice expressed increased amounts of mRNAs specific for cytokines IL-6 and IL-1, the acute phase reactant C-reactive protein, c-jun, and apoptosis-associated Rp-8 when compared to littermate and parental control animals. Levels of beta transforming growth factor, c-fos, proliferating cell nuclear antigen, and ornithine amino transferase transcripts were the same in livers from wasted mice and controls. These results suggest a relationship between an acute phase reactant response in wasted mice and temperature dysregulation. PMID- 8612294 TI - Interleukin 2 pseudomonas exotoxin (IL2-PE66(4)Glu) chimeric protein kills B cells from patients with Myasthenia gravis. AB - IL2-PE66(4)Glu is a chimeric cytotoxin consisting of interleukin 2 (IL2) fused to a mutant form of Pseudomonas exotoxin (PE66(4)Glu). The chimeric cytotoxin has been previously shown to be extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. To explore the possible clinical utility of IL2-PE66(4)Glu for autoimmune diseases, particularly in which B cells are involved, we tested fresh B cells from patients with myasthenia gravis for sensitivity to this chimeric cytotoxin. Seventy-six percent (16 of 21) of the B cells tested were markedly sensitive to IL2 PE66(4)Glu-mediated cytotoxicity, with inhibition of protein synthesis ranging from 20 to 92%. B cells from control donors were much less sensitive to IL2 PE66(4)Glu cytotoxicity. Moreover, a control protein lacking IL2 as the targeting moiety of the chimera had no effect toward all B cells tested, thus establishing its specific activity. Our results suggest that IL2-PE66(4)Glu could be an effective tool for selective targeted immunotherapy of myasthenia gravis patients. PMID- 8612296 TI - A regulatory role of c-Fos in the development of precursor B lymphocytes mediated by interleukin-7. AB - The proto-oncogene product c-Fos, a component of the transcription factor AP-1, plays a critical role in the expression of genes required for cellular proliferation and differentiation. The c-Fos is induced in early B lineage cells developed in the interleukin-7-dependent bone marrow (BM) cell culture from normal mice. In order to investigate a role of the c-Fos in early B cell development, we have used BM cells from two different transgenic mice carrying the exogenous c-fos gene controlled by the promoter of the H-2Kb gene (H2-c-fos) or the interferon alpha/beta (IFN)-inducible Mx gene (Mx-c-fosD). Development of B lineage cells was retarded in the BM cell culture from H2-c-fos mice. Although B lineage cells normally developed in the BM cell culture from Mx-c-fosD mice without IFN stimulation, the development was completely blocked in the Mx-c-fosD culture when transgenic c-fos was induced in BM cells by IFN stimulation. Furthermore, the IL-7-dependent proliferation of B lineage cells in Mx-c-fosD BM cells was also suppressed by the induction of c-Fos. These results suggest that the c-Fos plays a role as a negative regulator in the early B cell development. PMID- 8612297 TI - Immunological abnormality in C3H/HeJ mice with heritable inflammatory bowel disease. AB - To explore the immunological abnormality of a heritable inflammatory bowel disease developed in a new substrain of C3H/HeJ mice, we examined the expression of integrins beta7 and other cell adhesion molecules on normal and disease mice lymphocytes by flow cytometry. We also examined the cytotoxicity of small intestinal intraepithelial lymphocytes to epithelial cells and the proliferation and aggregation of intestinal lymphocytes. There are several significant changes in the expression levels of alpha4 and alphaM290beta7 integrins, CD11a, ICAM-1, CD45RB, CD4, CD44, and CD45 in different lymphocyte populations. The cytotoxicity of small intestinal intraepithelial lymphocytes from disease mice was higher than that from normal mice and could be stimulated by PHA and inhibited by mAb to alphaM290beta7. The proliferation of both normal and disease small intestinal intraepithelial lymphocytes was enhanced by costimulation by mAb to CD2 or CD3 and ProNectin. In comparison to disease mice, normal small intestinal intraepithelial lymphocytes proliferated at a significantly higher rate in response to sheep RBC and mAb to CD2 or CD3 and ProNectin costimulation. Homotypic aggregation of small intestinal intraepithelial lymphocytes isolated from disease mice was greater than in those from normal mice. The abnormality of expression of integrin beta7 and other cell adhesion molecules and cytotoxic, proliferative, and aggregative responses of lymphocytes from disease mice may play important roles in the pathogenesis of this heritable inflammatory bowel disease. PMID- 8612298 TI - Modulation of expression of class II MHC and CD40 molecules in murine B cells by various muramyl dipeptides. AB - Several compounds of the MDP (muramyl dipeptide) series which have the capacity to enhance the immune response to antigens exerted a comitogenic effect on murine splenic B cells. The expression of surface class II major histocompatibility and CD40 antigens was used to more accurately evaluate the comparative influence of the synthetic agents on mature B cells and on the pre-B cell line 70Z/3. MDP and two adjuvant-active analogs enhanced expression of both surface molecules and increased the response to lipopolysaccharide (LPS) or interferon-gamma (IFN gamma) in splenic B cells. The three synthetic adjuvants alone did not lead to expression of cell-surface I-Ad or CD40 in 70Z/3 cells, indicating that they were unable by themselves to achieve differentiation of pre-B cells to a mature B cell phenotype. However, they increased the CD40 level induced by treatment with LPS. In this cell line, the response (CD40 protein and mRNA) to IFN-gamma was strongly increased by MDP but not by the two other compounds. Actually, MDP was the only adjuvant among the three compounds to functionally activate the transcription factor NF-kappaB, to induce kappa transcription, and to stimulate surface kappa light-chain expression in 70Z/3 cells. The response to muramyl dipeptides in mature splenic B cells could appear independent of the transcription factor. PMID- 8612299 TI - Sensitivity of multidrug-resistant tumor cell lines to immunologic effector cells. AB - The ability of malignant cells to survive exposure to cytotoxic agents is a major obstacle to cure in patients with cancer. Multidrug resistance and the expression of P-glycoprotein are emerging as a cause of chemotherapy failure. Immunologic effector cells such as lymphokine-activated killer (LAK) cells or cytokine induced killer (CIK) cells are capable of killing a broad range of tumor cell lines or freshly isolated tumor cells. As demonstrated here, LAK, and CIK cells possess a high level of cytotoxic activity against tumor cell lines both resistant and sensitive to chemotherapeutic agents such as doxorubicin or vinblastine. CIK cells possessed a higher level of cytotoxic activity than LAK cells as determined by 51Cr release and a tumor colony assay. Monoclonal antibodies against P-glycoprotein did not block the lysis of tumor cells resistant to chemotherapy by CIK cells. In contrast, antibodies to LFA-1 and ICAM 1 blocked CIK cell-mediated tumor cell lysis. These data demonstrate that immunological approaches to cancer therapy may be useful in overcoming disease caused by drug resistance. PMID- 8612300 TI - 4-1BB is expressed on CD45RAhiROhi transitional T cell in humans. AB - Murine 4-1BB is an approximately 30-kDa glycoprotein expressed on activated T cells and plays a role in T-cell-mediated proliferative response. To date the majority of work on 4-1BB has been conducted in the mouse. To assess the role of 4-1BB in humans, mAbs were made against the recombinant human (rh) 4-1BB protein. One such mAb 4B4-1 specifically binds SF-21 insect cells expressing rh4-1BB but not irrelevant control protein as measured by flow cytometry (FCM). 4B4-1 mAb stains PMA- and ionomycin-stimulated CEM (human T lymphoma) cells and PHA stimulated peripheral blood T cells, but not resting cells. 4B4-1 mAb immunoprecipitates both approximately 32 and approximately 80 kDa protein from rh4-1BB expressing SF-21 cells and an approximately 39- and approximately 85-kDa protein from PMA-stimulated CEM cells under reducing conditions by SDS-PAGE. As added proof of its specificity, binding of FITC-labeled 4B4-1 mAb to PHA stimulated T cells was blocked by rh4-1BB protein. Together these data demonstrate that 4B4-1 is specific for 4-1BB in humans. Unlike in the mouse, 4 1BB is expressed much earlier (within 24 hr) peaking around 2-3 days following PHA stimulation. As in the mouse 4-1BB is induced on both CD4+ and CD8+ T cell subsets. 4-1BB expression is induced upon PHA stimulation in both the naive (CD45RAhi-CD45ROlo/-) and the memory (CD45RAlo/-ROhi) T cell populations. Virtually all CD45RAhiROlo/- cells upon culture in PHA give rise to an intermediate CD45RAhiROhi 4-1BB+ transitional cell and subsequently CD45RAlo/ ROhi 4-1BBlo/- and CD45RAlo/-ROhi 4-1BBhi cells. In contrast, approximately 27% of CD45RAlo/-ROhi 4-1BB- cells when cultured in PHA for 24 hr acquire 4-1BB expression and all remain CD45RAlo/-ROhi. PMID- 8612301 TI - Synergy between T cell Receptor and Fas (CD95/APO-1) signaling in mouse thymocyte death. AB - Administration of anti-TCR/CD3epsilon antibody in vivo or in thymic organ culture results in the apoptotic death of CD4+/CD8+ thymocytes. In contrast, purified thymocytes in suspension culture are resistant to TCR/CD3epsilon-induced apoptotic death. We show that induction of thymocyte death, in suspension culture, can be induced by the combination of TCR/CD3epsilon and Fas (CD95/Apo-1) signaling. No significant thymocyte death was observed after in vitro Fas cross linking unless TCR/CD3epsilon was simultaneously co-cross-linked or metabolic inhibitors such as actinomycin D were added. Furthermore, TCR/CD3epsilon and Fas synergy did not operate through upregulation of Fas but by facilitation of the Fas-mediated death signal. Both TCRmid/lo/HSAhi/CD4+/CD8+ (double positive) and TCRhi/HSAlo/CD4+/CD8- or CD4-/CD8+ (single positive) thymocytes were susceptible to death induced by co-cross-linking of TCR/CD3epsilon and Fas. Our results reveal a signaling synergy between the Fas and TCR/CD3epsilon complex that has important implications for our understanding of in vivo vs in vitro models of thymocyte deletion. PMID- 8612302 TI - SarCNU (2-chloroethyl-3-sarcosinamide-1-nitrosourea): a novel analogue of chloroethylnitrosourea that is transported by the catecholamine uptake2 carrier, which mediates increased cytotoxicity. PMID- 8612303 TI - AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. AB - 3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress. PMID- 8612304 TI - The integrity of cisplatin in aqueous and plasma ultrafiltrate media studied by 195Pt and 15N nuclear magnetic resonance. AB - 195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cisplatin in water and plasma ultrafiltrate (PUF). Cisplatin was found to be stable for at least 2, but no longer than 5 months in a reconstituted clinical formulation, as determined by 195Pt NMR. In aqueous solution, the cis PtCl2(NH3)2 195Pt and 15N NMR signal intensities decreased with time and the formation of [PtCl(H2O)(NH3)2]+ at PH values of 3.0, 6.5, 7.5 and 9.5 was observed within 24 h of sample preparation. In addition, [Pt(H2O)2(NH3)2]++ was observed at pH 3.0, and [PtCl(OH)(NH3)2] and [Pt(OH)2(NH3)2] were observed at pHs 7.5 and 9.5. During incubation of PUF with cisplatin for 35 h, 15N NMR signals for at least eight cisplatin derivatives appeared at different times, whereas only four were observed by 195Pt NMR. With our NMR protocols, the detection limit for quantifiable cisplatin derivatives is estimated at 500 microM using 195Pt NMR and < or = 200 microM using 15N NMR. In addition to providing useful information about the chemical stability of cisplatin and derivatives formed in aqueous solution, these magnetic resonance techniques, particularly 15N NMR, can provide useful information about the metabolism of cisplatin in biological regimes. PMID- 8612305 TI - In vitro schedule-dependent interaction between paclitaxel and cisplatin in human carcinoma cell lines. AB - The schedule-dependent interaction of paclitaxel and cisplatin was studied in four human carcinoma cell lines: non-small cell lung cancer, A549; breast cancer, MCF7; ovarian cancer, PA1; and colon cancer, WiDr cells. The cells were exposed simultaneously to the drugs for 24 h and sequentially to paclitaxel first for 24 h followed by cisplatin for 24 h, or vice versa, and then incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was then determined by the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the IC80 level were analyzed by the isobologram method. On simultaneous exposure to paclitaxel and cisplatin, additive and subadditive (slight antagonistic) effects were observed in A549, MCF7, and PA1 cells, while sub-additive and protective (antagonistic) effects were observed in WiDr cells. On sequential exposure to paclitaxel first, followed by cisplatin, additive effects were observed in all cell lines. On sequential exposure to cisplatin first, followed by paclitaxel, additive effects were observed in PA1 cells, while additive, sub-additive, and protective effects were observed in A549, MCF7, and WiDr cells. These findings suggest that the interaction of paclitaxel and cisplatin is schedule- and cell line-dependent. The optimal schedule of this combination may be paclitaxel first followed by cisplatin. PMID- 8612306 TI - Lipid-complexed camptothecin: formulation and initial biodistribution and antitumor activity studies. AB - Water-soluble derivatives of camptothecin, and active topoisomerase I inhibitor, have shown a broad spectrum of activity against human tumors. Early clinical trials with the water-soluble sodium salt of camptothecin were hindered by significant cystitis, gastroenteritis, and leukopenia. Furthermore, the sodium salt of camptothecin has been shown to have significantly less activity than the water-insoluble lactone form of the compound. We describe a formulation of lipid complexed CPT (LC-CPT; particle size range 20.8-208.1 nm) that is very easy to prepare and allows for intravenous administration in vivo in clinically relevant lipid-drug ratios (12.5:1 w/w). The lipid formulation had in vitro antitumor activity similar to that of CPT formulated without lipids and displayed similar cytotoxicity against MDR-1-negative and -positive tumor cells. The biodistribution of CPT was profoundly affected by lipid complexation; free CPT achieved the greatest concentration in the pulmonary parenchyma while LC-CPT achieved the highest concentration in the gastrointestinal tract. LC-CPT had significant antitumor activity in vivo against intraperitoneal L1210 and P338 leukemia and appeared to be more potent then free CPT. PMID- 8612307 TI - Penetration of intravesical doxorubicin in human bladders. AB - The bladder wall penetration kinetics of intravesical doxorubicin were examined in radical cystectomy patients, to provide insight into drug concentrations at target tumor sites. The dosing solution (40 mg/20 ml) was instilled just prior to the start of surgery and maintained for 60-115 min until just prior to bladder excision. The data showed considerable inter-patient variability in the peak plasma concentration (24-fold), urine concentration (7- fold), and tissue concentration (28-fold). The urine concentration at the time of tissue harvest was about 17% of the concentration in the dosing solution. This was due to the dilution by post-catheterization residual urine and urine produced during treatment. The doxorubicin concentration dropped by 32-fold across the urothelium, and declined semi-logarithmically with respect to depth in the capillary-perfused tissues beneath the urothelium with a 50% decrease over about 500 micromole. In three of six patients from whom tumor tissue was obtained, the doxorubicin concentration was higher than the adjacent non-tumor-bearing tissues of comparable tissue depth, whereas the reverse was seen in the remaining three tumors. The plasma concentrations were 0.02, 0.03, 0.05, 0.27, and 0.69% of the concentrations in the tumors, urothelium, lamina propria, superficial and deep muscle layers, respectively. These data indicate: (a) a considerable intra- and inter-patient variability in bladder tissue concentrations, in part due to the variability in the urine concentration; (b) the urothelium is an effective barrier to doxorubicin penetration; and (c) a targeting advantage of intravesical therapy for the treatment of superficial bladder cancer yielding superficial bladder tissue concentrations at least 2000-fold higher than in the systemic circulation. A comparison of the data of doxorubicin with our previously published data on mitomycin C shows similar bladder tissue pharmacokinetics for the two drugs, suggesting that there is no pharmacokinetic preference for either drug. PMID- 8612309 TI - On the mechanism of action of doxorubicin encapsulation in nanospheres for the reversal of multidrug resistance. AB - We had previously shown that doxorubicin encapsulation in polyisohexylcyanocrylate nanospheres could circumvent the P-glycoprotein-mediated multidrug resistance (MDR) exhibited by C6 rat glioblastoma in culture. We then investigated what could be the mechanism of such a circumvention. The cytotoxicity of free and encapsulated doxorubicin was evaluated in two MDR variants of the C6 cell line in a device allowing the separation of cells from drugs by a polycarbonate membrane of 0.2 micron pore size. We observed that the progressive disruption of the nanospheres allowed their doxorubicin content to reach the cell monolayer and exert its cytotoxicity in a fashion similar to that exhibited by free doxorubicin. However, no circumvention of MDR is obtained by doxorubicin encapsulation when drug-containing nanospheres are separated from the cells by the polycarbonate membrane. In addition, no effect on azidopine binding to P-glycoprotein-enriched membranes is exerted by unloaded nanospheres, even after their spontaneous degradation in cell-culture medium. Taken together, these results suggest that a physical contact between doxorubicin-containing nanospheres and the cells is required for the circumvention of MDR. The role of degradation products from the nanospheres as modulators of P-glycoprotein activity can be ruled out. PMID- 8612308 TI - Intracellular Localization and intercellular heterogeneity of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase. AB - O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy. PMID- 8612310 TI - A phase I/II study of high-dose cyclophosphamide, cisplatin, and thioTEPA followed by autologous bone marrow and granulocyte colony-stimulating factor primed peripheral-blood progenitor cells in patients with advanced malignancies. AB - The purpose of the present study was to determine the maximally tolerated dose of thioTEPA given with fixed high-dose cyclophosphamide (CPA) and cisplatin (cDDP) followed by autologous bone marrow (ABM) with or without granulocyte colony stimulating factor (G-CSF)-primed peripheral-blood progenitor cells (PBPCs) in patients with advanced malignancies. Patients were required to have histologically documented malignancies and adequate renal, hepatic, pulmonary, and cardiac function. CPA was given at 1,875 mg/m2 per day as a 1-h i.v. infusion for 3 consecutive days, and cDDP was given at 55 mg/m2 per day as a 24-h continuous i.v infusion over 3 days concurrently with CPA. ThioTEPA was given once as a 1-h i.v. infusion (300-900 mg/m2) either following (the first 13 patients) or prior to CPA and cDDP. In all, 31 patients received PBPCs. A total of 46 patients were treated. There were 6 deaths among the 15 patients who did not receive PBPCs (13 received thioTEPA following CPA and cDDP). Among the other 31 patients who received PBPCs (all of whom also received thioTEPA prior to CPA and cDDP), there were 4 deaths, all involving patients with refractory ovarian carcinoma. The main toxicities were mucositis, esophagitis, hepatotoxicity, and nephrotoxicity. The median time required to achieve an absolute neutrophil count of 500 microliter was 10 days (range, 9-12 days) for those who received PBPCs and 15 days (range, 15-34 days) for those who did not receive PBPCs. Altogether, 47% of the major organ toxicities (grades 3 and 4 renal, hepatic, and cardiac toxicities) occurred among the 15 patients who did not receive PBPCs, although these patients received thioTEPA at the lowest 2 dose levels. There were 3 complete responses and 22 partial responses among 35 evaluable patients (overall response rate, 71%), with the median duration of response being 3.5 months (range, 2-17 months). The maximally tolerated dose of thioTEPA was 600 mg/m2 given as a 1-h i.v. infusion on the day prior to CPA and cDDP administration, The combination of high-dose CPA, cDDP, and thioTEPA is a well-tolerated regimen when thioTEPA is given prior to CPA and cDDP and when the combination also includes PBPCs in addition to ABM. This regimen is active in a variety of malignancies. PMID- 8612312 TI - A comparison of hyperthermia cisplatin sensitization in human ovarian carcinoma and glioma cell lines sensitive and resistant to cisplatin treatment. AB - Two pairs of human tumor cell lines (glioma and ovarian carcinoma (OvCa) each having a parental cell line and cisplatin-resistant variant, were evaluated for (a) cisplatin response, (b) hyperthermia response, and (c) combined hyperthermia and cisplatin response. The two resistant lines had comparable resistant responses while for the parental lines, the OvCa was more sensitive than the glioma to cisplatin doses up to 14 microgram/ml. For the hyperthermia response, the OvCa parental line was more resistant than the variant line at low temperature hyperthermia (41 degrees C or 42 degrees C) but became more sensitive at high temperature (45 degree C). For the glioma, the parental line was more sensitive to hyperthermia at all temperatures tested. Hyperthermia caused sensitization to cisplatin in all cell lines but was generally greater in the glioma cell lines. In the OvCa system, hyperthermia had a slightly greater sensitizing effect on the resistant cell lines, while in the glioma the opposite was true. The degree of sensitization increased with hyperthermia temperature. In summary, the results showed that there is no cross- resistance for hyperthermia and cisplatin, that the degree of thermal sensitization is not reduced in cisplatin- resistant cell lines, and that cisplatin thermal sensitization is cell line and temperature dependent. Thus, hyperthermia can effectively improve tumor cell response to cisplatin and may be useful in overcoming resistance to cisplatin. PMID- 8612311 TI - Peripheral blood mononuclear cell dihydropyrimidine dehydrogenase activity in volunteers with and without diabetes mellitus. AB - It has been reported that cancer patients with diabetes mellitus receiving a continuous infusion of 5-fluorouracil (5-FU) have more toxicity and higher plasma 5-FU levels that patients without diabetes mellitus. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU. DPD activity in peripheral blood mononuclear cells has been reported to correlate inversely with 5-FU plasma levels in patients. We therefore undertook a study to compare the activity of DPD in peripheral blood mononuclear cells of human subjects with and without diabetes mellitus. The study groups comprised 43 volunteers with and 39 without diabetes mellitus, and peripheral blood mononuclear cell DPD activity was assayed on samples obtained between 8 a.m. and 11 a.m. DPD activity was not decreased in diabetic subjects. There was no relationship between DPD activity and gender body mass index, or race. There was a modest correlation between DPD activity and age (r=0.19, P =0.08). We conclude that increases in 5-FU-related toxicities in diabetics must be related to factors other than peripheral blood mononuclear cell DPD activity. PMID- 8612313 TI - A phase I and pharmacokinetic study of oral uracil, ftorafur, and leucovorin in patients with advanced cancer. AB - A phase I and pharmacokinetics study of oral uracil, ftorafur, and leucovorin was performed in patients with advanced cancer. Uracil plus ftorafur (UFT) was given in a 4:1 molar ratio in three divided doses for 28 consecutive days. Patient cohorts were treated at 200, 250, 300, and 350 mg/m2 of UFT daily. For all patients, 150 mg of leucovorin was given daily in three oral doses. A 1-week rest period followed each 28-day treatment course. Gastrointestinal toxicity, characterized by diarrhea, nausea, and vomiting, was dose-limiting at 350 mg/m2 UFT in patients who had received prior chemotherapy. Mild fatigue and transient hyperbilirubinemia were also common. In previously untreated patients, UFT at 350 mg/m2 was well-tolerated, suggesting this as an acceptable phase II dose in this schedule with leucovorin. Two of eight previously untreated patients with advanced colorectal cancer had partial responses with UFT (350 mg/m2) plus leucovorin. Pharmacokinetic parameters [ftorafur, uracil, 5-fluorouracil (5-FU), 5-methyltetrahydrofolate] showed wide interpatient variations. Plasma levels of 5 FU (Cmax 1.4 +/- 1.9 microM) were comparable to those achieved with protracted venous infusions, and folate levels (Cmax 6.1 +/- 3.6 microM) were sufficient for biochemical modulation. Ongoing study will determine if this convenient oral regimen will compare favorably in terms of efficacy, toxicity, and cost with intravenous fluoropyrimidine programs. PMID- 8612314 TI - A randomized trial comparing ftorafur alone with ftorafur plus tamoxifen in postoperative adjuvant therapy for breast cancer. Kinki Area Research Group for Postoperative Adjuvant Therapy for Breast Cancer. AB - A randomized study was performed in 35 centers in the Kinki area of Japan to determine the effectiveness of ftrorafur (FT) plus tamoxifen (TAM) compared with FT monotherapy in postoperative adjuvant therapy for breast cancer. Patients were randomized by the envelope method to receive either FT 600 mg/day or FT 600 mg/day plus TAM 20 mg/day orally for 1 year, starting on day 7 after mastectomy. Between April 1982 and January 1985, 628 patients were assigned to treatment with FT alone and 626 to treatment with FT + TAM. Of these, 571 (90.9%) and 539 (86.1%) patients, respectively, met the eligibility requirements for this study. There were no significant differences in major background factors between the two groups of eligible patients. Five-year survival rates were 91.4% for FT alone and 91.1% for FT+TAM (not significantly different). Five-year disease-free survival rates showed a tendency towards a better prognosis (P = 0.090) in the FT + TAM group, with observed rates of 83.0% for FT alone and 86.7% for FT + TAM. Stratified analysis showed that disease-free survival with FT + TAM is better than with FT alone for patients aged 50 years or more ( P = 0.048) and for patients with from one to three positive nodes (P = 0.064). PMID- 8612315 TI - Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine. AB - We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells. Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells. There were also differences in spermine and putrescine uptake rates between parental and MDR positive with the MDR-positive cells having a lower Vmax and a higher Km. The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA. The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX). Cytotoxicity was measured by the MTT [3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM. This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used. No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene. Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin. The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of [3H]-VBL and [3H]-TAX in the Dx5 but not MES-SA cells. Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells. DFMO pretreatment is known to upregulate the polyamine transporter(s). These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump. This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance. PMID- 8612316 TI - Transforming growth factor-beta in in vivo resistance. AB - The potential role of transforming growth factor-beta in in vivo resistance was examined by administration of transforming growth factor-beta-neutralizing antibodies to animals bearing the EMT-6/Parent tumor or the antitumor alkylating resistance tumors, EMT-6/CTX or EMT-6/CDDP. Treatment of tumor bearing animals with anti-TGF-beta antibodies by intraperitoneal injection daily on days 0-8 post tumor cell implantation increased the sensitivity of the EMT-6/Parent tumor to cyclophosphamide (CTX) and cisplatin (CDDP) and markedly increased the sensitivity of the EMT-6/CTX tumor to CTX and the EMT6/CDDP tumor to CDDP, as determined by tumor cell survival assay. Bone marrow granulocyte-macrophage colony-forming units (CFU-GM) survival was determined from these same animals. The increase in the sensitivity in the tumors upon treatment with the anti-TGF beta antibodies was also observed in increased sensitivity of the bone marrow CFU GM to CTX and CDDP. Treatment of non-tumor-bearing animals with the anti-TGF-beta regimen did not alter blood ATP or serum glucose level but did decrease serum lactate levels. This treatment also decreased hepatic glutathione, glutathione S transferase, glutathione reductase, and glutathione peroxidase in non-tumor bearing animals by 40-60% but increased hepatic cytochrome P450 reductase in these normal animals. Animals bearing the EMT-6/CTX and EMT-6/CDDP tumors had higher serum lactate levels than normal or EMT-6/Parent tumor-bearing animals; these were decreased by the anti-TGF-beta regimen. Treatment of animals bearing any of the three tumors with the anti-TGF-beta regimen decreased by 30-50% the activity of hepatic glutathione S-transferase and glutathione peroxidase, and increased by 35-80% the activity of hepatic cytochrome P450 reductase. In conclusion, treatment with transforming growth factor-beta-neutralizing antibodies restored drug sensitivity in the alkylating agent-resistant tumors, altering both the tumor and host metabolic states. PMID- 8612317 TI - Carboplatin and vinorelbine in advanced non-small-cell lung cancer. AB - A total of 32 patients with advanced non-small-cell lung cancer were treated with carboplatin (350 mg/m2, day 1) and vinorelbine (days 1 and 8) every 28 days. A response rate of 28% (95% confidence limits 12.5 - 43.7%) was observed. The activity of this combination was demonstrated in an outpatient setting with acceptable toxicity. PMID- 8612318 TI - Phase I study of ifosfamide plus high-dose epirubicin in advanced non-small-cell lung cancer. AB - The present phase I study was designed to determine the maximum tolerated dose (MTD) of epirubicin given in combination with ifosfamide at a dose of 3 g/m2, recycled every 4 weeks, in patients with advanced non-small-cell lung cancer (NSCLC). A total of 18 patients entered the study; they received the following four dose levels of epirubicin (i.v., day 1): 75 (6 patients), 90 (3 patients), 105 (3 patients, and 120 mg/m2 (6 patients). The MTD of epirubicin was 120 mg/m2, neutropenia being the dose-limiting toxicity. We observed 1/6, 1/3 1/3, and 2/6 partial responses (PRs) at epirubicin dose levels of 75, 90, 104, and 120 mg/m2, respectively. A phase II study of epirubicin given at a dose of 120 mg/m2, in association with conventional-dose ifosfamide in advanced NSCLC is in order. PMID- 8612319 TI - Is there a circadian variation in plasma concentrations of etoposide given by prolonged continuous infusion? AB - The prolonged continuous infusion of low-dose etoposide is a new approach to treating cancer. Whether or not a circadian variation in the plasma levels of etoposide existed was investigated in nine patients with non-small-cell lung cancer. Etoposide was infused for 14 days and blood samples were obtained every 4 h for 1 day. There was no significant circadian variation, and the observed small within-day variations seemed to lack clinical significance. PMID- 8612320 TI - Development of endothelial nitric oxide synthase in endothelial cells in the human cerebrum. AB - We demonstrate the distribution and development of endothelial nitric oxide synthase (eNOS) immunoreactivity in vessels of the human brain. eNOS-positive endothelial cells were recognized in small vessels in the parenchyma of the cerebrum, as well as in the large arteries and veins in the leptomeninges. By 10 13 weeks of gestation, eNOS-positive endothelial cells had already appeared in the vessels of the leptomeninges and deep white matter. Immunoreactivity for eNOS was noted in most vessels at 14-17 weeks in the intermediate white matter, at 18 21 weeks in the cortex, and at 23-26 weeks in the subcortical white matter. Such differences in regional development may be responsible in part for the topographical predilection for hemorrhagic or hypoxic-ischemic lesions in the developing brain. PMID- 8612321 TI - Evidence for dopamine receptor pruning between adolescence and adulthood in striatum but not nucleus accumbens. AB - Postnatal development of dopamine D1 and D2 receptor families in striatum and nucleus accumbens of rats was studied at 25, 35, 40, 60, 80, 100 and 120 days using autoradiography. These ages were selected to test the hypothesis that dopamine receptors were overproduced prior to puberty (day 40), and pruned back to adult levels thereafter. This hypothesis was confirmed in striatum but not nucleus accumbens. D1 receptor Bmax ([3H]SCH-23390) peaked at 40 days, with levels 67 +/- 21% greater than at 25 days. However, Bmax levels were at least 35% lower at 60-120 days than at 40 days. Similarly, D2 receptor numbers ([3H]YM 09151-2) increased 144 +/- 26% between 25 and 40 days, but were reduced by 34-38% between 60-120 days. In contrast, D1 and D2 receptor Bmax increase approximately 150% between 25 and 40 days in nucleus accumbens, levels fell slightly at 60 or 80 days, but were no different at 100 and 120 days then they were at 40 days. These findings suggest that these two major dopamine target regions follow different developmental strategies, and this has implications for etiological theories of schizophrenia that focus on anomalous receptor pruning. PMID- 8612322 TI - Developmental changes in the inducibility of fos-like immunoreactivity in primary embryonic spinal cord cultures. AB - The immediate early gene (IEG) transcription factor c-fos coordinates changes in the pattern of long term gene expression and, therefore, it may be involved in mediating epigenetic control during neurodevelopment. We used pharmacological treatments mimicking various environmental and intracellular signals and assessed the inducibility of fos-like immunoreactivity (LIR) at various stages of neurodifferentiation in a primary embryonic spinal cord culture system by immunohistochemistry. Constitutive fos LIR exclusively found in neurons, was driven by the onset and extent of spontaneous electrical activity, as it was blockable by tetrodotoxin (TTX) at all developmental stages. Phorbol myristate 13 acetate (PMA) increased the number of fos-LIR cells equally effectively at all stages, but the predominant cellular localization of fos-LIR changed through ontogeny. The effect of veratridine, kainate and serum-derived factors in significantly inducing fos-LIR was restricted to the earliest developmental stage (4 days in vitro; DIV) investigated; whereas forskolin, the GABAA antagonist picrotoxin and NMDA failed to induce fos-LIR at this stage, but increased the number of fos-LIR neurons at later stages. Dihydropyridine agonists of the voltage-sensitive calcium channels (VSCC) raised the number of fos-LIR neurons and also prevented TTX-mediated down-regulation; whereas antagonists markedly reduced fos-LIR at all ages. Either type of NMDA antagonists (AP5 and MK801) and the GABAA agonist muscimol significantly reduced fos-LIR at all ages. These findings demonstrate that the inducibility of fos-LIR is substantially different in embryonic neurons than in adult ones and that inducibility by various first and second messengers is dependent on the development stage. PMID- 8612323 TI - Developmental regulation of MAP2 variants during neuronal differentiation in vitro. AB - Microtubule-associated protein 2 (MAP2) is a major component of the neuronal cytoskeleton and is known to promote the assembly and stabilization of microtubules, functions which have important implications in neuronal differentiation. MAP2 consists of high molecular weight (HMW) proteins MAP2a, MAP2b and a low molecular weight (LMW) isoform MAP2c which are produced from a single gene by alternative splicing. In this study, we describe the expression of the various MAP2 mRNA isotypes and protein isoforms during the development of rat cerebellar granule cell neurons over a 21-day period in vitro. In situ hybridization was used to detect MAP2 mRNA isotypes which corresponded to HMW- and LMW-MAP2 proteins. The distribution of MAP2 mRNAs in the developing P7 cerebellar cortex was related to the different stages of granule neuron development in situ. During early stages of neuronal differentiation in vitro, high levels of MAP2c mRNA were observed which gradually decreased as development progressed. Throughout the period studied, MAP2ab mRNA concentrations remained low although a small transient rise was noted during the first 14 days in vitro (div). The profile of MAP2 protein variants showed further developmental regulation. The expression of the LMW-MAP2c isoform closely mirrored that of its mRNA whilst HMW-MAP2b protein concentrations rose during the first 10 div and were maintained in older cultures. HMW-MAP2a appeared after 4 div and gradually increased throughout the remainder of the study. Clearly, the outline of HMW-MAP2 protein did not relate to its encoding mRNA and such disparity may be due to the operation of different transcriptional and/or posttranslational mechanisms. Immunocytochemical analyses of MAP2 variants provided further information concerning their localization during neurite outgrowth. These results describe the developmental regulation of MAP2 mRNA and protein variants and that the profile of their expression relates to the formation of processes during the differentiation of granule neurons in vitro. PMID- 8612324 TI - The ontogeny of cardiac and neural A1 adenosine receptor expression in rats. AB - To provide insights into the sites and mechanisms of adenosine action during fetal life, the ontogeny of A1 adenosine receptor (A1AR) expression was studied in rats. Using in situ hybridization and receptor binding assays, A1AR expression was examined at gestational days (GD) 8, 11, 14, 17, and 21. At GD 8, A1AR mRNA expression was detected in the myocardium but not in other fetal structures. At GD 11, A1AR mRNA was present in the atria, but not in the ventricles or neural structures. At GD 14, A1AR mRNA was present in the atria and the pontine neuroectoderm, the thalamus, and the ventral horn of the spinal cord. At GD 14, A1ARs were first detectable using [3H]DPCPX (8-cyclopentyl-1,3-dipropylxanthine). Functional binding sites were highest in the atria and present at low levels in brain. When GD 17 was reached, patterns of A1AR expression in the brain were similar to those observed in adult animals. Sites of heavy labeling included the spinal cord, medulla, pons, midbrain, thalamus, and the hippocampus. When pre- and postnatal tissue concentrations of A1ARs were compared, cardiac A1AR receptor concentrations were similar. However, postnatal brain A1AR levels were considerably greater than in fetuses. These data identify the atria as a prominent site of fetal A1AR expression and show that the A1AR gene is one of the earliest expressed G protein-coupled receptor genes in the heart. A1AR expression in brain lags behind cardiac expression in early stages of gestation and exceeds cardiac A1AR expression with progressive maturation. A1ARs may therefore influence the heart and brain during critical periods of fetal development. PMID- 8612325 TI - Differentiation and maturation of rabbit retinal oligodendrocyte precursor cells in vitro. AB - The differentiation of oligodendrocytes from undifferentiated progenitor cells was studied in cultures obtained from the postnatal rabbit retina. 'Sandwich' cultures were established by turning the coverslips with adhering cells up-side down about 24 h after seeding. As a result O4-positive oligodendrocyte progenitors stop dividing and differentiate. Within 6 days in vitro they form extensive membranous sheets and acquire myelin associated glycoprotein (MAG), proteolipid protein (PLP), and myelin basic protein (MBP). O4-/MBP-positive oligodendrocytes and vimentin-positive/GFAP-negative Muller cells (a kind of modified astrocyte type in the retina), which are also present in these cultures, occupy distinct territories in vitro. When oligodendrocyte precursors were seeded onto a preformed Muller cell feeder layer they prefer to settle on the Muller cell free substrate poly-L-lysine, develop numerous processes but no membranous sheets and fail to acquire detectable amounts of MBP. In addition, culturing Muller cells and oligodendrocytes within the same medium, but without direct contact to each other, oligodendrocyte precursor cells fail to express MBP. The Muller cell factor(s) responsible for this interaction remains to be determined. PMID- 8612326 TI - Occipital cortico-pyramidal projection in hypothyroid rats. AB - It is well established that the progressive disappearance of a transient occipito spinal projection in neonatal rats involves the selective elimination of axonal collaterals. We studied whether the development of the occipito-spinal pathway was affected by hypothyroidism induced by treatment with the goitrogen 6n-propyl 2-thiouracil (PTU) beginning prenatally. Using both anterograde (biocytin and Dil) and retrograde (horseradish peroxidase and Fast Blue) tracing techniques in adult hypothyroid rats, we found that many cells with projections into the pyramidal tract are present in regions of visual cortex that are devoid of such cells in normal adult rats. Our results suggest that hypothyroidism induced by PTU treatment leads to the maintenance of occipito-spinal projections that are normally transient. PMID- 8612327 TI - Distribution of six transplasma membrane NADH-dehydrogenases in rat brain tissue. AB - Transplasma membrane redox plays a significant role in cellular activation and growth. Six isoenzymes could be prepared from purified rat brain synaptic plasma membrane. Polyclonal antibodies have been prepared against six transplasma membrane oxydoreductases (PMO-I to PMO-VI) and the tissue distribution of the various iso-enzymes have been investigated in adult rat brains by means of immunohistochemistry. PMO-I is densely observed in layers I, IV and V of the parietal cortex, in CA1 of the hippocampus (except for the molecular layer), in the caudate putamen, in the dorsal, granular and ventral parts of the auditory nuclei, in some loci of the vestibular nuclei as well as in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-II is mainly located in the polymorphic layer of the dentate gyrus and in the deep cerebellar nucleus and in the granular layer of the cerebellar cortex. PMO-III is abundant in the piriform cortex, in the pyramidal layers of both CA1 and CA2, in the diagonal band of the basal ganglia, in the supraoptic nucleus and in various loci of the magnetocellular paraventricular nucleus of the hippothalamus as well as in the vestibular nuclei from the brain stem. In addition PMO-III is also densely present in motor nuclei (oculomotor, facial, hypoglossal and ambiguus nuclei), in the reticular formation and in the deep cerebellar nucleus as well as in the Purkinje layer of the cerebellar cortex. PMO-IV has a similar location but is less abundant in the vestibular nuclei of the sensory brain stem and in the motor nucleus. PMO-V in contrast is poorly present in most brain areas compared to the other iso-enzymes, apart of the Purkinje layer of the cerebellar cortex. Finally PMO-VI is mainly present in the oriens layer and in the stratum radiatum of the hippocampus formation, in the supraoptic and lateral magnocellular nucleus of the hypothalamus, in the mesencephalic trigeminal nucleus, in the ventral auditory nucleus and in the facial nucleus of the brain stem as well as in red nucleus of the reticular formation and in the Purkinje layer of the cerebellar cortex. These data show that the iso-enzymes are located in specific brain nuclei. The significance of the results in respect to the yet very poorly defined function of PMO's is discussed. PMID- 8612328 TI - Developmental changes in the localization of the transplasma membrane NADH dehydrogenases in the rat brain. AB - The function of transplasma membrane oxidoreductases (PMO's) has been further studied by means of investigating the postnatal (PN) developmental changes in the tissue localization of six isoenzymes previously characterized (see accompanying paper). The changes were followed in the midbrain for PMO-I, -II, and -V and in the brainstem for PMO-III, -IV and -VI. PMO-I is not observed before PN5 and develops as long vertical fibers located mainly in the pontine nucleus and in the dorsal raphe nucleus until it merges all over the midbrain except for the aqueduct and the superior colliculus after PN10. At that stage it is highly expressed in the trigeminal nucleus and the dorsal raphe, but its expression then strongly decreases and PMO-I disappears almost completely later on. Similarly PMO II only develops around PN5, first in the dorsal and caudal linear raphe and later on (at PN7) also in the pontine nucleus and in the median raphe; at PN10 PMO-II gradually had vanished from these areas and strongly developed in the dorsal raphe and in the mesencephalic trigeminal nucleus. Later on PMO-II also decreases from these areas. PMO-III slowly develops within the gigantocellular reticular nucleus from PN1 to PN5 and later on reaches the facial nucleus (after PN5), the density of PMO-III in these regions at PN10 being much higher than in the adult. PMO-IV follows a similar developmental pattern in the midbrain, with an optimal density around PN10 also. PMO-V appears only at about PN5, first within the dorsal raphe in parallel fibers and in multipolar neurons. It disappears from the fibers around PN10 and remains present in neurons up to adulthood. PMO-VI appears at early stages within the gigantocellular reticular nucleus and after PN5 within the central gray in vertical fibers. At later stages PMO-VI is found in the spinal trigeminal nucleus, at first within the neuropil then in multipolar neurons that remain present up to adulthood. These datas suggested that the different isoenzymes are expressed at various stages in specific areas. The role of PMO's in neuronal development is discussed. PMID- 8612329 TI - Coloboma hyperactive mutant exhibits delayed neurobehavioral developmental milestones. AB - The coloboma mutation (Cm) is a neutron-irradiation induced gene deletion located on the distal portion of mouse chromosome 2. This deletion region includes a gene encoding the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25). The resulting mutation is semi-dominant with heterozygote mice exhibiting a triad of phenotypic abnormalities that comprise profound spontaneous hyperactivity, head bobbing and a prominent eye dysmorphology. Because the expression pattern of two SNAP-25 isoforms begins to change during the first postnatal week, neurobehavioral developmental milestones were examined in order to determine if the expression of the coloboma behavioral phenotype could be detected during this period of postnatal development. The early classification of coloboma mutant offspring may help to further describe the penetrance of this mutation as well as the contribution of developmental changes to the adult behavioral phenotype. The coloboma mutation resulted in delays in some tests of complex motor skills including righting reflex and bar holding. In addition, coloboma mutants were characterized by body weight differences (first appearance day 7) and hyperreactivity to touch (day 11) and head bobbing (day 14). These data demonstrate disruptions in the time course of attaining developmental milestones in coloboma mutants and provide further evidence supporting the hypotheses that alterations in Snap gene expression are associated with functional behavioral consequences in developing offspring. PMID- 8612330 TI - Perturbation of target-directed neurite outgrowth in embryonic CNS co-cultures grown in the presence of ethanol. AB - Studies were conducted to determine the influence of ethanol on target-directed fiber outgrowth in culture, using embryonic chick spinal cord-muscle, and fetal rat septal-hippocampal co-cultured explants. Process extension from the spinal cord and septal explants in control cultures was selectively oriented toward the appropriate target tissue. Ethanol in the culture medium (500 mg/dl) eliminated this target-oriented outgrowth in both systems, although the overall extent of neurite outgrowth was not affected. In an effort to further characterize the source of this disruption, target explants were grown alone, with and without ethanol, and the target-conditioned culture media was subsequently harvested and placed on newly plated spinal cord or septal explants, to determine whether ethanol decreased the target production of soluble substances. To determine whether deposition of substrate-bound materials by the target tissue was affected by ethanol, spinal cord or septal explants were plated in wells which had previously been occupied by the appropriate target tissue. These studies revealed that ethanol significantly inhibited production of soluble and substrate-bound materials by muscle explants, but not by hippocampal explants. It was concluded that the ethanol-induced loss of target-directed neurite outgrowth in the spinal cord explants could be accounted for primarily by the attenuated production of neurotropic/neurotropic substances by the muscle tissue. The loss of target directionality in the septal explants appeared to be due to other factors, possibly related to ethanol-induced compromise of the capacity of the septal neurons to respond appropriately to target-derived neurotrophic/neurotropic substances. The implications of these results for the fetal alcohol syndrome are considered. PMID- 8612331 TI - Development and distribution of gonadotropin-releasing hormone neuronal systems in the frog (Rana esculenta) brain: immunohistochemical analysis. AB - The ontogenesis of the gonadotropin-releasing hormone (GnRH) neuronal systems was studied in the brain of the frog, Rana esculenta. Attention was also focussed on the differential distribution of molecular forms of GnRH during development. The first GnRH-immunoreactive neurons appear in the mesencephalon of posterior limb stage tadpoles. These neurons are shown to contain only chicken [His5,Trp7,Tyr8]GnRH (cGnRH-II). Later in development, mammalian [Tyr5,Leu7,Arg8] GnRH (mGnRH)-like peptide-containing neurons appear simultaneously in the terminal nerve as well as in the anterior preoptic area of the telencephalon. Subsequently, only after metamorphosis, mGnRH-containing neurons appear in the medial septal area of the posterior telencephalon. It is here shown that neurons containing the two forms of GnRH are distributed in distinct brain areas during development and in the adult: mGnRH-immunoreactive neurons in the terminal nerve, olfactory bulb, mediobasal telencephalon, medial septal area, anterior preoptic area, ventrolateral thalamus and infundibulum, whereas cGnRH-II neurons are located in the mesencephalon. We hypothesize that the terminal nerve/forebrain located GnRH neurons express immunohistochemically late in development and originate extracranially migrating centrally, along the terminal nerve, during development, whereas those located in the mesencephalon express earlier and may have an intracranial site of origin. PMID- 8612332 TI - Alterations in geniculate ganglion proteins following fungiform receptor damage. AB - Previous anatomical studies in rat have shown that damage produced to fungiform receptors of the anterior tongue at postnatal age 2 (P2) alters the growth and ramification of primary gustatory axons in the rostral nucleus of the solitary tract (NST). Studies employing artificial rearing (AR) procedures, which functionally deprive rat pups of orochemical stimulation during critical periods of postnatal life, produce similar alterations in the development of primary gustatory axons in the NST. Therefore, orochemical stimulation during rat's early postnatal life is necessary for normal development of primary gustatory axons in the rostral NST. One hypothesis concerning receptor-damage effects and AR effects is that receptor damage during critical periods of development may alter the regulation (i.e. transcription/translation) and/or distribution (i.e. transport) of proteins in geniculate ganglion neurons, thereby affecting growth of primary gustatory axons in the rostral NST. Specific aims of the present experiments were to comprehensively examine electrophoretic profiles of geniculate ganglion proteins following P2 receptor damage and late (> P40) receptor damage. Results show that concentrations of particular geniculate ganglion proteins are differentially altered following P2 receptor damage and late receptor damage, and that early receptor damage and late receptor damage produces distinct effects on the electrophoretic profiles of particular classes of proteins. Between the ages of P7-P38, P2 receptor damage lowers ganglion concentration of an acidic membrane glycoprotein designated as A1, with an apparent M(r) of 64-67 kDa and a pI of 4.8 5.2 P2 receptor damage also lowers ganglion concentrations of GAP-43. P2 receptor damage produces transient decreases in ganglion concentrations of NF-160, NF-200, and 8 additional acidic proteins. Three of these proteins may correspond to peripheral nerve sheath proteins analyzed in previous studies of the sciatic nerve, and one of these proteins may correspond to a 24 kDa growth-associated protein characterized in regenerating optic nerve. The time-course for changes observed in ganglion proteins following P2 damage was consistent with that observed for normal anatomical development of primary gustatory axons in both the lingual epithelium and NST. Receptor damage produced at P40 and later yielded different patterns of changes in geniculate ganglion proteins. Late receptor damage produced a transient increase in ganglion concentrations of NF-160, NF 200, GAP-43 and four additional acidic proteins within the 29-57 kDa M(r) range. Late receptor damage also produced a transient decrease in the concentrations of protein A1 and a 30 kDa protein that was not affected by P2 damage. Therefore, proteins that were preferentially affected by P2 damage may be involved in the regulation of initial axonal growth within the lingual epithelium and NST, as opposed to the structural repair or maintenance of extant axons. Relationships between normal anatomical development in peripheral and central components of primary gustatory axons are discussed in relation to availability of particular cytoskeletal and growth-associated proteins. PMID- 8612333 TI - Investigation of neonatal brain cytochrome redox by NIRS. AB - Near Infrared Spectroscopy (NIRS) has been used to detect changes in cerebral blood and tissue oxygenation. Redox state of enzyme cytochrome aa3 (Cyt aa3 is thought to give an indication of tissue oxygenation. This information would be of value in exploring the aetiology of cerebral ischaemic lesions in preterm infants. However we have found that the change in cerebral cytochrome redox in infants following desaturation was inconsistent and insignificant. PMID- 8612334 TI - Alpha adrenoceptor gene expression in the rat iris during development and maturity. AB - We have examined the developmental profile of the alpha-1 and alpha-2 adrenergic receptor mRNA expression in the rat iris. The expression of the six mRNAs was studied using reverse transcription-polymerase chain reaction. At 4 weeks, the dominant mRNA transcripts in the rat iris were alpha-1B, alpha-1C and alpha-2A. This pattern of alpha adrenoceptor expression was seen from birth, throughout development, and maintained into old age. PMID- 8612335 TI - Developmental and regional changes in brain norepinephrine levels in diabetic C57BL/KsJ mice: effects of estradiol and progesterone. AB - Developmental and diabetes-associated changes in regional brain norepinephrine (NE) concentrations, and the influence of estradiol (E) and progesterone (P) on NE levels, were correlated with changes in blood glucose levels and body weight (obesity) in developing 4-16-week-old C57BL/KsJ (db/db) mice relative to corresponding age-matched control (+/?) parameters. Regional brain (i.e. amygdala, hypothalamus and medulla) NE levels were determined by high performance liquid chromatography. The (db/db) mice exhibited overt hyperglycemia and obesity relative to controls between 4 and 16 weeks of age. Hypothalamic NE levels in diabetics were chronically elevated as compared to those of age-matched controls by 8 weeks of age, and remained elevated through 16 weeks of age. Regional amygdaloid and medullary NE concentrations were comparable in (+/?) and (db/db) groups by 16 weeks. E-treatments normalized (db/db) hypothalamic NE concentrations to control levels between 8 and 16 weeks of age, but had no effect on amygdaloid or medullary values. In contrast, in 16 week old (db/db) mice, P treatments elevated hypothalamic and medullary NE levels compared to controls and expected diabetic levels. These data demonstrate that a marked modification in regional brain NE concentrations occurs in association with the overt expression of the diabetes mutation during development in this species. Observed changes in adrenergic influences in specific CNS loci may be therapeutically modulated by ovarian steroid hormones, especially in the hypothalamic locus which is recognized to possess steroid-concentrating neurons. The observed normalization of regional brain NE concentrations by E-therapy may be causally related to the ovarian steroid-modulation of overt hyperglycemia and diabetes-associated neuronal degeneration in (db/db) mice. PMID- 8612336 TI - Signal transduction mechanisms subserving activity-dependent release of neuronal proteoglycans. AB - We demonstrate here using dissociated hippocampal neurons that glutamate-induced release of proteoglycans which have been shown to have neurite growth-promoting activity is regulated by serine/threonine kinases of the protein kinase C and calcium/calmodulin type II kinase families, and that the state of phosphorylation of hippocampal neurons is a determinant of the magnitude and duration of the release response. Nitric oxide is also involved in mediating glutamate-induced PG release. PMID- 8612337 TI - Basic theory of mass spectrometry. PMID- 8612338 TI - Mass spectrometry in uremia. PMID- 8612339 TI - Mass spectrometry in diabetes mellitus. PMID- 8612340 TI - Mass spectrometry in Parkinson's disease. PMID- 8612341 TI - Mass spectrometry in hepatic diseases. PMID- 8612343 TI - Clinical mass spectrometry. Introduction. PMID- 8612342 TI - Mass spectrometry in disorders of organic acid metabolism. PMID- 8612345 TI - Development of the immunoglobulin repertoire. AB - In this Review we will include studies on the development immunoglobulin repertoire. We will discuss the pattern of V, (D), and J rearrangement in both normal B cells and autoimmune disorders. We will define the role of the recombination signal sequences and the importance of the nucleotide sequence of these highly conserved motifs. Whether deviations from the consensus recombination signal sequence will be tolerated by the recombination mechanism and the importance of the recombination-activating genes are also discussed. We will address the issue of whether pathogenic autoantibodies are generated as part of the normal immune repertoire and the importance of receptor editing as a means by which the immune system deletes autoreactive B cells. PMID- 8612344 TI - Procedures for MS analysis of clinically relevant compounds. PMID- 8612346 TI - Increased serum concentrations of the carboxy-terminal-cross-linked telopeptide of collagen type I in patients with acute Plasmodium falciparum malaria. AB - We determined serum levels of the carboxy-terminal-cross-linked telopeptide and carboxy-terminal propeptide of type I collagen (ICTP and PICP) in 24 patients with acute complicated Plasmodium falciparum malaria prior to and 7, 14, 21, and 28 days after therapy by radioimmunoassay in Bangkok, Thailand. Elevated levels of ICTP were observed in patients (mean +/- SD concentration 16.7 +/- 5.8 ng/ml), compared with normal controls (3.1 +/- 1.3 ng/ml), during the acute phase of the disease. In contrast, serum concentrations PICP were not different between patients and controls (168 +/- 63 and 144 +/- 57 ng/ml, respectively). After therapy serum ICTP concentrations decreased but remained elevated even 28 days after the malaria attack (10.3 +/- 2.9 ng/ml). These findings suggest an increased production or release of ICTP in P. falciparum malaria, which could implicate an alteration of extracellular matrix during P. falciparum malaria. PMID- 8612348 TI - Immunomodulation by an adenylate cyclase activator, NKH477, in vivo and vitro. AB - Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger which modulates T cell function. NKH477 is a direct adenylate cyclase activator derived from forskolin and now under clinical investigation as a positive inotropic agent. While the immunosuppressive effects of forskolin on lymphocytes have been reported, little is known about its effects in vivo. In this study, we investigated whether NKH477 has immunosuppressive effects in mice, namely on cardiac allograft survival, and on the generation of cytotoxic T lymphocytes (CTL), T cell proliferation in mixed lymphocyte reaction (MLR), and production of interleukin-2 (IL-2) in MLR and in mitogen response. We assessed the effects of standard immunosuppressant cyclosporin A (CsA) on IL-2 production and on allograft survival to estimate the intensity of rejection in this acute rejection model. Saline-treated C57BL/6 (H-2b) mice rejected DBA/2 (H-2d) cardiac allografts with a median graft survival time of 10 days. In contrast, median graft survival was prolonged to 12 and 15 days in mice treated with NKH477 at 1 and 3 mg/kg/day, respectively (P < 0.01 vs control). The equivalent dose of CsA (40 mg/kg/day) to the maintenance dose after clinical cardiac transplantation prolonged median graft survival time to 15.5 days, indicating that high dose of NKH477 was as efficacious as lower dose of CsA. Addition of NKH477 to the culture medium suppressed the generation of CTL, T cell proliferation in MLR, and production of IL-2 in MLR and in mitogen response. These results suggest that NKH477 exerts a beneficial effect on murine cardiac allograft survival by modulating T cell function. PMID- 8612347 TI - Enhanced CD3 monoclonal antibody induced proliferation of colonic mucosal T lymphocytes in Crohn's disease patients free of corticosteroid or immunosuppressor treatment. AB - Mucosal lymphocytes are increasingly activated in Crohn's disease. In order to investigate this phenomenon we tested the response of cultured colonic mucosal T lymphocytes to CD3 activation. These lymphocytes were obtained by biopsy from control subjects and Crohn's disease patients. T cells from mucosa involved in Crohn's disease showed a higher CD3-induced proliferative response compared to control T lymphocytes (P < 0.01). T cells from uninvolved areas showed a wide range of response, ranging from normal to very high proliferative indices. Our results suggest that the exaggerated response of mucosal T lymphocytes, due to the impairment of the downregulation of the CD3-dependent signaling process, may contribute to the pathogenesis of Crohn's disease. PMID- 8612349 TI - Adhesion and TNF priming in neutrophil-mediated cartilage damage. AB - Neutrophils predominate in the acute stages of rheumatoid arthritis and are implicated in the cartilage damage which is characteristic of this disease. In vitro neutrophils can be primed for increased ability to damage host tissues by a number of cytokines including tumor necrosis factor-alpha (TNF). The role of adherence in this process was investigated. Opsonization of cartilage with aggregated IgG (HAGG) and complement promoted neutrophil damage to cartilage. Adherence was increased by HAGG and TNF. Separation of neutrophils from cartilage markedly reduced the neutrophil-mediated injury and abolished the priming effect of TNF. Inactivation of complement or antibodies against CD11a or CD11b reduced neutrophil-mediated cartilage damage and markedly reduced TNF-priming of this damage and yet did not alter adherence of control or TNF-primed cells. These results suggest that neutrophil damage to cartilage is promoted by agents that favor adherence. The failure to block adhesion of neutrophils by complement inactivation or antibodies to CD11a or CD11b suggests that neutrophil adherence to cartilage occurs simultaneously through several different receptors. The massive reduction of TNF enhancement of neutrophil damage to cartilage by preventing adhesion suggests that adherence is required for this action of TNF. PMID- 8612350 TI - Downregulation of interleukin-2 and apha-chain interleukin-2 receptor biosynthesis by cisplatin in human peripheral lymphocytes. AB - To further investigate the mechanisms by which the antineoplastic agent cisplatin interferes with immune function, we studied its effect on the biosynthesis of interleukin-2 (IL-2) and its alpha-chain receptor (IL-2Ralpha). Normal human peripheral blood lymphocytes (PBL) were activated in vitro with phytohemagglutinin (PHA), and anti-CD3 antibody in the presence of various concentrations of cisplatin. Purified T cells were also cultured with anti-CD3 antibody and costimulated by CD80 (B7-1, B7/BB1)-transfected P815 mastocytoma cells in the presence of cisplatin. Tritiated thymidine incorporation assays, an enzyme-linked immunosorbent assay for soluble IL-2Ralpha determination, and RNA dot-blot analysis and hybridization with IL-2- and IL-2Ralpha-specific probes were used. PHA-induced and anti-CD3 antibody-induced proliferation of PBL were significantly inhibited by cisplatin at concentrations attainable in vivo. This inhibition was not due to direct cell death as shown by the absence of trypan blue uptake in the presence of high concentrations of cisplatin. Therapeutic concentrations of cisplatin (1 microgram/ml) also inhibited the IL-2-dependent proliferation of purified T cells, mediated via the CD28-CD80 costimulatory pathway. In addition, the amount of soluble IL-2Ralpha released in the T cell culture supernatants was decreased by cisplatin in a dose-dependent fashion, suggesting that inhibition of cell proliferation was associated with a parallel decrease in IL-2Ralpha production. These effects correlated with a specific cisplatin-induced downregulation of both IL-2 and IL-2Ralpha messenger RNA accumulation in PHA-stimulated PBL that was dependent on the concentration of the drug. These findings suggest that the immunomodulatory effects of cisplatin may result in part from its capacity to directly downregulate the IL-2/IL-2R system in activated lymphocytes. PMID- 8612352 TI - In vitro RNA selection of an autoimmune epitope on stem-loop II of U1 RNA. AB - Autoantibodies to U1 RNA occur frequently in sera from patients with SLE and SLE overlap syndromes. These autoantibodies have been previously shown to recognize major epitopes on stem-loops II and IV of U1 RNA. To further define these recognition sites, in vitro RNA selection was performed to identify the individual nucleotides which form the antibody binding site. Serum autoantibodies were used in this procedure to select synthetic variants from a library of RNA oligomers with a central region of 25 degenerate nucleotides in a linear context. A consensus sequence was derived from the autoantibody-selected RNA ligands that corresponded to a region of stem-loop II. It contained six continuous nucleotides (U63-C64-C65,-A66-X-U68) and one presumed discontinuous nucleotide (U79). In vitro selection of RNA ligands from simpler sublibraries consisting of oligomers with random loops and fixed U1 stem II sequences yielded no consensus sequence, suggesting that autoimmune recognition occurs independent of loop nucleotides. Competition assays confirmed the specificity of these binding reactions. The structural nature of this autoimmune U1 RNA epitope is compatible with a model of autoantibody production based on stimulation by native small nuclear ribonucleoprotein particles. PMID- 8612351 TI - Genetically programmed development of salivary gland abnormalities in the NOD (nonobese diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a potential trigger for sialoadenitis of NOD mice. AB - NOD (nonobese diabetic) mice develop chronic lymphocytic infiltrates of the salivary glands (sialoadenitis) that correlate with a temporal decline in saliva production. To differentiate autoimmune and nonautoimmune components in this decline, we evaluated glandular function in NOD-scid mice. Although saliva volumes and protein concentrations appeared normal, amylase and EGF activities declined 50 and 20%, respectively, in NOD-scid mice between 10 and 25 weeks of age. Salivary protein profiles on SDS-polyacrylamide gels showed a profound decline in two prominent proteins of 32 and 20 kDa, and the emergence of a new 27 kDa protein. All three proteins exhibited amino acid sequence homology to parotid secretory protein (PSP) and reacted with PSP-specific antibody, suggesting an age dependent alteration in PSP. In addition, there was an induced expression of proline-rich protein in the salivary glands and saliva of NOD and NOD-scid mice that was not detectable in mouse strains lacking autoimmune disease. Submandibular gland histology revealed selective loss of acinar tissue despite an absence of sialoadenitis. These changes in salivary protein composition and histology in the absence of detectable lymphocytic infiltration suggest that glandular defects in the NOD genetic background may contribute to the triggering of the autoimmune response in the salivary glands. PMID- 8612353 TI - IL-15 induces the release of soluble IL-2Ralpha from human peripheral blood mononuclear cells. AB - The recently identified and cloned cytokine IL-15 shares many of the T-cell and B cell stimulatory activities of IL-2 and utilizes the beta and gamma chains of the IL-2R for binding and signaling. The present report shows that, like IL-2, IL-15 in a concentration and time-dependent manner causes the release of sIL-2Ralpha from PHA-activated human peripheral blood mononuclear cells. This effect of IL-15 is largely direct and independent of IL-2. Blocking of the IL-2Rbeta chain with the antibody Mik-beta1 prevented the release of sIL-2Ralpha by IL-15 but not by IL-2. IL-7, another cytokine utilizing the gamma chain of the IL-2R, drove the release of sIL-2Ralpha as well. Several clinical conditions are associated with abnormal serum sIL-2Ralpha levels and are also monitored by the measurement of sIL-2Ralpha. The reason for sIL-2Ralpha release is not fully understood. In this study, IL-15, like IL-2 was shown to be a potent inducer of sIL-2Ralpha release in vitro. PMID- 8612354 TI - Intratumoral administration of sizofiran activates Langerhans cell and T-cell infiltration in cervical cancer. AB - In order to ascertain a correlation between infiltration of Langerhans cells (LCs) or T-cells in tumor tissues and the intratumoral administration of a biological response modifier, Sizofiran (SPG) was analyzed in cancer of the uterine cervix. Cancer specimens of 45 patients with stage II-III invasive cervical cancers were analyzed. SPG was administered to the cervical tumor at high and low concentrations (Strong SPG and weak SPG) as well as by intramuscular injection twice a week. LC and T-cell infiltrations to tumor tissues of the uterine cervix were studied immunohistochemically and electron microscopically. Of 10 patients with systemic but no local immunization, 1 (10.0%) showed an increase in LC infiltration and 2 (20.0%) showed a decrease. Of 15 patients with strong SPG immunization, 2 (13%) showed an increase and 5 (33%) showed a decrease. In contrast, of 20 patients with weak SPG immunization, the incidence of increase in LC infiltration was 55% (11 patients), significantly greater than the above-mentioned groups and none showed a decrease. Of the 20 patients with weak SPG administration, 3 (15%) showed T-cell infiltration before SPG administration, and 12 (60%) showed an increase in T-cell infiltration after SPG was given. Up on electron microscopy, Birbeck granules in the cytoplasm of LC significantly increased after SPG immunization, indicating activation of LC. In conclusion, the present study suggested that the LC and T-cell infiltrations in cancer tissues were augmented by intratumoral SPG administration at a certain concentration. Intratumoral administration of SPG may be applied to improve the prognosis after multidisciplinary treatment of advanced cervical cancer. PMID- 8612355 TI - Induction and regulation of NFkappaB during aging: role of protein kinases. AB - Aging is accompanied by significant declines in immune function. To understand the basis for this age-related decline we have investigated the induction and regulation of transcription factor NFkappaB in murine T cells. We report that anti-CD3 treatment of T lymphocytes from old mice resulted in significantly lower levels of NFkappaB than those observed in similarly treated T cells from young mice. Pretreatment of T cells with staurosporine reduced the induction of NFkappaB in anti-CD3-activated cells from young mice but had no effect in similarly treated T cells from old mice. However, pretreatment with H-89 had little or no effect on the induction of NFkappaB in T cells from young mice, but significantly enhanced the induction of NFkappaB in T cells from old mice. These results suggest that the impairment in the generation of NFkappaB during aging may reflect age-associated differences in the regulation of protein kinase A. PMID- 8612356 TI - Iron deficiency anemia in pregnancy. AB - Iron deficiency is seen commonly in women during their reproductive years and particularly during pregnancy. The physician who primarily cares for women should be aware of this and initiate iron supplementation when indicated. The association of poor pregnancy outcome is of concern when iron deficiency is present in the first trimester. It is unclear whether this is because of the anemia or other epidemiologic factors. It is also unknown whether correcting iron deficiency before conception improved neo-natal outcome. Until more is known concerning IDA, it is recommended that pregnant women receive nutritional assessment and prenatal care along with adequate iron supplementation to potentially decrease the risk of poor pregnancy outcome. PMID- 8612357 TI - Megaloblastic anemia in pregnancy. AB - Megaloblastic anemia is one of the acquired nutritional anemias that may complicate pregnancy. It is most often secondary to folic acid deficiency because folate requirements are increased during gestation. When the diagnosis of megaloblastic anemia is confirmed, appropriate therapy will initiate a rapid reversal of the anemia process. Because of the association between neural tube defects and folate deficiency, it is recommended that women of reproductive age take folic acid supplementation. PMID- 8612358 TI - Hemolytic anemias in pregnancy. AB - Polymorphism exists and complicates the diagnosis of inherited hemolytic anemias. However, with linkage DNA analysis and, on occasion, with characterization of the mutant gene, it is possible to make a diagnosis on the DNA level. This technique increases our understanding of the enzymatic defect and the relationship with clinical findings. In acquired hemolytic anemias, pregnancy is one of the most commonly associated conditions. The physiology is not yet entirely understood and, therefore, does not allow specific treatment. Supportive therapy is usually the rule in these cases. PMID- 8612359 TI - Pregnancy complicated by sickle hemoglobinopathy. AB - During the last decade it has been shown that patients with major sickle hemoglobinopathies can experience a normal reproductive outcome. This has been accomplished with early aggressive prenatal care, effective counseling, and appropriate intervention by providers with a high index of suspicion for factors that lead to untoward outcomes in such women. Because controversy surrounds the use of transfusion therapy for pregnant patients with sickle cell disease, individualization should depend on patient circumstances and provider experience because this is a key factor in the management of these women. New therapies for those with major sickle hemoglobinopathy are on the horizon, but their use in pregnancy awaits further evaluation. PMID- 8612361 TI - Inherited disorders of coagulation in pregnancy. AB - Normal physiology has been reviewed to serve as a basis for understanding the abnormalities leading to the inherited coagulation disorders. These abnormalities involve each of the three arms of the coagulation process: platelets, the coagulation cascade, and the clot prevention/lysis pathways. Pregnancy presents a unique challenge to women with these disorders. However, with close attention to the natural course of the disorder and appropriate therapy instituted in a timely fashion, many of these women can achieve successful pregnancies. PMID- 8612360 TI - Thalassemia in pregnancy. AB - The responsibility of the obstetrician is to identify during the first trimester women who are carriers for thalassemia. When she has been identified, the father of the baby should be screened and if he is also a carrier, the couple should be offered prenatal diagnosis. In the majority of cases, prenatal diagnosis can identify the homozygous fetus, thus allowing the family a choice in continuing the pregnancy. PMID- 8612362 TI - Management of disseminated intravascular coagulopathy. AB - Disseminated intravascular coagulopathy is a process in which aberrant activation of the clotting mechanism results in defective coagulation. Although infrequently encountered during pregnancy, disseminated intravascular coagulopathy is associated with several clinical situations unique to obstetrics. Management consists of treating the underlying cause, restoring and maintaining the circulating blood volume, and replacing deficient clotting factors as indicated. PMID- 8612363 TI - Management of the obstetric patient with thrombocytopenia. AB - Thrombocytopenia that complicates pregnancy can occur secondary to known maternal disease processes or may arise again during, and as a result of, gestation. A combination of maternal history, clinical presentation, and laboratory investigation usually leads the obstetrician to the proper diagnosis of the condition and, when necessary, dictates appropriate fetal intervention. Although the differential diagnosis of thrombocytopenia can be a frustrating and difficult exercise, especially in an emergent intrapartum environment, clinical persistence coupled with hematology consultation when appropriate usually will reward the provider and patient with the best chance to achieve optimal maternal-fetal outcome. PMID- 8612364 TI - Hematologic malignancies during pregnancy. AB - Most hematologic malignancies are rare during the childbearing years with the exception of Hodgkin's lymphoma. However, the continued spread of retroviruses, such as human immunodeficiency virus, in the heterosexual population may result in a substantial rise in viral-induced non-Hodgkin's lymphomas. Although pregnancy does not affect the natural course of these illnesses, adequate staging and therapy can be a difficult task. The obstetrician and the consultant hematologist/oncologist must weigh the benefit of immediate treatment while minimizing toxicity to the fetus. PMID- 8612365 TI - Transfusion therapy in pregnancy. PMID- 8612366 TI - Prenatal diagnosis of hematologic disorders. PMID- 8612367 TI - Assessing the effectiveness of cervical screening. AB - The mathematic models used to assess the benefits and cost-effectiveness of cervical screening reveal little consistency in the definition of disease status, the basic assumptions made, or the data used in the model. Conclusions derived from the models often are model and data dependent. Several authors have used simplified models and unrealistic assumptions, such as the failure to differentiate between different grades of dysplasia, or 100% sensitivity for the screening test. The Markov process assumes that the rate of transfer between states is independent of the duration of time spent in one state, and this assumption may be unsound. The difficulty with all models is in interpreting the appropriateness of the parameter values. Some are well documented, for example, stage-specific survival rates for treated patients or attendance for screening. Many, however, cannot be given a fixed value. The large number of factors that appear implicated in the incidence of and mortality from cervical cancer can lead to many feasible sets of parameter values that generate output that approaches the observed data. PMID- 8612368 TI - Every woman with an abnormal cervical smear should be referred for treatment: debate. AB - No dispute exists regarding the optimal management of women with moderate or severe dyskaryosis (high grade SIL). These women should have a colposcopic assessment and biopsy. Women with borderline nuclear abnormalities should have a repeat smear and undergo colposcopy only if the abnormality persists. Low-grade abnormalities (low-grade SIL) are common, and their management is relevant. We suggest that these women be referred for an immediate colposcopic assessment for the following reasons: a. a policy of cytologic surveillance allows an opportunity for default, and these women are at an increased risk of invasive cancer. b. a significant proportion of these women will have underlying CIN grade III that should be treated. c. a minority of these women revert to cytologic normality without a recurrent dyskaryotic smear and, therefore, eventually will be referred to colposcopy. d. a policy of immediate referral to colposcopy may be financially less expensive and avoid unnecessary anxiety to the woman during the period of undergoing repeated smears. PMID- 8612369 TI - Every woman with an abnormal cervical smear should not be referred for colposcopy: debate. PMID- 8612371 TI - Electrosurgery: principles and safety issues. AB - The use of monopolar electrosurgical energy has been the "gold standard" for the past 50 years. It has diverse capabilities, such as fulguration, precise vaporization, and coaptation of large vessels. Technologic advances in performance and safety have positioned this device as a useful tool in a surgeon's armamentarium. The adaptation of active electrode monitoring for stray energy as a result of insulation failure or capacitive coupling and the use of completely metal trocar cannulas will increase the confidence of the surgeon and the safety of his/ her patient. It is the author's opinion that its use will prove itself in laparoscopy. As with any surgical tool or energy source, education and skill are required. This introduction on the principal of the biophysics of electrical energy on tissue and the safety consideration is a start to further one's understanding of this surgical tool. PMID- 8612370 TI - Cervical neoplasia: are adjunctive tests to cervical cytology worthwhile? PMID- 8612372 TI - Large loop excision of the transformation zone. AB - During the early 1990s, the technique of LLETZ has acquired widespread approval throughout much of the colposcopic community, and its advantages over destructive methods of treatment have been described by several authors. 1. It allows for histologic audit of the colposcopic diagnosis. 2. It allows histopathologic examination to rule out microinvasion. 3. It allows excision of the dysplastic lesion and the transformation zone, which may be confirmed histologically. 4. It may be performed at the first (assessment) colposcopic examination. 5. It may be adapted to treat all cases of CIN, irrespective of the size and site of the transformation zone. 6. It is an easily learned technique. 7. It uses inexpensive, readily available equipment and has low operating costs. 8. It is usually an office or outpatient procedure performed using local anesthesia. The last five of these eight advantages are also potential disadvantages of the technique that may combine to increase the morbidity of the procedure. Women may be treated more easily and at a lower threshold of abnormality in the office with local anesthesia and with transformation zones of almost any dimension, situated on the ectocervix in the endocervical canal or both. If more women are treated (at a lower threshold of suspected abnormality), then procedure-related morbidity will increase. Because the technique allows sufficient flexibility to accommodate transformation zones of every site and dimension, it is inevitable that women who would otherwise have had a cone biopsy will now have a LLETZ procedure. The morbidity of a cone biopsy (LLETZ, laser, or cold knife) is related to the volume, and also probably the amount of endocervical tissue excised. It is important that the morbidity associated with removal of a long endocervical transformation zone be recognized as a consequence of the size and site of the transformation zone, rather than of the choice of excisional technique. EASE OF USE The method is technically straightforward and undemanding to an experienced and relatively dexterous colposcopist. COST When compared with the laser technique, the method is less expensive. However, the other destructive modalities are equally inexpensive (cold coagulation, cryocautery, and radical diathermy). Many of the electrosurgical units that are used for LLETZ also may be used for a variety of procedures in gynecology. EFFECTIVENESS It is evident that a method of excising the transformation zone will have the same likelihood of successfully treating women with CIN as do the destructive techniques, and this has been supported by the published series of patients treated by LLETZ. It is also true that LLETZ is unlikely to significantly improve on the success/failure rates of treatment achieved by the protagonists of each destructive method of treatment. This is because the success/failure rates of destructive methods are high when performed by experts. However, women with CIN cannot always be treated by experts of individual destructive techniques. Perhaps a more clinically important question is whether LLETZ is associated with a superior success/failure rate compared with destructive methods in which each are performed by the nonspecialized practicing gynecologist. PMID- 8612373 TI - Modern image capture and data collection technology. AB - Computer data collection technology is intended to facilitate patient management and clinic administration. The systems should be devised for ease of usage and careful consideration should be given to the core set of data required and the task to be performed by the system. Security of data is important, and it must cover not only the inadvertent access to confidential data, but also must stop corrupt data from entering the system. Audit is becoming increasingly important, and the ability to achieve this should be inbuilt into any data collection system. A staged introduction of these systems is suggested without trying to achieve too much at the initial attempt. Adaptability is important so that as new technologies are introduced, the old systems should not be made redundant. Eventually it should be possible to achieve the goals set, and include fields for image capture of the colposcopic findings, which although not an essential requirement for such systems, is certainly a desirable one. PMID- 8612374 TI - Endocervical curettage has no place in the routine management of women with cervical intraepithelial neoplasia: debate. PMID- 8612375 TI - Endocervical curettage: a technique in search of an indication?: debate. PMID- 8612376 TI - The pathology of cervical cancer. AB - The significant changes over the last decade in the CIN/cervical carcinoma area have involved the increasing recognition of many cervical carcinomas as adenocarcinomas, rather than squamous cell type, and their propensity for arising in young women. Another area of interest is the deployment of new molecular techniques that are unveiling the role of oncogene co-factors (e.g. HPV, p53) in the biologic evolution of CIN and hopefully glandular dysplasia. PMID- 8612377 TI - International standards for training in colposcopy. PMID- 8612378 TI - Publication and proprietary information. PMID- 8612379 TI - Diltiazem enhances the effects of triazolam by inhibiting its metabolism. AB - BACKGROUND: Triazolam is metabolized by CYP3A4. Diltiazem is an inhibitor of this isozyme and interacts with midazolam, another substrate of this enzyme. Therefore the possible interaction between triazolam and diltiazem is worth investigation. METHODS: A balanced, randomized, double-blind crossover study design was used, with an interval of 2 weeks between phases. Ten healthy volunteers were given 60 mg diltiazem orally or placebo three times a day for 2 days. On the second day they received a single 0.25 mg oral dose of triazolam, after which plasma samples were collected and effects of triazolam measured for up to 17 hours. RESULTS: Diltiazem increased the mean area under the triazolam concentration-time curve threefold (p < 0.001) and the elimination half-life (p < 0.001) and the peak plasma concentration of triazolam twofold (p < 0.005). The increased concentrations of triazolam during the diltiazem phase were associated with increased and prolonged pharmacodynamic effects. CONCLUSIONS: Diltiazem has a clinically significant interaction with oral triazolam. The data is highly suggestive that diltiazem inhibits the metabolism of triazolam during the first pass and elimination phases. Prescription of triazolam should be avoided if a patient is using diltiazem or other potent inhibitors of CYP3A. PMID- 8612380 TI - Determination and allelic allocation of seven nucleotide transitions within the arylamine N-acetyltransferase gene in the Polish population. AB - The frequency of various genotypes of arylamine N-acetyltransferase (NAT2) was investigated in 248 Polish unrelated children. Allele-specific polymerase chain reaction (PCR) was applied for mutation at 341 nucleotide (nt) of NAT2 coding sequence and PCR/restriction fragment length polymorphism for the other mutations. Genotypes coded for slow acetylation in 62.9% (56.6% to 68.9%). The frequency of specific NAT2 alleles was *4 (wild-type), 22.0%; *5A (341C, 481T), 5.2%; *5B (341C, 481T, 803G), 33.1%; *5C (341C, 803G), 6.0%; *6A (282T, 590A), 30.0%; *7B (282T, 857A), 3.4%; and *12A (803G), 0.2%. No mutations were found at 191, 434, and 845 nt. By a molecular-genetic procedure, genotypes *4/*6A were confirmed not to mask *6B/*13 (590A/282T). *6B and *13 were absent in a composite sample representative of 826 alleles (95% confidence limits, 0% to 0.45%). Five cases of genotype-phenotype discrepancy were sequenced and their mutation allocation confirmed; 21 further genotypes were confirmed by sequencing. This first evaluation of NAT2 genes among a Slavic population should provide a basis for clinical and epidemiologic investigations of NAT2 in the Polish population. PMID- 8612381 TI - Grapefruit juice alters terfenadine pharmacokinetics, resulting in prolongation of repolarization on the electrocardiogram. AB - OBJECTIVES: To establish whether the pharmacokinetics and electrocardiographic pharmacodynamics of terfenadine are affected by concomitant administration of grapefruit juice and to determine whether any effect of grapefruit juice is dependent on the timing of administration in relation to the dose of terfenadine. METHODS: Twelve healthy volunteers were studied in a prospective randomized trial. The primary end points were QT prolongation on the surface electrocardiogram and the pharmacokinetic parameters: area under the concentration-time curve (AUC), maximum concentration, and time to maximum concentration of terfenadine and its acid metabolite terfenadine carboxylate. All subjects received 60 mg terfenadine twice a day with 240 ml water for 7 days. They were then randomized to drink 240 ml of double-strength grapefruit juice simultaneously with terfenadine (simultaneous group) for an additional 7 days or to drink the same dose of grapefruit juice 2 hours after terfenadine for 7 days (delayed group). Twelve timed electrocardiograms and plasma terfenadine and metabolite levels were measured on days 7 and 14. RESULTS: None of the 12 subjects had quantifiable levels of terfenadine when the drug was administered with water. All six subjects who took terfenadine and drank grapefruit juice simultaneously had quantifiable terfenadine levels. Only two of six who drank grapefruit juice 2 hours after terfenadine had quantifiable levels. The AUC of the acid metabolite increased 55% (p < 0.05) in the simultaneous group and 22% (p = NS) in the delayed group. The mean QT interval increased from 420 to 434 msec (p < 0.05) in the simultaneous group and decreased from 408 to 407 msec (p = NS) in the delayed group. CONCLUSIONS: Administration of grapefruit juice concomitantly with terfenadine may lead to an increase in systemic terfenadine bioavailability and result in increases in QT interval. The clinical significance of an increase in QT interval of this magnitude is unclear. PMID- 8612382 TI - Ferrous sulfate does not reduce serum levels of famotidine or cimetidine after concurrent ingestion. AB - A series of randomized crossover studies were performed to determine whether there was a reduction in serum levels of cimetidine and famotidine when coingested with ferrous sulfate (300 mg). Coingestion of a ferrous sulfate tablet with cimetidine (300 mg) was associated with little reduction in serum cimetidine area under the curve (AUC) (mean versus mean, 20.8 versus 23.4 mumol.hr/L; mean percentage difference, -11%; 95% confidence interval [CI] of percentage difference, -26% to 4.2%) or peak concentration (Cmax) (mean versus mean, 5.1 versus 6.1 mumol/L; mean percentage difference, -16%; CI of percentage difference, -36% to 4%). Similarly, ferrous sulfate solution coingested with cimetidine caused little change in cimetidine AUC (mean versus mean, 19.9 versus 23.0 mumol.hr/L; mean percentage difference, -13%; CI of percentage difference, 34% to 7%) or Cmax (mean versus mean, 5.0 versus 5.0 mumol/L; mean percentage difference, 1%; CI of percentage difference, -18% to 20%). Concurrent ingestion of famotidine (40 mg) with a ferrous sulfate tablet did not result in significant reductions in serum famotidine AUC (mean versus mean, 1.78 versus 1.99 mumol.hr/L; mean percentage difference, -10%; CI of percentage difference, -34% to 13%) or Cmax (mean versus mean, 0.31 versus 0.32 mumol/L; mean percentage difference, -3%; CI of percentage difference, -27% to 22%). The formation of famotidine:iron(III) complexes was shown in methanol but was not observed in an aqueous buffer at pH 6.5. Ranitidine did not bind iron in an aqueous buffer and only weakly bound iron in methanol. Coingestion of ferrous sulfate with either cimetidine or famotidine does not cause a clinically relevant reduction in serum histamine H2-receptor blocker levels and, on the basis of in vitro binding experiments, iron is unlikely to interact with ranitidine. PMID- 8612383 TI - Dose released and absolute bioavailability of nicotine from a nicotine vapor inhaler. AB - In an open, randomized, three-way crossover study, 14 healthy smokers used one type of nicotine vapor inhaler intensely for 20 minutes every hour for 11 hours (12 administrations). Two different inhalation techniques were applied, shallow frequent sucking (buccal mode) and deep inhalations (pulmonary mode). The determination of nicotine was performed by capillary gas chromatography after single-step liquid-liquid extraction of the plasma sample. Nicotine was detected by means of a nitrogen-sensitive detector, giving high selectivity and sensitivity. The mean (+/- SD) nicotine dose released from each nicotine vapor inhaler unit was estimated at 4.00 +/- 0.60 mg (buccal mode) and 3.87 +/- 0.75 mg (pulmonary mode), inhaled with approximately 15 L of air. Mean (+/- SD) peak plasma level of the last dosing interval was 32.0 +/- 8.7 ng/ml and 34.2 +/- 8.9 ng/ml for the buccal and the pulmonary technique, respectively, achieved after 0.33 and 0.50 (median) hour, respectively. The mean (95% confidence interval [CI]) absolute bioavailability of nicotine was 51 (95% CI, 40 to 65) and 56 (95% CI, 47 to 67) when the buccal and pulmonary techniques were used, respectively. A significant correlation was found between systemically available dose and average steady-state nicotine plasma concentration. Based on the achievement of similar nicotine plasma levels, it may be concluded that the two modes of inhalation appear to be clinically equivalent. PMID- 8612385 TI - Determination of dextromethorphan metabolic phenotype by salivary analysis with a reference to genotype in Chinese patients receiving renal hemodialysis. AB - BACKGROUND: The polymorphic metabolism of debrisoquin and sparteine by cytochrome P450IID6 (CYP2D6) is genetically determined. Determination of the CYP2D6 metabolic phenotype with conventional urine analytic methods is not feasible in anuric patients with renal failure. The possibility of using salivary analysis, with dextromethorphan as a probe drug, to determine the CYP2D6 metabolic phenotype in patients with renal failure was evaluated. METHODS AND RESULTS: One hundred four Chinese patients with renal failure were recruited. All 104 patients were receiving hemodialysis. Saliva was collected before and at 3 hours after each patient took a capsule of dextromethorphan hydrobromide (30 mg). Four patients were excluded because of insufficient samples of saliva. The distribution of logarithms of the metabolic ratios (log[MR]) in the 100 patients appeared to be normal. Administration of quinidine sulfate (200 mg twice daily) to nine of the patients significantly and markedly increased the dextromethorphan metabolic ratios. The metabolic ratios of nine patients pretreated with quinidine were higher than any of the 100 patients with renal failure who did not receive quinidine pretreatment. A metabolic ratio of 33 separated these two groups. Genomic deoxyribonucleic acid was extracted from whole blood in a subset of patients. Polymerase chain reaction (PCR)-based methods were used to detect the CYP2D6 and B mutant genes. Mutant B alleles (which are common in white poor metabolizers) of CYP2D6 genes were not detected in any of the 47 subjects tested. A PCR-based test of cytosine (C188) to thymine (T188) polymorphism at 188 base pairs in exon 1 of CYP2D6 genes was performed in 61 patients. Subjects who were homozygous for C188 had significantly (p = 0.0067) lower log[MR] values than those who were homozygous for T188. CONCLUSIONS: Determination of dextromethorphan metabolic ratios in saliva is feasible in patients with renal failure requiring hemodialysis. All subjects in this study appeared to be "extensive metabolizer" phenotype for CYP2D6, and no poor metabolizer was identified. From the results with quinidine pretreatment, a metabolic ratio of 33 is suggested to be a tentative antimode for identification of poor metabolizers in patients with renal failure. PMID- 8612384 TI - Age-related difference in tamoxifen disposition. AB - PURPOSE: To investigate the pharmacokinetic aspects of tamoxifen, such as the pharmacokinetic-pharmacodynamic (toxicity and clinical response) relationship and the influence of hepatic dysfunction, age, treatment duration, and associated chemotherapy on tamoxifen pharmacokinetics. PATIENTS AND METHODS: Three hundred sixteen patients with breast cancer (247 postmenopausal women) were investigated. Mean age was 58 years (age range, 29 to 85 years). One hundred seventeen patients received tamoxifen as single therapy (adjuvant, 60; neoadjuvant, 17; metastatic, 40); 292 of 316 received 30 mg daily. We obtained 794 blood samples at steady state. Tamoxifen and metabolites, N-desmethyltamoxifen, N-desdimethyltamoxifen, primary alcohol, and 4-hydroxytamoxifen were measured by HPLC. RESULTS: Serum concentrations of tamoxifen and metabolites showed a wide asymmetrical distribution. Median and extremes were 347 nmol/L (not detectable [ND] to 1677) for tamoxifen, 572 nmol/L (ND to 3132) for N-desmethyltamoxifen, 109 nmol/L (ND to 795) for N-desdimethyltamoxifen, and 59 nmol/L (ND to 390) for primary alcohol. 4-Hydroxytamoxifen was detectable in 9.5% of the samples (ND to 162 nmol/L). Neither the absolute nor the relative concentrations of each compound showed significant variations during treatment. Chemotherapy concomitant with tamoxifen slightly increased the tamoxifen blood concentration. Hepatic dysfunction had no obvious effect on drug concentrations, an exception being a slight reduction in the relative proportion of tamoxifen. The influence of age revealed that concentrations of tamoxifen and metabolites increased significantly with age: women younger than 40 years had a tamoxifen plus metabolite median concentration of 802 nmol/L compared with 2428 nmol/L for women older than 80 years. In the 28 patients in whom tamoxifen-related side effects developed, the proportion of demethylated metabolites was higher than that in patients in whom toxicity did not develop. There was no difference in drug concentrations between responding and nonresponding patients. CONCLUSION: Despite the tremendous interpatient variability in drug concentrations, the present data show that tamoxifen and metabolite concentrations significantly increase with age. PMID- 8612386 TI - Iron-ovotransferrin preparation does not interfere with ciprofloxacin absorption. AB - Iron supplements can interfere with the bioavailability of a number of drugs, including thyroxine, tetracycline derivatives, penicillamine, methyldopa, levodopa, carbidopa, ciprofloxacin, and the newer fluoroquinolones. A new iron formulation was tested in which iron ions are bound to ovotransferrin, a protein that shares more than an 80% similarity with the sequence of human transferrin and apparently is less likely than the commonly used iron salts to reduce drug absorption. Ciprofloxacin was taken as a model drug, of wide use and restricted range of therapeutic levels, and its absorption was evaluated after the administration of the iron-ovotransferrin complex versus an iron-gluconate formulation in healthy volunteers. At variance with the iron gluconate formulation, which led to a reduction of about 50% of peak serum ciprofloxacin levels (Cmax; 1.0 +/- 0.2 versus 2.4 +/- 0.3 micrograms/ml; p < 0.01) and of the area under the serum concentration-time curve from time 0 to infinity [AUC(0 - infinity); 10.1 +/- 1.1 versus 18.3 +/- 1.0 mg.L-1.hr; p < 0.01], the iron ovotransferrin complex caused only modest, non significant changes in absorption with a minimal reduction of the AUC[0 - infinity) (17.3 +/- 1.0 versus 18.3 +/- 1.0 mg.L-1.hr; difference not significant) and a nonsignificant decrease in the Cmax (2.2 +/- 0.3 versus 2.4 +/- 0.3 microgram/ml; difference not significant). Iron was also well absorbed from the formulation in the presence of a fatty meal. The very common drug interactions with oral iron preparations can be effectively prevented by the use of the iron-ovotransferrin complex interacting to a minimal extent with a sensitive drug with a reduced margin of efficacy, such as ciprofloxacin. PMID- 8612387 TI - The CYP2D6 genotype predicts the oral clearance of the neuroleptic agents perphenazine and zuclopenthixol. AB - BACKGROUND: Most antidepressant and neuroleptic agents are metabolized by the polymorphic cytochrome P450 enzyme CYP2D6. This study evaluates the importance of the CYP2D6 genotype for the disposition of the neuroleptic agents perphenazine and zuclopenthixol. METHODS: Patients treated with neuroleptic agents (n = 36) were studied prospectively with regard to CYP2D6 genotype and neuroleptic plasma concentration during oral treatment. Because no patient provided enough samples for individual kinetic modeling, a bayesian approach was used for determination of the clearance. Population kinetic parameters for this procedure were collected from retrospective therapeutic drug monitoring data (n = 113) by use of a nonparametric approach. RESULTS: The CYP2D6 genotype significantly predicted the oral clearance of perphenazine and zuclopenthixol (p < 0.01 by multiple regression). The difference in clearance between homozygous extensive metabolizers and poor metabolizers was threefold for perphenazine and twofold for zuclopenthixol. CONCLUSION: The results show that the genotype for CYP2D6 is closely related to the oral clearances of perphenazine and zuclopenthixol. If this finding can be confirmed in a larger population, genotyping may become an important tool for the dosing of these two neuroleptic agents. PMID- 8612388 TI - The effects of menopause and hormone replacement therapies on prednisolone and erythromycin pharmacokinetics. AB - The pharmacokinetics of oral and intravenous prednisolone and intravenous erythromycin were examined in premenopausal women, postmenopausal women not undergoing hormone replacement therapy, postmenopausal women undergoing estrogen replacement therapy, and postmenopausal women undergoing estrogen and progestin replacement therapy. The unbound clearance of prednisolone was significantly lower in postmenopausal women (11.6 +/- 2.3 ml/min/kg) than in premenopausal women (16.6 +/- 3.5 ml/min/kg). A comparable difference was also observed in total clearance and in half-life. The bioavailability and volume of distribution of prednisolone were unaffected by menopausal status. Hormone replacement therapies did not significantly affect prednisolone pharmacokinetics. In contrast to prednisolone elimination, erythromycin elimination, as measured by the erythromycin breath test, was not significantly affected by either menopausal status or hormone replacement therapy. In addition, there was no correlation between prednisolone clearance and the erythromycin breath test result. Although cytochrome P450 3A4 (CYP3A4) has been implicated in steroid hormone metabolism, these results suggest that another enzyme system, which is decreased in menopause (rather than simply an age effect), is also involved in prednisolone metabolism. PMID- 8612390 TI - Inhibition of tacrine oral clearance by cimetidine. AB - Plasma tacrine, 1-hydroxytacrine, 2-hydroxytacrine, and 4-hydroxytacrine concentrations were measured in 12 healthy elderly subjects in this nonblinded two-period study to assess the effect of multiple doses of cimetidine on single dose tacrine pharmacokinetics. Subjects received 40 mg tacrine (Cognex) alone and during multiple-dose cimetidine (300 mg four times a day) administration. Overall, tacrine and cimetidine were well tolerated by healthy elderly subjects. After coadministration of cimetidine with tacrine, plasma tacrine concentrations were approximately one-third higher than values after administration of tacrine alone; metabolite concentrations were also higher. Mean tacrine oral clearance was reduced by 30%; however, mean absorption rate and elimination half-life values were not affected by cimetidine. It was concluded that cimetidine inhibits first-pass hepatic extraction of tacrine by cytochrome P450 enzymes but has little effect on systemic drug clearance. Clinical considerations may dictate a reduction in tacrine dosage when tacrine is coadministered with cimetidine. PMID- 8612389 TI - Population pharmacokinetics of carboplatin in children. AB - OBJECTIVES: In pediatric patients, administration of carboplatin according to body surface area results in a large variation in the area under the plasma ultrafilterable carboplatin concentration versus time curve. A population pharmacokinetic study using the NONMEM program was undertaken to determine the effects of a variety of covariates on the clearance of ultrafilterable carboplatin. PATIENTS: Plasma carboplatin pharmacokinetics were determined in 57 children (2 months to 18 years old, with serum creatinine levels ranging from 27 to 268 mumol/L) treated for various tumor types. RESULTS: The best fit corresponded to the formula: clearance (ml/min) = 2.85.weight.(1-0.00357.serum creatinine).(1-0.372.Np) + 8.7 (with serum creatinine in micromoles per liter, weight in kilograms, and Np = 1 or 0 for unilateral nephrectomy or not, respectively). The interindividual variability in clearance, as expressed by the coefficient of variation, decreased from 74% (no covariates) to 49% by taking account of weight, and to 29% under the final regression formula. CONCLUSION: The ability of this formula to predict carboplatin clearance in children should be evaluated prospectively and compared to a method based on the determination of the glomerular filtration rate. PMID- 8612391 TI - Simultaneous inhibition of catechol-O-methyltransferase and monoamine oxidase A: effects on hemodynamics and catecholamine metabolism in healthy volunteers. AB - OBJECTIVE: To evaluate the effects of simultaneous pharmacologic inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase type A (MAO-A) on hemodynamics and catecholamine metabolism in healthy volunteers at rest and during exercise. BACKGROUND: Entacapone, a COMT inhibitor, is studied as an adjunct to levodopa treatment in patients with Parkinson's disease. Moclobemide, an MAO-A inhibitor, is already in clinical use as an antidepressant. It is likely that entacapone and moclobemide will be used concomitantly in the future in patients who have both Parkinson's disease and depression. It was therefore considered to be important to investigate the tolerability of combined COMT and MAO-A inhibition with entacapone and moclobemide. DESIGN AND METHODS: This was a randomized, single-dose, double-blind crossover study of 12 healthy male volunteers. The treatments were either placebo, 200 mg entacapone, 150 mg moclobemide, or the combination of entacapone and moclobemide in single doses. Heart rate, blood pressure, impedance cardiography, and plasma concentrations of catecholamines and their metabolites were measured both at rest and during submaximal standardized bicycle exercise. RESULTS: Entacapone and moclobemide (either alone or in combination) did not change heart rate, blood pressure, or any hemodynamic parameter at rest or during exercise compared with placebo. Neither were the concentrations of norepinephrine and epinephrine in plasma influenced. Both drugs had the expected effects on catecholamine metabolite concentrations in plasma. The decrease in the concentration of 3-methoxy-4 hydroxyphenylglycol (MHPG) induced by moclobemide was not potentiated by entacapone. CONCLUSION: The combined use of therapeutic single doses of entacapone and moclobemide in healthy volunteers did not affect the hemodynamics or concentrations of unconjugated norepinephrine and epinephrine in plasma. Other mechanisms are capable of regulating the concentrations of norepinephrine and epinephrine in circulating blood (and apparently also at receptors in the heart and vascular tissue) when both COMT and MAO-A activity are inhibited to a significant extent. This was also the case during marked sympathetic stimulation. The changes in the catecholamine metabolite concentrations provide evidence of effective COMT and MAO inhibition. Concentrations of MHPG in plasma are determined mainly by MAO-A activity because COMT inhibition did not have an additional effect on the moclobemide-induced decrease in plasma MHPG. PMID- 8612392 TI - Phenytoin in the treatment of cocaine abuse: a double-blind study. AB - OBJECTIVES: To evaluate the effectiveness of phenytoin in the treatment of cocaine abuse. SUBJECTS AND METHODS: A 12-week, double-blind, placebo-controlled outpatient study of phenytoin in the treatment of cocaine abuse was conducted. Sixty cocaine-using subjects were randomly assigned to a daily fixed dose of 300 mg phenytoin or placebo. Forty-four subjects initiated treatment and returned for weekly visits. Primary measures of outcome included weekly quantitative and qualitative cocaine urinalysis, self-reported cocaine use, global functioning and improvement, craving intensity, and subject retention. RESULTS: Cocaine use, as measured both by weekly urinalysis and self-report, was significantly lower in the phenytoin group. The phenytoin group was also rated as significantly less impaired and more improved than the placebo group. Craving intensity was lower in the phenytoin group, but the difference was not statistically significant. Among phenytoin subjects, serum phenytoin levels above 6.0 micrograms/ml were associated with lower rates of positive cocaine urine specimens and longer cocaine-free periods. No differences were observed between groups in study retention. CONCLUSIONS: These findings suggest that phenytoin may be useful in the treatment of cocaine abuse. Further studies are needed to replicate these findings. PMID- 8612393 TI - Methylnaltrexone prevents morphine-induced delay in oral-cecal transit time without affecting analgesia: a double-blind randomized placebo-controlled trial. AB - Methylnaltrexone is a quaternary opioid antagonist with limited ability to cross the blood-brain barrier and the potential to antagonize the peripherally mediated effects of opioids. The effectiveness of methylnaltrexone in preventing morphine induced changes in gastrointestinal motility and transit without affecting analgesia was evaluated in humans. Twelve healthy volunteers were given intravenous placebo, placebo plus 0.05 mg/kg morphine, or 0.45 mg/kg methylnaltrexone plus 0.05 mg/kg morphine. Oral-cecal transit time was assessed by the pulmonary hydrogen measurement technique, and analgesia was measured with use of the cold-pressor test. Morphine significantly increased oral-cecal transit time from 104.6 +/- 31.1 minutes (mean +/- SD) to 163.3 +/- 39.8 minutes (p < 0.01). Methylnaltrexone prevented 97% of morphine-induced increase in oral-cecal transit time (106.3 +/- 39.8 minutes; not significant compared with baseline; p < 0.01 compared with morphine alone). Methylnaltrexone did not affect the analgesic effect of morphine on both pain intensity and pain bothersomeness ratings. At a higher dose of morphine (0.1 mg/kg), our preliminary results indicated that 0.45 mg/kg methylnaltrexone also prevented the morphine-induced delay in oral-cecal transit time, with no effect on analgesia. Methylnaltrexone may be a useful adjunct to opioids for the relief of opioid-induced constipation. PMID- 8612394 TI - Ineffectiveness of angiotensin converting enzyme inhibition (enalapril) on overt and silent myocardial ischemia in vasospastic angina and comparison with verapamil. PMID- 8612395 TI - Analytical information is mandatory for pharmacokinetic papers. PMID- 8612396 TI - Bioassay of steroids. PMID- 8612397 TI - Cytochrome P450 and atherosclerosis. PMID- 8612398 TI - Fuzzy sets for feature identification in biomedical signals with self-assessment of reliability: an adaptable algorithm modeling human procedure in BAEP analysis. AB - The analysis of biomedical signals has mostly been restricted to traditional signal-processing methods. This article proposes a different approach, applied to the evaluation of brainstem auditory evoked potentials. An automatic peak identification method is described which uses criteria applied by human evaluators in visual analysis. These criteria are defined as fuzzy sets and are combined using fuzzy operations, thus reflecting the weighting of different facts by humans. Membership values are interpreted as degrees of satisfaction which indicate the degree to which a sample satisfies a given criterion. The system judges its own performance in terms of degrees of reliability. Tests on a large set of clinical data showed high performance on good and average-quality curves. A substantial drawback was the assignment of too many peaks in poor potentials. The approaches presented here can easily be applied to similar one-dimensional (and higher) signal analysis tasks. PMID- 8612399 TI - Detection of spontaneous postsynaptic potentials. AB - The amplitude and time course of postsynaptic potentials (PSPs) recorded by intracellular techniques contain information that allow different synaptic events to be detected. In the present paper an algorithm to detect spontaneous PSPs is described. The algorithm is based on computation of approximations of first and second derivatives of the signals. The method was tested on both computer simulated potentials and on experimental data recorded from dissociated mouse spinal cord neurons in tissue culture. The receiver operating characteristics of the detection algorithm were computed. This method can be applied to investigations of dynamic changes in the activity of neural networks. PMID- 8612401 TI - Experiments in coding clinical information: an analysis of clinicians using a computerized coding tool. AB - We present data from a controlled experiment with computerized browsing and encoding tool. Eighteen practicing clinicians were asked to extract medical concepts from narrative exercise cases using two approaches--traditional and computer-assisted use of ICD-9. Our results indicate that completeness of coding can be improved by up to 55% using a computerized coding tool; enforcing mandatory as opposed to optional modifier codes results in lower rates of incomplete coding (0 vs 55%), higher rates of correct coding (41 to 92%), and no change in the number of incorrect codings; and manual coding takes 100% longer than coding with the help of the computerized coding tool. Furthermore, clinicians need 59% more time for processing the whole set of codes than is suggested by the sum of individual codes. We conclude that use of a computerized coding tool can save time and result in higher quality coding However, de facto time spent on coding may be underestimated when looking at individual coding times instead of looking at the whole task of processing a clinical scenario. PMID- 8612402 TI - Acute respiratory distress syndrome: consensus, definitions, and future directions. PMID- 8612400 TI - Digital angiographic impulse response analysis of myocardial perfusion: influence of heart rate and blood pressure changes on microcirculatory transit time measurement in the normal canine coronary circulation. AB - The study describes the effect of acute changes in aortic blood pressure and heart rate in the intact normal canine coronary circulation on digital angiographic measurements of the mean transit time of the microcirculation compartment and on direct flow meter measurement of resting and maximal hyperemic coronary blood flow. The mean transit time of the radiographic contrast material through the normal coronary circulation was calculated in 20 dogs by the impulse response analysis from serial digital coronary angiograms under systematically changed blood pressure and heart rate conditions. The mean aortic blood pressure (MAP) was controlled by the inflation of a balloon in the descending aorta (70 150 mmHg). The heart rate was altered by atrial pacing (90, 120, and 150/min). The mean transit times (T mu) of contrast material across the myocardial microcirculation which had been recently shown to correlate with coronary flow reserve (CFR) in stenosed arteries in the canine model were calculated under resting and hyperemic flow conditions from 428 selective coronary angiograms after a hand-injected contrast bolus, T mu was found to be heart rate independent. T mu-1 was linearly correlated with MAP (r = 0.7). There was no difference in T mu under resting and hyperemic flow conditions at comparable MAP. The corresponding distribution volume (V mu) of the microcirculation was calculated as the product of the mean transit time T mu and the measured coronary blood flow. Under resting flow conditions, V mu decreased with rising MAP. Furthermore, V mu increased with rising heart rate, V mu was constant during blood pressure or heart rate changes under hyperemic flow conditions. The data suggest that CFR whether by direct measurements of flow or by radiographic assessment of contrast material kinetics should be standardized with respect to hemodynamic conditions of heart rate and MAP. PMID- 8612403 TI - Fundamental critical care support: another merit badge or more? PMID- 8612404 TI - Regional blood flow alterations after bovine fumaryl beta beta-crosslinked hemoglobin transfusion and nitric oxide synthase inhibition. AB - OBJECTIVES: a) To determine whether isovolemic exchange transfusion with cell free, bovine fumaryl beta beta-crosslinked hemoglobin results in a different pattern of regional blood flow distribution than transfusion with a poor oxygen carrying, colloidal solution. b) Because of potential nitric oxide scavenging by plasma-based hemoglobin, to determine whether blood flow differences are reduced after nitric oxide synthase inhibition. DESIGN: A prospective, randomized design with repeated blood flow measurements within groups. SETTING: Experimental physiology laboratory in a university medical center. SUBJECTS: Pentobarbital anesthetized female cats. INTERVENTIONS: Three groups of eight cats were studied: a) a control group with no transfusion (hematocrit of 32%); b) an anemia group in which exchange transfusion with an albumin-containing solution reduced hematocrit to 18% over a 40- to 50-min period; and c) a group in which cell-free hemoglobin was exchanged transfused to reduce hematocrit to 18%, without a proportional reduction in oxygen-carrying capacity. Bovine hemoglobin was covalently crosslinked intramolecularly between the 81-lysine residues on the beta-subunits to stabilize the tetramer. Regional blood flow was measured by the radiolabeled microsphere technique before transfusion and at 10, 100, and 180 mins from the start of transfusion. At 190 mins, N omega-nitro-L-arginine methyl ester (L-NAME; 10mg/kg) was infused to inhibit nitric oxide synthase and blood flow was measured 30 mins later. MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure was unchanged in the control and albumin-transfused groups. However, mean arterial pressure increased rapidly in the hemoglobin-transfused group. With hemoglobin transfusion, there were marked reductions in blood flow to the intestines, kidneys and adrenal glands. Administration of L-NAME after hemoglobin transfusion failed to increase arterial pressure or cause further reductions in intestinal, renal, or adrenal blood flow. Administration of L-NAME to the control and albumin transfused groups increased arterial pressure and reduced intestinal, renal, and adrenal blood flows to values attained with hemoglobin transfusion. In contrast, in skeletal muscle and left ventricle, blood flow rates increased in the albumin transfused group and were greater than those values found in the control group and hemoglobin-transfused group. The greater flow in the albumin-transfused group persisted after L-NAME administration. There was no difference in renal sodium, potassium, or osmolar excretion, or in urine flow between groups. CONCLUSIONS: Transfusion with cell-free, bovine crosslinked hemoglobin in cats can selective reductions in blood flow in the intestines, kidneys, and adrenal glands without evidence of renal dysfunction by a mechanism consistent with nitric oxide scavenging. In skeletal and cardiac muscle, the increase in blood flow persisted after nitric oxide inhibition in the albumin group relative to the hemoglobin transfused group at equivalent hematocrit values. This finding is consistent with compensatory vasoconstriction with hemoglobin transfusion due to improved oxygenation by this oxygen carrier. PMID- 8612405 TI - Pyruvate dehydrogenase inactivity is not responsible for sepsis-induced insulin resistance. AB - OBJECTIVE: To determine whether activation of pyruvate dehydrogenase with dichloroacetate can reverse sepsis-induced insulin resistance in humans or rats. DESIGN: Prospective, controlled study. SETTING: Intensive care unit (ICU) and laboratory at a university medical center. SUBJECTS: Nine patients were admitted to the ICU with Gram-negative sepsis, confirmed by cultures. In addition, chronically instrumented, Sprague-Dawley rats, either controls or with live Escherichia coli-induced sepsis. INTERVENTIONS: Hyperinsulinemic euglycemic clamp, with or without coadministration of dichloroacetate. MEASUREMENTS AND MAIN RESULTS: In humans, a primed, constant infusion of [6,6-2H2]glucose was used to determine endogenous glucose production and whole-body glucose disposal. Septic humans exhibited impaired maximal insulin-stimulated glucose utilization (39.5 +/ 2.7 mumol/kg/min), despite complete suppression of endogenous glucose production. In rats, a primed, constant infusion of [3-3H]glucose was used to determine endogenous glucose production and whole-body glucose disposal. Tissue glucose uptake in vivo was determined by [14C]-2-deoxyglucose uptake. Maximal, whole-body, insulin-stimulated glucose utilization was 205 +/- 11 and 146 +/- 9 mumol/kg/min in control and septic rats, respectively. The defect was specific to skeletal muscle and heart. Stimulation of pyruvate dehydrogenase with dichloroacetate caused a 50% decrease in plasma lactate concentration but failed to improve whole-body insulin-stimulated glucose utilization in either the septic human or rat. Dichloroacetate reversed the impairment of insulin-stimulated myocardial glucose uptake in septic rats, but did not influence skeletal muscle glucose uptake either under basal conditions or during insulin stimulation. CONCLUSIONS: Activation of pyruvate dehydrogenase with dichloroacetate does not ameliorate the impairment of whole-body, insulin-stimulated glucose uptake in septic humans or rats, or reverse the specific defect in insulin-mediated skeletal muscle glucose uptake by septic rats. Therefore, the decreased pyruvate dehydrogenase activity associated with sepsis does not appear to mediate sepsis induced insulin resistance during insulin-stimulated glucose uptake at either the whole-body or tissue level. PMID- 8612406 TI - A descriptive study of skeletal muscle metabolism in critically ill patients: free amino acids, energy-rich phosphates, protein, nucleic acids, fat, water, and electrolytes. AB - OBJECTIVE: To characterize biochemical changes in skeletal muscle in critically ill patients. DESIGN: Survey of critically ill patients. SETTING: Intensive care unit (ICU) at a university hospital. PATIENTS: Critically ill patients (n = 20) subjected to trauma, surgical complications, and/or bacteremia who were treated in the ICU and showed no risk of bleeding complications were included. Reference groups of metabolically healthy volunteers and patients served as the control/reference groups. INTERVENTIONS: Percutaneous muscle biopsy was obtained from both patients and healthy volunteers. MEASUREMENTS AND MAIN RESULTS: Total free amino acids in skeletal muscle decreased 59% (p < .001) and skeletal muscle glutamine concentration decreased 72% (p < .001) in the critically ill patients. Basic amino acids decreased 49% (p < .001). Branch-chain amino acids increased 39% (p < .01), and aromatic amino acids increased 88% (p < .001) in the patients. Adenosine triphosphate (ATP) was reduced by 12% (p < .01). Total creatine concentration increased by 26% (p < .001) due to an 80% increase in free creatine (p < .001). The phosphorylated creatine fraction of total creatine decreased 22% (p < .001) in the patients. Alkali-soluble protein/DNA decreased 24% (p < .01) and fat free solid/DNA decreased 21% (P <.01) in patients sampled on or after ICU day 5 compared with the reference group. Muscle water increased 10% due to a doubling of the extracellular water fraction. CONCLUSIONS: Although critically ill patients are a very heterogeneous group from a clinical point of view, there is a remarkable homogeneity in many of the biochemical parameters regardless of the severity of illness and the length of the ICU admission. The three most consistent differences were the skeletal muscle low glutamine concentration, the decrease in protein content, and the increase in extracellular water in the patients. PMID- 8612407 TI - Low lipid concentrations in critical illness: implications for preventing and treating endotoxemia. AB - OBJECTIVES: To determine the prevalence and clinical significance of hypolipidemia found in critically ill patients, and whether the addition of a reconstituted lipoprotein preparation could inhibit the generation of tumor necrosis factor-alpha (TNF-alpha) in acute-phase blood taken from these patients. SETTING: Surgical intensive care unit (ICU) of a large urban university hospital. DESIGN: Prospective case series. PATIENTS: A total of 32 patients with a variety of critical illnesses had lipid and lipoprotein concentrations determined. Six patients and six age- and gender-matched control subjects had whole blood in vitro studies of the effect of lipoprotein on lipopolysaccharide mediated TNF alpha production. INTERVENTIONS: Blood samples were drawn on admission to the ICU and over a subsequent 8-day period. MEASUREMENTS AND MAIN RESULTS: Mean serum lipid and lipoprotein values obtained from patients within 24 hrs of transfer to the surgical ICU were extremely low: mean total cholesterol was 117 mg/dL (3.03 mmol/L), low-density lipoprotein cholesterol 71 mg/dL (1.84 mmol/L), and high density lipoprotein cholesterol 25 mg/dL (0.65 mmol/L). Only the mean triglyceride concentration of 105 mg/dL (1.19 mmol/L), and the mean lipoprotein(a) concentration of 25 mg/dL (0.25 g/L) were within the normal range. During the first 8 days following surgical ICU admission, there were trends toward increasing lipid and lipoprotein concentrations that were significant for triglycerides and apolipoprotein B. Survival did not correlate with the lipid or lipoprotein concentrations, but patients with infections had significantly lower (p = .008) high-density lipoprotein cholesterol concentrations compared with noninfected patients. Lipopolysaccharide-stimulated production of TNF-alpha in patient and control blood samples was completely suppressed by the addition of 2 mg/mL of a reconstituted high-density lipoprotein preparation. CONCLUSIONS: Patients who are critically ill from a variety of causes have extremely low cholesterol and lipoprotein concentrations. Correction of the hypolipidemia by a reconstituted high-density lipoprotein preparation offers a new strategy for the prevention and treatment of endotoxemia. PMID- 8612408 TI - Thyrotropin-releasing hormone in critical illness: from a dopamine-dependent test to a strategy for increasing low serum triiodothyronine, prolactin, and growth hormone concentrations. AB - OBJECTIVE: The aim of this study was to examine the effect of dopamine infusion on the thyrotropin (TSH), thyroid hormone, prolactin, and growth hormone responses to thyrotropin-releasing hormone (TRH) in critically ill patients. DESIGN: Prospective, randomized, controlled, open-labeled clinical study. SETTING: The intensive care unit, University Hospital Gasthuisberg, Leuven, over a 1-month period. PATIENTS AND INTERVENTIONS: In 15 critically ill patients receiving dopamine treatment (5 micrograms/kg/min) for a mean of 43.3 +/- 1.2 (SEM) hrs after trauma or cardiac surgery, we studied the TSH, thyroid hormone, prolactin, and growth hormone responses to the administration of two consecutive intravenous TRH boluses of 200 micrograms, with a 6-hr interval. The dopamine infusion was continued in the control group and discontinued in the study group. Serum concentrations of TSH, prolactin, and growth hormone were measured before and 20, 40, 60, and 120 mins after TRH administration. Serum concentrations of thyroxine (T4), triiodothyronine (T3), reverse T3, and thyroid hormone binding globulin were determined before and 120 mins after each TRH injection. MEASUREMENTS AND MAIN RESULTS: There was a > 100-fold interindividual variation in the baseline TSH concentration and in the TSH peak value after TRH administration. Two consecutive doses of TRH evoked a mean 16% increase in serum T4 concentration (p = .003) and a mean 47% increase in T3 (p = .001), whereas serum reverse T3 and thyroid hormone binding globulin values remain unaltered. Each of the TRH boluses increased serum growth hormone concentrations in the continued dopamine and discontinued dopamine groups, by a median of 60% (p = .001) and 68% (p = .001), respectively. Three hours after dopamine withdrawal, there was a three-fold increase of the peak TSH response (p = .001), a higher T3 response (p = .01), and a ten-fold increase of the peak prolactin value (p = .001) in response to TRH administration. CONCLUSIONS: The TSH response to TRH administration in critical illness presents a striking interindividual variation and dopamine dependent. Repeated TRH administration results in a repetitive increase of TSH, prolactin, growth hormone, T4, and T3, without increasing reverse T3. These observations point toward a potential for TRH as a strategy for reversing the euthyroid sick syndrome, growth hormone deficiency, and immune dysfunction associated with critical illness. PMID- 8612409 TI - Early prediction of outcome in score-identified, postcardiac surgical patients at high risk for sepsis, using soluble tumor necrosis factor receptor-p55 concentrations. AB - OBJECTIVE: To investigate the prognostic value of increased serum concentrations of soluble tumor necrosis factor (TNF) receptors in patients at high risk for sepsis. DESIGN: Prospective study. SETTING: Cardiac surgical intensive care unit in a University Hospital. PATIENTS: Those 27 of 870 consecutive postcardiac surgical patients who met a previously validated high-risk criterion for imminent sepsis (Acute Physiology and Chronic Health Evaluation II [APACHE II] score of > or = 24 on the first postoperative day [day 1]). In this population, systemic inflammatory response syndrome was present in 96% of the patients and the in hospital mortality rate was 30%. In addition, ten postcardiac surgical patients with an uncomplicated course (mortality rate 0%) were studied for comparison. INTERVENTIONS: Blood sampling for measurements of serum concentrations of TNF and soluble TNF receptors 55 kilodalton (TNF receptor-p55) and 75 kilodalton (TNF receptor-p75) on days 1, 2, 3, and 5. MEASUREMENTS AND MAIN RESULTS: Compared with the ten patients with an uncomplicated course (group A), the high-risk patients had significantly higher baseline (day 1) serum concentrations of soluble TNF receptor-p55 (9.2 vs. 4.2 ng/mL) and soluble TNF receptor-p75 (9.2 vs. 5.5 ng/mL). These high-risk patients could be further differentiated into two subgroups: one (B) with a prompt decrease in APACHE II score and a good prognosis (mortality rate 0%) and another (C) with a persisting high risk of sepsis and mortality rate (40%, p < .05). Although baseline APACHE II score was similar in both high-risk subgroups, soluble TNF receptor-p55 concentrations were significantly higher in subgroup C compared with subgroup B already at baseline (10.7 vs. 4.7 ng/mL). The receiver operating characteristic curve for subgroup classification by soluble TNF receptor-p55 was in a discriminating position with an area (0.773 +/- 0.096), confirming soluble TNF receptor-p55 as a predictor of mortality. TNF and soluble TNF receptor-p75 concentrations were less predictive at baseline. CONCLUSIONS: This study suggest that increased soluble TNF receptor p55 concentrations in the serum of postcardiac surgical patients allow earlier prognostication of subsequent hospital course than APACHE II scores alone. This study further suggests that the combination of physiologic scores and cytokine receptor measurements could improve the predictive power of early postoperative risk stratification. PMID- 8612410 TI - Nonrespiratory predictor of mechanical ventilation dependency in intensive care unit patients. AB - OBJECTIVE: To determine the role of serum albumin concentration as a predictor of mechanical ventilation dependency. DESIGN: Prospective, observation trial. SETTING: Multidisciplinary intensive care unit (ICU) in a university hospital. PATIENTS: One hundred forty-five consecutive patients who required mechanical ventilation for > 72 hrs. INTERVENTIONS: Patients were classified into five different groups based on the cause of respiratory failure. The following parameters were recorded daily: serum albumin concentration; Acute Physiology and Chronic Health Evaluation II (APACHE II) score; and fluid balance. Using multiple regression, multiple logistic regression analysis, and the Anderson-Gill proportional hazards model, we determined the metabolic factors that could help predict weaning success. MEASUREMENTS AND MAIN RESULTS: The mean length of ICU stay was 12.3 +/- 1.0 days. The duration of mechanical ventilation dependency was 10.5 +/- 1.0 days. The initial mean serum albumin concentration was 25.2 +/- 0.6 g/L. The APACHE II score on the first day of ICU stay was 19.1 +/- 0.6. Although albumin concentration was significantly lower and the APACHE II score was significantly higher in ICU nonsurvivors than in ICU survivors, albumin concentration on ICU admission was not a predictor of the length of time spent receiving mechanical ventilation. The profile of albumin concentration changes was different between weaned and mechanical ventilation-dependent patients. At the time of weaning patients from the ventilator, the median albumin concentration was higher than in those patients who continued to be supported by mechanical ventilation. This effect of albumin could not be attributed to patient fluid balance or to the severity of illness since each factor had an independent influence in predicting weaning, using the Anderson-Gill proportional hazards models. CONCLUSIONS: Initial serum albumin concentration did not necessarily predict weaning success. However, when serum albumin concentration was assessed on a daily basis, its trend was important in determining the relative chance of being successfully weaned from the ventilator. This finding suggests that albumin may be an index of the metabolic status of the patient, which could be important in determining the weanability of the patients who are mechanically ventilated for prolonged periods of time. PMID- 8612411 TI - Capnography facilitates tight control of ventilation during transport. AB - OBJECTIVE: We tested the hypothesis that Paco2 would be more tightly controlled if end-tidal CO2 monitoring was used during hand ventilation for transport of intubated patients. DESIGN: Randomized, prospective analysis of the no-monitor and monitor-blind groups (the monitor was on the bed during transport but only the investigator was aware of the end-tidal CO2 values). Nonrandomized, prospective analysis of the monitor group (ventilation controlled using end-tidal CO2 value from monitor). SETTING: University hospital operating room and intensive care unit (ICU). PATIENTS: Fifty intubated patients who were transported from the operating room to the ICU or from the ICU to the neuroradiology suite were assigned randomly to one of two groups: a) no-monitor group (n = 25); and b) monitor-blind group (n = 25). An additional group (monitor group, n = 10) was subsequently added to the study. INTERVENTIONS: Capnography was instituted in all patients in a blocked fashion. MEASUREMENTS AND MAIN RESULTS: Arterial blood gases and end-tidal CO2 values were measured before and after transport. When comparing overall group data, pre- and post-Paco2 values were similar: monitor 39 +/- 2 vs. 41 +/- 2 torr (5.2 +/- 0.3 vs. 5.5 +/- 0.3 no monitor 39 +/- 1 vs. 37 +/- torr (5.2 +/- 0.1 vs. 5.0 +/- 0.1 kPa). However, when comparing Paco2 values for individual patients, we found that there was significantly greater variability for Paco2 after transport when end-tidal CO2 was not used for control of ventilation during transport. CONCLUSIONS: These data do not support routine monitoring of end-tidal CO2 during short transport times in adult patients requiring mechanical ventilation. However, the monitor may prevent morbidity in patients requiring tight control of Paco2. PMID- 8612412 TI - Modulating effects of propofol on metabolic and cardiopulmonary responses to stressful intensive care unit procedures. AB - OBJECTIVE: Patients in the intensive care unit (ICU) undergo acute increases in metabolic and cardiopulmonary demands in response to routine care interventions, such as chest physical therapy. This study examined whether the short-acting drug, propofol, could blunt the responses to chest physical therapy. DESIGN: Prospective, randomized, crossover (placebo vs. drug) study. SETTING: University hospital surgical ICU. PATIENTS: Postoperative ICU patients being ventilated in the synchronized intermittent mandatory ventilation mode. INTERVENTIONS: Two groups of 16 patients were studied. Each patient received two successive sessions of chest physical therapy. In random fashion, one was preceded by the administration of placebo and the other by an intravenous bolus of propofol (0.75 mg/kg in one group and 0.35 mg/kg in the other group). Each session was preceded and followed by a period of rest. MEASUREMENTS AND MAIN RESULTS: The increases in oxygen uptake, CO2 elimination, oxygen delivery, heart rate, and systolic blood pressure associated with chest physical therapy were attenuated with the low dose and suppressed with the high dose of propofol. The Paco2 concentration was slightly increased during both placebo and drug administration. CONCLUSIONS: Propofol, in the doses administered in this study, significantly reduced the hemodynamic and metabolic stresses caused by chest physical therapy. PMID- 8612413 TI - Interhospital transfers: decision-making in critical care areas. AB - OBJECTIVES: To evaluate the training of clinical staff in the use of interhospital transfer guidelines and to examine the underlying decision-making behavior in organizing patient transfers between hospitals. DESIGN: Prospective assessment of clinical scenarios, given before (time 1), immediately after (time 2), and 3 months after (time 3) a program informing clinical staff about the use of interhospital transfer guidelines. SETTING: Three emergency departments and one intensive care unit at three hospitals and a medical retrieval service in Sydney, Australia. SUBJECTS: Physicians, nurses, and a paramedic working in critical care areas and at a medical retrieval service. MEASUREMENTS AND MAIN RESULTS: A questionnaire containing clinical scenarios was administered to clinical staff. There was a significant difference in mean scores for selecting the appropriate escort levels across time (F2,78 = 24.2; p < .01) and for participant's experience with interhospital transfer (F2,39 = 4.63; p = .02). Significant improvement in mean scores occurred between time 1 (7.55 +/- 1.84 and time 2 (9.48 +/- 1.47) (t41 = -6.21; p < .01). The improvement in selecting appropriate escorts was maintained at time 3 (mean score 9.86 +/- 2.01). The error rate for inappropriate assignment of low levels of escorts decreased from 35% (time 1) to 10% (time 2) and 14% (time 3). Using conjoint analysis, there were large variations in the decision-making behaviour between each time period. The relative importance of each factor in influencing the decision to organize an escort at time 3 were as follows: treatment (43%); physiology (29%); patient age (24%); and diagnosis (4%). The decision-making model observed at time 3 had a high predictive value (87%) as compared with the model at time 1 (48%). CONCLUSION: Clinical staff can make informed and appropriate decisions by using standardized guidelines when organizing interhospital transfers. PMID- 8612414 TI - Nociceptive somatic nerve stimulation and skeletal muscle injury modify systemic hemodynamics and oxygen transport and utilization after resuscitation from hemorrhage. AB - OBJECTIVE: To examine if either nociceptive somatic nerve stimulation or skeletal muscle injury modified systemic hemodynamics and oxygen transport and utilization after resuscitation from hemorrhage in anesthetized pigs. DESIGN: Prospective, randomized, controlled laboratory study. SETTING: Animal laboratory. SUBJECTS: Twenty isoflurane-anesthetized and mechanically ventilated large white pigs. INTERVENTIONS: Three groups of animals were instrumented with femoral arterial and thermodilution pulmonary artery catheters. One group of animals had bilateral brachial nerve electric stimulation before hemorrhage (brachial nerve stimulation + hemorrhage, n = 7). The second group of animals had bilateral hindlimbs skeletal muscle injury induced by firing a captive-bolt handgun with standard charges before hemorrhage (skeletal muscle injury + hemorrhage, n = 6). The third group had neither insult before hemorrhage (control, n = 7). Controlled bleeding was initiated to reduce the cardiac index and systemic oxygen delivery (Do2) by 50% in all animals. Animals were then left for 30 mins before resuscitation. All animals were resuscitated with 4.5% human serum albumin at 45 mL/kg and observed for 2 hrs. MEASUREMENTS AND MAIN RESULTS: Plasma volume, systemic hemodynamics, and oxygen transport variables were measured and calculated after resuscitation. Similar increases of plasma volume and supranormal cardiac index were observed in all groups immediately after resuscitation. The branchial nerve stimulation and hemorrhage group maintained higher heart rate, cardiac index, Do2, and oxygen consumption (Vo2) than the hemorrhage group. In contrast, the skeletal muscle injury and hemorrhage group had lower systemic mean arterial pressure and vascular resistance, and a tendency for decrease in Vo2, than the hemorrhage group, although heart rate, cardiac index, and Do2 were similar in both groups. Hemorrhage increased the arterial plasma lactate concentration, which was later normalized in all groups 60 mins after resuscitation. CONCLUSIONS: Neither nociceptive brachial nerve stimulation nor skeletal muscle injury attenuated the increase in plasma volume, cardiac index, or the repayment of systemic oxygen debt after resuscitation from hemorrhage. Brachial nerve stimulation was associated with augmented cardiac index, systemic Do2, and increased Vo2 requirements related to increased sympathetic nervous system activation. Skeletal muscle injury produced early systemic arterial hypotension and vasodilation, and a decrease in Vo2 that was suggestive of pathologic supply dependency on systemic Do2. PMID- 8612415 TI - Effects of intraluminal and extracorporeal inferior vena caval bypass on canine hemodynamics. AB - OBJECTIVE: To compare inferior vena cava-right atrial extracorporeal bypass with intraluminal atriocaval shunting during hepatic vascular isolation. DESIGN: Prospective, randomized, controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult mongrel dogs (n = 5) weighing 20 to 27 kg. INTERVENTIONS: Anesthetized dogs underwent laparotomy and sternotomy for vascular isolation. For atriocaval shunting, 20- and 24-Fr intraluminal shunts were inserted into the inferior vena cava via right atriotomy. For extracorporeal bypass, each animal underwent inferior vena cava, portal vein, and right atrial cannulation for venovenous bypass, utilizing a centrifugal pump. Hemodynamic data were recorded at baseline and at intervals after caval occlusion, Pringle maneuver, and caval occlusion with Pringle maneuver. MEASUREMENTS AND MAIN RESULTS: Isolated Pringle maneuver and caval occlusion with Pringle maneuver produced significant reductions in mean arterial pressure (MAP) and cardiac output, irrespective of pulmonary artery occlusion pressure. Extracorporeal bypass, including both caval and portal venous return, produced significant increases in MAP and cardiac output during caval occlusion with Pringle maneuver, while atriocaval shunting and extracorporeal bypass without portal venous return did not improve MAP or cardiac output. CONCLUSION: Venovenous extracorporeal bypass with portal return, acting as a right ventricular assist device, is superior to intraluminal atriocaval shunting in maintaining hemodynamic stability during hepatic vascular isolation. PMID- 8612416 TI - Characteristics of the intestinal epithelial barrier during dietary manipulation and glucocorticoid stress. AB - OBJECTIVES: a) To determine the significance of stress-induced alterations in intestinal permeability by measuring the transmucosal flux of formyl-methionyl leucyl-phenylalanine (f-MLP), a ubiquitous neutrophilic chemoattractant present in the human and rodent colon; and b) to determine whether stress and/or diet influence(s) bacterial adherence-induced changes in epithelial permeability by affecting the production of secretory immunoglobulin A (IgA), the main immune mechanism preventing bacterial adherence. DESIGN: Prospective, randomized, controlled study. SETTING: University animal research laboratory. SUBJECTS: Female Fischer rats. INTERVENTIONS: Rats were randomly assigned to four groups of seven animals each. Groups of animals were assigned to receive saline or dexamethasone (0.8 mg/kg ip) and were either starved (5% dextrose in water ad libitum) or fed (water and rat chow) for 48 hrs. MEASUREMENTS AND MAIN RESULTS: Mucosal barrier function was evaluated by measuring secretory IgA, bacterial adherence to the intestinal mucosa, and transepithelial electrical resistance, a measure of tight junction permeability. The f-MLP permeation across the mucosa was also determined in segments with significant permeability changes. Results indicate that starvation in dexamethasone-treated rats significantly impairs secretory IgA, promotes bacterial adherence to the mucosa, and results in increased intestinal permeability to f-MLP. These effects are significantly attenuated by the feeding of rat chow. CONCLUSIONS: Alterations in intestinal barrier function are characterized by depressed IgA, bacterial adherence to the intestinal mucosa, and permeation of clinically relevant proinflammatory luminal macromolecules (f-MLP). Enteral stimulation with foodstuffs is a necessary protective measure to prevent altered epithelial barrier function during glucocorticoid stress. PMID- 8612417 TI - Lidocaine attenuates the hypotensive and inflammatory responses to endotoxemia in rabbits. AB - OBJECTIVE: To assess the effects of lidocaine on the hemodynamic and inflammatory responses to Escherichia coli endotoxemia in rabbits. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University laboratory. SUBJECTS: Twenty-seven female Japanese rabbits, anesthetized with urethane and ventilated mechanically. INTERVENTIONS: Animals were randomly assigned to one of three groups: a) endotoxemic control group (n = 9), receiving intravenous Escherichia coli endotoxin (0.5 mg/kg bolus) via the mesenteric vein; b) laparotomy control group (n = 9), treated identically to the endotoxemic control group, except for substitution of 0.9% saline for endotoxin; and c) lidocaine treated group (n = 9), treated identically to the endotoxemic controls and additionally, intravenous lidocaine (3 mg/kg bolus, followed by infusion at 2 mg/kg/hr) was administered immediately after endotoxin MEASUREMENTS AND MAIN RESULTS: We compared hemodynamics, blood gases, and microscopic findings of lung tissue obtained at necropsy in each group. Laparotomy alone had a minimal effect on the parameters and findings. Endotoxin injection decreased mean systolic arterial pressure from 135 +/- 6 (SD) to 95 +/- 25 mm Hg (p < .05) and increased the mean base deficit from -1.2 +/- 1.8 to -14.4 +/- 4.2 mmol/L (p < .05), and caused the infiltration of neutrophils into the lungs. Lidocaine administration abolished the hypotension and attenuated the increase the base deficit to -9.5 +/ 2.1 mmol/L (p < .05) and the cellular infiltration in comparison with endotoxemic controls. CONCLUSIONS: Lidocaine attenuated the hemodynamic and inflammatory responses to endotoxemia in rabbits. Findings suggest that lidocaine administration may prevent the development of hypotension and metabolic acidosis during endotoxemia. PMID- 8612418 TI - Effect of jet ventilation on heart failure: decreased afterload but negative response in left ventricular end-systolic pressure-volume function. AB - OBJECTIVE: To examine the mechanism of cardiac assist with systolic jet ventilation, specifically effects on loading conditions and left ventricular pressure-volume function. Both systolic and diastolic jet ventilation were compared in the absence and presence of heart failure. DESIGN: Prospective, two factor, repeated-measures study. SETTING: Animal laboratory. SUBJECTS: Ten anesthetized, closed-chest dogs. INTERVENTIONS: The measurement protocol consisted of two phases: a) apnea, randomized jet ventilation (systole- and diastole-synchronized); b) postjet ventilation apnea, before and after heart failure, induced with a propranolol-imipramine-plasma expansion treatment. MEASUREMENT AND MAIN RESULTS: Systolic and diastolic jet ventilation was associated with mean airway pressures of approximately 7 mm Hg and intrapleural pressures of approximately 3 mm Hg in both heart conditions. In normal hearts, jet ventilation (either mode) decreased transmural left ventricular end-diastolic pressure by 40% to 60% (p < .05), left ventricular end-diastolic volume 25 +/- 8%, and stroke volume by 28% to 30%. Heart failure was associated with decreases (41 +/- 6%) in end-systolic pressure-volume function (i.e., pressure change/volume change or elastance), transmural left ventricular end-systolic pressure (22 +/- 3%), and stroke volume (16 +/- 4%), and increased transmural left ventricular end-diastolic pressure (139 +/- 6%). Application of jet ventilation (either mode) during heart failure did not affect stroke volume but significantly (p < .05) attenuated transmural left ventricular end-diastolic pressure by 30% to 40%, left ventricular end-diastolic volumes by 33 +/- 9%, and transmural left ventricular end-systolic pressure by 11% to 19% (p < .05). After jet ventilation, left ventricular elastance was decreased 36 +/- 8% in normal hearts and 35 +/- 11% in failing hearts. Stroke volume, however, returned to baseline levels because of increases in transmural left ventricular end-diastolic pressure in both heart conditions, and also in failing hearts, because transmural left ventricular end-systolic pressure remained decreased approximately 30% (p < .05). CONCLUSIONS: Jet ventilation did not decrease stroke volume in failing hearts because of the afterload-reducing benefit (decreased transmural left ventricular end-systolic pressure) of increased intrapleural pressure in dilated ventricles. Moreover, jet ventilation did not have positive effects on myocardial function and had negative effects on left ventricular elastance in the postjet ventilation period in both normal and failing hearts. Cardiac assist by jet ventilation was not cycle specific, suggesting no selective benefit of jet ventilation over conventional positive-pressure ventilation during heart failure. These studies demonstrate a negative inotropy associated with jet ventilation that, during heart failure, may compromise the general benefit of positive pressure-mediated increases in intrapleural pressure. PMID- 8612419 TI - Effect of conventional mechanical ventilation and jet ventilation on airway pressure in dogs and plastic models with tracheal stenosis. AB - OBJECTIVE: To evaluate the effect of jet ventilation on tracheal stenosis in dogs and plastic models. DESIGN: Prospective, randomized trial in dogs, and multitrial tests in tracheal stenosis models. SETTING: Animal laboratory in a university setting. INTERVENTIONS: Tracheal stenosis was surgically created around the middle of the trachea. Conventional mechanical ventilation and jet ventilation were compared at the same value of Paco2 in dogs and at the same tidal volume in tracheal stenosis models. SUBJECTS: Twelve mongrel dogs and four types of plastic models with combinations of short or long stenosis and fluid or nonfluid stenosis. MEASUREMENTS AND MAIN RESULTS: Canine Studies. Mean peak peak airway pressure values at the distal and proximal portion of the stenosis, and the end expiratory pressure at the distal portion of the stenosis, were significantly higher during conventional mechanical ventilation than during jet ventilation. The mean values of arterial pressure, pulmonary arterial pressure, central venous pressure, and cardiac output did not change significantly between conventional mechanical ventilation and jet ventilation, except for the pulmonary artery occlusion pressure valve. Plastic Mold Studies. peak airway pressure and end expiratory airway pressure at the poststenotic trachea during jet ventilation with the model lung were significantly lower than during conventional mechanical ventilation. The difference in peak airway pressure, and end-expiratory airway pressure values between jet ventilation and conventional mechanical ventilation increased more in short stenosis and nonfluid stenosis. CONCLUSIONS: The jet flow that struck the portion of the stenosed wall reversed direction, even during early expiration. Therefore, the expiration during jet ventilation was facilitated more by the reversed flow than by the expiration during conventional mechanical ventilation. This reversed flow may provide lower end-expiratory airway pressure at the poststenotic portion with jet ventilation than with conventional mechanical ventilation. We conclude that jet ventilation was a useful method of ventilation in cases with tracheal stenosis, especially nonfluid and short stenosis. PMID- 8612420 TI - Liver function and morphology after resuscitation from severe hemorrhagic shock with hemoglobin solutions or autologous blood. AB - OBJECTIVE: To test the effects of three hemoglobin solutions on liver function and hepatic morphology after resuscitation from severe hemorrhagic shock. DESIGN: Prospective study. SETTING: Laboratory. SUBJECTS: Thirty-three beagle dogs. INTERVENTION: Hemorrhagic shock was induced in anesthetized dogs by removal of blood at a rate of 2 mL/kg/min until systolic blood pressure (BP) reached 50 mm Hg. BP was maintained at this level 2 hrs by further withdrawing 5 to 10 mL aliquots whenever BP increased > 50 mm Hg. Resuscitation was then initiated with autologous whole blood (n = 7), 4% pyridoxalated-hemoglobin-polyoxyethylene conjugate (4% PHP [n = 6]), 8% pyridoxalated-hemoglobin-polyoxyethylene conjugate (8% PHP [n = 9], or 8% stroma-free hemoglobin (n = 7). Four dogs were managed identically but were not resuscitated. Gross necropsy and histologic examination of the liver were performed on all dogs after 7 days, or earlier if death occurred. MEASUREMENTS AND MAIN RESULTS: In vitro interferences of PHP and stroma free hemoglobin with liver function tests were determined and recommendations for interpretation of results from blood samples containing PHP and stroma-free hemoglobin were made. Blood was collected before, during, and after resuscitation from hemorrhagic shock. The dogs were then awakened and survivors were monitored daily with blood sampling until they were killed and necropsy was performed. After 7 days, the survival rate following hemorrhagic shock was 100% for whole blood and 4% PHP, 86% for stroma-free hemoglobin, and 33% for 8% PHP. Of the resuscitated dogs not surviving 7 days, all but one died within 27 hrs from coagulopathy. All dogs not resuscitated died within 1.75 hrs after 2 hrs of shock. Bilirubin, alkaline phosphatase, and lactic dehydrogenase concentrations could not be measured due to interferences of stroma-free hemoglobin and PHP. Aspartate (AST) and alanine (ALT) aminotransferase concentrations could be measured after dilution to overcome the interferences. Significant increases in AST and ALT values in all groups 24 hrs after resuscitation were attributed to hypoxic hepatocellular damage associated with the severity of the shock model rather than to the resuscitation fluid. Liver histology showed no changes attributed to toxic damage of hepatocytes in dogs resuscitated with stroma-free hemoglobin or PHP. However, changes, were less severe in dogs resuscitated with 4% PHP than in other groups. CONCLUSION: Morphologic studies at necropsy and liver function tests in dogs receiving hemoglobin solutions, compared with autologous blood, support the conclusion that the PHP and stroma-free hemoglobin solutions tested did not produce hepatic toxicity when used as resuscitation fluids in this model of severe shock. PMID- 8612422 TI - Mechanical ventilation and arterial blood gas measurements 24 hours postextracorporeal life support for survivors of pediatric respiratory failure. AB - OBJECTIVE: To summarize our institutional experience concerning mechanical ventilation support and blood gas measurements in the 24-hr period following extracorporeal life support (ECLS) for pediatric acute respiratory failure. DESIGN: Descriptive study. SETTING: A tertiary pediatric referral center. PATIENTS: Children aged 1 month to 18 yrs treated with ECLS for acute respiratory failure at University of Michigan Hospitals from November 1982 to June 1993. All patients aged 1 month to 18 yrs who received ECLS for acute respiratory failure were included. Patients who received ECLS primarily for cardiac support or who had a diagnosis of congenital gastrointestinal malformation (i.e. congenital diaphragmatic hernia) were excluded. INTERVENTIONS: ECLS for severe pediatric respiratory failure. MEASUREMENTS AND MAIN RESULTS: Forty-nine children were treated at our center with ECLS for acute respiratory failure 36 (73%) survived. Ventilator settings immediately after decannulation from ECLS for survivors were as follows: FIO2 0.53 +/- 0.18 (SD); intermittent mandatory ventilation (IMV) 29.6 +/- 1.18 breaths/min, positive end-expiratory pressure 5.3 +/- 1.6 cm H2O, mean airway pressure 12.6 +/- 2.9 cm H2O, and peak inspiratory pressure 31.7 +/- 5.5 cm H2O. Arterial blood gas measurements at decannulation were PaO2 89.4 +/- 30.9 torr (11.9 +/- 4.1 kPa), PaCO2 43.7 +/- 9.7 torr (5.8 +/- 1.3 kPa), and pH 7.39 +/- 0.07. Twenty-four hours after decannulation, ventilator settings and arterial blood gas measurements were as follows: FIO2 0.42 +/- 0.14, IMV 27.4 +/- 13.5 breaths/min, positive end-expiratory pressure 5.2 +/- 1.6 cm H2O, mean airway pressure 12.0 +/- 3.4 cm H2O, peak inspiratory pressure 31.1 +/- 6.5 cm H2O, PaO2 77.0 +/- 16.9 torr (10.3 +/- 2.3 kPa), PaCO2 44.9 +/- 8.4 torr (6.0 +/- 1.1 kPa), and pH 7.40 +/- 0.07. Variables associated with oxygenation status (P[A a]O2) and mean airway pressure (oxygenation index) improved during the immediate 24-hr period postbypass (p < .05). CONCLUSIONS: Successful decannulation from ECLS for > 24 hrs resulted in long-term survival in 97% (36/37) of children. Ventilator parameters and arterial blood gas measurements during the 24-hr period following bypass have been described for this population. Such conventional support may indicate safe levels of oxygen and mechanical ventilation pressures for the postbypass recovering lung. PMID- 8612421 TI - Prediction of outcome in patients with anoxic coma: a clinical and electrophysiologic study. AB - OBJECTIVE: To evaluate and compare the predictive powers of clinical examination, electroencephalography (EEG), and studies of short-latency somatosensory evoked potentials in determining the prognosis in anoxic coma. DESIGN: Prospective case series of patients in anoxic coma, whose prognoses were uncertain based on previously established clinical criteria. The clinical features, EEG, and somatosensory evoked potentials results were correlated with outcome. SETTING: A 40-bed intensive care unit in a university teaching hospital. PATIENTS: Thirty four consecutive patients admitted over a 2-yr period with anoxic coma as the principal diagnosis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Twenty seven (79%) patients never recovered consciousness and seven (21%) patients made a good recovery. One of six patients whose pupillary reflexes were present but whose other cranial nerve reflexes were absent on day 1 recovered, but none of the seven patients with these features still present on day 3 survived. None of the patients with motor responses of extension to painful stimuli or worse on days 1 or 3 recovered. The EEGs were classified into benign, uncertain, and malignant categories. The results of both EEG and somatosensory evoked potentials studies were strongly associated with outcome. With malignant EEG, the sensitivity was 74%, the specificity was 71%, and the positive predictive value was 90% [corrected] for the prediction of no recovery (death or persistent vegetative state). However, two patients with an initially malignant EEG eventually made a good recovery. The sensitivity for low amplitude or absent somatosensory evoked potentials for prediction of no recovery was 66%. There were no falsely pessimistic predictions with somatosensory evoked potentials, as all 18 patients with absent or low-amplitude responses had no recovery (specificity and positive predictive value were 100%). EEG and somatosensory evoked potentials studies were complementary to clinical examination in the determination of prognosis. Using a combined clinical and electrophysiologic approach, 63% of patients who had no recovery could be identified by day 3. Repeat EEG and somatosensory evoked potentials studies were of value in patients whose prognoses were uncertain, as their evolution invariably correlated with outcome. CONCLUSIONS: Based on the present data and a literature review, we propose that clinical examination combined with the results of EEG and somatosensory evoked potentials can be used to establish an early, definitive prognosis in a significant proportion of patients in anoxic coma. On day 3 or thereafter, patients with motor response of extension to pain or worse and malignant EEG, or those patients with flexor posturing or worse and bilaterally absent cortical somatosensory evoked potentials invariably have poor outcome. However, some patients with initially malignant EEG and normal somatosensory evoked potentials may recover and should be supported until their prognoses become more definitive. PMID- 8612423 TI - Association of pre mortem diagnosis and autopsy findings in pediatric intensive care unit versus emergency department versus ward patients. AB - OBJECTIVE: As part of the overall quality assurance program for the Department of Pediatrics, we determined whether there were differences in the rates of unexpected autopsy findings between pediatric intensive care unit (ICU), emergency department, and ward patients. DESIGN: Prospective, descriptive study. SETTING: Tertiary care children's hospital. PATIENTS: Pediatric deaths (n = 212). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Autopsies were obtained more frequently in emergency department patients (27/29 [93%]) compared with pediatric ICU (88/121 [73%] and ward (42/62 [68%]) patients (p = .03). The medical examiner's cases were more frequently from emergency department patients (22/27 [81%]) compared with pediatric ICU (39/88 [44%]) or ward (11/42 [26%]) patients (p < .001). We found unexpected autopsy findings in 19 (12%) of 157 patients. There were no unexpected findings from the medical examiner's cases. The most common unexpected findings were unidentified infections (n = 7 [three fungal, three viral, and one nonspecific]) and unrecognized cardiac malformations (n = 4). Unexpected findings occurred more frequently in pediatric ICU patients (16/88 [18%]) vs. emergency department (2/27 [7%]) or ward (1/42 [2%]) patients (p = .03). The occurrence rates of major unexpected findings (Class I and II) in pediatric ICU (7/79 [9%]), emergency department (2/27 [7%]), and ward (1/42 [2%]) patients were similar (p = .4). There were two Goldman's Class I unexpected findings in the pediatric ICU and emergency department patients, and one Goldman's Class I unexpected finding in the ward patients. CONCLUSIONS: Autopsies were performed more frequently in emergency department patients. Class I through IV unexpected findings occurred more frequently in pediatric ICU patients compared with emergency department or ward patients. Autopsy examinations are an especially valuable diagnostic tool for pediatric ICU patients and physicians. PMID- 8612424 TI - Adult respiratory distress syndrome: a systemic overview of incidence and risk factors. AB - OBJECTIVE: To determine the published incidence of adult respiratory distress syndrome (ARDS) as well as the clinical evidence supporting a casual association between ARDS and its major risk factors. DATA SOURCES: The National Library of Medicine MEDLINE database and the bibliographies of selected articles. STUDY SELECTION: Clinical studies were selected from the English literature, if they pertained to either the incidence of ARDS or its association with one or more commonly identified risk factors. DATA EXTRACTION: All relevant studies identified by the search were evaluated for strength of design, and risk factors were scored according to established criteria for the strength of causation. DATA SYNTHESIS: A total of 83 articles were considered relevant: six of incidence and 77 on risk factors. Only 49% of the 83 articles provided a definition of ARDS; a definition of risk factors was given in 64%, and 23% had no definition for either ARDS or risk factors. The published, population-based incidence of ARDS ranges from 1.5 to 5.3/10(5) population/yr. The strongest clinical evidence supporting a cause-effect relationship was identified for sepsis, aspiration, trauma, and multiple transfusions. The weakest clinical evidence was identified for disseminated intravascular coagulation. The following study types were represented by the 77 articles on risk factors: observational case-series (56%); cohorts (23%); case-controls (12%); nonrandomized clinical trials (5%); and randomized clinical trials (3%). Only a single study reported an odds ratio. CONCLUSIONS: The significant variation in the incidence of ARDS is attributed to differences in the type and strength of study designs, as well as definitions or ARDS. While a substantial body of evidence exists concerning a casual role of ARDS risk factors, such as sepsis, aspiration, and trauma, > 60% of clinical studies employed weak designs. The lack of reproducible definitions for ARDS or its potential risk factors in 49% of studies raises concerns about the validity of the conclusions of these studies regarding the association between ARDS and the supposed risk factors. PMID- 8612425 TI - Teaching critical care in Europe: analysis of a survey. AB - OBJECTIVE: To analyze the status of the teaching of critical care at the postgraduate and undergraduate levels in Europe. DATA SOURCES: Two types of questionnaires were sent in order to collect data. The first questionnaire was sent to the heads of the intensive care units (ICUs) of at least five of the major hospitals in each western country of the European Region of the World Health Organization and to the people responsible for specialist training in the Eastern countries. Countries in the former USSR and former Yugoslavia were excluded; 50 questionnaires (of 105) were returned. The second questionnaire was sent to the 374 medical schools in the European Region; 253 were returned although only 185 were selected for this article. RESULTS: Postgraduate and undergraduate training differ widely in terms of the content of, and disciplines in charge of, the teaching of critical care. Even countries of the World Health Organization's European Region, where critical care is a recognized clinical specialty, do not have an academic discipline of critical care. This lack of an established academic discipline of critical care contributes to the diversity in teaching critical care in medical schools in the countries we studied. Postgraduate training is more formally regulated, and objectives and guidelines have been established in many countries. The topics in medical schools that correspond to critical care medicine do not encompass a distinct body of knowledge and are distributed among more traditional disciplines, most frequently internal medicine, anesthesiology, and surgery. The critical care medicine experience afforded to undergraduates varies widely between and within countries. The repetition of critical care conditions for which patients receive critical care in several disciplines, as described by the responses, leads us to wonder whether these conditions are really included in the curriculum. CONCLUSIONS: The standardization of curriculum content on critical care medicine, the clear definition of competence (the combination of knowledge, attitudes, skills, and judgment necessary to practice) in medical schools, and better coordinated postgraduate training are needed to clarify an educational approach in the field. Practitioners of critical care medicine will have to participate actively on curriculum committees. The recognition of critical care medicine as a specialty or subspecialty and as an academic discipline will facilitate the achievement of a comprehensive critical care education program. PMID- 8612426 TI - Noninvasive mechanical ventilation in patients with acute respiratory failure. AB - OBJECTIVES: a) To describe the introduction of noninvasive means to provide positive-pressure ventilation in acute respiratory failure; b) to describe the physiologic response to noninvasive ventilation; c) to review the current published literature on using noninvasive ventilation in patients with acute hypercapnic and/or hypoxemic respiratory failure; d) to describe the technique of applying mask ventilation and current recommendations for using noninvasive ventilation and current recommendations for using noninvasive ventilation in patients with acute respiratory failure; and e) to discuss the advantages and disadvantages of noninvasive ventilation. DATA SOURCES: All relevant articles published in the English medical literature from 1988 through August 1994 were retrieved through a MEDLINE search, as well as from the author's experience. STUDY SELECTION: Studies were selected based on the use of positive-pressure mechanical ventilation delivered, using facial or nasal masks in various acute settings of respiratory failure. DATA EXTRACTION: The authors extracted all applicable data. DATA SYNTHESIS: Studies were analyzed according to the type of respiratory failure (hypercapnic vs. hypoxemic) and underlying conditions where noninvasive ventilation seemed to be a better alternative. The results were evaluated based on types of masks used and modes of ventilation. Outcome measures were compared based on studies that randomized patients with acute respiratory failure to receive noninvasive vs. conventional therapy. Complications of noninvasive ventilation, mainly local, were compared with those complications seen endotracheal intubation in acute respiratory failure patients. CONCLUSIONS: Noninvasive ventilation is a safe and effective means of ventilatory support for many patients with acute respiratory failure. Noninvasive ventilation is well tolerated, principally because it allows the patient to be in control and to continue verbal communication, and should be strongly considered in managing terminally ill patients with potentially reversible causes of respiratory failure. The duration of mechanical ventilation and its associated complications are significantly decreased in hypercapnic respiratory failure with noninvasive ventilation. PMID- 8612427 TI - Thermodilution cardiac output monitor. PMID- 8612428 TI - Neuromuscular blocking agents in the intensive care unit: a two-edged sword. PMID- 8612429 TI - Determining the expiratory time constant. PMID- 8612430 TI - Determining the expiratory time constant. PMID- 8612431 TI - Origin of endothelin in sepsis. PMID- 8612432 TI - Epidemiology and prevention of rabies. PMID- 8612433 TI - Specific induction of uncoiling in NORs of human acrocentric chromosomes by 5 azacytidine and 5-azadeoxicytidine. AB - The organization of rDNA-containing chromatin was analysed by transmission electron microscopy after treatment of cultured human lymphocytes with 5 azacytidine (ACR) or 5-azadeoxicytidine (AdCR). The number of observed acrocentric chromosomes with satellites was significantly increased after treatment with low doses of ACR or AdCR during the last 24 h of culture, whereas with exposures during the last 7 h the number remained normal. The results suggest that the incorporation of ACR and AdCR in the early period of the S-phase may have reverted the non-satellized to satellized chromosomes. The cytidine analogues may have become more visible during secondary constriction thus changing the NOR structure leading to an increased number of satellized chromosomes. PMID- 8612434 TI - Eastern white pine wood-drying condensate induced cytotoxicity and genotoxicity in human peripheral blood lymphocytes in vitro. AB - Condensate from eastern white pine, one of the commercially most important species of tree in the northeastern United States, was treated for potential cytotoxicity and genotoxicity in human peripheral blood lymphocytes in the absence of S-9 activation. Cytotoxicity was evaluated by mitotic index (MI) determination and proliferative rate index. Genotoxicity was measured by the chromosome aberration (CA) assay and sister chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were observed with laboratory-generated eastern white pine condensate. The MI data showed an inverse correlation between the MI and treatment dosage. A dose response curve was observed using the CA assay and also with the SCE analysis. The present findings thus corroborate the results from Ames testing and represent the only information currently available on the cytotoxic and genotoxic potential of these chemicals. PMID- 8612435 TI - Phospholipid and glutamic acid decarboxylase autoantibodies in diabetic neuropathy. AB - OBJECTIVE: To determine the prevalence and significance of phospholipid autoantibodies (PLAs) and glutamic acid decarboxylase (GAD) autoantibodies in the circulation of normal patients and diabetic patients with and without neuropathy. RESEARCH DESIGN AND METHODS: We measured PLAs in a total of 78 patients (a diabetic group with somatic or autonomic neuropathy [n = 40] another group without neuropathy [n = 38]), and GAD autoantibodies in a subset of 22 patients. RESULTS: PLAs are found in 2% of the general population. We found PLAs in 32% of the diabetic population without neuropathy, in 88% of those with neuropathy, in 55% of those with retinopathy, and in 25% of those with established nephropathy. The frequencies of immunoglobulins in the neuropathic group were: IgG = 78%, IgM = 33%, and IgA = 23%. There was no correlation between PLAs and microalbuminuria, macrovascular disease, fibrinogen, duration of diabetes, or neuropathy, but there was a strong correlation with total neuropathy score. Sera with high PLA IgG titers bound to the surface of neuroblastoma cells and inhibited cell growth. Antibodies to GAD65 were present in 32% and to GAD67 in 0% of patients. No titers of GAD65, GAD67, or the GAD65 ratio were associated with the degree of neuropathy of the presence of PLAs. CONCLUSIONS: PLAs occur frequently in the sera of patients with diabetes and correlate with the extent of neuropathy, suggesting a role for PLAs in the etiology thereof. The measurement of PLAs may constitute a marker for ongoing damage to nerves. PMID- 8612437 TI - Effects of a behavioral weight loss program stressing calorie restriction versus calorie plus fat restriction in obese individuals with NIDDM or a family history of diabetes. AB - OBJECTIVE: The aim of this randomized trial was to compare the effects of a behavioral intervention focusing on either calorie restriction alone or calorie plus fat restriction on weight loss and changes in lipids and glycemic control in individuals with non-insulin-dependent diabetes mellitus (NIDDM) or a family history of diabetes. RESEARCH DESIGN AND METHODS: We recruited 44 obese women with NIDDM and 46 obese women with a family history of NIDDM and randomly assigned these subjects to calorie restriction (CAL) or to calorie plus fat restriction (CAL + FAT). All subjects participated in a 16-week behavioral weight loss program, with training in diet, exercise, and behavior modification. Subjects assigned to the CAL condition were given a 1,000-1,500 kcal/day goal and self-monitored calories consumed. Subjects assigned to the CAL+FAT condition had the same calorie goal, but were also given a fat goal (grams of fat/day), to produce a diet with < 20% of calories from fat; this group monitored both calories and fat grams. RESULTS: Among NIDDM subjects, weight loss of the subjects in the CAL+FAT condition was significantly greater than subjects in the CAL condition (7.7 vs. 4.6 kg) and the CAL+FAT condition group also maintained their weight loss better at the 1-year follow-up (5.2 vs. 1.0 kg). Significant decreases in glucose, high-density lipoprotein (HDL) cholesterol, and total cholesterol were seen after 16 weeks of treatment among NIDDM subjects; these changes were similar in CAL and CAL+FAT groups, but a greater proportion of subjects in CAL condition required oral hypoglycemic medication. At the 1-year follow-up, all parameters had returned to baseline. No significant differences in weight loss or physiological changes were seen between CAL and CAL+FAT conditions in subjects with a family history of diabetes. CONCLUSIONS: These results suggest that using the combination of calorie and fat restriction may help promote weight loss in obese NIDDM patients. No other long-term benefits of this regimen were observed. PMID- 8612436 TI - Renal, metabolic, and hormonal responses to proteins of different origin in normotensive, nonproteinuric type I diabetic patients. AB - OBJECTIVE: Whether the differences in renal function found in vegetarian compared with omnivorous subjects are related to quantity or quality of the protein is unknown. We have studied the renal function of nine normotensive, nonproteinuric type I diabetic patients who were fed in random order for 4 weeks either an animal protein diet (APD) (protein intake 1.1 g . kg-1 . day-1) or a vegetable protein diet VPD (protein intake 0.95 g . kg-1 . day-1). The two diets were isocaloric. RESEARCH DESIGN AND METHODS: In a crossover study, we measured glomerular filtration rate (GFR) (inulin clearance), renal plasma flow (RPF) (p aminohippurate clearance), plasma amino acids, growth hormone, glucagon, insulin like growth factor I-(IGF-I), and microalbuminuria. RESULTS: GFR and RPF were lower with the VPD than with the APD (89.9 +/- 4.1 vs. 105.6 +/- 5.1 ml . min-1 . 1.73 m-2, P < 0.05, and 425.7 +/- 22.2 vs. 477.8 +/- 32.2 ml . min-1 1.73m-2, P < 0.05, respectively). Renal vascular resistance (RVR) was higher with the VPD than with the APD (101 +/- 25 vs. 91 +/- 10 mmHg . min-1 . ml-1, P < 0.05). Filtration fraction (FF) remained unchanged after either diet. Fractional clearance of albumin fell with the VPD to 2.0 +/- 0.65 from 3.4 +/- 1.15 x 10-6 (P < 0.05). At the end of the APD and VPD, the plasma levels of growth hormone and glucagon did not differ significantly. Plasma levels of IGF-I were higher with the APD than with the VPD (1.1 +/- 0.6 vs. 0.9 +/- 0.13 U/ml, P < 0.05). Plasma concentrations of valine and lysine were significantly higher with the APD than with the VPD (234.6 +/- 30.3 vs. 164.5 +/- 25.4 mm1/1, P < 0.05, and 565 +/- 45.1 vs. 430 +/- 56.1 mmol/l, P < 0.05, respectively), whereas plasma valine was strongly correlated to the GFR (r = 0.832, P < 0.01). No differences were found in other amino acids. CONCLUSIONS: A VPD has significantly different renal effects from an APD equal in protein intake in normotensive type I diabetic patients. This could be explained partly by differences in plasma concentrations of amino acids and IGF-I. PMID- 8612438 TI - Comparative validity of a food frequency questionnaire for adults with IDDM. AB - OBJECTIVE: To investigate the characteristics of a food frequency questionnaire (FFQ) in measuring dietary intake in an adult insulin-dependent diabetes mellitus (IDDM) population. FFQs have been widely used in developed countries to assess usual dietary intake; however, information regarding the application of the method to individuals advised to follow a specific dietary regimen (such as people with IDDM) is scarce. RESEARCH DESIGN AND METHODS: The measurement of energy and macronutrients by an interviewer-administered FFQ was assessed in 84 adults, who were representative of adults with IDDM in Tasmania, Australia, by comparing it with 2 days of weighed dietary records. Mean daily energy and macronutrient intakes by each method were compared, and Pearson correlation coefficients were calculated for both unadjusted and energy-adjusted macronutrient intakes. RESULTS: The ratio of within-person to between-person variance in dietary estimates from weighed records was 0.53 for energy, 0.70 for fat, 2.55 for protein, 0.50 for carbohydrate, 0.78 for saturated fat, and 3.56 for dietary cholesterol. The correlation coefficient between the FFQ and a 2-day weighed dietary record for the same nutrients ranged from 0.38 for protein intake to 0.60 for saturated fat intake. The correlation coefficient between the FFQ and true usual dietary intake was estimated to be 0.60 for energy-adjusted fat intake, 0.36 for energy-adjusted protein intake, 0.72 for energy-adjusted carbohydrate intake, 0.55 for energy-adjusted saturated fat intake, and 0.77 for energy-adjusted cholesterol intake. CONCLUSIONS: People with IDDM had a low ratio of within-person to between-person variance in daily energy, fat, carbohydrate, saturate fat, and cholesterol intake. The performance of the FFQ in the IDDM population is consistent with similar questionnaires in nondiabetic populations; however, there was a significant decrease in the dietary energy estimate upon readministration of the questionnaire. Validation studies such as this are important to provide information to guide the design and analysis of investigations that use dietary questionnaires. PMID- 8612439 TI - Prevalence estimates of diabetes and of other cardiovascular risk factors in the two largest Algonquin communities of Quebec. AB - OBJECTIVE: To compare the prevalence of non-insulin-dependent diabetes mellitus (NIDDM) in the two largest Algonquin communities of Quebec (Canada) with that of other native groups and to describe the different patterns of NIDDM and other cardiovascular risk markers in these communities (River Desert [RD] and Lac Simon [LS]). RESEARCH DESIGN AND METHODS: The population-based study targeted all residents aged 15 years and older. In the age-group considered here (30-64 years), there were 480 eligible subjects and 299 participants (50.8% in RD and 86.9% in LS). All except those with confirmed diabetes underwent an oral glucose tolerance test. Serum triglyceride and lipoprotein cholesterol levels, blood pressure, body mass index (BMI), and waist-to-hip ratio (WHR) were measured. RESULTS: The age-standardized (world population) prevalence of NIDDM in women was twice as high in LS as in RD (48.6% vs. 23.9%). In men, it was 23.9% in LS and 16.3% in RD. Upper-body obesity followed the same pattern. In contrast, high-risk serum low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels were significantly more prevalent in RD than in LS, particularly among men. The rate of high blood pressure was twice as high in men as in women, with little community differences. When we controlled for age, sex, diabetic, and obesity status, mean fasting serum glucose remained significantly higher triglycerides and LDL cholesterol lower in LS than in RD. There was also an independent community effect on WHR but no on BMI. CONCLUSIONS: The prevalence of NIDDM in LS women reaches the rate observed in Pima Indian women. The observed differences between two Algonquin communities suggest a highly heterogeneous pattern of NIDDM and cardiovascular disease risk factors in Amerindian populations, even within a given tribe and a limited geographic area. PMID- 8612441 TI - Subcutaneous glucose concentration in humans. Real estimation and continuous monitoring. AB - OBJECTIVE: To determine the real subcutaneous glucose concentration in healthy volunteers to help in the development of new calibration methods for subcutaneous glucosensors. RESEARCH DESIGN AND METHODS: We developed a new method to estimate the real subcutaneous glucose concentration based on the recirculation of phosphate-buffered saline (PBS) in a microdialysis probe inserted into the subcutaneous tissue. Tissue glucose diffuses into the probe until complete equilibration between the glucose concentration outside and inside the microdialysis probe is achieved. Later, the glucose content of the recirculated PBS is assessed in vitro. We applied the method in 10 healthy volunteers under fasting state and during a hyperglycemic clamp. In addition, we monitored the subcutaneous glucose with an enzymatic-amperometric glucosensor combined with a microdialysis probe. RESULTS: The subcutaneous glucose concentration measured by the recirculation method was 72 +/- 6 and 78 +/- 6% of the blood glucose measured in the fasting state and during the hyperglycemic clamps, respectively. On the other hand, the glucosensor's signal correlated significantly with the blood glucose. CONCLUSION: The recirculation method estimated the real subcutaneous glucose concentration, opening the way to develop new calibration procedures for subcutaneous glucosensors. However, a suitable calibration procedure is still lacking. PMID- 8612440 TI - Effect of chronic intermittent intravenous insulin therapy on antihypertensive medication requirements in IDDM subjects with hypertension and nephropathy. AB - OBJECTIVE: The prevalence of systemic hypertension is increased in patients with diabetes. In this prospective, randomized, crossover clinical trial, we assessed antihypertensive effects of chronic intermittent intravenous insulin therapy (CIIIT) on insulin-dependent diabetes mellitus (IDDM) subjects with hypertension and nephropathy by monitoring the amount of antihypertensive medication (AHM) required to maintain blood pressure (BP) < or = 140/90 mmHg. RESEARCH DESIGN AND METHODS: After a stabilization period, 26 hypertensive IDDM subjects were randomly assigned to a control or treatment phase for 3 months and then crossed over into the opposite phase for another 3 months. Addition of CIIIT during the treatment phase was the only procedural difference between the control and treatment phases. RESULTS: The AHM dosage requirements for maintenance of the baseline BP levels decreased significantly (46%; P < 0.0001) and linearly over time (P < 0.0058) during the treatment phase, while remaining essentially unchanged during the control phase. CONCLUSIONS: Our data suggest that CIIIT markedly improves BP control, as evidenced by the significantly reduced AHM dosage requirements in subjects with IDDM and hypertension, possibly through an improvement in vascular reactivity. PMID- 8612442 TI - Prevalence and determinants of glucose intolerance in a Dutch caucasian population. The Hoorn Study. AB - OBJECTIVE: To study the prevalence and determinants of glucose intolerance in a general Caucasian population. RESEARCH DESIGN AND METHODS: A random sample of 50- to 74-year old Caucasians (n = 2,484) underwent oral glucose tolerance tests. Multiple regression analyses were performed to study the association of 2-h postload plasma glucose values with potential determinants. RESULTS: Prevalence of known and newly detected diabetes and impaired glucose tolerance was 3.6, 4.8, and 10.3%, respectively. In women, but not in men, the association of body mass index with 2-h glucose was fully accounted for by the waist-to-hip ratio. Maternal history of diabetes was twice as prevalent as paternal history, but paternal history only was associated with 2-h glucose. In addition, paternal history was a stronger determinant in men than in women. An independent positive association with 2-h plasma glucose was found for alcohol use of > 30 g/day in women and for intake of total protein, animal protein, and polyunsaturated fatty acids in men. An independent inverse association with 2-h plasma glucose was demonstrated for height (both sexes), alcohol use of < or = 30 g/day (both sexes), energy intake (in men), and, unexpectedly, current smoking (in men). CONCLUSIONS: The prevalence of diabetes in elderly Caucasians was 8.3%. In men, dietary habits may unfavorably influence glucose tolerance independent of obesity. PMID- 8612443 TI - Glucose tolerance and physical activity in a Mexican indigenous population. AB - OBJECTIVE: To assess diabetes prevalence and physical activity among an indigenous population in Mexico. RESEARCH DESIGN AND METHODS: A total of 101 adult (mean age = 38 years) Mixtec Indians living on the outskirts of Huajuapan de Leon, Oaxaca, Mexico, volunteered to be measured for height, weight, blood pressure, glucose tolerance, and physical activity. RESULTS: According to World Health Organization criteria, 2 women were diabetic and 42 subjects (15 men, 27 women) had impaired glucose tolerance (IGT). Although the population was generally lean, with a mean +/- SD body mass index (BMI) of 23.0 +/- 2.7 kg/m2 for men (n = 47) and 22.8 +/- 3.0 kg/m2 for women (n = 54), the group with normal glucose tolerance had a significantly lower BMI than those with abnormal glucose tolerance (IGT or diabetes). Men were significantly more physically active than women, and a larger percentage of women (54%) than men (32%) had abnormal glucose tolerance. However, within gender groups, there was not a significant effect of physical activity on status of glucose tolerance. CONCLUSIONS: The Mixtec population may be genetically predisposed to non-insulin-dependent diabetes, although their current lifestyle provides a protective effect. Less of a protective effect is seen for females, however. It is expected that further environmental changes impacting energy balance will have the most damaging repercussions for diabetes prevalence among Mixtec women. PMID- 8612444 TI - The Jamaican Diabetes Survey. A protocol for the Caribbean. AB - OBJECTIVE: This study was designed to investigate the point prevalence of diabetes in Jamaica. RESEARCH DESIGN AND METHODS: A two-stage stratified random sampling design was used, and individuals aged 15 years and over were interviewed. Nonresponse was documented and factored into the final analysis of the survey data. The overall response rate obtained was 57.9%. All subjects with fasting blood glucose (FBG) greater than 6.1 mmol/1 (110 mg/dl) were brought back for an abbreviated glucose tolerance test. The data was analyzed using Epi 5, an advanced statistical program designed specifically for use with epidemiological data. RESULTS: The 2,109 subjects who participated were the basis for estimates of diabetes and IGT prevalence. Among those with previously diagnosed diabetes, diet therapy alone, oral hypoglycemic agents plus diet therapy, insulin therapy, and lack of treatment were reported. CONCLUSIONS: By the World Health Organization (WHO) criteria, Jamaica has a point prevalence of diabetes of 17.9% in the 15-and-over age-group. PMID- 8612445 TI - Increasing incidence of IDDM in Austrian children. A nationwide study 1979-1993. Austrian Diabetes Incidence Study Group. AB - OBJECTIVE: A nationwide population-based study was conducted to assess the insulin-dependent diabetes mellitus (IDDM) incidence in childhood over a 15-year period (1979-1993). RESEARCH DESIGN AND METHODS: A questionnaire was sent to all Austrian pediatric departments and diabetologists. The secondary data source was based on patient organization lists and hospital administration data. The data from 1979-1987 were collected retrospectively, while from 1988 to 1993 the registration of cases was performed prospectively in the same network. Estimates of probability of ascertainment were calculated according to the capture recapture method. RESULTS: The achieved ascertainment was 94% . The overall annual incidence was 7.9/100,000 person-years in children 0-14 years old. During the observation period, the incidence rose by 2.4% annually. CONCLUSIONS: The incidence of childhood IDDM in Austria, a European country with an intermediate risk for IDDM, showed a proportionally similar increase to that of Northern European countries over the past decade. The increase seems to be continuous, following mainly a linear trend with superimposed sudden outbreaks indicating environmental causative factors. PMID- 8612446 TI - Recurrent infection of continuous subcutaneous insulin infusion sites with Mycobacterium fortuitum. PMID- 8612448 TI - Simultaneous acute cerebral and pulmonary edema complicating diabetic ketoacidosis. AB - OBJECTIVE: To report on a child with diabetic ketoacidosis (DKA) who developed simultaneous acute cerebral edema (CE) and pulmonary edema (PE), required extracorporeal membrane oxygenation (ECMO), and yet survived without significant neurological or pulmonary handicap. CASE SUMMARY: A 3-year-old girl with DKA as the first manifestation of insulin-dependent diabetes mellitus (IDDM) sustained coincident acute CE and PE 9 h into therapy. The former responded to mannitol, but the latter matured into adult respiratory distress syndrome (ARDS) resistant to conventional management and requiring ECMO. CONCLUSIONS: CE, PE, and ARDS can complicate DKA. Survival without sequelae is possible with aggressive treatment. PMID- 8612447 TI - Lack of long-term diabetic complications in spite of poor glycemic control in twins with pure gonadal dysgenesis. AB - BACKGROUND: The prepubertal years have little effect on the chronic complications of diabetes. Animal studies have shown castration prevents diabetic complications. No previous data on untreated agonadal human diabetic subjects is available. CASE HISTORY: Clinical study of untreated agonadal twins with insulin dependent diabetes for presence of chronic complications of diabetes. In spite of the twins' poor glycemic control, no evidence of the chronic complications of diabetes was found. CONCLUSIONS: The absence of gonadal sex steroids in the diabetic patient may have a protective effect against the development of the chronic complications of diabetes. PMID- 8612449 TI - Program development to prevent complications of diabetes. Assessment of barriers in an urban clinic. PMID- 8612450 TI - Associations between insulin levels and cardiovascular disease are confounded by comorbidity. PMID- 8612451 TI - Is insulin really a heart disease risk factor. PMID- 8612452 TI - HbA1c determination on capillary blood sample: validity, stability, and potential usefulness. PMID- 8612453 TI - Response to Nathan. PMID- 8612454 TI - A simplified method of blood sampling for diabetes control. PMID- 8612455 TI - Simple photometric test of pupillary dysfunction. PMID- 8612456 TI - Diabetes care in emergency settings. PMID- 8612457 TI - Pneumomediastinum and subcutaneous emphysema in diabetic ketoacidosis. PMID- 8612459 TI - American Diabetes Association scientific sessions, 1995. Neuropathy and retinopathy. PMID- 8612458 TI - Cardiac sympathetic nervous dysfunction in mitochondrial cardiomyopathy and diabetes. PMID- 8612460 TI - [Subclinical hypercortisolism in incidentally detected adrenal adenoma]. AB - AIM OF STUDY: To define in a prospective study the prevalence and manifestations of subclinical hypercortisolism in a sizeable group of patients with incidentally detected adrenal adenoma. Such tumour - although usually clinically silent - may cause discrete endocrine dysfunctions. PATIENTS AND METHODS: Between 1990 and 1994, 85 consecutive patients with incidentally discovered adrenal adenoma were investigated (54 women, 31 men; mean age 54.1 +/- 13 [24-81] years). In addition to history and clinical examination all patients had a low-dose dexamethasone suppression test (2mg) and their morning basal plasma ACTH concentration was measured. The diagnosis of subclinical hypercortisolism was made if there was inadequate suppression of cortisol after dexamethasone administration, plasma ACTH level was decreased and there were no clinical signs of Cushing's syndrome. RESULTS: Five of the patients fulfilled the criteria of subclinical hypercortisolism (prevalence of 6%). One of them had oligomenorrhoea, another arterial hypertension, and three had mild histological proof of adrenal adenoma. Postoperatively the patient with oligomenorrhoea had normal menstrual cycles, while in all others with subclinical hypercortisolism excision of the tumour had no influence of their clinical state. CONCLUSIONS: Subclinical hypercortisolism is rare, but is one of the most common endocrine disorders in patients with incidental adrenal adenoma. It can be diagnosed only by hormonal analysis. Once the latent cortisol excess has been eliminated, those symptoms which are due to hormonal abnormality may regress, but nonspecific signs, such as obesity or hypertension, may not necessarily improve. PMID- 8612461 TI - [Endoscopic manometry in suspected dysfunction of the sphincter of Oddi]. AB - BASIC PROBLEM AND OBJECTIVE OF STUDY: Dysfunction of the sphincter of Oddi may be a cause of persistent problems after cholecystectomy. The aim was to find out whether various factors are of value in predicting abnormal manometric results and thus aid in deciding whether endoscopic manometry is indicated. PATIENTS AND METHODS: 97 patients were investigated prospectively (13 men, 84 women; mean age 50.2 [29-72] years) in which endoscopic cholangiopancreatography (ERCP) had not revealed any cause of the biliary complaints. The patients were divided into three types according to four criteria: (1) history (biliary colics); (2) biochemistry (cholestasis); (3) dilated biliary tract (at ERCP); (4) contrast retention in biliary tract (at ERCP). Type I: all four criteria present; type II: positive history and one or two other criteria; type III: biliary colic only. Endoscopic manometry (EM) was performed after classification of the patients. RESULTS: EM was successful in 83 of 97 patients (86%). All 15 patients of type I had sphincter of Oddi dysfunction (SOD) defined as basic sphincter pressure > 40 mm Hg. SOD was demonstrated in only 23 of 38 patients of type II (61%) and 15 of 30 patients of type III (50%) (P < 0.01). Increased rate of sphincter of Oddi contraction ("tachyoddi") was demonstrated in only four patients (4%), in two of them in combination with an increased basic sphincter pressure. Mild to moderate pancreatitis occurred within 24 hours of manometry in ten of 83 patients. CONCLUSIONS: Endoscopic manometry is not necessary in patients of type I for confirming the diagnosis. But it is obligatory for patients of type II and III, because the demonstration of SOD has therapeutic consequences, and should be performed in clinical studies, if possible. PMID- 8612463 TI - [Diagnosis of rheumatoid arthritis]. PMID- 8612462 TI - [Hydroa vacciniforme]. AB - HISTORY AND CLINICAL FINDINGS: For three years, each time the day after intensive exposure to the sun a 5-year-old girl developed moderately painful erythema and blisters on uncovered parts of her body which subsequently healed with scarring. There was no increased photosensitivity among members of her family. There were numerous blisters and bullae, some of them confluent, filled with serous fluid on her back, on her arms an in the face. There were varioliform scars on the extensor surfaces of her arms. INVESTIGATION: There were no porphyrins in red cells, serum or urine. The characteristic skin changes were reproduced on exposure to UV-A. These findings established the diagnosis of hydroa vacciniforme. TREATMENT AND COURSE: Prolonged sun exposure was to be avoided and protective ointment was prescribed together with administration of beta-carotene, 25 mg twice daily. CONCLUSION: In children light-induced skin changes which heal with scarring may be due to hydroa vacciniforme. PMID- 8612464 TI - [Varicose veins and migrating thrombophlebitis]. PMID- 8612465 TI - [Controversy over calcium antagonists: is there a rationally differentiating therapy?]. PMID- 8612466 TI - [Antibody activity before polio vaccination]. PMID- 8612467 TI - [Is esophagus and stomach biopsy mandatory in endoscopic diagnosis of reflux esophagitis]. PMID- 8612468 TI - [Non-cardiac chest pain--disorder of the esophagus?]. PMID- 8612470 TI - Levocabastine. An update of its pharmacology, clinical efficacy and tolerability in the topical treatment of allergic rhinitis and conjunctivitis. AB - Levocabastine is a potent and selective histamine H1-receptor antagonist which has been evaluated as a topical treatment (nasal spray and/or eyedrops) for allergic rhinitis and/or conjunctivitis. Data available at the time of the previous review in Drugs, together with more recent results, have clearly demonstrated that levocabastine nasal spray and eyedrops are clinically effective, have a rapid onset of action and are well tolerated in patients with nasal and/or ocular allergic conditions. Previous evidence indicating that topical levocabastine has efficacy similar to or better than that of topical sodium cromoglycate (cromolyn sodium) has been confirmed in more recent studies. Furthermore, results from a number of controlled clinical trials have also shown that topical levocabastine is at least as effective as oral terfenadine for the treatment of allergic rhinoconjunctivitis. Notably, topical levocabastine appears to be more effective than oral terfenadine in improving the severity of selected symptoms. Limited data indicating efficacy equivalent to that of oral loratadine, oral cetirizine or azelastine nasal spray will need to be confirmed. Data from several studies have shown that topical levocabastine has a tolerability profile similar to that of placebo, topical sodium cromoglycate or oral terfenadine. The main adverse events seen in patients treated with topical levocabastine are ocular irritation after application of eyedrops, and headache, nasal irritation, somnolence and fatigue after administration of the nasal spray. Administered doses of topical levocabastine, and subsequent plasma concentrations, are low, and the risk of systemic adverse events is therefore expected to be minimal. Thus, topical administration of levocabastine rapid and effective symptom relief with no apparent serious adverse events in patients with allergic rhinitis and/or conjunctivitis. Topical levocabastine is a useful alternative to topical sodium cromoglycate or oral terfenadine. Additional data supporting current evidence that topical levocabastine can provide more effective symptom relief than oral terfenadine, together with clarification of the relative efficacies of these agents in relation to varying pollen exposure, would help to further confirm its clinical potential. However, the results available to date suggest that the topical formulations of levocabastine are a valuable treatment option in patients with allergic rhinitis and/or conjunctivitis. PMID- 8612472 TI - What is the role of corticosteroids in meningitis? PMID- 8612471 TI - Tiaprofenic acid. A reappraisal of its pharmacological properties and use in the management of rheumatic diseases. AB - Tiaprofenic acid is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of patients with rheumatic diseases and other clinical conditions of pain and inflammation. Like other propionic acid derivatives, tiaprofenic acid is effective and generally well tolerated. Comparative studies in patients with rheumatoid arthritis or osteoarthritis receiving tiaprofenic acid 600 mg/day demonstrated improvements in pain intensity, duration of morning stiffness, articular index and other clinical variables which were similar to those achieved with alternative NSAIDs. Tolerability was also comparable between tiaprofenic acid and other NSAIDs in most trials; the most frequently reported adverse events involved the gastrointestinal tract. Some studies showed a trend towards fewer patient withdrawals because of adverse events with tiaprofenic acid than with NSAIDs such as indomethacin. Current evidence suggests that nonbacterial cystitis is more likely to be associated with tiaprofenic acid than with other NSAIDs. This reaction should, however, be considered in the perspective of its infrequent occurrence and its typical reversibility, and against the wider background of the established usage of tiaprofenic acid and its overall tolerability profile which is similar to that of other NSAIDs. Unlike indomethacin, tiaprofenic acid was not associated with increased cartilage degradation in a recently completed large clinical trial known as LINK, which evaluated the effects of long term administration in patients with osteoarthritis of the knee. Thus, tiaprofenic acid is an established option among the range of NSAIDs used in the treatment of patients with rheumatic diseases, with efficacy and tolerability profiles that are relatively well characterised. The availability of a sustained release dosage form of tiaprofenic acid, which has a similar efficacy and tolerability profile to the standard formulation, provides a convenient once daily dosage regimen. PMID- 8612469 TI - Amifostine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential as a radioprotector and cytotoxic chemoprotector. AB - Amifostine (WR-2721) was originally developed as a radioprotective agent. In animals, it protects normal tissues from the damaging effects of irradiation and, as shown in more recent studies, of several cytotoxic agents. Protection of tumours is generally reduced compared with that of normal tissues in animals, suggesting that amifostine may increase the therapeutic window of cytotoxic therapies. Clinical data concerning amifostine suggest that cytotoxic chemotherapy-induced haematological toxicity and cisplatin-induced neurotoxicity, nephrotoxicity and ototoxicity are decreased upon administration of amifostine prior to cytotoxic drugs. Similarly, amifostine reduces damage to normal tissues caused by radiotherapy. Available data show that this protection is achieved without adversely affecting tumour response or patient survival. In 1 large trial, the reduction in cyclophosphamide- and cisplatin-related toxicities manifested as a decrease in the incidence and severity of neutropenia-related fever and sepsis and in the number of patients with ovarian cancer who discontinue therapy before completion of treatment, thus improving the tolerability of this antineoplastic regimen. In addition, the incidences of cisplatin-induced nephro- and neurotoxicity were reduced. Increased doses of cytotoxic therapy have also been administered when amifostine was given prior to therapy, which may increase tumour response. The predominant adverse effect associated with amifostine are hypotension, nausea and vomiting, somnolence and sneezing. Thus, amifostine is likely to be a useful adjuvant to the treatment of patients with malignancy, particularly those receiving cyclophosphamide plus cisplatin. discontinued therapy before completion of treatment, thus improving the tolerability of this antineoplastic regimen. In addition, the incidences of cisplatin-induced. PMID- 8612474 TI - Risk and management of hypertension-related left ventricular hypertrophy. AB - Knowledge gained from epidemiological studies and clinical trials on hypertension has led to impressive reductions in morbidity and mortality, particularly from stroke and coronary heart disease (CHD) as complications of end-organ damage from untreated, prolonged systemic hypertension. Data on reductions in stroke when hypertension is treated have been clear and convincing from individual clinical trials. Most of these trials, however, have consistently shown only trends towards a reduction in CHD, and few have individually reported statistically significant reductions. A recent meta-analysis, however, suggests that a significant beneficial reduction in CHD exists when the overall data are examined, although at a lower magnitude of benefit and lesser degree of certainty than for stroke. The presence of left ventricular hypertrophy (LVH) increases the risk of subsequent cardiovascular disease events, cardiovascular mortality and all-cause mortality in hypertensive patients. Although echocardiography appears more sensitive than electrocardiography in diagnosing LVH, much of the information demonstrating risks from LVH is from electrocardiography data, and it is not clear how echocardiography will change the risk prediction. Some data from large clinical trials and populations studies suggest that LVH regresses, particularly if the hypertension is adequately treated. A meta-analysis of a large number of small clinical studies in hypertensive patients suggests that the 4 commonly used antihypertensive drug classes, beta-blockers, diuretics, calcium channel antagonists and ACE inhibitors, are all associated with significant reductions in left ventricular mass. While the primary indication for treatment is clearly the hypertension and not the LVH, the presence of the latter necessitates careful treatment and follow-up of these hypertensive individuals. PMID- 8612473 TI - Gene therapy. Clinical potential and relationship to drug treatment. PMID- 8612476 TI - Current guidelines for the eradication of Helicobacter pylori in peptic ulcer disease. AB - Pharmacological suppression of gastric acid secretion has traditionally been the most rational approach to healing ulcers successfully. However, ulcers initially healed using antisecretory therapy have a tendency to relapse after treatment is withdrawn. This tendency is altered definitively by eradication of Helicobacter pylori. Antimicrobial therapy should be given to all patients with documented duodenal and gastric ulcer associated with H. pylori infection. The optimal therapeutic regimen to eradicate H. pylori is still not completely clear. The requirement for treatment to be effective in more than 90% of patients makes monotherapy and dual therapy inappropriate. Bismuth-based triple therapy (bismuth, tetracycline and metronidazole) is highly efficacious if the H. pylori strain is sensitive to metronidazole and the patient is compliant, but adverse effects often occur. Triple therapy consisting of omeprazole and 2 antimicrobials (clarithromycin and/or amoxicillin and/or metronidazole) and quadruple therapy (bismuth-based triple therapy plus omeprazole) are both very effective and patient compliance may be better because of the shortened (1 week) course. Preliminary data indicate that the efficacy of the regimen is not influenced by imidazole resistance. Eradication of H. pylori prevents complications and relapse of peptic ulcer disease and is a cost-effective option compared with maintenance acid-suppressive therapy. PMID- 8612475 TI - Lazaroids. CNS pharmacology and current research. AB - The 21-aminosteroids (lazaroids) are inhibitors of lipid membrane peroxidation and appear to function as oxygen free radical scavengers. The therapeutic potential of the lazaroid tirilazad mesylate has been extensively studied in several CNS disorders. Tirilazad and related compounds have been found to be highly beneficial in spinal cord trauma. Spinal cord injury studies utilising tirilazad are currently underway to determine the optimal combination of medications. Tirilazad has also been found to be beneficial in experimental head injury models, however current clinical studies have failed to confirm this efficacy, due in part to difficulties in obtaining therapeutic drug concentrations. Clinical studies using tirilazad in subarachnoid haemorrhage have been more promising. It has been shown to be beneficial in terms of reducing vasospasm and cerebral infarction associated with subarachnoid haemorrhage, and has now been approved in several European countries in this indication. Results from US studies are expected shortly. Finally, tirilazad has also been extensively tested in a variety of stroke models. Although it appears to be highly beneficial in experimental models, the clinical studies to date have failed to confirm this efficacy. Again, this failure appears to be due largely to inadequate drug concentrations having so far been tested. PMID- 8612478 TI - ret protooncogene mutations and endocrine neoplasia--a story intertwined with neural crest differentiation. PMID- 8612477 TI - Guidelines for the drug treatment of hypertensive crises. AB - Hypertensive crises are a group of clinicopathological entities in which rapid reduction of hypertension is necessary to prevent serious end-organ damage. The diagnosis and treatment plan depends on the identification of specific end-organ dysfunction. The goal of treatment is to limit the progression of end-organ damage in patients with hypertensive crises. Several potent antihypertensive drugs, such as sodium nitroprusside, labetalol and urapidil, are available to produce an immediate fall in blood pressure. The choice of the drug should be made on the basis of its pharmacodynamic properties, clinical effects, advantages and contraindications. Additionally, rapid reduction of blood pressure carries a considerable risk, if it is performed in an uncontrolled manner, leading to further end-organ damage. The aim of the treatment is not just to reduce blood pressure, but to do so with minimal adverse effects while preserving organ function. PMID- 8612480 TI - Glycosylation: to what end for the glycoprotein hormones? PMID- 8612479 TI - Characterization of ret oncogenic activation in MEN2 inherited cancer syndromes. AB - Germline mutations of c-ret, encoding a receptor-type tyrosine kinase, were found to be associated with variants of multiple endocrine neoplasia type 2 (MEN2A, MEN2B), and familial medullary thyroid carcinoma. NIH/3T3 stable transfectants expressing RET with a mutation of MEN2A (MEN2A/RET) or MEN2B (MEN2B/RET) gained a transformed morphology, formed colonies in soft agar, and formed tumors in nude mice. These results confirmed that both MEN2A/RET and MEN2B/RET exert dominant transforming activities in NIH/3T3 cells. However, in contrast to their clinical manifestation, transfectants expressing MEN2A/RET exhibited a higher tumorigenicity in nude mice than transfectants expressing MEN2B/RET may depend on the presence of its ligand and/or substrates that are absent in NIH/3T3 cells. No change in the cellular localization of the mutated RET proteins was observed compared to c-RET. Interestingly, ret activation in NIT/3T3 cells appeared to be associated with up-regulation of homologous gap-junctional intercellular communication and increased expression of a gap-junctional protein, connexin43. PMID- 8612481 TI - Centrally administered neuropeptide FF inhibits arginine vasopressin release in conscious rats. AB - There is evidence indicating that neuropeptide FF (NPFF) is an endogenous modulator of opioid systems. In the present study, we investigated the effect of centrally administered NPFF on arginine vasopressin (AVP) release in conscious rats. The plasma AVP increase in response to either hyperosmolality [i.p. injection of hypertonic saline (600 mosmol/kg)] or hypovolemia [i.p. injection of polyethylene glycol (PEG)] was significantly blunted when NPFF was injected into the lateral ventricle so that the given drug could act at the hypothalamus and also reach the brain stem (hypertonic saline with 10 micrograms/rat NPFF, 3.28 +/ 0.48 pg/ml; hypertonic saline alone, 7.85 +/- 1.78 pg/ml; PEG with 10 micrograms/rat NPFF, 4.07 +/- 1.40 pg/ml; PEG alone, 8.25 +/- 1.90 pg/ml). The plasma AVP increase in response to PEG-induced hypovolemia was also attenuated significantly and more potently when NPFF was injected into the cisterna magna so that the given drug could be readily accessible to the dorsal medulla where the nucleus of solitary tract is located (10 micrograms/rat; 2.71 +/- 0.14 pg/ml). In contrast, the NPFF injected into the cisterna magna had no significant effect on hyperosmolality-induced AVP release. Treatment with naloxone (10 mg/kg BW, sc) significantly reversed the inhibitory effects of NPFF on AVP release. These results suggest that central NPFF might play an inhibitory role via the hypothalamus in the osmoregulation of plasma AVP and via both the hypothalamus and the nucleus of solitary tract in the baroregulation, and that the intrinsic opioid systems are involved in the action of NPFF. PMID- 8612482 TI - The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion. AB - We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland. PMID- 8612483 TI - 1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation. AB - 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation. PMID- 8612484 TI - Connexin-26 and connexin-43 are differentially expressed and regulated in the rat myometrium throughout late pregnancy and with the onset of labor. AB - Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of labor contractions. The expression of connexin-43 (Cx-43), the major myometrial gap junction protein, is increased during delivery (associated with an increase in the plasma estradiol/progesterone ratio) and after estradiol treatment of ovariectomized nonpregnant rats. However, Cx-43 is only 1 member of at least 16 proteins encoded by this family of gap junction genes. Using a RT-PCR method, we identified the presence of another member of this family, Cx-26, in laboring rat myometrium. The temporal expression pattern of Cx-26 was assessed using Northern and Western analyses. In contrast to Cx-43, whose expression is low throughout the pregnancy but increases immediately before the onset of labor (day 23), the expression of Cx-26 increased on day 17, reached maximal levels between days 19 21, and fell to low levels before the onset of labor. Treatment of pregnant rats with progesterone beginning on day 20 (which blocks both the increase in Cx-43 expression and the onset of labor) maintained the elevated expression of Cx-26. Induction of preterm labor in rats after ovariectomy on day 17 inhibited the normal preterm increase in Cx-26 transcripts. Progesterone treatment of these animals reversed the effects of ovariectomy. Immunofluorescence data identified Cx-26 antigen in the cell membranes of myometrial cells and in the luminal and glandular epithelium of the endometrium in the late pregnant (day 21) uterus. These data suggest that the role of gap junction formation in the myometrium in relation to the maintenance of pregnancy and the onset of labor is much more complex than previously recognized. Myometrial cell-cell communication is afforded by at least two different gap junction proteins, Cx-43 and Cx-26, that not only exhibit temporally distinct patterns of expression but are also subject to differential regulation. PMID- 8612485 TI - Stable expression of the nuclear vitamin D receptor in the human prostatic carcinoma cell line JCA-1: evidence that the antiproliferative effects of 1 alpha, 25-dihydroxyvitamin D3 are mediated exclusively through the genomic signaling pathway. AB - The secosteroid hormone 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to regulate the growth and differentiation of human prostate cancer cells, although the precise mechanisms mediating these effects have not been defined. 1,25-(OH)2D3 is capable of acting through both nongenomic signaling pathways involving a membrane-associated receptor and genomic pathways involving the nuclear vitamin D receptor (VDR). The primary purpose of this study was to directly evaluate the role of the nuclear VDR in mediating the growth inhibitory effects of 1,25-(OH)2D3 on human prostate cancer cells. The cell line JCA-1 was used because it fails to express detectable number of VDRs and is not measurable affected by 1,25-(OH)2D3 in growth studies. These cells were stably transfected with a wild-type VDR complementary DNA construct producing the following results: 1) the expression of high affinity nuclear VDRs, 2) the dose-dependent inhibition of growth by 1,25-(OH)2D3, and 3) a significant increase in 24-hydroxylase up regulation by 1,25-(OH)2D3 compared to that in controls. These data indicate that nuclear VDR expression is sufficient to mediate the antiproliferative effects of 1,25-(OH)2D3 on prostate cancer cells. In addition, because the stereoisomer 1 beta, 25-dihydroxyvitamin D3 failed to block these antiproliferative effects, we conclude that nongenomic mechanisms of action are not requisite for growth inhibition by 1,25-(OH)2D3. PMID- 8612486 TI - Splice variants of rat TR4 orphan receptor: differential expression of novel sequences in the 5'-untranslated region and C-terminal domain. AB - The use of rapid amplification of 5'-cDNA ends-PCR yielded two novel sequences for the rat orphan receptor, TR4, representing heterogeneity on the 5' untranslated region. Genomic structure analysis revealed that the 5'-untranslated region of the longer messenger RNA fragment, rTR4-1, contained three exons, alpha, beta, and gamma. The skipping of exon gamma gave rise to rTR4-2, indicating that rTR4-1 and rTR4-2 are products of alternative splicing. We isolated another novel rat TR4 splice variant, rTR4-NS, which was found to diverge from rTR4-2 at codon 504. rTR4-NS contained an unspliced intronic sequence with in-frame codons for eight amino acids followed by a termination codon. The three TR4 messenger RNA variants were differentially expressed. rTR4 NS appeared to be a rare transcript found in limited areas of the brain. In situ hybridization detect prominent TR4 signals in brain areas known to be involved in stress response. In cerebellar granule cells, the rise in TR4 expression correlated with the progression of neuronal maturation. N-Methyl-D-aspartate treatment triggered a marked increase in TR4 expression. These results suggest a possible role for TR4 in neuronal differentiation. PMID- 8612488 TI - Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum. AB - In this study we examined the prospect that the superoxide radical (SOR) is involved in the mechanism by which LH stimulates progesterone secretion in the rat corpus luteum (CL). Treatment of dispersed CL cells with low doses of LH or a SOR-generating system (xanthine-xanthine oxidase) resulted in a significant increase in progesterone release and SOR production. High doses of each treatment were inhibitory. SOR generation also decreased hCG binding. To determine whether SOR may be required for progesterone secretion, dispersed cells were electroporated with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and treated with either low (50 ng) stimulatory or high (20 micrograms) inhibitory doses of LH. At 50 ng LH, insertion of SOD or CAT dose-dependently inhibited progesterone secretion. However, at high doses of LH (20 micrograms), which are associated with high levels of SOR, electroporation of SOD or CAT produced the opposite response. This stimulatory response of SOD or CAT on progesterone release was also dose related. These results indicate that SOR may be involved in the mechanisms that stimulate as well as those that inhibit progesterone release. The effect on progesterone secretion appears to be dose related, with small increases associated with stimulation and high levels involved in inhibition of secretion. PMID- 8612487 TI - Cloning of rat 17 beta-hydroxysteroid dehydrogenase type 2 and characterization of tissue distribution and catalytic activity of rat type 1 and type 2 enzymes. AB - 17 beta-Hydroxysteroid dehydrogenases (17HSDs) are enzymes catalyzing the conversion between 17 beta-hydroxy- and 17-ketosteroids. Both estrogens and androgens possess their highest activity in the 17 beta-hydroxy form, and the enzymes, therefore, regulate the biological activity of sex hormones. In this study, we have characterized the complementary DNA (cDNA) for rat 17HSD type 2. The cDNA encodes a protein with a predicted mol wt of 42,010 Da. The protein has 77% similarity and 62% identity with the human 17HSD type 2 enzyme. Furthermore, the hydropathicity profiles of the enzymes are very similar. The two isozymes contain a putative transmembrane region close to the N-terminus. However, the rat isozyme lacks the two lysine-rich amino acid cluster present at the N- and C terminals of human 17HSD type 2. The tissue distribution of the rat 17HSD type 1 and type 2 enzymes is very similar to that of the human enzymes. The highest expression of 17HSD type 2 was detected in the placenta. In addition, a 1.5 kilobase messenger RNA for the enzyme was detected in the small intestine, liver, and kidney of both sexes. The two messenger RNAs for rat 17HSD type 1 (1.4 and 1.7 kilobases) were highly expressed only in the ovary, and at very low concentrations in the kidney of both sexes. Transiently expressed rat 17HSD type 2 showed oxidative activity almost exclusively in cultured human embryonic kidney 293 cells, converting estradiol into estrone and testosterone into androstenedione, whereas the opposite was observed for the rat type 1 enzyme. The data suggest that similarly to the corresponding human isoforms, rat 17HSD type 2 is mostly involved in the oxidation of 17 beta-hydroxysteroids into their relatively inactive keto derivative in peripheral tissues, whereas rat 17HSD type 1 is mainly involved in the glandular biosynthesis of estradiol. PMID- 8612489 TI - Somatostatin antisense oligodeoxynucleotide-mediated stimulation of lymphocyte proliferation in culture. AB - We have previously shown that somatostatin (SRIF) is synthesized in B and T lymphocytes of rat spleen and thymus and released into the medium of cultured lymphocytes. To determine the role of SRIF in the control of lymphocytes proliferation, the expression of SRIF in normal lymphocytes was inhibited using a 3'-terminal phosphorothioate-modified antisense oligonucleotide complementary to a sequence that includes the translation start site of the rat SRIF messenger RNA. Spleens were obtained from adult male rats, and their lymphocytes were cultured for 24 or 72 h to measure SRIF content and cell proliferation, respectively. For the proliferation studies, [3H]thymidine was incorporated during the final 18 h. The lymphocytes were incubated with 15-30 micrograms/ml SRIF antisense and control antisense. SRIF antisense (25 micrograms/ml) increased lymphocyte proliferation 15-fold (P < or = 0.001), reaching a plateau (25- to 30 fold increase) between 25-30 micrograms/ml SRIF antisense. SRIF was extracted from lymphocytes and measured by RIA. Levels of SRIF content were almost undetectable with 10 micrograms/ml antisense and were significantly lower (P < or = 0.01) with 25 micrograms/ml antisense. When RC 160 (10(-5) M), a SRIF agonist analog, was used in the incubation, the stimulation of cell proliferation exerted by the SRIF antisense was completely abolished. Control antisense had no effect on proliferation or SRIF content. These findings indicate that 1) lymphocytes in culture are able to incorporate SRIF antisense; and 2) SRIF antisense inhibits the expression of lymphocytic SRIF, which leads to lymphocyte proliferation. In conclusion, cell proliferation is dramatically increased by eliminating the expression of SRIF from the lymphocytes, which indicates that in vitro SRIF is acting in a paracrine and/or autocrine fashion to inhibit lymphocyte proliferation. PMID- 8612490 TI - Effects of interleukin-1 beta on thyrotropin secretion and thyroid hormone uptake in cultured rat anterior pituitary cells. AB - The effects of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) on basal and TRH-induced TSH release, and the effects of IL-1 beta on the uptake of [125I]T3 and [125I]T4 and on nuclear binding of [125I]T3 were examined. Furthermore, the release of other anterior pituitary hormones in the presence of IL-1 beta was measured. Anterior pituitary cells from male Wistar rats were cultured for 3 days in medium containing 10% FCS. Incubation were performed at 37 C in medium with 0.5% BSA for measurement of [125I]T3 uptake and with 0.1% BSA for measurement of [125I]T4 uptake. Exposure to IL-1 beta (1 pM-1 nM) or TNF alpha (100 pM) for 2-4 h resulted in a significant decline in TSH release, which was almost 50% (P < 0.05) for 1 nM IL-1 beta and 24% (P < 0.05) for 100 pM TNF alpha. Measurement of other anterior pituitary hormones (FSH, LH, PRL, and ACTH) in the same incubation medium showed that IL-1 beta did not alter their release. When the effects of IL-1 beta (1 pM-1 nM) and TNF alpha (100 pM) on TRH-induced TSH release were measured in short term experiments, the inhibitory effects had disappeared. The addition of 1-100 nM octreotide, a somatostatin analog, resulted in a decrease in TRH-induced TSH release up to 33% of the control value (P < 0.05). Exposure to dexamethasone (1 nM to 1 microM) affected basal and TRH-induced TSH release similar to the effect of IL-1 beta. The 15-min uptake of [125I]T3 and [125I]T4, expressed as femtomoles per pM free hormone, was not affected by the presence of IL-1 beta (1-100 pM). When IL-1 beta (100 pM) was present during 3 days of culture, TSH release was reduced to 88 +/- 2% of the control value (P < 0.05). This effect was not associated with an altered [125I]T3 uptake (15 min to 4 h) or with any change in nuclear T3 binding. We conclude that 1) IL-1 beta decreases TSH release by a direct action on the pituitary; 2) this effect is not due to elevated thyroid hormone uptake or increase T3 nuclear occupancy; 3) IL-1 beta does not affect TRH-induced TSH release or the release of other anterior pituitary hormones; and 4) TNF alpha affects basal and TRH-induced TSH release in the same way as IL-1 beta. PMID- 8612492 TI - Elimination of the carboxy-terminal sequences of parathyroid hormone-related protein 1-173 increases production and secretion of the truncated forms. AB - The endoproteolytic processing of polypeptides at basic residues into distinct biologically active peptides is a common theme in prohormone maturation and processing. PTH-related protein (PTHrP) 1-173 contains eight putative endoproteolytic consensus sites that include a mono-arginyl (R37), paired basic (RR154-155), and related basic residue motifs (RLKR-4 to -1, RRR19-21, KKKK88-91, KRK96-98, KKKRR102-106, and KKKK147-150). To analyze the primary structural determinants involved in the posttranslational processing and secretion of PTHrP 1-173, we constructed a series of nonsense mutants that code for carboxy-terminal truncated polypeptides. Since the basic residue motifs are probable sites of endoproteolysis, these sites and the residues downstream were serially eliminated, thereby creating PTHrP 1-152, 1-146, 1-101, 1-95, 1-87, 1-36, and 1 18. The wild type PTHrP 1-173 and nonsense mutant constructs were transiently transfected into two cell lines, COS-1 and SK-N-BE(2). The COS-1 cells have a constitutive secretory pathway, whereas the neuroblastoma-derived BE-2 cells have, in addition, a regulated secretory pathway. PTHrP was measured in the conditioned media and cell extracts of the transfected cells with two peptide specific RIAs. In COS-1 cells, PTHrP truncation mutants 1-152, 1-146, 1-101, 1 95, and 1-87 were present relative to wild type isoform 1-173, at 4.4-, 3-, 19-, 12-, and 57-fold excess, respectively; a similar pattern was also detected with BE-2 transfected cells, although the relative increases above the quantities of PTHrP 1-173 were not as dramatic. As the carboxy-terminal sequences were eliminated, the amount of total and secreted PTHrP increased, and the percentage found within the cell decreased. In COS-1 cells, 10.5% of the total PTHrP 1-173 was intracellular, whereas only 1% of the total PTHrP 1-87 was intracellular. In BE-2 cells, 54% of the total PTHrP 1-173 and only 9% of the total 1-87 mutant were intracellular. In COS-1 cells, a time course analysis demonstrated that PTHrP 1-87 and 1-95 were detectable in media 3 h after transfection, whereas 1 173 was barely detectable after 24 h. Our studies suggest that the carboxy terminal sequence of PTHrP 1-173 is responsible for the intracellular degradation of this polypeptide, which may be the endogenous cellular mechanism that regulates the amount of processed and secreted PTHrP. PMID- 8612491 TI - Proteolytic activity is involved in changes in intrafollicular insulin-like growth factor-binding protein levels during growth and atresia of ovine ovarian follicles. AB - In the sheep, follicular growth is characterized by both an increase and a decrease in the level of intrafollicular insulin-like growth factor-binding protein-3 (IGFBP-3) and IGFBPs less than 40 kDa (IGFBP-2, -4, and -5), respectively. In contrast, follicular atresia is associated with a decrease and a large increase in levels of IGFBP-3 and IGFBPs less than 40 kDa, respectively. To assess whether intrafollicular proteases are involved in such changes, follicular fluid from follicles of different sizes and degrees of atresia was incubated alone or with pure human IGFBP-3, -4, or -5 or serum (as a source of exogenous IGFBP-2) for 20 h at 37 C. Samples were then analyzed by Western ligand blotting and by immunoblotting using specific antisera. Ovine follicular fluid from different classes of follicles contained proteolytic activity degrading IGFBP-2, 3, -4, and -5. Degradation of IGFBPs was accompanied by the generation of small proteolytic fragments visualized by immunoblotting or after autoradiography using radiolabeled IGFBP-4. Moreover, follicular growth and atresia were characterized by changes in IGFBP proteolytic activity. Indeed, follicular growth (between 2 and 6 mm in diameter) was characterized by 1) a decrease in IGFBP-3 proteolytic activity and 2) a dramatic increase in proteolytic activity degrading IGFBP-4 and, to a lesser extent, IGFBP-2 and -5. Atresia, in contrast, was associated with a strong increase in IGFBP-3 proteolytic activity in small ( < 3-mm diameter) follicles and a decrease in IGFBP-4 and -5 proteolytic activity in large ( > 5-mm diameter) follicles. Regardless of the follicle class, IGFBP proteolytic activity was strongly inhibited by EDTA and 1,10-phenanthroline, but very slightly or not at all inhibited by tissue inhibitor of matrix metalloprotease-1 and-2 and BB-2116 (natural and synthetic inhibitors of matrix metalloproteases, respectively) as well as cysteine and serine proteases inhibitors, with the exception of phenylmethylsulfonylfluoride (1 mM) in atretic follicles. In addition, IGFBP proteolytic activity was dependent on the presence of zinc and calcium chloride. Zymography experiments showed the presence of 72- and 92- to 96-kDa gelatinases in follicular fluid; their levels were dramatically increased during follicular atresia. These results suggest that 1) changes in intrafollicular IGFBP proteolytic activity could be at least partly responsible for the changes in intrafollicular IGFBP levels that occur during follicular growth and atresia in the sheep; and 2) metalloprotease(s) in healthy and atretic follicles as well as serine protease(s) in atretic follicles are involved in IGFBP degradation. PMID- 8612493 TI - An oviposition-inducing peptide: isolation, localization, and function of avian galanin in the quail oviduct. AB - It is well established that avian oviposition is regulated, at least partly, by a neurohypophysial hormone, arginine vasotocin, and ovarian hormones, prostaglandins, are regulated through mechanisms of the induction of uterine contractions. Although abundant nerves are terminated in the musculature in the uterine and vaginal oviduct regions, limited information is available on the neuronal control mechanism of avian oviposition. To identify the oviduct factor that plays an important role in the induction of oviposition as a neurotransmitter or a neuromodulator, a study was conducted to isolate the bioactive substance involved in the vaginal and uterine contractions from mature oviducts of the Japanese quail. Acetic acid extracts of 200 quail oviducts were forced through disposable C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange HPLC purifications. A purified bioactive substance showed a single peak on the reversed-phase HPLC and was further subjected to amino acid sequence analysis and molecular weight presumption. The substance was identified as avian galanin, which was previously isolated from chicken intestine. The synthetic peptide enhanced contractions of both the vagina and the uterus in a manner similar to the native peptide, and the threshold concentrations were 10(-9) - 10(-8) M in the vagina and 10(-10) - 10(-9) M in the uterus. An intraperitoneal injection of the synthetic peptide significantly evoked quail oviposition during 5 min after injection. Immunohistochemical analysis with the anti-galanin serum revealed that abundant immunoreactive fibers were distributed in muscle layers of the vagina and the uterus. The immunoreaction examined in these regions was completely inhibited by preincubation of the antibody with synthetic peptide. These results suggest that avian galanin in the oviduct evokes oviposition through mechanisms of the induction of uterine and vaginal contractions. This peptide may contribute as a neurotransmitter or a neuromodulator to avian oviposition. PMID- 8612494 TI - Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. AB - alpha-Melanocyte stimulating hormone (alpha-MSH) and ACTH increase the proliferation and melanogenesis of cultured human melanocytes. To further analyze how melanotropins produce these biological effects, we investigated the regulation of the melanocortin receptor MC1R expression by alpha-MSH and ACTH using Northern blot analysis and determine the relative affinity of the receptor for the structurally similar peptides alpha-MSH, ACTH, beta-MSH, and gamma-MSH. We also determined the relative potencies of these hormones to stimulate cAMP formation, tyrosinase activity, and melanocyte proliferation. The order of affinity and potency of the noted melanotropins in these assays were alpha-MSH = ACTH > beta-MSH > gamma-MSH. Because the binding affinity of each of these melanotropins for the MC1R correlated with its ability to stimulate human melanocyte proliferation and melanogenesis, we conclude that these effects are mediated specifically by binding to and activation of the MC1R. gamma-MSH stimulated cAMP formation without affecting proliferation or melanogenesis. However, we found that relative to alpha-MSH, the effect of gamma-MSH on cAMP formation was transient. Our results suggest that alpha-MSH, ACTH, and possibly beta-MSH, but not gamma-MSH, are capable of a physiological role in regulating human pigmentation, and that melanocytes in human skin are a specific target for these hormones. PMID- 8612495 TI - Gonadotropin-releasing hormone regulation of pituitary follistatin gene expression during the primary follicle-stimulating hormone surge. AB - Follistatin is produced in the gonadotrope and folliculostellate cells of the pituitary gland and is thought to indirectly regulate FSH biosynthesis and secretion through its ability to bind activin. Recent measurements of follistatin gene expression during the rat estrous cycle revealed a marked increase in pituitary follistatin messenger RNA (mRNA) levels at the time of the preovulatory FSH surge. This finding suggests a role for follistatin in the regulation of FSH at this dynamic time of the cycle. The aim of the present study was to identify the hormonal control mechanisms responsible for stimulating follistatin gene expression on proestrus. In particular, the roles of estrogen (E) and GnRH were assessed using an in vivo ovariectomized (OVX) animal model in which steroid priming results in daily gonadotropin surges. Follistatin mRNA and serum FSH levels were unchanged throughout the day in untreated OVX rats. E priming of OVX rats elicited a 2-fold elevation in follistatin mRNA levels between 1600-2000 h coincident with the peak of the E-induced FSH surge. To determine whether this effect of E on follistatin mRNA levels was the result of the direct or indirect effects of E on the pituitary, follistatin mRNA levels were examined in E-primed OVX rats that had been treated with pentobarbital at 1430 h (to block hypothalamic neurosecretion). Pentobarbital treatment prevented the E-induced increase in follistatin mRNA levels, suggesting that the effects of E are mediated via GnRH or other hypothalamic factors. The effects of GnRH on follistatin gene expression were examined further using an in vitro perifusion model. Proestrous or metestrous pituitaries were perifused for 8 h with pulsatile GnRH (one pulse per h), continuous GnRH, or medium only. Continuous GnRH treatment resulted in a significant elevation in follistatin mRNA levels in both proestrous and metestrous pituitaries, whereas pulsatile GnRH had no effect at either cycle stage. These results suggest that the proestrous GnRH surge is responsible at least in part for the elevation in pituitary follistatin mRNA levels that is associated with the primary FSH surge. GnRH-induced follistatin production on proestrus probably plays a role in the dynamic regulation of FSH at this time of the ovulatory cycle. PMID- 8612496 TI - Glucokinase, hexokinase, glucose transporter 2, and glucose metabolism in islets during pregnancy and prolactin-treated islets in vitro: mechanisms for long term up-regulation of islets. AB - During pregnancy, islets undergo a number of up-regulatory changes to meet the increased need for insulin. One of the most important changes is an increase in glucose-stimulated insulin secretion with a reduction in the glucose-stimulated threshold. Similarly, placental lactogen and PRL induce the same changes in islets as pregnancy. In this study, we examined the effects of pregnancy and PRL treatment of islets in vitro on insulin secretion; glucokinase and hexokinase activities; glucokinase, hexokinase, and glucose transporter 2 protein levels; and rates of glucose utilization and oxidation. Glucokinase activity was 4.9 +/- 0.4 pmol glucose/ng DNA.h in control islets and was significantly increased by 50% in islets on day 15 of pregnancy and by 60% on day 20 of pregnancy. Hexokinase activity was 11.7 +/- 0.9 pmol glucose/ng DNA.h in control islets and was increased by 20% in islets on day 15 of pregnancy and by 90% on day 20 of pregnancy. In the in vitro studies, glucokinase activity was 7.4 +/- 0.89 pmol glucose/ng DNA.h in control islets. PRL treatment of islets in vitro increased glucokinase activity by 60%, an effect similar to that observed in the pregnancy islets. In contrast, hexokinase activity was nearly undetectable in cultured islets, whether control or PRL treated. Quantitative Western blot analysis of glucokinase and hexokinase was performed using equivalent number of protein per lane for all experimental groups. On a protein equivalency basis, glucokinase expression levels were the same in control islets on days 15 and 20 of pregnancy. Likewise, hexokinase levels were not different between control islets and islets on days 15 and 20 of pregnancy. Similarly, Western blot analysis of cultured islets indicated that there were not effect of PRL on glucokinase or hexokinase levels. However, when enzyme levels were normalized on the basis of DNA, the levels of expression appeared to be commensurate with their activities. In cultured islets, the very low level of hexokinase activity corresponded to the low level of hexokinase detected by Western blots. Glucose transporter 2, as determined by Western blot quantification, was increased 2-fold in pregnancy islets on day 15 and increased by 45% in pregnancy islets on day 20. Similar results were observed in cultured islets where glucose transporter 2 was increased 2-fold in PRL-treated islets. Islet glucose utilization and oxidation rates on day 15 of pregnancy were significantly greater than those in control islets at all glucose concentrations examined. This enhanced glucose sensitivity resulted in a shift of the glucose utilization and oxidation response curves to the left. Comparable results were obtained from islets on day 20 of pregnancy. PRL treatment of islets in vitro resulted in the same changes in glucose utilization and oxidation rates that were observed during pregnancy. These results demonstrate changes in glucokinase, hexokinase, and glucose transporter 2 levels and glucose metabolism that occur as islets adapt to an increased need for insulin secretion during pregnancy. The results also indicate that these same changes can be induced by PRL treatment of islets in vitro. This provides further evidence that the long term adaptive changes that occur under the normoglycemic conditions of pregnancy are mediated by lactogen-regulated events. PMID- 8612497 TI - The ontogeny of hepatic growth hormone receptor and insulin-like growth factor I gene expression in the sheep fetus during late gestation: developmental regulation by cortisol. AB - The effects of cortisol on hepatic GH receptor and insulin-like growth factor-I (IGF-I) gene expression were investigated in sheep fetuses during late gestation and after experimental manipulation of plasma cortisol levels by fetal adrenalectomy and exogenous infusion of cortisol. Hepatic GH receptor and IGF-I messenger RNA (mRNA) levels increased with increasing gestational age in parallel with the normal rise in fetal cortisol levels toward term (145 +/- 2 days). These increases in mRNA abundance toward term were prevented when the prepartum cortisol surge was abolished by fetal adrenalectomy and were stimulated prematurely in fetuses younger than 130 days by exogenous infusion of cortisol. Both the class 1 and class 2 transcripts of the IGF-I gene were increased when cortisol levels were elevated either endogenously or exogenously. However, there were no significant changes in fetal plasma IGF-I levels either with increasing gestational age or in response to experimental manipulation of the fetal cortisol level. When the data from all the fetuses were combined irrespective of treatment or gestational age, there were significant positive correlations between the log plasma cortisol concentration in utero and the abundance of GH receptor and IGF-I mRNA in the fetal liver. There was also a significant inverse relationship between log plasma cortisol and the ratio of class 1 to class 2 transcript abundance in the fetal liver. These findings show that cortisol is a physiological regulator of hepatic GH receptor and IGF-I gene expression in fetal sheep during late gestation and indicate that it preferentially increases the class 2 transcript of the IGF-I gene. The prepartum cortisol surge therefore appears to have an important maturational role in initiating the perinatal switch from the fetal to adult modes of somatotrophic regulation. PMID- 8612498 TI - Regulation of erythropoietin gene expression in fetal sheep by glucocorticoids. AB - Erythropoietin (Epo) gene expression in ovine fetal liver and kidneys was measured by competitive RT-PCR in situations in which fetal glucocorticoid status was altered. Bilateral adrenalectomy at 120 +/- 0.3 days gestation (term is 145 150 days) caused a significant (P < 0.05) 5-fold increase in renal Epo messenger RNA (mRNA) levels at 145 +/- 1 days compared to those in age-matched controls. With cortisol replacement in adrenalectomized fetuses, renal Epo mRNA levels dropped to 17% of this values (P < 0.05). Cortisol infusion (230 micrograms/h for 48 h) at 108 +/- 1 day decreased renal Epo gene expression significantly (P < 0.01) to 23% of the control value; dexamethasone treatment of the ewe at midgestation (0.76 mg/h for 48 h) also decreased fetal, but not adult, renal Epo mRNA levels (to 12% of control value; P < 0.01). Fetal and maternal liver Epo mRNA levels were unaffected by glucocorticoid status at any stage of pregnancy. Thus, glucocorticoids can influence fetal renal, but not maternal, Epo gene expression. In the presence of high concentrations of fetal glucocorticoids, plasma Epo values were consistently 4-5 mU/ml, close to the sensitivity of the assay, whereas in seven adrenalectomized fetuses, the plasma Epo value was 9.1 +/ 1.4 mU/ml. PMID- 8612499 TI - Species differences in the induction of time-dependent potentiation of insulin secretion. AB - The secretory responsiveness of the pancreatic beta-cell can be markedly improved by prior short term exposure to a stimulatory glucose level. Termed time dependent potentiation (TDP), priming, or sensitization, this phenomenon has been documented to occur in both human and rat islets and my involve, at least in part, information flow in the phospholipase C and protein kinase C (PKC) signal transduction pathway. In contrast to human and rat islets, however, mouse islets fail to exhibit TDP in response to priming with high glucose. In the present series of studies, we explored in more detail the conditions and stimulants necessary for the induction of TDP in mouse islets and compared these responses with those obtained in rat islets. In agreement with previous reports, high (15 mM) glucose alone primed the rat beta-cell, but not the mouse beta-cell, to subsequent restimulation with 15 mM glucose. However, muscarinic stimulation of mouse islets with carbachol (100 microM) in the presence of 15 mM glucose primed the beta-cell to a subsequent 15-mM glucose stimulus. In addition, prior exposure to 50 nM of the PKC activator tetradecanoyl phorbol acetate dramatically amplified the subsequent insulin secretory responses of mouse islets to 15 mM glucose. In contrast to its significant inhibitory effect on glucose-induced insulin release from rat islets, the PKC inhibitor staurosporine (50 nM) had not effect on 15 mM glucose-induced release from control or prior glucose-exposed mouse islets. However, staurosporine significantly reduced the priming effect of tetradecanoyl phorbol acetate or carbachol on 15 mM glucose-induced insulin secretion from mouse islets. These findings emphasize the dramatic species differences that exist in the capacity of prior high glucose stimulation to induce TDP in rat and, presumably, human islets, on the one hand, and mouse islets, on the other. They also serve to emphasize the role of phosphoinositide hydrolysis and PKC activation in the induction of TDP. PMID- 8612500 TI - Ovarian expression of insulin-like growth factor-I (IGF-I), IGF binding proteins, and growth hormone (GH) receptor in heifers actively immunized against GH releasing factors. AB - Active immunization against GRF at 6 months of age delays puberty in beef heifers. The objectives of the present study were to determine whether active immunization against GRF at an earlier age would affect normal onset of puberty and follicular growth and to determine whether these changes were related to alterations in ovarian insulin-like growth factor I (IGF-I) or IGF binding protein (IG-FBP) messenger RNA (mRNA) levels. Heifers were immunized against human serum albumin (HSAi; n = 15) or against GRF conjugated to HSA (GRFi; n = 18) at 3 months of age. A third group of heifers was not immunized (CON; n = 16). Immunization against GRF delayed puberty beyond 13 months of age in 75% of treated heifers. Unilateral ovariectomy at 191 days of age revealed that the delay in puberty was associated with a reduction in the number of large ( > or = 7 mm in diameter) follicles. Large follicles were present in only 22% of GRFi heifers compared to 77% of HSAi heifers. The number of small ( < or = 3 mm in diameter) and medium (4 to 6 mm in diameter) follicles was not affected by GRFi. The percentage of 1- to 3-mm follicles that were atretic was not different between HSAi (65%) and GRFi (62%) heifers. Unilateral ovariectomy had no effect on age at puberty. Immunization against GRF decreased (P < 0.01) concentrations of IGF-I in serum (23 +/- 2 ng/ml) compared to HSAi heifers (109 +/- 11 ng/ml). IGF-I levels in follicular fluid (FFL) of medium and small follicles were also decreased by GRFi from 82 +/- 3 ng/ml in HSAi heifers to 48 +/- 6 ng/ml (P < 0.01). Levels of IGFBP-3 (determined by ligand blot analysis) in serum and FFL of small follicles were decreased by GRFi (P < 0.01). In contrast, IGFBP-2 serum levels were increased from 422 +/- 32 ng/ml in HSAi heifers to 657 +/- 6 ng/ml in GRFi heifers (P < 0.05). Likewise, IGFBP-2 levels in FFL from small and medium follicles were increased from 785 +/- 44 ng/ml to 926 +/- 44 ng/ml (P < 0.05). Ligand blot analysis indicated that IGFBP levels were lower in FFL from large vs. small follicles. The band intensities of IGFBP-4 and -5 were drastically reduced ( > 80%) while the decreases in IGFBP-2 and -3 were less marked ( < 50%). The decreased levels of IGFBP-5 in FFL from large follicles was not associated with an increase in proteolytic fragments detectable by immunoblot analysis. While mRNA transcripts for IGF-I, GH receptor, and IGFBP-2, -3, -4, and -5 were readily detectable in ovarian tissue, GRFi had no effect on ovarian levels of mRNA for each of these proteins. This suggests that the decrease in follicular development associated with GRFi may be related to changes in circulating IGF-I and/or IGFBPs. PMID- 8612501 TI - Divergent prolactin and pituitary-adrenal activity in rats selectively bred for different dopamine responsiveness. AB - The present study explores the significance of brain dopamine phenotype for individual variation in the neuroendocrine stress response of the rat. For this purpose, we used two Wistar rat lines previously selected for high or low responsiveness of the dopamine system to apomorphine using the gnawing response as the selection criterion. Systemic administration of the drug evoked in apomorphine-susceptible (apo-sus) rats a vigorous gnawing response, whereas apomorphine-unsusceptible (apo-unsus) rats did not gnaw under these conditions. These two rat lines represent individuals displaying extreme differences in gnawing behavior that otherwise coexist in a normal Wistar population. In this study basal and stress-induced hypothalamic-pituitary-adrenal activity and PRL release were measured in chronically cannulated, freely moving rats that endured a conditioned emotional response. Tyrosine hydroxylase messenger RNA (mRNA), corticosteroid receptor mRNA, and in vivo retention of [3H]corticosterone were measured in rat brain sections using in situ hybridization and in vivo autoradiography. The result show that 1) apo-sus rats had a markedly reduced PRL response to stress compared to apo-unsus animals, whereas basal levels were not significantly different. A12 dopaminergic neurons in the arcuate nucleus expressed significantly higher levels of tyrosine hydroxylase mRNA in apo-sus rats, suggesting that the reduced stress-induced PRL release could be due to an increased inhibitory control by dopaminergic neurons; 2) in apo-sus rats, stress resulted in a sustained elevation of ACTH and free corticosterone levels, whereas the total corticosterone levels were not different between the two rat lines; 3) under basal morning conditions, apo-sus rats had significantly higher plasma ACTH, but, in contrast, lower free corticosterone than apo-unsus rats; total plasma corticosterone levels were not different; 4) the basal evening ACTH level was elevated in apo-sus rats; after removal of the adrenals in the morning, this increased ACTH level in apo-sus rats persisted into the afternoon 6 h postadrenalectomy; and 5) hippocampal mineralocorticoid (MR), but not glucocorticoid (GR), receptor capacity for the ligand comparable between the groups; the MR of apo-sus rats displayed an increased retention of [3H]corticosterone in all hippocampal cell fields measured 24 h adrenalectomy; MR and GR mRNA in hippocampus as well as GR mRNA in the paraventricular nucleus were not significantly different in the two rat lines. In conclusion, the data suggest a common genetic background for individual variation in stress responsiveness and dopamine phenotype. High dopamine reactivity is linked to a reduced PRL and an increased ACTH response after stress. These high dopamine responders display a hyporesponsive adrenal cortex and corticosteroid feedback resistance associated with altered brain corticosteroid receptor properties. PMID- 8612502 TI - Cortisol enhances the transcription of insulin-like growth factor-binding protein 6 in cultured osteoblasts. AB - Previous work indicate that glucocorticoids inhibit the synthesis of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IG-FBP-3), -4, and -5, but not IGFBP-6, in osteoblast cultures. IGFBP-6 binds IGF-II with high affinity and prevents IGF-II-mediated effects. As IGF-II is present at high concentrations in bone, we postulate that glucocorticoids may regulate IGF-II by altering IGFBP-6 synthesis. We tested the expression of IGFBP-6 in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Treatment of Ob cells with cortisol caused a time- and dose-dependent increase in IGFBP-6 messenger RNA levels, as determined by Northern blot analysis. The effect was maximal after 48 h of treatment and observed with cortisol concentrations of 10 nM to 1 microM. Treatment with cortisol also increased IGFBP-6 polypeptide levels in the medium, as determined by Western immunoblot analysis. Cycloheximide at 3.6 microM decreased IGFBP-6 transcripts and prevented the stimulatory effect of cortisol. Cortisol did not modify the decay of IGFBP-6 messenger RNA in transcriptionally arrested Ob cells. In addition, cortisol increased the rate of IGFBP-6 transcription, as determined by nuclear run-on assays. In conclusion, cortisol stimulates IGFBP-6 expression in Ob cells by transcriptional mechanisms. As IGFBP 6 binds to and prevents the effect of IGF-II, its increased synthesis could be relevant to the inhibitory actions of cortisol in bone. PMID- 8612503 TI - Involvement of pituitary adenylate cyclase-activating polypeptide in growth hormone secretion induced by serotoninergic mechanisms in the rat. AB - Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) stimulated GH secretion in superfused rat anterior pituitary cell in vitro and in conscious male rats in vivo. PACAP-38-induced GH secretion was inhibited by PACAP-(6-38), an N-terminal-deleted analog, at 100-fold concentrations of PACAP-38 both in vitro and in vivo. In contrast, a GH-releasing hormone antagonist did not affect the action of PACAP-38 to stimulate GH release in vitro. Plasma GH increase induced by i.v. injection of 5-hydroxy-L-tryptophan (1 mg/100 g BW), a precursor of serotonin, was blunted by PACAP-(6-38) (1 nmol/100 g BW, i.v.), whereas spontaneous pulsatile GH secretion in conscious male rats, which is governed by hypothalamic GH-releasing hormone and somatostatin, was not affected by repeated i.v. injection of PACAP-(6-36). These findings suggest that PACAP-(6-38) is a potent antagonist of PACAP-38 to stimulate GH secretion both in vivo and in vitro. Taken together with the facts that PACAP-38 is highly concentrated in the hypothalamus and that is released into the hypophysial portal blood, our present findings suggest that PACAP-38 might play a stimulatory role on GH secretion induced by serotoninergic mechanisms in the rat. PMID- 8612505 TI - Natriuretic peptide receptors on rat thymocytes: inhibition of proliferation by atrial natriuretic peptide. AB - Because the thymus expresses the natriuretic peptides (NP) as well as their respective receptors, an involvement of NP in the physiology of this organ has been suggested. To evaluate functional aspects of NP in the thymus, we looked for thymic cells bearing NP receptors (Npr). Furthermore, the regulation of Npr expression by activation of cells and the influence of NP on the proliferation of thymocytes was studied. Expression of receptor messenger RNAs (mRNAs) was examined by PCR and Northern blot. Existence of functional Npr was confirmed by measurement of cGMP, the second messenger of NP. Proliferation of thymocytes upon concanavalin A (Con A) stimulation was analyzed by incorporation of [3H]thymidine. We report her that thymocytes express mRNAs for the three Npr, namely Npra, Nprb, and Nprc and that activation of Npra and Nprb increases cGMP levels. Stimulation of thymocytes with Con A (1 microgram/ml, 48 h) resulted in an increase of mRNA coding for Npra, the receptor specific for atrial natriuretic peptide (ANP) and brain natriuretic peptide. Nprb and Nprc receptor expression was not altered under these conditions. In agreement with these data only ANP, but not the C-type natriuretic peptide, elicited increased cGMP response in Con A stimulated cells. ANP inhibited also the proliferation of Con A stimulated thymocytes, whereas C-type natriuretic peptide did not show this effect. These results suggest that ANP affects the complex mechanisms of thymocyte proliferation and differentiation. PMID- 8612504 TI - Prostaglandin E receptor subtypes in mouse osteoblastic cell line. AB - PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone. PMID- 8612506 TI - Suppression of endogenous corticosterone levels in vivo increases the steroidogenic capacity of purified rat Leydig cells in vitro. AB - In vitro studies have shown that corticosterone (B) directly inhibits testosterone (T) production by purified Leydig cells but does so only at high concentrations. 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) in Leydig cells oxidatively inactivates B, lowering its effective concentration, thus protecting against the suppressive effect of glucocorticoid on T production. The aim of the present study was to assess the significance of B at physiological levels in modulating T production and 11 beta-HSd activity in Leydig cells. To determine the effects of endogenous B on Leydig cell steroidogenesis, male rats (200-250 g body wt) were adrenalectomized (ADX), while control rats were subjected to sham surgery (SHAM). Seven days after surgery: T and LH were measured in serum; T production was measured in aliquots of spent culture media from 3-h incubations of purified Leydig cells; 11 beta-HSD activity and messenger RNA was measured in purified Leydig cells. ADX rats had elevated serum T (P < 0.05) in contrast to SHAM control or ADX rats that received B replacement (1 mg/100 g body wt per day, i.p., on the final 3 days). Serum LH levels were uninfluenced by ADX, with or without B replacement (SHAM), 0.45 +/- 0.16 ng/ml; ADX, 0.35 +/- 0.13 ng/ml; ADX + B, 0.61 +/- 0.09 ng/ml, NS, P > 0.05). This indicated that the alteration of T production was induced by a mechanism that is independent of LH. ADX nearly doubled LH-stimulated T production by purified Leydig cells, from 106.3 +/- 9.3 (SHAM) to 183.2 +/- 16.7 (ADX) ng/10(6) cells.3 h (mean +/- SEM for three replications of the experiment, P < or = 0.02). T production by Leydig cells from the ADX + B treatment group was suppressed to 53% of SHAM values, indicating that B inhibits T production after ADX. The oxidative activity of 11 beta-HSD in Leydig cells exceeded its reductive activity, and both activities declined after ADX. The decline in 11 beta-HSD activities after ADX was prevented by B replacement. Similarly, the steady state levels of 11 beta-HSD messenger RNA declined in Leydig cells after ADX, and this decline was prevented by B replacement. We conclude that physiological levels of B exert a tonic, negative control directly on Leydig cell steroidogenesis and also induce intracellular 11 beta-HSD activity, thereby protecting against B-mediated inhibition of T production. By modulating the level of active glucocorticoid in Leydig cells, 11 beta-HSD is thus a significant determinant of their steroidogenic capacity. PMID- 8612507 TI - Expression of growth hormone receptors in murine lymphoid cells analyzed by flow cytofluorometry. AB - We have analyzed the expression of GH receptors (GHR) in murine lymphoid organs using flow cytofluorometry with biotinylated bovine GH and specific fluorescein isothiocyanate-labeled monoclonal antibodies defining distinct lymphoid cell populations. GHR were widely expressed in al murine hematopoietic tissues and in fetuses, newborns, and 3- and 7-week-old animals. In the bone marrow, all hematopoietic lineages expressed variable levels of GH receptors, whereas in the thymus, this expression was mainly seen in CD4-, CD8-, CD4+CD8+, and CD8+ subpopulations. At the periphery, 50% of splenocytes and peripheral blood lymphocytes and 20% of lymph node cells were GHR positive, with a wider receptor expression on B cells and macrophages (approximately 50%) than on T cells (approximately 20%), where the labeling was seen on both CD4+ and CD8+ cell subsets. Interestingly, the proportion of GHR-bearing CD4+ and CD8+ splenocytes significantly increased after T cell activation with Concanavalin A or anti-CD3. Finally, we demonstrated that all peripheral T cells expressing GHR also expressed PRL receptors. Our study provides a molecular basis to study the factors controlling GHR expression and to better understand the influence of GH in the regulation of immune function. PMID- 8612508 TI - Suppression of cornified envelope formation and type 1 transglutaminase by epidermal growth factor in neoplastic keratinocytes. AB - Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF dependent reduction of cornified envelope formation. Only a modest ligand dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity. PMID- 8612509 TI - Peptide growth factor cross-talk with the estrogen receptor requires the A/B domain and occurs independently of protein kinase C or estradiol. AB - Modulation of steroid receptor-dependent transcription by extra- cellular ligands represents a novel mechanism of steroid receptor regulation. We have assessed the effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factor I (IGF-I) on transcription from consensus estrogen response elements (ERE) in estrogen receptor (ER)-positive BG-1 human ovarian adenocarcinoma calls. EGF, TGF alpha, IGF-I, and estradiol (E2) enhanced transcription in a dose-dependent manner using either a strong or a minimal promoter, and ICI 164,384, a specific ER antagonist, inhibited these responses. Combinations of E2 with TGF alpha or IGF-I induced synergistic activation of transcription from an ERE, whereas as additive response was observed with combinations of IGF-I and TGF alpha of EGF. Tetradecanoyl 12-phorbol 13-acetate (TPA), a protein kinase C (PKC) activator, stimulated ERE-mediated transcription, and this effect was inhibited by ICI 164,384. Bisindolylmaleimide, a relatively specific inhibitor of PKC, completely antagonized TPA-induced transcription, but did not affect the response to TGF alpha, IGF-I, or E2. The combination of TPA with E2 in transcriptional synergism was inhibited by ICI 164,384; conversely, the combination of TPA with either TGF alpha of IGF-I elicited a response only equal to the maximal TPA response. Thus, peptide growth factors elicit ER dependent transcription independently of PFC; however, there may be a common mechanistic component, as saturation of response was observed. Finally, activation of ERE-dependent transcription in Chinese hamster ovary cells by IGF-I was observed in the presence of a mutant receptor that lacks estrogen-binding activity. The effect of both IGF-I and E2 were dependent on the ability of the ER to bind to DNA. IGF-I elicited only weak transcriptional activation in the presence of a deletion mutant that lacked the entire A/B domain; however, synergism between IGF-I and E2 was observed with this mutant. Therefore, ligand independent activation of ER-dependent transcription by IGF-I is predominantly mediated through activation function I by a mechanism distinct from that of E2. PMID- 8612510 TI - Constitutive nitric oxide synthase in hypothalami of normal and hereditary diabetes insipidus rats and mice: role of nitric oxide in osmotic regulation and its mechanism. AB - Constitutive nitric oxide synthase (cNOS) was immunolocalized to study its role in osmotic regulation. Immunoreactivity was observed in all major hypothalamic osmoregulatory structures, the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, and supraoptic and paraventricular nuclei. These nuclei were compared in normal Long-Evans rats and homozygous Brattleboro rats with hereditary hypothalamic diabetes insipidus and in normal mice and mice with hereditary nephrogenic diabetes insipidus. About 50% of supraoptic neurons in Long-Evans rats and 90% in Brattleboro rats were cNOS immunopositive; a qualitatively similar difference occurred in the paraventricular nucleus. Mice with hereditary nephrogenic diabetes insipidus also showed a greater proportion of cNOS-positive supraoptic neurons (50%) than normal mice (20%). However, the number of cNOS-positive cells in the organum vasculosum laminae terminalis, subfornical organ, and median preoptic nucleus dis not differ significantly between diabetic and normal animals. The similar changes in cNOS in two mutant strains in which the only common feature is chronic osmotic stimulation shows that differences in vasopressin and oxytocin are not involved in the regulation of cNOS. The results suggest strongly that cNOS is involved in long term modulation of the hypothalamo-neurohypophysial system and, hence, body water and electrolyte homeostasis, and that cNOS is itself regulated by body osmotic status. PMID- 8612511 TI - Adrenal steroid precursors exert potent androgenic action in the hamster sebaceous glands of flank organs and ears. AB - To assess the effect of the androgen precursors dehydroepiandrosterone (DHEA) and androstenedione (4-ene-dione) on androgen-sensitive parameters in the skin of the hamster, these two steroids were released from SILASTIC implants inserted sc into castrated male hamsters. The pigmented area of the flank organs, the size of the underlying sebaceous glands, [3H]thymidine incorporation into these sebaceous glands, and the size of the ear sebaceous glands were measured. The decrease in flank organ size caused by orchidectomy was partially reversed by DHEA and completely reversed by 4-ene-dione, testosterone (T), or dihydrotestosterone (DHT) implants. Similarly, the size of the sebaceous glands of the flank organs was reduced 88.1% by castration, whereas DHEA, 4-ene-dione, T, and DHT implants reversed the effect of orchidectomy. Orchidectomy also decreased the sizes of the sebaceous glands of the ears; DHEA, 4-ene-dione, T, and DHT implants increased their sizes to 50.6%, 81.9%, 91.6%, and 105.8% of the values found in intact hamsters, respectively. Parallel results were observed on the labeling of flank organ sebaceous glands with [3H]thymidine as well as on prostate weight. Serum concentrations of T and DHT became undetectable in castrated hamsters and were increased to intact or slightly elevated values in animals receiving implants of DHEA, 4-ene-dione, or T. The present results show that DHEA and 4-ene-dione are potent stimulators of androgen-sensitive parameters in the sebaceous glands of both the flank organs and ears in the hamster and illustrate the importance of extragonadal or peripheral intracrine formation of active steroids. It is suggested that the castrated hamster supplemented with adrenal precursor steroids is a good model that can closely mimic the human situation, where adrenal steroids play an important role in androgen formation and action in peripheral tissues. PMID- 8612512 TI - Regulation of corticotropin-releasing factor receptor messenger ribonucleic acid in rat anterior pituitary. AB - Adrenalectomy (ADX) leads to a decrease in the number of CRF-binding sites in the rat anterior pituitary (AP). However, the molecular mechanisms of CRF receptor (CRF-R) regulation are unknown. In the present study, we analyzed the effects of ADX on pituitary CRF-R1 messenger RNA (mRNA) levels in vivo and the direct effects of CRF, arginine vasopressin (AVP), and glucocorticoid, the levels of which are altered by ADX, on CRF-R1 mRNA levels in vitro. The mRNA level was determined by Northern blot analysis using a rat brain CRF-R1 complementary RNA probe. The CRF-R1 level in AP fell to 20% of the sham level 1 day after ADX and returned to the sham level after 14 days. In cultured rat AP cells, treatment with CRF, AVP, and dexamethasone led to significant reductions in CRF-R1 mRNA, with maximal inhibition to 32%, 22%, and 37% of control levels, respectively. The time course of CRF-R1 mRNA reduction varied depending on the drug, with effects detectable as early as 1 h after treatment. These findings indicate that elevated portal CRF and AVP levels may contribute to the decrease in CRF-R1 mRNA soon after ADX. A decrease in mRNA levels, in turn, may lead to a decrease in CRF-R1 protein on corticotrophs. PMID- 8612513 TI - Proliferation and differentiation of a human colon cancer cell line (CaCo2) is associated with significant changes in the expression and secretion of insulin like growth factor (IGF) IGF-II and IGF binding protein-4: role of IGF-II. AB - The extent to which the insulin-like growth factor (IGF) system contributes to the initiation and progression of colon cancer remains poorly defined. We recently reported that a majority of human colon cancers express and secrete the potent mitogen IGF-II and at least two inhibitory binding proteins, IGFBP-2 and IGFBP-4. In the present study we measured the expression and secretion of IGF-II, IGFBP-2, and IGFBP-4 in relation to growth and differentiation of CaCo2 human colon cancer cells, which undergo spontaneous enterocytic differentiation in culture. Under the conditions of the present study, CaCo2 cells demonstrated an initial rapid phase of growth between Day 2 through days 7-9 of culture, followed by a significant retardation in the growth between days 9-13. Alkaline phosphatase (ALP) activity, a marker of enterocytic differentiation, progressively increased between Days 7-13 in culture, temporally correlating with post-confluent phase of negligible growth. These changes in growth and differentiation were accompanied by > 80% decline in the relative concentration of IGF-II messenger RNA (mRNA) between Days 2-13. In contrast, the relative mRNA concentrations of inhibitory binding proteins (IGFBP-2 and IGFBP-4) increased rapidly to 200% of Day 2 values by Days 5-7 before returning to baseline levels by Day 13. The relative protein concentrations of the three factors measured in the conditioned media of the cells followed a pattern very similar to that measured for the mRNA levels. While the changes in the relative protein concentrations and mRNA levels of IGF-II and IGFBP-4 were statistically significant, the changes measured in the RNA and protein levels of IGFBP-2 were not, as a result of large inter experimental variations. Thus these results suggested that CaCo2 cell differentiation may require an attenuation of IGF-II effects. To confirm the latter possibility, additional studies were conducted with a specific neutralizing antibody against IGF-II. Incubation of CaCo2 cells with anti-IGF-II antibodies from Day 0 through Day 7 significantly retarded the growth of the cells and was accompanied by a significant increase in the concentration of Alkaline phosphatase activity per 10(6) cells. Recently, we reported a potent inhibitory role of IGFBP-4 in the growth of colon cancer cells. In the present studies, a possible important role of IGF-II is illustrated not only in the growth but also in the differentiation of colonic cells. Our studies thus suggest that differential expression of IGF-II and IGFBPs may be playing a critical role in both proliferation and differentiation of colonocytes. PMID- 8612515 TI - Cell differentiation and endogenous cyclic adenosine 3',5'-monophosphate regulate osteopontin expression in human trophoblasts. AB - Integrin receptors and their extracellular matrix ligands have been implicated in the molecular and cellular mechanisms of trophoblast adhesion and migration. In the present series of experiments, the regulation of expression of osteopontin (OPN), a secretory extracellular matrix protein that mediates cell adhesion by binding to members of the alpha V family of integrins was investigated. Human chorionic villi were obtained during the mid and late first trimester, early second trimester, and late third trimester of pregnancy. In addition, cytotrophoblasts were isolated from chorionic villi, and the effects of exposure of cultured cytotrophoblasts to cAMP agonists or antagonists were assessed. OPN messenger RNA (mRNA) was expressed by human trophoblasts in the first trimester and throughout pregnancy. Using immunolocalization in prepared tissue sections, cytotrophoblasts stained intensely for OPN, but syncytial trophoblasts did not. This differentiation-dependent expression was confirmed in vitro by demonstrating that freshly isolated mononuclear cytotrophoblasts exhibited a high level of OPN mRNA, but as the cells aggregated and fused to form multinucleated syncytia in vitro, mRNA levels decreased dramatically. 8-Bromo-cAMP inhibited the expression of OPN mRNA, whereas the cAMP antagonist Rp-cAMP inhibited the OPN mRNA decrease during the in vitro differentiation of the cells. This study demonstrates the regulated expression of OPN by human trophoblasts. In addition, our data suggest that this expression is dependent upon the state of cellular differentiation of the trophoblasts and is regulated by endogenous cAMP. We speculate that binding of OPN to its alpha V beta 3-integrin receptor may be a critical signaling pathway contributing to the integrity of the chorionic villus and may also play a role in maternal-embryonic communication during the process of placentation. PMID- 8612514 TI - Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1. AB - In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2 specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP dependent pathway and that their promoter activity is dependent on the presence of Pit-1 PMID- 8612516 TI - Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. AB - Human endometrial stromal cells contain human CG (hCG)/LH receptors and in vitro, hCG/LH can promote stromal cells differentiation into decidua. In the present study, we tested the hypothesis that the treatment of stromal cells with exogenous hCG/LH to promote in up-regulation of cyclooxygenase-2 (COX-2) gene expression. The stromal cells from proliferative phase endometria were cultured for 10 days with 10 ng/ml estradiol and 100 ng/ml progesterone in the presence or absence of increasing concentrations of highly purified hCG. Northern blotting demonstrated that the cells contained a 4.4-kb COX-2 messenger RNA transcript whose levels significantly increased after treatment with hCG. Western blotting showed that the cells contained a 72-kDa COX-2 protein which also significantly increased after treatment with hCG. The effect of hCG on COX-2 messenger RNA and protein was seen at 10 ng/ml and higher concentrations sustained the increased levels. Although human LH mimicked hCG, human FSH, TSH, and isolated alpha- and beta-subunits of hCG had no effect on COX-2 protein levels suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. The effect of hCG on COX-2 protein paralleled the increase in media prostaglandin E2 levels indicating that the increased COX-2 gene and half-life of its transcripts to determine the molecular mechanism of hCG action. The results showed that hCG treatment had no significant effect on transcription rate of the COX-2 gene. On the other hand, treatment with hCG significantly increased the half-life of the COX-2 transcripts from 2.6 h in the control to 6.7 h after treatment. In summary, we conclude that treatment of human endometrial stromal cells with exogenous hCG to promote their differentiation into decidua results in an up-regulation of COX-2 gene expression by increasing the stability of the transcripts. PMID- 8612517 TI - Insulin-like growth factor I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide. AB - Although most insulin-like growth factor I (IGF-I) in the circulation is generated by the liver, the hormone is also produced locally by the vasculature, suggesting its potential importance in regulation of regional blood flow. Accordingly, we studied the effects of in vivo exposure to IGF-I (5.1 nmol, i.v.) as well as in vitro incubation (100 nM) on endothelium-intact rat tail artery contractile responses to KCl and norepinephrine (NE). Systemic administration of IGF-I resulted in transient lowering of blood pressure, with maximal reduction occurring at 15 min and a return to baseline by 60 min. Maximal contractility of rings removed from animals 90 min after a bolus injection of IGF-I, when blood pressure had returned to normal, was significantly reduced for both KCl (58%) and NE (51%) without a change in sensitivity. Similar data were obtained when rings from untreated animals were preincubated in vitro for 90 min; maximal contractility in response to KCl was decreased by 31% and that to NE by 22%. L Nitroarginine methyl ester, an inhibitor of nitric oxide (NO) production, administered in vivo before IGF-I or added to the bath buffer reversed the attenuation. The nearly identical in vivo and in vitro results suggest that the observed diminution in contractility is a direct effect of IGF-I on the vasculature, probably mediated in large part by the release of NO. This idea is supported by our observation that IGF-I stimulates NO production in intact vessels. Further, the latency required indicates that rather complex mechanisms involving actions common on both receptor- and nonreceptor-mediated events are initiated by IGF-I and/or NO. PMID- 8612518 TI - Short photoperiod-dependent down-regulation of thyrotropin-alpha and -beta in hamster pars tuberalis-specific cells is prevented by pinealectomy. AB - Hamster hypophyseal pars tuberalis (PT)-specific cells are characterized by the expression of common alpha-chain and TSH beta. Immunoreactivity for these subunits and the morphology of these cells are known to exhibit remarkable seasonal changes. The high density of melatonin (Mel) receptors on PT-specific cells leads to the supposition that fluctuations in circulating Mel levels induced by photoperiodic signals are a crucial factor for the morphological alterations. To more closely investigate transcriptional and translational activities in PT-specific cells, we cloned and sequenced hamster alpha and TSH beta complementary DNA fragments and assessed messenger RNA/protein formation by in situ hybridization and immunocytochemistry under short and long photoperiod and in pinealectomized animals kept in short photoperiod. Hamster common alpha chain and TSH beta exhibited high sequence homology with the corresponding rat hormones [94% (alpha-chain) and 90% (TSH beta) on the nucleotide level and 100% (alpha-chain) and 96% (TSH beta) on the amino acid level]. Immunocytochemical staining with antibodies directed against the common alpha-chain and TSH beta revealed a reduced immunoreactivity of PT-specific cells under short photoperiod, but this was not altered in pinealectomized animals exposed to short photoperiod. In situ hybridization against both hormonal subunits paralleled these changes, with a dramatic decrease in hormonal messenger RNA in short photoperiod. This regulatory influence was also blocked in pinealectomy. Taken together, these results demonstrate that transcription and translation of hormonal subunits are regulated by photoperiod in hamster PT-specific cells, whereas expression remained unchanged in short photoperiod if pinealectomy was performed. We, therefore, conclude that in hamsters, the Mel Signal not the light regimen per se, is a direct or indirect Zeitgeber for the transduction of photoperiodic information to the secretory activity in this pituitary cell type. PMID- 8612520 TI - Progesterone diminishes the sensitivity of gonadotropin-releasing hormone stimulated luteinizing hormone (LH) release and protects an LH pool from desensitization: actions opposed by cholera toxin. AB - In the present study we used characterized perifusion model for the development of homologous desensitization to GnRH in the pituitary gonadotrope. This system relies on immobilized primary pituitary cultures that are perifused with various pulse patterns and concentrations of GnRH; these patterns and concentrations are selected to maintain, inhibit, restore, circumvent the desensitization process. The data indicate that progesterone (P) inhibits LH release in response to pulsatile administration of GnRH; this effect is blocked by the P antagonist, RU486, and is opposed by cholera toxin, an agent that provokes the formation of cAMP. In multiple and rapid pulse administration of 5 nM GnRH, which is usually associated with desensitization, the area under the LH release curve is progressively diminished when cells are preincubated with medium only or with medium containing cholera toxin. In contrast, cells pretreated with P are less responsiveness to GnRH, but maintain a relatively constant level of LH release (area under the curve). These data suggest a modulatory role for P in the development of the desensitized state and indicate that P can functionally protect a pool of LH from the onset of desensitization. PMID- 8612519 TI - Calcitonin is a physiological inhibitor of prolactin secretion in ovariectomized female rats. AB - Calcitonin (CT) inhibits secretion of PRL when administered intravenously in rats and humans. It also inhibits PRL release from cultured rat anterior pituitary (AP) cells. Recent evidence suggests that CT-like immunoreactive peptide is synthesized and released from the AP gland. However, its physiological role in the regulation of PRL secretion has not been understood. Present studies tested the role of endogenous pituitary CT (pit-CT) in the regulation of PRL secretion in vivo by passive immunization. In the first group of experiments, ovariectomized (ovx) adult female rats were administered either preimmune or anti salmon CT (sCT) serum, and their serum PRL levels were analyzed at various time points up to 3 h. A second group of experiments examined the effects of anti-sCT serum and dopamine on PRL release from cultured rate AP cells. In the next group of experiments, the regional distribution of pit-CT secretion was examined in different sections of the AP gland. In the last set, CT-like activity of AP extract was tested in neonatal rat kidney cells, which respond to CT with an increase in cAMP accumulation. These experiments also tested whether anti-sCT serum reduces AP extract-induced increase in cAMP accumulation. The results suggest that anti-sCT serum dramatically increased serum PRL levels (by 5-fold) of ovx rats within 30 min of administration. The serum PRL levels declined gradually after the peak. However, a significant increase in serum PRL levels was maintained by the anti-sCT serum for the duration of the experiment. The anti serum also induced a significant increase in PRL release from cultured AP cells when added to the presence or absence of dopamine. The distribution profile of pit-CT within the AP gland suggests that the release of pit-CT immunoreactivity was significantly greater in the inner sections, and anti-sCT serum also caused greater increase in PRL release in these sections. Finally, AP extract and sCT stimulated cAMP accumulation in neonatal rat kidney cells, and anti-sCT serum significantly reduced AP extract-induced cAMP accumulation. These results demonstrate that pit-CT is an important regulator of tonic PRL secretion in female rats and can potently inhibit PRL secretion even in the presence of dopamine. PMID- 8612521 TI - Effects of thyroid hormone on carbonic anhydrase I concentration in human erythroid burst-forming unit-derived cells. AB - Individuals with hyperthyroidism exhibit red blood cell concentrations of carbonic anhydrase I (CAI) that reflect the integrated serum thyroid hormone concentration over the preceding few months. Furthermore, T3, at a physiological free concentration, decreases the CAI concentration in human erythroleukemic YN-1 cells. The effect of T3 on CAI concentration in burst-forming unit-erythroid (BFU E)- derived cells, obtained by culturing peripheral blood mononuclear cells with various cytokines, including erythropoietin, has now been investigated. BFU-E derived cells contained a high concentration of CAI (mean +/- SE, 4.8 +/- 0.8 x 10(-12) mol/10(6) cells; n = 8). The CAI in BFU-E-derived cells was immunologically identical to that present in mature red blood cells. T3 decreased the CAI concentration in BFU-E-derived cells in a dose-dependent manner (28%, 47% and 75% decreases at 3 x 10(-10), 1 x 10(-9), and 3 x 10(-9) mol/liter T3, respectively). These results suggest that BFU-E-derived cells may be used to study the effect of T3 on human red blood cell CAI. This system may prove useful in the tissue diagnosis of resistance to thyroid hormone. PMID- 8612522 TI - Molecular cloning and functional characterization of a rat pituitary G protein coupled adenosine triphosphate (ATP) receptor. AB - There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein linked ATP receptor found in the pituitary. PMID- 8612523 TI - Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1. AB - Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM). hGH induced cytosolic Ca2+ ([Ca2+]i) rises, which were transient, dependent on the presence of extracellular Ca2+, blocked by verapamil, calciseptine, and the hyperpolarizing agent diazoxide, suggesting that hGH stimulates Ca(2+)-influx through L-type Ca(2+)-channels. Similar effects on [Ca2+]i were observed with bGH or PRL. hGH caused membrane depolarization in a small proportion of the cells ( < 25%) as detected by cell-attached patch-clamp analysis. However, hGH failed to stimulate acute insulin secretion. hGH, bGH, and PRL promoted tyrosine phosphorylation of JAK2 tyrosine kinase. Verapamil inhibited neither [3H]thymidine incorporation nor JAK2 phosphorylation stimulated by hGH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is suggested because Rp-cyclic adenosine-3', 5'-monophosphorothioate, a competitive inhibitor of protein kinase A, abolished hGH-induced [Ca2+]i rises and DNA synthesis. cAMP appears to play a permissive role, although hGH failed to raise cellular cAMP levels. These results support the idea that activation of JAK2 is a major signaling event, whereas the [CA2+]i rise is not a prerequisite, for the mitogenic effects of GH and PRL in insulin-secreting cells. PMID- 8612524 TI - Insulin-like growth factor II modulates the routing of cathepsin D in MCF-7 breast cancer cells. AB - A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells. PMID- 8612525 TI - 1,25-Dihydroxyvitamin D3 enhances the enzymatic activity and expression of the messenger ribonucleic acid for aromatase cytochrome P450 synergistically with dexamethasone depending on the vitamin D receptor level in cultured human osteoblasts. AB - Not every postmenopausal woman with a low level of estrogen suffers from osteoporosis, and no correlation of bone density with serum estrogen level, but a significant correlation with adrenal androgens is often noted. Vitamin D3 has been reported to be osteoclastic in vitro, whereas the effectiveness of vitamin D3 for the treatment of osteoporosis is clinically relevant. To study the roles of these factors in the development of osteoporosis, we characterized aromatase activity converting androgens to estrogens in human osteoblasts, because postmenopausal women maintain considerable levels of adrenal androgens. Glucocorticoids at 10(-9)-10(-7) M transiently induced the expression and enzymatic activity of aromatase cytochrome P450 (P450AROM) in primary cultured osteoblasts, and the Km value for androstenedione (4.7 +/- 2.9 nM) was lower than that in adipose tissue and skin. Human osteoblasts showed a promoter specificity different from that found in other tissues. 1,25-Dihydroxyvitamin D3 [1,25 (OH)2D3] alone did not induce aromatase activity, but enhanced and maintained glucocorticoid-induced P450AROM gene expression. This synergistic effect was not observed by other sex steroids or retinoic acids. The enhancement of P450AROM activity by 1,25-(OH)2D3 varied from 0.94-fold (no enhancement) to 2.40-fold (maximal enhancement) among the individual human osteoblasts examined, but the magnitude of the enhancement was significantly correlated with the level of vitamin D receptor messenger RNA (P < 0.05). Cycloheximide did not abolish the synergistic effect of 1,25-(OH)2D3, suggesting that de novo protein synthesis is not required for the synergism with 1,25-(OH)2D3. These results suggest that bone tissue can synthesize estrogen from adrenal androgens by a unique aromatase activity depending on the level of vitamin D receptor expressed. PMID- 8612526 TI - Deoxyribonucleic acid-binding ability of androgen receptors in whole cells: implications for the actions of androgens and antiandrogens. AB - In whole cells, the effects of several androgens and antiandrogens on the in the induction of DNA binding for the human wild-type androgen receptor (AR) and a mutant receptor ARL (LNCaP mutation; codon 868, Thr to Ala) were examined and related to the transcription activation ability of these receptors. To study DNA binding, an AR expression vector was cotransfected in Chinese hamster ovary cells with a promoter interference plasmid cytomegalovirus-(androgen-responsive element)3-luciferase, containing one or more androgen-responsive elements between the TATA box of the cytomegalovirus promoter and the start site of luciferase gene transcription. Expression levels of the AR are up-regulated by some agonists, but receptor expression levels are comparable for all antiandrogens studied. In the presence of androgens, the wild-type AR is able to reduce promoter activity of the cytomegalovirus-(androgen-responsive element)3 luciferase plasmid, indicating androgen-dependent DNA binding of the AR. The full antagonists hydroxyflutamide, ICI 176.334, and RU 23908 block AR binding to DNA. The antagonists cyproterone acetate and RU 38486 induce approximately 50% of the DNA binding found for androgens. In a transcription activation assay, the RU 38646-bound receptor was almost inactive, and the receptor complexed with cyproterone acetate showed partial agonistic activity. Interaction of the antagonists cyproterone acetate, hydroxyflutamide, and RU 23908 with the mutant receptor ARL resulted in both DNA-bound and a transcriptionally active receptor. In conclusion, transformation of the AR to a DNA-binding state in whole cells is blocked by several antiandrogens. Furthermore, studies with the antiandrogens cyproterone acetate and RU 38486 show that DNA binding alone is not sufficient to accomplish full transcriptional activity. Full activity requires additional changes, presumably in the protein structure of the receptor. PMID- 8612527 TI - Tonic inhibition of renin secretion by the 12 lipoxygenase pathway: augmentation by high salt intake. AB - Recent evidence suggests that lipoxygenase (LO) metabolites inhibit renin production in vitro. However, the physiological significance of this effect has not been determined. This study examined the role of the LO pathway in the regulation of plasma renin concentration (PRC) in vivo. The acute administration of two structurally unrelated LO inhibitors, phenidone (30 and 60 mg/kg) and esculetin (60 mg/kg), resulted in suppression of platelet 12 hydroxyeicosatetraenoic acid (12HETE) production, reduction in systemic arterial pressure and a 2- to 3-fold increase in PRC. To determine whether the esculetin induced increase in PRC was secondary to hypotension, esculetin was also administered to rats preinfused with a pressor dose of norepinephrine. In these acutely hypertensive rats, esculetin still induced a 2.5-fold increase in PRC, whereas blood pressure remained over 40 mm Hg above basal levels. Further, esculetin (10(-6)M) increased renin release in renal slices from 150 +/- 10 to 310 +/- 20 ng/ml.h (P < 0.05) and this rise was entirely blocked in the presence of 12HETE (10(-7)M; 130 +/- 40 ng/ml.h). In rats placed on high salt intake, 12HETE concentration in renal slices from the outer cortex was considerably higher than in renal slices from salt-restricted rats (116.5 +/- 15.7 vs. 65 +/- 12 pg/mg protein; P < 0.05). Chronic administration of the LO inhibitor phenidone also resulted in an increase of PRC, which was independent of changes in blood pressure. On either high salt (3.15%0 or low salt (0.05%) diet phenidone-treated rats had higher PRC levels than the respective control groups [high salt 9.7 +/- 3.5 vs. 1.9 +/- 1.4 ng/ml.h; P < 0.05; low salt 33.2 +/- 5.3 vs. 19.4 +/- 3.10 ng/ml.h; P < 0.05]. The finding that LO blockers are potent stimulators of PRC in vivo suggests the existence of a physiological tonic inhibition of renin secretion by LO products that is operative under a wide range of salt intake. High salt intake enhances this inhibitory tone by increasing renal cortical 12 LO activity and, in fact, normal suppression of PRC during high salt diet does not occur in LO-blocked animals. Thus, the LO pathway exerts a tonic inhibitory effect on renin release, which appears particularly important for renin suppression during high salt intake. PMID- 8612529 TI - Type I procollagen production and cell proliferation is mediated by transforming growth factor-beta in a model of hepatic fibrosis. AB - Fibrosis is a significant component of advanced chronic inflammatory liver diseases and is caused by the accumulation of extracellular matrix, including type I procollagen. The mechanism by which fibrosis develops in liver tissue remains unknown. We tested the effects of transforming growth factor beta 1 (TGF beta), a cytokine that alters cell differentiation and proliferation, and bleomycin, a cytotoxic glycopeptide antibiotic, on cultured isolated rat hepatocytes. TGF-beta (1 ng/ml) inhibited radiolabeled thymidine incorporation 39% at 24 h and 69% at 48 h. Inhibition of hepatocyte proliferation was dose dependent. Bleomycin (1 microgram/ml) significantly inhibited radiolabeled thymidine incorporation at 48 h (44%). Neutralizing antibody to thymidine incorporation at 48 h (44%). Neutralizing antibody to TGF-beta (TGF-beta-Ab) attenuated the inhibition of proliferation by TGF-beta and bleomycin in a concentration-dependent manner. The addition of either TGF-beta or bleomycin increased immunostaining of type I procollagen in hepatocytes. The addition of TGF-beta-Ab alone increased cell proliferation, suggesting that neutralization of endogenous TGF-beta may attenuate the inhibition of hepatocyte proliferation. These data suggest that the hepatocyte contains type I procollagen and, under some conditions, produces TGF-beta. We propose that procollagen production in rat hepatocytes is induced by TGF-beta and may be related to endogenous production of this cytokine in response to cell injury. The cytotoxic effect of bleomycin is mediated by TGF-beta and inhibition of TGF-beta and bleomycin with TGF-beta-Ab attenuates the additive effects of those compounds on isolated rat hepatocytes. These data provide a model of collagen expression in isolated rat hepatocytes. PMID- 8612528 TI - Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system. AB - Leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) were found to be pleiotropic modulators of Sertoli cell and gonocyte development (both isolated from the neonatal rat testis) in a coculture system, whereas IL-6, another member of this cytokine family, had no effect on these cells. LIF and CNTF significantly enhanced the survival of the Sertoli cells in a dose- and time dependent manner. The effect of LIF on the Sertoli cells was significant at a concentration of 1 ng/ml after 3 or 6 days of culture, whereas CNTF had a significant effect at 10 ng/ml. Neither LIF nor CNTF had an effect on Sertoli cell proliferation. The survival of proliferating gonocytes (isolated from 3-day old rats testes) was also significantly higher in cultures to which LIF (7.5 ng/ml) or CNTF (10 ng/ml) was added. No effect of these cytokines was found on the mitotic activity of proliferating gonocytes. However, LIF (7.5 ng/ml) stimulated the proliferation of quiescent gonocytes (isolated from day 1 testes) after 3 days of culture. Combinations of LIF (or CNTF) with fibroblast growth factor 2 (10 ng/ml) and steel factor (50 ng/ml) did not further improve the long term culture of the gonocytes. LIf- and CNTF-like proteins of the expected molecular masses (32,000 and 22,000 daltons, respectively, under reducing conditions) were found by Western blotting in testicular extracts of 3-day-old rats. Taken together, these results indicate that LIF or CNTF may play a role at the start of the spermatogenesis. The characterization of receptors for LIF or CNTF on the gonocytes and/or neonatal Sertoli cells will aid in a better understanding of the physiological role of these cytokines in the reproductive system. PMID- 8612530 TI - The obese growth hormone (GH)-deficient dwarf rat: body fat responses to patterned delivery of GH and insulin-like growth factor-I. AB - We describe a new animal model of obesity and GH deficiency and report the effects on body fat of administering (GH) and insulin-like growth factor (IGF-I) in the model. Female GH-deficient dwarf rats fed a high-fat diet became obese and insulin-resistant compared with chow-fed controls. They were treated with recombinant human GH (rhGH 100-500 micrograms/day, s.c. for 14 days) by daily injection or minipump infusion with or without rhIGF-I (200 micrograms/day, sc infusion). Injections of rhGH increased body weight; infusions of rhGH caused weight loss. RhIGF-I by itself, or rhIGF-I plus GH injections had little effect, whereas rhGH infusions plus rhIGF-I caused a weight loss equivalent to the weight gained during the high-fat feeding and a decrease in fat pad weight. For some responses (serum IGF-1 and GHBP), the obese rats were GH resistant. Fat was lost from the internal fat pads when obese rats were returned to a chow diet, and injections of rhGH surprisingly attenuated this loss of fat. In obese dwarf rats, the lipolytic effects of rhGH are dose-regime dependent. By itself IGF-I is not insulin-like, but in the presence of GH it has antiinsulin actions causing a powerful net lipolysis. If GH plus IGF-I have similar effects in humans they may be useful for reducing body fat. PMID- 8612531 TI - Differential long-term effects of insulin-like growth factor-I (IGF-I) growth hormone (GH), and IGF-I plus GH on body growth and IGF binding proteins in hypophysectomized rats. AB - The long-term effects of recombinant human insulin-like growth factor-I (rhIGF-I) and GH (rhGH) on body growth and the IGF-I/IGF binding protein (IGFBP)/acid labile subunit (ALS) axis were investigated in hypophysectomized (hypox) rats given excipient, rhIGF-I (2 mg/kg.day s.c., minipumps), rhGH (2 mg/kg.day, s.c., daily injections), or rhIGF-I plus rhGH for 28 days. The relative growth promoting activity of the treatments was rhGH plus rhIGF-I more than rhGH more than rhIGF-I. Weight gain induced by rhIGF-I progressively declined after 4 days, compared with a more maintained effect of rhGH. At day 28, growth responses did not correlate with serum IGF-I levels [rhGH plus rhIGF-I (492 +/- 140) > rhIGF-I (322 +/- 75) > rhGH (85 +/- 26) > control (39 +/- 7 ng/ml)]. Serum ALS concentrations in hypox rats were remarkably low (0.42 +/- 0.04 micrograms/ml) but were restored toward normal by rhGH (12.55 +/- 4.78) or rhGH plus rhIGF-I (12.85 +/- 6.64) but not by rhIGF-I alone (0.85 +/- 0.25). Antibodies against rhGH were present at day 28, with titer being negatively related to weight gain, IGF-I, and ALS levels. All treatments increased serum IGFBP-3. The molecular size distribution of IGFBP-3 in rhGH-treated rats was similar to that of normal rats (IGFBP-3 in the 150K mol wt range), due to rhGH increasing serum ALS, but was altered by rhIGF-I (IGFBP-3 in the 200-300K and 44K mol wt range). In a GH deficient animal, restoring the IGF/IGFBP-3/ALS axis towards normal is associated with greater growth promotion. PMID- 8612532 TI - Osteogenic protein-1-mediated insulin-like growth factor gene expression in primary cultures of rat osteoblastic cells. AB - Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein family and has been shown to induce new bone formation in vivo. In the present study, we determined whether the expression of the IGF system, a significant growth factor system in bone, was altered by OP-1 in primary cultures of fetal rat calvarial cells. Levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF I), IGF-II, IGF-I receptor, and IGF-binding proteins (IGFBP-1 to -6) were determined after OP-1 treatment. The level of total IGF-I mRNA was elevated in an OP-1 concentration (0-1000 ng/ml)-dependent manner, with maximal stimulation of IGF-I mRNA of 2- to 3-fold apparent 24 h after treatment. The increase in the IGF I mRNA level involved a preferential stimulation of transcripts initiated at start site 2 in the exon 1 promoter. The level of IGF-II mRNA also increased by approximately 2-fold in OP-1 treated cells in a concentration-dependent manner. The level of IGF-I receptor mRNA was not altered by treatment. Whereas IGFBP-1 mRNA was not detected in these cells, IGFBP-2 mRNA was expressed, but the expression was not changed after treatment for 48 h in the concentration range (0 1000 ng/ml) tested. The IGFBP-3 mRNA level was increased slightly 48 h after OP-1 treatment in a concentration-dependent manner. The IGFBP-4, -5, and -6 mRNA levels decreased dramatically in an OP-1 concentration-dependent manner. In addition, coincubation of antisense oligonucleotides corresponding to IGF-I or II mRNA sequence with OP-1 reduced the OP-1 induced elevation in alkaline phosphatase activity. The present results suggest that the differentiation of rat osteoblastic cells in response to OP-1 is mediated in part by increased IGF-I -II gene expression and alterations in the gene expression of different IGFBPs. PMID- 8612533 TI - Mediation by epidermal growth factor of the estradiol-induced increase in cyclic guanosine 3',5'-monophosphate content in the rat uterus. AB - Estrogen treatment of immature or ovariectomized mature rats induces an increase in uterine cGMP content, with a peak 2-3 h after hormone administration. This response to estrogenic action also develops in vitro, in incubated uterine horns, thus excluding the intervention of another organ. Its function is still unknown. We show here that treatment of incubated uterine horns from immature or mature rats with 8 nM epidermal growth factor (EGF), exactly mimicked the effect of 1 nM estradiol on cGMP levels. The estradiol-induced increase in uterine cGMP was canceled in the presence of the phosphotyrosine kinase inhibitor genistein. Like the cGMP response to EGF, the estradiol-induced increase in uterine cGMP was completely suppressed in the presence of an antimouse EGF antibody. On the other hand, whereas the induction of cGMP accumulation by estradiol in vivo or in vitro was suppressed by prior treatment of the animals with the pure antiestrogen ICI 164,384, such pretreatment had no effect on the EGF-induced increase in uterine cGMP content. Together, these data support the concept that the uterine cGMP response to estrogens is entirely due to auto/paracrine mediation by the EGF-EGF receptor system. Considering reports from the literature showing that EGF can directly induce the phosphorylated active form of the estrogen receptor, we speculate that this might implicate its action on cGMP, with the latter then intervening as cofactor of the involved phosphokinase(s). PMID- 8612534 TI - Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia. AB - Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system. PMID- 8612535 TI - Evidence that gonadal steroids modulate nitric oxide efflux in the medial preoptic area: effects of N-methyl-D-aspartate and correlation with luteinizing hormone secretion. AB - Several lines of evidence suggest that nitric oxide (NO) is involved in the neuroendocrine control of reproductive function. This study was undertaken to determine 1) NO activity in the medial preoptic area (MPOA) where LHRH- and NO synthase-containing neurons are coextensive; 2) whether N-methyl-D-aspartate (NMDA) receptor activation, which stimulates LHRH release, augments NO activity in the MPOA; and 3) whether NO activation in the MPOA underlies the steroid dependency of NMDA-induced pituitary LH release. As extracellular levels of cGMP in discrete brain sites are a reliable index of basal and stimulated activity of NO, extracellular cGMP levels in the MPOA of freely moving, awake rats were measured by microdialysis in the current study. In the first experiment, the MPOA of intact and castrated male rats were microdialyzed with artificial cerebrospinal fluid at a rate of 5 microliters/min. The basal level of cGMP efflux was determined from the initial seven samples collected at 20-min intervals. The NO response to a single i.v. injection of NMDA (10 mg/kg) or saline was assessed in the next five samples. In the second experiment, the basal and NMDA-evoked NO effluxes in the MPOA of ovariectomized (ovx) and estrogen treated ovx rats were examined. Results showed that in both sexes, the absence of gonadal steroids resulted in significantly lower basal cGMP levels. Additionally, the cGMP response to NMDA was steroid dependent. Whereas in castrated rats it failed to affect cGMP efflux, NMDA in intact male rats promptly raised cGMP levels at 20 min, and these elevated levels were maintained through the duration of the experiment. This NMDA-induced cGMP response, observed selectively in intact rats, was also associated with stimulation of plasma LH levels. In female rats, NMDA similarly enhanced MPOA cGMP efflux and pituitary LH secretion in estradiol benzoate-treated, but not in oil-treated, ovx rats. The NMDA receptor antagonist D,L-amino-5-phosphoropentanoic acid and the NO synthase inhibitor, N omega-nitro-L-arginine, completely blocked the NMDA-induced cGMP and LH responses, thereby demonstrating the specificity of the NMDA receptor --> NO line of communication probably operating in the MPOA in the control of pituitary LH release. Therefore, these results show that gonadal steroids augment basal as well as NMDA-induced MPOA cGMP efflux in male and female rats. It is likely that facilitation of NO/cGMP activity in the MPOA may underlie the steroid dependency of NMDA-evoked LH hypersecretion in the rat. PMID- 8612536 TI - L-arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: involvement of neuropeptide Y. AB - Although estrogen can induce LH hypersecretion in ovariectomized (ovx) rats, the magnitude and duration of the LH surge are invariably far less than those of the preovulatory LH surge in cycling rats. Recently, the crucial roles of hypothalamic nitric oxide (NO) and neuropeptide Y (NPY) in the induction of LHRH and the LH surge have been recognized. In this study, we have tested the hypothesis that low grade stimulatory feedback effects of 17 beta-estradiol (E2) on LH secretion in ovx rats is due to a deficit in NO signaling. As the NO substrate, L-arginine (L-Arg), can enhance NO-dependent physiological responses, the effects of L-Arg on the LH surge in E2-primed ovx rats was studied. Ovx rats bearing permanent cannulas in the lateral cerebroventricle received 15-mm long SILASTIC capsules filled with E2 (300 micrograms/ml oil, s.c.) at 1000 h. Two days later, saline (SAL) or L-Arg (0.1 or 1 nmol in 5 microliters SAL) or D-Arg (1.0 nmol) was injected intracerebroventricularly every hour from 1100-1400. Blood samples were collected at 1100 h and at hourly intervals from 1400-1800 h via intraatrial cannulae implanted the day before. Whereas an expected moderate LH surge occurred in SAL-injected control rats, L-Arg, but not D-Arg, greatly augmented the magnitude and duration of the LH surge, with peak values attaining the range normally seen on proestrus. Furthermore, L-Arg (1 or 10.0 mM) and the NO donor, sodium nitroprusside (1 or 10.0 mM), stimulated the basal and K(+) induced in vitro release of LHRH and NPY from the hypothalami of ovarian steroid primed ovx rats. As NPY is stimulatory to LHRH release, we next tested the possibility that L-Arg/NO may initially stimulate NPY release in the hypothalamus, which, in turn, elicits LHRH discharge. E2-treated ovx rats receiving intracerebroventricular L-Arg were injected with antisense or missense oligodeoxynucleotides (oligos) to NPY-messenger RNA. Antisense, not missense, NPY oligos blocked the L-Arg-induced potentiation of the LH surge. These results revealed for the first time the neurochemical cause underlying the E2 feedback action and show that a deficiency in either L-Arg itself or L-Arg-based NO signaling is responsible for the low grade LH surge in ovx rats treated with estrogen alone. Additionally, the L-Arg-dependent potentiation of the LH surge may involve increased signaling in the NO and NPYergic systems, resulting in optimal LHRH hypersecretion from the hypothalamus. PMID- 8612537 TI - Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid epithelial cells. AB - Thyroglobulin (Tg) is the major secretory product of thyroid epithelial cells and is stored in the lumen of thyroid follicles at high concentrations. Thyroid hormone liberation is assumed to occur separately from this storage compartment within lysosomes. However, for the transfer of Tg to lysosomes, mechanisms to solubilize the luminal content must precede its endocytosis, because part of the luminal Tg occurs in a covalently cross-linked form. Here, by immunoprecipitation and immunoblotting we show that the majority of procathepsin B or L and a fraction of mature cathepsin B are released from porcine thyrocytes in vitro. Released cathepsins were detectable on the cell surface of the thyrocytes by immunocytochemistry and amounted to 27% of the total cathepsin B. Cytochemical studies revealed the proteolytic activity of cathepsin B at neutral pH on the cell surface of thyrocytes. Therefore, the possibility of extracellular proteolysis by cathepsins was investigated by incubating plasma membrane preparations, conditioned media, or lysosomes with Tg. The liberation of thyroid hormones was quantitated by RIA, and the degradation of Tg was determined by SDS PAGE. Extracellular and plasma membrane-associated proteases rapidly mediated up to 54% of the total T4 liberation by limited proteolysis of Tg at neutral pH under conditions where cysteine proteases were reactivated. We propose that released and proteolytically active cysteine protease i.e. cathepsins B and L, provide thyrocytes with a pathway of limited extracellular proteolysis of Tg before endocytosis. PMID- 8612538 TI - Growth regulation of rat thyrocytes (FRTL-5 cells) by the secreted ectodomain of beta-amyloid precursor-like proteins. AB - The TSH-dependent expression of amyloid precursor-like proteins and the secretion of their ectodomain (sAPP) in rat thyroids coincide with increased rates of thyrocyte proliferation. To analyze whether the secretion of sAPP and the proliferation of thyrocytes are regulatorily linked, we employed [3H]thymidine or 5-bromo-2'-deoxyuridine assays and found that conditioned culture medium stimulated the proliferation of FRTL-5 cells depending on the content of sAPP. These observations prompted experiments with sAPP-derived peptides known to stimulate the growth of APP-deficient fibroblasts. Using autoradiography and radiochemical assays, we observed that an iodinated 19-mer sAPP peptide was bound specifically to the surface of FRTL-5 cells. Binding of this peptide was followed by a 2- to 8-fold increase in cell proliferation, which reached a plateau at 1 nM. This effect was significant only when cells were cultured in nonconfluent monolayers, and contact inhibition did not interfere. Our observations indicate that sAPP and sAPP-derived peptides increased the proportion of proliferation competent FRTL-5 cells and suggest that sAPP may be a new member in the family of peptides involved in the growth regulation of thyrocytes. PMID- 8612539 TI - An antidiabetic thiazolidinedione potentiates insulin stimulation of glycogen synthase in rat adipose tissues. AB - Thiazolidinedione derivatives are a novel class of insulin-sensitizing agents that have demonstrated effective antidiabetic activity in vivo. Here, the effects of the potent thiazolidinedione derivative, AD-5075, on the rate-limiting enzyme of glycogen synthesis, glycogen synthase, were investigated in cultured rat adipose tissue. Short term preincubation of adipose tissue with AD-5075 potentiated acute insulin stimulation of I-form glycogen synthase activity in a concentration-dependent (EC50 approximately, 61nM) and time-dependent (t1/2 approximately, 2.3 h) manner. The thiazolidinedione derivative increased the responsiveness of I-form glycogen synthase activity to insulin stimulation at both maximal and submaximal insulin concentrations. In contrast, it had no effect on total glycogen synthase activity. Isoproterenol inhibited acute insulin activation of I-form glycogen synthase activity in a dose-dependent manner; maximal inhibition was attained at a concentration of 3 nM. AD-5075 antagonized isoproterenol inhibition of insulin's action. The concentration of glycogenolytic agent required to attain maximal inhibition was increased an order of magnitude in tissue treated with the antidiabetic agent. Short term preincubation of adipose tissue under hyperglycemic conditions (15 or 25 mM glucose) decreased insulin-stimulated I-form glycogen synthase activity. Concurrent treatment of the tissue with AD-5075 abrogated this glucose toxicity-induced inhibition of insulin action. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked insulin activation of glycogen synthase in a dose-dependent manner. Half-maximal inhibition was observed at approximately 0.3 microM, and maximal inhibition occurred at 1.0 microM. AD-5075 did not antagonize wortmannin's inhibitory action. These results indicate that thiazolidinediones can act directly on adipose tissues to augment an important metabolic effect of insulin and counteract the inhibitory effects of catecholamines or hyperglycemia. As insulin stimulation of glycogen synthase remains wortmannin inhibitable in the presence of AD-5075, the effects of thiazolidinediones on insulin signal transduction may be phosphatidylinositol 3-kinase-dependent. PMID- 8612540 TI - Transforming growth factor-beta 1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site. AB - Osteocalcin (OC), a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix, is down-regulated upon treatment with transforming growth factor (TGF)-beta 1. To address the potential role of OC gene expression in relation to TGF-beta 1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-beta 1 treatment. 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs demonstrated that TGF-beta 1 treatment repressed chloramphenicol acetyltransferase activity by 2.4-fold in transient transfections of ROS 17/2.8 cells. A 29-bp region between -162 and -134 identified as the TGF beta 1 response domain, conferred TGF-beta 1 responsiveness to the -108 to +24 rat OC basal promoter in an orientation dependent manner. Mutation of an activator protein-1/cAMP-response element-like motif (- 146 to -139) abolished TGF-beta 1 responsiveness of the construct. In vitro gel-mobility shift and competition assays using wild-type and mutated oligonucleotides and antibodies indicate that Fra-2, a Fos related transcription factor, binds to this motif. We show that Fra-2 is an activator of the OC promoter, and TGF-beta 1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF beta 1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-beta 1 mediated down regulation of the OC promoter activity. Together, these results demonstrate that TGF-beta 1 responsiveness of the rat osteocalcin gene in ROS 17/2.8 cells is mediated through an activator protein-1 like cis-acting element that interacts with Fra-2. Furthermore, our results are consistent with a critical role for TGF beta 1 induced phosphorylation of Fra-2 in the repression of OC gene transcription. PMID- 8612541 TI - Antagonistic effects of transforming growth factor-beta on vitamin D3 enhancement of osteocalcin and osteopontin transcription: reduced interactions of vitamin D receptor/retinoid X receptor complexes with vitamin E response elements. AB - Osteocalcin and osteopontin are noncollagenous proteins secreted by osteoblasts and regulated by a complex interplay of systemic and locally produced factors, including growth factors and steroid hormones. We investigated the mechanism by which transforming growth factor-beta (TGF beta) inhibits 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-enhanced expression of the osteocalcin (OC) and osteopontin (OP) genes. ROS 17/2.8 cells, in which both genes are expressed, were transfected with reporter constructs driven by native (i.e. wild-type) rat OC and mouse OP promoters. TGF beta abrogated the 1,25-(OH)2D3 enhanced transcription of both the OC and OP genes. The inhibitory TGF beta response for each requires vitamin D response element (VDRE) sequences, although there are additional contributions from proximal basal regulatory elements. These transcriptional effects were further investigated for contribution of the trans-activating factors, which interact with OC and OP VDREs, involving the vitamin D receptor (VDR) and retinoid X receptor (RXR). Gel mobility shift assays show that TGF beta significantly reduces induction of the heterodimers VDR/RXR complexes in 1,25 (OH)2D3-treated ROS 17/2.8 cells. However, Western blot and ligand binding analysis reveal that TGF beta does not affect nuclear availability of the VDR. We also show that activator protein-1 activity is up-regulated by TGF beta; thus, activator protein-1 binding sites in the OC promoter may potentially contribute to inhibitory effects of TGF beta on basal transcription. Our studies demonstrate that the inhibitory action of TGF beta on the 1,25-(OH)2D3 enhancement of OC and OP transcription in osteoblastic cells results from modulations of protein-DNA interactions at the OC and OP VDRE, which cannot be accounted for by changes in VDR protein levels. As OC and OP participate in bone turnover, our results provide insight into the contributions of TGF beta and 1,25-(OH)2D3 to VDR mediated gene regulatory mechanism operative in bone formation and/or resorption events. PMID- 8612542 TI - The insulin hypoglycemia-induced inhibition of gonadotropin-releasing hormone pulse generator activity in the rhesus monkey: roles of vasopressin and corticotropin-releasing factor. AB - Insulin-induced hypoglycemia (IIH) profoundly inhibits the activity of the hypothalamic GnRH pulse generator. The aim of this study was to determine the role of vasopressin and CRF in this response. Ovariectomized rhesus monkeys with chronically implanted recording electrodes in the mediobasal hypothalamus and with intracerebroventricular (icv) cannulas in the lateral ventricle were placed in primate chairs, and blood samples were taken every 10 min. Pulse generator activity was monitored electrophysiologically by detecting characteristic increases in hypothalamic multi-unit activity (MUA volleys) and by attendant LH pulses measured in peripheral blood. Arginine vasopressin (AVP) infused via the i.c.v. cannula (50 micrograms/60 microliters.h) in eight monkeys failed to decrease pulse generator activity, as measured by the frequency of MUA volleys, but decreased mean serum LH concentrations (P < 0.001) while increasing serum cortisol levels (P < 0.02). Central administration of an AVP antagonist ([deamino Pen1, O-Me-Tyr2-Arg8] vasopressin) in four monkeys at a rate (180 micrograms/60 microliters.h) that had previously been found to block the aforementioned effects of coadministered AVP failed to prevent the IIH-induced inhibition of GnRH pulse generator activity and LH secretion in the same animals. On the other hand, a CRF antagonist, [D-Phe12,Nle21,38,C alpha MeLeu37] rat CRF-(12-41), infused i.c.v. at a rate of 500 micrograms/60 microliters.h in four monkeys, delayed the inhibition of pulse generator frequency in response to IIH. These results suggest that AVP does not mediate the hypoglycemia-induced inhibition of GnRH pulse generator frequency in the rhesus monkey, but that CRF may be involved in this response. PMID- 8612543 TI - Normalization of insulin secretion by a selective alpha 2-adrenoceptor antagonist restores GLUT-4 glucose transporter expression in adipose tissue of type II diabetic rats. AB - Noninsulin-dependent mellitus is characterized by the coexistence of defective insulin secretion with insulin resistance in peripheral tissues. Therapeutic objectives are, therefore, to normalize glucose-induced insulin secretion and to restore normal glucose transport into insulin-sensitive tissues. In the present study we evaluate the effects of acute and subchronic administration (2 or 10 days) of the alpha 2-adrenoceptor antagonist SL 84.0418 on glucose tolerance in nondiabetic control rats and type I and type II diabetic rats and the level of the insulin-sensitive glucose transporter GLUT-4, which is exclusively expressed in white and brown adipose tissues, heart, and skeletal muscles. Glucose tolerance and insulin secretion were markedly impaired in type II diabetic rats (neonatal injection of streptozotocin) and were totally corrected by an acute i.p. injection of SL 84.0418. As a consequence of the chronic restoration of insulin secretion, GLUT-4 messenger RNA (mRNA) levels, initially decreased by 67% in white adipose tissue of type II diabetic rats, were normalized by subchronic (10 days), but not acute (2 days), treatment with SL 84.0418. The same results were obtained in brown adipose tissues of type II diabetic rats, whereas no modification of GLUT-4 mRNA levels remained very low in brown adipose tissues of type I diabetic rats (adult injection of streptozotocin) after acute or subchronic administration of SL 84.0418, suggesting that this drug acted by the restoration of insulin secretion. This study reports a decrease in GLUT-4 levels in insulin-sensitive tissues in this model of type II diabetes as well as its regulation after subchronic normalization of insulin secretion. We suggest a direct role for the alpha 2-adrenoceptor antagonist SL 84.0418 in pancreatic beta cells that allows normalization of glucose tolerance. PMID- 8612544 TI - Distinct responses of different populations of bone cells to mechanical stress. AB - To explore lineage-dependent responses to mechanical stress in bone cells, newborn rat calvarial cells, exhibiting differential characteristics of osteoblastic and osteocytic cells, were compared in their immediate and late responses to stretching. Seven fractions of sequentially prepared cells were cultured on Matrigel to promote their differentiation. By cyclically stretching the flexible bottom of culture plates, cells were exposed to a physiological stress of approximately 4000 microstrain on Matrigel. Cells in fractions IV, V and VI exhibited striking responses; the levels of cAMP and insulin-like growth factor I, bone Gla protein, and mineral accumulation were significantly elevated in the stretched cells. Also, proliferation was significantly inhibited regardless of the presence of 10(-6)M indomethacin. In contrast, osteoblasts in fraction III and osteocyte-like cells in fraction VII exhibited no significant response. Thus, these intermediate cells, very mature osteoblasts to young osteocytes, are likely to serve as a mechanosensor in bone, controlling the metabolic aspects of physical stress. We conclude that the responses of these young osteocytes to low level, physiological strain are transmitted in a manner different from the responses of osteoblasts to higher magnitude of strain in which PGE2 induces cell proliferation, as reported by others. PMID- 8612545 TI - Thyrotropin induces G1 cyclin expression and accelerates G1 phase after insulin like growth factor I stimulation in FRTL-5 cells. AB - We have investigated the mechanism by which TSH pretreatment potentiates insulin like growth factor I (IGF-I)-induced DNA synthesis in FRTL-5 cells. As previously described, pretreatment with TSH increased IGF-I-induced DNA synthesis, suggesting that the effect of TSH is mediated through the cAMP pathway. TSH and A kinase activators required at least 12 h to precondition cells to respond to IGF I stimulation. The presence of cycloheximide abolished the effect of TSH to increase IGF-I-induced DNA synthesis. When the time course of thymidine uptake after IGF-I addition was studied, TSH pretreatment increased the maximum DNA incorporation and shortened the G1 phase interval. These results indicated that some proteins induced by TSH are required for the effect of TSH on IGF-I activity, and the proteins are important for cell cycle progression. Cyclins are key regulators of the cell cycle; therefore, we investigated the expression of cyclins D1 and E after TSH stimulation. TSH- and A kinase-activating agents increased the expression of cyclins D1 and E after 24 h. The same amounts of cyclins D1 and E induced by IGF-I were increased after TSH pretreatment. TSH pretreatment induced the expression of G1 cyclin in FRTL-5 cells, and IGF-I caused the accumulation of enough G1 cyclins to drive the cell cycle from G1 to S phase in a short time, which accounts for the effect of TSH on IGF-I induced DNA synthesis. PMID- 8612546 TI - Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line. AB - It has been known for many years that adipocytes express high affinity ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, and that ACTH, alpha MSH, and beta-lipotropin are potent lipolytic hormones. We show here that the adipocyte response to the melanocortin peptides results from the expression of both the MC2 (ACTH) receptor as well as the newly discovered MC5 receptor. Using RT-PCR and Northern blot hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 receptor mRNA was found in a subset of these. Both receptors mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induced to differentiate into adipocytes. This cell line was then used to characterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of approximately 1 nM. An MSH binding site characterized in these cells is presumably the MC5 receptor, based on the observation that this is the only other melanocortin receptor mRNA detected in these cells. The MC5 receptor in the 3T3 L1 adipocyte activated adenylyl cyclase in response to alpha-MSH stimulation. Interestingly, Nle4, D-Phe7-alpha-MSH (NDP-MSH), a commonly used synthetic alpha MSH agonist, was a potent antagonist of the MC5 receptor expressed in the 3T3-L1 cell line. Although the agouti signaling peptide is a potent antagonist of NDP MSH binding to the MC1 and MC4 melanocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte. PMID- 8612547 TI - Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in 150-kilodalton IGFBP complexes by a cation-dependent protease activity in adult rat serum promotes the release of bound IGF-I. AB - Insulin-like growth factor I (IGF-I) circulates in plasma predominantly in a 150 kDa complex together with IGF-binding protein-3 (IGFBP-3), an approximately 40 kDa glycoprotein that binds IGF-I with high affinity, and an 85-kDa acid-labile subunit that does not bind IGFs. The 150-kDa complex serves as a potential reservoir of IGF-1 by sequestering the growth factor in the vascular compartment. Before IGF-I can reach the tissues, however, it must be released from the complex in a form that can cross the capillary-endothelial barrier. The present study demonstrates that proteolysis of IGFBP-3 in the 150-kDa complex occurs in vitro and results in the release of IGF-I. Specific IGFBP-3 protease activity in adult rat serum was demonstrated by incubating the serum at 37 C in the presence and absence of various protease inhibitors, followed by ligand blotting. Intact and truncated (30-kDa) IGFBP-3 were almost completely proteolyzed after 12 h of incubation, whereas IGFBP-2 and IGFBP-4 in the serum were unchanged even after 48 h incubation. The IGFBP-3 protease activity was inhibited by EDTA, indicating cation dependence, and by 4-(2-aminoethyl)-benzenesulfonyl fluoride, a serine protease inhibitor. Fractionation of the serum after incubation indicated that IGFBP-3 was proteolyzed while it was part of the 150-kDa complex. Proteolysis of human IGFBP-3 (hIGFBP-3) also occurred within 150-kDa complexes when ternary complexes that had been reconstituted from recombinant hIGFBP-3, rat acid-labile subunit, and IGF-I were incubated with rat serum. Release of IGF-I from ternary complexes after proteolysis of hIGFBP-3 was indicated by the observation that some of the [125I]IGF-I initially associated with reconstituted 150-kDa complexes was transferred to endogenous rat IGFBPs during the incubation. Similar proteolysis of IGFBP-3 within 150-kDa complexes in vivo would provide a mechanism for mobilizing IGF-I from the circulating reservoir in plasma as well as for the turnover of IGFBP-3. PMID- 8612548 TI - Susceptibility of avian ovarian granulosa cells to apoptosis is dependent upon stage of follicle development and is related to endogenous levels of bcl-xlong gene expression. AB - Studies were conducted to evaluate the susceptibility of avian ovarian granulosa cells to apoptosis when incubated in vitro and to relate this relative susceptibility to both the stage of follicle development from which granulosa cells were collected (atresia-prone vs. -resistant) and to the expression of a gene previously linked to the regulation of cell viability, bcl-xlong. Granulosa cells from slow growing, prehierarchal (4- to 8-mm diameter; atresia-prone) follicles were found to undergo rapid and progressively extensive apoptosis after incubation in defined medium for 6-24 h (P < 0.05 vs. unincubated controls). By contrast, cells from the largest preovulatory (F1) follicle, as well as from follicles most recently recruited into the follicle hierarchy (9- to 12-mm diameter), showed significantly less low molecular wt labeling at 6 h of incubation (P < 0.05 vs. 4- to 8-mm follicles). Furthermore, the amount of low molecular wt labeling did not significantly increase in cells from either stage of follicle development at 12 or 24 h of incubation (P < 0.05 vs. 6 h incubation). This biochemical indication of ongoing apoptosis in prehierarchal follicle granulosa cells was confirmed by an increased incidence of pyknotic nuclei detected by morphological analysis. Thus, increased susceptibility to apoptosis in incubated prehierarchal follicle granulosa cells is correlated with the high rate of follicle atresia that is known to occur at this stage of development in vivo. Recombinant human FSH (100 mIU) and transforming growth factor-alpha (3.3 nM) partially suppressed apoptosis in prehierarchal follicle granulosa cells after 6 h of incubation (by 46-57%; P < 0.05 vs. control), as did the cAMP analog, 8-Br-cAMP (1 mM; by 59%; P < 0.05). A single form of the bcl-2 related gene, bcl-x, was detected in hen ovarian tissues; this transcript corresponded to bcl-xlong, the death-suppressing form of bcl-x. The highest levels of bcl-xlong messenger RNA were found in granulosa tissue from preovulatory follicles, with significantly lower levels detected in prehierarchal follicle granulosa tissue (P < 0.05). Elevated expression of bcl-xlong in preovulatory follicles was correlated to increased resistance to the process of apoptosis, in vitro, and the virtual absence of follicle atresia at this stage of development, in vivo. We conclude that there is a direct relationship between the inherent susceptibility of avian granulosa cells to apoptosis and the high rate of follicle atresia in follicles not yet selected into the preovulatory hierarchy. Moreover, our results are consistent with the proposal that the expression of death-suppressing genes, including bcl-xlong, is capable of rendering cells resistant to the process of apoptosis. The findings reported herein provide the foundation for a novel model with which to further elucidate molecular mechanisms related not only to the initiation of follicle atresia, but also events associated with the process of follicle selection. PMID- 8612549 TI - Effects of pregnant human, nonpregnant human, and fetal bovine sera on human chorionic gonadotropin, estradiol, and progesterone release by cultured human trophoblast cells. AB - Explant and cell culture methodologies are frequently employed in the investigation of the mechanisms that mediate placental hormone production. Recent reports suggest the presence of unknown regulatory factors in maternal serum that may impact significantly on the regulation of these biosynthetic pathways. The present study, therefore, determined the effects of sera obtained from pregnant women in the second to third trimester (PWS), nonpregnant women (NPWS), and men (MS) as well as commercially prepared FBS on hCG, estradiol, and progesterone release into medium by cultured human trophoblast cells. Placental villous tissue was enzymatically dispersed, and cytotrophoblast cells were purified via density gradient centrifugation and cultured (37 C; 90% air-10% CO2) in DMEM with 10% PWS, NPWS, MS, or FBS. All cytotrophoblast cultures aggregated and progressed to syncytial forms, although cells cultured with PWS exhibited notably larger multinucleated syncytial elements by 48 h in culture than cells cultured with FBS. Significant increases (P < 0.05) occurred in hCG, estradiol, and progesterone release due to the progression of cytotrophoblasts into the syncytiotrophoblast phase in all cultures. The quantity of hCG release was unaffected by serum origin. Cells cultured with human serum released greater (P < 0.05) amounts of estradiol than cells cultured with FBS. Cells cultured with MS released more (P < 0.01) estradiol than cells cultured with either PWS or NPWS, in a ratio to the concentration of endogenous androgen precursor available. Progesterone release was greater (P < 0.01) for PWS-cultured cells than for FBS cultured cells. Progesterone release by NPWS- and MS-cultured cells was intermediate. Syncytiotrophoblasts cultured with PWS released approximately 3 fold more (P < 0.01) progesterone than syncytiotrophoblasts cultured with FBS and low density lipoprotein cholesterol, although the concentrations of available cholesterol substrate were similar. Culture of cells in steroid-depleted or lipoprotein-depleted PWS or FBS resulted in similar decreases (P < 0.01) in estradiol and progesterone release, respectively. In summary, increased estradiol release by placental cells cultured in intact human serum was attributed to aromatizable androgens, whereas enhanced progesterone release by cells cultured in human serum could be only partially attributed to higher concentrations of low density lipoprotein cholesterol substrate in human serum. Evidence of increased syncytial maturity and progesterone release by PWS-cultured cells may indicate the presence of undefined serum-borne regulators, which is enhanced during pregnancy. PMID- 8612550 TI - Estrogen and raloxifene stimulate transforming growth factor-beta 3 gene expression in rat bone: a potential mechanism for estrogen- or raloxifene mediated bone maintenance. AB - Estrogen or raloxifene (LY156758) prevent estrogen deficiency-induced bone loss in animals and humans. We demonstrated in the rat that a 22% reduction in bone mineral density generated by ovariectomy was associated with a 2-fold reduction of transforming growth factor-beta 3 (TGF beta 3) messenger RNA expression in the femur. Administration of 17 beta-estradiol or raloxifene to ovariectomized rats restored both bone mineral density and TGF beta 3 messenger RNA expression in the femur to levels measured in intact animals. In transient transfection assays, the promoter sequence from -38 to + 110 of the human TGF beta 3 gene, which contains no palindromic estrogen response element, was sufficient to mediate 17 beta estradiol or raloxifene induced-reporter gene expression in presence of the estrogen receptor. Raloxifene activated TGF beta 3 promoter as a full agonist at nanomolar concentrations. In the same cellular system, raloxifene inhibited the estrogen response element-containing vitellogenin promoter expression as a pure estrogen antagonist. In two well characterized osteoclast differentiation models, TGF beta 3 significantly inhibited the differentiation and bone-resorptive activities of murine and avian osteoclasts. These findings suggest that regulation of TGF beta 3 gene expression by raloxifene or estrogen in bone may be an important target to mediate bone maintenance. PMID- 8612551 TI - Increased epidermal growth factor expression produced by testosterone in the submaxillary gland of female mice is accompanied by changes in poly-A tail length and periodicity. AB - The level of mature EGF messenger RNA (mRNA) in the female submaxillary salivary gland (SMG) begins to rise after 3 days' treatment with testosterone (200 micrograms sc qod), and by 5 days it reaches a plateau of approximately 5 times baseline. Testosterone can increase the transcription of other genes in the SMG rapidly, so the lag in the EGF is not due to a slow androgen receptor response, and EGF mRNA can respond rapidly to other mediators, so the lag is not an innate characteristic of EGF transcription. Immunoreactive EGF levels reach a steady state several times greater than the plateau reached by EGF mRNA, suggesting that testosterone also enhances the efficiency of EGF mRNA translation. Because testosterone has been reported to alter poly-A polymerase activities and because the translation and stability of some mRNAs is affected by changes in their polyadenylation, we used 3' rapid amplification of complementary DNA ends (3' RACE) to determine whether testosterone affected this aspect of EGF RNA metabolism. We found that EGF transcripts in untreated female SMG occur indistinct size classes, with poly-A tails of approximately 20, 50, 70, 100, and 200 A's attached after the terminal polyadenylation signal. In contrast, EGF transcripts in male SMG have poly-A tails of less clearly defined lengths, being more heterogeneous, ranging from approximately 20-100 A's. Treating female mice with testosterone causes the poly-A pattern in the SMG to change to a more heterogeneous population ranging from approximately 20-100 A's, similar to the male pattern. This change in EGF transcript polyadenylation occurs concurrently with the changes observed in the levels of EGF mRNA. EGF transcripts from male or female kidney contain distinct poly-A tails of approximately 20, 50, 70, 100, and 200 A's: neither EGF mRNA levels nor polyadenylation was altered by testosterone. The tissue-specific increase in EGF mRNA levels and in translational efficiency produced by testosterone in the female mouse SMG could involve this posttranscriptional alteration in transcript polyadenylation. PMID- 8612552 TI - Inducible nitric oxide synthase (iNOS) in pancreatic islets of nonobese diabetic mice: identification of iNOS- expressing cells and relationships to cytokines expressed in the islets. AB - Inflammatory cytokines and nitric oxide (NO) are candidate mediators of pancreatic islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we used a semiquantitative PCR assay to measure levels of messenger RNA (mRNA) expression of the inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha, and interferon-gamma (IFN gamma), and of the inducible form of NO synthase (iNOS) in mononuclear leukocytes isolated from pancreatic islets of autoimmune diabetes-prone nonobese diabetic (NOD) female mice. We found that mRNA levels of iNOS, IL-1 alpha, and IFN gamma in islet mononuclear leukocytes increased from 5 weeks of age to onset of diabetes ( > 13 weeks of age). To determine whether increased iNOS, IL-1 alpha, and IFN gamma mRNA expressions were related to diabetes development, we compared mRNA levels of these molecules in mononuclear leukocytes from islets of 12 week-old diabetes prone NOD female mice and three groups of 12-week-old mice with low diabetes risk: NOD female mice injected with complete Freund's adjuvant at 4 weeks of age, NOD male mice, and BALB/c female mice that do not develop diabetes. We found that iNOS, IL-1 alpha, and IFN gamma mRNA levels were higher in mononuclear leukocytes from islets of diabetes-prone NOD female mice than in those from mice correlated with IL-1 alpha and IFN gamma mRNA levels. By using specific antibodies and immunohistochemical methods, we localized iNOS in macrophages as well as in beta cells of islets from diabetes-prone NOD female mice. These findings suggest that IL-1 alpha and IFN gamma may promote islet beta-cell destruction at least in part by up-regulating iNOS expression an No production by both macrophages and beta cells in the islets of autoimmune diabetes-prone NOD mice. PMID- 8612553 TI - Insulin-like growth factor II affects the appearance and glycogen content of glycogen cells in the murine placenta. AB - The phenotype of mice with a targeted disruption of the insulin-like growth factor II gene (IGF-II null mice) is growth retardation of both fetus and placenta during the last two thirds of gestation (1). We have compared the placenta of IGF-II null and wild-type mice from days 9-18 of gestation. No morphological differences were detected until after day 12 of gestation, when a new population of placental cells, the glycogen cells, normally first appears. Fewer glycogen cells were present in the null placenta compared to the wild-type placenta on days 13, 15, and 18 of gestation. By day 15, glycogen cells constituted approximately 50% of the basal zone cells in the wild-type placenta, but only 20% of the basal zone cells in the null placenta (P < 0.01). By contrast, spongiotrophoblasts constituted 40% of the basal zone cells in the wild type placenta and 70% of the basal zone cells in the null placenta. There were no differences in cell size at any time. These results suggest that glycogen cells do not efficiently differentiate in the absence of IGF-II. The differentiation of glycogen cells did not appear to be simply delayed in the null placenta, as the number of glycogen cells in the null placenta did not increase between days 15 18, and there was no change in the ratio of glycogen to spongiotrophoblasts in the basal zone (P > 0.50). The glycogen content of both spongiotrophoblasts and glycogen cells was significantly reduced in the null placenta, suggesting that IGF-II may be an important regulator of glycogen synthesis in the placenta. These results indicate that IGF-II regulates cell number in the placenta and may play an important role in the differentiation of glycogen cells and the production of glycogen by placental cells. PMID- 8612554 TI - Growth hormone receptor gene is expressed in neuropeptide Y neurons in hypothalamic arcuate nucleus of rats. AB - GH feeds back on the hypothalamus and regulates its own secretion. We have previously shown that systemic administration of GH induces expression of the c fos gene, a marker of neuronal activity, on the hypothalamic neuropeptide Y(NPY) and somatostatin neurons in rats. We argued that if GH were to act directly on NPY neurons, NPY neurons should express the GH receptor (GHR) gene. To test this hypothesis, coronal sections of the medial basal hypothalamus from adult male Wistar rats were processed by double label in situ hybridization using a 35S labeled NPY complementary RNA probe and a digoxigenin-labeled GHR complementary RNA probe. In the medial basal hypothalamus, NPY messenger RNA (mRNA) was observed in the arcuate nucleus (ARC) and the dorsomedial nucleus. The majority (95%) of NPY mRNA-containing cells in the ARC expressed the GHR gene, whereas no NPY mRNA-containing cells in the dorsomedial nucleus expressed the GHR gene. These findings suggest that NPY neurons in the ARC mediate the feedback effect of GH on the hypothalamus. PMID- 8612555 TI - Immunocytochemical localization of stanniocalcin cells in the rat kidney. AB - Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fishes, where it functions as a regulator of calcium and phosphate homoeostasis. Recently, complementary DNAs encoding human STC (hSTC) have been characterized, and recombinant hSTC has been synthesized in a bacterial expression system. In preliminary studies, STC-immunoreactive cells have already been identified in human kidney tubules with antibodies to recombinant hSTC. The purpose of this study was to map the overall spatial distribution of STC cells in mammalian kidney, using the rat as a model system. Immunocytochemistry was performed on fixed sections of rat kidney tissue using hSTC antiserum in conjunction with fluorescein isothiocyanate-conjugated second antibodies. STC-immunoreactive cells were found in cortical thick ascending limb, in macula densa, in distal convoluted tubules, and in the cortical and medullary collecting ducts. All cortical thick ascending limb cells contained immunoreactive STC. Most distal convoluted tubules cells contained STC, and these were identified as principal cells. The distribution of STC cells in cortical and medullary collecting ducts also corresponded closely to the known frequently of principle cells in these segments, suggesting that principal cells are the site of STC storage and/or synthesis in both distal convoluted tubules and collecting ducts. Some collecting duct intercalated cells contained STC as well, and these were tentatively identified as alpha-type intercalated cells. As all tubular segments containing STC are known to be involved in regulated ion transport, renally derived STC may be acting in an autocrine, paracrine and/or endocrine fashion to regulate one or more of these transport processes. PMID- 8612556 TI - Alterations of maternal estrogen levels during gestation affect the skeleton of female offspring. AB - Estrogens have important effects on bone turnover in both humans and experimental animals models. Moreover, the decreased level of estrogen after menopause appears to be one of the key factors in determining postmenopausal osteoporosis. The presence of estrogen receptor in both osteoblasts and osteoclasts has suggested a direct role of these steroid hormones on bone tissue. Thus, this tissue is now regarded as a specific estrogen target tissue. Exposure to estrogens during various stages of development has been shown to irreversibly influence responsive target organs. We have recently shown that transient developmental neonatal exposure (days 1-5 of life) of female mice to estrogen resulted in an augmented bone density in the adult animals. The aim of the present study was to evaluate whether short-term modification of maternal estrogen levels during pregnancy would induce changes in the skeleton of the developing fetuses and to identify any long-term alterations that may occur. Pregnant mice were injected with varying doses (0.1-100 micrograms/kg maternal BW) of the synthetic estrogen diethylstilbestrol (DES) from day 9-16 of pregnancy. Offspring were weaned at 21 days of age, and effects on bone tissue of the female mice were evaluated in adulthood (6-9 months of age). Prenatal DES treatment(s) did not significantly affect BW. However, a dose-dependent increase in bone mass, both in the trabecular and cortical compartments, was observed in the prenatal DES-exposed female offspring. Furthermore, long bones of DES-exposed females were shorter than controls. Normal skeletal mineralization accompanied these changes in the bone tissue, as shown by a parallel increase in skeletal calcium content. Double tetracycline labeling performed in 6-month-old DES-exposed animals showed an increase in mineral apposition rate in adult DES-exposed mice as compared with untreated control animals, although no significant difference in the circulating estrogen levels was found in animals of this age. Experiments were then performed to evaluate whether perturbation of the estrogen surge at puberty in these diethylstilbestrol (DES)-exposed mice could reverse the observed changes. Femur length was chosen as a marker of potential estrogenic effect. Prepubertal ovariectomy of the prenatally DES-treated animals could only partially reverse the effects observed in the skeleton of the DES-treated animals. Further experiments were performed to evaluate whether these changes could have occurred in utero. CD-1 pregnant female mice were injected with DES (100 micrograms/kg maternal BW) from days 9-15 of gestation. On day 16 of gestation, fetuses were examined and stained by a standard Alizarin Red S and Alcian Blue procedure to visualize calcified and uncalcified skeletal tissue. Estrogen treatment induced an increase in the amount of calcified skeleton as compared with untreated controls and also a decrease in the length of long bones, strongly suggesting a change in both endochondral ossification and endosteal and periosteal bone formation. In summary, these data show, for the first time, that alterations in the maternal estrogenic levels during pregnancy can influence early phases of fetal bone tissue development and subsequently result in permanent changes in the skeleton. Finally, the effect of this short-term estrogen treatment can be seen in the fetal skeleton, suggesting an estrogen-imprinting effect on bone cell programming in fetal life because treatment effects on bone cell turnover can be observed later in adult life. PMID- 8612557 TI - Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 during corpus luteum formation and luteolysis in the adult pseudopregnant rat. AB - Proteolytic activity generated by the plasminogen activator (PA) system is associated with many biological processes. Using an adult pseudopregnant rat model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1), are expressed temporally and spatially during different developmental stages of the corpus luteum (CL). Northern blot analysis, in situ hybridization, in situ zymography, and fibrin overlay were used to analyze the expression and distribution of tPA and PAI-1 messenger RNA (mRNA) as well as PA activity in CL of different ages. We demonstrated that during the luteinization period (approximately days 1-2), tPA mRNA was highly and evenly expressed in newly formed CL, whereas PAI-1 mRNA was mainly detected in the central part of the same CL. In accordance with these findings, proteolytic activity generated by tPA was detected in the outer region of newly formed CL by in situ zymography. During the luteotropic period (approximately days 3-10), tPA mRNA expression was very low. PAI-1 mRNA expression was also low, but increased on day 10. As expected, proteolytic activity was very low during this period. During functional luteolysis (days 13-14) and subsequent structural luteolysis, tPA mRNA was elevated. PAI-1 mRNA was also expressed during this period. Moreover, the net PA activity, as determined by fibrin overlay, was relatively high during this period. Our studies indicate that tPA and PAI-1 are coordinately expressed in the CL, resulting in increased proteolytic activities during the luteinization and luteolytic periods. PA-mediated proteolysis may, therefore, play a role in both CL formation and luteolysis in rats. PMID- 8612558 TI - Insulin-like growth factor I-enhanced renal expression of osteopontin after acute ischemic injury in rats. AB - Pretreatment of rats with insulin-like growth factor I (IGF-I) ameliorates the course of acute ischemic renal injury. Differential display PCR was used to identify genes that are expressed in kidney after induction of acute ischemic renal injury in rats pretreated with vehicle or IGF-I. One amplification product that showed enhanced expression in kidneys of rats rendered ischemic compared to kidneys of sham-operated rats was identified as osteopontin. Sequence analysis of full-length complementary DNAs revealed a single species. Renal tissue was obtained for study 12 h and 1, 2, 3, 5, 7, 14, and 28 days postinjury. Levels of whole kidney osteopontin messenger RNA (mRNA) in rats rendered ischemic 1 day previously were elevated approximately 18-fold compared to levels measured in sham-operated controls, as determined by Northern analysis. No differences were noted 12 h postinjury. Levels of osteopontin mRNA remained elevated for 14 days after ischemia, but were no longer elevated at 28 days. IGF-I pretreatment resulted in enhanced levels of osteopontin mRNA 12 h, 1 day, and 5 days postinjury. In situ hybridization demonstrated that the elevated expression of osteopontin 1 day postinjury was localized predominantly to cells in the distal tubule and medullary thick ascending limb of Henle's loop. Immunostaining showed an identical localization for elevated protein expression. Five days postinjury, osteopontin peptide and mRNA were clearly detected in regenerating proximal tubules in addition to distal tubule and medullary thick ascending limb. We propose that endogenous osteopontin serves to promote tissue regeneration and tissue remodeling within 1 day after acute ischemic injury of kidney. IGF-I enhanced expression of osteopontin at an earlier time postischemia may ameliorate the course of injury. PMID- 8612559 TI - In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage specific fashion. AB - The effects of FSH on stage-specific apoptosis and DNA synthesis in the adult rat seminiferous epithelium were studied in vitro. Seminiferous tubular segments from stages I, V, VIIa, and VIII-IX were cultured for 24, 48, and 72 h in different concentrations of FSH. Apoptotic cells were detected by in situ end labeling of DNA strands and quantified from squash preparations. After 48 h of culture, a FSH concentration of 2 ng/ml prevented apoptosis of early (steps 1-3) spermatids. In stage VIII-IX tubules cultured for 72 h, FSH decreased the apoptosis of pachytene spermatocytes. An apoptotic type of cell death of germ cells was confirmed by DNA laddering, electron microscopy, supravital acridine orange staining, and phase contrast microscopy of unstained living cells. The effects of FSH on stage specific DNA synthesis were studied using the same culture system. FSH increased [3H]thymidine incorporation specifically at stages I and VIII-IX, and autoradiography confirmed stimulation of mitotic and meiotic DNA synthesis in type B spermatogonia and preleptotene spermatocytes, respectively. Increased thymidine incorporation also suggested that FSH stimulated DNA synthesis of type A and intermediate spermatogonia. Most effects exerted by FSH were seen in stages containing high levels of FSH receptors and FSH-stimulated cAMP production. In conclusion, the results suggest that FSH, probably acting via Sertoli cells, has a regulatory function in spermatogenic apoptosis and DNA synthesis in stages previously demonstrated to be preferentially dependent on FSH stimulation. PMID- 8612560 TI - Distribution of follistatin messenger ribonucleic acid in the rat brain: implications for a role in the regulation of central reproductive functions. AB - Follistatin (FS), which binds to the inhibin/activin beta A- or beta B-subunit is localized with and modulates the biological actions of activin in many systems. However, in contrast to the wide distribution of the activin beta-subunit proteins and messenger RNAs (mRNA) in the brain, demonstration of FS mRNA signal has been limited to the olfactory tubercle and layer II of the frontal cortex. We have hypothesized a more extensive distribution of central FS gene expression and localization in regions coinciding with inhibin/activin beta-subunits and possible activin-mediated effects. In the present study, we examined the central distribution of FS mRNA expression in the normal adult male rat. With in situ hybridization analysis, using a 33P-labeled RNA probe specific for rat FS, gene expression is shown to be widely distributed throughout the brain. Abundant FS mRNA expression is localized in several areas of the olfactory bulb as well as the frontal cortex, a few thalamic nuclei, and in septal regions. Moderate FS mRNA is observed in the caudate putamen and various hypothalamic areas including the paraventricular, ventromedial, dorsomedial, and arcuate nuclei. Several brain stem regions are also found to express FS mRNA, including the medial vestibular and solitary tract nuclei. Notably, FS mRNA, including the medial vestibular and solitary septal/diagonal band region is localized in patterns that are highly correlative with those of GnRH gene expression and hence may serve to regulate possible activin-mediated effects in these areas. FS mRNA is also expressed in areas associated with the activin-oxytocin pathway (solitary tract nucleus and paraventricular nucleus) and is therefore in a position to modulate the role of activin in the solitary tract nucleus-paraventricular nucleus pathway (afferent system mediating the milk-ejection reflex). The results suggest that FS is centrally localized in sites compatible with a role in the regulation of central reproductive functions. PMID- 8612561 TI - A novel peptide from the growth hormone releasing hormone gene stimulates Sertoli cell activity. AB - The growth hormone releasing hormone (GHRH) gene is believed to produce a single functional peptide, GHRH (1). Although GHRH mRNA and peptide have been identified in various tissues, the only accepted function of GHRH is the stimulation of pituitary growth hormone. We have discovered that the GHRH mRNA encodes a second peptide that is abundant in testicular germ cells and specifically activates Sertoli cell expression of stem cell factor (SCF), a factor crucial for the normal progression of spermatogenesis (2). These observations suggest that the GHRH gene produces two distinct peptides, each with tissue-specific activities. PMID- 8612562 TI - Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. AB - Estrogen treatment of ovariectomized rats rapidly increases immunoreactivity for the phosphorylated form of the cAMP response element binding protein (CREB)in neurons of the preoptic area and the bed nucleus of the stria terminalis. These effects were detected within 15 minutes after estrogen exposure. Since the antisera used for these studies detect CREB phosphorylation at ser133, which is important for transcriptional activation these data provide a possible explanation for estrogen's effects on neuronal genes lacking estrogen response elements (EREs) but which contain cAMP response elements (CREs). These data also provide evidence for non-genomic effects of steroid hormones involving protein kinase associated signal transduction pathways traditionally associated with effects at the cell membrane. PMID- 8612564 TI - Preprothyrotropin-releasing hormone-(178-199) does not inhibit corticotropin release. AB - Pituitary ACTH synthesis and secretion are positively regulated by hypothalamic factors and negatively regulated by adrenal corticosteroids. Negative hypothalamic regulation of pituitary ACTH synthesis and secretion has been postulated, but not proved. The search for a hypothalamic corticotropin release inhibiting factor has recently focused on peptides derived from the prepro-TRH precursor of TRH. One of the peptides, prepro-TRH-(178-199), was reported to suppress basal and stimulated ACTH release. We examined the effects of prepro-TRH (178-199) alone and in combination with CRH and corticosterone, two known physiologic regulators of ACTH secretion. Prepro-TRH-(178-199) had no effect on basal, stimulated, or attenuated ACTH release from cells that responded normally to CRH and/or corticosterone. These results indicate that prepro-TRH-(178-199) is not a corticotropin release-inhibiting factor. PMID- 8612563 TI - Cloning and characterization of human urocortin. AB - Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33). PMID- 8612565 TI - The third cytoplasmic domain of the GLP-1[7-36 amide] receptor is required for coupling to the adenylyl cyclase system. AB - Truncated forms of glucagon-like peptide-1 (tGLP-1) are potent endogenous stimuli of insulin secretion from pancreatic beta cells and have powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the tGLP-1 receptor (R) that are required for its efficient coupling to the adenylyl cyclase (AC) system since it is well established that cAMP is the primary second messenger activated by tGLP-1. The predicted third intracellular loop (IC3) of the rat tGLP-1R was systemically scanned using a mutagenic based strategy. The resulting receptor mutants were expressed in COS-7 cells and examined for cAMP formation in response to tGLP-1 stimulation (10nM) and [125I] tGLP-1(7-36) amide binding. A single block deletion (IC3-1) within the N-terminal region of IC3 (K334-L335-K336) resulted in a dramatic reduction in the cAMP response to tGLP-1 (7.1 +/- 1.4% of the wild type (wt) tGLP-1R response, n = 3, p < or = 0.01), while displaying comparable levels of expression, (expressed as the %Bmax of the wt-tGLP-1R (101 +/- 13%, n = 3, p > or = 0.05). This receptor mutation was further analyzed by stable expression in CHO-K1 cells. In agreement with the COS model, IC3-1 displayed comparable levels of receptor expression (97 +/- 16% Bmax of wt tGLP-1R, n = 3, p > or = 0.05) and affinity for tGLP-1(Kd of 460 +/- 15pM vs. 450 +/- 12pM wt tGLP-1R, n = 3, p > or = 0.05), but was unable to effectively stimulate cAMP production (7.7 +/- 0.4% of wt tGLP-1R, n = 3, p < or = 0.01) in response to tGLP-1 (10nM), No other mutation examined within the IC3 domain displayed a lack of correlation between binding activity and cAMP accumulation. Further analysis of the K334-L335-K336 sequence by substitution analysis revealed that a K334 to A substitution was the only modification to result in a striking attenuation of the cAMP response (28 +/- 1.9% of wt tGLP-1, n = 3, p < or = 0.01). These results strongly suggest that within the IC3 domain the N-terminal KLK sequence or a portion thereof (specifically K-334) is required for the efficient coupling of the tGLP-1 receptor to the AC system. PMID- 8612566 TI - Specific immunoneutralization of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat. AB - Although requirement for follicle stimulating hormone (FSH) in the initiation of spermatogenesis is well documented, its role in adult spermatogenesis is still debated. In the present communication, we have investigated the effect of specific immunoneutralization of FSH on apoptotic cell death in the testicular germ cells both in immature and adult rats. The germ cells of control animals showed predominantly high molecular weight DNA while the antiserum (a/s) treated group showed DNA fragmentation characteristic of apoptosis. The pattern could be detected within 24 hours of a/s treatment, and became more pronounced after 48 hours. The germ cells were purified from FSH a/s treated rats by centrifugal elutriation and vulnerability of each cell type to undergo apoptosis on FSH neutralization was investigated. The pachytene spermatocytes were found to be most sensitive to absence of FSH, even in the adult animals suggesting the involvement of FSH in spermatogenesis. The in situ analysis of DNA strand breakage following FSH a/s treatment showed fragmentation of the DNA of the pachytene spermatocytes confirming this observation. The in situ analysis also showed that the spermatogonia undergo apoptosis in addition to the pachytene spermatocytes. These data clearly demonstrate the role of FSH in the adult rat spermatogenesis. PMID- 8612568 TI - Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact. AB - We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cells (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3 E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell and/or cell-to matrix contact. PMID- 8612567 TI - The tau mutation in the Syrian hamster alters the photoperiodic responsiveness of the gonadal axis to melatonin signal frequency. AB - This study investigated the role of the circadian timing system (CTS) in photoperiodic time measurement by examining the response of the tau mutant hamster to programmed infusions of melatonin. The mutation is a single Mendelian gene defect which accelerates circadian period from 24 h in the wild-type (WT) to 20 h in the homozygote. If the CTS does not contribute to the photoperiodic interpretation of the melatonin signal, then the tau mutation would not influence photoperiodic responses of pinealectomised (PX) animals to systemic infusions of melatonin (10 h) once every 20, 24 or 25 h, mimics short-daylengths and causes gonadal involution. More ( < 18 h) or less ( > 25 h) frequent signals are ineffective. In this study, taus which received melatonin (10 h) once every 16 or 20 h exhibited significant gonadal atrophy relative to saline controls, whereas infusions of melatonin every 24 or 28 h were ineffective. Serum concentrations of LH and PRL were also significantly reduced in both the 16 and 20 h, but not 24 and 28 h groups. The tau mutant hamster may therefore respond to a different and higher ranger of melatonin signal frequencies than those reported for WTs. The 4 h shift in the frequency-response function correlates with the altered circadian period and suggests that the CTS contributes to the photoperiodic interpretation of a series of melatonin signals. PMID- 8612569 TI - Use of heterologous DNA-based gene transfer to follow physiological, T3-dependent regulation of myosin heavy chain genes in Xenopus tadpoles. AB - In vivo gene transfer and RNase protection assay were used to follow thyroid hormone (T3)-dependent regulation of myosin heavy chain (myHC) genes in Xenopus tadpole dorsal muscle. One embryonic and one adult myHC form were measured by each approach. RNase protection assay showed that T3 decreased expression of endogenous embryonic mRNA (E3), but increased adult (A7) transcripts. Gene transfer showed that T3 exerted transcriptional effects on mammalian embryonic and adult myHc promoters injected into the same muscle. The kinetics and profiles of the transcriptional responses were superimposable on endogenous responses. The results strengthen the use of in vivo approaches for determining the roles of transcription factors and cis-regulatory sequences in integrated contexts. PMID- 8612570 TI - Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells. AB - Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP) SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle. PMID- 8612571 TI - A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae. AB - Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co translational pathway of protein transport. PMID- 8612572 TI - The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. AB - Calnexin, a membrane protein of the endoplasmic reticulum, is generally thought to function as a molecular chaperone, based on indirect or correlative evidence. To examine calnexin's functions more directly, we reconstituted the assembly of class I histocompatibility molecules in the absence or presence of calnexin in Drosophila melanogaster cells. Calnexin enhanced the assembly of class I heavy chains with beta 2-microglobulin as much as 5-fold. The improved assembly appeared largely due to more efficient folding of heavy chains, as evidenced by increased reactivity with a conformation-sensitive monoclonal antibody and by a reduction in the level of aggregates. Similar findings were obtained in mouse or human cells when the interaction of calnexin with class I heavy chains was prevented by treatment with the oligosaccharide processing inhibitor castanospermine. The ability of calnexin to facilitate castanospermine. The ability of calnexin to facilitate heavy chain folding and to prevent the formation of aggregates provides compelling evidence that calnexin functions as a bona fide molecular chaperone. PMID- 8612573 TI - The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide. AB - The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone. PMID- 8612574 TI - Structure of a neutralizing antibody bound bivalently to human rhinovirus 2. AB - The structure of a complex between human rhinovirus serotype 2 (HRV2) and the weakly neutralizing monoclonal antibody 8F5 has been determined to 25 A resolution by cryo-electron microscopy and 3-D reconstruction techniques. THe antibody is seen to be bound bivalently across the icosahedral 2-fold axis, despite the very short distance of 60 A between the symmetry-related epitopes. The canyon around the 5-fold axis is not obstructed. Due to extreme flexibility of the hinge region the Fc domains occupy random orientations and are not visible in the reconstruction. The atomic coordinates of Fab-8F5 complexes with a synthetic peptide derived from the viral protein 2 (VP2) epitope were fitted to the structure obtained by cryo-electron microscope techniques. The X-ray structure of HRV2 is not unknown, so that of the closely related HRV1A was placed in the electron microscopic density map. The footprint of 8F5 on the viral surface is largely on VP2, but also covers the VP3 loop centred on residue 3060. C alpha atoms of VP1 and 8F5 come no closer than 10 A. Based on the fit of the X ray coordinates to the electron microscope data, the synthetic 15mer peptide starts and ends in close proximity to the corresponding amino acids of VP2 on HRV1A. However, the respective loops diverge considerably in their overall spatial disposition. It appears from this study that bivalent binding of an antibody directed against a picornavirus exists for a smaller spanning distance than was previously thought possible. Also bivalent binding does not ensure strong neutralization. PMID- 8612575 TI - The transition of pre-BI to pre-BII cells is dependent on the VH structure of the mu/surrogate L chain receptor. AB - We have demonstrated previously that the majority ( > 90%) of VH12 B cells are absent from the adult peripheral repertoire, and that most that remain have the fourth position at the D-J function (designated 10/G4). We report here that most VH 12-expressing pre-B cells are lost during the transition from the pre-BI to the pre-BII cell stage in normal mice, and that pre-BII cell productive (P) rearrangements ar enriched in 10/G4 CDR3. This coincides with the initial expression of H chain and the generation of the mu/surrogate L chain (SL) receptor. In contrast, there is not enrichment for 10/G4 CDR3 in mu MT mice, and the frequency of P rearrangements is as expected from a random rearrangement mechanism, ruling out a biased rearrangement mechanism unique to VH12. We have also demonstrated that non-10/G4 mu chains can associate with SL and be expressed on the cell surface, suggesting that they are available on the cell surface for selection. Thus, transition of pre-BI to pre-BII cells is dependent on the structure of the VH domain. PMID- 8612576 TI - The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence. AB - Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of full-length murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an alpha-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the delta m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed. PMID- 8612577 TI - Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. AB - Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/- mice were normal as shown by glucose tolerance tests. Normal Insr-/- pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post-natal growth retardation (up to 30-40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6-fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr-/- mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed. PMID- 8612578 TI - Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor. AB - The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine-rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive-like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha-32P]GTP. The reaction was blocked by KA, glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a peptide containing the glycine-rich motif. Site-directed mutagenesis of residues K72 and Y73 within the glycine-rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine-rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine rich motif is the binding site at which guanine nucleotides inhibit the glutamate mediated responses of various members of the subfamily of glutamate ionotropic receptors. PMID- 8612580 TI - Interferon-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three. AB - Treatment of cells with interferon (IFN)-alpha caused phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The protein tyrosine kinase Jak1 was found to be necessary for the activation of cPLA2. Jak1 could be co immunoprecipitated with cPLA2 from cell extracts, indicating that a close physical interaction occurs between these two proteins. The induction of IFN stimulated gene factor three (ISGF3) by IFN-alpha, is blocked by cPLA2 inhibitors in cell cultures and in cell-free reconstituted systems. However, these inhibitors do not block IFN-alpha or gamma-induced binding of STAT1 to the inverted repeat (IR) element of the IFN regulatory factor 1 (IRF-1) gene. Thus, cPLA2 activations occurs as an early event in the IFN-alpha response and is selectively involved in ISGF3-dependent gene activation. PMID- 8612579 TI - Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation. AB - Interleukin-6 (IL-6) induces growth arrest and macrophage differentiation through its receptor in a murine myeloid leukaemic cell line, M1, although it is largely unknown how the IL-6 receptor generates these signals. By using chimeric receptors consisting of the extracellular domain of growth hormone receptor and the transmembrane and cytoplasmic domain of gp130 with progressive C-terminal truncations, we showed that the membrane-proximal 133, but not 108, amino acids of gp130 could generate the signals for growth arrest, macrophage differentiation, down-regulation of c-myc and c-myb, induction of junB and IRF1 and Stat3 activation. Mutational analysis of this region showed that the tyrosine residue with the YXXQ motif was critical not only for Stat3 activation but also for growth arrest and differentiation, accompanied by down-regulation of c-myc and c-myb and immediate early induction of junB and IRF1. The tight correlation between Stat3 activation and other IL-6 functions was further observed in the context of the full-length cytoplasmic region of gp130. The result suggest that Stat3 plays an essential role in the signals for growth arrest and differentiation. PMID- 8612581 TI - Proteolytic cleavage of the urokinase receptor substitutes for the agonist induced chemotactic effect. AB - Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s). PMID- 8612582 TI - The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products. AB - c-Abl is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Abl in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Abl C-terminus. c-Abl is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Abl can be directed by a pentalysine nuclear localization signal in the Abl C terminus. Here, we have identified two additional basic motifs in the Abl C terminus, either of which can function independently of the pentalysine signal to localize Abl to the nucleus. Using a quantitative transfection assay, we show that both c-Abl and transforming Abl proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. Further, we demonstrate that the Abl cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Abl inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggest a direct connection between p53 and Rb in this growth inhibitory pathway. PMID- 8612583 TI - Cell type-specific inhibition of p53-mediated apoptosis by mdm2. AB - The effect of excess mdm2 on p53-mediated apoptosis was investigated in two human derived cell lines, H1299 and HeLa. In H1299 cells, overexpression of mdm2 resulted in effective protection from apoptosis. This protective effect was seen only under conditions allowing the formation of p53-Mdm2 complexes. In contrast, excess mdm2 failed to abolish p53-mediated apoptosis in HeLa cells, despite a complete abrogation of p53-dependent sequence-specific transcriptional activation (SST). These data strongly support the contention that SST is dispensable for at least some types of p53-mediated apoptosis. Further, they suggest that one of the roles of mdm2 may be to modulate the apoptotic activity of p53, in a manner which is dictated by the pathway through which p53 induced apoptosis in a given cell type PMID- 8612584 TI - HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4. AB - Tax, a regulatory protein of human T-cell leukemia virus type 1 (HTLV-1), is an oncoprotein which immortalizes human T cells and induces tumors in transgenic mice. These effects may be due to its interaction with cellular proteins, consisting of several transcription factors including CREB, NF-kappa B and SRF, and the transcriptional inhibitor, I kappa B. Here, we found that Tax binds to a cyclin-dependent kinase inhibitor, p16INK4A, which has ankyrin motifs similar to I kappa B. p16INK4A binds to the cyclin-dependent kinases, CDK4 and CDK6, and inhibits their activity, resulting in suppression of G1 phase progression. The binding of Tax to p16INK4a induced a reduction in the p16INK4A-CDK4 complex, with subsequent activation of CDK4 kinase. Tax also suppressed p16INK4A-mediated inhibition of U2OS cell growth. The p16INK4A gene was frequently deleted in many T-cell lines, but not in HTLV-1-infected T-cell lines. Taking these findings together, the functional inactivation of p16INK4A by Tax through protein-protein interaction is suggested to contribute to cellular immortalization and transformation induced by HTLV-1 infection. PMID- 8612586 TI - A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression. AB - The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila. PMID- 8612585 TI - Functional interactions between p53 and the TFIIH complex are affected by tumour associated mutations. AB - The p53 tumour suppressor is mutated in the majority of human tumours. p53's proposed role as the guardian of the genome is reflected in its multiple effects on transcription genome stability, cell growth and survival. We show that p53 interacts both physically and functionally with the TFIIH complex. There are multiple protein-protein contacts, involving two regions of p53 and three subunits of TFIIH, ERCC2 (XPD), ERCC3 (XPB) and p62. p53 and its C-terminus (amino acids 320-393) inhibit both of the TFIIH helicases and in vitro transcription in the absence of TFIIH. Transcription inhibition is overcome by TFIIH. The N-terminal region of p53 (1-320), lacking the C-terminus, is inactive on its own, yet apparently affects the activity of the C-terminus in the native protein. Interestingly, mutant p53s that are frequently found in tumours are less efficient inhibitors of the helicases and transcription. We hypothesize that the interactions provide an immediate and direct link for p53 to the multiple functions of TFIIH in transcription, DNA repair and possibly the cell cycle. PMID- 8612587 TI - The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis. AB - In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1. PMID- 8612589 TI - White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. AB - The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc 1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation. PMID- 8612588 TI - Epidermal egr-like zinc finger protein of Drosophila participates in myotube guidance. AB - We have cloned and molecularly characterized the Drosophila gene stripe (sr) required for muscle-pattern formation in the embryo. Through differential splicing, sr encodes two nuclear protein variants which contain a zinc finger DNA binding domain in common with the early growth response (egr) family of vertebrate transcription factors. The sr transcripts and their protein products are exclusively expressed in the epidermal muscle attachment cells and their ectodermal precursors, but not in muscles or muscle precursors. The results suggest that sr activity induces a subset of ectodermal cells to develop into muscle attachment sites and to provide spatial cues necessary to orient myotubes along the basal surface of the epidermis to their targeted attachment cells. PMID- 8612590 TI - Synergistic enhancement of both initiation and elongation by acidic transcription activation domains. AB - The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation. PMID- 8612591 TI - Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. AB - We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single-stranded thymidines. We found that KMnO4-detectable opening of the AdML promoter requires the presence of the complete pre-initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the -9 to +1 region becomes single stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH-associated DNA helicases is to create a single stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond. PMID- 8612592 TI - Multiple regions of the Arabidopsis SAUR-AC1 gene control transcript abundance: the 3' untranslated region functions as an mRNA instability determinant. AB - The small-auxin-up-RNA (SAUR) transcripts are rapidly induced by auxin and are among the most short-lived mRNAs in higher plants. In this study, we investigate the regulation of SAUR-AC1, a well characterized SAUR gene of Arabidopsis. Be examining the expression of chimeric genes in transgenic tobacco, we demonstrate that the promoter region of SAUR-AC1 mediates auxin induction. Sequences downstream of the promoter region were found to limit mRNA accumulation in a manner that was independent of auxin treatment. Both the coding region and the 3' untranslated region (UTR) of SAUR-AC1 independently contribute to this limitation. Effects on mRNA stability were assayed using chimeric genes under the control of the tetracycline-repressible Top10 promoter. mRNA half-life analysis following tetracycline treatment showed that the SAUR-AC1 coding region does not contain elements that decrease mRNA stability. In contrast, the 3' UTR was found to act as a potent mRNA instability determinant. This finding and the general utility of the Top10 system should provide the means to elucidate mRNA decay pathways that are potentially novel and specific for certain unstable transcripts. PMID- 8612593 TI - Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts. AB - Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s). PMID- 8612594 TI - Structure of the C-terminal end of the nascent peptide influences translation termination. AB - The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes. PMID- 8612595 TI - A preference of histone H1 for methylated DNA. AB - We have identified a clear preference of histone H1 for CpG-methylated DNA, irrespective of DNA sequence. The conditions under which this preference is observed allowed cooperative binding of H1; the H1-DNA complexes formed were shown earlier to be 'tramlines' of two DNA duplexes bridged by an array of H1 molecules, and multiples of these. The preference for methylated DNA is clear in sedimentation assays, which also show that the preference is greater with increased methylation level, and in gel retardation assays with an oligonucleotide containing a single methyl-CpG pair; it is shared by the globular domain which also binds cooperatively to DNA. A small intrinsic preference of H1 for methylated DNA is also apparent in Southwestern assays where the immobilized H1 presumably cannot bind cooperatively. Methylated DNA in H1-DNA complexes was partially protected (relative to unmethylated DNA) against digestion by MspI but not by enzymes whose cutting sites were not methylated, consistent with a direct interaction of H1 with methylated nucleotides; this was also true of GH1-DNA complexes. H1 variants (spH1 and H5) from transcriptionally repressed nuclei have a stronger preference than H1 for methylated DNA, suggesting that this may be relevant to the stabilization of chromatin higher order structure and transcriptional repression. PMID- 8612596 TI - Gene conversion plays the major role in controlling the stability of large tandem repeats in yeast. AB - The genomic stability of the rDNA tandem array in yeast is tightly controlled to allow sequence homogenization and at the same time prevent deleterious rearrangements. In our study, we show that gene conversion, and not unequal sister chromatid exchange, is the predominant recombination mechanism regulating the expansion and contraction of the rDNA array. Furthermore, we found that RAD52, which is essential for gene conversion, is required for marker duplication stimulated in the absence of the two yeast type I topoisomerases. Our results have implications for the mechanisms regulating genomic stability of repetitive sequence families found in all eukaryotes. PMID- 8612597 TI - The mismatch repair system contributes to meiotic sterility in an interspecific yeast hybrid. AB - The mismatch repair system is the major barrier to genetic recombination during interspecific sexual conjugation in prokaryotes. The existence of this anti recombination activity has implications for theories of evolution and the isolation of species. To determine if this phenomenon occurs in eukaryotes, the effect of a deficiency of mismatch repair on the meiotic sterility of an interspecific hybrid of Saccharomyces cerevisiae and the closely related species Saccharomyces paradoxus was examined. The results demonstrate that the rare viable spores from these hybrids have high frequencies of aneuploidy and low frequencies of genetic exchange. Hybrids lacking mismatch repair genes PMS1 or MSH2 display increased meiotic recombination, decreased chromosome non disjunction and improved spore viability. These observations are consistent with the proposal that the mismatch repair system is an element of the genetic barrier between eukaryotic species. We suggest that an anti-recombination activity during meiosis contributes towards the establishment of post-zygotic species barriers. PMID- 8612598 TI - Experimental elongation of telomeres extends the lifespan of immortal x normal cell hybrids. AB - Hybrids between immortal cells that express telomerase and normal cells that lack telomerase have a limited lifespan. We demonstrate that telomerase is repressed in such hybrids. Treatment of immortal human cell lines with certain oligonucleotides resulted in telomere elongation. We took advantage of this observation to test the hypothesis that elongation of telomeres would extend the lifespan of cells in culture. An immortal human cell line was treated with an oligonucleotide to lengthen its telomeres and then was fused with mortal cells. The lifespan of these hybrid cells was longer than that of the hybrids in which telomeres had not been elongated. These observations provide the first direct evidence supporting the hypothesis that telomere length determines proliferative capacity of human cells. PMID- 8612600 TI - A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. PMID- 8612599 TI - The herpes simplex virus type 1 origin-binding protein carries out origin specific DNA unwinding and forms stem-loop structures. AB - The UL9 protein of herpes simplex virus type 1 (HSV-1) binds specifically to the HSV-1 oriS and oriL origins of replication, and is a DNA helicase and DNA dependent NTPase. In this study electron microscopy was used to investigate the binding of UL9 protein to DNA fragments containing oriS. In the absence of ATP, UL9 protein was observed to bind specifically to oriS as a dimer or pair of dimers, which bent the DNA by 35 degrees +/- 15 degrees and 86 degrees +/- 38 degrees respectively, and the DNA was deduced to make a straight line path through the protein complex. In the presence of 4 mM ATP, binding at oriS was enhanced 2-fold, DNA loops or stem-loops were extruded from the UL9 protein complex at oriS, and the DNA in them frequently appeared highly condensed into a tight rod. The stem-loops contained from a few hundred to over one thousand base pairs of DNA and in most, oriS was located at their apex, although in some, oriS was at a border. The DNA in the stem-loops could be stabilized by photocrosslinking, and when Escherichia coli SSB protein was added to the incubations, it bound the stem-loops strongly. Thus the DNA strands in the stem loops exist in a partially paired, partially single-stranded state presumably making them available for ICP8 binding in vivo. These observations provide direct evidence for an origin specific unwinding by the HSV-1 UL9 protein and for the formation of a relatively stable four-stranded DNA in this process. PMID- 8612601 TI - A nonribosomal system of peptide biosynthesis. AB - This review covers peptide structures originating from the concerted action of enzyme systems without the direct participation of nucleic acids. Biosynthesis proceeds by formation of linear peptidyl intermediates which may be enzymatically modified as well as transformed into specific cyclic structures. The respective enzyme systems are constructed of biosynthetic modules integrated into multienzyme structures. Genetic and DNA-sequence analysis of biosynthetic gene clusters have revealed extensive similarities between prokaryotic and eukaryotic systems, conserved principles of organisation, and a unique mechanism of transport of intermediates during elongation and modification steps involving 4' phospho-pantetheine. These similarities permit the identification of peptide synthetases and related aminoacyl-ligases and acyl-ligases from sequence data. Similarities to other biosynthetic systems involved in the assembly of polyketide metabolites are discussed. PMID- 8612602 TI - Association of Lyn tyrosine kinase with the nuclear matrix and cell-cycle dependent changes in matrix-associated tyrosine kinase activity. AB - The nuclear matrix isolated from HeLa cells and Rat2 fibroblasts harbors tyrosine kinase and tyrosine phosphatase activities. Polypeptides of 53, 56 and 60 kDa, associated with this subnuclear structure, were phosphorylated at tyrosine in vivo. By immunoblot and immunolabelling experiments, we identified one of the nuclear-matrix-associated tyrosine kinases as Lyn, a Src family member. Lyn was distributed as foci throughout the matrix. The p56 and p53 isoforms of Lyn remained firmly associated with the nuclear matrix after a variety of matrix preparation procedures, and were not detectable in the chromatin fraction of the nucleus. The tyrosine kinase activity associated with the nuclear matrix showed cell-cycle-dependent changes, maximum activity being observed at the G1/S transition phase. Polyoma-virus-transformed rat fibroblast cells showed sixfold higher tyrosine kinase activity in the nuclear matrix preparations compared to that in untransformed cells. These observations are consistent with the suggestion that tyrosine kinase activity associated with the nuclear matrix may be an important determinant of cellular proliferation. PMID- 8612603 TI - Nerve growth factor treatment of sensory neuron primary cultures causes elevated levels of the mRNA encoding the ATP synthase beta-subunit as detected by a novel PCR-based differential cloning method. AB - The mRNA encoding the rat ATP synthase beta-subunit was rapidly induced by nerve growth factor, within 60 min, in cultured adult rat dorsal root ganglion neurons. ATP synthase beta-subunit cDNA clones were isolated from a lambda library. The library was constructed using rat dorsal root ganglion mRNA that was differentially screened with cDNA-derived probes from untreated and nerve-growth factor-treated primary cultures of adult rat dorsal root ganglion sensory neurons. Radiolabelled probes were made from submicrogram quantities of RNA, by a novel PCR-based technique, which allows small amounts of primary tissue to be used for library screening. The use of this technique in isolating novel differentially expressed mRNAs is discussed. PMID- 8612604 TI - Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types. AB - Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27 bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation. PMID- 8612605 TI - Motifs resembling hepatocyte nuclear factor 1 and activator protein 3 mediate the tissue specificity of the human plasminogen gene. AB - Plasminogen is one of the key elements in the fibrinolytic process. Like most of the gene products that participate in such reactions and which interact with plasminogen, the site of its synthesis is mainly confined to the hepatocyte. Plasminogen RNA has additionally been detected in kidney and very low amounts also in testes. Deletional analysis has indicated that two 5' sequences located within 2.5 kb of the first ATG are responsible for the transcriptional activation and the tissue specificity of the expression of the gene. By DNase protection and gel mobility shift assays with HepG2 nuclear extracts, the two sequences were localized and found to be the recognition sites for the widely known hepatocyte nuclear factor 1 (HNF-1) a trans-acting factor, and a nuclear factor like activator protein 3 (AP-3). The first one lies in a rather unusual position, i.e. within the 5'-untranslated region. The latter is located further upstream in a region between --2200 and --2100 from the plasminogen mRNA cap site. Moreover, site-directed mutagenesis coupled by functional experiments in HepG2 cells has demonstrated a synergism between these two positively acting elements in controlling the transcription of the human plasminogen gene. PMID- 8612606 TI - The translational cis-regulatory element of mammalian ribosomal protein mRNAs is recognized by the plant translational apparatus. AB - The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth status of the cells and its control is mediated through a 5' terminal oligopyrimidine tract (5' TOP) common to all these mRNAs. In the present study, we demonstrate that the plant translational apparatus, as represented by wheat germ extract, discriminates against mammalian mRNAs containing this motif to the same extent as do quiescent mammalian cells. Moreover, mutations in the 5' TOP, which abolish the growth-dependent translational control of the respective mRNAs in mammalian cells, render these mRNAs refractory to discrimination in the plant cell-free system. This selective discrimination reflects neither the specific instability of 5' TOP-containing mRNAs during the incubation in vitro nor a lower competitive potential for the cap-binding protein. The lower in vitro translational efficiency of these mRNAs is an inherent feature which is independent of whether they were derived from polysomes or messenger ribonucleoprotein particles of the transfected mammalian cells. The conservation of the discriminatory property of the translational apparatus between the animal and plant kingdoms is discussed from mechanistic and evolutionary points of view. PMID- 8612607 TI - Purification and characterization of human topoisomerase I mutants. AB - A system for rapid purification and characterization of eukaryotic topoisomerase I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently. PMID- 8612608 TI - 1H-NMR-derived secondary structure and the overall fold of the potent anti-mammal and anti-insect toxin III from the scorpion Leiurus quinquestriatus quinquestriatus. AB - We describe the secondary structure and the overall fold of toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain toxin category and is highly toxic to both mammals and insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 psi, phi dihedral-angle restraints derived from NOE connectivities and 3JNH alphaH coupling constants using the HABAS program. This fold, which mainly consists of an alpha-helix packed against a small antiparallel three-stranded beta-sheet, and of several turns and loops, is similar to that of other long chain scorpion toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this toxin. The central core of toxin III is compared with the cysteine-stabilized alpha beta motif (an alpha-helix connected to a beta-sheet through two disulfide bridges) found in insect defensins and plant thionins. Defensins and thionins are small proteins (approximately 40--50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine stabilized alpha beta motif is a common core to a number of small proteins from different origins and having different activities. PMID- 8612609 TI - 1H NMR of high-potential iron-sulfur protein from the purple non-sulfurbacterium Rhodoferax fermentans. AB - Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed. PMID- 8612610 TI - Reduced B subunit of heat-labile enterotoxin associates with membranes in vivo. AB - The B subunit of heat-labile enterotoxin, a periplasmic protein of Escherichia coli has an internal disulfide bond that forms after the protein has been exported. The presence of 2.5 mM dithiothreitol in the medium prevents the formation of the disulfide bond and this causes the protein to rapidly bind to membranes, preferentially but not exclusively to the cytoplasmic membrane. The binding is irreversible in vivo but chaotropic agents disrupt the association between the non-native B subunit and the membranes in vitro. The fact that the reduced B subunit binds to both the cytoplasmic and outer membranes that enclose the periplasm suggests that it is exported normally to the periplasm and then, because it is unable to form its native structure, adsorbs to membranes in the vicinity. This is confirmed by the finding that when synthesised by spheroplasts, in which the outer membrane is disrupted, the majority of reduced B subunit, which is not now confined in the periplasm, is exported to the medium and is not associated with membranes. PMID- 8612611 TI - Molecular cloning of two different mannose-binding lectins from tulip bulbs. AB - Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N-acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor. PMID- 8612612 TI - A note on circular-dichroic-constrained prediction of protein secondary structure. AB - Circular dichroic (CD) spectra of bovine immunosuppressant binding proteins FKBP12 and FKBP25, and cyclophilins (peptidylprolyl isomerases) A (bCyP-18) and B(bCyP-20), the immunophilins which selectively bind the clinically useful immunosuppressants FK506, rapamycin and cyclosporin A, respectively, were analysed using the singular-value-decomposition algorithm augmented by a simplified variable selection method. The differences between the CD-estimated values of alpha-helix, beta-structure and beta-turn and those predicted by the Chou-Fasman algorithm were minimized using the CD data as constraints of an algorithm which utilizes the method of hierarchical updating of quasi equipotential peptide segments of the Chou-Fasman prediction. The method allows one to correct the Chou-Fasman prediction of secondary structures in globular proteins. PMID- 8612613 TI - Alterations of lipoprotein fluidity by non-esterified fatty acids known to affect cholesteryl ester transfer protein activity. An electron spin resonance study. AB - The aim of the present study was to investigate the effect of saturated, monounsaturated and polyunsaturated non-esterified fatty acids (NEFA) on lipoprotein fluidity by using the electron spin resonance (ESR) method. The fluidity of the lipid phase of lipoproteins was evaluated by calculating from ESR spectra the S parameter of three different positional isomers of spin-labeled stearic acid incorporated into the lipoprotein. In non-enriched lipoproteins, S values were higher in high-density lipoprotein 3 (HDL3) than in low-density lipoprotein (LDL) indicating that the surface of HDL3 was more ordered. Prior incubation of lipoprotein particles with NEFA significantly reduced S values, indicating an increased lipoprotein fluidity as compared with non-supplemented homologous samples. In NEFA-enriched lipoproteins, the modifications in fluidity were shown to be dependent on the structure of the NEFA acyl carbon chains. Medium-chain fatty acids [lauric (12:0) and myristic (14:0) acids] appeared to be better fluidizing molecules as compared with both shorter [octanoic (8:0) and decanoic (10:0) acids] and longer [palmitic (16:0) and stearic (18:0) acids] homologues. In addition, introducing at least one double bond in the acyl carbon chain significantly increased the ability of NEFA to reduce S as compared with saturated homologues. In both LDL and HDL3, the extent of the modifications of the molecular mobility at the lipoprotein surface was dependent on the final NEFA/lipoprotein ratio. In conclusion, these results suggest that the ability of NEFA to modulate the activity of the cholesteryl ester transfer protein might relate in part to alterations in fluidity at the lipoprotein surface. PMID- 8612614 TI - Cooperation of tyrosine kinases p72syk and p53/56lyn regulates calcium mobilization in chicken B cell oxidant stress signaling. AB - A chicken B cell line DT40 and its syk-negative or lyn-negative mutants were used to investigate the roles of protein-tyrosine kinases in oxidant stress signaling. The data presented here for wild-type cells demonstrate that hydrogen peroxide stimulates p53/56lyn-dependent tyrosine phosphorylation and activation of p72syk, and induces a rapid and prolonged elevation of intracellular calcium, which consists of calcium release from intracellular stores and influx from the extracellular space. Hydrogen-peroxide-triggered calcium mobilization was impaired in both syk-negative and lyn-negative cells, which was mainly due to the loss of calcium release from intracellular stores. Further studies indicated that inositol trisphosphate production was also abolished in both syk-negative and lyn negative cells, which is consistent with the loss of calcium release. Taken together, these observations suggest that the defect of p72syk or p53/56lyn was responsible for the abnormality of calcium mobilization in both lyn-negative and syk-negative cells, and that both p72syk and p53/56lyn might regulate calcium mobilization through the phosphatidylinositol pathway in B cell oxidant stress signaling. PMID- 8612615 TI - Purification and cDNA cloning of ferritin from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus. AB - Ferritin was purified from the hepatopancreas of the freshwater crayfish Pacifastacus leniusculus after injection of iron. It has the same size as horse spleen ferritin (440 kDa) and migrates as two bands, 19 kDa and 20 kDa, respectively, in SDS/PAGE under reducing conditions. Crayfish ferritin (20 kDa) was cloned from a hepatopancreas cDNA library. The deduced amino acid sequence of the crayfish ferritin shows a closer relationship to vertebrate ferritin heavy chains than to insect ferritin and contains the conserved H-specific residues for the ferroxidase centre found in vertebrate ferritin heavy chain. An IRE(iron responsive element)-like sequence with a predicted stem-loop structure was present in the 5' untranslated region of the crayfish ferritin mRNA. Crayfish ferritin does not share the atypical properties of insect ferritins, such as high molecular mass of intact protein, abundance in hemolymph, and export into vacuoles. We suggest that there are two different types of ferritins distributed in different species: insect-type or secretory ferritins which are predominant in the snail oocyte and insects, and vertebrate (crustacean)-type or cytosolic ferritins which are predominant in vertebrates and crustacea. PMID- 8612616 TI - The myeloid differentiation antigen CD14 is N- and O-glycosylated. Contribution of N-linked glycosylation to different soluble CD14 isoforms. AB - The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N- and O-linked carbohydrates from serum- and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membrane-bound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple forms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14. PMID- 8612618 TI - Energizing effects of illumination on the reactivities of lysine residues of the gamma subunit of chloroplast ATP synthase. AB - Incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the gamma subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s after the onset of illumination. Illumination of thylakoids increased the reactivities of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20-30 s after the onset of illumination. In the presence of 10 mM NH4Cl, illumination of thylakoids did not change the reactivities of these lysine residues. These results suggest that the Lys24- and Lys30-containing region of the gamma subunit changes its conformation rapidly in response to delta mu H+ and that the Lys222- and Lys231 containing region of the gamma subunit changes its conformation in two steps in response to delta mu H+ formation. PMID- 8612617 TI - Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction. AB - Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH -cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions. PMID- 8612619 TI - Dissociation of c-fos induction and mitogen-activated-protein kinase activation from the hepatocyte-growth-factor-induced motility response in human gastric carcinoma cells. AB - The function of hepatocyte growth factor/scatter factor (HGF/SF) is to increase proliferation as well as to stimulate motility and disperse cell colonies of epithelial cells. In this study, we examined the motogenic and mitogenic responses of two human gastric carcinoma cell types, MKN7 and MKN74. Cell motility of both cell lines was markedly stimulated by HGF/SF. In contrast, HGF/SF stimulated cell growth of MKN74 cells, but did not stimulate growth of MKN7 cells. To address the cause of the difference in response of these cells, which may reflect some differences in signaling pathways downstream from the HGF/SF receptor, c-Met, we investigated the induction of the proto-oncogene c fos. The level of c-fos mRNA increased and reached a maximum approximately 40 min after HGF/SF stimulation in MKN74 cells, and thereafter its level rapidly decreased. In contrast, the level of c-fos expression was very low irrespective of the stimulation in MKN7 cells. c-Fos protein was transiently induced only in MKN74 cells l h after treatment with HGF/SF, and its levels subsequently decreased. We subsequently examined the activation of mitogen-activated-protein kinase, which is a major mediator in the signaling pathway leading to the stimulation of c-fos transcription, after HGF/SF treatment in both cell lines. Mitogen-activated-protein kinase was markedly activated by this treatment in MKN74 cells, but was only slightly activated in MKN7 cells. These results suggest that although mitogen-activated-protein kinase activation and c-fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/SF. PMID- 8612621 TI - The crystal structure of transthyretin from chicken. AB - The crystal structure of chicken transthyretin has been solved at 290-pm resolution by molecular-replacement techniques. Transthyretin is the protein component of the amyloid fibrils found in patients suffering from either familial amyloidotic polyneuropathy or senile systemic amyloidosis. Familial amyloidotic polyneuropathy is an autosomal dominant hereditary type of amyloidosis which involves transthyretin with either one or two amino acid substitutions. The three dimensional structure of chicken transthyretin was determined in order to compare a non-amyloidogenic, species-variant transthyretin with wild-type and mutant transthyretin molecules. Of the 31 chicken-to-human residue differences, 9 occur at positions which in human transthyretin give rise to amyloidogenic variants although none corresponds to the appropriate side-chain substitutions. The model of chicken transthyretin has been refined to an R-factor of 19.9%. The overall fold of the protein is that of an all-beta protein. Compared with wild-type human transthyretin the avian transthyretin shows quite large differences in the region known to be involved in binding to retinol-binding protein, it has a much shorter helical component than the human protein and some of the monomer-monomer interactions are different. PMID- 8612620 TI - Molecular cloning of mouse 17 beta-hydroxysteroid dehydrogenase type 1 and characterization of enzyme activity. AB - The biological activity of certain estrogens and androgens is modulated by enzymes called 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs), which catalyze the interconversion between less active 17-oxosteroid and more active 17 beta-hydroxysteroid forms. In the present report, we describe cloning of mouse 17 beta-HSD type-1 cDNA from an ovarian library generated from 4,4'-(1,2-diethyl-1,2 ethenediyl)bisphenol-(diethylstilbestrol)-tr eated mice, and characterization of the corresponding enzyme. The open reading frame of the mouse 17 beta-HSD type-1 cDNA encodes a peptide of 344 amino acid residues with a predicted molecular mass of 36785 Da. The mouse 17 beta-HSD type-1 enzyme shares 63% and 93% overall identity with human and rat 17 beta-HSD type-1 enzymes, respectively, and the most striking differences between the mouse and human type-1 enzymes are between the amino acid residues 197 and 230 and in the carboxy terminus of the enzymes. Similarly to the human 17 beta-HSD type-1 enzyme, the mouse type-1 enzyme primarily catalyzes reductive reactions from 17-oxo forms to 17 beta-hydroxy forms in intact cultured cells, but unlike the human type-1 enzyme, the mouse enzyme does not prefer phenolic over neutral substrates. Thus, mouse 17 beta-HSD type 1 catalyzes reduction of androst-4-ene-3,17-dione (androstenedione) to 17 beta-hydroxyandrost-4-en-3-one (testosterone) as efficiently as 3 beta hydroxyestra-1,3,5(10)-trien-17-one (estrone) to estra-1,3,5(10)-triene-3 beta, 17 beta-diol (estradiol). 17 beta-HSD type 1 is predominantly expressed in mouse ovaries, in which it is located in granulosa cells. PMID- 8612622 TI - CCAAT/enhancer-binding protein beta (C/EBP beta) binds and activates while hepatocyte nuclear factor-4 (HNF-4) does not bind but represses the liver-type arginase promoter. AB - In an attempt to elucidate the mechanism governing liver-specific transcription of the arginase gene, we previously detected two protein-binding sites designated footprint areas A and B at positions around--90 and --55 bp, respectively, relative to the transcription start site of the rat arginase gene. Based on the finding that area A was bound by a liver-selective factor(s) related to CCAAT/enhancer-binding protein (C/EBP), we performed cotransfection assay and showed that C/EBP family members and a related factor, albumin D-element-binding protein (DBP) stimulate transcription from the arginase promoter. In addition to area A, a recombinant C/EBP beta protein bound to area B, which appeared to be primarily responsible for activation by C/EBPs. We unexpectedly found that the arginase promoter activity stimulated by C/EBPs and DBP was repressed by another liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4). Analysis of chimeras formed between the arginase promoter and the herpes simplex virus thymidine kinase promoter allowed us to delimit the negative HNF-4 responsive element into the region overlapping with footprint area B. However, no apparent binding of HNF-4 was observed in this negative element. We speculate that HNF-4 is involved in fine regulation of the arginase gene in the liver or shutdown of the gene in nonhepatic tissues without direct binding to the promoter region. PMID- 8612623 TI - The proteasome subunit, C2, contains an important site for binding of the PA28 (11S) activator. AB - The PA28, or 11S regulatory complex, stimulates the peptidase activities of the 20S proteasome. Monoclonal antibodies were screened for their ability to inhibit the activation by PA28 of proteasomes from rabbit reticulocytes. We identified one antibody that inhibited proteasome activation by PA28 and dissociated formed proteasome-PA28 complexes. A fourfold molar excess of antibody to proteasome markedly reduced the PA28 activation of three peptidase activities. Examination of proteasome-antibody mixtures by electron microscopy revealed that the antibody formed chains of proteasomes, and digital image analysis of individual proteasomes demonstrated that the antibody binds to the outer alpha rings. This antibody recognizes proteasome subunit C2, which we conclude contains an important contact site for the PA28 activator. However, the antibody did not block proteasome activation by PA700, or 19S regulator, which also associates with the alpha rings. Thus, these two regulators appear to bind to the proteasome at different sites. PMID- 8612624 TI - A factor derived from polymorphonuclear leukocytes enhances interleukin-1-induced synovial cell collagenase and prostaglandin E2 production in rats. AB - We found that short-term culture medium and homogenate of casein-induced rat peritoneal polymorphonuclear leukocytes (PMN) markedly induced collagenase and prostaglandin E2 (PGE2) production by normal rat synovial cells and these effects were abrogated by anti-(rat interleukin-1 alpha) (IL-1 alpha) polyclonal antibodies. However, collagenase activity and PGE2 induced by recombinant rat IL 1 alpha were less than those induced by rat PMN culture medium. It was also proved by radioimmunoassay that rat PMN culture medium contains a relatively small amount of IL-1 alpha. The introduction of IL-1 alpha-deleted PMN culture medium and recombinant rat IL-1 alpha together into the synovial cell culture system revealed that IL-1 alpha deleted PMN culture medium has a significant enhancing activity on IL-1 alpha-induced synovial cell collagenase and PGE2 production. This new factor, which was shown to be a negatively charged protein of about 80 kDa, may have important roles in connective tissue destruction and chronic inflammation in diseases such as rheumatoid arthritis. PMID- 8612626 TI - Different apoptotic pathways mediated by Fas and the tumor-necrosis-factor receptor. Cytosolic phospholipase A2 is not involved in Fas-mediated apoptosis. AB - Fas is a cell-surface receptor that belongs to the tumor-necrosis factor (TNF)/nerve growth factor receptor family. Fas can transduce an apoptotic signal through the death domain in the cytoplasmic region, which has similarity with the corresponding region of the TNF type-I receptor. Here, we expressed human Fas in mouse L929 cells or its subline (C12), which express extremely low levels of cytosolic phospholipase A2 (cPLA2). L929 cells were sensitive to the cytotoxic activity of TNF, while C12 cells were resistant. Cross-linking of human Fas with anti-(human Fas antibody) Ig killed both L929 transformants and C12 transformants expressing human Fas. Various inhibitors of the arachidonate metabolism significantly inhibited the TNF-induced cytotoxicity in L929 cells, but they did not have any effect on Fas-mediated apoptosis. These results indicated that cPLA2 is required for TNF-induced apoptosis, whereas it is dispensable for Fas-mediated apoptosis, and suggested that the TNF receptor and Fas use different signaling pathways for apoptosis. PMID- 8612625 TI - Role of insulin-like growth factors in autocrine growth of human retinoblastoma Y79 cells. AB - In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin-like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross linking procedure, that Y79 cells contain the type-I IGF receptor (IGF-IR). Furthermore, surface-bound IGF-I is internalised by the receptor, then degraded to amino acids. Insulin, IGF-I and IGF-II caused down-regulation of IGF-IR; the effect is concentration and time dependent. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF I while the apparent Kd value calculated for IGF-I binding was not significantly modified. IGF-I, IGF-II and insulin induced tyrosine phosphorylation of IGF-IR. Tyrosine phosphorylation of this receptor with, however, a less strong signal, was detectable even in cells cultured in serum-free medium without the addition of any exogenous growth factor. Similar results have been found concerning the tyrosine phosphorylation of insulin receptor substrate-1 (IRS 1). Tyrosine phosphorylation of both IGF-IR and IRS 1, either under basal conditions or after stimulation with growth factors, was strongly inhibited when alpha-IR3, a monoclonal antibody to IGF-IR, was added to the culture. IGF-I was capable of inducing Y79 cell proliferation and its effect was entirely inhibited by the addition of alpha-IR3. This antibody also markedly reduced the proliferation of Y79 cells cultured in serum-free medium not supplemented with stimulatory factors. Our results indicate that IGF-I and IGF-IR mediate an autocrine growth mechanism in Y79 cells. PMID- 8612627 TI - Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles. AB - The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross-reinnervation by a fast nerve of the slow twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast-twitch Semimembranous accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow-type (1S', 1S and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type 1 MyLC corresponding most probably to an embryonic MyLC. Five-month cross-reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co-expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type II fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast-type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98% type IIx/d and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow-type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast-twitch or slow-twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast-twitch SMa muscle, and is barely expressed in cross-reinnervated slow-twitch SMp muscle. In contrast, type IIa and type IIx/d are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I. PMID- 8612628 TI - A comparative study of the phosphotyrosyl phosphatase specificity of protein phosphatase type 2A and phosphotyrosyl phosphatase type 1B using phosphopeptides and the phosphoproteins p50/HS1, c-Fgr and Lyn. AB - The phosphotyrosyl phosphatase (PTPase) specificity of phosphotyrosyl-phosphatase activator-(PTPA)-stimulated protein phosphatase (PP)2A(D) (rabbit muscle) and a bona fide PTP-1B (Xenopus laevis oocytes) were examined in vitro using phosphotyrosine-containing peptides, derived from the phosphorylation sites of p34cdc2, p50/HS1 protein, Abl, c-Src and c-Fgr, as well as the intact phosphoprotein p50/HS1 and the Src-related tyrosine kinases, Lyn and c-Fgr. The local specificity determinants were found to be different for both PTPases. The length of the phosphopeptides is more important for PP2A(D) than for PTP-1B, C terminal acidic residues adjacent to the phosphotyrosine are detrimental for the PTPase activity of PP2A(D), but they do not affect the PTP-1B activity. Acidic residues at the --2 and --3 position relative to Tyr(P) primarily dictate dephosphorylation by PTP-1B. The higher-order structure of the protein substrates also differentially influences both enzymes: the phospho-octapeptide KDDEYpNPA, which reproduces the autophosphorylation site in c-Fgr (Tyr400), is only dephosphorylated by PP2A(D) if embedded in the intact protein, whereas the opposite is true for PTP-1B. Both the intact p50/HS1 phosphoprotein and the derived phosphopeptide are substrates only for PTP-1B and not for PP2A(D). Lyn and c-Fgr phosphorylated by C-terminal Src kinase (CSK) at their down-regulatory site are resistant to the action of both PTPases while the [Phe6]Src-(514-533) phosphopeptide, representing the highly similar site affected by CSK in c-Src, is readily dephosphorylated by both PTPases, although to a different extent. In vitro dephosphorylation of the c-Fgr Tyr400 site by PP2A(D) is correlated with a decreased tyrosine kinase activity towards exogenous substrates. Under experimental conditions in which both Tyr400 (autophosphorylation site) and Tyr511 (down-regulatory site) of c-Fgr are phosphorylated, PP2A(D) can reverse both phosphorylations. PMID- 8612629 TI - Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli. AB - Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro 5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth. PMID- 8612630 TI - Linking of isozyme and class variability patterns in the emergence of novel alcohol dehydrogenase functions. Characterization of isozymes in Uromastix hardwickii. AB - The nature of the isozyme differences in the class-I alcohol dehydrogenase structure from the lizard, Uromastix hardwickii, was determined and related to those in the human and horse enzymes, for which isozyme structures have also been established. The Uromastix isozymes differ much (at a total of 72 positions, 19%) but, in spite of this, have similar properties and were not obtained resolved. Their structures were analyzed in mixture, and the two sub-sets of peptides obtained could be distinguished by evaluation of the recovery ratios within the peptide pairs. The isozymes have class-I activities, with an ethanol dehydrogenase activity of 0.6 U/mg and no formaldehyde dehydrogenase activity, have typical class-I structures, and are composed of N-terminally acetylated 375 residue subunits (a and b). Importantly, variability patterns between the isozymes are reminiscent of those both in the other two lines with isozymes (primates and horse) and in the class distinctions of the enzyme. Hence, the variability pattern since the distant stage of class-I emergence is also visible within the more recent isozyme divergence, illustrating a continuity in the evolution of isozymes to classes (and then to enzymes). The pattern also links the different levels of multiplicity and may suggest an acceptability in common to duplications and mutations, compatible with the emergence of novel functions. PMID- 8612631 TI - Liver class-I alcohol dehydrogenase isozyme relationships and constant patterns in a variable basic structure. Distinctions from characterization of an ethanol dehydrogenase in cobra, Naja naja. AB - The major ethanol dehydrogenase of cobra liver was characterized in order to clarify isozyme relationships and functional motifs of the vertebrate enzyme. The cobra protein is a class-I form, most related to one of the isozyme subunits (the a form) in Uromastix (lizard) liver. This positions the isozyme duplication and defines the main-line alternative. The new structure also allows extensive correlations with structure/function relationships for alcohol dehydrogenases in general, of which 38 animal variants (still disregarding strain and allelic differences) now have been characterized. Architectural features are discerned, distinguishing the enzyme at large, the classes, and the functional interactions at the sites of substrate binding and coenzyme binding. Variability is greater at the substrate-binding site, with only one of 13 residues strictly conserved (His67, one of the active-site zinc ligands) but all other residues differing among and frequently within classes. However, many substrate-interacting residues are class preferential and may be used in predictive assignments. Class-I/III differences concern position 48 (typically Ser in class I, Thr in class III), position 93 (Phe versus Tyr), position 141 (branch-chained aliphatic residue versus methionine), position 57 (hydrophobic residue versus Asp), position 115 (Asp versus Arg), position 116 (Leu or Ile versus Val), position 306 (Met or Leu/Ile versus Phe), position 309 (Phe or Leu/Ile versus Val) and position 318 (Val or Ile versus Ala). In contrast, coenzyme binding is more conserved. A characteristic coenzyme-binging motif, covering only a 50-residue stretch, is defined as tVDiK (residues 178, 203, 223, 224, 228; capital letters for residues strictly conserved and small-cases letters for residues nearly so). This motif is class independent and unique to animal alcohol dehydrogenases. Therefore, the novel enzyme structure establishes class-I isozyme relationships, shows characteristic 'constant' residues also in the 'variable' class-I line, and defines residue-specific patterns which may have a predictive value in functional assignments of an increasing number of undefined further forms expected to result from gene projects. PMID- 8612632 TI - Comparative study of the sugar chains of alkaline phosphatases purified from rat liver and rat AH-130 hepatoma cells. Occurrence of fucosylated high-mannose-type and hybrid-type sugar chains. AB - The N-linked sugar chains of alkaline phosphatases, purified from rat AH-130 hepatoma and from normal rat liver, were released quantitatively as oligosaccharides by hydrazinolysis and were labeled by reduction with NaB3H4. A comparative study of their structures revealed that following structural differences are induced by hepatocyte carcinogenesis: complex-type tetraantennary sugar chains and hybrid-type sugar chains appear; outer-chain moieties of the sugar chains of the hepatoma enzyme contain exclusively the Gal(Beta 1-4)GlcNAc groups (type 2 chains) but those of the normal enzyme contain other Gal(Beta 1 )GlcNAc groups and type 2 chains; and novel fucosylated high-mannose-type sugar chains are found in the oligosaccharides of the hepatoma enzyme. PMID- 8612633 TI - Degradation of the light-stress protein is mediated by an ATP-independent, serine type protease under low-light conditions. AB - Green plants respond to light stress by induction of the light-stress proteins (ELIPs). These proteins are stable as long as the light stress persists but are very rapidly degraded during subsequent low light conditions. Here we report that the degradation of ELIPs is mediated by an extrinsic, thylakoid-associated protease which is already present in the membranes during light stress conditions. Partial purification of the protease by perfusion chromatography indicates that this proteolytic activity may be represented by a protein with an apparent molecular mass of 65 kDa. The ELIP-directed protease is localized in the stroma lamellae of the thylakoid membranes and does not require ATP or additional stromal factors for proteolysis. The protease has an optimum activity at pH 7.5 9.5 and requires Mg2+ for its activity. The ELIP-degrading protease show an unusual temperature sensitivity and becomes reversibly inactivated at temperatures below 20 degree C and above 30 degree C. Studies with protease inhibitors indicate that this enzyme belongs to the serine class of proteases. The enhanced degradation of ELIP in isolated thylakoid membranes after addition of the ionophore nigericin suggests that a trans-thylakoid delta pH or changes in ionic strength may be involved in the mechanism of protease activation. PMID- 8612635 TI - Anti-peptide immunoglobulins from rabbit and chicken eggs recognise recombinant human dihydroorotate dehydrogenase and a 44-kDa protein from rat liver mitochondria. AB - Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate. 312-bp and 983 bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32. The identity of the PCR products was verified by dideoxynucleotide sequencing. Transformation of Escherichia coli strain M15 resulted in expression of 13-kDa and 36-kDa proteins with an affinity tag consisting of six consecutive histidine residues; these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+-chelating resin. In immunoblotting analyses, the fusion proteins were recognised by polyclonal avian and mammalian anti peptide immunoglobulins. These were generated against synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDNA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabbits and laying hens were immunized with these peptides without conjugation to a carrier protein. Comparison of the anti-peptide immunoglobulins produced from egg yolk and rabbit serum demonstrated that avian anti-(dihydroorotate dehydrogenase) immunoglobulins may be considered a superior alternative to the mammalian equivalent; antibodies from both sources were applicable for all immunochemical purposes. Here, these antibodies were applied for identification of a 44-kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrogenase activity. PMID- 8612634 TI - Purification, metal cofactor, N-terminal sequence and subunit composition of a 5 aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'. AB - 5-Aminolevulinic acid dehydratase was purified to apparent homogeneity from Scenedesmus obliquus, mutant C-2A', starting with serial affinity chromatography according to Wang et al., followed by separation on DEAE-Cellulose DE 52, TSKgel Toyopearl HW-55 and FPLC on Mono Q. The enzyme was purified 117-fold compared with the initial crude soluble enzyme preparation and showed a final specific activity of 9.17 microkat/kg protein at pH 8.2 at a total recovery of 7%. Mg2+ was determined to be the metal cofactor of the enzyme. It can, to a certain extent, be substituted by other divalent cations. From the purified enzyme the first 15 amino acids of the N-terminus could be determined, showing a moderate similarity to 5-aminolevulinic acid dehydratases from spinach, pea, Escherichia coli and yeast. The molecular mass of the native protein was determined by gel filtration to be 282+/-5 kDa. 42+/-1 kDa were ascertained for the subunit size by SDS/PAGE. These investigations, supported by electron microscopy, revealed that the enzyme from Scenedesmus consists of six subunits arranged in a six-membered ring. Additionally, there is some evidence that two of the rings form a sandwich like complex. PMID- 8612636 TI - Transcriptional regulation of transferrin receptor expression during phorbol ester-induced HL-60 cell differentiation. Evidence for a negative regulatory role of the phorbol-ester-responsive element-like sequence. AB - The mechanism involved in the regulation of transferrin receptor (TfR) expression during phorbol-ester-induced HL-60 cell differentiation was investigated. The mRNA of the TfR was constitutively expressed in proliferating HL-60 cells. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h resulted in a gradual decrease in the expression of the TfR mRNA. Nuclear run-on assays revealed that the transcription of the TfR gene was inhibited by prior treatment of cells with PMA. The effect of PMA on the binding of nuclear proteins to the TfR gene promoter region was then investigated. Based on sequence similarity and previous footprinting data, the promoter region of the TfR gene seems to contain a sequence like that of the phorbol-ester-responsive element (TRE). Our results showed that the binding of nuclear extracts to the TfR gene promoter region containing the TRE-like sequence was increased in PMA-treated cells. This binding activity could be abolished by prior incubation of the nuclear extracts with a synthetic oligonucleotide containing the consensus TRE sequence. In vitro transcription assays revealed that prior incubation of the nuclear extracts of PMA-treated cells with excess consensus TRE oligonucleotide enhanced the gene transcription driven by the TfR gene promoter. These findings suggest that the TRE-like element may play a role in the inhibition of TfR gene transcription. PMID- 8612637 TI - A 13C-NMR study of the mechanism of bacterial metabolism of monomethyl sulfate. AB - Two different mechanisms have been proposed previously for initiating the biodegradation of monomethyl sulfate (MeSO4) in bacteria. For a Hyphomicrobium species, a sulfatase enzyme has been proposed to hydrolyse MeSO4 to methanol and inorganic sulfate. For an Agrobacterium sp., an alternative proposal involves monooxygenation of MeSO4 (hydroxylation) to produce methanediol monosulfate, which decomposes spontaneously to formaldehyde and inorganic sulfate. In the present study, 13C-NMR was used to monitor metabolic intermediates of [13C]MeSO4 in real time in each species in order to resolve the issue of mechanism of biodegradation. Agrobacterium sp. M3C grew on MeSO4 but not on methanol. MeSO4 grown cells catabolised [13C]MeSO4 but not [13C]methanol, and [13C]methanol did not accumulate from MeSO4 in the presence of a known inhibitor of methanol dehydrogenase (cyclopropanol). Hyphomicrobium MS223 grew on MeSO4 and, in contrast with the Agrobacterium sp., also on methanol. The normally rapid metabolism of [13C]methanol by methanol-grown cells was arrested by cyclopropanol, but metabolism of [13C]MeSO4 by MeSO4-grown cells was unaffected. Moreover there was no accumulation of [13C]methanol from [13C]MeSO4 under conditions in which methanol dehydrogenase was shown to be inactive. The results provided strong evidence against the intermediacy of methanol in the biodegradation of MeSO4 in either species, and thereby render untenable mechanisms involving sulfatase-mediated hydrolysis of MeSO4. The data are consistent with the hydroxylation of MeSO4 via a monooxygenation mechanism and subsequent spontaneous hydrolysis of the methanediol monosulfate intermediate. PMID- 8612639 TI - Regulation of olfactory signalling via cGMP-dependent protein kinase. AB - Strong odor stimuli elicit a slow and sustained increase of the cGMP concentration in isolated rat olfactory cilia. Elevated cGMP levels appear to attenuate the primary response to odorant stimulation. Incubating cilia with membrane-permeable cGMP derivates caused a significantly reduced cAMP signal in response to odorant stimulation. This inhibitory effect was mimicked by 8-(4 chlorophenlythio)-cGMP, a selective activator of cGMP-activated protein kinases; in contrast, a selective inhibitor, [8-(4-chlorophenylthio)-guanosine-3',5' cyclic monophosphorothioate] of cGMP kinases enhanced the reactivity to odorant stimulation. The data suggest that the responsiveness of olfactory sensory cells is governed by a cGMP-dependent protein kinase. Western-blot analysis using subtype-specific antibodies indicated that cytosolic type-I cGMP kinase, but not the membrane-associated type-II cGMP kinase, is expressed in olfactory sensory neurons. PMID- 8612638 TI - A two-hybrid system analysis shows interactions between 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase but not between other glycolytic enzymes of the yeast Saccharomyces cerevisiae. AB - The yeast two-hybrid system was used to investigate whether protein-protein interactions between enzymes of the early reactions of glycolysis exist in the yeast Saccharomyces cerevisiae. Various glycolytic enzymes were fused either to the GAL4 transcription-activating or DNA-binding domain and were tested for their ability to restore GAL4-dependent gene activation. All the different fusion proteins complemented the growth and enzymatic defects caused by the deletion of the respective genes, which indicates that these proteins are still functional. Interactions between the two phosphofructo-1-kinase subunits PFK1 and PFK2, interactions between the phosphofructo-2-kinase subunits, and dimerization of phosphoglucose isomerase were demonstrated. Dimerization of hexokinase 2, however, could not be demonstrated neither with N-terminal nor C-terminal fusions. A direct interaction between the hexose-6-phosphate interconverting enzymes hexokinase 2, phosphoglucose isomerase, and phosphofructo-1-kinase could also not be demonstrated. Nevertheless, our results indicate a weak interaction between phosphofructo-1-kinase and phosphofructo-2-kinase. PMID- 8612640 TI - Regulation of expression of the rat serine protease inhibitor 2.3 gene by glucocorticoids and interleukin-6. A complex and unusual interplay between positive and negative cis-acting elements. AB - The rat serine protease inhibitor 2.3 gene (spi 2.3) is almost completely silent in normal animals and is transiently expressed during acute inflammation. It encodes a potential anti-elastase which is likely to play a major physiological role for the host defense. Two well-known inflammatory mediators, glucocorticoids and interleukin-6 (IL-6) activate the spi 2.3 promoter and increase steady-state levels of mRNA in cultured hepatocytes. GC activation is mediated by a single glucocorticoid-response element which seems to act autonomously. A unique array of four functional IL-6-response sites was identified in the spi 2.3 promoter. Three of them (C-II--IV) bear structural identity to the CCAAT/enhancer-binding protein-binding site consensus sequence, whereas the fourth closely resembles the consensus kappa B nuclear factor recognition motif. The C-IV element, which is the most active, contains the motif 5'-CTGGGA and binds the IL-6-inducible acute phase response factor present in liver nuclear extracts from inflamed rats. Both basal and IL-6-dependent activities of each individual cytokine-response element tested separately are strongly down regulated by a recently identified regulatory sequence, located in the 3' untranslated region of the spi 2.3 gene. However, this repressor element does not significantly affect overall IL-6-dependent spi 2.3 promoter activity. This suggests that, in the context of the active gene in vivo, all four IL-6-response sites, which are largely redundant, cooperate to overcome the strong repressive effect of the 3' untranslated region silencer and are needed to bring about a maximal IL-6 response. These data reveal a novel type of regulation of an acute-phase gene involving different classes of IL-6-response elements controlled by a repressor and acting in conjunction with a glucocorticoid-response element. PMID- 8612641 TI - Interaction of the La (SS-B) autoantigen with small ribosomal subunits. AB - The La (SS-B) autoantigen is an evolutionarily conserved 47-kDa protein which binds to nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) messenger RNAs. Here, we show that La in the cytoplasm is associated with a subset of small ribosomal subunits, possibly by direct association with 18S ribosomal RNA. This association is likely to be related to the putative role of this protein in translation regulation. PMID- 8612642 TI - Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide. AB - The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ-proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse-phase chromatography, mass spectrometry and N-terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin-sensitive sites in a single step. The implications for production and recovery of various recombinant proteins are discussed. PMID- 8612643 TI - Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant. AB - Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced Mr: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The Mr is similar to those of soluble animal enzymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectability in mutants to about 50% of the wild-type levels in transformed cells. PMID- 8612644 TI - The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene. AB - Carbonic anhydrase 1 (CA1) is an abundant enzyme in colon epithelia. In the gastrointestinal tract, carbonic anhydrase is vital for NaCl resorption, alkalinization of gut contents, and absorption of short-chain fatty acids. The CA1 gene has two promoters, one of which is specifically active in colon epithelia and the other in erythroid cells. We are investigating the factors that regulate CA1 expression from the colon-specific promoter. Colon-specific deoxyribonuclease I hypersensitive sites (DHS) have been mapped close to the colon transcription initiation site (DHS6c) and in the upstream intron (DHS5c). Using electrophoretic mobility-shift assays to search the 650-bp region which contains DHS6c, we have identified sequences that bind a colon-specific factor (COF1) and by deletion analysis we have narrowed down the COF1-binding motif to a 17-bp sequence. A comparison of this motif with a protein-binding motif in the sucrase-isomaltase gene promoter, competition assays, and antibody studies indicate that COF1 is identical to the homeodomain protein Cdx-2. We propose that Cdx-2 plays an important role in the intestine-specific expression of CA1. PMID- 8612645 TI - Limited plasmin proteolysis of vitronectin. Characterization of the adhesion protein as morpho-regulatory and angiostatin-binding factor. AB - The adhesion protein vitronectin is associated with extracellular matrices and serves as cofactor for plasminogen-activator inhibitor-1. Limited proteolysis by plasmin converts vitronectin into defined fragments which are detectable at sites of inflammation and angiogenesis. The loss and gain of binding functions of vitronectin fragments for macromolecular ligands was characterized in the present study. The initially generated 61--63-kDa vitronectin-(1--348)-fragment serves as typical binding component for plasminogen and binding function was lost upon carboxypeptidase B treatment indicating the importance of a C-terminal lysine. Complementary binding sites reside in isolated plasminogen kringles 1--3 (designated angiostatin) as deduced from direct binding and ligand blotting experiments. A synthetic vitronectin-(331--348)-peptide from the C-terminus of the 61--63-kDa fragment could mimic plasminogen and angiostatin binding. Also, the immobilized peptide bound tissue plasminogen-activator and mediated plasmin formation, comparable to fibrinogen-derived peptides. The 61--63-kDa vitronectin fragment was indistinguishable in its adhesive properties to intact vitronectin and bound active but not latent plasminogen-activator inhibitor-1. Late plasminolysis of vitronectin resulted in the processing of the N-terminal region of the protein with the generation of 42 kDa/35-kDa fragments that had Gly89 as new N-terminus and that were ineffective in promoting cell adhesion. Thus, at sites of cell-matrix interactions which become proteolytically modified by plasmin during inflammatory and angiogenic processes, vitronectin serves as plasminogen/angiostatin-binding factor. Due to this differential change in functions particularly at sites of deposition in the vascular system or at wound sites vitronectin is considered to be an important morpho-regulatory factor. PMID- 8612646 TI - A high-potential soluble cytochrome c-551 from the purple phototrophic bacterium Chromatium vinosum is homologous to cytochrome c8 from denitrifying pseudomonads. AB - A minor cytochrome c-551 component of Chromatium vinosum was previously found to efficiently couple electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. We have now determined the amino acid sequence of this cytochrome c-551 and find that it is homologous to cytochrome c8 (formerly called Pseudomonas cytochrome c-551). It is most similar to Methylophilus methylotrophus, Rhodocyclus tenuis, and Azotobacter vinelandii cytochromes c8 (respectively, 57%, 52% and 51%). The C. vinosum cytochrome c8 has a single residue insertion relative to Pseudomonas and Azotobacter cytochromes c8. It has fewer charged residues than its homologs and is essentially neutral, which may explain why it is less soluble than the others. The cytochromes c8 are only very distantly related to the cytochromes c2 found in other species of purple bacteria which are much larger in size and which usually mediate electron transfer between the cytochrome bc1 complex and the reaction center. The photosynthetic pathway in Chromatium thus appears to be radically different from that in purple non-sulfur bacteria. PMID- 8612647 TI - 1H,13C-NMR and X-ray absorption studies of copper(I) glutathione complexes. AB - The tripeptide glutathione (gamma-L-Glu-L-Cys-Gly, GSH) is an important intracellular reducing agent for Cu(II) and complexation agent for Cu(I). We have studied the complexation of Cu(I) to GSH in aqueous solution at a range of pH values and Cu(I):GSH molar ratios by 1H-NMR and 13C-NMR spectroscopy and X-ray absorption spectroscopy. The NMR data are consistent with formation of a complex with approximate 1:1 stoichiometry [Cu(SG)] as the major species with only thiolate sulfur of GSH binding to Cu(I). The rate of exchange of GSH with GS-Cu was determined to 13 s(-1) at 283 K, pH 6.8. X-ray absorption spectroscopic measurements showed that Cu(I) is coordinated to 3.1+/-0.3 sulfur atoms at approximately 0.222 nm in solutions (and solids) containing GSH:Cu in 1:1 and 2:1 mol ratios. The possible structures of polymeric Cu(I)-glutathione complexes are discussed. The high thermodynamic stability of Cu(I)-S bonds in Cu(I)-glutathione complexes coupled with their kinetic lability may provide efficient and specific pathways for the transport of copper in cells. PMID- 8612649 TI - Probing the aromatic-donor-binding site of horseradish peroxidase using site directed mutagenesis and the suicide substrate phenylhydrazine. AB - The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C. PMID- 8612648 TI - Stereochemical course and reaction products of the action of beta-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. AB - Beta-Xylosidases are grouped in families 39 and 43 of a general classification of glycosyl hydrolases based on amino acid sequence similarities. The Beta xylosidase from Butyrivibrio fibrisolvens, which belongs to family 43, has been shown to operate by a molecular mechanism which results in the inversion of the anomeric configuration. Thermoanaerobacterium saccharolyticum B6A-RI Beta xylosidase which belongs to family 39 was purified as a recombinant enzyme from Escherichia coli. The stereochemistry of the hydrolysis of p-nitrophenyl Beta-D xylopyranoside was followed by 1H NMR. The spectrum recorded after 2 h hydrolysis showed a large signal centred at 4.47 ppm (J approximately 10 Hz) assignable to H1 of free Beta-xylose with a small amount of alpha-xylose (5.05 ppm, J approximately 3 Hz) attributable to mutarotation. This result indicates that T. saccharolyticum Beta-xylosidase operates with overall retention of the anomeric configuration. This result, with the lack of sequence similarity between the two families of Beta-xylosidases, suggests that these two families have major differences in their active-site geometries. Consistent with its retaining mechanism, Beta-xylosidase of T. saccharolyticum B6A-RI also displayed transglycosylating activity:reverse-phase HPLC showed approximately 30% conversion of p-nitrophenyl Beta-D-xylopyranoside into a number of higher nitrophenyl oligosaccharides after 5 min incubation with the enzyme. The structure of the most abundant oligosaccharides could be determined by total correlation spectroscopy NMR and showed that the enzyme can build Beta-1,4, Beta 1,3- and Beta-1,2-linked xylo-oligosaccharides. PMID- 8612650 TI - The importance of conserved residues in human liver UDPglucose pyrophosphorylase. AB - Comparison of the amino acid sequences of five eukaryotic UDPglucose pyrophosphorylases has identified a number of conserved residues that may be important for substrate binding or catalysis. Using the cloned cDNA for the human liver enzyme, we have investigated the role of several of these residues by site directed mutagenesis. Changing the single conserved cysteine (residue 123) to serine resulted in an active enzyme, as did mutating the single concerned histidine (residue 266) to arginine. The two conserved tryptophans were each altered to serine; W218S is active while W333S is not. In the latter case, the enzyme does not appear to fold correctly, and a similar result was obtained by mutation to lysine at one (residue 391) of the four conserved arginines. The other three arginines are not essential, as judged by the observation that R389H, R422Q and R445H are all active. The kinetic properties of each active mutant were investigated and in most cases were found to be similar to those of wild-type. The most dramatic change is a sevenfold increase in the Km for magnesium pyrophosphate with C123S. Overall, none of these conserved residues appears to be essential for activity, although such a role cannot be ruled out for W333 and R391 where mutation resulted in defective folding. PMID- 8612651 TI - Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis. AB - We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8 Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8 Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8 Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8 Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer. PMID- 8612652 TI - Comparative study of the metabolic pools of sphingomyelin and phosphatidylcholine sensitive to tumor necrosis factor. AB - The metabolism and localization of the pools of sphingomyelin and phosphatidylcholine (PtdCho) which are hydrolyzed upon activation of the sphingomyelin signal transduction pathway were studied in human skin fibroblasts treated with tumor necrosis factor alpha (TNF-alpha). In a first series of experiments, cellular phospholipids were labeled with [3H]choline under conditions that inhibit the vesicular traffic to the plasma membrane. Thus, in human fibroblasts metabolically labeled in the presence of brefeldin A, monensin or at 20 degree C, the arrival of newly synthesized sphingomyelin to the cell surface was prevented, supporting previous conclusions for a vesicular mechanism of sphingomyelin transport to the plasma membrane. Under these conditions, TNF alpha induced the hydrolysis of PtdCho but did not promote the hydrolysis of 3H labeled sphingomyelin, suggesting that the sphingomyelin signaling pool resides in a compartment distal to the Golgi apparatus, and possibly in the plasma membrane. TNF was also unable to trigger the breakdown of a radioactive sphingomyelin, [ceramide-3H]sphingomyelin, exogenously added to the cells to label the exoplasmic side of the cell surface. However, TNF caused PtdCho and sphingomyelin degradation in fibroblasts that had been treated with bacterial sphingomyelinase to degrade the sphingomyelin pool of the external leaflet of the plasma membrane. A similar result was obtained at 4 degree C, i.e. under conditions which inhibit endocytosis, thereby excluding the endosomes as a potential site for TNF-induced sphingomyelin hydrolysis. Altogether, these results strongly argue for a localization of the sphingomyelin signaling pool at the inner leaflet of the plasma membrane, but neither in the endolyso-somal nor the Golgi compartments. In addition, when [3H]choline-labeled fibroblasts were treated under non-lytic conditions with bacterial phospholipase C to degrade the external pool of PtdCho, TNF was still able to stimulate the hydrolysis of PtdCho. This demonstrates that the pool of PtdCho involved in TNF-alpha signaling (and which is hydrolyzed concurrently with sphingomyelin to generate diacylglycerol), is not located in the outer leaflet of the plasma membrane. PMID- 8612654 TI - Technetium-99m L,L-ethylenedicysteine clearance and correlation with iodine-125 orthoiodohippurate for the determination of effective renal plasma flow. AB - Technetium-99m L,L-ethylenedicysteine (99mTc-L,L-EC) is a new renal tubular tracer that allows the determination of the effective renal plasma flow (ERPF). The aim of this study was to derive simplified methods for the estimation of 99mTc-L,L-EC clearance using one or two plasma samples after bolus injection. Fifty-nine multiple plasma dual-tracer samples (nine samples from 5 to 120 min after injection) were obtained after injection of kit-formulated 99mTc-L,L-EC and iodine-125 orthoiodohippurate (OIH). The studies were performed in 25 stable and 24 unstable transplant recipients, in five patients with renal insufficiency (four on chronic haemodialysis) and in five normal volunteers. This allowed a wide range of renal function values to be covered, with ERPF (estimated by OIH clearance) ranging from 25.4 to 604.0 ml/min. The reference 99mTc-L,L-EC clearance, as calculated from the multisample model, could be estimated from two samples at 15 and 90 min with an error of 11.3 ml/min and from one sample at 90 min with an error of 17.8 ml/min. Using appropriate linear regression analysis, ERPF could be estimated by the two- and one-sample 99mTc-L,L-EC clearance with an error of 24.2 and 22.8 ml/min, respectively. In conclusion, 99mTc-L,L-EC clearance can be accurately estimated by simplified one- or two-sample methods. Moreover, these methods can be used to estimate ERPF with an error that remains acceptable for clinical purposes. PMID- 8612653 TI - Assessing anthracycline cardiotoxicity in the 1990s. PMID- 8612656 TI - Relationship between thallium-201 uptake and tumour proliferative ability in thyroid nodules. AB - To evaluate whether thallium-201 scan can reflect tumour proliferative activity in thyroid nodules. We compared the degree of 201Tl uptake with the tumour proliferative ability as assessed immunohistochmically by the labelling index of proliferating cell nuclear antigen (PCNA) in malignant and benign thyroid nodules. The case material comprised ten benign and 31 malignant surgically resected nodules from a total of 41 patients. 201Tl scan was performed 5 min (early scan) and 2h (delayed scan) after intravenous injection of 74 MBq of 201Tl. The degree of 201Tl uptake was visually divided into three grades [from ( ) to (++)], as compared with its uptake in normal adjacent thyroid tissue. Immunohistochemical staining of PCNA was performed using a monoclonal antibody for PC10 on paraffin-embedded specimens. On both the early and the delayed scans, the mean PCNA index in the nodules with an intense 201Tl, i.e. (++), was significantly higher than that in nodules with a lower or with negative 201Tl uptake. The correlation was higher on the delayed 201Tl scan (P=0.009) than on the early scan (P=0.019). Our results indicate that 201Tl uptake may reflect the tumour proliferative activity of thyroid nodules, and this is especially true with regard to the uptake on delayed scans. PMID- 8612655 TI - Evaluation of gamma camera-based measurement of individual kidney function using iodine-123 orthoiodohippurate. AB - Gamma camera-based clearance techniques which use the renal uptake ratio (RUR) of the radiotracer are available to estimate the effective renal plasma flow (ERPF) and glomerular filtration rate. To evaluate the accuracy of these techniques, we measured RUR by an optimized procedure and compared it with standard ERPF. Iodine 123 orthoiodohippurate (OIH) scintigraphy and simultaneous para-aminohippurate clearance study for measuring standard ERPF were performed in three hospitals in 24 patients with normal or mildly impaired renal function. 123I-OIH was injected intravenously and 10-s consecutive imaging of the kidneys was started when the abdominal aorta was seen. The attenuation coefficient for 123I was measured in each hospital using the same water-equivalent absorption materials and used for the attenuation correction. After subtracting background radioactivity, RURs were defined as the count ratios of fractional renal uptakes based on the integral from 1 to 2, 2 to 3, 1.5 to 2.5 and 1 to 3 min after the injection of 123I-OIH in relation to injected doses using the following three procedures in respect of attenuation correction: (1) RUR without attenuation correction, (2) RUR with fractional renal uptake corrected by the measured attenuation coefficient, (3) RUR with the total injected dose corrected by the absorption material. To decide upon the appropriate correction method and time interval, RURs were compared with standard ERPF. Among the three correction methods, procedure 2 showed the highest correlation between RUR and standard ERPF, but the correlation coefficient was low (r=0.75). No significant difference was observed among the RURs of each time interval. Individual kidney function measured from early renal uptake may be inaccurate even when appropriate correction is made for attenuation, background activity or time lag between injection and data acquisition. Gamma camera-based measurement of renal function using 123I-OIH is limited with regard to accuracy and reproducibility, though it is convenient and non-invasive. PMID- 8612657 TI - Kinetics of radioiodinated species in subcellular fractions from rat hearts following administration of iodine-123-labelled 15-(p-iodophenyl)-3-(R,S) methylpentadecanoic acid (123I-BMIPP). AB - It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S) methylpentadecanoic acid (123I-BMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1-120 min after intravenous injection of 123I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 123I-BMIPP was maximal at 5 min (6.81%+/-0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of 123I-BMIPP, 123I-p-iodophenylacetic acid (123I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite, 123I-p-iodophenyldodecanoic acid (123I-PIPC12), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of 123I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial alpha-oxidation followed by subsequent cycles of beta oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven. PMID- 8612658 TI - Improved left ventricular function after growth hormone replacement in patients with hypopituitarism: assessment with radionuclide angiography. AB - Prolonged growth hormone deficiency (GHD) leads to marked cardiac dysfunction; however, whether reversal of this abnormality may be achieved after specific replacement therapy has not yet been completely clarified. Fourteen patients with childhood-onset GHD (nine men and five women, mean age 27+/-4 years) and 12 normal control subjects underwent equilibrium radionuclide angiography under control conditions at rest. Patients with GHD were also studied 6 months after recombinant human (rh) GH treatment (0.05 IU/kg per day). Normal control subjects and patients with GHD did not differ with respect to age, gender and heart rate. In contrast, left ventricular ejection fraction (53%+/-9% vs 66%+/-6%, P <0.001), stroke volume index (41+/-11 vs 51+/-8 ml/m2, P <0.01) and cardiac index (2.8+/ 0.6 vs 3.+/-0.5 l/min/m2, P <0.001) were significantly lower in GHD patients than in normal control subjects. None of the GHD patients showed adverse or side effects during rhGH therapy; thus none required a reduction in GH dose during the treatment period. Heart rate and arterial blood pressure were not significantly modified by rhGH treatment. After 6 months of rhGH therapy a significant improvement in left ventricular ejection fraction (from 53%+/-9% to 59%+/-9%, P <0.01), stroke volume index (from 41+/-11 to 47+/-13 ml/m2, P <0.05) and cardiac index (from 2.8+/-0.6 to 3.3+/-0.8 l/min/m2, P <0.01) was observed in GHD patients. In conclusion, prolonged lack of GH leads to impaired left ventricular function at rest. Reversal of this abnormality may be observed after 6 months of specific replacement therapy in patients with childhood-onset GHD. PMID- 8612660 TI - Evaluation of cardiac sympathetic neuronal integrity in diabetic patients using iodine-123 metaiodobenzylguanidine. AB - Autonomic dysfunction is associated with increased mortality in diabetic patients. To evaluate the cardiac autonomic dysfunction in these patients, a prospective study was undertaken using iodine-123 metaiodobenzylguanidine (MIBG) single-photon emission tomography (SPET). The study groups consisted of ten diabetic patients with cardiac autonomic neuropathy (group I) and six without autonomic neuropathy (group II). Autonomic nervous function tests, thallium scan, radionuclide ventriculographic data including ejection fraction and wall motion study, and 24-h urine catecholamine levels were evaluated. 123I-MIBG SPET was performed at 30 min and 4h following injection of 3 mCi of 123I-MIBG in groups I and II and in normal subjects (n=4). On planar images, the heart to mediastinum (H/M) ratio was measured. Defect pattern and severity of MIBG uptake were qualitatively analysed on SPET. Compared with control subjects, diabetic patients had a reduced H/M ratio regardless of the presence of clinical autonomic neuropathy. There was no difference in H/M ratio between groups I and II. On SPET images, focal or diffuse defects were demonstrated in all patients in group I, and in five of the six patients in group II. The extent of defects tended to be more pronounced in group I than in group II. In conclusion, 123I-MIBG scan was found to be a more sensitive method than clinical autonomic nervous function tests for the detection of autonomic neuropathy in diabetes. PMID- 8612659 TI - The influence of vascular diathesis on the localization of inflammatory foci in renal allografts with a specific antigranulocyte antibody. AB - Immunoscintigraphy with technetium-99m labelled BW 250/183, a murine monoclonal antibody specific for granulocytes, yielded a false-positive result in a patient suspected of having an abscess in his renal graft. To substantiate the presumption that diathesis and unspecific accumulation of the antibody may have caused this result, ten selected patients were investigated who presented with chronic vascular graft rejection but without signs of bacterial infection. Scintiscans were recorded 4 and 24h after administration of 99mTc-labelled BW 250/183. Graft-background ratios (GBRs) were calculated for each transplant. These were compared with the mean of physiological kidney-background ratios (KBRs) and with bone marrow-background ratios (BMBRs). After removal, the grafts were examined with pathological and immunohistological methods. Seven transplants demonstrated 4-h GBRs (mean: 3.9+/-1.1, P <0.001) significantly outside the range of normal KBRs while three were within the normal range (mean: 1.8+/-0.4). The relation between 4-h and 24-h GBRs varied. After 24h five GBRs still remained increased (mean: 3.2+/-1.4, P <0.05). By contrast the BMBRs decreased uniformly by 18%+/-5%. After graft removal, histopathology demonstrated no dominant granulocyte accumulations but various degrees of chronic vascular and tubulo interstitial rejection. Immunohistochemical studies did not indicate cross reactivity of BW 250/183. Increased GBRs of long-standing renal allografts indicate the passage of the antibody through injured vascular walls rather than the presence of granulocyte accumulations. Therefore, variability of GBRs with time reflects changes in transitory concentrations of 99mTc-labelled BW 250/183 in the tissues. PMID- 8612661 TI - The clinical impact of thallium-201 reinjection for the detection of myocardial hibernation. AB - Thallium-201 reinjection improves detection of hibernating myocardium in about 30%-50% of persisting defects. The main goal of cardiac revascularization techniques is amelioration of clinical symptoms such as angina and dyspnoea; however, improvement in regional and global pump function is an additional and important target. The aim of this study was to investigate whether fill-in in the reinjection study is correlated with improved contractile function after treatment (percutaneous transluminal coronary angioplasty/aortocoronary bypass surgery). We studied 32 patients with coronary heart disease and impaired regional wall motion (RWM). RWM and ejection fraction (EF) were assessed by analysing ventriculographic images using the centreline method (values in standard deviations from mean values found in a healthy control group). Three 201Tl single-photon emission tomographic studies (stress, redistribution and reinjection) were performed prior to revascularization and analysed using a bull s-eye scheme. Patients were divided into two groups (group FI-=no fill-in, n=16; group FI+=fill-in, n=16). Fifty-six percent of all patients showed persisting defects, and 56% of these defects showed fill-in after reinjection. Fill-in in our patient group was independent of the size of the persisting defects. After revascularization RWM increased significantly in group FI+ (from -1.9 to 0.0 SD, P<0.001) whereas group FI- showed no significant change (from -1.6 to -1.8 SD). EF increased from -4.3 preoperatively to -2.1 SD postoperatively in group FI+ and did not change significantly in group FI- (-2.5 to -3.2 SD). The predictive value of reinjection for improvement of RWM was 88%. It is concluded that fill-in in the 201Tl reinjection image can predict recovery of RWM and EF after revascularization and should be used in all patients with impaired RWM and persisting defects independent of their extent. PMID- 8612662 TI - Technetium-99m labelled hydrazinonicotinamido human non-specific polyclonal immunoglobulin G for detection of infectious foci: a comparison with two other technetium-labelled immunoglobulin preparations. AB - Recently a new linker - hydrazinonicotinate (HYNIC) - was introduced for labelling of proteins and peptides with technetium-99m. HYNIC and other linkers have been used for labelling of human non-specific polyclonal immunoglobulin G (hIgG) with 99mTc for the detection of infections. In this study we compared the tissue distribution of three different 99mTc-hIgG preparations in groups of five Wistar rats with a focal intramuscular infection with Staphylococcus aureus. We compared 99mTc-HYNIC-hIgG with 99mTc-hIgG labelled via the so-called Schwarz method (reduction of disulphide bonds) and with the 99mTc-labelled commercially available Technescan-HIG. Unlike the HYNIC linker, in the two other labelling methods free sulph-hydryl groups are involved in the binding of 99mTc. High performance liquid chromatography analysis of the labelled preparations and of plasma samples revealed aggregate or polymer formation in all three agents; this was least pronounced in the product labelled by means of the Schwarz method. The tested preparations did not show signs of degradation in vitro. The difference in linker chemistry was reflected in the tissue distribution. Thus the biodistribution of 99mTc-HYNIC-hIgG was significantly different from the distribution of the two other preparations: abscess (1.4%+/-0.2%ID/g), muscle, liver, spleen, plasma, lung, bone marrow, and small intestine concentrations were higher at 24 h p.i.; kidney uptake (1.19%+/-0.08%ID/g) was significantly lower. The abscess-to-plasma and the abscess-to-muscle ratios (0.5 and 11, respectively), however, were in the same range for the three preparations tested. Quantitative analysis of the scintigraphs revealed that the total body clearance of 99mTc-HYNIC-hIgG was significantly slower than for the other agents. The abscess uptake of 99mTc-HYNIC-hIgG as a percentage of the remaining body activity was significantly higher. Based on its high abscess uptake, its low uptake in the kidneys and the high percentage of its abscess uptake in relation to the remaining body activity, we conclude that 99mTc-HYNIC-hIgG seems superior to the two other preparations tested for the detection of infections. PMID- 8612663 TI - Effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling. AB - The purpose of this study was to assess the effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling. To this end intravascular ligand kinetics between the free and a bound compartment in plasma and the exchange of tracer between the capillary space and tissue were analysed using a simple compartment model. The effect of non-equilibrated intravascular compartments on parameter estimates was evaluated in a computer simulation. It was found that three kinetic situations must be distinguished. If the intravascular compartments are fully equilibrated when the ligand reaches the target organ, intravascular binding simply acts as a scale factor for the transport-related parameter K1. If the intravascular kinetics is very slow, only minimal binding will occur. In between there is a range where ongoing equilibration leads to time variability of K1. Since tracer kinetic modelling usually does not account for such time variability, the parameter estimates become biased, the degree of the bias depending on the intravascular binding kinetics. Furthermore the bias may be dependent on receptor density, meaning that model-derived receptor estimates are not linearly related to the true receptor density. It is concluded that intravascular ligand binding can severely affect parameter estimates derived from tracer kinetic modelling. Especially disturbing are effects due to ongoing intravascular equilibration following the arrival of the ligand in the target organ. These can be avoided by letting the ligand equilibrate with blood in a syringe prior to injection. PMID- 8612665 TI - Influence of high-energy photons from cobalt-57 flood sources on scintillation camera uniformity images. AB - Cobalt-57 flood sources are often used for system flood-field uniformity checks of scintillation camera performance. Such sources are now available in large sizes and with high activities. Uniformity images using new 57Co sources have shown artefacts which are not present in uniformity images obtained from technetium-99m flood sources of the same activity. The high-energy photons emitted by 57Co and cobalt isotope impurities appear to influence the images obtained. This was investigated in three cameras using three 57Co sources of different age. The flood-field images obtained with the 57Co sources showed non uniformity patterns that were dependent on the age of the cobalt source, the distance of the source to the collimator, and the specific camera type. Quantification of the uniformity images reflected these findings. Energy spectra of a new 57Co source, obtained with an external 1024-channel analyser connected to the camera, showed a broad tail of high-energy photons above the 122-keV photopeak, due to Compton scatter and collimator penetration. This tail diminished with older sources and with increased source to collimator distance, indicating that in both situations fewer high-energy photons were being measured by the camera system. This tail of high-energy photons contributes to the total count rate, but because this is not obvious from the counts observed in the photopeak window, the camera can unsuspectingly be operated at too high a total countrate where pile-up effects become significant. Caution is therefore advised when using 57Co sources. Problems may be minimized by purchasing low-activity sources (certainly no more than 370 MBq), by placing the source at a distance from the collimator, e.g. 50 cm, by allowing a new source to decay, and by manufacturers producing 57Co sources without detectable radioactive contaminants. PMID- 8612664 TI - Differential kinetics of [123I]beta-CIT binding to dopamine and serotonin transporters. AB - Iodine-123-labelled 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid ([123I]beta CIT) labels both the dopamine transporter (DAT) and the serotonin transporter (5 HTT) and this ligand is able to clarify pathological changes in both dopaminergic and serotonergic systems. However, the differential kinetics of beta-CIT binding to DAT and 5-HTT has not been clarified fully. In this study we examined time activity curves of [123I]beta-CIT in individual regions in the rat brain. Using cerebellum as the reference region, k3 and k4 values were estimated by a two compartment kinetic analysis. In the striatum, the kinetics was slowest among all brain areas. In this area specific binding reached its peak 4 h after the injection. In the hypothalamus, specific binding reached its peak 1 h after the injection and its amount did not change until 4 h after the injection. In the occipital cortex, the binding and washout of the ligand were fastest among all brain regions. Estimated k3 values were 0.040+/-0.003 in the striatum, 0.019+/ 0.002 in the hypothalamus and 0.082+/-0.011 in the occipital cortex (min-1, mean +/-SD). Estimated k4 values were 0.0034+/-0.0005 in the striatum, 0.0071+/-0.0009 in the hypothalamus and 0.083+/-0.013 in the occipital cortex (min-1, mean +/ SD). Therefore binding kinetics of [123i]beta-cit in the region rich in dat is apparently different from that in the region rich in 5-HTT. These results will provide fundamental data to image both DAT and 5-HTT in one series of examinations with [123I]beta-CIT. PMID- 8612666 TI - Effects of low flow and hypoxia on myocardial retention of technetium-99m BMS181321. AB - The purpose of the present study was to determine whether graded levels of low flow ischemia would lead to graded differences in uptake and clearance of BMS181321. Using a perfused rat heart model, 7.4 MBq (200 microCi) of BMS181321 was infused over 20 min, followed by a 60-min clearance phase. Activity was monitored using an NaI detector. Four groups were studied using Krebs-Henseleit buffer perfusion using low flow or hypoxia: group 1=12 ml/min, group 2=3 ml/min, and group 3=1 ml/min during uptake and clearance phases, and group 4=12 ml/min with hypoxia during clearance. Control and low-flow groups were also perfused using red blood cells and albumin. There was a stepwise increase in peak myocardial uptake (% injected dose) as flow progressively decreased (group 1=2.4%+/-0.2% SEM, group 2=13.1%+/-0.7%, group 3=28.6%+/-2.4%, P <0.05). Group 3/group 1 mean peak activity ratio was 12:1. Mean 1-h fractional retention significantly increased in a stepwise manner as flow decreased (group 1=0.32+/ 0.02, group 2=0.43+/-0.03, group 3=0.59+/-0.05, P <0.05). Group 3/group 1 mean 1 h clearance activity ratio was 30:1. Groups 5 and 6 perfused with red blood cells and albumin demonstrated similar increases in peak uptake and 1-h retention in the low-flow hearts. This study demonstrates a stepwise increase in uptake and a stepwise increase in retention rate of BMS181321 with progressive reduction in flow. PMID- 8612667 TI - Biodistribution of iodine-125 tyramine transforming growth factor alpha antisense oligonucleotide in athymic mice with a human mammary tumour xenograft following intratumoral injection. AB - The Watson-Crick base pairing rule provides the underlying principle for the antisense (AS) approach to inhibiting gene expression. Transforming growth factor alpha (TGFalpha) was the first growth factor to be associated with tumorigenesis, thus making the TGFalpha (mRNA) a potential target for AS therapy and offering the potential for monitoring of the progression of malignancy by non-invasive imaging with radiolabelled AS phosphodiester. Probe labelling and biodistribution were studied in the present report. A 23-mer oligonucleotide sequence was synthesized and grafted in 5' with a tyramine group which was further radioiodinated. The radiolabelled AS was injected intratumorally in mammary tumour-bearing BALB/c mice (3 weeks after inoculation of 7.10(6)NS2T2A mammary cells). Biodistribution was monitored by sequential scintigraphy and organ radioactivity after autopsy. The 5' tyramine group allowed specific and stable radiolabelling of the AS with 125I. The 125I AS oligonucleotide was rapidly cleared from the tumour by intestine and kidneys. Four hours after intratumoral injection, 6.5%+/-1.5% of the dose was retained in the tumour as non-degraded 125I AS. It is concluded that 5' tyraminylated AS provides information on the biodistribution of AS oligonucleotide following intratumoral injection. These data will contribute to the pharmacology of AS oligonucleotides which can be used for therapy. PMID- 8612668 TI - Iodine-123 metaiodobenzylguanidine in the assessment of late cardiac effects from cancer therapy. AB - Recognition of adverse late cardiac effects from cancer therapy may enable identification of patients with risk of cardiotoxicity upon cancer retreatment. In this study the feasibility of using iodine-123 metaiodobenzylguanidine (123I MIBG) heart scintigraphy to detect abnormalities of the myocardial adrenergic neurone function in the late period after cancer therapy was evaluated in relation to the left ventricle ejection fraction (LVEF) in 18 cancer patients: 11 had undergone thoracic irradiation involving the heart, in five cases in combination with anthracycline therapy, 11-228 months (median 60 months) before radionuclide tests, while seven had not received previous anthracycline and/or radiotherapy (controls). The 123I-MIBG cardiac uptake, expressed as a heart-to mediastinum ratio on planar images after 4h, ranged from 1.21 to 1.76 (median 1.56) in cancer therapy patients, which was significantly decreased (P=0.0006) in comparison with controls (range 1.81- 2.06, median 1.9). The myocardial 123I-MIBG washout, calculated from planar images after 15 min and 4 h, and LVEF also showed significant differences, but with some overlap in individual cases. In cancer therapy patients, cardiac abnormalities seen on planar images and additional single-photon emission tomographic images varied from focal defects to diffusely reduced myocardial uptake. It is concluded that 123I-MIBG heart scintigraphy, which is able to identify cardiac adrenergic neurone abnormalities in the follow up period after cancer therapy, may help to identify relapsed patients who are at increased risk of developing cardiotoxicity during retreatment with cardiotoxic therapy modalities. PMID- 8612670 TI - Scintigraphic findings on 99mTc-MDP, 99mTc-sestamibi and 99mTc-HMPAO images in Gaucher's disease. AB - Gaucher s disease is an autosomal recessive lysosomal storage disease characterized by the specific deficiency of glucocerebrosidase that leads to accumulation of insoluble glucocerebroside in the reticuloendothelial system, particularly the bone marrow, liver, spleen and lymph nodes. Direct scintigraphic visualization of lipid deposits in Gaucher s disease has recently been described, based on the use of the lipid-soluble xenon-133. We report here on the use of the lipophilic cationic complex technetium-99m sestamibi (99mTc-MIBI), employed as an indicator of increased cellular density and metabolic activity, to evaluate Gaucher cell infiltrates in the bone marrow; 99mTc-hexametazime (99mTc-HMPAO) was also employed, as a pure indicator of lipidic infiltration in the bone marrow. A 67-year-old patient with known type 1 Gaucher s disease presented with a painful left hip and knee and difficulty in gait subsequent to traumatic fracture of the left femoral neck that had required implant of a fixation screw-plaque. Bone scan with 99mTc-methylene diphosphonate revealed reduced uptake at the distal metaphyseal-epiphyseal femoral region. In addition, whole-body maps and spot-view acquisitions of the thighs and legs were recorded at both 30 min and 2.5 h after the injection of 99mTc-MIBI: the scintigraphic pattern clearly showed increased uptake at several sites involved by Gaucher deposits in the bone marrow (both knees, with variable intensity in different areas), matching the bone changes detected by conventional x-ray. The target to non-target ratios slowly decreased with time, from an average value of 2.25 in the early scan to an average value of 2 in the delayed scan. The lipid-soluble agent 99mTc-HMPAO exhibited a superimposable scintigraphic pattern of accumulation at the involved sites, though with lower target to non-target ratios (1.27-1.48). The results obtained in this patient suggest a potential role of 99mTc-MIBI in the scintigraphic evaluation of Gaucher s lipid deposits in the bone marrow. If the results are confirmed in other patients, this radiopharmaceutical would offer clear advantages over 133Xe because of its wider availability and greater practicality (i.v. administration of 99mTc-MIBI versus inhalation of 133Xe, and use of a single gamma camera instead of two as with 133Xe). PMID- 8612669 TI - The uptake mechanisms of inflammation- and infection-localizing agents. AB - Over the past 30 years, a wide variety of radiopharmaceuticals have been proposed for the scintigraphic detection of inflammatory and infectious disease. All radiopharmaceuticals yield a functional image of a process placed somewhere in the cascade of reactions in inflammation, this being the common pathway of response to tissue injury. This paper discusses relevant aspects of the biodistribution, in vivo kinetics and mechanisms of uptake of both clinically used and experimental radiopharmaceuticals that have been proposed for the scintigraphic detection of focal inflammation and infection. PMID- 8612671 TI - Does leukocyte scintigraphy answer the need for a more widely available radiopharmaceutical that can detect and assess the proliferative state of lymphoma? PMID- 8612672 TI - Histone H1 and core histones in Leishmania and Crithidia: comparison with Trypanosoma. AB - The Trypanosomatidae family is characterized by flagellated protozoa presenting a kinetoplast. Several genera of this family contain species that are pathogenic to man and domestic animals. Their chromatin is not condensed into chromosomes during cell division. As a contribution to the understanding of basic aspects of their genome organization, we present a systematic characterization of the histones from three genera of the Trypanosomatidae family. Crithidia fasciculata and Leishmania mexicana show core nucleosomal histones with electrophoretic mobilities both similar to and different from those of Trypanosoma cruzi and higher eukaryotes. Another protein is extracted from the chromatin of these organisms by procedures designed to purify histone H1. This protein presents elution profiles by HPLC and amino acid composition of histone H1. Considering these data and the high mobility of this protein in Triton-acetic acid-urea polyacrylamide gel electrophoresis, as well as its position relative to the nucleosomal core histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we postulate that Crithidia and Leishmania possess a histone H1 shorter than that of higher eukaryotes as we have previously shown to be the case for T. cruzi. The possible presence of a shorter histone H1 in these trypanosomatids may explain the absence of chromatin condensation during cell division in these flagellates. PMID- 8612673 TI - Effects of microgravity on osteoblast growth activation. AB - Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts. PMID- 8612674 TI - TIMP-2 growth-stimulatory activity: a concentration- and cell type-specific response in the presence of insulin. AB - In addition to proteinase-inhibitory activities, growth-stimulatory activities have been described for all three known members of the tissue inhibitors of the metalloproteinase (TIMP) family, TIMP-1, TIMP-2, and ChIMP-3, believed to be the chicken homologue of TIMP-3. However, the mechanism by which the TIMPs stimulate cell growth is unclear. In this report we have demonstrated that rTIMP-2 was growth-stimulatory for human foreskin fibroblasts (HSF4, HSF43, HS68), lung adenocarcinoma cells (A549), human melanoma cells (WM115), and the Burkitt's lymphoma cell line RAMOS, and this stimulatory response was concentration dependent, with the greatest stimulation occurring a 10-30 pM rTIMP-2 in [3H]thymidine incorporation assays and at 20-100 pM in cell growth assays. Normal human colon (18Co) and lung (37Lu) fibroblasts showed no response to rTIMP-2. [3H]Thymidine incorporation was inhibited by rTIMP-2 treatment in the nonadherent cell line HL60. These studies also demonstrated that for the cell types tested, TIMP-2 alone was insufficient for a growth stimulatory response requiring, at a minimum, the presence of insulin. In the absence of any "co-factor(s)," such as insulin, TIMP-2 treatment was inhibitory. PMID- 8612675 TI - Integrin-dependent control of inositol lipid synthesis in vascular endothelial cells and smooth muscle cells. AB - Extracellular matrix (ECM) molecules, such as fibronectin (FN), regulate fibroblast sensitivity to soluble growth factors, in part, by controlling cellular levels of phosphatidylinositol bis-phosphate (PIP2), the substrate for phospholipase C-gamma (McNamee et al., 1993, J. Cell Biol. 121, 673-678). In the present study, we extended these investigations by exploring whether cells of the vascular wall also exhibit this response and analyzing the mechanism by which adhesion to ECM regulates intracellular PIP2 mass. Capillary endothelial cells, pulmonary vascular smooth muscle cells, and C3H 101/2 fibroblasts were all found to exhibit a similar two- to threefold increase in PIP2 mass within 3 h after binding to dishes coated with FN. Furthermore, similar effects were observed using dishes coated with a variety of different ECM molecules, including collagen types I and IV as well as a synthetic RGD-containing peptide. An increase in PIP2 mass also was produced when suspended cells bound to microbeads (4.5 micron diameter; coated with RGD-peptide or anti-integrin beta 1 antibody) that induce local integrin clustering and focal adhesion formation, independently of cell spreading. In contrast, neither binding of soluble FN nor binding of microbeads coated with ligands for other transmembrane surface receptors (e.g., acetylated low-density lipoprotein, antibodies against heparan sulfate) had any effect on PIP2 mass. While these results suggest that integrin clustering stimulates PIP2 synthesis, no change in total cellular or cytoskeletal-associated phosphatidylinositol-4-phosphate kinase (PIP kinase) activity could be detected when cells bound to immobilized integrin ligands. However, when focal adhesion complexes were isolated from these cells using a magnetic procedure (G. Plopper and D. E. Ingber, 1993, Biochem. Biophys. Res. Commun. 193, 571-578), this subfraction of the cytoskeleton was found to be enriched for PIP kinase activity by more than twofold relative to the whole cytoskeleton. These data suggest that ECM binding may increase PIP2 mass in vascular cells by clustering cell surface integrin receptors and activating cytoskeletal-associated PIP kinases locally within the focal adhesion complex. PMID- 8612676 TI - Heat shock induces differentiation of human embryonal carcinoma cells into trophectoderm lineages. AB - NCR-G3 cells were established from a testicular embryonal carcinoma and are highly multipotential, differentiating into trophectoderm cells upon exposure to retinoic acid. Differentiated NCR-G3 cells begin to produce human chorionic gonadotropin (hCG), a trophectoderm-specific hormone. We have previously isolated the up-regulated genes at the early stage of differentiation. One of them was found to be a heat shock protein gene. The heat shock protein gene (HSP90) is induced at the early stage of differentiation and decreases to the basal level or under the basal level at the later stage. We speculate that heat shock per se induces the differentiation of human EC cells. With exposure to heat, NCR-G3 cells began to express a series of differentiation markers such as cytokeratin and hCG. Heat, which is classically known to induce heat shock proteins, is able to differentiate an embryonal cell line into trophectoderm lineages, implying a new recognized function of a heat-like event in early differentiation. PMID- 8612677 TI - The 3' untranslated region of prohibitin and cellular immortalization. AB - We have been studying the role of the evolutionarily conserved prohibitin gene in cellular immortalization and tumor suppression. Immortalized human cells are classified into four complementation groups (A, B, C, and D) based on the ability of fusion hybrids to become senescent. The present study expands our preliminary evidence showing that the antiproliferative activity of prohibitin is only effective in immortalized Group B cells and normal cells. Data presented here show that the expression of a prohibitin mRNA with a long 3' untranslated region (3'UTR) and prohibitin protein is elevated in immortalized cells from all complementation groups. However, all immortalized cells classified in complementation Group B, and no cell lines in any of the other groups, are sensitive to the antiproliferative activity of wild-type prohibitin transcripts. All Group B cells are also homozygous for one of two human prohibitin alleles that are distinguishable by two distinct intron polymorphism restriction sites. Interestingly, sequence analysis of the prohibitin gene from representatives of each of the complementation groups showed that the 3'UTR from Groups A, C, and D matched wild type; however, the sequence from all four Group B cell lines differed from wild type. Functional inhibition assays on truncated wild-type mRNA transcripts as well as 3'UTR specific wild-type and mutated transcripts show that the antiproliferative activity of prohibitin resides, at least in part, in the 3'UTR. These data suggest that the prohibitin 3'UTR may function as a trans acting regulatory RNA (riboregulator) whose tumor suppressor activity has been inactivated by mutation in Group B cells. PMID- 8612678 TI - Processing of 125I-insulin by polarized cultured kidney cells. AB - Renal clearance of insulin is achieved by glomerular filtration and by passage from the postglomerular peritubular circulation into the renal interstitium. In the proximal tubule, filtered insulin binds to the apical membrane and is internalized and degraded while insulin in the interstitium is taken up by receptor-mediated endocytosis and degraded. To study these processes we have utilized cultured opossum kidney cells. These cells have proximal-like features and process insulin in a manner consistent with that described in vivo. To study apical and basolateral uptake and metabolism of insulin independently, cells were grown on filters suspended in culture wells. insulin was degraded to large insulin-size intermediates and low-molecular-weight products. This occurred whether the protein was internalized from the apical or basolateral pole of the cells. Analysis of the intermediate products by reverse-phase high-performance liquid chromatography revealed that products formed after apical or basolateral internalization were similar. Since products were preferentially released from the side of uptake, it is likely that apically and basolaterally internalized insulin is degraded in comparable organelles located in different regions of the cell. Most of the internalized insulin traversed the degradative pathway but some insulin followed a retroendocytic or minor transcytotic pathway. Degradation was inhibited by chloroquine, which also selectively increased the release of internalized insulin from the apical pole irrespective of the side of uptake. Thus while the polar degradative processes appear to be similar in nature, the polar exocytotic processes appear to be different. PMID- 8612679 TI - FGF6 modulates the expression of fibroblast growth factor receptors and myogenic genes in muscle cells. AB - Fgf6 is the only known member of the FGF family whose expression is restricted to the muscle cell lineage during development, suggesting it may have a role in myogenesis. Muscle satellite cells but not C2 myoblast cells were found to express Fgf6. We have used purified recombinant FGF6 protein to explore the effect of this factor on C2 cells in culture. FGF6 stimulated the proliferation of C2 myoblasts and, in combination with heparin, induced their morphological transformation. FGF6, added at 5 ng/ml and in the presence of heparin, increased the expression of a subset of muscle cell differentiation markers. In contrast, at 25 ng/ml, it down-regulated the expression of myogenic markers and myogenic transcription factors examined and delayed differentiation into myotubes of C2 cells. It also up-regulated the expression of FgfR1 and had an opposite effect on FgfR4. These results suggest that intramuscular FGF6 concentrations could influence the proliferation and differentiation processes taking place during development. PMID- 8612680 TI - Human Kupffer cell recognition and phagocytosis of apoptotic peripheral blood lymphocytes. AB - Cells undergoing apoptosis are recognized and rapidly phagocytosed by macrophages before their degradation, thus preventing the inflammatory reaction and protecting tissues from the damaging effects of released potentially harmful intracellular contents. In spite of growing interest in the mechanisms leading to the engulfment of apoptotic cells, the molecular bases by which an apoptotic cell is recognized are not entirely understood. Among the several potential mechanisms by which a macrophage can identify a cell as apoptotic, the data reported in the present paper support the idea that Kupffer cells phagocytose apoptotic cells by means of lectin-like receptors. Human Kupffer cells, which possess galactose specific binding sites, can recognize and phagocytose peripheral blood lymphocytes undergoing apoptosis after heat shock (43 degrees C) or cycloheximide treatment, but not normal living peripheral blood lymphocytes. The putative structure by which apoptotic peripheral blood lymphocytes are targeted as "edible" could be the molecular changes in the plasma membrane, In fact, our experiments indicate that the membranes of apoptotic peripheral blood lymphocytes express increased amounts of N-acetylgalactosamine, D-galactose, and mannose residues when compared with membranes of normal PBL. Phagocytosis was inhibited by adding to the culture medium sugar cocktail solution (glucose, N acetylgalactosamine, methyl mannopyranoside, fucose, 80 mM final concentration) or to a lower extent by desialylated glycoproteins (lactosylated bovine serum albumin, asialofetuin, 2 mg/ml final concentration), but not by nondesialylated glycoproteins (fetuin, 2 mg/ml final concentration, bovine serum albumin, 20% final concentration). In addition, phagocytosis of apoptotic peripheral blood lymphocytes by human Kupffer cells was a very rapid process, being almost entirely completed within 15 min of incubation. PMID- 8612681 TI - Morphological differentiation of N1E-115 neuroblastoma cells by dimethyl sulfoxide activation of lipid second messengers. AB - Quantitative changes in the lipid second messenger diacylglycerol (DAG) were studied in the rat neuroblastoma N1E-115 following exposure to the differentiating agent dimethylsulfoxide (DMSO). Relatively high basal levels of DAG are present in these cells, and addition of 2% DMSO elicited a biphasic increase in DAG levels, dependent on the presence of extracellular Ca2+. Exposure to DMSO also elicited a rapid increase in inositol phosphate and a slight increase in phosphatidic acid (PA), trailing that of DAG. The molecular species (MS) of DAG were analyzed. Within 60 s of DMSO application there were transient increases of DAG representative of phosphatidylinositol (PI) hydrolysis. At longer intervals, more DAG originated from phosphatidylcholine. The MS composition of newly formed PA resembled that of PI and native DAG. Inhibition studies indicated that DAG is formed in the DMSO-treated cells by phospholipases C and that PA formed later is a result of DAG phosphorylation and not activity of phospholipase D (PLD). Undifferentiated cells exhibited an active PLD pathway. In contrast, PLD in DMSO-differentiated cells was not active. In examining the involvement of the sphingomyelin pathway, DMSO exposure was found to increase ceramide levels with a concomitant decrease in sphingomyelin. Addition of the exogenous, soluble analog C6-ceramide to undifferentiated cells resulted in dramatic reductions in DAG and PA levels and PLD activity. These results indicate that DMSO treatment inactivates PLD while activating phospholipases C and the sphingomyelin pathway, suggesting a "switch" between signal transduction pathways in the undifferentiated and differentiated states of N1E-115. PMID- 8612682 TI - Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. AB - Fluorescence in situ hybridization and immunofluorescence have been used to visualize specific genomic DNA sequences and proteins in interphase nuclei treated with transcriptional inhibitors. The adenosine analog 5,6-dichloro-beta-D ribofuranosylbenzimidazole (DRB) and alpha-amanitin selectively inhibit transcription by RNA polymerase II at low doses. Upon exposure to DRB or alpha amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA genes. Similarly, blocks of tandemly repeated satellite DNAs dissociate into extended beaded strands. Localized (euchromatic) chromosome domains and even whole chromosome territories disperse throughout the nuclear interior. Treatment of cells with actinomycin D (AMD) at doses that block rRNA synthesis does not cause significant decondensation of nucleolar, heterochromatic, and interphase chromosome domains. Interestingly, both alpha amanitin and AMD cause coilin to associate with the nucleolar domain. In AMD treated cells, coilin is enriched in nucleolar caps abutting upon the residual nucleolus. After alpha-amanitin treatment, coilin is concentrated in numerous beads closely associated with individual rDNA transcription units within nucleolar necklaces. The changes in higher-order nuclear structure are reversible in cell cultures exposed to nontoxic doses of transcriptional inhibitors. It therefore may be concluded that nuclear topographic organization is dependent on a continued transcription of nuclear genes, but not of the rRNA genes. PMID- 8612683 TI - Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. AB - Earthworm coelomocytes (leukocytes) effect cytotoxicity at significantly high levels against the NK-sensitive, human tumor cell line, K562, and the NK resistant targets (U937, BSM, CEM). By cytofluorimetric analyses using mouse anti human monoclonal antibodies and by morphological evaluations, two types of coelomocytes were identified: (1) small (8-11 micron) electron-dense cells (SC): CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+, beta 2-m+ and Thy-1+; (2) large (12-15 micron) electron-lucent cells (LC) that are negative for these markers. Both cell types were negative for other CD and MHC class I and class II markers. SC were active during recognition, rapidly binding to targets; LC were phagocytic. Release of 51Cr revealed rapid, significant, and equal levels of killing at 4 degrees, 20 degrees, and 37 degrees C. We propose that primitive NK-like activity appeared early in evolution. PMID- 8612684 TI - Inhibition by Interleukin-4 of stromelysin expression in human skin fibroblasts: role of PKC. AB - This study explores novel aspects of the interaction between inflammatory mediators and extracellular matrix degradation. Here we have evaluated the effects of a T-cell cytokine interleukin-4 (IL-4) on the expression and activity of a metalloprotease, stromelysin, and its tissue inhibitor (TIMP-1) in human skin fibroblasts. IL-4 strongly decreased stromelysin mRNA levels and stromelysin producing activity induced by IL-1 beta-treated and untreated cells. Under the same experimental conditions, TIMP-1 mRNA expression was slightly modified. Phorbol ester (PMA), a PKC activator, induced stromelysin gene expression, an effect enhanced by the addition of IL-1 beta. IL-4 was not able to decrease the PMA and PMA + IL1 beta effects. Calphostin, a specific PKC inhibitor, inhibited stromelysin mRNA expression induced by IL-1 beta. Forskolin, a PKA activator, did not modify mRNA levels and was not able to reduce the effect of IL-4 on IL-1 beta induced stromelysin expression. These data suggest that in human dermal fibroblasts, activation of PKC abolishes the observed IL-4 effect on both basal and IL-1 beta-induced stromelysin gene expression. It therefore appears that lack of PKC activation is a prerequisite for the inhibitory effect of IL-4 in the system. PMID- 8612685 TI - Downregulation of protein kinase C suppresses induction of apoptosis in human prostatic carcinoma cells. AB - Protein kinase C (PKC) has been implicated in propagating signals for apoptosis. We have investigated the effect of pharmacological modulation of PKC activity in DU-145 human androgen-independent prostatic carcinoma cells. The apoptotic death of these cells is characterized by the acquisition of classical apoptotic morphology and generation of > or = 1-mbp and 450- to 600-kbp DNA fragments in attached preapoptotic cell populations prior to cellular detachment and accrual of 30- to 50-kbp DNA fragments. We found that induction of apoptosis was arrested by downregulation of PKC activity and not by transient activation or inhibition of the enzyme. Concentrations and durations of exposure to phorbol esters that downregulated PKC activity correlated with inhibition of VP-16 or melphalan induced morphological apoptosis and generation of the 30-to-50-kbp DNA fragments. Chronic exposure to phorbol-12,13-dibutyrate (PDBu) did not, however, suppress production of the > or = 1-mbp and 450- to 600-kbp DNA fragments found in preapoptotic cell populations, suggesting that PKC downregulation may interfere with the transition between a preapoptotic cell and an apoptotic cell. PKC isozyme analysis revealed that chronic PDBu treatment caused downregulation of PKC-alpha and -epsilon in DU-145 cells. Using concentrations of the PKC inhibitor UCN-01 that were consistent with PKC-alpha inhibition (but not PKC-epsilon inhibition), however, did not mimic the effects of chronic PDBu treatment, implying that downregulation of PKC-epsilon may be of particular importance. Together, these findings suggest that phorbol esters may act as tumor promoters by suppressing apoptosis. PMID- 8612686 TI - Expression of growth arrest-specific (Gas) genes in murine keratinocytes: Gas2 is specifically regulated. AB - In order to elucidate a possible role of growth arrest-specific (gas) genes in the regulation of tissue proliferation, we analyzed their expression in keratinocytes isolated from murine back skin. On the mRNA level gas1, gas5, and gas6 were found to be significantly expressed whereas there was a relatively low expression of gas2, gas3, and gas4. Using keratinocytes fractionated according to their density resulted in subpopulations of cells: differentiating suprabasal cells in fractions I and II; proliferative basal cells in fractions IIIa, III and IV. We found gas2 protein to be expressed more strongly in the proliferative cells than in the differentiating cells. Stimulation of hyperproliferation by 12 O-tetradecanoylphorbol-13-acetate (TPA) resulted in a transient increase of gas2 protein content concomitantly with the time of maximal cell renewal. In this respect the murine keratinocyte cell line MSCP5 resembled freshly isolated keratinocytes. There was a higher expression of gas2 protein during exponential growth than during growth arrest, induced either by serum starvation or by TGFbeta treatment. Since, in contrast to the results reported for 3T3 cells, growth arrest within these cells was not accompanied by an elevation of gas2 protein, we suggest a cell-specific regulation of its expression. PMID- 8612687 TI - Smooth muscle alpha-actin downregulation in cultured chick aortic smooth muscle and neural crest cells is associated with altered cell shape. AB - A modified CXL retrovirus was used to clone an antisense smooth muscle alpha actin ribozyme sequence adjacent to the reporter lacZ sequence. The virus was applied to downregulate alpha-actin expression in cultured smooth muscle cells obtained from chicken aortic arch and cultured neural crest cells. After infection with the ribozyme-containing CXL retrovirus both the smooth muscle and neural crest cells showed beta-galactosidase activity accompanied by a reduction of smooth muscle alpha-actin-positive fibers. Double staining of beta galactosidase and smooth muscle alpha-actin using immunohistochemistry revealed that single cells infected with the CXL/ribozyme showed little to no smooth muscle alpha-actin protein. The absence of smooth muscle alpha-actin was associated with a distinct change in cellular morphology of the cultured cells, suggesting that expression of smooth muscle alpha-actin in cultured neural crest cells may be associated with cytoskeletal elements rather than vascular smooth muscle phenotype. PMID- 8612688 TI - Expression of the integrin alpha 5 subunit in HT29 colon carcinoma cells suppresses apoptosis triggered by serum deprivation. AB - It is clear that certain integrins can regulate the growth of tumors, probably by contributing to signal transduction processes. In the present study we have used HT29 human colon carcinoma cells stably transfected with human cDNA for the integrin alpha 5 subunit and studied the effects of alpha 5 expression on the induction of apoptosis. We observe that apoptosis can be triggered in HT29 cells by removal of serum and that this process can be suppressed by the stable expression of full-length integrin alpha 5 subunits. While the mechanism underlying this effect is still unclear, these observations suggest that the alpha 5 beta 1 integrin plays an important role in modulating tumor cell responses to growth factors and nutrients. PMID- 8612689 TI - Dual regulation of beta-lactoglobulin/human serum albumin gene expression by the extracellular matrix in mammary cells from transgenic mice. AB - Mammary explants and epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously we demonstrated that mammary explants from virgin female transgenic mice synthesize and secrete high levels of HSA during the first day in culture. Here we present a detailed analysis of endogenous and transgene expression during the first 20 h of mammary explant cultures. We show that HSA genes as well as endogenous milk protein genes are rapidly induced upon explantation. Unexpectedly, HSA was synthesized also in mammary explants from strains that do not secrete HSA into the milk, indicating the existence of a cryptic potential to express the transgene. Histological examination revealed that some luminal epithelial cells detached from the underlying extracellular matrix (ECM) soon after explantation. Epithelial cell cultures from nonsecreting strains grown on plastic rapidly induced transgene expression and secreted higher levels of HSA into the medium compared to cells grown on collagen. These results suggest that tissue organization and most likely the interaction of epithelial cells with the ECM are intimately involved in the control of HSA transgene expression. PMID- 8612690 TI - An alpha 2 beta 1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube formation in three-dimensional collagen matrix. AB - Human endothelial cells, when suspended within three-dimensional collagen matrices, develop intracellular vacuoles that coalesce to form capillary lumens and tubes. Vacuole and lumen formation are completely dependent on the collagen binding integrin alpha 2 beta 1, while other endothelial cell integrins had no apparent influence. Vacuole formation occurs by a pinocytic process with internalization of plasma membrane and molecules from the extracellular space, such as fluorescent tracers. By immunofluorescence, vacuole membranes were found to contain associated cell surface proteins, proteins involved in endosomal trafficking (i.e., caveolin and annexin II), and F-actin. Furthermore, some vacuole compartments contained von Willebrand factor. Integrin-regulated vacuole formation and coalescence are major mechanisms controlling capillary lumen and tube formation within a three-dimensional extracellular matrix. PMID- 8612691 TI - Characterization of c-myc-transformed rat fibroblasts resistant to apoptosis induced ny growth factor deprivation. AB - Under appropriate conditions (e.g., growth factor withdrawal), the deregulated expression of c-myc in rodent fibroblasts leads to substantial cell death due to apoptosis. To better understand this process, we selected for c-myc-transformed Rat1A fibroblasts that were resistant to growth factor deprivation-induced cell death. One clonal isolate exhibited prolonged survival in serum-free medium and displayed reduced levels of apoptosis-related DNA fragmentation. These cells were also resistant to induction of apoptosis by the protein kinase inhibitor staurosporine. They retained a transformed cell phenotype and expressed the proviral human c-myc allele in an unaltered fashion, strongly indicating that the mutation of a cellular gene other than c-myc accounts for the apoptosis-resistant phenotype. The results of somatic cell hybrid analysis of this cell line are consistent with a recessive mutation. Our findings suggest a novel mechanism for abrogation of apoptosis in neoplastic cells and provide a model system for the study of its role in tumorigenesis and resistance to antineoplastic therapy. PMID- 8612692 TI - Molecular mechanisms of TNFalpha cytotoxicity: activation of NF-kappaB and nuclear translocation. AB - The murine fibrosarcoma cell line WEHI 164 is well known for its susceptibility to tumor necrosis factor (TNFalpha). We have studied the activation of the transcription factor NF-kappaB when WEHI 164 cells are challenged with TNFalpha. NF-kappaB is retained in the cytoplasm of unchallenged cells by its inhibitor IkappaB-alpha. Upon cellular stimulation, IkappaB-alpha is functionally inactivated and NF-kappaB translocated to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-kappaB show the same TNFalpha dependence. TNFalpha induces a rapid and transient activation of NF kappaB in WEHI 164 cells which is followed by a second, long lasting phase in which the amount of NF-kappaB complex in the nucleus remains at about 50% of maximum. Upon TNFalpha treatment, IkappaB-alpha is rapidly degraded. However, newly synthesized IkappaB-alpha can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-kappaB is an obligatory step for the cytotoxic effect to take place. Thus, WEHI 164 cells treated with TNFalpha for up to 6 h can be rescued as long as NF-kappa relocalizes to the cytoplasm in its inactive form. On the other hand, TNFalpha treatments as short as 15 min cause the cytotoxic effect provided that NF-kappaB remains in the nucleus. The activation of NF-kappaB is controlled by both phosphorylation and proteolysis. The activation of NF-kappaB can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of IkappaB-alpha does not lead to the dissociation of the inhibitor from NF-kappaB. Phosphorylation appears to regulate the inhibitory activity of IkappaB-alpha both positively and negatively. since inhibitors of protein kinases have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-kappaB and was synergistic with TNFalpha induced activation. Calphostin C, on the other hand, can block the activation of NF-kappaB by TNFalpha, also blocking its proteolytic degradation. PMID- 8612693 TI - Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation. AB - To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C alpha, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the delta isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C beta II, delta, and zeta and the appearance of protein kinase C epsilon and eta at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC epsilon and eta in serum stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the zeta isoform after mitogenic stimulation and for PKC alpha during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells. PMID- 8612695 TI - Growth and cell density-dependent expression of stathmin in C2 myoblasts in culture. AB - Stathmin is a 19-kDa, ubiquitous cytoplasmic phosphoprotein whose expression is strongly regulated during tissue development and maturation and which was proposed as a general relay integrating diverse intracellular signaling pathways. Since myoblasts tend to align and differentiate in vitro toward myotubes above a certain density in culture, we examined the expression of stathmin as a function of cell density in the C2 myogenic cell line. Whereas stathmin was hardly detectable in low-to medium-density cultures corresponding to less than 1 microgram soluble protein/cm2, it became expressed to a stable level above this threshold of cell density. This cell density effect on stathmin expression was not mediated by a diffusible factor, since myogenic C2 or fibroblastic 3T3 cells grown at low and high density within the same culture flask displayed the same pattern of density-dependent stathmin expression, significant stathmin levels being observed only in the dense moiety of the flask. Interestingly, culture conditions which indirectly perturb cell-cell contacts, such as low Ca2+ or incubation with cytoskeleton disrupting agents such as nocodazole or cytochalasin D, prevented the expression of stathmin in C2 cells even at high density. More directly, anti-E-cadherin immunoglobulins, interfering with direct cell-cell contacts of the E-cadherin expressing S180 sarcoma-derived 2B2 cells, also prevented the expression of stathmin in these cells even at high density. Altogether, our results indicate that cell-cell contacts, probably mediated by adhesion molecules such as cadherins, are responsible for the high-density induced expression of stathmin, which might then participate, in particular in the case of myogenic cells, in the control of the proliferation of cells and of their entry into the differentiation process. PMID- 8612694 TI - Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation. AB - Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX 2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published. PMID- 8612696 TI - Synthesis of the mammalian synaptic vesicle protein synaptophysin in insect cells: a model for vesicle biogenesis. AB - The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such an in vitro approach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively with n-octyl-beta-D glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular. PMID- 8612697 TI - Codominant regulation of keratin gene expression by cell surface receptors and nuclear receptors. AB - Epidermal keratinocytes are subject to a large variety of signals that modulate their differentiation in health and their activation in disease. Hormones and vitamins, which act via nuclear receptors, affect the differentiation process, whereas growth factors and cytokines, which act via cell surface receptors, affect keratinocyte activation and related events. Using expression of keratin genes as markers for keratinocyte phenotype, we examined the interaction between the nuclear receptor and cell surface receptor pathways. We expected to find dominance of one of the pathways. Surprisingly, we found that the two pathways are codominant. Specifically, while EGF induces expression of K6 and K16 keratin genes, retinoic acid suppresses their expression, and when both mediators are present simultaneously, the level of expression is intermediate, a product of both signals. Similar codominant effects were found on other keratin genes using interferon gamma, TGF beta, and thyroid hormone signaling molecules. These codominant effects are specific only for genes that are regulated by both pathways. Our results suggest that a judicious combination of hormones, vitamins, growth factors, and cytokines may be used to target specific expression of appropriate genes in the treatment of human epidermal diseases. PMID- 8612698 TI - Modulation of expression and assembly of vinculin during in vitro fibrillar collagen-induced angiogenesis and its reversal. AB - A model of collagen-induced in vitro angiogenesis was used to investigate the modulation of expression and assembly of focal adhesion plaque-associated proteins during the process of differentiation. Human umbilical vein endothelial cells (HUVEC), first attached on an adhesive substratum (gelatin-, fibronectin-, or laminin-coated dish) or adherent collagen gel and then covered by an overlaying collagen get, organized within 3-4 days in tube-like structures (TLS). Removing the overlaying collagen gel from fully differentiated HUVEC induced a reversion of the process and HUVEC returned to a monolayer pattern. Modulations of focal adhesion-associated proteins occurring in HUVEC during the in vitro differentiation process and its reversal were investigated by Western blot analysis. A significant decrease of expression of vinculin, the integrin alpha2 subunit, talin, alpha-actinin, and actin was observed in TLS whereas the amount of FVIII-related antigen did not vary as compared to control monolayer cultures. During reversal, all the reduced proteins were markedly reexpressed. Human skin fibroblasts (HSF), submitted to the same experimental conditions, did not form TLS. Most of the focal adhesion proteins in HSF were similarly modulated by an overlaying collagen gel with the exception of vinculin, which was not modified. This particular protein was therefore more thoroughly investigated. In a nondifferentiated monolayer of HUVEC, a significant proportion of vinculin was organized into a detergent-resistant juxtamembranous structure (focal adhesion plaque) which disassembled early in TLS formation and reassembled during the reversal of the process. The reduction of vinculin during TLS formation was preceded by a downregulation of its mRNA while this mRNA was upregulated during reversal of the morphotype. These results suggest that the modulations of the cytoskeletal and focal adhesion proteins and more specifically of vinculin coupled to its subcellular redistribution are critical and early events in the cascade of mechanochemical signaling during in vitro angiogenesis induced by fibrillar collagen. PMID- 8612699 TI - Characterization of cis-acting signals for nuclear import and retention of the La (SS-B) autoantigen. AB - The La (SS-B) autoantigen is a 47-kDa protein which binds to the 3' termini of nascent RNA polymerase III transcripts and to a number of viral leader RNAs. The La protein plays a direct role in the termination of RNA polymerase III transcription and recent findings have suggested an additional role in several aspects of translation of (viral) mRNAs. In this study we have addressed the intracellular trafficking of the La protein and characterized cis-acting elements involved in nuclear import and retention in Xenopus laevis oocytes by microinjection of in vitro translated La protein. The steady-state distribution of recombinant human La protein was, like the endogenous Xenopus La protein, mainly nuclear. Nuclear import of La appeared to be energy-dependent and is governed by a nuclear localization signal (NLS) located in the extreme C-terminal part of the protein, resembling the consensus bipartite NLS. Another sequence element in La, which completely corresponds to the bipartite NLS consensus, appeared to be nonfunctional in nuclear import of the La protein. Nuclear accumulation of La was found to be mediated by retention in the nuclear compartment. The N-terminal RNA binding domain of La is not involved in this retention, but sequence elements in the central region of the polypeptide (amino acids 165 to 337) appear to be required. Amino acids 266-269 as well as 313-337 were found to be of major importance for retention in the nucleus. PMID- 8612701 TI - Coexpression of stem cell factor and c-kit in embryonic and adult liver. AB - Stem cell factor and its receptor c-kit constitute an important signal transduction system implicated in survival, proliferation, and differentiation of stem cells in hematopoiesis, gametogenesis, and melanogenesis. In the present study we used both immunocytochemical methods and Western analysis to demonstrate the presence of this cytokine/receptor system in both embryonic and adult rat liver. Stem cell factor was present in the ductular cells around the portal vein during the late embryonic stage of the liver. In the adult liver both bile ducts and bile ductules were positive for stem cell factor and c-kit. When the activation of the liver stem cell compartment was induced by combining administration of acetylaminofluorene and partial hepatectomy, both stem cell factor and c-kit were expressed in the infiltrating oval cell population, but absent in the newly formed basophilic hepatocytes. Activation of oval cell proliferation following administration Of D-galactosamine also produced a similar but less prominent increase in the level of the stem cell factor. Our data suggest that the stem cell factor/c-kit signal transduction system is involved in the development of bile ducts and that it may also be an important member of the growth factor/receptor systems associated with the biology of liver stem cells. PMID- 8612700 TI - A vacuolar-type proton ATPase mediates acidification of plasmalemmal vesicles during potocytosis. AB - Previously we showed that the potocytosis of 5-methyltetrahydrofolate is dependent on the acidification of plasmalemmal vesicles created each time a caveolae, closes off from the cell surface. We now report that bafilomycin A1, which is a specific inhibitor of the V-type proton ATPase, inhibits 5 methyltetrahydrofolate uptake into MA104 cells (ED50 = 150 nM). The inhibitory effect was reversed within 30 min after removal of the drug from the cells. Bafilomycin A1 had no effect on the binding of folic acid to its receptor. A concentration of up to 200 nM bafilomycin A1 did not affect sequestration of folate receptors. Immunoblotting showed that the 70-kDa subunit of the V-type proton pump was localized to caveolae-rich fractions isolated from the plasma membrane of these cells. These results suggest that a V-type proton pump acidifies the lumen of plasmalemmal vesicles during potocytosis. PMID- 8612702 TI - Altered expression and regulation of the alpha 5beta1 integrin-fibronectin receptor lead to reduced amounts of functional alpha5beta1 heterodimer on the plasma membrane of senescent human diploid fibroblasts. AB - Previously, we reported that fibronectin (FN) mRNA was overexpressed in normal late-passage (old) and pre- maturely senescent Werner syndrome (WS) fibroblasts when compared to normal early-passage (young) cells (Murano et al. Mol. Cell. Biol. 11, 3905-3914, 1991). Therefore, we investigated the expression and function of the alpha5 beta1 FN receptor (FNR), a member of the integrin family, in young and senescent normal and WS cells. Levels of the alpha5 polypeptide, a unique subunit of the alpha5 beta1 FNR, were reduced in old cells, so that old cells produced fewer alpha5 beta1 heterodimers on the plasma membrane. The reduced levels of alpha5 polypeptide might be due to deficient translation and/or nonfunctional alpha5 mRNA since increased mRNA levels and unchanged polypeptide turnover were found in these cells. Moreover, the alpha5 polypeptides on the senescent cell surface were less accessible to monoclonal antibody, suggesting sequestration of this subunit, which might affect receptor-ligand binding. In contrast, beta1 subunit, a common subunit for the beta1 integrin subfamily, showed relatively stable levels during cellular aging, but underwent slower intracellular processing. Old cells exhibited reduced attachment to FN, which might be in part mediated by the alpha5 beta1 FNR. More importantly, old cells were deficient in response to FN-induced DNA synthesis and cell proliferation. This induction was pronounced in young cells, however, and could be completely inhibited by alpha5-specific monoclonal antibody, indicating mediation by alpha5 beta1 FNR. WS cells behaved like normal old cells in the above assays. Our results indicate that reduction of alpha5 beta1 FNR expression and its mediated effects are associated with the senescent phenotype of fibroblasts. These findings provide new insight into the mechanism(s) of replicative senescence in human fibroblasts. PMID- 8612703 TI - Establishment of long-term myogenic cultures from patients with Duchenne muscular dystrophy by retroviral transduction of a temperature-sensitive SV40 large T antigen. AB - We have established long-term human myogenic cultures from adult human skeletal muscle biopsies by infecting primary explant cultures with an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen, tsA58 U19. Infected myoblasts expressed the large T antigen and showed greatly enhanced proliferative capacity when cultured at 33 degrees C, compared with noninfected cells. When the infected cultures were incubated at 39 degrees C, the cells withdrew from cycle, aligned, and fused to form multinucleated myotubes which expressed certain antigens that are similarly expressed in nontransduced differentiating muscle cells. Myogenic clones with greatly increased proliferative capacity were generated, for the first time, from biopsies obtained from Duchenne muscular dystrophy patients as well as from normal, dystrophin positive individuals. Cell lines produced by this approach may prove valuable for in vitro studies of myogenesis and for investigating the cellular and molecular consequences of inherited muscle diseases. PMID- 8612704 TI - DNA repair fine structure in Werner's syndrome cell lines. AB - Werner's syndrome (WS) is a human segmental progerioid disorder with an autosomal recessive pattern of inheritance. Patients with WS exhibit a number of symptoms resembling a premature aging phenotype. We have examined the fine structure of the DNA repair of UV-induced cyclobutane pyrimidine dimers in Epstein-Barr virus (EBV)-transformed WS lymphoblastoid cell lines and in a primary WS fibroblast cell line. The repair was measured at the level of the gene and also in the general genome. Gene-specific and strand-specific DNA repair was measured in the actively transcribed genes dihydrofolate reductase (DHFR), c-myc, and p53, and in the transcriptionally inactive regions, delta globin and the X-linked 754 domain. Both gene-specific repair and strand-specific repair were deficient in the transformed WS lymphoblastoid cell lines compared to normal controls. In normal cells, repair in the transcribed strand was 25 (4 h), 43 (8 h), and 72% (24 h); in the WS cells on average, repair in the transcribed strand was 18 (4 h), 27 (8 h), and 44% (24 h). However, in the primary WS fibroblast cell line, we found a pattern of preferential gene repair which was similar to that in normal human cells. In contrast to cells from patients with the gene-specific repair deficient disease Cockayne's syndrome, which show greatly delayed RNA synthesis recovery after UV irradiation, the WS cells had normal recovery of RNA synthesis. The DNA repair results differ for the different cell types, and our findings thus do not establish a general DNA repair phenotype for WS cells. The fibroblasts had proficient repair, but in the WS lymphoblasts we find a deficiency in DNA repair which could contribute to the reported hypermutability in these cells. The lymphoblasts are, however, transformed cells, and it raises the concern that biological findings in transformed cells may not reflect the situation in primary cells. PMID- 8612706 TI - Generation of small fusion genes carrying phleomycin resistance and Drosophila alcohol dehydrogenase reporter properties: their application in retroviral vectors. AB - We have used the Drosophila ADH cDNA to engineer new fusion genes carrying both reporter activity and bleomycin/phleomycin resistance (Sh ble). Cassettes of ADH::Sh ble, Sh ble::ADH, or ADH::Sh ble::ADH with or without polyadenylation signals were constructed. Placed under the control of the strong CMV promoter, these constructs induced intense ADH substrate staining and phleomycin resistance, whatever the position of the ADH gene, in avian or mammalian cell lines. SW-based nonreplicative retroviral vectors were constructed and introduced into the appropriate packaging cell line. Titers up to 10(6) ADH forming units/ml of viral supernatant were obtained except for the ADH::Sh ble::ADH construct, which reached 10(5) ADH forming units. These retroviral vectors were inoculated to the E3 chick embryo via the coelom. Three days later, cells from different organs were put in culture for 24 h and stained to detect ADH activity. A large number of positive cells were found in cultures from all organs. The new fusion genes described here are, to our knowledge, the smallest (1.1 kb) published to date that carry both reporter and drug resistance properties. These genes represent the basis of a new retroviral vector model with three distinct properties in two genetic units; their advantage is to reduce the size and increase the efficiency of the vector. PMID- 8612705 TI - Establishment and characterization of cell line LSV5 that retains the altered adhesive properties of human junctional epidermolysis bullosa keratinocytes. AB - Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by hampered expression of the adhesion ligand laminin-5. Thus far, analysis of the processes underlying the epithelial-mesenchymal dysadhesion marking this disease has been limited by the reduced growth and adhesive capabilities of the epithelial cells derived from H-JEB patients. To overcome these difficulties, we used SV40 virus to immortalize H-JEB keratinocytes with a homozygous nonsense mutation in the gene that encodes the gamma2 chain of laminin-5. Cell lines (LSV) derived from infected keratinocytes maintain a stable karyotype, grow independent of 3T3 feeder layers and are not tumorigenic. Further analysis of clone LSV5 showed an increased secretion of laminin-6 and fibronectin compared to normal keratinocytes. Similar to parental H-JEB keratinocytes, these cells regenerate stratified epidermis in vitro and, in in vivo models, they synthesize a basement membrane lacking laminin-5. LSV cells show hypermotility and reduced adhesive properties resulting from an incomplete association with the underlying culture substrate. These results demonstrate that LSV5 cells retain the pathologic phenotype of H-JEB keratinocytes and can serve as a model system to study the adhesion processes mediated by laminin-5. PMID- 8612707 TI - Reversible large-body formation from nuclear bodies upon amino acid(s) starvation in T24 cells. AB - AP435 dot, a nuclear dot-like structure that is recognized by a monoclonal antibody AP435 MAb and that seems to correlate with perinuclear intermediate filaments, was identified as a nuclear body by double immunofluorescent staining with AP435 MAb and the nuclear-body-specific antibody alphaSp100 or mAb 5E10. In T24 cells, nuclear bodies usually appear as small entities with an apparent diameter ranging from 0.2 to 0.7 microm, and several to 20 or more of them are present per nucleus. After long culture without a change in the medium, however, nuclear bodies disappeared while one or more large doughnut-shaped bodies appeared, which had apparent outer diameters of 0.7-1.8 microm. When the medium was changed or medium components were added, large bodies disappeared and many nuclear bodies of normal size reappeared within several hours. Large-body formation was not related to the arrest of DNA synthesis, as revealed by double labeling with AP435 MAb and anti-cyclin antibody. Among the medium components, only an amino acid mixture induced the change from large bodies to nuclear bodies. Large-body formation was also observed in long-cultured HeLa cells. These results suggest that nuclear bodies reversibly aggregate or reorganized to form large bodies upon amino acid(s) starvation. PMID- 8612708 TI - Loss of deoxcytidine kinase expression and tetraploidization of HL60 cells following long-term culture in 1,25-dihydroxyvitamin D3. AB - Development of drug resistance is a major problem in attempts to control the growth of neoplastic cell populations. The resistance can he either inherent or acquired by an exposure to a chemotherapeutic drug. The available models for study of these phenomena have not led to major improvements in the therapy for human cancers. Therefore, in order to develop a new model for such studies, we have exposed human myeloid leukemia cells HL60 to increasing concentrations of 1,25-dihydroxyvitamin D3 (1,25D3) and characterized the emerging new phenotypes of these cells over a period of 4 years. During the stepwise development of resistance only cells which did not adhere to the flask walls were passaged. Beginning at 30 nM 1,25D3 the sublines became resistant to the differentiation inducing and growth-retarding properties of 1,25D3 even at 400 nM. Also, their growth rates in 1,25D3-free media increased. In addition, beginning at 40 nM 1,25D3 the sublines acquired resistance to 5-beta-D-arabinocytosine (araC) due to the lack of expression of the deoxycytidine kinase gene. The araC-resistant sublines were also near-tetraploid, as judged by their DNA content. When grown in 1,25D3-free long-term culture the phenotype was essentially stable. The development of cross-resistance to araC during growth in the presence of an unrelated compound (i.e., 1,25D3) shows that in some instances an apparently inherent drug resistance may in fact he due to a metabolic defect resulting from an exposure to another agent. PMID- 8612709 TI - Truncated activin type II receptors inhibit bioactivity by the formation of heteromeric complexes with activin type I. receptors. AB - Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors. PMID- 8612710 TI - Nerve growth factor rescues pigment cells from ultraviolet-induced apoptosis by upregulating BCL-2 levels. AB - Apoptosis plays an important role in eliminating dysfunctional damaged cells. For skin, the best characterized injurious environmental agent is ultraviolet (UV) irradiation. Most of the damaging UV irradiation is absorbed in the epidermis and leads to apoptosis of keratinocytes. However, epidermal melanocytes appear to be protected from UV-induced apoptosis. We now report that in pure cultures melanocytic cells undergo characteristic apoptosis after physiologic UV exposures. However, nerve growth factor (NGF) supplementation protects them from this programmed cell death. Furthermore, we show that NGF protects melanocytic cells from UV-induced apoptosis by upregulating BCL-2 protein in these cells and that prior downregulation of BCL-2 abrogates the NGF protective effect on melanocytes. Our data suggest that NGF, known to be constitutively produced by epidermal keratinocytes and induced in these cells after UV irradiation, may preserve the population of cutaneous melanocytes that would otherwise be depleted by casual sun exposure. PMID- 8612711 TI - The urokinase receptor is a major vitronectin-binding protein on endothelial cells. AB - We have previously demonstrated that vitronectin (VN), a morphoregulatory protein in the vessel wall, is internalized and translocated to the subendothelial matrix by an integrin-independent mechanism (J. Histochem. Cytochem. 41, 1823-1832, 1993). The cell surface component which mediates the initial contact of VN with endothelial cells is defined here. The specific binding of VN to endothelial cells demonstrated the following properties: a threefold increase after phorbol ester treatment; 85% inhibition by pretreatment of cells with phosphatidylinositol-phospholipase C to release glycolipid-anchored surface proteins; a 90% inhibition by urokinase (u-PA) receptor blocking antibody. u-PA increased VN binding to cells due to an eightfold increase in the affinity of VN for the u-PA receptor. Structure-function studies showed that the amino-terminal fragment of u-PA, devoid of any proteolytic activity, mediated this effect. Active plasminogen activator inhibitor-1 (PAI-1), but not inactivated PAI-1, inhibited VN binding to cells and displaced VN that was prebound to endothelial cell monolayers. Similarly, VN binding to purified (immobilized) u-PA receptor, but not to integrin, was enhanced by u-PA and inhibited by PAI-1. Hence, the binding of soluble VN to endothelial cell surfaces is mediated by the u-PA receptor, and the relative concentrations of u-PA and PAI-1 are able to regulate the strength of this interaction. Endothelial cell adhesion to immobilized VN was found to be integrin-mediated without any involvement of the VN-uPA-receptor system. Hence, the interaction of VN with the u-PA receptor may be involved in the regulation of cellular processes necessary for endothelial cell invasion and migration at VN-rich extracellular matrix sites. PMID- 8612712 TI - Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures. AB - The regulation of intestinal cell proliferation, migration, and differentiation has been the subject of numerous studies. However, in human, progress in this field has been traditionally hampered by the lack of normal epithelial cell models. The aim of the present study was to define conditions in order to isolate, and more importantly to grow in a continuous manner, human small intestinal epithelial cells. A number of mechanical and/or enzymatic dissociation methods have been tested to isolate viable epithelial cells from the fetal small intestine. Cultured cells were characterized by indirect immunofluorescence and Western blot analysis. It was found that the use of thermolysin (50 microgram/ml, 2-3 h at 37 degrees C) can be advantageously applied to the isolation of viable epithelial cells free from contaminating fibroblasts when obtained from the 17- to 19-week fetal ileum. Furthermore, this procedure allowed the generation of continuously growing human intestinal epithelial cell cultures, which retain the ability to express specific cytokeratins as well as intestinal cell markers over a number of passages. This study shows that normal epithelial cell cultures can be relatively easily and reproducibly generated from the human fetal small intestine. PMID- 8612713 TI - Nucleophilic distribution of metallothionein in human tumor cells. AB - Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 microM CdCl2 did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell. PMID- 8612715 TI - Differential expression of PTP1D, a protein tyrosine phosphatase with two SH2 domains, in a slow and fast skeletal muscle fibers. AB - We show that PTP1D, a protein tyrosine phosphatase that contains two SH2 domains, is preferentially expressed in slow skeletal muscle fibers. Immunohistochemical staining using polyclonal antibodies against PTP1D demonstrated that PTP1D was expressed in a subpopulation of rodent muscle fibers. These fibers were identified as slow Type I fibers based on histochemical ATPase assays and slow myosin heavy chain expression. Northern and Western analyses showed that PTP1D levels were higher in predominantly slow muscles than in predominantly fast muscles. This differential expression of PTP1D in slow muscle fibers appeared by birth. In cultures of mouse myogenic cells, PTP1D was expressed after MyoD and myogenin and appeared in myotubes derived from embryonic, fetal, and postnatal myoblasts. Remarkably, PTP1D was organized into sarcomeres in a pattern coincident with myosin heavy chain, suggesting that PTP1D associates with a component of the thick filament. These results show that PTP1D is preferentially expressed in slow muscle fibers. We speculate that PTP1D may play a role in slow muscle fiber function and differentiation. PMID- 8612717 TI - 27th Annual meeting of the Swiss Societies for Experimental Biology. Fribourg, March 30-31, 1995. Abstracts. PMID- 8612716 TI - Chick cartilage fibronectin differs in structure from the fibronectin in limb mesenchyme. AB - Fibronectin, present in the extracellular matrix of several tissues, is heterogeneous in structure. This heterogeneity is largely due to the alternative splicing of three exons (IIIB, IIIA, and V) during processing of the fibronectin primary transcript. Previously, we determined that the splicing patterns of fibronectin mRNAs change from B+A+ to B+A- during the differentiation of mesenchyme into cartilage (V. D. Bennett, K. M. Pallante, and S. L. Adams, J. Biol. Chem. 266, 5918-5924, 1991). Therefore, the structure of fibronectin at the protein level most likely changes during chondrogenesis as well. In order to characterize the fibronectin protein in chick limb prechondrogenic mesenchyme and cartilage, we generated a polyclonal antibody specific for the region encoded by exon IIIB in the chick fibronectin gene. Immunoblot and immunohistochemistry analyses with this antibody and an antibody specific for the region encoded by exon IIIA indicate that both antibodies react with the fibronectin present in prechondrogenic mesenchyme. In contrast, only the exon IIIB antibody reacts with the fibronectin present in chick cartilage. Quantitative ELISA assays with these antibodies indicate that approximately 96% of the fibronectin in chick embryonic cartilage contains exon IIIB while less than 3% of the fibronectin contains exon IIIA. These results corroborate the structures of the fibronectin isoforms present in prechondrogenic mesenchyme and cartilage that were predicted from our previous characterization of the fibronectin mRNAs in these tissues and indicate that essentially all of the fibronectin in cartilage is synthesized and secreted by cartilage chondrocytes. PMID- 8612714 TI - The cloning and characterization of chick tyrosinase from a novel embryonic cDNA library. AB - Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic chick melanocyte cDNA library. Neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. A cDNA library was constructed and screened with a mouse tyrosinase cDNA probe. Nineteen clones were obtained, seven of which cross-hybridized to a mouse tyrosinase cDNA on Southern blots. The longest of these clones, B8.3 (1.9 kb), was sequenced and found to share 99.7% nucleotide and 99.8% amino acid sequence homology to a reported chick tyrosinase cDNA. Both Northern blot analysis and in situ hybridization demonstrated that clone B8.3 was expressed in the retinal pigment epithelium of chick embryos. Our results suggest therefore that the cDNA library described here may allow the cloning of novel melanogenic genes. PMID- 8612718 TI - Proteoglycan alterations in the aortic intima-media of alloxan-diabetic rabbits: an ultrastructural and biochemical study. AB - Proteoglycans and glycosaminoglycans in the aortic intima of diabetic rabbits and age-matched controls were examined at 2 weeks and 3, 6, and 12 months after alloxan (or saline) treatment. Measurements were made by morphometric analysis of ruthenium red-stained large proteoglycan granules (LPG) in electron micrographs and by analysis of 35S-labeled glycosaminoglycans, extracted and purified from the intima-media of aortas of rabbits which had been injected with 35S-sulfate 18 hr before exsanguination. There was a progressive increase in the area of the aortic intima with time which was greater in diabetic than in control rabbits. The concentration of proteoglycan (LPG/microns 2) and the concentration of the 35S-glycosaminoglycans in diabetic intima-media were similar to respective values of control intima-media throughout the 12 months. However, the specific radioactivity of the [35S]glycosaminoglycan pool from intima-media of diabetic rabbits was significantly less than that from controls (P < 0.001) at 6 and 12 months. In addition, the staining intensity of LPG of the diabetic compared to control extracellular matrix was decreased at these times. The profile and electrophoretic mobility of the glycosaminoglycan types were similar in diabetic and control intima-media. We conclude that the onset of diabetes in the rabbit has altered the metabolic turnover but not the concentration, sulfate content or profile of aortic intima-media proteoglycan and glycosaminoglycans. PMID- 8612719 TI - Intestinal anastomosis healing in rat: collagen concentration and histochemical characterization by Picrosirius red staining and polarizing microscopy. AB - The study was designed to characterize collagen fibers in the healing intestinal anastomosis of rat by examining collagen concentration and recording the polarization colors of the collagen fibers in Picrosirius red-stained tissue sections. No significant changes in collagen concentration expressed by hydroxyproline content were noted at the anastomotic site at various postoperative time points. In the normal intestine, the predominant polarization colors were in the yellow to yellow-orange range, signifying tightly packed, matured collagen. During the first postoperative week, collagen fibers at the anastomotic region exhibited mainly polarization colors in the green to green yellow range, implying less packed intermediate collagen fibers. After 2 weeks, collagen fibers at the anastomotic region gradually reacquired their preoperative polarizing texture. These findings strongly suggest that the "quality" rather than the quantity of collagen fibers plays an important role in maintaining the tensile strength of the healing intestinal anastomosis. PMID- 8612721 TI - Na,K-ATPase activity in red blood cells from patients with Chediak-Higashi syndrome. AB - Na,K-ATPase activity of red blood cells from Chediak-Higashi syndrome (CHS) patients and relatives (gene heterozygous) was determined and compared to that of control, healthy, individuals. The enzyme activity was found to be strongly diminished in the CHS patients and slightly lower in their relatives. This reduced activity was due to a lower turnover number of the Na, K-ATPase as well as a decreased number of pumps. The reduced enzyme activity observed in these patients could be the result of an abnormal cell membrane fluidity, and the lowered number of Na, K-pumps could be explained as a consequence of an altered or deficient cell machinery caused by the CHS gene. PMID- 8612720 TI - Naturally occurring modified low density lipoproteins are similar if not identical: more electronegative and desialylated lipoprotein subfractions. AB - Previous studies demonstrated that more electronegative low density lipoprotein (LDL) isolated from human blood by ion exchange chromatography has a chemical composition and physical properties similar to desialylated LDL obtained by lectin chromatography (Avogaro et al., 1988; Orekhov et al., 1989). In this study, sialic acid content and percentage of desialylated LDL in the electronegative LDL (LDL-) subfraction have been investigated. The sialic acid content of the LDL- was 2- to 6-fold lower than that of native LDL and close to that of desialylated LDL. In the native LDL subfraction, 83% of lipoprotein particles did not bind to the Ricinus communis agglutinin, indicating lack of terminal galactose, presumably appearing as a result of desialylation of LDL carbohydrate chains. By contrast, a major proportion of human LDL- (81%) was bound to the lectin. It was also found that the desialylated LDL subfraction consists of 88% LDL-. Native LDL did not affect the contents of free and esterified cholesterol in intimal cells cultured from grossly normal human aorta, while LDL- and desialylated LDL induced a 1.5- to 3-fold increase in the intracellular content of cholesteryl esters. Thus, LDL- is desialylated LDL which induces lipid accumulation in intimal cells. Our findings suggest that the LDL- particle is similar if not identical to the desialylated LDL particle. PMID- 8612722 TI - Localization of retinoic acid receptors in anterior-human embryo. AB - Retinoic acid receptors (RARs) are nuclear transcription factors that are activated by all-trans-retinoic acid or 9-cis-retinoic acid and are found in all tissues but predominantly in developing fetus, dividing tumor cells, and adult skin. Three forms of these receptors, alpha, beta, and gamma, have been described. In this paper we report the presence of RAR alpha and beta determined by hybridization with anti-sense messenger RNA, and histochemical localization of the three forms of RARs using monospecific polyclonal antibodies in various tissues of early human embryos. In a 54-day-old embryo, RAR alpha was expressed primarily in the liver and the brain, with somewhat lesser expression in the intestine. RAR beta was the highest in the brain, followed by a restricted expression in the intestine and the liver. Other organs, i.e., adrenal, kidney, and testis, did not show measurable amounts of RAR beta. The immunohistochemical localization in anterior sections of a 43-day-old embryo indicated that RAR alpha was present in the neuroepithelial cells and in cells lining the primitive pharyngeal sac, dorsal aorta, and pericardium. RAR beta was somewhat less prevalent in same tissues, whereas the expression of RAR gamma was the lowest of the three RARs in any tissues examined. Results indicated that RAR alpha and beta appear at early stages of human embryonic development and their expression is restricted to certain types of tissues. PMID- 8612723 TI - Propolis protects against doxorubicin-induced myocardiopathy in rats. AB - Propolis (bee glue) is one of the major hive products of bees and is rich in flavonoids, which are known for antioxidant activities. Doxorubicin-induced myocardiopathy is the consequence of oxidative stress through the mediation of free radicals. The effect of intraperitoneal administration of propolis (50 and 100 mg/kg) was studied on cardiomyopathy produced by doxorubicin (10 mg/kg, i.v.) in rats. Serum creatine phosphokinase (CK), aspartate aminotransferase (AST), blood and tissue glutathione (GSH), and thiobarbituric acid reactive substances (TBARS) in heart were estimated to assess the status of heart muscle. An elevation of the levels of CK, AST, GSH, and TBARS was observed following doxorubicin treatment. Parallel experiments with a pretreatment of propolis significantly reduced the levels of these parameters . Biochemical observations were supplemented by histopathological examination of heart sections. The protective effect of propolis was compared with that of rutin, a known cardioprotective flavonoid. The study demonstrates the cardioprotective effect of propolis in doxorubicin-induced experimental cardiotoxicity. PMID- 8612724 TI - Nuclear accumulation of MDM2 protein in well-differentiated papillary thyroid carcinomas. AB - Papillary thyroid cancer is the most common endocrine malignancy. Of all solid cancers presenting in adults, papillary thyroid cancer generally carries the best long-term prognosis. However, very little is understood about the molecular pathogenesis of this neoplasm. We recently hypothesized that increased nuclear levels of MDM2 protein might occur in well-differentiated papillary thyroid carcinomas (Gerasimov et al., Exp. Mol. Pathol. 62, 52-62, 1995). MDM2 is known to complex with and inactive the p53 tumor suppressor protein. Since p53 inactivation by gene mutation has an established role in the pathogenesis of undifferentiated (anaplastic) thyroid carcinoma, we reasoned that abrogation of p53 function by nuclear MDM2 protein accumulation might participate in the pathogenesis of certain well-differentiated thyroid cancers such as papillary cancer. In the present report we present the first direct evidence of MDM2 protein accumulation in the nuclei of papillary thyroid carcinoma cells in a subset of tumors. Using the IF-2 monoclonal antibody, which reacts specifically with human MDM2 protein, we studied 24 well-differentiated papillary thyroid carcinomas and 26 benign lesions (nodular goiters, adenomas, thyroiditis). Nuclear staining was quantitated using the CAS computerized image analysis system. We found positive nuclear MDM2 immunoreactivity in 8 (33%) of the carcinomas. In contrast, MDM2 staining was negative in all benign lesions (P = 0.001, two-tailed Fisher exact test). Normal thyroid tissue was also negative. These data suggest that nuclear accumulation of MDM2 protein might be implicated in the pathogenesis of a subset of papillary carcinomas. Further studies to investigate this possibility are warranted. PMID- 8612725 TI - Tissue factor expression during coculture of endothelial cells and monocytes. AB - The role of monocytes as initiators of coagulation through the expression of tissue factor has been well documented in vitro, and the relationship of monocyte tissue factor to the thrombotic complications of atherosclerosis has been suggested. Tissue factor antigen has been identified in the plasma membranes of monocytes adherent to the vascular endothelium overlying atherosclerotic plaques and the presence of tissue factor in adherent mononuclear cells correlates with the polymerization of fibrin at these same sites. To further understand the relationship of cellular adhesion to tissue factor expression, human monocytes were cocultured for periods ranging from 30 min to 24 hr with endothelial cells isolated from human umbilical veins (HUVEC). Tissue factor antigen, as assayed by both ELISA and immunogold electron microscopy, was minimal on either monocytes or HUVEC maintained in homogeneous cultures or on the cells when cocultured for 1 hr or less. This was true whether the HUVEC were in a native state or if they had been stimulated with interleukin-1 (IL-1 beta) or lipopolysaccharide (LPS) prior to monocyte adhesion. Typically, less than 13% of the cells in short-term cocultures were positively labeled through anti-tissue factor immunogold microscopy. The level of tissue factor, however, was increased 3-fold above baseline when monocytes were cocultured with unstimulated HUVEC for 4 hr, and it was more than double this if the HUVEC had been exposed to IL-1 beta or LPS (7 fold increase). By 24 hr, the expression of tissue factor antigen was nearly 50 fold higher in cocultures involving stimulated HUVEC, and at later times greater than 70% of the cells were labeled with immunogold. Through the use of quantitative immunogold electron microscopy, the increase in tissue factor was most pronounced on monocytes which had three times greater increase in tissue factor than HUVEC in the same cultures. These studies document the stimulation of tissue factor expression by monocytes upon coculture with endothelial cells, and the data document an enhancement of this coculture effect upon HUVEC stimulation with cytokines. These observations have relevance to atherosclerotic disease by suggesting that interaction of monocytes with dysfunctional endothelial cells overlying atherosclerotic plaques would be sufficient to induce tissue factor and by so doing predispose to localized thrombotic events. PMID- 8612726 TI - Regulation of gene expression at a distance: the hypothetical role of regulatory protein-mediated topological changes of DNA. AB - A theoretical model is presented that a regulatory protein may activate the transcription of a promoter by interacting with a single remote operator. In response to an inducer molecule the regulatory protein bound to the operator undergoes a conformational change, and might mediate a B to Z-DNA conversion of the operator. This transition would remove both helical turns and supercoils from the intervening region between the operator and the promoter, resulting in the correct spatial arrangement of the -10 and -35 hexamers of the promoter, which therefore can be efficiently transcribed. PMID- 8612727 TI - Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase. AB - The effect of various oxidants on bovine aortic prostacyclin synthase was tested with 14C-labelled prostaglandin endoperoxide as substrate. No sensitivity against hydrogen peroxide, superoxide or hydroxyl radicals was observed but hypochlorite inhibited with an IC50 value of 7 microM. Among the reactive nitrogen species nitric oxide and nitrogen dioxide radicals were ineffective, but peroxynitrite irreversibly blocked prostacyclin biosynthesis with an IC50 value of 50 nM. Peroxynitrite acted within seconds whereas hypochlorite required up to 30 min for completion. Simultaneous generation of nitric oxide and superoxide also caused inhibition which suggested that under pathological conditions like ischemia reperfusion not only the vasodilatory effects of nitric oxide but also those of prostacyclin could be eliminated. PMID- 8612728 TI - Immunolocalization of X-arrestin in human cone photoreceptors. AB - X-arrestin is a recently identified retina-specific gene of unknown function. Affinity-purified anti-peptide antibody to human X-arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an approximately 47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X-arrestin immunoreactivity was compared with immunolabeling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S antigen. The results showed that X-arrestin is expressed in red-, green- and blue sensitive cones in the human retina. PMID- 8612729 TI - Specific association of phosphatidylinositol 3-kinase with the protooncogene product Cbl in Fc gamma receptor signaling. AB - A tyrosine-phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, when the enzyme was essentially activated upon ligand engagement of Fc gamma receptors (Fc gamma R) leading to engulfment of IgG-coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732 22737]. Here, the 115-kDa protein is identified as the product of human c-cbl protooncogene. Cross-linking of Fc gamma RII on the surface of THP-1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3 kinase that was immunoprecipitated with the anti-Cbl or the anti-phosphotyrosine antibody, and (c) specific association of Cbl with p85. Thus, Cbl functions in phagocytes as a result of its association with PI 3-kinase in response to Fc gamma R ligation. PMID- 8612730 TI - The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase. AB - The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1- mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa3 and of a cytochrome c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex. PMID- 8612731 TI - Facilitator oligonucleotides increase ribozyme RNA binding to full-length RNA substrates in vitro. AB - Primer extension arrest (PEA) studies have demonstrated that antisense oligonucleotides (beta 112C, beta 114C), which lie upstream of a ribozyme targeted to beta-amyloid peptide precursor (beta APP) mRNA, but not sense oligonucleotides (beta 112S, beta 116S) or a scrambled oligonucleotide, beta 116 M, affect ribozyme-mediated cleavage in vitro. Substrate dissociation experiments revealed that the ribozyme binding site in this mRNA was masked; PEA kinetics showed the association of the ribozyme and substrate was enhanced by antisense oligonucleotide binding. These studies suggest that masked ribozyme cleavage sites that may occur in disease-causing mRNAs can be targeted for degradation using "facilitator" oligonucleotides. PMID- 8612732 TI - Regulation of the H+/e- stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios. AB - This paper describes the effect of intramitochondrial ATP/ADP ratios on the H+/e- stoichiometry of reconstituted cytochrome c oxidase (COX) from bovine heart. At 100% intraliposomal ATP the H+/e- stoichiometry of the reconstituted enzyme is decreased to half of the value measured below 98% intraliposomal ATP (above 2% ADP), while it remains constant up to 100% ADP. The decrease is obtained with different COX preparations, independent of the absolute value of the H+/e- stoichiometry. Decrease of H+/e- stoichiometry is prevented by preincubation of the enzyme with a tissue-specific monoclonal antibody to subunit VIa-H (heart type). Tissue-specific regulation of the efficiency of energy transduction in COX of muscle mitochondria could have a physiological function in maintaining the body temperature at rest or sleep, i.e. at low ATP expenditure. PMID- 8612734 TI - Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti-neutrophil cytoplasmic autoantibodies. AB - Using the baculovirus/insect cells system, we have expressed a recombinant proteinase 3 (PR3) -- the neutrophil-derived serine protease autoantigen in Wegener's granulomatosis -- as a glycosylated intracellular and membrane associated protein. Oligosaccharides accounted for the difference in molecular weights between recombinant (34 kDa) and neutrophil-PR3 (29 kDa). Whereas rabbit anti-PR3 IgG recognized both recombinant and neutrophil-derived PR3, autoantibodies from Wegener patient sera recognized only neutrophil-derived PR3. Although oligosaccharides were not involved in PR3 epitope recognition, autoantibodies did not recognize the amino acid primary structure of recombinant PR3. Improper disulfide bond formation and/or lack of post-translational events in insect cells, may affect the conformation and/or lack of post-translational events in insect cells, may affect the conformation of PR3, precluding its reactivity with sera from WG patients. PMID- 8612733 TI - cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant. AB - A cDNA encoding variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full-length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gi proteins. Analysis of receptor transcripts by RT-PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors. PMID- 8612735 TI - An active-site cysteine of sorghum leaf NADP-malate dehydrogenase studied by site directed mutagenesis. AB - The chloroplast NADP-malate dehydrogenase is activated through the reduction of two different disulfides per subunit. The activated enzyme, as well as a permanently active mutant where all four regulatory cysteines were replaced are still sensitive to thiol reagents. This observation suggested the presence of an additional important cysteine at the active site. In an attempt to identify that cysteine, site-directed mutagenesis was performed on the cDNA encoding sorghum leaf NADP-malate dehydrogenase. The replacement of Cys-175 by an alanine yielded an enzyme whose sensitivity to thiol reagents was markedly decreased whereas its catalytic activity was enhanced. This finding suggests that Cys-175 has no catalytic function but is located close to the active site. PMID- 8612736 TI - The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo. AB - Purified flavohaemoglobin (HMP) of Escherichia coli reduces Fe(III) in a superoxide dismutase (SOD)-sensitive reaction, demonstrating superoxide anion generation during aerobic NADH oxidation. In vivo, sodA-lacZ fusion activity was increased 3-fold by introducing plasmid pPL341, containing the hmp gene, or by growth with paraquat. The effects were additive and SOXS-dependent. Thus HMP activity causes oxidative stress in vivo. Activities of sodA-lacZ and hmp-lacZ fusions were stimulated in a himA mutant, demonstrating repression of both promoters by integration host factor (IHF), but the effects of pPL341 on sodA lacZ activity were not due to titration of IHF by the hmp promoter. PMID- 8612737 TI - A link between ferritin gene expression and ribonucleotide reductase R2 protein, as demonstrated by retroviral vector mediated stable expression of R2 cDNA. AB - We have constructed a retroviral expression vector for the mammalian ribonucleotide reductase R2 component. Stable infectants, which express a myc epitope tagged R2 protein from the vector cDNA were obtained and described for the first time. Cells containing the recombinant protein exhibited increased ribonucleotide reductase activity, and were resistant to the antitumour agent hydroxyurea, which targets the R2 component of ribonucleotide reductase. Furthermore, a direct link between ferritin gene expression and R2 protein was observed, since cells containing vector expressed recombinant R2 protein exhibited increased H-chain and L-chain ferritin gene expression. PMID- 8612738 TI - Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants. AB - The casein kinase from the Golgi apparatus of lactating mammary gland (GEF-CK) is distinct from ubiquitous 'casein kinases' termed protein kinases CK1 and CK2 and appears to define a family of secretory pathways protein kinases that phosphorylate seryl residues followed by an acidic residue at position +2. In this report we show that a new synthetic peptide substrate derived from beta casein (beta[28-40]) is suitable for the fast, efficient and selective monitoring of GEF-CK, being unaffected by CK1 and CK2, and we define the consensus sequence of this protein kinase as being Ser-Xaa-Glu/SerP, distinct from that of CK2 (Ser/Thr-X-X-Glu/Asp/SerP/TyrP). In particular, the failure to recognize Asp as crucial specificity determinant prevents the phosphorylation of the specific CK2 peptide substrate RRRADDSDDDDD by GEF-CK. Thus, peptide substrates are now available for the fast and specific monitoring of all the three classes of 'casein kinases', CK1, CK2 and GEF-CK. PMID- 8612739 TI - Crystallization and preliminary X-ray diffraction analysis of boar seminal plasma spermadhesin PSP-I/PSP-II, a heterodimer of two CUB domains. AB - Boar spermadhesin PSP-I/PSP-II (M(r) 29 000-30 000), a non-covalent heterodimer of two CUB domains, was crystallized in two crystal forms. Complete diffraction data sets for hexagonal (space group P6(1,5)22) and trigonal (space group P3(1,2)21) crystals have been collected up to 2.9 and 2.5 angstrom resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are a=b=87.2 angstrom, c=152.4 angstrom, and a=b=96.2 angstrom, c=70.8 angstrom, respectively. The calculated packing parameters (Vm) are 2.8 and 3.2 angstrom(3)/DA for the hexagonal and trigonal crystal forms, respectively, indicating that, in both cases, the asymmetric unit is constituted by one PSP I/PSP-II heterodimer. This paper reports the first crystals of a protein built up by a CUB domain architecture. PMID- 8612740 TI - Identification of a third yeast mitochondrial Tom protein with tetratrico peptide repeats. AB - The mitochondrial outer membrane contains a protein complex with at least eight subunits responsible for recognition and translocation of preproteins synthesized in the cytosol. Two subunits, the receptors Tom20 and Tom70, contain tetratrico peptide repeats that are thought to be involved in protein-protein interactions. We have identified Saccharomyces cerevisiae Tom72, a new Tom protein expressed at a low level. Tom72 is homologous to Tom 70, including seven tetratrico peptide repeats. Tom72 is targeted to the mitochondrial outer membrane, forms a large domain exposed to the cytosol and loosely associates with the translocase complex of the outer membrane. These results suggest that Tom72 represents a ninth, weakly expressed component of the preprotein translocase of the mitochondrial outer membrane. PMID- 8612741 TI - Phase specific association of heterotrimeric GTP-binding proteins with the actin based cytoskeleton during thrombin receptor-mediated platelet activation. AB - Subcellular distribution of heterotrimeric GTP-binding proteins during thrombin receptor-mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2 alpha and Gs alpha shows first phase translocation to the low-speed pellet (15000 x g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2 alpha, Gs alpha, Gq alpha, and G beta translocate to the low-speed pellet, depending on platelet aggregation. These translocations correlated with the reorganization of the actin-cytoskeleton and were inhibited by cytochalasin D. Reconstitution experiments also revealed that G proteins are associated with the actin-cytoskeleton during platelet activation. PMID- 8612742 TI - An essential tyrosine residue of Aspergillus polygalacturonase. AB - Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms. PMID- 8612743 TI - Succinyl phosphonate inhibits alpha-ketoglutarate oxidative decarboxylation, catalyzed by alpha-ketoglutarate dehydrogenase complexes from E. coli and pigeon breast muscle. AB - Effects of a set of alpha-ketoglutarate phosphoanalogues on the activity of alpha ketoglutarate dehydrogenase (EC 1.2.4.2) complexes from E. coli and pigeon breast muscle, as well as on alpha-ketoglutarate dehydrogenase isolated from the pigeon breast muscle, have been studied. alpha-Ketoglutarate phosphoanalogues (succinyl phosphonate and its monomethyl ester) were found to be effective inhibitors of alpha-ketoglutarate oxidative decarboxylation, catalyzed by both muscle and bacterial alpha-ketoglutarate dehydrogenase complexes, as well as muscle alpha ketoglutarate dehydrogenase. The ability of glutamate phosphoanalogues to inhibit alpha-ketoglutarate oxidative decarboxylation has been shown in E. coli extract and a model system. PMID- 8612744 TI - Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of alpha subunit of E. coli-F(0)F(1). AB - Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E. coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36-->E, alpha-R40-->K) or unrelated amino acids (alpha D-36-->K, alpha R40-->G), respectively, by employing oligonucleotide-directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36-->K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36-->K mutation. In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine beta-R52. The effect of alpha-D36- >K replacement on catalysis and coupling thus may be due to an electrostatic repulsive effect in the crown region which alters the alpha and beta interaction. PMID- 8612745 TI - Antibacterial activity of peptides homologous to a loop region in human lactoferrin. AB - Human lactoferrin contains a 46 residue sequence named lactoferricin H thought to be responsible for its antimicrobial properties. Synthetic peptides HLT1, corresponding to the loop region of human lactoferricin (FQWQR-NMRKVRGPPVS) and HLT2, corresponding to its charged portion (FQWQRNMRKVR), exerted significant antibacterial effects against E. coli serotype O111 strains NCTC 8007 and ML35. The corresponding sequences in native human lactoferrin were shown to adopt a charged helix and hydrophobic tail within the N-lobe remote from the iron binding site. Sequence similarities between lactoferricin and dermaseptin and magainins suggest that lactoferricin may act as an amphipathic alpha helix. PMID- 8612746 TI - Inactivation of an NADPH-dependent succinic semialdehyde reductase by o phthalaldehyde. AB - Incubation of an NADPH-dependent succinic semi-aldehyde reductase from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics with the second-order rate constant of 28 M(-1) s(-1). The inactivation was prevented by preincubation of the enzymes with NADPH, but not by succinic semialdehyde. There was a linear relationship between isoindole formation and the loss of enzyme activity. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the formation of one isoindole derivative per molecule of enzyme, which was formed from the reaction of cysteine and lysine residues with o phthalaldehyde at or near the enzyme active site. PMID- 8612747 TI - Rabbit translation elongation factor 1 alpha stimulates the activity of homologous aminoacyl-tRNA synthetase. AB - Functional and structural sequestration of aminoacyl-tRNA has been recently found in eukaryotic cells and the aminoacyl-tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964-968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl-tRNA synthetase and homologous translation elongation factor 1 alpha (EF-1 alpha), the proteins which may play a role of sequential components involved in the transfer of the aminoacyl-tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl-tRNA synthetase activity by EF-1 alpha is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl-tRNA channeling process during eukaryotic protein synthesis. PMID- 8612748 TI - An oocytic membrane receptor for biotin-binding protein. AB - The chicken oocyte accumulates a biotin-binding protein (BBP) in the yolk that is distinct from the avidin in the 'egg white'. An identical BBP to that of the yolk is also present in the circulation of the laying hen. We report the first evidence for the existence of a BBP receptor in the oocyte vitelline membrane. Reduction of the 100 kDa receptor results in loss of BBP-binding activity; this suggests that a disulfide bonded region of the receptor is necessary for maintaining BBP-binding activity. We show further that the levels of serum BBP are strictly dependent on the presence of estrogen. As expected, BBP is not detected in significant quantities in rooster serum. Thus, these results suggest that circulatory BBP, like other estrogen-dependent components of serum, has a cognate binding activity on the oocyte membrane that may mediate its endocytosis. PMID- 8612749 TI - Plant chitinases use two different hydrolytic mechanisms. AB - Bacterial, fungal, animal, and some plant chitinases form family 18 of glycosyl hydrolases. Most plant chitinases form the family 19. While some chitinases also have lysozyme activity, animal lysozymes belong to different families. For glycosyl hydrolases, two reaction mechanisms are possible, leading to either retention or inversion of the anomeric configuration. We analyzed by HPLC the stereochemical outcome of the hydrolysis catalyzed by cucumber and bean chitinases, belonging to families 18 and 19, respectively. Cucumber chitinase used the retaining mechanism as known for bacterial chitinases and hen egg white lysozyme for which the mechanism has been determined. In contrast, bean chitinase catalyzed the hydrolysis of chitooligosaccharides with overall inversion of anomeric configuration. PMID- 8612751 TI - Sphingosine-1-phosphate inhibits actin nucleation and pseudopodium formation to control cell motility of mouse melanoma cells. AB - Sphingosine-1-phosphate (Sph-1-P), the initial product of sphingosine (Sph) catabolism, has been reported to inhibit motility of mouse melanoma B16/F1 and other types of cells at very low concentrations (10-100 nM). Sph-1-P (100 nM-1 microM) inhibited pseudopodium formation by blocking polymerization and reorganization of actin filaments in newly formed pseudopodia, and reduced F actin by approximately 25% in F1 cells. A pyrene-labeled actin nucleation assay revealed that Sph-1-P (100 nM) inhibits actin nucleation mediated by F1 cell plasma membranes. These results suggest that Sph-1-P interacts with molecules associated with actin nucleation to inhibit reorganization of pseudopodium formation and cell motility. PMID- 8612750 TI - Antioxidant properties of omeprazole. AB - Potential antioxidant properties of therapeutically achievable concentrations of the protonated, active form of omeprazole (OM) were investigated in vitro at specific acidic pH values to mimic intragastric conditions in the clinical setting. We found that OM is a powerful scavenger of hypochlorous acid (HOCl) even at a drug concentration of 10 microM at pH 5.3 or 3.5. This effect is also evident in the presence of the physiological HOCl scavenger ascorbate. Moreover, 10 and 50 microM OM inhibit significantly both iron- and copper-driven oxidant damage at pH 5.3 and 3.5, respectively. Since oxidative stress is involved the gastric injury of peptic ulcer and gastritis, it may be hypothesized that some therapeutical effects of OM could also be related to its antioxidant properties. PMID- 8612752 TI - Dictyostelium discoideum contains a family of calmodulin-related EF-hand proteins that are developmentally regulated. AB - A full-length genomic DNA fragment that codes for a novel EF-hand protein Dictyostelium discoideum was cloned and sequenced. The protein is composed of 168 amino acids and contains four consensus sequences that are typical for (Ca2+) binding EF-hand domains. The protein sequence exhibits only minor similarities to other calmodulin-type proteins from Dictyostelium. The genomic DNA harbors two short introns; their positions suggest that the gene is unrelated to the EF-hand proteins from the calmodulin group. Northern blot analysis showed that the mRNA level was significantly increased during development. Polyclonal antibodies raised against the recombinant protein recognized in Western blots a protein of about 20 kDa. Like the mRNA, also the protein was more abundant in developing cells. Overlay experiments with 45Ca2+ indicated that the EF-hands in fact have (Ca2+)-binding activity. The recent description of CBP1, another calmodulin-type Dictyostelium protein that is upregulated during development [Coukell et al. (1995) FEBS Lett. 362, 342-346], suggests that D. discoideum contains a family of EF-hand proteins that have specific functions during distinct steps of development. We therefore designate the protein described in this report as CBP2. PMID- 8612753 TI - The micelle to vesicle transition of lipids and detergents in the presence of a membrane protein: towards a rationale for 2D crystallization. AB - The assembly of two-dimensional membrane protein crystals in the presence of lipids was analyzed with quasielastic light scattering and electron microscopy. Mixtures of detergent-solubilized lipids and/or proteins were submitted to slow or rapid dilution while measuring the hydrodynamic radii of the aggregates. Lipids alone exhibited lambda-shaped dilution curves with intermediate rod-shaped particles that converted into small vesicles. Depending on the protein-protein and protein-lipid interactions, detergent-solubilized protein-lipid mixtures showed a sharp transition from micelles to large densely packed proteoliposomes. Electron microscopy revealed that formation of crystals occurred shortly after this phase transition. PMID- 8612754 TI - Chemical and functional studies on the importance of purple membrane lipids in bacteriorhodopsin photocycle behavior. AB - In native purple membrane (PM), there are approximately 1 squalene, 2 glycolipid sulfate (GLS), and 6 phospholipid (PL) molecules per bacteriorhodopsin (BR) monomer. Brief (approximately 2 min) exposure to 0.1% Triton X-100 removes about 25%, 20%, and 6% of squalenes, GLS, and PL, respectively (this paper) while causing profound changes in the BR photocycle, including the loss of 'photocooperativity'. The BR photocycle in Triton-treated PM can be restored to near normal behavior by reconstitution with native PM lipids. Isolated squalenes are not effective whereas PL alone partially restores normal photocycle characteristics. PMID- 8612755 TI - Solubility of artificial proteins with random sequences. AB - A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3-42 and RP3-45 and insoluble RP3-70 were purified. The solubility of the purified form is the same as that in the cell lysate. PMID- 8612756 TI - Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. AB - Suspension-cultured cells of Arabidopsis thaliana generated active oxygen species (AOS) (measured by luminol-dependent chemiluminescence) following challenge with the bacterial protein elicitor harpin or the protein kinase activator phorbol 12 myristate 13-acetate. These responses were blocked by inhibitors of superoxide dismutase (SOD), NADPH oxidase and protein kinase. Harpin treatment also resulted in an increase in cell death, a response reduced by inhibitors of AOS generation or AOS scavengers. Extracellular SOD activity was found to be present in cell culture medium. Immunoblotting of Arabidopsis extracts revealed the presence of proteins immunologically related to the human neutrophil NADPH oxidase complex, and cell-free reconstitution assays showed that human neutrophil cytosol combined with Arabidopsis membranes could initiate superoxide generation. These data suggest that the enzyme catalysing the generation of superoxide in elicited Arabidopsis cells is similar to the mammalian NADPH oxidase and that a signalling cascade leading to AOS generation involves protein phosphorylation. PMID- 8612757 TI - Redox control of gene expression involving iron-sulfur proteins. Change of oxidation-state or assembly/disassembly of Fe-S clusters? AB - Attention is drawn to a mechanism of redox control of gene expression involving Fe-S proteins which depends on the disassembly and reassembly of Fe-S clusters rather than a change in oxidation state. Iron Regulatory Protein (IRP)/aconitase and FNR are discussed as examples for such a mechanism. PMID- 8612758 TI - Aspirin-DNA interaction studied by FTIR and laser Raman difference spectroscopy. AB - The interaction of calf-thymus DNA with aspirin is investigated in aqueous solution at pH 7-6 with drug/DNA (phosphate) molar ratios of r = 1/40, 1/20, 1/10, 1/5, 1/2, 1 and 2. Fourier transform infrared (FTIR) and laser Raman difference spectroscopy are used to determine drug binding sites, sequence preference and DNA secondary structure, as well as the structural variations of aspirin-DNA complexes in aqueous solution. Spectroscopic evidence showed that at low aspirin concentration (r =1/40), drug-DNA interaction is mainly through the backbone PO2 groups and the A-T base pairs. Such interaction largely perturbs the phosphate vibration at 1222 cm(-1) and the A-T bands at 1663 and 1609 cm(-1) with no major helix destabilization. At higher drug concentration (r > 1/20), the participation of the G-C bases in drug-DNA complexation was evident by strong perturbations of the guanine and cytosine vibrations at 1717 and 1494 cm(-1), with a partial helix destabilization. A major alteration of the B-DNA structure towards A-DNA occurs on drug complexation. The aspirin interaction was through anion CO and COOCH3 donor atoms with those of the backbone PO2 group and DNA bases donor sites (directly or indirectly via H2O molecules). PMID- 8612759 TI - Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes. AB - Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo-3 signals were similar. During electrical pacing, transients of Fluo-3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation-contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically competent to regulate mitochondrial metabolism on a beat-to-beat basis. PMID- 8612760 TI - Generation of VV-hemorphin-7 from globin by peritoneal macrophages. AB - Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D. Analysis of resulting peptides, by reversed-phase high-performance liquid chromatography (RP-HPLC), showed the release of a bioactive peptide, VV-hemorphin 7. When a carboxyl proteinase inhibitor such as pepstatin A was added, no hemorphin was generated. Our results clearly demonstrated that VV-hemorphin-7 generation was principally due to cathepsin D. This study allowed us to hypothesize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages. PMID- 8612761 TI - Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR--an activator of AMP-activated protein kinase. AB - We determined whether the cell permeable molecule AICAR, whose metabolite activates AMP-activated protein kinase (AMPK) in cells, affected glycogen metabolism in rat soleus muscle preparations in vitro. The basal and insulin stimulated rates of radiochemical lactate formation, net lactate release and glycogen synthesis were determined. AICAR stimulated net lactate release (but not radiochemical lactate formation) only at a basal concentration of insulin. An increased rate of glycogenolysis was the likely cause of increased net lactate release as glycogen phosphorylase activity was significantly increased by AICAR. AICAR-stimulated net lactate release and phosphorylase activity were potently inhibited by insulin. PMID- 8612762 TI - Increase in activin beta A mRNA in rat hippocampus during long-term potentiation. AB - We have used mRNA differential display to isolate genes that are induced by neural activity in rat hippocampus. One of these encodes activin beta A subunit. Convulsive seizure caused by kainate significantly induced the expression of activin beta A mRNA. Furthermore high frequency stimulation (HFS) of perforant pathway, which produced a persistent long-term potentiation (LTP) (>10 h), caused a marked increase at 3 h in the level of activin beta A mRNA at the dentate gyrus of urethane-anesthetized rat. The increase was NMDA receptor-dependent. By contrast the level of inhibin alpha mRNA was not changed following the induction of LTP. THe results suggest a role for activin in maintenance of neural plasticity in the adult brain. PMID- 8612763 TI - Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP dependent protein kinase. AB - The critical function of the neurofibromatosis type 1 (NF1) gene product (neurofibromin) is not well defined except that neurofibromin has homology with a family of the GTPase-activating proteins (GAPs). In this study, we confirmed that neurofibromin is constitutively phosphorylated and detected kinase activities which specifically phosphorylate the cysteine/serine-rich domain and the C terminal domain of the neurofibromin in cell lysate. In vitro and in-gel kinase assays strongly indicated that cAMP-dependent protein kinase (PKA) is a candidate for the neurofibromin kinase. THe biological significance of the phosphorylation of neurofibromin is unclear at present, but we speculate that neurofibromin plays a crucial role in cellular function since it links the two major cellular pathways which are the GAP-ras and PKA-associated signals. PMID- 8612764 TI - Expression of recombinant pro-neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by 'prohormone thiol protease' (PTP). AB - The preference of the 'prohormone thiol protease' (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro-neuropeptide Y (pro NPY), proopiomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro-NPY. The processing of POMC by PTP was minimal. These results demonstrate PTP's preference for certain prohormone substrates. PMID- 8612765 TI - Localization and expression of the closely linked cyanelle genes for RNase P RNA and two transfer RNAs. AB - The genomic region encoding the RNA subunit of the cyanelle RNase P has been characterized. rnpB, which has no homologue in chloroplasts, is flanked by two tRNA genes on the complementary DNA strand. Transcriptional control elements of all three genes have been experimentally determined. Comparison of the sequenced region with the corresponding loci of chloroplast genomes from vascular plants suggests that major inversions may have led to a possible loss or severe truncation of the RNase P RNA coding region during the course of plastid evolution. PMID- 8612766 TI - Cytoplasmic topography of focal contacts. AB - To investigate the structure of focal contacts, the cytoplasmic faces of fibroblast membranes were examined in solution by scanning force and immunofluorescence microscopy. Focal contacts were identified in scanning force topographs by correlation with fluorescence images. Finer details were resolved in topographs of the focal contacts than in fluorescence micrographs. Increased separation of ventral plasma membranes from the substrate correlated with the duration of cell culture. The cytoplasmic projections of the focal contacts also increased with the cell culture period. These changes accompanied lateral spreading of fibroblasts during a period of several hours after seeding cells in culture medium. PMID- 8612767 TI - Evidence for trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein. AB - Analysis of the chromophore p-coumaric acid, extracted from the ground state and the long-lived blue-shifted photocycle intermediate of photoactive yellow protein, shows that the chromophore is reversibly converted from the trans to the cis configuration, while progressing through the photocycle. The detection of the trans and cis isomers was carried out by high performance capillary zone electrophoresis and further substantiated by 1H NMR spectroscopy. The data presented here establish the photo-isomerization of the vinyl double bond in the chromophore as the photochemical basis for the photocycle of photoactive yellow protein, a eubacterial photosensory protein. A similar isomerization process occurs in the structurally very different sensory rhodopsins, offering an explanation for the strong spectroscopic similarities between photoactive yellow protein and the sensory rhodopsins. This is the first demonstration of light induced isomerization of a chromophore double bond as the photochemical basis for photosensing in the domain of Bacteria. PMID- 8612768 TI - Comparative studies of gamma-interferon receptor-like proteins of variola major and variola minor viruses. AB - To study specific properties of the human gamma-interferon (gamma-IFN) receptor like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E. coli cells have been constructed. The recombinant viral gamma-IFN receptor-like proteins have been found to have high interferon-neutralising activity with regard to human gamma-IFN but not murine gamma-IFN and human alpha IFN. The variola major and variola minor proteins under study do not differ in the efficiency of human gamma-IFN antiviral activity inhibition. PMID- 8612769 TI - A calcium switch for the functional coupling between alpha (hslo) and beta subunits (KV,Ca beta) of maxi K channels. AB - KV,Ca beta subunit dramatically increases the apparent calcium sensitivity of the alpha subunit of MaxiK channels when probed in the micromolar [Ca2+]i range. Analysis in a wide range of [Ca2+]i revealed that this functional coupling is exquisitely modulated by [Ca2+]i. Ca2+ ions switch MaxiK alpha+beta complex into a functionally coupled state at concentrations beyond resting [Ca2+]i. At [Ca2+] < or = 100 nM, MaxiK activity becomes independent of Ca2+, is purely voltage activated, and its functional coupling with its beta subunit is released. The functional switch develops at [Ca2+]i that occur during cellular excitation, providing the molecular basis of how MaxiK channels regulate smooth muscle excitability and neurotransmitter release. PMID- 8612770 TI - Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast. AB - The yeast PMR2/ENA1 gene encodes an ATPase involved in sodium extrusion and induced by NaCl. At low salt concentrations (0.3 M) induction is mediated by the HOG-MAP kinase pathway, a system activated by non-specific osmotic stress. At high salt concentrations (0.8 M) induction is mediated by the protein phosphatase calcineurin and is specific for sodium. Protein kinase A and Sis2p/Hal3p modulate PMR2/ENA1 expression as negative and positive factors, respectively but Sis2p/Hal3p does not participate in the transduction of the salt signal. Salt stress decreases the level of cAMP and the resulting decrease in protein kinase A activity may contribute to HOG-mediated induction. PMID- 8612771 TI - Identification of RII-binding proteins in the mollusc Mytilus galloprovincialis. AB - Several proteins with M(r) > 70 kDa from various tissues of the sea mussel Mytilus galloprovincialis were specifically recognized in vitro by the regulatory subunit (type RII alpha) of cAMP-dependent protein kinase (cAPK) from porcine heart. However, none of these proteins interacted with the regulatory subunit of cAPK from the mollusc itself. The results suggest that, unlike mammalian RII, regulatory subunit from mussel lacks the specific residues responsible for interaction with R-binding proteins. Consequently, the identified molluscan RII alpha-binding proteins should play a distinct role from cAPK anchoring. PMID- 8612773 TI - Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL-60 cells. AB - Didemnin B induces rapid apoptosis in human promyeloid HL-60 cells with an optimal concentration of 1 microM (Grubb et al. (1995) Biochem. Biophys. Res. Commum. 215, 1130-1136), but little is known about how it does so. In order to determine whether protein tyrosine phosphorylation is involved in this rapid induction of apoptosis, HL-60 cells were pre-treated with tyrosine kinase inhibitors for 1 h before didemnin B treatment. Genistein, 2,5-dihydroxycinnamic acid methyl ester, and a range of tyrphostins inhibit didemnin B-induced apoptotic morphology in a concentration-dependent manner. DNA fragmentation induced by didemnin B is also inhibited by genistein, 2,5-dihydroxycinnamic acid methyl ester, and tyrphostins. PMID- 8612772 TI - Characterization of a short unique sequence in the yeast HO gene promoter that regulates HO transcription in a SIN1 dependent manner. AB - Recently it has become clear that general chromatin proteins as well as sequence specific DNA binding proteins are important in the control of gene expression. SIN1 in Saccharomyces cerevisiae is a chromatin component that regulates the transcription of a family of genes. Previously, we identified a 32 bp unique sequence (here termed XBS) in the promoter of one of those genes, HO, which specifically binds a protein that interacts with SIN1. We also found that this sequence can function as a weak UAS in a heterologous promoter that is dependent on the presence of SIN1. Here we report a relationship between the level of HO expression and the presence of the short sequence in situ in the HO gene. By comparing the expression of HO from wild type or XBS deleted HO promoters, we concluded that XBS serves as a weak UAS in situ in the HO gene, that it influences HO transcription via the SWI/SNF complex, and that sequences other than the XBS mediate the effect of SIN1 on HO transcription. In addition, we show that a portion of the SIN1 protein that has sequence similarity to mammalian HMG1 preferentially binds the XBS. PMID- 8612774 TI - Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I-binding loop: energy transfer from tryptophan to AEDANS. AB - Alteration of the actin polypeptide chain within the DNase I-binding loop by cleavage with E. coli A2 protease or subtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys 374. Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can be energy transfer donors. It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin. These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer. We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling. PMID- 8612776 TI - Peroxisomal remnants in peroxisome-deficient mutants of the yeast Hansenula polymorpha [corrected]. AB - We have analyzed the presence of peroxisomal remnants ('ghosts') in three peroxisome-deficient (per) mutants of the yeast Hansenula polymorpha, namely delta per4, delta per5 and delta per10. Under peroxisome-inducing growth conditions peroxisomal membrane proteins (PMPs) were normally synthesized in cells of these mutants. In addition, these cells contained clusters of small membraneous vesicles, which were absent in cells grown under peroxisome repressing growth conditions. These structures displayed typical peroxisomal properties in that they proliferated upon overproduction of Per8p, the H. polymorpha peroxisome proliferation factor. Moreover, in delta per4 and delta per5 these vesicles were susceptible to glucose-induced proteolytic degradation. PMID- 8612775 TI - 20S proteasome from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences. AB - 20S proteasomes of tissues from LMP7 knock out mice which show reduced MHC class I restricted antigen presentation were analyzed with regard to their subunit composition, peptide hydrolyzing activity and their ability to cleave a synthetic 25-mer polypeptide. LMP7 deficiency results in an enhanced incorporation of subunit MB1 and in a 2-3.8-fold increase in Vmax for the Suc-LLVY-MCA hydrolyzing activity. Since LMP7 deficiency also affects the cleavage site preference of 20S proteasomes the reduced MHC class I antigen presentation of LMP7 knock out mice is most likely due to an impairment in peptide generation. PMID- 8612777 TI - Production and crystallization of MHC class I B allele single peptide complexes. AB - Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution. PMID- 8612779 TI - Femtosecond kinetics of electron transfer in the bacteriochlorophyll(M)-modified reaction centers from Rhodobacter sphaeroides (R-26). AB - Formation of the vibronic wavepacket by 90-fs excitation of the primary electron donor P in bacteriochlorophyll(M)-modified reaction centers is shown to induce nuclear motions accompanied by (1) oscillation of the stimulated emission from excited primary electron donor P* and (2) wavepacket motions leading to electron transfer at 293 K from P* to bacteriochlorophyll (B(L)) and then to bacteriopheophytin (H(L)). The latter motions have low frequency (about 15 cm-1) and are related to protein-nuclear motions which are along the reaction coordinate. When the wavepacket approaches the intersection of the reactant (P*B(L)) and product (P+B(L)-) potential energy surfaces (approximately 1.5 ps delay), about 60% of P* is converted to the P+B(L)- state. The P+H(L)- state formation is delayed by approximately 2 ps with respect to that of P+B(L)-. It is suggested that the wavepacket is transferred to and moves also slowly on the P+B(L)- potential energy surface and approaches the intersection of the surfaces of P+B(L)- and P+H(L)- within approximately 2 ps (approximately 8 cm-1), indicating the electron transfer to H(L). PMID- 8612778 TI - Characterization of the dystrophin-syntrophin interaction using the two-hybrid system in yeast. AB - The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin, a cytoplasmic component of the dystrophin glycoprotein complex, by in vitro biochemical studies such as overlay assay or immunoprecipitation. Using the two-hybrid system, we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin, therefore confirming for the first time this interaction via an in vivo approach. In addition, we have reduced the interaction domain to the distal half of alpha1 syntrophin. PMID- 8612780 TI - Time-resolved Fourier-transform infrared studies of the cytochrome P-450cam carbonmonoxide complex bound with (1R)-camphor and (1S)-camphor substrate. AB - The CO-binding reaction of cytochrome P-450cam bound with (1R)-camphor and (1S) camphor are compared in the temperature region of 210-260 K using time-resolved Fourier-transform infrared spectroscopy with the CO stretch vibration as spectroscopic probe. For (1S)-camphor as substrate the association of CO is slowed down by a factor of 2, while the dissociation is accelerated by a factor of 3. The CO complex for the (1S)-camphor-bound P-450 is less stabilized (deltaG= 22 kJ/mol) compared to the natural substrate (1R)-camphor (deltaG=-30 kJ/mol). The data are interpreted by a smaller change of the mobility of the (1S)-camphor due to CO binding as compared to (1R)-camphor, which would indicate a higher mobility of (1S)-camphor already in the CO free reduced form of P-450cam. The higher mobility of (1S)-camphor in the heme pocket might explain the increased uncoupling rate (hydrogen peroxide formation) of 11% [Maryniak et al. (1993) Tetrahedron 49, 9373-9384] during the P-450cam catalyzed hydroxylation compared to 3% for the conversion of (1R)-camphor. PMID- 8612781 TI - Attempts to convert chymotrypsin to trypsin. AB - Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin --> trypsin specificity conversion. PMID- 8612782 TI - Stimulation of human peripheral blood lymphocytes by bioactive peptides derived from bovine milk proteins. AB - The in vitro modulation of the proliferation of human peripheral blood lymphocytes by different synthetic peptides derived from milk proteins was investigated. Therefore, proliferation changes were followed up after incorporation of BrdU into the DNA, and the influence on protein biosynthesis was measured using the [3H]leucine incorporation test. Tyr-Gly and Tyr-Gly-Gly significantly enhanced (maximal 90 and 35%, respectively) the proliferation of PBL. For beta-casomorphin-7 and beta-casomorphin-10,lymphocyte proliferation was suppressed at lower concentrations, but stimulated at higher concentrations (> or = 10(-7) mol/l). Protein synthesis was stimulated (maxima at 25%) only with Tyr Gly and Tyr-Gly-Gly. The findings point to a need for further studies on the possible function of peptides derived from milk proteins as orally bioavailable immunopotentiatory compounds. PMID- 8612783 TI - Isolation of a novel gene down-regulated by v-src. AB - We have isolated a novel gene which was expressed in normal rat cells, but completely suppressed in cells transformed by v-src. The molecularly cloned cDNA was about 1.8 kb in size, containing an open reading frame composed of 464 amino acid residues. DNA sequence analysis showed that there was no corresponding gene in the data bases. Besides the suppression of gene expression in the v-src transformed cells, its expression was also strongly suppressed in cells transformed by other oncogenes such as v-abl, v-fps, v-mos, v-sis, v-K-ras, and polyomavirus middle T, but not affected in cells transformed by human papillomavirus type 16 E6E7 and polyomavirus large T. We named the gene drs for a gene down-regulated by v-src. PMID- 8612784 TI - Novel effects of dendrotoxin homologues on subtypes of mammalian Kv1 potassium channels expressed in Xenopus oocytes. AB - We have examined the effects of two DTX homologues, toxin I and toxin K, on Kv1.1, Kv1.2 and Kv1.6 channels expressed in Xenopus oocytes. Toxin I blocked all three channels; in contrast, toxin K was selective for Kv1.1. Both toxins slowed channel activation and inactivation kinetics with 10 nM toxin I approximately doubling activation and inactivation time constants of Kv1.1. For the first time, we have demonstrated the selectivity of a DTX homologue for a single cloned Kv1 channel and suggest that these toxins may sterically hinder the conformational changes that occur during channel gating. PMID- 8612785 TI - Secondary structure of an armadillo single repeat from the APC protein. AB - The armadillo domain is a repeating sequence motif of a variety of proteins with different functions. Here we describe the structure of a synthetic single armadillo repeat solved by two-dimensional nuclear magnetic resonance spectroscopy. Our results indicate alpha-helical secondary structural elements in half of the residues. PMID- 8612786 TI - In vitro characterisation of Ro 46-8443, the first non-peptide antagonist selective for the endothelin ETB receptor. AB - We describe here Ro 46-8443, the first non-peptide endothelin ETB receptor selective antagonist. It displays up to 2000-fold selectivity for ETB receptors both in terms of binding inhibitory potency and functional inhibition. The observed parallel rightward shift of concentration-response curves with different antagonist concentrations is consistent with a competitive binding mode. Since R0 46-8443 selectively inhibits ETB receptor mediated responses, it is a valuable tool for clarifying the role of ETB receptors in pathology. PMID- 8612787 TI - The role of ETB receptors in normotensive and hypertensive rats as revealed by the non-peptide selective ETB receptor antagonist Ro 46-8443. AB - We used Ro 46-8443, non-peptidic antagonist selective of endothelin ETB receptors, to study the role of ETB receptors in rat hypertension models. In normotensive rats, Ro 46-8443 decreased blood pressure, but in SHR and DOCA rats, it induced a pressor effect, due to blockade of ETB-mediated release of nitric oxide since L-NAME prevented it. In rats rendered hypertensive by chronic L-NAME, Ro 46-8443 did not induce a pressor but depressor effect. Thus, in DOCA rats and SHR, Ro 46-8443 reveals a predominant influence of endothelial 'vasorelaxant' ETB receptors, while in normotensive rats the prevailing role of ETB receptors seems to be in mediating a vasoconstrictor tone. PMID- 8612788 TI - Legumin encoding sequences from the redwood family (Taxodiaceae) reveal precursors lacking the conserved Asn-Gly processing site. AB - We have cloned and sequenced two different cDNAs encoding legumins from Japanese red cedar (Cryptomeria japonica, Taxodiaceae). The derived amino acid sequences show between 34% and 55% identity when compared with legumins from angiosperms and from Pinaceae, respectively. The predicted precursors are unusual in that they contain potential glycosylation signals, and we have found the corresponding beta-polypeptides actually to be glycosylated. As most outstanding feature one of the precursors is lacking the Asn-Gly processing site which has been assumed to be highly conserved in legumin gene evolution. Legumin encoding sequences amplified from genomic DNA suggest that these unusual precursors are widespread if not ubiquitous in the Taxodiaceae family. From previous reports on legumin precursors with divergent processing sites, on the proteases involved in legumin precursor processing and from the results presented here it is concluded that the Asn-Gly processing site has been acquired rather than conserved during legumin gene evolution. PMID- 8612789 TI - Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein. AB - The AC2 protein of potato yellow mosaic geminivirus (PYMV) is by analogy with related geminiviruses thought to be a transcriptional activator protein. We have over-expressed the AC2 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. We have studied the interaction of the AC2 protein with DNA and from gel retardation assays shown that it binds both double-stranded (ds) and single-stranded (ss) DNA non-specifically. The binding to PYMV intergenic region ds DNA appeared to be independent of the presence of zinc ions and did not require the protein to be phosphorylated. PMID- 8612790 TI - Dual role of tropomyosin on chemically modified actin filaments from skeletal muscle. AB - Actin filaments copolymerized with both intact and chemically modified actin monomers restored their sliding activity when they were supplemented with tropomyosin extracted from skeletal muscle. In contrast, the ATPase activation of the copolymers was decreased when supplemented with tropomyosin. The results indicate that tropomyosin along with actin monomers may facilitate sliding activity of the entire actin filament but suppress ATPase activation of intact actin monomers themselves. Accordingly, tropomyosin molecules could be viewed as playing a dual role of both mechanical and chemical regulation of actin monomers. PMID- 8612791 TI - Calcium dependence of ryanodine-sensitive calcium channels from brain cortex endoplasmic reticulum. AB - Endoplasmic reticulum vesicles isolated from rat brain cortex and fused with lipid bilayers displayed ryanodine-sensitive calcium channels, with three cytoplasmic calcium dependences. A: Channels (n=5) stimulated by Ca2+ (K0.5=1.2 microM and nHill=1.9) and not inhibited up to 0.5 mM Ca2+. B: Channels (n=14) cooperatively activated (K0.5=6.9 microM and nHill=1.8), and inhibited by Ca2+ (K0.5=152 microM and nHill=1.8). C: Low Po (<0.1) channels (n=22), non cooperatively activated and inhibited with the same K0.5=26.3 microM Ca2+. These three types of responses to cytoplasmic [Ca2+] may underlie separate calcium release pathways in neurons of rat brain cortex. PMID- 8612792 TI - Acidic phospholipids inhibit the phospholipase D activity of rat brain neuronal nuclei. AB - An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 microM for PIP2 to 200 microM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity. PMID- 8612793 TI - Cloning and sequencing of the 5' region of the human glucose-6-phosphatase gene: transcriptional regulation by cAMP, insulin and glucocorticoids in H4IIE hepatoma cells. AB - We have cloned and sequenced the first 1.2 kb of the 5' region of the human glucose-6-phosphatase gene. Transfection of H4IIE hepatoma cells with the 1.2 kb fragment fused to a luciferase reporter gene demonstrated both basal and hormone responsive luciferase activity. Dexamethasone increased and insulin decreased luciferase activity. Insulin and dibutyryl cyclic AMP both significantly decreased activity in the presence of dexamethasone. PMID- 8612794 TI - Yeast aspartic protease 3 (Yap3) prefers substrates with basic residues in the P2, P1 and P2' positions. AB - The yeast aspartic protease Yap3 is localised to the secretory pathway and correctly cleaves pro-alpha-mating factor at its dibasic sites. We determined the specificity of Yap3 for mono-, di-, and multi-basic cleavage sites in the context of 15 residue synthetic proalbumin peptides. Yap3 cleaved after dibasic ArgArg and LysArg sites but not after monobasic Arg sites even when there was an additional arginine at -6 and/or -4. Yap3 did not cleave a tetra-arginine site and tri-basic sites (RRR and RRK) were poor substrates. Cleavage always occurred C-terminal to the last arginine in the di- or tri-basic sequence. The optimal cleavage site sequence was RR DR and this substrate was cleaved 8-9-fold faster than the normal RR DA sequence. In contrast to Kex2, Yap3 did not remove the propeptide from normal proalbumin or a range of natural or recombinant proalbumin variants. However at pH 4.0 Yap3 slowly cleaved proalbumin and albumin between domains 2 and 3. PMID- 8612795 TI - Hyaluronan-binding properties of human serum hemopexin. AB - Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan interacting hemopexin species. Hemopexin itself has no hyaluronidase activity. PMID- 8612796 TI - H2O2 renders cells accessible to lysis by exogenous phospholipase A2: a novel mechanism for cell damage in inflammatory processes. AB - Phospholipase A2 (PLA2) and H2O2, secreted from activated inflammatory cells, play a central role in the tissue damage occurring in inflammatory processes. However, while exogenous PLA2 alone does not cause cell lysis, it readily does so when acting with H2O2. We have found that H2O2 degrades cell surface proteoglycans, thus rendering the membrane PL accessible to hydrolysis by exogenous PLA2. This novel mechanism introduces a role for cell surface proteoglycans in protection of cells from damage by pro-inflammatory agents, and may assign a central role for the combined action of H2O2 and PLA2 in inflammatory and bacteriocidal processes. PMID- 8612797 TI - Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres. AB - The catalytic and spectroscopic properties of the reversible hydrogenase from the cyanobacterium Anabaena variabilis have been examined. The hydrogenase required reductive activation in order to elicit hydrogen-oxidation activity. Carbon monoxide was a weak (Ki=35 microM), reversible and competitive inhibitor. A flavin with the chromatographic properties of FMN, and nickel were detected in the purified enzyme. A. variabilis hydrogenase exhibited electron paramagnetic resonance (EPR) spectra in its hydrogen-reduced state, indicative of [2Fe-2S] and [4Fe-4S] clusters. Although no EPR signals due to nickel were detected, the results are consistent with the enzyme being a flavin-containing hydrogenase of the nickel-iron type. PMID- 8612798 TI - Induction of a novel cytochrome P450 (CYP93 family) by methyl jasmonate in soybean suspension-cultured cells. AB - We isolated a cDNA encoding a novel cytochrome P450 (CYP93A1) from soybean suspension-cultured cells that had been treated with methyl jasmonate (MeJA). The amino acid sequence of the gene product had 30-40% identity with those of other plant P450s. The protein contained the heme-binding domain which is highly conserved among plant P450s. Transcription of the cytochrome P450 gene in soybean cells was induced by 30 microM MeJA even in the presence of cycloheximide, and reached maximum level 6 h after MeJA treatment. This is the first report of a plant cytochrome P450 gene whose transcription is induced by MeJA even without protein synthesis. PMID- 8612799 TI - Significance of secondary structure predictions on the reactive center loop region of serpins: a model for the folding of serpins into a metastable state. AB - To address how serpins might fold so as to adopt the mechanistically required metastable conformation we have compared the predicted secondary structures of the reactive center loops (RCLs) of a large number of serpins with those of the equivalent regions of other non-serpin protein proteinase inhibitors. Whereas the RCLs of non-serpin inhibitors are predicted to be loop or beta-strand, those of inhibitory serpins are strongly predicted to be alpha-helical. However, non inhibitory serpins, which also adopt the metastable conformation, show no consistent preference for alpha-helix. We propose that the RCL primary structure plays little role in promoting the metastable serpin conformation. Instead we hypothesize that preference for the metastable state results from the incorporation of part of the RCL into beta-sheet C, which as a consequence precludes incorporation of the RCL into beta-sheet A to give the most stable conformation. Consequently the RCL must be exposed and by default will adopt the most stable conformation in this particular context, which is likely to be an alpha-helix irrespective of the primary structure. Thus the observed correlation between inhibitory properties in serpins and prediction of alpha-helix in the RCL may instead reflect a need for alanine residues between positions P12 and P9 for functioning as an inhibitor rather than a structural or mechanistic requirement for alpha-helix. PMID- 8612800 TI - Interaction between human amphipathic apolipoproteins and amyloid beta-peptide: surface plasmon resonance studies. AB - Several apolipoproteins including apoE and apoA-I are known to be associated with amyloid beta-peptide, a major component of senile plaques in Alzheimer's disease. In the present study the interaction between three human amphipathic apolipoproteins apoE3, apoA-I and apoA-II and immobilized amyloid beta-peptide (1 40) was quantified by plasmon resonance. The interactions were saturable and reversible. The results demonstrated a high affinity of the binding of amphipathic apolipoproteins to amyloid beta-peptide. On the other hand, only a small population of synthetic amyloid beta-peptide participated in the interaction. The apparent equilibrium dissociation constants K(D) were 10 nM for apoE3, 25 nM for apoA-I and 80 nM for apoA-II under physiological conditions. The affinity of the apoE3-amyloid beta-peptide binding was not affected by pH in the range 6.0-8.0 but was significantly increased by high salt concentration. ApoA-I mainly followed similar patterns. A major participation of hydrophobic forces in the binding of apoE3 and apoA-I to amyloid beta-peptide was suggested. PMID- 8612803 TI - The spatial distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus varies during the cell cycle. AB - Recently, we have developed a photocrosslinking approach which uses anthracene as a photoactivatable group and which allows us to determine the lateral distribution of lipids in membranes quantitatively. In synchronous cultures of the gram-positive bacterium Micrococcus luteus, this approach shows that the spatial distribution of phosphatidylglycerol and dimannosyldiacylglycerol, the two major lipids in the bacterial membrane, varies greatly during the cell cycle. Minimum heterogeneity was observed during cell growth while maximum heterogeneity was detected during cell division. PMID- 8612801 TI - Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation. AB - Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity. PMID- 8612802 TI - Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells. AB - In this paper we demonstrate that the expression of the multidrug resistance associated protein (MRP) in a variety of intact human tumour cells results in the ATP-dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide-glutathione conjugate. The MRP-dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross-inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP-dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross-inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug-resistance modulating agents. PMID- 8612804 TI - Human mineralocorticoid receptor interacts with actin under mineralocorticoid ligand modulation. AB - The human mineralocorticoid receptor of the steroid receptor family contains a modular structure with domain E which is considered to be a hormone binding domain. Recombinant protein approaches enabled us to clearly determine that this domain is also able to interact with F-actin (Kd about 2 microM) and G-actin. Moreover, it was revealed that this mineralocorticoid receptor domain/actin interaction was modulated by specific mineralocorticoid ligands. Agonist (aldosterone) steroid binding almost totally (91%) abolished the interaction with F-actin, while antagonist (progesterone) binding allowed more than 30% of this binding. Steroid modulation of the interaction between domain E and actin indicated that this actin binding is specific and could be essential for cellular mineralocorticoid receptor activity. PMID- 8612805 TI - An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity. AB - The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column. Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln 148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506. PMID- 8612806 TI - Metabolism of angiotensins by head membranes of the leech Theromyzon tessulatum. AB - Angiotensins (angiotensin I, angiotensin II, angiotensin II-amide) have been isolated in leeches and such peptides are involved in diuresis in these animals. To explore possible inactivation mechanisms of these peptides, angiotensins were incubated with head membranes of the leech T. tessulatum. Membranes derived from head parts of this leech are very rich in peptidases. They contain endopeptidase 24.11-like enzyme (NEP-like) associated with a battery of exopeptidase. The way that angiotensins are degraded by the combined attack of these membrane peptidases has been investigated. The contribution of individual peptidases was assessed by adding inhibitors (phosphoramidon, captopril and amastatin) to the membrane fractions, when they were incubated with the peptides. In the case of angiotensin I, the primary attack was performed by a combined action of the NEP like and the ACE-like enzymes, followed by aminopeptidase attacks. Angiotensin II and III were hydrolyzed by NEP-like enzyme at the same Tyr-Ile bond, whereas the N-terminal arginine residue of angiotensin III was removed by an arginyl aminopeptidase. These results show that angiotensins are efficiently degraded by membranes and that NEP-like enzyme plays a key role in this process. PMID- 8612807 TI - Is there an unidentified defence mechanism against infection in human plasma? AB - The total peroxyl radical scavenging capacity (TRAP) of human plasma was measured from pneumonia patients and controls. TRAP and its main components, ascorbic acid, alpha-tocopherol, uric acid or protein thiol groups, were unaltered, but the concentration of unidentified antioxidants in pneumonia patients was significantly reduced. Our results indicate that human plasma may contain so far unidentified antioxidants depleted in infection. PMID- 8612809 TI - Transduction signals induced in rat brain cortex astrocytes by the HIV-1 gp120 glycoprotein. AB - Cultures of rat brain cortex astrocytes were exposed to 10(-10)-10(-9)M of the HIV-1 envelope glycoprotein, gp120. No specific binding was revealed by the iodinated protein, suggesting expression of only a few sites onto the cells. In contrast, two transduction signals were rapidly induced by gp120: increased tyrosine phosphorylation of a approximately 56 kDa protein and increased [Ca2+]i. This latter effect, present in 1/3 of the investigated astrocytes, consisted in: discrete or biphasic peaks; slowly rising plateaus; and various types of oscillations. Moreover, in apparently unresponsive cells [Ca2+]i rose slowly (45 min) to double the resting levels. Rat brain cortex astrocytes thus appear highly sensitive to gp120. The induced array of signals might contribute to neurotoxicity during HIV infection. PMID- 8612808 TI - Effect of bile on the intestinal bile-acid binding protein (I-BABP) expression. In vitro and in vivo studies. AB - Enterocytes actively transport bile acids from the ileal lumen to the portal blood. This physiological process greatly contributes to maintaining the bile acid homeostasis. However, little is known about the molecular mechanisms involved in this transport system. The effect of bile on gene expression of the intestinal bile-acid binding protein (I-BABP) expressed in the enterocytes was studied in vivo, using the by-pass method, and in vitro, using organ culture of ileum explants and Caco-2 cell line. The low cytosolic I-BABP concentration and I BABP mRNA level found in diverted ileum was totally recovered when bile was added in the ileal lumen. Northern blot analysis of the ileal explants revealed a dose dependent increase in the I-BABP mRNA in the presence of bile. In Caco-2 cells, the I-BABP transcript was dramatically increased in the presence of human bile while it was undetectable in the control cultures. These data offer the first evidence that biliary components regulate the I-BABP gene expressed in the enterocytes. PMID- 8612810 TI - Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit. AB - To explore the structural basis required for the holoenzyme formation of cAMP dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation. PMID- 8612811 TI - Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase). AB - Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild-type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate. PMID- 8612812 TI - Modulation of microtubule shape in vitro by high molecular weight microtubule associated proteins MAP1A, MAP1B, and MAP2. AB - The effect of microtubule associated proteins on microtubule shape has been investigated in reconstitution experiments using purified tubulin and purified MAP1A, MAP1B, and MAP2. Microtubules assembled in the presence of these MAPs were fixed with 0.1% glutaraldehyde and, after negative staining, were examined by electron microscopy. The results show that MAP1A microtubules were generally short and "straight' while those assembled with MAP1B were longer and "bendy'. MAP2 microtubules showed both types of morphologies even though straight microtubules were more abundant. These data suggest that MAPs may modulate not only microtubule dynamics but also microtubule shape which may be important in their spatial distribution and/or role in specific neuronal areas. PMID- 8612813 TI - Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae. AB - Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A. We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387-amino acid protein from Saccharomyces cerevisiae. Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast. This gene was shown to be an intron-free, single-copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF-5A, derived from two distinct genes of yeast. PMID- 8612814 TI - Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli. AB - Methods are described for producing an active amino-terminal domain of tissue inhibitor of metalloproteinases-1 (N-TIMP-1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis. The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2 and 3 is 6-8-fold less than that of the recombinant full length protein, indicating that deletion of the C-terminal domain reduces the free energy of interaction by < 10%. PMID- 8612816 TI - Rice gibberellin-binding phosphoprotein structurally related to ribulose-1,5 bisphosphate carboxylase/oxygenase activase. AB - A gibberellin A (GA)-binding protein was identified from rice (Oryza sativa L.) leaves by a ligand-binding assay. The dissociation constant of GA-binding protein and GA complex was about 100 nM. This protein has a relative molecular mass of 47 000 and an isoelectric point of 5.1. The partial amino acid sequence of the protein was determined for 54 residues from both the N-terminal and internal regions. A sequence homology search indicated that the amino acid sequence of GA binding protein was homologous to that of the ribulose-1,5-biphosphate carboxylase/oxygenase activase from barley, Arabidopsis, spinach and Chlamydomonas. The GA-binding protein was immunologically detected in two polypeptides in the protein extract from leaves. The GA-binding protein identified was phosphorylated with Ca2+, Mg2+ and ATP in the leaf protein extracts of rice grown in the presence of exogenous GA. PMID- 8612815 TI - Nitric oxide induced poly(ADP-ribose) polymerase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl-2. AB - Endogenously generated or exogenously supplied nitric oxide causes cleavage of poly(ADP-ribose) polymerase (PARP) and apoptotic cell death in RAW 264.7 macrophages. With the use of NO donors such as S-nitrosoglutathione or spermine NO we established that PARP digestion occurs in parallel with DNA fragmentation, and is preceded by accumulation of the tumor suppressor gene product p53. PARP cleavage in response to lipopolysaccharide and interferon-gamma treatment is prevented by NG-monomethyl-L-arginine, thus proving a NO requirement. Endogenous NO generation, p53 accumulation, and PARP degradation occurred prior to the detection of significant chromatin condensation. In contrast, in stable Bcl-2 transfected cells, NO-initiated PARP cleavage was almost completely blocked. Our data implicate PARP as a proteolytic substrate during NO-mediated apoptotic cell death in RAW 264.7 macrophages and establish Bcl-2 as an efficient signal terminator in this process. PMID- 8612817 TI - C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation. AB - Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded. PMID- 8612818 TI - DNA affinity to biological membranes is enhanced due to complexation with hydrophobized polycation. AB - The interaction of negatively charged liquid phosphatidylcholine/cardiolipin liposomes with water-soluble negatively charged DNA/cetylpyridinium bromide and DNA/poly(N-alkyl-4-vinylpyridinium bromide) complexes was studied. It is shown that the DNA/cetylpyridinium bromide complex while interacting with the liposomes is destroyed, so that the cetylpyridinium cation is incorporated into the liposomal membrane and DNA remains in the solution. The DNA/poly-(N-ethyl-4 vinylpyridinium bromide) complex does not interact at all with the liposomes. On the contrary, the complex of DNA with the poly(vinylpyridinium) cation carrying a small amount of N-cetyl groups is adsorbed on the membrane as a whole. The data obtained indicate that complexation of DNA with hydrophobized polycations can be used for enhancing DNA affinity to biological membranes. PMID- 8612819 TI - Activation of mitogen-activated protein kinase by protein kinase C isotypes alpha, beta I and gamma, but not epsilon. AB - Treatment of CHO.T cells with either PMA or insulin led to the activation of MAP kinase by approximately 3-fold, and p90rsk by approximately 4-fold. Over expression of the alpha, beta I or gamma isoforms of protein kinase C caused a substantial enhancement of the effect of PMA on the activation of MAP kinase and p90rsk, however, the effect of insulin was unchanged. Over-expression of the epsilon isoform of protein kinase C did not alter the effect of either PMA or insulin on the activation of MAP kinase and p90rsk. The results suggest that protein kinase C isotypes, alpha, beta I and gamma, but not epsilon, can mediate MAP kinase activation by PMA, and strongly support the hypothesis that protein kinase C isoforms can initiate distinct signalling pathways. PMID- 8612820 TI - The 21-residue surfactant peptide (LysLeu4)4Lys(KL4) is a transmembrane alpha helix with a mixed nonpolar/polar surface. AB - The 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4) has been synthesized and analyzed regarding its secondary structure and orientation in lipid environments. Fourier transform infrared and circular dichroism spectroscopy shows that the peptide exhibits approximately 80% alpha-helical content both in dodecylphosphocholine micelles and in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) 7:3 (w/w) bilayers. The positively charged lysine residues are evenly distributed over the entire, otherwise nonpolar, circumference of the helix. This is in sharp contrast to the uneven distribution of polar and nonpolar residues in amphipathic helices. Fourier transform infrared spectroscopy of the peptide inserted in DPPC/PG bilayers shows that the helical axis is oriented parallel to the lipid acyl chains. These data do not support a previous hypothesis that the KL4 peptide interacts with peripheral parts of a phospholipid monolayer and mimics the pulmonary surfactant protein SP-B, which is composed of several amphipathic alpha-helices. KL4 accelerates the spreading of phospholipid mixtures at an air/water interface but does so less efficiently than other transmembranous helical polypeptides studied. PMID- 8612822 TI - Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting. AB - Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening. PMID- 8612821 TI - A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel alpha 1C subunit. AB - The well-characterized enhancement of the cardiac Ca2+ L-type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the alpha 1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the beta subunit. Phosphorylation of serine 1928 of alpha 1C may mediate the modulatory effect of PKA on the cardiac voltage-dependent ca2+ channel. PMID- 8612823 TI - The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor. AB - We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor. PMID- 8612824 TI - Studies on sarcolemma components may be misleading due to inadequate recovery. PMID- 8612825 TI - Messenger ribonucleic acid kinetics in human oocytes--effects of in vitro culture and nuclear maturational status. AB - OBJECTIVE: To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [MII]) and in vitro culture on the kinetics of maternal messenger ribonucleic acid (mRNA) in human oocytes. DESIGN: Molecular biology on excess oocytes obtained from our clinical IVF program. INTERVENTIONS: The oocytes, classified as either PI or MII at collection, were used as such or cultured in vitro for an additional 24 hours. The relative levels of c-mos and cyclin-B1 were measured using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The mean levels of c-mos and cyclin-B1 transcripts were indistinguishable between the PI, MII, PI oocytes matured in vitro, PI oocytes failing to mature, and MII oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the vitro culture. CONCLUSIONS: The level of c-mos and cyclin-B1 transcripts were not different in PI versus MII oocytes, therefore, differences seen in the clinical outcome of PI and MII oocytes may be unrelated to levels of these gene products. C-mos and cyclin B1 mRNA were maintained in vitro, thus degradation of maternal RNA is not activated in excess during the 24-hour culture. PMID- 8612826 TI - Semen analyses in 1,283 men from the United States over a 25-year period: no decline in quality. AB - OBJECTIVE: To determine whether semen quality has changed in the United States over the last 25 years. DESIGN: Retrospective review. SETTING: Three U.S. sperm banks, Cryogenic Laboratories, Inc. (Roseville, Minnesota), Idant Laboratories (New York, New York), and California Cryobank, Inc. (Los Angeles, California). INTERVENTION: None. MAIN OUTCOME MEASURES: Age at sample collection, sperm concentration, volume, motility, and days of abstinence before sample collection were determined for each man. Linear and multiple regression analyses were used to assess changes in these characteristics over time. RESULTS: Controlling for the effects of age and duration of abstinence, there was a slight but significant increase in mean sperm concentration but no change in either motility or semen volume over the 25-year period. Both sperm motility and semen volume decreased with increasing age at sample collection. Both sperm concentration and semen volume increased as a function of duration of abstinence. There were significant differences in mean (+/- SEM) sperm concentrations (10(6) sperm/mL) and motilities between the different sperm banks with California lowest (72.7 +/- 3.1, 51.4% +/- 1.1%, respectively), Minnesota higher (100.8 +/- 2.9, 56.0%, respectively), and New York highest (131.5 +/- 3.5, 58.2% +/- 0.5%, respectively). CONCLUSIONS: Our data show no decline in sperm counts over a 25 year period in 1,283 men who banked sperm before vasectomy at three distinct geographical sites in the United States. PMID- 8612827 TI - Data from men in greater Seattle area reveals no downward trend in semen quality: further evidence that deterioration of semen quality is not geographically uniform. AB - OBJECTIVE: To determine whether there has been a decline in semen quality in a group of healthy men during the past 21 years. DESIGN: Retrospective analysis of the relationship between time and changes in semen parameters over 21 years using regression analysis. SETTING: A tertiary university center. PATIENTS: Five hundred ten healthy, normal men who donated multiple semen samples as participants in clinical studies between 1972 and 1993. MAIN OUTCOME MEASURES: Sperm concentration, semen volume, total numbers of sperm per ejaculate, and percent spermatozoa with normal morphology. RESULTS: There was no decrease in sperm concentration, semen volume, total number of sperm per ejaculate, and percent normal sperm morphology in 510 healthy men studied between 1972 and 1993. CONCLUSION: We conclude that in this population of healthy young men there has not been any decline in semen quality in the past 21 years. PMID- 8612828 TI - Intraobserver, interobserver variation of sperm critical morphology: comparison of examiner and computer-assisted analysis. AB - OBJECTIVE: To examine intraobservational and interobservational variation in sperm critical morphology analysis between trained andrologists and a computer over the range of 0% to 15% normal forms. DESIGN: Retrospective. SETTING: Fertility center. PATIENTS: Twenty-four semen specimen slides were read five times in a randomized, blinded fashion by two andrologists and a computerized semen analyzer. Twenty-five samples were analyzed using a different stain and a centrifugation step. MAIN OUTCOME MEASURE: Sperm strict morphology. RESULTS: Mean average intraobserver deviation of normal forms was small (1.3% to 2.7%). Intraobserver deviation did not differ between andrologists, but both differed from the computer. Interobserver deviation for percent normal forms was similar for andrologists and computer. Mean average deviations were small over the variation was independent of the initial percent normal forms. The computer frequently evaluated significantly fewer cells than the andrologists. Updated software increased the number of cells read by the computer requiring more time than the andrologist. CONCLUSIONS: Trained andrologists and a computer determine sperm critical morphology similarly over the range of 0% to 15% normal forms. A single evaluation is highly predictive of multiple evaluations. Improvements in the speed of computer assessment are still necessary. PMID- 8612829 TI - Human papillomavirus deoxyribonucleic acid and ribonucleic acid in seminal plasma and sperm cells. AB - OBJECTIVE: To investigate the possible presence and expression of human papillomavirus viruses (HPV) in human plasma and sperm cells. DESIGN: Controlled clinical study. SETTING: A major medical center affiliated with a medical college. PATIENTS: Twenty-four randomly selected patients who attended Fertility Clinics at the Chang Gung Memorial Hospital. INTERVENTIONS: Specimens of semen were collected from volunteered patients MAIN OUTCOME MEASURE: The presence of HPV types 16 and 18 DNA and RNA sequences were examined by polymerase chain reaction. RESULTS: Human papillomavirus type 16 E6 and E7 DNA and RNA sequences were found in two and zero seminal plasma specimens, respectively, and in six and two sperm cells specimens, respectively. Deoxyribonucleic acid and RNA sequences of HPV type 18 were found in eight and two seminal specimens and in 11 and 5 sperm cells specimens, respectively. CONCLUSION: These results seem to suggest that HPV cannot only infect human sperm cells, certain HPV genes are expressed actively in infected sperm cells. The virus-infected sperm cells conceivably can behave as vectors or carriers for the transmission of HPV, to sexual partner during sexual contact, to fetuses through fertilized eggs, or both. PMID- 8612830 TI - Effects of hormonal therapies and dietary soy phytoestrogens on vaginal cytology in surgically postmenopausal macaques. AB - OBJECTIVE: To evaluate the effects of conjugated equine estrogens, medroxyprogesterone acetate (MPA), conjugated equine estrogens combined with MPA, tamoxifen, and soybean estrogens on vaginal cytology in surgically postmenopausal cynomolgus macaques (Macaca fascicularis). DESIGN: Randomized long-term experimental trial. SETTING: Cytologic samples were taken from animals in two long-term randomized studies of the effects of hormonal and dietary effects on atherosclerosis. PATIENTS: Surgically postmenopausal cynomolgus macaques. INTERVENTIONS: Conjugated equine estrogens at the doses given do not exert an estrogenic effect on the with MPA, tamoxifen, and soybean estrogens were given via the diet, at doses scaled from those given to women. MAIN OUTCOME MEASURE: Vaginal cytologic maturation index. RESULTS: Conjugated equine estrogens elicited a marked maturation effect, which was antagonized partially by the addition of MPA. Tamoxifen produced a lesser estrogenic response. The cytologic pattern in animals given soybean estrogens or MPA alone did not differ from that of controls. CONCLUSION: Soybean estrogens at the doses given do not exert an estrogenic effect on the vagina of macaques. Conjugated equine estrogens are potent inducers of vaginal keratinization in this model; tamoxifen has a lesser effect. Medroxyprogesterone acetate partially antagonizes the effects of conjugated equine estrogens, and has no effect when given alone. The results support the possibility that soybean estrogens may be a "tissue-selective" estrogen with minimal effects on the reproductive tract. PMID- 8612832 TI - Geographic variations in sperm counts: a potential cause of bias in studies of semen quality. AB - OBJECTIVE: To determine whether geographic variations in sperm counts might bias conclusions drawn from studies of semen quality. DESIGN: Reanalysis of published data from a meta-analysis of 61 studies from 1938 to 1990 that concluded a worldwide decline in semen quality over the last 50 years. MAIN OUTCOME MEASURES: Influence of geographic location on sperm counts. RESULTS: Of 61 studies in the meta-analysis, only 20 included > or = 100 men. These 20 studies collectively comprised 91% of the total men studied. We focused our reanalysis on these 20 studies. Of the studies before 1970, all were from the United States and 80% were from New York. These studies represented locations with the highest sperm counts. In contrast, after 1970, 80% of the studies were from locations not represented earlier, including five studies from third world countries, where sperm counts were low. CONCLUSIONS: Sperm counts vary dramatically among different geographic locations. PMID- 8612831 TI - Effects of acetylsalicylic acid (aspirin) and naproxen sodium (naproxen) on ovulation, prostaglandin, and progesterone production in the rabbit. AB - OBJECTIVE: To determine the effects of acetylsalicylic acid (aspirin) and naproxen sodium (naproxen) on ovulation, ovarian prostaglandins (PG), and P production in the rabbit via in vivo and in vitro studies. DESIGN: Aspirin and naproxen were administered i.v. 6.5 and 7 hours, respectively, after hCG administration to New Zealand White adult female rabbits. Laparotomy was performed 24 hours after hCG administration. For in vitro experiments, control animals underwent laparotomy 6.5 (aspirin) and 7 hours (naproxen) after hCG administration. The treated animal received aspirin and naproxen; laparotomy was performed 1 hour later. One ovary was perfused for 6 hours with aspirin or naproxen whereas the contralateral ovary served as a control and was perfused with control medium (M199; GIBCO, Grand Island, New York). Perfusate samples were collected at 1-hour intervals for PG and P determination. SETTING: A conventional laboratory setting. INTERVENTIONS: In vivo experiments used i.v. administration of 100 mg/kg aspirin and 10 and 50 mg/kg naproxen. In vitro perfusion was also carried out with 100 micrograms/mL aspirin and 10 and 50 micrograms/mL naproxen added to the perfusate. MAIN OUTCOME MEASURES: Ovulatory efficiency (no. of ovulations/no mature follicles) and ovarian vein PG and P concentration were determined. RESULTS: Ovulatory efficiency was 88% for control, 41% for in vivo aspirin-treated, and 40% (10 mg/kg) and 0% (50 mg/kg) for naproxen-treated rabbits. Aspirin and naproxen were associated with decreased ovulatory efficiency when administered in vitro to both in vivo control and in vivo treated ovaries (control-medium = 70%; control-aspirin = 14%; aspirin-medium = 34%; aspirin aspirin = 0%; control-naproxen = 25%; naproxen-medium = 38%; naproxen = 0% with 10 microgram/mL, and control-naproxen = 13%; naproxen-medium = 0%; naproxen = 0% with 50 micrograms/mL). Prostaglandin F2 alpha was undetectable in the perfusate of those ovaries perfused of those ovaries perfused either with aspirin or naproxen. Ovarian venous concentration of P in the perfusate was similar in all groups. CONCLUSIONS: Aspirin and naproxen significantly reduced ovulatory efficiency and PG production both in vivo and in vitro in hCG-treated rabbits. A critical period of 6.5 and 7 hours after hCG administration was established. PMID- 8612833 TI - Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. AB - OBJECTIVE: To report a normal pregnancy and the delivery of a healthy child after the combination of in vitro maturation of germinal-vesicle stage oocytes and intracytoplasmic sperm injection (ICSI) in a patient. SETTING: Procedures were performed in a tertiary IVF center coupled with an institutional research environment. MAIN OUTCOME MEASURES: Maturation rate of immature oocytes after in vitro maturation and intactness, fertilization, and developmental rates of oocytes after microinjection. RESULTS: Nine of 14 germinal-vesicle stage oocytes matured to the metaphase II stage after 30 hours of in vitro culture (64%). Seven of eight injected and intact oocytes fertilized normally (78%) and five of them cleaved with < 20% fragmentation (71%). Four embryos were transferred and a singleton pregnancy was obtained that ended in the delivery of a healthy child. CONCLUSION: In vitro maturation of immature oocytes together with ICSI can result in normal fertilization, embryo development, pregnancy, and the delivery of healthy child. PMID- 8612834 TI - Seminoma discovered in two males undergoing successful testicular sperm extraction for intracytoplasmic sperm injection. AB - OBJECTIVE: To describe two cases with testicular seminoma incidentally discovered at the time of successful testicular sperm extraction for intracytoplasmic sperm injection (ICSI)> DESIGN: Report of two cases. SETTING: Tertiary care academic center. PATIENTS: Two males with nonobstructive azoospermia with history of cryptorchidism. INTERVENTION: Testicular biopsy combined with ICSI; orchiectomy with radiotherapy. RESULTS: From two couples and in three different cycles, 17 of 55 (31%) metaphase II oocytes fertilized after microinjection. Eleven of these fertilized oocytes cleaved normally and all 11 were replaced. One twin pregnancy was established and was delivered successfully later. In both cases, a seminoma was discovered at the time of testicular biopsy. CONCLUSIONS: At the time of testicular biopsy for ICSI, a specimen for histopathology must be sent to exclude the presence of seminoma in males with nonobstructive azoospermia with a history of cryptorchidism. The effectiveness of testicular sperm extraction in combination with ICSI also has been demonstrated even against the background of testicular tumor. PMID- 8612836 TI - CA-125 is elevated in viable pregnancies destined to be miscarried: a prospective longitudinal study. AB - OBJECTIVE: To assess the predictive value of E2, hCG, CA-125, and morphometric parameters in early pregnancies with demonstrable fetal heartbeat, complicated by bleeding. DESIGN: Prospective longitudinal follow-up. SETTING: The emergency service of the Department of Obstetrics and Gynecology, "Rebecca Sieff" Hospital, Safed, Israel. PATIENTS: Twenty-five consecutive patients with vaginal bleeding during weeks 7 to 12 of pregnancy who had demonstrable fetal heartbeat. Ten women with normal pregnancies, serving as controls. MAIN OUTCOME MEASURE: The serum levels and the morphometric parameters were related to the outcome of pregnancy as revealed by future hospitalization, delivery, or telephone questioning. RESULTS: In the five patients who eventually aborted, the values of CA-125 were > 125 U/mL, whereas none of the successful pregnancies had a value > 93 U/mL. The mean values were 133 +/- 4.85 versus 36.95 +/- 23.1 U/mL for miscarriages and successful pregnancies, respectively. In normal pregnancies the respective value was 32.3 +/- 4.3 U/mL. Although mean E2 levels were lower in the serum of women who eventually aborted, there was a significant overlap of values with the successful pregnancies. All the other parameters measured had no correlation to the outcome of these pregnancies. CONCLUSION: CA-125 may serve as an accurate predictor of the outcome in early pregnancies with demonstrable fetal heartbeat, which are complicated by bleeding. PMID- 8612835 TI - Pregnancy outcome after early amniotic fluid leakage after transabdominal multifetal reduction. AB - OBJECTIVE: To assess the importance of amniotic fluid leakage in the early period ( < 14 days) after the fetal reduction procedure. DESIGN: Prospective clinical study. SETTING: Pregnant women who underwent fetal reduction to twins in an academic, tertiary perinatal department. PATIENTS: Sixty triplet and 31 quadruplet pregnancies were identified before the ninth postmenstrual week of gestation. All patients underwent transabdominal multifetal pregnancy reduction was performed within 14 days of the procedure. INTERVENTIONS: Transabdominal multifetal pregnancy reduction from triplets and quadruplets to twins. MAIN OUTCOME MEASURE: Amniotic fluid leakage in the early period after fetal reduction. RESULTS: Early PROM occurred in eight (13.3%) patients from the triplets group and in six (19.3%) patients in the quadruplet group. Three of 60 (5.0%) patients from the triplet group and 1 of 31 (3.2%) patients from the quadruplet group miscarried before 24 weeks gestation. However, none of these patients had had PROM during the early period after the procedure. Early PROM did not influence the incidence of premature contractions of delivery and the reduction procedure, and 59% (99/169) of those who completed 24 gestational weeks delivered between 25 and 37 weeks. CONCLUSION: The presence of amniotic fluid leakage in the period close to the multifetal reduction procedure, although alarming, is not ominous for the remaining fetuses. We see no reason for any intervention in these patients. PMID- 8612837 TI - Preoperative evaluation of intersex patients with pelvic magnetic resonance imaging in search of Y chromosome-bearing gonadal tissue. AB - OBJECTIVE: To illustrate the utility of preoperative magnetic resonance imaging in the evaluation of intersex patients who require gonadectomy. DESIGN: Case reports of two phenotypic females having karyotypes containing a Y chromosome whose preoperative evaluation included pelvic magnetic resonance imaging. SETTING: Tertiary academic referral center. MAIN OUTCOME MEASURE: Pelvic magnetic resonance imaging for gonadal localization. RESULTS: Preoperative evaluation with magnetic resonance imaging correctly identified the most appropriate surgical approach to gonadectomy. CONCLUSIONS: Pelvic magnetic resonance imaging can be useful in the preoperative evaluation of intersex patients having nonpalpable Y chromosome-bearing gonadal tissue. PMID- 8612838 TI - Ruptured tubo-ovarian abscess complicating transcervical cryopreserved embryo transfer. AB - OBJECTIVE: To present a rare infectious complication related to transcervical ET, without prior transvaginal puncture. DESIGN: Case report. SETTING: Hadassah University Hospital, IVF-ET unit. PATIENT: One patient undergoing cryopreserved thawed ET. INTERVENTIONS: Artificial preparation of the endometrium with E2 and P, followed by transcervical intrauterine cryopreserved-thawed embryo transfer. RESULTS: After ET, severe pelvic inflammatory disease (PID) with ruptured tubo ovarian abscess was diagnosed and treated. CONCLUSIONS: Severe PID including tubo ovarian abscess formation should be considered a potential complication after ET, even without transvaginal oocyte aspiration. PMID- 8612839 TI - A new method to study the process of implantation of a human blastocyst in vitro. AB - OBJECTIVES: To develop a "miniorgan" culture of human endometrium dated according to the LH peak in order to study the process of implantation of a human blastocyst in vitro. DESIGN: Preliminary results of a more extensive study with the same objectives. SETTING: Hospital-based unit of reproductive health and university-related reproductive research laboratories. PATIENTS: Six apparently healthy women with normal regular menstrual cycles providing endometrial material 4, 5 and 6 days after the LH peak. INTERVENTION: One part of the biopsy was used for histologic dating. The other part was incubated in culture medium RPMI-1640 and exposed to the contact with one to three embryos fertilized in vitro. MAIN OUTCOME MEASURE: Biopsy material before and after incubation was studied by morphometric methods in a light microscope. The histologic changes were recorded by photomicrographs. RESULTS: Implantation of an embryo obtained 4 days after IVF penetrated the lining endometrial epithelium of a biopsy obtained 4 days after the LH peak and kept in vitro for 24 hours. The embryo was placed on the lining epithelium of the endometrium within 3 hours after the biopsy was taken. CONCLUSION: A new method to study the process of implantation of a human embryo in vitro has been described, allowing the embryo to penetrate the lining endometrial epithelium in a biopsy obtained at LH +4/+5. PMID- 8612840 TI - Transmyometrial embryo transfer after difficult immediate mock transcervical transfer. AB - OBJECTIVE: To evaluate the place of ultrasound-directed transvaginal transmyometrial ET in a protocol using both the transcervical and transmyometrial routes in a step-wise fashion. DESIGN: A prospective descriptive clinical study. SETTING: A university-based assisted conception unit. PATIENTS: Thirteen patients who had difficult or impossible mock transcervical ET immediately before the real transfer. INTERVENTION: Ultrasound-directed transvaginal transmyometrial ET. MAIN OUTCOME MEASURES: Pregnancy and clinical pregnancy. RESULTS: Four patients became pregnant, including three with clinical pregnancies. CONCLUSIONS: In cases in which transcervical ET isd difficult or impossible, transvaginal transmyometrial ET is a viable option. The use of mock transcervical ET immediately before the real transfer would identify patients needing transmyometrial ET. PMID- 8612841 TI - An important technical detail for hysteroscopic GIFT! PMID- 8612842 TI - "Motility maintenance factor"--a measure of sperm stamina and tenacity! PMID- 8612843 TI - Progesterone concentrations--physiologic or pharmacologic? PMID- 8612844 TI - Ectopic pregnancies in subjects on progestin-only pills. PMID- 8612845 TI - Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. AB - OBJECTIVE: To describe the novel micromanipulation technique known as intracytoplasmic sperm injection (ICSI), which has been applied successfully to treat male factor infertility, even in patients with severely impaired sperm characteristics. This paper reviews the historical aspects that led to the development of ICSI in the animal model as well as the current experience in the human. DESIGN: Before using assisted fertilization techniques to enhance fertilization of human gametes, it is imperative that practitioners gain extensive experience in the animal model. In addition, criteria for accepting individuals for treatment with ICSI are discussed along with other applications of the procedure in infertile couples who do not benefit from standard IVF. RESULTS: Because ICSI resulted in limited success in animal models, it seemed unlikely that it would be successful in humans. Yet, ICSI now appears to be the most successful and significant innovation developed for dealing with male factor infertility since the emergence of IVF itself. To date, a relatively large group of healthy children have been born from this technology and there appears to be no increased incidence of congenital malformations. CONCLUSIONS: The consistently high success rate resulting from the application of ICSI to treat couples with male factor infertility is comparable to the results obtained using standard IVF techniques performed in nonmale factor couples. This finding indicates that spermatozoa obtained from subfertile men selected for intracytoplasmic injection are usually genotypically normal. PMID- 8612846 TI - "The debate continues"--the continuing debate over the possible decline in semen quality. PMID- 8612847 TI - Dose-response effect of depot leuprolide acetate on serum androgens in hirsute women. AB - OBJECTIVE: To determine the dose of leuprolide acetate (LA) needed to maximally suppress serum androgens in hirsute women. DESIGN: Prospective, dose-escalation study. SETTING: Outpatient endocrinology clinic. PATIENTS: Eight hyperandrogenic women with moderate to severe hirsutism. INTERVENTIONS: A LA dose-response study was done in women receiving depot LA plus estrogen-progestin replacement. MAIN OUTCOME MEASURES: Serum concentrations of T, androstenedione (A), and basal and GnRH-stimulated LH. RESULTS: The lowest LA dose (3.75 mg/mo) suppressed serum T by 62% +/- 6% and A by 56% +/- 7%. No further decrease in serum androgens was seen with doses up to 15 mg/mo. Maximal suppression of basal and stimulated LH was also seen with the lowest dose of LA. CONCLUSIONS: As opposed to results previously published in children with precocious puberty, the 3.75 mg dose of depot LA is sufficient to maximally suppress serum androgens in hyperandrogenic women. PMID- 8612848 TI - Serum creatine kinase does not predict ectopic pregnancy. AB - OBJECTIVE: To evaluate the discriminatory ability of maternal serum creatine kinase (SCK) as a test for ectopic pregnancy (EP). DESIGN: Serum creatine kinase concentrations were obtained prospectively from symptomatic patients being evaluated for early abnormal pregnancy. Serum creatine kinase concentrations from all patients and from a subset of these patients with maternal serum beta-hCG concentrations < 6,500 mIU/mL (conversion factor to SI unit, 1.00) were analyzed with descriptive statistics, receiver operator characteristic (ROC) curve analysis, and calculations of predictive values. SETTING: A university hospital emergency room. PATIENTS: Fifty-six patients with intrauterine gestations (25 with beta-hCG concentrations < 6,500 mIU/mL) and 23 patients with EP (20 with beta-hCG concentrations < 6,500 mIU/mL) were studied. RESULTS: For all patients and the subgroup with beta-hCG concentrations < 6,500 mIU/mL, mean SCK levels were not significantly different between ectopic and intrauterine gestations. For all patients and the subgroup with beta-hCG concentrations < 6,500 mIU/mL, the areas under the ROC curves did not demonstrate discriminatory ability of the SCK test. The highest positive predictive value of an elevated SCK for EP was 52% using the SCK concentration of 70 U/L, and this was seen in the subgroup of patients with beta-hCG values < 6,500 mIU/mL. CONCLUSIONS: Maternal SCK concentrations do not reliably predict EP. PMID- 8612849 TI - Failure of creatine kinase to predict ectopic pregnancy. AB - OBJECTIVE: To investigate serum creatine kinase (CK) levels in pregnant women with ectopic pregnancy (EP), spontaneous abortion, and normal pregnancy with comparable serum hCG concentrations. DESIGN: A retrospective, case-controlled study. SETTING: Helsinki University Central Hospital, Helsinki, Finland. PATIENTS: Forty-four patients with a suspicion of EP of which 15 had laparoscopically confirmed tubal pregnancies; 15 had a spontaneous abortion, a blighted ovum, or a missed abortion; and 14 patients a normal intrauterine pregnancy. INTERVENTIONS: The diagnosis was made by transvaginal sonography and serum hCG determinations either at admission or after repeated examinations. Ectopic pregnancy was confirmed and treated by laparoscopy. MAIN OUTCOME MEASURES: Serum CK and hCG levels. RESULTS: No significant differences in CK levels were observed between the groups by one-way analysis of variance and no correlation was found between serum CK and hCG levels within any group. CONCLUSIONS: Creatine kinase does not appear to be useful in the diagnosis of early EP. PMID- 8612851 TI - Epidermal growth factor and basic fibroblast growth factor in peritoneal fluid of women with endometriosis. AB - OBJECTIVE: To determine and compare the concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) in the peritoneal fluid of women with and without endometriosis. DESIGN: Prospective study. SETTING: Tertiary care center affiliated with university medical school. PATIENTS: Forty-two women with endometriosis and 34 women without endometriosis confirmed by laparoscopy. MAIN OUTCOME MEASURES: Epidermal growth factor and basic FGF concentrations were determined by highly sensitive enzyme immunoassays using monoclonal antibodies. RESULT: Approximately 50% of the PF samples had EGF concentrations of > 0.4 pg/mL (conversion factor to SI unit, 0.17) and 90% had basic FGF concentrations of > 1 pg/mL (conversion factor to SI unit, 0.058). Concentrations of EGF or basic FGF in PF did not differ significantly between the two groups. In normal women, there was a significant correlation between EGF and basic FGF during the luteal phase. In women with endometriosis, EGF levels were higher during the luteal phase, but there was no correlation of either EGF or basic FGF levels with the severity of endometriosis. CONCLUSIONS: The concentrations of EGF and basic FGF were highly variable in the PF of women with or without endometriosis and did not differ significantly. The concentrations were so low that neither EGF nor basic FGF in PF could bind to its receptor. PMID- 8612850 TI - Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. AB - OBJECTIVE: To evaluate basal (constitutive) and stimulated synthesis of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-8, IL-10 by peritoneal macrophages (PM) in women with endometriosis. DESIGN: Peritoneal macrophages were cultured in the presence or absence of lipopolysaccharide (LPS) for 24 hours. Peritoneal fluids (PF) and PM supernatants were assayed for cytokines using ELISA. SETTING: Institute for the Study and Treatment of Endometriosis and university-based research laboratories. SUBJECTS: Fertile controls undergoing tubal ligation (n = 8) and women with endometriosis (n = 17). INTERVENTION: Peritoneal fluid samples were obtained at the time of diagnostic laparoscopy (endometriosis group) or laparoscopy for tubal ligation; both were performed in the midluteal phase of the cycle. RESULTS: Both basal and LPS stimulated production of TNF alpha, IL-8, and IL-10 by the PM were elevated significantly in women with endometriosis as compared with the fertile controls. CONCLUSIONS: This study demonstrated that cytokines TNF alpha, IL-8, and IL-10 are synthesized at greater than normal levels by basal and stimulated PM from women with endometriosis. The levels of TNF alpha and IL-8 correlated with the levels in the PF, suggesting that PM are the principal source of these cytokines in the PF. PMID- 8612852 TI - Growth hormone (GH)-releasing hormone-induced GH response in hypothalamic amenorrhea: evidence of altered central neuromodulation. AB - OBJECTIVE: To evaluate the GH-releasing hormone (GH-RH)-induced response of GH in patients affected by hypothalamic amenorrhea. DESIGN: Patients affected by weight loss-related hypothalamic amenorrhea (n = 28) were studied and compared with 20 healthy controls. Among patients with weight-loss amenorrhea, both hypogonadotropic and normogonadotropic conditions were present. All subjects underwent a GH-RH test (GEREF, Sereno, Rome, Italy) (1 microgram/kg body weight IV). Plasma GH concentrations were determined using commercially available RIAs. Also, in selected samples insulin-like growth factor-I (IGF-I) levels were measured. RESULTS: Basal plasma IGF-I levels as well as body mass index (BMI) were lower in amenorrheic patients than in healthy controls. No significant correlation was found between BMI and IGF-I or E2 plasma levels or between LH and IGF-I plasma levels. The basal GH plasma levels were comparable in all groups of subjects. The GH-RH--induced GH response evaluated as maximal release and as area under the curve (AUC) was higher in amenorrheic patients than in control subjects. CONCLUSIONS: The amenorrheic condition associated with reduced BMI changes the GH-RH--induced GH response in hypothalamic amenorrhea, supporting a GH and a IGF-I disregulation in weight-loss--related amenorrhea. PMID- 8612853 TI - Empirical evidence of bias in infertility research: overestimation of treatment effect in crossover trials using pregnancy as the outcome measure. AB - OBJECTIVE: To determine whether crossover trials with simple pooling of data over different study periods leads to a different estimate of treatment effect compared with parallel group trials in infertility research using pregnancy as the outcome measure. DESIGN: An observational study using nine overviews that included trials with both crossover and parallel group designs. These overviews comprised 17 crossover and 17 parallel group trials. In total, there were 5,291 outcomes including 775 pregnancies. The association between study design and treatment effect estimate was analyzed using multiple logistic regression, controlling for differences in the therapeutic interventions and variations in the methodological quality of the trials. SETTING: Infertile patients in an academic research environment. PATIENTS: Infertile patients undergoing treatment efficacy evaluation in controlled trials. INTERVENTIONS: Random allocation to a variety of treatments including clomiphene citrate, hCG, IUI, tamoxifen, and bromocriptine. MAIN OUTCOME MEASURE: Estimate of bias between study designs, based on the interaction of study design and treatment in the logistic regression model. RESULTS: Crossover trials produced a larger average estimate of treatment effect compared with trials with a parallel group design, overestimating the odds ratio by 74% (95% confidence interval, 2% to 197%). CONCLUSION: The use of a crossover design for evaluating infertility treatments with outcomes that prevent patients from completing later phases of the trial should be avoided because it leads to exaggerated estimates of treatment effect and may result in erroneous inferences and clinical decisions. Furthermore, the type of study design should be taken into account when assessing the methodological quality of therapy trials in infertility. PMID- 8612854 TI - Can metformin reduce insulin resistance in polycystic ovary syndrome? AB - OBJECTIVE: To examine whether metformin is able to reduce insulin resistance in polycystic ovary syndrome (PCOS). DESIGN: Single-blind study comprising two successive periods of treatment: 8 weeks of placebo and 10 weeks of metformin (orally, 850 mg twice daily). SETTING: Clinic of endocrinology and metabolism of Cerrahpasa Medical Faculty at Istanbul University, Istanbul, Turkey. PATIENTS: Sixteen insulin-resistant women with PCOS. INTERVENTIONS: Insulin sensitivity (with an IV insulin tolerance test), plasma glucose and insulin levels during an oral glucose tolerance test (OGTT), serum androgens, and lipids were measured at baseline and after each treatment period. RESULTS: Insulin sensitivity, the mean fasting serum levels of glucose, insulin, total cholesterol, triglyceride, low density lipoprotein cholesterol, high-density lipoprotein cholesterol, total T, free T, androstenedione, DHEAS, and LH:FSH ratio, and the areas under the curve for plasma glucose and insulin during OGTT were not changed by either placebo or metformin treatment. CONCLUSION: Metformin does not decrease insulin resistance in PCOS. This finding suggests that cellular mechanism of insulin resistance in PCOS is different from other common insulin-resistant states such as non-insulin dependent diabetes mellitus and obesity. PMID- 8612855 TI - Ovarian hyperstimulation augments adrenal dehydroepiandrosterone sulfate secretion. AB - OBJECTIVE: To determine if factor(s) secreted by the ovaries during hyperstimulation potentiate basal and ACTH-stimulated adrenal androgen secretion. DESIGN: Retrospective and prospective and prospective clinical study. SETTING: University tertiary care center infertility clinic. PARTICIPANTS: Two hundred thirteen hyperstimulation cycles in endocrinologically normal women were identified from 92 patients with ovulatory infertility, aged 25 to 45 years. Further, seven endocrinologically normal infertile women, aged 22 to 37 years, who were undergoing empiric ovarian hyperstimulation for infertility were identified and studied. INTERVENTIONS: In the previously performed cycles, basal and peak serum DHEAS and cortisol (F) levels were assayed and compared with each other and to the extant E2 levels. Additionally, at the baseline and the peak of ovarian hyperstimulation cycles, a standard ACTH test was performed and serum was assayed for DHEAS, DHEA, and F. MAIN OUTCOME MEASURE: Basal and ACTH-stimulated serum DHEAS, DHEA (prospective part only), and F concentrations. Where applicable, mean peak values were generated and compared between the baseline and the peak of stimulation with or without a correction for intrapatient variability in F secretion. RESULTS: Basal serum DHEAS levels rose with ovarian hyperstimulation independent of F. Post-ACTH mean peak value concentrations rose with ovarian hyperstimulation for DHEAS but not DHEA or F. CONCLUSIONS: Ovarian hyperstimulation potentiates basal and ACTH perturbed adrenal DHEAS secretion. This implies the existence of a humoral ovarian factor(s) that mediate this ovarian-adrenal cross-talk. PMID- 8612856 TI - Transforming growth factor-beta 1 and -beta 2 induce inhibin and activin beta B subunit messenger ribonucleic acid levels in cultured human granulosa-luteal cells. AB - OBJECTIVE: To examine the effect of transforming growth factor-beta (TGF-beta) on inhibin and activin subunit messenger ribonucleic acids. DESIGN: Human granulosa luteal cell culture model. SETTING: Granulosa cells were obtained from women undergoing an IVF program in a private IVF clinic. PATIENTS: Regularly menstruating women undergoing oocyte retrieval for IVF because of either tubal obstruction or infertility of the spouse. INTERVENTIONS: For each experiment, cells of two to four patients were pooled, enzymatically dispersed, separated from red blood cells by centrifugation through Ficoll-Paque and cultured in vitro in the presence of TGF-beta 1 or TGF-beta 2 and/or hCG whereafter cellular RNA was extracted for Northern or dot blot filter hybridization with inhibin alpha-, beta A, and beta B-subunit complementary DNA probes. RESULTS: Both TGF-beta 1 and TGF-beta 2 induced the expression of a 4.8-kb inhibin and activin beta B-subunit messenger (mRNA) transcript in a time- and dose-dependent manner but had no effect on alpha- or beta A-subunit mRNA levels. Human chorionic gonadotropin alone did not affect beta B-subunit mRNA levels, but when administered together with TGF-beta s, it prevented the induction of beta B-subunit mRNAs. CONCLUSIONS: Our results suggest that in human ovary, granulosa, or thecal cell-derived TGF beta 1 or -beta 2 may eventually locally modulate in a paracrine or autocrine manner the relative expression levels of inhibin and activin subunits favoring the formation of the inhibin and activin dimers containing the beta B-subunit. The effect of TGF-beta is clearly different from that of gonadotropins, which potently induce the alpha- and beta A-subunit mRNAs, indicating that distinct components of the human ovarian inhibin and activin system are regulated differentially by endocrine and local factors. PMID- 8612857 TI - High-dose human menopausal gonadotropin stimulation in poor responders does not improve in vitro fertilization outcome. AB - OBJECTIVE: To study the outcome in poor responders to three ampules (225 IU) of hMG per day in subsequent IVF treatment cycles in which six ampules (450 IU)of hMG per day were administered. DESIGN: Retrospective chart review. SETTING: Academic tertiary center. PATIENTS: Between January 1988 and May 1995, 126 poor response patients had a first treatment cycle on three ampules and a second cycle on six ampules of hMG per day. MAIN OUTCOME MEASURES: Numbers of follicles, oocytes, and embryos, and pregnancy rates. RESULTS: On six ampules, patients had significantly more follicles and oocytes. The number of embryos did not differ significantly. The pregnancy rate on six ampules were low (3.2% pregnancies per cycle started). CONCLUSION: Poor responders do not benefit from high-dose hMG stimulation; their reproduction outcome is poor. PMID- 8612858 TI - Midluteal buserelin is superior to early follicular phase buserelin in combined gonadotropin-releasing hormone analog and gonadotropin stimulation in in vitro fertilization. AB - OBJECTIVE: To establish whether time to down-regulation and pregnancy and live birth rates were different when buserelin acetate was started in the midluteal phase or early follicular phase in IVF-ET patients. DESIGN: Prospective, controlled, randomized, parallel-group multicenter clinical study. SETTING: Women attending seven infertility clinics. PATIENTS: One hundred twenty-four women with tubal or unexplained infertility with normal menstruation and fertile partners. INTERVENTIONS: Intranasal buserelin acetate started in the midluteal or early follicular phase combined with standard hMG and hCG stimulation after achievement of down-regulation. Established IVF-ET methods. MAIN OUTCOME MEASURES: Duration of down-regulation; clinical pregnancy and live birth rates. RESULTS: Kaplan Meier estimations of the duration of down-regulation were 15.5 days when buserelin acetate was started in the early follicular phase (127 cycles) and 14.6 days when it was started in the midluteal phase (96 cycles). This difference was statistically significant. The pregnancy rates per first treatment cycle, treatment cycle, oocyte retrieval, and ET were significantly higher when buserelin acetate was started in the midluteal phase. The live birth rates were also higher, but only significantly so for the rate per first treatment cycle. CONCLUSIONS: Clinical pregnancy and live birth rates are better when buserelin acetate is started in the midluteal phase rather than the early follicle phase before hMG and hCG stimulation in preparation for IVF-ET. PMID- 8612859 TI - The hypo-osmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia. AB - OBJECTIVE: To determine the ability of the hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI). DESIGN: Nonrandomized, sequential comparative study. PATIENTS: Thirteen couples enrolled in our ICSI program had 16 cycles in which sperm preparations with 0% motility were obtained. Five cycles used cryopreserved epididymal sperm with complete asthenozoospermia. INTERVENTIONS: In eight cycles, the semen samples were washed through a Percoll gradient and sperm were selected randomly for ICSI. In another eight cycles, the washed sperm were placed in a hypo-osmotic solution (75 mM fructose; 25 mM sodium citrate dihydrate) and the sperm with curled tails taken up with the microinjection needle, rinsed, and used ICSI. MAIN OUTCOME MEASURES: Fertilization rate per oocyte injected as determined by the presence of two pronuclei at 18 hours after retrieval and embryo cleavage rate per oocyte injected at 48 hours after retrieval. RESULTS: With random sperm injection, the fertilization and cleavage rates were 26% and 23%, respectively. In contrast, after injection of sperm selected using the hypo-osmotic swelling test, fertilization and cleavage rates were significantly greater (43% and 39%, respectively). There were three pregnancies in the eight cycles with the hypo osmotic swelling test-selected sperm, including two from frozen epididymal sperm. CONCLUSION: Based on these preliminary observations, we believe that the hypo osmotic swelling test will prove to be valuable for increasing fertilization and cleavage rates and pregnancy rates in ICSI cycles where no motile sperm are recovered. PMID- 8612860 TI - Intracytoplasmic sperm injection for treatment of infertility due to acrosomal enzyme deficiency. AB - OBJECTIVE: To determine whether absence of fertilization in IVF associated with an acrosomal enzyme defect (hyaluronidase deficiency) results from a simple mechanical block to sperm penetration or from a more serious sperm abnormality. DESIGN: Nonrandomized, prospective study. SETTING: Toronto Center for Advanced Reproductive Technology, a tertiary referral center for infertility associated with The University of Toronto. PATIENTS: One hundred twenty-two couples about to undergo intracytoplasmic sperm injection (ICSI) were selected. Thirty-six of the studied couples had failed to fertilize in prior IVF cycles. INTERVENTIONS: Hyaluronidase activity was measured in the semen samples provided for ICSI using a zymogenic assay. Intracytoplasmic sperm injection was performed in all couples using standard techniques. RESULTS: Forty-eight of 122 semen samples had poor of absent semen hyaluronidase activity. All 48 samples resulted in successful fertilization with ICSI in the present study. The average fertilization rate per oocyte was 59.43% in couples in whom the partner had low semen hyaluronidase activity and 55.85% in whom the male had normal hyaluronidase activity. The ET rate per cycle was 100% and 95% and pregnancy rates per cycles were 26% and 25% in cycles with poor and normal semen hyaluronidase activity, respectively. Unlike routine IVF, no statistical correlation was found between semen hyaluronidase activity and the fertilization rate in ICSI. CONCLUSION: Our results indicates that semen hyaluronidase deficiency is associated with a simple mechanical block to fertilization. In addition, the measurement of semen hyaluronidase activity can provide a reliable means for selecting couples who would benefit from ICSI. PMID- 8612861 TI - Elevated progesterone levels in the late follicular phase do not predict success of in vitro fertilization-embryo transfer. AB - OBJECTIVE: To examine the effects of subtle elevation in P levels in late follicular phase on the outcome of IVF-ET cycles, using GnRH agonist (GnRH-a) and hMG +/- FSH protocol. DESIGN: A retrospective analysis of data. PATIENTS: Fifty four patients who completed 63 IVF-ET cycles were treated with midluteal GnRH-a, followed by hMG +/- pure FSH. Depending on serum P levels on the day of hCG administration, patients were divided in two groups. In group 1, P levels were < or = 0.9 ng/mL (conversion factor to SI unit, 3.180) and in group 2, the levels were > 0.9 ng/mL. RESULTS: Luteinizing hormone levels, on the day of hCG administration, as measured by RIA, were suppressed completely. In cycles with subtle P rise (71%), we observed a significantly higher serum E2 concentration, greater number of mature follicles, and greater number of oocytes retrieved. There were no differences between the two groups in fertilization rate, number of embryos transferred, clinical pregnancy rate, implantation rate, and miscarriage or delivery rates. CONCLUSIONS: We conclude that in IVF-ET cycles, when pretreated with GnRH-a, P levels may increase on the day of hCG administration despite LH suppression and such elevation may not affect adversely the final outcome. PMID- 8612862 TI - The effectiveness of sublingual progesterone administration during cryopreserved embryo transfer cycles: results of a matched follow-up study. AB - OBJECTIVE: To compare cryopreserved ET pregnancy rates in subjects receiving either sublingual parenteral P. DESIGN: Matched follow-up study. SETTING: University-based assisted reproduction program. PATIENTS: Women undergoing cryopreserved ET between January 1993, and December 1994. Cases received a hormone replacement protocol containing oral E2 and sublingual P and controls received a hormone replacement protocol containing oral E2 and parenteral P. Cases and controls were matched one-to-one according to age, number of embryos transferred, embryo grade, and route of ET. INTERVENTIONS: Cryopreserved embryos were thawed and transferred in all patients in an identical manner independent of the route of P administration. MAIN OUTCOME MEASURES: Clinical and ongoing pregnancy rates. RESULTS: Of 61 ET cycles performed in the sublingual P group, there were 16 clinical pregnancies (26.2%) and 12 ongoing pregnancies (19.7%). Of the 61 ET cycles in the parenteral P group, there were 14 clinical pregnancies (23.0%) and 11 ongoing pregnancies (18.0%). A chi 2 test revealed no significant differences in either clinical or ongoing pregnancy rates according to the route of P administration. CONCLUSIONS: This data suggests that sublingual P administration is an effective alternative to parenteral P administration in preparing the endometrium for the implantation of cryopreserved embryos. PMID- 8612863 TI - Comparison of intravenous albumin and transfer of fresh embryos with cryopreservation of all embryos for subsequent transfer in prevention of ovarian hyperstimulation syndrome. AB - OBJECTIVE: To compare the efficacy of administration i.v. albumin to prevent severe ovarian hyperstimulation syndrome (OHSS) in patients undergoing ovarian stimulation for IVF with a standard policy of cryopreserving all embryos and to assess the impact of the two methods of treatment on pregnancy rates (PRs). DESIGN: Prospective randomized study. SETTING: A tertiary referral center for assisted conception. PATIENTS: Twenty-six patients undergoing IVF treatment cycles who were considered to be at high risk of developing severe OHSS on the basis of their serum E2 concentrations on the day of hCG administration and the number of oocytes collected. INTERVENTION: In group 1 (n = 13) all the generated embryos were cryopreserved to be transferred subsequently in hormonally manipulated cycles. In group 2 (n = 13) patients received IV infusions of albumin on the day of oocyte retrieval and 5 days later. Patients in group 2 had transfers of fresh embryos. MAIN OUTCOME MEASURES: The total dosage of hMG used, total number of follicles developed, number of follicles > 14 mm in diameter, serum E2 concentrations and endometrial thickness on day of hCG administration, number of oocytes retrieved, number and quality of embryos generated, PRs per cycle commenced, and onset and degree of any OHSS developed. RESULTS: There were no significant differences in the above parameters between the two groups, except for PRs that were significantly higher in patients who had all embryos cryopreserved (38.6% versus 0%). CONCLUSION: The policy of cryopreserving all generated embryos appears as effective as the administration of i.v. albumin in preventing OHSS in high-risk patients and produces significantly higher PRs. PMID- 8612864 TI - Embryonic human leukocyte antigen-G expression: possible implications for human preimplantation development. AB - OBJECTIVE: To investigate further the association between human leukocyte antigen G (HLA-G) expression in human embryos and other factors known to influence IVF pregnancy outcome. SETTING: A university-based tertiary referral center (The Toronto Hospital). INTERVENTIONS: Nontransferred embryos at the two- to four-cell stage were obtained from patients undergoing IVF and were cultured in Ham's F-10 medium supplemented with 10% human sera or cocultured with ovarian cancer cells in the same medium. Embryos that reached blastocyst stage (n = 148) were analyzed by reverse transcriptase-polymerase chain reaction for HLA-G and beta 2 microglobulin (beta 2m) expression. Statistical analysis was performed to identify possible factors associated with variability of expression. RESULTS: Approximately 40% of studied blastocysts had detectable expression of both HLA-G and beta 2m messenger RNA. In 46% of blastocysts, beta 2m alone was observed. Interestingly, sibling embryos from patients that became pregnant were significantly more likely to express HLA-G than embryos from patients that did not conceive as a result of their IVF cycles. No association was found between HLA-G expression and culture conditions, patients age, or infertility diagnosis. CONCLUSION: The population of embryos obtained through IVF is heterogeneous in expression of HLA-G and beta 2m, which may reflect overall health of the embryos. Blastocysts showing positive HLA-G expression may have increased viability and implantation potential, although the underlying mechanisms remain to be elucidated. PMID- 8612865 TI - Spatio-temporal pattern of proliferation of immunoglobulin G-containing astrocytes in the injured mouse cerebral hemisphere. AB - Following a mechanical injury of the cerebral hemisphere in adult mice, [3H]thymidine was injected at different time intervals to reveal proliferating cells. Brain sections were double immunostained for immunoglobulin G (IgG) and glial fibrillary acidic protein (GFAP), and subjected to autoradiography. The procedure allowed to distinguish four cell types: (1) proliferating process bearing cells immunopositive for IgG but expressing no GFAP (GFAP-/IgG+ PB cells) (2) proliferating and (3) non-proliferating astrocytes immunopositive both for GFAP and IgG (GFAP+/IgG+), (4) proliferating astrocytes expressing GFAP and containing no IgG (GFAP+/IgG-). Thereafter, the distribution of each cell type within the injury area was recorded at each stage of the experiment and displayed as contour line maps. During the whole posttraumatic period, areas of very strong proliferation of GFAP-/IgG+ PB-cells were located within deep layers of the cerebral cortex and in the white matter around the bottom part of the lesion. Few proliferating GFAP+/IgG+ astrocytes were mainly located in the same regions. However, changes in the distribution of proliferating astrocytes containing no IgG followed different spatio-temporal pattern. On the 4th day after injury areas of their proliferative activity surrounded both the bottom and superficial parts of the lesion. It seems that the immediate contact with the extravasated blood serum does not determine IgG cellular uptake. The uptake appears to depend on the cell type and its role in re-establishing the extracellular space in the injured brain tissue. PMID- 8612866 TI - In vitro effect of Hepes buffer on maintaining the number of gap junction plaques in human myometrium at term. AB - In human myometrium, at various stages of labour and in correlation with the concentration of progesterone and estradiol in maternal blood the formation of gap junctions has been described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated human myometrium at term to Hepes buffer, on the number of gap junction plaques. Biopsies of myometrium were obtained from 5 pregnant women at term: they had an elective caesarean operation in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer, trimmed under a stereo microscope into small strips and kept in warm (37 degrees C) oxygenated Hepes buffer supplemented with glucose (0.01 mM). Then some strips of myometrium were incubated at 37 degrees C for 10, 20, 40, 55, and 180 min. The number of gap junction plaques were counted: it decreased in strips of myometrium after 10, 20, 40, 55, and 180 min of in vitro experiment (P < 0.01 vs control). In some of our experiments, the decrease in quantity of gap junction plaques was very dramatic and far below 50% of their numbers found in control specimens but never reached the zero value. PMID- 8612867 TI - Changes in actin cytoskeleton are involved in the cytopathic action of Candida albicans upon human skin fibroblasts. AB - The infection of human skin fibroblasts grown in culture with Candida albicans causes the death of infected cells, as assayed with the trypan blue exclusion test. The cell death is preceded by: (1) an attachment of germ tubes to the perinuclear surface of fibroblasts; (2) the disappearance of stress fibers and the disorganization of F-actin cytoskeleton and (3) cell rounding and detachment from the substratum. The results provide evidence for the role of F-actin cytoskeleton as a target in the cytopathic activity of C. albicans against human skin fibroblasts. PMID- 8612869 TI - Neuroendocrine cells in the gills of the bowfin Amia calva. An ultrastructural and immunocytochemical study. AB - Neuroendocrine (NE) cells were localized by electron microscopy and immunocytochemistry in the gill epithelium of bowfin Amia calva. The NE cells are dispersed in whole epithelium of the gill as solitary cells without intraepithelial innervation. All the observed NE cells do not reach the surface of the epithelium. The NE cells are characterized by a large nucleus with patches of condensed chromatin, numerous mitochondria, a well developed Golgi apparatus and a few dense core vesicles of various size scattered in the cytoplasm. Dense core vesicles range from 100 to 560 nm in diameter, while a clear space between the electron dense core ant the limiting membrane ranges from 20 to 240 nm. Immunocytochemical observations reveal the presence of general neuroendocrine markers such as neuro-specific enolase and bioactive substances: serotonin, leu enkephalin and met-enkephalin. we demonstrated the presence of endothelin - for the first time in fish - and suggested a local paracrine role for the NE cells. Some ultrastructural aspects and the immunocytochemical characteristics of NE cells of bowfin gills are common with those encountered in such cells of other lower vertebrate species. PMID- 8612868 TI - Occurrence and coexistence of some neuropeptides in nerve fibers supplying the bovine ovary and its extrinsic blood vessels. AB - Single- and double-labelling immunofluorescence was applied to investigate the occurrence and coexistence of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P (SP) in nerve fibers supplying the bovine ovary as well as its extrinsic blood vessels. We also studied the coexistence of these neuropeptides with tyrosine hydroxylase (TH) and neuropeptide Y (NPY) to determine features of "chemical coding" of nerve fibers innervating this organ. The bovine ovary was found to be supplied by VIP- and SP immunoreactive (VIP- and SP-IR) nerve fibers. No intraovarian CGRP-IR nerve terminals were detected. Immunoreactivity to VIP was shown in a moderate number of nerve fibers occurring in all ovarian regions - ovarian hilus, cortex and medulla. VIP-IR nerves were found to innervate blood vessels and ovarian follicles, especially primordial and primary ones. Immunoreactivity to SP was detected only in solitary intraovarian nerve fibers which were also encountered in all ovarian regions, but with special preference to ovarian medulla, where they innervated blood vessels. SP-IR nerve fibres were sometimes observed in close vicinity to primordial and primary follicles. Double-labelling immunofluorescence revealed that some VIP-IR nerve fibres supplying intraovarian blood vessels were simultaneously TH- or NPY-IR. Small numbers of VIP-IR fibres contained immunoreactivity to SP. All SP-IR nerve terminals were TH-or NPY negative. The ovarian artery as well as arteries of the paraovarian plexus were moderately supplied with VIP-, or by single CGRP- or SP-IR nerve fibres. Veins were innervated by only solitary nerve terminals containing these neuropeptides. The following patterns of coexistence were determined in nerve fibres supplying the investigated blood vessels: VIP, VIP/CGRP, VIP/SP, CGRP/SP, VIP/NPY. No distinct differences in the innervation of the ovary and its extrinsic blood vessels between juvenile and adult cows were found. PMID- 8612870 TI - Monoclonal antibodies recognized bovine leukocyte antigens used in immuno scanning electron microscopy. AB - Antibovine leukocyte monoclonal antibodies were tested in order to use them for scanning electron microscopy in backscattered electron imaging mode. The tests revealed that numerous antibovine leukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types. The results of these tests are presented and discussed. Clearcut differences in the surface morphology exist among bovine peripheral blood normal lymphocytes. Expression of IgM and IgG molecules was observed first of all on the surface of B lymphocyte microvilli. PMID- 8612871 TI - Histochemical demonstration of DNA in osteocytes from dinosaur bones. AB - Osteocytes were isolated from bones of dinosaur, Tarbosaurus bataar, aged approximately 80 million years. About 10% of sections of such osteocytes revealed the presence of DNA, the latter being demonstrated histochemically using Feulgen's method and staining with ethidium bromide. An attempt was made to explain why DNA remnants could be detected only in sectioned osteocytes which all originated from bones of two (out of ten) specimens. Scanning electron micrographs showed that while in the majority of osteocytes the metaloorganic sheath developed during fossilization was damaged and porous, in some it was well preserved and intact. It is suggested that an intact cell sheath enabled the preservation of DNA, though it simultaneously prevented the penetration of reagents and dyes into the cells in unsectioned material. In osteocytes with porous sheaths, the preservation of nucleic acids until today would not be possible. PMID- 8612873 TI - Regulation of calcitonin secretion by thyroid parafollicular cells in vitro. AB - The studies were performed on cultured TT cells originating from human thyroid medullary carcinoma (i.e., from parafollicular cells of the thyroid). The amount of released calcitonin was dependent upon calcium level in the medium. Moreover, calcitonin secretion might be regulated by medium supplementation with polypeptide hormones. Somatostatin inhibited while glucagon and pentagastrin stimulated calcitonin secretion to t he medium. Calcitonin secretion was also influenced by biogenic amines and their precursors. Dihydroxy-1-phenylalanine and serotonin augmented while 5-hydroxy-1-tryptophan and dopamine inhibited calcium secretion. This, calcitonin secretion may be controlled by different substances present in the healthy organism. This points to a complex control of calcium ion level in the blood. PMID- 8612872 TI - The diverse effect of topoisomerase I specific inhibitor (camptothecin) on normal and BCR/ABL-dependent hematopoietic cells proliferation: therapeutic implications. AB - Camptothecin (CPT), a specific topoisomerase I inhibitor, in the presence of hematopoietic growth factors exerted an antiproliferative effect against normal bone marrow cells (NBMC) as well as chronic myelogenous leukemia-chronic phase (CML-CP) and blast crisis (CML-BC) cells. In the absence of growth factors, however, only the colony formation by CML-BC cells was inhibited by CPT, leaving NBMC and CML-CP cells intact or much less affected. Analysis of the cellular DNA content revealed that CPT induced specific changes in cell cycle distribution: decrease in S and G2/M fraction with simultaneous accumulation of the cells in G1 phase and the appearance of "sub-diploid" (apoptotic) peak. To determine if CPT is able to exert selective antileukemic effect, 1:1 mixture of NBMC and CML-BC cells was exposed to CPT in the absence of growth factors and assayed for growth ability in clonogenic assay and for expression of BCR/ABL transcript in single colonies. BCR/ABL transcript was not detected in colonies incubated with CTP, in contrast, most of colonies arising from untreated cells possessed leukemic origin (BCR/ABL expression). Our results indicate that CPT is selectively effective in vitro against the leukemia cells. This offers the prospect of a novel and more selective treatment of CML. PMID- 8612874 TI - Visualization of accessory nuclei from oocytes of the sawfly Athalia rosae (Hymenoptera: Tenthredinidae) by a spreading technique. AB - Oocytes of hymenopterans are equipped with specific organelles termed accessory nuclei (AN). These organelles are surrounded by a double envelope and contain RNA positive inclusions. We have employed the spreading technique to analyse AN isolated from the ooplasm of the sawfly Athalia rosae. In electron microscopic spreading preparations AN appear as membranous, extensively folded structures. In their vicinity, numerous nuclear pore complexes have been found. This observation confirms the presence of pores in the envelopes of accessory nuclei. PMID- 8612876 TI - Localization of highly repetitive, species-specific EcoRI elements from Lupinus luteus L. AB - Using Southern, dot-blot and in situ hybridization, molecular and cytological localization of repetitive Lupinus luteus DNA sequence was shown. Under CsCl gradient centrifugation conditions CG-rich satellite fraction appeared. Dot-blot hybridization clearly indicated that 1070bp repetitive element being a member of previously described EcoRI fragments family appeared only in the main band. The use of that DNA fragment as an in situ hybridization probe revealed the hybridization signal in the euchromatin area. PMID- 8612877 TI - Practice goodwill will be good 'til? PMID- 8612875 TI - Restriction enzyme buffers and 5-azacytidine induce loss of protein and banding in plant metaphase chromosomes. AB - The incubation of root meristem of Vicia faba L. with 5-azacytidine for 72 h (5 cell cycles) resulted in shorter DNA fragments after digestion with a restriction enzyme Sau3A. Under the same condition in situ digestion of metaphase chromosomes with Sau3A revealed a stronger banding (C-banding-like) pattern in 5-azacytidine treated chromosomes than in the control. This difference could hardly be attributed to the euchromatin demethylation as 5-azacytidine caused a loss of chromosomal proteins; similar effect was found as a result of incubation with some restriction endonuclease buffers which did not diminish the labeling with 3H thymidine. After 5-azacytidine administration the entrance of cells into mitosis was delayed, the metaphase chromosomes were undercondensed and the pattern of DNA replication remained unchanged. PMID- 8612878 TI - Expect a rising tide of environmental regulation. PMID- 8612879 TI - Where do you stand? PMID- 8612881 TI - At home in a historic setting. PMID- 8612880 TI - Managed care: trends in the transfer of financial risk. PMID- 8612882 TI - Does your dental laboratory add value? PMID- 8612883 TI - Office identity. PMID- 8612884 TI - Apppearance-related dentistry--an entire portrait-analysis concept. PMID- 8612885 TI - A painless local-anesthetic technique that works. PMID- 8612886 TI - The joy of dentistry. PMID- 8612887 TI - Office reflects a growing practice. PMID- 8612888 TI - More practices are filing electronic claims. PMID- 8612890 TI - Thriving in the worst of times. PMID- 8612889 TI - Auxiliary pay continues to rise. PMID- 8612891 TI - Build your practice through referral-prospecting. PMID- 8612892 TI - Current developments in the esthetic inlay/onlay. PMID- 8612893 TI - Radiosurgery: the answer to quality dentistry and increased production. PMID- 8612894 TI - Increasing treatment acceptance with the intraoral camera. PMID- 8612895 TI - Doctor, you've got a gun to your head! PMID- 8612896 TI - Communicating with elderly patients. PMID- 8612897 TI - Six steps for promoting an urban practice. PMID- 8612898 TI - Leaders seek excellence. PMID- 8612899 TI - Hiring and firing in the 1990s. PMID- 8612900 TI - Become a humorous dentist! PMID- 8612901 TI - 'The practice I've always wanted'! PMID- 8612902 TI - Diagnosis for sealants. PMID- 8612904 TI - Excellence in complete dentures. PMID- 8612903 TI - A conservative technique for beautifying the smile. PMID- 8612905 TI - Wide implants for wide teeth. PMID- 8612906 TI - Advancing your practice with dental lasers. PMID- 8612907 TI - Are we playing to win or playing to keep from losing? PMID- 8612908 TI - Dentists more optimistic in 1995 practice survey. PMID- 8612910 TI - Business office is centrally located. PMID- 8612909 TI - High-income dentists see more patients. PMID- 8612911 TI - Thriving in dentistry. PMID- 8612912 TI - Are dental lasers coming of age? PMID- 8612914 TI - Decathlon dentists achieve fulfillment. PMID- 8612913 TI - The treatment coordinator. PMID- 8612915 TI - Don't sell your practice short! PMID- 8612916 TI - Nerve transpositioning to facilitate implant placement. PMID- 8612917 TI - Dynamic marketing for the implant practice. PMID- 8612918 TI - Clinical and statistical analysis of a comprehensive implant reconstructive practice. PMID- 8612919 TI - Implant placement in patients with atrophic jaws. PMID- 8612920 TI - Radiopaque surgical stents: a practical office technique. PMID- 8612921 TI - Esthetics and the argon ion laser. PMID- 8612922 TI - Kiss bad breath goodbye. PMID- 8612923 TI - The search for an ideal provisional material. PMID- 8612924 TI - Managing managed care--an ethical conundrum. PMID- 8612925 TI - Managed care--coming to you. PMID- 8612926 TI - Dental fees keep pace with low inflation rate. PMID- 8612927 TI - Quality or quantity--you decide. PMID- 8612928 TI - Key issues in office leasing. PMID- 8612929 TI - Designed for subtle elegance. PMID- 8612930 TI - Moving toward elective dentistry. PMID- 8612931 TI - Information overload. PMID- 8612932 TI - Proving negatives or pursuing positives. PMID- 8612933 TI - Writing an office manual. PMID- 8612934 TI - Office reflects the Southwest. PMID- 8612935 TI - Presenting the annual software systems review. PMID- 8612936 TI - Computer-based options for your dental practice. PMID- 8612937 TI - Dental high-tech equals dental high-touch. PMID- 8612938 TI - Dentistry with an intraoral camera. PMID- 8612939 TI - Choosing suture materials and needles. PMID- 8612940 TI - Ridge preservation--the future practice of dentistry. PMID- 8612941 TI - The reference denture system: simplified, uncompromised complete dentures. PMID- 8612942 TI - Supplies and the profit line. PMID- 8612943 TI - Infection control--no: patient protection--yes! PMID- 8612944 TI - Accurate dies and well-fitting crowns using three-in-one, all-plastic impression trays. PMID- 8612945 TI - The successful mandibular denture implant--part two. PMID- 8612946 TI - Hear that big noise? It's getting louder! PMID- 8612947 TI - The right time to discuss fees. PMID- 8612948 TI - Advanced cosmetic dentistry. PMID- 8612949 TI - Insurance coverage shouldn't affect diagnosis. PMID- 8612950 TI - Steps to successful retirement planning. PMID- 8612951 TI - A patient/client survey can yield valuable information. PMID- 8612952 TI - Are you mad at me? PMID- 8612953 TI - Combining art and dentistry. PMID- 8612955 TI - The developmental consequences of RNA processing. Introduction. PMID- 8612954 TI - Using the existing prosthesis as a provisional restoration. PMID- 8612956 TI - Translational control in spermatogenesis. AB - Translational silencing phenomena during spermatogenesis in the two model systems Drosophila and mouse are reviewed. Cis-acting sequences were identified in both species that are necessary for translational repression. While in Drosophila these elements so far have only been found in the 5' untranslated region (5' UTR), in the mammals such regions were identified both in the 5' as well as in the 3' UTR. In all cases, RNA-binding proteins interact with these regions, yet their specific role in the observed negative regulation of translation has to be established. PMID- 8612957 TI - The developmental consequences of alternate splicing in sex determination and differentiation in Drosophila. PMID- 8612958 TI - mRNA localisation during development. AB - Although there are many differences, mRNA localisations in the Xenopus oocyte show some tantalizing similarities to those occurring in Drosophila development. As in Drosophila, transcripts localise to opposite poles of the oocyte, this localisation is hierarchical and occurs in a multistep process in which localisation is followed by anchoring at the cortex. This distinction between initial transport and long-term maintenance reflects the dynamic nature of the cytoskeleton: the microtubule tracks form and reform according to the needs of the cell so that stable localisation must be mediated by a more constant structure--the cortex. A possible exception is the localisation of gurken mRNA where it is unknown whether there are separate mechanisms for transport to and maintenance at the oocyte nucleus. However, gurken is responsible for the transmission of a transitory signal; once this has been received, and the fate of the recipient follicle cells determined, there is no further need for localisation. It is possible that the time scale over which the localisation machinery is stable is sufficient for transmission of this signal without the need for a separate maintenance phase. The existence of a nanos homologue, Xcat-2 (Mosquera et al., 1993), associated with the Xenopus germ plasm is particularly interesting because of the morphological and functional similarities between Drosophila polar granules, Caenorhabditis P-granules, and Xenopus germ plasm. These electron-dense protein-RNA complexes are maternally supplied and in each case segregate with the germ line. These granules may represent a fundamental conserved pathway to germ-cell specification and it is now at least a possibility that they are also involved in establishing the embryonic axis through translational repression. In the case of Drosophila, this occurs through localised nanos acting (via Pumilio) on nanos response elements in hunchback mRNA. No such regulatory pair has yet been demonstrated in C. elegans or X. laevis, but each contains a candidate for one half of the interaction: glp-1 could be a target for an unidentified nanos-like protein; Xcat-2 may control translation of an unknown NRE-containing mRNA. Another common feature of mRNA localisation is that in every case where the targeting signal has been determined, it has been mapped to a region of the 3' UTR capable of forming an extensive secondary structure (e.g., David and Ish-Horowicz, 1991; Dalby and Glover, 1992; Gavis and Lehmann, 1992; Kim-Ha et al., 1993; Kislauskis et al., 1993, 1994; Lantz and Schedl, 1994). In several cases, translational control and transcript stability signals have also been mapped to these regions (Jackson and Standart, 1990; Standart et al., 1990; Standart and Hunt, 1990; Davis and Ish Horowicz, 1991; Wharton and Struhl, 1991; Dalby and Glover, 1993; Evans et al., 1994; Kim-Ha et al., 1995). The large secondary structures may provide a means for stably exposing sequence-specific regions of RNA to proteins. Due to the ease with which RNA forms base pairs, it is likely that rather than remaining single stranded, RNA within the cell forms some sort of secondary structure. The geometry of purely double-stranded RNA does not permit sequence specific interactions between proteins and the bases because the major groove is inaccessible to amino acid side chains (Weeks and Crothers, 1993). However, the distortions to the dsRNA helix provided by bulges, pseudoknots, and the single strand loop regions in stem-loop structures do present sequence information that can be "read" by proteins. The extensive 3'UTRs may produce a stable secondary structure which ensures that regulatory elements remain exposed in such regions rather than hidden in double-stranded stems. (ABSTRACT TRUNCATED) PMID- 8612959 TI - Developmental role of transcription factor isoforms generated by alternative splicing. AB - The production of transcription factor isoforms by developmentally regulated alternative splicing of pre-mRNAs is a widespread phenomenon. Frequently, differences in biochemical function among the isoforms can be predicted from sequence analysis, and in many instances such differences have been demonstrated in vitro or in cultured cells. A great variety of strategies for functional diversification can be classified into three main types: modulation of DNA binding specificity or affinity, production of activators and antagonists from the same gene, and modulation of dimerization properties. Despite obvious implications in many cases, the actual developmental consequences are understood only in a few instances. The roles inferred from these examples are diverse, ranging from developmental switches that have profound effects on pathways of differentiation to mechanisms that may optimize or fine-tune transcription factor function in different contexts. PMID- 8612960 TI - Heterochrony and the phylotypic period. AB - There has been a resurgence of interest in comparative embryology. It is now important to be able to compare gene expression in different species at similar developmental stages. One phenomenon which may make it difficult to compare embryos in this way is heterochrony--a change in developmental timing during evolution. It is not clear whether heterochrony can affect the intermediate stages of embryonic development, when many important genes involved in pattern formation are expressed. A prevalent view is that these so-called phylotypic stages are resistant to evolutionary change because they are when the body plan is laid down. Haeckel's famous drawings, which show different vertebrates developing from virtually identical somite-stage embryos, are still used to support this idea. I have reexamined the morphological data relating to developmental timing in somite-stage embryos. The data reveal striking patterns of heterochrony during vertebrate evolution. These shifts in developmental timing have strongly affected the phylotypic stage, which is therefore poorly conserved and is more appropriately described as the phylotypic period. This is contrary to the impression created by Haeckel's drawings, which I show to be inaccurate and misleading. The study of gene expression in embryos which show heterochrony could give important insights into evolutionary and developmental mechanisms. PMID- 8612961 TI - Cytoskeletal control of myogenesis: a desmin null mutation blocks the myogenic pathway during embryonic stem cell differentiation. AB - A differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates the in vivo cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation. PMID- 8612962 TI - The epidermis is a source of directional information for the migrating pronephric duct in Ambystoma mexicanum embryos. AB - In the urodele Ambystoma mexicanum, the pronephric duct (PND) is formed from a coherent group of cells that migrate from the pronephros to the cloaca along a pathway immediately ventral to the developing somites. The guidance cues used by the migrating PND primordium to find the cloaca are a local property of the migration substratum, are temporally regulated, and are both polarized and oriented. Since the pronephric duct migrates between two tissues--the underlying lateral mesoderm and the overlying epidermis--we performed a study to identify the tissue(s) in which PND guidance cues originate. Through direct manipulation of the epidermis overlying the duct pathway, we show that the migrating PND reads epidermally derived cues (1) along the anterior-posterior axis that direct migration from anterior to posterior and (2) along the dorsal-ventral axis that constrain migration to the duct pathway. Heterochronic grafting experiments reveal that the ability to direct PND migration is a stable property of flank epidermis throughout the period of PND migration. Epidermal cues are, therefore, not responsible for the observed temporal restrictions on PND migration. Thus, the region of the embryo within which the advancing PND tip can migrate actually represents an area where two distinct but required sets of PND migration cues overlap. The epidermis overlying the duct pathway provides directional information; temporal restriction of duct migration is hypothesized to be a property of the flank mesoderm. PMID- 8612964 TI - Fetal brain subdivisions defined by R- and E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. AB - We found that R-cadherin, a Ca2(+)-dependent cell--cell adhesion molecule, is expressed in restricted regions of the mouse fetal brain, as was found for E cadherin previously. R-cadherin delineated a subset of alar domains within forebrain neuromeres and certain future nuclei, while E-cadherin was expressed in another distinctive pattern. When cells were collected from various local regions of the fetal brain, dissociated, and reaggregated under the conditions in which only cadherins are active for cell aggregation, R-cadherin-positive and -negative cells segregated from one another. Similar results were obtained for E-cadherin. Such segregation of cells was, however, suppressed when the cadherins were inactivated either by Ca2+ depletion or with blocking antibodies. These results suggest that cadherins confer region-specific adhesiveness on fetal brain cells and that this process may take part in brain segmentation. PMID- 8612963 TI - Sequence and expression of grasshopper antennapedia: comparison to Drosophila. AB - We have cloned and characterized the Antennapedia (Antp) gene from the grasshopper Schistocerca americana. The Antennapedia protein contains seven blocks of sequence, including the homeodomain, that are conserved in the homologous proteins of other insects, interspersed with (usually repetitive) sequences unique to each species. There is no similarity between 1.8 kb of 3' untranslated sequence in grasshopper and Drosophila. We examined Antennapedia protein expression in grasshopper using an antibody raised against a grasshopper fusion protein and reexamined its expression in Drosophila using several different antibodies. Early patterns of expression in the two insects are quite different, reflecting differing modes of early development. However, by the germband stage, expression patterns are quite similar, with relatively uniform epithelial expression throughout the thoracic and abdominal segments which later retracts to the thorax. Expression is observed in muscle pioneers, the peripheral nervous system, and the central nervous system (CNS). In the CNS expression is initially limited to a few neurons, but eventually becomes widespread. Both insects show strong expression in certain homologous identified neurons and similar temporal modulation of expression. PMID- 8612965 TI - Hairless promotes stable commitment to the sensory organ precursor cell fate by negatively regulating the activity of the Notch signaling pathway. AB - In Drosophila imaginal discs, the function of the Hairless (H) gene is required at multiple steps during the development of adult sensory organs. Here we report the results of a series of experiments designed to investigate the in vivo role of H in sensory organ precursor (SOP) cell specification. We show that the proneural cluster pattern of proneural gene expression and of transcriptional activation by proneural proteins is established normally in the absence of H activity. By contrast, single cells with the high levels of achaete, scabrous, and neuralized expression characteristic of SOPs almost always fail to appear in H mutant proneural clusters. These results indicate that H is required for a relatively late step in the development of the proneural cluster, namely, the stable commitment of a single cell to the SOP cell fate. We also show that expression of an activated form of the Notch receptor leads to bristle loss with the same cellular basis--failure of SOP determination--as loss of H function and that simultaneous overexpression of H suppresses this effect. Finally, we demonstrate by epistasis experiments that the failure of stable commitment to the SOP fate in H null mutants requires the activity of the genes of the Enhancer of split complex, including groucho. Our results indicate that H promotes SOP determination by antagonizing the activity of the Notch pathway in this cell, thereby protecting it from inhibitory signaling by its neighbors in the proneural cluster. We propose a simple threshold model in which the principal role of H in SOP specification is to translate a quantitative difference in the activity of the Notch pathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision. PMID- 8612966 TI - The Ames dwarf gene is required for Pit-1 gene activation. AB - The tissue-specific POU domain transcription factor Pit-1 is required for phenotypic specification of three cell types in the anterior pituitary gland of mammals: somatotropes, thyrotropes, and lactotropes. Mutations in the Pit-1 gene, as first described in Snell and Jackson dwarf (dw) mice, led to pituitary hypoplasia due to lack of these three cell types, as well as hypothyroidism and dwarfism because of deficiency of thyroid-stimulating hormone and growth hormone, respectively. The Ames dwarf (df) exhibits a phenotype identical to that of Pit-1 mutated mice but the defective gene has been mapped to a locus on mouse chromosome 11, distinct from the Pit-1 gene located on mouse chromosome 6. Our studies indicate that initial activation of the Pit-1 gene is deficient in the Ames dwarf. This suggests that the df gene is required for activation of the Pit 1 gene and provides evidence for a hierarchy of tissue-specific factors required for cellular commitment in the anterior pituitary gland. PMID- 8612967 TI - Neuronal production of fibronectin in the cerebral cortex during migration and layer formation is unique to specific cortical domains. AB - The distribution of fibronectin (FN) changes rapidly during early development of the cerebral cortex, but its cellular source is not known. With in situ hybridization we find two spatially and temporally distinct periods of FN mRNA expression in the embryonic and early postnatal cortex of the mouse. Before and during formation of the preplate by the first postmitotic neurons, FN mRNA levels are high throughout the telencephalic vesicle, deep in the neuroepithelial proliferative zone that contains dividing cells and the cell bodies of radial glia; expression in the cortical proliferative zone is limited to the period of neurogenesis. Just after the cortical plate is formed within the preplate, FN mRNA is expressed in the intermediate zone, which contains migrating neurons, and in the cortical plate, where neurons migrate past their predecessors to form layers. Brefeldin A treatment of an organotypic slice preparation demonstrates FN production in the intermediate zone and cortical plate, in locations that correspond exactly to the distribution of FN mRNA by in situ hybridization. FN producing cells immunolabel with neuron-specific markers; in the intermediate zone and lower cortical plate they have morphological features characteristic of migrating neurons and are closely apposed to radial glia. FN mRNA expression and protein production continue in neurons of the cortical plate through the period of layer formation and then are downregulated. Examination of dissociated cortical cells by laser confocal microscopy confirms that FN accumulation after brefeldin A treatment is intracellular in neurons as well as in glia. Neuroepithelial expression of FN mRNA takes place throughout the telencephalon; FN produced by neurons is restricted to cells migrating toward and into specific cortical domains that include neocortex, insular and perirhinal cortex, and subiculum. Thus FN may be involved initially in supporting the cell division and fate determination that takes place in the neuroepithelium; later production by migrating neurons may play a role in the selection of radial glial pathways that lead to specific cortical regions, and in interactions between neurons as they form cortical layers within these regions. PMID- 8612968 TI - Different roles for fibronectin in the generation of fore and hind limb precartilage condensations. AB - Fibronectin expression and spatiotemporal distribution were examined in relation to the distinctive patterns of mesenchymal condensation and chondrogenesis seen in high-density serum-free cultures of chicken wing and leg bud precartilage cells. More fibronectin protein was produced on a per cell basis by leg than by wing mesenchyme, both in freshly isolated tissue and during the prechondrogenic condensation period in culture, where the difference was twofold. The quantitative difference in fibronectin expression in freshly isolated wing and leg mesenchyme was also seen at the level of total and poly (A)+ RNA. During the condensation phase, fibronectin was distributed in the wing and leg mesenchymal cultures in a way that prefigured the eventual distribution of cartilage in these cultures: in wing cultures condensations were broad and flat, and rich in diffusely organized fibronectin; in leg cultures, condensations were compact and spheroidal, and contained abundant deposits of fibronectin. In addition, the leg condensations were connected by long fibronectin-rich fibers. Transient treatment with TGF-beta early during the culture period led to increase in fibronectin production and expansion of condensations in both wing and leg cultures. Leg mesenchyme was more responsive to transforming growth factor-beta than wing mesenchyme with respect to fibronectin production, and this was reflected in a greater enhancement of cartilage formation in later cultures. Treatment of cultures with monoclonal antibody 304 directed against the amino-terminal heparin binding domain of fibronectin inhibited condensation formation and reduced chondrogenesis in wing mesenchyme, but left these two processes unchanged in leg mesenchyme, despite disruption by the antibody of the leg-specific fibronectin fibers. These studies indicate that for both wing and leg mesenchyme the morphology, extent, and spatiotemporal regulation of precartilage condensation and subsequent chondrogenesis closely parallels the deposition of fibronectin. But whereas the interaction between cells and fibronectin in wing bud mesenchyme is mediated in part by the protein's amino-terminal domain, this domain does not appear to be involved in analogous interactions in leg bud mesenchyme. PMID- 8612969 TI - Expressed ryanodine receptor can substitute for the inositol 1,4,5-trisphosphate receptor in Xenopus laevis oocytes during progesterone-induced maturation. AB - Two structurally related forms of intracellular calcium release channels that can mediate the release of intracellular calcium have been identified: the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate receptors (IP3R). Each channel responds to distinct pathways for activation. The IP3R is activated by IP3 and the RyR is thought to be activated by calcium or by another second messenger cADP ribose. It has been proposed that each type of channel subserves a specialized pool of intracellular calcium, and it is not understood why some cell types require more than one form of intracellular calcium release channel. The present study was designed to examine whether the RyR can substitute for the IP3R during oocyte maturation. IP3R expression was inhibited in Xenopus laevis oocytes using antisense oligonucleotides. These oocytes, with reduced levels of IP3R, demonstrated a marked delay in the time course of progesterone-induced maturation. The cloned skeletal muscle RyR1 was then expressed in X. laevis oocytes that were deficient in IP3R. Functional studies showed that the properties of the cloned RyR1, expressed in oocytes, were comparable to those of the native RyR1. X. laevis oocytes deficient in IP3R, but expressing RyR1, were able to undergo progesterone-induced maturation with a time course comparable to that seen in wild-type oocytes when caffeine was used to activate RyR and induce intracellular calcium release. These studies show that RyR1 can substitute for the IP3R as the intracellular calcium release channel required for Xenopus oocyte maturation and that intracellular calcium release is important for controlling the rate of progesterone-induced maturation. PMID- 8612970 TI - An ECM-bound, PDGF-like growth factor and a TGF-alpha-like growth factor are required for gastrulation and spiculogenesis in the Lytechinus embryo. AB - Growth factors and the extracellular matrix have been shown to fulfill vital developmental roles in many embryonic systems. Our hypothesis is that a developmental role played by the extracellular matrix in sea urchins may be the binding of a PDGF-like growth factor to promote signaling activity. We report here that anti-human PDGF-B antibodies and anti-human TGF-alpha antibodies immunoprecipitated specific proteins isolated from Lytechinus embryos. Addition of these antibodies to Lytechinus embryos inhibited gastrulation and spiculogenesis. The embryos are sensitive to the antibodies from the four-cell through the hatching blastula stages, which suggests that the TGF-alpha-like and PDGF-like ligands are required for the early differentiation of the gut and spicules. We present evidence that the PDGF-like growth factor depends on the extracellular matrix for signaling activity. Synthetic peptides representing the heparan sulfate proteoglycan binding sequence on human PDGF-B were added to Lytechinus embryo cultures to compete for binding sites with the endogenous PDGF like growth factor. The experimental peptide inhibited gastrulation and caused radially arranged multiple spicules to form. Development was unaffected by a control peptide. These studies support our hypothesis and suggest that TGF-alpha like and PDGF-like growth factors induce signaling events required for sea urchin gastrulation and spiculogenesis and suggest that an extracellular matrix associated PDGF-like growth factor is involved in differentiation along the oral aboral axis. PMID- 8612971 TI - Four-dimensional microscopic analysis of the filopodial behavior of primary mesenchyme cells during gastrulation in the sea urchin embryo. AB - During gastrulation of the sea urchin embryo, primary mesenchyme cells (PMCs) migrate from the vegetal pole to a site below the equator of the embryo where they form a ring-like structure and begin producing the larval skeleton. As these cells migrate, they extend and retract filopodia which appear to interact with the basal lamina and underlying ectoderm. To better characterize this behavior in vivo, we studied PMC migration using differential interference contrast (DIC) microscopy in combination with four-dimensional imaging (x, y, z space and time). We were able to determine the persistence and direction of extension of each filopodium and were also able to observe the dynamic behavior of each using colorized movie loops. This analysis showed that: (1) Most filopodia are quite transient, usually persisting for less than 0.5-6.0 min, during which time they continuously survey their surroundings; (2) PMCs extend an average of 121 filopodia/hr during migration; (3) the initial direction of extension of filopodia from the cell body is random, with just as many filopodia projecting toward as away from the direction of migration; (4) as a consequence of (2) and (3) above, each PMC explores the area surrounding its cell body approximately once every 5 min; (5) PMCs nearer to the target site migrate faster than those located farther away. To further investigate filopodial distribution, confocal microscopy was used to collect z series of PMCs transplanted to different locations in the embryo and fixed during migration. We found that more filopodia tended to be distributed toward the target site as cells approached the ring, suggesting that filopodial distribution may reflect regional differences in directional cues. PMID- 8612972 TI - Identification of inducing, responding, and suppressing regions in an experimental model of notochord formation in avian embryos. AB - The notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0-250 micrometer lateral to the streak and 500-750 micrometer caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250-500 micrometer lateral to the streak and 0-750 micrometer caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates. PMID- 8612973 TI - Changes in responsiveness to extracellular ATP in chick skeletal muscle during development and upon denervation. AB - Skeletal muscles in developing chick embryos were tested for responsiveness to adenosine 5'-triphosphate (ATP), a substance known to depolarize chick skeletal muscle in culture. The sensitivity to extracellular ATP was tested at various stages of development in five different muscles; pectoralis superficia, anterior latissimus dorsi, posterior latissimus dorsi, sartorious, and gastrocnemius. At the earliest time that muscles were tested (Embryonic Day 6, stage 30 of Hamburger and Hamilton, 1951) application of ATP(50-100 microM) elicited vigorous contractions in all five muscles, but within a few days (Embryonic Day 17, stage 43) none of the muscles contracted in response to ATP. Sensitivity declined at approximately the same time in all five of these muscles. Intracellular recordings made from muscle fibers near the time of hatching (Embryonic Days 18 21 or Postnatal Days 1-2) indicated that the loss of the ability to contract in response to ATP was due to the total loss of responsiveness to ATP. Surgical denervation of the anterior latissimus dorsi and posterior latissimus dorsi was performed in a series of chicks 1-2 days after hatching, and the ability of these muscles to contract in response to ATP was tested 3-10 days after the surgery. Contractions in response to ATP were present in many of the muscles. Thus denervation of muscles in newly hatched chicks led to the reappearance of sensitivity to ATP. The disappearance of ATP responsiveness shortly after muscles become innervated and the reappearance of ATP responsiveness following denervation suggest that the expression of ATP responsiveness is regulated by motor neurons. PMID- 8612974 TI - Dorsal and ventral cell types can arise from common neural tube progenitors. AB - To challenge the developmental potential of dorsal neural tube cells and test whether single neuroepithelial cells can give rise to the full range of neural tube derivatives, we grated a notochord lateral to the closing neural folds. This results in juxtaposition of dorsal and ventral cell types, by inducing floor plate cells and motor neurons dorsally. Clonal analysis with the vital dye lysinated rhodamine dextran showed that both "dorsal" and "ventral" neural tube derivatives can arise from a single precursor. Cells as diverse as sensory ganglion cells, presumptive pigment cells, roof plate cells, motor neurons, and floor plate cells were observed in the same clone. The presence of such diversity within single clones indicates that the responses to dorsal and ventral signals are not mutually exclusive; even in the early neural tube, neuroepithelial cells are not restricted to form only dorsal or ventral neural tube derivatives. PMID- 8612975 TI - trkC-mediated NT-3 signaling is required for the early development of a subpopulation of neurogenic neural crest cells. AB - Soon after they segregate from the neural tube, trunk neural crest cells disperse on two spatially and temporally distinct pathways. Only crest cells that migrate early and ventromedially give rise to neurons of the peripheral nervous system. It is also known that neural crest cell-derived populations require appropriate environmental cues early in development in order to generate neurons, and for the subsequent survival of differentiated neurons. We examined whether neurotrophin-3 (NT-3), a survival factor for subsets of peripheral neurons, is also involved in the regulation of neurogenesis by neural crest cells. First, we found that premigratory and migrating neural crest cells on the medial migration pathway of Embryonic Day 2.5 (E 2.5) embryos express mRNAs encoding multiple isoforms of the NT-3 receptor, trkC, as do cells in the neural tube and epithelial dermamyotome. On E4, a subpopulation of neurons in nascent sensory ganglia express trkC message. Second, we demonstrate that trkC mRNA is only expressed in neural crest cell populations that possess neurogenic potential. Third, we show that the presence of NT-3, during the initial development of cultured neural crest cells, is required for neurogenesis by a subpopulation of neurogenic neural crest derived cells. These results suggest that a subpopulation of neurogenic neural crest cells expresses functional trkC receptors and requires the timely availability of NT-3 for their development before reaching their final embryonic locations. We suggest that developmental heterogeneity exists in the identity and requirements of neural crest cell subsets that harbor neurogenic potential. We also suggest that the "paradoxical" expression of trkC receptors by the somitic dermamyotome may, in fact, play a role in the exclusive development of crest derived neurogenic precursors on the medial pathway by limiting the availability of NT-3 on the lateral pathway. PMID- 8612976 TI - Analysis of neural-responsive myogenin upstream sequences by myoblast implantation. AB - The expression of myogenin is suppressed during innervation and has been implicated in determining properties of skeletal muscle which are regulated by electrical activity. We previously reported that transcription driven by 3700 bp of the mouse myogenin upstream sequence (MYG3700) is activated by denervation in transgenic mice (Nucleic Acids Res. 21, 5684-5693, 1993). To extend our investigation of the activity dependence of the myogenin promoter, we have utilized myoblast implantation as a novel approach to in vivo reporter analysis. Myoblasts for hindlimb injections were generated by stable transfection of chloramphenicol acetyltransferase (CAT) reporters into a beta-galactosidase expressing line of C2 cells. In vitro characterization of stable myoblast clones carrying myogenin-CAT deletion constructs revealed that while the proximal myogenin 5'-flanking sequence confers myotube specificity, high-level expression requires a region upstream (-335 to -1102) which depends on chromosomal integration for its function. For analysis by implantation, incorporation of injected myoblasts into existing myofibers was confirmed by histochemical staining. Using clonal myoblasts harboring nicotinic receptor alpha-subunit (alpha 800) and myosin light chain receptors as positive and negative controls, respectively, for denervation responsiveness, we determined that the nuclei of injected myoblasts are susceptible to regulatory signals imposed by nerve-induced electrical activity of the myofiber into which they incorporate. In in vivo analysis of myogenin upstream sequence by implantation, CAT activities of MYG3700 and MYG1565 reporters in injected limbs increased up to fourfold within 4 days after denervation, whereas the activities of MYG1102 and MYG335 were unchanged. By 10 days after denervation, all myogenin reporters displayed denervation responsiveness. These implantation data suggest an early phase of denervation activation, one that is mediated by control elements residing within -1102 to 1565 of the myogenin upstream sequence. Thus, the combined analyses of stable reporter myoblast lines in vitro and in vivo by implantation provide an efficient means of evaluating regulatory regions for high-level expression and neural modulation of muscle gene transcription. PMID- 8612977 TI - Halves of epithelial somites and segmental plate show distinct muscle differentiation behavior in vitro compared to entire somites and segmental plate. AB - Medial and lateral halves of the somite are known to differ with respect to their developmental fates: Cells from the medial half of the somite give rise to the epaxial muscle of the back and cells from the lateral half of the somite give rise to the skeletal muscles of the limbs and the ventrolateral body wall. To get a better insight into myogenic determination of somite hemispheres, isolated entire somites as well as medial and lateral parts of somites and of segmental plate from 2 day chick embryos were explanted in vitro. These parts of the paraxial mesoderm were also cocultured in contact with somite surrounding tissues such as neural tube lacking floorplate, neural tube including notochord floorplate complex, and intermediate mesoderm, which were examined with respect to their muscle promoting or inhibiting influences. Skeletal muscle differentiation was monitored by the use of anti-myosin heavy chain antibody (MF20). It is shown that medial and lateral halves of segmental plate and epithelial somites are capable of undergoing myogenesis in the absence of axial organs. In contrast, cultures of intact segmental plate and epithelial somites from the same levels did not show muscle differentiation. Neural tube lacking floorplate promoted muscle differentiation in the medial halves especially of epithelial somites and also of segmental plate, but not in the lateral halves of the paraxial mesoderm at these levels. Intermediate mesoderm was found to inhibit muscle differentiation in medial and lateral halves of segmental plate and of epithelial somites. We further demonstrate that the arrangement of the myoblasts within tissue cultures is influenced by the presence or absence of axial organs. PMID- 8612978 TI - A developmental role for the heterotrimeric G protein Go alpha in a migratory population of embryonic neurons. AB - The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. We have investigated the potential role of one specific G protein, Go(alpha), in the control of neuronal migration. During embryogenesis of the moth, Manduca sexta, an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Previously, immunohistochemical studies indicated the presence of Go(alpha) related proteins in the EP cells during migration. We have now verified this result, using probes derived from the Go(alpha) gene in Manduca. A clone containing the full-length coding domain for Go(alpha) was sequenced from a Manduca cDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Go(alpha) gene during embryogenesis. Go(alpha)-specific transcripts could first be detected in the EP cells several hours before the onset of their migration. The level of Go(alpha) expression in all of the EP cells continued to increase during migration, but subsequently was down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Go(alpha)-related protein in the EP cells. We also used a semi-intact culture preparation of staged embryos to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Go(alpha)-and Gi(alpha)-related proteins, inhibited the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Go(alpha) and Gi(alpha)) or cholera toxin (a selective activator of Gs(alpha)) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocked the inhibitory effects of mastoparan alone. These results suggest that Go(alpha)-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo. PMID- 8612979 TI - Propagated and nonpropagated calcium transients during egg activation in the annelid, Chaetopterus. AB - Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface. PMID- 8612980 TI - Initial cell-type choice in a simple eukaryote: cell-autonomous or morphogen gradient dependent? AB - Dictyostelium discoideum is a simple eukaryote that lives as an amoeba until starvation triggers aggregation. The aggregate forms a slug which then develops into a fruiting body with two main cell types, stalk and spore cells. Two mechanisms have been proposed to explain cell-type differentiation. Studies using expression of the ecmA gene as a prestalk cell marker indicated that gradients of morphogens determine cell fate in the slug. However, studies using dyes or the cysteine proteinase 2 (CP2) gene product as a prestalk cell marker indicated that cell autonomous factors such as cell-cycle phase at the time of starvation cause an initial choice of cell fate. To help resolve these differences, we have used transformed cells containing the promoter of the prestalk gene ecmA fused to beta galactosidase (Jermyn and Williams, 1991) to study the differentiation of Dictyostelium cells at low cell density, at which cell-to-cell interactions and morphogen gradients are minimal. We find that under all conditions of low cell density in which express ion the ecmA fusion gene occurs, it is invariably detected in less than 25% of the cells from a clonal population. This suggests that a cell-autonomous mechanism is involved in ecmA expression. We then used double-labeled immunofluorescence to examine the ontogeny of the CP2-positive and the ecmA-positive cells. In developing aggregates, 9 to 12% of the cells are CP2 positive from 12 to 24 hr of development. The ecmA-positive cells are first detected at 16 hr as a subset of the CP2-positive cells and then increase in number. At approximately 20 hr, the CP2-positive cells and the ecmA-positive cells are almost completely overlapping sets. By late development, all of the CP2 positive cells are ecmA-positive and an additional 10% of the CP2-negative cells are also ecmA-positive. This indicates that up to 20 hr development, ecmA is expressed only in CP2-positive cells. The data thus suggest that cell-cycle phase at the time of starvation causes an initial choice of cell type and that during later development other factors influence cell fate. PMID- 8612981 TI - Imaging protein kinase C activation in living sea urchin eggs after fertilization. AB - The fluorescent dye NBD-phorbol acetate was used to visualize the activation of protein kinase C (PKC) in living Lytechinus pictus eggs during fertilization. The dye interacts directly with PKC as determined using a competitive binding assay. Quantitative image analysis of sequential images from laser-scanning confocal microscopy showed a significant reorganization of the signal in the vicinity of the cortical granules and the plasma membrane that began immediately following fertilization and persisted up to 1 hr (P<0.0001). At the concentrations employed, the NBD-phorbol dye was not capable of inducing a significant translocation of the fluorescent signal to the membrane, nor did it appear to interfere with the cell cycle. It therefore seems likely that the present in vivo results reflect the previously reported in vitro activation of protein kinase C immediately subsequent to fertilization. Such an interpretation is parsimonious with the results of parallel subcellular fractionation experiments using an N terminal polyclonal antibody to sea urchin PKC which showed a significant (P<0.037) translocation of the enzyme from the cytosolic fraction to the membrane fraction 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to fertilization occurs at or near the egg plasma membrane, perhaps in association with arachadonic acid-rich cortical granules. PMID- 8612982 TI - Polarized expression of the receptor protein tyrosine kinase Cek5 in the developing avian visual system. AB - Receptor protein tyrosine kinases of the Eph subfamily have been proposed to play roles in pattern formation based on their distribution during embryonic development. Cek5 (chicken embryo kinase 5) and Cek8 (chicken embryo kinase 8) are Eph-related kinases highly expressed in the chicken embryonic retina. To assess their potential roles in the development of the visual pathway, we examined their distribution by immunoperoxidase labeling. Cek8 is expressed throughout the pathway of the retinal ganglion cell axons, including the nerve fiber layer of the retina, optic nerve, optic chiasm, and stratum opticum of the tectum. Cek5 immunoreactivity is highly concentrated in only a portion of the optic nerve and optic chiasm, and in retinal cultures, Cek5 is detected in neurons. This prompted us to examine the regional distribution of Cek5 in the developing retina and led to the observation that Cek5 is most concentrated in the ventral aspect. RT-PCR established that the differential regulation of Cek5 expression in different portions of the retina occurs at the transcriptional level. Immunoblotting analysis revealed that this unusual expression pattern is distinctive for Cek5, as three other members of the Eph subfamily, Cek4, Cek8, and Cek9, are evenly expressed across the dorsal-ventral axis of the retina. Both Cek5 and Cek8 are distributed in manners which are consistent with their regulating the outgrowth of retinal ganglion cell axons to the tectum. Furthermore, Cek5 represents the first signal transduction molecule found to exhibit the polarized pattern of expression predicted for proteins that control the specificity of the retinotectal projections. PMID- 8612984 TI - A novel TGF-beta-like gene, fugacin, specifically expressed in the Spemann organizer of Xenopus. AB - Using a differential screening strategy, we have cloned a novel Xenopus gene, fugacin, related to the transforming growth factor beta superfamily. Transcripts were detected primarily in the dorsal marginal zone of late blastula. Thereafter, they became highly localized to the blastopore lip of early gastrula and were not observed at later stages. This gene, which is most homologous to the mouse gene nodal, displays a new pattern of cysteine residues. These findings highlight the potential role of these growth factors during early vertebrate development. PMID- 8612983 TI - TTF-1 regulates lung epithelial morphogenesis. AB - TTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. In vitro, TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in an in vitro model of embryonic mouse lung branching morphogenesis. This strategy suppressed TTF-1 translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis. PMID- 8612985 TI - Protein tyrosine kinase activity during egg activation is important for morphogenesis at gastrulation in the sea urchin embryo. AB - Fertilization triggers a series of preprogrammed events functioning to activate egg metabolism, incorporate the paternal genome, and initiate development. The activity of protein tyrosine kinases during egg activation is required for several steps leading to the first cell division. We now present evidence for an additional protein tyrosine kinase-mediated event that occurs between 30 and 45 min after insemination and is not required until gastrulation, which occurs over 24 hr later. Eggs treated with protein tyrosine kinase inhibitors within this window of time cleaved and formed normal blastulae but could not gastrulate or undergo further development to the pluteus stage. These findings provide the first evidence that some of the control mechanisms used in later development are established during a brief period of time in the fertilized egg and require the action of one or more protein tyrosine kinases. PMID- 8612986 TI - The receptor tyrosine kinase QEK5 mRNA is expressed in a gradient within the neural retina and the tectum. AB - In the retinotectal system, positional information has long been postulated to take the form of molecular gradients within both the retina and the tectum. Recent reports have implicated Mek4, a member of the Eph (also named class V) family of tyrosine kinase receptors (RTKs), and two ligands, RAGS and ELF-1, in this process. Here, we report the cloning and distribution pattern of QEK5, another member of the Eph family of RTKs, isolated from a quail cDNA library. During retinal differentiation, QEK5 transcripts accumulate in a ventral to dorsal gradient within the retinal neuroepithelium, where its expression becomes restricted to the ganglion and bipolar cell layers. Within the tectum, QEK5 transcripts are detectable in a posterior to anterior gradient in the ventricular layer and newly formed superficial layers. The pattern of QEK5 expression in the retina and tectum is distinct from that of Mek4, suggesting that complex patterns of Eph RTKs and their ligands may play a role in cell-cell interactions involved in retinotectal projections and differentiation of the central nervous system. PMID- 8612987 TI - Expanded CD4+CD45RO+ phenotype and defective proliferative response in T lymphocytes from patients with Crohn's disease. AB - BACKGROUND & AIMS: An abnormal immune response may play a pathogenic role in Crohn's disease. The aim of this study was to determine the role of regulatory T cells in Crohn's disease. METHODS: T-cell phenotype and function were studied in blood lymphocytes from patients with Crohn's disease and a control group consisting of healthy donors and patients with ulcerative colitis. RESULTS: Flow cytometric studies showed a significant increase in the percentage of CD3+DR+ and CD4+CD45RO+ T cells in patients with Crohn's disease. T cells from patients with Crohn's disease and ulcerative colitis showed a defective proliferative response after stimulation with surface mitogenic ligands (phytohemagglutinin and anti CD28 or anti-CD3 antibodies). Soluble interleukin-2 receptor alpha was augmented in the Crohn's disease and ulcerative colitis groups. In the Crohn's disease group, impairment of T-lymphocyte proliferation was normalized by exogenous interleukin 2, although endogenous interleukin-2 production and interleukin-2 receptor alpha expression were normal. CONCLUSIONS: An in vivo expansion of CD4+ T lymphocytes with memory phenotype and impaired T-cell proliferation that can be restored by pharmacological amounts of interleukin 2 was found in patients with Crohn's disease. There is a severe immunodisturbance in the T-cell compartment of patients with either clinically active or inactive Crohn's disease. PMID- 8612988 TI - Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis. AB - BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer is characterized by early-onset colorectal cancer and the occurrence of various other cancers. The recent isolation of four mismatch repair genes responsible for hereditary nonpolyposis colorectal cancer allows for the identification of carriers within affected families. The purpose of this study was to assess the age-specific cancer risk in a large series of gene carriers. METHODS: Thirty-four families were studied by mutation analysis. In 19 of these families, pathogenic mutations were found at hMSH2 or hMLH1. Of 382 relatives, 124 had a mutation in hMLH1 and 86 in hMSH2. RESULTS: The lifetime risk of colorectal cancer was the same in both groups of gene carriers (80%). The risk of endometrial cancer was greater in hMSH2 gene carriers compared with hMLH1 gene carriers (61% vs. 42%), but the difference was not statistically significant. A very high relative risk of cancer of the small bowel (relative risk of >100) was observed in carriers of either gene. Only the carriers of hMSH2 mutations had a significantly increased relative risk of cancer of the urinary tract (kidney and ureter) (relative risk of 75.3), stomach (relative risk of 19.3), and ovaries (relative risk of 8.0). CONCLUSIONS: This study provides estimates of cancer risk that may contribute to the appropriate management of gene carriers within families with hereditary nonpolyposis colorectal cancer. PMID- 8612989 TI - Colorectal polyp counts and cancer risk in familial adenomatous polyposis. AB - BACKGROUND & AIMS: In familial adenomatous polyposis, colorectal cancer prevention is by prophylactic colectomy, but dietary or chemopreventative strategies have been recently proposed in low-risk individuals. The aim of this study was to define predictive risk factors for colorectal cancer in familial adenomatous polyposis. METHODS: Between 1918 and 1993, 317 patients underwent colectomy. A multivariate analysis was performed to assess the relationship between colorectal cancer risk and polyp count, sex, and age at colectomy. RESULTS: The median polyp count was 842 (range, 78-7500), and cancer was found in 22% of patients. Polyp count and age, but not sex, predicted cancer risk. Patients with >1000 polyps had 2.3 times greater risk of cancer than those with <1000 polyps (P=0.006). Synchronous cancers increased with poly count (P<0.05). Each 10-year age group had a 2.4-fold difference in cancer risk (95% confidence interval, 1.9-3.2; P<0.001). Four cases of cancer occurred in patients at low risk (younger than 30 years of age, <1000 polyps; 3.3%). CONCLUSIONS: More adenomas and older age are associated with a higher risk of colorectal cancer. However, cancer does occur in low-risk individuals and may be missed by surveillance, making alternatives to prophylactic surgery inadvisable. PMID- 8612990 TI - Helicobacter pylori in the drinking water in Peru. AB - BACKGROUND & AIMS: An association between water sources and the prevalence of Helicobacter pylori infection in Peruvian children was shown previously. The aim of this study was to confirm the presence of H. pylori in drinking water in the same community. METHODS: Forty-eight drinking water samples from different locations in pueblo jovenes (new towns) near Lima were collected. Samples were frozen until technology advanced to the point to the point at which H. pylori might be reliably detected. Immunomagnetic beads coated with anti-H. pylori immunoglobulin Gs were used to concentrate H. pylori, and two polymerase chain reaction assays based on different H. pylori genes were used. One was a polymerase chain reaction for the detection of the H. pylori adhesin subunit encoding gene, and the second was a previously validated H. pylori 16S ribosomal RNA reverse transcriptase-polymerase chain reaction. RESULTS: The expected 375 base pair fragment from the adhesin gene was amplified from 24 water samples. The expected 500-base pair fragment of the 16S ribosomal RNA and the 375-base pair fragment of the adhesin gene were amplified from 11 of the samples. CONCLUSIONS: These results confirm the presence of H. pylori in drinking water in Peru and are consistent with conclusions from a previous epidemiological study of the same population. This provides additional evidence for waterborne transmission of H. pylori in some environments. PMID- 8612991 TI - Risk indicators of delayed gastric emptying of solids in patients with functional dyspepsia. AB - BACKGROUND & AIMS: Although gastric dysmotility and dyspeptic symptoms are often associated, their relationship remains unclear. The aim of this study was to evaluate the relationship between gastric emptying abnormalities and clinical features in functional dyspepsia. METHODS: In 343 patients with functional dyspepsia, the gastric emptying of solids was measured by a radioisotopic technique and four dyspeptic symptoms (epigastric pain and burning, postprandial fullness, nausea, and vomiting) were measured as absent, mild, relevant, and severe, according to their influence on patients' usual activities. RESULTS: Delayed gastric emptying was detected in 33.5% of dyspeptics. Delayed gastric emptying was particularly frequent in patients characterized by female sex, low body weight, presence of relevant and severe postprandial fullness, nausea, vomiting, and absence of relevant and severe epigastric pain. Logistic regression showed that delayed gastric emptying was invariably associated with female sex and postprandial fullness (odds ratio, 2.34; 95% confidence interval, 1.45-3.75) and vomiting (odds ratio, 4.04; 95% confidence interval, 1.30-12.54) when coded as severe and only postprandial fullness (odds ratio, 3.78; 95% confidence interval, 1.78-8.01) when coded as relevant and severe. CONCLUSIONS: Female sex, relevant and severe postprandial fullness, and severe vomiting are independently associated with delayed gastric emptying of solids in patients with functional dyspepsia seen in a referral center. PMID- 8612992 TI - Effects of aging and gastritis on gastric acid and pepsin secretion in humans: a prospective study. AB - BACKGROUND & AIMS: Recent studies suggesting that gastric secretion does not decrease with aging included few elderly individuals and measured only acid secretion. The aims of this study were to measure gastric acid and pepsin output in 206 health Americans (age range, 18-98 years) and to compare secretion rates with gastric histology. METHODS: Immediately after basal and pentagastrin stimulated acid and pepsin outputs were measured, oxyntic biopsy samples were obtained. RESULTS: Gastric acid and pepsin output rates were similar in young (age range, 18-34 years) and middle-aged (age range, 35-64 years) groups. Stimulated acid output was reduced approximately 30% in the elderly (age range, 65-98 years). However, after adjustment for histology, Helicobacter pylori infection, and other variables, age had no independent effect on acid output. The decline in acid secretion in the elderly was primarily related to a higher prevalence of chronic atrophic gastritis and a lower prevalence of smoking. Pepsin output was reduced by approximately 40% in the elderly. After adjustment for other variables, age remained a robust predictor of reduced pepsin output. CONCLUSION: Although advancing age has no independent effect on gastric acid secretion, it is associated with reduced pepsin output independent of atrophic gastritis, H. pylori infection, and smoking. PMID- 8612993 TI - Tumor necrosis factor microsatellites define a Crohn's disease-associated haplotype on chromosome 6. AB - BACKGROUND & AIMS: HLA class II associations have been described in Crohn's disease (CD) and ulcerative colitis (UC) and may be markers for other closely linked genes that are involved in disease pathogenesis. The tumor necrosis factor (TNF) locus, which contains the genes for TNF-alpha and TNF-beta, is located on chromosome 6 within the major histocompatibility complex loci. To investigate potential genetic associations in inflammatory bowel disease at the TNF locus, we studied 75 patients with CD, 73 patients with UC, and 60 ethnically matched controls using microsatellite markers. METHODS: Five TNF microsatellite loci (TNFa, TNFb, TNFc, TNFd, and TNFe) were typed using polymerase chain reaction. RESULTS: A CD-associated allelic combination, TNFa2b1c2d4e1, was found in 24% of patients with CD, 4.1% of patients with UC (P=0.001; odds ratio, 7.4; CD vs. UC), and 6.7% of control subjects (P=0.01; odds ratio, 4.4 CD vs. controls). This TNF haplotype was associated with the previously described HLA-DR1/DQ5 combination in CD. CONCLUSIONS: The TNFa2b1c2d4e1 allelic combination is the strongest genetic risk factor described in CD and, with HLA class II alleles, defines a group of markers on chromosome 6 that extends from HLA class II to upstream of the TNF beta gene. PMID- 8612994 TI - Mucosally activated circulating human B cells in diarrhea express homing receptors directing them back to the gut. AB - BACKGROUND & AIMS: Recirculation of mucosal lymphocytes has been established in animals but not in humans. Specific antibody-secreting cells in the blood of patients with diarrhea, initially activated in gut mucosa, are potential recirculating cells. The aim of this study was to determine whether these cells circulate back to gut by analyzing their homing receptors. METHODS: Blood mononuclear cells, separated with immunomagnetic cell sorting into receptor positive and receptor-negative populations, were assayed for pathogen-specific antibody-secreting cells and all immunoglobulin-secreting cells using enzyme linked immunospot assay. RESULTS: The gut mucosa homing receptor alpha4beta7 was expressed more frequently on pathogen-specific antibody-secreting cells than on immunoglobulin-secreting cells of healthy controls (P<0.001). Conversely, L selectin, a homing receptor for peripheral lymph nodes, was found on remarkably fewer antibody-secreting cells of the patients compared with immunoglobulin secreting cells of controls (32.9% and 70.3%, respectively; P<0.001). Three to 6 months after the disease, specific antibody-secreting cells had disappeared and frequency of L-selectin-and alpha4beta7-expressing cells had returned to control levels. CONCLUSIONS: Circulating mucosally activated antibody-secreting cells express a set of homing receptors guiding them back to the gut. This provides evidence for recirculation of mucosal lymphocytes in humans. PMID- 8612995 TI - Lysophosphatidylcholine increases 3-Hydroxy-3-methylglutaryl-coenzyme A reductase gene expression in CaCo-2 cells. AB - BACKGROUND & AIMS: The small intestine plays an important role in cholesterol homeostasis. The aim of this study was to examine the regulation of cholesterol synthesis by lysophosphatidylcholine in intestinal cells. METHODS: CaCo-2 cells cultured on semipermeable supports were incubated with taurocholate and lysophosphatidylcholine, and cholesterol synthesis rate, 3-hydroxy-3 methylglutaryl-coenzyme A (HMG-CoA) reductase activity, mass, and messenger RNA abundance were estimated. RESULTS: Lysophosphatidylcholine increased the rate of cholesterol synthesis as estimated by HMG-CoA reductase activity and acetate or water incorporation into sterols. Reductase was also increased by lysophosphatidylinositol or lysophosphatidylethanolamine but not by lysophosphatidylserine. Lysophosphatidylcholine increased HMG-CoA reductase messenger RNA and mass, suggesting that lysophosphatidylcholine regulated reductase at the level of gene expression. The various lysophospholipids caused the efflux of cellular cholesterol into the apical medium, and the amount effluxed correlated with the observed increase in reductase activity. Adding cholesterol to micelles containing lysophosphatidylcholine prevented the increase in HMG-CoA reductase activity and mass. CONCLUSIONS: Lysophosphatidylcholine increased cholesterol synthesis by increasing the expression of HMG-CoA reductase at the level of the gene and protein. Efflux of cellular cholesterol and the need to replace this lost cholesterol account for the observed changes in cholesterol metabolism. PMID- 8612996 TI - Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. AB - BACKGROUND & AIMS: Keratinocyte growth factor (KGF) is known to enhance tissue repair in the skin; however, its role in the gastrointestinal tract is largely unknown. The aim of this study was to evaluate the effects of exogenous KGF in an experimental model of colitis in rats. METHODS: KGF was administered before or after induction of colitis with 2,4,6-trinitrobenzenesulfonic acid/ethanol. In the first two study groups, KGF (5 mg/kg) was administered intraperitoneally 24 hours and 1 hour before induction of colitis; animals were killed 8 hours (n=10) and 1 week (n=10) after injury. In subsequent study groups, KGF or vehicle treatment was begun 24 hours after the induction of colitis at doses of 5 (n=20), 1 (n=10), and 0.1 (n=10) mg/kg intraperitoneally and continued once daily for 1 week. Colonic tissue samples were evaluated macroscopically and microscopically for mucosal injury and assayed for myeloperoxidase activity. RESULTS: Administration of KGF after but not before induction of colitis significantly ameliorated tissue damage. Macroscopic necrosis and microscopic ulcerations were reduced by 40%-50% at KGF doses of 1 and 5 mg/kg. CONCLUSIONS: Exogenous KGF has a key role in mucosal healing in an experimental model of colitis in rats. PMID- 8612998 TI - Polyunsaturated phosphatidylcholine prevents stricture formation in a rat model of colitis. AB - BACKGROUND & AIMS: Polyunsaturated phosphatidylcholine stimulates collagen breakdown in experimental models of liver cirrhosis. Bowel strictures are characterized by excess deposition of collagen in the intestinal wall. The aim of this study was to investigate the effect of polyunsaturated phosphatidylcholine in the prevention of bowel strictures. METHODS: Colitis was induced by trinitrobenzenesulfonic acid. On day 21, the presence of strictures was assessed in control rats, rats with colitis, and phosphatidylcholine-fed (100 mg/day) rats with colitis. Furthermore, serum transforming growth factor beta1, collagen deposition, and collagenase activity in colonic tissue were measured in all groups. RESULTS: None of the control rats but 12 of 16 rats with colitis developed colonic strictures. In contrast, only 2 of 15 phosphatidylcholine-fed rats with colitis showed strictures. Collagen content was much higher in rats with colitis than in phosphatidylcholine-fed rats with colitis and control rats. Phosphatidylcholine-fed rats showed significantly higher collagenase activity in colonic tissue than rats with colitis and control rats. In an ancillary study, free linoleic acid-fed rats showed no differences when compared with rats with colitis. Stimulation of transforming growth factor beta1 was similar in all rats with colitis. CONCLUSIONS: Oral supplementation with polyunsaturated phosphatidylcholine prevents the accumulation of collagen in inflamed intestinal tissue and the formation of strictures. This effect is associated with an enhanced collagen catabolism. PMID- 8612997 TI - Neurohormonal modulation of rat enterochromaffin-like cell histamine secretion. AB - BACKGROUND & AIMS: Gastric mucosal cells and nerve terminals contain at least acetylcholine, adrenergic agents, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, serotonin, gamma-amino-butyric acid, neurokinins A and B, neurotensin, neuropeptide Y, peptide YY, gastrin-releasing peptide, somatostatin, and [Met5]enkephalin. Although some of these agents have been implicated as regulators of gastric acid secretion, their site and mechanism of action is not well understood. The aim of this study was to investigate whether local gastric neurotransmitters modulate acid secretion by regulating basal and gastrin-driven enterochromaffin-like (ECL) cell histamine release. METHODS: The effects of the above agents were investigated in a short-term 90% 95% pure ECL cell culture system. Cells were incubated with either the neuromodulator alone or in combination with gastrin for 10-40 minutes, and histamine secretion was measured by enzyme immunoassay. RESULTS: Acetylcholine, isoproterenol, and vasoactive intestinal polypeptide significantly stimulated basal and gastrin-driven histamine secretion, whereas calcitonin gene-related peptide and somatostatin inhibited basal and gastrin-driven histamine secretion. The M1 muscarinic receptor antagonist pirenzepine dose dependently inhibited the action of acetylcholine, whereas the M3 receptor antagonist 4-diphenylacetoxy-N (2-chloroethyl)-piperidine hydrochloride had no effect. the rest of the evaluated agents had no effect on ECL cell histamine secretion. CONCLUSIONS: These data are consistent with the hypothesis that substantial neurohormonal modulation of ECL cell function exists. PMID- 8612999 TI - Carrier-mediated jejunal absorption of conjugated bile acids in the guinea pig. AB - BACKGROUND & AIMS: Conjugated bile acid absorption is known to occur in the jejunum in mammals, but the mechanism has not been well defined. The aim of this study was to define the mechanisms by which conjugated bile acids are absorbed from the jejunum. METHODS: The steady-state absorption of eight conjugated bile acids from a perfused jejunal segment was measured in the anesthetized biliary fistula guinea pig. Experiments defined the effect of bile acid structure, tested for competitive inhibition and saturation of transport, and compared the absorption rate of taurine conjugates with that of glycine conjugates at pH 7.6 or 5.0. RESULTS: Dihydroxy conjugates were absorbed twice as rapidly as cholyl conjugates from the perfused jejunum; glycine and taurine conjugates of a given bile acid were absorbed at a similar rate. Absorption of ursodeoxycholate taurine showed saturability and competitive inhibition by other conjugated bile acids. When intraluminal pH was decreased to pH 5.0, the absorption rate of glycine (but not taurine) conjugates increased, indicating passive absorption of the protonated species of glycine-conjugated bile acids. CONCLUSIONS: Uptake of glycine- or taurine-conjugated bile acids by the guinea pig jejunum occurs by at least two mechanisms: carrier-mediated transport (dihydroxy conjugates greater than trihydroxy conjugates) and passive absorption in protonated (uncharged) form of glycine conjugates. PMID- 8613001 TI - Outbreak of acute hepatitis C following the use of anti-hepatitis C virus- screened intravenous immunoglobulin therapy. AB - BACKGROUND & AIMS: Hepatitis C virus (HCV) infection has been associated with intravenous (IV) immunoglobulin (Ig), and plasma donations used to prepare IV Ig are now screened to prevent transmission. Thirty-six patients from the United Kingdom received infusions from a batch of anti-HCV antibody-screened intravenous Ig (Gammagard; Baxter Healthcare Ltd., Thetford, Norfolk, England) that was associated with reports of acute hepatitis C outbreak in Europe. The aim of this study was to document the epidemiology of this outbreak. METHODS: Forty-six patients from the United Kingdom treated with Gammagard (34 exposed and 12 unexposed to the batch) returned epidemiological questionnaires. RESULTS: Eighty two percent of the exposed patients (28 of 34) became positive for HCV RNA. Eighteen percent of the patients (6 of 34) who had infusions with this batch tested negative for HCV RNA, but 2 of the patients had abnormal liver function and subsequently seroconverted to anti-HCV antibody positive. Twenty-seven percent of the patients (9 of 34) developed jaundice, and 79% (27 of 34) had abnormal liver transferase levels. Virus isolates (n=21), including an isolate from the implicated batch, were genotype 1a and virtually identical by sequence analysis of the NS5 region, consistent with transmission from a single source. CONCLUSIONS: Hepatitis C infection can be transmitted by anti-HCV-screened IV Ig. Careful documentation of IV Ig batch numbers and regular biochemical monitoring is recommended for all IV Ig recipients. PMID- 8613000 TI - Long-term survival in patients with hereditary hemochromatosis. AB - BACKGROUND & AIMS: The course of hereditary hemochromatosis may depend on the degree of iron overload and the time of therapeutic intervention. This analysis evaluates the impact of early diagnosis and iron removal on survival and complications in hereditary hemochromatosis. METHODS: A Cohort of 251 patients with hemochromatosis was followed up for 14.1 +/- 6.8 years. RESULTS: Survival was reduced in the total group of patients when compared with a matched normal population. Survival in noncirrhotic and nondiabetic patients and in patients diagnosed between 1982 and 1991 was identical with rates expected. Survival was reduced in patients with severe iron overload vs. those with less severe overload. The percentage of early diagnoses increased threefold between 1947 and 1969 to that between 1970 and 1981; there was only a further 20%-25% increase in the last decade. Deaths caused by liver cancer, cardiomyopathy, liver cirrhosis, and diabetes mellitus were increased as compared with expected rates. Liver cancers were associated with cirrhosis and amount of mobilizable iron but not with hepatitis B or C markers. CONCLUSIONS: Prognosis of hemochromatosis and most of its complications, including liver cancer, depend on the amount and duration of iron excess. Early diagnosis and therapy largely prevent the adverse consequences of iron overload. PMID- 8613002 TI - Expression and function of integrin receptors for collagen and laminin in cultured human hepatic stellate cells. AB - BACKGROUND & AIMS: Human hepatic stellate cells (HSCs), liver-specific pericytes, are currently considered major producers of extracellular matrix (ECM) components and key elements in the development of liver fibrogenesis. However, little is known about the possible functional interactions between HSCs and the various ECM components. Therefore, the present study was designed to evaluate the expression of integrins, the major family of extracellular matrix receptors. METHODS: Integrin expression was evaluated by immunoprecipitation and confirmed by immunocytochemistry and flow cytometry. RESULTS: Human HSCs were shown to express alpha1beta1, alpha2beta1, alpha(v)beta1. Adhesion to type IV collagen, type I collagen, fibronectin, and laminin 1 was inhibited by anti-beta1 antibody identifying beta1-containing integrins as possible receptors for these components. In addition, we showed that HSCs express alpha6beta4, a heterodimer known to mediate adhesion of epithelial cells to laminin and not previously characterized in mesenchymal cells. Adhesion to laminin 1 was not inhibited by antibodies specific for alpha6 or beta4, thus establishing that laminin 1 is not a ligand for alpha6beta4 in this cell type. CONCLUSIONS: These findings represent the first description of integrin receptors in HSC and provide an attempt to cover the gap of information in the field of HSC-ECM interactions. PMID- 8613004 TI - Effects of hepatic stimulator substance, herbal medicine, selenium/vitamin E, and ciprofloxacin on cirrhosis in the rat. AB - BACKGROUND & AIMS: Cirrhosis is a potentially lethal condition for which there is no proven effective therapy. The aim of this study was to compare the effects of hepatic stimulator substance, traditional Chinese herbal medicine, selenium plus vitamin E, and ciprofloxacin treatment on biochemical and histological features of fibrosis in rats with carbon tetrachloride (CCl4)/ethanol-induced cirrhosis. METHODS: One hundred twenty adult Wistar rats were divided into six study groups (20 rats/group): healthy controls, CCl4/ethanol-injured rats left untreated, and CCl4/ethanol-injured rats treated for 4 weeks with either hepatic stimulator substance, traditional Chinese herbal medicine, a combination of selenium plus vitamin E, or ciprofloxacin. After the 4-week treatment, rats were killed and the following parameters of hepatic fibrosis were determined: hepatic hydroxyproline and proline levels, serum hyaluronic acid concentrations, and histological staining of hepatic tissue. RESULTS: Hepatic fibrosis was significantly improved in all four treated groups compared with the untreated CCl4/ethanol-injured controls. Improvements were most striking in the groups treated with traditional Chinese herbal medicine and ciprofloxacin. CONCLUSIONS: The data indicate that hepatic stimulator substance, traditional Chinese herbal medicine, selenium plus vitamin E, and ciprofloxacin significantly decrease the amount of hepatic fibrosis caused by CCl4/ethanol injury in rats. PMID- 8613003 TI - Expression of cadherins and alpha-catenin in primary epithelial tumors of the liver. AB - BACKGROUND & AIMS: Cadherins and their associated molecules, such as alpha catenin, have been shown recently to play a pivotal role in epithelial carcinogenesis. METHODS: The expression of E-cadherin, N-cadherin, and alpha catenin in 10 normal samples, 28 focal nodular hyperplasias, 9 liver cell adenomas, 65 hepatocellular carcinomas, and 9 cholangiocarcinomas was studied by immunohistochemistry and Western blotting. RESULTS: In the normal liver, hepatocytes expressed E-cadherin and a 129-kilodalton cadherin identified by the anti-N-cadherin antibody GC4. The expression level of alpha-catenin was low. Bile duct cells expressed only E-cadherin and showed high levels of alpha-catenin. The expression of cadherins and alpha-catenin was preserved in focal nodular hyperplasia. In liver cell adenomas, cadherins and alpha-catenin were heterogeneously expressed. In hepatocellular carcinomas, cadherin and alpha catenin expression was frequently reduced or absent. Alterations in cadherin expression correlated with large tumor size, low grade of histological differentiation, and occurrence of capsular and vascular invasion. In cholangiocarcinomas, neoplastic cells inconstantly expressed E-cadherin and alpha catenin. CONCLUSIONS: Alterations of cadherin and alpha-catenin expression are frequent in liver cell adenomas and primary liver carcinomas. Their incidence in hepatocellular carcinomas is of prognostic significance. PMID- 8613005 TI - Increased blood hemoglobin attenuates splanchnic vasodilation in portal hypertensive rats by nitric oxide inactivation. AB - BACKGROUND & AIMS: Nitric oxide, which is quenched by hemoglobin, has been implicated in the pathogenesis of portal hypertension. The aim of this study was to investigate the effects of increasing blood hemoglobin concentration by erythropoietin treatment on the gastrointestinal vasodilation associated with portal hypertension. METHODS: Portal-hypertensive and sham-operated rats treated with erythropoietin were studied 2 weeks after surgery. Hemodynamic and rheological parameters were measured in baseline conditions and after N(G)-nitro L-arginine methyl ester (L-NAME) or sodium nitroprusside treatment. RESULTS: In portal-hypertensive rats, erythropoietin attenuated the increase in gastric mucosal and superior mesenteric artery blood flows and the decrease in arterial blood pressure and splanchnic vascular resistances. Those parameters were not affected by erythropoietin in sham-operated rats. A direct vascular effect of erythropoietin was ruled out by the lack of changes in blood pressure or mesenteric blood flow after intravenous erythropoietin administration and by a similar in vitro relaxation to acetylcholine in mesenteric artery rings. In portal-hypertensive rats, erythropoietin blunted the blood pressure response to sodium nitroprusside and attenuated the gastric and mesenteric blood flow response to L-NAME. CONCLUSIONS: Gastrointestinal vasodilation associated with portal hypertension can be attenuated by increasing blood hemoglobin concentration. Inactivation of overproduced NO by hemoglobin may account for this effect. PMID- 8613006 TI - Energy metabolism and regeneration in transgenic mouse liver expressing creatine kinase after major hepatectomy. AB - BACKGROUND & AIMS: The catalysis of a creatinine/phosphocreatine system by creatine kinase is not expressed in the liver. The aim of this study was to examine the energy energy metabolism and regeneration after hepatectomy using transgenic mouse liver expressing creatine kinase to clarify the effects of phosphocreatine on liver regeneration. METHODS: Transgenic mice were divided into two groups: group 1 was fed normal chow, and group 2 was fed 10% creatine chow for 5 days. Hepatic energy metabolism was evaluated before and after hepatectomies. Changes in remnant liver weight gain and bromodeoxyuridine labeling index were measured after 70% and 80% hepatectomies. RESULTS: Hepatic adenosine triphosphate level 24 hours after 70% hepatectomy was significantly higher in group 2 than group 1 (P<0.05). In group 2, mitochondrial adenosine triphosphate synthesis was enhanced because of elevated intramitochondrial adenine nucleotide content before hepatectomy, leading to sufficient adenosine triphosphate synthesis after a 70% hepatectomy. Bromodeoxyuridine DNA labeling index 24 hours after a 70% hepatectomy was significantly higher in group 2 than group 1. Rapid liver weight gain was observed in group 2 after a 70% hepatectomy. CONCLUSIONS: Abundant phosphocreatine promotes liver regeneration by reinforced hepatic energy metabolism. Gene transfer of creatine kinase to the liver may be a potential method in liver surgery. PMID- 8613007 TI - Antigen-presenting function and B7 expression of murine sinusoidal endothelial cells and Kupffer cells. AB - BACKGROUND & AIMS: Inflammatory liver disease as well as rejection of liver allografts are thought to be mediated by resident antigen-presenting cells in the liver. At the same time, in vivo antigen presentation in the liver appears to be a more tolerogenic than systemic antigen challenge. The aim of this study was to show and characterize the antigen-presenting capability of sinusoidal endothelial cells and Kupffer cells. METHODS: Purified murine sinusoidal endothelial cells and Kupffer cells were studied for their ability to serve as accessory cells and antigen-presenting cells by proliferation assays. They were also studied for their expression of interleukin 1 and the B7 costimulatory molecules by Northern blotting, polymerase chain reaction, and flow cytometry. RESULTS: Both cell types expressed interleukin 1 messenger RNA and could serve equally well as accessory and antigen-presenting cells. B7-2 messenger RNA and surface expression on sinusoidal endothelial cells and on Kupffer cells was shown. Antibodies to the B7 molecules inhibited antigen presentation. Addition of interleukin 10 as a regulatory cytokine secreted by Kupffer cells was suppressive. CONCLUSIONS: Sinusoidal endothelial cells carry functional B7-2 molecules and can serve as effective antigen-presenting cells. However, antigen presentation by sinusoidal endothelial cells may be locally down-regulated by interleukin 10. PMID- 8613008 TI - Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells. AB - BACKGROUND & AIMS: When rat hepatocyte regeneration after partial hepatectomy is blocked by 2-acetylaminofluorene, a proliferation of biliary epithelia sends out ductules into the parenchyma. The ability of these neoductules to act as a significant progenitor compartment for hepatocytes is in dispute. This study aims to resolve this question by varying the amount of 2-acetylaminofluorene administered. METHODS: Rats were fed 2-acetylaminofluorene fr 6 days before and up to 7 days after partial hepatectomy was performed at a dose of either 2.5 (low) or 5 (high) mg/kg(-1)/day(-1). The response was monitored by the immunohistochemical expression of intermediate filaments and cytochrome P450 enzymes. RESULTS: No regeneration by mature hepatocytes occurred with either dose, and new ductules expressed the biliary cytokeratins 7, 8, 18, and 19 and, in addition, vimentin. At the high dose, hepatocytic differentiation was infrequent, whereas apoptosis and intestinal differentiation were common. At the low dose, almost all ductules differentiated into hepatocytes within 14 days of hepatectomy. CONCLUSIONS: Biliary epithelium is an effective and substantiative hepatocyte progenitor compartment under appropriate conditions. PMID- 8613009 TI - Role of altered beta-adrenoceptor signal transduction in the pathogenesis of cirrhotic cardiomyopathy in rats. AB - BACKGROUND & AIMS: Attenuated ventricular contractility has been documented in cirrhosis, but the pathogenesis remains unclear. The beta-adrenergic receptor system is critical in modulating cardiac contraction. Therefore, the aim of this study was to clarify beta-adrenoceptor signaling function in a rat model of cirrhosis. METHODS: Cirrhosis was induced by bile duct ligation, whereas controls underwent a sham operation. Myocardial contractility was studied by measuring isolated left ventricular papillary muscle contraction under isoproterenol stimulation. Beta-Adrenoceptor signaling was evaluated by measuring adenosine 3',5'-cyclic monophosphate generation after stimulation with isoproterenol, sodium fluoride, and forskolin. Guanosine triphosphate-binding protein expression from ventricular plasma membranes was determined by Western blots to measure G(s)alpha, Gi2alpha, and G(common)beta, respectively. RESULTS: Maximum papillary muscle contractile responses in control and cirrhotic rats were 113% +/- 3% and 70% +/- 2% of basal, respectively (P<0.01), with no significant differences in the dose-inducing half-maximal response. Adenosine 3', 5'-cyclic monophosphate generation after stimulation with all three agents was significantly lower in cirrhotic compared with control rat membranes. G(s)alpha and Gi2alpha expression was significantly reduced in cirrhotics compared with controls, but G(common)beta expression remained unchanged. CONCLUSIONS: These data showed cardiac contractile impairment in cirrhosis, associated with altered beta-adrenergic receptor signaling function and guanine nucleotide-binding protein expression. These factors may play an important role in the pathogenesis of cirrhotic cardiomyopathy. PMID- 8613010 TI - Sex steroid metabolism and receptor status in hepatic hyperplasia and cancer in rats. AB - BACKGROUND & AIMS: Both androgenic and estrogenic steroids have been implicated in the development and course of several liver diseases, including hepatocellular carcinoma. The aim of this study was to investigate temporal changes in hepatic estrogen and androgen receptors and hormone metabolism in a rat model of liver hyperplasia and carcinogenesis. METHODS: Rats were fed hepatocarcinogenic peroxisome proliferator agents for 3 days to 10 months. Livers were examined for proliferation markers, activity and cellular distribution of sex steroid receptors, and key enzymes in sex hormone homeostasis. RESULTS: At all times, liver weight and proliferation markers in treated rats were increased. Early exposure resulted in increased nuclear estrogen and androgen receptor activity in treated rats. Tumors that developed after 9-10 months showed a marked decrease in estrogen receptor activity and, in contrast, an increase in androgen receptor activity, as did liver surrounding the tumors. Both short-term and long-term exposure to the carcinogens resulted in dramatic reductions in steroid metabolism. CONCLUSIONS: This study supports the thesis that, in preneoplastic stages such as hyperplasia, there is an elevation of both receptor activities and that the progression from hyperplasia to cancer results in suppression of estrogen receptor expression but maintenance of androgen receptor. PMID- 8613011 TI - Pancreatic thread protein is mitogenic to pancreatic-derived cells in culture. AB - BACKGROUND & AIMS: Pancreatic thread proteins (PTPs) are acinar cell products and members of the regenerating gene (reg) family. reg expression increases during islet regeneration, is depressed during aging-related islet dysfunction, and may be important in beta-cell growth and maintenance. The aim of this study was to examine the genetic expression of reg in pancreatic-derived cells in vitro and the mitogenic effect of PTP/reg protein on these cells. METHODS: reg gene expression was measured by Northern analysis in three rat pancreatic cell lines: ARIP (ductal), AR42J (acinar), and RIN (beta-cell). PTP/reg protein was isolated from bovine and human pancreas. Cells were cultured with PTP/reg for 72 hours, and thymidine incorporation was measured. RESULTS: reg messenger RNA was detected in AR42J but not in ARIP or RIN. PTP/reg protein was mitogenic to RIN and ARIP in a dose-related fashion but not to AR42J. It was not mitogenic to cultured mature rat islets. CONCLUSIONS: reg messenger RNA is expressed in acinar but not in beta cell or ductal pancreatic cell lines. PTP/reg protein was mitogenic to both beta cell and ductal cell lines but not to mature, nondividing islets. This supports the hypothesis that PTP/reg protein is an acinar cell-derived mediator of beta cell growth and may be involved in modulating the duct-to-islet axis. PMID- 8613012 TI - Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in pancreatic tumors. AB - BACKGROUND & AIMS: Cell-cycle inhibitor and tumor-suppressor gene p16/MTS1 was found to be altered in a variety of human tumors. To directly investigate genetic alterations and expression of p16/MTS1 and p15/MTS2, this study surveyed pancreatic tumors. METHODS: Cell-cycle inhibitors were analyzed for genetic alterations and expression by polymerase chain reaction, DNA sequencing, reverse transcription polymerase chain reaction, and Western blotting. RESULTS: The analysis of pancreatic adenocarcinoma (19 cell lines and 3 xenografts) for p16/MTS1 and p15/MTS2 revealed homozygous deletions in 10 of 22 cases (46%) (7 cell lines and 3 xenografts) involving both genes. We show in these 7 cell lines as well as in 3 additional cases (10 of 19[53%]) loss of p16/MTS1 transcripts and in 2 further cases (12 of 19 [63%]) mutations leading to the loss of p16 protein. The frequency of mutations in p16/MTS1 was 56% (5 of 9). In contrast to p16/MTS1, p15/MTS2 transcripts were obtained in all cases exhibiting the p15/MTS2 gene (54%). Loss of expression was not observed for p27 and p18. CONCLUSIONS: These results support that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behavior of this tumor type. PMID- 8613013 TI - Ursodeoxycholic acid reduces protein levels and nucleation-promoting activity in human gallbladder bile. AB - BACKGROUND & AIMS: Ursodeoxycholic acid prevents gallstone formation in selected patients. The aim of this study was to examine whether decreased concentration and nucleation-promoting activity of various proteins contribute to this beneficial effect. METHODS: Gallbladder bile of 13 patients with cholesterol gallstones treated with ursodeoxycholic acid (10 mg/kg(-1)/day(-1)) and of 13 untreated patients were compared. RESULTS: Total protein concentration in gallbladder bile (2.8 +/- 0.6 vs. 6.7 +/- 1.3 mg/mL; P=0.008) and concanavalin A binding fraction (0.16 +/- 0.03 vs. 0.42 +/- 0.07 mg/mL; P=0.003) were strongly decreased by ursodeoxycholic acid therapy. Significant decreases were also found for gallbladder bile alpha1-acid glycoprotein, haptoglobin, immunoglobulin (Ig) A, IgG, gamma-glutamyl transpeptidase, and aminopeptidase N but not for IgM, mucin, or beta-glucuronidase. Decreases were most pronounced for proteins of canalicular membrane origin. Gallbladder bile total protein correlated with cholesterol saturation index (r=0.54; P=0.0047) but not with bile salt hydrophobicity index. Crystallization-promoting activity of the concanavalin A binding fraction (assessed by nephelometry and microscopic examination) was also significantly decreased by ursodeoxycholic acid. CONCLUSIONS: Ursodeoxycholic acid strongly decreases levels of various proteins and nucleation-promoting activity in bile. PMID- 8613014 TI - Cell death by apoptosis: basic concepts and disease relevance for the gastroenterologist. AB - We have reviewed in a conceptual manner with appropriate scientific detail the role apoptosis plays in diseases of the gastrointestinal tract. This information should help the clinician and clinician investigator to better understand and treat gastrointestinal diseases. Future investigations in apoptosis probably will provide further insight into the cell-signaling cascades regulating apoptosis, the mechanism(s) by which Bcl-2 inhibits apoptosis, and the precise role of apoptosis in cancer initiation, promotion, and progression. We anticipate that in the future, the practicing gastroenterologist will have therapeutic strategies available to inhibit apoptosis for various gastrointestinal diseases associated with cell death and to induce apoptosis for the treatment of gastrointestinal neoplasia. PMID- 8613015 TI - Relationship between Helicobacter pylori eradication and reduced duodenal and gastric ulcer recurrence: a review. AB - BACKGROUND & AIMS: The aim of this study was to determine whether the current literature supports the use of Helicobacter pylori cure as the primary efficacy end point in peptic ulcer clinical trials. This could potentially reduce the complexity of future trials. METHODS: Published articles containing information on both H. pylori eradication and ulcer recurrence were searched with MEDLINE. Abstracts were found by reviewing references from both primary and review articles. RESULTS: Fourteen duodenal ulcer and five gastric ulcer studies satisfied requisite inclusion criteria. Ulcer recurrence was significantly less common among H. pylori-cured patients vs. noncured patients (6% vs. 67% for patients with duodenal ulcers; 4% vs. 59% for patients with gastric ulcers). For H. pylori-cured patients, duodenal ulcer recurrence was higher in studies using two endoscopic tests compared with three tests (9% vs. 3%) and higher in abstracts compared with published articles (14% vs. 4%). Timing of H. pylori eradication (4 weeks vs. < / = 12 weeks) and ulcer recurrence assessment (6 months vs. < / = 12 months) was not significantly related to duodenal ulcer recurrence. CONCLUSIONS: The current literature strongly suggests that H. pylori eradication 4 weeks after therapy should be used as the primary efficacy end point for reduced gastric and duodenal ulcer recurrence for the purpose of clinical trial design. PMID- 8613016 TI - High-resolution chromoendoscopy for the diagnosis of diminutive colon polyps: implications for colon cancer screening. AB - BACKGROUND & AIMS: A visual, nonbiopsy technique that could reliably determine the histology of diminutive colorectal polyps could greatly reduce the cost of colon cancer screening. This study was designed to report our experience using a high-resolution colonoscope combined with indigo carmine dye to diagnosis diminutive colorectal polyps. METHODS: Colonoscopy using a Fujinon EC-400 HM/HL was performed in 36 patients with polyps <10mm in diameter. Polyps from the first 12 patients (phase 1) were sprayed with 10 mL of 0.2% indigo carmine dye, and a biopsy was performed or a specimen removed and submitted for histological analysis. The morphological data were used to predict polyp histology in the subsequent 24 patients (phase 2). RESULTS: Hyperplastic polyps had a characteristic surface "pit pattern" of orderly arranged "dots" that resembled the surrounding, nonpolypoid mucosa. Adenomatous polyps had surface "grooves" or "sulci." Sensitivity and specificity of our techniques in distinguishing adenomatous from nonadenomatous colorectal polyps were 93% and 95% respectively. CONCLUSIONS: High-resolution chromoendoscopy provides morphological detail of diminutive colorectal polyps that correlates well with polyp histology. If incorporated into colon cancer screening, these techniques may limit the need for biopsy and/or subsequent colonoscopy and ultimately decrease costs. PMID- 8613017 TI - Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. AB - BACKGROUND & AIMS: Multiple studies show that continuous use of nonsteroidal anti inflammatory drugs (NSAIDs) lowers the risk of colon cancer in humans and carcinogen-treated rodents. One target for NSAIDs is cyclooxygenase (COX), and two isoforms of this enzyme have been identified: COX-1 and COX-2. The present study was undertaken to determine if there is differential expression of COX in colonic tumors in azoxymethane-treated rats. METHODS: COX-1 and COX-2 messenger RNA levels were determined by Northern blot analysis of total RNA isolated from colonic tumors and normal adjacent mucosa. COX-2 protein levels were determined by Western blotting analysis. Quantitation of relative band densities was performed using standard densitometry scanning techniques. RESULTS: There was a marked increase in COX-2 RNA levels in six of six colonic tumors compared with paired normal mucosa. In contrast, there was equivalent intensity of the COX-1 RNA transcript between the normal mucosa and tumor in all of the specimens examined. Western blotting analysis showed an increase in the level of the COX-2 protein in four of five of the colonic tumor samples. CONCLUSIONS: COX-2 but not COX-1 gene expression is markedly elevated in most colonic tumors examined in azoxymethane-treated rodents. COX-2 may provide a target for chemopreventive strategies for colorectal cancer. PMID- 8613018 TI - The American Gastroenterological Association legislative and public policy program: advocating for the patient, the clinician, and the academician. PMID- 8613019 TI - Training the gastroenterologist of the future: the gastroenterology core curriculum. The Gastroenterology Leadership Council. PMID- 8613020 TI - Abuse and functional gastrointestinal disorders: what is the link and should we care? PMID- 8613021 TI - Hemochromatosis: the impact of early diagnosis and therapy. PMID- 8613022 TI - Is safe always safe? Pooled plasma derivatives and viral hepatitis. PMID- 8613023 TI - Neoformation of liver epithelial cells: progenitor cells, stem cells, and phenotypic transitions. PMID- 8613024 TI - Endoscopy or empirical treatment for peptic ulcer disease: decisions, decisions. . . PMID- 8613025 TI - Weight loss in acquired immunodeficiency syndrome: wasting or wanting not? PMID- 8613026 TI - The short-bowel syndrome: new vistas. PMID- 8613027 TI - Mismatch repair function and neoplasia in ulcerative colitis. PMID- 8613028 TI - Endoscopy, nonsteroidal anti-inflammatory drug, and omeprazole regimen in colorectal cancer prevention. PMID- 8613029 TI - Apoptosis and proliferation: the balance. PMID- 8613030 TI - Gastrin-releasing peptide, acid secretion, Helicobacter pylori, and duodenal ulcer: another epiphenomenon? PMID- 8613031 TI - Expression of transforming growth factors alpha and beta in colonic mucosa in inflammatory bowel disease. AB - BACKGROUND & AIMS: Transforming growth factors (TGFs) alpha and beta are key regulatory peptides that modulate mucosal cell populations critical to inflammatory bowel disease. The aim of this study was to assess TGF-alpha and TGF beta expression in human colonic mucosa. METHODS: TGF-alpha and TGF-beta expression was assessed in colonic mucosa from patients with ulcerative colitis, patients with Crohn's disease, and controls by Northern blot analysis, in situ hybridization, and bioassay. RESULTS: TGF-alpha messenger RNA expression localized to the villous tips of the small intestine and the surface epithelium of the colon. TGF-alpha expression was enhanced 2.3-fold in inactive ulcerative colitis mucosa relative to active ulcerative colitis, Crohn's disease, or normal controls. Enhanced expression correlated with duration of disease. TGF-beta expression was increased in affected mucosa from both patients with ulcerative colitis and Crohn's disease with active disease. TGF-beta1 messenger RNA expression in ulcerative colitis and Crohn's disease localized mostly to cells of the lamina propria with the highest concentration in inflammatory cells closest to the luminal surface. CONCLUSIONS: TGF-alpha may contribute to epithelial hyperproliferation and the increased risk of malignancy in long-standing ulcerative colitis. TGF-beta may be a key cytokine during periods of active inflammation, modulating epithelial cell restitution and functional features of cells within the lamina propria. PMID- 8613032 TI - Angiocentric T-cell lymphoma of the intestine: a distinct etiology of ischemic bowel disease. AB - BACKGROUND & AIMS: Bowel ischemia and perforation is an infrequent but ominous event in patients with malignant lymphoma. The underlying pathogenesis of this association remains to be clarified in most cases. Seven unusual cases of angiocentric T-cell lymphoma of the intestine presenting with bowel ischemia or perforation are reported. Their clinicopathologic features were analyzed. METHODS: Clinical records and histopathology were reviewed. Immunophenotypic studies and EBER1 in situ hybridization for Epstein-Barr virus were performed. RESULTS: All patients (3 men and 4 women; mean age, 42 years) presented with acute abdominal pain, bloody stool, or bowel perforation. The intestinal lesions were ulcerated and transmurally necrotic or gangrenous. The angiodestruction of the tumor cells appeared to be responsible for the presenting bowel ischemia. The neoplastic cells expressed a pan-T-cell antigen CD45RO; 4 of them contained Epstein-Barr virus transcripts EBER1. The prognosis was grave; 6 patients died within 3 months of onset. CONCLUSIONS: Intestinal angiocentric T-cell lymphoma represents a distinct etiology of bowel ischemia. The condition should be taken into consideration, particularly in young adults with ischemic bowel disease. PMID- 8613033 TI - Folate synthesized by bacteria in the human upper small intestine is assimilated by the host. AB - BACKGROUND & AIMS: Some intestinal flora are known to synthesize folate. The aim of this study was to determine whether folate synthesized by small intestinal flora is assimilated by the human host. METHODS: Subjects with atrophic gastritis and healthy volunteers were studied before and after omeprazole administration. A double-lumen perfusion tube was placed in the duodenum. 3H-labeled P-aminobenzoic acid, a precursor substrate for bacterial folate synthesis, was perfused. Downstream intestinal aspirates and a 48-hour urine collection were obtained. RESULTS: Atrophic gastritis and omeprazole administration were associated with increases in duodenal pH and in small intestinal flora. Bacterially synthesized folates were isolated from the intestinal aspirates. Tritiated 5 methyltetrahydrofolate, a major metabolite of folate, was isolated from the urine of omeprazole-treated subjects in greater quantities than from drug-free subjects (P<0.01); the quantity of tritiated 5-methyltetrahydrofolate in the urine of the subjects with atrophic gastritis was similarly elevated. CONCLUSIONS: (1) Mild bacterial overgrowth caused by atrophic gastritis and administration of omeprazole are associated with de novo folate synthesis in the lumen of the small intestine; (2) the human host absorbs and uses some of these folates; and (3) the contribution to folate nutriture from this source remains unclear. PMID- 8613035 TI - Our new president--Tadataka Yamada, M.D. PMID- 8613034 TI - Health status by gastrointestinal diagnosis and abuse history. AB - BACKGROUND & AIMS: Standardized assessment of health status by diagnosis (functional vs. organic) and the relative influence of abuse history on health status have not been studied previously. The aim of this study was to estimate the health status and abuse history for gastrointestinal diagnoses among patients seen in a tertiary-care gastroenterology clinic and to evaluate the relative predictive effects of diagnosis and abuse history on health status. METHODS: Standardized measures of sexual and physical abuse history and six health status measures were estimated for the patients by diagnosis. Analysis of covariance was performed to determine the relative contributions of diagnosis type and abuse history on the health status measures. RESULTS: Patients with functional gastrointestinal diagnoses had poorer health status and a higher frequency of severe types of abuse than patients with structural diagnoses. Independent of abuse history, functional diagnosis was significantly associated with greater pain severity and psychological distress and poorer daily function. Independent of diagnosis, abuse history significantly contributed to greater pain severity, more days in bed, more psychological distress, and poorer daily function. CONCLUSIONS: The type of diagnosis and abuse history independently contributed to the health status of this population. Therefore, medical symptoms alone may not be sufficient to understand patients' health status. Attention must also be paid to contributing psychosocial factors. PMID- 8613036 TI - Passing the torch: a look back at our editorship. PMID- 8613037 TI - Survival and cause-specific mortality in inflammatory bowel disease: a population based cohort study. AB - BACKGROUND & AIMS: A population-based cohort study of 1251 subjects with Crohn's disease and 1547 subjects with ulcerative colitis, diagnosed in Stockholm between 1955 and 1984, was performed to examine the survival, changes in survival over time, and cause-specific mortality. METHODS: The cohort of patients was followed up in the National Cause-of-Death register until 1990. National mortality rates were used for comparisons. RESULTS: The observed vs. expected survival rate after 15 years was 93.7% (95% confidence interval [CI], 91.8%-95.7%) for Crohn's disease and 94.2% (95% CI, 92.4%-96.1%) for ulcerative colitis. Overall, 174 deaths occurred vs 115.42 expected (standardized mortality ratio, 1.51; 95% CI, 1.29-1.75) in Crohn's disease. In ulcerative colitis, 255 deaths occurred compared with 186.78 expected (standardized mortality ration, 1.37; 95% CI, 1.20 1.54). Inflammatory bowel disease was the major contributor to the elevated mortality rate, but mortality from colorectal cancer, asthma, and non-alcohol related liver diseases was increased in ulcerative colitis; mortality from other gastrointestinal diseases was increased in ulcerative colitis as well as in Crohn's disease. CONCLUSIONS: Data in the present study are compatible with the hypothesis that subjects with inflammatory bowel disease have an increased mortality compared with the general population. PMID- 8613038 TI - Identification of subjects at risk for colorectal carcinoma through a test based on K-ras determination in the stool. AB - BACKGROUND & AIMS: The gold standard for screening for colorectal carcinoma is colonoscopy. The aim of this study was to compare endoscopic results with those obtained using the noninvasive screening test of K-ras determination in the stool in a large population of patients undergoing colonoscopy. METHODS: Two hundred thirty consecutive patients were studied by K-ras amplification on stool-derived DNA using polymerase chain reaction and oligomer-specific hybridization. RESULTS: Wild-type K-ras was amplified in 103 of 230 patients (44.8%), the rate of amplification being directly proportional to the presence of an organic disease of the intestine characterized by hyperproliferating mucosa. In 30 of these 103 patients (29.1%), a K-ras mutation was found. Four of 5 with early colorectal carcinoma, all who had K-ras mutations in the tumor, were identified. In first degree relatives of patients with colorectal carcinoma, all subjects either carrying adenomas > 1 cm in diameter or multiple smaller adenomas were identified. In patients with inflammatory bowel disease, the test identified the only patient with neoplastic transformation. CONCLUSIONS: The sensitivity and specificity of K-ras determination on stool-derived DNA in patients with colorectal carcinoma, in first-degree relatives of patients with colorectal carcinoma, and in patients with inflammatory bowel disease support the opportunity of a large-scale trial to validate its use as a screening test. PMID- 8613039 TI - Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis. AB - BACKGROUNDS & AIMS: Patients with mucinous colon cancers often have a poor prognosis. The aim of this study was to determine whether metastatic potential depends on specific alterations in mucin-associated carbohydrate structures. METHODS: A quantitative scoring system was used to examine the expression of mucin-associated carbohydrates in paired human primary colon cancers and metastases and in cecal tumors and liver metastases from an animal model of metastasis. Adhesion of metastatic cells to basement membrane and endothelial ligands was examined. RESULTS: Metastases expressed a decrease in mucin core structures Tn and T, a reciprocal increase in sialyl T and sialyl Tn, and an increase in peripheral sialyl Le(x) compared with the primary tumors from which they arose. Altered expression of sialylated mucin structures resulted from selective metastasis of cells that produce sialomucins. Antibodies to sialylated epitopes or desialylation inhibited adhesion of metastatic cells to basement membranes. Neutralizing antibody to endothelial-associated E-selectin (a ligand for sialyl Le(x)) inhibited adhesion of metastatic cells to cytokine-activated hepatic endothelial cells, and inhibition of sialomucin with antisense to the MUC2 gene inhibited adhesion to E-selectin. CONCLUSIONS: Increased sialylation of mucin-associated carbohydrates is characteristic of colon cancer cells that are most likely to metastasize. Sialylated carbohydrate structures on mucin play a role in adhesive interactions involving both basement membrane and endothelial associated ligands. PMID- 8613040 TI - Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients. AB - BACKGROUND & AIMS: Mucosal cell-mediated immune response is considered the central event in the pathogenesis of celiac disease. In cultured intestinal explants from celiacs in remission, we have characterized the early stages of gliadin-induced immune activation. METHODS: Intestinal biopsy specimens (15 treated celiacs and 13 controls) were cultured with gliadin or maize prolamine digests for 24 hours as well as for 1, 2, 4, 6, 8, and 12 hours in some subjects. The expression of immunologic markers was detected by immunocytochemistry. RESULTS: Gliadin challenge may initiate two parallel pathways, one of which leads to T-cell activation and another that precedes it. Epithelial cells overexpress DR molecules after 1 hour, and in a second stage T lymphocytes become fully activated. Moreover, T lymphocytes migrate in the upper mucosal layers. T lymphocytes that migrate in the higher lamina propria compartments are mainly CD4+ and show markers of activation; migrating intraepithelial lymphocytes are CD8+ and do not express these markers. CONCLUSIONS: In vitro gliadin challenge is a suitable model to reproduce various immunologic features of celiac lesions; these may be caused by different pathways. The comprehension of these phenomena is essential to clarify the distinctive pathogenic mechanisms leading to disease and may help in defining novel therapeutic approaches. PMID- 8613041 TI - Cholecystokinin-A receptors modulate gastric sensory and motor responses to gastric distension and duodenal lipid. AB - BACKGROUND & AIMS: The combination of duodenal lipid and gastric distention induces meal-like fullness followed by nausea in healthy subjects. The aim of this study was to assess the role of cholecystokinin (CCK) A receptors in these changes using a CCK-A antagonist loxiglumide. METHODS: Twelve healthy subjects were studied on four occasions, during which either 0.9% saline or 20% Intralipid was infused intraduodenally on two occasions each (1 mL/min) while the proximal stomach was distended with air (100 mL/min). During each duodenal infusion, subjects received intravenous loxiglumide (10 mg.kg-1.h-1) on 1 day and placebo on the other. Intragastric pressure changes were recorded, and the subjects reported gastric sensations (fullness, nausea). RESULTS: Loxiglumide did not influence gastric motility or sensitivity during duodenal saline infusion. Duodenal lipid reduced gastric tonic and phasic pressure activity during distensions and induced meal-like fullness and nausea; sensations were reported at similar volumes but lower intragastric pressures (P < 0.001 vs. saline). Loxiglumide partially restored gastric tonic and phasic activity during lipid infusion, reduced the occurrence of meal-like fullness and nausea, and increased the pressures at which sensations were reported (P < 0.001 vs. placebo). CONCLUSIONS: CCK-A receptors are involved in the induction of meal-like fullness and nausea associated with intraduodenal lipid and gastric distention. PMID- 8613042 TI - A mechanism by which Helicobacter pylori infection of the antrum contributes to the development of duodenal ulcer. AB - BACKGROUND & AIMS: Helicobacter pylori infection and duodenal ulcer disease are firmly correlated. However, the bacteria do mainly colonize the antrum, indicating an indirect pathogenic mechanism. The aim of this study was to test a concept claiming that H. pylori infection of the antrum selectively blocks normal inhibitory reflex pathways to gastrin and parietal cells. METHODS: The effect of antral distention was studied on gastric acid secretion stimulated by pentagastrin and on gastrin release stimulated by gastrin-releasing peptide in H. pylori-infected and noninfected patients with and without duodenal ulcer disease, as well as after eradication of the bacteria. RESULTS: The inhibitory effect on gastric acid secretion induced by antral distention was absent in H. pylori infected patients irrespective of whether or not they had duodenal ulcer disease. The inhibitory mechanism was restituted in 8 of 10 patients within 9 months after successful eradication of H. pylori infection. Similar results were obtained in studies on gastrin release. CONCLUSIONS: H. pylori infection blocks normal, physiological inhibitory mechanisms from the antrum to both the gastrin cells and to the parietal cell region, resulting in increased gastrin release and impaired inhibition of gastric acid secretion, which will probably lead to an increased duodenal acid load as a general prerequisite for the development of duodenal ulcer disease. PMID- 8613043 TI - Intestinal permeability changes in response to acetylsalicylic acid in relatives of patients with Crohn's disease. AB - BACKGROUND & AIMS: Presence of a familial intestinal permeability defect in Crohn's disease remains controversial despite numerous studies. The purpose of this study was to determine whether detection of a permeability defect in first degree relatives of patients with Crohn's disease can be enhanced using an acetylsalicylic acid provocation test. METHODS: Lactulose-mannitol ratio, a measure of intestinal permeability, and total sucrose excretion, a measure of gastroduodenal permeability, were determined before and after ingestion of acetylsalicylic acid in healthy controls, in patients with Crohn's disease, and in the first-degree relatives of patients with Crohn's disease. Subjects were classified as hyperresponders if their results were above the mean of + 2SD of the controls. RESULTS: First-degree relatives had a 110% increase in intestinal permeability after acetylsalicylic acid compared with an increase of 57% in controls (P = 0.001). Thirty-five percent of relatives were classified as hyperresponders. There was no significant difference in the change in sucrose excretion between relatives and controls (259% vs 198%; P < 0.05). CONCLUSIONS: First-degree relatives of patients with Crohn's disease have an exaggerated increase in intestinal but not gastroduodenal permeability in response to acetylsalicylic acid. This study supports a familial permeability defect in Crohn's disease, which may not be present in all families. PMID- 8613044 TI - Linkage analysis identifies gene carriers among members of families with hereditary nonpolyposis colorectal cancer. AB - BACKGROUND & AIMS: Uncertainty about genetic risk in hereditary nonpolyposis colorectal cancer (HNPCC) may lead to unnecessary screening. The aims of this study were to show how gene linkage findings can elucidate who is at risk and requires intensive screening and how cancer control can be enhanced by screening high-risk family members. This information can be useful given the public health magnitude of HNPCC. METHODS: An extended family with HNPCC was studied using formal linkage analysis with DNA extraction from blood samples, followed by genotyping with polymerase chain reaction technique for microsatellite markers. Sixty-one blood relatives of a family with HNPCC, 5 of whom had colorectal cancer, and 12 unrelated family members underwent DNA sampling for genetic analysis. RESULTS: Linkage analysis showed that all 5 affected individuals had a haplotype with the same alleles 10/7/9, which was also detected in 13 first degree healthy gene carriers and absent in the remaining 43 non-gene carriers. In the asymptomatic subjects screened, one incidental colorectal cancer and four adenomas were detected in 3 of 6 gene carriers. An adenoma was found in 1 of 17 noncarriers; the remaining 16 noncarriers have undergone 67 unnecessary colonoscopies. CONCLUSIONS: Linkage analysis can differentiate gene carriers from non carriers. Colorectal cancer screening should be restricted to gene carriers. PMID- 8613045 TI - Botulinum toxin for achalasia: long-term outcome and predictors of response. AB - BACKGROUND & AIMS: Botulinum toxin injection into the lower esophageal sphincter of patients with achalasia results in effective short-term relief of symptoms. The aims of this study were to examine the long-term outcome of these patients and to determine the predictors of response to this therapy. METHODS: Thirty-one patients with achalasia treated with botulinum toxin were followed up prospectively for a median duration of 890 days. RESULTS: Twenty-eight patients improved initially, but only 20 patients had sustained improvement beyond 3 months; the latter patients were classified as responders. The response rate was greater in patients older than 50 years of age (82% vs. 43% in younger patients; P = 0.03) and in patients with vigorous achalasia (100% vs. 52% with classic achalasia; P = 0.03). Duration of illness, previous dilation, and baseline radiological characteristics did not influence outcome. Nineteen responders eventually had relapse after a median duration of 468 days (range, 153 - 840 days). Fifteen of these patients received a second injection with satisfactory results obtained in the majority of patients. CONCLUSIONS: Botulinum toxin is an effective treatment for achalasia in about two thirds of patients, with a duration of response averaging 1.3 years. Age and type of achalasia seem to be important predictors of response. PMID- 8613046 TI - Methotrexate in chronic active ulcerative colitis: a double-blind, randomized, Israeli multicenter trial. AB - BACKGROUND & AIMS: Uncontrolled studies have suggested that methotrexate may be effective in patients with active ulcerative colitis. The aim of this study was to evaluate the effectiveness of oral methotrexate in chronic steroid-dependent ulcerative colitis in a randomized, double-blind multicenter trial. METHODS: Patients with active ulcerative colitis who have received steroids and/or immunosuppressives for at least 4 months during the preceding 12 months with a current Mayo Clinic score of > or = 7 were included in the study. Methotrexate (12.5 mg) or placebo was added to their treatment once weekly for 9 months. RESULTS: Sixty-seven patients were included (methotrexate, 30 patients, placebo, 37 patients). The proportion of patients entering first remission (methotrexate, 46.7%; placebo, 48.6%), the time to reach first remission (methotrexate, 4.1 +/- 1.9 months; placebo, 3.4 +/- 1.7 months), as well as the proportions of patients having a relapse after first remission (methotrexate, 64.3%; placebo, 44.4%) were not significantly different between the two groups. The mean Mayo Clinic score, the mean monthly steroid dose, and the proportion of abnormal laboratory results during the study were also similar. CONCLUSIONS: Methotrexate at a weekly oral dose of 12.5 mg was not found to be better than placebo in the induction or maintenance of remission in patients with chronic active ulcerative colitis. PMID- 8613047 TI - Esophageal bolus transit imaged by ultrafast computerized tomography. AB - BACKGROUND & AIMS: Conventional transit imaging techniques detect only the solid or liquid component of a swallowed bolus within the esophagus. This study aimed to dynamically image the composition, distribution, and propulsion of esophageal contents during swallowing. METHODS: Multiple adjacent cross-sectional images of the esophagus were obtained in 7 subjects using ultrafast computerized tomography. Images from two 10-mL swallows were synchronized and analyzed for bolus distribution and propulsion, cross-sectional area, intraluminal volume, and intraluminal content. RESULTS: Both liquid and air were swallowed, with the relative distribution varying among levels. Within the ampulla, air occupied 71% of the luminal cross-sectional area. Air was propelled ahead of the liquid bolus at 17 cm/s compared with 7 cm/s for fluid (P < 0.01) and accumulation within the ampulla. Mean bolus velocity was slower through the ampulla. A variable (8-32 mL) volume of air was ingested during swallowing. CONCLUSIONS: Ultrafast computerized tomography studies during transit of a swallowed bolus through the esophagus showed substantial aerophagia and partial bolus separation with air preceding fluid. The ampulla exhibited greater distention that the tubular esophagus because the bolus accumulated at this level before transfer across the hiatus. PMID- 8613048 TI - Bacteroides fragilis enterotoxin modulates epithelial permeability and bacterial internalization by HT-29 enterocytes. AB - BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis has been associated with diarrheal disease, and the enterotoxin has a cytopathic effect on cultured HT-29 enterocytes. Experiments were designed to determine the effect of B. fragilis enterotoxin on bacteria-enterocyte interactions. METHODS: Confluent HT-29 enterocytes were incubated for 1 hour with B. fragilis enterotoxin, followed by 1 hour of incubation with pure cultures of enteric bacteria, namely, Salmonella typhimurium (two strains), Listeria monocytogenes (three strains), Proteus mirabilis, Escherichia coli (three strains), and Enterococcus faecalis. Enterocyte viability was assessed using vital dyes, epithelial permeability was measured using transepithelial electrical resistance, enterocyte morphology and bacteria-enterocyte interactions were visualized using light and electron microscopy, and bacterial internalization was assessed using a quantitative culture of lysed enterocytes. RESULTS: B. fragilis enterotoxin did not affect enterocyte viability but decreased transepithelial electrical resistance, and individual enterocytes pulled apart. Enterotoxin pretreatment decreased internalization of L. monocytogenes (P < 0.01) but increased (P < 0.01) internalization of the other strains of enteric bacteria. Augmented bacterial internalization was associated with preferential bacterial adherence on the exposed lateral surface of enterotoxin-treated enterocytes. CONCLUSIONS: B. fragilis enterotoxin was associated with HT-29 cell rounding and with augmented internalization of selected strains of enteric bacteria that were preferentially adherent on the exposed enterocyte lateral surface. PMID- 8613049 TI - Modulation of ionic currents in isolated canine and human jejunal circular smooth muscle cells by fluoxetine. AB - BACKGROUND & AIMS: Fluoxetine is a commonly prescribed antidepressant with frequent gastrointestinal side effects. The aim of this study was to examine the effects of fluoxetine on isolated canine and human jejunal circular smooth muscle cells. METHODS: Patch clamp and dual wavelength ratio techniques were used. RESULTS: In amphotericin-perforated patch whole-cell recordings, fluoxetine at 100 nmol/L, 1 mumol/L, and 10 mumol/L concentrations decreased the outwardly delayed rectifier potassium current in canine cells by 12% +/- 3%, 27% +/- 12%, and 37% +/- 3%, respectively, and depolarized the membrane potential by 9.7 +/- 1.8 mV at 10 mumol/L. At 100 mumol/L and 1 mmol/L concentrations, fluoxetine increased the outward current by 88% +/- 40% and 475% +/- 270%, respectively. The increase in the outward current was blocked by charybdotoxin, suggesting an effect on the calcium-activated potassium current. In human cells, fluoxetine at 1 mumol/L decreased the outward potassium current by 26% +/- 4% and at 100 mumol/L increased the outward potassium current by 134% +/- 22%. CONCLUSIONS: Fluoxetine had direct effects on canine and human jejunal circular smooth muscle cells. Low concentrations decreased the outwardly delayed rectifier potassium current, and higher concentrations activated calcium-activated potassium channels. The results may in part explain the frequent gastrointestinal side effects of the drug. PMID- 8613050 TI - Increased gastroduodenal concentrations of transforming growth factor alpha in adaptation to aspirin in monkeys and rats. AB - BACKGROUND & AIMS: The mechanism by which gastric mucosa becomes more resistant to damage by repeated aspirin administration is not known. Transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) prevent drug-induced gastric injury. The aim of this study was to determine whether gastroduodenal tissue levels of TGF-alpha and EGF protein were altered during adaptation to aspirin-induced injury in monkeys and rats in vivo. METHODS: Animals were given aspirin daily for up to 28 days. Gross mucosal injury was assessed by computerized image analysis in rats and by endoscopy in monkeys. Mucosal concentrations of TGF-alpha and EGF were quantitated by radioimmunoassays from endoscopic biopsy samples in monkeys and from scraped mucosa in rats. RESULTS: Long-term administration of aspirin caused a significant increase in gastric and duodenal tissue levels of TGF-alpha in monkeys and rats; the increased levels of TGF-alpha significantly correlated with the decrease in aspirin-induced injury. No change in the gastroduodenal tissue levels of EGF was observed. Adaptation was not associated with any significant change in basal gastric acid secretion in monkeys and occurred despite a significant decrease in gastric mucin in rats. CONCLUSIONS: Adaptation of the gastric mucosa to the damaging effect of aspirin is associated with a significant and specific increase in TGF-alpha protein in the gastroduodenum. PMID- 8613051 TI - Activation of T lymphocytes by syngeneic murine intestinal smooth muscle cells. AB - BACKGROUND & AIMS: Intestinal smooth muscle cells (ISMCs) express major histocompatibility complex II (MCH II) and intercellular adhesion molecule 1 (ICAM-1) after exposure to interferon gamma (IFN-gamma). T lymphocytes invade the intestinal musculature during Crohn's disease or pseudoobstruction. The aim of this study was to determine whether ISMCs activate syngeneic T cells via MHC II and ICAM-1. METHODS: Cultured murine ISMCs were exposed to IFN-gamma for 72 hours and analyzed for Mac-1 (CD11B CD18) antigen, MHC II, and ICAM-1 expression using enzyme-linked immunosorbent assay and fluorescence-activated cell sorter scan. T lymphocytes from mesenteric lymph nodes of ovalbumin-sensitized mice were examined for their ability to proliferate after coculture with IFN-gamma pretreated and ovalbumin-pretreated ISMCs using [3H]thymidine incorporation. RESULTS: ISMCs expressed smooth muscle alpha-actin before and after IFN-gamma exposure. No macrophages were identified in these cultures. Exposure to IFN-gamma and ovalbumin for 72 hours induced MHC II and ICAM-1 expression; these treated ISMCs induced T-cell proliferation, whereas untreated ISMCs did not. T-cell proliferation was markedly enhanced by adding interleukin 2 and was blocked by antibodies against MHC II and ICAM-1. CONCLUSIONS: ISMCs activate T lymphocytes in an MHC II-linked manner and thus possess the ability to modulate immune function in the gut. PMID- 8613052 TI - Selective kallikrein-kinin system activation in inbred rats differentially susceptible to granulomatous enterocolitis. AB - BACKGROUND & AIMS: Crohn's disease is characterized by unrestrained inflammation with a genetic component. Genetic susceptibility and activation of the kalli krein-kinin (contact) system were investigated in experimental enterocolitis and extraintestinal inflammation induced by bacterial polymers. METHODS: Kinetics of inflammation in inbred Lewis and Buffalo rats injected subserosally with peptidoglycan-polysaccharide polymers were correlated with in vivo and in vitro activation of the contact system. RESULTS: Lewis rats had a biphasic course of enterocolitis. Acute inflammation peaked 1 day after injection, gradually decreasing until day 14 when intestinal inflammation spontaneously reactivated and persisted for 16 weeks, accompanied by arthritis, granulomatous hepatitis, anemia, and leukocytosis. Self-limited acute enterocolitis in Buffalo rats resolved by 24 days without extraintestinal involvement. Consumption of the precursor proteins prekalli-krein and high-molecular-weight kininogen indicated activation of the plasma contact system in Lewis rats and closely correlated with chronic intestinal inflammation. Contact system activation did not occur in Buffalo rats, even during acute inflammation. In vitro studies showed a decreased rate of kininogen cleavage in Buffalo plasma. CONCLUSIONS: Selective in vivo and in vitro activation of the contact system in susceptible Lewis rats suggests that this pathway is one determinant of genetic susceptibility to granulomatous enterocolitis and systemic complications. PMID- 8613053 TI - Mast cell-dependent neutrophil and mononuclear cell recruitment in immunoglobulin E-induced gastric reactions in mice. AB - BACKGROUND & AIMS: Immunoglobulin (Ig) E-dependent reactions elicit an immediate response and can also result in a late-phase reaction that is characterized by the infiltration of leukocytes. This study assessed whether IgE-dependent late phase responses can be elicited in the stomach wall of mice and examined the role of mast cells in this reaction. METHODS: IgE-dependent gastric inflammation was elicited in genetically mast cell-deficient KitW/KitW-v mice, the congenic normal (+/+) mice, and mast cell-deficient KitW/KitW-v mice that had undergone local and selective reconstitution of gastric mast cell populations. RESULTS: IgE-dependent gastric reactions were associated with mast cell degranulation and the infiltration of both neutrophils and mononuclear cells in normal mice, but no significant leukocyte infiltration was observed in mast cell-deficient KitW/KitW v mice. By contrast, in mast cell-reconstituted KitW/KitW-v mice, IgE-dependent reactions were associated with the infiltration of neutrophils and mononuclear cells. CONCLUSIONS: These results show that late-phase reactions can occur during IgE-dependent gastric inflammation in the mouse and that the infiltration of both neutrophils and mononuclear cells that are observed during this reaction are mast cell dependent. PMID- 8613054 TI - Fat-induced ileal brake in the dog depends on peptide YY. AB - BACKGROUND & AIMS: Fat in the distal gut inhibits transit through the proximal small intestine as the ileal brake. Although the mediator of this response is not established, peptide YY (PYY) has been considered the most likely peptide candidate because inhibition of intestinal motility by fat in the distal gut correlated with the release of PYY but not other distal gut peptides such as enteroglucagon or neurotensin. Although intravenous administration of PYY inhibits intestinal transit, the role of this peptide remains to be confirmed because systemic PYY may not exert its effect by the same regulatory pathway as fat-induced ileal brake. The aim of this study was to definitively test the hypothesis that PYY mediates fat-induced ileal brake using the technique of peptide immunoneutralization. METHODS: In a fistulated dog model, intestinal transit during perfusion of the distal gut with 60 mmol/L oleate (ileal brake) was examined after intravenous administration of 0.5 mg/kg of PYY antibody (anti PYY), nonspecific immunoglobulin G (control), or 0.15 mol/L NaCl. Intestinal transit result (cumulative percent recovery of 99mTc) was normalized within each animal against the transit result of the 0.15 mol/L NaCl experiment. RESULTS: Intestinal transit accelerated with PYY immunoneutralization, increasing cumulative percent recovery from 25.9 +/- 6.2 (control) to 81.2 +/- 6.3 (anti PYY). CONCLUSIONS: Fat-induced ileal brake depends on PYY. PMID- 8613055 TI - Primary sclerosing cholangitis is associated with nonsmoking: a case-control study. AB - BACKGROUND & AIMS: Cigarette smoking is thought to protect against the development of ulcerative colitis. The relationship between a related disease, primary sclerosing cholangitis, and smoking is unknown. The aim of this study was to determine if the relationship between smoking and sclerosing cholangitis is similar to that found between smoking and ulcerative colitis. METHODS: A stratified sample of 184 patients with primary sclerosing cholangitis and age- and sex-matched institutional control subjects were identified. Smoking information was obtained from a medical questionnaire completed at the time of visit. RESULTS: Eighty-one percent of the patients had associated inflammatory bowel disease. Only 4.9% of them were current smokers compared with 26.1% of the controls; 69.6% of the patients had never smoked vs. 46.7% of the controls. The estimated odds of having primary sclerosing cholangitis in current smokers compared with never-smokers was 0.13. The odds of having disease among former and current users of any tobacco relative to never-users was 0.41 regardless of the presence or absence of inflammatory bowel disease. CONCLUSIONS: The odds of having primary sclerosing cholangitis was significantly decreased among current smokers. Concomitant ulcerative colitis does not fully explain the association between nonsmoking and primary sclerosing cholangitis. Smoking may exert its protective effect in a systemic, rather than colonic, manner. PMID- 8613056 TI - Risk of primary sclerosing cholangitis is associated with nonsmoking behavior. AB - BACKGROUND & AIMS: Seventy percent of patients with primary sclerosing cholangitis (PSC) have concomitant ulcerative colitis. Smoking and previous appendectomy may protect against ulcerative colitis. The aim of this study was to examine these factors in patients with PSC. METHODS: Fifty-nine patients with PSC, 130 patients with ulcerative colitis and normal liver biochemistry, and 197 control subjects were interviewed about smoking behavior and history of appendectomy. RESULTS: There were less current smokers in the PSC and ulcerative colitis groups than in the control group (19%, 12%, and 38%, respectively). The resulting odds ratio for current smoking was 0.37 (95% confidence interval, (0.18 0.76) in the PSC group and 0.23 (95% confidence interval, 0.13-0.41) in the ulcerative colitis group. Percentage of persons who ever smoked was also significantly less in the PSC group (41% vs. 56% in the control group). Frequency of previous appendectomy in the PSC and ulcerative colitis groups was not significantly different from that of controls (19%, 9%, and 14%, respectively). CONCLUSIONS: Smoking but not previous appendectomy is associated with decreased risk of PSC. PMID- 8613057 TI - Microwave coagulation therapy for hepatocellular carcinoma. AB - BACKGROUND & AIMS: Surgical resection is not always feasible in patients with hepatocellular carcinoma. Microwave coagulation therapy has been used as an alternative to resection, and its efficacy has been evaluated. METHODS: Nineteen patients with unresectable hepatocellular carcinoma underwent microwave coagulation therapy through laparotomy (n = 12), laparoscopy (n = 5), or thoracotomy (n = 2) because of advanced liver cirrhosis and/or intrahepatic metastases. One nodule was treated in 13 patients, tumor size ranged from 5 to 90 mm. Patient outcomes were studied. RESULTS: Microwave coagulation therapy created a reproducible regional necrosis. Fourteen patients underwent potentially curative treatment; the remaining 5 patients underwent palliative treatment (n = 4) or incomplete tumor coagulation (n = 1). Of the 31 nodules treated, 28 underwent complete tumor ablation. Only 2 patients undergoing laparoscopic microwave coagulation therapy developed local recurrence. The coagulated area subsequently shrank. Patients showed rapid recovery without hepatic dysfunction. Thirteen patients, including 2 long-term survivors, are alive either without tumor (n = 10; 14-64 months) or with tumor (n = 3; 17-22 months). Six patients died of hepatocellular carcinoma (n = 4) or liver insufficiency (n = 2). CONCLUSIONS: This preliminary study suggests the efficacy of microwave coagulation therapy, including safety and potential curability, in patients with hepatocellular carcinoma with advanced liver cirrhosis and multifocal or central tumors. PMID- 8613058 TI - Effects of ursodeoxycholic acid on survival in patients with primary biliary cirrhosis. AB - BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) has been shown to be a safe and effective treatment for patients with primary biliary cirrhosis; however, its effect on patient survival is less certain. To study this issue, the survival of patients receiving long-term UDCA treatment was compared with that of a control group, adjusting for their risk scores based on the Mayo model. METHODS: One hundred eighty patients were randomized to receive either 13-15 mg.kg-1.day-1 UDCA (n = 89) of placebo (n = 91). After the study closure, the patients originally receiving placebo were switched to active drug, and prospective follow up was continued for 3 years. Patients were censored at the time of transplantation, voluntary withdrawal, of crossover of the placebo group (efficacy analysis). The survival of the two groups was adjusted for risk scores at the time of entry to the study. A secondary analysis was an intent-to-treat analysis, whereby patients were followed up regardless of their voluntary withdrawal or crossover. RESULTS: At the time of analysis, the patients receiving placebo had a significantly increased risk of death and/or requiring transplantation (relative risk, 2.6; P = 0.04) compared with the UDCA-treated patients CONCLUSIONS: UDCA should be considered as a safe, effective, and life extending treatment for patients with primary biliary cirrhosis. PMID- 8613059 TI - Effect of hepatocyte proliferation and cellular DNA synthesis on hepatitis B virus replication. AB - BACKGROUND & AIMS: Interrelationship between hepatitis B virus (HBV) replication and the stage of hepatocyte proliferation and differentiation may play an important role in the pathogenesis of HBV infection. The aim of this study was to assess the effect of hepatocyte proliferation and/or cell arrest on HBV replication. METHODS: Hepatoblastoma cells transfected with HBV were subjected to serum deprivation or treatment with aphidicolin or camptothecin. Cell cycle analysis was performed using flow cytometry, and cellular DNA synthesis was analysed by assessing 5-bromo-2'-deoxyuridine incorporation. Distribution of episomal HBV DNA and proliferating cell nuclear antigen in liver specimens was assessed by simultaneous in situ hybridization and immunohistochemistry. RESULTS: Serum deprivation inhibited cellular DNA synthesis and increased levels of HBV messenger RNA (mRNA). Aphidicolin treatment resulted in cell arrest in C1, with concomitant increases in levels of HBV mRNA and viral DNA. Cell entry into S phase inhibited expression of HBV mRNA. Camptothecin induced G2 cell arrest and inhibited cellular DNA synthesis with increased amounts of viral replication and levels of HBV mRNA. In vivo studies showed an inverse correlation between expression of proliferating cell nuclear antigen and presence of episomal HBV DNA in individual hepatocytes. CONCLUSIONS: HBV replication is cell cycle dependent, supporting the concept of enhanced viral replication in quiescent hepatocytes. The results may explain the mechanism of viral elimination during cell regeneration. PMID- 8613060 TI - Acute and chronic cyclooxygenase blockage in portal-hypertensive rats: influence in nitric oxide biosynthesis. AB - BACKGROUND & AIMS: Prostacyclin and nitric oxide have been involved in the hyperkinetic syndrome in portal hypertension. The aim of this study was to investigate the relative contribution and possible interaction between prostacyclin and NO in this circulatory abnormality. METHODS: Portal vein-ligated and sham-operated rats received indomethacin and vehicle either on a short-term (5 mg/kg subcutaneously) or long-term basis (5 mk.kg-1.day-1, continuous 7-day infusion with an osmotic minipump). Measurements of arterial pressure and superior mesenteric arterial flow (mL.min-1.kg-1, ultrasonic flow probe) were then performed before and after NG-nitro-L-arginine methyl ester (L-NAME) injection (13 mg/kg intravenously). RESULTS: Short-term or long-term indomethacin treatment had no effect in sham-operated rats but significantly decreased mesenteric arterial flow in portal-hypertensive rats. Mesenteric flow remained higher after long-term than after short-term indomethacin treatment (54.5 +/- 2 vs. 46.1 +/- 2; P = 0.01). This blunted response to long-term indomethacin treatment was associated with an enhanced response to L-NAME, shown by greater increments in arterial pressure (29% +/- 3%) and mesenteric arterial resistance (209 +/- 22%) in indomethacin-treated rats than in rats receiving vehicle (19% +/ 2% and 130% +/- 20%; P < 0.05). CONCLUSIONS: Both prostacyclin and NO contributed to splanchnic hyperemia in portal-hypertensive rats. There was an enhanced release of NO after long-term prostacyclin inhibition, suggesting that both vasodilatory systems interact, promoting splanchnic hyperemia in portal hypertension. PMID- 8613061 TI - Glycine prevents alcohol-induced liver injury by decreasing alcohol in the rat stomach. AB - BACKGROUND & AIMS: Inactivation of Kupffer cells prevents alcohol-induced liver injury, and hypoxia subsequent to a hypermetabolic state caused by activated Kupffer cells probably is involved in the mechanism. Glycine is known to prevent hepatic reperfusion injury. The purpose of this study was to determine whether glycine prevents alcohol-induced liver injury in vivo. METHODS: Male Wistar rats were exposed to ethanol (10-12 g.kg-1.day-1) continuously for up to 4 weeks via an intragastric feeding protocol. The effect of glycine on the first-pass metabolism of ethanol was also examined in vivo, and the effect on alcohol metabolism was estimated specifically in perfused liver. RESULTS: Glycine decreased ethanol concentrations precipitously in urine, breath, peripheral blood, portal blood, feces, and stomach contents. Serum aspartate amino transferase levels were elevated to 183 U/L after 4 weeks of ethanol-treatment. In contrast, values were significantly lower in rats given glycine along with ethanol. Hepatic steatosis and necrosis also were reduced significantly by glycine. Glycine dramatically increased the first-pass elimination of ethanol in vivo but had no effect on alcohol metabolism in the perfused liver. CONCLUSIONS: Glycine minimizes alcohol-induced liver injury in vivo by preventing ethanol from reaching the liver by activating first-pass metabolism in the stomach. PMID- 8613062 TI - Identification of betaine as an osmolyte in rat liver macrophages (Kupffer cells). AB - BACKGROUND & AIMS: Anisosmotic cell volume changes were identified recently as a critical factor in the regulation of eicosanoid formation in stimulated liver macrophages (Kupffer cells). Therefore, the aim of this study was to investigate whether osmolytes are involved in the control of Kupffer cell function. METHODS: Cultured rat liver macrophages (Kupffer cells) were studied with respect to their functional adaptation to hyperosmotic environments. RESULTS: Hyperosmotic exposure of lipopolysaccharide-stimulated Kupffer cells led to sevenfold stimulation of prostaglandin E2 formation caused by a strong induction of cyclooxygenase 2. Hyperosmotic exposure was accompanied by approximately a 10 fold increase of Na(+)-dependent betaine uptake and betaine transporter-messenger RNA levels. Conversely, hypoosmotic exposure decreased betaine uptake and stimulated betaine release from Kupffer cells and the perfused rat liver. Addition of betaine (1 mmol/L) to hyperosmotically exposed Kupffer cells abolished the strong induction of cyclooxygenase 2 and the increase of prostaglandin E2 formation. CONCLUSIONS: This study identifies the use of an osmolyte strategy in liver macrophages. Betaine serves as an osmolyte in Kupffer cells, whose transport is induced in response to increases of ambient osmolarity. Regulation of this transporter as well as betaine availability may represent a novel regulation site for Kupffer cell function. PMID- 8613063 TI - Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes. AB - BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. UDCA is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2+ agonist and stimulates biliary exocytosis and hepatocellular Ca2+ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. METHODS: The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accummulation of sn-1,2-diacylglycerol (DAG), the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. RESULTS: Immunoblotting studies showed the presence of four isoenzymes (alpha, delta, epsilon, and zeta). The phorbol ester phorbol 12-myristate 13-acetate (1 mumol/L) induced translocation of alpha PKC, delta-PKC, and epsilon-PKC from cytosol to a particulate membrane fraction, a key step for activation of PKC. TUDCA, but not taurocholic acid, selectively induced translocation of the alpha-PKC isoenzyme from cytosol to the membranes. In addition, TUDCA induced a significant increase in hepatocellular DAG mass and stimulated membrane-associated PKC activity. CONCLUSIONS: TUDCA might stimulate Ca(2+)-dependent hepatocellular exocytosis into bile in part by activation of alpha-PKC. PMID- 8613064 TI - Structure-function correlation of tight junctional impairment after intrahepatic and extrahepatic cholestasis in rat liver. AB - BACKGROUND & AIMS: Tight junctions, the only barrier between blood and bile, are crucial in bile formation. The aim of this study was to correlate changes in morphology and permeability by comparing structural parameters with marker secretion into normal and cholestatic rat bile. METHODS: Cholestasis was induced by bile duct ligation of 5 and 21 days of ethinylestradiol administration. Quantitated structural parameters induced junctional length, strand number, junctional depth, and spacing of junctional particles. Junctional permeability was probed with horseradish peroxidase and dextrans of increasing sizes. RESULTS: Junctional length was decreased slightly by ethinylestradiol (-16% after 21 days) but increased by ligation (77%). Mean strand number decreased from 4.6 to 3.7 after 21 days of ethinylestradiol and 3.4 after ligation associated with increased junctional depth. The proportions of morphologically horseradish peroxidase-positive junctions increased from 4% to 15% after 21 days of ethinylestradiol and to 56% after ligation. Horseradish peroxidase secretion was increased twofold by ethinylestradiol and 6.5-fold by ligation, paralleled by an increase of dextran size selectivity from 70,000 to 79,000 daltons after ethinylestradiol and to 266,000 daltons after ligation. CONCLUSIONS: Impairment of junctional integrity is paralleled with the degree of cholestasis, whereas correlation of morphological and physiological alterations shows a close structure-function relationship. PMID- 8613065 TI - Serum antibodies to carbonic anhydrase I and II in patients with idiopathic chronic pancreatitis and Sjogren's syndrome. AB - BACKGROUND & AIMS: Patients with idiopathic chronic pancreatitis and Sjogren's syndrome show immune responses against a 60-kilodalton protein isolated from human pancreas extracts. Monoclonal antibody SP3-1, raised against the 60 kilodalton protein, reacts with ductal cells in several exocrine organs and cross reacts with human carbonic anhydrase II. The present study evaluated serum from patients with these diseases for antibodies to human carbonic anhydrase I and II. METHODS: An enzyme-linked immunosorbent assay was used to quantify serum antibody against carbonic anhydrase I and II. RESULTS: Serum antibodies against carbonic anhydrase I and II were detected in 7 and 11 of 33 patients with idiopathic chronic pancreatitis, respectively, and in 8 and 13 of 21 patients with Sjogren's syndrome, respectively. The positive prevalence rates of patients with antibodies of carbonic anhydrase II were significantly different among patients with idiopathic chronic pancreatitis and Sjogren's syndrome compared with normal patients (1 of 21). There were no significant differences in the prevalence rates of patients with alcoholic chronic pancreatitis (3 of 20), gallstone-related chronic pancreatitis (0 of 7), and primary biliary cirrhosis (1 of 11). CONCLUSIONS: The results suggest that carbonic anhydrase II is one candidate target antigen recognized during the autoimmune pathophysiology observed in idiopathic chronic pancreatitis and Sjogren's syndrome. PMID- 8613066 TI - Diagnostic utility of K-ras mutations in fine-needle aspirates of pancreatic masses. AB - BACKGROUND & AIMS: Mutations at codon 12 of the K-ras gene are present in 65% 100% of carcinomas of human exocrine pancreas and could be used as a potential tumor marker at the tissue level. The purpose of this study was to assess, in large series of patients, the utility of K-ras mutation analysis to evaluate fine needle aspirates of pancreatic masses. METHODS: One hundred fifteen fine-needle aspirates obtained from 93 patients were evaluated retrospectively. Cytological analysis was based on the review of cell blocks. Mutations were detected by using artificial restriction fragment length polymorphisms using the Hphl and BstNl approaches. RESULTS: The sensitivity and specificity of cell block cytology was 64% and 100%, respectively, for the diagnosis of pancreatic carcinoma. K-ras mutations were detected in 41 pancreatic carcinomas (sensitivity, 59%) and in one mucinous cystic tumor; specificity of ras analysis alone was 100%. The sensitivity of cytology combined with K-ras mutations were 77.6% and 100%, respectively. CONCLUSIONS: The detection of K-ras mutations would have suggested the diagnosis of pancreatic cancer in 14 cases otherwise not detected by cytology alone. K-ras mutation analysis should be restricted to cell blocks containing suspicious, normal-appearing duct cells, or insufficient material in the cytological examination. PMID- 8613067 TI - gamma-Aminobutyric acid secretion from pancreatic neuroendocrine cells. AB - BACKGROUND & AIMS: Neuroendocrine cells and tumors derived therefrom contain hormone-storing large dense core vesicles and neuron-like small synaptic vesicle analogues with unknown function. The aim of this study was to characterize the small synaptic vesicle pathway in detail. METHODS: In human pancreatic neuroendocrine tumors and corresponding mammalian cell lines, the expression of key proteins if regulated secretion were detected by immunofluorescence microscopy. Using 3H-gamma-aminobutyric acid (GABA), uptake and release by small synaptic vesicle analogues were studied. RESULTS: Tumor tissues obtained from 14 patients expressed key proteins of neurosecretion such as synaptobrevin, syntaxins, and SNAP 25. These proteins were also found in the cell lines AR42J, BON, RIN, and INR. The cell lines specifically transported GABA by low-affinity plasma membrane transporter and showed an adenosine triphosphate-sensitive GABA uptake into an intracellular compartment. Stored GABA was released upon stimulation by regulated exocytosis. Electrophysiological analyses suggested that calcium-dependent secretion was mediated by activation of voltage-dependent calcium channels of mainly the L type, but also of the N and probably the T type. CONCLUSIONS: Small synaptic vesicle analogues in neuroendocrine cells and tumors can store and secrete GABA and probably other amino acid transmitters by regulated exocytosis comparable with neurons. PMID- 8613068 TI - Inhibitory effect of 220-oxa-1,25-dihydroxyvitamin D3 on the proliferation of pancreatic cancer cell lines. AB - BACKGROUND & AIMS: Effective chemotherapy for pancreatic cancer is urgently needed. The aim of this study was to compare the anti-proliferative activity of a new vitamin D3 analogue, 22-oxa-1,25-dihydroxyvitamin D3 (22-oxa-calcitriol), on pancreatic cancer cells lines with that of 1,25-dihydroxyvitamin D3 (calcitriol) with analysis of vitamin D receptor status. METHODS: Antiproliferative effects of both agents were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide method and by measuring the tumor size of xenograft inoculated into athymic mice. Vitamin D receptor contents by Scatchard analysis and mutational analysis of receptor complementary DNA were performed. RESULTS: In vitro, 22-oxa-calcitriol and calcitriol markedly inhibited the proliferation (3 of 9 cell lines) and caused a G1 phase cell cycle arrest by appearance of numerous domes. In vivo, 22-oxa-calcitriol inhibited the growth of BxPC-3 xenografts more significantly than calcitriol without including hypercalcemia. Hs 766T, showing no response to either agent, had the second highest receptor contents with no abnormalities in its primary structure deduced by receptor complementary DNA. CONCLUSIONS: 22-oxa-calcitriol may provide a more useful tool for the chemotherapy of pancreatic cancer than calcitriol. Also, the susceptibility of the cell lines to both agents is not well determined by evaluating either the contents or the mutation of vitamin D receptor. PMID- 8613069 TI - Early mucosal changes of the gallbladder in patients with anomalous arrangement of the pancreaticobiliary duct. AB - BACKGROUND & AIMS: Anomalous arrangement of the pancreaticobiliary duct (AAPBD) is frequently associated with gallbladder cancer. The aim of this study was to characterize the early epithelial changes of the gallbladder that might be associated with carcinogenesis in patients with AAPBD. METHODS: Routinely fixed tissue specimens obtained from pediatric patients with AAPBD and control patients were examined histologically with H&E, periodic acid-Schiff, and argyrophilic nucleolar organizer region (AgNOR) staining. RESULTS: Epithelial hyperplasia was frequently observed in the gallbladder of patients with AAPBD but not in patients without this anomaly. The incidence of epithelial hyperplasia was significantly higher in the gallbladder of patients in whom the major pancreatic duct joined the common bile duct (P-C type) than in patients in whom the common bile duct joined the pancreatic duct (C-P type). The mean AgNOR number in the gallbladder mucosa was significantly higher in patients with AAPBD than in patients without AAPBD. Patients with C-P type AAPBD had a significantly higher mean AgNOR number of the gallbladder mucosa than patients with C-P type AAPBD. CONCLUSIONS: Patients with AAPBD, especially those with the P-C type anomaly, have increased cellular proliferative activity of the gallbladder mucosa in early childhood. PMID- 8613070 TI - Hepatic cholesterol metabolism in patients with cholesterol gallstones: enhanced intracellular transport of cholesterol. AB - BACKGROUND & AIMS: Alterations of hepatic cholesterol metabolism in patients with cholesterol gallstones are still controversial. This study investigated whether hepatic cholesterol metabolism is altered in Japanese patients with cholesterol gallstones. METHODS: In this systematic study of 24 middle-aged nonobese and nondiabetic Japanese patients who had cholesterol gallstones and were undergoing elective cholecystectomy, an analysis of three regulatory enzymes in the cholesterol metabolism, as well as cytosolic total and free cholesterol levels and sterol carrier protein 2/nonspecific lipid transfer protein (SCP2/nsLTP) levels, was conducted using liver biopsy samples obtained during surgery. RESULTS: The activities of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, cholesterol 7 alpha-hydroxylase, and acyl-CoA/cholesterol acyltransferase were not significantly different between patients and controls. Nevertheless, patients with gallstones showed tendencies for elevated HMG-CoA reductase activity and protein content and decreased cholesterol 7 alpha hydroxylase activities. As anticipated, serum levels of 7 alpha hydroxycholesterol and squalene paralleled these findings. The patients with gallstones also had significantly increased cytosolic total and free cholesterol levels (P < 0.001), which correlated strongly with increased cytosolic levels of SCP2/nsLTP (r = 0.80, P < 0.001 and r = 0.81, P < 0.001, respectively). CONCLUSIONS: The results suggest that intracellular cholesterol transport is enhanced in patients with cholesterol gallstones. PMID- 8613071 TI - Multifocal stenosing ulcerations of the small intestine revealing vasculitis associated with C2 deficiency. AB - A patient with cryptogenic multifocal ulcerous stenosing enteritis characterized by repeated bouts of intestinal obstruction, ulcerative stenosis of the small bowel relapsing after surgical resection, and steroid sensitivity is described. Fourteen strictures of the jejunum were found at laparotomy. Despite resection, abdominal pain persisted. Steroid therapy was effective but led to dependence. In our patient, cryptogenic multifocal ulcerous stenosing enteritis was associated with fever, asthma, Raynaud's phenomenon, sicca syndrome, heterozygous type I C2 deficiency (28-base pair gene deletion), stenosis, and aneurysms in selective mesenteric angiography. It is hypothesized that cryptogenic multifocal ulcerous stenosing enteritis might be related to a particular form of polyarteritis nodosa with mainly intestinal expression or to a yet unclassified independent vasculitis. PMID- 8613072 TI - Mesenteric venous thrombosis treated with urokinase via the superior mesenteric artery. AB - A 77-year-old man with a history of multiple surgically treated malignancies presented with increasing abdominal pain after eating. Computerized tomographic scan showed superior mesenteric vein and portal vein thrombosis. The patient was treated with selective superior mesenteric artery infusion of urokinase resulting in clinical improvement and near complete resolution of the mesenteric venous thrombosis. An underlying gastric malignancy was found and is believed to be the cause of the patient's hypercoagulable state. Direct infusion of urokinase into the superior mesenteric artery for treatment of mesenteric venous thrombosis is an alternative to surgery in selected patients and an alternative to the much more complicated delivery systems presently used. PMID- 8613074 TI - Centennial report to American Gastroenterological Association members. PMID- 8613075 TI - Helicobacter pylori and perturbations in acid secretion: the end of the beginning. PMID- 8613073 TI - Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. AB - BACKGROUND & AIMS: While the lobular heterogeneity of hepatocytes is established, limited data suggest that bile duct epithelial cells, of cholangiocytes, are heterogeneous along the normal intrahepatic biliary tree. Thus, we tested the hypothesis that cholangiocytes are structurally and functionally heterogeneous in the biliary tract of normal rats. METHODS: A series of in situ and in vitro experiments was performed in normal rats using complementary morphometric, molecular, and functional approaches. RESULTS: In situ morphometry showed that (1) intrahepatic bile ducts are heterogeneous in external diameter (5-200 mum); (2) individual cholangiocytes lining bile ducts are heterogeneous in area (3-80 mum2); and (3) a significant relationship exists between bile duct diameter and cholangiocyte area. Using a novel approach developed by us, we isolated three subpopulations of small, medium, and large cholangiocytes and compared them at the molecular and functional level. The expression of two cholangiocyte-specific genes (gamma-glutamyl transpeptidase and cytokeratin 19) was similar among the three subpopulations. In contrast, secretion receptor, Cl-/HCO3- exchanger, and cystic fibrosis transmembrane conductance regulator messenger RNAs were differentially expressed, being present on medium and large but not small cholangiocytes. At the functional level, adenosine 3', 5'-cyclic monophosphate and intracellular pH responses and Cl-/HCO3- exchanger activity was enhanced by secretion in medium and large but not small cholangiocytes. CONCLUSIONS: These data indicate that cholangiocytes are morphologically and functionally heterogeneous along the normal intrahepatic biliary tree and suggest that secretion-regulated transport of water and electrolytes occurs principally in medium and large ducts. PMID- 8613076 TI - Botulinum toxin for achalasia: to be or not to be? PMID- 8613077 TI - Methotrexate and ulcerative colitis: wrong drug? Wrong dose? Or wrong disease? PMID- 8613078 TI - The role of mast cells in gastrointestinal inflammation. PMID- 8613079 TI - Protection from primary sclerosing cholangitis: smoke trails of just coattails? PMID- 8613081 TI - Colectomy as treatment for constipation. PMID- 8613080 TI - Leaky junctions and cholestasis: a tight correlation. PMID- 8613082 TI - CFTR: what will it do next? PMID- 8613083 TI - Measuring sugar absorption in unrestrained animals. PMID- 8613084 TI - Somatostatin receptor imaging in cholangiocarcinoma. PMID- 8613086 TI - The impact of immunonutrition. PMID- 8613085 TI - Immunonutrition: we are what we eat. PMID- 8613087 TI - Treatment of snoring with a nocturnal airway patency appliance (NAPA). Part II. PMID- 8613088 TI - Freedom to function. PMID- 8613089 TI - Multidisciplinary management of the cervicocranial/temporomandibular disorders- appreciation of the whole cube. PMID- 8613090 TI - Poor man's VTO--Part II. PMID- 8613092 TI - Interview with Dr. Terrance J. Spahl, 1992 Clinician of the Year. Interview by Dr. Craig C. Stoner. PMID- 8613091 TI - Achieving excellence in TMJ, functional orthopedics and case finishing: Part III- Cephalometric evaluation of vertical dimension. PMID- 8613093 TI - Treatment of snoring with a nocturnal airway patency appliance (NAPA): Part III. PMID- 8613094 TI - Finishing touches for beautiful smiles: orthodontist and general dentist coordinate efforts to achieve optimum esthetics--Part I. PMID- 8613095 TI - Functional forum: the standard of care. PMID- 8613096 TI - The cyclic nature of TMD, the headache patient, and the functional treatment rationale. PMID- 8613097 TI - The Pendulum Appliance. PMID- 8613098 TI - An introduction to modified twin block appliance therapy (M.T.B.A.T.). Part I: Sound basics. PMID- 8613099 TI - Functional orthodontics: past, present, future. PMID- 8613101 TI - Functional forum: do we treat the "asymptomatic" click? PMID- 8613100 TI - Achieving excellence in TMJ, functional orthopedics, and orthodontic case finishing: Part IV (Section I). Determinants of occlusion and vertical dimension. PMID- 8613102 TI - Achieving excellence in TMJ, functional orthopedics, and orthodontic case finishing: Part IV (Section 2). Determinants of occlusion and vertical dimension. PMID- 8613103 TI - Bone remodeling orthodontics: an early one-phase non-extraction therapy. PMID- 8613104 TI - Basic treatment precepts ensuant to a whiplash episode. PMID- 8613105 TI - An introduction to modified twin block appliance therapy (M.T.B.A.T.). Part II: putting the basics into practice. PMID- 8613106 TI - Functional forum: more meaningful cephalometrics. PMID- 8613107 TI - Joint vibration analysis--part I. PMID- 8613108 TI - Past, present and future of functional orthodontics. PMID- 8613109 TI - The golden number: applications to cranio-facial evaluation. AB - The Golden Number, expressing in numbers what the ancient Greeks called the "Divine Proportion" has a value of 1.618. This number is found in numerous natural phenomena, geometrical propositions and human architectural constructions. These proportions are worth comparing to those of the human face. The author shows that this "golden proportion" is found in many cephalometric measurements and in various stages of facial growth. PMID- 8613111 TI - What if the condyles are already down and forward? PMID- 8613112 TI - Bone remodeling orthodontics: an early, one-phase, non-extraction therapy--Part II. PMID- 8613113 TI - Latent dysfunctions resulting from unresolved trauma-induced head & neck injuries. PMID- 8613110 TI - Documented instance of restored conductive hearing loss. AB - The relationship between temporomandibular joint dysfunction and hearing disorders has long been recognized by some healthcare providers (1,2). Fonder reports that "chronic low-grade otitis media is a constant finding in patients who have a disturbance of the stomatognathical structures due to malocclusion" (3). Fingeroth stated that "a constricted maxillary dental arch frequently results in a decrease in nasal permeability...and within this environment a conductive hearing loss may be present" (4). Histological studies confirm the intimate relationship between the TMJ, the tympanic cavity and the eustachian tube (5,6). Nevertheless, craniomandibular origins are frequently overlooked in the medical profession as possible causes for hearing problems. The following case illustrates this point. PMID- 8613114 TI - The ALF appliance. PMID- 8613115 TI - Dentofacial orthopedics in the mixed dentition using fixed composite twin blocks. PMID- 8613116 TI - The twin block appliance. PMID- 8613117 TI - The 10 great laws of orthodontics. Part I: Laws I-V. PMID- 8613118 TI - A perspective on dental facial orthopedics. PMID- 8613119 TI - Alternative lightwire functionals (ALF). AB - The ALF appliance represents a major innovation in TMD and orthopedic/orthodontic therapy. Thanks to the genius and perseverance of Dr. Darick Nordstrom and laboratory technician Heather Ashton, the ALF appliances are custom designed and capable of correcting not only the various types of malocclusion but also cranial base and cranial distortions present in the chronic pain, TMD type patient. PMID- 8613121 TI - The Han appliance. PMID- 8613120 TI - Chronic, developmental TMJ trauma and a new rationale for multiple phase treatment. PMID- 8613122 TI - Tomographic proof of trauma-induced injury to the TMJoint and other sites in the body. PMID- 8613123 TI - Functional forum: AAFO online. PMID- 8613124 TI - An interview with Dr. James W. May, 1994 AAFO Clinician of the Year. Interview by Dr. Craig C. Stoner. PMID- 8613125 TI - Foraging synchrony in a group of Yakushima macaques (Macaca fuscata yakui). AB - The synchrony of behaviour among individuals in a group of Yakushima macaques was evaluated in relation to group size and food resources. The degree of synchrony was greater when the group was small (5-8 individuals) than when it was large (15 19 individuals). Diet did not affect the degree of synchrony. However, the duration of the 'active phase', in which most members of the group synchronized feeding and moving, was shorter when they fed mostly on fruit and seeds (fruit- and seed-eating season) than when they fed mostly on leaves and fallen seeds (leaf- and fallen-seed-eating season). When the group was large, the monkeys ranged over a greater area and foraged in fewer trees during the fruit-and seed eating season than during the leaf- and fallen-seed-eating season. However, this tendency was not so clear when the group was small. These results suggest that the extent to which the distribution of food resources determines patterns of foraging increases with group size and that monkeys in a larger group reduce levels of intragroup food competition in order to obtain sufficient food. PMID- 8613126 TI - Hand, mouth and eye preferences in the common marmoset (Callithrix jacchus). AB - Motor and sensory preferences were measured for 8 common marmosets (Callithrix jacchus). Three types of motor function were examined: spontaneous feeding, mouth use when chewing and visuospatial reaching. Eye preference was used as an index of sensory lateralization. The marmosets displayed individual preferences for the performance of the motor tasks, but no group biases were revealed. Correlations between preferences displayed on the motor tests suggest that one hemisphere may control spontaneous feeding and lateralized mouth use while the other controls visuospatial reaching, but no consistent preference was present. In an eye preference test, 7 subjects displayed right-eye preferences, suggesting hemispheric specialisation for this function. PMID- 8613127 TI - Maternal responsiveness to infant distress calls in stumptail macaques. PMID- 8613128 TI - Variability of trace element content in permanent teeth of Macaca mulatta--two different populations. PMID- 8613129 TI - Electromyography of 15 limb muscles in Japanese macaques (Macaca fuscata) during vertical climbing. PMID- 8613130 TI - Sperm length and sperm competition in primates: a rebuttal of criticism. PMID- 8613131 TI - Aggressive non-Hodgkin's lymphoma: T-cell versus B-cell. AB - This study is aimed to define the clinical characteristics, treatment of outcome and independent prognostic factors of 144 patients with T- and 357 B-cell non Hodgkin's lymphomas. Entities with well-defined immunophenotype and clinical characteristics were excluded. Patients with T-cell tumours were younger. T immunophenotype was associated with more advanced disease and presence of B symptoms. They were also less likely to have bulky disease. Primary nasal lymphomas were usually T-cell while lymphomas arising from the gastrointestinal tract and Waldeyer's ring were mostly B-cell. On univariate analysis, T immunophenotype was associated with lower CR rate, higher relapse rate and inferior overall survival. On the other hand, multivariate analysis revealed that advanced stage, presence of B symptoms, advanced age, high serum lactate dehydrogenase level and use of non-doxorubicin-containing regimens for induction were associated with poor prognosis. Immunophenotype was not a significant independent prognostic factor. PMID- 8613132 TI - Specificity of hematopoietic stem cell homing. AB - Homing of hematopoietic stem cells (HSC) may be defined as the cells' ability to seek marrow stroma selectively, to interact with it and subsequently to lodge within it to initiate hematopoiesis. This complex process is no doubt mediated through multiple recognition/adhesion events. Homing may proceed through one of several alternative mechanisms, however, such as through physical trapping of stem cells by marrow ultrastructural elements or through the providing of a selective survival and/or proliferative advantage by marrow. A third alternative that provides for the central element of stem cell homing--its high degree of specificity--is through the action of a specific homing protein in HSC. There are data to support this latter mechanism of stem cell homing as the correct one, and the nature of this protein may be similar to that of the lymphocyte homing receptors that are lectin-like molecules. Lectin-carbohydrate interactions are known to provide enormous specificity to cell recognition processes and to participate in cellular targeting. Leukemic cells have recently been demonstrated to home to marrow stroma and proliferate in the same way as normal stem cells. Thus, identification of proteins or other adhesion molecules that participate in normal and malignant cell homing could lead to more specific recruitment regimens for tumour-free collections. PMID- 8613133 TI - Acute myeloid leukemia following therapy of Hodgkin's disease with radiotherapy and ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine). AB - A 26-year-old man developed myelodysplasia rapidly progressing to acute myelomonocytic leukemia 3 years after receiving three cycles of ABVD chemotherapy and upper mantle and upper abdomen radiotherapy for stage IA Hodgkin's disease. This represents the fourth such case reported. The risk of secondary AML after ABVD or radiation therapy is discussed. PMID- 8613134 TI - Severe hemosiderosis post allogenic bone marrow transplantation. AB - Abnormal liver function persisting late after allogeneic BMT is usually attributed to chronic GvHD, viral hepatitis or drug toxicity. We describe a patient who had negative hepatitis serology, was on no hepatotoxic medication, had no evidence of GvHD but had abnormal liver function 15 months post MBT. She was diagnosed as having grade IV hemosiderosis of the liver. Her total red cell support had only been 52 units. We therefore postulate that in a proportion of patients receiving allogeneic BMT impaired intestinal iron absorption may be an important cause of hemosiderosis. PMID- 8613136 TI - Current awareness in hematological oncology. PMID- 8613135 TI - Long-term control of polycythemia rubra vera with recombinant alpha interferon. AB - The natural history of PRV is characterized by a prolonged period of myeloproliferation chiefly affecting the red cell series. It can be controlled by venesection and cytotoxic agents. In this case report the patient declined further 'standard' chemotherapy to control his disease and the use of relatively low doses of IFN-a was successful in our patient in controlling myeloproliferation and stabilizing the disease over a prolonged period. These results support the use of IFN-a, not only as a means of myelosuppression in PRV but also as an agent with the potential to prevent disease progression. PMID- 8613137 TI - Long-term survival of patients with HIV-related systemic non-Hodgkin's lymphomas. AB - BACKGROUND: the overall outcome of patients with HIV-related non-Hodgkin's lymphomas (HIV-NHL) is poor because of the adverse clinico-pathological features of HIV-NHL and the underlying HIV infection. However, the experience of physicians in the management of HIV-NHL has increased, in particular in the use of intensive chemotherapy regimens, leading to an improvement in the prognosis of some of these neoplasms. Because some patients with AIDS may survive up to 5 years, it is possible to evaluate the long-term efficacy of the treatment of patients with HIV-NHL. In the general population, aggressive NHL, that are those occurring in HIV patients, may be considered cured after 2 years of lasting complete remission (CR) after chemotherapy. PATIENTS AND METHODS: we reviewed our monoinstitutional case-series of 73 HIV-infected patients with systemic NHL, observed between April 1985 and February 1993. Two groups of patients were arbitrarily identified, the first one (group A) including patients with a CR lasting for at least 2 years (N = 13) and the other including all remaining patients (group B) (N = 60). RESULTS: the 13 patients of group A differed significantly from the other patients in terms of higher CD4+ cell count and performance status (PS) at the time of diagnosis of NHL. There was no significant difference in the histological subtypes of the HIV-NHLs. The overall survival of the 73 patients was 8 months. In a separate analysis on all patients, age less than 30 years, PS less or equal to 1, a CD4+ cell count equal to or higher than 100/mm3 and the absence of B symptoms were significantly associated with a longer survival. The median survival in patients of group A was 42 months, however none of these patients relapsed during a median observation time of 42 months (range, 24-90). CONCLUSIONS: long-term survival and possibly cure can be obtained in some patients with HIV-NHL, in particular in those with a better PS and a less advanced immune dysfunction. In fact some of these patients are alive without evidence of disease 4 to 7 years after therapy, and others died of causes related to underlying HIV infection, in particular opportunistic infections, rather than relapse of NHL. PMID- 8613138 TI - Structure and organization of pig MHC class II DRB genes: evidence for genetic exchange between loci. AB - The pig major histocompatibility complex DRB genes were studied by polymerase chain reaction (PCR) amplification of exon 2 from eight domestic pigs and two European wild boars. Sequence comparisons together with a phylogenetic analysis showed the existence of at least three DRB genes of which only one appears to be expressed. The two putative DRB pseudogenes contained deletions in exon 2, making it possible to confirm the presence of three non-allelic DRB genes by analyzing the length polymorphism of the amplified PCR products. The expressed gene shows allelic polymorphism at the same positions as in the human DRB1 gene. In addition, this pig gene shows extensive allelic polymorphism at positions 84-88, whereas, e.g., human DRB genes do not. Surprisingly, the two putative DRB pseudogenes also display a considerable amount of allelic polymorphism, albeit of a different character as compared with the expressed DRB gene. Short stretches of sequences are shared between individual alleles at different loci. These sequence similarities cannot be due to natural selection, since two of the three DRB genes involved are polymorphic pseudogenes constituting allelic series that have diverged after the inactivation event. Instead, the results indicate that the sequences have been exchanged between the DRB genes by intergenic recombination. PMID- 8613139 TI - H2-M polymorphism in mice susceptible to collagen-induced arthritis involves the peptide binding groove. AB - The ability to develop type II collagen (CII)-induced arthritis (CIA) in mice is associated with the major histocompatibility I-A gene and with as yet poorly defined regulatory molecules of the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. H2-M molecules are thought to be involved in the loading of antigenic peptides into the MHC class II binding cleft. We sequenced H2-Ma, H2-Mb1, and H2-Mb2 genes from CIA-susceptible and resistant mouse strains and identified four different Ma and Mb2 alleles and three different Mb1 alleles defined by polymorphic residues within the predicted peptide binding groove. Most CIA-resistant mouse strains share common Ma, Mb1, and Mb2 alleles. In contrast, H2-M alleles designated Ma-III, Ma-IV, Mb1-III, and Mb2-IV could be exclusively identified in the CIA-susceptible H2r and H2q haplotypes, suggesting that allelic H2-M molecules may modulate the composition of different CII peptides loaded into MHC class II molecules, presumably presenting "arthritogenic" epitopes to T lymphocytes. PMID- 8613140 TI - Identical genetic control of MLC reactivity to different MHC incompatibilities, independent of production of and response to IL-2. AB - The inbred strain STS/A exhibits a higher proliferative response in the mixed lymphocyte culture (MLC) to stimulator cells of all 11 tested inbred mouse strains with 10 different major histocompatibility complex (MHC) haplotypes, as well as to stimulation with IL-2 than does the strain BALB/cHeA. However, alloantigen-stimulated BALB/c cells produce more IL-2 than STS/A cells. To study the genetic basis of these differences, we used 20 recombinant congenic strains (RCS) of the CcS/Dem series. Each of these CcS/Dem RC strains contains a different subset of about 12.5% of genes from the STS/A strain and the remaining approximately 87.5% of BALB/c origin genes. As a result the multiple non-linked genes responsible for phenotypic differences between BALB/c and STS/A became separated into different CcS/Dem strains. The strain distribution pattern (SD) of high or low MLC response of individual CcS/Dem strains to stimulator cells of four different strains was almost identical, indicating that differences in responsiveness, rather than the alloantigenic difference itself, determine the magnitude of the response, and that the responsiveness to different alloantigens is largely controlled by the same genes. The SDP of IL-2 stimulation was different from that of MLC responsiveness. The differences in the proliferative responses observed among individual CcS/Dem strains were not due to differences in numbers of CD3+, CD4+ or CD8+ cells or to the observed differences in IL-2 production, and hence they likely reflect genetically determined intrinsic properties of T cells. These results show that a set of non-linked genes controls proliferative responses in MLC irrespective of the MHC haplotype of the stimulator cells, and that stimulation with IL-2 and production of IL-2 are controlled by different subsets of genes. Since the genomes of all RCS are extensively characterized by microsatellite markers, they can be used to map the genes controlling proliferative responsiveness to stimulation with alloantigens and IL-2. PMID- 8613141 TI - Trans-species polymorphism of class II Mhc loci in danio fishes. AB - A characteristic feature of the major histocompatibility complex (Mhc) polymorphism in mammals is the existence of allelic lineages shared by related species. This trans-species polymorphism has thus far been documented only in primates, rodents, and artiodactyls. In this communication we provide evidence that it also exists in cyprinid (bony) fishes at the class II A and B loci coding for the alpha and beta polypeptide chains of the class II alpha:beta heterodimers. The study has focused on three species of the family Cyprinidae, subfamily Rasborinae: the zebrafish (Danio rerio), the giant danio (D. malabaricus), and the pearl danio (D. albolineatus). The polymerase chain reaction was used to amplify and then sequence intron 1 and exon 2 of the class II B loci and exon 2 of the class II A loci in these species. Phylogenetic analysis of the sequences revealed the existence of allelic lineages whose divergence predates the divergence of the three species at both the A and B loci. The lineages at the B locus in particular are separated by large genetic distances. The polymorphism is concentrated in the peptide-binding region sites and is apparently maintained by balancing selection. Sharing of this unique Mhc feature by both bony fishes and mammals suggests that the main function of the Mhc (presentation of peptides to T lymphocytes) has not changed during the last 400 million years of its evolution. PMID- 8613142 TI - Identification and characterization of a new major histocompatibility complex class I gene in carp (Cyprinus carpio L.). AB - In this study we report the finding of three representatives of a new group of major histocompatibility complex class I sequences in carp: Cyca-12 (Cyca UA1*01), a full-length cDNA; Cyca-SP1 (Cyca-UAW1), a polymerase chain reaction (PCR) fragment from cDNA; and Cyca-G11 (Cyca-UA1*02), a partial genomic clone. Comparison of the amino acid sequences of Cyca-12, Cyca-SP1, and Cyca-G11 with classical and non-classical class I sequences from other species shows considerable conservation in regions that have been shown to be involved in maintaining the structure and function of class I molecules. The genomic organization of Cyca-12 has been elucidated by analysis of a partial genomic clone (Cyca-G11, in combination with PCR amplifications on genomic DNA of a homozygous individual. Although the genomic organization is similar to that found in class I genes from other species, the 3' untranslated region contains an intron which is unprecedented in class I genes, and intron 2 is exceptionally large (+/-14 kilobases). Southern blot analysis indicates the presence of multiple related sequences. In phylogenetic analyses, the Cyca-UA sequences cluster with class I genes from zebrafish and Atlantic salmon, indicating that the ancestral gene arose before the salmonid/cyprinid split, approximately 120 150 million years ago. The previously reported class I Cyca-Z genes from carp and Caau-Z genes from goldfish cluster as a completely separate lineage. A polyclonal antiserum (anti-Cyca12) was raised against a recombinant fusion protein containing most of the extracellular domains of Cyca-12. The antibodies showed substantial reactivity to the recombinant protein and an Mr 45000 protein in membrane lysates of spleen and muscle, as well as to determinants present on leucocytes in fluorescence-activated cell sorter analyses. Erythrocytes and thrombocytes were found to be negative. PMID- 8613144 TI - Genetic variability of the human CD4 V2 domain. PMID- 8613145 TI - Two Tcrb-V8.3 epitopes: one unique and the other shared by Tcrb-V8.2. PMID- 8613143 TI - Fine mapping of the human pentraxin gene region on chromosome 1q23. AB - The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P (SAP) protein (APCS),a nd a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the alpha-subunit of the IgE high-affinity Fc receptor and the IFI-16 gene, an interferon-gamma-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid alpha spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-IFI-16-CRP-CRPP1-APCS FCER1A- tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. PMID- 8613146 TI - Molecular cloning of the gene coding for pig alpha1-->2fucosyltransferase. PMID- 8613147 TI - Nucleotide sequence of the human MHC class I MICA gene. PMID- 8613148 TI - Correction of the HLA-Cw3 genomic sequence tentatively identifies it as HLA Cw*0304. PMID- 8613149 TI - Identification of the new HLA-DRB1*0812 allele detected by sequenced based typing. PMID- 8613151 TI - Neonatal cervical choristoma--a case report. PMID- 8613152 TI - A rare Rh phenotype in a north Indian family. PMID- 8613150 TI - Physical mapping of the E/C and grc regions of the rat major histocompatibility complex. AB - Alignment of class I-hybridizing cosmids from an R21 (AlBlDlEugrc+) genomic DNA library gave two contigs: one [150 kilobases (kb)] encompassed the E/C region, or a large part thereof, and the other (110 kb) contained the grc region which has genes influencing resistance to chemical carcinogens (rcc), fertility (ft), and growth (dw-3). Amplification of gene sequences in the four cosmids in the E/C region using Eu-specific and LW2 (RT1.C)-specific primers showed that each cosmid contained both Eu-like and C-like genes. They are clearly different but closely associated, and they show some variation from the prototypic E (Eu) and C (LW2) genes, respectively. Comparison of DNA from grc+ and grc- strains of rats showed that the deletion in the grc- strains was approximately 50 kb, and that it was located on two of the three cosmids in the grc-region contig. The use of specific class I probes showed that the grc region contained tandemly duplicated RT1.O RT1.N genes and that the RT.BM1 loci lay outside of the grc region. Neither contig reacted with probes specific for class II, TNFA, Hsp70, or RT1.M genes. The data presented here and the previous data in the literature (summarized in Gill et al. 1995) suggest that the gene order in the major histocompatibility complex (MHC) and MHC-linked region of the rat is: A-E/C-grc-M. PMID- 8613153 TI - Giant adrenal cysts. PMID- 8613154 TI - Primary pure squamous cell carcinoma of the breast. PMID- 8613155 TI - Nasopharyngeal teratoma with a difference. PMID- 8613156 TI - Chondroid hamartoma of the lung--an underdiagnosed entity in fine needle aspiration cytology. PMID- 8613157 TI - Multiple gall bladder adenomas. PMID- 8613158 TI - Mycobacterium xenopi causing renal tuberculosis: a case report. PMID- 8613159 TI - Malignant mixed mesodermal (Mullerian) tumour of ovary. PMID- 8613160 TI - Salmonella inguinal abscess in a patient with systemic lupus erythematosus. PMID- 8613161 TI - Autopsy report of a case of perinatal AIDS. PMID- 8613162 TI - Bombay 'O' group blood (Oh phenotype) in two subjects in a family at Calcutta. PMID- 8613164 TI - Massive oedema and fibromatosis ovary--a report of three cases. PMID- 8613163 TI - Squamous-cell carcinoma arising in the remnant of an extirpated interacerebral epidermoid cyst. PMID- 8613165 TI - Torulopsis glabrata cystitis--a report of three cases. PMID- 8613166 TI - Adenocarcinoma in situ of the gall bladder--an incidental finding. PMID- 8613167 TI - Cutaneous paragonimiasis: a case report. PMID- 8613168 TI - Pulmonary blastoma--a case and review of literature. PMID- 8613169 TI - Platelet parameters detected by cell counter in an unusual case of extreme thrombocytosis. PMID- 8613170 TI - Keratinising squamous metaplasia of the urinary bladder. PMID- 8613171 TI - Paraganglioma-like adenoma of thyroid. PMID- 8613172 TI - T-cell rich B-cell lymphoma. PMID- 8613173 TI - Occult medullary carcinoma of the thyroid. PMID- 8613174 TI - Pilomatrix carcinoma--calcifying epitheliocarcinoma of Malherbe (a case report). PMID- 8613175 TI - Mixed tumour of breast--report of a rare case. PMID- 8613176 TI - Paroxysmal nocturnal hemoglobinuria--a case report. PMID- 8613177 TI - Echinostomiasis in India. PMID- 8613178 TI - Epignathus: a case report with review of literature. PMID- 8613180 TI - Gastric carcinoma complicating Menetrier's disease--a case report. PMID- 8613181 TI - Primary non-Hodgkin's lymphoma of the prostate gland--a case report. PMID- 8613179 TI - Diffuse form of primary leptomeningeal gliomatosis--a case report. PMID- 8613182 TI - Adenomatoid tumour of the uterus--a case report. PMID- 8613183 TI - Carcinoma of the breast and carcinoma of the uterine cervix. A rare form of double malignancy. PMID- 8613184 TI - Fabry's disease--a case report. PMID- 8613185 TI - Chronic recurrent parotitis complicated by salmonellosis in a renal transplant patient. PMID- 8613186 TI - Gastric mucormycosis. PMID- 8613187 TI - Multiple intraductal papillomas and sclerosing adenosis in the male breast. PMID- 8613188 TI - Mechanisms for inducible control of angiotensinogen gene transcription. AB - The intravascular renin-angiotensin system is an endocrine system designed to maintain cardiovascular homeostasis in response to hypotension. Under normal conditions, angiotensinogen concentrations circulating in the plasma are rate limiting for the maximum velocity of angiotensin I formation. In the liver, the major site of circulating angiotensinogen synthesis, angiotensinogen expression is under exquisite hormonal control. We review the mechanisms by which hormones effect transcriptional control of angiotensinogen expression. Adrenal-derived glucocorticoids produce the translocation of the glucocorticoid receptor into the nucleus. It in turn binds to two glucocorticoid response elements and stimulates angiotensinogen gene transcription. Inflammation activates angiotensinogen transcription as a result of the macrophage-derived cytokines interleukin-1 and tumor necrosis factor-alpha. These cytokines change the abundance of two transcription factor families that bind a single regulatory site in the angiotensinogen promoter, the acute-phase response element. These proteins include the nuclear factor-kappaB complex and the CCAAT/enhancer binding protein family. Activation of the renin-angiotensin system, through production of angiotensin II, results in feedback stimulation of angiotensinogen synthesis (the "positive feedback loop"). We have discovered that the nuclear factor-kappaB transcription factor is regulated by angiotensin II, a finding that provides a mechanism for the transcriptional component of angiotensinogen gene synthesis in the positive feedback loop. These studies underscore the concept that induction of the angiotensinogen gene by diverse physiological stimuli is mediated through changes in the nuclear abundance of sequence-specific transcription factors. The intracellular convergence of cytokine- and angiotensin II-induced signaling pathways on the nuclear factor-kappaB transcription factor provides a point for "cross talk" between angiotensin- and cytokine-activated second messenger pathways. PMID- 8613189 TI - Importance of tyrosine phosphorylation in angiotensin II type 1 receptor signaling. AB - Angiotensin II is the major effector peptide of the renin-angiotensin system. In addition to its vasoconstrictor activity, angiotensin II stimulates smooth muscle cell growth in arterial hypertension and in models of vascular injury. The angiotensin II type 1 receptor is a seven-transmembrane receptor and is responsible for virtually all the physiological actions of angiotensin II. This class of receptor signals in part through its association with heterotrimeric G proteins. A newly developed concept for guanine nucleotide protein-coupled receptors is the activation of intracellular second-messenger proteins via tyrosine phosphorylation. For instance, angiotensin II stimulates the rapid tyrosine phosphorylation and activation of phospholipase C-gamma1. Also, angiotensin II stimulates the tyrosine phosphorylation of Janus kinases. In this review, we discuss early signaling events induced by angiotensin II with an emphasis on tyrosine phosphorylation. Understanding the importance of tyrosine phosphorylation in the signaling pathways of the angiotensin II type 1 receptor may lead to new treatment modalities for cardiovascular disease. PMID- 8613190 TI - Salt sensitivity of blood pressure in humans. AB - A variety of different techniques have been used for the assessment of the blood pressure response to changes in salt and water balance in humans. These have generally been found to be reproducible and to yield congruent results. This review surveys the characteristics of subjects identified as salt sensitive and salt resistant by different investigators from demographic and physiological perspectives. PMID- 8613191 TI - Functional analysis of human tissue kallikrein in transgenic mouse models. AB - Clinical studies show that an inverse correlation exists between blood pressure and urinary kallikrein levels. It has been postulated that the tissue kallikrein kinin system contributes to the maintenance of normal blood pressure. To test this hypothesis, we have established transgenic mice that overexpress human tissue kallikrein under the promoter control of the mouse metallothionein gene and a liver-targeted albumin gene. These animals secrete human tissue kallikrein in plasma at levels 10- to 40-fold higher than that found in normal human serum, and they are chronically hypotensive. This hypotensive effect can be reversed by the injection of aprotinin, a potent tissue kallikrein inhibitor, or Hoe 140, a specific bradykinin receptor antagonist. Transgenic mice overexpressing human tissue kallikrein show a sustained reduction in blood pressure throughout their life spans, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein. Somatic gene delivery of rat kallikrein binding protein by muscle injection increases the blood pressure of the hypotensive transgenic mice to levels comparable with those in normotensive control mice. These results indicate that a direct link exists between kallikrein gene expression and alterations in blood pressure. In addition, we have developed normotensive transgenic mice that harbor the human tissue kallikrein gene containing 801 bp of its native promoter. The tissue distribution pattern of human kallikrein in these transgenic mice is similar to that in human tissues, with the highest level in the pancreas and much lower levels in the kidney and salivary gland. These transgenic mice provide new animal models for investigating the tissue-specific regulation of tissue kallikrein and its role in altering blood pressure. PMID- 8613192 TI - Strategies for studying cardiovascular phenotypes in genetically manipulated mice. AB - Unraveling the pathogenesis of complex cardiovascular diseases, such as hypertension, requires the development of in vivo animal model systems. Although large-animal models have long served as the gold standard, recent advances in transgenic and gene-targeting approaches, mouse genetics, and microsurgical technology are initiating a revolution that has led to the unexpected coupling of in vivo molecular physiology with genetically engineered mice. This article discusses the design of strategies to study complex cardiovascular phenotypes in genetically modified mice, including both transgenic and gene-targeted animals. At this time, a number of strategies are used to address specific molecular or physiological questions, and examples are briefly highlighted. In addition, a number of potential problems in the generation and use of transgenic mice in the study of cardiovascular biology are presented. PMID- 8613193 TI - Role of transcriptional cis-elements, angiotensinogen gene-activating elements, of angiotensinogen gene in blood pressure regulation. AB - Results of recent genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension. Using antisense technology, we demonstrated that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation. In the present study, we examined how the angiotensinogen gene is regulated in vivo. The transcriptional cis-elements, angiotensinogen gene activating elements (AGE) 2 and 3, have been reported to regulate angiotensinogen production in human hepatocytes in vitro. To determine the critical transcriptional regulator of angiotensinogen production in vivo, we used synthetic double-stranded oligodeoxynucleotides (ODN) as "decoy" cis-elements to block the binding of nuclear factors to promoter regions of the targeted gene, resulting in the inhibition of gene transactivation. Here we examined whether AGE 2 and AGE 3 in the promoter region of the angiotensinogen gene have a pivotal role in hepatic angiotensinogen production in vivo. Hepatic angiotensinogen mRNA was decreased by the transfection of AGE 2 but not mismatched decoy ODN. Transfection of decoy but not mismatched ODN against AGE 2 resulted in a transient decrease in blood pressure of spontaneously hypertensive rats (SHR), accompanied by a reduction in plasma angiotensinogen and angiotensin II levels. In contrast, transfection of AGE 3 decoy ODN had little effect on blood pressure. Overall, our results demonstrate that transfection of decoy ODN against AGE 2, but not against AGE 3, of the angiotensinogen gene resulted in a transient decrease in high blood pressure of SHR, suggesting that the transcriptional cis element AGE 2, rather than AGE 3, has an important role in blood pressure regulation through the control of circulating angiotensinogen. PMID- 8613194 TI - Permanent inhibition of angiotensinogen synthesis by antisense RNA expression. AB - The renin-angiotensin system plays a pivotal role in blood pressure regulation. Recent molecular biological findings led to the new concept that in addition to the classic endocrine system, local tissue systems may also play an important role in cardiovascular diseases such as hypertension. In particular, the brain renin-angiotensin system was shown to influence the central control of blood pressure and is thought to contribute to the hypertensive phenotype of genetically hypertensive rat models. To identify the physiological role of these local systems, we established an antisense strategy to downregulate the expression of the precursor hormone angiotensinogen (AOGEN) in cell culture, which can also be used to establish transgenic rat lines. Plasmids encoding an RNA sequence complementary to the rat AOGEN mRNA under control of different viral and tissue-specific promoters were constructed and transfected into an AOGEN expressing cell line. A competitive reverse transcription-polymerase chain reaction method was established for the quantification of AOGEN mRNA. Depending on the level of antisense RNA, the expression of the AOGEN gene was reduced down to 22% of control levels. Furthermore, the secretion of AOGEN protein was totally abolished. These results clearly demonstrate that the antisense constructs used are functional in reducing the AOGEN gene expression in vivo and can be used for the production of transgenic rats. PMID- 8613195 TI - Cathepsin B is a prorenin processing enzyme. AB - Conversion of prorenin to renin results from proteolytic cleavage of a 43-amino acid prorenin prosegment in renal juxtaglomerular cells. The enzyme that performs this processing is not known. Of several enzymes proposed, cathepsin B is a candidate because it colocalizes with renin in juxtaglomerular cell secretory granules and accurately cleaves the prosegment of human prorenin in vitro. It is not known whether cathepsin B can perform this function in the cell. We examined this using secretory granule-containing rat GH4C1 cells transfected with a human preprorenin expression vector. When treated with secretagogue (KCl 50 mmol/L + forskolin 10 micromol/L), these cells secrete 95% prorenin and 5% active renin into the medium, indicating little prorenin processing activity. In contrast, when the cells are cotransfected with a vector that expresses human preprocathepsin B or mouse prohormone convertase 1, secretagogue-induced secretion of active renin increased to 12% and 16.5%, respectively. With antisera that recognize the prosegment and renin, prorenin and renin were identified as proteins of 47 and 43 kD, respectively, and an antibody specific to the prosegment precipitated only the 47-kD species. These results do not address whether cathepsin B is the authentic renal prorenin processing enzyme. However, the results do demonstrate that cathepsin B can localize to the appropriate subcellular compartment and process prorenin to renin in GH4C1 cells and are consistent with a role for this enzyme in prorenin processing. PMID- 8613196 TI - Expression of aminopeptidase A, an angiotensinase, in glomerular mesangial cells. AB - Glomerular mesangial cells are known to express angiotensin II type 1 receptors and contract in response to circulating and/or locally produced angiotensin II. In addition, stimulation of mesangial cell matrix protein synthesis by elevated levels of angiotensin II is known to contribute to the development of glomerulosclerosis. Previously, we reported that mesangial cells were positively immunostained with antiserum directed against aminopeptidase A, the principal angiotensinase in the metabolism of angiotensin II. Here we demonstrate directly that aminopeptidase A is expressed in mesangial cells cultured from rat kidney. First, cultured mesangial cells had measurable aminopeptidase A enzymatic activity. Second, immunoblots for aminopeptidase A were positive for isolated glomeruli and mesangial cells, although two bands were seen for mesangial cells (approximately 138 and 144 kD), and only the larger band was seen for isolated glomeruli and kidney. Third, Northern blot hybridizations of total RNA from mesangial cells or kidney were positive and labeled similarly sized bands. Fourth, reverse transcription-polymerase chain reaction amplification of mesangial cell total RNA yielded a partial cDNA of the expected size that was confirmed by sequencing to be identical to rat kidney aminopeptidase A. These results indicate that aminopeptidase A is expressed within mesangial cells. These results further suggest that metabolism of angiotensin II by aminopeptidase A could play a protective role in minimizing the adverse effects of angiotensin II stimulation of mesangial cells. PMID- 8613198 TI - Regulation of angiotensin II type 2 receptor gene by the protein kinase C-calcium pathway. AB - In the present study, rat angiotensin II type 2 (AT2) receptor expression was upregulated in confluence-arrested PC12 cells compared with expression in proliferating cells. Treatment with cycloheximide inhibited the increase in mRNA levels in confluent cells. The state of growth arrest by serum deprivation was associated with increased expression of the AT2 receptor, which was markedly suppressed by exposure to the active phorbol ester 12-O-tetradecanoylphorbol 13 acetate and the calcium ionophore A23187. Similar inhibitions were also observed in myocytes isolated from neonatal rat heart. The change in AT2 mRNA levels by serum deprivation was due to the increase in the gene transcription rate. The effect of 12-O-tetradecanoylphorbol 13-acetate was mediated through decreases in gene transcription and mRNA stability, whereas A23187 affected mRNA stability. Vasoactive substances with the protein kinase C-calcium pathway, such as norepinephrine and angiotensin II, also downregulated the AT2 mRNA level in myocytes. These findings indicate that the expression of AT2 receptor in PC12 cells is regulated in a growth state-dependent manner, which is involved in confluence-induced new protein synthesis, thus providing a means by which cells can modulate their responsiveness to external angiotensin II stimulus. The activation of protein kinase C or calcium mobilization modifies this regulatory mechanism, suggesting that neurotransmitters or vasoactive substances with the protein kinase C-calcium pathway at least in part affect neuronal activity or blood pressure control by downregulating AT2 receptor expression. PMID- 8613197 TI - Angiotensin-(1-7) dilates canine coronary arteries through kinins and nitric oxide. AB - Angiotensin-(1-7) [Ang-(1-7)] was recently recognized to have novel biological functions that are distinct from those of Ang II. In these studies, we determined the vasoactive effects of Ang-(1-7) together with the endothelium-dependent mediator(s) of these responses in canine coronary arteries. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers perfused with 95% O2/5% CO2 at 37 degrees C. Ang-(1-7) caused significant concentration-dependent vascular relaxation (2.73 +/- 0.58 micromol/L, EC50) of rings precontracted with the thromboxane A2 analogue U46,619. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (1 mol/L) abolished the vasodilator response to Ang-(1-7), whereas treatment with the cyclooxygenase inhibitor indomethacin (10 micromol/L) was without effect. The vasodilator response produced by Ang-(1-7) was blocked by 75% with the bradykinin B2 receptor antagonist Hoe 140 (1 micromol/L) or by 80% with the nonselective Ang II antagonist [Sar1,Thr8]-Ang II (1 micromol/L). In contrast, the selective AT1 or AT2 Ang II antagonists CV 11974 (1 micromol/L), and PD 123319 (1 micromol/L), respectively, were ineffective in inhibiting the Ang-(1-7)-elicited vasodilation. Furthermore, pretreatment of the coronary rings with 2 micromol/L Ang-(1-7) markedly potentiated the bradykinin response. These results suggest that Ang-(1 7) elicits coronary vasodilation that is specifically mediated by the endothelium dependent release of nitric oxide. These responses involve a B2 bradykinin receptor and a non-AT1, non-AT2, angiotensin receptor. These data suggest that increases in circulating levels of Ang-(1-7) accompanying long-term administration of converting enzyme inhibitors or Ang II receptor blockers may contribute to the cardioprotective actions of these drugs. PMID- 8613199 TI - Human renin-dependent hypertension in rats transgenic for human angiotensinogen. AB - To examine the utility of rats transgenic for human angiotensinogen in the study of human renin-induced hypertension, we first developed assays to measure both the human and rat renin-angiotensin systems in these rats. We used human and mouse renin, transgenic human angiotensinogen, and the human renin inhibitor Ro 42-5892 to determine human- and rat-specific plasma angiotensinogen concentrations, renin activity, and renin concentration. The assays were validated with rat and human plasma mixed in known amounts and with plasma from rats transgenic for human renin. We then tested the human angiotensinogen transgenic rats by infusing recombinant human renin over 10 days (50 ng/h, n=4) with osmotic minipumps. High human angiotensinogen transgene expression was found in the liver, brain, kidney, gastrointestinal tract, and aorta, whereas rat angiotensinogen gene expression was detected in the liver and brain. During human renin infusion, blood pressure increased to >200/150 mm Hg. Before infusion, human angiotensinogen was 100-fold greater than rat angiotensinogen (141 +/- 73 versus 1.2 +/- 0.16 microg angiotensin l/mL); the relation was not changed by renin infusion. Plasma renin activity increased 300-fold; human plasma renin concentration increased to very high levels (449 +/- 262 ng of angiotensin I per mL per hour), whereas rat plasma renin concentration decreased to undetectable levels. Thus, chronic human renin infusion resulted in severe hypertension with extreme plasma renin activity and plasma renin concentration. However, even at these levels, human angiotensinogen was not rate limiting and angiotensin II was not a significant stimulus for angiotensinogen production. We conclude that these transgenic rats represent a novel model of human renin-dependent hypertension. PMID- 8613200 TI - SA gene expression in the proximal tubule of normotensive and hypertensive rats. AB - Previous studies have shown that the SA gene is expressed at higher levels in the kidney of genetically hypertensive rats than in control strains and that in hybrid crosses of genetically hypertensive rats and normotensive controls, markers in or close to the SA gene cosegregate with blood pressure. The present studies examine the localization of the SA gene product in the kidney by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). cDNA was prepared from microdissected nephron segments from Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHRs), and Wistar-Kyoto (WKY) rats, and RT-PCR was performed using specific primers. In all three strains, SA gene mRNA was found to be abundantly expressed in proximal tubules. SA PCR product was occasionally detected at approximately 100-fold lower abundance in glomeruli, while no signal was obtained from the collecting duct, thick ascending limb of the loop of Henle, or arcuate artery. Within the proximal tubule of normotensive rats, distribution of SA mRNA was found to be strain dependent: in SD rats it was expressed at high levels in the proximal convoluted tubule, whereas in WKY rats it was restricted to the proximal straight tubule. In SHRs, SA PCR product was detected along the entire proximal tubule. Induction of hypertension by renal artery clamping (two-kidney, one-clamp Goldblatt model) did not alter the pattern of expression observed in the SD rat. These results indicate that an extension of SA gene expression to the full length of the proximal tubule accompanies spontaneous hypertension and that in nonhypertensive animals the pattern of gene product expression is more restricted but shows substantial strain variability. PMID- 8613201 TI - Genetic characterization of the "new" Harlan Sprague Dawley Dahl salt-sensitive rats. AB - In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (S(HSD)). To evaluate the genetic integrity and salt sensitivity of thse new S(HSD), we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 S(HSD), 16 "authentic" SS/Jr from the inbred colony of John Rapp (S(Rapp)), 9 Harlan Sprague Dawley salt-resistant SR/Jr (R(HSD)), and (genotyping only) 6 known "contaminated" Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between S(Rapp) and S* and 18 were polymorphic between S(Rapp) and R(Rapp), but none of the 22 markers revealed polymorphisms between S(Rapp) and the new S(HSD). The phenotyping studies showed that during an ultra-low salt diet, mean arterial pressure was higher (P < .05) in both authentic S(Rapp) (129 +/- 2 mm Hg; mean +/- SE) and new S(HSD) (120 +/- 2 mm Hg) than in R(HSD) (93 +/- 1 mm Hg). A high salt diet increased mean arterial pressure in every S(HSD) and S(Rapp). Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P < 0.05) greater in authentic S(Rapp) (+51 +/- 3 mm Hg) than in new S(HSD) (+39 +/- 3 mm Hg). In addition, salt-induced mortality was significantly greater in S(Rapp) (62.5%) than S(HSD) (8.3%) after 8 weeks (P < 0.01). S(HSD) were genotypically indistinguishable from S(Rapp), had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new S(HSD) supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in S(HSD) compared with S(Rapp). This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the S(HSD) and S(Rapp) may now constitute distinct substrains of Dahl SS/Jr. PMID- 8613202 TI - cDNA cloning and gene expression of human type Ialpha cGMP-dependent protein kinase. AB - The type I cGMP-dependent protein kinase (cGK) is one of the major pathways for the cGMP cascade and has been demonstrated to inhibit platelet aggregation, relax smooth muscle cells, and control cardiocyte contractility. There are two subtypes of the type I cGK, cGKIalpha and cGKIbeta. The former is more sensitive to cGMP than the latter. In humans, cGKIbeta cDNA was isolated, but the full structure and tissue-specific gene expression of cGKIalpha have not been determined. The significance of cGK in human cardiovascular diseases has not been investigated at the molecular level. In the present study, we isolated the full-length human CGKIalpha cDNA (-36 to +2177; the translation start site: +1) enclosing the 671 amino acid protein. Nucleotides +267 to +2177 of the isolated cDNA were identical to the corresponding nucleotides of human cGKIbeta cDNA. Southern blot analysis suggested that human cGKIalpha and cGKIbeta are generated by alternative splicing of a single gene assigned to chromosome 10. By Northern blot analysis, we detected abundant human cGKIalpha mRNA (7.0 kb) in the aorta, heart, kidneys, and adrenals. In contrast, human cGKIbeta mRNA (7.0 kb) was detected abundantly only in the uterus. In cultured vascular smooth muscle cells, the type I cGK mRNA concentration was reduced to 10% of the basal level by 4 x 10(-10) mol/L platelet derived growth factor. Angiotensin II (10(-8) mol/L), transforming growth factor beta (4 x 10(-11) mol/L), and tumor necrosis factor-alpha (6 x 10(-6) mol/L) also exhibited an inhibitory effect on type I cGK gene expression. These findings suggest a pathophysiological implication of the type I cGK in cardiovascular diseases, including hypertension and atherosclerosis. PMID- 8613203 TI - Polymorphisms of renin-angiotensin genes among Nigerians, Jamaicans, and African Americans. AB - Within the context of an international collaborative study of the evolution of hypertension in the black diaspora, we determined the allelic distribution of hypertension candidate genes for the renin-angiotensin system in three populations of African origin. The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) and the M235T and T174M variants of the angiotensinogen (AGT) gene were examined in individuals from Nigeria, Jamaica, and the United States. Large differences in the prevalence of hypertension were recorded in door-to-door surveys, ranging from 16% in Nigeria to 33% in the United States. The frequency of the D allele was similar in all groups (54%, 59%, and 63% in Nigeria, Jamaica, and the United States, respectively). The 235T allele of the AGT gene was found in 81% of US and Jamaican blacks and 91% of Nigerians; very little variation was seen for the T174M marker. Despite large differences in hypertension rates, genetic variation at the index loci among these groups was modest. Overall, the frequency of the ACE*D allele was only slightly higher than that reported for European and Japanese populations, whereas the AGT 235T allele was twice as common. Compared with blacks in the western hemisphere, Nigerians had a higher frequency of the 235T allele, which is consistent with 25% European admixture in Jamaica and the United States. The results indicate the potential for etiologic heterogeneity in genetic factors related to hypertension across ethnic groups while suggesting that environmental exposures most likely explain the gradient in risk in the comparison among black populations. PMID- 8613204 TI - Cytochrome P4504A genotype cosegregates with hypertension in Dahl S rats. AB - Recent studies indicate that the production of 20-HETE by a P4504A2 enzyme in the outer medulla of the kidney is reduced in Dahl salt-sensitive (SS/Jr) rats, but the contribution of this abnormality to the elevation in loop Cl- transport and development of hypertension in this model is unknown. THe present study found that alleles at the locus for the P4504A2 gene cosegregate with blood pressure in an F2 population (n=151) derived from a cross between SS/Jr and Lewis rats (P < .0001). The P4504A2 locus is located in a region on rat chromosome 5 where a blood pressure quantitative trait locus was previously detected. Systolic blood pressure averaged 201 +/- 6 mm Hg in rats with the SS genotype (n=36), 192 +/- 4 mm Hg in SL genotype rats (n=77), and 169 +/- 3 mm Hg in LL genotype rats (n=38). In further studies, we confirmed that there are phenotypic differences in the expression of the P4504A2 gene in the kidneys of SS/Jr and Lewis rats. Although the production of 20-HETE from 14C-arachidonic acid was similar in microsomes prepared from the renal cortex of SS/Jr and Lewis rats (54 +/- 3 versus 55 +/- 3 pmol/min/mg protein), the production of 20-HETE in microsomes prepared from the outer medulla (OM) was markedly reduced in SS/Jr rats (2.8 +/- 0.8 versus 6.7 +/- 1 pmol/min/mg protein). The diminished production of 20-HETE in the OM was due to a threefold reduction in the level of P4504A2 protein. These results suggest that an altered expression of the P4504A2 enzyme in the OM may contribute to the development of hypertension in SS/Jr rats. PMID- 8613205 TI - Angiotensin I-converting enzyme gene polymorphism and salt sensitivity in essential hypertension. AB - We undertook the present study in 66 Japanese patients with essential hypertension to identify genetic factors associated with salt sensitivity. Patients were classified into salt-sensitive or salt-resistant groups on the basis of changes in their mean blood pressures from a week of a low salt diet (50 mmol/d) to a week of a high salt diet (340 mmol/d). Salt sensitivity and resistance were studied in relation to a 287-bp insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme gene detected by a polymerase chain reaction method and the haptoglobin phenotype determined by polyacrylamide gel electrophoresis. Patients with the angiotensin I-converting enzyme gene genotype II were more apt to be salt sensitive than patients with the ID and DD genotypes, although plasma renin activity was similar in each group. The frequency of the I allele in the salt-sensitive group was significantly higher than that in the salt-resistant group (chi2 = 7.4, odds ratio = 2.78). However, there was no significant relationship between haptoglobin phenotype and salt sensitivity. These data suggest that an I/D polymorphism of the angiotensin I converting enzyme gene is a genetic factor associated with salt sensitivity of blood pressure independently of plasma renin activity in Japanese patients with essential hypertension. PMID- 8613206 TI - Salt-sensitive hypertension in (mREN-2)27 transgenic rats. AB - The (mREN-2)27 transgenic model of hypertension was developed to investigate the effect of genetic over activity of angiotensin II systems as a contributing factor in the development of arterial hypertension. In this model, transgene positive rats demonstrate elevated renin-angiotensin system activity not only in the circulatory system but also in adrenal gland, reproductive organs, and brain. Since evidence indicates that angiotensin peptides and osmotic stimuli interact synergistically to produce exaggerated behavioral, endocrine, and cardiovascular effects, we examined the effect of salt consumption on arterial pressure, plasma vasopressin, and body fluid balance in male (mREN-2)27 transgene-positive and negative rats. Four days of drinking 2% NaCl increased mean arterial pressure from 165 +/- 10 to 199 +/- 7 mm Hg in transgene-positive rats. In contrast, transgene-negative rats showed no change in arterial pressure (126 +/- 5 to 128 +/- 3 mm Hg). Plasma vasopressin levels were significantly elevated only in transgene-positive rats, whereas pituitary levels of vasopressin were significantly lower in transgene-positive rats compared with transgene-negative controls (18 +/- 3 and 118 +/- 14 ng, respectively). Although transgene-positive rats consumed significantly more 2% NaCl than did transgene-negative rats, during this period 24-hour sodium balance did not differ between the groups. Since fluid and electrolyte balance is similar between the two groups of rats, the data suggest that transgene-positive rats may be more sensitive to the effects of increased NaCl intake in terms of both endocrine and cardiovascular responses. PMID- 8613207 TI - Acute saline infusion decreases norepinephrine release in the anterior hypothalamic area. AB - Ingestion of a high NaCl diet elevates arterial pressure in spontaneously hypertensive rats, at least in part, by reducing the release of norepinephrine in the anterior hypothalamic area. The mechanism by which dietary NaCl excess alters anterior hypothalamic area norepinephrine release is unknown. Plasma Na+ is slightly elevated after ingestion of a meal; therefore, in the present study we tested the hypothesis that a small increase in plasma Na+ could reduce the release of norepinephrine in the anterior hypothalamic area and elevate arterial pressure. Male spontaneously hypertensive rats were randomized to be fed a diet containing either 1% (basal) or 8% (high) NaCl at age 7 weeks and were maintained on the diets for 2 weeks. Age-matched normotensive Wistar-Kyoto rats received a basal NaCl diet only. All rats were instrumented with a push/pull cannula, and 5 days later, the baseline release of 3-methoxy-4-hydroxyphenyl glycol (the major metabolite of norepinephrine in brain) was measured in awake, freely moving rats. Rats were then challenged with an intravenous infusion (75 microL/min) of hypertonic (2.7%) saline for 20 minutes. In spontaneously hypertensive rats fed a basal NaCl diet, the hypertonic saline infusion elevated mean arterial pressure by 12% and reduced the concentration of the norepinephrine metabolite in the anterior hypothalamic area by 19%; these alterations persisted after termination of the hypertonic saline infusion. Spontaneously hypertensive rats maintained on the high NaCl diet showed greatly reduced arterial pressure and norepinephrine metabolite responses. In normotensive control rats compared with the hypertensive rats fed the basal NaCl diet, the hypertonic saline had considerably less effects on arterial pressure and norepinephrine metabolite levels in the anterior hypothalamic area, and the responses were significantly shorter. Thus, a small elevation in plasma Na+ can reduce the release of norepinephrine in the anterior hypothalamic area. This response is greatly exaggerated in spontaneously hypertensive rats fed a basal (but not a high) NaCl diet, suggesting that a postprandial rise in NaCl could initiate the fall in norepinephrine and thereby contribute to the rise in arterial pressure in spontaneously hypertensive rats ingesting a high NaCl diet. PMID- 8613208 TI - Platelet activation in carotid sinuses triggers reflex sympathoinhibition and hypotension. AB - The carotid sinuses, one of the major sites of baroreceptor innervation, are also a common site of atherosclerotic lesions and platelet aggregation. The goal of the present study was to determine whether platelet activation in carotid sinuses causes reflex-mediated changes in renal sympathetic nerve activity and arterial pressure. Rabbit platelets were isolated, resuspended in Krebs' buffer, and activated by thrombin. Injection of activated platelets (3 x 10(8) platelets/mL) into the vascularly isolated carotid sinuses of anesthetized rabbits essentially eliminated sympathetic nerve activity and acutely decreased mean arterial pressure from 126 +/- 5 to 53 +/- 4 mm Hg (n=16; P < .05). Sympathetic activity and arterial pressure returned to control levels over a period of minutes despite sustained exposure to activated platelets. Injection of U-46619, a thromboxane analogue and vasoconstrictor, into carotid sinuses did not alter sympathetic activity or arterial pressure. However, serotonin (5-hydroxytryptamine [5-HT]), which is known to be released from activated platelets, and the 5-HT3 receptor agonist phenylbiguanide mimicked the effect of platelets. Furthermore, the platelet-induced reflex inhibition of sympathetic activity and hypotension were not altered by the cyclooxygenase inhibitor indomethacin but were attenuated significantly by 5-HT receptor antagonists. Platelet activation inhibited sympathetic activity to 5 +/- 2% of control in the absence of antagonists but to only 35 +/- 11 and 76 +/- 4% of control after selective blockade of 5-HT2 and 5 HT3 receptors with ketanserin and MDL-72222, respectively. The results indicate that (1) platelet activation in carotid sinuses triggers reflex inhibition of sympathetic nerve activity and hypotension; (2) the reflex is not caused by carotid vasoconstriction and is not mediated by prostanoids; and (3) the reflex is mediated by 5-HT acting primarily on 5-HT3 and to a lesser extent on 5-HT2 receptors. We speculate that this reflex may contribute to arterial pressure lability and susceptibility to stroke in patients with carotid atherosclerotic disease. PMID- 8613209 TI - Role of area postrema in transgene hypertension. AB - Transgenic [Tg(+)] rats carrying the mouse Ren-2d gene [(mRen-2d)27] are a newly established monogenetic form of experimental hypertension. To determine whether the area postrema contributes to the development of hypertension in mRen-2 Tg(+) rats, this circumventricular organ in the fourth ventricle was removed from 5 week-old Tg(+) rats. From weeks 4 through 9, systolic blood pressure was measured weekly by tail-cuff plethysmography in area postrema-lesioned and sham-lesioned Tg(+) rats. Although systolic blood pressure rose markedly in sham-lesioned Tg(+) rats, the increase in systolic blood pressure was significantly attenuated in area postrema-lesioned Tg(+) rats. At 9 weeks of age, a femoral artery was cannulated for the measurement of arterial pressure in awake rats. Mean arterial pressure (MAP) in area postrema-lesioned Tg(+) rats was significantly (P < .01) lower than that in sham-lesioned rats: 171 +/- 7 and 132.+/- 5 mm Hg, respectively. Baroreceptor reflex was evaluated by intravenous infusion of sodium nitroprusside. There was no significant difference in baroreceptor reflex sensitivity between the two groups. Intravenous pentolinium (5 mg/kg), used to produce sympathetic ganglionic block, caused significant decreases in MAP in both groups. However, the reduction of MAP in the sham-lesioned group was significantly (P < .05) greater than that in the area postrema-lesioned group: 73 +/- 4 and -48 +/- 6 mm Hg, respectively. The ratio of left ventricular weight to body weight in sham-lesioned Tg(+) rats was significantly larger than that of area postrema-lesioned rats. These results suggest that ablation of the area postrema markedly attenuates the development of hypertension in mRen-2d Tg(+) rats, and this attenuation may be attributed to decrease in sympathetic outflow. PMID- 8613210 TI - Hypotensive response to losartan in normal rats. Role of Ang II and the area postrema. AB - We have reported that the angiotensin II (Ang II) AT1 receptor antagonist losartan markedly lowers arterial pressure in sodium-replete, normotensive rats. We hypothesized that this action of losartan was mediated by its blocking the effects of endogenous Ang II. To test this hypothesis, rats were instrumented with arterial and venous catheters for measurement of arterial pressure and infusion of losartan, respectively. After 3 days of control measurements, losartan was infused for 10 days (10 mg/kg/d) in rats on a normal daily sodium intake (NNa; approximately 2 mmol/d, n=6) and rats on a high daily sodium intake (HNa; approximately 15 mmol/d, n=7) to suppress endogenous Ang II. Although basal plasma renin activity was markedly suppressed in HNa rats (0.9 +/- 0.4 ng Ang I/ mL/h) compared with NNa rats (4.0 +/- 0.3 ng Ang I/mL/h), control arterial pressure was not different between NNa (113 +/- 4 mm Hg) and HNa (113 +/- 2 mm Hg) rats. Losartan decreased arterial pressure from control levels in NNa rats on the first day of infusion (-12 +/- 2 mm Hg) but had no effect on arterial pressure in HNa rats (+4 +/- 4 mm Hg). Furthermore, by day 10 of losartan infusion, arterial pressure had decreased further from control levels in NNa rats (-32 +/- 2 mm Hg) but remained unchanged compared with control in HNa rats (+5 +/ 6 mm Hg). A second study was conducted to test the hypothesis that the area postrema, a circumventricular organ proposed to mediate the long-term neurogenic pressor activity of Ang II is a site of action for losartan. After 3 control days, losartan was administered for 10 days to area postrema-lesioned rats (APx; n=11) or sham-lesioned rats (n=10) consuming an NNa diet. Control arterial pressure was similar in sham (95 +/- 3 mm Hg) and APx (96 +/- 2 mm Hg) rats. Basal plasma renin activity was not different between groups (sham, 4.1 +/- 1.5 versus APx, 5.3 +/- 1.6 mm Hg Ang I/mL/h). On day 1 of losartan treatment, arterial pressure decreased to a significantly lower level in sham (80 +/- 2 mm Hg) compared with APx (90 +/- 3 mm Hg) rats. This trend continued through day 4 of losartan infusion, in which arterial pressure in sham rats (72.2 +/- 2 mm Hg) was significantly lower than in APx rats (83 +/- 4 mm Hg). However, during the remainder of the losartan infusion, there were no significant differences between groups with the exception of day 8 (sham, 72 +/- 2 mm Hg; APx, 84 +/- 2 mm Hg). Taken together, these results support the hypothesis that the hypotensive actions of losartan in sodium-replete, normotensive rats are due to blockade of the physiological effects of endogenous Ang II. Furthermore, an intact area postrema is essential for full expression of the hypotensive actions of losartan in normal rats. PMID- 8613211 TI - Bradykinin and protein kinase C activation fail to stimulate renal sensory neurons in hypertensive rats. AB - In normotensive rats, renal sensory receptor activation by increased ureteral pressure results in increased ipsilateral afferent renal nerve activity, decreased contralateral efferent renal nerve activity, and contralateral diuresis and natriuresis, a contralateral inhibitory renorenal reflex response. In spontaneously hypertensive rats (SHR), increasing ureteral pressure fails to increase afferent renal nerve activity. The nature of the inhibitory renorenal reflexes indicates that an impairment of the renorenal reflexes would contribute to the increased efferent renal nerve activity in SHR. We therefore examined whether there was a general decrease in the responsiveness of renal sensory receptors in SHR by comparing the afferent renal nerve activity responses to bradykinin in SHR and Wistar-Kyoto rats (WKY). In WKY, renal pelvic perfusion with bradykinin at 4, 19, 95, and 475 micromol/L increased afferent renal nerve activity by 1066 +/- 704, 2127 +/- 1121, 3517 +/- 1225, and 4476 +/- 1631% x second (area under the curve of afferent renal nerve activity versus time). In SHR, bradykinin at 4 to 95 micromol/L failed to increase afferent renal nerve activity. Bradykinin at 475 micromol/L increased afferent renal nerve activity in only 6 of 10 SHR. In WKY, renal pelvic perfusion with the phorbol ester 4beta phorbol 12,13-dibutyrate, known to activate protein kinase C, resulted in a peak afferent renal nerve activity response of 24 +/- 4%. However, 4beta-phorbol 12,13 dibutyrate failed to increase afferent renal nerve activity in SHR. These findings demonstrate decreased responsiveness of renal pelvic sensory receptors to bradykinin in SHR. The impaired afferent renal nerve activity responses to bradykinin in SHR may be due to a lack of protein kinase C activation or a defect in the intracellular signaling mechanisms distal to protein kinase C activation. PMID- 8613212 TI - Role of renal nerves in mediating the hypertensive effects of nitric oxide synthesis inhibition. AB - Recent studies suggest that enhanced renal sympathetic nervous activity plays an important role in mediating the renal hemodynamic and electrolyte excretion changes associated with acute inhibition of NO synthesis. The purpose of this study was to determine the importance of renal nerves in mediating the long-term hypertensive and renal actions of NO synthesis blockade. To achieve this goal, we infused N(G)-nitro-L-arginine methyl ester (L-NAME) at a rate of 25 microg/kg per minute for 2 weeks in control dogs and in bilaterally renal-denervated dogs. NO synthesis blockade in control dogs increased arterial pressure by 18%, from 94 +/ 3 to 111 +/- 4 mm Hg, and decreased heart rate from 74 +/- 4 to 57 +/- 4 beats per minute (bpm). L-NAME also decreased renal plasma flow from 195 +/- 18 to 166 +/- 18 mL/min while having no effect on glomerular filtration rate (67 +/- 7 versus 63 +/- 6 mL/min). In the renal-denervated dogs, inhibition of NO synthesis increased arterial pressure by 14%, from 92 +/- 4 to 105 +/- 5 mm Hg, and decreased heart rate from 80 +/- 4 to 65 +/- 5 bpm. Renal plasma flow in this group decreased from 195 +/- 20 to 165 +/- 20 mL/min, whereas glomerular filtration rate remained unchanged (66+/- 6 versus 64 +/- 6 mL/min). In addition, renal excretion of sodium and water in response to L-NAME was similar in each group. The results of this study indicate that the long-term hypertensive and renal effects of NO synthesis inhibition in the dog are not dependent on activation of the renal sympathetic nervous system. PMID- 8613213 TI - Acute sympathoinhibitory actions of metformin in spontaneously hypertensive rats. AB - Chronic treatment with the antihyperglycemic agent metformin prevents hypertension in spontaneously hypertensive rats. This effect has been ascribed to normalization of plasma insulin levels. However, whether metformin affects arterial pressure via changes in sympathetic nerve activity is unknown. Therefore, the objective of this study was to examine whether acute administration of metformin produces changes in mean arterial pressure, heart rate, or efferent renal sympathetic nerve activity in spontaneously hypertensive rats. Rats were anesthetized with alphaxalone-alphadolone (Saffan), paralyzed with pancuronium, and artificially ventilated. Intravenous administration of metformin (0, 1, 10, 100 mg/kg) produced dose-dependent reversible decreases in mean arterial pressure, heart rate, and efferent renal sympathetic nerve activity that were not affected by arterial or cardiopulmonary baroreceptor denervation, nitric oxide synthase inhibition by N(omega)-nitro-L-arginine methyl ester, or cyclooxygenase inhibition by indomethacin. Metformin given into the lateral cerebral ventricle (250, 500, 1000 microg) produced dose-dependent decreases in mean arterial pressure, heart rate, and efferent renal sympathetic nerve activity in doses that caused no changes when given intravenously. The sympathoinhibitory response to intracerebroventricular administration of metformin was not affected by alpha2-adrenoceptor blockade by intracerebroventricular yohimbine. We conclude that metformin has acute sympathoinhibitory effects (decreased arterial pressure, heart rate, and efferent renal sympathetic nerve activity) that are produced by a direct central nervous system site of action. PMID- 8613215 TI - Role of 20-HETE in elevating loop chloride reabsorption in Dahl SS/Jr rats. AB - In vivo tubular perfusion experiments were performed in normotensive Dahl salt sensitive (SS/Jr) and salt-resistant (SR/Jr) rats maintained from birth on a low salt (0.4% NaCl) diet to examine the role of 20-HETE in elevating loop Cl- transport in SS/Jr rats. Chloride reabsorption in the loop of Henle was significantly greater in SS/Jr than in SR/Jr rats (77 +/- 2% versus 57 +/- 3% of the perfused Cl- load). When the renal metabolism of arachidonic acid by P450 was blocked by the addition of 17-octadecynoic acid (10 micromol/L) to the perfusate, loop Cl- transport increased in SR/Jr rats to 70 +/- 2% of the delivered Cl- load, but it had no effect in SS/Jr rats. Conversely, addition of 20-HETE (10 micromol/L) to the perfusate lowered loop Cl- transport in S rats to 60 +/- 2% of perfused Cl- load, but it had no effect in SR/Jr rats. Addition of another endogenously formed HETE to the perfusate, 15-HETE (20 micromol/L), had no effect on Cl- reabsorption in the loop of Henle of SS/Jr rats. These findings indicate that endogenously produced P450 metabolites of arachidonic acid regulate Cl- transport in the loop of Henle of the rat in vivo and support the view that a diminished production of 20-HETE in the outer medulla of SS/Jr rats contributes to the elevation in loop Cl- transport and the resetting of the pressure natriuresis relation in these animals. PMID- 8613214 TI - Renal sympathetic neural mechanisms as intermediate phenotype in spontaneously hypertensive rats. AB - The borderline hypertensive rat, the F1 of a cross between a hypertensive spontaneously hypertensive rat (SHR) and a normotensive Wistar-Kyoto (WKY) rat, is a NaCl-sensitive model of genetic hypertension. In addition to hypertension, borderline hypertensive rats fed 8% NaCl develop characteristic alterations in the regulation of efferent renal sympathetic nerve activity and the neural control of renal function that are similar to those observed in the SHR parent. Like the normotensive WKY rat parent, borderline hypertensive rats fed 1% NaCl remain normotensive and do not exhibit these alterations in renal sympathetic neural mechanisms. These renal sympathetic neural mechanisms constitute a complex quantitative trait that may represent an intermediate phenotype. They have a plausible pathogenetic role in hypertension and are different between SHR and WKY rats. This study evaluated two aspects of this complex quantitative trait, enhanced renal sympathoexcitation with air-jet stress and enhanced renal sympathoinhibition with guanabenz, as a candidate intermediate phenotype. As neither of these aspects was observed in two-kidney, one clip Goldblatt hypertensive rats, this suggests that the trait is not secondary to hypertension from an acquired cause. In a backcross population (F1 x WKY) fed 8% NaCl for 12 weeks, both enhanced renal sympathoexcitation with air-jet stress and enhanced renal sympathoinhibition with guanabenz cosegregated with the hypertension. These results support renal sympathetic neural mechanisms as an intermediate phenotype in SHR. PMID- 8613216 TI - Cardiac insulin-like growth factor I and growth hormone receptor expression in renal hypertension. AB - The aim of the present study was to investigate the role of insulin-like growth factor I in the development of cardiac hypertrophy in two-kidney, one clip hypertension by relating growth hormone receptor and insulin-like growth factor I receptor mRNA levels to insulin-like growth factor I gene transcription using a solution hybridization/RNase protection assay. Two-kidney, one clip hypertension was induced in male Wistar rats, and experiments were performed 2, 4, 7, and 12 days after surgery. Systolic blood pressure was elevated 2, 7, and 12 days after clipping (P < .001). Left ventricular weights were increased 2, 4, 7, and 12 days after surgery (P < .01). Associated with the rise in blood pressure, left ventricular insulin-like growth factor I mRNA was increased 2, 7, and 12 days after surgery (P < .01). Furthermore, growth hormone receptor and insulin-like growth factor I receptor gene expression increased specifically in the left ventricle of renal hypertensive rats (P < .05 and P < .001, respectively). Left ventricular growth hormone receptor mRNA peaked 7 days after induction of renal artery stenosis. These results show that insulin-like growth factor I, growth hormone receptor, and insulin-like growth factor I receptor mRNA increase in the pressure-overloaded left ventricle of two-kidney, one clip rats, suggesting a role for insulin-like growth factor I and the growth hormone/insulin-like growth factor I axis in the development of cardiac hypertrophy. PMID- 8613217 TI - Renal response to L-arginine in salt-sensitive patients with essential hypertension. AB - This study examined whether disturbances in nitric oxide formation contribute to renal dysfunction in salt-sensitive essential hypertensive patients. We evaluated the effects of intravenous administration of L-arginine (500 mg/kg given over 30 minutes) on systemic and renal hemodynamics in 23 patients with mild essential hypertension during 1 week of a low NaCl diet (50 mmol/d) followed by 1 week of a high NaCl diet (340 mmol/d). Patients were classified as salt sensitive (n=10) or salt resistant (n=13) based on salt-induced changes in their blood pressures. Salt loading increased renal vascular resistance but not renal plasma flow in salt-sensitive patients. The L-arginine-induced renovascular relaxation was significantly reduced by a high NaCl diet (renal vascular resistance: low NaCl 12.4 +/- 2.3% versus high NaCl -7.1 +/- 1.8%, P < .001) in salt-sensitive patients, whereas it was unchanged in salt-resistant patients. The increase in plasma cGMP in response to L-arginine was also reduced by a high NaCl diet in the salt-sensitive patients (low NaCl 49 +/- 7% versus high NaCl 36 +/- 8%, P < .05) but not in the salt-resistant patients (low NaCl 51 +/- 6% versus high NaCl 58 +/ 6%). These findings suggest that NaCl loading in salt-sensitive patients with mild essential hypertension reduces the ability of L-arginine to produce nitric oxide in the endothelium of the renal vasculature. PMID- 8613218 TI - Influence of NaCl concentration at the macula densa on angiotensin II-induced constriction of the afferent arteriole. AB - The macula densa, a plaque of specialized tubular epithelial cells, monitors NaCl concentrations in tubular fluid and controls resistance of the glomerular afferent arteriole (AA). In vivo micropuncture studies suggest that there are significant interactions between angiotensin II (Ang II) and macula densa control of glomerular hemodynamics. We tested the hypothesis that Ang II causes stronger constriction of the AA when NaCl concentration at the macula densa is elevated. Rabbit AAs and the attached macula densa were simultaneously microperfused in vitro, and dose-response curves to Ang II were obtained when the macula densa was not perfused or was perfused with either low NaCl (Na+, 26 mEq/L; Cl-, 7 mEq/L) or high NaCl (Na+, 84 mEq/L; Cl-, 65 mEq/L). Ang II induced stronger constriction when the macula densa was perfused with high NaCl; the decrease in diameter at 100 pmol/L was 29 +/- 5.6% (n= 7) compared with 2.1 +/- 1.2% (n=8) for the nonperfused macula densa or 6.1 +/- 4.2% (n=7) for low NaCl (P < .002). However, there was no such difference in the action of norepinephrine. Adding furosemide (10 micromol/L) to the macula densa perfusate abolished the difference in Ang II action between low and high NaCl at the macula densa. Since AA tone is higher when the NaCl concentration at the macula densa is elevated, we tested whether augmented Ang II action is due to higher AA tone. Preconstriction of the AA by 20% with norepinephrine had no effect on Ang II action. Thus, our results demonstrate that sensitivity of the AA to Ang II increases when NaCl concentration at the macula densa is elevated. Such modulation of Ang II action by macula densa NaCl concentration may be important in the control of glomerular hemodynamics. PMID- 8613219 TI - Impaired renal vasodilation and urinary cGMP excretion in Dahl salt-sensitive rats. AB - We previously have shown that Dahl salt-sensitive rats increase renal vascular resistance in response to excessive salt feeding before total peripheral resistance increases and hypertension occurs. Failure of renal vasculature to dilate, as normally occurs in Dahl salt-resistant rats fed a high salt diet, may play a role in the development of hypertension in Dahl salt-sensitive rats. We also showed that renal vasculature in salt-sensitive rats is hyperreactive to vasoconstrictors and hyporeactive to vasodilators. Atrial natriuretic peptide, by stimulating cell-bound receptors, and nitroprusside, by generating nitric oxide, cause renal vasodilation by generating cGMP. Studies were undertaken to determine whether defective renal vasodilation in Dahl salt-sensitive rats is due to impaired production of cGMP. We examined the influence of nitroprusside infusion and salt intake on renal vascular resistance and cGMP excretion in salt-sensitive rats. Results demonstrate that salt feeding and nitroprusside infusion increase cGMP excretion and decrease renal vascular resistance in salt-resistant rats (P < .01), and, although this relationship was less clear in salt-sensitive rats, there was a reciprocal relationship between renal vascular resistance and cGMP excretion in all animals studied. Salt feeding and nitroprusside infusion caused less of an increase in cGMP excretion in salt-sensitive than in salt-resistant rats (P < .01). In conclusion, these studies support the concept that impairment in cGMP generation may play a primary role in the inability of the kidneys of Dahl salt-sensitive rats to vasodilate in response to increased salt intake. Such an impairment could contribute to salt retention and the development of hypertension. PMID- 8613220 TI - Renal accumulation of circulating angiotensin II in angiotensin II-infused rats. AB - Previous studies have demonstrated that low-dose angiotensin II (Ang II) infusion for 14 days mimics two-kidney, one clip Goldblatt hypertension and increases intrarenal Ang II levels. The objective of the present study was to determine whether the augmented intrarenal Ang II is due to intrarenal accumulation of the infused Ang II and/or to an increase in intrarenal formation of endogenous Ang II. Male Sprague-Dawley rats were uninephrectomized and divided into three groups: control (N=6), those infused with [Ile5]Ang II (endogenous form) (N=6), and those infused with [Val5]Ang II (n=8). [Ile5]Ang II or [Val5]Ang II was infused at 40 ng/min via an osmotic minipump implanted subcutaneously. By day 12, systolic blood pressure increased significantly in both [Val5]Ang II-infused rats (197 +/- 7 mm Hg) and [Ile5]Ang II-infused rats (173 +/- 3 mm Hg). Blood and kidney samples were harvested, subjected to high-performance liquid chromatography to separate [Val5]Ang II from [Ile5]Ang II, and then measured by radioimmunoassay. Plasma renin activity was markedly suppressed in both [Ile5]Ang II- and [Val5]Ang II-infused rats. Plasma Ang II levels were elevated in rats infused with both [Ile5]Ang II (121 +/- 24 fmol/mL) and [Val5]Ang II (119 +/- 14 fmol/mL) compared with controls (69 +/- 15 fmol/mL). Both [Ile5]Ang II- and [Val5]Ang II-infused rats exhibited an enhancement of total intrarenal Ang II. Only [Ile5]Ang II (358 +/- 53 fmol/g) was detected in the kidneys of rats infused with -Ile5-Ang II. In [Val5]Ang II-infused rats, a significant portion of total renal Ang II (371 +/- 57 fmol/g) was in the form of [Val5]Ang II (256 +/- 44 fmol/g). Renal [Ile5]Ang II levels were maintained in the [Val5]Ang II-infused rats (116 +/- 15 fmol/g) compared with control rats (116 +/- 11 fmol/g) despite marked suppression of renin release. These results support the hypothesis that infused circulating ANG II is bound to receptor or taken up intrarenally in a manner that protects against degradation. PMID- 8613221 TI - Interaction of adrenomedullin and platelet-derived growth factor on rat mesangial cell production of endothelin. AB - Adrenomedullin has recently been isolated from human pheochromocytoma. We designed the present study to examine the effect of adrenomedullin on the production of the vasoconstrictive and growth-promoting peptide endothelin-1 (ET 1) after stimulation with platelet-derived growth factor (PDGF) in cultured rat glomerular mesangial cells. PDGF stimulated ET-1 production in a concentration dependent manner. Rat adrenomedullin inhibited this stimulated ET-1 production in a concentration-dependent manner between 10(-7) and 10(-8) mol/L. Rat adrenomedullin also increased the cellular level of cAMP in a concentration dependent manner between 10(-7) and 10(-8) mol/L. Human adrenomedullin was less effective than rat adrenomedullin with respect to inhibiting ET-1 production and increasing cAMP levels. The addition of 8-bromo-cAMP (10(-3) and 10(-4) mol/L) reduced PDGF-induced ET-1 production. Furthermore, forskolin (10(-4) and 10(-5) mol/L), an activator of adenylate cyclase, reduced PDGF-induced ET-1 production. In contrast, the basal production of ET-1 was not significantly altered by rat and human adrenomedullin. These results indicate that adrenomedullin inhibits PDGF-induced ET-1 production in cultured rat mesangial cells, probably through a cAMP-dependent process. PMID- 8613222 TI - Acute Na+,K+-ATPase inhibition with bufalin impairs pressure natriuresis in the rat. AB - Although it has been reported that Na+,K+-ATPase inhibition with bufalin induces acute and chronic hypertension in the rat, the mechanisms mediating this response are unclear. To examine the role of the kidney in this process, glomerular filtration rate, renal blood flow, and pressure natriuresis were determined in rats treated with bufalin or vehicle during changes in renal perfusion pressure. Mean arterial pressure increased from 123 +/- 4 to 149 +/- 3 mm Hg (P < .05) after 40 minutes of intravenous bufalin and remained at this level. In control rats, glomerular filtration rate was well autoregulated. In bufalin-treated rats, glomerular filtration rate fell with decreasing renal perfusion pressure. Glomerular filtration rate autoregulatory index was greater in bufalin-treated than control rats (P < .05). Renal blood flow showed a similar pattern. Urine flow and sodium excretion were less in bufalin-treated than control rats at equivalent renal perfusion pressures. The slope of the line describing the relation between urine flow and renal perfusion pressure was greater (P < .05) in control than bufalin-treated rats. Similarly, the slope of the line relating sodium excretion to renal perfusion pressure was greater (P < .05) in control than bufalin-treated rats. Thus, acute increases in blood pressure during Na+, K+ ATPase inhibition are associated with impaired renal autoregulation and pressure natriuresis. This effect may be important in chronic hypertension associated with Na+,K+-ATPase inhibition in the rat. PMID- 8613223 TI - Nitric oxide release from kidneys of hypertensive rats treated with imidapril. AB - To examine whether endothelial dysfunction in hypertension is reversible or not, we studied the effects of imidapril, an angiotensin-converting enzyme inhibitor, on nitric oxide release in stroke-prone spontaneously hypertensive rats (SHR) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. After a 4-week treatment with imidapril (1 or 10 mg/d SC) or vehicle, acetylcholine-induced vasodilation and nitric oxide release in the isolated kidneys were determined. Nitric oxide release was measured by a chemiluminescense assay. Imidapril lowered blood pressure in stroke-prone SHR in a dose-dependent manner. Untreated stroke prone SHR exhibited significantly attenuated responses to acetylcholine (10(-8) mol/L) of both renal perfusion pressure (stroke-prone SHE 42 +/- 4% versus Wistar Kyoto rats [WKY] 58 +/- 4% [mean +/- SE], P < .01) and nitric oxide release (stroke-prone SHR +7.6 +/- 2.1 versus WKY +29.7 +/- 9.7 fmol/min per gram of kidney wt, P < .01). Imidapril at 10 mg/d significantly increased acetylcholine induced renal vasodilation and nitric oxide release in stroke-prone SHR (renal perfusion pressure, 56 +/- 3%; nitric oxide release, +27.1 +/- 6.4 fmol/min per gram of kidney wt; both P < .01 versus stroke-prone SHR treated with vehicle). On the other hand, imidapril neither decreased blood pressure nor changed nitric oxide release induced by acetylcholine in DOCA-salt hypertensive rats. Staining for endothelial nitric oxide synthase and brain nitric oxide synthase was clearly detected in the kidneys of both stroke-prone SHR and WKY, whereas staining intensity was weaker in DOCA-salt hypertensive rats. Inducible nitric oxide synthase immunoreactivity was barely noticeable in any type of rat. Thus, imidapril restored endothelial damage by pressure-dependent mechanisms. Most of the nitric oxide detected in the perfusate seemed to be derived from constitutive nitric oxide synthase. PMID- 8613224 TI - Mechanism of the nitric oxide-induced blockade of collecting duct water permeability. AB - Nitric oxide has a diuretic effect in vivo. We have shown that nitric oxide inhibits antidiuretic hormone-stimulated osmotic water permeability in the collecting duct; however, the mechanism by which this occurs is unknown. We hypothesized that inhibition of antidiuretic hormone-stimulated water permeability by nitric oxide in the collecting duct is the result of activation of cGMP-dependent protein kinase, which in turn decreases intracellular cAMP. To test this hypothesis, we microperfused cortical collecting ducts. Antidiuretic hormone-stimulated water permeability was 317 +/- 47 microm/s (P < .001). Addition of spermine NONOate, a nitric oxide donor, to the bath decreased water permeability to 74 +/- 38 microm/s (P < .002). In the presence of LY 83583, an inhibitor of soluble guanylate cyclase, spermine NONOate did not change water permeability. Addition of spermine NONOate increased cGMP production (P < .01). In the presence of the cGMP-dependent protein kinase inhibitor, spermine NONOate did not change water permeability. Since antidiuretic hormone increases water permeability by increasing cAMP, we hypothesized that nitric oxide inhibits water permeability by decreasing cAMP. In tubules pretreated with antidiuretic hormone, intracellular cAMP was 18.9 +/- 3.9 fmol/mm. In tubules treated with antidiuretic hormone and spermine NONOate, cAMP was 9.3 +/- 1.7 fmol/mm (P < .03). We also examined the effect of spermine NONOate on dibutyryl-cAMP-stimulated water permeability. In the presence of dibutyryl-cAMP, water permeability was 388 +/- 30 microm/s. Addition of spermine NONOate had no significant effect on water permeability. Time controls and inhibitors by themselves did not change antidiuretic hormone-stimulated water permeability. We concluded that nitric oxide decreases antidiuretic hormone-stimulated water permeability by increasing cGMP via soluble guanylate cyclase, activating cGMP-dependent protein kinase and decreasing cAMP. PMID- 8613226 TI - Influence of dietary sodium intake on renal medullary nitric oxide synthase. AB - We previously reported that chronic systemic treatment of rats with a nitric oxide synthase inhibitor leads to a selective decrease in renal medullary blood flow, retention of sodium, and the development of hypertension. In the present studies, we used protein blotting techniques to determine the whole tissue distribution and relative quantitation of the different nitric oxide synthase isoforms in the renal cortex and medulla of Sprague-Dawley rats maintained on a low (0.4% NaCl) or high (4.0% NaCl) dietary salt intake. Neural, endothelial, and inducible nitric oxide synthase were readily detectable in homogenized renal inner and outer medullas. Only endothelial nitric oxide synthase was detectable in the renal cortex. Densitometric comparison of Western blots from equal amounts of total inner medullary tissue protein indicated that endothelial, inducible, and neural nitric oxide synthase were increased by 145%, 49%, and 119%, respectively, in rats maintained on a high NaCl diet compared with rats on a low NaCl diet. No significant differences in nitric oxide synthase levels were detected in the outer medulla, renal cortex, or aorta of rats maintained on low and high NaCl diets. In separate studies, continuous intravenous infusion of N(G) nitro-L-arginine methyl ester (8.6 mg/kg per day) for 11 days in chronically instrumented rats increased mean arterial pressure 32 +/- 3 mm Hg in rats on a high NaCl diet (n=5) but only increased pressure 17 +/- 3 mm Hg in rats on a low NaCl diet (n=6). These data indicate that increased levels of renal medullary nitric oxide synthase may be important in the chronic adaptation to increased sodium intake. PMID- 8613225 TI - Mechanisms of action of atrial natriuretic factor and C-type natriuretic peptide. AB - After secretion by the heart, atrial natriuretic factor (ANF) circulates in plasma, whereas C-type natriuretic peptide (CNP), which is found in abundance in the endothelium, may regulate vascular tone in a paracrine manner. However, there is little information on the effect of CNP on renal microvessels. We hypothesized that CNP dilates the afferent arteriole via the nitric oxide pathway, whereas ANF acts directly on vascular smooth muscle cells. When we perfused rat kidneys with minimal essential medium and bovine serum albumin at 100 mm Hg and examined the juxtamedullary afferent arterioles, neither CNP nor ANF was found to have any effect. When the peptides were added to arterioles preconstricted with norepinephrine, CNP and ANF dilated them in a similar fashion; diameters increased by 25 +/- 4% (n=7) and 29 +/- 6% (n=6) at 10(-7) mol/L, respectively (P < .008). Pretreatment with 10(-4) mol/L N-nitro-L-arginine methyl ester (L-NAME) or 5 x 10(-6) mol/L indomethacin blocked CNP-induced dilation; dilation by ANF was unaffected by indomethacin (52 +/- 25%, n=5) and potentiated by L-NAME (73 +/ 14%, n=5). Thus, CNP dilates the afferent arterioles via the prostaglandin/nitric oxide pathway, whereas ANF dilates them directly. This difference may be important in controlling glomerular hemodynamics. PMID- 8613227 TI - Expression of the subtype 1A dopamine receptor in the rat heart. AB - The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined. PMID- 8613228 TI - Myocardial contractility is modulated by angiotensin II via nitric oxide. AB - We hypothesized that in cardiac muscles, angiotensin II partially inhibits the contractile response to beta-agonists. We studied the contractile response of isolated rat left ventricular papillary muscles to isoproterenol and the effect of angiotensin II on this response. We also investigated whether the effect of angiotensin II is mediated by bradykinin, prostaglandins, nitric oxide, and/or cGMP. Contractility of isolated papillary muscles was recorded with a force transducer, and rest tension, maximal developed tension (DT), maximal rate of rise in developed tension [T(+)], and maximal velocity of relaxation [T(-)] were measured (1) under basal conditions, (2) after pretreatment with various drugs, and (3) after cumulative doses of isoproterenol. Pretreatment groups included (1) vehicle (controls); (2) angiotensin II; (3) angiotensin II and N(omega)-nitro-L arginine, an inhibitor of nitric oxide release; (4) L-arginine, the substrate for nitric oxide synthase; (5) L-arginine and N(omega)-nitro-L-arginine; (6) 8-bromo cGMP, analogous to the second messenger of nitric oxide; (7) angiotensin II and icatibant (Hoe 140), a bradykinin B2 antagonist; and (8) angiotensin II and indomethacin, a cyclooxygenase inhibitor. There were no differences in contractile parameters before and after any of the pretreatments. Isoproterenol increased DT, T(+), and T(-), and these effects were attenuated by angiotensin II, L-arginine, and 8-bromo-cGMP. The effects of angiotensin II and L-arginine were blocked by inhibition of nitric oxide release with N(omega)-nitro-L arginine. Neither the bradykinin B2 antagonist nor the cyclooxygenase inhibitor altered the effects of angiotensin II. We concluded that angiotensin II partially inhibits the contractile response of cardiac papillary muscles to isoproterenol This effect is likely mediated by nitric oxide release, perhaps acting via cGMP. Kinins and prostaglandins do not appear to participate in the inhibitory effect of angiotensin II. Attenuation of the contractile effect of isoproterenol by angiotensin II may help explain why cardiac function improves in heart failure after blockade of the renin-angiotensin system. PMID- 8613229 TI - Mechanisms of interleukin-1beta regulation of nitric oxide synthase in cardiac myocytes. AB - Cytokines and endotoxin stimulate inducible NO synthase (iNOS) in different types of cells; however, little is known about regulatory mechanisms. Using the Griess reagent for nitric levels, Western blots for iNOS protein, Northern blots for iNOS mRNA, and transient transfection studies to monitor transcription, we determined potential mechanisms involved in interleukin-1beta stimulation of iNOS in cultured neonatal ventricular myocytes. When myocytes were treated with interleukin-1beta (5 ng/mL), nitrite levels increased, and this effect was inhibited 80% by the specific iNOS inhibitor aminoguanidine. Neither interferon gamma nor tumor necrosis factor-alpha alone stimulated nitrite production. Bacterial endotoxin alone stimulated nitrites and potentiated the effect of interleukin. To determine whether a tyrosine kinase-mediated signaling pathway was involved in interleukin action, we used the inhibitor genistein, which blocked interleukin-stimulated nitrites, iNOS protein, and iNOS mRNA. To determine the effect of activation of protein kinase C, we treated cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA decreased both interleukin-stimulated nitrites and iNOS protein by 40%. To determine the involvement of cyclic nucleotides, cells were treated with either dibutyryl cAMP or cGMP. cAMP (1 mmol/L) stimulated iNOS mRNA, protein, and nitrite production, whereas cGMP had no effect. To test for a direct effect of interleukin on transcription of the iNOS gene, we transfected the full-length mouse iNOS 5' regulatory sequences (-1592 to +160) coupled to a luciferase reporter gene ( 1592iNOSLuc). Interleukin stimulated luciferase activity 1.8 +/- 0.2-fold. To determine whether interleukin also affects iNOS mRNA stability, interleukin stimulated iNOS mRNA was allowed to decay in the presence of the transcription inhibitor actinomycin D. iNOS mRNA t1/2 (approximately 1 hour) was not affected by interleukin. Thus, our data suggest that (1) interleukin-1beta is the primary cytokine in myocyte iNOS regulation and acts predominantly at the transcriptional level; (2) interleukin stimulation of iNOS mRNA and protein is coupled to a tyrosine kinase-mediated signaling pathway; and (3) protein kinase C and cAMP can modify interleukin signaling by decreasing and increasing iNOS, respectively. PMID- 8613230 TI - Tissue-specific expression of the human brain natriuretic peptide gene in cardiac myocytes. AB - Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. To identify the cis-acting elements involved in regulation of the human BNP gene, we subcloned the full-length promoter (-1818 to +100) and deletions thereof upstream from a luciferase reporter gene and transiently transfected them into primary cultures of neonatal rat atrial and ventricular myocytes and myocardial fibroblasts. Luciferase activity of the full-length construct was higher in ventricular (39064 +/- 8488 relative light units, N=11) and atrial (11225 +/- 1907, N=17) myocytes than myocardial fibroblasts (329 +/- 113, n=5). Maximal promoter activity in ventricular and atrial myocytes was maintained by sequences positioned between -1818 and -1283 relative to the transcription start site. Deletion to -1175 resulted in a decrease, whereas further deletion to -500 effected an increase in reporter activity in both cell types. In ventricular and atrial myocytes, deletion from -500 to -40 reduced luciferase activity 20-fold and 2-fold, respectively, whereas in myocardial fibroblasts, deletion to -40 upregulated the BNP promoter 2-fold. Of note, deleting 16 bp between -127 and -111 reduced luciferase activity 7-fold and 4 fold in ventricular and atrial myocytes, respectively, but had essentially no effect on luciferase activity in fibroblasts. Placement of sequences lying between -127 and -40 upstream from a heterologous thymidine kinase promoter resulted in reporter expression that was 7.4-fold greater than the vector alone in ventricular myocytes, approximately 2-fold greater in atrial myocytes, and equivalent to the vector alone in fibroblasts. For study of activity of the human BNP promoter in adult myocytes, either 408 or 97 bp of 5' flanking sequence coupled to the luciferase reporter gene was injected into the apex of adult male Sprague-Dawley rat hearts. After 7 days, luciferase activity in the injected myocardium was 9.8-fold higher for the longer construct (70683 +/- 14744 versus 7223 +/- 3920, n=4, P < .01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricular versus atrial myocytes and essentially inactive in fibroblasts, (2) the distal BNP promoter contains both positive and negative regulatory elements, (3) a region of the proximal BNP promoter located between -127 and -40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart. PMID- 8613231 TI - Obesity hypertension is related more to insulin's fatty acid than glucose action. AB - Although resistance to insulin-mediated glucose disposal has emerged as a link between abdominal obesity and hypertension, abnormalities of nonesterified fatty acid metabolism may play a greater role. Analyses were performed on existing data from 17 abdominally obese subjects (11 hypertensive, 6 normotensive) to determine whether fatty acid concentration and turnover were related to blood pressure independently of hyperinsulinemia and resistance to insulin-mediated glucose disposal. Glucose utilization, fatty acid concentration, and fatty acid turnover were obtained fasting and during euglycemic hyperinsulinemia at 10 and 40 mU/m/min. Analyses were also performed on another group of 30 subjects with a wide range of risk factors who had blood pressure data as well as glucose and fatty acid measurements during an insulin tolerance test. Fatty acid concentration and turnover were markedly more resistant to suppression by insulin in obese hypertensive than in lean or obese normotensive individuals. In the 17 obese subjects, blood pressure measured at screening, in the laboratory, and over a period of 24 hours correlated significantly with fatty acid concentration and turnover but not with glucose disposal measured during the hyperinsulinemic clamp. These correlations remained significant after fasting insulin, the insulin area under the curve during an oral glucose tolerance test, and glucose disposal during the clamp were controlled for. In the second group of subjects, plasma fatty acids 15 minutes after intravenous insulin also correlated with blood pressure. These correlations remained significant after insulin and an index of sensitivity to insulin-mediated glucose disposal were statistically controlled for. The data indicate that blood pressure is related to the effects of insulin on fatty acid metabolism. The findings raise the possibility that resistance of hormone-sensitive lipase to insulin participates in elevating the blood pressure of abdominally obese hypertensive subjects by increasing fatty acid concentration and turnover. PMID- 8613232 TI - Hyperinsulinemia and left ventricular geometry in a work-site population in Japan. AB - The present study was designed to test whether hyperglycemia or hyperinsulinemia influences left ventricular mass and geometry. An echocardiogram and 75-g oral glucose tolerance test were performed in 210 normotensive and 180 mildly to moderately hypertensive male workers in a bus company who were free from cardiac diseases and were not taking medication for hypertension and diabetes mellitus. When we divided subjects into four groups according to the left ventricular geometric pattern using left ventricular mass index of 110 g/m2 and relative wall thickness (ratio of 2 x posterior wall thickness to end-diastolic left ventricular diameter) of 0.44, body mass index and systolic blood pressure were higher in those with concentric hypertrophy and eccentric hypertrophy. In addition, hemoglobin A(Ic) level and the sum of fasting and 2-hour postload serum glucose levels were higher in subjects with concentric hypertrophy. In subjects without diabetes mellitus (n=336), 2-hour postload serum insulin level and the sum of fasting and 2-hour postload serum insulin levels tended to be higher in those with concentric hypertrophy and concentric remodeling. In multiple regression analysis, the sum of glucose levels (or hemoglobin A(Ic) level) in all subjects and the sum of insulin (or 2-hour postload insulin) levels in subjects without diabetes mellitus significantly correlated with relative wall thickness, independent of age, systolic blood pressure, and body mass index. Neither glucose nor insulin levels correlated with left ventricular mass index. Our results suggest that hyperglycemia and hyperinsulinemia may promote concentric changes in the left ventricle in normotensive and mildly to moderately hypertensive men. PMID- 8613233 TI - Poor glycemic control induces hypertension in diabetes mellitus. AB - We conducted this study to test the hypothesis that hypertension is a primary consequence of poor glycemic control per se very early in insulin-dependent diabetes mellitus. Sprague-Dawley rats (n=15) were instrumented with artery and vein catheters, placed in metabolic cages, and sodium intake was clamped throughout the study. Mean arterial pressure was measured 24 h/d. After a precontrol period, streptozotocin (70 mg/kg IV) was administered, and 15 hours later a continuous intravenous infusion was begun at 4 U/rat per day. The insulin infusion was titrated on an individual rat basis to maintain good glycemic control, and after this 7-day control period, blood glucose, urinary sodium excretion, and mean arterial pressure were not different from precontrol values, averaging 8.8 +/- 0.6 mmol/L, 2.8 +/- 0.2 mmol/d, and 103 +/- 2 mm/Hg, respectively, for control days 5 through 7. Subsequently, a 4-day period of poor glycemic control was initiated by reducing the insulin infusion rate. Blood glucose, urinary sodium excretion, and mean arterial pressure began to increase on day 1; for diabetes days 3 and 4, they averaged 23.4 +/- 1.0 mmol/L, 3.6 +/- 0.1 mmol/d, and 110 +/- 2 mm Hg, respectively. All were significantly elevated. When insulin treatment was restored, all variables returned to control levels during the next 4 days. A second 4-day diabetic period yielded similar results. These results indicate that elevated blood pressure is a primary consequence of poor glycemic control in insulin-dependent diabetes, occurring before renal injury has had time to develop, and therefore, may be a factor contributing to the initiation of end-organ injury. PMID- 8613234 TI - Increased responsiveness and decreased expression of G proteins in deoxycorticosterone hypertension. AB - Deoxycorticosterone-salt (DOCA-salt) hypertension is characterized by elevated vasoconstriction to agonists that stimulate G protein-mediated activation of phospholipase C. However, the mechanisms responsible for the augmented responsiveness are unknown. This study tested the hypothesis that this augmented vascular responsiveness is due to elevated content of G(alpha)q, the G protein alpha-subunit that activates phospholipase C. Thoracic aortae from DOCA-salt hypertensive rats (systolic blood pressure 183 +/- 7 mm Hg) and normotensive controls (systolic blood pressure 115 +/- 2 mm Hg) were homogenized and G protein content determined. Western analysis revealed that G(alpha)i content was decreased in DOCA compared with control rats (1364 +/- 196 versus 2343 +/- 188 densitometry units, P < or = .05) with no differences observed for G(alpha)q or G(alpha)s. In addition, contractile responses in denuded femoral artery strips revealed a significant decrease in EC50 values in DOCA arteries to all of the agonists examined: aluminum fluoride (DOCA = 1.42, control = 2.34 mmol/L), mastoparan (DOCA = 0.51, control = 35 micromol/L), phenylephrine (DOCA = 0.08, control = 0.53 micromol/L), and serotonin (DOCA = 0.014, control = 0.04 micromol/L, EC20 values). Finally, arteries from DOCA rats contracted with aluminum fluoride had increased sensitivity to G protein antagonists but not to a phospholipase C inhibitor. The enhanced contractile responsiveness in the DOCA arteries may be mediated in part through decreased G(alpha)i levels. However, it is not caused by increased concentrations of G(alpha)q in the cell membrane or by increased phospholipase C sensitivity, and the increased constrictor response to G protein stimulators of phospholipase C appears to depend primarily on increased G protein sensitivity. PMID- 8613235 TI - Sexual dimorphism of cardiovascular responses to early blockade of bradykinin receptors. AB - To assess whether the cardiovascular effects induced by early blockade of bradykinin B2-receptors with Hoe 140 (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin) are influenced by sex, Wistar rats of both sexes received the antagonist (300 nmol/d per kilogram body wt) or vehicle from 2 days to 7 weeks of age by subcutaneous injection and then by intraperitoneal infusion. Compared with control rats, Hoe 140-treated female rats showed higher systolic blood pressure levels at 7 and 9 weeks of age (125 +/- 2 versus 111 +/- 2 mm Hg and 132 +/- 3 versus 116 +/- 2 mm Hg, respectively, P < .05), whereas in male rats a difference was found at 7 weeks (122 +/- 4 versus 108 +/- 4 mm Hg, P < .05) but not at 9 weeks. At this stage, the mean blood pressure of Hoe 140-treated rats was higher than that of control animals, and this difference was more pronounced at 12 weeks in female rats (121 +/- 2 versus 100 +/- 3 mm Hg in control animals, P < .01) compared with males (116 +/- 3 versus 104 +/- 2 mm Hg in control animals, P < .05). After the first week of life, body weight gain was greater in Hoe 140 treated female rats than in control rats, whereas a group-difference was detected in male rats only after weaning. In Hoe 140-treated female rats, heart weight was already increased at 9 weeks (330 +/- 6 versus 305 +/- 5 mg/100 g body wt in control rats, P < .05), whereas it was necessary to prolong Hoe 140 administration in male rats to develop heart hypertrophy (300 +/- 4 versus 275 +/ 4 mg/100 g body wt in control rats at 12 weeks, P < .05). Tissue kallikrein mRNA levels were higher in the kidney of adult female rats, whereas no sex difference was detected in the heart. The finding of a sexual dimorphism in the cardiovascular response to early blockade of bradykinin receptor suggests that endogenous kinins play a role in the regulation of cardiovascular function in both sexes, but they may be functionally more important in the female rat. PMID- 8613236 TI - Vascular smooth muscle polyploidy and cardiac hypertrophy in genetic hypertension. AB - We studied the mechanisms responsible for vascular and cardiac hypertrophy in hypertension (pressure load and humoral and genetic factors) in two experimental approaches: (1) We carried out a cosegregation analysis to correlate cardiac and vascular hypertrophy with subphenotypes of blood pressure in an F2 generation of a cross between stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats; (2) we treated 8-week-old SHRSP with perindopril, an angiotensin-converting enzyme inhibitor; losartan, an angiotensin type 1 receptor antagonist; or perindopril combined with a nitric oxide synthase inhibitor to investigate the relative contributions of blood pressure and angiotensin II to the pathogenesis of cardiac hypertrophy and vascular smooth muscle polyploidy. Vascular smooth muscle polyploidy was measured with flow cytometry DNA analysis. Cardiac hypertrophy was assessed by measuring the ratios of heart weight to body weight and left ventricle + septum weight to body weight. Blood pressure was measured with radiotelemetry in the F2 cosegregation experiment and with tail-cuff plethysmography in the pharmacological study. In the F2 rats, the best predictor of smooth muscle polyploidy by ANCOVA was systolic pressure (F=29.28, P < .0001). The ratio of left ventricle + septum weight to body weight had four major predictors: the male progenitor of the cross, sex, pulse pressure, and change in systolic pressure during salt (F=43.67, P < .0001; F=16.37, P < .0001; F=8.41, P=.0022; and F=12.39, P= .0003, respectively). The ratio of heart weight to body weight had similar predictors. In the pharmacological study, treatment with losartan alone, perindopril alone, or perindopril in combination with N(G)-nitro-L-arginine methyl ester prevented the development of smooth muscle polyploidy and cardiac hypertrophy. The prevention of cardiac hypertrophy was most marked in the SHRSP treated with perindopril plus N(G)-nitro-L-arginine methyl ester, despite blood pressure being higher in this group than in the two other treatment groups. We conclude that vascular and cardiac hypertrophy in this form of hypertension are regulated by different variables. However, suppression of the action of angiotensin II lessens hypertrophy of both types of muscle. PMID- 8613237 TI - Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. AB - We performed studies to further elucidate the mechanisms of angiotensin II (Ang II)-induced angiogenesis of the microvasculature. Rats were placed on a high salt diet (4% NaCl), and Ang II was infused at a subpressor rate (5 ng/kg per minute) for 3 days. Blood pressure was measured daily for 2 control and 3 infusion days. Microvessel density in the cremaster muscle was measured at the end of the infusion. Vessel density in rats that received subpressor Ang II infusion increased by 12.6% compared with rats that received vehicle infusion. When the angiotensin type 2 (AT2) receptor antagonist PD 123319 was coinfused with Ang II, blood pressure was elevated and vessel density increased above that observed with Ang II infusion alone (23% increase). When the AT1 receptor antagonist losartan was coinfused with Ang II, blood pressure was lower than control and vessel density was reduced compared with the Ang II group but was still greater than control (7.8% increase). In this study, Ang II stimulated angiogenesis in the rat cremaster muscle; this effect was enhanced by AT2 antagonism and inhibited by AT1 antagonism. Ang II infusion at a subpressor dose resulted in a pressor response with AT2 antagonism and a depressor response with AT1 antagonism. This suggests that in the microvasculature, the AT1 receptor mediates angiogenesis and vasoconstriction, and the AT2 receptor mediates an inhibition of angiogenesis and vasodilation. PMID- 8613238 TI - Smooth muscle cell-derived adenosine inhibits cell growth. AB - Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum-induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([3H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro 9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [3H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A2 adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation. PMID- 8613239 TI - cAMP signaling inhibits dihydropyridine-sensitive Ca2+ influx in vascular smooth muscle cells. AB - This study examines the role of the cAMP signaling pathway in the regulation of 45Ca influx in cultured vascular smooth muscle cells from the rat aorta. K+o induced depolarization of smooth muscle cells increased the rate of 45Ca uptake by twofold to threefold. This effect was completely abolished by the dihydropyridine derivatives nifedipine and nicardipine, with a Ki of 3 and 10 nmol/L, respectively. Activators of cAMP signaling (isoproterenol, forskolin, cholera toxin) increased cAMP content by 50- to 100-fold and decreased voltage dependent 45Ca uptake by 50% to 70%. Neither the dihydropyridines nor the cAMP activators affected basal 45Ca influx. Direct addition of the protein kinase inhibitor H-89 to the incubation medium in the 1- to 10-micromol/L range did not alter basal 45Ca uptake but completely abolished voltage-dependent Ca2+ transport. Preincubation of cells for 1 hour with 10 micromol/L H-89 did not modify basal and depolarization-induced 45Ca uptake in H-89-free medium but prevented forskolin-induced inhibition of voltage-dependent Ca2+ influx. The addition of cytoskeleton-active compounds reduced voltage-dependent Ca2+ transport and completely abolished its regulation by cAMP. Activation of cAMP signaling decreased the volume of smooth muscle cells by 12% to 15%. The same cell volume diminution in hyperosmotic medium did not alter voltage-dependent 45Ca uptake. Thus, data obtained in this study show that in contrast to cardiac and skeletal myocytes, in vascular smooth muscle cells, 45Ca influx, putatively due to L-type channels, is inhibited by cAMP. This regulatory pathway appears to be mediated via protein kinase A activation and cytoskeleton reorganization. PMID- 8613240 TI - 20-HETE requires increased vascular tone to constrict rabbit afferent arterioles. AB - Renal production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 dependent arachidonate metabolite, increases during development of hypertension in spontaneously hypertensive rats, and inhibition of its production prevents hypertension. Since 20-HETE is a potent vasoconstrictor, these findings suggest that 20-HETE may contribute to the development of hypertension by elevating renal vascular resistance. In this study we examined the direct action of 20-HETE on the afferent arteriole, a vascular segment crucial to the control of renal vascular resistance. Rabbit afferent arterioles were microperfused at 60 mm Hg in vitro, and 20-HETE was added to the lumen. Although 20-HETE (10(-10) to 10(-6) mol/L) had no effect on the diameter of non-treated afferent arterioles (n=6), it caused dose-dependent constriction when vascular tone was increased with norepinephrine (0.3 micromol/L); 20-HETE at 10(-6) mol/L decreased diameter by 43 +/- 4% (n=6, P < .001). This constriction was abolished by disrupting the endothelium (n=5). Moreover, pretreatment with the cyclooxygenase inhibitor indomethacin (50 micromol/L) or the thromboxane/endoperoxide receptor antagonist SQ29548 (1 micromol/L) significantly (P < .03) attenuated 20-HETE-induced constriction: 20-HETE at 10(-6) mol/L constricted norepinephrine-treated afferent arterioles by 28 +/- 3% (n=6) and 25 +/- 4% (n=5), respectively. These results demonstrate that an increase in afferent arteriolar tone is required for the vasoconstrictor action of 20-HETE, which is partly mediated by the endothelial cyclooxygenase pathway. THus, increased production of 20-HETE in the kidney and increase in afferent arteriolar tone, both of which often precede the development of hypertension, may synergistically contribute to the development of hypertension by elevating renal vascular resistance. PMID- 8613241 TI - Adenosine inhibits growth of rat aortic smooth muscle cells. Possible role of A2b receptor. AB - Abnormal growth of vascular smooth muscle cells (SMC) is frequently associated with hypertension and atherosclerosis, and homeostasis within a normal vessel is maintained by the balanced generation of both vasoconstrictors and vasodilators. Moreover, several endogenous vasoconstricting factors induce SMC growth, whereas several vasodilators inhibit SMC growth. Inasmuch as adenosine is a potent vasodilator, it is possible that it too could inhibit SMC growth. Hence, the effects of adenosine (10(-8) to 10(-3) mol/L), 2-chloroadenosine (a stable analogue of adenosine; 10(-8) to 10(-3) mol/L), and 8-bromo-cAMP (10(-8) to 10( 3) mol/L) on fetal calf serum (FCS; 2.5%)-induced growth of rat aortic SMC were evaluated. Growth was analyzed by assaying DNA synthesis (thymidine incorporation in SMC pulsed for 4 hours with 1 microCi/mL [3H]thymidine) and cell proliferation (change in cell number). Growth-arrested SMC were treated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine, or 8-bromo-cAMP for 24 hours for DNA synthesis or 4 days for cell proliferation. All three substances inhibited DNA synthesis and cell proliferation in a concentration-dependent manner. Compared with adenosine, 2-chloroadenosine was more potent in inhibiting growth. The inhibitory effects of 2-chloroadenosine were reversed by KF17837 (a specific A2 receptor antagonist) but not by DPCPX (a specific A1 receptor antagonist). Furthermore, the inhibitory effects of 2-chloroadenosine were not mimicked by CGS21680 (an A2a receptor agonist), and the effects of N6 cyclopentyladenosine (CPA; an A1 receptor agonist) were not markedly more potent than those of 2-chloroadenosine, suggesting that the inhibitory effects of adenosine are possibly mediated via A2b receptors. These studies provide evidence that adenosine inhibits SMC growth and suggest that a decrease in local levels of adenosine may initiate SMC growth and contribute to the vascular remodeling process observed in hypertension and atherosclerosis. PMID- 8613243 TI - Remodeling of the radial artery in response to a chronic increase in shear stress. AB - Chronic changes in large artery blood flow rates induce corresponding adjustments in arterial diameter, but little is known about structural adaptations of the vessel wall in humans. We used a high-resolution echo-tracking system to measure radial artery internal diameter, wall thickness, and mean blood flow on both arms of 11 patients with end-stage renal disease. Measurements were performed on the wrist side of the arteriovenous fistula. The contralateral radial artery was investigated as control. Wall cross-sectional area, circumferential wall stress, and mean wall shear stress were calculated. Results indicate a sixfold increase in blood flow on the side of the arteriovenous fistula compared with the control side, with a 1.4-fold increase in internal diameter. The diameter enlargement was sufficient to normalize wall shear stress. Changes in diameter were not associated with arterial wall hypertrophy because wall cross-sectional area was not increased and rather suggest a "remodeling" of the arterial wall. For the same level of blood pressure, circumferential wall stress was increased on the side of the arteriovenous fistula. These results suggest that the structural adaptations of the arterial wall to a chronic increase in blood flow normalize wall shear stress and overcome stretch-induced changes in the particular circumstance of arteriovenous fistula. PMID- 8613242 TI - Effects of endothelin receptor inhibition on cerebral arterioles in hypertensive rats. AB - The purpose of this study was to examine the effects of endothelin receptor inhibition on cerebral arterioles in stroke-prone spontaneously hypertensive rats (SHRSP). Structure and mechanics of cerebral arterioles were examined in untreated Wistar-Kyoto rats (WKY) and SHRSP that were either untreated or treated for 3 months with bosentan, an inhibitor of endothelin receptors (100 mg/kg per day). We measured pressure, external diameter, and cross-sectional area of the vessel wall (histologically) in maximally dilated (EDTA) arterioles on the cerebrum. Bosentan reduced but did not normalize arteriolar mean pressure (103 +/ 3 and 81 +/- 5 mm Hg in untreated and treated SHRSP versus 51 +/- 4 mm Hg in WKY, P < .05; mean +/- SEM) and pulse pressure (40 +/- 2 and 33 +/- 2 mm Hg in untreated and treated SHRSP versus 25 +/- 3 mm Hg in WKY, P < .05) in SHRSP. Cross-sectional area of the vessel wall (CSA) was increased in untreated SHRSP (1627 +/- 173 microm2), and CSA in treated SHRSP (1287 +/- 78 microm2) was similar to that in WKY (1299 +/- 65 microm2). Bosentan had no effect on reductions in external diameter (remodeling) of cerebral arterioles (104 +/- 7 and 96 +/- 4 microm in untreated and treated SHRSP compared with 126 +/- 7 microm in WKY, P < .05). Stress-strain curves indicate that bosentan had no significant effect on distensibility of arterioles on the cerebrum in SHRSP. The results suggest that endothelin-1 may contribute to the development of hypertrophy, but not remodeling or changes in distensibility, of cerebral arterioles in SHRSP. PMID- 8613244 TI - Reduced pulmonary artery vasoconstriction in methacholine in cholesterol-fed rabbits. AB - Alterations in vascular tone are well documented in hypercholesterolemia, yet little is known about the role of dietary cholesterol in endothelium-dependent contractions of pulmonary arteries. Methacholine and arachidonic acid cause endothelium-dependent contractions in normal rabbit pulmonary artery that are mediated by thromboxane A2. We tested the effect of these agonists on pulmonary arteries from rabbits fed standard rabbit chow or chow supplemented with 2% cholesterol for 2 weeks. Arachidonic acid-induced contractions did not differ in the groups. However, methacholine-induced contractions were significantly depressed in cholesterol-fed rabbits. Vascular thromboxane A2 production was similar in normal and cholesterol-fed rabbits. Pretreatment with the nitric oxide synthase inhibitor nitro-L-arginine had no effect on contractions observed with methacholine in normal rabbits but enhanced methacholine-induced contractions in cholesterol-fed rabbits. In norepinephrine-precontracted vessels, methacholine caused a small relaxation response in normal rabbits. In contrast, in cholesterol fed rabbits, methacholine produced enhanced relaxations, suggesting that cholesterol feeding augments relaxations and decreases contractions by increasing nitric oxide. However, nitric oxide synthase activity in pulmonary arteries from cholesterol-fed and normal rabbits was not different between the two groups. In an additional experiment, the calcium-dependent potassium channel blocker charybdotoxin had little effect on methacholine-induced contractions in cholesterol-fed rabbits. In summary, the present study demonstrates that hypercholesterolemia alters pulmonary artery vascular contractions and relaxations to methacholine. This effect is not mediated by a decreased production of thromboxane A2 or by an increased production of nitric oxide. Although the mechanisms mediating the altered vascular responses is still unknown, the results from this study clearly indicate that the regulation of vascular tone is different in normal and hypercholesterolemic vessels. PMID- 8613245 TI - Vascular endothelial growth factor suppresses C-type natriuretic peptide secretion. AB - Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7 +/- 5.5 versus 8.9 +/- 0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide. PMID- 8613246 TI - Contributions of vascular tone and structure to elastic properties of a medium sized artery. AB - Isobaric compliance and distensibility of the radial artery were recently reported to be normal or slightly increased in untreated hypertensive patients. However, these findings provide no information on the intrinsic mechanical properties of the wall material. To address this question, we determined intima media wall thickness, wall-to-lumen ratio, and incremental elastic modulus in the radial artery of 25 untreated hypertensive patients with blood pressure of 150 +/ 14/103 +/- 6 mm Hg (mean +/- SD) and 25 matched control subjects with blood pressure of 118 +/- 9/79 +/- 6 mm Hg. High-resolution echotracking for assessment of internal diameter and intima-media wall thickness was combined with measurements of blood flow velocity by Doppler and blood pressure by photoplethysmography. In addition, isobaric compliance and distensibility and incremental elastic modulus were measured at peak diameter during reactive hyperemia after a 5-minute brachial occlusion. No significant difference was found between the two groups for isobaric compliance or distensibility at baseline or during hyperemia. However, incremental elastic modulus at 100 mg Hg tended to be lower in hypertensive patients than control subjects (1.9 +/- 1.1 versus 2.5 +/- 1.2 mm Hg x 10(4), P = .1) in resting conditions. Hypertensive patients and control subjects had similar internal diameters (2.47 +/- 0.32 versus 2.41 +/- 0.35 microm), but intima-media wall thickness and wall-to-lumen ratio were significantly increased in hypertensive patients compared with control subjects (0.268 +/- 0.032 versus 0.236 +/- 0.025 mm -P < or = .01- and 0.220 +/- 0.038 versus 0.195 +/- 0.028 -P < or = .05-, respectively). Peak hyperemic blood flow response (hypertensive patients versus control subjects: 349% versus 360% increase from baseline) and reactive hyperemic dilation (7.2% versus 7.9%) were similar in amplitude and duration in the two groups. These results suggest that wall thickening is an adaptive process that reduces wall tension in hypertensive patients while preserving a normal mechanical behavior of the radial artery. This is most likely accomplished by modification of the incremental elastic modulus of wall components rather than by a change in vascular tone. PMID- 8613247 TI - Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. AB - Interleukin-1 induced a time-dependent release of high levels of nitric oxide from rat vascular smooth muscle cells up to 96 hours. A time-dependent release of lactate dehydrogenase was also induced by Interleukin-1 from 72 to 96 hours after its stimulation. In situ nick end-labeling assay revealed that incubation for 48 hours with interleukin-1 induced a positive staining of fragmented nuclei. However, NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, inhibited both lactate dehydrogenase release and DNA fragmentation induced by interleukin-1. Furthermore, sodium nitroprusside, a nitric oxide donor, also induced lactate dehydrogenase release and DNA fragmentation. Fluorescent staining of DNA revealed patches of irregularly dispersed, brightly staining, and condensed chromatin in rat vascular smooth muscle cells treated with sodium nitroprusside. Flow cytometric analysis with monoclonal antibody against human Fas revealed that expression of Fas was upregulated by sodium nitroprusside in human vascular smooth muscle cells. Methylene blue, an inhibitor of soluble guanylate cyclase, did not affect sodium nitroprusside-induced upregulation of Fas. Furthermore, 8-bromo-guanosine 3':5'-cyclic monophosphate, an analogue of cGMP, did not upregulate Fas expression. These findings indicate that nitric oxide released from vascular smooth muscle cells may induce apoptosis in vascular smooth muscle cells themselves and also induced upregulation of Fas via a cGMP independent mechanism. Thus, nitric oxide could trigger the remodeling of atherosclerotic plaques. PMID- 8613248 TI - Regulation of Na+,K+-ATPase alpha-subunit expression by mechanical strain in aortic smooth muscle cells. AB - We have previously demonstrated that vascular sodium pump activity is stimulated in several rat models of hypertension. In addition, others have reported an upregulation of mRNA for the Na+,K+-ATPase alpha1-subunit in hypertension. To test the effect of sustained, cyclic, stretch-relaxation stimuli on the expression of alpha1- and alpha2-subunits of Na+,K+-ATPase in vascular smooth muscle cells, we used the Flexercell strain unit to stretch rat aortic smooth muscle cells for several days on a collagen-coated silicone elastomer substratum. Six-second cycles of stretch-relaxation were applied to obtain 10% average surface elongation (22% maximum) for 4 days. Control cells were not stretched but were grown on a similar surface. The effect of Gd3+, a blocker of stretch activated channels, was also investigated. At the end of 4 days, protein expression of alpha1- and alpha2-subunits was determined by Western blot analysis. Intensity of the bands for alpha1- and alpha2-subunits was quantified with the use of a computerized image analyzer. In the stretched cells, both the alpha1- and the alpha2-subunit protein-band intensities were significantly increased compared with those of the non-stretched cells. Treatment with 50 micromol/L Gd3+ during the application of stretch prevented the upregulation of alpha2-expression but not that of alpha1-expression. Sodium pump activity, the functional counterpart of Na+,K+-ATPase, was inhibited as a result of stretch; Gd3+ had no effect on this variable. Our results suggest that in vascular smooth muscle, stretch may be a signal for the upregulation of both the alpha1- and alpha2-isoforms. However, a differential response of the two isoforms to the blocker of stretch-activated channels implies involvement of different mechanisms. This alteration in protein expression is not reflected in the function of the enzyme. PMID- 8613250 TI - 11th Annual Hoechst Marion Roussel Hypertension Research Clinical Fellowship Award 1995. PMID- 8613249 TI - Natriuretic peptide receptors in human artery and vein and rabbit vein graft. AB - Natriuretic peptides elicit their biological effects by elevation of cGMP through activation of two biologically active receptors: natriuretic peptide A receptor, which shows high affinity to atrial and brain natriuretic peptides, and natriuretic peptide B receptor, which is specific to C-type natriuretic peptide. To elucidate the implications of the natriuretic peptide system in arteries and veins, we examined the cGMP production in response to atrial and C-type natriuretic peptide and gene expressions of biologically active natriuretic peptide receptors in human gastroepiploic artery, internal mammary artery, and saphenous vein. Atrial natriuretic peptide augmented cGMP production more potently by one order of magnitude in arteries than in veins. C-type natriuretic peptide stimulated cGMP production weakly and equally in these vessels. Analyzed by reverse transcription-polymerase chain reaction, gene expression of natriuretic peptide A receptor was four times more abundant in arteries than in veins. Gene expression of natriuretic peptide B receptor was approximately the same between these vessels. We also studied the responsiveness to atrial and C type natriuretic peptide in rabbit jugular vein grafted into carotid artery. In arterialized vein grafts 4 weeks after operation, the effects of atrial and C type natriuretic peptides on cGMP production did not change from those in jugular veins. In conclusion, atrial natriuretic peptide stimulates cGMP production more potently in arteries than in veins due to the preferential expression of natriuretic peptide A receptor in arteries. These observations support the distinct roles of natriuretic peptides in cardiovascular homeostasis. PMID- 8613251 TI - Council for High Blood Pressure Research Lifetime Achievement Award 1995. PMID- 8613252 TI - CIBA Award for Hypertension Research 1995. PMID- 8613253 TI - CIBA Award for Hypertension Research 1995. PMID- 8613254 TI - Harry Goldblatt Award 1995. PMID- 8613255 TI - F. Merlin Bumpus Award 1995. PMID- 8613256 TI - Tumor necrosis factor activates angiotensinogen gene expression by the Rel A transactivator. AB - Angiotensinogen encodes the only known precursor of angiotensin II, a critical regulator of the cardiovascular system. Transcriptional control of angiotensinogen in hepatocytes is an important regulator of circulating angiotensinogen concentrations. Angiotensinogen transcription is increased by the inflammatory cytokine tumor necrosis factor (TNF)-alpha by a nuclear factor kappaB-like protein binding to an inducible enhancer called the acute-phase response element. By gel mobility shift assays, we observe two specific acute phase response element-binding complexes, C1 and C2. The abundance of C2 is not changed by TNF treatment. In contrast, C1 is faintly detected in untreated cells, and its abundance increases by fivefold after stimulation. We identify the nuclear factor-kappaB subunits in these complexes using subunit-specific antibodies in the gel mobility "supershift" assay. The transcriptionally inert nuclear factor-kappaB DNA-binding subunit NF-kappaB1 is present in both control and stimulated hepatocyte nuclei. Its abundance changes weakly upon TNF stimulation. In contrast, the potent transactivating protein Rel A is not found in unstimulated hepatocyte nuclei and is recruited by TNF-alpha into the C1 DNA binding complex. Overexpression of Rel A results in acute-phase response element transcription. Cotransfection of a chimeric GAL4-Rel A protein with GAL4 DNA binding sites is a strategy that allows for selective study of Rel A. The GAL4:Rel A chimera is a TNF-alpha-inducible transactivator. Deletion of the amino terminal 254 amino acids of Rel A produces a constitutive activator (that is no longer TNF-alpha inducible). The cytokine induction of Rel A, then, is mediated through its amino-terminal 254 amino acids. We conclude that Rel A:NF-kappaB1 is a crucial cytokine-inducible transcription factor complex regulating angiotensinogen gene synthesis in hepatocytes and may be involved in controlling the activity of the renin-angiotensin system. PMID- 8613257 TI - Evidence for cell-specific regulation of transcription of the rat alpha2A adrenergic receptor gene. AB - We investigated the transcriptional activity of the -131 to -92 region of the rat alpha2A-adrenergic receptor gene. In HT29 cells, this region has a positive effect on transcription, whereas in RINm5F cells, this region has a negative effect on transcription. The -131 to -92 region has a GC box (GGGGCGG) surrounded by overlapping GGAGG repeats. To analyze nuclear factor binding to this region, we made a series of sequence substitutions in the GGAGG repeats, the GC box, or both regions. Gel mobility shift assays indicated that most of the nuclear factor complexes formed between the wild-type -131/-92 sequence and either HT29 or RINm5F extracts were specific for SP1 or related proteins that recognize a GC box. Mutation of either the GGAGG repeats or the GC box did not eliminate the binding of Sp1 or related nuclear factors, suggesting that both the GGAGG repeats and the GC box could bind Sp1-related factors. Mutation of both these sites eliminated the binding of Sp1-related factors. In the absence of SP1 binding sites, this region had a negative effect on transcription in HT29 and a positive effect on transcription in RINm5F cells. These data support the notion that Sp1 and/or a related factor may control both positive and negative gene expression and suggest that the -131/-92 region may be involved in regulating tissue specific levels of alpha2A-adrenergic receptor gene expression. PMID- 8613258 TI - Mechanisms of transcriptional synergism of eukaryotic genes. The interferon-beta paradigm. AB - The virus-inducible enhancer of the human interferon-beta gene has served as an excellent example for the mechanisms controlling the activation and repression of transcription. This enhancer is activated by three different transcription factors that, with the help of the high mobility group protein HMG I(Y), assemble in a unique nucleoprotein complex that interacts as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcription factors may provide the specificity required for regulation of complex patterns of gene expression in higher eukaryotes. PMID- 8613259 TI - Aging progressively impairs endothelium-dependent vasodilation in forearm resistance vessels of humans. AB - Studies in experimental models suggest that endothelium-derived nitric oxide is reduced with aging, and this circumstance may be relevant to atherogenesis. The aim of this study was to determine whether increasing age resulted in altered endothelium-dependent vasodilation in the forearm resistance vessels of healthy humans. Forearm blood flow was measured in 119 healthy subjects, aged 19 to 69 years, by venous occlusion plethysmography. Brachial artery infusions of methacholine chloride (0.03 to 10.0 microgram/min) were used to assess endothelium-dependent vasodilation and of sodium nitroprusside (0.03 to 10.0 microgram/min) to assess endothelium-independent vasodilation. The slope of the dose-blood flow response relation was calculated in each subject for each drug. Univariate and multiple stepwise regression analyses were used to relate vascular reactivity to selected variables, including age, lipids, and blood pressure. Endothelium-dependent vasodilation was progressively impaired with increasing age, assessed as a reduction in slope from 2.25 +/- 0.16 to 0.34 +/- 0.11 (mL/100 mL tissue per minute)/(microgram/min) (P <.001). The decline in endothelium dependent vasodilation was already evident by the fourth decade (age 30 to 39 years). Endothelium-independent vasodilation did not change with age. Age, total cholesterol, and low-density lipoprotein cholesterol were univariate predictors of endothelium-dependent vasodilation. Age remained the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analysis. From these observations, it can be concluded that endothelium-dependent vasodilation declines steadily with increasing age in healthy human subjects. Age is a strong univariate and multivariate predictor of endothelium-dependent vasodilation. This finding may be a marker for more widespread endothelial dysfunction. PMID- 8613260 TI - Angiotensin mediates forearm glucose uptake by hemodynamic rather than direct effects. AB - Insulin sensitivity may be improved with the angiotensin-converting enzyme inhibitor captopril, suggesting that inhibition of angiotensin II (Ang II) improves insulin resistance. However, the administration of systemic Ang II has also been associated with an improvement in rather than worsening of glucose utilization. Since both stimulating and antagonizing the renin-angiotensin system improve glucose uptake and both angiotensin-converting enzyme inhibitors and intravenous Ang II elicit skeletal muscle vasodilation, it is conceivable that hemodynamic factors rather than a direct effect of either Ang II or angiotensin converting enzyme inhibitors on skeletal muscle metabolism modulate the increase in glucose utilization. The direct effects of Ang II on glucose extraction in intact human skeletal muscle have not been previously described. We investigated the effects of local infusion of Ang II on glucose uptake in the forearm of 20 healthy subjects. With the use of the isolated insulin-perfused forearm model, local plasma insulin values were raised to 100 mU/mL over fasting values and maintained there for a 90-minute infusion period. After the first 60 minutes of insulin alone, Ang II was infused into the brachial artery for the last 30 minutes. Intra-arterial Ang II infusion caused a 38% decrease in forearm blood flow (P <.05) and 59% increase in the arteriovenous glucose gradients (P <.05) to maintain a steady glucose utilization (a decrease of 4%, P=NS). Thus, local Ang II infusion does not impair insulin-stimulated glucose utilization. Furthermore, glucose extraction increases to compensate for the decrease in forearm blood flow (as the Fick principle would predict for freely diffusible substances). We conclude that the described increase in glucose utilization from systemic infusion of Ang II and during angiotensin-converting enzyme inhibitor treatment is mediated by hemodynamic factors rather than a direct effect of Ang II on skeletal muscle metabolism. PMID- 8613261 TI - Phorbol ester activation of the rat vascular myocyte Na(+)-H(+) exchanger isoform 1. AB - Vascular myocytes from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)-H(+) exchanger activity associated with increased cell proliferation and hyperresponsiveness to agonists such as phorbol esters. Since the Na(+)-H(+) exchanger isoform 1 (NHE-1) is stimulated by protein kinase C, we have investigated the effects of phorbol esters on NHE-1 activity and its phosphorylation in vascular myocytes of these rats. SHR cells demonstrated a larger alkalinization response to 12-O-tetradecanoylphorbol 13-acetate than Wistar-Kyoto rat (WKY) cells. Kinetic analyses indicated that whereas 12-O tetradecanoylphorbol 13-acetate increased the maximal transport capacity of NHE-1 in both cell types, affinity for H+ was increased in WKY cells and cooperativity for H+ at the internal modifier site was reduced in SHR cells. In neither cell type was the subcellular distribution of NHE-1 altered by phorbol ester stimulation. NHE-1 phosphorylation was markedly reduced in WKY cells stimulated by the phorbol ester, an effect abolished by inhibition of protein kinase C. In contrast, NHE-1 phosphorylation in quiescent SHR cells was approximately double that of WKY cells and was reduced after phorbol ester treatment. Inhibition of protein kinase C in SHR cells led to a marked elevation of NHE-1 phosphorylation that was not associated with a change in the exchanger activity, but WKY cells exhibited a small, insignificant rise in NHE-1 phosphorylation. Thus, the kinetic responses of NHE-1 to phorbol esters in vascular myocytes of these rat strains are different, the changes in exchanger kinetics of SHR resembling those described in human hypertension. NHE-1 phosphorylation has an inverse relationship with protein kinase C activity. However, modulation of NHE-1 phosphorylation may not be associated with concurrent alterations in activity, indicating a role for non-phosphorylation-dependent mechanisms. PMID- 8613262 TI - Regulation of angiotensin II receptor subtypes by dexamethasone in rat mesangial cells. AB - The objective of this study was to examine the role of dexamethasone on the expression of angiotensin II (Ang II) receptors in cultured rat mesangial cells. Dexamethasone caused concentration- and time-dependent decreases in 125I [Sar1,Ala8]Ang II binding that were prevented by glucocorticoid receptor inhibition with mifepristone. A lag time of 24 hours and a dexamethasone concentration of at least 10 nmol/L were necessary for this effect to occur. Dexamethasone-induced reduction of 125I-[Sar1,Ala8]Ang II binding resulted from decreased Ang II type 1 (AT1) receptor density. No change in the apparent dissociation constant was observed. Dexamethasone also markedly inhibited Ang II dependent inositol phosphate accumulation. Both reverse transcription-polymerase chain reaction and Northern blot analysis using specific short probes from the 3' noncoding region of the cDNA demonstrated the presence of AT1A and AT1B receptor mRNAs in rat mesangial cells, with a slight predominance of AT1B. Therefore, we studied the effect of dexamethasone on the expression of these two subtypes in rat mesangial cells. Dexamethasone produced a time-dependent decrease of AT1B receptor mRNA that was apparent after 6 hours of incubation, whereas AT1A receptor mRNA did not change. Mifepristone also suppressed the dexamethasone induced decrease in AT1B receptor mRNA. In conclusion, glucocorticoids diminish Ang II receptor density at the mesangial cell surface through a mechanism that implies successive interaction with the glucocorticoid receptor and specific reduction in AT1B receptor mRNA expression. This differential regulation of both AT1 receptor subtypes might allow glucocorticoids to exert adjusted effects in their various target tissues. PMID- 8613263 TI - Evidence for a physiological role of angiotensin-(1-7) in the control of hydroelectrolyte balance. AB - In this study we evaluated the possibility that angiotensin-(1-7) [Ang-(1-7)] acts as an endogenous osmoregulatory peptide by determining the effect of acute administration of its selective antagonist [D-Ala7]Ang-(1-7) (A-779) on renal function parameters in rats. In addition, we investigated the physiological mechanisms involved in the antidiuretic effect of Ang-(1-7). The antidiuretic effect of Ang-(1-7) (40 pmol/0.05 mL per 100 g BW) in water-loaded rats was completely blocked by A-779 (vehicle-treated, 3.34 +/- 0.43 mL/h; Ang-(1-7), 1.48 +/- 0.23; A-779, 2.72 +/- 0.35; Ang-(1-7) plus A-779, 3.26 +/- 0.49). In contrast, the antidiuretic effect of Ang-(1-7) was not significantly changed by a vasopressin V2 receptor antagonist in a dose that completely blocked the antidiuresis produced by an equipotent dose of vasopressin. In addition, Ang-(1 7) administration did not significantly change vasopressin plasma levels in water loaded rats. The antidiuretic effect of Ang-(1-7) in water-loaded rats was associated with a reduction of creatinine clearance (0.68 +/- 0.04 versus 1.38 +/ 0.32 mL/min in vehicle-treated rats, P <.05) and an increase in urine osmolality (266.8 +/- 32.7 versus 182.8 +/- 14 mOsm/kg in vehicle-treated rats, P <.05). An effect of Ang-(1-7) in tubular water transport was demonstrated in vitro by a fourfold increase in the hydraulic conductivity of inner medullary collecting ducts in the presence of 1 nmol/L Ang-(1-7). Subcutaneous administration of A-779 (2.3 to 9.2 nmol/100 g) produced a significant increase in urine volume (4.6 nmol/100 g, 0.45 +/- 0.12 mL/h; vehicle-treated rats, 0.16 +/- 0.03 mL/h; P <.05) comparable to that of acute administration of a vasopressin V2 receptor antagonist. The diuretic effect of A-779 was associated with an increase in creatinine clearance and decrease in urine osmolality. In contrast, no significant effects on urine volume were observed after systemic administration of angiotensin subtype 1 or 2 receptor antagonists (DuP 753 and CGP 42112A, respectively). These findings suggest that endogenous Ang-(1-7), acting on specific receptors, participates in the control of hydroelectrolyte balance by influencing especially water excretion. PMID- 8613264 TI - Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth. AB - Mesangial cell growth and accumulation of extracellular matrix proteins constitute key features of progressive glomerular injury. Endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictor agents, evoke a number of similar responses in mesangial cells. In rat mesangial cells, we compared ET-1 and Ang II effects on matrix protein production and cell proliferation as well as the potential interaction between the two hormones. When cells in 0.5% fetal calf serum were incubated for 24 hours with various concentrations of ET-1 or Ang II, both peptides stimulated, in a dose-dependent manner, fibronectin and type IV collagen mRNA expression, fibronectin synthesis, and mitogenesis. Incubation with specific receptor antagonists of both hormones demonstrated that endothelin subtype A (ETA) and angiotensin type 1 (AT1) receptors were involved. Preincubation of cells with two different protein kinase C inhibitors or with a neutralizing anti-transforming growth factor-beta antibody, but not an unrelated IgG, diminished the peptide-induced fibronectin synthesis. A dual interrelation seems to exist between ET-1 and Ang II. Thus, the AT1 receptor antagonist losartan and the angiotensin-converting enzyme inhibitors quinaprilat and captopril diminished the ET-1-mediated effects, whereas, the ETA receptor antagonist BQ-123 diminished the Ang II-induced fibronectin synthesis and mesangial cell proliferation. Our results suggest that ET-1 and Ang II stimulate matrix protein synthesis and mesangial cell mitogenesis through ETA and AT1 receptors, respectively, by complicated mechanisms, implicating protein kinase C activation, synthesis of transforming growth factor-beta, and release of one peptide by the other. These data could be important for a better understanding of the participation of vasoactive substances in the pathogenesis of glomerulosclerosis. PMID- 8613265 TI - L-arginine restores dilator responses of the basilar artery to acetylcholine during chronic hypertension. AB - The objective of this study was to test the hypothesis that administration of L arginine, a substrate for nitric oxide synthase, restores acetylcholine-induced dilatation of the basilar artery in chronically hypertensive rats. Basilar artery diameter was measured through a cranial window in anesthesized stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) aged 6 to 7 months (adult) and 12 months (older adult). Under control conditions, baseline basilar artery diameter was smaller in SHRSP (adult, 239 +/- 30 micron; older adult, 198 +/- 13 micron) (mean +/- SE) than in WKY (adult, 261 +/- 10 micron; older adult, 259 +/- 7 micron) (P <.05 versus SHRSP). Topical application of acetylcholine (10(-5) mol/L) produced dilatation of the basilar artery in WKY, which was impaired in both adult and older SHRSP (P <.05). Topical L-arginine (10(-3) mol/L for 30 minutes) did not affect responses to acetylcholine in adult SHRSP but enhanced vasodilatation in response to acetylcholine (10(-5) mol/L) in older SHRSP without affecting responses to sodium nitroprusside. In contrast, D arginine did not affect acetylcholine-induced vasodilatation in older SHRSP. These results suggest that impaired dilatation of the basilar artery in response to acetylcholine in older SHRSP is restored toward normal by L-arginine, a substrate for nitric oxide synthase. PMID- 8613266 TI - Angiotensin II is mitogenic for cultured rat glomerular endothelial cells. AB - Angiotensin II (Ang II) has growth-stimulatory properties on different renal cell types. However, possible growth effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvasculature have not been formally investigated. Therefore, we isolated and characterized primary cultures of rat glomerular endothelial cells. We used a simple technique in which collagenase treated glomeruli were sparsely plated in several 96-well culture plates and microscopically screened for cobblestone-like outgrowth. After two limiting dilutions, homogeneous cultures were obtained. Cells were characterized by positive staining for the endothelial markers factor VIII, CD 31, endothelial leukocyte adhesion molecule-1, and the lectin Bandeiraea simplificifolia. Ang II stimulated the synthesis and release of endothelin-1 in culture supernatants. Moreover, in contrast to syngeneic mesangial cells, glomerular endothelial cells expressed angiotensin-converting enzyme. Ang II stimulated a mild but significant proliferation of quiescent cells, as measured by [3H]thymidine incorporation and direct cell counting. This mitogenesis was transduced by losartan-blockade angiotensin type 1 receptors. Moreover, Ang II mediated phosphorylation of mitogen-activated protein kinase 2 and induction of transcripts for the immediate early gene Egr-1. Our results indicate that Ang II is a moderate mitogen for primary cultures of rat glomerular endothelial cells and activation of these metabolically active cells may play a role in the pathophysiology of several types of glomerulonephritis. Moreover, remodeling of glomerular endothelial cells by Ang II may be important in the progression of structural renal damage during the course of hypertensive injury. PMID- 8613267 TI - Differing metabolism and bioactivity of atrial and brain natriuretic peptides in essential hypertension. AB - Plasma concentrations of both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are elevated in severe hypertension, acute myocardial infarction, and heart failure. In the current study of individuals with essential hypertension, we have documented the hemodynamic, hormonal, and endocrine effects of infusions of these two peptides given alone or in combination in equimolar doses calculated to induce increments in plasma peptides to concentrations (30 to 60 pmol/L) observed in these disease states. The metabolic clearance rate of ANP (4.56 +/- 0.62 L/min) was greater than that for BNP (3.4 +/- 0.23 L/min, P <.001). Infusions of each cardiac hormone impaired the clearance of coinfused peptide. All peptide infusions enhanced natriuresis (17% to 70% above preinfusion levels versus placebo, 6%; P <.001), lowered blood pressure (10 to 18 mm Hg fall in mean arterial pressure below placebo levels; P <.001), increased hematocrit, suppressed the renin-angiotensin-aldosterone system, and enhanced plasma norepinephrine concentrations. The natriuretic and blood pressure-lowering effects of BNP were twofold to threefold those of ANP. In contrast, ANP-induced increments in plasma and urinary second messenger (cGMP) levels were greater than those for BNP. Both peptides suppressed the renin-angiotensin-aldosterone system (approximately one-third fall in renin activity and plasma aldosterone) and enhanced plasma norepinephrine concentrations (+30%) to a similar degree. Increments in plasma ANP and BNP that occur simultaneously in cardiovascular disease states appear capable of causing hemodynamic, endocrine, and renal effects that would tend to ameliorate conditions such as hypertension or heart failure. PMID- 8613268 TI - Antihypertensive mechanism of diuretics based on pressure-natriuresis relationship. AB - We analyzed the hypotensive mechanisms of a thiazide-type diuretic, mefruside, on the basis of the pressure-natriuresis relationship. We performed a 5-week study in eight patients with essential hypertension who were given a high sodium diet (15 to 18 g NaCl per day) during the 1st and 5th weeks, a severely sodium restricted diet (1 to 3 g/d) during the 2nd week, and a mildly sodium-restricted diet (5 to 7 g/d) during the 3rd and 4th weeks. Mefruside (25 mg/d) was administered during the 4th and 5th weeks. Urinary sodium excretion rate and mean arterial pressure were measured at the end of each week, and the pressure natriuresis relationship was drawn by plotting urinary sodium excretion rate on the ordinate and mean arterial pressure on the abscissa before and after mefruside treatment. Before treatment, the pressure-natriuresis relationship was linear, and mean arterial pressure was changed as a consequence of sodium intake alteration (1st week, 117 +/- 9 mm Hg; 2nd week, 105 +/- 7; 3rd week, 109 +/- 9). After treatment, however, the change in mean arterial pressure was very small (4th week, 102 +/- 8 mm Hg; 5th week, 104 +/- 7). Mefruside steepened the slope of the relationship (20.8 +/- 10.5 versus 143 +/- 85 [mmol/d]/mm Hg, P <.005) without significantly shifting the x intercept (104 +/- 6 versus 101 +/- 9 mm Hg, P=NS) of the relationship. The increase in the slope was greater in patients whose slope had been depressed and blood pressure was sodium sensitive before mefruside treatment. The hypotensive effect of mefruside during a high sodium diet correlated positively with both the hypotensive effect of sodium restriction (r=.84, P <.01) and the increase in the slope by mefruside (r=.83, P <.02). Thus, mefruside lowers blood pressure especially in patients with high sodium sensitivity mainly by making blood pressure sodium insensitive through its diuretic action. Strict sodium restriction seems unnecessary when diuretics are administered for blood pressure control. PMID- 8613269 TI - Erythrocyte sodium transport, intraplatelet pH, and calcium concentration in salt sensitive hypertension. AB - We evaluated changes in erythrocyte sodium transport systems, platelet pH, and calcium concentration induced by low and high salt intakes in a group of 50 essential hypertensive patients classified on the basis of their salt sensitivity. Patients received a standard diet with 20 mmol NaCl daily for 2 weeks supplemented in a single-blind fashion by placebo tablets the first 7 days and NaCl tablets the following 7 days. Salt sensitivity, defined as a significant rise (P <.05) in 24-hour mean blood pressure obtained by ambulatory blood pressure monitoring, was diagnosed in 22 (44%) patients. The remaining 28 (56%) were considered to have salt-resistant hypertension. In the entire group of hypertensive patients, high salt intake promoted a significant increase (P <.05) in the maximal rate of erythrocyte NA(+)-Li(+) countertransport (from 271 +/- 19 to 327 +/- 18 microM/(L cells/h) and of the Na(+)-dependent HCO3(-)-CL(-) exchanger (from 946 +/- 58 to 1237 +/- 92 microM/L cells/h) as well as in platelet pH (from 7.15+/-0 0.01 to 7.19+/-0.02 and calcium concentration (from 49+/-2 to 57 +/-2 nmol/L). Depending on salt sensitivity, high salt intake promoted opposing changes in some of the sodium transport systems studied. Salt sensitive patients increased the maximal rate of the erythrocyte Na(+)-K(+) pump (fom 7.0 +/- 0.4 to 8.8 +/- 0.4 mmol/(L cells/h), Na(+)-K(+)-Cl(-) cotransport (from 416 +/- 37 to 612 +/- 41 micromol/(L cells/h), Na(+)-Li(+) countertransport (from 248 +/- 20 to 389 +/- 17 micromol/(L cells/h) at the end of the high salt period. Conversely, salt-resistant patients decreased the Na(+)-K(+) pump (from 8.0 +/- 0.4 to 6.9 +/- 0.3 mmol/(L cells/h) and Na(+)-K(+)-Cl(-) cotransport (from 578 +/- 53 to 481 +/- 43 micromol/(L cells/h). We conclude that modulation of erythrocyte sodium transport systems by high salt intake depends on salt sensitivity. The Na(+)-K(+) pump, Na(+)-K(+)-Cl(-) cotransport, and Na(+)-Li(+) countertransport increase in salt-sensitive patients, whereas the activity of these sodium transport systems tends to decrease in salt-resistant patients. Independent of salt sensitivity, high salt intake promotes a significant increase in the erythrocyte Na(+)-dependent HCO3(-)-Cl(-) exchanger, platelet pH, and calcium concentration in essential hypertensive patients. PMID- 8613270 TI - Hypothalamic lesions induce obesity and sex-dependent glomerular damage and increases in blood pressure in rats. AB - Placement of two symmetrical lesions in the ventromedial hypothalamus of the rat causes massive overeating and obesity. We have studied male (n=8) and female (n=5) Munich-Wistar rats 7 months after induction of obesity and compared them with age-matched controls. Body weight and kidney weight were greater in control males versus females (396 +/- 7 and 1.5 +/- 0.1 g versus 229 +/- 4 and 1.0 +/- 0.1 g, respectively; both P <.001). Both obese males and females were heavier than lean counterparts (592 +/- 30 and 361 +/- 19 g, both P <.001), whereas kidney weight was similar between obese and control rats of each sex (obese males, 1.5 +/- 0.1 g; obese females, 1.1 +/- 0.1 g). Blood pressure was higher in obese versus control males; there was no differences between other groups. Single nephron glomerular filtration rate was similar in control females and males and obese females but depressed in obese males. Glomerular blood pressure was normal in all groups. Urinary protein excretion and the percentage of sclerosed glomeruli were similar in control females and males and obese females but elevated in obese males. Plasma triglyceride levels were elevated in obesity, particularly in males. We conclude that hypothalamic lesioning induces overeating and obesity and selectively in the male causes hypertension and glomerular damage as well as declines in renal function. This injury is not hemodynamically mediated (glomerular blood pressure is normal) but may be related to the elevation in plasma triglyceride levels, which has previously been causally linked to glomerular damage in genetically obese rats. PMID- 8613271 TI - Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. AB - ATP is coreleased with norepinephrine from sympathetic nerve endings and subsequently broken down to adenosine. In animal preparations, adenosine can inhibit norepinephrine release by stimulation of presynaptic receptors. We tested this feedback mechanism in humans by using a specific nucleoside transport inhibitor (draflazine) as a pharmacological tool to allow accumulation of endogenous adenosine in the synaptic cleft. In a dose-finding study on draflazine infusions into the brachial artery (n=10), we identified an optimal dose of 250 ng/min per deciliter of forearm tissue that induced considerable local nucleoside transport inhibition (approximately 40%) without systemic effects. In the main study, we investigated the effects of this draflazine dose on sympathetic mediated norepinephrine spillover during lower body negative pressure (-25 mm Hg) by the use of the [3H]norepinephrine isotope dilution technique (n=25). Lower body negative pressure induced a significant increase in total body norepinephrine spillover, forearm norepinephrine appearance rate, forearm vascular resistance, and heart rate. During draflazine infusion into the brachial artery, the responses to lower body negative pressure were preserved for all parameters, with the exception of the median increase in forearm norepinephrine appearance rate, which was reduced from 54% to 2% (P <.05). We conclude that accumulation of endogenous adenosine in the synaptic cleft during sympathetic stimulation can inhibit norepinephrine release from sympathetic nerve endings. PMID- 8613272 TI - Differential alteration of neuronal and cardiovascular responses to adenosine microinjected into the nucleus tractus solitarius of spontaneously hypertensive rats. AB - We previously reported that adenosine elicited site-dependent neuronal and cardiovascular responses in two subareas of the nucleus tractus solitarius (NTS) of normotensive rats. Pressor and tachycardic responses were obtained from the rostral NTS (adenosine pressor system), and depressor and bradycardic responses were obtained from the caudal NTS (adenosine depressor system). In both areas, adenosine inhibited the firing rate of barosensitive neurons. The present study investigated whether spontaneously hypertensive rats (SHR) exhibit abnormal neuronal and cardiovascular responses mediated by the adenosine pressor and depressor systems within the NTS. Male SHR and Wistar-Kyoto rats (WKY) were anesthesized with urethane and prepared for blood pressure and heart rate recording, stereotaxic microinjection of adenosine into the NTS, and extracellular recording of single-unit neuronal activity of NTS neurons. Chemical identification of the targeted neuronal pool was made by L-glutamate (5 nmol) and confirmed by histology. SHR exhibited significantly higher mean arterial pressure and firing rate of caudal NTS neurons (45.0 +/- 4.5 versus 27.3 +/- 4.7 spikes per 2.5 seconds, P <.05) but similar heart rate and neuronal firing rate of rostral NTS neurons compared with WKY. Adenosine (0.1, 1, and 10 nmol) elicited dose-related neuronal and cardiovascular responses in both strains. However, SHR exhibited differential alterations in both adenosine systems. Compared with WKY, SHR exhibited attenuated pressor, tachycardic, and neuronal responses mediated by the adenosine pressor system and exaggerated depressor, bradycardic, and neuronal responses mediated by the adenosine depressor system. In both strains, the responses elicited by adenosine were virtually abolished by theophylline (10 mg/kg IV), suggesting that these responses were mediated by adenosine receptors in the NTS. Furthermore, the theophylline-evoked increase in blood pressure was twofold higher in SHR (15.0 +/- 1.7 versus 6.9 +/- 1.5 mm Hg, P <.05); larger but nonsignificant increases in heart rate and neuronal firing rate also were evident in SHR compared with WKY. These findings suggest differential alterations in adenosine pressor and depressor systems in the NTS of SHR, which may be implicated in the pathophysiology of this model of hypertension. PMID- 8613273 TI - Hypertension promotes coronary calcium deposit in asymptomatic men. AB - Despite its important role in coronary disease, coronary atherosclerosis has been poorly investigated in uncomplicated hypertension. Therefore, we evaluated the presence and amount (score) of coronary calcium with ultrafast computed tomography in 73 pairs of age-matched asymptomatic hypertensive or normotensive men. We also estimated the extent of peripheral atherosclerosis as the number of arterial sites (carotid, aortic, femoral) with echographic plaque. Compared with normotensive men, hypertensive men had more frequent coronary calcium (63% versus 47%), a higher calcium score (57 +/- 111 versus 18 +/- 38), and an odds ratio of calcium deposit of 1.95 (with confidence intervals [CI] 95%, 1.01 to 3.79) for any score and of 2.38 (95% CI, 1.02 to 5.52) or 4.84 (95% CI, 1.53 to 15.3) for scores above 50 or 100, respectively. Hypertensive men showed correlations of calcium score with age and hypertension duration but not with the height of blood pressure, and the odds ratio of calcium deposit between extensive and minor peripheral atherosclerosis was 4.67 (95% CI, 1.41 to 15.45) for any score and 8.63 (95% CI, 2.10 to 35.5) or 8.13 (95% CI, 1.64 to 40.3) for scores above 50 or 100. Thus, high blood pressure and in particular its duration rather than its value promotes the presence and overall extent of coronary calcium, a potential predictor of sudden coronary death, in parallel with the extent of peripheral atherosclerosis. The mechanisms of the interaction of hypertension and coronary calcification may be multifactorial and not specific to hypertension. PMID- 8613274 TI - Abnormalities of insulin receptors in spontaneously hypertensive rats. AB - Insulin resistance is present in some strains of rats with genetic hypertension. To determine whether this abnormality is present at the level of the insulin receptor, we compared insulin sensitivity, insulin receptor binding, and mRNA levels in tissues of 10-week-old spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls. Because we have previously demonstrated an inverse relationship between dietary sodium intake and renal insulin receptor density and mRNA levels in normal Sprague-Dawley rats, the two rat strains in the current experiment were fed either low salt (0.07% NaCl) or high salt (7.5% NaCl) chow until the SHR became hypertensive. Fasting plasma glucose and plasma insulin levels did not differ between SHR and WKY and were not affected by salt intake. When the rats were maintained on the low salt diet, the rate of glucose infusion required to main euglycemia during a hyperinsulinemic clamp was significantly lower in SHR than WKY. High salt diet decreased the rate of glucose utilization during the hyperinsulinemic clamp in WKY but not SHR. During the low salt diet, insulin infusion decreased sodium excretion in both WKY and SHR. When the rats were maintained on the high salt diet, the antinatriuretic response to insulin was blunted in WKY but not SHR. Both the density and mRNA levels of insulin receptor were comparable in the kidney of WKY and SHR, but only WKY had the previously demonstrated decrease in receptor number and mRNA levels when fed the high salt chow. Hepatic insulin receptor mRNA levels were significantly lower in SHR than WKY fed the low salt diet. High salt diet decreased significantly insulin receptor mRNA levels in the liver of WKY but not of SHR. Thus, SHR appear to have lost the feedback mechanism that normally limits insulin-induced sodium retention when extracellular volume is expanded. A decreased expression of insulin receptor in the liver of SHR provides a possible explanation for the insulin resistance and decreased insulin clearance present in this strain. PMID- 8613275 TI - Menstrual cycle effects on catecholamine and cardiovascular responses to acute stress in black but not white normotensive women. AB - This study examined cardiovascular and catecholamine responses to two standardized laboratory stressors in 33 healthy age- and weight-matched black and white normotensive women (mean age, 32 years) during two phases of the menstrual cycle. Subjects were studied in a randomized order at the same time of day on two separate occasions approximately six weeks apart, once during the follicular phase (days 7 to 10 after menses) and once during the luteal phase (days 7 to 10 after the leutenizing hormone surge) of the menstrual cycle. Black women has higher systolic (P=.01) and diastolic (P=.01) pressures compared with white women. Black women showed greater diastolic pressure (P <.01) and plasma epinephrine (P <.05) responses to stress during the follicular compared with the luteal phase of the menstrual cycle; white women showed no significant changes in these variables. The findings extend the literature on race differences in responsivity to stress and indicate that in contrast to white women, reproductive hormones do influence cardiovascular and catecholamine responsivity to stress in black women. PMID- 8613276 TI - Community-based education classes for hypertension control. A 1.5-year randomized controlled trial. AB - Community-based hypertension control is important for primary prevention of cardiovascular disease. In this study, untreated men and women aged 35 to 69 years were randomly assigned to an intervention (n=56) or control (n=55) group in a 1.5-year community-based education program. Subjects had no evidence of hypertensive end-organ defects and had screening blood pressures of 140 to 179 mm Hg systolic and/or 90 to 109 mm Hg diastolic, with no difference in mean blood pressure between groups (148 to 150 mm Hg for mean systolic and 83 to 84 mm Hg for mean diastolic pressures). The intervention group took four education classes in the first 6 months and four classes during the next year, and the control group took two classes. Health education focused on reduced dietary sodium and increased milk intake, brisk walking, and, if necessary, reduction of alcohol and sugar intakes. Antihypertensive medication was started less often in the intervention than in the control group at 1.5 years (9% versus 24%, P <.05). Mean systolic pressure was 5 to 6 mm Hg less in the intervention than in the control group at both 6 months and 1.5 years (P <.05), with or without inclusion of those subjects who began antihypertensive medication. Diastolic pressure and body mass index did not change significantly between groups. Urinary sodium excretion declined in the intervention but not in the control group (differences between groups: P=.04 at 6 months and P=.07 at 1.5 years). According to a behavioral questionnaire, sodium reduction and milk increase were greater in the intervention than the control group (sodium: P <.01 at 6 months and P=.08 at 1.5 years; milk: P <.001 at 6 months and P <.01 at 1.5 years). Mean ethanol intake was reduced in the intervention but not the control group (P=.04 at 1.5 years). This community-based hypertension control program was effective in reducing systolic pressure levels by nonpharmacological means during the first 6 months and maintaining the reduction for 1.5 years. PMID- 8613277 TI - Genetic analysis of renin gene expression in rat adrenal gland. AB - We examined the mechanism of the increased renin mRNA concentration in the adrenal glands of spontaneously hypertensive rats (SHR). In 52 female F2 rats (25 to 27 weeks of age) derived from SHR and Wistar-Kyoto rats, we determined blood pressure, renin mRNA concentration in the adrenal gland, plasma renin activity, plasma aldosterone concentration, and genotype of the renin gene. Eighteen of the F2 rats were fed a high salt (8%) diet for 14 days. The renin mRNA concentration in the adrenal glands showed a significant correlation with the genotype of the renin gene in the normal salt diet group (P <.0001), whereas this relationship was not observed in the high salt group. Multivariate analysis revealed that the plasma aldosterone concentration in the normal diet group was significantly explained (P=.0004, R2=.454) by plasma renin activity (P=.0005), the renin mRNA concentration in the adrenal gland (P=.0496), and the genotype of the renin gene (P=.0236). The SHR allele of the renin gene was associated with a lower aldosterone concentration. On the other hand, in the high salt diet group, only the genotype of the renin gene showed a significant relationship with plasma aldosterone concentration (P=.0237). Again, the SHR allele of the renin gene was associated with a lower aldosterone concentration. We can conclude that the higher renin mRNA concentration in the SHR adrenal glands is governed by the SHR allele of the renin gene or renin gene locus. The renin mRNA concentration in the adrenal gland exerts a minor influence on aldosterone synthesis. Paradoxically, the SHR allele of the renin gene or renin gene locus confers a lower rate of aldosterone synthesis at 25 to 27 weeks of age, the mechanism of which remains to be determined. PMID- 8613278 TI - Transcriptional regulation of the genes involved in lipoprotein transport. The role of proximal promoters and long-range regulatory elements and factors in apolipoprotein gene regulation. PMID- 8613279 TI - Hepatitis E in children. PMID- 8613280 TI - Persistent diarrhea: management in a diarrhea treatment unit. AB - Five hundred ninety two children (6.0%) were diagnosed as persistent diarrhea (PD) out of a total attendance of 9795 cases in the Diarrhea Training and Treatment Unit (DTU) over a period of 1 year. Most of the cases were initially managed as outpatients on dietary advice and treatment of associated infections. Eighty eight per cent of the cases followed as outpatients from the DTU responded to treatment and only 11.5% of them had to be hospitalized. A total of 49/592 cases (8.3%) required to be hospitalized on account of treatment failure from outpatients and other indications. Clinical spectrum of hospitalized children included severe malnutrition (40.8%), pneumonia (40.8%), urinary tract infection (32.7%), lactose intolerance (32.7%), anemia (28.6%), septicemia (16.3%), dysentery (8.2%) and neck flop due to hypokalemia (4.1%). Dietary management included modifications in the diet already offered to hospitalized patients. Thirty eight children were fed on one of these diets. Of these 13/35 children (37.1%) were successfully managed with lactose reduced diet., 18/22 cases (81.8%) with lactose free diet and only in 2 cases carbohydrate free diet was given. In 3 cases, normal feeding was continued. Eleven cases were too sick to be offered any oral feeding. Eleven of forty nine cases (22.4%) expired. Mortality was highest in infants <6 months (31.6%). The causes of death included severe malnutrition (14.3%), septicemia (14.3%) and pneumonia (12.2%). Screening and treating cases of PD for associated infections like septicemia, pneumonia and urinary tract infection seems to be a key factor which determines morbidity and mortality in these cases. Feeding on a hospital food modified as lactose reduced/free diet can benefit majority of cases with PD and a very small proportion of cases may require carbohydrate free diet. PMID- 8613281 TI - Infected-associated hemophagocytic syndrome. AB - An epidemic of an infection associated with circulating hemophagocytes (HP) and activated monocytes (AM) was seen in Bombay. Although certain features overlapped with the well-defined entity of virus-associated hemophagocytic syndrome and familial hemophagocytic lymphohistiocytosis, it was distinct enough to place it in a separate category. Affected children were predominantly two days to two years of age. They had fever, altered sensorium, neurological symptoms, dyspnea, and/or diarrhea, and significant bleeding. Laboratory tests showed neutrophilia, AM and HP's in every blood smear, coagulopathy, normal cerebrospinal fluid, normal liver transaminases, hypertriglyceridemia, and hypoalbuminemia. Surgical cases were remarkable in that they had small bowel malformations. These cases were subdivided into four distinct groups based on age of presentation, neonates, infants, children and a surgical group. The clinical differences in each group are described. PMID- 8613282 TI - A comparative clinical trial of albendazole versus metronidazole in giardiasis. AB - The adverse effects and treatment failures to some of the currently recommended drugs for giardia infection have given rise to the need for alternative antigiardial agents. In an open, randomized parallel group study, the safety and efficacy of albendazole was compared with that of metronidazole for the treatment of giardiasis in children. Sixty two children aged between 2-12 years were randomized to receive either albendazole suspension 400 mg daily for 5 days or metronidazole suspension 7.5 mg/kg thrice daily for 5 days. The mean days required for cure, as evident by absence of cysts and/or trophozoites in the stool specimen, was 3.7 + 1.4 and 4.5 + 1.1 days, respectively for children on albendazole and metronidazole therapy. Six children on metronidazole therapy developed anorexia 2 to 4 days after the treatment. Albendazole proved as effective as metronidazole in the treatment of giardia infection in children with the added advantage of absence of anorexia. PMID- 8613283 TI - Testing compliance of drug taking--a simple bed side method. AB - Assessment of compliance in drug taking is a problem in a crowded Outpatient Department. Using riboflavin as a urinary marker is a simple and rational method. Identifying riboflavin in the urine by fluorescence on exposure to ultraviolet (UV) rays or torch light is being used in medical practice but not extensively. In this study, the validity and reliability of these methods were assessed. The sensitivity and specificity of this test by UV method was 86% and 82% for Reader I (medical person) and 82% and 94% for Reader II (paramedical person). For Reader I, the accuracy of reading by UV lamp was the same as torch light (85%) whereas for Reader II the accuracy was better with UV lamp (87%) than with torch (79%). In reading the fluorescence by UV lamp the crude agreement between the 2 readers was 82% and chance corrected agreement was 64%. UV lamp method appears to be a reliable way of assessing compliance both by medical and paramedical persons whereas torch method appears to be more reliable when used by a medical person than by a paramedical person. PMID- 8613284 TI - Serum amino acids and genesis of protein energy malnutrition. AB - Twenty four patients of classical marasmus and kwashiorkor along with equal number of healthy controls were selected for the study. Their serum amino acid patterns analysis revealed a mean ratio of glutamate to alanine in fasting samples of normal individuals to be 0.33, while it as 9.3 in kwashiorkor and 1.6 in marasmus. This differences in controls, kwashiorkor and marasmus was statistically significant. This observation may explain evolution of marasmus and kwashiorkor in children with similar diets. On the basis of the present observation it is postulated that in kwashiorkor, the conversion of pyruvate to alanine in presence of glutamate, an aminogroup donor does not proceed normally, resulting in accumulation of glutamate and low alanine. Thus the development of marasmus and kwashiorkor may not be related to dietary inadequacy alone but also to the transaminase function. This could be genetic in origin. PMID- 8613285 TI - Urodynamic study of children with voiding problems. AB - Twenty high risk children aged 5-12 years with various voiding problems were studied prospectively by urodynamics to evaluate the function of their urinary bladder and its continence mechanism. None of them had neuropathic bladder or any obstruction distal to bladder neck. Fourteen out of twenty (70%) had abnormal findings on urodynamics evaluation; 8 (40%) had non-neurogenic neurogenic bladder (NNNB); 3 (15%) had small capacity hypertonic bladder (SCHB); 2 (10%) had atonic bladder (AB) and 1 (5%) had hyperreflexic bladder (HB). We conclude that urodynamic abnormalities are as frequent in high risk Indian children as they are in developed countries. The high risk children should be subjected to urodynamic studies more frequently than being done hitherto and be directed to proper therapeutic modality. PMID- 8613286 TI - Assessing the cause of in-patients pediatric diarrheal deaths: an analysis of hospital records. AB - Records of all the diarrheal patients up to the age of 5 years who were admitted to and died in Dr. B.C. Roy Memorial Hospital for Children, Calcutta, between January and December 1990 were analyzed. The records were reviewed to assess the relative importance of three clinical types of diarrhea (acute watery diarrhea, acute dysentery and persistent diarrhea) as the causes of mortality. Annual hospital death rates of children suffering from acute watery diarrhea, dysentery and persistent diarrhea were 13.6%, 18.2% and 25.9%, respectively. Overall death rates in dysentery (p = 0.03) and persistent diarrhea groups (p < .00001) were significantly higher than watery diarrhea group. Maximum deaths occurred among children aged between 7 and 36 months in all categories of diarrhea. Shigella infected children had higher case fatality rate. In acute watery diarrhea, 30.9% cases were assigned to associated causes of death whereas the same could be assigned to 92.6% and 93.2% cases in dysentery and persistent diarrhea group, respectively. Deaths occurred in most of the cases who had bronchopneumonia as underlying cause, septicemia as immediate cause and protein calorie malnutrition as associated cause and these were most frequently associated in patients suffering from dysentery and persistent diarrhea. Only 2.0% children suffering from acute watery diarrhea had dehydration at the time of death. Significantly, a high percentage of deaths occurred among malnourished children who suffered from dysentery (54.4%) and persistent diarrhea. These data suggest that Diarrheal Disease Control Programme should also give emphasis on management of non watery, non-dehydrating type of diarrhea with complications. PMID- 8613288 TI - Changing pattern of childhood poisoning (1970-1989): experience of a large north Indian hospital. AB - The epidemiology of acute poisoning in children aged 0-15 years hospitalized between 1970-1989 was retrospectively investigated. Two hundred and seventeen children were admitted during this period with 134 in 1980-1989 vs 83 in 1970 1979. Two distinct patterns were observed: accidental poisoning in under 11's and adult pattern and self poisoning in children over 11's. More children belonged to urban areas (72.3%) as compared to rural areas. In both decades more than half of children belonged to middle income group followed by lower income group and least to upper income group. The overall mortality was low (12.5%), with majority of deaths (78%) occurring in older children. The incidence of kerosene ingestion was noticed to have dropped by more than half between 1980-1989 as compared to 1970 1979 (14.9% vs 42%). The study highlights the unacceptable high rate of preventable accidental poisoning in young children and suicide and parasuicide in older children (11-15 years age group). PMID- 8613287 TI - Impaired pancreatic bicarbonate secretion in chronic malnutrition. AB - Twenty three children with recurrent episodes of diarrhea and chronic malnutrition were studied for pancreatic duct function. Those children were subjected to pancreatic stimulation with pancreozymin and secretin. Grade I malnourished children, as per Gomez classification, formed the control group. The water output from pancreas increased in malnourished children (p < 0.05). It correlated significantly to cationic transport (p < 0.01). Sodium and potassium together accounted for significant proportion of water output in pancreatic fluid. Potassium transport increased with increasing severity of malnutrition and may be responsible for the hypokalemia observed in malnourished children. Pancreatic secretion of bicarbonate decreased in severe malnutrition inspite of increased flow rate of pancreatic secretion. This is probably due to defective bicarbonate secretion likely to be located at pancreatic duct epithelial cell membrane. PMID- 8613289 TI - Oral aluminium phosphide poisoning. PMID- 8613290 TI - Epidermal nevus syndrome. PMID- 8613292 TI - Psychiatric complications of chloroquine. PMID- 8613291 TI - Knowledge and attitudes of Anganwadi workers about infant feeding in Delhi. PMID- 8613293 TI - Tuberculin sensitivity in low birth weight and malnourished children. PMID- 8613294 TI - Asphyxiating thoracic dystrophy. PMID- 8613295 TI - Citrobacter sepsis in infants. PMID- 8613296 TI - Familial Noonan syndrome. PMID- 8613297 TI - Conjoined twins with jugular lymphatic obstruction sequence. PMID- 8613298 TI - Sporadic growth hormone insensitivity syndrome. PMID- 8613299 TI - Multifocal chloromas preceding acute myelogenous leukemia. PMID- 8613300 TI - Renal bruit due to aberrant renal vessels. PMID- 8613301 TI - Dandy Walker malformation: a cause of developmental retardation. PMID- 8613302 TI - Thin meconium stained liquor--what is the right approach? PMID- 8613303 TI - Hypernatremia and ORS. PMID- 8613304 TI - Steroids in bacterial meningitis. PMID- 8613305 TI - A concurrent comparison of a WHO-recommended 30-cluster survey and a modified version of it under Indian conditions in the estimation of immunization coverages. AB - A concurrent comparison of the WHO 30-cluster sample survey method for estimating immunization coverages (DPT, Polio, BCG, Measles) and an Indian modification of (GOI) was undertaken in five districts in South India. The essential difference between the two methods is the manner in which the first household is selected in the chosen clusters. With the WHO method, it is chosen clusters. With the WHO method, it is chosen at random, whereas with the GOI method it is often close to the village centre. Estimates with the required degree of precision, i.e., 95% confidence limits of +/- 10 percentage points, were provided in 18 (90%) of 20 instances by the WHO method and in 19 (95%) by the GOI method, findings which are in accordance with expectation. The estimated coverages were, however, higher by the GOI method than by the WHO method in two districts, lower in one district, and in the remaining two districts there was no clear pattern. On the average, there was a suggestion that the GOI method yielded slightly higher coverages, but the differences were not statistically significant. PMID- 8613306 TI - Typhoid vaccine. PMID- 8613307 TI - Nebulized steroids for mild-to-moderate croup. PMID- 8613308 TI - Pediatric HIV infection: recent advances. PMID- 8613309 TI - Routine antibiotic cover for newborns intubated for aspirating meconium: is it necessary? AB - A retrospective analysis was performed on 215 babies to evaluate the incidence of septicemia in babies intubated at birth for aspirating meconium from the trachea. Only term, appropriate for gestational age babies were included. Babies with any known perinatal risk factor for infection were excluded from the study and none of the babies had been put on "prophylactic antibiotics." There were 88 babies in the intubated group in a one year period from January 1991 to December 1991. One hundred and twenty seven babies were taken as controls. There was no significant difference in the incidence of early septicemia in the two groups. There were no deaths in either group. It is concluded that well term babies who are intubated for aspirating meconium need not be put on routine antibiotic cover. PMID- 8613310 TI - Furazolidone in typhoid fever--correlation of clinical efficacy with serum bactericidal activity. AB - Treatment of typhoid fever with furazolidone produces a high cure rate. This is a clinical curiosity, as furazolidone is described to be poorly absorbed. The present study examined whether furazolidone could produce unequivocal clinical response and, if so whether this was due to the drug producing bactericidal levels in the serum. Twenty one patients selected by defined criteria were treated with furazolidone and evaluated for definite clinical response in 5-7 days. Bactericidal activity of pre dose and post dose sera were estimated in seven patients showing definite clinical response. All the seven patients had a clinical cure without the drug producing significant bactericidal levels in the blood. Hence we concluded that the major site of action of furazolidone was in the intestine. It is our postulate that the organisms reaching the intestine in large numbers from bile are prevented from gaining re-entry into the circulation by the action of furazolidone in the intestine. After repeated cycles of entry of organisms into the intestine from bile and the simultaneous prevention of its re entry into the circulation, the number of organisms remaining in circulation comes down considerably, thus helping the immune system to bring about a cure. PMID- 8613311 TI - Contamination of weaning foods and transmission of E. coli in causation of infantile diarrhea in low income group in Chandigarh. AB - Samples of weaning foods and other sources of contamination, such as water, mother's nails, utensils and swab samples of feeding bottle nipple, mother's teats and child's hands were collected from a total of 100 houses of Low Income Group (LIG) in Chandigarh. A high incidence of E. coli isolation (72.3%) was noticed amongst the collected samples. Seventy nine per cent of storage containers of water exhibited the presence of E. coli. Eighty per cent of the children had diarrhea even when exclusively breastfed. Sixty six per cent children were weaned within 3-6 months; the ratio increasing with increase in the educational qualification of the mother. Eighty out of the total 100 households which had a history of infantile diarrhea exhibited 80.9% E. coli isolation. PMID- 8613312 TI - Flexible fibreoptic bronchoscopy in 582 children--value of route, sedation and local anesthetic. AB - The value of route, sedation and local anesthetic was studied in 582 children aged 50 days to 12 years who were subjected to flexible fibreoptic bronchoscopy (FFBS) at the Institutes of Child Health, Madras, during January 1989 to July 1993. Pentax 3.5 mm and Olympus 4.9 mm bronchoscopes were used. Bronchoscopy was performed with sedation and/or local anesthetic through nasal/oral route after premedication with atropine. It was successfully carried out through nasal route in 97.4% and only in 40% through oral route. As nasal route proved advantageous, the oral route was abandoned. PMID- 8613313 TI - Normal parameters of ventricular system in healthy infants. AB - Six hundred healthy inborn newborns and infants upto the age of 18 months were studied. Cranial sonography was performed by real time 2D scanner with 5 MHz transducer and images were obtained through anterior fontanelle and temporo squamal suture. Various parameters related to ventricular system were measured for different ages till the fontanelle remained open. These values will prove useful for diagnosing hydrocephalus at an early stage of the disease and also to find out the blocks at various levels in ventricular system. PMID- 8613314 TI - Continuing education: concepts and strategies. PMID- 8613315 TI - Febrile seizures: an update. PMID- 8613316 TI - Pediatric AIDS. PMID- 8613317 TI - Cytomegalovirus infection acquired through blood transfusions. PMID- 8613318 TI - Sarcoidosis. PMID- 8613319 TI - Calcification in renal tuberculosis. PMID- 8613320 TI - Resurgence of chloramphenicol sensitive Salmonella typhi. PMID- 8613321 TI - Cleidocranial dysplasia. PMID- 8613322 TI - Lissencephaly. PMID- 8613324 TI - The outpatient management of bloody diarrhea in young children. PMID- 8613323 TI - Availability of antenatal and perinatal care in an ICDS area. PMID- 8613325 TI - HIV serosurveillance--report from a medical college in Delhi. PMID- 8613326 TI - Hereditary pancreatitis with lithiasis. PMID- 8613327 TI - Dopamine dosing dilemma. PMID- 8613328 TI - Hepatitis B vaccine. PMID- 8613329 TI - Short course chemotherapy in childhood tuberculosis. PMID- 8613330 TI - Pulmonary function tests in normal Indian children and changes in respiratory disorders. AB - To document the normal values of pulmonary function tests in children and changes occurring in their values with various respiratory disorders, a study was carried over a period of one year in 95 healthy controls (39 females and 56 males) of 8 13 years of age and 51 cases with respiratory disorders (bronchial asthma-31, pneumonia-10, empyema-10) of matched age, sex and height distribution. The lung functions studied were FVC, FEV1, FEV1/FVC, PEFR and FEF25-75%. In children with bronchial asthma, the FEV1/FVC%, PEFR and FEF25-75% were reduced in accordance with the severity of the disease. A typical restrictive pattern of equivalent decrease in FVC and FEV1 along with insignificant lowering of flow rates, i.e., PEFR and FEF25-75% was observed in pneumonia whereas in patients of empyema a combined pattern of significantly decreased FVC and FEV1 along with mildly reduced FEV1/FVC%, PEFR and FEF 25 75% was observed. PMID- 8613331 TI - Timing and dose of BCG vaccination in infants as assessed by postvaccination tuberculin sensitivity. AB - Tuberculosis is an important public health problem in developing countries and BCG plays an important role in preventing serious form of the disease in children. BCG induced tuberculin sensitivity is a quantitative characteristic and has been used to compare vaccine efficacy. The standard dose of BCG vaccine is 0.1mg in 1 ml though manufacturers of certain strains of BCG, i.e., Copenhagen 1331 recommend half dose in infants. There are also varying ideas about the optimum time to vaccinate babies and some studies suggest that late vaccination confers a high degree of protection. This study was carried out to evaluate tuberculin sensitivity and side effects following 0.05 ml and 0.1 ml of BCG at birth and 0.1 ml of BCG at 4-6 weeks of age. Two hundred and thirty eight newborns were vaccinated randomly with 0.05 ml and 0.05 ml and 0.1 ml of BCG vaccine (Copenhagen 1331 strain) containing .69 million culturable particles per ml. One hundred and eight infants 4-6 weeks of age were vaccinated with 0.1 ml of BCG. One hundred and fifty five (44.7%) infants were evaluated by Mantoux test using 1TU PPD RT23 10-12 weeks after vaccination and 105 (30.6%) followed up till 6 months for any side effects. No significant difference in mean tuberculin reaction, tuberculin positivity and mean scar size was observed in groups receiving 0.1 ml at birth or 4-6 weeks of age. However, the group receiving 0.05 ml at birth had a significantly lower mean tuberculin reaction, tuberculin positivity and mean scar size. No locoregional side effects were observed. Hence the present practice of giving 0.1 ml of BCG at birth should be continued. PMID- 8613332 TI - Tuberculous meningitis in children--clinical profile, mortality and morbidity of bacteriologically confirmed cases. AB - One hundred and seven cases of tuberculous meningitis were registered as a part of a case-control study during the period 1990-1992. The CSF of all cases was positive for culture and/or smear for acid fast bacilli. Children were examined at the time of admission and at the time of discharge and they were contacted at the end of 1 year. Clinical picture, mortality and morbidity were analyzed. Mortality of children during the first month of illness was 22%. Some of the cases presented as acute neurological illness. We also came across CSF picture with minimal cytological and biochemical changes but with positive culture results. PMID- 8613333 TI - Knowledge of asthma among parents of asthmatic children. AB - The knowledge and attitude towards asthma, of parents, of 85 asthmatic children was assessed using a 17 item questionnaire. Results showed that 34.1% believed asthma to be contagious, 48.2% of the parents hesitated in referring to their child's illness as asthma. Other commonly held beliefs were that asthma is a life long illness (35.3%); food items are important precipitating factors for acute attacks (88.2%); mild exacerbations need to be treated with bronchodilators (6.3%); bronchodilators should be started at home before consulting a physician in case of an acute attack (61.2%); and cure of asthma is possible through modern drugs (30.6%) or through alternative systems of medicine (65%). Ninety one per cent of parents lacked an awareness of the side effects of anti-asthma medication. It is concluded that parental education through improved physician parent communication is necessary for enhancing the quality of care being provided to children with asthma, a fact also highlighted by the International Consensus Report on Management of Asthma. PMID- 8613334 TI - Skeletal dysplasias in a hospital in southern India. AB - A hospital based study of skeletal dysplasias was conducted over a period of 2 years in Davangere, Karnataka, in which 169 cases of skeletal dysplasias were studied. One hundred were osteochondrodysplasias and were grouped according to international classification of osteochondrodysplasias. Among the individual cases, osteogenesis imperfecta (13 cases) had the maximum representation. Several cases of rare disorders were also identified. Eighty eight cases of skeletal dysplasias were in the pediatric age group and of these 41 were newborns. The incidence of skeletal dysplasia among newborns was 19.6 per 10,000 deliveries and lethal dysplasias 5.2 per 10,000 deliveries. In 7 cases of skeletal dysplasia, an antenatal diagnosis was possible by ultrasonography. PMID- 8613335 TI - Profile of intersex children in south India. AB - Thirty five children with ambiguous genitalia admitted to our centre between January 1986 to December 1991, were followed up and their clinical, laboratory and management strategies were analyzed. Most of them presented between 1 month and 2 years of age and only 2 presented in the newborn period. Sixteen were female pseudohermaphrodites. Eighteen out of 31 children were assigned female sex. One genetic female with congenital adrenal hyperplasia was assigned male sex. We practised more than one type of clitoroplasty in our centre. Parents prefer the intersex children to be reared as male possibly because of the less social stigma attached to an impotent male than to sterile female, and because males are socially independent. PMID- 8613336 TI - Congenital Tuberculosis. PMID- 8613337 TI - Tuberculosis in BCG vaccinated children. PMID- 8613338 TI - Typhoid fever in a neonate. PMID- 8613339 TI - Widal reaction in Kala-azar. PMID- 8613340 TI - Progressive multifocal leukoencephalopathy in a case of acute lymphocytic leukemia. PMID- 8613342 TI - Wilms' tumor arising in a horseshoe kidney. PMID- 8613341 TI - Cerebrospinal fluid C-reactive protein in meningitis. PMID- 8613343 TI - Fungal peritonitis complicating peritoneal dialysis. PMID- 8613344 TI - Unusual complications of rickets. PMID- 8613346 TI - Typical facies in Duchenne muscular dystrophy. PMID- 8613345 TI - Can BCG be given along with DPT and polio? PMID- 8613347 TI - If the iris is absent? PMID- 8613348 TI - Suthi feeding: an experience. PMID- 8613349 TI - Hypertension in a child: diagnostic aspects. PMID- 8613350 TI - Hepatitis B vaccine. PMID- 8613351 TI - Growing out of asthma. PMID- 8613352 TI - Hyaluronate capsule and surface M protein in resistance to opsonization of group A streptococci. AB - The major virulence determinant of group A streptococci is the ability to resist opsonization and phagocytic ingestion. The present studies were performed to compare the mechanisms of resistance to opsonization of type 18 and type 24 streptococci and to determine the relative roles of M protein-fibrinogen interaction and the hyaluronate capsule in preventing phagocytic ingestion and killing. By use of parent strains and acapsular transposon mutants in the presence and absence of fibrinogen, we show that type 18 and type 24 streptococci rely on somewhat different mechanisms for resistance to opsonization. Type 24 streptococci bound fibrinogen avidly to their surfaces, and encapsulated organisms were completely resistant to opsonization only in the presence of fibrinogen. In contrast, type 18 streptococci bound 10-fold less fibrinogen than type 24 streptococci and were fully resistant to phagocytosis only when they expressed capsule. The general structural characteristics of the amino-terminal halves of type 18 and type 24 M proteins differed in that type 18 M protein contained only one complete B repeat, whereas type 24 M protein contained five complete B repeats, a structural difference which could potentially be related to the differences in fibrinogen binding between the two serotypes. Immunofluorescence assays of complement deposition were used in combination with 125I-C3 binding assays to show that encapsulated type 24 streptococci were fully resistant to opsonization by C3 only in the presence of plasma. Encapsulated and unencapsulated type 18 streptococci were equally opsonized by C3 in either plasma or serum, yet only encapsulated organisms resisted phagocytic killing in blood. The results of this study indicate that opsonization by C3 does not necessarily lead to phagocytic ingestion and that the hyaluronate capsule and M proteins are variably important in resistance to different group A streptococci to opsonization and phagocytic killing. PMID- 8613353 TI - Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys. AB - We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations. PMID- 8613355 TI - Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses. AB - Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses. PMID- 8613354 TI - Adenovirus-mediated transfer of a gene encoding acyloxyacyl hydrolase (AOAH) into mice increases tissue and plasma AOAH activity. AB - Although the host response to gram-negative bacterial infection follows largely from the interactions of bacterial lipopolysaccharides (LPS or endotoxin) with host cells, little information is available concerning the mechanisms by which the host eliminates or detoxifies LPS. Acyloxyacyl hydrolase (AOAH) is an enzyme, found in phagocytic cells, that catalyzes the enzymatic deacylation of the lipid A moiety of LPS. Enzymatically deacylated LPS is much less potent than LPS at inducing responses in human cells, and it can antagonize the ability of LPS to activate human macrophages, neutrophils, and endothelial cells. Despite these observations, the physiologic role of LPS deacylation remains undefined. To investigate the ability of AOAH to carry out LPS deacylation in vivo, we produced a recombinant adenovirus carrying a gene encoding (AOAH) (Ad.CMV-AOAH) and employed this vector to elicit transient overexpression of AOAH in mice. Mice infected with Ad.CMV-AOAH expressed high levels of the enzyme in plasma, liver, spleen, and kidney. Although adenovirus-induced hepatitis reduced hepatic uptake of intravenously injected [3H]LPS, animals expressing the transgene deacylated a larger fraction of the [3H]LPS taken up by their livers than did mice infected with a control adenovirus. These studies indicate that AOAH can catalyze the deacylation of LPS in vivo, and they provide evidence that the rates of hepatic LPS uptake and deacylation are not closely linked. PMID- 8613356 TI - Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess. AB - Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis. PMID- 8613357 TI - The Legionella pneumophila hel locus encodes intracellularly induced homologs of heavy-metal ion transporters of Alcaligenes spp. AB - We continued characterization of the Legionella pneumophila hel locus. Mutagenesis and DNA sequencing identified three genes similar to the czc and cnr loci of Alcaligenes eutrophus and the ncc locus of Alcaligenes xylosoxidans. On the basis of their similarity to these loci, we designated the L. pneumophila genes helC, helB, and helA. Mutations in the hel genes led to reduced cytopathicity towards U937 cells, although the mutant strains did not appear defective in other assays of virulence. Transcription of the hel locus was induced by the intracellular environment but was not induced by any of a variety of in vitro stress conditions. The function of the hel gene products remains to be determined. PMID- 8613358 TI - Cloning and characterization of a putative cytadhesin gene (mgc1) from Mycoplasma gallisepticum. AB - A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas. PMID- 8613359 TI - Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of helicobacter-induced carcinogenesis. AB - Helicobacter hepaticus causes hepatitis in selected strains of mice and in A/JCr mice is linked to liver cancer. To analyze whether H. hepaticus persists in specified ecological niches, to determine whether biomarkers of infection exist, and to analyze the influence of H. hepaticus on hepatocyte proliferation, a longitudinal study of H. hepaticus-infected A/JCr mice was undertaken. A/JCr mice were serially euthanatized from 3 through 18 months and surveyed by enzyme-linked immunosorbent assay; bacterial culture of liver, colon, and cecum; histology; electron microscopy; hepatocyte proliferation indices determined by using 5-bromo 2'-deoxyuridine; and measurement of the liver enzyme alanine aminotransferase. In infected animals throughout the 18-month study, H. hepaticus was consistently isolated from the lower bowel but only sporadically from the liver. By electron microscopy, H. hepaticus was noted infrequently and only in bile canaliculi. Infected mice, particularly males, showed chronic inflammation; oval cell, Kupffer cell, and Ito cell hyperplasia; hepatocytomegaly; and bile duct proliferation. The inflammatory and necrotizing lesion was progressive and involved the hepatic parenchyma, portal triads, and intralobular venules. Hepatic adenomas were noted only in male mice, whereas 5-bromo-2'-deoxyuridine proliferation indices were markedly increased in both sexes, but especially in males, compared to control A/J mice. Infected mice also developed sustained anti H. hepaticus serum immunoglobulin G antibody responses and elevated alanine aminotransferase levels. H. hepaticus, which persists in the lower bowels and livers of A/JCr mice, is associated with a chronic proliferative hepatitis, and hepatomas in selected male mice indicate that this novel bacterium may cause an increased risk of hepatic cancer induction in susceptible strains of mice. This murine model should prove useful in dissecting the molecular events operable in the development of neoplasms induced by bacteria belonging to this expanding genera of pathogenic Helicobacter species. PMID- 8613360 TI - Trypanosoma cruzi-induced immunosuppression: selective triggering of CD4+ T-cell death by the T-cell receptor-CD3 pathway and not by the CD69 or Ly-6 activation pathway. AB - In a model of experimental Chagas' disease induced with metacyclic forms of Trypanosoma cruzi, CD4+ but not CD8+ T cells undergo T-cell receptor (TCR)-CD3 mediated activation-induced cell death (AICD) in vitro. CD4+ T cells from T. cruzi-infected mice also developed unresponsiveness in proliferative responses to TCR-CD3-mediated stimulation. A linear correlation was found between extent of proliferative unresponsiveness and loss of CD4+ T-cell viability. CD4+ T-cell activation through the CD69 or Ly-6 A/E pathway, on the other hand, did not result in proliferative unresponsiveness compared with controls. Lack of suppression in proliferation assays correlated with lack of AICD by cells stimulated through the CD69 or Ly-6 A/E pathway. Concomitant stimulation through CD69, however, did not rescue CD4+ T cells from CD3-induced death. Flow cytometry study of cells stimulated in vitro showed no defect in interleukin-2 receptor expression by CD4+ T cells from infected donors, which escaped TCR-mediated AICD. In vivo injection of anti-CD3 into acutely infected mice, but not into control mice, led to splenocyte DNA fragmentation and failed to increase splenic CD4+ T cell numbers. These results show that TCR-CD3-mediated AICD is involved in CD4+ T cell unresponsiveness in vitro following infection with T. cruzi. In addition, successful activation of these cells through the CD69 and Ly-6 pathways is due to differences in the inability of these stimuli to trigger AICD. Since TCR-CD3 mediated AICD can be induced in vivo in infected mice, these findings may be relevant for the onset of immunological disturbances in the host. PMID- 8613361 TI - Coordinate induction of two antibiotic genes in tracheal epithelial cells exposed to the inflammatory mediators lipopolysaccharide and tumor necrosis factor alpha. AB - Peptides with potent broad-spectrum antibiotic activity have been identified in many animal species. Recent investigations have demonstrated that epithelial cells are a site of antibiotic peptide expression, suggesting that these peptides contribute to host defense at mucosal surfaces. Expression of tracheal antimicrobial peptide (TAP), a member of the beta-defensin family of peptides, is inducible in cultured tracheal epithelial cells (TEC) upon challenge with bacterial lipopolysaccharide (LPS) (G. Diamond, J.P. Russell, and C.L. Bevins, Proc. Natl. Acad. Sci. USA, in press). In this study, an anchored reverse transcriptase PCR strategy was used to determine if TAP was the sole beta defensin isoform expressed upon stimulation of the cells with LPS. In addition to TAP, a second class of cDNA clones which encoded lingual antimicrobial peptide (LAP), a beta-defensin peptide recently isolated from a different mucosal site, the bovine tongue, was identified (B.S. Schonwetter, E.D. Stolzenberg, and M. Zasloff, Science 267:1645-1648, 1995). Northern (RNA) blot analysis demonstrated in vivo expression of LAP mRNA in tracheal mucosa. Levels of LAP mRNA were higher in cultured TEC challenged with either LPS or tumor necrosis factor alpha than in control cells. Thus, a response of TEC exposed to inflammatory mediators is induction of antibiotic-encoding genes, including both TAP and LAP. This work complements the in vivo studies of Schonwetter et al. (cited above), which showed elevated levels of LAP mRNA in squamous epithelial cells of the tongue near sites of tissue injury and inflammation, by suggesting possible mediators of the in vivo observation. Together these lines of investigations support the hypothesis that inducible expression of endogenous antibiotic peptides by inflammatory mediators characterizes local defense of mammalian mucosal surfaces. PMID- 8613362 TI - Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice. AB - The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414 36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice. PMID- 8613363 TI - Colony-stimulating factor 1-dependent resident macrophages play a regulatory role in fighting Escherichia coli fecal peritonitis. AB - Osteopetrotic op/op mice have less than 5% of the normal number of macrophages in the peritoneal cavity (W. Wiktor-Jedrzejczak, A. Ahmed, C. Szczylik, and R.R. Skelly, J. Exp. Med. 156:1516-1527, 1982). Fecal peritonitis was induced by intraperitoneal injection of 0.5 ml of 5% autoclaved feces in saline along with Escherichia coli grown from feces of mice of the same colony and added in doses ranging between 10 and 10(6) CFU. Such infection led to a septic shock and either was lethal within 24 h or became cured without additional treatment of the mice. The op/op mice survived administration of 30-times-smaller doses of bacteria compared with their normal littermates. Analysis of the kinetics of cellular changes in the peritoneal cavity associated with such infection revealed that this increased susceptibility of macrophage-deficient mice cannot be explained by a direct role of macrophages in combating the infection. Instead, it appeared that the increased susceptibility to fatal fecal peritonitis was most likely due to delayed and impaired recruitment of neutrophils to the site of infection in mutant mice. The increased susceptibility of the op/op mice to E. coli fecal peritonitis was not due to their possible increased sensitivity to endotoxin, since the mutant mice tolerated lipopolysaccharide doses more than twice those tolerated by control littermates. On the other hand, their susceptibility to exogenous tumor necrosis factor alpha and interleukin-1 alpha was increased. Both mutant op/op and control mice were able to survive secondary challenge with 10(6) E. coli (administered along with feces) lethal for both types of mice on primary challenge. These data suggest that colony-stimulating factor 1-dependent resident peritoneal macrophages play a role in controlling primary infection by recruiting neutrophils and are not required for efficient response to secondary infection. PMID- 8613364 TI - Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells. AB - We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence. PMID- 8613366 TI - gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans. AB - Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens. PMID- 8613365 TI - Molecular composition of Clostridium botulinum type A progenitor toxins. AB - The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed. The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously. Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components. The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA. HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol. The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross linked by the 35-kDa subcomponent. The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol. It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA. This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture. We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with three types of progenitor toxins. PMID- 8613367 TI - Defining antibody targets in Streptococcus oralis infection. AB - Immunoblotting of sera from 12 neutropenic patients with Streptococcus oralis septicemia and 18 patients with endocarditis due to viridans group streptococci revealed immunodominant S. oralis antigens at 85 and 180 kDa. The former cross reacted with a mouse monoclonal antibody to hsp90. The latter was identified by sequencing positive clones obtained by screening a genomic expression library of S. oralis with pooled sera from patients who had been infected with S. oralis. Antibody eluted from one of these clones reacted with the 180-kDa antigen of S. oralis. Southern blotting confirmed the origin of the clone from S. oralis. The derived amino acid sequence showed 76.2% homology with the PAc protein precursor of Streptococcus mutans and 73.8% homology with the SpaA protein precursor of Streptococcus sobrinus. Epitope mapping of the derived amino acid sequence with sera from patients with viridans group streptococcal endocarditis delineated nine epitopes. Peptides 1 (TMYPNRQPGSGWDSS) and 2 (WYSLNGKIRAVDVPK), representing two of these epitopes, and peptide 3 (YEVEKPLEPAPVAPS), representing the repeat proline region, were synthesized. These three peptides were used to screen a phage antibody display library derived from a patient who had recovered from S. oralis infection. Two of the human recombinant antibodies produced (SORAL 3 and SORAL 4 against peptide 3) and a human recombinant antibody (B3.7) against the conserved epitope (LKVIRK) of hsp90 gave statistically significant protection, compared with control groups, in a mouse model of lethal S. oralis infection. PMID- 8613368 TI - Interleukin-1 alpha production during Rickettsia rickettsii infection of cultured endothelial cells: potential role in autocrine cell stimulation. AB - Rickettsia rickettsii infection results in numerous responses by cultured endothelial cells, among them a rapid, transient increase in steady-state levels of tissue factor mRNA (L.A. Sporn, P.J. Haidaris, R.-J. Shi, Y. Nemerson, D.J. Silverman, and V.J. Marder, Blood 83:1527-1534, 1994). In this study, production of interleukin-1 (IL-1) was measured during infection and its potential role in autocrine cell stimulation was investigated. A fivefold increase in levels of IL 1 alpha antigen was measured in cell lysate samples by enzyme-linked immunosorbent assay at 18 h of infection. The majority of IL-1 alpha remained cell associated, as no significant increase was detected in culture medium. No IL 1 beta antigen was detected in cell lysates or culture medium from either control or infected cultures. A dramatic increase in the levels of IL-1 alpha mRNA occurred following infection, as measured by reverse transcriptase PCR, which revealed the appearance of the expected 421-kb product with RNA extracted from cells infected for 4 h and no detectable product from control cell samples. The presence of functional, cell-associated IL-1 alpha activity in infected cells was confirmed, following disruption, by the ability of the infected cells to induce tissue factor expression in target endothelial cells. Such induction was eliminated by pretreatment of the disrupted cell samples with neutralizing antibodies against IL-1 alpha but not against IL-1 beta. To investigate whether endogenously produced IL-1 participates in the stimulation of tissue factor expression, neutralizing antibodies against IL-1 or the IL-1 receptor antagonist were added to culture medium during infection. Both anti-IL-1 alpha and the IL-1 receptor antagonist resulted in approximately 40% inhibition of tissue factor expression, thus implicating IL-1 alpha in autocrine cell stimulation. PMID- 8613369 TI - Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. AB - Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions. PMID- 8613370 TI - Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton. AB - Neisseria gonorrhoeae enters cultured human mucosal cells following binding of a distinct gonococcal opacity (Opa) outer membrane protein to cell surface proteoglycan receptors. We examined the route of internalization that is activated by Opa-expressing gonococci (strain VP1). Microscopy of infected Chang epithelial cells showed that gonococcal uptake was insensitive to monodansylcadaverine (150 microM), which interferes with clathrin-mediated endocytosis. Similarly, indirect immunofluorescence staining for clathrin in infected cells showed distribution of cellular clathrin unaltered from the distribution in noninfected cells. The microtubule inhibitors colchicine (50 microM) and nocodazole (20 microM) but not the microtubule-stabilizing agent taxol (10 microM) caused a moderate (30 to 50%) reduction in gonococcal entry without affecting bacterial adherence. The most dramatic effects were obtained with the microfilament-disrupting agent cytochalasin D (3 microM), which totally blocked bacterial entry into the cells. Double immunofluorescence staining of gonococci and actin filaments in infected cells demonstrated bacterium-associated accumulations of F-actin as an early signal of bacterial entry. The recruitment of F-actin was transient and disappeared once the bacteria were inside the cells. Cytochalasin D disrupted the actin cytoskeleton architecture but did not prevent the recruitment of F-actin by the bacteria. Adherent, noninvasive gonococcal Opa variants lacked the ability to mobilize F-actin. Recombinant Escherichia coli expressing the gonococcal invasion-promoting Opa of gonococcal strain MS11 (Opa50) adhered to the epithelial cells in an Opa-dependent fashion but was not internalized and did not recruit detectable amounts of F-actin. Coinfection with the E. coli recombinant strain and gonococci resulted in specific entry of the diplococci, despite the presence of large numbers of adherent E. coli cells. Together, our results indicate that Opa-mediated gonococcal entry into Chang cells resembles phagocytosis rather than macropinocytosis reported for Salmonella spp. and sequentially involves gonococcal adherence to the cell surface, Opa dependent and cytochalasin-insensitive recruitment of F-actin, and cytochalasin D sensitive bacterial internalization. PMID- 8613371 TI - Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin. AB - Fimbriae (the oligomeric form of fimbrillin) are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. In the present study, we have identified the structural domains of P. gingivalis fimbrillin that mediate the binding to salivary proline-rich protein 1 (PRP1) and statherin. A series of synthetic fimbrillin peptides were used to localize the active fimbrillin domains involved in the binding to PRP1 and statherin. The binding of 125I-labeled 41-r-Fim (whole-length recombinant fimbrillin, amino acid [aa] residues 1 to 337) to PRP1-coated hydroxyapatite beads (HAP) was strongly inhibited by the fimbrillin C-terminal peptides corresponding to aa residues 266 to 286 and 318 to 337 (peptides 266-286, and 318-337, respectively), while the binding to statherin was inhibited by C-terminal peptides 266-286, 293-306 and 307-326. Peptide 126-146 also showed a weak inhibitory effect, about half that of other active peptides, on the binding to both PRP1 and statherin. P. gingivalis whole-cell binding to PRP1- or statherin-coated HAP was inhibited by more than 80% by the same active peptides. To confirm that the C-terminal portion of fimbrillin includes domains responsible for the binding, two C-terminally truncated variants of recombinant fimbrillin were generated and purified. These were designated 34.5-r-Fim, corresponding to aa residues 1 to 286, and 32-r-Fim, corresponding to aa residues 1 to 265. 125I-34.5-r-Fim revealed 35 and 34% loss of binding ability to PRP1 and statherin, respectively. 125I-32-r-Fim had significantly less binding ability to PRP1 and statherin than 125I-34.5-r-Fim, which was reduced 78 and 73%, respectively. Whole-cell binding to PRP1-, statherin-, or whole saliva-coated HAP was inhibited up to 100% by 41-r-Fim, while 32-r-Fim also showed considerable inhibition, possibly due to the region of aa 126 to 146. Collectively, these results suggest that there are separate and multiple binding sites for PRP1 and statherin in the P. gingivalis fimbrillin, and the combination of all of these binding sites may be indispensable in establishing stable bacterial adherence to saliva-coated surfaces in the oral cavity. PMID- 8613372 TI - Upregulation of tumor necrosis factor alpha and interleukin-1 beta in Q fever endocarditis. AB - The occurrence of Q fever endocarditis likely involves some alterations in the responses of monocytes, the in vivo targets of Coxiella burnetii. To test this hypothesis, the production of the inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 was assessed in monocytes from patients with Q fever endocarditis. Spontaneous transcription and secretion of tumor necrosis factor and interleukin-1 were significantly higher in patient monocytes than in healthy controls. The interleukin-6 transcripts were also upregulated in patient cells. Moreover, in patients with recent endocarditis exhibiting high titers of immunoglobulin G directed to C. burnetii in phase I, monocytes released significantly higher levels of tumor necrosis factor and interleukin-1 than in patients with stabilized endocarditis. Immunoglobulin G titers and the overproduction of tumor necrosis factor and interleukin-1 were significantly correlated. Hence, the overproduction of inflammatory cytokines might be a marker of disease activity. PMID- 8613373 TI - Colonization in the rectum and uterine cervix with group B streptococci may induce specific antibody responses in cervical secretions of pregnant women. AB - We have studied the relationships between genital or rectal carriage of group B streptococci (GBS) with the levels of systemic and mucosal antibodies to GBS in 200 women at about week 17 of pregnancy. Secretions from the uterine cervix were collected with absorbent cylindrical wicks for quantification of antibody levels with whole cell enzyme-linked immunosorbent assay. GBS were cultured from the cervix (with or without concomitant rectal colonization) of 13.5%, from the rectum (with or without concomitant cervical colonization) of 12%, and from both culture sites of 8.5% of the women. Serotypes Ia, II, and III were predominant. Compared with culture-negative women, the group of women colonized rectally had markedly elevated levels of both immunoglobulin A (IgA) and IgG antibodies to GBS in cervical secretions and also had a moderate but significant elevation of IgA antibodies in sera. Women colonized only in the cervix had increases of specific IgA and IgG antibodies in cervical secretions, but their serum antibody levels were not elevated. In cervical secretions, the increase in antibody levels in the groups of colonized women was most pronounced for the IgG isotype, indicating a mucosal immune response involving IgG as well as IgA. A close correlation was found among the levels of antibodies to each of the three GBS serotypes tested. Evidence for such cross-reacting antibodies to different serotypes of GBS, as well as to group A streptococci, was also obtained from absorption experiments. Altogether, our results show that undiluted secretions for antibody determination can be easily collected from the uterine cervix with absorbent wicks and demonstrate that colonization of GBS in the rectum and the uterine cervix may induce a systemic as well as a pronounced local immune response in the female genital tract. The findings may have implications for the development of a mucosal vaccine against GBS disease. PMID- 8613374 TI - Role of YadA in resistance to killing of Yersinia enterocolitica by antimicrobial polypeptides of human granulocytes. AB - The virulence plasmid pYVe of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may protect this bacterium against killing by human granulocytes. Granulocytes kill ingested microorganisms by oxygen-dependent and oxygen independent mechanisms, the latter including antimicrobial polypeptides. The aim of this study was to determine whether virulent (pYVe+) Y. enterocolitica and plasmid-cured avirulent (pYVe-) Y. enterocolitica differ in susceptibility to antimicrobial polypeptides extracted from granules of human granulocytes. The acetic acid granule extract contained several polypeptides with antimicrobial activity against Y. enterocolitica as determined by gel overlay and radial diffusion assays. Two of these polypeptides were identified as lysozyme and defensins. pYVe+ Y. enterocolitica was less susceptible than pYVe- Y. enterocolitica to the antimicrobial activity of granule extract, lysozyme, and defensins as determined in a suspension assay, which indicated that the pYVe plasmid mediates a reduced susceptibility to these polypeptides. The role of YadA in the resistance to antimicrobial polypeptides was analyzed by using mutants of Y. enterocolitica that specifically lack or express YadA. The results demonstrated that YadA conferred resistance to the killing of Y. enterocolitica by the granule extract. Together, these results indicate that the plasmid-encoded factor YadA contributes to the resistance of Y. enterocolitica to the killing by antimicrobial polypeptides of human granulocytes. PMID- 8613375 TI - A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge. AB - The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections. PMID- 8613377 TI - An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies. AB - Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80 kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts. PMID- 8613376 TI - Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria. AB - NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria. PMID- 8613378 TI - De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells. AB - Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila. PMID- 8613380 TI - Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1. AB - The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E. coli BM2-1. This strain was shown to bind loosely, but massively, to HeLa cells. A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation. These alterations resulted in cell death starting about 5 days after interaction. The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum. Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter. These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation. On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE. In conclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells. PMID- 8613379 TI - A recombinant minigene vaccine containing a nonameric cytotoxic-T-lymphocyte epitope confers limited protection against Listeria monocytogenes infection. AB - We have previously shown that vaccines expressing virus-derived cytotoxic-T lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000 fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge. PMID- 8613381 TI - Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. AB - Intranasal or intramuscular (i.m.) immunization of mice and i.m. immunization of rabbits with formalinized staphylococcal enterotoxin B (SEB) toxoid in saline elicited higher anti-SEB serum immunoglobulin G (IgG) titers when the toxoid was formulated with proteosomes. In addition, intranasal immunization of mice with this proteosome-toxoid vaccine elicited high levels of anti-SEB IgA in lung and intestinal secretions, whereas the toxoid without proteosomes did not. Two i.m. immunizations with proteosome-toxoid plus alum also induced higher murine serum responses than alum-adjuvanted toxoid without proteosomes. Furthermore, proteosome-toxoid delivered intranasally in saline or i.m. with either saline or alum afforded significant protection against lethal SEB challenge in two D galactosamine-sensitized murine models of SEB intoxication, i.e., the previously described i.m. challenge model and a new respiratory challenge model of mucosal SEB exposure. Efficacy correlated with the induction of high serum levels of anti SEB IgG. In contrast, intranasal or i.m. immunization with toxoid in saline without proteosomes was not significantly protective in either challenge model. Proteosome-toxoid plus alum given i.m. also elicited more significant protection against respiratory challenge than the alum-adjuvanted toxoid alone. The capacity of proteosomes to enhance both i.m. and intranasal immunogenicity and efficacy of SEB toxoid indicates that testing such proteosome-SEB toxoid vaccines in the nonhuman primate aerosol challenge model of SEB intoxication prior to immunogenicity trials in humans is warranted. These data expand the applicability of the proteosome mucosal vaccine delivery system to protein toxoids and suggest that respiratory delivery of proteosome vaccines may be practical for enhancement of both mucosal and systemic immunity against toxic or infectious diseases. PMID- 8613383 TI - ADP-ribosylation of an approximately 70-kilodalton protein of Klebsiella pneumoniae. AB - An approximately 70-kDa protein in the culture supernatant of a human pathogenic strain of Klebsiella pneumoniae was labeled in the presence of [32P adenylate]NAD. Labeling was significantly increased by the addition of dithiothreitol ( > 1 mM) but prevented by treatment of the culture supernatant for 3 min at 56 degrees C. The addition of unlabeled NAD, but not of ADP-ribose, blocked labeling of the approximately 70-kDa protein. The radioactive label was released by formic acid but not by HgCl2 (1 mM) or neutral hydroxylamine (0.5 M). The addition of homogenates of human platelets, human neutrophils, rat brain, rat lung, or rat spleen tissues to the culture supernatant did not induce labeling of eukaryotic proteins. The data indicate that the K. pneumoniae strain produces ADP ribosyltransferase which modifies an endogenous protein. PMID- 8613382 TI - Interaction of verotoxin 2e with pig intestine. AB - In pigs with edema disease, verotoxin 2e (VT2e) is produced in the intestine and transported to tissues, but neither the mechanism by which toxin passes through the intestine nor its failure to induce an enterotoxic reaction is understood. Binding of VT2e to pig intestine was examined by enzyme-linked immunosorbent assay involving microvillus membranes (MVM) and crude mucus; thin-layer chromatographic overlay immunoassay with total lipids extracted from MVM; and indirect immunofluorescence of toxin bound to thin sections of jejunum, ileum, and colon. VT2e bound significantly to MVM from pig jejunum and ileum but not to crude mucus. Verotoxin 2e-binding glycolipids, globotetraosylceramide and globotriaosylceramide, were detected by thin-layer chromatographic overlay immunoassay in extracts of MVM from jejunum and ileum. Indirect immunofluorescence showed that VT2e bound to vessels within the submucosa and muscularis mucosa of the jejunum, ileum, and colon and to enterocytes at the lower portion but not at the tips of villi in the jejunum and ileum. Receptors for VT2e are therefore present in the intestine of the pig, but their role in absorption of VT2e is unclear since intraintestinal inoculation of pigs with large quantities of VT2e does not result in edema disease. Previously reported lack of enterotoxicity of verotoxins in pig intestine may be explained by the absence of toxin receptors in the villus absorptive enterocytes. PMID- 8613384 TI - A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model. AB - Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi. PMID- 8613385 TI - Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock. AB - The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope. PMID- 8613386 TI - Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by gamma/delta T lymphocytes. AB - Listeria monocytogenes is an intracellular bacterium which causes an acute infectious disease in mice. Initial host resistance depends on innate immunity mediated primarily by natural killer (NK) cells followed by specific alpha/beta T cells, which are central to acquired specific immunity. Gamma/delta T lymphocytes seem to provide a link between the innate and the specific immune response. All these lymphocyte populations produce gamma interferon (IFN-gamma), which, because of its macrophage-activating potential, is central to antibacterial protection. IFN-gamma from NK cells not only contributes to early host resistance but also promotes development of protective T-cell responses of helper T type 1 (Th1) type. Here, we show that innate resistance and early IFN-gamma production in listeriosis are markedly impaired in T-cell receptor (TCR)-delta-/- but not TCR beta-/- gene disruption mutant mice. By two-color cytofluorimetry, we demonstrate that NK cells rather than gamma/delta T lymphocytes are the major cellular source of IFN-gamma in immunocompetent mice and that IFN-gamma production by NK cells is impaired in the TCR-delta-/- mutants. Probably, reduced tumor necrosis factor production in listeria-infected TCR-delta-/- mutants contributed to impaired NK cell activation. Our data reveal a novel function of gamma/delta T cells as regulators of innate resistance against sublethal infection with an intracellular pathogen. PMID- 8613387 TI - A cloned major Schistosoma mansoni egg antigen with homologies to small heat shock proteins elicits Th1 responsiveness. AB - In schistosomiasis mansoni, soluble egg antigens of the worm induce chronic T cell-mediated granulomatous tissue responses. Since the first preparation of crude soluble egg antigen extract, a dearth of highly purified antigens has hampered the identification of granuloma inducer molecules. Here we report that a cloned 38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a Th1-type immune response, with strong interleukin-2 (IL-2) and gamma interferon secretion but no IL-4 and IL-10 secretion. Extensive cross-reactivity between these two polypeptides was evident. THis pattern was confirmed by reverse transcription-PCR that showed strong IL-2 and gamma interferon message expression but trace amounts of IL-4 message expression in r38-sensitized splenocytes. In mice, the polypeptide induced pulmonary mononuclear granuloma formation around antigen-coupled beads or worm eggs. We propose that the superior immunogenicity of r38 is linked to its relatedness to small heat shock proteins and that the 38 kDa polypeptide may induce the Th1 cytokine responses observed during the early development phase of the egg-induced granuloma. PMID- 8613389 TI - Human monocyte CD14 is upregulated by lipopolysaccharide. AB - Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first 3 h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5 fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with > or = 1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 micrograms/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor alpha, anti-interleukin-6, anti gamma interferon, and anti-LPS-binding protein. LPS-induced tumor necrosis factor alpha production was abolished after a second 4-h challenge. In contrast, the LPS induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents. PMID- 8613388 TI - Relative importance of three iron-regulated outer membrane proteins for in vivo growth of Vibrio cholerae. AB - Iron is an essential nutrient to support the growth of most bacterial species. However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme. In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced outer membrane proteins. Previous data on the role of iron acquisition systems for the intraintestinal growth of mucosal pathogens such as V. cholerae are conflicting. In this report, we isolated mutants of V. cholerae with TnphoA fusions in each of viuA, hutA, and irgA, as well as strains mutant in each pair of these genes and all three simultaneously, to analyze the role of these iron-induced outer membrane protein receptors for in vivo growth of V. cholerae. The fusion between hutA and TnphoA in a single copy on the chromosome allowed the study of in vitro regulation of hutA in response to iron, fur, and irgB; transcription of hutA was tightly iron regulated (70-fold) and dependent on a functional Fur but did not require IrgB. To investigate the effects of mutations in these iron-induced outer membrane proteins on in vivo growth, we inoculated ileal loops in a rabbit model of infection. This avoids exposure of organisms to the potential killing effects of gastric acid, allows several logarithmic increases in growth in the in vivo environment, and facilitates direct comparison of multiple strains in the same animal to avoid any differences between animals. We grew each mutant to be tested in competition with the wild type strain in the same loop, to provide an internal control. We confirmed that the inocula for these experiments were grown under conditions of iron stress prior to in vivo inoculation, by measuring the alkaline phosphatase activity of the iron-regulated fusion in each strain. The results confirmed that mutation of irgA produced a much more substantial in vivo growth defect than mutation of either hutA or viuA alone. Double mutants of irgA with either viuA or hutA, or the strain mutant in all three genes, showed an in vivo growth defect comparable to the strain mutant in irgA only, suggesting that mutation of irgA was the most relevant for in vivo growth. The strain mutant in both hutA and viuA was also markedly impaired for in vivo growth, suggesting that mutation of both of these iron uptake systems simultaneously can also produce a substantial in vivo growth defect. PMID- 8613390 TI - A factor from Trypanosoma cruzi induces repetitive cytosolic free Ca2+ transients in isolated primary canine cardiac myocytes. AB - An unusual 120-kDa alkaline peptidase contained in a trypomastigote soluble fraction (TSF) of Trypanosoma cruzi is associated with the induction of repetitive Ca2+ transients and subsequent invasion by the parasite of a number of mammalian cell lines, including tissue culture L6E2 myoblasts (B. A. Burleigh and N. W. Andrews, J. Biol. Chem. 270:5172-5180, 1995; S. N. J. Moreno, J. Silva, A. E. Vercesi, and R. Docampo, J. Exp. Med. 180:1535-1540, 1994; A. Rodriguez, M. G. Rioult, A. Ora, and N. W. Andrews, J. Cell Biol. 129:1263-1273, 1995; I. Tardieux, M. H. Nathanson, and N. W. Andrews, J. Exp. Med. 179:1017-1022, 1994). Using single cell spectrofluorometry and whole-cell patch clamping, we show that TSF produces rapid repetitive cytosolic Ca2+ transients (each associated with cell contraction) in primary cardiac myocytes isolated from dogs. The response of myocytes to TSF was dose dependent in that increasing numbers of cells responded to increasing concentrations of TSF. The TSF-induced Ca2+ transients could be obliterated when TSF was heated or treated with trypsin or the protease inhibitor leupeptin. Aprotinin, pepstatin A, and E-64 did not affect TSF activity. The TSF induced Ca2+ transients and trypomastigote cell invasion could not be inhibited by alpha (prazosin)- or beta (propanolol)-adrenergic blockers or L-type Ca2+ channel blockers (verapamil, nisoldipine, or cadmium) or by removal of extracellular Ca2+. However, inhibition of pertussis toxin-sensitive G proteins and Ca2+ release from the sarcoplasmic reticulum (with thapsigargin or ryanodine) prevented the TSF-induced Ca2+ transients and cell invasion by trypomastigotes. These data suggested that cardiac myocyte pertussis toxin-sensitive G proteins are associated with the regulation of TSF-induced Ca2+ transients and myocyte invasion by trypomastigotes but are independent of Ca2+ entry into the cytosol via L-type Ca2+ channels. The Ca2+ transients are dependent on release of Ca2+ from sarcoplasmic reticulum Ca2+ stores, but this release is not dependent on extracellular Ca2+ or on the classic model of Ca2+ -induced Ca2+ release in cardiac myocytes. Further, subthreshold depolarizations, together with cell contraction as demonstrated by whole-cell patch clamping, occurred with each Ca2+ transient. However, the depolarizations were of magnitude insufficient to generate an action potential, providing further evidence for a lack of dependence on L-type Ca2+ channels and other voltage-dependent channels (Na+ and K+ channels) in the generation of TSF-induced Ca2+ transients. Our findings suggest that primary canine cardiac myocytes respond to TSF and parasite invasion in ways similar to those of the in vitro cell lines studied to date. Since cardiac myocytes are primary targets for T. cruzi in the vertebrate host, our study indicates that TSF may play a role in the pathogenesis of Chagas' disease in humans. PMID- 8613392 TI - Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages. AB - We investigated the ability of a virulent wild-type parent strain and acapsular avirulent transposon mutants to enter and survive intracellularly within murine peritoneal macrophages. In the presence of normal or immune serum, the parent and mutant strains were both ingested; however, the number of ingested bacteria was three- to fourfold greater in the case of mutant strains than in the case of the parent strain. The parent strain, but not the mutant strains, survived and replicated intracellularly when ingested in the presence of normal serum, whereas both the parent and the mutant strains were readily killed when ingested in the presence of immune serum. To further investigate the mechanism by which the parent strain can survive and replicate within macrophages, we studied the oxidative burst response of macrophages to these strains by measuring chemiluminescence and intracellular reduction of Nitro Blue Tetrazolium dye. Challenge exposure of macrophages with either the parent strain preopsonized with immune serum or the mutant strains preopsonized with normal or immune serum induced a strong oxidative burst, whereas the level was very low when the parent strain was preopsonized with normal serum. Phagocytosis of either the parent strain, in the presence of immune serum, or the mutant strains, in the presence of normal or immune serum, by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium, whereas minimal amounts were reduced by the parent strain in the presence of normal serum. These results suggest that virulent E. rhusiopathiae can survive and subsequently replicate within murine macrophages when ingested in the presence of normal serum and that the reduced production of reactive oxidative metabolites by macrophages may, in part, be responsible for this occurrence. PMID- 8613391 TI - Evaluation of humoral and cell-mediated inducible immunity to Haemophilus ducreyi in an animal model of chancroid. AB - To study the mechanisms of inducible immunity to Haemophilus ducreyi infection in the temperature-dependent rabbit model of chancroid, we conducted passive immunization experiments and characterized the inflammatory infiltrate of chancroidal lesions. Polyclonal immunoglobulin G was purified from immune sera raised against H. ducreyi 35000 whole-cell lysate or a pilus preparation and from naive control rabbits. Rabbits were passively immunized with 24 or 48 mg of purified polyclonal immunoglobulin G intravenously, followed 24 h after infusion by homologous titered infectious challenge. Despite titratable antibody, no significant difference in infection or disease was observed. We then evaluated the immunohistology of lesions produced by homologous-strain challenge in sham immunized rabbits and those protectively vaccinated by pilus preparation immunization. Immunohistochemical stains for CD5 and CD4 T-lymphocyte markers were performed on lesion sections 4, 10, 15, and 21 days from infection. Lesions of pilus preparation vaccinees compared with those of controls had earlier infiltration with significantly more T lymphocytes (CD5+) and with a greater proportion of CD4+ T lymphocytes at day 4 (33% +/- 55% versus 9.7% +/- 2%; P = 0.002), corroborating earlier sterilization (5.0 +/- 2 versus 13.7 +/- 0.71 days; P < 0.001) and lesion resolution. Intraepithelial challenge of pilus-vaccinated rabbits with 100 micrograms of the pilus preparation alone produced indurated lesions within 48 h with lymphoid and plasmacytoid infiltration, edema, and extravasation of erythrocytes. We conclude that passive immunization may not confer a vaccine effect in this model and that active vaccination with a pilus preparation induces a delayed-type hypersensitivity skin test response and confers protection through cell-mediated immunity seen as an amplified lymphocytic infiltrate and accelerated maturation of the T-lymphocyte response. PMID- 8613393 TI - Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages. AB - The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection. However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly. Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice. Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro. Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene. These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine. PMID- 8613394 TI - Antigenic topology of the P29 surface lipoprotein of Mycoplasma fermentans: differential display of epitopes results in high-frequency phase variation. AB - Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important mechanism for altering the antigenic or functional surface topology of this mycoplasma and other wall-less mycoplasmas. PMID- 8613395 TI - Developmental changes in the expression of Leishmania chagasi gp63 and heat shock protein in a human macrophage cell line. AB - The ability of the protozoan Leishmania chagasi to infect a vertebrate host depends on its ability to survive intracellularly in a mammalian macrophage. Novel patterns of gene expression are probably important for conversion from the extracellular promastigote to the obligate intracellular amastigote parasite form. We found that the human macrophage-like cell line U937 provided an in vitro model of phagocytosis of L. chagasi promastigotes and intracellular conversion to amastigotes, allowing examination of parasite protein and RNA expression. The Leishmania surface protease gp63 assumed three isoforms during stage conversion, and a 64-kDa form of gp63 not present in promastigotes became the most prominent form in amastigotes. gp63 RNAs derived from the three different classes of msp genes (mspS, mspL, and mspC) were also differentially expressed. Infectious promastigotes contained mRNAs from mspS and mspC genes, whereas converting parasites expressed only mspL and mspC mRNAs. Sequence analysis of clones from an amastigote cDNA library confirmed the presence of gp63 mRNAs only from mspL and mspC class genes in tissue-derived amastigotes. Finally, 24 h after phagocytosis, there was a transient increase in the level of hsp70 and hsp90 proteins that subsequently decreased to baseline; this increase was not due to heat shock alone. We conclude that a unique pattern of selected L. chagasi proteins and RNAs is induced following phagocytosis by macrophages. PMID- 8613396 TI - Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids. AB - Clinical isolates of Pseudomonas aeruginosa were examined for binding interactions with phospholipids of corneal epithelium. Thin-layer chromatography (TLC) of lipids extracted from corneal epithelia followed by staining with an ammonium molybdate spray reagent revealed three phospholipid components, PL1, PL2, and PL3. The chromatographic mobility of PL1 was similar to that of the phospholipid standards phosphatidylinositol (PI) and phosphatidylserine (PS), which were not well resolved from one other; PL2 and PL3 comigrated with the standards phosphatidylcholine and phosphatidylethanolamine, respectively. By use of a TLC-bacterial overlay procedure, 35S-labeled P. aeruginosa organisms were shown to bind to PL1 but not to PL2 or PL3. P. aeruginosa binding to PL1 was concentration dependent. Alkaline methanolysis abolished the binding. PL1 was separated into two components, PL1-I and PL1-S, by chromatography on borate treated TLC plates. Both PL1-I and PL1-S contained binding sites for P. aeruginosa. Mass spectral analysis identified PL1-I and PL1-S as PI and PS, respectively. Radiolabeled P. aeruginosa organisms were subsequently shown to bind to commercially available bovine PI and PS and synthetic dipalmitoyl-PS but not to other phospholipid standards, including bovine SM and PC or synthetic dioleoyl- and distearoyl-PC. A control Escherichia coli strain did not bind to either PS or PI. Tetramethylurea, a disrupter of hydrophobic associations, did not influence the binding of P. aeruginosa to PS or PI. P. aeruginosa bound to the monolayers of corneal epithelial cells. P. aeruginosa binding to the monolayer cultures as well as to rabbit corneas pretreated with exogenous PS and PI was significantly higher than that to those preincubated with PC or medium alone. The data suggest that phospholipids PS and PI present in mucus or on the cell surface may function as P. aeruginosa receptors and contribute to selective bacterium-host interactions responsible for initial colonization. PMID- 8613397 TI - Lacto-N-fucopentaose III (Lewis x), a target of the antibody response in mice vaccinated with irradiated cercariae of Schistosoma mansoni. AB - Carbohydrates on soluble egg antigens are major epitopes for the antibody responses of patients and mice infected with Schistosoma mansoni. Recently, protective sera of mice vaccinated with irradiated cercariae were shown to recognize carbohydrate epitopes on schistosomal glutathione S-transferase. The present study demonstrates that carbohydrate epitopes are major targets of sera from C57BL/6J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni. Antibody titers to carbohydrate epitopes increased with the number of vaccinations and were considerably higher in C57BL/6J mice than in CBA/J mice. The specificity of this anticarbohydrate response was determined by measuring antibody binding to defined oligosaccharide residues known to be present on the parasite. A predominant target of the humoral anticarbohydrate response of vaccinated mice was lacto-N-fucopentaose III, a molecule relevant for cell trafficking. We observed no binding to its nonfucosylated homolog, lacto-N neotetraose, or to oligosaccharides present on keyhole limpet hemocyanin. The strongest antibody response to lacto-N-fucopentaose III was observed for C57BL/6J and CBA/J mice repeatedly vaccinated with 15-kilorad-irradiated cercariae, which also achieve the highest levels of protection. Immunoglobulin M was the predominant antibody class binding to lacto-N-fucopentaose III. We conclude that in the irradiated-cercariae vaccine model, C57BL/6J and CBA/J mice produce anticarbohydrate antibodies against various stages of S. mansoni and that the oligosaccharide lacto-N-fucopentaose III is one target of this response. Lacto-N fucopentaose III and its specific antibodies may profoundly affect host resistance and parasite homing. PMID- 8613399 TI - Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model. AB - A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies. PMID- 8613398 TI - Identification of neutralization and diagnostic epitopes on PIM, the polymorphic immunodominant molecule of Theileria parva. AB - The polymorphic immunodominant molecule (PIM) of Theileria parva is expressed by the schizont and sporozoite stages of the parasite. We have recently cloned the cDNA encoding the PIM antigen from two stocks of the parasite: the cattle-derived T. parva (Muguga) stock and a buffalo-derived stock. The cDNAs were used in transient-transfection assays to assess the reactivity of the antigen with monoclonal antibodies (MAb) previously raised against schizont-infected cells and used for parasite strain identification. We demonstrate that 19 of the 25 MAb are specific for PIM. Antibody reactivities with deletion mutants of a fusion protein containing PIM and Pepscan analysis of the Muguga version of the molecule with 13 of the MAb indicate that there are at least 10 different epitopes throughout the molecule. None of the MAb react with a tetrapeptide repeat present in the central region of the molecule, probably because of an inability of BALB/c mice to produce antibodies to this repeat. In contrast, sera from infected cattle react strongly with the repeat region, suggesting that this region alone may be useful as a diagnostic reagent. Previous studies showed that MAb to PIM inhibit sporozoite infectivity of bovine lymphocytes in vitro, which suggests that the antigen may be useful in immunizing cattle against T. parva infection. Pepscan analysis revealed that sera from infected cattle reacted with peptides recognized by the neutralizing MAb, as did sera from cattle inoculated with a PIM-containing recombinant protein. The latter sera did not, however, neutralize sporozoite infectivity in vitro. These results will be useful in exploiting the strain identification, diagnostic, and immunizing potentials of this family of antigens. PMID- 8613400 TI - Spectrum of Legionella species whose intracellular multiplication in murine macrophages is genetically controlled by Lgn1. AB - We examined the intracellular growth of 20 strains within six species of Legionella in thioglycolate-elicited peritoneal macrophages from A/J and C57BL/6 mice and a congenic strain derived from them (A.B Lgn1). With the exception of Legionella pneumophila Togus-1 and Bloomington-2, the intracellular growth of the 15 L. pneumophila strains tested was controlled by Lgn1. However, the intracellular growth of five Legionella species other than L. pneumophila was not under Lgn1's control. PMID- 8613401 TI - Streptococcus mitis cell walls and lipopolysaccharide induce lethality in D galactosamine-sensitized mice by a tumor necrosis factor-dependent pathway. AB - Purified cells walls of Streptococcus mitis induced tumor necrosis factor in vitro in whole blood of both lipopolysaccharide (LPS)-sensitive OF1 and LPS resistant C3H/HeJ mice. They were as effective as heat-killed bacteria in inducing death in both strains of mice sensitized with D-galactosamine. Lethality was suppressed by anti-tumor necrosis factor antibodies. The histopathophysiological findings in mice after challenge with LPS or gram positive cell walls were indistinguishable. PMID- 8613403 TI - Cryptosporidium parvum infection in T-cell receptor (TCR)-alpha- and TCR-delta deficient mice. AB - Mice deficient in either alpha beta or gamma delta T cells were more susceptible to infection with the protozoan parasite Cryptosporidium parvum than were control mice. Both neonatal and adult alpha beta-T-cell-deficient mice developed chronic infection. Gamma delta-T-cell-deficient neonatal mice were more susceptible than control mice but were able to clear the infection. Adult gamma delta-T-cell deficient mice were not susceptible to infection. These data indicate that alpha beta T cells are important for resistance to C. parvum while gamma delta T cells have a less critical role. PMID- 8613402 TI - Cloning, sequencing, and expression of a gene from Campylobacter jejuni encoding a protein (Omp18) with similarity to peptidoglycan-associated lipoproteins. AB - A Campylobacter jejuni genomic plasmid library was screened with antiserum generated against whole C. jejuni, revealing two immunoreactive clones. Sequence analysis of the recombinant plasmids revealed a common open reading frame of 498 nucleotides encoding a protein of 165 amino acids with a calculated molecular mass of 18,018 Da. The recombinant product partitioned to the outer membrane fractions of Escherichia coli transformants and has been designated Omp18. The deduced amino acid sequence of the cloned C. jejuni gene exhibits considerable similarity to peptidoglycan-associated lipoproteins from other gram-negative bacteria. PMID- 8613404 TI - Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells. AB - In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors. PMID- 8613405 TI - Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium. AB - The role of the Salmonella typhimurium fimbrial operons, lpf, fim, and pef, in adhesion to and invasion of epithelial cell lines was investigated. An S. typhimurium lpfC mutant was unable to adhere to or to invade HEp-2 cells, while an S. typhimurium fim deletion mutant did not attach to or enter HeLa cells. These results suggest that adhesion is a prerequisite for invasion and that distinct fimbrial adhesins select different target cells for invasion by S. typhimurium. PMID- 8613406 TI - Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load. AB - After systemic infection with the yeast Candida albicans, inbred mice show substantial differences in mortality, organ colonization, and severity of tissue damage. To examine the relationships between these variables, which are not directly correlated with each other, fungal colonization of the kidneys and brain was enumerated in six inbred strains that exhibit different patterns of tissue damage and mortality. Mice lacking the fifth component of complement (C5) are highly susceptible to lethal challenge, and A/J and DBA/2 mice, both C5 deficient, showed the highest colony counts in the kidneys after challenge with 10(5) blastoconidia. In contrast, colony counts in the brain of all six strains were equivalent at this challenge dose. A/J and DBA/2 mice died after challenge with 3 x 10(5) blastoconidia, but other strains showed an increase in kidney colonization, and strain-dependent differences in clearance from the brain became evident. The data suggest that mortality in A/J and DBA/2 mice is related to an unusual susceptibility of the kidneys to colonization by C. albicans and that there may be tissue-specific differences in host protective mechanisms. PMID- 8613407 TI - Peptide epitopes from noncytosolic Listeria monocytogenes can be presented by major histocompatibility complex class I molecules. AB - Listeria monocytogenes is an intracellular pathogen which escapes the phagosome and resides in the cytosol of the host cell. Using Listeria innocua and a mutant strain of L. monocytogenes (listeriolysin O negative), which do not enter the cytosol of the host cell, we demonstrate class I presentation of an epitope of p60, a protein secreted by L. monocytogenes, to a class I-restricted CD8+ cytotoxic T lymphocyte clone. PMID- 8613408 TI - Absence of siderophore-like activity in Legionella pneumophila supernatants. AB - Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore. Hence, we rigorously examined the reported siderophore like activity using the chrome azurol S indicator. Although chrome azurol S reactivity was detected in supernatants, control experiments indicate that it was due to cysteine in the media. When bacteria were cultured in media without cysteine, no siderophores were detected. PMID- 8613409 TI - Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus. AB - The adherence characteristics in vivo and virulence of two isogenic strains of Staphylococcus aureus differing in fibronectin binding were compared in a rat model of catheter-induced infective endocarditis. No differences were found between the two strains. The results strongly point to the multifactorial nature of bacterial adherence to damaged heart valves and suggest that other binding functions can compensate for the lack of fibronectin binding in S. aureus. PMID- 8613410 TI - Serum antibody responses of cattle following experimental infection with Escherichia coli O157:H7. AB - Oral inoculation of calves and steers with 10(10) CFU of Escherichia coli O157:H7 induced prompt and sustained increases in serum antibodies to O157 lipopolysaccharide. Neutralizing antibodies to verotoxin 1 also increased rapidly in most steers but more gradually in calves. None of the animals developed neutralizing antibodies to verotoxin 2. These serological responses were not correlated with elimination of infection in calves or steers or protection of calves against reinfection with the same strain. PMID- 8613411 TI - Magnetic resonance signal abnormalities along the pyramidal tracts in amyotrophic lateral sclerosis. AB - Magnetic resonance imaging (MRI) studies of the brain were reviewed in 16 patients with amyotrophic lateral sclerosis (ALS), representative of a large and homogeneously studied series, 11 of whom showed signal abnormalities along the pyramidal tracts. These were more frequent in patients with more severe upper motor neuron signs but did not correlate with disease severity. Our study suggests that MRI signal abnormalities along the pyramidal tracts are common in ALS and may reflect the severity of pyramidal tract degeneration. PMID- 8613412 TI - Variable response of the Mongolian gerbil to unilateral carotid occlusion: magnetic resonance imaging and neuropathological characterization. AB - In the present investigation, we estimated both the evolution and the severity of ischemic damage following unilateral carotid occlusion (UCO) in Mongolian gerbils by using conventional magnetic resonance imaging (MRI, i.e. T2 weighted imaging) and histological techniques. Immediately after UCO, the animals showed different clinical effects. The mortality (46%) detected within the first 48h was considered an "stroke-sensitivity", the "stroke-resistant" animals showed wide variability in terms of both temporal evolution and the extent of ischemic damage. The signal hyperintensity and negative MRI observed during the first 30h after UCO did not always correlate with the cerebral damage presented after 14 days, although a close correlation was established between the T2 weighted images taken more than 30h after UCO and neuropathology: the gerbils negative to imaging showed no morphological changes, whereas an enhanced signal was always prognostic of ischemic injury. Moreover, late MRI documented ventricular dilatation. Histopathology showed that the ischemic damage differed among the stroke resistant gerbils and was often bilateral. The present study confirms the differences in gerbil susceptibility to hemispheric infarction after permanent UCO and suggests that conventional MRI may be a useful non-invasive method for i) identifying the stroke-resistant animals prone to mature ischemic injury and ii) monitoring the evolution of therapeutic efficacy without sacrificing animals. PMID- 8613413 TI - Primary intracranial hypotension: pathogenetic and neuroradiological considerations. AB - Primary or spontaneous intracranial hypotension (SIH) is a rare syndrome which causes postural headache associated with spinal fluid hypotension. We report three cases of SIH, characterised on magnetic resonance imaging (MRI) by pachymeningeal enhancement not only at cerebral level, but also in the cervical spinal cord, which subsequently resolved completely and spontaneously. We discuss the possible pathogenetic mechanisms of the dural alterations and underline the radiological aspects. PMID- 8613414 TI - Lowering body temperature with a cooling suit as symptomatic treatment for thermosensitive multiple sclerosis patients. AB - A cooling system (Mark VII Microclimate System) was used to give six thermosensitive multiple sclerosis patients two 45-minute daily coolings for a period of one month. Before the first cooling, a baseline clinical and electrophysiological examination was performed. The same tests were repeated after the first application and after the thirtieth cooling day, thus providing information relating to acute and chronic efficacy. A clinical improvement was observed after both acute and, more unexpectedly, chronic cooling, whereas a significant improvement in central somatosensory conduction was recorded only under acute conditions. Our data suggest that cooling with this device leads to an improvement in some functional performances (mainly fatigue and strength) of about two hours' duration in thermosensitive patients. PMID- 8613415 TI - Topographic EEG mapping of 3/s spike-and-wave complexes during absence seizures. AB - Topographic color mapping has recently been introduced for the study of ictal EEG manifestations of absences. We recorded 2-4 3/s spike-and-wave (sw) bursts in 12 patients with absence epilepsy, and performed a spectral analysis of the EEG under baseline conditions and during the 2 sec preceding the 3/s sw bursts. An increase in delta and theta bands was found in preseizure conditions. Moreover, the serial mapping of the sw complexes showed a different field distribution of the various components when the first sw was compared with the subsequent ones. In particular, the negative peak of the spike was mainly frontal during the first complex, with a tangentially oriented dipole, but it became better represented over the midline in the following sw, with a radially oriented dipole. We conclude that EEG changes can be detected before the onset of 3/s sw activity in absences, and that they are probably related to the strong inhibitory mechanisms acting in this type of seizure; moreover a frontal cortical mechanism seems to be prevalently active at the beginning of the seizure, which is soon captured by a cortico-subcortical process as the attack progresses. PMID- 8613416 TI - Parallel fluctuations of psychiatric and neurological symptoms in a patient with multiple sclerosis and bipolar affective disorder. AB - The case of a female patient affected by simultaneously onsetting multiple sclerosis and bipolar affective disorder at age 33 is reported. Over the following years, the patient displayed minor mood fluctuations but, at the ages of 41 and 42 years, respectively, she suffered from a major depressive and a manic episode, both of which were concomitant with a marked worsening in her neurological condition. PMID- 8613417 TI - Transient hyperacute allodynia in Schneider's syndrome: an irritative genesis? AB - A case of hyperacute allodynia in a patient with Schneider's syndrome is described. Allodynia was completely relieved by surgery: an irritative genesis could explain such a finding. Central pain ensued a short time later. PMID- 8613418 TI - Dyskinesia of vascular origin. Clinical data and response to therapy in 7 cases. AB - Seven patients with dyskinesia due to cerebrovascular lesions are described. They presented hemichorea, hemiballism and focal dystonia; the site(s) of the lesion responsible, as defined by MRI (magnetic resonance imaging) or CT (computerized tomography) scan were the putamen, the caudatus, the thalamus and the subthalamic body of Luys. Data of similar cases in the literature are reviewed with reference to the location of the responsible lesions, which can aid in predicting the outcome of illness or prescribing treatment. PMID- 8613420 TI - Unusual pattern of mental deterioration in a patient with signs of corticobasal degeneration. PMID- 8613419 TI - Reliability of death certificates in the study of stroke mortality. A retrospective study in a Sicilian municipality. AB - The subjects who died in the Sicilian municipality of Riposto between 1985 and 1992, and whose death certificates reported diagnoses of cerebrovascular disease, were re-evaluated with the aim of verifying the reliability of the certificates themselves. The relatives of the deceased were interviewed to confirm or exclude stroke, and about 35% of the cases proved to be false positives. Among the causes reported on the death certificates, "stroke", "cerebral hemorrhage" and "cerebral thrombosis" presented the smallest number of false positives. Our results show that the sensitivity and specificity of the death certificates was poor, and there would also seem to be a large number of false negatives. However, the official mortality rates for cerebrovascular disease are probably not very far from the truth. PMID- 8613421 TI - Fak and focal adhesions. PMID- 8613422 TI - The U.S.-Japan Cooperative Cancer Research Program: some highlights of seminars, interdisciplinary program area, 1981-1996. AB - Thirty-one seminars have been held in the 16 years since 1981. A principal interest from the beginning was the genetics of cancer, well before this subject became widely popular. This interest arose in part because of marked binational differences in type-specific cancer rates, such as the very low rates among Japanese for Hodgkin's disease in the young, testicular cancer, Ewing's sarcoma, superficial spreading melanoma, chronic lymphocytic leukemia, and Wilms' tumor (half the U.S. frequency). Three seminars were devoted to the seeming reciprocal relationship between B-cell lymphoma (low in Japan) and certain autoimmune diseases (high in Japan), which is perhaps similar in origin to the male/female differences in the rates for these diseases. A seminar on Li-Fraumeni syndrome led to the recognition of cases among Japanese pedigrees brought to the meeting, and generated a study of its occurrence in Japanese families with adrenocortical carcinoma in a child. Another seminar revealed a marked clustering of rare cancers in Werner's (premature aging) syndrome in Japan, and led to a binational study and analysis of case-reports worldwide. Three seminars on pathology heightened appreciation of the importance of subclassifying cancer by subsite and subtype for racial and other comparisons. Four seminars on biostatistics in cancer research generated a substantial exchange of specialists and trainees in this field. PMID- 8613423 TI - Induction of OX40, a receptor of gp34, on T cells by trans-acting transcriptional activator, Tax, of human T-cell leukemia virus type I. AB - gp34, which we had identified as a target molecule of the trans-activation by Tax of human T-cell leukemia virus type I (HTLV-I), has been found to bind OX40, a member of the tumor necrosis factor receptor family, resulting in growth stimulation of activated T cells. We here demonstrate that not only gp34 (OX40L), but also OX40 can be transcriptionally activated by Tax. Three Tax-producing human T-cell lines carrying the HTLV-I genome expressed OX40 on their surfaces. Furthermore, Tax-induced transcriptional activation of OX40 was shown in Tax inducible JPX-9 cells. These results demonstrate that both OX40 and its ligand (gp34) are constitutively expressed on the surfaces of Tax-expressing T lymphocytes, suggesting that the OX40L/OX40 system contributes to growth stimulation of the virus-infected T cells. PMID- 8613424 TI - Second primary neoplasms in thyroid cancer patients. AB - To determine risk patterns for second primary neoplasms after the occurrence of thyroid cancer, we conducted a retrospective cohort study of 3321 thyroid cancer patients who were operated and histologically confirmed at the Noguchi Thyroid Clinic and Hospital Foundation between 1946 and 1985. They were followed from the date of operation through the end of 1990 with an observation period from 45 to 5 years. The average observation period of the patients was 13.4 years and the follow-up rate reached 98%. The standardized mortality ratio (SMR) was computed to assess possible risk increase by cancer site. In this computation, the time period less than 5 years after operation was omitted to reduce the influence of deaths related to the original thyroid cancer. A total of 103 deaths from malignant neoplasms other than thyroid cancer were observed during this time period (SMR = 1.6, 95% confidence interval [CI] = 1.3-2.0). Analyses of site specific cancer mortality revealed significantly elevated risks for the central nervous system (SMR = 16.1, CI = 5.2-37.6) and respiratory organs (SMR = 2.6, CI = 1.5-4.1). Based on a review of available medical records with histological findings, we concluded that the risk increases for these sites were most likely to be attributable to second primary neoplasms. Whether or not the patients had received radiotherapy was not significantly associated with elevated risk. Further investigations are needed to clarify the risk factors responsible for the above findings. PMID- 8613425 TI - Modifying influence of swine-serum-induced liver fibrosis on development of preneoplastic lesions in rat liver. AB - Modifying effects of fibrosis or a cirrhotic state, caused by treatment with swine serum (SS), on the induction of preneoplastic focal lesions were assessed in a rat medium-term liver bioassay model for the detection of environmental carcinogens, in which the test compound is administered during the promotion phase after initiation with diethylnitrosamine. In experiment I, repeated intraperitoneal administration of SS concomitantly with the hepatopromoting agent deoxycholic acid (DCA) or phenobarbital (PB) resulted in a cirrhotic state and a significant increase in the number or size of preneoplastic glutathione S transferase placental form (GST-P)-positive liver cell foci as compared to the corresponding DCA or PB alone groups. In experiment II, SS was given prior to commencement of the same medium-term bioassay system, in which a known hepatopromoting agent, DCA, 17-alpha-ethynylestradiol, or 2-acetylaminofluorene, was applied. In this case, the liver did not show obvious cirrhotic change and, rather than any enhancement, slight inhibition of promotion occurred. The results indicate that a coexisting, but not a pre-existing, cirrhotic condition acts to increase growth pressure on GST-P+ preneoplastic foci, and suggest that concomitant administration of SS with the promoting agent could be applied to improve the sensitivity of the assay protocol. PMID- 8613426 TI - Analysis of early initiating event(s) in radiation-induced thymic lymphomagenesis. AB - Since the T cell receptor rearrangement is a sequential process and unique to the progeny of each clone, we investigated the early initiating events in radiation induced thymic lymphomagenesis by comparing the oncogenic alterations with the pattern of gamma T cell receptor (TCR) rearrangements. We reported previously that after leukemogenic irradiation, preneoplastic cells developed, albeit infrequently, from thymic leukemia antigen-2+ (TL-2+) thymocytes. Limited numbers of TL-2+ cells from individual irradiated B10.Thy 1.1 mice were injected into B10.Thy 1.2 mice intrathymically, and the common genetic changes among the donor type T cell lymphomas were investigated with regard to p53 gene and chromosome aberrations. The results indicated that some mutations in the p53 gene had taken place in these lymphomas, but there was no common mutation among the donor-type lymphomas from individual irradiated mice, suggesting that these mutations were late-occurring events in the process of oncogenesis. On the other hand, there were common chromosome aberrations or translocations such as trisomy 15, t(7F;10C), t(1A;13D) or t(6A;XB) among the donor-type lymphomas derived from half of the individual irradiated mice. This indicated that the aberrations/translocations, which occurred in single progenitor cells at the early T cell differentiation either just before or after gamma T cell receptor rearrangements, might be important candidates for initiating events. In the donor type lymphomas from the other half of the individual irradiated mice, microgenetic changes were suggested to be initial events and also might take place in single progenitor cells just before or right after gamma TCR rearrangements. PMID- 8613427 TI - Genetic and epigenetic resistance of SL/Ni mice to lymphomas. AB - The murine spontaneous B lymphoma is etiologically related to the expression of endogenous ecotropic murine leukemia virus (ETV). Although both SL/Kh and SL/Ni mouse strains show a high level of expression of ETV from early in life, the former is a pre-B lymphoma-prone strain and the latter is rather lymphoma resistant. In order to identify the host background difference related to the lymphomagenesis, we performed a genetic cross study between these two strains. In the reciprocal F1 generation, the length of the lymphoma latent period was slightly but significantly longer in (SL/Ni xSL/Kh)F1 than in (SL/KhxSL/Ni)F1(P < 0.05). The incidence of overall lymphomas and that of acute pre-B lymphomas was lower in (SL/NixSL/Kh)F1 than in (SL/KhxSL/Ni)F1, although the difference was not statistically significant. These observations indicate that an epigenetic maternal resistance mechanism of SL/Ni mice plays a role in the lymphoma resistance. Furthermore, in the backcross combinations without maternal influence of SL/Ni, we observed a genetic mechanism of lymphoma resistance: an SL/Ni derived recessive lymphoma-resistance gene mapped in the proximal segment of Chr. 4. We named this gene nir-1 (SL/Ni-lymphoma resistance-1). Thus, we have demonstrated epigenetic and genetic mechanisms of lymphoma resistance of the SL/Ni mouse with the high expression of endogenous ETV. PMID- 8613428 TI - Genomic organization of human papillomavirus type 18 in cervical cancer specimens. AB - It has been reported that cervical cancers positive for human papillomavirus (HPV) 18 have a poorer prognosis than those with other HPV types. To gain a better understanding of the aggressive character of HPV 18-positive cancers, we examined the difference in genomic organization between HPV 18 and HPV 16 harbored in cervical cancers. We amplified E1 and E2 genes from 9 HPV 18-positive and 31 HPV 16-positive cervical cancers by polymerase chain reaction (PCR). At least one of the two early genes was missing in 3 out of 9 HPV 18-positive cancers, while both PCRs were positive in all 31 HPV 16-positive cancers (P < 0.05). We then analyzed the 9 HPV 18-positive cancers by 15 contiguous polymerase chain reactions covering collectively the whole HPV 18 genome. In addition to the three with a deletion of the E1 or E2 gene, one had a deletion in the E5 and L2 genes and one had an insertion in the long control region. The frequent alterations in genomic organization, especially preferential deletion of the E1 or E2 gene, may be related to the more aggressive properties of HPV 18-positive cervical cancers. PMID- 8613429 TI - Establishment of an undifferentiated leukemia cell line (Kasumi-3) with t(3;7)(q27;q22) and activation of the EVI1 gene. AB - A novel human leukemia cell line (Kasumi-3) was established from the blast cells of a 57-year-old man suffering from myeloperoxidase-negative acute leukemia. The cell line had five distinctive features, as follows. 1) Flow cytometric analyses showed cell surface expression of CD7, CD4, CD13, CD33, CD34, HLA-DR and c-Kit. This phenotype is compatible with that of acute myelocytic leukemia cells with the M0 subtype in the French-American-British classification. 2) Kasumi-3 cells carried chromosomal abnormalities of t(3;7)(q27:q22), del(5)(q15), del(9)(q32), and add(12)(p11). The breakpoint of 3q27 was located near the EVI1 gene, and a high level of expression of the EVI1 gene was observed. 4) Kasumi-3 cells treated with TPA showed maturation to monocytic lineage. 5) Treatment with either interleukin (IL)-2, IL-3, IL-4, granulocyte-macrophage colony-stimulating or stem cell factor induced the proliferation of Kasumi-3 cells. Thus, the Kasumi-3 cell line shows the characteristic features of undifferentiated leukemia. It should, therefore, be useful both for studying the biological characteristics of acute myelogenous leukemia M0 subtype and for investigating the role of the EVI1 gene in leukemogenesis. PMID- 8613430 TI - Absence of a mutation of the p21/WAF1 gene in human lung and pancreatic cancers. AB - The expression of a negative regulator of the cell cycle, p21WAF1 protein, is trans-activated by wild-type p53, but not by the mutant protein. Therefore, mutations of the p53 and WAF1 genes may be complementary. We examined DNAs from 70 human primary lung (63 of NSCLC and 7 of SCLC) and 24 pancreatic cancers (19 primary cancers and 5 cell lines) for mutations of the WAF1 gene. No mutations were detected in any samples examined, regardless of the mutational state of the p53 gene. The results suggested that aberrations of the coding sequence of the WAF1 gene are not associated with carcinogenesis in lung and pancreatic cancers. PMID- 8613431 TI - Loss or somatic mutations of hMSH2 occur in hereditary nonpolyposis colorectal cancers with hMSH2 germline mutations. AB - Hereditary nonpolyposis colorectal cancer (HNPCC) is a major cancer susceptibility syndrome known to be caused by the inheritance of mutations in DNA mismatch repair genes, such as hMSH2, hMLH1, hPMS1 and hPMS2. To investigate the role of genetic alterations of hMSH2 in HNPCC tumorigenesis, we analyzed 36 Japanese HNPCC kindreds as to hMSH2 germline mutations. Moreover, we also examined somatic mutations of hMSH2 or loss of heterozygosity at or near the hMSH2 locus in the tumors from the hMSH2-related kindreds. Germline mutations were detected in five HNPCC kindreds (5/36, 14%). Among them, three were nonsense mutations, one was a frameshift mutation and the other was a mutation in an intron where the mutation affected splicing. Loss of heterozygosity in four and somatic mutations in one were detected among the eight tumors with hMSH2 germline mutations. All these alterations were only detected in genomic instability(+) tumors, i.e., not in genomic instability(-) ones, indicating that mutations of hMSH2 were responsible for at least some of the tumors with genomic instability. These data establish a basis for the presymptomatic diagnosis of HNPCC patients, and constitute further evidence that both DNA mismatch repair genes and tumor suppressor genes may share the same requirement, i.e., two hits are necessary to inactivate the gene function. PMID- 8613432 TI - Expression of thymidine phosphorylase in human gastric carcinoma. AB - The activity of thymidine phosphorylase (dThdPase) has been reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immunostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a x400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P < 0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P < 0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P < 0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P < 0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P < 0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P < 0.001) or lymph node metastasis (P = 0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease. PMID- 8613433 TI - Distribution of transforming growth factor-beta and its receptors in gastric carcinoma tissue. AB - The distribution of the three mammalian isoforms of transforming growth factor (TGF)-beta (TGF-beta 1, -beta 2, and -beta 3) as well as their signaling receptors, TGF-beta type I and type II receptors (T beta R-I and T beta R-II, respectively), in gastric carcinoma tissue was examined by immunohistochemistry using specific antibodies. Tissue specimens were obtained from 25 cases of gastric carcinoma, which were classified into two groups according to Lauren's classification, i.e. 15 cases of diffuse carcinoma and 10 cases of intestinal carcinoma. In normal gastric mucosa apart from carcinoma nests, all of TGF-beta 1, -beta 2, -beta 3, T beta R-I and T beta R-II were clearly demonstrated in fundic glands. In sharp contrast, none of them was detectable in surface mucous cells. In carcinoma cells, strong staining for TGF-beta 1, -beta 2 and -beta 3 was obtained only in diffuse-type carcinoma. In particular, carcinoma cells scattered as single cells or small nests had a tendency to show strong staining for TGF-betas. The receptors tended to be distributed concomitantly with the ligands, and diffuse-type carcinoma showed stronger receptor staining than intestinal-type carcinoma. In cancer stroma, TGF-betas and receptors were detected in both diffuse and intestinal types, but the area with positive staining was wider and more dispersed in diffuse-type carcinoma than in intestinal carcinoma. These results suggest that TGF-beta may contribute in part to the variety of histogenesis and mode of progression of gastric carcinoma. PMID- 8613435 TI - Sonodynamically induced antitumor effect of a gallium-porphyrin complex, ATX-70. AB - The sonodynamically induced antitumor effect of a gallium-porphyrin complex, ATX 70, was evaluated in mice bearing colon 26. In order to find the optimum timing for the ultrasonic exposure after the administration of ATX-70, the ATX-70 concentrations in the plasma, skin, and tumor were measured and analyzed. Antitumor effect was estimated by measuring the tumor size. When used alone, ultrasound showed a slight antitumor effect, which became increasingly significant as the dose of ATX-70 was increased, while use of ATX-70 alone had no significant effect. At an ATX-70 dose of 2.5 mg/kg or higher, the average tumor size decreased to smaller than a half by three days after the ultrasonic exposure. This was smaller than a third of the size of the untreated tumors on the same day. From these results, it is concluded that ATX-70 significantly sensitizes tumors to ultrasound, demonstrating a synergistic antitumor effect. PMID- 8613434 TI - Flat serrated adenomas of the colorectal mucosa. AB - A total of 47 flat serrated neoplasias of the colorectal mucosa are presented: 44 were flat serrated adenomas and the remaining 3 flat serrated adenocarcinomas arising in flat serrated adenomas. These lesions were found among 600 flat mucosal lesions removed at colonoscopy during a 3-year period (1992 and 1994) at the Karolinska Hospital. Thirty-five of the 47 patients (74%) were males and the remaining 12 (26%), females. Depending upon the degree of cellular dysplasia within the epithelium, serrated adenomas were divided into those with low-grade dysplasia (LGD), when the dysplastic nuclei were present in the deeper half of the epithelium, and those with high-grade dysplasia (HGD), when the dysplastic nuclei were found even in the upper half of the epithelium. LGD was present in 37 (84.1%) of the 44 serrated adenomas and HGD in the remaining 7 (15.9%). Depending upon the topographic distribution of the dysplastic epithelium within the crypts, flat serrated adenomas were divided into type I, when the dysplastic epithelium was limited to the lower half of the serrated crypts, and type II, when the dysplastic epithelium was even present in the superficial half of the serrated crypts. Of the 44 serrated adenomas, 38 (86.1%) were type I and the remaining 6 (13.9%) type II. The dysplastic epithelium seemed to originate at the base of the crypts and to progress upwards, replacing the scalloped, serrated epithelium of the sides of the crypts. Invasive adenocarcinomas (i.e., with submucosal extension) were seen to arise from flat serrated adenomas with LGD type I (n = 2) or with HGD type II (n = 1). This preliminary survey suggests that flat serrated adenomas of the colorectal mucosa may be lesions with a propensity to evolve into invasive adenocarcinoma, irrespective of the degree of the epithelial dysplasia or of their extension along the crypts. PMID- 8613437 TI - Hydroxyapatite-coated femoral stems. A matched-pair analysis of coated and uncoated implants. AB - Fifty-two pairs of patients who had had a total hip arthroplasty with a porous coated femoral implant were studied in a retrospective, matched-pair analysis. Half of the patients had received a femoral component coated with hydroxyapatite and the other half (the controls), an identical component but without hydroxyapatite. The patients were matched for age, sex, weight, diagnosis, Charnley class, operative approach, and duration of follow-up. Identical uncoated hemispherical acetabular implants were used in both groups. At the time of follow up, at a mean of 2.2 years (range, two to 3.4 years) after the operation, the mean Charnley scores for pain, function, and motion were 5.6, 5.5, and 5.6 points, respectively, in the group that had received a hydroxyapatite-coated femoral component and 5.6, 5.6, and 5.6 points, respectively, in the group that had received a non-coated component; none of these differences were significant (p = 0.86, 0.89,, and 0.80, respectively). There were no revisions in either group. Radiographs indicated stable fixation in both groups and no differences in the radiographic parameters of loosening between the two groups. Within the relatively short time-frame of this study, there appeared to be no clinical or radiographic advantage to the use of hydroxyapatite in primary total hip arthroplasties. However, these results should be considered as preliminary. Longer follow-up may reveal unrecognized advantages or disadvantages. PMID- 8613436 TI - Usefulness of ultrasonography combined with conventional physical examination in mass screening for breast cancer: a retrospective study of Yamanashi Health Care Center results from 1989 to 1994. AB - We retrospectively analyzed the records for 34,474 women who participated in mass screening for breast cancer by physical examination with or without ultrasonography (US) at Yamanashi Health Care Center between April, 1989 and March, 1994 to evaluate the usefulness of US in mass screening. In one group (15,935 women) conventional physical examination with inspection and palpation alone had been performed, and in another (18,539 women) both conventional physical and US examinations were performed. Breast cancer was detected in 27 of the women (0.08% of the total group screened), 22 of whom were in the group examined by US; moreover, 16 of these 22 women had early breast cancer, which was a non-palpable tumor in 13. Half of the 22 women were examinees under the age of 50 years. Of the 22 tumors detected in the groups examined by US, 16 (73%) were early breast cancer. The overall detection of early breast cancer (0.09%) in the US group was significantly higher than that (0.01%) in the group examined by conventional methods (P < 0.05). Of the tumors detected in the US group, 59.1% were non-palpable. These results suggest that early and non-palpable breast cancer can be detected using US, and the incidence of detection of such tumors in women under the age 50 years is increased in mass screening including US examination. This examination is effective in mass screening for breast cancer, especially for early and non-palpable breast cancer tumors. PMID- 8613438 TI - Revision total hip arthroplasty with use of so-called second-generation cementing techniques for aseptic loosening of the femoral component. A fifteen-year-average follow-up study. AB - We reviewed the results in a consecutive series of forty-three unselected hips (forty-one patients) after revision of the femoral component, because of aseptic loosening, with use of so-called second-generation cementing techniques. This series was previously reported on after average follow-up intervals of six and 11.7 years; we now report the results after an average duration of follow-up of 15.1 years (range, 14.2 to 17.5 years). None of the eight patients (eight hips) who had died before this review had had a reoperation. Over the course of the study period, repeat revision was done after four (11 per cent) of the thirty-six index procedures that were the first femoral revision and after three of the seven that were a second or third revision. Of the thirty-five hips in the thirty three surviving patients, seven (20 per cent) had a repeat revision of the femoral component because of aseptic loosening. The average age at the time of the index revision for this group of patients was fifty-one years. This young age has been associated with distinctly poorer results after revision. In two additional hips (two patients), there was radiographic evidence of loosening of the femoral component. Therefore, the rate of loosening of the femoral component was 26 per cent (nine of thirty-five hips) at an average of 15.1 years. These results support the concept that so-called second-generation cementing techniques have decreased the prevalence of aseptic loosening after femoral revision, compared with the shorter-term results that have been reported after revision with use of so-called first-generation cementing techniques. PMID- 8613439 TI - Revision of the femoral component of a total hip arthroplasty with the calcar replacement femoral component. Results after a mean of 10.8 years postoperatively. AB - Patients who have major loss of bone in the region of the medial aspect of the femoral neck, shortening of the limb, or a high center of the hip joint constitute a special challenge for surgeons performing revision total hip replacements. The use of a so-called calcar-replacement femoral component is one approach to these problems. Of forty-eight hips (forty-four patients) that had been treated consecutively with a total revision arthroplasty with insertion of a calcar-replacement femoral component with cement, thirty-eight hips (thirty-five patients) were followed for a mean of 10.8 years (range, 5.8 to 16.6 years). Ten of the forty-eight hips did not qualify for this study, including nine hips in eight patients who had died before the minimum five-year duration of follow-up and one hip in a patient who had refused follow-up. Of the thirty-eight hips that were followed, seven (18 per cent) had had a repeat revision because of aseptic loosening of the femoral component, one (3 per cent) had been revised again because of lysis around a well fixed femoral component, and an additional four (11 per cent) had a component that was loose according to radiographic criteria. Thus, twenty-six (68 per cent) of the thirty-eight index femoral components were rigidly fixed according to radiographic criteria, and thirty (79 per cent) were still in place. The clinical results were very good for the thirty hips that had not been revised. The mean Harris hip-rating for these patients increased from 50 points preoperatively to 84 points at the most recent follow-up evaluation. PMID- 8613440 TI - The prosthesis-bone interface adjacent to tibial components inserted without cement. Clinical and radiographic follow-up at nine to twelve years. AB - We evaluated the radiographic and functional status of a one-piece composite tibial component, designed to be inserted without cement, that was developed and used only at our institution. Thirty-six patients (thirty-seven knees) were managed with the component between November 1981 and September 1983, and none were lost to follow-up. When they were last seen, thirty-four (92 per cent) of the thirty-seven knees had a well-fixed tibial component, both clinically and radiographically. Eight patients (eight knees; 22 per cent) died. Eleven patients (eleven knees; 30 per cent) had a revision at an average of sixty-five months (range, four to ninety-five months) postoperatively. The reason for the revision was failure of a metal-backed patellar component in three knees, excessive wear of the polyethylene of the tibial component in one, a hematogenous infection in four, aseptic loosening of the tibial component in two, and chronic synovitis in one. The remaining seventeen patients (eighteen knees; 49 per cent) were seen for clinical and radiographic follow-up at an average of eleven years (range, nine to twelve years) after the operation. With removal of the implant as the end point, the cumulative rate of survival was 83 per cent at fifty-six months and 67 per cent at 108 months. Some of the failures were secondary to features of the prosthetic design that currently are considered to be inadequate, including a metal-backed patellar component and carbon-fiber-reinforced polyethylene. In the twenty-six knees in which the prosthesis had been retained, the implant was stable and the prosthesis-bone interface was unchanged as seen radiographically at the time of the most recent follow-up examination. This finding demonstrates that a porous ingrowth surface is capable of providing a secure interface for biological fixation over the long term. PMID- 8613441 TI - Comparison of the findings of triple-injection cinearthrography of the wrist with those of arthroscopy. AB - Fifty consecutive patients who had a history and clinical findings consistent with internal derangement of the wrist were prospectively entered into a study to compare the findings of triple-injection arthrography with those of arthroscopy of the wrist with use of three portals. Twenty-six patients were men, and twenty four were women. They had an average age of thirty-six years (range, eighteen to seventy years). The average duration of symptoms in the wrist was eight months (range, one to twenty-four months). The arthrograms of the wrist, which included cineradiographs, were all made and evaluated by the same radiologist. The arthroscopic evaluation of the wrists was performed by two hand surgeons who had previous knowledge of the arthrographic findings. The abnormal findings included in this study were limited to those that should be detectable with both arthrography and arthroscopy. These were full-thickness tears of the scapholunate ligament, the lunotriquetral ligament and the triangular fibrocartilage. The findings of arthrography were normal in eighteen wrists, demonstrated a single lesion in twenty-one, and demonstrated multiple lesions in eleven. Twelve wrists were noted to have a tear of the scapholunate ligament; fifteen, a tear of the lunotriquetral ligament; and eighteen, a tear of the triangular fibrocartilage. The arthroscopic findings were normal in six wrists, demonstrated a single lesion in twenty-five, and demonstrated multiple lesions in nineteen. Twenty-two wrists were noted to have a tear of the scapholunate ligament; fifteen, a tear of the lunotriquetral ligament; and thirty, a tear of the triangular fibrocartilage. When compared with arthroscopy of the wrist, the sensitivity, specificity, and accuracy of triple-injection cinearthrography in detecting tears of the scapholunate ligament, lunotriquetral ligament, and triangular fibrocartilage, as a group, were 56, 83,and 60 per cent. Although arthrography of the wrist is a well accepted diagnostic modality in the evaluation of pain in the wrist, this study suggests that normal arthrographic findings do not necessarily rule out the possibility of internal derangement of the wrist. PMID- 8613442 TI - Intracarpal soft-tissue lesions associated with an intra-articular fracture of the distal end of the radius. AB - Sixty patients who had a displaced intra-articular fracture of the distal end of the radius were managed with manipulative reduction and internal fixation performed under both fluoroscopic and arthroscopic guidance. According to the AO/ASIF classification system, seven fractures were type B1, two were type B2, three were type B3, thirteen were type C1, twelve were type C2, and twenty-three were type C3. Forty-one patients (68 per cent) had soft-tissue injuries of the wrist, including tears of the triangular fibrocartilage complex (twenty-six patients), the scapholunate interosseous ligament (nineteen), and the lunotriquetral interosseous ligament (nine). Thirteen patients had two soft tissue injuries. Intracarpal soft-tissue injuries were identified most frequently in association with fractures involving the lunate facet of the distal articular surface or the radius. PMID- 8613443 TI - Experimental models for the study of drug resistance in osteosarcoma: P glycoprotein-positive, murine osteosarcoma cell lines. AB - P-glycoprotein is an adenosine triphosphate-dependent drug-efflux pump that extrudes drugs from cells and causes drug-resistance. P-glycoprotein is believed to mediate drug-resistance in a wide variety of tumors. In this study, we developed two P-glycoprotein-positive, murine osteosarcoma cell lines that were resistant to Adriamycin (doxorubicin) (MOS/ADR1 and MOS/ADR2). We created the cell lines by short-term pulse exposures of the parent cell line to Adriamycin followed by single-cell cloning. The MOS/ADR1 and MOS/ADR2 cells were sevenfold and eighteenfold more resistant to Adriamycin than the cells from the parent line. Expression of P-glycoprotein, as examined with an immunofluorescence method, was detected in most of the MOS/ADR1 and MOS/ADR2 cells but not in the parent cells. After the cells had been incubated with Adriamycin for one hour, there was less accumulation of the drug in the resistant cell lines than in the parent cell line. The reduced accumulation was due to the increased efflux of Adriamycin. The Adriamycin-resistant cell lines demonstrated greater alkaline phosphatase activity than the parent cell line and produced more differentiated osteoblastic sarcomas in mice. Dose survival studies with use of a tetrazolium colorimetric assay showed that the MOS/ADR1 cells were cross-resistant to vincristine, vinblastine, etoposide, bleomycin, mitomycin C, and actinomycin D but not to dacarbazine, cisplatin, carboplatin, cytosine arabinoside, carmustine, cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil. Although the MOS/ADR2 cells exhibited a similar spectrum of cross-resistance, they were more resistant than the MOS/ADR1 cells. We also tested the effect of three different resistance-modifying agents on the reversal of resistance to Adriamycin. We found that verapamil and trifluoperazine substantially reversed resistance to Adriamycin in the P-glycoprotein positive cell lines, whereas cyclosporin A was relatively ineffective. Because these cell lines retain the histological and biochemical features of bone-producing sarcomas and display the multidrug resistant phenotype, they may be useful models for additional investigations of drug resistance in osteosarcoma. PMID- 8613444 TI - Allograft reconstruction of segmental defects of the humeral head for the treatment of chronic locked posterior dislocation of the shoulder. AB - The recognized options for the treatment of chronic locked posterior dislocation of the shoulder are dependent on the size of the anteromedial defect of the humeral head. Transfer of the lesser tuberosity with its attached subscapularis tendon into the defect is recommended for defects that are smaller than approximately 40 per cent of the joint surface. Prosthetic replacement is preferred for larger defects. Four consecutive patients who had a chronic locked posterior dislocation of the glenohumeral joint associated with a defect of the humeral head that was at least 40 per cent of the articular surface were managed with reconstruction of the shape of the humeral head with use of an allogeneic segment of the femoral head. Stability was restored and maintained in each patient at an average of sixty-eight months (range, sixty to seventy-six months) after the procedure. Three patients reported little or no pain and no or slight functional restrictions in the activities of daily living, and they considered the result to be satisfactory. The fourth patient had mild pain and moderate-to severe dysfunction secondary to avascular necrosis of the remaining portion of the humeral head after a symptom-free period of six years. PMID- 8613445 TI - Clinical screening for congenital dislocation of the hip. AB - The hips of 20,417 newborns were examined within the first three days after birth and at the age of two to three months, from 1985 to 1990. One hundred and twenty four children had abnormal findings in one or both hips and treatment was instituted. After an average duration of follow-up of six years (range, four to nine years), 122 (98 per cent) of the children had normally developed hip joints. One child had unilateral deformity of the femoral head due to ischemia, and another had bilateral anteversion as the only abnormal finding. A survey of the medical records of the only orthopaedic clinic in Stara Zagora that has a pediatric ward in which patients with hip problems are managed revealed no cases of congenital dislocation of the hip diagnosed after the age of three months in the infants born from 1985 to 1990. Clinical screening for congenital dislocation of the hip was effective in this group of patients, and a good result was noted in all but two patients who were treated for such a dislocation. Thus, a screening program is of value in the examination of all infants. PMID- 8613446 TI - Magnetic resonance imaging of skewfoot. AB - Magnetic resonance imaging was used to visualize the ossified and unossified portions of the bones and soft tissues of the feet in order to evaluate the tarsometatarsal anatomy in sixteen children, three months to six years old (mean, fifteen months old), who were seen in the orthopaedic clinic with a suspected diagnosis of skewfoot. Twenty-seven feet were clinically abnormal and five were normal. Of the abnormal feet, twenty-six had a radiographic diagnosis of skewfoot and one, of simple metatarsus adductus. Of the skewfeet, seven had a talocalcaneal angle of 45 degrees or more as measured on the lateral radiograph and six had a talocalcaneal angle of 45 degrees or more as measured on the anterior radiograph. Valgus deformity of the hindfoot was not apparent on clinical examination in any of the children. The talocalcaneal angles measured on the magnetic resonance images corresponded poorly with those measured on the radiographs, possibly because it is not possible to simulate weight-bearing during magnetic resonance imaging or because the effect of partial volume averaging on thin sections. However, magnetic resonance imaging demonstrated the shapes of the bones and the positions of the unossified portions of the bones. Magnetic resonance imaging showed lateral subluxation of the navicular in twenty four skewfeet, plantar subluxation in ten, and medial subluxation of the first metatarsal on the medial cuneiform in twenty-five. The alignment of the lateral margin of the calcaneus and cuboid on the magnetic resonance images was normal in all patients. Magnetic resonance imaging has the unique ability to show the cartilaginous and ossified portions of the developing bones of the foot. PMID- 8613447 TI - Acute slipped capital femoral epiphysis. Review of outcomes and rates of avascular necrosis. AB - One hundred and forty-nine patients (208 hips) were managed for slipped capital femoral epiphysis at the Hospital for Sick Children in Toronto from 1980 through 1990. We retrospectively reviewed the records of twenty-four patients (twenty-six hips) who had an acute slipped capital femoral epiphysis to evaluate the outcome of treatment and possible risk factors for avascular necrosis associated with this condition. The patients were evaluated with the Iowa hip-scoring system, a physical examination, and anteroposterior and lateral radiographs. The severity of degenerative joint disease was assessed on the radiographs with use of the system described by Boyer et al. Eighteen slips were classified as grade 1; seven, as grade 2; and one, as grade 3, according to the system of Southwick. Twenty-three hips were treated with in situ pinning and three, with reduction and pinning. The mean duration of follow-up was 5.9 years (range, 2.1 to 12.8 years). Poor Iowa hip scores and more severe degenerative changes were related to the development of avascular necrosis and to the severity of the slip. Avascular necrosis developed in four hips (15 per cent), two of which had had a reduction. Multiple logistic regression analysis, which included all 150 slips (both acute and chronic) for which there was adequate follow-up, showed that the rate of avascular necrosis was related to both the severity and the acute nature of the slip. PMID- 8613448 TI - Orientation of the lumbar facet joints: association with degenerative disc disease. AB - The orientation of the lumbar facet joints was studied with magnetic resonance imaging in 140 subjects to determine if there is an association between facet tropism and intervertebral disc disease or between the orientation of the facet joints and degenerative spondylolisthesis. The 140 subjects were divided into four groups: sixty-seven asymptomatic volunteers, forty-six of whom did not have a herniated disc on magnetic resonance scans (Group I) and twenty-one who did (Group II); forty-six symptomatic patients who had a herniated disc confirmed operatively (Group III); and twenty-seven patients who had degenerative spondylolisthesis at the interspace between the fourth and fifth lumbar vertebrae (Group IV). Axial scans were made at each lumbar level and digitized, and the facet joint angle was measured by two independent observers with use of image analysis software in a personal computer. The technique of measurement of the facet angles on magnetic resonance scans was validated with a subset of subjects who also had computed tomography scans made. Similar values were obtained with the two methods (r = 0.92; p = 0.00001). For the forty-six asymptomatic volunteers who did not have a herniated disc on the magnetic resonance scans (Group I), the median facet tropism was 5 to 6 degrees and was more than 10 degrees in 24 per cent (eleven) of the subjects. There was no association between increased facet tropism and disc degeneration. At the level of the fourth and fifth lumbar vertebrae, the median facet tropism was 10.3 degrees in the symptomatic patients who had a herniated disc at the same level and 5.4 degrees in the asymptomatic volunteers (Group I) (p = 0.05). The mean orientation of the lumbar facet angles relative to the coronal plane was more sagittal at all levels in the patients who had degenerative spondylolisthesis. The greatest difference was at the level of the fourth and fifth lumbar vertebrae (p = 0.000001). The mean facet angle was 41 degrees (95 per cent confidence interval, 37.6 to 44.6 degrees) in the asymptomatic volunteers and 60 degrees (95 per cent confidence interval, 52.7 to 67.1 degrees) in the patients who had degenerative spondylolisthesis. Furthermore, both the left and the right facet joints were more sagittally oriented in the patients who had degenerative spondylolisthesis. An individual in who both facet-joint angles at the level of the fourth and fifth lumbar vertebrae were more than 45 degrees relative to the coronal plane was twenty-five times more likely to have degenerative spondylolisthesis (95 per cent confidence interval, seven to ninety-eight times). The increase in facet angles at levels other than that of the spondylolisthesis suggests that increased facet angles represent variations in anatomy rather than a secondary result of spondylolisthesis. PMID- 8613449 TI - Experimental impact injury to the cervical spine: relating motion of the head and the mechanism of injury. AB - The purpose of this study was to analyze, with use of an impact model, the relationships among motion of the head, local deformations of the cervical spine, and the mechanisms of injury; the model consisted of the head and neck of a cadaver. Traditionally, the mechanisms of injury to the cervical spine have been associated with flexion and extension motions of the head and neck. However, the classification of the mechanisms is not always in agreement with the patient's account of the injury or with lacerations and contusions of the scalp, which indicate the site of the impact of the head. Eleven specimens were dropped in an inverted posture with the head and neck in an anatomically neutral position. Forces, moments, and accelerations were recorded, and the impacts were imaged at 1000 frames per second. The velocity at the time of impact was on the order of 3.2 meters per second. The angle and the padding of the impact surface varied. Observable motion of the head did not correspond to the mechanism of the injury to the cervical spine. Injury occurred 2.2 to 18.8 milliseconds after impact and before noticeable motion of the head. However, the classification of the mechanism of the injuries was descriptive of the local deformations of the cervical spine at the time of the injury. Accordingly, it is a useful tool in describing the local mechanism of injury. Buckling of the cervical spine, involving extension between the third and sixth cervical vertebrae and flexion between the seventh and eight cervical vertebrae, was observed. Other, more complex, buckling deformations were also seen, suggesting that the deformations that occur during impact are so complex that they can give rise to a number of different mechanisms of injury. PMID- 8613450 TI - Lead poisoning from an intra-articular shotgun pellet in the knee treated with arthroscopic extraction and chelation therapy. A case report. PMID- 8613451 TI - Compression of the medial half of the deep branch of the ulnar nerve by an anomalous origin of the flexor digiti minimi. A case report. PMID- 8613452 TI - Open reduction of traumatic atlanto-axial rotatory dislocation with use of the extreme lateral approach. A report of two cases. PMID- 8613453 TI - Reimplantation and lengthening with use of the Ilizarov apparatus after a traumatic amputation of the leg. A case report. PMID- 8613454 TI - Osteochondritis dissecans. PMID- 8613455 TI - Treatment of Langerhans-cell histiocytosis in children. Experience at the Children's Hospital of Nancy. PMID- 8613456 TI - The role of antibiotic-loaded cement in the treatment of an infection after a hip replacement. PMID- 8613457 TI - Non-traumatic avascular necrosis of the femoral head. PMID- 8613459 TI - Retinoylation of the type II cAMP-binding regulatory subunit of cAMP-dependent protein kinase is increased in psoriatic human fibroblasts. AB - Previously, we have reported a defect in the cAMP-dependent protein kinases (cAMP PK) in psoriatic cells (i.e., a decrease in 8-azido-[32P]cAMP binding to the regulatory subunits and a decrease in phosphotransferase activity) which is rapidly reversed with retinoic acid (RA) treatment of these cells. This led us to examine a possible direct interaction between retinoids and the RI and RII regulatory subunits through retinoylation. Retinoylation of RI and RII present in normal and psoriatic human fibroblasts was analysed by [3H]RA treatment of these cells, followed either by chromatographic separation of the regulatory subunits or by their specific immunoprecipitation. These studies indicated that RI and RII can be retinoylated. [3H]RA labeling of the RII subunit was significantly (P < 0.005) greater in psoriatic fibroblasts (nine subjects; mean 7.47 relative units +/- 1.37 SEM) compared to normal fibroblasts (eight subjects; mean 2.46 relative +/- 0.49 SEM). [3H]RA labeling of and the increase in 8-azido-[32P]-binding to the RI and RII subunit in psoriatic fibroblasts showed a similar time course. This suggests that the rapid effect of retinoic acid treatment to enhance 8-azido [32P]-cAMP binding to the RI and RII in psoriatic fibroblasts may be due, in part, to covalent modification of the regulatory subunits by retinoylation. PMID- 8613458 TI - Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin. AB - Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2. By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2. Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent. PMID- 8613460 TI - 16,16-Dimethyl Prostaglandin E2 modulation of endothelial monolayer paracellular barrier function. AB - Prostaglandins prevent gastrointestinal mucosal injury and promote healing following mucosal injury by various noxious agents. Preservation or repair of microvascular function appears to be crucial in these processes. The processes involved in prostaglandin-mediated repair and preservation of endothelial function are unclear. In the present study, we investigated the role of prostaglandins on endothelial paracellular barrier function using the filter grown bovine aortic endothelial cell monolayers. Endothelial paracellular barrier function as assessed using a paracellular marker, mannitol. Prostaglandin analogs 16,16-dimethyl prostaglandin E2 (DMPGE2) and prostaglandin I2 (PGI2) caused an enhancement of endothelial monolayer paracellular barrier function as evidenced by a dose-dependent decrease in endothelial paracellular permeability. DMPGE2 induced enhancement of endothelial paracellular barrier function correlated directly with increasing intracellular cAMP levels. Agents which increase intracellular cAMP levels at different stages of cAMP amplification cascade including phosphodiesterase inhibitor (3-isobutyl-1 methylxanthine [IBMX]), membrane permeable cAMP (8-bromo cAMP), and adenylate cyclase activators (isoproterenol and forskolin) also produced enhancement in endothelial paracellular barrier function. DMPGE2 enhancement of paracellular barrier function correlated with dense accumulation of actin microfilaments near the intercellular junctions. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also produced similar changes in endothelial actin microfilaments. Cytochalasin B prevented the DMPGE2 enhancement of paracellular barrier function. Indomethacin (INDO), a cyclooxygenase inhibitor, caused a dose-dependent increase in endothelial paracellular permeability. Pharmacologic doses of INDO resulted in condensation and disruption of actin microfilaments with formation of large paracellular openings or gaps between the adjacent cells. Pretreatment of endothelial monolayers with DMPGE2 prevented INDO-induced disturbance of actin microfilaments and paracellular barrier function. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also prevented INDO-induced changes in actin microfilaments and paracellular barrier function. These findings indicate that DMPGE2 has a paracellular barrier enhancing effect on filter-grown endothelial monolayers. This effect appears to be mediated through intracellular cAMP and actin microfilaments. PMID- 8613461 TI - cGMP accumulation and gene expression of soluble guanylate cyclase in human vascular tissue. AB - Gene expression of soluble guanylate cyclase (sGC) and cGMP accumulation in response to sodium nitroprusside (SNP) were studied in cultured human vascular cells and freshly harvested vascular tissue. As revealed by reverse transcriptase polymerase chain reaction, cultured smooth muscle and endothelial cells, as well as freshly isolated human vascular tissue, express mRNA for the alpha 3 and beta 3 subunits but not for the alpha 2 and beta 3 subunits is evident even in the absence of increased cGMP accumulation in response to SNP. cGMP accumulation in human cells cultured from different vascular beds typically increased two- to five-fold (maximum of 11.4-fold) over baseline following stimulation with 100 microM SNP. Bovine, murine, canine, and avian vascular smooth muscle cells accumulated similar or lower amounts of cGMP than human cells, whereas porcine, rat, and rabbit smooth muscle cells accumulated greater amounts of cGMP. In freshly harvested human vessels, cGMP accumulation in response to SNP was found to increase fifteen-fold over baseline. In contrast to the SNP-induced cGMP accumulation, cGMP levels in response to particulate guanylate cyclase activator atriopeptin II were equal to or greater in cultured human cells than in fresh human vascular tissue. We conclude that human vascular cells (fresh and cultured) express the mRNA for both a large (alpha 3) and a small (beta 3) sGC subunit and that fresh human cells are more sensitive to SNP stimulation. PMID- 8613462 TI - Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes. AB - Pb2+ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanisms and thiol groups in Pb2+ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb2+ to stimulate potassium efflux as measured with 86Rb. The most potent stimulation of 86Rb efflux by Pb2+ occurred when conditions were optimal for the AE mechanism--that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO4 or NaNO3 was used. Furthermore, 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfuonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the 86Rb efflux by Pb2+. These conditions or inhibitors did not affect stimulation of the 86Rb efflux by ionomycin plus Ca2+. A role for Ca2+ channels was dismissed because the inorganic Ca2+ channel blockers, Cd2+ or Mn2+, did not prevent stimulation of 86Rb efflux by Pb2+ but did inhibit stimulation by ionomycin plus Ca2+. 86Rb efflux was more sensitive to Pb2+ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca(2+)-dependent process by Pb2+. PMID- 8613463 TI - Fibroblast growth factor and heparin protect endothelial cells from the effects of interleukin 1. AB - Vascular endothelium is involved in both active and passive processes in haemostasis, but inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF) have been reported to convert the comparatively inert endothelial cell to an inflammatory state. Acidic fibroblast growth factor (aFGF) in the presence of heparin has effects opposite to IL-1 on cultured human umbilical vein endothelial cells (HUVEC); therefore, we have investigated the modulation of IL-1-induced effects by the c combination of aFGF and heparin (aFGF/heparin). First passage HUVEC were cultured for 6 days in the presence of 20% human serum with and without the addition of 625 pM human recombinant aFGF (hr aFGF) and 7 microM heparin. On day 5, recombinant IL-1 beta was included for 24 h. The following day the cells were washed and measurements made of the release of prostacyclin, von Willebrand factor, plasminogen activator inhibitor type 1, and thrombospondin, both in the resting state and following stimulation for 60 min with 1 U/ml thrombin. Tissue-type plasminogen activator was assayed in HUVEC lysates. Similar experiments were performed to assess effects on the expression of vascular adhesion molecule, intracellular adhesion molecule, and E selectin using an ELISA on cells in situ. This study indicates that aFGF/heparin in the culture medium of HUVEC abrogates the measured responses to IL-1. These data imply that routine endothelial cell culture with aFGF/heparin may cause artefacts, the effects of FGF and Il-1 may involve common pathways, and FGF/heparin may offer an approach to design therapeutics to counter the adverse effects of IL-1. PMID- 8613464 TI - Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: role of P36. AB - Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6 bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD --> NADH direction (malate --> oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms. PMID- 8613465 TI - Nuclear accumulation of multiple protein kinases during prolactin-induced proliferation of Nb2 rat lymphoma cells. AB - Intracellular kinases play important roles in signal transduction and are involved in the surface receptor-mediated regulation of cellular functions, including mitogenesis. In the present study, we examined the possible involvement of various protein kinases in the passage of a mitogenic signal from the cell surface to the nucleus of Nb2 cells, a rat nodal lymphoma cell line in which prolactin is a mitogen. Following a prolactin challenge, various kinase activities were monitored at short intervals in different cellular fractions over a 60 min period. Protein kinase C (PKC) activity in the cytosolic fraction rapidly declined to 50% of its original activity within the first 30 min, while PKC activity in the nuclear fractions increased sharply, reaching its highest level by 30 min following a prolactin challenge. There were also increases in both casein kinase and protein tyrosine kinase (PTK) activities in the nuclear fractions during the first 30 min following a prolactin challenge that paralleled PKC activity. The activities of all three kinases declined thereafter, reaching levels close to their respective basal values by 60 min following initiation of prolactin treatment. These observations suggest the possibility that multiple protein kinases may be involved in mitogenic signal transduction for prolactin in Nb2 cells. PMID- 8613466 TI - Fibroblast growth factor-1-inducible gene FR-17 encodes a nonmuscle alpha-actinin isoform. AB - Polypeptide growth factor binding to cell surface receptors activates a cytoplasmic signaling cascade that ultimately promotes the expression of specific nuclear genes. As an approach to investigate the molecular mechanism of fibroblast growth factor (FGF)-1 mitogenic signaling, we have begun to identify and characterize FGF-1-inducible genes in murine NIH 3T3 cells. Here we report that one of these genes, termed FGF-regulated (FR)-17, is predicted to encode a nonmuscle isoform of alpha-actinin, an actin cross-linking protein found along microfilaments and in focal adhesion plaques. FGF-1 induction of alpha-actinin mRNA expression is first detectable at 2 h after mitogen addition and is dependent on the novo RNA and protein synthesis. Maximal alpha-actinin mRNA expression, corresponding to an approximately nineteenfold level of induction, is present after 12 h of FGF-1 stimulation. Western blot analysis indicated that FGF 1-stimulated cells also produce an increased amount of alpha-actinin protein. The FGF-1-related mitogen FGF-2, calf serum, several of the polypeptide growth factors present in serum, and the tumor promoter phorbol myristate acetate can also induce alpha-actinin mRNA expression. Finally, nonmuscle alpha-actinin mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in adult mouse intestine and kidney. These results indicate that nonmuscle alpha-actinin is a serum-, polypeptide growth factor-, and tumor promoter-inducible gene in mouse fibroblasts. PMID- 8613467 TI - Thyroid hormone analogues potentiate the antiviral action of interferon-gamma by two mechanisms. AB - L-thyroxine (L-T4) potentiates the antiviral activity of human interferon-gamma (IFN-gamma) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN-gamma (1.0 IU/ml), 2) for 24 h cotreatment with IFN-gamma, 3) for 4, after 20 h cell incubation with IFN gamma, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN-gamma. The antiviral effect of IFN-gamma was then assayed. L-T4 potentiated the antiviral action of IFN-gamma by a reduction in virus yield of more than two logs, the equivalent of a more than 100-fold potentiation of the IFN's antiviral effect. 3,3 of the IFN's antiviral effect. 3,3',5-L-triiodothyronine (L-T3) was as effective as L-T4 when coincubated for 24 h with IFN-gamma but was less effective than L-T4 when coincubated for only 4 h. D-T4, D-T3, 3,3',5 triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5 diiodothyronine (T2) were inactive. When preincubated with L-T4 for 24 h prior to IFN-gamma treatment, tetrac blocked L-T4 potentiation, but, when coincubated with L-T4 for 4 h after 20 h IFN-gamma, tetrac did not inhibit the L-T4 effect. 3,3',5 L-triiodothyronine (rT3) also potentiated the antiviral action of IFN-gamma, but only in the preincubation model. Furthermore, the effects of rT3 preincubation and L-T3 coincubation were additive, resulting in 100-fold potentiation of the IFN-gamma effect. When L-T4, L-T3, or rT3, plus cycloheximide (5 micrograms/ml), was added to cells for 24 h and then removed prior to 24 h IFN-gamma exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN-gamma potentiation by 4 h of L-T4 or L-T3 coincubation was not inhibited by cycloheximide (25 micrograms/ml). These studies demonstrate two mechanisms by which thyroid hormones can potentiate IFN-gamma's effect: 1) a protein synthesis-dependent mechanism evidenced by enhancement of IFN-gamma's antiviral action by L-T4, L-T3, or rT3 preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis-independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L-T4 or L-T3, but not rT3, with IFN-gamma. The protein synthesis-dependent pathway is responsive to rT3, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN-gamma can be regulated has not previously been described. PMID- 8613468 TI - Role of extracellular Ca2+ in acetylcholine-induced repetitive Ca2+ release in submandibular gland acinar cells of the rat. AB - Acetylcholine (ACh) caused repetitive transient Cl- currents activated by intracellular Ca2+ in single rat submandibular grand acinar cells. As the concentration of ACh increased the amplitude and the frequency of the transient Cl- currents increased. These responses occurred also in the absence of extracellular Ca2+ but disappeared after several minutes. Repetitive transient Cl currents were restored by readmission of Ca2+ to the extracellular solution. The higher the concentration of extracellular Ca2+ readmitted, the larger the amplitude of the transient Cl- currents. Ca2+ entry through a store-coupled pathway was detected by application of Ca2+ to the extracellular solution during a brief cessation of stimulation with ACh. In these experiments too, the higher the concentration of Ca2+, the larger the transient Cl- currents activated by Ca2+ released from the stores. The time course of decrease in total charge movements of repetitive transient responses to ACh with removal of extracellular Ca2+ depended on a decrease in charge movements of each transient event rather than a decrease in frequency of the repetitive events. The decrease of charge movements of each transient event was due to a decrease in its amplitude rather than its duration. The results suggest that in this cell type and amplitude modulated mechanism is involved in repetitive Ca2+ release and that Ca2+ entry is essential to maintain the repetitive release of Ca2+. The results further suggest that the magnitude of Ca2+ entry determines the number of unitary stores filled with Ca2+ which can synchronously respond to ACh. PMID- 8613469 TI - Cellular changes during cold acclimatation in adipose tissues. AB - Cold exposure is a well-known physiological stimulus that activates the sympathetic nervous system and induces brown adipose tissue (BAT) hyperplasia. The effects of cold exposure or cold acclimatation have been extensively studied in interscapular BAT (IBAT). However, it has been recently shown that studied adipocytes are present in adipose deposits considered as white fat such as periovarian (PO) fat pad. We have investigated the kinetic of brown precursor recruitment in adipose tissues using DNA measurement and specific marker expression. In IBAT, cold exposure induces proliferation of precursor cells and differentiation into preadipocytes characterized by the expression of A2COL6, a marker specific to early steps of the differentiation process. A chronic stimulation of the tissue is necessary to observe the full effect. In PO fat pad, no proliferation can be detected, whereas differentiation of brown preadipocytes and maybe phenotypic conversion of white adipocytes seems to be promoted. In conclusion, these data demonstrated that 1) the same stimulus (cold exposure) does not induce the same response in terms of preadipocyte proliferation and differentiation in periovarian and brown adipose tissues, although both contain brown adipocytes, and 2) preadipocyte recruitment in adipose tissues after cold exposure depends on the predominant type of fat cells. PMID- 8613470 TI - Interleukin-4 and interferon-gamma discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets. AB - Pulmonary fibrosis is a potentially fatal consequence of treatments for malignancy and is an increasing problem in bone marrow transplant patients and in cases of allogenic lung transplant. The fibrotic response is characterized by increases in lung fibroblast number and collagen synthesis. This laboratory previously isolated stable, functionally distinct, murine lung fibroblast subsets (Thy-1+ and Thy-1-) to study the contribution of fibroblast subpopulations in lung fibrosis. The fibroblast fibrotic response may be induced by cytokines secreted by infiltrating cells such as T lymphocytes and mast cells. In the current study two key regulatory cytokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), were investigated for their effects on the collagen synthesis of murine lung fibroblast subsets. IL-4 and IFN-gamma are putatively characterized as fibrogenic and anti-fibrogenic cytokines, respectively, and are found in repairing lung tissue. Stimulation with recombinant IL-4 induced a100% increase in total collagen production only by Thy-1+ fibroblasts. Types I and III collagen mRNA were increased in the Thy-1+ fibroblasts, unlike the Thy-1- subset. In contrast, IFN-gamma decreased constitutive collagen production by more than 50% in Thy-1+ and Thy-1- fibroblasts. Interestingly, the two subsets utilized their collagen production machinery (collagenase, tissue inhibitors of metalloproteinases) differently to further regulate collagen turnover in response to IL-4 and IFN-gamma. Overall, our data support the hypothesis that IL-4 is fibrogenic and IFN-gamma is anti-fibrogenic. Moreover, selective expansion of IL 4 responsive fibroblasts (e.g., Thy-1+) may be important in the transition from repair to chronic fibrosis. In addition, these data suggest that an inflammatory response dominated by IL-4-producing Th2 lymphocytes and/or mast cells will promote fibrosis development. PMID- 8613471 TI - Interferon-gamma induced cell death in a cultured human salivary gland cell line. AB - Increased levels of several cytokines, including interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), have been demonstrated in the salivary gland microenvironment of patients with Sjogren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of the disease is not well understood. This study examined the role of IFN-gamma +/- TNF-alpha on cellular death in a cultured human salivary gland cell line (HSG). Cells treated long-term with IFN-gamma +/- TNF-alpha demonstrate a profound antiproliferative effect with a decrease in cell number to below that initially plated. Treatment of HSG cells with TNF-alpha alone did not have any significant effects on growth but did increase the expression of the IFN-gamma receptor. Cells labelled with propidium iodide and anti-digoxigenin dUTP/dATP were examined by flow cytometry to determine the percentage of cells exhibiting low DNA content and DNA strand breaks. The percentage of cells exhibiting subdiploid DNA and DNA strand breaks increased with increased time of exposure to the cytokines. The maximum percentage of cells exhibiting DNA degradation at 12 days was 58% for cells treated with IFN-gamma + TNF-alpha, 31% for IFN-gamma treated cells, and < 5% for TNF-alpha-treated and untreated cells. The cells with subdiploid ( < 2n) DNA were subsequently demonstrated to represent two populations, both with evidence of increased DNA strand breaks but with differing light scatter characteristics. One population had features of cells undergoing necrosis, whereas the second population exhibited features of apoptosis. These findings were confirmed by transmission electron microscopy. Cells not exposed to cytokines did not exhibit significant evidence of either death process. We conclude that long-term exposure of a human salivary gland epithelial cell line to IFN-gamma +/- TNF-alpha leads to increased DNA degradation and subsequent cell death. This suggests a potential SS disease mechanism and implicates the role of the epithelial cell in this disease as an important area for future study. PMID- 8613472 TI - Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (Leishmania) amazonensis. AB - Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22 degrees C) to the temperature of the mammalian host (37 degrees C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34 degrees C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [alpha 32P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms. PMID- 8613473 TI - Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion. AB - We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7 fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells. PMID- 8613474 TI - Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts. AB - In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNF alpha) and gamma interferon (IFN gamma) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN-induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose-related (10-10 M EGF, half-maximal) and proceeds via the interruption of an early step in the cytokine-induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNF alpha, and IFN gamma may determine the rate of trophoblast growth and renewal during gestation. PMID- 8613475 TI - Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells. AB - Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13 acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of human plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP 1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP mediated activation of proMMP-2. PMID- 8613476 TI - Quantitative analysis of lactase-phlorizin hydrolase expression in the absorptive enterocytes of newborn rat small intestine. AB - At birth, the mammalian small intestine displays regional differences in morphology as well as complex proximal-to-distal (horizontal) patterns of protein distribution. Lactase-phlorizin hydrolase (LPH), an enterocyte-specific disaccharidase crucial for the digestion of lactose in milk, reveals a characteristic horizontal pattern of expression at birth. However, it is not certain whether this topographic pattern is due to variations in epithelial structure along the length of the small intestine or to regional differences in the transcription of the LPH gene. In order to understand the mechanisms that regulate the regionalization of LPH at birth, we characterized the epithelial structure along the horizontal axis using stereologic techniques and correlated these data with the patterns of lactase activity and LPH mRNA abundance in the small intestine of unsuckled, newborn rats. Epithelial volume and microvillar surface area per unit of intestinal length decreased three-to fourfold from duodenum to distal ileum. In contrast, lactase activity and LPH mRNA abundance were highest in proximal jejunum and lowest in the most proximal and distal ends of the small intestine. Mean lactase activity per cell in proximal duodenum, proximal jejunum, and distal ileum was estimated at 12.0, 26.7, and 5.6 nU/absorptive enterocyte, respectively, and paralleled the concentration of LPH mRNA in the same segments: 20, 45, and 15 molecules of LPH mRNA/absorptive enterocyte. Our data indicate that horizontal gradients of lactase activity in the newborn rat intestine do not depend on epithelial organization or on enteral factors, since the horizontal gradient is established before suckling. Each absorptive enterocyte along the small intestine expresses lactase activity in a position-dependent manner which is controlled at the level of mRNA abundance. PMID- 8613477 TI - Acute hypoxia increases intracellular L-arginine content in cultured porcine pulmonary artery endothelial cells. AB - Exposure to hypoxia (0% O2) for 4-24 h resulted in increased intracellular L arginine content and increased activity of calpain, a calcium-dependent neutral cysteine protease, in pulmonary artery endothelial cells. Calpain-inhibitor I abolished the increased L-arginine content in hypoxic cells. When endothelial cell proteins were labeled with [3H]-L-arginine and the cells exposed to hypoxia, we observed an increase in free [3H]-L-arginine and a decrease in [3H]-L-arginine labeled proteins. Once again, calpain-inhibitor I prevented the increases in free [3H]-L-arginine and the decreases in [3H]-L-arginine-labeled proteins in hypoxic cells. Hypoxia also inhibited the synthesis of L-arginine-containing proteins. Thus, the increase in intracellular L-arginine content in hypoxic pulmonary artery endothelial cells is caused by an increase in proteolysis secondary to calpain and a decrease in protein synthesis. These results indicate that hypoxia can modulate the availability of free intracellular L-arginine, the exclusive precursor of nitric oxide (NO) and the primary substrate of NO synthase, by affecting the synthesis and degradation of cellular proteins. PMID- 8613478 TI - Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562. AB - The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na(+)-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5 dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume activated taurine influx and efflux, while [dihydroindenyl)oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. PMID- 8613479 TI - Minocycline impairment of both osteoid tissue removal and osteoclastic resorption in a synchronized model of remodeling in the rat. AB - In addition to their antibacterial effects, tetracyclines may inhibit interstitial collagenase activity and bone resorption. These properties were assessed morphometrically using minocycline (25 and 50 mg/kg/day given by the IM route) in a rat model of synchronized remodeling in which osteoclastic resorption peaks 4 days after the activating event (the extractions of the upper molars) along the antagonist mandibular cortex, a zone undergoing physiologically active formation. During the first 2 days of activation, minocycline at the two doses impaired very significantly the disorganization of both the osteoid seam and the layer of osteoblasts, a prerequisite to give osteoclasts access to the mineralized bone surface. The number of readily identifiable osteoblasts decreased slightly during this period, suggesting that minocycline prevented their transformation into lining cells. Their synthetic activity, as estimated by the size of the cells and their nucleus, appeared relatively preserved too, mostly with the higher dose. AT the peak of osteoclasia, the bone surfaces undergoing remodeling were significantly decreased in the minocycline-treated groups. The resorption surface was reduced (P < 0.0003) as well as the number of osteoclasts (P < 0.0007), which were also significantly smaller. Their resorbing activity was dramatically affected as well: they excavated lacunae whose area was significantly reduced by over 70%. In addition, formation was still a prominent activity in the treated animals. These data are compatible with the inhibition at the early stages of activation of an osteoblast-secreted collagenase whose action may be the elimination of the osteoid seam. The inhibition of an osteoclast collagenase and/or of a bone matrix bound-collagenase may be responsible for the reduction in lacunar size. A direct effect of minocycline on osteoclast resorptive activity may also participate in the low resorption profile, as tetracyclines are known to interfere with the intracellular [Ca2+]. PMID- 8613480 TI - The role of superantigens in disease and animal models as instructional paradigms of human disease. Proceedings of a meeting. Tucson, Arizona, April 7-10, 1994. PMID- 8613481 TI - Gi2 alpha protein deficiency: a model of inflammatory bowel disease. AB - Mice deficient for the G protein subunit Gi2 alpha were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 Gi2 alpha-deficient mice with colitis also developed adenocarcinomas of the colon. Gi2 alpha-deficient thymocytes displayed two- to fourfold increases in mature CD4+8- and CD4-8+ phenotypes, an approximately threefold increase in high-intensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of Gi 2 alpha-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumour necrosis factor, and interferon-gamma production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that Gi 2 alpha-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies. PMID- 8613483 TI - The potential role of bacterial superantigens in the pathogenesis of Kawasaki syndrome. AB - Kawasaki syndrome is an acute multisystem vasculitis of infancy and early childhood associated with high fever, mucocutaneous inflammation, and the development of coronary artery abnormalities. Despite the widely held belief that Kawasaki syndrome is an infectious disease, investigations have failed to identify a causal organism. Previous studies have demonstrated that this illness is associated with marked activation of monocyte/macrophages and the selective expansion of V beta 2-, less so, of V beta 8.1/8.2-expressing T cells in the peripheral blood from Kawasaki syndrome patients during the acute phase of their illness. These immunologic features are characteristic of diseases that are caused by bacterial toxins which act as superantigens. Staphylococcal enterotoxins and streptococcal exotoxins are prototypic superantigens which stimulate large populations of T cells expressing particular T-cell receptor beta chain variable (V beta) gene segments. Using the V beta 2+ T-cell expansion as an "immunologic footprint" for a superantigen, we have extended these observations to the identification and isolation of a novel clone of toxic shock syndrome toxin-1-producing Staphylococcus aureus in the majority of patients with Kawasaki syndrome and streptococcal pyrogenic exotoxin B/streptococcal pyrogenic exotoxin C-producing streptococci in a minority of Kawasaki syndrome patients. Toxic shock syndrome toxin-1, streptococcal pyrogenic exotoxin B, and streptococcal pyrogenic exotoxin C are known to stimulate V beta 2+ T cells. These observations support the hypothesis that the activation of V beta 2+ T cells during the acute phase of Kawasaki syndrome is caused by bacterial superantigen(s). PMID- 8613482 TI - Transgenic mice that express different forms of the influenza virus hemagglutinin as a neo-self-antigen. AB - We have generated transgenic mouse lineages that express the influenza virus hemagglutinin in different physical forms. One kind expresses the full-length hemagglutinin molecule as a cell surface glycoprotein and can be recognized by hemagglutinin-specific B and T cells. The other expresses a truncated polypeptide corresponding to the N-terminal third of the hemagglutinin molecule. This polypeptide encodes known hemagglutinin-specific T-cell determinants; however, it contains no native B-cell epitopes, since these depend on the conformation of the fully folded protein. In each case, the hemagglutinin transgenic mice display ubiquitous expression of transgenic messenger RNA and induce T-cell tolerance to the transgene-encoded T-cell determinant site 1. Thus, the hemagglutinin is a neo self-antigen in both kinds of hemagglutinin transgenic mice and should provide a useful system for understanding the factors and mechanisms that govern tolerance and autoimmunity to self-antigens. PMID- 8613484 TI - Animal models for complement deficiencies. AB - The complement system plays a key role in host defense and in the development of autoimmunity. Three types of animal models of complement-mediated disease have traditionally been used: they involve normal animals, animals with spontaneously arising genetic deficiency, and animals treated with complement-inactivating agents. All of these approaches have had partial success in our attempts to understand complement mechanisms. Most animal models of genetic deficiency have been studied relatively little, as the availability of such animals is limited. C4, C2, and partial C3 deficiency in the guinea pig are well characterized, although only C4 deficiency in the guinea pig has been exclusively studied. C3 deficiency in the dog and C6 deficiency in the rabbit are well described, although studies are limited in number. C6 deficiency in the rat has been described recently and C5 deficiency in inbred mice strains has been studied fairly extensively. Factor H deficiency in the Yorkshire pig has also been described. Relatively few agents that inhibit complement are in use. Most widely used in animal studies is cobra venom factor. This inactivates the alternative complement pathway in the fluid phase and thereby depletes complement protein levels. The antigenicity of this protein, purified from the venom of cobras, limits its duration of use in most animal models. Complement-inhibiting agents are rare and, as yet, not widely used. We recently described the use of intravenous immune globulin for inhibiting complement in animal studies and present data on its use in animals, including discordant xenograft rejection, and its potential use in human disease. New developments in molecular biology provide the potential for a vast new array of deficiency models. A limited number of laboratories are actively engaged in the production of animals with inactivated genes. For example, gene knockout mice with no C3, and with no factor B, have been generated. Several complement control proteins have been prepared by genetic molecular biological techniques. Most promising among these is CR1, which limits complement damage in several animal models. Transgenic animals, which complement regulatory proteins expressed on their cells, have been prepared. As complement control proteins tend to be more efficient at regulating complement of the same species type as the regulatory protein, these animals may be useful in such areas as xenograft transplantation. The various animal models are reviewed and their potential application to understanding of human disease is emphasized. PMID- 8613485 TI - Modulation of adhesion molecules by cytokines in vivo using human/severe combined immunodeficient (SCID) mouse chimeras. AB - Endothelial cell-leukocyte interactions involve multiple cell adhesion molecules acting in a programmed and sequential manner to create a leukocyte-endothelial cell adhesion cascade. To understand this process fully, in vivo models are needed. To accomplish this, we have transplanted pieces of normal human tissues onto immunodeficient mice to create chimeric animals. In one model, human skin is grafted and closely resembles normal skin histologically. The grafts retain their human vasculature and show low baseline expression of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1. After intradermal injection of human cytokines, these cell adhesion molecules are markedly upregulated and an active inflammatory reaction ensues, with migration of murine leukocytes. Intravenous injection of an anti-human E-selectin antibody completely inhibits leukocyte accumulation induced by tumor necrosis factor-alpha but only partially inhibits leukotriene B4-induced inflammation. In a second model, human bronchus was successfully transplanted heterotopically into severe combined immunodeficient mice. Injection of tumor necrosis factor induced upregulation of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 in the submucosal microvessels, with slightly different kinetics than in the skin. In conclusion, human-severe combined immunodeficient chimeric mice represent a useful model system to study the regulation and function of human cell adhesion molecules in an in vivo setting. PMID- 8613486 TI - Distinct rate and patterns of human CD4+ T-cell depletion in hu-PBL-SCID mice infected with different isolates of the human immunodeficiency virus. AB - The most fundamental question about infection with the human immunodeficiency virus is the mechanism by which infection leads to depletion of CD4+ T lymphocytes, a critical cell type for the regulation of both cellular and humoral immunity. We have studied this issue using a unique small-animal model that is highly susceptible to infection with human immunodeficiency virus. Severe combined immune deficient mice are transplanted with human peripheral blood leukocytes to create hu-PBL-SCID mice, which maintain human T and B lymphocytes and some elements of functional immunity. The hu-PBL-SCID mice respond to human immune deficiency virus infection by the relatively rapid loss of human CD4+ T cells, while other human cells remain unaffected. In this paper, we review evidence showing that different isolates of human immunodeficiency virus-2 cause different rates of CD4+ T-cell depletion and that these rates reflect differences in local spread of infection with lymphoid organs. PMID- 8613487 TI - The HIV-1 reservoir in distinct V beta subsets of CD4 T cells: evidence for a putative superantigen. AB - Human immunodeficiency virus-1 (HIV-1) replicates more efficiently in T cells expressing T-cell receptors using certain V beta genes, V beta 12 in particular. This V beta specificity was consistent with an HIV-1-associated superantigen. In addition, T cell-depleted peripheral blood mononuclear cells from HIV-positive donors potently stimulated V beta 12 cell lines to proliferate in culture, but not control B beta 6.7a cell lines, thus indicating the presence of a V beta selective mitogen. The targeted V beta subsets were not deleted. It was therefore possible that these subsets might represent a viral reservoir in vivo. Viral load was assessed by quantitative polymerase chain reaction (with HIV-1 gag primers) and with an infectivity assay to measure competent virus. It was shown that the tiny V beta 12 subset (1-2% of T cells) has a higher viral load than other V beta subsets in about 65% of infected individuals. Selective HIV-1 replication in V beta 12 cells was also observed 6-9 days after in vitro infection of peripheral blood T cells from several normal HIV-1-negative donors. In summary, a superantigen-like activity appears to promote V beta-selective HIV-1 replication in vitro and in vivo in patients infected with HIV-1. New therapeutic approaches are suggested based on these findings. PMID- 8613488 TI - The role of superantigens in virus infection. AB - Murine mammary tumor viruses are retroviruses which encode superantigens capable of stimulating T cells via superantigen-reactive T-cell receptor V beta chains. Murine mammary tumor viruses are transmitted to the suckling offspring through the milk. We have established that B cell-deficient pups which were foster-nursed by virus-secreting mice do not transfer infectious murine mammary tumor viruses to their offspring. No murine mammary tumor virus proviruses could be detected in the spleen and mammary tissue of these mic. We conclude that B cells are essential for the completion of the viral life cycle in vivo. This indicates that B cells are infected first and that viral amplification takes place only if infected B cells present the murine mammary tumor virus superantigen on their surface, which, in turn, results in activation of T cells expressing the appropriate T-cell receptor B beta chains. These activated T cells secrete factors which stimulate B cells, enabling viral replication. PMID- 8613489 TI - B-cell superantigens: definition and potential impact on the immune response. AB - Superantigens have been extremely helpful tools in exploring fundamental questions in immunobiology including mechanisms of cell activation, tolerance, and autoimmunity. Until recently, attention has been focused exclusive on T-cell superantigens. However, new data suggest that there are superantigens that directly activate B cells. By definition, these agents (1) stimulate a high frequency of B cells, (2) target B cells that have restricted usage of VH or VL family genes, and (3) bind to immunoglobulins outside the sites that bind conventional antigens. A candidate B-cell superantigen that has received considerable attention in this laboratory is staphylococcal protein A. This agent is best known to the immunologist because of its ability to bind to the Fc fragment of IgG. This binding has been localized to two alpha-helical structures on each of four or five homologous regions that comprise the extracellular domain of protein A. However, it is now clear that protein A contains a second site that binds to determinants on the Fab regions of certain immunoglobulins independently of their heavy-chain isotype. In man this so-called alternative site appears to bind only to immunoglobulins that utilize heavy-chain genes of the VH3 subfamily. In the mouse this type of binding is restricted to immunoglobulins using heavy chains belonging to the S107 and J606 VH families. In this review, we examine the growing list of microbial products that dominate B-cell superantigenic properties. Using staphylococcal protein A as a model for a B-cell superantigen, we consider the potential impact of this novel class of antigens on the immune response. We focus on the ability of B-cell superantigens to influence the expression of the B-cell repertoire. In addition, we consider the hypothesis that the interaction of a B-cell superantigen with its reactive serum immunoglobulins activates the classical complement cascade and thus represents a powerful stimulant of tissue inflammation. PMID- 8613490 TI - Modification of immediate hypersensitivity responses by staphylococcal enterotoxin B. AB - The staphylococcal enterotoxins have been termed superantigens based on their ability to stimulate polyclonal proliferative responses of murine and human T lymphocytes expressing particular T-cell receptor V beta gene products. Certain of these toxins have been shown both to activate and to induce anergy in reactive T cells. Staphylococcal enterotoxin B is known to interact with murine T cells bearing V beta 3, -7, -8.1, -8.2, -8.3, and -17. In BALB/c mice V beta 3+ and V beta 17+ T cells are deleted; V beta 7+ T cells are low in frequency. BALB/c mice sensitized to ovalbumin via the skin and airways develop immediate hypersensitivity including IgE/IgG1 antiovalbumin antibodies, immediate cutaneous reactivity to ovalbumin and, increased airway responsiveness. In both in vitro and in vivo studies, the development of these responses has been associated with the V beta 8+ subset of T cells and controlled by V beta 2 + T cells. In view of the central role of V beta 8+ T cells in these responses, we tested the effects of staphylococcal enterotoxin B on the development of immediate hypersensitivity in this system. Intradermal injection of staphylococcal enterotoxin B prevented the development of these responses in the absence of a major deletion of V beta 8+ T cells. The data suggest that the administration of staphylococcal enterotoxin B prevented the antigen-induced expansion of V beta 8+ T cells resulting in a state of responsiveness or anergy, thus preventing the manifestations of immediate hypersensitivity. Bacterial toxins may provide a novel approach to intervention in allergic or autoimmune diseases. PMID- 8613491 TI - Molecular structure of staphylococcus and streptococcus superantigens. AB - Staphylococcus aureus and streptococci, notably those belonging to group A, make up a large family of true exotoxins referred to as pyrogenic toxin superantigens. These toxins cause toxic shock-like syndromes and have been implicated in several allergic and autoimmune diseases. Included within this group of proteins are the staphylococcal enterotoxins, designated serotypes A, B, Cn, D, E, and G; two forms of toxic shock syndrome toxin-1 also made by Staphylococcus aureus; the group A streptococcal pyrogenic exotoxins, serotypes A, B, and C; and recently described toxins associated with groups B, C, F, and G streptococci. The nucleotide sequences of the genes for all of the toxins except those from the groups B, C, F, and G streptococcal strains have been sequenced. The sequencing studies indicate that staphylococcal enterotoxins B and C and streptococcal pyrogenic exotoxin A share highly significant sequence similarity; staphylococcal enterotoxins A, D, and E share highly significant sequence similarity; and toxic shock syndrome toxin-1 and streptococcal pyrogenic exotoxin B and C share little, if any, sequence similarity with any of the toxins. Despite the dissimilarities seen in primary amino acid sequence among some members of the toxin family, it was hypothesized that there was likely to be significant three-dimensional structure similarity among all the toxins. The three-dimensional structures of three of the pyrogenic toxin superantigens have been determined recently. The structural features of two of these, toxic shock syndrome toxin-1 and enterotoxin C3, are presented. Toxic shock syndrome-1 exists as a protein with two major domains, referred to as A and B. The molecule begins with a short N-terminal alpha-helix that then leads into a clawshaped structure in domain B that is made up of beta strands. PMID- 8613492 TI - Use of intravenous immune globulin in the therapy of children with rheumatological diseases. AB - Traditional treatment of dermatomyositis often fails and is associated with severe side effects, especially in children. Similarly, some patients with systemic juvenile rheumatoid arthritis continue to develop crippling arthritis despite the currently used modalities of therapy. We have used monthly infusions of intravenous immune globulin (2 g/kg/month) in patients with dermatomyositis or systemic juvenile rheumatoid arthritis who have not responded to traditional treatment. We show that intravenous immune globulin is beneficial in improving muscle strength and skin manifestations of dermatomyositis. In a similar fashion, intravenous immune globulin infusions were very effective in ameliorating systemic manifestations of systemic juvenile rheumatoid arthritis but less effective in controlling long-lasting arthritis (more than 1 year). We established prognostic criteria early after disease onset which predict the development of severe joint disease and should help in patient selection for future studies. PMID- 8613493 TI - A mutation in zap-70 protein tyrosine kinase results in a selective immunodeficiency. AB - We have previously described a new type of selective T-cell deficiency characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that selective T-cell deficiency patients carry a mutation of zap-70 protein tyrosine kinase, resulting in a loss of the activity of this kinase. The thymus of zap-70(-1-1) patients shows the presence of CD4CD8 double-positive cells in the cortex, however, only CD4, and not CD8, single positive cells are present in the medulla. Peripheral CD4+ T cells from the zap 70(-1-1) patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T-cell receptor stimulation by mitogens or antigens. Thus zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for the signal transduction and function of single-positive CD4 T cells. PMID- 8613494 TI - Inflammatory neuropathies--pathogenesis and the role of intravenous immune globulin. AB - The inflammatory neuropathies may be subdivided into an acute form, Guillain Barre syndrome, and a chronic form referred to as chronic inflammatory demyelinating polyneuropathy. More recently a chronic, asymmetrical pure motor neuropathy with multifocal conduction blocks has been described. All three neuropathies are considered to be immune-mediated. Their response to therapy is discussed, with special emphasis on high-dose intravenous immune globulin. For Guillain-Barre syndrome the efficacy of intravenous immune globulin has been proven in a randomized clinical trial. In chronic inflammatory demyelinating polyneuropathy a response rate of over 60% in newly diagnosed patients is suggested. Clinical prognostic criteria, however, seem to be very important to predict the effect of intravenous immune globulin. In multifocal motor neuropathy intravenous immune globulin is at present the only alternative to cyclophosphamide. PMID- 8613495 TI - Update on the use of intravenous immune globulin in the treatment of patients with inflammatory muscle disease. AB - The inflammatory myopathies consist of three distinct groups: dermatomyositis, polymyositis, and inclusion body myositis. Dermatomyositis is distinguished by its characteristic rash, while polymyositis is a diagnosis of exclusion. Inclusion body myositis is characterized by early involvement of distal muscle groups and the quadriceps. Definitive diagnosis is made by muscle biopsy, which demonstrates histological features characteristic for each disorder. Immune mechanisms play a role in the pathogenesis of the inflammatory myopathies. A complement-mediated microangiopathy is seen in dermatomyositis, while there is evidence for a T cell-mediated process in polymyositis and inclusion body myositis. Treatment with prednisone is helpful to a majority of patients for a period of time. Immunosuppressive drugs have met with limited success. We describe a group of patients with dermatomyositis, resistant to available therapies, whose muscle strength, skin changes, and muscle biopsies improved significantly during treatment with intravenous immune globulin. The treatment of polymyositis and inclusion body myositis with intravenous immune globulin is currently under study. PMID- 8613496 TI - Treatment of systemic and renal-limited vasculitic disorders with pooled human intravenous immune globulin. AB - Idiopathic crescentic glomerulonephritis is characterized by an absence of immunohistological evidence of immune deposits, often with evidence of segmental glomerular necrosis. Such pauciimmune crescentic glomerulonephritis is the most common renal manifestation seen in patients with Wegener's granulomatosis, polyarteritis nodosa, and glomerulonephritis associated with other systemic vasculitic disorders (i.e., Churg-Strauss syndrome). Recently, the idiopathic crescentic glomerulonephritides, either in renal-limited form or in association with other systemic vasculitic disorders, were found to have in common a serologic marker, antineutrophil cytoplasmic autoantibodies. These cytoplasmic and perinuclear antineutrophil cytoplasmic autoantibodies are specific for constituents of neutrophil primary granules and monocyte lysosomes. As serologic markers for vasculitic disorders, they are also felt to be directly involved in the pathogenesis of necrotizing vascular injury. In vitro, both perinuclear and cytoplasmic antineutrophil cytoplasmic autoantibodies are capable of causing cytokine-primed neutrophils to undergo degranulation and respiratory burst, releasing toxic oxygen species and lytic enzymes. Anti-idiotype antibodies which inhibit antineutrophil cytoplasmic autoantibodies in vitro, in a V region dependent manner, are found in pooled human gamma-globulin preparation. Intravenous immune globulin infusions in vivo have produced dramatic improvements in the necrotizing vascular injury produced by antineutrophil cytoplasmic autoantibodies, and a rapid reduction in these autoantibody levels is seen post intravenous immune globulin infusion in most patients. The proposed mechanisms of action of intravenous immune globulin in vasculitic disorders include Fc dependent mechanisms, and F(ab')2-dependent mechanisms are likely important. Intravenous immune globulin infusions appear to have an important place in the management of the necrotizing vascular injury. Blinded, randomized, placebo controlled trials will be necessary to establish definitely intravenous immune globulin as a therapeutic option in vasculitic disorders. PMID- 8613498 TI - Successful second bicuspid bonding. PMID- 8613497 TI - The antiphospholipid-protein syndrome. AB - The pathogenesis of the antiphospholipid syndrome remains uncertain. Antibodies that react with phospholipids may not be directly responsible for cellular injury, but may be part of the immune network through which autoantibodies with pathogenic potential are generated. The latter may recognize proteins such as beta 2-glycoprotein I that form complexes with phospholipids, proteins whose functions depend upon interaction with phospholipids such as protein C and its cofactors, altered lipoproteins such as oxidized low-density lipoproteins, or other molecules that share only antigenic similarity. Thus, a spectrum of autoantibodies that recognize different lipid-protein complexes may develop in these patients and contribute to the observed clinical heterogeneity of the syndrome. Current techniques do not permit identification of the subset of patients with antiphospholipid antibodies at risk for thrombosis or abortion and there are no prospective, controlled trials addressing the prophylaxis or treatment of affected individuals. Identification of the cellular targets of antibodies to lipid-protein moieties is needed to identify patients at risk for these complications and as a means to monitor therapy. PMID- 8613500 TI - Converting observation recalls to new starts. PMID- 8613499 TI - The orthodontic 'Grasshopper'. PMID- 8613502 TI - Taking construction bites for functional appliances. PMID- 8613501 TI - A universal retraction spring. PMID- 8613503 TI - A fixed-removable Herbst appliance. PMID- 8613504 TI - Soffit treatments. PMID- 8613505 TI - An instant photo record system. PMID- 8613506 TI - Vertical elastics for correction of anterior open bite. PMID- 8613507 TI - Effectiveness of thera-bite wafers in reducing pain. PMID- 8613508 TI - Comparison of NiTi coil springs vs. elastics in canine retraction. PMID- 8613509 TI - Current Herbst appliance therapy. PMID- 8613510 TI - Employment in orthodontic offices: a JCO opinion survey. PMID- 8613511 TI - Frictional forces with the friction-free edgewise bracket. PMID- 8613512 TI - Comparison of in-office vs. commercial laboratories. PMID- 8613513 TI - Scanatron enhancement of radiographic contrast. PMID- 8613516 TI - Distalization with 'driftodontics'. PMID- 8613514 TI - Bell-shape bonded lingual splint. PMID- 8613515 TI - Enamel microabrasion for removal of smooth surface decalcification lesions. PMID- 8613517 TI - Permanent marking of bite registration records. PMID- 8613518 TI - Update on bonding brackets: an in vitro survey. PMID- 8613519 TI - Improve profitability with new technology and labor efficiency. PMID- 8613520 TI - The new patient relationship. PMID- 8613521 TI - Publish your own practice newsletter. PMID- 8613522 TI - Update on the Bass appliance system. PMID- 8613523 TI - Specific genetic deficiencies of the A and B isoenzymes of monoamine oxidase are characterized by distinct neurochemical and clinical phenotypes. AB - Monoamine oxidase (MAO) exists as two isoenzymes and plays a central role in the metabolism of monoamine neurotransmitters. In this study we compared the neurochemical phenotypes of previously described subjects with genetically determined selective lack of MAO-A or a lack of both MAO-A and MAO-B with those of two subjects with a previously described X chromosome microdeletion in whom we now demonstrate selective MAO-B deficiency. Mapping of the distal deletion breakpoint demonstrates its location in intron 5 of the MAO-B gene, with the deletion extending proximally into the Norrie disease gene. In contrast to the borderline mental retardation and abnormal behavioral phenotype in subjects with selective MAO-A deficiency and the severe mental retardation in patients with combined MAO-A/MAO-B deficiency and Norrie disease, the MAO-B-deficient subjects exhibit neither abnormal behavior nor mental retardation. Distinct neurochemical profiles characterize the three groups of MAO-deficient patients. In MAO-A deficient subjects, there is a marked decrease in deaminated catecholamine metabolites and a concomitant marked elevation of O-methylated amine metabolites. These neurochemical changes are only slightly exaggerated in patients with combined lack of MAO-A and MAO-B. In contrast, the only biochemical abnormalities detected in subjects with the MAO-B gene deletion are a complete absence of platelet MAO-B activity and an increased urinary excretion of phenylethylamine. The differences in neurochemical profiles indicate that, under normal conditions, MAO-A is considerably more important than MAO-B in the metabolism of biogenic amines, a factor likely to contribute to the different clinical phenotypes. PMID- 8613524 TI - Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. AB - We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. PMID- 8613525 TI - Effect of anti-interleukin 12 treatment on murine lyme borreliosis. AB - The effect of anti-interleukin (IL-12 treatment on Lyme borreliosis in C3H/HeN (C3H) mice was assessed because other studies have implicated CD4+ T cell helper (Th) type 1 responses in the genesis of disease caused by Borrelia burgdorferi. Infection of inbred mice with B. burgdorferi results in varying degrees of arthritis: BALB/c mice develop mild disease and C3H mice develop severe arthritis that is most pronounced 2-4 wk after infection. Since IL-12 is a major inducer of Th1 responses, we blocked this cytokine in vivo in B. burgdorferi infected C3H mice, and evaluated the effects of treatment on the development of arthritis at the peak of acute joint inflammation (14 d) and in the resolution phase (60 d) of disease. As expected, intraperitoneal administration of an anti-IL-12 monoclonal antibody (mAb) to C3H mice resulted in a decrease in both IFN-gamma and B. burgdorferi-specific IgG2a in serum, indicative of diminished Th1 responses. No IL-4 production was detected in serum of anti-IL-12 mAb treated or control mice. IgG1 and IgG2b levels did not increase in B. burgdorferi infected mice treated with anti-IL-12 mAb compared with controls suggesting that Th2 responses were not affected. Nevertheless, CD4+ T cells from both control and anti-IL-12 mAb treated mice had similar in vitro responses to B. burgdorferi antigens. Treatment with anti-IL-12 mAb produced a significant reduction in peak arthritis severity, and an increase in the number of spirochetes in ear tissue. These data show that treatment of B. burgdorferi infected mice with anti-IL-12 mAb results in a reduction of the Th1 and/or innate immune responses in vivo and a reduction in the severity of acute murine Lyme arthritis. PMID- 8613526 TI - Nuclear retention of COL1A1 messenger RNA identifies null alleles causing mild osteogenesis imperfecta. AB - Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone fragility. Most cases of severe OI result from mutations in the coding region of the COL1A1 or COL1A2 genes yielding an abnormal collagen alpha chain. In contrast, many patients with mild OI show evidence of a null allele due to a premature stop mutation in the mutant RNA transcript. We have previously described a null allele arising from a splice donor mutation where the transcript containing the included intron was sequestered in the nucleus. Here we demonstrate that transcripts from null alleles arising from premature stop mutations are also present in the nucleus and absent in the cytoplasm. Using reverse transcriptase-PCR and single-strand conformational polymorphism of COL1A1 mRNA from patients with mild OI, we describe three patients with distinct null producing mutations identified from the mutant transcript within the nuclear compartment. A fourth patient with a Gly--->Arg expressed point mutation exhibits the mutant transcript in both compartments. Defining the distribution of allelic variants of COL1A1 mRNA in the nuclear and cytoplasmic compartments gives further insight into cell biology of OI and provides a strategy for investigating potential causes of a null allele. PMID- 8613527 TI - Chronic exposure of betaTC-6 cells to supraphysiologic concentrations of glucose decreases binding of the RIPE3b1 insulin gene transcription activator. AB - We have shown previously that chronic exposure of HIT-T15 cells to supraphysiologic glucose concentrations causes decreased insulin gene transcription and decreased binding activities of two beta-cell specific transcription factors, STF-1 and the RIPE3b1 activator, and have suggested that these events may provide a mechanism for glucose toxicity on beta-cell function. However, this contention can be criticized because it is not clear whether these observations are unique to the HIT-T15 cell or generalizable to other beta-cell lines and the islet. Therefore, we cultured betaTC-6 cells for up to 41 wk in either 11.1 or 0.8 mM glucose. We observed a passage-dependent decrease in insulin content and insulin mRNA levels in betaTC-6 cells chronically cultured in 11.1 mM glucose. Cells chronically cultured in 0.8 mM glucose had higher insulin mRNA levels than cells chronically cultured in 11.1 mM glucose. The relative activity of a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5' regulatory region of the human insulin gene was decreased in late passage betaTC-6 cells chronically cultured in 11.1 mM glucose, but was preserved in late passages of cells chronically cultured in 0.8 mM glucose. Electromobility shift assays demonstrated that binding of a specific nuclear protein that recognizes the RIPE3b1 binding site of the insulin gene was markedly diminished in late passage cells chronically exposed to 11.1 mM glucose, whereas binding activities of STF-1 and ICE activators were unchanged. RIPE3b1 binding activity was preserved in late passage cells chronically exposed to 0.8 mM glucose. Mutation of the RIPE3b1 binding site almost completely abolished insulin gene transcription as well as binding activity. We conclude that chronic exposure of betaTC-6 cells to high glucose concentrations paradoxically decreases insulin gene transcription, in part, by decreasing activity of the trans-activating factor which binds to the RIPE3b1 sequence. This study uniquely demonstrates that altered binding to the RIPE3b1 sequence mediates glucose toxicity in betaTC-6 cells, thus reinforcing the importance of this cis-acting element in the regulation of insulin gene transcription. We conclude that the phenomenon of glucose toxicity decreasing binding of transcription factors and thereby reducing insulin gene expression is not a feature solely of HIT-T15 cells and may be demonstrable generally in beta-cell lines. PMID- 8613528 TI - Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes. AB - We have generated a transgenic model consisting of both the human renin and human angiotensinogen genes to study further the role played by the renin-angiotensin system in regulating arterial pressure. Transgenic mice containing either gene alone were normotensive, whereas mice containing both genes were chronically hypertensive. Plasma renin activity and plasma angiotensin II levels were both markedly elevated in the double transgenic mice compared with either single transgenic or nontransgenic controls. The elevation in blood pressure caused by the human transgenes was independent of the genotype at the endogenous renin locus and was equal in mice homozygous for the Ren-1c allele or in mice containing one copy each of Ren-1c, Ren-1d, or Ren-2. Chronic overproduction of angiotensin II in the double transgenic mice resulted in a resetting of the baroreflex control of heart rate to a higher pressure without significantly changing the gain or sensitivity of the reflex. Moreover, this change was not due to the effects of elevated pressure itself since angiotensin-converting enzyme inhibition had minimal effects on the baroreflex in spontaneously hypertensive BPH-2 control mice, which exhibit non-renin-dependent hypertension. This double transgenic model should provide an excellent tool for further studies on the mechanisms of hypertension initiated by the renin-angiotensin system. PMID- 8613529 TI - Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. AB - Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions. PMID- 8613530 TI - Impaired actions of insulin-like growth factor 1 on protein Synthesis and degradation in skeletal muscle of rats with chronic renal failure. Evidence for a postreceptor defect. AB - The actions of insulin-like growth factor 1 (IGF-1) on protein turnover and of the IGF-1 receptor (IGF-1R) were examined in skeletal muscle of rats with chronic renal failure (CRF) and sham operated (SO), pair-fed controls. Acidemia was prevented in CRF rats with NaHCO3. Serum IGF-1 and skeletal muscle IGF-1 and IGF 1 mRNA were reduced in CRF rats. Dose-response studies revealed impaired stimulation of protein synthesis and suppressed inhibition of protein degradation by IGF-1 in epitrochlearis muscle of CRF rats. Neither IGF-1 analogues with low affinity to IGF binding proteins nor proteinase inhibitors obliterated the IGF-1 resistance. In CRF rats, skeletal muscle IGF-1R mRNA was increased; displacement ligand binding studies and affinity labeling of the IGF-1R alpha subunit indicated increased total skeletal muscle IGF-1R number with normal affinity. However, both autophosphorylation of the IGF-1R beta subunit (i.e., IGF-1R tyrosine kinase) and the IGF-1R tyrosine kinase activity towards exogenous insulin receptor substrate-1, a natural substrate for IGF-1R tyrosine kinase, were reduced in CRF fats. These data indicate that in skeletal muscle of CRF rats there is resistance to the IGF-1 effects on protein synthesis and degradation and decreased IGF-1 and IGF-1 mRNA levels; IGF-1R mRNA and number are increased; but activity of IGF-1R tyrosine kinase is impaired. This postreceptor defect may be a cause of the skeletal muscle resistance to IGF-1 in CRF. PMID- 8613531 TI - Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro. AB - Net HCO3- transport in the rabbit kidney cortical collecting duct (CCD) is mediated by simultaneous H+ secretion and HCO3- secretion, most likely occurring in a alpha- and beta-intercalated cells (ICs), respectively. The polarity of net HCO3- transport is shifted from secretion to absorption after metabolic acidosis or acid incubation of the CCD. We investigated this adaptation by measuring net HCO3- flux before and after incubating CCDs 1 h at pH 6.8 followed by 2 h at pH 7.4. Acid incubation always reversed HCO3- flux from net secretion to absorption, whereas incubation for 3 h at pH 7.4 did not. Inhibition of alpha-IC function (bath CL- removal or DIDS, luminal bafilomycin) stimulated net HCO3- secretion by approximately 2 pmol/min per mm before acid incubation, whereas after incubation these agents inhibited net HCO3- absorption by approximately 5 pmol/min per mm. Inhibition of beta-IC function (luminal Cl- removal) inhibited HCO3- secretion by approximately 9 pmol/min per mm before incubation, whereas after incubation HCO3- absorption by only 3 pmol/min per mm. After acid incubation, luminal SCH28080 inhibited HCO3- absorption by only 5-15% vs the circa 90% inhibitory effect of bafilomycin. In outer CCDs, which contain fewer alpha-ICs than midcortical segments, the reversal in polarity of HCO3- flux was blunted after acid incubation. We conclude that the CCD adapts to low pH in vitro by downregulation HCO3- secretion in beta-ICs via decreased apical CL-/base exchang activity and upregulating HCO3- absorption in alpha-ICs via increased apical H+ -ATPase and basolateral CL-/base exchange activities. Whether or not there is a reversal of IC polarity or recruitment of gamma-ICs in this adaptation remains to be established. PMID- 8613532 TI - Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor. AB - Extracellular calcium concentrations (Cao) > 0.1 mM are required for the differentiation of normal human keratinocytes in culture. Increments in Cao result in acute and sustained increases in the intracellular calcium level (Cai), postulated to involve both a release of calcium from intracellular stores and a subsequent increase in calcium influx through nonspecific cation channels. The sustained rise in Cai appears to be necessary for keratinocyte differentiation. To understand the mechanism by which keratinocytes respond to Cao, we measured the acute effects of Cao on Cai and calcium influx in keratinocytes at various stages of differentiation. We then demonstrated the existence of the calcium receptor (CaR) in keratinocytes and determined the effect of calcium-induced differentiation on its mRNA levels. Finally, we examined the role of Cai in regulating both the initial rise in Cai after the switch to higher Cao and the activity of the nonspecific cation channel through which calcium influx occurs. Our data indicate that the acute Cai response to Cao is lost as the cells differentiate and increase their basal Cai. These data correlated with the decrease in CaR mRNA levels in cells grown in low calcium. However, calcium influx as measured by 45Ca uptake increased with differentiation in 1.2mM calcium, consistent with the increase in CaR mRNA in these cells as well as the calcium-induced opening of the nonspecific cation channels. We conclude that the keratinocyte contains a CaR that regulates both the initial release of Cai from intracellular stores and the subsequent increase in calcium flux through nonspecific calcium channels. A rising level of Cai may turn off the release of calcium from intracellular stores while potentiating the influx through the nonspecific cation channels. Differentiation of keratinocytes appears to increase the CaR, which may facilitate the maintenance of the high Cai required for differentiation. PMID- 8613533 TI - Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy. AB - The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP 9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability. PMID- 8613534 TI - Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases. PMID- 8613535 TI - The expression of TNF alpha by human muscle. Relationship to insulin resistance. AB - TNFalpha is orverexpressed in the adipose tissue of obese rodents and humans, and is associated with insulin resistance. To more closely link TNF expression with whole body insulin action, we examined the expression of TNF by muscle, which is responsible for the majority of glucose uptake in vivo. Using RT-PCR, TNF was detected in human heart, in skeletal muscle from humans and rats, and in cultured human myocytes. Using competitive RT-PCR, TNF was quantitated in the muscle biopsy specimens from 15 subjects whose insulin sensitivity had been characterized using the glucose clamp. technique. TNF expression in the insulin resistant subjects and the diabetic patients was fourfold higher than in the insulin sensitive subjects, and there was a significant inverse linear relationship between maximal glucose disposal rate and muscle TNF (r = -0.60, P < 0.02). In nine subjects, muscle cells from vastus lateralis muscle biopsies were placed into tissue culture for 4 wk, and induced to differentiate into myotubes. TNF was secreted into the medium from these cells, and cells from diabetic patients expressed threefold more TNF than cells from nondiabetic subjects. Thus, TNF is expressed in human muscle, and is expressed at a higher level in the muscle tissue and in the cultured muscle cells from insulin resistant and diabetic subjects. These data suggest another mechanism by which TNF may play an important role in human insulin resistance. PMID- 8613536 TI - In vivo neutralization of eosinophil-derived major basic protein inhibits antigen induced bronchial hyperreactivity in sensitized guinea pigs. AB - This study examines the effect of purified rabbit antiguinea pig eosinophil derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig. PMID- 8613537 TI - Interleukin (IL)-10 inhibits long-term IL-6 production but not preformed mediator release from rat peritoneal mast cells. AB - Mast cells have been implicated in a number of diseases involving chronic inflammation including asthma, rheumatoid arthritis, and inflammatory bowel diseases. They are a potent source of several cytokines, including IL-6 and TNF alpha. Freshly isolated rat peritoneal mast cells will produce IL-6 in response to anti-IgE, LPS, PGE1, or PGE2; however, the mechanisms by which such cytokine production is regulated are poorly understood. IL-10 is recognized as an important immunoregulatory cytokine with effects on T cell development and the production of inflammatory cytokines. IL-10 has previously been described to enhance mast cell development in the context of IL-3 and IL-4. In the current study, we have examined the ability of IL-10 to modulate rat peritoneal mast cell IL-6 and TNF-alpha production in response to a variety of stimuli. We have observed that recombinant murine IL-10 can inhibit the production of both IL-6 and TNF-alpha by mast cells without altering the degree of histamine release in response to anti-IgE. Concentrations of IL-10 as low as 0.2 ng/ml were sufficient to inhibit IL-6 production by LPS- or anti-IgE-activated cells significantly. IL 10 also inhibited PGE1- and PGE2-induced IL-6 production. The relative potency of IL-10 as an inhibitor of mast cell IL-6 production was highly dependent upon the stimulus used, with a 10-fold difference in the IC50 for LPS- or anti-IgE activated cells (0.21 ng/ml) and cells activated with a combination of LPS and PGE2 (2.29 ng/ml). This suggests that prostanoids may limit the ability of IL-10 to modulate mast cell IL-6 production in the context of inflammation. These data have important implications for the regulation of mast cell IL-6 in inflammatory diseases involving prostanoid production and the effects of treatment with cyclooxygenase inhibitors. Our results also demonstrate a dual role for IL-10 on mast cells as a growth factor and inhibitor of cytokine production. PMID- 8613539 TI - Mast cell tryptase: hoisted by its own petard? PMID- 8613538 TI - The effect of docosahexaenoic acid on aggression in young adults. A placebo controlled double-blind study. AB - 41 students took either docosahexaenoic acid (DHA)-rich oil capsules containing 1.5-1.8 grams DHA/day (17 females and 5 males) or control oil capsules containing 97% soybean oil plus 3% fish oil (12 females and 7 males) for 3 mo in a double blind fashion. They took a psychological test (P-F Study) and Stroop and dementia detecting tests at the start and end of the study. The present study started at the end of summer vacation and ended in the middle of mental stress such as final exams. In the control group extraggression (aggression against others) in P-F Study was significantly increased at the end of the study as compared with that measured at the start (delta = +8.9%, P = 0.0022), whereas it was not significantly changed in the DHA group (delta = -1.0%). The 95% CI of differences between the DHA and control groups were -16.8 to -3.0%. DHA supplementation did not affect the Stroop and dementia-detecting tests. Thus, DHA intake prevented extraggression from increasing at times of mental stress. This finding might help understand how fish oils prevent disease like coronary heart disease. PMID- 8613540 TI - Molecular insights into Fanconi anemia. PMID- 8613541 TI - Is idiopathic hypoparathyroidism an autoimmune disease? PMID- 8613542 TI - Genes and physiology: molecular physiology in genetically engineered animals. PMID- 8613543 TI - Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. AB - Acquired hypoparathyroidism (AH) has been considered to result from an autoimmune process but the self-antigens have not been identified. We studied 25 patients with AH, of which 17 had type I autoimmune polyglandular syndrome and 8 had AH associated with autoimmune hypothyroidism. Five of 25 (20%) AH sera reacted to a membrane-associated antigen of 120-140 kD in human parathyroid gland extracts using immunoblot analysis. This is the exact size of the calcium sensing receptor (Ca-SR). The AH sera were then tested by immunoblot using a membrane fraction of HEK-293 cells transfected with Ca-SR cDNA. Eight of 25 (32%) AH sera reacted to a 120-140-kD protein, which closely matched that recognized by the anti-Ca-SR IgG raised in rabbits. The Ca-SR cDNA was translated in vitro into two parts in order to identify the antigenic epitopes. By using this technique, 14 of 25 (56%) AH sera were positive to the extracellular domain of the Ca-SR, whereas none of the AH patients sera reacted to the intracellular domain. The reactivity of the positive sera was completely removed after pre-absorption with the Ca-SR containing membranes. Sera from 50 patients with various other autoimmune diseases as well as 22 normal controls were also tested, and none of them was positive. In conclusion, the Ca-SR has been identified as an autoantigen in AH. PMID- 8613544 TI - Interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness. AB - To address the role of IL-11 in viral airways dysfunction, we determined whether infectious agents that exacerbate asthma stimulate stromal cell IL-11 production, determined whether IL-11 could be detected at sites of viral infection and evaluated the effects of IL-11 on airway physiology. Respiratory syncytial virus (RSV), parainfluenza virus type 3 (PIV3), and rhinovirus (RV) 14 were potent stimulators while cytomegalovirus and adenovirus only weakly stimulated and herpes simplex virus type 2 and bacteria did not stimulate IL-11 elaboration. IL 11 was not detected or barely detected in nasal aspirates from children without, but was detected in aspirates from children with viral upper respiratory tract infections. The levels of IL-11 were highest in patients with clinically detectable wheezing. IL-11 also caused nonspecific airways hyperresponsiveness in BALB/c mice. These studies demonstrate that three major causes of viral-induced asthma, RSV, RV, and PIV, in contrast to other viruses and bacteria, share the ability to induce stromal cell IL-11 production. They also demonstrate that IL-11 can be detected in vivo during viral respiratory infections, that the presence of IL-11 correlates with clinical bronchospasm and that IL-11 is a potent inducer of airways hyperresponsiveness. IL-11 may be an important mediator in viral airways disorders. PMID- 8613545 TI - The molecular basis of hereditary complement factor I deficiency. AB - The molecular basis of hereditary complement factor I deficiency is described in two pedigrees. In one pedigree, there were two factor I-deficient siblings, one of whom was asymptomatic and the other suffered from recurrent pyogenic infections. Their factor I mRNA was analyzed by reverse transcription of fibroblast RNA followed by amplification using the polymerase chain reaction. Both siblings were homozygous for the same transversion (adenine to thymine) at nucleotide 1282 in the cDNA. This mutation causes histidine-400 to be replaced by leucine. The altered histidine is a semi-conserved residue within the serine proteinase family, although no function has been ascribed to it. The proband of the second pedigree studied was found to be a compound heterozygote. One allele had the same mutation as the first family, the second allele had a donor splice site mutation that resulted in the deletion of the mRNA encoded in the fifth exon (a low-density lipoprotein receptor domain) from its transcript. PMID- 8613546 TI - Endogenous growth hormone (GH)-releasing hormone is required for GH responses to pharmacological stimuli. AB - The roles of hypothalamic growth hormone-releasing hormone (GHRH) and of somatostatin (SRIF) in pharmacologically stimulated growth hormone (GH) secretion in humans are unclear. GH responses could result either from GHRH release or from acute decline in SRIF secretion. To assess directly the role of endogenous GHRH in human GH secretion, we have used a competitive GHRH antagonist, (N-Ac-Tyr1,D Arg2)GHRH(1-29)NH2 (GHRH-Ant), which we have previously shown is able to block the GH response to GHRH. We first tested whether an acute decline in SRIF, independent of GHRH action, would release GH. Pretreatment with GHRH-Ant abolished the GH response to exogenous GHRH (0.33 microgram/kg i.v.) but did not modify the GH rise after termination of an SRIF infusion. We then investigated the role of endogenous GHRH in the GH responses to pharmacologic stimuli of GH release. The GH responses to arginine (30 g i.v. over 30 min), L-dopa (0.5 g orally), insulin hypoglycemia (0.1 U/Kg i.v.), clonidine (0.25 mg orally), or pyridostigmine (60 mg orally) were measured in healthy young men after pretreatment with either saline of GHRH-Ant 400 microgram/kg i.v. In every case, GH release was significantly suppressed by GHRH-Ant. We conclude that endogenous GHRH is required for the GH response to each of these pharmacologic stimuli. Acute release of hypothalamic GHRH may be a common mechanism by which these compounds mediate GH secretion. PMID- 8613547 TI - Hepatic and neuromuscular forms of glycogen storage disease type IV caused by mutations in the same glycogen-branching enzyme gene. AB - Glycogen storage disease type IV (GSD-IV) is an autosomal recessive disease resulting from deficient glycogen-branching enzyme (GBE) activity. The classic and most common form is progressive liver cirrhosis and failure leading to either liver transplantation or death by 5 yr of age. However, the liver disease is not always progressive. In addition, a neuromuscular type of the disease has been reported. The molecular basis of GSD-IV is not known, nor is there a known reason for the clinical variability. We studied the GBE gene in patients with various presentations of GSD-IV. Three point mutations in the GBE gene were found in two patients with the classical presentation: R515C, F257L, and R524X. Transient expression experiments showed that these mutations inactivated GBE activity. Two point mutations, L224P and Y329S, were detected in two separate alleles of a patient with the nonprogressive hepatic form. The L224P resulted in complete loss of GBE activity, whereas the Y329S resulted in loss of approximately 50% of GBE activity. The Y329S allele was also detected in another patient with the nonprogressive form of GSD-IV but not in 35 unrelated controls or in patients with the more severe forms of GSD-IV. A 210-bp deletion from nucleotide 873 to 1082 of the GBE cDNA was detected in a patient with the fatal neonatal neuromuscular presentation. This deletion, representing the loss of one full exon, was caused by a 3' acceptor splicing site mutation (ag to aa). The deletion abolished GBE activity. Our studies indicate that the three different forms of GSD-IV were caused by mutations in the same GBE gene. The data also suggest that the significant retention of GBE activity in the Y329S allele may be a reason for the mild disease. Further study of genotype/phenotype correlations may yield useful information in predicting the clinical outcomes. PMID- 8613548 TI - Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation. AB - Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation. PMID- 8613549 TI - Induction of Fanconi anemia cellular phenotype in human 293 cells by overexpression of a mutant FAC allele. AB - The polypeptide encoded by the Fanconi anemia (FA) complementation group C gene, FAC, binds to a group of cytoplasmic proteins in vitro and may form a multimeric complex. A known mutant allele of FAC resulting from the substitution of Pro for Leu at codon 554 fails to correct the sensitivity of FA group C cells to mitomycin C. We reasoned that overexpression of the mutant protein in a wild-type cellular background might induce the FA phenotype by competing with endogenous FAC for binding to the accessory proteins. After stable transfection of 293 cells with wild-type and a mutant FAC allele containing the L554P substitution, four independent clones that expressed four-to-fifteen fold higher levels of transcript from the mutant transgene relative to the endogenous FAC gene showed hypersensitivity to mitomycin C. By contrast, both parental and FAC overexpressing cells maintained their relative resistance to mitomycin C. No differences in the biosynthesis, subcellular localization and protein interactions of the normal and mutant proteins were detected. The induction of the FA phenotype in this system is compatible with the competition hypothesis and provides support for a functional role of the FAC-binding proteins in vivo. PMID- 8613551 TI - Volume regulation in the bovine lens and cataract. The involvement of chloride channels. AB - The purpose of this study was to investigate volume regulation in the lens and its involvement in lens opacification (cataract) and the role of chloride channels in these processes. Single, isolated lens fiber cells from the lens were whole cell patch clamped. When exposed to hypotonic solution, and outwardly rectifying whole-cell current was activated. The current increased from 1.0 to 32.6 pA/pF, reversed at the chloride reversal potential (Ec1 = O mV), and was blocked by the chloride channel blockers 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB) and tamoxifen. Replacing all but 5mM of the external chloride with gluconate caused the reversal potential to shift +33 mV, consistent with a CL- current with a gluconate/chloride permeability ratio of 0.26. When the whole lens of the eye was exposed to hypotonic solution, there was an initial increase in anterior-posterior diameter (5-8 min), representing lens swelling of 6.5%. This was followed by a decrease in volume to a new steady state value that lasted for up to 2 h. In the longer term (> or = 2h), the lenses began to swell again. The simultaneous exposure to hypotonic solution and tamoxifen or NPPB caused swelling and prevented this volume regulation. Lenses incubated in hypotonic solution and hypotonic solution containing tamoxifen becane ipaque after a 2-h incubation period. We conclude that the lens is able to volume regulate. It possesses volume-activated Cl- channels, the inhibition of which results in inhibition of volume regulation, lens swelling and opacification. Our data suggest the long-term prophylactic use of tamoxifen may make the patient more susceptible to cataract. PMID- 8613550 TI - Regulatory effects of endogenous interleukin-1 receptor antagonist protein in immunoglobulin G immune complex-induced lung injury. AB - IL-1 receptor antagonist (IL-1Ra) has regulatory effects on IL-1 activity both in vitro and in vivo. In the IgG immune complex model of lung injury in rats, exogenously administered human IL-1Ra suppressed neutrophil recruitment and ensuing lung injury. In this study, we sought to determine if endogenous rat IL 1Ra might regulate this lung-inflammatory response. By Northern blot analysis of lung mRNA and Western analysis of bronchoalveolar lavage (BAL) fluids, rat IL-1Ra expression was found to increase during development of inflammation in IgG immune complex-mediated alveolitis. By immunostaining, alveolar macrophages and recruited neutrophils were the apparent sources of IL-1Ra. In vivo blocking of endogenous IL-1Ra resulted in a 53% increase in lung vascular permeability and a 180% increase in BAL fluid neutrophils. In companion studies, a significant increase in IL-1beta was found, whereas no significant change in TNF-alpha activity was observed. Whereas the in vivo regulatory effects of IL-1R appear to be limited to IL-1beta, IL-10 regulates both IL-1beta and TNF-alpha in this model, reflected by a 48% increase in BAL IL-1beta in rats treated with anti-IL 10. These findings suggest that IL-1Ra is an intrinsic regulator of inflammatory injury after deposition of IgG immune complexes and that it regulates production of IL-1beta. PMID- 8613552 TI - Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. AB - Highly reactive oxygen species rapidly inactivate nitric oxide (NO), and endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P selectin. Mixing experiments showed that the patients'platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H2O2 generation in one patient and a 3.4-fold increase in the second patient, both compared woth control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans. PMID- 8613553 TI - A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I. AB - Tryptase is the major protein constituent of human mast cells, where it is stored within the secretory granules as a fully active tetramer. Two tryptase genes (alpha and beta) are expressed by human mast cells at the level of mRNA and protein, each with a 30 amino acid leader sequence. Recombinant precursor forms of human alpha- and beta-tryptase were produced in a baculovirus system, purified, and used to study their processing. Monomeric beta-protryptase first is shown to be intermolecularly autoprocessed to monomeric beta-pro'tryptase at acid pH in the presence of heparin by cleavage between Arg-3 and Val-2 in the leader peptide. The precursor of alpha-tryptase has an Arg-3 to Gln-3 mutation that precludes autoprocessing. this may explain why alpha-tryptase is not stored in secretory granules, but instead is constitutively secreted by mast cells and is the predominant form of tryptase found in blood in both healthy subjects and those with systemic mastocytosis under nonacute conditions. Second, the NH2 terminal activation dipeptide on beta-pro'tryptase is removed by dipeptidyl peptidase I at acid pH in the absence of heparin to yield an inactive monomeric form of tryptase. Conversion of the catalytic portion of beta-tryptase to the active homotetramer at acid pH requires heparin. Thus, beta-tryptase homotetramers probably account for active enzyme detected in vivo. Also, processing of tryptase to an active form should occur optimally only in cells that coexpress heparin proteoglycan, restricting this pathway to a mast cell lineage. PMID- 8613554 TI - Role of K+ ATP channels and adenosine in the regulation of coronary blood flow during exercise with normal and restricted coronary blood flow. AB - Regulation of coronary vasomotor tone during exercise is incompletely understood. We investigated the contributions of K+ ATP channels and adenosine to the coronary vasodilation that occurs during exercise in the normal heart and in the presence of a coronary artery stenosis. Dogs that were chronically instrumented with a Doppler flow probe, hydraulic occluder, and indwelling catheter on the left anterior descending coronary artery were exercised on a treadmill to produce heart rates of approximately 200 beats/min. By graded inflation of the occluder to produce a wide range of coronary stenosis severities, we determined the coronary pressure-flow relation. K+ atp channel blockade with intracoronary glibenclamide (10-50 microgram/kg per min) decreased coronary blood flow during exercise at coronary pressures within and below the autoregulatory range, indicating that coronary K+ ATP channel activation is critical for producing coronary vasodilation with either normal arterial inflow or when flow is restricted by a coronary artery stenosis. Adenosine receptor blockade with intravenous 8-phenyltheophylline (5 mg/kg) had no effect on coronary flow at pressures within the autoregulatory range but decreased flow at pressures < 55 mmHg. In contrast, in the presence of K+ ATP channel blockade, the addition of adenosine receptor blockade further decreased coronary flow even at coronary pressures in the autoregulatory range, indicating increased importance of the vasodilator influence of endogenous adenosine during exercise when K+ atp channels are blocked. Intracoronary adenosine (50 microgram/kg per min) increased coronary flow at perfusion pressures both within and below the autoregulatory range. In contrast, selective K+ ATP channel activation with intracoronary pinacidil (0.2-5.0 microgram/kg per min) increased flow at normal but not at lower coronary pressures (< 55 mmHg). This finding demonstrates that not all K+ ATP channels are activated during exercise at pressures in the autoregulatory range, but that most K+ ATP channels are recruited as pressures approach the lower end of the autoregulatory plateau. Thus, K+ ATP channels and endogenous adenosine play a synergistic role in maintaining vasodilation during exercise in normal hearts and distal to a coronary artery stenosis that results in myocardial hypoperfusion during exercise. PMID- 8613555 TI - The width of radiologically-defined attached gingiva over deciduous teeth. AB - The aim of the present cross-sectional study was to radiologically investigate the continuous eruption process in the deciduous dentition by assessing the difference in the width of radiologically-defined attached gingiva (RAG) in 6 year-old and 10-year-old children. The group of 6-year-olds had predominantly deciduous teeth and the 10-year-olds were in their mixed dentition period. The mucogingival junction was revealed with Schiller's iodine solution and marked mid labially along the long axis of each tooth with a piece of metal wire prior to taking panoramic radiographs. The width of RAG over deciduous canines and molars was measured from the cementoenamel junction to the mucogingival junction. A significant increase in the width of RAG was found from 6 to 10 years of age. PMID- 8613556 TI - Ultrastructure of hypersensitive and non-sensitive dentine. A study on replica models. AB - Patients with cervical dentine exposures do not always complain of dentine hypersensitivity. The identification of morphological features connected to symptoms is important for both prevention and management of dentine hypersensitivity. The aim of the study was to determine the relationship between the dentine ultrastructure and clinical symptoms in patients with cervical dentine exposures. Replica models for a total of 28 teeth (from 10 patients) with cervical non caroius lesions and dentine exposures were obtained before and after acid etching. The replica models were studied blindly in the scanning electron microscope (SEM). 12 of these were classified clinically as hypersensitive and 16 as non-sensitive. Because of the morphological heterogeneity of all specimens, a grid was used to isolate smaller, more homogeneous areas. 120 (75 non-sensitive and 45 hypersensitive areas) grid areas randomly selected from the 28 dentine surfaces were analyzed. The presence and morphology (amorphous or crystalline) of smear layers and the density and diameter of dentine tubules were recorded. The chi 2 test was used for statistical analysis. In unetched specimens, the surfaces classified as non-sensitive were frequently coated by an amorphous smear layer (88.0%) and occasionally by a crystalline smear layer (2.7%). Only a few and narrow tubules were observed (9.3%). On the other hand, the unetched specimens of the hypersensitive dentine exhibited less frequently (31.3%) an amorphous smear layer. The presence of crystalline smear layer (33.3%), many and wider patent tubules and, sometimes, loss of intertubular dentine manifested as grooves between tubules, were observed (35.6%). The differences were significant (p < .001). In hypersensitive dentine, the acid etching always removed the smear layer whereas removal in non-sensitive dentine was partial or absent. These morphological findings underline the role of smear layer in reducing permeability of dentine in patients with dentine sensitivity. PMID- 8613557 TI - Crevicular fluid osteocalcin and pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) as markers of rapid bone turnover in periodontitis. A pilot study in beagle dogs. AB - The objective of this study was to correlate the levels of 2 putative markers of bone metabolism, namely osteocalcin and pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), to the progression of experimental alveolar bone loss in the beagle dog. 36 control sites and 36 experimental sites in 2 beagle dogs were assessed longitudinally at 2-week intervals for gingival crevicular fluid (GCF) osteocalcin and ICTP levels during a 6-month observation period. Analysis of osteocalcin and ICTP in GCF was performed by RIA. During the study, bone-seeking radiopharmaceutical uptake (BSRU) of 99mTc-MDP was assessed monthly; standardized radiographs were taken at 2-week intervals. The results showed osteocalcin and ICTP levels in GCF increased significantly (p < 0.05) by 2 weeks following initiation of disease. This increase preceded significant increases in BSRU by 2 weeks and radiographic evidence of bone loss by 4 weeks. BSRU was significantly elevated (p < 0.05) at experimental sites as compared to controls at 4 and 8 weeks post-disease initiation. Osteocalcin in GCF peaked 8 and 10 weeks after ligature placement in experimental sites at levels nearly 10 fold greater than contralateral paired control sites. ICTP levels in GCF remained elevated throughout the entire disease progression phase. Following the removal of ligatures, both GCF osteocalcin and ICTP levels dropped precipitously approaching control values. Osteocalcin revealed overall a positive predictive value (PPV) and negative predictive value (NPV) for future bone loss during disease progression of 0.87 and 0.34, respectively, while ICTP showed both high PPV and NPV of 0.87 and 0.91 respectively. Results from this study in the dog model indicate that osteocalcin and especially ICTP relate to indices of active periodontal bony destruction and suggest that these molecules may serve as predictive markers for future alveolar bone loss. PMID- 8613558 TI - Management of furcation-involved teeth. A retrospective analysis. AB - In the present study, data from more than 550 periodontally diseased patients with more than 1100 furcation invasions were retrospectively analysed. There were apparent differences in the distribution of different furcation degrees in patient populations treated by 2 differently experienced operators. However, treatment modality patterns were rather similar. Scaling during periodontal flap surgery was the most often performed treatment procedure in degree I (97-98%) and II (75-83%) involvements. About 44% of degree III involved teeth were extracted. In order to determine the influence of degree of furcation involvement, tooth type and operator variability on treatment modality, logistic regression analysis was applied. Degree of furcation involvement was an important indicator variable in all models. Scaling as a sole measure was mainly performed in relation to degree I of furcation involvement. With every increase in degree, the odds of scaling decreased by factor 12.7. The odds of root resection was upper 1st molars 46 x higher than in wisdom teeth or lower 2nd molars with the same degree of involvement, but only 3.3 x higher than in lower 1st molars. Tunnel preparation as well as regenerative procedures were mainly confirmed to lower molars. Operator variability was only introduced as a covariate in the extraction model. hence, despite of different operator skill and severity of periodontal disease in treated populations, decision for one or the other treatment modality seems to depend essentially on degree of furcation involvement as well as tooth type. PMID- 8613559 TI - Intra - and inter-examiner reproducibility in constant force probing. AB - This study evaluated intra- and inter-examiner reproducibility for a conventional manual probe versus a computer-interfaced force-controlled periodontal probe. 2 examiners recorded probing depths (PD) and relative attachment levels (AL) at 1128 sites in 15 periodontal maintenance patients. Each site was evaluated 2x, 7 to 10 days apart by both examiners. Probing force for the electronic probe was 15 g. PD intra-examiner reproducibility (within +/- 1.0 mm) for shallow sites (PD < or = 3 mm) was 98.6% versus 91.5% for the conventional versus the electronic probe for examiner 1 and 98.5% versus 88.7% for examiner 2. Corresponding values for deeper sites (PD > 3 mm) were 96.4% versus 85.9% for examiner 1 and 95.1% versus 77.0% for examiner 2. Generally, AL intra-examiner reproducibility was 1 to 3% lower than for PD. PD inter-examiner reproducibility (within +/- 1.0 mm) was 99.2% versus 90.7% for the conventional versus the electronic probe, respectively, for shallow sites and 95.4% versus 76.9% for deeper sites. AL inter examiner reproducibility (within +/- 1.0 mm) was 1 to 5% lower than for PD. Both intra- and inter-examiner reproducibility was higher for anterior than for posterior sites. Mean PD and AL were similar for both examiners. However, the electronic probe consistently recorded 0.1 to 0.2 mm higher values than the conventional probe. Standard deviations indicated a greater variability for electronic than for manual probing. The results suggest that intra- and inter examiner reproducibility may not necessarily be higher with an electronic, force controlled periodontal probe than with a conventional manual probe. PMID- 8613560 TI - The comparative tea staining potential of phenolic, chlorhexidine and anti adhesive mouthrinses. AB - Staining of teeth and mucous membranes is a well-known side-effect with chlorhexidine mouthrinses in which dietary chromogens play an important role. The purpose of this study was to determine whether a co-polymer anti-adhesive agent would prevent staining by a low concentration chlorhexidine solution. Additionally, the possibility that an essential oil/phenolic rinse product may cause staining was investigated. The rinses studied were the anti-adhesive alone and combined with 0.02% chlorhexidine and the essential oil/phenolic rinse. These were positioned against a positive control rise, 0.2% chlorhexidine, and a negative control rinse, water. The study was a single blind 5-treatment, randomised Latin square cross-over design, incorporating balance for carry-over effects. 15 volunteers participated and on Day 1 of each study period were rendered stain free by scaling and polishing of the teeth. Oral hygiene was suspended and 8 x per day subjects rinsed under supervision, firstly with the allocated formulation and then with 10 ml of warm black tea. On Day 4, tooth and tongue staining was scored by area and intensity (colour). A washout period of at least 3 1/2 days was permitted between treatment periods when oral hygiene was resumed. Before the study and during washouts, volunteers practised tongue brushing. Tooth and tongue staining was significantly increased with 0.2% chlorhexidine compared to the essential oil/phenolic rinse which in turn was significantly increased compared to the other 3 rinses. The antiadhesive/chlorhexidine rinse produced no more staining than the anti-adhesive or water rise. However, the parallel plaque regrowth study suggests this inhibition of staining resulted from the vitiation of the chlorhexidine activity by the antiadhesive. The methodology would appear a simple and quick way of assessing the propensity of mouthrinses to cause extrinsic staining. PMID- 8613561 TI - The comparative effects on plaque regrowth of phenolic chlorhexidine and anti adhesive mouthrinses. AB - The inhibition of bacterial attachment to the tooth surface is one possible approach to plaque control. This study evaluated in vivo the plaque inhibitory action of a novel copolymer reported to have considerable antiadhesive properties in vitro. The study was a single blind, 5-treatment, randomised Latin square crossover design, incorporating balance for carry-over effects. The rinses were the antiadhesive (1%), the antiadhesive with 0.02% chlorhexidine, a 0.2% chlorhexidine rinse product, an essential oil/phenolic rinse product and water. 15 volunteers participated and on day 1 of each study period were rendered plaque free, ceased toothcleaning and rinsed 2 x daily, under supervision, with the allocated formulation. On day 5, plaque was scored by index and area. Washout periods were 2 1/2 days. Alone or combined with chlorhexidine, the antiadhesive agent showed no effects greater than water. The chlorhexidine rinse was significantly more effective than the essential oil/phenolic rinse which in turn was significantly more effective than the other rinses. PMID- 8613562 TI - Neutrophil elastase in crevicular fluid: comparison of a middle-aged general population with healthy and periodontitis groups. AB - Neutrophil elastase (NE) was measured in crevicular fluid (GCF) collected from 3 subject groups. GCF was harvested at a single visit of subjects with periodontal health (n = 21) and with periodontitis (n = 28). Samples were obtained from 132 middle-aged, middle-class health conscious patients of a health maintenance organization (HMO) at baseline and 1 year later. GCF NE was higher in periodontitis than in health. Mean GCF NE of HMO subjects was much closer to health than to periodontitis. Few members of the HMO population had enzyme levels typical of periodontitis. Subjects and sites of the HMO population were segregated into 3 categories based on enzyme levels of the healthy and periodontitis subjects. Most HMO subjects and sites were in the activity category corresponding to healthy subjects. Only a small portion were in the activity category common in periodontitis. Enzyme levels in the highest activity category at both samplings were infrequent. High enzyme levels in the HMO population were not associated with attachment loss. Thus, assay of GCF NE provided little evidence of disease in a middle-aged, middle-class health conscious general population. This finding confirms an analysis of epidemiological surveys which concluded that a population such as studied here would not benefit from periodontal diagnostic testing. PMID- 8613563 TI - The pharmacokinetics of phenytoin in gingival crevicular fluid and plasma in relation to gingival overgrowth. AB - The aim of this study was to determine whether phenytoin (PHT) could be detected in gingival crevicular fluid (GCF), and to relate its concentration to both plasma level and degree of gingival overgrowth. 23 patients medicated with phenytoin for at least 6 months were clinically examined for signs of periodontal disease and gingival overgrowth. 12 patients out of these demonstrated clinically significant overgrowth and their plaque scores and gingival inflammation were greater than for the non-overgrowth group (p < 0.001). Phenytoin concentrations were determined by high performance liquid chromatography, and was detected in GCF. There was a significant correlation between the GCF and plasma phenytoin concentrations (p < 0.05), but it was not related to the extent of gingival overgrowth. Inflammation increased the GCF volume, but was not a determinant of GCF phenytoin concentration. It is concluded that effusion of phenytoin into GCF is regulated by the plasma levels of the drug, but its concentration in GCF is not related to the incidence of gingival overgrowth. PMID- 8613564 TI - Periodontal healing and periopathogenic microflora in smokers and non-smokers. AB - The aim of the present study was to monitor the clinical and microbiological effects of non-surgical therapy in smokers and non-smokers. The subject material included 32 patients (age range 32-61 years), 11 men and 21 women with moderate to severe periodontitis. 17 patients were smokers ( > or = 15 cigarettes/day) and 15 non-smokers. All patients were subjected to non-surgical periodontal therapy performed by a dental hygienist. Periodontal variables (plaque index, gingival index and probing depth) were registered and bacterial samples were collected before and 2 months after treatment. The treatment resulted in significant reductions towards very low plaque and gingival indices in smokers and non smokers alike (p < 0.05). Although probing depth was reduced in both smokers and non-smokers, the probing pocket depth reduction was significantly smaller in smokers than non-smokers (p < 0.05). Microbiologically, the same therapeutical efficacy was attained in both smoking groups, indicating an almost total eradication of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Concerning Prevotella intermedia, out of 14 smokers and 10 non-smokers positive at baseline, 9 and 5, respectively, remained positive after treatment. The results suggest a less favourable clinical outcome of non-surgical therapy in smokers than non-smokers in spite of the fact that the therapy was equally effective with regard to reducing the alleged periopathogens A. actinomycetemcomitans, P. gingivalis and P. intermedia. PMID- 8613565 TI - Effect of gingival wall on resistance to probing forces. AB - This study was conducted to determine whether the gingival tissue lateral to the periodontal sulcus contributes resistance to the advancement of a periodontal probe tip into the sulcus under different applied pressures. An electromechanical device was used to advance a probe tip 0.6 mm in diameter into the facial sulcus at a constant speed until resisting forces of 0.70 N were encountered. The device registered the resisting force and probe advancement simultaneously. The gingiva of all 2nd incisors, 2nd premolars, and 1st molars of 4 young adult male beagle dogs were tested. After the first measurement, the buccal gingiva of experimental sites were incised mesiodistally from the gingival margin to the alveolar crest and the sulcus was reprobed. 2 experimental and control quadrants were selected randomly resulting in 6 sets of both experimental and control data from each animal. The data were analyzed with analysis of variance. The analysis demonstrated significant variation from site to site, and dog to dog; therefore, only changes between the 1st and 2nd probings at the same sites could be compared. Less variability of probing distance in different animals occurred at higher forces; however, the incision had a significant effect on probing distance at these forces. The pressure at which probing distance had less variability among animals and least affected when the gingival sulcular wall was incised was estimated to be 106 N/cm2. This corresponds to 30 g force on the 0.6 mm diameter probe. PMID- 8613566 TI - Future dental human resource needs--a qualitative approach. AB - As part of the National Oral Health Survey, dentists in private practice were asked to give their views and opinions on the future planning of human resource needs for South Africa. The data, which were qualitative in nature, yielded 2600 responses. After computing a frequency distribution of these responses it was possible to classify these data into five major categories. These were a) "too many dentists/fewer should be trained" b) "more dentists for developing areas" c) "more auxiliaries in general" d) "greater need for preventive and health education services" and e) "high cost of running and maintaining private dental practices". The predominant view was that fewer dentists and more auxiliaries should be trained, while the need for preventive and health education services received considerably less support. PMID- 8613568 TI - Intra-oral camera systems. PMID- 8613567 TI - Oral health, oral health care and dental services--the consumer perspective. AB - As part of a National Oral Health Survey conducted in 1988/89, community knowledge of and attitudes towards oral health and oral health care were examined in the various population groups in South Africa. A wide range of issues were explored. These included amongst others, help-seeking and oral health behaviour, sources of health information and attitudes to dentists and dental care. Given the major political change that has recently occurred in the country, the results of the survey suggest that these findings could profitably be used in future dental personnel planning as well as in the re-structuring of the health services that is currently taking place. PMID- 8613569 TI - Communication...the basis of good practice management. PMID- 8613570 TI - Policy statement on dental amalgam. PMID- 8613571 TI - A new cohesive gold foil: two case reports. PMID- 8613572 TI - Private dental practitioners--perceptions of needs and barriers to dental care. AB - The purpose of this paper was to examine needs and barriers to dental care as perceived by private dental practitioners in South Africa. In 1988/89 a National Oral Health survey was conducted. This included a questionnaire sent to 3200 dental practitioners. The items examined in this paper were part of this questionnaire. Fifty six and 29 per cent of practitioners considered that there was a shortage of dentists for black and coloured persons respectively and only 4 per cent perceived a shortage for whites. Twenty two per cent of respondents believed that there was no shortage of dentists for black patients. There was however, a high percentage of "don't know" responses. An analysis of the type of treatment provided indicated that 60 per cent of dentists spend more than 60 per cent of their time on performing extractions for black patients and minimal time on prevention. The main barriers to dental care were seen as attitudinal, structural and dentist-related. Dentist perceptions of these barriers highlight the role non-dental factors play in dentistry and oral health. These factors need to be seriously considered in order to improve the nature and quality of, and accessibility to dental care. PMID- 8613573 TI - Private dental practitioners in South Africa--a professional profile. AB - A questionnaire submitted to dentists in private practice was part of the first National Oral Health Survey 1988/89. The questionnaire investigated demographic and professional characteristics. Completed questionnaires were returned by 2224 dentists (69.5 per cent). Most respondents were located in the 5 main metropolitan areas. There was a paucity of Black and Coloured dentists. Male dentists dominated the profession with almost 74 per cent under 50 years of age. General practitioners made up over 90 per cent of the profession and orthodontists were the largest group of specialists. Conservation was ranked as the most frequently practised service and preventive procedures were ranked low. Medical aid systems were seen to have some disadvantages. Fees and working conditions were felt to be better in private practice than in the public sector. PMID- 8613574 TI - Disorders of the temporomandibular joint: a review of anatomical concepts. PMID- 8613575 TI - Prevalence and severity of fluorosis in the primary and permanent dentition using the TSIF. AB - This paper reports on the prevalence and severity of dental fluorosis in the primary and permanent teeth and the tooth surfaces of 262 school children aged 6 18 years from two communities in KwaNdebele (South Africa), the first with 8-9 ppm F-, and the second with 0.6-1.6 ppm F- in the drinking water. The Tooth Surface Index of Fluorosis (TSIF) was used to measure the degree of fluorosis. In both areas, the population prevalence, for primary and permanent teeth was more than 90 per cent. For the lower F- area, 40 per cent of tooth surfaces were free of fluorosis compared with only 22 per cent in the high F- area. Of the surfaces affected by fluorosis in the high F- area, 37 per cent scored in the categories 4 7, compared with 6 per cent in the lower F- area. This difference in severity was found to be statistically significant (P<0.001). This study has shown that, in two areas with significantly different levels of fluoride in the water supply, the population prevalences of fluorosis were similar, but significant differences existed in severity. The World Health Organization 1984 guidelines for Drinking water Quality recommend that 1.5 ppm F- is the maximum acceptable concentration in drinking water but this may not be appropriate for South Africa. PMID- 8613576 TI - Re-establishing occlusal anatomy: a new technique for direct tooth coloured restorations. PMID- 8613577 TI - Sandblasting and tin-plating techniques to enhance bonding to cast metal restorations. PMID- 8613578 TI - Improving practice profits. Part I ... A resume. PMID- 8613579 TI - Cephalometric analysis of a Sotho-Tswana group. AB - A cephalometric study was conducted on 50 male and 54 female Sotho-Tswana children 11-16 years of age. The subjects were selected for excellence of occlusion and balance of facial proportions. Various analyses, including Steiner, Wits, and McNamara, were used for the evaluations. The mean measurements were compared with those of other Southern African Black groups and Caucasoids. Our study shows that cephalometric analyses for other groups cannot be used with confidence for the Sotho-Tswana people and our data provides norms for them. PMID- 8613580 TI - Water fluoridation in South Africa: will it be effective? AB - The National Oral Health Survey showed that differences in the prevalence and experience of dental caries occurred between racial groups and between geographical areas according to fluoride levels in the domestic water supply that varied from <0.05 ppm in the coastal areas to 0.35 ppm in the Bloemfontein area. The Orange Free State (OFS) Goldfields were not included in the survey. The fluoride content of the water supply to this community varies from 0.2 to 0.9 ppm, with a long term average of 0.54 ppm. A survey was conducted in this area to determine the effect of the raised fluoride level on the caries levels in Black and White children. The results show that caries levels in Black children in the OFS Goldfields are >50 per cent and >80 per cent lower than that found in the Bloemfontein and coastal areas respectively. In the OFS Goldfields 39 per cent of White and 16 per cent of Black 12 year old children presented with dental fluorosis on the maxillary central incisors. The results of this study seem to indicate that Black children will benefit more from water fluoridation than White children. PMID- 8613581 TI - Effectiveness of two school-based caries preventive programmes. AB - Although the efficacy of school-based preventive programmes is well established, their effectiveness under conditions prevailing in the community require further exploration. A longitudinal study was conducted to assess the community effectiveness of two school-based caries preventive programmes. A group of 150 primary school pupils, 10-15 years old were randomly selected from each of three primary schools (Dagbreek, Vergesig, De Villiers) in the Boland town of Robertson. The sample comprised 75 10-12 year-olds and 25 in each of the 13, 14 and 15 year age groups. The 75 (10-12 year-olds) constituted the experimental cohort, which was to be followed up over a period of 3 years and compared to the 13-15 year-olds, the control or baseline cohort. The experimental cohort in Vergesig was exposed to a daily toothbrushing programme with a fluoride dentifrice, the Dagbreek children were exposed to the same regimen plus a weekly 0.2 per cent fluoride rinse and the De Villiers cohort acted as a control group for contamination and co-intervention. The DMFT index (WHO, 1987) was used as a caries measure. Dagbreek showed a reduction of 0.96 DMFT (14.2 per cent) and Vergesig of 1.85 DMFT (24.5 per cent). The difference is attributed to a higher baseline DMFT at Vergesig. The weekly fluoride rinse did not confer any additional benefit. Although the caries reduction was lower than that generally found in clinical trials, the two intervention programmes were cost-effective. PMID- 8613582 TI - Failure of dental local anaesthetic cartridges under pressure. AB - The objective of this study was to determine the pressures that caused cartridge failure (leakage or fracture) of six different dental local anaesthetics available in South Africa. Forty glass cartridges of each local anaesthetic were placed in an apparatus and subjected to pressure testing using two different plungers. Failure of the cartridges was observed visually as well as on the computer screen of the testing apparatus. In addition the compressibility of local anaesthetic rubber stoppers (bungs) was measured. The data obtained was analysed by means of a two-way analysis of variance and a chi-square test. The results from this study showed that there were statistically significant differences in the mean pressures that caused failure of local anaesthetic cartridges available on the South African market. PMID- 8613583 TI - Treatment of severe fluorosis with the aid of modern techniques: a case report. PMID- 8613584 TI - Bond strength values: should they be considered in material selection? AB - This study determined shear bond strengths of 4 glass ionomer cements under standardized conditions and compared the results with data derived from the product manufacturers. Flat dentine planes were prepared on the buccal surfaces of 40, unerupted, surgically removed, human third molars. Ten cylinders of Fuji II LC (FII), Aqua Ionofil (AI), Variglass VLC (VA) and Vitremer (VI) were bonded to dentine surfaces. The specimens were stored in distilled water for 96 hours and their shear bond strengths were then determined using an Instron testing machine. FII LC achieved the highest (15 MPa) and VA the lowest (2 MPa) mean shear bond strength values and those for Aqua Ionofil and Vitremer were 5 to 6 MPa. The manufacturers have reported lower bond strengths for FII and AI and higher values for VI and VA. This study showed that in view of differences in test methods bond strength test values are not comparable and we recommend that they should not be used as the only criterion for material selection. PMID- 8613585 TI - A profile of South African orthodontists: NOHS 1988/89. AB - The response by orthodontists to a 1988/89 National Oral Health Survey questionnaire is reported. The 54 predominantly white male respondents reflected a balanced age distribution. The majority were in solo private practice. Almost half employed oral hygienists, 96 per cent worked between 40 and 49 weeks per year and 61 per cent worked between 35 and 44 hours per week. Half were satisfied with their busyness and the remainder were evenly divided between being too busy or not busy enough. The majority of patients were white. It is recommended that imbalances in race and gender amongst orthodontists be redressed. PMID- 8613586 TI - Influencing the delivery of primary oral health care at community, district, provincial and national levels. PMID- 8613587 TI - Salvation of a failed three unit ceramo-metal bridge. PMID- 8613588 TI - The use of tricure glass ionomer cement as an apical sealant after apicoectomy. AB - The adaptation and sealing ability of a tricure glass ionomer material (Vitremer), used in a retrograde cavity was assessed and compared with amalgam. Fifty single-rooted, extracted teeth were prepared and filled endodontically. All teeth underwent root resection and retrograde cavities were prepared. The teeth were divided into two groups. One group of 10 teeth received a layer of varnish and an amalgam filling in a retrograde cavity, while the other group of 40 teeth received a layer of primer and a glass ionomer filling. All the teeth were placed in an aqueous solution of Procion Brilliant Blue for 7 days whereafter ground sections were prepared. Micro leakage was determined according to the extend of dye penetration using an image analysis system. The results showed that significantly less dye penetration was observed in teeth filled with the glass ionomer cement than in those with the amalgam. PMID- 8613589 TI - The resistance to deformation forces of endodontic posts manufactured from six different materials. AB - Twenty posts of 1.25 mm diameter and 10 of 1.0 mm diameter were cast in 4 different metals, namely, Argenco 4, Argenco 9, Wiron 99 and Rexillium V (5 each). These posts as well as pre-formed wrought stainless steel and titanium posts (5 each of the same diameter) were tested in an Instron machine to a deformation point of one millimetre. Of the cast posts, those of Rexillium proved to be the most rigid but could only withstand 68 per cent of the force that the wrought posts could resist. No statistically significant differences were found between the wrought posts of stainless steel and titanium and between gold and Argenco 9 in the 1.25 mm category and between stainless steel and titanium and Wiron 99, Argenco 9 and gold in the 1.0 mm category. PMID- 8613590 TI - WHO/FDI consensus statement on dental amalgam. PMID- 8613592 TI - Exploring the applications of the Internet: a guide for the first time user. PMID- 8613591 TI - A team approach to hypersensitive dentine and gingival recession. PMID- 8613593 TI - The cost of cross infection control. PMID- 8613594 TI - Private medical schemes: dental claims during 1989 and 1990. AB - In this investigation, claim patterns of members of 6 private medical aid schemes were studied. The number of members per scheme, the maximum benefits per member, the number of members with no claims, the age distribution of members with no dental claims and the percentage of total paid out per discipline are shown. The percentage of principal members who made no dental claims from these 6 medical schemes ranged between 53.6 - 75.5 per cent in 1989 and between 67.1 - 85 per cent in 1990. In this article we seek possible explanations for these unexpected findings. PMID- 8613595 TI - Dental status of rural school children in a sub-optimal fluoride area. AB - Past investigations by us have shown high levels of caries experience in rural coloured school children of the Western Cape. These studies were all undertaken in fluoride poor areas. In contrast studies of children in areas with optimal and higher concentrations of fluoride in their drinking water have shown lower DMFT scores. On a previous visit to the town of Fraserburg in the North Western Cape we observed fluorosis in children, although the fluoride content of the municipal drinking water was sub-optimal (0.68-0.78 ppm F-). The purpose of this study was therefore to measure the dental status of 6, 12 and 15 year old school children using the dmft, DMFT and Dean's Fluorosis Index according to the 1987 WHO guidelines. The examinations were done by two calibrated examiners using portable equipment. Results showed low mean dmft values of 3.31 +/- 3.90 and 0.22 +/- 0.86 for the 6 and 12 year olds respectively. The corresponding DMFT scores including that for 15 year olds were 0.08 +/- 0.35; 1.45 +/- 1.81 and 1.00 +/- 1.60 respectively and no significant statistical gender differences were observed (p>0.05). The mean fluorosis scores for 6, 12 and 15 year olds were 1.68 +/- 1.05; 2.78 +/- 1.34 and 2.90 +/- 1.58 respectively. For both the 12 and 15 year olds the severity of fluorosis ranged from no fluorosis to severe mottling and corrosion. It can be concluded that the results even at a sub-optimal fluoride level show a high similarity to the dental status of children in rural fluoride rich areas. Defluoridation of the Fraserburg municipal drinking water therefore becomes imperative and a concentration of 0.4 ppm F- is suggested. Furthermore the children have no access to dental services and this has led to accumulated needs which demand urgent addressing. PMID- 8613596 TI - Hygienic maintenance of dental handpieces and turbines. PMID- 8613597 TI - FDI/WHO agreed plan for co-operation. PMID- 8613598 TI - The danger of unplanned extractions. PMID- 8613599 TI - Preferred provider contracts and the law. PMID- 8613600 TI - Stents for intracoronary placement: current status and future directions. AB - The technique of intracoronary stenting has achieved remarkable progress over the last few years. Improved stent deployment techniques and optimization of postprocedural management have dramatically improved the safety of intracoronary stent placement. At present, the incidence of early vessel closure after stenting is even lower than that after standard angioplasty and, as most operators no longer prescribe aggressive anticoagulation, bleeding complications are uncommon. Stenting has become an extremely effective treatment for abrupt or threatened vessel closure or for any suboptimal angiographic result during conventional angioplasty. Furthermore, large prospective trials have demonstrated that its efficacy is superior to that of conventional angioplasty for primary restenosis prevention in focal lesions of some native coronary arteries. Ongoing trials tend to extrapolate these conclusions to saphenous vein graft lesions. Mechanical support of the vessel wall explains the sustained angiographic benefit observed after stenting. Future developments may include the use of stents as a vehicle for local drug delivery in an attempt to further reduce the incidence of restenosis. In view of these results, coronary stents should be considered a new standard therapeutic modality in interventional cardiology. PMID- 8613601 TI - Reocclusion: the flip side of coronary thrombolysis. AB - Since the introduction of thrombolytic therapy for acute myocardial infarction, the incidence of coronary artery reocclusion has been intensively studied. Also, the prediction and diagnosis of reocclusion by angiographic and clinical variables, as well its invasive and pharmacologic prevention, have gained much attention. By angiographic definition, reocclusion requires three angiographic observations: one with an occluded artery, one with a reperfused artery and a third for the assessment of subsequent occlusion (true reocclusion). Since the introduction of early intravenous reperfusion therapy, most studies use only two angiograms: one with a patent and one with a nonpatent infarct-related artery. A search for all published reocclusion studies revealed 61 studies (6,061 patients) with at least two angiograms. The median time interval between the first angiogram after thrombolysis and the second was 16 days (range 0.1 to 365). Reocclusion was observed in 666 (11%) of 6,061 cases. Interestingly, the 28 true reocclusion studies showed an incidence of reocclusion of 16 +/- 10% (mean +/- SD), and the 33 studies with only two angiograms 10 +/- 8% (p=0.04), suggesting that proven initial occlusion of the infarct-related artery is a risk factor for reocclusion after successful thrombolysis. The other predictors for reocclusion are probably severity of residual stenosis of the infarct-related artery after thrombolysis and perhaps the flow state after lysis. Reocclusion is most frequently seen in the early weeks after thrombolysis. The clinical course in patients with reocclusion is more complicated than in those without this complication. Left ventricular contractile recovery after thrombolysis is hampered by reocclusion. Routine invasive strategies have not been proven effective against reocclusion. In the prevention of reocclusion, both antiplatelet and antithrombin strategies have been tested, including hirudin and hirulog, but the safety of these agents in thrombolysis is still questionable. Thus, reocclusion after thrombolysis is an early phenomenon and is more frequent after proven initial occlusion of the infarct-related artery. Reocclusion can be predicted by angiography after thrombolysis. Because reocclusion is detrimental, strategies to prevent it should be developed and carried out after thrombolytic therapy for acute myocardial infarction as soon as they are deemed safe. PMID- 8613602 TI - Circadian variations of onset of acute myocardial infarction and efficacy of thrombolytic therapy. AB - OBJECTIVES: The present study investigated whether the onset of acute myocardial infarction and resistance to thrombolysis have similar circadian variations. BACKGROUND: Circadian variations of the onset of acute myocardial infarction and resistance to thrombolysis in the early morning have been reported. Some studies have also reported a secondary peak incidence in late evening; however, it is not known whether the resistance to thrombolysis has a similar circadian variation in these patients. METHODS: Six hundred eight Japanese patients with an acute myocardial infarction were the subjects of the study. Two hundred forty-four of the 608 patients were treated with thrombolysis within 12 h of the onset of symptoms. One hundred thirteen patients received urokinase, and 131 patients received tissue-type plasminogen activator (t-PA) over 60 min. Patency of the infarct-related artery, the primary end point of the study, was evaluated at 60 min after the initiation of thrombolytic therapy, and Thrombolysis in Myocardial Infarction (TIMI) grade 0, 1 or 2 was defined as resistant to thrombolysis. RESULTS: The onset of acute myocardial infarction and resistance to thrombolysis showed circadian variations with early morning and late evening peaks (p<0.001 and p<0.05, respectively). These circadian patterns showed similar distributions as evaluated with Spearman's method (r=0.70, p<0.05), although resistance to thrombolysis showed a phase difference of about 2 h earlier than the infarction incidence. The circadian variation of the resistance to thrombolysis was independent of the types of thrombolytic agents (urokinase or t-PA). CONCLUSIONS: These findings suggest that adjustment of treatment based on the time of the onset of symptoms may be warranted for the patients with acute myocardial infarction. PMID- 8613603 TI - Development and validation of a Bayesian model for perioperative cardiac risk assessment in a cohort of 1,081 vascular surgical candidates. AB - OBJECTIVES: This study sought to develop and validate a Bayesian risk prediction model for vascular surgery candidates. BACKGROUND: Patients who require surgical treatment of peripheral vascular disease are at increased risk of perioperative cardiac morbidity and mortality. Existing prediction models tend to underestimate risk in vascular surgery candidates. METHODS: The cohort comprised 1,081 consecutive vascular surgery candidates at five medical centers. Of these, 567 patients from two centers ("training" set) were used to develop the model, and 514 patients from three centers were used to validate it ("validation" set). Risk scores were developed using logistic regression for clinical variables: advanced age (>70 years), angina, history of myocardial infarction, diabetes mellitus, history of congestive heart failure and prior coronary revascularization. A second model was developed from dipyridamole-thallium predictors of myocardial infarction (i.e., fixed and reversible myocardial defects and ST changes). Model performance was assessed by comparing observed event rates with risk estimates and by performing receiver-operating characteristic curve (ROC) analysis. RESULTS: The postoperative cardiac event rate was 8% for both sets. Prognostic accuracy (i.e., ROC area) was 74 +/- 3% (mean +/- SD) for the clinical and 81 +/- 3% for the clinical and dipyridamole-thallium models. Among the validation sets, areas were 74 +/- 9%, 72 +/- 7% and 76 +/- 5% for each center. Observed and estimated rates were comparable for both sets. By the clinical model, the observed rates were 3%, 8% and 18% for patients classified as low, moderate and high risk by clinical factors (p<0.0001). The addition of dipyridamole-thallium data reclassified >80% of the moderate risk patients into low (3%) and high (19%) risk categories (p<0.0001) but provided no stratification for patients classified as low or high risk according to the clinical model. CONCLUSIONS: Simple clinical markers, weighted according to prognostic impact, will reliably stratify risk in vascular surgery candidates referred for dipyridamole-thallium testing, thus obviating the need for the more expensive testing. Our prediction model retains its prognostic accuracy when applied to the validation sets and can reliably estimate risk in this group. PMID- 8613604 TI - Meta-analysis of intravenous dipyridamole-thallium-201 imaging (1985 to 1994) and dobutamine echocardiography (1991 to 1994) for risk stratification before vascular surgery. AB - OBJECTIVES: This study evaluated the prognostic value of abnormal test results with pharmacologic stress with regard to perioperative and long-term outcomes in a large population of candidates for vascular surgery. BACKGROUND: Although numerous studies have demonstrated the prognostic value of dipyridamole-thallium 201 myocardial perfusion and dobutamine echocardiography in vascular surgery candidates, a synopsis of predictive estimates is difficult because of individual study variability in pretest clinical risk, sample size and study design. METHODS: A systematic review of published reports on preoperative pharmacologic stress risk stratification from the MEDLINE data base (1985 to 1994) identified 10 reports on dipyridamole-thallium-201 myocardial perfusion (1,994 patients) and 5 on dobutamine stress echocardiography (446 patients). Random effects models were used to calculate summary odds ratios and 95% confidence intervals. RESULTS: Summary odds ratios for death or myocardial infarction and secondary cardiac end points were greater for dobutamine echocardiographic dyssynergy (14- to 27-fold) than for dipyridamole-thallium-201 redistribution (4-fold); wider confidence intervals were noted with dobutamine echocardiography. Pretest coronary disease probability was correlated with the positive predictive value of a reversible thallium-201 defect (r=0.70), increasing sixfold from low to high risk patient subsets. Cardiac event rates were low in patients without a history of coronary artery disease (1% in 176 patients) compared with patients with coronary disease and a normal or fixed-defect pattern (4.8% in 83 patients) and one or more thallium-201 redistribution abnormality (18.6% in 97 patients, p=0.0001). CONCLUSIONS: Meta-analysis of 15 studies demonstrated that the prognostic value of noninvasive stress imaging abnormalities for perioperative ischemic events is comparable between available techniques but that the accuracy varies with coronary artery disease prevalence. PMID- 8613605 TI - Cardiac risk for vascular surgery. PMID- 8613606 TI - Adenosine technetium-99m sestamibi myocardial perfusion SPECT in women: diagnostic efficacy in detection of coronary artery disease. AB - OBJECTIVES: This study sought to assess the diagnostic efficacy of adenosine technetium-99m sestamibi myocardial perfusion single-photon emission computed tomography (SPECT) in a consecutive series of female patients. BACKGROUND: The utility of adenosine myocardial perfusion SPECT for the detection of coronary artery disease is not well defined in women because most studies have described a predominantly male population with a high prevalence of coronary artery disease. METHODS: Of the 201 consecutive female patients in the study group who had undergone adenosine Tc-99m sestamibi myocardial perfusion SPECT, 130 had coronary angiography within 2 months of the nuclear test, and the other 71 had a low likelihood (<10%, mean [+/-SD] 5 +/- 3%) of coronary artery disease. The SPECT protocol used separate acquisition of rest thallium-201 and adenosine Tc-99m sestamibi and was visually analyzed in 20 segments with a semiquantitative five point scoring system (0=normal; 4=absent uptake). RESULTS: The normalcy rate in patients with a low likelihood of coronary artery disease was 93% (66 of 71). Among the catheterized group, the overall sensitivity, specificity and predictive accuracy of adenosine sestamibi SPECT for detecting coronary artery disease (> or = to 50% diameter stenosis) were 93% (87 of 94), 78% (28 of 36) and 88% (115 of 130), respectively. In the 103 patients without a prior myocardial infarction, the sensitivity, specificity and predictive accuracy were 91% (61 of 67), 78% (28 of 36) and 86% (89 of 103), respectively, for detecting > or = to 50% diameter stenosis. Of particular interest, the sensitivity and specificity were as high in patients with nonanginal symptoms (93% and 69%, respectively) as in patients with angina (92% and 83%, respectively, p=NS). The sensitivity and specificity among patients with a relatively low (<25%), intermediate (between 25% and 75%) or high prescan likelihood of coronary artery disease (>75%) were similar: 82% and 82%, 93% and 73%, and 95% and 100%, respectively. The sensitivity and specificity for detecting individual diseased vessels (> or = to 50% diameter stenosis) were, respectively, 76% and 81% for the left anterior descending coronary artery, 44% and 90% for the left circumflex coronary artery and 75% and 77% for the right coronary artery. CONCLUSIONS: Adenosine Tc-99m sestamibi SPECT is an efficient protocol with high sensitivity and specificity for the detection of coronary artery disease in women irrespective of presenting symptoms or pretest likelihood of coronary artery disease and a high normalcy rate. These findings are of particular clinical relevance because chest pain, anginal or otherwise, has been shown to be a frequent but a less specific marker for coronary artery disease among female patients. PMID- 8613607 TI - Effects of intraaortic balloon pumping on septal arterial blood flow velocity waveform during severe left main coronary artery stenosis. AB - OBJECTIVES: We sought to evaluate the effect of intraaortic balloon pumping on the phasic blood velocity waveform into myocardium with severe coronary artery stenosis. BACKGROUND: In the presence of severe coronary artery stenosis, it is not clear whether intraaortic balloon pumping augments intramyocardial inflow during diastole or changes systolic retrograde blood flow from the myocardium to the extramural coronary arteries. METHODS: Using anesthetized open chest dogs (n=7), we introduced severe stenosis in the left main coronary artery to reduce the poststenotic pressure to approximately 60 mm Hg (>90% diameter stenosis). Septal arterial blood flow velocities were measured with a 20-MHz, 80-channel ultrasound pulsed Doppler velocimeter. Left anterior descending arterial flow, aortic pressure and poststenotic distal coronary pressure were measured simultaneously. The diastolic anterograde flow integral and systolic retrograde flow integral were compared in the presence and absence of intraaortic balloon pumping. RESULTS: Although intraaortic balloon pumping augmented diastolic aortic pressure, this pressure increase was not effectively transmitted through stenosis. Septal arterial diastolic flow velocity was not augmented, and left anterior descending arterial flow was unchanged during intraaortic balloon pumping. CONCLUSIONS: In the presence of severe coronary artery stenosis, intraaortic balloon pumping failed to increase diastolic inflow in the myocardium and did not enhance systolic retrograde flow from the myocardium to the extramural coronary artery. Thus, the major effect of intraaortic balloon pumping on the ischemic heart with severe coronary artery stenosis may be achieved by reducing oxygen demand by systolic unloading. PMID- 8613608 TI - Intraaortic balloon counterpulsation: deciphering its effects on coronary flow. PMID- 8613609 TI - Neutrophil and platelet activation at balloon-injured coronary artery plaque in patients undergoing angioplasty. AB - OBJECTIVES: This study sought to investigate changes in the expression of activation-dependent adhesion receptors on neutrophils and platelets after exposure to the balloon-injured coronary artery plaque. BACKGROUND: Activation of blood cells at the balloon-injured coronary artery plaque may contribute to abrupt vessel closure and late restenosis after percutaneous transluminal coronary angioplasty. METHODS: In 30 patients undergoing elective coronary angioplasty, blood specimens were obtained through the balloon catheter proximal to the plaque before dilation and distal to the plaque after dilation. Simultaneous blood samples obtained through the guiding catheter served as control samples. Total surface expression of the inducible fibrinogen receptor (CD41) and surface expression of the activated fibrinogen receptor (LIBS1) on platelets as well as Mac-1 (CD11b) and L-selectin (CD62L) surface expression on neutrophils were assessed by flow cytometry. RESULTS: After exposure to the dilated coronary artery plaque, surface expression of LIBS1 on platelets increased by 40.5 +/- 11.0 mean (+/-SE) fluorescence (p=0.001) and that of CD11b on neutrophils increased by 20.1 +/- 4.4 mean fluorescence (p=0.018). Concomitantly, anti-CD62L binding on neutrophils decreased by 6.6 +/- 2.4 mean fluorescence (p=0.022). In contrast, surface expression of the adhesion receptors did not change significantly between the coronary ostium and the prestenotic coronary segment. CONCLUSIONS: The results of this study demonstrate neutrophil and platelet activation at the balloon-injured coronary artery plaque. This cellular activation may serve as a target for pharmacologic interventions to improve the outcome of coronary angioplasty. PMID- 8613611 TI - The dilemma of diagnosing coronary calcification: angiography versus intravascular ultrasound. AB - OBJECTIVES: We sought to determine whether careful examination of angiograms in conjunction with other clinical information could reliably detect, quantitate and localize target lesion calcification before a coronary intervention. BACKGROUND: The presence, extent and location of calcium in coronary artery lesions are important determinants of outcome after coronary intervention. Intravascular ultrasound is proposed as a superior technique for identifying patients with coronary artery calcification. However, the precise role of this costly and invasive method has not yet been established. METHODS: Target lesion calcification was assessed in 183 patients (155 men; mean [+/-SD] age 58 +/- 10 years) by angiography and intravascular ultrasound before a planned percutaneous coronary intervention. RESULTS: Ultrasound detected calcium in 138 patients (>90 degrees in 56, 91 degrees to 180 degrees in 52, 181 degrees to 270 degrees in 22 and > 270 degrees in 8), whereas angiography showed calcification in 63 (1+ in 32, 2+ in 27 and 3+ in 4). The two techniques agreed in 92 patients and disagreed in 91. Sensitivity and specificity of angiography were 40% and 82%, respectively. The arc of calcium by ultrasound was greater in patients with angiographically visible calcification (175 degrees +/- 85 degrees vs. 108 degrees +/- 71 degrees, p=0.0001). The depth of calcification by ultrasound was superficial in 61 patients (44%), deep in 68 (49%) and mixed in 8 (7%). The sensitivity of angiography in identifying superficial calcium was 35%. Of 120 patients without angiographically visible calcium at the target lesion site, 83 showed calcium by ultrasound. The only predictor of ultrasound calcium in these 120 patients was angiographic calcification elsewhere in the coronary tree (p=0.0001). The probability of any calcium and superficial >90 degrees calcium were 60% and 12%, respectively, in the 90 patients without angiographic calcifications anywhere in the coronary tree. CONCLUSIONS: Despite poor sensitivity, angiography may help identify patients requiring intravascular ultrasound. When it is angiographically visible, the arc of calcium is likely to be large and superficial. Angiographic calcification at a remote site is a predictor of angiographically undetected target lesion calcium. Patients without angiographic calcification in the coronary tree may not need routine ultrasound examination, as the likelihood of >90 degrees superficial calcium is low. PMID- 8613610 TI - Atheroma morphology and distribution in proximal left anterior descending coronary artery: in vivo observations. AB - OBJECTIVES: This study sought to examine, in vivo, the shape and position of atheroma in the proximal left anterior descending coronary artery. BACKGROUND: The prevalence, shape and location of atheromas involving the proximal left anterior descending artery have implications regarding the role of disturbed shear forces in the genesis of atherosclerosis. However, no data are available regarding in vivo findings or advanced disease. METHODS: Forty-two consecutive high quality intravascular ultrasound images were examined from patients with atherosclerotic disease in the proximal left anterior descending artery just distal to the left main bifurcation. Lesion percent area stenosis and maximal, minimal and flow divider intimal-medial thickness were measured at the region immediately after the circumflex takeoff. The angle formed by the midpoint of the flow divider, the human center of gravity and the maximal plaque thickness were determined. RESULTS: Eccentricity of vessel wall atheroma was observed such that the maximal wall thickness (1.42 +/- 0.50 mm [mean +/- SD]) differed significantly from minimal wall thickness (0.17 +/- 0.098 mm). Further, the region of vessel wall manifesting maximal thickness was greater than the flow divider thickness (0.26 +/- 0.16 mm). Maximal plaque thickness spared the region of the flow divider in 100% of cases and was positioned at a mean angle of 193 +/ 49 degrees from the center of the flow divider. Eccentric morphology was maintained across the 24% to 80% range of area stenosis. CONCLUSIONS: Atheromas in the very proximal left anterior descending artery are located opposite the circumflex takeoff, spare the flow divider and maintain eccentricity across a wide range of vessel stenoses. These in vivo morphologic data support the potential role of fluid dynamic mechanical factors in atherogenesis and have implications regarding the success of catheter-based interventional procedures at the site. PMID- 8613612 TI - Dichotomous pattern of coronary atherosclerosis 1 to 9 years after transplantation: insights from systematic intravascular ultrasound imaging. AB - OBJECTIVE: The aim of this study was to evaluate the extent and distribution of coronary atherosclerosis after transplantation. BACKGROUND: Transplant coronary artery disease is an important cause of death after cardiac transplantation. Unlike coronary angiography, intravascular ultrasound is a sensitive tool for detection and quantitation of this disease. METHODS: We performed intravascular ultrasound imaging in 132 (106 men, 50 +/- 10 years) patients, 1 to 9 years after transplantation using a 30-MHz ultrasound catheter. RESULTS: All three coronary arteries were visualized in 49, two in 62 and one in 21 patients. Of the 1,188 coronary artery segments, 706 were imaged (74% proximal, 64% mid- and 40% distal). At least one site with atherosclerosis (intimal thickness > or = to 0.5 mm) was found in 83% of patients. Atherosclerosis was noted in 64% of proximal, 43% of mid- and 26% of distal segments. Disease was diffuse in 48% and focal in 52%, circumferential in 66% and noncircumferential in 34%. Focal atherosclerosis was more common in proximal (59%) than mid- (48%) and distal segments (27%) (p=0.001). Noncircumferential plaques were more common in the proximal (42%) than mid- (28%) and distal segments (12%) (p=0.001). This pattern of focal and noncircumferential disease proximally, diffuse and circumferential disease distally, was observed irrespective of the time from transplantation. CONCLUSION: Atherosclerosis was detected in more than 80% of patients, with proximal segments most frequently involved. Diffuse and circumferential atherosclerosis was more common in mid- and distal segments. However, focal and noncircumferential involvement was more frequent proximally, a similar pattern to native atherosclerosis. These findings suggest that transplant coronary artery disease has a dual etiology based on the dichotomous pattern of atherosclerosis seen by intravascular ultrasound. PMID- 8613613 TI - Transient sympathovagal imbalance triggers "ischemic" sudden death in patients undergoing electrocardiographic Holter monitoring. AB - OBJECTIVES: The aim of this study was to investigate the relation between "ischemic" sudden death (arrhythmic death preceded by ST segment shift) and autonomic nervous system activity. Background. Mechanisms precipitating sudden death are poorly known despite the importance of detecting functional factors that may contribute to such a fatal event. METHODS: We analyzed the tapes of eight patients (seven men and one woman with a mean age of 66 +/- 8 years) who had ischemic sudden death during ambulatory electrocardiographic (Holter) monitoring. Four patients had unstable and four had stable angina; none was taking antiarrhythmic drugs. Twenty patients with angina and transient myocardial ischemia during Holter monitoring served as control subjects. Arrhythmias, ST segment changes and heart rate variability were analyzed by a computerized interactive Holter system. RESULTS: Five patients had ventricular tachyarrhythmias (ventricular fibrillation in three, ventricular tachycardia in two), and three had bradyarrhythmias (atrioventricular block in two, sinus arrest in one) as the terminal event; all eight patients showed ST segment shift (maximal change 0.46 +/- 0.16 mV; with ST elevation in two) that occurred 41 +/- 34 min (mean +/- SD) before sudden death. The standard deviation of normal RR intervals (SDNN) was 89 +/- 33 ms during the 10 +/- 6 h of Holter monitoring; 5 min before the onset of the fatal ST shift, SDNN measurements were significantly lower than during the initial 5-min period (48 +/- 10 vs. 29 +/- 9 ms; p=0.002). In control patients, the SDNN was 102 +/- 39 ms during Holter monitoring, whereas it measured 56 +/- 30 ms 5 min before the most significant episode of ST shift (p<0.01 vs. 29 +/- 9 ms [corrected] in the group with sudden death). CONCLUSIONS: Autonomic dysfunction, as detected by a marked decrease in heart rate variability, is present in the period (5 min) immediately preceding the onset of the ST shift precipitating ischemic sudden death. These data suggest that sympathovagal imbalance may trigger fatal arrhythmias during acute myocardial ischemia, thus resulting in sudden death. PMID- 8613614 TI - "Pseudodisappearance" of atrial electrogram during orthodromic tachycardia: new criteria for successful ablation of concealed left-sided accessory pathways. AB - OBJECTIVES: This study sought to analyze two new criteria along with other known predictors of success of radiofrequency ablation. Background. Although the overall success rate of radiofrequency ablation of accessory pathways is high, the individual predictive value of each of the established criteria is low. METHODS: We prospectively studied the local electrograms obtained before the application of radiofrequency energy in 33 patients with a left-sided concealed accessory pathway successfully ablated. Two new criteria ("pseudodisappearance" during tachycardia of a bipolar atrial electrogram visible during sinus rhythm and the presence of an "atrial notch" in the ascending limb of the unipolar ventricular electrogram during tachycardia) were studied along with other known predictors. Electrograms recorded at a total of 157 sites were analyzed (33 successful applications, 124 failures). RESULTS: Electrogram characteristics that were predictive of success during ablation on the basis of univariate analyses were a pseudodisappearance criterion (p<0.001), the presence of a Kent potential (p<0.005) and the presence of an "atrial notch" (p<0.005). After adjustment for between-patient differences, logistic regression analysis showed that only the "pseudodisappearance" criterion (odds ratio [OR] 7.2, 95% confidence interval [CI] 1.2 to 42.5, p<0.03) and the presence of a Kent potential (OR 2.4, 95% CI 1.01 to 5.79, p<0.05) had independent predictive value. CONCLUSIONS: The pseudodisappearance during tachycardia or ventricular pacing of a bipolar atrial electrogram present during sinus rhythm is associated with a good outcome during radiofrequency ablation of concealed accessory pathways. These observations may help to ablate accessory pathways and to avoid missing appropriate sites for ablation when the atrial activation is not clearly visible at the local electrogram. PMID- 8613615 TI - Radiofrequency catheter ablation of common atrial flutter: comparison of electrophysiologically guided focal ablation technique and linear ablation technique. AB - OBJECTIVES: The purpose of this study was to study electrophysiologic characteristics and compare the electrophysiologically guided focal ablation technique and linear ablation technique in patients with common atrial flutter in a prospective randomized fashion. Background. Catheter ablation of the common atrial flutter circuit can be performed with different techniques. To date, these two techniques have not been compared prospectively in a randomized study. METHODS: Sixty patients with drug-refractory common atrial flutter were randomly assigned to undergo radiofrequency catheter ablation performed with the electrophysiologically guided focal ablation (Group I) or linear ablation technique (Group II). In Group I, radiofrequency energy was delivered to the site characterized by concealed entrainment with a short stimulus-P wave interval (<40 ms) and a postpacing interval equal to the atrial flutter cycle length. In Group II, continuous migratory application of radiofrequency energy was used to create two linear lesions in or around the inferior vena cava-tricuspid ring isthmus. Serial 24-h ambulatory electrocardiographic (Holter) and follow-up electrophysiologic studies were performed to assess recurrence of tachycardia and possible atrial arrhythmogenic effects. RESULTS: Successful elimination of the flutter circuit was achieved in 28 of 30 patients in Group I and 29 of 30 patients in Group II. More atrial premature beats and episodes of short run atrial tachyarrhythmias in the early period (within 2 weeks) after ablation were found in Group II. Recurrence rate (2 of 28 vs. 3 of 29) and incidence of new sustained atrial tachyarrhythmias (3 of 28 vs. 3 of 29) was similar in the two groups. Occurrence of recurrent atrial flutter and new sustained atrial tachyarrhythmias was related to associated cardiovascular disease and atrial enlargement in both groups. However, in Group II, the procedure time (104 +/- 17 vs. 181 +/- 29 min, p<0.01) were significantly shorter than those in Group I. CONCLUSIONS: Radiofrequency ablation of the common atrial flutter circuit was safe and effective with either the electrophysiologically guided focal ablation or linear ablation technique. However, the linear ablation technique was time saving. PMID- 8613616 TI - Radiofrequency catheter ablation of right ventricular outflow tachycardia in children and adolescents. AB - OBJECTIVES: The current study reviews the safety and efficacy of radiofrequency catheter ablation for the treatment of right ventricular outflow tachycardia in children and adolescents and describes a modified method for mapping the tachycardia focus. BACKGROUND: Although radiofrequency catheter ablation has proved highly effective for the treatment of supraventricular tachycardia during childhood and adolescence, its application in children with idiopathic right ventricular outflow tachycardia has been limited. METHODS: Six children (mean [+/ SD] age 10.6 +/- 2.4 years, range 6 to 16) with right ventricular outflow tachycardia underwent seven radiofrequency catheter ablation procedures. The mean tachycardia cycle length was 323 +/- 24 ms (range 300 to 360). Two multipolar catheters were positioned in the right ventricular outflow tract to map the tachycardia focus. RESULTS: Radiofrequency catheter ablation was successful in five (83%) of the six children (95% confidence interval 36% to 99%). At successful ablation sites, local endocardial activation time preceded the surface QRS onset by 46 +/- 5 ms (range 37 to 57), and there was concordance of the 12 lead pace map and the electrocardiogram (ECG) in 11 (one patient) to 12 ECG leads (four patients). One patient developed complete right bundle branch block during radiofrequency catheter ablation. There were no additional complications and no clinical recurrences over a mean follow-up period of 12.7 +/- 3.8 months (range 9 to 22). CONCLUSIONS: These results suggest that radiofrequency catheter ablation is a safe and effective treatment for right ventricular outflow tachycardia during childhood and adolescence. In addition, tachycardia mapping may be enhanced by use of a multipolar right ventricular outflow catheter technique. PMID- 8613617 TI - Radiofrequency catheter ablation for idiopathic right ventricular tachycardia: first, last or only therapy--who decides? PMID- 8613618 TI - "Crochetage" (notch) on R wave in inferior limb leads: a new independent electrocardiographic sign of atrial septal defect. AB - OBJECTIVES: This study sought to determine the clinical significance of a "crochetage" pattern--a notch near the apex of the R wave in electrocardiographic (ECG) inferior limb leads--in secundum atrial septal defect. BACKGROUND: Atrial septal defect is often overdiagnosed on the basis of classical clinical features. Thus, more specific signs on the ECG for screening are needed. Methods. We searched for a crochetage pattern in 1,560 older children and adults: 532 with secundum atrial septal defect, 266 with ventricular septal defect, 146 with pulmonary stenosis, 110 with mitral stenosis, 47 with cor pulmonale and 459 normal subjects. RESULTS: This pattern was observed respectively in 73.1%, 35.7%, 23.3%, 6.4%, 10.6% and 7.4% of these groups (p<0.001). In atrial septal defect, its incidence increased with larger anatomic defect (p<0.0001) or greater left-to right shunt (p<0.0001), even in the presence of pulmonary hypertension. By multiple regression analysis, only shunt size (p<0.0006) and defect location (p<0.0001) were the determinants of its presence. In all groups, the specificity of this sign for the diagnosis was remarkably high when present in all three inferior limb leads (> or = to 92%), even when comparison was limited to patients with an incomplete right bundle branch block (> or = 95.2%). Early disappearance of this pattern was observed in 35.1% of the operated-on patients although the right bundle branch block pattern persisted. CONCLUSIONS: A crochetage pattern of the R wave in inferior limb leads is frequent in patients with atrial septal defect, correlates with shunt severity and is independent of the right bundle branch block pattern. Sensitivity and specificity of this sign are remarkably high when it is associated with an incomplete right bundle branch block or present in all inferior limb leads. PMID- 8613619 TI - Doppler echocardiography reliably predicts pulmonary artery wedge pressure in patients with chronic heart failure with and without mitral regurgitation. AB - OBJECTIVES: This study was performed to assess whether the combination of multiple echocardiographic and Doppler variables can provide a reliable estimation of pulmonary artery wedge pressure in patients with chronic heart failure. BACKGROUND: In patients with chronic heart failure a high pulmonary artery wedge pressure is associated with poor prognosis, more severe symptoms and low exercise tolerance. Several Doppler echocardiographic indexes have been shown to be related to pulmonary artery wedge pressure, but the dispersion of data has generally not allowed a quantitative assessment of this important variable. METHODS: Simultaneous Doppler echocardiographic examinations and right heart catheterizations were performed in 231 patients with chronic heart failure due to dilated cardiomyopathy. Mitral and pulmonary venous flow velocity variables, left atrial volumes, mitral regurgitation jet area and left ventricular ejection fraction were correlated with pulmonary artery wedge pressure by both single and multilinear regression analysis. The reliability of the obtained multilinear equations was then tested in a separate group of 60 patients. RESULTS: By univariate analysis, the deceleration rate of early diastolic mitral flow and the systolic fraction of pulmonary venous flow showed the strongest correlations (r=0.78 and =-0.76, respectively). Stepwise regression analysis led to two multilinear equations for predicting pulmonary artery wedge pressure in the whole population: the first included only two-dimensional echocardiographic and mitral flow velocity variables (r=0.84) and the second also included pulmonary venous flow variables (r=0.87). The highest correlation was obtained (r=0.89) by a third equation in the 73 patients without significant mitral regurgitation. Correlation coefficients between estimated and measured pulmonary artery wedge pressure were 0.91 (SEE=2.7 mm Hg) and 0.97 (SEE=1.8 mm Hg) when the first and the second equation, respectively, were applied to the testing group. CONCLUSIONS: These results indicate that, in patients with chronic heart failure due to dilated cardiomyopathy, pulmonary artery wedge pressure can be reliably estimated even when mitral regurgitation is present by combining Doppler echocardiographic variables of mitral and pulmonary venous flow. PMID- 8613620 TI - Left ventricular echocardiographic and histologic changes: impact of chronic unloading by an implantable ventricular assist device. AB - OBJECTIVES: We studied the effects of chronic left ventricular unloading by a ventricular assist device and assessed left ventricular morphologic and histologic changes. BACKGROUND: The implantable left ventricular assist device has been effective as a "bridge" to cardiac transplantation. Although there are reports documenting its circulatory support, little is known about the effects of chronic left ventricular unloading on the heart itself. METHODS: We performed intraoperative transesophageal echocardiography at the insertion and explanation of a HeartMate left ventricular assist device in 19 patients with end-stage heart failure. They were supported by the assist device for 3 to 153 days (mean [+/-SD] 68 +/- 33). Measurements were taken retrospectively to obtain left atrial and ventricular diameters and interventricular septal and posterior wall thicknesses. Histologic examinations were made from the left ventricular myocardial specimens of 15 patients at the times of insertion and explanation for heart transplantation. Insertion and explanation specimens were compared qualitatively (0 to 3 scale) for wavy fibers, contraction band necrosis and fibrosis, with quantitative measurement of minimal myocyte diameter across the nucleus. RESULTS: Left atrial and left ventricular diastolic and systolic diameters decreased immediately after insertion of the left ventricular assist device (from 46 to 35, 63 to 41 and 59 to 36 mm, respectively, all p < 0.001). Left ventricular wall thickness increased from 10 to 14 mm (p < 0.001) for the interventricular septum and from 10 to 13 mm for the posterior wall (p<0.001). No echocardiographic measurements showed significant subsequent changes at the chronic stage. Myocardial histologic findings demonstrated a reduction in myocyte damage (from 1.9 to 0.5, p<0.001, for wavy fiber and from 1.3 to 0.2, p<0.01, for contraction band necrosis) and an increase in fibrosis (from 1.3 to 1.9, p<0.05), but without significant change in myocyte diameter (from 15.6 to 16.8 micrometer, p=0.065). CONCLUSIONS: Left ventricular unloading with the implantable assist device induces an immediate increase in wall thickness, consistent with the reduction in chamber size, thereby decreasing wall stress. Chronic unloading allows myocardial healing and fibrosis without evidence for ongoing myocyte damage or atrophy. Left ventricular assist device insertion may have a role in "resting" the ventricle for selected patients with heart failure. PMID- 8613621 TI - Amelioration of severity of myocardial injury by a nitric oxide donor in rabbits fed a cholesterol-rich diet. AB - OBJECTIVES: This study compared the effect of a nitric oxide donor on limiting the size of infarct resulting from myocardial ischemia-reperfusion between atherosclerotic and nonatherosclerotic models. BACKGROUND: Endothelial-derived relaxation in coronary arteries affected by ischemia is substantially impaired after reperfusion, and this impairment may exacerbate the myocardial ischemia reperfusion injury. In animals with experimental atherosclerosis, release of endothelial-derived relaxing factor is also decreased, and the propagation of myocardial infarction could be exacerbated. METHODS: We examined the extent of myocardial injury induced by ischemia (30 min) and reperfusion (48 hr) in rabbits fed a cholesterol-rich (1%) or normal diet for 10 weeks. We also evaluated the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP], a nitric oxide precursor (L-arginine) or a degradation product of SNAP (N acetylpenicillamine) on infarct size in these models. RESULTS: Severity of myocardial injury was significantly exacerbated in cholesterol-fed rabbits (75.2 +/- 4.4% [mean +/- SEM]) compared with that in non-cholesterol-fed rabbits (53.2 +/- 5.2%). This exacerbation was prevented by treatment with SNAP (50.2 +/- 6.4%) but not with L-arginine (70.5 +/- 6.0%) or N-acetylpenicillamine (70.4 +/- 4.8%) in cholesterol-fed-rabbits. However, SNAP did not limit infarct size in non cholesterol-fed rabbits (60.8 +/- 4.2%). The rate-pressure product was similar during the course of the experiment in all the groups. CONCLUSIONS: Myocardial damage induced by ischemia-reperfusion was significantly exacerbated in rabbits fed a long-term cholesterol-rich diet but was effectively reversed by treatment with a nitric oxide donor. However, this agent did not limit infarct size in normal rabbits. Thus, a nitric oxide donor reduces myocardial infarct size in atherosclerotic but not in nonatherosclerotic rabbits. PMID- 8613623 TI - Dimensionality of flow data. PMID- 8613622 TI - Guidelines for perioperative cardiovascular evaluation for noncardiac surgery. Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Perioperative Cardiovascular Evaluation for Noncardiac Surgery). PMID- 8613624 TI - Zatebradine and exercise tolerance. PMID- 8613625 TI - Dobutamine stress echocardiography in orthotopic heart transplant recipients. PMID- 8613626 TI - Preinfarction angina as a major predictor of left ventricular function and long term prognosis after a first Q wave myocardial infarction. PMID- 8613627 TI - Update on cytokines. PMID- 8613628 TI - Urinary leukotriene E4 in the assessment of nocturnal asthma. AB - BACKGROUND: Urinary leukotriene E4 (LTE4) is a marker of the body's production of cysteinyl LTs, important mediators of airway inflammation. The role of the latter in nocturnal asthma is a topic of increasing interest. OBJECTIVE: This investigation was aimed at determining whether nighttime attacks are associated with increased release of LTs, expressed by urinary LTE4, and the relationship between the two phenomena. METHODS: Three groups were studied: group A, seven control subjects; group B, nine asthmatic patients without nocturnal attacks; and group C, nine asthmatic patients with a comparable daytime FEV1 but who were experiencing nocturnal exacerbations (morning dips in peak expiratory flow greater than 20%). Urine was collected over 24 hours in three samples (9:00 AM to 3:00 PM; 3:00 PM to 9:00 PM; and 9:00 PM to 9:00 AM). LTE4 was measured by high performance liquid chromatography and radioimmunoassay and expressed as nanograms per millimole of creatinine. RESULTS: No significant differences between urinary LTE4 were noticed within groups A and B. Conversely, in group C urinary LTE4 at night (geometric mean with 95% confidence interval; 35.16 with 28.77-42.85) was significantly higher than that of the other samples (respectively 23.12 with 17.78-30.06, p less than 0.05; and 25.18 with 21.03-30.13, p less than 0.02); it was also significantly higher than in all the samples of other groups. A significant (p less than 0.02) linear correlation was observed between morning dip in peak expiratory flow and the log urinary LTE4 in the nocturnal sample. CONCLUSION: These results indicate the role of LTs in nocturnal asthma and suggest that urinary LTE4 may be a useful marker of this condition. PMID- 8613629 TI - Decrease in hospitalization for treatment of childhood asthma with increased use of antiinflammatory treatment, despite an increase in prevalence of asthma. AB - BACKGROUND: During the past 15 years, the prevalence of asthma in children in Sweden has doubled. However, since 1985, antiinflammatory treatment with inhaled steroids has increased continuously. OBJECTIVE: The aim of this study was to analyze the net effect of these changes in terms of hospitalization of children for treatment of asthma. METHODS: The numbers of hospital days, admissions, and individual patients admitted to the Children's Hospital in Goteborg because of acute asthma were recorded from 1985 through 1993. all the in-patient treatment of children is centralized at this hospital (i.e., the study was population based). Goteborg has half a million inhabitants. Hospitalization policies were not altered during the study period. RESULTS: In children aged 2 to 18 years, the number of hospital days per year gradually decreased to less than a third (r = 0.9; p less than 0.001), and admissions decreased by 45% (r = 0.7; p less than 0.05). The decrease in hospitalization was most marked in the group older than the age of 5 years in which hospital days were reduced to one fifth (r = 0.9; p less than 0.0001) and admissions were halved (r = 0.8; p less than 0.05). A decreasing trend in number of hospital days was also seen in the 2- to 5-year-old group. The number of individual patients admitted did not show a statistically significant decreasing trend. In children under the age of 2 years, the number of hospital days fluctuated, and there was no clear-cut change with time. CONCLUSION: Although increased concentration on the education of parents and patients may have been a contributing factor, the major reason for the decrease in hospitalization in the group of children aged 2 to 18 years is most probably antiinflammatory treatment with inhaled steroids. The results suggest that this is a very cost-effective therapeutic approach. PMID- 8613630 TI - Comparison of triamcinolone acetonide nasal inhaler with astemizole in the treatment of ragweed-induced allergic rhinitis. AB - BACKGROUND: Few clinical trials have directly compared the efficacy of antihistamines with topical nasal corticosteroids. OBJECTIVE: The study was performed to compare the efficacy and safety of triamcinolone acetonide nasal spray at a dose of 110 micro g in each nostril once daily with 10 mg of oral astemizole once daily for the treatment of seasonal allergic rhinitis. METHODS: A multicenter, double-blind, parallel-group study was conducted in 239 patients who were randomized to receive either triamcinolone acetonide or astemizole. A 5-day, drug-free, lead-in period was followed by 4 weeks of double-blind treatment. One hundred four patients treated with triamcinolone acetonide and 105 patients treated with astemizole could be evaluated. RESULTS: Overall, triamcinolone acetonide was more effective than astemizole in reducing total nasal symptoms, nasal stuffiness, nasal itching, and sneezing (p 1 Gy between cell lines: cells would be intrinsically radiosensitive because they have a diminished inducible response. PMID- 8613678 TI - Participation of gap-junctional cell communication on the adaptive response in human cells induced by low dose of X-rays. AB - To investigate the radioadaptive response of normal cells to low-dose radiation, we irradiated human embryonic (HE) cells and HeLa cells with low-dose X-rays and examined the changes in sensitivity to subsequent high-dose X-irradiation using the trypan blue dye-exclusion test. When HE cells were irradiated by 200 cGy, the growth ratio of the living cells 5 days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a conditioning irradiation of 10-20 cGy 4 h before the irradiation of 200 cGy, the relative growth ratios increased significantly to 45-53%, and a peak was reached at a conditioning dose of 13 cGy to the cells. This conditioning effect was not observed in LeLa cells. When the HE cells were suspended in a Ca2+ ion-free medium or TPA added to the medium while receiving the conditioning irradiation of 13 cGy, the effect of the conditioning dose was not observed. This indicates that normal cells show an adaptive response to low-dose radiation and become more radioresistant. These results suggest that gap junctional intercellular communication may play a role in radioadaptive responses in human cells. PMID- 8613679 TI - Increased micronucleus frequency in the progeny of irradiated Chinese hamster cells. AB - V79 Chinese hamster cells were irradiated with doses of 1-12 Gy 300 kV X-rays. Their colony-forming ability and the frequency of micronuclei in binucleate cells after treatment with cytochalasin B was determined at various times after irradiation. The frequency of micronuclei determined within the first 24 h after irradiation increased with doses up to 4 Gy and decreased as the dose increased further. Up to 4 Gy there was a close correlation between surviving fraction and the fraction of cells without micronuclei although the surviving fraction was 2-3 times lower than the fraction of cells without micronuclei. Six, 10 or 13 days after irradiation with either 9 or 12 Gy the plating efficiency and the frequency of micronuclei after cytokinesis block with cytochalasin B was determined in the irradiated, but surviving, cells. The delayed plating efficiency of irradiated cells was significantly decreased. The proportion of binucleated cells was in the normal range at 6-13 days after irradiation, indicating that the proliferative activity of irradiated but surviving cells was not depressed at that time. The micronucleus frequency, however, was significantly increased at all times after irradiation. There was little heterogeneity of plating efficiency and micronucleus frequency among 12 clones which had been isolated for irradiated cultures, 3 weeks after 12 Gy. PMID- 8613680 TI - Radiation-enhanced differentiation of erythroid progenitor cells and its relation to reproductive cell death. AB - Terminally differentiated cells usually do not divide and are, thus, reproductively dead. To elucidate the significance of radiation-enhanced differentiation to reproductive cell death, murine erythroid progenitor cells were gamma-irradiated in plasma clot cultures and the development of haemoglobinized clones was studied thereafter. If irradiation occurred when the cells had resumed proliferation, the total numbers of haemoglobinized clones and, in parallel, the numbers of newly haemoglobinized clones were elevated above control levels 6-24 h after 10-30 Gy and 24-48 h after 1 Gy respectively. Thereafter, clone numbers decreased below controls. This decrease was faster with the newly haemoglobinized clones, indicating that both the accumulation of haemoglobinized clones and fast exhaustion of the pool of more primitive precursors in the cultures are due to accelerated differentiation. The haemoglobinized clones appearing after irradiation were reduced in size without indication of direct cell death. We conclude that the reproductive cell death occurring in our system is due to enhancement of differentiation. Enhancement of differentiation is expressed by omission of cell cycles normally passed through by the cell progeny before terminal differentiation is reached. Dependence of differentiation enhancement on the presence of cycling cells at the time of irradiation indicates involvement of growth of essential cytoplasmic constituents during mitotic delay as observed in other cell systems. PMID- 8613681 TI - ERCC1/ERCC4 5'-endonuclease activity as a determinant of hypoxic cell radiosensitivity. AB - In this study, the relationships between cellular oxygen enhancement ratios (OER) and nucleotide excision repair capability were examined using the UV20 mutant cell line (which has a defective ERCC1 gene). Using a clonogenic survival assay, the OER for the killing of wild-type AA8 cells was 3.2 +/- 0.1, whereas that for UV20 cells was only 2.35 +/- 0.05; the decreased OER of UV20 cells was the result of their significantly greater radiosensitivity relative to wild-type cells under hypoxic conditions. In AA8 cells, hypoxia protected against DNA double-strand break (dsb) induction (determined by pulsed-field gel electrophoresis) by a factor 3.5 +/- 0.3; i.e. to a similar extent that it modulated cell killing. However, this correlation was not apparent in UV20 cells, where hypoxia protected against dsb induction to a similar extent as in wild-type cells (approximately 3.2-fold). Stably transfected UV20 cells over-expressing a full-length ERCC1 cDNA clone displayed a normal OER (3.5 +/- 0.1) in addition to wild-type resistance to UV light. Our data suggest that the hypoxic radiosensitivity of UV20 cells is a direct result of their ERCC1 deficiency and reflects their inability to process some type of DNA damage (not dsbs) that is induced preferentially in hypoxic cells. PMID- 8613682 TI - A radioresistant variant cell line, NMT-1R, isolated from a radiosensitive rat yolk sac tumour cell line, NMT-1: differences of early radiation-induced morphological changes, especially apoptosis. AB - A radioresistant variant cell line, NMT-1R, was isolated by repeated radiation exposure of a radiosensitive rat yolk sac tumour cell line, NMT-1, producing alpha-fetoprotein, with the potential for lymphatic metastasis in the inbred Wistar rat. Cultured NMT-1R cells showed more cobblestone-like appearances, although the morphological features were almost the same as radiosensitive NMT-1 cells reported previously. The doubling time of NMT-1R cells was 13.6 h, being shorter than that of NMT-1 cells (16.0 h). For NMT-1R cells, D0 for radiation sensitivity was 165 +/- 3 cGy, 1.7 times as large as for NMT-1 cells. The extrapolation number, n, was 1.48 +/- 0.17 for NMT-1R cells although that for NMT 1 cells was 1.08 +/- 0.15. The surviving fractions at 2 Gy (SF2) were 0.42 for NMT-1R cells and 0.28 for NMT-1 cells. The population of G2-M phase for NMT-1R cells was larger than for NMT-1 cells (32.5 versus 26.8%) in exponentially growing cells. Although a clear G2 delay was observed after irradiation with a dose of 182 cGy for both cell lines, NMT-1R cells had a shorter recovery time from G2 block than NMT-1 cells, G1 arrest was observed in NMT-1 cells. NMT-1 cells showed much higher incidence of early morphological changes, especially apoptosis, after irradiation with a dose > 500 cGy compared with NMT-1R cells. PMID- 8613683 TI - Lethality of radiation-induced chromosome aberrations in human tumour cell lines with different radiosensitivities. AB - In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity. PMID- 8613684 TI - Methylene blue sensitizes E. coli cells to X-rays. AB - Methylene blue (MB) is shown to sensitize E. coli cells to the effects of X-rays. This sensitization is dependent on factors such as dye concentration, incubation temperature, membrane permeability, and repair capacities, suggesting that the binding and/or penetration of the dye into the cells determines the potentiation of the lethal effects of X-rays by MB. It is also demonstrated that the presence of the polymerase 1 enzyme is essential for this sensitization to take place. Since MB is known to penetrate, accumulate, and be retained preferentially in some malignant tissues, the possibility of using this dye as a specific sensitizer to X-rays in the radiotherapy of some cancers is discussed. PMID- 8613685 TI - A multiple-radical model for radiation action on DNA and the dependence of OER on LET. AB - We have developed a multiple-radical model of the chemical modification reactions involving oxygen and thiols relevant to the interactions of ionizing radiations with DNA. The treatment is based on the Alper and Howard-Flanders equation but considers the case where more than one radical may be involved in the production of lesions in DNA. This model makes several predictions regarding the induction of double strand breaks in DNA by ionizing radiation and the role of sensitizers such as oxygen and protectors such as thiols which act at the chemical phase of radiation action via the involvement of free radicals. The model predicts a decreasing OER with increasing LET on the basis that as radical multiplicity increases so will the probability that, even under hypoxia, damage will be fixed and lead to lesion production. The model can be considered to provide an alternative hypothesis to those of "interacting radicals' or of "oxygen-in-the track'. PMID- 8613686 TI - Use of the alkaline comet assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC, UVB, UVA and gamma-rays. AB - The alkaline single cell gel electrophoresis (comet) assay applied to human fibroblasts allowed us to analyze the response to components of the solar spectrum (UVB and UVA) in comparison with the well-established response to UVC and gamma-rays. DNA strand breaks related to nucleotide excision repair of DNA photoproducts were produced 1 h after exposure to UVB or UVC in the normal cell line but not in the repair deficient XPD and TTD-2 cell lines. In contrast, the immediate production of DNA strand breaks observed in all cell lines after exposure to UVA or gamma-rays was followed by restitution of high molecular weight DNA upon post-exposure incubation. These results imply that (1) fibroblasts as well as lymphocytes can be analysed by the comet assay and (2) the comet assay clearly distinguishes cellular nucleotide excision repair capacity without the use of inhibitors of DNA synthesis. PMID- 8613687 TI - UV-B-induced cell cycle perturbations, micronucleus induction, and modulation by caffeine in human keratinocytes. AB - UV-B-induced perturbations of cell cycle progression in asynchronous human keratinocytes were analysed during two cell cycles with respect to their cell cycle stage at the time of irradiation using BrdUrd/Hoechst flow cytometry. Exponentially growing SCL-2-keratinocytes exposed to UV-B radiation showed a short delay in G1-phase exit and were blocked in the S and G2/M phases of the first cell cycle. UV-A wavelengths did not show any detectable effect on cell cycle progression. In contrast, 137Cs-irradiation of these cells induced a temporary G2 block only. Micronucleus frequency increased in gamma-irradiated cells as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine starting immediately after 137Cs gamma-irradiation prevented accumulation of cells in G2 phase, but did not influence the frequency of micronuclei. In UV-B-irradiated keratinocytes, however, the damage-induced cell cycle perturbations were merely reduced by caffeine, but not eliminated. Compared with gamma-irradiation a moderate induction of micronuclei was observed in UV-B-irradiated cells. Caffeine, however, potentiated the induction of micronuclei by UV-B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV-B- and gamma-irradiation that may be repaired through different pathways. PMID- 8613689 TI - Membrane lipid peroxidation as a mechanism of sonodynamically induced erythrocyte lysis. AB - Sonodynamically induced lipid peroxidation with haematoporphyrin (Hp) was studied using rat erythrocytes. Both suspensions of erythrocyte ghosts and of intact erythrocytes were exposed to ultrasound in the same way in the presence and absence of Hp (80 microM). The lipid peroxidation in erythrocyte ghost membranes was estimated by measuring the amount of reactive substance produced from thiobarbituric acid added immediately after the exposure. Haematoporphyrin multiplied the ultrasonically induced lipid peroxidation by three to five times, while Hp alone showed no peroxidation. A 24-h interval between the exposure and the preparation for measurement did not increase the measured amount of peroxide. In the presence of Hp the estimated peroxidation rate and the rate of erythrocyte lysis correlated quite well for each acoustic condition and for each chemical condition such as the presence or absence of active oxygen scavengers in the suspension. The sonodynamically induced lipid peroxidation with Hp was doubled by deuterium oxide substitution for suspension medium and was significantly reduced by histidine, by sodium azide, and also by nitrogen substitution for saturation gas, whereas superoxide dismutase and mannitol showed no significant inhibitory effect. These results are consistent with a hypothesis that lipid peroxidation in membranes by singlet oxygen is the primary mechanism of sonodynamically induced erythrocyte lysis with Hp. PMID- 8613688 TI - Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA. AB - The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.0, 2.2 and 2.5 kb were isolated that corrected several aspects of the phenotype of cells from patients with the human genetic disorder ataxia-telangiectasia, characterized by hypersensitivity to ionizing radiation. The criteria used to assess correction included cell viability, induced chromosome aberrations, G2 phase delay and induction of p53 after exposure to radiation. One cDNA (2.5 kb) was identified as the complete sequence of the RNA helicase p68, which was capable of correcting radiosensitivity based on two of the above four criteria, with p53 induction post irradiation being partially corrected. The 2.2 kb cDNA was shown to correspond to the complete sequence of arginyl tRNA synthetase and the other two cDNAs were identical to the 3' untranslated regions (UTR) of the transcription factor TFIIS (1.6 kb) and phospholipase A2 (2.0 kb) respectively. Additional transfections with the 3'UTR (198 nucleotides) of p68 RNA helicase and its inverse sequence revealed that the 3'UTR had the same complementation capacity as the full-length cDNA, whereas the inverse construct failed to complement radiosensitivity. These data provide additional support for a novel role for 3'UTRs in the regulation of gene expression. PMID- 8613690 TI - Considerations about field-size effects after irradiation in pig epidermis as detected by a colony assay in situ. PMID- 8613691 TI - Regulation of cytokine gene expression by reactive oxygen and reactive nitrogen intermediates. AB - Reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), and cytokines are frequent companions at sites of acute inflammation. Previous work has established a clear link between the production of cytokines and the subsequent generation of ROI and RNI. However, more recent data indicates that ROI and RNI not only serve as end-stage effector molecules of pathogen destruction and tissue injury, but also as initiators of acute inflammation. Specifically, ROI and RNI will upregulate cytokine gene expression since antioxidants inhibit interleukin 8 (IL-8) production and do not decrease production of other cytokines. Treatment with hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) will decrease the production of IL-8 in stimulated human whole blood, fibroblasts, type II epithelial cells, and hepatoma cells, but not other cytokines. Addition of exogenous ROI will increase IL-8 production in these same cells. Inhibition of nitric oxide synthase will decrease production of IL-8, whereas addition of nitric oxide (NO)-generating compounds will increase production of IL-8. The hydroxyl radical appears to be the final common pathway of cell activation for IL-8 synthesis, since DMSO will inhibit the NO-driven production of IL-8. Our data indicate that ROI and RNI can serve as intracellular second messengers to induce IL-8 gene expression. PMID- 8613692 TI - IL-15: the role of translational regulation in their expression. AB - We previously reported that the human T cell lymphotropic virus type I (HTLV-I) associated adult T cell leukemia (ATL) line HuT-102 produces a cytokine designated interleukin (IL)-T. Using anti-cytokine antibodies we demonstrated that IL-T is identical to the simultaneously described IL-15. The observation of a discordance between IL-15 message expression and IL-15 synthesis led us to examine normal and aberrant IL-15 mRNA for post-transcriptional controls that inhibit protein production at the level of RNA translation. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 T cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5' untranslated region (UTR) of IL-15. R segment introduction eliminated over 200 nucleotides of IL-15 5' UTR including 8 of 10 upstream AUGs that are present in the normal IL-15 message. On analysis of the 5' UTR of normal IL-15, we demonstrated that these 10 upstream AUGs interfere with IL-15 mRNA translation. Thus, IL-15 synthesis by the ATL cell line HuT-102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I-R fusion message that lacks many upstream AUGs. PMID- 8613693 TI - Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation. AB - Using adenoviral-mediated gene transfer techniques, the murine granulocyte macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others. PMID- 8613694 TI - The inflammatory response in interleukin-1 beta-deficient mice: comparison with other cytokine-related knock-out mice. AB - Interleukin-1 (IL-1) plays a crucial role in the development of the pathophysiological responses to infection and inflammation. However, the relative contributions of IL-1 alpha and IL-1 beta remain to be clarified. IL-1 beta deficient mice are a powerful tool to investigate the specific role of IL-1 beta in various experimental conditions. In this report, we summarize the response of IL-1 beta deficient mice to two different inflammatory stimuli, turpentine and endotoxin. Although IL-1 beta-deficient mice respond normally to the systemic administration of lipopolysaccharide (LPS), they do not develop an acute-phase response in the localized tissue damage model of turpentine injection. The results obtained using the IL-1 beta-deficient mice are compared here with those observed in the IL-1 beta-converting enzyme-deficient, IL-6-deficient, tumour necrosis factor-receptor p55-deficient, and interferon-gamma-receptor-deficient mice. PMID- 8613695 TI - Dendritic cells from different tissues induce production of different T cell cytokine profiles. AB - The precise role of antigen-presenting cells (APC) in regulating the balance of T helper type 1 (Th1) and T-helper type 2 (Th2) cytokine production is unclear. Dendritic cells (DC), the most potent APC for activation of naive T cells, were found to regulate Th1 and Th2 cytokine profiles in a fashion dependent upon their tissue of origin. Spleen (systemic) DC induce mainly Th1 cytokines and Peyer's patch (mucosal) DC induce predominantly Th2 cytokines. These findings support the current concept that different tissues, each with its distinct microenvironment of cytokines, hormones, and cellular elements, are involved in the selection, promotion, and/or maintenance of different immune responses. With regard to DC, it is apparent that the tissue of DC origin determines the cytokine profiles produced by T cells and that DC from different tissues favor either cellular versus humoral immune responses by influencing T cell cytokine production. PMID- 8613696 TI - Control of the production of soluble interleukin-4 receptors: implications in immunoregulation. AB - Soluble cytokine receptors (sCR) are generated in vivo through proteolytic cleavage of the membrane-bound receptors or by direct translation of mRNAs specifically encoding the soluble forms. Despite their widespread presence in biological fluids, the physiological role of endogenous sCR as immunoregulatory molecules is not yet well understood. In vivo, exogenous soluble interleukin-4 receptors (sIL-4R) have been shown to have both agonistic and antagonistic effects on IL-4 responses, depending on the relative concentration ratios of sIL 4R to IL-4. In an effort to elucidate the potential role of endogenous sIL-4R in the regulation of IL-4 responses, the mechanisms controlling the production of sIL-4R have been investigated. Although many cell types are able to constitutively produce low levels, production of sIL-4R is significantly up regulated in vitro by T cell activation and IL-4. The ability of splenic cells to produce sIL-4R and the serum levels of sIL-4R have consistently been found to be increased during immune responses characterized by T cell activation and IL-4 secretion (Th2 responses). In agreement, clones of Th2, but not Th1, cells were found to significantly up-regulate sIL-4R production following antigenic stimulation. However, the production of sIL-4R by Th2 cells appears to be independent from that of IL-4 and can also be induced by cell contact and/or IL-1 dependent pathways. Taken together, these observations suggest that the production of sIL-4R in vivo is closely associated with the secretion of IL-4, and are consistent with the notion that endogenous sIL-4R are involved in the regulation of IL-4 activity during immune responses. PMID- 8613697 TI - Immunoregulation by interleukin-12. AB - Interleukin-12 (IL-12) is a heterodimeric cytokine produced primarily by antigen presenting cells (monocytes, macrophages, dendritic cells, and B cells). Its production is stimulated by bacteria, bacterial products, and intracellular parasites and enhanced by priming with granulocyte-macrophage colony-stimulating factor (CM-CSF) and interferon-gamma (IFN-gamma) or inhibited by IL-10. The major biological activity of IL-12 is on T and natural killer (NK) cells in which it increases cytokine production, proliferation, and cytotoxicity. Its production occurs several hours after exposure to infections agents, which induces a rapid production of IFN-gamma by NK and later by T cells. This IFN-gamma potentiates antigen-presenting cell functions important in clearing infections agents (phagocytosis, oxidative burst, and production of nitrous oxide) and also increases further production of IL-12. IL-12 has been clearly demonstrated to be important in the generation of CD4 and CD8 type 1 T cells both in vivo and in vitro. Our data reveals that IL-12 primes naive T cells for high IFN-gamma and IL 10 production, whereas IL-4 is required for IL-4 priming, thus suggesting that these genes and possibly others are independently regulated. IL-12 is therefore involved in the skewing of cytokine production toward a type 1 and has been implicated in being involved in selective mechanisms of established T cells. It is now becoming clear that the IL-12 acts as both a proinflammatory cytokine and an immunomodulator and therefore bridges the innate and adaptive immune responses. PMID- 8613698 TI - Role of the type I interferons in allograft rejection. AB - Type I interferons are potent immuno-modulatory cytokines that enhance expression of the major histocompatibility complex (MHC) class I antigens, T-cell cytotoxicity, and natural killer (NK) cell activity, all of which are implicated in graft rejection. A monoclonal antibody (mAb) directed against the extracellular domain of the human interferon gamma (IFN-gamma) receptor (IFN alpha R), which inhibits both the binding and biological activity of all the type I IFNs tested, exerted a dose-dependent inhibition of the mixed lymphocyte reaction and induced permanent survival of skin allografts in MHC-divergent Cynomologus monkeys treated with a subeffective dose of cyclosporin A. Marked differences were observed in the composition of T lymphocyte subpopulations in anti-IFN-alpha R mAb-treated animals relative to the various control groups. Skin biopsies from animals treated with anti-IFN-R Mab + cyclosporin A revealed very low levels of MHC class I and class II antigen expression and the absence of histological signs of rejection, whereas skin biopsies from control animals exhibited high levels of MHC antigen expression and the histological signs of acute rejection, including a pronounced lymphocytic infiltrate, edema, and necrosis. No monkey antibodies (IgG) to the mouse anti-human IFN-alpha R mAb were detected in the serum of any of the animals treated with the anti-IFN-alpha R mAb either alone or together with cyclosporin A. Treatment of lethally irradiated Cynomologus monkeys with the anti-IFN-alpha R mAb together with a subeffective dose of cyclosporin A was also found to markedly enhance the survival of animals grafted with allogeneic bone marrow cells from donors differing in both MHC class I and class II antigens. These results show that selective and lasting immunosuppression can be obtained by the short-term administration of an IFN alpha antagonist together with a subeffective dose of cyclosporin A, and may have important implications for the therapy of human allograft rejection. PMID- 8613699 TI - Dissection of the pathologies induced by transmembrane and wild-type tumor necrosis factor in transgenic mice. AB - With increasing awareness that seemingly diverse immune-mediated diseases involve similar pathogenetic mechanisms, and the identification of a growing number of key effector molecules, it is becoming possible to design and generate effective transgenic models for such diseases. Tumor necrosis factor (TNF) plays a prominent role in immune and host defense responses and there is strong evidence that abnormal TNF production contributes to disease initiation and progression in rheumatoid arthritis, systemic inflammatory response syndrome, diabetes, multiple sclerosis, and many other immune-mediated disorders. The generation of TNF transgenic mice, in which TNF production is deregulated, has provided us with direct evidence that, in vivo, this cytokine can indeed trigger the development of such complex disease phenotypes. Transgenic mice that have been engineered to overexpress human or murine TNF molecules in peripheral joints, T cells, or neurons of the central nervous system represent important animal models for human rheumatoid arthritis, systemic inflammation, and multiple sclerosis, respectively. In addition to establishing a central role for TNF in such diseases, these animal models have already proved valuable for identifying additional important disease-effector molecules, and for gaining an insight into the complex in vivo mechanisms that are involved in disease pathogenesis. For example, in the case of arthritis, TNF has been found to transmit its pathogenic effects entirely through interleukin-1, which may therefore represent an additional important target for therapeutic intervention in the human disease. In summary, TNF transgenic models of human disease are expected to serve as important in vivo tools for defining details of disease pathogenesis, potential targets for therapeutic intervention and for evaluating the possible involvement of additional genetic and environmental factors on the disease state. PMID- 8613700 TI - Neutrophil oxidative metabolism and killing of P. brasiliensis after air pouch infection of susceptible and resistant mice. AB - The oxidative burst of polymorphonuclear neutrophils (PMN) and their ability to inhibit Paracoccidioides brasiliensis growth was studied in susceptible (B10.A) and resistant (A/J) mice. The cells were obtained after subcutaneous inoculation in air pouches, yielding highly pure PMN preparations; the number of cells was similar for both strains at 24 h and five times higher in the resistant strain at 15 days. The oxidative metabolism of these PMN was evaluated by the luminol and lucigen-enhanced chemiluminescence upon stimulation with PMA or killed P. brasiliensis (Pb). At 24 h of infection PMN from both strains showed similar responses. However, at 15 days a great enhancement of the Pb-stimulated luminol enhanced chemiluminescence was observed only in PMN from resistant mice. Such increase was markedly inhibited by the addition of catalase. Independent of the mouse strain or time of infection of lucigen-enhanced chemiluminescence showed the same intensity. The lucigen-enhanced chemiluminescence of PMN without stimuli from resistant mice did not change with the time of infection, however, after 15 days of infection a significantly lower chemiluminescence was detected with PMN from susceptible mice. At 15 days of infection the PMN from B10.A were unable to kill P. brasiliensis yeast cells in vitro. Because the lucigenin- and luminol enhanced chemiluminescence detects, respectively, the O2- production and the myeloperoxidase/hydrogen peroxide halide system, the present data show parallels between deficiency in the production of oxygen-reactive species by PMN and lower fungicidal activity. PMID- 8613701 TI - Alveolar macrophage kinetics and multinucleated giant cell formation after lung injury. AB - Multinucleated giant cells (MGC) are a prominent feature of some chronic inflammatory states in the lung. These cells are formed by macrophage fusion, but how this process relates to the kinetics of alveolar macrophage (AM) production and proliferation is not clear. In this serial study, we compare AM kinetics and MGC formation after instilling carbon, silica, asbestos, bleomycin, and saline into the lungs of mice. Animals were killed up to 16 weeks later with [3H]thymidine injected 1 h before death. Counts of AM and MGC were carried out after bronchoalveolar lavage, and cell labeling was assessed by autoradiography. All test substances induced an inflammatory response with equal AM numbers recovered up to 2 weeks. Subsequently, the number returned to normal after carbon but remained elevated in the other groups. After carbon the lung structure was normal, there was no increase in AM label, and no MGC formed. Bleomycin-injected lungs progressed to fibrosis with only a brief, small increase in AM labeling and no MGC formation. After silica, and particularly asbestos, the lungs showed fibrosis, and many granulomas with large MGC were seen. Lavaged AM from these lungs showed a significant increase in DNA synthesis after 2 weeks, followed by higher numbers of MGC, none of which were labeled. Labeled AM tended to be free of particles, whereas MGC after 4 weeks contained many particles. The results indicate a relationship between AM proliferation and fusion, whereby AM growth appears to be prerequisite for cell infusion and MGC formation as a feature of granulomatous disease. PMID- 8613702 TI - In vitro culture of murine peritoneal and alveolar macrophages modulates phagocytosis of Pseudomonas aeruginosa and glucose transport. AB - Phagocytosis by murine peritoneal macrophages (PM phi) of unopsonized Pseudomonas aeruginosa is a novel, glucose-dependent process occurring in concert with glucose or mannose transport via the GLUT1 facilitative transporter. The mechanism by which this transport triggers phagocytosis is not understood. The purpose of these investigations was to improve our understanding of this mechanism by delivery of an alternative sugar (fructose) to PM phi and to murine alveolar macrophages (AM phi). Fructose-cultured PM phi developed fructose dependent phagocytosis of P. aeruginosa with increased glucose-dependent phagocytosis, GLUT1 expression, and [14C]glucose transport. Freshly explanted AM phi, which were unable to transport [14C]glucose or to ingest P. aeruginosa acquired the ability to transport glucose and to phagocytose P. aeruginosa with culture in either glucose or fructose. Both fructose- and glucose-cultured AM phi remained viable but incapable of measurable fructose transport or fructose dependent phagocytosis. These studies suggest that an intracellular metabolite of fructose, glucose, and mannose is involved in triggering macrophage phagocytosis of P. aeruginosa. We demonstrate that delivery of appropriate substrates can substantially improve AM phi phagocytic function and may therefore possibly improve pulmonary host defense against P. aeruginosa. PMID- 8613703 TI - Serglycin-binding proteins in activated macrophages and platelets. AB - The major proteoglycan in macrophages and platelets is the chondroitin sulphate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulphate-Sepharose was used to isolate proteoglycin-binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein-1 alpha (MIP-1 alpha) precipitated a 14-kDa 35S-methionine-labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of approximately 10 and 14 kDa were precipitated by an antiserum against the murine MIP-1 alpha. Protein sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4 sulphate. Amino-terminal sequencing of two proteins from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PE4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparin sulfate in blocking the binding of [3H] chondrotin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1 alpha, and lysozyme play important role in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response. PMID- 8613704 TI - Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. AB - Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of CD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increase. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-beta, PMA caused a strong increase in PKC-delta and a weak increase in PKC-alpha, PKC-epsilon, and PKC-zeta expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation. PMID- 8613705 TI - Estrogen regulation of JE/MCP-1 mRNA expression in fibroblasts. AB - We have recently demonstrated that 17 beta-estradiol (E2) inhibits peritoneal adhesion formation. Because macrophages play a central role in inflammation and wound healing, we chose to investigate whether the E2 could inhibit the expression of JE, the murine monocyte chemoattractant protein-1 (MCP-1). To accomplish this, murine fibroblasts were cultured with physiological concentrations of E2 (3-300 pg/ml) with or without inducers of JE/MCP-1 mRNA expression. Untreated cells failed to express the message, but, following stimulation, a marked increase in JE/MCP-1 mRNA expression was observed. At 10-30 pg/ml, E2 had no effect on JE/MCP-1 mRNA expression in stimulated fibroblasts. In contrast, lower and higher doses of E2 inhibited the expression of JE/MCP-1 mRNA in stimulated fibroblasts. Treatment with tamoxifen reversed the E2-inhibition of expression of the message. These data demonstrate that JE/MCP-1 mRNA expression is controlled, in part, by estrogen and suggest that macrophage recruitment may be affected by circulating levels of E2. PMID- 8613707 TI - Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma. AB - The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO. PMID- 8613706 TI - Interleukin-6 stimulates neutrophil production of platelet-activating factor. AB - Interleukin-6 (IL-6) is an integral mediator of the acute phase response to injury and infection; an exaggerated IL-6 response has been associated with adverse clinical events. The precise role of IL-6 is unclear, but it appears capable of modulating the functional repertoire of mature neutrophils (PMNs). Our previous work demonstrated that IL-6 -stimulated PMNs are primed by lower concentrations of platelet-activating factor (PAF) than nonstimulated PMNs. Recently, we have found that IL-6 suppresses PMN apoptosis via a PAF-like mechanism. We hypothesized that IL-6 stimulates PMNs to produce PAF. PMNs isolated from healthy human donors were incubated with IL-6 (0.1-100 ng/ml) at 37 degrees C. Lipid production was measured by use of thin-layer chromatography, and PAF quantitated with a scintillation proximity assay. IL-6 (1 and 10 ng/ml) stimulated PMNs to produce increase quantities of PAF. PAF production was associated with an increase in PMN cytosolic calcium. These data may provide mechanistic insight into IL-6 regulation of PMN-mediated cytotoxicity and the role of PAF in mediating IL-6 effects on PMNs. PMID- 8613708 TI - IL-4-dependent proliferation of BA/F3 cells expressing a growth-negative mutant of the human IL-4 receptor is restored by enforced expression of Bcl-2. AB - We have studied the regulation of growth and apoptosis in murine BA/F3 cells stably expressing cytoplasmic deletion mutants of the human interleukin-4 receptor (hIL-4R). Previously, we showed that BA/F3 cell transfectants expressing a cytoplasmic deletion mutant of the hIL-4R that lacks the region between Thr(462) and Ala(580), referred to as delta R3, fails to proliferate in the presence of hIL-4. Here we report that supertransfection of delta R3-expressing cells with a constitutively active murine bcl-2 gene results in prolonged survival of the delta R3/bel-2 double transfectants in the absence of cytokines. More importantly, however, the constitutive expression of Bcl-2 restored their capacity to grow permanently with hIL-4. This may provide an explanation for the discrepancy with previous reports showing growth mediation by hIL-4R truncated at position 367. PMID- 8613709 TI - Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils. AB - In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function. PMID- 8613710 TI - Transcriptional and post-transcriptional regulation of GM-CSF-induced IL-1 beta gene expression in PMN. AB - Polymorphonuclear leukocytes (PMN) play an important role in inflammation, immune responses, and tissue repair by secreting interleukin- 1 beta (IL-1 beta). We investigated the regulation of IL-1 beta gene expression in human PMN treated with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced IL 1 beta mRNA accumulation at 0.1 ng/ml and maximal induction was observed at 1 ng/ml. IL- 1 beta mRNA levels reached a maximum with 1-2 h after stimulation with GM-CSF and returned to baseline levels by 4-6 h. The time course of IL-1 beta mRNA induction by GM-CSF was more protracted than previously reported for PMN stimulated with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml). Nuclear run-on analysis indicated that GM-CSF, like TNF, increases IL-1 beta transcription. Kinetic studies with the RNA synthesis inhibitor, actinomycin D, showed that GM CSF induces stable IL-1B mRNA. Cycloheximide enhanced the IL-1 beta mRNA accumulation by GM-CSF at the level of mRNA stabilization, but blocked IL-1 beta mRNA expression by TNF. Thus, GM-CSF increases IL-1 beta message accumulation in PMN at both the transcriptional and post-transcriptional levels by mechanisms that are different from TNF induction of IL-1 beta gene expression. PMID- 8613711 TI - The relationship between hospital per diem billing and DRG reimbursement for urban trauma patients. AB - STUDY OBJECTIVE: To study the relationship between a trauma center per diem charges and medicare DRG reimbursement. DESIGN: Retrospective comparison of charges ($630/day, $1500/ICU day) and hypothetical DRG reimbursement using medical records ICD-9 N and P codes and version 5.0 of grouper. SETTING: An urban level I trauma center that participates in a trauma system that serves a population of 3 million people. PATIENT POPULATION: Trauma patients > or = 16 years old (mean age of 32 years) admitted and discharged between 1/1/88 and 9/30/88. The group was 85% male, 75% black, with a blunt mechanism of injury in 64%. The mean ICU stay was 0.9 days, and the mean total length of stay was 5.0 days. RESULTS: Total per diem charges were $8,652.159, and DRG reimbursement was $8,636,505, causing a net loss of $15,654, or 0.2% of charges. Mean charges and reimbursement did not differ for the entire group. The mean loss per patient was "8. Mean charges and reimbursement differed in penetrating trauma patients (mean loss = $138), as well as those with different lengths of stay. The correlation between charges and reimbursement was 0.42; for penetrating trauma patients, the correlation was 0.58 (p < .001). CONCLUSION: If DRG reimbursement were provided for all admitted trauma patients, the amount would equal per diem rates. Trauma centers with similar patients and lengths of stay can use these per diem rates to estimate DRG reimbursement. PMID- 8613712 TI - The frequencies of disease names with the natural language used in the hospital information system. AB - The statistical behavior of disease names referred by physicians with the natural language in a large hospital information system is little known despite the theoretical and practical interest. To address this issue, we reviewed and investigated the usage-frequencies of 18,274 disease names, 10,288 for outpatient care and 7986 for inpatient care, referred from October 1983 to June 1992 with the notation of the natural language in Japanese by the use of the registration retrieval system of disease names at Fukui Medical School, Japan. Consequently, we found that the investigated distributions did not conform to the Poisson distribution, but conformed well to the Polya-Eggenberger distribution in both cases of outpatient and inpatient care. It implies that the disease names with the natural language are possibly referred by physicians with some interrelations. PMID- 8613713 TI - A managed care workstation for support of ambulatory care in Veterans Health Administration medical centers. AB - This paper describes the development of a Managed Care Workstation for implementation in a Department of Veterans Affairs hospital. Each VA hospital information system contains a wealth of information in a comprehensive and well integrated M database, however, a clinician's access to the information is hampered by a lack of usable database tools. The Managed Care Workstation is designed to enhance access to VA databases. The workstation uses M (Mumps) and SQL to present the VA's M hierarchical database as relational tables on the workstation. This work reveals the benefits of using a SQL-M workstation to access data contained in an M based hospital information system and demonstrates how the workstation architecture supports the information model necessary for management of patient care outcomes. PMID- 8613714 TI - LabLink--the key to multi-computer interfacing. AB - The growing complexity of computing environments requires creative solutions to prevent the gain in productivity promised by computing advances from being swallowed up by the necessity of moving information from one environment to another. LabLink is an interfacing tool for accomplishing such transfers in a clinical laboratory environment. It handles the automated data transfer among four systems, as well as providing substantial back-up capacity for critical operations when systems are down for saves or maintenance. PMID- 8613715 TI - A time domain mean frequency estimation algorithm of two-dimensional real-time Doppler imaging system. AB - A time domain mean frequency estimator is proposed for use in the two-dimensional real-time Doppler ultrasound blood flow imaging system. Since the frequency domain techniques require a long observation period to resolve the frequency content of the Doppler signal spectrum, it is unsuitable for real-time two dimensional applications. Performance characteristics of the Doppler mean frequency estimator are obtained using a computer simulation, which models the Doppler signal and the signal processing network. PMID- 8613716 TI - Mental health case management and technical efficiency. AB - The purpose of the study was to assess whether Community Mental Health Centers (CMHC) operate at different levels of technical efficiency in the production of case management services. A nonparametric technique, Data Envelopement Analysis (DEA), was used to compare 39 CMHC case management programs. The CMHCs, located in Virginia, were compared on the basis of actual performance as reported in annual statistical reports. Efficient utilization of resources in the delivery of case management services was present in only six (15%) of the CMHCs. As a group, CMHCs with local inpatient services have lower technical efficiency than CMHCs without local inpatient services. The provision of local inpatient care is costly; thereby, giving CMHCs without such services a competitive edge on average technical efficiency. This suggests that the decision to provide local hospital care might be a philosophical one, rather than one that is financially driven. PMID- 8613717 TI - Transduction and adaptation in sensory receptor cells. AB - Sensory transduction shares common features in widely different sensory modalities. The purpose of this article is to examine the similarities and differences in the underlying mechanisms of transduction in the sensory receptor cells for vision, olfaction, and hearing. One of the major differences between the systems relates to the nature of the stimulus. In both the visual and olfactory systems a quantal mechanism of detection is possible, because the absorption of a photon or the binding of an odorant molecule provides an energy change significantly greater than the thermal noise in the receptor molecule. In hearing, on the other hand, the energy of a phonon is far lower, and detection occurs by a "classical" mechanism. For vertebrate photoreceptors and olfactory receptor cells, sensory transduction employs a G protein cascade that is remarkably similar in the two cases, and that is closely homologous to other G protein signaling cascades. For auditory and vestibular hair cells, transduction operates via a mechanism of direct coupling of the stimulus to ion channels, in a manner reminiscent of the direct gating of post-synaptic ion channels in various synaptic mechanisms. The three classes of sensory receptor cell share similarities in their mechanisms of adaptation, and it appears in each case that cytoplasmic calcium concentration plays a major role in adaptation. PMID- 8613718 TI - The migration of luteinizing hormone-releasing hormone neurons in the developing rat is associated with a transient, caudal projection of the vomeronasal nerve. AB - Luteinizing hormone-releasing hormone (LHRH) neurons originate in the olfactory placode and vomeronasal organ and migrate to the brain from embryonic day 14 (E14) in the rat. We investigated the development of the vomeronasal nerve and its role as a guide for migrating LHRH neurons. Using fluorescent, lipophilic dye tracing methods, we observed axons that emerge from the vomeronasal organ and cross the nasal septum as several large fascicles. At E14-15, these fascicles converge as they enter the region of the cribriform plate and subsequently disperse, projecting dorsally and caudally across the olfactory bulb and rostral forebrain. At E16, the dorsal branch of the vomeronasal nerve forms a more tightly fasciculated projection; the caudal fibers remain dispersed, extending along the medial forebrain. The number of caudally directed axons decreases during development, leaving four or five present at postnatal day 4 (P4). Immunohistochemical studies indicate that the vomeronasal nerve can be divided into four spatially distinct subpopulations of fibers. One subset, composed of caudal fibers that terminate in the lamina terminalis, selectively expresses TAG 1, a transient axonal surface glycoprotein and PSA-N-CAM, a highly polysialated form of neural cell adhesion molecule. The extension and subsequent retraction of this branch of the vomeronasal nerve corresponds spatially and temporally with the migration of LHRH neurons from the nasal cavity to the brain. Our studies show that between E14 and E18, LHRH neurons migrate in contact with the TAG-1+, PSA-N-CAM+ caudal branch of the vomeronasal nerve. PMID- 8613719 TI - Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion. AB - Most nicotinic ACh receptors (AChRs) studied to date in the embryonic chicken ciliary ganglion are recognized either by monoclonal antibody (mAb) 35 or by alpha-bungarotoxin (alpha-Bgt). Previous studies found that mAb 35-AChRs are found at both synaptic and extrasynaptic sites, while alpha-Bgt-AChRs are found exclusively at extrasynaptic sites. To gain a three-dimensional understanding of the distribution of these two AChR classes and their spatial relationship to synaptic sites, we have visualized mAb 35-AChRs and alpha-Bgt-AChRs immunofluorescently using laser scanning confocal microscopy and have compared their distribution with that of the synaptic vesicle antigen SV2. Both mAb 35 AChR and alpha-Bgt-AChRs are found in clusters that are widely distributed over the surface of embryonic chicken ciliary ganglion neurons. Some mAb 35-AChRs are located at synaptic sites, but the bulk of them are located extrasynaptically in well-defined patches measuring 1-4 micron in diameter. alpha-Bgt-AChRs are found almost exclusively in these extrasynaptic sites, which thus contain both AChR types. These sites are often surrounded by elements of the synaptic calyx but are themselves largely free of SV2 antigen. In 14 week chickens the relationship between mAb 35-AChRs, alpha-Bgt-AChRs, and synaptic sites is similar to that in embryos except that in this instance individual synaptic boutons are often surrounded by AChR-containing patches. These results suggest that most surface AChRs in both embryonic and mature chicken ciliary neurons are perisynaptic, which raises questions about the function of these AChRs. PMID- 8613720 TI - Regional patterns of c-fos mRNA expression in rat hippocampus following exploration of a novel environment versus performance of a well-learned discrimination. AB - Previous studies using c-fos cRNA in situ hybridization demonstrated a differential involvement of hippocampal subfields CA1 and CA3 in the acquisition of an olfactory discrimination (Hess et al., 1995). The present experiments employed the same method to examine changes in neuronal activity associated with two related behaviors: (1) initial exploration of the training apparatus and (2) performance of a well-learned odor discrimination. Rats in the two groups had similar labeling patterns within hippocampus indicating increased expression in all three major subfields with the greatest effect being in CA1. This pattern of "CA1 dominance" was notably different from that produced during early stages of two-odor discrimination learning in prior experiments. Hippocampal labeling in exploration and performance rats differed in that (1) hybridization was greater in CA1, CA3, and dentate gyrus in the former group and (2) a tendency for labeled cells to occur in clusters was more evident in exploration animals. Levels of c fos mRNA in olfactory and visual structures were not predictive of expression patterns within hippocampus although labeling in piriform cortex and dentate gyrus was correlated in rats performing a well-practiced discrimination. Moreover, the pattern of hybridization in olfactory bulb was found to be behaviorally dependent. These results, together with those from previous studies, indicate that hippocampus has multiple patterns of regional activation but that one of these is common to very different behavioral circumstances. It is hypothesized that this common pattern emerges whenever the animal responds to distant cues using species-specific or well-learned behaviors and involves coordinated temporal convergence of sensory and septal/brainstem inputs. PMID- 8613721 TI - Intrastriatal implantation of fibroblasts genetically engineered to produce brain derived neurotrophic factor prevents degeneration of dopaminergic neurons in a rat model of Parkinson's disease. AB - Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of the dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although various treatments are successfully used to alleviate the symptoms of PD, none of them prevents or halts the neurodegenerative process of the disease. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of proteins, supports the survival and the differentiation of dopaminergic neurons. BDNF also prevents the death of dopaminergic neurons in vitro, which suggests that it may be of possible use in the development of neuroprotective therapies for PD. To determine whether BDNF is neuroprotective for SNpc dopaminergic neurons in the adult brain, we used a rat model of PD in which degeneration of 60-70% of these neurons was induced by an intrastriatal injection of 6-hydroxydopamine (6-OHDA). We report here that intrastriatal grafts of fibroblasts genetically engineered to produce BDNF partially prevent the loss of nerve terminals and completely prevent the loss of cell bodies of the nigrostriatal dopaminergic pathway that is induced by the intrastriatal injection of 6-OHDA. In contrast, the implantation of control fibroblasts that did not produce BDNF failed to protect nerve terminals and cell bodies against 6-OHDA induced damage. Our observation that grafts of BDNF-producing fibroblasts protect against 6-OHDA-induced degeneration of SNpc dopaminergic neurons in the adult rat brain opens new perspectives for treatments aimed at the prevention of neurodegeneration in PD, using gene therapy and neurotrophic factors such as BDNF. PMID- 8613722 TI - Regional, cellular, and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain. AB - The pathways governing signal transduction in the mesocortical and nigrostriatal dopamine systems of the brain are of central importance in a variety of drug actions and neurological diseases. We have analyzed the regional, cellular, and subcellular distribution of the closely related D1 and D5 subtypes of dopamine receptors in the cerebral cortex and selected subcortical structures of rhesus monkey using subtype specific antibodies. The distribution of D1 and D5 receptors was highly differentiated in subcortical structures. In the neostriatum, both D1 and to a lesser extent D5 antibodies labeled medium spiny neurons, while only D5 antibodies labeled the large aspiny neurons typical of cholinergic interneurons. In the caudate nucleus, D1 labeling was concentrated in the spines and shafts of projection neurons, whereas D5 antibodies predominantly labeled the shafts, and less commonly, the spines of these cells. The D1 receptor was abundantly expressed in the neuropil of the substantia nigra pars reticulata while the D5 antibodies labeled only a few scattered cell bodies in this structure. Conversely, D5 antibodies labeled cholinergic neurons in the basal forebrain more intensely than D1 antibodies. Within the cerebral cortex and hippocampus, D1 and D5 antibody labeling was prominent in pyramidal cells. Double-label experiments revealed that the two receptors were frequently coexpressed in neurons of both structures. Ultrastructurally, D1 receptors were especially prominent in dendritic spines whereas dendritic shafts were more prominently labeled by the D5 receptor. The anatomical segregation of the D1 and D5 receptors at the subcellular level in cerebral cortex and at the cellular level in subcortical areas suggest that these closely related receptors may be preferentially associated with different circuit elements and may play distinct regulatory roles in synaptic transmission. PMID- 8613723 TI - Increased production of 4 kDa amyloid beta peptide in serum deprived human primary neuron cultures: possible involvement of apoptosis. AB - The etiology of the amyloid beta peptide in sporadic Alzheimer's disease (AD) is not known. Amyloid beta peptide (A beta), a proteolytic product of the amyloid precursor protein (APP), is deposited in the senile plaques and cerebrovascular tissues of individuals with either sporadic or familial AD (FAD). Increased A beta production from mutant APPs in FAD fosters the hypothesis that overexpression of A beta plays a primary role in the pathogenesis of AD. The absence of APP mutations in sporadic AD which displays identical pathological features than FAD such as synapse and neuronal loss, senile plaques and neurofibrillary tangles, suggests other causes for overexpression and/or deposition of A beta. To investigate the effect of neuronal death on APP metabolism and A beta secretion, human primary neuron cultures were induced to undergo apoptosis by serum deprivation. Serum deprived neurons display shrunken and rounded morphology, contain condensed chromatine and fragmented DNA, which are characteristic of apoptosis. In serum deprived neurons, metabolism of APP through the nonamyloidogenic secretory pathway is decreased to 20% from 40% in control cultures whereas 4kDa A beta is increased three- to fourfold. The results suggest that human neurons undergoing apoptosis generate excess A beta and indicates a possible mechanism for increased A beta in the absence of APP mutations. PMID- 8613724 TI - Excitotoxic cell death and delayed rescue in human neurons derived from NT2 cells. AB - The excitotoxic response of NT2-N cells, a clonal line of human teratocarcinoma cells that are terminally differentiated into neuron-like cells, was examined using several endpoints. A 15 min exposure to glutamate produced a dose-dependent toxicity with a maximal cell loss of 80-90% in 6 week old cells. The rapidly triggered excitotoxicity induced by glutamate was blocked by NMDA selective antagonists, was calcium dependent and pH sensitive and could be mimicked by NMDA but not by non-NMDA agonists, AMPA, kainate or quisqualate. The non-NMDA agonists however caused toxicity on prolonged exposure. The NMDA receptor modulators glycine and spermidine enhanced glutamate-mediated toxicity whereas ifenprodil potently and completely inhibited toxicity suggesting that the toxic response is mediated by the NR1/NR2B combination of NMDA subunits. These cells can be rescued from death up to 1 hr after removal of glutamate by NMDA receptor blockade, removal of extracellular Ca2+ or lowering of pH. The extent of rescue is directly related to the time elapsed before intervention. Blockage of NMDA receptor activity for 1 hr immediately after removal of glutamate is both necessary and sufficient for complete rescue. Glutamate-mediated toxicity was not prevented by nitric oxide synthase inhibitors nor was nitric oxide synthase detected in NT2-N cells indicating that nitric oxide is not required for glutamate-mediated excitotoxicity. In summary, NT2-N cells exhibit a robust excitotoxic response and represent a novel model system in which to study the molecular basis of excitotoxic cell death. PMID- 8613725 TI - ATP-induced cytoplasmic calcium mobilization in Bergmann glial cells. AB - ATP receptor mediated Ca2+ signaling was recorded from Bergmann glial cells in cerebellar slices obtained from mice of different ages (postnatal days 6 to 45). To measure the cytoplasmic concentration of Ca2+ ([Ca2+]in), either individual cells were loaded with the Ca(2+)-sensitive probes using the whole cell patch clamp technique or slices were incubated with the dye and the microfluorimetric system was focused on individual cells. Signals were recorded either with single detector microfluorimetry of the dye fura-2 or by confocal laser scanning microfluorimetry (fluo-3-based recordings). Extracellular application of 100 microns ATP caused a transient elevation of [Ca2+]in, which amplitude was significantly higher in Bergmann glial cell processes as compared with their soma. The rank order of potency for the purinoreceptor agonists was: ADP > or = ATP > UTP >> AMP = adenosine = alpha, beta-methylene-ATP. ATP-triggered Ca2+ transients were reversibly inhibited by the P2 purinoreceptor agonist suramin (100 microM). The involvement of P2 metabotropic receptors is inferred by the observation that ATP mediated cytoplasmic Ca2+ transients were not associated with a measurable change in membrane conductance. The [Ca2+]in increase was due to release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular stores since responses were still observed in Ca(2+)-free extracellular solutions and were irreversibly blocked by the inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase, thapsigargin, and by the competitive inhibitor of the InsP3-gated intracellular Ca2+ channels heparin. Intracellular dialysis altered the refilling process of the InsP3-sensitive stores, suggesting that cytoplasmic factors control ATP mediated Ca2+ signalling. PMID- 8613726 TI - Nerve injury enhances rat neuronal glutamate transporter expression: identification by differential display PCR. AB - An increase in neuronal glutamate transporter expression after nerve injury was demonstrated by means of differential display PCR (DD-PCR) coupled with in situ hybridization. DD-PCR was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the rat. The expression of several gene fragments were found to be increased following nerve injury; the full length cDNA corresponding to one fragment was cloned by subsequent rat cDNA library screening. The close homology of glutamate transporters with our rat cDNA led us to conclude that this clone corresponds to the rat neuronal glutamate transporter (rat EAAC1). We speculate that the upregulation of this glutamate uptake system may increase the resistance of these cells against neurotoxic glutamate accumulation during the process of nerve regeneration. PMID- 8613727 TI - Fibroblast growth factor 2 increases Otx2 expression in precursor cells from mammalian telencephalon. AB - Dissociated primary cultures from rat telencephalon at different developmental stages were used to study the effect of basic fibroblast growth factor (FGF2) on Otx2, Dlx1, and Emx1, three homeobox genes expressed in different regions of the developing mammalian forebrain. At embryonic day (E)13.5. the regional pattern of expression of Otx1, Otx2, Dlx1, Dlx2, Dlx5, and Emx1 is maintained in primary culture, suggesting that cells are already committed to a regional identity at this stage. In these cultures, Otx2 is expressed by precursor cells, whereas Dlx1 and Emx1 are predominantly expressed by postmitotic cells. We found that FGF2 increased Otx2 expression within precursor cells and the total number of Otx2 expressing cells. This effect was gene-specific, dose-dependent, and temporally regulated, with larger effects at earlier stages of development (E11.5). At E13.5, the effect of FGF2 on Otx2 expression was restricted to the basal telencephalon. Our results suggest that a restricted population of neuroblasts respond to FGF2 in a temporally regulated fashion by proliferating and increasing Otx2 expression. This interaction between FGF2 and Otx2 may be important for the regulation of neurogenesis in the forebrain. PMID- 8613729 TI - Junctional and extrajunctional glutamate receptor channels in Drosophila embryos and larvae. AB - Glutamate receptor channels in Drosophila embryos and larvae were examined with the patch-clamp technique in various configurations. In the cell-attached mode, only one type of channel was observed in the extrajunctional region at any stages. The burst duration histogram was fit with three exponentials. The burst duration of long component lengthened with increasing glutamate concentration. In excised outside-out patches the unitary channel current was 7.1 pA at -60 mV and direction of current reversed at zero membrane potential. In contrast, junctional receptor channels had different properties. In the whole-cell configuration, spontaneous synaptic currents with steps on the falling phase were observed. The step amplitudes had two discrete values of 9.4 and 18.5 pA at -60 mV, due to openings of junctional glutamate receptor channels. Synaptic currents changed amplitudes linearly with the membrane potential in the negative potential range but nonlinearly above zero. With 1 mM glutamate in the bath, synaptic currents were no longer observed. Instead, there were single channel events with the current amplitude varying between 8 and 12 pA at -60 mV. Their long burst duration depended on glutamate concentration indicating that they are glutamate receptor channel events. The extrapolated reversal potential of these channel currents was around +12 mV. These junctional receptor channels were strictly localized at the junction. Our findings suggest that the channel conversion mechanism in Drosophila is different from that observed in vertebrates. Further close examination of other intermediate steps during neuromuscular junction formation is needed. PMID- 8613728 TI - Visual responses in the lateral geniculate nucleus of dichromatic and trichromatic marmosets (Callithrix jacchus). AB - New-world primates such as the marmoset (Callithrix jacchus) show polymorphism for the middle- to long-wavelength sensitive cone pigments. Each X-chromosome carries a gene for only one of three possible pigments. All males are thus dichromats, but some females will be trichromats. We have investigated the responses of cells of the parvocellular (PC) and magnocellular (MC) systems within animals from a single marmoset family. The middle- to long-wavelength pigment of dichromats was identified physiologically. Trichromats could readily be distinguished from dichromats by the presence of a high proportion of red green opponent PC-cells. The physiological classification of phenotypes was confirmed with genetic analysis. The pattern of inheritance was consistent with current genetic models. In trichromatic females, the properties of cells resembled in detail those of cells from the PC- and MC-pathways of the macaque. In dichromats, cell responses resembled those of trichromats (except for the lack of opponency in PC-cells); PC-cells showed sustained and MC-cells transient responses, with a lower contrast gain for the former type. One difference was that a proportion of PC-cells in dichromats showed strong rod input even at high levels of retinal illuminance. Thus, in trichromatic marmosets the presence of two middle- to long-wave pigments appears to permit the elaboration of all the physiological properties associated with trichromacy. PMID- 8613730 TI - Transiently selective activation of phosphoinositide turnover in layer V pyramidal neurons after specific mGluRs stimulation in rat somatosensory cortex during early postnatal development. AB - Biochemical analysis of muscarinic- and metabotropic-glutamate receptor stimulated phosphoinositide (PI) turnover in rat cortical preparations during the first three weeks of postnatal development indicates the existence of a transiently increased accumulation of labeled inositol polyphosphates during the first postnatal week (Gonzales and Crews, 1985; Dudek et al., 1989). We now report for first time the visualization of those neurons responding with increased PI turnover to glutamatergic or cholinergic-receptor stimulation in rat somatosensory cortex during early postnatal development utilizing a recently described method (Bevilacqua et al, 1994). Three, 7, 10, 14, and 21 d old rats were studied. Carbachol in the presence of lithium stimulates 3H-CMP-PA accumulation throughout the cortex at all ages studied. In comparison labeled neurons responding to t-ACPD in the presence of lithium were located exclusively in layer V at P3 and P7, but were found labeled throughout the cortex at P10. Given that glutamate and cholinergic agonist stimulation are both necessary but not sufficient for cortical plasticity to occur, and that muscarinic and mGluRs stimulation both induce a peak in PI turnover response during the same period of experience-dependent neocortical plasticity, PI derived second messengers signals might be involved in the regulation of the molecular mechanisms of neuronal plasticity. Furthermore, our results show the anatomical correlate of receptor specific PI turnover activation, and indicate that specific agonist induced PI responses are age, and layer specific. PMID- 8613732 TI - A quantitative analysis of presynaptic calcium dynamics that contribute to short term enhancement. AB - Augmentation and posttetanic potentiation--two forms of short-term synaptic enhancement produced by repetitive presynaptic action potentials--are dependent on the buildup and decay of nerve terminal residual calcium that occurs on the seconds to minutes time scale. With the goal of providing a quantitative understanding of these kinetics, we measured the buildup and decay of calcium ions in nerve terminals at the crayfish neuromuscular junction under a variety of intracellular buffer conditions and stimulation paradigms. The calcium extrusion process in the terminals was characterized by analysis of calcium levels reached during long stimulus trains as a function of action potential frequency. The extrusion was linearly dependent on the free calcium ion concentration. Using this result, we developed a mathematical model and computer simulation of the residual calcium kinetics. The model demonstrates the experimentally observed dependence of decay rate on exogenous calcium buffer concentration, and can be explicitly solved to provide an expression for the limiting exponential time course of calcium decay following trains in terms of calcium buffer and extrusion characteristics. Methods to determine the calcium influx per action potential, characteristics of endogenous buffer, and the rate of calcium extrusion are suggested by our analysis and demonstrated experimentally. PMID- 8613731 TI - Brain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brain. AB - A pathology of brain serotonergic (5-HT) systems has been found in psychiatric disturbances, normal aging and in neurodegenerative disorders including Alzheimer's and Parkinson's disease. Despite the clinical importance of 5-HT, little is known about the endogenous factors that have neurotrophic influences upon 5-HT neurons. The present study examined whether chronic pain parenchymal administration of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NGF could prevent the severe degenerative loss of serotonergic axons normally caused by the selective 5-HT neurotoxin p chloroamphetamine (PCA). The neurotrophins (5-12 micrograms/d) or the control substances (cytochrome c or PBS vehicle) were continuously infused into the rat frontoparietal cortex using an osmotic minipump. One week later, rats were subcutaneously administered PCA (10 mg/kg) or vehicle, and the 5-HT innervation was evaluated after two more weeks of neurotrophin infusion. As revealed with 5 HT immunocytochemistry, BDNF infusions into the neocortex of intact (non-PCA lesioned) rats caused a substantial increase in 5-HT axon density in a 3 mm diameter region surrounding the cannula tip. In PCA-lesioned rats, intracortical infusions of BDNF completely prevented the severe neurotoxin-induced loss of 5-HT axons near the infusion cannula. In contrast, cortical infusions of vehicle or the control protein cytochrome c did not alter the density of serotonergic axons in intact animals, nor did control infusions prevent the loss of 5-HT axons in PCA-treated rats. NT-3 caused only a modest sparing of the 5-HT innervation in PCA-treated rats, and NGF failed to prevent the loss of 5-HT axon density. The immunocytochemical data were supported by neurochemical evaluations which showed that BDNF attenuated the PCA-induced loss of 5-HT and 5-HIAA contents and 3H-5-HT uptake near the infusion cannula. Thus, BDNF can promote the sprouting of mature, uninjured serotonergic axons and dramatically enhance the survival or sprouting of 5-HT axons normally damaged by the serotonergic neurotoxin PCA. PMID- 8613733 TI - Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth. AB - B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons. PMID- 8613734 TI - Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of alpha 7 by membrane depolarization through a Ca2+/calmodulin-dependent kinase pathway. AB - We have examined the regulation of neuronal nicotinic ACh receptor (nAChR) genes and ACh-evoked currents by neonatal rat sympathetic neurons developing in culture. These neurons contain 5 nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4. When developing in culture, the neurons express 4 of these transcripts, alpha 3, alpha 5, beta 2, and beta 4, at levels similar to those in neurons developing in vivo: alpha 3 mRNA levels increase two- to threefold over the first week, whereas the levels for alpha 5, beta 2, and beta 4 remain essentially constant. In contrast, alpha 7 mRNA levels drop by 60-75% within the first 48 hr and remain low. We show that during the first week, the ACh-evoked current densities on these cultured neurons increase twofold and correlate well with the increase in alpha 3 mRNA levels. Depolarizing the neurons with 40 mM KCl for 1-2 d upregulates the alpha 7 gene; this specific change in alpha 7 mRNA level correlates with an increase in alpha-bungarotoxin (alpha-BTX) binding on the surface of the neurons. Depolarization has little effect on the expression of the other four transcripts, or on the magnitude or kinetics of the ACh-evoked currents. Furthermore, activators or inhibitors of protein kinase A (PKA), protein kinase C (PKC), or tyrosine kinase do not affect nAChR transcript levels in these cultured neurons. The effect of membrane depolarization on alpha 7 expression is a result of Ca2+ influx through L-type Ca2+ channels, and we show that alpha 7 is upregulated through a Ca2+/calmodulin-dependent protein kinase (CaM kinase) pathway. The identification of CaM kinase as a link between activity and neurotransmitter receptor expression may indicate a novel mechanism that underlies some forms of synaptic plasticity. PMID- 8613735 TI - Role of ventrolateral medulla catecholamine cells in hypothalamic neuroendocrine cell responses to systemic hypoxia. AB - Systemic hypoxia stimulates the release of vasopressin (VP) and adrenocorticotropin hormone (ACTH). To examine the involvement of catecholamine cell groups of the ventrolateral medulla (VLM) in the neuroendocrine responses, we have used the c-fos activity mapping technique to compare the effects of hypoxia on VLM catecholamine cells to those on neurosecretory VP and putative corticotropin releasing factor (CRF) containing cells. A limited degree of catecholamine cell activation was evident at predominantly mid-VLM levels at 12% oxygen in the inspired air. Further reduction in inpsirate oxygen levels enhanced recruitment of caudally located VLM catecholamine cells considered to form part of the A1 noradrenergic cell group. Threshold for activation of VP and putative CRF cells occurred at the 10% oxygen level. Unexpectedly, this stimulus also activated neurosecretory oxytocin (OT) cells. With increasing hypoxic severity the number of activated supraoptic VP and OT cells was not significantly different to that observed at the 10% level. However, paraventricular neuroendocrine responses continued to increase with putative CRF containing cells of the medial parvocellular zone having nearly double the level of activity (as measured by the number of cells within this region displaying Fos-like immunoreactivity; FLI) at 6% compared to that apparent to the 10% level of hypoxia. Paraventricular VP cells displaying FLI were also increased at the most severe levels of hypoxia but this effect was much less marked than the medial parvocellular response. Consistent with a role for VLM catecholamine cells in generation of neuroendocrine cell responses to hypoxia, unilateral VLM lesions, restricted to the caudal two thirds of the catecholamine cell column, resulted in significant reductions in the responses of all three cell types. These results, in addition to establishing a role for VLM catecholamine cells in neuroendocrine cell responses to systemic hypoxia, have important general implications for catecholamine cell group involvement in neuroendocrine regulation. PMID- 8613736 TI - Multiple Shaker potassium channels in a primitive metazoan. AB - Voltage-gated potassium channels are critical elements in providing functional diversity in nervous systems. The diversity of voltage-gated K+ channels in modern triploblastic metazoans (such as mollusks, arthropods and vertebrates) is provided primarily by four gene subfamilies (Shaker, Shal, Shab, and Shaw), but there has been no data from the ancient diploblastic metazoans until now. Diploblasts, represented by jellyfish and other coelenterates, arose during the first major metazoan radiation and are the most structurally primitive animals to have true nervous systems. By comparing the K+ channels of diploblasts and triploblasts, we may determine the fundamental set of K+ channels present in the first nervous systems. We now report the isolation of two Shaker subfamily cDNA clones, jShak1 and jShak2, from the hydrozoan jellyfish Polyorchis penicillatus (Phylum Cnidaria). JShak1 and jShak2 express transient outward currents in Xenopus oocytes most similar to Shaker currents from Drosophila in their rates of inactivation and recovery from inactivation. The finding of multiple Shaker subfamily genes is significant in that multiple Shaker genes also exist in mammals. In Drosophila, multiple Shaker channels are also produced, but by a mechanism of alternative splicing. Thus, the Shaker K+ channel subfamily had an established functional identity prior to the first major radiation of metazoans, and multiple forms of Shaker channels have been independently selected for in a wide range of metazoans. PMID- 8613737 TI - Cellular processing of temporal information in medial vestibular nucleus neurons. AB - Quantitative descriptions of the cellular transformations from behaviorally relevant inputs into temporal patterns of firing are crucial for understanding information processing in systems of neurons and for incorporating biological properties of neurons into models of the neural control of behavior. To understand how neurons that mediate vestibulo-ocular behavior transform their inputs into temporal patterns of firing, we examined responses of medial vestibular nucleus (MVN) neurons to current injected intracellularly. MVN neurons recorded from avian brain slices fired spontaneously. Sinusoidal modulation of input current produced precisely sinusoidal modulation of firing rate. The transformation between input current and firing rate was remarkably linear: firing rate scaled linearly as a function of current amplitude, and the responses to steps of input current were predicted accurately from the linear superposition of responses to sinusoidal modulation of input current. Over the physiological range of head movement frequencies, from 0.1 to 10 Hz, peak-to-peak modulation of firing rate was relatively constant or increased slightly in most neurons. In contrast, when hyperpolarizing current was used to keep neurons below threshold for action potentials, the frequency response of the membrane potential behaved like a low-pass filter. These results imply that the membrane conductances that are active when MVN neurons fire compensate for the low-pass characteristics of the membrane to allow faithful transmission of high frequency head movement signals. PMID- 8613738 TI - Two voltage-dependent K+ conductances with complementary functions in postsynaptic integration at a central auditory synapse. AB - The medial nucleus of the trapezoid body (MNTB) relays auditory information important for sound source localization. MNTB neurons faithfully preserve the temporal patterning of action potentials (APs) occurring in their single giant input synapse, even at high frequencies. The aim of this work was to examine the postsynaptic potassium conductances that shape the transfer of auditory information across this glutamatergic synapse. We used whole cell patch techniques to record from MNTB neurons in thin slices of rat brainstem. Two types of potassium conductance were found which had a strong influence on an MNTB neuron's postsynaptic response. A small low voltage threshold current, Id, limited the response during each EPSP to a single brief AP. Id was specifically blocked by dendrotoxin (DTX), resulting in additional APs during the tail end of the EPSP. Thus DTX degraded the temporal fidelity of synaptic transmission, since one presynaptic AP then led to several postsynaptic APs. A second conductance was a fast delayed rectifier with a high voltage activation threshold, that rapidly repolarised APs and thus facilitated high frequency AP responses. Together, these two conductances allow high frequency auditory information to be passed accurately across the MNTB relay synapse and separately, such conductances may perform analogous functions elsewhere in the nervous system. PMID- 8613739 TI - Facilitation of olfactory learning by a modulator of AMPA receptors. AB - The effects of a benzoyl-piperidine drug (BDP) that facilitates AMPA receptor mediated synaptic responses were tested on the acquisition and retention of long term memory at dosages that had no detectable effects on a variety of performance measures. BDP-12 produced a dose-dependent suppression of exploratory activity in rats with statistically reliable effects occurring at 50 mg/kg (i.p.). The drug had no effects on balance beam performance at 30 mg/kg but at 45 mg/kg reduced the number of crossings made within a session; it did not, however, affect the time required to perform a traversal. The performance of well-trained rats presented with a familiar pair of odors (correct and incorrect) was not not detectably altered by BDP-12 at 30 mg/kg; however, the number of correct responses made in a five-trial test was reduced at 45 mg/kg. These results indicate that the AMPA receptor modulator at 30 mg/kg has little influence on arousal, motivation, sensori-motor processing, and attention; higher dosages cause a depression of learned and unlearned prepotent responses. The effects of the lower concentration were tested on two-odor discrimination learning in rats that had extensive training on the task. The animals (n = 20) were given three or five acquisition trials with novel odor pairs immediately after an injection of drug or vehicle and then tested 1-3 d later for retention in five unrewarded probe trials. Retention performance was not significantly better than chance 52.6 +/- 4.5% correct) for odors learned on vehicle injection days but was well above chance for odors learned on drug injection days (70.6 +/- 4.2% correct). Within subject comparisons confirmed the memory enhancing effect of BDP-12 (p < 0.01). Analyses of performance during five training trials indicated that the rats made more correct responses on days on which they were given the drug than on days on which they were injected with vehicle (p < 0.02). Within-subject differences in acquisition were correlated with differences in retention (r = 0.70). There were no evident effects of the drug on response latencies during acquisition. These results suggest that AMPA receptor modulators reduce the amount of training needed for the formation of long-term memory and do so at dosages which have little effect on variables that secondarily influence acquisition. Possible reasons for this selectivity are discussed. PMID- 8613740 TI - Opioid and adenosine peripheral antinociception are subject to tolerance and withdrawal. AB - The selective mu-opioid agonist, D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAMGO), or the selective A1-adenosine agonist N6-cyclopentyladenosine (CPA), when coinjected intradermally with prostaglandin E2 (PGE2), dose-dependently inhibited PGE2 induced mechanical hyperalgesia in the rat hindpaw, as determined by the Randall Selitto paw-withdrawal test. Repeated (hourly x 3) intradermal injections of DAMGO or CPA produced tolerance to the antinociceptive effect of a fourth injection 1 hr later. Furthermore, repeated (hourly x 3) intradermal injections of DAMGO produced cross-tolerance to the antinociceptive effect of CPA, and repeated (hourly x 3) intradermal injection of CPA produced cross-tolerance to the antinociceptive effect of DAMGO. The demonstration of the bidirectional cross tolerance between the peripheral antinociceptive effects of DAMGO and CPA supports the hypothesis that both these agents produced antinociception by acting on the same cell, presumably the primary afferent nociceptor, and that the development of tolerance involves changes downstream to activation of mu-opioid and A1-adenosine receptors. The opioid antagonist naloxone, which had no effect on paw-withdrawal threshold in normal paws, produced withdrawal threshold in normal paws, produced withdrawal hyperalgesia in DAMGO-tolerant paws. Furthermore, naloxone elicited a cross-withdrawal hyperalgesia response in CPA tolerant paws. Similarly, the A1-adenosine antagonist 1,3-dipropyl-8-(2-amino-4- chlorophenyl)-xanthine (PACPX), which had no effect on paw-withdrawal threshold in normal paws, elicited a withdrawal hyperalgesia response in CPA-tolerant paws and cross-withdrawal hyperalgesia responses in DAMGO-tolerant paws. These cross dependence and cross-withdrawal responses suggest that the development of dependence to mu-opioid and A1-adenosine agonists involves changes in the same second messenger system downstream to both mu-opioid and A1-adenosine receptor activation. PMID- 8613741 TI - Synchronized oscillations in hippocampal CA3 neurons induced by metabotropic glutamate receptor activation. AB - The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3 dicarboxylic acid (ACPD) at concentrations above 60 microM produced stereotypic oscillatory activity in CA3 pyramidal cells of rat hippocampal slices. This oscillatory activity consisted of trains of depolarizations with overriding action potentials. On average, individual trains lasted 7 sec and recurred at intervals of 24 sec. During each train, the constituent depolarizations achieved a maximum frequency of 27 Hz, then slowed to 8 Hz toward the end of the train. Extracellular and dual intracellular recordings suggested that this ACPD-induced oscillatory activity occurred synchronously in the CA3 population. The oscillations persisted in the presence of GABAA, GABAB, and NMDA receptor antagonists. In contrast, the oscillations were blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10-30 microM). Likewise, the oscillations were blocked by the metabotropic glutamate receptor antagonists (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG; 1 mM], (S)-4 carboxy-3-hydroxyphenylglycine [(S)-4C3HPG; 1mM] and (S)-4-carboxyphenylglycine [(S)-4CPG; 1 mM]. The results suggest that activation of metabotropic glutamate receptors can result in a permissive state that allows AMPA/kainate receptor mediated conductances to mediate synchronized activity among hippocampal CA3 neurons. PMID- 8613742 TI - Differential effects of glycosaminoglycans on neurite growth on laminin and L1 substrates. AB - Glycosaminoglycans (GAGs), the carbohydrate moieties of proteoglycans, are thought to be positive and negative regulators of axonal growth. The physiological role of GAGs is controversial as some studies have shown that GAGs inhibit cell adhesion and neurite elongation (Exp Neurol 109:111, 1990) whereas other studies have reported a growth stimulatory effect of GAGs (Development 114:17, 1992). These and other studies have examined the effects of GAGs using different types of neurons and different substrate conditions thereby making a direct comparison of the experimental data difficult. To resolve the controversy concerning the ability of exogenous GAGs to modulate neurite growth, we examined the effects of a panel of structurally different GAGs on the growth of postnatal rat cerebellar granule neurons and embryonic rat dorsal root ganglia (DRG) neurons on substrates of either laminin or the L1 glycoprotein. Here we show that chondroitin 4-sulfate (CS4), chondroitin 6-sulfate (CS6), and keratan sulfate (KS) inhibit neurite growth from both cerebellar and DRG neurons on laminin coated surfaces. On L1 surfaces, however, these GAGs are either extremely weak inhibitors of neurite extension or, in the case of CS4, a modest stimulator of neurite growth. Heparan sulfate (HS) and dermatan sulfate (DS) inhibited the growth of cerebellar neurons but not the growth of DRG neurons on L1-coated surfaces. On laminin surfaces, DS and HS had no effect on neurite growth from both cerebellar and DRG neurons. These results demonstrate a cellular and a substrate specificity to the effects of exogenous GAGs on neurite extension in vitro. They suggest that while CS and KS GAGs may not exert strong negative influences over axonal growth in regions of the developing CNS where the L1 glycoprotein is abundant, these GAGs are capable of inhibiting the growth of axons that extend within an environment rich in laminin. PMID- 8613743 TI - Laminin alpha 2 chain (M chain) is found within the pathway of avian and murine retinal projections. AB - Laminin-1 is found at the end-feet of neuroepithelial cells along the outer margin of the optic pathway during early stages of development. Prior to the establishment of most retinal projections in vivo, laminin-1 expression becomes restricted to basement membranes associated with the eye and optic pathway. We report that, in contrast to the alpha 1, beta 1, and gamma 1 chains of laminin-1, laminin alpha 2 chain (formerly laminin M chain) is expressed within the pathway of avian and murine retinal ganglion cell (RGC) growth cones as they extend into the optic nerve, across the optic chiasm and into the brain. Expression of laminin alpha 2 chain is reduced soon after formation of the visual projections but nevertheless maintained at non-basal lamina sites within the adult optic nerve. Laminin alpha 2 chain, in contrast to laminin-1 chains, is also highly expressed in the developing avian tectobulbar pathway. Chick optic nerve derived type-1 astrocytes in culture express laminin alpha 2 chains as extracellular fibrils on their surface. Laminin alpha 2 chain was also detected on the surface of cultured embryonic retinal neurons and developing RGCs. These results suggest that astrocytes and/or RGCS may synthesize laminin alpha 2 chain along the developing optic pathway, and imply that laminin alpha2--in a complex with non beta1 and non-gamma1 laminin chains-may serve as an adhesive substrate and possibly as a guidance cue for elongating RGC growth cones in vivo. PMID- 8613744 TI - Large-scale reorganization at multiple levels of the somatosensory pathway follows therapeutic amputation of the hand in monkeys. AB - Reorganization of somatosensory cortex after peripheral nerve damage typically has been attributed to cortical plasticity. Here we provide evidence that much of the large-scale cortical reorganization that occurs after a major loss of peripheral inputs reflects the sprouting or expansion of afferents from the remaining forelimb into deprived territories of the spinal cord and brainstem. We examined sensory afferent terminations in the spinal cord and brainstem, and determined the somatotopic organization of cortical area 3b in three adult monkeys with previous hand or forearm amputation, as veterinary treatment of forelimb injuries. In each monkey, the distribution of labeled sensory afferent terminations from the remaining parts of the fore-limb was much more extensive than the normal distribution of inputs from the forelimb, and extended into portions of the dorsal horn of the spinal cord and the cuneate nucleus of the brainstem related to the amputated hand. In the same animals, tactile stimulation of the forelimb activated much of the deprived hand representation in area 3b of cortex; the lateral portion of the deprived region in area 3B appeared to be reactivated by inputs from the face. These data provide important new evidence that one of the mechanisms subserving large scale reorganization in cortex is a relay of topographic changes that occur subcortically. Presumably, the expanded primary sensory inputs activate postsynaptic neurons that are normally driven by inputs from the hand so that the neurons now have receptive fields on the forearm. Since the topographic representation of the body is greatly magnified in the relay to cortex, the subcortical changes can result in dramatic cortical map changes. PMID- 8613745 TI - A distinct subset of tenascin/CS-6-PG-rich astrocytes restricts neuronal growth in vitro. AB - Astrocytes provide an optimal surface for attachment, migration, and growth of CNS neurons. Nonetheless, not all astrocytes are alike: our previous work demonstrated a heterogeneity in the ability of cultured astrocyte monolayers to support neuronal growth. Areas displaying a fibrous, uneven surface ("rocky" astrocytes) were shown to be restrictive substrates, whereas surrounding, flat areas were permissive substrates. However, whether these cell types are in fact different cannot be addressed using mixed cultures. Therefore, in the current study we used morphological criteria to isolate the two subpopulations from mixed astrocyte cultures established from the cerebral cortex of neonatal rats. Following isolation, the purified populations only produced progeny with the same phenotype as the parent cells. We then measured production of several extracellular matrix molecules putatively involved in neuronal guidance during development and quantitatively assessed neuronal behavior on the purified populations. Immunocytochemistry and immunoblotting showed that rocky astrocytes were enriched in tenascin and chondroitin-6- sulfate-containing proteoglycans, but not in laminin or fibronectin. In addition, these astrocytes, as well as their isolated matrix, were a less permissive substrate for neuronal growth than flat astrocytes/matrix. Neurite outgrowth was significantly increased on rocky astrocytes following treatment with chondroitinase ABC or AC, but not heparitinase or hyaluronidase. These data support a critical role for matrix bound chondroitin-6-sulfate-containing proteoglycans. We hypothesize that rocky astrocytes represent a subtype of cells which form barriers to neuronal growth during cortical development. PMID- 8613746 TI - A homeodomain protein selectively expressed in noradrenergic tissue regulates transcription of neurotransmitter biosynthetic genes. AB - In order to characterize the specificity of expression of the neurotransmitter biosynthetic gene dopamine beta-hydroxylase (DBH), the identification of proteins that interact with the DB1 enhancer was initiated. A homeobox-containing cDNA was isolated from a PC12 expression cDNA library screened with the DB1 enhancer. The homeodomain is a member of the paired-like class, and is encoded by several nonidentical cDNAs. The cDNAs contain the same sequence in the homeodomain and 3' coding and noncoding sequences, but diverge in sequence 5' to the homeodomain. This family of homeobox-containing cDNAs is named Arix. Arix mRNA transcripts are found only in noradrenergic, DBH-positive tissues, and in cell lines derived from those tissue. The DB1 enhancer contains two binding sites for the Arix homeodomain, and both sites contribute to basal activity of the DBH promoter. When introduced into tissue culture, Arix regulates the transcriptional activity from the DBH promoter, and also from the promoter of the tyrosine hydroxylase gene, encoding the initial enzyme of the catecholamine biosynthetic pathway. The pattern of expression of the Arix transcripts, the presence of the homeodomain, and the transcriptional regulatory properties suggest that this family of proteins may be involved in the specificity of expression of the catecholamine biosynthetic genes. PMID- 8613747 TI - Uncoupling of GABAA/benzodiazepine receptor alpha 1, beta 2, and gamma 2 subunit mRNA expression in cerebellar Purkinje cells of staggerer mutant mice. AB - The mammalian GABAA/benzodiazepine (GABAA/BZ) receptor is comprised of several subunit isoforms: alpha 1-6, beta 1-13, gamma 1-3 and delta. In the present studies, the expression of alpha 1, beta 2, and gamma 2 subunit mRNAs was examined in cerebellar Purkinje cells and deep cerebellar neurons of staggerer mutant mice during postnatal development. In control animals, the three subunit mRNAs were present at high density in Purkinje cells which, in adult animals, form a monolayer at the interface of the granule cell and molecular layers. The number of Purkinje cells in the staggerer cerebellar cortex is reduced; the majority of those that remain are retained within the granule cell layer and are unable to receive normal afferent synapses from granule cells. The three subunit mRNAs were ex pressed at similar levels in both staggerer and control Purkinje cells until postnatal day 9. After this time, although the alpha 1 subunit mRNA was maintained at control levels in staggerer Purkinje cells, the expression of beta 2 and gamma 2 subunit mRNAs decreased, and was largely absent by postnatal day 20. The loss of beta 2 and gamma 2 mRNA expression in staggerer was specific to Purkinje cells, since all three mRNAs were present throughout postnatal development in other brain regions, including the deep cerebellar nuclei. The present studies indicate that in cerebellar Purkinje cells, the GABAA/BZ receptor alpha 1, and beta 2, and gamma 2, subunit mRNAs are regulated by distinct mechanisms which are differentially affected by the staggerer mutation. PMID- 8613748 TI - Nerve growth factor facilitates cholinergic neurotransmission between nucleus basalis and the amygdala in rat: an electrophysiological analysis. AB - Treatment of rats in vivo with NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain cholinergic neurons. We showed recently (Williams et al., 1993) that NGF-induced stimulations of the cholinergic markers ChAT and high-affinity choline uptake are reflected in an enhanced synthesis and release of ACh in terminals fields of basal forebrain cholinergic neurons. The objective of the present study was to determine whether such effects translate into an enhancement in neurotransmission between nucleus basalis neurons and postsynaptic target cells, and therefore are likely to be of physiological significance. Changes in cholinergic neurotransmission after NGF were assessed by comparing the ability of cholinergic pathway activation, produced by electrical stimulation of nucleus basalis or the external capsule, to elicit intracellularly recorded muscarinic responses in basolateral amygdaloid (BLA) neurons in ventral forebrain slice preparations from NGF-treated and control Fischer 344 adult rats. Chronic infusion of NGF for 3 weeks (1.2 micrograms/d, i.c.v.) increased the likelihood of eliciting cholinergic slow depolarizations (slow EPSP) via stimulation of cholinergic pathways in the slice. In addition, the frequency-response curves for generation of the cholinergic slow EPSP by nucleus basalis or external capsule stimulation were shifted approximately twofold to the left and the EF50 values significantly reduced in neurons from NGF-treated slices, compared to those in preparations from vehicle treated or untreated controls. Treatment with NGF also resulted in a leftward shift in the frequency-response curve for cholinergic pathway-induced blockade of the slow afterhyperpolarization, without change in the maximal inhibitory effect. The NGF-induced enhancement in cholinergic synaptic effectiveness was not accompanied by alterations in the resting membrane properties or intrinsic excitability of BLA pyramidal neurons. Nor did treatment with NGF affect their chemosensitivity or responsiveness to direct postsynaptic applications of the cholinergic carbachol. We conclude from these results that chronic administration of exogenous NGF can facilitate neurotransmission within basal forebrain cholinergic projections in normal adult brain, presumably as a consequence of its ability to stimulate presynaptic mechanisms involved in synthesis and/or release of ACh. PMID- 8613749 TI - At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats. AB - Removal of the superior colliculus (SC) in neonatal Wistar rats results in a rapid loss of retinal ganglion cells (RGCs). There is an early twofold increase in RGC death 4-8 hr postlesion (PL) followed by a later 10-11-fold increase in pyknosis about 24 hr PL. We have now used neurotrophic factors (BDNF, NT-4/5, NT 3, NGF, LIF), glutamate receptor antagonists (MK-801, DNQX, CNQX), an antioxidant (N-ace-tyl-L-cysteine), and an NOS inhibitor (L-NAME) to determine whether the early and late phases of lesion-induced RGC death involved similar or different mechanisms. Normal and pyknotic nuclei of tectally projecting RGCs were visualized by injecting the left s.c. of 2 d old rats with diamidino yellow (DY). Two days later the injection site was removed. In most rats, right eyes were injected with factors immediately after the s.c. ablation. Rats were perfused either 6 or 24 hr PL. In the latter group a second intravitreal injection of the appropriate factor was sometimes made 12 hr PL. NT- 4/5 and BDNF significantly decreased RGC pyknosis 6 and 24 hr PL, whereas NT-3 was only protective 6 hr PL. LIF slightly reduced RGC death 24 hr PL, but NGF had no influence on RGC survival at either time point. NT-4/5 also reduced the rate of naturally occurring RGC death. MK-801, DNQX, CNQX, N-acetylcysteine, and L-NAME all prevented the early lesion-induced increase in RGC death but had no significant effect on RGC death measured 24 hr PL; none of these factors significantly reduced the rate of naturally occuring RGC death. Cycloheximide, shown previously to reduce RGC pyknosis 24 hr PL, did not prevent RGC death 6 hr PL. The data indicate that there are at least two mechanisms involved in RGC death after neonatal target ablation. The early increase is related to excitotoxic effects mediated by glutamate receptors and involves NOS and the production of free radicals. We found no evidence that RGC death measured 24 hr PL is dependent on these processes, but the later death does require protein synthesis and, most likely, the activation of an endogenous suicide program. NT-4/5 and BDNF protected RGCs from both types of lesion-induced death. PMID- 8613750 TI - Expression of mu-, delta-, and kappa-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation. AB - Recently, antisera that recognize unique epitopes of the cloned mu-, delta-, and kappa-opioid, receptors (MOR, DOR, KOR, respectively) have been developed. In the present study MOR-, DOR-, and KOR-like immunoreactivities (LIs) were examined in rat dorsal root ganglia (DRGs, L4-5) after injection of carrageenan (CAR) into the hindpaw. In normal control rats 20.9%, 13.5%, and 9% of the DRG neurons contained MOR-, DOR-, KOR-LI, respectively. A marked upregulation in MOR-LI was observed in DRG neurons 1 and 3 d after inflammation. In contrast, CAR induced a distinct downregulation in DOR- and KOR-LIs. MOR-, DOR-, and KOR-LIs were preferentially localized in small DRG neurons. MOR-LI was often located in patches in the cytoplasm, and in some cells close to the somatic plasmalemma. However, DOR- and KOR-LIs mainly showed a diffuse staining pattern within cytoplasm. Two or even all three receptors could sometimes be found to coexist in DRG neurons. In the spinal cord, these receptors were mainly confined to the superficial dorsal horn, with a somewhat diffuse staining which was strong for MOR-LI, and weak for KOR-LI. DOR-LI had distinctly punctate, varicose distribution. CAG induced-alterations in opioid receptor staining in spinal cord were much less pronounced than those in the DRGs with a small increase in MOR-LI and a slight decrease in DOR-LI ipsilaterally. There was an accumulation of all three types of receptors in the sciatic nerve both proximal and distal to the ligation site as early as 2 hr, indicating both antero- and retrograde transport of multiple opioid receptors. However, DOR-LI accumulation was stronger than that of MOR- and KOR-LIs. Taken together, these results suggest that all three opioid receptors are involved in the response to inflammation and that they may play different roles in this pathological state. The coexistence of MOR, DOR, and KOR in at least some primary sensory neurons provides a substrate for functional interactions between these receptors. PMID- 8613751 TI - D1 and D2 dopamine receptor function in the striatum: coactivation of D1- and D2 dopamine receptors on separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons. AB - D1- and D2-dopamine receptor-mediated regulation of immediate early gene levels in identified populations of neurons in the striatum was examined with quantitative in situ hybridization histochemical techniques. Levels of messenger RNA (mRNA) encoding the immediate early genes zif268 and c-fos were examined in two experiments in rats with unilateral lesions of the nigrostriatal dopamine pathway. In a dose-response study, animals were treated with doses of 0.5, 1.0, and 1.5 mg/kg of the D1 agonist SKF-38393 either alone or in combination with the D2 agonist quinpirole (1 mg/kg). Levels of immediate early gene mRNAs 60 min following drug treatments showed a dose-related increase to the D1 agonist alone and a potentiation to combined D1 and D2 against treatment. In a second experiment, in animals receiving 1 mg/kg SKF-38393 either alone or in combination with 1 mg/kg quinpirole, the level of zif268 mRNA was measured with a double labeling method in striatal neurons containing enkephalin mRNA, a marker of D2 containing neurons, and in neurons not containing enkephalin, putative D1 containing neurons. In the dopamine-depleted striatum, D1 agonist treatment alone did not affect enkephalin-positive neurons but significantly elevated zif268 mRNA levels in nearly all enkephalin-negative neurons. Combined D1 and D2 agonist treatment further increased zif268 mRNA levels in this population of enkephalin negative neurons and decreased zif-268 mRNA levels in enkephalin-positive neurons. These data indicate that the synergistic response to combined D1- and D2 receptor stimulation is mediated by interneuronal interactions involving the activation of D1 and D2 receptors on separate populations of striatal neurons. PMID- 8613752 TI - Role of neural activity during synaptogenesis in Drosophila. AB - This article reveals a novel aspect in the regulation of synaptic connectivity in Drosophila. Reducing neural activity genetically or pharmacologically disrupts the normally precise embryonic and larval neuromuscular connections. In third instar larvae with mutations that affect sodium channel function or expression such as no action potential, temperature-induced paralysis E, or seizure1, foreign neuromuscular synapses, arising from inappropriate nerve sources, are observed on muscle fibers throughout the abdominal body wall. Their frequencies increase as neural activity is further reduced in double mutant combinations. These foreign connections are first observed during late embryogenesis as filopodial-like contacts, but critical period analysis suggests that neural activity must be reduced during both late embryogenesis and the first larval instar to promote the differentiation of these embryonic contacts into foreign motor synapses. In addition, the loss of electrical activity in the motoneuron, as opposed to the loss of postsynaptic potentials in the muscle fibers, appears to be responsible for these changes in connectivity. Our experiments suggest that neural activity may function during development by preventing inappropriate connections and thereby maintaining the precise connectivity achieved during nerve outgrowth and target selection. PMID- 8613753 TI - Peripheral target specification of synaptic connectivity of muscle spindle sensory neurons with spinal motoneurons. AB - The source of environmental cues determining the central connections of muscle sensory neurons was investigated by manipulating chick embryos so that sensory neurons supplied a duplicate set of dorsal thigh muscles. These neurons projected out ventral nerve pathways and along motor axons that normally project to ventral muscles but their ultimate target tissue was the duplicate set of dorsal muscles. The central connections of these sensory neurons to motoneurons supplying normal dorsal muscles were then determined with intracellular recordings in isolated spinal cord preparations. Sensory neurons supplying individual duplicate dorsal muscles made the same connections as those supplying the corresponding normal dorsal muscles; the pattern of these connections was different than that made by afferents supplying ventral muscles. Sensory neurons thus made synaptic connections appropriate for their target muscle rather than for their more proximal ventral environment. These findings suggest that the target muscle is the source of cues that determine the central connections of the sensory neurons projecting to it. Motoneurons forced to innervate novel muscle received many of the same sensory inputs they would normally receive, suggesting that motoneurons are less influenced by their target tissue than sensory neurons. PMID- 8613754 TI - The central nucleus of the amygdala contributes to the production of morphine antinociception in the rat tail-flick test. AB - Current models of endogenous pain control circuitry emphasize neural substrates within the brainstem and spinal cord. We have recently shown, however, that the central nucleus of the amygdala (Ce) contributes to morphine-induced suppression of formalin-induced nociceptive behaviors. In the four experiments reported here, we investigated the possibility that the Ce also contributes to morphine-induced suppression of simple, spinally mediated nociceptive reflexes. Bilateral N-methyl D-aspartate (NMDA)-induced lesions of the rat Ce, but not bilateral lesions centered on either the basolateral or medial amygdaloid nucleus, abolished the antinociception produced by 2.5 mg/kg morphine sulfate in the noxious heat-evoked tail-flick test. Bilateral Ce lesions also abolished the antinociception produced by 2 or 4 mg/kg morphine sulfate, but a relatively large dose of morphine sulfate (10 mg/kg, s.c.) resulted in partial reinstatement of antinociception. It is unlikely that these effects were due to secondary, seizure-induced damage following NMDA injection (e.g., to areas outside the amygdala) since bilateral inactivation of the Ce with the local anesthetic lidocaine also reliably attenuated morphine antinociception. It is also unlikely that these effects were artifacts of lesion-induced hyperalgesia, since Ce lesions failed to result in reliable thermal hyperalgesia, even at baseline tail-flick latencies of 10-12 sec. These data are the first to provide direct evidence that systemically administered morphine requires the integrity of a forebrain area in order to suppress spinally mediated nociceptive reflexes. It is argued that the present results, together with recent evidence linking the Ce to the production of several forms of conditioned and unconditioned environmentally induced antinociception, warrant incorporation of the Ce into current models of endogenous pain control circuitry. PMID- 8613755 TI - Direct demonstration of a physiological role for carbon monoxide in olfactory receptor neurons. AB - Recent evidence suggests that, like nitric oxide (NO), carbon monoxide (CO), another activator of soluble guanylyl cyclase, may serve as an intercellular messenger in the brain. Heme oxygenase, which converts heme to biliverdin and CO, is abundantly expressed in the brain and is localized to discrete neuronal populations. However, evidence for the actual generation of CO by neurons is lacking. Heme oxygenase-2 immunoreactivity is abundantly present in olfactory receptor neurons where it essentially colocalizes with immunoreactivity to soluble guanylyl cyclase, the target of CO action. To examine the generation of CO by neurons, we measured CO production directly and determined its relationship to cyclic GMP levels in cultured rat olfactory receptor neurons. This system has the advantage of not having measurable NO production, which could confound results since NO is a more potent activator of guanylyl cyclase than CO. Metabolic labeling experiments permitted the direct measurement of 14CO production by neurons in vitro. CO release parallels endogenous cyclic GMP concentrations with its peak at the immature stage of neuronal differentiation in culture. Cyclic GMP production is inhibited by zinc protoporphyrin-9 and zinc deuteroporphyrin IX 2,4-bis glycol, inhibitors of heme oxygenase, indicating that CO is an endogenous regulator of soluble guanylyl cyclase activities in these cells. Transforming growth factor-beta 2, an olfactory neurogenic factor, specifically shows a negative effect on CO release in olfactory receptor neurons. These results indicate that CO may serve as a gaseous neuronal messenger linked to cyclic GMP production and suggests its involvement in developmental processes of the olfactory receptor neuron. PMID- 8613756 TI - Inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury. AB - The present study characterized whether inflammatory leukocytic infiltration is temporally and regionally correlated with neuronal degeneration and/or blood brain barrier (BBB) breakdown resulting from traumatic brain injury. Adult rats were sacrificed at 5 min, 2, 4, 12, 24, and 72 hr after lateral fluid percussion brain injury. BBB breakdown, neuronal degeneration and leukocyte infiltration were assessed using immunocytochemistry, silver impregnation and toluidine blue and eosin staining. BBB breakdown and neuronal degeneration occurred concomitantly in injured cortex, hippocampus, and along the dorsolateral quadrant of the diencephalon. However, neuronal degeneration within deep diencephalic structures transpired in the absence of IgG extravasation. Neutrophils were observed only in regions exhibiting BBB damage and were first apparent in injured cortex and hippocampus between 2-12 hr posttrauma lining the vasculature and filling subarachnoid/subdural spaces. Neutrophils then migrated from damaged vasculature into traumatized cortical and hippocampal parenchyma by 24 hr after lateral fluid percussion injury. Macrophages were also observed within cortical parenchyma at 24 hr and completely filled the cortical lesion site by 72 hr after injury. Macrophages were not as abundant throughout hippocampal parenchyma and were found only in hippocampal regions exhibiting focal hemorrhage at 72 hr. Finally, neutrophils did not migrate to deep diencephalic structures that showed no BBB damage despite extensive neuronal degeneration. Indeed, lateral fluid percussion elicits inflammatory leukocytic recruitment only in regions experiencing concomitant BBB damage and neuronal degeneration. In summary, inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury. PMID- 8613757 TI - Axonal sprouting in layer V pyramidal neurons of chronically injured cerebral cortex. AB - We performed experiments to determine whether axonal sprouting occurs in neurons of chronic neocortical epileptogenic lesions. Partially isolated somatosensory cortical islands with intact pial blood supply were prepared in mature rats. Neocortical slices from these lesions, studied 6-39 d later, generated spontaneous and/or evoked epileptiform field potentials (Prince and Tseng, 1993) during which neurons displayed prolonged polyphasic excitatory and inhibitory synaptic potentials/currents. Single electrophysiologically characterized layer V pyramidal neurons in control and epileptogenic slices were filled with biocytin using sharp and patch-electrode techniques, their axonal arbors reconstructed and compared quantitatively. Neurons in injured cortex had a 56% increase in total axonal length, a 64% increase in the number of axonal collaterals and more than a doubling (115% increase) of the number of axonal swellings. The presumed boutons were smaller and more closely spaced than those of control cells. In some neurons the main descending axon had hypertrophic segments from which branches arose. These highly significant changes were most marked in the perisomatic region of layer V. The axonal sprouting was associated with a decrease in somatic area but no significant change in dendritic arbors. Results suggest that a significant degree of axonal reorganization takes place in the chronically injured cortex where it might be an adaptive mechanism for recovery of function after injury, or might be maladaptive and play an important role in the generation of epileptiform events by increasing the numbers and density of synaptic contacts between neurons. PMID- 8613758 TI - Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals. AB - The protein kinase inhibitor staurosporine inhibited, and often abolished, activity-dependent destaining of frog motor nerve terminals that had been preloaded with the fluorescent dye FM1-43. Staurosporine did not, however, block synaptic transmission; staurosporine treated muscles twitched in response to nerve stimulation, and the amplitudes of evoked end plate potentials were reduced only slightly, and in some cases not at all. The blockade of FM1-43 destaining was not reversed by washing, although treatment with black widow spider venom caused complete destaining. Nerve terminal pretreated with staurosporine could subsequently be stained with FM1-43 (and then destained by black widow spider venom). Thus, staurosporine blocked destaining but not staining of nerve terminals. Staurosporine treatment had little effect on the ultra-structure of resting terminals, the main difference we noted being a somewhat closer packing of synaptic vesicles after exposure to staurosporine. However, staurosporine blocked completely the ultrastructural changes produced by prolonged nerve stimulation, such as depletion of synaptic vesicles, appearance of intraterminal cisternae, and the uptake of horseradish peroxidase. The effects of staurosporine were not mimicked by KN-62, H7, calmidozolium, or trifluoroperazine. These and other observations are consistent with, but do not prove the hypothesis that, after exposure to staurosporine, the exocytotic fusion pore behaved like a valve, letting FM1-43 in, but not out, as if staurosporine interfered with the postexocytotic collapse of synaptic vesicles into the surface membrane. PMID- 8613759 TI - Three distinct axonal transport rates for tau, tubulin, and other microtubule associated proteins: evidence for dynamic interactions of tau with microtubules in vivo. AB - Microtubule-associated proteins (MAPs), such as tau, modulate neuronal shape and process outgrowth by influencing the stability and organization of microtubules. The dynamic nature of MAP-microtubule interactions in vivo, however, is poorly understood. Here, we have assessed the stability of these interactions by investigating the synthesis and axoplasmic transport of tau in relation to that of tubulin and other MAPs within retinal ganglion cells of normal adult mice in vivo. Using immunoprecipitation and Western blot analysis with anti-tau monoclonal and polyclonal antibodies, we unequivocally identified in optic axons a family of 50-60 kDa tau isoforms and a second 90-95 KDa tau family, the members of which were shown to contain the domain of tau encoded by exon 4A. To measure the rates of translocation of tau proteins in vivo, we injected mice with 35S methionine intravitreously and, after 6-30 d, quantitated the radiolabeled tau isoforms immunoprecipitated from eight consecutive 1.1 mm segments of the nerve and optic tract and separated by electrophoresis. Linear regression analysis of protein transport along optic axons showed that the tau isoforms advanced at a rate of 0.2-0.4 mm/d, and other radiolabeled MAPs, identified by their association with taxol-stabilized microtubules, moved three- to fivefold more rapidly. By contrast, tubulins advanced at 0.1-0.2 mm/d, significantly more slowly than tau or other MAPs. These studies establish that tau is not cotransported with tubulin or microtubules, indicating that associations of tau with microtubules within axons are not as stable as previously believed. Our findings also reveal differences among various MAPs in their interactions with microtubules and provide evidence that assembly and reorganization of the microtubule network is an active process even after axons establish connections and fully mature. PMID- 8613760 TI - Differential paired pulse depression of non-NMDA and NMDA currents in pyramidal cells of the rat frontal cortex. AB - Excitatory postsynaptic currents (EPSCs) were induced in layer II-V pyramidal cells in the frontal cortex of the young rat (postnatal day 14-21) by stimulation of layers II/III in the presence of bicuculline using the whole-cell patch-clamp technique. EPSCs usually consisted of fast and slow components that were sensitive to CNQX and APV, respectively. In response to paired stimuli of identical strength, paired pulse depression (PPD) was seen for these EPSCs. The PPD of fast EPSCs was most pronounced at an interstimulus interval (ISI) of 200 300 msec and ceased to occur at ISIs greater than 3-5 sec, while the PPD of slow EPSCs became most pronounced at an ISI of 500-1000 msec and ceased to occur at ISIs greater than 10 sec. The PPD of fast EPSCs was attenuated by (-)-baclofen (1 5 microM) and removed by 2-hydroxy-saclofen (0.2-0.4 mM). By contrast, the PPD of slow EPSCs consisted of early and late components that were attenuated by (-) baclofen and muscarine (1-5 microM), respectively. The late PPD of slow EPSCs induced in the presence of baclofen was removed by pirenzepine (1-3 microM). Thus, fast and slow components of glutamatergic EPSCs displayed two distinct PPDs. These results suggest that a part of the glutamatergic afferents likely arising from layer II/III pyramidal cells may terminate predominantly on NMDA receptors in pyramidal cells of the frontal cortex and receive distinct presynaptic inhibition through at least the muscarinic receptors. PMID- 8613761 TI - Connexin32 is a myelin-related protein in the PNS and CNS. AB - We have examined the expression of a gap junction protein, connexin32 (Cx32), in Schwann cells and oligodendrocytes. In peripheral nerve, Cx32 is found in the paranodal myelin loops and Schmidt-Lanterman incisures of myelinating Schwann cells, and the levels of Cx32 protein and mRNA change in parallel with those of other myelin-related genes during development, Wallerian degeneration, and axonal regeneration. In the central nervous system, Cx32 is found in oligodendrocytes and their processes, but not in compact myelin, and the levels of Cx32 protein and mRNA increase during development in parallel with those of the other myelin genes. Thus, Cx32 is expressed as part of the myelinating phenotype of both Schwann cells and oligodendrocytes, indicating that this gap junction protein plays in important role in the biology of myelin-forming cells. PMID- 8613762 TI - Deficits in radial arm maze performance in kindled rats: evidence for long lasting memory dysfunction induced by repeated brief seizures. AB - Repeated activation of neural pathways by kindling induces brief seizures, permanent increases in seizure susceptibility, neuronal loss in the hippocampus, and mossy fiber sprouting in the dentate gyrus. Because kindling induces permanent cellular alterations in hippocampal pathways that have been implicated in memory, it was of interest to determine if kindling also induces long-lasting impairments in a spatial memory task in rats. In this study, the effects of kindling on memory were investigated by assessing kindled rats in a radial arm maze behavior that is impaired by hippocampal damage. Kindled rats studied at 1 month after the last of 30-134 evoked generalized tonic-clonic seizures acquired competence in the performance of the radial arm maze task at a rate that was indistinguishable from age-matched normal controls, but demonstrated a deficit in the ability to repeat the task on consecutive days. The repetition deficit was not observed in rats that experienced three afterdischarges or three generalized tonic-clonic seizures, and the severity of the deficit varied directly with the number of evoked kindled seizures. Repeated brief seizures evoked by kindling induced a long-lasting deficit in a radial arm maze task that is a rodent model of memory. The observation of a long-lasting deficit in radial arm maze performance in kindled rats suggests that the cellular alterations induced in the hippocampus by seizures could contribute to the memory dysfunction in human epilepsy. PMID- 8613763 TI - Regulation of GABA transporter activity and mRNA expression by estrogen in rat preoptic area. AB - This study has examined whether estrogen regulates GABA transporter synthesis and activity in the female rat brain. In the first experiment in situ hybridization studies examined the effects of ovariectomy on cellular GABA transporter-1 (GAT 1) mRNA content. A 25% decrease in GAT-1 mRNA expression was detected within the medial preoptic area (MPOA) but not the parietal cortex, magnocellular preoptic nucleus (Mg-POA) or caudate-putamen (C-P). Estrogen replacement for 7 d returned GAT-1 mRNA content of MPOA cells to levels observed in intact rats. In the second experiment, the effect of increased brain GABA concentrations on GAT-1 mRNA expression was investigated by treating rats with gamma-vinyl GABA, a GABA transaminase inhibitor. Although resulting in a twofold increase in tissue GABA content, in situ hybridization experiments revealed no changes in GAT-1 transcript expression. A third series of experiments examined GABA transporter activity in vitro using a 3H GABA uptake assay in MPOA, cortex, and C-P punches. Nipecotic acid (10 microM) reduced specific 3H GABA uptake in all three brain regions while 100 microM beta-alanine only reduced uptake in the MPOA. Estrogen treatment for 7 d resulted in a significant increase in 3H GABA uptake in the MPOA but not the cortex or C-P. The presence of a putative estrogen response element in the GAT-1 gene and the effects demonstrated here on GAT-1 mRNA content and GABA transporter activity indicate that estrogen may influence GAT-1 gene transcription to alter GABA transporter function within the MPOA but not the C-P or cortex. PMID- 8613764 TI - NCAM-associated polysialic acid on ciliary ganglion neurons is regulated by polysialytransferase levels and interaction with muscle. AB - NCAM in its high polysialic acid (PSA) form is expressed on chick hindlimb motoneurons during their growth, and then decreases at about the time that synaptogenesis is completed. In order to characterize this regulation at the cell and molecular level, the present studies use the chick ciliary ganglion (CG) system, which constitutes a homogeneous and developmentally synchronized population of motoneurons that can be used for in vitro studies. Levels of PSA in the CG were evaluated both by SDS-PAGE immunoblot analysis of total NCAM and by pulse radiolabeling of newly synthesized NCAM. Up- and downregulation of PSA expression on newly synthesized NCAM in the CG was found to be closely correlated with in vivo innervation and synaptogenesis, respectively. Moreover, the downregulation observed at synaptogenesis was prevented by in vivo blockade of neuromuscular activity with alpha-bungarotoxin. The developmental regulation of PSA expression was found to coincide precisely with an increase and decrease in levels of specific polysialyltransferase activity. By contrast, the expression of the VASE exon in NCAM, which in CNS is temporally correlated with PSA downregulation, was not expressed in the CG. Cocultures of CG neurons with myotubes were used to provide direct evidence that neuron-muscle interaction can cause a specific downregulation of both neuronal PSA and polysialyltransferase activity. PMID- 8613765 TI - G protein-coupled receptors mediate a fast excitatory postsynaptic current in CA3 pyramidal neurons in hippocampal slices. AB - Synaptic activation in the presence of competitive (D,L-APV,CNQX) and noncompetitive (MK-801,GYKI-52466) ionotropic glutamate receptor antagonists induced fast (10-90% rise time of 15-30 msec) postsynaptic responses in CA3 pyramidal neurons from acute and cultured hippocampal slices. Postsynaptic currents were studied extensively in slice cultures, and displayed a linear current-voltage relationship, with a reversal potential between 0 mV and +10 mV, suggesting the activation of a nonselective cationic conductance. Inhibition of the GTPase cycle by intracellular perfusion with the nonhydrolyzable analog of GDP, GDP beta S, blocked the fast postsynaptic responses evoked in ionotropic antagonists, as well as baclofen-mediated outward K+ currents, known to be mediated by G protein-coupled GABAB receptors. Intracellular perfusion with GDP beta S did not affect the AMPA/kainate component of the synaptic currents. Irreversible activation of G proteins by intracellular perfusion with the nonhydrolyzable analog of GTP, GMP-PNP, occluded the baclofen responses, and evoked an inward current, consistent with the synaptically mediated conductance. Incubation of the slice cultures in pertussis toxin for 72 hr blocked baclofen induced outward K+ currents, while the fast postsynaptic currents remained. The metabotropic glutamate receptor (mGluR) agonists 1S,3R-ACPD and 1S,3S-ACPD induced an inward current in the presence of the ionotropic antagonists, and occluded the fast EPSCs. The fast EPSCs were partially blocked by the mGluR antagonists L-AP3 and (+)MCPG, but there was differential antagonists sensitivity in two pathways stimulated (CA3 stratum radiatum vs CA3 stratum oriens). These data suggest that fast postsynaptic responses evoked in the presence of ionotropic glutamate receptor antagonists are mediated by G protein-coupled mGluRs linked to nonselective cationic channels. PMID- 8613766 TI - Neurofilaments help maintain normal morphologies and support elongation of neurites in Xenopus laevis cultured embryonic spinal cord neurons. AB - Neurofilament number and subunit composition, which are highly regulated during development, have been proposed to help regulate axonal diameter and stability. From experiments on dissociated cell cultures of Xenopus laevis embryonic spinal cord, we have obtained direct evidence that neurofilaments help maintain the structural integrity of newly developing axons. An anti-neurofilament monoclonal antibody specific for Xenopus NF-M and the cell lineage tracer, lysinated FITC dextran, were coinjected into a single blastomere of 2-cell stage embryos. Within neurons descended from the injected blastomere, this antibody specifically confined neurofilaments to the cell body for the first two days of culture, as assayed by immunocytochemical staining with antiserum against the low molecular weight neurofilament protein XNIF. Although whole IgGs and Fab fragments both affected neurofilament distribution, the whole IgGs were more effective. For the first 9 hr of culture, neurites containing anti-NF-M developed normally. By 21 hr, they were shorter than those of sibling control neurons within the same dish, and many became morphologically abnormal. Defects included large variations in diameter, poorly defined separations between the growth cone and neurite, and more collateral branching. Despite these abnormal features, neurons containing anti-NF-M had normal distributions of alpha-tubulin immunoreactivity and phalloidin-stained F-actin. These latter observations argued that defects resulted from the absence of neurofilaments rather than from interference of the movement of other structural materials essential for axonal growth. These results support the hypothesis that neurons use neurofilaments to help maintain the characteristic shapes of axons against the increasing structural demands placed upon the elongating process. PMID- 8613767 TI - Spontaneous CNS remyelination in beta 2 microglobulin-deficient mice following virus-induced demyelination. AB - Animal models with selective genetic immunodeficiencies are useful tools to identify pathogenic mechanisms of disease. Resistant (C57BL/6F 129/J) (H-2b) mice are rendered susceptible to Theiler's murine encephalomyelitis virus-induced demyelination by genetic disruption of the beta 2 microglobulin gene [beta 2 m(-l )]. The absence of beta 2 m prevents the expression of major histocompatibility complex class I molecules and normal levels of functional CD8+ T cells. We tested whether genetic depletion of beta 2 m would permit CNS remyelination after chronic demyelination induced by the Daniel's strain of Theiler's virus. In contrast to the minimal spontaneous remyelination observed in SJL/J mice after infection with the Daniel's strain of Theiler's virus, chronically infected beta 2 m(-I-) mice showed extensive and progressive spontaneous CNS remyelination at 6, 12, and 18 months after infection. Spontaneous remyelination by both oligodendrocytes and Schwann cells occurred despite the presence of persistent virus antigen and RNA, but was associated with diminished virus-specific humoral and delayed-type hypersensitivity responses. These experiments support the hypothesis that the immune response inhibits myelin regeneration after virus induced CNS demyelination. PMID- 8613768 TI - Differential cholinergic regulation of dopamine release in the dorsal and ventral neostriatum of the rat: an in vivo microdialysis study. AB - We used in vivo microdialysis to investigate the effects of local perfusion with the AChE inhibitor neostigmine on the basal and haloperidol evoked increase in dialysate dopamine levels in the dorsolateral and fundus striata of the bilaterally implanted halothane anaesthetized rat. In the absence of neostigmine basal dopamine was consistently higher in the dorsolateral striatum compared with the fundus striati. Local perfusion with neostigmine (10 and 100 microM) increased basal dopamine in the fundus striati compared to the contralateral (control) side but not in the dorsolateral striatum. In the absence of neostigmine haloperidol (0.05-0.5 mg/kg, s.c.) increased dopamine release in both the dorsolateral and fundus striata. However, local perfusion with neostigmine (10 microM) attenuated this increase in the dorsolateral striatum at all doses of haloperidol while only the effect of the highest (0.5 mg/kg) dose of haloperidol was counteracted in the fundus striati. Both the basal and haloperidol (0.25 mg/kg) induced increase in dopamine release in the control (no neostigmine) and neostigmine (+10 microM) treated dorsolateral striata were abolished following local perfusion with tetrodotoxin (1 microM). The data demonstrate that the introduction of neostigmine into the neostriatum selectively increases basal DNA levels in the fundus striati and strongly counteracts the haloperidol evoked DA release in the dorsolateral striatum and thus provide strong evidence for a differential cholinergic regulation of striatal DA release in vivo. In addition, we demonstrate that the stimulatory and inhibitory effects of neostigmine operate independently and have a regional specificity within the neostriatum. PMID- 8613769 TI - Glutaminase-positive and glutaminase-negative pyramidal cells in layer VI of the primary motor and somatosensory cortices: a combined analysis by intracellular staining and immunocytochemistry in the rat. AB - Pyramidal neurons in layer VI of the primary motor and somatosensory cortices were examined by a combined method of intracellular recording, biocytin injection, and immunocytochemistry using in vitro slice preparations of rat brain immunofluorescence staining revealed that biocytin-injected pyramidal cells in layer VI were separated into glutaminase (PAG)-immunopositive and PAG immunonegative cells. Although the two groups of pyramidal cells showed no statistically significant differences in passive membrane properties and spike characteristics, a clear difference was found in spike afterpotentials. Ten of 12 PAG-positive pyramidal cells showed no or a small fast afterhyperpolarization (fAHP), whereas 10 of 11 PAG-negative pyramidal cells displayed a large fAHP. Depolarizing afterpotentials were observed only in PAG-positive pyramidal cells than in PAG-negative cells. In contrast, the arborization of basal dendrites was more developed in PAG-positive pyramidal cells than in PAG-negative cells. The main axons of all the pyramidal cells entered the subcortical axons of all the pyramidal cells entered the subcortical white matter. The local axon collaterals of PAG-positive pyramidal cells were widely spread in the horizontal direction, whereas those of PAG-negative cells were distributed vertically along the dendritic tree. Since PAG is considered to be a marker of glutamatergic neurons in the cerebral cortex, the present results indicate that layer VI pyramidal cells are separated into glutamatergic and nonglutamatergic neurons that have different electrical properties and input-output organizations. Thus, cortical outputs from layer VI are suggested to use at least two distinct systems. PMID- 8613770 TI - Targeted neocortical cell death in adult mice guides migration and differentiation of transplanted embryonic neurons. AB - Local expression of cellular and molecular signals is required for normal neuronal migration and differentiation during neocortical development and during periods of plasticity in the adult brain. We have previously shown that neonatal and juvenile mice that induction of apoptotic degeneration in neocortical pyramidal neurons by targeted photolysis provides an altered environment that directs migration and differentiation of transplanted embryonic neurons. Here we employ the same paradigm in adult mice to test whether targeted photolysis induces the reexpression in the mature brain of developmental signals that control migration, differentiation and integration of embryonic neurons. We examined both the time course of migration and the morphologic and immunocytochemical differentiation of embryonic neurons transplanted into regions of targeted photolytic cell death. Pyramidal neurons in neocortical lamina II/III underwent photolytically induced apoptosis after retrograde incorporation of the photoactive chromophore chlorine e6 and transdural exposure to 674 nm near infrared laser energy. Embryonic day 17 neocortical neurons were prelabeled with fluorescent nanospheres and the lipophilic dye PKH26, transplanted into regions of ongoing neuronal degeneration in adult mice, and examined histologically and immunocytochemically. Transplanted neurons began migration into regions of neuronal death within 3 d and differentiated into large pyramidal neurons similar to those degenerating. In contrast, neurons transplanted into intact cortex did not migrate, and they differentiate into small presumptive interneurons. Migration up to 430 microM in experimental mice was complete by 2 weeks; approximately 45% of the donor neurons migrated greater than 3 SDs beyond the mean for neurons transplanted into intact neocortex of age-matched adult hosts. Following migration, dendrites and axons of many donor neurons were properly oriented toward the pial surface and corpus callosum, indicating integration into the host parenchyma. Neurofilament and neuron-specific enolase staining further support appropriate differentiation and integration. These results indicate that signals guiding neuronal migration and differentiation in neocortex are reexpressed in adult mice well beyond the period of corticogenesis within regions of targeted photolytic cell death. Elucidating the molecular mechanisms underlying these events by comparison with adjacent unperturbed regions will contribute to efforts toward future therapeutic transplantation and control over endogenous plasticity. PMID- 8613771 TI - Spinohypothalamic tract neurons in the cervical enlargement of rats: descending axons in the ipsilateral brain. AB - Spinohypothalamic tract (SHT) cells are spinal cord neurons with axons that project directly to or through the contralateral hypothalamus. Frequently, SHT axons decussate in the posterior optic chiasm, turn posteriorly and descend to unknown locations in the ipsilateral brain. We attempted to determine the course and the termination of these descending axons. Sixty neurons in the cervical enlargement of rats were antidromically activated initially from the contralateral hypothalamus and then from multiple anterior-posterior levels in the ipsilateral brain. Fifty-three (88%) were backfired with low currents at increased latencies from the ipsilateral brain. The axons of 35 neurons were surrounded with electrode penetrations from which high currents could not activate the neuron antidromically, suggesting the examined axons terminated in the surrounded areas. Seven SHT axons that were surrounded (20%) appeared to terminate in the contralateral hypothalamus, 5 (14%) in the ipsilateral hypothalamus, and 9 (26%) in the ipsilateral thalamus. Fourteen SHT axons (40%) ended in the ipsilateral midbrain mainly in the superior colliculus, cuneiform nucleus or nucleus brachium inferior colliculus. An additional 11 axons were followed even further posteriorly into the ventral pons or rostral medulla. Each of the 26 neurons that could be physiologically classified responded either preferentially or specifically to noxious mechanical stimuli. These results indicate that SHT axons course through a surprisingly long and complex path. After decussating in the hypothalamus, the axons of many SHT neurons descend into the ipsilateral posterior thalamus, midbrain, pons, or even rostral medulla. These axons may provide nociceptive information to a variety of nuclei throughout the diencephalon and brainstem bilaterally. PMID- 8613772 TI - Mutations in the Caenorhabditis elegans Na,K-ATPase alpha-subunit gene, eat-6, disrupt excitable cell function. AB - We have cloned a Na,K-ATPase alpha-subunit gene from Caenorhabditis elegans and discovered that it is identical to the gene eat-6, eat-6 mutations cause feeble contractions and slow, delayed relaxations of pharyngeal muscle. The resting membrane potential of eat-6 mutant pharynxes is consistently depolarized compared to wild-type. The action potentials are smaller, and the return to resting potential is slower. To explain these abnormalities, we propose that a reduction of Na,K-ATPase activity in eat-6 mutants leads to a reduction of the ion concentration gradients that power membrane potential changes. PMID- 8613774 TI - A strongly inwardly rectifying K+ channel that is sensitive to ATP. AB - We have cloned an inwardly rectifying K+ channel from the hamster insulinoma cDNA library and shown that it is inhibited by cytoplasmic ATP. The channel is 90.97% identical to the IRK3 channels cloned from other species, and its mRNA is found primarily in the brain. When expressed in Xenopus oocytes, the channel displays strong inward rectification typical of inward rectifiers. The channel is inhibited reversibly by physiological concentrations of ATP via a mechanism that does not appear to involve ATP hydrolysis, as shown by studies of channels in excised inside-out membrane patches. This effect is antagonized by ADP, again in the physiological range, implying that this channel is sensitive to the index of metabolic state, i.e., the intracellular [ATP]/[ADP] ratio. This channel is different from previously known ATP-sensitive K+ channels, although it may also be stimulated by MgATP, as are other ATP-sensitive K+ channels. The potential physiological significance of these ATP-dependent regulations will be discussed. PMID- 8613773 TI - Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo. AB - Nitric oxide may be a key mediator of excitotoxic neuronal injury in the central nervous system. We examined the effects of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) on excitotoxic striatal lesions. 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 7-NI attenuated secondary striatal excitotoxic lesions produced by the succinate dehydrogenase inhibitor malonate, and the protection was reversed by L arginine but not by D-arginine, 7-NI produced nearly complete protection against striatal lesions produced by systemic administration of 3-nitropropionic acid (3 NP), another succinate dehydrogenase inhibitor, 7-NI protected against malonate induced decreases in ATP, and increases in lactate, as assessed by 1H magnetic resonance spectroscopy. 7-NI had no effects on spontaneous electrophysiologic activity in the striatum in vivo, suggesting that its effects were not mediated by an interaction with excitatory amino acid receptors. 7-NI attenuated increases in hydroxyl radical, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. The present results implicate neuronal nitric oxide generation in the pathogenesis of both direct and secondary excitotoxic neuronal injury in vivo. As such they suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role. PMID- 8613775 TI - Expression and characterization of Aplysia protein kinase C: a negative regulatory role for the E region. AB - The Aplysia nervous system contains two phorbol ester-activated protein kinase C isoforms, the Ca(2+)-activated Apl I and the Ca(2+)-independent Apl II. Short term applications of the facilitatory transmitter serotonin (5-HT) activates Apl I, but not Apl II. In contrast, Apl II, but not Apl I, can form an autonomous kinase. To investigate the biochemical characteristics of the Aplysia kinases that might underlie their differential activation, we expressed Apl I, Apl II, and two derivatives of Apl II with deletions in the amino-terminal 150 amino acid E region in insect cells using the baculovirus system. Similar to nervous system extracts, expressed Apl II has more autonomous activity than Apl I. Removal of the E region lowered the amount of phosphatidylserine required for activation of Apl II, but did not remove the autonomous kinase activity. In addition, phosphatidylserine vesicles could sediment fusion proteins containing the E region, consistent with a role for the E region in lipid interactions. A partial deletion of the E region modifies activation of Apl II by phorbol esters and oleic acid, suggesting that in the intact enzyme the E region interacts with the phorbol ester-binding domain of the kinase. These results introduce a model whereby the E region acts as a negative regulator of Apl II activation and suggest that this inhibition may explain the inability of short-term applications of 5-HT to activate Apl II. PMID- 8613776 TI - The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. AB - Although three distinct subunits seem to be sufficient to form a functional pentameric GABAA receptor channel, cerebellar granule cells express nRNA for nine subunits. They receive GABAergic input from a relatively homogenous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of granule cells or whether some of them have differential subcellular distribution resulting in distinct types of synaptic and/or extrasynaptic channels. Antibodies to different parts of the alpha 6 and alpha 1 subunits of the GABAA receptor and electron microscopic immunogold localization were used to determine the precise subcellular distribution of these subunits in relation to specific synaptic inputs. Both subunits were present in the extrasynaptic dendritic and somatic membranes at lower densities than in synaptic junctions. The alpha 6 and alpha 1 subunits were colocalized in many GABAergic Golgi synapses, demonstrating that both subunits are involved in synaptic transmission in the same synapse. Synapses immunopositive for only one of the alpha subunits were also found. The alpha 6, but not the alpha 1, subunit was also concentrated in glutamatergic mossy fiber synapses, indicating that the alpha 6 subunit may have several roles depending on its different locations. The results demonstrate a partially differential synaptic targeting of two distinct GABAA receptor subunits on the surface of the same type of neuron. PMID- 8613777 TI - Calcium-dependent inactivation of calcium current in synaptic terminals of retinal bipolar neurons. AB - Giant synaptic terminals (approximately 10 micrometer diameter) of bipolar neurons from goldfish retina were used to directly investigate calcium-dependent inactivation of presynaptic calcium current. During sustained depolarization, calcium current was initially constant for a period lasting up to several hundred milliseconds and then it declined exponentially. The duration of the initial delay was shorter and the rate of inactivation was faster with larger calcium current. The fastest time constant of inactivation (in the range of 2-5 sec) was observed under weak calcium buffering conditions. Inactivation was attenuated when external Ca2+ was replaced with Ba2+ and when terminals were dialyzed with high concentrations of internal BAPTA. Elevation of intracellular calcium concentration ([Ca2+]i) by application of the calcium ionophore ionomycin or by dialysis with pipette solutions containing buffered elevated [Ca2+] produced inactivation of calcium current. The rate of recovery from inactivation was not determined by the recovery of [Ca2+]i to baseline after a stimulus. The results demonstrate that the presynaptic calcium current in bipolar neurons is inactivated by elevated [Ca2+]i, but the inactivation is approximately 100-fold slower than previously described calcium-dependent inactivation in other types of cells. PMID- 8613778 TI - Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H1 receptors and cyclic nucleotides. AB - Activating direct olfactory (glutamatergic) inputs to supraoptic nucleus (SON) neurons increases interneuronal coupling in slices from lactating but from not virgin or male rats. Studied here were influences on coupling of another monosynaptic input to SON, the histaminergic tuberomammillary nucleus (TM) projection, activation of which selectively excites phasically firing (putative vasopressin) cells. Effects of TM stimulation and its possible downstream consequences on Lucifer yellow (LY) dye coupling among putative vasopressin cells were determined in male rat SONs. In unstimulated slices, 12 LY injections (1 cell/SON) yielded eight single and four pairs of coupled neurons. In slices in which TM was stimulated for 10 min at 10 Hz, 13 injections yielded 4 single and 28 coupled cells, with groups of 2 to 4 cells coupled to the injected neuron, a threefold increase in the number of coupled cells per injection (p < 0.02). Bathing slices in medium containing 10 microM pyrilamine (H1 antagonist) blocked this stimulation-induced coupling increase, suggesting mediation by activation of guanylate cyclase-cGMP to which H1 receptors often are linked . Bathing slices in medium containing 0.5-1 mM 8-bromo-cGMP yielded results similar to those of TM stimulation, a 2.5-fold increase over control in the number of coupled cells per injection. Effects of TM stimulation on coupling also were blocked by bathing slices in a guanylate cyclase inhibitor (10 microM LY83583). In contrast to cGMP, 1 mM 8-bromo-cAMP significantly reduced coupling. We conclude that synaptically released histamine increases coupling via cGMP-dependent mechanisms. PMID- 8613779 TI - Rise in intracellular calcium via a nongenomic effect of allopregnanolone in fetal rat hypothalamic neurons. AB - This study examines the early effects of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone on cytosolic free calcium concentration ([Ca2+]i in primary cultures of fetal rat hypothalamic neurons. Microspectrofluorimetry of fluorescent Ca2+(-)sensitive indicator Fura-2 was used to quantify these changes. Allopregnanolone (1 pM to 100 nM) increased [Ca2+]i within 2-3 sec, in a dose dependent manner, with an EC50 of 10 +/- 4 nM. The stimulatory effect of allopregnanolone was attributable principally to a Ca2+ influx, as shown by the strong inhibition of external Ca2+ removal or by the calcium channel blocker nifedipine. The effect was stereospecific because the allopregnanolone isomer 3 beta-hydroxy-5 alpha-pregnan-20-one had no effect on [Ca2+]i. Among two other steroids examined, progesterone had no effect on [Ca2+]i, but 17 beta-estradiol evoked a rise in [Ca2+]i, although to a lesser extent than allopregnanolone. The allopregnanolone-induced [Ca2+]i rise was inhibited by picrotoxin and bicuculline but was unaffected by tetrodotoxin or by pretreatment of neurons with pertussis toxin. These results are consistent with a membrane site of action for allopregnanolone associated with GABAA receptors, leading to rapid changes in [Ca2+]i in fetal rat hypothalamic neurons. PMID- 8613780 TI - Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system. AB - Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, synaptobrevin I and II were colocalized in many terminals. However, labeling for synaptobrevin I was more intense whereas labeling for synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only synaptobrevin I. In the sciatic nerve, synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that synaptobrevin I is present predominantly in motor neurons whereas synaptobrevin II is present in adrenergic and sensory neurons. We conclude that synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons. PMID- 8613781 TI - Localization of L-type Ca2+ channels at perisynaptic glial cells of the frog neuromuscular junction. AB - The presence of L-type Ca2+ channels at the frog neuromuscular junction (nmj) was studied by monitoring changes in intracellular Ca2+ evoked in presynaptic terminals and perisynaptic Schwann cells (PSCs) and by studying the distribution of Ca2+ channels using a monoclonal antibody directed against the alpha 2/delta subunit of L channels. L-type Ca2+ channel agonist and antagonist had no effect on resting level of fluorescence and nerve-evoked Ca2+ responses in presynaptic terminals. However, depolarization of PSCs induced by KCl (25 mM) produced entry of Ca2+, which was prevented by L-type Ca2+ channel blockers, in (+)R Bay K 8644 of nimodipine. Labeling of Ca2+ channels revealed an intracellular epitope with an irregular and spotty distribution along the endplate. Similar results were obtained with a fluorescent phenylalkylamine [(-)DM-BODIPY-PAA], a blocker of L type Ca2+ channels. Ca2+ channel labeling remained in absence of nerve terminals but was absent after mechanical removal of nerve terminals and PSCs. Most Ca2+ channel spots were distributed in between bands of cholinergic receptors labeled with alpha-bungarotoxin-TRITC. Cross sections of motor endplates revealed that labeling of Ca2+ channels was found only at the level of the synaptic cleft and not all around the PSCs. We conclude that L-type Ca2+ channels are located in perisynaptic glial cells in an appropriate location to sense depolarization induced by neurotransmitters and thus may support possible roles of glial cells on synaptic function. PMID- 8613782 TI - Acid efflux from retinal glial cells generated by sodium bicarbonate cotransport. AB - Sodium bicarbonate cotransport was studied in freshly dissociated Muller cells of the salamander retina. Variations in intracellular and extracellular pH evoked extracellular potassium concentration ([K+]o were recorded. Intracellular pH was measured by standard ratio imaging of the pH-sensitive dye BCECF, whereas extracellular pH was monitored by imaging BCECF fixed to coverslips under dissociated cells. Increasing [K+]o from 2.5 to 50 mM resulted in an intracellular alkalinization. The rate of alkalinization, 0.047 pH units/min, was reduced to 42% of control when HEPES was substituted for HCO3- and was reduced to 36% of control by the addition of 0.5 mM DIDS, a Na+/HCO3- cotransport blocker. The K(+)-evoked alkalinization was Cl(-)-independent and was not substantially reduced by amiloride or bumetanide. Increasing [K+]o to 50 mM also produced a rapid extracellular acidification, 0.01 to 0.05 pH units in amplitude. HEPES substitution and addition of 0.5 mM DIDS reduced the acidification to 7-8% of control, respectively. These results confirm the presence of a Na+/HCO3- cotransport system in salamander Muller cells and provide definitive evidence that glial cells can generate an extracellular acidification when [K+]o is increased. The K(+)-evoked extracellular acidification measured beneath cell endfeet was 304% of the amplitude of the acidification beneath cell somata, confirming that cotransporter sites are preferentially localized to the endfoot. The carbonic anhydrase inhibitor benzolamide (2 x 10(-5) M), which is poorly membrane permeant, increased the K(+)-evoked extracellular acidification to 269% of control, demonstrating that salamander Muller cells possess extracellular carbonic anhydrase. PMID- 8613783 TI - In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. AB - Thalamic reticular (RE) neurons are involved in the genesis of synchronized thalamocortical oscillations, which depend in part on their complex bursting properties. We have investigated the intrinsic properties of RE cells using computational models based on morphological and electrophysiological data. Simulations of a reconstructed RE cells were compared directly with recordings from the same cell to obtain precise values for the passive parameters. In a first series of experiments, the low-threshold calcium current (I(Ts)) was studied via voltage clamp in acutely dissociated RE cells that lack most of their dendrites. Simulations based on a cell with truncated dendrites and Hodgkin Huxley kinetics reproduced these recordings with a relatively low density of I(Ts). In a second series of experiments, voltage-clamp recordings obtained in intact RE cells in slices showed a higher amplitude and slower kinetics of I(Ts). These properties could be reproduced from the reconstructed cell model assuming higher densities of I(Ts) in distal dendrites. In a third series of experiments, current-clamp recordings were obtained on RE cells in vivo. The marked differences with in vitro recordings could be reconciled by simulating synaptic bombardment in the dendrites of RE cells, but only if they contained high distal densities of I(Ts). In addition, simpler models with as few as three compartments could reproduce the same behavior assuming dendritic I(Ts). These models and experiments show how intrinsic bursting properties of RE cells, as recorded in vivo and in vitro, may be explained by dendritic calcium currents. PMID- 8613784 TI - Properties of the endosomal-lysosomal system in the human central nervous system: disturbances mark most neurons in populations at risk to degenerate in Alzheimer's disease. AB - Specific antibodies and cytochemical markers combined with several imaging and morphometric techniques were used to characterize the endosomal-lysosomal system in mature neurons of the normal human central nervous system and to quantitate changes in its function in Alzheimer's disease. Compartments containing cathespin D (Cat D) and other acid hydrolases included a major subpopulation of mature lysosomes lacking mannose-6-phosphate receptors (MPR) and smaller populations of late endosomes (MPR-positive) and lipofuscin granules (MPR-negative). Antibodies to the pro-isoform of Cat D decorated perinuclear vacuolar compartments corresponding to late endosomes. Neurons and glia contained lysosomes with differing complements of acid hydrolases, implying different processing capabilities. Endosome/lysosome number per unit volume of cytoplasm was relatively well conserved within populations of normal neurons. By contrast, in morphometric analyses of Alzheimer's disease brains, 80-93% of pyramidal cells in the prefrontal cortex (laminae III or V) and hippocampus (CA2, CA3) displayed two to eightfold higher numbers of hydrolase-positive vacuolar compartments than did corresponding cell populations in age-matched normal brains. Only 5-10% of cerebellar Purkinje cells, a less vulnerable population, showed the same statistically significant elevations. Most affected in these brain regions and in subcortical areas seemed otherwise normal by conventional histological staining and ultrastructural inspection. That both lysosomal and pro-Cat D- and MPR positive endosomal compartments increased in number demonstrates that the endosomal-lysosomal system is activated markedly in vulnerable neuronal populations of Alzheimer's disease brains and implies that endocytosis or autophagy or both are accelerated persistently at an early stage of cellular compromise, greatly surpassing the degree of activity associated with normal aging. Early activation of the endosomal-lysosomal system represents a biological event potentially linking major etiological factors in Alzheimer's disease, including defective membrane proteins, apolipoprotein E function, and altered amyloid precursor protein processing. PMID- 8613786 TI - AMPA receptor activation is rapidly toxic to cortical astrocytes when desensitization is blocked. AB - Although cultured astrocytes express functional glutamate receptors, they are generally resistant to excitotoxic cell death. We explored the role of receptor desensitization in glutamate-mediated astrocyte injury. In cultures of type 1 astrocytes from mouse neocortex, brief application of AMPA evoked small, rapidly desensitizing inward currents, whereas kainate evoked small, sustained currents. Neither agonist increased cytosolic calcium, and astrocyte toxicity occurred only after 24 hr exposure to high (500-1000 microM) concentrations of kainate but not to AMPA or glutamate. Cyclothiazide, a drug that selectively blocks AMPA receptor desensitization, greatly potentiated AMPA- or kainate-gated currents and intracellular calcium elevation. Coapplication of 10-100 microM cyclothiazide with glutamate, AMPA, or kainate produced widespread astrocyte cell death within 2 hr or application. The enhancement of toxicity by cyclothiazide, which alone was not toxic, was concentration-dependent for each of the tested agonists (EC50 30-100 microM) and was blocked by further addition of the selective AMPA/kainate antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX). NMDA caused no injury even in the presence of cyclothiazide. Cyclothiazide-enhanced injury varied with the age of astrocyte cultures; the maximal effect occurred at approximately 2 weeks in vitro, and little death was seen after 4 weeks. Type 1 astrocytes express AMPA-type glutamate receptors that are unmasked by reducing their desensitization with cyclothiazide. Although overactivation of AMPA receptors can be rapidly lethal to astrocytes, rapid desensitization normally limits this toxicity. The extent of AMPA receptor desensitization may be an important determinant of glial vulnerability to excitotoxic insults. PMID- 8613785 TI - Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics. AB - NMDA receptor antagonists can induce a schizophrenia-like psychosis, but the role of NMDA receptors in the pathophysiology of schizophrenia remains unclear. Expression patterns of mRNAs for five NMDA receptor subunits (NR1/NR2A-D) were determined by in situ hybridization in prefrontal, parieto-temporal, and cerebellar cortex of brains from schizophrenics and from neuroleptic-treated and nonmedicated controls. In the cerebral cortex of both schizophrenics and controls, mRNAs for NR1, NR2A, NR2B, and NR2D subunits were preferentially expressed in layers II/III, Va, and VIa, with much higher levels in the prefrontal than in the parieto-temporal cortex. Levels of mRNA for the NR2C subunit were very low overall. By contrast, the cerebellar cortex of both schizophrenics and controls contained very high levels of NR2C subunit mRNA, whereas levels for the other subunit mRNAs were very low, except NR1, for which levels were moderate. Significant alterations in the schizophrenic cohort were confined to the prefrontal cortex. Here there was a shift in the relative proportions of mRNAs for the NR2 subunit family, with a 53% relative increase in expression of the NR2D subunit mRNA. No comparable changes were found in neuroleptic-treated or untreated controls. These findings indicate regional heterogeneity of NMDA receptor subunit expression in human cerebral and cerebellar cortex. In schizophrenics, the alterations in expression of NR2 subunit mRNA in prefrontal cortex are potential indicators of deficits in NMDA receptor-mediated neurotransmission accompanying functional hypoactivity of the frontal lobes. PMID- 8613787 TI - MN20, a D2 cyclin, is transiently expressed in selected neural populations during embryogenesis. AB - Although the regulation of proliferation and differentiation during brain development has long been considered to be interrelated, the mechanisms that coordinate the control of cell division and histogenesis are poorly understood. The cell cycle is a dynamic process that is governed by the concerted action of numerous cell cycle regulatory proteins in response to signals both intrinsic and extrinsic to the cell. Thus, proteins that regulate the cell cycle are well suited to provide a link between processes that control neuroblast proliferation and differentiation. We reported previously the isolation from brain of a message form of D2 cyclin, one of several cyclin proteins known to promote the progression from G1 to S phase. This MN20/D2 cyclin mRNA is expressed in highly restricted neural populations at embryonic (E) day 15 and postnatal (P) day 6 in the mouse. To gain insight into the role(s) this cyclin may serve in brain formation, the spatial and temporal pattern of MN20/D2 cyclin expression was examined by in situ hybridization at 48 hr intervals from E10.5 to P8. MN20 mRNA was detected in developing cerebellum, dorsal mesencephalon, cerebral cortex, and epithalamus, but not hippocampus, striatum, or thalamus. Comparison with 5 bromodeoxyuridine labeling of cells in S phase indicated that MN20 expression in embryonic cerebellum and cerebral cortex was most pronounced in young neurons that recently had become postmitotic. Although expressed in other embryonic cerebellar neurons, MN20 was detected in granule precursors only postnatally, after their migration from the rhombic lip to the external germinal layer. This indicates that MN20/D2 cyclin is induced in cerebellar granule precursors as they become competent to differentiate. The spatial distribution of MN20 expression in the developing brain suggests that regional differences in cell cycle regulation depend in part on the selective use of cyclin proteins. Moreover, detection of MN20 mRNA in postmitotic neural cells indicates that cyclin D2 expression has effects beyond promoting cell cycle progression and may also have a role in the response of the neural precursor to terminal differentiation signals as the cells exits from proliferation. PMID- 8613789 TI - Differential regulation of adrenergic receptor development by sympathetic innervation. AB - Rat sweat glands provide an interesting model system for a developmental study of adrenergic receptor expression because their sympathetic innervation undergoes a switch from a nonadrenergic to cholinergic and peptidergic phenotype. alpha 1B, alpha 2B, and beta 2 receptors are expressed in rat footpads; alpha 1 and beta 2 receptors are localized specifically to sweat glands, and alpha 2 receptors also are expressed in other tissues. alpha 1 and, to a lesser extent, beta 2 receptors decrease during development, whereas alpha 2 levels remain relatively constant. Decreased receptor expression is accompanied by the loss of alpha 1-stimulated inositol phosphate accumulation, but no change in beta-stimulated cAMP production. The number of alpha 1 and beta 2 receptors decreases after P21, when the sympathetic innervation no longer produces catecholamines. Neonatal sympathectomy causes a partial failure of alpha 1 downregulation, but has no effect on beta 2 or alpha 2 receptor levels. Therefore, at least two distinct mechanisms regulate development of adrenergic receptors in sweat glands. Innervation-independent processes control developmental expression of alpha 1, beta 2, and alpha 2 receptors, and an additional, innervation-dependent mechanism influences expression of alpha 1 receptors. Denervation at postnatal day 20, when the sympathetic innervation is cholinergic and peptidergic, results in retention of alpha 1 receptors, but cholinergic blockade begun at P20 does not. These results indicate that regulation of receptor expression in sweat glands is complex, and suggest that the innervation-dependent factors that decrease alpha 1 levels during development act through a nonadrenergic, noncholinergic mechanism. PMID- 8613788 TI - Persistent dentate granule cell hyperexcitability after neonatal infection with lymphocytic choriomeningitis virus. AB - Infection of neonatal Lewis rats with lymphocytic choriomeningitis virus (LCMV) produces distinct retinal, cerebellar, and hippocampal neuropathology. To understand the neurophysiological consequences of LCMV-induced hippocampal pathology, we studied evoked monosynaptic potentials and electro-encephalographic (EEG) activity in the dentate gyrus and CA1 and CA3 subfields of the hippocampus in vivo. Lewis rats were inoculated intracerebrally with LCMV at postnatal day 4. In rats studied 84-107 d postinfection, virus was cleared from the dentate gyrus and the number of dentate granule cells was decreased by 70%. No viral antigen or cell loss was apparent in CA1 or CA3. The hippocampal EEG of LCMV-infected rats 84-102 d postinfection was dominated by continuous theta. Although evoked potentials elicited in CA1 and CA3 by monosynaptic afferent stimulation revealed no differences between sham- and LCMV-infected rats, there was a site-specific dissociation of synaptic [population excitatory postsynaptic potential (pEPSP)] and cellular (population spike) responses and a suppression of GABA-mediated recurrent inhibition in the dentate gyrus of LCMV-infected rats. These findings indicate that GABA-mediated inhibition was markedly decreased in LCMV-infected rats. In support of this, parvalbumin-immunoreactive cell bodies and neuronal processes were decreased in LCMV-infected rats, suggesting that a subpopulation of GABA interneurons was affected. These findings indicate that abnormalities in synaptic function persist after clearance of infectious virus from the central nervous system and suggest that decreased inhibition subsequent to pathological sequence in a subpopulation of GABA interneurons may be implicated in the hyperexcitability of dentate granule cells. PMID- 8613790 TI - The molecular composition of neuronal microfilaments is spatially and temporally regulated. AB - The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm-5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, beta-actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of beta-actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta-actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and beta-actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation. PMID- 8613791 TI - Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. AB - We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide. PMID- 8613792 TI - Sequence of stress-induced alterations in indices of synaptic and transcriptional activation in parvocellular neurosecretory neurons. AB - Immediate-early genes (IEGs) are widely used to mark endocrine hypothalamic neurons that are activated in response to stress, yet their relationship to the transcriptional control of relevant effector molecule expression is unclear. Acute ether stress provokes increased adrenocorticotropic hormone (ACTH) and corticosterone secretion that peaks at 5 and 30 min, respectively, after the challenge. Using probes complementary to intronic sequences of genes encoding ACTH secretagogues in parvocellular neurosecretory neurons of the paraventricular nucleus, we found these events to be accompanied by rapid and transient increases in corticotropin-releasing factor heteronuclear RNA (CRF hnRNA; peak at 5 min) and by a delayed upregulation of arginine vasopressin (AVP) hnRNA (120 min). To identify candidate mechanisms regulating peptide expression, we followed the timing of ether effects on representatives of three transcription factor classes: IEGs [c-fos and nerve growth factor I-B (NGFI-B)], a POU-domain factor (Brn-2), and the cAMP response element-binding protein (CREB), using antisera specific to its transcriptionally active, phosphorylated form (pCREB). After ether exposure, c-fos and NGFI-B mRNA induction were maximal at 30--60 min, whereas Fos protein peaked at 60--120 min. Brn-2 mRNA was expressed constitutively in the PVH and was unresponsive to stress. By contrast, pCREB was induced in parvocellular neurons with a time course parallel to that of CRF hnRNA expression. Stress-induced transcriptional activation of the CRF and AVP genes in hypophysiotropic neurons follows distinct time courses that are compatible with control mechanisms involving phosphorylation events and de novo protein synthesis, respectively. PMID- 8613793 TI - Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents. AB - Behavioral and electrophysiological evidence suggests that glutamatergic neurotransmission plays an important role in some of the long-term effects of cocaine and other drugs of abuse on brain function. We therefore examined the effect of repeated cocaine treatment on glutamate receptor subunit expression in central dopamine (DA) pathways implicated in many of cocaine's behavioral actions. By immunoblotting procedures using subunit-specific antibodies, we found that repeated, but not acute, cocaine treatment increased the levels of immunoreactivity of GluR1 (an AMPA receptor subunit) and NMDAR1 (an NMDA receptor subunit) in the ventral tegmental area (VTA), a nucleus containing mesolimbic DA neurons. In contrast, chronic cocaine treatment did not alter levels of GluR2 (an AMPA receptor subunit), NMDA2A/B (NMDA receptor subunits), or GluR6/7 (kainate receptor subunits) in this brain region. Moreover, GluR1 and NMDAR1 levels were not regulated in other regions of the mesolimbic or nigrostriatal DA pathways, including the substantia nigra. Because several drugs of abuse and stress can elicit common and cross-sensitizing effects on mesolimbic DA function, we next examined whether repeated morphine and stress treatments would regulate these proteins similarly in the VTA. Although morphine delivered by subcutaneous pellet implantation had no significant effect on subunit levels, morphine delivered intermittently by subcutaneous injections of escalating doses elevated GluR1 levels in the VTA. Repeated restraint stress also paradigm (2 stressors/d under variable conditions) increased both GluR1 and NMDAR1 levels in this brain region. Unlike cocaine, morphine, and stress, repeated treatment with other psychotropic drugs (haloperidol, raclopride, sertraline, and desipramine) that lack reinforcing or sensitizing properties did not regulate GluR1 or NMDAR1 subunit levels in the VTA. Increased glutamate receptor subunit expression in the VTA may represent an important molecular mechanism by which drugs of abuse and stress exert common, long-term effects on mesolimbic DA function. PMID- 8613794 TI - Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study. AB - To assess the significance of GABAA-receptor heterogeneity, which is based on a family of at least 15 subunits, the cellular localization and subunit composition of GABAA-receptor subtypes were analyzed immunohistochemically in the rat spinal cord. The distribution of subunits alpha 1, alpha 2, alpha 3, alpha 5, beta 2,3, and gamma 2 was investigated with subunit-specific antibodies, and their colocalization within individual neurons was visualized by double immunofluorescence staining. The results reveal a widespread expression of the subunits, alpha 3, beta 2,3, and gamma 2 in the spinal cord, whereas the three other alpha subunits displayed a more restricted, lamina-specific distribution. The alpha 1 and alpha 5 subunits were most abundant in the intermediate zone, whereas the alpha 2 subunit was predominant in the superficial layers of the dorsal horn and in somatic and preganglionic motoneurons. From colocalization studies, seven subunit combinations could be identified (alpha 3/beta 2,3/gamma 2; alpha 2/beta 2,3/gamma 2; alpha 1/beta 2,3/gamma 2; alpha 5/beta 2,3/gamma 2; alpha 1/alpha 5/beta 2,3/gamma 2; alpha 2/gamma 2; alpha 2/alpha 5/gamma 2) that correspond presumably to distinct receptor subtypes. Although most neurons expressed the subunit triplet alpha x/beta 2,3/gamma 2, the beta 2,3 subunits could not be detected in motoneurons that may thus possess "atypical" receptor subtypes (alpha 2/gamma 2 and alpha 2/alpha 5/gamma 2). ON the subcellular level, aggregates of immunoreactivity, suggestive of postsynaptic GABAA receptors, typically were seen on the surface of neuronal somata and proximal dendrites. In addition, an intense diffuse staining was observed in laminae I--III for the subunits alpha 2, alpha 3, beta 2,3, and gamma 2, presumably localized on primary afferent terminals. The localization of GABAA-receptor subtypes in distinct laminar compartments of the spinal cord suggests that GABAA-receptor heterogeneity is of relevance for the modulation of sensory inputs, nociception, and motor control at segmental levels. PMID- 8613795 TI - The synaptic drive from the spinal locomotor network to motoneurons in the newborn rat. AB - The nature of the synaptic drive from the locomotor spinal network onto the motoneurons was studied in the newborn rat. For this purpose, an in vitro isolated spinal cord preparation of newborn rat was used. The recording chamber was partitioned with Vaseline walls to separate the L1/L2 lumbar segments, in which the spinal locomotor network is located, from the motoneurons in the lower lumbar segments. Locomoter-like activity was induced by bath-applying a mixture of serotonin and NMDA to segments L1/L2. In this way, the synaptic activity could be modified at the lower lumbar level without affecting the motor pattern. The drive elicited onto the motoneurons during sequences of locomoter-like activity, which was monitored by performing intracellular recordings, consisting of an inhibitory component followed by an excitatory component. The inhibitory synaptic volley was reversed at a membrane potential of --60 mV with K acetate electrodes, whereas it was shifted toward positive values with KCl electrodes. The glycinergic blocker strychnine, bath-applied to segments L3/L5, blocked the inhibitory drive without affecting the rhythmic activity, whereas it disrupted the locomoter-like activity when bath-applied to segments L1/L2. The inhibitory part of the drive was more sensitive than the excitatory part to changes in the membrane potential. The excitatory phase was mixed and consisted of an NMDA and a non-NMDA component, which were sensitive to 2-amino-5-phosphonovaleric acid and 6 cyano-7-nitroquinoxaline-2,3-dione, respectively. It was concluded that the locomotor network located in segments L1/L2 sends a biphasic projection to the various groups of motoneurons located along the lumbar spinal cord. PMID- 8613796 TI - A dendritic GABAA-mediated IPSP regulates facilitation of NMDA-mediated responses to burst stimulation of afferent fibers in piriform cortex. AB - Studies in a number of cortical systems have shown that the NMDA component of the EPSP is strongly regulated by GABAA-mediated inhibition. The present study explored the possibility that specificity in inhibitory circuitry could allow such regulation to occur during normal function without increasing the propensity for epileptiform bursting, which occurs with indiscriminate GABAA blockade. Specifically, the hypothesis was tested that a dendritic GABAA-mediated IPSP is present which strongly modulates the NMDA component and can be activated independently of the somatic IPSP. The experiments were performed on slices of piriform cortex in which the NMDA component of the EPSP was pharmacologically isolated by bath-applied 6,7-dinitroquinoxaline-2,3-dione. A facilitation of NMDA responses to burst stimulation of afferent fibers is described, which required GABAA blockade and served as an assay for the presence of a functionally significant GABAA input. When bicuculline was applied focally in the somatic region, the feedback IPSP was blocked with little or no increase in the NMDA component of the response to burst stimulation of afferent fibers. In contrast, when bicuculline was applied focally in the dendritic region, the NMDA-mediated response to burst stimulation was facilitated with minimal effect on the somatic IPSP, confirming the hypothesis. PMID- 8613797 TI - mRNA expression of KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, KIF5, and cytoplasmic dynein during axonal regeneration. AB - Mouse brain expresses multiple kinesin superfamily proteins (KIFs), which are involved in vesicle transport. The expression of KIFs is developmentally regulated, and both the mRNA and proteins of KIF2 and KIF4 are expressed abundantly in the juvenile brain. To elucidate the role of individual kinesin superfamily motor proteins during regenerative outgrowth of axons, we examined the mRNA expression of KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 in adult mouse dorsal root ganglion cells after sciatic nerve crush. Seven to fourteen days after the nerve crush, the mRNA expression pattern of neurofilament and beta tubulin isotypes suggested that the regenerative outgrowth of axons was active. At these stages, levels of mRNA for KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 were 50.80% of control. The levels of mRNA for KIF4, which are detected in juvenile brain but not in the adult, were under the detection limit in both control and regenerating dorsal root ganglion cells. Because mRNA of neither KIF2 nor KIF4 increased significantly, the results suggest that the gene expression of KIFs during regeneration does not recapitulate the embryonic development and support the hypothesis that different series of events take place during the regenerative and embryonic outgrowths of axons. In contrast, mRNA for cytoplasmic dynein was slightly increased, up to 140%. This is consistent with the hypothesis that retrograde transport plays critical roles in regeneration such as the transport of neurotrophic factors. PMID- 8613798 TI - High-speed optical imaging of afferent flow through rat olfactory bulb slices: voltage-sensitive dye signals reveal periglomerular cell activity. AB - Fast, multiple-site optical recording and video imaging techniques were combined to visualize the olfactory processing stream as it flowed through rat olfactory bulb slices stained with the voltage-sensitive dye RH155. A 464 element photodiode detector array was used to record the voltage-sensitive dye signals. Focal electrical stimulation of the olfactory nerve layer evoked relatively large optical responses in the olfactory nerve and glomerular layers but only small responses within the external plexiform layer. With paired-pulse stimulation, glomerular attenuation was evident in signals recorded from the glomerular and external plexiform layers but not from the olfactory nerve layer. At very high recording speeds ( < 0.2 msec/frame), the presynaptic component of the olfactory processing stream could be followed as it flowed through the olfactory nerve layer and into the glomerular layer, where its amplitude rapidly declined. This decline was followed by a reciprocal rise in a postsynaptic depolarization that was largely restricted to the glomerular layer. Spatiotemporal interactions between overlapping afferent streams within the glomerular layer were observed and partially characterized. The optically recorded glomerular layer response was largely resistant to bath application of GABAA receptor antagonists but was sensitive to manipulations of external chloride concentration and to bath application of a stilbene derivative, 4-acetamido-4'isothiocyanatostilbene-2,2' disulfonic acid known to block Cl- conductances. It is suggested the the voltage sensitive dye signals recorded from the glomerular layer reflect activity in periglomerular cells and that Cl- efflux through non-GABAA chloride channels contributes to the postsynaptic depolarization of these cells after olfactory nerve stimulation. PMID- 8613799 TI - The effect of efferent stimulation on basilar membrane displacement in the basal turn of the guinea pig cochlea. AB - Tone-evoked basilar membrane (BM) displacements were measured with a laser diode interferometer from the basal turn of the guinea pig cochlea. The olivocochlear bundle (OCB) was electrically stimulated for 60--80 msec periods at rates of < 200 sec-1 via electrodes placed at the point at which the OCB crosses the floor of the fourth ventricle. For tones close to the best or characteristic frequency (CF), OCB stimulation tended to linearize the highly compressive displacement level functions and to displace the steep, low-level region toward higher intensities along the intensity axis by < 27 dB sound pressure levels. This shift resulted in a desensitization of the tip of the BM displacement tuning curve that was associated sometimes with downward shifts in the tuning curve CF of < 500 Hz. OCB-induced suppression of the BM response was not associated with a consistent broadening of the tuning curve or with major changes in the phase of the BM response. At frequencies in the low-frequency tail of the tuning curve, OCB stimulation had no observable effect on the motion of the BM. The effect of OCB stimulation on the BM response was blocked by perfusing the scala tympani with 1 microM strychnine. Thus, the effect of OCB stimulation on the frequency tuning of the BM is very similar to the effect of OCB stimulation on the sensitivity and frequency tuning of afferent fibers and inner hair cells. The results indicate that the postsynaptic action of the OCB may cause a change in gain of the voltage dependent outer hair cell motility without observable changes in the stiffness of the cochlear partition or the position of the BM. PMID- 8613800 TI - Medial preoptic area afferents to periaqueductal gray medullo-output neurons: a combined Fos and tract tracing study. AB - We have shown recently that the medial preoptic area (MPO) robustly innervates discrete columns along the rostrocaudal axis of the midbrain periaqueductal gray (PAG). However, the location of PAG neurons responsive to MPO activation is not known. Anterograde tract tracing was used in combination with Fos immunohistochemistry to characterize the MPO --> PAG pathway anatomically and functionally within the same animal. Focal electrical or chemical stimulation of MPO in anesthetized rats induced extensive Fos expression within the PAG compared with sham controls. Fos-positive neurons were organized as 2-3 longitudinal columns. The organization and location of these columns overlapped remarkably well with the distribution of fibers and terminals in PAG labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injected into the same MPO stimulation site. This indicates that MPO inputs may terminate on the soma or proximal dendrites of neurons exhibiting elevated Fos. A second series of experiments investigated whether MPO stimulation excited PAG neurons with descending projections to the medulla. Retrograde labelling of PAG neurons projecting to the medial and lateral regions of the rostroventral medulla (RVM) was combined with MPO-induced Fos expression. The results showed that a substantial population (37-53%) of Fos positive PAG neurons projected to the ventral medulla. This indicates that MPO stimulation engages PAG-medullary output neurons. Taken together, these results suggest that the MPO --> Pag --> RVM projection constitutes a functional pathway. This circuit may coordinately regulate neuroendocrine, motor, and autonomic adjustments necessary for the elaboration of sexual behaviors. PMID- 8613801 TI - The interaction of imposed and inherent olfactory mucosal activity patterns and their composite representation in a mammalian species using voltage-sensitive dyes. AB - From amphibian data, two mechanisms that could underlie the encoding of odorants by the mucosal activity patterns they engender are as follows (1) receptors with similar odorant selectivities could be aggregated spatially on the mucosa (inherent patterns); (2) in analogy to gas chromatography, as odorants are drawn along the surface of the mucosa the strongly sorbed ones could be deposited preferentially upstream, whereas the weakly sorbed ones could be distributed more evenly (imposed patterns). Do both of these possible coding mechanisms operate in mammals and, if so, how do they interact in giving composite patterns (imposed + inherent)? Fluorescence changes in di-4-ANEPPS applied to rat mucosas were monitored by a 10 x 10 pixel photodiode array. To observe the inherent patterns, three odorants of varying sorbabilities first were puffed uniformly onto the entire mucosa mounted in a Delrin chamber. To bring out the imposed patterns, the chamber was then sealed to replicate anatomically the rat's nasal cavity, and these same odorants were drawn at three flow rates along the mucosal flow path. The results demonstrated for the first time the existence of imposed patterns in a mammal. The strongly sorbed odorants, unlike the weakly sorbed one, showed marked imposed patterns. Within physiological limits, increasing the flow rate decreased the magnitude of the imposed patterns. One might consider strategies that the olfactory process could use either to negate or to take advantage of the chromatographic effect, because the lability of the composite patterns with changing stimulus conditions raises questions about their role in odorant encoding. PMID- 8613802 TI - Hippocampal ensemble activity during spatial delayed-nonmatch-to-sample performance in rats. AB - Multiple-cell recording from specially designed arrays of microwire electrodes allowed analysis of anatomically defined ensemble activity from 10 different locations within the hippocampus of rats (n = 7) performing a two-lever operant version of a spatial delayed-nonmatch-to-sample task (DNMS). Application of population analysis procedures to ensembles of single-neuron activity within the CA1 and CA3 fields revealed firing patterns related to task-relevant events within a DNMS trial. The patterns were extracted via a canonical discriminant analysis in the form of "roots" that represented sources of variance in firing within the ensemble, such as phase of the task (Sample or Nonmatch), spatial position of the lever press response (left or right), and correct versus error trials. Comparison of the ensemble firing on correct versus error trials revealed important insight into ensemble information encoding, such as "miscoding" of the response position and lack of distinct encoding of the response in the Sample phase, which became increasingly vulnerable to error as a function of the duration of delay interval. The extracted discriminant scores were reflective of multiple representations within ensembles and suggested that "conjunctions" of task-relevant features could be represented effectively by small numbers of hippocampal neurons. The findings support the long-held supposition that hippocampal neurons play a critical role in the encoding and retrieval of information in recognition memory tasks. PMID- 8613803 TI - Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons. AB - Metabotropic glutamate receptor (mGluR) modulation of voltage-gated Ca2+ channels was examined in isolated deep layer frontoparietal cortical neurons under conditions designed to isolate calcium-independent modulatory pathways. Trans-1 aminocyclopentane-1,3-dicarboxylate (t-ACPD), a nonspecific mGluR agonist, produced rapid and reversible inhibition of Ca2+ channels. This effect was mimicked by agonists for group I and group II, but not group III, mGluRs. Effects of group I and II agonists often were observed in the same neurons, but separate subgroups of neurons were unresponsive to the group I agonist quisqualate or the group II agonist 2-(2,3-dicarboxycyclopropyl) glycine (DCG-IV). Inhibition by quisqualate and DCG-IV was nonocclusive in neurons responding to both agonists. These agonists thus appear to act on different mGluRs. The mGluR antagonist alpha methyl-4-carboxylphenylglycine attenuated inhibition by t-ACPD, quisqualate, and DCG-IV. Inhibition by quisqualate and DCG-IV was voltage-dependent. Although the effects of both agonists were greatly reduced by N-ethylmaleimide (NEM), inhibition by DCG-IV was more sensitive to NEM than inhibition by quisqualate. t ACPD-induced inhibition was reduced by omega-conotoxin GVIA (omega-CgTx) and omega-agatoxin IVA (omega-AgTx) but was affected little by nifedipine. Inhibition by DCG-IV and quisqualate also was reduced by omega-CgTx. We conclude that multiple mGluR subtypes inhibit Ca2+ channels in cortical neurons and that N- and possibly P-type channels are inhibited. Modulation is via a rapid-onset, voltage dependent mechanism that likely involves a pertussis toxin (PTX)-sensitive G protein. Type I mGluRs may work via additional PTX-insensitive pathways. PMID- 8613804 TI - NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine. AB - The present study was performed to assess the role of excitatory amino acid and dopamine receptors on associative functions of the prefrontal cortex (PFC) of the rat. Spatial delayed alternation was used as a PFC-sensitive cognitive task. In addition, in vivo microdialysis was used to assess the release of dopamine in the PFC. The noncompetitive NMDA antagonists ketamine (10-30 mg/kg) and MK-801 (0.1 and 0.5 mg/kg) dose-dependently impaired the spatial delayed alternation performance compared with the saline-treated control group. Administration of the dopamine antagonists raclopride (0.1 and 0.5 mg/kg), SCH-23390 (0.1 mg/kg), or haloperidol (0.1 mg/kg) was without a significant effect. However, haloperidol and raclopride (but not SCH-23390) reversed the disruptive effect of 30 mg/kg ketamine on spatial delayed alternation performance. Microdialysis studies revealed that this dose of ketamine preferentially increased the release of dopamine in the PFC compared with the striatum. These findings indicate that attenuation of glutamatergic neurotransmission at the NMDA receptor impairs PFC dependent cognitive functions. Furthermore, activation of dopamine neurotransmission contributes, at least in part, to this impairment. PMID- 8613805 TI - Neuronal circuitry for comparison of timing in the electrosensory lateral line lobe of the African wave-type electric fish Gymnarchus niloticus. AB - An African wave-type electric fish, Gymnarchus, compares timing on the order of microseconds of sensory feedback from from its high-frequency (approximately 400 Hz) electric organ discharges (EODs) received at different parts of its body surfaces. This capability is essential for and demonstrated by the jamming avoidance response (JAR). The organization of the timing comparison mechanisms was identified in the electrosensory lateral line lobe (ELL) in the hindbrain by field potential, extra- and intracellular recordings, and intracellular labeling with biotinylated agents. Timing of phase of the EOD feedback is carried by action potentials of S-type primary afferent fibers that project to the inner cellular layer (ICL) of the medial zone of the ELL and to the giant neurons in the ELL. The giant neurons bilaterally project to the ICL, where neurons sensitive to phase differences between different parts of the body occur. Although sensitive to dynamic phase changes of several microseconds, these differential-phase-sensitive neurons showed adaptation to steady-state changes of phase difference over a wide range (greater than +/- 100 microseconds) and continued to respond to small modulations after the mean difference was shifted. Gymnarchus and an independently evolved South American electric fish, Eigenmannia, exhibit nearly identical JARs and share a rather complex but identical set of computational algorithms for JAR. This study showed that one of the computational steps, the timing comparison between body surfaces, occurs in the hindbrain in Gymnarchus, in contrast to the midbrain in Eigenmannia. Thus, similar systems with a similar overall function may have evolved differently in different genera by assigning a subfunction to different substructures within the brain. PMID- 8613806 TI - Synchronization of fast (30-40 Hz) spontaneous cortical rhythms during brain activation. AB - We investigated the synchronization of fast spontaneous oscillations (mainly 30 40 Hz) in anesthetized and behaving cats by means of simultaneous extra- and intracellular recordings from multiple neocortical areas. Fast Fourier transforms, auto- and cross-correlations, and spike- or wave-triggered averages were used to determine the frequency and temporal coherence of fast oscillations that outlasted the stimulation of ascending activating systems or that occurred naturally during behavioral states of waking and rapid eye movement (REM) sleep but also appeared during the depolarizing phases of slow sleep oscillations. In 90% of microelectrode tracks, the fast oscillations did not show field reversal at any depth of the cortex and were not observable in the underlying white matter. The negative field potentials of the fast oscillations were associated at all depths with neuronal firing. This field potential property of fast oscillations was in sharp contrast to the reversal of slow sleep oscillation or evoked potentials at depths of 0.25-0.5 mm. The coherence of fast spontaneous rhythms was spatially limited, being confined within a cortical column and among closely located neocortical sites, in contrast to the long-range synchronization of slow sleep rhythms. Depolarizing current pulses elicited spike-bursts (200-400 Hz) recurring at a frequency of 30-40 Hz. Our experiments demonstrate that the conventional notion of a totally desynchronized cortical activity upon arousal should be revised as fast rhythms are enhanced and synchronized within intracortical networks during brain activation. Spontaneously occurring, subthreshold membrane potential depolarizing oscillations may bias cortical and thalamic neurons to respond synchronously, at fast frequencies, to relevant stimuli in the wake state or to internally generated drives in REM sleep. PMID- 8613807 TI - The selective neuronal NO synthase inhibitor 7-nitro-indazole blocks both long term potentiation and depotentiation of field EPSPs in rat hippocampal CA1 in vivo. AB - The membrane-permeant gas NO is a putative intercellular messenger that has been proposed on the basis of previous in vitro studies to be involved in synaptic plasticity, especially the induction of long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus and cortex. In the present study, the role of NO in synaptic plasticity has been investigated in vivo. In particular, the action of the novel and selective neuronal NO synthase (nNOS) inhibitor 7-nitro-indazole (7-NI) has been investigated on the induction of LTP and depotentiation (DP) of field EPSPs in CA1 of the hippocampus in vivo. Unlike previously studied nonselective NOS inhibitors, 7-NI does not increase arterial blood pressure. In vehicle-injected rats, high-frequency stimulation consisting of a series of trains at 200 Hz induced LTP. However, LTP induction was strongly inhibited in 7-NI (30 mg/kg, i.p.)-treated animals. The inhibitory effect of 7-NI on the induction of LTP was prevented by pretreatment with L-arginine, the substrate amino acid used by NOS. In control animals, low-frequency stimulation consisting of 900 stimuli at 10 Hz induced DP of previously established LTP, whereas in 7-HI-treated animals only a short-term depression was induced. This effect of 7-NI also was prevented by D-arginine. The LTP and DP induced in control animals in this study were NMDA receptor-dependent, the NMDA receptor antagonist 3-(R,S)-2-carboxypiperazin-4-yl-propyl-1- phosphonic acid inhibiting the induction of both forms of synaptic plasticity. The present experiments are the first to demonstrate that an NOS inhibitor blocks the induction of the synaptic component of LTP and DP in vivo and, therefore, these results strengthen evidence that the production of NO is necessary for the induction of LTP and DP. PMID- 8613808 TI - Calcium-independent activation of the secretory apparatus by ruthenium red in hippocampal neurons: a new tool to assess modulation of presynaptic function. AB - The functional plasticity of the nervous system may result in part from the direct modulation of the effectiveness of the release machinery of synaptic terminals. To date, direct modulation of secretion in neurons has proven difficult to study because of the lack of a suitable tool to probe the release machinery independently of calcium influx. We report that the polyvalent cation ruthenium red (RR) directly evokes rapid and reversible calcium-independent quantal secretion in hippocampal neurons by binding to external sites on the presynaptic terminal membrane. This binding can be displaced by heparin and is not associated with ultrastructural damage to the synaptic terminals. The use of RR-evoked release as a tool has allowed us to detect a direct modulation of the secretory apparatus after activation of A1 adenosine receptors on hippocampal neurons. PMID- 8613809 TI - Components of glial responses to exogenous and synaptic glutamate in rat hippocampal microcultures. AB - Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling. PMID- 8613810 TI - 11 beta-Hydroxysteroid dehydrogenase in cultured hippocampal cells reactivates inert 11-dehydrocorticosterone, potentiating neurotoxicity. AB - 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyzes the conversion of the glucocorticoid corticosterone (cortisol in humans) to inert 11 dehydrocorticosterone (cortisone). 11 beta-HSD activity is present in the hippocampus, where it is induced by glucocorticoids and stress in vivo, prompting suggestions that the enzyme may attenuate the deleterious effects of chronic glucocorticoid excess on neuronal function and survival. Two isoforms exist: 11 beta-HSD1, a bidirectional NADPH-dependent enzyme, and 11 beta-HSD2, an NAD(+) dependent exclusive 11 beta-dehydrogenase (corticosterone-inactivating enzyme). In this study, 11 beta-HSD1 activity and mRNA synthesis were demonstrated in primary fetal hippocampal cell cultures. Unexpectedly, the reaction direction in intact hippocampal cells was 11 beta-reduction (reactivation of inert 11 dehydrocorticosterone), although homogenization revealed that the enzyme was capable of 11 beta-dehydrogenation when removed from its normal cellular context. Dexamethasone (10(-7) M) increased 11 beta-HSD activity in homogenates of hippocampal cultures (102% increase). In intact hippocampal cells, dexamethasone induced 11 beta reductase, not dehydrogenase. To determine the functional relevance of hippocampal 11 beta-reductase, glucocorticoid potentiation of kainic acid neurotoxicity was examined. Pretreatment of hippocampal cells with corticosterone reduced survival on kainate exposure. Hippocampal cell 11 beta-HSD activity was potently inhibited by carbenoxolone. Carbenoxolone had no effect on cell survival after kainate alone and did not alter the effect of corticosterone. 11-Dehydrocorticosterone also potentiated kainate neurotoxicity; this effect was lost, however, if 11 beta-HSD was inhibited with carbenoxolone. Thus, hippocampal 11 beta-HSD seems to be a functional 11 beta-reductase in intact cells. Measures to attenuate hippocampal 11 beta-reductase may reduce neuronal vulnerability to glucocorticoid toxicity. PMID- 8613811 TI - In vitro ischemia promotes calcium influx and intracellular calcium release in hippocampal astrocytes. AB - The intracellular calcium concentration ([Ca2+])i of astrocytes within rat hippocampal slices was measured during simultaneous hypoxia and hypoglycemia to examine the early intracellular signaling events induced by this in vitro model of ischemia. Hypoxia-hypoglycemia for 3.3-7.5 min evoked [Ca2+]i increases in astrocytes iontophoretically loaded with calcium orange (11/14 slices; 2.5 min to peak [Ca2+]i, 5 min to > 60 min duration). Calcium elevations also were observed in the absence of extracellular calcium ([Ca2+]o) (4/4 slices), indicative of Ca2+ release from internal stores. Hypoxia-hypoglycemia depolarized astrocytes (51 +/- 16 mV), suggesting additional contribution from voltage-gated Ca2+ influx. Depolarization of a similar magnitude (51 +/- 4 mV) by 50 mM extracellular potassium ([K+]o triggered [Ca2+]i increases (20/24 slices), which were blocked by removal of [Ca2+]o (8/8 slices) indicating that depolarization promoted Ca2+ influx. Voltage-gated Ca2+ influx and internal release were measured in accurately isolated astrocytes during in vitro ischemia to examine these processes in the absence of surrounding neurons. Hypoxia-hypoglycemia (7.5 34.0 min) induced only modest, slow increases in the basal [Ca2+]i of Fura-2 loaded isolated astrocytes (average 12% increase in Fura-2 ratio R340/380 after 10 min) that were blocked by [Ca2+]o removal. Voltage-gated Ca2+ influx was still functional under ischemia, however, as 50 mM [K+]o evoked [Ca2+]i increases (14/14 cells, delta R340/380 of 48%) approximately equal to preischemic responses. Isolated neurons displayed large irreversible increases in basal [Ca2+]i after 1.5-6.5 min in vitro ischemia (10/12 cells; average delta R 340/380 of 152%). The absence of significant basal [Ca2+]i increases on isolated astrocytes indicates that ischemia-induced Ca2+ influx and internal release in astrocytes within slices depend on signals released from neurons (K+, neurotransmitters). Ischemic [Ca2+]i elevations may constitute a signaling mechanism for postischemic reactive responses. PMID- 8613812 TI - Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. AB - Rohon-Beard (RB) spinal neurons of Xenopus larvae are depolarized by GABA. To study the mechanisms underlying this distinctive response, intracellular and patch-clamp recordings were made from RB neurons in situ. The intracellularly recorded GABA reversal potential (EREV) was near -30 mV in normal saline and was approximately 25 mV more negative in Na(+)-free saline. Whole-cell recordings from RB neurons and from neighboring dorsolateral interneurons (DLi) revealed that GABA responses of both cells were mediated by GABAA receptors. Currents elicited by GABA were mimicked by muscimol and reversibly blocked by bicuculline, and EREV shifted with changes in Cl- concentration ([Cl]) in agreement with Cl- selectivity. In perforated patch recordings, EREV for RB cells was significantly more positive than for DLi cells (-38 vs -63 mV), indicating that intact RB cells maintain higher levels of intracellular Cl-. Replacement of external Na+ or exposure to the Cl- transport inhibitor bumetanide (100 microM) shifted RB cell EREV to move negative values, consistent with Na+(-)dependent Cl cotransport contributing to higher internal [Cl]. In contrast, these treatments did not change DLi cell EREV. The results indicate that a Na+(-)dependent Cl- transport mechanism underlies GABAA receptor-mediated depolarizing Cl- conductances in RB neurons. Thus, both inhibitory and excitatory GABA responses appear to be present during the same developmental period in vivo. GABA may stimulate Ca2+ influx in RB neurons because the intracellular GABA EREV is above the threshold for low voltage-activated Ca2+ channels. PMID- 8613813 TI - Taurine induces a long-lasting increase of synaptic efficacy and axon excitability in the hippocampus. AB - The physiological role of taurine, one of the most abundant free amino acids in the mammalian brain, is still poorly understood. We have found that bath application of the amino acid taurine induces two opposite actions on field excitatory synaptic potentials (fEPSP) recorded in the CA1 area of hippocampal slices: a decrease in fEPSP slope prevented by GABAA antagonists, and a long lasting potentiation of fEPSP independent of GABAA or NMDA receptor activation. Two long-lasting processes account for this taurine-induced potentiation: (1) an increase in synaptic efficacy that is accompanied neither by modifications in the basic postsynaptic membrane electrical properties nor by those presynaptic changes involved in fEPSP paired-pulse facilitation; and (2) an increase in the axon excitability revealed by a reduction on the threshold for antidromic action potential activation. In addition, taurine perfusion also induces a long-lasting increase in intracellularly recorded EPSPs and monosynaptically activated IPSPs. A number of experimental observations such as temperature dependence, extracellular Na+ concentration dependence, and saturation studies, although they are not unequivocally conclusive, suggest that the taurine uptake system is required for the taurine-induced fEPSP potentiation. Our data describe a new taurine action defined as a potentiation of synaptic transmission due in part to an increment in presynaptic axon excitability and in synaptic efficacy. PMID- 8613814 TI - The transcondylar approach to extradural nonneoplastic lesions of the craniovertebral junction. AB - Ventral extradural lesions at the craniovertebral junction are commonly exposed through the transoral or transmaxillary approach. The disadvantages of these approaches include: 1) difficulty in reaching laterally located lesions; 2) ineligibility of patients with an intradental distance of less than 25 mm or severe macroglossia; 3) the need for a separate procedure for stabilization and fusion; and 4) the risk of infection from transgressing a contaminated field. In this report, the authors describe the use of the transcondylar approach to extradural nonneoplastic lesions of the anterior craniovertebral junction for decompression and stabilization. Advantages of this approach include: 1) a short distance to the lesion; 2) a wide surgical envelope; 3) direct visualization of the dural sac, eliminating manipulation of the brainstem or upper spinal cord; 4) easy identification and control of the ipsilateral vertebral artery; 5) direct visualization and preservation of the lower cranial nerves; and 6) a sterile field. In addition, occipitocervical fusion and instrumentation can be performed during the same procedure. The transcondylar approach, based on anatomical studies in cadavers, was used to treat eight patients with ventral nonneoplastic lesions at the craniocervical junction. The technique and results are described. PMID- 8613816 TI - Pott's puffy tumor: the forgotten entity. Case report. AB - Pott's puffy tumor is a subperiosteal abscess of the frontal bone associated with underlying frontal osteomyelitis. The introduction of antibiotic medications has diminished the incidence of complications of frontal sinusitis. As a result, Pott's puffy tumor has become a rarity. In this communication a case of Pott's puffy tumor secondary to antecedent frontal sinusitis in an otherwise healthy adult man is described. PMID- 8613815 TI - Multilevel anterior thoracic discectomies and anterior interbody fusion using a microsurgical thoracoscopic approach. Case report. AB - A video-assisted thoracoscopic microsurgical approach was performed to treat a myelopathic patient with a severe kyphotic deformity caused by chronic nonunion of compression fractures of the T7-9 vertebrae. The kyphotic deformity was treated by combined operative procedures. First, an anterior release was performed using a thoracoscopic technique, sectioning the anterior longitudinal ligament and performing multilevel thoracic discectomies. Next, a posterior reduction and internal fixation of the deformity was achieved using hook-rod instrumentation. Finally, bone graft harvested during the posterior approach was used for interbody fusion via a thoracoscopic approach. Microsurgical thoracoscopic techniques potentially can be used in a variety of spinal surgeries. Compared to transthoracic and posterolateral approaches, this technique presents distinct advantages to treatment of anterior spinal pathology. The small incisions made into the intercostal spaces without retracting the ribs may reduce postoperative pain, shorten the length of hospitalization, and allow early return to activity. The operative techniques used in this case are described in detail. This report demonstrates that thoracoscopic discectomies and interbody fusion are technically feasible and can be effectively performed with acceptable results. PMID- 8613817 TI - Dural arteriovenous fistula of the posterior fossa developing after surgical occlusion of the sigmoid sinus. Report of five cases. AB - In this article, the authors present five cases of dural arteriovenous fistula (AVF) that developed in the transverse-sigmoid sinus 2 to 6 years after sacrifice of the sigmoid sinus because of tumor involvement. The original tumor was meningioma in two patients and neurinoma, glomus jugulare tumor, and ameloblastoma in one patient each. The involved sigmoid sinus was resected along with the tumor and ligated at the normal edge; all that remained of the sigmoid sinus was a small stump on the retrosigmoid portion. Serial angiography performed before and after tumor surgery revealed no abnormal arteriovenous communications or dural AVF in any of the cases. Although many reports have suggested that sinus thrombosis is a precipitating factor in the pathogenesis of dural AVFs, this has been difficult to verify because of the small number of cases in which serial angiography was performed before the development of a dural AVF. In all of the cases presented in this article, surgical resection of a dural AVF and histological examination were performed. Subintimal fibrous thickening was marked, and the sinus wall was found to contain many dural vessels. Organized thrombosis with neovascularization was seen in only two patients. These cases demonstrate that subintimal fibrous thickening a hypertrophied sinus wall secondary to increased intrasinus pressure or sinus thrombosis occurring after sinus occlusion can provoke the development of a dural AVF within the course of a lifetime. Maintenance of intrasinus blood flow may be very important to prevent this late postoperative complication. PMID- 8613819 TI - Use of bleomycin in intratumoral chemotherapy for cystic craniopharyngioma. Case report. AB - The authors present a case of a gigantic cystic craniopharyngioma that was treated with intratumoral injections of bleomycin. The mass had eroded the skull base and extended to the sphenoid bone. A total of eight intratumoral injections through an Ommaya reservoir were given. Six months after treatment, there was complete regression of the lesion and improvement in both visual and endocrinological symptomatology. PMID- 8613818 TI - A cervical dural arteriovenous fistula in a patient presenting with radiculopathy. Case report. AB - A 51-year-old man presenting with radiculopathy a rare cervical dural arteriovenous fistula (AVF) is reported. Angiography revealed that the cervical dural AVF was fed mainly by the left C-3 and C-4 radicular arteries and drained into the internal vertebral venous plexus with no communication with intradural structures. The dural AVF was treated surgically after embolization therapy. Although the AVF showed mass effect on computerized tomography (CT) scanning, abnormal vessels, which were suspected to drain the AVF, were observed intraoperatively to compress the left C-4 and C-5 nerve root sleeves. After resection of these abnormal epidural vessels, monoparesis of the left proximal upper extremity was markedly improved. In this patient, dynamic CT scanning was useful in the initial diagnosis, and the preoperative embolization therapy was very effective. PMID- 8613820 TI - A tentorial sling in microvascular decompression for trigeminal neuralgia. Technical note. AB - The key to the successful microvascular decompression of the trigeminal nerve is the maintenance of the separation between the trigeminal nerve and the offending vessel. The authors describe a technique whereby a dural sling is fashioned from the tentorium cerebelli (the anatomy of which is briefly reviewed), which is used to suspend the vessel away from the nerve and is secured by a single hemoclip. PMID- 8613821 TI - A hybrid clinical-research depth electrode for acute and chronic in vivo microelectrode recording of human brain neurons. Technical note. AB - For several decades, important scientific information has been gained from in vivo microelectrode recordings of individual human cerebral cortical neurons in patients with epilepsy. The experimental methods used, however, are technically complex and require a highly skilled intraoperative team. There are also significant experimental time limitations, as well as constraints on the type of behavioral tests conducted, and the brain regions that may be safely studied. In this report, a new method is described for obtaining in vivo microelectrode recordings using a hybrid depth electrode (HDE). High-impedance research recording contacts are interspersed between low-impedance clinical electroencephalographic (EEG) contacts along the HDE shaft. The HDE has the same external physical properties as a standard clinical depth electrode (DE). Following preclinical laboratory testing, 15 HDEs were used in the evaluation of six patients with medically refractory epilepsy. High-quality EEG recordings were obtained in all cases (two acute intraoperative, four from the chronic epilepsy monitoring unit). Action potentials from individual neurons were successfully recorded during all experimental sessions; however, the chronic preparations were clearly superior. Chronic HDEs are placed using a standard stereotactic system, and the locations of recording contacts are documented on a postimplantation imaging study. The quality of the chronic research recordings was excellent over study periods ranging from 5 to 14 days. The patients rested comfortably on the ward and were able to cooperate with complex experimental instructions. Basic neuroscientists participated fully in all aspects of the chronic investigations. The use of an HDE in place of a standard clinical DE may now allow detailed physiological investigations of any brain region targeted for clinical DE implantation. PMID- 8613822 TI - William Macewen and the treatment of brain abscesses: revisited after one hundred years. AB - The year 1993 marked the centennial of the publication of Sir William Macewen's monograph, Pyogenic Infective Diseases of the Brain and Spinal Cord, and its accompanying volume, Atlas of Head Sections. As Harvey Cushing noted, the text on pyogenic diseases of the brain was a landmark in surgery of the nervous system. At the time of its publication, Macewen's work was the most comprehensive study of pyogenic brain diseases. In this paper the author reviews the state of knowledge of brain abscess existing in the 19th and 20th centuries, with particular emphasis on the late 19th century, and elucidates factors contributing to Macewen's remarkable success. His thorough knowledge of the natural history of pyogenic diseases of the temporal bone and nasal sinuses, in addition to his clear description of cranial anatomy, as illustrated in his Atlas of Head Sections, were especially important in developing his successful treatment of brain abscess. The x-ray had not yet been discovered; Macewen's diagnosis was based on clinical findings superbly illustrated by his three clinical stages of brain abscess development. His clinical observations are as relevant today as when he described them 100 years ago. Macewen recorded 25 cases of brain abscess. Nineteen of these patients came to his attention in time to undergo surgery, resulting in 18 recoveries. All five of his patients with extradural abscess recovered. These results were achieved in the era known as "the most glorious period in British surgery." Neurosurgery was in its infancy; nevertheless, even as the 20th century closes, Macewen's results still have not been surpassed. PMID- 8613823 TI - Surgical treatment of epidermoid cysts of the cerebellopontine angle. AB - A total of 40 patients with epidermoid cysts of the cerebellopontine angle (CPA) underwent surgery between 1980 and 1993. Total resection was achieved in 30 cases (75%); in 10 cases (25%) parts of the cyst capsule were left because they adhered to the brainstem and vascular structures of the CPA. One patient with very large bilateral epidermoid cysts, who underwent complete bilateral resection in one stage, died of pulmonary aspiration and infection. As of their latest clinical and radiological follow-up examinations (mean 5.7 years), 93% of the patients are able to lead useful lives. Three cases of cyst regrowth have been observed thus far. Modern radiological tools and microsurgery techniques have considerably improved the completeness of cyst resection and reduced postoperative mortality and morbidity rates; however, there still are some cases in which complete resection is impossible without producing severe neurological deficits. PMID- 8613824 TI - Insulin reduction of cerebral infarction. PMID- 8613825 TI - Radiation necrosis or recurrence. PMID- 8613826 TI - Ischemic optic neuropathy. PMID- 8613827 TI - Decompression for spinal metastasis. PMID- 8613828 TI - Growth of meningiomas. PMID- 8613829 TI - Delayed hyponatremia. PMID- 8613830 TI - Spontaneous intracranial hypotension. PMID- 8613831 TI - Petroclival meningiomas: surgical experience in 109 cases. AB - The surgical removal of petroclival meningiomas has historically been associated with a high incidence of morbidity and mortality. The 109 consecutive patients included in the present retrospective study represent a combined series of tumors operated on by the four authors during a period from 1980 to 1992. The series is composed of 40 men and 69 women ranging in age from 25 to 75 years (mean 51 years). Surgical approaches to tumors in this series included simple retromastoid (60 cases), combined supra- and infratentorial petrosal (22), transtemporal (primary transsigmoid retrolabyrinthine, translabyrinthine, or transcochlear (12), subtemporal (11), and frontotemporal transcavernous (eight). Gross-total removal was achieved in 75 patients (69%). Recurrence or progression of disease occurred in 14 patients (13%) over a 6.1-year mean follow-up period, and it was found within the cavernous sinus in 12 of these cases. Four recurrent cases demonstrated histological compatibility with malignant meningioma. Perioperative death occurred in four patients, and there were 56 significant complications in 35 other patients. Review of this series, with the attendant complications, has facilitated the authors' decision-making when considering the risk of gross-total removal in selected patients with asymptomatic cavernous sinus invasion or tumor adherent to the brainstem. PMID- 8613832 TI - Psychosocial functioning and quality of life in patients with primary brain tumors. AB - Perceived quality of life (QOL) was evaluated in a group of 50 patients with primary brain tumors. Participants completed two QOL measures and a demographic profile. Age was found not to be an important factor in differentiating QOL in these patients. All areas of QOL were found to be affected adversely by one or more of five factors: being female, being divorced, having bilateral tumor involvement, having received chemotherapy, and having a poor performance status. Pearson product-moment correlations conducted on the subscale and total scores of the two QOL instruments revealed that the two scales measure different QOL aspects and overlap in only a few areas. This may be related to the different measurement paradigms that these two instruments represent. This study is one of the first to evaluate the multidimensional aspects of QOL in patients with primary brain tumors, an understudied group. A prospective study of QOL in this group, already underway at the authors' institution, is needed to evaluate comprehensively the effect of different treatments and interventions on the QOL functioning of primary brain tumor patients. Additionally, this study shows that the choice of QOL instruments is very important and needs to be driven by the research question. PMID- 8613833 TI - Ultra-early rebleeding in spontaneous subarachnoid hemorrhage. AB - To determine the incidence of, and risk factors for, the occurrence of rebleeding between admission and early operation (ultra-early rebleeding) in patients with spontaneous subarachnoid hemorrhage (SAH), the authors reviewed the cases of 179 patients admitted within 24 hours after their last attack of SAH. Thirty-one (17.3%) of these patients had ultra-early rebleeding despite scheduling of early operation (within 24 hours after admission). The incidence of rebleeding significantly decreased as the time interval between the last attack and admission increased. Patients with rebleeding before admission, high systolic blood pressure, intracerebral or intraventricular hematoma, those in poor neurological condition on admission, and those who underwent angiography within 6 hours of the last SAH were significantly more likely to have ultra-early rebleeding than those without these factors. The incidence of rebleeding also significantly increased as levels of enhancement of platelet sensitivity and thrombin-antithrombin complex increased. Multivariate analysis revealed that the following three factors were independently associated with ultra-early rebleeding: the level of enhancement of platelet sensitivity; the time interval between the last attack and admission; and the level of thrombin-antithrombin complex. On the basis of these findings, the authors suggest that many of the risk factors for ultra-early rebleeding are interrelated. A particularly high risk of ultra-early rebleeding was observed in those patients 1) who had platelet hypoaggregability; 2) who were admitted shortly after their last SAH; and 3) whose thrombin-antithrombin complex levels were extremely high and were thus in severe clinical condition. PMID- 8613834 TI - Gender-related differences in aneurysmal subarachnoid hemorrhage. AB - Female gender is a recognized risk factor for the occurrence of aneurysmal subarachnoid hemorrhage. In the present study the authors analyzed differences in admission characteristics and outcome between 578 women (64%) and 328 men (36%) who were enrolled in a recently completed clinical trial. The female-to-male ratio was nearly 2:1. The women in the study were older than the men (mean age 51.4 years vs 47.3 years, respectively, p<0.001). Female patients harbored aneurysms of the internal carotid artery more frequently than male patients (36.8% vs. 18.0%, p<0.001) and more often had multiple aneurysms (32.4% vs. 17.6%, p<0.001). On the other hand, anterior cerebral artery aneurysms were more commonly encountered in men (46.1% in men vs. 26.6% in women, p<0.001). Other baseline prognostic factors were balanced between the gender groups. Surgery was performed equally in both sexes (98%), although the time to operation was shorter for women (mean 3.6 days for women vs. 5.3 days for men, p = 0.0002). In the placebo group, the occurrence of vasospasm was not statistically different between the two groups. Primary causes of death and disability were the same, and favorable outcome rates at 3 months were not statistically different between the genders (69.7% for women vs. 73.4% for men, p = 0.243). The odds of a favorable outcome in women versus one in men were not statistically significant either before of after adjustment for age. These observations lead the authors to suggest that although women are older and harbor more aneurysms, the 3-month outcome for women and men who experience aneurysmal subarachnoid hemorrhage is the same. PMID- 8613835 TI - Predicting outcome following surgical treatment of unruptured intracranial aneurysms: a proposed grading system. AB - Surgical treatment of unruptured aneurysms is gaining increased support owing to the recently defined poor long-term natural history of these aneurysms. The benefit of treatment ultimately depends on the relative risk of subsequent aneurysm rupture in untreated patients versus the risk of surgery. To identify those patients at a higher risk from surgery, the authors reviewed the management of 172 patients with unruptured intracranial aneurysms treated at their institution. The size of the aneurysms ranged from 3 to 45 mm (mean 13.7 mm). Twenty-two patients (12.8%) had aneurysms in the posterior circulation, and 32 (18.6%) of these were giant aneurysms. Major morbidity occurred in 12 patients (6.9%) and five patients (2.9%) died. Multivariate logistic analysis of several risk factors revealed that aneurysm size and location had an independent correlation with surgical outcome and that patient age approached statistical significance. Patients presenting with ischemic cerebrovascular disease, in particular, did not have a higher risk of a poor outcome. A simple classification for predicting patients at high risk from surgical morbidity and mortality is proposed. Preoperative grading is based on the size and location of the aneurysm and patient's age. The lowest grade is given to young patients with small anterior circulation aneurysms, and the highest grade includes elderly patients with complex giant posterior circulation aneurysms. A retrospective analysis of this classification demonstrated a strong correlation with postoperative outcome. The incidence of poor outcome progressively increased with a higher grade, ranging from 0% in Grade 0 to 66.6% in Grade VI. An analysis of this classification on 50 consecutive surgically treated patients with unruptured aneurysms not included in the analysis also validated the predictive value of this system. Along with predicting outcome, this classification should provide a standardized format for comparison of results from different clinical centers as well as different therapeutic techniques (surgical vs. endovascular) without omission of significant risk factors found to influence outcome. PMID- 8613836 TI - Treatment of large and giant fusiform intracranial aneurysms with Guglielmi detachable coils. AB - Results in nine patients with large or giant fusiform intracranial aneurysms that were treated with Guglielmi detachable coils (GDCs) are reported. There were six males and three females between the ages of 12 and 63. Four patients presented with subarachnoid hemorrhage (SAH) and four with mass effect; in one patient the aneurysm was asymptomatic and located in an arterial feeder of an arteriovenous malformation. Five aneurysms were supratentorial and four were in the posterior fossa. Five were giant and four were large. Selective occlusion with preservation of the parent artery was attempted in three cases, and complete occlusion of the aneurysm and the parent artery was performed in six patients. The tolerance to parent artery occlusion was assessed by angiography, balloon test occlusion, and amytal testing. Six aneurysms were permanently occluded and two partially recanalized. In one case, GDC embolization was not possible. The four patients who presented with SAH made an excellent clinical recovery. Three of the four patients presenting with mass effect recovered completely and one remained unchanged. The patient with an incidental aneurysm remained asymptomatic. There were no permanent complications. In conclusion, GDCs were useful for the occlusion of large and giant intradural fusiform aneurysms. Occlusion of the aneurysm and the parent artery afforded the greatest opportunity for a complete cure. Advantages of GDCs compared to balloons include: occlusion of a shorter segment of normal artery, no traction on the parent vessel, and safer and easier catheterization techniques. PMID- 8613837 TI - Intraoperative endovascular surgery for cerebral aneurysms. AB - The application of a number of procedures that can be considered intraoperative endovascular neurosurgery has enhanced our ability to treat cerebral aneurysms from the abluminal surface. This study identifies a role for these techniques in the management of difficult aneurysms. A review of the last 1202 aneurysms undergoing direct clipping by the authors disclosed that these methods were used in 62 cases. Of these aneurysms, 36 arose from the internal carotid artery, 12 from the middle cerebral artery, eight from the vertebrobasilar distribution, and six from the anterior cerebral artery. The indications for applying these methods were large size (12-60 mm), intraluminal thrombus, broad neck, plaque at the neck, the potential compromise of branches at the base of the aneurysm, or a combination of these problems. The most frequently chosen intraoperative technique was suction decompression with direct removal of plaque and thrombus using suction, dissection, and/or ultrasonic aspiration. The application of temporary clips was required in all cases in which the aneurysm was opened before definitive clipping. No special pharmacological cerebral protective regimen was used. In one case in which a greater occlusion time was anticipated, cardiopulmonary bypass with profound hypothermia was performed. A favorable outcome was achieved in 73% of these difficult cases. An increased neurological deficit after surgery was seen in 11%, and the mortality rate was 8%. These methods should be considered and can be anticipated before surgery for unusual aneurysms. Many cases now being considered for embolization may be more suitable for definitive surgical obliteration. PMID- 8613838 TI - Presurgical identification of the primary sensorimotor cortex by functional magnetic resonance imaging. AB - The ability of functional magnetic resonance (MR) imaging to detect a selective sensorimotor cortex activation in healthy subjects and the feasibility of motor activation in patients with lesions around the central sulcus were investigated. Twenty-five healthy volunteers performed 100 motor activation trials, using a variety of motor tasks, which were monitored by several image analysis methods. The functional images were obtained using a 1.5-tesla standard MR imaging system magnet with blood oxygenation level-dependent contrast. Four patients were assessed using functional MR imaging and invasive cortical mapping. Rolandic cortex activation was observed in 98% of the trials performed on healthy subjects in which no head motion occurred. Nevertheless, the cortical response was not selective in a task-rest analysis due to concurrent activation of neighboring regions. Across-task comparison analyses were useful in cancelling nonrelevant activity in most cases (86%). In the patient group, the region identified as the sensorimotor cortex by invasive means corresponded accurately to the area that was activated in functional MR imaging. Present data support the feasibility of detecting selective activation of the rolandic cortex, even in the clinical setting, leading the authors to suggest the usefulness of this widely available technique in surgical planning. PMID- 8613839 TI - Nitric oxide mediation of chemoregulation but not autoregulation of cerebral blood flow in primates. AB - The authors sought to develop a model for assessing in vivo regulation of cerebral vasoregulation by nitric oxide (NO), originally described as endothelial derived relaxing factor, and to use this model to establish the role of NO in the regulation of cerebral blood flow (CBF) in primates. By using regional intraarterial perfusion, the function of NO in cerebral vasoregulation was examined without producing confounding systemic physiological effects. Issues examined were: whether resting vasomotor tone requires NO; whether NO mediates vasodilation during chemoregulation and autoregulation of CBF; and whether there is a relationship between the degree of hypercapnia and hypotension and NO production. Twelve anesthetized (0.5% isoflurane) cynomolgus monkeys were monitored continuously for cortical CBF, PaCO2, and mean arterial pressure (MAP), which were systematically altered to provide control and experimental curves of chemoregulation (CBF vs. PaCO2) and autoregulation (CBF vs. MAP) during continuous intracarotid infusion of 1) saline and 2) an NO synthase inhibitor (NOSI), either L-n-monomethyl arginine or nitro L-arginine. During basal conditions (PaCO2 of 38-42 mm Hg) NOSI infusion of internal carotid artery (ICA) reduced cortical CBF from 62 (saline) to 53 ml/100 g/per minute (p<0.01), although there was no effect on MAP. Increased CBF in response to hypercapnia was completely blocked by ICA NOSI. The difference in regional (r)CBF between ICA saline and NOSI infusion increased linearly with PaCO2 when PaCO2 was greater than 40 mm Hg, indicating a graded relationship of NO production, increasing PaCO2, and increasing CBF. Diminution of CBF with NOSI infusion was reversed by simultaneous ICA infusion of L-arginine, indicating a direct role of NO synthesis in the chemoregulation of CBF. Hypotension and hypertension were induced with trimethaphan camsylate (Arfonad) and phenylephrine at constant PaCO2 (40 +/- 1 mm Hg). Autoregulation in response to changes in MAP from 50 to 140 mm Hg was unaffected by ICA infusion of NOSI. In primates, cerebral vascular tone is modulated in vivo by NO; continuous release of NO is necessary to maintain homeostatic cerebral vasodilation; vasodilation during chemoregulation of CBF is mediated directly by NO production; autoregulatory vasodilation with hypertension is not mediated by NO; and increasing PaCO2 induces increased NO production. PMID- 8613840 TI - Relationship between transcranial Doppler-determined pulsatility index and cerebrovascular resistance: an experimental study. AB - Clinical studies with transcranial Doppler suggest that the pulsatility of the flow velocity (FV) waveform increases when the distal cerebrovascular resistance (CVR) increases. To clarify this relationship, the authors studied animal models in which the resistance may be decreased in a controlled manner by an increase in arterial CO2 tension, or by a decrease in cerebral perfusion pressure (CPP) in autoregulating animals. Twelve New Zealand white rabbits were anesthetized, paralyzed, and ventilated. Transcranial Doppler basilar artery FV, laser Doppler cortical blood flow, arterial pressure, intracranial pressure, and end-tidal CO2 concentration were measured continuously. Cerebrovascular resistance (CPP divided by laser Doppler cortical flux) and Gosling Pulsatility Index (PI, defined as an FV pulse amplitude divided by a timed average FV) were calculated as time dependent variables for each animal. Four groups of animals undergoing controlled manipulations of CVR were analyzed. In Group I, arterial CO2 concentration was changed gradually from hypocapnia to hypercapnia. In Group II, gradual hemorrhagic hypotension was used to reduce CPP. In Group III, the short-acting ganglion blocking drug trimetaphan was injected intravenously to induce transient hypotension. Intracranial hypertension was produced by subarachnoid saline infusion in Group IV. During the hypercapnic challenge the correlation between the cortical resistance and Doppler flow pulsatility was positive (r = 0.77, p<0.001). In all three groups in which cerebral perfusion pressure was reduced a negative correlation between pulsatility index and cerebrovascular resistance was found (r = -0.84, p<0.001). The authors conclude that PI cannot be interpreted simply as an index of CVR in all circumstances. PMID- 8613841 TI - Significance of proliferating cell nuclear antigen in predicting recurrence of intracranial meningioma. AB - It is well known that the histological appearance of meningiomas often fails to predict accurately the clinical behavior of the tumor. Therefore, attention has turned from tumor histology to tumor biology. Proliferating cell nuclear antigen (PCNA), a cell cycle-regulated protein, has been recently characterized as the cofactor of DNA polymerase-delta, an enzyme required for DNA replication. The rate of synthesis of PCNA directly correlates with the proliferative state of cells. Immunohistochemical labeling of this antigen is now possible with monoclonal antibodies that allow for its demonstration in routinely fixed, paraffin-embedded specimens. In this study, the PCNA labeling index (LI) was determined for 83 meningiomas, including tumors with both benign and malignant clinical courses and with benign, atypical, and malignant histologies, apparent after total or subtotal resections. No statistical difference was found between the LI on recurrence and that found at initial presentation. In addition, stepwise multivariate regression analysis failed to identify any combination of factors (age, gender, race, age of specimen, tumor histology, Simpson grade of resection) that contributes to the predictive strength of the PCNA LI for tumor recurrence. However, for LIs less than 2%, only one of 26 gross totally resected tumors recurred (mean follow up 53 months); for LIs more than 7%, five of 13 gross totally resected tumors recurred (mean follow up 55 months). The difference in recurrence rates between gross totally resected meningiomas with PCNA LIs less than 2% and those with PCNA LIs more than 7% achieved statistical significance with a Fisher's exact probability equaling 0.011. The authors conclude that quantitative PCNA labeling of meningiomas is a promising technique that can provide meaningful prognostic information. PMID- 8613843 TI - Characterization of edema by diffusion-weighted imaging in experimental traumatic brain injury. AB - The objective of this study was to use diffusion-weighted magnetic resonance imaging (DWI) to help detect the type of edema that develops after experimental trauma and trauma coupled with hypotension and hypoxia (THH). Reduction in the apparent diffusion coefficients (ADCs) is thought to represent cytotoxic edema. In a preliminary series of experiments, the infusion edema model and middle cerebral artery occlusion models were used to confirm the direction of ADC change in response to purely extracellular and cytotoxic edema, respectively. The ADCs increased (p<0.05) in the case of extracellular edema and decreased (p<0.001) in cytotoxic edema. Following these initial experiments, a new impact acceleration model was used to induce traumatic brain injury. Thirty-six adult Sprague-Dawley rats were separated into four groups; sham, trauma alone, hypoxia and hypotension (HH), and THH. Following trauma, a 30-minute insult of hypoxia (PaO2 of 40 mm Hg) and hypotension (mean arterial blood pressure (MABP) of 30 mm Hg) were imposed and the animals were resuscitated. The DWI was carried out at four 1-hour intervals postinjury, and MABP, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and cerebral blood flow (CBF) were monitored. The ADCs in the control and HH groups remained unchanged. The ADCs in the THH group rapidly decreased from a control level of 0.68 +/- 0.05 x 10(-3) mm2/second to 0.37 +/- 0.09 x 10(-3) mm2/second by 3 hours posttrauma (p < 0.001). In this group, the decreased CBF and CPP during secondary insult remained low despite resuscitation, with the ICP increasing to 56 +/- 7 mm Hg by 3 hours. In the trauma alone group, the rise in ICP reached a maximum value (28 +/- 3 mm Hg) at 30 minutes with a significant and sustained increase in CBF despite a gradual decrease in CPP. The ADCs in this group were not significantly reduced. The data lead the authors to suggest that the rise in ICP following severe trauma coupled with secondary insult in this model is predominately caused by cytotoxic edema and that ischemia plays a major role in the development of brain edema after head injury. PMID- 8613842 TI - Edema from intracerebral hemorrhage: the role of thrombin. AB - The mechanism by which intracerebral hemorrhage leads to the formation of brain edema is unknown. This study assesses the components of blood to determine if any are toxic to surrounding brain. Various solutions were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; brain edema and ion contents were measured. Whole blood caused an increase in brain water content and ion changes consistent with brain edema. Concentrated blood cells, serum from clotted blood, and plasma from unclotted blood all failed to provoke edema formation when infused directly into the brain. On the other hand, activation of the coagulation cascade by adding prothrombinase to plasma did produce brain edema. The edema response to whole blood could be prevented by adding a specific thrombin inhibitor, hirudin, to the injected blood. This study indicates that thrombin plays an important role in edema formation from an intracerebral blood clot. PMID- 8613844 TI - Management strategies for tumors of the axis vertebra. AB - Primary and metastatic tumors of the axis vertebra pose difficult surgical problems. The authors report 14 cases of patients with axis tumors who underwent surgery between 1970 and 1993. The eight male and six female patients ranged in age from 9 to 70 years (mean 46 years). Presenting symptoms and signs included pain in 12, myelopathy in nine, radiculopathy in four, and cranial nerve deficits in two patients. Preoperative instability of the cervical spine was present in eight patients. The goal of surgery was to achieve diagnosis, decompression, and stabilization; the choice of operative intervention was based on tumor location and patient prognosis. Six transoral-transpalatal resections, two lateral extrapharyngeal approaches, four posterolateral decompressions, and 11 dorsal decompressions were performed. Eleven fusion procedures were required to achieve spinal stability. Patients with aggressive tumors requiring concomitant radiotherapy underwent fusion using contoured loop instrumentation or methyl methacrylate, whereas those with benign lesions underwent fusion using traditional techniques. With maximum medical and surgical intervention, 10 patients are alive and their tumors are in remission. In eight of these 10 patients, gross-total resection was achieved. Four patients died from tumor progression, and in three of these cases gross-total resection was not possible. There were no operative deaths and morbidity was minimal. Development of new surgical approaches to the axis has allowed resection of previously inoperable tumors. The extent of resection correlated with the duration of survival. A classification system for axis tumors is proposed to facilitate selection of appropriate surgical approaches that will maximize the extent of resection while achieving or maintaining spinal stability. PMID- 8613845 TI - Posterior instrumentation of the unstable cervicothoracic spine. AB - Fractures, tumors, and other causes of instability at the cervicothoracic junction pose diagnostic and treatment challenges. The authors report on 23 patients with instability of the cervicothoracic region, which was treated with posterior plate fixation and fusion between the lower cervical and upper thoracic spine. During operation AO reconstruction plates with 8- or 12-mm hole spacing were affixed to the spine using screws in the cervical lateral masses and the thoracic pedicles. Postoperative immobilization consisted of the patient's wearing a simple external brace for 2 months. The following parameters were analyzed during the pre- and postoperative treatment period: neurological status, spine anatomy and reconstruction, and complications. Follow up consisted of clinical and radiographic examinations (mean duration of follow up, 15.4 months; range, 6-41 months). No neurovascular or pulmonary complications arose from surgery. All patients achieved a solid arthrodesis based on flexion-extension radiographs. There was no significant change in angulation during the postoperative period, but one patient had an increase in translation that was not clinically significant. There were no hardware complications that required reoperation. One patient requested hardware removal in hopes of reducing postoperative pain in the cervicothoracic region. One postoperative wound infection required debridement but not hardware removal. The authors conclude that posterior plate fixation is a satisfactory method of treatment of cervicothoracic instability. PMID- 8613846 TI - Posterior occipitoaxial fusion for atlantoaxial dislocation associated with occipitalized atlas. AB - Between 1989 and 1994, 50 patients suffering from congenital atlantoaxial dislocation with either an assimilated atlas or a thin or deficient posterior arch of the atlas were treated with occipitocervical fusion using the technique described by Jain and colleagues in 1993 with a few modifications. An artificial bridge created from the occipital bone along the margin of the foramen magnum was fused to the axis using sublaminar wiring and interposed strut and lateral onlay bone grafts. Ten patients (20%) also underwent atlantoaxial lateral joint fusion by intraarticular instillation of bone chips. In 22 patients (44%) with irreducible dislocation, posterior fusion was preceded by transoral odontoidectomy. In seven patients (14%) with ventral compression, who showed marked clinical improvement on traction despite radiological evidence of persisting atlantoaxial dislocation, occipitocervical fusion was performed without ventral decompression. Seven patients (14%) underwent a single-stage transoral odontoidectomy and posterior fusion. There was no perioperative mortality and the osseous fusion rate was 88%. Of the 43 patients available at follow-up examination (range 3-12 months), 31 patients (72.09%) improved, seven (16.28%) remained the same, and five (11.6%) deteriorated in comparison with their preoperative status. Hence, this technique achieves a stable occipitocervical arthrodesis without supplemental external orthoses and facilitates early postoperative mobilization. PMID- 8613847 TI - Anterior decompression and stabilization using a microsurgical endoscopic technique for metastatic tumors of the thoracic spine. AB - It is well accepted that the treatment of spinal tumors that threaten neurological integrity comprises resection, vertebral body reconstruction, and stabilization if the patient's condition is suitable. In spite of the excellent results reported using thoracotomy, the majority of investigators recommend posterolateral techniques because of lower morbidity, shorter hospitalization time, and the possibility of performing dorsal stabilization via the same incision. To overcome some of the disadvantages of thoracotomy, the authors developed an anterior procedure that permits vertebrectomy, reconstruction, and stabilization to be performed entirely by endoscopic technique. Microsurgical endoscopy and stabilization were performed in four patients with metastatic disease of the thoracic spine. All were ambulatory after surgery and at follow up; preoperative neurological and neurophysiological deficits improved as well. No complications occurred in this small series. Microsurgical endoscopy achieves a substantial reduction in trauma, use of analgesic medications, and hospitalization time. Early results seem to indicate that adequate decompression, stabilization and reduction of surgical morbidity can be achieved with this technique. PMID- 8613848 TI - Pediatric transoral surgery: indications, complications, and long-term outcome. AB - Knowledge of the role and hazards of transoral surgery has expanded rapidly, but the application of this technique in children has been limited. To assess its usefulness, 27 pediatric patients who underwent transoral surgery between 1985 and 1994 were studied. Transoral surgery was performed for irreducible anterior neuraxial compression at the craniovertebral junction caused by basilar impression, atlantoaxial subluxation with pseudotumor, or chordoma. The patients ranged in age from 3 to 17 years. Symptomatic presentation varied widely, but 89% had significant neurological deficits before surgery. No patient with normal strength deteriorated after surgery. Of the 16 patients with a preoperative motor deficit, nine improved rapidly, three were unchanged, and four significantly worsened in the perioperative period. Those with mobile atlantoaxial subluxation were most vulnerable to surgically related neurological morbidity. Twenty-four patients were alive for long-term follow-up study (average 5.7 years, range 1-9.2 years). Of those with preoperative weakness, nine improved one Frankel grade, four remained the same, and one deteriorated from Frankel Grade D to C. Swallowing and speech worsened in five patients; this occurred only after resection of lesions above the foramen magnum (p<0.05) when rostral pharyngeal disruption resulted in velopharyngeal dysfunction. This study, unlike previous reviews of pediatric transoral operations, leads the authors to suggest that although transoral surgery can be effective, it also carries a significant risk of neurological injury in patients with symptomatic spinal cord compression and it is also associated with long-term swallowing and speech difficulties. PMID- 8613849 TI - Cranial chordomas in children and adolescents. AB - Chordomas are rare tumors that usually occur in adults. This report describes four cases of intracranial chordomas treated in patients 20 years of age or younger by the senior author (O.A.M.) during a 4-year period. The authors also reviewed the literature on pediatric patients, which revealed that the clinical presentations, histological patterns, and behaviors of these tumors differ considerably depending on whether the patient is younger or older than 5 years of age. The younger population had a wider range of presenting symptoms, a greater prevalence of atypical histological findings with aggressive behavior, a greater range of cellularity than the classic chordomas, and a higher instance of metastasis; it showed no chondroid component compared to a 17.1% instance in the older patients. The prognosis for patients with a chordoma is related directly to the histological pattern of the tumor; the atypical chordoma carries a poor prognosis. The prognoses for children older than 5 years of age with a classic or chondroid tumor were not significantly different (p=0.788). At follow up, the difference in survival rates between patients undergoing surgery plus radiation therapy and those who had surgery alone was statistically significant (p=0.00446). No correlation was found between radical resection or radiation therapy and an improved prognosis for patients younger than 5 years of age who had a tumor with an atypical histological pattern. This study identifies and delineates the distinction between these age groups and provides a review of the potential prognostic factors. PMID- 8613850 TI - Pigmented villonodular synovitis of the spine: a clinical, radiological, and morphological study of 12 cases. AB - Cases of pigmented villonodular synovitis (PVNS) that affect the axial skeleton are rare and thus information regarding its natural history, treatment, and prognosis remains limited. To characterize this lesion more fully, the authors reviewed their experience with 12 cases of PVNS of the spine (one of which had been previously reported), then reviewed the 11 cases that previously had been reported, and obtained additional follow-up data in six of them. On the basis of the cumulative data provided by these 22 cases, PVNS of the spine appears to occur over a wide range of ages (21-81 years) and, contrary to what was previously suggested, does not show definite gender predilection (12 women and 10 men). The lesion affects the posterior elements of the vertebrae at all levels and involves the facet joints in 89% of cases. Extension into the epidural spine is frequent (70%). Surgical resection appears to be the treatment of choice. Although PVNS of the spine tends to recur locally (18%), repeat surgical excision appears to be curative. PMID- 8613851 TI - Spontaneous spinal cerebrospinal fluid leaks and intracranial hypotension. AB - Spinal cerebrospinal fluid (CSF) leaks are often implicated as the cause of the syndrome of spontaneous intracranial hypotension, but they have rarely been demonstrated radiographically or surgically. The authors reviewed their experience with documented cases of spinal CSF leaks of spontaneous onset in 11 patients including their surgical observations in four of the patients. The mean age of the six women and five men included in the study was 38 years (range 22-51 years). All patients presented with a postural headache; however, most had additional symptoms, including nausea, emesis, sixth cranial-nerve paresis, or local back pain at the level of the CSF leak. All patients underwent indium-111 radionucleotide cisternography or computerized tomographic (CT) myelography. The location of the spontaneous CSF leak was in the cervical spine in two patients, the cervicothoracic junction in three patients, the thoracic spine in five patients, and the lumbar spine in one patient. The false negative rate for radionucleotide cisternography was high (30%). Subdural fluid collections, meningeal enhancement, and downward displacement of the cerebellum, resembling a Chiari I malformation, were commonly found on cranial imaging studies. In most patients, the symptoms resolved in response to supportive measures or an epidural blood patch. Leaking meningeal diverticula were found to be the cause of the CSF leak in four patients who underwent surgery. In three patients these diverticula could be ligated with good result but in one patient an extensive complex of meningeal diverticula was found to be inoperable. Two patients had an unusual body habitus and joint hypermobility, and two other patients had suffered a spontaneous retinal detachment at a young age. In conclusion, spontaneous spinal CSF leaks are uncommon, but they are increasingly recognized as a cause of spontaneous intracranial hypotension. Most spinal CSF leaks are located at the cervicothoracic junction or in the thoracic spine, and they may be associated with meningeal diverticula. The radiographic study of choice is CT myelography. The disease is usually self-limiting, but in selected cases our experience with surgical ligation of leaking meningeal diverticula has been satisfactory. An underlying connective tissue disorder may be present in some patients with a spontaneous spinal CSF leak. PMID- 8613852 TI - Neurochemical monitoring using intracerebral microdialysis in patients with subarachnoid hemorrhage. AB - The authors have developed a method for routine monitoring of disturbances in brain energy metabolism and extracellular levels of excitatory amino acids using intracerebral microdialysis in 10 patients with subarachnoid hemorrhage. Microdialysis was conducted for periods ranging from 6 to 11 days after ictus. Altogether, 16,054 chemical analyses from 1647 dialysate samples were performed. Concentrations of the energy-related substances lactate, pyruvate, glucose, and hypoxanthine were measured, and the lactate/pyruvate ratio was calculated. The excitatory amino acids glutamate and aspartate were measured. The microdialysis data were matched with computerized tomography findings, clinical course, and outcome. The results support the concepts that microdialysis is a promising tool for chemical monitoring of the human brain and that extracellular fluid levels of lactate, lactate/pyruvate ratio, glucose, hypoxanthine, and glutamate are useful markers of disturbances in brain energy metabolism in neurointensive care patients. These results have generated a working hypothesis that the pattern of these extracellular markers may help differentiate between various causes of energy perturbations, such as hypoxia and different degrees of ischemia. The correlation between the dialysate levels of excitatory amino acids and outcome supports the concept of glutamate receptor overactivation in acute human brain injury. PMID- 8613853 TI - The significance of bacteriologically positive ventriculoperitoneal shunt components in the absence of other signs of shunt infection. AB - The purpose of this study was to determine the significance of "asymptomatic bacteriological shunt contamination" (ABSC), defined as a positive bacteriological culture found on a ventricular shunt component in the absence of bacteria in the cerebrospinal fluid (CSF) culture and/or clinical evidence of infection. Of 174 ventriculoperitoneal shunt revisions, 19 cases of ABSC were identified and reviewed retrospectively. In all but one case, no antibiotic medications were instituted because of the positive bacteriological culture. The most common infecting organisms were coagulase-negative staphylococci (seven) and propionibacteria (eight). A comparison of the 19 study cases with the authors' overall shunt experience, as documented in the British Columbia's Children's Hospital shunt database for the time period of the study, lead the authors to suggest that ABSC was not of significance in causing the shunt failure at which contamination was identified and, more importantly, did not increase the risk of future shunt malfunction. The results of this study indicate that in the absence of clinical evidence of shunt infection or a positive bacteriological culture from CSF, bacteria in a shunt component removed at revision in a child almost always represents a contaminant that may be ignored. Therefore, the authors advise that routine culture of shunt components removed at revision of a shunt is not indicated. PMID- 8613854 TI - Comparison of syringopleural and syringosubarachnoid shunting in the treatment of syringomyelia in children. AB - Case records from the Montreal Children's Hospital containing the diagnosis of shunted syringomyelia were retrospectively reviewed. From 1984 to 1994, 31 patients had their syrinx treated by either syringopleural (19 cases, Group A) or syringosubarachnoid (13 cases, Group B) shunting. One patient was included in both groups. Associated diagnoses included: in Group A, two cases of Chiari I and 14 of Chiari II malformations, 14 cases of shunted hydrocephalus, 13 cases of spina bifida aperta, and three cases of spina bifida occulta; Group B, four cases of Chiari I and two of Chiari II malformations, four cases of shunted hydrocephalus, two cases of spina bifida aperta, and five cases of spina bifida occulta. Eight Group A and six Group B patients had undergone prior posterior fossa decompression. Motor deficits predominated in both groups and arachnoiditis was a uniform operative finding. Neurological follow-up examinations showed 11 Group A patients improved and eight stabilized, whereas on magnetic resonance imaging, 12 cavities appeared to have collapsed, five were markedly reduced, and one had increased. One patient underwent reoperation for pleural effusions and one for shunt displacement. In Group B, one patient improved, eight stabilized, three worsened neurologically, and one was lost to follow-up review. Radiologically, one cavity appeared to have collapsed, six were significantly reduced, two were unchanged, and three had enlarged. The authors conclude that syringopleural shunting is a valuable option for controlling syringomyelia in patients without Chiari malformation or in patients who have previously undergone a craniovertebral decompression or are otherwise asymptomatic from their Chiari malformation. PMID- 8613855 TI - Effects of prednisone on ventriculoperitoneal shunt function in hydrocephalus secondary to cysticercosis: a preliminary study. AB - In a prospective open study, 15 patients with hydrocephalus secondary to cysticercosis who required insertion of a ventriculoperitoneal (VP) shunt were treated with 50 mg of prednisone given orally three times a week. Treatment began in the 1st postoperative week, with isoniazid and pyridoxine administered daily as antituberculous chemoprophylaxis. The drug regimen was continued with close follow up for 24 months. Clinical status, Karnofsky performance status (KPS) scores, and postoperative course in the prednisone-treated group were compared with 30 control patients with hydrocephalus due to cysticercosis. The control patients were matched by age and sex, underwent surgical shunting in the same period, and were followed routinely by the neurosurgery staff. Lumbar cerebrospinal fluid (CSF) was studied in 2, 16, and 32 weeks postoperatively in the prednisone group. At 24-month follow up two (13%) of 15 patients in the prednisone group and 19 (60%) of 30 patients in the control group required surgical shunt revisions for symptomatic shunt obstruction (p=0.002). Follow-up studies of CSF performed at 32 weeks in the prednisone group revealed improvement of abnormal values with statistically significant differences for glucose (p<0.02). Serial imaging studies in the prednisone group revealed persistence of cysticercal cysts with no change in size. Mean initial KPS scores were similar in both groups. At the end of the follow-up period, the mean KPS score was significantly higher in the prednisone group (p=0.003). Prednisone and chemoprophylactic drugs were well tolerated. These results suggest that in selected patients with hydrocephalus secondary to cysticercosis, intermittent long-term prednisone therapy after VP shunting may reduce shunt malfunction and improve the functional status of the patients. PMID- 8613856 TI - Risks in using siphon-reducing devices in adult patients with normal-pressure hydrocephalus: bench test investigations with Delta valves. AB - To elucidate the reason for malfunction of Delta valves in patients with normal pressure hydrocephalus, the authors applied a new concept of the bench test and simulated intracranial pressure (ICP) to measure the resultant flow with a complete shunt system. Subcutaneous pressure on the valve, valve implantation site, and postural hydrostatic differential-pressure changes were simulated in this bench test designed for adult patients with normal-pressure hydrocephalus. Subcutaneous pressure ranged within 6.4 +/- 0.09 cm H2O (mean +/- standard deviation) on the 7th day after the implantation of the valve in rats. A linear correlation between valve closing pressures and the external pressure was observed: gradual increase of the latter resulted in an approximately equal rise in the closing pressure. Closing pressure ranged within the physiological variation of ICP (10 +/- 5 cm H2O) in the supine position. In the erect position it was higher than the physiological variation (+5 to -5 cm H2O) when the valve was placed at the level of the foramen of Monro and lower when placed at the level of the clavicle. This observation indicates that the subcutaneous pressure around the Delta valve significantly affects its closing pressure. An excessive reduction of flow or a functional obstruction occurs when the patient with a Delta valve implanted at the foramen of Monro assumes an erect posture. The authors conclude that Delta valves are not recommended for implantation at the level of the foramen of Monro in patients with normal-pressure hydrocephalus because of the risk of underdrainage. The mastoid process or clavicle may be the alternative levels for its implantation to prevent underdrainage; however, the possibility of shunt malfunction should be kept in mind because subcutaneous pressure is variable among patients and that might affect the performance characteristics of these valves. PMID- 8613857 TI - Brain tissue pressure gradients created by expanding frontal epidural mass lesion. AB - A porcine model was used to study the regional intracranial pressure (ICP) differences caused by a frontal mass lesion. Intraparenchymal ICP monitors were placed in the right and left frontal lobes, right and left temporal lobes, midbrain, and cerebellum. A frontal epidural mass lesion was created by placing a balloon catheter through a burr hole into the right frontal epidural space. A computer was used to acquire data from all monitors at 50-msec intervals. The balloon was expanded by 1 cc over a period of 1 second every 5 minutes and maximum pressure immediately before and during expansion was determined for each balloon volume at each site. Prior to expansion of the mass, the morphology of the cerebellum pressure tracing was different from that seen in all supratentorial regions. Also, pressures in the midbrain, at baseline, were slightly but significantly lower than pressures in the frontal and temporal regions. During expansion of the mass, a pressure differential that increased as the size of the mass increased developed between intracranial regions. Furthermore, the regional pressures were found to vary in a consistent fashion expressed by the formula RF=LF>RT=LT>MB>CB, in which RF and LF are the right and left frontal lobes, RT and LT are the right and left temporal lobes, MR in the midbrain, and CB is the cerebellum. The study shows that an expanding epidural mass reproducibly results in a gradient of brain parenchymal pressure. This gradient results in parenchymal pressures that are significantly different in each region of the brain depending on the proximity of that region to the epidural mass. The results of this study have implications for clinical ICP monitoring and therapy. PMID- 8613858 TI - Loss of nitric oxide synthase immunoreactivity in cerebral vasospasm. AB - To determine the distribution of nitric oxide synthase (NOS) in the primate cerebral artery nervi vasorum and to examine the potential role of NOS in cerebral vasospasm after subarachnoid hemorrhage (SAH) in primates, the distribution of NOS immunoreactivity (NOS-IR) in the major cerebral arteries was examined immunohistochemically in cynomolgus monkeys by the use of whole, mounted preparations of the circle of Willis. In four normal monkeys, NOS-IR was localized to the endothelial and adventitial layers of the large cerebral arteries. On the abluminal side, NOS-IR staining was densely concentrated in perivascular nerve fibers (nervi vasorum) of the anterior circulation. Staining was less prominent in the posterior circulation. In six monkeys with vasospasm on Day 7 after placement of preclotted arterial blood to form an SAH around the right middle cerebral artery (MCA) (42% +/- 8.3% decrease of MCA area, mean +/- standard deviation), NOS-IR was virtually absent in nerve fibers around the spastic right MCA but was normal on the contralateral side. In five monkeys in which vasospasm resolved by Day 14 after SAH (36% +/- 14% decrease of right MCA area on Day 7, and 5% +/- 14% decrease on Day 14), NOS-IR was also absent in the right MCA adventitial nerve fibers and remained normal in the left MCA. Adventitial NOS-IR was also normal in cerebral vessels of a sham-operated, nonspastic monkey. These findings provide further evidence that nitric oxide (NO) functions as a neuronal transmitter to mediate vasodilation in primates and indicate a role for adventitial NO in the pathogenesis of cerebral vasospasm after SAH in humans. PMID- 8613859 TI - Combined fetal neural transplantation and nerve growth factor infusion: effects on neurological outcome following fluid-percussion brain injury in the rat. AB - This study was designed to evaluate the histological and behavioral impact of fetal neural transplantation with and without neurotrophin infusion in rats subjected to traumatic brain injury using a clinically relevant model of lateral fluid-percussion brain injury. Adult male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1-2.3 atm). Twenty-four hours after injury, minced fetal cortical grafts (E16) were stereotactically transplanted into the site of injury cavity formation (in 32 rats). Ten control animals received injections of saline. A third group of 29 animals that received transplants also underwent placement of a miniosmotic pump (immediately after transplantation) to continuously infuse nerve growth factor (NGF) directly into the region of graft placement for the duration of the experiment. A fourth group of eight animals underwent transplantation of fetal cortical cells that had been dissociated and placed in suspension. Animals were evaluated at 72 hours, 1 week, and 2 weeks after injury for cognitive function (using the Morris water maze), posttraumatic motor dysfunction, and transplant survival and morphology (using Nissl and modified Palmgren's silver staining techniques). Robust survival of whole-tissue transplants was seen in 65.5% of animals and was not increased in animals receiving NGF infusion. Animals receiving transplants of cell suspension had no surviving grafts. Brain-injured animals receiving transplants showed significant cognitive improvements compared with controls at the 2-week evaluation. Significantly improved memory scores were seen at all evaluation times in animals receiving both NGF and transplants compared with injured controls and compared with animals receiving transplants alone at the 72-hour and 1-week evaluations. Neurological motor function scores were significantly improved in animals receiving transplants alone and those receiving transplants with NGF infusion. Histological evaluation demonstrated differentiation of grafted cells, decreased glial scarring around transplants when compared with control animals, and the presence of neuronal fibers bridging the interface between graft and host. This study demonstrates that fetal cortical cells transplanted into the injured cortex of the adult rat can improve both posttraumatic cognitive and motor function and interact with the injured host brain. PMID- 8613860 TI - Recurrent transient quadriparesis after minor cervical trauma associated with bilateral partial agenesis of the posterior arch of the atlas. Case report. AB - The case of a 33-year-old woman with bilateral partial agenesis (type D) of the posterior arch of the atlas and recurrent transient quadriparesis due to contusion of the spinal cord after minor cervical trauma is described. At least some patients with type C or D congenital anomalies of the posterior arch of the atlas are prone to transient quadriparesis; thus a more aggressive management is advocated for them. Radiological and surgical findings showing the possible causative mechanism are presented and a review of the literature is given. PMID- 8613861 TI - Longitudinal atlantoaxial dislocation with type III odontoid fracture. Case report and review of the literature. AB - Odontoid fractures are a common traumatic upper cervical spine injury. Treatment of Type III odontoid fractures includes skeletal traction for realignment and halo vest immobilization. The authors report an unusual case of severe atlantoaxial ligamentous disruption accompanying a traumatic Type III odontoid fracture. Five pounds of skeletal traction was associated with marked neurological deterioration from unanticipated longitudinal instability. Radiographic findings were identified that were suggestive of extensive ligamentous disruption. Recommendations for individualized patient management are given in the context of related literature. PMID- 8613863 TI - Acquired cerebral arteriovenous malformation in a child with moyamoya disease. Case report. AB - The authors report a unique case involving a 2-year-old child with idiopathic moyamoya disease who presented with cerebral infarctions and seizures. On initial evaluation, computerized tomography (CT) showed a left parietal infarct and angiograms demonstrated early moyamoya disease with no evidence of arteriovenous malformation (AVM). Approximately 9 years later, angiography and magnetic resonance (MR) imaging revealed an AVM centered on the same region of the left parietal lobe. Angiographic, CT, and MR images are presented that demonstrate the progression of moyamoya disease and de novo development of the AVM in the infarct site. The possible role of angiogenesis in the etiology of acquired AVMs and moyamoya disease is discussed. PMID- 8613864 TI - Aneurysmal subarachnoid hemorrhage in a patient with bilateral A1 fenestrations associated with an azygos anterior cerebral artery. Case report and literature review. AB - Fenestration of the proximal anterior cerebral artery (A1 segment) is a rare occurrence. This vascular anomaly is often associated with aneurysms and other abnormalities. The current article describes the case of a 33-year-old man who presented with a subarachnoid hemorrhage secondary to a ruptured aneurysm originating from the proximal end of an A1 fenestration. This patient also had a contralateral A1 fenestration as well as an azygos anterior cerebral artery. This is the first report of such an unusual vascular anatomy. The literature is reviewed and possible embryological mechanisms are discussed. PMID- 8613862 TI - Nerve allotransplantation following severe tibial nerve injury. Case report. AB - The successful recovery of sensibility across a long peripheral nerve allograft in a 12-year-old boy who sustained a severe posterior tibial nerve injury is reported. The historical clinical experience with nerve allotransplantation is also reviewed. It is concluded that in the carefully selected patient with severe nerve injury, consideration for nerve allotransplantation can be given. PMID- 8613866 TI - Paradoxical aggravation of vasospasm with papaverine infusion following aneurysmal subarachnoid hemorrhage. Case report. AB - Reports of intraarterial papaverine infusion as treatment for cerebral vasospasm are few and documented complications are uncommon. The authors report the case of a patient with paradoxical aggravation of cerebral arterial narrowing during selective intraarterial papaverine infusion intended to treat vasospasm following aneurysmal subarachnoid hemorrhage (SAH). A 48-year-old man presented to the authors' service with symptomatic vasospasm 10 days after experiencing an SAH. The ruptured anterior communicating artery aneurysm was surgically obliterated the following day, and thereafter maximum hypervolemic and hypertensive therapies were used. However, the patient remained lethargic, and a stable xenon computerized tomography (CT) cerebral blood flow (CBF) study revealed CBF to be 15 cc/100 g/minute in the left anterior cerebral artery (ACA) and 25 cc/100 g/minute in the right ACA territories. Cerebral arteriography demonstrated diffuse severe left ACA and mild left middle cerebral artery (MCA) vasospasm. In response intraarterial papaverine was infused into the internal carotid artery just proximal to the ophthalmic artery. During the infusion the patient became aphasic and exhibited right hemiplegia. Arteriography performed immediately after the intraarterial papaverine infusion revealed diffuse exacerbation of vasospasm in the distal ACA and MCA territories. A repeat xenon-CT CBF study showed that CBF in the left ACA and the MCA had drastically decreased (2 cc/100 g/minute and 10 cc/100 g/minute, respectively). Despite aggressive management, infarction ultimately developed. This is the first clinical case to illustrate a paradoxical effect of intraarterial papaverine treatment for vasospasm following aneurysmal SAH. The possible mechanisms of this paradoxical response and potential therapeutic reactions are reviewed. PMID- 8613865 TI - Chromaffin cell survival and host dopaminergic fiber recovery in a patient with Parkinson's disease treated by cografts of adrenal medulla and pretransected peripheral nerve. Case report. AB - A 55-year-old woman with severe Parkinson's disease was treated by cografting adrenal medulla with pretransected peripheral nerve into the bilateral caudate nuclei. The patient showed modest improvement of her akinesia; this effect persisted for 1 year after transplantation, when she suddenly died from upper gastrointestinal bleeding unrelated to the grafting procedure. At autopsy, a large number of tyrosine hydroxylase-immunoreactive chromaffin cells were observed within the caudate graft sites and a dense network of host dopaminergic fibers was visualized. This autopsy finding is very important for the field of experimental and clinical chromaffin cell grafting because it is the first evidence that cografts using pretransected peripheral nerve might enhance the survival of chromaffin cells and the recovery of host dopaminergic fibers in humans suffering from Parkinson's disease. PMID- 8613867 TI - Percutaneous endoscopic treatment of suprasellar arachnoid cysts: ventriculocystostomy or ventriculocystocisternostomy? Technical note. AB - The use of an endoscope in the treatment of suprasellar arachnoid cysts provides an opening of the upper and lower cyst walls, thereby allowing the surgeon to perform a ventriculocystostomy (VC) or a ventriculocystocisternostomy (VCC). To discover which procedure is appropriate, magnetic resonance (MR)-imaged cerebrospinal fluid (CSF) flow dynamics in two patients were analyzed, one having undergone a VC and the other a VCC using a rigid endoscope. Magnetic resonance imaging studies were performed before and after treatment, with long-term follow up periods (18 months and 2 years). The two patients were reoperated on during the follow-up period because of slight headache recurrence in one case and MR imaged CSF flow dynamics modifications in the other. In each case surgery confirmed the CSF flow dynamics modifications appearing on MR imaging. In both cases, long-term MR imaging follow-up studies showed a secondary closing of the upper wall orifice. After VCC, however, the lower communication between the cyst and the cisterns remained functional. The secondary closure of the upper orifice may be explained as follows: when opened, the upper wall becomes unnecessary and tends to return to a normal shape, leading to a secondary closure. The patent sylvian aqueduct aids the phenomenon, as observed after ventriculostomy when the aqueduct is secondarily functional. The simplicity of the VCC performed using endoscopic control, which is the only procedure to allow the opening in the cyst's lower wall to remain patent, leads the authors to advocate this technique in the treatment of suprasellar arachnoid cysts. PMID- 8613868 TI - Intraoperative ultrasound for monitoring anterior cervical vertebrectomy. Technical note. AB - The authors describe the use of intraoperative ultrasonography with a small high frequency (15 mHz) probe for evaluation of the extent of lateral bone removal during anterior cervical vertebrectomy. The relationship of the bone resection margins to the lateral aspect of the spinal cord was visualized. Postoperative computerized tomography scans revealed the extent of bone removal to be similar to that demonstrated by ultrasound. Intraoperative ultrasonography may be useful during anterior cervical surgery to assure adequate decompression of the spinal canal and spinal cord. PMID- 8613869 TI - A new method for inserting the atrial end of a ventriculoatrial shunt. Technical note. AB - This technical note describes a simple percutaneous mechanism for placement of the atrial end of ventriculoatrial shunts. The method is fast, efficient, and involves no neck dissection. No special equipment is required. Placement involves the technique of central line insertion familiar to all surgeons. This new method has been used successfully in one adult and one pediatric patient. PMID- 8613871 TI - Complications of a ventriculoatrial shunt necessitating thoracic surgery. Case illustration. PMID- 8613870 TI - Resolution of spinal syringes and Chiari I malformation in a child. Case illustration. PMID- 8613872 TI - Symptomatic Rathke's cleft cysts in identical twins. Case illustration. PMID- 8613873 TI - Carotid-cavernous fistula and jugular venous oxygen saturation. PMID- 8613874 TI - Catheter implant systems for intrathecal drug delivery. PMID- 8613875 TI - Errors in statistical analysis. PMID- 8613876 TI - Academic neurosurgery. PMID- 8613877 TI - Economic abuses. PMID- 8613878 TI - Dietary saturated fatty acids and LDL receptor activity. PMID- 8613879 TI - Remembering Hamish Munro (1915-1994). PMID- 8613880 TI - Emil Abderhalden: his contribution to the nutritional biochemistry of protein. PMID- 8613881 TI - Oxidized ethyl linoleate induces mucosal hypertrophy of the large intestine and affects cecal fermentation of dietary fiber in rats. AB - Oxidized ethyl linoleate (OEL) was prepared by aeration at low temperature. Peroxide value (POV, mEq/kg lipid) of OEL was 1400; the major oxidized compounds were 9-hydroperoxy-cis, trans- and 13-hydroperoxy-trans, cis-octadecadienoate. Rats fed fiber-free or sugar-beet fiber (SBF, 100g/kg diet) diets were divided into three groups for each diet, and administered OEL (high OEL group), OEL diluted with ethyl linoleate (low OEL group, POV 700) and nonoxidized ethyl linoleate (EL group) through gastric tubes each day at 1400-1600 h (2.5 g/kg body wt) for 16 d. The relative wet weight, and DNA and protein contents of the cecal mucosa were higher in the high OEL groups than in the low OEL and EL groups in rats fed the fiber-free diet and in rats fed the SBF diet except for mucosal protein content. Spermidine concentration in cecal mucosa of rats fed the fiber free diet was greater in the high OEL group than in the EL group. These results suggest that metabolism related to mucosal proliferation of the cecum was affected by the high dose of OEL. The total short-chain fatty acid (SCFA) concentration in the cecal contents of SBF-fed rats was 100% higher than the concentration in rats fed the fiber-free diet in the EL group, but the administration of low dose and high dose OEL lowered the SCFA concentration in fiber-fed rats to that of rats fed the fiber-free diet. Butyric acid concentration was markedly lowered by ingestion of OEL in a dose-dependent manner in rats fed the SBF diet. In contrast, the isobutyric acid concentration was higher in the OEL-treated groups than in the EL groups. We conclude that a low dose of OEL depresses cecal fermentation of dietary fiber with changes in SCFA composition, and that a high dose of OEL induces mucosal hypertrophy in the cecum. These data show that dietary oxidized lipids affect cecal metabolism and may be associated with colon cancer. PMID- 8613882 TI - Aspects of cardiomyopathy are exacerbated by elevated dietary fat in copper restricted rats. AB - The objective of this study was to determine if a high fat diet having a 2:1 saturated-polyunsaturated fatty acid ratio exacerbates signs of copper deficiency. Male weanling Long-Evans rats were randomly placed into one of the following treatment groups: adequate copper low fat or deficient copper high fat. The levels of fat used were 31 or 12% of daily energy, and copper concentrations were 94.5 micromol/kg and <15.8 micromol/kg in the copper-adequate and copper deficient diets, respectively. Cardiac hypertrophy as well as lower liver copper levels and superoxide dismutase activity were observed in both groups of copper deficient rats. Irrespective of copper level, consumption of the high fat diet resulted in the thickening of the interventricular septum and left ventricular free wall. Electrocardiograms revealed that the copper-deficient high fat diet led to a significantly smaller QT interval compared with all other groups. Significantly greater S-wave voltage due to copper deficiency was observed. Significantly lower heart cytochrome c oxidase (CCO) activity was found in the copper-deficient groups with the copper deficient high fat group showing the lowest activity. Western blots of the cardiac non-myofibrillar fraction demonstrated lower amounts of CCO nuclear encoded peptides in the copper deficient groups, with the least amount seen in the copper-deficient high fat treatment. These data suggest that a high level of dietary fat exacerbates some of the signs of copper deficiency. PMID- 8613883 TI - Dietary components modify the ability of garlic to suppress 7,12 dimethylbenz(a)anthracene-induced mammary DNA adducts. AB - Various dietary components were evaluated as factors influencing garlic's ability to depress rat mammary cell DNA adducts resulting from 7,12 dimethylbenz(a)anthracene (DMBA) treatment. Diets with or without garlic powder (20 g/kg) were provided for 2 wk before DMBA treatment (25 mg/kg body weight). Rats fed diets containing 36 g casein/100 g diet had 31% fewer (P < 0.05) mammary cell DNA adducts than those fed 12 g/100 g. Garlic supplementation significantly (P < 0.05) reduced DNA adducts in rats fed either 12 or 36 g casein/100 g by 35 and 32% respectively. In the absence of dietary garlic, DNA adducts were 23% lower (P < 0.05) in rats provided a diet containing supplemental L-methionine at 0.9 g/100 g than at 0.3 g/100 g. However, adduct inhibition by garlic supplementation was greater in rats fed the lower (P < 0.05) amount of methionine (54 vs. 26% inhibition). Adduct levels in rats fed diets with 20 g corn oil/100 g were twice those occurring in rats fed 5 g/100 g (P < 0.05), regardless of adjustment for energy density. Garlic supplementation prevented the increase in DNA adducts caused by increasing dietary corn oil. Combining dietary supplements of garlic, selenite (0.5 mg/kg diet) and retinyl acetate (328 mg/kg diet) inhibited the occurrence of DNA adducts to a greater degree than when each was supplied individually. These studies demonstrate that while dietary garlic can reduce DNA adduct formation in mammary tissue caused by DMBA, this protection is influenced by several dietary components. PMID- 8613884 TI - Transgenic mice that overexpress metallothionein-I resist dietary zinc deficiency. AB - Transgenic mice that overexpress metallothionein-I (MT-I) accumulate mo re MT-I and zinc in major organs than do control mice. The effects of overexpression of MT-I on resistance to dietary zinc deficiency were examined by feeding transgenic and control mice a zinc-deficient (0.5-1.5 micron/gram) or a zinc adequate (50 micron/g) diet and by measuring effects on pregnancy. When pregnant mice were maintained under conditions of dietary zinc deficiency, the number of resorptions and teratogenic defects of fetuses was greatly reduced in transgenic compared with control mice. Differences between transgenic and controls were not apparent at d 8 of pregnancy (d 1 = vaginal plug) but were apparent by d 14. This result suggests that the larger maternal zinc pool in the transgenic females allows fetal development to progress normally for a longer period of time. However, neither transgenic nor control zinc-deficient mice could complete pregnancy. Pancreatic MT concentrations were the greatest in zinc adequate transgenic mice. Moreover, there was >10-fold more MT per gram wet weight in the pancreas of transgenic mice than in any other organ examined. Pancreatic MT concentrations were an exceptionally sensitive indicator of zinc deficiency. Pancreatic MT declined 99.8% and zinc declined to basal levels by d 14 of pregnancy when transgenic and control mice were fed a zinc-deficient diet, whereas MT concentrations in other organs decreased only modestly. We suggest that the larger pool of zinc MT in the transgenic mice provides a biologically important labile pool of zinc during periods of zinc deficiency. PMID- 8613885 TI - 1,25-Dihydroxycholecalciferol regulates rat intestinal calbindin D9k posttranscriptionally. AB - To determine whether calbindin D9k (CaBP) is subject to posttranscriptional control, 6-wk-old Sprague Dawley-derived rats were fed one of three purified diets, 1.5% Ca and 3.0% Ca, mostly as carbonate, and 2.9% Ca, mostly as gluconate. Two weeks later, 5-cm segments of duodenum, jejunum, ileum, cecum and colon were obtained and analyzed for CaBP and CaBP-mRNA. Analysis of the steady state distribution of CaBP-mRNA and of CaBP revealed a statistically significant (r = 0.95; P < 0.01) linear relationship between CaBP-mRNA and CaBP. When, however, animals that had been fed the 1.5% Ca diet received by intrajugular injection 1.2 nmol 1,25-dihydroxycholecalciferol [1.25-(OH)2-D3] and their CaBP mRNA and CaBP were analyzed as a function of time after 1,25-(OH)2-D3 administration, the kinetic response of the two molecules differed. The CaBP-mRNA increased linearly by approximately 68% for 4 h after administration and then declined over the next 6 h to a concentration below the preinjection value. Thus, appearance and disappearance of CaBP-mRNA approximated 17% x h(-1). The CaBP, however, increased steeply to 80% above preinjection concentration until 2 h postinjection, i.e., at a rate of 40% x h(-1). Thereafter, CaBP decreased to 35% above the preinjection value between 5 and 10 h postinjection (2.5% x h(-1)). These findings are consistent with a 1,25-(OH)2-D3-mediated posttranscriptional regulation of CaBP concentrations, because the 1,25-(OH)2-D3-mediated increase in CaBP-mRNA is not reflected in an immediately changed CaBP level. PMID- 8613886 TI - Dietary zinc deficiency alters 5 alpha-reduction and aromatization of testosterone and androgen and estrogen receptors in rat liver. AB - We studied the effects of zinc deficiency on hepatic androgen metabolism and aromatization, androgen and estrogen receptor binding, and circulating levels of reproductive hormones in freely fed, pair-fed and zinc deficient rats. Hepatic conversion of testosterone to dihydrotestosterone was significantly less, but formation of estradiol from testosterone was significantly greater in rats fed the zinc-deficient diet compared with freely fed and pair-fed control rats. There were significantly lower serum concentrations of luteinizing hormone, estradiol and testosterone in rats fed the zinc-deficient diet. No difference in the concentration of serum follicle-stimulating hormone was observed between the zinc deficient group and either control group. Scatchard analyses of the receptor binding data showed a significantly higher level of estrogen receptor in zinc deficient rats (36.6 +/- 3.4 fmol/mg protein) than in pair-fed controls (23.3 +/- 2.2 fmol/mg protein) and a significantly lower level of androgen binding sites in rats fed the zinc-deficient diet (6.7 +/- 0.7 fmol/mg protein) than in pair-fed control rats (11.3 +/- 1.2 fmol/mg protein). There were no differences in hepatic androgen and estrogen receptor levels between freely fed and pair-fed controls. These findings indicate that zinc deficiency reduces circulating luteinizing hormone and testosterone concentrations, alters hepatic steroid metabolism, and modifies sex steroid hormone receptor levels, thereby contributing to the pathogenesis of male reproductive dysfunction. PMID- 8613887 TI - The CD4/CD8 ratio in the blood does not reflect the response of this index in secondary lymphoid organs of weanling mice in models of protein-energy malnutrition known to depress thymus-dependent immunity. AB - A low ratio of cellular numbers within CD4+ (helper/inducer) relative to CD8+ (suppressor/cytotoxic) thymic lymphocyte subsets (low CD4/CD8 ratio) is widely accepted as fundamental to the depression in thymus-dependent immunocompetence associated with wasting protein-energy malnutrition (PEM). The objective of this investigation, therefore was to determine the CD4/CD8 ratio in peripheral lymphoid compartments of diverse murine models of protein-energy malnutrition which produce systemic wasting (loss of approximately 1.8% of initial body weight per day), lymphoid involution and (as shown in many previous studies) depression in thymus-dependent immunocompetence. In the first of two experiments, male and female weanling mice of disparate inbred strains, CBA/J and C57BL/6J, were allocated to a zero-time control group (23- and 19-d-old, respectively), or to groups fed for 14 d as follows: ad libitum intake of a complete purified diet (19% crude protein, 17 kJ/g gross energy), restricted intake of the complete diet, or ad libitum intake of an isocaloric low protein diet (0.6% crude protein). In a supplementary experiment, (0.6% crude protein). In a supplementary experiment, male and female C57BL/6J weanling mice were fed the complete diet or the low protein diet for either 6 or 21 d. CD4+ and CD8+ thymic lymphocytes were enumerated by flow cytometry in mononuclear cell suspensions from blood, spleen and mesenteric lymph nodes. A low CD4/CD8 ratio is common in the blood in wasting protein-energy malnutrition, but appears uncharacteristic of the profoundly involuted lymphoid organs which generate acquired immune responses. The CD4/CD8 ratio is irrelevant to the thymus-dependent immunoincompetence previously demonstrated in the rodent models used in this investigation. PMID- 8613888 TI - Apolipoprotein B mRNA editing is preserved in the intestine and liver of zinc deficient rats. AB - Apolipoprotein B (apo B) mRNA editing is a site-specific, post transcriptional cytidine deamination reaction that generates apo B48 in the mammalian small intestine and in the liver of certain animals. This reaction is mediated by an enzyme complex that includes the catalytic subunit apobec-1, a zinc-dependent cytidine deaminase. To determine the importance of zinc status to apo B mRNA editing in vivo, we examined the effects of experimentally induced zinc deficiency in rats upon hepatic and serum lipid levels and several indices of apo B gene expression. Rats were either given unlimited access to or were pair-fed a semipurified zinc-supplemented (30 mg Zn/kg) diet or were fed a zinc-deficient diet (approximately 1 mg Zn/kg) for 17 d. Significant differences were detected in the ratio of serum apo B100/B48 in the unlimited access, zinc-supplemented group compared with either zinc-deficient rats or pair-fed controls. There were no alterations in hepatic triglyceride and cholesterol concentrations, hepatic apo B mRNA abundance or apo B mRNA editing in either the small intestine or liver. Taken together, these data suggest that the altered ratios of serum apo B isomorphs seen in zinc deficiency are not mediated through changes in hepatic or intestinal apo B mRNA editing. PMID- 8613889 TI - Insulin increases lipogenic enzyme activity in human adipocytes in primary culture. AB - Studies with human adipose tissue have demonstrated the presence of key enzymes of fat synthesis. However, long-term regulation of these enzymes has not been reported. To address this issue, we used human adipocytes in primary culture. Human adipose tissue was obtained from abdominal fat of patients undergoing abdominal surgery. Adipocytes were isolated by collagenase digestion and cultured in media supplemented with 1% fetal bovine serum. To evaluate metabolic activity of cultured cells, we assessed the following during the culture: DNA pattern, cell size, glucose consumption and activities for two lipogenic enzymes, fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH). Analysis of DNA pattern showed that human adipocytes cultured under the above condition did not undergo cell apoptosis. In addition, no significant change in the cell size occurred during 22 d of culture. Glucose consumption by cultured cells was also constant during the culture and was 60% greater in the presence of 10 nmol/L of insulin. Treatment of cultured human adipocytes with insulin for 3-22 d increased GPDH and FAS activity by 60% and 2.8-fold, respectively, compared to cells cultured without insulin. Furthermore, the increase in FAS activity due to insulin treatment was dose dependent and maximal at 10 nmol/L. Our studies show for the first time that human adipocytes can be maintained viable and metabolically active for 2-3 wk in culture. Interestingly, cultured cells remain responsive to insulin. Therefore, this system will allow further characterization of long-term regulation of lipogenesis in human adipocytes and will be useful for developing pharmacological treatments of obesity. PMID- 8613890 TI - A diet high in wheat fiber decreases the bioavailability of soybean isoflavones in a single meal fed to women. AB - The absorption of some dietary components may be inhibited by dietary fiber. To study the effect of dietary fiber on the bioavailability of Isoflavones, seven healthy women were randomly assigned in a crossover design to a control diet containing 15 g dietary fiber or a wheat fiber-supplemented diet containing 40 g dietary fiber, both fed with a single dose of 0.9 mg isoflavones/kg body weight from tofu or texturized vegetable protein (TVP). The fiber-rich diet produced 55% lower plasma genistein at 24 h after soy dosing (P < 0.05) and reduced total urinary genistein by 20% (P < 0.03). Urinary daidzein was not significantly related to fiber intake. Highly insoluble, dietary wheat fiber reduced the absorption of genistein probably by its bulking effect and hydrophobic binding to this compound. Urinary genistein was greater by 23% after tofu than after TVP consumption (P < 0.02), but the percentage of ingested genistein recovered in urine was not affected by soy product intake. The higher urinary genistein after tofu consumption compared with TVP was apparently due to differences in amount of genistein between these soy foods, not the different forms of genistein present in these two soy food products. PMID- 8613891 TI - Proline is synthesized from glutamate during intragastric infusion but not during intravenous infusion in neonatal piglets. AB - Glutamate is considered the primary precursor amino acid for proline synthesis in mammals. Evidence exists, however, suggesting that proline may be a dietary indispensable amino acid for 2.5-kg piglets due to inadequate synthesis. This hypothesis was tested by intravenous and intragastric infusion of radiolabeled amino acids in vivo. Piglets (3 to 4 d old) were surgically implanted with catheters in the femoral (infusion) and jugular (sampling) veins and in the stomach (feeding and infusion). Piglets were fed hourly, via the stomach catheter, a semi-purified diet containing 10% dried skim milk, 15% corn oil, amino acids, vitamins and minerals. Experiment 1 was a 2 X 2 factorial design, with 24 piglets adapted to either low or supplemental proline diets (1.3 and 16.4 g proline x kg(-1) respectively) for 7 d, then intravenously infused with either [U-14C]glutamate or [U-14C]proline (185 k Bq x kg(-1) prime; 370 kBq x kg(-1) x h(-1) constant) for 4 h. Experiment 2 followed similar protocols, with eight piglets adapted to the low proline diet for 7 d and [U-14C]glutamate or [U 14C]proline infused into the stomach catheter. Piglets infused intravenously with [U-14C]glutamate did not convert glutamate to proline. Radioactive label was recovered in proline in all of the piglets receiving intragastric infusion of [U 14C]-glutamate. The fractional synthesis rate of proline from intragastric glutamate was 125 microgram ol kg(-1) x h(-1), accounting for approximately 40% of the proline accumulated. These data provide conclusive evidence that intravenously infused glutamate is not used as a precursor for proline synthesis and that, although conversion of glutamate to proline occurs in the gastrointestinal tract, the rate is not sufficient to provide the proline accumulated. PMID- 8613892 TI - Dietary long-chain polyunsaturated fatty acids influence tissue fatty acid composition in rats at weaning. AB - We studied the fatty acid composition of plasma, plasma phospholipids, erythrocyte membrane lipids, liver microsomal phospholipids and brain lipids in rats fed three different diets varying in their (n-3) and (n-6) long-chain polyunsaturated fatty acid (LCP) concentrations for 0, 2 and 4 wk after weaning. The three diets contained 10% fat; diet HO had a high-oleic acid proportion; diet FO was enriched in n-3 LCP provided by fish oil; and diet FO + BPL contained n-3 and n-6 LCP supplied by fish oil and a brain phospholipid concentrate. At 2 and 4 wk after weaning the proportions of oleic acid in all tissues, except in liver microsomes of the FO + BPL group, were significantly higher than in weanling rats. The absence of (n-3) LCP intake resulted in significantly lower levels of docosapentaenoic [20:5(n-3)] and 22:6(n-3) acids in plasma, plasma phospholipids, erythrocyte membrane lipids and liver microsomal phospholipids but not in brain lipids compared with rats at weaning. Dietary supplementation with (n-3) LCP (FO and FO + BPL groups) for 4 wk led to higher levels of 22:6(n-3) in all tissues compared with rats fed the HO fat. The proportions of 20:4(n-6) and total (n-6) LCP were significantly lower in all tissues from rats fed the FO diet than in rats at weaning and rats fed the HO diet. After 2 and 4 wk, rats fed the FO + BPL diet had significantly higher levels of 20:4(n-6) and total (n-6) LCP in plasma, plasma phospholipids, erythrocyte lipids and liver microsomal phospholipids; the brain also showed a higher content of those fatty acids after 4 wk. Our results suggest that dietary supplementation with 20:4(n-6) and 22:6(n-3) influences the concentration of 20:4-(n-6) and 22:6(n-3) in body tissues of rats after weaning. PMID- 8613894 TI - Protein and lipid refeeding changes protein metabolism and colonic but not small intestinal morphology in protein-depleted rats. AB - In this study, we fed rats a 2% casein AIN 76 diet for 2 wk to produce protein malnutrition. We determined in these animals the effects of different concentrations of dietary protein refeeding (2% and 20% casein) on recovery and gut mucosal repletion and the potential role of type of dietary fat in the regulation of protein metabolism and mucosal growth by providing conventional long-chain triglyceride (LCT), a structured lipid composed of long-, medium- and short-chain fatty acids (SC/SL), or a physical mixture of the same components present in the structured lipid given as individual pure triglycerides (SC/PM) along with adequate amounts of protein and energy. The results confirmed that protein malnutrition can be reversed rapidly by protein refeeding, as indicated by an increase in body weight, positive nitrogen balance, liver growth and elevations in plasma concentrations of insulin-like growth factor-1, leucine and albumin. In the colon, crypt cell number, crypt depth and number of crypt cells in the rapidly proliferating fraction of the colon were greater in rats fed the higher protein diet. However, the general architecture of small intestinal mucosa, including duodenum, jejunum and ileum, was not affected by protein malnutrition. Although the number of colonic cells was similar with fat refeeding, there were significantly fewer displaying the proliferating cell nuclear antigen in the colonic epithelium when rats were fed SC/PM compared with SC/SL. Therefore, changes in colonic mucosal proliferation were only seen with repletion by adequate protein and by SC/SL feeding. PMID- 8613895 TI - Short-term metabolic responses do not differ between neonatal piglets fed formulas containing hydrolyzed or intact soy proteins. AB - Hydrolyzed soy protein (SH) could be used as a protein source in formulas for infants with intolerance to cow's milk protein and may be preferable to intact soy protein (SI). However, metabolic responses to SH are poorly defined. Because of their partially hydrolyzed nature, nonphysiological elevations in either plasma amino acids or regulatory hormones may occur. Therefore, we evaluated effects of SH on plasma nutrient and pancreatic hormone (insulin, glucagon) concentrations. In Experiment 1, 24 newborn pigs were fitted with umbilical arterial and portal catheters, fed formula for 36 h and food deprived for 12 h. Pigs were then fed formula including either SH or SI with glucose polymers or casein-whey proteins (CW) containing lactose, and serial blood samples were taken for 2 h postprandially. Peak portal exceeded peak arterial amino acid concentrations within each treatment, and peak amino acid concentrations in CW fed pigs exceeded those of SH- and SI-fed pigs. However, only SH formula-fed piglets had higher postprandial portal minus arterial amino acid concentrations (P < 0.05) throughout Experiment 1, suggesting that SH was well digested and absorbed. In Experiment 2, arterial catheters were inserted in 24 piglets. Previous procedures were followed except dietary carbohydrate was standardized to glucose polymers for all three diets, and sampling was extended to 3 h. Overall, portal or arterial nutrient and hormone concentrations were not different in the SI and SH groups (P > 0.05), indicating that hydrolyzed soy protein did not cause abnormal plasma concentrations. In conclusion, hydrolyzed soy protein did not result in elevated nutrient concentrations or hormone responses compared with intact soy or cow's milk protein. PMID- 8613893 TI - Bone growth rather than myofibrillar protein turnover is strongly affected by nutritional restriction at early weaning of calves. AB - Our previous study revealed that weaning of calves aged 5 wk (early weaning) resulted in transient nutritional restriction with subsequent repletion. The present study was conducted to examine age-related changes in nitrogen balance, bone growth and myofibrillar protein degradation after early weaning in calves. At weaning, calves used in Experiment 2 had more severe nutritional restriction than those used in Experiment 1 due to a shorter duration of the suckling period (3 vs. 4 wk), a lesser amount of daily milk replacer (500 vs. 600 g) and a shorter period when given solid feed (for the last week vs. throughout the suckling period). In both experiments, nitrogen and calcium retentions were lower at weaning and transiently increased at 6 wk after weaning. However, detrimental effects on nitrogen and calcium retention immediately after weaning were more evident, and compensatory increases of nitrogen and calcium retention after weaning were more pronounced and continuous in Experiment 2 than in Experiment 1. The changes in plasma intact osteocalcin concentrations and urinary hydroxyproline excretion after weaning were similar to those for nitrogen and calcium retention in Experiment 2, whereas fractional degradation rate of myofibrillar protein was constant throughout the study in both experiments. These results suggest that the extent of nutritional restriction at weaning affects subsequent enhanced utilization of nitrogen and calcium, and that age-related changes in nitrogen retention after early weaning partly reflect changes in bone growth regulated by both forming and resorbing activities, independent of myofibrillar protein degradation. PMID- 8613896 TI - Changes in gut and liver glucose, lactate, insulin, and oxygen flux in mature ewes during mesenteric or abdominal vena cava glucose infusion. AB - The objective of the study was to determine whether exogenous glucose infusion affects endogenous glucose production in ewes. Mature ewes with catheters placed in the abdominal aorta, two mesenteric veins, the hepatic portal vein and the hepatic vein were used to determine the effect of exogenous glucose infusion on net glucose, insulin, lactate and oxygen flux by the portal-drained viscera (PDV) and liver of conscious sheep. Net hepatic glucose release was determined, and glucose was subsequently infused into either a mesenteric vein or an abdominal vena cava at an equal rate (mmol/L) for 240 min, and net fluxes were determined every 30 min. Arterial concentrations of glucose and insulin increased over time, whereas lactate concentration decreased over time (P < 0.05). Hepatic glucose release decreased linearly over time when glucose was infused into the mesenteric vein (P = 0.03) and tended to decrease linearly when glucose was infused into the abdominal vena cava (P = 0.06). Net PDV and splanchnic plasma insulin release during the vena cava infusion increased linearly over time (P < 0.02). Net PDV blood lactate release did not differ between infusion sites (P = 0.75), nor did the release rate change over time (P > 0.10). Net splanchnic lactate release in the abdominal vena++ cava infusion, however, did decrease over time (P = 0.05). Net hepatic oxygen consumption decreased quadratically over time (P = 0.004) when glucose was infused into the mesenteric vein. In conclusion, infusion of glucose results in a decrease in hepatic glucose production. PMID- 8613897 TI - Dietary restriction induces biochemical and morphometric changes in the small intestine of nursing piglets. AB - The purpose of this study was to evaluate the biochemical and morphometric changes in the small intestine of nursing piglets caused by 60% dietary restriction, and to ascertain whether this model reproduces the intestinal alterations caused by malnutrition in human infants. Piglets subjected to dietary restriction had significantly lower levels of mucosal DNA and protein, and significantly reduced segmental disaccharidase and leucine aminopeptidase activities compared with age-matched, freely fed controls. However, greater disaccharidase-specific activities were observed in duodenum and jejunum of diet restricted piglets compared with controls. Other findings included significantly lower thickness of the mucose, villous height and width, and villous surface area, a significantly lower number of goblet cells, and significantly greater mucosal crypt depth, intraepithelial leucocyte number, and infiltrated cells per area of lamina propria. The model reproduces most of the biochemical and morphometric changes observed in the small intestine of young human infants with chronic diarrhea and malnutrition, and may be useful in further investigations of the biochemical and molecular mechanisms of intestinal alterations caused by primary malnutrition in early infancy. PMID- 8613898 TI - Somatotropin treatment does not affect nonesterified fatty acid response to adrenergic injections in underfed or overfed nonlactating cows. AB - This experiment was conducted to determine the respective effects of bovine somatotropin (bST) and energy balance on the in vivo responses of plasma nonesterified fatty acids and glucose to isoproterenol (a nonselective beta agonist) or epinephrine injection in nonlactating nonpregnant cows. Two groups of adult Holstein cows were either underfed (n = 4) at 75%, or overfed (n = 5) at 150% of maintenance energy requirement, respectively. Cows received or did not receive a subcutaneous injection of Sometribove (500 mg) during two experimental periods (cross-over design). Adrenergic or placebo injections (4 nmol/kg body weight of epinephrine or isoproterenol or 4 mL of sterile saline) were administered intravenously on d 7-9 after bovine somatotropin injection, 1 h before 3.5 h after feeding for under- or overfed cows, respectively. Glucose and nonesterified fatty acid responses to each challenge were calculated as area under the response curve and above the base line, from the time of challenge until 60 min postchallenge. Basal plasma nonesterified fatty acids and their response to adrenergic injections were enhanced by underfeeding. Responses of nonesterified fatty acids to isoproterenol injection were higher than they were to epinephrine injection. Basal plasma glucose was enhanced by bovine somatotropin treatment, which increased the glucose response at 5 min after adrenergic injections. Response of plasma glucose was higher after epinephrine than after isoproterenol injection. Treatment with bovine somatotropin did not change plasma nonesterified fatty acid responses to epinephrine or isoproterenol injection in under- or overfed cows, at constant energy intake, whereas underfeeding modified these responses markedly. PMID- 8613899 TI - Gut endogenous nitrogen and amino acid excretions in adult domestic cats fed a protein-free diet or an enzymatically hydrolyzed casein-based diet. AB - Ileal and fecal gut endogenous nitrogen and amino acid excretions in adult domestic cats were determined. Ileal digesta were collected (10 cm of terminal ileum) from the cats fed either a protein-free diet or an enzymatically hydrolyzed casein-based diet (free amino acids and peptides < 10,000 Da) for 1 wk. Chromic oxide was included in each diet as an indigestible marker. The relative contribution of the hindgut to total endogenous gut excretion was investigated in a separate study by feeding cats a protein-free diet with or without added antibiotics for 10 d. Endogenous ileal nitrogen and amino acid nitrogen excretions of (mean +/- SEM 2.4 +/- 0.27 and 1.9 +/- 0.13 mg/g food dry matter intake, respectively, were found for the cats fed the protein-free diet, whereas higher excretions of 3.6 +/- 0.73 (P = 0.12) and 3.6 +/- 0.76 (P = 0.03) mg/g food dry matter intake were obtained in cats fed the enzymatically hydrolyzed casein. Significantly (P < 0.05) higher endogenous ileal amino acid excretions, for the enzymatically hydrolyzed casein-fed cats compared with those fed the protein-free diet, were found for methionine, aspartic acid, serine, glutamic acid, proline, valine and isoleucine, with the differences in excretions of glycine, alanine, leucine and histidine being significant at the 6% level. Most of the endogenous fecal amino acid excretions were unaffected by the inclusion of the antibiotics in the protein-free diet, although bacterial numbers were significantly lower (69%). Antibiotics addition led to significantly higher fecal endogenous excretions of nitrogen, taurine, threonine, serine and histidine but significantly lower excretions of methionine and lysine. Cats, like other simple-stomached mammals, excrete higher amounts of endogenous amino acids at the terminal ileum when the diet contains peptides. PMID- 8613900 TI - Kinetic model of molybdenum metabolism developed from dual stable isotope excretion in men consuming a low molybdenum diet. AB - The aim of this study was to develop a compartmental model of molybdenum metabolism based on stable isotope excretion patterns. Molybdenum (Mo) is an essential trace element in humans, with an estimated safe and adequate daily dietary intake (ESADDI) of 75-250 micrograms Mo/d. Four adult men were fed low molybdenum diets, 22 micrograms Mo/d for a period of 102 d. 97Mo+ and 100Mo stable isotopes, in intravenous and oral doses, respectively, were administered at selected intervals. The resulting 6-d cumulative urinary and fecal isotope excretion data were used to model molybdenum metabolism using SAAM/CONSAM software. A kinetic model, including gastrointestinal (GI), plasma, slow-turnover tissue and fast-turn-over tissue compartments, accurately simulated the observed pattern of urinary and fecal excretion for both stable isotopes in all four subjects. Residence time for molybdenum in the GI tract was estimated at 1.7 +/- 0.4 d. Predicted residence time for plasma molybdenum was 22 +/- 4 min, whereas slow-turnover tissue (possible hepatic) retention averaged 58 +/- 16 d. The model thus permitted estimation of kinetic parameters for molybdenum metabolism in tissues not readily accessible or measurable in humans. PMID- 8613902 TI - The riboflavin requirement of adult dogs at maintenance is greater than previous estimates. AB - A study was conducted to determine the riboflavin requirement of adult dogs at maintenance. Twenty adult mixed breed dogs were fed a semipurified meal with one of five riboflavin concentrations: Diet 1, 1.7 mg/kg; Diet 2, 2.7 mg/kg; Diet 3, 3.7 mg/kg; Diet 4, 4.7 mg/kg; and Diet 5, 5.7 mg/kg. The erythrocyte glutathione reductase activity coefficient (EGRAC) was used to determine biochemical riboflavin deficiency. Dogs fed Diet 1 had a greater (P < 0.05) EGRAC (1.24) on d 56 of the trial compared with that of dogs fed Diet 5 (1.11), indicating marginal riboflavin deficiency in dogs fed Diet 1. On d 84 the mean EGRAC for dogs fed Diet 1 (1.36) was different from EGRAC obtained for dogs fed the other diets (1.19, P < 0.05). The difference in mean EGRAC was still present of d 112 (1.59 vs. 1.27; P< 0.01). There was no difference in d 112 mean EGRAC for dogs fed Diets 2, 3, 4 and 5 (P < 0.05). The broken line requirement estimate for the adult dog at maintenance was determined to be 66.8 microgram riboflavin x kg body wt(-1) x d(-1) using the d 112 EGRAC as the basis for assessing biochemical riboflavin deficiency. PMID- 8613901 TI - Long-term administration of high dose vitamin A to rats does not cause fetal malformations: macroscopic, skeletal and physicochemical findings. AB - A rat model was used to investigate whether high oral doses of vitamin A lead to fetal malformations and to what extent retinyl esters (RES) are transferred from the mother to the fetuses. Retinol and RES concentrations in plasma behave similarly in rats and humans. When high concentrations of vitamin A are administered, plasma retinol concentrations remain relatively constant, whereas plasma RES increased in parallel with the dose. To achieve an elevation from approximately 150 to > 1525 nmol x L(-1) in the experimental group before mating, female Ibm: RORO (spf) rats were fed a maintenance diet enriched with 15.2 x 10(3) retinol equivalents (RE) x kg(-1) at the start and increased stepwise to 52.5 x 10(3) for a total of 8 mo. A parallel subgroup was maintained to measure progress in experimental rats without interference by blood taking. Rats of the control group received the basal diet analyzed to contain 4.5 x 10(3) RE x kg( 1). Before mating the mean body weights of experimental and control rats were not significantly different. All-trans, 13-cis, 4-oxo-all-trans and 5,6-epoxy-all trans retinoic acid (RA) concentrations were determined in maternal and fetal plasma. With high vitamin A intake, 4-oxo- and 5,6-epoxy RA concentrations were significantly higher in the fetuses than in their mothers. Although these high intakes of vitamin A by the rat dams resulted in high maternal and fetal plasma concentrations of vitamin A and its metabolites, fetal malformations were not observed. This may be due to the fact that circulating RES are not teratogenic and that after crossing the placental barrier, they are stored mainly in fetal liver. PMID- 8613903 TI - Feeding of potato, tomato and eggplant alkaloids affects food consumption and body and liver weights in mice. AB - Reduced liver weight was used to evaluate the potential toxicity in mice of four naturally occurring steroidal glycoalkaloids: alpha-chaconine and alpha-solanine, alpha-tomatine and solasonine. Increased liver weights was used to evaluate the three corresponding steroidal aglycones: solanidine, tomatidine, and solasodine and the non-alkaloid adrenal steroid dehydroepiandrosterone (DHEA). Adult female Swiss-Webster mice were fed diets containing test compound concentrations of 0 (control), 1.2, 2.4 or 4.8 mmol/kg diet for 7, 14 or 28 d. Absolute liver weights (LW) and relative liver weights (liver weight/body weight x 100, %LW/BW) were determined at autopsy. The %LW/BW was lower than that of controls in mice fed the potato glycoalkaloid alpha-chaconine (-10%, P < or = 0.05) for 7 d with the 2.4 mmol/kg diet dose. Under these same conditions, %LW/BW was greater than that of controls in mice fed two aglycones: solanidine (27%, P < or = 0.001) and solasodine (8%, P < or = 0.01). Relative liver weight increases induced by the aglycones were determined under time and dose conditions in which differences in body weight and food consumption were not significant (2.4 mmol/kg diet for 28 d). Under these conditions, the observed %LW/BW increases relative to the controls were as follows: solanidine (32%, P < or = 0.001), solasodine (22%, P < or = 0.001) and DHEA (16%, P < or = 0.001). Solanidine, solasodine and DHEA were equally potent and were more potent than tomatidine. We also observed that the greater %LW/BW in mice fed 2.4 mmol/kg diet solasodine or solanidine for 14 d declined to near control values if they were fed control diets for another 14 d. The increase in relative liver weight induced by solanidine and solasodine is a reversible adaptive response. These findings and the apparent effects of structure on biological activity should serve as a guide for the removal of the most toxic ++compounds from plant foods. The implications of the results for food safety and health are discussed. PMID- 8613904 TI - Structure and function of P2 purinocepters. PMID- 8613905 TI - Summary of the ASPET-sponsored Colloquium: Alpha-2 adrenergic receptors: structure, function, and therapeutic implications, October 25-27, 1995. PMID- 8613906 TI - Dexamethasone metabolism by human liver in vitro. Metabolite identification and inhibition of 6-hydroxylation. AB - The metabolism of the synthetic glucocorticoid dexamethasone in human liver microsomal incubations has been studied. Metabolites were analyzed by radiometric high-performance liquid chromatography and were identified by liquid chromatography-mass spectrometry; in addition, the major metabolite 6beta hydroxydexamethasone was identified by cochromatography with a chemically synthesized standard. A total of 17 human livers were used in this study and the following metabolites were identified: 6beta-hydroxydexamethasone, 6 alpha hydroxydexamethasone, 6-hydroxy-9 alpha-fluoro-androsta-1,4-diene-11 beta-hydroxy 16 alpha-methyl-3,17-dione (6-hydroxy-9 alpha-F-A) and 9 alpha-fluoro-androsta 1,4-diene-11 beta-hydroxy-16 alpha-methyl-3,17-dione (9 alpha-F-A). Dexamethasone underwent side-chain cleavage to form 9 alpha-F-A. This metabolite was then a substrate for 6-hydroxylation. There was considerable interindividual variability in metabolic profiles. Mean (+/-S.D.) K(m) values for 6 beta- and 6 alpha hydroxydexamethasone formation were 23.2 +/- 3.8 and 25.6 +/- 1.6 microM (n = 4), respectively. The corresponding V max values were 14.3 +/- 9.9 and 4.6 +/- 3.1 pmol x min(-1) mg protein (-1). Ketoconazole (3 microM) completely inhibited 6 alpha- and 6 beta-hydroxylation, indicating that formation of both metabolites was catalyzed by CYP3A4. This was confirmed in studies of correlations between the rate of metabolite formation and the relative expression of CYP3A4: r = 0.74 for 6 beta-hydroxydexamethasone, P = .003; r = 0.70 for 6 alpha hydroxydexamethasone, P = .006. In addition to ketoconazole, both ellipticine and gestodene caused marked inhibition of 6-hydroxylation. Ellipticine is clearly not a selective CYP1A inhibitor as has been stated previously. However, furafylline (CYP1A inhibitor), tolbutamide (CYP2C substrate), and sulfaphenazole (CYP2C inhibitor) were essentially noninhibitory. The relatively simple metabolic profile of dexamethasone compared to other steroids may point to this being a potentially useful in vivo probe for CYP3A4 in humans. PMID- 8613907 TI - Haloperidol and apomorphine differentially affect neuropeptidase activity. AB - In addition to their well characterized effects at dopamine receptors, neuroleptic drugs have been shown to affect the level and in vitro metabolism of neuropeptides. In the present study, the effect of acute and subchronic administration of the neuroleptic haloperidol and the nonselective, dopamine agonist apomorphine on neuropeptidase activity was determined in regional, rat brain P2 membranes. Subchronic administration of haloperidol decreased the activity of aminopeptidase N in the frontal cortex and caudate-putamen. In contrast, subchronic administration of apomorphine increased aminopeptidase N activity in the frontal cortex and caudate-putamen. Neutral endopeptidase 24.11 also was affected differentially in the caudate-putamen, but both subchronic haloperidol and apomorphine decreased neutral endopeptidase 24.11 activity in the frontal cortex. Metalloendopeptidase 24.15 activity was decreased in the caudate putamen after acute haloperidol and increased in the frontal cortex after acute apomorphine administration; however, no effect was noted after subchronic administration of either drug. Angiotensin converting enzyme was not affected by any treatment. Therefore, neuroleptic-induced alterations in aminopeptidase N, neutral endopeptidase 24.11 and metalloendopeptidase 24.15 activity may account for previously reported alterations in neuropeptide degradation. In view of the interaction between mesocorticolimbic dopamine neurons and neuropeptides, e.g., substance P, neurotensin and enkephalins, neuroleptic-induced alterations in the activities of neuropeptidases, and thus neuropeptide metabolism can, in turn, play a role in modulating midbrain dopaminergic activity. PMID- 8613908 TI - Felbamate inhibits dihydropyridine-sensitive calcium channels in central neurons. AB - The effect of the antiepileptic drug felbamate (FBM) on high-voltage-activated Ca++ currents was studied in cortical and neostriatal neurons acutely isolated from adult rats. Patch-clamp recordings in the whole-cell configuration were performed. Ba++ ions as the charge carrier for Ca++ channels were used. In pyramidal cortical cells, FBM dose-dependently reduced high-voltage-activated Ca++ currents in all the tested neurons. At concentrations of 30 to 100 nM, FBM already produced a significant inhibition of high-voltage-activated Ca++ currents (-6/-15%). At saturating concentrations (1-3 microM), FBM-mediated inhibition averaged 44%. The responses were fully reversible. The dose-response curves revealed IC50 of 504 nM. In striatal neurons, FBM decreased the same conductances by about 28%; the threshold dose was 1 to 2 microM, with an IC50 of 18.7 microM. In both structures, the observed inhibitions were unaffected by omega-conotoxin GVIA and omega-agatoxin IVA, suggesting that N-like channels and P-Like channels were not involved in the FBM-mediated responses. In addition, when omega conotoxin GVIA and omega-agatoxin IVA (100 nM) were coapplied, the FBM-mediated inhibition on the remaining Ca++ currents averaged 87%. The FBM responses were occluded by micromolar concentrations of nifedipine, supporting a direct interference with dihydropyridine-sensitive channels. It is concluded that the described effect of FBM might represent an efficacious mechanism for either controlling spike discharge from epileptic foci or protecting neurons from excessive Ca++ loading. In both cases, FBM would act as a broad spectrum neuroprotective agent. PMID- 8613909 TI - Carvedilol, a new beta adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits. AB - Oxygen-derived free radicals play a critical role in atherogenesis and reperfusion injury. The present experiment evaluated the effects of carvedilol, a new beta adrenoreceptor blocker with potent free radical-scavenging activity, on myocardial ischemia and reperfusion injury in a hypercholesterolemic rabbit model. New Zealand rabbits were fed a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with 1200 ppm carvedilol or propranolol. Eight weeks later, the rabbits were subjected to 60 min of myocardial ischemia followed by 60 min of reperfusion. The nontreated cholesterol-fed animals experienced greater cardiac damage after ischemia and reperfusion than rabbits fed a normal diet (necrosis 51% +/- 4% vs. 28% +/- 3% in the normal-diet group, P < .01). In addition, nontreated cholesterol-fed rabbits showed a significantly decreased vasorelaxant response to ACh in U-46619-precontracted aortic rings (56% +/- 5% vs 90% +/- 3% in the control group, P < .001). Treatment with propranolol neither preserved endothelial function after cholesterol feeding nor reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. Propranolol treatment did significantly decrease HR, pressure-rate index and infarct size (necrosis 33% +/- 4%). Despite their having essentially identical effects on HR and pressure-rate index, carvedilol exerted more profound cardiac protective effects than propranolol (necrosis 19% +/- 3%). Moreover, carvedilol treatment significantly preserved aortic endothelial function and markedly reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. These results indicate that in addition to its beta blocking activity, the antioxidant and endothelial protective activities of carvedilol contributed significantly to its cardiac protective effects after ischemia and reperfusion. PMID- 8613910 TI - Effects of the novel antipsychotic agent 7-(4-[4-(2,3-dichlorophenyl)-1 piperazinyl]butyloxy)-3,4-dihydro -2(1H)-quinolinone (OPC-14597) on prolactin release from the rat anterior pituitary gland. AB - The effects of a novel antipsychotic agent, 7-(4-[4-(2,3-dichlorophenyl)-1 piperazinyl]butyloxy)-3,4-dihydro-2 (1H)-quinolinone (OPC-14597, generic name aripiprazole), on prolactin (PRL) release from isolated rat anterior pituitary slices and on the serum PRL levels were investigated in male rats. In in vitro experiments on the isolated anterior pituitary, the spontaneous PRL release was decreased by talipexole, a dopamine D2 receptor full agonist, in a dose-dependent manner to 36% of the basal release, and the decrease was antagonized by haloperidol, a D2 receptor antagonist. OPC-14597 also decreased the release of PRL at the same concentration range with a maximal decrease to 78%, the potency being weaker than that of talipexole. The decrease in PRL release induced by OPC 14597 was completely antagonized by haloperidol. Moreover, OPC-14597 antagonized the inhibition of PRL release induced by talipexole. In in vivo experiments, haloperidol increased the serum PRL levels to 8 times the basal PRL level, whereas talipexole decreased the levels to 49% of the basal level. OPC-14597 increased the serum PRL levels by 2-fold and also antagonized the talipexole induced decrease. The hyperprolactinemia induced by estrogen, which was inhibited by talipexole but enhanced by haloperidol, was enhanced by OPC-14597, whereas the hyperprolactinemia induced by reserpine, which was inhibited by talipexole but elevated by haloperidol, was inhibited by OPC-14597. In addition, the OPC-14597 induced inhibition was antagonized by haloperidol. These results suggest that OPC 14597 has a mixed agonist/antagonist profile at D2 receptors on lactotroph cells and thereby exerts either an antagonistic or an agonistic action, depending on the preexisting tone of the dopaminergic neuronal activities. PMID- 8613911 TI - Protein kinase C mediates spontaneous tone in the cat lower esophageal sphincter. AB - The intracellular pathways responsible for maintenance of tone in the lower esophageal sphincter (LES) are not well understood. We show that the protein kinase C (PKC) antagonists (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride) and calphostin C reduce spontaneous resting tone in LES muscle strips, whereas the calmodulin antagonist N-(6-aminohexyl-5-chloro-1 naphthalenesulfonamide hydrochloride) has no effect, which suggests that LES tone is maintained by a PKC-mediated mechanism. In addition, U73122, an inhibitor of phosphatidylinositol-4,5-bisphosphate (PIP2)-specific phospholipase C, and D609, an inhibitor of phosphatidylcholine-specific phospholipase C, reduced diacylglycerol formation and LES tone in a concentration-dependent manner. Finally diacylglycerol levels and PKC activity were reduced during relaxation of the LES induced by the inhibitory neurotransmitter vasoactive intestinal peptide. These data suggest that resting LES tone is associated with elevated diacylglycerol levels and PKC activity, which are reduced during relaxation. Diacylglycerol is derived from at least two different sources. Hydrolysis of PIP2 by PIP2-specific phospholipase C produces equimolar amounts of inositol 1,4,5 triphosphate and diacylglycerol, which may interact synergistically to activate PKC and develop tone. Furthermore, PKC-mediated contraction may be augmented by additional diacylglycerol production arising from the hydrolysis of phosphatidylcholine by phosphatidylcholine-specific phospholipase C. PMID- 8613912 TI - Cyanide-induced neurotoxicity involves nitric oxide and reactive oxygen species generation after N-methyl-D-aspartate receptor activation. AB - To study oxidative mechanisms in cyanide toxicity, cyanide-induced generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescence dye 2,7 dichlorofluorescin. KCN produced a concentration-dependent (25-200 microM) generation of intracellular oxidant species that was blocked by N-methyl-D aspartate receptor antagonists (MK-801 or AP5) or by removal of extracellular Ca++ from the incubation medium. To determine the relative contribution of NO and reactive oxygen species (ROS) to the increase of cellular fluorescence after KCN, a selective inhibitor of nitric oxide synthase, a NO scavenger and enzymes that metabolize ROS were added to the incubation medium. Interference with the nitric oxide system (reduced hemoglobin as a NO scavenger or [N(G)-nitro-L-arginine methyl ester [L-NAME] reduced fluorescence by 50%). Addition of enzymes that metabolize peroxide (catalase or superoxide dismutase [SOD]) also reduced fluorescence by nearly 50%. Combination of SOD with hemoglobin or L-NAME provided additional attenuation of the fluorescence and it was concluded that both NO and ROS are generated concurrently after KCN. Furthermore a correlation was observed between NO and ROS formation and levels of malonaldehyde (MDA), a marker of lipid peroxidation. Pretreatment with MK-801 blocked KCN-induced MDA formation, whereas L-NAME partially diminished MDA production. Treatment with a combination of SOD/catalase and L-NAME blocked the KCN-induced lipid peroxidation. In cytotoxicity studies cyanide-induced cell death was blocked by MK-801, whereas partial attenuation was produced by L-NAME; SOD/catalase treatments did not protect the cells. However, significant protection from cyanide-induced cytotoxicity was observed when L-NAME was combined with SOD/catalase. It is concluded that cyanide activates N-methyl-D-aspartate receptors to simultaneously generate both NO and ROS, which may lead to formation of the cytotoxic peroxynitrite anion. PMID- 8613913 TI - Activation of ATP-dependent K+ channels and inhibition of insulin release: effect of BPDZ 62. AB - The present study was undertaken to characterize the effects of BPDZ 62, an original pyridothiadiazine derivative structurally related to both diazoxide and pinacidil, on ionic and secretory events in the rat pancreatic islet cells. BPDZ 62 increased the rate of 86Rb outflow from islets perfused in the presence or absence of extracellular glucose. These effects persisted in the absence of extracellular Ca++ but were abolished by glibenclamide. Such data support the view that BPDZ 2 activates ATP-sensitive K+ (K(ATP)) channels. This proposal was substantiated by the finding that the drug enhanced the flow of current through K(ATP) channels in excised inside-out membrane patches. BPDZ 62 markedly decreased 45Ca uptake, 45Ca outflow and insulin output from islets incubated in the presence of 16.7 mM glucose. By contrast, the drug did not affect the increase in 45Ca outflow and 45Ca uptake mediated by K+ depolarization. In single B cells, BPDZ 62 inhibited the glucose but not the KCl-induced rise in [Ca++]i. It is concluded that the inhibitory effect of BPDZ 62 on the insulin-releasing process results from the activation of K(ATP) channels leading to a decrease in Ca++ influx and [Ca++]i. Last, BPDZ 62 was shown to be five times more potent than diazoxide at inhibiting the insulin-releasing process. This suggests that BPDZ 62 could be a valuable pharmacological tool for further characterization of B-cell K(ATP) channels. PMID- 8613914 TI - Mechanism of cardiac inotropy by phenamil, and epithelial sodium channel blocker. AB - Phenamil, an amiloride derivative, is a potent inhibitor of epithelial type sodium channels and a relaxant of smooth muscle. In canine cardiac ventricular trabeculae, which do not express epithelial type sodium channels, phenamil produces positive inotropy and prolongs twitch duration. Sarcoplasmic reticulum does not appear to be essential for phenamil-induced inotropy, because cyclopiazonic acid and ryanodine do not abolish this effect. Furthermore, in tissues made to contract biphasically with 90 to 98% substitution of calcium with strontium, phenamil enhanced the second phase of the contraction which is transsarcolemmal-calcium dependent. Phenamil did not alkalinize or acidify the cytosol (measured with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, BCECF) during the induction of positive inotropy, therefore the sodium-hydrogen exchange is not affected. Sodium-calcium exchange, as assessed by twin rapid cooling contractures, was not inhibited by phenamil. Direct inhibition of this exchanger is therefore not necessary for the inotropic action of phenamil. Phenamil did not inhibit the sodium pump in smooth muscle. Unlike ouabagenin, it significantly prolonged the action potential duration at 90% repolarization. We have demonstrated recently that prolongation of cardiac action potential duration with phenamil is due to inhibition of the inwardly rectifying potassium current without any direct effect on cardiac calcium channels. The resulting delay in repolarization of the terminal part of the action potential reduces the driving force for the forward mode of sodium-calcium exchange. This indirectly reduces the activity of the sodium-calcium exchanger and slows the extrusion of calcium from the cell at the end of the action potential leading to a gradual development of positive inotropy. PMID- 8613915 TI - BW755C, a dual lipoxygenase/cyclooxygenase inhibitor, reduces mural platelet and neutrophil deposition and vasoconstriction after angioplasty injury in pigs. AB - Arachidonic acid metabolism through the lipoxygenase and cyclooxygenase pathways in neutrophils and platelets may be involved in the pathophysiological response to arterial injury in vivo. Therefore, we investigated the effects of 3-amino-1 [m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), a dual lipoxygenase/cyclooxygenase inhibitor, on mural platelet and neutrophil deposition, and the vasoconstrictive response that followed carotid arterial injury by angioplasty in pigs. 51Cr platelet deposition at the site of deep arterial wall injury averaged 55.4 +/- 12.2 x 10(6)/cm2 in the vehicle-treated group (n = 10). BW755C (10 mg/kg i.v., n = 10) significantly reduced this mural platelet deposition by more than 50% to 25.4 +/- 5.3 x 10(6)/cm2 (P < .05) and decreased 111In neutrophil deposition from 240.6 +/- 30.4 x 10(3)/cm2 in the control group to 114.4 +/- 20.4 x 10(3)/cm2 (P < .005) in the treated group. BW755C had no hemodynamic effects. However, the angiographic vasoconstrictive response at the site of endothelial injury distally was significantly attenuated by the dual inhibitor from 45.4 +/- 2.6% to 29.7 +/- 4.3% (P < .005). These beneficial effects of BW755C were associated with inhibition of neutrophil mediated but not platelet-mediated whole-blood aggregation and with a significant reduction in neutrophil superoxide anion generation. These results indicate that the dual lipoxygenase/cyclooxygenase inhibitor BW755C prevents the acute thrombotic and vasomotor responses related to arterial injury by angioplasty, predominantly through inhibition of neutrophil function. PMID- 8613916 TI - d-Sotalol terminates reentry by two mechanisms with different dependence on the duration of the excitable gap. AB - We used eight adjustable preparations in which the canine atrial tricuspid rings were cut and reconnected electronically by sensing activation on one side of the cut and pacing the other after an adjustable delay. A long delay resulted in a long cycle length (CL) and excitable gap (EG) during reentry. Decreasing delay decreased CL and EG. d-Sotalol (4 mg/l) significantly increased effective refractory period (ERP) and action potential duration with no effects on conduction time during constant 400-msec pacing. During reentry, d-sotalol increased action potential durations more than CLs, so it decreased diastolic intervals. It decreased EG by increasing ERP more than CL. Although d-sotalol increased action potential duration more at longer delays with longer CLs, showing reverse use-dependence, it terminated sustained tachycardias by increasing ERP only for the short delays when the initial EG was short. In 5 of 8 experiments, longer equilibration with d-sotalol produced fixed block at a vulnerable site, so reentry could not be induced at any delays. Fixed block could be transiently reversed by ACh and resolved after washout of d-sotalol. We conclude that d-sotalol terminated reentry by two mechanisms: 1) It terminated sustained reentry by increasing ERP when the initial EG was sufficiently short. 2) In some preparations, it caused fixed block at a vulnerable site, which prevented reentry regardless of the initial EG. PMID- 8613918 TI - A1, but not A2A, adenosine receptors modulate electrically stimulated [14C]acetylcholine release from rat cortex. AB - Adenosine A1 receptors are known to be widely distributed in various regions of the brain. A2A receptors are enriched in the dopamine-rich areas of the brain, but are also present in rat cortex. Electrically stimulated, perfused rat cortical slices were used to examine the influence of interactions between A1 and A2A receptors on the release of acetylcholine (ACh) from cortical cholinergic nerves. The A1-selective agonist, N6-cyclopentyladenosine (CPA) caused a dose dependent inhibition of ACh release, which was attenuated in the added presence of the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 microM). The inhibitory effects of CPA were unaltered in the added presence of the A2-selective antagonist (E)-8-(3,4-dimethyloxystyryl)-1,3-dipropyl-7 methylxanthine (KF 17837; 1 microM). The A2A-selective agonist 2-[p (carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), over the concentration range 1 nM to 10 microM, did not significantly alter ACh release when given alone or in the presence of DPCPX or KF 17837. These data suggest that the A(2A) receptors previously identified in rat cortex are not functionally coupled to modulation of ACh release in this tissue. This does not exclude that these receptors may regulate the release of other neurotransmitters. PMID- 8613917 TI - Inhibition of dopamine synthesis by dopamine D2 and D3 but not D4 receptors. AB - The goal of the current study was to determine which of the D2-like receptors (D2, D3 or D4) are involved in autoreceptor regulation of dopamine synthesis. We have derived a model system utilizing a mouse mesencephalic cell line, MN9D, which both synthesizes and releases dopamine, to characterize the modulation of tyrosine hydroxylase activity, the rate limiting enzyme in the conversion of tyrosine to dopamine, by the D2-like receptors. Previously, we have shown that stimulation of D2 and D3, but not D4, dopamine receptors transfected into MN9D cells inhibited the release of dopamine. In the current study, we show that quinpirole stimulation of transfected D2 and D3, but not D4, dopamine receptors inhibited K+-stimulated tyrosine hydroxylase activity in a pertussis toxin sensitive manner, strongly suggesting G-protein coupling as a mechanistic pathway. The D2 receptor effect could be maintained for at least 60 min, whereas the D3 receptor effect desensitized. Treatment with 10 microM forskolin, which raises cyclic AMP levels or with 100 nM okadaic acid, a potent phosphatase inhibitor, had no effect on the D2-or D3-mediated inhibition, suggesting that these effects may be independent of both cyclic AMP- and okadaic acid-sensitive phosphatase activity. Taken together, these data confirm the hypothesis that dopamine D2 and D3 receptors can perform dual roles in autoreceptor regulation. PMID- 8613919 TI - Effects of benztropine on behavioral and toxic effects of cocaine: comparison with atropine and the selective dopamine uptake inhibitor 1-[2 (diphenylmethoxy)ethyl]-4-(3-phenyl-propyl)-piperazine. AB - Behavioral effects of cocaine that are relevant to its abuse have been associated with pharmacological actions at the dopamine uptake carrier. Benztropine (Cogentin) is an antiparkinson agent that has limited abuse despite its ability to block dopamine uptake, and has been suggested as a candidate for the treatment of cocaine dependence. Preclinical studies were conducted to assess the behavioral and toxic effects of benztropine alone and in conjunction with cocaine. Because of the mixed pharmacology of benztropine which includes antimuscarinic as well as dopaminergic actions, results obtained from parallel experiments with atropine and the selective dopamine uptake inhibitor, GBR 12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-propyl)-piperazine), were performed. All of the drugs stimulated locomotor activity of mice, but atropine and benztropine had much lower efficacy. Nonstimulatory doses of GBR 12935 enhanced the locomotor stimulant effects of cocaine, whereas benztropine and atropine did not share this effect. GBR 12935, benztropine and cocaine increased fixed interval responding, whereas atropine decreased fixed-interval response rates in rats. Only GBR 12935 and cocaine increased responding during timeout periods. GBR 12935, but not benztropine or atropine, fully reproduced the discriminative stimulus effects of cocaine (10 mg/kg). GBR 12935 and atropine augmented the discriminative stimulus effects of lower cocaine doses in rats. Only GBR 12935 and cocaine had convulsant effects and only GBR 12935 significantly enhanced the convulsant effects of cocaine in mice. These results document a behavioral and toxicity profile for benztropine distinct from that of classical dopamine uptake blockers. The data underscore further the potential of benztropine as a candidate for clinical evaluation in the treatment of cocaine dependence. PMID- 8613920 TI - K-7259, a novel dilazep derivative, and d-propranolol attenuate H2O2-induced cell damage. AB - We studied the effects of dilazep, K-7259 (a novel derivative of dilazep) and d propranolol on the change in cell shape and accumulation of nonesterified fatty acids (NEFA) induced by hydrogen peroxide (H2O2) in isolated rat cardiac myocytes. Myocytes were incubated in a Krebs-Ringer bicarbonate buffer containing 2 mM diethyltriamine pentaacetic acid (DETAPAC) and 2mM FeSO4 for 10 min, and then treated with 2mM H2O2 for 50 min. Before the treatment with H2O2, the percentage of the number of rod-shaped cells to that of total cells was 66 +/- 2%, and decreased to 35 +/- 3%, 25 +/- 4% and 14 +/- 2%, after 30, 40 and 50 min of the H2O2 treatment, respectively. The levels of NEFA (lauric, myristic, palmitoleic, arachidonic, linoleic, palmitic, oleic and stearic acids) increased after the treatment with H2O2. In the absence of FeSO4 and DETAPAC, however, H2O2 did not have these effects, and therefore all the experiments with drugs were performed in the presence of Fe2SO4 and DETAPAC. K-7259 (30 microM) and d propranolol (50 microM) attenuated both the changes in cell shape and accumulation of NEFA induced by H2O2, whereas dilazep (30 or 50 microM) did not. N-(2-mercaptopropionyl)glycine (2 mM), an .OH scavenger, inhibited the H2O2 induced changes completely. These results suggest that K-7259 and d-propranolol attenuate the H2O2-induced changes in cell shape and accumulation of NEFA, probably because of their .OH-scavenging effect. PMID- 8613922 TI - Pharmacokinetics of alpha-methyl-L-tryptophan in rhesus monkeys and calculation of the lumped constant for estimating the rate of serotonin synthesis. AB - We have determined several kinetic and pharmacokinetic parameters of L-tryptophan (Trp) and alpha-methyl-L-tryptophan (alphaMTrp) in the rhesus monkey from which the lumped constant for the alphaMTrp method of estimating serotonin synthesis rates is calculated. AlphaMTrp was isolated from DL-alphaMTrp using a chiral separation column with high performance liquid chromatography. AlphaMTrp (50 microgram/kg) was administered i.v. to four adult male rhesus monkeys and arterial blood samples were collected for a 4-hr period. Plasma concentrations, as determined by high performance liquid chromatography with electrochemical detection, were best fitted by a tri-exponential equation. Plasma protein binding of Trp and alphaMTrp was determined by measuring concentrations in ultrafiltrates obtained at 30 degrees C. After a 2-hr adjusted rate infusion of alphaMTrp designed to establish steady-state plasma concentrations, three adult male rhesus monkeys were killed by exsanguination with perfused ice-cold saline. Brain/arterial plasma concentration ratios of Trp and alphaMTrp and the Michaelis Menten parameters for tryptophan hydroxylase, EC 1.14.16.4, with Trp and alphaMTrp as initiating substrates, were determined for seven brain regions. The lumped constants determined for the different brain regions were not significantly different from each other and indicate, that for modeling purposes, the brain may be treated as a homogeneous area and the lumped constant given a single value, 0.18 +/- 0.05. PMID- 8613921 TI - Chronic d-amphetamine or methamphetamine produces cross-tolerance to the discriminative and reinforcing stimulus effects of cocaine. AB - These experiments tested the hypothesis that chronic administration of d amphetamine (d-A) or methamphetamine (METH) would produce cross-tolerance to the discriminative and/or reinforcing effects of cocaine. One group of rats (n = 20) was trained to detect cocaine (10.0 mg/kg; i.p.) from vehicle; cocaine (1.0-17.8 mg/kg) dose dependently substituted for the training dose. Chronic administration of d-A or METH (0.32, 1.0 and 3.2 mg/kg/12 hr for 7 days) resulted in cross tolerance to the discriminative stimulus effects of cocaine. A second group of rats (n = 12) was implanted with indwelling jugular catheters and were trained to self-administer cocaine under a fixed-ratio 2 schedule of reinforcement. This group of rats also received chronic d-A or METH (0.32, 1.0 and 3.2 mg/kg/12 hr for 7 days. In this group, chronic administration of the highest dose of d-A and of METH (3.2 mg/kg) resulted in cross-tolerance to the self-administration of cocaine. A third group of rats (n = 15) was implanted with indwelling jugular catheters and were trained to self-administer cocaine under a progressive-ratio schedule of reinforcement. Chronic administration of d-A and METH (3.2 mg/kg/12 hr for 7 days) resulted in cross-tolerance to the self-administration of cocaine under this progressive-ratio schedule. The data obtained from these experiments demonstrate that chronic treatment with central nervous system stimulants of the amphetamine type (d-A or METH) produces cross-tolerance to both the discriminative and reinforcing effects of cocaine. PMID- 8613923 TI - The selectivity in vitro of the stereoisomers of the beta-3 adrenoceptor agonist BRL 37344. AB - The stimulation by BRL 37344 of lipolysis in rat adipose tissues, and of relaxation of the rat distal colon, is mediated by the beta-3 adrenoceptor. The stereochemical requirements of the beta-3 adrenoceptor are poorly understood. The activities of the four stereoisomers of BRL 37344 (i.e., two pairs of diastereoisomers) on three beta-3 adrenoceptor-mediated responses (brown and white adipose tissue lipolysis and relaxation of distal colon) have been determined and compared with those responses mediated by beta-1 adrenoceptors (increase in atrial rate) and beta-2 adrenoceptors (uterine relaxation). The potency order for the stereoisomers (RR>RS=SR>>SS) was the same for all tissues, regardless of whether the response was mediated by beta-1, beta-2 or beta-3 adrenoceptors. These results indicate that both chiral centers are determinants of agonist potency at all three subtypes of the beta adrenoceptor. Furthermore, agonist activity at beta-1, beta-2 and beta-3 adrenoceptors resides predominantly with the RR enantiomer. Finally, the RR enantiomer of BRL 37344 was a more potent agonist in brown adipocytes (EC50 = 3.3 +/- 0.8 nM) than in white adipocytes (EC50 = 5.7 +/- 0.9 nM) or colon (EC50 = 27.5 +/- 7.7 nM). PMID- 8613924 TI - Effects of cocaine on pulsatile activity of hypothalamic-pituitary-adrenal axis in male rhesus monkeys: neuroendocrine and behavioral correlates. AB - Cocaine stimulates the hypothalamic-pituitary-adrenal (HPA) axis in rodents and in humans. This study examined the acute effects of cocaine (0.4 and 0.8 mg/kg) and saline placebo on pulsatile adrenocorticotropic hormone (ACTH) and cortisol release in seven male rhesus monkeys. Pulsatile ACTH and cortisol release were evaluated with an intensive (2-min) venous blood sampling procedure and cluster analysis. In addition, the behavioral responses to cocaine were analyzed to assess the relationship between HPA axis activation and behavior. Although analysis of group data revealed significant (P < .05) increases in pulse amplitude and incremental peak height of ACTH and cortisol release after cocaine (0.8 mg/kg) administration, examination of individual data indicated that this effect was not consistent across all monkeys. Cocaine (0.8 mg/kg) increased ACTH plasma levels within 4.7 +/- 1.3 min (P < .05) and amplitude-related characteristics (P < .05) of pulsatile ACTH and cortisol release only in those animals that subsequently showed behavioral stimulation (high responders: n = 3). The frequency of pulsatile ACTH and cortisol remained unchanged by cocaine. Cocaine (0.8 mg/kg) decreased the mean amplitude of ACTH peaks with no changes in pulsatile cortisol release in the four monkeys that showed no behavioral stimulation (low responders). These differences in pulsatile ACTH and cortisol release patterns after cocaine could not explained by different plasma cocaine levels. Peak plasma cocaine levels averaged 63.1 +/- 13.4 and 78.0 +/- 21.4 ng/ml within 2 min after lower dose and 183.3 +/- 52.3 and 204.3 +/- 50.8 ng/ml after higher dose of cocaine in high- and low responder groups, respectively (P > .05; N.S.). Base-line cortisol, but not ACTH, levels were higher (P < .05) in low responders before administration of 0.8 mg/kg of cocaine. Peak and valley characteristics of base-line cortisol release were higher in low responders than in high responders and an inverse relationship was found between basal cortisol levels and postcocaine ACTH release and behavior. In summary, cocaine stimulated the pulsatile ACTH and cortisol release by increasing the amplitude of secretory episodes in behaviorally responsive monkeys. PMID- 8613925 TI - Alpha-1 adrenergic regulation of lactate production by white adipocytes. AB - The production of lactate from glucose and lactate release have recently been recognized as a novel function of adipose tissue. It has been demonstrated that catecholamines are able to induce lactate production; nevertheless, the adrenoceptor subtype involved has not yet been clearly identified. We studied the adrenergic regulation of lactate production by epididymal adipocytes. We showed that lactate production was enhanced by noradrenaline (a nonselective adrenergic agonist) and phenylephrine (an alpha-1 adrenergic agonist). On the other hand, isoproterenol (a nonselective beta agonist) and UK 14304 (an alpha-2-adrenergic agonist) did not stimulate glycolysis. Moreover, we observed that the maximal glycolysis induced by 10(-7) M phenylephrine was not significantly different from the maximal lactate production induced by 10(-5) M norepinephrine (0.43 +/- 0.03 vs. 0.39 micromol lactate/10(6) cells. 15 min.). The sensitivity of adipocytes to glycolytic stimulation by catecholamines was much higher for phenylephrine (pD2 = 9.66 +/- 2.30) than for norepinephrine (pD2 = 7.35 +/- 0.113, P < .05). Finally, the stimulation of lactate production by norepinephrine was totally inhibited by the presence of prazosin (10(-6)M) in the incubation medium. Our findings suggest that lactate production by epididymal adipocytes is under the control of alpha-1 adrenoceptors. PMID- 8613926 TI - The validity of spot urine samples for low-level occupational mercury exposure assessment and relationship to porphyrin and creatinine excretion rates. AB - Hg and porphyrin levels in single void urine specimens (spot samples) were compared with calculated 24-hr urine levels in 35 (20 [correction of 25] male and 15 female) practicing dentists who had been occupationally exposed to low levels of elemental Hg. The study aimed to: 1) determine the individual variability for Hg and porphyrin concentrations in spot samples over a 24-hr period; 2) test for the presence of diurnal variation in urinary Hg and porphyrin concentrations; and 3) determine the time of day at which a spot sample would give a Hg concentration closest to the 24-hr average concentration. Results confirmed previous reports of a first-order diurnal pattern with a mid-morning peak for Hg concentration (P < .001). A second-order model best described creatinine excretion (P = .0089), with peaks at about 5:00 and 19:00. The use of creatinine adjustment for Hg concentration significantly reduced the intraindividual variation around the diurnal curve. No diurnal patterns were found for any of the porphyrins examined. We recommend that, for small clinical studies using urinary Hg concentration, 24 hr sampling would be ideal, but that for mass screenings and cross-sectional studies, spot samples may be useful because they correlate fairly well with 24-hr averages (creatinine adjusted, r = 0.61; unadjusted, r = 0.74). Because of the existence of diurnal variation, for all cases using serial sampling attention should be paid to time of day. PMID- 8613927 TI - Alpha-1 adrenoceptor subtypes involved in mediating adrenergically induced antinatriuresis and antidiuresis in two kidney, one clip Goldblatt and deoxycorticosterone acetate-salt hypertensive rats. AB - This study characterized the alpha-1 adrenoceptor subtypes involved in adrenergically induced antinatriuresis and antidiuresis in pentobarbital anesthetized deoxycorticosterone acetate (DOCA)-salt and two kidney, one clip (2K1C) Goldblatt hypertensive rats. In the DOCA-salt rats, phenylephrine infusion (100 microgram kg(-1) hr(-1) close renal arterially) caused increases in blood pressure of 5 to 10%, decreases in renal blood flow of 5 to 12%, whereas there were large reversible decreases in urine flow and absolute and fractional sodium excretions of 55 to 70%. The presence of chloroethylclonidine (10 microgram kg( 1) hr(-1) to block alpha-1B adrenoceptors) had no effect on the magnitude of the phenylephrine-induced excretory responses, but after 5-methylurapidil (10 microgram kg(-1) hr(-1) to block alpha-1A adrenoceptors) they were abolished. Infusion of phenylephrine in the 2K1C hypertensive rats caused small hemodynamic and renal blood flow responses with marked 55 to 70% reductions in urine flow and absolute and sodium excretion. The phenylephrine-induced antinatriuresis and antidiuresis, which was probably due to activation of alpha-1 adrenoceptors present on the renal tubular epithelial cells, and the fact that these tubular responses were blocked by 5-methylurapidil but not by chloroethylclonidine in both DOCA-salt and 2K1C Goldblatt hypertensive models, means that the alpha-1A adrenoceptors was the major functional subtype present. PMID- 8613928 TI - Befloxatone, a new reversible and selective monoamine oxidase-A inhibitor. I. Biochemical profile. AB - Befloxatone, a novel oxazolidinone derivative, inhibited selectively and competitively monoamine oxidase (MAO)-A in human and rat brain, heart, liver and duodenum homogenates with Ki values ranging from 1.9 to 3.6 nM for MAO-A and from 270 to 900 nM for MAO-B. In vitro, befloxatone was more potent at inhibiting MAO A activity than reference compounds (befloxatone > harmaline > brofaromine > BW 137OU87 > RS 8359 > toloxatone > moclobemide). The inhibition of MAO-A by befloxatone was time-dependent and fully reversible after dilution. After p.o. administration, befloxatone induced a dose-dependent and selective inhibition of rat brain and duodenum MAO-A activities ex vivo with ED50 values of 0.06 and 0.025 mg/kg, respectively. Befloxatone (0.5 mg/kg p.o.) decreased MAO-B activity by only 20% in both tissues. In the brain, liver and duodenum, the inhibition of MAO-A activity by befloxatone was short lasting. Twenty-four hours after administration of befloxatone (0.75 mg/kg p.o.), a full recovery of MAO-A activity was observed in the brain, but the enzyme activity was still decreased by 38 and 56% in the duodenum and liver, respectively. In the rat brain, befloxatone (0.75 mg/kg p.o.) increased levels of norepinephrine, dopamine and 5 hydroxytryptamine and decreased levels of their respective deaminated metabolites. These variations were dose-dependent and reversed 24 hr after administration. In addition, befloxatone (0.75 mg/kg p.o.) decreased free 3,4 dihydroxyphenylethylene glycol levels in the brain and plasma. Befloxatone (10 microM) did not modify the activities of diamine or benzylamine oxidase and did not interact with monoamine uptake mechanisms or with a variety of neurotransmitter or drug receptor sites. In conclusion, the neurochemical profile of befloxatone demonstrates that this compound is a selective, competitive, potent and reversible MAO-A inhibitor. PMID- 8613929 TI - Befloxatone, a new reversible and selective monoamine oxidase-A inhibitor. II. Pharmacological profile. AB - The pharmacological profile of befloxatone, a reversible, selective and competitive inhibitor of monoamine oxidase-A has been investigated in rodents. In mice, befloxatone was more active at potentiating generalized tremors induced by L-5-hydroxytryptophan (ED50, 0.21 mg/kg p.o.) than phenylethylamine-induced stereotypies (ED50, 58 mg/kg p.o.), indicating a very high in vivo selectivity for inhibition of the A form of monoamine oxidase. Befloxatone showed potent activity in behavioral models in rodents predictive of antidepressant activity (forced swimming test, learned helplessness and reserpine reversal) with minimal effective doses of 0.1 to 0.2 mg/kg p.o. In these tests, befloxatone was much more potent (10- to 500-fold) than reference antidepressant compounds (reversible and irreversible monoamine oxidase inhibitors and monoamine reuptake inhibitors). In rats, befloxatone increased rapid eye movement sleep latency and decreased rapid eye movement sleep duration, without rebound effects. Potential anxiolytic activity was observed in the elevated-plus maze test in rats (minimal effective dose, 1-2 mg/kg p.o.). Befloxatone had no effect on motor performance, did not induce sedative or stimulant activity up to doses of 200 mg/kg p.o. and was devoid of anticholinergic activity in mice. Interaction studies with p.o. dietary tyramine (12 mg/kg), carried out in freely moving rats, demonstrated that, in contrast to irreversible monoamine oxidase inhibitors, befloxatone did not potentiate the pressor effect of this amine in the range of doses which showed pharmacological activity in antidepressant behavioral models. Furthermore, of the compounds tested (moclobemide, brofaromine, nialamide and phenelzine), comparison of doses active in antidepressant models and doses potentiating the pressor effects of tyramine demonstrated that befloxatone had the best therapeutic index. The results suggest that befloxatone will show clinical antidepressant activity at low doses and will be devoid of the side effects associated with irreversible monoamine oxidase inhibitors. PMID- 8613930 TI - Blockade of the serotonin and norepinephrine uptake processes by duloxetine: in vitro and in vivo studies in the rat brain. AB - In in vitro uptake experiments, duloxetine inhibited [3H]5-hydroxytryptamine (5 HT) and [3H]norepinephrine (NE) uptake in hippocampus slices of control rats with IC50 values of 28 and 46 nM, respectively. The uptake of both[3H]5-HT and [3H]NE was equipotently inhibited in hippocampus slices prepared from rats treated for 2 days with different doses of duloxetine (5, 10, 15 and 20 mg/kg/day s.c.). In in vivo electrophysiological experiments in the hippocampus, the effects of duloxetine on the suppression of CA3 pyramidal neuronal firing activity by microiontophoretically applied 5-HT and NE were examined with two modes of administration. Five successive i.v. injections (2 mg/kg each) significantly and dose-dependently prolonged the recovery time of the firing activity of hippocampus CA3 pyramidal neurons from the 5-HT applications. A 2-day treatment (10, 15 and 20 mg/kg/day s.c.) also increased the recovery time in a dose dependent manner. Whereas the recovery time from NE applications was unaffected by low doses of duloxetine (2 mg/kg i.v.; 10 mg/kg/day for 2 days), it was prolonged significantly by higher doses (8 and 1 0 mg/kg iv.; 20 mg/kg/day for 2 days). Acute i.v. injections of duloxetine suppressed the spontaneous firing activity of dorsal raphe 5-HT and locus ceruleus NE neurons with ED50 values of 99 and 475 microgram/kg, respectively. Taken together, the present results confirmed that duloxetine is a dual 5-HT/NE uptake inhibitor. Furthermore, the results obtained in in vivo experiments indicate that duloxetine has a preferential inhibitory effect on the 5-HT transporter. PMID- 8613931 TI - Reversible inhibition of rat gastric H+/K+-ATPase by T-330, 2-[2 dimethylaminobenzyl)sulfinyl]-1-(3-methylpyridine-2-yl)imidazole. AB - The effect of 2-[(2-dimethylaminobenzyl)sulfinyl]-1-(3-methylpyridine-2 yl)imidazole (T-330), on rat gastric H+/K+-ATPase was studied in vitro and in vivo in comparison with the irreversible proton pump inhibitor omeprazole. T-330 and omeprazole inhibited rat gastric H+/K+-ATPase at pH 6.1, and their IC50 values were 75 microM and 4.6 microM, respectively. Recovery from the T-330 inhibited H+K/+-AtPase activity was effected by beta-mercaptoethanol at concentrations above 10 microM, whereas significant recovery from the inhibition by omeprazole required 100 mM. When intraduodenally administered, T-330 (0.6-10 mg/kg) induced a more potent yet shorter-lasting inhibition of both gastric H+/K+ -ATPase activity and gastric acid secretion than did omeprazole (2.5-40 mg/kg). Recovery of the gastric H+/K+-ATPase activity depressed by omeprazole was completely blocked by an inhibitor of protein synthesis, cycloheximide, whereas that by T-330 was not prevented. This indicates that restoration of the enzyme activity after inhibition in vivo by T-330 does not require de novo synthesis of the enzyme in contrast to inhibition by omeprazole. beta-Mercaptoethanol (1 mM) fully restored the H+/K+-ATPase activity inhibited in vivo by T-330 but not by omeprazole. These observations indicate that T-330 reversibly inhibits gastric H+/K+-ATPase, resulting in a short-lasting antisecretory effect, and that the sulfhydryl groups of the enzyme are involved in the action of T-330. PMID- 8613932 TI - Effects of caffeine and theophylline on acetaminophen pharmacokinetics: P450 inhibition and activation. AB - Metabolism of acetaminophen (APAP) to its reactive metabolite N-acetyl-p benzoquinoneimine (NAPQI) is mediated by cytochrome P450. A pharmacokinetic study was conducted to quantitate changes in the formation clearance (Cl(f)) of NAPQI to assess in vivo the activation and inhibition of NAPQI formation by methylxanthines. Cl(f) of NAPQI was unaltered by methylxanthine administration in saline-pretreated rats. In phenobarbital-induced rats receiving a nontoxic dose of APAP (100 mg/kg i.v.), a single dose of caffeine (100 mg/kg i.p.) co administered with APAP increased the Cl(f) of NAPQI formation from 0.58 +/- 0.47 to 2.08 +/- 1.1 1 ml/min/kg (P = .01). Unlike caffeine, theophylline (93 mg/kg i.p.) had no effect on the Cl(f) of NAPQI in phenobarbital-induced rats. The increase in the Cl(f) of NAPQI immediately after a single dose of caffeine demonstrates that P450 activation by caffeine can occur in vivo, as we observed previously in microsomes. The same dose of APAP and methylxanthines also was administered to rats induced with methylcholanthrene. The co-administration of either a single dose of caffeine or theophylline diminished the Cl(f) of NAPQI by 86% (P = .01) and 52% (P = .03), respectively. These in vivo results agree with our previous studies of the effects of the methylxanthines on the formation of NAPQI in rat liver microsomes. PMID- 8613933 TI - Phencyclidine metabolite irreversible binding in the rat: gonadal steroid regulation and CYP2C11. AB - These studies were conducted to determine the effect of hormones on sex-related differences in phencyclidine (PCP) metabolite irreversible binding and to determine the cytochrome P450 isoform(s) involved in this process. Sprague-Dawley male rats were castrated or administered estradiol and Sprague-Dawley female rats were ovarectomized or ovarectomized and given testosterone. Liver microsomal metabolism studies demonstrated that PCP metabolite binding to proteins was significantly altered by testosterone and estrogen administration. Castration of male rats decreased metabolite binding to 57% of sham-operated male levels, and administration of testosterone to ovarectomized female rats increased metabolite binding to 41 % of normal male levels. No metabolite adducts could be detected in microsomes from male rats administered estradiol or from sham-operated females given vehicle. These hormone-induced changes in metabolite binding closely matched the hormone-induced changes in CYP2C11 function and expression in these same microsomes. PCP metabolite irreversible binding to microsomal proteins was highly correlated with CYP2C11 function (as assessed by the formation of 2alpha OH-testosterone, r = 0.91) and with CYP2C11 expression (as assessed by Western blot analysis, r = 0.95). In addition, an anti-CYP2C11 monoclonal antibody almost completely inhibited PCP metabolite binding (down to 7% of control male values) in an antibody concentration-dependent manner. These data strongly implicate CYP2C11 as an isoform involved in PCP metabolism and the formation and/or binding of a reactive PCP metabolite to microsomal proteins. PMID- 8613934 TI - A pharmacological analysis of receptor subtypes and the mechanisms mediating the biphasic response induced by kinins in the rat stomach fundus in vitro. AB - Bradykinin (BK), des-Arg9-BK (DABK) and related kinins caused biphasic response (BR) in rat stomach fundus (RSF) precontracted with BaCl2. The B2 receptor antagonist HOE 140 (3-30 nM) produced parallel rightward shifts of the contractile concentration-response curve (CRC) for BK, yielding a pA(2) value of 9.07 +/- 0.27 and slope of 0.99, but caused only discrete rightward shift of the relaxant CRC for BK, leaving the BR CRC to DABK unaffected. The B1 receptor antagonist des-Arg9-NPC 17761 (10 nM to 1 microM) caused graded rightward shifts of the relaxant (but not the contractile) CRC to DABK, yielding a pA2 value of 8.35 +/- 0.05 and slope of 0.59, but had no effect on BK-induced BR. BK- and DABK (100 nM) induced relaxation were almost suppressed by apamin (1 microM) or by nifedipine (1 nM), but were unaffected by nitric oxide synthase inhibitors, methylene blue, lipo and cyclooxygenase inhibitors, selective receptor antagonist for histamine (H1 and H2), nicotine, platelet activating factor, tachykinins (both NK1 and NK2, calcitonin gene-related peptide, vasoactive intestinal peptide and ganglion blocking agent. BK- and DABK-mediated relaxations were reduced in Ca2+ -free medium plus EGTA, although BK-mediated contraction was more resistant. Escherichia coli endotoxin treatment (10 microgram /rat), 24 hr before, potentiated DABK-induced relaxation, but not contraction, and reduced BK-mediated relaxation (P < .05). It is concluded that RSF express both B1 and B2 receptors. BK-induced contraction involves activation of B2 receptors, although DABK-induced relaxation is mediated by B1 receptors. Both B1 and B2 receptors in RSF are constitutive, but LPS treatment caused induction of B1 and down-regulation of B2 receptors. Finally, kinin-mediated relaxation in RSF are coupled to activation of Ca2+ activated K+ channels, and rely on Ca2+ influx via L-type voltage-sensitive channels. PMID- 8613935 TI - The role of specific eicosanoids in mediating the acute narcotic effects of ethanol. AB - The pharmacological effects of ethanol may be in part due to increased membrane fluidity, resulting in increased phospholipase A2 activity and the subsequent conversion of released arachidonic acid (AA) into pharmacologically relevant eicosanoids (prostaglandins/leukotrienes). A significant correlation between the in vivo and in vitro potency of prostaglandin synthetase (PES) inhibitors to antagonize PES activity and their ability to antagonize the acute narcotic and rate-depressant effects of ethanol support this hypothesis. However, inhibition of PES not only decreases the production of at least five prostaglandins and thromboxane but may shunt free AA into the lipoxygenase cascade with subsequent formation of leukotrienes. The purpose of the present study was to systematically investigate numerous points in the AA cascade to determine the eicosanoid products relevant to the acute narcotic effects of ethanol. Ethanol-induced loss of the righting reflex in mice was used as the behavioral endpoint. The relative importance of PES metabolites vs. lipoxygenase metabolites was determined via administration of specific enzyme inhibitors (phospholipase, lipoxygenase and dual PES/lipoxygenase inhibitors) and receptor agonists/antagonists (prostaglandin and leukotriene). Pretreatment with specific lipoxygenase inhibitors, dual PES/lipoxygenase inhibitors and leukotriene antagonists suggest a negligible role for lipoxygenase metabolites in acute ethanol-induced narcosis. Pretreatment with prostaglandin agonists and antagonists suggest a significant role for prostaglandin E (PGE) and a minor role for prostaglandin D in mediating the acute effects of ethanol. The PGE agonist 16,16-dimethyl PGE significantly enhanced the narcotic effects of ethanol (> 400%), whereas the prostaglandin receptor antagonist L-640,035 significantly decreased the effects of ethanol ( 54%). These results further support the hypothesis that eicosanoid formation is important in the biochemical and behavioral effects of ethanol. PMID- 8613936 TI - Basolateral glutarate transport by isolated S2 segments of rabbit kidney proximal tubules. AB - The properties of tubular glutarate uptake and the coupling to p-aminohippurate (PAH) transport were studied on isolated nonperfused S2 segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were totally collapsed, the tubular glutarate uptake may be assumed to represent the quantity transported across the basolateral membrane. The results show that the S2 segments effectively accumulated 14C glutarate (500 micron). The cell/bath 14C-glutarate concentration ratio reached maximum values of about 20 after a 20-min incubation period. The tubular 14C glutarate accumulation could be markedly depressed by lithium (5 mM) but not by probenecid (1 mM), which, however, inhibited tubular 3H-PAH (1 microM ) uptake. External PAH (0.1 mM) stimulated efflux of 14C-glutarate from S2 segments preloaded with 14C glutarate (500 microM), and external glutarate stimulated tubular uptake of 3H PAH (1 microM), providing evidence for glutarate-PAH countertransport in proximal S2 segments. The phorbol ester, phorbol 12-myristate 13-acetate (0.1 microM), did not affect steady-state cell/bath 14C-glutarate concentration ratio nor the initial 14C-glutarate transport rate. Protein kinase C may, therefore, not be a regulator of basolateral glutarate transport in renal S2 proximal tubules. PMID- 8613937 TI - Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. AB - Cytochrome P450 chemical inhibitors are widely used to define the role of individual cytochrome P450 isozyme(s) in a metabolism process. In this study, cytochrome P450 isoform-dependent reactions were investigated on our human liver microsomes bank (n = 34) and characterized for both KM and VMAX values (n > or = 3). These metabolic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogenation (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (Ki) and the inhibition-type toward their specific substrate. Results were as follows: alpha-naphthoflavone (CYP1A1; mixed-type interaction with a Ki = 0.01 microM), furafylline (CYP1A2; competitive-type interaction with a Ki = 3 microM when microsomes were incubated with both furafylline and phenacetin; noncompetitive-type interaction with a Ki = 0.6 microM when microsomes were preincubated with furafylline and NADPH), pilocarpine (CYP2A6; competitive-type interaction with a Ki = 4 microM), sulfaphenazole (CYP2C9; competitive-type interaction with a Ki = 0.3 microM), quinidine (CYP2D6; competitive-type interaction with a Ki = 0.4 microM, diallyldisulfide (CYP2E1; noncompetitive-type interaction with a Ki = 150 microM on an aniline concentration range of 10-60 microM; competitive-type interaction with a Ki = 100 microM on an aniline concentration range of 80-2000 microM) and ketoconazole (CYP3A4; mixed-type interaction with a Ki = 0.015 microM). Once the inhibitors' potency was determined, the selective effects of these inhibitors were evaluated after incubation of human hepatic microsomes with isoform selective substrates in the presence of the different chemical inhibitors. Up to 10 times the Ki value toward the isoform-selective probe, pilocarpine, sulfaphenazole, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacetin and 7-ethoxyresorufin O-deethylation processes, a consequence of the absence of CYP1A1 in noninduced human liver. Diallyldisulfide exhibited broad and nonspecific inhibitory effects. When used in their "window of selectivity," ie., up to 10-fold the Ki value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isoform-specific reactions when analyzed at their KM values. PMID- 8613938 TI - Protection of ischemic and reperfused rat myocardium by the nonglucocorticoid 21 aminosteroid U-74389G, a new inhibitor of lipid peroxidation. AB - We studied the effects of the aminosteroid U-74389G (21-[4-(2, 6-di-1 pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-pregna-1,4,9(11)- triene-3,20-dione(2) 2-butenenedionate), a putative inhibitor of lipid peroxidation, which protects the rat myocardium after ischemia and reperfusion. Pentobarbital-anesthetized (50 mg/kg) rats were subjected to 60 min of occlusion of the left main coronary artery followed by 60 min of reperfusion. Myocardial ischemia/reperfusion produced a large cardiac necrosis (81 +/- 8.6% of the area at risk and 65 +/- 14.8% of the total left ventricle), polymorphonuclear infiltration in the jeopardized tissue (myeloperoxidase activity = 4.2 +/- 2.1 U X 10(-3)/g tissue in the area at risk and 7.0 +/- 3.6 U X 10(-3)/g tissue in the necrotic area), hydroxyl radical (OH.) formation (0.55 +/- 0.16 nmol/ml), increased plasma malonylaldehyde (40.2 +/- 3.9 nmol/ml) and lactate dehydrogenase (431 +/- 30 mIU/ml) and caused a decrease in the survival rate. Treatment with U-74389G (15 and 30 mg/kg i.v.) at the onset of reperfusion caused a reduction of necrotic area expressed as a percentage of either the area at risk (76 +/-7.4% with 15 mg/kg and 69 +/- 13.5% with 30 mg/kg; P < .05) or the total left ventricle (53 +/ 13.6% with 15 mg/kg and 46 +/- 16.8% with 30 mg/kg; P < .05). Treatment U-74389G reduced the myeloperoxidase activity, evaluated as an index of neutrophil infiltration, both in the area at risk (2.7 +/- 1.1 and 2.2 +/- 1.7 U X 10(-3)/g tissue with the doses of 15 and 30 mg/kg, respectively; P < .05) and in the necrotic area (4.3 +/- 2.4 and 3.8 +/- 2.9 U X 10(-3)/g tissue with 15 and 30 mg/kg, respectively; P < .05); decreased OH. formation (measured indirectly by the administration of the trapping agent salicylic acid); and analyzing the hydroxylation product 2,5-dihydroxybenzoic acid during reperfusion (0.35 +/- 0.12 and 0.32 +/- 0.15 nmol/ml with the doses of 15 and 30 mg/kg, respectively; P < .005). Treatment inhibited lipid peroxidation by blunting plasma malonylaldehyde (26.7 +/- 3.1 and 20.8 +/- 3.3 with the doses of 15 and 30 mg/kg, respectively; P < .001), prevented cellular disruption by reducing the increase of plasma lactate dehydrogenase (288.6 +/- 28 and 201.3 +/- 16 mIU/ml with the doses of 15 and 30 mg/kg, respectively; P < .001). Finally, U-74389G enhanced the survival rate evaluated at the end of the experiment (from 40 to 87%). These outcomes suggest that the drug may have potential for cardioprotective use in acute myocardial infarction. PMID- 8613939 TI - Testosterone causes direct relaxation of rat thoracic aorta. AB - Several recent studies have provided evidence that gonadal steroid hormones can exert acute (nongenomic) effects on both neural and vascular tissues. This study examines the acute effects of testosterone (T) on vascular reactivity of the rat thoracic aorta. Aortic rings from male Sprague-Dawley (SD) rats with (+ENDO) and without (-ENDO) endothelium were prepared for isometric tension recording. In (+ENDO) male aortae precontracted with phenylephrine (PE), T produced dose dependent relaxation from 25 microM (30.3 +/- 7.1%) to 300 microM (99.4 +/- 0.4%), whereas T vehicle (< or = 0.5% ethanol) had no effect. Pretreatment of (+ENDO) aortae with T (50 microM; 10 min) attenuated subsequent contractile responses to PE. Both maximal contraction and sensitivity to PE were reduced by T. Pretreatment of (+ENDO) aortae with both T and N omega-nitro-L-arginine methyl ester (250 microM) reversed in part the attenuating effects of T alone; however, both maximal response and sensitivity to PE were still reduced compared to control rings (without T or N omega-nitro-L-arginine methyl ester). Pretreatment of (-ENDO) aortae with T reduced sensitivity to PE, but had no effect on maximal contraction. T pretreatment (50 microM; 10 min) of both (+ENDO) female SD aortae and (+ENDO) male testicular-feminized rat aortae reduced maximal contraction and sensitivity to PE in both groups to a similar extent as in (+ENDO) male SD aortae. These data suggest that T has a direct vasodilating effect on the rat aorta, which involves endothelium-dependent (enhanced NO release) and independent mechanisms and is gender- and intracellular androgen receptor independent. PMID- 8613940 TI - The effects of LY293111Na, a leukotriene B4 receptor antagonist, on the pulmonary neutrophilia and CD11b expression caused by inhalation of a leukotriene B4 aerosol in rhesus monkeys. AB - The dose-response relationship between aerosolized leukotriene B4 (LTB4 and pulmonary neutrohilia was examined in a group of five rhesus monkeys. The effects of an oral dose of LY293111Na on LTB4-aerosol-induced pulmonary neutrophilia were also examined. Ex vivo expression of CD11b receptors on polymorphonuclear leukocytes from bronchoalveolar lavage fluid and peripheral whole blood were also assessed. Up-regulation of CD11b adhesion receptors by LTB4 was assessed ex vivo on the peripheral whole blood. Pulmonary neutrophilia was linearly associated with dose of inhaled LTB4. LY293111Na, at 10 mg/kg, significantly blocked the profound bronchoalveolar lavage neutrophilia produced by LTB4 aerosol inhalation. A large (48%), but not statistically significant, reduction was seen for CD11b expression on bronchoalveolar lavage polymorphonuclear leukocytes after pretreatment with LY293111Na. LY293111Na did not significantly change the number of white blood cells in peripheral blood. LY293111Na did significantly attenuate the LTB4-induced up-regulation of CD11b receptors on peripheral blood neutrophils. We conclude that LY293111Na may be an effective oral treatment for diseases that involve neutrophilic inflammation. PMID- 8613941 TI - Skeletal sarcoplasmic reticulum dysfunction induced by reactive oxygen intermediates derived from photoactivated rose bengal. AB - We investigated the role of reactive oxygen intermediates generated from photoactivation of xanthene dye rose bengal on skeletal sarcoplasmic reticulum (SR) function, which plays a major role in the regulation of intracellular Ca++ and thereby in the generation of force. We used SR microsomes of canine masseter muscle as a model system in which to explore the effect of oxidation by determining oxalate-supported Ca++ uptake, Ca++, Mg++-adenosine triphosphatase (Ca++-ATPase) activity and Ca++ permeability of the SR vesicles. Skeletal SR vesicles exposed to rose bengal (50 nM) illuminated at 560 nm resulted in significant inhibition of Ca++ uptake velocity and Ca++-ATPase activity and in stimulation of Ca++ permeability. The observed effect afforded by illuminated rose bengal was dependent on intensity of light. Most reactive oxygen species scavengers tested had no protective effect; histidine (a powerful quenching agent for singlet oxygen), however, significantly protected the effect of illuminated rose bengal on Ca++ uptake velocity and Ca++-ATPase activity. The illumination of rose bengal also caused histidine-inhibitable loss of total sulfhydryl groups of SR. The increased Ca++ permeability elicited by illuminated rose bengal was blunted by a cocktail of histidine-catalase, but not by histidine alone. Generation of reactive oxygen species (singlet oxygen, superoxide and hydroxyl radical) from photoactivation of rose bengal was studied by electron spin resonance spectroscopy by use of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP). We found that illumination of rose bengal formed a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct, which was effectively blunted by hydroxyl radical scavenger, dimethyl sulfoxide, and by superoxide scavenger, superoxide dismutase. The results of electron spin resonance study also showed that singlet oxygen was produced by photoactivation of rose bengal was detected as singlet oxygen-TEMP product (TEMPO); 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of TEMPO signal was strongly inhibited by histidine. Similarly, we could detect hydrogen peroxide production from illuminated rose bengal. It is suggested that photoactivation of rose bengal generated singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radical, and the data obtained from the present study indicate that singlet oxygen, rather than superoxide, hydrogen peroxide and hydroxyl radical, to be the active agent in the Ca++ transport system of SR; the observed effect of singlet oxygen may be due to sulfhydryl group oxidation. Our results are also consistent with the view that singlet oxygen does not appear to be an exclusive species that increases Ca++ permeability of SR vesicles, but the increased Ca++ permeability may be caused in part by hydrogen peroxide as well as singlet oxygen. PMID- 8613942 TI - Both dynorphin A(1-17) and [Des-Tyr1]dynorphin A(2-17) inhibit adenylyl cyclase activity in rat caudate putamen. AB - In this study, the ability of a series of dynorphin peptides to inhibit adenylyl cyclase (AC) activity was determined. The endogenous ligand of the kappa opioid receptor, dynorphin A(l-17) (Dyn A(l-17)), produced a significant concentration dependent inhibition of AC activity in membranes prepared from the caudate putamen (CPu) of naive Fischer 344 rats. The opioid receptor antagonist, naloxone (10(-5)M), which is predominantly mu opioid receptor directed, but with modest kappa and delta receptor activity, partially blocked this inhibition. Nor Binaltorphimine (10(-5)M), the selective kappa receptor antagonist, also blocked the effect of Dyn A(l-17), but to a lesser degree. [Des-Tyr1]Dyn A(2-17), a major nonopioid biotransformation product of Dyn A(l-17) with known biological activities, also inhibited AC in rat CPu membranes. Dyn A(l-13) inhibited AC, as did its major opioid biotransformation product, Dyn A(l-12). One of the major nonopioid biotransformation products of Dyn A(l-13), Dyn A(4-12), showed no activity. Dyn A(2-12), another nonopioid product of Dyn A(l-13), showed limited activity. Dyn A(l-6), a minor biotransformation product of both Dyn A(l-17) and Dyn A(l-13), also inhibited AC activity. These findings suggest that, in rat CPu membranes, the inhibition of AC activity by Dyn A(l-17) is mediated in part by kappa and mu opioid receptors. In addition, Dyn A(2-17), and to a lesser extent Dyn A(2-12), may bind to a yet unidentified site that is also coupled to the AC enzyme in rat CPU. PMID- 8613943 TI - The effect of protein binding on the deep tissue penetration and efflux of dermally applied salicylic acid, lidocaine and diazepam in the perfused rat hindlimb. AB - Doses of radiolabeled water, salicylic acid, lidocaine and diazepam were applied in 2 ml of phosphate buffer saline to the exposed dermis on the thigh of a perfused rat hindlimb. Hindlimbs were perfused with Krebs-Heinseleit buffer at 37 degrees C containing 4% albumin or 2.5% dextran 40 at a constant rate of 4 ml/min. Clearance of solutes from dermal sites was monitored by frequent sampling at the dermal site and of the outflowing perfusate. In addition, the concentration of solutes in the various tissues below the treated and contralateral sites were determined at the end of a perfusion after dissection, solubilization and scintillation counting of the individual tissues. Estimates of the absorption rate constant from the dermis and the elimination rate constant from the perfused limb were made by the simultaneous fitting of absorption and efflux data with a one-compartmental pharmacokinetic model. Diazepam was cleared fastest through the dermis, followed by water, salicylic acid and lidocaine. The efflux of diazepam and salicylic acid from the hindlimb was significantly enhanced by the presence of albumin in the perfusate relative to no albumin (P < .05), whereas the efflux of water and lidocaine were unaffected by changes in albumin content. Analysis of individual tissues at the end of the perfusion showed that water had the highest relative tissue concentrations with diazepam the least, high concentrations appearing to partition into and remain associated with the dermis. The presence of albumin in the perfusate significantly increased the concentration of diazepam found in contralateral skin samples (P < .05) and showed the same trend for salicylic acid and lidocaine. These findings show that protein binding significantly affects the deep tissue penetration and distribution of dermally applied solutes and that this effect is more prominent for highly protein-bound solutes. PMID- 8613944 TI - Determination and metabolism of dithiol chelating agents. XVII. In humans, sodium 2,3-dimercapto-1-propanesulfonate is bound to plasma albumin via mixed disulfide formation and is found in the urine as cyclic polymeric disulfides. AB - The binding of 2,3-dimercapto-1-propanesulfonate (DMPS) in plasma was determined in three healthy young adults after a single 300-mg p.o. dose. By 5 hr after DMPS administration, 62.5% of the total plasma DMPS was bound to proteins. The remainder consisted of nonprotein associated DMPS disulfides (36.6%) and unaltered DMPS (0.9%). Protein-bound DMPS consisted of a DMPS-albumin complex (84%) and a higher molecular weight protein complex (16%), perhaps albumin aggregates. DMPS was released from the isolated DMPS-albumin complex after treatment with dithiothreitol, indicating that it was bound via a disulfide linkage. The half-life of unaltered DMPS was 1.8 hr, whereas that of altered DMPS was 20 hr, suggesting that the DMPS-albumin disulfide complex is stable and that DMPS was released from it slowly. In addition, the biotransformation of OMPS to disulfide forms was extensive. By 9 hr after administration, 10% of the total urinary DMPS was unchanged drug and 90% was altered DMPS. The latter was converted to DMPS by dithiothreitol, indicating that the altered DMPS consisted of disulfides. In 2- to 4-hr urine, DMPS disulfides included cyclic polymeric DMPS disulfides (97%), DMPS-cysteine (1:2) mixed disulfide (2.5%) and acyclic DMPS disulfide (0.5%). The cyclic polymeric DMPS disulfides were present in a major (91.5%) and minor (5.5%) form. DMPS-albumin mixed disulfide and nonprotein DMPS disulfides may prolong the heavy metal mobilizing activity of DMPS and thus may represent reservoirs of DMPS which can be released by disulfide reduction in vivo. PMID- 8613945 TI - Dose-dependent transitions in nucleus accumbens cell firing and behavioral responding during cocaine self-administration sessions in rats. AB - At the initiation of cocaine self-administration sessions, neurons in the rat nucleus accumbens (NA) exhibit a spontaneous transition in firing rate from activity unrelated to the reinforced response to one of four types of patterned discharges. This transition in NA activity is accompanied by a shift from high response rates at the start of the session ("Load-Up" behavior) to a lower rate for the remainder of the session. In this study, the relationship between transitions in behavioral responding and NA activity was examined further by changing the dose of cocaine per session (0.66, 0.50, 0.33, 0.16 or 0.08 mg/inf). Results show that the number of Load-Up responses significantly increased at lower cocaine doses [0.16, 0.08 mg/infusion (inf)] and decreased at higher doses (0.33, 0.50, 0.66 mg/inf). Thereafter, animals responded either with regularly spaced interinfusion intervals (INTs) at high doses (0.33, 0.50 and 0.66 mg/inf), or frequent bursts and pauses in responding at low doses (0.16 and 0.08 mg/inf. NA neurons exhibited a spontaneous transition in firing rate that was significantly correlated with this shift in behavioral responding across different doses of cocaine. Pretreatment with the dopamine D1 receptor antagonist SCH23390 (5 or 10 microgram/kg) prolonged the onset of NA patterned discharges, similar to responding for low doses of cocaine (0.08 and 0.16 mg/inf. These findings are discussed in terms of a functional role of the NA in mediating the reinforcing properties of cocaine. PMID- 8613946 TI - The actions of prostaglandin E2 on potassium currents in rat tail artery vascular smooth muscle cells: regulation by protein kinase A and protein kinase C. AB - In vascular smooth muscle cells (VSMCs) of rat tail artery, prostaglandin E2 (PGE2) inhibited a voltage-dependent, delayed rectifier K channel current (Ik). The inhibition was concentration-dependent, via a receptor-mediated mechanism involving the activation of G protein(s) (Ren et al., 1995). In this study, we show that the PGE2-induced inhibition of Ik was mediated by activation of protein kinase A (PKA) and possibly protein kinase C (PKC). Pretreatment of the cells with cyclic adenosine 3',5'-monophosphothioate Rp-isomer (Rp-cAMPs), an inhibitor of adenosine 3', 5'-cAMP-dependent protein kinase (PKA), almost completely abolished the PGE2-induced inhibition. Forskolin, dibutyryl cAMP (Db-cAMP) and cyclic adenosine 3',5'cyclic monophosphothioate Sp-isomer (Sp-cAMPs), activators of adenylate cyclase and PKA, mimicked the effect of PGE2 on Ik. Phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine did not alter the PGE2-induced inhibition of Ik. Moreover, we also found that phorbol myristate acetate (PMA), a PKC activator, significantly suppressed Ik. Both the kinase inhibitor staurosporine and down-regulation of PKC by prolonged exposure of the cells to PMA blocked the PGE2-induced inhibition of Ik, but had no effects on the forskolin, Db-cAMP or SpcAMP-induced effect on Ik. Pretreatment of the cells with Rp-cAMPs only partially diminished the degree of Ik inhibition evoked by PMA. Assay of cAMP content indicated that both PGE2 and PMA induced cAMP accumulation. These results strongly suggest that the modulation of Ik by PGE2 in rat tail artery VSMCs involves signal transduction through both PKA and PKC activation. The activation of PKC may potentiate the cAMP-PKA stimulation, whereas the cAMP PKA cascade did not seem to affect the PKC pathway. These observations suggest that "cross talk" between the two second-messenger systems is involved in the mechanisms that mediate the effect of PGE2. PMID- 8613947 TI - Serotonin-mediated increase in prefrontal cortex dopamine release: pharmacological characterization. AB - Interactions between serotonin (5-HT) and dopamine (DA) neuronal systems in the prefrontal cortex (PFC) may be important in the pathophysiology of cognitive disorders such as schizophrenia. We have examined the effect of 5-HT, applied locally through a microdialysis probe, on extracellular DA in the PFC, and compared the response to that observed in the striatum. 5-HT in concentrations of 1 to 10 microM increased extracellular DA dose-dependently to a greater extent in the PFC than in the striatum. The PFC response was pharmacologically characterized to determine the 5-HT receptor subtype mediating the increase in DA levels. The coperfusion of selective 5-HT2A and 5-HT3 antagonists MDL 100,907 ((R (+)-(2,3-dimethoxyphenyl)-1-[2(4-flourophenylethyl)]-4- piperidine-methanol) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate), respectively, with 5-HT failed to significantly attenuate the 5-HT induced increase of extracellular DA. Furthermore, the local application of the 5-HT2A/2C agonist (+/-)-1-(2,5 dimethoxy-4-iodophenyl aminopropane did not yield an increase in extracellular DA. On the other hand, coperfusion of the selective 5-HT1B/1D antagonist GR 127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1 ,2,4-oxadiazol-3-yl)-[1,1-biphenyl]-4-carboxamide)) with 5-HT completely blocked the effect of 5-HT alone. Infusion of the selective 5-HT1B agonists CP 93,129 (3 (1,2,5,6-tetrahydro-4-pyridyl)pyrrolo[3,2-b]pyrid-5-one) and CP 94,253 (3 (1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrolo[3,2-b]pyridine) resulted in a significant increase in extracellular DA and the effect of CP 93,129 was attenuated by coperfusion of GR 127935. The results obtained demonstrate a functional interaction between DA and 5-HT pathways in the PFC, with evidence of potential mediation by the 5-HT1B receptor subtype. PMID- 8613948 TI - Budesonide inhibits T cell-initiated epithelial pathophysiology in an in vitro model of inflammation. AB - Recognition of the therapeutic value of glucocorticosteroids in the treatment of inflammation has preceded awareness of the mechanism(s) of action of these drugs. We recently showed that coculture of human T84 epithelial monolayers for 2 days with anti-CD3 activated peripheral blood mononuclear cells (A-PBM) led to impaired ion transport responses and reduced barrier function. We tested the hypothesis that budesonide, as a member of the new generation of more topically selective steroids, could prevent these immune-mediated epithelial abnormalities. Budesonide added to the coculture system dose-dependently inhibited the following functional T84 abnormalities measured in Ussing chambers: reduced transport responses (decreased short-circuit current changes to carbachol (raises [Ca2+]i) and forskolin (raises cAMP, cyclic adenosine monophosphate(i)); and increased permeability (decreased resistance and increased fluxes of 3H-mannitol and 51CrEDTA). For the beneficial effects of budesonide to be observed, PBM pretreatment (> or = 3 hr) and daily addition (for 2 days) to the coculture system was necessary. Budesonide (10(-7) M) dramatically reduced A-PBM proliferation (measured by 3H-thymidine incorporation) and cytokine (IL-1beta, IL 2, IL-6, IFN-gamma, TNF-alpha) production, but was not cytotoxic to immune cells. Budesonide treatment of T84 epithelial cells alone did not directly affect epithelial physiology, nor did it prevent epithelial abnormalities evoked by subsequent exposure to A-PBM or conditioned media from immune cells. Our studies showed that budesonide prevents epithelial dysfunction in this model by inhibiting activation of both T cells and monocytes. PMID- 8613949 TI - Inhibition of rho1 receptor GABAergic currents by alcohols and volatile anesthetics. AB - We studied the effects of alcohols and anesthetics on homomeric gamma aminobutyric acid (GABA) receptors formed of rho1 subunits expressed in Xenopus laevis oocytes. This subunit shares considerable amino acid sequence homology with the GABA(A) receptor subunits. In contrast to our previous findings with a variety of GABA(A) receptors, ethanol (10-100 mM) significantly inhibited the current induced by a low concentration (400 nM) of GABA, in an apparently competitive manner. Butanol (2-40 mM), hexanol (0.5-4 mM), heptanol (0.3-1 mM), octanol (0.055-1 mM) and nonanol (45 and 113 microM) also inhibited these GABAergic currents. Although efficacious positive modulators of GABA(A) receptor function, the volatile anesthetics enflurane, halothane and isoflurane inhibited the function of rho1 receptors. All of these drug effects were fully reversed by a 6-min washout period. When higher concentrations of GABA were used (5 microM) producing approximately 80-90% of a maximal response) neither the alcohols nor enflurane had any effect. In agreement with previous reports, pentobarbital (50 and 200 microM) was ineffective at any GABA concentration tested. Alphaxalone and propofol were also without effect. Furthermore, none of these compounds produced any significant effects when applied to oocytes in the absence of exogenously added GABA. The opposite effects of alcohols and anesthetics on rho1 and GABA(A) receptors, despite their significant amino acid sequence homology, may help in the identification of the particular amino acids responsible for the actions of these compounds on these receptors. PMID- 8613950 TI - Trimetazidine reverses calcium accumulation and impairment of phosphorylation induced by cyclosporine A in isolated rat liver mitochondria. AB - When applied to rat liver mitochondria in contact with Ca++, cyclosporine A (CsA) induced both an accumulation of this ion and a decrease in oxidative phosphorylation. Trimetazidine (TMZ) reversed both phenomena in a dose-dependent manner. These two effects were demonstrated in separate experiments. A decrease in oxidative phosphorylation was observed with succinate as substrate. V3 and P/O (ratio corresponds to the number of ADP molecules added in the medium per oxygen atom consumed during phosphorylation and represents the yield of ATP synthesis) were simultaneously decreased by CsA (1 microM) and restored by TMZ. Ca++ accumulation in mitochondria was observed when it was added to the mitochondrial suspension; its uptake was followed by a new equilibrium. CsA prolonged its duration, whereas TMZ reduced it in a dose-dependent manner. The same phenomenon was observed when ADP was used instead of CsA. Ca++ efflux from mitochondria could be induced by TMZ without the addition of CsA. It was immediate and always partial and followed by a reuptake process only observed at concentrations of TMZ of >1 microM. Compared with ruthenium red, which blocks Ca++ uniporter, TMZ seemed to act on Ca++ efflux mechanisms. Interestingly, low TMZ doses promote a Ca++ efflux process without activating reentry mechanism, which may explain the correction of deleterious effect of CsA on V3 and P/O. As nephrotoxicity observed in humans after CsA chronic administration is considered to be related, at least in part, to an alteration of Ca++ intracellular homeostasis, TMZ seems to be a candidate for alleviation of CsA nephrotoxic effects in humans. PMID- 8613951 TI - Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes. AB - The HIV-1 protease inhibitor ritonavir (ABT-538) undergoes cytochrome P450 mediated biotransformation in human liver microsomes to three major metabolites, Ml, M2 and M11, with wide interindividual variation in the rates of metabolite formation. The structures of these metabolites were determined with the use of electrospray ionization mass spectrometry. Chemical inhibition, metabolic correlation, immunoinhibition and metabolism by microsomes derived from specific CYP cDNA-transfected B-lymphoblastoid cell lines indicated that the CYP3A subfamily of enzymes was the major contributor to the formation of M1 and M11, whereas both CYP3A and CYP2D6 contributed to the formation of M2. None of the typical CYP3A substrates/inhibitors (e.g., ketoconazole, troleandomycin) were able to completely inhibit ritonavir metabolism, even at high concentrations. Ritonavir was found to be a potent inhibitor of CYP3A-mediated biotransformations (nifedipine oxidation, IC50) = 0.07 microM; 17alpha-ethynylestradiol 2 hydroxylation, IC50 = 2 microM; terfenadine hydroxylation, IC50 = 0.14 microM). Ritonavir was also found to be an inhibitor of the reactions mediated by CYP2D6 (IC50 = 2.5 microM) and CYP2C9/10 (IC50 = 8.0 microM). The results of this study indicate the potential for in vivo inhibition of the metabolism by ritonavir of drugs that are CYP3A, CYP2D6 and, to a lesser extent, CYP2C9/10 substrates. PMID- 8613952 TI - Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. IV. Regulation by external Ca++ of alpha-bungarotoxin-sensitive receptor function and of rectification induced by internal Mg++. AB - Applying the whole-cell mode of the patch-clamp technique to cultured hippocampal neurons, we demonstrated that extracellular Ca++ modulates the activation and inactivation of type IA nicotinic currents, ie., the currents subserved by alpha bungarotoxin (alpha-BGT)-sensitive, alpha-7-containing nicotinic acetylcholine receptors (nAChRs). The rundown profile of acetylcholine (ACh)-induced type IA currents that were obtained using patch pipettes filled with F(-)-based internal solution had two components: the first component of rundown was counteracted by a more physiological internal solution containing an organic anion (malate or aspartate), suggesting energy dependence; the second component exhibited dependence on concentration of CaCl2 added to the external solution ([Ca++]o), with rundown minimized at 0.32 mM. The inward rectification of Ach-elicited type IA currents, induced by intracellular Mg++ was augmented by lowering [Ca++]o (from 2 to 0.32 mM). Moreover, extracellular Ca++ (0.01-10 mM) acted in a concentration-dependent manner (IC50 = 0.26 mM) to decrease the cooperativity induced by ACh (nH was reduced from 2.7 to 1). Extracellular Ca++ (EC50 = 0.1 mM) also increased the efficacy of ACh, but exposure to [Ca++]o from 1 to 32 mM decreased the efficacy of ACh and inactivated the alpha-BGT-sensitive nAChRs (IC50 = 11 mM). In conclusion, Ca++ regulates agonist efficacy and cooperativity at the alpha-BGT-sensitive neuronal nAChR, modulates rundown and counteracts Mg++ -dependent inward rectification of type IA currents. It is suggested that the regulation by Ca++ of the alpha-BGT-sensitive nAChR activity could modulate many neuronal functions. PMID- 8613953 TI - Modulation of sodium channels of rat cerebellar Purkinje neurons by the pyrethroid tetramethrin. AB - The pyrethroid insecticides are known to slow the kinetics of the activation and inactivation gates of sodium channels. This results in prolonged openings of individual sodium channels and prolonged flow of whole-cell sodium current, which in turn cause hyperexcitation in animals. The aim of the present study was to solve three important remaining questions. First, the percentages of the sodium channels modified by the pyrethroid tetramethrin were measured and compared with the threshold concentration to initiate repetitive discharges in rat cerebellar Purkinje neurons. Tetramethrin at 0.1 microM modified only 0.6% of the sodium channels and generated repetitive afterdischarges. Thus, the pyrethroid toxicity is greatly amplified from the sodium channel to the whole animal. The pyrethroid sensitivity of Purkinje neuron sodium channels was lower than that of invertebrate sodium channels by a factor of > or = 10. Chloramine-T at 200 microM removed the sodium channel inactivation and increased the percentage of sodium channel modification by tetramethrin through open channel modification. Second, temperature had a profound effect on the ability of tetramethrin to cause repetitive afterdischarges; at 0.1 to 0.3 microM tetramethrin, repetitive discharges were induced at l5 degrees C and 20 degrees C, but this effect subsided at 25 degrees C to 35 degrees C. This negative temperature dependence could be explained by an increase in charge movement during slow tail current as temperature was lowered. The Q10 value for the charge movement during tail current was 0.22 between 20 degrees C and 30 degrees C. Third, the selective toxicity of pyrethroids between mammals and insects could be explained quantitatively on the basis of sodium channel factors that include temperature dependence, intrinsic sensitivity and recovery rate and detoxication factors. PMID- 8613954 TI - Characterization and modulation of acute tolerance to nicotine in mice. AB - Acute tolerance to the effects of nicotine is believed to play an important role in the development and maintenance of dependence to this drug. The objective of this study was to investigate and characterize the development of acute tolerance to nicotine after systemic and intrathecal administrations. Acute tolerance developed to several centrally mediated pharmacological effects of nicotine after systemic (motor coordination, body temperature, antinociception) and intrathecal (antinociception) injection of the drug. The appearance and the magnitude of acute tolerance varied depending on the response measured. Development of acute tolerance to nicotine-induced hypothermia and motor impairment was blocked after intraperitoneal pretreatment with nimodipine. Similarly, an intrathecal injection of nimodipine blocked the development of acute tolerance to nicotine-induced antinociception. On the other hand, intrathecal administration of calcium and thapsigargin enhanced the acute tolerance to nicotine-induced antinociception. Characterization of the acute tolerance to nicotine in several animal models revealed time and dose dependencies that are consistent with receptor-mediated events. More importantly, acute tolerance was modulated by agents that influence cellular calcium homeostasis. PMID- 8613955 TI - Respiratory effects of opioid full and partial agonists in rhesus monkeys. AB - Respiratory and behavioral effects of the mu selective opioids levorphanol (0.01 3.0 mg/kg), methadone (0.03-5.6 mg/kg) and codeine (0.3-30.0 mg/kg) and the mixed action opioids buprenorphine (0.003-10.0 mg/kg), butorphanol (0.003-0.3 mg/ kg) and nalbuphine (0.03-30.0 mg/kg) were studied in rhesus monkeys. In respiratory experiments, awake, seated monkeys wore plastic masks through which they breathed air or differing concentrations of CO2 mixed in air. Exposure to CO2 in air stimulated ventilation in a concentration-dependent manner. All opioids produced dose-dependent decreases in ventilation that were more pronounced as the concentration of CO2 increased. The highest doses of levorphanol, methadone, and butorphanol reduced the ventilatory stimulant effects of 5% CO2 in air by 85 to 90%, whereas the highest doses of nalbuphine, buprenorphine and codeine reduced the effects of 5% CO2 in air by only 50 to 75%. After presession butorphanol (0.01-0.1 mg/kg) or nalbuphine (1.0-3.0 mg/kg), levorphanol further reduced the ventilatory stimulant effects of 5% CO2 mixed in air, with some evidence of dose effect flattening. However, pretreatment with buprenorphine (1.0-10.0 mg/kg) and the highest dose of nalbuphine (10.0 mg/kg) attenuated the effects of 10.0 mg/kg levorphanol, indicative of antagonism. In behavioral experiments, all drugs produced dose-related decreases in responding under a 30-response fixed-ratio schedule. The highest doses of levorphanol, methadone, codeine, and butorphanol nearly abolished responding in all subjects, whereas buprenorphine and nalbuphine most often reduced response rates by no more than 50%. Except for codeine, rank orders of potency were the same in behavioral and respiratory experiments. The lesser effects of nalbuphine and buprenorphine on ventilation, in conjunction with their levorphanol-antagonist effects, suggest their limited mu agonist efficacy at sites mediating the respiratory-depressant effects of opioids. PMID- 8613956 TI - Prolonged central effects of quinpirole on cardiovascular regulation. AB - Central cardiovascular effects of the dopamine D2 receptor agonist quinpirole were studied in conscious rats. The i.v. injection of 0.3 mg/kg of quinpirole in spontaneously hypertensive rats (SHR) caused a rapid but short-lasting increase in blood pressure. Heart rate showed little change. Pretreatment with the centrally acting selective dopamine D2 receptor antagonist raclopride, but not the D1 antagonist SCH23390, completely prevented the rise in blood pressure. A second injection of quinpirole, 30 min after the first injection, induced little change in blood pressure, although at 4 or 24 hr after quinpirole treatment, we observed partial and complete recovery of the pressor response, respectively. This pattern of desensitization was similar to that seen after administration of the dopamine D2 receptor agonists N-propylnorapomorphine (0.3 mg/kg) or quinelorane (0.1 mg/kg), and was similar in spontaneously hypertensive rats, Wistar Kyoto and Sprague-Dawley rats. At 30 min after treatment with quinpirole, the hypotension induced by i.v. injection of clonidine (0.01 mg/kg) or of 8 hydroxy-dipropylaminotetralin (0.1 mg/kg) was markedly reduced when compared to that in saline-pretreated spontaneously hypertensive rats, suggesting a prolonged effect of quinpirole at the level of sympathetic regulation. The rapid fall in blood pressure caused by i.v. injection of the ganglion blocker pentolinium (10 mg/kg) was slightly, but significantly enhanced by treatment with quinpirole, which suggests an overall prolonged increase in resting sympathetic vasomotor tone. This would be difficult to reconcile with an inhibition of the action of sympatholytic drugs, unless it is hypothesized that the increase in sympathetic vasomotor tone was differential between different sympathetic beds or different neuronal populations in the brain. This may prohibit any additional pressor responses and, through a central feedback mechanism, may inhibit the action of sympatholytic drugs. No evidence was found for lasting changes in circulating levels of vasopressin, angiotensin or atrial natriuretic factor, nor were there changes in hematocrit. Cardiac sympathetic tone appeared to be enhanced, although vagal tone was normal and no major changes in baroreflex sensitivity were observed. PMID- 8613957 TI - Novel guanylyl cyclase inhibitor potently inhibits cyclic GMP accumulation in endothelial cells and relaxation of bovine pulmonary artery. AB - The aim of the present study was: 1) to determine the effects of the novel selective inhibitor of guanylyl cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3, a]quinoxaline-1-one) on basal and agonist-stimulated cyclic GMP levels in cultured porcine aortic endothelial cells and bovine pulmonary artery strips; 2) to determine its effects on agonist-induced relaxations of bovine pulmonary artery strips; and 3) to compare pulmonary artery cyclic GMP levels with vessel relaxation. ODQ (1 nM-30 microM) inhibited cyclic GMP accumulation in endothelial cells stimulated with S-nitrosoglutathione, nitroprusside and 3-morpholine sydnonimine at IC50 values of 40 to 100 nM. Complete suppression of cyclic GMP generation was observed at approximately 10 microM. Relaxation of pulmonary artery strips induced by S-nitrosoglutathione, nitroprusside, glycerol trinitrate, nitrite (all endothelium-independent), bradykinin and the Ca++ ionophore A23187 (endothelium-dependent) were antagonized by ODQ (1-10 microM) in a concentration-dependent way. A consistent feature of the inhibitor was that maximal relaxant effects also were reduced. Basal levels and agonist-induced increases in arterial tissue cyclic GMP were inhibited in the same concentration range. However, tissue cyclic GMP production correlated poorly with pulmonary artery relaxation in that relaxations induced by S-nitrosoglutathione were only inhibited in part (50%), whereas rises in cyclic GMP were abolished completely by ODQ (10 microM). Furthermore, at 1 microM, ODQ had no effect on relaxation induced by endothelium-dependent agonists, but prevented entirely stimulation of cyclic GMP accumulation in arterial tissue. These results suggest that ODQ inhibits nitrovasodilator-induced and endothelium-dependent relaxation through inhibition of guanylyl cyclase activation, but also point to the presence of a cyclic GMP-independent component of relaxation in bovine pulmonary artery. PMID- 8613958 TI - Effects of naloxone and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance in mice. AB - Previous studies measuring opioid inhibition of cyclic adenosine monophosphate in SH-SY5Y cells supported the hypothesis that continuous agonist stimulation causes a gradual conversion of the mu opioid receptor to a sensitized or constitutively active state termed mu*. Conversion to mu* was prevented by the kinase inhibitor H7, but not its close analog H8. Naloxone was proposed to act as a negative antagonist (inverse agonist) blocking mu* activity, whereas D-Phe-Cys-Tyr-D-Trp Arg-Thr-Pen-Thr-NH2 (CTAP) appeared to act as a neutral antagonist having no effect on mu* activity. Initial in vivo results indicated that mu* activity may play a role in narcotic tolerance and dependence (Wang et al., Life Sci. 54: PL339-PL350 1994). Our study explores the pharmacology of H7 and H8, naloxone and CTAP in mice after induction of acute tolerance and dependence induced by a single s.c. dose of morphine (100 mg/kg). Physical dependence was defined by withdrawal jumping induced by i.p. naloxone injections 4 hr after the morphine dose, the time of maximal physical dependence. Neither H7 nor H8 (50 nmol or less) induced jumping, affected morphine antinociception or produced significant behavioral effects, when injected by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. When given 30 min before the naloxone challenge, H7, but not H8, significantly reduced naloxone jumping by i.c.v. injection. Administration of naloxone into the central nervous system, rather than by i.p. administration, required coinjection by both i.c.v. and i.th. routes to elicit full withdrawal jumping (30 nmol at each site). In contrast, the putative neutral antagonist CTAP caused little withdrawal jumping when coinjected i.c.v. and i.th., as expected if modulation of mu* activity played a role in dependence. However, CTAP was capable of partially reversing naloxone (i.p.) induced jumping when given either i.c.v. or i.th., indicating that CTAP competes with naloxone at mu*. Moreover, these results demonstrate that both spinal and supraspinal sites are required for full opioid withdrawal jumping in mice. Antinociceptive tolerance was also evaluated by determining the response to morphine in the 55 degrees C warm-water tail-flick test. Morphine pretreatment (100 mg/kg, s.c., -5 hr) produced antinociceptive tolerance as shown by a 2.7-fold increase in the calculated morphine A50 value. Tolerance was reversed by H7, but not H8, treatment (50 nmol, i.c.v., -30 min). These results are consistent with the hypothesis that a sensitized or constitutively active mu* state plays a role in narcotic tolerance and dependence. PMID- 8613959 TI - Characterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated "knock-down" of opioid receptor in vivo. AB - Pharmacological studies in vivo and in vitro have suggested the existence of subtypes of the delta opioid receptor termed delta1 and delta2 (delta1 and delta2). The hypothesis of subtypes of delta receptors was further explored by assessing the effects of administration of antisense or mismatch oligodeoxynucleotides (ODN) in vivo to the cloned DOR, or to a conserved region of the cloned opioid receptors, on the antinociceptive responses elicited by selective mu, ku and delta opioid receptor agonists in mice. Additionally, the density of opioid delta receptors in brain after delta opioid receptor (DOR) ODN treatment was investigated. Repeated twice daily intracerebroventricular (i.c.v.) administration of DOR antisense, but not mismatch, ODN, produced a dose- and time related blockade of i.c.v. [D-Ala2, Glu4]deltorphin (delta2 agonist), but not [D Pen2, D-Pen5]enkephalin (delta1 agonist), antinociception. The antinociceptive responses to selective mu and kappa opioid agonists were unaffected by DOR antisense or mismatch ODN treatments. The antinociceptive effect of an A90 dose of [D-Ala2, Glu4]deltorphin was significantly reduced by the third day of DOR antisense ODN administration and persisted over a treatment period of 6 days with recovery by the third posttreatment day. Saturation studies in mouse whole brain preparations with the selective delta-radioligand [3H]naltrindole showed that DOR antisense, but not mismatch, ODN treatment produced a significant time-related reduction in Bmax values of approximately 30 to 40% by day 6, without changing the Kd value. The reduction in DOR density was reversible and returned to control levels within 3 days after cessation of antisense ODN treatment. The i.c.v. administration of an antisense, but not mismatch, ODN directed to a conserved region of the cloned opioid receptors, termed common opioid receptor antisense ODN, inhibited the antinociceptive effects of i.c.v. mu, kappa and delta agonists, including [D-Pen2, D-Pen5]enkephalin. These data further support the hypothesis of subtypes of opioid delta receptors. PMID- 8613960 TI - Antithrombotic effects of TAK-029, a novel GPIIb/IIIa antagonist, in guinea pigs: comparative studies with ticlopidine, clopidogrel, aspirin, prostaglandin E1 and argatroban. AB - The antithrombotic and bleeding time (BT)-prolonging effects of TAK-029, a novel glycoprotein IIb/IIIa antagonist, were characterized and compared with those of conventional antithrombotic agents in guinea pigs. TAK-029 potently inhibited the binding of fibrinogen and von Willebrand factor to purified human GPIIb/IIIa with IC50 values of 0.67 +/- 0.03 and 0.33 +/- 0.04 nM; it also inhibited human platelet aggregation induced by various aggregating agents with IC50 values of 29 to 38 nM. The in vitro antiplatelet effect of TAK-029 was potent in humans, guinea pigs and monkeys. When TAK-029 was given p.o. to guinea pigs, severe prolonging of BT (>1800 sec) was not observed with plasma concentrations of TAK 029 that inhibited ex vivo platelet aggregation by < 100%. The p.o. administration of TAK-029, ticlopidine and clopidogrel prolonged BT to the same extent, in parallel with their inhibition of ex vivo platelet aggregation. TAK 029 inhibited ex vivo platelet adhesion and thrombus formation in an arteriovenous shunt model more strongly than ticlopidine, clopidogrel and aspirin at doses causing similar prolongations of BT. In a balloon catheter-induced carotid thrombosis model, i.v. administration of TAK-029 significantly inhibited thrombus formation without prolonging BT. At doses that caused an incomplete antithrombotic effect, PGE1-alpha-cyclodextrin and argatroban produced hypotension and prolongation of BT, respectively. TAK-029 may be effective in patients suffering from arterial thrombotic diseases, which are refractory to these conventional antithrombotic agents. PMID- 8613961 TI - Uptake of [N-methyl-11C]propionyl-L-carnitine (PLC) in human myocardium. AB - We studied the uptake of propionyl-L-carnitine from plasma by the myocardium in 10 human subjects using positron emission tomography. Propionyl-L-carnitine was labeled in the N-methyl position with carbon-11 (T1/2 = 20.4 min) and administered i.v. in trace amounts. The uptake of the radiolabel by the myocardium was then scanned over a period of 1 1/2 h. The activity-time course of the tracer in blood and plasma and the exchange of the label in plasma between propionyl carnitine, acetyl carnitine and free carnitine was followed during the scans. Myocardial blood flow was also measured in the same subjects. The results show an exchange of the tracer between the myocardium and plasma, and they show an apparently irreversible component of uptake, a result consistent with the incorporation of the label into relatively large intracellular carnitine pools. PMID- 8613962 TI - Myenteric ganglionic 5-hydroxytryptamine(1P) signal transmission is mediated via Go protein. AB - The role of G proteins in mediating the signal transduction of the guinea pig myenteric ganglionic 5-hydroxytryptamine (5-HT)1P receptors was examined. Stimulation of ganglionic membranes with 5-HT in the presence of [35S]GTPgammaS or [alpha 32P]GTP increased guanine nucleotide binding to G(alpha)o but not to G(alpha)s, G(alpha)i or G(alpha)q in a concentration-dependent fashion. Pertussis toxin pretreatment markedly reduce this 5-HT induced response. Similarly, the 5 HT1P receptor-mediated slowly developing and long-lasting depolarizing response is potentiated by GTPgammaS and is inhibited by GDPbetaS or pertussis toxin. The activation of G(alpha)o by 5-HT also was mimicked by the 5-HT1P agonist, 5 hydroxyindalpine and was blocked by the selective 5-HT1P antagonist, N-acetyl-5 hydroxytryptophyl-5-hydroxytryptophan amide. These data provide compelling evidence to suggest that transmembrane signaling for the 5-HT1P receptors in isolated myenteric ganglia is transduced by the trimeric Go protein. PMID- 8613963 TI - Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures. AB - Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure. PMID- 8613964 TI - Extended in vivo half-life of human soluble complement receptor type 1 fused to a serum albumin-binding receptor. AB - A new approach has been used to extend the T(1/2) of human soluble complement receptor type 1 (sCR1) in rats. The albumin-binding domains B2A3 (BA) and B1A2B2A3 (BABA) from Streptococcal protein G were fused to the carboxyl terminus of sCR1, and the recombinant genes were expressed and amplified in Chinese hamster ovary cells. Western blot analysis and surface plasmon resonance measurements demonstrated the binding of rat serum albumin to both sCR1-BA and sCR1-BABA but not to sCR1. The in vitro complement inhibitory activity of the fusion proteins was shown to be similar to that of sCR1, indicating that neither the albumin-binding domains nor the presence of bovine serum albumin interfere with sCR1 function. Pharmacokinetic analysis showed that the T(1/2) of the distribution phase (T(1/2alpha)) was 3.3, 20.0 and 6.0 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The T(1/2) of the elimination phase (T(1/2beta)) was 103, 297 and 170 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The plasma elimination of sCR1-BA and sCR1-BABA was significantly (P < .05) prolonged as compared to sCR1. The proteins showed similar tissue distribution; at 4-hr postdosing, the highest levels of 125I-radioactivity per gram of tissue were localized in the urine, blood, liver, stomach, and small intestine. PMID- 8613965 TI - Negative inotropic action of denbufylline through interfering with the calcium channel independently of its PDE IV inhibitory activity in guinea pig ventricle papillary muscles. AB - The inotropic actions of xanthine derivatives with long alkyl chains were investigated in guinea pig ventricular papillary muscle. A potent and nonselective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine, elicited a positive inotropy and inhibited the negative inotropic effects of calcium channel inhibitors, as did a selective PDE III inhibitor, amrinone, and these effects were canceled by a protein kinase inhibitor, N-[2-(p bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). However, 1,3-di-n butyl-7-(2'oxopropyl)xanthine (denbufylline) and 1-n-butyl-3-n-propylxanthine (XT 044), which have potent and selective PDE IV-inhibitory activities, showed negative inotropic actions that became more potent in the presence of H-89. Denbufylline abolished the late restoration phase induced by ryanodine. This xanthine derivative attenuated the effects of both the calcium channel acting agents Bay K 8644 and verapamil, without interaction with caffeine and dihydropyridine calcium channel inhibitors, and denbufylline had little direct influence on the specific binding of [(3)H]azidopine and [(3)H]desmethoxyverapamil to cardiac membranes. A nonxanthine PDE IV inhibitor, Ro 20-1724, did not affect the inotropic actions of calcium channel inhibitors. The attenuation by denbufylline or XT-044 of the negative inotropic action of verapamil was not influenced by treatment with H-89. These results suggest that in the ventricular papillary muscle, these xanthine derivatives elicit negative inotropy by acting on a verapamil-sensitive site of the calcium channel without involving their PDE-inhibitory activity. PMID- 8613967 TI - Tolerance of hypothalamic beta-endorphin neurons to mu-opioid receptor activation after chronic morphine. AB - The mu-opioid receptor is an autoreceptor on hypothalamic beta-endorphin neurons that when activated inhibits cell firing via increasing an inwardly rectifying potassium conductance. The membrane hyperpolarization to DAMGO ([D-Ala2, N-Me Phe4, Gly-ol5]-enkephalin) in beta-endorphin and other arcuate (ARC) neurons was investigated in hypothalamic slices from control and morphine-treated, ovariectomized guinea pigs. Chronic morphine treatment caused both a decreased potency (EC50 220 +/- 10 nM vs. 64 +/- 3 nM in controls) and a decreased efficacy (Vmax: -7.1 +/- 1.1 mV vs. -10.7 +/- 0.6 mV in controls) of DAMGO in a population of ARC neurons including beta-endorphin neurons. In another population of ARC neurons from morphine-treated animals, DAMGO was less potent (EC50: 110 +/- 4 nm) than in controls (EC50: 64 +/- 3nM), but there was not a significant change in the efficacy of DAMGO. Twenty percent of ARC neurons did not exhibit any signs of tolerance. The density of mu-opioid receptors labeled with the antagonist radioligand [3H]diprenorphine was found to be significantly decreased in the ARC and surrounding mediobasal hypothalamus after morphine treatment (Bmax: 217 +/- 9 vs. 276 +/- 16 fmol/mg protein in controls), which is consistent with the altered response in beta-endorphin neurons. In summary, chronic morphine treatment decreases mu-opioid receptor density and the functional coupling of mu-opioid receptors to K+ channels in ARC neurons. This expression of morphine tolerance by beta-endorphin (ARC) neurons may serve as a homeostatic mechanism to maintain opioid control of a variety of systems ranging from reproduction to motivation and reward. PMID- 8613966 TI - Effects of wortmannin analogs on bone in vitro and in vivo. AB - The possible importance of phosphatidylinositol (PI) 3-kinase activity in bone resorption activity in vitro and in vivo were evaluated with synthetic wortmannin analogs in two in vitro bone resorption assays, two in vitro assays for PI 3 kinase activity and for the first time, in two in vivo rat models. Wortmannin and LY301497 were shown to be potent, dose-dependent inhibitors of the bone resorption activity of differentiating chicken osteoclast-like cells and isolated rat osteoclasts. A similar structure/activity profile and potency relationship was observed for the inhibition of osteoclastic activity and of bovine PI 3 kinase activity with purified enzyme, as well as direct inhibition of the PI 3 kinase activity of chicken osteoclast lysates. These in vitro data identified LY301497 as an inhibitor of bone resorption that is 10-fold more potent than wortmannin itself, and the most potent inhibitor of PI 3-kinase activity identified thus far. Wortmannin and analogs also lowered the osteoclast-dependent serum calcium levels like salmon calcitonin in a rat model of secondary hyperparathyroidism. More directly, oral administration of wortmannin analogs prevented the estrogen deficiency-induced loss of trabecular bone in the metaphysis of proximal tibiae from ovariectomized rats. Wortmannin, and especially LY301497, compared favorably in potency in vivo to orally administered estrogen. Taken together, these data are strong evidence to show that wortmannin analogs directly block osteoclastic activity in vitro and in vivo, and confirm that PI 3-kinase activity is a necessary step in the regulation of bone resorption. PI 3-kinase activity appears to be an important component of ovariectomy-stimulated bone loss in rats. This mechanism is supported by the finding that wortmannin had little effect on the activity of myosin light chain kinase in intact osteoclasts. The use of LY301497 should prove useful in elucidating specific molecular interactions important in bone resorption and other PI 3-kinase-mediated cell processes. These data also suggest the possible therapeutic utility of wortmannin analogs to treat conditions characterized by excessive bone loss, such as hyperparathyroidism or hypercalcemia of malignency. PMID- 8613969 TI - Effects of napsagatran (Ro 46-6240), a new synthetic thrombin inhibitor and of heparin in a canine model of coronary artery thrombosis: comparison with an ex vivo annular perfusion chamber model. AB - Napsagatran, a new synthetic direct thrombin inhibitor, was compared with heparin in a canine model of coronary thrombosis and concomitantly in an ex vivo perfusion chamber model. Occlusive thrombosis of the left circumflex coronary artery was induced by electrical injury. In parallel, arterial subendothelium was exposed to native blood using an annular perfusion chamber for 5, 10 and 20 min at a wall shear rate of 650/s. Dogs received saline, heparin (40 and 70 U/kg/h) or napsagatran (3 and 10 microgram/kg/min). Heparin (40 U/kg/h) and napsagatran (3 microgram/kg/min) delayed or prevented in vivo thrombotic occlusion, but only napsagatran (10 microgram/kg/min) significantly decreased the intracoronary thrombus when compared with saline. High-dose heparin (70 U/kg/h) or napsagatran (10 microgram/kg/min) decreased the platelet-rich thrombus after a 20-min chamber perfusion. Neither heparin nor napsagatran decreased the thrombus volume after a 5-min perfusion. Heparin (70 U/kg/h) and napsagatran (10 microgram/kg/min) prolonged the activated partial thromboplastin time differently (>x6 and x1.4, respectively, P<0.01), whereas the activated clotting time was prolonged equally (x2.5). Thus napsagatran in this model shows arterial antithrombotic effects similar to those of heparin. The chamber experiments suggest that neither compound affects the initiation of platelet thrombus formation. In arterial thrombosis, the activated clotting time has a higher predictive value than the activated partial thromboplastin time when a direct thrombin inhibitor is compared with heparin. PMID- 8613968 TI - The positive inotropic effect and the hydrolysis of phosphoinositide induced by endothelin-3 in rabbit ventricular myocardium: inhibition by a selective antagonist of ET(A) receptors, FR139317. AB - Endothelin-3 (ET-3), an isopeptide of ET, had a concentration-dependent positive inotropic effect (PIE) on rabbit papillary muscle. The maximal inotropic response to ET-3 was 65% of the maximal response to isoproterenol. ET-1 elicited a PIE below 10(-9) M, namely, over a concentration range at which ET-3 did not elicit a PIE. The selective ET(A) antagonist FR139317 effectively antagonized the PIE of ET-3. FR139317 abolished the PIE induced by ET-1 (<10(-9)M) but did not inhibit the PIE induced by high concentrations of ET-1. FR139317 also antagonized the PIE of sarafotoxin S6c. ET-3 caused a time- and concentration-dependent increase in [3H]inositol phosphates (inositol monophosphate, inositol bisphosphate and inositol trisphosphate). FR139317 at 10(-5) M decreased the ET-3-induced increase in inositol phosphates by about 60%, whereas it attenuated the increase in [3H]IP1 induced by ET-1 (3 x 10(-8)M) by only 20%. Thus, in the presence of FR139317, the PIEs of ET-3 and ET-1 were partially dissociated from the PI hydrolysis that was induced by these isopeptides. FR139317 inhibited the specific binding of [125I]ET-1 and of [125I]ET-3, and it was apparent that FR139317 had a high-affinity and a low-affinity site for competing for specific binding with each ligand. These findings indicate that different subtypes of the ET receptor are involved in the induction of the PIEs of ET-3 and ET-1. The PIE of ET-3 may be mediated predominantly by ET(A1) receptors that are susceptible to FR139317 and BQ-123 and partially by ET(B) receptor that are inhibited by RES-701-1. Both ET(A1) and ET(A2) receptors may be responsible for the PIE of ET-1, depending on the concentration in rabbit ventricular myocardium. PMID- 8613970 TI - Placental transfer of valproic acid after liposome encapsulation during in vitro human placenta perfusion. AB - Valproic acid (VPA) is an antiepileptic drug that crosses the placenta freely. Because its use in pregnancy is associated with an increased incidence of fetal malformation and toxic effects, this study was designed to check whether the placental transfer of VPA entrapped in liposomes was reduced. VPA was encapsulated in dehydrated-rehydrated liposomes prepared with equimolar concentrations of phosphatidylcholine, cholesterol and alpha-tocopherol. Liposomes were analyzed for their physicochemical characteristics, their stability and percentage of encapsulation of VPA. A system of dual perfusion of an isolated lobule of term human placenta was used. Six placentas were perfused with liposome-VPA and six with free VPA for 180 min using recirculating maternal and fetal circuits. The rate of transfer and time to reach equilibrium of VPA was similar in placentas perfused with free VPA and with liposome-encapsulated VPA. Liposomes significantly reduced VPA transplacental transfer and placental uptake. This was confirmed by FMM at equilibrium, that was 0.548 +/- 0.058 in free VPA and 0.393 +/- 0.075 in liposome-VPA. The ratio of fetal to maternal concentrations at equilibrium was 0.90 +/- 0.10 in controls and 0.66 +/- 0.13 in liposome-VPA. The amount of VPA recovered in fetal circulation and in placental tissue were 28 +/- 4 and 7 +/- 3% in controls and 19 +/- 4 and 3 +/- 2% in liposome-VPA. In conclusion, our data indicate that encapsulating VPA in liposomes significantly reduces the fetal amount and exposure, and further in vitro and in vivo investigations are needed to optimize the use of liposomes, particularly in pregnancy. PMID- 8613971 TI - Behavioral and biochemical characterization of benzodiazepine receptor partial agonists in pigeons. AB - The ability of benzodiazepine receptor partial agonists to exhibit full efficacy in preclinical anxiolytic tests, in conjunction with initial clinical results, has suggested the possibility of a reduced clinical side-effect profile compared to benzodiazepine receptor full agonists like diazepam. Because punished behavior of pigeons has been useful in detecting effects of novel anxiolytic drugs, effects of imidazobenzodiazepine and beta-carboline benzodiazepine receptor partial agonists and some related compounds were evaluated in this species. The abilities of these compounds to substitute for the discriminative stimulus effects of the full agonists midazolam also was determined. Intrinsic efficacy was assessed by the degree to which gamma-aminobutyric acid increased ligand potency to displace [(3)H]Ro15-1788 (flumazinil) from membranes of pigeon cerebrum, and ranged from full agonist-like efficacy (Ro 19-5470; 7-(3 cyclopropyl-1,2,4-oxodiazol-5-yl)-5,6-dihydro-5-methyl-4H- imidazo[1,5a] thieno[3,2-f]diazin-4-one) to minimal gamma-aminobutyric acid potentiations close to that of the antagonist flumazenil (abecarnil and Ro 41-7812; 7-chloro-4,5 dihydro-3-(3-hydroxy-1-propynyl)-5-methyl-6H-imidazo[1,5-a] -[1,4 ]benzodiazepine 6-one). Punished responding was increased markedly by midazolam and by all partial agonists, except Ro 41-7812 and Ro 42-8773 (7-chloro-3-[3 (cyclopropylmethoxy)-1-propynyl]-4,5-dihyro-5 -methyl-6H-imidaz o[1,5 a][1,4]benzodiazepine-6-one), at doses that did not affect nonpunished responding. In contrast to the full substitution generally observed in mammals, all of the partial agonists produced incomplete substitution (40-70%) in the midazolam drug discrimination procedure in pigeons. A positive relationship was observed between the degree of substitution and intrinsic efficacy. The benzodiazepine antagonists, flumazenil and ZK 93,426 (ethyl-5-isopropoxy-4 methoxymethyl-beta-carboline-3-carboxylate), neither increased punished responding nor substituted for midazolam. The results of the present study suggest that benzodiazepine receptor partial agonists and related compounds may provide full anxiolytic activity at doses that do not fully reproduce the subjective effect profile of full agonists. PMID- 8613972 TI - LY353433, a potent, orally effective, long-acting 5-HT(4) receptor antagonist: comparison to cisapride and RS23597-190. AB - Although many 5-HT (serotonin, 5-hydroxytryptamine)(4) receptor antagonists have been described, none possess the requisite oral activity and duration of action for a clinically effective therapeutic agent. The present report identifies LY353433 (1-(1-methylethyl)-N-[2-[4-[tricyclo[3.3.1.1(3,7)]dec-1-ylcarbo nyl) amino]-1-piperidinyl]ethyl]-1H-indazole-3-carboxamide), an indazole amide, as a high affinity antagonist at the 5-HT(4) receptor in the rat esophagus. LY353433 (10(-8), 3 x 10(-8), 10(-7) M) inhibited 5-HT-induced relaxation of carbamylcholine-contracted esophagus with greater potency than cisapride or RS23597-190, a known 5-HT(4) receptor ligand. Furthermore, RS23597-190 possessed marked agonist activity as did cisapride, whereas LY353433 did not relax the rat esophagus in concentrations up to 10(-5) M. LY353433 (up to 10(-5) M) did not possess appreciable affinity for adrenergic, dopaminergic, histaminergic, muscarinic or GABAergic receptors and, thus, was a highly selective 5-HT(4) receptor antagonist. In addition, LY353433 only slowly associated with an dissociated from the 5-HT(4) receptor, an attribute that conferred long-lasting 5 HT(4) receptor antagonist activity, in contrast to RS23597-190, which rapidly dissociated from the 5-HT(4) receptor. LY353433 dose-dependently inhibited the 5 HT(4) receptor-mediated ex vivo relaxation in the rat esophagus after either i.v. (0.1, 0.3 and 1.0 mg/kg) or p.o. (0.1, 0.3, 1.0 and 3.0 mg/kg) administration. Furthermore, the p.o. to i.v. dose ratio was approximately one, suggesting that LY353433 was well absorbed with excellent pharmacodynamics in the rat. LY353433 (0.3 mg/kg p.o.) blocked esophageal 5-HT(4) receptors ex vivo through 6 hr after p.o. dosing with responses returning to control by 16 hr, indicative of long duration receptor blockade. Lastly, in rats LY353433 was exceptionally safe because acute doses up to 300 mg/kg p.o. did not result in either symptoms or deaths. Thus, LY353433 is a potent, selective, orally effective, long-acting and safe 5-HT(4) receptor antagonist that is highly suitable for clinical use. PMID- 8613973 TI - Nonclassical mucosal antibodies predominate in genital secretions of HIV-1 infected chimpanzees. AB - To identify mucosal immunity in HIV-infected chimpanzees, IgG, IgA, and IgM from plasma, saliva, rectal swabs, vaginal washes, semen, and urethral washes were tested from four male and three female HIV-1IIIB infected chimpanzees. The level of HIV infections in the seven chimpanzees were classified as high, intermediate and low depending on the number of HIV-1 infected cells per 10(7) peripheral blood mononuclear cells (PBMC). One male chimpanzee had a relatively high viral load, two males and two females had moderate viral loads and one male and one female had low levels of infection. All seven animals had plasma antibody. The principal finding was that nonclassical mucosal antibodies of the IgG isotype were the predominant antibody in the saliva, rectal swabs, vaginal washes, semen, and urethral washes of infected animals. All plasma and mucosal samples were negative for IgM antibodies. The results show that HIV-1 specific IgG responses and not sIgA predominate at mucosal surfaces of HIV-1IIIB infected chimpanzees. A trend was observed in which high viral loads correlated with high plasma IgG, IgA and sIgA titers. An overall correlation between relatively high virus loads and high amounts of mucosal IgG was also found. PMID- 8613974 TI - A restraining system for rhesus monkeys used in space research. AB - A new chronic restraining system has been designed specifically for male rhesus monkeys (8-13 kg) housed in weightlessness for scientific purposes. The restraining system consists of a coat (jacket and skirt) and a chair. The system separates the thorax from the lower part of the body, and allows large movements of the upper part of the body. The macaque can feed and groom itself, and can also sleep in close-to-normal position. PMID- 8613975 TI - Chemotherapy-induced hemolytic uremic syndrome: description of a potential animal model. AB - Hemolytic uremic syndrome (HUS) is an uncommon complication of chemotherapy that contributes to the morbidity of oncology and bone marrow transplant patients. The pathogenesis is not well understood and no established clinical animal model exists. We studied four rhesus monkeys (RM) that developed fatal HUS following high-dose chemotherapy. Microangiopathic hemolytic anemia (pre-Hct 40% and day 5 8 Hct 31% (P < .05), increased BUN (168 mg/dl), creatinine (8.2 mg/dl), and lactate dehydrogenase (1458 IU/L) (mean day 5-8 measurements) were observed. Platelets counts decreased to 39 +/- 15 x 10(9)/l from a mean of 397 +/- 31 x 10(9)/L (P < .0001). vWF, ATIII, thrombin:anti-thrombin complex (T:AT) and prothrombin fragment F1.2 levels were not different from a control group (N = 2). The data presented describe chemotherapy-induced HUS with typical clinical and laboratory features which may provide an animal model for the study of this important syndrome. PMID- 8613976 TI - Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA. AB - One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey. PMID- 8613977 TI - MHC expression of African green monkeys of Barbados is limited in heterogeneity. AB - Major histocompatibility complex expression of activated peripheral blood lymphocytes of captive African green monkeys from Barbados and from Africa were analyzed biochemically; class I molecules by one-dimensional isoelectric focusing and class II DR molecules by one-dimensional nonequilibrium pH gradient electrophoresis. Much less diversity was observed in the major histocompatability molecule expression of the African green monkeys of Barbados than in the African cohort. PMID- 8613978 TI - Magnetic resonance imaging in vivo monitoring of T2 relaxation time: quantitative assessment of primate brain maturation. AB - This work describes quantitative MRI assessment of primate brain maturation. Nine young baboons were followed from the age of one to 30 months. Assessment of myelination was based on the gray/white matter contrast on MR images and the evolution of T2 relaxation time respectively. The brain maturation began in the posterior fossa and progressed to the olfactory bulbs corresponding to decreasing white matter T2 values. Relaxation parameters provide new opportunities to trace the myelination process in vivo. PMID- 8613979 TI - Tumor necrosis factor-alpha in serum of macaques during SIVmac251 acute infection. AB - TNF secretion was explored in sera during acute SIV-infection of cynomolgus macaques. A peak of TNF was detected in sera of animals in concomitance with SIV replication. Likewise, AZT treatment delayed and reduced peaks of viral replication and TNF production. Thus, SIVmac251-infected monkey could be an excellent model to explore the interdigitation existing between HIV and TNF in acute and chronic infection and to develop new therapeutic strategies that target the production of this cytokine or its inductive effects. PMID- 8613980 TI - Structural bases for sulfide recognition in Lucina pectinata hemoglobin I. AB - The X-ray crystal structure of the sulfide derivative of ferric Lucina pectinata hemoglobin component I (HbI) has been determined at 1.9 A resolution (R-factor 0.186). The heme pocket structural organization of HbI is in keeping with its ligand binding properties. The fast sulfide association rate constant can be related to the presence of Gln(64)E7, as the heme distal residue, together with the protein structural properties in the CD-E distal region. Moreover, the very high sulfide affinity for HbI is reflected by the exceptionally slow ligand dissociation rate. The stabilization of the heme-bound sulfide molecule is achieved through hydrogen bonding to Gln(64)E7, as well as by finely tuned aromatic-electrostatic interactions with the clustered residues Phe(29)B10, Phe(43)CD1 and Phe(68)E11. Such a peculiar arrangement of phenylalanyl residues at the distal ligand binding site has not been observed before in the globin family, and is unique to HbI, a protein functionally devoted to sulfide transport. PMID- 8613981 TI - Fibrillin-1: organization in microfibrils and structural properties. AB - To investigate the microfibrillar organization and structural properties of fibrillin-1, we produced overlapping recombinant peptides in human cells which altogether span the fibrillin-1 molecule. The peptides were purified under non denaturing conditions and extensive characterization indicated correct folding. The purified proteins were used to map monoclonal antibodies 26, 69 and 201. The binding sites are located at the N-terminal end between amino acid residues 45 and 450 (mAb 26), 451 and 909 (mAb 201) and at the C-terminal end between residues 2093 and 2871 (mAb 69). Immunolocalization of these antibodies to extended beaded structures (microfibrils) demonstrated that the N- and C-terminal ends of fibrillin-1 are located in proximity and on opposite sides of the beads, and more central parts of the molecule are located between the beads. Each epitope is present once between each bead. These data allow two possible models for the organization of fibrillin in microfibrils. However, comparison of distances between antibody binding sites on the recombinant peptides and labeling events in tissue suggests that fibrillin molecules are compacted within their tissue form as microfibrils. Additional analysis of the recombinant peptides provide new information regarding the eight-cysteine motif, a novel domain present in fibrillins and TGF beta binding proteins, and suggest that fibrillins are processed at their N-and C-terminal ends. PMID- 8613982 TI - The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator. AB - Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315 Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity. PMID- 8613983 TI - Structure and dynamics of a protein assembly. 1H-NMR studies of the 36 kDa R6 insulin hexamer. AB - The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers. PMID- 8613984 TI - Transcription activation parameters at ara pBAD. AB - We studied the formation of open complexes of RNA polymerase and promoter DNA as activated by the AraC protein at the Escherichia coli araBAD promoter pBAD and by the cyclic AMP receptor protein at the galKTE promoter P1. The DNA migration retardation assay was demonstrated to be suitable for the detection and quantitation of open complexes by the correspondence in the properties of open complexes in solution and retarded complexes observed in gels. These included, on the ara promoter, heparin resistance, lifetime, DNAseI footprinting, exonuclease III footprinting, permanganate footprinting and disappearance upon transcription, and on the gal promoter, the correspondence between the kinetic parameters Kd and k2 obtained with established techniques and those obtained with the migration retardation assay. On the pBAD promoter we obtained kinetic parameters of Kd = 0.3 nM and K2 = 1 minute(-1). The unusually tight binding of polymerase in the presence of AraC suggests that AraC binds polymerase tightly. PMID- 8613985 TI - Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors. AB - The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2. PMID- 8613986 TI - The solution structure of bovine ferricytochrome b5 determined using heteronuclear NMR methods. AB - The solution structure of a recombinant form of cytochrome b5 containing 104 amino acid residues has been determined using three-dimensional NMR spectroscopy. Using protein enriched in 15N the majority of the polypeptide backbone resonances have been assigned to reveal numerous chemical shift differences to those reported previously for smaller fragments of cytochrome b5. By using 3D NMR methods the extensive spectral overlap of resonance cross-peaks in 2D NMR spectra could be satisfactorily resolved. The large number of sequence-specific assignments made for this form of the protein allowed the identification of over 1130 NOEs, giving an average of 14 NOEs per assigned residue, and 52 dihedral angles (phi). This data was used in an ab initio simulated annealing protocol to determine the solution structure for bovine microsomal cytochrome b5. A series of 50 structures was generated using distance restraints derived from the magnitude of the NOE and torsional angles based on the measured JHN-HA coupling constants. From an initial round of simulated annealing a family of 36 structures was selected on the basis of good covalent geometry and minimal restraint violations. A single cycle of simulated annealing refinement produced 36 converged structures that exhibited an average r.m.s.d. of 0.73 A for the backbone atoms. The determination of the solution structure of cytochrome b5 is the first using NMR methods for any form of this protein. It is also the only cytochrome whose structure has been determined in the oxidised or paramagnetic state. The results show that despite significant line broadening and pseudocontact shifts for resonances lying close to the paramagnetic haem centre, and despite extensive spectral overlap that prevents complete resonance assignment, the topology of the polypeptide backbone can be derived. The conformation for cytochrome b5 determined in this study reveals several small, but significant, differences in structure to that determined previously by crystallography for a smaller fragment of this protein. For example, NMR data do not support a short beta strand as the first element of secondary structure at the N terminus nor is it likely that a beta-bulge structure forms between residues 75 to 79. The data obtained in this study are more consistent with a turn in this region of the protein linking helices 5 and 6 and leads to cytochrome b5 containing only three clearly defined beta strands. Four of the six helices together with the antiparallel beta strands make up a haem binding pocket in which the solvent-accessible area of the protoporphyrin IX centre remains very similar to that found in the crystal structure. The remaining helices and the beta strands form a second structural domain on which the four helix bundle that surrounds the haem is based. THe derivation of the solution structure of cytochrome b5 will allow a greater understanding of the functional properties of cytochrome b5 including its role in biological electron transfer and molecular recognition together with insight into haem protein folding and stability. PMID- 8613987 TI - Effect of sodium on the energetics of thrombin-thrombomodulin interaction and its relevance for protein C hydrolysis. AB - Measurements of the apparent affinity constant for thrombomodulin (TM) binding to human alpha-thrombin as a function of both NA+ and temperature at constant ionic strength (0.15 M) showed that TM affinity increases in the presence of Na+ and vice versa. Moreover, this experimental strategy allowed us to accurately split the free energy of sodium binding into its entropic and enthalpic components for both the TM-free and TM-bound enzyme. Namely, at 25 degrees C, the value of delta G of sodium binding was found equal to -2.4 kcal/mol in the absence of TM and 3.6 kcal/mol for the thrombin-TM complex. The enthalpic contribution to the free energy of sodium binding is equal to -27 kcal/mol and -21 kcal/mol in the TM-free and TM-bound thrombin forms, respectively. Finally, the entropy change for sodium binding was also affected by TM, being equal to -83 cal/(mol deg) and -58 cal/(mol deg) in TM-free and TM-bound thrombin species, respectively. Moreover, the thermodynamic parameters for TM binding to Na+-free thrombin species were solved. TM binding is characterized by an enthalpy and entropy change equal to 10 kcal/mol and 2 cal/(mol deg), respectively, for Na+-free thrombin. It is well known that Na+ binding to thrombin causes conformational transitions and functional activation of the enzyme molecule. The finding that binding of thrombomodulin enhances thrombin affinity for sodium and vice versa raises the question as to whether the change of Na+ ligation induced by TM binding could contribute to the change in thrombin specificity for the hydrolysis of Protein C. Therefore, the effect of sodium binding to thrombin on the hydrolysis of human Protein C was extensively investigated. At both 25 and 37 degrees C the value of kcat/Km for Protein C hydrolysis by thrombin in the absence of TM was found to be enhanced by Na+ over a concentration ranging from 0 to 150 mM. Application of thermodynamic principles demonstrated that the Na+-thrombomodulin linkage contributes, under physiological conditions of sodium activity and temperature, to reduce significantly the transition-state stabilization free energy for Protein C hydrolysis. PMID- 8613988 TI - From structure to sequence and back again. AB - With a simple lattice model and sequence design algorithm, we can design sequences to fit arbitrary compact globular structures. We judged the success of the design algorithm by performing exhaustive conformational searches to determine if a designed sequence's lowest energy conformation matched the target for which it was designed. Designed sequences tend to be much better optimized for their targets than a natural sequence is optimized for its lowest energy model conformation. We examined the effect of varying the number of available amino acid types on the success of the design method. It was more difficult but not impossible to successfully design discriminating sequences using fewer amino acid types. PMID- 8613989 TI - Compensating effects of opposing changes in putrescine (2+) and K+ concentrations on lac repressor-lac operator binding: in vitro thermodynamic analysis and in vivo relevance. AB - Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI. PMID- 8613990 TI - In vivo and in vitro studies of TrpR-DNA interactions. AB - The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively using in vitro and in vivo methods. DNA sequence requirements for 1:1 and tandem 2:1 (TrpR:DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is 3'CNTGA5'5'GNACT3', consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2:1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2:1 complex, or with an 8-base pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprinting in vitro of 1:1 and 2:1 complexes formed sequentially on the two unequal half-site pairs of the trpEDCBA operator from Serratia marcescens indicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting of Escherichia coli operators in vivo showed that, over a wide range of intracellular TrpR concentration, the trpEDCBA operator is occupied by three repressor dimers, aroH is occupied by two dimers, and the 1:1 binding mode is used on the trpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of approximately -2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varies with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation. PMID- 8613991 TI - Antibiotic-induced oligomerisation of group I intron RNA. AB - Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era. PMID- 8613992 TI - Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement. AB - Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations. PMID- 8613993 TI - Surface labelling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding. AB - The type IC methyltransferase M.EcoR124I consists of a specificity subunit (HsdS) and two methylation subunits (HsdM). Using chemical modifications, we have investigated the accessibility of lysine residues in the free enzyme and in the complex with its DNA recognition sequence. A total of 41 of the 109 lysine residues in the enzyme are susceptible to modification, of which 19 are located in the HsdS subunit and 11 in each of the two HsdM subunits. DNA binding results in extensive protection of lysine residues in the HsdS subunit, while those in the HsdM subunit are only protected weakly. The DNA binding activity of the methylase is abolished when a small fraction of the accessible lysine residues are modified. Peptide mapping and N-terminal sequencing has been used to locate the rapidly modified lysine residues in HsdS that are critical for DNA binding. Highly modified residues (K297, K261 and K327) are found in the C-terminal variable domain that is responsible for DNA recognition, but others (K196, K203 and K210) are found in the conserved regions that had not previously been implicated in DNA binding. PMID- 8613994 TI - Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants. AB - Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo. Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions. We used maltose binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspecific hydrophobic interactions. Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol. Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3. However the on-rate is decreased by at least two orders of magnitude. Therefore, the rebinding reaction is prevented and folding occurs in solution. PMID- 8613995 TI - Freeze fracture electron microscopy of lyotropic lipid systems: quantitative analysis of the inverse micellar cubic phase of space group Fd3m (Q227). AB - An inverse micellar cubic phase of cubic aspect 15 formed by dioleoylglycerol/dioleoylphosphatidylcholine mixtures has been studied by freeze fracture electron microscopy. The structure was well preserved after freezing samples which had been hydrated either in pure water or in 30 vol% aqueous glycerol solutions. Electron microscopy images of high quality and resolution have been obtained. Four types of fracture planes, perpendicular to the [111], [110], [311] and [100] crystallographic axes, were identified by optical diffraction of the images from selected areas of the replicas. This is the largest number of different fracture planes yet observed in any lipid mesophase by electron microscopy. These planes are also perpendicular to the directions of the lowest order, and most intense reflections in the X-ray patterns from this cubic phase. The images were filtered using correlation averaging techniques, and they revealed the presence of mirror planes, which establishes that the space group is Fd3m (Q227) rather than Fd3. The interpretation of the images was aided by the novel use of standard deviation (s.d.) information obtained from the averaging procedures. The results are easily interpreted with the structure model deduced from X-ray diffraction and consisting of a complex packing of two different sizes of quasi-spherical inverse micelles located at positions (a) and (d) of the Fd3m unit cell. The results also show clearly that the fracture pathways always coincide with the regions of high CH3 concentration, located between the crystallographic planes containing the larger inverse micelles located at positions (a). PMID- 8613996 TI - Evaluating the clinical and economic trade-offs of hepatic arterial infusion. PMID- 8613998 TI - New technologies yield insight into how cells go awry. PMID- 8613997 TI - Tamoxifen induction of apoptosis in estrogen receptor-negative cancers: new tricks for an old dog? PMID- 8613999 TI - A burst of research activity follows BRCA1 finding. PMID- 8614000 TI - Fate of Canadians' health warnings on tobacco products is uncertain. PMID- 8614001 TI - Beta carotene's fall from grace draws mixed reactions. PMID- 8614002 TI - Molecular mechanisms underlying hereditary nonpolyposis colorectal carcinoma. AB - MutS and MutL are bacterial genes that have critical roles in DNA repair and recombination. Mutations in homologues of these genes cause hereditary nonpolyposis colorectal carcinoma and are implicated in some sporadic (nonhereditary) colorectal cancers. Molecular functions of these genes have been defined through extensive work in bacteria and yeast. This article reviews and explores molecular events that require MutS and MutL, including mismatch repair, homologous recombination, and gene conversion. The mechanisms of action of eukaryotic MutS and MutL homologues are compared with those of their prokaryotic counterparts, and the relevance of these mechanisms to tumorigenesis is discussed. PMID- 8614004 TI - Reductase enzyme expression across the National Cancer Institute Tumor cell line panel: correlation with sensitivity to mitomycin C and EO9. AB - BACKGROUND: Many antitumor drugs require metabolic activation to exert their cytotoxic or cytostatic effects. The so-called bioreductive compounds, whose conversion into active antitumor agents is catalyzed by reductase enzymes, are examples of such drugs. The identification of specific enzymes involved in the activation of these compounds is important in understanding cellular factors that may influence drug antitumor activity. PURPOSE: We measured expression levels of three different reductase enzymes-DT-diaphorase [NAD(P)H (i.e., reduced nicotinamide adenine dinucleotide, with or without phosphate): quinone oxidoreductase]; NADPH:cytochrome P-450 reductase; and NADH (i.e., reduced nicotinamide adenine dinucleotide): cytochrome-b5 reductase- in 69 cell lines (most of the National Cancer Institute [NCI] human tumor cell panel) to see if relationships could be established between the activities of these enzymes and cellular sensitivities to the bioreductive compounds mitomycin C and EO9. METHODS: For all 69 cell lines, the activity of each enzyme was determined using cellular extracts and photometric assays involving the reduction of cytochrome c. Western blot analysis was used to measure the relative amount of DT-diaphorase protein in each extract, and coupled reverse transcription and polymerase chain reactions were employed to assess DT-diaphorase and NADPH:cytochrome P-450 reductase messenger RNA (mRNA) levels in a subset of the cell lines. The cytotoxic and/or cytostatic activities of mitomycin C and EO9 toward the cell lines were determined under aerobic conditions. Relationships between enzyme activity levels and drug sensitivities were assessed by use of the COMPARE program and Pearson correlation coefficients. RESULTS: In general, DT-diaphorase activity levels were higher than those observed for the other two reductases across the entire cell line panel. Measured activities for all three enzymes varied among cell lines derived from the same tissue as well as between lines derived from different tissues; however, tissue-specific patterns of expression could be discerned. Differences in the activity levels of individual enzymes appeared to reflect differences in corresponding enzyme protein and/or mRNA levels. A relationship between enzyme activity and chemosensitivities to mitomycin C and EO9 was observed only for DT-diaphorase (Pearson correlation coefficient = .424 [two-sided P<.0005] for mitomycin C and .446 [two-sided P< or = to .0013] for EO9). CONCLUSIONS: Reductase enzyme expression is heterogeneous across human tumor cell lines, and tissue-specific patterns of expression are apparent. DT-diaphorase activity levels correlate with sensitivities to mitomycin C and EO9, supporting a role for this enzyme in the bioactivation of these anticancer compounds. IMPLICATIONS: Comparison of biochemical, molecular biological, and chemosensitivity data obtained from screening a large number of cell lines (e.g., the NCI tumor cell line panel) may facilitate investigation of factors influencing drug antitumor activity. The knowledge gained may be of value in the development of new anticancer agents or in the selection of patients to receive specific therapies. PMID- 8614003 TI - Reappraisal of hepatic arterial infusion in the treatment of nonresectable liver metastases from colorectal cancer. AB - BACKGROUND: Metastases confined to the liver cause substantial morbidity and mortality for patients with colorectal cancer. The results of several randomized clinical trials conducted to study the effectiveness of hepatic arterial infusion (HAI) of fluoropyrimidines for the treatment of such patients have suggested that this treatment, as compared with systemic administration of fluoropyrimidines, increases the likelihood of tumor response. However, the impact of HAI on survival is unclear. PURPOSE: A meta-analysis was carried out to provide an objective and quantitative appraisal of the benefits of HAI in terms of tumor response rate and overall patient survival. METHODS: The meta-analysis was based on individual data provided by the principal investigators of six individual trials and on summary data for one trial. Of the seven trials, five compared HAI with floxuridine (5-fluoro-2'-deoxyuridine; FUDR) and intravenous chemotherapy (IVC) with FUDR (three trials) or fluorouracil (5-FU) (two-trials), and two compared HAI with FUDR and an ad libitum control group in which some patients could be left untreated. Response data were analyzed by use of a Mantel-Haenszel test on all randomized patients. Survival data were analyzed by the use of stratified logrank test. Multivariate analyses were performed with use of the logistic regression model for tumor response and the Cox regression model for survival. All P values resulted from two-sided statistical tests. The analyses were performed by an independent secretariat and were reviewed by the collaborators. RESULTS: The tumor response rate was 41% for patients allocated to HAI with FUDR or 5-FU (CR, 2%; PR, 12%). This difference was highly significant, with a response odds ratio of 0.25 (95% confidence interval = 0.16-0.40; P < 10 ( 10)). Survival analyses showed a statistically significant advantage for HAI with FUDR compared with control when trials were taken into account (P = .0009) but not when the survival analysis was restricted to trials comparing HAI with FUDR and IVC with FUDR or 5-FU (P = .14). CONCLUSION: These results confirm that HAI can achieve much higher tumor response rates than systemic chemotherapy in patients with liver metastases from colorectal cancer. IMPLICATIONS: The therapeutic benefit of use of HAI with FUDR in these patients should be judged together, with an overall evaluation of this therapy in terms of convenience, toxicity, and costs. These end points should be considered in addition to tumor response and survival in further trials involving HAI. PMID- 8614005 TI - Radiotherapy, alkylating agents, and risk of bone cancer after childhood cancer. AB - BACKGROUND: Individuals who had cancer in childhood are at higher risk of developing bone cancer than any other type of second primary cancer. PURPOSE: Using the population-based National Registry of Childhood Tumours in Britain, we investigated the incidence and etiology of second primary bone cancer after childhood cancer in a cohort study and in a case-control study. METHODS: A cohort study of 13,175 3-year survivors of childhood cancer diagnosed in Britain between 1940 and 1983 revealed 55 subsequent bone cancers. A largely nested case-control study comprised 59 case subjects developing second primary bone cancer, and 220 control subjects were selected and matched for sex, type of first cancer, age at first cancer, and interval between diagnosis of first cancer and subsequent bone cancer. Outcome measures were the incidence of bone cancer after childhood cancer, the cumulative dose of radiation received at the site of the second cancer in the case subject and at the corresponding anatomic site in the matched control subjects, and the cumulative dose of alkylating agents and vinca alkaloids received by case and control subjects. RESULTS: The percentage of 3 year survivors developing bone cancer within 20 years did not exceed 0.9%, except following heritable retinoblastoma (7.2%), Ewing's sarcoma (5.4%), and other malignant bone tumors (2.4%). The risk of bone cancer increased substantially with increased cumulative dose of radiation to the bone (P< .001, linear trend). At the highest levels of exposure, however, the risk appeared to decline somewhat (P=.065, nonlinearity). Exposure to less than 10 Gy was at worst, associated with only a small increased relative risk (RR) of bone cancer (RR= 0.7; 95% confidence interval = 0.2-2.2). The risk of bone cancer increased linearly (P= .04, one tailed test) with increased cumulative dose of alkylating agents. IMPLICATIONS: This population-based study provides grounds for reassurance of the majority of survivors in that their risk of developing bone cancer within 20 years of 3-year survival did not exceed 0.9%. The higher risks found for bone cancer following the other specific rare types of childhood cancer provide a rational basis for surveillance. The RRs reported for bone cancer after specified levels of exposure to radiation should help in making decisions concerning future treatment protocols. PMID- 8614006 TI - Role of c-myc in tamoxifen-induced apoptosis estrogen-independent breast cancer cells. AB - BACKGROUND: The antiestrogen tamoxifen (TAM) is effective in the treatment of estrogen receptor (ER)-positive as well as some ER-negative breast cancers. However, the precise mechanism of action of TAM, especially in estrogen independent cells, remains unclear. Previous work by our laboratory has demonstrated that TAM induces the morphologic and biochemical changes that are characteristic of apoptosis in both ER-positive and ER-negative cells. PURPOSE: We compared the effect of TAM at a clinically achievable concentration on cell growth and apoptosis with the effect of TAM on c-myc (also known as C-MYC) messenger RNA (mRNA) and protein expression in ER-negative MDA-231 cells. METHODS: MDA-231 cells were treated for up to 72 hours with 1.0 microM TAM alone or in the presence of 50 microM c-myc antisense or nonsense oligonucleotides. c myc mRNA expression was determined by northern blot analysis, protein expression by western blot analysis, cell growth inhibition counts, and DNA cleavage by agarose gel electrophoretic analysis. Differences between the mean values from different treatment groups were compared with the use of the two-sided Wilcoxon Ranksum test. RESULTS: TAM treatment for 72 hours increased c-myc mRNA five-fold (from a relative radiolabeled hybridization signal intensity of 17 +/- 4 up to 93 +/- 10; P<.05) and c-MYC protein threefold (from a relative immunofluorescence signal intensity of 28 +/- 7 up to 83+/-21; P< .05). The induction of c-myc by TAM was accompanied by internucleosomal DNA cleavage characteristic of apoptotic cell death. Addition of c-myc antisense oligonucleotide (5'CACGTTGAGGGGCAT-3') to MDA-231 cells resulted in a nearly twofold decrease of basal c-myc mRNA (P< .05) and a sevenfold decrease of basal c-Myc protein (P< .05) expression. Addition of c-myc antisense oligomer also antagonized the TAM-induced increase in c-myc mRNA (P< .05) and protein expression (P< .05) and inhibited TAM-induced cytostasis (P< .01) and apoptosis. In parallel experiments, addition of the nonsense oligomer had no effect on any of the measured parameters. CONCLUSIONS: These results indicate that the effects of TAM on ER-negative MDA-231 cells may be mediated through c-myc overexpression. c-myc may play a critical role in the growth and progression of MDA-231 breast cancer cells. PMID- 8614007 TI - Estrogen receptor variants in normal human mammary tissue. AB - BACKGROUND: Several estrogen receptor (ER) variant messenger RNAs (mRNAs) have been identified previously in human breast cancer biopsy samples and cell lines. The relative levels of certain ER variant mRNAs have been observed to increase with breast tumor progression. In vitro assays of the function of polypeptides encoded by some of these variant mRNAs have led to speculation that ER variants may be involved in the progression from hormone dependence to independence in breast cancer. PURPOSE: We set out to establish if ER variant mRNAs are present in normal human breast tissues and, if so, to compare levels of these variants between normal and neoplastic human breast tissues. METHODS: Four human breast tissue samples from reduction mammoplasties and five samples from tissue adjacent to breast tumors were analyzed. The tissue samples were confirmed to be normal (i.e., not malignant) by histopathologic analysis. RNA was extracted immediately from adjacent frozen sections. Human breast tumor specimens originally resected from 19 patients were acquired from a tumor bank and processed in the same way as the normal tissue samples. The RNAs were then reverse transcribed and subsequently amplified with the use of the polymerase chain reaction (PCR). PCR primer sets were designed to detect several different exon-deleted ER variants and a truncated ER variant (i.e., clone 4). A semiquantitative PCR-based method was used to determine the relative expression of exon 5- and exon 7-deleted variants to wild-type ER mRNAs in the nine normal tissues and in 19 ER-positive breast tumor tissues. The Mann-Whitney rank sum test (two-sided) was used to determine P values. RESULTS: ER variant mRNAs corresponding to the clone 4 ER truncated variant and to variants deleted in either exon 2, exon 3, exons 2-3, exon 5, or exon 7 were detected in all normal samples. The results were confirmed by restriction enzyme analyses and sequencing of the PCR products. The expression of exon 5-deleted ER variant relative to the wild-type ER mRNA was significantly lower (P< .001) in normal tissue than in tumor tissue. A similar trend was noted for expression of the exon 7-deleted ER variant mRNA; however, the difference did not achieve statistical significance (P= .476). CONCLUSION: Several ER variant mRNAs are present in normal human breast tissue, but the level of expression of some of these variants may be lower in normal tissue than in tumor tissue. IMPLICATION: These data suggest that the mechanisms generating ER variant mRNAs exist in normal breast tissue and may be deregulated in breast cancer tissues. Further investigation of the role of variant ER expression in development and progression of human breast cancer appears warranted. PMID- 8614008 TI - Serum sex hormone levels after menopause and subsequent breast cancer. AB - BACKGROUND: High levels of androgens and estrogens have been reported to be associated with breast cancer. However, the multiplicity of factors that influence hormone levels and methodologic issues complicate the study of the relationship between steroid sex hormones and breast cancer. PURPOSE: Using an improved study design, we assessed prospectively the relationship between the principal steroid sex hormones in serum and the subsequent occurrence of invasive breast cancer in postmenopausal women. METHODS: Four thousand fifty-three healthy postmenopausal women aged 40-69 years, were enrolled from June 1987 through June 1992 in a prospective investigation of hormones and diet in the etiology of breast tumors (ORDET study) as part of a larger volunteer cohort of 10 788 premenopausal and postmenopausal women from Varese Province, northern Italy. At recruitment, blood samples were taken between 8:00 AM and 9:30 AM (after overnight fasting), and sera were preserved in -80 degree Celsius freezers. Women who had received hormone treatment in the 3 months prior to enrollment, who had bilateral ovariectomy, or who had a history of cancer or liver disease were not recruited. Twenty-five women in the final eligible cohort of postmenopausal women developed histologically confirmed, invasive breast cancer during the first 3.5 years of follow-up for the cohort (13 537 women-years). For each case subject, four control subjects were randomly chosen after matching for factors possibly affecting hormone preservation in serum. One case subject and eight control subjects were excluded because premenopausal hormonal patterns were found; thus, after also excluding the four control subjects matched to the ineligible case subject, we included 24 case and 88 control subjects. In the spring of 1994, stored sera of case and control subjects were assayed in a blinded manner for dehydroepiandrosterone sulfate and estradiol (E2) by in-house radioimmunoassay and for total and free testosterone and sex hormone-binding globulin by commercially available nonextraction iodination kits. Mean differences in risk factors were tested by analysis of variance for paired data. Relative risks (RRs) were estimated by conditional logistic regression analysis. All P values resulted from two-sided tests. RESULTS: Age-adjusted mean values of total testosterone, free testosterone, and E2 were significantly higher in case subjects than in control subjects: total testosterone, 0.34 ng/mL versus 0.25 ng/mL (P<.001); free testosterone, 1.07 pg/ml versus 0.77 pg/mL (P= .006); and E2, 25 pg/mL versus 22 pg/mL (P= .027). Age-adjusted RRs for breast cancer in increasing tertiles were as follows: for total testosterone, 1.0, 4.8, and 7.0 (P for trend =.026); for free testosterone, 1.0, 1.8, and 5.7 (P for trend=.005); and for total E2, 1.0, 7.1, and 5.5 (P for trend= .128). CONCLUSIONS AND IMPLICATIONS: This prospective study provides further evidence in support of the already established association between elevated estrogen levels and breast cancer. Even more importantly, it provides new evidence that high serum testosterone levels precede breast cancer occurrence. PMID- 8614009 TI - Non-Hodgkin's lymphoma and sunlight. PMID- 8614010 TI - Multiple neoplasias: an oncologic reality. PMID- 8614011 TI - Inflammatory bowel disease associated with levamisole and fluorouracil chemotherapy for colon cancer. PMID- 8614012 TI - Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis. AB - The aim of this study is to assess the ability of three methods, alkaline elution (AE), nick translation (NT), and single-cell gel electrophoresis (SCGE), to detect DNA single-strand breaks (ssb) in human peripheral blood lymphocytes (HPBL) exposed in vitro to three genotoxic agents; gamma-rays, ethyl methanesulfonate (EMS) and benzo[a]pyrene diol epoxide (BPDE). The ultimate objective is to select the most feasible, sensitive, and reproducible method for the monitoring of populations exposed to genotoxic agents. AE and NT do not seem suitable assays. AE is able to detect DNA lesions induced by the three compounds, but only at relatively high doses (2 Gy, 5 mM EMS and 20 microM BPDE). With NT, DNA alterations induced by gamma-rays are not detected and ssb are only evidenced after exposure to EMS (80 mM), which already alters the viability of the lymphocytes. Nick translation is able to detect ssb induced by 10 microM BPDE. Compared to the other assays, the sensitivity of the SCGE assay is significantly higher since statistically significant changes were detected after incubation with 0.5 mM EMS and 1.25 microM BDPE. SCGE is a relatively simple method, not time-consuming and applicable to a large number of samples per working day. In conclusion, on the basis of the results of this in vitro comparison, SCGE seems a promising method for the monitoring of populations exposed to genotoxic chemicals. PMID- 8614013 TI - Flow cytometric and histological assessment of 1,2:3,4-diepoxybutane toxicity on mouse spermatogenesis. AB - The effects of diepoxybutane (DEB) on mouse reproductive cells have been investigated by flow cytometric and histological description of testicular cell populations and alterations of sperm chromatin packaging. Mice were treated with single intraperitoneal injections of DEB, with doses ranging between 8.5 and 78 mg/kg (100-900 microM), and were killed after 7, 14, 21, 28 or 35 d. Dose dependent reductions of tetraploid cells, round spermatids, and elongated spermatids were detected at 7, 21, and 28 d, respectively, reflecting cytotoxic damage on the differentiating spermatogonia compartment. The dose necessary to reduce the number of differentiating spermatogonia to half the control value was estimated equal to 650 microM or 55 mg/kg. Stem cells were not affected by this treatment. Histological section of seminiferous tubules showed depletion of spermatids and reduction of the secondary spermatocyte layers. In addition, a high although not statistically significant frequency of sperm with altered chromatin packaging was detected after DEB treatment. DEB is one of the key metabolites of butadiene, which is a compound of high environmental and occupational concern. These results contribute to the assessment of the reproductive health impact of butadiene in humans. PMID- 8614014 TI - Mutagenic potential of binary and complex mixtures using different enzyme induction systems. AB - A series of experiments was conducted to investigate the mutagenic potential of binary and complex mixtures in the presence and absence of inducible liver enzyme systems prepared with several different chemical inducers. Liver homogenate (S9, or 9000 x g supernatant) fractions were obtained from Sprague-Dawley rats induced with either Aroclor 1254 (AR), phenobarbital (PB), 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD), or corn oil (UI). the mutagenic potential of test samples was measured with Salmonella typhimurium strain TA98 using each of the various S9 fractions. Test samples included benzo[a]pyrene (BaP), pentachlorophenol (PCP), a binary mixture of BaP and PCP, two five-component mixtures, a methylene chloride extract of wood preserving waste-amended soil, and a methanol extract of coal gasification waste. At a dose of 25 micrograms/plate, BaP produced 55, 83, 217, and 161 net revertants per plate with UI-, PB-, AR-, and TCDD-induced S9, respectively. The complex mixture extracted from the wood preserving waste amended soil induced approximately equal responses with all four S9 mixes. At a dose of 250 micrograms/plate, the methanol extract of a coal gasification waste produced 56 net revertants using the uninduced S9; however, when Ar- and TCDD induced S9 was used, 129 and 67 net revertants were observed, respectively. These data demonstrate the relative importance of the various induced cytochrome P-450 isozymes for the metabolism of mutagenic chemicals and complex mixtures. PMID- 8614015 TI - Disposition, metabolism, and toxicity of methyl tertiary butyl ether, an oxygenate for reformulated gasoline. AB - Studies of the toxicology of methyl tertiary butyl ether (MTBE) were reviewed as a possible information base for evaluating the health effects of evaporative emissions from reformulated gasoline (RFG). The major metabolites of the oxidative demethylation of MTBE in vivo were methanol and tertiary butyl alcohol (TBA), whereas formaldehyde and TBA were the principal products of hepatic microsomal oxidation by cytochrome P-450. Pharmacokinetic studies in rats treated with intragastric MTBE in corn oil gave an initial disposition T1/2 for MTBE of 0.32 h. The decline in the serum drug versus time curve for MTBE in rats was accompanied by a progressive increase in serum methanol concentrations to levels more than 50-200 times those of the parent compound. Repeated exposure of MTBE vapor by inhalation in rats resulted in dose-dependent increases in MTBE in the blood, brain, and adipose tissue compartments. Blood concentrations of TBA were also dose dependent and provided an estimate of the total amount of MTBE distributed to peripheral drug metabolizing compartments. Perirenal fat/blood MTBE concentration ratios ranged from 9.7 to 11.6 after 15 wk of intermittent exposure. During an oxyfuels program in Fairbanks, AK, blood levels of occupationally exposed workers were 0.2-31.5 microgram/L MTBE and 1.6 to 72.2 microgram/L TBA with a mean TBA:MTBE blood concentration ratio of 4.2. In patients who received MTBE by percutaneous, transhepatic puncture for the dissolution of cholesterol gallstones, concentrations of MTBE in fat tissue reached 60 and 300 microgram/g at a treatment time when mean blood MTBE was less than 20 microgram/ml. The results of laboratory and clinical studies indicate that metabolites of MTBE may contribute to the nephropathy, neoplasms, and other pathological changes associated with repeated exposure to MTBE in experimental animals. It is concluded that such studies can provide a well-defined database for quantitative safety comparisons and health risk-benefit analyses of MTBE and other oxygenates in RFG. PMID- 8614016 TI - Distinctive profile of alveolar macrophage-derived cytokine release induced by fibrogenic and nonfibrogenic mineral dusts. AB - Groups of 7 Wistar rats each received a single intratracheal instillation of either saline (control), UICC chrysotile B asbestos (5 mg), or very short 4T30 chrysotile asbestos fibers (5 mg). Five animals in each group were killed at 1, 3, and 6 wk posttreatment and analyzed by bronchoalveolar lavage (BAL) for BAL cell populations and cytokine production in conjunction with histopathological assessment of lung tissue. Chrysotile B and short 4T30 chrysotile fibers induced chronic inflammatory reactions characterized by alveolar macrophage (AM) accumulation that resulted, respectively, in lung fibrosis and resolving granuloma. Alveolar macrophages (AM) obtained from rats treated with UICC chrysotile B and short 4T30 chrysotile produced enhanced levels of interleukin-1 (IL-1) and interleukin-6 (IL-6), both spontaneously and in response to lipopolysaccharide (LPS). A different pattern of response was observed for tumor necrosis factor-alpha (TNF-alpha). Fibrogenic chrysotile B caused biphasic changes characterized by significant inhibition of LPS-induced TNF-alpha release by AM 1 and 3 wk after treatment, followed by stimulation of spontaneous and LPS induced TNF-alpha at 6 wk. In contrast, no significant change in spontaneous and LPS-induced TNF-alpha release was seen with AM from animals with resolving granuloma (4T30 group). Thus, modulation of AM-derived TNF-alpha was correlated under these conditions with the fibrogenic potential of asbestos dusts. These data support a role for TNF-alpha in fibrosis and suggest that TNF-alpha may represent a useful marker of lung damage induced by fibrogenic dusts. PMID- 8614017 TI - Role of decomposition products in sodium methyldithiocarbamate-induced immunotoxicity. AB - Sodium methyldithiocarbamate (SMD) is a widely used agricultural agent that causes immunological changes in B6C3F1 mice. The most prominent effects of SMD include a decrease in thymus weight and percentage of CD4+CD8+ thymocytes, an increase in spleen weight, an increase in the percentage of neutrophils in the blood, a decrease in the percentage of lymphocytes in the blood, and a decrease in natural killer (NK) cell activity in the spleen. The mechanism by which SMD causes these changes is unknown, and the relative importance of the parent compound and its decomposition products is not known. In addition, it is not known if these effects are unique to mice, or if other mammals are affected similarly. This prompted the present investigation of the major decomposition product of SMD, methylisothiocyanate (MITC), and two minor products, methylamine and carbon disulfide, in mice. Equimolar dosages of methylamine and carbon disulfide caused minimal immunological changes, and these changes were not characteristic of those noted for SMD. In contrast, MITC significantly decreased thymus weight and cellularity and changed peripheral white blood cell populations in a manner similar to that noted for an equimolar dosage of SMD. However, MITC did not significantly affect NK cell activity or increase spleen weight. Thus, MITC is probably responsible for some of the immunological changes noted in SMD treated mice. The remaining changes are not produced by MITC, methylamine, or carbon disulfide. Thus, it is likely that the parent compound or a synergistic action of the parent compound with one or more of the decomposition products is responsible for these remaining changes (increased spleen weight and decreased splenic NK cell activity). Data are also presented that indicate that SMD-induced thymic atrophy occurs in rats as well as mice and that the dosage required to decrease thymus weight by 50% is lower for rats than for mice. Investigations of other mammals are needed to indicate SMD's potential as a human immunotoxicant and to compare the role of MITC in the immunotoxic effects of SMD in different species. PMID- 8614018 TI - Effects of carbamate insecticides in a rat medium-term bioassay for hepatocarcinogens. AB - Ethiofencarb and pirimicarb, widely used insecticides, were investigated for hepatocarcinogenesis-modifying effects using a medium-term bioassay system for carcinogens. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and then, starting 2 wk later, received ethiofencarb in the diet at concentrations of 500 or 250 ppm, pirimicarb at 400 or 200 ppm, or a combination of ethiofencarb (250 ppm) and pirimicarb (200 ppm) for 6 wk. Control groups received DEN (200 mg/kg) or carbamate insecticides alone as stated earlier, without DEN. All rats were subjected to two-thirds partial hepatectomy at wk 3 and were killed at wk 8. Development of preneoplastic lesions, glutathione S-transferase placental form-positive foci, in the liver was significantly increased in terms of number receiving 500 ppm ethiofencarb. The results thus indicate that ethiofencarb at high dose possesses promoting activity for rat liver carcinogenesis. PMID- 8614019 TI - Subchronic toxicity study of L-isoleucine in F344 rats. AB - A subchronic toxicity study with L-isoleucine was conducted using F344 rats. Groups of 10 rats of each sex were given diet containing 0, 1.25, 2.5, 5.0, or 8.0% L-isoleucine for 13 wk. No treatment-related effects were observed in terms of body weight change, food consumption or hematology. In both sexes given 8.0% L isoleucine, increased or a tendency for increased urine volume and relative kidney weights were observed. Furthermore, the high-dose L-isoleucine treatment brought about an elevation of urinary pH and variations in serum electrolytes. However, histopathological alterations related to these changes were not observed in any organs of either sex. In conclusion, the present study demonstrated that L isoleucine possessed minimal toxicity at dietary levels of 5.0% and 8.0%, while it did not exert any adverse affects at a dietary level of 2.5% or less. PMID- 8614020 TI - Exogenous catalase may potentiate oxidant-mediated lung injury in the female Sprague-Dawley rat. AB - Enhancement of lung antioxidant capacity has been proposed in the therapy of acute lung injuries involving local accumulation of reactive oxygen species (ROS). We have studied in the female Sprague-Dawley rat the effect of intratracheal administration of catalase (CAT) on the acute lung response induced by different ROS generating systems. The lung response was assessed at several time intervals (60-360 min) by monitoring in bronchoalveolar fluid (BALF) the activity of lactate dehydrogenase and the levels of total protein, albumin, and glucose. While CAT (50,000 IU/rat) significantly reduced the biochemical changes induced by hydrogen peroxide produced by a glucose/glucose oxidase system, it markedly exacerbated the lesions induced by phorbol myristate acetate (PMA). Several observations indicate that a particular chemical species formed during the catalase inactivation process is responsible for this effect. Parallel to the development of the lung damage, we noted a rapid reduction of CAT activity (80%) in the BALF of animals treated with PMA and CAT. In vitro an inhibition of CAT activity was observed in the presence of a superoxide anion generating system, and this inhibition was prevented by superoxide dismutase (SOD). A dose of 10,000 IU superoxide dismutase did not prevent the development of the lung lesions induced by PMA plus CAT. Administered alone or in association with PMA, CAT inactivated by heat or 3-aminotriazole also caused severe lung damage. In conclusion, the present study indicates that exogenous catalase may not always protect against the inflammatory reaction resulting from an oxidative stress. In the presence of superoxide anions, catalase may aggravate the lesions, and this possibility should be kept in mind when considering an antioxidant therapy. PMID- 8614021 TI - Exposure to environmental tobacco smoke results in an increased production of (+) anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide in juvenile ferret lung homogenates. AB - Six-week-old ferrets were exposed head-only to clean air or environmental tobacco smoke (ETS) at an average particulate concentration of 38 +/- 13 mg/m3 for 2 h/d, 5 d/wk for up to 15 wk. Twenty four hours after last exposure, the ferrets were sacrificed and the metabolism of benzo[a]pyrene and (-)-7R-trans-benzo[a]pyrene 7,8-dihydrodiol was studied in lung homogenates. The results show that after ETS exposure total metabolism of benzo[a]pyrene, measured by the accumulation of hexane nonextractable radioactivity, was increased by 35% in the males and 66% in the females (p < .05), respectively, of that observed with air-exposed controls. With (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol as substrate, the formation of both benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetraol and (+)-anti-benzo[a]-pyrene 7,8-dihydrodiol-9,10-epoxide-derived tetraols by lung homogenates of ETS-exposed male and female ferrets was significantly increased compared to the air-exposed controls (p < .01). DNA-bound radioactivity was significantly increased in both the males (p < .01) and females (p < .05) compared to the air-exposed ferrets. PMID- 8614022 TI - Effect of radon on the immune system: alterations in the cellularity and functions of T cells in lymphoid organs of mouse. AB - Exposure to radon and its progeny induces significant damage to the cells of the respiratory tract and causes lung cancer. Whether a similar exposure to radon would alter the functions of the immune system has not been previously investigated. In the current study, we investigated the effect of exposure of C57BL/6 mice to 1000 or 2500 working-level months (WLM) of radon and its progeny by inhalation, on the number and function of T lymphocytes in lymphoid organs. The control mice received uranium ore dust carrier aerosol by inhalation. Exposure to radon induced marked decrease in the total cellularity of most lymphoid organs such as thymus, peripheral lymph nodes (PLN), and lung-associated lymph nodes (LALN), when compared to the controls. The percentage of T cells increased, while that of non-T cells decreased, in all peripheral lymphoid organs at both the doses of radon. In the thymus, particularly at 2500 WLM of radon exposure, there was a marked decrease in CD4+CD8+ T cells and an increase in the immature CD4-CD8- T cells. Such alterations in both the numbers and percentages of lymphocytes and macrophages in radon-exposed mice may have resulted from the cell killing by the alpha particles as the immune cells were migrating through the lungs, or it may have been caused by altered migration of cells, inasmuch as expression of CD44, a molecule involved in migration and homing of immune cells, was significantly altered on cells found in different lymphoid organs. In the LALN, where one would predict the largest number of damaged cells to be present, there was a significant decrease in the T-cell responsiveness to mitogens while the B-cell response was not affected. Such alterations may have resulted from the direct effect of alpha-particle exposure on the migrating lymphocytes, altered percentage of lymphocytes as seen in secondary lymphoid organs, or altered expression of adhesion molecules involved in cell activation such as CD44 and CD3. Interestingly, radon exposure caused and increase in the T- and B-cell responsiveness to mitogens in the spleen and PLN. Since there is little evidence of direct radiation dose from radon in lymphoid organs, our studies demonstrating immunological alterations suggest an indirect effect of radon exposure that may have significant repercussions on the development of hypersensitivity and increased susceptibility to infections and cancer in the lung. PMID- 8614023 TI - Characterization of 4-aminobiphenyl-hemoglobin adducts in maternal and fetal blood-samples. AB - The maternal-fetal exchange of the potent tobacco-related human carcinogen 4 aminobiphenyl was studied in women smokers during pregnancy. The number of cigarettes smoked per day by each of the women in the study was assessed via questionnaire and by measurement by immunoassay of serum and urine cotinine in maternal and fetal blood samples. Maternal and fetal blood samples were classified as coming from nonsmokers (n = 74), individuals smoking less than 1 pack of cigarettes per day (n = 16), individuals smoking 1 pack of cigarettes per day (n = 19), individuals smoking 1-2 packs of cigarettes per day (n = 19), and individuals smoking greater than 2 packs of cigarettes per day (n = 20). Both maternal and fetal blood samples were obtained at the time of delivery. 4 Aminobiphenyl was extracted from both maternal and fetal blood samples using organic extractions and the released amine was qualitatively and quantitatively characterized by analysis of the samples by gas chromatographic and mass spectrometric analysis. Background levels of 4-aminobiphenyl-hemoglobin adducts were detected in maternal nonsmokers (18.3 +/- 12.7 pg 4-aminobiphenyl/g hemoglobin, mean +/- SD) and in fetal samples (8.88 +/- 5.8 pg/g hemoglobin). Increasing levels of 4-aminobiphenyl-hemoglobin adducts were found as the smoking status of the women increased, ranging from 144 +/- 22.2 ( < 1 pack/d) to 633 +/- 87.9 ( > 2 packs/d). A corresponding increase in the presence of fetal 4 aminobiphenyl-hemoglobin adducts was also detected (74 +/- 17.8, < 1 pack/d, to 319 +/- 50.5, > 2 packs/d). This study confirms that the potent tobacco-related carcinogen 4-aminobiphenyl crosses the human placenta and binds to fetal hemoglobin in significantly higher concentrations in smokers when compared to nonsmokers. PMID- 8614024 TI - Rat strain- and gender-related differences in neurobehavioral screening: acute trimethyltin neurotoxicity. AB - Trimethyltin (TMT) produces unique pathological and behavioral changes after a single dose. In this study, TMT was used to examine the ability of a neurobehavioral screening battery (functional observational battery and motor activity) to characterize these behavioral changes in rats. The behavioral profile of TMT was obtained using these tests in male Long-Evans (LE) and Fischer 344 (F344) rats, to assess the influence of rat strain, and in LE males and females to evaluate gender-related differences. All rats were tested before dosing and again at 1, 7, 21, and 42 d after a single dose of either 0, 4, 6, or 8 mg/kg TMT-hydroxide (intravenously). In general, the characteristic syndrome of tremor, increased reactivity, and hyperactivity was observed; however, the magnitude and time course of these effects were much greater in F344 rats. Significant strain- but not gender-related differences were obtained when comparing TMT effects on different domains of neurological function. Comparisons of predosing data between male LE and F344 rats, as well as between male and female LE rats, revealed significant differences in baseline values for about half of the measures of the test battery. These preexisting differences, however, could not account for the observed dissimilarities in treatment effects. Quantitative and qualitative differences were evident to a greater extent when comparing LEs and F344s than between males and females. Therefore, conclusions based on these types of neurobehavioral screening data would be influenced considerably more by the differences between rat strains. PMID- 8614025 TI - Fungal metabolism of 2-nitrofluorene. AB - Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) are direct-acting mutagens and carcinogens that are considered a risk to human health. We investigated the metabolism of 2-nitrofluorene by the fungus Cunninghamella elegans ATCC 36112. At 144 h of incubation, C. elegans had metabolized about 81% of the [9-14C]-2 nitrofluorene, resulting in 6 metabolites. The major metabolites were separated by reversed-phase high-performance liquid chromatography and identified by 1H NMR, ultraviolet (UV)-visible, and mass spectral analyses as 2-nitro-9-fluorenol, 2-nitro-9-fluorenone, 6-hydroxy-2-nitrofluorene, and sulfate conjugates of 7 hydroxy-2-nitro-9-fluorenone and 7-hydroxy-2-nitrofluorene. 2-Nitro-9-fluorenol accounted for about 62% of the total metabolism. For comparison with the microbial system, experiments with liver microsomes of rats pretreated with 3 methyl-cholanthrene were conducted. Microsomal incubations indicated formation of phenolic and ring-hydroxylated products of 2-nitrofluorene. 2-Nitrofluorene and hydroxylated metabolites have been previously implicated as direct-acting mutagens in bacterial assays and have shown sister chromatid exchanges in vivo in bone marrow cells and in vitro in ovary cells and unscheduled DNA synthesis in mammalian studies. Previous studies with other PAHs using C. elegans have shown that the phenols and glucoside and sulfate conjugates of phenols are generally less mutagenic than the parent. The results from the metabolism of 2 nitrofluorene by C. elegans suggests the detoxification potential of this fungus. PMID- 8614026 TI - Acute-phase response in rat to carbon tetrachloride-azathioprine induced cirrhosis and partial hepatectomy of cirrhotic liver. AB - Irreversible liver cirrhosis was induced in rats by supplementing their diet with 0.02% azathioprine and intubating them twice a week with carbon tetrachloride in corn oil. Over period of 3 mo, intoxicated rats showed an atypical acute-phase reaction (APR). The relative concentrations of haptoglobin, beta-lipoprotein, alpha-1-antitrypsin, an unknown peak "X, " and transferrin increased exponentially following a mild initial drop, while albumin, C3c + C3, alpha-1 acid glycoprotein, alpha-1-lipoprotein, and macroglobulin declined continually during the experiment. The accumulated peritoneal fluid was found to contain a similar spectrum of APR proteins. On the other hand, histological examination revealed gradual liver damage manifested as a gradual increase in the areas of collagen separating liver cells, and at the end of the experiment, severe liver damage was evident with isolated hepatocytes in a matrix of collagen. The available data point to the disparity that exists between the physical status of hepatocytes and their biochemical function, which suggests that the remaining metabolically fatigued hepatocytes of the cirrhotic liver continue to biosynthesize and release elevated concentrations of some secretable APR proteins and less of others. Changes in the spectrum of APR plasma components during the progression of inflammatory or physical lesion remain a valid biochemical measure of the pathological function of the acutely intoxicated liver. Partial hepatectomy (PH) of cirrhotic liver displayed a mute APR and no regenerative activity of the remnant hepatic tissue, most likely due to the substantial depletion of hepatic DNA and possible chemical damage to DNA of the remaining viable hepatocytes. A possible cause for the depressed APR to the surgical insult of PH is that the initial azathioprine-CCl4 intoxication had maximally affected APR gene expression and a second injury would then elicit minimal further changes in mRNA levels. Thus, in a compounded pathological condition, the initial inflammatory stimulus on various pre-rRNAs, rRNAs, and mRNAs is rate-limiting to the hepatic biosynthesis and secretion of APR proteins and may not respond linearly, if at all, to a second stimulus. PMID- 8614027 TI - Postinjury multiple organ failure: a bimodal phenomenon. AB - To better define the epidemiology of postinjury multiple organ failure (MOF), we prospectively evaluated 457 high-risk trauma patients who survived more than 48 hours. Overall, 70 (15%) developed MOF. In 27 (39%) patients, the occurrence was early, while in 43 (61%) patients the presentation was delayed. At presentation, early MOF had more cardiac dysfunction, while late MOF had greater hepatic failure. Indices of shock were more critical risk factors for early MOF, while advanced age was more important for late MOF. While early and late MOF had a similar high incidence of major infections, these appeared to be more important in precipitating late MOF. Finally, while mortality is similar, early MOF patients appear to succumb faster. In conclusion, postinjury MOF remains a significant challenge and appears to present in at least two patterns (i.e., early versus late). Better understanding of the relative roles of the dysfunctional inflammation and infections in early MOF versus late MOF may facilitate the development of new strategies for the prevention and treatment of morbid syndrome. PMID- 8614028 TI - Utility of illness severity scoring for prediction of prolonged surgical critical care. AB - OBJECTIVE: To determine whether APACHE III and multiple organ dysfunction syndrome scores can predict a prolonged length of stay for critically ill surgical patients in the intensive care unit. DESIGN: Prospective, inception cohort study. SETTING: Surgical intensive care unit (SICU) of an urban, tertiary care hospital. PATIENTS: 2,295 consecutive admissions for critical surgical illness, postoperative complications, or postoperative monitoring in 2,058 patients. INTERVENTIONS: Calculation of Acute Physiology and Chronic Health Evaluation (APACHE) II and APACHE III scores 24 hours after admission to the SICU. Serial quantitation of organ dysfunction for the duration of hospitalization according to the multiple organ dysfunction score. Patients were stratified by survival and time intervals for the duration of critical care, and followed until discharge or death. MAIN OUTCOME MEASURES: Hospital mortality and length of stay in the SICU. RESULTS: The mean APACHE II and APACHE III scores were 14.0 +/- 0.2 and 45.2 +/- 0.6 points, respectively (mean +/- SEM). The incidence of organ dysfunction was 43%, and the hospital mortality was 9.7%. The mean ICU length of stay was 6.1 +/- 0.2 days, but decreased progressively from 6.8 +/- 0.5 days in 1991 to 5.3 +/- 0.6 days in 1995 (p < 0.01) with no change in either illness severity or the number of admissions. By univariate analysis, increased length of stay in the ICU was associated with increasing APACHE scores, an increased incidence of emergency admissions, and the incidence and magnitude of organ dysfunction (all p < 0.01). Severity indices appeared to plateau in magnitude in patients whose ICU stay ultimately exceeded 21 days. By multivariate analysis of variance (MANOVA), independent predictors of a prolonged stay in the SICU were APACHE III (p = 0.0023), emergency admission (p = 0.0007), and the magnitude of organ dysfunction (p < 0.00001), but not APACHE II. Only an emergency admission (p = 0.0005) and the magnitude of organ dysfunction (p < 0.00001) predicted a prolonged stay independently in survivors. In contrast, only the admission APACHE III score(p = < 0.0001) and the magnitude of organ dysfunction (p = 0.0001) were independently predictive of mortality by MANOVA. CONCLUSIONS: The development of multiple organ dysfunction syndrome is a powerful predictor of a prolonged ICU course in critical surgical illness, even in survivors. Increased risk of a prolonged stay in the ICU plateaued at 21 days, making 21 days an appropriate definition of prolonged care for future studies. Predictive models should account for organ dysfunction and very long stays in future estimations. The combined use of APACHE III and the multiple organ dysfunction score may provide improved prediction of a prolonged stay in the ICU, but further enhancements are needed before prediction of outcome in individual patients is reliable. PMID- 8614029 TI - Evaluation of the medical management and preventability of death in 137 road traffic fatalities in Victoria, Australia: an overview. Consultative Committee on Road Traffic Fatalities in Victoria. AB - OBJECTIVES: In 1992 a multidisciplinary committee was established to identify problems in the management of road fatalities in Victoria, Australia, to assess their contribution to death, and to identify preventable deaths (preventable: survival probability more than 75%; potentially preventable: 25 to 75%). METHODS: For 1992 and 1993 all 137 fatality cases surviving until arrival of ambulance services were evaluated by analysis and discussion of their complete prehospital, hospital, and autopsy records. RESULTS: 1,012 problems were identified in 509 admissions to the various areas of care. Six hundred eighty-five (68%) were management errors and 217 (21%) were system inadequacies. Technique errors (45 (4%)), diagnosis delays (25 (2%)), and diagnosis errors (40 (4%)) were less frequent. The emergency department (ED) accounted for 537 (53%) problems, followed by prehospital (200 (20%)) and intensive care unit (118 (12%)). Four hundred seventy (46%) problems were assessed as contributing to death. Two hundred twenty-eight (49%) occurred in the ED, 90 (19%) were prehospital problems, and 63 (13%) occurred in the intensive care unit. Management errors comprised 326 (69%) problems contributing to death, and system inadequacies 88 (19%). Resuscitation problems accounted for 82 (49%) of the 167 ED management errors contributing to death. Eighty-five (62%) deaths were assessed as nonpreventable, 7 (5%) as preventable, and 45 (33%) as potentially preventable. CONCLUSION: Organizational and educational counter measures are required to reduce the high frequency of problems in emergency services and clinical management. PMID- 8614030 TI - Influence of a statewide trauma system on location of hospitalization and outcome of injured patients. AB - OBJECTIVE: Evaluate the influence of implementing the Oregon statewide trauma system on admission distribution and risk of death. DESIGN: Retrospective pre- and posttrauma system analyses of hospital discharge data regarding injured patients with one or more of the following injuries: head, chest, spleen/liver, pelvic fracture, and femur/tibia fracture. MATERIALS AND METHODS: Risk-adjusted odds ratio of admission to Level I or II (tertiary care) trauma centers, and odds ratio of death were determined using hospital discharge abstract data on 27,633 patients. Patients treated in 1985-1987, before trauma system establishment, were compared to patients treated in 1991-1993 after the trauma system was functioning. MEASUREMENTS AND MAIN RESULTS: After trauma system implementation, the odds ratio of admission to Level I or II trauma centers increased (odds ratio 2.36, 95% confidence interval 2.24-2.49). In addition, the odds ratio of death for injured patients declined after trauma system establishment (odds ratio 0.82, confidence interval 0.73-0.92). CONCLUSIONS: The Oregon trauma system was successfully implemented with more patients with index injuries admitted to hospitals judged most capable of managing trauma patients. The Oregon trauma system also appears beneficial since trauma system establishment is associated with a statewide reduction in risk of death. PMID- 8614032 TI - Prostaglandin E2 production by endotoxin-stimulated alveolar macrophages is regulated by phospholipase C pathways. AB - BACKGROUND: Eicosanoids play an important role in many aspects of systemic inflammatory responses and host defense. Although the synthesis of eicosanoids by different enzymes has been elucidated, the regulatory mechanism of eicosanoid production is not clear. We designed this study to investigate the hypothesis that PGE2 production by endotoxin (lipopolysaccharide; LPS)-stimulated macrophages (MO) is dependent on phospholipase C (PLC) signaling pathways. METHODS: Rabbit alveolar macrophages (MO) were obtained by bronchoalveolar lavage. MO were suspended in RPMI-1640 medium at 1 x 10(6)/mL and were exposed to Escherichia coli LPS (10 ng/mL) +/- various agonists and antagonists of PLC and its secondary messengers. After 24 hours of incubation, prostaglandin E2 (PGE2) production was measured by ELISA. RESULTS: LPS-activated MO produced four times as much PGE2 as did control unstimulated MO. The increase in PGE2 production was inhibited by PLC inhibitors (U73122 or D609) and a low-molecular-weight PLA2 inhibitor, manoalide. An increase in intracellular calcium and activation of both the calmodulin and protein kinase C kinase pathways increase PGE2 production. CONCLUSIONS: PGE2 production is intimately dependent on several phospholipases. Production is not only dependent on low-molecular-weight PLA2 cleavage of arachidonic acid from membrane phospholipids, but also by-products of PLC activation. PLC-dependent intracellular Ca-calmodulin signaling and protein kinase C activation provide significant modulation of PGE2 production. PMID- 8614031 TI - Thoracic aorta injuries: management and outcome of 144 patients. AB - Rupture of the thoracic aorta from blunt injury is often lethal. Methods of operative repair vary, based on the surgeon's preference and circumstances. The primary hypothesis of this study was that operative management choices would correlate with outcome. Data on demographics, injury mechanism, initial evaluation, diagnostic procedures, operative treatment, and outcome were obtained from chart review at the state's eight trauma centers. Rates of paraplegia and survival were compared for different methods of operative repair. Of 63,507 hospitalized trauma patients, 144 patients sustained thoracic aortic injury (incidence = 0.23%). Sixty-four died (44.1%), most of whom died in the emergency department (26) or the operating room (12). Eighty-six patients had complete operative data for analysis, including cross-clamp time and methods of repair. No patient in the group with a cross-clamp time of less than 35 minutes developed paraplegia (p = 0.02). For the patients with longer cross-clamp times, 6 of 14 patients (42.9%) undergoing clamp and sew repair developed paraplegia, as compared to 2 of 37 patients (5.4%) repaired on bypass (p = 0.005). This study suggests that the rate of paraplegia after repair of thoracic aortic injury can be minimized with short cross-clamp times or the use of bypass when long cross clamp times can be anticipated. PMID- 8614033 TI - Role of CD14 in hemorrhagic shock-induced alterations of the monocyte tumor necrosis factor response to endotoxin. AB - OBJECTIVE: To determine if the shock-induced alterations in whole blood monocyte tumor necrosis factor (TNF) response are mediated by the CD14 receptor. DESIGN: Prospective controlled animals experiments. MATERIALS AND METHODS: New Zealand White rabbits (n = 15) were subjected to hemorrhage and resuscitation. Blood samples obtained before shock and 24, 72, and 120 hours after shock were stimulated with lipopolysaccharide in the presence or absence of the anti-CD14 monoclonal antibody, 63D3. Tumor necrosis factor was assayed using L929 cells. MEASUREMENTS AND MAIN RESULTS: There are no detectable TNF activity in unstimulated blood. The CD14 inhibition resulted in a 55% reduction in baseline TNF activity. After shock, there was a marked increase in TNF activity with lipopolysaccharide stimulation. Addition of 63D3 resulted in a dose-dependent 95% reduction in TNF activity at 24 and 72 hours after shock, (p < 0.05). CONCLUSION: The enhanced whole blood monocyte TNF response after hemorrhage is CD14 dependent. PMID- 8614034 TI - Is sepsis-induced apoptosis associated with macrophage dysfunction? AB - Apoptosis (A O) is a pathological process by which cells undergo a form of inducible nonnecrotic cellular suicide. In vitro studies suggest that changes in the rate of macrophage (Mo) A O may be associated with elevated proinflammatory cytokine secretory capacity, such as interleukin-1 beta (IL-1 beta) (via IL-1 converting enzyme activation). Furthermore, it has been reported that Mo are activated during early (0-4 hours) experimental septic insult to act as sources of proinflammatory cytokines, such as IL-1. However, with the progression of sepsis, these same cells become refractory to further stimulation (appearing dysfunctional). Nonetheless, it remains unknown if this acquired immunosuppression (dysfunction) is associated with an acceleration in macrophage A O. To determine this, male C3H/HeN mice were subjected to sepsis (cecal ligation and puncture, CLP) or sham-CLP and 4 or 24 hours thereafter Mo were isolated from the peritoneum (PMo) and liver (KMo). Macrophage monolayers were lysed either after stimulation with lipopolysaccharide (LPS) (10 microgram/mL, 24 hours) in vitro or immediately (ex vivo) before LPS stimulation and the cytoplasmic cell fraction was retained. The extent of A O was determined using a cell-death enzyme-linked immunosorbent assay, which detects the presence of cytoplasmic oligonucleosomes and changes in the propidium iodide staining intensity. The results indicate that, early after CLP (4 hours) only PMo stimulated with LPS in vitro showed evidence of increasing A O. At 24 hours (late) after the onset of sepsis, the ex vivo extent of A O in PMo was increased but it was decreased in KMo. However, the addition of LPS in vitro results in a marked increase in both septic PMo and KMo A O. This latter result suggests that the inability of Mo to release cytokines in response to stiumulants, such as LPS during late sesis (24 hours), may be because of induciton of accelerated A O in these Mo populations. PMID- 8614035 TI - Interleukin-6 delays neutrophil apoptosis via a mechanism involving platelet activating factor. AB - BACKGROUND: Interleukin (IL)-6, an integral mediator of the physiologic acute phase response to injury, has been associated with adverse postinjury complications when present in excessive concentrations. The precise role of IL-6 is unclear, but may involve exacerbation of polymorphonuclear neutrophil leukocytes (PMN)-mediated hyperinflammation. We have shown that IL-6 delays PMN apoptosis, thereby inhibiting the resolution of inflammation. More recently we have found that IL-6 stimulates PMNs to generate platelet-activating factor (PAF). Given the evidence for PAF involvement in postinjury hyperinflammation, we hypothesized that IL-6 delayed apoptosis via a mechanism involving PAF. METHODS: PMNs were isolated from healthy human donors using plasma-Percoll gradients and were cultured in enriched RPMI 1640 media at 2 x 10(7) PMNs/mL for 24 hours (37 degrees C, 5% CO2). Subgroups were treated with IL-6 (0.1-10 ng/mL) or PAF (0.1 10 ng/mL) or pretreated with the PAF receptor antagonist WEB 2170 (20 microM) before IL-6 or PAF. Morphologic assessment and quantitation of apoptosis was performed with acridine orange/ethidium bromide stain. RESULTS: Both IL-6 and PAF suppressed PMN apoptosis. Pretreating PMNs with WEB 2170 abrogated the effects IL 6 as well as PAF. CONCLUSION: Interleukin-6 delays PMN apoptosis via a mechanism involving PAF. These observations may help elucidate the mechanisms of IL-6 and PAF in mediating postinjury hyperinflammation and secondary organ dysfunction, ultimately leading to effective therapeutic targets in patients at risk for multiple organ failure. PMID- 8614036 TI - Correlation of Doppler derived velocity change with cardiac index. AB - Doppler ultrasound may be used as a screen to determine the need for placement of a pulmonary artery catheter. We tested the utility of Doppler derived acceleration, in correlation with pulmonary artery catheter derived cardiac index, as a screen for pulmonary artery catheter placement in 40 trauma patients. We found the expected quadratic relationship between acceleration and cardiac index. We also found that acceleration less than 200 cm/s2 correlates well with cardiac index less than 3.0 liter/min/m2. PMID- 8614037 TI - Is immediate decompression of high voltage electrical injuries to the upper extremity always necessary? AB - OBJECTIVE: To determine if immediate decompression is required for all high voltage injuries to the upper extremity. DESIGN: Retrospective review. MATERIALS AND METHODS: Charts reviewed of 62 patients who had upper extremity contact with >1,000 volts of electricity over a 10-year period. MAIN RESULTS: One hundred upper extremities were treated. Twenty-two percent were decompressed within 24 hours because of progressive nerve dysfunction, clinical compartment syndrome, or failure of resuscitation. This group required a mean of 4.2 operations with an amputation rate of 45%, similar to other series. Thirty-five percent of burned extremities had their first operative procedure delayed until resuscitation was complete. This group required a mean of 2.1 operations with no amputations. Forty three percent of extremities did not require operations to achieve healing. Overall results show a 10.0% amputation rate and mean hospital stay of 27 days. CONCLUSIONS: We conclude that the need for amputation and multiple operations is determined by the injury itself and that immediate decompression is only required for the usual clinical signs of compartment syndrome. Selective decompression may actually preserve tissue and decrease the need for eventual amputation because fasciotomy can lead to soft tissue dessication by exposing viable tissue. PMID- 8614038 TI - Trauma pneumonectomy revisited: the role of simultaneously stapled pneumonectomy. AB - OBJECTIVE: The aim of this study was to compare simultaneously stapled pneumonectomy (SSP) with individual ligation (IND) as a method for performing urgent pneumonectomy (Py) for trauma. METHODS: Twelve patients who required Py were reviewed. SSP was performed in nine cases and IND in three cases. The two groups had statistically similar injury severity scores, presenting systolic blood pressures, and Trauma and Injury Severity Score derived probabilities of survival. An animal model of Py was developed, in which seven animals underwent SSP and seven underwent IND methods. Burst pressures of the pulmonary artery and bronchus were calculated after 14 days. RESULTS: There were no differences noted in survival rates between SSP (5 (56%)) and IND (1 (33%)), nor in incidence of bronchopleural fistula. The SSP group had a significantly shorter operative time compared with that of IND (88.9 +/- 14.3 minutes vs 213 +/- 57.8 minutes, respectively, p - 0.01). The animal study revealed no difference in burst pressures of the bronchus (SSP = 662.9 +/- 169.9 mm Hg vs. IND = 591.4 +/- 193.2 mm Hg, p = 0.752) or of the pulmonary artery (SSP = 554.3 +/- 195.1 mm Hg vs. IND = 477.7 +/- 247.5 mm Hg, p = 0.529). CONCLUSION: Survival after pulmonary injuries that require Py depends upon the rapidity of hilar control and of the procedures itself. Simultaneously stapled pneumonectomy is an effective and rapid method of dealing with such rare injuries. PMID- 8614039 TI - Lower limb trauma with injury to the popliteal vessels. AB - A retrospective analysis of blunt trauma to the lower extremity with injury to the popliteal vessels was undertaken in an attempt to determine the major predictors of outcome and to expose the shortcomings of our management. Thirty one patients with lower extremity trauma including a popliteal artery injury were admitted to our clinic between 1979 and 1993. Two patients died of hemorrhagic shock or from associated lesions. Amputation of the leg was performed primarily in one patient because of massive tissue damage and secondarily in five patients because of uncontrolled local infection (two patients), excessive tissue damage (two patients), and persistent ischemia (one patient who later died). A peripheral neurologic deficit resulted in 12 of 24 non-amputated extremities. Three additional patients suffered sequelae from bone and joint damage. In all, nine patients recovered completely from their limb injury. Severe ischemia of the leg was found to be an indicator of major limb damage and was a strong determinant of poor outcome. Of 18 patients with severe ischemia, two died (one after amputation), five were amputated, and eight were left with a peripheral neuropathy. Only two patients recovered completely. Of 13 patients with relative ischemia, five recovered completely and four sustained a peripheral neuropathy. The deleterious effects of delayed revascularization were evident in four patients who developed a peripheral neuropathy secondarily. Morbidity from the ischemic insult could have been reduced in seven patients: the diagnosis was missed in two, its seriousness not realized in one, and a non-optimal management led to an excessive ischemic time in four. The magnitude of skeletal and soft tissue injury, alone or in combination, was also strongly associated with an increased morbidity. Most patients with blunt lower limb trauma and popliteal vascular injury are left with serious sequelae from associated neuro-musculo skeletal damage and from ischemia. Although the magnitude of the first variable is determined by initial trauma and cannot be altered, a constant awareness of possible arterial injury in lower limb trauma, and adherence to a plan of management according to the ischemic state of the leg, should help avoid the additional deleterious effects of prolonged ischemia. PMID- 8614040 TI - Violence prevention involvement among trauma surgeons: description and preliminary evaluation. AB - Violence has become a primary focus for the national agenda and a growing public health concern in the medical community. Although prevention is a major component of public health policy, it is unclear what contribution trauma surgeons and trauma centers are making toward violence prevention. PURPOSE: The purpose of this study was to assess the extent of violence prevention activity at trauma centers, the involvement and attitudes of trauma surgeons toward violence prevention, and the perceived need for a formal violence prevention curriculum. METHODS: Self-report postal surveys were sent to trauma directors and associate directors at 430 Level I and Level II trauma centers throughout the United States. A descriptive analysis was performed using the 230 (53%) returns. RESULTS: 55% of Centers reported an active violence prevention program with surgeons participating in these existing programs 47% of the time. Overall, only 26% of surgeons reported being active in violence prevention activities, although 71% thought that violence prevention should be an integral part of trauma center activity. Trauma surgeon involvement in violence prevention and the presence of an active institutional program was demonstrated significantly (p <0.001, chi2). Lack of available time and "not knowing where to start" were cited as the most common reasons for lack of involvement in violence prevention activity. CONCLUSION: There is strong support among trauma surgeons for violence prevention programs and for the integration of these programs into the trauma center. A relatively small number of surgeons actually are engaged in violence prevention activity, but most (69%) are willing to become personally involved. The data suggest that established violence prevention programs facilitate involvement of trauma surgeons in violence prevention activity. The discrepancy between actual involvement in, and general support for, violence prevention efforts may be explained by a lack of established roles and previous experience for surgeons and by limited guidance outside of existing programs. PMID- 8614041 TI - 1,000 consecutive ultrasounds for blunt abdominal trauma. AB - Diagnostic peritoneal lavage (DPL) and computed tomography (CT) are the primary diagnostic modalities used in the evaluation of patients with suspected blunt abdominal trauma (BAT). DPL is fast and accurate but is associated with complications. CT is also accurate, yet requires stability and transportability of the patients. Ultrasound (US) has been suggested as an aid in evaluating BAT. We evaluated US in the initial assessment of BAT in 1000 patients. Patients were eligible for the study if they met specified trauma criteria and had suspected BAT. We then followed the outcome of the patients and their further work-up. US showed a sensitivity of 88%, a specificity of 99%, and an accuracy of 97% for detecting intraabdominal injuries. We conclude that emergency ultrasound may be used as the initial diagnostic modality for suspected blunt abdominal trauma. PMID- 8614042 TI - Interleukin-10 is associated with the development of sepsis in trauma patients. AB - Interleukin-10 (IL-10) is a potent regulator of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1, IL-6, and interferon-gamma. We retrospectively evaluated 66 severely injured patients for detectable plasma IL 10. the presence or absence of IL-10 was correlated with clinical parameters. Forty of 66 patients had detectable levels of IL-10. Plasma IL-10 was associated with admission hypotension (p < 0.01) and the development of sepsis (p < 0.05). There was no difference between IL-10-positive and -negative patients with respect to age, mechanism or severity of injury, blood transfusion, operative interventions, or the subsequent development of ARDS, hepatic dysfunction, or renal insufficiency. We conclude that IL-10 can be detected in the plasma of some severely injured patients and that it is associated with the development of sepsis. Further investigation of the immunoregulatory effects of IL-10 after trauma is indicated. PMID- 8614043 TI - The effects of positive end-expiratory pressure of intrapulmonary shunt and ventilatory deadspace in nonhypoxic trauma patients. AB - Controversy exists regarding the routine use of positive end-expiratory pressure (PEEP) in mechanically ventilated patients. We hypothesized that nonhypoxic patients receiving 5-cm H2O PEEP would have improved shunt and PaO2/F10(2) ratios (P/F), without an increased dead space to tidal volume ratio (VD/VT) versus patients receiving no PEEP. Forty-four trauma patients were randomized to receive 5-cm H2O PEEP (PEEP) or 0-cm H2O PEEP (ZEEP). Shunt VD/VT and P/F were measured at 0, 12, 24, 36, and 48 hours after intubation and after extubation. PEEP and ZEEP comparisons used Student's t test and the General Linear Models procedure. Shunt was significantly increased at t = 0 and at extubation in the PEEP group. At extubation, the PEEP group demonstrated significantly higher VD/VT and poorer P/F ratios. After correction for baseline values, no statistically significant differences were noted in spite of a trend toward worsening pulmonary function in all measured parameters. These results suggest that routine use of 5-cm H2O PEEP in mechanical ventilated trauma patients is not necessary. PMID- 8614044 TI - Posttraumatic inflammatory response, secondary operations, and late multiple organ failure. AB - The objective of this study was to determine the role of surgical procedures as secondary inflammatory insults in the development of late multiple organ dysfunction syndrome in patients with multiple trauma and to evaluate both specific and nonspecific indicators of the inflammatory response in their ability to indicate the risk of severely injured patients to develop organ failure after secondary operations. In a prospective study of 106 severely injured patients (ISS 40.6) who underwent secondary operations (> 3 days after trauma), we compared the level of preoperative inflammation with the sequelae of surgical trauma. The interventions included facial reconstructions; osteosynthesis of the pelvic girdle, long bones, and spine; and others. Group 1 consisted of 40 patients (38%) who developed respiratory, renal, or hepatic failure, or combinations thereof, within 2 days after the operation or whose preexisting organ dysfunction worsened by more than 20% from baseline. The remaining 66 patients (62%) with an uneventful recovery formed group 2. The preoperative levels of neutrophil elastase (92.2 vs. 61.3 ng/dL), C-reactive protein (12.4 vs. 7.6 mg/dL), and platelet count (118,000 vs. 236,000/microL) were significantly more abnormal in the patients of group 1. PO2/FiO2 ratio was also somewhat lower in group 1 patients (305.5 vs. 351), whereas other parameters (e.g., blood pressure, heart rate, bilirubin, creatinine, urinary output, lactate, pH, and coagulation) did not allow preoperative differentiation between groups 1 and 2. An increased state of inflammation (neutrophil elastase > 85 ng/mL, C-reactive protein > 11 mg/dL, platelet count < 180,000/microL) predicted postoperative organ failure with an accuracy of 79% (sensitivity, 73%; specificity, 83%). We conclude that secondary operations may act as a second insult and may precipitate late multiple organ dysfunction syndrome if they are performed in patients with multiple trauma while they still have an increased level of posttraumatic inflammation. However, future investigations have to show whether postponing surgery until inflammation has subsided or the use of less invasive surgical techniques will decrease the rate of postoperative organ failure in the trauma patient. PMID- 8614045 TI - Analysis of deaths within 24 hours of injury: cost-benefit implications for organ and tissue donations. AB - OBJECTIVE: To determine useful predictors of successful organ donation in patients who die within 24 hours of injury (early deaths). DESIGN: Retrospective review of a 3-year experience at a Metropolitan Level I Trauma Center. MATERIALS AND METHODS: All 223 early deaths among 5,719 trauma patients in a 3-year period were reviewed. This group represented 62% of all trauma deaths. RESULTS: Forty six patients (21%) donated 102 vascularized organs and made 66 donations of tissues. Patients with isolated severe head injuries had the highest rate of successful donation (33%). Those with severe head injury and another severe organ injury had a lower rate of donation (13%), and donation was rare (1%) among patients with severe organ injury in the absence of head injury (p < 0.001). There were no organ donors among victims >65 years old or in 64 of 65 patients with a Revised Trauma Score of <2.2. The Revised Trauma Score was significantly higher in organ donors (3.39 vs. 3.07, p < 0.05). The cost-benefit ratio for early deaths was $6,512 per organ/tissue recovered. CONCLUSIONS: Decisions regarding the resuscitation of trauma patients who have characteristics associated with a recognized low rate of organ donation should be made exclusive of the potential for organ recovery. PMID- 8614047 TI - Contact lens care in the unconscious. AB - We present a case of gradual visual loss in a patient after a severe road traffic accident. The cause of visual loss was prolonged contact lens wear. We stress the importance of excluding contact lens wear and suggest a method for bedside examination of an unconscious patient to detect the presence of contact lenses. PMID- 8614046 TI - Patterns of injury and disability caused by forklift trucks. AB - Over a 7-year period, 34 patients were treated at the Southern New Jersey Regional Trauma Center for forklift-related injuries, ranging from minor contusions to multiple organ-system trauma. Hospital and rehabilitation courses were prospectively evaluated, documenting long term impairment of function and disability. Patients injured by falling from forklifts generally had less severe injuries, requiring fewer surgical procedures, shorter hospital stays, and less overall disability, than patients who received crush-type (object-oriented) injuries. The more serious injuries were most frequently caused by a forklift striking or running over the patient. There were strong correlations between the Injury Severity Score assessed upon initial evaluation and subsequent length of hospitalization, degree of disability, and extent of functional impairment after recovery. These findings support the enforcement of existing safety precautions for the operation of forklift trucks. PMID- 8614048 TI - Left atrial appendage rupture caused by a seat belt: a case report and review of the literature. AB - Motor vehicle accidents account for most cases of blunt chest trauma; seat belts have been implicated as the cause of a few of these injuries. We report a case of a seat-belt-induced fatal isolated left atrial appendage rupture in a 40-year-old female. PMID- 8614050 TI - Penetrating trauma of the posterior fossa resulting in Vernet's syndrome and internuclear ophthalmoplegia. AB - We report the case of a young male assaulted with a screwdriver, which was embedded in the posterior fossa, causing Vernet's syndrome and internuclear ophthalmoplegia. His hospital course and the cranial nerve deficits sustained as a result of the injury with the relevant neuroanatomy are discussed. PMID- 8614049 TI - Toxic megacolon caused by cytomegalovirus colitis in a multiply injured patient. AB - Cytomegalovirus (CMV) colitis is a rare event that has been described mainly in immunocompromised patients with immunosuppressive medication or HIV infection. An association with severe trauma has not been described previously. We report a formerly healthy, multiply injured 75-year-old male who subsequently developed what appeared to be pseudomembranous colitis. By the time the diagnosis of toxic megacolon on the basis of CMV colitis was established, he had succumbed to multiple organ failure. Whenever pseudomembranous colitis is clinically suspected but not confirmed in a critically ill formerly healthy patient, CMV colitis should be excluded. Once the diagnosis is confirmed, generous resection of all affected colon is mandatory in view of the limited benefit of antiviral therapy in CMV-induced toxic megacolon. PMID- 8614051 TI - Shotgun slug injuries: case report and literature review. AB - Shotguns are usually used to fire multiple pellets; however, they are capable of firing a single projectile in the form of a slug. Although rare, shotgun slug injuries are severe, producing wounds comparable to those inflicted by high velocity weapons with the potential for even more tissue destruction because of the slug's size and mass. We report the first survivor of a close range shotgun slug injury, with a review of this weapon's unique projectile ballistics and the relevant literature. PMID- 8614052 TI - Traumatic spinal subdural hematoma: rapid resolution after repeated lumbar spinal puncture and drainage. AB - A 15-year-old boy developed back pain and sciatica after a minor trauma. Magnetic resonance imaging revealed lumbar spinal subdural hematoma. After repeated lumbar spinal puncture and drainage of hemorrhagic fluid, spinal subdural hematoma was resolved completely. The benefits of conservative treatment by lumbar spinal puncture are discussed. PMID- 8614053 TI - Gunshot injuries of the sacrum. AB - Gunshots to the sacrum are unusual and present several management problems. Associated injuries and particularly sacral bleeding are troublesome. Conventional methods of hemostasis are not suitable in this setting as the spinal blood supply is very complex because it is largely derived from the longitudinal spinal arteries originating intracranially. Attempts at proximal control are difficult and could lead to neurological injury. We successfully managed brisk bleeding in three patients with sacral gunshots. After the major intra abdominal hemorrhage had been controlled, attention was turned to the sacral wounds that had been packed with sponges up to that time. The sacral defect was closed with bone wax to control bleeding definitively. Methyl cellulose was then put over the bone wax and the periosteum of the sacrum and posterior peritoneum (mobilized if necessary), sutured over the methyl cellulose. Post operatively the patients are carefully monitored for developing neurological deficit that would necessitate immediate sacral laminectomy and decompression. We advocate tamponading of the sacral wound with bone wax, covered by methyl cellulose and kept in place and held firm by the periosteum and posterior peritoneum sutured over it as a successful interim or definitive form of therapy. PMID- 8614054 TI - "Gamekeeper's thumb" variant, complicated by reflex sympathetic dystropy. AB - A case of gamekeeper's thumb resulting from a motor vehicle accident is presented. This injury was complicated by the onset of reflex sympathetic dystrophy. An overview of the presentation and management of these disorders, which may result in severe disability without prompt recognition and therapy, is submitted for discussion. PMID- 8614055 TI - Bilateral Smith fracture of the radius caused by airbag deployment. AB - A passenger-side occupant of a car involved in a collision suffered bilateral Smith's fracture after extending both arms for protection and being hit by the deploying airbag. PMID- 8614056 TI - Use of ultrasonography for the evaluation of pregnant trauma patients. AB - The expeditious diagnosis and management of the pregnant trauma patient is essential for the survival of both the mother and fetus. The rapid trauma ultrasound examination, which has been accurately utilized by trauma surgeons and emergency physicians, may have a tremendous impact on the timely identification of acute intraperitoneal injuries and, potentially, on the evaluation of fetal viability in the pregnant trauma patient. This report describes our experience with the rapid trauma ultrasound examination in the management of three pregnant trauma patients and outlines the potential advantages and limitations of the procedure. PMID- 8614057 TI - Blunt popliteal vascular injuries: combined trauma to integument, skeleton, muscle, nerve, and vessels. PMID- 8614058 TI - Trauma surgeons on violence prevention: ready, willing, and able? PMID- 8614059 TI - Neuroleptic malignant syndrome in a trauma patient. PMID- 8614060 TI - Safe and normothermic massive transfusions by modification of an infusion warming and pressure device. PMID- 8614061 TI - A new means of communication among trauma caregivers on the Internet. PMID- 8614062 TI - Significance of intraabdominal extraluminal air detected by CT scan in blunt abdominal trauma. PMID- 8614063 TI - Does the potential for organ donation justify scene flights for gunshot wounds to the head? PMID- 8614064 TI - Neither dopamine nor dobutamine reverses the depression in mesenteric blood flow caused by positive end-expiratory pressure. AB - Positive end-expiratory pressure (PEEP) has been shown to cause a depression of mesenteric blood flow (MBF) and redistribution of blood flow away from the mesenteric vascular bed. OBJECTIVE: We sought to determine whether two commonly used vasoactive agents, dopamine, a known mesenteric vasodilator and inotrope, and dobutamine, with its inotropic properties, would correct the MBF depression caused by PEEP. DESIGN, MATERIAL, AND METHODS: Sprague-Dawley rats, 180 to 250 g, were treated with mechanical ventilation and either no PEEP (control group) or increasing levels (0, 10, 15, and 20 cm of H2O pressure) of PEEP (PEEP group). Also, we evaluated PEEP's effect on MBF and cardiac output (CO) under the influence of a continuous infusion of 2.5 or 12.5 microgram/kg/min of dopamine or 2.5 or 12.5 microgram/kg/min of dobutamine. Cardiac output and, using in vivo videomicroscopy, mesenteric A1, A2, and A3 arteriolar intraluminal radii and A1 arteriolar optical Doppler velocities were measured. After 20 cm of H2O pressure PEEP was attained, two boluses of 2 mL of 0.9 normal saline were given. The MBF was calculated from vessel radius and red blood cell velocity. MEASUREMENTS AND MAIN RESULTS: There were no significant changes from baseline in mean arterial pressure or A2 or A3 diameters in any of the groups. Both MBF and CO were unchanged over time in the control group. The MBF was reduced 78% (p < 0.05) and the CO was reduced 31% (p < 0.05) from baseline at 20 cm of H2O pressure PEEP. After 4 mL of normal saline, the MBF was still 53% below baseline (p < 0.05), while the CO had returned to baseline in the PEEP group. Low-dose dopamine partially ameliorated both the decrease in CO and MBF caused by PEEP, but 4 mL of normal saline was required in addition to the low-dose dopamine to return MBF to baseline levels while on 20 cm of H2O pressure PEEP. High-dose dopamine with the addition of 4 mL of normal saline returned CO to baseline on 20 cm of H2O pressure PEEP, but MBF remained approximately 46% below baseline despite fluid boluses. Neither low-dose nor high-dose dobutamine, with or without fluid boluses, had an appreciable positive effect on CO or MBF. CONCLUSIONS: It is clear that inotropes are not a replacement for adequate fluid loading to correct the depression in cardiac output and mesenteric blood flow associated with the use of mechanical ventilation and PEEP. Low-dose dopamine may serve as an adjunct to adequate fluid resuscitation to improve MBF. PMID- 8614065 TI - Starvation and endotoxin act independently and synergistically to coordinate hepatic glutamine transport. AB - OBJECTIVE: Because hepatic glutamine transport is markedly enhanced during critical illness, we tested the hypothesis that nutrient starvation and endotoxemia act coordinately to augment transport activity. DESIGN: Fed or starved (48 hours) rats received Escherichia coli endotoxin (LPS, 10 mg/kg of body weight, intraperitoneally) or saline before hepatocyte isolation for measurement of glutamine transport. MATERIALS AND METHODS: Hepatocytes were isolated from fed or fasted rats 4 hours after LPS treatment. [3H]glutamine uptake was measured and normalized to cellular protein. Data (mean +/- standard deviation, three separate determinations) were analyzed by Student's t test and analysis of variance. MAIN RESULTS: Starvation induced a 1.6-fold increase in glutamine transport, while LPS treatment of fed rats increased transport activity 2.6-fold. Treatment of fasted animals with LPS induced a sixfold increase in glutamine transport. Kinetically, this effect in endotoxemic starved rats was mediated by both an increase in System N Vmax and the induction of a high affinity System A amino acid carrier which transports glutamine. CONCLUSIONS: Starvation and endotoxemia regulate hepatocyte glutamine transport independently and synergistically. This hepatic response provides glutamine and other amino acids to support key metabolic pathways in the liver during critical illness. PMID- 8614066 TI - Inhibition of the biologic activity of tumor necrosis factor maintains vascular endothelial cell function during hyperdynamic sepsis. AB - BACKGROUND AND OBJECTIVE: Although vascular endothelial cell function (i.e., the release of endothelium-derived nitric oxide) decreases and plasma tumor necrosis factor (TNF) increases during sepsis, it is not known whether the elevated TNF is responsible for the depression of endothelial cell function under such conditions. The aim of this study, therefore, was to determine if inhibition of TNF biologic activity by polyethylene glycol dimerized conjugate of the recombinant human form of the p55 soluble TNF receptor (PEG-(rsTNF-R1)2) maintains endothelial function during sepsis. DESIGN, MATERIALS AND METHODS: Rats were subjected to sepsis by cecal ligation and puncture (CLP). Immediately before the onset of sepsis, 600 microgram/rat PEG-(rsTNF-R1)2 or an equal volume of saline was infused intravenously. At 10 hours after CLP (i.e., hyperdynamic sepsis), the thoracic aorta was isolated, cut into rings, and placed in organ chambers. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG), were determined. Endothelial cell structure was examined by transmission electron microscopy. RESULTS: Endothelium-dependent vascular relaxation was depressed at 10 hours after the onset of sepsis. Administration of PEG-(rsTNF-R1)2 before CLP, however, maintained ACh-induced relaxation. In contrast, no significant difference in NTG-induced relaxation was seen, irrespective of administration of PEG-(rsTNF-R1)2 Furthermore, the deterioration in endothelial structure during sepsis was prevented by PEG-(rsTNF-R1)2 pretreatment. CONCLUSION: Since administration of PEG-(rsTNF-R1)2 maintains vascular endothelial cell structure and function, it can be concluded that TNF plays a pivotal role in producing endothelial dysfunction during sepsis. Thus, pharmacologic agents that inhibit TNF biologic activity and/or its production may be useful for protecting endothelial cells during sepsis. PMID- 8614067 TI - Immunosuppression after endotoxin shock: the result of multiple anti-inflammatory factors. AB - OBJECTIVES: Endotoxin induced suppression of cellular immune function is thought to contribute to septic complications in trauma patients. A rabbit model of endotoxemia was used to determine the relative roles of the anti-inflammatory factors interleukin-4 (IL-4), interleukin-10 (IL-10), transforming growth factor beta1 (TGFbeta1), and prostaglandin E2 (PGE2) in addition to other factors, in inducing immunosuppression. DESIGN: T-cell suppressive factors (TSF) in serum ultrafiltrates were separated and tested for the presence of the known suppressive factors PGE2, IL-4, IL-10, and TGFbeta1. MATERIAL AND METHODS: New Zealand rabbits were injected with 50 microg/kg of purified Escherichia coli lipopolysaccharide. Animals were exsanguinated after 48 hours and serum was separated by ultrafiltration (cutoff 50 kd), TSK HW-40 size exclusion chromatography, and Q-Sepharose anion exchange chromatography. TSF activities of chromatographic fractions and serum samples were measured with a mitogen induced in vitro T-cell proliferation assay. Levels of PGE2, IL-4, IL-10, and TGFbeta1 were measured with enzyme immunoassays. MEASUREMENTS AND MAIN RESULTS: Serum TSF activity, and levels of PGE2, IL-4, IL-10, and TGFbeta1 were increased after endotoxemia. Size exclusion chromatography revealed three major fractions (TSF1 3) with up to 600 times more TSF activity compared with controls. IL-4 and IL-10 were found in TSF1 and TSF3. Further separation of TSF1 by anion exchange chromatography revealed a total of eight different T-cell suppressive factors. TGFbeta1 probably remained in the retentate after ultrafiltration, while PGE2 eluted at a higher retention time. The known anti-inflammatory factors TGFbeta1, IL-10, IL-4, and PGE2 only accounted for 13% of the total serum TSF activity of 614 U/mL. CONCLUSIONS: Lipopolysaccharide shock results in the release of multiple T-cell suppressive factors in addition to known immunosuppressive factors, all of which contribute to the anti-inflammatory response. PMID- 8614068 TI - Hemodynamic and fibrinolytic consequences of intermittent pneumatic compression: preliminary results. AB - OBJECTIVE: To elucidate the time course and magnitude of hemodynamic and fibrinolytic changes associated with sequential gradient intermittent pneumatic compression (SGIPC). DESIGN: Two-phase, intervention and response investigation in normal volunteers. MATERIALS AND METHODS: Subjects were assigned to control (phase I) or compression (phase II) groups. Serial blood samples were obtained via femoral venous catheters for tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1), tPA-PAI-1 complex (tPA-PAI), and euglobulin lysis time (ELT) from all subjects and for fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) from phase II subjects. Duplex venous scanning was carried out on phase II subjects before and during SGIPC. RESULTS: Catheter placement caused elevations in PAI-1 and tPA-PAI, which stabilized within 4 hours of catheter insertion. In phase II, SGIPC induced significant increases in FbDP, FgDP, and tPA-PAI and decreases in ELT and PAI-1, all of which quickly reverted to baseline on termination of compression. Femoral venous blood flow increased by more than 100% with SGIPC. CONCLUSIONS: Sequential gradient intermittent pneumatic compression induces prompt, but short-lived, alterations in both fibrinolytic and hemodynamic function. Noncontinuous SGIPC may result in suboptimal thromboembolic prophylaxis. PMID- 8614069 TI - Changes in granulocyte colony-stimulating factor concentration in patients with trauma and sepsis. AB - OBJECTIVE: To better understand the role of granulocyte colony-stimulating factor (G-CSF) after the inflammatory response. DESIGN: Serum G-CSF concentrations were measured serially in 19 trauma and 15 sepsis patients. Changes in G-CSF concentration were compared with those in the neutrophil ratio, phagocytic and bactericidal activities, and other cytokines. MEASUREMENTS AND MAIN RESULTS: G CSF concentrations in trauma patients were elevated on day 1, but quickly decreased within 7 days. G-CSF reached its maximum 3 hours after injury, parallel with peaks of interleukin-6 (IL-6) and IL-8, but not of tumor necrosis factor alpha (TNF-alpha). In sepsis patients, G-CSF as well as TNF-alpha, IL-6, and IL-8 concentrations were markedly elevated at diagnosis and remained high during the course of the illness. These levels decreased significantly in the 11 survivors. Up to 3 days after the trauma, nonsegmented neutrophil ratios were higher than those thereafter. Neutrophil phagocytic and bactericidal activities remained normal during the course of disease in both conditions. CONCLUSIONS: These results suggest that G-CSF plays an important role in the maturation and maintenance of function of neutrophils during the inflammatory response to trauma and sepsis. PMID- 8614070 TI - Granulocyte colony-stimulating factor for gastrointestinal perforation in patients with leukopenia. AB - OBJECTIVE: Leukopenia in the setting of widespread infection may predispose to sepsis, which is associated with a poor prognosis. Granulocyte colony-stimulating factor (G-CSF), which restores polymorphonuclear leukocyte function and count, has been shown to have protective effects in animal models of sepsis and burns. The aim of this retrospective study was to determine whether G-CSF can reduce the morbidity and mortality gastrointestinal perforation with leukopenia. PATIENTS AND METHODS: The studied subjects were 31 patients who had reduced leukocyte and neutrophil counts before undergoing surgery for gastrointestinal perforation, including six gastroduodenal, nine small intestinal, and 16 colonic perforations from 1986 to 1994. The patients were divided into two groups: a G-CSF(+) group (n = 8) that received G-CSF subcutaneously (150 microgram/day) during the perioperative period, and a G-CSF(-) group which did not. MAIN RESULTS: No significant difference was found in the preoperative and operative factors of the two groups. The postoperative increase in the leukocyte and polymorphonuclear cell counts of the G-CSF(+) group was significantly higher than that of the G CSF(-) group (p <0.01). Renal, hepatic, and gastrointestinal insufficiency was significantly less common in the G-CSF(+) group than in the G-CSF(-) group. The mean number of organs that failed was reduced from 4.00 +/- 2.50 in the G-CSF(-) group to 1.88 +/- 2.03 in the G-CSF(+) group. One of the eight patients who received G-CSF died of sepsis because of panperitonitis. In contrast, in the G CSF(-) group, 15 of 23 patients died of sepsis because of panperitonitis. The cause-specific survival rate of the G-CSF(+) group was better than that of the G CSF(-) group (p <0.05). CONCLUSION: These results suggested that G-CSF reduced the morbidity and mortality of gastrointestinal perforation in patients with leukopenia and encouraged a prospective randomized study in future. PMID- 8614071 TI - Validation of trauma and injury severity score in blunt trauma patients by using a Canadian trauma registry. AB - OBJECTIVE: To compare outcomes in blunt trauma by using Trauma and Injury Severity Score (TRISS) models derived from the Major Trauma Outcome Study (MTOS) and the Ontario Trauma Registry (OTR) as well as to evaluate the role of the Revised Trauma Score within the TRISS model. METHODS: Consecutive blunt trauma cases from 11 Level I trauma centers over a 4-year period were identified from the OTR. Coefficients of the Revised Trauma Score were modified using the Ontario data and this score was tested by using the Hosmer-Lemeshow Goodness of Fit Test. Two Ontario-specific TRISS models were developed with revised coefficients. The first used the standard Revised Trauma Score and the second used the Revised Trauma Score with regenerated coefficients. The accuracy of mortality predictions for all models were compared by using a Hosmer-Lemeshow Goodness of Fit procedure. Additionally, each TRISS models performance characteristics and receiver operating characteristic (ROC) curves were used to evaluate their discriminative capabilities. RESULTS: A total of 5,436 cases were incorporated in the analysis. Patients with all component TRISS variables had a significantly lower mortality compared to all blunt trauma patients (7.0% vs. 15.5%,p < 0.01). Use of the Revised Trauma Score led to the exclusion of 40% of cases because of absent data necessary to compute the score. The Hosmer-Lemeshow Goodness of Fit statistic for the Revised Trauma Score was 79.45 (p = 0.0001). The Hosmer Lemeshow Goodness of Fit Statistic ranged from 11.42, p = 0.175 and 13.1, p = 0.125 for the Ontario TRISS models compared to 25.62, p < 0.005 for the MTOS TRISS model. Sensitivity of all three TRISS models ranged from 98% to 99% with specificity ranging from 24% to 35%. ROC curves were identical for all three TRISS models. CONCLUSIONS: TRISS demonstrated satisfactory performance in a Canadian blunt trauma population. Although revision of coefficients led to a better fit on the Hosmer-Lemeshow statistic, ROC curves demonstrated virtually identical performance of the MTOS and Ontario-based TRISS models. The poor performance of the Revised Trauma Score and the observation that its use led to the exclusion of 40% of cases with a higher mortality raises concerns regarding its use in the TRISS model. PMID- 8614072 TI - Direct current reduces wound edema after full-thickness burn injury in rats. AB - OBJECTIVE: To observe the effect of 4 and 40 microA direct current (DC) on edema formation after burn injury in rats. DESIGN, MATERIALS, AND METHODS: Silver-nylon wound dressings were used as either anodes (-) or cathodes (+) on 20% total body surface area full-thickness scalds in anesthetized male Sprague-Dawley rats. Untreated burned rats and rats treated with silver-nylon dressings without current were used as controls. MEASUREMENTS AND MAIN RESULTS: Immediately applied, continuous DC reduced burn edema by 17 to 48% at different times up to 48 hours postburn (p < 0.001). Neither reversal of electrode polarity nor change in current density had any significant effect on the results of treatment. Starting treatment during the first 8 hours postburn produced the least edema accumulation, but the reduction was significant even when DC was applied 36 hours afterburn. If started immediately after injury, treatment had to be continued a minimum of 8 hours to be most effective. CONCLUSIONS: Direct electric current has a beneficial effect in reducing wound edema after burn injury. PMID- 8614073 TI - Transplantation of cryopreserved cultured epidermal allografts. AB - OBJECTIVE: To optimize cryopreservation methods for cultured epidermal allografts (CEAs) for transplantation onto wounds. DESIGN: Conditions were determined to optimize the cryopreservation of CEAs. Cryopreserved CEAs were then grafted onto 16 donor sites in a double blind randomized trial. MATERIALS AND METHODS: CEAs were grown in culture. Viability of cryopreserved CEAs was determined by: 1) trypan blue dye exclusion; 2) histology; 3) flow cytometry; and 4) acid lipase activity. Cryopreserved CEAs were grafted onto 16 donor sites. Wound healing assessments included 1) visual assessment of healing; 2) histologic assessment of re-epithelialization and differentiation; and 3) visual scar assessment. MEASUREMENTS AND MAIN RESULTS: CEAs were cryopreserved using a controlled rate freezer with an experimentally determined setting of -7 degrees C for the CEA freezing point. Such freezing conditions resulted in retention of approximately 92% of the original viability, no loss in basal keratinocytes as determined by flow cytometry, and no change in acid lipase activity. CEAs cryopreserved according to this method were grafted onto 16 donor sites in a double blind randomized trial. All donor sites underwent complete healing. Histologic examination of biopsies taken from the center of the wound beds showed that CEA treated wounds significantly accelerated the rate of re-epithelialization (7.8 +/ 0.6 days vs. 9.2 +/- 0.9 days for CEA- and control-treated wounds, respectively; p = 0.039) and epithelial differentiation (p = 0.023) compared with control sites. Longterm results showed that CEA-treated wounds were comparable to control sites with regard to: 1) pigmentation; 2) scar height; 3) scar pliability; 4) vascularity; and 5) pain. Wounds treated with cryopreserved CEAs remained durable and not prone to blistering after healing. CONCLUSIONS: CEAs can be successfully cryopreserved for long-term storage. Upon retrieval from storage, CEAs may be used to treat partial thickness wounds. PMID- 8614074 TI - Internal carotid artery gunshot wounds. AB - OBJECTIVE: To review a series of patients who sustained internal carotid artery (ICA) gunshot wounds. DESIGN, MATERIALS, AND METHODS: We retrospectively studied the demographics and clinical presentation, angiographic findings, methods of treatment, and outcome of 38 consecutive patients who had ICA injury identified by angiography. RESULTS: Thirty-four of 38 patients were symptomatic with neck hematomas (32 patients), active hemorrhage (12 patients), and/or neurologic deficit (10 patients). Angiography showed active bleeding in 22 patients and occlusion in 16 patients. Twelve patients were treated operatively by ligation (seven patients), repair (four patients), or intracranial/extracranial bypass (one patient). Twenty-six patients were managed nonoperatively either by angioplasty (one patient), embolotherapy (17 patients), or observation alone (eight patients). Percutaneous balloon catheters were also used in three patients for vascular control of the ICA before operative repair or as a method of assessing intracranial collateral circulation. The mortality of 18.4% was largely related to strokes. CONCLUSIONS: Penetration of the ICA is a very severe injury with a high mortality. The major cause of death in this series was related to neurologic damage associated with carotid injury and shock. However, neurologic deficit among the survivors was uncommon and often resulted from emboli. Interventional radiology can play an important role in the management of these wounds and often obviates the need for operative exploration. PMID- 8614075 TI - Transcervical gunshot injuries: mandatory operation is not necessary. AB - BACKGROUND: It has been suggested that all transcervical gunshot wounds should be explored surgically because of the high incidence of injuries to vital structures. The present prospective study investigated the clinical presentation, the role of various diagnostic investigations, and the need for surgery in patients with transcervical gunshot injuries. METHODS: Ninety-seven patients sustained gunshot injuries to the neck and 33 of them (34%) were transcervical. All victims were assessed clinically according to a written protocol and subsequently were evaluated angiographically, and, in the appropriate case, by means of endoscopy and esophagography. RESULTS: Overall, 24 (73%) of the 33 patients with transcervical gunshot wounds had injuries to cervical structures. Vascular injuries were found in 48%, spinal cord injuries in 24%, and aerodigestive tract injuries in 6% of patients with transcervical injuries. In the 64 patients without midline crossing, the incidence of cervical structure injuries was 31%. Despite the high incidence of injuries to cervical structures in transcervical wounds, only 21% of the patients had a therapeutic operation. The overall mortality was 3%. There were no in-hospital deaths or local complications in the nonoperatively managed group. CONCLUSIONS: The results of the present study do not support the current recommendations of mandatory operation for all transcervical gunshot wounds. A careful clinical examination combined with the appropriate diagnostic investigations should determine the treatment modality. About 80% of these patients can safely be managed nonoperatively. PMID- 8614076 TI - Mortality and prognostic factors in penetrating injuries of the aorta. AB - PURPOSE: This study was designed to investigate the epidemiology and prognostic factors determining survival in penetrating injuries of the aorta. PATIENTS AND METHODS: This was a retrospective analysis of all patients with penetrating aortic injuries, admitted to a large, level I trauma center. The following factors were analyzed for their role in determining survival: mechanism of injury, anatomical site of the aortic injury, initial blood pressure on admission, need for emergency room thoracotomy, and the introduction of a dedicated trauma program with an attending surgeon in-house. RESULTS: There were 93 patients with penetrating aortic injuries over a 5-year period. The abdominal aorta was injured in 67 patients (72%) and the thoracic aorta in 26 (28%). Most of the victims (82.5%) were admitted in shock and 41% had an unrecordable blood pressure on admission. Victims with injury to the thoracic aorta were more likely to have an unrecordable blood pressure on admission than patients with abdominal aortic injuries (73% vs 28.4%), and more likely to require an emergency room thoracotomy (76.9% vs 20.9%). Thirty-four patients (36.6%) required an emergency room thoracotomy and there were no survivors. The overall mortality was 80.6% (87.5% for gunshot injuries, 64.7 % for knife injuries). Patients with abdominal aortic injuries were three times more likely to survive than those with thoracic aortic injuries (23.9% vs 7.7%). The introduction of a dedicated trauma program, which resulted in significant reduction of mortality in other types of severe trauma, had no effect on the outcome in aortic injuries. CONCLUSIONS: Penetrating aortic injuries still have a very high mortality rate with no improvement in survival despite improved trauma services. Injury to the thoracic aorta, gunshot wounds, unrecordable blood pressure on admission, and the need for emergency room thoracotomy, are important predictors of high mortality. PMID- 8614077 TI - Hypoxemia and arterial hypotension at the accident scene in head injury. AB - OBJECTIVE: To quantify the occurrence of arterial hypotension and arterial oxygen desaturation in a series of patients with head trauma rescued by helicopter. DESIGN: Prospective, observational study. MATERIALS AND METHODS: Arterial HbO2 was measured before tracheal intubation at the accident scene in 49 consecutive patients with head injuries. Arterial pressure was measured using a sphygmomanometer. MAIN RESULTS: Mean arterial saturation was 81% (SD 24.24); mean arterial systolic pressure was 112 mm Hg (SD 37.25). Airway obstruction was detected in 22 cases. Twenty-seven patients showed an arterial saturation lower than 90% on the scene, and 12 had a systolic arterial pressure of less than 100 mm Hg. The outcome was significantly worse in cases of hypotension, desaturation, or both. CONCLUSIONS: Hypoxemia and shock are frequent findings on patients at the accident scene. Hypoxemia is more frequently detected and promptly corrected, white arterial hypotension is more difficult to control. Both insults may have a significant impact on outcome. PMID- 8614078 TI - Radiographic cervical spine evaluation in the alert asymptomatic blunt trauma victim: much ado about nothing. AB - OBJECTIVE: To evaluate the hypothesis that alert nonintoxicated trauma patients with negative clinical examinations are at no risk of cervical spine injury and do not need any radiographic investigation. DESIGN: Prospective study. SETTING: A university-affiliated teaching county hospital. PATIENTS: Five hundred and forty nine consecutive alert, oriented, and clinically nonintoxicated blunt trauma victims with no neck symptoms. RESULTS: All patients had negative clinical neck examinations. After radiographic assessment, no cervical spine injuries were identified. Less than half the patients could be evaluated adequately with the three standard initial views (anteroposterior, lateral, and odontoid). All the rest needed more radiographs and/or computed tomographic scans. A total of 2,27 cervical spine radiographs, 78 computed tomographic scans and magnetic resonance imagings were performed. Seventeen patients stayed one day in the hospital for no other reason but radiographic clearance of an asymptomatic neck. The total cost for x-rays and extra hospital days was $242,000. These patients stayed in the collar for an average of 3.3 hours (range, 0.5-72 hours). There was never an injury missed. CONCLUSIONS: Clinical examination alone can reliably assess all blunt trauma patients who are alert, nonintoxicated, and report no neck symptoms. In the absence of any palpation or motion neck tenderness during examination, the patient may be released from cervical spine precautions without any radiographic investigations. PMID- 8614079 TI - The effect of age on peripheral motor nerve function after crush injury in the rat. AB - OBJECTIVE: To determine the ontogeny of functional recovery after peripheral nerve crush injury. DESIGN: Comparative study in rats of varying ages. MATERIAL AND METHODS: Sixty-second crush injury was performed on the left posterior tibial nerve. Control animals underwent either nerve transection or sham procedure. Nerve function was evaluated 2, 4, and 8 weeks following injury by walking track analysis. Print length ratio (PLR), (ratio of normal right-sided print length to experimental left-sided print length), was used to evaluate functional recovery. MEASUREMENTS AND MAIN RESULTS: Two weeks after crush injury, adult rats experienced significantly greater functional impairment than both 4-day-old and 3 week-old animals (p < 0.05). Four weeks after injury, the difference in function between 4-day-old and adult rats and between 3-week-old and adult rats became insignificant. Complete recovery had been achieved by 8 weeks in all groups. CONCLUSIONS: These results demonstrate faster functional recovery after nerve injury in immature rats than in adults. PMID- 8614080 TI - Surgical neck fractures of the proximal humerus: a laboratory evaluation of ten fixation techniques. AB - OBJECTIVE: A biomechanical cadaver study was performed to compare the stability and ultimate strength of ten standard fixation techniques used for the treatment of surgical neck fractures of the proximal humerus. DESIGN: One hundred twenty (60 fresh frozen, 60 embalmed) proximal humerus specimens were selected and divided into two groups: fresh frozen specimens represented a nonosteopenic group and embalmed specimens an osteopenic group. Simulated fractures were created at the level of the surgical neck, reduced, and randomly assigned to one of ten methods of fixation (six fresh frozen and six embalmed specimens per fixation group). These constructs were then mechanically tested with the humeri oriented to create primarily shear loading of the fixation. RESULTS AND CONCLUSIONS: The T plate and screws provided significantly stronger fixation (p < 0.005) in the fresh frozen specimens than all other methods. The Ender nails/tension band construct was the second strongest fixation technique, providing significantly stronger fixation (p < 0.01) than all the remaining techniques. Four Schanz pins with one pin placed through the greater tuberosity followed by the T-plate and screws provided the strongest fixation in embalmed specimens. Tension band fixation in both humeral groups was shown to provide the least effective fixation. PMID- 8614082 TI - Force transmission and stress distribution in a computer-simulated model of the kidney: an analysis of the injury mechanisms in renal trauma. AB - Injury mechanisms in renal trauma were investigated by analyzing the stress distributions within a two-dimensional computer-simulated model of the kidney. In biomechanics, damage to biological tissue is primarily caused by stresses resulting in tissue deformation beyond recovery limits. Segmental surface force was applied to the model and the resulting stress distributions were analyzed. Maximum stress concentrations were found at the periphery of the kidney model. Stresses were caused by the combined effect of the applied force and the reaction generated by the liquid-filled inner compartment as a function of its hydrostatic pressure. Maximum stress concentrations corresponded to typical injury sites observed clinically. Our findings suggest that a similar mechanism may play a crucial role in renal trauma. Renal injuries as well as the higher trauma susceptibility of hydronephrotic kidneys and renal cysts could thus be explained. The role of computer models in injury biomechanics research is discussed. PMID- 8614081 TI - Internal fixation in pelvic fractures and primary repairs of associated genitourinary disruptions: a team approach. AB - Associated urological and orthopedic injuries of the pelvic ring are complex with numerous potential complications. These patients are treated optimally using a team approach. The combined expertise is not only helpful initially when managing these difficult patients, but also later as problems develop. This study describes a treatment protocol and reports the early results of 23 patients with unstable pelvic fractures and associated bladder or urethral disruptions, or both, treated surgically with open reduction and internal fixation of the anterior pelvic ring injuries at the same anesthetic and using the same surgical exposure as the urethral realignments or bladder repairs or both. Early complications occurred in four patients (17%): one patient sustained a fifth lumbar nerve injury caused by the pelvic reduction procedure, and three patients had anterior pelvic internal fixation failures. Late complications occurred in eight patients (35%). There was one deep wound infection (4.3%) that presented 6 weeks after injury. Late urological complications occurred in seven patients (30%). Four of the nine male patients with urethral disruptions had urethral stricture after their primary urethral realignments (44%). Three of the 18 male patients admitted to impotence (16.7%). One of the three had a residual thoracic paraplegia caused by a burst fracture. One of the five female patients had urinary incontinence and required a bladder suspension operation to restore normal function (20%). A low infection rate can be expected despite the use of internal fixation. Early urethral "indirect" realignments avoid more difficult delayed open repairs; however, late urological complication rates are still high. Early "direct" bladder repairs are easily performed at the time of anterior pelvic open reduction and internal fixation. Suprapubic tubes are not necessary to adequately divert the urine when large diameter urethral catheters are used in these patients. PMID- 8614083 TI - Early placement of prophylactic vena caval filters in injured patients at high risk for pulmonary embolism. AB - OBJECTIVE: Pulmonary embolism (PE) is a major problem in patients with multiple injuries. We present our experience with early placement of prophylactic vena caval filters (VCFs). DESIGN: Prospective study group with historical control. MATERIALS AND METHODS: From March 1993 to December 1993, VCFs were placed in 40 consecutive patients with three or more risk factors for PE and had demographic, physiologic, venous thromboembolic prophylaxis, and outcome data collected prospectively (VCF group). They were compared to 80 injured patients admitted between November 1991 and February 1993 who survived > 48 hours and who were matched with the VCF group for mechanism of injury and risk factors for PE (NO VCF group). MEASUREMENTS AND MAIN RESULTS: VCF placement affected a significant reduction in the incidence of PE (2.5% vs. 17%) and a clinical reduction in PE related mortality. Embolic trapping was suggested by a 10% incidence of documented vena caval thrombi and although two patients developed significant venous stasis disease, no other VCF-related morbidity was noted. CONCLUSIONS: In spite of long-term morbidity, early prophylactic VCF placement is safe and should be considered in the prophylaxis of PE in the high-risk injured patients. This intervention may be effective in eliminating PE as a major cause of posttrauma morbidity and mortality. PMID- 8614084 TI - Comparison of accident and emergency with police road injury data. AB - This paper examines the consistency of police and hospital reporting of outcomes of road traffic crashes using a database of linked police crash reports and accident and emergency department data. The database used consisted of linked records of road traffic crashes in Western Australia for the period of October 1, 1987 to December 31, 1988 from police reported casualty crashes, the discharge records from all hospital admissions in Western Australia, the Registrar General's death records, and records for each ambulance trip as a result of a road crash in the metropolitan area of Perth. The results suggest that police records of hospital admissions from the group of accident and emergency attendances underestimated the total by approximately 15%. PMID- 8614085 TI - Gang warfare: the medical repercussions. AB - Gang related violence in Los Angeles County has increased, with homicides increasing from 205 in 1982 to 803 in 1992. This study examines the medical and financial consequences of such violence on a level I trauma center. Of 856 gunshot injuries over a 29-month period, 272 were gang related. There were 55 pediatric and 217 adult patients. Eighty-nine percent were male and 11% were female. Trauma Score averaged 14.7 +/- 3.1, Glasgow Coma Scale average score was 13.7 +/- 3.4, and the mean Injury Severity Score was 10.8 +/- 14. Twenty-two percent of the gunshots were to the head and neck, 20% to the chest, 20% to the abdomen, 6% had a peripheral vascular injury, and 33% sustained an extremity musculoskeletal injury. Emergency surgery was performed on 43%, including laparotomy 58 (49%), craniotomy 16 (13%), laparoscopy 14 (12%), vascular procedures 10 (8%), orthopedic procedures 6 (5%), head and neck endoscopies 4 (3%), thoracotomies 2 (2%), and 10 (8%) unspecified. There were 25 deaths (9%), primarily caused by head injuries and exsanguinating hemorrhage. Eighty-six percent entered the hospital during the hours of minimal staffing that preempted the use of facilities for other emergent patients. Charges totaled $4,828,828 (emergency room, surgical procedures, intensive care, and surgical ward stay) which equated to $5,550 per patient per day. Fifty-eight percent had no third party reimbursement, 22% had Medi-Cal, and 20% had medical insurance. Because of dismal reimbursement rates, the costs of gang violence are passed on to the tax payer. The cost of gang related violence cannot be derived from hospital charges only, because death, disability, and pain are not entered into the calculation. Education, increased social programs, and strict criminal justice laws and enforcement may decrease gang related violence and the drain it has on financial and medical resources. PMID- 8614086 TI - The status of the trauma coordinator position: a national survey. AB - The trauma coordinator (TC) position is a vital link in the development and operations of trauma care systems. In 1992 and 1993, the American Trauma Society conducted a national survey of TCs to describe the roles and characteristics of the persons who hold those positions. Of 354 trauma coordinators identified in 46 states, more than three-fourths were employed by large hospitals designated as trauma centers. The typical TC was a woman 26 to 59 years old who held at least a bachelor's degree in nursing. Although new as TCs (mean, 3 years as TCs), the respondents averaged 14 years experience in nursing. Both full-time and part-time TCs worked longer hours than scheduled, often had supervisory responsibilities, and generally were in the nursing administration or the emergency department structure. Most TCs worked with computerized trauma registries that were used routinely in quality of care reviews. PMID- 8614087 TI - Biomechanical epidemiology: a new approach to injury control research. AB - Injury control studies, from inception and design to dissemination of results, tend to remain within individual discipline. This is largely because each of the disciplines has a unique language and approach to research. Collaborative research is often performed serially with one discipline presenting the results of that discipline's studies to another discipline. Epidemiologists and clinicians tell engineers to design a safety technology to prevent a specific injury. Engineers tell lawyers what is feasible to include in standards. As a result, epidemiological studies lack mechanical data needed by the engineers and engineering studies lack generalizability. The procedure for incorporating the best of multiple disciplines throughout the performance of injury control studies has not existed until recently and is presented conceptually in this manuscript. This new approach, Biomechanical Epidemiology, is an exciting enhancement to current injury control research. PMID- 8614088 TI - Bilateral inferior glenohumeral dislocations. PMID- 8614089 TI - Straight pin aspiration in young women. AB - Straight pins are used extensively for securing facial scarves in women and girls in the Middle East. Accidental aspiration often results in the pin lodging in the segmental bronchus with the sharp end pointing cephalad. Flexible bronchoscopy is superior to rigid bronchoscopy in retrieving these pins. We report on five cases successfully treated by fiberoptic bronchoscopy under general anesthesia. PMID- 8614090 TI - Big cat attack: a case study. AB - A 28-year-old man was attacked by a large female tiger at an exotic animal farm, sustaining penetrating injuries to the neck and pharynx as well as a cervical spine fracture. This case and review of the literature demonstrates the ability of these animals to cause significant trauma and occult injuries. Furthermore, this case demonstrates the need for a high index of suspicion when treating these patients, as serious underlying bony and soft tissue damage can easily be overlooked. PMID- 8614091 TI - Left anterior descending artery occlusion after blunt chest trauma. PMID- 8614092 TI - Embolization of splenic artery branch pseudoaneurysm after blunt abdominal trauma. PMID- 8614093 TI - Acute suprarenal aortogastric fistulae caused by gunshot wounds: the importance of early recognition in directing surgical strategy. AB - Penetrating suprarenal aortic injuries carry high mortality rates. Difficulties in surgical exposure and bleeding control in this area add to the ominous prognosis. In rare occasions, synchronous injury to the adjacent upper part of stomach may lead to an acute aortogastric fistula, resulting in aortic bleeding into the stomach instead of the peritoneal cavity. Filling of the stomach with blood may temporarily tamponade the aortic perforation. Distortion of this delicate communication during dissection before proximal and distal vascular control is achieved could result in catastrophic hemorrhage. Therefore, recognition of the importance of a fully distended stomach at the suspicion of aortic injury is essential in directing a particular surgical strategy that aims to achieve an unrestricted operative exposure and successful bleeding control. PMID- 8614094 TI - Renal autotransplantation after horseshoe kidney injury: a case report and literature review. AB - We present a patient with lap seatbelt trauma to a previously unsuspected horseshoe kidney. Preoperative single film intravenous pyelography did not suggest the presence of this renal anomaly or define the extent of renal injury. Because of the severity of the injury, nephrectomy was life-saving, but inadvertently rendered the patient anephric. Recognition of horseshoe kidney anatomy, microvascular back-bench reconstruction, and renal autotransplantation allowed the salvage of both the patient and her renal function. PMID- 8614095 TI - Difficult diagnosis of laryngeal blunt trauma. PMID- 8614096 TI - Facial injury by mercury from a broken thermometer. AB - The case of a 2-year-old girl with a facial soft-tissue injury from an accident with a mercury thermometer is presented. All tissues containing droplets of metallic mercury should be removed immediately and careful examination for signs of mercury poisoning should be conducted over the long term. PMID- 8614098 TI - A piece of my mind. Standards of Care. PMID- 8614097 TI - Prospective, randomized trial of survivor values of cardiac index, oxygen delivery, and oxygen consumption as resuscitation endpoints in severe trauma. PMID- 8614099 TI - New ideas on pathology of restenosis. PMID- 8614100 TI - Changes in practice bring cardiologists conflicts. PMID- 8614102 TI - From the Veterans Health Administration. PMID- 8614101 TI - Nicorette finds new place in smoking cessation. PMID- 8614103 TI - From the Centers for Disease Control and Prevention. World Health Organization Consultation on public health issues related to bovine spongiform encephalopathy and the emergence of a new variant of Creutzfeldt-Jakob disease. PMID- 8614104 TI - From the Centers for Disease Control and Prevention. HIV/AIDS education and prevention programs for adults in prisons and jails and juveniles in confinement facilities--United States, 1994. PMID- 8614105 TI - From the Centers for Disease Control and Prevention. Diphtheria outbreak- Saraburi Province, Thailand, 1994. PMID- 8614106 TI - Preferences for CPR and life-sustaining treatment among nursing home residents. PMID- 8614107 TI - Preferences for CPR and life-sustaining treatment among nursing home residents. PMID- 8614108 TI - What is adequate and appropriate pain treatment? PMID- 8614109 TI - What is adequate and appropriate pain treatment? PMID- 8614110 TI - What is adequate and appropriate pain treatment? PMID- 8614111 TI - What is adequate and appropriate pain treatment? PMID- 8614112 TI - Measles elimination in the Americas. PMID- 8614113 TI - Childhood immunization registries. PMID- 8614114 TI - Reducing choking deaths in children. PMID- 8614115 TI - Reducing choking deaths in children. PMID- 8614116 TI - Diet and risk of non-Hodgkin lymphoma in older women. AB - OBJECTIVE: To test whether high dietary intakes of fat, protein, and milk are associated with the development of non-Hodgkin lymphoma in older women. DESIGN: Prospective cohort study with a 7-year follow-up period. SETTING: General community. PARTICIPANTS: Sample of 35 156 Iowa women aged 55 to 69 years with no prior history of cancer who returned the 1986 baseline questionnaire. MAIN OUTCOME MEASURE: Non-Hodgkin lymphoma (104 incident cases). MAIN RESULTS: After controlling for age, marital status, residence, total energy intake, and transfusion history, the relative risks (RRs) for the highest tertile of intake compared with the lowest were 2.00 (95% confidence interval [CI], 1.21-3.30; P for trend = .01) for animal fat, 1.69 (95% CI, 1.07-2.67; P for trend = .02) for saturated fat, and 1.90 (95% CI, 1.18-3.04; P for trend = .01) for monounsaturated fat, and there was no association with vegetable fat or polyunsaturated fat. Greater intake of animal protein (RR = 1.52; 95% CI, 0.94 2.44; P for trend = .08), but not vegetable protein, was associated with elevated risk, and this was mainly explained by greater consumption of red meat (RR = 1.98; 95% CI, 1.13-3.47; P for trend = .02) and hamburger in particular (RR = 2.35; 95% CI, 1.23-4.48; P for trend = .02). Milk and dairy product consumption were not associated with elevated risk. There was also a decreased risk of non Hodgkin lymphoma with greater consumption of fruits (RR = 0.64; 95% CI, 0.40 1.05; P for trend = .07). CONCLUSIONS: A high-meat diet and a high intake of fat from animal sources is associated with an increased risk of non-Hodgkin lymphoma in older women. PMID- 8614117 TI - Using admission characteristics to predict short-term mortality from myocardial infarction in elderly patients. Results from the Cooperative Cardiovascular Project. AB - OBJECTIVE: To develop a prediction model of death within 30 days of hospital admission for Medicare patients with acute myocardial infarction that would permit use of risk-adjusted mortality rates as hospital quality measures. DESIGN: Retrospective cohort study using data created from medical charts and administrative files. SETTING: All acute care hospitals in Alabama, Connecticut, Iowa, or Wisconsin. PATIENTS: A cohort of 14,581 patients with acute myocardial infarction covered by Medicare in 1993. RESULTS: The unadjusted 30-day mortality rate was 21%, ranging from 18% in Connecticut to 23% in Alabama. The 4 largest contributors to variability in mortality rates were mean arterial pressure, age, respiratory rate, and serum urea nitrogen level. The area under the receiver operator characteristic curve was 0.79 in a developmental sample of 10 936 patients and 0.78 in a validation sample of 3645 patients. Based on admission variables, we were able to explain 27% of the variability in 30-day mortality rates. During the index admission, aspirin, beta-blockers, angiotensin-converting enzyme inhibitors, and thrombolytic agents were used in 72%, 39%, 32%, and 15% of patients, respectively. Explained variation increased by 6 percentage points to 33% when drug therapies and revascularization procedures performed during the index admission were added to the model predictors. CONCLUSIONS: Short-term mortality remains high for elderly patients with acute myocardial infarction, and a large percentage of variation remains unexplained after controlling for admission severity. Part of the unexplained variability can be explained by the location of the admitting hospital; some of the remaining unexplained variation may reflect differences in quality of care or unmeasured differences in disease severity. Researchers should develop quality indicators based on process measures for acute myocardial infarction and should incorporate these measures into mortality models to determine whether quality accounts for variation in 30-day mortality rates beyond that explained by clinical status at admission. PMID- 8614118 TI - Survival from early, intermediate, and late stages of HIV infection. AB - OBJECTIVE: To estimate expected survival time among homosexual men infected with the human immunodeficiency virus type 1 (HIV-1) by (1) the calendar period before (1985-1988) and after (1989-1993) the widespread availability of acquired immunodeficiency syndrome (AIDS) treatments with antiretroviral and prophylactic interventions, and (2) stage of HIV disease. DESIGN: A prospective cohort study. A group of HIV-1-infected homosexual men were followed from July 1985 through June 1993 and evaluated every 6 months for the presence of clinical symptoms and measurement of the CD4 cell count. To measure the effectiveness of AIDS therapies in this nonrandomized study, we used 2 calendar periods as proxy measures of relative intensity of exposure to antiretroviral therapy. Stage of infection was defined by CD4 cell count and presence of HIV-related clinical symptoms or AIDS. SETTING AND STUDY PARTICIPANTS: Homosexual men infected with HIV-1 from the Multicenter AIDS Cohort Study. MAIN OUTCOME MEASURE: Survival time based on stage of HIV infection. RESULTS: The percentage of HIV-1-infected individuals free of AIDS and clinical symptoms at baseline who survived 2.5 years according to baseline CD4 cell counts of 0 to 0.100, 0.101 to 0.200, and 0.201 to 0.350 x 10(9)/L was 22%, 53%, and 83%, respectively, for the 1985-1988 calendar period, compared with 54%, 71%, and 91%, respectively, for men in the 1989-1993 calendar period. Among men free of AIDS with CD4 cell counts of greater than 0.350 x 10(9)/L, the relative hazard of mortality was 1.6 to 2.3 times higher for those with clinical symptoms compared with those free of clinical symptoms. CONCLUSIONS: Survival of AIDS-free HIV-1-infected individuals with CD4 cell counts of less than 0.350 x 10(9)/L has improved since antiretroviral and HIV prophylactic treatments have become available, but the long-term prognosis remains poor. PMID- 8614119 TI - Development of cardiomyopathy in female carriers of Duchenne and Becker muscular dystrophies. AB - OBJECTIVE: To characterize the presence and behavior of the dystrophinopathic myocardial damage in female carriers of a gene defect at the Xp21 locus of the X chromosome that causes Duchenne and Becker muscular dystrophies (DMD and BMD). DESIGN: Cohort study from April 1, 1985, to April 30, 1995, with cardiologic follow-up performed yearly for a minimum of 3 to a maximum of 10 years. SETTING: Counseling center for genetic muscular disorders. PATIENTS: A total of 197 women and girls aged 5 to 60 years ascertained to be carriers of the DMD (n = 152) or BMD (n = 45) gene. MAIN OUTCOME MEASURES: Cardiac status at yearly examinations as determined by 12-lead electrocardiogram (ECG), 24-hour ambulatory ECG, M-mode and 2-dimensional echocardiography, and carotid pulse tracing. Myocardial scintigram was performed on each individual at least twice during the study. Immunohistochemical analysis of dystrophin from myocardium and/or skeletal muscle biopsy was performed in 12 carriers. RESULTS: Preclinical or clinically evident myocardial involvement was found in 166 cases (84.3%), without significant differences in percentage and behavior between DMD and BMD carriers. Its occurrence increased significantly with age, from 54.5% (18 cases) in carriers aged between 5 and 16 years to 90.2% (148 cases) in carriers older than 16 years. Dystrophin anomalies were detected at the membrane level of the myocardial fibers in all endomyocardial biopsy specimens. CONCLUSIONS: Genetic anomalies can be considered the primary cause of myocardial damage in carriers of dystrophinopathic myopathies; myocardial damage shows the same behavior already described in DMD and BMD patients and progresses from preclinical to dilated cardiomyopathy, passing through stages of myocardial hypertrophy or dysrhythmias. PMID- 8614120 TI - Use of terfenadine and contraindicated drugs. AB - OBJECTIVE: To assess changes in concurrent use of products containing terfenadine and contraindicated macrolide antibiotics (erythromycin, clarithromycin, troleandomycin) and imidazole antifungals (ketoconazole, itraconazole) following reports of serious drug-drug interactions and changes in product labeling. DESIGN: Retrospective review of computerized pharmacy claims. SETTING: A large health insurer in New England. PATIENTS: Health plan members with 1 or more paid pharmacy claims for products containing terfenadine between January 1990 and June 1994. MAIN OUTCOME MEASURES: Among persons with paid claims for terfenadine in any given month, percentage with a prescription for any contraindicated drug that alternatively was dispensed on the same day as ("same-day dispensing") or had therapy days that overlapped those of ("overlapping use") a prescription for terfenadine. RESULTS: Concurrent use of terfenadine and contraindicated drugs declined over the study period. The rate of same-day dispensing declined by 84% from an average of 2.5 per 100 persons receiving terfenadine in 1990 to 0.4 per 100 persons during the first 6 months of 1994, while the rate of overlapping use declined by 57% (from 5.4 to 2.3 per 100 persons). Most cases involved erythromycin. CONCLUSIONS: Despite substantial declines following reports of serious drug-drug interactions and changes in product labeling, concurrent use of terfenadine and contraindicated macrolide antibiotics and imidazole antifungals continues to occur. PMID- 8614121 TI - A meta-analytic evaluation of the polymerase chain reaction for the diagnosis of HIV infection in infants. AB - OBJECTIVE: To evaluate the sensitivity and specificity of the polymerase chain reaction (PCR) for the diagnosis of infection with human immunodeficiency virus (HIV) in infants. DATA SOURCES: We used studies published between 1988 and 1994 identified in a literature search of 17 databases, including MEDLINE. STUDY SELECTION: Studies were included if DNA amplification by PCR was performed on peripheral blood mononuclear cells from infants or children. DATA EXTRACTION: Two investigators independently extracted data. The study design was assessed independently by 2 investigators who were blinded to study results. DATA SYNTHESIS: Thirty-two studies met the inclusion criteria and were analyzed. The median reported sensitivity was 91.6% (range, 31%-100%), and the median specificity was 100% (range, 50%-100%). A summary receiver operating characteristic curve based on all 32 studies indicated that PCR has a maximum joint sensitivity and specificity between 93.2% and 94.9%. Subgroup analysis indicated that the joint sensitivity and specificity was significantly (P = .04) higher in older infants (98.2%) than in neonates (aged < or = 30 days; 93.3%). For infants at low risk of perinatal transmission (probability of transmission, 8.3%), the positive predictive value for PCR is 55.8% in neonates and 83.2% in older infants. A negative PCR result reduces the probability of HIV infection to less than 3%. No studies met all criteria for study design. CONCLUSIONS: Although PCR is one of the best available tests for diagnosis of HIV infection in neonates and infants, it is not definitive. Therefore, PCR should be interpreted with the aid of careful clinical follow-up examinations. The sensitivity and specificity of PCR in neonates is lower than in older infants, which results in a low positive predictive value; however, negative tests are informative. Delaying the use of PCR until after the neonatal period or repeating PCR on independent samples obtained 30 to 60 days later will reduce test errors. PMID- 8614122 TI - Chemoprevention of breast cancer. PMID- 8614123 TI - Health care needs of gay men and lesbians in the United States. Council on Scientific Affairs, American Medical Association. PMID- 8614124 TI - DNA polymerase chain reaction for the diagnosis of vertical HIV infection. PMID- 8614125 TI - Physicians accessing the Internet, the PAI Project. An educational initiative. PMID- 8614126 TI - The patient's perspective. PMID- 8614127 TI - Medical school tuition and the cost of medical education. PMID- 8614128 TI - When the military foots the bill. PMID- 8614129 TI - Health care policy education in medical school. PMID- 8614130 TI - Do referral practices affect the results of coronary artery surgery? PMID- 8614131 TI - Is referral source a risk factor for coronary surgery? Health maintenance organization versus fee-for-service system. AB - We began performing coronary artery bypass grafting for a large health maintenance organization (HMO) in 1974, as the sole provider of their cardiac surgery. The outcomes of our HMO group of patients were compared with those of our patients treated on a fee-for-service (FFS) basis. The HMO system entails preintervention and multidisciplinary screening conferences and is devoid of self referral and personal financial incentives. Since 1985, the operative mortality for HMO patients has been consistently lower than for FFS patients. There were 8483 operations during this study period: 3168 (37%) were in the HMO group, with an overall operative mortality of 2.7%, and 5315 (63%) were in the FFS group, with an operative mortality of 4.6% (p=0.00002). This difference was investigated with univariate and multivariable analyses. Sixteen factors were found to univariately affect the risk of operative mortality; for five of these risk correlates there was a significant maldistribution between the HMO and FFS patients. Logistic regression was used to explore the influence of this imbalance in risk factors. The model found seven independent risk factors (left ventricular failure, emergency coronary bypass, redo bypass, nonuse of the internal thoracic artery, unstable angina, age, and diabetes) that significantly affected operative mortality. The FFS group variable closely approached independent risk significance at p=0.059. This multivariable model explained only one third of the observed differences in actual mortality between the HMO and FFS groups. The system-wide angioplasty/coronary bypass ratio, which could not be used in a patient-specific model, was 0.6 in the HMO system and 1.5 in the FFS group. Other factors related to the operating structure of a mature, large HMO may account for the remainder of the difference. The HMO referral system, through a powerful selection process, resulted in fewer emergencies, redo bypass operations, and catheterization complications that, in turn, yielded lower operative mortality than a noncoordinated FFS system of cardiovascular management. PMID- 8614133 TI - Risk factors for postoperative morbidity. AB - OBJECTIVE: Analysis of outcomes after coronary artery bypass grafting has focused on risk factors for operative mortality. Nonfatal perioperative morbidity is far more costly and more common after operation. To identify the risk factors that lead to postoperative morbidity, we evaluated 938 patients undergoing coronary artery bypass grafting at Albany Medical Center Hospital during 1993. METHODS: Multivariate statistical analysis was performed on preoperative patient variables to identify risk factors for either serious postoperative morbidity or increased hospital length of stay. Variables were considered both individually and in combination. For example, age was considered individually or in combination with other variables, including parameters of blood volume (i.e., age divided by red blood cell volume or Age/RBCVOL). Similar multivariate analysis was performed to identify independent risk factors for hospital mortality. RESULTS: In order of decreasing importance, the following patient variables were significantly associated with increased length of stay by stepwise Cox regression analysis: Age/RBCVOL, history of congestive heart failure, hypertension, femoral-popliteal peripheral vascular disease, chronic obstructive lung disease, and renal dysfunction. The combination variable, Age/RBCVOL, was an important risk factor for both increased length of stay and serious postoperative morbidity. Variables that were significant independent predictors of increased mortality, such as preoperative shock, and redo operation, were not risk factors for either serious morbidity or increased length of stay. CONCLUSIONS: We conclude that risk factors for postoperative morbidity are different from those for postoperative mortality. These results suggest that older patients with preoperative anemia and low blood volume who also have other comorbidities (congestive heart failure, stroke, chronic obstructive pulmonary disease, or hypertension) are at increased risk for postoperative complications. This allows identification of a high-risk cohort of patients who are likely candidates for interventions to lessen postoperative morbidity. PMID- 8614132 TI - Extensive calcification of the mitral valve anulus: pathology and surgical management. AB - Extensive calcification of the mitral valve anulus is a pathologic entity frequently associated with degenerative valvular disease. The calcification process remains localized to the anulus in 77% of the cases. It may extend, however, to the underlying myocardium. Whenever an operation is necessary for an associated valve insufficiency, the question arises whether it is preferable to repair or to replace the valve and how to manage the calcification. In the first part of this paper the pathology of this disease is studied, and in the discussion a mechanism is proposed to explain the development of the process of calcification. In the second part, a new operation is described, which comprises the temporary detachment of the leaflets, en bloc resection of the calcium deposit, annular reconstruction, and valve repair. For patients in whom the calcification extends to the myocardium a "sliding atrioplasty" of the left atrium is described, which allows the area of exposed muscular fibers to be covered. Between 1986 and 1994, among 68 patients with extensive calcification of the anulus and severe mitral valve insufficiency, 67 benefited from these repair techniques. Ages ranged from 18 to 82 years (mean 62 years). Thirty-two patients had a billowing mitral valve (Barlow), 27 a fibroelastic deficiency, and two Marfan's disease. The calcification involved more than one third of the anulus in 88% of the patients, the posterior anulus in 10.5%, and the whole anulus in 1.5%. The calcification process extended to the myocardial wall in 12% of the patients and to the papillary muscles in 4.5%. In the group of 67 valve repairs, there were two hospital deaths (2.9%), no instances of anulus dehiscence, and no early reoperations. The follow-up period extended from 4 months to 8 years (mean 3 years 8 months). There were two late deaths, 2 and 17 months after the operations, for an actuarial survival of 93% at 7 years. Late reoperation (6 to 62 months) was necessary in four patients (6.4%) for residual mitral valve incompetence (n=2), hemolysis (n=1), or endocarditis (n=1). In one of these patients a new repair was possible, whereas the three other patients required a valve replacement. All patients but one survived the reoperation. Actuarial freedom from reoperation was 87% at 7 years. All 60 patients with valve repair were reviewed for this study by clinical examination and echocardiography. All but one were in functional class I or II. There was no incompetence or trivial residual mitral valve incompetence in 55 patients and moderate incompetence in five. Two thromboembolic events have been recorded for a linearized rate of 1%/pt yr. This study shows that complete anulus decalcification and valve repair can be done safely in patients with mitral valve insufficiency and extensive calcification of the anulus, even when the calcification process deeply involves the myocardium. It also demonstrates that an initially good result remained stable up to 7 years. PMID- 8614134 TI - The additional hospital costs generated in the management of complications of pacemaker and defibrillator implantations. AB - The rapid approach of capitated reimbursement mandates that providers examine their practice patterns associated with all surgical procedures. Documentation of (1) the complications associated with these procedures and (2) the additional hospital costs associated with the management of these complications is critical for comprehensive fiscal accountability. This study analyzed (1) the feasibility of obtaining accurate hospital cost data specific for complications and (2) the outcome in terms of fully loaded hospital costs generated in the management of the most common surgical complications associated with pacemaker and nonthoracotomy implantable defibrillator therapies. Between July 1989 and September 1994, a total of 1031 pacemaker and 105 implantable defibrillator procedures were performed by a cardiac surgeon in a tertiary-level teaching hospital setting. The additional fully loaded hospital costs were determined by (1) correlating clinical data from the complete medical record with complete hospital charge data for the admission(s) related to the complication, (2) carving out complication-related charges based on the clinical data, (3) converting complication-related charges to fully loaded costs based on conversion factors in effect at the time of service, and (4) correlating cost with hospital net reimbursement and payor source. The feasibility study determined that accurate and reliable cost data specific to complications can be obtained, although the process was cumbersome and difficult. The outcomes study determined that mean fully loaded complication costs were $4345 +/- $1540 for pacemaker lead revision and $4879 +/- $3167 for implantable defibrillator lead dislodgement, $24,459 +/- $14,585 for pacemaker infection, and $13,736 +/- $12,505 for defibrillator generator system malfunction. The one infected defibrillator cost $57,213 to treat. Costs exceeded reimbursement for almost all Medicare patients with complications in this study, suggesting that similar shortfalls would occur under a capitation scheme. This information is critical to a complete understanding of the financial impact of interventional procedures in a capitated reimbursement environment. PMID- 8614135 TI - Effective control of pulmonary vascular resistance with inhaled nitric oxide after cardiac operation. AB - Increased pulmonary vascular resistance may greatly complicate the perioperative management of cardiac surgical patients. Inhaled nitric oxide may be a promising new therapy to selectively lower pulmonary vascular resistance. The purpose of this study was to examine the effects of inhaled nitric oxide on pulmonary and systemic hemodynamics in cardiac surgical patients. Twenty patients (age 57 +/- 6 years) were studied in the operating room after weaning from cardiopulmonary bypass. Mean pulmonary artery pressure, pulmonary vascular resistance, systemic vascular resistance, and mean aortic pressure were determined at four points of data collection: before nitric oxide, with 20 ppm nitric oxide, with 40 ppm nitric oxide, and after nitric oxide. Statistical analysis was by analysis of variance; significance was accepted for p < 0.05. Inhaled nitric oxide produced selective pulmonary vasorelaxation. Pulmonary vascular resistance was lowered from 343 +/- 30 before nitric oxide to 233 +/- 25 dynes.sec-1.cm-5 with 20 ppm nitric oxide. Pulmonary vascular resistance was not further lowered by 40 ppm nitric oxide (p < 0.05). Mean pulmonary arterial pressure was 29 +/- 1 mm Hg before nitric oxide and was lowered to 22 +/- 1 mm Hg by 20 ppm nitric oxide and 21 +/- 1 mm Hg by 40 ppm nitric oxide (p < 0.05). Both pulmonary vascular resistance and mean pulmonary arterial pressure returned to baseline after withdrawal of inhaled nitric oxide. Inhaled nitric oxide produced no changes in either systemic vascular resistance or mean aortic pressure. We conclude that nitric oxide may be used as an effective pulmonary vasodilator after cardiac operations. It may be particularly valuable for selectively lowering right ventricular afterload in patients with right ventricular dysfunction. PMID- 8614137 TI - Transplant candidate's clinical status rather than right ventricular function defines need for univentricular versus biventricular support. AB - We have studied our experience since 1988 with 31 patients who required a mechanical circulatory bridge to transplantation and also had biventricular failure (mean right ventricular ejection fraction 11.8%) to better define the need for biventricular or total artificial heart support versus univentricular support. Clinical factors including preoperative inotropic need, fever without detectable infection, diffuse radiographic pulmonary edema, postoperative blood transfusion, and right ventricular wall thickness were compared with hemodynamic parameters including cardiac index, right ventricular ejection fraction, central venous pressure, mean pulmonary arterial pressure, and total pulmonary resistance for ability to predict need for mechanical or high-dose inotropic support for the right ventricle. Patients were grouped according to need for right ventricular support after left ventricular-assist device implantation: none (group A, 14) inotropic drugs (group B1, 7), and right ventricle mechanical support (group B2, 10). There were no differences in preimplantation hemodynamic variables. Groups B1 and B2 had significantly lower mixed venous oxygen saturation (39.2% vs 52.5% in group A; p < 0.001), greater level of inotropic need (p < 0.02), greater impairment of mental status, and lower ratio of right ventricular ejection fraction to inotropic need (0.37 vs 0.56 for group A; p < 0.02) before left ventricular-assist device implantation. A significant discriminator between groups B1 and B2 was the presence of a fever without infection within 10 days of left ventricular-assist device implantation (43% in group B1 vs 70% in group B2). Group B2 had more patients with preimplantation pulmonary edema seen on chest radiography and a greater requirement for postoperative blood transfusion (5 units of cells in group B1 vs 14.8 units in group B2. Right ventricular wall thickness at left ventricular-assist device explantation was 0.83 cm in group B2 vs 0.44 cm in group B1 (p < 0.05). Transplantation rates after bridging were 100% in group A, 71% in group B1, and 40% in group B2. Clinical factors that reflect preimplantation degree of illness and perioperative factors that result in impairment of pulmonary blood flow or reduced perfusion of the right ventricle after left ventricular-assist device implantation are now considered to be more predictive of the need for additional right ventricular support than preimplantation measures of right ventricular function or hemodynamic variables. PMID- 8614138 TI - Continuous warm reperfusion during heart transplantation. AB - From April 1991 to January 1993, 37 orthotopic heart transplantations were performed at our institution. Conventional preservation technique with cold crystalloid cardioplegia++ and topical hypothermia during storage and implantation was used in the first 15 cases (group A). After January 1992, for the next 22 patients (group B), we administered a first dose of hyperkalemic blood cardioplegia on arrival of the graft and thereafter instituted continuous warm reperfusion by infusion of oxygenated blood with added potassium. The groups were compared retrospectively, and significant differences were observed. In group B, the ischemic time was shortened by 31 minutes, the suture time lasted 12 minutes longer, sinus rhythm recovered spontaneously, the duration of inotropic support was reduced, postoperative arrythmias decreased, length of intensive care and hospital stays were reduced, there was less ischemic damage in the first endomyocardial biopsy sample, and right ventricular pressures a month after operation were lower. Continuous warm reperfusion during implantation of the donor heart is technically feasible and seems to provide enhanced myocardial preservation. PMID- 8614136 TI - A prospective trial of tacrolimus (FK 506) in clinical heart transplantation: intermediate-term results. AB - Between January 1, 1989, and December 31, 1994, we have treated 122 primary heart recipients with FK 506 (group I) and 121 with cyclosporine (group II). Fifty patients in the cyclosporine (CyA) group received no lympholytic induction (CyA alone) and 71 others received lympholytic induction with either rabbit antithymocyte globulin or OKT3 (CyA+LI). The mean follow-up was longer in the FK 506 group than in the CyA groups (3.2 +/- 1.3 vs 2.3 +/- 1.8 years; p< 0.01). Patient survival did not differ on the basis of the type of immunosuppression used. At 3 months after transplantation, the freedom from rejection in the FK 506 group was higher than that of the CyA-alone group (47% vs 22%, p < 0.01) but similar to that of the CyA+LI group (47% vs 53%). The linearized rejection rate (episodes/100 patient-days) of the FK 506 group (0.09 episodes) was lower (p < 0.05) than that of the CyA-alone group (0.26) and the CyA+LI group (0.13). The requirement for pulsed steroids to treat rejection was less in common in the FK 506 group than in either CyA group. Eighteen patients in the CyA group had refractory rejections; all resolved with FK 506 rescue. Two patients in the FK 506 group had refractory rejection that resolved with total lymphoid irradiation (n=1) and methotrexate therapy (n=1). Patients receiving FK 506 had a lower risk of hypertension and required a lower dose of steroids. Although the mean serum creatinine concentration at 1 year was higher in the FK 506 group, this difference disappeared after 2 years. No patients required discontinuation of FK 506 because of its side effects. Our intermediate-term results indicate that FK 506 compares favorably with CyA as a primary immunosuppressant in heart transplantation. PMID- 8614140 TI - Combined thoracoscopic/laparoscopic staging of esophageal cancer. AB - Unlike mediastinoscopy in lung cancer, there exists no standard minimally invasive test to stage esophageal cancer. If it were possible to obtain exact preoperative staging in esophageal cancer, patients could be separated prospectively to receive neoadjuvant therapy appropriately. We studied the feasibility and efficacy of thoracoscopic and laparoscopic lymph node staging in esophageal cancer. Thoracoscopic staging was performed in 45 patients with biopsy proven carcinoma of the esophagus. Laparoscopic staging was done in the last 19 patients. Thoracoscopic staging was aborted in three patients because of adhesions. Thoracic lymph node stage was N0 in 39 patients and N1 in three; celiac lymph nodes were normal in 13 and diseased in six. Esophageal resection was performed in 30 patients after thoracoscopic staging; 17 of these underwent laparoscopic staging. Thoracoscopic staging showed N0 lymph node status in 28 patients and N1 in two patients. Two of the 28 patients (7%) with N0 disease were found at resection to have paraesophageal lymph node involvement (N1); thus the disease was understaged by thoracoscopic staging. Thoracoscopic staging was accurate in detecting the presence of diseased thoracic lymph nodes in 28 of 30 cases (93%). Laparoscopic staging detected normal celiac nodes in 12 patients and diseased lymph nodes in five patients. After esophagectomy, the final pathology report in the 12 patients with N0 disease was N0 in 11 and diseased lymph nodes in one patient. Thus laparoscopic staging was accurate in detecting lymph node metastases in 16 of 17 patients (94%). Thoracoscopic and laparoscopic staging are more accurate than existing staging methods. Six of 19 patients in whom laparoscopic staging was used had unsuspected celiac axis lymph node involvement that had been missed by standard noninvasive techniques. One of three patients with thoracic lymph nodes and three of six with celiac lymph nodes were downstaged after preoperative chemotherapy/radiotherapy. The role of thoracoscopy and laparoscopy in staging esophageal cancer should be further evaluated in a multiinstitutional trial. PMID- 8614139 TI - Transmyocardial laser revascularization: clinical experience with twelve-month follow-up. AB - We are investigating a new technique for myocardial revascularization in which an 800 W carbon dioxide laser is used to drill 1 mm diameter channels into a beating heart after left thoracotomy. Clotting occludes the channels on the subepicardium, and in the long-term setting, blood from the left ventricular cavity flows through these channels to perfuse the ischemic subendocardium. To test the efficacy of this technique in a preliminary clinical trial, we used it as sole therapy for 21 consecutive patients. All patients had hibernating myocardium, reduced coronary flow reserve, or both, had distal diffuse coronary artery disease, and had angina refractory to normal therapy. Eight patients were excluded from follow-up because of death (n=5), rerevascularization (n=2), or diaphragmatic paralysis resulting in postoperative respiratory incapacity (n=1). In the remaining 13 patients available for follow-up, the mean angina class (Canadian Cardiovascular Society) was 3.7 +/- 0.4 before operation and 1.8 +/- 0.6 12 months after operation (p < 0.01). Mean resting left ventricular ejection fraction was 48% +/- 10% before operation and 50% +/- 8% at 12-month follow-up. At 12 months, resting mean subendocardial/subepicardial perfusion ratio had increased by 20% +/- 9% in septal regions treated by laser but decreased by 2% +/ 5% in untreated regions (n=11, p <.001). These results suggest that revascularization by this laser technique positively affects subregional myocardial perfusion and may result in clinical benefits for patients with reversible myocardial ischemia. Studies to date have not demonstrated significant changes in global and regional ventricular contractile function. PMID- 8614141 TI - Results and prognostic factors in resections of primary tracheal tumors: a multicenter retrospective study. The French Society of Cardiovascular Surgery. AB - To determine long-term survival and prognostic factors, 208 patients with primary tracheal tumors were evaluated in a retrospective multicenter study including 26 centers. Ninety-four patients had squamous cell carcinoma, four had adenocarcinoma, 65 had adenoid cystic carcinoma, and 45 patients had miscellaneous tumors. The following resections were performed: tracheal resection with primary anastomosis, 165; carinal resection, 24; and laryngotracheal resection, 19. Postoperative mortality rate was 10.5% and correlated with the length of the resection, the need for a laryngeal release, the type of resection, and the histologic type of the cancer. Fifty-nine percent of patients with tracheal cancer and 43% of patients with adenoid cystic carcinomas had postoperative radiotherapy. The 5- and 10-year survivals, respectively, were 73% and 57% for adenoid cystic carcinomas and 47% and 36% for tracheal cancers (p < 0.05). Among patients with tracheal cancers, survival was significantly longer for those with complete resections than for those with incomplete resections. On the other hand, the presence of positive lymph nodes did not seem to decrease survival. Postoperative radiotherapy increased survival only in the case of incompletely resected tracheal cancers. Long-term prognosis was worsened by the occurrence of second primary malignancies in patients with tracheal cancers and by the occurrence of late pulmonary metastases in patients with adenoid cystic carcinomas. PMID- 8614142 TI - The importance of surgical staging in the treatment of malignant pleural mesothelioma. AB - OBJECTIVES: Progress in the therapy of malignant pleural mesothelioma is limited by the lack of an adequate staging system and controversy about prognostic factors. This surgical series was analyzed to determine whether a new TNM staging system proposed by the International Mesothelioma Interest Group and certain prognostic factors could stratify patients in future clinical trials. METHODS: Thoracotomy was performed if computed tomographic scans showed resectable tumor confined to one hemithorax. Pleurectomy/decortication was done if visceral pleural tumor was minimal, and extrapleural pneumonectomy was done for more locally advanced disease. Complete resection was defined as no gross residual tumor. Adjuvant therapy was given as required by serial clinical trials. Patients had computed tomographic scans every 3 months until death. Prognostic factors were examined by log-rank and Cox regression analyses. RESULTS: From October 1983 to July 1994, a total of 131 thoracotomies were performed, resulting in 101 resections, 72 of which were complete. Extrapleural pneumonectomy was done in 50 patients and pleurectomy/decortication in 51. The ratio of men to women was 108:23. Median age was 63 years (range 32 to 80 years). Operative mortality was five of 131 patients (3.8%), three of 50 in the group having extrapleural pneumonectomy (6%). Ninety-five of the 131 tumors were epithelial. Fifty-one of 89 patients (57%) having node dissections had diseased nodes, 45 (50%) N2. By univariate analysis, type of resection, T and N status, stage, histologic type, and adjuvant therapy, but no gender or age, significantly affected survival. Type of resection, stage, and histologic type were significant in a multivariate analysis. Local recurrence occurred mainly after pleurectomy/decortication, and distant metastases developed after extrapleural pneumonectomy. CONCLUSIONS: (1) N2 nodal disease is more frequent than previously reported; (2) the prognostic importance of histologic type is confirmed; (3) both T and N status influence outcome, and the International Mesothelioma Interest Group staging system successfully identifies patients whose prognosis is poor; (4) despite more locally advanced disease in most patients with extrapleural pneumonectomy, that approach provided better local control than pleurectomy/decortication but failed to improve survival because of distant metastatic disease. Contrary to past practice, future clinical trials should stratify for histologic type, must control for TNM stage, and must consider the impact of type of surgical resection on the pattern of relapse. PMID- 8614143 TI - Molecular genetic differentiation between primary lung cancers and lung metastases of other tumors. AB - When solitary pulmonary tumors are observed in patients with a history of cancer, differentiation between metastasis and primary lung cancer is crucial for appropriate therapy. Assuming that p53 mutations are conserved in metastases, mutation analysis of the p53 gene would be a valuable tool in differentiating metastases from primary carcinomas of the lung. In nine of 267 resected lung tumors, the origin of the lung tumor could not be defined histologically. Five patients had a history of colorectal carcinoma, one had a history of breast carcinoma, one had a history of soft-tissue carcinoma, and one had a history of head and neck carcinoma. One patient with a clear cell carcinoma of the lung had been surgically treated for both renal and thyroid cancer. Material from one patient with adenocarcinoma of the lung, histologically defined regional lymph nodes, and distant brain metastasis served as a control. We extracted deoxyribonucleic acid from the snap-frozen tissue of the unclassified lung tumors, from paraffin-embedded tissue of the previously removed primary cancers, and also from peripheral blood of the patients. Exons 2 to 11 of the p53 gene were amplified in separated polymerase chain reactions and directly sequenced. In all cases, the presence of germline mutations was excluded by analysis of peripheral blood deoxyribonucleic acid. The p53 mutation detected in the deoxyribonucleic acid of the lung tumor of the control patient proved to be conserved in the lymph nodes as well as in the brain metastasis. In two cases, the lung tumors exhibited a p53 mutation not present in the previously removed primary tumor and were therefore classified as new primary lung cancers. In five cases, the lung tumors proved to be metastases of the first tumor, exhibiting the identical p53 mutation. One of these lung tumor samples could be identified as a metastasis from the renal cancer, but the corresponding thyroid cancer material was different. For two cases, molecular analysis remained inconclusive. In one case, no p53 mutation could be found in the compared samples; in the other, no deoxyribonucleic acid could be extracted. Analysis of p53 mutations allowed exact classification in tumors for which standard methods failed to distinguish between metastasis or primary tumor. More than two thirds of lung tumors in patients with previous gastrointestinal carcinoma were revealed to be metastases, but second primary lung cancer could also be diagnosed. This diagnosis allowed correct surgical and adjuvant treatment of these patients. PMID- 8614144 TI - Staged operation to Fontan increases the incidence of sinoatrial node dysfunction. AB - Morbidity and mortality of total cavopulmonary connection (modified Fontan procedure) may be decreased in many patients with single ventricle in whom the risk of surgery is high by performing the operations in a staged fashion. Each operative intervention, however, exposes the sinoatrial node region to risk of injury, and a multistaged approach may increase the risk of altered sinoatrial node function in these patients. The purpose of this study was to compare the prevalence of perioperative arrhythmias in patients undergoing either a primary or staged approach to the Fontan operation. Records were retrospectively reviewed for all patients having a Fontan procedure between January 1988 and December 1992. Of 324 patients undergoing a Fontan operation, 227 had a Fontan operation without a prior cavopulmonary shunt (group 1) and 97 had a cavopulmonary shunt before a Fontan operation (group 2). Arrhythmias were classified as altered sinoatrial node function, supraventricular tachycardia, or atrioventricular block. The prevalence of both transient (resolving before hospital discharge) and fixed (persisting until hospital discharge) altered sinoatrial node function was similar for the two groups after cavopulmonary shunt or primary Fontan despite a heterogeneous patient population (group 1: 10.6%/4.4%; group 2: 10.3%/3.1%; p=0.28). Conversion from cavopulmonary shunt to Fontan in group 2 resulted in a higher prevalence of altered sinoatrial node function in the early postoperative period (transient: 23.7%; fixed: 23.7%; p < 0.001) and on follow-up (group 1: 7.7%; group 2: 16.7%; p < 0.02). In group 2, 40 of 82 patients without arrhythmia after first intervention (cavopulmonary shunt) had an arrhythmia after the second intervention (Fontan) (49%); of 14 with an arrhythmia after the first operation, 10 (71%) had one at the second intervention (p < 0.01). In conclusion, a multistaged operative pathway to Fontan reconstruction is associated with a higher early risk of altered sinoatrial node function. The occurrence of altered sinoatrial node function after cavopulmonary shunt is itself a risk factor for arrhythmia after the Fontan operation. Longer follow-up is needed to assess the full impact of this finding. PMID- 8614145 TI - A one-way valved atrial septal patch: a new surgical technique and its clinical application. AB - Patients who undergo surgical repair of congenital heart defects, characterized by a hypoplastic right ventricle or high pulmonary vascular resistance, are at high risk for the development of postoperative right heart failure. This risk may discourage the surgical team from carrying out a biventricular or complete repair in such patients. To reduce the risk for right heart failure, we developed a one way, valved, atrial septal patch to serve as an artificial one-way foramen ovale and tested it in an animal model. By permitting right-to-left shunt, this device decompresses the failing right ventricle and maintains systemic cardiac output. The device has been used in 15 patients divided into three different groups: group 1 (n = 8), patients with a hypoplastic right ventricle and pulmonic stenosis or atresia, seven of whom underwent a biventricular repair; group 2 (n = 5), patients with evidence of pulmonary disease after longstanding left-to-right shunt caused by a correctable atrial or ventricular septal defect, all of whom had a complete repair; group 3, two patients with acute right heart failure in whom the device was used as a last option of treatment to wean them from cardiopulmonary bypass. This article presents our data in regard to the use of the one-way, valved, atrial septal patch and the indications for its clinical use. PMID- 8614146 TI - Truncus arteriosus repair: influence of techniques of right ventricular outflow tract reconstruction. AB - Fifty-six consecutive patients underwent total correction of truncus arteriosus. Median age at repair was 41 days, with a range of 2 days to 8 months. In 71% the operation was done in the first 2 months of life. Nine patients had complex forms of truncus and 11 patients had aortic insufficiency. The truncal aortic root was transected, which provides a clear exposure of the coronary ostia. The aorta was reconstructed by direct end-to-end anastomosis, and the truncal valve was preserved in every case. Several different techniques were used for pulmonary reconstruction, including three types of anatomic reconstruction of the pulmonary valve with a trisigmoid leaflet system and two types of nonanatomic reconstruction. The anatomic techniques included use of 33 Dacron valved conduits, eight homograft valved conduits, and one porcine aortic root bioprosthesis. The nonanatomic reconstructions included direct anastomosis to the right ventricle in nine patients and insertion of autologous pericardial valved conduits in five. The hospital mortality was 16% (9/56; 95% confidence limits, 2% to 30%). Multivariate analysis outlines two independent incremental risk factors for hospital death: nonanatomic pulmonary valve reconstruction techniques and age younger than 1 month. The hospital mortality was 7.1% in the group with anatomic pulmonary valve reconstruction versus 43% in the group with nonanatomic pulmonary valve reconstruction (p = 0.015). The hospital mortality was 5.7% in those older than 1 month versus 33% in those younger than 1 month of age (p = 0.04). There were two late deaths. The actuarial freedom from reoperation and angioplasty at 7 years was 100% for patients receiving pericardial conduits, 80% for those undergoing direct anastomosis, 77% for those receiving Dacron conduits, and only 43% for those receiving homografts (p = 0.02). In conclusion, anatomic reconstruction of the pulmonary valve seems important at the time of the operation, age younger than 1 month remains an incremental risk factor, and the truncal valve can be preserved. PMID- 8614147 TI - Chronic nonpulsatile blood flow. II. Hemodynamic responses to progressive exercise in calves with chronic nonpulsatile biventricular bypass. AB - We investigated the effects of stepwise treadmill exercise on animal (calf) hemodynamic variables during chronic nonpulsatile biventricular bypass with ventricular fibrillation. Seven days was allowed for recovery from the effects of anesthesia and surgery; each animal's natural heart was then fibrillated. The pump flows were maintained at nominal rates of 90, 100, and 120 ml.kg-1.min-1 for 1 week each, with the order varying from experiment to experiment. A total of 30 incremental exercise tests were performed on five animals. No significant changes in mean aortic pressure were observed during nonpulsatile perfusion at the three nominal flow rates of nonpulsatile flow either before or during exercise. The systemic vascular resistance decreased significantly during exercise (from 705 +/ 22 to 547 +/- 81 dyne.sec.cm-5, p < 0.01, and from 604 +/- 25 to 510 +/- 15 dyne.sec.cm-5, p < 0.05, at nominal flow rates of 100 and 120 ml.kg-1.min-1, respectively). There were also significant (analysis of variance, Scheffe test, p < 0.05) differences in systemic vascular resistance among three nominal flow rates both before and during exercise. These results suggest that the autonomic nerve reflex control of the cardiovascular system in physical exercise was functioning normally in animals with chronic nonpulsatile blood flow. PMID- 8614148 TI - Chronic nonpulsatile blood flow. III. Effects of pump flow rate on oxygen transport and utilization in chronic nonpulsatile biventricular bypass. AB - The relationship between blood flow and oxygen transport was studied in five calves with chronic nonpulsatile biventricular bypass. Seven days was allowed for recovery from the effects of anesthesia and operation; the natural heart was then fibrillated. Pump flows were maintained at nominal rates of 90, 100, or 120 ml.kg 1.min for 1 week each, with the sequence varied from experiment to experiment. Venous and arterial blood samples were taken at rest for blood gas analysis. Serum lactate analysis was done twice a week, on the third and seventh days after each pump flow change. Serum catecholamine levels were assayed on the seventh day of each flow rate. Progressive exercise tests were also conducted during each test segment. Basal oxygen consumption of a 4-month-old calf was 6.3 +/- 0.3 ml.kg-1.min-1. The mixed venous oxygen tension decreased when pump flow rate was reduced (29.6 +/- 1.0, 28.3 +/- 1.2, and 23.8 +/- 0.9 mm Hg at 120, 100, and 90 ml.kg-1.min-1 of pump flow, respectively), and oxygen extraction increased linearly when pump flow rate was reduced. Hemoglobin concentration significantly affected oxygen extraction rate. Serum lactate concentration increased significantly at a 90 ml.kg-1.min-1 perfusion compared with concentrations at other pump flow rates (7.81 +/- 2.42 mEq/L at 90 ml.kg-1.min-1 vs 0.71 +/- 0.19 and 0.73 +/- 0.81 mEq/L at 100 and 120 ml.kg-1.min-1, respectively; p < 0.01, analysis of variance, Scheffe F test). Maximum oxygen extraction during exercise was 78%. These results suggest that a critical flow level between 90 and 100 ml.kg-1.min-1 maintains oxidative metabolism in the calf with chronic nonpulsatile flow. The resulting oxygen delivery was slightly higher than that indicated in the literature. Maximal oxygen extraction was normal. PMID- 8614149 TI - Controlled limb reperfusion in patients having cardiac operations. AB - HYPOTHESIS: Severe limb ischemia in patients having cardiac operations may occur after intraaortic balloon pump insertion, prolonged femoral vessel cannulation, percutaneous cardiopulmonary bypass, dissecting aneurysms, or emboli. Normal blood reperfusion can cause a postischemic syndrome that increases morbidity and mortality. This clinical study is based on an experimental infrastructure patterned after controlled cardiac reperfusion. (1) It tests the hypothesis that controlled limb reperfusion (i.e., modifying the composition of the initial reperfusate and the conditions of reperfusion) reduces the local and systemic complications seen after normal blood reperfusion. (2) It reports initial clinical application of this strategy in three cardiac surgery centers. METHODS: Controlled limb reperfusion was applied to 19 patients with signs of severe prolonged unilateral or bilateral ischemia (including paralysis, anesthesia, and muscle contracture); six patients (32%) were in cardiogenic shock. The mean ischemic duration was 26 +/- 6 hours. The reperfusion method includes a 30-minute infusion into the distal vessels of a normothermic reperfusate solution mixed with the patient's arterial blood (obtained proximal to the obstruction) in a 6:1 blood/reperfusate ratio. Data are mean +/- standard error of the mean. RESULTS: Sixteen patients (84%) survived with salvaged and functional limbs at the time of discharge. No renal, cardiac, pulmonary, cerebral, or hemodynamic complications developed in the survivors. The three deaths occurred in patients undergoing controlled limb reperfusion while in profound postoperative cardiogenic shock; neither postischemic edema nor contracture developed in any of them. CONCLUSIONS: These findings show that controlled limb reperfusion can be applied readily with standard equipment that is used for cardiac surgery and may salvage limbs while reducing postreperfusion morbidity and mortality. PMID- 8614150 TI - Effect of sodium nitroprusside on complement activation induced by cardiopulmonary bypass: a clinical and experimental study. AB - Complement activation and leukocyte stimulation were prospectively studied during and after cardiopulmonary bypass in 16 children receiving sodium nitroprusside--a nitrovasodilator releasing nitric oxide--for vasodilation during the cooling and rewarming periods of extracorporeal circulation. Results were compared with those in 29 patients who were not treated with sodium nitroprusside during the operation. Patients treated with sodium nitroprusside had significantly less C3 conversion during cardiopulmonary bypass as measured by the ratio C3d/C3 (p <0.05) and significantly less C5a liberation immediately after cardiopulmonary bypass (p < 0.005) than patients not treated with sodium nitroprusside. C4 was not overtly consumed in our series. Leukocyte count during the rewarming period of cardiopulmonary bypass, but not leukocyte elastase release during cardiopulmonary bypass, was significantly reduced in patients treated with sodium nitroprusside (p <0.05). In vitro experiments were conducted to analyze the effect of sodium nitroprusside on complement hemolytic activity initiated by the classic and the alternate pathways and on zymosan-induced C3 conversion by the activation of the alternate pathway. The in vitro experiments clearly demonstrate inhibition of complement hemolytic activity by sodium nitroprusside in the sera tested. The 50% inhibitory concentration of sodium nitroprusside on the available complement hemolytic activity was less through the alternate pathway than through the classic one (4.2 +/- 0.8 mmol/L and 14.0 +/- 2.88 mmol/L, respectively). The decrease of complement hemolytic activity measured was dose-dependent and was enhanced by the sodium nitroprusside preincubation of the sera tested. This effect was related to the duration of preincubation. Sodium nitroprusside photodegradation (enhancing nitric oxide release) increased the anticomplementary effect of the drug, reducing the 50% inhibitory concentration on complement hemolytic activity to 0.24 to 0.02 mmol/L for the alternate pathway and 2.74 o 0.3 mmol/L for the classic pathway. the zymosan-induced C3 conversion was inhibited by sodium nitroprusside. Nitroglycerin and isosorbide dinitrate (other nitric oxide donors) had in vitro effects on complement hemolytic activity similar to those of nonphotodegraded sodium nitroprusside at similar concentrations (1 mmol/L). Our results suggest that sodium nitroprusside, both in vitro and in vivo, has an inhibiting effect on complement activation initiated by both classic and alternate pathways and that this effect is mediated by nitric oxide release from sodium nitroprusside. This is the first report on the anticomplementary effect of sodium nitroprusside by nitric oxide release. PMID- 8614151 TI - Long-term results of surgical treatment for mitral regurgitation with severe left ventricular dysfunction after myocardial infarction caused by Kawasaki disease. PMID- 8614152 TI - Mitral regurgitation caused by ruptured chordae tendineae in Kawasaki disease. PMID- 8614153 TI - Late failure of internal thoracic artery grafts caused by sequelae of mediastinitis or its treatment. PMID- 8614154 TI - Volume reduction of the native lung after single-lung transplantation for emphysema. PMID- 8614155 TI - Influence of perfusion technique and pH management strategy during coronary artery bypass surgery. PMID- 8614156 TI - Anomalous origin of the left coronary artery from the pulmonary artery: is reconstruction of a double coronary artery system always necessary? PMID- 8614157 TI - Massive bleeding at resternotomy despite a polytetrafluoroethylene surgical membrane. PMID- 8614158 TI - Relationship between intelligence and duration of circulatory arrest with deep hypothermia. PMID- 8614159 TI - Relief of aortic obstruction in (S,L,L) hearts with rudimentary right ventricle and restrictive bulboventricular foramen. PMID- 8614160 TI - Historical perspectives of the American Association for Thoracic Surgery. Samuel J. Meltzer (1851-1920). PMID- 8614161 TI - Assessing the culture of medical group practices. AB - This study was designed to identify the relevant components of the organizational culture of medical group practices and to develop an instrument to measure those cultures. Building on the work of industrial psychologists and organizational sociologists, a 35-item instrument was developed through an iterative process with more than 100 medical groups. The final instrument was tested using responses from physicians practicing in two very different medical groups: one a prepaid group practice with salaried physicians and the other, until recently, a fee-for-service practice. Using stepwise discriminant analysis of the responses to this instrument, more than 90% of the physicians were able to be placed in the appropriate practice setting. PMID- 8614162 TI - The benefits and risks of over-the-counter availability of nicotine polacrilex ("nicotine gum"). AB - Nicotine polacrilex ("nicotine gum") is effective in helping persons to quit smoking cigarettes. Because many persons try to quit without formal assistance, it may be an appropriate product for over-the-counter (OTC) purchase. Some smokers, however, might use such a product in lieu of more effective methods of cessation, and still others might use it to cope with enforced periods of nicotine abstinence (eg, at the work place) and thereby delay their decision to quit. The study's objective was to assess the public health benefits and risks of OTC availability of nicotine gum. A Markov model was developed and used to contrast two alternative policy scenarios: one in which nicotine gum was assumed to remain available only by prescription, and another in which it was assumed to be made available for OTC purchase. Various data sources were used to estimate the model, including the Health Promotion and Disease Prevention Supplement to the 1991 National Health Interview Survey and the 1986 Adult Use of Tobacco Survey. Primary outcome measures included the numbers of persons who would try to quit smoking, the numbers who would use various methods of smoking cessation, including OTC nicotine gum, and the numbers of current adult smokers who would be abstinent at the end of 10 years. Findings suggest that an average of 3 million persons each year would use OTC nicotine gum. As a consequence of OTC availability, an additional 450,000 smokers would be abstinent at the end of 10 years. These results are sensitive to assumptions regarding the effectiveness of OTC nicotine gum, as well as to the effect of OTC availability on the use of other methods of smoking cessation. The number of persons who would quit smoking, however, increases under a fairly wide range of assumptions. Over-the-counter availability of nicotine gum may confer significant public health benefits. PMID- 8614163 TI - The sex of the general practitioner: a comparison of characteristics, patients, and medical conditions managed. AB - In Australia an increasing proportion of active general practitioners (GPs) are women. Overseas research showing differences between male and female GPs in practice style, the reasons patients consult them, or in the nature of the medical conditions they manage has failed to adjust for confounders. In Australia, such differences have never been investigated. This study assessed differences between male and female GPs in terms of their personal characteristics, patient mix, patient reasons for consultation, and the medical conditions they manage. It also considered the extent to which differences are accounted for by the effect of confounders. A secondary analysis was done of data from the Australian Morbidity and Treatment Survey 1990 to 1991 (n = 113,000 general practice encounters). In addition, univariate analysis was followed by multivariate analysis, with adjustments for GP and patient characteristics and (in analysis of conditions managed) for patient reasons for encounter. Significant differences were found in the work patterns and patient mix of male and female GPs. Patients' selectivity in the problems presented to the two groups remained after adjustment for confounders. Female GPs managed more female specific, endocrine, general, and psychosocial problems even after multivariate adjustment. Although male GPs managed more cardiovascular, musculoskeletal, male genital, skin, and respiratory problems at the univariate level, these differences were no longer apparent after adjustment. Male and female GPs manage different types of medical conditions. Although some differences are due to their patient mix and to patient selectivity, others are inherent to the sex of the physician. Extrapolation of results to Australian general practice suggests that these two groups of GPs could become semispecialized. PMID- 8614164 TI - Assessing oral health-related quality of life: findings from the normative aging study. AB - The contribution of oral health to health-related quality of life (HQOL) has seldom been examined. This study was designed to develop and validate a measure of oral health-related quality of life (OHQOL), examine relations between OHQOL and HQOL, and explore OHQOL's relation to problem-based dental care utilization in a sample of 1,242 older men, using data gathered by a mail survey. Factor analysis of eight oral health items revealed that three items related to the impact of oral conditions on daily functioning defined a factor labeled OHQOL. Factor analysis of the HQOL items and these three OHQOL items showed that OHQOL represents a separate and independent factor. Correlational analyses supported the construct validity of the OHQOL measure: Men with better OHQOL scores reported less dental pain or discomfort, fewer eating problems, and less problem based dental care utilization. Logistic regression analysis showed that the measures of dental pain and oral discomfort were related positively to utilization, whereas OHQOL was related negatively. These results suggest that OHQOL represents a separate and distinct facet of HQOL that is associated with dental care utilization. Thus they support the validity of the OHQOL construct and suggest its use in future studies of HQOL. PMID- 8614165 TI - Migration of obstetrician-gynecologists into and out of rural areas, 1985-1990. AB - This study sought to determine if county-level demographic, health care resource, policy, and competitive factors are associated with the movement of obstetrician gynecologists (ob-gyns) into and out of rural areas. County-level descriptive data from the Area Resource File, the American Medical Association Physician Masterfile, and the American Hospital Association Guide were used for hospital descriptions. This was a correlational study that measured the association of ecologic indicators of nonmetropolitan counties with indicators of gain or loss of ob-gyns. Descriptive statistics characterize the supply and movement of ob gyns by size and location of the counties. Multinomial logistic regression models describe the net effect of the ecologic indicators on physician movement. During the period 1985 to 1990, a total of 962 patient care ob-gyns moved out of 531 nonmetropolitan counties, and 979 ob-gyns moved into 528 counties. Counties in the southern Atlantic states experienced the greatest net inflow, whereas Illinois, Missouri, and Texas had the greatest net outflow. Counties that retained ob-gyns during this period were in the mid-range of population. Positive correlates of outward migration were adjacency to a metropolitan county and loss of hospital bed supply; negative correlates with outward migration were the supply of hospital beds and total population. Inward migration was positively correlated with retention or gain of county family physicians and with adjacency; negative correlates were overall population and total family physician supply. The movement of ob-gyns in nonmetropolitan counties is influenced by state policies, local resources, and relative location. No clear evidence shows that there are competitive relations between family physician supply and ob-gyn supply. PMID- 8614166 TI - Minority physicians serving in rural National Health Service Corps sites. AB - Providing National Health Service Corps (NHSC) scholarships to under-represented minorities has been an important federal mechanism to bolster the numbers of minority physicians. Little is known about how minorities fare during their NHSC commitment periods. In 1991, questionnaires were mailed to all primary care physicians placed in rural communities from 1987 through 1990 in the NHSC scholarship program, in a retrospective cohort study. One hundred and twenty-two of the 398 eligible NHSC physician respondents (31%) indicated they were minorities. National Health Service Corps physicians were found to be well matched by race to the sites where they served, and minority NHSC physicians worked in counties and practices with greater proportions of minority inhabitants and patients. Minorities among rural NHSC physicians were less likely to have been raised in rural areas and were less interested in rural practice during medical school and when placed in their rural NHSC sites. The relative urban preferences of minority physicians in large part explains why this group was more dissatisfied with their work and personal lives while serving their obligations. Minority physicians also reported lower satisfaction for their families. Minority and nonminority NHSC physicians reported comparable acceptance by their communities, and demonstrated similarly low retention rates. The NHSC plays a significant role in the careers of many young minority physicians and in promoting the temporary availability of minority physicians for rural health professional shortage areas. However, as of 1991, many minority NHSC physicians placed in rural areas would have preferred urban sites, which resulted in their lower satisfaction. PMID- 8614167 TI - The impact of practicing in multiple hospitals on physician profiles. AB - Although physicians are all too familiar with the psychologic impact of having multiple responsibilities, the associated impact on practice styles has not been examined systematically. To provide some data on the effects of "work dispersion," we examined the hypothesis that the inpatient resource use of physicians would rise with the number of hospitals in which they work. Data for 1991 from Medicare's National Claims History File were used to profile a sample of attending physicians (n = 33,756) in seven states. The attending physician "profile" was the case mix-adjusted relative value of all physician services (regardless of who delivered them) that were delivered during each patient's hospital stay. Relative value was measured in relative value units, used by Medicare in determining physician payments. The authors then categorized physicians in terms of the number of hospitals to which they admitted patients. Physician profiles were adjusted further to control for geography, physician specialty, and characteristics of the physician's primary (ie, most used) hospital. One third of the physicians in the sample had admissions to more than one hospital. Physicians working in one hospital had inpatient practice profiles 2.1% below the sample mean. Additional hospital affiliations were associated with progressively higher profiles: two hospitals, 2.3% above the mean; three hospitals, 4.5% above; four hospitals, 8.2% above; and five or more hospitals, 11.5% above (all P < 0.01). The practice of medicine in more than one hospital is associated with higher inpatient profiles and shows a dose-response relationship. Physicians and policy makers will need to consider carefully whether there are any associated benefits to justify the increased cost. PMID- 8614168 TI - Total quality management in hospitals: the contributions of commitment, quality councils, teams, budgets, and training to perceived improvement at Veterans Health Administration hospitals. AB - Studies of total quality management as a means of improving health care quality to date have relied on case studies of individual teams or hospitals. The Total Quality Improvement Registry Project surveyed quality coordinators (n = 36) and quality improvement team leaders (n = 228) to collect both site-level and team level data on quality improvement in Veterans Health Administration hospitals. Usable responses were received from 100% of quality coordinators and 73.7% (168) of team leaders. Site-level data include hospital structural characteristics and measures of training and commitment, as well as features and activities of the hospital quality councils. Team-level data include size, membership, task, age, activities, and a proxy measure of quality improvement. The authors report on the relations between levels of commitment to total quality management principles, training levels, activities of quality councils, and team formation and success. These data provide support for a model of commitment to quality improvement that involves four realms of influence within the medical centers: (1) management, (2) physician leadership, (3) physician staff and middle management, and (4) nurses and employees. The authors also report on the activities of quality councils and the relation of their activities to commitment and perceived improvement. Using bivariate correlation and multiple regression, the authors found that the age of the quality council, overall facility commitment to total quality management philosophy, and physician commitment are the most critical variables in explaining numbers of teams, training intensity, and total perceived improvement at this sample of medical centers. Specifically, we find that commitment to total quality management philosophy and the number of active teams explains 41% of the observed variation in quality improvement. In future articles, the authors will report details of team activities and the development of teams over time. PMID- 8614169 TI - Minimal increase in use of breast-conserving surgery from 1986 to 1990. AB - Substantial geographic and hospital-based variations have been documented in the use of breast-conserving surgery (BCS) in 1986. The authors studied the patterns of adoption of this procedure from 1986 to 1990. National Medicare inpatient claims were used to study women aged 65 to 79 who underwent an operation for local or regional breast cancer in 1986 (38,679 patients) or 1990 (43,083 patients). Breast-conserving surgery was used for 5,509 (14.1%) of the Medicare patients in 1986 and 6,476 (15.0%) in 1990. The only region with an increase in BCS use from 1986 to 1990 was New England. Many hospitals had low volumes of operations, with a median of six to seven patients annually. Ten percent of the hospitals performed 55% of the conservative operations. Large hospitals, urban hospitals, and those with higher patient volumes or a cancer center were somewhat more likely to have increased use of BCS by 1990. Despite the substantial evidence supporting BCS as an alternative to mastectomy, the overall use of BCS in Medicare inpatients increased minimally from 1986 to 1990. Many patients are treated in hospitals with little experience with BCS. Hospitals using more BCS in 1986 were somewhat more likely to increase the use of BCS by 1990. PMID- 8614170 TI - Comorbidity-adjusted complication risk: a new outcome quality measure. AB - The measurement of inpatient complications his received substantial attention in recent years because mortality rates and other outcome measures often appear unable to discriminate superior from inferior hospital care. Complication measurement holds out the promise of being more sensitive to variations in patient care because complications occur more frequently than do mortalities, and because complications are more direct consequences of the process of care. The authors developed a new measure of complications that seeks to give insight into the patient care given by different hospitals or physicians by using commonly available data. Specifically, this measure is based on a decision-theoretic model that estimates the probability of a complication for combinations of admitting and secondary International Classification of Diseases, 9th Revision, Clinical Modification diagnoses. The measure can be evaluated at the patient level, or aggregated and risk-adjusted for the population of a given care provider (eg, physician or hospital). When applied to a set of patient-level UB- 82/92 data, this measure estimates the risk of complication for any member of a population, controlling for comorbidity, and hence is designated comorbidity-adjusted complication risk (CACR). The authors describe the development of CACR and its testing and validation using data acquired from the states of Pennsylvania, California, and Florida, as well as facility data obtained directly from hospitals. The data set includes 480,000 patients from 50 Pennsylvania hospitals, 300,000 patients from 33 Florida hospitals, 370,000 patients from 35 California hospitals, and 37,000 patients from six validation hospitals. Comorbidity adjusted complication risk is constructed from widely available data common to most patient cases. Comorbidity-adjusted complication risk can be adjusted for its case mix, but such risk adjustment has much less effect on CACR than on other adverse outcomes such as mortality and morbidity. Comorbidity-adjusted complication risk varies widely across the hospitals in this sample, yet it is stable across time and is correlated with other known quality outcomes, including such accepted "gold standards" as hospital-documented adverse event rates and chart review determinations of complications. PMID- 8614171 TI - Managed care: past, present, and future. AB - The authors have examined managed care from several perspectives in this article. A look at the origins of managed care provided a historical perspective. The current state of managed care and its issues were presented. Finally, the authors speculated what the future may bring in terms of both continued current trends and significant changes in managed care. The authors hope that this presentation provides some insight to the practicing physician on how to work in a world with managed care and what the future may bring. Managed care brings changes in the way physicians practice, but it also offers physicians many opportunities to improve the quality and cost-effectiveness of health care. Managed care is the wave of the present and of the future. It is the health care market's choice to address the challenges that health care faces in quality, cost-effectiveness, accountability, access, and choice. Although there are numerous proposed, alternative, and theoretic solutions, managed care has insurmountable momentum. It will be molding the shape of health care as society enters the twenty-first century. PMID- 8614172 TI - Managed care, accountability, and the physician. AB - The 1990s is truly the era of increased accountability in the practice of medicine. Through the methods of cost and quality measurement and the introduction of a manager (i.e., the MCO), society as a whole will benefit from a medical delivery system that focuses on linking the outcomes of care delivered to the processes of the care provided. Report cards serve an important tool by which information about quality and costs can be quantified and shared with the purchasers and users of the medical delivery system. Purchasers and patients are asking for greater accountability from payers and providers. The increased accountability of physicians to health plans, IDSs, hospitals, and patients with whom they interact has major implications on the definition of success in the managed medical delivery system of today. The theme of accountability has been described by the examples of the HEDIS Quality Report Card for health plans and Quality Report Cards for hospitals, PCPs, and specialists. Physicians must provide high-quality care to each patient they see but must also develop the mindset and structures to manage an entire population of patients. The expectations of each of the entities with whom they interact must be understood, and physicians need to develop the skills and infrastructure to put total quality management and information technology to work to help them facilitate the delivery of high-quality care in a cost-effective manner. Everyone involved in the health care system--from purchasers to payers to consumers--shares the same goals as physicians: provide the highest-quality care and achieve the best possible outcomes in the most cost-effective manner. PMID- 8614173 TI - Ethics and managed care. AB - Managed care presents physicians with potential ethical dilemmas different from dilemmas in traditional fee-for-service practice. The ethical assumptions of managed care are explored, with special attention to the evolving dual responsibilities of physicians as patient advocates and as entrepreneurs. A number of proposals are described that delineate issues in support of and in opposition to managed care. Through an understanding of how to apply basic ethics principles to managed care participation, physicians may yet hold on to the basic ethic of the fiduciary doctor-patient relationship. PMID- 8614174 TI - Practice management, health maintenance, and the use of computers in today's medical office. AB - It is clear that a successful medical practice needs a competent caring physician as well as office personnel who understand how to manage an office. The successful management of a practice helps achieve the goals that the physician and practice manager have established. This article has introduced ideas about how to manage and evaluate a practice. Adoption of some or all of these ideas helps provide more comprehensive and satisfying care for the physician and his or her patients. PMID- 8614175 TI - Office gynecology for the primary care physician, part I: vaginitis, the Papanicolaou smear, contraception, and postmenopausal estrogen replacement. AB - Many routine gynecologic problems can be managed easily in the primary care physician's office. Most cases of vaginitis are due to one of several organisms that are easy to diagnose and treat with information from the history, physical examination, and wet mount. The technique of performing a Papanicolaou smear and the interpretation of results are described, as are various methods of birth control and their efficacies and risks. Last, the risks and benefits of estrogen replacement therapy and modalities for beginning therapy are discussed. PMID- 8614176 TI - Office gynecology for the primary care physician, part II: pelvic pain, vulvar disease, disorders of menstruation, premenstrual syndrome, and breast disease. AB - Approaches to patients with pelvic pain, vulvar disease, disorders of menstruation, premenstrual syndrome, and breast diseases are addressed. In the great majority of cases, it is appropriate for the primary care physician to initiate evaluation and management of these problems. It is hoped that the brief introductions contained here suggest a diagnostic approach to each disorder and guide referral to consultants as needed. PMID- 8614177 TI - Preconception counseling for the primary care physician. AB - All health care providers that interact with women of childbearing age should understand the potential benefits of preconception counseling and to approach the evaluation in a thorough manner during routine health maintenance visits. With the increased number of patients enrolled in managed care programs, health maintenance visits provide the unique opportunity to educate women contemplating pregnancy regarding the potential influences of their lifestyle and health status on the future pregnancy. It is becoming increasingly apparent that interventions made during the preconception period are just as crucial as the subsequent 9 months of prenatal care to achieve an optimal maternal-fetal outcome. Some guidelines for the preconception evaluation have been provided, and the implications of chronic medical illness on pregnancy have been discussed. PMID- 8614178 TI - Primary care of the HIV patient: standard practice and new developments in the era of managed care. AB - It is easy when taking care of the patient with AIDS to lose sight of the whole person and become focused on the details of micromanagement, distracted by the array of new therapies, and overwhelmed by the financial risks of the disease. It is therefore critical that a healing and respectful relationship is developed with patients and they are engaged in the decisions regarding their care. Physicians must also continue the search for new therapies and struggle to ensure that patients have access to state-of-the-art treatment. In this, the primary care physician plays a critical role, through identifying study centers, becoming an investigator in expanded access programs, and using referrals to clinical trials to provide new therapies to patients and improve understanding of HIV treatment. Finally, quality of life must be at the forefront of physicians' medical conscience. Ultimately, the physician must know when the best treatment he of she can offer is the assurance of a dignified and comfortable death. PMID- 8614179 TI - The diagnosis and management of gastroesophageal reflux disease. AB - GERD is a common clinical problem. Generally, its clinical presentation and management are straightforward. Greater awareness of the numerous extraesophageal manifestations of the disease aids patients and physicians in appropriate recognition and treatment. Medical therapy is effective in the majority of cases but often requires long-term medication for acceptable symptom control. A small, but significant proportion of patients presents with or develops complications of GERD, most importantly Barrett's esophagus. Although the logistics of long-term surveillance of persons with Barrett's esophagus is unclear, the association of this metaplastic change with esophageal adenocarcinoma underscores the importance of regular follow-up. PMID- 8614180 TI - Management of depression in the outpatient office. AB - Current practice suggests that primary care physicians are in the best position to identify initially and treat depression in the ambulatory setting. Educating primary care physicians about depression is essential for identifying and adequately caring for depressed patients. Depression is commonly seen and easily treated in primary care settings. A structured, consistent approach to the depressed patient is essential and should include patient education; eliciting information about symptoms; clinical observation about the examination; a history of previous psychiatric episodes; family history of affective disorders; history from relatives, other providers, or other clinics if necessary; aggressive use of medication alone or in combination with psychotherapeutic techniques; and appropriate referral. Depression is associated with significant suffering and disability and increased utilization of health care services. Depressed patients can present with a variety of somatic and cognitive symptoms. Primary care physicians should be alert for denial and minimization of symptoms as well as for the presence of stigmatization. The biologic, psychological, and social aspects of depression should be expanded on as needed according to the patient's illness beliefs. A medical model such as that used for explaining diabetes or coronary artery disease may be useful. Primary care physicians miss the diagnosis of depression in as any as two out of three cases, and when the diagnosis is made, patients are often undertreated and given antidepressants at subtherapeutic dosages and for only short periods of time. Treatment of depression can be extremely satisfying and gratifying for patients and physicians, who need to have a high index of suspicion for depression. All antidepressants have the same efficacy, and the selection is based primarily on their side-effect profile. The new generation of antidepressants are better tolerated, relieve symptoms of depression, improve the quality of life, and probably improve morbidity and mortality. In many cases, depression is chronic and recurrent, and treatment should be long-term to prevent relapses. As with other chronic diseases, if a patient requires treatment that the primary care provider cannot render alone, it needs to be coordinated. In particular, consultants may need to be called in for psychotherapy. Primary care physicians should feel confident that, given enough time and cooperation, they can almost always find a treatment regimen that succeeds in alleviating and perhaps preventing the great suffering and risk occasioned by depression in medically ill ambulatory patients. PMID- 8614181 TI - Preventing complications in diabetes mellitus: the role of the primary care physician. AB - Many Americans, knowingly or unknowingly, are afflicted with diabetes. Because of a lack of awareness or a disbelief that aggressive treatment benefits patients on the part of both patients and physicians, diabetes, particularly NIDDM, remains underdiagnosed and undertreated despite complications that can dramatically diminish quality of life. Increasing evidence that good glycemic control forestalls if not prevents these outcomes makes it the primary care physician's imperative to diagnose diabetes before complications develop. Physicians, through targeted screening and aggressive treatment of patients in whom they diagnose this chronic disease, can markedly reduce diabetes-related morbidity and mortality. PMID- 8614182 TI - Outpatient anticoagulation issues for the primary care physician. AB - Increasingly, primary care providers are caring for patients who require anticoagulation. In this article the indications for, complications of, and methods of dosing and monitoring warfarin in the outpatient setting are reviewed. Heparin use among ambulatory patients also is discussed. PMID- 8614183 TI - Cervical thymic cyst: case reports and review of the literature. AB - Due to its rarity, cervical thymic cyst is seldom included in the differential diagnosis of a neck mass. Approximately 80 cases have been reported thus far, and most of these cases have occurred asymptomatically in children and adults. Only 5 cases have involved patients younger than 1 year of age. The authors report four new patients with thymic cyst in the neck. Two of the patients were newborns in whom the cyst caused airway obstruction and dysphagia. All four patients underwent successful resection of the lesions, with complete resolution of symptoms. The embryology, histopathology, and differential diagnosis of cervical thymic cysts are also reviewed. The authors recommend that despite its infrequent occurrence cervical thymic cyst should be considered in the evaluation of neck masses in children. PMID- 8614184 TI - Extranodal Rosai-Dorfman disease of the head and neck. AB - Rosai-Dorfman disease is a rare, idiopathic, benign histiocytic proliferation usually seen in younger patients. Massive lymphadenopathy most commonly involves the cervical lymph nodes, with a predominant infiltration of sinusoidal histiocytes. Nearly half of the patients will have extranodal involvement, 75% occurring in sites in the head and neck. Three cases of extranodal Rosai-Dorfman disease of the head and neck involving the nose, paranasal sinuses, and parotid gland are presented. The clinical presentation, histologic characteristics, radiographic findings, and treatment of the disease are discussed. Because of the scarcity of cases, the clinical and histopathologic features of this disease may be overlooked. Familiarity with its relatively frequent clinical manifestations in the head and neck, as well as with the diagnostic histopathology, should preclude confusion with other disease entities. PMID- 8614185 TI - Laser photochemotherapy with anthracyclines on cultured human squamous carcinoma cells. AB - A new treatment for cancer has been tested in vitro using light-sensitive anthracyclines followed by laser photoactivation, as described by several investigators. We previously reported 10-fold enhanced laser killing after 2 hours of incubation with daunomycin by cultured human carcinoma cells. This short term uptake leads to drug localization in cytoplasmic and membrane sites prior to nuclear accumulation and topoisomerase inhibition. In the present study, daunomycin was incubated for 2 or 24 hours with P3 squamous carcinoma cells to directly compare cytoplasmic vs. nuclear drug targeting before and after KTP-532 laser activation. Monolayer cultures of the P3 cells sensitized with daunomycin for 2 hours, then chilled (4 degree C), and exposed to the KTP laser (532 nm, 94.2 J/cm2) had a 2- to 10-fold increased therapeutic response compared with drug or laser alone when measured by MTT tetrazolium assays. After 24 hours of incubation with daunomycin, the chemotherapeutic response of P3 tumor cells was amplified 2-fold by laser exposure. The results suggest that daunomycin and laser treatment can be combined for improved therapy of human cancer. PMID- 8614186 TI - Advances in the development of the interferometric otoscope. AB - Present measurement techniques for middle ear function have inherent limitations because they are either spatially insensitive (acoustic immittance) or descriptive and qualitative in nature (otoscopy). By integrating advances in electrooptic technology (fiber optics, miniature diode lasers, solid-state detector arrays) and digital processing, further advances are possible. On the basis of measurements taken with electronic speckle-pattern interferometry on human temporal bones and models, we demonstrate quantitative static and dynamic vibration/displacement characteristics of the tympanic membrane with high spatial resolution. Our presentation emphasizes advantages of optically based methods and demonstrates computerized signal processing capable of fringe localization, enhancement, and counting. Miniaturization and real-time digital image processing in the clinical setting is the goal of this research. PMID- 8614188 TI - A combined endoscopic Co2 laser and external approach for treatment of glottic cancer involving the anterior commissure: an animal study. AB - Endoscopic laser resection usually has been discouraged for anterior commissure (AC) carcinoma because of inadequate exposure and close proximity to underlying cartilage. A new technique combining endoscopic CO2 laser incision and an external approach, creating a window in the thyroid cartilage, was tested in this in vivo study of six dogs. An en bloc specimen including adjacent cartilage was excised while preserving the thyroid framework. The glottic reconstruction was accomplished with external placement of a sternohyoid muscle flap, by either using a bipedicled muscle flap with overlying skin or a unipedicled muscle flap with a graft of free mucosa. The results showed a satisfactory anatomic reconstruction and acceptable functions. The bipedicled muscle flap was superior to the unipedicled muscle flap due to a better AC reconstruction. It is believed that this new combined technique may overcome limited access with endoscopic technique and excessive cartilage resection with the external partial laryngectomy. PMID- 8614187 TI - Pulmonary atelectasis after reconstruction with a latissimus dorsi myocutaneous flap. AB - Atelectasis is the most common postoperative complication encountered in head and neck surgery. Risk factors include preexisting pulmonary disease, type of surgery performed, and the length of anesthetic. It is controversial whether reconstruction of defects with regional myogenous flaps predisposes to atelectasis. The latissimus dorsi myocutaneous flap requires the patient to be placed on his side for a period of time. Whether it is the position or the surgery that contributes to the development of atelectasis has not been examined. Eighteen patients underwent latissimus dorsi myocutaneous flap reconstruction following major ablative procedures for head and neck cancer. The cutaneous area transferred ranged from 70 to 225 cm2 (mean, 128 cm2). The flap size ranged from 7 x 10 to 15 x 15 cm. The majority of flaps were 10 x 15 cm or greater. These patients were compared to 18 patients who did not undergo pedicled myocutaneous chest flap reconstruction. Patients were matched for age, sex, length of operation, site of primary, and stage of disease. Postoperative atelectasis was radiographically detected in 89% of flap patients vs. 79% of controls. Major atelectasis was encountered in 16% of patients undergoing flap surgery vs. 11% of patients in the control group. Patients with large cutaneous paddles on the flaps (> 120 cm2) had significantly more atelectasis than patients with smaller cutaneous paddles (P<.05, chi-squared). The incidence of radiographic postoperative atelectasis in patients having a latissimus dorsi myocutaneous flap is high. The size of the skin paddle harvested as well as the position change may contribute to this. PMID- 8614189 TI - The effects of keratinocyte growth factor on healing of tympanic membrane perforations. AB - Tympanic membrane (TM) perforations heal by reepithelialization and fibrous layer proliferation. The rat TM model may be used to study growth factors that promote epithelialization and fibroblast proliferation, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). The authors previously evaluated the effects of FGF on tympanic membrane perforations and showed an enhanced rate of wound healing with preservation of normal structure and function. The same model was used to test keratinocyte growth factor (KGF, also called FGF-7). This growth factor has been shown to stimulate the migration and proliferation of keratinocytes. This is the first study investigating KGF in the tympanic membrane perforation model. Our results show that in contrast to FGF and EGF, KGF does not enhance the rate of wound healing, but rather results in a more organized wound repair process. PMID- 8614191 TI - Prediction of the clinical outcome of acute frontal sinusitis with ventilation measurement of the nasofrontal duct after trephination: a long-term follow-up study. AB - Acute frontal sinusitis has become an increasing clinical problem in the region that is served by the hospital represented by the authors of this study. The standard surgical treatment protocol after the failure of conservative therapy has been to perform trephination of the involved frontal sinus. More sophisticated procedures have been used in patients with prolonged or recurrent disease. The authors have developed a simple ventilation test of the nasofrontal duct that can be used to determine which patients require further surgery. In a long-term follow-up study of 85 patients, this ventilation test was shown to predict the cases that would heal uneventfully after trephination and the cases that would require further surgery because of an obstructed nasofrontal duct. The findings of this study are of special interest for modern functional endoscopic sinus surgery, the purpose of which is to open up the nasofrontal region. PMID- 8614190 TI - Basic innervation pattern and distribution of classic autonomic neurotransmitters in human nasal mucosal vasculature. AB - The neural control of human nasal vasculature is still not completely understood. This study was performed to demonstrate the innervation pattern of the different vessel types and to distinguish between nor-adrenergic and cholinergic structures. General innervation was demonstrated using antibodies to neuron specific enolase and S-100 protein. Autonomic structures were shown by using antibodies to tyrosine hydroxylase and choline acetyltransferase (ChAT). In addition, choline acetyltransferase (AChe) histochemistry was performed. Nasal vasculature is controlled by a dense innervation that increases with the thickness of the tunica media. While all larger vessels show a mixed autonomic innervation, sympathetic structures seem to predominate in veins. These findings demonstrate that classic neurotransmitters play a major role in the regulation of nasal vasculature. The stronger innervation of arteries and cushion veins underlines their central position in the control of nasal air flow. PMID- 8614192 TI - Effects of botulinum toxin type A injections on aerodynamic measures of spasmodic dysphonia. AB - The effects of botulinum toxin type A (BOTOX) injections on aerodynamic measures of phonation were examined in 30 patients with adductor spasmodic dysphonia. Patients received bilateral injections of 2.5 U of botulinum toxin type A in each thyroarytenoid muscle. Measures of air pressure, average airflow, coefficient of variation (CV) of airflow, and laryngeal resistance were obtained before the injections and at 2 and 10 weeks after the injections. These measures were also obtained from 12 normal subjects. Compared with normal subjects, the spasmodic dysphonia patients had significantly higher preinjection values for air pressure, CV of airflow, and laryngeal resistance. At 2 and 10 weeks after the botulinum toxin type A injections, the spasmodic dysphonia patients showed a significant increase in the average airflow values and a significant decrease in the CV of airflow values and the laryngeal resistance values. At 10 weeks after the injections, the values for the three measures began to approach the preinjection values. It is concluded that these aerodynamic measures of phonation can provide useful measures of treatment outcome in adductor spasmodic dysphonia. PMID- 8614193 TI - Laryngotracheal reconstruction using microplates in a porcine model with subglottic stenosis. AB - Current techniques of laryngotracheal reconstruction require a choice between prolonged stenting (conventional technique) or short-term stenting with maintenance of sedation and paralysis until the airway has stabilized (single stage laryngotracheal reconstruction). An alternative method is proposed using microplates to provide immediate airway stabilization without stenting. This study was designed to evaluate the long-term effects of microplate repair of stenosis of the subglottis and trachea on the growing larynx. Subglottic stenosis was produced in piglets using a transoral endoscopic technique. Eight animals underwent repair of the stenosis using an anterior cricoid split with microplate distraction and stabilization of the cricoid cartilage and first tracheal ring. The distraction was maintained and airway growth continued for the duration of this study. However, with growth of the larynx the plates migrated away from their original position. In 50% of the animals followed up for 90 days the plates migrated into the airway lumen. This study suggests that rigid distraction of the stenotic airway with microplates is a viable alternative to more traditional methods of repair. However, plate removal at some interval after surgery is required in the growing larynx in order to prevent migration of the plate into the airway. PMID- 8614194 TI - Endoscopic treatment of tracheal stenosis using the carbon dioxide laser and the Gianturco stent: indications and results. AB - The self-expandable Gianturco stent was used in 11 patients with tracheal stenosis who where treated from September 1992 through July 1994. Under optical control, the stent was placed through a bronchoscope with an outside diameter of 9 mm after cross-section and dilation of the stenosis were performed with the carbon dioxide laser according to the Shapshay technique. The follow-up period varied from 3 to 22 months, with a mean of 12 +/- 6 months. Pulmonary function tests showed a mean improvement of the peak expiratory flow (50%), from 0.95+/ 0.45 L/sec before the operation to 2.13+/-0.86 L/sec after the procedure. Radiologic and fibroscopic controls showed prosthesis stability. Three patients needed a second endoscopic procedure because of malposition of the stent, the formation of granuloma at the superior end of the prosthesis, and the development of a mucous membrane webbing between the two loops of a stent. Due to the lack of long-term follow-up, this technique is reserved for the treatment of severe tracheal stenosis with cartilage impairment in patients who have contraindications for external reconstructive surgery. PMID- 8614195 TI - Malignant melanoma metastatic to the tonsil. AB - Metastasis to the oral cavity from melanoma arising in distant sites is very rare. The authors present three cases of melanoma metastatic to the palatine tonsil: two were cutaneous melanomas and one arose from the mucous membrane of the hard palate. Fewer than 30 cases of metastatic melanoma to the palatine tonsil have been reported, and none have been from mucosal melanoma originating from another mucosal primary site in the head and neck. From this perspective, one of the cases in this article is unique. The authors present clinical histories of the three cases of melanoma metastatic to the palatine tonsil. Two patients were still alive a substantial time after treatment. The case presentations indicate that careful examination of the head and neck should be part of the routine follow-up examination in all melanoma patients. PMID- 8614196 TI - Elevated arginine vasopressin levels in squamous cell cancer of the head and neck. AB - The reported effectiveness of single tumor markers (TMs) associated with squamous cell cancer of the head and neck ranges from 15% to 71%, with most studies reporting sensitivity no higher than 50%. An increased incidence of the syndrome of inappropriate secretion of antidiuretic hormone or arginine vasopressin (SIADH) in patients with head and neck cancer has been reported. Serum arginine vasopressin (AVP) was studied as a possible TM in these patients. Sixty-three patients with squamous cell carcinoma of the head and neck determined as potentially curable were prospectively evaluated before treatment and compared to 17 patients with apparent cure of head and neck squamous cell cancer who served as controls. Serum AVP levels were obtained and determined by radioimmunoassay in the preoperative period and 1 week postoperatively in 15 patients. Thirty-four patients were staged as T4, 26 as T3, and 3 as T2. Twenty-one (33%) of the 63 patients had no neck involvement. Twenty-four (38%) of 63 patients had elevated serum AVP levels corrected for serum osmolarity. Of the 15 patients evaluated before and after surgery, 8 (53%) had elevated serum AVP levels preoperatively. Of these 8 patients, 3 had reduction in AVP levels and 5 had complete normalization after 1 week. The result obtained for serum AVP do not exceed results of other TMs reported. AVP may also not be as specific as other TMs for cancer of the head and neck. Our group with AVP sampled postoperatively is too small for us to draw conclusions, but reduction of its levels after treatment in all patients may be significant. These preliminary results indicate that further evaluation of AVP during the posttreatment course in a larger number of cases, and perhaps with other TMs as well, is warranted. PMID- 8614197 TI - Wound healing: effects of closing tension, zyplast, and platelet-derived growth factor. AB - Previous studies have shown that beginning 5 to 7 days from wounding, the tensile strength of wounds closed under tension in rats is significantly higher than the tensile strength of wounds closed without tension. This study evaluated the effects of increased closing tension, zyplast implant (ZI), and human recombinant platelet-derived growth factor (PDGF) on the tensile strength of wounds. Six groups of 12 rats each were divided into two main subsets: wounds were closed without tension in three groups and with high tension of approximately 100 g in the three other groups. In both no-tension and high-tension groups, one subgroup received no intervention (controls), one subgroup received ZI (zyplast controls), and one subgroup received ZI-PDGF (ZI used as a carrier for PDGF). Healing was evaluated by tensile strength determinations at 5 days. For the controls, wounds closed under tension showed a trend toward higher tensile strength, but statistical significance was not reached. Compared with controls, tensile strength was 45% lower in the wounds with ZI and closure with tension (P = .0063) and 38% lower in the wounds with ZI and closure without tension (P = .007). Treatment with ZI-PDGF resulted in 69% higher tensile strengths (P = .049) as compared with ZI controls for wounds closed with tension. This study demonstrated the beneficial effect of PDGF in accelerating healing in wounds closed under tension. Although ZI was able to deliver PDGF to the wound, the use of this substance as a carrier is questioned since it was detrimental to healing. PMID- 8614198 TI - Intratemporal facial nerve perineurioma. AB - Forty-two cases of perineurioma have been reported in the literature. This report adds the first intratemporal facial nerve perineurioma to the literature and reviews the others. Unlike schwannoma and neurofibroma, the histological features of perineurioma demonstrate onion bulb-like structures with a strong positive immunoreactivity for epithelial membrane antigen. The clinical history of gradual facial nerve paresis was 15 years in the case presentation and the clinical diagnosis of tumor was overlooked. PMID- 8614199 TI - Risk of transmission of human immunodeficiency virus infection during tympano ossicular homograft: an experimental study. AB - It is generally agreed that middle ear reconstructive surgery performed with tympano-ossicular homografts produces superior functional results compared with prosthetic material, especially with respect to extrusion rate. The use of homografts, though, has been seriously hampered recently by the fear of transmission of human immunodeficiency virus (HIV) infection. In HIV-infected patients, the virus is primarily found in the cells of the lymphoid and monocytic lineage. The nature of the tissues in the eardrum and ossicles, mostly fibrous tissue and compact bone without marrow, suggests that little virus load should be found in homografts. Indeed, culturing minced homograft tissue from two HIV infected donors with acquired immune deficiency syndrome (AIDS) in a sensitive culture system with PHA-stimulated lymphoblasts produced no virus. Before use, homografts undergo a fixation procedure in 5% formaldehyde and then are kept in a solution containing Cialit as a preservative. The authors therefore examined the capacity of formaldehyde and Cialit to reduce the infectivity of HIV in models of infected tissue as measured in vitro. The reduction of in vitro infectivity due to these treatments was at least 10(5)-fold and 10(2)-fold, respectively. Coupled with the low virus burden in tympano-ossicular tissue, our data suggests that the fixation procedure affords such a reduction in infectivity that the risk of HIV transmission, even from an HIV-infected donor, is vanishingly low. PMID- 8614200 TI - SPECT gallium scintigraphy in malignant external otitis: initial staging and follow-up. Case reports. AB - Malignant external otitis (MEO) is a recognized entity characterized by a stubborn Pseudomonas external otitis; it has been most frequently observed in elderly diabetic patients. Early diagnosis is necessary for successful treatment but, despite a widespread inflammatory response, routine plain x-ray studies and computed tomography (CT) scanning show no abnormalities in its early stage. In this report, the clinical value of gallium 67 (Ga 67) single photon emission CT (SPECT) is studied in three patients suspected of having MEO, and the results are compared with findings on CT scan and laboratory tests. These results confirm that the high sensitivity of GA 67 SPECT in the initial recognition of MEO provides a more adequate technique than CT scan. Furthermore, Ga 67 scintigraphy appears to be highly accurate for follow-up evaluation of these patients. PMID- 8614201 TI - Late sequelae of cochlear infection. AB - Inflammatory reactions within the inner ear are deleterious to cochlear function and can result in server hearing loss that does not recover. This study investigated a guinea pig model of long-term cytomegalovirus infection. At high doses active inflammation was still present after 35 days. At lower doses some ears showed partial resolution while others were still inflamed. Hearing was totally lost in all cases of persistent inflammation. There was some residual hearing in the cases that had resolved. Cochlear structures including the organ of Corti, stria vascularis, and spiral ganglion were partially degenerated. Fibrotic matrix within scala tympani was ossified in many cases. These changes are consistent with those described for human cochleas following putative viral infections. PMID- 8614202 TI - Structural characterization of persistent tympanic membrane perforations in man. AB - In the present structural study the authors investigated the border of permanent tympanic membrane (TM) perforations in patients selected for myringoplasty. Furthermore, a panel of monoclonal antibody markers that recognize different epitopes within glycosaminoglycans as well as antibodies to epidermal growth factor and fibronectin were applied to the sections. In half of the specimens the epithelial junction ended at the inside of the perforation border, whereas in the other half it was located at the perforation border itself. In the junctional area the keratinocytes were covered by a thick keratin layer which protruded as a spur centripetally in order to bridge the perforation. Epidermal cells formed papillae and contained remnants of keratinocyte nuclei that showed similarities to those of the skin in inflammatory conditions. The connective tissue layer was fibrous and showed areas containing sclerotic plaques. The inner epithelium of the TM had abundant ciliae, thus supporting the concept that cells of the mucosal lining of the TM are able to differentiate in inflammatory conditions into ciliated cells and secretory cells. The immunoreactivity of hyaluronan and other glycosaminoglycans, the immunoreactivity of epidermal growth factor, and immunoreactivity of fibronectin, all of which are known to occur in healing wounds, were only scantily demonstrated; this could be one reason for the arrested healing and a reason why the natural drive to complete a mature closure is abandoned. PMID- 8614203 TI - Development of the 12-item Cross-Cultural Smell Identification Test (CC-SIT). AB - The development of the 12-item Cross-Cultural Smell Identification Test (CC-SIT), based upon items from the University of Pennsylvania Smell Identification Test (UPSIT), is described. In developing this test, the authors initially selected UPSIT items that are familiar to most persons from North American, European, South American, and Asian cultures. The CC-SIT was then administered to 198 people ranging in age from 5 to 96 years, and the the test scores were compared to analogous items from UPSITs administered to 198 age-, sex-, race-, and smoking habit-matched control subjects. Since the pattern of test scores did not differ for the two groups, the authors developed normative data for the 12-item test using equivalent UPSIT items sampled from a database containing UPSIT scores for 3760 subjects. Norms are provided for determining the percentile ranks of a given patient's score as a function of age and gender. The CC-SIT provides, for the first time, a self-administered means for reliably assessing olfactory function in less than 5 minutes. PMID- 8614204 TI - Oral lichen planus and its association with squamous cell carcinoma: an update on pathogenesis and treatment implications. AB - Lichen planus of the oral mucosa (OLP) is characterized by lymphocytic mucositis, basal cell lysis, and lymphocyte transmigration into the epithelial compartment. Some reports have suggested a high incidence of oral squamous cell carcinoma (OSCCA) in OLP patients and have implicated OLP as a premalignant lesion. We describe five cases of OSCCA arising in patients with preexisting OLP. At our institution, the incidence of OSCCA in patients with OLP approximates that reported in other series. The immunopathologic basis for OLP, its potential association with malignancy, and the variable clinical picture of OSCCA in patients with OLP are reviewed. Specific recommendations are given for treatment and follow-up of lesions, including the role of future testing with viral and oncogene markers. PMID- 8614205 TI - The use of sliding genioplasty for treatment of failed chin implants. PMID- 8614206 TI - The M-meatoplasty of the external auditory canal. PMID- 8614207 TI - Safety of ototopical antibiotics. PMID- 8614209 TI - Directory of Otolaryngological Societies. PMID- 8614208 TI - Snare dissection uvulopalatoplasty. PMID- 8614210 TI - John J. Conley, MD. An issue of special recognition. PMID- 8614211 TI - The stylomastoid area: anatomic-histologic study and surgical approach. AB - This three-part study explored the difficult problem of dissecting the facial nerve through the soft tissues of the mastoid tip. The anatomic relationships of the mastoid tip tissues were measured in 20 specimens. At the stylomastoid foramen, the facial nerve lies an average of 9 mm from the digastric muscle, 11 mm from the external canal, and 21 mm from the nerve's parotid bifurcation. The histologic relationships between the nerve sheath and the mastoid tip fascial tissues were examined in 10 specimens. The tight adherence of sheath and fascial fibers prevent their easy separation. By applying this new anatomic and histologic knowledge on cadaver specimens, the author refined a technique for the safe dissection of the facial nerve through mastoid tip tissues. This technique has proven successful in the operating room. PMID- 8614212 TI - Ototoxicity resulting from combined administration of cisplatin and gentamicin. AB - The hypothesis that cisplatin can augment the ototoxicity of gentamicin was tested. Seven groups of 11 guinea pigs each were given a single dose of cisplatin either alone or 14 days before, at the beginning, midway through, or at the end of a course of gentamicin administered daily for 14 days. Blood and perilymph gentamicin and cisplatin concentrations were determined in three of the animals from each group. Auditory damage was determined in the remaining 8 animals electrophysiologically by measuring the compound action potential and alternating current cochlear potential. Hair cell damage was determined using the surface preparation technique. An augmented ototoxic effect occurred when the cisplatin was given early in the 14-day course of gentamicin and did not occur when it was given at the end of treatment. PMID- 8614213 TI - Laparoscopic gastrostomy versus open gastrostomy in head and neck cancer patients. AB - Alimentation in the surgically treated head and neck cancer patient frequently requires bypassing the upper aerodigestive tract. The laparoscopic gastrostomy fulfills this criterion. The authors compared 25 laparoscopic gastrostomies (group 1) with 18 open gastrostomies (group 2) performed on head and neck cancer patients. The length of operation, morbidity, mortality, and cost were evaluated. Operative time was significantly shorter in group 1 (40 +/- 2 minutes) than in group 2 (56 +/- 4 minutes), with P=.003. The major complication rate was 9% for group 1 and 11% for group 2. There was no procedure-related mortality in group 1, but 1 patient died in the immediate postoperative period in group 2. The cost was not significantly different. It is concluded that the laparoscopic gastrostomy is a safe and cost-effective alternative to open gastrostomy in this patient group. PMID- 8614214 TI - Image-guided surgery in a new magnetic resonance suite: preclinical considerations. AB - Surgical procedures require correct identification of exposed anatomy with concomitant localization amidst contiguous structures. In endoscopic procedures the surgeon is provided a real-time endoscopic view and is prepared with radiologic images. Here we present an overview of a methodology of localization using intraoperatively acquired magnetic resonance (MR) images in preparation for magnetic resonance imaging-guided endoscopic sinus surgery. The methodology centers around a unique prototype imaging device and operating environment. An "open" 0.5 Tesla MR unit has been created that allows complete access to the patient's head and neck while concomitant images are obtained. Illustrative examples of localization technique from cadaver experiments are presented, as well as insights into the host of concerns for anesthesia, equipment, surgical instrumentation, communications, and documentation. PMID- 8614215 TI - Erbium laser in middle ear surgery: in vitro and in vivo animal study. AB - Cadaveric human temporal bones, cadaveric rabbits, and live rats were used to demonstrate the utility and safety of the erbium:yttrium-scandium-gallium-garnet (Er:YSGG) laser for otologic applications. The shallow penetration in water of this wave-length and its ability to ablate bony tissue with minimal collateral thermal effects spare underlying and adjacent structures and make it appealing for stapedotomy. The authors were able to satisfactorily perform small fenestra stapedotomy, atticotomy facial nerve decompression, and mastoidectomy. Temperature measurements from the round window area during Er:YSGG stapedotomy recorded an elevation of less than 2 degrees C, which is well within acceptable limits. An acoustic shock produced at the impact site is the major disadvantage and requires further in vivo investigation of the transient and sustained deleterious effects away from the application site. This work supports further investigation into potential applications of the Er:YSGG laser in otology. PMID- 8614217 TI - Identifying obstructive sleep apnea in patients presenting for laser-assisted uvulopalatoplasty. AB - The authors report on a series of 850 patients with snoring who were evaluated for laser-assisted uvulopalatoplasty (LAUP). Stepwise multivariate linear regression was employed to correlate patient symptoms and characteristics to the respiratory disturbance index (RDI). Body mass index, falling asleep while driving, snoring every night, and stopping breathing during sleep were found to correlate strongly with an increasing RDI (variance of 25%). Logistic multivariate linear regression analysis was used to predict the outcome of apnea (RDI>10). This model selected all of the above variables, as well as age, male sex, and the total number of symptoms, as being strong predictors of apnea. A receiver operating characteristic curve was used to describe the ability of this model to predict apnea. The authors conclude that otolaryngologists play an important role in the evaluation of OSAS, especially when a snoring patient may undergo LAUP, and they present an algorithm for the evaluation of such a patient. The authors believe that the clinical assessment, including a thorough history and a complete physical examination, remains extremely important in this evaluation. At present, the authors strongly recommend referral for a PSG if there is any suspicion of OSAS. PMID- 8614216 TI - Galvanic-induced postural movements as a test of vestibular function in humans. AB - Galvanic stimulation produces a postural sway and eye movements in humans. Since galvanic currents are thought to exert their effect at the trigger zone of the vestibular nerve, an intact vestibular nerve should be necessary to produce a response. We have used galvanic stimulation in humans to test the hypothesis that intact vestibular nerve fibers are required to obtain a postural away response. Experimental subjects included normal subjects, patients who had undergone resection of an acoustic neuroma, and patients who had undergone vestibular neurectomy and surgical labyrinthectomy. Our results support the hypothesis that an intact vestibular nerve is necessary to produce a response. Moreover, two patients with recurrent vertigo following vestibular neurectomy and labyrinthectomy, who had absent ice-water caloric test responses in the operated ears, were found to have a positive galvanic response. This result suggested that their recurrent vertigo was based on intact residual vestibular nerve fibers. Although previous research has not yielded a routine clinical use for galvanic stimulation, our results suggest that galvanic stimulation of the vestibular system can provide unique and valuable diagnostic information. PMID- 8614218 TI - Quantitative assessment of the variation within grades of facial paralysis. AB - A completely objective, unambiguous outcome measure of facial function is now available. A new automated computer-assisted clinimetric system combines the crucial detection capabilities of the human observer and the unique capacity of the computer to quantify the image light reflectance difference observed during facial expression. The new system was applied to 27 patients with a variety of diseases affecting the facial nerve. All subjects could be individually and objectively ranked, and disease-specific profiles could be constructed. These tasks are not possible with the House-Brackmann scale, because of the wide variation within grades and the ambiguity between grades. With the automated objective, unambiguous outcome measure, it may be possible to define individual case progression, recovery, and outcome over the course of disease. PMID- 8614219 TI - Intraoperative electrophysiologic monitoring of the recurrent laryngeal nerve. AB - Intraoperative electrophysiologic monitoring of the recurrent laryngeal nerve was performed with a commercially available device consisting of an endotracheal tube with integrated stainless-steel-wire surface EMG electrodes positioned at the level of the true vocal cords. Forty-two recurrent laryngeal nerves were successfully monitored with this system in 31 patients undergoing thyroidectomy or parathyroidectomy. In all cases, evoked EMG responses were elicited by direct electrical recurrent laryngeal nerve stimulation. Stimulus thresholds for evoked responses ranged from 0.2 to 0.6 mA (mean 0.3 mA) for the 37 nerves with preoperative ipsilateral normal vocal cord mobility. Mechanically evoked potentials with acoustic signals were also detected during the surgical procedures related to recurrent laryngeal nerve manipulation. It may be concluded that surface electrode monitoring of the recurrent laryngeal nerve with this system provides a simplified, noninvasive technique that is as sensitive as monitoring with intramuscular laryngeal electrodes. PMID- 8614220 TI - Mucociliary clearance following segmental tracheal reversal. AB - Ciliated tracheal epithelium is arranged in a polarized pattern oriented according to the inferior-superior axis of the trachea and is responsible for the transport of mucus toward the larynx. In this study, ciliary beat orientation and the influence of external factors on mucociliary clearance direction were studied in rabbit inverted cervical tracheas. The animals displayed normal respiration postoperatively. After 16 weeks, airway clearance was studied by observation of the movement of silicone particles placed in the inverted segment and in normal parts of the ciliated epithelium. Cilia exhibited unidirectional and coordinated movement within inverted tracheal segments. As shown by the direction of effective flow produced by beating cilia and by scanning electron microscopy, the cilia in the inverted segment beat in the opposite direction from the cilia in the remainder of the trachea. This study demonstrated that ciliary orientation is irreversibly determined, but the reversal of ciliary beating within the cervical trachea had no adverse effects on the survival of the animals. PMID- 8614221 TI - Epidermal growth factor receptor expression by acoustic neuromas. AB - Antibodies directed against epidermal growth factor receptor (EGFr) impede proliferation and induce differentiation of EGFr-positive cancers. To explore the effectiveness of anti-EGFr monoclonal antibodies on acoustic neuromas (ANs), we first sought to evaluate EGFr expression by ANs. The records of all patients with the diagnosis of AN at our institution from January 1989 to July 1994 were reviewed. Immunohistochemical analysis for EGFr was performed on formalin-fixed, paraffin-embedded archival surgical specimens. Skin, liver, and placenta were used as positive tissue controls. Purified rabbit immunoglobulin G replacing the experimental antibody acted as a negative control, and normal eighth cranial nerve was evaluated for background staining. Slides were scored as 0, +, ++, or and for percentage of positive cells by two pathologists, with Antoni A and Antoni B areas scored separately. Results demonstrate that most tumors are revealed to be EGFr positive with a mild degree of staining. Antoni A areas generally have greater staining than Antoni B regions, while normal eighth cranial nerves demonstrate minimal background staining. These results suggest that ANs express low levels of EGFr, with Antoni A areas having the highest levels. While further studies may more accurately quantitate EGFr levels in these tumors, the clinical efficacy of anti-EGFr-based therapies for ANs seems doubtful. PMID- 8614222 TI - Pathogenesis of middle ear adhesions. AB - Middle ear adhesions are well-known to the ear surgeon, although data on etiology, pathogenesis, and significance are lacking in current literature. This study on experimental acute otitis media presents histopathological data on these aspects. Pneumococci were inoculated in the right middle ear bulla of 25 rats; the left ear served as control. At days 4, 8, 16, 90, and 180, respectively, 5 rats were decapitated, and the bullae were removed, opened, and stained with periodic acid-Schiff (PAS)/alcian blue. The entire middle ear mucosae were dissected from the bone, embedded as whole mounts in colophonium chambers, and examined by light microscopy. Representative parts of the mucosae were sectioned and examined in the same way. All inoculated ears from day 8 and later (20 in total), contained mucosal adhesions of various sizes, shapes, and locations. None were found in control ears. The site of predilection for the development of adhesions was the hypotympanum, followed by the anterior epitympanum, the attic, the drum, the interossicular spaces, and the tubal orifice. Based on present histopathological findings, we conclude that the middle ear adhesion is a pathological phenomenon caused by infection, and we propose a six-stage hypothesis of pathogenesis: 1. Localized epithelial rupture; 2. Prolapse of subepithelial tissue; 3. Epithelialization of the prolapse; resulting in a polypous/fold-like prominence; 4. Growth and elongation of the prominence; 5. Fusion of the end/tip of the prominence with another part of the mucosa; 6. Formation of an adhesion. PMID- 8614223 TI - Epidermal differentiation in the human external auditory meatus. AB - The differentiation of epidermis in the various parts of the human ear canal was documented on the basis of cytokeratin (Ck) expression patterns. Immunohistochemistry was performed on cryostat sections of normal meatal skin using a comprehensive panel of monospecific Ck antibodies representing the main lines of epithelial differentiation. The epidermis of the cartilaginous part showed a Ck profile characteristic of normal skin type differentiation. The deep meatal skin, including the tympanic membrane, showed a peculiar type of differentiation: in addition to epidermal Cks, hyperproliferation-associated Cks 6, 16, and 17 were expressed in the suprabasal cells, while the simple epithelia cell marker Ck 19 was found in the basal cells. The presence of hyperproliferative Cks in the deep meatal skin could only partly be related to areas of proliferative activity. Keratinocytes, which express markers of hyperproliferation, are migratory. Therefore, their presence in the meatal skin is likely to be related to the peculiar pattern of keratinocyte migration, the purpose of which is to keep the meatus free from desquamation products. PMID- 8614224 TI - A positional maneuver for treatment of horizontal-canal benign positional vertigo. AB - Horizontal-canal benign positional vertigo (HC-BPV) is characterized by brief attacks of intense vertigo that are induced by mainly rolling over in bed. Examination shows a burst of purely horizontal nystagmus beating toward the undermost ear when the head is turned from supine to either lateral position. Two patients with typical HC-BPV were treated by a new positional procedure that aims to clear particles from the affected canal. The maneuver starts with the patient in the supine position and consists of three 90-degree head rotations toward the unaffected ear. Both patients had immediate and sustained relief of their attacks. No positional nystagmus could be elicited after the maneuver. The rapid cessation of positional vertigo and nystagmus adds evidence that HC-BPV is caused by dense particles that move within the canal whenever its orientation toward gravity is changed. PMID- 8614225 TI - Hypericin: a new laser phototargeting agent for human cancer cells. AB - Laser activation of anthracycline-related drugs combines chemotherapy with photoablation for improved treatment. Hypericin, a structurally related anthraquinone, was tested for laser activation and cytotoxicity in human cancer cells. Viability of P3 squamous cell carcinoma cells incubated with 1 to 20 microgram/mL hypericin was reduced by more than 95% after 1 minute exposure at 4 degrees C to an argon laser (514 nm, 5 W), a KTP-532 laser (532 nm, 5 W), or a 20 A xenon lamp. Viability was reduced over 90% in six human carcinoma, sarcoma, and melanoma cell lines by this combined treatment, but only trace toxicity was seen after separate exposure to hypericin or light alone. These results show that hypericin is a sensitive agent for phototherapy of human cancer cells in vitro and indicate that this drug may be useful for tumor targeting via minimally invasive imaging-guided laser fiber optics. PMID- 8614226 TI - Laryngeal muscle activity during speech breaks in adductor spasmodic dysphonia. AB - To determine the laryngeal muscle activation abnormalities that are associated with speech symptoms in adductor spasmodic dysphonia (ADSD), electromyographic measures of extrinsic and intrinsic laryngeal muscles during speech compared 1) muscle activity when ADSD patients had breaks in words with when they produced the same words without breaks; and 2) muscle activity in ADSD patients during speech without voice breaks with normal control producing phonetically similar words. Simultaneous electromyographic recordings were made from the thyroarytenoid (TA), cricothyroid (CT), sternothyroid (ST), thyrohyoid (TH) and the posterior cricoarytenoid (PCA) muscles during speech testing in 11 ADSD patients and 10 control subjects. Speech breaks were identified and mean muscle activity measured starting 100 ms preceding a voice break and for the remainder of the word. Mean muscle activity level was significantly greater on break than non break words in ADSD patients only for the thyroarytenoid muscle (p<.001). No significant differences were found between the ADSD and control subjects during non break words for any of the laryngeal muscles studied. The results demonstrated that 1) only the thyroarytenoid, of the muscles tested, was affected in ADSD, 2) that muscle activation abnormalities were spasmodic, only appearing when symptoms occurred and 3) no imbalances of muscle tone were evident when speech disruptions did not appear. PMID- 8614227 TI - Primary closure of pharyngeal remnant after total laryngectomy and partial pharyngectomy: how much residual mucosa is sufficient? AB - After total laryngectomy with or without partial pharyngectomy, the remaining pharyngeal defect can be repaired either by primary closure or with additional tissue, depending on the amount of pharyngeal tissue remnant available. The aim of this study was to determine the minimum width of the pharyngeal remnant that could be safely closed primarily without causing difficulty in swallowing. A total of 52 consecutive patients who underwent total laryngectomy were entered into the study. The relaxed and stretched widths of the pharyngeal remnant were measured after removal of the specimen. The widths of the pharyngeal mucosa ranged from 1.5 to 5.0 cm relaxed (mean, 3.24 cm) and from 2.5 to 8.0 cm stretched (mean, 4.83 cm). All neopharynx was reconstructed by closing the pharynx primarily. Seven of the 52 patients developed recurrent tumor with concomitant dysphagia. Two of the 45 patients without recurrence presented with acute dysphagia from food bolus obstruction, and 1 patient developed benign inflammatory stricture following an episode of fish-bone impaction. The narrowest widths of the pharyngeal remnant in this group of 45 were 1.5 cm relaxed and 2.5 cm stretched. As these patients do not have swallowing difficulty, we conclude that in the absence of tumor recurrence, this amount of residual pharyngeal tissue is sufficient both for primary closure of the pharynx and in restoring swallowing function. PMID- 8614228 TI - Supracricoid partial laryngectomy after failed laryngeal radiation therapy. AB - Twelve patients managed with supracricoid partial laryngectomies (SCPLs) after failed laryngeal radiation therapy (RT) were evaluated. None of the recurrent tumors were amenable to vertical or horizontal partial laryngectomy. Results were analyzed for tracheostomy decannulation, oral alimentation, morbidity, local control, and survival. Major complications included perichondritis (2 patients), laryngeal stenosis (2 patients), and pneumonia from aspiration (1 patient). Mucocutaneous fistula and cricoarytenoid joint ankylosis were not encountered. Margins of resection were uninvolved in all cases. The Kaplan-Meier 3-year actuarial survival and local control estimate was 83.3%. Salvage total laryngectomy allowed for an overall 100% local control rate and a 75% laryngeal preservation rate. This preliminary report suggests that, in patients with failed laryngeal RT not amenable to vertical or horizontal partial laryngectomy, the SCPL procedures should be discussed before advocating salvage total laryngectomy. Further series analyzing the role and limitations of the various SCPL procedures after failed laryngeal RT are warranted. PMID- 8614229 TI - A new method of applying external laryngeal pressure during microlaryngeal surgery: the laryngeal strap. PMID- 8614230 TI - Transnasal endoscopic repair of cerebrospinal fluid rhinorrhea: an interposition technique. PMID- 8614231 TI - Mandibular body bone in facial plastic and reconstructive surgery. PMID- 8614232 TI - The "two-hole" ossiculoplasty technique. PMID- 8614233 TI - The necrosed incus in stapedectomy revision. PMID- 8614234 TI - Directory of otolaryngological societies. PMID- 8614235 TI - Significant differences in the activity of midbrain dopamine neurons between male Fischer 344 (F344) and Lewis rats: an in vivo electrophysiological study. AB - There is a significant difference between the Lewis and Fischer 344 rats regarding their propensity to self-administer drugs of abuse. This study compares the number and the firing pattern of spontaneously active dopamine (DA) neurons in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) in these two strains of rats. This was accomplished using the technique of in vivo extracellular recording. The number of spontaneously active DA neurons in the SNC and VTA of the Lewis rat were significantly lower compared to that of the F344 rats. Furthermore, a greater percentage of SNC and VTA DA neurons in the Lewis rats exhibited a burst firing pattern compared to F344 rats. These findings indicate that F344 and Lewis rats possess differences in mesolimbic DA neuronal activity and this may be a factor in explaining the different propensities between the two strains to self-administer drugs. PMID- 8614236 TI - Different atypical beta-adrenoceptors mediate isoprenaline-induced relaxation in vascular and non-vascular smooth muscles. AB - Atypical beta-adrenoceptors mediating smooth muscle relaxation were compared in several rat tissues including the distal colon, fundic strip, thoracic aorta and common carotid artery. Isoprenaline, CGP 12177 and BRL 37344 concentration dependently relaxed longitudinal strips of the distal colon and fundus precontracted with carbachol (10(-6)M) as well as ring segments of the aorta and carotid artery precontracted with noradrenaline (10(-7)M). The rank order of potency was isoprenaline = BRL 37344 > CGP 12177 in the distal colon, isoprenaline = CGP 12177 > BRL 37344 in the aorta and carotid artery segments and isoprenaline > BRL 37344 > CGP 12177 in the fundic strip. Pretreatment with BRL 37344 induced a marked desensitization of the distal colon and fundic strips but not the aorta and carotid artery to isoprenaline. In the fundus and distal colon, pretreatment with CGP 12177 (10(-4)M abolished the effect of isoprenaline. Cyanopindolol (10(-6)M) shifted the isoprenaline curve to the right, without reducing the maximum response, in the distal colon fundic strip. -logKB values were 7.44 +/- 0.08 and 7.53 +/- 0.10 in the distal colon and fundic strip respectively. The same concentration of cyanopindolol did not inhibit the relaxant effect of isoprenaline in the aorta and carotid artery segments. It was therefore concluded that atypical beta-adrenoceptors in these preparations were not identical, indicating heterogeneity of atypical beta-adrenoceptors. PMID- 8614237 TI - A possible role for nitric oxide but not for prostaglandin E2 in basal and interleukin-1-beta-induced PRL release in vitro. AB - In previous experiments we have shown that nitric oxide (NO) was able to modulate CRH and ACTH release from cultured rat hypothalamic and anterior pituitary cells, in vitro. Now, we show experimental evidence of an involvement of NO in basal and interleukin-1 beta-induced prolactin (PRL) release. L-NG-nitro-arginine, an inhibitor of nitric oxide synthetase, and hemoglobin, a NO scavenger, impaired basal and interleukin-1-beta-induced PRL release, while molsidomine, a NO donor, was able to release PRL and to amplify interleukin-1-beta-induced PRL release, confirming a modulatory role for nitric oxide in pituitary hormone secretion. On the other hand, no evidence regarding a possible role of prostaglandin E2 (PGE2) in IL-1beta-induced PRL release came out from our experiments. PMID- 8614238 TI - Buprenorphine exerts its antinociceptive activity via mu 1-opioid receptors. AB - The mechanism of the antinociceptive effect of buprenorphine was assessed by administering selective mu-, mu1--, delta- and kappa-opioid receptor antagonists in mice. Intraperitoneal administration of buprenorphine, at doses of 0.3 to 3 mg/kg, produced dose-dependent antinociception in the tail-flick test. The antinociceptive activity of buprenorphine did not result from the activation of kappa- or delta-opioid receptors, since treatment with either nor binaltorphimine, a selective kappa-opioid receptor antagonist, or naltrindole, a selective delta-opioid receptor antagonist, was completely ineffective in blocking buprenorphine-induced antinociception. However, the antinociceptive effect of buprenorphine was significantly antagonized by beta-funaltrexamine, a selective mu-opioid receptor antagonist. Moreover, selective mu1-opioid receptor antagonist, naloxonazine and naltrexonazine, also significantly antagonized the antinociceptive effect of buprenorphine. Co-administration of kappa- and delta opioid receptor antagonist with the mu-opioid receptor antagonists had no significant effect on the antagonistic profiles of the mu-opioid receptor antagonists on the antinociceptive effect of buprenorphine. These results suggest that buprenorphine acts selectively at mu1-opioid receptors to induce antinociceptive effects in mice. PMID- 8614239 TI - MCI-154 activates the Ca(2+)-activated K+ channel of vascular smooth muscle cells. AB - The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca2+-activated K+ channel (KCa channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 microM MCI-154 activated the KCa channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100 microM MCI-154 to the cytosolic side activated the KCa channel directly, suggesting that the Ca2+ sensitivity of the KCa channel itself is modulated. Though extracellular application of 100 microM amrinone, a phosphodiesterase inhibitor, activated the KCa channel in the cell-attached patch configurations, application of 100 microM amrinone to the cytosolic side could not activate the KCa channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca2+ sensitivity of the KCa channel in vascular smooth muscle cells. PMID- 8614240 TI - Histogranin, a modified histone H4 fragment endowed with N-methyl-D-aspartate antagonist and immunostimulatory activities. AB - Histogranin is a naturally-occurring pentadecapeptide with a structure 80% homologous with that a fragment-(86-100) of histone H4. First isolated from bovine adrenal medulla, the peptide was also shown to be present in the pituitary, brain, adrenal glands, blood plasma, lungs and spleen. At the subcellular level, histogranin is concentrated in secretory vesicles and it is released from perfused bovine adrenal glands 15-35 min after stimulation with carbamylcholine as opposed to catecholamines and [Leu5]enkephalin which are released immediately after stimulation. Rat brain membranes possess specific binding sites for [125I][Ser1]histogranin with characteristics of a receptor, namely high affinity, saturability, reversibility and sensitivity to heat and proteolytic enzyme treatments. Intracerebroventricular injections of synthetic histogranin (10-100 nmol) in mice protect them against N-methyl-D-aspartate (NMDA)-induced convulsions without affecting convulsions induced by (R,S)-alpha amino-3-hydroxy -5-methyl-4-isoxazole-propionate (AMPA), kainate and bicuculline. The peptide also binds to specific sites on human peripheral blood mononuclear cells and it evokes the release of tumor necrosis factor-alpha (TNF), interleukin 1 (IL-1) and interleukin-6 (IL-6) from isolated rat macrophages in culture. Since the structure of histone H4 is considered as one of the most conservative, it is presumed that histogranin possesses its own precursor and that its gene is distinctly expressed. PMID- 8614241 TI - Short-term exposure to lipopolysaccharide is associated with microvascular contractile dysfunction in vivo. AB - The purpose of this study was to determine whether short-term exposure of resistance arterioles to lipopolysaccharide in situ is associated with changes in vasomotor tone. Using intravital microscopy, we found that suffusion of Escherichia coli lipopolysaccharide (3 micrograms/ml) over hamster cheek pouch arterioles for 1 h was associated with a significant immediate biphasic response: vasoconstriction followed by vasodilation (p < 0.05). The former was attenuated by indomethacin, and the latter by SK&F 108566, a selective, non-peptide angiotension II receptor antagonist (p < 0.05). The nitric oxide synthase inhibitor, NG-L-nitro arginine, had no significant effects on lipopolysaccharide induced responses. Allopurinol, a scavenger of reactive oxygen species, significantly attenuated lipopolysaccharide-induced vasodilation. Acetylcholine- and nitroglycerin-induced vasodilation were significantly potentiated after lipopolysaccharide. These responses were recorded in the absence of any significant changes in systemic arterial blood pressure. Collectively, these data suggest that short-term exposure of the peripheral microcirculation to lipopolysaccharide in situ is associated with an ischemia-reperfusion-like injury. These changes may contribute to end organ failure observed several hours after exposure to lipopolysaccharide. PMID- 8614242 TI - Endothelin analogs which distinguish vasoconstrictor and vasodilator ETB receptors. AB - [Pen 1,11, Nle7, Glu9, Ala18]-Sarafotoxin S6b (BMS-184696) and [Pen1,11, Nle7, Glu9, Leu18]-sarafotoxin S6b (BMS-184697) were synthesized with the aim of preparing ETB receptor antagonists. BMS-184696 was a potent ETA antagonist, an extremely potent vasoconstrictor ETB agonist, and a non-competitive vasodilator ETB antagonist with no agonist activity. BMS-184697 was a potent ETA antagonist, a potent vasoconstrictor ETB agonist, and a vasodilator ETB agonist with moderate potency. The ability of BMS-184696 to activate the vasoconstrictor ETB receptor but not the vasodilator ETB receptor, despite having high affinity binding to the vasodilator ETB receptor as evidenced by its antagonist activity, strongly suggests the existence of ETB receptor subtypes. PMID- 8614243 TI - A rapid, high capacity assay for basic fibroblast growth factor binding. AB - A rapid, high capacity assay for the binding of basic fibroblast growth factor has been developed. Rat lung tissue was selected as the optimum source of membranes and 2M sodium chloride used to remove endogenous growth factor. The assay has been adapted to the Millipore Multi-Screen system so that it can be run in 96-well format with a volume of 300 microliters. The assay has been validated through the demonstration of inhibition by standard inhibitors such as suramin and protamine sulfate. The assay has proven useful for the screening of random compounds as well as the more detailed examination of suspected inhibitors. By running compounds in the presence and absence of a mid-range concentration of unlabeled bFGF, an estimate of the proportion of the inhibition due to high affinity binding can be obtained. Suramin and protamine sulfate show no selectivity and inhibit high affinity binding and overall binding with similar potencies. Another inhibitor, dimercaptothiadiazole, is more potent against high affinity binding. PMID- 8614244 TI - Direct effects of fentanyl on canine coronary artery rings. AB - The potent opioid fentanyl, is commonly used as a general anesthetic for coronary artery bypass surgery. Experiments were designed to determine the direct effects of fentanyl on unstimulated coronary artery tissue. Isolated, endothelium denuded canine epicardial rings were suspended in physiologic tissue baths. Changes in tension were measured as the concentration of fentanyl was increased. Fentanyl caused increases in ring tension at concentrations of 10(-6)M-10(-4)M, then caused a decrease in tension at 10(-3) M. Calcium channel blockade by 10(-7)M nifedipine abolished all increases in contractile responses to fentanyl and prevented the relaxation in tension produced by fentanyl. The fentanyl dose response curve was unchanged by opioid receptor blockade with 10(-6)M naloxone and by alpha and beta adrenoceptor blockade produced by 10(-6)M prazosin and 10( 6)M propranolol. Muscarinic blockade with 10(-6)M atropine and cyclooxygenase inhibition by 10(-6)M indomethacin attenuated the constrictor response to fentanyl. The opioids alfentanil, sufentanil, morphine, and naloxone all produced a dose-response similar to fentanyl that varied only in amplitude. These findings indicate that increasing concentration of the anesthetic opioid fentanyl can cause biphasic changes in basal canine epicardial coronary artery ring tension. These responses are calcium dependent and may be characteristics of other opioid agonists and antagonists. PMID- 8614245 TI - The effects of sigma ligands on protein release from lacrimal acinar cells: a potential agonist/antagonist assay. AB - Sigma receptor antagonists have been proposed as leading clinical candidates for use in various psychotic disorders. Prior to clinical testing, it is imperative that a new agent be correctly identified as an antagonist and not an agonist since the latter may worsen the psychosis. For sigma-ligands many behavioral and pharmacological assays have been developed in an attempt to classify agonist/antagonist activity. These assays evaluate a response or a behavior in an animal model that can be related to clinical efficacy. However, is the action by the presumed antagonist a consequence of sigma-receptor activity? Previously we have identified sigma-receptors in acinar cells of the main lacrimal gland of the New Zealand white rabbit and have measured protein release after the addition of various N,N-disubstituted phenylalkylamine derivatives known to be sigma-ligands by receptor binding studies. Although protein release from acinar cells has been attributed to either muscarinic or alpha-adrenergic stimulation, protein release from sigma-receptor stimulation was also confirmed. In the reported studies here, we isolated and incubated acinar cells with varying concentrations of known sigma ligands and measured protein concentration. A knowledge of the receptor profile for the disubstituted phenylalkylamines permitted experiments to be designed in which various alpha, muscarinic, serotonergic, and dopaminergic antagonists could be added in equimolar concentrations. Under the conditions of these experiments, statistically significant increases in protein release for sigma-ligands could be attributed to stimulation of sigma-receptors. Haloperidol, an apparent sigma antagonist, caused a statistically significant decrease in protein release and also inhibited protein release when tested with a known sigma-ligand, AF2975 [N,N dimethyl-2-phenylethylamine]. In this system, stimulation and inhibition of protein release were defined as agonist and antagonist behavior, respectively. Of particular interest were the results for BMY14802 and +/- pentazocine, both of which were found to be agonists. Various antipsychotic and antidepressant drugs were measured for their agonist/antagonist behavior. Because of multireceptors present in acini, their agonist or antagonist behaviour could not be attributed solely to interaction with the sigma-receptor unless specific antagonists were added. PMID- 8614247 TI - Serotonin 5-HT2 receptor availability in chronic cocaine abusers. AB - Serotonin 5-HT2 receptor availability was evaluated in chronic cocaine abusers (n = 19) using positron emission tomography and F-18 N-methylspiperone and was compared to control subjects (n =19). 5-HT2 Receptor availability was measured in frontal, occipital, cingulate and orbitofrontal cortices using the ratio of the distribution volume in the region of interest to that in the cerebellum which is a function of Bmax/Kd. 5-HT2 Receptor availability was significantly higher in cingulate and orbitofrontal cortices than in other frontal regions or occipital cortex. The values were not different in normal subjects and cocaine abusers. These results did not show any changes in 5-HT2 receptor availability in cocaine abusers as compared to the control subjects. PMID- 8614246 TI - The role of p56lck in CD4-mediated suppression of CD3-induced early signaling events in T lymphocytes. AB - Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3xCD4 co crosslinking. By comparison with CD3 crosslinking alone, CD3xCD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, P120 was only minor phosphorylated on tyrosine upon receptor crosslinking in A2.01/CD4(-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3xCD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p120 phosphorylation. These results suggest that p120 is a p56lck substrate playing an important role during T-cell activation. PMID- 8614248 TI - Metabolism of capsaicinoids: evidence for aliphatic hydroxylation and its pharmacological implications. AB - A new metabolic oxidation pathway of capsaicin (N-[(4-hydroxy-3-methoxyphenyl) methyl]-8-methyl-(E)-6 -nonenamide), a major pungent and pharmacologically active principle of hot peppers, was investigated. Incubation of capsaicin with phenobarbital-induced rat liver postmitochondrial supernatant enriched with NADPH generating system produced N-(4,5-dihydroxy-3-methoxybenzyl)-(E)-6 -nonenylamide and a more polar metabolite. The latter metabolite was spectrophotometrically and chromatographically identical to authentic omega-hydroxycapsaicin. This new metabolite was also detected in the urine of rabbits given capsaicin by gastric intubation. Other analogs of capsaicin, such as dihydrocapsaicin and nonivamide, also formed similar metabolites via aliphatic hydroxylation. When tested for antinociceptive activity as well as pungency, the above polar metabolites were found to be inactive while their parent compounds exhibited strong sensory effects. Capsaicin interacted irreversibly with heptic drug metabolizing enzymes, thereby inhibiting their activity as indicated by prolongation of pentobarbital sleeping time in rats. Such inhibition of drug metabolism was not observed with omega-hydroxycapsaicin. These findings suggest that metabolism of capsaicinoids via hydroxylation of their side chains plays an important role in the detoxification of these pharmacologically active substances. PMID- 8614250 TI - Proconflict effect of carbon dioxide inhalation in rats. AB - The effect of brief inhalation of carbon dioxide (CO2) was studied in a conflict situation (Vogel test) in the rat. This treatment, which inhibits gamma aminobutyric acid (GABA)-mediated transmission in rat brain and induces anxiety and panic attacks in humans, elicited a proconflict effect. Exposure of rats for 1 min to CO2 decreased by approximately 40% the number of licking periods in the test. This effect was abolished by prior administration of alprazolam (0.5 mg per kilogram of body mass, i.p.). Although these results may support a role for GABA mediated transmission in the anxiogenic effect of CO2 inhalation, the possibility that different neurotransmitters other than GABA are involved in the action of CO2 can not be ruled out. PMID- 8614249 TI - Low concentrations of nonsteroidal anti-inflammatory drugs affect cell functions. AB - The effect of 10(-14)-10(-4)M ibuprofen and aspirin both on arachidonic acid metabolism in peritoneal murine macrophages and on the concanavalin A-induced proliferation of murine splenocytes were investigated. It was shown that 10(-7) 10(-4)M ibuprofen inhibits the arachidonic acid metabolism. On the other hand, 10(-12)-10(-11)M ibuprofen causes pronounced activation of arachidonic acid metabolism. The low concentration (10(-14)-10(-10)M) effects also take place when non-steroidal anti-inflammatory drugs influence other functions of the immune system: that is, they activate the splenocyte mitogen-induced proliferative response. These results are in accord with our suggestion that the low concentration effects of these drugs do not depend upon cell types and may have an important physiological significance. PMID- 8614251 TI - Silicone breast implants: immunotoxic and epidemiologic issues. AB - Silicone gel implants for breast augmentation and reconstruction have been in use since 1962. Significant local complications include capsular contracture, rupture, gel "bleed", and spread of the implant material to regional lymph nodes (1-7) as well as histologic findings of foreign body granulomas in the capsular tissue and in lymph nodes (7-9). Through magnetic resonance spectroscopy and atomic emission spectroscopy, silicon compounds were found in the blood of some women with silicone breast implants; silicone and silica have also been found in liver (10). Well-publicized case reports have raised significant concerns regarding an association between implants and systemic disease. However, despite the availability of silicone implants for over 30 years, controlled epidemiological studies were not carried out until 1992. Currently available epidemiologic data are extremely limited. In part, because the majority of implants were used after 1981, the incidence of long-term problems is not yet known. In 1992, due to the unavailability of studies demonstrating the safety of implants, the U.S. Food and Drug Administration advised that silicone breast implants should be used only in reconstructive surgery and as part of clinical trials (11). This decision spurred a wave of research on the bioreactivity of silicone and clinical observations of patients with implants. Herein, we review the adverse immune effects following contact with silicone as well as the epidemiologic data available. PMID- 8614253 TI - The nitric oxide synthase inhibitor NW-nitro-L-arginine methylester attenuates brain catalase activity in vitro. AB - Nitric oxide has been implicated in mediating the neurotoxic effects of ischemia in the brain. However, studies of the effects of nitric oxide inhibition with nitric oxide synthase inhibitors have provided controversial results. One of the reasons for the controversy may be related to the specificity of the nitric oxide synthase inhibitors, such as Nw-nitro-L-arginine methylester (L-NAME), which has recently been questioned. The present work investigated the possible interaction of L-NAME with the enzyme catalase in vitro. Catalase is an iron containing enzyme which could potentially interact with the iron-binding groups of L-NAME. Since the normal function of catalase in the brain is to remove excess hydrogen peroxide, the inhibition of this process could have potentially toxic effects. L NAME was found to attenuate the catalase inhibiting effects of the known catalase inhibitor cyanamide in vitro, suggesting a competition between cyanamide and L NAME for catalase. In addition, L-NAME by itself attenuated catalase activity in vitro. These results indicate that in addition to inhibiting nitric oxide synthase, L-NAME may have effects on catalase activity. PMID- 8614252 TI - Different neurotransmitter systems are involved in the development of esophageal achalasia. AB - Clinical and pharmacological evidence suggests that several neurotransmitters are involved in the control of the esophageal motility; in fact, besides the well known cholinergic and sympathetic innervation, Vasoactive Intestinal Polypeptide (VIP)-containing fibers as well as dopamine (DA)-containing nerve endings have been identified within the esophageal wall. Lower Esophageal Sphincter (LES) achalasia is a neuromuscular disorder characterized by the absence of peristalsis in the body of the esophagus and by the failure of the LES to relax in response to swallowing. Stimulation of both VIP receptors and D-2 DA receptors induce a decrease in LES pressure, while D-1 receptors mediates LES contractions. In the present study we show that both VIP and DA system is disregulated in LES achalasia. In particular, this disease is associated not only with the lack of VIP nerves in the LES, but also with a failure in the responsiveness of postsynaptic receptors to VIP stimulation. Furthermore, we demonstrate a selective functional loss of the D-2 DA receptor component, without changes in the D-1 DA receptor mediated responses. PMID- 8614254 TI - Role of glutamate in regulating hypothalamic proglucagon-derived peptide secretion in vitro. AB - We have previously demonstrated expression of the proglucagon gene and synthesis and secretion of the proglucagon-derived peptides (PGDPs) in the fetal rat hypothalamus. The excitatory amino acid glutamate has been found to be a key regulator of hypothalamic neuroendocrine hormone secretion. Therefore, the effects of glutamate on hypothalamic PGDP secretion and synthesis were examined in the present study, using the hypothalamic culture model. Glutamate (10 microM) significantly stimulated PGDP secretion (P < 0.01), but had no effect on the total PGDP content of the cultures over 24 hr of incubation. Similarly, the metabotropic/ionotropic glutamate receptor agonist, quisqualic acid (10 microM) stimulated PGDP secretion only ( P < 0.05). In contrast, the ionotropic glutamate receptor agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 microM) and antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), had no effects on either secretion or content of the hypothalamic PGDPs. These findings suggest that excitatory amino acids, and glutamate in particular, regulate secretion but not synthesis of the PGDPs in the hypothalamus. PMID- 8614256 TI - Is hypothalamic prostaglandin E2 involved in avian fever? AB - In chickens, the effect of Escherichia coli lipopolysaccharide (LPS) on body temperature and ex vivo hypothalamic prostaglandin E2 (PGE2) production was examined to test the possible involvement of PGE2 in mechanisms of avian fever. PGE2 is reported to be the major central mediator of fever in mammals; it has not been examined in birds. An intraperitoneal injection of LPS caused an elevation of body temperature but not an elevation of hypothalamic PGE2 production. It seems that: (a) hypothalamic PGE2 is not involved in the development of the febrile response in birds; (b) central mechanisms of avian fever differ from those in mammals. PMID- 8614255 TI - Tissue culture supernatants from human islets of Langerhans activate the oxidative burst response of human monocytes in vitro. AB - Macrophages play a major role in the pathogenesis of insulin-dependent diabetes mellitus in animals. These cells are the first to invade the pancreas and macrophage-eradicating treatments reduce the incidence of the disease. In humans, however, their role is less clear. In this study we investigated the hypothesis that the pancreatic environment per se could activate macrophages. Tissue culture supernatants from human islets of Langerhans were tested for chemotactic activity and oxidative burst response in monocytes isolated from healthy adults. Preincubation with the supernatants enhanced the oxidative burst response evoked by fMLP (up to 379%) and opsonized zymosan (up to 173%). The activity decreased by dilution and was no longer detectable at 1:16. No increased activity was seen in supernatants from a number of other human endocrine and non-endocrine primary cells, suggesting a factor specific for islet tissue. The increased oxidative burst response could partially be eliminated by heat- and proteinase K treatment, suggesting that the activity could be of polypeptide nature. The factor could not be absorbed by polyvalent rabbit antibodies directed towards a variety of cytokines not by a mixture of high-titer anti-cytokine antibodies. It is possible that islet factors could also promote such monocyte activation in vivo in monocytes attracted to the islets of Langerhans by other means. This could contribute to the development of insulin-dependent diabetes in humans. PMID- 8614257 TI - Intracerebroventricular growth-hormone-releasing peptide-6 stimulates eating without affecting plasma growth hormone responses in rats. AB - The purpose of this study was to determine the effect of intracerebroventricular (i.c.v.) injections of a synthetic, opioid-related hexapeptide, growth-hormone releasing peptide-6 (GHRP-6), on stimulation of eating by rats and to correlate this aspect of feeding behavior with the peripheral plasma growth hormone (GH) response to the administered peptide. GHRP-6 dissolved in 5 microL of saline was injected into the lateral ventricles of sated, adult, male, Sprague-Dawley rats in doses from 0 pmol (saline only) to 1000 pmol. For 1 hour after injection, the occurrence of eating was noted, and specimens of arterial blood were collected at 0, 15, 30, and 60 minutes. The plasma was assayed for GH. A nearly linear, statistically significant (p < 0.01) dose-response relationship between the dose of GHRP-6 and the incidence of eating was noted. The mean change from baseline of plasma GH during the 60 minutes after injection was not dose-related (p > 0.2, p > 0.1, and p > 0.1 at 15, 30, and 60 minutes, respectively). We conclude that GHRP-6 given intracerebroventricularly to sated, adult, male, Sprague-Dawley rats stimulates eating and suggest that it does so by some mechanism that is independent of its GH-releasing property. PMID- 8614258 TI - Neurotensin stimulates neutrophil adherence to bronchial epithelial cells in vitro. AB - Neutrophils and neuropeptides have both been implicated in airway inflammation. We hypothesized that neurotensin, a neuropeptide found in the airways, would stimulate neutrophil adherence to bronchial epithelial cells. Adherence was assessed using 51Cr-labelled human neutrophils added to confluent monolayers of bovine bronchial epithelial cells. Neurotensin added to bronchial epithelial cells produced a time-and concentration-dependent increase in adherence which was maximal at 4 h and 10(-10)M (17.6 +/- 1.4% vs. 6.1 +/- 0.4%, p < 0.01). Conversely, neurotensin stimulation of neutrophils induced a concentration dependent and rapid ( < 5 min) increase in adherence which was also maximal at 10(-10)M (27.1 +/- 1.9% vs. 10.1 +/- 1.4%, p < 0.01). The effects were reproduced by the carboxy portion of the molecule. Anti-CD11a, -CD11b or -ICAM-1 antibodies significantly decreased the neurotensin-induced increase in adherence. These data suggest an important role for neurotensin in modulating airway inflammation. PMID- 8614259 TI - Augmentation of dietary fat preference by chronic, but not acute, hypercorticosteronemia. AB - Numerous studies have documented a role for corticosterone in appetitive behavior, including caloric intake and dietary fat preference. In the present study, we have examined the mechanism(s) underlying modulation of dietary fat preference by corticosterone. The results of these studies show a) an increased fat preference with increased basal urinary output, or decreased stimulation of corticosterone output on fasting, b) elevation of fat preference following chronic, but not acute, hypercorticosteronemia produced by exogenous corticosterone administration, and c) emergence of hypercorticosteronemia prior to the development of increased fat preference in developing rats. These observations have led us to suggest that increased fat preference after chronic hypercorticosteronemia may be secondary to changes in the levels or actions of agents known to affect fat intake. PMID- 8614260 TI - Stimulation of the secretion of dehydroepiandrosterone by melatonin in mouse adrenals in vitro. AB - Adrenals of young adult male mice kept on a LD 12:12 lighting regimen for three weeks prior to study and harvested at four different circadian stages were incubated for 2 hours with 0.4 IU synthetic ACTH in 2 ml Krebs-Ringer buffer (KR), or with 50, 150, and 450 microM of melatonin in KR containing 0.4 IU ACTH. The addition of melatonin to ACTH leads to a dose dependent stimulation of production and/or secretion of DHEA into the incubation medium irrespective of the circadian stage of harvesting of the adrenals. This relationship is of interest in view of the simultaneous decrease of dehydroepiandrosterone and melatonin in the course of aging, and the effects of these compounds upon aging related changes. PMID- 8614261 TI - Ascorbate administration to normal and cholesterol-fed rats inhibits in vitro TBARS formation in serum and liver homogenates. AB - We have recently shown that ascorbate has a hypocholesterolemic and hypotriglyceridemic effect on rats fed a diet enriched with 1.5% cholesterol and 25% hydrogenated coconut oil (Nath diet). In this study we evaluated the effect of intraperitoneal ascorbate administration on susceptibility to lipoperoxidation either in rats fed standard or Nath diet. In normal rats ascorbate treatment decreased (p<0.05) the susceptibility to lipoperoxidation induced by incubation of serum for 24 hours with 2.2 mM Cu++, without altering the normal serum fatty acid profile. In rats fed Nath diet we observed a reduced susceptibility of serum to CU++-induced lipoperoxidation (36%), according with their low levels of serum unsaturated fatty acids (40% less than rats fed standard diet). In these animals ascorbate administration affects serum fatty acid profile leading to a decrease of S/U ratio from 1.6 to 1.2 without significantly modifying the susceptibility of serum to lipoperoxidation. Moreover, the production of spontaneous lipid peroxides in liver homogenates, measured as TBARS levels, was strongly inhibited by ascorbate (p<0.01) in rats fed either standard or Nath diet. These data indicate that ascorbate administration exerts an antioxidant effect and that in hypercholesterolemic rats, in addition to a lipid lowering effect, ascorbate exerts a protective role against the peroxidative damage of lipids. PMID- 8614262 TI - The developmental increase of the AT1A, but not the AT1B, receptor mRNA level at the preoptic area in spontaneously hypertensive rats. AB - To determine the possible involvement of the central angiotensin system in hypertension, the angiotensin II type-1 receptor subtype mRNA levels in the preoptic area (POA) and hypothalamus were measured by means of a reverse transcriptase/polymerase chain reaction in spontaneously hypertensive rats (SHR), and the results were then compared with the findings in age-matched normotensive Wistar-Kyoto rats (WKY). In 4-week-old (prehypertensive stage) and 7-week-old (evolving stage) SHR, the AT1A and AT1B receptor subtype mRNA levels in the POA and hypothalamus did not show any significant difference between the SHR and WKY. However, in the 16-week-old SHR (hypertensive stage), AT1A receptor subtype mRNA at POA was approximately 2-fold higher than in the WKY, while the AT1B receptor subtype mRNA showed no difference. On the other hand, neither the AT1A nor the AT1B mRNA level at the hypothalamus were different between the 16-week-old SHR and WKY. These results suggest that the AT1A mRNA level, but not the AT1B mRNA level, increases in the POA in hypertensive stage of SHR and the increase may therefore be related in some way to the state of hypertension. PMID- 8614263 TI - Effects of streptozotocin-induced diabetes and insulin treatment on substance P of the rat arterial wall. AB - Substance P (SP) is present in perivascular nerves throughout the mammalian vasculature. Reports of diminished SP levels in nerve and gastrointestinal tissues of diabetic rats led us to examine SP-like immunoreactivity (SP-LI) in large arteries by RIA. Six weeks after inducing diabetes with streptozotocin (STZ), SP-LI was measured in the thoracic aorta, abdominal aorta, and the proximal superior mesenteric artery. In diabetics we measured a doubling (P<0.01) of SP-LI in all three artery wall preparations. This finding was verified in a second experiment which included a subset of diabetics treated daily with insulin for the sixth week of the holding period. Again, we measured a two-fold or greater increase of SP-LI (P<0.01-0.05) in arteries from the diabetics and found that insulin treatment significantly reduced SP-LI (P<0.05). In contrast to reports of diminished SP content in other tissues of diabetic rats, our findings demonstrate that the artery wall experiences at least a two-fold increase of SP LI in the diabetic state. Furthermore, this elevation of SP-LI is reduced by insulin. We speculate that these changes of arterial wall SP-LI may contribute to altered regulation of the vascular system in the diabetic state. PMID- 8614264 TI - The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death. AB - The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms. PMID- 8614265 TI - Potent inhibition of the inducible isoform of nitric oxide synthase by aminoethylisoselenourea and related compounds. AB - The generation of nitric oxide (NO) by nitric oxide synthase (NOS) can be inhibited by certain guanidines and S-alkylisothioureas. In particular, aminoethylisothiourea (AE-TU) shows selectivity towards the inducible isoform (iNOS) over the endothelial isoform (ecNOS). Here we report on the effects of the selenium analog of AE-TU, aminoethylisoselenourea (AE-SeU), and its homologue, aminopropylisoselenourea (AP-SeU), on the activities of iNOS and ecNOS. AE-SeU and AP-SeU inhibited the conversion of L-arginine to L-citrulline in homogenates of lung taken from endotoxin-treated rats (a model of iNOS acitivity) with potencies (EC50=1.1, and 0.1 microM, respectively) greater than that of N(G) methyl-L-arginine (L-NMA) (22 microM). In contrast, AE-SeU and AP-SeU were weaker than or similar to L-NMA at inhibiting ecNOS activity in homogenized bovine endothelial cells (EC50 values = 104, 15, and 16 microM, respectively). AE-SeU and AP-SeU potently inhibited nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values of 10 and 4 microM respectively. The corresponding EC50 value for L-NMA was 160 microM. The inhibition was dose-dependently reduced by increasing concentrations of L arginine in the medium. In vivo, AE-SeU had only modest effects on blood pressure when given as a bolus to anesthetized rats, suggesting only a small effect on ecNOS in vivo, whereas AP-SeU had potent pressor effects similar to those of L NMA. We found that both AE-SeU and AP-SeU were unstable in aqueous solution at pH values above 6. Their disappearance from solution was accompanied by the appearance of a reductive species, probably free selenol. These findings suggest that AE-SeU and AP-SeU exert their inhibitory effects through intramolecular rearrangement to yield selenoethylguanidine and seleno-propylguanidine. Thus, selenoalkylguanidines are novel inhibitors of iNOS. PMID- 8614266 TI - Pharmacology of L-744,453, a novel nonpeptidyl endothelin antagonist. AB - L-744,453 ((+/-)3-[4-(1-carboxy-1-(3,4-methylenedioxyphenyl)methoxy)-3,5-diprop ylphenyl methyl]-3H-imidazo[4,5-c]pyridine) is an endothelin (ET) receptor antagonist from a new structural class, the dipropyl-alpha-phenoxyphenylacetic acid derivatives. L-744,453 competitively and reversibly inhibits [125I]-ET-1 binding to Chinese Hamster Ovary cells expressing cloned human ET receptors (K(i)s: hET(A)=4.3 nM; hET(B)=232 nM), and is selective for endothelin receptors compared to other peptide receptors. It is an antagonist of ET-1 stimulated phosphatidyl inositol hydrolysis in rat uterine slices (IC50=220 nM) and exhibits no agonist activity. This compound also inhibits ET-1 stimulated contraction of rat aortic rings with a K(b) value of 50 nM. L-744,453 protects against ET-1 induced lethality in mice after i.v. (AD50=13 mg/kg i.v.) or oral administration. This compound also antagonizes ET-1 induced increases in diastolic blood pressure in conscious normotensive rats (AD50=0.67 mg/kg i.v.) and anesthetized ferrets (AD50=1.6 mg/kg i.v.). L-744,453 is a potent, selective, orally active endothelin antagonist which may be useful in elucidating the role of endothelin in normal and pathophysiological states. PMID- 8614267 TI - An iodinated derivative of moclobemide as potential radioligand for brain MAO-A exploration. AB - In vivo evaluation of MAO-A would be of great value for the diagnosis and follow up of therapy of depression. In order to perform this exploration by SPECT, we developed an iodinated derivative of the reversible MAO-A inhibitor, moclobemide. Ro 11-9900 was synthesized and analysed by IR, NMR and HPLC. Radioiodination was carried out by nucleophilic exchange of [125I] on the brominated precursor, and yielded [125I]-Ro 11-9900 with high specific activity. In vitro experiments on rat brain homogenates showed that Ro 11-9900 had poor inhibitory activity on MAO A, as already described for moclobemide. By contrast, in vivo biodistribution in the rat brain showed that [125I]-Ro 11-990 accumulated in a region corresponding to the localization of locus coeruleus known for its high density of MAO-A. Moreover, preinjection of the irreversible MAO-A inhibitor clorgyline (10 mg.kg 1) prevented accumulation of radioactivity by 40 to 60% and we found that the radioactivity in the brain corresponded exclusively to [125I]-Ro 11-9900 and not to a metabolite. [125I]-Ro 11-9900 was highly accumulated in the pineal gland, both on MAO-A and on MAO-B sites. We concluded that the unmetabolized iodinated derivative of moclobemide, Ro 11-9900, preferentially labeled MAO-A in vivo in the rat brain. This compound would therefore be a potential tracer for evaluation of MAO-A by SPECT. PMID- 8614269 TI - Functional coupling of the Na+/Ca2+ exchanger with Ca2+ release from intracellular stores in cultured smooth muscle cells of guinea pig ileum. AB - The mechanism of increase in intracellular Ca2+ concentration ([Ca2+]i) by removal of extracellular Na+, which phenomena were reported previously (Japan. J. Pharmacol. 63 83-91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca2+]i. The pretreatment of thapsigargin (0.5 microM), but not nicardipine (10 microM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca2+ (nominally Ca2+-free solution), the Na+-free induced transient increase was observed, but neither the second cell exposure to the Na+-free solution nor the following application of histamine increased [Ca2+]i, indicating that removal of extracellular Na+ releases Ca2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP3)-releasable pools. The Na+-free induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca2+ and releasable Ca2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca2+ by the reverse-mode action of the Na+/Ca2+ exchanger can release Ca2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools. PMID- 8614268 TI - Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice. AB - We studied the chronic effects of hyperprolactinemia, induced by ectopic pituitary grafting, on blood glucose and lipid levels in adult male mice. For one year after pituitary grafting, we measured the blood levels of prolactin, growth hormone (GH), insulin, glucose and free fatty acid (FFA) at various intervals. The graft caused consistent hyperprolactinemia without changes in the serum GH levels. Hypoglycemia developed at 1 and 3 months after grafting but was not accompanied by any changes of the serum insulin levels. Thereafter, the blood glucose and serum insulin levels began to increase in the pituitary-grafted (PG) mice, and at 12 months after the operation, both levels became significantly higher in PG mice than controls. The serum FFA levels and the weight of epididymal fat bodies were significantly lower in PG mice than controls from 3-12 months after the grafting. Thus, hyperprolactinemia leads to persistent hypolipidemia and biphasic changes in the blood glucose level. PMID- 8614271 TI - Potentiation of glucagon-like peptide 1 insulinotropic action by succinic acid dimethyl ester. AB - The glucagon-like peptide 1 (7-36) amide (GLP-1, 1.0 nM) was administered to isolated rat pancreases perfused either in the absence of exogenous nutrient or presence of 10 mM succinic acid dimethyl ester (SAD). In the absence of any exogenous nutrient, GLP-1 failed to affect either insulin or glucagon release. The administration of SAD caused a biphasic stimulation of insulin output and inhibited glucagon secretion. In the presence of SAD, GLP-1 still failed to affect glucagon release, but markedly enhanced insulin secretion. These findings indicate that GLP-1 is not truly a glucose-dependent, but rather nutrient dependent insulin secretagogue. They also suggest that non-glucidic nutrients, such as SAD, could be used to optimalize the B-cell secretory response to GLP-1 in non-insulin-dependent diabetes mellitus. PMID- 8614270 TI - Central administration of 8-OH-DPAT and mCPP stimulates prolactin secretion in ovariectomized, estrogen-treated rats: lack of an effect on tuberoinfundibular dopaminergic neuron activity. AB - The effects of central administration of two serotonin receptor agonists, 8-OH DPAT and mCPP, on tuberoinfundibular dopaminergic (TIDA) neuron activity and serum prolactin (PRL) levels in ovariectomized, estrogen-treated rats were determined. 8-OH-DPAT dose-dependently (0.1-10 microgram/rat, icv) stimulated serum PRL levels, and depressed serotonergic neuron activity in 30 min. However, the TIDA neuron activity was not affected at all. Similar treatment of mCPP was less effective than 8-OH-DPAT: only the highest dose of mCPP (10 microgram) stimulated PRL secretion and inhibited serotonergic neuron activity. No change in TIDA neuron activity was observed either. We conclude that central serotonin acts on 5-HT1 receptors to stimulate the PRL secretion, which may not involve the TIDA neurons. PMID- 8614272 TI - Recombinant insulin-like growth factor-1 modulates aggregation in human platelets via extracellular calcium. AB - Insulin like growth factor-1 (IGF-1) potentiated aggregation of human platelets induced by thrombin-, collagen- and ADP in a dose-dependent manner over the range 30-300 nM. IGF-1 (100 nM) reduced EC50 values for thrombin, collagen and ADP induced aggregation by 19.6%, 53.6% and 22.8% respectively. Potentiation by IGF-1 was dependent on the presence of extracellular Ca2+ and was inhibited by verapamil or nifedipine. Further, IGF-1 enhanced the elevation in free intraplatelet Ca2+ induced by the platelet agonists collagen and thrombin. PMID- 8614273 TI - Safety/tolerability trial of SDZ ENA 713 in patients with probable Alzheimer's disease. AB - SDZ ENA 713 (ENA 713) is an acetylcholinesterase inhibitor being developed as a potential treatment for Alzheimer's disease (AD). A prior Phase II safety and efficacy study used an upper dose limit of 6 mg/day ENA 713. The present study was designed to assess the safety and tolerability of higher doses of ENA 713 in probable AD patients. Fifty AD patients (22M; 28F, mean age 68 yrs, range 45-90) were assigned to a fixed, nine-week dose escalation schedule in which they were randomized to receive up to 12 mg/day of ENA 713 bid (n=20) or tid (n=20), or placebo (n=10) followed by a one-week washout. Mg/day dose escalation for the bid and tid ENA 713 groups was identical, beginning with 2 mg/day on Days 1 to 3 and escalating to 12 mg/day in Weeks 8 and 9. Doses through 12 mg/day were well tolerated. Most adverse events were mild to moderate in severity and of limited duration, most commonly headache, nausea, dizziness, and diarrhea. Three of forty patients on ENA 713 discontinued, all due to adverse events. Two experienced nausea and vomiting; the third experienced an unrelated mild atrial fibrillation. PMID- 8614274 TI - Simultaneous brain and blood microdialysis study with a new removable venous probe. Serotonin and 5-hydroxyindolacetic acid changes after D-norfenfluramine or fluoxetine. AB - A removable intravenous microdialysis probe was developed and simultaneously used with a removable microdialysis probe placed in the lateral hypothalamus (LH). Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) changes in blood and brain dialysates were measured by HPLC-EC after an i.p. injection of 5 mg/kg d norfenfluramine (dNF) or 10 mg/kg fluoxetine (FLU) in freely moving rats. 5-HT in the LH significantly increased after both drugs, but the rise was larger and faster with dNF [F(7,28)=4.0 p<0.05] than with FLU [F(5,20)=5.0 p<0.01]. By contrast, in venous blood 5-HT increased after FLU [F(5,20)=2.96 p<0.05] but not after dNF. 5-HIAA after both drugs continued decreasing significantly in the LH [dNF F(7,28)=11.4 p<0.01; FLU F(5,20)=22.8 p<0.01], but it did not change in blood. Simultaneous dialysis in brain and blood allowed evaluation of the differential effects of dNF and FLU on 5-HT and 5-HIAA in the two places. Removable venous probes prevented the inflammatory reaction that may occur around permanently implanted probes, and the dialysis could be more efficient and with less risk of clogging. PMID- 8614275 TI - Desensitization of melanotropin receptors in COS-7 cells. AB - Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle(4)-D-Phe(7)]-alpha-MSH (NDP alpha-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP-alpha-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of alpha-MSH, beta-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems NDP-alpha-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-alpha-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-alpha-MSH also occurred more rapidly than desensitization induced by the other MSH analogs. PMID- 8614276 TI - Role of nitric oxide in the maintenance of resting cerebral blood flow during chronic hypertension. AB - The influence of nitric oxide (NO) on basal vascular tone varies with different hypertensive models or vascular beds. The goal of the present study was to examine the role of NO in the maintenance of resting cerebral blood flow (CBF) during chronic hypertension. In 9-10 months old Wistar-Kyoto (WKY) rats (n=47) and spontaneously hypertensive rats (SHR;n=47) anesthetized with pentobarbital sodium (60 mg/kg i.p.), regional CBF of the right parietal cortex was monitored by laser-Doppler flowmetry. Reductions in CBF in response to intravenous infusion of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 1, 3, 10, and 30 mg/kg) were similar between WKY rats (17 +/- 6 approximately 43 +/- 6%; means +/- SE) and SHR (15 +/- 6 approximately 48 +/- 6%) while arterial blood pressure was maintained on the baseline level by controlled hemorrhage. Effects of L-NAME (3 mg/kg i.v.) on arterial blood pressure and CBF were almost completely inhibited by L-arginine (300 mg/kg i.v.), but not by D-arginine (300 mg/kg i.v.). In addition, intravenous infusion of L-arginine (300 mg/kg) alone did not affect resting CBF in both WKY rats and SHR. Thus, these findings suggest that 1) NO plays an important role in the maintenance of resting CBF in both normotensive and chronically hypertensive rats and 2) the contribution of NO to the maintenance of resting CBF is not altered during chronic hypertension. PMID- 8614277 TI - Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain. AB - SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo. PMID- 8614278 TI - Extrapyramidal effects of methanandamide, an analog of anandamide, the endogenous CB1 receptor ligand. AB - Recent evidence has demonstrated that arachidonylethanolamide ("anandamide", AEA), the major endogenous ligand of CB1 receptors, inhibits motor behavior in rats, as does (-)delta 9-tetrahydrocannabinol (THC), the prototypical tricyclic cannabinoid derived from Cannabis sativa preparations. However, its effects were of shorter duration, as compared to THC, likely due to its rapid breakdown by an amidase activity. The present work has been designed to examine the motor effects of AM356(R-methanandamide), an analog of AEA that possesses higher metabolic stability to amidase hydrolysis. We have studied the dose-response and time course effects of R-methanandamide, i.p. administered, on ambulatory activity, frequency of stereotypy and time spent in inactivity measured in an open-field test. Results were as follows. R-Methanandamide, as THC and AEA, inhibited motor behavior. Thus, it decreased ambulation and stereotypy and increased the time spent in inactivity, usually in a dose-related manner, 10 min after administration. However, the motor deficit caused by the highest dose of R methanandamide was usually more pronounced than that caused by a similar dose of AEA. These inhibitory effects persisted 30 min after the administration of R methanandamide, as occurred with AEA and THC. Interestingly, at 60 min after administration, the effects of AEA disappeared, likely because of its breakdown to arachidonic acid and ethanolamine, but this did not occur with R methanandamide whose effects persisted even until 180 min after treatment as occurred with THC. In summary, R-methanandamide inhibits motor behavior in a manner (its effects were persistent) that resembles the effects of THC rather than the effects of AEA (its effects were of rapid onset but shorter duration). This fact supports the use of R-methanandamide as a valuable tool for studying the physiological roles of the anandamidergic system. PMID- 8614279 TI - Plasma endothelin-1 level in athletes after exercise in a hot environment: exercise-induced dehydration contributes to increases in plasma endothelin-1. AB - We investigated whether dehydration due to exercise contributes to the increase in plasma endothelin-1 (ET-1) concentration. We measured the plasma concentration of ET-1 before and after exercise in a hot environment (about 30 degrees C). Five male intercollegiate Kendo (Japanese fencing) players entered the present study. Each athlete participated in 15 min of Kendo fighting, followed by 5 min of rest and another 15 min of Kendo fighting (i.e., total exercise 30 min), with or without oral intake of 700 ml of water. Body weight and left atrial diameter, a parameter that reflects changes in circulating plasma volume, were significantly decreased after exercise under both conditions. However, the decreases in both values were significantly greater after exercise without water intake than after exercise with water intake, indicating that dehydration and decreased circulating plasma volume were more marked after exercise without water intake. The extent of the increase in plasma ET-1 concentration appeared to be closely related to the extent of exercise-induced dehydration; the greater the dehydration, the greater the increase in plasma ET-1 concentration. These findings suggest that exercise induced dehydration may contribute to increases in plasma ET-1 concentrations. PMID- 8614280 TI - Isolation of anti-leukemia compounds from Citrus reticulata. AB - In vitro effects of medicinal plant extracts from the pericarpium of Citrus reticulata (cv Jiao Gan) (PCRJ) on the growth and differentiation of a recently characterized murine myeloid leukemic cell clone WEHI 3B (JCS) were investigated. Extracts of PCRJ not only inhibited the proliferation of JCS cells in a dose dependent manner, but also induced differentiation of JCS cells into macrophages and granulocytes. Morphological differentiation of PCRJ treated JCS cells was associated with an increase in phagocytic activity of the cells. Furthermore, both in vitro clonogenicity and in vivo growth of PCRJ treated JCS leukemic cells in syngeneic BALB/c mice were significantly reduced. The survival rate of mice receiving PCRJ treated JCS tumour cells was also increased. Using 1H-NMR, 13C NMR, and GC/MS, two active components isolated from PCRJ were identified as nobiletin and tangeretin. PMID- 8614282 TI - Significantly different fatty acid profiles in various phospholipid head groups of neuroblastoma cell lines. AB - We studied lipid profiles of hybrid cells derived from fusion of sympathetic ganglia and neuroblastoma cells (NCB 20, F 11) and also CHO (Chinese hamster ovary) cells. The proportion of saturated to unsaturated fatty acids changed significantly in different phospholipid fractions. We observed 35.4% of total fatty acids as saturated fatty acids in the phosphatidyl ethanolamine (PE) fraction of NCB-20 cell lines while 47.2% of total fatty acids were saturated in the phosphatidyl inositol (PI) fraction. In general, in neuroblastoma cell lines, we observed the lowest proportion of saturated fatty acids in the PE fraction while the other lipid fraction showed a much higher proportion of saturated fatty acids. On the other hand the PE fraction of a non neuronal cell line, CHO, showed 57.4% saturated fatty acids in the PE fraction. This significant difference in saturated to unsaturated fatty acid ratios in different phospholipid head groups may be linked to the different biological functions of those hybrid cells. PMID- 8614281 TI - Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. AB - Signal transduction cascades initiated by the neuronal kappa opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The kappa opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-kappa, harboring the kappa-opioid receptor cDNA. Positive clones (HN2 kappa 24 and CHO kappa 18) from both lines showed high expression of the kappa opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2 kappa 24 and CHO kappa 18. Scatchard analysis revealed the presence of high affinity kappa opioid receptors in both engineered cell lines (KD=1.3 nM for HN2 kappa 24 and 2.1 nM for CHO kappa 18). Functional coupling to adenylate cyclase was displayed by 1 microM U-69,593 mediated inhibition (55-63%) of prostaglandin E1-stimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed kappa opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 microM) treatment stimulated PL-C, but not PL-D, in HN2 kappa 24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHO kappa 18 cells. Our results using the model neuronal system, HN2 kappa 24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL C cascade, which regulates neuronal firing. PMID- 8614283 TI - Enhancement of postischemic myocardial stunning by calcium overload in hearts of diabetic rats. AB - The effects of Ca2+ concentration on postischemic myocardial stunning were studied in isolated working hearts of rats with streptozotocin-induced diabetes and of age-matched control rats. During reperfusion after 10 min of ischemia, hearts from control rats showed complete recovery of cardiac function of Ca2+ concentrations of 1.25, 1.88, and 2.50 mmol/L, while the recovery of diabetic rats was decreased only at a Ca2+ concentration of 2.50 mmol/L. Although myocardial Na+ and Ca2+ concentrations were comparable between control and diabetic rats, only diabetic rats showed increases in myocardial concentration of Na+ during ischemia and Ca2+ during reperfusion at a Ca2+ concentration of 2.50 mmol/L. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning via an overload of calcium. PMID- 8614284 TI - Inhibition of hypoxic coronary vasoconstriction by pinacidil. AB - Acute hypoxia causes constriction of isolated coronary arteries from several species. The present study was designed to test whether pinacidil, a potassium channel opener, inhibits hypoxia-induced contraction of porcine isolated coronary arteries. Coronary arterial rings were suspended in organ baths for isometric tension recording. Hypoxic contractions were evoked by rapidly changing gas mixture from 95% O2/5% CO2 to 95% N2/5% CO2 in preparations partially contracted with KCl. Pretreatment with pinacidil (10(-6) to 10(-4) M) caused concentration dependent inhibition of the contractile response to hypoxia. The inhibitory effect of pinacidil was attenuated by the K ATP channel blocker, glibenclamide (10(-6) M). In rings contracted with acetylcholine, glibenclamide caused a rightward shift in the concentration-response curve to pinacidil while having no effect on the vasorelaxant responses to sodium nitroprusside and diltiazem, thus confirming the specificity of glibenclamide for potassium channel opener-mediated responses, Taken together, the data indicate that pinacidil prevents hypoxia induced contraction of porcine coronary arteries, and that the effect of pinacidil may be mediated by the opening of glibenclamide-sensitive potassium channels. PMID- 8614286 TI - Insulin-like growth factor-I given subcutaneously reduces clinical deficits, decreases lesion severity and upregulates synthesis of myelin proteins in experimental autoimmune encephalomyelitis. AB - To extend our evaluation of insulin-like growth factor-1 (IGF-1) treatment for human demyelinating diseases, we compared effects of s.c. and i.v. IGF-1 in an in vivo model with lesions resembling those seen in multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats with an emulsion containing guinea pig spinal cord and treatment with placebo or with s.c. or i.v. IGF-1 was started when definite clinical weakness was present. IGF-I given subcutaneously significantly reduced clinical deficits and lesion severity. The clinical improvement, as measured by clinical deficit scores, stride lengths and exercise wheel rotations, was evident in 48 hrs and was comparable to that produced by the same IGF-I dose administered intravenously. Subcutaneously administered IGF-I also increased relative mRNA levels of myelin basic protein (MBP), proteolipid (PLP) and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP), thereby promoting myelin regeneration. We conclude that s.c. IGF-I produces dramatic improvement in acute, demyelinating EAE. Our results also suggest that this growth factor may be useful in treating multiple sclerosis patients with active demyelination. PMID- 8614285 TI - The estrogen antagonist tamoxifen inhibits carrageenan induced inflammation in LEW/N female rats. AB - Carrageenan induces a measurable inflammatory response in susceptible animals, and mature females are more responsive to carrageenan, than males. In the present study, we tested whether the estrogen antagonist tamoxifen influences carrageenan induced inflammatory responses. Female LEW/N rats were treated with tamoxifen and compared to a control group of animals injected with vehicle. Tamoxifen significantly reduced estrous phase of estrous cycle during treatment, consistent with its functional anti-estrogen effects. Moreover, tamoxifen significantly decreased exudate volume but did not significantly influence relative white blood cell counts in the exudate. Interestingly, tamoxifen induced differential dose dependent alterations in peripheral blood lymphocyte subpopulations. Low dose of tamoxifen increased CD25 cells. The high tamoxifen dose significantly increased CD8 blood lymphocytes counts. Our data indicate that tamoxifen treatment decreases carrageenan-induced inflammatory response in female LEW/N rats and suggest therefore that this inflammatory response is, at least in part, estrogen related. Moreover, our results suggest a possible role for tamoxifen in treatment of inflammatory disorders. PMID- 8614287 TI - Decreased affinity of K-receptor binding during reperfusion following ischaemic preconditioning in the rat heart. AB - The effects of ischaemic preconditioning with three cycles of ischaemia of 3 min and reperfusion of 5 min each cycle on ventricular fibrillation threshold (VFT) and ventricular fibrillation (VF), and binding properties of tritiated U69,593, a selective kappa opioid-receptor (k-receptor) agonist, during subsequent ischaemia and/or reperfusion were studied in the rat heart. It was found that ischaemic preconditioning significantly enhanced the VFT values during ischaemic and reperfusion. VF during the subsequent reperfusion period was also significantly reduced. The Kd of the [3H]U69,593 binding sites in the sarcolemma of the heart at 5 min of reperfusion was significantly increased following ischaemic preconditioning. The Bmax was, however, not altered after the preconditioning. The study provides evidence for the first time suggesting that the cardioprotective effects of ischaemic preconditioning may be related to a reduction in affinity of the K-receptor binding. PMID- 8614288 TI - Extracts of Ginkgo biloba leaves inhibit monoamine oxidase. AB - Extracts of Ginkgo biloba leaves produce reversible inhibition of rat brain monoamine (MAO). Both MAO-A and -B types were inhibited to a similar extent. The MAO inhibitory compound(s) were present in dried or fresh Ginkgo biloba leaves as well as in commercially available capsules of Ginkgo biloba and appear to be heat stable with relatively low molecular weight. MAO inhibition by Ginkgo biloba may be a mechanism underlying reported anti-stress and anxiolytic activities of this natural product. PMID- 8614289 TI - Relationship between cAMP induced inhibition of human meningioma cell proliferation and autocrine secretion of interleukin-6. AB - Previous studies have suggested that activation of the adenylyl cyclase - cAMP system in meningiomas results in decreased mitosis. We have used meningioma cell culture to further investigate this phenomenon and to examine the potential role played by interleukin-6 (IL6). Incubation of cultured meningioma cells for 4-6 days with cholera toxin and theophylline, both of which increase intracellular cAMP levels, markedly stimulated IL6 secretion and inhibited cell growth rate. Similar effects were observed with 8-bromo-cAMP. In contrast, a neutralising polyclonal antibody against IL6 significantly stimulated meningioma proliferation and reduced the inhibitory effects of 8-bromo-cAMP. These results support the concept that IL6 acts as an autocrine / paracrine inhibitory factor for meningioma proliferation, and that the inhibition exerted by elevated intracellular cAMP levels may be at least partially mediated via increased secretion of the cytokine. PMID- 8614290 TI - Serum type III, IV collagens and TIMP in patients with type II diabetes mellitus. AB - We investigated serum levels of type III procollagen aminopeptide (CIII), 7S type IV collagen (CIV), and tissue inhibitor of metalloproteinase (TIMP) in 33 patients with type II diabetes mellitus (DM) without uremia (serum creatinine less than 1.5 mg/dl). The patients were divided into three groups based on measurement of the urinary albumin excretion (UAE) index obtained during two morning outpatient clinic visits: non-proteinuric patients (n = 11), UAE index less than 2.26 mg/mmol Cr; patients with microalbuminuria (n = 15), UAE index of 2.26 - 22.6 mg/mmol Cr; and patients with proteinuria (n = 7), UAE index more than 22.6 mg/mmol Cr. Serum levels of CIV and TIMP in patients with microalbuminuria and proteinuria were significantly higher than non-proteinuric patients (ANOVA, p <0.05). Serum levels of CIII in patients with proteinuria were significantly higher than those in non-proteinuric patients (p < 0.05). There was a significant positive correlation between CIV and TIMP (r = 0.502, p < 0.003), but no correlation was observed between CIII and TIMP. These results demonstrated that serum CIII and CIV increases as diabetic nephropathy progresses in terms of increasing proteinuria in type II DM patients, suggesting feasibility and usefulness of measuring serum CIV and CIII in assessing diabetic nephropathy. The increase in TIMP may be, at least in part, a possible cause for the increase in serum CIV in type II DM patients. PMID- 8614291 TI - Long-term effects of inhaled nicotine. AB - Tobacco smoking has been reported to be associated with increased risk of cardiovascular disease and cancer, particularly of the lungs. In spite of extensive research on the health effects of tobacco smoking, the substances in tobacco smoke exerting these negative health effects are not completely known. Nicotine is the substance giving the subjective pleasure of smoking as well as inducing addiction. For the first time we report the effect on the rat of long term (two years) inhalation of nicotine. The rats breathed in a chamber with nicotine at a concentration giving twice the plasma concentration found in heavy smokers. Nicotine was given for 20 h a day, five days a week during a two-year period. We could not find any increase in mortality, in atherosclerosis or frequency of tumors in these rats compared with controls. Particularly, there was no microscopic or macroscopic lung tumors nor any increase in pulmonary neuroendocrine cells. Throughout the study, however, the body weight of the nicotine exposed rats was reduced as compared with controls. In conclusion, our study does not indicate any harmful effect of nicotine when given in its pure form by inhalation. PMID- 8614292 TI - N-docosahexaenoyl, 3 hydroxytyramine: a dopaminergic compound that penetrates the blood-brain barrier and suppresses appetite. AB - Fatty acids with varying chain lengths (2-22 carbon atoms long) and degrees of unsaturation (0-6 double bonds) were used to synthesize dopaminergic compounds for a study of the carrier mediated transport of dopamine (DA) to the brain. The most active carrier was the all cis C22:6 fatty acid [docosahexaenoic acid, (DHA)]which increased DA uptake through the blood-brain barrier by greater than 7.5 fold. The DHA-DA compound, NMI 8739, depressed the general locomotor activity of mice in a dose dependent manner. It also suppressed the appetite of Balb c mice and Charles River rats by 50% and 95% respectively at a dose of 10 mg/kg. Daily administration of NMI-8739 for a three week period did not induce tolerance. These results demonstrate DHA's potential for the carrier mediated transport of small molecules to the brain. PMID- 8614293 TI - Effects of inhalation anesthetics of Kainate-induced glutamate release from cerebellar granule cells. AB - The mechanisms of inhalation anesthesia may include inhibiting non NMDA excitatory amino acid neurotransmission. This possibility was addressed by measuring the effect of three anesthetics at clinically relevant concentration on kainate-induced glutamate release from cerebellar granule cells. Cerebellar granule cells were obtained from 8-day-old SD rats and maintained in vitro for 9 14 days. Medium glutamate concentrations were measured by HPLC after 90 minutes incubation with kainate or NMDA. Inhalation anesthetics were introduced by holding the cells under a continuous flow of air/anesthetic mixtures. All anesthetics tested did not effect NMDA-induced glutamate release. Halothane (1 MAC), isoflurane (1 MAC) inhibited kainate-induced glutamate release from the cultured cells whereas enflurane (1 MAC) had no effect on kainate-induced glutamate release. The difference between enflurane and the other anesthetics tested suggests that anesthesia is dependent on more than one process and the extent at which each function is perturbed is dependent on the specific anesthetic used. Halothane inhibition of kainate-induced glutamate release was not reversible by increasing kainate concentration, indicating halothane does not directly compete with kainate at its receptor. PMID- 8614294 TI - The vesamicol receptor ligand (+)-meta-[125I]iodobenzyltrozamicol [(+)-[125I] MIBT] reveals blunting of the striatal cholinergic response to dopamine D2 receptor blockade in the 6-hydroxydopamine (6-OHDA)-lesioned rat: possible implications for Parkinson's disease. AB - Previous studies of radiolabelled vesamicol receptor (VR) ligands suggest that the latter may be used, in conjunction with dopamine D2 antagonists, to measure changes in striatal cholinergic function in vivo. In the present study, the radiolabelled VR ligand (+)-meta-[125I]iodobenzyltrozamicol {(+)-[125I]MIBT} was used to assess striatal cholinergic function in the unilateral 6-hydroxydopamine (6-OHDA)-treated rat. In control animals, the levels of this radiotracer monitored at 3 hr post injection displayed bilateral symmetry in the striatum, cerebral cortex and cerebellum. However, in animals pretreated with the dopamine antagonist spiperone (2 mg/kg ip), the radiotracer concentration in the striatal hemisphere ipsilateral to 6-OHDA lesion increased by 23% (p = 0.068) while the concentration in the contralateral striatum was elevated by 87% (p < 0.0001). Since the nigrostriatal dopaminergic system modulates striatal cholinergic function, and dopamine D2 receptor blockade is known to result in increased striatal cholinergic function, the refractoriness of striatal cholinergic neurons following the loss of nigrostriatal dopaminergic innervation confirms the existence of a dopaminergic-cholinergic imbalance in Parkinson's disease. Therefore the combination of a D2 antagonist and radiolabelled VR ligand may provide a potentially useful method for assessing the effects of dopamine depletion in Parkinson's disease. PMID- 8614295 TI - Effects of halothane and isoflurane on rat ventricular action potentials recorded in situ. AB - The effects of halothane and isoflurane on ventricular intracellular action potentials recorded in situ in pentobarbital anesthetized rats were studied. Halothane (0.5, 1 and 2 vol.%) and isoflurane (0.75, 1.5 and 3 vol.%) did not have identical effects on rat epicardial action potentials recorded by floating microelectrodes. However, over the concentration range tested, both anesthetics reduced blood pressure and heart rate to a similar extent. Isoflurane did not effect the maximum rate of rise of the action potential amplitude. However, 3 vol.% isoflurane reduced the resting membrane potential from -72+/-2 to -65+/-3 mV (mean+/-SEM, p<0.05) while the highest concentration of halothane had no effect. Halothane (2 vol.%) reduced action potential amplitude from 74+/-4 to 65+/-3 mV (p<0.05) and reduced the maximum rise rate of action potential from 175+/-21 to 133+/-8 V/s (p<0.05). Both isoflurane and halothane prolonged action potential duration at 10, 25 and 50% repolarization while only halothane significantly shortened action potential duration at 75% repolarization, Thus the effects of halothane and isoflurane on ventricular transmembrane action potentials were similar, but not identical. The relevance of such observations to the antiarrhythmic actions of halothane, but not isoflurane in this species is not clear. PMID- 8614296 TI - Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14 alpha-demethylation (P45014DM) in hamster liver. AB - To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450 lanosterol 14 alpha-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL 6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein showed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14 alpha-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14-alpha-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital- and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals. PMID- 8614297 TI - Multiple forms of the enzyme glycerophosphodiesterase are present in human brain. AB - Brain levels of glycerophosphodiesters, including glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), are altered in many human central nervous system disorders. Although much information is available on the enzymes responsible for the formation of these phospholipid metabolites, little information is known regarding their catabolism, by glycerophosphodiesterases, in human brain. In both autopsied and biopsied temporal cortex, a phosphocholine producing glycerophosphodiesterase activity was observed. In the presence of 1 mM EDTA, the enzyme possessed a pH optimum of 9.0, while the addition of 5 mM zinc acetate shifted the pH optimum to 10.5. When assayed at pH 9.0 in the absence of zinc acetate, the Km and Vmax were 104 +/- 2 microM and 77 +/- 18 nmol/h/mg protein, respectively, while assaying at pH 10.5 in the presence of 5mM zinc acetate yielded a Km of 964 +/- 56 microM, and a Vmax of 534 +/- 114 nmol/h/mg protein. Furthermore, whereas submillimolar concentrations of zinc acetate stimulated the activity of the enzyme in a dose-dependent manner when assayed at pH 10.5 (EC50 =20.3 +/- 3.0 microM), this did not result in a reciprocal inhibition of glycerophosphocholine phosphodiesterase (GPC PD) activity when assayed at a more acidic pH. This may suggest that human brain contains two phosphocholine-producing GPC PD activities, differentiable by their sensitivity to zinc ions. An activity capable of hydrolyzing GPE to form phosphoethanolamine could not be detected in either biopsied or autopsied brain. However, a choline/ethanolamine-producing glycerophosphodiesterase activity could be readily detected in biopsied, but not autopsied brain. this novel enzyme possessed a neutral pH optimum and was dependent upon divalent cations for activity. In conclusion, human brain contains at least two different glycerophosphodiesterases, a phosphocholine, and a choline/ethanolamine-producing activity, only one of which can be detected in autopsied tissue. The results of previous studies measuring brain glycerophosphodiesterase activity in degenerative brain conditions may need to be reevaluated in the light of these observations. PMID- 8614299 TI - Functional and ultrastructural effects of essential fatty acid deficiency in kidney epithelial cells. AB - Madin-Darby canine kidney (MDCK) epithelial cells were grown in culture medium supplemented with 1% fetal bovine serum (FBS) to provide a cell culture model of essential fatty acid deficiency (EFAD). 5,8,11-Eicosatrienoic acid (20:3n-9) accumulated in cellular phospholipids, and arachidonic acid (20:4) decreased. A large increase in cellular cholesterol/phospholipid ratio was observed. Hemicyst formation was greatly reduced from normal levels in the EFAD-MDCK cells. Scanning and transmission electron microscopy revealed that EFAD-MDCK were much flatter than their normal counterparts. They had much less dense surface microvilli, mitochondria and other organelles were very sparse, except in the perinuclear area, and much of the peripheral cytoplasm was amorphous. The EFAD was rapidly reversed by the addition of as little as 10 microM linoleic or arachidonic acid to the medium. Cells supplemented with 10% FBS, the usual culture condition, displayed borderline EFAD, with intermediate levels of 20:3n-9 and 20:4 and hemicyst formation. These studies suggest that EFAD reduces water and electrolyte transport in renal tubular epithelium. PMID- 8614298 TI - Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines. AB - Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C acetate incorporation, in response to the following treatments in lipoprotein deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in subconfluent cells. Evaluation of LDL receptor activity through 125I-LDL binding and internalization studies demonstrated LDL receptor expression was reduced by 37% in normal density cells relative to the low density cultures. In contrast to cholesterol synthesis, exogenous LDL could inhibit LDL receptor activity at both densities. Thus subconfluent growing colonic adenoCA cell lines retain the capacity for sterol repression, but, in contrast to normal fibroblasts, exhibit a high endogenous cholesterol synthesis which LDL cannot regulate. PMID- 8614300 TI - The essential oil from Tagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain. AB - The hypothesis that the essential oil from Tagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude members from chick brains. The essential oil from T. minuta L. inhibited [3H]FNTZ specific binding to chick brain members. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (Kd) = 2.47 +/- 0.32 nM, maximal binding (Bmax) = 556 +/- 5 fmoles/mg protein, and Hill coefficient (n) = 1.00 +/- 0.07 in the absence, and Kd = 6.73 +/- 1.4 nM, Bmax = 583 +/- 69 fmoles/mg protein, and n = 1.02 +/- 0.08 in the presence of 29 microgram/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 microgram/mL. The marked increase in [3H]FNTZ nonspecific binding starting at 60 micrograms of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with members. [3H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oil's micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oil's components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oil's CMC suggests that this effect may be due to oil's partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface. PMID- 8614302 TI - Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: an experimental and model study. AB - Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5 degrees C) and summer (25 degrees C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids--[2-, 12(N-9-anthroyloxy)stearic acid and 16(N 9-anthroyloxy)palmitic acid--embedded in native and synthetic (16:0/16:0, 16:0/22:6, 18:0/22:6, 18:1/22:6, 20:4/20:4, 22:6/22:6 phosphatidylcholines and 16:0/18:1, 18:1/22:6 phosphatidylethanolamines) phospholipid vesicles was also determined between -30 and 30 degrees C and 5 and 30 degrees C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5 degrees C downshift in the solid-gel to liquid-crystalline phase transition temperature (-8 vs. -13 degrees C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warm-adapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16:0/22:6 phosphatidylcholine (75%) and either 18:1/22:6 phosphatidylethanolamine (25%) vs. 16:0/18:1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16:0/22:6 and 100% 18:1/22:6 phosphatidylcholine vesicles. Mixing either 16:0/18:1 (25%) or 18:1/22:6 (25%) phosphatidylethanolamines to 18:0/22:6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18:0/22:6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination with cis delta 9 monounsaturated fatty acids in the position sn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature. PMID- 8614301 TI - Effect of marine oils supplementation on coagulation and cellular activation in whole blood. AB - A study was performed to explore the effects of supplemental intake of various marine oils known to be part of the Eskimo diet. Healthy men and women (134) were randomly selected to consume 15 mL/d of oil from blubber of seal, cod liver, seal/cod liver, blubber of Minke whale, or no oil for ten weeks. Total cholesterol was unchanged in the oil groups, whereas high density lipoprotein cholesterol increased 7% in the seal/cod liver oil (CLO) group (P < 0.05) and 11% in the whale oil group (P < 0.005). Triacylglycerol was significantly reduced in the CLO group only. The concentration of prothrombin fragment 1 + 2 was reduced 25% (P < 0.05) after whale oil supplementation. No change in fibrinogen or factor VIIc was detected. Tumor necrosis factor generation in lipopolysaccharide (LPS) stimulated blood was 30% reduced after whale oil (P < 0.05), but was unaffected by intake of seal or CLO. The LPS-induced tissue factor activity in monocytes was reduced to a significant degree only in the seal/CLO group (34%) and whale oil group (35%) (P < 0.05). The most dramatic change in thromboxane B2 in LPS stimulated blood was seen after whale oil intake with 44% reduction (P < 0.01). Supplementation of a regular diet with a combination of seal oil and CLO and especially with whale oil seems to have beneficial effects on several products thought to be associated with cardiovascular and thrombotic diseases. PMID- 8614303 TI - Composition of phospholipids of white muscle of six tuna species. AB - A comparative study of the phospholipids of white muscle of six of the commercially utilized tuna species, including quantitative analyses of phospholipid classes and studies of the acyl composition of the major components. Plasmalogen compounds also were identified and quantified. Choline and ethanolamine glycerophospholipids were the most abundant classes in all the samples, as well as the only molecules containing plasmalogens (16:0, 18:0, and 18:1 alkenylether chains). The patterns of fatty acid distribution within each of the phospholipid classes showed general similarities in the species analyzed. However, ratios between certain saturated and polyunsaturated fatty acids in different phospholipid classes showed remarkable differences. The high content of n-3 polyunsaturated fatty acids in the principal phospholipids, such as the plasmalogens, and taking into account the fatty acids possible importance in human nutrition, indicates that the white muscle of tuna species may be a potentially important dietary item. PMID- 8614304 TI - Effect of tocotrienols on the growth of a human breast cancer cell line in culture. AB - The tocotrienol-rich fraction (TRF) of palm oil consists of tocotrienols and some alpha-tocopherol (alpha-T). Tocotrienols are a form of vitamin E having an unsaturated side-chain, rather than the saturated side-chain of the more common tocopherols. Because palm oil has been shown not to promote chemically-induced mammary carcinogenesis, we tested effects of TRF and alpha-T on the proliferation, growth, and plating efficiency (PE) of the MDA-MB-435 estrogen receptor-negative human breast cancer cells. TRF inhibited the proliferation of these cells with a concentration required to inhibit cell proliferation by 50% of 180 microgram/mL whereas alpha-T had no effect at concentrations up to 1000 microgram/mL as measured by incorporation of [3H]thymidine. The effects of TRF and alpha-T also were tested in longer-term growth experiments, using concentrations of 180 and 500 microgram/mL. We found that TRF inhibited the growth of these cells by 50%, whereas alpha-T did not. Their effect on the ability of these cells to form colonies also was studied, and it was found that TRF inhibited PE, whereas alpha T had no effect. These results suggest that the inhibition is due to the presence of tocotrienols in TRF rather than alpha T. PMID- 8614305 TI - Oxidative susceptibility of low density lipoprotein from rabbits fed atherogenic diets containing coconut, palm, or soybean oils. AB - The oxidative susceptibilities of low density lipoproteins (LDL) isolated from rabbits fed high-fat atherogenic diets containing coconut, palm, or soybean oil were investigated. New Zealand white rabbits were fed atherogenic semisynthetic diets containing 0.5% cholesterol and either (i) 13% coconut oil and 2% corn oil (CNO), (ii) 15% refined, bleached, and deodorized palm olein (RBDPO), (iii) 15% crude palm olein (CPO), (iv) 15% soybean oil (SO), or (v) 15% refined, bleached, and deodorized palm olein without cholesterol supplementation [RBDPO(wc)], for a period of twelve weeks. Total fatty acid compositions of the plasma and LDL were found to be modulated (but not too drastically) by the nature of the dietary fats. Cholesterol supplementation significantly increased the plasma level of vitamin E and effectively altered the plasma composition of long-chain fatty acids in favor of increasing oleic acid. Oxidative susceptibilities of LDL samples were determined by Cu2(+)-catalyzed oxidation which provide the lag times and lag-phase slopes. The plasma LDL from all palm oil diets [RBDPO, CPO, and RBDPO(wc)] were shown to be equally resistant to the oxidation, and the LDL from SO-fed rabbits were most susceptible, followed by the LDL from the CNO-fed rabbits. These results reflect a relationship between the oxidative susceptibility of LDL due to a combination of the levels of polyunsaturated fatty acids and vitamin E. PMID- 8614306 TI - Dietary lipid modification of myocardial eicosanoids following ischemia and reperfusion in the rat. AB - Several different edible oils were compared for their ability to modify eicosanoid biosynthesis following experimentally-induced myocardial ischemia and reperfusion in the rat. Two types of palm oil [neutralized, bleached, and deodorized (NBDPO) and refined, bleached, and deodorized (RBDPO)] and partially hydrogenated soybean oil (SBO) were tested against a diet supplemented with sunflower seed oil (SSO) rich in n-6 polyunsaturated fatty acids (PUFA). Fish oil (FO) rich in n-3 PUFA, with its known cardioprotective actions, served as an internal reference point for the study. Test oils were fed as a 12% (w/w) supplement for nine months before the induction of myocardial ischemia and reperfusion. Palm oil diets exerted effects indistinguishable from the SBO group against cardiac arrhythmia, which occurred following alterations to coronary blood flow. Arrhythmic potentials, as expressed by a hierarchical scale (0-9) of arrhythmia score, were: SSO, 1.5 +/- 0.5; FO, 0.9 +/- 0.4; SBO, 3.1 +/- 0.5*; NBDPO, 3.2 +/- 0.5*; RBDPO, 3.3 +/- 0.6*; *P < 0.05 vs. SSO. Following ischemia and reperfusion, both SSO and RBDPO groups tended to show an increase in myocardial prostacyclin, with the effect being more prominent in the RBDPO group (SSO, 10%; RBDPO, 25%). Thromboxane production was reduced in the FO group. Interestingly, cardiac muscle from both FO and palm oil groups displayed a reduced capacity to produce 12-hydroxyeicosatetraenoic acid SSO, 591 +/- 95.8; SBO, 375.5 +/- 48.9; NBDPO, 287.2 +/- 64.7*; RBDPO, 230.9 +/- 80.2**; FO, 203.7 +/- 81.4** (ng/g dry wt, *P < 0.05, **P < 0.01). No clear relationship was seen between the availability of 20:4n-6 in myocardial phospholipids and eicosanoid profile. Data suggests that fatty acid composition of edible oils is not the only determinant of arrhythmic vulnerability and eicosanoid production. PMID- 8614308 TI - Palmolein and groundnut oil have comparable effects on blood lipids and platelet aggregation in healthy Indian subjects. AB - Substitution of palmolein (POL) for groundnut oil (GNO) doubles saturated fatty acids and decreases by half the linoleic acid (18:2n-6) content of Indian diets. The effects of this substitution on selected parameters of cardiovascular risk and membrane functions were studied in middle-aged subjects. Both metabolic (short-term) and "in-home" (long-term) studies were conducted, and the subjects were crossed over from GNO to POL or vice versa. During both studies and in both sexes, blood pressure, plasma levels of total cholesterol and triglycerides and their distributions in various lipoprotein fractions were not altered. The lower 18:2n-6 and higher 16:0 intakes were reflected in fatty acid compositions of cholesteryl esters and triglycerides. However, the plasma and platelet phospholipid fatty acid patterns did not shift toward saturation. The observation that the levels of long-chain polyunsaturated fatty acids in phospholipids were similar at the end of GNO and POL regimens indicates that 18:2n-6 furnished during POL regimen may be sufficient to maintain the levels of arachidonic acid in cell membranes. Platelet aggregation, erythrocyte membrane fluidity, and activity of Na+, K+ ATPase, a membrane-bound enzyme, were essentially similar at the end of the two oil regimens. These results indicate that POL is comparable to GNO and may not induce hypercholesterolemia in Indian subjects consuming cereal based diets containing 30% total fat calories and low cholesterol. PMID- 8614307 TI - Plasma lipids are affected similarly by dietary lauric or palmitic acid in gerbils and monkeys. AB - To compare the relative impact of dietary lauric acid (12:0) and palmitic acid (16:0) on plasma lipids, two fat-sensitive species, Mongolian gerbils and cebus monkeys, were fed cholesterol-free, purified diets enriched with either 12:0-rich or 16:0-rich fats, while all other fatty acids were held constant by selective blending of up to five natural fats or oils. The two gerbil diets (40 en% from fat) allowed for an 8 en% exchange between 12:0 and 16:0, and the monkey diets (31 en% from fat) allowed for 6 en% exchange between these two fatty acids. Eight gerbils received the diets for eight weeks, and 12 cebus monkeys were fed each diet in a cross-over design for up to 22 wk. Both diets resulted in similar plasma cholesterol, triglyceride, and high density lipoprotein cholesterol concentrations within each species. Additionally, separation of cebus lipoproteins by discontinuous density-gradient ultracentrifugation failed to show any dietary differences in concentration or composition of the three major lipoprotein classes (d < 1.019, 1.019-1.055, and 1.055-1.168 g/mL). Thus, in two species sensitive to manipulations in dietary fat while consuming cholesterol free diets, 16:0 was not hypercholesterolemic relative to 12:0. PMID- 8614309 TI - Response of hypercholesterolemic subjects to administration of tocotrienols. AB - The cholesterol-suppressive actions of Palmvitee and gamma-tocotrienol were assessed in hypercholesterolemic subjects after acclimation to the American Heart Association Step 1 dietary regimen for four and eight weeks, respectively. The four-week dietary regimen alone elicited a 5% decrease (P < 0.05) in the cholesterol level of the 36 subjects. Subjects continuing on the dietary regimen for a second four-week period experienced an additional 2% decrease in their cholesterol levels. Dietary assessments based on unanticipated recalls of 24-h food intake records suggest that significant reductions in energy and fat, predominantly in saturated fat, intakes are responsible. The subjects experienced significant Palmvitee- and gamma-tocotrienol-mediated decreases in cholesterol. The group of subjects acclimated to the dietary regimen for four weeks responded to Palmvitee (a blend of tocols providing 40 mg alpha-tocopherol, 48 mg alpha tocotrienol, 112 mg gamma-tocotrienol, and 60 mg delta-to-cotrienol/day for four weeks) with a 10% decrease in cholesterol (P < 0.05). Dietary assessments showed no further change in energy and fat intakes. alpha-Tocopherol attenuated the cholesterol-suppressive action of the tocotrienols. The second group of subjects, acclimated to the dietary regimen for eight weeks, received 200 mg gamma tocotrienol/d for four weeks. The cholesterol-suppressive potency of this alpha tocopherol-free preparation was calculated to be equivalent to that of the mixture of tocotrienols (220 mg) used in the prior study. Cholesterol levels of the 16 subjects in the second group decreased 13% (P < 0.05) during the four-week trial. Plasma apolipoprotein B and ex vivo generation of thromboxane B2 were similarly responsive to the tocotrienol preparations, whereas neither preparation had an impact on high density lipoprotein cholesterol and apolipoprotein A-1 levels. PMID- 8614311 TI - Bed rest induces neural and contractile adaptations in triceps surae. AB - The electromechanical adaptations of the triceps surae muscle were examined in one subject confined to bed for 5 wk due to strong orthostatic headaches. The torque produced by the triceps surae and the electromyographic (EMG) activities of the slow soleus (Sol) and fast lateral gastrocnemius (LG) were studied in response to voluntary and electrically induced contractions. Bed rest caused a 46% decrease in the maximal voluntary contraction (MVC) torque. This loss of strength was associated with a deficit of 33% in central activation and 19% in the estimated muscle force-generating capacity. The EMG activity during MVC was found to be reduced in both muscles, but to a greater extent in the LG (-51%) than in the Sol (-32%). The decrease in amplitude of the maximal M wave (Mmax) recorded in response to supramaximal electrical stimulation was greater in Sol ( 28%) than in LG (-12%). After bed rest, the twitch time to peak torque was slightly prolonged (+9%) and the maximal rate of tension development (dT/dt) was reduced by 31% in electrically induced and 24% in voluntary contractions. Although muscle fatigability during a 60-s sustained MVC was not affected by the bed rest period, the relative contributions of the two muscles differed from control. These results indicate that muscular adaptations were more prominent in Sol while neural adaptations were more significant in LG. Furthermore, the neural changes almost completely recovered within 2 wk, whereas the contractile alterations required at least 5 times longer to return to the control values. PMID- 8614310 TI - Antioxidant effects of tocotrienols in patients with hyperlipidemia and carotid stenosis. AB - Antioxidants may have a role in the prevention of atherosclerosis. In the present trial, we investigated the antioxidant properties of Palm Vitee, a gamma tocotrienol-, and alpha-tocopherol enriched fraction of palm oil, in patients with carotid atherosclerosis. Serum lipids, fatty acid peroxides, platelet aggregation and carotid artery stenosis were measured over an 18-month period in fifty patients with cerebrovascular disease. Change in stenosis was measured with duplex ultrasonography. Ultrasound scans were done at six months, twelve months, and yearly thereafter. Bilateral duplex ultrasonography revealed apparent carotid atherosclerotic regression in seven and progression in two of the 25 tocotrienol patients, while none of the control group exhibited regression and ten of 25 showed progression (P < 0.002). Serum thiobarbituric acid reactive substances, an ex vivo indicator of maximal platelet peroxidation, decreased in the treatment group from 1.08 +/- 0.70 to 0.80 +/- 0.55 microM/L (P < 0.05) after 12 mon, and in the placebo group, they increased nonsignificantly from 0.99 +/- 0.80 to 1.26 +/- 0.54 microM/L. Both tocotrienol and placebo groups displayed significantly attenuated collagen-induced platelet aggregation responses (P < 0.05) as compared with entry values. Serum total cholesterol, low density lipoprotein cholesterol, and triglyceride values remained unchanged in both groups, as did the plasma high density lipoprotein cholesterol values. These findings suggest that antioxidants, such as tocotrienols, may influence the course of carotid atherosclerosis. PMID- 8614312 TI - Effects of long-distance running on serum bilirubin. AB - This study was undertaken to determine the effects of long-distance running on serum bilirubin fractions. Thirteen male ultramarathon runners participating in a 100-km race volunteered for the study. Venous blood samples were obtained shortly before and immediately after the race. Decreased serum haptoglobin levels (-66%) indicated the presence of hemolysis. After accounting for plasma-volume loss, significant post-race increases were found for creatine kinase (+20-fold), creatine kinase-MB (+252%), alanine aminotransferase (+42%), aspartate aminotransferase (+193%), gammaglutamyl transpeptidase (+56%), and glutamate dehydrogenase (+58%) serum activities, suggesting that running causes alterations of both muscle and liver tissues. Serum concentration of total bilirubin was significantly elevated (+106%) following the race, with changes corresponding to both unconjugated (+96%) and conjugated esters (+283%) of the pigment and significant increases in the ratio of esterified to total bilirubin. Our data show that long-distance running causes increases in the different serum bilirubin fractions which can be accounted for both hemolysis and hepatic disturbances. PMID- 8614313 TI - The prevalence and significance of post-exercise (postural) hypotension in ultramarathon runners. AB - A consistent finding in exercise-associated collapse is a marked postural fall in the systolic blood pressure associated with a tachycardia. The prevalence and significance of these post-exercise (postural) changes in blood pressure among noncollapsed ultradistance athletes has not been well documented. The aim of this study was to compare pre- and post-race changes in systolic and diastolic blood pressures with changes in body weight and plasma volume and with symptoms of post exercise hypotension, including the effects of posture, among a group of 31 runners competing in an 80-km footrace. During the race, runners developed a mean (+/- SD) weight loss of 3.5 (+/- 1.2) kg and plasma volume change of 12.8% (+/- 9.1). Asymptomatic postural hypotension defined as a fall in systolic blood pressure of greater than 20 mm Hg from the supine to the erect position without syncopal symptoms was present in two runners (7%) before the race and in 21 runners (68%) afterward. The degree of postural variation in systolic blood pressure was unrelated to changes in body weight or a fall in plasma volume. We conclude that (i) all runners were dehydrated by the race activity with a range of 1% to 7% and an average of 4.6%; (ii) asymptomatic post-exercise (postural) hypotension developed in the majority (68%) of ultramarathon runners in this study; (iii) the post-exercise hypotension is likely of multifactorial origin and is not entirely related to whole body dehydration or a reduction in plasma volume; and (iv) despite marked levels of dehydration among our sample of runners, their cardiovascular status in the supine position was not greatly compromised. PMID- 8614314 TI - Left ventricular ejection fraction during incremental and steady state exercise. AB - Despite extensive study of left ventricular (LV) function during incremental exercise (INC), there is little known about LV function during steady state exercise typical of that used during exercise training. In this study we evaluated LV ejection fraction (LVEF) during upright cycle ergometer exercise using first-pass radionuclide angiography (RNA). Healthy volunteers (N = 10) were studied during both INC and steady state. INC studies were performed at rest, at the ventilatory threshold (VT), and at maximal exercise. During steady state studies were performed after 10, 20, and 30 min of exercise at VT. During INC LVEF increased from rest (61% +/- 5%) to exercise at the VT (73% +/- 5%). There was no further change in LVEF at maximal exercise (73% +/- 5%). During steady state, LVEF increased from rest (61% +/- 5%), to exercise at VT (73% +/- 5%), with further increases after 20 (78% +/- 6%) and 30 (79% +/- 3%) min of exercise. The results suggest that LVEF is nearly maximal during submaximal exercise at VT. During steady state LVEF continues to increase with continuation of steady state exercise. PMID- 8614315 TI - Effects of carbohydrate type and concentration and solution osmolality on water absorption. AB - We studied intestinal absorption of solutions containing either one (glucose, Glu, or maltodextrin, Mal) or two (fructose, Fru, and Glu or sucrose, Suc) transportable carbohydrate (CHO) substrates using segmental perfusion technique in eight healthy male subjects. These CHO were either free or directly transportable monosaccharides (Glu, Fru), bound as the disaccharide (sucrose, Suc), or as oligomers (maltodextrins, Mal). [CHO] was varied from 6% to 8% (120 444 mmol.1(-1)). All solutions contained low [Na+] (15-19 mEq) and [K+] (3-4 mEq). Solutions osmolalities varied from 165 to 477 mOsm.kg(-1). Osmolalities in the test segment ranged from 268 to 314 mOsm.kg(-1). The regression line of osmolality with water absorption differed for single as compared with multiple substrate solutions. The significantly different intercepts of these two regression lines suggest that solutions with multiple substrates produce greater water absorption at a given osmolality than those with one. Comparing all solutions, test segment solute flux (partial r = 0.69) was more important than mean osmolality (partial r = 0.32). In conclusion, solutions with multiple substrates stimulate several different solute absorption mechanisms yielding greater water absorption than solutions with only one substrate. PMID- 8614317 TI - Expired air temperature during steady-state running. AB - While some metabolic measurement systems measure expiratory temperature to standardize gas volumes, other systems use only an estimate. This study investigated the effect of prolonged exercise on expiratory temperature near the pneumotachometer to provide a basis for its estimation when actual measurement is unavailable. Seven active females each performed two 45-min treadmill runs at identical speeds (64.5% +/- 11.8% of VO2max) in which the pneumotachometer heater control was either set to 37 degrees C or turned off. Expired air temperatures were monitored with thermocouples at the nonrebreathing valve (VAL) and 1 cm upstream (UPS) and downstream (DNS) from the pneumotachometer screens. There were no temperature differences over time for any of the conditions, and there were no differences in the VAL or UPS temperatures between the heated and unheated conditions. DNS temperature was higher during the heated condition at all time periods (P < 0.01). Mean DNS temperatures for the heated and unheated condition were 30.2 +/- 1.0 degree C and 27.9 +/- 1.1 degrees C, respectively. We conclude that expired air temperatures near the pneumotachometer remain stable during extended steady-state exercise regardless of whether the pneumotachometer is heated or not. PMID- 8614316 TI - Plasma catecholamine and ventilatory responses to cycling after propranolol treatment. AB - The purpose of the present study was to examine the relationship between minute ventilation (VE) and plasma concentrations of epinephrine (EPI) and norepinephrine (NE) during incremental cycling (20 W.2 min-1) performed under conditions of beta-adrenergic blockade (80 mg of propranolol) and placebo in six untrained male subjects. No significant differences existed between treatments in O2 uptake, CO2 output, blood lactate, pH, or VE during the submaximal work stages of incremental exercise common to both treatments (20 - 220 W). During exercise with beta-blockade, EPI, and NE concentrations were both significantly elevated compared with control levels at every submaximal work stage. Significant positive correlations between VE and plasma levels of EPI and NE were found during both beta-blockade (r = 0.98 and 1.00) and control conditions (r = 0.98 and 0.96). Although the high correlations were unchanged during exercise with beta-blockade, the slopes of the regression lines for the VE-EPI and the VE-NE relationships were both significantly reduced compared with control conditions. Beta-blockade resulted in elevated plasma levels of both EPI and NE compared with control conditions without causing a change in exercise VE. These findings suggest that catecholamines may not be important substances in regulating breathing during exercise. PMID- 8614318 TI - Physical inactivity and glucose intolerance in the multiethnic island of Mauritius. AB - The island nation of Mauritius, located in the southwest Indian Ocean, has a high prevalence of non-insulin-dependent diabetes mellitus (NIDDM) among all of its ethnic groups (Hindu and Muslim Indians, African-origin Creoles, and Chinese). These high rates of NIDDM among groups of varying genetic background provide strong support for the importance of environmental components in the etiology of the disease. Research in Mauritius using a simple activity scale has suggested that physical inactivity may be one of these components. The current investigation further examined the association between glucose tolerance and physical activity in middle-aged nondiabetic residents of Mauritius using a more extensive physical activity questionnaire (the Modifiable Activity Questionnaire). Excluding individuals with NIDDM, a statistically significant (P < 0.05) inverse relationship between physical activity and 2-h post-load glucose concentration was found for both males (rho = -0.14) and females (rho = -0.11). Stratifying by ethnic group, similar inverse correlations were observed in Hindu, Creole, and Chinese males, and in Hindu females (P < 0.05), as well as weaker relationships in Muslim males and Creole females (P < 0.10). Total physical activity remained an independent predictor of 2-h post-load glucose concentration after controlling for body mass index, waist-hip ratio, age, and family history of NIDDM. These data are supportive of a potentially important role of physical activity in the prevention of NIDDM in middle-aged inhabitants of Mauritius. PMID- 8614319 TI - Ethnicity affects aerobic fitness in US adolescent girls. AB - The purpose of this investigation was to determine whether aerobic fitness (VO2max) differed between black (N = 40) and white (N = 53) adolescent girls who were similar in age (13.5 yr) and percent body fat (24.6%). Expired gases were collected continuously while each girl performed a standard Bruce protocol to volitional exhaustion on a motorized treadmill (TM). Heart rates (HR) were measured during the exercise testing via telemetry. Fat-free mass (FFM) was estimated with total body electrical conductivity (TOBEC). Average (+/- SD) maximum HR (black = 194 +/- 7; white = 198 +/- 8) and respiratory exchange ratios (black = 1.17 +/- 0.08; white = 1.22 +/- 0.09) did not differ between subject groups. Aerobic fitness was significantly lower (P < 0.01) lower in black versus white girls when VO2max was expressed relative to body weight (31.8 +/- 5.8 vs 38.5 +/- 6.8 ml.kg(-1).min(-1)) and body weight raised to the 0.67 power (120.9 +/- 19.5 vs 138.5 +/- 20.7 ml.kg(-0.67).min-1). Treadmill time to exhaustion was significantly less (P < 0.01) in the black (8.49 +/- 1.30 min) versus white (9.41 +/- 1.60) subjects. Also, black subjects demonstrated less ability to utilize O2 during maximal exercise at a given FFM. This suggests the black girls' FFM contained a smaller percentage of skeletal muscle mass that could be utilized during treadmill exercise. It is possible that lower aerobic fitness values seen in the black girls are related to a combination of anatomical, physiological and/or behavioral factors. PMID- 8614320 TI - Physical activity and the initiation of high-risk health behaviors in adolescents. AB - The association of physical activity to the initiation of health risk behaviors was examined in a 3-yr prospective study of a population-based cohort of 1245 adolescents aged 12-16 yr. Four hundred thirty-seven students (35% of the cohort) were identified at baseline via self-report survey as never having smoked cigarettes, consumed alcohol, used marijuana, or carried a weapon. Three measures of physical activity were obtained at baseline: leisure-time physical activity (LTPA), level of aerobic fitness (AF), and participation in competitive athletics. Significant associations, with notable gender differences, were observed between physical activity and the initiation of cigarette smoking and alcohol use. The cumulative proportion of male students initiating alcohol use was 48%, 42%, and 24% for high, moderate, and low LTPA, respectively (P < 0.01). Males who participated in competitive athletics were significantly more likely than nonathletes to initiate alcohol use (44% vs 17%, P < 0.01). The cumulative proportion of female students initiating cigarette use was 10%, 23%, and 22% for high, moderate, and low LTPA, respectively (P < 0.05) and 7%, 28%, and 16% for high, moderate, and low AF, respectively (P < 0.05). No association was found between physical activity and weapon carrying. These results indicate that in this cohort of adolescents, the most active or most fit females were less likely to initiate cigarette smoking. In contrast, the most active males or males who participated in competitive athletics appeared more at-risk for initiating alcohol consumption than their less active counterparts. PMID- 8614321 TI - Associations among baseline physical activity and subsequent cardiovascular risk factors. AB - To determine stability of cross-sectional associations between physical activity and cardiovascular risk factors and provide information regarding possible independent effects of physical activity on reduced cardiovascular disease, this report examined associations among baseline physical activity and risk factors measured over 15 yr. Subjects were 1,379 Honolulu Heart Program participants who were evaluated at baseline and three subsequent examinations. For men initially 45-54 yr, higher physical activity level was significantly associated cross sectionally and at 2 yr with lower diastolic blood pressure, body mass index, and skinfold thicknesses, and at 5 yr with higher high density lipoprotein (HDL) cholesterol. By the 15-yr examination, only associations between physical activity level and skinfold thicknesses remained significant. For men 55-68 yr, significant cross-sectional and 2-yr associations were found between higher physical activity level and lower skinfold thicknesses, and higher HDL cholesterol at 5 yr. Higher physical activity continued to be associated with lower skinfold thicknesses and was related to lower systolic blood pressure by the 15-yr examination. Results suggest that most cross-sectional associations between physical activity and risk factors diminish over time, providing support for prospective investigations that find physical activity has a beneficial effect on reduced cardiovascular disease partially independent of traditional risk factors. PMID- 8614322 TI - The influence of a strength-sprint training sequence on multi-joint power output. AB - The purpose of this study was to determine whether adaptation to single- versus multi-joint strength training and sprint training was different and whether sequencing strength prior to sprint training was beneficial for increasing power. Thirty-two untrained males were assigned to control (C), sprint-sprint (SS), multi-joint (MJS), or single-joint (SJS) strength-sprint groups. Subjects were tested before training, after 8 wk of strength or sprint training, and after an additional 6 wk of sprint training. By mid-training both SJS and MJS increased 10 repetition maximum strength, but this was not transferable to isometric or isokinetic strength or rate of torque development. SS showed no improvement in these variables. All training groups increased cycle ergometer power output by 8 wk and had similar fiber hypertrophy with no EMG changes. Subsequent sprint training continued to increase maximum power with no further hypertrophy. Tibial nerve conduction velocity increased in all training groups. These results indicate little difference in adaptation to single- and multi-joint strength training. Strength or power improvements caused by training in these models does not transfer to isometric or isokinetic movements. Further, sequenced strength spring training provided no additional power gain over sprint training alone. PMID- 8614323 TI - Involvement of eccentric muscle actions in giant slalom racing. AB - Joint angular movements and muscle activation (EMG), were determined in male elite racers while performing the giant slalom. Movement cycles averaged 3.5 +/- 0.6 s (left plus right turn), and knee angle ranged 66-114 degrees (180 degrees = straight leg). Knee extensor muscle use was dominated (rectified EMG; P < 0.05) by the leg controlling the outside (downhill) ski during the turn. Time spent while decreasing knee angle (eccentric muscle action) of outside leg averaged 1.0 +/- 0.2 s. This phase was longer (P < 0.05) than the average push-off (concentric muscle action) phase of 0.5 +/- 0.1 s. Moreover, EMG activity of the outside leg during eccentric muscle actions exceeded (P < 0.05) that of concentric actions and was similar to that attained during maximum isometric knee extension in laboratory tests. Knee and hip angular movement ranged 20-50 degrees. Average joint velocities equalled 20-40 degrees.s(-1) during the turning phase. Thus, competitive giant slalom skiing is dominated by slow eccentric muscle actions performed at near maximum voluntary force. Because of their greater ability to generate force, eccentric muscle actions may be warranted or even required to resist the G-forces induced during the turn phase. PMID- 8614324 TI - Electrical stimulation and swimming performance. AB - The purpose of the study was to examine the influence of a 3-wk period of electrostimulation training on the strength of the latissimus dorsi m. and the swimming performances of 14 competitive swimmers divided into 7 electrostimulated (EG) and 7 control swimmers (CG). The peak torques registered during the flexion extension of the arm was determined with the help of an isokinetic dynamometer at different velocities (from -60 degrees.s(-1) to 360 degrees.s(-1)). Performances were measured over a 25-m pull buoy and a 50-m freestyle swim. For EG, a significant increase of the peak torques was measured in isometric, eccentric, and concentric conditions (P < 0.5). The swimming times declined significantly (P < 0.01) by 0.19 +/- 0.14 s, for the 25-m pull-buoy, and by 0.38 +/- 0.24 s, for the 50-m freestyle. For CG, no significant difference was found for any of the tests. For the whole group, the variations of the peak torques, measured in eccentric condition (-60 degrees.s(-1)) were related to the variations of the performances (r = 0.77; P < 0.01). These results showed that an electrostimulation program of the latissimus dorsi increased the strength and swimming performances of a group of competitive swimmers. PMID- 8614325 TI - Treadmill roller ski test predicts biathlon roller ski race results of elite U.S. biathlon women. AB - This study evaluated ski-specific laboratory testing and the relationship of test parameters to biathlon race performance. Since the ski skating technique used exclusively during biathlon racing requires a large upper body contribution, treadmill roller ski test parameters may provide relevant information for ski specific training and ski performance. Seven top U.S. female biathlon skiers performed lactate threshold and VO2 peak tests running (TR) and roller skiing (skating technique, RS) on a motor driven treadmill. In addition, a constant workload (14 km.h(-1), 6% grade) roller ski test to exhaustion at race pace (ET) on a motor driven treadmill, and a short-term double pole power test (DP) were also performed. Results indicate that lactate threshold VO2 and HR were significantly lower during RS than TR, DP power (w.kg(-1)) was related to RS VO2 peak (r = 0.90), but not to TR parameters. ET time to exhaustion was related to gross efficiency during ET (r = -0.86), RS VO2 peak (r = 0.80) and DP HR (% peak HR, r = 0.80). These results suggest that ski-specific laboratory testing provides better insight to ski performance than treadmill run testing. PMID- 8614326 TI - Evaluation of a new air displacement plethysmograph for measuring human body composition. AB - A new air displacement plethysmograph, the BOD POD (BP), was evaluated in comparison to hydrostatic weighing (HW). Sixty-eight adult subjects (26 F, 42 M) varying widely in age (range 20-56 yr), ethnicity, and fatness participated in this study. Same-day test-retest reliability was assessed in a subsample of 16 subjects (9 F, 7 M) and validity was assessed in all subjects (N = 68). The test retest coefficients of variation (CV) for %FAT measured by BP (%FATBP) and HW (%FATHW) were not significantly different (1.7% +/- 1.1% and 2.3% +/- 1.9% for BP and HW, respectively (mean +/- SD)), indicating excellent reliability for both methods. Validity of percent fat measured by the BP (%FATBP) was also excellent. The mean difference in %FAT (BP - HW) was -0.3 +/- 0.2 (SEM), with a 95% confidence interval of -0.6 to 0 %FAT. The regression equation (%FATHW = 1.86 + 0.94 %FATBP; r2 = 0.93, SEE = 1.81) was not significantly different from the line of identity (%FATHW = %FATBP), and did not differ by gender. These findings indicate that the BOD POD is a highly reliable and valid method for determining %FAT in adult humans in comparison to HW. This new method has several advantages over HW in that it is quick, relatively simple to operate and may be able to accommodate special populations such as the obese, elderly, and disabled. PMID- 8614327 TI - A new air displacement method for the determination of human body composition. AB - A new device based on the plethysmographic measurement of body volume has been developed for the purpose of estimating human body composition. The device, the BOD POD Body Composition System, uses the relationship between pressure and volume to derive the body volume of a subject seated inside a fiberglass chamber. Derivation of body volume, together with measurement of body mass, permits calculation of body density and subsequent estimation of percent fat and fat-free mass. Critical issues which have hampered prior plethysmographic approaches are discussed. The present system's ability to measure the volume of inanimate objects was evaluated for accuracy, reliability, and linearity. Twenty successive tests of a known volume (50,039 ml) on two separate days produced values of 50,037 +/- 12.7 ml and 50,030 +/- 13.5 ml (mean +/- SD) for each day, respectively. The CV for these series were 0.025% and 0.027%. Further testing across a wide range of volumes approximating human size (25-150 1) produced the following regression equation where y = measured volume (1) and x = actual volume (1): y = 0.9998x - 0.0274, r2 = 1.0, SEE = 0.004 1. The resultant device is likely to enhance opportunities for the quick, simple and noninvasive measurement of body composition for both research and clinical applications. PMID- 8614328 TI - Gas mixing apparatus for determining cardiac output by CO2 rebreathing. AB - The carbon dioxide rebreathing technique is widely used for determination of cardiac output during exercise. The equilibration method of Collier et at. (J. Appl. Physiol. 9:25, 1956) is generally preferred over the exponential method of Defares (J. Appl. Physiol. 13:159, 1968). However, the equilibration method requires the volume and initial CO2 percentage in the rebreathing bag to be adjusted according to the work rate. A device for mixing two gases (100% O2 and 20% CO2/80% O2) was constructed for this purpose. Multistage regulators are attached to the gas tanks and connected to a medical gas mixer via high-pressure air hoses. A variable time-delay switch causes a solenoid valve to open for 1.0 10s to deliver a predetermined gas volume. The device was found to accurately deliver a preset volume and concentration of gas to the rebreathing bag. A gas mixing apparatus simplifies the equilibration CO2 rebreathing technique by allowing the investigator to easily select the initial volume and percentage of CO2. PMID- 8614329 TI - The start in speed skating: from running to gliding. AB - The purpose of this study was to describe the push-off kinematics in speed skating using three-dimensional coordinates of elite male sprinters during the first part of a speed skating sprint. The velocity of the mass center of the skater's body VC, is decomposed into an "extension" velocity component VE, which is associated with the shortening and lengthening of the leg segment and a "rotational" velocity component Vr, which is the result of the rotation of the leg segment about the toe of the skate. It can be concluded that the mechanics of the first strokes of a sprint differ considerably from the mechanics of strokes later on. The first push-offs take place against fixed location on the ice. In these "running-like" push-offs the contribution of Vr in the forward direction is larger than the extension component Ve. Later on, the strokes are characterized by a gliding push-off in which Ve increases. In these gliding push-offs no direct relation exists between forward velocity of the skater and the extension in the joints. This allows skaters to obtain much higher velocities than can be obtained during running. PMID- 8614330 TI - The hospice movement matures. PMID- 8614331 TI - Haemophilia--darkest hours before the dawn. PMID- 8614332 TI - Genetic screening and primary health care. PMID- 8614333 TI - Continuity of care: in search of the Holy Grail of general practice. PMID- 8614334 TI - Limited adverse occurrence screening: using medical record review to reduce hospital adverse patient events. AB - OBJECTIVES: To determine whether continuous detection of adverse patient occurrences followed by analysis and medical intervention can alter the rate of adverse occurrences. DESIGN AND PARTICIPANTS: 15912 patients discharged from one hospital were reviewed in two stages. Medical records were screened retrospectively by medical records staff for one or more of eight general patient outcome criteria. Those that screened positive for the criteria were reviewed by one of four doctors. If an adverse occurrence was confirmed, further analysis and recommendations for action to prevent its recurrence were made at meetings of the four doctors, and forwarded to a committee of visiting medical officers who decided on the appropriate course of action. SETTING: A rural base hospital in Horsham, Victoria, between July 1991 and June 1994. MAIN OUTCOME MEASURES: The rate and severity of adverse patient occurrences in each year. RESULTS: 1465 records were screened positive for one or more criteria, and an adverse patient occurrence was confirmed in 155. 88 cases were determined to be minor or not preventable and further action (mostly by changes to hospital policies) was recommended for the remaining 67. Over the three years, the number of adverse occurrences fell from 69 (1.35% of all patient discharges in the first year) to 33 (0.58% of all patient discharges in the third year) (P < 0.0001) and there was no significant change in severity. CONCLUSIONS: The rate of adverse patient occurrences can be significantly reduced by their continuous detection using retrospective screening in conjunction with review, analysis and action to prevent recurrences. PMID- 8614335 TI - Sequential continuity of care by general practitioners: which patients change doctor? AB - OBJECTIVE: To identify individual and social characteristics of patients making sequential visits to a different rather than the same general practitioner (GP). METHOD: Data for this study were extracted from the "Record Linkage Pilot Study" of the National Centre for Epidemiology and Population Health, which linked information from personal interviews with Health Insurance Commission and National Heart Foundation Risk Factor Survey data. Each sequence of visits (any two consecutive visits) made by each participant to the same or a different GP from January 1991 to December 1992 was treated as an event. PARTICIPANTS: 521 subjects aged between 23 and 72 years who gave consent to release of Health Insurance Commission data. MAIN OUTCOME MEASURE: A visit to the same GP or a different GP from the one seen at the last visit. RESULTS: Logistic regression analysis showed that younger age, good physical functioning, good self-rated health, normal body mass index, shiftwork and a longer time interval between visits were significantly associated with less continuity of care. CONCLUSIONS: Our study raises questions about the relationship between chronological continuity and quality of care. For example, if infrequent visits (associated with less continuity) are for distinct illnesses, is quality of care affected by information or treatment from a previous visit? Our results also suggest that some GPs, because of the demography of their practices (more young people, a higher proportion of shift workers), may be disadvantaged by continuity-based reward systems. Moreover, because of lack of continuity young people may miss out on GPs' health promotional activities. PMID- 8614336 TI - Suspected snakebite in children: a study of 156 patients over 10 years. AB - OBJECTIVE: To describe the epidemiology and clinical features of children presenting to an emergency department with suspected snakebite. DESIGN: A retrospective study of patient records. SETTING: An emergency department of a children's teaching hospital (Princess Margaret Hospital) in Perth, Western Australia. PARTICIPANTS: All children attending the emergency department from 1984 to 1993 with suspected snakebite. MAIN OUTCOME MEASURE: Clinical and laboratory evidence of envenomation. RESULTS: Over the decade studied, 156 children (mean age, six years and eight months) presented with suspected snakebite; over two-thirds (68%) were boys. In at least 31% of cases, no appropriate first aid had been applied. Only 14 children were envenomed according to clinical and laboratory criteria: 10 of these had coagulopathy; one of the 10 also had rhabdomyolysis. A Venom Detection Kit was used in 117 children. The test gave a positive result in 21 children (13%). Antivenom was given to 18 children, 14 of whom were definitely envenomed. Four of the envenomed children returned a negative result of Venom Detection Kit testing at all sites tested, and in five patients not clinically envenomed the urine specimen tested positive with the Venom Detection Kit (presumably a false positive result or subclinical envenomation). Of the 156 children, 130 were admitted to hospital, and 26 were discharged directly from the emergency department. All children recovered completely. CONCLUSIONS: (i) Many children did not receive appropriate first aid for snakebite; (ii) Most children with suspected snakebite presenting to the emergency department were not envenomed; (iii) Envenomation was best diagnosed by clinical features and laboratory investigations, with the Venom Detection Kit being used to determine the appropriate antivenom; (iv) Discharging children directly from the emergency department is not recommended. PMID- 8614337 TI - Cystic fibrosis carrier screening in two New South Wales country towns. AB - OBJECTIVE: To assess the feasibility of offering community testing for carrier status of delta F508, a gene mutation associated with cystic fibrosis (CF). DESIGN: Prospective pilot survey. SETTING: General practice, the two main high schools and workplaces in the country towns of Young and Harden (combined population, 14,940; with 7707 people aged 16-55 years) in New South Wales (NSW). PARTICIPANTS: Individuals aged 16 years and over. MAIN OUTCOME MEASURES: Number of delta F508 carriers, test uptake rates, mode of learning about the testing, motivation for testing, retention of knowledge about CF, and test results and emotional effects of knowledge about carrier status. RESULTS: We tested 610 people (8% of the population aged 16-55 years) and identified 47 carriers (20% of the expected number in the 7707 people aged 16-55 years). Testing in schools had the highest uptake. Retention of knowledge was high; all delta F508-positive individuals recalled their carrier status accurately. Anxiety was transient among carriers; over 90% of all respondents felt they had made the right decision to be tested. CONCLUSIONS: We recommend community testing for carrier detection and suggest targeting those with a family history of CF and girls aged over 16 in high schools. PMID- 8614339 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.8 HIV and musculoskeletal disease. AB - Prompt and accurate diagnosis of musculoskeletal disease in HIV infection should result in effective therapy, although some of the usual treatments are contraindicated. PMID- 8614338 TI - Locally acquired Brachylaima sp. (Digenea: Brachylaimidae) intestinal fluke infection in two South Australian infants. AB - Eggs of a small intestinal trematode were found in the faeces of two 21-month-old children from the same rural district of South Australia who presented 18 months apart with mild abdominal pain and diarrhoea. Treatment with praziquantel resulted in egg clearance and resolution of gastrointestinal symptoms. A brachylaimid intestinal trematode involving the common house mouse, poultry and introduced European helicid snails is well established in South Australia. Both infants had been seen eating raw snails, and snails from their environment were found to be infected with metacercariae of a brachylaimid trematode. PMID- 8614340 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.9 HIV-related cardiovascular disease. AB - Heart disease is common in HIV, but often asymptomatic. As improved therapies prolong the lives of HIV-infected patients, an increase in symptomatic heart disease can be expected. PMID- 8614341 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.10 HIV and renal disease. AB - Renal parenchymal and fluid and electrolyte abnormalities may complicate hospitalisation in 20% of patients. HIV-specific renal abnormalities are being defined, but most follow opportunistic infection or drug nephrotoxicity. Principles of treatment are largely the same as for patients without HIV. PMID- 8614342 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.11 HIV and eye disease. AB - Blindness is a threatening complication of HIV infection, but one that can be prevented by correct diagnosis and management of ocular disease. The general practitioner can play a vital role in diagnosis by being familiar with the types of ocular disease seen at different stages of HIV infection. PMID- 8614343 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.12 HIV and haematological disease. AB - Patients with HIV infection may present with symptoms and/or signs of anaemia, neutropenia or thrombocytopenia at all stages, including seroconversion. PMID- 8614344 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.13 HIV-related lymphoma and other malignancies. AB - Treating systemic malignancies requires specialist skill and experience, with active treatment often providing the best initial palliation of symptoms. A full management plan involving general practitioner, patient and others is essential at this stage of HIV disease. PMID- 8614345 TI - Non-melanoma skin cancer. PMID- 8614346 TI - The safety of topical beta-blockers in glaucoma treatment. AB - While adverse effects are uncommon in patients who are otherwise fit and well, doctors should be aware of the implications of the systemic effects of these drugs, particularly the non-selective types, and particularly in the elderly. PMID- 8614347 TI - Trauma in pregnancy and cerebral palsy: is there a link? PMID- 8614348 TI - The Professional Indemnity Review: what did it accomplish? AB - In a previous article,* Richard Tjiong criticised the Professional Indemnity Review's specific recommendations for reform of professional indemnity insurance. But the Review covered many other issues, particularly to do with identifying, evaluating and reducing adverse outcomes of medical procedures. Charlotta Blomberg highlights some of the key findings (and failings) of the Review's Final Report. PMID- 8614349 TI - Laparoscopic cholecystectomy: what national benefits have been achieved and at what cost? PMID- 8614350 TI - Is a syringe exchange feasible in a prison setting? PMID- 8614351 TI - Screening for diabetic retinopathy. PMID- 8614352 TI - Starting SSRI antidepressant therapy: its effect on tricyclic antidepressant and benzodiazepine prescribing. PMID- 8614353 TI - Abnormal folate metabolism and neural tube defects. PMID- 8614354 TI - Hepatitis B surface antigenaemia in a child after vaccination. PMID- 8614355 TI - Rural practice: is the correct message getting through? PMID- 8614356 TI - After-hours service availability. PMID- 8614357 TI - Fasciola hepatica liver infection in a Victorian dairy farmer. PMID- 8614358 TI - Serious envenomation from the bite of a "harmless" snake. PMID- 8614359 TI - Mysteclin V in the treatment of rheumatoid arthritis. PMID- 8614360 TI - Snakes and ladders: getting patients into metropolitan public hospitals. PMID- 8614361 TI - To smell a rose. PMID- 8614362 TI - Treatment outcome and dose-response relationship in infants younger than 1 year treated for retinoblastoma with primary irradiation. AB - Thirty-three infants ( < 1 year at diagnosis) were treated for retinoblastoma with primary irradiation at St. Jude Children's Research Hospital (SJCRH) between 1963 and June 1992. Staging of the 44 treated eyes was as follows: Reese Ellsworth (R-E) Groups I (n = 20), Group II (n = 9), Group III (n = 6), Group IV (n = 2), Group V (n = 7). Irradiation was delivered using either a single anterior field (31 eyes) or lens-sparing techniques (13 eyes). Total doses ranged from 21-45 Gy (median = 36 Gy) in fractions of 150-180 cGy (n = 34) or > 180 cGy (n = 10). One child died of metastatic disease at 42 months. Three patients have developed second malignant neoplasms; two have succumbed at 88 and 125 months post-RB diagnosis; the remaining patients are alive at 6-259 months postdiagnosis (median follow-up = 127 months). Local control with irradiation alone and supplemented cryotherapy given within 2 months (n = 2) was maintained in 29 eyes, with no statistical difference seen for total doses < or = 36 Gy (21/8 eyes) vs. > 36 Gy (8/16). Of 15 eyes that required salvage therapy, tumor control has been maintained in 13. Enucleation was required for four patients, two with recurrent retinoblastoma and one with a phthisical eye. Cataract formation was documented in 23 eyes (87.5%) treated with lens-sparing techniques developed cataract. At last follow-up, 23 of 30 patients tested (77%) had visual acuity of 20/100 or better. This experience confirms early observations in that doses > or = 36 Gy do not appear to improve local control with irradiation alone in infants ( < 365 days) with retinoblastoma. PMID- 8614363 TI - Prognostic variable in patients with advanced colorectal cancer treated with fluorouracil and leucovorin-based chemotherapy. AB - Possible prognostic variables for tumor response, time to progression (TTP), and survival in 141 patients with advanced colorectal cancer treated with fluorouracil and leucovorin-based chemotherapy were analyzed. None of the variables examined for their possible influence on tumor response attained significance in the stepwise logistic regression. In the univariate analysis, variables found to be strongly associated with TTP were performance status (PS) (P = 0.0301), liver involvement (P = 0.030), and the initial values of WBC (P = 0.0319), lactic dehydrogenase (LDH; P = 0.0053), gamma-glutamyl-transpeptidase (gamma-GT; P = 0.0013), alkaline phosphatase (ALP; P = 0.0186), albumin (P = 0.0004), and carcinoembryonic antigen (CEA; P = 0.0014). In the Cox analysis, liver involvement (P = 0.0553), albumin (P = 0.0181), PS (P = 0.484), and ALP (P = 0.0553) were retained as independently significant variables. When only patients with liver metastases were included in the analysis, then only albumin (P < 0.001) demonstrated a prognostic significance. Also, in the univariate analysis, variables predicting survival were PS (P = 0.0230), grade (P = 0.00600), liver involvement (P = 0.0002), LDH (P = 0.0001), gamma-GT (P < 0.001), ALP (P = 0.0006), albumin (P = 0.0309), and CEA (P = 0.005). With the multivariate analysis, gamma-GT (P = 0.0004), albumin (P = 0.0634), and CEA (P = 0.0804) were selected as significant. In those patients who presented with liver involvement, variables predicted survival were gamma-GT (P = 0.0041), albumin (P = 0.0442), and the percentage of involved liver parenchyma (P = 0.0690). These results could be helpful for the stratification of future trials in advanced colorectal cancer. PMID- 8614364 TI - Survival of childhood leukemia in Singapore. AB - The objective of this study was to evaluate the treatment outcome of children with acute leukemias at a university hospital in Singapore. Between January 1988 and January 1994, 66 children were treated, comprising 13 cases of acute myeloid leukemia (AML) and 53 acute lymphoblastic leukemia (ALL). The 2-year disease-free survival (DFS) was computed according to the Kaplan-Meier method. The results showed that the survival of AML was poor, with a 2-year DFS of only 30%. The major cause of death for AML was leukemia and leukemia-related complications, such as hemorrhage and severe infections. In contrast, a 62% 2-year DFS was achieved for ALL. It was found that marked hepatosplenomegaly (enlarged liver and/or spleen > or = 10 cm below the costal margin) at presentation correlated with a significance shortened survival in our patients with ALL. The major cause for treatment failure in ALL was recurrence of disease. We conclude that the DFS for our patients with ALL at 2 years was fair. The treatment results for AML were poor, but the numbers are too small to make any definite conclusions. PMID- 8614365 TI - Compensatory hypertrophy and progressive renal damage in children nephrectomized for Wilms' Tumor. AB - Clinical, biochemical, and sonographic evaluation of the remaining kidney function and size was performed in 34 patients, 12 males and 22 females, ages 2.1 19.6 years, nephrectomized (NP) for Wilms' tumor (WT) at least 2 years before (mean 8.6). All patients had normal blood pressure and serum bicarbonates. Two of them had microhematuria, four proteinuria 4 mg/m2/hr, and 11 microalbuminuria (MA) > 20 mg/24 hr. Only one patient had reduced creatinine clearance and maximum bipolar length (MBL) as well as kidney volume (KV) < 100% of expected. In the other patients, average MBL was 128 +/- 11% (P = 0.0001). MBL, but not KV, was inversely correlated (P = 0.04) to age at NP. KV, but not MBL, was directly correlated (P = 0.009) to MA. Average MA was 48 +/- 94 mg/24 hr and was correlated to the time from NP (P = 0.026). The remaining kidney increases in volume much more than in length. The increase in KV is related to the degree of MA, whereas the increase in MBL is higher in subjects younger at NP. The high prevalence of significant MA, which is in turn related to the time from NP and to the KV, raises some concerns about the long-term renal prognosis of children NP for WT. PMID- 8614366 TI - Monitoring neovascularity as an indicator to response to chemotherapy in osteogenic and Ewing sarcoma using magnetic resonance angiography. AB - Histologic studies on resected specimen have shown that tumor neovascularity is related to prognosis and response to therapy in a variety of human neoplasms. In nine patients with osteogenic or Ewing sarcoma, we evaluated the use of magnetic resonance angiography (MRA) to assess neovascularity non-invasively in vivo and to monitor response to chemotherapy. Seven patients with osteosarcoma and two patients with Ewing sarcoma were studied before and after chemotherapy by MRA (2 D time-of-flight gradient-echo sequence, TR = 50 msec, TE = 9.5 msec, theta = 50 degrees, acquisition time 13 min). MR angiograms were assessed for chemotherapy induced changes in neovascularity. MRA showed both feeder vessels and neovascularity. Six patients responded to chemotherapy ( > or = 90% histologic tumor necrosis). MRA demonstrated marked reduction in neovascularity in all responders. Three patients did not respond to chemotherapy ( < 90% histologic tumor necrosis). MRA demonstrated persistent or increased neovascularity in the non-responders. MRA provides a unique opportunity to study tumoral neovascularity noninvasively in vivo and helps to assess response to chemotherapy in patients with osteogenic or Ewing sarcoma. These general principles may be applicable to other human tumors. PMID- 8614367 TI - Langerhans cell histiocytosis in children under 2 years of age. AB - This is a retrospective study of 55 children under the age of 2 years diagnosed with Langerhans cell histiocytosis (LCH). They were classified according to age and organ function and dysfunction following Lahey's criteria. The studied population was divided into four groups by age of diagnosis (0-6, 7-12, 13-18, and 19-24 months). Statistical analysis showed no significant difference in outcome between age groups, although the population under 6 months had a 81.3% fatality rate. The presence of organ dysfunction was a major cause of death in all age groups, being statistically significant in outcome (P > 0.005) compared with patients without organ dysfunction. The presence of thrombocytopenia and/or respiratory dysfunction was also highly associated with a fatal outcome. In the surviving population, no second malignancies have been reported. The late secondary effects of therapy include endocrine, orofacial, and osseous pathologies. PMID- 8614368 TI - Errors involving pediatric patients receiving chemotherapy: a literature review. AB - A review of mishaps involving pediatric patients receiving anticancer chemotherapy was undertaken in order to assist intervention. Although the case literature is too sparse to provide definite recommendations, suggestions for management are made in the event of an error with a high risk (based on the case literature) of life-threatening toxicities. It is recommended that all incidents be reported in the literature in order to provide a basis for devising standard treatment protocols. It is also suggested that studies using animal models continue to be done in order to provide more experimental data about toxicities and potentially beneficial rescue therapies. PMID- 8614369 TI - Nasopharyngeal carcinoma in children. PMID- 8614370 TI - Secondary malignancies in a child with Hodgkin's disease: T-cell lymphoma and myelodysplastic syndrome evolving into acute nonlymphoblastic leukaemia. AB - Hodgkin's disease (HD) has been linked to an increase risk of second malignant neoplasms (SMN), especially non-Hodgkin's lymphoma (NHL) and acute nonlymphoblastic leukaemia (ANLL). The mutagenic property of cytotoxic therapy as well as defective immunity have been implicated as playing a major role in the development of SMN in patients previously treated for HD. We report a case of a 14-year-old girl with HD who developed two different second malignancies within a latent period of 28 months following HD diagnosis. The patient presented initially with bilateral cervical and supraclavicular as well as mediastinal and paraaortic lymphadenopathy. She was staged as IIIA, nodular sclerosing type HD, and was given eight alternative cycles of MOPP-ABVD followed by "mantle" field radiotherapy to a total dose of 3.3 Gy plus 0.4 Gy to the upper mediastinum. Within 8 months following the completion of therapy, a period of myelodysplasia and progressive severe immune deficiency, considered as a result of initial treatment, occurred. Eighteen months after HD diagnosis while the patient was continuously neutropenic and heavily immunocompromised, a peripheral T-cell lymphoma of the angiocentric immunoproliferative lesion type (AIL) Grade III, appeared in both lungs within and beyond the radiation field, with no evidence of HD in biopsy specimens. After institution of a new chemotherapy regimen (L17M), a satisfactory response regarding NHL lesions was noted. However, 10 months later the myelodysplastic syndrome (MDS) accompanied by complex chromosomal abnormalities evoluted to frank ANLL with a rapid fatal course. This case supports the hypothesis that combined modality treatment accompanied by severe immunodeficiency may result in the development of multiple second malignancies even within a very short latent period, especially in a subgroup of HD patients who may be particularly increased risk of second cancers. PMID- 8614371 TI - Diffuse plasmacytosis in a child with brainstem glioma following multiagent chemotherapy and intensive growth factor support. AB - The use of granulocyte colony-stimulating factor (G-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) in order to abrogate chemotherapy induced neutropenia has become a routine part of many cancer treatment regimes. However, there are still very few data available about possible complications related to repeated or prolonged use of these agents in patients with malignant solid tumors. The authors report a child with brainstem glioma who received repeated cycles of multiagent chemotherapy with G- or GM-CSF support. During this period of 10 months, no clinical side effects were observed that could have been attributed to growth factor administration. However, postmortem histological examination revealed the presence of diffuse plasmacytosis, a rare hematological disorder in childhood. Undifferentiated plasma cells of nonmonoclonal origin could be demonstrated infiltrating bone marrow, lungs, and lymph nodes of the patient. Based on previously published in vitro and in vivo evidence on the interleukin-6 (IL-6)-mediated stimulatory effect of G- and GM-CSF on myeloma cell proliferation, the authors suggest a possible link between extensive growth factor support and the development of plasmacytosis in this patient. PMID- 8614372 TI - Second malignant tumours in childhood Hodgkin's disease. AB - This study was undertaken to determine the treatment-specific incidence of second malignant tumours (SMT) in childhood Hodgkin's disease. The institutional databases at The Hospital for Sick Children, the Princess Margaret Hospital, and the Toronto-Bayview Regional Cancer Centre were reviewed for the years 1958-1993. Three hundred and forty-three consecutive newly diagnosed children were evaluated. The overall 30 year cumulative SMT incidence was 31%. The 20 year SMT incidence was greater for patients who relapsed (n = 129), 27%, compared with patients who remained relapse free (n=214), 13%. For patients with stage 1-3B disease who remained relapse free, the 10 year SMT rate was 7% for patients who were surgically staged and treated with extended field radiation treatment (EF RT) (35 G), compared with 3% in clinically staged patients treated with MOPP (six cycles) and EF RT (25-30 G). To date there is no significant difference in the oncogenicity of these treatment protocols. However, EF RT alone was less effective in disease control. For stages 1-3B, 62% of patients relapsed after EF RT alone compared with 18% after bimodal treatment. Therefore treatment intensification due to relapse was more frequent in the former group. The overall 10 year SMT incidence for patients treated with these protocols was 11% and 3%, respectively. The 20 year SMT incidence following EF RT alone was 24%. We conclude that SMTs were a common late complication in childhood Hodgkin's disease and are a limiting factor in the achievement of cure. The incidence of SMTs was increased in children who required retreatment and was minimal in children who remained in a first complete remission. Therefore the initial treatment strategy in childhood Hodgkin's disease must be to minimize the risk of relapse, in order to avoid the morbidity and mortality associated with both relapse and SMT induction, and to achieve this objective with a primary treatment protocol of low oncogenicity. PMID- 8614373 TI - Granulocyte-colony-stimulating factor after allogeneic and autologous bone marrow transplantation in children. AB - We evaluated the use of granulocyte CSF (G-CSF) after both allogeneic BMT (allo BMT) and autologous BMT (ABMT) in children. After allo-BMT, G-CSF was used in 15 children who were compared with 20 historical controls. The ABMT patients were two sequential groups: the G-CSF group of 13 children and 11 historical controls. The patients were conditioned with different high-dose chemotherapy regimens with or without total body irradiation. G-CSF was administered at 5 micrograms/kg/day s.c. and was continued until an absolute neutrophil count (ANC) of 1,000 x 10(6)/l was reached. Following allo-BMT, G-CSF accelerated myeloid engraftment with a difference of 5 days at the ANC level of 500 x 10(6)/l (P<0.02) and 9 days at 1,000 x 10(6)/l (P<0.001). In the ABMT patients, G-CSF also accelerated myeloid engraftment. The difference between the G-CSF group and the control group was 6 days at ANC 200 (P<0.05), 11 days at ANC 500 (P<0.02) and 17 days at ANC 1,000 (P<0.005). In the ABMT patients, benefit by G-CSF was also observed in a smaller number of days with fever and days on antibiotics. We conclude that G-CSG significantly accelerated myeloid engraftment, after both allogeneic and autologous BMT in children, and also decreased the duration of febrile illness in the ABMT patients. PMID- 8614374 TI - Treatment of patients with pineoblastoma with high dose cyclophosphamide. AB - The outcome for patients with pineoblastoma has historically been very poor, with most patients dying of disseminated disease despite irradiation. Furthermore, the low incidence of this tumor has hindered progress toward defining better treatment strategies. Here we report the activity and toxicity of cyclophosphamide administered as a single agent at a dose schedule of 2 g/m2/day for 2 successive days at monthly intervals for a maximum of four courses. Eight patients were evaluated, six newly diagnosed and two recurrent. Amongst the six newly diagnosed patients, there were three patients demonstrating partial responses, and three had stable disease throughout the cyclophosphamide treatment period. All six patients are alive and disease free after further therapy. One patient with recurrent disease demonstrated tumor progression on cyclophosphamide, and the other had stable disease throughout the cyclophosphamide treatment period. Both patients subsequently died of progressive disease. The major toxicity of high dose cyclophosphamide was hematopoietic, with one patient requiring a dose reduction after three courses due to prolonged thrombocytopenia. One patient was also withdrawn from treatment with cyclophosphamide due to impaired pulmonary function. This study demonstrates the activity of high dose cyclophosphamide in the treatment of pineoblastoma and may serve as basis for the design of future studies of this tumor. PMID- 8614375 TI - Pain palliation with strontium-89 in children with metastatic disease. AB - We report two cases of children with metastatic bone disease who received strontium-89 intravenously. An 11-year-old boy with stage IV neuroblastoma received 50 microCi/kg of strontium-89. He had a good response, and his pain abated to the point that he could be taken off IV Dilaudid and was discharged from the hospital. A 7-year-old girl with the diagnosis of squamous cell carcinoma of the lung disclosed minimal increased uptake on a bone scan. Following the strontium-89 therapy, she did not have any significant improvement in pain, probably due to the minimal osteoblastic activity evidenced by the minimal abnormalities on the bone scan. Until this report there has been no reported case of using strontium-89 in the treatment of children with metastatic disease. PMID- 8614376 TI - Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia. AB - The importance of the cellular pharmacokinetics of cytarabine triphosphate (ara CTP) with regard to therapeutic efficacy is well established. In vitro and in vivo monitoring of pharmacokinetic parameters of leukemic blast cells were initiated in order to contribute to the pharmacological basis of optimal ara-C treatment strategies. Peripheral or bone marrow blast cells from 66 leukemic patients [51 acute myelogenous leukemia (ALL), 15 acute lymphoblastic leukemia (AML) were separated and incubated with ara-C for 1 hour and in ara-C-free medium for another 3 hours, and the intracellular formation and retention of ara-CTP was measured. In eight children who received continuous ara-C infusion for induction treatment, the ara-CTP concentration in circulating blast cells was monitored in vivo. The in vitro values observed in this assay corresponded to the cellular levels monitored in vivo. The ara-CTP retention differed clearly among the individual groups, as classified by immunophenotype at the time of the initial diagnosis: non-T-ALL 67+/-25% (x+/-SD, n=33), T-ALL 37+/-15% (n=8), and AML 34+/ 18% (n=14). The difference in ara-CTP retention between non-T-All and AML (P<0.05) as well as T-ALL (P<0.05) was significant. There was a tendency toward lower ara-CTP retention in relapsed as compared with newly diagnosed ALL, but the difference was not significant. The maximal accumulation of ara-CTP (after 1 hour incubation) was comparable in AML, T-ALL, non-T-ALL, and blast cells from children in relapse. The observed similarity of cellular accumulation in all groups and the significantly more rapid decrease in T-ALL and AML provide the pharmacokinetic rationale supporting the prolonged infusion duration for ara-C in these subgroups as an alternative to the intensification by high-dose ara-C schedules with short-term infusion. PMID- 8614377 TI - Prevention of indwelling central venous catheter sepsis. AB - In an attempt to decrease the incidence of central venous catheter sepsis in children with cancer, we conducted a study to evaluate the benefit of adding broad-spectrum antibiotics to the catheter "flush solution." In a prospective, placebo-controlled, double-blinded, randomized trial, 69 children with different types of malignancies were studied. The central venous catheters in these children were flushed with either the standard solution (normal saline + 100 U/ml of heparin) or the study solution (25 microgram/ml of both amikacin and vancomycin added to the standard solution). At the conclusion of the study, 64 children with a total of 67 indwelling central venous lines were assessable. The total catheter days on study were 20,700 days, with a median of 323 catheter days per patient. We documented 10 events of catheter-related infections (0.49 events/1,000 catheter days at risk). Five of these events were catheter-related sepsis (0.24 sepses/1,000 catheter days): two were fungal and three were bacterial. Due to the low incidence of catheter-related sepsis in this study, no statement regarding the prophylactic use of antibiotics could be made. The extremely low rate of catheter-related sepsis reported herein may be retrospectively attributed to continuous staff education regarding aseptic techniques in handling these catheters. Staff education is essential, and probably the most effective factor in preventing catheter-related sepsis. PMID- 8614378 TI - Alveolar rhabdomyosarcoma of the lip in an infant. PMID- 8614379 TI - Rothmund-Thomson syndrome and osteosarcoma. AB - The Rothmund-Thomson syndrome (RTS), also called poikiloderma congenitale is a rare autosomal recessive disease first described in 1868. This syndrome includes most frequently seen skin lesions (atrophy, telangiectases, pigmentation), cataracts and bone defects (dysostosis, dysplasia). Some authors describe an association with malignancy. We report three cases of Rothmund-Thomson syndrome associated with osteosarcoma. After cutaneous epithelioma, osteosarcoma is the most frequent malignancy. Thus, patients with RTS need a careful survey. The treatment did not differ from sporadic osteosarcoma. Chemosensitivity and toxicity are also not different. PMID- 8614380 TI - Acute tumor lysis syndrome in high grade lymphoblastic lymphoma after a prolonged episode of fever. AB - Acute tumor lysis syndrome occurs in rapidly growing lymphoid malignancies, usually as a consequence of chemotherapy or corticosteroids. We present what appears to be the first reported case of tumor lysis occurring after a sustained episode of high fever of 42.0 degrees C in a patient with high grade lymphoblastic lymphoma and a high tumor burden. PMID- 8614381 TI - Treatment of pediatric malignant thymoma: long-term remission in a 14-year-old boy with EBV-associated thymic carcinoma by aggressive, combined modality treatment. AB - Malignant thymoma, including thymic carcinoma, is extremely uncommon in the pediatric population. It is known to have a very poor outcome. We report on a 14 year-old boy with Epstein-Barr virus (EBV)-associated thymic carcinoma. Sections of the original tumor were analyzed for EBV by in situ hybridization to confirm the histological diagnosis of a lymphoepithelioma-like subtype. High copy numbers of EBV RNA were detected in the tumor tissue, suggesting an etiological role of EBV in our case. Intensive treatment resulted in long-term remission over 12 years. In order to facilitate the difficult management of the rare child with malignant thymoma, a literature search was initiated. Forty well-documented pediatric cases of malignant thymoma were found in the literature. Histological characteristics, clinical features, and therapeutic regimens were reviewed. Having the very limited experience with malignant thymoma in childhood in mind, it is concluded that its aggressiveness makes the most intensive treatment necessary. Long-term remission can be achieved by application of radical surgery, high-dose irradiation, and multiagent chemotherapy. The combination of cisplatinum, etoposide, and ifosfamide seems to be promising. PMID- 8614382 TI - Summary of the International Symposium on Childhood Non-Rhabdomyosarcoma Soft Tissue Sarcomas, Padua, Italy, February 10-12, 1994. PMID- 8614383 TI - "Chemotherapy for unresectable pancreatoblastoma". PMID- 8614384 TI - SIOP Working Committee on Psychosocial issues in pediatric oncology: guidelines for care of long-term survivors. AB - This is the third official document of the SIOP Working Committee on Psychosocial issues in pediatric oncology constituted in 1991. This document develops another topic already discussed and approved by the SIOP committee. The topic: "Care of long-term survivors" is addressed to the pediatric oncology community as guidelines that could be followed for considering this issue in a more appropriate way. PMID- 8614385 TI - Randomized comparison of moderate-dose methotrexate infusions to oral methotrexate in children with intermediate risk acute lymphoblastic leukemia: a Childrens Cancer Group study. AB - Methotrexate (MTX) infusions of 500--1,000 mg/m2 over 24 hours may improve survival and prevent relapse in children with acute lymphoblastic leukemia (ALL). Childrens Cancer Group (CCG) Study 139 compared weekly oral methotrexate 20 mg/m2/week (oral MTX) to MTX 500 mg/m2 infused over 24 hours (IV MTX) three times during consolidation and every 6 weeks during maintenance in 164 children with intermediate-risk ALL, i.e., those patients over age 1 year with white blood cell count 10,000 to 49,999/ml and no bulky extramedullary disease. Median follow-up for CCG-139 exceeded 75 months. Thirty-four events occurred among 80 patients receiving IV and oral MTX and 36 events among 84 patients receiving oral MTX. Two children died during induction and one did not enter remission. Remission induction rate is 98%. There have been 26 marrow relapses, 11 combined marrow and extramedullary relapses, 24 CNS relapses, and five testicular or other relapses. The frequency and distribution of relapses does not differ between the two regimens. For the entire group, overall event-free survival (EFS) at 6 years is 57.9% (standard deviation=4.0%) and actuarial survival is 80.0% (standard deviation =3.3%). Of the 29 patients with isolated extramedullary relapse, 18 survive free of a second event, a median of 42 months from relapse. In contrast to other trials, this trial does not show that IV MTX in this dose and schedule offers an advantage over standard therapy for this group of children. PMID- 8614386 TI - Elevated circulating levels of interleukin-1 receptor antagonist but not IL-1 agonists in hemophagocytic lymphohistiocytosis. AB - The familial form of hemophagocytic lymphohistiocytosis (HLH) is an inherited disease with disturbed immunomodulation and characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, and coagulopathy, i.e., findings which are similar to many of the reported biological effects of the inflammatory cytokines. Due to the previously shown hypercytokinemia in active HLH with elevated levels of interleukin (IL)-6, tumor necrosis factor-alpha, and interferon-gamma, it has been suggested that cytokine dysregulation may be of pathophysiological importance. Here we have assayed the serum levels of the members of the IL-1 ligand family, the two agonists IL-1 alpha and IL-1 beta and the antagonist IL-1 receptor antagonist (IL-1ra), in nine children with HLH and cerebrospinal fluid (CSF) specimens from four children. Serum IL-1ra was elevated in all patients with active disease to a degree which correlated well with disease activity. Furthermore, the levels decreased day by day during treatment of a patient who suffered a relapse. Moreover, high levels of IL-1ra were also detected in CSF during active disease. However, IL-1 beta levels were all within normal limits and circulating IL-1 alpha levels were normal in all but two patients. PMID- 8614387 TI - Ototoxicity in children with malignant brain tumors treated with the "8 in 1" chemotherapy protocol. AB - BACKGROUND: Adjuvant chemotherapy has improved the outcome of childhood malignant brain tumors in large randomized trials. With increasing survival rates, treatment toxicity has become a matter of concern. Radiation therapy and cisplatinum are known to be ototoxic. METHODS: We evaluated the incidence and factors predisposing to ototoxicity in children treated with the "8 in 1" chemotherapy protocol in Finland during 1986--1993. Thirty-five of the 82 children survived for at least 1 year after diagnosis. Thirty of these children were old enough to have an audiogram. RESULTS: Seventeen of the 30 children had normal hearing, seven had hearing loss at high frequencies, and six (20%) had severe hearing loss in the speech range. The risk factors for severe hearing loss were young age, a high cumulative dose of cisplatinum, and deteriorating renal function. In the presence of these factors, the risk of severe hearing loss was over 50%. Hearing loss at high frequencies could occur after low cumulative doses of cisplatinum, but severe hearing loss correlated with high cumulative doses. CONCLUSIONS: Cisplatinum-induced hearing loss at high frequencies is common, but hearing loss in the speech range also occurs, particularly in children with predisposing factors, and may progress insidiously and rapidly. Therefore a hearing test before each "8 in 1" course is important. PMID- 8614388 TI - Der(16)t(1;16)(q21;q13) in Wilms' tumor: friend or foe. AB - The der(16)t(1;16)(q21;q13) chromosomal abnormality has been reported rarely in Wilms' tumor. This abnormality has also been found in several other pediatric and adult neoplasms, and may imply a poor prognosis in at least some of these solid tumors. To investigate its clinical significance in Wilms' tumor, we examined the records of 65 consecutive children with Wilms' tumor whose tumor cells were successfully karyotyped. The t(1;16) was present in seven patients (10%) whose ages ranged from 2.5 to 4.7 years (median 3.5 years) at diagnosis. Six of the seven patients were female. All four stages of Wilms' tumor were represented (two patients had stage IV disease). No patient had bilateral disease. All tumors were of "favorable histology." All seven patients are alive and off therapy with a median follow-up of 3.2 years (range, 2 to 8.5 years). One patient with this abnormality developed brain metastases within 4 months of completion of therapy. Comparison of these patients with the remaining 58 Wilms' tumor patients revealed no significant differences with regard to disease stage, histology, survival, or relapse-free survival. Cytogenetic evidence of der(16)t(1;16)(q21;q13) in Wilms' tumor does not appear to portend an adverse clinical outcome, although only a larger study that includes molecular detection methods can provide more conclusive evidence. PMID- 8614389 TI - Successful treatment of childhood pilocytic astrocytomas metastatic to the leptomeninges with high-dose cyclophosphamide. AB - Leptomeningeal dissemination of childhood pilocytic astrocytoma (PA) is a rare event with little information available regarding therapy. We report here four children with disseminated PA whom we treated with high doses of cyclophosphamide with clinical benefit. The patients were aged 2.5 to 8 years. Three patients presented with PA localized in the posterior fossa, initially treated with surgical resection (n = 3) and radiotherapy (n = 1). Leptomeningeal dissemination occurred at 32, 44, and 8 months from diagnosis, respectively. The fourth patient presented with an optic pathway tumor with leptomeningeal dissemination at diagnosis. At commencement of cyclophosphamide therapy, disease was present in the subarachnoid space (intracranial, n = 2; spinal, n = 4), cerebral ventricles (n = 2), and primary site (n = 3). Histology was identical at diagnosis and recurrence in the two biopsied cases and cerebrospinal fluid was negative in all cases. Treatment was with cyclophosphamide 4-5 g/m2/cycle given every 4 weeks for a total of two cycles (n = 1) and four cycles (n = 3). One patient achieved disease stabilization (duration 27 months at the time of publication) and three patients experienced significant reductions in tumor burden. Subsequent intrathecal therapy was administered to two patients. Two patients developed disease progression at 10 and 9 months from cessation of chemotherapy. The one re treated patient responded to further, lower dose, cyclophosphamide. This is the first report of the use of high dose cyclophosphamide for disseminated PA. The recurrence of disease in two cases with a further response to lower dose cyclophosphamide has implications for the optimal duration of therapy for these low grade, aggressive tumors. PMID- 8614390 TI - Exploration of a self-protective strategy in pediatric oncology staff. AB - Oncology care is considered a stressful occupation. Little is known about the coping strategies oncology staff members use to deal with the stress of their work. The purpose of this study was to determine whether a self-protective strategy found in parents of children with cancer is also present in pediatric oncology staff. To explore this assumption, staff members' ratings were compared with the parents' ratings on the need for support and with the children's ratings on experienced pain. We also explored the characteristics which staff members attribute to the children and the parents. A total of 76 staff members, 84 children with cancer, and their 163 parents participated in the study. Both gender and number of years working in oncology care were positively associated with increased self-protective reactions in staff members. Male staff members rated medical procedures and the pain children experience in general as less painful than did female staff members. Their judgements about experienced pain in general and the lumbar puncture procedure in particular tended to be lower than the children's ratings. Female staff members attributed more positive characteristics to children with cancer and their parents than did male staff members. Staff members with more years of experience in oncology tended to rate all three medical procedures as less painful than those with less years of experience, and they also attributed more positive characteristics to the children. Staff members should be observant to their perceptions of children and parents, especially if their experience increases. PMID- 8614391 TI - Hyperbaric oxygen in the management of osteoradionecrosis. PMID- 8614392 TI - Secondary lymphoid malignancy in two children with neuroblastoma. AB - Secondary lymphoid malignancy, particularly acute lymphoblastic leukemia (ALL), is rare. We report one case of ALL and another case of mediastinal lymphoblastic lymphoma developed after treatment for neuroblastoma. The secondary ALL characterized by short latency period and an 11q23 translocation apparently was induced by etoposide treatment. The pathogenesis of the secondary lymphoma is less certain and may be related to previous treatment with cyclophosphamide and radiotherapy, host susceptibility, or chance occurrence. One child died of progressive lymphoma and the other remains in remission 1 year following allogeneic bone marrow transplantation. Additional studies are needed to determine the risk, pathogenesis, and optimal treatment for secondary lymphoid malignancy. PMID- 8614393 TI - Ifosfamide nephrotoxicity in children: histopathological features in two cases. AB - We report on two children with rhabdomyosarcoma who received ifosfamide as part of their chemotherapy schedule. Both children subsequently developed severe ifosfamide-induced nephrotoxicity, necessitating electrolyte supplementation. We describe the histopathological findings of renal biopsies performed in these children after the onset of renal dysfunction and comment on the possible mechanisms involved in ifosfamide nephrotoxicity. PMID- 8614394 TI - Ecthyma gangrenosum occurring at sites of iatrogenic trauma in pediatric oncology patients. AB - We report two cases of ecthyma gangrenosum which occurred at sites of iatrogenic trauma. The first case developed due to metastatic seeding with Pseudomonas aeruginosa during an episode of septicaemia and the second case occurred as a primary skin lesion. Both required prolonged courses of antibiotics and one patient died. The different pathogenic mechanisms and outcomes associated with this condition are discussed. PMID- 8614395 TI - Aluminum toxicity following intravesical alum irrigation for hemorrhagic cystitis. AB - Mental status changes in an immunosuppressed child can be due to a variety of causes; aluminum toxicity is rarely considered. We report a teenage girl with acute lymphoblastic leukemia who developed mental status changes, speech disturbance, coarse tremor, and abnormal EEG findings following intravesical 1% alum irrigation and administration of aluminum-containing antacids. Her serum aluminum levels were mildly elevated (14-22 milligram(s), normal 0-6 milligram(s)), and bone marrow biopsy specimens demonstrated aluminum deposition on special staining (Krueger's method). All abnormalities resolved after a nine week course of intravenous deferoxamine. PMID- 8614397 TI - Tobacco use and usual source of cigarettes among high school students--United States, 1995. AB - Approximately 90% of all initiation of tobacco use occurs among persons aged < or = 18 years, and the prevalence of tobacco use among adolescents is increasing. Despite laws prohibiting the sale of tobacco to minors in all states and the District of Columbia, most minors are able to purchase tobacco products. To determine current prevalences of the use of cigarettes and smokeless tobacco products (i.e., chewing tobacco and snuff) by high school students, the usual source of cigarettes among those who smoked, and the percentage of students who were asked to show proof of age when buying cigarettes, CDC analyzed data from the 1995 Youth Risk Behavior Survey (YRBS). This report summarizes the results of the analysis, which indicate a higher prevalence of smoking among high school students in 1995 than in 1993 and 1991, a doubling of the prevalence of current smoking among non-Hispanic black male students during 1991-1995, and that most high school students aged < or = 17 years who buy cigarettes from stores are not asked to show proof of age. PMID- 8614396 TI - Adjuvant chemotherapy of childhood posterior fossa ependymoma: cranio-spinal irradiation with or without adjuvant CCNU, vincristine, and prednisone: a Childrens Cancer Group study. AB - In 1975, members of The Children's Cancer Group (CCG) initiated a trial for patients with infratentorial medulloblastomas and ependymomas. Patients, all of whom received post-operative cranio-spinal irradiation (CSI), were randomized to receive or not receive adjuvant chemotherapy (CT) with lomustine (CCNU), vincristine, and prednisone for 1 year. Thirty-six of the 42 patients with ependymoma entered on study were suitable for analysis; 22 received combined modality therapy and 14 irradiation (RT) alone. The failure-free survival (FFS) for the entire sample at 10 years is 36% and overall survival (OS) 39%, with no difference in outcomes between the two regimens. Survival was better for females (73%) than males (21%) and for those older than 10 years (51% vs. 31%). There were two toxic deaths in the group receiving CT. We conclude from this study with long-term follow-up that the CT used was not effective in improving the outcome in children with ependymoma. PMID- 8614398 TI - Compliance with the Clinical Laboratory Improvement Amendments of 1988 for hemoglobin screening--California, 1995. AB - The Clinical Laboratory Improvement Amendments of 1988 (CLIA) established standards for improving the quality of clinical laboratory testing in the United States. One intent of CLIA was the regulation of smaller, provider-based laboratories, such as those operated by health-care providers in the Child Health and Disability Prevention (CHDP) program. In 1995, in conjunction with an assessment of county-specific variations in prevalence rates of anemia, the California Department of Health Services conducted a mail survey of CHDP providers to assess compliance with CLIA regulations for hemoglobin screening. This report summarizes the results of that survey, which indicate that, in California, many CHDP providers do not comply with CLIA-mandated quality assurance practices for hemoglobin screening in their clinical laboratories. PMID- 8614399 TI - Mercury exposure among residents of a building formerly used for industrial purposes--New Jersey, 1995. AB - Potential sources of elemental mercury in residential settings include mercury switches, mercury-containing devices (e.g., thermostats and thermometers), and mercury obtained from laboratories, dental offices, or other industrial sources. In January 1995, pools of elemental mercury were found in a five-story factory building that had been converted to residential use in Hoboken, New Jersey; the building previously had been used to manufacture mercury vapor lamps. This report summarizes the investigation by the New Jersey Department of Health (NJDOH), the U.S. Environmental Protection Agency (EPA), the Agency for Toxic Substances and Disease Registry (ATSDR), the Hoboken Board of Health, and the Hudson Regional Health Commission (HRHC), which identified high levels of mercury vapor in the building and indicated that residents had been exposed to high levels of mercury. PMID- 8614400 TI - Coordination of transcription of the human 17 beta-hydroxysteroid dehydrogenase type 1 gene (EDH17B2) by a cell-specific enhancer and a silencer: identification of a retinoic acid response element. AB - Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes primarily the reductive reaction of estrone to the biologically more active form, estradiol. The enzyme is highly expressed in the human placenta and the ovary and, in addition, in certain estrogen target cells, such as breast epithelial cells. To elucidate the transcriptional control of the EDH17B2 gene, the gene encoding 17HSD type 1, we fused a series of 5'-deletion mutants of the EDH17B2 gene into chloramphenicol acetyl transferase reporter gene vectors. An enhancer region was identified within the bases -661 to -392 and it increased, in both orientations, thymidine kinase promoter activity more than 200-fold in JEG-3 choriocarcinoma cells. This enhancer region was also functional in another choriocarcinoma cell line, JAR, although to a lesser extent. In BT-20 and T-47D breast cancer cells the enhancer region increased thymidine kinase promoter activity to some degree but not as efficiently as expected on the basis of endogenous enzyme expression. No such enhancer activity was observed in 17HSD type 1 nonexpressing cell lines. The retinoic acid responsive element, which was located between bases -503 and -487 in the EDH17B2 enhancer, bound retinoid acid receptor alpha retinoid X receptor alpha complex and transmitted retinoic acid induction on transcription in JEG-3 and T-47D cells. Finally, a silencer, functional in all the cell lines tested, was localized in the region from -392 to -78. Deletion of the region lad to a 4-fold increase in reporter gene expression. Altogether, our findings suggest that transcriptional control of the EDH17B2 gene is coordinated by the cell-specific enhancer and the silencer. PMID- 8614401 TI - Functional comparison of the Mus musculus molossinus and Mus musculus domesticus Sry genes. AB - The Sry gene functions as a genetic switch initiating testicular development of the indifferent mammalian gonad. The Mus musculus molossinus Sry open reading frame (ORF) encodes a 395-amino acid transcription factor (mSry) that specifically binds and bends DNA through its N-terminal HMG domain and activates transcription through its long C-terminal (residues 144-366) glutamine/histidine rich activation domain. The M. m. domesticus Sry ORF encodes a highly homologous, truncated protein (dSry) of approximately 230 amino acids, and the molecular basis for truncation is a point mutation that creates an amber stop codon within the activation domain. The mSry protein activates transcription of a Sry responsive reporter gene in HeLa cells, but dSry does not. Gene swapping and in vitro DNA binding experiments revealed that lack of transcriptional activation by dSry was not the result of polymorphisms within the first 137 amino acids of the protein. Direct analysis of the C-terminal glutamine/histidine-rich domain revealed that dSry lacked a functional transcriptional activation domain. Fusion of the GAL4 DNA-binding domain to the C-terminal deletion mutants of the GAL4 mSry chimeric protein indicated that residues 263-345 of the glutamine/histidine rich domain were necessary for high level transactivation. Furthermore, readthrough of the premature amber stop codon by transfer RNA suppression resulted in a strong GAL4-dSry transactivator. This demonstrated that the premature stop codon is the only polymorphism responsible for the inability of the dSry glutamine/histidine-rich region to transactivate. PMID- 8614402 TI - Hybridization of the complementary mRNAs for P450c21 (steroid 21-hydroxylase) and tenascin-X is prevented by sequence-specific binding of nuclear proteins. AB - The CYP21 gene that encodes the steroid 21-hydroxylase, P450c21, is overlapped on the opposite strand of DNA by the TX-X gene encoding the extracellular matrix protein, tenascin-X. These transcripts contain perfectly complementary segments of 299 bases at their 3'-ends. As these genes are tandemly duplicated and are transcribed in the adrenal cortex, we investigated whether these self complementary transcripts formed RNA-RNA hybrids in vivo. Formation of heterogeneous nuclear ribonucleoprotein complexes between nascent RNA transcripts and nuclear proteins might modulate such potential RNA-RNA interactions. Using a double RNase protection assay, we found that these RNAs form very small amounts of double-stranded RNA-RNA hybrids in adrenal cells in vivo. To understand why these mRNAs fail to hybridize in vivo, we studied the actions of nuclear proteins on the binding and annealing of their complementary regions in vitro. The nucleation of interstrand annealing was kinetically favored over binding and was efficiently promoted by nuclear extracts. However, RNA-RNA strand zippering was inhibited, suggesting that protein binding and/or stable RNA secondary structures contribute to discontiguous base pairing. Increasing concentrations of nuclear proteins increased the relative proportion of these RNAs in perfect RNase resistant duplexes but reached only about 20% of the total available RNA strands at saturating concentrations of nuclear proteins. Preincubation of either of the two single-stranded RNAs with nuclear proteins strongly inhibited the nucleation step of annealing, whereas preincubation of both strands abolished the annealing. RNase footprinting of the wild type and mutagenized overlapping transcripts suggested that sequence-specific binding of nuclear proteins is limited to the 5' half of each RNA strand. These results indicate that the transcription of complementary, opposite-strand RNAs is not a mechanism for the regulation of the abundance of adrenal P450c21 mRNA and suggest that nuclear proteins strongly interfere with interstrand RNA base pairing in vitro as well as in vivo. PMID- 8614403 TI - Constitutive expression of the orphan receptor, Rev-erbA alpha, inhibits muscle differentiation and abrogates the expression of the myoD gene family. AB - Rev-erbA alpha is an orphan steroid receptor that is expressed in skeletal muscle. Rev-erbA alpha binds to single/tandem copies of an AGGTCA motif, is transcribed on the noncoding strand of the c-erbA- alpha gene locus, and is postulated to modulate the thyroid hormone (T3) response. T3 induces terminal muscle differentiation and regulates fiber type composition via direct activation of the muscle-specific myoD gene family (e.g. myoD, myogenin). The myoD gene family can direct the fate of mesodermal cell lineages and activate muscle differentiation. Hence we investigated the expression and physiological role of Rev-erbA alpha during myogenesis. We observed abundant levels of Rev-erbA alpha mRNA in dividing C2C12 myoblasts, which were suppressed when the cells differentiated into postmitotic multinucleated myotubes. This decrease in Rev erbA alpha mRNA correlated with the appearance of muscle-specific mRNAs (e.g. myogenin and alpha-actin). Constitutive overexpression of full length Rev-erbA alpha cDNA in the myogenic cells completely abolished differentiation, suppressed myoD mRNA levels, and abrogated the induction of myogenin mRNA. We then demonstrated that 1) GAL4-REV-erbA alpha chimeras that contain the 'AB' region and lack the 'E' region activated transcription of GAL4 response elements in the presence of 8-Br-cAMP and 2) the ligand-binding domain (LBD) contains an active transcriptional silencer. Overexpression of Rev-erbA alpha (delta AB) in myogenic cells had no impact on the ability of these cells to morphologically or biochemically differentiate. Furthermore, this orphan receptor 1) down-regulated thyroid hormone receptor (TR)/T3 mediated transcriptional activity from the myogenin promoter and thyroid hormone response element (TRE) an 2) disrupted TR homodimer and TR/retinoid X receptor (RXR) heterodimer formation on a number of TREs found in the myoD gene family. In conclusion, Rev-erbA alpha functions as a negative regulator of myogenesis by targeting the expression of the myoD gene family. The mechanism of action may involve inhibition of functional TR/RXR heterodimer formation on critical TREs and dominant trans-repression of gene expression. PMID- 8614404 TI - TOR: a new orphan receptor expressed in the thymus that can modulate retinoid and thyroid hormone signals. AB - Vitamin A and other fat-soluble hormones and vitamins have important roles as modulators of essential biological processes such as homeostasis, development, differentiation, and oncogenesis and also as regulators of the immune system. The active form of vitamin A, retinoic acid, as well as vitamin D3 and thyroid hormones exert their actions by binding to specific nuclear receptors that represent one subfamily of the steroid/thyroid hormone receptor superfamily. To identify new members of the retinoid/thyroid hormone receptor subfamily that could play a role in the immune system, a screening of a T cell cDNA library was performed using a retinoid X receptor probe. A clone was isolated encoding a novel nuclear receptor expressed mainly in the thymus and T cell lines. This new receptor, TOR (thymus orphan receptor), is most closely related in both its DNA binding domain and ligand-binding domain, 90% and 53%, respectively, to ROR alpha/RZR alpha and clusters with these two receptors and RZR beta in a phylogenetic tree, when both the DNA-binding domain and the ligand-binding domain sequences of nuclear receptors are compared. Thus, TOR is part of a subgroup of receptors, one of which has recently been reported to be activated by melatonin. TOR binds specifically to a direct repeat of the half-site sequence 5'-AGGTCA-3' with a four- or five-nucleotide spacer, DNA sequences that also serve as binding sites for thyroid hormone (TR), and retinoic acid receptors (RAR). In transient transfection experiments TOR does not activate a reporter gene carrying these sequences in the absence or the presence of any known nuclear receptor ligands. TOR, however, is able to repress TR and RAR activity on DR-4-TREs or DR-5-RAREs, respectively. Therefore, our data suggest that TOR, similar to COUP-TF, can negatively regulate retinoic acid and thyroid hormone signals. However, the response elements recognized by TOR and COUP-TF differ as do the expression patterns of these receptors. Thus, one important role of TOR could be to modulate retinoid and thyroid hormone signals in the thymus. PMID- 8614405 TI - The isolation and characterization of MINOR, a novel mitogen-inducible nuclear orphan receptor. AB - The nuclear (steroid/thyroid/retinoid) receptor superfamily is a set of evolutionarily related ligand-inducible regulators of transcription. One subgroup within this family has been termed the orphan receptors because the potential ligands required for their activity have not been identified. We have cloned a novel orphan receptor, MINOR, which is mitogen inducible in a variety of cell types. Unlike NGFI-B/Nur77, another mitogen-inducible orphan receptor, MINOR gene expression is inhibited in Jurkat cells by the immunosuppressant cyclosporin A, suggesting that it is regulated by distinct second messenger pathways. The conservation of the DNA-binding domain between MINOR and other orphan receptors is reflected in the fact that they are able to bind to the same sequence, AAAG GTCA [termed the NBRE (NGFI-B response element)]. The marked divergence in other domains, particularly the N-terminal putative transactivation domain, may result in qualitative or quantitative differences in other functions among these proteins. One of these differences may be the apparent squelching of peak levels of MINOR-mediated transcription by supraoptimal levels of MINOR expression, an effect not obtained with NGFI-B/Nur77. When MINOR i coexpressed with submaximal levels of NGFI-B/Nur77, synergistic or additive levels of reporter gene expression are obtained. However, at maximal levels of NGFI-B/Nur77 expression, MINOR antagonizes the level of reporter gene expression in a dose-dependent fashion. These cooperative/competitive interactions, together with the nonidentical expression patterns of MINOR and NGFI-B/Nur77, suggest complexity in the regulation of genes responsive to orphan receptors which bind to the NBRE. PMID- 8614406 TI - The proline-rich region of the GH receptor is essential for JAK2 phosphorylation, activation of cell proliferation, and gene transcription. AB - Mutational analysis of the proximal transmembrane region of the cytoplasmic domain of the GH receptor (GHR) allowed us to characterize box 1, a proline-rich sequence of eight amino acids, which has been shown to be critical for signal transduction of many cytokine receptors. Mutants of the box 1 region of the rat GHR were studied for their ability to initiate the phosphorylation of JAK2 and the proliferation of stably transfected BAF B03 cells and also the activation of Spi 2.1 gene transcription in transiently transfected Chinese hamster ovary (CHO) cells. Convergence of effects of the box 1 mutants on JAK 2 phosphorylation, cell proliferation, and gene transcription was found. Our results suggest that no single amino acid in the box 1 sequence is essential for signaling and that the last two prolines (PXP motif) and the hydrophobic residues are necessary for integrity of box 1. Box 1 represents a structural determinant, potentially able to provide an interaction between JAK2 and the receptor; this interaction could be direct or indirect via an adaptor protein. PMID- 8614407 TI - Importance of extracellular domains for ligand binding in the thyrotropin releasing hormone receptor. AB - The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor. PMID- 8614408 TI - Functional expression of yoked human chorionic gonadotropin in baculovirus infected insect cells. AB - hCG is a glycoprotein hormone composed of an alpha-subunit, common to all gonadotropins and to TSH, and a hormone-specific beta-subunit. The non-covalent association of the two subunits is an obligatory step for the formation of biologically active hormones. The correct assembly of the heterodimer is also important for efficient secretion of the hormone, receptor binding, and signal transduction. Herein, we have demonstrated that expression of the two subunits from independent promoters present in a single recombinant baculovirus resulted in subunit association and secretion of biologically active holoprotein by the insect cells. To determine whether the active conformation of heterodimer could be achieved when the two subunits were synthesized in tandem on a single polypeptide chain, two single chain or yoked hCG1, the C-terminus of the complete beta-subunit (145 amino acid residues) was conjoined to the N-terminus of the alpha-subunit. Yoked hCG2 was similar, except that it contained the N-terminal 123 amino acid residues of the beta-subunit. Both yoked hCG molecules bound LH/CG receptor with high affinity and stimulated adenylate cyclase and progesterone levels in transformed mouse Leydig (MA-10) cells. Therefore, the alpha- and beta subunits are able to fold into a biologically active conformation when covalently linked. Interestingly, when compared with urinary hCG, the hormone expressed in baculovirus-infected insect cells binds to the LH/CG receptor with higher affinity, but exhibits diminished signaling, thus providing another example of a partial dissociation between receptor binding and activation. PMID- 8614409 TI - Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not of the lutropin/choriogonadotropin receptor. AB - It has recently been reported that Asp 397 of the rat lutropin/ choriogonadotropin receptor (rLHR) may be involved in transducing the signal from hormone binding to the stimulation of cAMP production. We examined the analogous region in the rat follitropin receptor (rFSHR) by substituting the Asp at position 404 (D404) of the rFSHR with either Glu (D404E), Ala (D404A), or Lys (D404K). Both in intact 293 cells and in detergent-solubilized extracts of 293 cells transiently transfected with the rFSHR constructs, only the wild type rFSHR exhibited detectable binding activity. Although the D404-substituted rFSHR mutants were visible on Western blots, in contrast to the wild type rFSHR which is present on Western blots as both mature and immature forms, only a single band comigrating with immature receptor was observed for the mutants. Furthermore, these mutants were sensitive to endoglycosidase H (Endo H), thus indicating that the mutant receptor proteins were retained intracellularly in the endoplasmic reticulum. To test whether the lack of binding of the D404-substituted rFSHR mutants was due to a perturbation of a binding site or to the intracellular retention of the mutants, a truncated rFSHR(t637) mutant, containing a cytoplasmic truncation that should not directly affect FSH binding, was examined. As with the D404-substitution mutants, rFSHR(t637) was stably expressed but sensitive to Endo H. Significantly, detergent-soluble extracts of cells expressing rFSHR(t637) were unable to bind FSH. From these results, we conclude that substitution of D404 of the rFSHR prevents hormone binding as a result of the intracellular retention of the mutants in the endoplasmic reticulum presumably in an incompletely folded state, as opposed to disruption of a hormone binding site at D404. Comparable rLHR substitution (D397K) and truncation (t616) mutants were constructed and used to transfect 293 cells. For both rLHR(D397K) and rLHR(t616), human CG (hCG) binding to intact cells was not detectable, but high affinity hCG binding was observed in detergent-soluble extracts of the cells. Therefore, the rLHR differs from the rFSHR in that mutants of the rLHR that are retained in the endoplasmic reticulum have already been folded correctly and can bind hCG with high affinity as long as a hormone-binding site has not been perturbed by the mutation. In contrast, mutants of the rFSHR that are retained in the endoplasmic reticulum have not yet folded into a conformation that can bind hormone. This suggests a difference in the temporal pattern of folding between the two structurally related gonadotropin receptors. Our studies also demonstrate how mutagenesis studies of the rFSHR must be interpreted with caution, as FSHR mutants that are expressed but are retained intracellularly will most likely not be able to bind FSH even when a hormone-binding site has not been altered. PMID- 8614410 TI - Repression of glycoprotein hormone alpha-subunit gene expression and secretion by activin in alpha T3-1 cells. AB - The alpha T3-1 cell line, a GnRH-responsive gonadotroph cell line developed by targeted oncogenesis in transgenic mice, was used to study regulation of the glycoprotein hormone alpha-subunit by activin. Transient transfection assays established that activin suppressed transcription of both the human and mouse alpha-subunit genes. Initial studies demonstrated that activin decreased transcription of -846 and -180 human alpha-subunit-luciferase constructs by about 30%, but that inhibin and follistatin were without effect. Subsequent studies to localize sequences mediating responses to activin were carried out using a series of 5'-deletions (-507 to -133) of the mouse alpha-subunit promoter fused to luciferase. The luciferase activity of the -507-base pair construct was decreased by 60-70% in the presence of activin, and follistatin prevented this decrease. There were significant stepwise losses of activin responsiveness when sequences between -507 and -424, -424, and -288, and -288 and -205 base pairs were eliminated. Clustered point mutations of the mouse alpha-subunit gene, shown previously to reduce basal expression and GnRH responsiveness, were tested to further identify sequences mediating activin repression. Constructs containing a mutated (-337 to -330) pituitary glycoprotein hormone basal element (PGBE) showed significant loss of activin responsiveness in the context of both the native promoter (-507 to +46) and a minimal promoter downstream of the -507 to -205 region of the mouse alpha-subunit gene, whereas mutation of sequences (-406 to 399) in the GnRH-response element had no effect. Multimers of the PGBE element ( 344 to -300) were insufficient to mediate a full activin response when linked to a minimal promoter. When added together with GnRH to transfected cells, activin abolished the stimulatory effect of GnRH on alpha-transcription. Secretion of free alpha-subunit by alpha T3-1 cells decreased 10-50% after exposure to activin for approximately 20 h, and steady state levels of alpha-subunit messenger RNA (mRNA) decreased by about 20-25% after 24-72 h. As changes in activin sensitivity could modulate its action, activin receptor II mRNA levels were measured by Northern blot hybridization at various times after activin (or inhibin) treatment. The three species of ActRII mRNA present in alpha T3-1 cells (approximately 6, 3, and 0.5 kilobases) were unaffected up to 72 h by these treatments. These observations provide the first demonstration that activin regulates a gonadotropin subunit gene at the level of transcription. Suppression of transcription of the mouse alpha-subunit gene by activin appears to involve several segments of the alpha-promoter, one of which is in the region of the PGBE. Thus, alpha T3-1 cells may provide a favorable system to further identify the DNA sequences and nuclear factors through which activin acts to alter transcription. PMID- 8614411 TI - Transduction of prolactin's (PRL) growth signal through both long and short forms of the PRL receptor. AB - The genes for the long form of the human and the short form of the mouse PRL receptors were transfected independently into NIH 3T3 cells. Reverse transcriptase-polymerase chain reaction indicated that the transfectant designated LFH contained message for only the long form and the transfectant designated SFM had message for only the short form of the receptor. Both transfectant cell lines specifically bound lactogenic hormones with high affinity and responded to PRL in culture with a 2- to 3-fold increase in cell number preceded by transient activation of mitogen-activated protein kinase. After a PRL responsive casein-chloramphenicol acetyl transferase (CAT) construct was introduced into both LFH and SFM cells, CAT activity was induced by PRL only in the LFH-CAT cells. Thus, while the long form of the receptor can transduce the differentiation signal, both the long and the short forms of the receptor can signal the cells to grow. PMID- 8614412 TI - Vascular endothelial growth factor and its receptors, flt-1 and flk-1, are expressed in normal pancreatic islets and throughout islet cell tumorigenesis. AB - Endocrine organs, such as the pancreatic islets of Langerhans, contain permeable, fenestrated endothelium that allows direct access of endocrine cells to the blood stream. Factors that control differentiation and maintenance of this highly specialized endothelium remain unknown. Vascular endothelial growth factor (VEGF) is a multifunctional growth factor that may be responsible for the homeostasis of endocrine endothelium; it is a selective mitogen for endothelial cells and is able to permeabilize endothelium. We have analyzed the expression of VEGF mRNA and protein in pancreatic islet cells of normal mice and during the different stages of tumor progression in a transgenic mouse model of beta-cell carcinogenesis. The 120-amino acid and the 164-amino acid isoforms of VEGF are expressed in normal islets of Langerhans and are moderately up-regulated during the stages of tumor development. Two high-affinity receptors for VEGF, flt-1 and flk-1, are expressed by endothelial cells both in normal islets and in the stages of tumorigenesis; these receptors are not up-regulated during this process. Our data raise the possibility that VEGF is involved in the maintenance of permeable endothelium in islets of Langerhans, an observation that may have implications for islet cell physiology and diabetes. While VEGF may also play an important role in the growth of new blood vessels during islet cell tumorigenesis, it cannot be the only factor required for the activation of tumor angiogenesis. PMID- 8614413 TI - CCAAT/enhancer-binding protein-alpha-dependent transactivation of CYP2C12 in rat hepatocytes. AB - Expression of the rat CYP2C12 gene is liver specific and is induced by GH at the transcriptional level. In primary cultures of rat hepatocytes, GH inducibility of CYP2C12 and the presence of C/EBP alpha protein were demonstrated to be equally dependent on attachment of the cells to an extracellular matrix gel (Matrigel). Transient transfection of a C/EBP alpha expression vector into hepatocytes, cultured without Matrigel, increased the cellular P4502C12 messenger RNA levels 10-fold. Cotransfection studies using deletion constructs of the CYP2C12 promoter fused to the luciferase reporter gene localized the C/EBP alpha response to the region -250 to -180. Sequence comparisons and deoxyribonuclease I footprinting using rat liver nuclear extracts indicated two potential C/EBP binding sites in this region. Mutagenesis of the most upstream element (-229 to -207) abolished transactivation by C/EBP alpha. Using gel mobility supershift assays, this element was demonstrated to bind C/EBP alpha and C/EBP beta in liver nuclear extracts and in lysates from hepatocytes cultured on Matrigel. GH treatment of the cells did not alter the C/EBP protein levels or the C/EBP-binding activity to this element. Neither did GH increase the expression of CYP2C12 reporter gene constructs regardless of the presence of different amounts of cotransfected C/EBP alpha. We conclude that C/EBP alpha is a potent transactivator of the CYP2C12 gene and most likely contributes to its liver-specific expression. Although the results presented here do not exclude the possibility of a GH-enhanced transactivating ability of C/EBP alpha, the mechanism of GH-induced levels of P4502C12 is not through increased levels of C/EBP alpha or via enhanced DNA binding activity of this transcription factor. PMID- 8614414 TI - Posttranscriptional regulation of primate Ldhc mRNA by its AUUUA-like elements. AB - The Ldhc locus encodes the testis-specific isozyme of lactate dehydrogenase in mammals. In our efforts to understand the regulatory mechanisms involved in expression of Ldhc, we recognized the possibility that this gene could be post transcriptionally regulated in certain species as the 3'-untranslated region (3' UTR) of Ldhc in primates, but not rodents, contains a number of AU-rich motifs and is conserved. To determine whether the primate Ldhc mRNA is posttranscriptionally regulated, comparison of baboon and mouse Ldhc mRNA stability was made in a cell-free system. The results indicated that the baboon mRNA is labile, while that of mouse, which does not contain the AU-rich motifs, is highly stable. Consistent with these results, the steady state level of primate Ldhc was found to be 8 to 12 fold lower than that of the mouse. We show that in a transformed murine germ cell line, the human Ldhc mRNA is moderately unstable, and removal of its 3'-UTR leads to stabilization of the mRNA. Mutations disrupting the AU-rich motifs of human Ldhc result in stabilization of the mRNA in vitro. On the basis of these observations, we conclude that stability of the primate Ldhc transcript is regulated by dispersed AU-rich elements found in its 3'-UTR. Because AU-rich motifs similar to these are found in many mRNAs, these findings may have broad implications. PMID- 8614415 TI - Effect of PML and PML-RAR on the transactivation properties and subcellular distribution of steroid hormone receptors. AB - PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid. PMID- 8614416 TI - Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation. AB - To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G1 cyclins and their cyclin-dependent kinase (CDK) partners were investigated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophosphorylated, growth inhibitory form of the retinoblastoma protein (pRB). This change began at 4-6 h, preceding effects on S phase. This suggests that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progression. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprecipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained constant over 24 h. Cyclin D1 messenger RNA and protein were down-regulated by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27KIP1 and p21WAF1/CIP1 at later times. These observations are compatible with the hypothesis that antiestrogens block entry of cells into S phase and inhibit cell proliferation as the consequence of an early decline in pRB phosphorylation contributed to by reduced cyclin D1/Cdk4 activity. At later times, increased CDK inhibitor abundance may act to repress Cdk2 and Cdk4 activities, causing additional reductions in pRB phosphorylation, thus maintaining the antiestrogen blockade of cell cycle progression. PMID- 8614417 TI - Vitamin D3-retinoid X receptor dimerization, DNA binding, and transactivation are differentially affected by analogs of 1,25-dihydroxyvitamin D3. AB - A number of analogs of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been synthesized that act as more potent inducers of cellular differentiation and inhibitors of cell growth than the natural ligand; at the same time, many of the analogs have reduced hypercalcemic properties. This combination makes these compounds attractive candidates for clinical use. The mechanism by which the analogs act, however, is unclear. Potentially, the analogs could be taken up more readily, be more slowly catabolized, or have higher binding affinities for the vitamin D receptor (VDR). Analogs of 1,25-(OH)2D3 could also differentially modulate one or more of the activities of VDR, namely dimerization, DNA binding, and/or transcriptional regulation. To directly examine this latter possibility, we used a sensitive assay for the kinetics of dimerization and DNA binding, surface plasmon resonance, and report here that three 1,25-(OH)2D3 analogs, 1,25 (OH)2-16-ene-23-yne-D3, 1,25-(OH)2-16-ene-23-yne-26,27-di home-D3, and 1,25-(OH)2 26,27-hexafluoro-16-ene-23-yne-D3, all confer distinct rate and equilibrium constants for VDR-retinoid X receptor heterodimerization and DNA binding to a specific vitamin D response element relative to the natural ligand. In response to the hexafluoro analog, the apparent Kd for DNA binding by VDR was significantly lower than that for 1,25-(OH)2D3, and correspondingly, in vivo transactivation from a responsive reporter was greater. Interestingly, solution heterodimerization was not affected by this analog. These results suggest that vitamin D analogs do indeed confer biological effects by acting directly and differentially at the level of VDR, and that specific vitamin D analogs can act on distinct receptor functions. PMID- 8614418 TI - Influence of hormone antagonists on chromatin remodeling and transcription factor binding to the mouse mammary tumor virus promoter in vivo. AB - Steroid hormones act via a group of high affinity receptors that regulate transcription by binding to hormone response elements located in the promoters of hormone-inducible genes. Our understanding of these processes has been greatly enhanced by the use of steroid hormone antagonists in both clinical and experimental procedures. However, despite their usefulness in these applications, much about their mechanisms of action remains to be elucidated. Using in vivo analysis techniques, we investigated the influence of type I (ZK98299) and type II (RU486 and ZK112993) steroid hormone antagonists on glucocorticoid-regulated transcription from the mouse mammary tumor virus promoter. Both type I and type II antagonists substantially reduced glucocorticoid-induced expression from the mouse mammary tumor virus promoter stably maintained as chromatin. Concurrent treatment with glucocorticoid and type I or type II antagonists reduced the receptor-dependent chromatin remodeling and loading of transcription factor NF1 that are signature responses of this promoter in the context of chromatin. Treatment with either type I or type II antagonists alone did not induce chromatin remodeling or transcription factor loading. Although type II antagonist occupied receptor can bind DNA, our results show that this binding is not functionally equivalent to that of agonist-occupied receptor, as it can not interact productively with the cellular apparatus required to open a repressive chromatin structure. PMID- 8614419 TI - The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group. AB - BACKGROUND: Controlled clinical trials have shown that beta-blockers can produce hemodynamic and symptomatic improvement in chronic heart failure, but the effect of these drugs on survival has not been determined. METHODS: We enrolled 1094 patients with chronic heart failure in a double-blind, placebo-controlled, stratified program, in which patients were assigned to one of the four treatment protocols on the basis of their exercise capacity. Within each of the four protocols patients with mild, moderate, or severe heart failure with left ventricular ejection fractions < or = 0.35 were randomly assigned to receive either placebo (n = 398) or the beta-blocker carvedilol (n = 696); background therapy with digoxin, diuretics, and an angiotensin-converting-enzyme inhibitor remained constant. Patient were observed for the occurrence death or hospitalization for cardiovascular reasons during the following 6 months, after the beginning (12 months for the group with mild heart failure). RESULTS: The overall mortality rate was 7.8 percent in the placebo group and 3.2 percent in the carvedilol group; the reduction in risk attributable to carvedilol was 65 percent (95 percent confidence interval, 39 to 80 percent; P < 0.001). This finding led the Data and Safety Monitoring Board to recommend termination of the study before its scheduled completion. In addition, as compared with placebo, carvedilol therapy was accompanied by a 27 percent reduction in the risk of hospitalization for cardiovascular causes (19.6 percent vs. 14.1 percent, P = 0.036), as well as a 38 percent reduction in the combined risk of hospitalization or death (24.6 percent vs, 15.8 percent, P < 0.001). Worsening heart failure as an adverse reaction during treatment was less frequent in the carvedilol than in the placebo group. CONCLUSIONS: Carvedilol reduces the risk or death as well as the risk of hospitalization for cardiovascular causes in patients with heart failure who are receiving treatment with digoxin, diuretics, and an angiotensin converting-enzyme inhibitor. PMID- 8614420 TI - The sequencing of chemotherapy and radiation therapy after conservative surgery for early-stage breast cancer. AB - BACKGROUND: Patients with early-stage breast cancer who are at substantial risk for systemic metastases are increasingly treated with breast-conserving therapy and adjuvant chemotherapy. However, the optimal sequencing of chemotherapy and radiation therapy is not clear. METHODS: Two hundred forty-four patients with stage I or II breast cancer who were at substantial risk for distant metastases were randomly assigned to receive a 12-week course of chemotherapy either before or after radiation therapy. All had had breast-conserving surgery. The median length of follow-up in surviving patients was 58 months (range, 10 to 124). RESULTS: The five-year actuarial rates of cancer recurrence at any site and of distant metastases in the radiotherapy-first group and the chemotherapy-first group were 38 percent and 31 percent (P = 0.17) and 36 percent and 25 percent (P = 0.05), respectively. Overall survival was 73 percent and 81 percent (P = 0.11), respectively. The five-year crude rates of first recurrence according to site in the radiotherapy-first and chemotherapy-first groups, respectively, were 5 percent and 14 percent for local recurrence and 32 percent and 20 percent for distant or regional recurrence or both. This difference in the pattern of recurrence was of borderline statistical significance (P = 0.07). CONCLUSIONS: This study suggests that for patients ar substantial risk for systemic metastases, it is preferable to give a 12-week course of chemotherapy followed by radiation therapy, rather than radiation therapy followed by chemotherapy. PMID- 8614421 TI - Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection. AB - BACKGROUND: Chlamydia trachomatis is a frequent cause of pelvic inflammatory disease. However, there is little information from clinical studies about whether screening women for cervical chlamydial infection can reduce the incidence of this serious illness. METHODS: We conducted a randomized, controlled trial to determine whether selective testing for cervical chlamydial infection prevented pelvic inflammatory disease. Women who were at high risk for disease were identified by means of a questionnaire mailed to all women enrollees in a health maintenance organization who were 18 to 34 years of age. Eligible respondents were randomly assigned to undergo testing for C. trachomatis or to receive usual care; both groups were followed for one year. Possible cases of pelvic inflammatory disease were identified through a variety of data bases and were confirmed by review of the women's medical records. We used an intention-to screen analysis to compare the incidence of pelvic inflammatory disease in the two groups of women. RESULTS: Of the 2607 eligible women, 1009 were randomly assigned to screening and 1598 to usual care. A total of 645 women in the screening group (64 percent) for chlamydia; 7 percent tested positive and were treated. At the end of the follow-up period, there had been 9 verified cases of pelvic inflammatory disease among the women in the screening group and 33 cases among the women receiving usual care (relative risk, 0.44; 95 percent confidence interval, 0.20 to 0.90). We found similar results when we used logistic regression analysis to control for potentially confounding variables. CONCLUSIONS: A strategy of identifying, testing, and treating women at increased risk for cervical chlamydial infection was associated with a reduced incidence of pelvic inflammatory disease. PMID- 8614422 TI - Brief report: hepatic dysfunction as a complication of adenosine deaminase deficiency. PMID- 8614423 TI - Images in clinical medicine. Inflammatory autobullectomy. PMID- 8614425 TI - Theophylline in asthma. PMID- 8614424 TI - The role of critical care nurses in euthanasia and assisted suicide. AB - BACKGROUND: Euthanasia and assisted suicide has received considerable attention recently in medical literature, public discussion, and proposed state legislation. Almost all the discussion in this area has focused on the role of physicians. However, nurses--especially critical care nurses--may be in a special position to understand the wishes of patients and to act on this understanding. METHODS: I mailed a survey to 1600 critical care nurses in the United States, asking them to describe anonymously any requests from patients, family members or others acting for patients (surrogates), or physicians to perform euthanasia or assisted suicide, as well as their own practices. RESULTS: Of the 1139 nurses who responded (71 percent), 852 said they practices exclusively in intensive care units for adults in the United States. Of these 852 nurses, 141 (17 percent) reported that they had received requests from patients or family members to perform euthanasia or assist in suicide; 129 (16 percent of those for whom data were available) reported that they had engaged in euthanasia; and an additional 36 (4 percent) reported that they had hastened a patient's death by only pretending to provide life-sustaining treatment ordered by a physician. Some nurses reported engaging in these practices without the request for advance knowledge of physicians or others. The method of euthanasia most commonly described was the administration of a high dose of an opiate to a terminally ill patient. CONCLUSIONS: As public debate continues about euthanasia and assisted suicide, some critical care nurses in the United States are engaging in the practice. PMID- 8614426 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 16-1996. A 36-year-old woman with bilateral facial and hand weakness and impaired truncal sensation. PMID- 8614427 TI - Beta-adrenergic blockers and survival in heart failure. PMID- 8614428 TI - The changing approach to the treatment of early breast cancer. PMID- 8614429 TI - Screening for chlamydia--a key to the prevention of pelvic inflammatory disease. PMID- 8614430 TI - Euthanasia and nursing practice--right question, wrong answer. PMID- 8614431 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614432 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614433 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614434 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614435 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614436 TI - Clinical problem-solving: identifying Addison's disease. PMID- 8614437 TI - Tissue plasminogen activator for acute ischemic stroke. PMID- 8614438 TI - Tissue plasminogen activator for acute ischemic stroke. PMID- 8614439 TI - Tissue plasminogen activator for acute ischemic stroke. PMID- 8614440 TI - Low-molecular-weight heparin for the treatment of acute ischemic stroke. PMID- 8614441 TI - Cardiac myxomas. PMID- 8614442 TI - Cardiac myxomas. PMID- 8614443 TI - Prenatal screening for Down's syndrome in Maine, 1980 to 1993. PMID- 8614444 TI - Chernobyl's legacy to science. PMID- 8614445 TI - Biotech industry woos MEPs to ease regulatory burden. PMID- 8614446 TI - Block grant opposed for NIH clinical centre. PMID- 8614447 TI - Chernobyl damage 'underestimated'. PMID- 8614448 TI - US broadens scientific base of controls on carcinogens. PMID- 8614449 TI - AIDS advisers disagree over events in HIV blood scandal. PMID- 8614450 TI - Interest ferments in yeast genome sequence. PMID- 8614451 TI - MRC faces negligence claims over growth hormone victims. PMID- 8614452 TI - Controversial AIDS vaccine fails to delay disease. PMID- 8614453 TI - 'Gene hunting' in India. PMID- 8614454 TI - 'Gene hunting' in India. PMID- 8614455 TI - 'Gene hunting' in India. PMID- 8614456 TI - Life in the hot zone around Chernobyl. PMID- 8614457 TI - How the galaxy keeps its halo. PMID- 8614458 TI - Medial temporal lobe imaging. PMID- 8614459 TI - Phylogenesis of prion protein. PMID- 8614460 TI - Role of leptin in fat regulation. PMID- 8614461 TI - Human minisatellite mutation rate after the Chernobyl accident. AB - Germline mutation at human minisatellite loci has been studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation was found to be twice as high in the exposed families as in the control group. Mutation rate in the Mogilev families was correlated with the level of caesium 137 surface contamination, consistent with radiation induction of germline mutation. PMID- 8614462 TI - A galactic chimney in the Perseus arm of the Milky Way. AB - Galaxies are surrounded by large haloes of hot gas which must be replenished as the gas cools. This has led to the concept of galactic 'chimneys'--cavities in the interstellar medium, created by multiple supernova explosions, that can act as conduits for the efficient transport of hot gas from a galaxy's disk to its halo. Here we present a high-resolution map of atomic hydrogen in the Perseus arm of our galaxy, which shows clear evidence for the existence of such a chimney. This chimney appears to have been formed by the energetic winds from a cluster of young massive stars, and may currently have reached the stage of bowing out into the halo. PMID- 8614463 TI - High levels of genetic change in rodents of Chernobyl. AB - Base-pair substitution rates for the mitochondrial cytochrome beta gene of free living, native populations of voles collected next to reactor 4 at Chernobyl, Ukraine, were estimated by two independent methods to be in excess of 10(-4) nucleotides per site per generation. These estimates are hundreds of times greater than those typically found in mitochondria of vertebrates, suggesting that the environment resulting from this nuclear power plant disaster is having a measurable genetic impact on the organisms of that region. Despite these DNA changes, vole populations thrive and reproduce in the radioactive regions around the Chernobyl reactor. PMID- 8614464 TI - Essential role of the posterior morphogen nanos for germline development in Drosophila. AB - In many animal groups, factors required for germline formation are localized in germ plasm, a region of the egg cytoplasm. In Drosophila embryos, germ plasm is located in the posterior pole region and is inherited in pole cells, the germline progenitors. Transplantation experiments have demonstrated that germ plasm contains factors that can form germline, and germ plasm also directs abdomen formation. Genetic analysis has shown that a common mechanism directs the localization of the abdomen and germline-forming factors to the posterior pole. The critical factor for abdomen formation is the nanos (nos) protein (nanos). Here we show that nos is also essential for germline formation in Drosophila; pole cells lacking nanos activity fail to migrate into the gonads, and so do not become functional germ cells. In such pole cells, gene expression, which normally initiates within the gonad, begins prematurely during pole-cell migration. Premature activation of genes in germline precursors may mean that these cells fail to develop normally. A function for nos protein in Drosophila germline formation is compatible with observations of its association with germ plasm in other animals. PMID- 8614465 TI - Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4. AB - A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro. PMID- 8614466 TI - Activation of medial temporal structures during episodic memory retrieval. AB - Medial temporal lobe structures have been implicated in human episodic memory. Patients with medial temporal lesions show memory deficits, and functional neuroimaging studies have revealed activation in this region during episodic encoding and retrieval when data are averaged over a sample of subjects. The relevance of such observations for memory performance has remained unclear, however. Here we have used positron emission tomography (PET) to examine cerebral blood flow related to verbal episodic retrieval. We observed strong positive correlations between retrieval and blood flow in left medial temporal structures in individual normal human subjects. In addition, multivariate analysis showed that regions in the left medial temporal lobe were dominant components of a pattern of brain regions that distinguished a high-retrieval condition from conditions of lower retrieval. These results suggest that medial temporal activity is related to retrieval success rather than retrieval attempt, possibly by reflecting reactivation of stored patterns. PMID- 8614467 TI - Prevention and reversal of renal allograft rejection by antibody against CD45RB. AB - Rejection continues to be the single largest impediment to successful organ transplantation. Antilymphocyte globulin, which contains antibodies that react with the leukocyte common antigen known as CD45, has proved to be one of the most effective agents for preventing rejection. We have shown earlier that a monoclonal antibody directed against the RB isoform of CD45 substantially inhibits the alloreactivity of human CD4+ lymphocytes in vitro. Here we investigate whether CD45RB could be an appropriate target for preventing renal allograft rejection in mice. Mice treated with two injections of a monoclonal antibody (MB23G2) raised against CD45RB protein all survived and had normal renal function. Furthermore, this antibody reversed acute rejection when therapy was delayed until day 4, and the mice survived for their natural lifespan. The immunosuppression achieved may find application in the prevention and treatment of transplant rejection in man. PMID- 8614468 TI - Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. AB - The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation. PMID- 8614469 TI - Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. AB - Binding of Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas bearing cells. The interleukin-1Beta-converting enzyme (ICE) is a cysteine protease that is involved in apoptosis induced by various stimuli, including Fas mediated apoptosis. Several ICE homologues have been identified, and these are subdivided into three groups (ICE-, CPP32-, and Ich-1-like proteases). We show here that specific inhibitors of ICE- or CPP32-like proteases can inhibit Fas mediated apoptosis. Transient ICE-like activity was found in the cytosolic fraction of Fas-activated cells, whereas ICE-dependent, CPP32-like activity gradually accumulated in the cytosol. Cell lysates from mouse lymphoma supplemented with either recombinant ICE or CPP32 induced apoptosis of nuclei. The CPP32 inhibitor inhibited ICE- or CPP32-induced apoptosis in the cell-free system, whereas the ICE-inhibitor only inhibited ICE-induced apoptosis. Cell extracts from thymocytes from ICE-null mice induced apoptosis in the cell-free system when it was supplemented with CPP32. These results indicate that Fas sequentially activates ICE- and CPP32-like proteases, and that downstream CPP32, together with a component(s) in the cytoplasm, causes apoptosis of nuclei. PMID- 8614470 TI - Structure of the UmuD' protein and its regulation in response to DNA damage. AB - For life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response'. For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD' and UmuC (umu: UV mutagenesis). Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD'. To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD' mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual Beta-structure associate to form molecular dimers, and extended amino-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response. PMID- 8614472 TI - Long-term correction of rat model of Parkinson's disease by gene therapy. PMID- 8614471 TI - Context-dependent secondary structure formation of a designed protein sequence. AB - Protein secondary structures have been viewed as fundamental building blocks for protein folding, structure and design. Previous studies indicate that the propensities of individual amino acids to form particular secondary structures are the result of a combination of local conformational preferences and non-local factors. To examine the extent to which non-local factors influence the formation of secondary structural elements, we have designed an 11-amino-acid sequence (dubbed the 'chameleon' sequence) that folds as an alpha-helix when in one position but as a beta-sheet when in another position of the primary sequence of the IgG-binding domain of protein G (GB1). Both proteins, chameleon-alpha and chameleon-beta, are folded into structures similar to native GB1, as judged by several biophysical criteria. Our results demonstrate that non-local interactions can determine the secondary structure of peptide sequences of substantial length. They also support views of protein folding that favour tertiary interactions as dominant determinants of structure. PMID- 8614473 TI - Role of hormonal-cytokine interactions in the formation of the humoral immune response. PMID- 8614474 TI - Neurophysiologic correlates of the decision-making processes in the cerebral cortex of monkeys during visual recognition. AB - The impulse activity of groups of neurons of the visual, prefrontal, and inferotemporal cortex was recorded simultaneously in behavioral experiments on monkeys solving a problem of delayed visual differentiation of variously colored stimuli. The neurophysiological correlates of the decision-making processes were studied. Erroneous motoric reactions were accompanied in all monkeys by significant reorganizations of the patterns of impulse activity of the neurons. In the case of correct solutions of the monkeys, synchronization in time and cross-correlation which was significant in magnitude between the activity of the groups of neurons were observed. Incorrect solutions were accompanied by desynchronization and by a decrease in cross-correlation between the activity of these groups of neurons. Possible decision-making mechanisms of the causes of the desynchronization of the informational processes during erroneous reactions are discussed. PMID- 8614475 TI - Circadian rhythm of fluctuations in the level of gonadoliberin in the hypothalamus of rats and the influence on it of various xenobiotics. AB - There are no regular patterns in the fluctuations of the content of gonadoliberin in relation to the time of day in the preoptic region and the median eminence in females and males. However, the existence of a well-marked circadian rhythm of the fluctuations of this neurohormone has been identified in females in the vascular organ of the lamina terminalis. Its level is extremely low in the morning; its level increases five-fold immediately after noon, and ten-fold at 1700-1800 h. This rhythm does not depend on the stage of the estrous cycle and is manifested identically both in diestrus and in proestrus. Circadian fluctuations in the content of gonadoliberin in the vascular organ of the lamina terminalis were not observed in males. The chronic exposure to various xenobiotics in low concentrations (toloul, benzol, dioxan, styrol, and lead) leads to a break in the circadian rhythm of the gonadoliberin content in the vascular organ of the lamina terminalis in females; this is expressed first and foremost in a significant increase in the level of this neurohormone in the morning hours. This is apparently associated with attenuation of inhibitory tone; this is indirectly confirmed by a simultaneous increase in the level of prolactin in the blood. PMID- 8614476 TI - Influence of leukotrienes B4 and C4, 12-hydroxyeicosatetraenoic acid, and erythro epimers of hepoxilin B3 on the plasticity of cholinoreceptors of neurons of the common snail. AB - The influences of a number of acyclic eicosanoids which are formed from arachidonic acid under the influence of 5- and 12-lipoxygenases on the plasticity of cholinoreceptors of neurons of the common snail, which was assessed on the basis of the degree of frequency depression of the amplitude of the inward current induced by local application of acetylcholine to the soma, were investigated in identified RPa3 and LPa3 neurons of the common snail using the method of bielectrode recording of the potential on the membrane. The plasticity of the cholinoreceptors was enhanced by leukotrienes B4 and C4, and was not altered by 12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid, or by the threo and erythro epimers of hepoxilin B3. An inference is drawn regarding the regulation of the plasticity of cholinoreceptors by leukotrienes B4 and C4, which are formed under the influence of 5-lipoxygenase. One of these pathways includes inositol-1,4,5-triphosphate-dependent mobilization of deposited Ca2+. PMID- 8614477 TI - Genetic features of the spatial organization of the human cerebral cortex. AB - The within-pair similarity of the topographical maps of the spatial synchronization of the cerebral cortical potentials was studied in 11 pairs of monozygotic twins and 20 genetically unrelated subjects in the resting state and during four types of intellectual activity. It was demonstrated that the degree of similarity of the topographical maps is higher in the resting state in the monozygotic twins than in the genetically unrelated subjects. No frequency band of the EEG which exerts a special influence on the formation of the high degree of similarity of the spatial synchronization of the potentials was found. The maximal similarity in the topographical maps of the monozygotic twins is observed in the anterior regions of the left hemisphere and in the temporoparietal regions of the right. Differences between groups are recorded in both regions; however they are especially distinct in the right hemisphere. The data obtained make it possible to draw an inference regarding the influence of a genetic factor not only on individual components of the EEG (which had been previously demonstrated by a number of authors), but also on the systemic organization of the cortical processes. The right-hemispheric activity apparently relates to the most controllable genetic processes. The results of the analysis of the similarity of topographical maps during various types of activity confirm this: the coefficient of similarity reaches the level of significance in the majority of pairs of monozygotic twins only in a "right-hemispheric" test (simultaneous analysis of nonverbal material). PMID- 8614478 TI - Topography of afferent and efferent flows in the mechanisms of auditory selective attention. AB - The auditory evoked potentials (AEP) were recorded in 10 healthy subjects under the conditions of simple rhythmic stimulation and in an attention task. The mid- (10-60 msec) and long-latency (up to 400 msec) AEP were analyzed by mapping the evoked potentials and by assessment of their spatial-temporal characteristics. It is hypothesized that the interaction of the parietal and left-sided temporal and frontal divisions exert an efferent influence on the brainstem structures in the 15-35 msec time segment. The amplitude of the P90 and P250 waves is reduced in the attention task. The amplitude of the late component P350 increases mainly in the central and anterior divisions of the brain. PMID- 8614480 TI - Varied influence of damage to the motor cortex on precision avoidance and escape reactions in dogs. AB - The influence of local extirpation of the representation of the forelimb in the motor area of the cortex on the performance of precision elevations of this limb was investigated. Even imprecise avoidance reactions to an acoustic signal did not recover spontaneously. Irregular motoric reactions which were insufficient in amplitude could easily be restored in the course of retraining, but precise movements (both rapid fused, as well as relatively slow stepwise movements) did not recover for at least a half year after the operation. By contrast with this, precise rapid reactions in response to electrical stimulation of a "working" limb (inducing unconditioned reflex flexion) remained essentially preserved, i.e., the capacity to stop the movement in the correct (previously learned) position of the limb was preserved. The capacity to fix the position of the raised limb was not lost, but was persistently impaired. The capacity to overcome the unconditioned reflex flexion during the performance of extensor "slow" escape reaction was reduced sharply and persistently. Limb presentation reactions regulated by vision were essentially spontaneously and nearly completely restored within a month. PMID- 8614479 TI - Influence of neuropeptides on the processes of higher nervous activity in primates. The action of cyclic analogs of enkephalins on the behavior of the lower monkeys. AB - The effect of two cyclic enkephalin analogs (IVS-43 and IVS-46) on the group and individual behavior of rhesus macaques and hamadryas baboons has been studied. A pronounced influence of IVS-46 on competitive and operant goal-directed behavior of the monkeys was identified. PMID- 8614481 TI - Dynamic shifts in the parameters of the traditional frequency range of the EEG during learning in dogs. AB - The parameters of the electrical activity of various regions of the neocortex (NC) and the olfactory bulbs (OB) of dogs were studied by means of correlation spectral analysis during the development of a food-dispenser paw pedal-pressing motor skill; a wide band of frequencies (1-100 Hz), which included both the traditional range (1-20 Hz) as well as high frequencies (HF), were used. Differences were demonstrated in the dynamics of the parameters in the NC and OB. The energy level of the potentials of the NC increased in the HF range (beta 3 and gamma), while in the OB it increased in the HF and alpha range. The coherence phase characteristics of the NC potentials suggest that more stable (by comparison with resting wakefulness) temporal relationships between the oscillations not only within the limits of HF, but also in the alpha band, are formed in the presence of the developed skill. At the same time, greater phase shifts are characteristic for the interrelationships of the OB potentials and those of the NC, and the increase in coherence relates only to the HF range, whereas the oscillations of the traditional range are characterized by a decrease in coherence. The possible role of the alpha range (along with the HF) in the processing of information which supports appropriate behavior is stressed. PMID- 8614482 TI - Influence of dilantin on the reactions of neurons of the sensorimotor cortex and hippocampus during learning. AB - The probability of movements in response to light flashes, the conditional signal of a defense reflex, did not alter against the background of the action of dilantin (D) in a dose which, according to published data, attenuates posttetanic potentiation (PTP). A weak but stable disinhibition of the motor reaction was observed in response to flashes turned on against the background of conditional inhibition, continuous light. The activation of neurons of sensorimotor cortex in response to reinforced and nonreinforced light flashes against the background of D was intensified in the same way as was observed previously as exemplified by the reactions of neurons of the visual cortex [3], while it was diminished in response to painful reinforcement. The acceleration of impulses in response to reinforced light flashes under the influence of D became weaker in the hippocampus, by contrast with neurons of the new cortex. It can be concluded on the basis of the data obtained that PTP does not participate in the storage in the new cortex of long-term memory of the biological significance of an activating stimulus. PMID- 8614483 TI - An investigation of hippocampal theta rhythm of rats as a nonlinear dynamic process. AB - The results of an investigation of the hippocampal theta rhythm of rats in the paradoxical phase of sleep and during orienting behavior by the method of estimation of attractor dimension are presented in this article. It has been demonstrated that the hippocampal theta rhythm consists of a regular constituent and an irregular constituent of high dimension, the interrelationship of which varies within wide limits depending upon the state of the animal. Frequency components of the regular and irregular constituents were identified. The mechanisms of their formation are considered and some hypotheses regarding their biological nature are proposed. PMID- 8614484 TI - Food-procuring behavior of rats under the conditions of chronic activation and blockade of the neostriatal dopaminergic system. AB - Using the technique of intracerebral microinjections, the features of food procuring behavior (the realization of a situational instrumental conditioned reflex in a Skinner box) were investigated in rats in experiments under the conditions of a pharmacological influence on the dopaminergic system of the neostriatum. Amphetamine in a dose of 15 and 45 micrograms and haloperidol in a dose of 5 mg were injected daily over the course of three weeks bilaterally into the rostral division of the neostriatum. A amphetamine dose of 15 micrograms was ineffective, but a increase in motor hyperactivity, a behavioral stereotypy, and a substantial acceleration of food-procuring movements were observed against the background of the injection of 45 micrograms of amphetamine. Stimulation of the dopaminergic system of the neostriatum qualitatively altered the behavioral strategy in animals with an initially low level of realization of the reflex; this fostered a stable activation of the instrumental skill enduring even after the cessation of the microinjections. Chronic blockade of dopamine receptors by haloperidol induced the reverse effect. PMID- 8614485 TI - Effectiveness versus efficacy of treatment of hypertension for stroke prevention. PMID- 8614486 TI - The neuromythology of silicone breast implants. AB - OBJECTIVE: To define neurologic problems that may occur in women with silicone breast implants. BACKGROUND: The association between silicone breast implants (SBIs) and certain rheumatologic disorders has been discussed since the 1980s. Recent uncontrolled case series have reported neurologic problems believed to be associated with SBIs. DESIGN: Case series based on a retrospective data analysis of medical records from 131 women diagnosed as having a neurologic problem related to SBIs. METHODS: Data extracted from the medical records and analyzed included neurologic symptoms, neurologic examination findings, and a variety of laboratory studies. Symptoms, examination findings, and laboratory studies were analyzed using methods that would purposely overreport false-positive results in order to negate possible bias accusations. Finally, prior diagnoses made by evaluating physicians and thought to be related to SBIs were also recorded. An independent assessment was also made for alternative diagnoses using standards accepted by the medical and neurologic communities which did not necessarily accept a causative link between SBIs and their alleged complications. RESULTS: Neurologic symptoms were frequently endorsed, including fatigue (82%), memory loss and other cognitive impairment (76%), and generalized myalgias (66%). Despite multiple complaints, most patients (66%) had normal neurological examinations. Findings reported as abnormal were mild and usually subjective, including sensory abnormalities in 23%, mental status abnormalities in 13%, and reflex changes in 8%. No pattern of laboratory abnormalities was seen, either in combination or in attempts to correlate them with the clinical situation. Laboratory studies appeared to be random without an attempt to confirm or correlate with a particular diagnosis. Diagnoses by physicians endorsing the concept that SBIs cause illness included "human adjuvant disease" in all cases, memory loss and other cognitive impairment ("silicone encephalopathy") and/or "atypical neurologic disease syndrome" in 73%, "atypical neurologic multiple sclerosis-like syndrome" in 8%, chronic inflammatory demyelinating polyneuropathy in 23%, and some other type of peripheral neuropathy in 18%. There was no coherence in making these diagnoses; the presence of any symptoms in these women was sufficient to make these diagnoses. Alternatively, after review of the data, no neurologic diagnosis could be made in 82%. Neurologic symptoms could be explained in some cases by depression (n=16), fibromyalgia (n=9), radiculopathy (n=7), anxiety disorders (n=4), multiple sclerosis (n=4), multifocal motor neuropathy (n=1), carpal tunnel syndrome (n=1), dermatomyositis (n=1), and other psychiatric disorders (n=3). CONCLUSIONS: There is no evidence that SBIs are causally related to the development of any neurologic diseases. Methods of diagnosis that have been used to make the diagnosis of neurologic disease in these patients are contrary to standards accepted by the neurologic community. Several possible explanations exist for the neurologic and other symptoms in women with breast implants. PMID- 8614487 TI - Optic neuropathy. PMID- 8614488 TI - Brain damage and postoperative hyponatremia: the role of gender. PMID- 8614489 TI - Testing cognition may contribute to the diagnosis of movement disorders. PMID- 8614490 TI - From enigmatic to problematic: the new molecular genetics of childhood spinal muscular atrophy. PMID- 8614491 TI - Hemorrhagic transformation of brain infarct: predictability in the first 5 hours from stroke onset and influence on clinical outcome. AB - OBJECTIVE: To identify, in the first 5 hours of acute brain infarct, clinical and radiologic predictors of subsequent hemorrhagic transformation (HT), and to evaluate its influence on the clinical course. BACKGROUND: The identification of early predictors of HT might be important to plan antithrombotic or thrombolytic treatments. PATIENTS: One hundred fifty consecutive patients with cerebral anterior circulation infarct systematically underwent a first CT within 5 hours of onset. During the first week after stroke, we performed a repeat CT or autopsy to look for HT. Outcome measures were early neurologic deterioration within the first week of onset and 30-day case fatality rate and disability. RESULTS: HT was observed in 65 patients (43%): 58 (89%) had a petechial HT and seven (11%) a hematoma. Among initial clinical an CT findings, the only independent predictor of HT was early focal hypodensity. Its presence was associated with subsequent HT in 77% of cases (95% CI, 68 to 86%), whereas its absence predicted the absence of subsequent HT in 94% of cases (95% CI, 89 to 99%). No baseline clinical or CT characteristic differentiated patients with petechial HT from those with hematoma. Antithrombotic and antiplatelet agents did not influence the occurrence of either type of HT. The frequency of early neurologic deterioration and of 30 day death or disability in HT patients was twice as high as in those without HT. However, a large-sized infarct and the presence of mass effect at the repeat CT or autopsy were the only factors independently linked to both the outcome events, irrespective of the development of HT. Clinical evolution of HT patients given antithrombotics was comparable with that of HT patients not receiving these drugs. CONCLUSIONS: HT of a brain infarct is a common event that occurs independently of anticoagulation and can be reliably predicted as early as 5 hours from stroke onset by the presence of focal hypodensity at CT. Apart from the infrequent cases of massive hematoma, HT does not influence prognosis, whereas a poor outcome in HT patients is correlated with a higher frequency of large edematous infarcts in this subgroup. The clinical course and final outcome of HT in anticoagulated patients does not differ from that of non-anticoagulated HT patients. PMID- 8614492 TI - Cerebellar hemorrhagic infarction. AB - We investigated 17 patients with 26 cerebellar hemorrhagic infarcts for their vascular anatomy, stroke mechanisms, and clinical course. Sixteen infarcts involved the superior cerebellar artery, nine the posterior inferior cerebellar artery, and one the anterior inferior cerebellar artery territories. The infarcts involved the full territory of the supplying arteries in 19 of 26 infarcts (73%). Sixteen of 17 patients were stable or improving when the hemorrhagic infarction was detected. All but one patient had an imaging study at the time of presentation that was negative for blood; hemorrhagic infarction was detected on routine serial scans performed during the first 15 days. Nine of the 17 patients were on anticoagulants when the cerebellar hemorrhagic infarct was detected; anticoagulation was maintained in eight of them with no clinical worsening. The stroke mechanism in all patients was considered embolic from cardiac and intra arterial sources. The causes, imaging findings, and consequences of hemorrhagic infarcts in the posterior circulation are similar to those in the anterior circulation. PMID- 8614493 TI - Population-based study of seizure disorders after cerebral infarction. AB - We performed the first population-based study that determined the magnitude of the risk and identified the factors predictive of developing seizure disorders after cerebral infarction. Five hundred thirty-five consecutive persons without prior unprovoked seizures were followed from their first cerebral infarctions until death or migration out of Rochester, Minnesota. Thirty-three patients (6%) developed early seizures (within 1 week), 78% of which occurred within the first 24 hours after infarction. Using multivariate analysis, the only factor predictive of early seizure occurrence was anterior hemisphere location of infarct (odds ratio 4.0; 95% CI 1.2 to 13.7). Twenty-seven patients developed an initial late seizure (past 1 week), whereas 18 developed epilepsy (recurrent late seizures). Compared with the population in the community, the risk during the first year was 23 times higher for initial late seizures and 17 times higher for epilepsy. The cumulative probability of developing initial late seizures was 3.0% by 1 year, 4.7% by 2 years, 7.4% by 5 years, and 8.9% by 10 years. Independent predictive factors on multivariate analysis for initial late seizures were early seizure occurrence (hazard ratio of 7.8 [95% CI 2.8 to 21.7]) and stroke recurrence (3.1 [1.2 to 8.3]). Both early seizure occurrence (16.4 [5.5 to 49.2]) and stroke recurrence (3.5 [1.2 to 10.5]) independently predicted the development of epilepsy as well. We also found that early seizure occurrence predisposed those with initial late seizures to develop epilepsy. PMID- 8614494 TI - Cranial nerve palsy in spontaneous dissection of the extracranial internal carotid artery. AB - Cranial nerve palsy was present in 23 of 190 consecutive adult patients (12%) with spontaneous dissection of the extracranial internal carotid artery. Ten patients (5.2%) had a syndrome of lower cranial nerve palsies (with invariable involvement of cranial nerve XII with or without additional involvement of cranial nerves XI, X, and IX), seven (3.7%) had palsy of cranial nerve V, and five (2.6%) had a syndrome of ocular motor palsies. Palsy of cranial nerve VIII and ischemic optic neuropathy occurred in one patient each. Three patients had dysgeusia without other cranial nerve involvement, presumably due to involvement of the chorda tympani nerve. Headache or face pain (often unilateral) was present in 83% of patients. Other associated manifestations were cerebral ischemic symptoms, bruits, or oculosympathetic palsy. In one patient, cranial nerve palsy was the only manifestation of internal carotid artery dissection, and in another patient, the disease presented only as a palsy of cranial nerve XII and oculosympathetic palsy. In six patients, a syndrome of hemicrania and ipsilateral cranial nerve palsy was the sole manifestation of internal carotid artery dissection. Cranial nerve palsy is not rare in internal carotid artery dissection. Compression or stretching of the nerve by the expanded artery may explain some but not all of the palsies. An alternative mechanism is likely interruption of the nutrient vessels supplying the nerve. PMID- 8614495 TI - Does the central sulcus divide motor and sensory functions? Cortical mapping of human hand areas as revealed by electrical stimulation through subdural grid electrodes. AB - To clarify the exact anatomic relationship of electrically identified hand areas to the central sulcus, we constructed cortical surface renderings of magnetic resonance images (MRI) to locate the central sulcus accurately and measured the distances of stimulated points from the central sulcus and the Sylvian fissure. We obtained hand responses in 33 patients who underwent implantation of subdural grid electrodes for evaluation and surgical treatment of intractable epilepsy and analyzed these responses according to the presence of motor, sensory, mixed motor and sensory, and arrest responses. Hand motor responses occurred not only in the precentral gyrus but also in the postcentral gyrus, with great variability in superior-to-inferior distribution. Sensory responses also occurred in both the precentral and postcentral gyri with a distribution more ventral than that of motor responses. Mixed motor and sensory responses tended to be limited to the middle part of the central sulcus. Sites where electrical stimulation arrested simple hand repetitive voluntary movements occurred widely throughout the premotor and primary sensorimotor cortices. These data indicate a marked variability in the location of the human cortical hand area, and suggest that motor and sensory hand cortices overlap and are not divided in a simple manner by the central sulcus. PMID- 8614496 TI - Cerebrospinal fluid in acute optic neuritis: experience of the optic neuritis treatment trial. AB - The Optic Neuritis Treatment Trial (ONTT) is a prospective study of corticosteroid treatment of acute optic neuritis (ON), with subsequent longitudinal follow-up to determine development of clinically definite multiple sclerosis (CDMS). We analyzed the CSF of 83 patients with clinically isolated ON who underwent lumbar puncture within 24 hours of enrollment into the ONTT to determine the value of CSF changes in ON, especially regarding diagnostic utility, immunologic changes, MRI correlations, and progression to CDMS. All patients had baseline MRI scans graded for changes typical of MS. CSF measurements included immunoglobulin G (IgG) synthesis, IgG ratio, myelin basic protein, IgG kappa light chains, and oligoclonal banding. No patients had their diagnosis or management altered as a result of CSF findings. Except for oligoclonal bands, few patients showed any abnormalities on CSF tests, and no tests correlated with the 2-year development of CDMS. Oligoclonal banding, present at baseline in 11 of 13 patients who developed CDMS, did predict progression to CDMS, but this was not independent of MRI abnormalities. Two patients with oligoclonal bands and a normal MRI did progress to CDMS. We conclude that CSF analysis may not be necessary in the routine evaluation of patients presenting with a typical clinical profile of acute ON, and that most CSF tests add little additional information to MRI results for predicting the 2 year development of CDMS. However, the precise role of oligoclonal banding in the analysis of such patients awaits longer follow-up of this cohort. PMID- 8614497 TI - Serial gadolinium-enhanced MRI of the brain and spinal cord in early relapsing remitting multiple sclerosis. AB - Although serial MRI studies of the brain in relapsing-remitting MS have demonstrated frequent asymptomatic disease activity, less is known about the spinal cord. We carried out monthly gadolinium-enhanced brain and spinal cord MRI scans over 1 year in 10 patients with relapsing-remitting MS. Six of the patients had a total of 11 clinical relapses, eight of which involved the spinal cord. A total of 167 active (enhancing or new nonenhancing) lesions in the brain and 19 in the spinal cord were present. Only one active brain lesion was symptomatic compares with six spinal cord lesions. Overall, one-third of new spinal cord lesions were symptomatic, and three-quarters of clinical spinal cord relapses were associated with a new MRI lesion in a location appropriate to the symptoms. Activity in both the spinal cord and brain was more common around the time of relapse. There was a strong association between the spinal cord and brain MRI activity. We did not detect progressive spinal cord atrophy from measurements of a spinal cord cross-sectional area. We conclude that, in relapsing-remitting MS, imaging of the brain alone will detect 90% of active lesions; spinal cord MRI using current technology will therefore provide only modest gains in treatment trials in which lesion activity is the primary outcome measure. The lack of ++progressive spinal cord atrophy in these patients, suggesting that significant axonal loss has not occurred, is in keeping with their good recovery after relapse. That brain and spinal cord lesions occur concurrently implies a systemic trigger for disease activity. PMID- 8614498 TI - Comparison of triple dose versus standard dose gadolinium-DTPA for detection of MRI enhancing lesions in patients with MS. AB - We studied whether a triple dose of gadolinium-DTPA alone or in combination with delayed scanning increases the sensitivity of brain MRI for detecting enhancing lesions in patients with MS. We obtained T1-weighted brain MRI scans in two sessions for 22 patients with clinically definite MS. In the first session, we obtained one scan 5 to 7 minutes after the injection of 0.1 mmol/kg gadolinium DTPA (standard dose). In the second session, 6 to 24 hours later, we obtained one scan before the two scans 5 to 7 minutes (for all patients) and one hour (for 11 patients) after the injection of 0.3 mmol/kg gadolinium-DTPA (triple dose). We detected 83 enhancing lesions in 14 patients when the standard dose of gadolinium DTPA was used. The numbers of enhancing lesions increased to 138 (average increase 66%; p = 0.001) and the numbers of patients with such lesions to 18 (increase 28%) when we used the triple dose of gadolinium-DTPA. In addition, the total area per patient occupied by such lesions was greater (p < 0.0001) and lesion signal intensity higher (p = 0.0001) on the triple-dose scans than the standard-dose scans. There was an increase in the number of large enhancing lesions (p = 0.03) in the scans obtained 1 hour after the injection of the triple dose of gadolinium-DTPA. These data indicate that in patients with MS, a triple dose of gadolinium-DTPA can reveal many more enhancing lesions, which also appear larger. This suggests that the pathologic nature of "active" lesions in MS is heterogeneous, which might have impact on planning clinical trials. PMID- 8614499 TI - Comparison between multiple sclerosis in India and the United States: a case control study. AB - The prevalence of MS in India is low, and it is unclear whether the manifestations of the disease in India are similar to the United States. We carried out a case-control study to compare the disease in the two populations and used clinical, evoked potential, and MRI criteria to assess similarities and differences. Our results indicate that the rate of disease progression and frequency of involvement of the cerebral hemispheres, cerebellum, spinal cord, and brainstem were similar in the two populations. The visual system was more frequently involved in Indian patients. No Indian patient had a family history of MS; this suggests an environmental disease-triggering agent. PMID- 8614500 TI - Delayed emergence of a parkinsonian disorder in 38% of 29 older men initially diagnosed with idiopathic rapid eye movement sleep behaviour disorder. AB - We report longitudinal data on a group of 29 male patients 50 years of age or older who were initially diagnosed as having idiopathic REM sleep behavior disorder (RBD) after extensive polysomnographic and neurologic evaluations. Thirty-eight percent (11/29) were eventually diagnosed as having a parkinsonian disorder (presumably Parkinson's disease) at a mean interval of 3.7 +/- 1.4 (SD) years after the diagnosis of RBD+, and at a mean interval of 12.7 +/- 7.3 years after the onset of RBD. To date, only 7% (2/29) of patients have developed any other neurologic disorder. At the time of RBD diagnosis, data from the RBD group with eventual Parkinson's disease (n = 11) and the current idiopathic RBD group (n = 16) were indistinguishable, with two exceptions: the RBD-Parkinson's disease group had a significantly elevated hourly index of periodic limb movements of non REM sleep and an elevated REM sleep percentage. RBD was fully or substantially controlled with nightly clonazepam treatment in 89% (24/27) of patients in both groups. Thus, RBD can be the heralding manifestation of Parkinson's disease in a substantial subgroup of older male RBD patients. However, a number of presumed Parkinson's disease patients may eventually be diagnosed with multiple system atrophy (striatonigral degeneration subtype). Our findings indicate the importance of serial neurologic evaluations after RBD is diagnosed and implicate the pedunculopontine nucleus as a likely site of pathology in combined RBD Parkinson's disease, based on experimental and theoretical considerations rather than on autopsy data. PMID- 8614501 TI - The clinical spectrum of narcolepsy and idiopathic hypersomnia. AB - To better define the clinical spectra of narcolepsy and idiopathic hypersomnia, we retrospectively compared clinical and polygraphic findings and questionnaire results in groups of subjects with narcolepsy with or without cataplexy, idiopathic hypersomnia, insufficient sleep syndrome, mild sleep apnea, and excessive daytime sleepiness not otherwise specified. Sleep paralysis and sleep related hallucinations were most frequent in narcolepsy-cataplexy, but their frequency did not differ between narcolepsy without cataplexy and idiopathic hypersomnia. Mean durations of nocturnal sleep, daytime naps, and morning grogginess were not increased in idiopathic hypersomnia compared with other groups. Among subjects without cataplexy, symptoms of sleep paralysis and sleep related hallucinations were equally common in subjects with and without frequent sleep-onset REM periods. These findings suggest that the occurrence of these symptoms in subjects without classical narcolepsy-cataplexy is a function of factors other than a propensity for early onset of REM sleep and indicate a need to reevaluate diagnostic criteria for narcolepsy and idiopathic hypersomnia. PMID- 8614502 TI - Risk for neuropathologically confirmed Alzheimer's disease and residual aluminum in municipal drinking water employing weighted residential histories. AB - We investigated a possible relation between aluminum concentration ([Al]) in public drinking water and Alzheimer's disease (AD), with AD cases and controls defined on the basis of strict neuropathologic criteria. Using the case/control odds ratio as an estimate of relative risk and [Al] > or = 100 microgram/L as the cutoff point, elevated risks for histopathologically verified AD were associated with higher [Al]. Comparing all AD cases with all non-AD controls, and using the [Al] of public drinking water at last residence before death as the measure of exposure, the estimated relative risk associated with [Al] > or = 100 microgram/L was 1.7 (95% CI: 1.2-2.5). Estimating aluminum exposure from a 10-year weighted residential history resulted in estimates of relative risk of 2.5 or greater. The public health implications of the observed relationship between [Al] in drinking water and AD prevalence in the population depend in large measure on population exposure characteristics. In Ontario, it is estimated that 19% of the population was exposed to residual [Al] greater than or equal to 100 microgram/L. Based on the estimated relative risk and the assumption of causality, this translates to an etiologic fraction of 0.23. Although the potential contributions of confounding and mitigating factors are not defined in this report, the merit of limiting residual aluminum in drinking water supplies deserves serious attention. PMID- 8614503 TI - Familial and sporadic Alzheimer's disease: neuropathology cannot exclude a final common pathway. AB - Whether all etiologic forms of Alzheimer's disease (AD) share a final common pathway is a major issue. We determined the severity and regional distribution of neuronal loss, amyloid plaques, neuritic plaques (NPs), and neurofibrillary tangles (NFTs), and calculated the ratio of neuronal loss to NPs and NFTs in brains of 19 familial AD (FAD) patients with linkage to chromosome 14, six AD patients with mutations of chromosome 21 (codon 717 of the beta-amyloid percursor protein gene), and 11 sporadic AD (SAD) patients. There was no difference in the pattern of distribution of the various pathologic features or in the ratio of neuronal loss to NPs or NFTs in any AD group. However, FAD groups could be distinguished from SAD by the greater severity and the lack of influence of apolipoprotein E genotype on pathology. These differences may reflect differences in age at onset rather than different etiopathologic mechanisms. The similarity of pathologic findings in the different AD groups provides evidence for a final common pathophysiologic pathway in AD. PMID- 8614504 TI - Clinical and neuropsychological characteristics in familial and sporadic Alzheimer's disease: relation to apolipoprotein E polymorphism. AB - Alzheimer's disease (AD) is a heterogeneous entity presenting as sporadic and familial disease. In familial AD, there is evidence for genetic linkage to a yet undefined gene on chromosome 14 in early-onset pedigrees and on chromosome 19 in late-onset pedigrees. In a few early-onset kindreds, there were mutations in the amyloid precursor gene on chromosome 21. There is an increased frequency of apolipoprotein E (ApoE) epsilon4 allele in patients with late-onset AD. We studied the clinical presentation and profile of cognitive deficits in 58 AD patients at the early stage of the disease. We divided the AD patients into subgroups of sporadic late-onset (SLO) (> or = 65 years), familial late-onset (FLO) (> or = 65 years), sporadic early-onset (SEO) (<65 years), and familial early-onset (FEO) (<65 years) patients and into three subgroups according to their ApoE genotype zero epsilon4, one epsilon4, and two epsilon4 alleles. The AD subgroups did not differ in the global clinical severity of dementia or the duration of the disease. SLO, FLO, SEO, and FEO subgroups did not differ in clinical characteristics such as occurrence of rigidity, hypokinesia, tremor, myoclonus, hallucinations, delusions, or epileptic seizures nor in the profile of deficits on tests assessing memory, language, visuospatial, executive, and praxic functions. The epsilon4++ allele frequency was 0.43 for all AD patients and did not differ across subgroups divided according to the familial aggregation and age of onset. Patients with two epsilon4 alleles had earlier age at onset of dementia than those with no epsilon4 allele (63 +/- 9 versus 68 +/- 9 years), but otherwise the clinical symptoms and signs were not related to the ApoE genotype. However, the AD patients with two epsilon 4 alleles had lowest scores on memory tests and differed significantly from those with one or zero epsilon4 allele in the delayed list learning (p<0.05) and from those with zero epsilon4 allele in the immediate and delayed story recall. In contrast, verbal functions were better preserved in two epsilon4 patients than in those with other ApoE genotypes. This study failed to confirm the earlier reports of severe aphasia, agnosia, and apraxia in familial AD patients, but the clinical phenotype was similar irrespective to the familial aggregation. However, AD patients with two epsilon4 alleles are characterized by more severe memory loss and earlier age of onset than those without the epsilon4 allele. PMID- 8614505 TI - d-Cycloserine enhances implicit memory in Alzheimer patients. AB - We tested the ability of d-cycloserine, a partial glycine agonist acting at the N methyl-D-aspartate (NMDA) receptor complex, to improve implicit memory in Alzheimer patients in a parallel-group, placebo-controlled, double-blind study. One-hundred eight patients with probable Alzheimer's disease of mild to moderate severity received d-cycloserine (5, 15, or 50 mg) or placebo twice daily for 10 weeks. We then evaluated their ability to identify perceptually degraded words, some of which were repeated over multiple trials across 3 days. Implicit memory performance of words repeated across trials was significantly enhanced for the patients who received 15 mg d-cycloserine compared with those who received placebo. These findings support development of NMDA receptor-mediated glutamatergic interventions for the treatment of Alzheimer-related memory disorders. PMID- 8614506 TI - Prospective neuropsychological assessment of nondemented patients with biopsy proven senile plaques. AB - Senile plaques are characteristic of Alzheimer's disease but also occur as an age related change in some neurologically normal individuals at autopsy. The significance of these "incidental" senile plaques, with regard to the development of dementia, is unknown and cannot be assessed by postmortem studies. Patients with biopsy proven senile plaques offer an important opportunity for prospective followup. We identified senile plaques in temporal lobectomy specimens, removed in the surgical treatment of intractable epilepsy, from 11 patients. Review of preoperative neuropsychological test results showed no suggestion of dementia in any of the patients with senile plaques and no significant difference compared with controls. Postoperative followup ranged from 2 to 7 years (mean, 3.7 yr). There was no evidence of generalized cognitive deterioration in any of the study patients. These findings indicate that an abundance of senile plaques may be present without associated dementia and without cognitive deterioration for at least several years. PMID- 8614507 TI - Progressive familial leukodystrophy of late onset. AB - We report a family in which three siblings developed dementia between the ages of 40 and 70 years. Two of the siblings developed symptoms of depression, abnormal behavior, and an inability to function, progressing to severe dementia. The third sibling had a severe dementia, the clinical details of which are not available. In the two deceased siblings neuropathologic examinations demonstrated severe demyelination, axon loss, and gliosis in cerebral white matter. Cerebellar and brainstem white matter were unaffected. Cerebral gray matter was negligibly affected. The disorder, histopathologically classified as a pigmented orthochromatic leukodystrophy, is extremely rare. Its etiology is unknown, but the pathology and familial occurrence imply that it represents a genetic defect in a function localized in the cerebral white matter. PMID- 8614508 TI - Chemotherapy without radiation therapy as initial treatment for primary CNS lymphoma in older patients. AB - Chemotherapy plus radiation therapy (RT) for primary CNS lymphoma (PCNSL) has significantly improved patient survival over RT alone, but there are late neurologic sequelae of RT, particularly in the elderly. We treated 13 patients over age 50 years (mean age 74 years) with chemotherapy alone as initial treatment for PCNSL. All received methotrexate (MTX) and procarbazine; in addition, five received thiotepa, four vincristine, and four vincristine and cytarabine. Ten achieved a complete response (CR), 2 a partial response (PR), and 1 progressed through treatment. Two patients with ocular lymphoma responded to MTX, procarbazine, and vincristine. Four of six patients who relapsed after achieving a CR or PR were treated with additional chemotherapy or RT; three achieved a CR and one a PR. Five patients remain in CR at 7.5 to 30 months, one is alive at 35 months but with progressive disease, six died of PCNSL at 5 to 30.5 months, and one died in CR of sulfur allergy 2 months after diagnosis. The Karnofsky Performance Status improved in 11 to 13 patients with treatment. Cognitive deficits were present in nine patients at diagnosis and improved in eight of these nine after chemotherapy. Only one patient developed new cognitive deficits, due to progressive tumor and possibly MTX leukoencephalopathy. Chemotherapy alone for PCNSL is effective in the elderly and eliminates the risk of RT-related neurotoxicity. RT can salvage those who relapse after chemotherapy. PMID- 8614509 TI - T-cell infiltration of primary CNS lymphoma. AB - We stained 13 primary CNS lymphomas (PCNSLs) (six from patients with AIDS, seven from immunocompetent patients) with a panel of antibodies to T cells (pan T cell [CD3], T helper cell [CD4], T suppressor cell [CD8], delta/delta cell [CD4-8-]), B cells (CD20), hematopoietic cells (T200), and NK cell (CD56). We estimated the percentage of tumor cells staining with each antibody. All tumors were B-cell lymphomas. The non-AIDS tumors showed a significant infiltration with CD3+ cells (mean of 10.82% of total cells). The AIDS patients' tumors showed a smaller percentage of CD3+ infiltrating cells (mean, 4.88% of total cells) (p<0.01). CD4+ cells were 9.11% of the total hematopoietic cells in the non-AIDS patients and 3.13% in AIDS patients (p<0.01). AIDS patients showed some CD8+ cells (0.3%), which was significantly higher than in immunocompetent patients (0%) (p<0.05). Very few tumor cells stained with the NK cell and delta/delta cell markers. Both immunocompetent and AIDS patients with PCNSL exhibit significant CD3+ and CD4+ cell infiltration of their tumors; this infiltration is significantly lower in AIDS patients. AIDS patients show a minor CD8+ cell infiltration of their tumors. These results on PCNSL are different from systemic lymphomas, which show a higher CD4 and CD8 cell infiltration, and may offer insights into the more aggressive nature of AIDS-related PCNSL. PMID- 8614511 TI - Ocular motor dysfunction in HIV-1-infected subjects: a quantitative oculographic analysis. AB - We recorded eye and head movements in 13 human immunodeficiency virus type-1 (HIV 1)-infected patients with CD4 counts of less than or equal to 500 cells/mm3 using magnetic search coil oculography. Horizontal and vertical saccades, smooth pursuit, and vestibular smooth eye movements were recorded, as were horizontal antisaccades and vestibular memory-guided saccades. Rightward and leftward and upward and downward responses were analyzed separately. Compared to normal control subjects, HIV-1--infected patients performed the antisaccade test poorly, making the initial antisaccade in the correct direction (away from the target) in only 33% of trials. The mean final gaze position achieved during the vestibular memory-guided saccade task was less accurate for HIV-1-infected patients than for control subjects, and this correlated with inaccuracies on the antisaccade task. Horizontal saccades, horizontal and vertical smooth pursuit, and vestibular smooth eye movements were quantitatively normal. However, smooth pursuit showed directional asymmetries, vertically more than horizontally; horizontal and vertical unpredictable saccades were more inaccurate than predictable saccades; and vertical saccade latencies were prolonged. In patients with HIV-1 infection, abnormalities in vertical eye movements and relative asymmetries in smooth pursuit gains, both horizontally and vertically, are more sensitive and consistent indicators of CNS dysfunction than are horizontal eye movement abnormalities or measurements of absolute smooth pursuit gain and phase. PMID- 8614510 TI - Prognosis and response to therapy of cytomegalovirus encephalitis and meningomyelitis in AIDS. AB - Effective diagnosis and treatment of cytomegalovirus infection of the nervous system in AIDS patients has been limited by a lack of sensitive diagnostic measures. Retrospective series suggest a poor prognosis for cytomegalovirus encephalitis with rapid mortality. Polymerase chain reaction amplification of cytomegalovirus DNA allows detection in CSF that appears specific for CNS infection. In this series of seven patients with CNS cytomegalovirus infection in AIDS, four patients responded to therapy. Serial determinations of cytomegalovirus DNA in CSF in five patients revealed persistent detection in two treatment failures and absence of detection in three responders on subsequent CSF samples. A prospective trial to determine optimal therapy and to confirm the utility of cytomegalovirus DNA in CSF as a marker of the course of cytomegalovirus infection in the CNS is warranted and should consider prior therapy for cytomegalovirus, prior opportunistic infections, and leukoencephalopathy as potential prognostic variables. PMID- 8614512 TI - Familial migraine with vertigo and essential tremor. AB - We report a family with dominantly inherited migraine headaches, episodic vertigo, and essential tremor. All symptoms improved with the use of acetazolamide. Linkage analysis ruled out linkage to markers on chromosome 19p, known to be linked to the genetic defect in families with the clinically similar syndromes of hemiplegic migraine and periodic ataxia. This genetic heterogeneity of migraine syndromes could result from defects in a family of genes coding proteins with similar properties. PMID- 8614513 TI - Cognitive dysfunction as the major presenting feature of Becker's muscular dystrophy. AB - We report four patients, currently aged 15, 17, 19, and 42 years, with X-linked dystrophinopathy who presented with mental retardation (IQ range, 60-68) and psychiatric disturbance in the absence of muscle weakness. All patients had elevated serum creatine kinase and dystrophic changes on muscle biopsy. There were alterations in the size and abundance of dystrophin on immunohistochemistry and immunoblotting in all cases, consistent with a molecular diagnosis of Becker's muscular dystrophy. Two patients had deletions of the dystrophin gene on DNA analysis. These findings suggest that Becker's muscular dystrophy may be associated with a predominantly neuropsychiatric presentation and that dystrophinopathy should be considered in the differential diagnosis of unexplained cognitive or psychiatric disturbance in males. Serum creatine kinase may provide an adequate screening test in this clinical situation. PMID- 8614514 TI - Valproic acid hepatic fatalities. III. U.S. experience since 1986. AB - We report the results of a third retrospective study of the U.S. experience with fatal hepatotoxicity associated with valproic acid (VPA). In the United States, over one million patients received new prescriptions for VPA during the years 1987 to 1993, and 29 patients developed fatal hepatotoxicity. Decreased alertness, jaundice, vomiting, hemorrhage, increased seizures, anorexia, and edema were the most common presenting signs. Risk factors included young age, polytherapy, developmental delay, and coincident metabolic disorders. Patients less than 2 years old receiving VPA as polytherapy were at the greatest risk (1:600) of developing this complication. PMID- 8614515 TI - "Grievous bodily harm:" gamma hydroxybutyrate abuse leading to a Wernicke Korsakoff syndrome. AB - Gamma-hydroxybutyrate (GHB) is a naturally occurring GABA-like drug used illicitly by bodybuilders. Although there are reports of several cases of GHB abuse, with a variety of nervous system complications, we present the first case associated with a Wernicke-Korsakoff syndrome. PMID- 8614516 TI - Associated postural adjustments are impaired by a lesion of the cortex. AB - A ballistic movement of one arm may be accompanied by contractions of contralateral postural muscles known as "associated postural adjustments." Ballistic movements are considered to involve the motor cortex, but it is not known where associated postural adjustments are generated. We recorded the EMG activity in the deltoid and latissimus dorsi muscles during ballistic abduction of the arm in 10 normal subjects and five subjects with lesions of the motor cortex. When subjects with cortical lesions abducted the unaffected arm, the associated postural adjustment in the contralateral latissimus dorsi was significantly smaller than normal. We argue that postural adjustments, like ballistic movements, require the participation of the motor cortex. PMID- 8614517 TI - Blepharospasm in association with a lower pontine lesion. AB - A patient with neurofibromatosis type I and breast carcinoma developed a bilateral but asymmetric blepharospasm and paresis of the left abducens muscle over a 2-week course. MRI disclosed a small lesion in the left dorsomedial lower pontine region. Electrodiagnostic investigations revealed bilateral R1 responses after stimulation of the left supraorbital nerve and enhancement of R1 and R2 recovery curves. We concluded that lesions in the lower pontine tegmentum may cause blepharospasm. PMID- 8614518 TI - Preliminary clinical observations on a new trigeminal reflex: the trigemino cervical reflex. AB - Short latency trigemino-cervical reflexes can be recorded from sternocleidomastoid muscle after stimulation of the infraorbital branch of the trigeminal nerve. We studied the trigemino-cervical reflexes and the conventional blink reflex in three patients with an isolated lesion in the medulla oblongata, eight patients with multiple sclerosis, and two patients with supratentorial ischemic lesion. The trigemino-cervical response was abnormal in the patients with an isolated lesion in the medulla oblongata and in all multiple sclerosis patients, whereas both components of the blink reflex were preserved in the patients with a lesion in the medulla oblongata and in half of the patients with multiple sclerosis. The trigemino-cervical reflex was preserved in patients with supratentorial lesions, whereas the late component of the blink reflex was abnormal. These findings suggest that central pathways generating the trigemino cervical reflex are confined to the medulla oblongata and that they are independent from those generating the long latency (R2) component of the blink reflex. The trigemino-cervical reflex may help in disclosing and localizing brainstem lesions. PMID- 8614519 TI - Involvement of the white matter in hypomelanosis of Ito (incontinentia pigmenti achromiens). AB - We report our clinical and neuroradiologic findings in 13 patients affected by hypomelanosis of Ito. Seven patients were boys and six were girls; their ages ranged from 11 months to 16 years. Neurologic signs were present in all but two cases, and they consisted of language disabilities, seizures, hypotonia, mental retardation, and autistic behavior. MRI was performed in all patients. We observed anomalies of the white matter in seven of the 13 patients; all but one of these seven had neurologic signs that included seizures, hypotonia, language disabilities, and mental retardation. The abnormal signals in the white matter were mostly located in the parietal periventricular and subcortical regions of both hemispheres. Moreover, we found asymmetry of the cerebral hemispheres in one of our 13 patients and atrophy of the cerebellar vermis in another patient, with no involvement of the white matter in either. In the remaining four of the 13 patients results of MRI appeared normal. There was a relationship between the anomalies in the central nervous system at MRI, as a whole, and the neurologic manifestations, even though two patients with apparently normal images on MRI had partial and generalized tonic clonic seizures, respectively. A correlation was also found between white matter anomalies and neurologic signs; extended and deep changes in white matter images were associated with more severe neurologic abnormalities and delayed language milestones appeared to be a constant finding in this group of patients. These anomalies of the white matter, which did not progress over time, resembled those seen in other neurocutaneous syndromes. The hypothesis is presented that underlying disarray of cortical lamination or neuronal loss with subsequent wallerian degeneration and altered or delayed myelination could be the cause of the abnormal findings on MRI. PMID- 8614520 TI - Manganese intoxication in the rhesus monkey: a clinical, imaging, pathologic, and biochemical study. AB - We gave three adult rhesus monkeys seven IV injections of manganese chloride at approximately 1-week intervals. We evaluated neurologic status by serial clinical examinations and performed a levodopa test if the animal developed features of basal ganglia dysfunction. After the animals were killed, we performed neuropathologic, neurochemical, and laser microprobe mass analysis (LAMMA) studies. Two of three animals developed a parkinsonian syndrome characterized by bradykinesia, rigidity, and facial grimacing suggestive of dystonia but not tremor. Neither animal responded to levodopa. Autopsy demonstrated gliosis primarily confined to the globus pallidus (GP) and the substantia nigra pars reticularis (SNr). We detected focal mineral deposits throughout the GP and SNr, particularly in a perivascular distribution. LAMMA studies noted that mineral deposits were primarily comprised of iron and aluminum. The severity of pathologic change correlated with the degree of clinical dysfunction. These studies demonstrate that, in contrast to Parkinson's disease (PD) and MPTP induced parkinsonism, manganese primarily damages the GP and SNr and relatively spares the nigrostriatal dopaminergic system. Further, the results suggest that Mn-induced parkinsonism can be differentiated from PD and MPTP-induced parkinsonism by the clinical syndrome and response to levodopa. The accumulation of iron and aluminum suggests that iron/aluminum-induced oxidant stress may contribute to the damage associated with Mn toxicity. PMID- 8614521 TI - Lateralized effect of rapid-rate transcranial magnetic stimulation of the prefrontal cortex on mood. AB - We studied the effects of rapid-rate transcranial magnetic stimulation (rTMS) of different scalp positions on mood. Ten normal volunteers rated themselves before and after rTMS on five analog scales labeled "Tristeza" (Sadness), "Ansiedad" (Anxiety), "Alegria" (Happiness), "Cansancio" (Tiredness), and "Dolor/Malestar" (Pain/Discomfort). rTMS was applied to the right lateral prefrontal, left prefrontal, or midline frontal cortex in trains of 5 seconds' duration at 10 Hz and 110% of the subject's motor threshold intensity. Each stimulation position received 10 trains separated by a 25-second pause. No clinically apparent mood changes were evoked by rTMS to any of the scalp positions in any subject. However, left prefrontal rTMS resulted in a significant increase in the Sadness ratings (Tristeza) and a significant decrease in the Happiness ratings ("Alegria") as compared with right prefrontal and midfrontal cortex stimulation. These results show differential effects of rTMS of left and right prefrontal cortex stimulation on mood and illustrate the lateralized control of mood in normal volunteers. PMID- 8614522 TI - Cutaneous electromyographic silent period findings in brachial dystonia. AB - In this study we investigated the physiologic mechanisms in primary brachial dystonia by analyzing the cutaneous EMG silent period during isometric contraction of the opponens pollicis muscle. Results from the affected and unaffected arms of 11 patients with dystonia were compared to 7 patients with Parkinson's disease and 16 age-matched normal individuals (controls). The silent period onset latency, degree of EMG suppression during the silent period, and EMG rebound at the end of the silent period did not differ significantly between patients with dystonia and any other group. The duration of the silent period (the S-X interval), however, was significantly prolonged in dystonia (p<0.005) and in Parkinson's disease (p<0.001) in both affected and unaffected arms compared with controls. These findings suggest that mechanisms responsible for the initiation of the cutaneously induced silent period and the subsequent suppression depth of EMG activity are not affected in brachial dystonia, but the abnormally prolonged S-X intervals may reflect dysfunctional basal ganglia timing influences over spinal circuitry common to both dystonia and Parkinson's disease. PMID- 8614523 TI - Supplementary motor area seizures: propagation pathways as studied with invasive recordings. AB - We studied propagation of epileptic discharges in five patients with supplementary motor area (SMA) seizures with subdural grid electrodes implanted over the dorsolateral frontal neocortex and in the interhemispheric fissure. We found that both interictal and ictal epileptic discharges occurred synchronously in the SMA and the primary cortex. The actively involved electrodes were separated by silent electrodes. The time lag between the SMA and the primary motor cortex averaged 25 msec for interictal and 100 msec for ictal discharges. Cortical stimulations of the affected electrodes showed motor effects in corresponding body parts. All patients underwent resections of the EEG onset zone within the SMA while sparing the primary motor cortex and experienced a significant (>90%) reduction of seizure frequency. We conclude that epileptic activity is propagated between the SMA and the primary motor cortex by a somatotopically organized monosynaptic pathway. PMID- 8614524 TI - Craniotopic defects of smooth pursuit and saccadic eye movement. AB - I recorded smooth pursuit and saccadic eye movements in six patients with unilateral cerebral infarction. By comparing responses within the hemiranges of eye position to the right and left of the orbital midline and in rightward and leftward directions, I quantified craniotopic and directional ocular motor deficits. Two patients had ipsiversive gaze deviation and severe craniotopic defects in which they could not generate smooth pursuit or saccadic eye movement into the contralateral orbital hemirange. Three patients without gaze deviation generated worse smooth pursuit in the contralateral hemirange than in the ipsilateral hemirange, but each had symmetric saccades according to eye position. All patients with craniotopic pursuit defects also had directional smooth tracking asymmetries in which eye velocities were lower for targets moving ipsilaterally than for targets moving contralaterally. Craniotopic and directional defects were associated with damage in the frontal eye field region. Orbital position is taken into account by cerebral circuits that govern smooth pursuit and saccades. PMID- 8614525 TI - 311C90, a new central and peripherally acting 5-HT1D receptor agonist in the acute oral treatment of migraine: a double-blind, placebo-controlled, dose-range finding study. AB - 311C90 is a novel, centrally and peripherally, acting 5-hydroxytryptamine1D receptor agonist. We investigated the efficacy and safety of 1, 5, and 25 mg of oral 311C90 in the acute treatment of migraine in a randomized, double-blind, placebo-controlled, parallel-group clinical trial involving 84 patients. The proportion of patients in whom the headache improved within 2 hours from moderate or severe to mild or no pain (primary efficacy measure) was 15% for placebo treated patients and 27% (1 mg), 62% (5 mg), and 81% (25 mg) for patients treated with 311C90. Treatment differences compared with placebo were 12% (95% CI - 12, 37; p = 0.460) for 1 mg 311C90, 47% (CI 21, 73; p < 0.005) for 5 mg 311C90, and 66% (CI 43,89; p < 0.001) for 25 mg 311C90. Photophobia and nausea also showed improvement after 311C90. Adverse events were generally mild and transient in all treatment groups. There were no clinically significant changes in ECG recordings, blood pressure, or laboratory tests. Oral 311C90 (5 and 25 mg) is highly effective and well tolerated in the acute treatment of migraine. The response rates and treatment differences compared with placebo in this study suggest possible superiority over existing antimigraine therapies. This needs to be confirmed in formal comparative trials. PMID- 8614526 TI - Trinucleotide repeat length and clinical progression in Huntington's disease. AB - We examined the relationship between length of the trinucleotide (CAG) repeat at IT-15 and clinical progression of Huntington's disease in 46 mildly to moderately affected patients over a 2-year interval. Patients were divided into those with short mutations (37 to 46 repeats; n = 25) and those with long mutations (> or = 47 repeats; n = 21). Patients with long repeat lengths had earlier age at onset and were younger and less functionally impaired than those with short repeats at the initial visit, but the groups did not differ in severity of neurologic or cognitive impairment. However, the long-repeat group displayed significantly greater decline in both neurologic and cognitive functioning over the 2-year follow-up period. The length of the CAG repeat correlated highly with age at onset (r = -0.72, p < 0.001) and was a strong predictor of decline in both neurologic and cognitive function. The mechanism of gene action, and the means by which longer expansions result in a more malignant disease process, remain to be elucidated. PMID- 8614527 TI - Experimental transmission of Creutzfeldt-Jakob disease and related diseases to rodents. AB - Sporadic Creutzfeldt-Jakob disease (CJD) with 129M/M, and iatrogenic and familial CJD with E200K and M232R, showed similar clinicopathologic features, a synaptic type deposition of PrPCJD, and high transmission frequencies to mice. Sporadic patients with 129M/V or 129V/V, and mutation cases with V180I, showed slightly different features and low or null transmission frequencies to mice. Hereditary cases with P102L, P105L, A117V, Y145stop, and insertions had different features but all demonstrated a long clinical duration and the presence of PrP plaques. The experimental transmission to mice of these mutant forms was difficult, except for one-third of the cases with P102L. CJD and related diseases, even those that are hereditary, may thus be divided into two different groups, those that are easily transmissible and those that are either difficult to transmit or nontransmissible. PMID- 8614529 TI - Psychogenic status epilepticus induced by a provocative technique. AB - We report a patient who developed sustained psychogenic seizures after undergoing a provocative technique (PT) for the diagnosis of psychogenic seizures. Because patients are at risk for severe agitation with PT, these diagnostic maneuvers should be used selectively, and the clinician should be prepared to deal with this complication. PMID- 8614528 TI - Immunoglobulins reactive with myelin basic protein promote CNS remyelination. AB - We tested the hypothesis that immunoglobulins directed against a CNS autoantigen, myelin basic protein, may promote remyelination in the course of a chronic, immune-mediated demyelinating disease. SJL/J mice infected chronically with Daniel's strain of Theiler's virus served as an experimental model of MS. The spinal cords of these mice exhibit extensive primary demyelination and inflammation with minimal spontaneous remyelination. Treatment with whole antiserum or affinity-purified mouse immunoglobulins directed against rat or rabbit myelin basic protein increased new myelin synthesis as measured by quantitative morphometry. Electron microscopy revealed numerous oligodendrocytes in remyelinated CNS lesions and a relative lack of inflammatory cells. Viral antigen persisted in the spinal cord despite enhanced CNS-type remyelination. These findings indicate that immunoglobulins reactive with myelin autoantigens have the potential to promote myelin repair. PMID- 8614530 TI - Stroke with sensory symptoms mimicking myocardial ischemia. AB - OBJECTIVE: To report five stroke patients with sensory deficits including prominent chest discomfort mimicking angina. BACKGROUND: Chest wall sensory discomfort, as a part of unilateral sensory dysfunction, has seldom been recognized as a potential imitator of cardiac ischemia. METHODS: A retrospective review of stroke patients with sensory symptoms from the New England Medical Center Stroke Registry. RESULTS: As a part of an acute stroke that included unilateral sensory symptoms and signs, five patients had chest pain or discomfort, which prompted cardiac evaluation for potential coronary artery disease. In two patients, the primary presentation was chest discomfort. In the other three, chest discomfort was part of a more extensive stroke syndrome. The symptoms were described as "burning," "hot feeling," "flashes," "tightness," and "cold." In three patients, an MRI or CT scan showed an infarct in the thalamus, corona radiata, or lateral medulla. Cardiac evaluation was negative in all but one patient who had single vessel percutaneous transluminal coronary angioplasty without resolution of sensory symptoms. Chest discomfort fluctuated but persisted for months or years after presentation. CONCLUSION: Chest discomfort mimicking cardiac ischemia may be a prominent sensory symptom in acute stroke. PMID- 8614531 TI - Side effect profile of interferon beta-1b in MS: results of an open label trial. AB - To determine which patients are prone to side effects from interferon beta-1b and which side effects are most troublesome, we studied 72 patients with clinically definite MS who were started on interferon beta-1b after its release and found that the side effects significantly associated with treatment included skin reactions, flu-like symptoms, fatigue, leukopenia, new or worsened depression, and new or worsened headache. Of these, only fatigue and depression were significantly associated with discontinuance of therapy. Moreover, the course of disease before initiation of treatment also had a significant impact on the likelihood of discontinuing medication. Thus, despite an apparently similar therapeutic benefit (as judged by the similarly reduced attack rate in each group), patients with a secondary chronic progressive course were more likely to discontinue treatment (63%) than patients with either a relapsing/progressive course (18%) or a remitting/relapsing course (7%). Indeed, in the final regression equation, the only factors significantly related (r = 0.875) to the discontinuance of therapy were fatigue (p < 0.0001), a fatigue-depression interaction (p < 0.0001), and a chronic progressive course of disease (p<0.0001). Thus, if future clinical trials are to provide useful information on the value of interferon beta-1b in progressive MS, the side effects of fatigue and depression will need to be ameliorated to limit the drop-out rate from such trials. PMID- 8614532 TI - Idiopathic hypertrophic cranial pachymeningitis associated with hydrocephalus and myocarditis: remarkable steriod-induced remission of hypertrophic dura mater. AB - We describe a patient with idiopathic hypertrophic cranial pachymeningitis (IHCP) associated with hydrocephalus and myocarditis in whom steroids caused remission of the hypertrophic dura and enlarged ventricles. Myocarditis induced complete atrioventricular block that responded to steroids initially but later required permanent pacemaker implantation. Serum ANA and anti-dsDNA antibody were positive. Our case suggests that IHCP and myocarditis may share a common autoimmune etiology. PMID- 8614533 TI - Trimethoprim and sulfonamide-associated meningoencephalitis with MRI correlates. AB - Early recognition of trimethoprim and sulfonamide-induced aseptic meningitis is important because drug cessation leads to rapid clinical improvement. We present clinical and laboratory findings in two typical cases. In both cases, MRI revealed previously undescribed diffuse white matter abnormalities that resolved within a few months. These MRI findings are important because they may aid in early diagnosis of this condition in the appropriate clinical setting. In addition, the white matter abnormalities suggest an encephalitic component in addition to the meningitis. PMID- 8614534 TI - Immune brachial plexus neuropathy: suggestive evidence for an inflammatory-immune pathogenesis. AB - We report brachial plexus biopsy findings from two Australian and two American patients with brachial plexus neuropathy. There were florid multifocal mononuclear inflammatory cell infiltrates. Present evidence suggests that these brachial neuropathies have an immune basis. PMID- 8614535 TI - Multifocal motor neuropathy with conduction block and Campylobacter jejuni. AB - We describe a patient with acute multifocal motor neuropathy with conduction block (MMNCB) and high titers of immunoglobulin G anti-GM1 antibodies after Campylobacter jejuni enteritis. Treatment with intravenous immune globulin led to rapid improvement with return of normal function by 6 weeks. This is the first report of C. jejuni enteritis preceding MMNCB. PMID- 8614536 TI - Extraocular muscle involvement in Becker muscular dystrophy. AB - We report limitation of gaze and slow saccadic eye movements by clinical examination and video-oculography in a patient with Becker muscular dystrophy. This rare association suggests that a dystrophinopathy should be considered in a patient with features characteristic of Becker muscular dystrophy even when mild impairment of eye movements is present. PMID- 8614537 TI - Facioscapulohumeral muscular dystrophy with chromosome 9p deletion. AB - We report a 31-year-old man with facioscapulohumeral muscular dystrophy who had congenital anomalies and mental retardation. Southern blot analysis, using the probe p13E-11, displayed an abnormal EcoRI DNA fragment that reflect DNA rearrangements in facioscapulohumeral muscular dystrophy. In addition, high resolution cytogenetic study revealed an interstitial deletion of the short arm chromosome 9: 46,XY,del(9)(p.22.1p24.1). PMID- 8614538 TI - Genetic heterogeneity in Charcot-Marie-Tooth neuropathy type 2. AB - Charcot-Marie-Tooth neuropathy type 2 (CMT2) is a common inherited axonal neuropathy. The locus for one form of CMT2 (CMT2A) is assigned to the short arm of chromosome 1. There is genetic heterogeneity in CMT2 because additional pedigrees do not demonstrate linkage to chromosome 1 and are designated as CMT2B. Further clinical heterogeneity is suggested by CMT2 pedigrees with diaphragm and vocal cord weakness and are designated as CMT2C. To address the possible genetic distinction between CMT2A and CMT2C, we tested markers from the CMT2A locus for linkage in a large CMT2C pedigree. There was no evidence to support linkage of the CMT2C gene to the region of the CMT2A locus on chromosome 1. CMT2C is not an allelic variant of CMT2A. This analysis provides further evidence for genetic heterogeneity within inherited axonal neuropathies. PMID- 8614540 TI - Epilepsy in the nevoid basal-cell carcinoma syndrome (Gorlin syndrome): report of a case due to a focal neuronal heterotopia. AB - We present a case of uncontrolled temporal lobe epilepsy due to a focal left temporal neuronal heterotopia associated with the nevoid-basal-cell carcinoma syndrome. This is the first pathologically described lesion associated with temporal lobe epilepsy in this syndrome. Because the patient's seizures resolved after a modified left anterior temporal lobectomy, this case illustrates that epilepsy in patients with the nevoid basal-cell carcinoma syndrome is potentially curable and should be investigated appropriately. PMID- 8614539 TI - Japanese triplets with cerebrotendinous xanthomatosis are homozygous for a mutant gene coding for the sterol 27-hydroxylase (Arg441Trp). AB - We present the first case of triplets with cerebrotendinous xanthomatosis (CTX). A C-to-T base change identified in the genomic DNA and cDNA encoding the sterol 27-hydroxylase led to replacement of arginine by tryptophan at position 441 (Arg441Trp) in the triplets. The triplets were homozygous and their mother was heterozygous for this mutant gene. The triplets exhibited an identical phenotypic expression, which was different from that of a sporadic CTX case with the same mutation. PMID- 8614541 TI - Research priorities for syringomyelia: a National Institute of Neurological Disorders and Stroke workshop summary. PMID- 8614542 TI - Indomethacin and Alzheimer's disease. PMID- 8614543 TI - Treatment of opsoclonus-myoclonus with high-dose intravenous immunoglobulin. PMID- 8614545 TI - Another kind of knapsack palsy. PMID- 8614544 TI - Treatment of Vogt-Koyanagi-Harada syndrome with intravenous immunoglobulin. PMID- 8614546 TI - Subacute measles virus encephalitis: a new and fatal opportunistic infection in a patient with AIDS. PMID- 8614547 TI - Stroke risk. PMID- 8614548 TI - Treatment of cryoglobulinemic neuropathy with alpha-interferon. PMID- 8614549 TI - Safety of heparin in acute ischemic stroke. PMID- 8614550 TI - Safety of heparin in acute ischemic stroke. PMID- 8614551 TI - Safety of heparin in acute ischemic stroke. PMID- 8614552 TI - Brainstem AVM. PMID- 8614553 TI - Language, Alzheimer's disease, and gender. PMID- 8614554 TI - Nitric oxide regulates excitatory amino acid release in a biphasic manner in freely moving rats. AB - The effect of altering hippocampal nitric oxide levels on basal and N-methyl-D aspartate (NMDA) receptor-evoked release of glutamate and aspartate has been studied in freely moving rats. NMDA increased extracellular glutamate and aspartate in a concentration-dependent manner. The nitric oxide synthase inhibitor L-nitro-arginine-methyl ester (L-NAME; 100 microM) increased basal glutamate and aspartate release, and also enhanced release of these amino acids evoked by NMDA (100 microM) compared with the same concentration of NMDA infused alone. L-NAME at 200 microM increased basal dialysate glutamate, but not aspartate, to a lesser extent than the 100 microM concentration of the drug, and the NMDA-induced release of glutamate and aspartate was decreased. L-NAME at 1.0 mM did not significantly alter basal extracellular glutamate but significantly decreased dialysate aspartate, while abolishing the NMDA-evoked release of both amino acids. The actions of L-NAME were not mimicked by its much less active isomer D-nitro-arginine-methyl ester. The nitric oxide donor drug S-nitroso-N penicillamine decreased dialysate glutamate and aspartate at a 500 microM concentration but increased the extracellular level of both amino acids when infused at 1.0 mM and 2.0 mM concentrations. These data suggest that nitric oxide may mediate both excitatory and inhibitory functions, according to the level of nitric oxide production in vivo. PMID- 8614555 TI - Fibrillogenesis of synthetic amyloid-beta peptides is dependent on their initial secondary structure. AB - Synthetic peptides containing the sequence of Alzheimer's amyloid-beta peptide (A beta) spontaneously form amyloid-like fibrils in vitro, and have been extensively used to study the factors that modulate fibrillogenesis. Contradictory observations have been reported regarding the neurotoxicity of A beta and the influence of some A beta-binding proteins on in vitro A beta amyloid formation. In this study, we show that A beta 1-40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties. No differences were detected in the chemical structure or in the initial assembly state by mass spectroscopy, reverse-phase high performance liquid chromatography and denaturing or non-denaturing gel electrophoresis. However, there was a direct correlation between the ability of soluble peptides to form amyloid and their percentage of beta-sheet structure, as determined by electron microscopy, fluorescence associated to thioflavine T bound to amyloid, and circular dichroism. The data suggest that the determinant factor of A beta fibrillogenesis is the secondary structure adopted by the peptide in its soluble state. PMID- 8614556 TI - Apoptotic cell death in the cerebellum of mutant weaver and lurcher mice. AB - Apoptosis in the central nervous system of mutant weave (wv) and lurcher (lc) mice was studied using in situ DNA end-labeling. The number of apoptotic cells in the external granule cell layer of weaver (wv/+) mice at postnatal day 9 was increased six- to eight-fold compared to (+/+) littermates. Purkinje cells and Bergmann glia cells were not affected. No labeled cells were found in the substantia nigra. In lurcher (lc/+) mice, labeled nuclei of large cells were found in the Purkinje cell layer, suggesting that Purkinje cells of mutant lurcher mice undergo apoptosis. Apoptotic granule cell nuclei were found in the internal granule cell layer. In contrast to weaver (wv/+) mice, the number of apoptotic cells in the external granule cell layer was not increased. Apoptosis appears to be the common pathway of cell death in the degenerative processes induced by the weaver and the lurcher gene, respectively. These mutant mice offer the opportunity to study the mechanisms that underlie inappropriate apoptotic cell death in vivo. PMID- 8614557 TI - Persistent increase in dopamine release following activation of metabotropic glutamate receptors in the rat nucleus accumbens. AB - Intracerebral microdialysis technique was utilized to study the effect of the metabotropic glutamate receptor agonist, 1-aminocyclopentane-1,3-dicarboxylic acid (ACDP), on extracellular dopamine concentration in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 mM (1S, 3R)-ACPD, a selective metabotropic glutamate receptor agonist, through the microdialysis probe caused a significant and persistent increase in extracellular dopamine level in the nucleus accumbens, which disappeared 2 h after perfusion of (1S, 3R)-ACPD was discontinued. On the other hand, a temporary increase in dopamine overflow was observed when 1 and 3 mM N-methyl-D-aspartate (NMDA), an ionotropic glutamate receptor agonist, was perfused into the nucleus accumbens. Application of 1 mM (1R, 3S)-ACPD, an inactive isomer, into the nucleus accumbens had no effect on the extracellular dopamine concentration. The metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) (5 mM) did not affect the basal dopamine level, but it attenuated the (1S, 3R)-ACPD-evoked dopamine overflow in the nucleus accumbens when applied concurrently with 1 mM (1S, 3R) ACPD. These results suggest that a long-lasting dopamine overflow following activation of metabotropic glutamate receptors contrasts with a transient one in response to NMDA receptor activation in the nucleus accumbens. PMID- 8614558 TI - Expression of a MADS box gene, MEF2D, in neurons of the mouse central nervous system: implication of its binary function in myogenic and neurogenic cell lineages. AB - MEF2D, a member of myocyte-specific enhancer binding factor 2 (MEF2) gene family, was shown by Northern blot hybridization to be strongly expressed in the head portion of mouse embryos at later stages of ontogenesis, in the cerebellum and the cerebrum of adult mice, in cultured cell lines of neuronal origin, and in skeletal and cardiac muscles. During ontogenesis, MEF2D transcripts were detected by in situ hybridization in the olfactory bulb, entorhinal cortex, pyriform cortex, and hippocampus, in Purkinje and granule cells, and in large neurons in both the ventral and dorsal horns of spinal cord. Adult mice continued to express MEF2D in these limited areas of the central nervous system. Thus, MEF2D seems to be involved in either the differentiation process or the function of these neurons. PMID- 8614559 TI - cDNA sequence, ligand biding, and regulation of galanin/GMAP in mouse brain. AB - The cDNA encoding the 29 amino acid-long neuropeptide galanin and its flanking peptide galanin message associated peptide (GMAP), has been cloned and sequenced from mouse hypothalamic cDNA. The primary sequence of mouse galanin is GWTLNSAGYLLGPHAIDNHRSFSDKHGLT, followed by an amidation signal GKR. There are now 12 galanin sequences known: human, porcine, dog, rat, bovine, chicken, sheep, alligator, bowfin, dogfish, trout and mouse. The N-terminal 14 amino acids are identical in all of these species and the whole primary sequence of mouse galanin is identical to that of rate galanin. The mouse C-terminal flanking peptide, the GMAP, which is encoded on the same mRNA as galanin, shows a high degree of homology with all other known GMAP sequences but is not identical to any of them and it is more charged than the other GMAP sequences. Synthetic mouse galanin was found to displace [125I]mono-iodo-Tyr26 galanin (porcine) from receptors in the mouse hypothalamic membranes with high affinity (KD = 0.9 nM). Estrogen treatment of mice (0.1 mg/kg i.p.) for 6 h, which elevates the rat pituitary galanin mRNA levels, does not affect the galanin mRNA levels in mouse hypothalamus and pituitary. Neither does a subchronic glucocorticoid treatment (dexamethasone, 0.5 mg/kg i.p. for 7 days) affect these mRNA levels. PMID- 8614560 TI - A novel serpin-like protein, B-43, exists in both neurons and astrocytes: an immunohistochemical study in the parietal region of the bovine brain. AB - The presence of a novel member of serine proteinase inhibitor, B-43, was immunohistochemically indicated in both neurons and astrocytes in the parietal region of the bovine brain. B-43-like immunoreactivity was detected in pyramidal cells in the cortex and GFAP-positive astroglial cells in the white matter. The processes of B-43 may play a cooperative role with glia-derived nexin/protease nexin-1 and alpha 1-antichymotrypsin in the brain. PMID- 8614561 TI - DNA single-strand breaks in postischemic gerbil brain detected by in situ nick translation procedure. AB - Using an in situ nick translation procedure, DNA single-strand breaks (SSB) in postischemic gerbil hippocampus were investigated after 15-min forebrain ischemia followed by 0-4h of recirculation. In the control group, increased SSB were noticed in the ependymal cell layer and the dentate gyrus. After 15-min ischemia without recirculation, no remarkable changes in SSB were observed. However, after 1 h of recirculation, a marked increase in SSB was recognized throughout the hippocampus, especially in the cells in CA1 subfield and the dentate gyrus. After 4 h of recirculation, SSB decreased to a level near that of the control group. The results of the present study indicate that ischemic insults may injure intranuclear DNA during postischemic recirculation periods. Although many factors may be involved, activated endonuclease due to an intracellular Ca2+ rise, free radicals, and postischemic hyperthermia appear to be involved in this phenomenon. PMID- 8614562 TI - Glyceraldehyde-3-phosphate dehydrogenase is over-expressed during apoptotic death of neuronal cultures and is recognized by a monoclonal antibody against amyloid plaques from Alzheimer's brain. AB - The age-induced apoptotic death of cerebellar neurons in culture is associated with over-expression of a 38-kDa particulate protein identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both the age-induced apoptosis and the 38-kDa protein overexpression were effectively suppressed by the presence of tetrahydroaminoacridine, an antidementia drug, or aurintricarboxylic acid. This over-expressed 38-kDa protein and purified GAPDH were found to react with a monoclonal antibody (mAb), Am-3, which was raised against amyloid plaques from Alzheimer's brain, but not with mAb, AmT-1, which was produced using synthetic amyloid beta peptide. These results raise the possibility that GAPDH is also involved in the neurodegeneration during the development of Alzheimer's disease. PMID- 8614563 TI - Effects of superoxide dismutase on nitric oxide production during reperfusion after focal cerebral ischemia is rats. AB - To assess the interaction of nitric oxide (NO) and superoxide during reperfusion after cerebral ischemia, we studied the dose effects of superoxide dismutase (SOD) on the levels of nitrosyl hemoglobin and plasma nitrite + nitrate which were increased at 30 min reperfusion after 2 h middle cerebral artery occlusion in rats. SOD was administered at 10 min before reperfusion. With 1, 5 and 10 mg/Kg of SOD, plasma nitrite + nitrate level was decreased by 25 mg/kg of SOD. The same amount of apo enzyme was without effect. These results suggest effect of superoxide in the NO level released during reperfusion after cerebral ischemia. PMID- 8614564 TI - Effects of losigamone on synaptic potentials and spike frequency habituation in rat entorhinal cortex and hippocampal CA1 neurones. AB - Losigamone is an anticonvulsant both in vivo and in vitro. We here studied possible mechanisms for such effects with conventional intracellular recordings from pyramidal cells of area CA1 and entorhinal cortex in combined hippocampal entorhinal cortex slices. Losigamone reversibly reduced the number of action potentials elicited by 1 s long depolarising current injections. In addition, the drug moderately reduced EPSP amplitudes while monosynaptic fast and slow IPSPs were unaffected. PMID- 8614565 TI - Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor alpha stimulation. AB - This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression. PMID- 8614567 TI - Selegiline protects dopaminergic neurons in culture from toxic factor(s) present in the cerebrospinal fluid of patients with Parkinson's disease. AB - The cerebrospinal fluid (CSF) of patients with Parkinson's disease (PD) contains substance(s) that inhibit the growth and functions of dopaminergic neurons. Further, selegiline, a monoamine oxidase B (MAO) inhibitor (0.125-0.250 microM) enhanced the number of tyrosine hydroxylase (TH)-positive neurons, augmented the high affinity uptake of dopamine (DA), and averted the neurotoxic effects of CSF of PD patients on rat mesencephalic neurons in culture. The neuroprotective effects of selegiline may be related either to its ability to inhibit MAO B, preventing the generation of free radicals, or to neuronal rescue property due to unknown mechanisms. PMID- 8614566 TI - Regional quantification of dopamine transporter mRNA in rat brain using a ribonuclease protection assay. AB - We describe the regional distribution of dopamine transporter (DAT) mRNA in selected regions of rat brain using a highly sensitive and specific nuclease protection assay. This method determines the absolute quantity of mRNA expressed in the brain regions surveyed. DAT mRNA level varied widely between brain regions, and was detected only in cell body regions of the major dopaminergic pathways. Highest expression was seen in substantia nigra/ventral tegmentum (SN/VTA). Lower but detectable expression of a protected mRNA of the expected size was observed within hypothalamus. Expression could not be detected by this method in other brain regions studied. Our results indicate that this method is sufficiently sensitive to allow study of mRNA expression in individual animals. PMID- 8614568 TI - c-fos and HSP70 gene expression in rat brains in high gravitation-induced cerebral ischemia. AB - Previous studies have shown that brief exposures of rodents to high gravitational forces (+Gz) in a specifically designed centrifuge cause global cerebral ischemia. In the present study, the effect of +Gz exposure to +22.5Gz for 15 to 60 s on c-fos and HSP70 gene expression was examined. Northern and RT-PCR analyses to total RNA isolated from brains of rats in different post-exposure times revealed a significant, time-dependent increase in the c-fos mRNA level which returned to near normal by 180 min. The HSP70 mRNA level was increased two fold at 30 min post exposure, and remained elevated until 180 min. The transient stimulation of c-fos and HSP70 gene expression should serve as useful biomarkers for hypergravic stress on the brain. The present results should aid in design of future experiments in our understanding of the pathophysiology of the high +Gz challenges. PMID- 8614569 TI - The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress. AB - A mild thermal stress protects primary cultures of dorsal root ganglion (DRG) neurons against a subsequent lethal heat stress as well as to a lesser extent against a subsequent lethal ischaemia. In contrast, a mild ischaemic stress protects DRG neurons only against a subsequent severe thermal stress and not against severe ischaemia. A greater induction of heat shock protein (hsp) synthesis was observed in these cells following mild temperature stress compared to mild ischaemia. This suggests that the protective effect observed is dependent on hsp synthesis resulting in the observed cross-protective effect and does not involve a particular pre-stress specifically protecting against a subsequent, more severe application of the same stress. Moreover, a particular level of hsp induction produces a better protective effect against lethal heat stress than against lethal ischaemia. PMID- 8614570 TI - Differential cellular localization of oestrogen receptor immunoreactivity and oxytocin mRNA and immunoreactivity in the rat preoptic area. AB - Oestrogen influences both oxytocin mRNA and peptide immunoreactivity in the preoptic area and the rat oxytocin gene contains functional oestrogen response elements. However, using combinations of immunocytochemistry and in situ hybridization for oxytocin and oestrogen receptor, we found that preoptic oxytocin neurons do not possess oestrogen receptors. This finding implies oestrogen actions on oxytocin synthesis in preoptic neurones are unlikely to be mediated directly. PMID- 8614573 TI - Anatomic considerations of the peripheral nerve in compressive neuropathies of the upper extremity. PMID- 8614574 TI - Dynamics and pathophysiology of nerve compression in the upper extremity. AB - Nerve compression syndromes are common disorders. The distinctive pathologic changes in the nerves are directly related to the physiologic changes that occur. The symptoms from which people suffer are due to these physiologic changes. This article describes the relevant pathologic and physiologic changes of compressed nerves and their relation to the signs and symptoms that result. PMID- 8614572 TI - Temporal link between plasma thyrotropin levels and electroencephalographic activity in man. AB - Plasma thyrotropin (TSH) levels have been previously shown to be associated with the internal sleep structure determined by conventional scoring of sleep stages. This temporal relationship was re-evaluated using spectral analysis of the sleep electroencephalogram (EEG). Eight healthy male subjects underwent two randomized night studies after having received either placebo or 5 mg ritanserin, a selective 5-HT2 receptor antagonist known to increase slow-wave sleep. Delta relative power and TSH levels, determined at 10 min intervals, were found to be inversely related with an average cross-correlation coefficient highly significant (P < 0.0001) in both experimental conditions. Alpha slow-wave index, an estimator of awakenings, and TSH pulses exhibited a significant temporal association in both conditions. These results demonstrate that TSH fluctuations are linked to the sleep EEG activity in man. PMID- 8614571 TI - Vasoactive intestinal peptide regulates extracellular adenosine levels in rat cortical cultures. AB - Adenosine is an important inhibitory neuromodulator in the cerebral cortex, yet it remains unclear how extracellular adenosine concentrations are regulated. Recently, it has been shown that beta-adrenergic receptor stimulation in rat cortical cultures causes the accumulation of extracellular adenosine derived by enzymatic hydrolysis from adenosine cyclic monophosphate (cAMP) transported into the extracellular space. In this study we show that vasoactive intestinal peptide (VIP), in addition to activating adenylyl cyclase and promoting the accumulation of intracellular cAMP in rat cortical cultures, also causes transport of cAMP and accumulation of extracellular adenosine. We further show that the extracellular accumulation of adenosine in response to VIP can be blocked by inhibition of cAMP transport, cyclic nucleotide phosphodiesterase activity, and 5'-nucleotidase, indicating that extracellular cAMP is the source of the adenosine. Cyclic AMP transport may be a general mechanism by which a variety of neuromodulators that act upon receptors coupled to adenylyl cyclase might regulate extracellular adenosine levels and thereby inhibitory tone in the cerebral cortex. PMID- 8614575 TI - Clinical diagnosis of peripheral nerve compression in the upper extremity. AB - Compression neuropathies are common in clinical practice. This article is a review of the clinical features of the common entrapment neuropathies affecting the upper extremity. The frequently found entrapment syndromes are discussed in detail. Uncommon syndromes are also briefly discussed. PMID- 8614576 TI - Electrodiagnosis in compression neuropathies of the upper extremities. AB - Compression neuropathies may occur at several points along the course of a nerve. Electrodiagnostic studies are helpful in the evaluation of nerve compression. Nerve conduction studies are the most useful of these techniques in determining the site of compression. Compression neuropathies of the upper extremities are common, and a well planned study is important to localize the site of involvement and the severity of the nerve damage. PMID- 8614578 TI - Cervical Radiculopathy. AB - This article discusses the relevant anatomy, clinical presentation, diagnosis and surgical treatment for cervical radiculopathy. The etiology of cervical radiculopathy can play a role in the subsequent treatment of this problem. Both anterior and posterior surgical management is discussed. PMID- 8614577 TI - Differential diagnosis and pitfalls in electrodiagnostic studies and special tests for diagnosing compressive neuropathies. AB - The differential diagnosis of compressive neuropathies in the arms includes syndromes involving the nerve roots and brachial plexus, as well as the peripheral nerves. Often these conditions coexist. Nerve conduction velocity studies as well as electromyography have a role along with the clinical evaluation in differentiating these conditions. Limitations in routine electrodiagnostic testing are present, which necessitate several specialized techniques for identifying compressive neuropathies. PMID- 8614579 TI - Thoracic outlet compression syndrome. AB - This article is concerned with thoracic outlet compression syndrome (TOCS), one of the most controversial subjects in medicine. It may also be the most underrated, overlooked, misdiagnosed, and probably the most important and difficult to manage peripheral nerve compression in the upper extremity. Contents of the chapter include the historical aspect, anatomy, etiology and incidence, pathophysiology, symptomatology, diagnosis, conservative and surgical treatment, other conditions associated with TOCS, and results of TOCS surgical treatment. PMID- 8614580 TI - Radial nerve entrapment. AB - The radial nerve is frequently more involved in entrapment syndromes than the ulnar and median nerves. Common sites of compression are the juncture of the middle and distal third of the arm (especially with fractures of the humerus), just distal to the elbow (radial tunnel), and proximal to the wrist between the brachioradialis and extensor carpi radialis longus. Often in entrapment syndromes involving the radial nerve, the true diagnosis is not evident and is arrived at only by exclusion, which sometimes delays initiation of effective treatment. Radial tunnel syndrome is rare, but decompression when indicated, can provide relief. Radial sensory nerve entrapment in the forearm (distal third) does occur, but patients often respond to temporary thumb spica splinting. PMID- 8614581 TI - Ulnar nerve anatomy and compression. AB - Compression of the ulnar nerve can be understood in terms of the anatomic and dynamic factors. Although the ulnar nerve may be compressed at any point along its course, it is particularly susceptible at the elbow and the wrist. Clinically relevant anatomy will be reviewed in an attempt to provide the reader with a logical framework for successfully diagnosing and managing typical and atypical ulnar nerve compression lesions. PMID- 8614582 TI - Intraepineurial constriction of nerve fascicles in pronator syndrome and anterior interosseous nerve syndrome. AB - In PS or AINS, if obvious epineurial compression deformity of the median nerve or the AIN is not found at known sites of compression, IeCNF should be considered. IeCNF may occur in one or more nerve fascicles of the median nerve at one of multiple levels in the distal upper arm and proximal forearm. Decompression of the nerve fascicles is achieved by epineurotomy, microsurgical interfascicular dissection, and removal of the constricting outer layer of the perineurium above and below the elbow. Resection of the constricted segments of the nerve fascicles is not necessary. Intraepineurial exploration of the nerve trunk also may be considered if, after surgical decompression of PS and AINS, expected recovery has not occurred and there is no evidence of axonal degeneration. PMID- 8614584 TI - Newer techniques of carpal tunnel release. AB - In this article, after a brief account of the history and evolution of endoscopic carpal tunnel release, limited incision techniques, and an overview of the relevant anatomy, the authors describe various methods and instrumentation. The results, complications, and contraindications are noted. Subsequent discussion of the objections to and the special requirements of new techniques leads to judgement on the choice of methods by the authors. PMID- 8614583 TI - Carpal tunnel syndrome. AB - Patients with carpal tunnel syndrome should be told that it is a progressive condition that, if not treated, probably will worsen as time goes on. When release is performed properly, they have an excellent chance for substantial improvement, although some always may have a degree of residual numbness at the fingertips. Initial relief of pain is rapid, with subsequent improvement in numbness and weakness occurring more slowly. Carpal tunnel syndrome is a very common problem. Although there may be a distinct cause in some patients, the underlying reason for the increased bulk of synovium is not known in most. Conservative treatment gives temporary relief, but surgical release remains the most effective treatment. Complications are not common, and proper attention to details minimizes them. The results generally are excellent. PMID- 8614585 TI - Pitfalls of endoscopic carpal tunnel release. AB - The article details various pitfalls of endoscopic carpal tunnel release. Highlighted are the two-portal Chow technique and the single-portal Agee technique. PMID- 8614587 TI - Unusual compressive neuropathies of the upper limb. AB - Compression neuropathies are one of the most common causes of upper extremity pain. This article has addressed the diagnosis and treatment of some of the more unusual neuropathies. As always, a thorough knowledge of anatomy and its valuation, and a careful and impulsive physical examination will guide the clinician to the appropriate diagnosis. Electrical studies, done carefully and with the clinical diagnosis as a label, will often be confirmatory. In cases not responsive to nonoperative measures, surgery to decompress the nerve and to restore missing function will usually proved a modicum of patient improvement. PMID- 8614586 TI - Multiple compression neuropathies and the double-crush syndrome. AB - Multiple compressions along a nerve have a cumulative effect on condition, both antegrade and retrograde. This nerve compromise renders the nerve more susceptible to a second source of compression. The proximal source of compression may be subclinical yet partially responsible for the cumulative compression syndrome. The primary "crush" may be anatomic or metabolic in origin. Strict attention to the patient's history and physical findings permit separation of the multiple compressions, making the choice of surgical decompression easier. Provocative testing and work simulation are important in examination, as is EMG testing. Complete medical evaluation can aid in diagnosing patients presenting with peripheral nerve compression and additional risk factors. Surgical decompression of a nerve with multiple levels of compression may not relieve all symptoms. Dynamic compressions in the work place and other nonsurgical types of compression must be identified so the prognosis for complete recovery is improved. PMID- 8614588 TI - Persistence of symptoms after surgical release of compressive neuropathies and subsequent management. AB - Persistent symptoms following surgical release for entrapment neuropathy are a source of great frustration for both the surgeon and patient. Fundamentals of management require review of the patient's history and examination in an attempt to confirm the initial diagnosis and to rule out elements of the differential diagnosis. Persistent symptomatology may be a reflection of an incorrect diagnosis or double crush syndrome. If the initial diagnosis can be reconfirmed, then it is reasonable to reinstitute conservative management and to objectively re-evaluate the entrapment neuropathy by electrodiagnostic testing. This electrodiagnostic testing must be comprehensive in order to evaluate all potential entrapment neuropathies within the differential diagnosis. Individuals who demonstrate a transient response to conservative management or evidence of further deterioration on electrodiagnostic testing may be considered candidates for revision surgery. These individuals may be found to have had an incomplete release, error of technique, or iatrogenic compression. Persistence of symptoms on the basis of end-stage disease must be recognized to avoid further surgery that is unlikely to be of benefit to the patient. If surgical intervention is chosen, the procedure must address issues of residual compression, preservation of nerve vascularity, prevention of neurodesis, and protective padding in the presence of nerve hypersensitivity. PMID- 8614589 TI - Congenital nephropathies and uropathies. AB - Although congenital nephropathies and uropathies only represent a fraction of possible genitourinary diseases detected during childhood, they have serious and sometimes avoidable morbidity and mortality. Advances in genetic, molecular, and cellular biology research continue to better define embryologic insults to normal organogenesis and offer the promise that many of these conditions might be avoided in the future. For now, awareness of these entities and their varied presentations and manifestations is crucial so that prompt evaluation and aggressive multidisciplinary management assures the affected child optimal growth and development. PMID- 8614590 TI - Sodium and water homeostasis. AB - Disorders of sodium and water homeostasis are common occurrences in pediatric practice. They reflect distinct problems in the regulation of total body sodium balance and water distribution, respectively. Each of these groups of disorders has separate afferent and efferent mechanisms that are activated during disease states. Optimal therapy of children with fluid and electrolyte problems requires accurate delineation of the ECF volume and water distribution disturbance and the design of therapeutic regimens that account for each component of the clinical condition. PMID- 8614591 TI - The kidney in acid-base balance. AB - The practitioner's approach to the pediatric patient with metabolic acidosis begins with calculation of the serum anion gap, which allows the clinician to place the patient in one of two categories of acid-base disturbance: a normal anion gap acidosis or high anion gap acidosis. Likewise, the patient with metabolic alkalosis can be categorized by urinary chloride concentration and the response to chloride replenishment as either chloride-responsive or chloride resistant. The disease states associated with each category are reviewed in this article. PMID- 8614592 TI - Renal calcification in the first year of life. AB - The introduction of renal ultrasound technology has shown renal calcification to be more common in infancy than was previously believed. Understanding the role of inhibitors and promoters in crystal formation helps elucidate the pathophysiology of nephrocalcinosis. Identification of the presence or absence of hypercalcemia and hypercalciuria is an effective way to direct the diagnostic work-up of infants with nephrocalcinosis. The sonographic image of renal calcification resolves spontaneously in many infants. Whether microscopic nephrocalcinosis persists below the threshold of ultrasonographic detection is unknown. Renal calcification can be associated with persistent renal function abnormalities if hypercalciuria continues, such as in VLBW infants who receive long-term furosemide therapy after discharge from the hospital. Renal calcification may also progress to renal failure, such as in infants with primary hyperoxaluria, owing to the persistence of hyperoxaluria, a potent promoter of calcium crystal formation. PMID- 8614593 TI - Interactions of drugs with the developing kidney. AB - Interactions between drugs and the kidney are necessary for renal drug elimination, metabolism, and occasionally for therapeutic effect. These interactions may result also in renal toxicity. Understanding the kidney's role in drug-handling helps the clinician to be aware of potential drug interactions and toxicity. Drug disposition, elimination, and toxicity may differ with development and are to be considered when prescribing drugs for children. Nephrotoxicity associated with drugs, although common, is usually reversible with discontinuation of the drug; however, when drug therapy with a well-known nephrotoxic drug (e.g., cisplatin) is necessary, pharmacologic modulators may play a role in limiting the associated nephrotoxicity. PMID- 8614594 TI - Urinary tract infections. Old and new concepts. AB - The recommendations for evaluation and management of pediatric patients with UTIs are summarized in Table 5. These recommendations were designed to minimize the risk of kidney damage in children with UTIs based on current perceptions of the pathogenesis of renal injury. The children at greatest risk for kidney damage are the infants and young children with febrile UTIs in whom effective treatment is delayed, those with gross VUR, and those with anatomic or neurogenic urinary tract obstruction. These recommendations likely will be modified as more is learned about the pathogenesis of renal injury associated with UTIs, as new therapeutic approaches are developed, and as imaging technology improves. PMID- 8614595 TI - Management of the difficult nephrotic patient. AB - Most children with nephrotic syndrome do well, usually with multiple relapses and remissions. Some children require high doses of oral steroids to sustain a remission and develop significant steroid toxicity. These patients frequently can be managed with oral alkylating agents or with cyclosporine. A few nephrotic children to not respond to oral prednisone. The most common biopsy finding in steroid-resistant patients is focal segmental glomerulosclerosis. Many patients with this condition progress to chronic renal failure. Evidence suggests that the outcome is improved with either cyclosporine or with a protocol using pulse intravenous methylprednisolone and oral alkylating agents. PMID- 8614596 TI - HIV nephropathy in children. AB - The evaluation and management of children with HIV nephropathy provide a great challenge to the pediatric nephrologist caring for patients with this tragic illness, of which the nephropathy is only part of their problem and extends beyond the patient to the family and all of the supportive team involved. Care decisions should be based on a multi-disciplinary approach in which the infectious disease component is included. There are many areas that require a better understanding, particularly the pathogenesis of HIV nephropathy and the approach to treatment of the nephropathy with specific drugs. Early identification of children affected with this condition and an aggressive approach to management seem essential to the improvement of the outcome of these patients. PMID- 8614598 TI - The hemolytic uremic syndrome. AB - HUS is the most common cause of acute renal failure in infants and young children and follows a diarrheal prodrome about 90% of the time. Persuasive evidence shows that virtually all of postdiarrheal cases are caused by EHEC infections, and that the great majority of cases in the United States are caused by the EHEC serotype O157:H7. Mortality is approximately 5%, and approximately 10% of survivors are left with severe sequelae. A much larger number (30%-50%) experience mild chronic renal damage. Public health strategies, including zero tolerance for fecal contamination in slaughter houses and additional public education on proper food handling and cooking, does much to decrease the prevalence of the syndrome. Efforts to further dissect the postdiarrheal pathogenic cascade should continue, and an animal model needs to be developed. Only then will researchers be positioned to develop effective intervention strategies. Preventing life threatening extrarenal complications, especially of the CNS, is a major challenge. Idiopathic nondiarrheal HUS accounts for approximately 10% of cases and comprises a poorly understood composite of HUS subsets. Research directed toward a better understanding of these mysterious variants also is a priority for the years ahead. PMID- 8614597 TI - Chronic glomerulonephritis in childhood. Membranoproliferative glomerulonephritis, Henoch-Schonlein purpura nephritis, and IgA nephropathy. AB - The chronic glomerulonephritides that lead to permanent loss of renal function may present with an acute nephritic syndrome, nephrotic syndrome, or asymptomatic hematuria and proteinuria. Membranoproliferative glomerulonephritis, Henoch Schonlein purpura nephritis, and IgA nephropathy are common childhood glomerulonephritides that may lead to chronic renal failure. Their clinical manifestations, natural history, and long-term prognosis are distinct. This article reviews various presentations of these common childhood glomerulonephritides and an approach to management and potential therapy. PMID- 8614599 TI - Bone and mineral metabolism in childhood end-stage renal disease. AB - Renal osteodystrophy represents a spectrum from high to low turn-over lesions of bone, and the specific pattern may change during the evolution of chronic renal failure and as a consequence of specific therapeutic interventions. Although secondary hyperparathyroidism remains the predominant histologic lesion in patients undergoing maintenance dialysis, recent evidence indicates higher frequency of adynamic lesion not associated with aluminum intoxication. The different factors involved in the development of each of the histologic subtypes have been described together with the clinical manifestations of renal bone disease in childhood. Avoidance of aluminum-containing medications and the intermittent administration of calcitriol are effective approaches for the management of the renal bone diseases. The long-term consequences of the adynamic lesion remain to be determined. PMID- 8614600 TI - New hormones in the therapeutic arsenal of chronic renal failure. Growth hormone and erythropoietin. AB - Although the benefits of rhGH and r-HuEPO therapy in children with CRF and on dialysis are already significant, further study of these new additions to the therapeutic arsenal remains necessary. Data on the final adult height achieved in patients who receive rhGH are extremely important information that is as yet unavailable. The risks and benefits of raising the target hematocrit to a "normal" value in patients receiving r-HuEPO remains under study. Only when these and other issues are soundly evaluated will the full impact of these medications be understood. PMID- 8614601 TI - Principles of renal replacement therapy in children. AB - Much has changed in pediatric renal replacement therapy during the past decade. Even the smallest critically ill patients can be temporarily supported, and chronic peritoneal and hemodialysis in young children has become routine. Although improved technical capabilities often may raise difficult ethical dilemmas, the health care team must know that such therapeutic modalities are available for the pediatric patient. PMID- 8614602 TI - Pediatric renal transplantation. AB - Renal transplantation in children is a most rewarding treatment that dramatically changes the overall health and lifestyle of children with ESRD. Complexities in different aspects of renal transplantation in children are obvious. Optimum technical conditions and drug therapy must be provided for the success of renal transplantation. Application of recent advances in immunology and long-term care to clinical transplantation continue to improve graft and patient survival rates. Optimization of growth and development also can be improved with the use of rhGH. PMID- 8614603 TI - Chronic diarrhea and malabsorption. AB - Diarrhea is one of the major causes of infant morbidity and mortality worldwide. Major advances in understanding the pathophysiology of chronic diarrhea and malabsorption have taken place during the past three decades. Analysis of absorptive and secretory functions of the intestine has provided some insight into the possible causes of diarrhea. This article summarizes some of the specific causes of malabsorptive diarrhea in infancy and childhood, with emphasis on pathophysiology and approaches to therapy. PMID- 8614604 TI - Secretory diarrhea in children: newly recognized toxins and hormone-secreting tumors. AB - This review of bacterial toxins and hormones that stimulate diarrhea shows that we have learned much concerning the pathophysiology of these diarrheal states; however, we have much yet to learn. It is hoped that research will continue to enlighten us with regard to the pathophysiology, diagnosis, and treatment for these disease processes. PMID- 8614606 TI - Chronic nonspecific diarrhea of childhood: pathophysiology and management. AB - Chronic nonspecific diarrhea, or toddler's diarrhea, is a frequently encountered disorder of defecation in otherwise healthy children. Although the precise pathophysiology remains to be elucidated, evidence suggests that toddler's diarrhea primarily is a gut motility disorder, modulated by dietary factors. Although the role of low-fat diets has since long been established, the liberal consumption of fruit juices and soft drinks is considered an equally important factor. Normalization of the child's diet, especially with regard to fat, fiber, fluids, and fruit juices, usually suffices to attain resolution of the diarrhea. PMID- 8614605 TI - Gluten-sensitive enteropathy. AB - Gluten-sensitive enteropathy is induced by dietary wheat gliadin and related proteins in genetically susceptible individuals. Most evidence suggests that the mucosal lesion represents an immunologically mediated injury triggered by gluten in the context of a particular assortment of major histocompatibility complex genes. The amino acid residues of gliadin and related proteins responsible for toxicity have not been identified; in vitro systems are available, but definitive conclusions must rely on in vivo jejunal challenges. At a conservative estimate, symptomatic gluten-sensitive enteropathy affects approximately 1 in 1000 individuals in Europe; however, it is now becoming clear that a greater proportion of individuals has clinically silent disease, and probably many others have a minor form of the the enteropathy. In most countries, the clinical presentation has changed over the past few years coming closer to the adult type of the disease, and the age of onset of symptoms is shifting upward. Liver, joint, hematologic, dental, and neurologic symptoms are increasingly being recognized. Several diseases are associated the gluten-sensitive enteropathy, such as IgA deficiency, insulin-dependent diabetes mellitus, and a range of other autoimmune diseases. Tests based on the measurement of antigliadin and antiendomysium antibodies have gained success as noninvasive screening tests; however, the ultimate diagnosis still is based on the finding of a severe histologic lesion of the jejunum while the patient is on a gluten-containing diet and on its disappearance once the gluten is excluded from the diet. A lifelong, strict GFD is mandatory for celiac children. Among other long-term problems, an increased risk of intestinal lymphoma has been reported in patients on a normal gluten-containing diet. PMID- 8614607 TI - Infectious gastroenterocolitides in children: an update on emerging pathogens. AB - The recognition that bacterial infections induce signal transduction responses in infected epithelial cells also provides new avenues to consider as novel forms of therapy. For example, the chemokine interleukin-8, which attracts neutrophils to sites of mucosal infection, is produced by epithelial cells of gastric and intestinal origin in response to bacterial infection. Inhibitors of chemokine production or inhibition of the biologic effects of neutrophil chemoattractants have the potential to reduce both mucosal inflammatory responses and the attendant clinical sequelae. Eukaryotic cells also respond to infection with elevations in cytosolic second messengers, including inositol triphosphate (IP3) and calcium ([Ca2+]i). In intestinal epithelium, these second messengers can mediate the diarrheal response to infection. Calcium/calmodulin inhibitors may have a beneficial effect in treating those gastrointestinal infections mediated through changes in the level of cytosolic free calcium. DuPont and colleagues showed, for example, that oral therapy with zaldaride maleate relieves symptoms of disease and shortens the duration of diarrhea in travelers with ETEC-induced diarrhea. Evaluation of additional signal transduction responses to microbial infections should provide both new insights into the pathogenesis of gastrointestinal infectious diseases and novel approaches to consider for the prevention and therapy for these human illnesses. PMID- 8614608 TI - Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. AB - During the past two decades, necrotizing enterocolitis has emerged as a major cause of mortality and morbidity in premature infants. The specific cause of the disease remains enigmatic, but several putative risk factors provide clues to a pathophysiology that seems to be multifactorial. With the use of newly developed scientific tools, an understanding of the basic pathophysiologic cascade that causes necrotizing enterocolitis is emerging, providing hope for improved treatment and prevention. PMID- 8614609 TI - Electrolyte fluxes in the gut and oral rehydration solutions. AB - This article provides a brief overview of the normal physiology of water and electrolyte fluxes across the gut as a prerequisite for understanding the pathologic disturbances occurring with diarrheal illnesses. In turn, the rationale for the use of oral rehydration solutions in diarrheal disorders is explored. PMID- 8614610 TI - The role of glutamine, short-chain fatty acids, and nucleotides in intestinal adaptation to gastrointestinal disease. AB - Important first steps have been taken towards establishing how some nutrients interact with genes and affect intestinal adaptation. These mechanisms may be typical of how other nutrients influence cell function and turnover and help to maintain intestinal integrity. The dietary effects of nucleotides on intestinal cell mucosa act at the gene transcription level. The dietary effects of nucleotides on immune suppression also may act through similar mechanisms. The effects of the other trophic agents may interact at this level or at other levels. Scientific interest in how the various tropic factors work to maintain and repair the gastrointestinal tract is manifested by a growing body of research that demonstrates potential mechanisms for nutrient-gene interaction and how much interactions affect intestinal development and turnover. It seems clear that intestinal gene transcription and the activity of transcription factors are at least sometimes directly related to nutrition. The techniques of molecular biology now permit the exploration and explanation of how dietary factors, such as glutamine, SCFAs, and nucleotides, affect normal and pathologic intestinal mucosal development, function, adaptation, and repair. PMID- 8614611 TI - Antioxidants in pediatric gastrointestinal disease. AB - Oxidant stress seems to be involved in the pathogenesis of several important gastroenterologic disorders in infants and children. The question can still be asked, in most circumstances, whether the oxidant stress precedes, and therefore is involved in, tissue or cellular injury or is a result of injury and not of clinical importance. The data favor the former situation in several inflammatory conditions of the bowel and in a variety of liver diseases. Experimental and clinical testing of this possible basic mechanism of tissue injury over the next few years will shed light on the role of antioxidants in treating gastrointestinal disorders. PMID- 8614612 TI - Gastrointestinal gas formation and infantile colic. AB - Gastrointestinal gas causes distress in many patients and their parents. Most often, patients do not have an actual increase in gastrointestinal gas volume, but rather their complaints derive from a misunderstanding of normal physiology, a misinterpretation of symptoms (colic), or an increase in intestinal sensitivity (irritable bowel syndrome). Symptoms from actual increases in intestinal gas volume are seen most frequently in children who swallow excessive amounts of air, have a dysmotility syndrome, or consume foods containing poorly absorbed carbohydrates. Although many therapies are used in the treatment of gas-related symptoms, under close scrutiny, the commonly recommended agents (e.g. simethicone) do not have proven efficacy. An understanding of the physiology of gas production and disposal is of practical use to pediatricians in determining the appropriate method of intervention for patients with these complaints. PMID- 8614613 TI - Intestinal motility during ontogeny and intestinal pseudo-obstruction in children. AB - From the fragmentary studies performed to date of both functional obstruction and the ontogeny of intestinal motility, several conclusions can be drawn. The development of motor activity that is primarily dependent on neuronal activity, such as sucking, swallow-induced peristalsis of the esophagus, and cyclic fasting small intestinal motor activity, occurs according to a timetable that is similar in all species studies is gestationally dependent, but its timing during gestation is species specific. Postprandial events, including gastric emptying and continuous postprandial motor activity in the small intestine, in contrast seem to be dependent on the nature of the humoral response to food, provided that the musculature of the gut and enteric nerves are able to respond to it. Although these activities are primarily determined by neuronal development, there is a limited amount of information to suggest that their appearance may be pharmacologically manipulated to advance the timing and rate of development. It is commonplace now for cortisol to be used to induce the production of surfactant in mothers of babies who are at risk of hyaline membrane disease. A limited amount of information suggests that cortisol may have similar effects in inducing duodenal motor activity, and, thus, it may be possible to advance the timing and rate of development of intestinal motility in the very preterm infant by its use. There are, however, no studies to date to prove this. It is now clear that intestinal pseudo-obstruction is a heterogeneous disorder associated with various pathologies, some of which are intrinsic to the gut, whereas others are multisystem diseases affecting the intestine. In addition, although intestinal pseudo-obstruction was originally thought to involve the small intestine, it is now realized that it may not affect only one region, as in achalasia or Hirschsprung's disease, but also it may present diffusely throughout the gut. The motility changes caused by the disease processes are nonspecific, although neuropathic and myopathic changes are distinct and due to the destruction of the particular motor control system involved. The mechanisms of the ontogenic and disease changes described in this article are, however, almost totally unexplored. The recent upsurge of interest in the area with the development of advances in cellular and molecular biology, at least the early events, is now beginning to unravel. At the present time, the stage is being set for probably the most exciting phase of understanding of the nature of the ontogeny of intestinal motor activity, which the author expects will enable us to manipulate motor activity in normal preterm infants and, it is hoped, congenital dysmotile states. PMID- 8614614 TI - Short bowel syndrome in children and small intestinal transplantation. AB - This article discusses the management of short bowel syndrome from the time of intestinal resection until the patient either recovers free of supplemental parenteral and enteral nutrition or progresses to the point of needing intestinal transplantation. The importance of aggressive use of enteral feedings is emphasized, especially in relation to the process of intestinal adaptation. An approach to the various complications of short bowel syndrome, especially small bowel bacterial overgrowth, is discussed. Surgical options short of transplantation also are described. Intestinal transplantation and its present and future roles in the management of patients with short bowel syndrome are discussed. PMID- 8614615 TI - Development of immune function in the intestine and its role in neonatal diseases. AB - This review has traced the ontogeny of the human mucosal immune system, speculating that appropriate gut immune responses are essential in preventing many significant neonatal enteric diseases. Because the gastrointestinal tract serves as the portal of entry for many potential antigens, its mucosal immune function is essential in controlling antigenic responses and ensuring systemic tolerance. A thorough under standing of the development of the entire immune system is essential in defining intestinal mucosal immune function. From the protective barrier covering the enterocyte to the intraepithelial T lymphocytes, these components work together to limit antigen passage from the gut lumen to the underlying immune cells and, thus, promote normal immunity and tolerance. When abnormalities exist or when this immune barrier has not matured fully, conditions afflicting newborns, especially preterm infants, occur. Necrotizing enterocolitis, milk-protein enteropathy, and enteric bacterial infections are only three clinical examples of how aberrant gut immune-mediated defenses may have a significant role in their pathogenesis. In clinical practice, it is not only important to recognize these conditions at their onset but also to understand the basis for the underlying illness and identify newborns who are at an increased risk of acquiring them. PMID- 8614616 TI - Gastrointestinal and nutritional problems in children with immunodeficiency and AIDS. AB - Immunodeficient children pose a challenge to clinicians because of the interrelationship between infectious disease, metabolism, gastrointestinal tract function, psychosocial problems, and immune function. The interplay between these factors is not always clear, and frequently the best course of therapy is obscured because of an inability to determine which factors have the greatest impact on child health. To optimize therapeutic intervention, a multidisciplinary health care team must be involved with the management of children and their families. PMID- 8614618 TI - The use of non-uniform deuterium labelling ['NMR-window'] to study the NMR structure of a 21mer RNA hairpin. AB - The first synthesis of a non-uniformly deuterium labelled 21mer RNA is reported using our 'NMR-window' concept, showing its unique application in the unambiguous NMR assignment of the non-exchangeable aromatic and sugar protons. PMID- 8614617 TI - A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands. AB - We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID- 8614619 TI - Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine. AB - Translation of an mRNA encoding a selenoprotein requires that at least one UGA codon in the reading frame is recoded as a site for the insertion of selenocysteine. In eukaryotes, the termination codon recoding event is directed by a cis-acting signal element located in the 3' untranslated region of the gene. This 'selenocysteine insertion sequence' (SECIS) comprises conserved sequences in a region of extensive base-pairing. In order to study the structure-function relationships of the SECIS structure, we have applied a newly developed reporter gene system which allows analysis of stop codon suppression in animal cell lines. This system obviates the need for enzymatic or immunological estimation of selenoprotein synthesis, relying instead on the simple quantification of translational readthrough from the lacZ gene into the luciferase gene. The 3'-UTR of the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene was shown to contain a highly active SECIS element. Mutations in the base-paired sequences of other SECIS elements were used to analyse the significance of primary structure, secondary structure and pairing stability in the stem regions. The results demonstrate that the exact sequences of the paired nucleotides are comparatively unimportant, provided that a consensus combination of length and thermodynamic stability of the base-paired structures is maintained. PMID- 8614620 TI - The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context. AB - African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced. PMID- 8614621 TI - Sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in human cells: the role of transcription units. AB - DNA sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in HeLa cells have been cloned and 106 representative clones sequenced; 10 clones containing random genomic fragments served as controls. As chromatin is prone to rearrangement, care was taken to isolate sequences using 'physiological' conditions that did not create additional attachments. Comparison (by Southern blotting) of the concentration of each cloned sequence in 'total' and 'attached' fractions of DNA showed that most clones did contain attached sequences, but even highly-attached sequences were not attached in all cells in the population. Results demonstrated that 28% of clones were derived from three specific parts of the mitochondrial genome and 22% from different parts of the alu repeat. In addition, 41% of clones contained unique nuclear sequences; these contained no more of the motifs found attached to nuclear scaffolds or matrices (ie SARs or MARs) than would be expected from their base composition. No other attachment motif(s) could be identified by sequence analysis. However, Northern blotting showed that all the mitochondrial clones and 76% of clones containing unique sequences were transcribed; the degree of attachment correlated with transcriptional activity. These results are consistent with transcription being responsible for ever-changing attachments in both nuclei and mitochondria. PMID- 8614623 TI - Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei. AB - The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs. PMID- 8614622 TI - A nucleolar RNA helicase recognized by autoimmune antibodies from a patient with watermelon stomach disease. AB - Watermelon stomach is characterized by prominent stripes of ectatic vascular tissue in the stomach similar to stripes on a watermelon; in patients with this disorder chronic gastrointestinal bleeding occurs and approximately half of these patients have associated autoimmune disorders. In the serum of one patient, an antinucleolar antibody titer of 1:25 600 was found; the antibodies specifically recognized an approximately 100 kDa nucleolar protein, which we referred to as the 'Gu' protein. Its cDNA was cloned and sequenced. The Gu protein is a member of a new subgroup of RNA helicases, the DEXD box family. Gu protein fused with glutathione S-transferase contains ATP-dependent RNA helicase activity which preferably translocates in the 5'-->3' direction. Its RNA folding activity, RNA dependent ATPase and dATPase activities, and its translocation direction are similar to those of RNA helicase II [Flores-Rozas, H. and Hurwitz, J. (1993) J. Biol. Chem. 268, 21372-21383]. Sequencing of 209 amino acids of RNA helicase II peptides showed 96.7% identity with the cDNA-derived amino acid sequence of the Gu protein. The precise biological roles of this RNA helicase in the biogenesis of ribosomal RNA and the pathogenesis of watermelon disease and autoimmune disorder require further study. PMID- 8614624 TI - Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities. AB - Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4 (spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications. PMID- 8614625 TI - Drosophila immunity: a comparative analysis of the Rel proteins dorsal and Dif in the induction of the genes encoding diptericin and cecropin. AB - In Drosophila, bacterial challenge induces the rapid transcription of several genes encoding potent antibacterial peptides. The upstream sequences of the diptericin and cecropin Al genes, which have been investigated in detail, contain two, respectively one sequence element homologous to the binding site of the mammalian nuclear factor kappaB. These elements have been shown to be mandatory for immune-induced transcription of both genes. Functional studies have shown that these kappaB-related elements can be the target for the Drosophila Rel proteins dorsal and Dif. Here we present a comparative analysis of the transactivating capacities of these proteins on reporter genes fused to either the diptericin or the cecropin kappaB-related motifs. We conclude from our results: (i) the kappaB motifs of the diptericin and cecropin genes are not functionally equivalent; (ii) the dorsal and Dif proteins have distinct DNA binding characteristics; (iii) dorsal and Dif can heterodimerize in vitro; (iv) mutants containing no copies of dorsal and a single copy of Dif retain their full capacity to express the diptericin and cecropin genes in response to challenge. PMID- 8614626 TI - Identification and analysis of the rnc gene for RNase III in Rhodobacter capsulatus. AB - The large subunit ribosomal RNA of the purple bacterium Rhodobacter capsulatus shows fragmentation into pieces of 14 and 16S, both fragments forming the functional equivalent of intact 23S rRNA. An RNA-processing step removes an extra stem-loop structure from the 23S rRNA [Kordes, E., Jock, S., Fritsch, J., Bosch, F. and Klug, G. (1994) J. Bacteriol., 176, 1121-1127]. Taking advantage of the fragmentation deficient mutant strain Fm65, we used genetic complementation to find the mutated gene responsible for this aberration. It was identified as the Rhodobacter homologue to mc from Escherichia coli encoding endoribonuclease III (RNase III). The predicted protein has 226 amino acids with a molecular weight of 25.5 kDa. It shares high homology with other known RNase III enzymes over the full length. In particular it shows the double-stranded RNA-binding domain (dsRBD) motif essential for binding of dsRNA substrates. The Fm65 mutant has a frame shift mutation resulting in complete loss of the dsRBD rendering the enzyme inactive. The cloned Rhodobacter enzyme can substitute RNase III activity in an RNase III deficient E. coli strain. Contrary to E. coli, the Rhodobacter mc is in one operon together with the lep gene encoding the leader peptidase. PMID- 8614627 TI - Comparative analysis of ribonuclease P RNA structure in Archaea. AB - Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro. PMID- 8614628 TI - In vitro generation of a circular exon from a linear pre-mRNA transcript. AB - Recent findings have firmly established the existence of circular exons in vivo. We were interested in the possible splicing mechanism by which these unusual mRNA molecules could be created in vitro, though no biological relevance has been attached to their existence as yet. In this report we demonstrate that a modified synthetic linear yeast ACT1 transcript whose sequence begins with the 3'-part of its original intron, is continued by 247 nt of exon sequence and terminates with the 5'-part of its intron will generate a circular exon when introduced to standard in vitro splicing reactions in whole cell splice extracts from Saccharomyces cerevisiae. The formation of a circular exon was found to be independent of specific circular or secondary structures of the pre-mRNA transcript. We hypothesize that circular exons which are found in vivo may be generated from pre-mRNAs which derive from rare events of transcription initiation within an intron. PMID- 8614630 TI - Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions. AB - Various oligonucleotides containing 8-methylguanine (m8G) have been synthesized and their structures and thermodynamic properties investigated. Introduction Of M8G into DNA sequences markedly stabilizes the Z conformation under low salt conditions. The hexamer d(CGC[M8G]CG)2 exhibits a CD spectrum characteristic of the Z conformation under physiological salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m8G]CG)2 adopts a Z structure with all guanines in the syn conformation. The refined NMR structure is very similar to the Z form crystal structure of d(CGCGCG)2, with a root mean square deviation of 0.6 between the two structures. The contribution of m8G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m8G-containing oligomers. The presence of m8G in d(CGC[m8G]CG)2 stabilizes the Z conformation by at least deltaG = -0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was further stabilized by increasing the number of m8Gs incorporated and destabilized by incorporating syn-A or syn-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the chemically less reactive m8G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate syn-G conformation. PMID- 8614629 TI - Characterization of a UV endonuclease gene from the fission yeast Schizosaccharomyces pombe and its bacterial homolog. AB - From the fission yeast Schizosaccharomyces pombe, a cDNA fragment was isolated, which confers UV resistance on repair deficient Escherichia coli host cells. The cloned cDNA encodes a protein of 68,815 Da, which has a 36.6% identity of amino acid sequence with the previously identified 74 kDa UV endonuclease of the filamentous fungus Neurospora crassa. Analysis of several truncated gene constructs shows that only the C-terminal two thirds region, which has 54% identity of amino acid sequence with the C-terminal region of the Neurospora homolog, is necessary for complementing activity of UV-sensitivity in the E. coli host cells. Purified recombinant protein from E. coli host cells incises both UV induced cyclobutane pyrimidine dimers and (6-4) photoproducts at the sites immediately 5' to the DNA damage in the same fashion as the Neurospora protein. Furthermore, a bacterial homologous sequence was isolated from Bacillus subtilis and shows a similar complementing activity of UV sensitivity in E. coli host cells, indicating a wide distribution of this alternative excision repair mechanism in life. PMID- 8614631 TI - Incomplete factorial and response surface methods in experimental design: yield optimization of tRNA(Trp) from in vitro T7 RNA polymerase transcription. AB - We have studied the yield of Escherichia coli tRNA(Trp) obtained from in vitro T7 RNA polymerase transcription using incomplete factorial and response surface methods. Incomplete factorial experiments were first used to estimate the relative impact of six variables on the yield of tRNA(Trp). Fifteen trials were performed according to a balanced and randomized design. The correlation between observed yield and all experimental variables was identified by stepwise multiple linear regression analysis. The concentrations of T7 RNA polymerase, DNA template, NTP and MgCl2 proved to be significantly correlated with the yield of tRNA(Trp). We then optimized the yield with respect to each of these four variables simultaneously with a designed, response surface experiment based on the Hardin-Sloane minimum prediction variance algorithm. Twenty experiments were performed, in duplicate, to sample the quadratic surface relating the yield to the four significant variables. Coefficients of the quadratic function with all two-factor interactions were evaluated by stepwise regression using least squares, and significant coefficients were retained. Partial differentiation of the resulting quadratic model showed it to possess an optimum. Transcription performed at the corresponding conditions yielded 6-fold more tRNA(Trp) than the initial conditions, confirming the predictive value of the experimentally determined response surface. PMID- 8614632 TI - The width of the minor groove affects the binding of the bisquaternary heterocycle SN-6999 to duplex DNA. AB - A complex between d(GGGAAAAACGG).d(CCGTTTTTCCC) and the minor groove binding drug SN-6999 has been studied by 1H nuclear magnetic resonance spectroscopy. The drug is found to bind in the d(A)5 tract, but with interactions extending one residue in the 3'-direction along each strand. Doubling of resonances in the complex indicates slow to intermediate exchange between two binding modes. An orientational preference (7:3) is found, the first such example in an SN-6999 complex. Furthermore, the upper limit of the lifetime for the major species is longer than was found for SN-6999 with other DNA duplexes. The preferred orientation of SN-6999 has the pyridinium ring near the 5'-end of the (+) strand; the minor binding mode has the reverse orientation. The orientational preference and slower exchange rate relative to other SN-6999 complexes is attributed to increased stabilization from van der Waals interactions due to better shape complementarity between the DNA duplex and ligand. The comparison of these results with studies of SN-6999 complexed to other DNA duplexes reveals the sensitivity of the binding properties to the delicate interplay between the molecular structure of the ligand and the specific characteristics of the DNA minor groove. PMID- 8614633 TI - The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human. AB - The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres. PMID- 8614634 TI - DNA damage and DNA sequence retrieval from ancient tissues. AB - Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens. PMID- 8614635 TI - Differential discrimination of DNA polymerase for variants of the non-standard nucleobase pair between xanthosine and 2,4-diaminopyrimidine, two components of an expanded genetic alphabet. AB - Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms. Experiments were performed with templates containing 2' deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c7dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA). Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4 diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c7dX was significantly lower and also independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E. coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c7dX. PMID- 8614636 TI - Core sequences and a cleavage site wobble pair required for HDV antigenomic ribozyme self-cleavage. AB - The secondary structures proposed for the cis-acting hepatitis delta virus (HDV) ribozymes contain four duplex regions, three sequences joining the duplexes and two hairpin loops. The core and active site of the ribozyme could be formed by portions of the joining sequences, J1/4 and J4/2, together with one of the hairpin loops, L3. To establish the core region and define essential bases within this putative active site 28 single base changes at 15 positions were made and tested for effects on ribozyme cleavage. At 14 of the 15 positions all of the changes resulted in detectable decreased rates of cleavage. At seven of the positions one or more of the changes resulted in a 500-fold or greater decrease in the observed rate constant for cleavage. Mutations that resulted in 10(3)-fold effects were found in all three regions hypothesized to form the core. At the cleavage site substitutions of the cytosine 5' of the site of cleavage did not provide strong support for a sequence-specific interaction involving this nucleotide. In contrast, an A-C combination was the most effective substitution for a potential G-U pair 3' of the cleavage site, suggesting a requirement for a wobble pair at that position. PMID- 8614637 TI - The yeast UME6 gene is required for both negative and positive transcriptional regulation of phospholipid biosynthetic gene expression. AB - In Saccharomyces cerevisiae, regulation of the phospholipid biosynthetic genes, INO1, CHO1, CHO2 and OPI3, is known to occur at the level of transcript abundance. Derepression in response to inositol deprivation requires the INO2 and INO4 regulatory genes. Repression in response to inositol supplementation requires the OPI1 regulatory gene. Here, we examined the role of the UME6 global negative regulatory gene in expression of the phospholipid biosynthetic genes. These studies were stimulated by the finding that the INO1 promoter included a UME6 cognate cis-acting regulatory sequence (URS1). We found that the UME6 negative regulatory gene was involved in regulation of phospholipid biosynthetic gene expression through two distinct regulatory pathways. One pathway was the direct repression of INO1 expression through the URS1 element. Surprisingly, the UME6 gene was also required for derepression of CHO1, CHO2 and OPI3 gene expression. Consistent with this observation, the UME6 gene was required for wild type levels of expression of the INO2 positive regulatory gene. Therefore, the UME6 gene has both a negative and a positive role in regulating phospholipid biosynthesis. PMID- 8614638 TI - Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19. AB - Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well. PMID- 8614639 TI - An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron. AB - On the basis of the nucleotide sequence of Tetrahymena group I intron, we constructed a 31 residue RNA that has the P7 stem and the 3'-terminal guanosine residue (3'-G) with a putative stem-loop structure (P9.0) intervening between them. For this model RNA (P7/P9.0/G), four residues around the guanosine binding site (GBS) in the P7 stem were found to exhibit much lower sensitivities to ribonuclease V1 than those of a variant RNA having adenosine in place of the 3' G, suggesting that the 3'-G contacts around the GBS. NMR analyses of the imino proton resonances of the P7/P9.0/G RNA indicated that the base pairing in the GBS is retained on the interaction with the 3'-G, and that the two base pairs of the putative P9.0 stem-loop are definitely formed. Comparison of the RNA with its variants with either A (3'-A) or a deletion in place of the 3'-G suggested that the stability of the P9.0 stem-loop is affected by the GBS-3'-G interaction. The melting temperatures of the P9.0 stem-loop were determined from the UV absorbances of these RNAs, which quantitatively indicated that the P9.0 stem-loop is significantly stabilized by the interaction of the GBS with the 3'-G, rather than the 3'-A, and also by direct interaction with divalent cations (Mg2+, Ca2+ or Mn2+). Upon replacement of the G-C base pair by C-G in the GBS of the P7/P9.0/G RNA, the specificity was switched from 3'-G to 3'-A, as in the case of the intact intron. PMID- 8614640 TI - Elk-1 can recruit SRF to form a ternary complex upon the serum response element. AB - The initial genomic response to serum growth factors is the transcriptional activation of a set of immediate-early genes. Serum-induced transcriptional activation of several of these genes involves the formation of a ternary complex that includes the serum response factor (SRF), a 62 kDa ternary complex factor (TCF) and a serum response element (SRE). TCF alone does not bind the SRE of the protooncogene c-fos, but requires the prior assembly of the SRF-SRE binary complex for it to be recruited into a ternary complex. Here we show that this SRF SRE binary complex is not an obligatory prerequisite for the formation of a serum responsive ternary complex. We demonstrate that Elk-1, which has properties of TCF can recruit SRF into a ternary complex on elements that do not support formation of the SRF-DNA binary complex. We also show that for two immediate early genes, pip92 and nur77, formation of the ternary complex may occur without the prior assembly of SRF-DNA binary complex. Finally, we show that the ability of different sequences to support formation of Elk-l-SRF-DNA ternary complex in vitro correlates with their ability to respond to serum growth factors in vivo. Our results suggest that a much broader range of DNA sequences than the consensus SRF and TCF binding sites can support ternary complex formation, and by inference, serum induction. Possible implications of these results are discussed. PMID- 8614641 TI - Folding of the HDV antigenomic ribozyme pseudoknot structure deduced from long range photocrosslinks. AB - A trans-acting system has been designed in order to explore the three-dimensional structure of the anti-genomic HDV ribozyme. In this system, the substrate (SANT) is associated by base-pairing to the catalytic RNA (RzANT) forming helix H1. RzANT is able to cleave specifically the RNA substrate as well as a deoxysubstrate analogue containing a single ribocytidine at the cleavage site (position -1). This demonstrates that such deoxysubstrate analogues are valuable tools for structural studies of this ribozyme domain. They form however weak complexes with RzANT which is due in part to their ability to fold as stable hairpins unlike the RNA substrate. Using a set of full deoxy or of mixed deoxy ribo substrate analogues site-specific substituted with the photoaffinity probe deoxy-4-thiouridine, ds4U, at a defined position, we were able to determine a number of long range contacts between the substrate and the ribozyme core. In particular, crosslinks between substrate position -1 and position -2 with residues C15, G19 and C67, thought to be involved in the ribozyme catalytic site, were detected. A three dimensional model of the antigenomic ribozyme system, derived from the structure proposed by Tanner et al. [Current Biol (1994) 4, 488 498] for the genomic system was constructed. Apart from residue deletion or insertion, only minor accommodations were needed to account for all photocrosslinks but one which is attributed to an alternative hybridization of the substrate with the ribozyme. This study therefore further supports the structure proposed by Tanner et al. for the pseudoknot model. PMID- 8614643 TI - Fully edited and partially edited nad9 transcripts differ in size and both are associated with polysomes in potato mitochondria. AB - Two classes of nad9 transcripts are present at different abundances in the steady state RNA pool of potato mitochondria. The 5'- and 3' termini of the transcripts were determined by primer extension and S1 nuclease protection analyses respectively. Using two primer pairs that will either specifically amplify the larger transcript or amplify both the larger and the smaller transcripts in RT PCR analyses it was found that the larger nad9 transcripts are partially edited, while the smaller transcripts are fully edited. Both the larger and the smaller transcripts were found to be associated with mitochondrial polysomes. The polysome association was found to be sensitive to EDTA and puromycin treatment. Therefore, both fully and partially edited nad9 transcripts appear to be engaged in translation. PMID- 8614642 TI - Positive elements in the laminin gamma 1 gene synergize to activate high level transcription during cellular differentiation. AB - Transcription of the murine laminin gamma 1 gene is activated during retinoic acid/cAMP induced differentiation of F9 embryonal carcinoma cells. Positive transcription control elements associated with two DNase I hypersensitive regions in the large first intron of the gene have been identified which confer a differentiation response on the laminin gamma 1 promoter. However, the kinetics of transcriptional activation suggest each DNA region interacts with transcription factors appearing at different times during differentiation. Synergy between the two regions in cis causes high level activation. PMID- 8614645 TI - Rapid site-directed mutagenesis by a method that selects for full length mutated DNA. PMID- 8614646 TI - Kinetics of transcription in a minute column. AB - Immobilized enzymes or nucleic acids are widely used to analyze association and dissociation reactions. A pseudo-rapid kinetic method for studying transcription was developed by using an immobilized template DNA packed in a minute column, in a micro-scale liquid chromatography system. This method, which has a dead-time of only several seconds, is quick enough to allow analysis of the release of product RNA in transcription by Escherichia coli RNA polymerase. The method could be applied to most enzymes that interact with DNA. PMID- 8614644 TI - Particle-mediated delivery of recombinant expression vectors to rabbit skin induces high-titered polyclonal antisera (and circumvents purification of a protein immunogen). AB - Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants. PMID- 8614647 TI - A novel method of identifying living transfected cardiac myocyte. PMID- 8614649 TI - [Evaluation of bronchoscopic examinations in Poland]. AB - The aim of the study was to assess the state of bronchoscopical examinations in Poland. The analysis was based on a quantitative method using a questionnaire. It encompassed the year 1986. 22,936 examinations were performed in 76 centers (61.0 per 100,000 population, 1.19 per one hospital bed per year). 239 physicians performed bronchoscopies annually. The mean was 96 bronchoscopies ranging from 14 835. Only 20 out of 76 centers were well equipped, 17-satisfactory. In 39 the equipment was judged to be below satisfactory level. The number of examinations is well below the level needed. If one would narrow down the number of patients requiring a bronchoscopy to only two groups--those with malignancy and unconfirmed tuberculosis the number of bronchoscopies per one patient is only 0.9. The authors suggest means of improving this situation in Poland. PMID- 8614648 TI - Rapid selection and classification of positive clones generated by mRNA differential display. PMID- 8614650 TI - [Effect of indomethacin on bronchial reactivity to supernatant of mononuclear cell cultures from patients with bronchial asthma]. AB - In 27 asthmatic patients a cell culture was made from mononuclear cell stimulated by PHA. Indomethacin was added to part of the cultures. After 2 days the culture was terminated and the supernatant was prepared for bronchoprovocation studies. Patients underwent baseline respiratory function studies, after which bronchial provocation was carried out with different concentrations of the supernatant. Bronchial provocation using supernatant from cells cultured with indomethacin produced significant respiratory function alterations in comparison with tests carried out with supernatant from cells stimulated with PHA. PMID- 8614652 TI - [Causes of delayed treatment of patients with small cell lung cancer]. AB - An analysis of factors producing advanced small cell lung cancer in 57 patients from the Chemotherapy Department of the Oncological Center in Cracow. The main reason for delay in chemotherapy in small cell lung cancer were diagnostic mistakes. Almost 40% of the patients were treated with improper diagnoses for a mean period of 6 months. Although 28.3% seeked non-medical help this did not effect nor delay chemotherapy. PMID- 8614651 TI - [Neuropathies in small cell lung cancer]. AB - A routine neurological examination, electromyography studies and conductance in sensory and motoric fibres of upper and lower extremity peripheral nerves, was carried out in 65 subjects with small cell lung cancer prior instituting chemotherapy. None of the patients demonstrated metabolic changes nor toxic injury to the neurological system. The results of the neurological examination led to suspicion of neuropathy in 22 (34%) which was later confirmed by the electromyographic studies. In 12 subjects only EMG abnormalities were found allowing to diagnose a subclinical phase of neuropathy. Altogether 52% of the subjects demonstrated injury of the peripheral nervous system. Sensory neuropathy was observed in 6 patients, motor-sensory in 7, motoric neuropathy in 12. In one of the subjects from the latter group a myasthenic syndrome of the Eaton-Lambert type was found. In 7 patients the EMG results suggested injury of the anterior horn cells, in two further patients the clinical and EMG data suggested injury of the peripheral and spinal column. PMID- 8614653 TI - [The course of allergic alveolitis based on personal experience]. AB - The course of allergic alveolitis was observed in 19 patients. The diagnosis was based on medical history, exposition to organic dust, clinical findings, respiratory function and in selected cases on results of transbronchial lung biopsy. All patients presented restrictive patterns of lung function abnormalities. Although a clinical improvement was seen, in none of the patients was a normalization of respiratory function observed. The results of treatment were more promising in patients with a shorter time between the onset of symptoms and making of diagnosis. Isolation from allergens resulted in better effect without prophylactic treatment with prednisone. Steroid therapy did not effect the relapses nor worsening of the respiratory function. PMID- 8614654 TI - [Cavernous changes as a result of pulmonary tissue hyperreactivity to Aspergillus fumigatus infection]. AB - A case report is presented of a 74 year old male with chronic bronchitis in whom fever, malaise and sputum hypersecretion was observed. Inflammatory changes in the right upper lobe were found on a chest radiogram. Broad spectrum antibiotics and antituberculous agents did not produce any improvement. Due to the fact that Aspergillus fumigatus organisms were cultured from the sputum and the precipitin test was positive to aspergillin the diagnosis of Aspergillus fumigatus infection was made. 5-fluorocytosine--Ancotil did not bring any improvement. After initiating steroid therapy with 30 mg of prednisone an improvement was observed. This allowed to diagnose hyperreactivity to Aspergillus fumigatus as the cause of the disease. PMID- 8614655 TI - [Sarcoidosis with multiple bronchial stenoses and neoplastic changes in the lungs]. AB - A case report of a 45 year old female with a high suspicion of a lung tumor based on radiological and bronchoscopic findings is presented. Typical sarcoid changes were seen in biopsies from bronchial mucosa. Steroid therapy was instituted but due to intolerance had to be terminated. The patient was treated with chlorambucil, cyclophosphamide and azathioprine. Therapy was monitored with bronchoscopy. The best response was seen after steroid therapy, less to chlorambucil and cyclophosphamide. Azathioprine was least effective. PMID- 8614656 TI - [Usefulness of bronchoalveolar lavage for diagnosis of pulmonary tuberculosis. Case report]. AB - The case of a 57 year old male with Wegener's granulomatosis is reported in whom the course of cyclophosphamide and prednisone treatment a subacute mycobacterial tuberculosis pneumonia evolved. The diagnosis was based on culture of Mycobacterium tuberculosis organisms from the bronchoalveolar lavage fluid. The authors discuss the role of bronchoscopy and bronchoalveolar lavage in the diagnosis of pulmonary tuberculosis. PMID- 8614657 TI - [Mycobacteriosis--diagnosis and treatment]. PMID- 8614658 TI - [Paraneoplastic syndromes of the nervous system and muscles during the course of small cell lung carcinoma]. PMID- 8614659 TI - [Increased activity of phagocytes in cigarette smokers--supposed connection with lung emphysema]. PMID- 8614660 TI - [Some aspects of humoral immunity in tuberculosis]. PMID- 8614661 TI - [Bronchoalveolar lavage in patients with pulmonary sarcoidosis and advanced radiologic changes]. AB - The study material was made up of 20 patients with stage I pulmonary sarcoidosis in different state of activity. BAL was carried out through a bronchofiberscope using four aliquots 50 ml each, immediately aspirated after introduction into siliconized vials. BAL induced side effects were not seen. The returned fluid was centrifuged and the cell differential count was carried out. 11 patients (55%) demonstrated a high lymphocytic count. The degree of lymphocytosis did not correlate with abnormalities in respiratory function. The degree of lymphocytosis did slightly correlate with disease activity. Thus serial BAL studies might be useful in therapy monitoring. The high lymphocytosis should be carefully interpreted and can not be the only indication for instituting steroid therapy. PMID- 8614662 TI - [Significance of exposure tests in diagnosis of occupational asthma]. AB - Twenty-eight patients aged from 32 to 60 years, including 18 female and 10 male, were examined because of bronchial asthma suspicion. They were employed in different professions, but more of them worked as baker or nurse. An occupation time was from 11 to 32 years, but first symptoms of bronchial asthma were noticed after 7-28 (mean 14 yrs.) years of work. At all patients the exposure test (inhalatory challenge) with some substances selected by disease history was performed. This above mentioned substances were mechanically polluted in air to simulate an occupational circumstances. The exposure was persisted from 5 to 30 minutes, according to observed disease symptoms. Spirometric tests were performed in 2 and 30 minutes after exposure, but in baker group also in 4 and 8 hours. At 26 patients a decrease higher than 20% of starting values of FEV1 was determined. In most cases there were immediate reactions, and at baker group--dual reactions. At two examined subjects no response of bronchial tree was observed but only burning of mucosa and cutaneous pruritus. For this reason at this group a diagnose of allergy to examined substances was established. Other ones 26 patients were diagnosed as occupational bronchial asthma. PMID- 8614663 TI - [Long term results of treatment with cytostatic drugs on respiratory function tests in children]. AB - In 47 children after a 5 year remission of treated acute lymphoblastic leukemia respiratory function was assessed. In 11 a decrease of VC was found, while in 12 signs of obturation of lower airways were present. These results imply the presence of restrictive changes suggesting an interstitial lung disease. These children are at risk of developing a chronic pneumopathy. PMID- 8614664 TI - [The role of diabetes as a factor for increased risk of infection with tuberculosis]. AB - The number of freshly registered sputum positive cases of pulmonary tuberculosis coexistent with diabetes mellitus was calculated from data of the Central Register of Tuberculosis in Poland. The incidence rate for this group in 1985 and 1986 was only 2.6%, while the calculated rate was only higher by 1/3 in comparison with rates of the general population. This leads to a conclusion that diabetes mellitus does not increase the risk of developing tuberculosis. Treatment did not differ from standard therapeutical regimes. Early results were comparable with those from a general population. The mortality rate analyzed in both years was three fold higher in the group with coexistent diabetes mellitus than in the general population treated for tuberculosis. This implies that tuberculosis coexistent with diabetes mellitus is a great clinical problem but epidemiologically is insignificant. PMID- 8614665 TI - [Evaluation of respiratory tract function in workers employed in the production of Enolophos]. AB - An analysis of the respiratory function of workers of a chemical factory producing Enolophos was carried out. All workers were included in the study-41 men (age range 23-59 years, mean working duration 4.9 years). In 48.8% chronic bronchitis was diagnosed. Spirometric analysis was carried out using an electronical spirometer with a steady volume plethysmograph. The following along with other pulmonary mechanics parameters were assessed: compliance, airway resistance. An increase of the FEV1%VC index was seen in 34.2% and RV/TLC in 40%. Compliance was increased in 30%. Airway resistance was increased especially in subjects with chronic bronchitis and a decrease in FEV1, FEV1%VC, FEF50, FEF25 rates. In patients with decreased erythrocyte acetylcholinesterase activity only increased airway resistance was seen. The duration of working in this factory did not affect respiratory function. The results demonstrate a harmful effect of Enolophos to the lungs and airways. PMID- 8614666 TI - [Echinococcal cysts of the lung and spleen]. AB - Pulmonary echinococcosis (hydatid disease) is not rare in endemic regions. In Poland it is rare, seen mainly in subjects returning from endemic areas. The intermediate host includes a variety of mammals-sheep, cows, and also humans. The definite host is the dog and is the main source of ova. A case of a 52 year old male is presented who lived in Argentina for a few years. Radiological examination disclosed two pulmonary cysts. Additionally one was also present in the spleen. The diagnosis was made basing of findings form radiological examinations. CT-scan, and serological studies. It was further confirmed during the surgical removal of the affected lung parenchyma. Leaving the spleen cysts is a risk factor it may be the source for further seeding of ova. Mebendazol therapy was instituted, which will be continued in the future. The patient will remain under observation. PMID- 8614667 TI - [Recurrent inflammation of the bronchial tree in the course of a congenital tracheo-esophageal fistula]. AB - A case of a 7 year old boy with chronic inflammatory changes of the bronchial tree in the course of a type H congenital tracheo-esophageal fistula is presented. The diagnosis was made basing of findings during bronchofiberoscopy. Radiological examination of the upper GI tract did not reveal the abnormality. The fistula was closed surgically. PMID- 8614668 TI - [Nobel prize in physiology and medicine in 1989]. PMID- 8614669 TI - [Metabolism of elastin and pulmonary emphysema]. PMID- 8614670 TI - [Mechanisms of obturation in chronic bronchitis]. PMID- 8614671 TI - [Respiratory pattern in newborn infants with acute respiratory insufficiency]. AB - The respiratory pattern was evaluated in 32 newborn infants (21--premature infants). In 17 cases the cause of the respiratory insufficiency was the respiratory distress syndrome, in 15 pneumonias. Basing on registered spirograms the mean inspiratory time (Ti), the duration of the respiratory cycle (Ttot), time component (Ti/Ttot), respiratory frequency (f), normalized respiratory volume according to weight (Vt/kg) and Vt/Ti ratio were calculated. The authors did not find any differences in breathing pattern components between premature and at term infants nor between infants with RDS and pneumonias. In infants with RDS a shortened duration of inspiration was seen during the initial four days. Statistically significant differences were found in components of the respiratory pattern between studied and healthy infants. PMID- 8614672 TI - [Results of bronchoprovocation tests with the allergen Erwinia herbicola in persons with occupational exposure to organic dust]. AB - The study was carried out on 290 males with occupational organic dust exposition. In all bronchoprovocation test with Erwinia herbicola antigen were carried out. A positive test was seen in 16.2% of the studied subjects. Bronchial hyperreactivity to Erwinia herbicola allergen depended on living environs, occupational exposure and symptoms characteristic of allergic alveolitis. PMID- 8614673 TI - [Occupational allergic respiratory disorders in farmers]. AB - The authors present their original experience in epidemiological and clinical diagnosis of allergical respiratory disorders in farm workers in Tschechoslovakia in the years 1966-1985. Bronchial asthma was seen in 21 subjects, allergical rhinitis in 5, allergical alveolitis (Farmer's lung) in 17. Most often with exposure while working with animals were effected. Skin prick tests were most often positive to grass, dust and cat fur allergens. Allergic alveolitis was most often of the subacute and chronic type. In 17 a positive serum aspergillin test was found. PMID- 8614674 TI - [Activity of PZ-peptidase in blood serum of workers from a pesticide dust production factory]. AB - In 31 subjects with an occupational exposure to dust pesticides the levels of PZ peptidase, alpha-1-antitrypsin, alpha-2-macroglobulin were determined. A decrease of PZ-peptidase and an increase of alpha-1-antitrypsin, alpha-2-macroglobulin were found in comparison with a control group of 30 healthy volunteers. PMID- 8614675 TI - [Surgical therapy results in patients with mediastinal cysts]. AB - During the years 1968-1988 270 patients with tumors and mediastinal cysts were treated surgically. In 48 (17.8%) cysts were found (26 males, 22 females, age ranged 10-63 years, mean 40.1 years). In 24 pericardial cysts were found. In 24 the cysts derived from the forgut. 23 subjects (47.9%) were symptomless. In all the cysts were removed. Mortality was not seen. 47 patients are included in long term observation. PMID- 8614676 TI - [A case of lymphangioleiomyomatosis]. AB - A case report of lymphangioleiomyomatosis is presented. The authors discussed the clinical picture, radiological and functional changes and the best therapeutical methods in such disorders. PMID- 8614677 TI - [A case of skin cryptococcosis in systemic lupus erythematosus]. AB - Here is presented a case of woman treated by immunosuppressive preparations because of systemic lupus erythematosus with ski manifestations as tubercles and ulcerations on skin of trunk and extremities. On the basis of histological examination of tubercle skin specimens and mycological examinations of material obtained from skin ulcerations cryptococcosis was diagnosed. Disease was limited to skin that was an entry of infection. Patient was treated by Amphotericin B administered intravenously and Flucitosine per os. Amphotericin B was also applied topically. The results of cultures became gradually negative, up to total disappearance of fungus cells in direct specimens, prepared from examined material. After treatment continuing for 5 months only discoloured scars were observed on sick skin. PMID- 8614678 TI - [Respiratory system changes in persons infected with the HIV virus]. PMID- 8614679 TI - [Chronic cor pulmonale]. PMID- 8614680 TI - [Lymphangioleiomyomatosis]. PMID- 8614681 TI - Growth, immunoresponsiveness, and disease resistance of diverse stocks of chickens reared under two nutritional regimens. AB - Growth, immunocompetence, and disease resistance were measured in a commercial broiler stock (BC), a commercial white-egg-layer stock (LC), and a White Plymouth Rock line selected for high juvenile BW (HW) under dietary regimes differing in protein and energy content. Diet E had 20% more protein and 17% more ME than Diet A. Stock by sex interactions were significant for BW at 28 and 36 d of age because sexual dimorphism occurred at younger ages in the meat than in the layer stocks. When inoculated with .1 mL of a .25 or 2.50% suspension of SRBC, diet by stock interactions were significant for antibody titers 6 d postinoculation (PI) at the lower dosage. Interactions were not present at the higher dosage or for either dosage 13 d PI. Diet by stock interactions were significant at both dosages for change in SRBC titers from 6 to 13 d PI. The interactions resulted from a significantly smaller decline in BC than HW or LC chicks fed Diet A, whereas all stocks responded similarly when fed Diet E. A significant decline in rate of BW gain was observed in chicks inoculated with marble spleen disease virus (MSDV), with the increase in relative weight of spleen significantly greater for LC than HW or BC chicks 6 d PI. When heterophil to lymphocyte ratios were used as an assay of response to MSDV challenge, rations were significantly higher for HW chicks fed Diet A than for chicks fed Diet E and for LC chicks fed Diet E than for chicks fed Diet A, but not different for BC chicks on the two diets. PMID- 8614682 TI - Heterosis and overdominance following long-term selection for body weight in Japanese quail. AB - Although nonadditive genetic variation is more important in crosses among laying stocks than among meat stocks, development of special sire and dam lines to control the release of primary lines results in broilers being the product of three- or four-way crosses. Therefore, nonadditive genetic effects are important in meat stocks because of opportunities to combine stocks that complement each other. Four experiments were conducted to investigate heterosis and overdominance arising from crossing Japanese quail lines originating from the same base population and selected long-term ( > 85 generations) for high 4-wk BW under different selection environments. The different selection environments were established by feeding different levels of CP (28 and 20%) and different levels of thiouracil (0 and.2%). Estimates of heterosis were high for hatch of total eggs ( > 25%) and liveability (12 to 47%), and low for hatch weight (2 to 4%). Overdominance estimates followed similar patterns but were of lower magnitude. Percentage heterosis estimates for BW were high at 1 wk (20 to 30%) and declined linearly to 5 to 10% at 4 wk when quail were fed 28 and 24% CP diets. Contrariwise, when fed low-CP (20%) diets, heterosis values were low initially and remained stable or increased across age. Data obtained from measuring feed intake and feed efficiency indicated that heterosis for BW was closely related to change in feed intake. Heterosis values for feed intake closely followed those for BW, including the decline across age. Conversely, heterosis appeared to be absent for feed efficiency, except at 1 wk. Similar to heterosis estimates, overdominance estimates were high initially and declined across age and were more clearly demonstrated under the 24 and 28% diets. It was concluded that considerable heterosis is present for BW in Japanese quail following the crossing of lines selected long-term for high BW, but that it is dependent on both environment (diet) and age. PMID- 8614683 TI - Responses of laying hens to forced molt procedures of variable length with or without light restriction. AB - The importance of the length of the rest period and of photoperiod as components of the forced molt procedures was evaluated in 650- and 560-d-old Lohman hens. The procedure included an 8-d feed withdrawal phase and rest periods varying from 0 to 35 d, during which the birds were fed for maintenance only. In the first trial, forced molt was applied with or without omission of artificial illumination. The rest period varied between 2 and 20 d. In the second trial, all treatments included omission of artificial illumination, and a variable rest period between 0 and 35 d. Egg production ceased, after 4 to 5 d of feed withdrawal and resumed 8 to 15 d after the end of the rest period, without any consistent response to its length. Forced molt stimulated egg production rate and diminished its age-dependent rate of decline, reduced the proportion of broken and shell-less eggs, and improved shell quality. Following wide oscillations proportional to the length of the rest period, egg weight stabilized at levels similar to that of the unmolted controls. Feed intake was stimulated by forced molt to levels exceeding those of the control hens. Body weight increased during the postmolt period to levels slightly exceeding those of unmolted controls. Results of one trial show that omission of artificial illumination was essential for the full expression of the molt responses. In the other trial, production rate and shell quality were improved and the percentage of broken eggs was reduced when the length of the rest period was increased. Maximal improvements appears to have been reached with rests period of 14 to 21 d. PMID- 8614684 TI - Effects of population density on layer performance. AB - Four experiments were conducted to study the effects of population density on layer performance. in each experiment, 20-wk--old White leghorn pullets were housed in 30.5 x 50.8 cm cages at the rate of one, two, three, and four birds per cage for Treatments 1, 2, 3, and 4, respectively. in Experiment 4, there were three additional treatments: for Treatments 5, 6, and 7, birds were placed into identical cages (30.5 x 50.8 cm) at the rate of three, two, and three birds per cage, respectively, at 20 wk of age. At 28 wk of age, one bird was removed from each cage in Treatment 5, and one bird was added to each cage in Treatments 6 and 7. In the first three experiments, all eggs that were collected for 2 consecutive d every 8 wk starting at 28 wk of age were used to measure egg traits. In Experiment 4, egg traits were determined once at 52 week of age. Birds at the highest population density had the lowest percentage hen-day egg production and had one of the poorest feed efficiencies. Egg production was negatively correlated (P < or = .01, Experiments 1, 2, 3; P < or = .05, Experiment 4) with population density. Feed required to produce a dozen eggs was positively correlated (P < or = .01, Experiments 1, 2; P < or = .05, Experiment 3) with population density in three out of the four experiments. The addition or removal of a cage mate to or from a multiple-bird cage in Experiment 4 did not (P > .05) affect egg production or feed efficiency. Final BW, mortality, and egg weight were not (P > .05) affected by population density. Only in one out of the four experiments was feed consumption (Experiment 1), eggshell thickness (Experiment 2), or albumen height (Experiment 3 lowered (P < or = .05) by having more than one bird per cage. This study showed that increasing population density decreased laying performance of the birds. PMID- 8614685 TI - Developmental stability in relation to population density and breed of chickens Gallus gallus. AB - Bilaterally symmetrical morphological characters with fluctuating asymmetry usually have small, random deviations from symmetry that reflect the ability of individuals to cope with genetic and environmental stress. A comparison of the level of fluctuating asymmetry in two fast-growing breeds (ScanBrid, Ross 208) with that of a slow-growing breed (La Belle Rouge) and wild jungle fowl from India revealed a positive relationship between growth rate and asymmetry. Fast growing chickens kept at three densities (20, 24, and 28 chickens per square meter) revealed a positive association between fluctuating asymmetry and density. Large individuals generally had higher levels of fluctuating asymmetry than small individuals. Tonic immobility, which is often used as a measure of fearfulness, was weakly positively associated with the degree of fluctuating asymmetry (P < .05). Measures of fluctuating asymmetry may provide information on levels of stress experienced by domesticated animals. PMID- 8614686 TI - Effects of supplemental ascorbic acid on the performance of broiler chickens exposed to multiple concurrent stressors. AB - An experiment was conducted to determine whether ascorbic acid (AA) increases resistance of female Hubbard x Hubbard broiler chicks to multiple concurrent stressors. Stressors imposed from 10 to 17 d posthatch included 2 x 2 x 2 factorial combinations of beak trimming [(B), sham-operated or beak-trimmed and cauterized], coccidiosis [(C), gavage with 0 or 3 x 10(5) sporulated Eimeria tenella oocysts], and heat stress [(H), 28 vs 33 C]. A starter diet was supplemented with AA to provide 0, 150, or 300 ppm (milligrams per kilogram). This resulted in a 2 x 2 x 2 x 3 factorial design with two six-chick replicates of each of the 24 treatment combinations. Data were analyzed using ANOVA and a level of 95% significance. Ascorbic acid increased feed intake and lowered plasma corticosterone and heterophil:lymphocyte ratios. Heat depressed weight gain and feed intake and elevated heterophil:lymphocyte ratios. Heat and AA interacted to improve weight gain and feed intake and lower heterophil:lymphocyte ratios. Coccidiosis depressed weight gain, feed efficiency, and heterophil:lymphocyte ratios. Coccidiosis and AA interacted to increase feed intake and lower plasma corticosterone and heterophil: lymphocyte ratios. Beak trimming increase heterophil:lymphocyte ratios. Beak trimming and AA interacted to increase feed intake and lower heterophil: lymphocyte ratios. Weight gain and feed efficiency decreased whereas heterophil:lymphocyte ratios increased linearly in unsupplemented birds as a function of stressor "order" (the number of stressors imposed simultaneously) indicating an additive effect of systematically increasing the number of stressors. No changes in feed efficiency or heterophil:lymphocyte ratios were detected as a function of stressor order when AA was provided. Ascorbic acid reduced the slope of the regression equation describing the relationship between weight gain and stressor order. It was concluded that AA, particularly at 150 ppm, enhanced performance of broiler chicks exposed to multiple concurrent environmental stressors. PMID- 8614688 TI - Prevalence of the poultry red mite, Dermanyssus gallinae, in different types of production systems for egg layers in Sweden. AB - This study was undertaken between May and December 1994 to investigate the prevalence of hematophagous mites in Sweden. A particular aim was to establish the relationship between occurrence of mites and certain types of production systems for egg layers. Initially a postal questionnaire study was performed. Some months later this was followed up by inspections, sampling, and interviews on farms. The only mite species found was the red poultry mite, Dermanyssus gallinae. Although this species was found to be present in cage batteries, deep litter systems, and backyard flocks, it was less prevalent among birds housed in cages. About 4% of the cage systems were found to be affected both according to the results of the postal inquiry and from the field study. However, discrepancies in prevalence were found when comparing the results obtained with these two investigations regarding alternative, deep-litter systems and backyard flocks. According to the inquiry, 21% of the deep-litter flocks were afflicted whereas 33% were found to be afflicted in the field investigation. Among the backyard flocks, 19% were afflicted whereas as many as 67% of the flocks were found to be afflicted upon inspection. These discrepancies were probably due to the fact that the situation had actually changed in some farms during the study period, but possibly also to inherent methodological errors. Farmers were also interviewed with focus on methods of husbandry and control strategies. Most farmers answered that they were combatting the mites by cleaning the empty houses and using chemicals such as organophosphorous compounds, carbamates, and synthetic pyrethroids. In several instances the problems were still not solved despite these curative or preventive measures. A control program for D. gallinae is suggested by regular screening and certification of the ectoparasite status of the birds before their transfer to production sites. PMID- 8614687 TI - Eimeria tenella infection induces recrudescence of previous Salmonella enteritidis infection in chickens. AB - Four experiments were conducted to examine the effect of Eimeria tenella infection on the recrudescence of Salmonella enteritidis in previously infected chickens. Significant recrudescence of cecal colonization in the birds challenged with E. tenella was observed at 3 wk after S. enteritidis infection in Experiment 1, at 3 and 4 wk in Experiment 2, and at 3, 4, and 5 wk in Experiments 3 and 4. The recrudescence was indicated by .81 to 6.31 logs increase in cecal S. enteritidis counts and by higher percentages of ceca that were culture-positive. The possible prolonged cecal S. enteritidis shedding from chickens infected with coccidia into the environment might be important for the perpetuation of S. enteritidis infectious cycle. Except for Experiment 1, in which a significant higher culture-positive rate of the liver was detected in the coccidia-infected group, no significant difference of culture-positive rate of liver and spleen between the treatments was observed. The recrudescence of previous S. enteritidis infection caused by E. tenella infection was obviously related to the initial S. enteritidis dose size and time of exposure to coccidia. PMID- 8614689 TI - Experimental reproduction of proventriculitis using homogenates of proventricular tissue. AB - Proventriculitis is a problem affecting the processing of broiler carcasses, particularly those processed at 4 to 5 wk of age. The proventriculus and the gastric isthmus connecting the proventriculus to the gizzard are enlarged and swollen and often rupture during processing, causing carcass contamination. This study suggested that a filterable agent found in homogenated proventriculi can cause lesions similar to those seen in field cases. Proventriculitis was produced independently of an effect on growth, and only unfiltered homogenate caused stunting. Field birds with severe proventriculitis were shown to have increased body weights compared with birds without proventriculitis or with milder lesions. Intestinal weakness was not associated with proventriculitis and field birds with the most severe proventriculitis had stronger intestines. Although infectious proventriculitis has generally been reported as one of the lesions associated with stunting syndrome, these data suggest that it may have an independent etiology. PMID- 8614690 TI - Effects of pelleting, lactose level, polyethylene glycol 4000, and guar gum compared to pectin on growth performances, energy values, and losses of lactose, lactic acid, and water in chickens. AB - Five mash and two pelleted diets were tested in broiler chickens (7 to 19 d). Mash diets consisted of a basal fraction diluted with either .5% pectin or .5% guar gum. Mash pectin and guar gum diets contained either 3% lactose (PL3m and GL3m diets, respectively) or 6% lactose (PL6m and GL6m diets, respectively). Compositions of pelleted diets (PL3p and GL3p) were those of PL3m and GL3m diets, respectively. All diets contained .5% polyethylene glycol 4000 (PEG) except the PL3m0 diet. The latter diet differed from PL3m diet by the PEG content, only. The real applied viscosities of pectin and guar gum diets were 1.48 and 4.94 mL/g, respectively, No effect of PEG was detected on growth performances, and excreta losses of lactose, lactic acid, and water. No negative effect of guar gum compared to pectin was observed on body weight (19 d), except with pelleted diets (P < .05). Feed:gain ratios for guar gum diets were 7% higher (P < or = .001) that those of pectin diets. The AMEn values of guar gum diets were 4% lower (P < or = .001) than those of pectin diets. For mash diets, lactose digestibilities were lower (P < .05) with guar gum than with pectin. Increasing lactose level from 3 to 6% did not affect (P > .05) AMEn values, feed: gain ratios, and body weights (19 d) but reduced (P > .001) lactose digestibilities from 78 to 64%. The positive effects of pelleting on body weights (19 d) were much less pronounced with guar gum than with pectin (P < .05). The AMEn values of pelleted diets (PL3p) and GL3p) were, on average, 2.5% lower (P = .005) than their mash counterparts (PL3m and GL3m). Water losses related to feed intake were greater with guar gum than with pectin (P < .001) and with 6% lactose than with 3% (P = .001) but were not affected (P > .05) by pelleting. Lactic acid losses related to feed intake were increased by guar gum compared with pectin (P < .001), with more pronounced effects induced by high lactose level (P < .05) and pelleting (P < .05). In many respects, the effects of guar gum seemed similar to those observed in an acid liquid diarrhea. PMID- 8614691 TI - An evaluation of various response criteria in assessing biological availability of phosphorus for broilers. AB - The relative bioavailability of P from seven sources was determined in relation to a standard dicalcium phosphate dihydrate (CaHPO4.2H2O) in a 21-d assay involving 1,320 broiler male chicks using several response criteria. The seven sources (Lucaphos-48, Lucaphos-40, Rukana, Cefkaphos-N, phosphoric acid, monocalcium phosphate monohydrate, or Biophos) were added to the basal diet (.40% total P and 1.10% Ca) at levels to supply .05, .08, .12, .17, .23, and .32% P. Two additional levels (.44 and .66%) of P from dicalcium phosphate dihydrate were included for the standards. The criteria selected to evaluate included tibia ash, tibia specific gravity, tibia shear force, toe shear force, and metatarsal shear force. Other criteria: weight, length, diameter, and volume of tibia; weight, volume, and specific gravity of metatarsus; and weight, volume, and specific gravity of toe were not selected because their response to increasing P levels were inconsistent and quite variable. Nonlinear (asymptotic and sigmoidal) regression equations were fitted to the data than linear equations. The ratios of regression coefficients were used to determine the bioavailability of various test phosphates relative to the reference standard. The results indicated that the response criteria used for the determination will considerably influence the relative bioavailability estimates of a P source. Body weight gain and toe ash percentage were found to be an equally or a more sensitive criteria for assessment of P availability than tibia ash. Tibia specific gravity, tibia shear force, toe shear force, and metatarsal shear force were of limited value as response criteria in P bioavailability assays based on standard error and difference required for significance. PMID- 8614692 TI - Improving phosphorus availability in soybean meal for broilers by supplemental phytase. AB - A 21-d experiment was conducted with day-old male broilers (n=840) to evaluate the effectiveness of supplemental phytase for improving the availability of phytate P in soybean meal when varying levels of P were fed. The semi-purified basal diet (.18% phytate P) contained soybean meal as the only protein source. Seven levels of phytase (0, 200, 400, 600, 800, 1,000, and 1,200 U/kg diet) were added to diets formulated to contain .20, .27, or .34% nonphytate P (nP; or .38, .45, and .52% total P, respectively). The desired levels of nP in the three basal P diets were achieved by adding varying amounts of defluorinated phosphate. A 2:1 Ca:total P ratio was maintained in all diets. Body weight gains and feed intake were improved (P < .001) by phytase at all nP levels, but the magnitude of response was greatest at low nP levels, resulting in an nP by phytase interaction (P < .01). Gain:feed was unaffected by phytase addition. A high mortality (35 to 45%) was observed for the .20 and .27% nP diets without added phytase, but this declined to normal levels with the addition of 200 to 400 U phytase/kg diet. Ash percentage of toes and tibia and shear force and stress of tibia increased with added phytase. These responses clearly show that the phytate-bound P in soybean meal was made more available to broilers by microbial phytase, and the total response was related to the phytase and nP/total P levels. Based on the high R2 values for the second order translog equations, BW gain, feed intake, and toe ash percentage were the most sensitive indicators to assess P availability, followed by tibia force and ash percentage. Derived nonlinear and linear equations for BW gain and toe ash percentage at the two lower nP levels were used to calculate P equivalency values of phytase for inorganic P. Using the average function of P released ( gamma ) by microbial phytase ( chi ) derived with nP levels of .20 and .27% for BW gain and toe ash percentage, gamma = 1.120 - 1.102e-.0027chi, 1 g of P could be released with 821 U of phytase. The amount of P released increased with increasing levels of phytase, but the amount of P released per 100 U of phytase decreased. Released P ranged from 31 to 58% of phytate P for 250 to 1,000 U of phytase/kg of diet. PMID- 8614693 TI - Response of turkey poults to tiered levels of Natuphos phytase added to soybean meal-based semi-purified diets containing three levels of nonphytate phosphorus. AB - A 3-wk feeding trial using 920 day-old turkey poults was conducted to evaluate the addition of seven levels of phytase (Natuphos; 0, 200, 400, 600, 800, 1,000, and 1,200 U/kg of diet) to diets containing three levels of nonphytate P (nP) (.27, .36, and .45%). A positive control diet contained .60% nP. Semi-purified basal diets contained soybean meal as the only protein source. The increase in BW gain from added phytase was greatest for the lowest nP diet (nP by phytase interaction, P < .001). At .27% nP, gains improved (P < .001) to 800 U of phytase/kg of diet and then reached a plateau. At .36 and .45% nP, increases in gains were observed only for 200 U of phytase/kg of diet. The highest phytase addition to.36 and .45% nP diets produced gains equal to those of the positive control diet. Feed intake increases paralleled those of BW gains. Gain:feed was lowest for the .27% nP diets without phytase, but improved (P < .001) to 800 U of phytase/kg of diet and then reached a plateau. The high incidence of leg disorders and high mortality (40%) observed for the poults fed the .27% nP diet without added phytase declined with the addition of 200 to 400 U of phytase/kg of diet. Ash percentage of toes and tibias increased as the levels of nP (P < .001) and phytase (P < .01) increased; the magnitude of the response to phytase decreased as nP in the diet increased, resulting in an nP by phytase interaction (P < .001). Tibial shear force and stress responded in a similar manner to increasing levels of nP and added phytase. Results show that 652 U of microbial phytase is equivalent to 1 g of P from defluorinated phosphate in turkey starter diets using soybean meal as the only source of phytate P. The response per 100 U of phytase decreased as the total amount of phytase added was increased. PMID- 8614694 TI - Development of a genetic map of the chicken with markers of high utility. AB - Microsatellites are tandem duplications with a simple motif of one to six bases as the repeat unit. Microsatellites provide an excellent opportunity for developing genetic markers of high utility because the number of repeats is highly polymorphic, and the assay to score microsatellite polymorphisms is quick and reliable because the procedure is based on the polymerase chain reaction (PCR). We have identified 404 microsatellite-containing clones of which 219 were suitable as microsatellite markers. Primers for 151 of these microsatellites were developed and used to detect polymorphisms in DNA samples extracted from the parents of two reference populations and three resource populations. Sixty, 39, 46, 49, and 61% of the microsatellites exhibited length polymorphisms in the East Lansing reference population, the Compton reference population, resource population No. 1 (developed to identify resistance genes to Marek's disease), resource population No. 2 (developed to identify genes involved in abdominal fat), and resource population No. 3 (developed to identify genes involved in production traits), respectively. The 91 microsatellites that were polymorphic in the East Lansing reference population were genotyped and 86 genetic markers were eventually mapped. In addition, 11 new random amplified polymorphic DNA (RAPD) markers and 24 new markers based on the chicken CR1 element were mapped. The addition of these markers increases the total number of markers on the East Lansing genetic map to 273, of which 243 markers are resolved into 32 linkage groups. The map coverage within linkage groups is 1,402 cM with an average spacing of 6.7 cM between loci. The utility of the genetic map is greatly enhanced by adding 86 microsatellite markers. Based on our current map, approximately 2,550 cM of the chicken genome is within 20 cM of at least one microsatellite marker. PMID- 8614695 TI - Quantitative determination of spermatozoa penetration of the perivitelline layer of the hen's ovum as assessed on oviposited eggs. AB - A technique was developed to assess the number of cock spermatozoa penetrating the perivitelline layer (PL) in oviposited eggs in vivo. Two trials were conducted to test this technique and to establish correlation values between fertility and sperm penetration (SP). First, three Athens Canadian Randombred males, previously tested as having high fertility (100%), were each housed with seven hens. Sperm penetration was determined from eggs laid over a 3-d period (n = 41) with the mean number of spermatozoa penetrating the PL overlying the germinal disc (GD; 1.35 mm2 area) and nongerminal disc (NGD) areas being 162.8 and 8.4, respectively. Following removal of the males, SP was monitored to establish its duration with an average of 4.6 eggs analyzed per male per day. Mean sperm penetration during this period declined from 167.0 to .2 and from 9.2 to 0 for the GD and NGD regions, respectively. The mean duration of SP was 15.7 and 11.3 d for the GD and NGD PL, respectively. The duration of fertility was also established to be 14.0 d. There was a positive correlation between sperm penetration of the GD PL and fertility from eggs laid by naturally mated hens (r = .89, P < .001). In the second trial, three groups (1, 2, or 3) of 16 hens (35 wk of age) each were artificially inseminated weekly for 4 consecutive wk with either 100, 50, or 25 million sperm/50 microL, respectively. Inseminations were repeated weekly for 12 consecutive wk. Mean values were obtained from each of three 4-wk periods and used as replicates. Mean SP values from the GD PL for Groups 1, 2, and 3 were 402, 19.5, and 14.1, with fertility values of 95.8, 92.4, and 83.3%, respectively. Each replicate mean was obtained from approximately 24 eggs per group per day postinsemination. A significant correlation between SP of the GD PL and fertility (r = .90, P < .001) was established using artificial insemination of hens. PMID- 8614696 TI - The effects of potassium sorbate and lactic acid on the shelf-life of vacuum packed chicken meats. AB - In this research, the effects of 5% potassium sorbate (PS) and 3% lactic acid (LA) applications on total mesophylic aerobic bacteria, total psychrotrophic aerobic bacteria, lactic acid bacteria, staphylococci and coliform bacteria, pH values, thiobarbituric acid (TBA) numbers, and sensorial properties of vacuum packed chicken leg and breast meats were investigated during storage at 4 +/- 1 C. In addition, residual sorbate was examined. A decrease in bacterial counts of chicken leg and breast meats was observed in the periods following the treatments of PS and LA; however, towards the end of the storage period, the effectiveness of PS was greater than that of LA. Although no effect was observed on pH values of samples treated with PS, LA caused a decrease in pH values in chicken meats. Both PS and LA treatments resulted in high TBA numbers. Although the shelf-life periods of samples treated with PS and LA were about 30 d, vacuum-packed controls and unsealed controls (both of which were untreated) lost their edibility on the 18th and the 6th d, respectively. Sensory analysis panel members could not distinguish between PS samples and control samples. However, samples containing LA had lower scores than others. Both PS and LA treatments were considered to be acceptable. Quantities of sorbic acid found in the samples treated with PS were below the Acceptable Daily Intake established by the Food and Agriculture Organization/World Health Organization. PMID- 8614697 TI - The effects of continuously or diurnally fed melatonin on broiler performance and health. AB - An experiment was designed to investigate the effects of dietary melatonin (MEL) on the performance and health of broiler chicks. Chicks raised under constant lighting were fed practical broiler diets in mash form containing 0, 20, or 40 ppm MEL from 0 to 3 wk. A fourth treatment (0 ppm MEL: 6h/d; 20 ppm MEL: 18 h/d) was used to mimic a diurnal light:dark cycle. Commercial broiler grower diets without MEL supplementation were fed from 3 to 6 wk of age. A small dose-related decrease was observed in weight gain (0 to 14 d) and feed consumption (0 to 21 d) due to continuous feeding of MEL. Continuously fed MEL also improved feed efficiency in the first 3 wk vs the control (0 ppm). Growth and feed consumption from 0 to 21 d were not affected by the diurnally fed diet when compared with the control treatment. No effects were found for mortality. However, the incidence of sudden death syndrome (SDS) was numerically less for the diurnal (3.56%) and 40 ppm (3.76%) diets from 0 to 42 d than for the control treatment 5.56%. Skeletal disease was generally unaffected, with the exception of the incidence of crooked toes at 42 d (0 ppm MEL: 8.67% vs diurnal: 4.89%). Dietary MEL fed either continuously or in a diurnal pattern had a minor effect on the performance and health of broiler chickens. PMID- 8614698 TI - Ability of broilers to resist heat following neonatal exposure to high environmental temperature. AB - Two trials were conducted to determine whether heat exposure of neonatal broilers had any effect on their growout performance or on their mortality during heat exposure later in life. Broiler chicks, 5 or 6 d old, were exposed to 36.1 C for 24 h in environmental chambers. At 47 d in Trial 1 and at 42 d in Trial 2, the broilers were exposed to 37.8 C for 6.5 h in Trial 1 and 8 h in Trial 2. Early heat exposure did not influence resistance to later heat exposure as measured by mortality. Also, early heat exposure did not affect body weight gain, feed efficiency, or growout mortality. PMID- 8614699 TI - Enrichment of cecal species of avian Eimeria in a mixed culture by transfer of sporozoites from foreign host to natural host birds. AB - An effective method for the enrichment of two cecal species of the avian Eimeria in mixed cultures has been developed. The method involves the transfer of sporozoites of Eimeria tenella and Eimeria adenoeides from their specific site of invasion in a foreign host bird to the natural host, and markedly increases the numbers of the cecal species in relation to the other species in the mixture. Donor turkeys and chickens were inoculated with mixed cultures containing equal numbers of oocysts of the chicken coccidia, E. tenella + Eimeria acervulina, or the turkey coccidia, E. adenoeides + Eimeria meleagrimitis, respectively. After 8 to 24 h, the cecal tissues of the donor birds were scraped into phosphate buffered saline and transferred, per os, to the natural host. On Day 6 to 7 posttransfer, numerous oocysts of E. tenella and E. adenoeides were found in the ceca of the recipient chickens and turkeys, respectively. In contrast, only occasional oocysts were found in a few birds in other areas of the intestine. PMID- 8614700 TI - Serotonergic mechanisms in the brain: a model for the integrative investigation of behavior and psychopharmacology. PMID- 8614701 TI - Outward and inward directed aggressiveness: the interaction between violence and suicidality. AB - This paper examines the relations and interactions between suicidality and violence. It reviews the recent literature indicating that more than 40 variables have been identified as risk factors for suicide. Evidence is presented to indicate that the differences between suicide attempters and suicide completers are a matter of degree, and that most conclusions that apply to one also apply to the other. A review of the recent literature on violence revealed that 37 variables are risk factors for violence and that 23 of them are risk factors for suicide as well. Evidence indicates that at least 17 variables have been identified as protective factors that decrease the risk of both suicide and violence. These considerations and others have led to the development of a Two Stage Model of Countervailing Forces that systematically relates suicide to violence. PMID- 8614702 TI - Experimental models for aggression and inventories for the assessment of aggressive and autoaggressive behavior. AB - This paper reviews principles realized in questionnaires for the assessment of aggression as well as in experimental models suitable for inducing aggression for the validation of questionnaire scales and for providing experimental models for testing aggression-reducing drug effects. Existing self-rating scales based on factor analysis were shown to measure certain parameters of reactions concerning modes of expression of aggression and its objects. In observer rating scales situations are usually also specified. A final scale containing 9 situations and 17 reactions grouped into seven factors is presented. It could be shown to differentiate between certain types of aggression provoking situations. Furthermore, models suitable for eliciting aggression were developed in three different departments of psychology. They are based on frustration by blockade of goals and critique or subtraction of positive reinforcers ("Tower of Hanoi" and "Superball Game" in Wurzburg, "Unsolvable Maze Computer Task" in Berlin) and by a competitive condition combined with application of aversive stimuli by a coplayer ("Modified Buss Machine" in Giessen). All experimental conditions were suitable for inducing anger and emotional arousal, negative ratings of confederates or experimenters, and partly also physiological changes. The results seem promising enough to test the relationship between artificially induced aggression and pathologiocal aggression in further research. PMID- 8614703 TI - The significance of aggression and impulsivity for self-mutilative behavior. AB - Self-mutilative behavior (SMB) is presented as a specific form of inwardly directed aggressiveness which is thought to be associated with problems of impulse control. Conceptual problems concerning impulsivity and impulsive aggression are discussed. Among different forms of dyscontrolled behavior, SMB is of special heuristic interest because of distinction can be made between patients committing impulsive and those committing premeditated self-harming actions. Psychometric and biological measures of impulsivity and aggressiveness were assessed in self-mutilators in comparison to depressives and normal probands. Self-mutilators were differentiated into two subgroups, those with an impulsive (ISMB) and those with a premeditated (PSMB) form of SMB, and depressives were also differentiated into two subgroups, those with a history of suicide attempts and those without. Only patients with ISMB (and depressives with a history of suicide attempts) showed an enduring tendency towards dyscontrolled patterns of behavior and cognition; therefore, SMB cannot be generally regarded as an indicator of high impulsivity or an impulse control disorder. Measures of impulsivity and aggressiveness did not behave analogically to each other and aggressive or autoaggressive modes of behavior should not be generally used as an index of impulsivity. A reduction of serotonergic activity, proved by a blunted prolactin response to D-fenfluramine, was found in all patient-groups in comparison to normal probands. Prolactin response after D-fenfluramine challenge turned out to be most blunted among self-mutilators with ISMB and among depressives with a history of suicide attempts. PMID- 8614704 TI - Psychopharmacology of central serotonergic systems. AB - Serotonin neurons in the rostral and caudal brainstem raphe nuclear groups give rise to collateralized ascending and descending projections which provide modulatory input into most networks throughout the entire neuraxis. The rostral raphe system is interconnected with target forebrain areas through reciprocal limbic-midbrain loops, which suggests that serotonin has a role in the regulation of complex intelligent adaptive behavior. Serotonergic pathways sensitize brainstem and spinal cord central rhythmic pattern generators which organize repetitive autonomic and motor activities, e.g. oral-buccal and nutritive behaviors, facilitate tonically active motor neurons innervating antigravity muscles, and disfacilitate somatosensory information processing. Serotonin effects are mediated by multiple receptor subtypes with distinct pre- and postsynaptic localization and regional distribution pattern. They belong to the G protein superfamily, coupling to adenylate cyclase (5-HT1,4,5,6,7) or phospholipase C (5-HT2), and to the ligand-gated ion channel superfamily (5-HT3). Drugs acting at these receptors are known to modulate various aspects of cooperative social behavior and responding latency, i.e. impulsivity, in a variety of experimental models of anxiety and depression. The clinical efficacy of the so-called selective serotonin reuptake inhibitors (SSRIs) in disorders characterized by poor impulse control, e.g. bulimia nervosa, obsessive-compulsive disorder (OCD) and violent suicidal or homicidal behavior, may likewise be due to improved responding latency. PMID- 8614705 TI - Serotonin receptors and animal models of aggressive behavior. AB - Various models of rodent agonistic behavior are described, which differentiate between offensive and defensive/flight models. Particular attention is given to one male and one female paradigm for offensive aggression, i.e. resident/intruder or territorial aggression (RI) and maternal aggression (MA). After an overview of the serotonin (5-HT) system in the CNS, a description is given of the ligands available. Subsequently, the effects of various drugs affecting serotonergic transmission in the RI- and MA-paradigms are described. The 5-HT1A receptor agonists busipirone, ipsapirone, and 8-OH-DPAT decreased aggression in RI and MA, but simultaneously led to a marked decrease in social interest and activity, indicative of a nonspecific antiaggressive profile. Nonselective 5-HT1 receptor agonists, such as RU24969, eltoprazine, and TFMPP reduced aggression quite specifically, did not decrease social interest or exploration, and sometimes even increased these behaviors. In RI and MA, the behavioral effects of these drugs were roughly similar. In contrast, MA was more sensitive to treatment with the 5 HT reuptake blocker fluvoxamine, which blocked RI aggression nonspecifically at the highest dose only. DOI, a 5-HT2A/2C# receptor agonist, decreased aggressive behavior and increased inactivity, without affecting social interest and exploration in RI as well as MA. This was, however, accompanied by "wet dog shaking" characteristic of 5-HT2 receptor stimulation. The nonspecific 5-HT receptor agonist (and 5-HT2 receptor antagonist) quipazine also induced "wet dog shaking" at doses which suppressed aggression, social interest, and exploration but increased inactive behaviors (sitting and lying). The discussion delineates a specific role for 5-HT1B receptor-subtype involvement in the modulation of aggression, with the restrictions we clearly face with regard to the lack of specific serotonergic agonists and antagonists for certain receptor subtypes. By and large, male and female rats react similarly to treatment with serotonergic drugs, and this fact underlines the consistent role of 5-HT in different forms of aggression. PMID- 8614707 TI - Decrease in circulating tryptophan availability to the brain after acute ethanol consumption by normal volunteers: implications for alcohol-induced aggressive behaviour and depression. AB - Acute ethanol consumption by fasting male volunteers decreases circulating trytophan (Trp) concentration and availability to the brain as determined by the ratio of (Trp) to the sum of its five competitors ([Trp]/[CAA]ratio). These effects of alcohol are specific to Trp, because levels of the 5 competitors are not increased. The decrease in circulating (Trp) is not associated with altered binding to albumin and may therefore be due to enhancement of hepatic Trp pyrrolase activity. It is suggested that, under these conditions brain serotonin synthesis is likely to be impaired and that, as a consequence, a possible strong depletion of brain serotonin in susceptible individuals may induce aggressive behaviour after alcohol consumption. The possible implications of these findings in the relationship between alcohol and depression are also briefly discussed. PMID- 8614706 TI - The flesinoxan 5-HT1A receptor challenge in major depression and suicidal behavior. AB - The prevailing neurochemical theory about biological correlates of suicidal behavior focuses on the serotonergic system. In this study, we assessed the cortisol, ACTH, GH, prolactin and temperature responses to flesinoxan, a5-HT1A agonist, in 30 DSM-III-R major depressed inpatients subgrouped into suicide attempters (n = 15) and nonattempters (n = 15). The patients were assessed after a drug-free period of at least 3 weeks. A subsample of 16 patients completed the Buss-Durkee Hostility Inventory as a measure of impulsive aggressive behavior. Mean delta cortisol responses to flesinoxan were significantly lower in the group of depressed patients with a history of suicide attempts than in the group without history of suicidal behavior: for the delta cortisol values 14.5 +/- 16.3 micrograms/l vs 101 +/- 94 micrograms/l (F = 8.9, df = 5.25, p = 0.006). There was also a very significant difference between suicide attempters and nonattempters for the temperature (delta T degrees) responses to flesinoxan: 0.20 +/- 0.24 degrees C vs. 0.60 +/- 0.24 degrees C (F = 18.1, df = 5.25, p = 0.0003). Hormonal and temperature responses to flesinoxan were not correlated with BDHI irritability or assault subscale scores. The results of the present study support the implication of the serotonergic system, particularly 5-HT1A receptors, in the control of self-directed aggressive behavior. Moreover, in depressed patients, serotonergic abnormalities do not appear to be related to aggressive behavior. PMID- 8614708 TI - [La Radiologia Medica in the future]. PMID- 8614709 TI - [The role of magnetic resonance angiography in the assessment of arterial stenosis in the transplanted kidney]. AB - Artery stenosis in the transplanted kidney is the most frequent vascular complication; hypertension onset or worsening may be associated and, at an end stage, also renal insufficiency. The diagnosis must be early and accurate and provide guidelines for medical, interventional or surgical therapy. To assess the diagnostic reliability or MRA, 27 patients were examined. On the basis of clinical, biochemical, pharmacological (Captopril test) and instrumental (color Doppler US) examinations, the artery of the transplanted kidney was considered normal in 6 patients and stenotic in 21. In the control group, MRA results were in agreement with color-Doppler findings. On the contrary, in 8 of 21 abnormal conditions, the two techniques were in disagreement. Digital angiography, considered as the gold standard, was performed in any questionable case, confirming a slight overestimation of the stenoses by MRA (3 cases) and 2 false positives by color-Doppler US. The authors believe color-Doppler US to be a reliable technique for screening stenosed arteries in the transplanted kidney. MRA is proposed as a complementary investigation useful to define stenosis type and to provide guidelines for treatment. PMID- 8614710 TI - [A comparative multicenter study of the efficacy of single- and double-lumen catheter drainage]. AB - Our study was aimed at comparing the therapeutic efficiency of single and double lumen catheters in the drainage of abdominal fluid collections. We report the results of in vitro and in vivo studies carried out to assess the usefulness of each catheter type based on its therapeutic results. In the in vitro study the aspiration efficacy of the catheters was tested in a simulated cavity. In the in vivo study 188 patients with 206 fluid collections in the abdomen were examined; the patients had been treated percutaneously with single or double lumen catheters, randomly. In each patient we studied drainage efficiency related to aspiration efficacy, the time the catheter rested in the cavity, patient's discomfort and finally the cost of both the device and hospitalization. Some patients underwent surgery for specific reasons, but nevertheless their clinical conditions were absolutely improved thanks to percutaneous drainage. The results of this study yield useful clinical data to choose the most suitable catheter for the percutaneous treatment of abdominal fluid collections. Single lumen catheters work better than double lumen ones, the latter being also more expensive. Hospitalization time is also reduced when single lumen catheters are used. PMID- 8614711 TI - [The information management of a radiology department: the development of a new type of software for the archiving of alphanumeric data and images]. AB - The authors report the main characteristics and the goals of the development of the RIS of the II Chair of the Institute of Radiology, La Sapienza University, Rome. The system was developed with a commercial software (4th Dimension), for use with an Ethernet network and Macintosh Apple computers. One of the main problems was to obtain a user-friendly system. The main options of our system are: booking, registration, exam execution, reporting, archives and statistics and system administration. The main characteristics of our RIS is that it allows important images to be archived in limited number and at low resolution. The aim is to use images for consultation and teaching purposes, not for diagnosis which is made on the original images. Low resolution images permit to use limited storage space. Image quality is very similar to that of the original images for the equipment connected on line with the RIS--i.e., US and MR units in our institute. Conventional radiographic and CT images are digitalized by two scanners with maximum resolution of 4k x 4k x 11 bits. To date, good results have been obtained. Our RIS has been used by the medical and non-medical staffs, without any particular instruction and has allowed us to organize and make faster department management and reporting. PMID- 8614712 TI - [High-dose-rate brachytherapy in esophageal carcinoma: the Italian experience]. AB - The results are reported of HDR intracavitary brachytherapy in 134 esophageal carcinoma patients (110 men and 24 women) treated in 10 Italian centers. Forty one patients received radical treatment and brachytherapy was often combined with external irradiation and/or chemotherapy. Clinical response rates follow: 56% complete remissions, 34% partial remissions, 10% no response/disease progression and not assessed. Ninety-three patients underwent palliative treatment: dysphagia was reduced in 80% of them and pain was reduced in 71% of them. Treatment-induced esophageal damage consisted in G3-G4 esophagitis (5% of patients), strictures (10%) and fistulas (3%). Complication rates were correlated with fraction dose (9.5% complications for fraction doses < 500 cGy, 20% with doses ranging 500-800 cGy and 38% with fraction doses > 800 cGy). Moreover, the esophagus was more severely injured when small tubes were used (24% with tubes phi < 2 mm, 19% with tubes phi 2-6 mm and 5% with tubes phi > 6 mm). When external irradiation was combined with brachytherapy, dysphagia was more relieved than with brachytherapy alone (89% vs. 71%), with no increase in complication rates. Also the chemotherapy-brachytherapy combination improved swallowing more than brachytherapy alone (88% vs. 79%) and once again complication rates did not increase. To conclude, in the radical treatment of esophageal carcinoma, HDR brachytherapy permits higher radiation doses to be delivered, with fair complication rates. As for palliative treatment, HDR brachytherapy is safe, has low morbidity and provides adequate relief of dysphagia in 80% of patients. We suggest the use of tubes phi > 6 mm and fraction doses ranging 5-6 Gy. PMID- 8614713 TI - [An in-vivo dosimetric study of the scattered radiation during the treatment of breast carcinoma]. AB - In the last decade, radiation therapy has been increasingly used to treat breast cancer conservatively and some authors showed their concern about the radiogenic effects of irradiation outside the treated area. Our aim was to measure the scattered dose to the contralateral breast, thyroid and gonads during radiation therapy after conservative breast surgery. Thermoluminescent dosimeters, LiF Mn Ti (3 x 3 x 0.9 mm3), were used to measure scattered radiations outside the treated area. They were positioned on the skin of the patients in small (2 cm in diameter and 1 cm thick) perspex holders. Each measurement was performed once per treatment and six dosimeters were used--two on the contralateral breast, one on the midpoint of the medial side and one on the midpoint of the lateral side; two on thyroid lobes and two on the pelvis. The minimum dose to the contralateral breast (p < 0.002) and the maximum dose to the gonads (p < 0.003) were significantly higher in the 60Co group than in the LINAC6 group. The mean values of the minimum doses to the contralateral breast were significantly different (p < 0.007) in the whole series when beam area values were lower or higher than the median value (176 cm2). In the whole series the beam area and the minimum dose to the contralateral breast were correlated (Pearson, p < 0.001); the beam area and the minimum dose to the contralateral breast (p < 0.005), the gantry angle for the lateral beam and the minimum dose to the contralateral breast (Spearman, p < 0.001) exhibited statistically significant correlations. Of 30 patients, 16 were irradiated with 60Co (group A) and 14 with LINAC6 (group B). In group A the average scattered dose to the contralateral breast ranged 0.53-2.15 Gy, 0.66-1.91 Gy in the thyroid, 0.14-0.19 Gy in the gonads. In group B the average scattered dose to the contralateral breast ranged 0.36-2.07 Gy, 0.53-0.98 Gy in the thyroid and 0.08-0.12 Gy in the gonads. In the LINAC6 group the beam area and the minimum dose to the thyroid exhibited a statistically significant correlation (p < 0.009), as well as the beam area and the maximum dose to the gonads (p < 0.006), the beam area and the minimum dose to the gonads (p < 0.02), the gantry angle of the lateral beam and the minimum dose to the contralateral breast (p < 0.02). In the 60Co group no correlation was statistically significant, except for the correlation between the beam area and the minimum dose to the gonads (p < 0.001). Our experience confirms the scattered dose to depend on head treatment, beam area, gantry angle and wedge angle. Finally, the literature on this subject is reviewed. PMID- 8614714 TI - [Giant-cell tumor of the carpal tunnel, a color Doppler echographic study. A case report]. PMID- 8614715 TI - [Elastofibroma dorsi. A report of a case diagnosed with magnetic resonance]. PMID- 8614716 TI - [A digital radiology method for assessing vertebral osteoporosis]. AB - The radiologic identification of vertebral fractures is usually subjective and reproducibility is poor. This paper describes a new digital radiologic method to perform vertebral morphometry, i.e. osteoradiometry (ORM). Lateral radiographs of the thoracic and lumbar spine were obtained in 50 premenopausal women and digitalized by means of a video camera. A special computer software enables to calculate the anterior (Ha), middle (Hm), and posterior (Hp) heights of vertebral bodies (T4-L5) and the morphometric indices of vertebral fractures. ORM reproducibility was assessed by comparing repeated measurements made by two radiologists: the intra- and interobserver variation coefficients (CV) were respectively 1.5% and 2.3% for Hp; 1.3% and 2% for Hm; 1.4% and 2.1% for Ha. The normal range for vertebral dimensions was therefore established. The anterior and posterior heights increased from T4 to L2, but for L3-L5 the posterior height was lower than the anterior height (Ha/Hp > 1). Vertebral heights positively correlated with the standing heights of the subjects (r = 0.2, p < 0.05). Weight and the body mass index (BMI) were not correlated with vertebral heights. These normal values, compared with those found in osteoporosis patients, will allow to assess ORM diagnostic efficacy in identifying vertebral fractures. PMID- 8614717 TI - [Simple lymphangioma of the breast. A case report]. PMID- 8614718 TI - [Hodgkin's lymphoma: an unusual pulmonary location. A case report]. PMID- 8614719 TI - [A pseudomass of the right atrium: an incidental echographic and magnetic resonance finding in a man over 100]. PMID- 8614720 TI - [The diagnosis of mediastinal hemangioma with magnetic resonance. A case report]. PMID- 8614721 TI - [Torsion of the greater omentum. A case report]. PMID- 8614722 TI - [The imaging diagnosis of a rare case of inflammatory pseudotumor of the spleen]. PMID- 8614723 TI - [Testicular microlithiasis. A pediatric case report]. PMID- 8614724 TI - [Recanalization of 2 suprahepatic veins with metal stents via a percutaneous transhepatic approach in a case of the Budd-Chiari syndrome]. PMID- 8614726 TI - [Chronic pain in the hindfoot: computed tomography as a complement in clinico radiographic evaluation. A study of 38 cases]. AB - A series of 38 patients affected with a chronically painful hindfoot underwent CT as a complement to conventional radiography and/or clinics for suspected bone and joint disorders. In all, CT depicted 20 bone abnormalities, i.e., 7 talar osteochondrites, 1 talar osteoid osteoma, 3 subtalar coalitions, 1 calcaneal bone lipoma, 5 posttraumatic talar deformities and 3 posttraumatic calcaneal deformities. Conventional radiography missed 6 of these abnormalities, i.e., 3 talar osteochondrites and 3 subtalar coalitions. In 5 cases CT gave additional information, useful both for the diagnosis (demonstration of fat in the bone lipoma) and to clarify the painful symptoms (2 patients with subtalar osteoarthrosis and 2 with peroneal tendon impingement). Twelve of 15 clinically suspected soft tissue abnormalities were also demonstrated, namely, 2 tibiotalar ligament injuries, 1 sprain of the interosseous ligament of tarsal sinus, 2 cases of accessory soleus muscle, 4 peroneal tendon injuries, 2 posterior tibial tendon injuries and 1 long flexor tendon injury. In the extant 3 patients with suspected bone disorders, both radiography and CT found nothing to confirm the clinical suspicion. To conclude, the authors believe CT to play a major diagnostic role, thanks to its capabilities in demonstrating the abnormal changes underlying chronically painful hindfoot conditions. PMID- 8614725 TI - [Computed tomography and magnetic resonance in the follow-up to interventions for the prosthetic replacement of the carpal bones]. AB - The authors examined with CT and MRI 12 patients submitted to prosthetic replacement of necrotic carpal bones--7 of them because of scaphoid proximal pole posttraumatic osteonecrosis and 5 because of lunate Kienbock's disease. The prosthetic implants were autologous in all patients: they were taken from palmaris gracilis tendon and modified to give them a rounded shape, to adapt them to the new anatomic site. All patients exhibited postoperative limitation in flexion-extension movements; 5 of them reported associated wrist pain. The authors investigated the anatomic reasons of the postoperative symptoms and tried to assess CT and MR diagnostic capabilities in depicting these conditions. CT was performed with thin sections (1.5 mm) and multiplanar reconstructions, with a Philips Tomoscan LX unit. MRI was performed with a GE MR Max Plus unit at 0.5 T and a Medical Advances transmit-receiving extremity coil, on the axial, coronal and sagittal planes, with T1- and T2-weighted sequences. All patients had been submitted to conventional radiography of the wrist. In 6 patients CT and MRI showed severe synovial reaction in the surgical site, with new ligament absorption in 5 of them. In 5 of these patients CT identified some nodular calcifications, while MRI better depicted the fibrotic portion, if present. MRI demonstrated the carpal dorsal intercalated instability which was present in all the patients submitted to scaphoid proximal pole resection; in the patients operated on for Kienbock's disease, volar tilt of the scaphoid was increased. Both kinds of carpal instability were correlated with scaphoid-lunate surgical dissociation. These instabilities were greatly correlated with the postoperative symptoms. Currently, MRI is the gold standard in studying carpal instability and it is therefore fundamental in investigating the complex anatomic and biomechanical features of these patients postoperatively. PMID- 8614727 TI - [The echographic and clinical follow-up of patients operated on for subcutaneous rupture of the Achilles tendon]. AB - Thanks to its good long-term results, surgery is the method of choice to treat subcutaneous ruptures of the Achilles tendon. Reconstructed tendons present typical morphological and functional US patterns which depend partly on the kind of surgical reconstruction and partly on the time passed since surgery. The authors report the results of the clinical and US follow-up of a series of 62 surgical patients treated in 7 years for the subcutaneous rupture of the Achilles tendon. The patients were 55 men and 7 women, whose mean age was 36 years (range: 25-65 years). The left-hand side was affected in 38 patients and the right-hand side in 24 patients. All patients were operated on using an end-to-end suture and reinforcement plastic surgery pulling down a gastrocnemius tendon flap. To homogenize the results, all the US exams were performed by the same operator, in the presence of the orthopedic specialist and under the same conditions: both the involved and the contralateral Achilles tendons were studied, longitudinal and transverse scans were performed with the foot in max. plantar and dorsal flexion and, whenever possible, dynamic scans were also performed making the sural triceps contract against resistance. The following parameters were studied clinically: pain (which was absent in 39 patients, occasional in 11, after stress in 9 and on walking in 3 patients), skin scar trophism (which was eutrophic in 53.23% of patients, keloid in 27.42% and hypertrophic in 19.35% of patients), ankle joint excursion (plantar flexion was impaired in 32.3% and dorsal flexion in 36% of patients), walking on tiptoe (in all, 22.6% of patients complained of difficulties walking on tiptoe) and, finally, work activity resumption (which all patients achieved). US depicted the surgical tendons as much bigger than the contralateral ones (3-4 times on the average), which increase in volume lasted throughout the follow-up. In 75% of patients the echo structure of the surgical tendons was inhomogeneous, with scattered hypoechoic and hyperechoic areas. In the extant 25% of patients, nearly all of them followed-up for over 6 years, US depicted a clear-cut hyperechoic area whose size and echo structure were similar to the healthy tendons'. Our results strongly suggest that tenorrhaphy and flap plastic surgery be used to repair subcutaneous ruptures of the Achilles tendon. US proved to be the most reliable and feasible method also in the follow-up. The US images of the patients submitted to surgery more than 6 years earlier revealed fibrillate reorganization patterns and tendon restructuring. These processes involve both ends of the sutured tendon and not the reinforcement flap, which further confirms the exclusively mechanical, and not biological, function of the latter. PMID- 8614728 TI - [The anatomy of the temporal region viewed by magnetic resonance]. AB - The study of the temporal region has been improved by technologic progress from conventional radiography to linear tomography, then to pluridirectional tomography, CT and, finally, MRI. Twenty-five patients were examined with MRI--50 temporal regions in all. MRI was performed with a brain coil, SE T1-weighted sequences (TR 500 ms, TE 25 ms, FOV 180 mm, matrix 256 x 512, 2 acquisitions, slice thickness: 3 mm, acquisition time: 5.30 minutes) before and after Gd-DTPA administration. SE T2-weighted sequences (TR 2000 ms, TE 20-80 ms, FOV 180 mm, matrix 256 x 256, 1 acquisition, slice thickness: 4 mm, acquisition time: 8.30 minutes), 3D CISS sequences (TR 20 ms, TE 8 ms, FOV 180 mm, matrix 256 x 256, acquisition time: 2.46 minutes) and MRA 3D TOF TONE sequences (TR 33 ms, TE 8 ms, FOV 180 mm, matrix 256 x 256, acquisition time: 9.30 minutes) were performed. Neither the temporal bone, made of compact tissue, nor the air spaces (mastoid, antrum, middle ear cavity) exhibit any MR signal. On the contrary, the fluid filling the membranous labyrinth (endolymph and perilymph) and the cerebrospinal fluid filling the internal acoustic canal and the cerebello-pontine angle have high signal. This pattern allows a detailed study, unfeasible with CT, of the inner ear structures (cochlea, vestibule and semicircular canal) and of the internal acoustic canal. Moreover, MRI allows the whole VII and VIII cranial nerves to be studied from their origin at the brain stem to the internal acoustic porus and into the internal acoustic canal. Finally, MRI permits the noninvasive study of temporal region vessels. To conclude, MRI yields new anatomical details of the temporal region useful to address major diagnostic issues--e.g., labyrinth, internal acoustic canal and cerebello-pontine angle lesions. PMID- 8614730 TI - [The diagnostic accuracy of a digital mammography system with photostimulable storage phosphors used with automatic reading]. AB - Even though digital mammography might potentially yield major advantages in management, biology and diagnosis, state-of-the-art digitalization is still in a clinical experimental phase. To assess the diagnostic accuracy of a digital mammography system with storage phosphors, we analyzed 320 digital and 320 conventional mammograms acquired in lateral-oblique projection in the same patients. Digital mammography capabilities in identifying and characterizing breast lesions were compared with those of conventional mammography; the presence/absence of lesions and their benign/malignant nature were investigated. Complete conventional mammography was our gold standard. The digital system did not miss any malignant lesion but it did miss some benign lesions (focal masses) which had been depicted with conventional mammography, especially small low contrast opacities. Microcalcifications were better depicted on digital images which showed, at the same time, tissues of different density, thanks to their wider dynamic range. Digital mammography yielded a false positive result on a cluster of microcalcifications, because its spatial resolution is lower. The diagnostic yield of digital mammography was poorer only in the detection of small low-contrast lesions and in the characterization of microcalcifications. To conclude, in our experience, the storage phosphor system seems to be suitable for clinical mammography, but only with careful monitoring; in contrast, we think it is not yet suitable for screening purposes. PMID- 8614729 TI - [The usefulness of computer tomography in planning cochlear implant surgery]. AB - In recent years, technical progress has created new complex acoustic implants which send an electrical stimulus to the eighth cranial nerve through one or more electrodes inserted through the round window into the scala tympani of the cochlea. The abnormal--mostly osteosclerotic--processes which cause deep hearing loss may prevent electrode insertion. Therefore, internal ear anatomy must be detailed, which is essential to assess the feasibility of surgery and, if surgery is indicated, to plan it properly. High resolution CT (HRCT) was performed on 79 patients to study cochlear patency, round window shape and patency, degree of temporal bone pneumatization and the proximity of vascular structures (carotid artery and jugular vein). On the basis of HRCT results, 14 of 79 patients were excluded from surgery. Comparing HRCT with surgical findings, the authors conclude that HRCT is the method of choice to examine the candidates to cochlear implant thanks to its high spatial resolution and excellent depiction of even the smallest structures. Its only limitation is that it fails to assess the lack of patency of the cochlear canal due to fibrosis, which is not associated with demonstrable density changes (3 of 19 surgical patients). This problem may be solved by submitting the potential surgical candidates to MRI. PMID- 8614731 TI - [Do clinical or physiopathological parameters exist for rationalizing the demand for thoracic radiograms in patients admitted to an intensive care unit]. AB - To rationalize the demand of chest radiography in Intensive Care Unit patients, 423 radiographs of 35 patients were studied prospectively. The radiologic findings were compared with clinical data, blood gas analysis and laboratory data. No significant correlation was demonstrated between pulmonary radiographic changes and patient clinical conditions classified as unchanged, improving and worsening. In contrast, a good correlation (R = 0.65; p < 0.001) was observed between the chest radiographic score of pulmonary changes and the PaO2/FiO2 ratio. Changes in this parameter may be a good reason to perform bedside chest radiographs, whereas this exam appears rather useless when PaO2/FiO2 ratio is unchanged. PMID- 8614732 TI - [The peripheral circulation: ECG-gated magnetic resonance angiography]. AB - This work was aimed at comparing magnetic resonance angiography (MRA) with and without ECG gating in the study of peripheral vessels. Ten volunteers, mean age 27.8 years, were examined with MRA of the femoral, popliteal and tibial segments. MRA was performed with a 1.5-T superconductive magnet, a transmit head coil and the TOF 2D technique. In all cases MRA was performed without cardiac gating and with different times of trigger delay (0, 20, 40, 70 and 200 ms). When comparing the different acquisitions, the number of vessels and the signal-to-noise (S/N) ratio were calculated. In the femoral segment, MRA without cardiac gating showed a mean of 7.7 vessels with 2.79 S/N ratio; MRA with 0 ms of trigger delay showed a mean of 13.1 vessels with 1.51 S/N ratio; MRA with 20 ms trigger delay showed a mean of 13.1 vessels with 1.52 S/N ratio; MRA with 40 ms trigger delay showed a mean of 13.2 vessels with 1.52 S/N ratio; MRA with 70 ms trigger delay showed a mean of 13.5 vessels with 1.50 S/N ratio; finally, MRA with 200 ms trigger delay showed a mean of 13.4 vessels with 1.50 S/N ratio. In the popliteal segment, the corresponding values were 6.4 vessels and 2.51 S/N ratio, 11.4 vessels with 1.54 S/N ratio, 11.3 vessels and 1.54 S/N ratio, 11.6 vessels and 1.52 S/N ratio, 11.8 vessels and 1.52 S/N ratio and, finally, 11.9 vessels and 1.52 S/N ratio. In the tibial segment, the corresponding values were 8.5 vessels and 1.84 S/N ratio, 14.4 vessels and 1.14 S/N ratio, 14.5 vessels and 1.17 S/N ratio, 14.5 vessels and 1.14 S/N ratio, 14.3 vessels and 1.17 S/N ratio and, finally, 14.5 vessels and 1.19 S/N ratio. To conclude, MRA with cardiac gating better visualized peripheral vessels whatever the trigger delay. PMID- 8614733 TI - [Hand injuries due to fireworks explosion]. AB - Two different series of patients with burst injuries of the hand were retrospectively reviewed: one series included 23 men, 15-55 years old, referred to the emergency department for the first-instance assessment of injuries of the right hand (13 patients) and left hand (10 patients). The other series included 44 patients (42 men and 2 women, aged 7-61 years) referred to our department for second-instance examinations: the latter patients had burst injuries involving only the hands in 43 cases and both the hand and the foot in one case. We report the radiologic patterns of traumatic bone injuries (fractures and amputations) and of musculotendinous and cutaneous injuries and discuss their mechanisms and pathogenesis. The two series were compared and the results follow: in both series the right hand was more frequently involved, metacarpal bones were most often fractured and phalanges most often amputated. In the first series, in the right hand the carpal bones were involved in one patient only, the 2nd and 3rd metacarpal bones were most frequently fractured and the 2nd finger was most frequently involved. In the left hand, the carpal bones were never affected, the 1st metacarpal bone was most often amputated and the 5th metacarpal bone most often fractured; the 2nd finger was most frequently involved. In the second series, in the right hand, the 1st metacarpal bone was most frequently fractured and the 2nd metacarpal bone most often amputated; the 2nd finger was most frequently involved. In the left hand, the 4th metacarpal bone was most frequently fractured and the 5th metacarpal bone most often amputated. The severity of the above injuries and the extent of tissue damage depend on several factors, including firecracker speed, shape, size, weight and characteristics. Radiologic exams are very useful for the accurate study of these traumatic injuries, providing indirect information about musculotendinous and cutaneous involvement. Prompted by the relative lack of information on the management of these injuries, we suggest that radiologic exams be quickly performed to help choose the most appropriate surgical approach for best cosmetic and functional results. PMID- 8614734 TI - [Ocular color Doppler echography: the examination technic, identification and flowmetry of the orbital vessels]. AB - We studied the main ocular and retrobulbar vessels with color-Doppler US and report on examination technique, detectability of the vessels and their flow characteristics to define the normal ranges of Doppler spectra for each artery and vein. We comparatively examined both eyes of 20 healthy subjects. Of each eye we studied the ophthalmic artery, the central retinal artery, the ciliary artery, the central retinal vein and the superior ophthalmic vein. The following flow parameters were considered: peak systolic velocity, end-diastolic velocity and resistive index for arteries; maximum and minimum velocity for veins. The examination lasted about 10 minutes per eye--20 minutes for each subject. In all subjects the five investigated vessels were identified in both eyes, and adequate Doppler spectra were obtained. The average peak systolic velocities of ophthalmic, central retinal and ciliary arteries were respectively about 35, 12 and 10 cm/s, with low resistance patterns (resistive index: 0.75, 0.72 and 0.68, respectively). The venous flow, which is usually continuous, may be sometimes influenced by cardiac and respiratory activities: the maximum velocities of superior ophthalmic and central retinal veins were about 6 and 5 cm/s, respectively. Color-Doppler US noninvasively visualizes both ocular and retrobulbar vessels, providing major hemodynamic information from different flow parameters; the knowledge of these parameters in normal conditions can be the basis of hemodynamic studies in many abnormal orbital conditions. PMID- 8614735 TI - [The 3rd national workshop on defecography: the functional radiology of (neo) rectal ampullae (ileal reservoir, colo-anal anastomosis, continent perineal colostomy)]. AB - A survey was made in 13 Italian centers with a questionnaire concerning the (a) indications, (b) postoperative complications, (c) functional results and (d) diagnostic imaging modalities related to the making of an ileal or colonic (neo) rectum. Ulcerative colitis (100%), familial polyposis (61.5%) and Crohn's disease (15.3%) were the most common indications for an ileal pouch; rectal cancer (7.96%), chronic inflammatory diseases (15.3%), diverticulosis, rectal prolapse, redundant colon and imperforate anus (7.6% each) were the most common indications for a colonic pouch. Postoperative complications included pelvic abscess (14%), sinus tract/dehiscence (10%) and bowel obstruction (9%). When compared with the S and W variants, the J-shaped ileoanal pouch proved superior because urgency and fecal retention rates were lower (18.4% vs. 44.4% and 23% vs. 28.6%, p < 0.01 and p < 0.05, respectively), despite slightly more frequent staining episodes (15.8% vs. 11.1%; p < 0.05). As for colonic ampullae, fecal retention and provoked evacuation were more frequent in the J pouch and after gracileplasty; urgency and incontinence in the straight colo-anal anastomosis (33.3% vs. 22.2% and 41.6% vs. 33.3%, respectively). The functional outcome was assessed by anal endosonography (available in 4/13 centers), defecography and anorectal manometry. Abnormal findings included: (a) reduced capacity, barium leakage, anal gaping, sphincter damage (urgency and incontinence); (b) barium retention, pouch dilatation, split evacuation, knobs and strictures (fecal retention). PMID- 8614736 TI - [Defecography in the diagnosis of fecal incontinence: an analysis of the receiver operating characteristic (ROC)]. AB - Two groups of patients with altered bowel habit and pelvic floor dysfunction, but comparable epidemiologic characteristics (i.e. n = 105 each; mean age and SD 47.3 +/- 15.8 yrs vs. 54.9 +/- 16.7 yrs; range 15-80 yrs vs. 9-88 yrs; F/M ratio 28:1 vs. 2.6:1) with the exception of the absence (or presence) of fecal incontinence, were examined with defecography, taking into account criteria other than anorectal angle values and anorectal junction mobility. At the Receiver Operating Characteristic (ROC) analysis a "barium leak sign", occurring either at rest or on straining was found to be a highly reliable index of fecal incontinence (specificity: 100% and 92-93%, respectively, intraobserver agreement K value = 0.82, Z = 21.58, p < 0.001). A false negative rate of 14.2% was limited to "minor" incontinence only, i.e., incontinence to gas and/or occasional staining episodes. In the search for an etiologic diagnosis, useful adjunctive criteria included (a) anal diameter > 10 mm at rest; (b) poor stop test (inability to interrupt the barium stream); (c) rectal diameter > 6.5 cm and < 4 cm (abnormally increased and reduced compliance, respectively). Defecography is a useful diagnostic tool in fecal incontinence and should precede anal endosonography, manometry and electromyography for proper therapeutic decision-making and in risk conditions, e.g., in the patients about to undergo elective pelvic surgery. PMID- 8614738 TI - [The role of portography with computed tomography in the diagnosis of liver metastases from colorectal neoplasms]. AB - This study was performed to confirm the high sensitivity of CT during arterial portography (CTAP) versus US and dynamic CT in the diagnosis of liver metastases from colorectal cancer. Ninety patients with 108 colorectal cancers underwent US, dynamic CT and CTAP to investigate the presence of liver metastases. US depicted 39 metastases in 26 patients, dynamic CT 46 metastases in 29 patients and CTAP 54 lesions in 34 patients. CTAP detected 8 metastases missed at dynamic CT; 4 of them were < 1 cm in diameter, 3 ranged 1-2 cm and one metastasis > 2 cm in diameter. After preoperative investigations only 7 patients were considered for hepatic resection. At surgery, palpation and intraoperative US of the liver detected two more metastases in the same patients. Our experience, in agreement with recent studies, confirms CTAP as the most sensitive preoperative method in detecting liver metastases and its superiority to be most apparent in lesions < 1 cm in diameter. Therefore, we consider CTAP an essential imaging technique for planning the correct treatment of liver metastases. PMID- 8614737 TI - [Magnetic resonance imaging of the small intestine and colon in Crohn's disease]. AB - Three normal volunteers and 20 patients with known Crohn's disease were examined with MRI--at 0.5 Tesla and with a superconductive magnet. Coronal T1-weighted GE images were mainly acquired, before and after i.v. Gd-DTPA injection in breath hold (TR 70 ms, TE 13 ms, FA 70 degrees). MR findings were compared with the results of small and large bowel enema. In 6 patients (30%) the abnormal loops were missed. In the other 14 patients (70%) MRI did depict the affected loops in the same sites as depicted by conventional radiography. The bowel wall was thickened (4-10 mm) in all patients. In 10 patients the thickened wall was markedly enhanced after Gd-DTPA injection. In 6 patients MRI demonstrated disease complications--i.e., stenoses, fistulae and abscesses--missed by conventional radiography. In 7 patients MRI showed the bowel to be more involved than demonstrated by conventional studies. Bowel wall thickening appeared to be a constant and reliable sign of disease. Wall enhancement was a less frequent sign but, when present, it was considered as characteristic as wall thickening. In the staging of Crohn's disease, MRI yields more pieces of information than conventional radiography and depicts the involvement of the intestinal wall and of its surrounding spaces. PMID- 8614739 TI - [Dynamic computed tomography in the characterization of focal hepatic lesions]. AB - The main goal of our study was to test dynamic CT capability to characterize focal liver lesions. We examined 57 patients: 6 were affected with focal nodular hyperplasia (FNH), 19 with hepatocellular carcinoma (HCC), 1 with a regenerating nodule on cirrhosis; 14 patients had metastases, 3 focal fatty infiltration, 1 a necrotic nodule, 1 a non-Hodgkin's lymphoma, 1 a cysto-adeno-cholangiocarcinoma and 11 hemangiomas. All lesions were identified with US and the diagnosis was confirmed with the gold standard technique--that is, biopsy or surgery, and red blood cell SPECT for hemangiomas. All lesions were studied with a CT multiphase protocol consisting of a single-level dynamic phase followed by an incremental dynamic phase and finally by a delayed phase to study prolonged and delayed enhancement. Single-level dynamic bolus CT requires an injection of 60 ml nonionic contrast agent administered with a power injector into a cubital vein, at a rate of 5 ml/s. Scanning begins 10 seconds after the injection and consists of 6 series of 2 scans each; each scan lasts 2 seconds and is obtained during the same respiratory apnea, with a 5-second interscan pause. In this phase, 12 scans 5 mm thick are obtained, lasting 24 seconds in all, with pauses lasting 25 seconds--in all, 49 seconds. The next phase is the dynamic incremental scanning, to study the whole liver: this phase requires a 50-ml contrast agent injection at a rate of 4 ml/s, followed by 70 ml at a rate of 1 ml/sec, using 5 mm slice thickness and 8 mm scan interval. This results in 16 scans, beginning 20 seconds after the injection, with a scan time of 2 seconds and 4 seconds of interscan delay, 92 seconds in all. In the last phase, scanning begins 5 minutes after the injection, with a maximum delay of 10-15 minutes. Enhancement variations in both the lesions and the surroundings parenchyma, as related to time, were collected together with morphological data. Time density curves were grouped according to histologic classification and red blood cells SPECT findings; the curves were analyzed with the regression analysis. The results were obtained by analyzing a series of equations describing the different densities of the lesion and the surrounding parenchyma at fixed time intervals, integrated with morphological data, and then comparing the groups of lesions with each other. The regression analysis of the density curves and of the morphological data allowed us to correctly differentiate the 4 most frequent types of lesions--that is, hemangioma, HCC, FNH and metastasis--in 89% of the patients. PMID- 8614740 TI - Formulae and tables for the determination of sample sizes and power in clinical trials for testing differences in proportions for the two-sample design: a review. AB - This paper is a compendium of exact and asymptotic formulae and tables for estimating the sample size in a clinical trial with two treatment groups and a dichotomous outcome. The paper provides separate formulae for equal and unequal treatment group sizes, formulae for the calculation of power given the sample size, and complete references for all formulae and tables cited. PMID- 8614741 TI - Practical p-value adjustment for optimally selected cutpoints. AB - This paper concerns a series of simulations undertaken to examine the effects of two data features--number of cutpoints and true marker prognostic effect size--on three methods of p-value adjustment (asymptotic, P(acor); improved Bonferroni, P(bon); and empirical permutation, P(emp)). H(o) rejection rates for P(emp) and P(bon) are almost indistinguishable from those for an independent validation sample (P(vld)), while those of P(acor) are somewhat conservative, especially when the number of cutpoints is small. Analysis of a new breast cancer prognostic marker, heat shock protein 70, illustrates the methods. These results underscore many of the problems associated with data-derived cutpoints in general, and the need for p-value adjustment. PMID- 8614742 TI - A personal view of some controversies in allocating treatment to patients in clinical trials. PMID- 8614743 TI - Modelling nutrition knowledge, attitudes, and diet-disease awareness: the case of dietary fibre. AB - Understanding how nutrition knowledge and attitudes vary across different population groups is critical for designing and evaluating nutrition education programmes and monitoring the nation's progress toward dietary goals. In this paper we use the Diet and Health Knowledge component of the USDA Continuing Survey of Food Intakes by Individuals to examine consumer knowledge of dietary fibre, fibre consumption attitudes and the awareness of fibre-related health problems. We use a latent variable probit model to estimate the relationships between an individuals's socio-demographic characteristics and his or her fibre knowledge, attitude and disease-awareness. The results suggest that the demographic profile of persons least knowledgeable about the fibre content of foods is low income, male, Black, Hispanic, smoker and low education levels. Add to this list younger individuals and one has a good description of those who lack information on the importance of eating plenty of grain products as well as those who lack awareness of fibre/disease links. The research suggests that messages about increasing fibre intake may have greatest success when targeted to individuals with some or all of these characteristics. PMID- 8614744 TI - A tree-based method of analysis for prospective studies. AB - Prospective studies often involve rare events as study outcomes, and a primary concern is to identify risk factors and risk groups associated with the outcomes. We discuss practical solutions to risk factor analyses in prospective studies and address strategies to determine tree structures, to estimate relative risks, and to manage missing data in connection with some important epidemiologic problems. Some of the basic ideas for our strategies follow from work of Breiman, Friedman, Olshen, and Stone, although we propose extensions to their methods to resolve some practical problems that arise in implementation of these methods in epidemiologic studies. To illustrate these ideas, we analyse low birthweight associated risk factors with use of a data set from the Yale Pregnancy Outcome Study. PMID- 8614745 TI - The homogenetic estimate for the variance of survival rate. AB - The homogenetic estimate for the variance of survival rate is proposed based on generalization and reduction between the complement of the empirical distribution function and the Kaplan--Meier or Berkson--Gage estimate. It reduces to the binomial variance estimate when there is no censoring. A Monte Carlo simulation study was carried out under various sample sizes, survival and censoring configurations, number of tied observations, and confidence levels with 2000 replications. It verifies that the commonly employed Greenwood estimate understimates, and the Simon and Lee expression for the Peto estimate strictly overestimates, the variance of survival rate to an extent dependent on the censoring distributions. The conclusions are identical with those of Peto et al. (1977) and Slud et al. (1984). The bias of the homogenetic estimate is less than that of both the Greenwood estimate and the Simon and Lee expression for the Peto estimate. The homogenetic estimate slightly overestimates when there are no ties and becomes unbiased and then slightly underestimates as the number of tied observations increases. PMID- 8614746 TI - Polynomials with asymptotes for longitudinal data. AB - I use Laguerre polynomials to model growth curves or time--response curves known to approach an asymptote as time approaches infinity. An example is with measurements on a variable or variables from subjects recovering from surgery. These variables can often vary in a non-monotonic fashion for which a functional form of the curve is unknown. Using a longitudinal data mixed model, one can include in the model random subject effects, within-subject serial correlation and fixed or time varying covariates. I present two examples that involve groups of subjects recovering from surgery. PMID- 8614747 TI - Statistical analysis of zidovudine (AZT) effect on CD4 cell counts in HIV disease. AB - We fit a class of random effects linear growth curve models for the square root of CD4 count to serial marker data from 164 HIV-positive individuals with known (or accurately estimated) dates of seroconversion and at least 10 CD4 measurements each (median 16). We do so by adopting a Bayesian viewpoint and using the Markov chain Monte Carlo technique Gibbs sampling. In particular, we examine the effect of the antiretroviral treatment zidovudine on the square root of CD4 series for the 136 patients who took the drug. Treatment effects are modelled by positing recoveries in square root of CD4 level proportional to current immuno-competence and changes in slope proportional to current rate of square root of CD4 loss. Both fixed and random treatment effects are considered and models are criticized and compared using Bayesian predictive methodology and checking data which comprise 424 new observations. Results indicate re-elevation of square root of CD4 level is associated with treatment but the effect, though significant, is mostly of small magnitude and is possibly transient; models neglecting consideration of treatment fit the checking data almost as well. Best overall model estimates mean rate of square root of CD4 loss per annum to be 2.1 (standard error 0.12); mean seroconversion value of square root of CD4 is 28.4 (SE 0.65). The estimated variance of individual slopes is 1.9 (SE 0.28), there being considerable individual variation in rate of CD4 loss, and a recovery in level of 0.047 (SE 0.014) times current square root of CD4 level is estimated at treatment uptake. PMID- 8614748 TI - Estimation of expected quality adjusted survival by cross-sectional survey. AB - To compare both mortality and quality of life (QOL) across different illnesses, we propose an estimator to calculate the expected quality adjusted survival (QAS) by multiplying the QOL into the survival function. While the survival function can be determined by the usual life table method, the QOL data can be collected by a cross-sectional survey among patients who are currently surviving. The area under the QAS curve is thus the expected utility of health of the specific illness, which may take a common unit of quality adjusted life year ready for outcome evaluation and policy decision. A simulation is performed to demonstrate that the proposed estimator and its standard error are relatively accurate. The limitations and guidelines for using this estimator are also discussed. PMID- 8614749 TI - Robust procedures for analysing a two-period cross-over design with baseline measurements. AB - Patel analysed a two-period cross-over design with baseline measurements assuming bivariate normality for the joint distribution of the period responses. In this paper, we propose non-parametric methods for analysing this design, including the use of the Wilcoxon rank sum test to derive the preliminary tests from the baseline measurements. We fit a robust regression line of the treatment response on baseline for each period and compute residuals. We also fit a robust locally weighted regression as an alternative method for computing residuals. Then, following Koch's procedure, we analyse the residuals for testing the significance of the treatment x period interaction and the treatment difference. We provide a numerical example to illustrate the methods. PMID- 8614750 TI - The analysis of incomplete data in the three-period two-treatment cross-over design for clinical trials. AB - The additional time to complete a three-period two-treatment (3P2T) cross-over trial may cause a greater number of patient dropouts than with a two-period trial. This paper develops maximum likelihood (ML), single imputation and multiple imputation missing data analysis methods for the 3P2T cross-over designs. We use a simulation study to compare and contrast these methods with one another and with the benchmark method of missing data analysis for cross-over trials, the complete case (CC) method. Data patterns examined include those where the missingness differs between the drug types and depends on the unobserved data. Depending on the missing data mechanism and the rate of missingness of the data, one can realize substantial improvements in information recovery by using data from the partially completed patients. We recommend these approaches for the 3P2T cross-over designs. PMID- 8614751 TI - Associations of types of lens opacities between and within eyes of individuals: an application of second-order generalized estimating equations. The Framingham Eye Studies Group. AB - The lens opacity characteristics of individuals constitute multivariate data. Our goal was to estimate the associations between the three main types of age-related lens opacities (nuclear, cortical, posterior subcapsular) both between and within eyes of individuals using cross-sectional data from the Framingham (Massachusetts) Eye Studies. We describe use of a recently proposed extension of the generalized estimating equations approach to marginal logistic models (GEE2), and we demonstrate that a variety of research problems can be investigated with this methodology. For example, in our data, there were strong associations of the same opacity types between the two eyes of individuals and weak associations between different types of opacities. We also note that estimation of such associations may be limited in other epidemiologic settings. PMID- 8614752 TI - An empirical comparison of expert-derived and data-derived classification trees. AB - Classification trees provide an attractively transparent discrimination technique, and may be derived from both expert opinion and from data analysis. We consider a real and complex problem concerning the diagnosis of babies with suspected critical congenital heart disease into one of 27 classes. A full loss matrix for all possible misclassifications was obtained from clinical assessments. A tree derived from expert opinion was compared with those derived from analysis of 571 past cases, both for the full problem and for a subset of 6 diseases. Automatic methods for tree creation and pruning were found to have problems for rare diseases, and hand-pruning was carried out. Inclusion of costs led to much improved clinical performance, even for trees that had originally been constructed to minimize classification errors. The expert tree showed a specific building strategy that could not be reproduced automatically. The expert tree generally outperformed those derived from data, particularly in the ability to identify important composite features. PMID- 8614753 TI - Confidence intervals for median survival times under a piecewise exponential model with proportional hazards covariate effects. AB - Brookmeyer and Crowley derived a non-parametric confidence interval for the median survival time of a homogeneous population by inverting a generalization of the sign test for censored data. The 1-alpha confidence interval for the median is essentially the set of all values t such that the Kaplan--Meier estimate of the survival function at time t does not differ significantly from one-half at significance level alpha. Here I extend the method to incorporate covariates into the analysis by assuming an underlying piecewise exponential model with proportional hazards covariate effects. Maximum likelihood estimates of the model parameters are obtained via iterative techniques, from which the estimated (log) survival curve is easily constructed. The delta method provides asymptotic standard errors. Following Brookmeyer and Crowley, I find the confidence interval for the median survival time at a specified value of the covariate vector by inverting the sign test. I illustrate the methods using data from a clinical trial conducted by the Radiation Therapy Oncology Group in cancer of the mouth and throat. It is seen that the piecewise exponential model provides considerable flexibility in accommodating to the shape of the underlying survival curve and thus offers advantages to other, more restrictive, parametric models. Simulation studies indicate that the method provides reasonably accurate coverage probabilities. PMID- 8614754 TI - Assessment of small health risks based on exact sample sizes. AB - Exact sample sizes and critical numbers of cases for the rejection of a known event probability (10(-2) to 10(-6)) in favour of an increased probability (1.5- to 50-fold) at levels -alpha;beta- = -0.05; 0.10- and -alpha;beta- = -0.10;0.05- are presented. The numbers are thoroughly validated using the characteristics of the confidence interval for the unknown true event probability. Equivalence is shown to be obtainable for the tolerated maximal value of the relative risk and the upper limit of the confidence interval for the true event probability. Also demonstrated is the use of the tables for planned actions to reduce given empirical risks. In addition, use of the tables is shown for judging results from given data sets. PMID- 8614755 TI - Characterization of HIV incubation distributions and some comparative studies. AB - In this paper we use a general stochastic model to characterize the HIV incubation distributions. We generate some Monte Carlo data under different conditions and compare the fitting of HIV incubation distributions by some well known parametric models and some non-parametric methods. The parametric models include most of those that have appeared in the literature. The non-parametric methods include the Kaplan--Meier method, the EMS method, the spline approximation and the Bacchetti method. The comparison criteria are the chi square statistic, the residual sum of squares, the AIC and the BIC. We show that the non-parametric methods, especially the EMS method, provide excellent fits in almost all cases; for the parametric models, the generalized log-logistic distributions with three and with four parameters fit better than other parametric models. PMID- 8614756 TI - The shape of the distribution of the number of sexual partners. AB - Information on the number of sexual partners that people have is necessary for predicting the likely long term course of the AIDS epidemic. More information is required than is provided by simply stating the mean and variance of the number of partners. It is useful to find a family of curves which approximate data on the proportion of people in the various 'number of partners' categories. Two Australian surveys of the sexual behaviour of first year university behavioural science students were analysed. It was found here that log-normal distributions gave good approximations to the distribution of number of partners amongst people who had more than one partner. Gamma and negative binomial distributions gave less satisfactory fits and the truncated normal and Poisson distributions were clearly unable to match the data. PMID- 8614757 TI - The influence of running patterns on running injuries. PMID- 8614758 TI - Injuries and bungee jumping. AB - Bungee jumping is a recreational sport that has gained world-wide popularity since its inception in 1955. Over 2 million individuals have performed bungee jumps since that time. The injuries and deaths which have occurred have made safety an integral issue in the practice of the sport. Although early reports of significant injuries are infrequent, more recent investigations have indicated severe sequelae, including ocular haemorrhage, peroneal nerve palsy and quadriplegia. Reports of minor trauma have also been numerous. Aetiology includes natural forces, impact, technician error, equipment failure and repetitive stress. Free-falling approximately 60 to 120m (200 to 400ft) and then being jerked to safety at the last minute creates a certain amount of unavoidable, and almost desired risk. A reduction in acute trauma may be possible with immediate changes in equipment, technique and regulations. Further studies are warranted to determine the future direction and safety of this recreational sport. PMID- 8614760 TI - Anxiety and panic in recreational scuba divers. AB - Scuba diving is a high-risk sport; it is estimated that 3 to 9 deaths per 100,000 divers occur annually in the US alone, in addition to increasing numbers of cases of decompression illness each year. However, there has been a tendency within the diving community to de-emphasise the risks associated with scuba diving. While there are numerous factors responsible for the injuries and fatalities occurring in this sport, there is general consensus that many of these cases are caused by panic. There is also evidence that individuals who are characterised by elevated levels of trait anxiety are more likely to have greater state anxiety responses when exposed to a stressor, and hence, this sub-group of the diving population is at an increased level of risk. Efforts to demonstrate that selected interventions such as hypnosis, imagery, mediation and relaxation can reduce stress responses in anxious divers has not yielded consistent findings, and there is a need for systematic research dealing with the efficacy of selected intervention strategies. PMID- 8614761 TI - The evaluation of pelvic injury in the female athlete. AB - The differential diagnosis of pelvic pain and possible injury in the female athlete is quite broad and must include gastrointestinal and genitourinary aetiologies, as well as musculoskeletal injuries. These considerations reflect the anatomical complexity of the female pelvis. The pelvic bones house the lower gastrointestinal and genitourinary viscera and transmit stress from the lower extremities to the upper body. The innervation of the pelvic structures also complicates evaluation and diagnosis when somatic and visceral afferent information affects the athlete's interpretation of pain. An algorithmic approach can facilitate evaluation and rehabilitation of pelvic injuries in the female athlete in the contest of previously described mechanisms of musculoskeletal injury. PMID- 8614763 TI - Wegener's granulomatosis with gangrene of toes. AB - The cutaneous manifestations of Wegener's granulomatosis (WG) are very variable. Gangrene of digits is extremely rare. We report here one such patient of WG with gangrene of toes. PMID- 8614762 TI - Cervical spinal stenosis with cord neurapraxia and transient quadriplegia. AB - Cervical cord neurapraxia is a transient, totally reversible phenomenon that results from compressive deformation of the spinal cord. It occurs as a result of developmental narrowing of the cervical canal, either as isolated entity or in combination with degenerative changes, instability or congenital abnormalities. Uncomplicated stenosis of the cervical canal in an individual with a stable spine does not predispose to permanent neurological injury. Our data does not indicate a correlation between developmental narrowing and permanent neurological sequelae in a spine rendered unstable by football-induced trauma. However, there are data indicating that the occurrence of an episode of cervical cord neurapraxia is not a harbinger, or an indication of susceptibility to permanent neurological sequelae. Nevertheless, we recommended that continued participation in collision activities be restricted in individuals who have a documented episode of cervical cord neurapraxia associated with (i) ligamentous instability; (ii) intervertebral disc disease with cord compression; (iii) significant degenerative changes; (iv) magnetic resonance imaging evidence of cord defect or swelling; (v) symptoms of positive neurological findings lasting more than 36 hours; and (vi) more than one recurrence. PMID- 8614764 TI - Colchicine myopathy. AB - Colchicine myopathy typically presents as painless muscle weakness. This report describes a post renal-transplant patient who presented with pain in both extremities bilaterally that was refractory to narcotic analgesics. He had been on low dose daily colchicine for 7 years. Laboratory, EMG, and biopsy findings all supported the diagnosis of colchicine myopathy. Symptoms subsided after discontinuation of colchicine. Myalgia as chief complaint is an unusual presentation of colchicine myopathy. PMID- 8614765 TI - Bilateral carpal tunnel syndrome secondary to tophaceous compression of the median nerves. AB - A 65-year-old man with long-term gouty arthritis developed bilateral carpal tunnel syndrome. At surgery a chalky substance, which showed negative birefringence on polarized microscopy, was found infiltrating around the intensely inflamed transverse carpal ligaments. In differential diagnosis of carpal tunnel syndrome, tophaceous compression over the median nerve should be taken into consideration. PMID- 8614766 TI - Pulmonary sarcoidosis coexisting with systemic lupus erythematosus. AB - A 23 year old female presented with fatigue, Raynaud's syndrome, symmetric polyarthritis, lymphocytopenia, hypocomplementemia, IgG elevation, and antinuclear antibodies with anti-U1RNP specificity. A diagnosis of systemic lupus erythematosus (SLE) was made. Disseminated micronodular infiltration in the chest X-ray, a pattern of lymphocytic alveolitis by bronchoalveolar lavage, histologic demonstration of non-caseating epitheloid microgranulomas, and rapid reversal of lung disease upon low-dose methylprednisolone treatment led to a diagnosis of concomitant sarcoidosis. Clues to the diagnosis of sarcoidosis coexisting with SLE are discussed. PMID- 8614767 TI - An aseptic inflammation of the clavicle in a patient with Crohn's disease. A potential manifestation of the SAPHO syndrome. AB - A female patient with Crohn's disease developed an septic osteoarticular involvement of the left clavicle and sterno- and acromioclavicular regions. Repeated surgical revisions combined with a broad-spectrum antimicrobial treatment could not prevent the progress of the disease. However, the patient started to improve after the diagnosis of the SAPHO (synovitis, acne, pustulosis, hyperostosis and osteomyelitis) syndrome was made and the dose of her immunosuppressive therapy increased. This patient reminds of the existence of extraintestinal aseptic infections in association with inflammatory bowel disease (IBD). Moreover, it may provide further evidence on the significant association between SAPHO and IBD. PMID- 8614768 TI - Anti-neutrophil cytoplasmic antibodies in primary small vessel vasculitides. AB - There is no doubt that anti-neutrophil cytoplasmic antibodies (ANCA) as a tool for diagnosing and monitoring a particular group of patients with small vessel vasculitides has come into wide use in many clinical specialties of medicine. However, heterogeneity and overlap of disease definitions and nomenclature for such conditions, as well as lack of standardized methods for detecting ANCA, have obscured scientific communication so much that clear conclusions on the significance of ANCA in clinical practice have not been reached. With these shortcomings in mind this review will primarily deal with the clinical aspects of ANCA serology and efforts to overcome the limitations. PMID- 8614769 TI - Acute sarcoid arthritis: a favourable outcome? A retrospective survey of 49 patients with review of the literature. AB - Forty-nine patients, 30 males and 19 females with acute sarcoid arthritis admitted to three different hospitals in Norway were studied retrospectively. All patients had peripheral arthritis and hilar adenopathy, and 87.8% also presented with erythema nodosum (EN). Mean duration of arthritis was 3.7 months (0.5-12 months), but in 26% of the cases, duration of the inflammatory joint disease exceeded three months. Radiological bony erosions were not seen. Two patients had recurrence of acute sarcoid arthritis, 14 months and 10 years after the initial episode, respectively. Two other patients developed chronic myalgia and fibromyalgia. Four patients, one female and three males, developed chronic pulmonal sarcoidosis. Of these, two patients had simultaneous onset of acute sarcoid arthritis and parenchymal disease while two patients developed chronic lung disease three months after onset of acute sarcoid arthritis. We thus tentatively suggest that although acute sarcoid arthritis is usually a self limiting joint disease, recurrences may occasionally occur and some cases develop chronic sarcoidosis of the lungs. PMID- 8614759 TI - The effects of exercise on growth. AB - The way in which exercise influences statural, hypertrophic and reparative growth is examined from the perspective of the human lifespan. Statural growth depends on a neuroendocrine programme which channels nutrient energy towards increments in lean body mass. Exercise can facilitate statural growth and is a necessary stimulus for reparative growth through its stimulatory effects on secretion of growth hormone (GH) and other anabolic hormones. An exercise-associated increase in GH secretion is a response to acute or prolonged exercise-induced fuel shortage that directs metabolism towards utilisation of lipids and promotes growth. Exercise can transiently block the expression of statural growth by competitively removing the necessary nutritional support for growth. Statural growth retardation can be corrected by catch-up growth, but stunting may also be permanent (depending on the timing and magnitude of the energy drain). Hypertrophic growth is less dependent on hormonal and nutritional support than statural growth, and exercise provides the necessary mechanical stress for growth and remodelling of the musculoskeletal system. Excessive mechanical strain may suppress hypertrophic growth. The intermittent nature of exercise provides temporal organisation that is necessary for the normal operation of cellular growth process. Exercise by pregnant women does not appear to influence fetal growth. Evaluation of the effect of exercise on growth of children and adolescents is complicated by nonrandom selection of individuals for participation in organised sports, and by lack of information on the magnitude of exercise-induced energy drain. Exercise is essential for regulation of body composition in adulthood. It provides mechanical and metabolic stimuli that are necessary for hypertrophy of the musculoskeletal system and increased GH secretion for reparative growth. PMID- 8614770 TI - Magnetic resonance imaging of the femoral head to detect avascular necrosis in active rheumatoid arthritis treated with methylprednisolone pulse therapy. AB - Efficacious management of patients with avascular necrosis of bone (AVN) necessitates the identification of patients with a high risk of collapse of the femoral head. In this prospective study we imaged both hips of 10 patients with active rheumatoid arthritis, who were treated with methylprednisolone pulse therapy. MRI and conventional radiography were performed before MP-pulse therapy and 6 and 12 months thereafter. Two patients showed unilateral changes, compatible with AVN. One patient became symptomatic and revealed characteristic radiographic abnormalities. The other patient remained asymptomatic and the MRI appearance returned to normal after 6 months. PMID- 8614772 TI - Red cell Na+/H+ exchange and B cell alloantigen 883 (D8/17) in patients with acute rheumatic fever and inactive rheumatic heart disease. AB - The association of Na+/H+ antiport with 883 alloantigen was studied in patients with rheumatic disease. Simultaneous measurements were made of red cell Na+/H+ exchange and 883 alloantigen in microlymphocytotoxic test with D8/17 monoclonal antibodies in 20 patients with acute rheumatic fever, 20 patients with inactive rheumatic heart disease, 20 patients with atherosclerotic heart disease (stable anginal syndrome), and 20 healthy subjects. The number of 883(+) B cells and the Na+/H+ antiport activity were increased in rheumatic fever compared to healthy controls: 24.8 +/- 0.4 vs. 11.1 +/- 1.0% cells, 431 +/- 43 vs 121 +/- 12 micromol H+/l cells in min, respectively; p < 0.001; and in patients with rheumatic heart disease compared to patients with atherosclerotic heart disease; 25.7 +/- 1.8 vs. 9.8 +/- 1.1% cells, 482 +/- 73 vs. 124 +/- 12 micromol H+/l cells in min, respectively; p < 0.001. PMID- 8614771 TI - Effects of aerobic exercise versus stress management treatment in fibromyalgia. A 4.5 year prospective study. AB - To determine and compare short- and long-term effects of aerobic exercise (AE), stress management treatment (SMT), and treatment-as-usual (TAU) in fibromyalgia, 60 patients were randomized to 14 weeks of treatment by either AE, SMT or TAU. Outcome measures at baseline, midway through treatment, at treatment completion, and at 4 year follow up included a patient made drawing of pain distribution, dolorimetry of tender points, ergometer cycle test, global subjective improvement, and VAS registrations of: pain, disturbed sleep, lack of energy, and depression. Both AE and SMT showed positive short-term effects. AE was the overall most effective treatment, despite being subject to the most sceptical patient attitude prior to the study. At follow up, there were no obvious group differences in symptom severity, which for AE seemed to be due to a considerable compliance problem. PMID- 8614773 TI - Chemiluminescence responses and chemotaxis of monocytes from patients with early rheumatoid arthritis. AB - We studied chemiluminescence (CL) responses and chemotaxis of monocytes of patients with early rheumatoid arthritis (RA) before starting anti-rheumatic drug treatment and correlated the data to 2-year prognosis of the patients. Luminol enhanced CL responses of RA monocytes to N-formylmethionyl-leucyl-phenylalanine or to phorbol myristate acetate, and lucigenin-enhanced responses to opsonized zymosan particles were significantly higher than those of healthy control subjects. Distances of chemotactic, chemokinetic and random migration of RA monocytes were similar to those of control cells. High CL responsiveness correlated with seropositivity but not with presence of HLA-DR4 or that of erosions at 2-year follow-up. The results give credence to the view that monocytes are metabolically activated in early untreated RA, but increased respiratory burst activity does not predict early development of erosions. PMID- 8614775 TI - Deep hypothermia and circulatory arrest in the surgical management of renal cell carcinoma with vena caval involvement. AB - Six patients operated on for renal cell carcinoma with vena caval involvement were prospectively studied. The mean age of the four men and two women was 58 (range 51-77) years. In four of them the tumour was excised during cardiopulmonary bypass and deep hypothermic circulatory arrest. The operation was radical in three of these patients and palliative in one. There were no major complications or deaths during hospitalisation averaging 9 (7-17) days. The mean follow-up was 9 (4-14) months, during which two patients had died of metastatic disease. The surgical approach with cardiopulmonary bypass and deep hypothermic circulatory arrest is well tolerated and can be used to facilitate complete tumour thrombectomy, with low operative risk. Need for caval tumour thrombectomy was found in 5% of all patients with renal cell carcinoma during the study period. PMID- 8614774 TI - Hyperprolactinemia in systemic lupus erythematosus? AB - The objective of this project was to determine the prevalence of hyperprolactinemia (HPRL) in systemic lupus erythematosus (SLE) and to evaluate the role of prolactin (PRL) as a disease-activity marker. We determined PRL in a cohort of 182 patients with SLE. Basal prolactin levels and clinical data (disease-activity index, sero-activity, medications) were analyzed in all patients. The findings show only mildly elevated PRL levels in four SLE-patients and normaprolactinemia in 98% (n = 178), without any association with disease activity. Our results differ from previous studies. HPRL appears not to be prevalent in SLE, nor to be associated with disease activity. PMID- 8614776 TI - Survival in thoracic or thoracoabdominal aortic aneurysm. Comparison between patients with or without surgical treatment. AB - The surgical mortality among 22 patients treated for thoracic or thoracoabdominal aneurysm was compared with the mortality in 47 patients managed without surgery. Surgical mortality ( < 30 days) was low (1/13) in ascending aortic aneurysm, but higher (3/8) in aneurysm of the descending or thoracoabdominal aorta (including both acute and elective operations). Of the 20 non-surgically managed patients in the latter group, 15 died after a mean of 1.1 year. The only patient operated on for aortic arch aneurysm died of cerebral ischaemia 2 days postoperatively. Most of the 19 non-operated patients with aneurysm of the arch or total aorta (mean age 76 years) were never considered for surgical treatment. The analysis supports aggressive management of patients with aneurysm of the ascending, descending or thoracoabdominal aorta. Many of our patients with aneurysm of the arch or involving most of the aorta were old and had other, concomitant diseases, and in such cases an aggressive treatment strategy does not seem justified. PMID- 8614777 TI - Repeated repair of tetralogy of Fallot. Report of 11 cases and review of the literature. AB - Eleven patients underwent late repeated correction of tetralogy of Fallot in 1991 1993. The previous operation was repair of simple Fallot's tetralogy in seven cases, repair plus transannular patch in one case and repair of tetralogy and pulmonic atresia in three cases. The indications for reoperation were residual ventricular septal defect, right ventricular outflow tract (R.V.O.T.) obstraction, residual branch pulmonary artery stenosis, aneurysmal dilatation of R.V.O.T. Patch or combination of any of the above. At reoperation these defects were corrected. The post operative course was uneventful in eight patients. Two required mechanical ventilation for 2-3 days, and one underwent another operation for residual branch pulmonary artery stenosis. The functional and haemodynamic results were good in ten patients, and one had residual distal pulmonary artery stenosis. There were no death during 2 years of follow-up. Repeated correction of tetralogy of Fallot thus had low postoperative morbidity and good haemodynamic results. For the relatively few patients initially found to have tetralogy of Fallot and pulmonic atresia, the outcome may be less favorable. PMID- 8614778 TI - Impaired neutrophil chemotaxis after cardiac surgery. AB - Chemotaxis of circulating peripheral neutrophils was studied in 12 patients undergoing elective cardiac surgery. Tests of neutrophil chemotaxis were made preoperatively and 16-20 hours postoperatively, using a multipore filter assay. Statistically significant postoperative decrease was demonstrated in both stimulated and random neutrophil migration (p = 0.012 and p = 0.009, respectively). Although the peripheral neutrophil count showed a statistically significant postoperative increase (p = 0.001), the increase did not correlate to the decrease in neutrophil chemotaxis. Nor could correlation be demonstrated between the altered neutrophil chemotaxis and any per- or postoperative variables. PMID- 8614779 TI - Management of primary chest wall tuberculosis. AB - Primary tuberculosis of the chest wall is rare and its clinical presentation may resemble pyogenic abscess or tumour. The diagnosis is difficult, since smears or cultures of aspirate frequently fail to show tubercle bacilli. Seven cases of primary chest-wall tuberculosis treated between 1973 and 1992 are described. All presented with a progressively enlarging mass. The diagnosis was based on bacteriologic and histologic findings, but definitive diagnosis was obtained before treatment in only two cases. Satisfactory results were obtained with surgical debridement and specific chemotherapy in six cases and with chemotherapy alone in one case. From this limited experience, we suggest that primary chest wall tuberculosis should initially be treated with a combination regimen of antituberculous chemotherapy, which should take more than 9 months. If the lesion progressively enlarges or secondary infection occurs, however, adequate surgical debridement is also required. PMID- 8614780 TI - Early decortication for postpneumonic empyema in children. Effect on pulmonary perfusion. AB - Early pulmonary decortication was performed on 66 of 137 children with postpneumonic empyema, while 71 received conventional treatment. The mean age of the 66 patients with decortication was 5.5 years (range 6 months-14 years). The empyema was left-sided in 34 and right-sided in 32. Decortication was performed when lung expansion was not obtained after 10-12 days of intercostal tube drainage, antibiotic therapy (guided by sensitivity tests of pleural fluid) and pleural irrigation. Scintigraphy showed loss of pulmonary perfusion on the side of empyema to be 65% +/- SD 20 (25-98)% before decortication in the 23 tested patients. In ten of them the test was repeated after surgery and showed significant (p < 0.001) diminution of the perfusion defect, from 57 +/- 6.8 (25 84)% to 4 +/- 2.6 (0-8)%. The hospital stay was significantly (p < 0.001) shorter for the surgically treated than for the classically managed patients, viz. 19.5 +/- 4 (13-36) days vs 73.6 +/- 14 (34-110) days. Early decortication thus had beneficial effects on pulmonary perfusion and hospital stay. PMID- 8614781 TI - Late sudden deaths after repair of tetralogy of Fallot. Electrocardiographic findings associated with survival. AB - Of 141 hospital survivors after intracardiac repair of tetralogy of Fallot, eight died suddenly 6-23 years later. Compared with the other 133 patients, these eight were older at operation, with higher post-repair systolic right ventricular pressure and more often complete atrioventricular block; ventricular arrhythmia was diagnosed before death in three cases. In follow-up totalling 2255 patient years, the linearized rate of sudden death was 0.35%/year. The instantaneous risk of sudden death showed continuous increase with the length of follow-up. Of 80 survivors electrocardiographically evaluated 13-26 (median 20) years postoperatively, none had complete block, but 79 had complete right bundle branch block, including seven with left anterior hemiblock. Ventricular extrasystoles were recorded in 1% at rest, in 34% during exercise and in 83% during 24-hour ambulatory monitoring, with Lown Grade > or = II in 27%. Old age and possibly presence of fibrosis and/or fibroelastosis in right ventricular outflow Lown Grade. A patient with Lown grade III died suddenly 2 years after our follow-up. Old age at repair thus was associated with increased risk of late sudden death and with frequent ventricular arrhythmia in long-term survivors. PMID- 8614782 TI - Conservative approach to granular cell tumour of the oesophagus. Three case reports. AB - Granular cell tumour is an uncommon and generally benign lesion. In oesophageal location it is often asymptomatic and incidentally diagnosed at endoscopy. Three cases of granular cell oesophageal tumour are reported, with multiple location in one. In two cases the tumour was removed endoscopically by multiple biopsies. PMID- 8614783 TI - Reconstruction of chest wall after full-thickness resection. Two case reports. AB - Full-thickness reconstruction to repair defects in the chest wall was performed with a new method, using Marlex mesh, artificial bones and musculocutaneous flap. The artificial bones were constructed from ceramic iliac crest spacers connected by wires and methyl methacrylate. The advantage of the method is that the artificial bone can be created in various lengths and curves, tailored for the individual defect. PMID- 8614784 TI - Pancytopenia associated with thymoma resolving after thymectomy and immunosuppressive therapy. Case report. AB - In a 43-year-old woman, pancytopenia accompanying thymoma persisted after thymectomy, requiring weekly blood transfusions, and did not respond to prednisone 50 mg/day. Cyclosporine 10 mg/kg/day plus prednisone 20 mg/day for a month gradually corrected the blood parameters. Thirty months later the patient is well and haematologically stable. PMID- 8614785 TI - Primary cardiac echinococcosis in childhood. Case report. AB - Cardiac echinococcosis is rare and usually occurs in adults. In a 12-year-old boy a left ventricular hydatid cyst was diagnosed by two-dimensional echocardiography and computed tomography and the cyst was surgically removed. PMID- 8614786 TI - Gene therapy in New Zealand. PMID- 8614787 TI - Eye evolution. PMID- 8614788 TI - NIH Regional Primate Centers. PMID- 8614789 TI - Scientific misconduct. Panels look for common ground. PMID- 8614790 TI - Genome researchers take the pledge. PMID- 8614792 TI - Microbiology careers. ASM report sees a mixed future. PMID- 8614791 TI - Chemicals behind Gulf War syndrome? PMID- 8614793 TI - Yeast genome sequence ferments new research. PMID- 8614794 TI - The cerebellum: movement coordinator or much more? PMID- 8614795 TI - Tobacco money lights up a debate. PMID- 8614796 TI - AIDS pathogenesis: a finite immune response to blame? PMID- 8614797 TI - p75NTR: a receptor after all. PMID- 8614798 TI - Fluorescent hydroxyl emissions from Saturn's ring atmosphere. AB - Just before earth passed through Saturn's ring plane on 10 August 1995, the Hubble Space Telescope Faint Object Spectrograph detected ultraviolet fluorescent emissions from a tenuous atmosphere of OH molecules enveloping the rings. Brightnesses decrease with increasing distance above the rings, implying a scale height of about 0.45 Saturn radii (Rs). A spatial scan 0.28Rs above the A and B rings indicates OH column densities of about 10(13) cm(-2) and number densities of up to 700 cm(-3). Saturn's rings must produce roughly 10(25) to 10(29) OH molecules per second to maintain the observed OH distribution. PMID- 8614799 TI - Role of type I myosins in receptor-mediated endocytosis in yeast. AB - Type I myosins are thought to drive actin-dependent membrane motility, but the direct demonstration in vivo of their involvement in specific cellular processes has been difficult. Deletion of the genes MYO3 and MYO5, which encode the yeast type I myosins, almost abolished growth. A double-deleted mutant complemented with a MYO5 temperature-sensitive allele (myo5-1) showed a strong defect in the internalization step of receptor-mediated endocytosis, whereas the secretory pathway remained apparently unaffected. Thus, myosin I activity is required for a budding event in endocytosis but not for several other aspects of membrane traffic. PMID- 8614800 TI - Evaluating electrostatic contributions to binding with the use of protein charge ladders. AB - Electrostatic interactions between charges on ligands and charges on proteins that are remote from the binding interface can influence the free energy of binding (delta Gb). The binding affinities between charged ligands and the members of a charge ladder of bovine carbonic anhydrase (CAII) constructed by random acetylation of the amino groups on its surface were measured by affinity capillary electrophoresis (ACE). The values of delta Gb derived from this analysis correlated approximately linearly with the charge. Opposite charges on the ligand and the members of the charge ladder of CAII were stabilizing; like charges were destabilizing. The combination of ACE and protein charge ladders provides a tool for quantitatively examining the contributions of electrostatics to free energies of molecular recognition in biology. PMID- 8614801 TI - Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection. AB - The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution. PMID- 8614802 TI - Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. AB - Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activated the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3. PMID- 8614803 TI - Cerebellum implicated in sensory acquisition and discrimination rather than motor control. AB - Recent evidence that the cerebellum is involved in perception and cognition challenges the prevailing view that its primary function is fine motor control. A new alternative hypothesis is that the lateral cerebellum is not activated by the control of movement per se, but is strongly engaged during the acquisition and discrimination of sensory information. Magnetic resonance imaging of the lateral cerebellar output (dentate) nucleus during passive and active sensory tasks confirmed this hypothesis. These findings suggest that the lateral cerebellum may be active during motor, perceptual, and cognitive performances specifically because of the requirement to process sensory data. PMID- 8614805 TI - Interactions between electrical activity and cortical microcirculation revealed by imaging spectroscopy: implications for functional brain mapping. AB - Modern neuroimaging techniques use signals originating from microcirculation to map brain function. In this study, activity-dependent changes in oxyhemoglobin, deoxyhemoglobin, and light scattering were characterized by an imaging spectroscopy approach that offers high spatial, temporal, and spectral resolution. Sensory stimulation of cortical columns initiates tissue hypoxia and vascular responses that occur within the first 3 seconds and are highly localized to individual cortical columns. However, the later phase of the vascular response is less localized, spreading over distances of 3 to 5 millimeters. PMID- 8614804 TI - Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13. AB - Hox genes regulate patterning during limb development. It is believed that they function in the determination of the timing and extent of local growth rates. Here, it is demonstrated that synpolydactyly, an inherited human abnormality of the hands and feet, is caused by expansions of a polyalanine stretch in the amino terminal region of HOXD13. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. The mutations identify the polyalanine stretch outside of the DNA binding domain of HOXD13 as a region necessary for proper protein function. PMID- 8614806 TI - Ocular dominance plasticity under metabotropic glutamate receptor blockade. AB - Occluding vision through one eye during a critical period in early life nearly abolishes responses to that eye in visual cortex. This phenomenon is mimicked by long-term depression of synaptic transmission in vitro, which may require metabotropic glutamate receptors (mGluRs) and is age-dependent. Peaks in mGluR expression and glutamate-stimulated phosphoinositide turnover during visual cortical development have been proposed as biochemical bases for the critical period. Pharmacological blockade of mGluRs specifically prevented synapse weakening in mouse visual cortical slices but did not alter kitten ocular dominance plasticity in vivo. Thus, a heightened mGluR response does not account for the critical period in development. PMID- 8614807 TI - Transcription-coupled repair deficiency and mutations in human mismatch repair genes. AB - Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC), and a subset of sporadic cancers. Here, several mismatch repair deficient tumor cell lines and HNPCC-derived lymphoblastoid cell lines were found to be deficient in an additional DNA repair process termed transcription-coupled repair (TCR). The TCR defect was corrected in a mutant cell line whose mismatch repair deficiency had been corrected by chromosome transfer. Thus, the connection between excision repair and mismatch repair previously described in Escherichia coli extends to humans. These results imply that deficiencies in TCR and exposure to carcinogens present in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair. PMID- 8614808 TI - Linkage of replication to start by the Cdk inhibitor Sic1. AB - In Saccharomyces cerevisiae, three G1 cyclins (Clns) are important for Start, the event committing cells to division. Sic1, an inhibitor of C1b-Cdc28 kinases, became phosphorylated at Start, and this phosphorylation depended on the activity of Clns. Sic1 was subsequently lost, which depended on the activity of Clns and the ubiquitin-conjugating enzyme Cdc34. Inactivation of Sic1 was the only nonredundant essential function of Clns, because a sic1 deletion rescued the inviability of the cln1 cln2 cln3 triple mutant. In sic1 mutants, DNA replication became uncoupled from budding. Thus, Sic1 may be a substrate of Cln-Cdc28 complexes, and phosphorylation and proteolysis of Sic1 may regulate commitment to replication at Start. PMID- 8614809 TI - Heart attacks: gone with the century? PMID- 8614810 TI - Risks from low doses of radiation. PMID- 8614811 TI - Risks from low doses of radiation. PMID- 8614812 TI - Risks from low doses of radiation. PMID- 8614813 TI - Safety of hepatitis B vaccine. PMID- 8614814 TI - Immunology taught by Darwin. PMID- 8614815 TI - DNA Patents. Patent office faces 90-year backlog. PMID- 8614816 TI - Wiley drops IQ book after public furor. PMID- 8614817 TI - Physicists advance into biology. PMID- 8614818 TI - New gene forges link between fragile site and many cancers. PMID- 8614819 TI - "Smart" genes use many cues to set cell fate. PMID- 8614820 TI - New skeleton gives path from trees to ground an odd turn. PMID- 8614822 TI - Finding new drugs to treat stroke. PMID- 8614821 TI - Uganda may host AIDS vaccine trial. PMID- 8614823 TI - Moscow cardiology institute battles for its life. PMID- 8614824 TI - New devices are helping transform coronary care. PMID- 8614825 TI - Molecular pathways controlling heart development. AB - Heart formation requires complex interactions among cells from multiple embryonic origins. Recent studies have begun to reveal the genetic pathways that control cardiac morphogenesis. Many of the genes within these pathways are conserved across vast phylogenetic distances, which has allowed cardiac development to be dissected in organisms ranging from flies to mammals. Studies of cardiac development have also revealed the molecular defects underlying several congenital cardiac malformations in humans and may ultimately provide opportunities for genetic testing and intervention. PMID- 8614826 TI - Molecular genetics of human blood pressure variation. AB - Hypertension is a common multifactorial vascular disorder of largely unknown cause. Recognition that hypertension is in part genetically determined has motivated studies to identify mutations that confer susceptibility. Thus far, mutations in at least 10 genes have been shown to alter blood pressure; most of these are rare mutations imparting large quantitative effects that either raise or lower blood pressure. These mutations alter blood pressure through a common pathway, changing salt and water reabsorption in the kidney. These findings demonstrate the utility of molecular genetic approaches to the understanding of blood pressure variation and may provide insight into the physiologic mechanisms underlying common forms of hypertension. PMID- 8614827 TI - Molecular genetic insights into cardiovascular disease. AB - The recent application of molecular genetic tools to inherited forms of cardiovascular disease has provided important insight into the molecular mechanisms underlying cardiac arrhythmias, cardiomyopathies, and vascular diseases. These studies point to defects in ion channels, contractile proteins, structural proteins, and signaling molecules as key players in disease pathogenesis. Genetic testing is now available for a subset of inherited cardiovascular diseases, and new mechanism-based therapies may be available in the near future. This remarkable progress and the implications it may have for more common forms of cardiovascular disease are reviewed here. PMID- 8614828 TI - Mouse models of atherosclerosis. AB - As a species the mouse is highly resistant to atherosclerosis. However, through induced mutations it has been possible to develop lines of mice that are susceptible to this disease. For example, mice that are deficient in apolipoprotein E, a ligand important in lipoprotein clearance, develop atherosclerotic lesions resembling those observed in humans. These lesions are exacerbated when the mice are fed a high-cholesterol, high-fat, Western-type diet. Other promising models are mice that are deficient in the low density lipoprotein receptor and transgenic mice that express human apolipoprotein B and transdominant mutant forms of apolipoprotein E. These models are now being used to study the pathogenesis of atherosclerotic lesions, as well as the influence of genetics, environment, hormones, and drugs on lesion development. PMID- 8614829 TI - Molecular therapies for vascular diseases. AB - Vascular disease is the most common cause of death in the industrialized world. Although significant progress has been made in treating these disorders, more therapeutic agents must be developed that effectively prevent, arrest, or reverse this disease. Recent insights into the pathogenesis of vascular disease have opened up a new frontier of molecular therapies that target molecules as diverse as adhesion molecules and transcription factors. The biological rationale for these new therapies and their prospects for success are discussed. PMID- 8614830 TI - Diabetes complications: why is glucose potentially toxic? PMID- 8614831 TI - Direct measurement of coupling between dendritic spines and shafts. AB - Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms. PMID- 8614832 TI - Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. AB - Signal transducers and activators of transcription (STAT) proteins can be conditionally activated in response to epidermal growth factor (EGF) and interferon (IFN)-gamma. STAT activation was correlated with cell growth inhibition in response to EGF and IFN-gamma. Activated STAT proteins specifically recognized the conserved STAT-responsive elements in the promoter of the gene encoding the cyclin-dependent kinase (CDK) inhibitor p21 WAF1/CIP1 and regulated the induction of p21 messenger RNA. IFN-gamma did not inhibit the growth of U3A cells, which are deficient in STAT1, but did inhibit the growth of U3A cells into which STAT1 alpha was reintroduced. Thus, STAT1 protein is essential for cell growth suppression in response to IFN-gamma. The STAT signaling pathway appears to negatively regulate the cell cycle by inducing CDK inhibitors in response to cytokines. PMID- 8614833 TI - In vitro development of primitive and definitive erythrocytes from different precursors. AB - During mouse embryogenesis the production of "primitive" erythrocytes (EryP) precedes the production of "definitive" erythrocytes (EryD) in parallel with the transition of the hematopoietic site from the yolk sac to the fetal liver. On a macrophage colony-stimulating factor-deficient stromal cell line OP9, mouse embryonic stem cells were shown to give rise to EryP and EryD sequentially with a time course similar to that seen in murine ontogeny. Studies of the different growth factor requirements and limiting dilution analysis of precursor frequencies indicate that most EryP and EryD probably developed from different precursors by way of distinct differentiation pathways. PMID- 8614834 TI - Homologous association of oppositely imprinted chromosomal domains. AB - Human chromosome 15q11-q13 encompasses the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) loci, which are subject to parental imprinting, a process that marks the parental origin of certain chromosomal subregions. A temporal and spatial association between maternal and paternal chromosomes 15 was observed in human T lymphocytes by three-dimensional fluorescence in situ hybridization. This association occurred specifically at the imprinted 15q11-q13 regions only during the late S phase of the cell cycle. Cells from PWS and AS patients were deficient in association, which suggests that normal imprinting involves mutual recognition and preferential association of maternal and paternal chromosomes 15. PMID- 8614835 TI - Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. AB - The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities. PMID- 8614836 TI - A mouse model of familial hypertrophic cardiomyopathy. AB - A mouse model of familial hypertrophic cardiomyopathy (FHC) was generated by the introduction of an Arg 403 --> Gln mutation into the alpha cardiac myosin heavy chain (MHC) gene. Homozygous alpha MHC 403/403 mice died 7 days after birth, and sedentary heterozygous alpha MHC 403/+ mice survived for 1 year. Cardiac histopathology and dysfunction in the alpha MHC 403/+ mice resembled human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fibrosis increased with age. Young male alpha MHC 403/+ mice showed more evidence of disease than did their female counterparts. Preliminary results suggested that exercise capacity may have been compromised in the alpha MHC 403/+ mice. This mouse model may help to define the natural history of FHC. PMID- 8614837 TI - The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). AB - The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells. PMID- 8614838 TI - Requirement for BMP signaling in interdigital apoptosis and scale formation. AB - Interdigital cell death leads to regression of soft tissue between embryonic digits in many vertebrates. Although the signals that regulate interdigital apoptosis are not known, BMPs--signaling molecules of the transforming growth factor-beta superfamily--are expressed interdigitally. A dominant negative type I BMP receptor (dnBMPR-IB) was used here to block BMP signaling. Expression of dnBMPR in chicken embryonic hind limbs greatly reduced interdigital apoptosis and resulted in webbed feet. In addition, scales were transformed into feathers. The similarity of the webbing to webbed duck feet led to studies that indicate that BMPs are not expressed in the duck interdigit. These results indicate BMP signaling actively mediates cell death in the embryonic limb. PMID- 8614839 TI - Origin of replication of Mycoplasma genitalium. PMID- 8614840 TI - THe genetics of familial breast cancer. AB - Because studies of breast cancer patients and their relatives provide statistical evidence for involvement of autosomal dominant genes, the identification of specific genetic effects has long been the focus of efforts to identify women at exceedingly high risk. BRCA1, a gene that confers greatly increased susceptibility to breast and ovarian cancer, was isolated in 1994, capping an intense analysis by a large number of groups of a complex phenotype. BRCA1 is a large gene and shows only limited homology to other known genes. Near the amino terminus of the predicted protein is a RING finger motif. In addition, a leucine heptad repeat appears in the interior of the sequence. Several groups have looked extensively for somatic BRCAI mutations in breast and ovarian tumors. The frequency of somatic mutations in ovarian tumors is low, and to date no somatic mutations have been found in breast tumors. More research is needed to define the role of BRCA1 in sporadic tumors. A second locus associated with predisposition to early onset breast cancer, BRCA2, has been localized to chromosome 13q. Positional cloning of this gene is well advanced and analysis of its biology and mutation spectrum is eagerly awaited. As the BRCA1 and BRCA2 genes are characterized further, a diagnostic test for breast cancer susceptibility becomes feasible. PMID- 8614841 TI - The role and extent of surgery in early invasive breast cancer. AB - The role of surgery in early invasive breast cancer is reviewed. Technologic developments and widespread use of screening mammography have allowed detection of smaller breast cancers that can be biopsied with minimally invasive procedures. Current treatment strategies include breast conservation therapy for the majority of early invasive breast cancers with local recurrence rates as low as 2%. The role of axillary dissection is being questioned in these early cancers. We review the current scientific data, which may lead to women with early breast cancers being treated with lumpectomy alone in the future, with axillary dissection being done in a scientific and highly selected fashion for the 10% to 30% of women who will benefit from that procedure. PMID- 8614842 TI - Selection of patients with early stage invasive breast cancer for treatment with conservative surgery and radiation therapy. AB - The combination of breast-conserving surgery and radiation therapy is now accepted as a standard treatment option for most women with clinical stage I or II invasive breast cancer. The great majority of individuals with early stage breast cancer are potential candidates for treatment with conservative surgery and radiation therapy. Nonetheless, for a few patients, such treatment may be absolutely or relatively contraindicated due to toxicity concerns, poor anticipated cosmetic results, or a heightened risk of local failure. This article will discuss the patient, clinical, and pathologic factors relevant to selecting patients for this treatment approach. PMID- 8614843 TI - The role of primary chemotherapy in early breast cancer. AB - Primary chemotherapy was first attempted in the early 1970s in an attempt to improve local control and survival in patients with large breast tumors. While it is now clear that primary chemotherapy can achieve high response rates and allow more conservative surgery, it is less apparent if survival is improved in these patients. It is logical, then, to consider moving this form of treatment to an earlier stage of disease in which the probability of resistant clones is less in patients with lower tumor burden. Many questions still remain unanswered in the evaluation of the role of primary chemotherapy in the treatment of early breast cancer: Is the best method of diagnosis fine needle aspiration or incisional biopsy? What diagnostic tests are important before chemotherapy? Can better conservative local treatment be achieved using the primary modality of chemotherapy? Is local control, disease response, and/or survival improved by using chemotherapy before local measures? Several important studies have been performed in the treatment of early stage disease with primary chemotherapy and will be discussed with these questions in mind. In addition, investigation of markers that predict response to chemotherapy may help us better select patients who would benefit from primary chemotherapy. Those patients who are determined to have a poorer prognosis would be candidates for novel investigational treatments. PMID- 8614844 TI - A computer program to assist in making breast cancer adjuvant therapy decisions. AB - This report describes a computer program designed to assist health care professionals in making projections of the average benefit of systemic adjuvant therapy for individual breast cancer patients. It requires as input patient age (used to make projections of natural mortality), an estimate of breast cancer related mortality at 5 years (used to make projections of breast cancer-specific mortality), and the proportional risk reduction for breast cancer mortality expected for the adjuvant therapy (with included tables from the Early Breast Cancer Trialist's 1992 meta-analysis). The program uses life table analytical techniques to make projections of outcome in three scenarios: that the breast cancer never occurred, that the breast cancer patient received definitive local therapy but no adjuvant systemic therapy, and that the patient received adjuvant therapy. The outcome projections are given for total, natural (non-breast cancer related), and breast cancer-related mortality at several time points and also of total remaining life expectancy. These estimates are currently widely made by clinicians by nonnumerical techniques. Computer-based tools can serve as valuable aids in physician and patient education and in the process of informed decision making. PMID- 8614845 TI - Adjuvant therapy for early stage breast cancer. AB - Metastatic disease in women who have undergone appropriate local therapy remains the leading ultimate cause of death in breast cancer. Therapy for overt metastatic disease is unlikely to offer curative potential for any but a small proportion of patients. Therapy for micrometastatic disease is offered in the hope that patients with a smaller tumor burden will be more amenable to cure. This report will examine the extent to which this hope has been fulfilled and discuss the future of adjuvant therapy. The initial adjuvant therapy trials used chemotherapy to treat women with lymph node-positive breast cancer. These trials clearly demonstrated a disease-free and overall survival benefit for women receiving combination chemotherapy in the adjuvant setting. These benefits were subsequently extended to women with lymph node-negative disease, and (in the form of adjuvant hormonal therapy) to women with steroid receptor-positive breast cancer. Current adjuvant therapy clearly improves disease-free and overall survival in patients with micrometastatic disease. It is less clear whether these improvements translate to a "cure" in the sense of total elimination of micrometastatic disease. Numerous questions remain to be answered regarding the dose intensity of chemotherapy, the appropriate sequencing of chemotherapy agents, and the use of novel chemotherapy agents, such as the taxanes. These and other questions are the subject of ongoing clinical trials. PMID- 8614846 TI - Chemotherapy for advanced breast cancer: a current perspective. AB - The systemic management of patients with hormone-refractory breast cancer metastatic to sites beyond regional axillary lymph nodes is classically characterized by the thoughtful, step-wise administration of cytotoxic chemotherapeutic agents. At various junctures in the continuum of care, a patient/physician analysis of the potential risks and benefits of chemotherapy may lead to the decision to proceed with subsequent systemic therapy, to consider an investigational strategy, or to opt for supportive care alone. The emergence of new active agents such as the taxanes has provided a valuable alternative option for such patients. While other cytotoxic agents in development may ultimately prove useful in the management of stage IV breast cancer, including some with novel antineoplastic mechanisms, there is evidence for optimism that the ability to perturb autocrine and paracrine growth factor pathways (eg, with monoclonal antibodies) may afford a viable alternative systemic approach, perhaps with a greater therapeutic index than "conventional" agents. Throughout the process of drug development, an increasing focus on the impact of new systemic regimens on quality of life, and costs associated with the palliative care of metastatic breast cancer should optimize the care of women with this disease. PMID- 8614847 TI - Duct carcinoma in situ: biological implications for clinical practice. AB - Duct carcinoma in situ (DCIS) has become an important, however controversial, focus of breast cancer management only since the advent of effective film mammography and the development of an increased interest and utilization of breast conservation therapy. Prior to 1975, DCIS remained an infrequent biopsy finding in patients who presented with a palpable mass, nipple discharge, or clinical Paget's disease. The vast majority of such patients harbored extensive noninvasive disease and frequently were found to have occult invasive breast cancer at mastectomy, which was the only method of available treatment. The significance of small foci of DCIS as detected mammographically and the implications of DCIS in conjunction with invasive carcinoma for breast conservation therapy were slowly learned over the next two decades. This paper reviews current studies of DCIS with a particular focus on practical applications for management. PMID- 8614849 TI - The role of taxanes in the treatment of breast cancer. AB - The taxanes paclitaxel and docetaxel are undergoing extensive evaluation in women with breast cancer in the United States and in Europe. Their dose-limiting toxicity is neutropenia. Paclitaxel also causes peripheral neuropathy, while docetaxel can cause unpredictable and severe skin toxicity, as well as edema and effusions due to a capillary leak syndrome. Due to threshold pharmacodynamics and nonlinear pharmacokinetics, tolerability of paclitaxel is schedule dependent. Single-agent paclitaxel was very active in multiple phase II trials in patients with different numbers and types of prior chemotherapy and disease extent (20% to 60% complete plus partial responses). Effective doses ranged from 135 to 250 mg/m2. Activity was observed with all infusion schedules (1, 3, and 24 hours) and in women with anthracycline-resistant tumors (25% to 38%). The use of a 96-hour infusion schedule was very active in anthracycline-refractory patients (48%) and in women who failed short infusion taxanes. The drug is undergoing extensive evaluation in combination with doxorubicin, cyclophosphamide, cisplatin, and antimetabolites. Very promising efficacy was observed for paclitaxel by 3-hour infusion plus bolus doxorubicin (approximately 40% complete responses and 50% partial responses). The combination also caused a high incidence of clinically reversible congestive heart failure(14% to 18%). Docetaxel also has very good efficacy in breast cancer, with approximately 70% major responses in untreated patients and more than 50% in anthracycline-resistant tumors. There is no evidence that efficacy and tolerability are schedule dependent as is the case for paclitaxel. At recommended doses (100 or 75 mg/m2 by 1-hour infusion every 3 weeks), docetaxel causes a fluid retention syndrome that may affect quality of life. Its common onset after multiple cycles may limit the use of docetaxel for palliation in metastatic breast cancer. These results clearly indicate that the taxanes will become a standard component of initial chemotherapy for women with breast cancer. The definition of their actual role still requires an answer to the unresolved questions of their optimal dose and combination with other anticancer agents. Most importantly, the drugs should be prospectively evaluated in a randomized study using comparable doses and schedules to assess which of the two has the better therapeutic index in breast cancer. PMID- 8614848 TI - High-dose chemotherapy with autologous hematopoietic progenitor cell support for metastatic and high-risk primary breast cancer. AB - High-dose chemotherapy (HDCT) is being evaluated in the treatment of metastatic and high-risk primary breast cancer. The actuarial 2-year disease-free survival rate for patients with stage IV breast cancer is approximately 15% to 20%. A single metastatic site and achievement of a complete response to induction chemotherapy may be favorable prognostic indicators for disease-free survival after HDCT. Phase II studies in patients with primary breast cancer and > or = 10 and four to nine involved axillary nodes are encouraging. Prospective randomized trials are ongoing in these patient groups. Various strategies are being investigated to reduce the incidence of relapse after HDCT. PMID- 8614850 TI - Implications of the new biology for therapy in breast cancer. AB - It is a truism that a better understanding of the biology of breast cancer should lead to improvements in diagnosis and therapy. Despite this, our significantly improved grasp of breast cancer biology has had little direct therapeutic impact to date. The technologies used to treat breast cancer (surgery, radiation therapy, chemotherapy, and hormonal therapy) were in general developed decades ago. With the exception of the prognostic factor area, advances in biological knowledge have not translated into either new therapies or altered outcomes. Fortunately, this now appears to be changing. This paper will focus on three emerging areas in which the new biology is most likely to have a therapeutic impact. The first area involves growth factors and their receptors. It is now clear that the growth of breast cancer is regulated by growth factor receptors (eg, EGFR and Her-2/neu), and that their upregulation is associated with impaired prognosis. Growth factors and their receptors represent a promising therapeutic target, both alone and in combination with other standard agents. Recent evidence suggests that growth factors and their receptors may be important for regulating programmed cell death (apoptosis) in breast cancer. A second emerging area involves matrix metalloproteinases. Breast cancer invasion and metastasis involves and requires activation of enzymes capable of dissolving natural barriers to spread. The most heavily studied of these are the matrix metalloproteinases, for which considerable in vitro and in vivo evidence suggests an important role in breast cancer. The recent advent of compounds with matrix metalloproteinase inhibitory activity makes a therapeutic intervention against matrix metalloproteinases appear reasonable. In vivo laboratory evidence suggests that their use may occur in the very near future. A third emerging area revolves around the question of neoangiogenesis (new blood vessel formation) in breast cancer. Breast cancers (indeed, all solid tumors) are incapable of growth beyond a certain critical diameter without new blood vessel formation. Recent work has demonstrated that breast cancers produce angiogenic factors stimulating this new growth. Therapies aimed at blocking this stimulation, and preventing neovascularization represent a promising therapeutic target. Numerous agents capable of preventing new blood vessel formation have been discovered in the laboratory and are poised to enter clinical trials. PMID- 8614851 TI - Breast cancer in older women. AB - By the end of the 20th century, more than 50% of new breast cancer patients will be greater than 65 years old. Until recently, research focused on this older group of women has been minimal. Previous studies have indicated that the elderly are less likely to be screened, and have lesser and frequently inferior treatment. In contrast, the few clinical trials focusing on the elderly suggest that they do as well with surgery, radiation, and standard chemotherapy regimens as their younger counterparts. Comorbidity is, however, more common in older women, and must be factored into treatment and prognosis. The available data indicate that mammography should be used on a yearly to every-other-year basis for screening older women, including those older than 70 years and in fair to good health. Older women should offered breast preservation options and should receive breast radiation following lumpectomy. Tamoxifen may be used as initial therapy for frail women with early to late stage breast cancer, but is inferior to surgery in achieving long-term local control. Adjuvant tamoxifen should be considered for all high-risk node-negative and all node-positive patients regardless of receptor status. Adjuvant chemotherapy is reasonable for high-risk node-negative and node-positive patients in good health and with reasonable life expectancy (>5 years), although clinical trials have not established efficacy for women in this age group. Older women with metastatic breast cancer are good candidates for endocrine therapy. Chemotherapy should be offered to those with progressive disease after endocrine therapy. There are inadequate numbers of older women enrolled in breast cancer clinical trials. Both physicians and patients should explore and define the barriers to clinical trial participation and develop successful interventions to overcome them. PMID- 8614852 TI - Women's psychological reactions to breast cancer. AB - Breast cancer is the cancer most studied in terms of psychological and psychosocial aspects because of the high prevalence and mortality of the disease and the psychological effects of surgery on an organ rich in meaning. The diagnosis of breast cancer, treatment, and treatment sequelae are major stressors for any woman; however, the psychological impact of the diagnosis and women's emotional responses vary considerably depending on medical parameters of the disease, the patient's psychological make-up and coping abilities, and the availability of emotional and financial support. We document the experience of breast cancer at all phases of the disease for a majority of psychologically healthy women and describe the emotional responses of women who are at high genetic risk of developing breast cancer. Having a comprehensive understanding of psychologically healthy women's reactions to breast cancer is necessary as we attempt to define standards of care and treatment guidelines for both psychologically healthy women with breast cancer and for those with psychiatric disorders that antedate or complicate breast cancer treatment. PMID- 8614853 TI - Economic and cost-effectiveness issues in breast cancer treatment. AB - The need for cost-effectiveness analyses is based on the unfortunate but universal situation of limited financial resources that ideally should be used to maximal benefit. Formal cost-effectiveness analyses assume a societal utilitarian perspective with the objective of maximizing net health benefit for members of a population within a limited level of resources. This societal perspective is in stark contrast to the clinician's perspective, whose goal is to maximize his or her patient's health status (no matter what effect those decisions have on other patients or resources). This difference in perspective and objectives explains why many clinicians object to the use of cost-effectiveness analysis in setting policies. When considering the natural history of breast cancer from screening, evaluation of suspicious lesions, primary therapy, staging, adjuvant therapy, monitoring, metastatic disease, and palliative care, it is striking that most cost-effectiveness studies have been related to screening or the use of adjuvant drug therapies. In prior work our group has shown that the use of chemotherapy in node-negative breast cancer and of tamoxifen alone or in combination with chemotherapy in premenopausal women are cost-effective compared with other common medical treatments. Given the increasing pressure to contain costs in contemporary medicine, one should remember that cost effectiveness is related to value, value defined as quality/costs. Examples are discussed when the controversy focuses on increasing quality (eg, valued outcomes, such as additional years of life or years of breast preservation) and on controlling costs (eg, integrating multidisciplinary care, minimizing superfluous testing, or reducing surgical biopsy rates). Efforts should be directed at both sides of this ratio. PMID- 8614854 TI - Imaging the diabetic foot. AB - Early and accurate diagnosis of infection or neuropathy of the diabetic foot is the key to successful management. Angiopathy leads to ischemia which, in combination with peripheral neuropathy, predisposes to pedal skin ulceration, the precursor of osteomyelitis. Chronic hyperglycemia promotes production of glycosylated end products which accumulate on endothelial proteins, causing ischemia of the vasa nervorum. When combined with axonal degeneration of the sensory nerves, the result is hypertrophic neuroarthropathy. Should the sympathetic nerve fibers also be damaged, the resultant loss of vasoconstrictive impulses leads to hyperemia and atrophic neuroarthropathy. Plain radiography, although less sensitive than radionuclide, magnetic resonance (MR), and computed tomographic examinations, should be the initial procedure for imaging suspected osteomyelitis in the diabetic patient. If the radiographs are normal but the clinical suspicion of osteomyelitis is strong, a three-phase 99mTc-MDP scan or MR imaging is recommended. An equivocal 99mTc-MDP scan should be followed by MR imaging. To exclude osteomyelitis at a site of neuroarthropathy, a 111In white blood cell scan is preferable. To obtain a specimen of bone for bacteriological studies, percutaneous core biopsy is the procedure of choice, with the entrance of the needle well beyond the edge of the subjacent ulcer. PMID- 8614855 TI - Tumoral calcinosis: radiologic-pathologic correlation. AB - OBJECTIVE: Tumoral calcinosis is a frequently misdiagnosed disorder. This study details the radiologic and pathologic characteristics of tumoral calcinosis that distinguish it from most other entities. DESIGN: Radiologic and pathologic findings, and medical records of 12 patients with tumoral calcinosis were reviewed and compared with equivalent information about 5 patients with other calcified lesions. PATIENTS: The 12 patients ranged in age from 15 months to 62 years. Six had idiopathic tumoral calcinosis and 6 had secondary tumoral calcinosis. RESULTS AND CONCLUSIONS: A consistent radiologic finding for tumoral calcinosis was a dense calcified mass that was homogeneous except for a "chicken wire" pattern of lucencies, which correlated histologically with thin fibrous septae. Other characteristics of tumoral calcinosis included fluid-calcium levels, demonstrated in four patients, and smooth osseous erosions adjacent to the mass, demonstrated in three patients. Five cases of tumoral calcinosis were originally confused with other calcified lesions; however, the radiologic findings were characteristic of tumoral calcinosis in retrospect. PMID- 8614856 TI - Synovial hemangioma of the knee: MRI findings in two cases. AB - The findings in two patients with histologically proven synovial hemangioma of the knee are described. Both cases emphasize the typical appearance of this unusual tumor on magnetic resonance imaging. Additional radiologic findings, such as adjacent osseous involvement, are discussed. PMID- 8614857 TI - Synovial hemangioma: imaging features in eight histologically proven cases, review of the literature, and differential diagnosis. AB - OBJECTIVE: This study was undertaken to describe the imaging characteristics of synovial hemangioma, with the goal of improving the disappointing rate (22%) of clinical diagnosis of this condition. A review of the literature and the differential diagnosis of intra-articular lesions, including synovial osteochondromatosis and pigmented villonodular synovitis, are also presented. PATIENTS: The subjects of the study were 8 patients (4 males, 4 females; age range: 5-47 years; mean age: 19 years) with histologically confirmed synovial hemangioma involving the knee (n = 7) or wrist (n = 1). We retrospectively examined the imaging studies performed in these patients, including plain radiography (n = 8), magnetic resonance imaging (MRI; n = 4), angiography (n = 3), arthrography (n = 2), and contrast-enhanced computed tomography (CT; n = 2). RESULTS: Plain radiographs showed a soft tissue density suggesting either joint effusion or a mass in all patients. Phleboliths and bone erosions on plain films in four patients with extra-articular soft tissue involvement pointed to the correct diagnosis. Angiography, showing fine-caliber, smooth-walled vessels, contrast pooling in dilated vascular spaces, and early visualization of venous structures, was diagnostic in two patients. Neither arthrography nor CT yielded specific enough findings. MRI was consistently effective in allowing the correct diagnosis to be made preoperatively, showing an intra-articular or juxta articular mass of intermediate signal intensity on T1-weighted images and of high signal intensity of T2- or T2*-weighted images with low-signal channels or septa within it. A fluid-fluid level was found in two patients with a cavernous-type lesion. CONCLUSION: Despite the limited nature of this study, it shows clearly that MRI is the procedure of choice whenever an intra-articular vascular lesion such as synovial hemangioma is suspected. Nonetheless, phleboliths and evidence of extra-articular extension of plain radiographs point to angiography as an effective procedure of first resort because it can be combined with embolotherapy. PMID- 8614858 TI - Ultrasonographic imaging of the hand and wrist in rheumatoid arthritis. AB - OBJECTIVE: To assess ultrasound findings in patients with rheumatoid arthritis affecting the hand and wrist compared to normal volunteers. DESIGN: Metacarpophalangeal and carpal articulations were imaged ultrasonically. Two readers reviewed static images for synovial, cartilaginous, and bony abnormalities using severity and probability scales. Ultrasound findings were correlated with disease activity. PATIENTS: Ten normal volunteers and 29 patients with known rheumatoid arthritis. RESULTS: Synovial abnormalities and erosions were most commonly identified in the rheumatoid hand and wrist (p < 0.01). Criteria used for normal and abnormal cartilage did not predict normal and disease states. Significant differences in synovial abnormalities and erosions were observed between the inactive and mildly active disease groups as well as the active and mildly active disease groups (p < 0.01). CONCLUSION: Ultrasound can detect abnormalities of the hand and wrist in patients with rheumatoid arthritis compared to normal volunteers. Normal articular anatomy is well demonstrated ultrasonically. PMID- 8614859 TI - Scintigraphic and radiographic patterns of skeletal metastases in breast cancer: value of sequential imaging in predicting outcome. AB - OBJECTIVE: To determine whether temporal changes in scintigraphic and bone radiographic findings have prognostic significance in patients with skeletal metastases from breast cancer. DESIGN: Clinical information and films were retrospectively reviewed in 101 randomly selected patients with skeletal metastases. Images from sequential bond scans and bone radiographs were correlated with survival after detection of the metastases. RESULTS: Time to detection of skeletal metastases and the length of time for which patients were classified as radiologically stable after development of skeletal metastases correlated with survival (r = 0.843; r = 0.821, respectively). Failure to develop a radiographically and scintigraphically stable pattern after treatment was associated with significantly decreased survival compared with the rest of the patients (mean survival 2.1 +/- 1.3 years vs 4.3 +/- 2.3 years; p < 0.001). Scintigraphic regression of metastases was associated with significant survival benefit and longer stabilization of disease compared to all other patterns (mean survival 5.0 +/- 2.7 years of regressive disease vs 3.7 +/- 1.9 years for stable disease and 2.2 +/- 1.3 years for progressive disease; p < 0.001). CONCLUSIONS: Sequential scintigraphic and radiographic imaging is useful in breast cancer patients not only for detection of metastases and monitoring of treatment effect, but also because these studies provide valuable prognostic information. PMID- 8614860 TI - Magnetic resonance imaging of hereditary hernias of the peroneus longus muscle. AB - Herniation of the left peroneus longus muscle was present in three male members of the same family, being the first reported case of this condition in a familial setting. The hernias were differentiated from other mass lesions and varices by magnetic resonance imaging. The images demonstrated a fascial defect originating in the area where vessels and nerves penetrate the fascia, suggesting that the three men had a congenital weakness in the fascia. PMID- 8614861 TI - Fatigue fracture of the sacral bone associated with septic arthritis of the symphysis pubis. AB - Fractures of the pubic rami are almost invariably associated with fractures of the posterior arch of the pelvic ring. Two women, aged 50 and 67 years, with septic arthritis of the symphysis pubis attended by severe low back pain, were followed with CT and MR imaging of the pelvis, as well as bond scintigraphy in one patient. In the first patient sacral fractures with severe displacement were revealed, prompting stabilizing symphysiodesis. In the second patient an undisplaced fatigue fracture was confirmed in the right half of the sacrum. In patients with pelvic laxity following arthritis of the symphysis and post traumatic osteolysis associated with low back pain, displaced or occult fractures of the bones adjacent to the sacroiliac joints should be considered. PMID- 8614862 TI - Periarticular heterotopic ossification following pharmacologically induced paralysis. PMID- 8614863 TI - Bilateral stress fracture of the clavicle. PMID- 8614864 TI - Dedifferentiated chondrosarcoma arising in an osteochondroma. PMID- 8614865 TI - Atypical skeletal tuberculosis. AB - Tuberculosis remains a major cause of skeletal infection in many parts of the world. With the advent of acquired immune deficiency syndrome (AIDS) and mass emigration from underdeveloped to industrialized nations this past decade, a new patient population is being encountered in which extrapulmonary tuberculosis is not uncommon and atypical presentations may confuse the diagnosis. Our recent experience with an unusual presentation has prompted this report. PMID- 8614866 TI - Christian brachydactyly in a father and two daughters. PMID- 8614867 TI - Fracture of the os peroneum and rupture of the peroneus longus tendon as a complication of diabetic neuropathy. PMID- 8614868 TI - Infantile myofibromatosis. AB - An unusual case of infantile myofibromatosis with foci of calcifications was presented, with atypical age of onset and location. The radiological features of this tumor are not always evocative, and histological examination is necessary for diagnosis. PMID- 8614869 TI - Osteosarcoma after chemotherapy for neuroblastoma. PMID- 8614870 TI - Epithelioid sarcoma presenting as chronic synovitis and mistaken for osteosarcoma. PMID- 8614871 TI - Rapidly destructive hip disease. PMID- 8614872 TI - Established adult respiratory distress syndrome successfully treated with corticosteroids. AB - In 1990, we described the treatment of established adult respiratory distress syndrome (ARDS) with a sustained course of adrenocortical steroids (ACS). This report updates our experience with an uncontrolled prospective series. Patients with ARDS of more than 3 days' duration were initially given ACS in intravenous dosages of 125 to 250 mg every 6 hours, with tapering every 3 to 4 days. ARDS was present for 3 to 40 days before treatment. The PO2/FIO2 ranged from 45 to 211 (mean, 109) and the injury index from 2 to 4 (mean, 3.25). Nineteen gallium Ga 67 citrate lung scans were obtained in 18 patients. The scans showed diffuse abnormality in all 19 studies, but added no new diagnostic information. We have treated 26 patients, and overall survival was 81% (21/26). When complicating features were present, survival was 64% (9/14). Our uncontrolled, observational experience in treating established ARDS suggests that a sustained course of ACS may improve survival in these severely ill patients. PMID- 8614873 TI - Ingestion of angel's trumpet: an increasingly common source of toxicity. AB - Angel's trumpet is a hallucinogenic plant commonly cultivated as an ornamental in the southeastern United States. Consumption of any part of the plant may result in severe anticholinergic toxicity. In past years, toxicity has largely resulted from accidental ingestion, but in 1994 there was a ten-fold increase in the number of reported ingestions throughout the state of Florida, largely by persons experimenting with the plant for its hallucinogenic effects. We report three such cases and review the literature on diagnosis, treatment, and sequelae of angel's trumpet intoxication. PMID- 8614874 TI - Causes of gastrointestinal hemorrhage in neonates and children. AB - The causes of gastrointestinal hemorrhage in children were detailed in 1964 by Spencer. We investigated the causes of gastrointestinal hemorrhage in 165 children seen at our institution over a 13-year period. The most common causes of gastrointestinal hemorrhage in the hospitalized patients were necrotizing enterocolitis (44%), anal fissures (23%), and guaiac-positive stools of unknown cause (16%). The mortality rate was 5.4%. Necrotizing enterocolitis was the most common cause of death. Only 16 patients required surgery. Gastrointestinal hemorrhage is an infrequent cause of mortality and morbidity in hospitalized pediatric patients. PMID- 8614875 TI - Headache prevention in retrosigmoid vestibular nerve section. AB - Most disorders of the inner ear resulting in vertigo can be managed medically with good results. Meniere's disease, one of the more common causes of recurring vertigo, does not respond adequately to medical management in 5% of the cases. These patients become candidates for surgical management. Selective vestibular nerve section has become the procedure of choice when one desires to preserve residual hearing. This paper reviews 22 cases of vestibular nerve section via a retrosigmoid approach. The disease entities treated, treatment results, operative morbidity, and complications are tabulated. The surgical technique is discussed in detail. The goals in reviewing these cases were to evaluate the impression that the incidence of severe and/or prolonged postoperative headache was infrequent and to compare surgical results with those reported by others in the literature. PMID- 8614876 TI - Autopsy evaluation of asbestos exposure: retrospective study of 135 cases with quantitation of ferruginous bodies in digested lung tissue. AB - In recent years, there has been increased interest in documenting asbestos exposure. The pathologic sine qua non of asbestos exposure has been the presence of "asbestos bodies" in lung parenchyma. In this retrospective study of 135 autopsies done to determine asbestos exposure, ferruginous bodies in digested lung tissue were quantitated by a simplified sodium hypochlorite procedure on fresh or fixed tissue. Of the 131 digested specimens, 26 (20%) showed no ferruginous bodies, 34 (26%) had <5 ferruginous bodies per slide, 7 (5%) had 5 to 10 ferruginous bodies per slide, and 64 (49%) had >10 ferruginous bodies per slide. Ferruginous bodies were identified in hematoxylin-eosin stained sections of lung tissue in only 41 cases (30%). The digestion method described is a simple, reliable, and inexpensive method to assess ferruginous bodies. PMID- 8614877 TI - Low incidence of hemodynamic instability in patients with gastrointestinal hemorrhage. AB - Patients with gastrointestinal hemorrhage are frequently admitted to critical care units, in large part to be observed for signs of hemodynamic instability. All patients admitted with gastrointestinal bleeding to our medical intensive care unit over a 1-year period (n=108) were retrospectively reviewed to determine the incidence of hemodynamic instability. In an elderly patient population with predominantly nonvariceal bleeding, only 13% of those admitted had documented hypotension that led to an intervention. Only 7% had clinically significant hypotension after the first 5 hours of admission. Admission clinical criteria were analyzed by multivariate analysis but could not reliably predict patients at increased risk for hemodynamic instability. However, patients without significant comorbid illness who have been endoscopically shown to have a low-risk lesion can be considered for early transfer to a regular bed after a short period of close observation. This could lead to better resource utilization and cost savings without jeopardizing patient care. PMID- 8614878 TI - Optimization of radiation dose and image quality in mammography: a clinical evaluation of rhodium versus molybdenum. AB - Mammography was done with a conventional molybdenum anode/filter combination and either a molybdenum/rhodium (Mo/Rh) or rhodium/rhodium (Rh/Rh) combination to compare the contrast characteristics and penetrating ability of the different techniques. Each pair was rated as being equal, one slightly better than the other, or one much better than the other. The "preferred" image was also selected. The entrance surface exposure and the mean glandular dose were determined for each exposure. Mo/Mo was compared with Mo/Rh in 26 patients and to Rh/Rh in 23. Mo/Mo was judged to produce better or equal contrast and penetration in all cases except one using the Mo/Rh combination. Mo/Rh was the preferred image only once and Rh/Rh not at all. In comparison to the conventional Mo/Mo technique, the average dose was reduced by approximately 25% with Mo/Rh and by 50% with Rh/Rh. PMID- 8614879 TI - Firearm-related hunting fatalities in North Carolina: impact of the 'Hunter Orange' Law. AB - In the decade spanning 1983 through 1992, 68 people were killed by firearms while hunting in North Carolina (average of 1.66 fatalities/100,000 licenses issued). Of these, 58 deaths involved two parties, a shooter and a victim. In 22% of the incidents the victims were mistaken for game. During the 1987-1988 hunting season a "Hunter Orange" law was initially enforced. This law requires sportsmen to wear an article of bright orange clothing while hunting. After enactment of this law, a reduction in the incidence of hunters being killed because they were "mistaken for game" has proven statistically significant. The present study documents that legally mandating bright orange clothing has resulted in fewer firearms-related fatalities due to the victim's being mistaken for game while hunting. The North Carolina experience implies that governmental intervention can influence the incidence of accidental deaths during recreational hunting. PMID- 8614880 TI - Diagnostic value of clinical examination and various imaging techniques for breast implant rupture as determined in 81 patients having implant removal. AB - To determine sensitivity and specificity of magnetic resonance imaging (MRI) and ultrasonography (US) in the detection of breast implant rupture, and also to determine the relative merits of clinical examination and mammography, we studied 81 patients (160 implants). All patients had implants removed, thus allowing confirmation of the presence or absence of rupture. Clinical examination positively identified only one patient with implant rupture, and mammography detected only two implant ruptures (both extracapsular). The sensitivity for US was 70% and specificity was 90%, while for MRI it was 75.6% and 94%, respectively. These differences between MRI and US were not statistically significant. Combining the results of US and MRI did not seem to add to the diagnostic discrimination. The most cost-effective method of diagnosing implant rupture was US in our study. PMID- 8614882 TI - Pneumocystis carinii pneumonia with an atypical granulomatous response. AB - Pneumonia due to Pneumocystis carinii may present atypical radiographic and pathologic findings. We report the case of a 32-year-old man with acquired immune deficiency syndrome (AIDS) whose chest roentgenogram showed a bilateral diffuse nodular pattern suggestive of miliary tuberculosis. Diagnosis ultimately required open lung biopsy, which showed evidence of an atypical granulomatous reaction with Pneumocystis carinii in the absence of other organisms. P carinii pneumonia must be considered in the differential diagnosis of patients with AIDS with nodular pulmonary densities and evidence of an atypical granulomatous reaction on pathologic specimens. PMID- 8614881 TI - The oldest reported patient with fibromuscular dysplasia of the renal artery. AB - An 80-year-old woman was brought to the emergency department with accelerated hypertension. Multiple medical regimens failed, including an angiotensin converting enzyme inhibitor, which caused acute renal failure. Results of a renal scan for captopril were negative; renal arteriogram revealed fibromuscular dysplasia of the right renal artery. Percutaneous transluminal angioplasty was successful in dilating the renal artery and making blood pressure easier to control. Although more common in younger people, fibromuscular dysplasia should be considered in patients who are elderly and have hypertension that is difficult to control. Transluminal angioplasty should be attempted to preserve renal function and control blood pressure. PMID- 8614883 TI - Early recognition of risk factors for persistent effects of vecuronium. AB - Renal failure and hepatic failure are important risk factors for persistent effects of vecuronium. We report a case in which vecuronium therapy after cardiac arrest led to prolonged paralysis due to severe renal and hepatic dysfunction not apparent until after vecuronium therapy. Biochemical and electrophysiologic examinations confirmed that neuromuscular blockade due to accumulation of one active metabolite of vecuronium was the primary cause of the patient's paralysis. This case highlights the importance of recognizing progressive renal and hepatic dysfunction after cardiac arrest when contemplating treatment with vecuronium. PMID- 8614884 TI - Spontaneous resolution of a lung mass due to infection with Mycobacterium terrae. AB - A 61-year-old smoker had a large right lung mass suggestive of bronchogenic carcinoma. Bronchoscopy showed what appeared to be tumor occlusion of a right middle lobe subsegment. Biopsies were nondiagnostic for malignancy. A scheduled percutaneous needle aspiration biopsy 1 week later was canceled because of a significant reduction in size of the lung mass. Acid-fast bacilli were subsequently found in bronchoscopic specimens, and treatment was started with isoniazid, rifampin, pyrazinamide, and ethambutol. The organism was identified as Mycobacterium terrae. In vitro resistance was noted to isoniazid. The early reduction in size of the mass before antimycobacterial therapy suggested that spontaneous regression may be part of the natural history of pulmonary infection with Mycobacterium terrae. PMID- 8614885 TI - Persistent pain associated with long-term intrathecal morphine administration. AB - A 32-year-old man with chronic intractable right lower extremity pain unresponsive to multiple neurosurgical and pharmacologic treatments, including intrathecal morphine administration, was successfully treated with sciatic nerve block, discontinuance of opioid therapy, and psychologic interventions. Plasma and urine ratios of morphine metabolites morphine-3-glucuronide and morphine-6 glucuronide were analyzed at the beginning of our interventions, and the results indicated that morphine-3-glucuronide levels were significantly higher than morphine-6-glucuronide levels. The possible association between the observed morphine metabolite ratio and the intractable pain in patients resistant to opioids may have potential clinical implications. PMID- 8614886 TI - Successful double bypass grafting in a child with coronary artery obstruction due to Kawasaki disease. AB - We describe the clinical evaluation and surgical treatment of a 7-year-old child who had severe coronary artery obstructions that occurred as a sequela of previously diagnosed Kawasaki disease. PMID- 8614887 TI - Chronic myeloid leukemia manifested during megakaryoblastic crisis. AB - We describe a 38-year-old man with a chronic myeloproliferative syndrome characterized by elevated white blood cell and platelet counts and increased blasts in the peripheral blood. Bone marrow aspiration was a "dry tap" and the biopsy specimen was hypercellular with numerous blasts, atypical megakaryocytes, and increased reticulin fibrosis. The blasts exhibited cytochemical reactivity for nonspecific esterase and PAS and immunohistochemically were positive for factor VIII, supporting megakaryoblastic lineage. Cytogenetic studies of peripheral blood revealed the t(9;22)(q34;q11). We interpreted these findings to be most consistent with chronic myeloid leukemia (CML) manifested at the time of megakaryoblastic crisis. Although the initial complete blood count showed leukocytosis and thrombocytosis, the patient subsequently had pancytopenia with clinical and pathologic findings consistent with acute myelofibrosis (AMF). Cytosine arabinoside and etoposide chemotherapy induced remission of the acute leukemia. We conclude that CML infrequently presents itself in megakaryoblastic crisis and that such cases may result in the clinicopathologic syndrome of AMF. The success of chemotherapy in this case also suggests that intensive antileukemic therapy may be useful in other patients with either CML-blast crisis or the clinicopathologic syndrome of AMF. PMID- 8614888 TI - Laryngoceles and saccular cysts. AB - Laryngoceles and saccular cysts are related, benign abnormalities of the larynx. The pathophysiology of both lesions can involve congenital as well as acquired factors, and the appearance of both may range from incidental findings on laryngoscopy to symptoms such as hoarseness, dysphagia, dyspnea, and laryngeal discomfort. The surgical techniques used for treatment of laryngoceles and saccular cysts are controversial. We present two cases for comparison and discuss the history, diagnosis, and surgical treatment of laryngoceles and saccular cysts. Emphasis is on the surgical approach, which in both cases transected the thyrohyoid membrane externally and provided excellent visualization and exposure without significant morbidity. PMID- 8614889 TI - Cutaneous lesions due to Pleurophoma (Phoma) complex. AB - We report a case of striking and disfiguring facial mycosis due to an organism belonging to the Pleurophoma complex of Coelomycetes. This condition was likely acquired through repeated contact with infected soil and exacerbated by prolonged applications of topical corticosteroid preparations. Antifungal drug susceptibility testing was used to direct a therapeutic choice in this unusual infection. PMID- 8614890 TI - Massive asymmetric virginal breast hypertrophy. AB - Massive unilateral virginal breast hypertrophy in a 13-year-old girl was treated by right breast reduction, with removal of 2,968 g of breast tissue. A year later, the patient had enlargement of the left breast, and contralateral reduction mammoplasty was done. Two-year follow-up has shown no further enlargement. This case lends itself to a discussion with regard to the differential diagnosis of unilateral breast hypertrophy in adolescence. True virginal gigantomastia is relatively rare. Surgical reduction mammoplasty plays a pivotal role in the treatment of massive virginal breast hypertrophy. PMID- 8614891 TI - Wegener's granulomatosis mimicking a sternal abscess. AB - Bone destruction as a manifestation of Wegener's granulomatosis has been reported, but these cases were limited to the head and face. We present a case in which a sternal abscess was the initial manifestation of Wegener's granulomatosis. We believe this is the first reported case of bone destruction due to Wegener's occurring in a location other than the head and face. PMID- 8614892 TI - Coxsackievirus-associated pancreatitis mimicking metastatic carcinoma. AB - The origin of idiopathic acute pancreatitis has not been well defined. Several enteroviruses, including coxsackievirus, have been implicated. We report the case of a 21-year-old woman with severe, acute pancreatitis with no classic risk factors. Initial evaluation failed to show choledocholithiasis. Her condition improved with conservative treatment, but symptoms recurred after resumption of oral intake. Computed tomography (CT) of the abdomen revealed an atypical pseudocyst and omental nodules suggestive of a metastatic malignancy. An acute coxsackievirus B5 titer delayed CT-guided omental biopsy. The patient showed improvement, but progression of the omental nodules prompted open laparotomy. No malignant or inflammatory process was identified. This is the first reported case of presumed viral pancreatitis causing reactive omental nodules. This case shows a benign process mimicking malignancy, requiring an extensive, potentially dangerous workup. It also emphasizes the need to consider viral causes for unusual presentations of common problems. PMID- 8614893 TI - Renal cell carcinoma and autoimmune hemolytic anemia. AB - A previously healthy man who became bedridden because of malaise, fatigue, and weakness was found to have an autoimmune hemolytic anemia (AIHA). In the course of his evaluation for the AIHA, he was found, coincidentally, to have a renal cell carcinoma. The AIHA was marginally responsive to therapy with corticosteroids, but it resolved promptly after excision of the cancer. This case represents probably a rarely observed association between a nonhematologic malignancy and autoimmune hemolytic anemia. PMID- 8614894 TI - Probable transfusion-associated graft-versus-host disease in a patient with tuberous sclerosis. AB - Acute transfusion-associated graft-versus-host disease (TA-GVHD) is rare but often fatal. Graft-versus-host disease that occurs as a complication of bone marrow transplantation is more common but has lower mortality. Features of TA GVHD are nonspecific and can be confused with viral infections and drug reactions. We present a case of probable TA-GVHD in a patient with tuberous sclerosis who survived and was subsequently given irradiated blood without any further adverse events. PMID- 8614895 TI - Adult respiratory distress syndrome: learning lessons from the past. PMID- 8614896 TI - "False-positive" factitious disorder by proxy. PMID- 8614897 TI - Poorly differentiated adenocarcinoma in a gastric hyperplastic polyp. PMID- 8614898 TI - Class II restorations in six different posterior composite resins: five-year results. AB - Class II restorations of six light cured posterior composite resin materials (the intermediates Occlusin, P 30, Fulfil, Profile and the microfine Heliomolar and Distalite) were followed for five years. The results from three years have been reported earlier (Lundin et al 1990). At baseline twenty-four dentists from seven different clinics of the Public Dental Health Services in the country of Bohuslan placed 247 Class II restorations on 213 patients of the ordinary clientele visiting the clinics. Before starting the operators were instructed and trained in performing the restorations according to a standardized clinical procedure. The restorations were evaluated, according to a specially designed assessment form using the USPHS criteria, after one week, three years and five years. Stone casts were used to quantitatively categorize the occlusal wear according to the Leinfelder method. After five years the failure rate (USPHS rating Charlie) was 27 restorations out of assessed 176, i.e. 15%. The most common reason for failure was secondary caries. Fulfil had the highest failure rate among the intermediate materials (Chi-Square, p < 0.05). Assessment of wear showed that Profile, compared to the other intermediate materials, had worn the most and the two microfine materials Heliomolar and Distalite, compared to all the intermediate materials had worn the least. This study confirms the results from three years, that clinically acceptable results can be obtained with posterior composite resin materials when used in a proper and scheduled manner. PMID- 8614899 TI - Children's dental health in Europe. Clinical calibration of dental examiners in eight EU countries. AB - An epidemiological investigation has been initiated from Sweden with the aim to study and compare dental health, dental treatment needs and attitudes to dental care in two well-defined age-groups, children of 5 and 12 years of age, in eight EU countries. To ensure comparability of the clinical registrations, data collection was preceded by clinical calibrations of the examiners from the participating countries. All the examiners participated in a workshop with initial calibration exercises. Agreement, expressed as sensitivity, was measured between the Swedish examiner acting as the reference examiner and each of the other examiners in turn, and assessed separately for the two age-groups. For DMFS/dmfs, agreement ranged from 44.3% to 82.2%. These results were discussed and where necessary the criteria were modified and/or made more stringent, so that they were clearcut and could be adhered to consistently. In a second calibration between the Swedish and the national examiner undertaken in each of the seven countries, the inter-examiner agreement (sensitivity) varied between 85.4% and 100%. The mean sensitivity for DMFS/dmfs after the total calibration procedures was 89.5% for the 12-year olds and 91.7 for the 5-year olds. The mean sensitivity for both age-groups together was 90.6% and the corresponding value for specificity was 98.9%. PMID- 8614900 TI - A comparison of oral health in 70-year-old city cohorts in Umea northern Sweden in 1981 and 1990: oral problems, dental and periodontal status. AB - A comparison of oral health in two different samples of 70-year-old men and women living in the city of Umea in 1981 and 1990 showed that a higher frequency of dental visits among men could be expected in the city population in 1990 (95% CI). The frequency and pattern of reported oral problems was similar in 1981 and 1990. Total edentulousness among men and women in 1981/1990 was 31.3/21.4 and 53.5/35.7% respectively. The lower frequency in 1990 was not statistically significant on the population level. The mean number of teeth was lower in dentated men (12.8 +/- 6.0 V.S. 17.4 +/- 5.4) (p < 0.05) and women (16.9 +/- 6.3 V.S. 13.9 +/- 8.4) (n.s.) in 1990 compared with in 1981 and the functional index according to Eichner showed no difference. Thus, the lower frequency of edentulousness in 1990 mainly seemed to be the result of another distribution of about the same total amount of teeth, on more people. The bleeding index showed values around 30% in both cohorts. Both men and women showed less recurrent caries in 1990 (p < or = 0.05). Men showed a higher number of missing teeth (p < or = 0.01) and of decayed root surfaces (p < or = 0.05) in 1990. The latter might be explained by a larger exposed root surface area which was indicated by a higher frequency (n.s.) of surfaces with an attachment level > 3 mm in the cohort examined in 1990. Although, the compared 70-year-old city cohorts in Umea examined in 1981 and 199 showed a higher frequency of dental visits and of dentated subjects, no improvement in reported oral health or dental status could be found in the 70-year-old in Umea in 1990. PMID- 8614901 TI - Removable partial denture design habits in general dental practice in Sweden. AB - An investigation was made concerning Swedish general dental practitioners' habits in removable partial denture design and their opinion of the undergraduate training programme in this matter. The investigation showed that many dentists failed to provide adequate prescriptions and that they relied heavily on the dental technician for partial dental design. A clear majority of the participants, educated at Umea dental school, considered the undergraduate training programme to be adequate, whilst a similar percentage educated at Gothenburg dental school was dissatisfied. The faculties in Stockholm and Malmo were placed between Umea and Gothenburg in this respect. The majority of the participants in this survey declared that the opportunity to attend small group postgraduate courses in the subject was desirable. A prerequisite for dentists to have control of the treatment they are responsible for, is that they at least have the necessary knowledge. This knowledge may have to be achieved after qualification instead of during their undergraduate training. PMID- 8614902 TI - Is caries prevalence underestimated in today's caries examination? A study on 16 year-old children in the county of Bohuslan, Sweden. AB - When caries prevalence was higher than today incipient lesions constituted 70% of carious lesions. Incipient lesions were often neglected in caries recordings and hence the true caries prevalence was underestimated. Moreover, it has been shown that there were evident differences in caries status between different areas in the same county though the preventive measures were supposed to be the same (Moberg Skold et al 1984). To find out if the conditions were the same when caries prevalence has decreased a total number of 240 16-year-old children were examined concerning caries in 1987 and 1990. The results showed that the caries prevalence was still underestimated. Incipient lesions constituted 80% of the total number of carious lesions which was approximately the same as when caries prevalence was higher. The difference in caries status within the country had decreased compared with the study in 1984 (Moberg Skold et al 1984). PMID- 8614903 TI - Number of applicants and examination frequency at the dental schools in Sweden 1968-1992. AB - The number of applicants to the dental schools in Sweden 1968-1987 has been documented. It was shown that there was a large number of applicants up to 1980. Then the number dropped dramatically. It was at its lowest in 1985 but increased slowly after this date. The number of applicants was related to other variables: the birth rate, education capacity, real wage and unemployment. A correlation and time series analysis shows that only real wage has an impact on the number of applicants. The number of applicants has also been related to the examination rate. The results show that the examination rate decreases when the application rate is going down. PMID- 8614904 TI - Uptake of 203Hg2+ in the olfactory system in pike. AB - Inorganic mercury (203Hg2+) was applied to the olfactory chambers or was given i.v. to pike (Esox lucius) and the uptake of the metal in the olfactory system and the brain was examined by autoradiography and gamma spectrometry. Application of 203Hg2+ in the olfactory chambers resulted in an accumulation of the metal in the olfactory nerves and the anterior parts of the olfactory bulbs of the brain. The levels of 203Hg2+ in other brain areas, such as the telencephalon, the optic tecti and the cerebellum, remained low. Application of 203Hg2+ in only one olfactory chamber resulted in an uptake of the metal only in the ipsilateral olfactory nerve and olfactory bulb. Intravenous injection of the 203Hg2+ resulted in a labelling of the olfactory system and the brain, which was much lower than of the blood. These results indicate that the 203Hg2+ is taken up in the olfactory neurones from the olfactory receptor cells in the olfactory rosettes and is transported to the terminal parts of the olfactory neurones in the olfactory bulbs. The uptake of mercury as well as some other metals in the olfactory system may result in noxious effects and this may be an important component in the toxicology of metals in fish. PMID- 8614905 TI - Psychological and somatic subjective symptoms as a result of dermatological patch testing with metallic mercury and phenyl mercuric acetate. AB - Sixty patients with a history of malaise over the ensuing weeks following the drilling out of old amalgam fillings were included in the study. They were tested epicutaneously weekly (standard procedure) with either 0.5% metallic mercury in petrolatum or 0.01% phenyl mercuric acetate in water, and, on 2 separate occasions, with only saline or petrolatum as a control according to a randomized double-blind protocol. The presence or absence of an allergic patch test response was read on day 3. Two patients showed allergic cutaneous responses towards metallic mercury and 1 to phenyl mercuric acetate. There was a concurrent 7-day self-registration of subjective psychological and somatic symptoms, using a validated visual analogue scale (minor symptom evaluation profile; MSE). In the group analysis it was clearly shown that the patients reacted with subjective symptoms to phenyl mercuric acetate. A reaction to test doses of metallic mercury seems to exist but could only be visualized when a scoring system was elaborated to individually define those subjects with a psychological and somatic response to test doses of mercury. This psychosomatic reactivity, named intolerance, seems to be unrelated to the cutaneous delayed allergic skin response. Thus, it might be possible to identify patients intolerant to small test doses of percutaneously penetrating mercury (previously considered innocuous). These findings may have a bearing on the systemic side-effects attributed to mercury released from amalgam tooth fillings. PMID- 8614907 TI - Expression of heat shock protein 27 in developing and adult human kidney. AB - The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state. PMID- 8614906 TI - Effects of dicoumarol on cytotoxicity caused by tert-butylhydroquinone in isolated rat hepatocytes. AB - The effects of dicoumarol, an inhibitor of DT-diaphorase, on the cytotoxicity of tert-butylhydroquinone (tBHQ) were studied in freshly isolated rat hepatocytes. Addition of tBHQ (0.5 mM) to hepatocytes resulted in a time-dependent cell death accompanied by depletion of intracellular ATP, glutathione (GSH), and protein thiols. Pretreatment of hepatocytes with dicoumarol (30 microM) did not affect cell viability or cellular levels of ATP, GSH, or protein thiols during the incubation period; however, dicoumarol did promote the appearance of cell blebs and the depletion of ATP and protein thiols induced by tBHQ and ultimately enhanced the cytotoxicity of tBHQ. PMID- 8614908 TI - Benzo[a]pyrene and nicotine alter prostaglandin E2 receptor and its functions in hamster buccal mucosa. AB - This study investigated the in vivo effect of benzo[a]pyrene (BP) and nicotine (NC) on prostaglandin E2 (PGE2) receptor and its cellular responses in hamster buccal mucosa. Adult male Syrian hamsters were treated by apical swabbing the pouch with mineral oil (control), or 1 mM BP + NC, twice/day, 3 times/week, for 8 weeks. The BP + NC treatment significantly decreased the specific binding of PGE2 to buccal mucosal membranes (12.8 +/- 0.14 vs 8.7 +/- 1.13 fmol/mg protein; control vs. BP + NC; mean +/- S.D., n=6, P < 0.05). Scatchard analysis revealed that the reduction of receptor binding might be due to an alteration of the receptor binding properties, such as increase of the dissociation constant (Kd, 1.08 vs. 4.20 nM; control vs. BP + NC) and elimination of the low-affinity binding site. The addition of 16,16-dimethyl PGE2 (DMPGE2) into the control buccal tissue caused a 36% decrease of the overall protein synthesis. In contrast, DMPGE2 enhanced the [35S]methionine incorporation into several major proteins (i.e., 150, 120, 90, 75, 55, 38 and 26 kDa) in the BP + NC group. DMPGE2 showed no effect on DNA synthesis in both groups. Our data demonstrated that BP + NC in vivo altered PGE2 receptor properties and cellular functions in hamster buccal pouch. PMID- 8614909 TI - Platelet activating factor receptor blockade alone or in combination with leukotriene synthesis inhibition does not ameliorate alpha-naphthylisothiocyanate induced hepatotoxicity. AB - Alpha-naphthylisothiocyanate (ANIT) is a cholangiolitic hepatotoxicant that causes periportal edema, hepatic parenchymal and biliary epithelial cell necrosis, and cholestasis in the rat. A hallmark of ANIT hepatotoxicity is periportal inflammation that includes neutrophil infiltration. Neutrophils are requisite for the expression of ANIT-induced liver injury; however, the mechanism(s) of neutrophil accumulation in the liver and the role of these cells in ANIT hepatotoxicity is incompletely understood. Platelet activating factor (PAF) is a proinflammatory agent that is both a chemoattractant for and an activator of neutrophils. Therefore, we evaluated the role of PAF in ANIT-induced liver injury. Rats were treated with the PAF receptor antagonist, WEB 2086 (WEB), to determine if it afforded protection from ANIT hepatotoxicity. In a separate study, a combination of WEB and the leukotriene synthesis inhibitor, Zileuton (ZIL), was used to address the possible interaction of PAF and leukotrienes in ANIT-induced liver injury. Treatment of rats with WEB, alone or in combination with Zileuton, did not attenuate ANIT-induced liver injury as assessed by increases in plasma alanine aminotransferase or gamma-glutamyl transferase activities. In addition, neither treatment ameliorated ANIT-induced cholestasis assessed as increased plasma bilirubin concentration. These results suggest that PAF, alone or in combination with products of 5-lipoxygenase, does not contribute to ANIT-induced liver injury. PMID- 8614910 TI - The detection of S-glutathionation of hepatic carbonic anhydrase III in rats treated with paraquat or diquat. AB - Protein S-glutathionation has been demonstrated to be one of the cellular responses under oxidative stress and may be involved in many cellular metabolisms. In this study, the effect of redox cycling bipyridylium compounds, paraquat and diquat, on this protein modification was investigated. Male Sprague Dawley rats were administered i.p. either paraquat at 20 or 40 mg/kg body wt. or diquat at 85 or 170 mg/kg body wt., respectively. The liver was examined at different time points for taking the measurement of the S-glutathionation of carbonic anhydrase III (CA III), thiobarbituric acid-reactive substances (TBARS), vitamin E depletion, glutathione (GSH) and glutathione disulfide (GSSG) contents. The extent of S-glutathionation of CA III was chosen as a marker and was determined by a method combining isoelectric focusing analysis with immunoblotting. Those results indicated that paraquat and diquat significantly increased the generation of TBARS and showed a time-dependent response. The significant effect on vitamin E depletion was only obtained in rats treated with a high dose of diquat for 2 h. Hepatic cellular GSSG contents did not increase but tended to decrease all of the treatments. Although oxidative damage was actually generated in liver, based on the increase of TBARS generation and vitamin E depletion, no increase of CA III S-glutathionation was observed. We propose that the reason for this observation under this circumstance is probably due to the reversible characteristic of CA III S-glutathionation, which has been demonstrated in our previous study (Chai et al., 1991) Arch. Biochem. Biophys. 384, 270-278) and named as dethiolation. PMID- 8614911 TI - Aspirin wars: the optimal dose of aspirin prevent stroke. PMID- 8614912 TI - Aspirin dose in stroke prevention: beautiful hypotheses slain by ugly facts. PMID- 8614913 TI - Nimodipine neuroprotection in cardiac valve replacement: report of an early terminated trial. AB - BACKGROUND: We conducted a double-blind, randomized clinical trial in patients undergoing cardiac valve replacement to determine whether nimodipine, a dihydropyridine calcium antagonist, reduced the risk of new neurological, neuro ophthalmologic, or neuropsychological deficits-common complications associated with cardiac surgery-1 week after surgery. METHODS AND RESULTS: Enrollment for a total of 400 patients started in May 1992 and was stopped in September 1994, with 150 patients randomized to the study. Nimodipine was given to the patients during the perioperative period. Patients underwent examinations before surgery and at approximately 1 week, 1 month, and 6 months after surgery. Major adverse events, including deaths and strokes, were monitored monthly. The trial was terminated early because of both an unexpected disparity in death rates between groups and a lack of evidence of a beneficial effect of nimodipine. New deficits were observed in 72% of the placebo group versus 77% of the nimodipine group (p=.55). In the 6 month follow-up period, 8 deaths (10.7%) occurred in the nimodipine group (n=75) compared with 1 death (1.3) in the placebo group (n=74) (p=.02). Major bleeding occurred in 10 patients in the nimodipine group versus 3 in the placebo group (13.3% versus 4.1%; P=.04). Six (46.2%) of the 13 patients with major bleeding died compared with 3 deaths (2.2%) among the 136 patients without major bleeding. CONCLUSIONS: Our findings add to the growing evidence that calcium antagonists have a prohemorrhagic effect in some patients and suggest that nimodipine use should be restricted perioperatively in patients scheduled for cardiac valve replacement. PMID- 8614915 TI - Magnetic resonance imaging correlates of transient cerebral ischemic attacks. AB - BACKGROUND AND PURPOSE: MRI of patients with a transient ischemic attack (TIA) may provide more detailed morphological insights than CT. We therefore studied the frequency and type of TIA-related infarcts shown by MRI, examined the utility of intravenous contrast material, and searched for potential predictors of infarct occurrence. METHODS: We performed 1.5-T MRI of the brain of 52 patients (age range, 28 to 93 years; mean, 61 years) with a hemispheric TIA. Contrast material (Gd-DTPA) was given to 45 individuals. We recorded type, number, size, and location of ischemic brain lesions and related the presence of acute infarction to features of clinical presentation and probable causes for the TIA. RESULTS: MRI showed focal ischemic lesions in 50 patients (81%), but an acute TIA associated infarct was seen in only 19 subjects (31%). In patients with an acute lesion, the infarcts were smaller than 1.5 cm in 13 (68%), purely cortical in 11 (58%), and multiple in 7 (37%) individuals. Contrast enhancement contributed to the delineation of an acute lesion in only 2 of 45 patients (4%). Acute infarction was unpredictable by clinical TIA features, but the frequency of identifiable vascular or cardiac causes was significantly higher in those patients with TIA-related morphological damage (odds ratio, 5.2 [95% confidence interval, 1.6 to 17.3]). CONCLUSIONS: More than two thirds of TIA patients showed no associated brain lesion even when MRI and contrast material were used, but the overall frequency of ischemic damage was high. TIA-related infarcts on MRI were mostly small and limited to the cortex and tended to consist of multiple lesions. A positive MRI underscores the need for comprehensive diagnostic workup since evidence of infarction appears to be associated with a higher frequency of significant vascular or cardiac disorders. PMID- 8614914 TI - Prolonged persistence of substantial volumes of potentially viable brain tissue after stroke: a correlative PET-CT study with voxel-based data analysis. AB - BACKGROUND AND PURPOSE: The existence in humans of brain tissue at risk for infarction but potentially viable (eg, the penumbra) remains unproven. One retrospective operational definition of such tissue includes its final infarction despite a relatively preserved or even normal cerebral metabolic rate of oxygen (CMRO2) in the early hours after stroke onset. Although previous positron emission tomography (PET) studies identified tissue whose CMRO2 declined from the acute to the subacute stage, in principle compatible with deteriorating penumbra, they all lacked a coregistered CT scan mapping of final infarct and an objective three-dimensional PET data analysis, while many patients were studied in the subacute (up to 48 hours) phase. We have evaluated whether tissue with CMRO2 ranging above a threshold for presumably irreversible damage in the first 18 hours of middle cerebral artery territory stroke, but below it in the chronic stage, could be retrospectively identified within the final infarct volume. METHODS: Our data bank comprises 30 consecutive patients with first-ever middle cerebral artery territory stroke prospectively studied with PET within the first 18 hours after clinical onset; the 15O equilibrium method was used to measure cerebral blood flow and CMRO2. All survivors with the following criteria were eligible for the present study: (1) technically adequate chronic-stage PET performed in the same stereotaxic conditions, (2) coregistered CT scan also performed in the chronic stage, and (3) an infarct of sufficient dimension (>16mm diameter) on late CT. Corresponding CT scan cuts and PET slices were exactly realigned, and the outlines of CT hypodensities were superimposed on the corresponding CMRO2 matrix. Infarcted voxels with CMRO2 values less than or greater than 1.40 mL/100 mL per minute (ie, the generally accepted threshold for irreversible damage) were automatically identified and projected on matrices of all other PET parameters and for both PET studies. RESULTS: Eight patients (mean age, 78 Years) were eligible for the present study. The acute-stage PET study was performed 7 to 17 hours after stroke onset and the chronic-stage PET 13 to 41 days later. Within the final infarct, mean CMRO2 fell significantly from the acute- to the chronic-stage PET study (P<.001). Eventually infarcted voxels with acute-stage CMRO2 values above the threshold were found in each of these eight patients; they were most often situated near the infarct borders and constituted 10% to 52% (mean, 32%) of the final infarct volume. The acute-stage CMRO2 in these voxels ranged up to 4.13 mL/100 mL per minute but fell below 1.40 mL/100 mL per minute in 93% of them at the chronic-stage PET. in 7 of 8 patients the acute stage mean cerebral blood flow ranged from 10 to 22 mL/100 mL per minute, and the mean oxygen extraction fraction was markedly increased (>0.70) in these voxels, consistent with a penumbral state. CONCLUSION: In a strictly homogeneous sample of prospectively studied patients, we have identified, up to 17 hours after stroke onset, substantial volumes of tissue with CMRO2 well above the assumed threshold for viability that nevertheless spontaneously evolved toward necrosis. This tissue exhibited penumbral ranges of both cerebral blood flow and oxygen extraction fraction and thus could represent the part of penumbra that might be saved with appropriate therapy. PMID- 8614917 TI - Cerebral blood flow alteration by acetazolamide during carotid balloon occlusion: parameters reflecting cerebral perfusion pressure in the acetazolamide test. AB - BACKGROUND AND PURPOSE: We attempted to clarify the role of the acetazolamide reactive mechanism in cerebral hemodynamic autoregulation and to establish a useful method of estimation using the acetazolamide test. METHODS: We examined 18 patients whose cerebral hemodynamics were considered to be normal and whose cerebral blood flow (CBF) was maintained during the balloon occlusion test (BOT) of the internal carotid artery. We measured the mean stump pressure (MSTP) and the mean CBF in the middle cerebral arterial territory using a xenon-enhanced CT system during BOT with and without acetazolamide activation. We obtained the asymmetry ratio (AR=occluded CBF/contralateral CBF) and the increased CBF parameters caused by acetazolamide activation expressed as an absolute value (delta CBF) and a percentage (%delta CBF) for the occluded side. RESULTS: AR during BOT with and without acetazolamide activation differed significantly (P<.001, paired t test) despite the lack of significant MSTP changes. Furthermore, although there was no significant correlation between MSTP and AR without acetazolamide activation, a positive significant correlation was detected with acetazolamide activation (r=.634, P=.005, linear regression analysis). There were significant correlations between delta CBF and MSTP (r=.574, P=.013) and %delta CBF and MSTP (r=.640, P=.004). CONCLUSIONS: We consider that the acetazolamide-reactive mechanism functions as autoregulation at the lower end of the autoregulatory range. The acetazolamide test, using %delta CBF or delta CBF as parameters (which both directly reflect MSTP), is useful for estimating the cerebral perfusion pressure decrease and presence of hemodynamic compromise. PMID- 8614916 TI - Improvement in cerebral hemodynamics after carotid angioplasty. AB - BACKGROUND AND PURPOSE: Carotid percutaneous transluminal angioplasty ( PTA) may offer an alternative treatment to carotid endarterectomy. However, in contrast to carotid endarterectomy, which has been shown to normalize impaired cerebral hemodynamics, the effects of carotid PTA are unknown. Therefore, we prospectively studied the effect of carotid PTA on both perioperative and postoperative cerebral hemodynamics. METHODS: Eleven patients undergoing carotid PTA for symptomatic carotid artery stenosis were prospectively studied. Transcranial Doppler recordings from the ipsilateral middle cerebral artery (MCA) were performed during the procedure. In addition, MCA blood flow velocity and CO2 reactivity were determined before PTA and at 2 days, 1 month, and 6 months after procedure. The results were compared with those in 11 similar patients undergoing carotid endarterectomy in whom measurements were performed before and 1 month after the operation. RESULTS: During carotid PTA, in 2 of 11 patients during passage of the balloon catheter through the stenosis, MCA blood flow velocity fell transiently. In 6 of 11 patients there was a reduction in flow velocity (>50%) during balloon deflation, but this lasted only a few seconds. After the procedure there was a significant improvement in ipsilateral hypercapnic reactivity: preoperative value, 59.8+/-42.2% (mean+/-SD); 2 days, 77.9+/-31.4%; 1 month, 88.7+/-45.0%; 6 months, 89.8+/-33.9%; and (ANOVA P=.003) preoperative value versus 1 month, P<.02; versus 6 months, P<.02. In all cases in which reactivity was significantly impaired preoperatively, it returned to the normal range. Pulsatility index also increased significantly: preoperative value, 0.827+/-0.251 (mean+/-SD); 2 days, 0.992+/-0.262 (P=.002). Contralateral MCA hypercapnic reactivity also improved after carotid PTA. There was a similar improvement in ipsilateral hypercapnic reactivity after carotid endarterectomy. CONCLUSIONS: Carotid PTA results in a normalization of impaired hemodynamics, as assessed by CO2 reactivity. The degree of improvement is similar to that seen after carotid endarterectomy. PMID- 8614918 TI - Recurrent spontaneous arterial dissections: risk in familial versus nonfamilial disease. AB - BACKGROUND AND PURPOSE: Among patients with spontaneous cervical artery dissections, the risk of recurrent arterial dissection is relatively low at 1% per year, but this risk may be higher for patients with a family history of arterial dissections. We compared the risk of a recurrent arterial dissection in patients with familial versus non familial disease. METHODS: Long-term follow-up was established in 200 patients (104 women and 96 men with a mean age of 44.9 years) with spontaneous cervical artery dissections evaluated at a single institution between 1970 and 1990. RESULTS: Among the 200 patients, 10 (5%) were identified who had a family history of spontaneous arterial dissections. In a multivariate analysis, family history was the only significant variable associated with the risk of recurrent dissection (X2=15.51; P=.0001). A recurrent arterial dissection was identified in 5 (50%) of the 10 patients with familial disease compared with 11 (5.8%) of the 190 patients with nonfamilial disease, with an estimated relative risk of 6.3 (95% confidence interval, 2.2 to 18.3; P=.0007). CONCLUSIONS: Among patients with spontaneous cervical artery dissections, a family history of arterial dissection is an important risk factor for the development of a recurrent arterial dissection. PMID- 8614919 TI - Incidence of subarachnoid hemorrhage: role of region, year, and rate of computed tomography: a meta-analysis. AB - BACKGROUND AND PURPOSE: The incidence of subarachnoid hemorrhage (SAH) has been estimated for many years at 10 to 15 per 100 000 person-years, but the most recent studies yield lower figures, of 6 to 8 per 100 000 person-years. To investigate the cause of this apparent decline, we studied the influence of year to study, rate of CT,and region. RESULTS: Eighteen studies fulfilled predefined inclusion criteria. In three Finnish studies, the pooled incidence was 21.4 per 100 000 person-years (95% confidence interval [CI], 19.5 to 23.4); in 15 non Finnish studies, it was 7.8 per 100 000 person-years (95% CI, 7.2 to 8.4). With univariate analysis, in non-Finnish studies the incidence decreased .96% for each percentage point increase of patients investigated with CT (rate ratio, -0.9904; 95% CI, 0.9878 to 0.9930). With 100% CT scanning, the incidence of SAH outside Finland is estimated at 6 per 100 000 person-years. The rate ratio for year of study was 0.952 (95% CI, 0.935 to 0.969) for each later year in the period 1960 to 1994. In multivariate analysis, only the use of CT was independently related to SAH incidence. For the Finnish studies, the rate ratios for use of CT and year to study were not statistically significant. We also found in six studies that incidence for women was 1.6 (95% CI, 1.1 to 2.3) times higher than that for men (7.1 [95% CI, 5.4 to 8.7] and 4.5 [95% CI, 3.1 to 5.8], respectively). CONCLUSIONS: The actual incidence of SAH has remained stable over the last three decades; the apparent decline in incidence is entirely explained by the greater proportion of patients investigated with CT. The incidence of SAH in Finland is almost three times as high as in other parts of the world. PMID- 8614920 TI - Validation of family history in subarachnoid hemorrhage. AB - BACKGROUND AND PURPOSE: In 6% to 9% of patients with subarachnoid hemorrhage (SAH), familial aggregation occurs; truly familial cases carry a worse prognosis than sporadic cases and raise the question of screening. If relatives have died from SAH, the family history is often the only available clue to the diagnosis, but the sensitivity and predictive value of such a history for SAH are unknown. METHODS: We contacted a next of kin for a consecutive series of patients who had died in the hospital of subarachnoid hemorrhage (n=20), intracerebral hemorrhage (n=22), or ischemic stroke (n=23) between 3 and 5 years previously, and we compared the diagnosis based on the history from this next of kin with the medical diagnosis confirmed by a CT scan. RESULTS: The positive predictive value of the diagnosis of "probable SAH" from the history in our study sample was 0.7; when adjusted for incidence rates in the general population it was 0.6 (95% confidence interval, 0.3 to 0.8). The sensitivity of the diagnosis based on the history was 0.5 (95% confidence interval. 0.3 to 0.7); 10 of the 20 cases of SAH were not identified. CONCLUSIONS: The family history of SAH, without confirmation from medical documents, is an insufficiently accurate tool to prove or disprove the diagnosis of familial SAH. PMID- 8614921 TI - Computed tomographic brain scans and antiplatelet therapy after stroke: a study of the quality of care in Dutch hospitals. AB - BACKGROUND AND PURPOSE: We sought to develop a measure ("quality weight" that indicates the severity of a deviation from optimal care with respect to secondary prevention with antiplatelet treatment after stroke. We also sought to estimate the effects that efforts to improve the quality of secondary prevention may have on health outcome and healthcare costs in the Netherlands. METHODS: First, we developed quality weights with decision analysis techniques. These quality weights express the excess risk of vascular events in the first 2 years after stroke compared with the optimal strategy (CT brain scan in all patients and aspirin in case of cerebral infarction). Second, these weights were applied in a follow-up study of 738 stroke patients older than 45 years. The number of stroke patients admitted to a hospital in 1991 in the Netherlands was used to estimate nationwide effects. We used data from 23 neurological departments and from the Information Center for Health Care in the Netherlands. RESULTS: The 2-year excess risk of fatal and nonfatal vascular events caused by omitting CT brain scan and giving aspirin to all patients is rather small (on average, 0.6%). The 2-year excess risk caused by not giving aspirin to a patient with cerebral infarction is much higher (4.1%). The follow-up study indicated that only 6% of the admitted patients had not been evaluated with a CT brain scan and that 14% of the patients with cerebral infarction proven by CT scan did not get antiplatelet treatment at discharge. Efforts to improve the quality of secondary prevention after stroke may prevent 74 vascular events annually in the Netherlands at an expense of 6200 Dutch guilders per prevented event (1 Dutch guilder=0.53 US dollar, 1991). CONCLUSIONS: Efforts to improve the quality of secondary prevention with antiplatelet treatment might reduce the number of new vascular events within the first 2 years after stoke by approximately 3%. The total costs related to the extra diagnostic and therapeutic activities are approximately 0.2% of the total annual hospital costs for acute stroke patients in teh Netherlands (250 million Dutch guilders). PMID- 8614922 TI - Results of a computerized screening of stroke patients for unjustified hospital stay. AB - BACKGROUND AND PURPOSE: Effective methods to monitor length of stay can help reduce unnecessary hospital stay without adversely affecting the quality of care. In this study a clinical algorithm for assessing unjustified hospital stay in stroke patients was computerized and tested. METHODS: An algorithm was developed by the authors to estimate the number of medically justified and unjustified hospital days for patients admitted with a primary diagnosis of ischemic stroke. Data for the algorithm were obtained from 177 stroke patients from an acute-care teaching hospital. The performance of the algorithm was evaluated on a subset of 46 patients by comparing the number of medically unjustified hospital days determined by the algorithm with the consensus determination of two neurologists. RESULTS: The algorithm classified 68% of the 177 patients as having some unjustified hospital days and 41% of all hospital days as unjustified. With the neurologists as the gold standard, the sensitivity of the algorithm was .89 and the specificity was .91. The correlation between the number of unjustified days determined by the algorithm and the neurologists was .76. CONCLUSIONS: There is considerable unjustified length of stay for stroke patients. Physicians can develop simple clinical algorithms for detecting unjustified hospital stay in stroke patients that provide a reasonable approximation of complex clinical judgment. PMID- 8614923 TI - Cigarette smoking is correlated with the periventricular hyperintensity grade of brain magnetic resonance imaging. AB - BACKGROUND AND PURPOSE: A new studies have observed a significant inverse correlation between cigarette smoking or lipid abnormalities and periventricular hyperintensities (PVHs) on T2-weighted magnetic resonance imaging (MRI) scans of the brain, which is surprising because smoking and hyperlipidemia are considered risk factors for cerebrovascular disease. We investigated the relation between smoking and lipid abnormalities and PVHs on T2-weighted MRIs. METHODS: MRI scans were performed in 253 patients over the age of 40 years, and PVHs were assessed retrospectively by use of a five-point scale. Patients who were receiving medical treatment for hyperlipidemia were excluded. Serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were determined in the fasting state by an automated enzymatic procedure. The low-density lipoprotein (LDL) cholesterol level was calculated by use of Friedewald's equation. Age, sex, hypertensive status, antihypertensive treatment, presence or absence of diabetes mellitus, and history of stroke were included in the analysis. RESULTS: Multiple linear regression analysis showed that age, hypertension, smoking, and antihypertensive treatment were significantly and independently correlated with the PVH score. The standard partial regression coefficients were .39 (P<.0001) for age, .33 (P<.0001) for hypertension, .16 (P=.0062) for smoking, and -.18 (P=.0124) for antihypertensive treatment. Hypercholesterolemia (total cholesterol level >220 mg/dL), HDL hypocholesterolemia (HDL cholesterol level <40 mg/dL, LDL hypercholesterolemia (LDL cholesterol level > 130 mg/dL), hypertriglyceridemia (triglyceride level >150 mg/dL), sex, diabetes mellitus, and a history of stroke were not correlated with the PVH score. CONCLUSIONS: Cigarette smoking was a weak but significant positive predictor of the PVH score and was independent of age, hypertension, and antihypertensive treatment. Lipid abnormalities were not related to the PVH score. PMID- 8614924 TI - Evaluation of intracranial and extracranial carotid steno-occlusive diseases in Taiwan Chinese patients with MR angiography: preliminary experience. AB - BACKGROUND AND PURPOSE: We attempted to evaluate the location of vascular lesions in cases of cerebrovascular steno-occlusive diseases in Chinese persons living in Taiwan. METHODS: With three-dimensional time-of-flight magnetic resonance angiography (MRA) as a screening tool, 108 symptomatic patients with cerebrovascular steno-occlusive diseases were examined. Cardioembolic disease and cerebral hemorrhage cases were excluded. The degrees of stenosis of bilateral cervical carotid arteries and their major intracranial tributaries were recorded. They were categorized as nonsignificant stenosis (0% to 49%), significant stenosis (50% to 99%), and total occlusion. RESULTS: Our data revealed that 32.4% of the cases were normal in either cervical carotid arteries or their intracranial tributaries. In 24.1% of the cases, significant extracranial carotid stenosis or occlusion was the only finding on MRA. In 25.9% of the cases, only significant intracranial-tributary stenosis was found. In 17.6% of them, significant lesions were found in both extracranial and intracranial carotid artery tributaries. CONCLUSIONS: A racial difference between Chinese and white patients in location of lesion in cerebrovascular steno-occlusive diseases was confirmed. About one third of symptomatic Chinese patients living in Taiwan showed small-vessel disease. Approximately 24% of patients had only extracranial carotid disease, and about 26% had only intracranial carotid tributary disease. We need a larger series of patients to confirm these findings. However, MRA might be a good screening tool for steno-occlusive cerebrovascular disease, especially in persons of a race with more intracranial carotid disease, such as the Chinese. PMID- 8614925 TI - Factors of carotid arterial enlargement in a population aged 59 to 71 years: the EVA study. AB - BACKGROUND AND PURPOSE: Factors of carotid arterial enlargement are not well known in general populations. The purpose of this study was to assess the cross sectional associations of arterial wall lesions and cardiovascular risk factors with carotid lumen diameter in elderly subjects. METHODS: B-mode ultrasound examinations of the carotid arteries and risk factor assessment were made in 1272 participants in the EVA (Etude sur le vieillissement arteriel) Study, a longitudinal study designed to evaluate vascular and cognitive aging in men and women aged 59 to 71 years. Ultrasound examinations included measurements of intima-media thickness (IMT) and interadventitial and lumen diameters of the common carotid arteries and quantification of atherosclerotic plaques in extracranial carotid arteries. RESULTS: Men showed greater IMT interadventitial and lumen diameters of the common carotid arteries than did women. In both sexes, common IMT and plaque score were positively associated with common interadventitial and lumen diameters. Stepwise multiple regression analysis showed that male sex, body height and weight, common IMT, plaque score, systolic blood pressure, and alcohol consumption were positively and independently related to lumen diameter. On the other hand, an independent negative association was observed between low density lipoprotein cholesterol and lumen diameter. CONCLUSIONS: In 59- to 71-year-old subjects, increased IMT and atherosclerotic plaques were accompanied by an increase in lumen diameter of the common carotid arteries, indicating an overcompensation. Luminal enlargement observed with several risk factors and with high blood pressure in particular might be partially counteracted by high lipid levels. PMID- 8614926 TI - Long-term prognosis of first-ever lacunar strokes. A hospital-based study. AB - BACKGROUND AND PURPOSE: Information concerning the long-term prognosis of lacunar strokes is still limited and has shown different results. The aim of this study was to investigate the long-term prognosis of first-ever lacunar strokes and the possible role of clinical prognostic factors and different pathogenic mechanisms. METHODS: Between March 1990 and November 1993, a cohort of consecutive patients presenting with first-ever lacunar infarcts was prospectively evaluated after stroke onset at day 0 to 3 and/or day 7, every 3 months up to 1 year, and every 6 months thereafter. All patients were studied according to a protocol that included demographic and clinical data, neurological examination, Toronto Stroke Scale, Barthel Index, Rankin Scale, CT scan, routine laboratory workup, electrocardiogram, carotid duplex scanning, and echocardiogram. More recently, patients have also been evaluated with transcranial Doppler ultrasonography. Recurrent strokes, myocardial infarction, and death were registered through direct observation, chart review, or interviews with the attending physician or family members. RESULTS: One hundred forty-five patients-94 (65%) with pure hemiparesis, 33 (23%) with sensorimotor stroke, 11 (8%) with ataxic hemiparesis, 5 (3%) with pure sensory stroke, and 2(1%) with dysarthria-clumsy hand syndrome were followed for a median period of 39 months. During follow-up ther were 17 deaths (3 vascular), 30 recurrent strokes (1 fatal), and 4 myocardial infarctions. Five-year survival rate free of recurrent stroke was 63% (95% confidence interval [CI], 52% to 73%), while 5-year survival rate was 86% (95% CI, 78% to 91%). Cox proportional hazards analysis showed that age (p=.02) was the only significant predictor of survival free of recurrent stroke. Age (P<.001) and the degree of neurological dysfunction and functional disability at 7 days after the index stroke measured by the Toronto Stroke Scale (P=.05) and a Barthel Index score <40 (P=.04) were the only significant predictors of death. The 5-year probability rate of stroke-free recurrence was 72% (95% CI, 60% to 81%). Sixty three percent of the first recurrent strokes were lacunar infarcts. When clinical, laboratory, and CT data as well as possible etiopathogenic mechanisms of lacunar strokes were considered, Cox proportional hazards analysis could not identify any predictor of stroke recurrence. CONCLUSIONS: Our study confirms that lacunar infarcts are associated with low stroke recurrence and mortality rates. In our series, the majority of first recurrent strokes were also lacunar infarcts. Age, degree of neurological dysfunction, and functional disability at day 7 after the index stroke were significant predictors of death. PMID- 8614927 TI - Incidence of transient ischemic attacks and minor ischemic strokes in Segovia, Spain. AB - BACKGROUND AND PURPOSE: The aim of this study was to determine the incidence of transient ischemic attacks (TIAs) and minor ischemic strokes (MISs) in Segovia, Spain. METHODS: A 2-year prospective community-based register of TIAs and MISs established in Segovia from February 16, 1992, to February 15, 1994. Every patient underwent underwent a complete clinical evaluation and cranial CT scan. Sex- and age-specific incidence rates with 95% confidence intervals (CIs) were calculated for all ages. RESULTS: The total series included 235 patients; 103 suffered TIAs and 132 suffered MISs. Mean age was 70.8 years (range, 29 to 96 years); 92 were women and 143 were men. The crude annual incidence was 0.80/1000 (95% CI, 0.70 to 0.90): 0.35/1000 (95% CI, 0.28 to 0.42) for TIAs and 0.45/1000 (95% CI, 0.37 to 0.53) for MISs. The incidence of TIAs and MISs increased with age. Approximately 78 of TIAs and MISs were in the carotid distribution, 19% were vertebrobasilar, and 3% were considered of uncertain vascular distribution. Cranial CT scan was performed in all patients. CT showed cerebral infarcts in 30.1% (31/103; 95% CI, 21% to 39%) of TIAs and 70% (92/132; 95% CI, 62% to 78%) of MISs (P<.00001). CONCLUSIONS: Our study is the first community-based register that provides sex-and age-specific rates for MISs and in which a CT scan was obtained in all patients. The incidence of TIAs in Segovia is comparable to that in other previous similar studies. PMID- 8614929 TI - Discriminative sensory dysfunction after unilateral stroke. AB - BACKGROUND AND PURPOSE: Although sensory deficits caused by stroke have been reported occasionally, dysfunction of discriminative sensation has seldom been studied in patients with unilateral stroke. The frequency and modality of bilaterally impaired discriminative sensation also remain to be clarified. METHODS: With the use of specifically designed methods, we tested discriminative sensations including texture discrimination, two-point discrimination, stereognosis, point localization, and position sense in 67 patients with acute unilateral stroke. The locations of the lesions were identified with the use of CT and/or MRI. Thirty-two age- and sex-matched healthy subjects were used as a control. RESULTS: Impaired discriminative sensation was common in patients with unilateral stroke (detected in 57 of the 67 patients) regardless of lesion location except for patients with lateral medullary infarction. Discriminative sensation remained intact in only 3 of 25 patients who were initially diagnosed as having pure motor stroke on the basis of conventional sensory tests. Point localization and stereognosis were bilaterally impaired in 17 of 39 patients and 7 of 38 patients, respectively, regardless of the laterality of the lesion. Dysfunction of other sensory modalities was observed exclusively on the side contralateral to the lesion. CONCLUSIONS: Discriminative sensory disturbances, which often occur bilaterally in some modalities, are common in patients with unilateral stroke even in those with intact sensory function on routine examination. The subtle disturbances of this sensation may explain, at least in part, the clumsiness of the patients that is not readily explained by conventional neurological tests. PMID- 8614928 TI - Risk factors for death from stroke in middle-aged Lithuanian men: results from a 20-year prospective study. AB - BACKGROUND AND PURPOSE: Although in eastern Europe, mortality from stroke at present is the highest in the world, no previous prospective study of the risk factors for stroke has been reported from this part of the world. The aim of our study, therefore, was to evaluate the relations between blood pressure, serum total cholesterol, glucose intolerance, body mass index, and cigarette smoking and the risk of death from stroke in middle-aged men in Kaunas, Lithuania. METHODS: We conducted a prospective study with an average follow-up of 17.5 years of 2295 men who had participated in risk factor surveys within the framework of the world Health Organization Kaunas-Rotterdam Intervention Study from 1972 to 1974. Risk factors included in the current analyses were smoking, blood pressure, serum total cholesterol, glucose intolerance, diabetes, and body mass index. Age and risk factor adjusted relative risks (RR) for death of stroke were determined by use of the Cox proportional hazards model. RESULTS: The strongest risk factors for death from stroke in middle-age men were systolic blood pressure (RR=1.02; P=.0001), diabetes (RR=4.17; P=.02), and smoking (RR=2.01; P=.004). Serum cholesterol, impaired glucose tolerance, and body mass index were not related to the risk of death from stroke. Twenty-five percent and 19% of stroke deaths were attributed to hypertension and smoking, respectively. CONCLUSIONS: Prevention and effective control of hypertension, smoking, and diabetes are the key elements in primary prevention of stroke in eastern Europe, where stroke mortality remains high. PMID- 8614930 TI - A novel technique for identification of doppler microembolic signals based on the coincidence method: in vitro and in vivo evaluation. AB - BACKGROUND AND PURPOSE: The applicability of a novel differentiation technique in embolus detection based on the coincidence principle and using a multigate probe was evaluated in this study. METHODS: According to the coincidence method, high intensity transients should only be classified as microembolic signals if they appear sequentially in the two sample volumes monitored and within a defined time window calculated from the blood velocity and the spatial distance between the insonation depths. Part A: microbubbles were introduced in a continuous flow bench model of the middle cerebral artery to evaluate the accuracy of the multigate probe in embolus detection. Part B: in the subjects and patients, the minimal and maximum time delays in the appearance of microembolic signals in the two middle cerebral artery sample volumes were calculated as 0.01 second and set at 0.1 second, respectively. The multigate probe was used to monitor (1) 5 normal volunteers in whom 1008 artifact signals were produced,(2) 2 patients undergoing aortic valve replacement surgery, and (3) 12 patients with potential cardiac or carotid embolic sources. RESULTS: In the bench model, 95.5% of microembolic signals produced by microbubbles appeared in the two sample volumes with a time delay between 0.02 and 0.05 second, while in the remaining 4.5% a shorter passage time of 0.01 second was measured. A total of 1968 high-intensity signals were recorded in subjects and patients. All but 20 of these (99%) appeared in both monitoring channels within the above time frame. To summarize, 996 (98.8%) of the 1004 artifact signals and 943 (98.1%) of the 961 microembolic signals were correctly classified. CONCLUSIONS: Application of the coincidence theory to distinguish microembolic signals from artifacts provides a promising new technique with high sensitivity and specificity that could decisively improve the validity of embolus detection. PMID- 8614931 TI - Effect of time and cerebrovascular symptoms of the prevalence of microembolic signals in patients with cervical carotid stenosis. AB - BACKGROUND AND PURPOSE: High-intensity transient signals (HITS) detect ed by transcranial Doppler ultrasonography correspond to microemboli in intracranial arteries. The aim of this study was to determine the time course of cerebral microembolism in patients with symptomatic internal carotid artery stenosis and to assess its relation to specific symptoms of cerebral ischemia. METHODS: On the basis of criteria established a priori, 69 middle cerebral arteries were selected from a series of consecutive studies obtained at our neurovascular laboratory. All patients had radiological evidence of cervical internal carotid artery disease and had corresponding symptoms. A TC-2000 instrument equipped with special software for microembolus detection was used. Accepted signals were unidirectional from baseline, had a chirping sound, were 9 dB higher than the surrounding blood, and lasted 25 milliseconds or more. RESULTS: HITS were identified in 20 of 69 (29%) arteries. The median interval between onset of symptoms and time of testing was 4 days for HITS-positive arteries and 12 days for those that were HITS negative (P=.0046). Fourteen of 32 (44%) arteries with transient ischemic attacks and 6 of 37 (16%) arteries with cerebral infarction were HITS positive (P=.012). CONCLUSIONS: In patients with symptomatic carotid stenosis, HITS are detected more frequently when patients are tested soon after symptoms of cerebral ischemia. HITS are also more prevalent in the territories of arteries with transient ischemic attacks rather than cerebral infarction. These findings may have diagnostic and therapeutic implications. PMID- 8614932 TI - Transesophageal echocardiography in patients with focal cerebral ischemia of unknown cause. AB - BACKGROUND AND PURPOSE: Identification of cardioembolic stroke is often limited by the difficulties involved in the detection of thrombi or the risk factors for thrombus formation. Patients in sinus rhythm with nondiagnostic transthoracic echocardiogram (TTE) and without carotid artery stenosis represent a subset in whom diagnostic recommendations are lacking. The aim of this study was to determine the value of transesophageal echocardiography (TEE) for diagnosis and therapy in this population. METHODS: Thirty patients with the characteristics described above were studied prospectively with the use of blood analysis, TTE, TEE, and Holter monitoring (in patients with atrial thrombus). RESULTS: TTE was abnormal in 16 of 30 patients, but no embolic source was identified. TEE disclosed left atrial appendage thrombus in 3 of 30 patients, atrial septal aneurysm in 2 of 30 patients, patent foramen ovale in 7 of 30 patients, and aortic plaques in 19 of 30 patients. The Tee resulted in a change of therapy in 3 of the 30 patients studied. CONCLUSION: This study demonstrates that TEE is a helpful diagnostic tool in delineating the risk of cardioembolic stroke and demonstrates significant pathology even in a subset of patients deemed to be at low risk. This additional new information led to a substantial change in therapy in 3 of 30 patients. PMID- 8614933 TI - Determination of duplex Doppler ultrasound criteria appropriate to the North American Symptomatic Carotid Endarterectomy Trial. AB - BACKGROUND AND PURPOSE: The North American Symptomatic Carotid Endarterectomy Trail (NASCET) demonstrated the benefit of carotid endarterectomy for symptomatic patients with > or = 70% carotid stenosis. Screening for detection of significant carotid occlusive disease has relied on duplex Doppler imaging. However, traditional duplex categories (50% to 79%, 80% to 99%) are not directly applicable to NASCET. We sought to evaluate duplex criteria for determination of > or = 70% carotid stenosis. METHODS: Duplex scan and arteriograms of 110 patients (210 carotids), performed within 1 month of each other, were reviewed by blinded readers. Arteriographic stenosis was determined by the NASCET method. Duplex measurements of peak systolic and end-diastolic velocity (PSV, EDV) were recorded, and ratios of velocities in the internal and common carotid arteries (ICA, CCA) were calculated. Receiver-operator characteristic (ROC) curves of sensitivity, specificity, positive and negative predictive values (PPV, NPV), and accuracy were determined. RESULTS: Interobserver agreement for measurement of arteriographic stenosis was "almost perfect" (kappa=0.86). The criteria chosen for detection of > or = 70% stenosis were PSVICA>210 cm/s (sensitivity, 94%; specificity, 77%; PPV, 68% NPV, 96% accuracy, 83%) EDVICA>70 cm/s (sensitivity, 92%; specificity, 60%; PPV, 73%; NPV, 86%; accuracy 77%), PSVica/PSVCCA >3.0 (sensitivity, 91%; specificity, 78%; PPV, 70%; NPV, 94%; accuracy, 83%), and EDVICA/EDVCCA>3.3 (sensitivity, 100%; specificity, 65%; PPV, 65% NPV, 100%; accuracy, 79%). CONCLUSIONS: We conclude that > or = 70% carotid stenosis can be reliably determined by duplex Doppler ultrasound. Individual vascular laboratories must validate their own results. PMID- 8614934 TI - Validity of B-mode ultrasonographic findings in patients undergoing carotid endarterectomy in comparison with angiographic and clinicopathologic features. AB - BACKGROUND AND PURPOSE: Determining factors for performing carotid endarterectomy (CEA) are the severity of carotid stenosis and the presence of plaque ulcerations. Precise detection of these factors is important. The aim of this study was to assess the applicability of using B-mode ultrasonography for diagnosing carotid lesions in patients undergoing CEA. METHODS: B-mode examinations were performed on 64 Japanese patients (68 arteries) who subsequently underwent CEA. In each case, the appearance of plaque on B-mode was classified into one of two types: heterogeneous or homogeneous; plaque echogenicity was expressed as hypoechoic, isoechoic, or hyperechoic. Surface characteristics such as ulceration also were examined, and the degree of carotid artery stenosis was calculated. The B-mode findings were compared with angiographic and pathological features. RESULTS: B-mode accurately visualized macroscopic ulceration and surface irregularity in 93.8% of the lesions examined, which was superior to angiography. Fifty-four lesions (79.4%) were of the heterogeneous type and 14 lesions (20.6%) were of the homogeneous type on B-mode. Microscopically, 57.4% of the heterogeneous-type lesions demonstrated fibrous change. The frequency of calcification was higher in the heterogeneous lesions than in the homogeneous lesions. CONCLUSION: B-mode ultrasonography findings can provide information about macroscopic and microscopic characteristics of carotid lesions. PMID- 8614935 TI - 'Medial defects' in the prenatal human cerebral arteries: an electron microscopic study. AB - BACKGROUND AND PURPOSE: Fine structural studies were performed to investigate the histogenesis of human intracranial arteries. Special attention was paid to whether "medial defects" exist in these arteries. METHODS: Segments of the intracranial extracerebral arteries of normal human embryos (n=6) were examined with transmission electron microscopy. RESULTS: Focal defects of the medial smooth muscle cells were disclosed at every bifurcation of the developing arteries. This configuration persisted until the arteries obtained enough muscle coat. These areas, in which an absence of medial smooth muscle cells (ie, a medial defect) existed, were occupied by fibrous connective tissues of elastin and collagen. CONCLUSIONS: The medial defect observed at the arterial bifurcation of the embryos seems to be a development process that accompanies human ontogenesis rather than a congenital anomaly, supporting a possible pathogenesis for intracranial saccular aneurysms. PMID- 8614936 TI - Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo. AB - BACKGROUND AND PURPOSE: We previously demonstrated that a monoclonal antibody (MoAb) with anti-CD31, anti-platelet-endothelial cell adhesion molecule (PECAM) like properties delayed platelet adhesion/aggregation at a site of minor endothelial injury. To our knowledge, this was the first in vivo demonstration of an effect of anti-CD31. There was no exposure of collagen or basal lamina at the injured site, and the modulation of adhesion/aggregation at such sites has not received much study. The present investigation attempted to replicate the first with the use of a different MoAb, definitely characterized as anti-PECAM. In addition, an ex vivo investigation was performed to see whether the in vivo action of anti-PECAM could have been caused by an effect of the MoAb on the platelets rather than on the endothelium. METHODS: A helium-neon laser, in the presence of intravascular Evans blue, was used to injure the endothelium of arterioles on the surface of the mouse brain. Intravital microscopy was used to determine the number of seconds required for the light to initiate the first recognizable platelet aggregate forming at the injured site. Mice injected with vehicle were compared with mice injected with 2 mg/kg anti-PECAM through the tail vein. The injection was 10 minutes before challenge with the laser. Additional studies were performed of aggregation produced in vitro by arachidonate and by ADP added to platelet-rich plasma (PRP) prepared from blood taken from MoAb treated and vehicle-treated mice. RESULTS: Aggregation latency was significantly prolonged (P<.02) by anti-PECAM (121+/-59 versus 65+/-26 seconds in controls; n=10 each). Aggregation ex vivo was not affected. CONCLUSIONS: PECAM is an important modulator of platelet adhesion/aggregation at sites of minor endothelial damage in brain arterioles. The data are consistent with the hypothesis that PECAM sites on the endothelium are involved and may be exposed by the injury to promote adhesion/aggregation. Since the endothelial cell layer is intact at these sites, mechanisms such as this offer important alternatives to the more commonly studied pathways of platelet activation, which require exposure of collagen and are not applicable in this model. PMID- 8614938 TI - Alterations of regional cerebral blood flow and oxygen saturation in a rat sinus vein thrombosis model. AB - BACKGROUND AND PURPOSE: The pathophysiology of sinus-vein thrombosis (SVT) in patients and experimental animals is still poorly understood. This study was designed to examine and further elucidate the pathophysiological sequence of events, especially the relationship between local and regional blood flow and hemoglobin oxygen saturation (HbSO2) detected at identical locations. The use of both parameters as outcome indicators should be compared. METHODS: SVT was induced by ligation of the superior sagittal sinus (SSS) and slow injection of kaolin-cephalin suspension into the SSS in rats. Regional cerebral blood flow (rCBF) was assessed by laser-Doppler flowmetry together with regional HbSO2, which was measured by a microspectrophotometric technique at 48 identical locations for 90 minutes after SVT using a scanning technique. Fluorescence angiography was performed before and 30 and 90 minutes after SVT induction. After 48 hours the animals were killed for histology. RESULTS: The fluorescence angiographic findings could divide animals into three groups: (1) group A, with a solitary SSS thrombus (n=8); (2) group B, with a thrombosis of SSS and cortical veins (n=10); (3) group C, animals that had undergone sham operation (n=5). Decreases of rCBF and HbSO2 and brain damage were seen in group B but not in group A. The reduction of local HbSO2 preceded the flow decrease after sagittal sinus ligation but before thrombosis. Blood pressure in group A was found to be significantly higher after SVT than in groups B and C. CONCLUSIONS: The brain with acute extension of thrombus from the SSS into cortical veins experiences a critically reduced supply of blood and oxygen. CBF, local HbSO2, and repeated angiography can be helpful monitors for the early detection of critical conditions after SVT. Local HbSO2 has a greater sensitivity to predict outcome than lCBF. Moreover, therapies directed to improve perfusion pressure or reduce vascular resistance may open further therapeutic windows during SVT progression. PMID- 8614937 TI - Brain capillary tissue plasminogen activator in a diabetes stroke model. AB - BACKGROUND AND PURPOSE: Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury. METHODS: Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion. RESULTS: All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively. CONCLUSIONS: Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury. PMID- 8614939 TI - Impaired pial arteriolar reactivity to hypercapnia during hyperammonemia depends on glutamine synthesis. AB - BACKGROUND AND PURPOSE: Acute hyperammonemia causes glutamine and water accumulation in astrocytes and loss of the cerebral blood flow response selectively to CO2. We tested whether extraparenchymal pial arterioles not subjected directly to mechanical compression by swollen astrocyte processes also lose hypercapnic reactivity and whether any such loss can be attenuated by inhibiting glutamine synthesis during hyperammonemia. METHODS: Pentobarbital anesthetized rats were pretreated intravenously with either saline vehicle, methionine sulfoximine (0.83 mmol/kg), which inhibits glutamine synthetase and potentially gamma-glutamylcysteine synthetase, or buthionine sulfoximine (4 mmol/kg), which inhibits gamma-glutamylcysteine synthetase. Three hours after pretreatment, cohorts received an intravenous infusion of either sodium or ammonium acetate for 6 hours. Pial arteriolar diameter was measured with radiolabeled microspheres during normocapnia and 10 minutes of hypercapnia. RESULTS: With sodium acetate infusion, pial arteriolar diameter increased during hypercapnia in groups pretreated with vehicle (23+/-3% [mean+/-SE]; n=6), methionine sulfoximine (37+/-11%; n=5), and buthionine sulfoximine (32+/-3%; n=5). With ammonium acetate infusion, pial arteriolar diameter increased only in the group pretreated with methionine sulfoximine (31+/-4%; n=8) but not in those pretreated with vehicle (-2+/-4%; n=8) or buthionine sulfoximine (4+/-4%; n=6). Methionine sulfoximine, but not buthionine sulfoximine, also prevented loss of the cerebral blood flow response to hypercapnia, an increase in cortical tissue water content, and an increase in pressure under the cranial window during normocapnia in hyperammonemic rats. In contrast to hypercapnia, hypoxemia increased arteriolar diameter 30+/-7% (n=5) during ammonium acetate infusion. CONCLUSIONS: Loss of the blood flow response to hypercapnia during acute hyperammonemia is not due simply to swollen astrocyte processes passively impeding blood flow because extraparenchymal resistance arterioles also lose their reactivity selectively to hypercapnia. Lost reactivity depends on glutamine synthesis rather than on ammonium ions per se and may reflect indirect effects of astrocyte dysfunction associated with glutamine accumulation or possibly effects of glutamine on nitric oxide production. PMID- 8614940 TI - Characterization of the cardiac effects of acute subarachnoid hemorrhage in dogs. AB - BACKGROUND AND PURPOSE: We know that significant cardiac involvement can occur in patients with acute intracranial hemorrhage, particularly in those with subarachnoid hemorrhage. These patients may present with electrocardiographic abnormalities that were previously thought to be benign. However, many die of cardiovascular sequelae, which suggests more serious cardiac problems. To characterize the cardiac, rhythmic, and myocardial disturbances that occur 2 to 4 hours after subarachnoid hemorrhage, we conducted an experimental study using autologous blood (7.9+/-0.3 mL) injected into the right frontal lobe and subarachnoid space in canines. METHODS: Nine adult mongrel dogs were anesthetized with isoflurane and their rectal temperatures maintained at 37 degrees C. Electrocardiogram, heart rate, mean arterial pressure, mean pulmonary artery pressure, and intracranial pressure were continuously measured. Transesophageal echocardiography was performed to assess myocardial wall motion changes and aortic and pulmonary flow velocities before, immediately after, and 2 and 4 hours after intracranial hemorrhage. Blood samples were collected and analyzed for catecholamines and cardiac enzymes, and cardiac output was measured. Animals were killed at 2 to 4 hours after subarachnoid hemorrhage, and a piece of the myocardium was freeze-clamped for analysis of tissue catecholamines. Light and electron microscopy were used for histopathologic assessment. RESULTS: Subarachnoid hemorrhage produced significant increases in intracranial pressure, cardiac output, and aortic and pulmonary flow velocities. Also, significant changes in creatine kinase and catecholamines were observed. Electrocardiographic recordings showed changes of tachycardia, ST-segment depression, inverted T wave, and premature ventricular contractions in four animals at 1 to 5 minutes after injection, and echocardiographic changes were evident in all animals at 20 to 240 minutes. Microscopic examination of the heart showed evidence of myocardial changes in one animal with the use of light microscopy and in nine with the use of electron microscopy. CONCLUSIONS: This study demonstrates the high incidence of cardiac involvement, specifically wall motion abnormalities, that occur after subarachnoid hemorrhage and suggests the importance of continuous cardiac monitoring, particularly echocardiographic measurements, in those patients. PMID- 8614941 TI - Effect of age and sex on N-methyl-D-aspartate antagonist-induced neuronal necrosis in rats. AB - BACKGROUND AND PURPOSE: Although N-methyl-D-aspartate (NMDA) antagonism may be a useful therapeutic approach in stroke treatment, it has been found that these pharmacological agents cause neuronal necrosis in restricted cortical regions of the rodent brain. METHODS: To test the hypothesis that age and sex influence NMDA antagonist-induced neuronal necrosis, male and female rats were studied at 2 months (young), 12 months (middle-aged), and 24 months (old) of age. A dose of 5 mg/kg MK-801 was administered, followed by quantitation of neuronal necrosis at nine coronal levels in the cingulate and retrosplenial cortex at 1 week of survival. RESULTS: Mortality was dependent on age but not sex and was higher in the old rats (P<.01). The number of necrotic neurons per hemisphere was greater in female than in male rats at all ages (P<.0001). Female rats also showed increasing neuronal necrosis with age (P<.05). CONCLUSIONS: The results indicate a major sex difference in neuronal cytotoxicity caused by NMDA antagonists and a minor increase in susceptibility with increasing age in females. The findings may be relevant to development of drugs with NMDA antagonist properties for use in human stroke. PMID- 8614942 TI - Ketamine antagonizes nitric oxide release from cerebral cortex after middle cerebral artery ligation in rats. AB - BACKGROUND AND PURPOSE: Ischemia or hypoxia activates N-methyl-D-aspartate (NMDA) receptors and results in nitric oxide (NO) production. The purpose of this study was to investigate whether an NMDA channel blocker can inhibit NO production during ischemia. METHODS: Temporary cerebral ischemia was induced by middle cerebral artery ligation while common carotid arteries were clamped bilaterally for 40 minutes in urethane-anesthetized rats. Extracellular NO concentration in the cortex was recorded through Nafion- and porphyrine-coated carbon fiber electrodes. Ketamine, and NMDA channel blocker, was administered (50 mg/kg) intraperitoneally 15 minutes before the cerebral artery ligation. RESULTS: During middle cerebral artery ligation, cortical NO was increased to its peak (18.76+/ 3.36 nmol/L) in 7 minutes and then declined. The overflow of NO can be antagonized by pretreatment with ketamine, dizocilpine maleate (MK801), or N(G) nitro-L-arginine methyl ester (L-NAME). Local application of nitroprusside also induced NO production. However, this effect was not antagonized by ketamine. CONCLUSIONS: These findings demonstrated that NO release induced by short-term cerebral ischemia can be attenuated by pretreatment with NMDA antagonists. PMID- 8614944 TI - Aspirin in ischemic cerebrovascular disease. How strong is the case for a different dosing regimen? AB - BACKGROUND: A vast consensus exists in defining a narrow range of recommended daily doses of aspirin, ie, 75 to 160 mg, for the prevention of myocardial infarction, stroke, and vascular death in patients with different manifestations of coronary hearth disease. In contrast, for patients with cerebrovascular disease, a much larger degree of uncertainty still exists, with recommendations ranging from 30 to 1300 mg daily. SUMMARY OF COMMENT: The contention that higher doses of aspirin (650 to 1300 mg) are more effective than lower doses in stroke prevention is based on indirect and selective comparisons of different trial data, mini-meta-analyses, or subgroup analyses of individual trials. In the absence of definitive evidence from direct randomized comparisons of low-dose versus high-dose aspirin in trials of adequate size to detect a moderate difference between the two, the biological hypotheses that underpin the suggestion of greater efficacy of higher aspirin doses in cerebrovascular disease patients are reviewed and disputed. Practical implications of the use of higher doses of aspirin are also assessed on the basis of theoretical calculations of absolute benefits and risks. CONCLUSIONS: Until additional information from ongoing trials is available, good clinical practice should dictate the use of the lowest dose of aspirin shown effective in the prevention of stroke and death in patients with ischemic cerebrovascular disease, ie, 75 mg daily. PMID- 8614943 TI - Recurrent right hemiplegia associated with progressive ipsilateral carotid artery stenosis. AB - BACKGROUND: Ipsilateral sensory motor symptoms associated with carotid artery stenosis are rare, and few reports are available in the literature. CASE DESCRIPTION: We report the case of a 50-year-old man who presented with right hemiplegia that recurred 14 months later. A left hemisphere watershed infarction was detected. Repeated angiograms showed a left internal carotid occlusion and a right internal carotid stenosis that initially measured 50% and worsened to 80% after the second stroke. CONCLUSIONS: Repeated quantitative measurements of cerebrovascular reserve demonstrated the hemodynamic mechanism of the strokes and the role of a right internal carotid lesion in causing the recurrence of right hemiplegia. PMID- 8614945 TI - Ischemic stroke and incomplete infarction. AB - BACKGROUND: The concept of selective vulnerability or selective loss o f individual neurons, with survival of glial and vascular elements as one of the consequences of a systemic ischemic-hypoxic insult (eg, transient cardiac arrest or severe hypotension), has been recognized for decades. In contrast, selective neuronal death as one of the lesions that may develop in the brain after occluding an intracranial artery is an idea not readily acknowledged in the current medical literature dealing with human stroke. SUMMARY OF REVIEW: A review of pertinent publications reveals that selective neuronal injury after middle cerebral artery occlusion was observed in autopsy specimens over 40 years ago, although its pathogenesis remains unclear. Recent observations in both humans and animals suggest that selective neuronal necrosis (rather than infarct) is the consequence of either a short-term arterial occlusion or permanent occlusion accompanied by ischemia of moderate severity. During the acute and subacute states of an ischemic stroke, the loss of a limited number of neurons (ie, incomplete infarction) does not result in structural changes discernible by either CT or conventional MRI. However, the loss of a selected number of neurons may be demonstrable in vivo by calculating the corresponding loss of benzodiazepine receptors. The use of specific radiotracers in combination with single-photon emission CT or positron emission tomography allows demonstration of a decrease in gamma-aminobutyric acid-ergic receptor sites at places where many neurons have been lethally injured. CONCLUSIONS: We aim to alert physicians to the potential development of incomplete brain infarctions in patients with intracranial arterial occlusions. Recognizing incomplete infarcts is particularly important in the context of stroke therapy with thrombolytic and neuroprotective agents. This brain lesion is likely to be the consequence of an arterial occlusion with a resultant ischemia of moderate severity (eg, regional blood flows in the range of 15 to 20 mL x 100 g-1 x min-1). PMID- 8614946 TI - Frequency of resistance to activated protein C due to factor V mutation in young patients with ischemic stroke. PMID- 8614947 TI - Can the presence of TCD-detected microemboli select a high-risk group of <70% carotid stenosis for appropriate prophylactic surgical treatment? PMID- 8614948 TI - Transcranial Doppler-detected microemboli in patients with acute stroke. PMID- 8614949 TI - Skepticism and carotid ultrasonography. PMID- 8614950 TI - Accuracy of carotid ultrasound. PMID- 8614951 TI - Which ingredient in tobacco smoke is the culprit? PMID- 8614952 TI - Clinical stroke scores in Chinese population. PMID- 8614953 TI - Agreement of disease-specific criteria for DNR orders in acute stroke. PMID- 8614954 TI - Visually evoked hyperpathia due to thalamic hemorrhage: a variant of Dejerine Roussy syndrome. PMID- 8614955 TI - Failure of isradipine in cerebral ischemia models. PMID- 8614956 TI - Transfusion-associated graft-versus-host disease in the neonate-expanding the spectrum of disease. PMID- 8614957 TI - The RoHar antigenic complex is associated with a limited number of D epitopes and alloanti-D production: a study of three unrelated persons and their families. AB - BACKGROUND: the RoHar antigenic complex has been characterized serologically by difficulties in D typing, weak e expression, lack of G antigen, presence of Rh33, a low-frequency Rh antigen, and, more recently, a second low-frequency antigen, FPTT. Allocation to one of the partial D catagories was not considered because of the unuaual reactions of RoHar cells and because anti-D production was not observed in RoHar persons. STUDY DESIGN AND METHODS: Three unrelated RoHar donors and their families were studied in detail with special emphasis on D epitope mapping, e and G typing, and screening for antibodies. RESULTS: Only D epitopes 5 and 6/7 were demonstrable, and D epitopes 1, 2, 3, 4, 8, and 9 seem to be absent in the RoHar complex. In one individual, the presence of alloanti-D with limited specificity, not reacting with RoHar red cells of other individuals, was found 6 months after a second D+ pregnancy. CONCLUSION: The finding of alloanti-D in an RoHar r person supports the concept that the D characteristic of this phenotype is a partial D antigen, which is consistent with the presence of the limited number of D epitopes found in epitope mapping. As has been suggested for other partial D antigens, RoHar individuals should be regarded as D- for the receipt of blood, and pregnant RoHar women who have had D+ pregnancies should receive anti-D prophylaxis. PMID- 8614958 TI - Evaluation of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in the inhibition of rouleaux formation. AB - BACKGROUND: DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibits the formation of serum-or plasma-induced rouleaux through its ability to bind to band 3 on red cell membranes. This property of DIDS was evaluated in the serologic testing of specimens exhibiting rouleaux. STUDY DESIGN AND METHODS: Optimal test conditions for DIDS treatment of reagent red cells were determined by varying the volume and concentration of DIDS solution and the incubation temperature and duration and comparing the results of antibody screening procedures using specimens that exhibit macroscopically visible rouleaux. Blind titration studies compared untreated and DIDS-treated red cells to evaluate the maintenance of antigen integrity. The use of DIDS-treated red cells for antibody detection and identification was evaluated by comparing the results in donor specimens containing antibodies with those in untreated and DIDS-treated selected panel cells. In addition, 4-percent (wt/vol) dextran in serum was used to induce rouleaux formation as a way of determining the capability of DIDS to resolve ABO serum grouping discrepancies. RESULTS: Complete inhibition of rouleaux formation occurred when reagent red cells were treated by incubation at 37 degrees C for 10 minutes with 150 microliter (approx. 5 drops) of 0.05 mg per mL of DIDS in 0.9 percent NaCl. There were no significant differences in titration scores of untreated and DIDS-treated red cells tested with the 19 antisera used to assess antigen integrity. Antibody identification studies showed that DIDS-treated reagent red cells reacted similarly to untreated reagent red cells. In addition, DIDS resolved dextran-induced ABO serum grouping discrepancies. CONCLUSION: DIDS effectively resolved the serologic problems associated with the presence of rouleaux, without affecting the results of the test system itself. Implementation of this method to inhibit the rouleaux-forming properties of serum and plasma specimens can be useful in serologic testing. PMID- 8614959 TI - Differentiation between human red cells of Pk and p blood types using Pseudomonas aeruginosa PA-I lectin. AB - BACKGROUND: The red cells of almost all human beings bear P antigen. Type P1 cells (around 75% of the population) contain P1 antigen in addition to P, and type P2 cells (around 25% of the population) contain only P. The red cells of only a few individuals are devoid of P: these cells may be of either Pk-positive (P1k and P2k[Pk]) or p type (the latter lack all the above-described antigens of the P system). Differentiation between them is of clinical importance, but there is a shortage of specific reagents. This article offers reliable means for differentiation. STUDY DESIGN AND METHODS: Agglutination of washed, papain treated red cells of all the P types by PA-1 and soybean lectins and adsorption of the lectins onto the red cells were examined. RESULTS: PA-1 strongly agglutinated papain-treated red cells. Examination of its interactions with red cells having different P system antigens revealed that Pk (both P1k and P2k) red cells of O, A, and B blood types were agglutinated significantly faster than p red cells. The agglutination intensities of Pk red cells of types O and A (most people) was considerably stronger than those of p red cells. P1 and P2 type red cell agglutination was intermediate (P1>P2). Adsorption tests with all the red cells, exhibited the same order of PA-1 affinities for the P system blood types: Pk>P1,>P2>p. The soybean lectin exhibited opposite behavior (p>P2>P1>Pl). CONCLUSION: The galactose-binding lectins PA-1 and soybean may facilitate the determination of Pk and p red cells. PMID- 8614961 TI - Interviews of individuals diagnosed as anti-human immunodeficiency virus-positive through the screening of blood donations in the Paris area to 1994: reflections on the selection of blood donors. AB - BACKGROUND: One of the aims of the medical interview routinely preceding each blood donation is the identification of individuals with a risk factor for infection with the human immunodeficiency virus (HIV). STUDY DESIGN AND METHODS: Interviews were performed with individuals diagnosed as being seropositive for HIV through the systematic biologic screening of blood donations in the Paris area to establish, first, the circumstances allowing HIV-seropositive individuals to pass through the predonation medical interview and, second, the motivation of these individuals as blood donors. Risk factors of 30 HIV-infected donors identified between 1991 and 1994 were determined. RESULTS: When asked whether they recognized the eventual risk to recipients of donated blood, 14 (47%) of 30 answered positively. Fifteen (50%) admitted having given their blood to determine their HIV status. CONCLUSION: These individuals did not exclude themselves from blood donation and probably hid their risk factor(s) at the predonation interview in order to be accepted as blood donors. PMID- 8614960 TI - Posttransfusion graft-versus-host disease in Japanese newborns. AB - BACKGROUND: Posttransfusion graft-versus-host disease (PT-GVHD) is underdiagnosed and underreported. Risk factors predisposing to PT-GVHD in newborn recipients, the clinical manifestations of the disease in newborns, and its mechanism are not well characterized. STUDY DESIGN AND METHODS: A literature review identified 27 cases of PT-GVHD in newborns in Japan. Detailed information on volume of blood transfused, donor(s), and clinical course was analyzed. Infants with known immunodeficiency were excluded. RESULTS: Of 27 newborns, 20 were premature and 7 were full-term. Thirteen premature neonates were transfused frequently because of anemia; in 10 cases, exchange transfusion was performed. Fresh blood ( 3 log increase in titer. However, from 100 to 135 days, titers were restricted to plateau levels of approximately 10(8)/g. In comparison, previous studies show no appreciable eclipse phase and > or = 100-fold higher brain titers in hamster scrapie. Our calculations also revealed an effective doubling time (ti) of 7.6 days in CJD and a markedly different ti of approximately 3 days in scrapie. Thus different initiation and replication programs are encoded by each of these strains. The most pertinent host molecular responses included early astrocytic activity by 54 days, unaccompanied by morphological or behavioral changes. Changes in host PrP were minimal until 87 days when titers were already 2 x 10(7)/g. In the next 20 days 60% of brain PrP became resistant to limited proteolysis but total PrP did not increase. These fulminant PrP changes preceded viral arrest and subsequent spongiform degeneration. Because these and other data are not consistent with PrP itself being the infectious agent, we discuss a model in which progressive PrP and glial activation are part of a final host strategy to contain a virus that is innocuous at low titers. This strategy is flawed because PrP is an independent provocateur of self-destruction in the brain. However, in the periphery this strategy may eliminate rare infected cells. PMID- 8615007 TI - Pepscan mapping and fusion-related properties of the major phosphatidylserine binding domain of the glycoprotein of viral hemorrhagic septicemia virus, a salmonid rhabdovirus. AB - The binding of labeled phosphatidylserine (PS) to a collection of synthetic 15 mer peptides covering full-length glycoprotein G (G) of viral hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus, showed three dominant overlapping reactive peptides. This major PS-binding region was contained in a 28 mer peptide (p2; aa 82-109) with consecutive hydrophobic amino acid a-d heptad repeats (putative amphipathic alpha-helix) and 2 carboxy-terminal arginines. This 28-mer peptide showed a 10-fold higher apparent specific activity for PS binding than the 15-mer peptides. Binding to PS was also detected with virion-purified protein G but was not detected with other viral proteins. The highest apparent specific activity for PS binding was found with purified VHSV particles by both solid-phase and liquid assays. In contrast to the pH-independent PS binding to peptide p2, binding to virions was optimal at pH 5.6. PS binding to purified VHSV was greatly reduced by protease or detergent treatments that removed protein G, by treatment at pH 7.6, or by anti-p2 mouse antibodies at pH 5.6. The PS-binding region seems to be related to viral-host cell fusion since anti-p2 mouse antibodies inhibited VHSV-infected cell to cell fusion (fusion from within) and the pH profile of the VHSV-infected cell to cell fusion was similar to the pH profile of PS binding to VHSV. Comparative analysis showed that sequences similar to the major PS-binding domain of VHSV were also present in other fish rhabdoviruses and in rabies and vesicular stomatitis viruses. PMID- 8615009 TI - Hepatitis B virus X gene expression is activated by X protein but repressed by p53 tumor suppressor gene product in the transient expression system. AB - Hepatitis B virus (HBV) X gene is known to exhibit a transcriptional activation function and is considered to play a major role in hepatocarcinogenesis. We determined a 20-bp promoter element for the HBV X gene transcription and found a binding protein to this promoter element, designated as an X-PBP. We then examined the effects of HBV X protein and p53 tumor suppressor gene product on X gene transcription from the 20-bp promoter element using the transient expression technique. Activity of the X gene promoter was stimulated by X protein expression, but, in contrast, was repressed by transfected normal p53 gene. On the other hand, mutant p53 gene product exhibited no repression. Moreover, the p53 repression of X gene transcription was canceled by X protein coexpression. Thus, the effects of X protein and normal p53 product appear to be mutually antagonistic in the regulation of X gene expression. However, mutated promoter elements which failed to bind to X-PBP still responded to X protein or p53, indicating that the process of X transactivation or p53 repression may be independent of X-PBP binding to the promoter element. Our data suggest that X protein could disrupt function of normal p53 protein in X gene-transfected cells. PMID- 8615008 TI - Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. AB - The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins into Escherichia coli, isolating the E. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4 M NaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either in E. coli or in planta from RNA 3 of CMV was tagged at its C terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni(2+)-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified from E. coli and zucchini squash, both were able bind CMV RNA 3 in vitro. PMID- 8615010 TI - Sequences within the early and late promoters of archetype JC virus restrict viral DNA replication and infectivity. AB - Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype's inactivity relative to that of rearranged forms was due to differences in the promoter-enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter-enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s). PMID- 8615011 TI - Mutations in the poliovirus 3CD proteinase S1-specificity pocket affect substrate recognition and RNA binding. AB - Sequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases. In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition. Single amino acid substitutions at 3C residues Thr-142, His-161, Gly 163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3C cis cleavage activity. Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure. An in vivo analysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication. In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5' terminal sequences. PMID- 8615013 TI - Analysis of HPV1 E4 complexes and their association with keratins in vivo. AB - The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1-E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers. PMID- 8615012 TI - Trans-activation of a cell housekeeping gene promoter by the IE1 gene product of baculoviruses. AB - Protein IE1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. This protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral DNA replication. To test whether the IE1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of Bombyx mori nuclear polyhedrosis virus (BmNPV) into silkworm Bm5 tissue culture cells together with expression cassettes directing expression of chloramphenicol acetyl transferase or juvenile hormone esterase under the control of the cytoplasmic actin A3 gene promoter of B. mori. Cotransfection with the ie1 gene resulted in a dramatic increase in the amount of the two enzymes expressed in the transfected cells. The increased enzyme activities correlate with an increased accumulation of the corresponding mRNAs, and the latter is caused by an increase in the rate of transcription directed by the cytoplasmic actin gene promoter. The chromosomal cytoplasmic actin gene of Bm5 cells is also upregulated upon transfection of the cells with the ie1 gene. However, infection of cells with BmNPV does not cause an increase in the level of expression of the endogenous cytoplasmic actin gene. Thus, the effect of IE1 on the transcriptional properties of the cytoplasmic actin gene vary depending on whether IE1 is expressed in isolation or in the context of a viral infection. The trans-activating effects of BmNPV ie1 gene expression on the silkmoth actin promoter are also evident in Spodoptera frugiperda Sf21 and Choristoneura fumiferana Cf1 tissue culture cells. Finally, the ie1 gene of Autographa californica nuclear polyhedrosis virus can substitute for its BmNPV counterpart in all cell lines tested. PMID- 8615014 TI - Transfer of an esterase-resistant receptor analog to the surface of influenza C virions results in reduced infectivity due to aggregate formation. AB - A synthetic sialic acid, N-acetyl-9-thioacetamidoneuraminic acid (9 ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but in contrast to the natural receptor determinant, N-acetyl-9-O-acetylneuraminic acid-is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer. PMID- 8615015 TI - Resistance to V3-directed neutralization caused by an N-linked oligosaccharide depends on the quaternary structure of the HIV-1 envelope oligomer. AB - A conserved N-glycan present within the V3 loop of gp120 modulates the sensitivity to neutralization by antibodies directed to the V3 loop. A glycan deficient mutant of HIVLAI, designated HIVA308, displayed a 100-fold increase in sensitivity to neutralization by anti-V3 MAb NEA-9205 compared to wild-type HIVLAI. This difference in sensitivity was not caused by an alteration of the antibody binding site itself, as NEA-9205 had equal affinity for both wild-type and mutant monomeric gp120. In contrast, virion-associated wild-type gp120 was immunoprecipitated less efficiently with NEA-9205 than virion-associated mutant gp120. This difference was completely abrogated, if immunoprecipitation were carried out in the presence of detergent. Furthermore, treatment of virion preparations with detergent exposed the C-terminal D7324 epitope, which is inaccessible on virion-associated gp120 but readily accessible on monomeric, soluble gp120. Finally, both wild-type and mutant monomeric, soluble gp120 were precipitated equally efficiently by NEA-9205 in the absence of detergent. Thus, the NEA-9205 epitope was readily accessible on monomeric gp120 regardless of the presence of the 306N-glycan, and inaccessibility of the NEA-9205 epitope imparted by the 306N-glycan was observed only on the intact envelope oligomer. PMID- 8615016 TI - Endogenous mouse mammary tumor virus Mtv-17 is involved in Mtv-2-induced tumorigenesis in GR mice. AB - Mtv-2 is an endogenous mouse mammary tumor virus (MMTV) that is responsible for the induction of mammary gland tumors in the high mammary gland tumor-incidence strain GR. GR animals inherit four different endogenous MMTVs in addition to Mtv 2: Mtv-3, Mtv-7, Mtv-8 and Mtv-17. In this study we analyzed the involvement of these nonpathogenic endogenous proviruses in the mammary gland tumors caused by Mtv-2. We showed that Mtv-17 is expressed in the mammary gland of GR mice, efficiently packaged into virions, and shed into milk. DNA isolated from both mammary gland tumors and the nonmalignant mammary gland tissues of GR mice contained amplified copies of both newly acquired Mtv-2 and recombinant proviruses with the env gene derived from Mtv-17. A small percentage of these tumors contained predominantly recombinant viruses. These findings suggest a role for Mtv-17 in the mammary gland tumors induced by Mtv-2 in GR mice. PMID- 8615017 TI - RNA phage KU1 has an insertion of 18 nucleotides in the start codon of its lysis gene. AB - We have determined the nucleotide sequence of group II RNA phage KU1. The most conspicuous difference in the comparison with other group II members such as GA and JP34 is the presence of an insertion in the start codon of the lysis gene. In GA and JP34, the coat and lysis genes overlap by one nucleotide in the configuration UAAUG. The 18-nt insertion in KU1 is positioned between the A and the U of the start codon. It does not affect the coat reading frame, but it destroys the AUG start codon and separates the previously overlapping genes by 17 nts. The insert creates a UUG codon at its 3' border which serves as the start site for lysis protein synthesis in KU1. We also show that analogous to the group I phages, such as MS2 and fr, expression of the lysis gene in KU1 and JP34 is coupled to termination of translation at the coat gene. RNA secondary structure models for the central parts of KU1 and JP34 are suggested which can account for the insertion as a separate stem-loop structure. PMID- 8615018 TI - Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions. AB - A highly conserved baculovirus late gene called odvp-6e was shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for both Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) and Cydia pomonella granulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPV odvp-6e gene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18-120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV from Autographa californica NPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of the odvp 6e gene from the baculoviruses Choristoneura fumiferana NPV (CfMNPV), AcMNPV, and Helicoverpa zea NPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins. PMID- 8615019 TI - Vpx association with mature core structures of HIV-2. AB - Vpx is a virion-incorporated protein encoded by HIV-2 and certain strains of SIV which is necessary for efficient infection of lymphocytes and macrophages. To determine the location of Vpx within the HIV-2 particle, core structures were obtained from mature HIV-2 virions. Analysis of purified cores reveal the presence of monomeric Vpx as well as a nondenaturable and nonreducible higher order Vpx complex. Characterization of the core-associated Vpx complex suggests it to be a Vpx homodimer, indicative of high concentrations of Vpx. The incorporation and concentration of Vpx in HIV-2 cores support the hypothesis that it functions within the viral preintegration complex. PMID- 8615020 TI - Use of lysolecithin-permeabilized infected-cell extracts to investigate the in vitro biochemical phenotypes of poxvirus ts mutations altered in viral transcription activity. AB - Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiation in vitro on plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. The in vitro system was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observed in vitro which could be elevated by the addition of pure mRNA capping enzyme. VTF dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observed in vivo and demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme. PMID- 8615022 TI - Regulation of neighboring gene expression by the herpes simplex virus type 1 thymidine kinase gene. AB - The herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of the gH (UL22) gene with its 3' end overlapping the gH promoter, and it overlaps the UL24 gene's regulatory and coding sequences at its 5' end in a head to-head orientation. Thus, tk expression could affect gH expression by promoter occlusion and UL24 expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects of tk promoter mutations that reduce tk expression on gH and UL24 expression. For gH, tk promoter mutations did not increase the accumulation of gH mRNA or the rate of gH transcription. Thus, tk expression does not appear to down-regulate gH expression. In contrast, we found that decreased tk expression correlated with increased accumulation of UL24 mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression of tk, but not at late times when tk mRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation of UL24 mRNA accumulation requires tk expression in cis. Nuclear run-off experiments did not detect differences in UL24 transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentially in cis. PMID- 8615021 TI - Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death. AB - The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR) TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients. PMID- 8615023 TI - Temporal regulation of herpes simplex virus type 1 UL24 mRNA expression via differential polyadenylation. AB - Using Northern blot, primer extension, and S1 nuclease analyses of wild-type and deletion-containing herpes simplex type 1 viruses, we found that UL24 sequences are contained in six different transcripts that originate from three previously identified mRNA start sites. Thus, the six UL24 transcripts represent three different pairs of 5' coterminal mRNAs. Each transcript pair consists of a short species whose 3' end corresponds to a polyadenylation signal located just downstream of the UL24 open reading frame, and a longer species whose 3' end corresponds to a polyadenylation signal located downstream of the UL26 gene. Maximal accumulation of the short UL24 transcripts was at early times during infection, while accumulation of the longer species did not decrease at late times. Consistent with early kinetics, the short transcripts were less sensitive to drugs that inhibited viral DNA replication than the longer transcripts which exhibited leaky-late kinetics. Quantitative S1 nuclease analysis indicated that 3' ends corresponding to the UL24 polyadenylation site were significantly more abundant at early times than at late times. Thus, differential polyadenylation determines the kinetics of accumulation of different UL24 transcripts. PMID- 8615024 TI - Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus. AB - We have sequenced the complete genome of a hepatitis B virus (HBV) strain that was transmitted from a gibbon with chronic hepatitis B to a chimpanzee that subsequently developed acute hepatitis B. The genome was 3,182 nucleotides long and had a genetic organization identical to and including the characteristics of other mammalian hepadnaviruses. Thus, the regulatory elements, the direct repeats, and the four open reading frames (ORFs) of this virus were all maintained, although there were amino acid substitutions affecting all the ORFs. Within the S gene encoding for the hepatitis B surface antigen (HBsAg), the subtype could be deduced as ayw3 in accordance with previous serological results. There were 25 amino acid substitutions affecting the P gene, 12 of which were within the spacer region. This region, which was the most divergent part of the genome compared to other HBV strains, also encodes for the pre-S proteins. A comparison with sequences of other hepadnaviruses revealed that the genome of gibbon HBV was unique as compared to previously described HBV genotypes. It was most similar to the chimpanzee HBV strain with which it shared 90.3% nucleic acid homology at the level of the complete genome and 96.3% homology at the level of the S-gene region corresponding to HBsAg, although being a distinct genotype as compared to the latter virus. Analyses performed using five different algorithms for phylogenetic tree construction showed more than 99% bootstrap support for the gibbon and the chimpanzee HBV to be grouped within the human HBV strains and that they represented later offshoots than the HBV strains of genotype F. However, in most of the dendrograms both the gibbon and the chimpanzee strains represented early lineages, indicating that these viruses are indigenous to their respective hosts and not recent acquisitions from man. PMID- 8615025 TI - Biological properties of recombinant HIV envelope synthesized in CHO glycosylation-mutant cell lines. AB - N-glycosylation of the human immunodeficiency virus type-1 envelope (Env) glycoprotein precursor (gp160) occurs by transfer of Glc3Man9GlcNAc2 onto the nascent protein. Maturation then occurs via cleavage of the three Glc residues, which starts during translation. These events are considered necessary to create Env functional conformation: treatment with "alpha"-glucosidase inhibitors, but not alpha-mannosidase inhibitors (i) impairs gp160 cleavage into gp120 and gp41, (ii) diminishes the accessibility of gp120 V3 region, (iii) prevents gp120 binding to its CD4 receptor, and (iv) prevents gp41-mediated membrane fusion. These inhibitors are of therapeutic interest. Here, using a collection of parent and mutant CHO cells that possess mutations in different steps of glycosylation, we reassessed the role of glycans in both the processing and the properties of recombinant gp160 expressed from a vaccinia virus vector. Mutant cells were as follows: Lec23 (which lacks alpha-glucosidase I activity) produces a collection of triglucosylated structures (Glc3Man7-9GlcNAc2); LEC10 (which has increased GlcNAc transferase III activity) produces complex glycans with a bisected GlcNAc residue; Lec1 (which lacks GlcNAc transferase I) and Lec3.2.8.1 (which lacks GlcNAc transferase I and has decreased activity of CMP-NeuNAc and UDP-Gal translocases) produce Man5GlcNAc2 glycans at complex or hybrid sites. As expected, glycosylation of Env produced from mutants was affected but, irrespective of the glycosylation phenotype, (i) similar quantities of Env were synthesized, (ii) the immunoreactivity of V3 was similar, (iii) gp160 was efficiently cleaved into gp120 and gp41, (vi) Env was exposed at the cell membrane, (v) secreted gp120 bound CD4, and (vi) membrane gp41 was able to induce membrane fusion with CD4+ cells. Thus, the glycosylation alterations examined are dispensable for Env processing and biological activity in CHO cells. In particular, removal of the three outer Glc residues was not required per se for Env folding in this system because functional Env is obtained from Lec23 cells: it appears therefore that lack of modification is not equivalent to drug inhibition of modification. These data are discussed in the light of previous reports describing the use of glycosidase inhibitors to alter glycosylation. PMID- 8615026 TI - Control of p53 expression by adenovirus 12 early region 1A and early region 1B 54K proteins. AB - The level of p53 is markedly increased in human cells in response to expression of the Ad12 E1A proteins and, quite separately, to the Ad12 E1B 54K protein. The behaviour of p53 in these two circumstances has been examined using A549 cells infected with Ad12 dl620 (a mutant virus which does not express the larger E1B protein and is replication-defective) and human skin fibroblasts expressing the Ad12 E1B 54K protein (HSF 54K). In normal and E1A-expressing A549 cells, p53 is located predominantly in the nucleus, whereas in the HSF 54K cells it is primarily cytoplasmic as is the Ad12 E1B 54K protein. The half-life of p53 is increased in Ad12 dl620-infected A549 cells from about 10 min (in uninfected cells) to 2 hr. The half-life of p53 in HSF 54K cells is even longer-probably in excess of 48 hr. The capacity of p53 to regulate transcription was assessed using a transfected CAT construct linked to p53-responsive elements. p53 transcriptional activity was very low in the HSF 54K cells and in human embryo kidney cells expressing the Ad12 E1B 54K protein (and p53) at high level. It was, however, dramatically increased in response to the p53 expressed as a result of E1A expression. Additionally, MDM2 was present at low level in the HSF 54K cell lines, whilst, as we have previously shown, it is overexpressed in response to infection with Ad12 dl620. We conclude that there are two distinct mechanisms for up-regulation of p53 attributable to the adenovirus E1 proteins. When E1A only is present the p53 is nuclear and transcriptionally active and can probably induce apoptosis in the absence of the E1B 19K protein. When the E1B 54K protein is present, however, p53 is transcriptionally inactive and does not induce apoptosis. PMID- 8615027 TI - Expression of the myxoma virus tumor necrosis factor receptor homologue and M11L genes is required to prevent virus-induced apoptosis in infected rabbit T lymphocytes. AB - Myxoma virus is a leporipoxvirus that causes a highly lethal virulent disease known as myxomatosis in the European rabbit. An important aspect of myxoma virus pathogenesis is the ability of the virus to productively infect lymphocytes and spread to secondary sites via lymphatic channels. We investigated the infection of the CD4+ T lymphoma cell line RL-5 with myxoma virus and Shope fibroma virus, a related but benign leporipoxvirus, and observed that myxoma virus, but not Shope fibroma virus, was able to productively infect RL-5 cells. We also discovered that infection of RL-5 cells with Shope fibroma virus or attenuated myxoma virus mutants containing disruptions in either the T2 or the M11L gene resulted in the rapid induction of DNA fragmentation, followed by morphological changes and loss in cell integrity characteristic of cell death by apoptosis. Purified exogenous T2 protein was unable to prevent apoptosis, suggesting that T2 functions intracellularly. Thus, myxoma virus T2, originally described as a secreted homologue of the tumor necrosis factor receptor, and M11L, a novel transmembrane species with no known cellular homologue, function to extend virus host range for replication in rabbit T lymphocytes through the inhibition of apoptosis in infected T lymphocytes. PMID- 8615028 TI - Genotypic and phenotypic characterization of a neutralization-resistant breakthrough population of HIV-1. AB - Certain antibody neutralization escape mutants of HIV-1 map outside of the antibody recognition epitope, thereby suggesting the presence of nonlinear conformational domains. In an effort to begin to define the interacting regions of the HIV envelope proteins, a neutralization-sensitive clone of HIV-1, HXB2/BH10Sal-Bam, was passaged in the presence of the V3-specific monoclonal antibody 0.5beta. DNA sequence analysis of the V3 domain of the breakthrough viral populations revealed one population that retained the parental V3 genotype. Quantitative DNA sequence analysis of this breakthrough population revealed the presence of mutational "hotspots" in several envelope domains that are noncontiguous with V3. Mutations were seen throughout gp41 and the C1, V1/V2, C2, and C5 domains of gp120. In contrast, other regions of gp120, C3, V4, C4, and V5 remained totally unchanged. Within V1, three residues within a 14-amino acid stretch experienced five substitutions and in C5 three residues within a 7-amino acid stretch experienced four substitutions. This finding, that certain residues clustered within particular domains (V1/V2, C5, and gp41) experienced multiple substitutions under a defined environmental stressor, suggests that the degree of adaptive plasticity exhibited by the HIV envelope is limited. Based on this observation it may be possible, using a set of antibodies to various envelope epitopes, to discern a set of rules which explain the interactions of the various envelope domains with each other and with their environment. The insight gained into the physiologic constraints that the envelope proteins are subject to may be useful in developing therapeutic and vaccination strategies. PMID- 8615029 TI - The viral ubiquitin gene of Autographa californica nuclear polyhedrosis virus is not essential for viral replication. AB - The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a protein with significant homology to ubiquitin. To study the role of viral ubiquitin in infection, a recombinant virus was constructed with a frameshift mutation within the coding sequence of the viral ubiquitin gene, v-ubi. This recombinant, named Vubi-FS, was viable, indicating that viral ubiquitin is not essential for replication in tissue culture. However, the yields of infectious budded virus were decreased 5- to 10-fold in single step growth curves, and the production of total budded virions was reduced to a similar extent. The mutant virus particles contained the phospholipid-modified form of ubiquitin (Pt-Ub), and amino acid sequence analysis revealed that only host ubiquitin was packaged into virions. Together, these results suggest that viral ubiquitin is a nonessential protein that may confer a slight growth advantage under certain conditions. PMID- 8615030 TI - Raccoon poxvirus feline panleukopenia virus VP2 recombinant protects cats against FPV challenge. AB - An infectious raccoon poxvirus (RCNV) was used to express the feline panleukopenia virus (FPV) open reading frame VP2. The recombinant, RCNV/FPV, was constructed by homologous recombination with a chimeric plasmid for inserting the expression cassette into the thymidine kinase (TK) locus of RCNV. Expression of the VP2 DNA was regulated by the vaccinia virus late promoter P11. Southern blot and polymerase chain reaction (PCR) analyses confirmed the cassette was in the TK gene of the RCNV genome. An immunofluorescent antibody assay using feline anti FPV polyclonal serum showed the expressed viral antigen in the cytoplasm of infected cells. Radioimmunoprecipitation with the same antiserum detected a 67 kDa VP2 protein which exactly matched the migration of the authentic FPV VP2 protein by SDS-polyacrylamide gel electrophoresis. Nine five-month-old cats were vaccinated and 21 days later were boosted with the recombinant virus. Peroral FPV challenge 2 weeks after the booster showed that the cats were fully protected as measured by examining clinical signs and total white blood cell counts in peripheral blood. Cats not immunized developed low to very low leukocyte counts following peroral FPV challenge. The nine vaccinated cats showed high FPV neutralization antibody prior to challenge, whereas nonvaccinated cats formed anti-FPV antibodies only after challenge. PMID- 8615031 TI - Different hemagglutinin cleavage site variants of H7N7 in an influenza outbreak in chickens in Leipzig, Germany. AB - The hemagglutinin (HA) genes from four avian H7N7 influenza A isolates, from a single outbreak, were shown to possess different cleavage sites that contain varying numbers of basic amino acid residues (KKKKR, KRKKR, KKRKKR, KKKKKKR). All four variants are highly pathogenic in chickens and share an immediate common ancestral HA with A/tern/Potsdam/342-6/79 (H7N7) and A/swan/Potsdam/63-6/81 (H7N7). These viruses are nonpathogenic and contain no extra basic amino acids at the cleavage site of their HA. During evolution a common precursor virus acquired different sequences at the cleavage site of the HA and became highly pathogenic in chickens. In vitro assays revealed that the HA from A/chicken/Leipzig/79 with KKKKR at the cleavage site was only partially cleaved (41%), compared to 93-100% cleavage of the other HAs. Since all four viruses were highly pathogenic in chickens, these findings confirm that the degree of pathogenicity in vivo is not exclusively determined by the degree of HA cleavability. PMID- 8615032 TI - Covalently crosslinked complexes of human immunodeficiency virus type 1 (HIV-1) gp120 and CD4 receptor elicit a neutralizing immune response that includes antibodies selective for primary virus isolates. AB - Specific conformational changes in the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1) may be critical for eliciting a broadly neutralizing immune response against primary virus isolates. Since the interaction of gp120 with its receptor, CD4, induces conformational perturbations in both molecules, gp120-CD4 complexes should present unique immunogenic features that may include novel epitopes for broadly neutralizing antibodies. To test this hypothesis, we raised polyclonal antiserum against covalently crosslinked gp120 CD4 complexes in a goat and examined the ability of the anti-complex antibodies to neutralize primary and laboratory-adapted HIV-1 isolates. In cell-free neutralization assays with HIV-1MN, the antiserum demonstrated the ability to neutralize primary virus more effectively than the laboratory-adapted isolate. The neutralizing capacity of the anti-complex serum extended to primary isolates from distant genetic clades A, D, and E, although the degree of neutralization was found to vary among the clades. The neutralizing activity of the serum was composed of two components. The first component included anti-CD4 antibodies that recognized epitopes outside the gp120 binding site; the second was independent of CD4 reactivity and was retained after removal of cell surface anti-CD4 reactivity by repeated absorption with CD4-positive cells. These results demonstrate that gp120-CD4 complexes can elicit a unique polyclonal antibody response that is relevant to the neutralization of primary isolates of HIV-1. PMID- 8615033 TI - Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement. AB - Geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infect Nicotiana benthamiana systemically, is confined to the inoculated leaves of Nicotiana tabacum or Datura stramonium. We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves of N. benthamiana. In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus-host adaptation. TGMV is well adapted to N. benthamiana and does not require CP to infect this host systemically, whereas BGMV is poorly adapted to N. benthamiana and requires CP. Analysis of TGMV-BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement in N. benthamiana. Thus, bipartite geminivirus movement in planta can be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted. PMID- 8615034 TI - Glycoprotein incorporation and HIV-1 infectivity despite exchange of the gp160 membrane-spanning domain. AB - We have examined the role of the membrane-anchoring domain of the HIV-1 glycoproteins in viral glycoprotein function, glycoprotein incorporation, and viral infectivity. For this purpose, we initially exchanged the entire membrane spanning region with that from a cellular glycoprotein (CD22). Subsequently, the strictly conserved arginine in the central position of the transmembranal alpha helix was replaced by a neutral residue (R696 --> I696). We have further examined the requirements within the cytoplasmic C-terminus for glycoprotein incorporation and replaced this region of gp160 with the long cytoplasmic C-terminus (118 amino acids) from CD22. Our results show that the specific amino acid sequence of the membrane-spanning region of gp160 is not necessary for viral infectivity, thus making it unlikely that this region is specifically involved in membrane fusion, in glycoprotein incorporation, or in infectivity of the cell lines tested. In contrast, recombinant gp160 with the CD22 C-terminal region, although present at the cell surface and membrane fusion-competent, was excluded from incorporation into particles. This could indicate that steric exclusion, and no pseudotyping, occurs when the heterologous, cytoplasmic C-terminal region is too long and not fitting. PMID- 8615035 TI - No intermolecular interaction between the large hepatitis delta antigens is required for the secretion with hepatitis B surface antigen: a model of empty HDV particle. AB - The large delta antigen (LDAg) of hepatitis D virus (HDV), which is similar to the small delta antigen (SDAg), except it has 19 additional amino acids and an isoprenylation signal at the C-terminus, is crucial for interacting with hepatitis B surface antigen (HBsAg) to form a mature virion of HDV. Previous studies indicated that the LDAg alone, but not SDAg, can interact with HBsAg to form an empty particle. However, no evidence yet shows whether the intermolecular interaction of LDAg is necessary for forming an empty HDV particle. By cotransfection of plasmids encoding deletion or isoprenylation-negative mutants of LDAg with a plasmid encoding HBsAg into human hepatoma cells, we demonstrated that (i) the isoprenylation-negative LDAg cannot be secreted, (ii) the coiled coil domain-deleted LDAg retains the secretion capability, (iii) the isoprenylation-negative LDAg can neither cosecrete with isoprenylation-positive LDAg nor suppress its secretion, and (iv) an intermolecular interaction between LDAgs is unlikely required for secretion. A hypothetical model of empty HDV particle containing HBsAg with isoprenylated LDAgs, which are probably present in a singular form, was then proposed. PMID- 8615036 TI - Syncytium formation by recombinant HTLV-II envelope glycoprotein. AB - Infection by human T-cell leukemia viruses (HTLV-I and HTLV-II) induces syncytium formation in certain cell types in vitro. Recombinant vaccinia viruses expressing the HTLV-II envelope (Env) gp61-II were used to infect human cells, and syncytium formation was observed, demonstrating that HTLV-II Env expression was sufficient to mediate syncytium formation in appropriate cells. This syncytium formation could be blocked by sera from HTLV-II-infected individuals. Infection with these recombinant vaccinia viruses caused fusion in mouse/human hybrid cells containing only human chromosome 17, whereas there was no obvious syncytium formation in mouse cells. This fusogenic phenotype of recombinant gp61-II is likely a property of the specific interaction between HTLV-II envelope protein and the HTLV cellular receptor, encoded by a gene that was previously indicated to be on human chromosome 17. On this basis, we developed a sensitive syncytium formation assay, using lacZ gene activation, for detecting the presence of the HTLV receptor(s). We used this fusion assay to test for HTLV receptor activity on a variety of cells. Our findings indicate that the HTLV receptor is widely distributed among species and cell types, including, to a limited extent, murine cells. PMID- 8615037 TI - Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus. AB - A recombinant baculovirus harboring the hemagglutinin (HA) gene of African swine fever virus, with homology to the T-lymphocyte surface antigen CD2, was constructed. The efficient expression of the HA gene was determined by immunofluorescence and Western blot studies on insect cells infected with the recombinant baculovirus. The baculovirus-expressed HA showed hemadsorption and erythrocyte-agglutinating activities characteristic of the CD2 homolog protein induced by the virus in infected macrophages. Pigs immunized with the recombinant HA developed hemagglutination-inhibition and temporary infection-inhibition antibodies that recognize a 75-kDa structural protein and were protected against lethal infection. PMID- 8615038 TI - A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety. AB - Successful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in vivo gene delivery. PMID- 8615039 TI - Mode of interaction between pseudorabies virus and heparan sulfate/heparin. AB - It has been demonstrated that the efficient attachment of pseudorabies virus (PrV) is mediated by an interaction between glycoprotein C (gC) and a cellular heparin-like substance (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and t. Ben-Porat, J. Virol. 64, 278-286, 1990). According to the prevalent concept, this interaction is likely to occur between clusters of basic residues of PrV gC and the negatively charged sulfate esters and carboxylate groups of heparan sulfate/heparin. To elucidate which of the three major types of sulfate groups of heparan sulfate/heparin are involved in the interaction with PrV, we used selectively N-, 2-O-, and 6-O-desulfated samples and other modified heparins as competitors in virus-attachment assays. PrV exhibited limited preference for the specific sulfate groups of heparan sulfate/heparin in accordance with a hierarchy of 6-O- > 2-O- > N-sulfates. In addition, since selective removal of any of the specific sulfates had only a slight effect on the competition capacity of heparin, it is likely that the combination of any two of three types of sulfate groups could contribute to an interaction with PrV with an efficiency nearly equal to native, fully sulfated heparin. When tested on different cell lines the pattern of PrV requirement for the specific O-sulfate groups, i.e., 6-O sulfates > 2-O-sulfates, remained the same. However, different minimum lengths of heparin fragments were required to inhibit PrV attachment to different cell lines, suggesting a relative virus flexibility in accommodation to different forms of heparan sulfate. PMID- 8615040 TI - Homotypic interaction and multimerization of hepatitis C virus core protein. AB - Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and may possess multiple functions. In this study, we demonstrated the homotypic interaction and multimerization of HCV core protein in vitro and in vivo. By using a yeast two-hybrid system, we showed that the amino-terminal hydrophilic portion (amino acids 1-115) of the core protein could interact with itself. Deletion analysis mapped the interacting domain within amino acid residues 36-91. The homotypic interaction of the core protein was also confirmed by in vitro protein-protein blotting assay using the recombinant HCV core proteins and by its binding to the glutathione S-transferase core fusion protein. The biological significance of the core protein self-interactions was demonstrated by the detection of multimeric forms of the core protein in mammalian cells. The domain responsible for multimerization was determined to be within the amino-terminal hydrophilic region (amino acids 1-115). Both the membrane-bound and the free core proteins exist in dimeric and multimeric forms, suggesting that multimerization of the HCV core protein occurred at an early stage of viral assembly and that the multimer forms may be involved in multiple functions of the core protein. PMID- 8615041 TI - The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. AB - We have studied the production and release of infectious DI-particles in vaccinia T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3' end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity. PMID- 8615042 TI - Poliovirus recombinants expressing hepatitis B virus antigens elicited a humoral immune response in susceptible mice. AB - Expression of foreign antigens in the context of poliovirus vectors may provide a plausible approach to vaccine development. Poliovirus recombinants were constructed by fusing preS surface or core HBV proteins to the poliovirus polyprotein as previously described (Andino et al., Science, 265, 1448-1451, 1994). All recombinant viruses replicated with near wild-type efficiency in tissue culture cells and stably expressed high levels of the HBV antigens. The kinetics of recombinant RNA synthesis were indistinguishable from that of wild type poliovirus. Exogenous proteins were not incorporated into the poliovirus particles, but HBV core proteins self-assembled into 100S particles composed of free HBV core proteins and fusions with poliovirus capsid proteins. Mice susceptible to poliovirus infection were inoculated with recombinant virus and elicited humoral immune responses against the HBV antigens. PMID- 8615043 TI - NF-kappaB activation Is delayed in mouse L929 cells infected with interferon suppressing, but not inducing, vesicular stomatitis virus strains. AB - Vesicular stomatitis virus (VSV) mutant T1026R1 of the Indiana (IN) serotype is a good inducer of interferon (IFN). This mutant was used to study the activation of NF-kappaB, a transcription factor necessary for IFN induction, in mouse L929 cells that were stably transfected with a chimeric gene containing the human IFN beta gene promoter attached to the chloramphenicol acetyltransferase (CAT) coding sequence. NF-kappaB DNA binding activity was detected as early as 30 min after virus adsorption in nuclear extracts, increased up to 4 hr, and then remained constant for at least 6 additional hr. The kinetics of CAT expression correlated with the kinetics of NF-kappaB nuclear DNA binding activity. Virus entry and delivery of viral components into the cytoplasm were required for NF-kappaB activation. Exposure of T1026R1 to one hit of UV irradiation nearly completely reduced NF-kappaB activation. In cells infected with wild-type (wt) VSV (IN), a noninducer of IFN, NF-kappaB DNA binding activity in the nucleus was delayed for several hours after virus adsorption. Coinfection of wt VSV and T1026R1 resulted in the reduction of T1026R1-promoted NF-kappaB activation. This inhibitory activity of wt VSV was abolished by one hit of UV irradiation. Under similar conditions expression of the CAT gene was more UV resistant, suggesting that IFN gene expression is regulated at multiple levels. PMID- 8615044 TI - Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions. AB - The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S. I) of both viral origin regions, whereas SVT preferentially bound B.S. I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo. PMID- 8615045 TI - Mutational analysis of the fusion peptide of the human immunodeficiency virus type 1: identification of critical glycine residues. AB - The ability of human immunodeficiency virus type 1 (HIV-1) to fuse its membrane with the membrane of the target cell is a function of a approximately 23-amino acid amino-terminal segment of the gp41 subunit of the envelope glycoprotein complex, known as the fusion peptide. The sequence of the fusion peptide is highly conserved among different variants of HIV-1 and is also very similar to that of HIV-2 and SIV. The fusion peptide is very hydrophobic and has a high content of glycine and alanine residues. Representation of the fusion peptide of HIV-1 as an alpha-helix predicts that most glycine residues would be found on one face of the alpha-helix. To assess the importance of the glycine residues for the fusogenic activity of the envelope glycoprotein complex, we mutagenized each glycine residue in the fusion peptide individually to a valine residue. The mutant envelope constructs were tested for their ability to induce syncytia (cell/cell fusion) and to mediate infection (virus/cell fusion) of CD4-positive cells. The results of our analyses show that two glycine residues (G10 and G13) located within the sequence FLGFLG in the middle of the fusion peptide are critical for syncytium formation and for the establishment of a productive infection, whereas other glycine residues (G3, G5, and G20) are more permissive to substitutions. Mutation of each of the two phenylalanines (F8 and F11) of the FLGFLG sequence to valine also decreased fusion, although to a lesser extent than mutation of G10 and G13. These observations demonstrate that G10 and G13 are critical elements of the fusion peptide and suggest that, in addition to hydrophobicity, the exact amino acid composition and structure of the fusion peptide are critical for function. PMID- 8615046 TI - Multinucleate cell angiohistiocytoma. A review and report of four cases. AB - Multinucleate cell angiohistiocytoma (MCA) was first characterized by Smith & Wilson-Jones. Although only a few cases have been published, this very characteristic benign tumor is probably not rare. The clinical pictures are firm circumscribed papules, mainly of the hands, which progress slowly over the years. The histologic features show an increase of capillaries and venules in the reticular dermis, overlaid by epidermal hyperplasia. Bizarre-shaped multinucleate cells are present between the vessels, and factor XIIIa-positive interstitial cells are increased in number. MCA can be easily recognised if the pathologist is aware of the diagnosis. We present here the clinical, histologic and immunopathologic features of 4 new cases and review the literature. PMID- 8615047 TI - A study of the steroid sulfatase gene in families with X-linked ichthyosis using polymerase chain reaction. AB - We have studied the steroid sulfatase (STS) gene in three Japanese families with X-linked ichthyosis (XLI), using polymerase chain reaction (PCR). PCR was performed using three sets of intraexonic primers covering exons 1, 5 and 10. In affected individuals from two of the families, DNA was not amplified in any of the three exons, suggesting that XLI in these families was due to the complete deletion of the STS gene. In affected individuals in the remaining family, DNA was amplified in predicted sizes in exons 1 and 5, but not in exon 10, suggesting that XLI in this family was due to partial deletion of the STS gene including exon 10. These results suggested that STS gene deficiency is heterogeneous in Japanese families with XLI. PCR is useful for the rapid diagnosis of XLI, the differentiation of XLI from ichthyosis vulgaris, and genetic counseling of XLI families. The PCR method was not applicable for carrier detection. PMID- 8615048 TI - Human in vivo cutaneous microdialysis: estimation of histamine release in cold urticaria. AB - A novel bioanalytical in vivo sampling technique, cutaneous microdialysis, was used to follow the chronology of skin histamine release in 3 patients with cold urticaria and in 2 healthy volunteers. Laser Doppler perfusion imaging was used simultaneously to monitor the skin circulatory response. Microdialysis samples were collected at 10-min intervals and analysed by radioimmunoassay technique. Fifty minutes after probe insertion, the ventral forearm skin in the area of the dialysis membrane was provoked for 5-15 min with a 25 x 40 mm ice cube covered with plastic foil. In the cold urticaria patients, an up to 80-fold increase of histamine was observed, with peak levels 20-30 min after challenge. Histamine levels then fell to reach "baseline" levels within 50 min. In the healthy subjects, the histamine increase was earlier, less pronounced and of shorter duration. Cutaneous microdialysis and laser Doppler imaging offer new possibilities for the chronological multiparameter assessment of inflammatory skin disorders in vivo. PMID- 8615049 TI - Histamine-induced itch and alloknesis (itchy skin) in atopic eczema patients and controls. AB - Alloknesis ("itchy skin") after histamine iontophoresis was studied together with itch sensations and skin reactions in 19 atopic eczema patients and 20 controls at the forearm and at the scapular area. Compared to controls, atopic eczema patients showed significantly reduced alloknesis or total lack of it in the area around a skin site to which histamine had been iontophoretically applied, although histamine elicited itching in most patients. As previously demonstrated, patients with atopic eczema also developed significantly smaller flares. However, covariance analysis revealed that the smaller alloknesis areas in atopic patients were not statistically related to the smaller flares. Our results suggest that in atopic eczema a diminished responsiveness of primary afferent nerves to histamine is not compensated by a higher central nervous sensitivity reflected in more vivid alloknesis responses to histamine. Therefore, we conclude that histamine is probably not the key factor of the spontaneous itch experienced by patients with atopic eczema. PMID- 8615050 TI - Modulation of CD3-dependent lymphocyte proliferation by extracellular matrix proteins in atopic dermatitis. AB - Patients with atopic dermatitis were found to have an about 7-fold increased spontaneous proliferative response of peripheral blood lymphocytes and an about 4 fold elevation of CD3-dependent lymphocyte transformation as compared to normal controls. The CD3-dependent lymphocyte response in patients with severe atopic dermatitis lesions was increased to a lower degree than in those with mild skin lesions. Despite a highly increased CD3-dependent lymphocyte response, the extracellular matrix proteins could induce further co-stimulation of lymphocytes in patients with atopic dermatitis, similar to that in normal controls. However, co-activation by type IV collagen was markedly increased in patients with severe lesions, whereas co-stimulations by both type I collagen and fibronectin were decreased in patients with mild lesions. This finding reflects presumably the changes in lymphocyte subpopulations and their activities related to the recirculation of these cells through the active skin lesions and to the contact of T cells with extracellular matrix proteins. The percentage of CD26-positive lymphocytes was also significantly (p < 0.05) increased in patients with severe atopic dermatitis. These data indicate that helper T cells are excessively activated in atopic dermatitis and that the function of beta-1-integrin receptors underlying the extracellular matrix protein-mediated co-activation of CD3 dependent lymphocyte responses is modified by disease severity. PMID- 8615051 TI - Pityrosporum ovale extracts increase interleukin-4, interleukin-10 and IgE synthesis in patients with atopic eczema. AB - Evidence for a possible role of the lipophilic yeast Pityrosporum ovale in the pathophysiology of atopic eczema has been found both in laboratory and therapeutical studies. Positive type I prick test reactions to P. ovale correlate with the intensity of eczematous skin lesions in the head and neck regions of patients with atopic eczema. Furthermore, antifungal treatment has been shown to be helpful in atopic eczema. In the present study the effect of P. ovale on IgE synthesis and cytokine production (IL-2, IFN gamma, IL-4, IL-10) was investigated in patients with atopic eczema, in vitro. Eight patients with atopic eczema were studied; of these, 5 patients had specific IgE antibodies against P. ovale, as determined by fluoroimmunoassay (RAST). The control group consisted of 5 healthy non-atopic, P. ovale IgE-antibody-negative volunteers. Freshly isolated peripheral blood mononuclear cells (PBMC) were incubated in the presence of different antigen concentrations (0.01, 0.1, 1.0, 10 micrograms/l x 10(6) cells) of P. ovale. IgE contents in the cell culture supematants were significantly elevated in RAST(+) patients with atopic eczema (p < 0.05), compared with RAST(-) atopic eczema patients and healthy volunteers. Coincubation of P. ovale stimulated PBMC with IL-4 (50 U/l/ 1 x 10(6) cells) resulted in a significantly higher IgE synthesis only in the RAST(+) atopic eczema patients. Additionally, incubation of PBMC from RAST(+) patients with atopic eczema led to an elevated synthesis of the TH2 related cytokines IL-4 and IL-10. Within the atopic eczema group, two subgroups differed markedly in their response to P. ovale antigen stimulation with a good correlation to the presence of specific IgE in serum and in vitro IL-4 and IL-10 production. The data support the assumption that P. ovale antigens might play a role in skin inflammation in at least a subgroup of patients with atopic eczema characterized by the presence of specific IgE antibodies to P. ovale. PMID- 8615052 TI - Intracutaneous transport of orally administered fluconazole to the stratum corneum. AB - Fluconazole administered at 150 mg/week for 1-5 weeks is effective orally against dermatophytes and yeast in stratum corneum. Clinical and mycological cure rates approach 90%, but the precise distribution of the drug within various layers of skin is uncertain. We administered fluconazole at 150 mg/week for 2 weeks to 5 volunteers. Distribution of fluconazole in biopsies of skin was imaged by energy dispersive analysis of X-rays (EDX) and transmission electron microscopy, and in cells by electron energy-loss spectroscopy (EELS). Eight hours after a second dose, EDX showed fluconazole highest and homogeneously distributed in stratum corneum, lower in the rest of the epidermis, and lowest in dermis. The highest fluconazole levels detected by EELS were in cytoplasmic inclusions of sweat and sebaceous glands and less in keratinocytes and dermal collagen. We conclude that fluconazole delivered to stratum corneum by direct diffusion from capillaries and in sweat is also in all likelihood transported in sebum. PMID- 8615053 TI - The effect of calcipotriol on lesional fibroblasts from patients with active morphoea. AB - We examined the responsiveness of cultured dermal fibroblasts from biopsies of 6 patients with active morphoea and a similar number of matched controls to the cell proliferation inhibition activity of calcipotriol. Cultured fibroblasts from controls showed no significant response to calcipotriol (at concentrations of 1 x 10(-8) to 1 x 10(-4) M). However, calcipotriol did inhibit the proliferation response of morphoea fibroblasts at all concentrations when compared with controls. There was 4- to 20-fold inhibition in 2 of the morphoea patients when compared with control samples. Four other morphoea samples showed inhibition but to a lesser extent compared with controls. PMID- 8615054 TI - The stimulatory effects of PDGF and TGF-beta 1 on dermal fibroblast attachment. AB - We investigated the effects of various growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha), keratinocyte growth factor (KGF)) on fibroblast attachment to plastic plates. It is thought that cell attachment to plastic plates in vitro may represent the step between cell migration and proliferation in vivo during wound healing. Among the growth factors examined, only PDGF and TGF-beta 1 significantly increased fibroblast attachment to both uncoated and collagen-coated plates in a concentration dependent manner. The addition of anti-PDGF antibody abolished the enhancing effect of PDGF but not that of TGF-beta 1, suggesting that the effect of TGF-beta 1 is not through the autocrine induction of PDGF-related activities secreted by the fibroblasts themselves. These data suggest that PDGF and TGF-beta 1 regulate fibroblast attachment to the suitable environment in the process of dermal wound healing in vivo. PMID- 8615055 TI - Raimer's bands: case report with a review of solar elastosis. AB - Raimer's solar elastotic bands of the forearm are a rarely described clinical variant of severe solar elastosis. We report a case of Raimer's bands on the forearm of a 43-year-old woman. Light microscopy demonstrated solar elastosis and electron microscopy revealed activated fibroblasts appearing to secrete the elastotic material. The clinical variants of solar elastosis are outlined and the possible pathogenesis of the elastotic material is discussed. PMID- 8615056 TI - High-frequency ultrasound for torture-inflicted skin lesions. AB - Objectivity, reproducibility and non-invasiveness are required for a medico-legal evaluation of skin lesions in torture victims. In this report we describe the use of the high-frequency method for imaging of scars presumed to be inflicted by torturing. The extent of scarring and the size of the lesions could be determined precisely and objectively. We suggest that skin ultrasonography may be a useful supplementary method for evaluation of skin changes in torture victims. PMID- 8615057 TI - Effects of gemfibrozil (Lopid) on hyperlipidemia in acitretin-treated patients. Results of a double-blind cross-over study. AB - Hyperlipidemia is a common side-effect of oral retinoid treatment, which sometimes interferes with long-term therapy. To evaluate the safety and efficacy of the lipid-lowering drug gemfibrozil on retinoid-associated hyperlipidemia, we studied the clinical response and the plasma lipoprotein levels in 22 acitretin treated (0.25-0.75 mg/kg) patients mainly suffering from psoriasis. Gemfibrozil or placebo was given in a double-blind cross-over fashion to 14 patients, who after 8 weeks of acitretin therapy and dietary advice exhibited hyperlipidemia (triglyceride levels > or = 50% above baseline and/or > or = 2.0 mmol/l). Serum triglycerides remained high (3.7 +/- 2.4 mmol/l) during placebo treatment but were reduced after 8 weeks of gemfibrozil treatment (p < 0.01). The total cholesterol level decreased slightly (p < 0.05) during gemfibrozil treatment, but the LDL/HDL ratio did not change significantly. No untoward effects of gemfibrozil on acitretin dose-response and clinical side-effects were noted. Gemfibrozil thus appears useful in patients prone to retinoid-induced hyperlipidemia unresponsive to dietary treatment and acitretin dose reductions. PMID- 8615058 TI - Topical treatment of psoriatic plaques with 1 alpha, 24 dihydroxyvitamin D3: a multiparameter flow cytometrical analysis of epidermal growth, differentiation and inflammation. AB - The clinical efficacy and tolerability of the vitamin D3 analogues calcitriol, calcipotriol and 1 alpha, 24 dihydroxyvitamin D3 in the treatment of psoriasis have been assessed in various clinical studies. In vitro and in vivo investigations have shown interference of these compounds with epidermal growth, keratinisation and inflammation. In this study we quantified the in vivo cell biological effects during treatment of psoriatic plaques with 1 alpha, 24 dihydroxyvitamin D3. By using a flow cytometric triple labelling procedure, we could discriminate different epidermal subpopulations, permitting precise assessment of epidermal cell cycle kinetics. Twenty patients with plaque-type psoriasis were treated in a double-blind placebo-controlled left-right comparative study with 1 alpha, 24 dihydroxyvitamin D3 ointment (4 micrograms/g applied once daily) for 8 weeks. Epidermal cell suspensions prepared from keratotome biopsies taken before and after treatment were stained with TO-PRO-3 iodide (a new DNA fluorochrome) and monoclonal antibodies against keratin 10 (as a marker for differentiation) and vimentin (as a marker for inflammation), simultaneously. The flow cytometric analyses showed a significant decrease of proliferating basal keratinocytes in verum-treated lesions, whereas such a decrease was not observed in placebo-treated lesions. The amount of keratin 10 positive keratinocytes increased and the presence of vimentin-positive cells decreased in cell suspensions derived from both verum- and placebo-treated lesions, but these effects were not significant. We conclude that multiparameter flow cytometry promises to be an adequate approach to assess the interference of antipsoriatic treatments with cutaneous inflammation, epidermal proliferation and keratinisation. Topical 1 alpha, 24 dihydroxyvitamin D3 seems to exert its in vivo antipsoriatic effect mainly through an inhibition of epidermal growth. PMID- 8615059 TI - UVB versus UVB plus calcipotriol (MC 903) therapy for psoriasis vulgaris. AB - It is well known that UVB therapy of psoriasis vulgaris is potentiated by the association with topical and oral drugs, while till now there have been very few reports on the association between UVB and calcipotriol. In order to evaluate the efficacy and tolerance of this association, we studied 19 patients with psoriasis vulgaris of mild severity (PASI: 5-10). Each patient was treated with UVB and invited to apply calcipotriol 50 micrograms/g ointment twice a day on one lesion usually on elbows or knees in order to compare it with the opposite side. The evaluation of each lesion was performed before and after 4 weeks of therapy. Our data show that the association between UVB and calcipotriol is significantly more effective than UVB therapy alone: 17 out of 19 patients (89%) showed a greater improvement with UVB plus calcipotriol as compared to UVB alone. PMID- 8615060 TI - The hair root pattern after calcipotriol treatment for scalp psoriasis. AB - Scalp psoriasis is associated with hair loss and an increased telogen/anagen ratio. Topical treatment of scalp psoriasis (with corticosteroids, dithranol or tar) results in decreased scaling, induration and erythema of the plaques. Calcipotriol is effective in the treatment of psoriasis vulgaris. However, the potent growth-inhibiting potential of this compound might theoretically induce hair loss. A study was designed to find out to what extent calcipotriol treatment modulates the percentage of anagen and telogen hair during treatment of scalp psoriasis. A group of 26 patients participated in a placebo-controlled dose finding study on the efficacy of calcipotriol in scalp psoriasis. Hair plucks before and after treatment were taken. The telogen/anagen ratio remained unaffected during 6 weeks of calcipotriol treatment. No correlation was demonstrated between efficacy of treatment and quantification of telogen/anagen ratio. It can be concluded that the growth-inhibiting potential of calcipotriol is not reflected in the in vivo hair growth pattern during calcipotriol treatment. PMID- 8615061 TI - Methotrexate-linked ventricular arrhythmias. AB - A 36-year-old male, who 1 year previously had survived a large anterior myocardial infarction, followed by cardiac arrest, was treated a few months for psoriasis with oral methotrexate, at single weekly oral doses of up to 10 mg, when he had to be hospitalized due to anginal pain and palpitation. Repeated 24 hour electrocardiogram recordings revealed ventricular ectopy up to 580 premature beats per hour. The ventricular premature beats were almost completely abolished after a few days' discontinuation of methotrexate therapy but recurred a few hours after an attempt to restart it had been made. A coronary angiogram showed only minimal wall abnormalities. Electrophysiological testing and endomyocardial biopsy were normal. PMID- 8615062 TI - Interruption of long-term methotrexate treatment in psoriasis. Evaluation of clinical course and laboratory parameters after discontinuation and reintroduction of weekly oral methotrexate. AB - The effects of interval treatment were evaluated in 10 psoriatic patients on long term treatment with low-dose oral methotrexate (MTX). In all patients, the dosage of MTX had already been tapered off as much as possible. After interruption of MTX treatment, the clinical course and changes in laboratory parameters were evaluated. The mean MTX-free period was 17 weeks, and the mean reduction in cumulative MTX dose was 76 mg (p = 0.05). However, only 3 patients preferred interval treatment to a continuous schedule. During the first 3 weeks of discontinuation, a significant decrease in the serum transaminases was observed, indicating a direct toxic influence of MTX on the liver parenchym. We conclude that interruption of long-term MTX treatment leads to a substantial reduction of the cumulative MTX dose and reduces the hepatotoxic load of MTX. It is necessary to motivate patients on long-term MTX treatment for regular treatment interruptions to establish a further reduction in their cumulative MTX dose. PMID- 8615063 TI - ELISA inhibition test using monoclonal antibody specific for Treponema pallidum as the serologic test for syphilis. AB - Monoclonal antibodies (Mabs) specific for Treponema pallidum were produced using hybridoma technology. In this study an ELISA inhibition test based on competitive inhibition by antibodies from human test sera of the binding of the enzyme labelled Mabs to T. pallidum was performed. Inhibition of the seroreactivity of Mabs was decreased according to the dilution of rabbit antiserum to T. pallidum. Seropositivity was found in 100% of secondary and early latent syphilis patients, 75% of primary syphilis patients, and 80% of late latent syphilis patients. The mean percentage inhibition was significantly higher in each syphilis group than in the controls, and statistically significant differences were shown between all the syphilis groups. VDRL and TPHA titers were correlated with the percentage inhibitions. Therefore the ELISA inhibition test using Mab specific for T. pallidum might well be a suitable tool as a new serologic test for syphilis. PMID- 8615065 TI - Histopathology of nail sarcoidosis. PMID- 8615064 TI - Cutaneous complications in heart transplant recipients in Norway 1983-1993. AB - All Norweigian heart transplant recipients with more than one year's survival (n = 140) were investigated for dermatological disorders. Observation time after transplantation was 1-10 years (mean 5.0 years). Patients alive at the end of 1993 (n = 122) were examined clinically, and medical records for all patients were reviewed. The histopathological diagnoses of excised skin specimens were reevaluated. Malignant skin tumours (i.e. basal cell carcinoma, squamous cell carcinoma and malignant melanoma) and/or premalignant skin tumours (i.e. morbus Bowen (carcinoma in situ), solar keratosis and keratoacanthoma) were found in 34 patients (24.3%), of which 18 patients (12.9%) had malignant skin tumours. Seventeen lesions diagnosed as keratoacanthoma and two lesions diagnosed as morbus Bowen had primarily been diagnosed as squamous cell carcinoma. Five patients (3.6%) had multiple keratoacanthomas. Other frequent dermatological diagnoses included hypertrichosis (86.9%), steroid-induced skin features (59.8%), common warts (42.6%) and seborrheic skin disorders (20.5%). This study demonstrates the importance of dermatological surveillance in the follow-up of heart transplant recipients. PMID- 8615066 TI - Epidermolytic hyperkeratosis of the nails in keratosis palmoplantaris nummularis. PMID- 8615067 TI - Perforating granuloma annulare and HIV. PMID- 8615068 TI - Segmental neurofibromatosis versus giant nevus spilus. PMID- 8615069 TI - Cutaneous vasculitis with lesions mimicking degos' disease and revealing Crohn's disease. PMID- 8615070 TI - Transient acantholytic dermatosis associated with superior vena caval syndrome in a patient with carcinoid tumour of the thymus. PMID- 8615071 TI - Successful treatment of papillomatosis cutis lymphostatica with acitretin. PMID- 8615072 TI - Apoptosis in normal skin. PMID- 8615073 TI - Isotretinoin-induced pemphigus. PMID- 8615074 TI - Paired helical filament tau (PHFtau) in Niemann-Pick type C disease is similar to PHFtau in Alzheimer's disease. AB - Niemann-Pick Type C disease (NPC) is a cholesterol storage disease with defects in the intracellular trafficking of exogenous cholesterol derived from low density lipoproteins. In NPC cases with a chronic progressive course, neurofibrillary tangles (NFTs) that consist of paired helical filaments (PHFs) have been reported. To determine if NPC tangles contain abnormal tau proteins (known as PHFtau) similar to those found in Alzheimer's disease (AD) tangles, we examined the brains of five NPC cases by immunohistochemical and Western blot methods using a library of antibodies to defined epitopes of PHFtau. We show here that PHFtau in tangle-rich NPC brains is indistinguishable from PHFtau in AD brains. We speculate, that the generation of PHFtau in NPC may induce a cascade of pathological events that contribute to the widespread degeneration of neurons, and that these events may be similar in NPC and AD. PMID- 8615075 TI - Structural changes of anterior horn neurons and their synaptic input caudal to a low thoracic spinal cord hemisection in the adult rat: a light and electron microscopic study. AB - Structural changes in lumbosacral ventral horn neurons and their synaptic input were studied at 3, 10, 21, 42, and 90 days following low thoracic cord hemisection in adult rats by light microscopic examination of synaptophysin immunoreactivity (SYN-IR) and by electron microscopy. There was an ipsilateral transient decrease in SYN-IR at the somal and proximal dendritic surfaces of anterior horn neurons which extended caudally from the site of injury over a postoperative (p.o.) period of 42 days. Concomitantly, at 21 days p.o., perineuronal SYN-IR started to recover in upper lumbar segments. By 90 days p.o., a normal staining pattern of SYN was noted in upper and mid lumbar segments, but the perineuronal SYN-IR was still slightly below normal levels in low lumbar and sacral segments. Electron microscopy revealed ultrastructural changes coincident with the alterations in SYN-IR. At 3 days p.o., phagocytosis of degenerating axon terminals by activated microglial cells was observed at the somal and proximal dendritic surfaces of ventral horn neurons. These changes were most prominent up to two segments caudal to the lesion. At 10 days p.o., advanced stages of bouton phagocytosis were still detectable in all lumbosacral motor nuclei. Additionally, abnormal axon terminals, with a few dispersed synaptic vesicles and accumulations of large mitochondria, appeared at the scalloped somal surfaces of anterior horn neurons. At 21 days p.o., several large lumbosacral motoneurons had developed chromatolysis-like ultrastructural alterations and motoneuronal cell bodies had become partially covered by astrocytic lamellae. At 42 days p.o., there was a transient appearance of polyribosomes in some M-type boutons. In addition, at 42 and 90 days p.o., a few degenerating motoneurons were detected in all lumbosacral segments, but most displayed normal neuronal cell bodies contacted by numerous intact synapses as well as by astrocytic processes. In contrast to these striking alterations of synaptic input at somal and proximal dendritic surfaces of motoneurons, relatively few degenerating boutons were detected in the neuropil of motor nuclei at all the p.o. times studied. We suggest that the preferential disturbance of the predominantly inhibitory axosomatic synapses on ventral horn neurons may be involved in the mechanisms which influence the well-established increase in motoneuronal excitability after spinal cord injury. PMID- 8615076 TI - Bovine herpesvirus meningoencephalitis association with infectious bovine rhinotracheitis (IBR) vaccine. AB - During 1992, on a farm in the Tokachi district of Hokkaido, Japan, approximately 20 Holstein-Friesian calves showed neuroparalysis and died within 7-10 days after routine vaccination. Six male calves, aged about 1.5 months, were submitted to our laboratory for pathological examination and diagnosed as acute or subacute necrotizing meningoencephalitis due to bovine herpes virus (BHV) infection. The main necropsy findings included a few hemorrhages or clots, and malacic lesions localized in the cortical to subcortical area of the cerebrum. Histopathological brain lesions were characterized by laminar or focal necrosis of neurons, accompanying macrophages, polymorphonuclear cell infiltration, severe astrogliosis, and perivascular cuffing in all six calves. Nuclear basophilic inclusion bodies, which showed positive reaction with immunocytochemical staining of BHV antigen, were observed in the necrotic neurons, astroglia and oligodendroglia in five affected calves. BHV antigens were also seen in the cell bodies and cell processes of the necrotic neurons, which was indicative of cell to-cell propagation of infection. There was a general tendency for more severe lesions to be located at the cortex to subcortex of the cerebrum. Milder lesions were observed in the cerebellum and brain stem. These findings suggest that the infectious route to the cerebrum in the present cases was through the olfactory bulbs and/or along the meninges beginning from the ethmoid bone, rather than through the trigeminal ganglia route as had been emphasized in studies dealing with experimental infection. PMID- 8615077 TI - Structural and immunocytochemical features of olivopontocerebellar atrophy caused by the spinocerebellar ataxia type 1 (SCA-1) mutation define a unique phenotype. AB - Neuropathological investigations performed on autopsied brain and spinal cords from 11 patients showed that spinocerebellar ataxia type 1 (SCA-1) can be distinguished from autosomal dominant spinocerebellar ataxia linked to SCA-2 and 3 loci on chromosomes 12 and 14, spinopontine, and the multisystem atrophies. The major diagnostic criteria were: absence of significant pars compacta nigral and locus coeruleus lesions, severe degeneration of olivocerebellar and dentatorubral pathways, extensive loss of Purkinje cells with partial sparing of flocculonodular lobes, severe atrophy of specific cranial nerve nuclei, mostly the third and 12th, extensive loss of motor neurons in anterior horns and Clarke's columns, and lack of oligodendroglial or neuronal cytoplasmic cytoskeletal inclusions. None of the brains displayed any significant immunoreactivity for the amyloid precursor protein or beta-amyloid peptide throughout the cortex. In conclusion, the type and neuroanatomical distribution of structural lesions were similarly reproduced in all probands at the end stage of SCA-1, to the point that they appeared to constitute a unique phenotype. PMID- 8615078 TI - Early endothelial damage and leukocyte accumulation in piglet brains following cardiac arrest. AB - This study examined the early microvascular and neuronal consequences of cardiac arrest and resuscitation in piglets. We hypothesized that early morphological changes occur after cardiac arrest and reperfusion, and that these findings are partly caused by post-resuscitation hypertension. Three groups of normothermic piglets (37.5 degrees - 38.5 degrees C) were investigated: group 1, non-ischemic time controls; group 2, piglets undergoing 8 min of cardiac arrest by ventricular fibrillation, 6 min of cardiopulmonary resuscitation (CPR) and 4 h of reperfusion; and group 3, non-ischemic hypertensive controls, receiving 6 min of CPR after only 10 s of cardiac arrest followed by 4-h survival. Immediately following resuscitation, acute hypertension occurred with peak systolic pressure equal to 197 +/- 15 mm Hg usually lasting less than 10 min. In reacted vibratome sections, isolated foci of extravasated horseradish peroxidase were noted throughout the brain within surface cortical layers and around penetrating vessels in group 2. Stained plastic sections of leaky sites demonstrated variable degrees of tissue injury. While many sections were unremarkable except for luminal red blood cells and leukocytes, other specimens contained abnormal neurons, some appearing irreversibly injured. The number of vessels containing leukocytes was higher in group 2 than in controls (3.8 +/- 0.6% vs 1.4 +/- 0.4% of vessels, P < 0.05). Evidence for irreversible neuronal injury was only seen in group 2. Endothelial vacuolization was higher in groups 2 and 3 than in group 1 (P < 0.05). Ultrastructural examination of leaky sites identified mononuclear and polymorphonuclear leukocytes adhering to the endothelium of venules and capillaries only in group 2. The early appearance of luminal leukocytes in ischemic animals indicates that these cells may contribute to the genesis of ischemia reperfusion injury in this model. In both groups 2 and 3 endothelial cells demonstrated vacuolation and luminal discontinuities with evidence of perivascular astrocytic swelling. Widespread microvascular and neuronal damage is present as early as 4 h after cardiac arrest in infant piglets. Hypertension appears to play a role in the production of some of the endothelial changes. PMID- 8615079 TI - Dystrophin-positive muscle fibers following C2 myoblast transplantation into mdx nude mice. AB - To determine when and how the dystrophin-positive muscle fibers are formed after myoblast transplantation into dystrophin-negative muscles, the tibialis anterior (TA) muscle from mdx nude mouse was chronologically examined after C2 myoblast transplantation by immunohistochemical and glucose 6-phosphate isomerase (GPI) isoenzyme analyses. The host TA muscle transplanted with C2 myoblasts became necrotic with accumulation of basic fibroblast growth factor in the necrotic areas. This may stimulate concomitant proliferation of the host satellite cells and C2 myoblasts. Small dystrophin-positive muscle fibers appeared in the necrotic areas 3 days after transplantation. This TA muscle contained two different kinds of homodimer GPI isoenzymes but did not contain the heterodimer, suggesting rare fusion of host and donor cells. The dystrophin-positive muscle fibers in the necrotic areas rapidly increased in number and in size by 7 days, but they were smaller than the original host muscle fibers. They had central nuclei, indicating that they were regenerating fibers. The presence of heterodimer GPI isoenzyme in these muscles indicated that the regenerating fibers were mosaic host/donor muscle fibers. The dystrophin-positive muscle fibers are probably formed first by fusion of donor cells with each other and then later by the fusion of host satellite and donor cells. PMID- 8615081 TI - Immunohistochemical study of apolipoprotein E in human cerebrovascular white matter lesions. AB - In the brains of ine cases with cerebrovascular disease, one with mixed dementia, one with amyloid angiopathy and two non-neurological controls, we found three cases with focal accumulation of apolipoprotein E (apo-E) in dystrophic axons and accompanying macrophages. Since amyloid precursor protein (APP) and chromogranin A (CgA) accumulate after axonal damages, and are sensitive markers of the white matter lesions, the regional distribution of apo-E was compared to that of APP and CgA. apo-E-immunoreactive axons were present in the periphery of an infarction with neighboring macrophages, but not in mild white matter lesions that contained APP- or CgA-immunoreactive fiber bundles. The results suggest a role of apo-E in recycling cholesterol and other membrane components via macrophages into remodeling neurites in the brain, but this phenomenon is restricted to the periphery of infarction and may be less prominent than in the peripheral nervous system. PMID- 8615080 TI - The membrane attack complex of complement mediates peripheral nervous system demyelination in vitro. AB - The present study used cocultures of rat dorsal root ganglia (DRG) and peritoneal macrophages to define the role of activated complement components during demyelination. The complement cascade was activated in vitro by treatment of the cultures with natural rat serum and lipopolysaccharides. Complement activation was examined by detection of the membrane attack complex of complement (MAC) with an antibody directed against rat C5-9. Detection of MAC in vitro by immunoelectron microscopy was associated with morphological changes of the myelin sheath. The sheath's regular structure was disrupted. Myelin lamellae were split and showed signs of decompaction. These changes were followed by a selective macrophage attack on myelin sheaths resulting in demyelination. Schwann cell viability was not affected by complement activation. Axons and sensory ganglion cells also survived this attack. The specificity of the complement effect was tested in experiments using treatment regimens with natural rat serum or lipopolysaccharides alone. In these experiments, no morphological changes of the myelin sheath were observed as well as no macrophage attack on myelin. PMID- 8615083 TI - Thorn-shaped astrocytes: possibly secondarily induced tau-positive glial fibrillary tangles. AB - Argyrophilic and tau-positive abnormal structures occurring in glial cells are called glial fibrillary tangles. In the astrocyte, a conspicuous tau-positive structure is known to appear in progressive supranuclear palsy (PSP). In this report, another type of argyrophilic and tau-positive astrocytes is reported. The morphology of this new type is quite different from that of the previously reported tau-positive astrocyte in PSP and they are designated here as thorn shaped astrocytes (TSA). TSA have an apparently argyrophilic cytoplasm with a few short processes and often have a small eccentric nucleus, whose appearance resembles that of a reactive astrocyte. Immunohistochemically, TSA are positive to anti-tau antibodies but are negative for ubiquitin. Simultaneous immunostaining revealed the coexistence of tau and glial fibrillary acidic protein epitopes in the same cytoplasm. Electron microscopically, bundles of 15 nm straight tubules were included in the cytoplasm together with abundant glial filaments. In the vicinity of a cluster of TSA, related structures of perivascular or subpial tau-positive linings, which correspond to astrocytic end feet, are sometimes observed. In almost all cases, a few TSA are generally located in a confined area of subpial and subependymal regions. Although TSA appear to be intimately associated with some diseases, they are also found in a wide range of cytoskeletal disorders including the aged brain with neurofibrillary tangles. TSA are presumed to be a secondary induced product in relation to astrocytic reaction. PMID- 8615082 TI - Achromatic neurons in the cortex of progressive supranuclear palsy. AB - Achromatic neurons (AN) in the cerebral cortex are a characteristic feature of several neurodegenerative conditions including Pick's disease, corticobasal degeneration and some cases of primary progressive aphasia. Although AN are occasionally seen in some other diseases, their presence in progressive supranuclear palsy (PSP) has not been previously documented. We found significant numbers of AN in the cerebral cortex of five out of seven cases which had been diagnosed pathologically as PSP. The identification of AN was greatly facilitated by the use of neurofilament immunohistochemistry. The entorhinal and transentorhinal cortices were most frequently involved, but in several cases AN were also seen throughout the neocortex. The presence and number of AN roughly correlated with a history of clinical dementia. This suggests that cortical AN may be a common and important pathological finding in PSP. PMID- 8615084 TI - Monoamine oxidase-B-positive granular structures in the hippocampus of aged senescence-accelerated mouse (SAMP8). AB - We examined the histochemical localization of monoamine oxidase in the hippocampus of young and old senescence-accelerated mouse (SAM). We found a monoamine oxidase-B-positive granular structure (MGS) in the hippocampus of old SAMP8, an accelerated senescence-prone line of SAM. The MGS was a round-shaped granular structure of 0.5 to 5 microns diameter and usually formed a cluster, the largest diameter of which ranged from 50 to 150 microns. No MGS were found in the hippocampus of young SAMP8 or of young SAMR1, an accelerated senescence resistant line of SAM, and only few, if any, were seen in old SAMR1. A monoamine oxidase positive astrocyte was usually observed in the central area of each cluster of MGS. Furthermore, the MGS was in close anatomical relationship with monoamine oxidase-positive astrocytic processes. The enzyme inhibition experiments showed that monoamine oxidase activities localized in the MGS and astrocytes were both predominantly of type B. These findings suggest MGS occurs at least partly in monoamine oxidase-B-positive astrocytes. Furthermore, the MGS was similar to a periodic acid-Schiff-positive granular structure, a polyglucosan body previously documented in the brains of old SAMP8 and some other aged mice strains including C57BL/6 and nude mice, in terms of their size, morphological appearances and topographical distribution in the hippocampus. Thus, the present results suggest that monoamine oxidase type B is a proteinaceous component of the periodic acid Schiff-positive granular structure in aged mice brains, and might provide some clues for clarifying the mechanisms of age-related occurrence of periodic acid Schiff-positive granular structures in mice brains. PMID- 8615085 TI - Astroglial localization of cholesteryl ester transfer protein in normal and Alzheimer's disease brain tissues. AB - Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that facilitates the transfer of cholesteryl esters, phospholipids and triglycerides between the lipoproteins, and regulates plasma high-density lipoprotein levels. We examined CETP-like immunolabeling in non-neurological and Alzheimer's disease (AD) liver and brain tissues. The anti-CETP antibodies showed positive staining in round cells in the liver sinusoid and in brain astrocytes. In the brains of non-neurological cases, positively stained astrocytes were preferentially distributed in the white matter. In AD tissue, many reactive astrocytes in the gray matter as well as the white matter astrocytes had CETP-like immunoreactivity. CETP-positive astrocytes may play a role for AD pathology such as tissue repair. PMID- 8615087 TI - Charcot-Marie-Tooth disease type 1A: morphological phenotype of the 17p duplication versus PMP22 point mutations. AB - Charcot-Marie-Tooth disease type 1A (CMT1A) or hereditary motor and sensory neuropathy type Ia (HMSN type Ia) is an autosomal dominant demyelinating polyneuropathy, which may result from duplications as large as 1.5 Mb on chromosome 17p 11.2-p12 encompassing the gene for the peripheral myelin protein PMP22, or from point mutations in this gene. In general, it is not possible to distinguish, by clinical and neurophysiological criteria, the cases associated with the duplication mutation from those associated with point mutations of the PMP22 gene, although the latter tend to be more severe. In this study we demonstrated that the two genotypes exhibit different morphological characteristics. In the PMP22 duplicated cases the mean g-ratio (axon diameter versus fibre diameter) is significantly lower than normal, while in cases of PMP22 point mutations nearly all myelinated fibers have an extremely high g ratio. In cases with point mutations, onion bulbs are abundantly present from an early age, whereas onion bulbs in the duplicated cases develop gradually in the first years of life. Increase in total transverse fascicular area is most pronounced in the point mutation cases. The differences in pathology between these two very different types of mutations involving the same gene likely reflect differences in pathogenesis and may offer clues in understanding the function of PMP22. PMID- 8615086 TI - Hypophysitis in surgical and autoptical specimens. AB - We present the clinical and histological findings of 11 cases of inflammatory anterior pituitary lesions, 8 of which were obtained during surgery and 3 of which were obtained from autopsies. Additionally, we extended the conventional classification of pituitary inflammatory disease by the new entity " secondary hypophysitis". Of the surgically obtained specimens 5 consisted of inflammatory extension into the pituitary gland out of the surrounding tissue. In all of these patients the inflammation originated from an additional tumor in the sellar region (4 craniopharyngiomas, 1 prolactinoma). These will be referred to as "secondary hypophysitis", an entity which has not yet been mentioned in the literature. Of the remaining 6 cases, 2 were granulomatous hypophysitis, 2 pituitary abscesses, 1 lymphocytic hypophysitis, and 1 showed extensive scarring of the anterior pituitary lobe due to preceeding lymphocytic hypophysitis. At histological examination the basic structure of the anterior pituitary was maintained in all cases. Relative counts of hormone-producing cells were normal. In secondary hypophysitis, the affected area was composed of fibrous tissue and granulation tissue. B and T lymphocytes were present in equal amounts. Granulomas were not found. Inflammatory infiltrates, granulation tissue and fibroses were seen in different proportions. Based on our results and three other cases reported in the literature so far, we think that the presently used classification of pituitary inflammatory diseases lacks an entity which describes a non-abscess-forming inflammation of the pituitary gland originating from an associated pathological process. Therefore, we introduced the term secondary hypophysitis to describe this fourth entity of pituitary inflammatory disease. PMID- 8615089 TI - Primary germinoma of the spinal cord: a case report with 28-year follow-up and review of the literature. AB - Germ cell neoplasms occur in extra-gonadal midline locations of the retroperitoneum, mediastinum, pineal gland, areas of the suprasellar cistern, and rarely in the spinal cord. We recently reviewed a case of an unresectable lumbar spinal cord tumor in a 16-year-old female previously diagnosed as "metastatic poorly differentiated carcinoma." An extensive evaluation for a primary neoplasm at that time was unsuccessful and the patient was treated with local radiation therapy. Recently, additional histochemical and immunocytochemical studies were performed on the archival formalin-fixed paraffin-embedded material and the clinical history was reviewed. These ancillary studies (including positive immunohistochemical staining for placental alkaline phosphatase) support a diagnosis of intramedullary germinoma of the conus medullaris. This patient has enjoyed 28 years of disease free survival which reflects the radiosensitive nature of this neoplasm. These data lend support to the existence of a primary germinoma in the spinal cord and illustrate the utility of using histochemical stains and immunohistochemistry to assist in diagnosing this treatable neoplasm. PMID- 8615088 TI - Congenital muscular dystrophy and severe central nervous system atrophy in two siblings. AB - Severe degenerative features of the nervous system of a hitherto unknown kind, associated with a neuromuscular disorder with histopathological features of congenital muscular dystrophy, are reported in two female siblings. The clinical profile was characterized by generalized hypotonia followed by spastic tetraplegia, contractures, polyneuropathy, lack of cognitive development and progressive microcephaly. There as no involvement of the eyes. Neuropathological examination of the brain of one sibling, who died at the age of 30 months, revealed subtotal loss of neurons in the cerebral and cerebellar cortex and in the ventral pons, and secondary loss of myelin in the cerebral and cerebellar subcortical white matter. Sural nerve biopsy in the other sibling, who had a similar neurological affection, showed a lack of large myelinated fibers. PMID- 8615091 TI - Can safe be safer? Hip arthroplasty by hypotensive epidural anesthesia. PMID- 8615090 TI - High serum creatine kinase levels associated with cylindrical spirals at muscle biopsy. AB - We studied the muscle biopsy from an asymptomatic patient with high serum creatine kinase values. Subsarcolemmal and intermyofibrillar granular inclusions were seen at the light microscopy level. Ultrastructural observation showed clusters of cylindrical spirals (CS). CS are nonspecific, morphological finding, so far reported only in a few cases, presenting with a wide variety of clinical phenotypes. The case we describe is peculiar because of the complete lack of clinical symptoms. The nature of the CS is unknown; we studied a possible alteration of cytoskeletal proteins using a set of different antibodies against these structures, but none of them reacted with CS. Also, since CS have been described in association with mitochondrial abnormalities, and since in our case CS were strongly positive when stained for succinate dehydrogenase, we performed specific immunohistochemical and genetic studies which ruled out any major mitochondrial alterations. PMID- 8615092 TI - Impacted morsellized allograft and cement for revision total knee arthroplasty: a preliminary report of 3 cases. AB - In 3 cases we have revised a failed knee arthroplasty with intramedullary impaction of morsellized allograft similar to the technique described for the hip. Follow-up after 18, 21 and 28 months showed good clinical and radiographic results. PMID- 8615093 TI - Boneloc cement. PMID- 8615094 TI - Pain reduction after anteromedial displacement of the tibial tuberosity: 5-year follow-up in 21 knees with patellofomoral arthrosis. AB - We performed anteromedial displacement of the tibial tuberosity in 21 knees of 16 patients having patellofemoral arthrosis with lateral subluxation. The mean age of the patients was 53 (47-65) years and they were followed for 5(2-13) years. Preoperatively, all knees were painful on descending or ascending stairs; pain was relieved after operation in 20. Retropatellar crepitations disappeared in 2 of 20 knees, but retropatellar pain when squeezing the patella against the femur disappeared in 16 of 17 knees. Patellar subluxation diminished in all knees. PMID- 8615095 TI - Metallic or absorbable implants for ankle fractures: a comparative study of infections in 3,111 cases. AB - Absorbable fracture fixation has been in clinical use since 1984. Our study compares the infection rates and some infection parameters between metallic (2073 patients) and absorbable fracture fixation devices (1012 patients) in displaced ankle fractures. The infection rate associated with metallic fixation was 4.1%, compared with 3.2% absorbable fixation (p 0.3). The patients who had a wound infection were older when metallic fixation was used (p 0.01). They also had a bi or trimalleolar fracture more often than did patients treated with absorbable fracture fixation, but this difference did not have a significant effect on the wound infection rate (p 0.2). The infections were mostly caused by microorganisms of the Staphylococcus species. Deep infections were equally common with both fixation methods (0.4%), but there was some variation in the bacterial spectrum. PMID- 8615096 TI - Concurrent ipsilateral fractures of the hip and femoral shaft: a meta-analysis of 659 cases. AB - 659 cases of concurrent, ipsilateral fractures in the hip and femoral shaft reported in 59 studies were analyzed. The causes were a road traffic accident in 78% and other types of high-energy traumas in 13% of the patients. This injury combination was rare in children. The median age was 34 years. 78% of the patients were men. One-third had multiple injuries, one-half had injuries of the ipsilateral knee and one-half had other lower limb injuries. The femoral neck fractures were most often basilar and the reported rate of avascular necrosis was 3%. The trochanteric fractures were intertrochanteric transverse, and seldom comminuted. The important factors in reducing morbidity were an early diagnosis of all injuries and efficient treatment of the shaft fractures. Locked intramedullary nails yielded results which were superior to combinations of plates or unlocked nails and separate hip screws. Reconstruction nails (cephalomedullary nails) gave results equal to those of customary locked nails and separate hip screws. The rate of healing of the hip fracture was over 99%, the treatment of the shaft fracture being of main importance for the outcome. PMID- 8615097 TI - Heterotopic ossification after hip arthroplasty: a randomized double-blind multicenter study tenoxicam in 147 hips. AB - 147 patients due to have a cemented total hip arthroplasty were randomized to 4 groups. They received either tenoxicam 20 mg or 40 mg, or placebo, for 5 days or morphine on the day of operation and placebo for 4 days. During the first 5 days 14 patients were excluded. The patients were followed for 1 year, during which another 10 patients were excluded. At follow-up, significantly fewer patients had heterotopic ossifications in the tenoxicam groups than in the placebo and morphine groups. There was no significant difference between the 2 tenoxicam treated groups, and we therefore conclude that tenoxicam 20 mg for 5 days postoperatively can reduce heterotopic ossification after cemented total hip arthroplasty. PMID- 8615098 TI - BMP-2 for intramuscular bone induction: effect in squirrel monkeys is dependent on implantation site. AB - Demineralized bone matrix (DBM) reproducibly induces extraskeletal bone formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce bone, although the same implants were effective in rats. DBM implants augmented with recombinant human bone morphogenetic protein-2 (rhBMP-2) induced intramuscular bone formation in squirrel monkeys. However, the amount of induced bone was often minimal and sometimes absent. One explanation of this weak and unpredictable effect could be reactions to cellular components of the allogeneic DBM that was used as carrier. Therefore, we now repeated the experiment, using bovine type 1 collagen as carrier. 48 collagen discs (10 mm diameter), containing 0, 10, 40 or 200 microgram rhBMP-2, were implanted in 6 monkeys and 6 rats. The BMP-2 implants induced dose-dependent amounts of intramuscular bone in rats whereas, in squirrel monkeys, macroscopic bone induction occurred in less than half of the BMP-2 implants. There was no dose-dependency. Whether bone was formed or not was significantly influenced by individual variation among the monkeys, and by implant location within the muscle. Implants close to the muscle aponeurosis more often induced bone than did purely intramuscular ones. In this small series, we could not demonstrate a significant effect of BMP-2, as compared to control implants. Presumably there were too few cells expressing BMP-2 receptors in the only minimally traumatized muscle of these monkeys. PMID- 8615099 TI - Femoral shortening in total arthroplasty for completely dislocated hips: 3-7 year results in 25 cases. AB - During the years 1988-1991, we performed 25 total hip replacements for completely dislocated hips in 15 women and 4 men with a median age of 54 (17-67) years. In all cases, femoral shortening at the subtrochanteric level was performed to obtain reduction of the hip. The patients have been followed for 3-7 years. 1 patient experienced sciatic nerve palsy, 1 a delayed union and 1 a malunion at the osteotomy site. There were no signs of mechanical failure. All patients were satisfied. According to the Charnley hip score, function was excellent in 15 cases, good in 9 and fair in 1. The median Harris hip score improved from 43 at the time of operation to 93 at follow-up. 7 hips had a positive and 18 a negative Trendelenburg test. Before operation, all patients had a Trendelenburg limp. Our intermediate results indicate that femoral shortening at the subtrochanteric level is a suitable adjunct to total arthroplasty for a completely dislocated hip. PMID- 8615100 TI - Proximal femoral replacement prosthesis for salvage of failed hip arthroplasty: complications in a 2-11 year follow-up study in 19 elderly patients. AB - We reviewed 19 elderly patients who underwent revision total hip arthroplasty with a proximal femoral replacement prosthesis for aseptic loosening and severe proximal femoral bone loss. The mean interval from initial hip replacement to revision arthroplasty was 8 (2-20) years. The mean age of the patients was 78 (63 87) years. 2 patients died within 2 years postoperatively and 1 patient was lost for follow-up evaluation. The remaining 16 patients were assessed clinically and radiographically after a mean follow-up period of 5 (2-11) years. All patients had local pain relief, but they all needed a crutch or another walking aid. According to the Merle d'Aubigne hip-rating scale there were no excellent results, 1 very good, 8 good, 5 fair, 2 poor and no bad results. 4 patients had an intraoperative fracture, 7 had a dislocation, 2 had a deep infection, and 3 patients had progressive loosening of the screws fixing the greater trochanter to the femoral component. Our series demonstrates that revision of a failed hip prosthesis, using a proximal femoral replacement prosthesis, presents complex problems. PMID- 8615101 TI - Radiographic risk signs for loosening after cemented THA: 61 loose stems and 23 loose sockets compared with 42 controls. AB - The 1-year radiographs in 61 stems and 23 sockets, later revised because of aseptic loosening, were carefully examined for changes such as 1) separation of the lateral side of the stem from the cement, 2) radiolucent zones at the bone cement interface, 3) fracture of the cement, 4) endosteal cavitation and 5) migration. They were compared with 42 clinically and radiographically successful primary total hip arthroplasties with 12-16 years follow-up. We found an increased risk of loosening in hips where radiolucencies appeared within the first postoperative year, whereas an unchanged radiographic appearance after 1 year strongly indicated that the risk of later loosening was small. Patients at risk should be followed indefinitely to detect progressive loosening and concomitant bone resorption in time. PMID- 8615102 TI - Hydroxyapatite-coated hip prostheses: difficulties with revision in 4 cases. AB - The introduction of hydroxyapatite ceramic (HAC) coating on hip implants in 1985 (Furlong and Osborn 1991) was hailed as a major advancement for fixation of uncemented protheses. A problem that is now becoming evident is the extraction of these securely fixed prostheses for purposes of revision. We report on 4 patients who have had HAC-coated protheses implanted (2 as revision procedures and 2 at primary hip replacement) who had either continuing pain or a proven infection, so that it became necessary to carry out a revision hip arthroplasty. The prostheses were well-fixed and difficult to remove. A transfemoral, longitudinal osteotomy was used in 3 cases. PMID- 8615103 TI - The incidence of physiolysis of the hip: a population-based study of 175 patients. AB - We studied the epidemiology of physiolysis of the hip (or slipped capital femoral epiphysis) between 1946 and 1992 in 175 patients from a well-defined population. The incidence of physiolysis of the hip was calculated using the attack rate method, i.e., the sum of the annual incidences in each 1-year group. The risk of developing physiolysis was 1:998 for boys, 1:1756 for girls and 1:1267 for both sexes and did not change significantly during this period. The average age at diagnosis was 13.6 (7-17) years in boys and 12.1 (8-15) years in girls and did not change to a significant degree during this period. The difference in the boy:girl ratio (1.8:1) and in the preponderance of the left hip being affected first (64% for boys and 52% for girls) did not change significantly during this period. No significant monthly variation was found in the onset of symptoms of physiolysis of the hip. PMID- 8615104 TI - Total hip arthroplasty with Boneloc: loosening in 102/157 cases after 0.5-3 years. AB - We report the outcome of 177 consecutive primary Charnley total hip arthroplasties inserted with Boneloc cement between November 1991 and November 1993. There were 107 women and 70 men. The mean age at the time of the operation was 71 years. 11 patients (13 hips) died during the follow-up period and 3 patients were too weak to attend a follow-up examination. Of the 161 remaining hips, 4 had been revised because of deep infection. The mean follow-up time for the remaining 157 hips was 2 (0.5-3) years. 24 hips had been revised and 6 are waiting for revision because of stem loosening. Of the remaining 127 hips, 72 showed radiographic signs of stem loosening and 2 hips were probably loose. Osteolysis was seen around the femoral component in 56 hips. PMID- 8615105 TI - Osteotomy for cubitus varus: a simple technique in 10 children. AB - 10 children with cubitus varus deformity after supracondylar fractures were operated on with a supracondylar lateral closing wedge osteotomy. The medial cortical periosteal hinge was left intact and the osteotomy stabilized with two Kirschner wires and a tension-band wire loop. The osteotomies healed within 2 months, without any complications or recurrence of the deformity. The outcome was satisfactory as regards both cosmesis and mobility. PMID- 8615106 TI - Posterolateral lumbosacral fusion with transpedicular fixation: 63 consecutive cases followed for 4 (2-6) years. AB - We analyzed the clinical, vocational and radiologic outcomes of 63 consecutive posterolateral lumbosacral fusions performed with transpedicular fixation. The indication for surgery was long-standing intractable lumbar and/or radiating pain with spondylolysis-olisthesis in 31 cases, degenerative disc disease and/or facet joint arthrosis in 23 cases and pain after laminectomy/decompression in 9 cases. Radiographic union was finally achieved in 30 out of the 63 cases. Fixation device-related complications, such as screw misplacement, breakage, bending and loosening, occurred in 33 cases. 15 patients underwent refusion. 43 patients obtained good pain relief. There was no correlation between bony healing and a good clinical outcome. 28/49 preoperatively employed patients returned to work. There was no correlation between relief of pain and return to work. 20 patients retired on a full disability pension. The clinical results were best in the spondylolysis-olisthesis group. Only 2/15 patients with markedly reduced spondylolisthesis maintained the reduction. In 3 patients, progressive disc degeneration above the level of fusion was observed. We conclude that posterolateral lumbosacral fusion with transpedicular fixation provides a satisfactory clinical outcome in patients with spondylolysis-olisthesis, but the high incidence of complications related to the fixation device in the other indications studied is a serious drawback of the method. PMID- 8615107 TI - Transient osteoporosis of the hip in pregnancy complicated by femoral neck fracture: a case report. PMID- 8615109 TI - Acute compartment syndrome in the thigh after revision hip arthroplasty: a case report. PMID- 8615108 TI - Continuous passive motion compared to active physical therapy after knee arthroplasty: similar hospitalization times in a randomized study of 68 patients. AB - 68 consecutive patients who had primary knee arthroplasties because of arthrosis were randomized to postoperative continuous passive motion (CPM) or active physical therapy (APT). Rehabilitation in both groups was initiated on the first postoperative day. The CPM group sustained less postoperative knee swelling with more rapid initial improvement in knee flexion than did the APT group, but there were no differences between the groups in knee flexion at discharge. Postoperative pain rating and hospitalization times were similar in the two groups. PMID- 8615110 TI - Aneurysm complicating high tibial osteotomy: a case report. PMID- 8615112 TI - Osteoid osteoma of the third lumbar vertebra: sequential observations with MRI--a case report. PMID- 8615111 TI - Arterial complications after knee arthroplasty: 4 cases and a review of the literature. AB - We reviewed 40 cases of arterial complications after total knee replacement from the literature and report 4 of our own cases. The incidence is low (0.03%-0.2%), but the prognosis is poor, with death or amputation in one fourth of the cases. Preexisting arterial insufficiency, absent pedal pulses, arterial calcification on plane radiographs, femoro- popliteal/distal grafts are all predisposing factors. Therefore, preoperative risk factor evaluation as well as rapid and adequate handling in case of bleeding and/or ischemic complications pre- and postoperatively are crucial. Cooperation with a vascular surgeon is also essential. PMID- 8615113 TI - Misleading magnetic resonance imaging in spinal osteoid osteomata: a report of 3 children. PMID- 8615114 TI - Hallux valgus. PMID- 8615115 TI - Hypotensive epidural anesthesia for total hip arthroplasty: a review. AB - Hypotensive epidural anesthesia provides arterial hypotension to maintain a mean arterial pressure of 50 mmHg and it can be used to reduce blood loss during total hip replacement. The technique combines an extensive epidural blockade with an intravenous infusion of low-dose epinephrine. This results in arterial hypotension, but with preservation of central venous pressure, heart rate, stroke volume, cardiac output, and an augmentation of blood flow to the lower extremity. The technique does not appear to adversely affect cardiac, renal, or cerebral function and is used safely in patients with hypertension, ischemic heart disease, and in the elderly. Intraoperative blood losses during primary total hip replacement are between 100 and 300 mL. Perioperative transfusions have declined with the introduction of the technique. Radiological evidence of improved fixation of cemented acetabular components has been observed. Rates of deep-vein thrombosis are low: 2-3% proximal deep-vein thrombosis with an overall rate of 10%. In-hospital mortality is 0.1%; lower than previously published rates. In conclusion, hypotensive epidural anesthesia is safe and provides a number of advantages over conventional anesthetic techniques for total hip replacement. PMID- 8615116 TI - Parkinson's disease. The apoptosis hypothesis. PMID- 8615117 TI - From basal ganglia to motoneurons. Probable involvement of pathways relaying in the medulla. PMID- 8615118 TI - Basal ganglia disorders. An electrophysiologic approach. PMID- 8615119 TI - Characteristics of pallidal neuronal discharges in Parkinson's disease patients. PMID- 8615120 TI - Pathophysiology of Parkinson's disease rigidity. Role of corticospinal motor projections. PMID- 8615121 TI - Prevalence of Parkinson's disease in a population of Uruguay. Preliminary results. PMID- 8615122 TI - The performance of rapid arm movements in Parkinson's disease. A review. PMID- 8615123 TI - Molecular biology of catecholamine neurons in relation to Parkinson's disease. PMID- 8615124 TI - Immune-mediated cell death in neurodegenerative disease. PMID- 8615125 TI - Neurotoxicity and the mechanisms of cell death in Parkinson's disease. PMID- 8615126 TI - Excitatory amino acids and MPTP toxicity. PMID- 8615127 TI - Molecular mechanisms for neurodegeneration. Synergism between reactive oxygen species, calcium, and excitotoxic amino acids. PMID- 8615128 TI - Etiology of Parkinson's disease. The role of environment and heredity. PMID- 8615129 TI - Is dopaminergic cell death accompanied by concomitant nerve plasticity? PMID- 8615130 TI - Glutamic acid decarboxylase mRNA expression in medial and lateral pallidal neurons in the MPTP-treated monkey and patients with Parkinson's disease. PMID- 8615131 TI - Electron microscopy of Lewy bodies in the amygdala-parahippocampal region. Comparison with inclusion bodies in the MPTP-treated squirrel monkey. PMID- 8615132 TI - Dopamine-induced, genotoxic activation of programmed cell death. A role in nigrostriatal neuronal degeneration in Parkinson's disease? PMID- 8615134 TI - Pulsatile administration of D1 but not D2 dopamine agonists induces behavioral tolerance in MPTP-treated monkeys. PMID- 8615133 TI - The dopamine transporter. The cloned target site of parkinsonism-inducing toxins and of drugs of abuse. PMID- 8615135 TI - Pharmacologic evidence for D2 dopamine receptor heterogeneity. PMID- 8615136 TI - The epidemiology of progressive supranuclear palsy. PMID- 8615137 TI - Human substantia nigra in vitro as a bioassay for neurotrophic molecules of clinical significance. PMID- 8615138 TI - Nuclear magnetic resonance spectroscopy characterization and iron content determination of human mesencephalic neuromelanin. PMID- 8615139 TI - Failure of brain and skeletal muscle energy metabolism in multiple system atrophy shown by in vivo phosphorous MR spectroscopy. PMID- 8615140 TI - Peripheral markers in Parkinson's disease. An overview. PMID- 8615141 TI - The neuropathologic diagnosis of secondary parkinsonian syndromes. PMID- 8615142 TI - Parkinson's disease: progression and mortality in the L-DOPA era. PMID- 8615143 TI - Progression and prognosis in Parkinson's disease in relation to concomitant cerebral or peripheral vasculopathy. PMID- 8615144 TI - Pathologically diagnosed diffuse Lewy body disease and Parkinson's disease. Do the parkinsonian features differ? PMID- 8615145 TI - The clinicopathologic spectrum of Lewy body disease. PMID- 8615146 TI - Visual evoked potentials and spatiotemporal contrast sensitivity changes in idiopathic Parkinson's disease and multiple system atrophy. PMID- 8615147 TI - Early-onset parkinsonism with dystonia. Clinical and biochemical differences from hereditary progressive dystonia or DOPA-responsive dystonia. PMID- 8615148 TI - Recent advances in the genetics of Parkinson's disease. PMID- 8615149 TI - A profile analysis of demented and nondemented Parkinson's disease patients. PMID- 8615150 TI - The evolution and profile of dementia in Parkinson's disease. PMID- 8615151 TI - Image generation from long-term memory in Parkinson's disease. PMID- 8615152 TI - Switching abilities in Parkinson's disease. PMID- 8615153 TI - Biochemical and pharmacologic assessment of autonomic function. AB - Autonomic failure produces distinct pathophysiological abnormalities that differ according to the site and nature of the lesion(s). Although the anatomic organization and processes mediating chemical neurotransmission in the autonomic nervous system facilitate clinical investigation, limited access to the central compartment hampers evaluation of central neurotransmitter metabolism and neuropeptide function. As illustrated in the discussions of noradrenergic and cholinergic function, several indirect strategies have been used to assess the biochemical and neuropharmacologic consequences of autonomic dysfunction. The methods validated in patients with autonomic failure can be applied to investigate autonomic function in other clinical disorders including Parkinson's disease. The results of such studies may help to guide therapy and develop improved strategies for managing those patients with autonomic insufficiency. PMID- 8615154 TI - Autonomic nervous system dysfunction and adrenoceptor regulation in Parkinson's disease. Clinical and pharmacological consequences. PMID- 8615155 TI - Disorders affecting autonomic function in parkinsonian patients. AB - This chapter deals with certain aspects of autonomic dysfunction in parkinsonian patients. It provides a classification of autonomic disorders and a brief description of autonomic manifestations. An outline of autonomic investigations, including those to diagnose the Shy-Drager syndrome (multiple system atrophy), is provided. It concludes with comments on terminology and on whether the parkinsonian syndromes with autonomic failure comprise single or multiple entities. PMID- 8615156 TI - The clinical diagnosis of multiple system atrophy presenting as pure parkinsonism. PMID- 8615157 TI - The cognitive syndrome of progressive supranuclear palsy. PMID- 8615158 TI - Differential diagnosis and clinical diagnostic criteria of progressive supranuclear palsy. PMID- 8615159 TI - Cloning, expression, and comparison of the binding characteristics of the known human dopamine receptors. PMID- 8615160 TI - Multiple system atrophy. PMID- 8615161 TI - Instrumental diagnosis of multiple system atrophy. PMID- 8615162 TI - The rate of progression of Parkinson's disease. A longitudinal [18F]DOPA PET study. PMID- 8615163 TI - Basal ganglia function during normal and parkinsonian movement. PET activation studies. PMID- 8615164 TI - Functional positron emission tomographic studies of striatal dopaminergic activity. Changes induced by drugs and nigrostriatal degeneration. PMID- 8615165 TI - Fluorodopa PET scanning in Parkinson's disease. AB - In summary, FD PET is a valid method for assessing the functional state of the nigrostriatal dopaminergic pathway. It is particularly useful for studies requiring repeated measures such as examinations of the course of a disease and the effect of treatment. PMID- 8615166 TI - Benzodiazepine receptor binding in the cerebellum in multiple system atrophy and olivopontocerebellar atrophy studied with positron emission tomography. PMID- 8615167 TI - Striatal dopamine D2 receptor alterations and response to L-DOPA in Parkinson's disease. A [123I]IBZM SPET study. PMID- 8615168 TI - Controversies in the therapy of Parkinson's disease. AB - Only by adequate controlled studies can we be certain of the value of the different therapeutic choices available for the treatment of patients with PD. What may look favorable on preliminary open-label trials may not be confirmed on more rigorous controlled, randomized trials. This appears to be the case when reviewing the evidence for using dopamine agonists in early PD. Patients and clinicians await the results of the large double-blind, multicenter trial that compared bromocriptine alone, L-DOPA alone, and the two drugs in combination. Similarly, we await the results of a large multicenter, controlled trial comparing standard Sinemet and Sinemet CR in de novo patients with Parkinson's disease. The DATATOP study provided proof that selegiline can delay the need for L-DOPA, but it also showed for the first time that this drug has symptomatic effects in early PD. DATATOP failed to find conclusively a protective effect from selegiline. The long-running controversy whether L-DOPA is the culprit or the bystander in producing motor response fluctuations and the more recent concern as to whether it can hasten the progression of PD by producing increasing oxidative stress need to be answered; a prospective, controlled clinical trial might be able to provide these answers. PMID- 8615169 TI - Treatment of Parkinson's disease with controlled-release Carbidopa/L-DOPA. PMID- 8615170 TI - Effects of catechol-O-methyltransferase (COMT) inhibition on the pharmacokinetics of L-DOPA. PMID- 8615171 TI - Contribution of dopaminergic and glutamatergic mechanisms to the pathogenesis of motor response complications in Parkinson's disease. PMID- 8615173 TI - New drugs in the treatment of Parkinson's disease. An introduction. PMID- 8615172 TI - Effects of L-DOPA on spatiotemporal contrast sensitivity in Parkinson's disease. PMID- 8615175 TI - The molecular biology of xenobiotic enzymes and the predisposition to idiopathic Parkinson's disease. PMID- 8615174 TI - A study on the effect and tolerance of lisuride on Parkinson's disease. AB - Seventeen idiopathic parkinsonian patients ranging between 47 and 75 years of age were included in this study to investigate the effect and tolerance of lisuride on PD. The duration of this simple clinical study, which had no control group was 12 weeks. There was 50% relief in disability scores and ADL in 13 patients in the first group in the combined therapy for 12 weeks with lisuride added to L-DOPA plus benserazide (p < 0.01). Optimal lisuride doses added to L-DOPA plus benserazide varied between 0.1 and 0.8 mg (mean 0.5 +/- 0.2 mg). With the addition of lisuride to treatment, the L-DOPA plus benserazide dose was reduced in 6 of 13 by 38%. Monotherapy with lisuride resulted in 56% to 57% improvement in disability scores and 47% in relief in ADL. Dry mouth, nausea, weakness, postural hypotension, and headache were the most frequently encountered side effects of lisuride. These adverse effects disappeared in 3 or 4 days, depending on a slight decrease and following increase in the dose of lisuride and/or the development of tolerance. Not only will such a combined therapy contribute to the reduction of the end-of-dose inadequacies, on-off phenomena, wearing off, peak dose dyskinesias, and similar motor fluctuations, it may also play a prophylactic role in their prevention or delay. PMID- 8615176 TI - A placebo-controlled study of ropinirole, a new D2 agonist, in the treatment of motor fluctuations of L-DOPA-treated parkinsonian patients. PMID- 8615177 TI - Preclamol. A "designer drug" in the treatment of advanced Parkinson's disease. PMID- 8615178 TI - Ondansetron, a 5-HT3 antagonist for visual hallucinations and paranoid delusional disorder associated with chronic L-DOPA therapy in advanced Parkinson's disease. PMID- 8615179 TI - Involvement of serotonin in clinical features of Parkinson's disease and complications of L-DOPA therapy. PMID- 8615180 TI - Effects of conventional and sensory-enhanced physiotherapy on disability of Parkinson's disease patients. PMID- 8615181 TI - Does stereotactic treatment for Parkinson's disease slow the progression of the disease? PMID- 8615182 TI - Does thalamotomy alter the course of Parkinson's disease? PMID- 8615183 TI - Effects of posteroventral pallidotomy on Parkinson's disease. PMID- 8615184 TI - Subthalamic nucleus stimulation alleviates akinesia and rigidity in parkinsonian patients. PMID- 8615185 TI - An open study with reversible MAO-A inhibitors in complicated Parkinson's disease. PMID- 8615186 TI - Gene-environment interaction in parkinson's disease. The case of CYP2D6 gene polymorphism. PMID- 8615187 TI - Dopaminergic regulation of peptide gene expression in the striatum of normal and parkinsonian monkeys. PMID- 8615188 TI - Glutamate receptor genes in Parkinson's disease. PMID- 8615190 TI - Debrisoquine hydroxylation genotype in familial forms of idiopathic Parkinson's disease. PMID- 8615189 TI - Genetic linkage studies in autosomal dominantly inherited L-DOPA responsive parkinsonism. Evaluation of candidate genes. PMID- 8615191 TI - The supplementary sensorimotor area. An overview. PMID- 8615192 TI - Functional specialization of the supplementary motor area in monkeys and humans. PMID- 8615193 TI - Generation of movement-related potentials in the supplementary sensorimotor area. PMID- 8615194 TI - Generation of movement-related potentials and fields in the supplementary sensorimotor area and the primary motor area. PMID- 8615195 TI - Generation of movement-related potentials in the supplementary sensorimotor area. PMID- 8615196 TI - Generation of contingent negative variation in the supplementary sensorimotor area. PMID- 8615197 TI - Functional magnetic resonance imaging and the supplementary sensorimotor area. PMID- 8615198 TI - Neurophysiology of the human supplementary motor area. Positron emission tomography. PMID- 8615199 TI - Historical overview. PMID- 8615200 TI - Electrical stimulation of the supplementary sensorimotor area. PMID- 8615201 TI - Cortical stimulation of the supplementary sensorimotor area. PMID- 8615202 TI - What stimulation of the supplementary motor area in humans tells about its functional organization. PMID- 8615203 TI - Electrical stimulation of the supplementary sensorimotor area. PMID- 8615204 TI - Surgical ablations of the mesial frontal lobe in humans. PMID- 8615205 TI - Lesions of the supplementary motor area. PMID- 8615206 TI - Neurocognitive studies in patients with supplementary sensorimotor area lesions. PMID- 8615207 TI - Functional organization of the human supplementary motor area and dorsolateral premotor cortex. PMID- 8615208 TI - Supplementary sensorimotor area epilepsy in adults. PMID- 8615209 TI - Supplementary sensorimotor area epilepsy in adults. AB - Based on stimulation studies, observation of seizure phenomena in patients with epilepsy, and diagnostic studies documenting SSMA anatomic and functional abnormalities, there is evidence that epileptic involvement of the SSMA results in a set of clinical phenomena that include asymmetric tonic posturing of the extremities, abduction and elevation of the contralateral upper extremity, and speech arrest with preserved consciousness. Auras and vocalization occur less frequently. These seizures tend to be brief and to have an abrupt onset and cessation. They are more common during sleep than during wakefulness, and they tend to occur multiple times per night. The abolition of seizures following a resection limited to the SSMA provides conclusive evidence that the SSMA is the epileptogenic zone in some patients with this type of seizure. On the other hand, there is variation in seizure phenomena among patients whose seizures originate in the SSMA. In addition, there are patients with seizures suggestive of SSMA involvement whose seizures originate in a broad epileptogenic zone that includes the SSMA, other patients whose seizures originate outside the SSMA and spread to the SSMA, and still other patients whose seizures originate outside the SSMA and remain outside the SSMA. To better understand the phenomena associated with SSMA seizure onset and to determine the specificity of certain phenomena to SSMA involvement, additional data are needed from structural and functional imaging studies, video and EEG monitoring studies with noninvasive and invasive electrodes, and pathologic and surgical outcome studies. Since one cannot be certain at this time of the specificity of seizure phenomena to SSMA involvement, and because localization for possible surgery is the primary rationale for detailed study of these patients, we recommend that the term SSMA seizure refer only to those seizures that originate in the SSMA and that SSMA epilepsy refer only to epilepsies in which the patient's seizures originate in the SSMA. Since the present classification of epileptic seizures is not of value in describing the major phenomena of a partial seizure or in localization of the site of onset, a descriptive classification is needed to improve communication concerning seizure phenomena and to provide clues to localization. PMID- 8615210 TI - Anatomy and transmitter receptors of the supplementary motor areas in the human and nonhuman primate brain. PMID- 8615211 TI - Unusual features of supplementary sensorimotor area epilepsy: cyclic pattern, unusual sensory aura, startle sensitivity, anoxic encephalopathy, and spontaneous remission. PMID- 8615212 TI - Supplementary sensorimotor area seizures in children and adolescents. PMID- 8615213 TI - Noninvasive electroencephalography in supplementary sensorimotor area epilepsy. PMID- 8615215 TI - Magnetic resonance imaging and supplementary motor area epilepsy. PMID- 8615214 TI - Invasive electroencephalography in the evaluation of supplementary motor area seizures. PMID- 8615216 TI - Epilepsy of the human supplementary motor area: positron emission tomography. PMID- 8615217 TI - Supplementary sensorimotor area seizure and ictal single-photon emission tomography. AB - Focal tonic seizures [supplementary motor seizures in the terminology of Penfield and Jasper (1) are often difficult to diagnose in terms of this chapter was undertaken to investigate whether ictal single-photon emission CT (SPECT) scanning could contribute to the diagnostic accuracy of this epileptic condition. In 15 patients (mean age, 21.3 years; ranges, 3 to 39 years) suffering from focal tonic seizures as the initial and most prominent seizure type, ictal SPECT scans were obtained after injection of 99mTc HMPAO within 30 seconds after clinical seizure onset. In 6 patients (40%; group I), ictal SPECT scans showed a focal hyperperused area concordant with results of other tests of the preoperative work up (prolonged EEG/video monitoring, MRI, and positron emission tomography PET) using [18F]fluoro-2-deoxy-D-glucose (18FDG)] In 6 patients (group II), results of the ictal SPECT scans also showed findings of other tests of the clinical work up. In 3 patients (group III), no ictal hyperperfusion could be observed. Results of the other presurgical tests in groups II and III were not sufficient to identify the seizure onset zone with the certainty needed for surgical resection. Ictal SPECT with HMPAO revealed confirmatory results in 40% of the patients with focal tonic seizures. In the remaining patients, results either were not congruent with other findings or did not show any change at all. This may have a twofold explanation: First (and probably most important), the interval between seizure onset and injection was too long considering the usually short duration of this seizure type, and second, the spatial resolution of SPECT imaging might not be sufficient to reveal a small seizure focus. PMID- 8615218 TI - Invasive investigation and surgery of the supplementary motor area. The Cleveland Clinic experience. PMID- 8615219 TI - Surgical strategies for patients with supplementary motor area epilepsy. The Grenoble experience. PMID- 8615220 TI - Surgical strategies for patients with supplementary sensorimotor area epilepsy. The Japanese experience. PMID- 8615221 TI - Surgical strategies for patients with supplementary sensorimotor area epilepsy. The Medical College of Georgia experience. PMID- 8615222 TI - Surgical strategies for patients with supplementary sensorimotor area epilepsy. The Montreal experience. PMID- 8615223 TI - Surgical management of patients with intractable supplementary motor area seizures. The Yale experience. PMID- 8615224 TI - The classic supplementary motor area is formed by two independent areas. PMID- 8615225 TI - Quantitative evaluation of the motor deficit after supplementary sensorimotor area resection. PMID- 8615226 TI - Psychogenic seizures and the supplementary sensorimotor area. PMID- 8615227 TI - Paroxysmal choreoathetosis. PMID- 8615228 TI - Dystonia and the supplementary sensorimotor area. PMID- 8615229 TI - The functions of the supplementary motor area. Summary of a workshop. PMID- 8615230 TI - Workshop on the anatomic definition and boundaries of the supplementary sensorimotor area. PMID- 8615231 TI - Corticospinal efferents of the supplementary sensorimotor area in relation to the primary motor area. PMID- 8615232 TI - Evolutionary and comparative neurobiology of the supplementary sensorimotor area. PMID- 8615233 TI - Is the supplementary motor area a bilaterally organized system? PMID- 8615234 TI - Contrast of neuronal activity between the supplemental motor area and other cortical motor areas. PMID- 8615235 TI - Malpractice issues in radiology. Possessing ordinary knowledge. PMID- 8615236 TI - A computer utility for automated retrieval of radiology reports. AB - OBJECTIVE: Many radiology information systems (RISs) installed during the 1980s have a user interface that is crude by today's standards. We explored improving this user interface by using the local processing power of the personal computer. In this article we describe a computer program that significantly enhances the ease of report retrieval by automating much of the interaction with our RIS, which is IDXrad. MATERIALS AND METHODS: The program, named DERVISH, runs under DOS on International Business Machines-compatible personal computers having an 80286 or faster processor, an enhanced graphics adapter or video gate array display, two serial ports, a mouse, and a bar code wand. It is written in the C++ programming language. Input may be keyboard, mouse, or bar code wand, and reports may be displayed singly or multiply and printed to a local printer or an RIS printer or copied to a disk file. DERVISH not only performs many of the routine tasks of an RIS session such a logging on and off but also provides a menu-driven environment for report display, including optional restriction of the report list to related prior reports. Keyboard use is minimized in favor of a mouse and a bar code wand. DERVISH emulates a standard RIS terminal for functions other than report retrieval. RESULTS: Originally intended to help retrieve the prior radiology reports of a patient at hand, DERVISH has found use in our quality management effort as well, simplifying comparison of reports from related radiologic techniques. An early version suffered poor acceptance because of its nonstandard interface and frequent malfunctions. The current version is presently in use in our emergency radiology department. CONCLUSION: DERVISH serves not only as a useful utility in itself but also as a demonstration of the ability of the local processing power of a desktop computer to improve the user interface of an aging RIS and to extend its functional life. PMID- 8615237 TI - Acute rupture of the diaphragm due to blunt trauma: diagnostic sensitivity and specificity of CT. AB - OBJECTIVE: The purpose of this study was to determine the diagnostic sensitivity and specificity of CT in detecting acute rupture of the diaphragm after blunt trauma. MATERIALS AND METHODS: Abdominal CT scans taken before surgery of 11 patients with diaphragmatic rupture (eight left and three right) and 21 patients with intact diaphragms after major acute blunt abdominal trauma were independently reviewed by three observers who were unaware of surgical findings. Retrospective note was made of diaphragmatic discontinuity, intrathoracic herniation of abdominal contents, waistlike constriction of bowel ("collar sign"), and associated findings. Right and left hemidiaphragms were graded as intact or ruptured, and these findings were correlated with surgical findings. Individual and average observer sensitivity and specificity in detecting acute diaphragmatic rupture were calculated. RESULTS: Of the 11 cases of diaphragmatic rupture, diaphragmatic discontinuity was seen in eight, visceral herniation was seen in six, and the "collar sign" was seen in four cases. Hemoperitoneum of hemothorax completely obscured visualization of the ruptured diaphragm in three cases. Individual diagnostic sensitivity for detecting diaphragmatic rupture was 54-73% and specificity was 86-90%. Average sensitivity for the three observers was 61% (95% confidence interval, 41-81%), and average specificity was 87% (95% confidence interval, 76-99%). CONCLUSION: CT is highly specific in diagnosing acute diaphragmatic rupture and detects approximately two thirds of acute diaphragmatic ruptures after blunt trauma. PMID- 8615238 TI - Pneumomediastinum: old signs and new signs. AB - Pneumomediastinum, also known as mediastinal emphysema, represents extraluminal gas in the mediastinum. Pneumomediastinum can lead to pneumothorax, pneumopericardium, pneumoperitoneum, or pneumoretroperitoneum. There are many causes of pneumomediastinum (Table 1) and several radiographic signs: pneumopericardium, continuous diaphragm sign, continuous left hemidiaphragm sign, Naclerio's V sign, V sign at confluence of brachiocephalic veins, ring-around-the artery sign, thymic spinnaker-sail sign, and extrapleural air sign. We review the common and uncommon signs of pneumomediastinum and present some new signs. We also address the problem of distinguishing pneumomediastinum from pneumothorax and pneumopericardium. PMID- 8615239 TI - Pneumothorax after small-bore catheter placement for malignant pleural effusions. AB - OBJECTIVE: The objective of this study was to evaluate the incidence and significance of pneumothorax after small-bore chest tube placement for symptomatic malignant pleural effusions. SUBJECTS AND METHODS: Over a 2-year period, 90 patients with a known primary malignant tumor and symptomatic pleural effusion were referred to the radiology service at Duke University Medical Center. All patients underwent placement of a small-bore chest tube with fluid drainage in preparation for intrapleural sclerotherapy. Two of these patients were excluded because of coexisting empyema (n=1) and thoracentesis (n=1). The remaining 88 patients (30 men and 58 women; 26-86 years old [mean, 60 years old], who had 90 chest tubes placed, formed our study group. The incidence, duration, and clinical significance of their pneumothoraces and the amount of pleural effusion drained were recorded. RESULTS: Among the 88 patients with 90 chest tubes, 27 patients with 28 chest tubes (31%) were found to have pneumothorax after the procedure. For 23 patients with 24 chest tubes, pneumothorax was evident on chest radiographs taken immediately after tube insertion and fluid drainage. Four patients with four chest tubes were found to have pneumothorax on chest radiographs taken the next day. No significant difference in the amount of fluid drained during the procedure was noted for patients with or without pneumothorax (831 ml versus 853 ml). No relationship between the size of each pneumothorax and the size of each drainage catheter was seen. The duration of pneumothorax ranged from 2 hr to 18 days (average, 3.5 days). Resolution of pneumothorax was seen in 22 (79%) of 28 cases; the remaining six cases of pneumothorax (21%) were stable, and the patients showed eventual fluid reaccumulation after chest tube removal and no sclerotherapy. No patient developed tension pneumothorax, respiratory distress, or other complications. CONCLUSION: Pneumothorax should be recognized as a common finding after chest tube placement and immediate fluid drainage for malignant pleural effusions. We suggest that this finding is related to rapid removal of fluid from a relatively stiff, noncompliant lung. Patients whose lungs do not fully re-expand in several days will probably not benefit from sclerotherapy. Their tubes may be removed without risk of an enlarging tension pneumothorax. PMID- 8615240 TI - Evidence of expiratory CT scans of small-airway obstruction in sarcoidosis. PMID- 8615241 TI - Castleman's disease of the lung: radiographic, high-resolution CT, and pathologic findings. PMID- 8615242 TI - Articular muscle of the knee: a muscle seldom recognized on MR imaging. AB - OBJECTIVE: We studied the MR imaging characteristics of the articular muscle of the knee, an important but seldom recognized structure of the distal thigh and knee. SUBJECTS AND METHODS: We used a 1.5-T system to prospectively evaluate the articular muscle of the knee in five healthy volunteers (10 knees); then we retrospectively studied the knee examinations of 24 patients (30 knees). One reader made duplicate readings to prospectively assess the sagittal proton density-weighted and axial T1-weighted images of the healthy volunteers. Three independent readers retrospectively evaluated the sagittal proton density weighted images of the 24 patients. RESULTS: The articular muscle of the knee was identified in 100% of prospective studies (10/10) and in 83% of retrospective studies (25/30). For the prospective study group, measurements (mean +/- SEM) were as follows: number of bundles, 2.4 +/- 0.68 (range, 1-4); length of uppermost bundle, 47.2 +/- 4.06 mm; width of origin, 8.6 +/- 0.79 mm; angle of origin, 11.4 degree +/- 1.29 degree; and cross-sectional bundle width, 19.8 +/- 2.05 mm2. CONCLUSION: The articular muscle of the knee can usually be identified by MR imaging as an independent entity, separate from the great (vastus) muscle group. PMID- 8615243 TI - MR evaluation of rotator cuff pathology using T2-weighted fast spin-echo technique with and without fat suppression. AB - OBJECTIVE: This study was designed to compare MR imaging findings from T2 weighted fast spin-echo images with and without fat suppression and then to compare surgical results with those MR imaging interpretations in the evaluation of rotator cuff disease. SUBJECTS AND METHODS: T2-weighted fast spin-echo images- with and without fat suppression--of 177 shoulder MR imaging studies were reviewed separately in a retrospective, randomized, and blinded fashion. Both sets of images were read in conjunction with the corresponding proton density weighted images. The diagnosis of normal tendon, tendinosis, degeneration, and partial- and full-thickness tears was made using established criteria. RESULTS: T2-weighted fast spin-echo techniques with and without fat suppression showed excellent agreement in the diagnosis of normal tendon (kappa=.90) amd full thickness tears (kappa=.98), good agreement for partial tears (kappa=.70) and moderate agreement for the combined group of tendinosis and degeneration (kappa=.53). MR imaging and surgical correlation in 43 patients showed 86% specificity (95% confidence interval, 65-96%) for intact tendons, and 100% sensitivity (95% confidence interval, 82-100%) for full-thickness tears on T2 weighted fast spin-echo imaging--both without and with fat suppression. For partial tears, MR imaging showed a sensitivity of 92% (95% confidence interval, 65-99%) with fat suppression and 67% (95% confidence interval, 39-86%) without fat suppression. CONCLUSION: T2-weighted fast spin-echo imaging--with or without fat suppression--is a highly sensitive technique in the diagnosis of normal tendons and complete tears of the rotator cuff. Because of increased lesion conspicuity, fat suppression tends to perform better in the diagnosis of partial tears. PMID- 8615245 TI - An acetabular fracture with superior gluteal artery disruption. PMID- 8615244 TI - Evaluation of selective wrist arthrography of contralateral asymptomatic wrists for symmetric ligamentous defects. AB - OBJECTIVE: The objective of this investigation was to study the role of selective wrist arthrography of the asymptomatic wrists of patients with unilateral wrist pain and the efficacy of three-compartment and selective-compartment injections of contrast medium into the asymptomatic wrist in demonstrating symmetric and asymmetric intercarpal ligament and triangular fibrocartilage communicating defects. SUBJECTS AND METHODS: Wrist arthrography with bilateral three compartment injections was performed for 62 patients with unilateral wrist pain. The numbers of bilateral intercarpal ligament and triangular fibrocartilage communicating defects were recorded. The results obtained with three-compartment injections in each wrist of these patients were compared with those obtained with single-compartment injections. RESULTS: Bilateral three-compartment injections identified 110 communicating defects (59 in the symptomatic and 51 in the asymptomatic wrists). Midcarpal injections showed all 36 scapholunate and lunatotriquetral ligament defects that were also shown by three-compartment injections in asymptomatic wrists. However, only 26 (72%) of these 36 ligament defects were shown by radiocarpal injections. No single-compartment injection showed all triangular fibrocartilage defects that were shown by three-compartment injections. Ten bilateral symmetric triangular fibrocartilage communicating defects were shown by three-compartment injections. All 10 triangular fibrocartilage communicating defects in asymptomatic wrists were shown by radiocarpal injections. However, only five of the 10 triangular fibrocartilage communicating defects in asymptomatic wrists were shown by injection of the distal radioulnar joints. CONCLUSION: Selective midcarpal injection of an asymptomatic wrist showed all matching defects in that wrist when only intercarpal ligament defects were found in the symptomatic wrist. Similarly, selective radiocarpal injection of an asymptomatic wrist showed all matching defects of the triangular fibrocartilage in that wrist. In either situation, routine injection of all three compartments of the asymptomatic wrist should not be necessary. PMID- 8615246 TI - MR imaging of occult traumatic fractures and muscular injuries of the hip and pelvis in elderly patients. AB - Recent studies have shown the usefulness of T1-weighted MR imaging for identifying fractures of the proximal femur in elderly patients with negative radiographs after acute trauma [1-4]. The advent of fast spin-echo and fast inversion recovery sequences allows for the rapid identification of a variety of other musculoskeletal injuries of the hip and pelvis that may explain a patient's symptoms but that may not be seen on T1-weighted images. This pictorial essay shows the types of lesions encountered. Lesion prevalence is given for 20 consecutive patients examined. PMID- 8615247 TI - MR imaging of the foot: utility of complex oblique imaging planes. AB - Successful MR imaging of the foot presents special challenges to the radiologist. Accurate and confident diagnosis presupposes the ability to produce high resolution images of obliquely oriented, relatively small structures. Orienting the foot within an appropriate local coil to bring such structures into an orthogonal imaging plane, or even into a conventional oblique plane, may be impossible or intolerably uncomfortable for the patient. The frequent result is motion artifacts, which are accentuated when using a small field of view. However, when patients are comfortably positioned, the anatomy of interest often lies in a plane that is not orthogonal to any of the conventional imaging planes. Fortunately, commercially available MR imaging equipment can produce images in complex oblique planes with relative ease. In this pictorial essay, we discuss the technical considerations for expedient diagnostic MR imaging of the complex anatomy of the foot and illustrate our experiences with this technique. PMID- 8615248 TI - CT evaluation of acute cholecystitis: findings and usefulness in diagnosis. AB - OBJECTIVE: The purpose of our study was to describe the CT findings of acute cholecystitis and apply previously proposed CT criteria for its diagnosis. MATERIALS AND METHODS: We retrospectively reviewed CT scans of 29 patients with proven acute cholecystitis. Scans were reviewed for gallstones, gallbladder distension, bile density, wall thickening, pericholecystic fluid, subserosal edema, pericholecystic stranding, and sloughed membranes. Previously published criteria were applied to determine the percentage of patients that met positive criteria for acute cholecystitis. RESULTS: The most common CT findings, in decreasing order of frequency, were wall thickening (n = 17), pericholecystic stranding (n = 15), distension (n = 12), pericholecystic fluid (n = 9), subserosal edema (n = 9), high-attenuation bile (n = 7), and sloughed membranes (n = 1). Of the 29 cases of acute cholecystitis, 15 met previously published CT criteria. CONCLUSION: CT can be useful in diagnosing acute cholecystitis. Common CT findings of acute cholecystitis include wall thickening, pericholecystic stranding, distention, high-attenuation bile, pericholecystic fluid, and subserosal edema. When these findings are present, the diagnosis of acute cholecystitis can be suggested. However, previously published CT criteria failed to identify a significant number of patients with acute cholecystitis. PMID- 8615249 TI - Low attenuation of acute traumatic hemoperitoneum on CT scans. AB - OBJECTIVE: This study was undertaken to determine the incidence of low attenuation values in intraperitoneal hemorrhage, which could be confused with ascites. MATERIALS AND METHODS: We retrospectively analyzed the CT scans of 42 consecutive patients with hepatic and splenic lacerations and intraperitoneal fluid after blunt abdominal trauma. Patients were excluded if they had prior peritoneal lavage, bladder or bowel injury, or low hematocrit values. Intraperitoneal fluid was categorized by the site of accumulation (perihepatic, perisplenic, Morison's pouch, paracolic gutters, or pelvis). The amount of fluid in each intraperitoneal location was categorized as small, moderate, or large. Attenuation values were obtained from each intraperitoneal site, and overall mean attenuation values were determined for each patient. We correlated the size of each fluid collection with the attenuation value. We also compared attenuation values at locations adjacent to the site of each injury with those at other intraperitoneal sites. We then evaluated technical factors that could have lowered attenuation values, including CT miscalibration, volume averaging, and beam-hardening artifacts. RESULTS: For the 42 patients, we measured 131 separate attenuation values. Attenuation values ranged from 0 to 80 H, with attenuation of 24% of sites (32/131) measuring less than 20 H. Only 16% of sites (21/131) had attenuation values greater than 45 H. Attenuation at the remaining 78 sites (60%) measured from 20 to 45 H. All intraperitoneal locations except the pelvis had mean attenuation values significantly lower then 40 H. Mean attenuation values (determined by averaging measurements from different intraperitoneal sites) were also calculated for each patient. Only 6 (14%) of 42 patients had mean attenuation values greater than 40 H, whereas 4 (10%) of 42 patients had mean attenuation values less than 20 H. The remaining 32 patients (76%) had mean attenuation values between 21 and 40 H. Patients with hepatic lacerations showed no significant difference (p = .3509) in attenuation between perihepatic fluid and the remainder of intraperitoneal fluid. However, in patients with splenic lacerations, perisplenic fluid had a significantly higher (p = .0013) attenuation value (43 H) than did fluid at other intraperitoneal locations. CONCLUSION: Low attenuation measurements for acute hemoperitoneum represented a common finding that was not attributable to technical factors or underlying anemia. Fluid with attenuation values less than 20 H in acute trauma should not be dismissed as ascitic fluid. PMID- 8615250 TI - Clostridial infection of the abdomen: CT findings in two successfully treated patients. PMID- 8615251 TI - CT findings in primary vascular tumors of the spleen. PMID- 8615252 TI - Angiographic selection criteria for living related liver transplant donors. AB - OBJECTIVE: The purpose of this study is to better define arteriographic selection criteria for living related liver transplantation (LRLT) based on literature review, technical and theoretical considerations, and correlation of patterns of variation in hepatic artery anatomy with recipient and donor outcomes. MATERIALS AND METHODS: Visceral angiograms of 92 consecutive living related liver transplant donors were retrospectively reviewed by two radiologists and one transplant surgeon. Arterial configurations were categorized. Recipient and donor outcomes were determined by a review of transplant surgery and radiology records. RESULTS: Anomalous hepatic artery anatomy was identified in 67% of potential donors. A left hepatic artery (LHA) with a diameter of less than 2 mm was identified in 1%, and with a diameter of 2-3 mm, in 5%. A dual LHA supply to the left lateral segment was identified in 11%. Two subtypes were defined. Bifurcation of the LHA into branches entering segment II and segment III less than 1 cm from the LHA origin was present in 8%. A replaced LHA from the left gastric artery (17%) and complex, aberrant branching of the LHA (4%) were identified. Vital LHA supply to tissue other than the left lateral segment was present in 21%, including the cystic artery as a branch of the LHA (4%), significant supply of the right lobe from the LHA (5%), and large branches from the LHA entering segment IV (13%). All three donors with significant supply of the right lobe from the transplanted LHA had complications. CONCLUSION: Absolute exclusionary criteria for LRLT are an LHA diameter of less than 2 mm, dual arterial supply to liver segments II and III, indeterminate arterial anatomy, preexisting vascular disease in donor liver, and a significant LHA supply to the right lobe. Relative exclusionary criteria are an LHA diameter of 2-3 mm, early bifurcation of the LHA, and arterial supply of segment 4 exclusively from the LHA. PMID- 8615253 TI - Cholangiographic features of biliary strictures after liver transplantation for primary sclerosing cholangitis: evidence of recurrent disease. AB - OBJECTIVE: Biliary strictures occur more frequently after liver transplantation for primary sclerosing cholangitis (PSC) than for other diseases. A hypothesized cause is recurrence of PSC in the liver graft. In our study, we compared cholangiographic features of biliary strictures after transplantation for PSC to those after transplantation for other diseases. MATERIALS AND METHODS: A study group of 32 PSC grafts in adults with biliary strictures was compared with a control group of 32 non-PSC grafts with strictures. Both groups were matched for the type of biliary anastomosis (choledochojejunostomy) and for the time interval between transplantation and stricture diagnosis. We then performed a blind retrospective review of cholangiograms in these 64 cases to evaluate for features of PSC. RESULTS: Location, number, and length of strictures and ductal dilatation were similar in the PSC and non-PSC groups. Mural irregularities of bile ducts were present in 15 of 32 (47%) PSC grafts compared with four of 32 (13%) in the control group (p=.005). Diverticulum-like outpouchings occurred in six of 32 (19%) PSC graft compared with one of 32 (3%) in the control group. An overall resemblance to PSC was observed in eight of 32 (25%) grafts in the PSC group compared with two of 32 (6%) in the control group. CONCLUSION: Mural irregularity and diverticulum-like outpouchings--findings suggestive of PSC--and an overall appearance resembling PSC occur more frequently in PSC transplants than in transplants for other diseases. These findings are consistent with the hypothesis that PSC may recur in liver transplants. PMID- 8615254 TI - Detection of focal hepatic lesions with MR imaging: prospective comparison of T2 weighted fast spin-echo with and without fat suppression, T2-weighted breath-hold fast spin-echo, and gadolinium chelate-enhanced 3D gradient-recalled imaging. AB - OBJECTIVE: The purpose of this study was to compare breath-hold three-dimensional (3D) rapid gradient-echo (GRE) MR imaging obtained before and after gadolinium chelate injection with T2-weighted fast spin-echo and T2-weighted breath-hold fast spin-echo (BHFSE) MR imaging in the detection of focal hepatic masses. SUBJECTS AND METHODS: Fifty-three patients with 108 focal hepatic masses had, prospectively, MR of the liver at 1.5 T. T2-weighted fast spin-echo (6000/117 [TR/effective TE]; echo train length=16; acquisition time = 3 min 12 sec) images obtained with and without fat suppression, T2-weighted BHFSE (2700/105; echo train length = 20; acquisition time = 22 sec), and 3D rapid GRE images (10.1/1.9/30 degrees [TR/TE/alpha]) obtained during one breath-hold (12 scan locations in 21 sec or 20 scan locations in 32 sec) before and after injection of gadolinium chelate were blindly and independently analyzed in consensus by three readers. RESULTS: Gadolinium chelate-enhanced 3D rapid GRE images allowed depiction of more focal hepatic masses (90 of 108, sensitivity = 83%) than did T2 weighted fast spin-echo with fat suppression images (76 of 108, sensitivity = 70%), T2-weighted fast spin-echo without fat suppression images (74 of 108, sensitivity = 69%), T2-weighted BHFSE images (73 of 108, sensitivity = 68%), and unenhanced 3D rapid GRE images (54 of 108, sensitivity = 50%) (p < .01). No difference in sensitivity was found between the three T2-weighted sequences. CONCLUSION: Gadolinium chelate-enhanced 3D rapid GRE imaging is superior to T2 weighted fast spin-echo images obtained with or without fat suppression for the detection of focal hepatic masses. T2-weighted BHFSE is similar to T2-weighted fast spin-echo images in detecting focal hepatic lesions. PMID- 8615255 TI - Hepatic metastases from granulosa cell tumor of the ovary: CT and sonographic findings. PMID- 8615256 TI - Helical (spiral) CT angiography for identification of crossing vessels at the ureteropelvic junction. AB - OBJECTIVE: The purpose of this study was to determine the feasibility of imaging crossing vessels at the ureteropelvic junction (UPJ) with helical (spiral) CT angiography for planning surgical repair of symptomatic UPJ obstruction. SUBJECTS AND METHODS: Twenty-four consecutive patients with symptomatic UPJ obstruction were imaged with dual-phase, contrast-enhanced helical CT (collimation, 3 mm; pitch, 1.3-1.7; reconstruction interval, 2 mm; early phase, 20-42 sec; and delayed phase, 90-112 sec after initiation of IV contract material injection [125 ml of ioversol containing 320 mg of iodine per ml, delivered at 4-5 ml/sec]). All imaging data were viewed interactively on an imaging workstation. Prospective on line interpretations were correlated with subsequent surgical and clinical findings at laparoscopy (n=3), open surgical repair (n=2), or ureteronephroscopic endopyelotomy (n=11). Vessels at the UPJ that were 2 mm or more in diameter were believed to be significant. Review of the transaxial images was performed to determine qualitatively the relative usefulness of the early versus the delayed phases for distinguishing arteries from veins. Multiplanar reformations also were retrospectively reviewed and compared with direct pyelograms to determine the accuracy with which the location of the UPJ and the proximal ureteral course were depicted with helical CT. RESULTS: Eleven of 24 (46%) patients collectively had 11 anterior and three posterior vessels (> or = 2 mm in diameter) crossing the UPJ on helical CT. Distinction between arteries and veins was significantly better on early-phase than on delayed-phase images (p=.01). Visualization of the UPJ and the proximal ureteral course was good or excellent for 18 (78%) of 23 patients for whom pyelograms were available, regardless of the presence of a ureteral stent (p>.05). Laparoscopy and open surgery findings were in agreement with helical CT angiograms for five of five patients. Uncomplicated endopyelotomy was performed for 11 patients in whom no significant vessels were seen posterior or posterolateral to the UPJ. CONCLUSION: Helical CT angiography can depict vessels crossing the UPJ and is valuable in planning surgical management. PMID- 8615257 TI - Detection of main renal artery stenosis using phase-contrast cine MR angiography. AB - OBJECTIVE: The purpose of our study was to assess the ability of phase-contrast cine MR angiography to detect the presence of main renal artery stenosis. SUBJECTS AND METHODS: We prospectively evaluated 75 hypertensive patients form main renal artery stenosis using phase-contrast cine MR angiography. Each main renal artery was evaluated as normal or abnormal. Thirty-seven of the 75 patients underwent conventional arteriography or intraarterial digital subtraction arteriography; these results were compared with the MR angiographic interpretations. Only those patients who had confirmatory arteriography were included in the statistical analysis. RESULTS: Thirty-six main renal arteries interpreted as normal by MR angiography were found to be without a focal stenosis on invasive arteriography. MR angiography suggested 32 main renal artery stenoses; invasive arteriography showed 29 of these as stenoses. Three main renal arteries that were interpreted as having focal stenoses by MR angiography were shown to be not stenotic by invasive arteriography. Three other patients had diffusely narrowed main renal arteries bilaterally without a focal stenosis on MR angiography; bilateral proximal renal artery stenoses were seen at arteriography in two of these patients, and diffusely narrowed main renal arteries were seen in the third patient. Thus, the sensitivity of phase-contrast cine MR angiography for detecting a focal stenosis or abnormal main renal artery was 100% (95% confidence interval, 88-100%) and the specificity was 93% (95% confidence interval, 80-99%). The kappa coefficient was 0.85 with a standard error of 0.08. CONCLUSION: Phase-contrast cine MR angiography had a high degree of accuracy and a high negative predictive value in detecting the presence of main renal artery stenoses and may be a good screening technique for renovascular hypertension. PMID- 8615258 TI - Abdominopelvic MR imaging in the nonobstetric evaluation of pregnant patients. AB - Managing a pregnant patient with a suspected nonobstetric abdominopelvic disorder is challenging. Information from imaging affects the treatment options, which range from operating emergently and risking miscarriage to terminating the pregnancy or delaying treatment until after delivery. The most common indications for MR imaging during pregnancy are disorders of the central nervous and cardiovascular systems. In the evaluation of the abdomen and pelvis, sonography plays the pivotal role; however, sonography may be inconclusive because of its limited specificity in tissue characterization and technical difficulties caused by the enlarged uterus. For these reasons MR imaging has emerged as a valuable adjunct to sonography. In this pictorial essay we describe a variety of nonobstetric abdominopelvic disorders during pregnancy in which MR imaging contributed to patient management. PMID- 8615259 TI - Adenomyosis: specificity of 5 mm as the maximum normal uterine junctional zone thickness in MR images. AB - OBJECTIVE: Our objective was to investigate the specificity of the criterion stating that a diagnosis of adenomyosis can be made confidently from MR images of the uterus when the junctional zone is thicker than 5 mm. SUBJECTS AND METHODS: Twenty clinically normal women volunteers (chosen by strict criteria) underwent MR imaging of the uterus. Images were independently evaluated by two experienced observers who measured the greatest thickness of the junctional zone in the anterior, posterior, right and left walls of the uterine corpus and at the top of the fundus. RESULTS: The mean value of the greatest junctional zone thickness (averaged for both observers) was 4.4 +/- 2.1 mm (mean +/- SD, range, 1-9 mm) for the anterior and posterior walls of the corpus, 4.3 +/- 2.1 mm (range, 1-10 mm) for the right and left walls, and 2.3 +/- 1.1 mm (range, 1-5 mm) for the fundus. At least one region of the junctional zone was found to be thicker than 5 mm by at least one observer in 13 (65%) of the 20 subjects; at least one region was found to be thicker than 5 mm by both observers in eight (40%) subjects. At least two regions were measured as thicker than 5 mm by both observers in six (30%) subjects. Serial studies of three volunteers showed transient irregular focal or diffuse thickening (up to 12 mm) of the junctional zone and focal myometrial bulging, probably due to myometrial contractions. CONCLUSION: Our results suggest that if a diagnosis of adenomyosis is based solely on junctional zone thickness in MR images, 5 mm should not be assumed as the upper limit of normal, because this assumption may result in a high false-positive rate. PMID- 8615260 TI - Detection of pseudocapsule of renal cell carcinoma with MR imaging and CT. AB - OBJECTIVE: A pseudocapsule surrounding a renal cell carcinoma is a pathologic feature seen frequently in the early stages of this disease. Partial nephrectomy or simple enucleation may be indicated when a pseudocapsule is detected. The purpose of this study is to analyze the roles of MR imaging and CT in showing the pseudocapsule in renal cell carcinomas. SUBJECTS AND METHODS: MR imaging and CT appearances of the kidneys in 52 patients with 54 renal cell carcinomas--and 40 patients with 45 other renal masses for comparison--were prospectively analyzed and correlated with pathologic results. The frequency of a pseudocapsule in renal cell carcinomas and in various renal tumors was analyzed. We compared the detectability of a pseudocapsule in renal cell carcinomas with MR imaging on T1 weighted spin-echo, T2-weighted spin-echo, and contrast-enhanced T1-weighted spin echo sequences, and on contrast-enhanced CT. RESULTS: At pathologic evaluation, a pseudocapsule was seen in 66% (19 of 29) of tumors 4 cm or less in diameter and in 28% (7 or 25) of tumors larger than 4 cm in diameter. All tumors with a pseudocapsule were low histologic grade. Large renal cell carcinomas and other tumors appeared expansile but not encapsulated. A rim corresponding to the pseudocapsule was seen around the tumor in seven lesions on T1-weighted images, 26 lesions on T2-weighted images, and 11 lesions on postcontrast T1-weighted images. T2-weighted imaging was the most sensitive technique for visualization of the pseudocapsule (sensitivity, 68%; specificity, 91%). All pseudocapsules detected on T1-weighted images or postcontrast T1-weighted images could be detected on T2-weighted images. With postcontrast images, enhancement of the pseudocapsule resulted in poor contrast relative to the surrounding tissue. At contrast-enhanced CT, the pseudocapsule was not visible in any tumors. In all MR imaging sequences and in CT, a pseudocapsule were not found in other pathologic conditions except oncocytoma. CONCLUSION: A pseudocapsule was seen in 66% of renal cell carcinomas 4 cm in diameter or smaller. T2-weighted MR imaging is the most sensitive technique for showing this feature. PMID- 8615261 TI - Hormonal ablation of prostatic cancer: effects on prostate morphology, tumor detection, and staging by endorectal coil MR imaging. AB - OBJECTIVE: The purpose of our study was to evaluate the effect of androgen deprivation therapy on the MR imaging findings of prostate gland anatomy and cancer pathology in men with prostatic cancer treated with hormonal ablation before radical prostatectomy. MATERIALS AND METHODS: Twenty-two patients (mean age, 66 years old) were divided into two groups: in group I (n=10), MR imaging studies were done before and after hormonal treatment; in group II (n=12), MR imaging studies were done only after hormonal treatment. MR imaging was performed on a 1.5 T-scanner (Signa; General Electric Medical Systems, Milwaukee, WI) and included transverse plane phased-array coil T1-weighted images (TR/TE, 600/12), combined endorectal phased-array coil transverse plane T1-weighted images, fast spin-echo T2-weighted (4000/102), and coronal plane fast spin-echo T2-weighted images. Image evaluation was by consensus and included assessment of the gland size, signal intensity, tumor depiction, extracapsular extension, seminal vesicle invasion, and overall staging accuracy (Jewett and Whitmore classification). MR imaging findings were correlated with pathologic findings of step section radical prostatectomy. RESULTS: After hormonal therapy, the volume of the prostate gland showed a mean decrease of 33.5% +/- 19.6% SD (range, 0-64%). Volume reduction in the transition zone (mean 29.2% +/- 22% SD) was less than in the peripheral zone (mean, 55.8% +/- 25.8% SD) (p < .05). On T2-weighted images, the peripheral zone showed homogeneous decreases in signal intensity in 13 of 22 (58%) patients. Compared with pathologic findings, the accuracy of tumor detection by MR imaging was 74% (98 of 132 sites). Tumor presence was overestimated in 32 of 132 (24%) sites. Overall staging accuracy after hormonal ablation was 68% (15 of 22). The positive predictive value and negative predictive value for extracapsular extension were 57% (13 of 23 sites) and 90% (19 of 21 sites), respectively, and for seminal vesicle invasion were 80% (8 of 10 sites) and 97% (33 of 34 sites), respectively. CONCLUSION: As detected by MR imaging, hormonal ablation caused a decrease in size and signal intensity of the prostate gland and seminal vesicles and overestimation of tumor presence and extracapsular extension. PMID- 8615262 TI - Radiologic-Pathologic Conferences of the Massachusetts General Hospital. Necrotizing fasciitis of the scrotum (Fournier's gangrene) PMID- 8615263 TI - CT findings after laparoscopic pelvic lymph node dissection and transperineal radioactive seed implantation for prostatic carcinoma. AB - OBJECTIVE: The purpose of this paper is to describe the CT findings in patients undergoing transperineal sonographically guided radioactive seed implantation with and without laparoscopic lymph node dissection for treatment of carcinoma of the prostate gland. MATERIALS AND METHODS: Ninety-six CT scans of the pelvis were retrospectively reviewed in patients who had undergone radioactive seed implantation of the prostate gland. The CT readings were correlated with the type of procedure each patient had and the pathologic results of laparoscopic lymph node dissection. RESULTS: Of 73 patients who underwent laparoscopic lymph node dissection, 42 were found at CT to have low-attenuation masses along lymph node chains. These masses resembled necrotic nodes. Only one patient who did not undergo laparoscopic lymph node dissection had similar findings. Fourteen patients had fluid collections within the pelvis, 13 of whom had undergone laparoscopic lymph node dissection. Other imaging findings included ectopic seeds (10 patients) and subcutaneous and intrapelvic air (11 patients). All except two patients had negative pathology reports for the removed lymph nodes. CONCLUSION: A wide variety of imaging findings may be seen after radioactive seed implantation and laparoscopic lymph node dissection, including masses that resemble necrotic lymph nodes. Radiologists should be aware of these findings in asymptomatic patients. PMID- 8615264 TI - Use of a variation of loop snare for manipulation of ureteral stents. PMID- 8615265 TI - Self-expandable stents for the treatment of iliac artery obstructive lesions: long-term success and prognostic factors. AB - OBJECTIVE: The purpose of our study was to report long-term (more than 2 years of follow-up) angiographic patency after self-expandable stent implantation in the iliac artery and to identify patient- or procedure-related prognostic factors of angiographic patency. SUBJECTS AND METHODS: Ninety-five consecutive patients (101 arteries) underwent Wallstent implantation to treat claudication (n=95 limbs), rest pain (n=2), and nonhealing ulcer (n=3). Another patient was asymptomatic but was treated for acute occlusion of the iliac artery after coronary angioplasty. After implantation of self-expandable stents, we followed up by examining clinical and angiographic records at 6 months, 1 year, and annually thereafter. The Kaplan-Meier survival curve was used to determine primary and secondary patency rates. Primary patency was that achieved after the initial procedure only. Secondary patency was defined as that achieved after one or more successful additional percutaneous procedures within the stent or beyond the stent. Multivariate analysis using the Cox proportional hazard model was performed to identify predictive factors of angiographic failure, defined as restenosis of 50% or greater or occlusion. RESULTS: Four-year patency rates of 61% (primary) and 86% (secondary) were found (mean follow-up, 29 months). The following five factors were associated with long-term angiographic failure: occlusion of the superficial femoral artery (relative hazard = 5.21), absence of hypertension (relative hazard = 4.85), a stent diameter of less than 8 mm (relative hazard = 4.45), two or more stents implanted (relative hazard = 3.56), and current tobacco consumption (relative hazard = 2.46). CONCLUSION: Improved patency rates may be obtained by selecting patients for Wallstent implantation in the iliac artery based on five factors shown to be prognostically important. PMID- 8615266 TI - MR angiography for mapping potential central venous access sites in patients with advanced venous occlusive disease. AB - OBJECTIVE: Patients who depend on long-term central venous catheter support frequently develop thrombi in central veins. An accurate, noninvasive technique is needed to find patent central veins for future access. We evaluated the suitability of MR angiography for this use. SUBJECTS AND METHODS: Using five healthy volunteers and 19 patients who had malfunctioning central venous catheters and a history of central venous occlusive disease, we tested the ability of MR angiography to assess central vein status. Three radiologists experienced in MR angiography blindly interpreted both source images and three dimensional reconstructed images. RESULTS: In the volunteers, MR angiography provided diagnostic-quality images of the internal and external jugular, innominate, subclavian, axillary, femoral, and iliac veins, and of the superior and inferior venae cavae. Interobserver interpretations did not vary. In the patients, MR angiography of venous patency was confirmed by venography in 27 segments, by sonography n 32 segments, and by attempted line placement in 21 placements. Images were diagnostically adequate in 206 of 216 segments (95%). For detection of occlusion, sensitivity was 97% and specificity was 94%. MR angiography predicted 100% of successful line placements and 80% of failures. Interobserver interpretations varied by 44%. MR angiography directly influenced therapy in 19 of 21 studies. CONCLUSION: We conclude that MR angiography provides risk-free, thorough, relatively accurate, and clinically useful assessment of most available central venous access sites, although interpretation may prove difficult in patients with extensive occlusions because of complex collateral drainage patterns. PMID- 8615268 TI - Stereotaxic large-core biopsy after failed surgical excision. PMID- 8615267 TI - Status of residency training in mammography. AB - OBJECTIVE: The purpose of this study was to survey the status of residency training in mammography, including mammography quality assurance, and to compare the results with previous surveys from 1990 and 1992. MATERIALS AND METHODS: A telephone interview was conducted with 213 chief residents and 11 other senior residents representing all 224 accredited residency programs in diagnostic radiology. RESULTS: Compared with 1990, more programs had exclusive mammography rotations (74% versus 40%). The number of fellowships in breast imaging increased from 17 to 76. More training programs differentiated screening from diagnostic examinations (50% versus 35%). Compared with 1992, the percentage of residents who knew recommended frequencies of quality control procedures increased by 30% and the percentage who knew the maximum allowable radiation dose increased by 50%. Residents lacked fundamental knowledge about conducting medical audits, such as calculating the positive predictive value for biopsies. CONCLUSION: More time is being devoted to breast imaging in diagnostic radiology residency training, and residents show greater knowledge about mammography quality assurance. PMID- 8615270 TI - Acute fracture of the distal tibial physis: role of gradient-echo MR imaging versus plain film examination. AB - OBJECTIVE: Recent reports indicate that, compared with MR imaging, plain film radiography often underestimates the extent of injury in children with physeal fracture-separation. Such underestimation may have significant therapeutic and prognostic outcomes. We performed this study to assess the benefit of MR imaging compared with plain film radiography in the diagnosis and immediate treatment of acute fractures of the distal tibial physis. SUBJECTS AND METHODS: Twenty-nine patients with acute fractures, including 15 Salter-Harris II, four Salter-Harris III, four Salter-Harris IV, and six triplane fractures, were prospectively examined by MR imaging with gradient-echo sequences. The MR images were compared with plain film radiographs. All cases were reviewed in a blind fashion by two experienced radiologists to determine the Salter-Harris classification on the basis of first plain film radiographs and then MR images. Two experienced pediatric surgeons were asked to propose treatment on the basis of first plain film radiographs and then MR images. Both the radiologists and the surgeons were asked to rate the two techniques in terms of degree of confidence and overall diagnostic effectiveness. RESULTS: Only 1 or 29 fractures (3%) was misclassified by plain film radiography. MR imaging never caused the treatment plan to be modified. However, the position of fracture fragments in Salter-Harris IV and triplane fractures was always better appreciated on MR images, facilitating more accurate surgical treatment. Except for those in the misclassified fracture, all other fracture lines were seen on both plain film radiographs and MR images but were more easily seen on MR images. CONCLUSION: Gradient-echo MR imaging allowed easier assessment of fracture lines than did plain film radiography, but the latter technique remains the primary means of evaluating epiphyseal injuries. For acute fractures of the lower extremity of the tibia, gradient-echo MR imaging should be limited to complex fractures and to cases in which the classification of a fracture on the basis of plain film evaluation is uncertain. PMID- 8615269 TI - Fetal congenital diaphragmatic hernia: accuracy of sonography in the diagnosis and prediction of the outcome after birth. AB - OBJECTIVE: This study evaluated the diagnostic accuracy of prenatal sonography in congenital diaphragmatic hernia (CDH) and assessed sonographic predictors of postnatal outcome. MATERIALS AND METHODS: Sonograms and medical records of 43 fetuses with CDH were retrospectively reviewed. Sonographic features were correlated to clinical evolution and surgical and pathologic findings. RESULTS: CDH was diagnosed prenatally with sonography in 40 cases. Intrathoracic stomach and liver were routinely identified. Ipsilateral lung tissue could not be differentiated from herniated content. Contralateral lung was identified in all cases except two. The overall survival rate was 43% after excluding terminated pregnancies. Besides associated malformations and chromosomal anomalies, the only statistically significant predictor of survival was the quantification of the contralateral lung area at the level of an axial four-chamber view: The survival rate was 86% when the contralateral lung area was equal to or greater than one half the area of the hemithorax. CONCLUSION: Sonography is highly accurate for prenatal diagnosis od CDH. Sonography also assists the prognostication of postnatal outcome in isolated CDH by allowing quantification of the contralateral lung area on a four-chamber view. PMID- 8615271 TI - A regional approach to classic metaphyseal lesions in abused infants: the distal tibia. AB - OBJECTIVE: The purpose of this study was to analyze systematically the spectrum of morphologic alterations of classic metaphyseal lesions (CML) involving the distal tibia of abused infants and to identify features that assist in the radiologic diagnosis and assessment of healing. MATERIALS AND METHODS: Thirty-one infants who died with evidence of inflicted injury were studied with high detailed skeletal surveys, resected-specimen radiography, and histopathologic analysis. The number of fractures identified, the portions of the distal tibial metaphyses involved, and the age of the lesion were assessed. RESULTS: A total of 16 CML of the distal tibia were noted. Eight infants had unilateral injury that always involved the left side, and four had bilateral lesions. When a CML was visible radiographically, it was always seen along the medial aspect of the metaphysis; lateral metaphyseal involvement was evident with more extensive injuries and always accompanied medial involvement. Fractures tended to be less conspicuous when acute and were more easily recognized with healing, especially with specimen radiography. Radiographically, the typical fracture separated a medial fragment that was tall and triangular; this appearance was histologically related to the undercutting of the long subperiosteal bone collar. In healing lesions, extension of hypertrophic chondrocytes from the growth plate into the region of the fracture was a consistent finding. CONCLUSION: CML of the distal tibia have distinctive radiologic and histopathologic characteristics that relate to the anatomy of the region. Visualization of these fractures depends on the quality of the radiographs obtained. An understanding of the radiologic and histopathologic features of CML should aid in the recognition of this strong indicator of infant abuse. PMID- 8615272 TI - Optimizing imaging parameters for MR evaluation of the spine with titanium pedicle screws. AB - OBJECTIVE: This study examined the contribution and interdependence of multiple imaging parameters in clinical imaging sequences to aid practicing radiologists in minimizing artifacts during MR imaging of the spine after implantation of titanium pedicle screws. MATERIALS AND METHODS: A lumbar spine specimen with titanium pedicle screws implanted in the pedicle was imaged with a 1.5-T scanner. Sequence type, voxel volume, TE, and bandwidth varied. Different voxel volumes were achieved by altering section thickness, field of view (FOV), and matrix size. Artifact size was measured on sagittal and axial images at the midpedicle level. Artifact size was expressed as a percentage of actual screw size, and mean artifact size was calculated for each sequence. Analysis of variance without replication was done. RESULTS: Mean artifact size ranged from 231% to 364% of actual screw size. Artifact size was independent of voxel volume for voxels greater than 3 MM3 (p<.001). Artifact size decreased significantly (p<.001) when voxel volume was less than 1 mm3. When we increased slice thickness and maintained a constant voxel volume, artifact size decreased. Decreases in artifact size correlated with a reduction in the ratio of the FOV to the number of pixels in the frequency-encoding direction (Nx). Artifact sizes were smallest when fast spin-echo sequences were used. Conventional spin-echo sequences produced artifacts that were smaller than the artifacts produced by the gradient echo sequences. Decreasing the TE did not diminish artifact size for conventional spin-echo images in larger voxel volume. CONCLUSION: Although voxel volume has been recognized as a factor that affects artifact size, the role of other contributing factors--slice thickness, number of phase-encoding steps, and FOV/Nx -has not been evaluated before. Artifact reduction proved to be dependent only on FOV/Nx. Artifact size was reduced by the use of fast spin-echo sequences. With conventional spin-echo sequences, TE should be minimized, although other technical factors may outweigh the gain in artifact reduction. PMID- 8615274 TI - What is the role of intraarterial vasodilators during angioplasty? Which and how much vasodilator should we use? PMID- 8615273 TI - Dental disease: a frequently unrecognized cause of maxillary sinus abnormalities? AB - OBJECTIVE: Periodontal disease may be a frequently unrecognized cause of maxillary sinus disease. The purposes of this study were to determine if maxillary sinus disease is more prevalent in patients with periodontal disease than in an age-and-sex-matched control group and to show radiographically an association of focal maxillary sinus disease with periodontal disease. MATERIALS AND METHODS: Maxillary DentaScans (General Electric Medical Systems, Milwaukee, WI) of 84 patients (168 maxillary sinuses) with periodontal disease were retrospectively evaluated for the simple presence or absence of maxillary sinus disease. This group was compared with an age-and-sex-matched control population of 84 patients who were referred for head or neck CT scans in which the maxillary sinuses (including their inferior aspects) were visualized. For the likelihood of sinus disease in patients compared with controls, an odds ratio and a 95% confidence interval were calculated using the SYSTAT version 5.2 (SYSTAT, Evanston, IL). In the second portion of the study, the subject population alone was graded in the following fashion to establish a causal relationship: grade 0, no sinus disease; grade 1, focal sinus disease not adjacent to periodontal disease (unlikely to be caused by periodontal disease); grade 2, nonfocal sinus disease (complete opacification, air-fluid levels, or diffuse mucoperiosteal thickening; indeterminate cause), and grade 3, focal sinus disease adjacent to periodontal disease (likely to be caused by periodontal disease). RESULTS: In the subject population--patients with periodontal disease who were referred for DentaScans--100 of 168 (60%) sinuses had sinus disease; in the control population, only 49 of 168 (29%) sinuses had sinus disease. The odds ratio for maxillary sinus disease in the patient population compared with controls was 3.6 (95% confidence interval, 2.3-5.6; p<.0001). The grading results of the subject population in the second portion of the study were grade 0, 68 sinuses (41%); grade 1, four sinuses (2%); grade 2, 32 sinuses (19%); and grade 3, 64 sinuses (38%). CONCLUSION: We have demonstrated a twofold increase in maxillary sinus disease in patients with periodontal disease and have shown a causal relationship. Recognition of this relationship may have an impact on the clinical management of patients, particularly those planning implant surgery. PMID- 8615275 TI - Emergency room radiographs of the facial bones for trauma show no abnormality except for an opaque maxillary antrum on the side of the injury. Is it necessary to investigate further for evidence of fracture? PMID- 8615276 TI - CT as the initial diagnostic tool for small-bowel obstruction. PMID- 8615277 TI - CT angiography: shaded surface versus maximum intensity projection. PMID- 8615278 TI - Sensitivity or specificity? PMID- 8615279 TI - Atypical ductal hyperplasia from stereotactic core biopsy of the breast. PMID- 8615280 TI - Re: making black-and-white slides of radiographs using Kodak Ektachrome film and a custom-made view box. PMID- 8615281 TI - Asymptomatic peripheral embolism during balloon angioplasty. PMID- 8615282 TI - Spinal tumors in patients with neurofibromatosis type 2: MR imaging study of frequency, multiplicity and variety. PMID- 8615283 TI - Preserving our images. PMID- 8615284 TI - Accessing intuition through imagery for stress management, creative problem solving, and diagnosis. PMID- 8615285 TI - Popliteal cysts on MR imaging: value as an ancillary sign of meniscal tears. PMID- 8615286 TI - MR imaging of the knee: findings in cat-scratch disease. PMID- 8615287 TI - An ossifying carcinosarcoma of the gallbladder: radiologic findings. PMID- 8615288 TI - Treatment of critical pulmonary valve stenosis by balloon dilatation in the neonate. AB - Critical pulmonary valve stenosis represents an emergency, and immediate treatment is mandatory. The purpose of this study was to evaluate the immediate and medium-term results of pulmonary valve dilatation. We report 18 neonates in whom pulmonary valvuloplasty was attempted. The procedure could be accomplished in 14 patients. The angiographically determined diameters of the pulmonary and tricuspid valve at the time of procedure were 5.6 +/- 1.5 mm and 14.0 +/- 5.4 mm. The mean Doppler gradient decreased from 71 +/- 27 mm Hg to 27 +/- 14 mm Hg. Perforation of the right ventricular outflow tract was the major complication in three patients with one fatal event. Infusion of prostaglandin E1 could be discontinued 1 to 5 days after the procedure. On follow-up three children required a second balloon dilatation with good results. Seven patients monitored for more than 9 months with a mean follow-up time of 34.4 +/- 16 months had a residual gradient of 11.6 +/- 6.7 mm Hg. In spite of a hypoplastic pulmonary valve annulus in seven of the patients, results were good and surgery could be avoided. PMID- 8615290 TI - Effectiveness of a thermal shape-memory patent ductus arteriosus occlusion coil. AB - A new shape-memory coil for transcatheter closure of the PDA was developed and evaluated in a dog PDA model. The coil is sensitive to temperature (composed of thermal shape-memory nickel-titanium). The coil was delivered to and implanted in the PDA model with a 5F catheter through the femoral artery. The PDA model was made in 14 dogs by infrarenal aorta banding (diameter 2.0 +/- 0.6 mm; length 2.9 +/- 0.1 mm). Coils were implanted in 10 dogs, whereas the other 4 dogs served as controls. Complete occlusion of the PDA model was confirmed by angiography 24 hours after the coil implantation in 7 of 10 dogs, whereas small residual flow was noted even 2 weeks after the coil implantation in the other 3 dogs. The PDA model internal diameter in the dogs with complete occlusion (1.8 +/- 0.2 mm) was significantly smaller than the diameter in dogs with residual flow (2.9 +/- 0.4 mm, P < 0.05). In the 4 control dogs, the PDA model remained patent 2 weeks after the banding. We conclude that the transcatheter occlusion with the shape-memory coil was effective in the small-diameter PDA model. PMID- 8615289 TI - Roles of percutaneous transluminal coronary angioplasty and bypass graft surgery for the treatment of coronary artery disease. PMID- 8615291 TI - Renin inhibition: a novel therapy for cardiovascular disease. AB - The renin-angiotensin system (RAS) is a major contributor in the regulation of blood pressure, and pharmacologic manipulation of this system has resulted in a beneficial class of therapeutic agents, which include angiotensin-converting enzyme (ACE) inhibitors. However, ACE inhibitors are not specific for RAS, and in addition, they can affect bradykinin and prostaglandin, which can also cause changes in vascular tone. Under development are renin inhibitors that are specific for angiotensinogen and act at the initial, rate-determining step of the RAS cascade. The various pharmacologic approaches to renin inhibition include specific renin antibodies, synthetic derivatives of the prosegment of renin precursor, pepstatin analogs, and angiotensinogen analogs. The last approach is the most promising for patient therapy. Multiple studies have shown the effectiveness of the renin inhibitors in both primates and human beings. Further research is now directed toward the development of an agent with good oral bioavailability for patient treatment and as a biologic probe for helping to understand the role of the RAS in control of blood pressure and blood volume. PMID- 8615292 TI - Ventricular pacing: a cause of reversible severe mitral regurgitation. PMID- 8615293 TI - Familial long QT syndrome: electrical storm and implantable cardioverter device therapy. PMID- 8615294 TI - Acute traumatic ventricular septal rupture. PMID- 8615295 TI - Isolated innominate artery in asplenia syndrome with aortic atresia: newly recognized cardiovascular complex. PMID- 8615296 TI - Cryoglobulinemia: a reversible cause of dilated cardiomyopathy. PMID- 8615297 TI - Munchausen's syndrome by telemetry. PMID- 8615298 TI - Successful heart failure therapy is effective antiarrhythmia therapy. PMID- 8615299 TI - Cyclic flow variations after angioplasty: a rare phenomenon predictive of immediate complications. DEBATE Investigator's Group. AB - Experimental studies and studies in human beings have indicated that cyclic flow variation (CFV) is a reliable marker of the formation of platelet aggregates and thus may be predictive of immediate complications after coronary angioplasty. In this study, the incidence and clinical relevance of CFV in a large patient population after elective percutaneous transluminal coronary angioplasty (PTCA) was assessed. One hundred two patients with one-vessel disease and no previous Q wave myocardial infarction underwent angiographically successful PTCA of a single lesion of a major coronary artery. A Doppler guide wire inserted distal to the stenosis was used to monitor flow velocity continuously after PTCA for 15 minutes. In 94 (92%) of 102 patients, a stable and reliable Doppler signal could be recorded for 15 minutes after the procedure. CFV, defined as gradual decline in flow over several minutes followed by a sudden restoration to higher values, was observed in 4 patients. In 3 of these 4 patients, the occurrence of CFV was predictive of immediate complications (2 intracoronary thrombosis and 1 acute closure), whereas none of the patients without CFV (n = 90) showed acute closure during hospital stay. In conclusion, CFVs in patients after angiographically successful elective PTCA are rare (4.3%) but highly sensitive for the prediction of abrupt occlusion. PMID- 8615300 TI - Superoxide dismutase activity as a predictor of myocardial reperfusion and salvage in acute myocardial infarction. AB - We attempted to predict successful myocardial reperfusion and salvage in acute myocardial infarction (AMI) by using measurements of plasma superoxide dismutase (SOD), a plasma free-radical scavenger, activity. Forty-nine patients with AMI were studied within 6 hours of symptoms onset. In group 1 (n = 26), primary percutaneous transluminal coronary angioplasty (PTCA) was undertaken, and plasma SOD activity was measured for 8 hours by the nitrite method. Left ventricular (LV) angiography was assessed before and 3 months after PTCA by computer LV contraction analysis. In group 2 (n = 23), TPA was infused intravenously over a 60-minute period, and plasma SOD activity was measured before and immediately after TPA infusion. In group 1, occluded coronary arteries were successfully dilated in 24 of 26 patients, and plasma SOD activity increased from 3.20 +/- 0.17 microns/ml to 4.66 +/- 0.29 microns/ml at 1 hour after PTCA (p < 0.001), returning to the basal level by 8 hours after PTCA. Plasma SOD activity did not significantly change patients with unsuccessful PTCA or those with the no-reflow phenomenon. The maximal increase in plasma SOD activity was significantly correlated with the grade of improvement in LV contraction (r = 0.852, p < 0.001). In group 2, the sensitivity and specificity of predicting coronary recanalization was 86% and 89%, respectively. In conclusion, myocardial reperfusion and salvage in AMI can be predicted by changes in plasma SOD activity. PMID- 8615301 TI - C-reactive protein as a predictor of cardiac rupture after acute myocardial infarction. AB - Although cardiac rupture is the second most common cause of death after ventricular failure in acute myocardial infarction, no diagnosis has ever been made before an episode of clinical compromise, and no significant predictive factors have been described. This study was designed to determine whether high serum C-reactive protein (CRP) levels could predict the incidence of subacute cardiac rupture after acute myocardial infarction. Nine consecutive patients with cardiac rupture were compared retrospectively with 28 consecutive control patients without rupture after acute myocardial infarction. In the rupture group, peak serum CRP levels increased rapidly and markedly after infarction, reaching more than 20 mg/dl on day 2, and persisted at high levels compared with those in the control group. However, the time course and levels of serum creatine phosphokinase were not significantly different between the two groups. High serum CRP levels ( > 20 mg/dl) had a high diagnostic sensitivity (89%) and specificity (96%) for cardiac rupture. Patients with persistently high serum CRP levels, particularly above 20 mg/dl, might have high probability of occurrence of sub acute cardiac rupture after acute myocardial infarction. PMID- 8615302 TI - Reliability of electromechanical dissociation in the diagnosis of left ventricular free wall rupture in acute myocardial infarction. AB - The reliability of electromechanical dissociation (EMD) in diagnosing acute left ventricular free wall rupture (LVFWR) was assessed in 479 consecutive patients with acute myocardial infarction (AMI). EMD was the mechanism of death in 193 patients, 140 without heart failure (group A, 74%), and 53 with heart failure (group B, 26%). Autopsies performed on 121 patients with EMD showed LVFWR in 81 (95%) of 85 from group A and in 7 (17%) of 36 from group B. Of the 106 patients without EMD (group C) autopsied, 5 (4.7%) had LVFWR. Excluding the eight patients with associated septal rupture, LVFWR occurred in 79 (95.2%) of 83 patients from group A, 4 (12.1%) of 33 from group B, and 2 (1.9%) of 103 from group C. Predictive accuracy of EMD for LVFWR in group A was 95% but only 17% in group B. Moreover, in 13 consecutive cases with a first AMI without heart failure and EMD, emergency surgery demonstrated LVFWR in all. Thus EMD has a highly predictive accuracy in diagnosing LVFWR in patients with a first AMI without overt heart failure. PMID- 8615303 TI - Pathogenesis of mitral regurgitation in acute myocardial infarction: importance of changes in left ventricular shape and regional function. AB - The pathogenesis of mitral regurgitation (MR) was determined by quantitative echocardiography in 188 patients with acute myocardial infarction (AMI) within 48 hours after admission. MR was classified, by using color Doppler, as significant (grades 3 to 4) or trivial (grades 0 to 2). Left ventricular (LV) function (global and regional), volume, and shape, as well as mitral valvular features, were measured and analyzed by stepwise logistic regression. Significant MR occurred in 25 (13%) patients. Univariately, recurrent infarction (p < 0.01), LV dilation (p < 0.001) and sphericity (p < 0.001), inferoposterolateral asynergy (p < 0.001), mitral annular dilatation (p < 0.005), and mitral leaflet restriction (p < 0.05) were associated with significant MR. In regression analysis, only recurrent infarction (odds ratio 5.08), LV sphericity index (odds ratio 1.12), and inferoposterolateral asynergy (odds ratio 6.07) were independently associated with significant MR, whereas none of the mitral valvular features examined had an independent association. In conclusion, changes in LV shape and regional function and not mitral valvular changes are prime determinants of significant MR after AMI. PMID- 8615304 TI - Pharmacologic profile of survivors of acute myocardial infarction at United States academic hospitals. AB - Optimal drug therapy for patients with acute myocardial infarction (AMI) is well described in the medical literature. However, data on the actual pharmacologic management of patients surviving AMI at academic hospitals is unavailable. The purpose of this study was to document treatment profiles in 500 patients surviving AMI at 12 academic hospitals in the United States. These profiles were compared with established guidelines and were evaluated for trends. Overall, thrombolytics (streptokinase > or = tissue-type plasminogen activator) were administered in 29% of the patients, with a greater proportion of patients receiving beta-blockers than calcium channel antagonists in the initial 72 hours (61% vs 40%; p < 0.005) and at discharge (51% vs 35%; p < 0.005). Further, women were less likely than men to receive thrombolytic therapy (odds ratio [OR] = 0.61; confidence interval [CI], 0.54 to 0.69) or beta-blocker therapy within the first 72 hours (OR = 0.61; CI, 0.55 to 0.67) or at hospital discharge (OR = 0.53; CI, 0.48 to 0.58). Overall, improvements could still be made in the number of patients who receive thrombolytic and acute and chronic beta-blocker therapies after AMI, particularly in women. Changes in treatment profiles may be a reflection of the publication of large clinical trials. PMID- 8615306 TI - Influence of vessel dilatation on restenosis after successful percutaneous transluminal coronary angioplasty. AB - The aim of this study was to evaluate the influence of vessel dilation on restenosis after successful percutaneous transluminal coronary angioplasty (PTCA) on the basis of quantitative angiographic analysis. To have the best comparison possible, we restrospectively studied a homogenous series of patients from the early 1980s treated according to a standardized PTCA procedure. The study group consisted of 86 patients with stable angina pectoris and single-vessel disease, all of whom underwent successful PTCA for a short concentric lesion in proximal vessel parts. The overall restenosis rate was 27%. Angiographically measured balloon size remained below specifications. The size of the inflated balloon at the site of minimal lumen diameter averaged 2.6 +/- 0.5 mm, and nominal balloon size was 3.3 +/- 0.4 mm (p < 0.001). In 22 patients with an oversized balloon (mean balloon/artery ratio 1.1 +/- 0.16) the restenosis rate was 5% compared with 34% in the corresponding group (p = 0.02). Minimal lumen diameters that were similar after the procedure (2.4 +/- 0.3 vs 2.3 +/- 0.4, NS) were 2.3 +/- 0.4 mm and 1.8 +/- 0.7 mm, respectively, at follow-up (p = 0.002). Multivariate analysis revealed balloon/vessel size ratio (p < 0.001), postprocedure diameter stenosis (p = 0.02), and percentage diameter increase produced by PTCA (p = 0.04) as independent correlates of the late outcome. Postangioplasty minimal lumen diameter was not related to restenosis. The strongest and most significant predictor of late PTCA outcome both by univariate and multivariate analysis was balloon/vessel size ratio, especially when balloon expansion at the site of minimal lumen diameter was regarded. In patients with continued success at follow up, the ratio was 0.81 +/- 0.15 compared with 0.60 +/- 0.11 in patients with restenosis (p < 0.001). Our results suggest that the late angiographic outcome of PTCA is strongly influenced by procedural factors. It appears that in a selected group of patients, an increased balloon/artery ratio, supposedly associated with increased vessel wall stretch, favorably affects the restenosis process. PMID- 8615305 TI - Rotational atherectomy with adjunctive balloon angioplasty versus conventional percutaneous transluminal coronary angioplasty in type B2 lesions: results of a randomized study. AB - A randomized pilot study was performed comparing conventional balloon angioplasty (percutaneous transluminal coronary angioplasty [PTCA] group) and rotational atherectomy (RA) with a medium size burr (50% to 70% burr/artery ratio) with systematic adjunctive balloon angioplasty (RA group) in type B2 stenosis. A total of 64 patients were included. Primary success was 93.7% in the RA group and 87.5% in the PTCA group (p = NS). Technical failure with no complication occurred once in each group. Acute complications occurred in three patients in the PTCA group and in one in the RA group. Angiographic restenosis rates were similar (RA group: 39%, PTCA group: 42%, p = NS) with a follow-up rate of 93%. In type B2 lesions, when compared with conventional angioplasty, RA with systematic balloon angioplasty does not seem to increase procedural success, and the restenosis rate remains comparable. However, these results must be confirmed in a larger series of patients. PMID- 8615307 TI - Microparticle deposition in periarterial microvasculature and intramural dissections after porous balloon delivery into atherosclerotic vessels: quantitation and localization by confocal scanning laser microscopy. AB - Local delivery of pharmacologic or genetic agents with a porous balloon catheter offers a potential therapeutic approach to reducing restenosis and atherosclerosis and minimizing undesirable systemic toxicity. However, the delivery efficiency and intramural retention of liquid agents is low. The local intramural delivery and prolonged retention of 5 microns microparticles (MP) has been described previously. The current study was designed to evaluate the distribution of locally delivered MPs and to determine the effects of MP size and infusion pressure on intramural delivery efficiency. A 1% suspension of fluorescent, latex MPs (1 or 4.5 microns in diameter) was infused at either 3 or 6 atm into atherosclerotic rabbit femoral arteries (n = 32) immediately after angioplasty. Four groups of arteries were evaluated: 1) 1 micron MPs infused at 3 atm; 2) 1 micron MPs at 6 atm; 3) 4.5 microns MPs at 3 atm; and 4) 4.5 microns MP at 6 atm. The location of MPs was evaluated by fluorescent and light microscopy and confocal laser scanning microscopy. The tissue was dissolved and the delivered MPs quantified. All groups manifested numerous MPs within the vasa vasorum and periadventitial microvasculature, with a substantially lesser number within the neointimal and medial layers. The intramural deposition of the MPs was associated with dissection within the intima or media caused by the antecedent angioplasty or local delivery, indicating that an intact vessel wall is an anatomic barrier to MP delivery. The median values of fractional intramural delivery, defined as the percentage of infused MPs retained within the arterial wall, were 0.059%, 0.071%, 0.047%, and 0.062% for the groups 1 through 4, respectively (p not significant [NS]). The values of intramural particle concentration, expressed as the total number of MPs per weight of arterial tissue, were 55, 65, 1.5, and 1.2 x 10(4) MPs/mg for groups 1 through 4, respectively (p < 0.001 for 1 micron vs 4.5 microns MPs). Although more 1 microM MPs were delivered than 4.5 microns MPs, the fractional intramural delivery was unaffected by particle size or infusion pressure. The local delivery of MPs at atherosclerotic sites after angioplasty is characterized by fractional intramural delivery values similar to values of nonparticulate agents, with few MPs deposited into intima or media in the absence of a dissection caused by the antecedent angioplasty or the delivery procedure itself. PMID- 8615308 TI - In vitro atherosclerotic plaque and calcium quantitation by intravascular ultrasound and electron-beam computed tomography. AB - The purpose of this investigation was to compare the accuracy of intravascular ultrasound (IVUS) and electron-beam computed tomography (EBCT) in quantitating human atherosclerotic plaque and calcium. In experiment 1, 12 human atherosclerotic arterial segments were obtained at autopsy and imaged by using IVUS and EBCT. The plaque from each arterial segment was dissected and a volume measurement of the dissected plaque was obtained by water displacement. The plaque from each arterial segment was ashed at 700 degrees F, and the weight of the remaining ashes was used as an estimate of the calcium mass. In experiment II, 11 calcified arterial segments were obtained at autopsy and imaged by using IVUS at one site along the artery. A corresponding histologic cross section stained with Masson's trichrome was prepared. In experiment I, the mean plaque volume measured by water displacement was 165.3 +/- 118.4 microliters. The mean plaque volume calculated by IVUS was 166.1 +/- 114.4 microliters and correlated closely with that by water displacement (r = 0.98, p < 0.0001). The mean calcium mass measured by ashing was 19.4 +/- 15.8 mg. The mean calculated calcium mass by EBCT was 19.9 mg and correlated closely with that by ashing (r=0.98, p<0.001). The mean calculated calcium volume by IVUS was 18.6 +/- 11.2 microliters and correlated linearly with the calcium mass by ashing (r = 0.87, p < 0.0003). In experiment II, the mean cross-sectional area of the calcified matrix was 1.71 +/- 0.66 mm2 by histologic examination compared with 1.44 +/- 0.66 mm2 by IVUS. There was a good correlation between the calcified cross-sectional area by histologic examination and IVUS (r = 0.76, p < 0.007); however, IVUS may underestimate the amount of calcium present depending on the intralesional calcium morphologic characteristics. In conclusion, IVUS accurately quantitates atherosclerotic plaque volume as well as the cross-sectional area and volume of intralesional calcium, especially if the calcium is localized at the base of the plaque. IVUS underestimates the amount of calcium present because of signal drop-off when the calcium is too thick for the ultrasound to completely penetrate. In comparison, EBCT accurately quantitates calcium mass regardless of the intralesional calcium morphologic characteristics; however, EBCT does not accurately quantitate plaque volume and will miss noncalcified atherosclerotic lesions. PMID- 8615309 TI - Left ventricular cavity size determined by preoperative dipyridamole thallium scintigraphy as a predictor of late cardiac events in vascular surgery patients. AB - We hypothesized that left ventricular (LV) cavity size measured on dipyridamole thallium scintigraphy identifies patients at risk for late nonfatal myocardial infarction and cardiovascular death. Accordingly, we retrospectively evaluated the predictive value of clinical and scintigraphic variables, including transendocardial LV cavity measurement performed on formatted images, in 335 vascular surgery patients. A nonhomogeneous perfusion pattern and enlarged LV cavity size were the most significant predictors of late events, and the interaction between these two variables was more predictive than was either variable alone. Life-table analysis demonstrated that patients with normal perfusion patterns had the lowest incidence of late events regardless of cavity size (p < 0.0005). Conversely, patients with a nonhomogeneous perfusion pattern and the largest LV cavity measurements were at the highest risk for late cardiac events (p < 0.0001). Therefore, this study demonstrated that a measurement of LV scintigraphic cavity size can provide important risk stratification for late cardiac events. PMID- 8615310 TI - Patterns of aspirin use in middle-aged adults: the Atherosclerosis Risk in Communities (ARIC) Study. AB - To determine correlates of and recent trends in aspirin use in middle-age men and women, we analyzed data from population-based samples selected in four U.S. communities. Aspirin use (during a 2-week period preceding the study examination) was more prevalent in whites than in blacks (30% vs 11%; p < 0.001) and in men than in women among whites (31% vs 28%; p < 0.002) but not blacks (10% in both sexes). In all four race and sex groups, there was a graded positive relation between estimated coronary heart disease (CHD) risk and age-adjusted prevalence of aspirin use. For example, 33% of CHD-free white men who reported diagnoses of hypercholesterolemia and hypertension and had ever smoked reported aspirin use as compared with 25% of their risk factor-free counterparts (p < 0.001). Among men with symptomatic CHD or at high risk for CHD, aspirin use increased by four percentage points between 1987 and 1989 in conjunction with the publication of results from the aspirin primary prevention trials. However, nearly 50% of participants reporting a history of myocardial infarction apparently did not take aspirin regularly. PMID- 8615311 TI - Dipyridamole technetium-99m sestamibi myocardial tomography in patients evaluated for elective vascular surgery: prognostic value for perioperative and late cardiac events. AB - Dipyridamole thallium-201 myocardial imaging can provide information regarding risk of perioperative cardiac events in patients being considered for vascular surgery. The value for this purpose of myocardial imaging with technetium-99m sestamibi (MIBI), a radiotracer with biokinetic and imaging properties different from thallium-201, has not been established. To this end the prognostic value of dipyridamole MIBI tomography for perioperative and late cardiac events was evaluated in 229 consecutive patients being considered for elective vascular surgery. Vascular surgery was done < or = 3 months after testing in 197 of these patients. Perioperative cardiac events (cardiac death, nonfatal myocardial infarction, unstable angina, or ischemic pulmonary edema) occurred in 9 (5%) patients. The rate of such events was 3% in patients with normal MIBI results, 5% in those with abnormal results, and 6% in patients with a reversible MIBI defect (both p = NS). When patients with abnormal MIBI results who had preoperative cardiac interventions (coronary revascularization or an increase in antiischemic medical therapy) were compared with with those who did not, no significant differences in the occurrence of perioperative cardiac events were found between these two groups either. A group of 172 medically treated patients who survived vascular surgery and did not have a nonfatal perioperative cardiac event was then monitored (mean 21 +/- 14 months) for the occurrence of a serious late cardiac event (nonfatal myocardial infarction or cardiac death). Event-free survival (Mantel-Cox) was significantly less in patients with abnormal studies compared with those with normal scan results. Late cardiac events occurred in 26 (15%) patients, with those having an abnormal MIBI result showing a significantly greater event rate than those with normal results (26% vs 4%, p < 0.0001). The rate of late cardiac events was 33% in patients with a reversible MIBI defect (p < 0.001) and 23% in those with a fixed defect (p < 0.03). Independent Cox multivariable predictors of increased risk of late cardiac events were a history of diabetes mellitus (relative risk [RR] 2.2, 95% confidence interval [CI] 1.0 to 4.9), an abnormal MIBI study (RR 3.7, 95% CI 1.2 to 11.4), and a reversible MIBI defect (RR 2.7, 95% CI 1.2 to 6.1). We conclude that, although its ability to assess increased perioperative cardiac risk remains uncertain, dipyridamole MIBI tomography does provide important prognostic information regarding the risk of serious cardiac events in patients having vascular surgery. The presence of an abnormal MIBI result, specifically one demonstrating a reversible perfusion defect, is associated with significantly increased risk. PMID- 8615312 TI - Idiopathic right ventricular outflow tract tachycardia: narrowing the anatomic location for successful ablation. AB - Pace mapping used to locate the site for ablation of idiopathic right ventricular outflow tract (RVOT) ventricular tachycardia remains difficult and time consuming. A method to facilitate pace mapping and the most common site of ablation of this tachycardia are reported. In 18 consecutive patients with RVOT ventricular tachycardia, electrocardiographic criteria based on the QRS orientation in lead 1 and the R wave progression in the precordial leads were used to find pace maps matching the arrhythmia. Identical pace maps were obtained on the septum of the RVOT in 16 patients and resulted in successful ablations. These sites were concentrated in the anterior superior aspect of the RVOT determined by fluoroscopic imaging. In the remaining two cases identical pace maps could not be found in this area. The results of this study narrow the anatomic location for radiofrequency ablation of idiopathic RVOT ventricular tachycardia. This is the first description of an electrocardiography-guided approach to finding an identical pace map in the RVOT. PMID- 8615313 TI - Localization of the origin of the atrioventricular junctional rhythm induced during selective ablation of slow-pathway conduction in patients with atrioventricular node reentrant tachycardia. AB - During radiofrequency catheter ablation of slow atrioventricular node pathway conduction in patients with atrioventricular node reentrant tachycardia, an atrioventricular junction rhythm is frequently observed. The origin and relation to ablation success of this junctional rhythm was examined in this study. By using standard intracardiac electrophysiology techniques, we studied the radiofrequency energy-induced atrioventricular junctional rhythm in 43 consecutive patients with atrioventricular node reentrant tachycardia undergoing selective ablation of slow-pathway conduction. The frequency of atrioventricular junctional activity was correlated with successful and unsuccessful attempts at ablation of slow-pathway conduction. Also, we compared the sequence of retrograde atrial activation of radiofrequency energy-induced atrioventricular junctional beats in a subgroup of 22 patients with the retrograde activation sequence observed during pacing from the right ventricular apex and the site of successful ablation of slow-pathway conduction. A total of 201 radiofrequency-energy applications was delivered in 43 patients with > or = 5 atrioventricular junctional beat(s) induced during 110 (55%) of 201 ablation attempts. Atrioventricular junctional activity was noted during 98% of successful ablations but only 43% of the unsuccessful attempts (sensitivity, 98%; specificity, 57%; negative predictive value, 99%). The mean time to appearance of atrioventricular junctional beats was 8.8 +/- 4.1 sec (mean +/- SD) after the onset of radiofrequency-energy application. In 22 (100%) of 22 patients in whom detailed atrial mapping was performed, the retrograde atrial activation sequence of the radiofrequency-induced atrioventricular junctional beats was earliest in the anterior atrial septum, identical to that seen during pacing from the right ventricular apex. Earliest retrograde atrial activation was at the posterior septum in all patients during pacing from the successful ablation site, a markedly different activation pattern compared with that seen during either radiofrequency ablation or ventricular pacing. Whereas the occurrence of atrioventricular junctional activity during radiofrequency ablation does not necessarily herald a successful ablation of slow atrioventricular node pathway conduction, its absence strongly suggests that the energy is being applied in an unsuccessful fashion. Furthermore, it appears that radiofrequency energy-induced atrioventricular junctional beats originate not from the endocardium in contact with the ablating catheter tip but instead appear to exit remotely from the anterior atrial septal region. This finding supports the existence of specialized tissues in the atrioventricular junction that preferentially transmit the effects of radiofrequency energy to an anterior exit site, possibly identical to the atrial exit site of the retrograde fast atrioventricular node conduction pathway. PMID- 8615314 TI - Effect of bundle branch block on local electrogram morphologic features: implications for arrhythmia diagnosis by stored electrogram analysis. AB - Analysis of stored local ventricular electrogram recordings is a useful diagnostic tool in the evaluation of patients with implantable cardioverter defibrillators. Visual analysis of local electrogram morphologic features has been demonstrated to be useful in distinguishing ventricular tachycardia from supraventricular rhythm. The effect of bundle branch block (BBB) aberration during supraventricular tachycardia on local electrogram morphologic features is not entirely clear. Erroneous diagnoses resulting from a change in electrogram morphologic features with BBB may occur. To determine whether the development of BBB can produce a change in local electrogram morphologic features and whether this change is dependent on the site of recording, we retrospectively reviewed local electrogram recordings from 23 patients who had intermittent BBB during electrophysiologic evaluation of documented or suspected supraventricular tachycardia. Local electrogram recordings from catheters placed in the right ventricular apex and coronary sinus during supraventricular tachycardia with BBB aberrancy were compared with recordings during narrow complex supraventricular tachycardia or normal sinus rhythm. Bipolar recordings were made with a 5 mm interelectrode distance with filter settings at 40 to 400 Hz. Three independent blinded observers defined the paired electrograms as the same or distinctly different. During right BBB a change in electrogram morphologic features was demonstrated in 11 (85%) of 13 recordings from the right ventricular apex and in only 1 (8%) of 12 recordings from the coronary sinus. In contrast, during left BBB a change in electrogram morphologic features was seen in 6 (100%) of 6 recordings from the coronary sinus and in only 1 (8%) of 13 recordings from the right ventricular apex. These results demonstrate that when the described recording techniques are used, a change in local ventricular electrogram morphologic features BBB is predominantly manifest in recording sites ipsilateral to the BBB, whereas recording sites contralateral to the BBB are relatively unaffected. This information may have implications regarding interpretation of stored electrograms when an attempt is made to establish a rhythm diagnosis leading to implantable cardioverter defibrillator therapy. PMID- 8615315 TI - Usefulness of tilt table test with normal saline infusion in management of neurocardiac syncope in children. AB - Head-up tilt test has been useful in evaluating children with neurocardiac syncope. In this study patients with positive baseline and isoproterenol tests had repeat tilt testing done after normal saline infusion. If the symptoms persisted, the test was then repeated with phenylephrine infusion. Of the 101 patients studied, 58 (57%) had a positive tilt sign. Normal saline infusion was given to 53 patients. Three patients were excluded because of structural heart disease. Fifty patients (aged 14 +/- 4 years) comprised the study group. Forty two (84%) of 50 patients had a negative repeat tilt sign after normal saline infusion, and these patients were treated with 0.5 to 1 gm of salt three times a day and/or fludrocortisone (20 patients). Of the 8 (16%) patients who did not respond to normal saline infusion, 5 had negative results when given phenylephrine and were treated with pseudoephedrine. Follow-up data on 42 patients (range 4 to 40 months, median 18 months) showed that all 35 patients who responded to normal saline were either asymptomatic or had improved. Two patients were successfully treated with pseudoephedrine; however, two patients in this group required pacemaker therapy. We conclude that (1) normal saline infusion mitigates the hemodynamic effects of neurocardiac syncope, (2) high-salt diet treatment in these patients was economical and effective, and (3) failure to respond to normal saline test may indicate a less favorable prognosis. PMID- 8615316 TI - Learning curve for radiofrequency catheter ablation in pediatrics at a single institution. AB - We examined the learning curve for radiofrequency ablation in pediatrics at a single institution. The first 146 cases were retrospectively reviewed, including patients < or = 21 years old with a single tachycardia diagnosis who were undergoing radiofrequency ablation for the first time. Data regarding demographics, electrophysiologic properties of the tachycardia, and procedural characteristics were tabulated. Data were then analyzed for evidence of association between these characteristics, success, and experience. Results revealed that success rates improved significantly with experience, reaching 85% success for all cases after < 100 cases attempted. Success for accessory pathway tachycardias alone reached > 93%. The number of cases of nonpathway tachycardias undertaken significantly increased as experience was gained. Fluoroscopy time improved to 34 +/- 27 minutes after < 100 cases. In conclusion, as experience was gained, (1) success rates showed a steep improvement; (2) the population undergoing radiofrequency ablation clearly shifted to include more difficult diagnoses; and (3) fluoroscopy time significantly decreased. PMID- 8615317 TI - Prospective randomized comparison of anodal monophasic shocks versus biphasic cathodal shocks on defibrillation energy requirements. AB - Biphasic shocks are believed to be superior to monophasic shocks. Monophasic anodal shocks, as opposed to cathodal shocks, are associated with improved defibrillation energy requirements (DERs). However, it is unclear how the DER of anodal monophasic shocks compare with conventional biphasic shocks. Therefore the purpose of this study was to prospectively compare the DER of an anodal monophasic shock with that of a cathodal biphasic shock. A transvenous defibrillation lead with distal and proximal shocking electrodes was used. The subjects of this study were 20 consecutive patients with a mean age of 64.2 +/- 10.5 years ( +/- SD) and a mean left ventricular ejection fraction of 0.36 +/- 0.18. Six had had cardiac arrest. The DER, defined as the lowest energy that converted ventricular fibrillation to sinus rhythm, was determined twice with a step-down protocol (25 J, 20 J, 15 J, 10 J, 5 J, 3 J, 1 J). If the DER was > or = 25 J, then a subcutaneous patch was deemed necessary for system implantation. In random order the DER was determined with a monophasic anodal shock (distal electrode positive) and then with a cathodal (first phase, distal electrode negative) biphasic shock. The mean DER with anodal monophasic shocks was 15.1 +/- 8.5 J compared with 13.6 +/- 8.1 J with cathodal biphasic shocks (p = 0.4). A DER > or = 25 J was present in three patients with the monophasic waveform and in three patients with the biphasic waveform (p = NS). In conclusion, the DER and frequency of subcutaneous patch use with an anodal monophasic waveform is comparable to that obtained with cathodal biphasic waveform. PMID- 8615319 TI - Mitral valve repair is superior to valve replacement for the early preservation of cardiac function: relation of ventricular geometry to function. AB - The immediate effect or mitral valve repair (MVP) or replacement (MVR) on cardiac function was compared in patients with mitral regurgitation in relation to the changes in left ventricular (LV) function and geometry by using intraoperative transesophageal echocardiography in 29 patients with MVP and 21 patients with MVR, before and immediately after cardiopulmonary bypass. The LV volumes, ejection fraction, and long-axis and short-axis lengths and eccentricity index (ratio of long axis to short axis) at end-systole and end-diastole were measured. After both MVP and MVR, there were significant decreases in LV end-diastolic volume (p < 0.0001). However, the ejection fraction did not change after MVP, whereas it decreased after MVR (p < 0.0001). After MVP, there was an increase in eccentricity index at end-systole (p < 0.0001). After MVR, there was no decrease in end-systolic volume, and the eccentricity index was lower than that after MVP (p < 0.0001). The change in LV ejection fraction correlated with the changes in eccentricity index at end-systole (r = 0.55; p < 0.0001) and end-diastole (r = 0.42; p < 0.0003). Immediate intraoperative LV function is preserved after MVP but is depressed after MVR for mitral regurgitation. The changes in ejection fraction correlate with changes in ventricular geometry. PMID- 8615318 TI - Principal results of the Hypertension and Lipid Trial (HALT): a multicenter study of doxazosin in patients with hypertension. AB - The Hypertension and Lipid Trial (HALT) was undertaken to assess the efficacy and safety of doxazosin, a selective alpha 1- adrenergic blocker, in patients with hypertension in a clinical practice setting. The effects of doxazosin on office blood pressure, changes in lipid profiles, and theoretic coronary disease risk were studied. In an open, noncomparative, multicenter trial, 851 patients were studied for a maximum of 16 weeks. Doxazosin significantly reduced mean sitting systolic blood pressure (SBP) and diastolic blood pressure (DBP) by 15.2/12.5 mm Hg and standing SBP and DBP by 16.1/12.7 mm Hg in the total study population (n = 807; p = 0.0001), with no significant effect on heart rate. Mean total cholesterol levels were significantly reduced by 2.7%, low-density lipoprotein cholesterol levels by 2.4%, and mean triglyceride levels by 3.4% (all p values < 0.05). High-density lipoprotein (HDL) cholesterol levels were essentially unchanged. The mean ratio of total to HDL cholesterol was significantly reduced (p < 0.05). Mean predicted 5-year coronary disease risk was significantly reduced with doxazosin therapy by 14.7% in previously untreated patients (p < 0.0001) and by 1.7% in patients who were previously receiving antihypertensive therapy (p < 0.05). The drug was well tolerated. This study demonstrates that antihypertensive therapy with doxazosin can favorably affect coronary disease risk factors and reduce predicted coronary disease risk. PMID- 8615320 TI - Assessment of mitral annular dynamics during diastole by Doppler tissue imaging: comparison with mitral Doppler inflow in subjects without heart disease and in patients with left ventricular hypertrophy. AB - The purpose of this study was to determine the normal pattern and magnitude of mitral annular velocities in diastole by Doppler tissue imaging (DTI) and to assess whether this is altered in patients with left ventricular hypertrophy. Mitral annulus velocities were measured by DTI. Peak and time-velocity integral were measured from the DTI tracings and the timing of the velocities in relation to electrocardiogram. DTI was compared with M-mode echo of the annulus and mitral inflow Doppler velocities. Integrated annular velocities by DTI correlated with the annular displacement. Early diastolic velocities decreased with age and in patients with left ventricular hypertrophy. In the hypertrophy group, early diastolic velocities were significantly lower than normal even after correcting for age. Patients with left ventricular hypertrophy also showed a delay in peak early diastolic mitral annular velocity (5.5 +/- 21 msec after the E wave). In conclusion, mitral annular velocity in diastole is readily recorded by DTI. The magnitude and the pattern of these velocities are significantly altered by age and by left ventricular hypertrophy. This method provides a new insight into diastolic filling events and may prove useful in detecting abnormal diastolic function. PMID- 8615322 TI - Increased plasma adrenomedullin levels in chronic congestive heart failure. AB - Adrenomedullin is a potent vasodilator peptide and occurs in circulating blood of human beings and experimental animals. Because it is produced in intact aorta of rats and in cultured vascular endothelial cells, adrenomedullin seems to participate in regulation of local vascular tone. To determine the pathophysiological roles of adrenomedullin, we investigated its plasma concentrations in 49 patients with heart failure. Plasma adrenomedullin levels increased significantly with advancing severity of the disease (New York Heart Association functional class I, 4.1 +/- 1.0; II, 5.6 +/- 1.6; III, 6.4 +/- 0.8; IV, 13.2 +/- 6.8 (fmol/l). Plasma adrenomedullin was correlated with pulmonary artery pressure (r = 0.44, p = 0.0114) and pulmonary capillary wedge pressure (r = 0.53, p = 0.0002). These findings indicate that adrenomedullin may play some important role in the pathophysiologic makeup of heart failure by its vasodilating effects against the concomitant exaggeration of humor pressor agents such as catecholamine and the renin-angiotensin system. Hemodynamic changes in pulmonary circulation may have some influence on the increased synthesis and secretion of plasma adrenomedullin in chronic congestive heart failure. PMID- 8615321 TI - Enhanced sensitivity of the failing human myocardium to cardiac glycosides and Na(+)-channel activators. AB - Cardiac glycosides and Na+ -channel activators increase intracellular Na+ and thereby enhance the transport rate of the sarcolemmal Na+/Ca2+ exchanger. We tested the hypothesis of whether increased expression of the Na+/Ca2+ exchanger in failing human myocardium is accompanied by enhanced sensitivity of the failing human myocardium toward cardiac glycosides and Na+ -channel activators. We studied the positive inotropic effects of the new Na+ -channel activator BDF and the cardiac glycoside ouabain in human failing (New York Heart Association [NYHA] functional class IV, heart transplants for dilated cardiomyopathy, n = 11) and nonfailing (donor hearts, n = 5) myocardium on electrically driven left ventricular papillary muscle strips (1 Hz, 37 degrees C). The effectiveness of ouabain and BDF to increase force of contraction was similar in human nonfailing and failing myocardium. BDF was more potent to increase force of contraction in failing than in nonfailing tissue (p < 0.05). The time until maximal inotropic effect developed after ouabain was significantly shorter in NYHA IV (mean 150 +/- 16 min) than in nonfailing myocardium (mean 240 +/- 20 min). These results suggest that human failing myocardium exerts and enhanced sensitivity to cardiac glycosides and Na+ -channel activators, possibly because of enhanced expression of the Na+/Ca2+ exchanger or because of an altered intracellular Na+ homeostasis. PMID- 8615324 TI - Evaluation of a chemical spot-test kit for the detection of airborne particulate lead in the workplace. AB - A commercial rhodizonate-based test kit was evaluated for its potential use in the detection of lead in airborne particulate samples at work sites. Over 350 air samples were collected at abrasive blasting lead paint abatement sites using cellulose ester membrane filters and personal sampling pumps. The filter samples were tested with the chemical spot test and then analyzed by graphite furnace atomic absorption spectrophotometry. No positive readings were recorded for lead masses below 1.3 micrograms Pb/filter, and no negative readings were observed for lead amounts above 8.1 micrograms Pb/filter. Experimental data were statistically molded in an effort to estimate the performance parameters of the spot test kit. The identification limit of the kit was found to be approximately 3.6 microgram/filter sample. For lead mass values above approximately 10 micrograms Pb/filter, 95% confidence of a positive reading was found, while 95% confidence of a negative reading was found for lead masses below approximately 0.6 micrograms Pb/filter. Based on the results of this study the rhodizonate-based test kit for lead demonstrates potential for use in field screening for lead in personal breathing zone and area air samples. PMID- 8615323 TI - Angiotensin-converting enzyme inhibition, autonomic activity, and hemodynamics in patients with heart failure who perform isometric exercise. AB - Effects of angiotensin-converting enzyme inhibition (ACEI) on autonomic responses and hemodynamics in patients with congestive heart failure (CHF) subjected to isometric exercise have not been studied. We tested whether acute ACEI might influence the effects of isometric exercise in patients with CHF. In the first part of the study we showed that isometric exercise increased blood pressure in the control group and in the CHF group, whereas cardiac output increased only in the control group. Stroke volume remained unchanged in the control group, whereas it decreased significantly in CHF group. We next analyzed the effect of acute ACEI (5 mg ramipril) on the decrease in cardiac output during isometric stress in patients with CHF. During isometric exercise mean blood pressure and heart rate increased similarly in both groups. However, cardiac output decreased during placebo by -0.48 +/- 0.12 L/min (p < 0.01) but not during ACEI. Spectral analysis of blood pressure showed an increase (p < 0.01) in the high-frequency parasympathetic component from 7.3% +/- 3.6% to 18.1% +/- 9.5% after ACEI. norepinephrine plasma levels increased after isometric stress in the placebo group, whereas other hormones did not change. ACEI prevented the norepinephrine increase after isometric stress. Thus the decrease in cardiac output during isometric exercise in patients with CHF was prevented by acute ACEI. The effect of ACE inhibition may be related to reduced sympathetic activity. PMID- 8615325 TI - Field performance measurements of half-facepiece respirators--foundry operations. AB - A series of field studies was conducted to measure workplace protection factors (WPF) provided by elastomeric and disposable half-facepiece respirators against different particulate contaminants. The research protocol developed for the study has been described in a previous article. This article presents results from dust and fume exposures at three foundries. The major components of the airborne exposures in these foundries were zinc, lead, and silicon. The major components of the in-facepiece samples were zinc, chlorine, and lead. Significant differences were observed in ambient zinc and lead concentration levels among foundries; however, no significant difference was observed in the in-facepiece concentrations of these elements among foundries. Respirator performance varied within each foundry, but there was no difference in performance when pooling all foundry data. The 5th, 10th and 50th percentile estimates for the pooled foundry and respirator WPF data were about 9, 16, and 114 respectively. The infacepiece concentration data clearly indicate that dust-fume-mist (DFM) class half facepiece respirators, when conscientiously used, worn, and maintained, in conjunction with other existing controls in these foundries, provided effective worker protection. PMID- 8615326 TI - Technology advancements in hearing protection circa 1995: active noise reduction, frequency/amplitude-sensitivity, and uniform attenuation. AB - Conventional hearing protection devices represent a mature technology that has been widely used since the late 1950s. When worn consistently and correctly such devices can provide suitable hearing protection in many, if not most noise hazardous or aurally annoying situations. However, such devices have often been implicated in compromised auditory perception, degraded signal detection, and reduced speech communication abilities. In some instances this can create hazards for the wearer, or at the very least, resistance to use by those in need of hearing protection. Recent technological developments have been used to augment hearing protectors in an attempt to alleviate these problems for the user while providing adequate attenuation. Operational characteristics, design alternatives, performance data, and applications for active noise reduction, active sound transmission, frequency selectively, adjustable attenuation, amplitude sensitivity, and uniform attenuation features in hearing protectors are discussed, and recommendations are provided. PMID- 8615327 TI - Exposure to airborne metals in the manufacture and maintenance of hard metal and stellite blades. AB - This study evaluated the exposure of Finnish workers to airborne metals. It was conducted in 16 workplaces; 8 tool manufacturing companies (3 of which specialize in the maintenance of hard metal blades) and 8 blade repair shops in the mechanical wood-processing industry. The main emphasis of the study was on exposure in servicing (resharpening), especially involving wet-tip grinding processes. The results show that workers grinding hard metal blades may be exposed to high levels of airborne cobalt even when airborne total dust concentrations are low. The respirable proportion of the total dust was found to be high, and the total dust concentrations correlated well with the cobalt concentrations. Most of the airborne cobalt was water soluble (ionized), but there was also solid particles containing cobalt and other materials in the air of the workplaces. The mean concentration of cobalt in the grinding coolants was high, though it varied considerably. Concentrations of all the other metals measured in the air (tungsten, chromium, cadmium, silver, and nickel) and in the coolants (tungsten and chromium) were low. To reduce workers' exposure to cobalt, it is recommended that grinding machines should be enclosed and equipped with local exhausts. Use of coolants that dissolve less cobalt is also recommended, and the coolants should be changed more often. Brazing should be done in a well ventilated place, and only cadmium-free materials should be used. PMID- 8615328 TI - Apparent versus real exposures of nuclear medicine technologists during aerosol lung ventilation scanning. AB - The exposure of nuclear medicine technologists to airborne radiocontaminants is generally far below the compliance limits. Most of the contamination is external rather than internal deposition. Higher contamination levels approaching the allowable limits are rare. Personal respiratory protection is unwarranted and may have undesirable impressions on patients undergoing lung scanning. Exposures are well controlled by suitable work practices that are in keeping with as low as reasonably achievable (ALARA) principles. PMID- 8615329 TI - American Health Foundation Roundtable on Healthy Weight. Proceedings of an expert panel discussion. New York, New York, September 20, 1994. PMID- 8615330 TI - Fractionating healthy weight. AB - Any discussion of healthy weight necessarily starts with body composition and its relation to long-term mortality and morbidity and then goes on to consider the limited range of ages for which we have epidemiologic data, the time span involved, the criteria used to define "healthy," and the possibility that a weight that is advantageous with respect to one outcome criterion may be disadvantageous for another. One may therefore ask, weight of what? healthy weight for whom? by what criteria? and question whether a simple weight-for height ratio is sufficiently effective for the task. PMID- 8615331 TI - Policy and healthy weight. AB - "Policy" is defined as a definite course of action adopted for reasons of expedience, prudence, wisdom, or common sense. What can be said about policy and healthy weights? We must draw conclusions from the evidence offered for the comorbidities associated with overweight. Then we must ask, what is a healthy weight? How much gain or loss will suffice to arrive at a healthy weight? What other issues such as economics and mental health costs affect policy? To facilitate the process, I will review the policy issues for which there is already a consensus and then outline the evidence available from which this group might gain perspective for formulating practical public health recommendations. PMID- 8615333 TI - Body weight and blood pressure regulation. AB - Excess body weight correlates closely with increased blood pressure. Virtually every prospective study of factors that influence blood pressure regulation has identified weight, or body mass index, as the strongest predictor of human blood pressure. In normotensive persons, longitudinal and intervention studies have documented that weight gain, over as short a period as 1-2 mo., is associated with predictable rises in arterial pressure. Conversely, in borderline hypertensive persons, similar types of studies have reported that weight reductions of as little as 4-5 kg result in normalization of blood pressure. During periods of weight loss, decreases in blood pressure are evident within the first 2-3 wk; the greatest proportionate blood pressure change occurs during the earliest phases of weight reduction. The decline in blood pressure with weight loss has been shown to be unrelated to variations in nutrient intake. However, the association between blood pressure decreases and increased amounts of physical activity, which generally accompany weight loss efforts, is less clear. For many overweight individuals, reductions in blood pressure that occur with weight loss may be due specifically to the initiation of a regular physical exercise regimen. Weight loss in hypertensive persons has been shown to dramatically reduce, and in many cases eliminate, antihypertensive medication requirements. It is estimated that in up to 50% of the adults in the United States whose hypertension is being pharmacologically managed, the need for drug therapy could be alleviated with only modest reductions in body weight. PMID- 8615332 TI - Effect of weight on cardiovascular disease. AB - Involuntary weight gains worsen all elements of the cardiovascular risk profile, including dyslipidemia, hypertension, insulin-resistant glucose intolerance, left ventricular hypertrophy, hyperuricemia, and elevated fibrinogen. On the basis of data from the Framingham Heart Study and from other studies, it can be concluded that the degree of overweight is related to the rate of development of cardiovascular disease. After 26 y of follow-up in the Framingham study, each SD increment in relative weight was associated with 15% and 22% increases in cardiovascular events in men and women, respectively. Avoidance of weight gain after the age of 25 y is advisable to reduce cardiovascular mortality. There is a great potential benefit to weight loss, suggesting that weight control as a means for preventing and lessening cardiovascular disease become a national health priority. The optimal weight for avoidance of cardiovascular disease and prolonging life corresponds to a body mass index of 22.6 for men and 21.1 for women. PMID- 8615334 TI - Weight and non-insulin-dependent diabetes mellitus. AB - I review the effect of weight on the prevalence of non-insulin-dependent diabetes mellitus (NIDDM) and of its associated conditions. Weight loss decreases morbidity in diabetic patients. The effect of weight loss on mortality also is favorable, although the number of reports in which this has been studied is small. The recommendation is made that the persons with NIDDM who have a body mass index (BMI, in kg/m2) > 25 should try to lose weight to at least that level. Those with BMIs between 20 and 25 should make every effort to maintain their present weight throughout life. PMID- 8615335 TI - Weight and osteoarthritis. AB - Osteoarthritis is the most common form of arthritis. It increases in prevalence with age. About 5% of the US population is affected with hip or knee osteoarthritis; 9.5% of adults aged > 62 y have knee osteoarthritis. Because of its frequency and associated pain and disability, osteoarthritis accounts for much of the disability in lower extremities in the elderly. More than 70% of total hip and knee replacements are for osteoarthritis. Because osteoarthritis is so common, the modification of factors that increase osteoarthritis risk could prevent substantial pain and disability in the elderly and the use of costly health care services. Overweight persons are at high risk of osteoarthritis in the knee and probably also in the hips and hands. The mechanism by which overweight causes osteoarthritis is poorly understood; a contribution from both local increased force across the joint and systemic factors is likely. Better evidence is needed on the effects of weight loss, but preliminary studies suggest that weight loss can both prevent the onset of symptomatic disease and alleviate symptoms when present. PMID- 8615336 TI - Putting body weight and osteoporosis into perspective. AB - Osteoporosis is characterized by a reduction in bone mineral density (BMD). Women more than men are at risk for osteoporosis-related fractures, especially in the wrists, lumbar spine, and hips. Numerous diet and lifestyle factors, including body weight, influence BMD, and in turn, fracture risk. BMD in the total body, hip, lumbar spine, and radius is weakly to moderately correlated to body weight, fat mass, and lean body mass in adolescent, perimenopausal, and elderly women, possibly as the result of stress on the skeleton from the mechanical loading of body weight alone. In addition, greater lean body mass may be a cause. Other explanations include increased hormonal circulation in obese women and greater conversion of adrenal androgens to estrogens linked to greater mass of adipose tissue. Currently no value is agreed on for weight-to-height versus osteoporosis and related fracture risk, but some extra fat mass yielding a body mass index > 26-28 does confer limited protection, whereas a slender figure yielding a body mass index < 22-24 increases risk. PMID- 8615337 TI - Body weight: estimation of risk for breast and endometrial cancers. AB - Consistent, positive, and independent associations between body weight or body mass index (BMI), weight gain, and various measures of central adiposity and the incidence of endometrial cancer exist. Increases in relative risks of 2-3.5 are reported for women with BMIs (in kg/m2) > or = 28-30, for women in the fourth compared with the first quartile of measures of central adiposity, and for women with weight gains from young adulthood to middle age of > or = 27 kg. Furthermore, endometrial cancer mortality is increased in heavier and taller women. Associations between breast cancer incidence and these measures of adiposity vary by age and menopausal status at the time of diagnosis. Heavier women appear to be at decreased risk for developing premenopausal breast cancer; relative risks of approximately 0.6 were reported for women with BMIs > or = 26 27. Conversely, heavier women are at increased risk of developing and dying from postmenopausal breast cancer. Although contradictory findings have been observed in cohort studies, modest increases in relative risks on the order of 1.2-1.5 were reported in older postmenopausal women with BMIs of > or = 28-30. Furthermore, adult weight gain and increased central adiposity have been consistently and independently associated with an increased risk for postmenopausal breast cancer. No significant associations have been observed between weight loss and postmenopausal breast cancer incidence. These findings indicate that avoidance of weight gain and accumulation of central body fat during adult life may reduce risk of both endometrial and postmenopausal breast cancer. PMID- 8615338 TI - Body weight and colon cancer. AB - In 1994, there were approximately 160,000 new cases of colon cancer in the United States with 58,000 fatalities, making this form of cancer the second most common cause of cancer deaths. Up to 50% of colon cancers may have a strong inherited factor, but in the remaining cases, diet and lifestyle factors are thought to play essential roles in the carcinogenic process. Various epidemiologic studies have examined the relation between obesity and colon cancer. The largest prospective study of 750,000 men showed that mortality from colorectal cancer was significantly elevated in men who were > or = 40% overweight. No such increase was found in women. Subsequent studies reported conflicting results. Overweight is likely a surrogate. Other risk factors include a high-fat, energy-dense diet; inadequate consumption of fruit and vegetables; and lack of physical activity, which have been associated with a high incidence of colon cancer. PMID- 8615339 TI - Morbidity and mortality associated with elevated body weight in children and adolescents. AB - The immediate and long-term risks associated with overweight in childhood and adolescence are best considered separately. Short-term mortality is rarely associated with overweight of the young. The greatest health burden of overweight in children and adolescents arises from long-term consequences. Long-term follow up studies of children and adolescents indicate that the risk of adult overweight is about twofold greater for individuals who were overweight as children compared with individuals who were not overweight. Persistence is greatest for extreme overweight and when overweight is carried through late adolescence. The few studies of long-term health consequences in adolescent males find that even moderate overweight is associated with excess mortality in adulthood. Females have been studied infrequently; it appears that overweight confers long-term health risks, but these are less severe. However, because of concern about weight preoccupation in this age group and lack of data of sufficient precision to support specific recommendations, weight guidelines for children and adolescents are not now warranted. PMID- 8615340 TI - Healthy body weights: an alternative perspective. AB - The primary purpose of this roundtable discussion of the American Health Foundation is to establish healthy weight standards for adults. In most large, long-term, well-designed studies, the lowest morbidity and mortality rates occurred in adults at weights that yielded BMIs (in kg/m2) between 19 and 25. Best body fat percentages averaged between 12% and 20% for men and 20% and 30% for women. However, statistical values are not applicable to everyone and their strict application may be counterproductive. We believe that most weight associated health problems result from a cascade of events associated with abnormal blood concentrations of insulin, glucose, or lipids that occur when fat cells become full and insulin-insensitive, and lose their protective functions. Indexes associated with high risk in obese persons often return to normal with appropriate physical activities, dietary habits, and a small weight loss even when body weight and percentage body fat remain above recommended amounts. We believe that statistically derived standards for body weight and percentage body fat are appropriate for use as a screening test but should be downplayed as strict guidelines for all. PMID- 8615341 TI - Does weight cycling present a health risk? AB - I review research on the hypothesis that weight fluctuations caused by unsuccessful dieting are hazardous to one's health. Recent epidemiologic findings show that weight variability over time is associated with increased total and cardiovascular mortality (relative risk: 1.5-2.0), independent of a variety of possible confounding variables. Although these findings are consistent across studies, methodologic limitations of a lack of a uniform or standard definition of weight cycling, and the linking of weight variability to unsuccessful dieting raise serous questions about whether these findings should be interpreted as supporting the weight-cycling hypothesis. The absence of data identifying a plausible biological mediator for weight fluctuation per se as a health hazard is also a problem. It is concluded that, although epidemiologic data on weight variability and health are intriguing, they are at present insufficient to alter public health recommendations regarding weight control. PMID- 8615342 TI - Increasing physical activity: alterations in body mass and composition. AB - I provide an overview of the role of physical activity in the prevention of overweight and obesity and in the treatment of overweight and obese individuals. A secondary focus of this paper is on the potential mechanisms responsible for changes in body composition consequent to physical activity. The use of the term "physical activity" is preferred to the term "exercise" to better reflect a broader scope of movement, not limited to formal exercise regimes. A brief review of prospective studies that investigated alterations in body composition consequent to physical activity alone is presented, along with a discussion of the influence of genetics, macronutrients in the diet, and characteristics of exercise programs on the magnitude of change observed in these studies. This is followed by a review of the specific role of chronic physical activity on energy intake, resting metabolic rate, the thermic effect of feeding, and the thermic effect of activity. Finally, I discuss the role physical activity plays in preventing overweight and obesity and the most appropriate use of exercise in the management of overweight. PMID- 8615344 TI - Social and economic effects of body weight in the United States. AB - Given that overweight is clearly associated with increased risk of many major chronic diseases, the United States could have saved approximately $45.8 billion or 6.8% of health care expenditures in 1990 alone if obesity were prevented. The question then arises, economically and socially, what is a healthy body weight? Using a prevalence-based approach to cost of illness, we estimated the economic costs (1993 dollars) associated with illness at different strata of body mass indexes (BMIs, in kg/m2) and varying increments of weight gain to address the questions: At what body weight do we initiate preventive services? What are the direct costs associated with weight gain? Second, using the 1988 National Health Interview Survey (NHIS), we evaluated the marginal increase in certain social indexes reflective of functional impairment and morbidity (ie, restricted activity days, bed days, and work-loss days) as well as physician visits associated with different strata of BMI. With respect to economic and social indexes, a healthy body weight appears to be a BMI < 25, and weight gain should be kept to < 5 kg throughout a lifetime. PMID- 8615343 TI - Psychosocial consequences of weight reduction: how much weight loss is enough? AB - We reviewed the psychosocial consequences of weight reduction and concluded that weight loss is usually associated with improvements in mood in significantly obese individuals (> or = 20% overweight) who are treated by diet and lifestyle modification. Less is known about the psychologic effects of weight loss in mildly overweight individuals who reduce their weight on their own but the limited data suggest similarly positive effects. We recommend that significantly obese individuals seek a 10% reduction in body weight, a loss that is likely to be associated with improvements in psychologic as well as physical health. A loss of this magnitude is typically produced by 16-20 wk of treatment by diet and behavior modification. Persons who ae mildly overweight (particularly those with health complications) are encouraged to reduce their weight by increasing their physical activity. Exercise is associated with modest but long-term weight losses and with improvements in mood and physical health. The recommendation that overweight Americans seek a healthier weight should be combined with efforts to promote healthier attitudes toward weight and shape in normal-weight women and girls. PMID- 8615345 TI - Overweight and health: communications challenges and opportunities. AB - Overweight is associated with multiple adverse health consequences, many of which have been addressed by the experts participating in this Roundtable on Healthy Weight organized by the American Health Foundation. While research and vigorous debate continue on the etiology, treatment, and prevention of obesity, health care professionals face a crucial challenge: effectively communicating current knowledge of the links between overweight and adverse health outcomes. Communications challenges that must be overcome include the clutter of diverse messages, distrust of experts, the anti-diet movement, public confusion, and misunderstandings about scientific reports. An effective communications strategy needs to focus on simple, friendly messages that are consistent with scientific evidence yet understandable to individuals in ways that promote acknowledgment of personal responsibility and promote action. PMID- 8615346 TI - Summary and recommendations from the American Health Foundation's Expert Panel on Healthy Weight. PMID- 8615347 TI - Position paper on trans fatty acids. ASCN/AIN Task Force on Trans Fatty Acids. American Society for Clinical Nutrition and American Institute of Nutrition. AB - This report addresses the current controversy about possible health hazards of dietary trans fatty acid isomers, which are created during hydrogenation of unsaturated fats to change their textural properties and melting points. Estimates of intakes are approximations based on limited data and problematic analytic techniques. Major contributors in the diet are fried and baked foods and margarine, in which partially hydrogenated vegetable oils may replace fat sources richer in saturated fatty acids and cholesterol. Consumption of trans fatty acids in the United States has been relatively constant, and new food technologies are yielding decreases in the trans fatty acid content of commercially prepared foods. When intake of trans fatty acids (as hydrogenated fat) is compared with that of saturated fat, total and low-density-lipoprotein (LDL)-cholesterol concentrations in blood are lower, but both trans fats and saturated fats increase total and LDL concentrations when compared with cis fatty acids or native unhydrogenated fat. Epidemiologic data are conflicting with respect to cardiovascular disease outcomes. We cannot conclude that the intake of trans fatty acids is a risk factor for coronary heart disease nor can we expect that substituting trans- for cis-containing fats will reduce the risk of coronary heart disease. Few rigorous studies have dealt with biomedical effects of trans fatty acids and possible mechanisms relevant to human health and diseases. The nutrition labeling issue is unresolved. The options, recommendations, and research suggestions in this report should outline for nutrition scientists the database needed before any new dietary recommendations or changes in nutrition policy concerning trans fatty acids can be made. The debate about trans fatty acids should not detract from dietary recommendations to limit the intake of saturated fat and total fat. PMID- 8615348 TI - Screening for nutrition interventions: the risk or the differential-benefit approach? AB - The risk approach has been promoted to improve screening for nutrition interventions on the premise that indicators of risk also predict greater response to interventions. This study tested whether the determinants of the risk of poor growth (eg, low length-for-age) at 36 mo of age were the same as the determinants of differential benefit from food supplementation. The sample included 460 Guatemalan children who were exposed to either a high-energy, high protein drink (atole) or a low-energy, no-protein drink (fresco) during their first 36 mo of life [INCAP (Institute of Nutrition of Central America and Panama) supplementation trial]. Low maternal stature, poor socioeconomic status, inadequate home diet, high diarrhea rates, and low anthropometry scores at 3 or 6 mo were all determinants of the risk of poor growth. Only indicators of child's thinness at 3 or 6 mo of age (low weight-for-age, weight-for-length, or midupper arm circumference) were determinants of differential benefit from supplementation. Thus, the development of screening indicators should be based on analyses of the predictors of differential benefit, not on conventional risk factor analysis. PMID- 8615349 TI - Analysis of body-composition techniques and models for detecting change in soft tissue with strength training. AB - The purpose of this study was to compare the ability of various body-composition assessment techniques to detect changes in soft tissue in older, weight-stable women (50-70 y of age) completing a 1-y randomized, controlled trial of progressive resistance training. The intervention group (n = 20) performed high intensity strength-training 2 d/wk with five different exercises; the control group (n = 19) was untreated. Hydrostatic weighing, 24-h urinary creatinine, computed tomography of thigh sections, total body potassium, and tritium dilution techniques were used to measure increases in total fat-free mass (FFM) and the muscle and water components of FFM. A decrease in fat mass (by hydrostatic weighing) was seen in the strength-trained women compared with the control subjects (P - 0.01-0.0001). Anthropometry, bioelectric impedance, dual-energy X ray absorptiometry, and total body nitrogen and carbon did not measure any significant change in soft tissue. The choice of a body-composition technique is important when designing a study expected to affect soft tissue, because not all techniques available are precise enough to detect small changes. PMID- 8615350 TI - Effect of long-chain n-3 fatty acid supplementation on visual acuity and growth of preterm infants with and without bronchopulmonary dysplasia. AB - Healthy preterm infants fed formula with long-chain n-3 fatty acids (n-3 LCFAs) from marine oil have better early visual acuity but lower plasma phosphatidylcholine (PC) arachidonic acid (AA) and growth than infants fed formula containing linolenic acid (LLA) as the sole n-3 fatty acid. This randomized, double-blind trial was designed to study the effects of a different source of n-3 LCFAs and a shorter feeding interval on visual acuity (by Teller Acuity Card) and growth of preterm infants (n = 59; 747-1275 g birth wt), some of whom required long periods of supplemental oxygen and developed bronchopulmonary dysplasia (BPD). Infants were studied at 0, 2, 4, 6, 9, and 12 mo past term. Plasma PC AA, and normalized weight, length, and head circumference were not influenced by BPD or n-3 LCFAs except that n-3 LCFA-supplemented infants weighed less at 6 (P<0.05) and 9 (P<0.01) mo and had smaller head circumferences at 9 mo (P<0.05). Compared with control infants, however, those fed n-3 LCFAs had lower weight-for-length at 2, 6, 9, and 12 mo (P<0.0003, P<0.0114, P<0.0008, and P<0.006, respectively). n-3 LCFAs improved early (2-mo) but not later acuity among infants without BPD (P<0.02). Regardless of diet, infants with BPD had poorer grating acuity at 2 (P<0.0002) and 4 (P<0.04) mo but not thereafter. PMID- 8615351 TI - Linoleic acid intake and susceptibility of very-low-density and low density lipoproteins to oxidation in men. AB - Lipoprotein peroxidation is thought to play an important role in atherogenesis. In the Kuopio Atherosclerosis Prevention Study (KAPS) the intake of fat and fatty acids, the oxidation susceptibility of the plasma very-low-density + low-density lipoprotein (VLDL+LDL) fraction (by induction with copper or hemin and hydrogen peroxide), and concentrations of plasma antioxidants, serum lipids, and lipoproteins were measured in 393 men. In the multivariate-regression model dietary linoleic acid was the most important determinant of the maximal oxidation velocity for the hemin assay (standardized regression coefficient = 0.294, P<0.0001). In the copper assay the association of dietary linoleic acid and maximal oxidation velocity was second in order of strength (standardized regression coefficient = 0.324, P< 0.0001). We conclude that high linoleic acid intake is associated with increased oxidation susceptibility of atherogenic lipoproteins in men. PMID- 8615353 TI - Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. AB - The influence of dietary fructooligosaccharide (neosugar) on the fecal flora and activities of reductive enzymes was studied in 12 healthy, adult human subjects fed a controlled diet for 42 d and given 4 g neosugar/d between days 7 and 32. Fecal samples were collected before, during, and after supplementation with neosugar to enumerate total anaerobes, aerobes, bifidobacteria, and enterobacteria, and to assay for beta-glucuronidase, nitroreductase, and glycocholic acid hydroxylase. Although the controlled diet caused an increase in total anaerobes and bifidobacteria, the highest densities occurred during supplementation with neosugar. Total aerobes and enterobacteria were less affected by diet and neosugar. Neosugar caused beta-glucuronidase and glycocholic acid hydroxylase activities to decrease 75% and 90%, respectively; both increased after supplementation with neosugar was stopped. Nitroreductase activity declined 80% after the control diet was started, but was not affected by neosugar. These findings indicate that 4 g neosugar/d alters the fecal flora in a manner perceived as beneficial by decreasing activities of some reductive enzymes. PMID- 8615352 TI - Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation. AB - Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose 4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium. PMID- 8615355 TI - Long-term oral vitamin E supplementation in cystic fibrosis patients: RRR-alpha tocopherol compared with all-rac-alpha-tocopheryl acetate preparations. AB - To investigate the efficacy of three different vitamin E preparations for optimizing vitamin E status in cystic fibrosis (CF patients long-term, 29 patients (aged 0.7-29.8 y) were randomly assigned to receive 400 IU of either RRR alpha-tocopherol (A: 268 mg, n = 10) or all rac-alpha-tocopheryl acetate as a fat soluble (B: 400 mg, n = 10) or water-miscible preparation (C: 400 mg, n = 9) and were followed for 6 wk. In the whole study group, plasma alpha-tocopherol concentrations increased from baseline (10.5 +/- 4.6 micromol/L) to 3 wk (25.7 +/ 6.5 micromol/L; P < 0.001), but not further between 3 and 6 wk; concentrations at 3 and 6 wk did not differ from those of age-matched control subjects (23.6 +/- 3.9 micromol/L). There was no significant difference in the increase from baseline to 6 wk among preparations A (17.75 +/- 8.43 micromol/L), B (14.0 +/- 9.4 micromol/L), and C (15.5 +/- 7.1 micromol/L). Because of differences in body weight, the dose administered ranged from 5.5 to 47.4 IU x kg-1 x d-1; it correlated positively with the increase in plasma alpha-tocopherol concentrations (P < 0.001). There was no significant difference in the increase in plasma alpha tocopherol concentrations between patients with CF-associated liver disease (n = 8) who received 10.2 +/- 3.8 IU x kg-1 x d-1 and those without liver disease taking comparable doses. We conclude that CF patients can be efficiently supplemented with 400 IU/d of any one of the three vitamin E preparations and plasma values of healthy control subjects can be achieved. PMID- 8615354 TI - Response to a single oral dose of all-rac-alpha-tocopheryl acetate in patients with cystic fibrosis and in healthy individuals. AB - Biochemical vitamin E deficiency and low plasma lipids are frequent findings in patients with cystic fibrosis (CF). The response to a single oral dose of all-rac alpha-tocopheryl acetate [100 IU (100 mg)/kg body wt] was studied over 24 h in 25 CF patients with exocrine pancreatic insufficiency and in 23 healthy individuals. Patients received pancreatic enzymes together with the vitamin E test dose. At baseline, plasma alpha-tocopherol concentrations correlated with cholesterol concentrations; both were lower in patients than in control subjects, as were erythrocyte alpha-tocopherol concentrations (all P < 0.0001). Plasma and erythrocyte alpha-tocopherol concentrations were significantly higher than baseline concentrations from 3 and 6 h onward, respectively, and peaked most frequently at 6 and 12 h, respectively, in both patients and control subjects. Maximum increases and areas under the concentration time curves for plasma alpha tocopherol concentrations were smaller in patients than in control subjects (P < 0.0001). When ratios of plasma alpha-tocopherol to cholesterol (to correct for differences in cholesterol concentrations) or erythrocyte alpha-tocopherol concentrations were applied, patients were shown to respond as efficiently as control subjects. On the basis of these results, we recommend vitamin E supplements in doses high enough to achieve vitamin E status in CF patients well within the range of healthy individuals; these supplements should be given with appropriate amounts of pancreatic enzymes. However, for long-term supplementation much lower doses than those used in this test situation may be sufficient. PMID- 8615356 TI - Bioavailability of a natural isomer mixture compared with synthetic all-trans beta-carotene in human serum. AB - The unicellular alga Dunaliella bardawil was shown previously to contain very high concentrations of beta-carotene composed of about equal amounts of the all trans and 9-cis isomers, which differ in their physicochemical features and antioxidative activity. The uptake of alpha- and beta-carotenes, oxycarotenoids, and other lipophilic substances from a basal diet supplemented with synthetic beta-carotene or dry D. bardawil power was studied in humans. Subjects were given a basal diet supplemented daily with 40 mg beta-carotene, synthetic or natural, for a relatively short period of 14 d. Serum analyses at the end of this period detected mainly oxycarotenoids, and to a lesser extent all-trans beta-carotene and alpha-carotene, but not 9-cis beta-carotene. Retinol was increased by the all trans beta-carotene diet. A high amount of oxidized dienic, lipophilic polar products was exhibited in HPLC predominantly in sera from the placebo and synthetic all-trans beta-carotene groups by strong, short ultraviolet absorbance peaks of 232 nm. The preferential serum absorption of all-trans beta-carotene over 9-cis beta-carotene, in parallel with the appearance of a high concentration of oxidized dienic products with supplementation of the basal diet with all-trans beta-carotene compared with the low concentration of serum-oxidized dienic products with supplementation with a natural beta-carotene source, suggests that 9-cis beta-carotene acts as an in vivo lipophilic antioxidant more efficiently than does all-trans beta-carotene. PMID- 8615357 TI - Sodium excretion in relation to calcium and hydroxyproline excretion in a healthy Japanese population. AB - To evaluate whether habitual excess sodium intake is a significant risk factor for calcium loss, we studied the relation between calcium excretion and sodium excretion in 410 male and 476 female Japanese aged 20-79 y. They were apparently healthy, free-living, and consuming diets of their own choosing. We divided the subjects into two groups: 20-49 y olds and 50-79 y olds. In each group, we observed significant positive correlation between daily calcium excretion and daily sodium excretion in both sexes. Multivariate analyses revealed that in each age group the relation was still significant after sex, age, body weight, and protein, calcium, and phosphorus intakes were adjusted for. The increases in urinary calcium excretion were estimated to be approximately 0.6 and 1.0 mmol for a 100-mmol increment in urinary sodium excretion for the 20-49 y olds and 50-79-y olds, respectively. We also observed significant positive correlations between daily hydroxyproline excretion and daily sodium excretion in both sexes for both age groups. The relation was still significant after sex, age, body weight, and protein intake from meat and fish were adjusted for. The results suggest that individuals with high sodium intake may lose more calcium in their urine than those with low sodium intake. PMID- 8615358 TI - Epidemiologic analysis of Crohn disease in Japan: increased dietary intake of n-6 polyunsaturated fatty acids and animal protein relates to the increased incidence of Crohn disease in Japan. AB - We examined the correlation between the incidence of Crohn disease and dietary change in a relatively homogeneous Japanese population. The incidence and daily intake of each dietary component were compared annually from 1966 to 1985. The univariate analysis showed that the increased incidence of Crohn disease was strongly (P < 0.001) correlated with increased dietary intake of total fat (r = 0.919). animal fat (r = 0.880), n-6 polyunsaturated fatty acids (r = 0.883), animal protein (r = 0.908), milk protein (r = 0.924), and the ratio of n-6 to n-3 fatty acid intake (r = 0.792). It was less correlated with intake of total protein (r = 0.482, P < 0.05), was not correlated with intake of fish protein (r = 0.055, P > 0.1), and was inversely correlated with intake of vegetable protein (r = -0.941, P < 0.001). The multivariate analysis showed that increased intake of animal protein was the strongest independent factor with a weaker second factor, an increased ration of n-6 to n-3 polyunsaturated fatty acids. The present study in association with reported clinical studies suggests that increased dietary intake of animal protein and n-6 polyunsaturated fatty acids with less n-3 polyunsaturated fatty acids may contribute to the development of Crohn disease. PMID- 8615359 TI - Effect of linoleic acid intake on growth of infants with cystic fibrosis. AB - Essential fatty acid deficiency as a result of inadequate linoleic acid impairs growth in healthy infants and is common in infants with malabsorption due to cystic fibrosis (CF). We investigated the effect of dietary linoleic acid intake on the growth of infants with CF. In this study, predigested formula preparations A and B, with linoleic acid contents of 12% and 7% of energy, respectively, were fed before and after 1989 to infants enrolled in the evaluation and treatment protocol of the Wisconsin CF Neonatal Screening Project. Outcome was assessed from height-for-age (HAZ) and weight-for-age (WAZ) Z scores on follow-up exams during the first year. Baseline characteristics did not differ significantly between groups A (n = 43) and B (n = 33). At diagnosis, 53% of the enrolled infants (n = 76) showed low plasma linoleic acid concentrations and 22% had a high ratio of triene to tetraene. After correcting for the effect of potentially confounding variables, we found that HAZ (by .27, P < 0.05) and WAZ (by 0.26, P = 0.081) were higher in group A than in group B. This occurred despite a significantly higher energy intake in group B. This difference was most pronounced between 6 and 9 mo of age. Our results suggest that a high linoleic acid content in formula benefits infants with CF because it optimizes nutrition, growth, and feeding efficiency. PMID- 8615360 TI - RRR-alpha-tocopheryl acetate supplementation at pharmacologic doses decreases low density-lipoprotein oxidative susceptibility but not protein glycation in patients with diabetes mellitus. AB - Patients with diabetes mellitus have an increased risk of premature atherosclerosis, which may be due in part to increased oxidizability of low density lipoprotein (LDL). Numerous studies have shown that alpha-tocopherol can reduce the oxidative susceptibility of LDL in normoglycemic subjects; however, there are few studies in persons with diabetes. In addition, alpha-tocopherol may reduce the extent of protein glycation. Therefore, the objective of the present study was to assess the effect of RRR-alpha-tocopheryl acetate supplementation on LDL oxidizability and protein glycation in persons with diabetes without evidence of vascular disease. Twenty-eight persons with insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) were randomly assigned to receive either placebo or 1632 mg (1200 IU) RRR-alpha-tocopherol/d, as tocopheryl acetate, for 8 wk. Plasma and LDL antioxidant concentrations and LDL oxidizability were assessed at both 0 and 8 wk. Plasma and LDL concentrations of alpha-tocopherol were significantly increased in the supplemented group only. Compared with the placebo group, the alpha-tocopherol-supplemented group had significant reductions in LDL oxidizability at 8 wk, as shown by the time-course curves of conjugated diene and lipid peroxide formation. Also, alpha-tocopherol supplementation produced a significant prolongation in the lag phases of both assays, which was evident in both the NIDDM and IDDM subgroups. However, there were no significant changes in glycated hemoglobin or in glycated plasma proteins after alpha-tocopherol supplementation. Thus, alpha-tocopherol supplementation may be beneficial in reducing LDL oxidizability in patients with diabetes. PMID- 8615361 TI - Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma of critically ill patients. AB - Plasma concentrations of the antioxidant vitamin ascorbic acid were measured by high-performance liquid chromatography in critically ill patients in whom the excessive generation of reactive oxygen species could compromise antioxidant defense mechanisms. Median concentrations of both total vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid in these patients were < 25% (P < 0.001) of the values found in healthy control subjects and in subjects in two other disease groups (diabetes, gastritis) in which reactive oxygen species are reported to be increased. The low values could not be explained by age, sex, intake, or treatment differences, but were associated with the severity of the illness and were not prevented by the use of parenteral nutrition containing ascorbic acid. In addition, the vitamin was less stable in blood samples taken from critically ill patients than in similar samples from subjects in the other groups. The findings indicate that antioxidant defenses could be considerably compromised in these very sick patients. If this reduces the patient's capacity to scavenge reactive species, then the potential of these species to damage DNA and lipid membranes could be increased and compromise recovery. PMID- 8615362 TI - Resistant starch lowers fecal concentrations of ammonia and phenols in humans. AB - We investigated the effect of resistant starch (RS) on markers of colonic protein metabolism. Eleven subjects participated in a randomized crossover study in which they consumed either high-RS (39 +/- 3 g/d, -chi +/- SEM) or low-RS (5 +/- 0.4 g/d) diets for 3 wk. All other macronutrients were kept constant. During the high RS diet daily excretion of fecal nitrogen increased from 1.84 +/- 0.15 to 2.86 +/ 0.42 g/d (P < 0.01) and excretion of fecal phenols fell from 9.2 +/- 1.4 to 5.3 +/- 0.8 mg/d (P < 0.01). Fecal concentrations of ammonia decreased from 397 +/- 33 to 278 +/- 49 microgram/g (P < 0.01) and phenols decreased from 69 +/- 8 to 39 +/- 10 microgram/g (P < 0.001). Daily output of urinary ammonia, urea, phenols, and total nitrogen did not change significantly, but pH decreased from 6.4 +/- 0.1 to 6.2 +/- 0.1 (P < 0.05) during the high-RS period. These results suggest that RS significantly attenuates the accumulation of potentially harmful byproducts of protein fermentation in the human colon. PMID- 8615364 TI - Learning disabilities and poor motivation to achieve due to prolonged iodine deficiency. AB - The effect of prolonged iodine deficiency on learning and motivation was studied. One hundred male children--matched for age, socioeconomic status, and formal education--were selected from both severely iodine-deficient (SID) and mildly iodine-deficient (MID) villages. Mean urinary iodine excretion was significantly lower in the SID than in the MID group (219.84 +/- 57.52 compared with 449.14 +/- 32.31 nmol/L, P < 0.001). The serum thyroxine concentration was significantly lower (90.36 +/- 6.46 compared with 123.70 +/- 15.42 nmol/L, P< 0.001) and serum thyroid-stimulating hormone (TSH) was significantly higher in the SID group than in the MID group (6.23 +/- 0.34 compared with 4.85 +/- 0.28 mU/L, P< 0.01). The children were administered maze, verbal, and pictorial learning tasks and a test of motivation. The results showed that SID children are slow learners compared with MID children. In both groups the rate of learning over trials was superior in younger (aged 9-12 y) children although the initial performance of older (aged 12-15 y) children was better (P < 0.01). SID children scored significantly lower than MID children on the achievement motivation scale (P < 0.01). The results are suggestive of neural impairment as well as poor sociopsychologic stimulation, resulting in learning disability and lowered achievement motivation. Unless iodine nutrition is improved in the community as a whole, these abnormalities may prevent millions of children from the SID areas from achieving their full potential even if learning opportunities are made available to them. PMID- 8615363 TI - Effect of vitamin A supplementation on the growth of young children in northern Ghana. AB - The effect of prophylactic vitamin A supplementation on child growth was studies in two randomized, placebo-controlled trials carried out in adjacent areas of northern Ghana between 1989 and 1991. In the Health Study, the midupper arm circumference (MUAC) and weight of the approximately 1500 children (aged 6-59 mo) in the trial were measured every 4 wk for up to 52 wk. In addition, MUAC, weight, and height were measured at each of the four potential vitamin A or placebo dosing times, which were at 4-mo intervals. In the Survival Study, MUAC and weight were measured at 4-mo intervals at each of seven dosing rounds in the approximately 15 000 children currently in the trial. Overall, there were > 90 000 observations of weight and MUAC in > 25 000 children, and 3347 observations of length/height in 1546 children. Within each study, the mean monthly weight, MUAC, and gains in length/height in each treatment group were compared by using multilevel modeling. There were no significant differences in either MUAC or gains in length/height. The only significant difference in weight gain was in the Survival Study: children in the vitamin A-supplemented group who were > or = 36 mo of age had a mean weight gain that was 3 g lower per month (95% CI: 0.4, 5.0, P = 0.02) than that in the placebo group; a difference that was unlikely to be functionally important in this age group. Vitamin A supplementation did not lead to any increased growth in this population of young children, in whom supplementation reduced mortality and severe morbidity substantially. PMID- 8615365 TI - Dietary prevention of osteoporosis: are we ignoring the evidence? PMID- 8615366 TI - Copper as an essential nutrient. AB - Animal and human studies have shown that copper is involved in the function of several enzymes. Studies have also shown that copper is required for infant growth, host defense mechanisms, bone strength, red and white cell maturation, iron transport, cholesterol and glucose metabolism, myocardial contractility, and brain development. Copper deficiency can result in the expression of an inherited defect such as Menkes syndrome or in an acquired condition. Acquired deficiency is mainly a pathology of infants; however, it has been diagnosed also in children and adults. Most cases of copper deficiency have been described in malnourished children. The most constant clinical manifestations of acquired copper deficiency are anemia, neutropenia, and bone abnormalities. Other, less frequent manifestations are hypopigmentation of the hair, hypotonia, impaired growth, increased incidence of infections, alterations of phagocytic capacity of the neutrophils, abnormalities of cholesterol and glucose metabolism, and cardiovascular alterations. Measurements of serum copper and ceruloplasmin concentrations are currently used to evaluate copper status. These indexes are diminished in severe to moderate copper deficiency; however, they are less sensitive to marginal copper deficiency. Erythrocyte superoxide dismutase and platelet cytochrome c activities may be more promising indexes for evaluating marginal copper deficiency. PMID- 8615367 TI - Copper biochemistry and molecular biology. AB - In this review, our basic and most recent understanding of copper biochemistry and molecular biology for mammals (including humans) is described. Information is provided on the nutritional biochemistry of copper, including food sources, intestinal absorption, transport, tissue distribution, and excretion, along with descriptions of copper binding proteins and other factors involved and their roles in these processes. The metabolism of copper and its importance for the functions of a roster of vital enzymes is detailed. Its potential toxicology is also addressed. Alterations in copper metabolism associated with genetic and nongenetic diseases are summarized, including potential connections to inflammation, cancer, atherosclerosis, and anemia, and the effects of genetic copper deficiency (Menkes syndrome) and copper overload (Wilson disease). Understanding these diseases suggests new ways of viewing the normal functions of copper and provides new insights into the details of copper transport and distribution in mammals. PMID- 8615368 TI - Liver copper storage and transport during development: implications for cytotoxicity. AB - Copper is an essential trace element for many biological processes. Its functions range from influencing specific gene expression to serving as a cofactor or prosthetic group for several enzymes. Intakes of copper at doses that exceed physiologic demands are normally met with efficient homeostatic mechanisms. Ceruloplasmin, albumin, and transcuprein, and to a lesser extent certain amino acids, are major copper-transporting constituents in circulating plasma. After its hepatic uptake, copper may be stored within hepatocytes, secreted into plasma, or excreted in bile. The biliary route represents the major excretory pathway of copper and largely accounts for its hepatic turnover. Copper retained by hepatocytes is mostly bound to specific metal-binding proteins, primarily metallothionein, or incorporated into several cuproenzymes. Copper incorporation into metallothionein and certain cuproproteins appears to require prior binding of copper to glutathione, thus defining a relation between copper metabolism and the intracellular availability of glutathione. Hepatic metallothionein concentrations can be modulated by dietary copper; changes in metallothionein and in copper status are significant throughout development. Binding of copper to metallothionein provides a temporary storage for cytoplasmic copper, preventing it from occurring as (potentially toxic) free ionic metal. In its unbound form, copper can generate hydroxyl radicals. Because metallothionein exhibits a high reactivity toward these radicals, it is increasingly recognized to play a protective role against copper-induced cytotoxicity. We discuss some of the possible toxicologic implications that may arise from changes in hepatic copper and metallothionein status during development. PMID- 8615369 TI - Bioavailability of copper. AB - Copper intakes of infants and adults are often much lower than current recommendations. Copper status, however, appears adequate in most populations. This suggests that copper requirements may be lower than believed earlier, except those for premature infants, who have high requirements as a result of low prenatal stores. Infants, in general, constitute a risk group because milk is low in copper. Bioavailability of copper from human milk is high, whereas it is lower from cow milk and infant formula. Protein source, amino acids, carbohydrates, and ascorbic acid can affect copper availability, whereas phytate, zinc, and iron appear to have little influence on copper absorption, at least physiologic intakes. PMID- 8615370 TI - Present interpretation of the role of copper in Indian childhood cirrhosis. AB - A common killer disease of the past, Indian childhood cirrhosis (ICC), which became preventable and treatable in the early 1990s, is now rare. ICC must be clearly distinguished in Indian children from other chronic liver disorders including Wilson disease. Grossly increased hepatic, urinary, and serum copper concentrations are characteristic of ICC. These increased concentrations are easily demonstrated histologically with orcein-rhodanine staining. Environmental ingestion of copper appears to be the most plausible explanation for ICC, as shown by feeding histories, the prevention of ICC is siblings and in the Pune district by a change in feeding vessels, and the dramatic reduction in incidence of ICC throughout India. The nature and role of a second factor in the causation of ICC remains unclear, although an inherited defect in copper metabolism is strongly suspected. ICC, however, does not appear to be a straightforward early onset of Wilson disease because ceruloplasmin is consistently normal and clinical and histologic recovery is maintained in the long term despite withdrawal of D penicillamine therapy. Descriptions of an ICC-like illness in the West suggest that different mechanisms (environmental, genetic, or both) can lead to the same end-stage liver disease: copper-associated childhood cirrhosis. ICC probably represents a specific form of copper-associated childhood cirrhosis that requires high environmental copper ingestion for its full expression. PMID- 8615371 TI - Genetic and molecular basis for copper toxicity. AB - Recent studies resulted in the cloning of the genes responsible for Menkes syndrome and Wilson disease. Despite the distinct clinical phenotypes of these disorders, each gene encodes a highly homologous member of the cation-transport P type ATPase family. The remarkable evolutionary conservation of these proteins in bacteria, yeast, plants, and mammals reveals a fundamental protein structure essential for copper export in all life forms. Characterization of a molecular defect in the rat homologue of the Wilson ATPase in the Long-Evans Cinnamon rat identifies an animal model of Wilson disease and will permit experimental analysis of the precise role of this ATPase in copper transport, the effects of specific inherited mutations on transport function, and the cellular and molecular mechanisms of tissue injury resulting from copper accumulation. Finally, recent molecular genetic analysis of a distinct group of patients with low serum ceruloplasmin and basal ganglia symptoms identified a series of mutations in the ceruloplasmin gene. The presence of these mutations in conjunction with the clinical and pathologic findings clarifies the essential biological role of this abundant copper protein in metal metabolism and identifies aceruloplasminemia as a novel autosomal recessive disorder of iron metabolism. PMID- 8615372 TI - Wilson disease and idiopathic copper toxicosis. AB - The pathogenic agent of both Wilson disease (WD) and non-Indian childhood cirrhosis (which we term idiopathic copper toxicosis, or ICT) is copper accumulating to excess in the liver. Inheritance of a pair of alleles of an autosomal recessive gene on chromosome 13 is necessary and sufficient to cause such copper accumulation in WD; reducing the dietary intake of copper cannot prevent the development of WD. In contrast, the lethal accumulations of copper in children with ICT have been attributed primarily to an increased dietary intake of copper. However, 64 124 child-year exposures of children under the age of 6 y to drinking water containing a copper concentration of approximately 125.9 micromol/L (8 mg/L) produced no deaths from any form of liver disease. Moreover, the ICT of seven infants was attributed primarily to drinking water containing < 110.2 micromol Cu/L (7 mg/L) despite evidence of the presence of a genetic defect in three of the patients, one of whom was exclusively breast-fed. These data suggest that ICT cannot be caused solely by increased dietary intake of copper and occurs only in children with an identified genetic defect. PMID- 8615373 TI - Limits of metabolic tolerance to copper and biological basis for present recommendations and regulations. AB - Acute copper toxicity is infrequent in humans. The evidence for chronic toxicity is derived principally from patients with Wilson disease and cases of infantile cirrhosis that were related to excessive copper intakes. The evaluation of the safety of a nutrient requires toxicologic studies to determine the limits of safe exposure. The acceptable daily intake (ADI) is calculated by determining the highest no-observed-adverse-effect level (NOAEL). When it is not possible to identify the NOAEL, the lowest observed-adverse-effect level (LOAEL) may be used. For the calculation of human ADI, the NOAEL or LOAEL obtained is divided by an arbitrary safety factor to provide an adequate margin of security. Drinking water standards have been adopted by the United States, the European Community, the World Health Organization, and other countries. The upper limits of copper concentration in water are based on organoleptic considerations and on debatable toxicity information. Given the importance of copper as an essential mineral for human health, it is conceivable that this and other essential minerals with health significance should be approached differently from nonessential minerals. PMID- 8615374 TI - From the editor. PMID- 8615375 TI - Effect of acute ketoacidosis on the myocardium in diabetes. AB - Abnormalities of left ventricular function are often present in patients with diabetes who are in a stable metabolic state. To determine whether acute metabolic abnormalities may contribute to pathogenesis, patients with diabetes and ketoacidosis (Group 1) or hyperglycemia without ketosis (Group 2) were studied. They were assessed noninvasively for evidence of acute injury or dysfunction of the myocardium. Left ventricular function was assessed on admission and after clinical recovery. Myocardial enzyme release was examined during the acute phase. In Group 1, plasma glucose averaged 32 mM/L and carbon dioxide content 12.4 mEq/L. On echocardiography, the initial circumferential shortening velocity of 1.85 + 0.07 circumferences per second was significantly higher than the final circumferential shortening velocity of 1.31 + 01 (P < 0.005). The systolic time interval ratio, pre-ejection period/left ventricular ejection time, was significantly lower on the initial day compared with the second study. These data are consistent with enhanced ventricular performance. In group 2, plasma glucose averaged 29 mM/L, and carbon dioxide content was normal. The initial circumferential shortening velocity of 1. 1 circumferences per second and pre-ejection period/left ventricular ejection time ratio of 0.38 were normal and remained unchanged. There was no significant alteration of heart rate or arterial pressure in either group. In both groups, total serum lactate dehydrogenase and creatinine phosphokinase levels, as well as their cardiac isoenzymes, were within normal limits. Therefore, the initial increase of myocardial performance and subsequent restoration to normal, as well as the lack of cardiac enzyme increase in plasma, support the view that shortterm ketoacidosis does not contribute to the abnormalities of ventricular function in diabetes. PMID- 8615376 TI - Suppression of cellular proliferation using p53 DNA recognition site-related oligonucleotides. AB - A number of oligonucleotides were designed to bind through Hoogsteen triple helix or Watson-Crick hydrogen bonds to the p53 consensus sequence homology localized within the human nontranscribed rRNA spacer region. The oligomers, which bind in vitro to the consensus sequence homology, function as p53 analogues in cells deficient in wild-type p53 protein. Oligomers suppress proliferation of human colon cancer cells by three to eightfold, but only suppress proliferation of normal human mesangial cells or lung fibroblasts by less than 50%. On the basis of these studies, p53 analogues may be used therapeutically to selectively modify proliferation of transformed cells. PMID- 8615377 TI - 1,25(OH)2D3 and cAMP synergistically induce complement 5a receptor messenger RNA. AB - Complement 5a receptor (C5aR) mediates both acute and chronic participation of monocytes in the immune response. In the human U937 monoblast, C5aR is maximally expressed 4 days after treatment with 1,25(OH)2D3 (or cycloheximide) and prostaglandin E2 combined. The authors asked whether these agents altered expression of C5aR messenger RNA (mRNA). Unstimulated U937 cells expressed neither C5aR mRNA nor C5a binding. Complement 5aR mRNA rose 3 hours after prostaglandin E2 application and fell to basal levels by 12 hours. This early rise in C5aR mRNA did not cause an acute rise in C5a binding, which gradually increased between 1 and 4 days. Neither 1,25(OH)2D3 nor cycloheximide induced expression of C5aR mRNA in the absence of prostaglandin E2 but did enhance prostaglandin E2-stimulated C5aR mRNA expression and C5a binding. The authors observed a late increase in C5aR mRNA at day 3 in treated cells. Inhibition of this late rise in mRNA with 5,6-dichlorobenzimidazole riboside attenuated C5a binding by 65%, indicating its importance in the generation of C5a binding sites. The expression of functional C5aR is, therefore, a complex process involving regulation at transcriptional and posttranscriptional levels. PMID- 8615378 TI - Case report: interaction of rifampin and nortriptyline. AB - Rifampin is well documented to cause numerous clinically significant drug interactions because of its induction of the hepatic cytochrome P450 system. The metabolism of nortriptyline and other tricyclic antidepressants is induced and inhibited by this enzyme system. To the authors' knowledge, this is only the second case report in the literature regarding a possible interaction between rifampin and a tricyclic antidepressant. Nortriptyline serum concentrations were not detectable while rifampin was given concurrently. Two weeks after discontinuation of rifampin, nortriptyline concentrations increased dramatically. Further study of this possible interaction is warranted. PMID- 8615379 TI - Case report: ofloxacin-induced hypersensitivity vasculitis. AB - Since its introduction in 1985, the new fluoroquinolone antibiotic ofloxacin has gained widespread use, and much information has accumulated about its possible adverse effects. Skin reactions have been uncommon, and there have been very few reports about hypersensitivity vasculitis directly related to ofloxacin. The authors report such a case, in which the patient needed plastic surgery because of severe vasculitis in both legs. PMID- 8615380 TI - Case report: severe hyperkalemia in a geriatric patient receiving standard doses of trimethoprim-sulfamethoxazole. AB - A case is reported of a 96-year-old woman with congestive heart failure, hypertension, and chronic obstructive pulmonary disease who presented with altered mental status and severe hyperkalemia, a serum potassium 9.3 meq/L, and electrocardiograph changes. The patient was discharged 1 week prior, with a normal serum potassium, receiving trimethoprim-sulfamethoxazole for urinary tract infection and pneumonia. Serum potassium measurements returned to normal after discontinuation of the drug. Other causes of hyperkalemia were ruled out. Mild hyperkalemia due to trimethoprim-sulfamethoxazole was first reported in 1983 in a 69-year-old woman in whom leukemia with leukopenia developed. In literature to date, mild hyperkalemia in younger geriatric patients has been described. Trimethoprim is thought to act by inhibiting amiloride sensitive sodium channels in the distal nephron and impairing renal potassium secretion in a dose dependent manner. The authors report the case, review the literature, and discuss age related reduction in renal function as a possible etiology. PMID- 8615381 TI - Case series: hyponatremia associated with moderate exercise. AB - Exercise-induced hyponatremia is commonly believed to be associated only with extraordinary physical efforts, or particularly strenuous exercise. Hyponatremia complicating moderate exercise has not been described previously. The authors describe the characteristics of seven patients with life-threatening hyponatremia associated with mild to moderate exercise. All patients suffered from nausea, vomiting, agitation, and confusion, appearing during or after moderate physical activity. Grand mal convulsions occurred in five of the patients. In laboratory results, hyponatremia was as low as 115 mEq/L, with a relatively high sodium concentration in the urine. High serum creatine kinase activity levels were found in most of the patients. All patients were discharged in good condition, without neurologic sequela. The authors conclude that hyponatremia is a possible complication of moderate exercise, and not only of endurance sports, and that exercise-induced hyponatremia can produce severe neurologic manifestations. The mechanism of the hyponatremia is unclear, but may be due to a hemodynamically inappropriate stimulus for antidiuretic hormone secretion. PMID- 8615383 TI - Review: Postoperative thrombocytopenia: with etiologic, diagnostic, and therapeutic consideration. PMID- 8615382 TI - Case report: immunoglobulin M-mediated, temperature-dependent neutrophil agglutination as a cause of pseudoneutropenia. AB - The authors report the case of a 77-year-old man in whom intermittent neutropenia developed during a prolonged hospitalization. Laboratory records revealed wide variations in the patient's routine leukocyte counts. Examination of the peripheral smear revealed clumps of 2-3 polymorphonuclear leukocytes throughout the slide and large aggregates (> 50 cells) along the smear edges. Neutrophil clumping also was observed in blood anticoagulated with citrate or heparin. Replacement of patient plasma with saline prevented agglutination. Addition of patient plasma or serum to normal cells induced neutrophil agglutination. Holding the patients blood at 37 degrees C prevented agglutination, which occurred spontaneously and more exuberantly as the temperature was reduced to 22 degrees C. Flow cytometry revealed immunoglobulin M on neutrophil surfaces. Spontaneous agglutination resolved 6 months after resection of the patient's subsequently diagnosed colon cancer. This is the first report of immunoglobulin M-induced neutrophil clumping occurring in the setting of malignancy, and the first reported immunoglobulin M not to require ethylenediaminetetraacetic acid as a cofactor for agglutination. PMID- 8615384 TI - Paracellular transport of water and carbohydrates during intestinal perfusion of protamine in the rat. AB - With these experiments, the authors' purpose was to determine whether the intestinal perfusion of protamine would successfully block paracellular transport without causing significant change in cardiovascular function. In anesthetized (50 mg x kg-1 sodium pentobarbital) rats (n=12), heart rate and mean arterial blood pressure were measured during perfusion (0.5 mL x min-1) of a carbohydrate electrolyte solution through the small intestine. The carbohydrate-electrolyte solution contained 150 mM glucose, 150 mM fructose, 10 mM lactulose, 17 mEq sodium, 3 mEq potassium, and either 0.0, 0.1, 1.0, or 10 mg x mL-1 protamine. Osmolality of the 4 solutions ranged from 363 +/- 2 to 365 +/- 3 mOsm x kg-1. Core temperature was maintained at 37 degrees C in an environmental chamber. Heart rate and mean arterial blood pressure were constant during all intestinal perfusions. Forty-one percent of the perfused lactulose was absorbed. Absorption of glucose, fructose, and lactulose was significantly inhibited by 0.1 mg x mL-1 protamine, while water absorption was decreased 41 percent by 1.0 mg x mL-1 protamine. Water and lactulose absorption fell 75% with protamine, and glucose and fructose absorption fell 50%. Lactulose and fructose absorption did not decrease further when protamine dose rose to 10 mg x mL-1. These results indicate that 1) perfusion of protamine into the small intestine in doses that significantly affect intestinal transport does not significantly affect heart rate and mean arterial blood pressure; and 2) if the primary effect of protamine is to block paracellular movement of water and solute, the greater protamine inhibition of water and lactulose absorption is consistent with a greater paracellular transport of water and lactulose than for glucose and fructose. PMID- 8615385 TI - Decreased insulin-mediated but not non-insulin-dependent glucose disposal rates in glucose intolerance and type II diabetes in African (Ghanaian) immigrants. AB - The authors evaluated the significance of beta cell function, non-insulin dependent glucose disposal (glucose effectiveness [Sg]), and insulin-dependent glucose disposal (insulin sensitivity) in African immigrants with varying degrees of glucose tolerance. Thirty-two African immigrants residing in Franklin County, Ohio, were studied. There were 16 subjects with normal glucose tolerance (NGT), 11 with intermediate glucose tolerance (IGT), and 5 with type II diabetes mellitus (DM). Insulin sensitivity index and Sg were measured by an insulin modified, frequently sampled intravenous glucose tolerance test. The mean fasting and post-glucose serum glucose levels were lowest in the NGT, intermediate in the IGT group, and highest in the DM group. Mean serum insulin and c-peptide responses rose briskly by threefold to a peak in the NGT and the IGT groups. In the DM group, mean serum insulin and c-peptide responses were severely blunted to glucose stimulation. The sensitivity index was highest in the NGT (3.09 +/- 0.27), intermediate in the IGT (1.81 +/- 0.47), and lowest in the DM (0.48 +/- 0.28 x 10(-2).mins-1 (microU/ml)-1). The Sg was identical in the NGT (2.78 +/- 0.22) and IGT (2.78 +/- 0.27) groups but was slightly but not significantly lower in the DM (2.20 +/- 0.35 x 10(-2).mins-1). In addition, the glucose decay constant was not statistically different in the NGT (3.00 +/- 0.38) and IGT (2.25 +/- 0.19) group, but the mean values were significantly greater than in the patients with diabetes (0.78 +/- 0.15 percent/mins). The mean disposition index (sensitivity index X beta cell function as assessed by both insulin and c peptide) was significantly greater in NGT than in the IGT (P<0.05) and in the diabetic group (P<0.001). In summary, the authors demonstrate that, in native African immigrants, type II diabetes is associated with significant reduction in beta cell function, insulin sensitivity, and glucose decay constant, but not in Sg. In patients with intermediate or impaired glucose tolerance, there is moderate insulin resistance and evidence of inadequate compensation by beta cell, but the Sg, the Sg at theoretical insulin concentration, and glucose decay constant remain normal. They conclude that, unlike other ethnic and racial groups, in glucose intolerant native African patients, alterations in Sg or non insulin dependent glucose disposal (ie, tissue glucose sensitivity) do not appear to play a significant role in the impairment of glucose tolerance and type II diabetes in African immigrants. PMID- 8615386 TI - The internal markets of the British National Health Service: prospects and problems. AB - Since the creation of the British National Health Service in 1948, it has provided a full range of medical services free at the point of demand. By the mid 1980s, the National Health Service was perceived as suffering from a chronic crisis of an increasingly limited supply of medical resources but unlimited demand. The response of the Conservative government was to attempt to increase efficiency by introducing competitive dynamics with the creation of an internal market within the National Health Service. Health-care providers were to compete effectively with one another by price and quality. In this article, the author examines the prospects and problems inherent in such an internal market. The creation of fundholding general practitioners is scrutinized within the context of ideologic and financial goals of the British government and the potential consequences for patients. PMID- 8615387 TI - Ethnicity and cardiovascular disease: The Evans County heart study. AB - A long term study of diversity between two ethnic groups was developed in Evans County, Georgia. The findings are predicated on the genotypic-phenotypic interactions, with the multitude of environmental factors. The genetic environmental interaction ultimately determines the individual's state of health or disease. For example, coronary heart disease prevalence and incidence rates were extremely low for blacks in Africa and four times lower than whites in rural South Georgia in the 1960s. Excessive hypertension and diabetes mellitus, and greater cerebrovascular disease mortality in black men, is now well known. Blood pressure levels studied in rural Africa were normal and did not rise with age, whereas blacks, conversely, demonstrated twice as much hypertension in South Georgia as whites and demonstrated an inverse relation between education and blood pressure (ie, the lower the education the higher the blood pressure). Cultural adaptation has accelerated hypertensive disease and strokes in blacks, while there remains an excess of atherosclerotic coronary heart disease in white men. Secular trends suggest that coronary heart disease is decreasing among white men but may be increasing in black men. Studies of ethnicity and biracial populations provide important cardiovascular disease associations with clinical risk factor studies. PMID- 8615389 TI - Case report: splenic sequestration and multiorgan failure as the presenting manifestation of hemoglobin SC disease. AB - Acute splenic sequestration, a well recognized complication of the various sickle cell syndromes, is characterized by increasing splenomegaly and a sudden fall in hemoglobin concentration. In this article, the authors describe a 21-year-old woman with previously undiagnosed hemoglobin SC disease whose initial presentation was that of acute, severe splenic sequestration. Despite the severity of her illness, prompt diagnosis and appropriate therapy led to a complete recovery. The splenic sequestration in this case was apparently exacerbated by a recent hepatitis B infection. To date, this presentation of hemoglobin SC disease has not been described in the medical literature. PMID- 8615388 TI - Case report: olfactory function in a fertile eunuch with Kallmann syndrome. AB - The olfactory and gonadal dysfunction in Kallmann syndrome share a common embryologic pathophysiology. To characterize further the linkage between the hypogonadotropic hypogonadism and anosmia, the authors performed a detailed evaluation of olfactory function in a patient with Kallman Syndrome having the rare variant of partial gonadotropin deficiency (fertile eunuch). The subject was seen initially at age 16 years because of delayed puberty. He received testosterone replacement therapy and subsequently completed pubertal development. As an adult, while untreated, he had subnormal levels of serum testosterone, low gonadotropins, and normal response to luteinizing hormone- releasing hormone. He also had impotence that was reversible with testosterone therapy, and a normal sperm count. Despite the mild degree of hypogonadism, olfactory function was completely absent, and the response to nasal trigeminal stimulants was markedly attenuated. Complete anosmia may therefore be associated with gonadotropin deficiency that is only partial; the presence of anosmia does not predict the need for gonadotropin therapy to attain fertility. PMID- 8615390 TI - Case report: reversible mitral regurgitation and congestive heart failure complicating thyrotoxicosis. AB - Congestive heart failure is a relatively uncommon manifestation of thyrotoxic heart disease, and different mechanisms have been proposed. The authors present a possible explanation of congestive heart failure in some cases of thyrotoxicosis. A 39 year-old woman with Graves' disease was hypermetabolic, in atrial fibrillation, and had signs of congestive heart failure. She had a loud murmur of mitral regurgitation, clinical cardiomegaly, accentuated pulmonic sound, and peripheral edema. Propranolol reduced the heart rate to 60 beats per minute, but the loud mitral regurgitation murmur persisted. Echocardiographic and angiographic data were consistent with moderate to severe mitral regurgitation, serious enough to consider mitral valve replacement. As the patient's hyperthyroid state came under control, weight increased and the cardiac murmur resolved. After radioactive iodine treatment and the return to a eumetabolic state, an echocardiogram revealed only trace mitral regurgitation, with near normal left ventricular function and pulmonary arterial systolic pressures. These findings were confirmed by subsequent cardiac catheterization. The authors believe that mitral regurgitation, perhaps secondary to intrinsic papillary muscle dysfunction from hyperthyroidism, was the major cause of reversible congestive heart failure in this case. Valvular disease may play a more substantive role in thyrotoxic heart disease than previously recognized. PMID- 8615391 TI - Case report: reversal of decreased bone mass by antibiotic treatment in a patient with Whipple's disease. AB - Whipple's disease is a rare systemic illness, with the main clinical feature being severe malabsorption syndrome. Bone involvement in the disease has rarely been described in previous articles. The authors report a case in which an extensive skeletal evaluation was carried out. Even though osteomalacia is generally considered the most typical metabolic bone disease of malabsorption syndromes, bone biopsy demonstrated that osteoporosis was the prominent histologic feature in this patient. On the basis of serial bone mineral density measurements, antibiotic treatment was able to reverse the initial reduced bone mass. PMID- 8615393 TI - Protective Role of TNF-alpha in respiratory syncytial virus infection in vitro and in vivo. AB - Respiratory syncytial virus (RSV) infection causes substantial morbidity in young children and immunocompromised adults, yet its pathogenesis is poorly understood. Because the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) may be important in host response to viral infection, HEp-2 cells were treated with TNF-alpha and mice were given TNF-alpha antibody before RSV infection. Pretreatment of HEp-2 cells with TNF-alpha inhibited RSV replication as determined by cytopathic effect. Respiratory syncytial virus-infected BALB/c mice treated with antibody to TNF-alpha had greater maximal weight loss and slower recovery time than control mice. These results suggest a protective role for TNF alpha in RSV infection. PMID- 8615392 TI - Case report: diabetic microangiopathic hemolytic anemia and thrombocytopenia with antiphospholipid syndrome. AB - A 38 year-old man with a 12-year history of noninsulin-dependent diabetes mellitus with rapidly progressive diabetic complications presented with microangiopathic hemolytic anemia and thrombocytopenia. He had no disorders that could induce microangiopathic hemolytic anemia other than diabetic microangiopathy. In addition, there was a significant negative correlation between serum lactate dehydrogenase levels and peripheral platelet counts, which suggested that the hemolysis and thrombocytopenia occurred through the same mechanism. Activated partial thromboplastin time was slightly prolonged, and lupus anticoagulant and antiphospholipid immunoglobulin G antibodies were positive. Both the hemolysis and the thrombocytopenia spontaneously improved after the initiation of hemodialysis. This is a unique case of diabetic microangiopathic hemolytic anemia and thrombocytopenia in which antiphospholipid syndrome also may be involved. PMID- 8615395 TI - Fractal analysis of mammographic lesions: a feasibility study quantifying the difference between benign and malignant masses. AB - The increased use of screening mammography has led to increased pressure to differentiate between benign and malignant lesions. Even those lesions considered "suspicious" by qualitative radiologists' interpretations may prove malignant in less than 30% of cases. Fractal analysis is a mathematical technique that quantifies complex shapes. The hypothesis tested is that fractal analysis can quantify the difference between the shapes of benign and malignant lesions as imaged by mammography. Ten mammograms from patients with biopsy-proven invasive ductal carcinoma and 10 mammograms from patients with biopsy-proven benign disease were compared using the box-counting technique of fractal analysis. The fractal dimension of the mediolateral and craniocaudal views were added together to derive the composite fractal dimension. Statistical analysis was done using the Mann-Whitney U test. The median composite fractal dimension for benign lesions was 1.831 (range 1.359-2.009) and for malignant lesions 2.477 (range 2.084-3.158) (P < 0.0001). In addition, all benign lesions had fractal dimensions < or = 2.009, and all malignant lesions had fractal dimensions > or = 2.084. In this sample of 10 mammograms of malignant lesions versus 10 mammograms of benign masses, the composite fractal dimension was perfectly discriminatory. Fractal analysis may be useful to evaluate mammographically discovered breast masses. A blinded, prospective trial will be needed to determine its ultimate usefulness. PMID- 8615394 TI - Dehydroepiandrosterone reduces plasma plasminogen activator inhibitor type 1 and tissue plasminogen activator antigen in men. AB - Dehydroepiandrosterone (DHEA) may help prevent heart disease in men. To test the hypothesis that DHEA might exert its effects by enhancing endogenous fibrinolytic potential, a double-blind, placebo-controlled study was conducted that assessed the effects of DHEA administration on plasma plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (tPA) antigen. Eighteen men received 50 mg DHEA orally and 16 men received a placebo capsule thrice daily for 12 days. Serum DHEA-sulfate and plasma PAI-1 and tPA antigen were measured before and after treatment. In the DHEA group, serum DHEA-sulfate (from 7.5 +/- 1.2 micromol/L to 20.2 +/- 1.5 micromol/L (P < 0.0001), androstenedione (from 2.6 +/- 0.2 nmol/L to 4.0 +/- 0.4 nmol/L; P < 0.005) and estrone (from 172 +/- 21 pmol/L to 352 +/- 28 pmol/L; P < 0.005) increased, whereas plasma PAI-1 (from 55.4 +/- 3.8 ng/mL to 38.6 +/- 3.3 ng/mL; P < 0.0001) and tPA antigen (from 8.1 +/- 1.9 ng/mL to 5.4 +/- 1.3 ng/mL; P < 0.0005) decreased. In the placebo group, serum DHEA-sulfate declined slightly from 8.0 +/- 3.3 micromol/L to 7.3 +/- 3.4 micromol/L (P < 0.05), but no other measured steroid changed. Plasma PAI-1 and tPA antigen did not change in the placebo group. These findings suggest that DHEA administration reduces plasma PAI-1 and tPA antigen concentrations in men. PMID- 8615396 TI - Unanticipated diagnoses found at autopsy in an urban public teaching hospital. AB - The authors set out to evaluate the use of the autopsy in an urban public teaching hospital setting during the AIDS era. Demographic and length of hospital stay data were obtained from weekly mortality review reports on all patients dying on the medicine service between 1/1/92 and 12/31/93. Clinical and autopsy diagnoses were compared for those patients who had autopsies. The autopsy rate was 16% (152/974). Significant, unsuspected diagnoses were found in 35% (53/152) of the cases, with infections, pulmonary emboli, and myocardial infarctions being most common. Human immunodeficiency virus-infected patients had a greater percentage of unsuspected findings (55%, 23/42), and many of these also were from an infectious etiology. The authors conclude that valuable, unsuspected information frequently can be obtained from autopsies in this clinical setting. PMID- 8615397 TI - Deep negative T waves and abnormal cardiac sympathetic image (123I-MIBG) after the Great Hanshin Earthquake of 1995. AB - The authors report the increased incidence of patients with deep negative T waves without Q wave after the Great Hanshin Earthquake of 1995. Subjects underwent cardiac metaiodobenzyl guanidine (123I-MIBG) imaging, 201Tl scintigraphy, and coronary angiography. Among 2,756 inpatients of the preceding 5-year period, 33 (1.2%) showed the deep negative T waves, whereas 6 of 94 (6.4%; P < 0.001) showed it after the earthquake. Four of six patients had an episode of chest pain. Cardiac metaiodobenzyl guanidine imaging revealed the extent defects in all six patients despite a minimal change of 201Tl image. In addition, cardiac metaiodobenzyl guanidine imaging washout rate was hastened not only in the defect area but also in the nondefect area, which suggested augmented sympathetic activation. Natural disasters can affect the frequency of deep negative T waves, which relate abnormal cardiac sympathetic imaging. PMID- 8615398 TI - Review: diabetic ketoacidosis and hyperglycemic hyperosmolar nonketotic syndrome. PMID- 8615399 TI - Review: corticosteroid usage: observations at a community hospital. AB - Corticosteroids have revolutionized the medical treatment of many diseases. However, their value is limited by substantial potential adverse effects. Therefore, the authors reviewed their clinical use at their community medical center by comparing physician decisions about therapeutic management with the indications published in the current medical literature. They retrospectively reviewed 100 consecutive charts of patients from 1993 to 1994 who received corticosteroid therapy during their hospital stay. They found that 36 patients received appropriate corticosteroid intervention, 49 received partially appropriate corticosteroid intervention, and 15 received inappropriate corticosteroid intervention when compared with current medical literature recommendations for patients with the corresponding diagnoses. Ninety-five percent of patients given "partially appropriate" corticosteroid therapy and 73% given "inappropriate" corticosteroid therapy experienced medication-related side effects compared with 19% of patients given appropriate corticosteroid therapy intervention (P < 0.05). No patient records addressed the possibility of steroid related osteoporosis. There were no significant differences in the clinical course of the three groups of patients treated with corticosteroids. On the basis of these data, the majority of hospitalized patients given corticosteroids did not receive them entirely in accordance with literature recommendations; these patients experienced increased medication toxicity and did not receive prophylaxis for osteoporosis. These limited observations suggest opportunities to improve patient care. PMID- 8615400 TI - Case Report: amyopathic dermatomyositis associated with transformed malignant lymphoma. AB - Amyopathic dermatomyositis is a disease of unknown origin characterized by the specific skin lesions of dermatomyositis but without clinical or laboratory evidence of myopathy. During the past 15 years, a great controversy between the different reports concerning a possible association of dermatomyositis with malignancy has been noted. In this report, the authors describe a patient with amyopathic dermatomyositis who presented first with a benign hyperplasia of the lymph node, which finally transformed into frank malignant lymphoma. In addition to follow-up care, screening tests to search for occult malignancy in patients with amyopathic dermatomyositis (or dermatomyositis) are recommended. PMID- 8615401 TI - Case report: splenic hamartoma with hematologic disorders. AB - Hamartomas of the spleen are rare benign tumors usually detected as asymptomatic, incidental findings at laparotomy or autopsy. By review of the literature, there are only 16 well-documented cases of symptomatic splenic hamartomas associated with hematologic disorders. The authors report on an additional case of splenic hamartoma in a 34-year-old man with thrombocytopenia. Findings by ultrasonography and computed tomography suggested multiple confluent splenic lesions. Because of progressively increasing thrombocytopenia, elective splenectomy was performed. Final pathology confirmed diagnosis of multiple hamartomas of the red pulp. Platelet count returned to normal within 1 month postoperatively. Differential diagnosis, diagnostic procedures, and pathologic findings of splenic hamartomas will be discussed. Hamartomas should be kept in mind in the differential diagnosis of splenic tumors. Splenectomy is indicated in cases where malignancy cannot be excluded and in cases of associated hematologic disorders. PMID- 8615402 TI - Body and soul. PMID- 8615403 TI - Images in neuroscience. Neuroimaging, VIII. Magnetic resonance imaging. PMID- 8615404 TI - Pharmacologic management of psychiatric illness during pregnancy: dilemmas and guidelines. AB - OBJECTIVE: Given concerns about use of psychotropic medication during pregnancy, the authors reviewed the literature regarding the effects of prenatal exposure to psychotropic medications on fetal outcome. METHOD: A MEDLINE search of all articles written in English from 1966 to 1995 was performed to review information on the effects of psychotropic drug use during pregnancy on fetal outcome. Where sufficient data were available and when methodologically appropriate, meta analyses were performed to assess risk of fetal exposure by psychotropic medication class. RESULTS: Three primary effects are associated with medication use during pregnancy: 1) teratogenicity, 2) perinatal syndromes (neonatal toxicity), and 3) postnatal behavioral sequelae. For many drug classes there are substantial data regarding risk for teratogenicity. Tricyclic antidepressants do not seem to confer increased risk for organ dysgenesis. The available data indicate that first-trimester exposure to low-potency phenothiazines, lithium, certain anticonvulsants, and benzodiazepines may increase the relative risk for congenital anomalies. However, the absolute risk of congenital malformations following prenatal exposure to most psychotropics is low. CONCLUSION: Exposure to certain psychotropic drugs in utero may increase the risk for some specific congenital anomalies, but the rate of occurrence of these anomalies even with the increased risk remains low. Use of psychotropic medications during pregnancy is appropriate in many clinical situations and should include thoughtful weighing of risk of prenatal exposure versus risk of relapse following drug discontinuation. The authors present disorder-based guidelines for psychotropic drug use during pregnancy and for psychiatrically ill women who wish to conceive. PMID- 8615405 TI - Research update on the psychosocial treatment of schizophrenia. AB - OBJECTIVE: This review is an update on the research evidence supporting psychosocial treatment for schizophrenia. It extends previous review articles by summarizing the literature on social skills training, family interventions, cognitive rehabilitation, and coping with residual positive symptoms. METHOD: The authors reviewed controlled treatment outcome studies of social skills training and family interventions. Different models of family therapy were contrasted. The current literature on cognitive rehabilitation and coping with residual positive symptoms was also examined. RESULTS: Social skills training produces improvement on specific behavioral measures, although changes in symptoms and community functioning are less pronounced. Family interventions (i.e., family psychoeducation and behavioral family therapy) are highly effective for reducing families' expressed emotion and improving patients' relapse rates and outcomes. Furthermore, family interventions are also associated with reduced family burden. Cognitive rehabilitation and training in coping with positive symptoms appear to be promising interventions, but more controlled, group trials are needed before definite conclusions can be drawn. CONCLUSIONS: The efficacy of a variety of different family intervention models, as well as social skills training, is supported by a large body of research. Future work needs to address improving delivery of existing psychosocial interventions, integrating these interventions with other psychosocial approaches (e.g., vocational rehabilitation and case management), identifying which patients will benefit from which treatments, isolating the "active" ingredients of family interventions (i.e., psychoeducation versus behavioral intervention), and identifying the amount of treatment (e.g., number of sessions) needed before treatment response is expected. PMID- 8615406 TI - Children's memories in the wake of Challenger. AB - OBJECTIVE: The Challenger spacecraft explosion of Jan. 28, 1986, offered an opportunity to study the memories of normal latency and adolescent children of different emotional involvements following one sudden and distant disaster. How would children of various levels of concern express their memories? And if studied over time, how would these narratives change? Would there be developmental differences? And would there be false details of memory? METHOD: The authors set out to compare the memories of 153 children from Concord, N.H. (who watched the explosion on television), and Porterville, Calif. (who heard about it). The structured-interview responses of involved and less involved children; latency-age versus adolescent children; and those seen initially (5-7 weeks after the explosion) versus those same children seen later (at 14 months) were statistically compared. RESULTS: The vast majority of children's memories of Challenger were clear, consistent, and detailed, with highlighting of personal placement, who else was there, and personal occurrences linked to the event. Those children who were less emotionally involved demonstrated significantly less clarity, consistency, and correct ordering of sequences and were less likely to remember personal placement, other people who were there, and related personal incidents. About 30% of all children in this study misunderstood something about Challenger and incorporated these misunderstandings into their memories as false details. Latency-age children continued to harbor false details for 14 months, as opposed to the adolescents. CONCLUSIONS: Childhood memories of the Challenger space shuttle explosion appeared predictable, were related to patterns of memory that have been observed following single, unrepeated traumas, and reflected age and stage differences. PMID- 8615407 TI - Relation of stressors and depressive symptoms to clinical progression of viral illness. AB - OBJECTIVE: The aim of this research was to determine whether and in whom stressors and depressive symptoms facilitate clinical recurrence of herpes simplex virus (HSV) and progression of HIV. METHOD: Meta-analytic techniques were used to review the relations of stressors and depressive symptoms to clinical recurrence of HSV in 16 published studies and to indicators of HIV progression in 19 published studies. The authors calculated average effect sizes, performed fixed effect and random effect inferential analyses, tested for heterogeneous findings, and identified potential moderating variables. RESULTS: Depressive symptoms were associated with a slightly increased risk of HSV recurrence and increased reports of HIV-related symptoms, whereas stressors were not. However, depressive symptoms were not associated with objective indicators of accelerated HIV progression. Stressor studies, especially those that ascertained population specific life events, found numerical and functional decrements in circulating natural killer cell populations. The candidate moderators identified include, for HSV recurrence, age, sex, and medication status, and for HIV-related symptoms, age, race, disease stage, and co-infection with HSV. CONCLUSIONS: Depressive symptoms, but not stressors, increase the risk of HSV recurrence generally. Depressive symptoms do not appear to accelerate HIV progression ubiquitously, although they are associated with increased reporting of HIV-related symptoms. Future studies that ascertain population-specific stressors should determine whether reductions in cytotoxic lymphocytes influence HIV disease progression. Moreover, researcher should investigate the role of the identified moderators and recognize psychoimmune moderators in existing and novel study groups. These analyses could confirm that certain individuals are especially susceptible to the effects on disease progression of stressors, depressive symptoms, or both. PMID- 8615408 TI - Occurrence, recognition, and outcome of psychological disorders in primary care. AB - OBJECTIVE: The authors' goal was to cross-validate the earlier finding of the Groningen Primary Care Study that recognition of psychological disorders was associated with better patient outcomes. METHOD: The 12-item General Health Questionnaire was used to screen 1,271 consecutive primary care patients. A stratified sample of 340 of these patients participated in the second-stage baseline series of interviews, which included the Composite International Diagnostic Interview, the occupational role section of the Social Disability Schedule, the 28-item General Health Questionnaire, and the SCL-90. Three months later 209 of the patients completed the 28-item General Health Questionnaire and the SCL-90, and 12 months later 213 of the patients completed the second-stage baseline series of interviews. The study was carried out in six primary care practices (11 general practitioners) in the northern part of The Netherlands. RESULTS: Recognition of psychological disorders was associated with higher initial severity of psychopathology and occupational disability and with a psychological reason for the medical encounter. Recognition rates were higher for anxiety than for depression. Patients whose psychological disorders were recognized did not have better outcomes than those whose psychological disorders were not recognized. CONCLUSIONS: Recognition of psychological disorders was not associated with better outcome. Recognition is a necessary but not a sufficient condition for delivery of treatment according to clinical guidelines. Increasing recognition is likely to improve outcomes only if general practitioners have the skills and resources to deliver adequate interventions. PMID- 8615409 TI - Description and evaluation of the Iowa Depression Awareness, Recognition, and Treatment Program. AB - OBJECTIVE: The National Institute of Mental Health developed the Depression Awareness, Recognition, and Treatment Program to provide up-to-date information and training to health, mental health, and social service professionals regarding the identification and treatment of depression. This study was undertaken to evaluate a series of these programs for professionals who provide services to rural residents in the Midwest. METHOD: The 18 2-day training programs were attended by a total of 1,221 participants, physicians, psychologists, social workers, and nurses. Participants' knowledge regarding depression was assessed both before and after each program. Practice characteristics and perceived ability to assess and treat depression were assessed. Finally, 6-month follow-up evaluations of the usefulness of the training to the participants were undertaken. RESULTS: Following the programs, participants evidenced significant increases in levels of knowledge of depression and a high degree of satisfaction with most elements of the program. Six-month follow-up evaluations indicated a continued positive evaluation of the program. CONCLUSIONS: These outcomes suggest that the goals of the Depression Awareness, Recognition, and Treatment Program were met and provide support for the wider dissemination of these training programs. PMID- 8615410 TI - The Melbourne Family Grief Study, I: Perceptions of family functioning in bereavement. AB - OBJECTIVE: The aim of this study was to identify patterns of family functioning in adult families after the death of a parent. METHOD: One hundred fifteen families completed measures of family functioning, grief, psychological state, and social adjustment 6 weeks (time 1), 6 months (time 2), and 13 months (time 3) after the death of a parent (a total of 670 individual responses). Cluster analytic methods were applied to develop a typology of perceptions of family functioning during bereavement. RESULTS: Five types of families emerged from dimensions of cohesiveness, conflict, and expressiveness on the Family Environment Scale. Thirty-six percent of the families were considered supportive because of their high cohesiveness, and another 23% resolved conflict effectively. Two types were dysfunctional: hostile families, distinguished by high conflict, low cohesiveness, and poor expressiveness, and sullen families, who had more moderate limitations in these three areas; they declined in frequency from 30% at time 1 to 15% at time 3. The remaining type (26%), termed intermediate, exhibited midrange cohesiveness, low control, and low achievement orientation. The typology at time 1 predicted typologies at time 2 and time 3. There were no age or gender differences among the family types, but offspring, as compared with spouses, were overrepresented in the hostile families. CONCLUSIONS: Family types can be identified, allowing at-risk families to be helped to prevent complications of grief. Screening with the family relationship index of the Family Environment Scale would facilitate such a family-centered approach. PMID- 8615411 TI - The Melbourne Family Grief Study, II: Psychosocial morbidity and grief in bereaved families. AB - OBJECTIVE: The aim of this study was to describe the intensity of grief, the psychosocial morbidity, and the coping patterns in members of families classified according to a typology of family functioning comprising supportive, conflict resolving, intermediate, sullen, and hostile classes. METHOD: One hundred fifteen families were assessed longitudinally 6 weeks (time 1), 6 months (time 2), and 13 months (time 3) after the death of a parent (constituting 670 individual responses) on measures of grief intensity, psychological state, social adjustment, and family coping. A previously described typology of perceptions of family functioning was applied. Repeated measures multivariate analysis of variance based on both individuals and families and post hoc comparisons of significant results were undertaken. RESULTS: Sullen families displayed the most intense grief and the most severe psychosocial morbidity. Well-functioning families (supportive and conflict-resolving) resolved their grief and adjusted more adaptively than their dysfunctional counterparts (intermediate, sullen, and hostile families). There were no cluster-by-time interactions. The clusters accounted for 15.7% of the variance in depression (Beck Depression Inventory) and 27.9% of the variance in social functioning (Social Adjustment Scale). Well functioning families used more family coping strategies (Family Crisis Oriented Personal Evaluation Scales). CONCLUSIONS: More intense grief and greater psychosocial morbidity are found in sullen, hostile, and intermediate bereaved families than in the more adaptive supportive and conflict-resolving types. At risk families are identifiable and could be treated preventively to reduce morbidity. PMID- 8615412 TI - Suicide among subjects with personality disorders. AB - OBJECTIVE: There have been few psychological autopsy studies of suicide among individuals with personality disorders. The possible specificity of characteristics of suicide among such individuals has been little investigated. METHOD: A random sample of 229 subjects who committed suicide, representing all suicide victims in Finland within a 12-month period, were comprehensively examined by using the psychological autopsy method and were diagnosed according to DSM-III-R criteria. Within this random sample the authors investigated all subjects with axis II personality disorders (N = 67) and divided them into clusters B (N = 43), C (N = 23), and A (N = 1). Individuals with clusters B and C personality disorders were separately compared with sex- and age-matched suicide victims without personality disorder, in terms of sociodemographic characteristics, comorbid axis I and III syndromes, treatment histories, previous suicide attempts, communication of suicide intent, and suicide methods. RESULTS: All suicide victims with a personality disorder received at least one axis I diagnosis. In 95% this included a depressive syndrome, a psychoactive substance use disorder, or both. Individuals with cluster B personality disorders were more likely than comparison subjects to have psychoactive substance use disorders (79% versus 40%) and previous nonfatal suicide attempts (70% versus 37%) and were less likely to have axis III physical disorders (29% versus 50%). Subjects with cluster C personality disorders were not found to differ from the control subjects in any of the variables examined. CONCLUSIONS: Suicide victims with personality disorders were almost always found to have had current depressive syndromes, psychoactive substance use disorders, or both. Suicide victims with cluster B personality disorders differed from other suicide victims in several characteristics, while those with cluster C personality disorders did not. PMID- 8615413 TI - Suicidality among patients with mixed and manic bipolar disorder. AB - OBJECTIVE: Patients with mixed or manic bipolar disorder were studied to determine whether suicidality in mania is associated with increasing depression in general (i.e., a dimensional construct based on symptom severity) or with the presence of a mixed state per se (i.e., a categorical construct requiring the presence of a full depressive syndrome). METHOD: Diagnostic (categorical) assessments of 91 consecutively hospitalized patients with DSM-III-R mixed or manic bipolar disorder were made by using the Structural Clinical Interview for DSM-III-R. Symptom (dimensional) assessments were made by using the Hamilton Depression Rating Scale, the Young Mania Rating Scale, and the Scale of the Assessment of Positive Symptoms. Logistic regression models were employed to test the relative associations between suicidality and dimensional and categorical depression measures. Receiver operating characteristics analysis was performed to identify a potential threshold depression rating that predicted suicidality. RESULTS: Suicidality was more common in mixed than manic bipolar disorder. However, when adjusted for total depression scores, this association with affective state did not persist, but the statistical model significantly improved. In the improved model, depressive symptom ratings were significantly associated with suicidality. Hamilton depression scale ratings greater than 18 identified over 90% of the patients with suicidality, although there was also a high rate of false positives (32%). CONCLUSIONS: It is the severity of concurrent depressive symptoms in mania, rather than the presence of a depressive syndrome per se (i.e., mixed state), that is associated with suicidality in bipolar patients. PMID- 8615414 TI - Psychiatric disorders in African American nursing home residents. AB - OBJECTIVE: High prevalence rates of psychiatric illness and high levels of behavioral disturbance have been reported in studies of nursing home residents; however, the populations evaluated have been predominantly Caucasian. The aims of the present study were to identify prevalence rates of psychiatric disorders and behavioral disturbances in a sample of African American nursing home residents. METHODS: The authors evaluated 106 African American nursing home residents, aged 65 and over, from a representative sample of nursing homes. The evaluation included informant interview with nursing home staff, cognitive assessment, and a psychiatric interview that included a physical and neurological examination. Consensus diagnoses were reached by using DSM-III-R criteria. RESULTS: Of the 106 subjects, 90% received at least one primary psychiatric diagnosis, and 71% had at least one behavioral problem; dementia was the most common psychiatric diagnosis (68%). Thirty -one percent of the patients were treated with neuroleptic medication; most of these patients received diagnoses of dementia or schizophrenia. Fifteen percent of the patients had been in physical restraints, which correlated with physical disability. CONCLUSIONS: The prevalence of psychiatric illness in this sample of African American nursing home residents is similar to that reported in previous studies with predominantly Caucasian populations. Behavioral disturbances, while commonly reported, were somewhat less frequent than reported in previous studies. PMID- 8615415 TI - Stability of diagnosis in schizophrenia. AB - OBJECTIVE: The authors investigated factors associated with change in diagnosis from schizophrenia to other disorders and from other disorders to schizophrenia, as well as the time elapsed before diagnostic change. METHODS: Using a longitudinal study design, they examined data collected over a 7-year period at an urban acute care psychiatric hospital. The subjects were 936 inpatients who had been hospitalized at least four times during the study period. Changes to and from a diagnosis of schizophrenia over the 7 years were investigated in relation to demographic variables, socioeconomic factors, and clinical features. RESULTS: Fifty-six (21.9%) of the 256 subjects with a diagnosis of schizophrenia at the beginning of the study received a different diagnosis during a subsequent hospitalization. Females and subjects of Hispanic origin were more likely to undergo a diagnostic change from schizophrenia. Two hundred twenty-three (32.8%) of the 680 subjects who initially had a diagnosis other than schizophrenia were later diagnosed with schizophrenia. Males and African Americans had significantly higher rates of change to a diagnosis of schizophrenia than females and other ethnic groups. In addition, socio-economic factors and clinical features were associated with a change in diagnosis from another disorder to schizophrenia. CONCLUSIONS: The diagnosis of schizophrenia, in current practice, is not static. Patients' characteristics interact with longitudinal clinical changes to produce shifts in diagnosis. Longitudinal follow-up is necessary to validate diagnoses. PMID- 8615416 TI - Selective deficits in visual perception and recognition in schizophrenia. AB - OBJECTIVE: The purpose of this study was to evaluate the performance of patients with schizophrenia on tests of visual discrimination and recognition of different stimulus features. METHOD: Thirteen medicated male schizophrenic patients and 13 normal comparison subjects were tested on four stimulus features: spatial frequency, pattern, location, and trajectory. Subjects had to make both discrimination and recognition judgments at three levels of stimulus disparity. RESULTS: The responses of the patient group were slower and less accurate than those of the comparison group on both the discrimination and recognition tasks. The patients were less accurate than the comparison subjects in processing spatial features of the stimuli, particularly trajectory, but were unimpaired in processing form attributes (high spatial frequencies and patterns). When the results of pattern and trajectory tasks were matched against the accuracy performance of the comparison group, the patients were less accurate on trajectory than on pattern judgments and less accurate on recognition than on discrimination performance. CONCLUSIONS: Schizophrenia may be accompanied by impaired visual spatial perception and representation. In schizophrenia, deficits in trajectory discrimination may reflect a disturbance of the dorsal pathway of the visual system, while disturbances of trajectory recognition performance may reflect a deficit in prefrontal systems involved in visual working memory operations. PMID- 8615417 TI - Exploration of failure on the subspecialty examination for child and adolescent psychiatry. AB - OBJECTIVE: The purposes of this study were to compare first-time performance on the oral examination for certification in child and adolescent psychiatry with first-time performance on the written and oral examinations for certification in general psychiatry and to identify factors that contribute to failure on the child and adolescent psychiatry oral examination. METHOD: To address the first question, two successive cohorts of first-time child and adolescent psychiatry candidates were identified, and chi-square analyses were used to explore the relationship between first-time performance on the different examinations. For the second question, a special checklist was developed and completed for all candidates who failed any section of the oral examination for child and adolescent psychiatry three successive times. RESULTS: There was no relationship between passing or failing the written and oral examinations for general psychiatry on the first attempt and passing or failing the child and adolescent psychiatry oral examination on the first attempt. The checklist results suggested that the failing candidates had difficulty in organizing and integrating case material. Other weaknesses were related to the specific sections of the oral examination. CONCLUSIONS: The results suggest that the knowledge and skills that are required to pass the child and adolescent psychiatry oral examination are different from those of the general psychiatry examination. The information about weaknesses should be of interest to general and child and adolescent psychiatric educators, candidates, and service delivery agencies. PMID- 8615418 TI - A 23-year-old woman with panic disorder treated with psychodynamic psychotherapy. PMID- 8615419 TI - Posttraumatic stress disorder in survivors of the Brooklyn Bridge shooting. AB - OBJECTIVE: The authors documented the frequency of posttraumatic stress disorder (PTSD) in civilian victims of urban terrorism. METHOD: A recent shooting attack on a van of Hasidic students provided a unique opportunity to document responses of survivors in this targeted group. Eleven of 14 survivors were compared with age-matched subjects on a variety of questionnaires and clinical evaluations. RESULTS: Of the 11 survivors, four were diagnosed with PTSD (all of whom also had concurrent major depressive disorder), one with major depressive disorder, and two with adjustment disorder. CONCLUSIONS: Findings are interpreted in the context of unique factors contributing to the heightened vulnerability of this group. PMID- 8615420 TI - Circadian pattern of acute, neuroleptic-induced dystonic reactions. AB - OBJECTIVE: This study examined the timing of acute dystonic reactions in 200 patients taking neuroleptic medication for the first time. METHOD: Two hundred patients received a twice-daily regimen of low-dose neuroleptic. RESULTS: Over 80% of the episodes of acute dystonia occurred between 12:00 noon and 11:00 p.m. The observed circadian variation was not accounted for by sleep, fatigue, or time elapsed from the last dose of medication. CONCLUSIONS: The finding of a diurnal pattern in one of the recognized side effects of neuroleptic medication suggests that the therapeutic efficacy of neuroleptics might similarly vary over the course of the day. PMID- 8615421 TI - Trends in pharmacotherapy of Schizoaffective and bipolar affective disorders: a 5 year naturalistic study. AB - OBJECTIVE: The authors' goal was to determine if the actual treatment of schizoaffective and bipolar affective disorders had changed in light of recent clinical drug trials that have suggested that valproate and carbamazepine may be equivalent in efficacy to lithium. METHOD: Medication utilization rates for each 6-month period from July 1, 1989, to June 30, 1994, were compiled from the clinical database of the Palo Alto Veterans Affairs Medical Center. RESULTS: The use of valproate and valproate plus lithium was negligible in 1989. by 1994, these medication regimens accounted for 25% of the standard antimanic treatments used for bipolar affective disorder and 38% of the treatments used for schizoaffective disorder. Regimens of carbamazepine and carbamazepine plus lithium dropped from 24% of antimanic treatments in 1989 to 18% in 1994. From 1989 to 1994, there was a decline in the rate of lithium monotherapy for treatment of bipolar affective disorder (from 84% to 43%) and schizoaffective disorder (from 100% to 53%). CONCLUSIONS: In the past 5 years, valproate monotherapy has increased as a percentage of total antimanic pharmacotherapies, while lithium monotherapy has declined. PMID- 8615422 TI - Use of methods from chaos theory to quantify a fundamental dysfunction in the behavioral organization of schizophrenic patients. AB - OBJECTIVE: This study aimed to quantify the complexity of behavioral sequences of patients with schizophrenia and comparison subjects by using methods from nonlinear dynamical systems theory. METHOD: A simple choice task consisting of predicting 500 random right or left appearances of a stimulus was used to obtain binary response sequences in 22 patients with schizophrenia and 16 comparison subjects. Dynamical entropy was measured and the fluctuation spectrum of local subsequence entropies calculated to quantify the degree of interdependency between consecutive responses of patients and comparison subjects. RESULTS: The response sequences generated by the schizophrenic patients exhibited a higher degree of interdependency than those of comparison subjects. Moreover, schizophrenic patients exhibited significantly less consistency in their response selection and ordering, characterized by a greater contribution of both highly perseverative and highly unpredictable subsequences of responses within a test session. CONCLUSIONS: The result of the biological abnormality underlying schizophrenia may not be a simple increase or decrease of neuropsychological or neurobiological functions. Instead, the observed abnormalities in behavioral patterns reflect a quantifiable dysregulation and disorganization of these functions. PMID- 8615424 TI - Dynamic susceptibility contrast MRI of regional cerebral blood volume in Alzheimer's disease. AB - OBJECTIVE: The purpose of this study was to investigate the potential effectiveness of dynamic susceptibility contrast magnetic resonance imaging (MRI) to discriminate elderly patients with Alzheimer's disease from normal matched comparison subjects. METHOD: Images of regional cerebral blood volume (CBV) were generated from echo-planar MRI with the dynamic susceptibility contrast method in 13 Alzheimer's disease patients and 13 comparison subjects group-matched on age and gender. RESULTS: Temporoparietal cerebral blood volume, expressed as a percentage of the cerebellum value, was reduced 17% bilaterally in the patients with Alzheimer's disease. Blood volume in sensorimotor regions was reduced only 8.5% in the patients. Discriminant function analysis based on left and right temporoparietal measures correctly classified 88.5% of the subjects as patients or comparison subjects. Temporoparietal CBV was reduced even in mildly affected Alzheimer's disease patients (Mini-Mental State scores > 24). CONCLUSIONS: Dynamic susceptibility contrast MRI of regional CBV is promising as a nonradioactive, potentially lower-cost alternative to other functional neuroimaging methods for evaluating Alzheimer's disease. PMID- 8615423 TI - Low plasma gamma-aminobutyric acid levels during the late luteal phase of women with premenstrual dysphoric disorder. AB - OBJECTIVE: Plasma gamma-aminobutyric acid (GABA) levels have been reported to be low in some patients with major depressive disorder. Premenstrual dysphoric disorder is often associated with major depressive disorder. Therefore, the authors sought to determine whether women with premenstrual dysphoric disorder with or without prior major depressive disorder also had low plasma GABA levels. METHOD: Plasma GABA levels were measured in 27 women with premenstrual dysphoric disorder and 21 comparison women during the the mid-follicular and late luteal phases of the menstrual cycle. RESULTS: In comparison women, plasma GABA levels increased from the mid-follicular to the late luteal phase. Women with premenstrual dysphoric disorder and a past history of major depressive disorder had low plasma GABA levels during both phases. In women with premenstrual dysphoric disorder but no past major depressive disorder, plasma GABA levels decreased from the nonsymptomatic, mid-follicular phase to the symptomatic, late luteal phase. CONCLUSIONS: Decreased GABA function may represent a common biological link between subtypes of depressive and premenstrual dysphoric disorders. A trait in major depressive disorder and a state-dependent decrease in premenstrual dysphoric disorder might imply a possible continuum between the two disorders. PMID- 8615425 TI - Seizure and transient SIADH associated with sertraline. PMID- 8615426 TI - Treatment of dysphoric mania with nefazodone. PMID- 8615427 TI - Nefazodone-induced carbamazepine toxicity. PMID- 8615428 TI - Neuroleptic-induced tardive dyskinesia. PMID- 8615429 TI - Risperidone-induced tardive dyskinesia. PMID- 8615430 TI - Risperidone and bullous pemphigoid. PMID- 8615431 TI - Agranulocytosis after addition of risperidone to clozapine treatment. PMID- 8615432 TI - Koro in an immigrant from Guinea with brief psychotic disorder. PMID- 8615433 TI - Venlafaxine for the treatment of chronic pain. PMID- 8615434 TI - Diabetic ketoacidosis from clozapine and lithium cotreatment. PMID- 8615435 TI - Clinical effects of clozapine on autistic disorder. PMID- 8615436 TI - Elevated clozapine plasma concentrations after fluvoxamine initiation. PMID- 8615437 TI - Pathophysiology of alcoholism. PMID- 8615438 TI - Pathophysiology of alcoholism. PMID- 8615439 TI - Pathophysiology of alcoholism. PMID- 8615440 TI - Day-to-day egg count fluctuation in Schistosoma mansoni infection and its operational implications. AB - In a study group of 183 people in a Schistosoma mansoni-endemic area in Burundi, stool examinations were performed with duplicate 25-mg Kato-Katz slides on seven occasions (days 1, 3, 5, 8, 10, 32, and 37). Point prevalences detected by single examinations of 25 mg and 50 mg of stool varied from 41.0% to 57.9% and from 55.7% to 63.9%, respectively. The cumulative prevalence for all seven measurements was 82.0%. The individual day-to-day variation in egg output was important. The majority of infections missed by the examination of single slides and specimens were light ones. The Kato-Katz method applied on a single stool specimen is more suitable for morbidity control, but less suitable for control of infection. When a precise quantitative diagnosis on the individual level is required, several measurements on different days are necessary. The data presented validate recently developed statistical models and charts predicting true prevalences. PMID- 8615441 TI - Detection of very low level Plasmodium falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan. AB - We have used the nested polymerase chain reaction (PCR) to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination. This revealed a substantial group of asymptomatic, submicroscopically patent infections within the population of a Sudanese village present throughout the year although clinical malaria episodes were almost entirely confined to the transmission season. In our September, January, April, and June surveys, the PCR-detected prevalences were 13%, 19%, 24%, and 19%, respectively. These figures reveal a much higher prevalence of dry season infection than previous microscopic surveys have indicated. Furthermore, 20% of a cohort of 79 individuals were healthy throughout the September to November transmission season but were PCR-positive for P. falciparum in a least one of a series of samples taken in the ensuing months. Levels of exposure to P. falciparum infection were therefore higher than was previously believed in this region, highlighting the fact that many individuals were infected but healthy for most of the year. The reservoir parasite population was thus larger and more stable than previously thought, a finding that is consistent with the high levels of genetic variation at polymorphic loci reported from analysis of P. falciparum parasites in this area. PMID- 8615442 TI - Seroepidemiology of La Crosse virus infection in humans in western North Carolina. AB - On the Cherokee Indian Reservation and surrounding area of western North Carolina, an area-wide serosurvey was conducted to determine the prevalence of neutralizing antibody to La Crosse (LAC) virus. A questionnaire was used to identify risk factors important in exposure to virus-infected mosquitoes in populations near the reservation. Of 1,008 serum samples tested, 9.6% were positive for LAC virus antibody. For samples solely collected from on (n = 311) or off (n = 697) the reservation, the prevalence of seropositive samples was 20.6% on the reservation and only 4.7% off the reservation. Seropositivity increased directly with age, indicating that transmission of LAC virus was highly endemic. Age and location residence (on versus off the reservation) were significant risk factors for exposure to LAC virus. Persons on the reservation were 5.5 times more likely to have been exposed to LAC virus than were people who reside off the reservation. An additive increase in risk of 1.5 times over each age group was found, so that the oldest age group ( > or = 75 years) was 7.5 times more likely to have been exposed to LAC virus than was the youngest age group ( < 1-14 years). PMID- 8615443 TI - La Crosse viremias in white-tailed deer and chipmunks exposed by injection or mosquito bite. AB - To further understand the role of wild mammals in the maintenance of La Crosse virus (LACV) in nature, we investigated the effects of inoculation method and virus source on the duration and amplitude of LACV viremia in vertebrate hosts. Earlier work suggested that deer are not sufficiently susceptible to LACV to play an important role in its maintenance. We re-evaluated the susceptibility of deer since subsequent studies showed that they constitute 65% of Aedes triseriatus blood meals, and thus would be exposed frequently to the virus. In our study, deer developed higher and longer viremia following exposure to LACV by infected Ae. triseriatus than those previously reported by inoculation with needle and syringe. However, susceptible Ae. triseriatus that fed on these viremic animals did not become infected. Because a large number of uninfected mosquitoes can feed upon a viremic deer in nature, we believe that deer should not be disregarded completely as a possible amplifier in the LACV transmission cycle. We also infected chipmunks to determine if there were significant differences in viremia response from mosquito delivery of virus to the chipmunk host, compared with artificial exposure by injection. Chipmunks exposed to infected mosquitoes had higher and longer viremias than the ones produced by intramuscular injection of an LACV suspension. These findings show the importance of using LACV infected mosquitoes for transmission experiments in mammals. PMID- 8615444 TI - The pattern of pediatric solid malignant tumors in western Kenya, east Africa, 1979-1994: an analysis based on histopathologic study. AB - This study analyzed histopathologic specimens of 600 pediatric solid malignant tumors seen during the period 1979-1994 at the histopathology laboratories of the Rift Valley Provincial General Hospital in Nakuru, the Nyanza Provincial General Hospital in Kisumu, and the Uasin Gishu Hospital in Eldoret in western Kenya. The crude incidence rate of each malignancy per 100,000 children per year was calculated. The patterns of malignancies were examined with a focus on tumor incidence, age, sex, geographic, and ethnic distribution to relate the tumors to putative environmental and genetic causative factors. The six common tumors were Burkitt's lymphoma (33.5%), non-Hodgkin's lymphoma (21.8%), retinoblastoma (11.5%), Kaposi's sarcoma (6.1%), nephroblastoma (4.5%), and Hodgkin's disease (4.1%). Significantly high crude incidence rates for lymphomas and Kaposi's sarcoma showed a characteristic ethnogeographic distribution. The majority of the tumors were found concentrated around Lake Victoria and showed decreasing occurrence as one moved towards the semi-arid and highland areas. We concluded that environmental factors seem to play a major role in childhood tumors in western Kenya. PMID- 8615445 TI - Fluctuation of schistosome circulating antigen levels in urine of individuals with Schistosoma mansoni infection in Burundi. AB - We studied the fluctuations of schistosome circulating antigens in urine as compared with fecal egg counts in 60 Burundese individuals infected with Schistosoma mansoni. Levels of circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in the urine were determined by quantitative enzyme-linked immunosorbent assays. Fecal samples were simultaneously collected and examined with duplicate Kato-Katz slides. Significant correlations were consistently found between circulating antigen levels in urine and fecal egg counts. Although both antigen levels and egg output fluctuated, there was less fluctuation of CCA levels in urine than of fecal egg counts. All individuals had CCA in at least one urine sample and 82% were at least once positive for egg counts. Positive CCA levels were found in at least one urine sample in 75% of all individuals, but levels were low. Our results show that detection of CCA in urine is a sensitive, quantitative, and reliable method for noninvasive diagnosis and screening of S. mansoni infections, due to the relatively low fluctuations. PMID- 8615446 TI - Field trial of the coproantigen-based diagnosis of Taenia solium taeniasis by enzyme-linked immunosorbent assay. AB - A microplate-type enzyme-linked immunosorbent assay for the detection of Taenia species antigen in human feces was tested in field studies undertaken in two Guatemalan communities. The test was based on immunoglobulin G antibodies from a rabbit hyperimmunized to Taenia solium proglottides. Comparison was made with microscopy and patient interviews as a means of diagnosis. The coproantigen test result was positive in 79 of the 1,582 fecal samples examined. Parasitologic confirmation was made in 55 of these cases. The coproantigen test was the most sensitive technique used, detecting 2.6 times as many confirmed cases of taeniasis as microscopy, which diagnosed 21 cases. Only one case was detected by interviewing. Microscopy revealed on false-negative coproantigen result. Mass treatment of the population did not result in the detection of any additional cases. Twelve coproantigen-positive results were categorized as unconfirmed and an additional 12 as putative false-positive results, giving an overall specificity of 99.2% for the coproantigen test. Of the 34 taeniid tapeworms identified to the species level, all were T. solium. The practicalities of the use of such a test in epidemiologic studies on human taeniasis are discussed. PMID- 8615448 TI - A rapid, highly sensitive method for the detection of Francisella tularensis in clinical samples using the polymerase chain reaction. AB - We have developed a highly sensitive method for detection of Francisella tularensis in clinical samples based on a nested polymerase chain reaction (PCR) for the FopA gene. Mice infected with F. tularensis were killed at 24-hr intervals, and the DNA from blood and spleens was extracted by a variety of methods and analyzed by PCR. The best method, based on the ability of DNA to bind to silica in the presence of guanidine thiocyanate, yielded amplifiable DNA without dilution of the murine tissue samples. Francisella tularensis in infected murine spleens and culture-positive blood samples was reliably detected by nested PCR following this extraction procedure. We believe this technique has significant advantages over traditional methods for diagnosing F. tularensis infection in terms of speed, ease of use, reproducibility, and safety. PMID- 8615447 TI - A polymerase chain reaction assay for detection of the parasite Wuchereria bancrofti in human blood samples. AB - To identify Wuchereria bancrofti DNA sequences that could be used as the basis for a simple and rapid parasite detection assay, a genomic library of W. bancrofti was constructed and screened for highly repeated DNA. The repeat found with the highest copy number was 195 basepairs (bps) long, 77% AT, and 300 copies per haploid genome. This sequence was designated the Ssp I repeat because it has a unique recognition site for that restriction endonuclease in all or most of the repeat copies. The Ssp I repeat DNA family is dispersed, genus-specific, and exists in all of the different geographic isolates of W. bancrofti tested. Based on DNA sequence analysis of this repeat, we have developed an assay to detect very small quantities of W. bancrofti DNA using the polymerase chain reaction (PCR). With this PCR assay, the Ssp I repeat was detected in as little as 1 pg of w. bancrofti genomic DNA (about 1% of the DNA in one microfilaria) added to 100 microliters of human blood. The PCR assay also amplified Ssp I repeat DNA from geographic isolates of W. bancrofti from around the world but not from other species of filariae or from human or mosquito DNA. Microfilaria-positive human blood samples collected in Mauke, Cook Islands were shown to be Ssp I PCR positive, while microfilaria-negative samples were PCR-negative. The specificity and sensitivity of the Ssp I PCR assay indicates that this approach has significant potential for improved screening of large human populations for active W. bancrofti infection. PMID- 8615449 TI - Hantavirus antigen detection using human serum immunoglobulin M as the capturing antibody in an enzyme-linked immunosorbent assay. AB - An enzyme-linked immunosorbent assay (ELISA) was developed to detect different hantavirus antigens in cell culture; i.e. Puumala (PUU), Hantaan (HTN), and Dobrava (DOB) viruses. The assay was based on binding human serum immunoglobulin M (IgM) antibodies to the solid phase by use of goat anti-IgM antibodies. The captured IgM antibodies were present in the acute phase serum from two patients: one infected in Sweden and the other in Bosnia. Antigens being bound to the solid phase by the human anti-PUU and anti-DOB/HTN IgM antibodies were detected by a broadly reacting polyclonal rabbit anti PUU-recombinant nucleocapsid protein antiserum. The IgM isotype was proven to be at least five times more efficient than IgG when used as the capturing antibody. The sensitivity of the PUU antigen ELISA was approximately 0.5 ng/ml, as measured by titration with a PUU recombinant nucleoprotein antigen. Cell-associated PUU antigen in tissue culture was seen after 48 hr by the PUU-ELISA and after 96 hr by immunofluorescent assay. When tested for capacity to discriminate between PUU, DOB, and HTN viruses, significant differences were found: the Swedish serum detected PUU antigen at high titers, whereas no reactivity was found against DOB and HTN; the Bosnian serum detected both DOB and HTN at high titers but had a low reactivity to PUU. The method was also tested for its usefulness in detecting PUU antigen in bank vole (clethrionomys glareolus) lungs. Of 59 animals captured from the surroundings of patients with nephropathia epidemica, three became positive with a high activity in the PUU-ELISA, but with low reactivity in the DOB/HTN-ELISA. It is concluded that a sensitive ELISA has been developed to detect different hantaviruses in cell culture and lungs of bank voles. PMID- 8615450 TI - The Santa Lucia strain of Plasmodium falciparum as a model for vaccine studies. I. Development in Aotus lemurinus griseimembra monkeys. AB - The Santa Lucia strain of Plasmodium falciparum and the Aotus lemurinus griseimembra monkey are proposed as models for the testing of sporozoite vaccines and transmission-blocking vaccines. Approximately 85% of splenectomized monkeys were infected when fed upon by 10 or more heavily infected Anopheles freeborni mosquitoes. Sporozoite-induced infections in monkeys with or without previous infection with P. vivax readily infected mosquitoes, thus making them candidates for testing transmission-blocking vaccines. PMID- 8615451 TI - The Santa Lucia strain of Plasmodium falciparum as a model for vaccine studies. II. Development of Aotus vociferans as a model for testing transmission-blocking vaccines. AB - The Santa Lucia strain of Plasmodium falciparum and the Aotus vociferans monkey were studied as models for the testing of transmission-blocking vaccines. Virulence developed early in the passage history. Despite the use of only small quantities of chlorguanide and/or quinine to control infection coupled with the use of small inocula and delays in splenectomy, mosquito infection was markedly reduced from that seen during primary passage to this species of Aotus. It appears that the model may be most useful during its initial passage from the primary species, Aotus lemurinus griseimembra. PMID- 8615452 TI - Brugia pahangi-induced contractility of bovine mesenteric lymphatics studied in vitro: a role for filarial factors in the development of lymphedema? AB - Products generated by filarial nematodes depress vascular reactivity by mechanisms involving endothelial cells. We hypothesized that comparable filarial induced alterations might occur in lymph vessels. Experiments were designed to test the hypothesis that spontaneous contractions of bovine mesenteric lymphatics studied in vitro are altered by the filarial parasite Brugia pahangi. Rings of bovine mesenteric lymphatics were suspended in tissue baths and spontaneous contractions were evaluated for rate, rhythm, and amplitude. Rings that met inclusion criteria (rate > 1.8/min, regular rhythm, and an amplitude > 500 mg) were randomly exposed to B. pahangi or used as controls. Parasites were added to the bath at time zero. Changes in rate, rhythm, and magnitude of spontaneous contractions were evaluated every 10 min. Comparisons were made within control or Brugia-infected groups over time and between groups (B. pahangi versus controls). The presence of B. pahangi significantly depressed the frequency of spontaneous contractions when compared with controls. Control rings were stable over time, without changes in rate, rhythm, or amplitude. However, B. pahangi alter both the rate and rhythm of spontaneous contractions. Since spontaneous contractile activity is likely to be important in the propulsion of lymph, alterations of contractile activity could result in lymphedema. Thus, filarial factors may be responsible, in part, for altered lymphatic function seen in lymphedema. Pharmacologic intervention aimed toward influencing host-parasite metabolic interactions may, in this complicated scenario, prove useful. PMID- 8615453 TI - Effective, single-dose treatment or porcine cysticercosis with oxfendazole. AB - The pig is a vital link in the transmission cycle of Taenia solium, the cestode responsible for human-porcine cysticercosis. Infected pigs also represent an important source of economic loss to farmers in developing countries. Past efforts to find an adequate therapeutic regimen to treat this parasite disease in swine have failed because of low efficacy, high cost, side effects, or the need for multiple doses. In this randomized, no treatment-controlled study, the efficacy and safety of oxfendazole and praziquantel for the treatment of porcine cysticercosis were evaluated in 16 naturally infected pigs. Four groups of four pigs were treated with oxfendazole, praziquantel, oxfendazole plus praziquantel, or untreated. The pigs were humanely killed 12 weeks post-treatment, the number of cyst was counted, and parasite viability was assessed by cyst evagination. No detectable side effects were seen in any of the pigs. Praziquantel treatment alone appeared to reduce the number of cysts, but did not decrease the viability of the remaining parasites. Treatment with oxfendazole alone or oxfendazole plus praziquantel killed all of the parasites, and left only microcalcifications in the meat. Oxfendazole provides, for the first time, a practical, effective, inexpensive, and single-dose therapy for porcine cysticercosis. PMID- 8615454 TI - DNA probes for the Anopheles punctulatus complex. AB - Genomic DNA probes were made for two recently identified members of the Anopheles punctulatus complex; Anopheles sp. near punctulatus from Papua New Guinea and Anopheles farauti No. 7 from the Solomon Islands. The probes are species-specific and with the use of 32P labeling sensitive enough so that a squash blot of only a small segment of the mosquito is required for identification. The 119-basepair (bp) probe for An. sp. near punctulatus and the 1,106-bp probe for An. farauti No. 7 have been sequenced in full and the probes have been tested on field collected specimens. These probes now make it possible to distinguish An. sp. near punctulatus and An. farauti No. 7 from the other eight members of the An. punctulatus complex. A pan-species probe was also made from the 18S ribosomal DNA that binds to DNA from all members of the complex. These three probes complete the set required for distinguishing all known members of the An. punctulatus complex by DNA hybridization. PMID- 8615455 TI - Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus). AB - Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years. PMID- 8615456 TI - Analysis and ultrastructural localization of Ehrlichia chaffeensis proteins with monoclonal antibodies. AB - Ehrlichia chaffeensis, an obligately intracellular bacterium with tropism for monocytes, is the etiologic agent of human monocytic ehrlichiosis. To determine the nature and ultrastructural location of E. chaffeensis antigens, monoclonal antibodies (MAbs) to E. chaffeensis were developed. The MAbs were used for immunofluorescence and Western immunoblotting analysis of the antigens of density gradient-purified ehrlichiae. Monoclonal antibody 6A1 recognized an epitope of a 30-kD protein. This antibody reacted with a strain-specific epitope of E. chaffeensis, Arkansas strain, and did not cross-react with any other ehrlichia tested. Monoclonal antibodies 3C7 and 7C1-B recognized Arkansas strain proteins of 30 and 29 kD and reacted with E. chaffeensis (strain 91HE17) proteins of 31 and 29 kD and an E. canis protein of 30 kD. Lack of reactivity of these two MAbs with E. sennetsu and E. risticii suggests that the epitope is group-specific. Monoclonal antibody 5D11 recognized a 58-kD protein of both strains of E. chaffeensis as well as E. canis, apparently a group-specific, conformation independent epitope. Monoclonal antibody 7C1-C reacted with 58- and 88-kD proteins of both the Arkansas and 91HE17 strains. Trypsin treatment destroyed the reactivity of E. chaffeensis antigens with all the MAbs when tested by Western immunoblotting, indicating that these antigens are proteins with trypsin sensitive epitopes. Immunoelectron microscopy of negatively stained intact E. chaffeensis organisms showed that the 30- and 29-kD proteins are present on the surface of the ehrlichial cell wall along with the previously localized 28-kD protein. PMID- 8615457 TI - Genotypic identification of murine typhus Rickettsia in rats and their fleas in an endemic area of Greece by the polymerase chain reaction and restriction fragment length polymorphism. AB - Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe. PMID- 8615458 TI - Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi. AB - Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest by Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme '1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications. PMID- 8615459 TI - Leishmania RNA viruses in Leishmania of the Viannia subgenus. AB - Karyotype analysis of 69 strains of Leishmania belonging to three species of the Viannia subgenus originating from the southeastern and southwestern regions of Colombia revealed approximately 5.3-kb RNAs in four strains of L. braziliensis and also in the World Health Organization reference strain L. guyanensis IWHI/BR/78/M5313. The RNA element in this reference strain and in L. braziliensis strains isolated from cutaneous and mucosal lesions of four patients hybridized with RNA probes prepared from cDNA of the RNA virus present in L. guyanensis strain CUMC-1-1A (LRV1-1). These strains also contained an 80-kD protein that reacted with polyclonal antibody prepared against a recombinant fragment of the coat (capsid) protein of LRV1-1. In addition, another Colombian strain of L. braziliensis was found to contain an approximately 3.5-kb RNA that did not hybridize with LRV1-1 probes. Contrasting with the strains containing the 5.3-kb RNA, a total lysate of this strain did not contain material reactive with antiserum to the capsid protein fragment. All Leishmania containing LRV1-related viruses identified to date have originated in the Amazon River basin. Karyotype analyses and biological characterization of 17 clones obtained from the highly metastatic L. guyanensis strain 5313 revealed retention of the approximately 5.3 kb RNA in all clones and no segregation of the virus with the metastatic trait. The restricted distribution of LRV1-related viruses among some strains of L. braziliensis and L. guyanensis circulating in the Amazon River basin makes these elements potential epidemiologic markers. PMID- 8615460 TI - Quantification of Plasmodium falciparum sporozoites by ribosomal RNA detection. AB - We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed. PMID- 8615461 TI - What about old surgeons? PMID- 8615462 TI - Pancreas Club Meeting. May 14, 1995 San Diego, California. PMID- 8615463 TI - Blood-flow augmentation of intermittent pneumatic compression systems used for prevention of deep vein thrombosis prior to surgery. AB - PURPOSE: To compare, using Duplex ultrasonography, different intermittent pneumatic compression (IPC) systems to augment venous blood flow for deep venous thrombosis (DVT) prevention during and after surgery and during periods of immobility. METHODS: This cross-over study randomly assigned 26 young, healthy, adult subjects, without history of DVT, hypertension, diabetes, stroke. vascular or cardiac pathologies, to an order of knee-high, foam, single-pulse IPC device and thigh-high, vinyl, sequential-pulse pneumatic compression systems. Prior to making the flow measurement, the girth of the calf and thigh and length of the leg of each subject were determined. The right leg was used in this evaluation. RESULTS: The average flow augmentation, which is a direct measure of the amount of femoral vein blood flow velocity increase over the base, was 107%+/-49% with the knee-high system, and 77%+/-35% with the thigh-high IPC system (P<0.002). Augmentation was higher for 62% of the subjects with knee-high IPC, and for 23% of the subjects with the thigh-high system. Overall, the blood was actively moving through the vein during the decompression phase. On occasion, the velocity during the decompression phase would fall to zero for short intervals with both systems, indicating complete emptying of the vessel. Variation in limb anatomy did not significantly affect blood-flow augmentation with the knee-high IPC, but augmentation decreased with increase in girth with the thigh-high IPC. CONCLUSIONS: The study indicates that the knee-high, foam, single-pulse IPC device produces a significantly higher venous blood-flow augmentation than the thigh-high, vinyl, sequential-pulse system. PMID- 8615464 TI - The effect of intravenous erythromycin on esophageal motility in healthy subjects. AB - BACKGROUND: It has been confirmed that erythromycin has gastrokinetic properties of enhancing gastric emptying both in health and disease. The objective of the present study was to investigate any possible effect of erythromycin on esophageal motility. METHODS: In 14 healthy subjects, standard esophageal manometry was performed before and after the intravenous administration of 200 mg of erythromycin. The calculated manometric parameters of esophageal motility were the lower esophageal sphincter (LES) pressure; the amplitude and duration of peristalsis at 5, 10, and 15 cm proximal to the LES; and the velocity and strength of peristalsis at 5 cm proximal to the LES. RESULTS: Erythromycin significantly increased the LES pressure (P<0.001), and the amplitude (P=0.002), duration (P=0.003), strength (P=0.014) and velocity (P=0.008) of peristalsis at 5 cm proximal to LES. Erythromycin also increased the amplitude of peristalsis at 10 cm proximal to the LES (P=0.035). CONCLUSION: Erythromycin affects the motility of the distal esophagus. PMID- 8615466 TI - A cost analysis of autologous and allogeneic transfusions in hip-replacement surgery. AB - PURPOSE: To analyze the cost consequences of autologous versus allogeneic transfusions. METHODS: Costs were determined when allogeneic transfusions were given in addition to, or instead of, autologous transfusions. Hospital charges were used to estimate costs for hip-replacement surgery. The main outcome measure was estimated incremental hospital costs per unit transfused. RESULTS: Among donors of autologous blood, mean total charges were $7,200 greater for recipients of both autologous and allogeneic transfusions than for recipients of autologous transfusion only (P=0.0001). Each allogeneic transfusion was associated with additional costs of $1,480. In a second cohort of patients receiving identical amounts of either allogeneic or autologous blood (mean=2.3 units), total hospital charges were a mean of $4,800 greater (P=0.0001) for allogeneic recipients. The per unit excess costs associated with each unit of allogeneic blood cohort were $1,043. CONCLUSIONS: Allogeneic transfusions are associated with incremental hospital costs of about $1,000 to $1,500 per unit transfused when compared with costs for similar patients receiving no transfusions or 1 to 5 units of autologous blood. PMID- 8615465 TI - Stapled ileal pouch anal anastomoses are safer than handsewn anastomoses in patients with ulcerative colitis. AB - BACKGROUND: One of the theoretic advantages of using a stapled versus handsewn ileal pouch anal anastomosis (IPAA) in restorative proctocolectomy is a reduction in septic complications. We performed this study to compare the incidence of early septic complications in patients undergoing restorative proctocolectomy with stapled or handsewn IPAA. PATIENTS AND METHODS: A chart review of 692 patients undergoing restorative proctocolectomy for treatment of ulcerative colitis was performed. The incidence of early septic complications in patients having stapled IPAA was compared to that in patients having handsewn IPAA. Follow up studies included an annual questionnaire and physical examination. RESULTS: Of the 692 patients, 238 had handsewn IPAA and 454 had stapled IPAA; these two groups were similar in sex, duration of disease, age at surgery, and type of surgical procedure performed. In the handsewn IPAA group, 25 patients (10.5%) had 32 septic complications, and 24 required 89 reparations. In 7 patients, the pouch was excised. In the stapled IPAA group, 21 patients (4.6%) had 23 septic complications, and 14 required 40 reparations. One patient needed pouch excision. There were more patients (P=0.0001) with early septic complications, and more (P<0.0001) pouch excisions because of these complications, in patients with handsewn IPAA than in patients with stapled IPAA. The sepsis-related reoperation rates did not differ significantly. CONCLUSIONS: The stapled technique for IPAA has fewer septic complications and results in fewer sepsis-related pouch excisions, in our hands, than the handsewn technique technique, for treating patients with ulcerative colitis. PMID- 8615467 TI - A clinical trial of wound closure by constant tension approximation. AB - BACKGROUND: Wounds that heal slowly may pass through a subacute phase and proceed to reach a chronic situation. The latter may heal slowly, if at all. These wounds often heal by secondary intention with a single layer of epithelium that has a tendency to break down. Methods that expedite healing by full-thickness skin usually involve operative procedures. A nonoperative method of achieving coverage of tardy wounds with full-thickness skin, partial or complete depending on the wound, would be valuable. PATIENTS AND METHODS: A device has been designed that approximates the wound margins by applying constant low-grade tension over a period of days or weeks. One or more devices have been applied to the wounds of 25 patients to date. RESULTS: Healing has been obtained in those patients (20) where the devices were used optimally. In 5 patients, optimal use was not possible, mostly for socioeconomic reasons; the latter included noncompliance, insurance problems, economic difficulties, and personal considerations. In these 5 cases, healing was obtained in the 3 that were available for follow-up, full thickness skin coverage being estimated by grid photography to be in the 80% to 90% range. One patient was lost to follow-up after the wound was 95% covered by full-thickness skin. One patient whose wound reduced in size declined further treatment after 12 days of treatment. CONCLUSIONS: It appears that when constant tension, low-grade force is applied to subacute and chronic wounds, healing is accelerated and, depending on the wound, a considerable number of ulcers can be closed by full-thickness skin. PMID- 8615468 TI - Constituent analysis may permit improved diagnosis of intra-abdominal abscess. AB - BACKGROUND: Intra-abdominal abscesses (IAA) often fail to resolve with intravenous antibiotics alone and frequently require drainage. Diagnosis of IAA in postoperative patients with other likely sources of infection is very difficult. PATIENTS AND METHODS: In order to characterize IAA and identify parameters that might facilitate diagnosis, we prospectively examined peripheral blood and pus of 15 consecutive patients with IAA and compared them to samples from 34 consecutive patients with soft-tissue abscesses (STA). RESULTS: Serum interleukin (IL)-10 was elevated in IAA patients, while abnormally detectable serum IL-4 was demonstrated in the pus of both IAA and STA patients. IL-10 in IAA pus was more than 11-fold higher than in STA, whereas IL-4 in pus was similar in both types of abscesses. Both IL-4 and IL-10 were 4- to 10-fold higher in IAA and STA pus than in corresponding patient serum. Serum lysozyme was, however, significantly elevated in all abscess patients. CONCLUSIONS: The presence of IL-4 and IL-10 may indicate a T-helper 2 lymphocyte response in the etiology of abscess formation and persistence, although precise determination of T-helper 1 related cytokines is needed to verify this. Serum lysozyme and IL-10 may be reliable and relatively inexpensive diagnostic aids. PMID- 8615469 TI - Protective effect of nitric oxide in an endotoxin-induced septic shock. AB - BACKGROUND: Calcium ion (Ca++)-independent nitric oxide (NO) synthase activity in animals was markedly induced by treatment with endotoxin, but NO levels in various tissues removed from endotoxin-treated animals have not been reported. The role of NO during an endotoxin-induced septic shock remains controversial. METHODS: ICR mice, randomly divided into one of six treatment groups, received intraperitoneal injections as follows: phosphate-buffered saline; Escherichia coli LPS (LPS); N(omega)-nitro-L-arginine (L-NNA); N(omega)-nitro-D-arginine (D NNA); LPS plus L-NNA; and LPS plus D-NNA. The mice were either monitored for mortality or killed for nitrite/nitrate assays and histologic analysis. RESULTS: NO levels in many tissues were markedly increased by injection of LPS, and administration of L-NNA increased mortality rates of LPS-treated mice, in association with an increase in tissue damage in the lung, liver, and kidney. CONCLUSIONS: The endogenous NO generated during LPS-mediated septic shock could be protective. PMID- 8615470 TI - Prognostic evaluation of preoperative combined treatment for advanced cancer in the lower rectum with radiation, intraluminal hyperthermia, and 5-fluorouracil suppository. AB - BACKGROUND: Since 1977, we have studied preoperative treatments using 5 fluorouracil (5-FU) suppositories alone or in combination with radiation to reduce local recurrence and improve survival of patients with rectal cancer. PATIENTS AND METHODS: We developed a novel preoperative therapy combining radiation, intraluminal hyperthermia, and 5-FU suppositories. Thirty-five patients with rectal cancer underwent surgery after this treatment, and 41 patients underwent surgery without pretreatment. RESULTS: The patients who underwent preoperative combination treatment showed significant tumor reduction; 2 achieved microscopically complete regression. In comparison with the patients who underwent surgery without pretreatment, the preoperative treatment group had a 16.7% lower local recurrence rate (10.4% versus 27.1%) and an improved survival rate (81.8% versus 67.6%). CONCLUSIONS: Preoperative combination therapy is a promising modality for the patients with respectable advanced cancer in the lower rectum. PMID- 8615471 TI - Hepatic resection with vascular isolation and routine supraceliac aortic clamping. AB - BACKGROUND: Hepatic resection with total vascular isolation has been reported to reduce hemorrhage. Addition of supraceliac aortic clamping putatively avoids hemodynamic instability, but may increase morbidity. METHODS: This technique was used in 99 major liver resections utilizing scalpel division and suture hemostasis. RESULTS: Livers were normal in 86 patients, cirrhotic with no portal hypertension in 5, and cirrhotic with portal hypertension in 8. There was 1 death in 91 patients with no portal hypertension due to hepatic failure or bleeding esophageal varices. There were 59 hemihepatectomies and 40 segmentectomies. Median operating time was 145 and 110 minutes, respectively, and mean transfused blood was 4 and 0 units, respectively, with minimal morbidity. CONCLUSIONS: Use of total hepatic vascular isolation with routine supraceliac aortic clamping is a safe and expedient method of hepatic resection that limits blood loss and maintains hemodynamic stability, but does not increase morbidity. However, the presence of portal hypertension precludes safe resection. PMID- 8615472 TI - Results of needle localized breast biopsy in women under age 50. AB - BACKGROUND: The use of screening mammography in women aged 40 to 50 years is controversial because of a relative lack of data demonstrating survival benefit for this segment of the population. PATIENTS AND METHODS: The charts of 809 consecutive women who underwent needle localized breast biopsy over a 6 1/2-year period were reviewed to determine the effect of age on the biopsy results. Patient ages ranged from 27 to 91 years (mean 56). RESULTS: Two hundred nineteen (27%) of the 809 needle localized breast biopsies were malignant, with a mean tumor diameter of 1.46 cm. This procedure identified malignancy in 3 (5%) of 60 patients age 40, and in 32 (15%) of 207 patients aged 40 to 49 years. Malignancy was significantly more likely in patients 50 years of age or older and was found in 184 (34%) of the 542 (P<0.001). Overall, 33% of all patients undergoing the procedure were under age 50. CONCLUSIONS: Without screening mammography, the diagnosis of breast cancer would have been delayed in 32 women aged 40 to 50, 15% of the total diagnosed with nonpalpable breast cancer in this study. We recommend the use of screening mammography in this age group. PMID- 8615474 TI - Malignant tumors missed at laparoscopic cholecystectomy. AB - BACKGROUND: Laparoscopic cholecystectomy (LC) is now the treatment of choice for gallstone disease. The wide acceptance of LC resulted in increased cholecystectomy rates and entailed specific drawbacks such as missed malignant tumors of other organs. PATIENTS AND METHODS: The prospective follow-up of patients who underwent LC was studied, and all patients treated for malignant disease were included regarding a history of LC. RESULTS: Of 838 LCs performed, 5 patients underwent reoperation for missed carcinoma of the pancreas (n=2) and the right colon (n=3). Two other patients with carcinomas of the pancreas and the right colon had a history of LC performed elsewhere. All 7 patients (median age 72 years) complained of recent atypical pain at the time of the LC. Five tumors were resected (2 palliatively); 2 patients died. CONCLUSIONS: This study emphasizes the necessity of making a careful semiological analysis of the pain and associated symptoms before performing an LC. PMID- 8615473 TI - Evaluation of surgical resection for small hepatocellular carcinomas. AB - BACKGROUND: The surgical results for small hepatocellular carcinomas (HCCs) are not necessarily satisfactory. The resectional therapy for small HCC was evaluated to elucidate its benefits and limitations. PATIENTS AND METHODS: Data were analyzed concerning 52 patients having small HCC of <3 cm in diameter and <3 nodules, who underwent hepatic resections from 1978 to 1989. RESULTS: Cumulative and disease-free survival at 5 years after resection were 57% and 37%, respectively. Histologic features, such as differentiation of HCCs, pseudocapsular invasion, and ploidy pattern of nuclear DNA, appeared to be good prognostic indicators. The survival rate of the large-resection group (number of resected segments greater than number of tumor-occupying segments) was significantly better than that in the small-resection group (84% versus 48% at 5 years, P<0.05). CONCLUSIONS: Surgical resection of small HCCs is a safe and effective therapy. A large resection appears to provide better patient survival and tumor-free survival compared to a small resection. PMID- 8615475 TI - Laparoscopic repair of recurrent inguinal hernias. AB - BACKGROUND: Repair of recurrent inguinal hernias is associated with recurrence rates as high as 30% and complication rates higher than for primary hernias. PATIENTS AND METHODS: In a prospective study, results were evaluated after laparoscopic transabdominal preperitoneal hernia repair in 192 patients with 200 recurrent inguinal hernias. A total of 132 hernia repairs followed one previous repair, 41 followed two repairs, 17 followed three repairs, 6 followed four, 3 followed five, and 1 followed six previous repairs. The surgical technique is described. RESULTS: Follow-up ranged from 9 to 31 months (mean 18.4). Twelve patients (6%) had groin seromas or hematomas; 3 (1.5%) had transient thigh numbness. One patient (0.5%) underwent laparoscopy a second time because of a large hematoma. In 1 patient (0.5%), a staple on the n. cutaneus femoris lateralis was removed laparoscopically. Patients described postoperative pain as being much less severe compared with their previous operation. Of the total group, 76% of patients were able to return to work within 2 weeks of surgery. One recurrence (0.5%) occurred after 6 months because of too small a prosthetic mesh. CONCLUSIONS: This laparoscopic technique can be applied to recurrent hernias, even in difficult cases, with low morbidity rates. Recurrence rates as low as for laparoscopic repair of primary hernias can be expected. PMID- 8615477 TI - A simple technique for repositioning the intra-aortic balloon catheter associated with ipsilateral limb ischemia. AB - Occasionally, ipsilateral ischemia develops following the groin insertion of an intra-aortic balloon catheter. Various treatment options have evolved, and include replacing the catheter in the opposite groin, removing it completely, or performing a femorofemoral bypass to deliver blood flow below the catheter. Outlined in this paper is a simple method to restore blood flow to a threatened limb, during femoral artery exploration, in the presence of an intra-aortic balloon. This method is also appropriate for optimal positioning of the balloon catheter prior to femorofemoral bypass. PMID- 8615479 TI - Management of adhesive small-bowel obstruction. PMID- 8615478 TI - Fungal infection in surgical patients. AB - Invasive fungal infections have become a major source of morbidity and mortality in the modern surgical intensive care unit. Patients at risk for invasion and dissemination are common, and are not as ill as thought previously. Severity of illness (APACHE II score > 10, ventilator use for >48 hours), antibiotics, central venous lines, total parenteral nutrition, burns, and immunosuppression are the most common risk factors. Recognition of these risk factors should arouse a high index of suspicion for the diagnosis of invasion or dissemination. Unfortunately, laboratory tests alone lack sensitivity and specificity. Therefore, the diagnosis of invasion and dissemination in the majority of cases requires the acquisition and proper interpretation of clinical evidence. Once the diagnosis is made, early systemic treatment is warranted. Reported toxicity and efficacy supports the use of fluconazole for most patients with invasive fungal infections. However, for the most critically ill patients amphotericin B remains the treatment of choice. PMID- 8615476 TI - A new method of creating an arteriovenous graft access. AB - Since the initial description of the radial arteriovenous (AV) shunt, hemodialysis has played a primary role in the survival of patients with chronic renal failure. However, it is not unusual to be confronted with patients on chronic hemodialysis who have exhausted all superficial venous access sites. We describe a technique using the deep veins of the arms the outflow system during AV graft construction. By anastomosing the venous limb of the graft to the larger of the two brachial veins, we have been able to give patients AV access when the superficial veins have been exhausted. We have performed this technique with good success in 12 patients. Further experience and longer follow-up are needed. PMID- 8615480 TI - Medical guidelines by other societies: should we ignore or influence them? PMID- 8615481 TI - Hemodilution tolerance in elderly patients without known cardiac disease. AB - Hemodilution tolerance is not well defined in elderly patients. In 20 patients older than 65 yr and free from known cardiovascular disease, hemodynamic variables, ST segment deviation, and O2 consumption were determined prior to and after 6 and after 12 mL/kg isovolemic exchange of blood for 6% hydroxyethyl starch. The mean age of the patients was 76 +/- 2 yr (mean +/- SEM, range 66-88 yr). During hemodilution, hemoglobin decreased from 11.6 +/- 0.4 to 8.8 +/- 0.3 g/dL (P < 0.05). With stable filling pressures, cardiac index increased from 2.02 +/- 0.11 to 2.19 +/- 0.10 L.min-1.m-2 (P < 0.05) while systemic vascular resistance decreased from 1796 +/- 136 to 1568 +/- 126 dynes.s.cm-5 (P < 0.05) and O2 extraction increased from 28.0% +/- 0.9% to 33.0% +/- 0.8% (P < 0.05) resulting in a stable O2 consumption during hemodilution. No alterations in ST segments were observed in lead II during hemodilution. In lead V5, ST segment deviation became slightly less negative during hemodilution from -0.03 +/- 0.01 to -0.02 +/- 0.01 mV (P < 0.05). The moderate decrease in hemoglobin was fully compensated by both an increase in cardiac index and in O2 extraction. Electrocardiographic signs of myocardial ischemia were not observed in this population. In conclusion, isovolemic hemodilution to a hemoglobin value of 8.8 +/- 0.3 g/dL is well tolerated in elderly patients free from known cardiac disease at the ages of 65-88 yr. PMID- 8615482 TI - Hemodilution tolerance in patients with coronary artery disease who are receiving chronic beta-adrenergic blocker therapy. AB - Hemodilution tolerance is not well defined in patients with coronary artery disease receiving beta-adrenergic blockers chronically. Ninety patients scheduled for coronary artery bypass graft (CABG) surgery were randomized to a hemodilution (n = 60) and a control group (n = 30). During midazolam-fentanyl anesthesia, hemodynamic variables, ST segment deviation, and O2 consumption were determined prior to and after 6 and 12 mL/kg isovolemic exchange of blood for 6% hydroxyethyl starch. Hemoglobin decreased from 12.6 +/- 0.2 to 9.9 +/- 0.2 g/dL (mean +/- SEM, P < 0.05). With stable filling pressures, cardiac index increased from 2.05 +/- 0.05 to 2.27 +/- 0.05 L.min-1.m-2(P < 0.05) and O2 extraction from 27.4% +/- 0.6% to 31.2% +/- 0.7% (P < 0.05), resulting in stable O2 consumption. No alterations in ST segments were observed in leads II and V5 during hemodilution. Individual increases in cardiac index and O2 extraction were not linearly related to age and left ventricular (LV) ejection fraction (P = 0.841, P = 0.799). We conclude that isovolemic hemodilution to a hemoglobin value of 9.9 +/- 0.2 g/dL is well tolerated and fully compensated in patients with coronary artery disease receiving beta-adrenergic blockers chronically. Within the investigated ranges, the compensatory mechanisms during hemodilution are largely independent of age (35-81 yr) and LV ejection fraction (26%-83%). PMID- 8615483 TI - Myocardial ischemia and reperfusion are associated with an increased stiffness of remote nonischemic myocardium. AB - During and after an ischemic injury, maintenance and recovery of cardiac function may critically depend on remote nonischemic myocardium. Graded myocardial ischemia is associated with an approximately 50% increase in stiffness of nonischemic myocardium. We determined whether this increase in stiffness is unique to the ischemic period or persists during reperfusion. Ten anesthetized (isoflurane 1.0% vol/vol) open-chest dogs were instrumented to measure left ventricular pressure and dimensions (sonomicrometry) in ischemic and nonischemic myocardium. Regional chamber stiffness and myocardial stiffness were assessed using the end-diastolic pressure-length relationship which was modified by stepwise infusion and withdrawal of 200 mL of the animals' own blood during baseline, 45 min low flow ischemia (systolic bulge), and 60 min after the onset of reperfusion. In remote nonischemic myocardium, regional myocardial ischemia was associated with a significant (P < 0.05) increase in chamber stiffness (+44%) and myocardial stiffness (+48%). Sixty minutes after the onset of reperfusion, chamber stiffness (+54%, P < 0.05 versus baseline) and myocardial stiffness (+55%, P < 0.05 versus baseline) remained increased. Thus, the ischemia-induced increase in stiffness of remote nonischemic myocardium persists for at least 60 min after reperfusion. PMID- 8615484 TI - The effects of alpha 2-adrenergic stimulation with mivazerol on myocardial blood flow and function during coronary artery stenosis in anesthetized dogs. AB - The central sympatholytic effect of alpha 2 agonists may be beneficial during myocardial ischemia, but could be opposed by their peripheral vasoconstrictive effect. We studied the effects of mivazerol during periods of moderate coronary artery stenosis in anesthetized dogs. Mivazerol decreased heart rate (from 125 +/ 6 to 106 +/- 6 bpm) and cardiac output (from 4.4 +/- 0.6 to 1.8 +/- 0.2L/min) under normal conditions, while mean arterial pressure did not change. Mivazerol reduced blood flow in nonischemic myocardium and in the ischemic epicardial layer, but blood flow was preserved in the ischemic midmyocardial and subendocardial layer. Mivazerol had no effect on myocardial oxygen extraction during the stenoses, and regional myocardial oxygen consumption was unchanged. However, mivazerol decreased myocardial oxygen demand from 4.51 +/- 0.51 to 3.17 +/- 0.24 mumol.min-1.g-1, thereby reducing oxygen deficiency of ischemic myocardium to values significantly lower than in the placebo group (from 1.07 +/- 0.32 to 0.47 +/- 0.41 mumol.min-1.g-1). Mivazerol had no effect on myocardial lactate production during the stenoses. We conclude that mivazerol reduced myocardial oxygen demand while blood flow was preserved in the inner layers of ischemic myocardium. PMID- 8615485 TI - Myocardial protection during coronary artery bypass graft surgery: a randomized, double-blind, placebo-controlled study with trimetazidine. AB - We conducted a randomized, double-blind, placebo-controlled study to assess the cardioprotective effects of trimetazidine (TMZ), an antiischemic drug, on left ventricular function using transesophageal echocardiography (TEE) after coronary artery bypass grafting (CABG). Forty patients undergoing elective CABG received either TMZ or a placebo (PCB). The primary measures of efficacy were serial measurements of fractional area change (FAC), percent of systolic wall thickening (SWT), and malonedialdehyde (MDA) production. The two groups were similar for the following variables: number of vessels revascularized (2.5 +/- 0.2 in the TMZ group and 2.8 +/- 0.1 in the PCB group), duration of aortic clamping (46 +/- 4 min in the TMZ group and 48 +/- 3 min in the PCB group), and bypass time (63 +/- 4 min in the TMZ group and 70 +/- 4 min in the PCB group). FAC increased by 12% in both groups 20 min after aortic unclamping (P < 0.05) and remained above the initial value at the sixth postoperative hour. SWT was 23.8% +/- 1.6%, 25.4% +/- 1.9%, then 21.6% +/- 1.5% in the TMZ group and 22.8% +/- 1.6%, 23.8% +/- 1.4%, then 22.3% +/- 1.6 % in the PCB group, after induction of anesthesia and 1 and 6 h after aortic unclamping (not significant). MDA increased by 24% in the PCB group and 25% in the TMZ group 20 min after aortic unclamping (P < 0.01). Lactate levels were lower in the TMZ group (P < 0.05) and patients from the TMZ group received less intravenous calcium before aortic clamping (P < 0.02) and less calcium channel entry blocking drugs in the early phase after aortic unclamping (P < 0.01) compared to the PCB group. We conclude that in patients with good preoperative ejection fraction undergoing CABG, TMZ as administered did not demonstrate clinically significant cardioprotective effects on left ventricular performance and lipid peroxidation compared to PCB. PMID- 8615486 TI - Volume-controlled ventilation is made possible in infants by using compliant breathing circuits with large compression volume. AB - We studied the weight dependency of set tidal volume (VTset) during volume controlled ventilation of 80 infants (ASA physical status I-IV, 0.7-20 kg), including prematures, neonates, and exprematures, who were anesthetized for major and minor surgery, including abdominal, thoracic, and neurosurgical procedures. After neuromuscular blockade and endotracheal intubation, infant's lungs were ventilated with an Ohmeda 7800 volume-limited ventilator and either a pediatric or adult circle breathing system (PC or AC) or a Bain circuit (Ba) and a pediatric- or adult-sized bellows (PB or AB). Except for larger and older infants in the ACAB group, body weight, age, peak inspiratory pressure (PIP), ETCO2, and SPO2 did not differ among groups. Compression volume loss was large in the five circuits tested. We found that VTset/kg varied with weight in a curvilinear relationship where y represents volume added and x represents PIP (y = 175.02x 0.87; r2 - 0.87), whereby VTset is approximately 150-200 mL/kg for a 1-kg infant and approximately 25 mL/kg for infants > or = 10 kg. Ventilation was adequate in each infant, except for one with extremely poor pulmonary compliance. We conclude that large compression volumes associated with compliant breathing systems make possible the use of volume-controlled ventilators in small infants. PMID- 8615487 TI - Environmental tobacco smoke: a risk factor for pediatric laryngospasm. AB - Adult patients who smoke are known to have airway complications during general anesthesia. The objective of this study was to explore the relationship between environmental tobacco smoke (ETS) exposure in the home and laryngospasm during general anesthesia in pediatric patients. A retrospective, cohort study was performed on pediatric ambulatory patients in the day surgery center and main operating room of a university hospital. We studied 310 consecutive pediatric patients (all ASA physical status I) who underwent an outpatient elective ear, nose, and throat or urologic surgical procedure in the spring and summer of 1994, and received inhalation induction by mask with halothane. Laryngospasm was identified from quality management and anesthetic records, and included only those patients whose records indicated that succinylcholine was given because of oxygen desaturation and inability to ventilate. Patients' families were questioned within 1 wk after surgery as to the number of smokers in each child's household. Of 96 children with ETS exposure, 9 (9.4%) developed laryngospasm. Of the 214 patients without domestic ETS exposure, 2 (0.9%) developed laryngospasm. The relative risk for developing laryngospasm was 10 times higher in the ETS exposed patients compared with the non-ETS-exposed group (95% confidence interval = 2.2-45.6; P < 0.001). We conclude that ETS exposure is a strong risk factor for laryngospasm in infants and children during general anesthesia. PMID- 8615488 TI - Dimenhydrinate decreases vomiting after strabismus surgery in children. AB - Dimenhydrinate, an H1-receptor antagonist, has been used to both prevent and treat postoperative vomiting (POV) in children for several decades. However, its effectiveness for POV after strabismus surgery remains anecdotal. This study was designed to determine the effectiveness and side effects of dimenhydrinate for the prevention of POV in children after strabismus surgery. Eighty ASA physical status I or II children, ages 1-12 yr inclusive, who were undergoing strabismus surgery, were prospectively and randomly allocated to receive either dimenhydrinate 0.5 mg/kg intravenously (n = 40) or placebo (n = 40) at induction of anesthesia. The incidence of POV and the times to arousal (and discharge from the recovery room and hospital) were recorded postoperatively in a double blinded manner. For 24 h after discharge from the hospital, all emetic episodes and medications given were recorded by the parents. Demographic data did not differ between the groups. Children who received dimenhydrinate had significantly less POV both inhospital (10%) and overall (30%) than those who received placebo (in hospital 38%, P < 0.008; overall 65%, P < 0.003). The times to arousal and discharge from the hospital did not differ between the two groups. Dimenhydrinate (0.5 mg/kg) is an effective, safe, and inexpensive antiemetic in children undergoing strabismus surgery. It significantly reduces the incidence of vomiting for 24 h postoperatively and is not associated with prolonged sedation or other adverse effects. PMID- 8615489 TI - Intraoperative ketorolac has an opioid-sparing effect in women after diagnostic laparoscopy but not after laparoscopic tubal ligation. AB - Ketorolac tromethamine (Toradol) is a parenteral, nonsteroidal antiinflammatory drug that is being extensively used to provide postoperative analgesia. This study evaluated whether intraoperative ketorolac would act synergistically with fentanyl to decrease postoperative analgesic requirements in outpatients undergoing gynecologic procedures. The patients studied were adult ASA physical status I or II females scheduled for diagnostic laparoscopy (DL) (n = 80) or laparoscopic tubal ligation (TL) (n = 46). Each patient received fentanyl 2 micrograms/kg intravenously (i.v.) before induction, followed by a standardized propofol anesthetic and 2 mL of saline or ketorolac 60 mg i.v. in a randomized double-blind fashion 30 min before the anticipated end of the operative procedure. Patients were assessed for postoperative pain via a 10-cm visual analog scale (VAS) (0 = no pain; 10 = severe pain) before analgesic treatment in the postanesthesia care unit (PACU). Severe postoperative pain (VAS or 5 or more) was treated with incremental doses of fentanyl, 25-50 micrograms i.v. by a blinded PACU nurse. Ibuprofen or acetaminophen with codeine was administered for pain control once the patient tolerated oral medications. This study showed that intraoperative ketorolac (60 mg i.v.) with fentanyl (2 micrograms/kg i.v.) administered at the induction of anesthesia resulted in significant opioid sparing and a diminution in pain in the DL sample but not in the TL sample. The analgesic regimen was also associated with a lower incidence of nausea and vomiting and resulted in earlier discharge, which was not seen after TL. These results demonstrate that pain after TL is far greater than that after DL, which suggests that these procedures should be considered separately when designing analgesic regimens. PMID- 8615490 TI - The effects of propofol on hemodynamics and renal blood flow in healthy and in septic sheep, and combined with fentanyl in septic sheep. AB - Sepsis is characterized by myocardial depression and systemic vasodilation, both of which are most likely mediated by nitric oxide. Propofol inhibits nitric oxide synthase and may therefore be beneficial in sepsis. On the other hand, renal blood flow, known to be only minimally affected by propofol in healthy subjects, may be drastically reduced in septic individuals, because the renal microvasculature is known to be very sensitive to nitric oxide. In this study, the effects of propofol in healthy and in septic sheep, and in combination with fentanyl, were analyzed and compared with nonanesthetized septic sheep. In healthy sheep, propofol caused only minor hemodynamic changes. In septic sheep, however, hemodynamics deteriorated. Renal blood flow was reduced to 60% +/- 10% of the preseptic baseline and to 39% +/- 4% of the septic value. This reduction was selective, since the cardiac output decreased significantly less. These adverse effects of propofol on hemodynamics and renal blood flow were reduced when propofol was combined with fentanyl. PMID- 8615491 TI - Variability of motor-evoked potentials recorded during nitrous oxide anesthesia from the tibialis anterior muscle after transcranial electrical stimulation. AB - When recorded as a compound muscle action potential (CMAP), the motor-evoked potential (MEP) is affected by volatile anesthetics and nitrous oxide. However, MEPs recorded using epidural electrodes in the presence of nitrous oxide are highly reproducible from trial to trial. We wished to establish the reproducibility over time of the CMAP produced by supramaximal transcranial electrical stimulation of the human motor cortex. Cascades of 100 successive CMAPs were recorded from the tibialis anterior muscles of six anesthetized patients undergoing scoliosis surgery, in response to transcranial electrical stimuli of > 500 V. Satisfactory CMAPs could be recorded in the presence of nitrous oxide, but not isoflurane. Latencies and amplitudes were reproducible in repeated sequences of 100 responses. However, amplitude and, to a lesser extent, latency, were highly variable within a sequence. In addition, occasional individual stimuli, although rarely successive ones, failed to evoke a CMAP. CMAPs have a much higher trial-to-trial variability than corticospinal volleys recorded from the epidural space. Using the present methodology it would be difficult to rely on CMAP recordings as an indicator of corticospinal function in the clinical monitoring situation. PMID- 8615492 TI - Pulmonary function changes during epidural anesthesia for cesarean delivery. AB - Although changes in pulmonary function in parturients are documented, little is known about effects of regional anesthesia on these changes. This study was undertaken to determine if two local anesthetics, often used for epidural anesthesia for cesarean delivery, have different effects on pulmonary function testing. Nineteen ASA physical status I parturients undergoing elective cesarean delivery with epidural anesthesia were randomly assigned in double-blind fashion to receive either 0.5% bupivacaine or 2% lidocaine with epinephrine (1/200,000). Pulmonary function tests were measured using a calibrated spirometer with computer-recorded flow volume loops. Peak inspiratory pressure and peak inspiratory flow rate, peak expiratory pressure (PEP) and peak expiratory flow rate, forced vital capacity (FVC), and forced expiratory volume in 1 s (FEV1) were measured. Measurements were taken prior to epidural placement and at T-10 and T-4 levels. Peak inspiratory pressure, FEV1/FVC, FEV1, FVC, peak expiratory flow rate, and peak inspiratory flow rate did not differ from baseline in either group. Patients receiving lidocaine showed a significantly greater decrease in PEP at both T-10 and T-4 levels. Pep is largely dependent on abdominal musculature. If a denser motor block is provided by 2% lidocaine with epinephrine than by 0.5% bupivacaine, these muscles would be more affected, resulting in a greater decrease in PEP. These results may have implications regarding choice of local anesthetic for epidural anesthesia in parturients with some degree of respiratory compromise undergoing cesarean delivery. PMID- 8615493 TI - Postoperative analgesia with continuous epidural sufentanil and bupivacaine: a prospective study in 614 patients. AB - To assess the efficacy and safety of postoperative analgesia with continuous epidural sufentanil and bupivacaine, we performed a prospective study in 614 patients undergoing major surgery. Before surgical incision, all patients received an initial dose of 50 micrograms sufentanil in 6-10 mL bupivacaine 0.125% via a lumbar or thoracic catheter. After 1 h, a continuous infusion was started with 50 micrograms sufentanil in 50 mL bupivacaine 0.125% at a rate of 6 10 mL/h. The infusion was continued postoperatively for 1-5 days or longer, depending on the type of operation and the patient's analgesic need. In the majority of patients, adequate pain relief was obtained at rest and during movement. Late respiratory depression was observed in three patients; in most patients only minor side effects were seen. Technical complications during epidural puncture or insertion of the catheter were 4% and 3%, respectively. We conclude that continuous epidural sufentanil and bupivacaine is safe and effective. PMID- 8615494 TI - Epinephrine decreases postoperative requirements for continuous thoracic epidural fentanyl infusions. AB - Epidural thoracic fentanyl infusions provide effective preoperative analgesia after thoracotomy; however, side effects can limit the effectiveness of this technique. This study evaluates epinephrine as an adjunct to continuous thoracic epidural fentanyl infusions after thoracotomy. Thirty-eight patients were studied in a prospective, randomized, double-blind trial comparing fentanyl alone to fentanyl with epinephrine 1:300,000. Epidural infusion rates were titrated to equivalent pain relief using a visual analog scale. With the addition of epinephrine, there was a significant reduction in fentanyl requirements (0.82 +/- 0.07 vs 1.19 +/- 0.11 micrograms.kg-1.h-1, P = 0.005, repeated-measures analysis of variance) and in plasma fentanyl concentrations (steady state: 0.91 +/- 0.13 vs 1.65 +/- 0.23 ng/mL, P = 0.007, repeated-measures analysis of variance). There were no differences in pain scores, side effects, spirometry, patient satisfaction scores, or hemodynamic variables. This study demonstrates that adding epinephrine 1:300,000 to continuous thoracic epidural infusions decreases fentanyl requirements titrated for effective analgesia. The reduction in fentanyl requirements was associated with reduced fentanyl plasma concentrations. PMID- 8615495 TI - Lidocaine inhibits blood coagulation: implications for epidural blood patch. AB - Lidocaine in the epidural space, through inhibitory effects upon coagulation, may contribute to inefficacy of epidural autologous blood patch (EBP). This study was undertaken to evaluate the effect of achievable epidural concentrations of lidocaine on blood coagulation as a step in testing this hypothesis. Ex vivo blood coagulation using whole blood (n = 20) was studied with computerized thrombelastography (TEG). Each blood specimen was exposed to serial dilutions of lidocaine hydrochloride or saline to form end-concentrations of 0.0 mM, 2.3 mM, 4.6 mM, 9.2 mM, 18.5 mM, and 36.9 mM lidocaine. Statistical analysis using analysis of variance for repeated measures revealed that the three highest lidocaine concentrations tested caused hypocoagulable and/or fibrinolytic changes as compared with controls. Achievable epidural admixtures of lidocaine and whole blood will impair coagulation. Therefore, residual lidocaine in the epidural space may contribute to failures of immediate or early EBP. PMID- 8615496 TI - Metabolism of compound A by renal cysteine-S-conjugate beta-lyase is not the mechanism of compound A-induced renal injury in the rat. AB - Compound A [CF2 = C(CF3)OCH2F], a vinyl ether produced by CO2 absorbents acting on sevoflurane, can produce corticomedullary junction necrosis (injury to the outer stripe of the outer medullary layer, i.e., corticomedullary junction) in rats. Several halogenated alkenes produce a histologically similar corticomedullary necrosis by converting glutathione conjugates of these alkenes to halothionoacetyl halides. To test whether this mechanism explained the nephrotoxicity of Compound A, we blocked three metabolic steps which would lead to formation of a halothionoacetyl halide: 1) we depleted glutathione by administering dl-buthionine-S, R-sulfoximine (BSO); 2) we blocked cysteine S conjugate formation by administering acivicin (AT-125); and 3) we inhibited subsequent metabolism by renal cysteine conjugate beta-lyase to the nephrotoxic halothionoacetyl halides by administering aminooxyacetic acid (AOAA). These treatments were given alone or in combination to separate groups of 10 or 20 Wistar rats before their exposure to Compound A. We hypothesized that blocking these metabolic steps should decrease the injury produced by breathing 150 ppm of Compound A for 3 h. However, we found either no change or an increase in renal injury, suggesting that this pathway mediates detoxification rather than toxicity. Our findings suggest that the cysteine-S-conjugate-mediated pathway is not the mechanism of Compound A nephrotoxicity and, therefore, observed interspecies differences in the activity of this activating pathway may not be relevant in the prediction of the nephrotoxic potential of Compound A in clinical practice. PMID- 8615497 TI - Factors affecting production of compound A from the interaction of sevoflurane with Baralyme and soda lime. AB - Various alkali (e.g., soda lime) convert sevoflurane to CF2=C(CF3)OCH2F, a vinyl ether called "Compound A, " whose toxicity raises concerns regarding the safe administration of sevoflurane via rebreathing circuits. In the present investigation, we measured the sevoflurane degradation and output of Compound A caused by standard (13% water) Baralyme brand absorbent and standard (15% water) soda lime, and Baralyme and soda lime having various water contents (including no water). We used a flow-through system, applying a gas flow rate relative to absorbent volume that roughly equaled the rate/volume found in clinical practice. Both absorbents, at similar water contents, temperatures, and sevoflurane concentrations, produced roughly equal concentrations of Compound A. Dry and nearly dry absorbents produced less Compound A early in exposure to sevoflurane, and more later, than standard absorbents. Increases in temperature and sevoflurane concentration increased output of Compound A. Both absorbents, especially when dry, also destroyed Compound A, the concentration exiting from absorbent resulting from a complex sum of production and destruction. We conclude that the variability of concentrations of Compound A found in clinical practice may be largely explained by the inflow rate used (i.e., by rebreathing), sevoflurane concentration, and absorbent temperature and dryness. The effect of dryness is complex, with fresh dry absorbent destroying Compound A as it is made, and with dry absorbent that has been exposed to sevoflurane for a period of time providing a sometimes unusually high output of Compound A. PMID- 8615499 TI - Norepinephrine does not potentiate porcine malignant hyperthermia. AB - Malignant hyperthermia (MH) is a rare genetic trait characterized by potential life-threatening episodes of hypermetabolism, hyperthermia, and muscle rigidity when susceptible humans or animals are exposed to triggering drugs. The role of norepinephrine (NE) in triggering MH is controversial. The purpose of this study was to show that NE does not initiate nor speed the onset of MH in susceptible swine exposed to known triggering drugs. Three groups of MH susceptible (MHS) pigs were exposed to two times the minimum alveolar anesthetic concentrations (MAC) halothane (2%) for 60 min and monitored continuously until a PaCO2 of 70 mm Hg was obtained as an end point for fulminant MH. This dose of halothane is associated with significant hypotension which was addressed by three modalities: no treatment; NE infusion at 8 micrograms.kg-1.min-1; and intraaortic balloon pump (IABP) with 1:1 augmentation (7.0-mL balloon catheter). NE and epinephrine (Epi) plasma levels were determined at 15-min intervals and at trigger time. All animals developed signs of MH during the study. There was no difference in pHa, lactate, PaCO2, or temperature at control or trigger times between groups. Time to trigger was longer in the untreated group compared to both the NE and the IABP groups which were equal. The NE group had greater NE and Epi plasma levels at all times than either the untreated or IABP group and the levels increased at each sample time. The IABP group had increased NE levels at time of trigger compared to control time period, however, Epi levels did not increase. In the untreated group, individual animals had marked increases in NE levels, but extreme variability in response prevented achievement of a single mean change. This group showed no increase in plasma Epi levels throughout the study. There was no difference in NE levels between the untreated and IABP groups. Three animals in the untreated group died prior to trigger due to complications of hypotension. In conclusion, addition of exogenous NE in high doses did not enhance triggering of MH. The large dose NE infusion resulted in increased total catecholamines throughout the study in the NE group with no effect on time to MH trigger compared to animals where mean arterial pressure (MAP) was maintained by IABP. Animals in all three groups with times to trigger of less than 30 min had significantly higher MAPs at control, 15 min, and trigger time than those with times to trigger of greater than 30 min. We conclude that NE does not trigger MH and that severe reduction of MAP delays the the onset of MH in susceptible swine. PMID- 8615498 TI - Thermal response in acute porcine malignant hyperthermia. AB - This study was designed to evaluate how vital organ and skin-surface temperatures correlate with other clinical signs of a malignant hyperthermia (MH) episode. Six susceptible swine were anesthetized with thiopental and nitrous oxide and kept normothermic (approximately equal to 38 degrees C). After a 30-min control period, halothane (1 minimum alveolar anesthetic concentration) was administered, followed in 5 min by a bolus of succinylcholine (2 mg/kg intravenously). Monitoring included: 1) ETCO2; 2)PaO2, PaCO2, pHa; 3) cardiovascular function; 4) core temperatures (esophagus, pulmonary artery, and rectum); 5) organ temperatures (brain, kidney, liver, and four skeletal muscles); and 6) skin temperatures (forehead, neck, and axilla). Within 10 min after exposure to halothane and succinylcholine, all animals developed fulminant MH. Kidney, liver, and brain temperatures increased more rapidly than pulmonary artery temperature with the onset of MH. Temperatures significantly increased in the visceral organs prior to the detection of contractures within skeletal muscles. The masseter, longissimus dorsi, quadriceps, deltoid, and extensor digiti II intramuscular temperatures were 1-2 degrees C less than pulmonary artery and esophageal temperatures during the episodes, whereas those of the kidney, liver, and brain were the same or slightly greater. When it occurs, core hyperthermia during acute MH results largely from heat produced in central organs, not in skeletal muscle per se. In these swine, changes in axilla skin surface temperatures correlated well with core temperature trends, whereas those of the neck and forehead did not. Unless a skin-surface probe can be placed in close proximity to a major vessel, cutaneous temperatures should not be substituted for measurements at an appropriate core site. PMID- 8615500 TI - Intravenous lecithin-coated microcrystals of dantrolene are effective in the treatment of malignant hyperthermia: an investigation in rats, dogs, and swine. AB - Dantrolene effectively treats malignant hyperthermia (MH) hut the current form, Dantrium, must be dissolved to a 0.33 mg/mL, pH 9.5 solution. This study describes lecithin-coated microcrystal formulations of sodium dantrolene (MC-NaD) and neutral dantrolene (MC-D) which reconstitute to 200 mg/mL within 1 min. In rats, the pharmacokinetics and pharmacodynamics of MC-NaD and Dantrium were similar: half-lives of 3.1 h, volume distributions of 0.54 and 0.59 L/kg, and 95% effective dose (ED95) values for depression of skeletal muscle twitch height (ED95T) of 2.6 +/- 0.7 and 2.8 +/- 0.5 mg/kg. In swine, the ED95T values for MC NaD and Dantrium were also similar (2.8 +/- 0.4 vs 2.7 +/- 0.6 mg/kg), but MC-D and Dantrium were only similar at doses more than 2.5 mg/kg (ED95T: 3.5 +/- 0.4 vs 2.7 +/- 0.5 mg/kg). In susceptible swine, MC-NaD successfully treated five of six MH episodes and prevented MH in three of four swine. However, MC-NaD caused marked pulmonary hypertension in swine, while MC-D caused only a mild response that was eliminated by filtration. Likewise, MC-D caused no pulmonary response in dogs. These observations suggest that MC-D has potential to improve the treatment of MH. PMID- 8615501 TI - Prior hypothermia attenuates malignant hyperthermia in susceptible swine. AB - This study was designed to determine the extent by which mild or moderate hyperthermia attenuates the triggering of malignant hypothermia (MH) induced by the combined administration of halothane and succinylcholine. Sixteen susceptible swine were initially anesthetized with nontriggering drugs and then either kept normothermic (approximately equal to 38 degrees C, n = 6) or cooled to induce mild (approximately equal to 35 degrees C, n = 6), or moderate (approximately equal to 33 degrees C, n = 4) hypothermia. Next, after a 30-min control period, the normothermic and mildly hypothermic animals were administered 1 minimum alveolar anesthetic concentration (MAC) halothane followed by a bolus dose of succinylcholine (2 mg/kg). Within 10 min all normothermic animals developed fulminant MH, whereas the onset of MH was slowed or was absent in the mildly hypothermic group. To test whether moderate hypothermia could more effectively minimize the signs of a MH episode, this group of animals was exposed to 1.5 MAC halothane followed 10 min later by a 3-mg/kg bolus of succinylcholine. MH was not induced and anesthesia was then changed to nontriggering drugs (ketamine and pancuronium). The animals were then aggressively rewarmed to 38 degrees C: a slight increase in the ETCO2 was detected, but MH episodes did not spontaneously occur. Subsequently, the readministration of halothane and succinylcholine rapidly provoked fulminant MH. We concluded that the induction of mild hypothermia impairs triggering and reduces the progression of MH induced by the combined administration of halothane and succinylcholine, whereas moderate hypothermia was completely protective and thus could be considered for prophylaxis. PMID- 8615502 TI - Mild intraoperative hypothermia reduces production of reactive oxygen intermediates by polymorphonuclear leukocytes. AB - Mild hypothermia directly impairs numerous immune functions in vitro. However, the in vivo effects of mild hypothermia on neutrophil phagocytosis and oxidative killing remain unknown. We tested the hypothesis that mild intraoperative hypothermia decreases neutrophil phagocytic capacity and generation of reactive oxygen intermediates (a measure of oxidative killing). Additionally, we evaluated the effects of in vitro temperature manipulations on each function. Thermal management was randomly assigned in 10 surgical patients, causing intraoperative core temperatures to range from 33 to 37 degrees C. Production of reactive oxygen intermediates and neutrophil phagocytosis were evaluated using flow cytometry at ambient temperature. Phagocytic capacity was assessed by uptake of fluorescein isothiocyanate-labeled Escherichia coli. Reactive oxygen production was estimated by the intracellular conversion of dihydrorhodamine 123 to rhodamine 123. Blood samples were obtained preoperatively, 1 h after surgery started, and 2 h postoperatively. Blood was also obtained from 10 matched control subjects and tested at 32, 37, and 40 degrees C. Neutrophil oxidative and phagocytic capacities were significantly reduced intraoperatively, compared with preoperative and postoperative values. Intraoperative production of reactive oxygen species was linearly related to core temperature. In contrast, there was no correlation between core temperature and phagocytic activity. In vitro production of reactive oxygen intermediates increased sixfold from 32 to 40 degrees C. In vitro phagocytic capacity increased fourfold in this temperature range. Production of oxidative intermediates was most closely related to intraoperative core temperature, decreasing nearly fourfold over a 4 degree C range. This in vitro temperature dependence was matched in vitro. Impaired neutrophil oxidative killing may contribute to the observed hypothermia-induced reduction in resistance to infection. PMID- 8615503 TI - The sedative and sympatholytic effects of oral tizanidine in healthy volunteers. AB - Tizanidine, an imidazoline derivative with alpha 2-receptor-mediated central muscle relaxant activity, is in widespread clinical use for the treatment of spasticity. To evaluate its possible role in anesthesia we assessed the sedative and sympatholytic effects of orally administered tizanidine in a double-blind, placebo-controlled, randomized, cross-over study in six healthy male volunteers. Three different doses of tizanidine (4, 8, and 12 mg) were tested and compared to clonidine 150 micrograms. The sedative and sympatholytic effects of tizanidine 12 mg were comparable in magnitude to those of clonidine 150 micrograms, but the effects of clonidine were longer lasting. Similarly, the observed decreases in arterial blood pressure (diastolic, 13% and 19%; systolic, 10% and 8% for tizanidine and clonidine, respectively) and salivation were comparable in magnitude but of shorter duration after tizanidine 12 mg than after clonidine. Clonidine and tizanidine 12 mg had also similar effects on the secretion of growth hormone. Our results indicate that the effects of a single 12-mg oral dose of tizanidine resemble those of 150 micrograms oral clonidine, but are of shorter duration. Tizanidine may thus be a useful alternative to clonidine as an orally active, short-acting alpha 2-adrenoceptor agonist in the perioperative period. PMID- 8615504 TI - The effects of sevoflurane, isoflurane, halothane, and enflurane on hemodynamic responses during an inhaled induction of anesthesia via a mask in humans. AB - A rapid increase in isoflurane or desflurane concentration induces tachycardia and hypertension and increases-plasma catecholamine concentration. Little information is available as to whether sevoflurane, halothane, and enflurane induce similar responses during anesthesia induction via mask. Fifty ASA physical status I patients, aged 20-40 yr, and scheduled for elective minor surgery, received one of four volatile anesthetics: sevoflurane, isoflurane, halothane, or enflurane. Anesthesia was induced with thiamylal, followed by inhalation of 0.9 minimum alveolar anesthetic concentration (MAC) of the anesthetic in 100% oxygen via mask. The inspired concentration of anesthetic was increased by 0.9 MAC every 5 min to a maximum of 2.7 MAC. Heart rate (HR) and systolic blood pressure (SBP) were measured before and every minute for 15 min during anesthetic inhalation. In the sevoflurane and isoflurane groups, venous blood samples were drawn to determine the concentrations of plasma epinephrine and norepinephrine 3 min after each increase in anesthetic concentration. Sustained increments in HR were observed after increases in inspired isoflurane concentration to 1.8 MAC and 2.7 MAC (peak changes of 15 +/- 3 and 17 +/- 3 bpm, respectively). Isoflurane also increased SBP transiently after the inspired concentration was increased to 2.7 MAC (peak change of 10 +/- 4 mm Hg). Enflurane increased HR after the inspired concentration was increased to 2.7 MAC (peak change of 9 +/- 2 bpm). In contrast, changes in sevoflurane and halothane concentrations did not induce hyperdynamic responses. Plasma norepinephrine concentration in the isoflurane group was significantly higher than that in the sevoflurane group during 2.7 MAC (P = 0.022). We propose that there is a direct relationship between airway irritation of the anesthetic and immediate cardiovascular change during an inhaled induction of anesthesia. PMID- 8615505 TI - Hemodynamic changes after retroperitoneal CO2 insufflation for posterior retroperitoneoscopic adrenalectomy. AB - Intraoperative complications and hemodynamic alterations during posterior capnoretroperitoneoscopic adrenalectomy in the prone position were investigated in 16 consecutive patients using invasive hemodynamic monitoring. Under general anesthesia with propofol and fentanyl, hemodynamic changes were made before (M1) and during retroperitoneal CO2 insufflation (15 mm Hg) [M2]; 20 mm Hg [M3]. Retroperitoneal insufflation resulted in a significant increase of cardiac output (+72%), stroke volume (+42%), mean arterial pressure (+39 %), and mean pulmonary arterial pressure (+36%). Although retroperitoneal inflation was accompanied by a significant increase of central venous pressure (+37%), an increase of preload may have lead to higher filling pressures. Heart rate, systemic vascular resistance, and pulmonary vascular resistance did not show significant changes. One pneumothorax and two cutaneous emphysemas occurred. We have demonstrated, in a small number of patients, that retroperitoneal CO2 insufflation for posterior capnoretroperitoneoscopic adrenalectomy in the prone position results in hemodynamic changes without apparent adverse effects. PMID- 8615506 TI - A comparison of lansoprazole, omeprazole, and ranitidine for reducing preoperative gastric secretion in adult patients undergoing elective surgery. AB - Acid aspiration syndrome of induction of anesthesia is a life-threatening complication whose severity is affected by both pH and volume of the aspirated gastric juice. We compared the effects of two proton pump inhibitors (PPIs), lansoprazole and omeprazole, and an H2 blocker, ranitidine, on gastric secretion in a prospective, randomized, double-blind fashion in 200 adult patients of ASA physical status I undergoing elective surgery. The patients were divided into eight groups (n = 25 each) according to their premedication. The patients received lansoprazole-lansoprazole (Group L-L), lansoprazole-placebo (Group L-P), placebo-lansoprazole (Group P-L), omeprazole-omeprazole (Group O-O), omeprazole placebo (Group O-P), placebo-omeprazole (Group P-O), placebo-ranitidine (Group P R), or placebo-placebo (Group P-P), as the first and second medications. The dose of the study drug was 30 mg for lansoprazole, 150 mg for ranitidine, and 80 mg for omeprazole. The first medication was administered orally at 9:00 PM on the night before surgery and the second at 5:30 AM in the morning on the day of the surgery. Each patient fasted overnight and took the drug with 20 mL of water. After tracheal intubation, gastric fluid was aspirated via an orogastric tube and the volume and pH of the aspirate were measured. The pH of the aspirated gastric fluid was higher in Groups P-R, L-L, P-L, O-O, and O-P than in Group P-P (P < 0.05). The volume of the gastric contents was similar in Groups P-0 and P-P, and the other groups had smaller gastric volume than Group P-P (P < 0.05). Gastric fluid from patients in Group P-R was the least acidic (pH 6.1 +/- 1.2) and had the least volume (0.09 +/- 0.06 mL/kg). Group L-L was comparable with Group P-R in both pH and volume, whereas Groups P-L and O-O were similar to Group P-R only in volume. The proportion of patients at risk according to the traditional criteria (pH < 2.5 and volume 0.4 mL/kg) was significantly lower in Groups L-L (0%), P-L (4%), O-O (4%), and P-R (0%) than in Group P-P (48%) (P < 0.05). We concluded that two consecutive doses of lansoprazole or a morning dose of ranitidine seemed to be the most effective preanesthetic medication for reducing gastric acidity and volume. PMID- 8615507 TI - Mechanisms of action of isoflurane on contraction of rabbit conduit artery. AB - Isoflurane may cause differential effects on different vascular beds of the same animal species. The mechanisms of this action have not been elucidated. Accordingly, we compared in rabbit aorta and femoral artery the effects of isoflurane (1-3.3%) in isolated rings (endothelium denuded) activated by norepinephrine, and isoflurane effects on Ca2+ fluxes from the sarcoplasmic reticulum in skinned strips. When < 30 nM norepinephrine was used to cause ring contraction, isoflurane increased the force of contraction in aortic rings, but decreased force in femoral arterial rings. At 30 nM norepinephrine stimulation, 3.3% isoflurane decreased the force and, in the presence of verapamil, isoflurane actually increased the force in both arterial types. In skinned strips of both arterial types, isoflurane present during Ca2+ uptake decreased the caffeine induced tension transients, whereas isoflurane present during Ca2+ release enhanced the transients. Isoflurane potentiated the depression of the tension transients by ryanodine. Isoflurane directly caused contracture even in the absence of caffeine. Thus, isoflurane has similar cellular mechanisms of action in the aortic and femoral arterial smooth muscle: inhibiting Ca2+ influx through the sarcolemma, decreasing Ca2+ uptake by the sarcoplasmic reticulum, and enhancing caffeine-induced Ca2+ release from the sarcoplasmic reticulum. PMID- 8615508 TI - A comparison of the effect of halothane on N-methyl-D-aspartate and non-N-methyl D-aspartate receptor-mediated excitatory synaptic transmission in the hippocampus. AB - Halothane depresses synaptic transmission in the rat brain. First we determined the concentration of halothane which decreased the amplitude of the population spike recorded in the CA1 region of the hippocampus to 50% of the control value (105 +/- 4.9 micrograms/mL [0.53 mM] halothane). Hippocampal glutamate receptors are divided into N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4 isoxazole proprionate (AMPA) and kainate (non-NMDA) subtypes. The NMDA and non NMDA receptors were blocked with (+/-)-2-amino-5-phosphonopentanoic acid (AP5) (30 microM), and 6,7-dinitroquinoxaline-2,3-dione (DNQX) (10 microM), respectively, to allow observation of the effects of halothane on the NMDA and non-NMDA receptors, respectively. gamma-Aminobutyric acid type A (GABAA) receptors were blocked in all studies with picrotoxin (PTX) (40 microM). When the non-NMDA receptors were blocked a halothane concentration of 38.1 +/- 5.6 mg/mL was required to produce a further 50% decrease in population spike amplitude. When NMDA receptors were blocked with AP5 or only GABAA receptors were blocked the halothane concentrations needed to produce 50% block were higher than needed for the control (160.8 +/- 17.8 and 190.2 +/- 12.1 microgram/mL, respectively). These studies indicate that the NMDA receptors are more sensitive to the effects of halothane than the non-NMDA receptors. PMID- 8615509 TI - The onset of rocuronium, but not of vecuronium or mivacurium, is modified by tourniquet inflation. AB - A previous investigation showed that inflation of a tourniquet did not interrupt onset of vecuronium neuromuscular block. To test the hypothesis that this effect depended on potency, twitch tension was measured in an arm with a tourniquet inflated during onset and compared with a control arm in 30 patients under fentanyl-thiopental-nitrous oxide-isoflurane anesthesia. Patients were randomly allocated to receive either vecuronium 0.1 mg/kg (n = 10), rocuronium 0.6 mg/kg (n = 10), or mivacurium 0.2 mg/kg (n = 10). The electromyographic response of the first dorsal interosseus to single twitch stimulation of the ulnar nerve every 10 s was recorded in both arms. When neuromuscular block was 20% (i.e., twitch tension was 80% of control), the tourniquet was inflated to a pressure of 300 mm Hg. It was deflated 5 min later. In the vecuronium and mivacurium groups, the tourniquet did not influence onset of block. In the rocuronium group, maximum neuromuscular block was (mean +/- SD) 79% +/- 10% in the tourniquet arm, compared with 96% +/- 4% in the perfused arm (P < 0.05). The maximum rate of onset was half that of the perfused arm. The difference in maximum neuromuscular block between arms was 17% +/- 7%, 5% +/- 5%, and 0% +/- 2% in the rocuronium, vecuronium, and mivacurium groups (P < 0.05). To explain that onset of block continues in spite of interruption of blood flow, drug molecules must gain access to the neuromuscular junction via routes other than the circulation. The results of this investigation are consistent with the hypothesis that there is redistribution of drug from extrajunctional to junctional areas during onset of action of muscle relaxants and this process is more important for the more potent drugs (vecuronium and mivacurium) than for rocuronium. PMID- 8615510 TI - Tracheal diameter predicts double-lumen tube size: a method for selecting left double-lumen tubes. PMID- 8615511 TI - The cocaine-abusing parturient is not at an increased risk for thrombocytopenia. PMID- 8615512 TI - The effective diameter and airflow resistance of the individual lumens of left polyvinyl chloride double-lumen endobronchial tubes. PMID- 8615513 TI - Plexus irritation caused by interscalene brachial plexus catheter for shoulder surgery. PMID- 8615514 TI - Spinal anesthesia and permanent neurologic deficit after interscalene block. PMID- 8615515 TI - Perioperative presentation of seizures in neonates. PMID- 8615516 TI - Acute facial, cervical, and thoracic subcutaneous emphysema: a complication of fiberoptic laryngoscopy. PMID- 8615517 TI - Failure to ventilate through a double-lumen tube due to carinal shift during lung volume reduction surgery. PMID- 8615519 TI - Prone position and oxygenation. PMID- 8615518 TI - Monitoring of ventilation on and off cardiopulmonary bypass: two standards or one? PMID- 8615520 TI - Autologous platelet-rich plasmapheresis in repeat cardiac surgery. PMID- 8615521 TI - The use of the laryngeal mask airway in children with bronchopulmonary dysplasia. PMID- 8615522 TI - A two-head stethoscope. PMID- 8615524 TI - Synergistic combinations of muscle relaxants may reduce costs. PMID- 8615523 TI - The effect of abdominal massage on the onset of epidural blockade in laboring women. PMID- 8615525 TI - Propofol-opioid mixtures during prolonged infusions. PMID- 8615526 TI - High-frequency jet ventilation during fiberoptic laser resection of tracheal granuloma in a small child. PMID- 8615527 TI - Ketorolac and strabismus surgery. PMID- 8615528 TI - Autologous platelet-rich plasmapheresis: risk versus benefit in repeat cardiac operations. PMID- 8615529 TI - Electroencephalogram as an indicator of cerebral ischemia. PMID- 8615530 TI - Continuous ventilation during trans-laryngeal mask airway fiberoptic bronchoscope aided tracheal intubation. PMID- 8615531 TI - Vecuronium for emergency intubations? PMID- 8615532 TI - Recovery properties of cisatracurium and vecuronium in intensive care unit patients. PMID- 8615533 TI - How far should a catheter be inserted into the epidural space? PMID- 8615534 TI - The optimal distance that a multiorifice epidural catheter should be threaded into the epidural space. PMID- 8615535 TI - Rectal flumazenil to reverse midazolam sedation in children. PMID- 8615536 TI - Pulmonary edema after transjugular intrahepatic portosystemic shunt. PMID- 8615537 TI - Inhalation agents: a young anesthetist's dilemma. PMID- 8615538 TI - [Significance of chronotropic exertion effects on the heart in diagnosing physical fitness in children practising swimming]. AB - Regarding the peculiar character of exercise adaptation in developmental age, two questions have been stated; in what degree heart rate (HR) changes after physical effort reflect and diagnose the physical fitness in children and if these values can be the base of indirect method of maximal oxygen intake (VO2max). The practical problem was to choose the method, which could make the data for the comparative analysis of the heart's chronotropic reaction in children practising swimming professionally and in non-practising ones possible to obtain. The research method should not be connected with excessive workload for children. The heart's chronotropic reaction has been thoroughly analysed in different phases of submaximal progressive effort, according to the methods specific for the PWC170 index estimation; its diagnostic value would determine its differentiation in the age groups of swimmers and non-practising children and correlation between its indexes and real oxygen efficiency estimated using maximal oxygen intake values obtained in direct method (VO2maxEZ). 186 girls and 225 boys aged 10 to 15, practicing systematically since the 10th year of life, were investigated. During six years of the study 1308 exercise trials have been performed, including 654 tests with submaximal workload specific for the PWC170 index estimation (Physical Work Capacity), and 654 spiroergometric tests (TSE) with maximal workload and direct measurement of oxygen intake. Data obtained from the test with submaximal workload were compared with those noted in the same age groups of non-practicing children (246 girls and 256 boys), who formed the control groups for male and female swimmers. Those children from primary school had never practised any organized form of sport apart from obligatory physical exercises at school. Comparative analysis of physical efficiency indexes followed the estimation of basic morphologic features in studied children (body weight, fat-free body weight, body height). The heart's chronotropic reaction has been characterized in a traditional way as PWC170 and PWC150 indexes; it has been also thoroughly analyzed during following stages of the test estimated by workload changes, as well as the resultant for the whole workload dynamics of the HR changes, which has characterized the physiologic chronotropic expense of one watt of the workload applied. In the same time relationship between values obtained directly before the beginning of the exercises (HRW) have been analyzed. Although it has been found that discussed PWC indexes can differentiate in mean falues the groups of practising and non-practising children in the same age, but in significant percentage their values were nearing in both groups; it has been distinctly shown in the value distribution in percentile scores (Fig 1). This situation undoubtedly reduces the diagnostic advantages of the discussed indexes. HRW values have influenced the course of the test and its final result, and especially PWC indexes (significant in some groups of children), which has been emphasized. In the research a problem is discussed: in what degree the changes of the heart rate can describe the general physical fitness of people studies. Comparative analysis of the PWC and TSE tests performed in swimmers show no relationship between physical oxygen efficiency and values of PWC170 and PWC150 indexes. In the longitudinal research the PWC170 test results have significantly fluctuated during progressively increasing values of maximal oxygen intake measured directly, which has been pointed out. Maximal oxygen intake should be evaluated critically on the base of the heart's chronotropic reaction after exercise in children. It seems that more reliable in estimating the exercise adaptation is to describe the heart's chronotropic reaction using its direct indexes. It is possible to use PWC170 results, but it is necessary to regard the initial HR values when starting the exercise. PMID- 8615539 TI - [Effect of contrast media used in hysterosalpingography (HSG) on phagocytosis of spermatozoa by peritoneal macrophages in female rats]. AB - It is apparent from numerous communications that hysterosalpinogography (HSG), besides its diagnostic value, may also claim therapeutic activity. After such examinations, more frequent pregnancies were recorded in previously infertile women without resorting to supplementary treatment. Pregnancies were more numerous when applying oil-based contrast media than water-based contrast ones. Hypothesis has been put forward as to the possibility of modulating activity of these agents exerted upon the peritoneal and oviductal macrophages. Increased amount of these cells as well as excessive phagocytosis of spermatozoa was shown in case of endometriosis, one of the most frequent causes of infertility. It is supposed that excessive phagocytosis of spermatozoa by peritoneal macrophages (PM) may be the cause of infertility. Studies were performed to determine the effect of oil- and water-soluble contrast media, on sperm phagocytosis by PM in vitro. PM were obtained from Wistar female rats. Subsequently, the cells were centrifuged, washed and suspended in culture medium. Next 1 million PM were allowed to attach to the cover glass for one hour under standard conditions of incubation. Dilutions of 0.25%, 0.5%, 1.0% Lipiodol Ultra--Fluid (Byk Gulden Konstanz) and Uropolinum 60% (Polfa) were added to chambers with incubated PM for next one hour. The controls were cultured in the same conditions without contrast medium added. Sperm were isolated from the epididymal cauda of male rats and subsequently suspended in culture medium at a concentration 1 million/ml and added to equivalent volume of cultured macrophages for 1.5 hours. After the exposure time the cultures were washed and stained. The spermiophagic index (SPI) was determined. SPI = number of phagocytosed sperm cells/number of macrophages x 100. Statistical analysis was performed by means of Student t-test. Additional histochemical reactions were made and scanning as well as transmission electron microscopy studies were accomplished. The established results (Fig. 1-9) indicate that lipiodol inhibits phagocytosis of spermatozoa by PM stronger (1% lipodol SPI = 1.99 + 0.94 < 0.0001) than uropolinum (1% uropolinum SPI = 5.07 +/- 1.02 p < 0.0001). In control studies, without contrast medium added, SPI was equal to 14.66 +/- 3.12 (p < 0.00001). Marked inhibition involving phagocytosis of spermatozoa was detected already after the treatment with lipiodol in low concentration (0.25% lipiodol SPI = 3.73 +/- 0.89 p < 0.0001). It failed to be so distinct after treating macrophages with uropolinum in low concentration (0.25% uropolinum SPI = 8.34 +/- 1.50 p < 0.0001). Scanning electron microscopy studies have disclosed that in the cultures of macrophages with spermatozoa, the macrophages spread well over the glass bottom. The macrophages' cytoplasmic membrane was highly folded with numerous protrusions different in size and shape. They covered the digested parts of spermatozoon causing its fragmentation. In cultures of macrophages with spermatozoa and 1% uropolinum, the structure of macrophages was similar to that in the control group. Accumulations and conglomerations of a large number of macrophages with spermatozoa were additionally revealed. In cultures of macrophages with spermatozoa and 1% lipiodol, the macrophages were not spread, but rather spherical. Also the cytoplasmic membrane protrusions were small, short, fine and appeared in smaller number. Occasionally areas of smooth cytoplasmic membrane without any folds were seen. Such macrophages did not phagocytose sperm. Transmission electron microscopy studies of macrophages cultured with sperm have disclosed that the macrophages' cytoplasmic membrane was highly folded with numerous protrusions different in size and shape. The latter encircled and closed parts of sperm (head, midpiece or tail) inside inside endosomes, finally linking the latter with lysosomes. The ultrastructure of macrophages cultured with spermatozoa and 1% uropolinum was s PMID- 8615540 TI - [Activity of choline esterases in blood cells and serum and cobalt activated acylase in toxic and non-toxic liver damage]. AB - Organic insecticides used in agriculture and drugs often taken without medical control remain an important source of intoxication. The significance of the problem has been discussed in American medical literature, where toxic and posttoxic damage to the liver is rated second (25%), just after viral damage (40%), like in other highly developed countries. It can be assumed that the leading position of viral damage is the result of difficulties in ascertaining the diagnosis in each case of the disease. Clinical diagnosis of liver damage, in spite of many laboratory methods described and in use, leaves a margin of uncertainty and error. The diagnosis relies heavily on the activity of enzymes characteristic for morphologic and functional status of the liver. The determination of enzyme activities in plasma is also one of the basic diagnostic tools in the toxicology clinic in all cases of intoxication by hepatotoxic compounds. Toxicological recognition of acute intoxications relies on a wide range of biochemical tests and enzyme indices to evaluate the extent of damage by xenobiotics to organs. Usually the tests are selected in a clinical setting when the cause of intoxication is known. The present data were supplemented with activities of AspAT and AlAT determined during hospitalization of the patients. This choice acknowledged the wide-spread use of these tests and their organ specificity. All parameters in the control group were within normal limits described in the literature. For patients with phosphoroorganic or carbaminate intoxications (Ia) the activity of AECh and ECh proved to be a specific and sensitive parameter. In these cases both activities showed large, significant drops, confirming earlier reports advocating the use of these tests in insecticide intoxications. (Tab. 1-3). Among clinical symptoms frequently accompanying intoxication by ethylene glycol (Ib) one would mention metabolic acidosis, kidney damage and narcotic action of the glycol on the CNS. Little attention has been devoted to the hepatotoxic action of this compound. In the present study activities of the aminotransferases deviated slightly from normal. The DeRitis index was also within normal limits. However, the activity of AACo in this group of patients differed significantly from the control values. In the group of patients with Amanita phalloides poisoning (Id) the most sensitive indicator of liver damage proved to be AACo. The activity of this enzyme was significantly higher than in the remaining groups. The rise of activity of this enzyme was significantly higher than in the remaining groups. The rise of activity preceded by a few hours the appearance of increased aminotransferase activity which was observed near the end of the second day after intoxication. In the group of patients with chronic active hepatitis (II) the only parameter within normal limits was ECh. This finding supports earlier reports as to the relatively late decrease in the rate of synthesis of this enzyme in liver diseases. The use of toxogonine in vitro to discriminate between ECh and AECh in intoxications and liver diseases was decided upon basing on numerous reviews describing the clinical applications of this agent. The results of agent in the toxogonine test are in line with the disruptive action to toxogonine on the enzyme-insecticide complex in vitro, probably similar to the situation in the living organism. The observed elevation in AECh activity after the enzyme was inhibited by the phosphoro-organic pesticide, observed 30 minutes since the addition of toxogonin, averaged 86% +/- 25%. In the case of carbaminate insecticides the addition of toxogonine was followed by a further decrease in esterase activity and only a few samples showed an increase, but not exceeding 25%. No effect of toxogonine was seen in the case of serum cholinesterase and after 30 minutes the activity continued to fall. PMID- 8615541 TI - [Evaluation of the usefulness of autogenic bone grafts in reconstruction of the mandible]. AB - The treatment of reconstructions of mandible by means of autogenic bone graft in 64 patients has been presented (Fig. 1). In 55 cases the bone material was taken from the iliac crest, in 5--the skin-muscle flap, in 4--the free bone graft from VIII rib and tibial bones. The resection in 47 patients was performed because of malignant neoplasms, in 4 cases--oral and mandibular benign neoplasms, in 3 cases a part of the mandible was resected because of the osteodystrophy, in 3 cases due to osteoradionecrosis, in 3 cases--congenital anomalies, in 2 cases because of a cyst, while in other 2--after an injury. Clinical parameters were evaluated: cosmesis, mastication, deglutition, diet, speech, and the use of dentures. The positive result was obtained in 84% cases. Cosmesis was improved in patients who had immediate restoration of mandibular reconstruction due to previous scarring. Mastication was poorer in the reconstructed group due to scarring, loss of the muscles of mastication, and inhibition of compensation of mandibular motion. Deglutition was not improved in patients who had mandibular continuity resorbed. Problems with deglutition were related to soft tissue resection. Speech and prosthetic rehabilitation was poor in both groups in patients. In 5% of patients, there was a slight extraoral graft denudation and the development of organic inflammatory process in the surrounding tissues and the graft itself. Application of the appropriate pharmacological and local treatments was sufficient for the maintenance of the graft and its normal healing. Negative result of the mandible reconstruction was observed in 11% of cases. In the patients with immediate reconstruction, more than 50% of the bone grafts resorbed. PMID- 8615543 TI - [Comparison of the effectiveness of anti-ulcer drugs and their influence on angiogenesis in healing of experimental chronic gastric ulcer]. AB - This study compares ulcer healing properties of pirenzepine (10 mg/kg), famotidine ) 4 mg/kg), colloidal bismuth subcitrate (200 mg/kg), misoprostol (0.2 mg/kg) in rats. Colloidal bismuth subcitrate (CBC), sucralfate, famotidine and pirenzepine given orally were equally effective in the healing of chronic acetic gastric ulcers, while misoprostol was less effective in this model. In addition, the influence of these drugs on angiogenesis is believed to play a significant role in the healing process of chronic gastric ulcer. Newly formed capillaries play an important role in early stages of healing, reabsorption and granulation. Abundant formation of new capillaries is especially characteristic for granulation. During scar formation these newly formed capillaries disappear. Taking into account the existence of capillaries during healing one can evaluate a quantity of newly born capillaries to describe the healing. The capillary system in histologic cross-sections has been visualized employing an enzymatic test reaction the activity of alkaline phosphatase and ATPase, which are very active in the capillary endothelium. The increased number of capillaries and their surface have been shown using computed analysis of microscopic pictures in animals that had received antiulcer drugs. (Fig. 1-4). Conclusions. (1) Histochemical methods and computed analysis of microscopic pictures used here given an objective evaluation of angiogenesis in the healing process of chronic gastric ulcers. (2) The antiulcer drugs investigated stimulate angiogenesis which is an important element of their accelerating effect on the healing of chronic gastric ulcers. PMID- 8615542 TI - [Evaluation of rabies prophylaxis in people of the Szczecin region in the years 1986-1991]. AB - The aim of the study was the analysis of epizootic and epidemiological situation of rabies in the region of Pomerania in 1986-1991 and the evaluation of correctness in determining indications for antirabies vaccination. In analysed period of time 441 cases of rabies in domestic and wild animals were noted. Altogether 2941 subjects were admitted to the Rabies Prophylaxis Outpatient Center (Fig. 1, Tab 1-7). Indications for vaccination were analysed in each case as well as the possibility to reduce the number of vaccinated patients in respect of economical benefits was considered. Vaccinated patients were inquired about side effects of Human Diploid Cell Vaccine (HDCV) (Merieux France). Among 2941 patients admitted to the out-patient clinic indications for rabies vaccination were given in 933 cases (31.7%). Most often they were determined due to dogs bite or another contact with dog's saliva. Postvaccinal reactions were observed in 61 cases (6.5%), mostly topical ones. Neurological complications were not noticed. CONCLUSIONS: 1. Despite the fact that the epizootic situation in domestic and wild animals has changed for worse, the number of indications for prophylactic vaccination decreased in last six years. 2. Proper evaluation of the indications for prophylactic vaccinations against rabies is the most important factor influencing the reduction in the number of vaccinated people. 3. Rationalisation of indications for vaccination against rabies justified claims to be economical one. 4. HDCV (Merieux) is a safe vaccine and its usage reduced the side effects to minimum degree. PMID- 8615544 TI - [The effect of various illumination sources on dynamics of selected visual functions]. AB - The ever-increasing use of sodium discharge lamp, being most economical among all of this type has evoked a wave of studies on the action of this lighting source. The aim of the paper was to evaluate the effect of illumination provided by this new type of high-pressure sodium discharge lamp with improved coefficiency of transmitting the colours (HPSM) on selected visual functions, as well as to compare with results having been established in illumination by filament lamps (GL) and standard sodium discharge lamp (HPS). HPSM are more closer to white light, thus more profitable for sight physiology system and central nervous system than standard sodium discharge lamps. The intention fo the paper was to obtain the answer whether in comparison with filament and standard high-pressure sodium discharge lamp, the new mode of illumination provides better psychophysical conditions complying with the used ergonomic principles, and if there is any possibility for HPSM to be applied for illuminating the closed spaces. The study involved 63 persons, aged from 17 to 54 years. The comparative group comprised 50 persons, who were subjected to the tests with filament and standard sodium illumination. All the studied subjects were divided into 3 age groups (I-to 30 years, II-31-40 years, III-over 41 years). The following visual functions were studied: acuity of far looking vision, proximity visual point, field of vision, time of conscious reaction minimal, medium and maximal, and the number of mistakes during the performed test, convergent, divergence fusion for distance and proximity as well as manual spatial visual localization. All the said examinations were carried out three times within morning hours and in this way gaining the possibility of evaluating the dynamics of functional changes in time (Fig. 1-9). The obtained results were compared with those established after having performed the same tests under standard sodium lighting and filament illumination. Conclusions resulting from the performed studies: 1. The obtained results provide confirmation of stimulating effect of both sources of sodium lighting exerted on the central nervous system. 2. This effect was expressed by: improved visual acuity for distance and reduced number of mistakes at the time of reaction as well as selection in all the age groups, increased divergence fusion for proximity point in persons of I group as well as pertaining to the peripheral visual field in all the studied age groups. 4. All of the above features become evident particularly strongly during the evaluation of the dynamics of the cited functions. 5. The obtained results suggest that the use of HPSM for illuminating rooms could be worth trying. PMID- 8615545 TI - [Address given by the rector of the Pomeranian Medical Academy at inauguration of the academic year 1994/1995]. PMID- 8615546 TI - [Effect of various methods of supplementing magnesium on health status of children under special care]. AB - Fifty pupils, aged 18 years, with minor mental handicap have been examined. The aim of the work was to evaluate the levels of magnesium, calcium, zinc and copper in serum and in hair in the examined population, and also the levels of magnesium, calcium, zinc and copper in serum and in hair in the examined population, and also the correlation between supplementation and pupils learning effects, perception and ability of concentration. Supplementation method, kind of preparations and their doses were analyzed and estimated accordingly. Investigations dealt with assessment of magnesium supplementation after Dolomit (320 mg for 7 intensive days of each month) and Lactomag (140 mg every day) for 6 months. Effects of supplementation were assessed by results of Vocational Power Test and by average marks before and after treatment. Deficiency of all the examined bioelements both in serum and hair was recorded prior to treatment. After magnesium supplementation, levels of bioelements were significantly changed except magnesium (Tax. 1,2,3,4,). That was due most probably to high deficiency of magnesium before the treatment. The use of these drugs evidently improved mental power of adolescents. There was no difference between the effects exerted by Dolomit and Lactomag, except the higher calcium level in hair after Dolomit treatment. The conclusions is that pupils with mental handicap should be provided with magnesium preparations because their effects are highly positive. PMID- 8615547 TI - [Evaluation of cartilaginous wrist development in the human during the prenatal period]. AB - The aim of the work was to assess the development of human chondral wrist in the prenatal period with regard to cartilaginous buds topography, histologic structure and development of article fissures in each month of intrauterine life. The performed examination covered 89 fetuses from 2nd to 9th month of prenatal life. Altogether 178 wrists were examined. The age of the fetuses was estimated on the clinical data, body mass, and crown-rump length base. Fetuses with developmental anomalies were excluded. At the beginning the material for microscopy was fixed and decalcified, next it was embedded in paraffin and cut on the microtome in a frontal plane. Preparations were stained by Van Gieson method, hematoxylin, and eosin. They were microscopied. I found that most of 2 month-old fetuses had 8 wrist bones buds. They were arranged in two rows: proximal and distal. Some of the examined fetuses had accessory bud that joined navicular bone during development process. Occasionally, 3, 4, 5, and 6 month-old fetuses revealed one chondral element with 2 or 3 differentiation foci in place of navicular, lunar and triangular bones. I disclosed no differences in histological development of wrist bone buds. (Fig. 1-4). There was a layer of thickened cells with mitotic figures at buds periphery in 2 and 3 month-old fetuses. A large amount of intercellular substance was in the central part. A reduced density of cells was seen in peripheral parts as well as in central parts, being viewed in intercellular substance, of buds in the 4th month of intrauterine life. Plane mesenchymal cells were observed on the surface of the buds, they created tracts forming layers, which may correspond to synovial membrane structure. The homogeneous substance staining yellow by Van Gieson method, what may be synovial membrane secretion, was found in 5 month-old fetuses. Reduction of cells from the periphery of buds had continued in 6th month. Cells forming buds resembled chondral, had light cytoplasm and central karyon, and fell into pairs. During further evolution it was observed that there was an increase of intracellular substance, formation of pairs and groups of chondral cells up to high differentiation. In 8th and 9th month of intrauterine life cells appeared like mature hyaline cartilage. Also chondral canaliculi including blood vessels were detected in the buds. Article fissures forming processes were observed already from 2nd month of intrauterine life and terminated their action in 5th month in the majority of fetuses. The evolution of distal radioulnar articular disc was seen, too. Mesenchyma being rich in cells was not differentiated in 2 month-old fetuses but during the evolution it changed into connective tissue band adhering to styloid process of ulna from one side and to head of radius from the other. Among forming article discs connective tissue fibres were found to be typical of chondral cells in 7, 8 and 9 month-old fetuses. PMID- 8615548 TI - [Treatment of lateral cross-bite with active plates in conditions of raised dental occlusion and by the quad-helix method]. AB - The purpose of the paper was to estimate the effects of the fixed elevation of occlusion on lateral cross-bite treatment. There was also comparative studies in the group of children treated with the quad-helix appliances. Own results were obtained from the studies of 36 children with lateral cross-bite whose occlusions were regulated. The children were divided into 3 groups:--in the first group 13 children had the occlusion arised with the metal dental crowns widened by metal flat surface on the buccal side. They were cemented on first permanent upper molars (Fig. 1);--in the second group 12 children had the occlusion arised by the mass type "Composite" covering first permanent lower molar teeth (Fig. 2); in both groups children were treated by upper active plate;--in the third group 11 children had the appliance quad-helix used to widen the upper dental arch (Fig. 3). The obtained results (Tax. 1-4) show that stable elevation of the occlusion makes the treatment shorter, especially in the children where the cooperation is worse. For the patients with bigger narrowness of the upper dental arch it is directed to raise the occlusion with the metal dental crowns with flat widened surfaces. In the cases when the narrowness is smaller it is sufficient to raise the occlusion by the mass type "Composite" (Fig. 2). The quad-helix appliance is the most effective method of the treatment in the case of deciduous and mixed teeth, because the time of treatment is short. In the treatment of lateral cross bites the most important matter is to liquidate the disproportion between the width of the upper and lower dental arches (by widening the maxillae) and to obtain the regular bite. PMID- 8615549 TI - [Evaluation of periodontium status and periodontal needs in persons aged 35-44 years in Poland]. AB - The aim of this study was providing current, objective and comparable with the world literature data on the frequency of occurrence and the intensity of periodontal diseases in the age group 35-44 years. During the surveys the WHO card from 1986 was used. The condition of periodontium the periodontal treatment needs were assessed by CPITN (Community Periodontal Index of Treatment Needs) with use of 621 WHO probe. One thousand three hundred and eighty persons in nine districts of Poland were examined, with regard to three living areas: big cities, small towns and the rural ones. The results of surveys accomplished in Poland indicate that the achievement of the second health purpose, designed by World Health Organization for the year 2000 in the field of periodontal tissues is unattainable. This is because, according to the second health purpose, 75% of the population at the age of 35-44 years ought to have three health sextants, and deep picketing can occur in maximum 5% of the population. Unfortunately, only in 7.1% of persons examined at least three healthy sextants were found. They were disclosed considerably more often in women (9.1%) than in men (5.1%). Deep periodontal pocketing of 6 mm and more were found in 15% of persons examined. They were detected considerably more frequently in men (18.4%) than in women (11.3%). By comparison of data pertaining to individual districts (Tab. 1-5), it turned out that the worst conditions of periodontium occurred in Bialostockie, where there was not a single person with healthy periodontium. Better results were recorded in Gdanskie, which must be the effect of systematical health education. Periodontal treatment needs among the persons examined are enormous. Almost 100% of persons require both instruction of brushing teeth and time consuming professional procedures of removal of the concrement on the teeth. Fifteen per cent of the persons need specialized treatment. These proportions varied differently in the surveyed districts, only 5% in Dganskie and from 21.7% to 23.9% in Bialostockie, Wrocllawskie and Krakowskie. However the proportions of those needs in Poland are much bigger than in other European countries, and differ considerably from the marked second periodontal health purpose for the year 2000. PMID- 8615550 TI - [Analytical evaluation of urinary calculi mineral composition]. AB - Urolithiasis is the most frequent disease of urinary system. It affects about 3% of people of the productive age. One half of the hospitalized in Departments of Urology is made up of patients with urinary calculi. In some regions of the globe, urolithiasis is a very common pathological disorder. Upper Silesia being such a region in Poland. The objective of the paper is: 1) systematization of calculi according to their mineral composition with regard to eventual prophylaxis of urolithiasis, 2) comparison of mineral components of calculi in the aspect of pollutions for Upper Silesia and West Pomerania territories, and the effect of external factors on the constitution of the formed calculi, 3) making use of X-ray microanalysis in complex determinations of mineral compositions of stones, particularly the trace elements. For that purpose a total of 185 urinary calculi stemming from individuals of both sexes, aged 24 to 82 years were used. The content of calcium, magnesium, zinc and iron was determined by atom absorption method, that of aluminium, chloride, sulphur and copper by X ray microanalysis method, fluoride by gas chromatographic method, phosphates and urates by colorimetric method. The content of cystine was defined by thermic decomposition and characteristic smell. The results were subjected to statistical analysis. With reference to the first task, the following chemical classification of stones has been suggested, namely; class I: ammonium stones being magnesium ammonium phosphate, class II: stones with elevated calcium content, calcium oxalate, class III: stones with elevated magnesium content, class IV: calculi rich in calcium and magnesium, class V: calculi deficient in calcium and magnesium, class VI: organic uric stones, class VII: organic cystine stones. The second task consisted in comparing the mineral composition of stones from subjects inhabiting two remote from each other regions-industrialized Silesia and Pomerania (Tab. 10). The statistical analysis has revealed differences being statistically significant only with regard to calcium and magnesium in Silesia and Pomerania groups. Undoubtedly that is of importance when one takes into consideration that calcium and magnesium display high affinity to fluoride, the presence of which was detected in all the stones and which may influence the physicochemical properties of the urinary calculi, first of all their hardness and solubility. The third task consisted in evaluating the possibility of applying the X-ray microanalysis for searching the stone for further elements principally microelements. The performed study comprised 10 stones: Silesia region and Pomerania region, whose composition was compared pairwise. The pairs were selected in such a manner that they should have some features in common found out by previously described atom absorption methods. The established results concerning calcium, magnesium and phosphorus showed in general compatibility of results obtained by the two methods. However, by X-ray microanalysis method it was possible to detect further elements: sulphur, aluminium, chloride and copper. The following conclusions have been drawn, namely: 1) analytic examinations of mineral composition of urinary calculi are the base of their chemical composition with regard to eventual prophylaxis of urolithiasis, 2) a varied composition of urinary calculi from two different regions of Poland may be the reflection of actual state of natural environment pollution in those regions, 3) presence of fluoride in all the urinary calculi allows us to suppose that it is permanent element of urinary calculi. PMID- 8615551 TI - [Development of the mandible and its vascularization in human fetuses in light of morphologic, microangiographic and gnathometric studies]. AB - Studies were carried out on 75 mandibles of human fetuses from the 4 to 9th month of fetal life. There were 31 female and 44 male fetuses. The material came from the Institute of Normal Anatomy--Pomeranian Medical Academy. Investigations consisted in filling the external carotid artery and its branches with Micropag. After filling the arteries, the head of the fetus was isolated and fixed in formalin solution in concentration--1.11 mol/dm3 for the period of 3 weeks. Next the mandibles with articular capsule were excised, soft tissue removed and cut at the site of symphysis. Some of the mandibles were decalcified in the solution of nitric acid in concentration--1.16 mol/dm3 for the period of two weeks. All the mandibles were X-rayed employing Unipan 401 equipped with a micro focus X-ray tube for structural examinations. Radiogram photo copies were equipped with a micro focus X-ray tube for structural examinations. Radiogram photo copies were made by the positive method to obtain additional blow up. The decalcified mandibles were X-rayed again. In order to perform a thorough study of vascularization, the decalcified mandibles were cut into specimens (transverse longitudinal axis) about 500 microns thick, thereafter structural pictures were taken--enlarged 9, 11, 17, 22 and 25 times. The decalcified mandibles were exploited to prepare histological specimens in longitudinal cross-section (along the longitudinal axis of mandible). Further observations referred to the growth of mandibles in the particular age groups. Therefore antropometric measurements were made on X-ray pictures and their results were subjected to statistical analysis (Fig. 1-2). The results of studies (Fig. 3-7) proved that in the radiological photo of human fetuses from 4 to 9th month of fetal life; morphological elements characteristic of extrafetal mandibles were found, (such as: body, angle, ramus, mentum, coronoid process, condylar process, alevoluses and teeth buds). The bone structure of mandible showed trabecular structure with clearly seen borders. The structure of condylar process was of isosceles triangle with different trabecular structure. With age and texture of the triangle shriveled. Vascularization of the body of the mandible with alveolar process derived mainly from the inferior alveolar artery. The condylar process was supplied with rich vascular rete derived from arteries of temporomandibular joint. The coronoid process was vascularized by the adjacent arteries. The condylar process stroma formed cartilaginous model surrounded by trabeculae osseae cuff resembled, by the look and structure, the epiphysis of long bones. The analysis of measurement of the fetal mandibles proved that both halves of the fetal mandible revealed statistic identity. The comparison of the results of mandible measurement in both sexes did not show any significant differences. The mandible parameters presented slow, successive and constant growth month after month, and the greatest dynamics of growth in all mandible parameters was present, however, in the 7th month of fetal life a growth inhibition was observed. The gonial angle was constant, its mean value -148 degrees. PMID- 8615552 TI - [Variability of the course of the uterine artery and its branches within the broad ligament and vascularization of uterine walls depending on a woman's age in light of anatomical, radiologic and microangiographic studies]. AB - The aim of the study was to perform uterus measurements in order to demonstrate variability of size, shape and proportions of each part of uterus. Topography, shape and dimensions of the uterine artery and its organic branches were also analyzed. The main part of the study was to analyze the arterial system in the walls of the human uterus. The study has been performed in order to find correlation between anatomic variety and age, and obstetric history of the investigated females. Internal genital organs taken at autopsy from 105 female cadavers (only unchanged pathologically) aged from 3 to 85, underwent evaluation (Table 1). Material consisted of 8 non menstruating girls, 30 nulliparas (20 premenopausal and 10 postmenopausal) and 67 multiparas (32 and 35, respectively). During postmortem examination topography of the origin sites of the uterine artery originating from the internal iliac artery, its diameters and its position towards ureter have been estimated. External measurements of the uterus have been performed. After excision of the uterus, uterine arteries have been injected with radiologic contrast medium. Radiography of the uterus has been taken using roentgenographic apparatus for structural research, and then arrangement of the uterine arteries in myometrium and their organic branches have been assessed. Uteri have been sectioned horizontally, and finally enlarged radiographs of each layer have been taken to estimate the arrangement of vessels at different levels of the myometrium. Fractionation itself made accurate measurements of the walls of the uterus possible at different levels. It has been stated that shape and size of the uterus changed considerably during the whole individual's life. Changes occurred not only within time, but they were connected with motherhood as well. Variability of the external dimensions of the uterus was always closely connected with variability of the uterus wall's thickness at different levels. Angiographic studies revealed that uterine arteries were present in every case, on both sides, regardless of the age and obstetric history. Topographic investigations proved that the vessels always crossed the ureter superficially on both sides. Considering each of the three segments of the uterine artery, it was stated that the length of the descending part of the vessel varied only within age, while the transverse and ascending parts were elongated within age and also after pregnancies. The number of the uterine artery branches running towards the body and cervix of the uterus was significantly variable and depended not only on age, but on pregnancies as well. Radiographic analysis revealed that connections between uterine vessels on both sides of the same uterus were not constant. Their absence was associated with the presence of the hypovascular, or even avascular zone in the medial part of the fundus and body of the uterus. This situation was evident at any age, regardless of the obstetric history. Artery of the fundus uteri, which had been previously described in many studies, was a rarely existing vessel. In the investigated material no azygos uterine artery has been found. Microangiographic studies (Fig. 1-4) revealed that angioarchitectonics of blood vessels in consecutive layers of the wall of the human uterus at different levels was variable to a great degree. It has been stated that situation depended not only on age, but also on past pregnancies. PMID- 8615553 TI - [Comparative studies of rhombencephalon vascularization in rabbit and man in light of anatomic and microangiographic studies]. AB - A comparative study of a microvascularization of the rhombencephalon, especially pre- and capillary system, in rabbits and in humans was performed. It was intended to find out whether there were any similarities and differences in rhombencephalon microvascularization of a rabbit and man. The study was carried out on 75 (50--female, 25--male) mixed-race rabbits aged 4 months, as well as on 30 (15--females, 10--males) human brains that had been dissected free from cadavers, aged from 20 up to 70 years. Angiography of sections of the rhombencephalon in rabbits and cadavers was performed. The vessels were investigated after the introduction of Micropaque contrast medium, under controlled pressure, into an investigated vessel. Intravital injection of the contrast into rabbit's vessel was beneficial for the experiment. Radiography of intact brains as well as sections of the frontal plane was accomplished. Large arterial vessels were measured, with the results being subjected to statistical analysis (Table 1-2, Fig. 1-7). Microvascularization of the medulla oblongata and of the pons in rabbits were similar to those in its human counterparts, whereas the vascular system of the cerebellum was different. The results of micrographic studies were not always the same as described by Bochenek et al. The anatomical forms that had not been previously described were noted: para--medial arteries to the medulla oblongata and the pons; lateral arteries to the medulla oblongata and the pons; posterior arteries to the medulla oblongata; branches of the upper artery of the cerebellum branching and the simple ones with few lateral branches. The central or medial artery that follows the central sulcus was separate. PMID- 8615554 TI - [Influence of caffeine on toxicity and pharmacokinetics of paracetamol]. AB - The aim of this study was an experimental assessment of the influence of caffeine on the symptoms of the toxic action of paracentamol in mice as well as a detailed analysis if paracetamol pharmacokinetics in men receiving caffeine at the same time. The toxicologic investigations were performed in 620 Swiss mice. The LD50 and LD100 were determined after an administration of paracetamol intraperitoneally. The effects of two doses of caffeine on the survival time and number of animal deaths were investigated. The degree of hepatic damage was assessed on the basis of biochemical serum criteria, i.e. alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and concentration of bilirubin in serum, as well as on the basis of biochemical investigations of liver homogenates, estimating the concentration of reduced glutathione and P-450 cytochrome in the liver. The anatomicopathologic liver evaluation was also performed, including histological and histopathological examinations (glycogen, lipids). The pharmacological investigations were performed in 9 healthy volunteers in two randomized subgroups with the use of a cross-over method twice at one week intervals. The blood paracetamol level was determined according to the method of Thoma et al. The course of changes of paracetamol plasma levels was described with a one-compartment model for extravascular administration of the drug. The biexponential equation, describing the assumed model, was solved with the method of the smaller squares, using non linear approximation. (Tab 1-6, Fig. 1-3). The experimental studies demonstrated a decrease in both the acute toxicity and hepatotoxic action of paracetamol administered in combination with caffeine, which was indicated by a significant decrease in aminotransferase and alkaline phosphatase activity and in concentration of bilirubin as well as by an increase in the concentration of P 450 cytochrome and GSH in the liver which decreased after administration of paracetamol alone and also by limitation or lack of hepatic necrosis. The pharmacokinetic calculations in men demonstrated an interaction between paracetamol and caffeine which was indicated by a decrease in plasma paracetamol levels, by a smaller surface under the curve of changes of paracetamol levels indicating faster elimination of the drug after simultaneous administration with caffeine. Therefore, paracetamol preparations with caffeine may be less toxic than paracetamol alone. PMID- 8615555 TI - [Measured and predicted oxygen uptake in highly qualified athletes]. AB - Applied methods of maximal oxygen uptake (VO2max) estimation can be generally divided into direct ones, based on oxygen uptake measured at maximal load level, and indirect ones, connected with submaximal load levels. It has been observed that maximal oxygen uptake in adults and people well adapted to physical exercise undergoes only small changes, whereas reaction of the circulatory system at submaximal load is variable and may influence the results achieved by indirect methods of oxygen uptake estimation, based on referring heart rate to the load level. Maximal oxygen uptake values measured directly were compared with foreseen values using two indirect methods: Astrand-Rhyming nomogram and von Dobeln formula. Studies were performed in highly qualified oarsmen, cyclists and swimmers (Tb. 1), representatives of sport disciplines significantly different as far as the type of physical exercise is concerned, which may cause different developing adaptation mechanisms. In groups we carried out spiroergometric test which can provide reports of the patient's heart rates (HR) and direct measurements of oxygen uptake (VO2) in steady state at different load levels. At the maximal exercise load we measured VO2max and HRmax. HR values were used to estimate VO2max by indirect methods. Following questions have been asked: 1. What is the convergence between VO2max values measured directly and those estimated by Astrand-Rhyming and von Dobeln methods? 2. What is the diagnostic usefulness of the indirect VO2max estimation in highly qualified groups of sportsmen? At the end of the test VO2max values were in oarsmen 5.139 +/- 0.06 1/min, in cyclists 5.085 +/- 0.05 l/min, in swimmers 4.689 +/- 0.01 l/min. Average VO2max values in indirect Astrand-Rhyming method (Tab. 2) in all groups estimated using HR values taken at lower exercise loads (100 W, 150 W, 200 W), were statistically lower than those measured directly (P < 0.01, P < 0.001, while values taken at 250 W were situated close to. VO2max estimated directly and indirectly being correlated in oarsmen at 200 W (0.316 < r < 0.411), in cyclists at 150 W and 200 W (0.343 < r < 0.599), and in swimmers at 150 W and 200 W--only for A3-VO2max (r = 0.3 and r = 0.37). Maximal oxygen uptake estimated by using von Dobeln formula (Tab. 3) was in all cases significantly lower (P < 0.001) then measured (values ranged from 72% to 79% VO2max of those measured directly). Significant correlation was observed at all applied exercise loads in oarsmen and cyclists (0.284 < r < 0.597) and in swimmers only at 200 W and 250 W (0.245 < r < 0.370). CONCLUSIONS: 1. Indirect VO2max estimation based on load level and heart rate in Astrand Rhyming method gives results compatible with direct measurements of oxygen uptake only using significant loads, adequate to physical efficiency in investigated people (HR near 160 beats per min., 70-80% Hrmax; oxygen uptake near 80% VO2max. 2. Estimating VO2max by Astrand-Rhyming method one should apply the correction regarding maximal heart rate, in case of unknown maximal heart rate the age factor should be applied, which is of lower significance. 3. In highly qualified sportsmen one should't estimate VO2max using von Dobeln method; values achieved are significantly lower than those estimated in direct oxygen uptake measurements. PMID- 8615557 TI - Laparoscopic ventral hernia repair: a community hospital experience. AB - From October 1993 to April 1994, laparoscopic ventral hernia repair was performed on 10 patients, all of whom had a history of failed ventral hernia repair and at least two prior ventral hernia repair procedures. Patients presented with complaints of abdominal discomfort, painful mass at the hernia site, or vague abdominal discomfort. No operative deaths occurred. Two patients had minor complications: a seroma at the repair site, which resolved spontaneously, and a superficial wound infection at a trochar site, which responded to an oral cephalosporin. Six patients were discharged within 24 hours of surgery and one patient was operated on as an outpatient and discharged the same day. Follow-up of all patients ranged from 10 to 17 months. No evidence of hernia recurrence has been noted. Some recurrent ventral hernias are amenable to laparoscopic repair, and this technique may be preferable in some patients, especially those who have had an earlier failed open repair with mesh. We do not advocate use of our technique for the first repair of a ventral hernia. Long-term follow-up is still needed to determine recurrence rates compared with conventional open techniques. PMID- 8615556 TI - Experience with three prosthetic materials in temporary abdominal wall closure. AB - There are circumstances that make abdominal wall closure unsafe and technically impossible after laparotomy for trauma. In these difficult cases, prosthetic materials may be necessary to temporarily close the abdominal wall. To determine the optimal prosthetic in these instances, a retrospective chart review was conducted in our urban Level I trauma center. Twenty-five patients received 31 abdominal wall prostheses over a 4-year period. There were 7, 8, and 10 patients with 7 Marlex, 9 Dexon, and 15 Goretex prostheses, respectively. Each patient had only one type of prosthesis placed. The average age was 30.7 +/- 12.0 years, injury severity score was 20.3 +/- 7.4, and abdominal trauma index was 35.9 +/- 18.0; there was no significant difference in these values between groups. Eight patients died soon after the prosthesis was placed (average, 12.9 days) secondary to ongoing shock or multiple organ failure. Three of the seven surviving Goretex patients (43%) were intentionally left with small hernias. Three of the six Dexon patients (50%) were left with hernias; one of these eviscerated on day 150 and subsequently died, and the others have disabling gigantic hernias. Three of the four Marlex patients (75%) developed fistulae as a result of erosion into the small bowel or colon. One Marlex patient suffered with a chronically draining abdominal wound for 398 days prior to definitive closure. Goretex appears to be the best prosthetic for temporary abdominal wall closure because it causes less inflammatory reaction because of its smooth surface. It is therefore easier to retrieve at the time of definitive closure and carries less risk of fistula formation than other prostheses. Our Dexon patients suffered with gigantic hernias and one died because of complications of evisceration. We have abandoned the use of Marlex in abdominal wall closure because of the high incidence of fistula formation. We advocate the use of Goretex in temporary abdominal wall closure in this challenging group of patients. PMID- 8615558 TI - Pudendal canal decompression for the treatment of fecal incontinence in complete rectal prolapse. AB - Our recent studies have attributed fecal incontinence (FI) when it is associated with complete rectal prolapse (CRP) to pudendal neuropathy caused by pudendal canal syndrome (PCS). Herein we present the results of pudendal canal decompression (PCD), performed for the treatment of FI in 21 patients whose CRP was corrected by Ivalon sponge rectopexy 5.2 years before presentation. Thirteen patients had partial and eight complete FI. Examination revealed perianal hypoesthesia, diminished rectal neck pressure, reduced electromyographic (EMG) activity of both the external anal sphincter (EAS) and levator ani (LA) muscle, as well as prolonged pudendal nerve terminal motor latency (PNTML). PCD was performed with a mean follow up of 14.8 months. Postoperatively, seven (53.8%) of the patients with partial FI showed full fecal control with normalization or improvement of the perianal hypoesthesia, rectal neck pressure, EMG of EAS and LA, as well as PNTML. The remaining six patients were failures. Five (62.5%) of the eight patients with complete FI showed full fecal control, two partial improvement, and one failure. The degree of response of FI to PCD seems to be related to the degree of pudendal nerve damage. Nonimprovement may be due to irreversible pudendal nerve damage or incomplete PCD. In conclusion, PCD is effective in the treatment of FI associated with CRP, provided it is performed before complete nerve damage occurs. PMID- 8615559 TI - The many faces of aortoenteric fistulas. AB - Aortoenteric fistulas represent a life-threatening complication of abdominal aortic surgery that is becoming increasingly well-recognized. The presentation is often subtle, with a herald bleed followed by a period of grace, followed by an exsanguinating hemorrhage, and resulting in cardiovascular collapse. The diagnosis is often difficult, even with modern modalities of endoscopy, arteriography, and CAT scanning. A high index of suspicion is critical for making a successful diagnosis. The fistulas most commonly occur between the proximal aortic suture line and the duodenum after abdominal aortic surgery for aneurysmal or occlusive disease. Typically they occur years after this procedure. However, over the last several years, we have seen 12 cases with extremely unusual presentations that illustrate the wide spectrum of possible presentations. Included in this group was a primary aortoduodenal fistula, and two fistulas occurring just months after the initial surgery. These cases are reported with attention to the details of the presentation to emphasize the wide range of presentations of this serious complication. A brief review of this literature is also included in the report. PMID- 8615560 TI - Effects of reconstituted high-density lipoprotein in persistent gram-negative bacteremia. AB - Reconstituted high-density lipoproteins (rHDL) have been shown bind bacterial LPS and reduce its toxic effects. Since the effect of rHDL on LPS in vitro cannot be directly extrapolated to the in-vivo picture of Gram-negative septic shock, we have investigated the effects of rHDL in a rabbit model of Gram-negative bacteremia. Rabbits were anesthetized, ventilated, and invasively monitored for 6 hours. Escherichia coli (4 x 10(9) CFU/kg) were infused over 2 hours in rabbits given rHDL (75 mg/kg) before the bacterial challenge. Antibiotics were not used in this model. The bacterial infusion resulted in a bacteremia that persisted until the end of the study. The sepsis-induced TNF peak was significantly lowered by rHDL treatment (10 +/- 3 ng/mL in rHDL treated versus 33 +/- 5 in controls, P = 0.001). Blood pressure, although not statistically significant, tended to be higher in the rHDL group. Acidosis was significantly attenuated up to 3 hours after the beginning of the bacterial challenge (7.39 +/- 0.05 versus 7.27 +/- 0.05 in controls, P = 0.041). rHDL treatment produced some transient beneficial effects in this model of persistent Gram-negative bacteremia. Additional studies, investigating the effects of rHDL in combination with antibiotics, are warranted. PMID- 8615561 TI - Hepatic hemangiomas in infants and children: a review of 30 cases. AB - From 1958 through 1992, 30 patients with hepatic hemangiomas were seen and treated at Children's Hospital Los Angeles. The majority of the patients were less 1 month of age (90 % younger than the first year of life) and there was no difference in sex distribution. Patients presented with coagulopathy, heart failure, abdominal mass, and respiratory distress. Eleven patients (33%) had hemangiomas in other sites. Fourteen patients were treated with steroid therapy. Of these, eight patients did not respond and received radiotherapy. Eleven patients who had the hemangioma confined to an anatomical lobe had resection of the hemangioma by liver lobectomy. Five of the most recent patients were successfully treated with hepatic artery embolization. Two other patients who were seen many years ago underwent diagnostic laparotomy and biopsy of the lesion before treatment with steroids. In one patient who presented with ruptured hepatic hemangioma, hepatic arterial ligation was performed. In another patient, seen recently, treatment with interferon alpha-2 was initiated, but the patient died. There were six deaths in the series. Four patients died of intractable congestive heart failure, steroids are given first. Course of steroid therapy may be repeated if necessary. Whereas formerly radiation therapy was added to the treatment of a patient resistant to steroids, therapeutic hepatic arterial embolization is a very good alternative for these patients. Surgical excision of the lesion can be performed by liver lobectomy if there is a solitary hemangioma within the boundaries of the surgical excision. Recently, in massive hemangioma with intractable thrombocytopenia, interferon alpha-2 therapy has been used, but so far our experience of this mode of therapy is limited. PMID- 8615562 TI - Technical results from the eversion technique of carotid endarterectomy. AB - A total of 167 carotid endarterectomies by the eversion technique were completed in 158 patients at a teaching hospital during the 6-year period ending July 1995. The average patient age was 66 years with a range of 39 to 89 years, and 99 (63%) were male. General anesthesia was employed routinely, and temporary indwelling shunts, were not used. Indications for endarterectomy included hemispheric transient ischemic attack (43), amaurosis fugax (20), stroke (41), and asymptomatic stenosis (63). Associated patient risk factors were not significantly different for men and women, and included diabetes mellitus (22%), tobacco abuse (72%), hypertension (69%), hypercholesterolemia (76%), cardiac disease (54%), and renal disease (21%). One (0.6%) permanent operative stroke and two (1%) 30-day hospital deaths occurred. Vascular laboratory follow-up was accomplished by duplex scanning with a documented sensitivity of 98 per cent in detecting a > or = 40 per cent stenosis. Eighty-nine per cent (148) of the 167 endarterectomies were tested at least once postoperatively. Overall laboratory follow-up averaged 17 months and ranged from one to 69 months. Residual stenosis, included perioperative thrombosis, occurred in 8 (5%) arteries. Recurrent stenosis was detected in four (2%) cases at 9, 24, 54, and 66 months after endarterectomy. Statistical analyses failed to implicate any specific patient risk factor, age, sex, or operative indication relevant to recurrent stenosis. Residual stenosis was correlated with younger patient age (P = 0.002), female gender (P = 0.12), and endarterectomy on the right side (P = 0.008). Carotid eversion endarterectomy appears to be a universally applicable, safe, and durable operative technique. PMID- 8615563 TI - Popliteal artery injury after total knee arthroplasty. AB - Total knee arthroplasty (TKA) is a commonly performed orthopedic procedure. The incidence of vascular complications after TKA is low. However, these complications may be debilitating, limb-threatening, and potentially avoidable. Our first patient, who had no preexisting vascular occlusive disease, developed an ischemic extremity after TKA. The second patient underwent TKA and was diagnosed with a severely ischemic limb 48 hours postoperatively. Both patients underwent above-knee to below-knee popliteal artery saphenous vein bypass grafting. Although limb salvage was obtained in both cases, one had a significantly neuropathic foot. In conclusion, long-term morbidity can be avoided by early identification and treatment of ischemia by bypass grafting. PMID- 8615564 TI - Small bowel obstruction from gastrointestinal histoplasmosis in acquired immune deficiency syndrome. AB - Disseminated histoplasmosis is a rare condition that is associated with an underlying immune disorder in approximately 25 per cent of patients. It often leads to GI histoplasmosis, but when the disease predominantly affects the GI tract few, if any, pulmonary symptoms appear. Although histoplasmosis of the gastrointestinal system has been described, it rarely causes a small bowel obstruction. In fact, a recent review of the English literature revealed 77 cases of gastrointestinal histoplasmosis, with only none having clinical presentation solely involving of the jejunum and ileum in acquired immune deficiency syndrome (AIDS) patients. At the time of urgent abdominal exploration, both patients had several areas of bowel strictures with subjacent mesenteric adenopathy. They required resection of small bowel segments. Pathology examination established the diagnosis of histoplasmosis, and both patients were discharged home after antifungal therapy. PMID- 8615565 TI - Are routine blood cultures effective in the evaluation of patients clinically diagnosed to have nosocomial pneumonia? AB - Blood cultures are a routine part of the evaluation of patients with clinically diagnosed nosocomial pneumonia; however, their utility has not been proven. We performed a retrospective chart review of 77 consecutive trauma patients diagnosed clinically to have nosocomial pneumonia at our level 1 trauma intensive care unit. Routine blood cultures did not alter the therapy of patients clinically diagnosed to have nosocomial pneumonia. We found the incidence of gram negative pneumonia was 71.4 per cent (55/77 patients) and gram positive pneumonia was 40.3 per cent (31/77 patients). Routine blood cultures taken at the time of diagnosis of nosocomial pneumonia fail to alter therapy. PMID- 8615566 TI - Major vascular injury as a complication of laparoscopic surgery: a report of three cases and review of the literature. AB - We retrospectively reviewed all 2201 laparoscopic procedures performed at our community teaching hospital in the 3-year period between 1992 and 1995. There were three major vascular injuries, for an incidence of 0.14 per cent. Vessels injured were the left common iliac vein, the right common iliac artery, and the left internal iliac artery. Two of the injuries were believed to involve the 10 mm trocar, whereas the third involved the Veress needle. Two of the injuries were All injuries were successfully repaired primarily. We believe such major vascular injuries can be avoided by strict use of proper techniques of needle and trocar insertion, and by using the open technique to create a pneumoperitoneum in patients with previous abdominal operations. Laparoscopic surgeons should be aware of the potential for injury to major vascular structures and constantly be prepared to identify and treat this potentially life-threatening complication. PMID- 8615567 TI - Sigmoid volvulus in pregnancy. AB - During pregnancy, intestinal obstruction due to sigmoid volvulus is extremely rare. Only 73 cases have been reported. A 24-year-old black woman, gravida 2, para 1, presented during Week 36 of an otherwise uneventful pregnancy, with intermittent abdominal pain and constipation, and no history of nausea, vomiting, fever, chills, previous medical problems, or prior abdominal surgery. Her previous pregnancy was a spontaneous vaginal delivery of a normal full-term neonate. On examination, she was afebrile, with abdominal tenderness. Laboratory studies revealed elevated WBC count of 13,500. She was admitted and given a Fleet enema, with no result or change in abdominal pain. Pain worsened; reexamination of her cervix revealed 3 cm dilation. After Pitocin augmentation, a viable male infant with Apgars of 7 and 9 was delivered. Postpartum, abdominal pain continued, with worsening abdominal distention. Radiograph revealed a massively distended colon. Physical examination 12 hours postdelivery indicated peritonitis. Exploratory laparotomy revealed volvulated, gangernous, massively distended sigmoid colon. The sigmoid colon was resected and Hartmann's colostomy performed. She was discharged on postoperative Day 4. Sigmoid volvulus complicating pregnancy is an uncommon and potentially devastating development that should be suspected with worsening abdominal pain and evidence of bowel obstruction. Prompt intervention is necessary to minimize maternal and fetal morbidity. PMID- 8615568 TI - Hydatid disease: a rare cause of adrenal cyst. AB - A case of hydatid cyst in the adrenal gland is presented. Eight cases have been previously reported. The diagnosis of an adrenal cyst is usually incidental, and the diagnosis of hydatid cyst is seldom made preoperatively. Surgical excision of the gland including the cyst is the treatment of choice. PMID- 8615569 TI - Laparoscopic cholecystectomy in the geriatric population. AB - Although the role of laparoscopic cholecystectomy (LC) as a safe and cost effective procedure has been ascertained, its role in the geriatric population, the majority of whom present with coexistent diseases, has yet to be defined. We retrospectively reviewed outcome parameters of 144 consecutive patients over age 65 undergoing LC, for both acute cholecystitis and symptomatic cholelithiasis. These results were compared with 72 patients having open cholecystectomy (OC) during the same time period and in the year preceding the introduction of LC. Groups were well matched with respect to age, age distribution indication for surgery, and underlying comorbid illnesses. Of those with symptomatic cholelithiasis, LC did not prolong operative time when compared with OC, but resulted in significantly earlier discharge (1.8 +/- 2.9 vs. 6.7 +/- 5.7 days (P < 0.0001)), with comparable hospital costs and with no increase in postoperative complications. With respect to acute cholecystitis, LC significantly prolonged operative time (105.8 +/- 40.8 vs. 78.1 +/- 28.5 minutes (P < 0.05)), but when successful, significantly reduced postoperative stay (4.2 +/- 3.8 vs. 7.5 +/- 2.3 days (P < 0.05)). There was no increase in postoperative complications in those having LC, and hospital costs were comparable with OC. Seven patients were converted from LC to OC; 4 of these (16%) were for acute cholecystitis versus a 2.5 per cent incidence of conversion for symptomatic cholelithiasis, and these resulted in prolonged hospital stays and costs. There was no incidence of hypotension/hypercarbia, despite a 64 per cent incidence of cardiopulmonary cardiopulmonary diseases in those having LC. There was a 14 per cent incidence of cardiopulmonary complications in those having LC in contrast to a 43 per cent incidence in OC. LC in the geriatric population is a safe procedure for symptomatic cholelithiasis. The procedure should be undertaken with caution in those with acute cholecystitis with a low threshold for either early conversion or primary OC. Finally, our results suggest that extensive hemodynamic monitoring is not indicated. PMID- 8615571 TI - Nonpalpable versus palpable invasive breast tumors treated with breast-conserving surgical management. AB - Most mammographically detected breast cancers are small, nonpalpable malignancies that should be amenable to cure by definitive breast-conserving therapy (BCT) consisting of tumor excision and postoperative radiation. We examined this hypothesis by retrospectively comparing the incidence of local recurrence and the rate of survival in breast cancer patients undergoing BCT for nonpalpable versus palpable lesions. Between 1982 and 1991, 345 patients at the John Wayne Cancer Institute, a large referral center for breast diseases, underwent BCT for invasive ductal and/or invasive lobular breast carcinomas: 120 (35%) had nonpalpable lesions detected by mammography (MG group), and 225 (65%) had palpable lesions detected by physical exam (PE group). The clinical and pathologic tumor status and the clinical outcome were recorded in each case. Median tumor size was significantly larger in PE than MG patients (2 cm versus 1 cm, P < 0.001). Only 29 percent of MG patients were premenopausal, compared with 51 percent of PE patients (P < 0.05). Axillary node involvement was more frequent in PE than MG patients (46% versus 19%, P < 0.01). Over a median follow-up of 58 months, local recurrence rates were 8 per cent for both MG and PE patients. In both groups, the incidence of local recurrence increased significantly when tumor was found in the margins of the resected breast specimen. In the MG group, the risk of local recurrence was significantly higher in premenopausal patients (P < 0.05). Survival was similar in both groups. The rate of local recurrence after BCT is the same for nonpalpable and palpable breast tumors. However, nonpalpable lesions have a lower rate of regional node metastases, which may improve survival. Both local recurrence and metastases seem to be related to tumor size. Tumor-free operative margins are the best predictor of local control. PMID- 8615570 TI - Ischemic preconditioning improves post-ischemic skeletal muscle function. AB - Ischemic preconditioning (IP), using one or more brief periods of ischemia before a sustained ischemia, represents a new approach to reduce tourniquet ischemia induced skeletal muscle damage. The aim of this study was to investigate the effect of IP on skeletal muscle function and high-energy phosphate tissues levels in a rodent model. IP protocols using one, two, or three preconditioning cycles were compared. IP was found to significantly improve force, performance, endurance, and contractility of postischemic skeletal muscle. The efficacy of IP induced protection was correlated with the number of preconditioning cycles. Preconditioning with three cycles resulted in a more effective protection as compared to one or two cycles. Three cycles of IP significantly improved force (409 +/- 63 versus 240 +/- 47 mN), performance (2546 +/- 481 versus 1081 +/- 242 mN*sec), endurance (46.7 +/- 5.0 versus 29.6 +/- 3.4 sec) and contractility (59.9 +/- 4.2 versus 38.7 +/- 5.1) in postischemic m.extensor dig. long. when compared to nonpreconditioned muscles. In contrast, high-energy phosphate tissue levels remained unchanged after three cycles of preconditioning. Altogether, this study describes, for the first time, the efficacy of IP to improve postischemic muscle function. The respective clinical potential warrants further exploration. PMID- 8615572 TI - Patient characteristics, treatment, and outcome of unknown primary melanoma in the United States for the years 1981 and 1987. AB - The American College of Surgeons performed a patient care and evaluation study of malignant melanoma for years 1981 and 1987 to determine the presenting symptoms, methods of evaluation, clinical management, and disease outcome. Previous reports on malignant melanoma of the skin, mucous membrane, and eye have been published. This report details the findings for 58 patients with malignant melanoma of an unknown primary diagnosed in 1981 and 87 patients diagnosed in 1987. The total number of patients was relatively small in comparison to all malignant melanoma patients. These patients were younger than the skin, ocular, and mucous membrane melanoma patients. There were significantly more males than females. When the anatomical site of a pathological positive node was known, it most frequently was in the axilla. Surgery, radiation therapy, and chemotherapy were frequently used in treatment of these patients. Although the overall prognosis is poor, some patients will have long term survival, and aggressive therapy should be considered for at least some of these patients. PMID- 8615573 TI - Simultaneous acute appendicitis in monozygotic twins: coincidence or genetic? AB - Acute appendicitis is the most common cause of an acute surgical abdomen. The authors present the first case of simultaneous acute appendicitis in monozygotic twins in the English literature and the fifth case reported in the world literature. Twin sisters, whose homozygosity is proven by DNA testing, presented with symptoms of acute appendicitis within 16 hours of each other. Both had acute appendicitis, one with free intraperitoneal perforation. These cases are presented, the world literature reviewed, and possible etiologic factors are discussed. PMID- 8615574 TI - Takayasu's arteritis: the middle aortic syndrome. AB - An isolated, long segment abdominal aortic coarctation is an infrequent occurrence. Although different names have been given to this entity, it is believed that all represent different stages of nonspecific inflammatory arteritis, or Takayasu's disease. Usually involving the arch and descending aorta, occasionally an isolated segment of the abdominal aorta is involved. Morbid presentation reflects the involvement with narrowing of visceral branches and aortic occlusive disease, as illustrated in this case. Operative intervention with aortic bypass and visceral revascularization is the treatment of choice for recurrent or extensive aortic involvement. PMID- 8615575 TI - Preoperative needle-localized parathyroidectomy for persistent secondary hyperparathyroidism. AB - Several preoperative localization techniques have been developed to assist the surgeon, with varying degrees of accuracy, in identifying the offending gland during reoperative parathyroid surgery. This is a case report of persistent secondary hyperparathyroidism that was treated with preoperative, computed tomography-guided needle localization followed by surgery. The patient underwent successful resection of a hyperplastic fifth parathyroid gland, and her calcium level decreased appropriately postoperatively. This case demonstrates a perioperative localization scheme that reduces operative time, reduces the risk of injury to surrounding structures, and helps to assure cure. PMID- 8615576 TI - Pneumothoraces secondary to blunt abdominal trauma: aids to plain film radiographic diagnosis and relationship to solid organ injury. AB - The objective was to identify subtle clues to the diagnosis of small pneumothoraces (PTX) in victims of blunt abdominal trauma (BAT) and to determine the relationship of PTX to solid organ injury. We retrospectively reviewed 1374 abdominal CT scans performed after BAT and assessed each for the presence of PTX and solid organ injury. In patients positive for PTX, the interpretation of the initial portable chest radiograph (PCXR) was noted and the film subsequently reviewed for subtle signs of PTX, presence of subcutaneous emphysema (SQE), and rib fractures. The initial PCXR of 50 consecutive blunt trauma admissions without CT evidence of PTX were reviewed for comparison. Eighty-four patients displayed PTX on CT, of whom 52 had initial PCXR available for review. Eight of 52 (15%) radiographs were initially interpreted as positive for PTX, whereas in our retrospective analysis, an additional 8 were discovered (total 31%). Of these, 23 of 52 (44%) had rib fractures, and 13 of 52 (25%) had SQE. Sixty-four of 1290 patients (5%) without CT findings of PTX sustained solid organ injury, whereas 15 of 84 (18%) with PTX had solid organ injury (significant by chi square analysis, P < 0.001). Although a large number of trauma-related pneumothoraces seen on CT will not be seen on admission PCXR, the search for rib fractures and SQE will enhance the sensitivity of detection. This has prognostic value, as the presence of PTX is related to a significantly increased incidence of abdominal solid organ injury. PMID- 8615577 TI - Endoscopic nasoenteral feeding tube placement following cardiothoracic surgery. AB - Our purpose was to evaluate the safety and efficacy of nasoenteral feeding tube placement in the cardiothoracic surgery patients. This is a retrospective analysis of 15 critically ill cardiothoracic surgery patients who underwent endoscopic placement of an enteral feeding tube beyond the proximal duodenum for maintenance of nutrition. Twenty-five entriflex 10-F nasoenteral tubes were placed endoscopically using a modified technique far into the distal duodenum, and the placement was confirmed radiographically. Mean patient age was 71 years. Seven were males and 8 were females. Eleven had undergone coronary artery bypass surgery, two aortic valve replacement, and two aortic aneurysm repair. The mean duration of tube function was 8.5 days and mean duration of tube feeding was 15.7 days. Of the total 15 patients, 7 required replacement due to various reasons, the most common being self extubation by the patient and malpositioning after initial placement. No cardiac complications or any other complications were noted directly related to the endoscopic procedure. In eight patients, the mean serum albumin level did not change [before: 2.5mg/dL, after: 2.6mg/dL] for the short time (avg. 8.5 days) the tube was functional. CONCLUSIONS: 1) Endoscopic placement of the nasoenteral tubes is a safe method of providing enteral nutrition in critically ill cardiothoracic surgery patients. 2) Benefits of nasoenteral tubes compared to nasogastric tubes remain unproven, and frequent repositioning of nasoenteral tubes is required. 3) A prospective comparison of nasoenteral and nasogastric tubes is warranted. PMID- 8615578 TI - Yellow fever in New Orleans. PMID- 8615579 TI - Pediatric exercise-induced laryngomalacia. AB - Laryngomalacia is a well-recognized cause of airway obstruction and inspiratory stridor in infants. As children grow and become more active, laryngomalacia may manifest in different, unexpected ways. Otherwise healthy athletes may generate enough inspiratory force to draw the aryepiglottic folds into the endolarynx, causing a subtotal glottic obstruction. This problem may be overlooked or attributed to asthma, lack of fitness, or functional abnormalities. The purpose of this report is to review the prevalence, diagnosis, and treatment of exercise induced laryngomalacia (EIL) in children and young adults. To study the incidence and diagnosis of this disorder, we examined 10 healthy volunteers. Fiberoptic laryngoscopy was used to videotape each subject's larynx during active exercise on a stationary bicycle. All volunteers demonstrated altered laryngeal dynamics with exercise, and 1 of the 10 volunteers developed laryngomalacia. Anatomically, it appears that the aryepiglottic fold serves as the critical point of obstruction. When symptomatic, laryngomalacia may be treated with supraglottoplasty. We have had experience with 2 EIL patients in the last 12 months who have undergone carbon dioxide laser microlaryngoscopy. Both patients benefited significantly from surgery. We conclude that EIL is underdiagnosed but responds well to treatment. PMID- 8615580 TI - Endoscopic laser-assisted reshaping of collapsed tracheal cartilage: a laboratory study. AB - Repair of anterior tracheal wall collapse is a common and troublesome problem encountered by the head and neck surgeon. The standard treatment calls for an open procedure with or without stenting, depending on the extent of the damage. To avoid the morbidity of the open procedure, a new concept of endoscopic cartilage reshaping was investigated in a laboratory animal study. It involved the application of 1.44-micron pulsed neodymium:yttrium-aluminum-garnet (Nd:YAG) laser at relatively low power to restructure without devitalizing cartilage. An in vivo study was done in six dogs to determine appropriate laser dosimetry in a model of tracheal wall collapse created by a tracheotomy. The deformed cartilage was treated endoscopically with a noncontact 1.44-micron Nd:YAG laser, at 2 to 4 W of power with a repetition rate of 20 Hz, in three animals. As a control, three animals had endoscopic cartilage incisions followed by stent placement. Six weeks postoperatively, both groups had an adequate airway lined by healthy mucosa. In the animals with stenting, however, there was stenosis formation due to scarring at both ends of the stent, with significant inflammatory response in the local area. This study shows that it is possible to use low-power laser energy to reshape cartilage without destroying its viability, and to restore the tracheal wall to a normal contour without ablation or vaporization. The reshaped cartilage will tend to retain its shape with functional elastic force, as seen in in vitro studies. These preliminary results are encouraging, and it seems reasonable to consider using the technique in selected clinical cases as an alternative to conventional open surgery. PMID- 8615581 TI - Vocal fold atrophy and its surgical treatment. AB - The effectiveness of surgical treatment for vocal fold atrophy of various causes was examined. Type I thyroplasty was performed on 31 patients with vocal fold atrophy, often bilaterally and occasionally combined with type III thyroplasty. Of these 31 patients, 3 patients underwent the operation twice, for a total of 34 operations. The surgery was found to be effective in improving the voice quality and the ease of phonation. When a scar or sulcus was present, the results were not as satisfactory. The intraoperative decision-making process as to which procedure to perform is extremely important for achieving an optimal voice, and should be based on the intraoperative voice quality, fiberoptic findings, and manual tests. In operations for vocal fold atrophy in which the vocal folds are mobile, a silicone shim should be firmly fixed to prevent migration. Overcorrection is generally recommended. No complications were encountered. It was often the increased ease of phonation rather than the improved voice quality that patients appreciated after surgery. PMID- 8615583 TI - Treatment of head and neck hemangiomas with recombinant interferon alpha 2B. AB - Fifteen patients with head and neck hemangiomas were treated with systemic recombinant interferon alpha 2b (rIFNalpha2b, Schering). There were 14 infants and 1 adult in the group, ranging in age from 5 weeks to 24 years old. Of the 15 patients in the group, 5 had involvement of the airway. Three of the patients had previously failed alternative systemic therapy. Twelve patients have had a beneficial response. Ten patients have completed the therapy and have been off interferon from 6 to 53 months without reappearance or progression of the disease. Two patients are currently on the therapy with resolving lesions. Three patients had minimal response and underwent successful surgical resection. No major toxicity was encountered during the therapy. Our experience demonstrates that rIFNalpha2b is a well-tolerated and effective therapy for hemangiomas of the head and neck that require intervention. PMID- 8615582 TI - Sulcus vocalis: a rational analytical approach to diagnosis and management. AB - The term sulcus vocalis has been applied to a spectrum of disorders ranging from minor vocal fold indentations to destructive lesions causing severe dysphonia. To clarify the pathophysiology and to develop a more rational approach to treatment, we report a series of sulcus patients including 20 surgical cases. Clinical and histopathologic analysis produced a clinically useful classification: type 1 is a physiologic variant accentuated by atrophy but with intact lamina propria; types 2 (sulcus vergeture) and 3 (sulcus vocalis) are characterized by severe dysphonia, loss of vibratory activity, and destruction of the functional superficial lamina propria. These latter cases respond favorably to microsurgery designed to remove destroyed tissue, release scar contracture, and promote mucosal redraping by regional undermining. Further study of the extracellular matrix of the superficial lamina propria (Reinke's space) might indicate a common pathway in the pathogenesis of sulcus deformities and other related benign vocal fold lesions. PMID- 8615584 TI - Laryngeal electromyography findings in idiopathic congenital bilateral vocal cord paralysis. AB - Children with idiopathic congenital bilateral vocal cord paralysis (BVCP) were investigated by electromyography (EMG) of the posterior cricoarytenoid and thyroarytenoid muscles to determine whether laryngeal EMG findings had diagnostic or prognostic significance. Four children between 3 weeks and 33 months of age were studied. Three had abductor paralysis and were tracheostomy-dependent, while the fourth had adductor paralysis requiring a feeding gastrostomy. Two of these patients also had other anomalies. Motor unit potentials showing phasic bursts with respiration were found in all four cases, while three children developed a full interference pattern on lightening of the anesthetic. Follow-up for between 37 and 52 months showed no significant clinical improvement in any of the patients. While the diagnosis of idiopathic congenital BVCP can represent a heterogeneous group of conditions, the findings suggest that normal laryngeal EMG findings may be a feature of idiopathic congenital BVCP but do not imply a favorable prognosis for early recovery. They may, however, have implications to explain the likely site of lesion in idiopathic congenital BVCP. PMID- 8615585 TI - Analysis of T cell receptor beta chain repertoire in middle ear effusions. AB - In order to elucidate the immune response in otitis media with effusion (OME), the polymerase chain reaction was employed to examine T cells in middle ear effusions in patients with OME for utilization of T cell receptor (TCR) variable region genes. Specimens of RNA were extracted from 13 ears of 12 patients (9 children and 3 adults). Oligonucleotide primers specific for individual TCR Vbeta gene families were used to amplify TCR gene products in each sample. Although the number of Vbeta families utilized by each sample varied from 1 family to 21, a few significant trends emerged. Eleven ears out of 13 expressed Vbeta7, which was the most frequently utilized (84.6%) Vbeta family among the 24 Vbeta families. In 5 of the 13 samples, the number of Vbeta families utilized was restricted to 1, which was Vbeta7 in all 5 samples. This result indicates the possibility that Vbeta7-bearing T cells in the middle ear are responding to a certain common antigen in some cases of OME. PMID- 8615586 TI - Postural changes in respiratory airflow pressure and resistance in nasal, hypopharyngeal, and pharyngeal airway in normal subjects. AB - We investigated the effect of posture on nasal and pharyngeal resistance in 12 healthy subjects studied during wakefulness. Airway pressure and airflow were measured with subjects seated and in dorsal and left lateral recumbency, during inspiration and expiration. We found that pharyngeal resistance was approximately four to six times lower than the nasal resistance. Only pharyngeal resistance was significantly increased upon assumption of a supine posture, from 0.02 +/- 0.01 Pa/mL per second when seated to 0.06 +/- 0.05 Pa/mL per second in dorsal recumbency and to 0.05 +/- 0.04 Pa/mL per second in left lateral recumbency. Mean nasal and pharyngeal resistances doubled upon assumption of a supine posture, but this difference was not statistically significant. There was no significant difference in pharyngeal resistance between inspiration and expiration. Finally, there was a strong linear relationship between pharyngeal pressure and pharyngeal resistance (r = .98, p<.0001). We concluded that in normal awake subjects 1) pharyngeal resistance increases with assumption of a supine posture, 2) the walls of the pharynx are not compliant enough to alter their resistance in response to inspiratory and expiratory pressure changes, and 3) it may be possible to infer pharyngeal resistance from measurements of pressure alone, without measurement of airflow. PMID- 8615587 TI - Effect of oxymetazoline nose drops on acute sinusitis in the rabbit. AB - This investigation was designed to evaluate the effect of the common topical nasal decongestant oxymetazoline hydrochloride on the early local tissue defense in an experimental bacterial infection. For that purpose, a bilateral infection of the maxillary sinus was induced in 14 rabbits. Nose drops (oxymetazoline) were instilled in one nasal cavity, and placebo in the other. After 48 hours, the degree of infection was judged by using a semiquantitative inflammatory score to evaluate histologic preparations of the maxillary sinuses. On the oxymetazoline treated side, we found a significantly higher degree of inflammation. We conclude that oxymetazoline nose drops, commonly used in acute rhinitis and sinusitis, interfere with the normal defense mechanisms during bacterially induced sinusitis, possibly by a decrease in mucosal blood flow. PMID- 8615588 TI - Schneiderian-type mucosal papillomas of the middle ear and mastoid. AB - Five cases of schneiderian-type mucosal papillomas arising in the middle ear space are reported. The patients were all women, ranging in age from 19 to 57 years (median, 31 years). Clinical complaints--unilateral conductive hearing loss, pain, or otorrhea--ranged from those lasting several months to recurrent problems spanning 20 years. All of the patients had a history of chronic otitis media predating the development of the papillomas; none of the patients had a history of sinonasal or nasopharyngeal schneiderian-type papillomas. Clinically, three patients had intact tympanic membranes, while the other two patients had perforated tympanic membranes through which a bulging polypoid mass was identified. Radiographic studies showed opacification of the middle ear space without evidence of osseous destruction. The intraoperative findings were of polypoid lesions filling the middle ear space, including involvement of the eustachian tube orifice. Histologically, the tumors were identical to sinonasal schneiderian papillomas. Immunohistochemical evaluation for human papillomavirus was negative. Surgical excision is the treatment of choice. In four of the patients, recurrent tumor was identified, necessitating additional surgery. In only one patient did the initial surgery result in complete ablation of the tumor. All patients are alive and free of recurrent disease over periods ranging from 6 months to 120 months (median, 84 months). PMID- 8615589 TI - Experimental otitis media with Moraxella (Branhamella) catarrhalis. AB - Two hundred fifty gerbils and 7 chinchillas were utilized in 11 experiments to determine the effect of inoculating viable and heat-killed suspensions of Moraxella (Branhamella) catarrhalis into the middle ear cavity. Development of otitis media was observed by otoscopy and histopathology. Gerbils were found to be susceptible to 2 x 10(4) viable M catarrhalis cells. Depending on the number of cells inoculated, the resulting untreated inflammation was a very mild to moderately severe, self-limiting disease with no permanent sequelae except in animals inoculated with high [10(6) to 10(7)] numbers of bacteria. Viable bacteria could not be isolated from the middle ears 24 hours after inflammation was induced. Heat-killed cells produced less severe acute inflammation with no permanent sequelae. We conclude 1) gerbils and chinchillas are susceptible to a self-limited inflammation caused by M catarrhalis, 2) no infection occurs, since viable bacteria cannot be recovered from middle ear aspirates, and 3) viable cells produce more severe inflammation than heat-killed cells. PMID- 8615590 TI - Human immunodeficiency virus infection presenting with lymphoepithelial cysts in a six-year-old child. PMID- 8615592 TI - Are papillary adenomas endolymphatic sac tumors? PMID- 8615591 TI - "Early" cancer of the larynx: the concept as defined by clinicians, pathologists, and biologists. AB - Since a great deal of confusion surrounds the different uses made by clinicians, pathologists, and biologists of the term, this paper clarifies the concept of "early" cancer of the larynx. Clinically, this is usually a glottic neoplasm in which full cordal mobility is still present; early supraglottic cancer is infrequent, and usually a chance finding. Whatever the site, early laryngeal cancer is a minimally invasive neoplastic lesion that does not invade the muscle or cartilage, but is still capable of metastasis. Being confined to the lamina propria, it is more than a carcinoma in situ but less than a deeply infiltrating carcinoma. Superficial extending carcinoma is therefore an early cancer. Biologically, early cancer belongs to stage II. PMID- 8615593 TI - Lack of suppression of tumor cell phenotype by overexpression of TIMP-3 in mouse JB6 tumor cells identification of a transfectant with increased tumorigenicity and invasiveness. AB - BACKGROUND: We have recently cloned mouse tissue inhibitor of metalloproteinases 3 (mTIMP-3) by the differential display technique and found that mTIMP-3 was expressed in preneoplastic but not in neoplastic mouse JB6 epidermal cells (Sun et at. Cancer Res. 54:11139, 1994). This down regulation of the gene is attributable at least in part to alteration in gene methylation (Sun et al., J. Biol. Chem., 270:19312, 1995). METHODS: To examine the potential role of TIMP-3 in two JB6 tumor cell model, we overexpressed mouse TIMP-3 in two JB6 tumor cell lines lacking endogenous mTIMP-3 expression. Stable transfectants from each line were selected and assayed for possible changes in tumor cell phenotype. RESULTS: Our results showed overexpression of mTIMP-3 in these two tumor lines did not change their ability to grow in soft agar, an assay for anchorage-independent growth, nor in nude mice, an in vivo tumorigenicity assay, nor to penetrate matrigel, an assay for invasiveness We, however, isolated a clone which is highly malignant was demonstrated by a) very short latent period for tumor formation; b) very fast tumor growth; and c) highly invasive in the matrigel assay. CONCLUSION: We conclude from this study that although TIMP-3 is not expressed in mouse JB6 tumor cells, overexpression by DNA transfection did not reverse tumor cell phenotype, suggesting a complex role for TIMP-3 in tumorigenesis. The highly malignant transfectant isolated by this study can be used as a tool for the cloning of dominant oncogenes as well as tumor suppressor genes. PMID- 8615594 TI - p53 involvement in activation of the cytokeratin 8 gene in tumor cell lines. AB - We show that the expression of the human cytokeratin 8 (CK8) gene is regulated by wild-type p53. DNA sequence data indicate that the 5' untranslated region of the CK8 gene contains a putative p53-like binding site. In this study we focused on the effect of the p53 protein on the regulation and expression of the CK8 gene. Cotransfection of the H358 p53-negative human lung cancer cell line with a CK8 promoter CAT expression vector and a plasmid expressing the wildtype p53 indicated that p53 induces CK8 expression. A transient assay in which a p53 negative cell line was cotransfected with a CK8 promoter CAT expression vector and a plasmid expressing wildtype or mutant p53 indicated that only wildtype p53 induces the CK8 promoter. Deletion of the putative p53-binding site from the CK8 promoter or introduction of mutations in the p53-binding sequences abolished the wild-type p53-mediated transactivation of CK8. A gel-retardation assay was used to measure DNA binding by the wild-type p53 protein. A 24-bp oligonucleotide corresponding to the putative p53 binding site was used for this assay. The wild type p53 protein bound weakly to this DNA sequence but much more strongly when three tandem repeat of the binding sequences was used. These studies suggest that the CK8 gene is a downstream target whose expression is regulated by wild-type p53. PMID- 8615595 TI - In vitro and in vivo evaluation of carboranyl uridines as boron delivery agents for neutron capture therapy. AB - The purpose of the present study was to evaluate 2' and 5'-O-(o-carboran-1 ylmethyl)uridine (CBU-2' and CBU-5') as delivery agents for Boron Neutron Capture Therapy (BNCT) of brain tumors. The in vitro cellular uptake, persistence, subcellular distribution and cytotoxicity, and in vivo biodistribution of CBU-2' have been studied as follows. Cellular uptake studies were carried out with the F98 rat glioma, U-87 MG human glioma, B16 melanoma, SP2/0 myeloma and MDCK fibroblasts. All tumor and non-tumor cell lines had high uptake of CBU-2' (46-75 ppm), indicating that uptake was not selective for neoplastic cells and was independent of cell proliferation. In vitro persistence studies showed high cellular retention of CBU-2' compared to sodium borocaptate (BSH), when cells were transferred from boron-containing to boron-free medium and cultured for an additional 24-48 hours. Subcellular fractionation revealed 75.6% of the recoverable boron was cell membrane associated, 15.6% was in the cytosol, and 8.8% was in the nuclear fraction, but no boron was detectable in the RNA and DNA fractions. F98 glioma cells were cultured in the presence of 3 metabolic inhibitors (rotenone, dipyridamole and NBMPR ?6-[(4-nitrobenzyl)thio]-9-beta-D ribofuranosylpurine?) and none of these blocked the cellular uptake of CBU-2' suggesting that uptake was neither energy nor nucleoside transport dependent. In vivo studies in F98 glioma bearing rats showed that CBU-2' in tumor attained concentrations of 8.0 +/- 2.1 micrograms B/g tissue, which was 13 x greater than that in normal brain of the ipsilateral and contralateral cerebral hemispheres (0.6 +/- 0.2 microgram B/g). The B levels, however, were still lower than the minimum 20-35 microgram B/g, which are required for in vivo BNCT. In summary, our in vitro and in vivo data indicate that CBU-2' was not sufficiently selective for in vivo targeting of brain tumors. However, CBU-2' and CBU-5' were highly toxic for F98 glioma cells in vitro (IC50 = 3 - 13 x 10(-5) M), as determined by measuring the uptake of 3H-thymidine, and the survival of F98 glioma cells using a clonogenic assay, which suggests that these compounds should be further evaluated as potential cytoreductive chemotherapeutic agents. PMID- 8615596 TI - The influence of sequential, in vitro passage on secretion of matrix metalloproteinases by human brain tumour cells. AB - Matrix metalloproteinases (MMP) are a family of zinc-dependent enzymes which degrade various components of the extracellular matrix (ECM) and play an important role in facilitating tumour cell invasion of the normal brain. The family includes the gelatinases, stromelysins and collagenases. Preliminary studies have shown that there is a differential expression four metalloproteinases in human brain tumour cell lines derived from neoplasms of various histological types and grades of malignancy. Morphological and antigenic changes in human glioma-derived cell lines over many serial in vitro passages have been reported in earlier studies. When established cell lines are maintained in culture over a long period, it is possible that the secretion of enzymes such as metalloproteinases may differ according to the passage level examined. This report presents a study on the secretion of four matrix metalloproteinases - interstitial collagenase (MMP-), 72-kDa and 92-kDa gelatinases (MMP-2 and MMP-9 respectively), and stromelysin (MMP-3) - in three human brain tumour-derived cell lines at sequentially increasing passage numbers, ranging from passage 2 to passage 50; foetal astrocytes were used as a positive control. Reverse zymography and substrate degradation analysis were employed to demonstrate the presence of these enzymes in cell- conditioned culture medium. Aminophenyl mercuric acetate (APMA) was used to activate latent zymogen. Results demonstrate that there is no definite pattern of change in the levels of enzyme secretion common to all cell lines studied. Instead, the fluctuations in APMA- activated metalloproteinase activity in serial passage seems to vary considerably depending on the cell line and the type of enzyme studied. The variation in metalloproteinase expression observed on serial passage may be due to in vitro selection processes or karyotype evolution where the transcription of either the enzyme and/or its inhibitor may be affected. Thus an imbalance of the two products could be occurring in serial passage. Ideally, experiments requiring the measurement of relative enzyme activities should use cultures as near to the biopsy stage as possible, i.e. very low passages, to avoid artifacts that may arise on prolonged culturing. PMID- 8615597 TI - Tenascin expression in adenoid basal cell carcinoma of the skin. AB - The immunoreactivity of tenascin, an extra cellular matrix glycoprotein, spatially and temporarily expressed in a site restricted manner during embryogenesis, wound healing and various benign and malignant tumours, was evaluated in 24 cases of adenoid basal cell carcinoma of the skin. The expression of tenascin showed three distinct patterns: firstly, the expression was confined to the stroma surrounding the tumour cells, secondly, to the stromal tissues of epithelial tumour foci and lumens of cribriform or cyst-like epithelial structures, and finally, a mixed pattern of these two was seen. It is suggested that in addition to the stromal reactivity, epithelial tumour cells may produce tenascin to deposit into the cribriform cystic lumens in the adenoid basal cell carcinoma in the skin. PMID- 8615598 TI - In vitro assessment for neurotoxicity of antitumor agents before local administration into central nervous system. AB - In vitro assays for neurotoxicity with the aid of cultured mouse fetal neurons and glial cells were applied to investigate neurotoxicity of recombinant murine interferon-beta (rMuIFN-beta). These data were compared with those for MTX, ADR, and ACNU. The range of concentrations of the drugs used in these experiments spanned their clinically achievable concentrations in patient serum (IFN-beta: 1 x 10(4) IU/ml, MTX: 100 micrograms/ml, ADR: 20 micrograms/ml, ACNU: 20 micrograms/ml). rMuIFN- beta damaged both neurons and glial cells at concentrations of more than 1 x 10(5) IU/ml but did not damage them at 1 x 10(4) IU/ml or less. Microtubule-associated protein 1A (MAP1A) staining was decreased in rMuIFN-beta-treated (more than 1 x 10(5) IU/ml) neutrons. In conclusion, since IFN-beta may have some neurotoxic effects at concentrations higher than 1 x 10(5) IU/ml, it should be administered carefully, as should other antitumor agents, into the tumor cavity in the CNS following surgery. PMID- 8615599 TI - Reduction of adriamycin cardiotoxicity by enoximone. AB - Several non catecholamine, non glycoside cardiotonic drugs have been described recently. New compounds include amrinone, sulmazole, milrinone and pimobendan. In an attempt to alleviate or prevent anthracycline toxicity, we have reported that these compounds reduce the negative effects of adriamycin, 4-epiadriamycin and esorubicin in isolated guinea pig atria. The present study reports the effects of a new cardiotonic agent: enoximone. Enoximone was administered after adriamycin (100 micrograms/ml) on the isolated and spontaneously beating atria, and on electrically driven left atria of guinea pig-in normodynamic and hypodynamic conditions. Exposure for 60 minutes to the antitumor drug causes a depression of contractile force (g) and its derivative versus time (dF/dt, as maximal rate of contractile force). The negative effects of adriamycin are antagonised by enoximone (100, 200 micrograms/ml). PMID- 8615600 TI - Cytofluorimetric analysis of gut-intraepithelial and mesenteric lymph node lymphocytes of tumour bearing mice fed with egg-white lysozyme. AB - The effects of the oral administration of 100 mg/kg/day egg-white lysozyme (EWL) on the expression of CD3, CD4, CD8 and CD25 antigens of lymphocytes harvested from IEL and mesenteric lymph nodes (MLNL) were tested in mice bearing MCa mammary carcinoma. Lysozyme, after oral administration, retains its enzymatic activity along the entire small bowel and almost 10% of the administered dose is recovered 1 hr after treatment in the middle of the jejunum. Correspondingly, the number of cells expressing the test antigens in MLNL is greater than in controls after a few days of treatment and is maintained high up to the end of treatment but returns to control values after treatment withdrawal; CD4:CD8 ratio is decreased by EWL in favour of CD8 positive cells. Treatment with EWL does not modify the ratio between CD4+ and CD8+ cells vs controls in IEL nor does it change the % of CD3 positive cells or the expression of IL-2 receptor at this level. These data support the existence of the induction of an immunity communication by EWL along the axis GALT-mesenteric lymph nodes which is in agreement with the reported effects of the oral administration of EWL on tumour growth in experimental systems and on host immunity in humans. PMID- 8615601 TI - Suppression by coffee cherry of the growth of spontaneous mammary tumours in SHN mice. AB - We previously found that coffee cherry (CC), residue after removal of coffee beans, significantly suppressed the development of spontaneous mammary tumours of mice. In this paper, the effects of CC on the growth of the palpable size of this type of tumour was examined. Free access as drinking water of 0.5% solution of the hot water extract of CC for 10 days resulted in a marked inhibition of the tumour growth: The percent changes of tumour sizes were 53.8 +/- 11.7% and 13.8 +/- 10.9% in the control and the experimental groups, respectively. Associated with this, thymidylate synthetase activity in the mammary tumours was significantly lower in the experimental group than in the control. Normal and preneoplastic mammary gland growth, body weight change and weights and structures of endocrine organs were only slightly affected by the treatment. The findings indicate that CC is promising as an antitumour agent. PMID- 8615602 TI - The glycosylation profile of interleukin-2 activated human lymphocytes correlates to their anti-tumor activity. AB - BACKGROUND: Natural killer cells display spontaneous, non-MHC-restricted cytotoxicity against tumour cells, which is strongly enhanced after incubation with IL-2. The molecular background of the increased anti-tumour activity of these lymphokine-activated killer cells is still only partly understood. MATERIALS AND METHODS: In this paper, investigation has been made of the correlation between cell surface glycosylation and anti-tumour activity of LAK cells by stimulating peripheral blood lymphocytes with interleukin-2, in the presence of inhibitors of N- and O-glycosylation. RESULTS: Inhibition of N- or O glycosylation of proteins during IL-2 activation leads to a 70-80% decrease in the cytolytic activity of LAK cells against K562 and Daudi tumour cells, coinciding with drastic alterations in their cell surface carbohydrate profile. CONCLUSION: The conclusion is drawn that there is a clear correlation between the glycosylation of LAK cell glycoproteins and their anti-tumour activity which points to the involvement of cell surface glycoconjugates in the development of LAK activity. PMID- 8615603 TI - Effects of interferon-beta on steroid receptors, prostaglandins and enzymatic activities in human endometrial cancer. AB - Steroid receptors, prostaglandin output and enzymatic activities were determined in explants derived from human endometrium exposed to natural interferon-beta (IFN-beta). Receptors and cell metabolism were evaluated before culturing the tissue fragments and after a 3-day treatment with varying concentrations of IFN beta. Total steroid receptor levels were unchanged when explants were set up, but there was a redistribution of both estrogen and progesterone receptors (ER and PR). A decrease in cytoplasmic receptors corresponded to an increase in receptor molecules within the nucleus. Treatment with low concentrations of IFN-beta caused a significant enhancement (p < 0.05) of ER and PR in neoplastic endometrium. In basal conditions the ratio between prostaglandin F2 alpha (Pgf2 alpha) and prostaglandin E2 (PgE2) was higher in normal than in neoplastic endometrium. The addition of low concentrations of IFN-beta to the culture medium determined a significant increase (p < 0.02) in PgF2 alpha and a parallel increase in the above ratio in neoplastic tissue, while no variation was found in normal endometrium. Analysis of the results concerning the variations in hormone related enzymatic activities due to IFN-B revealed a significant increase (p < 0.05) in 17 beta-hydroxy-steroid-dehydrogenase (17 beta-HSD) activity. The data presented here indicate that treatment with IFN-beta modifies those biological characteristics of neoplastic cells which are involved in hormone-responsiveness. PMID- 8615605 TI - Identification with monoclonal antibody 140.240 of a structural variant of melanotransferrin shed by human melanoma cell lines in vitro. AB - Shedding by cultured human melanoma cells of a well-characterised cell- surface glycoprotein antigen known as "melanotransferrin" was studied with two monoclonal antibodies, 140.240 and 96.5. By means of [35S]-cysteine metabolically-labelled melanoma cells and immunoprecipitation studies, identification was made, by 140.240 in the spent media of two of six melanoma cell lines, of a new molecule of 100-kDa, aside from the 88-kDa molecule. Only the 88-kDa shed molecule was detected in the remaining four melanoma cell lines with both antibodies. None of nine clonal sublines derived from the two melanoma cell lines were found to shed the 100-kDa or 88-kDa molecule exclusively. Both shed antigens were released spontaneously to the medium from the live melanoma cells rather than as a result of cell death and lysis, since there was no obvious cell death or debris in the spent medium nor in the monolayer cells detected at the time of spent medium collection. Digestion of the isolated 100-kDa and 88-kDa shed molecules with N glycanase followed by SDS-polyacrylamide gel electrophoresis resulted in the appearance of a single band of the 77-kDa molecule, which is deduced to be the polypeptide precursor of the cell-associated 87-kDa antigen. It is concluded that some melanoma lines shed the variant 100-kDa molecule, in addition to the 88-kDa molecule, and that both shed molecules and their cellular counterpart 87-kDa differ in their degrees of glycosylation. PMID- 8615604 TI - Effects of retinoic acid on cell differentiation and reversion toward normal in human endometrial adenocarcinoma (RL95-2) cells. AB - BACKGROUND: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation. METHODS: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin. RESULTS: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth. CONCLUSION: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype. PMID- 8615606 TI - Sensitivity to taxoid derivatives of a newly established human endometrioid ovarian adenocarcinoma radioresistant cell line. AB - An endometrioid ovarian adenocarcinoma cell line CAVEOC-2 was characterized. Maintained in monolayered culture, CAVEOC-2 cells exhibited a 33-hr doubling time. When xenografted into nude mice, these cells produced fast growing tumors. Colony-forming efficiency in agar was 50%. DNA index was 1.5 and cytogenetic analysis showed a triploid karyotype. CAVEOC-2 cells did not express mdr-1 gene and were chemosensitive to doxorubicin (IC50 = 1.82 +/- 0.76 mumol/l), paclitaxel (IC50 = 3.33 +/- 0.26 nmol/l) and docetaxel (IC50 = 0.68 +/- 0.28 nmol/l), while they showed an intermediate sensitivity to cisplatin (IC50 = 9.40 +/- 1.02 mumol/l). CAVEOC-2 cells seemed highly radioresistant (SF2 = 0.81, alpha = 0.02 Gy-1, beta = 0.025 Gy2, and MID = 4.31 Gy). Activities of glutathione S transferase and gamma-glutamyl transpeptidase were respectively 23.5- and 3.4- fold higher than those of sensitive A2780 cell line. These characteristics make the CAVEOC-2 cells a suitable model for the study of human endometrioid ovarian adenocarcinoma. PMID- 8615607 TI - Measurement of apoptotic fragments in growing hair follicles following gamma-ray irradiation in mice. AB - The usefulness of apoptotic fragments assay for investigating radiation response of hair follicles was examined. Frequency was defined as the ratio of the total number of apoptotic fragments to the number of hair follicles per section examined. The curve of dose-effect relationship for the data of apoptotic fragments was obtained by fitting the linear-quadratic model y= a+bD+cD2. When plotting on a linear scale against radiation dose, the line of best fit was y= 0.549 +/- 1.775) + 3.578 +/- 1.236)D + (-0.124 +/- 0.139)D2. The dose-response curves were linear-quadratic and a significant relationship was found between the frequency of apoptotic fragments and dose. The morphological findings of the irradiated groups were typical apoptotic fragments in the matrix region of hair follicles, but the spontaneous occurrence of apoptotic fragments was not observed. Since the apoptotic fragments was not observed. Since the apoptotic fragment assay is simple and reproducible in the whole body irradiation range of 0.5 to 8 Gy, it may be a good tool for evaluating the dose response of low dose radiation in vivo and provide a potentially valuable biological dosimeter for dose distribution determinations following accidental exposure. PMID- 8615609 TI - DNA ploidy, P53 expression, and cellular proliferation in normal epithelium and squamous dysplasia of non-cancerous and cancerous human oesophagi. AB - Ki67 expression, S-phase fraction, p53 immunoreactivity and DNA content were examined in morphologically normal mucosa and squamous dysplasia of both cancerous and non-cancerous human oesophagi in order to understand possible early events in the development of esophageal squamous cell carcinoma. 103 different foci from cancerous esophagi including 17 non-pathological epithelium, 10 mild, 17 moderate and 15 severe dysplasia, 14 intraepithelial carcinomas and 30 invasive squamous cell carcinomas were examined. Also studied were 57 biopsy specimens from cancer-free individuals, including 12 normal epithelia, 15 oesophagitis, and 16 mild, 11 moderate and 3 severe dysplasia. Areas of squamous dysplasia from both cancer-free and cancerous oesophagi were morphologically indistinguishable and both demonstrated increased cellular proliferation compared to normal or non-pathological epithelia. However, squamous dysplasia in cancerous oesophagi demonstrated significantly larger ki67 labelling indices and smaller S phase fractions than dysplasia in cancer-free patients. Squamous dysplasia in cancerous and non-cancerous oesophagi demonstrated an non-diploid DNA histogram in 67.9% and 43.3% respectively. However, dysplasia from cancer-free individuals demonstrated a non-diploid pattern with one or more peaks (Type I non-diploid histogram) and that from oesophageal cancer patients predominantly exhibited non diploid histograms without any distinctive peaks (Type II non-diploid histogram). Significant differences in the frequency of p53 positive foci were observed between dysplasia of cancer-free (23.3%) and cancerous (56.8%) oesophagi. IN cancerous oesophagi, dysplasia associated with Type II non-diploid histograms had a significantly larger number of p53-positive foci than those with diploid histograms or Type I non-diploid histograms. These results indicated that the biological features of squamous dysplasia were different between cancerous and non-cancerous human oesophagi despite indistinguishable morphological features. In addition, the combination of p53 immuno-histochemistry and DNA ploidy analysis may contribute to identify possible high-risk squamous dysplasia of the oesophagus. PMID- 8615608 TI - Transcatheter arterial embolization with cisplatin: apoptosis in VX2 tumour uterus transplants. AB - Effects of chemoembolization with cisplatin on gynecological malignancies were investigated using rabbit uterine tumour. 4 weeks after inoculation, the 35 rabbits were divided into 3 groups. In the experimental groups, the tumour tissue specimens were stained with hematoxylin and eosin, ApopTag stain and proliferating cell nuclear antigen (PCNA) dye. In two days the Pt level of tumour tissue in the transcatheter arterial chemoembolization (TACE) group was 1.97 times more than that in the intra-arterial (IA) infusion group (p < 0.001). Apoptotic index was 1.03% in the IA group versus 5.9% in the TACE (p < 0.001). The PCNA index was 86.6% in the IA group, compared with 8.6% in the TACE group (p < 0.001). Experiments reported here have revealed enhanced effects of the simultaneous action of nutrient restriction and increase concentration of drug caused by the vascular occlusion on cell death. PMID- 8615610 TI - Reversal of multidrug resistance by amitriptyline in vitro. AB - Amitriptyline, a tricyclic antidepressant, was able to reverse the multidrug resistance efflux pump of human colon cancer subline SW 620 and multidrug resistant (mdr) mouse lymphoma cells by decreasing rhodamine 123 efflux. The inhibitory effect of amitriptyline on the efflux pump was dose dependent. An investigation was made of the effects of mouse tumour necrosis factor (TNF) alpha and interferon (IFN) gamma on the efflux pump activity of mdr cells together with amitriptyline compared to the par cells (mdr-). After long-term cytokine pretreatment of mdr cells, the amitriptyline was more effective, due to some synergism between the amitriptyline and TNF-alpha. PMID- 8615611 TI - Interrelationships between microvessel density, expression of VEGF and resistance to doxorubicin of non-small lung cell carcinoma. AB - It has been shown that hypoxia can induce resistance to a number of antineoplastic agents. Since vessel density may be considered as an indirect measure of the oxygenation of tumours, in this study we analysed the relationship between tumour vascularity or vascular endothelial growth factor (VEGF) expression and drug resistance. Tumour specimens of 152 non-small cell lung carcinomas (NSCLC) of previously untreated patients were analysed for microvessel density by staining with factor VIII (von Willebrand factor) antibody and for expression of vascular endothelial growth factor (VEGF) using an anti-VEGF antibody. Both proteins were determined by immunohistochemistry and the expression was compared with the resistance to doxorubicin measured in vitro. Microvessel density was significantly reduced in resistant tumours when compared with sensitive tumours. Of the 98 tumours with low microvessel density 83 (85%) were resistant; whereas of the 54 tumours with high microvessel density only 34 (63%) were resistant (p = 0.004). Expression of VEGF was significantly lower in resistant than in sensitive lung carcinomas. Of the 102 tumors with low expression of VEGF, 87 (85%) were resistant; whereas of the 50 tumors with high expression only 30 (60%) were resistant (p = 0.0009). Corresponding results were obtained when the analysis was restricted to squamous cell lung carcinomas or adenocarcinomas of the lung. Analysis of microvessel density or VEGF expression and clinical data (stage, histology, metastasis) revealed no significant interrelationships. These data show clearly that poor microvessel density (vascularisation) and reduced expression of VEGF are linked with resistance to doxorubicin. PMID- 8615612 TI - Soluble interleukin-2 receptors in sera of children with primary malignant neoplasms. AB - PURPOSE: T lymphocyte activation regulates the autocrine type secretion of interleukin-2, a T cell growth factor and the de novo expression of its cell surface receptor (IL-2R). Afterwards, a special, truncated form of the IL-2R is secreted into the serum as a soluble molecule. Soluble interleukin-2 receptor (sIL-2R) in serum is non-specific marker of common activation of the cellular immune regulation. This study reports the results of an early diagnostic observation of sIL-2R level in the sera of 18 primary solid pediatric malignancies. PATIENTS AND METHODS: 18 children between the ages of 1 and 12 represented the Study Group of solid tumor bearing patients. 12 healthy children between the ages of 1 and 12 represented the Control Group. Concentrations of sIL 2R were detected in the sera of the children employing a double-antibody sandwich enzyme-linked immunosorbent assay (T Cell Sciences). RESULTS: Control Group concentrations of sIL-2R were in the range of 101.9 to 255.3 IU/mL with a mean value of 178.6 IU/mL. Serum levels of sIL-2R were markedly elevated in the sera of children with solid tumors (Study Group) to levels between 223.8 IU/mL and 927 IU/mL with a mean value of 575.4 IU/mL. CONCLUSIONS: 1) The shedding of cell surface receptors of immunological effector cells is a common physiological mechanism; 2) Presence of sIL-2R represents a sign of an overall activation of the effector elements of the host's cellular immune system; 3) An increase in the levels of sIL-2R represents an active phase of progression; conversely, decreasing levels indicate tumor regression; 4) Serum levels of sIL-2Rs may represent a useful laboratory parameter in choosing an efficient anti-cancer therapy. Thus, the level of sIL-2R in childhood solid tumor patients has a prognostic significance. PMID- 8615613 TI - DNA lesion-recognizing proteins and the p53 connection. AB - A great deal of the energy and time of a cell is invested in DNA repair activities. The first step in DNA repair pathways is recognition of the lesion on the DNA. The classical lesion-recognizing proteins interact with other repair proteins to form multiprotein complexes most notable of which are those that function in Nucleotide Excision Repair (NER). Proteins involved in lesion recognition include HMG1 and 2 recognizing cisplatin adducts but also maintaining active nucleosome structures and interacting with loops in cruciforms; HMG-box nuclear proteins; XPA and XPC lacking in xeroderma pigmentosum patients and involved in lesion recognition during NER; p53 recognizing strand breaks and insertion/deletion mismatches and causing arrest in the cell cycle; MSH2 mismatch repair protein identified as the human colon cancer gene product; and others including the transcription factor YB-1 that binds to depurinated DNA with a higher affinity compared with undamaged DNA. Other type of lesion-recognizing proteins are also repair enzymes like the O(6)-methylguanine-DNA methyltransferase and DNA glycosylases. Lesion recognition is an important process and might be the rate-limiting step in the overall repair process. PMID- 8615614 TI - In vitro antitumor activity of 4'-O-tetrahydropyranyladriamycin on human gastric cancer cells. AB - The antitumor activity of 4'-O-tetrahydropyranyladriamycin (THP-ADM) was assessed in established gastric cancer cell lines, including MKN-28, moderately differentiated adenocarcinoma and KATO-III, signet ring cell carcinoma and freshly excised human gastric cancer cells, using the MTT assay. The inhibition rates of THP-ADM were identical to those of adriamycin (ADM) in established gastric cancer cell lines, and the chemosensitivity of MKN-28 was higher than KATO-III. In fresh human gastric cancer cells obtained from 27 patients the inhibition rates of THP-ADM were identical to those of ADM, and there was a significant correlation of inhibition rates ADM and THP-ADM. These results indicate that THP-ADM should be a potent candidate to replace ADM in cancer chemotherapy. PMID- 8615615 TI - FK506 completely inhibits adriamycin efflux in the adriamycin-resistant strain of Ehrlich ascites tumor cells. AB - Adriamycin (ADR) is frequently used in the clinical treatment of cancer. ADR resistance is one of the most serious problems in cancer chemotherapy. Several factors have been demonstrated to be related to ADR resistance. Among these factors, the influx of ADR into cells and the efflux of ADR out of cells are important aspects of its effectiveness. In this study, we investigated the influence of FK506 on the ADR accumulation and retention in the ADR-resistant strain of Ehrlich tumor cells. The ADR accumulation in the ADR--resistant strain was about 40% of that of wild EAT cells. The addition of 50 muM FK506 inhibited the efflux of ADR completely. ADR accumulation with the addition of 50 muM FK-506 to the ADR-resistant strain was about 80% of that found in the wild strain. Thus, it was suggested that the increase in ADR accumulation by the ADR resistant strain by about 40%, when 50 muM FK-506 was added might be due to complete inhibition of ADR efflux by P-glycoprotein, that the decrease of about 20% in ADR accumulation of the ADR-resistant strain compared to that in parent cells might be caused by problems with ADR influx. Therefore, it is necessary that the mechanisms which cause the decrease of ADR influx in the ADR -resistant strain be clarified. PMID- 8615617 TI - In vitro cytotoxicity, pharmacokinetics and ex vivo pharmacodynamics of a new platinum compound, cis-malonato [(4R,5R-4,5-bis(aminomethyl)-1,3-dioxolane-2 spiro-1'-cyclopentane] platinum (II). AB - The in vitro cytotoxicity of a new platinum complex, cis-malonato [(4R, 5R)-4,5 bis(aminomethyl)-1,3-dioxolane 2- spiro 1' cyclo-pentane]platinum(II) (SKI 2054R) and cisplatin (CDDP) was evaluated against two human stomach adenocarcinoma cell lines (MKN-45 and KATO III) and a human lung adenocarcinoma cell line (PC-14). The in vitro 50% inhibitory concentration (IC50) values of SKI 2054R and CDDP against MKN-45, KATO III, and PC-14 were 1.21 and 0.51, 2.11 and 0.83, and 2.90 and 0.77 micrograms/ml, respectively. The pharma-cokinetic and ex vivo pharmacodynamic studies on SKI 2054R and CDDP were performed in beagle dogs. Equitoxic doses of SKI 2054R and CDDP (7.0 and 3.0 mg/kg, respectively) were administered i.v. bolus to beagle dose in a randomized crossover study. Plasma samples were analyzed for platinum by flameless atomic absorption spectrophotometry. Plasma concentrations of total and ultrafiltrable platinum for the two drugs declined in a biexponential fashion. The mean area under the concentration-tine curve (AUC(0)--> infinity) determined for ultrafiltrable platinum derived from SKI 2054R, as an active component was 6.61 +/- 2.34 micrograms . h/ml (mean +/- S.D.), with an initial half-life of 0.26 +/- 0.14 h, a terminal half-life of 1.57 +/- 0.71 hour, a total clearance of 17.65 +/- 4.99 ml/min/kg, and a steady-state volume of distribution of 1.46 +/- 0.11 l/kg. The ex vivo antitumor activity of SKI 2054R was assessed using the ultrafiltrable plasma against MKN-45, KATO III, and PC-14 by tetrazolium-dye (MTT) assay and was compared with that of CDDP using the antitumor index (ATI) determined from the ex vivo pharmacodynamic results of inhibition rates (%) versus time curves. The mean ATI value recorded for SKI 2054R was higher than that noted for CDDP; however, statistical difference was not observed between SKI 2054R and CDDP, suggesting that the antitumor activity of SKI 2054R is comparable to that of CDDP. These results suggest that SKI 2054R is a new platinum complex which is worth being evaluated further. PMID- 8615616 TI - The non-variation in radiosensitivity of different proliferative states of human glioma cells. AB - The radiosensitivity of the human glioma cell line U-343MGa, while growing as spheroids and as conventional monolayers, was studied. The spheroids were first irradiated with 60Co photons, and the radiosensitivity was then analyzed in different cell layers with varying proliferative activity. The different cell layers were isolated by automated sequential trypsinization. A relatively high radioresistance was found in all analysed cell layers, and the inner, mainly quiescent cells, were as resistant as the outer proliferating cells. It was, in parallel experiments on monolayers, shown that serum starved quiescent cells were as resistant as proliferating cells, and that direct cellular cloning did not give lower cell survival values than delayed cloning, as has been shown for other cell types. Thus, the studied glioma cells were equally radioresistant under all tested proliferative conditions when conventional low-LET radiation was applied. The spheroids were then irradiated with cyclotron accelerated helium ions with an intermediate ionization density, LET approximately equal to 55 keV/microns, and this gave a clear decrease in survival in all cell layers. PMID- 8615619 TI - Activin A inhibits growth and hexamethylene bisacetamide-induced differentiation in human retinoblastoma Y-79 cells. AB - Activin regulates the growth and differentiation of a variety of cells and is a member of the transforming growth factor-beta (TGF-beta) family. Previously, we found that the retinoblastoma cell line Y-79 expresses both activins and activin receptors, suggesting that activin may have an autocrine function in these cells. In this study, the effects of exogeneous activin A on cultured Y-79 cells were examined. The results demonstrate that activin A inhibits hexamethylene bisacetamide (HMBA) -induced Y-79 cell differentiation in both serum-containing and serum-free medium. Activin A also inhibits Y-79 cell growth in serum containing medium but not in serum-free medium. PMID- 8615618 TI - Differences in reactivity to cyclosporin A and interferon-gamma of normal and HPV transformed keratinocytes. AB - In humans, cyclosporin A (CsA) avoids organ allograft rejection but induces skin carcinomas after long term immunosuppressive treatment; some of these lesions contain human papillomavirus (HPV) DNA. Interferon-gamma (IFN-gamma) is sometimes used in local treatment of persistent or recurrent lesions in normal population. In vivo, both drugs have an effect on keratinocytes which remains unclear. Therefore, their effect was studied on in vitro models of normal or HPV transformed epithelial cell cultures. After exposure of proliferating cells for 1 3 days to 0.5-16 micrograms/ml CsA and 5-160 U/ml IFN-gamma, no cytotoxicity was observed; cell growth was inhibited; cell morphology was altered with CsA and cytoplasmic vacuoles were seen in some cells. Changes in the cell cycle were mainly obtained after treatment with 8 micrograms/ml CsA or 160 U/ml IFN-gamma, with an accumulation in S-phase especially in HPV-transformed cells. Thus, CsA and IFN-gamma affected, normal and HPV-transformed epithelial cells, differently. PMID- 8615620 TI - Growth regulation of multidrug resistant ovarian cancer cells by 1D7 monoclonal antibody. AB - The small molecular weight protein (p7) was overexpressed in the human ovarian carcinoma cell lines, SKVLB600 (selected for vinblastine resistance from SKOV3 cells). OVCAR 4/ADR100 (selected for doxorubicin resistance from NIH:OVCAR4) and (OVCAR4/VBL200 (selected for vinblastine resistance from NIH:OVCAR4). Trace amounts of the protein were also found in the parent cell lines, SKOV3 and NIH: OVCAR4. An anti-p7 monoclonal antibody (1D7) specifically inhibited the proliferation of the drug resistant cancer cells. In order to assess the function of p7, we established a new cell line, SKVLB600R, which was maintained in drug free medium for 16 months. This cell line stopped expressing Pgp that is responsible for its resistance to cytotoxic drugs, but continued to overexpress p7. The proliferation of SKVLB600R was also inhibited by 1D7. Our results indicate that p7 may be specifically involved in the proliferation of multidrug resistant cells. PMID- 8615621 TI - Expression of cytokeratin 8 in basal cell carcinoma: a comparative immunohistochemical and immunoelectron microscopy study. AB - The comparative study reported here was undertaken in order to resolve the discrepancies in the detection of cytokeratin (Ck) 8 reported in previous studies. The expression of Ck 8 was compared in 6 basal cell carcinomas (BCCs) using immunohistochemical and immunoelectron microscopic techniques and a panel of 4 different commercially available monoclonal antibodies (MoAbs). The results of this comparative study demonstrated not only that the consistent expression of Ck 8 using one of the MoAbs in immunohistochemistry was confirmed by immunoelectron microscopy, but that the inconsistent expression of Ck 8 observed using two other MoAbs was also confirmed. One of the MoAbs did not show any staining at all. The inability of this MoAb to detect the expression of Ck 8 using either of the techniques also indicated that this MoAb may be directed against an epitope of Ck8 that is not detectable in BCC in situ. PMID- 8615622 TI - Coexpression of epidermal growth factor receptor and TGF-alpha and survival in upper aerodigestive tract cancer. AB - In order to find a prognostic marker for the course of disease in head and neck cancer we hypothesized that patients with rapid disease progress would produce increased levels of transforming growth factor alpha (TGF-alpha) and its cell surface receptor, epidermal growth factor receptor (EGFR). Using molecular biological techniques, we examined the incidence of TGF-alpha and EGFR overexpression in 43 patients with tumors of the head and neck. The expression data was correlated with the course of disease in a 4 year follow-up. The tumors were classified into four groups according to the EGFR status: Group 1, no expression for EGFR (15 samples); group 2, expression level 10 for EGFR (18 samples); group 3, expression level 50 for EGFR (7 samples) and group 4, expression level 100 for EGFR (3 samples). Expression for the TGF-alpha protein was only detected in group 4. There was a significant correlation with EGFR/TGF alpha overexpression in group 4 and survival compared when with group 3 (p < 0.01) and group 1 (p < 0.05). The mean survival for group 1 to 4 was 27, 23, 34 and 10 months, respectively. The analysis of all patients revealed that the patients who expressed EGFR as well as TGF-alpha had the poorest prognosis. Increased production of TGF-alpha and EGFR in tumors of the head and neck may serve both as a marker for tumor progression and as a target for therapy (e.g. inhibition of the autocrine loop, blockage of TGF-alpha binding). PMID- 8615623 TI - Protein kinase C isoenzymes, p53, accumulation of rhodamine 123, glutathione-S transferase, topoisomerase II and MRP in multidrug resistant cell lines. AB - We investigated whether the expression of protein kinase C (PKC) isoenzymes, topoisomerase II alpha, II beta, multidrug resistance associated protein (MRP), p53 or the activity of glutathione-S- transferase (GST) are additional factors contributing to the resistance mediated by multidrug resistance gene 1 (mdr 1). the cell lines employed for these studies were human lymphoblastoid CCRF cells selected for resistance with actinomycin D, vincristine and adriamycin, KB-3-1 and matched resistant KB-8-5 and KB-C1 cells (selected with colchicine), and a HeLa cell line, in which the resistance was obtained by transfection with the mdr1-gene. Analysis of PKC isozymes showed that there is no correlation of a specific isoenzyme with resistance, although minor differences in the expression were observed. In vincristine and adriamycin selected cells, topoisomerase II alpha- and II beta-MRNA levels were reduced, and in vincristine selected cells the MRP-mRNA was elevated compared with the sensitive line. In KB cells the levels of topoisomerase II alpha and II beta mRNA were increasing with the resistance. Expression of p53 did not correlate with Pgp levels. In summary, MRP and topoisomerase II may contribute to the mdr1 -mediated resistance in some cell lines, but PKC, p53 and GST seem to be of minor or no importance. PMID- 8615624 TI - Effect of heat-drug sequences on thermoenhancement and uptake of cis-DDP in human pharyngeal carcinoma. AB - To discover the point of maximum interactive effect, we examined the time sequence of high (above (42.5 degrees C) or low (below 42.5 degrees C) hyperthermia and cis-diammine dichloroplatinum (II) (CDDP). Simultaneous or post hyperthermic CDDP (0.5 micrograms/ml) treatment at 43 degrees C resulted in a slight synergistic effect, with thermoenhancement ratios (TER) of 1.42 or 1.38, respectively, and there was a significant increase CDDP uptake after both combinations compared with pre-hyperthermic CDDP treatment. However, at 42 degrees C, the maximal interaction (TER = 8.57) was obtained when KB cells were simultaneously heated with CDDP, there was also a significant increase of CDDP uptake by simultaneous procedures compared with pre or post-hyperthermic CDDP treatment. These results indicate that simultaneous or post-hyperthermic CDDP treatment for high-hyperthermia and simultaneous CDDP treatment for low hyperthermia are the most effective means of CDDP thermochemotherapy with hyperthermia. PMID- 8615625 TI - Enhancement of antitumor effects of new analogue of platinum, cis-diammine (glycolato) platinum (254-S) combined with hyperthermia in vivo. AB - Cis-diammine (glycolato) platinum (254-S) is a second generation platinum complex with reduced nephrotoxicity. In this study the antitumor effect of 254-S combined with hyperthermia in vivo in mice was studied. On the 6th day after inoculation of the Ehrlich ascites tumour cells, 254-S (15mg/kg) was administered intraperitoneally (i.p.) and hyperthermia was induced by using a circulating water bath at 42.5 degrees C for 45 min. The antitumour effect was evaluated by relative tumour volume. Furthermore, platinum concentration in tumour tissue was determined by using atomic absorption spectrophotometry. The most effective condition was found to be the combination of 254-S with hyperthermia. A significantly higher concentration of platinum in the tumour tissue was observed when treatment with 254-S was combined with hyperthermia, than with treatment using 254-S alone. Our study suggested that the accumulation of 254-S in the tumour tissue, and its retention at a high concentration within the tumour tissue long term was one of the reasons for the enhancement of antitumour effect of 254 S treatment when combined with hyperthermia. PMID- 8615627 TI - Inhibitory effects of plant growth regulators on xanthine oxidase. AB - Several plant hormones and analogues were tested for their inhibitory effects on xanthine oxidase. The flavoprotein enzyme, xanthine oxidase, catalyses the oxidation of hypoxanthine to xanthine and then xanthine to uric acid which has lambda max 295 nm. Uric acid was thus the basis for a spectrophotometric assay of the activity of xanthine oxidase. The results showed that trans-zeatin displayed the strongest activity (IC50 = 23.5 muM) on xanthine oxidase inhibition, followed by indole-3-acrylic acid (IC50 = 136.0 muM) and then by the mixed isomers of zeatin (trans-zeatin and cis-zeatin) (IC50 = 198.65 muM). Trans-zeatin induced an uncompetitive inhibition of the enzyme with respect to the substrate xanthine and the apparent inhibition constant (Ki) was 5.09 muM. However, zeatin riboside was inactive. Since xanthine oxidase serum levels are increased in hepatitis, mild hepatic intoxication, tumours brain tissues, and DNA damage induced by cytotoxic agents, it is expected that trans-zeatin may be useful for the treatment of these diseases as well as gout which is caused by deposition of uric acid in the joints and oxidative damage of tissue caused by generation of superoxide anion radical. PMID- 8615626 TI - Lectin-affinity electrophoresis for the detection of AFP microheterogeneities in patients with hepatocellular carcinoma. AB - Our aims were to ascertain the analytical performance and diagnostic efficiency of a new lectin-affinity electrophoresis and antibody-affinity blotting method for separating and identifying fucosylated AFP. We also compared the diagnostic efficiency of fucosylated AFP with that of total AFP in the early diagnosis of neoplastic liver diseases. In 32 patients with hepatocellular carcinoma (HCC) and in 19 subjects with benign liver diseases, total AFP was assayed using an enzyme immunoassay, while AFP fractions (L1, L2 and to cathode L3 fucosylated) were separated using lectin-affinity electrophoresis followed by antibody-affinity blotting. No significant difference was found between the total AFP values of patients with HCC and those of patients with benign liver disease (p > 0.05). In patients with HCC the fucosylation index of AFP (L3%) was significantly higher (p < 0.0001) than that in patients with benign liver disease. Our findings suggest that fucosylated AFP is of diagnostic value in differentiating between hepatocellular carcinoma and benign hepatic diseases, and that its measurement may be useful in the early detection of hepatocarcinoma in patients with chronic liver disease. PMID- 8615628 TI - Effect of paclitaxel and Cremophor EL on mast cell histamine secretion and their interaction with adriamycin. AB - The effect of paclitaxel and of its solvent Cremophor EL, a polyoxyethylated castor oil on histamine release in rat peritoneal mast cells has been tested. Paclitaxel dissolved in Cremophor EL/ethanol and Cremophor EL alone induced a moderate histamine release; this secretory activity is provoked by the solvent Cremophor EL but is so scanty that it seems most unlikely to be responsible of the severe hypersensitivity reactions frequently caused by this drug. Since in a number of protocols paclitaxel is used in combination with anthracyclines, and since anthracyclines are well known histamine releasing agents, the effect of the combination of these substances was also studied. Cremophor EL did not increase the exocytotic activity of adriamycin; on the contrary, the release of histamine induced by this anthracycline was significantly limited. This is an interesting observation since it has been suggested that anthracycline-induced histamine release is involved in the pathogenesis of the cardiotoxicity caused by these drugs. Low concentrations of paclitaxel in Cremophor EL and Cremophor EL alone inhibited adriamycin uptake into the granules of mast cells, a process mediated by an active transport system, recently identified with the P-170 glycoprotein pump. PMID- 8615630 TI - Effects of gallium chloride on changes in action potentials and contraction in guinea pig ventricular muscle. AB - The electrophysiological effects of gallium chloride (Ga ) and its activity on arrhythmias induced by digitalis were investigated in guinea pig papillary muscle. KCl microelectrodes were used to record transmembrane electrical activity from Purkinje cells from the papillary muscle of guinea pig hearts during superfusion and electrical stimulation in vitro at 37 degrees C. Myocardial contractility was continuously monitored. Ga was superfused alone in cumulative concentrations(4.5 . 10(-5) to 3.6 . 10(-4) M). Arrhythmias were induced by a superfusion of Digitoxin (7.5 . 10 (-7) M). A superfusion of Ga (4.5 . 10(-5), 9 . 10(-5), 1.8 . 10(-4) M and 3.6 . 10(-4) M) was started 20 min later and maintained for 70 min. Ga alone produced a dose dependent reduction of action potential duration and contractility. Ga potentiated the decrease in the amplitude and duration of the action potential induced by Digitoxin. The incidence of arrhythmias was immediately reduced by two concentrations of Ga (4.5 . 10(-5) and 9 . 10(-5) M) in the digitalized papillary muscles. It is concluded that Ga inhibits calcium movements and has negative inotropic and antidysrhythmic effects. PMID- 8615629 TI - Tumorigenic behaviour of c-Ha-ras oncogene transfected CaCo 2 cells is associated with increased proteolytic potency. AB - BACKGROUND: Point mutations within the family of the ras genes are detected in approximately 50% of human colorectal adenomas and carcinomas. Therefore, it is generally accepted that the occurrence of ras-point mutations constitute an important step in colorectal carcinogenesis. In addition, many studies have demonstrated that the tumorigenicity of the human colorectal carcinoma cell line, CaCo 2, strongly increases after transfection with the c-Ha-ras oncogene. This cell line is suitable for gaining more insight into the mechanism of c-Ha-ras induced tumorigenesis. MATERIAL AND METHODS: Proliferation, differentiation, and proteolytic capacity of c-Ha-ras oncogene transfected CaCo 2 cells were studied in vitro. RESULTS: It was found that gelatinolytic capacity and production of urokinasetype plasminogen activator increased, whereas the production of tissue type plasminogen activator was similar. Proliferative activity, as measured by the potential doubling time, did not alter. The expression of the differentiation markers sucraseiso-maltase, mucin, and chromogranin A was not different from that of the parental CaCo 2 cell line, which indicates that an increased tumorigenic capacity of c Ha-ras oncogene transfected CaCo 2 cells is not accompanied by loss of differentiation. CONCLUSION: These data demonstrate that the highly increased tumorigenic capacity of c Ha-ras oncogene-transfected CaCo 2 cells is associated with an enchanced proteolytic capacity. PMID- 8615631 TI - Novel trans platinum complexes: comparative in vitro and in vivo activity against platinum-sensitive and resistant murine tumours. AB - Two pairs of cis/trans platinum complexes, JM118 (cis-ammine(cyclohexylamine) dichloro platinum(II)) and its trans counterpart, JM334 and JM149 (cis ammine(cyclohexylamine) dichloro-dihydroxo platinum(IV)) and its trans counterpart JM335 have been evaluated (both in vitro and in vivo) against two murine tumour models of historical importance in the discovery of novel platinum drugs; the ADJ/PC6 plasmacytoma and the L1210 leukaemia and sublines selected for resistance to platinum drugs. In vitro, results showed that the trans complexes induced comparable growth inhibitory properties to those observed for cisplatin and their respective cis isomers. Moreover, retention of activity was observed in a series of 5 acquired platinum drug (cisplatin, carboplatin, iproplatin, tetraplatin and JM149)-resistant L1210 sublines whereas at least partial cross resistance was observed to the cis isomer JM149 in the acquired carboplatin and iproplatin-resistant lines (in addition to being 11-fold resistant in the line selected for resistance to JM149 itself). In vivo, JM355 showed activity against both the ADJ/PC6 and L1210 models of acquired cisplatin resistance. Furthermore, JM355 was active against an ADJ/PC6 subline possessing resistance to iproplatin and a L1210 subline possessing resistance to its cis isomer JM149. Interestingly, the trans platinum(II) counterpart of JM335(JM334) was inactive in vivo. These data indicate that the trans platinum(IV) complex JM335 possess several in vitro growth inhibitory- and in vivo antitumour properties which are distinct from those observed for cisplatin (or its cis isomer). Thus, JM335 contravenes the original structure-activity rules determined for platinum-containing compounds and, because of its level of activity against cisplatin-resistant tumours, establishes the complex as of interest in the search for new platinum drugs active against cisplatin-resistant disease. PMID- 8615632 TI - Chlorpromazine-induced hypothermia: effect on tumour progression and survival in mice. AB - The effect of chlorpromazine on tumour progression and survival, when used as a modifier of chemotherapy response and toxicity, was studied in mice. The dosage was 15 mg/kg i.p., tested to produce rectal hypothermia within 1 hour, lasting for at least 8 hours, at an ambient temperature of 28 degrees C. The drug or saline was given to inbred C57Bl/6J mice four days previously inoculated i.v. with MCG101-AA sarcoma cells. Little acute toxicity, except for the intended hypothermia, was observed. No influence on survival could be discerned, nor any influence upon distribution of tumour deposits. In this tumour model system, chlorpromazine can be used as a chemotherapy response modifier without significant effects per se. PMID- 8615634 TI - Loss of heterozygosity in chromosome 4q12-q13 in hepatocellular carcinoma in southern African blacks. AB - Paired samples of hepatocellular carcinoma and non-tumorous hepatic tissue from 12 southern African blacks were examined for loss of heterozygosity at two loci (D4S409 and D4S392) in chromosome 4q12-q13 (mapping to the same region as the locus for the gene for alpha-fetoprotein). Deoxyribonucleic acid was extracted from carcinoma and non-tumorous tissues and amplified using the polymerase chain reaction. The primers used were based on flanking regions of minisatellite DNA specific for the loci. Products of PCR amplification were separated by denaturing polyacrylamide gel electrophoresis and analysed for allelic losses. At the D4S409 locus one of seven informative samples showed loss of heterozygosity, whereas none of eight informative samples showed loss of heterozygosity at the D4S392 locus. If these findings are considered together with those previously reported in hepatocellular carcinomas from black Africans, the prevalence of loss heterozygosity in chromosome 4q12-q13 in hepatocellular carcinoma in southern African blacks (33 per cent) is the same as that in tumours from Japanese patients. Loss of heterozygosity at chromosome 4q12-q13 is infrequent during hepatocellular carcinogenesis in southern African blacks. PMID- 8615633 TI - Calcipotriol (MC 903), a synthetic derivative of vitamin D3 stimulates differentiation of squamous carcinoma cell line in the raft culture. AB - We investigated whether calcipotriol, a synthetic derivative of vitamin D3 has the ability to correct defects in the control of proliferation and differentiation of human squamous carcinoma cells using the raft culture of SCC 13 cell line. Calcipotriol treatment at concentrations of 10(-8)-10(-6) M considerably enhanced terminal differentiation of SCC 13 cells, as shown by the appearance of enucleated-eosinophilic cells as well as granular cells in their upper cell layers. Immunohistochemical staining showed marked increases in the differentiation of marker proteins such as keratin 1, involucrin, or filaggrin expressing cells in their upper layers. The elevated expression at protein level was confirmed by immunoblotting analysis. Furthermore, calcipotriol also stimulated basal cell marker proteins such as keratin 14 and EGF receptor. However, the numbers of basal marker expressing cells within the architecture of SCC 13 raft culture were markedly reduced upon calcipotriol treatment, and their localization was mainly restricted in the innermost cell layer. In addition, calcipotriol stimulated EGF receptor biosynthesis for the first 16 hours post treatment and subsequently inhibited [3H]-thymidine incorporation of SCC 13 cells at 24 hours. In this study, we have clearly demonstrated that the long term application of calcipotriol considerably improves the complex defects in the regulation of proliferation and differentiation of SCC 13 cells, as supported by morphological and biochemical observations. This provides an evidence that calcipotriol can be applied clinically as a potent differentiation inducer in the treatment of human squamous cell carcinoma. PMID- 8615635 TI - Microwave-enhanced immunohistochemical staining of a formalin-sensitive antigen laminin receptor. AB - Many potentially useful antigens have been difficult to detect in formalin-fixed, paraffin-embedded tissues. Recently a number of pathological and research laboratories have demonstrated that some antigens masked by formalin fixation could be restored to detectability by microwave heating. Previously, we were unable to demonstrate laminin receptor in cells processed by the routine fixation. Our results showed that microwave heating together with trypsin produced the best immunohistochemical staining for this receptor. Nevertheless, no significance was found in the levels of 67 kD LR in high and low metastatic tumor cell lines. PMID- 8615636 TI - Systemic administration of a synthetic lipid A derivative, DT-5461a, reduces tumor blood flow through endogenous TNF production in hepatic cancer model of VX2 carcinoma in rabbits. AB - Intravenous administration of DT-5461a, synthetic low-toxicity lipid A derivative, significantly inhibited the growth of VX2 tumor transplanted in the liver of rabbits. DT-5461a induced high levels of TNF activity in tumor tissue from 30 to 60 min after the administration, while no TNF activity was detected in the adjacent nontumorous liver tissue. Simultaneous measurement of microcirculatory blood flow in both tumorous and nontumorous regions in the liver by laser doppler velocimetory revealed that intravenous administration of DT 5461a caused a significant blood flow reduction in tumor region, but not in nontumorous counterparts. Tumor blood flow was significantly reduced by 40 to 60% at 30 to 90 min after the DT-5461a administration as compared with preadministration value. In contrast, local administration of human recombinant TNF alpha through the hepatic artery induced blood flow reduction not only in tumor region but also in nontumorous liver tissue. These results suggest that systemic administration of DT-5461a induced selective tumor microcirculatory blood flow reduction via local endogenous TNF production. PMID- 8615637 TI - Comparative evaluation of the intracellular accumulation and DNA binding of idarubicin and daunorubicin in sensitive and multidrug-resistant human leukaemia K562 cells. AB - BACKGROUND: Idarubicin is a new anthracycline, more potent in in vitro models than the common anthracyclines doxorubicin and daunorubicin. The intracellular accumulation and DNA binding of daunorubicin and idarubicin have been studied in human leukaemia K562 cells and their doxorubicin-resistant variant, which presents all features of multidrug resistance. METHODS: This was achieved by measuring the decrease of total drug fluorescence during short-term incubation of cell suspensions, directly in the cuvette of a spectrofluorometer; this quenching is due to intercalation of the drug into DNA and represents an evaluation of the active intracellular concentration of the drugs. RESULTS: Accumulation of both anthracyclines was reduced in resistant cells when compared to sensitive ones. Accumulation of idarubicin was significantly higher than that of daunorubicin in both cell types. The difference in anthracycline accumulation between sensitive and resistant cells was lower for idarubicin than for daunorubicin. Verapamil increased the accumulation of both anthracyclines in resistant cells, with a more pronounced effect for idarubicin. Preincubation of the cells with either drug did not influence the accumulation of the other one. CONCLUSION: It has been shown, with this very rapid technique, that the high potency of idarubicin could be explained, at least in part, by its high level of accumulation, both in sensitive and multidrug-resistant leukaemia cells. This is not due to a lack of activity of P-glycoprotein on idarubicin, but rather to an enhanced influx of this drug compared to daunorubicin. PMID- 8615638 TI - Interaction of retinoic acid and interferon-alpha in breast cancer cell lines. AB - Retinoids and Interferons have been demonstrated to synergistically amplify the inhibition of proliferation in cultured breast cancer cells. Recently we reported that interferon-gamma (IFN-gamma) modulates the action of retinoic acid (RA): IFN gamma increased expression of retinoic acid receptor-gamma (RAR-gamma) and suppressed the increase of retinoic acid binding protein type II (CRABP-II) expression. To improve the understanding of mechanism mediating synergism we extended our studies to the type I interferon-alpha. Synergistic inhibition of proliferation could be detected also by IFN-alpha and RA in BT-20 and SKBR-3 breast cancer cell lines but not in MCF-7 cells. Neither IFN-alpha nor any retinoid tested alone were able to increase RAR-gamma message, only the combination of both had this ability. In MCF-7 breast cancer cell lines the combination of any retinoid with IFN-alpha increased CRABP II expression level compared with the retinoids alone. In contrast with SKBR-3 and BT-20 cells a combination of ATRA with IFN-alpha markedly reduced ATRA mediated CRABP II induction. These results suggest that two factors may be responsible for synergistic action of RA and IFN-alpha: the inhibition of the CRABP II expression and an IFN-alpha/RA mediated upregulation of RAR-gamma. PMID- 8615640 TI - Antitumor effect of carboplatin combined with hyperthermia on Ehrlich-ascites tumor in vivo. AB - We examined the optimal timing for the enhancement of antitumor effects of CDDP analogues, cis- diammine-1,1- cyclobutane dicarboxylate platinum (II); CBDCA combined with hyperthermia against the CD-1 mice bearing Ehrlich ascites tumor (EAT) cells in vivo. The prolonged tumor doubling time was observed when two modalities were combined. The longest tumor doubling time was obtained by simultaneous administration of CBDCA combined with hyperthermia. The findings indicated that the most effective condition was the simultaneous combination of CBDCA with hyperthermia. An increase in intratumoral platinum concentration was observed by the treatment of CBDCA simultaneous combined with hyperthermia. PMID- 8615639 TI - The synthetic phosphagen cyclocreatine phosphate inhibits the growth of a broad spectrum of solid tumors. AB - BACKGROUND: The brain isoform of creatine kinase (CKBB), an enzyme involved in energy metabolism, has been implicated in cellular transformation process. Cyclocreatine (CCr), a creatine kinase (CK) substrate analogue, was shown to inhibit the growth of a broad spectrum of solid tumors expressing high levels of CK. Cyclocreatine phosphate (CCrP) generated by CK, was proposed to be the active form responsible for growth inhibition. MATERIALS AND METHODS: We synthesized CCrP and tested its cellular uptake and anti tumor activity in stem cell assays and in athymic mouse models. RESULTS: CCrP seems to be taken up by cells and inhibits the growth of solid tumors with high levels of CK. CCr and CCrP have similar specificity and potency. CONCLUSION: The observation that only high-CK cell lines were responsive to CCrP, similar to CCr, indicates that the enzyme requirement was not bypassed. We propose that CK is a target for CCrP, and is involved in mediating its antiproliferative activity. PMID- 8615641 TI - Identification and tissue-specific expression of a NADH-dependent activity of dihydropyrimidine dehydrogenase in man. AB - Homogenates of human liver and human fibroblasts were able to convert thymine into dihydrothymine in the presence of NADH whereas almost no NADH-dependent activity could be detected in human lymphocytes. The different tissue distribution of the NADH-dependent activity suggests that different types of human dihydropyrimidine dehydrogenase exist. Both types of human liver dihydropyrimidine dehydrogenase showed a comparable affinity towards thymine, NADH and NADPH. Only a ten-fold lower Vmax value was observed for the NADH dependent enzyme. During partial purification of the NADPH-dependent enzyme, on a 2', 5' - ADP Sepharose 4B column, the NADH-dependent activity was completely lost. Neither type of activity was retained on a 5' - AMP Sepharose 4B column. PMID- 8615643 TI - MAPK activity is down-regulated in human colon adenocarcinoma: correlation with PKC activity. AB - Cellular growth is regulated by a cascade of kinases, among which mitogen activated protein kinase (MAPK) is an integral member of the Ras-mediated pathway, while protein kinase C (PKC) is recognized as the intracellular receptor of tumor promoters. To assess the role of these two signal transduction enzymes in colonic carcinogenesis, we measured MAPK and PKC activities in cytosol and membrane compartments of: 1) normal colon, 2) colon adenocarcinoma, and 3) histologically normal colonic tissue taken from the margin of resection of neoplastic colon. Both MAPK and PKC activities were down-regulated in solubilized membranes from the cancers. MAPK activity was also down-regulated in the tissue adjacent to the cancer and designated as histologically normal (by routine histopathology). Therefore, MAPK activity appears to be more suitable than PKC as a marker for early detection of colonic transformation. PMID- 8615642 TI - The ability of polyamine analogues to induce Z-DNA structure in synthetic polynucleotides in vitro inversely correlates with their effects on cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in human brain tumor cell lines. AB - We studied the effects of 72 h pretreatment with five polyamine analogues on the cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in U-251 MG and SF-188 human brain tumor cells. A colony forming efficiency assay showed that the pretreatment with clinically important analogues 1,11-bis(ethylamino)-4, 8 diazaundecane (BE-3-3-3), 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4), and 1,l9-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) increased the cytotoxicity of CDDP by 1.3 to 2.3-fold; 1,19-diamino-5,10, 15-triazanonadecane (4-4-4-4) did not affect CDDP cytotoxicity, and 1,11-diamino-4,8-diazaundecane (3 3-3) protected cells from the cytotoxic effects of CDDP. An alkaline elution assay detected a small increase in DNA interstrand crosslinks accompanying the enhancement of CDDP cytotoxicity only in cells pretreated with BE-3-3-3. This study is the first to show that the Z-DNA inducing abilities of the polyamine analogues in synthetic polynucleotides in vitro correlates inversely with their effects on CDDP cytotoxicity in human tumor cells in culture. PMID- 8615645 TI - Alterations of vinblastine influx in multidrug-resistant lymphoblastic leukaemic CEM cells. AB - Typical multidrug-resistant (MDR) CEM/VLB100 cells exhibited reduced uptake of vinblastine (VLB) compared to their sensitive CEM counterparts mean results were respectively, 3.19 and 35.4 pmol per 10(6) cells. In CEM cells, the efflux of drug reached a steady state after 10-15 min while in CEM/VLB100 cells, the typical P170-mediated efflux was still efficient after 40 min. Nevertheless, the most striking difference observed in CEM/VLB100 cells was a dramatic decrease in early (30 sec) influx of drug which was 10 times lower than in sensitive cells, a characteristic still observed in the presence of Na azide and absence of glucose. MDR cells without little or no effect on sensitive cells. The Q10 entry of VLB into sensitive cells was 1.2 while it was 2.2 in CEM, suggesting that the mode of entry of VLB into MDR cells differed from that of CEM cells. Verapamil or nigericin, which rapidly increased the accumulation of VLB raised the Q10 to >2. These results suggest that the primary defect in MDR cells would be an inhibition of influx, which might involve interactions with P170 through a reversible process. PMID- 8615644 TI - Differential EGF action on nuclear protooncogenes in human endometrial carcinoma RL95-2 cells. AB - Our aim was to analyze EGF action on nuclear protooncogenes in RL95-2 since it has not been documented so far. Synchronization and partial' growth arrest were obtained by maintaining cells for 15 hours in L-methionine-free medium. After this depletion, EGF transiently increased fos and jun mRNAs: the expression peaked at 45 minutes for c-fos (5.5 fold induction) and at 60 minutes for c-jun and jun-B (3 fold induction) and the mRNA levels returned to the basal value within 3 hours. Upon EGF addition, c-myc mRNAs peaked at 12 hours (7.6 fold induction) and surprisingly remained higher than the control up to 48 hours. Unlike fetal calf serum, EGF did not increase the cell number and this could be linked to steadily induced c-myc expression. These data provide evidence for a differential EGF action on fos/jun and c-myc in RL95-2 cells. PMID- 8615646 TI - Assessment of the metastatic ability of rat hepatoma cells in chick embryos by the polymerase-chain reaction. AB - To elucidate the degree of malignancy or the host-killing ability of rat ascites hepatoma AH66F, AH66 and AH130, the chick embryo-polymerase chain reaction system was used to measure the metastasis of these hepatoma cells. When inoculated into the chorioallantoic membrane vein of 10-day fertilized chicken eggs, AH66F cells metastasized in the lung and liver in an inoculum size-dependent manner up to 10(6) cells, but AH66 and AH130 cells only a little even at 10(6) cells. The adhesion of AH66F cells, but not other hepatoma cells, to rat mesentery-derived mesothelial cells was inhibited by protein kinase C inhibitors NA-382 and H-7, but not by other protein kinase inhibitors, and the life-span of rats inoculated with AH66F cells was also prolonged by treatment with Na-382. AH66F cells, pretreated with protein kinase inhibitors were inoculated into the fertile eggs and micrometastasis was assayed. Metastasis of AH66F cells in the chick embryo was clearly decreased by treatment with NA-382. The chick embryo-polymerase chain reaction system is a sensitive method to measure the metastatic ability of mammalian tumor cells, and the high metastatic ability of AH66F cells is probably related to their adhesion to target cells mediated by the protein kinase C pathway. PMID- 8615647 TI - Effect of lonidamine on the mitochondrial potential in situ in Ehrlich ascites tumor cells. AB - The effect of lonidamine (LND) on the mitochondrial membrane potential, in situ, of Ehrlich ascites tumour cells was investigated by using the safranine method. LND, because of its ability to inhibit electron transport from endogenous substrates to respiratory carriers, induced a de-energization of mitochondria. Addition of glucose to rotenone-treated cells induced mitochondrial membrane potential as shown by the spectral shift similar to that which occurred upon energization of mitochondria. The build-up of membrane potential in rotenone treated cells was due to glycolytically-generated ATP because the response to glucose was abolished by LND which inhibited the glycolysis of neoplastic cells by affecting the mitochondria bound hexokinase. PMID- 8615648 TI - Effect of flavone acetic acid on endothelial cell proliferation: evidence for antiangiogenic properties. AB - Flavone acetic acid (FAA) causes regression of a range of slow growing solid tumors implanted subcutaneously in mice. Although its precise mechanism of action is unknown, vascular collapse has been shown to precede tumor growth delay and regression. The aim of this study was to determine whether or not endothelial cell function was directly affected by clinically relevant concentrations of FAA. FAA at 100-250 micrograms/ml inhibited endothelial cell proliferation in vitro, but did not compromise cellular function or viability. FAA abolished tubule formation in an in vitro angiogenesis assay and reduced vascular development of the chick embryo chorioallantoic membrane. In addition to targeting established tumor vasculature, FAA may also affect proliferating endothelium which may be involved in mediating the reduced tumor growth rate or stasis often often observed after drug exposure. The chorioallantoic membrane of the chick embryo may represent an important model to elucidate more clearly the effect of FAA on a growing vascular network. PMID- 8615649 TI - Growth inhibition of breast cancer cell lines by combinations of anti-P185HER2 monoclonal antibody and cytokines. AB - The p185HER2 oncogene is associated with poor prognosis in patients with breast cancer. Both anti-p185HER2 MAb and cytokines have been shown to downregulate p185HER2 expression. We investigated the effect of combinations of humanized 4D5, and anti-p185HER2 MAB, and the cytokines IFN-alpha, IFN-gamma or TNF-alpha on the growth of three p185HER2 positive human abreast carcinoma cell lines, SK-BR-3, BT 474, and MDA-MB-453. All three cell lines were treated with IFN-alpha (1000 U/ml) or IFN-gamma (1000 U/ml) or TNF-alpha (100 U/ml) with or without huMAb 4D5 (100 microM), and cell growth was determined on days 3-7. While IFN-alpha, IFN-gamma, TNF-alpha, and huMAb 4D5 all inhibited growth, an apparently additive inhibitory effect occurred using combinations of huMAb 4D5 plus cytokine, with huMAb 4D5 plus TNF-alpha causing the greatest growth inhibition. These results demonstrate the potentiation of growth inhibition of breast cancer cell lines by the combination of anti p185HER2 MAb and cytokines, and suggest that combinations of anti-growth factor receptor MAb and cytokines may be useful in the treatment of breast cancer. PMID- 8615650 TI - Interleukin-1 receptor antagonist inhibits subcutaneous B16 melanoma growth in vivo. AB - BACKGROUND: Previously we demonstrated that injection of Interleukin-alpha (IL-1) stimulated B16 melanoma tumour growth in vivo, associated with increased intercellular adhesion molecule-1 (ICAM-1) expression on the tumour cells (Photodermatol Photoimmunol Photomed 1994; 10: 74-79, ). METHODS: In the present study, we examined the effect of blocking IL-1 receptors - by addition of recombinant Interleukin-1 receptor antagonist (IL-1RA) - on melanoma tumour growth in vivo and in vitro. RESULTS: Subcutaneous co-injection of IL-1RA with B16 tumour cells into C57BL/6 mice, followed by injection of IL-1RA every second day reduced tumour growth significantly from day 18 to day 33 post-injection. Treatment with IL-1RA appeared to have a bi-phasic effect, low doses being more effective than dosed > 1 microgram/mouse. After 33 days, mice treated with 1.0 or 0.1 micrograms/2nd day had significantly smaller tumours than controls (p < 0.05), however, mice treated with 10 micrograms had tumours no different in size from those of untreated controls. However, survival of IL-RA-treated mice was not significantly different between treatment groups. Addition of IL-1RA to B16 cultures in vitro caused a small dose-dependent decrease in cell growth. Maximum inhibition (64% of control numbers) was observed after 48h in media containing > or = 10 ng/ml Il-1RA (p < 0.05). Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we examined the expression of ICAM-1 message in B16 cells treated in culture with 10 ng/ml IL-1RA or 10 ng/ml IL-1 alpha for 6h or 24h. In IL-1-treated cultures ICAM-1 mRNA expression was increased to 161% of control levels. After 24h, ICAM-1 message was 198% control levels (p = 0.0008). Treatment with IL-1RA reduced the constitutive ICAM-1 expression to 75% of basal after 6h and to 68% of basal after 24h 9 (p = 0.011). CONCLUSION: Abrogation of IL-1 activity, in combination with other systemic therapies may prove useful in the treatment of melanoma. PMID- 8615651 TI - Energetic characteristics of cisplatin resistant ovarian carcinoma cells. AB - An elevated mitochondrial membrane potential coincides with cisplatin resistance in the 2008, C13* and RH4 ovarian carcinoma cell system. Since the mitochondrial membrane potential is a component of the overall proton motive force which drive mitochondrial ATP production, we evaluated the energetic characteristics of these cells with biochemical and pharmacologic assays. Our data shows that the cisplatin resistant C13* cells have reduced mitochondrial respiratory function and enhanced sensitivity to oligomycin, a specific inhibitor of oxidative phosphorylation, compared to the cisplatin sensitive 2008 parental line. This suggests that the chronic cisplatin exposures used to generate C13* cells debilitated mitochondrial functions. This characteristic, however, seems unrelated to cisplatin resistance since the impairment persist in the RH4 variant despite its returned cisplatin sensitivity. We suggest that mitochondrial membrane potential influences cellular processes distinct from energy production and that these processes are relevant to cisplatin resistance. PMID- 8615652 TI - Cathepsin D and PAI-1 expression in human head and neck cancer. AB - In order to determine possible overexpression of cathepsin D and PAI-1 (plasminogen activator inhibitor) in head and neck tumours, cytosols from 92 primary squamous cell carcinomas (SCC), 19 lymph node metastases and 24 adjacent normal tissue samples were examined. For both cathepsin D and PAI-1, significantly elevated concentrations in SCC compared to normal tissue were found, even in pairwise comparison. Moreover, significantly higher cathepsin D and PAI-1 concentration in lymph node metastases than in corresponding normal tissue were also present. The conclusion was drawn that cathepsin D and PAI-1 may play a specific role in the biology of head and neck SCC. PMID- 8615653 TI - Regulation of TNF-alpha and IL-1 gene expression during TPA-induced differentiation of "Malignant histiocytosis" DEL cell line t(5;6) (q35:p21). AB - The production of TNF-alpha and IL-1 alpha and beta molecules has been shown to be associated with the proliferation and activation of cells of the monocyte/macrophage series, the intermediate steps in the synthesis of these molecules have been less investigated. Unstimulated and TPA stimulated DEL cells (a CD30-positive, t(5;6)(q35;p21) malignant histiocytosis cell line) were used to study the expression of TNF-alpha and IL-1 genes and to evaluate, by nuclear run on assay and biological measurements, the control of their transcription and the level of protein production. To refine this analysis, the effects of cycloheximide and actinomycin D were also evaluated in this investigation. Following TPA stimulation, transcription of TNF-alpha (constitutively present) increased threefold as early as 30 mins and started decreasing by 24h. Cycloheximide superinduced the expression of TNF-alpha mRNA and, accordingly, the release of its protein. By contrast, transcription of IL-1 molecules appeared de novo and did not result in a biologically detectable protein. Measurements of RNA half line after actinomycin D indicated that TNF-a and IL-1 alpha mRNAs are not as stable as that of IL-1 beta. These results indicate that, despite their common synergistic activity, the transcriptional and post-transcriptional mechanisms regulating the synthesis of TNF-alpha and IL-1 alpha and IL-1 beta involve different pathways. PMID- 8615654 TI - Microdissection as a means to verify allelic imbalance in tumour biology samples. AB - Allelotypes (TP53, AFM051xd10 and alu-i1) in normal DNA and in DNA from paraffin embedded tumours of a patient with a p53 germ-line mutation were compared in order to demonstrate LOH. Microdissection was applied in order to overcome difficulties with the interpretation of LOH data from a pelvic recurrence of a primary malignant histiocytoma. Furthermore, a rapid and simple boiling method was developed in order to reduce the loss of DNA usually occurring during traditional methods for DNA extraction. The conclusion drawn is that it is of utmost importance to use highly enriched fractions of tumour cells when performing LOH-studies. It is also shown that a rapid and simple boiling procedure is sufficient to release enough DNA of microdissection-enriched tumour cells for microsatellite analysis by PCR to detect allelic imbalance. PMID- 8615655 TI - Induction of benign and malignant pulmonary tumours in mice with benzo(a)pyrene. AB - Most of the pulmonary tumors induced by chemical carcinogens in mice are pulmonary adenomas. Because of the morphological and biological characteristics, the site of origin and the spontaneous occurrence of this type of tumour, the pulmonary adenoma system is generally not considered to be an adequate model for studies designed to elucidate the pathogenesis of human bronchogenic carcinoma. The present study was carried out to observe the histopathological changes in the lung of 4 strains of mice intratracheally instilled with benzo(a)pyrene(BP). On the other hand, for the carcinogenesis experiments using animals, a method studying the incidence of pulmonary adenoma in newborn mice has been generally adopted, but this method still needs more than 24 weeks. This experiment was one of the attempts to make such an experimental period shorter. For the latter experiment, both inbred A/J and noninbred N:GP(s) newborn mice were used. In the group given intratracheal instillation, squamous cell carcinomas and adenocarcinomas were induced. Tumous were induced in high incidence in the lung of A/J and C57Bl/6 mice. Squamous cell carcinomas especially were well differentiated in the A/J mice with a higher incidence than C57BL/6. In the group given subcutaneous injection, adequate combinations at week 9 for the application following anticarcinogenesis assay were the groups of A/J treated with 40 micrograms (71.0 % adenoma incidence) and N:GP(s) mice treated with 500 micrograms (49.4 % adenoma incidence) of BP. These findings suggest that mice appear adequate for studies on the pathogenesis of malignant lung tumours and detecting anticarcinogens. PMID- 8615656 TI - Differential expression of cerbB3 in naevi and malignant melanomas. AB - 28 naevi, 43 malignant melanomas and 16 lymph nodes with melanoma metastases were stained immunohistochemically for the expression of cerbB3 oncoprotein. using antibody RTJ1. Positive reaction was found in 60% of naevi and in 25% of melanoma cases. The difference in frequency of the reaction occurrence between naevi and melanomas was statistically highly significant (p = 0.0017). In naevi, the percentage of cerbB3 positive cells (56.5%) was also significantly higher (p = 0.021) as compared with that of melanomas (37%). The cerbB3 positive cells were found only in cases without metastases. Positive reaction was also observed in 4 out of 16 investigated lymph nodes with melanoma metastases. The role of cerbB in the development of melanoma is discussed. PMID- 8615657 TI - Endometrial cancer cell lines are sensitive to paclitaxel. AB - We have previously reported a 24 fold difference in the cisplatin sensitivity and a 12 fold difference in carboplatin sensitivity of endometrial carcinoma cell lines. In this study as evaluate paclitaxel sensitivity of the same cell lines. We tested nine endometrial cancer cell lines with the 96-well plate clonogenic assay using limiting dilution. The chemosensitivity was expressed as IC50 value, the drug concentration causing 50% inhibition of clonogenic survival. IC50 values were obtained from dose-response curves after fitting the data to the linear quadratic equation. The IC50 values for paclitaxel were 0.49 - 2.3 nM showing only a 4.7 fold difference between various cell lines. No correlation could be demonstrated between in vitro paclitaxel and platinum analog sensitivities of endometrial adenocarcinoma cell lines. The variance in paclitaxel sensitivity of different cell lines was little. Our results suggest that endometrial adenocarcinoma cell lines tested with the same methods. The clinical efficacy of paclitaxel in the treatment of endometrial cancer should further be evaluated in clinical trials. PMID- 8615658 TI - MCF-7 cell cycle arrested at G1 through ursolic acid, and increased reduction of tetrazolium salts. AB - The effect of ursolic acid on the proliferation of MCF-7 human breast tumor cells was studied. During investigations of the anti-proliferative effects of this triterpene, we observed a clear difference between MTT colorimetric assay and direct cell counting, particularly 24 h after drug treatment. The MTT assay showed a stimulation of formazan production in the first 24 h exposure of cells to drug. The maximum stimulation was obtained with 15 and 20 microM of ursolic acid (about 30 - 40% of increase with respect to control); however, the number of cells was not increased as revealed by direct cell counting. Ursolic acid is a potent inhibitor of MCF-7 cell proliferation. This triterpene exhibits both cytostatic and cytotoxic activity. It exerts an early cytostatic effect at G1 followed by cell death. Cell cycle analysis is performed by propidium iodide staining and flow cytometry technique. These results suggest that alterations in cell cycle phase redistribution of MCF-7 human breast cancer, by ursolic acid, may significantly influence MTT reduction to formazan. PMID- 8615659 TI - The expression of myc and ras oncogene protein in childhood lymphomas. AB - Paraffin sections from 21 cases of Hodgkin's disease (HD), 18 cases of non Hodgkin's lymphomas (NHL) and 15 cases of reactive lymphadenitides occurring in children 3-15 years old were examined by immunohistochemistry for the presence of c-myc and pan-ras oncogene products. In all cases of childhood lymphomas studied c-myc protein was expressed. The highest percentage was in HD where in 29% of cases the percentage of positive cells was greater than 20%, while in only 6% of NHL cases this percentage was noted. In all cases of reactive lymphadenitides the number of cells where c-myc was expressed was less than 5%. The ras oncogene was also found in HD but with a lesser degree of positivity than c-myc. In 48% of these cases the number of positive cells ranged between 2 and 10%, while in NHL and in reactive lymphadenitides there was no positivity. The above results indicate a frequent expression of c-myc protein in childhood lymphomas. This could reflect either an implication of c-myc oncogene in the pathogenesis of these tumor or an epiphenomenon of lymphomagenesis reflecting the proliferation rate of tumor cell population. Molecular biology studies are needed to clarify this issue. PMID- 8615660 TI - Charge polymorphism in human lung cell pro-cathepsin B. AB - Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 human lung-fibroblast cells was pre-purified by ion exchange chromatography and investigated by 2D gel electrophoresis. Four (Wi-38), six (HS-24) and ten (alveolar macrophages) polypeptides differing in charge, but with the same molecular mass of 45 kDa, were found. The isoelectrical points of these polypeptides ranged from 5.43 to 6.57. Deglycosylation reduced the mass (7 kDa) but did not change the charge pattern. This investigation established cell type-specific patterns of secreted pro-cathepsin B-forms, but only parts of these may be cell type-specific forms depending on differentiated expression of mRNA and post-translational modification. PMID- 8615661 TI - Transient blockage of proliferative signalling: a novel strategy for protective chemotherapy. AB - An intact proliferative signalling pathway is essential to the growth of all normal cells, but is often not required by tumor cells. This fact was used to devise a protective chemotherapeutic protocol potentially applicable to all tissues. Four treatments were chosen to temporarily disrupt proliferative signalling. They acted either upstream, at, or downstream of cellular ras activity. As expected, the cell cycle progression of normal cells was temporarily interrupted, while those cells transformed by tumor genes, or tumor cells themselves often were not affected. During these cell cycle blocking treatments the cells were exposed to the topoisomerase inhibitor m-AMSA. This anti-cancer drug is selectively toxic to cycling cells. In each case the tumor cells were selectively killed as judged either by their ability to incorporate labeled thymidine, replate, or grow. These studies suggest new ways to utilize current drugs or search for new ones. PMID- 8615662 TI - Potentiation of radiotherapy by nontoxic pretreatment with combined vitamins C and K3 in mice bearing solid transplantable tumor. AB - BACKGROUND: The effect of intraperitoneal and oral pretreatment with combined vitamins C and K3 on the single dose radiotherapy of a transplantable solid mouse tumor have been investigated. MATERIALS AND METHODS: Groups of mice bearing intramuscularly transplanted liver tumors, were orally and parenterally pretreated with combined vitamins C and K3 and locally irradiated with single doses of 20, 30, or 40 Gy of X-rays. After this treatment tumor dimensions were measured twice weekly and the approximate tumor volume in groups of pretreated vitamins and irradiated mice was compared to the groups of mice only irradiated and to the absolute control groups without any therapy. RESULTS: This nontoxic pretreatment produced statistically significant potentiation of radiotherapy induced by 20 to 40 Gy of X-rays doses in groups of 11 to 20 mice. Combined vitamins C with K3 most probably constitute a redox-cycling system producing hydrogen peroxide and other active oxygen species to which cancer cells are selectively sensitive due to their frequent deficiency in enzymatic defense system against free oxyradicals agression. CONCLUSIONS: A possible introduction of such nontoxic and selective potentiation procedure into classical protocols of human cancer therapy appears to be generally accessible and without any additional risk for patients. PMID- 8615663 TI - Elevated nascent DNA content of peripheral blood mononuclear cell compartment in lymphoma patients. AB - The kinetics of DNA synthesis and the DNA pattern of isolated peripheral blood mononuclear cells from control subjects and lymphoma patients prior to drug treatment were studied as a possible tool in the early diagnosis of lymphoma. Thymidine and [H3]-dTTP incorporation represented the measure of replicative DNA synthesis in permeable murine thymocytes and HT-168 human melanoma cells as described earlier. The kinetic parameters of nucleotide incorporation were compared with the ploidity parameters of the Feulgen-stained smears examined by the DNASK TV-cytophotometric system. For better evaluation and visualization of the S-phase population, the method of silver impregnation of the Nucleolar Organizer-Region in combination with the Feulgen technique was used. Significant differences were observed among the five determined parameters of healthy and lymphoma patients. Our results allow to explain some of the facts related to T cell function deficiency in lymphoma. PMID- 8615664 TI - Telomeres, telomerase and cancer: is the magic bullet real? AB - Nature recruited telomerase to compensate for the incomplete replication of chromosomal ends (telomeres). In higher organisms, telomeres are eroded at each cell division. Cancer cells frequently show chromosomal instability resulting in ring chromosomes, telomeric associations, and dicentric chromosomes. As a consequence of telomeric erosion, the ribonucleoprotein complex termed "telomerase" is reactive in a subpopulation of cells. Telomerase adds a hexameric repeat of the sequence 5' TTAGGG 3' to the ends of the chromosomes and hence stabilizes the telomeric length. Telomerase is active in vertebrates mostly in germ cells and the early stage embryo but is inactivated or repressed in somatic cells. Detection of telomerase activity in the overwhelming majority of advanced and metastatic human cancers but not in most somatic cells implies that telomerase-dependent immortalization could contribute to the malignancy. Future studies on the expression and regulation of the individual components of telomerase may enable us to clarify the diagnostic and therapeutic potential of telomerase in cancer. PMID- 8615665 TI - Immunophenotypically varied cell subpopulations in primary and metastatic human melanomas. Monoclonal antibodies for diagnosis, detection of neoplastic progression and receptor directed immunotherapy. AB - During a systematic, immunocytochemical screening of 40 human cutaneous melanomas (30 primary and 10 metastatic) for immunophenotype (IP) heterogeneity, we employed a library of 20 well characterized, commercially available mono- and polyclonal antibodies. The use of the sensitive, indirect, four to six step immunoperoxidase or alkaline phosphatase conjugated streptavidin-biotin antigen detection technique provided excellent results. The immunocytochemically most characteristic IP for primary cutaneous melanoma, as detected by us was: HMB45+, S-100+, CEA+, vimentin+, cytokeratin 19+, p53+, Rbgene+, nm23+, HLA-DR+, HL.A DP+, c-erbB3/HER-3+/-, cytokeratin 10/13+/-, HLA-DQ-, cytokeratin 5/8-, EMA-, c myc-, and actin-. During melanoma progression, a tendency toward poor differentiation (dedifferentiation) and an increase in c-myc expression have both been observed, the latter downregulating HLA-A,B,C expression and consequently diminishing the possibility of melanoma cell Iysis by powerful CD8+, cytotoxic T lymphocytes (CTL) or other cytotoxic cells which requires HLA class I antigens. The development of the metastatic potential in melanomas caused an increase in CEA expression, eliminated the presence of nm23, and prompted the appearance of actin among the intermediate filaments, composing the cytoskeleton of these malignant tumor cells. The most characteristic IP for MMs, identified by this study was HMB45+, S-100+, CEA+, EMA+, vimentin+, HLA-DR+, HLA-DP+, cytokeratin 19+, actin-, c-erbB3/HER-3+, p53+, cytokeratin 10/13+/-, c-myc+/-, c-erbB2/HER 2+/-, HLA-DQ-, cytokeratin 5/8-, Rb gene-, nm23-. It has been observed that adhesion molecules and integrins play a significant role in the complex process of melanoma metastasis and thus we propose a blocking of these de novo expressed molecules with the appropriate antibodies as a form of immunotherapy of PMs and early stages of MMs. PMID- 8615667 TI - Endoluminal instillation of epidoxorubicin as adjuvant treatment for upper urinary tract urothelial tumours. AB - Intravesical use of chemotherapeutic agents has been documented as effective and beneficial in the treatment and prophylaxis of bladder urothelial cancer. Because of the success achieved with these agents, several investigators have proposed these same drugs as adjuvant treatment after conservative procedures for urothelial tumours of the upper urinary tract. Experiences concerning topical therapy for upper tract urothelial neoplasms are still limited, but suggest a great potential benefit in terms of improved tumour control. Advances in endourologic techniques and increasing interest in the conservative management of upper urinary tract urothelial cancer may lead to more frequent use of topical chemotherapy. Specific guidelines and surveillance protocols, similar to those with superficial bladder cancer, are required to determine the role of this adjuvant therapy in the treatment of urothelial tumours of the upper urinary tract. This report shows our experience gained on endoluminal instillation of epidoxorubicin in a selected group of 9 patients who underwent local excission of ureteral or renal pelvis tumours. PMID- 8615666 TI - Carboplatin-based chemotherapy in patients undergoing hemodialysis. AB - In the present study, we investigated the pharmacology of carboplatin in two patients with chronic renal failure undergoing hemodialysis. In Case 1, squamous cell carcinoma of the epipharynx was treated with 265 mg/m2 of carboplatin alone. In Case 2, adenocarcinoma of the lung was treated with 300 mg/m2 of carboplatin in combination with 50 mg/m2 of etoposide. All agents were injected intravenously. Hemodialysis was initiated 30 minutes after carboplatin administration. Most platinum was excluded by hemodialysis in a pattern similar to that in patients with normal renal function. These observations suggest that carboplatin-based chemotherapy can be considered as a treatment modality for malignant neoplasia in patients with chronic renal failure undergoing hemodialysis. PMID- 8615668 TI - Combination therapy with subcutaneous interleukin-2 and interferon-alpha in advanced renal cancer patients with poor prognostic factors. AB - Sixteen patients with metastatic renal cell carcinoma were treated with a combination of low-dose subcutaneous interleukin-2 (IL-2) and recombinant interferon (IFN)-alpha. One treatment course included 6 weeks of treatment followed by a 2-week rest. Patients received therapy as outpatients. All patients were assessable for toxicity and response assessment. Nine patients experienced severe toxicities resulting in dosage modification. The major treatment-limiting side effects were gastrointestinal, cutaneous, fever and flu-like symptoms. One patient (6%) had partial remission and six patients (37.5%) had disease stabilization. Overall median survival was 8 months. In our study IL-2 and IFN alpha showed a low activity and a quite high toxicity. It seems that the prognostic factors per se rather than treatment options might impact the treatment results in advanced renal cancer patients. PMID- 8615669 TI - A plasma cell variant of Castleman's disease treated successfully with cimetidine. Case report and review of the literature. AB - Castleman's disease is a rare benign condition of uncertain etiology, with several forms. It is postulated that a dysregulated inflammatory response may play a role in the pathogenesis of this disease. We report on a case of Castleman's disease treated with the histamine receptor-2 antagonist cimetidine with favourable response, suggesting that cimetidine may be helpful in the treatment of this disease. PMID- 8615670 TI - Inhibition of cellular chemotactic invasion by a vinca alkaloid, conophylline. AB - K-ras-NIH3T3 cells were more invasive than NIH3T3 cells in a chemotactic invasion assay. Conophylline, a new vinca alkaloid isolated as a ras function inhibitor, inhibited the invasion of K-ras-NIH3T3 cells, while it showed no effect on NIH3T3 cells. Conophylline did not increase expression of fibronectin but induced E cadherin expression in K-ras-NIH3T3 cells. Mouse melanoma B16/F10 is a highly metastatic cell line. Conophylline was found to induce flat morphology in B16/F10 cells and it again inhibited the invasion of the cells to the matrigel membrane. It induced fibronectin expression but not E-cadherin expression in B 16/F10 cells. Thus, conophylline lowered invasiveness of K-ras-NIH3T3 and B 16/F10 cells by reversing neoplastic phenotypes. PMID- 8615671 TI - Transcription factor decoy approach to decipher the role of NF-kappa B in oncogenesis. AB - Antisense inhibition of the RelA subunit of NF-kappa B transcription factor (but not the NFKB1 subunit) causes pronounced inhibition of tumor cell growth in vitro and in vivo. Inhibition of either subunit, however, results in inhibition of the heterodimeric NF-kappa B complex in antisense-treated cells. Either of the subunits of NF-kappa B can form homo- or heterodimers with other members of the Rel oncogene family. In an effort to decipher the role of homo- vs heterodimeric NNF-kappa B in regulating tumor cell growth, we have used a decoy approach to trap these complexes in vivo. Using double-stranded phosphorothioates as a direct in vivo competitor for homo- vs heterodimeric NF-kappa B, we demonstrate that decoys more specific to RelA inhibit growth tumor cell growth in vitro. We demonstrate that RelA, either as a homodimer or a heterodimer with some other members of the Rel family and not the classical NF-kappa B (RelA/NFKB1), is involved in the differential growth control of tumor cells. Our results indicate that such transcription factor decoys can be a non-antisense tool to study the function of DNA-binding transcription factors. PMID- 8615672 TI - Antitumor effect of TNP-470 on glial tumors transplanted in rats. AB - Anti-angiogenic agents were be clinically useful for the therapy of solid tumors, as the growth and proliferation of tumors require neovascularization. Previous reports have been made on the antitumor effect of TNP-470, an angiogenic inhibitor, on medulloblastomas implanted subcutaneously into nude mice. The present study was undertaken for the purpose of investigating possible antitumor effects on two model subcutaneous and intracranial tumors transplanted into rats. 9-L gliosarcomas were transplanted into the left femoral region subcutaneously and the left frontal region of Fisher 344 female rats, and the TNP-470 was then administered subcutaneously. The rats were divided into two treatment groups and a control group. TNP-470 resulted in dose-dependent inhibition of 9-L gliosarcoma growth and also prolonged survival in treatment groups. Therefore TNP-470 was considered to be effective for the treatment of malignant brain tumors, although it caused dose-dependent decreases in body weight. PMID- 8615673 TI - The immunohistochemical expression of cathepsin D in colorectal cancer. AB - It has been suggested that cathepsin D expression in stromal cells affects the prognosis of breast cancer. With reference to colon and breast cancer, this study verified cathepsin D immunostaining in epithelial and stromal cells of primary tumours and lymph-node metastases from 46 colorectal carcinomas. Eight of 46 cases (17%) were cathepsin D+ both in cancer and stromal cells. No staining was found either in cancer or stromal cells of the remaining cases. The results presented here suggest the possible paracrine influence of another diffusible factor produced by cancer cells which stimulates cathepsin D production in stromal and cancer cells. PMID- 8615674 TI - Plasminogen activation system in active even in thyroid tumors; an immunohistochemical study. AB - The plasminogen activation system is one of the prominent factors in tumour progression since it enhances both distant metastasis and direct invasion. In this study we investigated the expression and localization of this system in clinical thyroid tumours. It was found that urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor type 1 (PAI-1) were expressed diffusely in most thyroid carcinomas. The lesions positive for the latter two were equal to or sometimes even a litter more extensive than those positive for uPA. The immunoreactivity of plasminogen activator inhibitor type 2 (PAI-2), however, was often partially or even entirely absent, and only 56% of the carcinomas expressed it diffusely, indicating that the two types of inhibitors in this carcinoma have quite different characteristics. No relationship between the expression of these proteins and clinicopathological parameters could be determined. THese findings suggest that the plasminogen activation system is functionally active in thyroid carcinoma but that is has hardly any effect on its general characteristics, that is, slow growth and excellent prognosis of usual types and the exceptionally sudden progression of the anaplastic type. In benign tissues, too, this system was often expressed and appeared to enhance their growth to some extent. PMID- 8615675 TI - Epidermal Langerhans cells from mice bearing a granulocyte macrophage-colony stimulating factor-producing mammary tumor display impaired accessory functions. AB - A progressive depression of delayed type hypersensitivity reactions occurs during development of mammary tumors in BALB/c mice. This tumor constitutively produces prostaglandin E2 (PGE2) and granulocyte-macrophage colony stimulating factor (GM CSF). Epidermal Langerhans cells were found to have a decreased responsiveness to bacterial superantigen and to defined antigens in tumor-bearing mice, and also showed an impaired ability to induce proliferative responses in syngeneic or allogeneic responder T cells. Flow cytometric analysis revealed that the Langerhans cells of tumor bearers had decreased densities of the Ia molecule on their surfaces. No defects were observed in the potential of keratinocytes from tumor bearers to produce granulocyte-macrophage colony stimulating factor or to support the activation of syngeneic T cells. Incubation of normal Langerhans cells with tumor derived factors depressed their capacity to stimulate T cell syngeneic responses. Addition of indomethacin and anti-prostaglandin E2 did not reverse this depressed activity. These results indicate that epidermal Langerhans cells from tumor-bearing mice possess a functional deficit in acquiring accessory properties in vitro, which cannot be ascribed to a lack of GM-CSF in the local microenvironment or to production of inhibitory cytokines by their keratinocytes. The functional deficit of epidermal Langerhans cells of tumor-bearing mice may account for the depressed delayed hypersensitivity displayed by these mice, and factors elaborated by the tumor may be responsible for the deficiencies observed. PMID- 8615676 TI - Immunohistochemical localisation of aromatase and its correlation with progesterone receptors in ovarian epithelial tumours. AB - Aromatase cytochrome P-450 was immunohistochemically localised exclusively in the cytoplasm of neoplastic cells of benign and malignant nonfunctional ovarian tumours, whilst (progesterone receptors (PR) and estrogen receptors) (ER) were localised exclusively inn the nuclei of neoplastic cells. Aromatase activity and PR were detected in 68% (17/25) and 32% of the malignant tumours and 82% (22/27) and 67% of the benign tumours, respectively. In postmenopausal tumours, the positivity for PR in malignant tumours was less frequent (p < 0.01) than that in benign tumours. The tumours in which both aromatase and PR were positive, were less frequent (p < 0.05) in malignant than in benign tumours. Aromatase activity was detected in 100% (8/8) of the PR-positive tumours but in only 68% (9/17) of the PR-negative tumours. With postmenopausal malignant tumours, there was a positive correlation between aromatase activity and PR level (r = 0.77, p < 0.001). However, there was no significant difference in the positivity of ER. The serum steroid levels did not correlate with the tumour levels of aromatase activity, PR or ER. These findings suggest that aromatase activity is correlated with PR in ovarian tumours of postmenopausal women. In addition to steroid receptor status, aromatase activity may be a useful factor in ovarian cancer. PMID- 8615677 TI - Modulation of the rat tumor-associated shedding antigen (CE7) and augmentation of immunogenicity by irradiation. AB - We have previously reported that rat fibrosarcoma KMT-17 cells and their in vitro counterparts, cloned A3 cells, shed a tumor-associated antigen (TAA), termed CE7, from the cell surface on vesicular membranes, under growth-enhancing conditions. This study shows that irradiation (1 approximately Gy) from a 60Co source, inhibited A3 cell growth dose-dependently and correspondingly increased CE7 expression by A3 cells as determined by anti-CE7 monoclonal antibody using flow cytometry. CE7 expression gradually increased with increasing doses of irradiation and reached a peak level at 30Gy. After 30Gy irradiation, CE7 expressing A3 cells were fixed with 1% paraformaldehyde and were used to intradermally immunize syngenic rats. Immunized rats developed transplantation resistance to the parent KMT-17 cells as compared to rats immunized with unirradiated A3 cells. Rat MHC class 1 antigen expression was slightly decreased by irradiation and therefore, resistance to tumor transplantation appeared to arise solely due to the enhancing effects of irradiation on TAA expression which increases the antigenicity of the tumor cells coverting them to an effective stimulator of antitumor effector cells. This phenomenon may offer a possibility of the resistance to the re-emergence and metastasis of the tumor like a KMT-17 through the induction of antitumor memory cells. PMID- 8615678 TI - Nitric oxide (NO) in apoptotic versus necrotic RAW 264.7 macrophage cell death: the role of NO-donor exposure, NAD+ content, and p53 accumulation. AB - Nitric oxide (NO)-releasing compounds cause apoptotic cell death in RAW 264.7 macrophages. This is confirmed morphologically by chromatin condensation and biochemically by DNA laddering. With use of spontaneously decomposing NO donors known as NONOates we show that the integral of concentration over time accounts for the NO-donor damaging ability. A 30-min exposure to the rapidly decomposing NO-donor diethylamine-nitric oxide complex (DEA-NO) causes irreversible damage and apoptotic cell death after 6 to 8 h. For intermediate NO releasers like sodium nitroprusside, S-nitrosoglutathione (GSNO), and spermine-NO removal of the NO-donating compound halts fragmentation to a certain degree. The relatively stable diethylenetriamine-nitric oxide complex initiates fragmentation only after prolonged exposure. NO-mediated apoptotic signaling in macrophages neither decreases cellular NAD+, nor causes a drop in APT. Consistently, membrane integrity measured by lactate dehydrogenase release is preserved and inhibitors of poly(ADPribose) polymerase, like 3-aminobenzamide, are noneffective. The level of the tumor suppressor p53 increases in response to NO donors like GSNO and effectively senses NO intoxication in macrophages. GSNO removal concomitantly allows p53 to decline with only a small percentage of cells showing DNA fragmentation. Contrary, massive damage initiated by a 1-h exposure to DEA-NO is irreversible, with persistent p53 levels. NO-mediated apoptotic cell death in RAW 264.7 macrophages correlates with the degree of p53 accumulation, probably sensing the integrity of the genome. PMID- 8615679 TI - Chemical modification and inactivation of rat liver arginase by N bromosuccinimide: reaction with His141. AB - Treatment of rat liver arginase with N-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzynska (1979) Acta Biochim. Pol. 26, 103 114). In order to probe the chemistry of N-bromosuccinimide inactivation and the role of tryptophan residues in catalysis, the two tryptophan residues of rat liver arginase, Trp122 and Trp164, have been separately mutated to phenylalanine using site-directed mutagenesis of the protein expressed in Escherichia coli. Both single Trp -> Phe mutant enzymes have kinetic parameters nearly identical to those for the wild-type enzyme. Treatment of native, wild-type, and each of the Trp -> Phe mutant enzymes with N-bromosuccinimide results in loss of absorbance at 280 nm and is accompanied by a loss of catalytic activity. However, treatment of the wild-type enzyme with N-bromosuccinimide in the presence of the arginase inhibitors NG-hydroxy-L-arginine or the combination of L-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases with N-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated by N-bromosuccinimide, indicating that His141 is the critical target for the N-bromosuccinimide inactivation of the enzyme. PMID- 8615680 TI - Recombinant human cytochrome P450 1A2 and an N-terminal-truncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase. AB - Previous work from this laboratory indicated that the N-terminus of recombinant human cytochrome P450 (P450) 1A2 expressed in Escherichia coli is blocked (P. Sandhu, Z. Guo, T. Baba, M. V. Martin, R. H. Tukey, and F. P. Guengerich, (1994) Arch. Biochem. Biophys. 30, 168 -177). A modification of this construct was done to insert an extra 12 residues containing a thrombin-sensitive site just beyond the most N-terminal hydrophobic segment, and the protein was expressed, purified, and cut with thrombin. Treatment of E. coli membranes in which the P450 1A2 with 12 extra residues was present with thrombin did not release the truncated form, suggesting that the added thrombin site may be imbedded in the membrane. The N terminal of the recombinant proteins were blocked but mild acid hydrolysis generated the expected Met residues as analyzed by Edman degradation. Laser light scattering studies indicated that purified thrombin-cleaved P450 1A2 (devoid of the usual N-terminal 25 residues or the first 36 residues of the wild-type protein) was still aggregated in the absence of detergent and that some nondenaturing detergents could reduce the apparent size to that of a tetramer. The N-terminal truncated protein was as catalytically active as full-length P450 1A2 but required a higher concentration of NADPH-P450 reductase. P450 1A2 exhibited catalytic activity in E. coli cells, and activity of the purified enzyme could be supported by E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase could also substitute for NADPH P450 reductase. These artificial electron donors did not require phospholipid for oxidation reactions; however, phospholipid was required for optimal activity when either P450 1A2 or the truncated form was used with NADPH-P450 reductase. Rates of oxidation of 7-ethoxyresorufin were considerably higher for both P450 1A2 and the truncated form when NADPH-P450 reductase was replaced with the "oxygen surrogate" iodosylbenzene, indicating that P450 reduction and oxygen activation are normally limiting in this P450 1A2 reaction. PMID- 8615681 TI - Interaction of platelet-activating factor with rat hepatocytes: uptake, translocation, metabolism, and effects on PAF-acetylhydrolase secretion and protein tyrosine phosphorylation. AB - In the present study, the interaction of the phospholipid mediator platelet activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions. PMID- 8615682 TI - Reaction of wild-type C365S, and C458S saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with fluorescent iodoacetamide derivatives. AB - The reactivities of Cys365 and Cys458 of ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase against a range of sulfhydryl reagents have been investigated. The effect of pH on the second order reaction constants of N-(1-pyrenyl)maleimide with mutant C458S and C365S PEP carboxykinases allowed the determination of pKa values of 9.4 and 9.1 for Cys365 and Cys458, respectively. The analysis of the inactivation rates of C458S and C365S mutant enzymes by several sulfhydryl reagents of different hydrophobicity showed that the microenvironment of these residues is rather polar. Anisotrophy measurements and acrylamide quenching experiments carried out with N-(iodoacetyl)-N'-(5-sulfo 1-naphthyl)ethylenediamine-labeled mutant enzymes indicated a higher rotational freedom and solvent exposure for the probe linked to Cys458 than to Cys365. These findings point to differences in the protein microenvironments around Cys365 and Cys458 in S. cerevisiae PEP carboxykinase. A comparison of the results obtained with published data for GTP-dependent PEP carboxykinases, suggest significant differences for the protein region around the reactive cysteinyl residues in these enzymes. PMID- 8615683 TI - Molecular cloning and characterization of a novel casein kinase II substrate, HASPP28, from rat brain. AB - HASPP28 (heat- and acid-stable phosphoprotein of 28 kDa) has been purified to near homogeneity from the acid-stable protein fraction of rat brain extract. Based on the N-terminal 40 amino acid sequence, a pair of highly degenerate primers was used to generate a 107-bp probe from rat brain RNA by RT-PCR. From the rat brain lambda gt11 library, this probe identified two positive clones that together provided a cDNA of 837 bp with an open reading frame of 546 bp. This cDNA was extended by 3'RACE to 1.2 kb that included a polyadenylation signal and a poly(A) tail. The 180-amino-acid sequence derived from the open reading frame, which did not correspond to any known protein, was predicted to have phosphorylation sites for protein kinase C, casein kinase II (CKII), and protein kinase A. Indeed, both the purified rat brain HASPP28 and the recombinant HASPP28 (rHASPP28) can be phosphorylated by these kinases. Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kidney, liver, heart, and muscle, in decreasing order of abundance. Phosphopeptide analysis of rHASPP28 phosphorylated in vitro by various kinases showed different tryptic patterns on two-dimensional mapping and isoelectric focusing gels. From [32P]PO4-labeled N1E115 neuroblastoma cells, HASPP28 can be immunoprecipitated with a polyclonal antiserum raised against rHASPP28. The immunoprecipitated protein showed a phosphopeptide pattern similar to that of rHASPP28 phosphorylated by CK II in vitro. Furthermore, the immunoprecipitates from cells treated with phorbol 12-myristate 13-acetate or 8 bromo-cAMP did not show any increased phosphorylation over those of untreated ones, and the phosphopeptide patterns of the immunoprecipitates again were similar to that of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinases in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28. PMID- 8615684 TI - Biogenesis and metabolic fate of docosahexaenoic and arachidonic acids in rat uterine stromal cells in culture. AB - To gain some insight into the mechanisms involved in the opposing effects of arachidonic acid and docosahexaenoic acid on the growth of rat uterine stromal cells (UIII cells), the dynamics of the uptake, conversion, and incorporation of labeled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3), and 22:6(n-3) into lipid pools and phospholipid subclasses were examined. A very active and time-dependent conversion of [14C]18:3(n-3) to higher homologs was observed; 64.7 +/- 0.7 and 11.5 +/- 0.4% of the [14C] radioactivity incorporated in cellular lipids was recovered as 22:5(n-3) and 22:6(n-3) after 72 h incubation, respectively. The distribution of labeled fatty acids obtained after 72 h incubation with [3H]20:5(n-3) was not significantly different from that observed with 18:3(n-3). Arachidonic acid was the major fatty acid formed from [14C]18:2(n-6) and only trace amounts of 22:5(n-6) were detected. When cells were incubated for 72 h with 20:4(n-6), more than 75% of the radioactivity was recovered as arachidonate and slightly higher amounts of 22:4(n-6) and 22:5(n-6) were formed compared to those obtained after incubation with 18:2(n-6). Using both [14C]- and [3H]22:6(n-3), no significant retroconversion of labeled 22:6(n-3) occurred in the cells. More than 90% of labeled 20:4(n-6) and 22:6(n-3) taken up by the cells were esterified into phospholipids, but significant differences in their distribution among phospholipid classes and subclasses were observed. Docosahexaenoic acid was more rapidly and efficiently incorporated into phosphatidylethanolamine than 20:4(n-6) and was principally recovered in plasmalogens. Arachidonic acid was mainly incorporated in the diacyl subclasses of phosphatidylcholine and phosphatidylethanolamine and in phosphatidylinositol. The divergent profiles of these two fatty acids within the phospholipid compartments provide some information for the mechanisms of their opposite effects on UIII cell growth. PMID- 8615685 TI - Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase. AB - The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase. PMID- 8615686 TI - Ser-268 plays an important role in ligand binding of prostaglandin E2 receptor EP3alpha subtype. AB - Functional mouse prostaglandin E2 (PGE2) receptor EP3alpha subtype has been expressed in insect cells using the baculovirus system (C. Huang and H. H. Tai, 1995,Biochem . J. 307, 493-498). Site directed mutagenesis was carried out at Thr 221, Ser-268, and Ser-272. These hydroxy amino acid residues are highly conserved among prostaglandin receptors of many species and could potentially form hydrogen bonds with the hydroxyl group or the keto group of the receptor ligand PGE2. The mutant EP3alpha receptors again were expressed in insect cells and were studied by [3h]PGE2 binding assay. The results indicated that (1) replacement of Ser-272 by alanine did not cause any significant change in PGE2-binding activity, (2) replacement of Thr-221 by alanine also did not cause any significant change in PGE2-binding activity, and (3) replacement of Ser-268 by alanine almost abolished the PGE2-binding activity, while replacement by a threonine did not cause significant change in the PGE2-binding activity but did alter subtype specificity. These results suggest that Ser-268 of EP3alpha receptor plays an important role in ligand binding. PMID- 8615687 TI - Role of residue 480 in substrate specificity of cytochrome P450 2B5 and 2B11. AB - The role of residue 480 as a determinant of the specificities of cytochrome P450 2B5 and 2B11 toward androstenedione, progesterone, 2,2',4,4',5,5' hexachlorobiphenyl (245-HCB), and benzyloxyresorufin has been investigated. Two reciprocal mutants at position 480, 2B5 Val-480 -> Leu and 2B11 Leu -> Val, two hybrid enzymes, H1B (amino acid residues 1-370 from P450 2B11, 371-491 from P450 2B5) and H2B (amino acid residues 1-370 from P450 2B5, 372-494 from P450 2B11) (Kedzie et al., 1993, Biochim. Biophys. Acta 1164, 124-132), and two hybrid back mutants, H1B Val-480 -> Leu and H2B Leu-480 -> Val, were constructed and expressed in Escherichia coli, and compared with the wild-type enzymes P450 2B5 and 2B11. For androstenedione metabolism, the Leu-480 -> Val mutation in P450 2B11 resulted in an increase in the 16 beta-OH:16 alpha-OH ratio from 1.2 to 3, whereas the Val-480 -> Leu mutation in hybrid H1B decreased the 16 beta-OH:16 alpha-OH ration from 4.2 to 1.1 In the case of progesterone, the Leu-480 -> Val mutant of P450 2B11 displayed 3-fold higher 16 alpha- and 21-hydroxylase activities than the wild-type 2B11. In the absence of cytochrome b5, 2B11 L480V displayed half of the benzyloxyresorufin O-dealkylase (BROD) activity of 2B11 wild-type, whereas H1B V480L showed 5,6-fold higher activity than H1B. Therefore, residue 480 seems to play an important role in steroid and benzyloxyresorufin metabolism by P450 2B11. In contrast, the mutation Leu-480 -> Val did not have a significant effect on the 245-HCB hydroxylase activity of P450 2B11, and the mutation Val-480 -> Leu in P450 2B5 had no notable effect on its progesterone hydroxylase activity. Cytochrome b5 caused marked stimulation of the BROD activity of P450 2B11 and H1B and their mutants. Furthermore, P450 2B5 exhibited different progesterone metabolite profiles in the absence or presence of cytochrome b5. PMID- 8615688 TI - Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalysed by a human placental 17beta-hydroxysteroid dehydrogenase. AB - The human placental 17beta-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17beta-estradiol and other estrogens. Since 19 alpha-estradiol is a competitive inhibitor of 9,10 phenanthrenequinone by the 17beta-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17beta-hydroxysteroid dehydrogenase, the equilibrium between 17beta estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17beta hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans dihydrodiols tested. PMID- 8615690 TI - Characterization of the pH titration shifts of ribonuclease A by one- and two dimensional nuclear magnetic resonance spectroscopy. AB - One- and two-dimensional 1H NMR experiments were used to determine the chemical shifts of resonances arising from 29 residues of RNase A in H2O at 17 pH values ranging from 1.2 to 7.9 Nearly all resonances displaying pH-induced changes in chemical shift greater than 0.1 ppm were monitored. Individual plots of the chemical shift as a function of pH were fit by nonlinear least squares methods to Henderson-Hasselbalch models yielding pK alpha values which were then analyzed using a set of criteria to determine their reliability. The criteria included statistics from the curve fitting analysis as well as the distance of the reporter proton to ionizing groups. Only the most reliable pK alpha values were assigned to specific ionizing groups within RNase A based upon the proximity of the reporter proton to the ionizing group as determined from the X-ray crystal structure. Only 2 of the 15 groups expected to undergo ionization within the pH range investigated could not be assigned pK alpha values within the highest two levels of reliability. Of the 11 carboxylate groups, 5 have pK alpha values less than 3.0. Many of the low pK alpha values can be interpreted as resulting from favorable hydrogen bonds between the carboxylate group and other moieties within the protein. The pK alpha values for the four histidine residues are similar to earlier literature reports. Two resonances underwent particularly large pH induced shifts of approximately 2.3 ppm and corresponded to nitrogen-bound protons involved in hydrogen bonds with carboxylate groups. PMID- 8615689 TI - Difference between guinea pig and rat in the liver peroxisomal response to equivalent plasmatic level of ciprofibrate. AB - Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between the guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat. (1) Pharmacokinetic results show equivalent in guinea pig and rat when guinea pigs are treated with ciprofibrate at 30 mg/kg twice a day and rats are treated at 3 mg/kg once a day. (2) The treatment of guinea pigs at 30 mg/kg twice a day for 2 weeks leads to a significant increase in the liver peroxisomal palmitoyl-CoA oxidase activity (x 1.6) and also in the microsomal omega-laurate hydroxylase activity (x 1.8). These increases are in accordance with the changes in polypeptide patterns of isolated liver peroxisomes as well as in the immunoblotting of acyl-CoA oxidase. It is deduced that a weak, but significant, peroxisome proliferation can occur in guinea pig liver after a ciprofibrate treatment at dosages corresponding to equivalent plasmic concentrations of the drug between guinea pig and rat. (3) The hybridization of guinea pig liver RNA with the rat liver-inducible acyl-CoA oxidase cDNA probe shows a decrease in the corresponding heterologous mRNA content after treatment with ciprofibrate at 30 mg/kg twice a day. This result contrasts with the slight increase observed in immunodetection and in enzymatic assays, suggesting the existence of at least two different acyl-CoA oxidases in guinea pig liver peroxisomes. PMID- 8615691 TI - The effect of veratryl alcohol on manganese oxidation by lignin peroxidase. AB - The extracellular peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium have been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported peroxidase isozyme H2 can also oxidize Mn2+ (Khindaria et al., 1995, Biochemistry 34, 7773-7779). This lignin peroxidase isozyme oxidized Mn2+ with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H1O) also readily oxidized Mn2+, but that the rate of Mn2+ oxidation by compound II was extremely slow. Compound III rapidly the presence of Mn2+, oxalate, and H2O2. However, upon the addition of veratryl alcohol, the results indicated that veratryl alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn2+ oxidation was observed. The rate of Mn2+ oxidation was the same as for the reduction of compound II by veratryl alcohol. The dependence of the rate of Mn2+ oxidation on the concentration of veratryl alcohol was consistent with a mechanism in which Mn2+ is oxidized by compound I and veratryl oxidized by compound II. Therefore, under physiologically relevant conditions, in which both veratryl alcohol and Mn2+ are present, all lignin peroxidase isozymes would be capable of oxidizing Mn2+ to Mn3+ which can serve as a diffusible oxidant. PMID- 8615692 TI - Functional expression of uridine 5'-diphospho-glucose 4-epimerase (EC 5.1.3.2) from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli. AB - It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants. Searching a database of expressed sequence tags, a cDNA from Arabidopsis thaliana (clone 108G20T7) with sequence similarity to UDP-glucose epimerase was identified and further analyzed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein sequence showed a high degree of similarity to UDP-glucose epimerase sequences from bacteria, rat, and yeast. Complementation of the Saccharomyces cerevisiae gal1O mutant and expression of an active enzyme in Escherichia coli demonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It showed a broad pH optimum of 7.0 to 9.5 and a Km of 0.11 mM. The UDP-glucose epimerase activity was not dependent on the addition of the cofactor NAD+ and was only moderately inhibited by high salt concentrations. Tissue-specific Northern analysis showed that the gene is expressed in all tissues of A. thaliana with highest expression levels in the stems and roots. Based on Southern analysis, there seems to be a single gene encoding UDP-glucose epimerase in A. thaliana. The cDNA analyzed during this study is the first known to encode a sugar-nucleotide modifying enzyme from higher plants. Its availability provides the means to investigate the role of UDP-glucose epimerase for the biosynthesis of UDP-galactose as precursor of galactolipids and cell wall polysaccharides. PMID- 8615693 TI - Retarded and aberrant splicings caused by single exon mutation in a phosphoglycerate kinase variant. AB - The molecular abnormality of a phosphoglycerate kinase variant which was associated with severe tissue enzyme deficiency and episodes of muscle contractions and myoglobinuria was examined. Analysis of the patient's DNA showed the existence of a nucleotide transversion A/T - C/G in exon 7. No other nucleotide change was detected in the coding region of the variant gene. The mutation should produce a single amino acid substitution Glu - Ala at protein position 251 counting from the NH2-terminal acetyl serine residue. The protein abnormality caused by the amino acid substitution cannot explain the enzyme deficiency. Northern blot hybridization indicated that the PGK mRNA content of the patient's lymphoblastoid cells was only about 10% of that of normal. Nucleotide sequence analysis revealed the existence of two PGK mRNA components in the patient's cells. The major component corresponds to the normal PGK mRNA except for A - C change at nucleotide position 755 counting from adenine of the chain initiation codon. The minor component contains 5' region (52 bases) of intron 7 between exon 7 and exon 8. An inframe chain termination codon exists in the minor mRNA component, and the COOH-terminal half is expected to be deleted in the translation product. These results indicate that the low PGK activity in the patient's tissues is mainly due to retarded and aberrant pre-mRNA splicings caused by the change of the consensus 5' splice sequence AGgt to a nonconsensus sequence CGgt at the junction between exon 7 and intron 7 of the variant gene. PMID- 8615694 TI - The diurnal variation of hepatic HMG-CoA reductase activity is due to changes in the level of immunoreactive protein. AB - The diurnal variation in levels of hepatic HMG-CoA reductase immunoreactive protein and enzyme activity were determined in rats. Immunoreactive protein levels changed together with enzyme activity. Thus the catalytic efficiency of HMG-CoA reductase was not significantly changed. The data suggest that the diurnal variation in hepatic HMG-CoA reductase activity is due to changes in enzyme protein levels rather than changes in phosphorylation state of the enzyme, for example, which would cause changes in catalytic efficiency. PMID- 8615695 TI - Purification and characterization of a (R)-3-hydroxybutyrate dehydrogenase deletion mutant. Evidence for C-terminal involvement in enzyme activation by lecithin. AB - (R)-3-Hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) is a lipid-requiring enzyme with a specific requirement of phosphatidylcholine for optimal function. The purified enzyme, devoid of lipid, can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. In order to obtain insight into the mechanism of lipid activation, a C-terminal deletion mutant was constructed which contained 18 amino acids less than BDH. The purified deletion mutant had low, but detectable catalytic activity in the absence of phospholipid. However, the addition of either soluble lecithin or phospholipid vesicles containing lecithin had no effect on enzymatic function. Further experiments were conducted to determine if the deletion mutant had also lost its ability to bind to phospholipid vesicles and natural membranes. Our findings indicate that the mutant enzyme binds to both liposomes and rat liver microsomes. These results suggest that the binding of BDH to the phosphatidylcholine head group is independent of its interaction with the apolar core of the phospholipid bilayer. PMID- 8615696 TI - Differential stimulation of NAD kinase and binding of peptide substrates by wild type and mutant plant calmodulin isoforms. AB - Calmodulin from Arabidopsis thaliana consists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins in Escherichia coli, purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinase in vitro. The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparent Vmax it elicited and the K0.5 activation. These results are consistent with the hypothesis that the Arabidopsis CaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface when wt CaM-6 binds Ca2+. CaM 6M-activated NAD kinase displayed an apparent V. that was reduced 40% and a K0.5 that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+ dependence of CaM-GM to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, alpha-helical 12-residue c-myc epitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparent Vmax values within 10% of those obtained with the wild-type CaM proteins. PMID- 8615698 TI - Alkylation-induced oxidative cell injury of renal proximal tubular cells: involvement of glutathione redox-cycle inhibition. AB - The nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is an alkylating agent that causes oxidative stress and subsequently death of renal proximal tubular cells (PTC). In this paper the role of inhibition of the glutathione redox cycle (GSH-reductase (GRd) and -peroxidase (GPx) in the development of DCVC-induced oxidative cell injury is described. DCVC inhibited both GRd and GPx activity in PTC. Inhibition occurred already after 10 min incubation while at that time point lipid peroxidation and cell death had not started yet; the antioxidant N,N diphenyl-p-phenylenediamine did not prevent inhibition of GRd and Gpx- inhibition of L-cysteine S-conjugate beta-lyase-mediated formation of reactive metabolites using aminooxyacetic acid, which prevented covalent binding to cellular macromolecules, was associated with prevention of the DCVC-induced inhibition of both enzymes. Covalent binding of reactive metabolites of [35S]DCVC to several cellular proteins was found, including proteins which had molecular weights identical to subunits of GPx and GRd. An inhibitor of GRd, 1,3-bis(2-chloroethyl) 1-nitrosourea (BCNU), potentiated the oxidative cell injury caused by DCVC, whereas BCNU itself did not use oxidative stress and cell death. The thiol reducing compound dithiothreitol prevented the oxidative cell injury whereas oxidation of cellular thiols with diamide potentiated the DCVC-induced oxidative stress and cell death. Moreover, incubation with (R,S)-3-hydroxy-4-pentenoic acid (HPA), which depletes mitochondrial GSH, potentiated the DCVC-induced oxidative cell injury. Neither diamide nor HPA affected the covalent binding and inhibition of the GSH-redox cycle. Together, the data suggest that the inhibition of GRd and GPx, presumably caused by binding of reactive metabolites of DCVC, impairs the cellular antioxidant system, which seems causally related to DCVC-induced oxidative cell injury. PMID- 8615697 TI - Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin. AB - The amino acid sequence established for human thyroglobulin (hTG) from its cDNA sequence contains 20 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate. In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined. Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC. The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin. Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation. Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein. Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine. Five sites (at positions 1200, 1329, 1993, 2275, and 2562) contain high mannose type units and two sites (at positions 179 and 1345) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures. In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence. A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites. The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin (bTG) (27) and as has been recently reported for bTG, no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG. Only four of the 20 putative sites the sequence of hTG, at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation. PMID- 8615699 TI - Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin. AB - The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretary granules, and the interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase-SL interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme-inhibitor complex. The dissociation equilibrium constant (determined in reactions containing 0.18 M or 1.0M NaCl (pH 8.0, 25 degrees C) was 5 X 10(-8) and 2 x 10( 8) M, respectively. Addition of heparin to the low-salt reaction decreased the Ki approximately 10-fold to a value of 3 x 10(-9) M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10 fold increase in the association rate constant (kass) from 2 X 10(4) to 3 X 10(5) M-1 s-1. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitor in vivo, especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI. PMID- 8615700 TI - Oxidized aldose reductase: in vivo factor not in vitro artifact. AB - Characterization of aldose reductase purified from human placenta confirms that activation, as first analyzed in detail for the bovine enzyme, also occurs in humans. Routinely between 5 and 20% of the aldose reductase activity freshly purified from human placenta exhibits kinetic properties and insensitivity to aldose reductase inhibitors (ARIs) characteristic of the activated or oxidized enzyme form, as determined using a sensitive Sorbinil titration assay. In confirmation of previous studies, the amount of aldose reductase activity and the ratio of aldose to aldehyde reductase activity show wide patient to patient variability, with aldose reductase accounting for between 30 and 95% of the total aldo-keto reductase activity. The kinetic behavior described for enzyme isolated from human tissues (e.g., biphasic Dixon plots for ARI inhibition) can be reproduced exactly using mixtures of native and oxidized recombinant human aldose reductase and is not restricted to DL-glyceraldehyde. Measurement of substrate (NADPH versus NADPD and solvent (H2O versus D2O) deuterium isotope effects indicates that the ARI-resistant form is altered in a manner that perturbs the relative rates of steps along the normal reaction pathway. These results suggest that not only the level of enzyme activity, but also the extent of activation of human aldose reductase in vivo, may be an important factor in determining susceptibility to diabetic complications and responsiveness to ARI therapy. PMID- 8615701 TI - A novel invertase from a thermophilic fungus Thermomyces lanuginosus: its requirement of thiol and protein for activation. AB - Unlike the invertases from the mesophilic fungi and yeasts, invertase from a thermophilic fungi, Thermomyces lanuginosus, was unusually unstable both in vivo and in vitro. The following observations suggested that the unstable nature of the enzyme activity in the cell-free extracts was due to the oxidation of the cysteine residue(s) in the enzyme molecule: (a) the addition of dithiothreitol or reduced glutathione stabilized invertase activity during storage of the extracts and also revived enzyme activity in the extracts which had become inactive with time; (b) N-ethylmaleimide, iodoacetamide, oxidized glutathione, cystine, or oxidized coenzyme A-inactivated invertase; (c) invertase activity was low when the ratio reduced/oxidized glutathione was lower and high when this ratio was higher, suggesting regulation of the enzyme by thiol/disulfide exchange reaction. In contrast to the activation of invertase by the thiol compounds and its inactivation by the disulfides in the cell-free extracts, the purified enzyme did not respond to these compounds. Following its inactivation, the purified enzyme required a helper protein in addition to dithiothreitol for maximal activation. A cellular protein was identified that promoted activation of invertase by dithiothreitol and it was called "PRIA" for the protein which helps in restoring invertase activity. The revival of enzyme activity was due to the conversion of the inactive invertase molecules into an active form. A model is presented to explain the modulation of invertase activity by the thiol compounds and the disulfides, both in the crude cell-free extracts and in the purified preparations. The requirement of free sulfhydryl group(s) for the enzyme activity and, furthermore, the reciprocal effects of the thiols and the disulfides on invertase activity have not been reported for invertase from any other source. The finding of a novel invertase which shows a distinct mode of regulation demonstrates the diversity in an enzyme that has figured prominently in the development of biochemistry. PMID- 8615702 TI - Hantaviruses in the United States. PMID- 8615703 TI - Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease): new insights in pathogenesis, complications, and treatment. AB - Hereditary hemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu disease, is a hereditary disorder leading to easily bleeding telangiectases on skin and mucosal surfaces, and it is associated with the presence of arteriovenous malformations (AVMs) in multiple organ systems. These AVMs may cause serious complications when they are located in the lungs, liver, or brain. The prevalence of AVMs in patients with HHT might be higher than previously estimated. Nowadays, treatment is often possible. In some families, mutations have been shown in the gene encoding for a transforming growth factor receptor, endoglin. Genetic heterogeneity has been demonstrated, suggesting involvement of other transforming growth factor receptors. This might explain the variable clinical expression of the disease. In view of the high prevalence of pulmonary and cerebral AVMs, all patients with HHT should be screened for their presence, and relatives of patients with HHT should be investigated for presence of the disease. PMID- 8615704 TI - Human and rodent hantavirus infection in New York State: public health significance of an emerging infectious disease. AB - BACKGROUND: A case of hantavirus pulmonary syndrome with possible exposure in New York and/or Rhode Island was confirmed in February 1994. OBJECTIVE: To conduct four studies to determine the historical and geographic distribution of human and small-mammal infection with hantaviruses in New York State. METHODS: Enzyme linked immunosorbent assays were performed on serum samples obtained from 130 humans during a 1978 babesiosis survey, 907 small mammals collected in New York State since 1984, 12 rodents collected in 1994 near the residences of the patients with hantavirus pulmonary syndrome, and 76 New York patients with acute respiratory distress syndrome-like illness (as suspected cases of hantavirus pulmonary syndrome). RESULTS: None of the human serum samples from the 1978 serosurvey showed evidence of hantavirus exposure by enzyme-linked immunosorbent assay. Statewide historical serum samples from white-footed mice showed evidence of Sin Nombre virus infection in 12.0% (97/809) and Seoul-like virus infection in 9.6% (78/809). Site-specific seropositivity rates were as high as 48.5% with Sin Nombre virus during 1 year (1984). Two of 12 mice captured near the residences of a human patient were positive for Sin Nombre virus by enzyme-linked immunosorbent assay, yet were negative for viral RNA by polymerase chain reaction. None of the patients with suspected hantavirus pulmonary syndrome was serologically reactive for Sin Nombre virus. CONCLUSIONS: We provide serologic evidence of small-mammal infection with hantaviruses in New York State as long ago as 1984. Human cases of hantavirus pulmonary syndrome are rare in New York, and data indicate that transmission to humans is probably infrequent. A unique set of host, agent, and environmental factors may be necessary to cause hantavirus pulmonary syndrome in humans. PMID- 8615705 TI - Cholesterol-reduction intervention study (CRIS): a randomized trial to assess effectiveness and costs in clinical practice. AB - BACKGROUND: The 1988 US National Cholesterol Education Program Expert Panel Report recommended initial treatment with niacin or bile acid sequestrants, followed by other agents if needed, to lower low-density lipoprotein cholesterol (LDL-C) levels in hypercholesterolemic patients who require drug therapy. It is unknown how the effectiveness and costs of such an approach ("stepped care") compare in typical clinical practice to those of initial therapy with lovastatin. PATIENTS AND METHODS: We randomly assigned 612 patients, aged 20 to 70 years, who met 1988 National Cholesterol Education Program guidelines for drug treatment of elevated LDL-C level and had not previously used cholesterol-lowering medication, to either a stepped-care regimen or initial therapy with lovastatin (both n=306). The study, conducted at Southern California Kaiser Permanente, was designed to approximate typical practice: provider compliance with treatment plans was encouraged but not enforced, and patients paid for medication as they customarily would. RESULTS: At 1 year, the decline in mean LDL-C level was significantly greater among patients assigned to initial treatment with lovastatin (22% vs 15% for stepped care; P<.001), as was the number who attained goal LDL-C level (.1). However, patients of female providers were more likely to receive comprehensive screening than patients of male providers. Specifically, 95% of women seen by female attending physicians or fellows had comprehensive screening vs 67% for male attending physicians or fellows and 61% for residents (P=.008). CONCLUSIONS: Mammography may be replacing CBE especially among patients of male providers. Interventions targeted to these providers could help improve the use of CBE and mammography. PMID- 8615707 TI - Psychosocial interventions for patients with coronary artery disease: a meta analysis. AB - BACKGROUND: Narrative review strategies and meta-analyses have shown that drug treatment and exercise rehabilitation regimens can reduce psychological distress and postmyocardial infarction mortality and recurrence. OBJECTIVE: To question whether the addition of psychosocial interventions improves the outcome of a standard rehabilitation regimen for patients with coronary artery disease. METHODS: We performed a statistical meta-analysis of 23 randomized controlled trials that evaluated the additional impact of psychosocial treatment of rehabilitation from documented coronary artery disease. Anxiety, depression, biological risk factors, mortality, and recurrence of cardiac events were the clinical end points that were studied. Mortality data were available from 12 studies, and recurrence data were available from 10 of the 23 studies. RESULTS: The studies had evaluated 2024 patients who received psychosocial treatment vs 1156 control subjects. The psychosocially treated patients showed greater reductions in psychological distress, systolic blood pressure, heart rate, and cholesterol level (with effect size differences of -0.34 [corrected], -0.24, 0.38, and -1.54, respectively). Patients who did not receive psychosocial treatment showed greater mortality and cardiac recurrence rates during the first 2 years of follow-up with log-adjusted odds ratios of 1.70 for mortality (95% confidence interval [CI], 1.09 to 2.64) and 1.84 for recurrence (CI, 1.12 to 2.99). CONCLUSIONS: The addition of psychosocial treatments to standard cardiac rehabilitation regimens reduces mortality and morbidity, psychological distress, and some biological risk factors. The benefits were clearly evident during the first 2 years and were weaker thereafter. At the clinical level, it is recommended to include routinely psychosocial treatment components in cardiac rehabilitation. The findings also suggest an urgent need to identify the specific, most effective types of psychosocial interventions via controlled research. PMID- 8615708 TI - Acute care costs of the oldest old: they cost less, their care intensity is less, and they go to nonteaching hospitals. AB - BACKGROUND: The cost of acute hospital care is often believed to increase with age among older persons. Our clinical experience in the acute hospital setting suggested that people aged 90 years and older may be a distinct cohort who have different health care needs and who use health resources differently from younger aged groups. METHODS: To determine the cost of care for the oldest old and younger old patients in Massachusetts acute care hospitals, 1992 and 1993 discharge data from all nonfederal Massachusetts hospitals were examined (678 954 discharges) according to five age groups: 60 to 69 years (n=210 270), 70 to 79 years (n=256 781), 80 to 89 years (n=171 725), 90 to 99 years (n=39 170), and 100 or more years (n=1008). Average estimated total and ancillary costs per discharge and per diagnosis related group were calculated. Differences by gender and survivorship were also examined. RESULTS: Hospitalization costs peaked in the 70- to 79-year age group and declined with age thereafter. Case mix was an important determinant of this trend. Despite lower cost per stay, average length of stay was longer for the oldest age groups. Ancillary costs accounted for 53% of the total costs per stay among the 60- to 69-year-olds and only 32% among the 100 or more-year- olds. For hospitalizations during which the patient died, the average cost per discharge decreased 61%, from $16886 for 60- to 69-year-olds to $6523 for centenarians. Costs were greater for decedents than for survivors, although these differences decreased dramatically with increasing age. Those aged 80 years and older tended to be hospitalized in nonteaching hospitals. CONCLUSIONS: In the acute hospital setting, the oldest old cost less per admission than younger elderly patients. This finding must be considered when future health care costs are predicted for this fastest growing segment of our population. PMID- 8615709 TI - Knowledge and attitudes of hospital-based physicians and trainees about HIV infection in the United States, Canada, India, and Thailand. AB - OBJECTIVE: To examine the attitudes and knowledge of health care professionals regarding human immunodeficiency virus (HIV) infection in countries with a varying prevalence of HIV infection to assist in the development of acquired immunodeficiency syndrome (AIDS) educational programs. DESIGN: Anonymous questionnaire with four sections: demographics, attitudes, knowledge, and an open ended question investigating feelings about the potential impact that HIV infection may have on respondents' practices. PARTICIPANTS: Final-year medical students, house staff, and attending physicians at teaching hospitals in India, Thailand, Canada, and the United States. RESULTS: From January to October 1992, 819 health care professionals completed the questionnaire: 340 from India, 196 from Canada, 155 from the United States, and 128 from Thailand. The percentage of respondents who had previous contact with patients with HIV/AIDS varied from 30% to 98%; it was lowest in India, followed by Thailand and then Canada, and highest in the United States. Percentages of respondents uncomfortable performing a physical examination on a patient with HIV/AIDS were 24%, 25%, 9%, and 4%, respectively. Mean HIV/AIDS knowledge scores were 83%, 84%, 92%, and 93%, respectively. Most respondents correctly identified modes of transmission of HIV infection. Only 67% of Indian health care professionals understood the concept of a false-negative screening serologic test, compared with 98% of Canadian health care professionals. In Canada and the United States, only 78% and 76%, respectively, understood the concept of a false-positive screening serologic test. Awareness of an asymptomatic stage of HIV infection ranged from 32% in India to 74% in Canada. Despite their concerns of becoming infected, health care professionals in countries with a lower prevalence of HIV infection reported a strong ethical duty to care for these patients. CONCLUSIONS: Level of comfort in caring for HIV-infected patients and HIV/AIDS knowledge scores varied directly with the amount of previous contact with these patients. Disturbing numbers of health care professionals from all four countries did not understand the potential problems of the enzyme-linked immunosorbent assay serologic test and a significant percentage were unaware of the asymptomatic stage of HIV infection. There is a universal need for increased education of health care professionals about HIV infection and AIDS. PMID- 8615710 TI - Adverse 5-year outcome after coronary artery bypass surgery in blacks. AB - BACKGROUND: Coronary heart disease is the leading cause of death among blacks, but little is known about the late results of coronary artery bypass surgery in this population. It is not known whether differences in preoperative medical characteristics or medical health insurance affect outcome. We studied the effects of medical risk factors on survival outcome after coronary artery bypass surgery in a population of medically insured black and white patients. METHODS: Racial status and outcomes from surgery were determined in 3728 consecutive patients who had coronary artery bypass surgery at the authors' institution from January 1, 1984, to June 30, 1992. Coronary artery bypass surgery (excluding valve replacement) was performed in 115 black and 3113 white patients. RESULTS: Late survival probability was worse for blacks than whites at 1 year (84% vs 92%) and at 5 years (64% vs 82%, P=.001, Wilcoxon test). Most deaths were due to cardiac events in both groups (68% in blacks vs 67% in whites). Blacks had more hypertension (84% vs 54%), diabetes mellitus (36% vs 23%), and more were current smokers (21% vs 14%) (all P<.05, Fisher's exact test). Medical insurance coverage for blacks and whites was as follows: Medicare (60% vs 57%), private (38% vs 42%), and Medi-Cal (2% vs 2%). Operative mortality (30 days) was similar (5.2% for blacks vs 4.1% for whites; P=.48, Fisher's exact test). In a Cox regression model, race predicted long-term survival and persisted as an important risk factor after adjusting for preoperative factors related to patient survival (adjusted hazard ratio, 2.10; 95% confidence interval, 1.43 to 3.07). CONCLUSIONS: In this group of predominantly medically insured patients undergoing coronary artery bypass surgery, the risk of death in blacks at 5 years was twice that of whites. PMID- 8615711 TI - Effects of anatomic site, oral stimulation, and body position on estimates of body temperature. AB - BACKGROUND: A prior investigation characterized the range of body temperature in healthy young adults and established the importance of diurnal variations in defining the febrile state. METHODS: Sequential rectal, oral, and tympanic membrane temperature measurements were performed on 22 healthy subjects to determine the quantitative effects of anatomic site, oral stimulation, and body position on estimates of body temperature. RESULTS: Mean rectal temperatures exceeded concurrent oral readings by 0.4 degrees C +/- 0.4 degrees C (0.8 degrees F +/- 0.7 degrees F), which, in turn, exceeded concurrent tympanic membrane readings (obtained with a digital thermometer [IVAC Corp, San Diego, Calif]) by 0.4 degrees C +/- 1.1 degrees C (0.7 degrees F +/- 2.0 degrees F). Tympanic membrane readings were significantly more variable (both intrasubject and intersubject) than rectal or oral readings, especially when cerumen was present in the external ear canal being examined (P<.05). Mastication and smoking both caused significant increases in oral temperature that persisted for greater than 20 minutes. Drinking ice water caused a significant but more transient decrease in oral temperature. Of these activities, only mastication appeared to influence tympanic membrane readings. Body position exerted a modest effect on rectal temperature readings, but did not significantly affect oral or tympanic membrane readings. CONCLUSIONS: These findings indicate that, in addition to diurnal fluctuations in body temperature, the effects of anatomic site, oral stimulation, and body position should be considered in establishing criteria for the febrile state. PMID- 8615712 TI - Who decides? Physicians' willingness to use life-sustaining treatment. AB - BACKGROUND: Physician specialty training is associated with variations in the use of medical treatment for specific diseases. OBJECTIVE: To examine whether physicians' specialties predict differences in willingness to use life-sustaining treatments. METHODS: One hundred fifty-eight physicians (response rate, 85%) who cared for 378 hospitalized patients with end-stage congestive heart failure, chronic obstructive pulmonary disease, malignant neoplasms, or hepatic cirrhosis were interviewed to assess their thresholds for use of specific life-sustaining treatments. Their patients were then followed up to determine whether decisions were made to use or withhold cardiopulmonary resuscitation, ventilator support, or intensive care. Physicians' attitudes, their stated thresholds for treatment use, and their use of these treatments in daily practice were compared by specialty group. RESULTS: Physicians recommended cardiopulmonary resuscitation and ventilator support for patients with end-stage congestive heart failure or chronic obstructive pulmonary disease if the chance for survival was at lease 48%, but they required a predicted survival of at least 74% for patients with cancer. For a patient with end-stage congestive heart failure or chronic obstructive pulmonary disease, cardiologists were consistently more willing than other physicians to use life-sustaining treatments. In practice, decisions to use or withhold such treatments were made for 151 patients with end-stage diseases. Compared with other physicians, cardiologists were least likely to issue orders to withhold treatment and most likely to use life-sustaining treatments for patients they treated. Oncologists rarely used such treatments and issued orders to withhold these treatments much more often. CONCLUSION: Physician specialty is associated with differences in willingness to use, and in actual use of, life sustaining treatments. PMID- 8615713 TI - Procedure-specific do-not-resuscitate orders. Effect on communication of treatment limitations. AB - BACKGROUND: Do-not-resuscitate (DNR) orders are often inaccurately communicated between physicians and nurses or residents. Structured, procedure-specific DNR order forms have been suggested to improve communication, but no data exist to support this impression. METHODS: The level of agreement between attending physicians and nurses or residents in their understanding of the DNR orders of critically ill patients was measured before and after instituting a structured DNR order form. Caregivers were asked (1) about the clinical events to which the DNR order applied, (2) whether the DNR order withheld all elements of cardiopulmonary resuscitation, and (3) whether other treatments were to be withheld. Results were reported as kappa +/- SE. RESULTS: Nurses (n=41) and residents (n=34) showed only fair to moderate agreement with attending physicians (n=53) for the 76 evaluable patients before initiation of the DNR order form. After initiation of the structured DNR order form, nurses showed higher levels of agreement for the second (0.67 +/- 0.14) and third (0.69 +/- 0.13) components but not the first (0.39 +/- 0.15) component of the DNR order. Residents showed higher levels of agreement for the second (0.90 +/- 0.10) and third components (0.81 +/- 0.13) but not the first (0.57 +/- 0.17) component. Nurses compared with residents had lower levels of agreement with attending physicians for most aspects of the DNR order. CONCLUSION: A structured DNR order form improves agreement in understanding of some but not all components of the DNR order. PMID- 8615714 TI - Adherence to national guidelines for drug treatment of suspected acute myocardial infarction: evidence for undertreatment in women and the elderly. AB - BACKGROUND: Evidence-based guidelines for the treatment of patients with acute myocardial infarction (AMI) have been published and disseminated by the American College of Cardiology and the American Heart Association. Few studies have examined the rates of adherence to these guidelines in eligible populations and the influence of age and gender on highly effective AMI treatments in community hospital settings. METHODS: Medical records of 2409 individuals admitted to 37 Minnesota hospitals between October 1992 and July 1993 for AMI, suspected AMI, or rule-out AMI, and meeting electrocardiographic, laboratory, and clinical criteria suggestive of AMI were reviewed to determine the proportion of eligible patients who received thrombolytic, beta-blocker, aspirin, and lidocaine hydrochloride therapy. The effects of patient age, gender, and hospital teaching status on the use of these treatments were estimated using logistic regression models. RESULTS: Eligibility for treatment ranged from 68% (n=1627) for aspirin therapy, 38% (n=906) for lidocaine therapy, and 30% (n=734) for thrombolytic therapy to 19% (n=447) for beta-blocker therapy. Seventy-two percent of patients eligible to receive a thrombolytic agent received this therapy; 53% received beta-blockers; 81% received aspirin; and 88% received lidocaine. Among patients ineligible for lidocaine therapy (n=1503), 20% received this agent. Use of study drugs was lower among eligible elderly patients, especially those older than 74 years (thrombolytic agent: odds ratio, 0.2; 95% confidence interval, 0.1 to 0.4; aspirin: odds ratio, 0.4, 95% confidence interval, 0.3 to 0.6; beta-blocker: odds ratio, 0.4; 95% confidence interval, 0.2 to 0.8). Female gender was associated with lower levels of aspirin use among eligible patients (odds ratio, 0.7; 95% confidence interval, 0.6 to 0.9); and there was a trend toward lower levels of beta-blocker and thrombolytic use among eligible women. CONCLUSIONS: Use of lifesaving therapies for eligible patients with AMI is higher than previously reported, particularly for aspirin and thrombolytic use in nonelderly patients. Lidocaine is still used inappropriately in a substantial proportion of patients with AMI. Increased adherence to AMI treatment guidelines is required for elderly patients and women. PMID- 8615715 TI - 'Travelers' thyrotoxicosis'. Transitory thyrotoxicosis induced by iodinated preparations for water purification. AB - Two young adults presented with thyrotoxicosis after increased iodide ingestion in the course of backpacking trips to Central and South America, during which each of them used an iodinated preparation for water purification. In both cases, serologic tests were positive for antithyroid peroxidase antibodies, and one had a family history of immune thyroid disease. We suggest that in these two cases the condition was a variant of iodide-induced thyrotoxicosis, reflecting an inadequate response to iodide excess in association with a presumed preexisting asymptomatic immune thyroid disease. This observation adds a new consideration for travelers' consultation. PMID- 8615716 TI - Isoniazid preventive therapy. PMID- 8615717 TI - Neurobiology of insomnia and respiratory symptoms. PMID- 8615718 TI - Technical considerations in endoscopic cervicothoracic sympathectomy. AB - OBJECTIVE: To evaluate the technique and results of videoendoscopic cervicothoracic sympathectomy in patients who have reflex sympathetic dystrophy or hyperhidrosis of the upper extremity. DESIGN: Clinical case series. The cohort underwent diagnostic evaluation and surgical intervention, and had a mean postoperative follow-up of 14 months. SETTING: An urban, university-affiliated tertiary referral medical center. PATIENTS: A consecutive, referred sample. Seven of the nine patients had reflex sympathetic dystrophy and two had bilateral upper extremity hyperhidrosis. Five were women and four were men, with a mean age of 44 years. INTERVENTIONS: Ten thoracoscopic sympathectomies, encompassing the lower third of the stellate ganglion to the fourth thoracic ganglion, in nine patients. The technique is performed under general anesthesia, using three 1-cm incisions for instrument placement. Patients had bilateral hand temperature probes intraoperatively. Six of the procedures were in the left hemithorax, four in the right. MAIN OUTCOME MEASURES: Relief of the symptoms for which the patient was referred. Perfection and alteration of the technique also were measured. RESULTS: The average operating time was 91 minutes. The average length of hospital stay was 3.5 days. The mean increase in skin temperature was 2.4 degrees C. Nine of 10 patients had partial or complete relief of symptoms. One patient with severe dystrophic reflex sympathetic dystrophy has persistent symptoms. One patient had a pneumothorax for 48 hours. Horner's syndrome did not develop in any patient. CONCLUSION: Endoscopic cervicothoracic sympathectomy is an effective, minimally invasive therapy for upper extremity reflex sympathetic dystrophy and hyperhidrosis. PMID- 8615719 TI - Interleukin-6 production by rat hepatocellular carcinoma cells is associated with metastatic potential but not with tumorigenicity. AB - BACKGROUND: The phenotypic characteristics that allow some tumor cells to metastasize have not been fully identified. The production and/or response of tumor cells to various growth factors have been shown to distinguish cells of differing metastatic potentials. OBJECTIVES: To determine (1) whether rat hepatocellular carcinoma cell lines produce interleukin-6 (IL-6) and (2) whether production of IL-6 correlates with either metastatic potential or tumorigenicity. METHODS: The clonal cell lines 1682.C.2.9.L0 (poorly metastatic) and 1682.C.2.9.L10 (highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet and adapted to growth in vitro. RESULTS: Both cell lines resulted in primary tumors with equal frequency and developed a 40-mm nodule in a similar period time, when an inoculum of 5 X 10(6) cells was injected subcutaneously; however, only L10 cells metastasized to the lung. These cell lines did not demonstrate differential expression of several antigens noted to correlate with metastatic potential, including CD44 variant glycoprotein, p53, transferrin receptor, and E-cadherin. In contrast, L0 cells produced less than 10 U of IL-6 per milliliter in culture (as determined by bioassay using 7TD1 cells), whereas L10 cells released more than 95 U of this cytokine per milliliter under identical culture conditions (P<.01, Student's t test). In addition, serum concentrations of IL-6 were elevated in animals bearing L10-induced primary tumors but not in those with L0 induced tumors of comparable mass. Exogenous addition of IL-6 to both tumor cell lines had no effect on the rate of growth in vitro, supporting the similar the tumorigenic potentials observed in vivo. CONCLUSION: Excess IL-6 production appears to identify cells with metastatic potential and does not appear to be essential to the establishment of a primary tumor. PMID- 8615720 TI - Defining the criteria for local resection of ampullary neoplasms. AB - OBJECTIVES: To delineate factors determined preoperatively, which predict successful local resection of ampullary neoplasms. DESIGN: Retrospective review of case series of the author's experience from 1988 through 1995. The median follow-up of patients with malignancies was 29 months. SETTING: Tertiary care university teaching hospital. PATIENTS: Twenty-seven patients underwent surgery. The decision to perform either an ampullectomy or pancreaticoduodenectomy (PD) was based on the size of the lesion, the presence of a "field defect" (ie, familial polyposis), depth of invasion determined by preoperative endoscopic ultrasound, and extent of pancreatic and bile duct involvement seen on endoscopic retrograde cholangiopancreatography. INTERVENTIONS: Fourteen patients underwent ampullectomy, 12 patients underwent PD, and one patient had a retroperitoneal node biopsy performed without resection of the primary tumor. MAIN OUTCOME MEASURES: Resectability, morbidity, and mortality. RESULTS: Depth of invasion was accurately determined in nine of 12 patients studied by preoperative endoscopic ultrasound. Preoperative endoscopic biopsy specimens were obtained in 21 patients and were inaccurate in seven of 21 cases. The length of stay following local resection was 10.5 +/- 3.7 days vs 15.4 +/- 5.8 days following PD (P=.02). One patient died following PD, and there were no deaths following ampullectomy. Six of 12 patients undergoing PD had postoperative complications vs two of 14 patients undergoing local resection. CONCLUSIONS: Ampullectomy is the procedure of choice for resecting benign lesions smaller than 3 cm, small neuroendocrine tumors, and T1 carcinomas of the ampulla. While endoscopic ultrasonography is helpful in identifying stage T1 lesions suitable for local resection, no preoperative test proved accurate enough to substitute for clinical judgment and intraoperative pathological confirmation. PMID- 8615721 TI - Risks of synchronous gastrointestinal or biliary surgery with splenectomy for hematologic disease. AB - BACKGROUND: The addition of splenectomy to a gastrointestinal (GI) operation may have an adverse effect on mortality, morbidity, and even survival. OBJECTIVE: To determine the risks of the converse: synchronous GI surgery appended to splenectomy for hematologic diseases. DESIGN: Retrospective cohort. SETTING: Multiple hospitals comprising an affiliated surgical training program. PATIENTS: Consecutive sample of 207 adults (mean age, 49 years) with splenectomies for hematologic diseases. INTERVENTION: Splenectomy and concomitant GI or biliary surgery (group 1, n=19) and splenectomy alone (group 2, n=188). MAIN OUTCOME MEASURES: Length of hospital or intensive care unit stay, later operations, postoperative infections, postoperative abdominal abscess, major complications, and death. RESULTS: Preoperative and intraoperative factors were similar in both groups. Operative mortality was 3 of 19 in group 1 and 8 of 188 in group 2 (p=.07). The mean number of major complications tended to be higher in group 1 (1.5 vs 0.5, P=07). Despite no difference between the incidences of overall postoperative infections, patients in group 1 were much more likely to develop an abdominal abscess (4 of 19 vs 3 of 188, P=.002). Logistic regression established that patients undergoing splenectomy and synchronous GI or biliary surgery were 25 times more likely to develop an intra-abdominal abscess than were patients with splenectomy alone, even controlling for confounding factors (odds ratio, 24.7; 95% confidence interval, 3.1 to 196; P=.002). CONCLUSIONS: Synchronous GI or biliary surgery with splenectomy for hematologic disease increases the risk of intra-abdominal abscess and should be avoided. Complication and mortality rates may also be increased. PMID- 8615722 TI - Moose-motor vehicle collisions. An increasing hazard in northern New England. AB - OBJECTIVE: To analyze the epidemiology and epizootiology of moose-motor vehicle collisions (MMVC) and outcomes in severely injured patients to identify variables that might be modified to reduce the impact of this mutually deleterious interspecies interaction. DESIGN: Wildlife and Traffic Safety databases permitted retrospective, population-based assessment of MMVC epidemiology. A case series compiled from hospital trauma registries characterized morbidity and mortality from MMVC. SETTING: New Hampshire and Maine area. PATIENTS: All victims of MMVC (1980 through 1991) were included in population-based analyses. Twenty-three patients hospitalized at three rural trauma centers (January 1990 through June 1994) were included in the case series. MAIN OUTCOME MEASURES: Location, time of day and seasonal occurrence of MMVC were determined. Injury patterns and Injury Severity Scores were analyzed in 23 representative patients. Maine's 1991 traffic and medical data were linked, and factors predictive of injury from MMVC were identified using multivariate logistics. RESULTS: Most MMVC occur from April through October after dark. Of 23 subjects, 70% sustained head and/or face injuries and 26%, cervical spine injuries. Mortality was 9%. Mean Injury Severity Score was 15.7 (SD=9.0). Safety belt use, rear seat location, and light truck occupancy were associated with reduced injury (p<.05). CONCLUSIONS: Moose-motor vehicle collisions are increasing in rural regions. Prevention programs should emphasize defensive driving and seat belt use, especially during high-risk periods. Injury patterns in MMVC suggest a need for automobile design modifications that better protect the passenger compartment form direct impact. PMID- 8615723 TI - Bile duct injuries, 1989-1993. A statewide experience. Connecticut Laparoscopic Cholecystectomy Registry. AB - OBJECTIVE: To review the incidence of major bile duct injuries (MBDI) during the shift from open (OC) to laparoscopic cholecystectomy (LC). DESIGN: Cohort analysis; minimum 15-month patient follow-up. SETTING: Acute care Connecticut hospitals. PATIENTS: Medical records of 30211 patients with cholecystectomy (OC or LC) reviewed; 47 cases of MBDI confirmed. MAIN OUTCOME MEASURE: Rate of MBDI. RESULTS: The incidence of MBDI in Connecticut hospitals rose from 0.04% in 1989 to 0.24% in 1991, then decreased to 0.11% in 1993. The increase was due to increased numbers of cholecystectomies and the initial increased risk of injury with LC. The 1990-through-1993 trend of decreasing incidence of LC MBDI was statistically significant (P=.02). By 1993, the difference between LC and OC was no longer significant (P=.81). Acute cholecystitis (odds ratio, 3.3) and gallstone pancreatitis (odds ratio, 3.6) increased the risk of MBDI during LC (P<.001). The LC MBDI more commonly were ductal excision or transections and often were not diagnosed intraoperatively. Intraoperative cholangiography facilitated intraoperative recognition and repair. Most patients (89%) underwent definitive management of the MBDI at the hospital of origin; of those, 5% required further interventions. CONCLUSIONS: Surgeries for acute cholecystitis and gallstone pancreatitis are associated with an increased risk for MBDI. Ductal anatomy, the timing of recognition of injury, and the method of repair dictate patient outcomes. Most patients are successfully managed at the hospital of origin, with good long-term results. Late bile duct strictures appear rare. PMID- 8615724 TI - Simultaneous bacteriologic assessment of bile from gallbladder and common bile duct in control subjects and patients with gallstones and common duct stones. AB - OBJECTIVES: To determine the simultaneous prevalence of bacteria in bile from the gallbladder and common bile duct and to determine the influence of the number of stones present on bacteriologic findings. METHODS: A prospective study was performed in 467 subjects divided into seven groups: 42 control subjects with normal biliary tracts, 221 patients with symptomatic gallstone disease, 12 patients with hydropic gallbladder, 52 patients with acute cholecystitis, 67 patients with common bile duct stones without cholangitis, 49 patients with common bile duct stones and acute cholangitis, and 24 patients with previous cholecystectomy and common bile duct stones. In all except controls, bile samples from the gallbladder and common bile duct were taken simultaneously for aerobic and anaerobic cultures. RESULTS: Control subjects had no bacteria in gallbladder bile. Patients with gallstones, acute cholecystitis, and hydropic gallbladder had similar rates of positive cultures in the gallbladder and common bile duct, ranging from 22% to 46%, but the rate was significantly higher in patients with common bile duct stones without cholangitis (58.2%). Patients with cholangitis or previous cholecystectomy had a high rate of positive cultures of common duct bile (93% to 100%). Age greater than 60 years had a significant influence on the rate of positive bile cultures. There was no relationship between the number of stones in the gallbladder or common bile duct and the percentage of positive cultures. In 98% of the patients, the same bacteria were isolated from gallbladder and common duct bile. CONCLUSIONS: In normal subjects, no bacteria were present in the biliary tract. Among patients with common bile duct stones, there was an increasing percentage of positive cultures according to the severity of the disease. Age had an important influence, but sex and the number of common bile duct stones had no influence on positive cultures. PMID- 8615725 TI - Telesurgery. Acceptability of compressed video for remote surgical proctoring. AB - OBJECTIVE: To determine the clinical acceptability of various levels of video compression for remote proctoring of laparoscopic surgical procedures. DESIGN: Observational, controlled study. SETTING: Community-based teaching hospital. PARTICIPANTS: Physician and nurse observers. INTERVENTIONS: Controlled surgical video scenes were subjected to various levels of data compression for digital transmission and display and shown to participant observers. MAIN OUTCOME MEASURES: Clinical acceptability of video scenes after application of video compression. RESULTS: Clinically acceptable video compression was achieved with a 1.25-megabit/second data rate, with the use of odd-screen 43.3:1 Joint Photographic Expert Group compression and a small screen for remote viewing. CONCLUSION: With proper video compression, remote proctoring of laparoscopic procedures may be performed with standard 1.5-megabit/second telecommunication data lines and services. PMID- 8615726 TI - The role of venous reflux and calf muscle pump function in nonthrombotic chronic venous insufficiency. Correlation with severity of signs and symptoms. AB - OBJECTIVE: To determine the lower-limb venous hemodynamics in patients with varying grades of chronic venous insufficiency (CVI), not due to deep vein thrombosis, when matched for age and duration of disease. DESIGN: Case-control study. SETTING: Normal volunteers and patients with different grades of CVI referred to the vascular unit of a university hospital. MATERIALS: Fifty-one legs (40 patients) with CVI, but without a history of deep vein thrombosis, and 24 normal legs (20 volunteer subjects). These selected legs were grouped according to the severity of CVI (classes 0 - through 3) so that each class was matched for age (all study participants <60 years) and duration of signs and symptoms (<10 years). INVESTIGATIONS: Air plethysmography and color flow duplex imaging. MAIN OUTCOME MEASURES: Venous volume, venous filling index, and outflow, ejection, and residual volume fractions were assessed in all limbs with air plethysmography. The presence of reflux was confirmed by the results of color flow duplex imaging. RESULTS: Ten (42%) of 24 limbs in class 0 had no reflux. Twenty-five (57%) of the 44 limbs in classes 0 and 1 had superficial reflux alone, while all the limbs in class 1 had some degree of reflux in the superficial veins. The sites of reflux in these limbs were similar. The patterns of reflux in classes 2 and 3 were more complex. Eight (26%) of the 31 limbs had superficial reflux alone, whereas 10 (32%) had all three systems involved (superficial, deep, and perforating). The venous volume, venous filling index, and residual volume fraction worsened with progression of CVI. Significant statistical differences could, however, only be demonstrated between classes 0 and 1 vs classes 2 and 3. No changes could be found in the ejection and outflow fractions. CONCLUSIONS: Patients (age <60 years) with CVI of less than 10 years' duration and with no history of deep vein thrombosis had venous hemodynamic changes that correlated well with the clinical severity of the disease. This was owing to the increased reflux, as the ejecting ability of the calf muscle pump remained intact, and the venous outflow was normal. PMID- 8615727 TI - An alternative approach of choledocholithotomy via laparoscopic choledochotomy. AB - OBJECTIVE: To evaluate the safety and feasibility of laparoscopic choledocholithotomy via choledochotomy for the treatment of choledocholithiasis. DESIGN: A prospective series of 1332 consecutive patients who underwent laparoscopic cholecystectomies, with a mean follow-up of 21.2 months. SETTING: University-affiliated referral center. PATIENTS: Forty-three patients (3%) with documented common bile duct stones from January 1991 to February 1995. INTERVENTIONS: Laparoscopic choledocholithotomy with choledochotomy and T tube drainage were performed in 40 patients. Postoperative endoscopic sphincterotomy after laparoscopic cholecystectomy was performed in three patients. MAIN OUTCOME MEASURES: Documented removal of common bile duct stones and procedure-related complications. RESULTS: Laparoscopic choledocholithotomy via choledochotomy was successful in 35 (88%) of 40 patients in whom this procedure was attempted. The mean (+/- SD) operation time was 191.3 +/- 75.4 minutes, and the mean (+/- SD) length of postoperative stay was 10.4 +/- 2.7 days. Seven complications (18%) were recorded, including three major complications (8%) and two retained stones (5%). CONCLUSIONS: Laparoscopic choledocholithotomy via choledochotomy can be performed safely, without increasing the morbidity rate as compared with that of open choledocholithotomy. Thus, some of the advantages of minimally invasive surgery are preserved. PMID- 8615728 TI - Survey analysis of the American Board of Surgery In-Training Examination. AB - OBJECTIVE: To determine how other program directors use the American Board of Surgery In-Training Examination (ABSITE) scores in the resident evaluation process. DESIGN: A cover letter and a printed one-page survey of eight questions about individual residency programs, the use of ABSITE scores in the evaluation process, minimum score for advancement, and actions taken, if any, for failure to meet required scores; space was provided for comments. SETTING: Two hundred seventy directors of surgical residency programs. PARTICIPANTS: Two hundred thirty-one (86%) directors of general surgery residency programs. MAIN OUTCOME MEASURES: The responses received in this questionnaire were similar to those received in the 1983 survey given by the American Board of Surgery. RESULTS: The majority of directors require their residents to take the ABSITE, but they differ greatly in their methods to retain or dismiss a resident, to evaluate the program and the cognitive knowledge of the residents, and to measure resident performance. CONCLUSIONS: The actions taken by program directors in their use of ABSITE scores vary widely, with a noticeable difference found when comparing the methods between the university and community hospital directors. Our survey findings show that there is still no uniform standard or agreement as to how the scores should be used, even though the ABSITE has been in existence for 20 years. PMID- 8615729 TI - Does patient-controlled analgesia achieve better control of pain and fewer adverse effects than intramuscular analgesia? A prospective randomized trial. AB - OBJECTIVE: To compare three analgesic regimens in patients undergoing colon resection: patient-controlled morphine sulfate analgesia (PCA), intramuscular (IM) morphine, and IM ketorolac tromethamine. DESIGN: Prospective randomized case series. SETTING: Rural, private teaching hospital. PATIENTS: All patients (307) scheduled to undergo a major colon resection between January 1, 1992, and December 31, 1993, were eligible to participate. Of these, 10 (3%) were missed in the screening process, 132 (43%) declined participation, 73 (24%) were excluded, and 92 (30%) were enrolled and randomly assigned to a treatment group. INTERVENTIONS: Ninety-two patients were enrolled in the study. Two patients never received the medication to which they were assigned, owing to administrative error; their data was not analyzed. Of the remaining patients, 31 were randomized to the PCA morphine group, 31 were randomized to the IM morphine group, and 28 were randomized to the IM ketorolac group. The randomly assigned drug was first administered in the post-anesthesia care unit. On arrival on the postoperative ward, the patient was asked to rate his or her pain using both a numerical rating scale and a visual analog scale at 30 minutes; 1, 2, 3, 4, and 6 hours after arrival on the ward; and every 4 hours throughout the first 5 postoperative days. The Mini-Mental State Examination (MMSE) was administered the day before surgery and then daily for the first 5 postoperative days. The first day the patient passed flatus after surgery was also recorded. MAIN OUTCOME MEASURES: The end points analyzed were adverse effects, duration of postoperative ileus, degree of pain control, length of hospitalization, and development of postoperative confusion as measured on serial MMSEs. RESULTS: Only two patients, both in the PCA group, reported adverse effects; neither required a change in analgesia group. Significantly more patients assigned to IM ketorolac broke protocol and required alternative analgesia than did patients in the morphine groups (32% ketorolac vs 16% IM morphine and 0% PCA). The ketorolac group had a significantly shorter duration of ileus than either morphine group (P<.0l). The ketorolac group also had significantly lower pain scores (P<.04) and less postoperative confusion than the morphine groups (P<.03), although these results are limited by missing values. The ketorolac group had a significantly shorter length of stay than either morphine group (P<.01), while there was no significant difference between the morphine groups (P=.75). CONCLUSIONS: While it appears that ketorolac provides a better postoperative course than either IM or PCA morphine in terms of pain control, postoperative confusion, length of stay, and duration of ileus, 18% of our patients assigned to ketorolac required additional analgesia, and there was a strong patient preference for PCA. Most patients should probably be managed with PCA narcotics, but the addition of ketorolac might reduce narcotic dose and resultant adverse effects. Those patients particularly prone to adverse effects should receive primarily ketorolac. PMID- 8615730 TI - Multimodal-therapy breast salvage in the urban poor with locally advanced cancer. AB - OBJECTIVES: To determine whether economically disadvantaged urban women with locally advanced breast cancer (American Joint Committee on Cancer stages IIB to IIIB) have rates of response to sequential neoadjuvant chemotherapy and radiation, breast salvage rates, overall survival rates, and disease-free survival rates comparable with those previously reported in other socioeconomic groups and to compare these variables in different ethnic groups within the study population. DESIGN: Prospective, nonrandomized, case series. SETTING: Urban county hospital. PATIENTS: Thirty-seven women with locally advanced breast cancer who came to the breast clinic at Cook County Hospital, Chicago, Ill, during a 3 year interval. INTERVENTION: Sequential chemoradiation followed by surgery in selected patients. MAIN OUTCOME MEASURES: Comparison of clinical response rates, disease-free survival rates, and breast salvage rates between different ethnic groups in the study population. RESULTS: In the entire group, the overall response rate to neoadjuvant chemotherapy was 73%, with a complete response rate of 32%. Twenty-five percent of patients whose tumors responded incompletely to chemotherapy had a complete response after subsequent radiation. With a mean follow-up of 18.7 months, 65% of patients had no evidence of disease, and breast salvage without evidence of recurrent disease was achieved in 38% of patients. No differences in overall response rates, breast salvage rates, or early disease free survival rates were observed within different ethnic groups in the study population, and these results are generally comparable with previously reported results in other socioeconomic groups. CONCLUSION: These results do not show significant differences in responses to sequential chemotherapy and irradiation, in breast salvage rates, or in survival between different ethnic groups in this study population. PMID- 8615731 TI - Nipple-areolar preservation during breast-conserving therapy for subareolar breast carcinomas. AB - OBJECTIVE: To determine if women with subareolar breast carcinoma can be successfully treated by breast-conserving therapy consisting of segmental mastectomy that preserves the nipple-areolar complex, axillary lymph node dissection, and postoperative irradiation. DESIGN: Prospective study. SETTING: Tertiary care cancer center. PATIENTS: Twenty-five patients; median age, 56 years; median tumor diameter, 1.4 cm; and median follow-up, 48 months. INTERVENTION: Breast-conserving therapy for subareolar primary breast carcinoma. RESULTS: Two patients had positive surgical margins of resection, and another patient underwent simple mastectomy after developing a local recurrence. Nipple areolar distortion was the most common cosmetic deformity after breast-conserving therapy, but overall cosmesis was good. At the most recent follow-up, all patients were free of disease. CONCLUSION: Patients who have small subareolar primary breast carcinomas without evidence of nipple involvement are candidates for breast-conserving therapy with nipple-areolar preservation. PMID- 8615732 TI - Monocyte tumor necrosis factor receptor levels as a predictor of risk in human sepsis. AB - OBJECTIVE: To assess peripheral blood monocyte tumor necrosis factor receptor (TNFR) levels and plasma soluble tumor necrosis factor receptor (sTNFR) concentrations in critically ill patients with sepsis syndrome. DESIGN: Prospective, descriptive cohort study with no interventions. SETTING: Surgical intensive care unit of a tertiary-care hospital associated with a university medical school. PATIENTS: Twenty-one patients with a documented source of infection who met currently accepted criteria for sepsis syndrome/septic shock. MAIN OUTCOME MEASURES: Plasma sTNFR p55 and p75 values were quantified by enzyme linked immunosorbent assay, and monocyte TNFR levels were assessed by fluorescence flow cytometry after the monocytes were stained with biotinylated human recombinant TNF-alpha and streptavidin-phycoerythrin. RESULTS: Compared with healthy controls, plasma sTNFR p55 and p75 values were significantly higher (P <.01) in both surviving and nonsurviving patients with sepsis; in nonsurviving patients with sepsis, however, only sTNFR p55 values were significantly (P < .05) higher than in surviving patients with sepsis. By contrast, monocytes from the nonsurviving patients with sepsis manifested a significant (P < .01) and sustained (up to 4 days) decrease in cell surface TNFR values compared with either the normal controls or the surviving patients with sepsis. CONCLUSIONS: Assessment of monocyte surface TNFR values may provide a rapid prognostic indicator for patients with sepsis who are at increased risk of death. PMID- 8615733 TI - The effect of leukocytes on adhesion molecules. An explanation of blood transfusion enhancement of tumor growth. AB - OBJECTIVE: To determine the mechanism of the adverse relationship between perioperative blood transfusion and lung cancer recurrence, by focusing on endothelial cell adhesion molecules (CAMs), which are thought to play a role in distant tumor cell implantation. DESIGN AND OUTCOME MEASURES: Murine endothelial cells were cocultured with allogeneic leukocytes, syngeneic leukocytes, and syngeneic lung carcinoma cells for 60 hours. The percentage of endothelial cells expressing vascular CAMs (VCAMs) and intercellular CAMs (ICAMs) was quantified during this time using indirect immunofluorescence and flow cytometry. Tumor cell adhesion to the endothelium was quantified for 6 hours using cells labeled with sulfur 35 and a scintillation counter. SETTING: Laboratory. MATERIALS: C57/BL and Balb/C mice. RESULTS: Vascular CAM was not expressed on the endothelium, but ICAM was preferentially expressed without stimulation. Tumor-cell adhesion and endothelial ICAM expression were inversely related. After 15 hours of coculture, tumor cell adhesion was four-fold greater in the experimental group than in the control, and coincident endothelial ICAM expression was fourfold lower. CONCLUSION: Endothelial cell ICAM expression is negatively correlated with metastatic potential. PMID- 8615734 TI - Multiple-system organ damage resulting from prolonged hepatic inflow interruption. AB - BACKGROUND: It has been reported that patients undergoing major hepatectomy tolerated 90 and 127 minutes of continuous hepatic inflow interruption with no evidence of permanent damage to the liver. We questioned the safety and feasibility of the interruption beyond 90 minutes in normothermic human beings. We also postulated that, besides injury to the liver per se, extended continuous hepatic inflow interruption would cause extrahepatic multiple-system organ damage in subjects exposed to continuous hepatic inflow interruption for 90 or 120 minutes. DESIGN: Fifty Sprague-Dawley rats were divided into three groups. Group 1 served as controls that had only laparotomy. Group 2 underwent continuous hepatic inflow interruption for 90 minutes, and group 3 was subjected to continuous hepatic inflow interruption for 120 minutes. Scanning electron microscopy and transmission electron microscopy were used to evaluate ultrastructural alterations in the liver, lung, heart, and intestine. SETTING: Lund (Sweden) University Hospital and Top Cancer Institute, Lund. INTERVENTIONS: Intraoperative and postoperative infusion and blood transfusion were given in all experimental animals. MAIN OUTCOME MEASURES: Animal survival and manifestations of multiple-system organ failure. RESULTS: In rats with continuous hepatic inflow interruption for 90 or 120 minutes, scanning electron microscopy showed a necrotic surface of the liver cells together with fibrin exudation. Hepatic sinusoids and intrahepatic nerves also had severe injury. Destruction of pulmonary structures and breakdown of microcirculation in the lung were characterized by thinned and ruptured walls of alveoli and a greatly decreased number of capillaries in the damaged alveolar wall. Transmission electron microscopy showed four types of ultrastructural changes, ie, necrosis of epithelial cells, extremely swollen mitochondria in intestinal cells, death of mucosal cells, and increased permeability of vessels in the injured intestine. The affected heart manifested highly enlarged mitochondria in myocardial cells, thickened vascular walls, and scattered necrotic lesions in myocardial tissue. CONCLUSIONS: Multiple-system organ failure resulting from ischemia-reperfusion injury and obstacle of portal hemodynamics in a subject subjected to an extended continuous hepatic inflow interruption is an unrecognized new disorder that may cause a high mortality rate. Our preliminary results indicated that animals subjected to continuous hepatic inflow interruption for 90 or 120 minutes developed various injuries to the liver, lung, heart, and gut. Therefore, we believe that continuous hepatic inflow interruption exceeding 90 minutes could also be hazardous in human beings. PMID- 8615735 TI - The saline ballooning method for peritoneal dissection during laparoscopic herniorrhaphy. AB - To improve the safety and speed of the peritoneal dissection, we developed a saline ballooning method in which a dull needle is inserted lateral to the inferior epigastric vessels under direct laparoscopic visualization to a tissue plane just superficial to the peritoneum. Then, saline solution is injected. As a result, the peritoneum is easily separated from the spermatic cord and inferior epigastric vessels. Furthermore, bleeding is minimized during dissection of the peritoneum. PMID- 8615736 TI - The crime of saving lives: the FDA, John Najarian, and Minnesota ALG. PMID- 8615737 TI - A denture swallowed. Case report. AB - Swallowing and aspiration of dental foreign objects is often reported in the literature. Swallowing seems to be more common than aspiration, and is observed most often in the elderly. Too often the size and configuration of these objects compounds their impaction and removal. A 67-year-old man swallowed a unilateral removable partial denture which became lodged in his mid-oesophagus. The denture was located by radiographic examination of his chest and removal by a rigid oesophagoscopy was performed under general anaesthetic. PMID- 8615738 TI - Cleidocranial dysplasia. Case report. AB - A 29-year-old Caucasian woman who presented with short stature and multiple unerupted supernumerary teeth is described. Radiological investigations of her cranial and skeletal abnormalities revealed cleidocranial dysplasia. Because of the advanced age of the patient and contraindication for orthodontic treatment, only surgical and prosthetic treatment were performed. The characteristics and treatments of this rare autosomal dominant disorder are discussed. PMID- 8615739 TI - Central calcifying odontogenic cyst. Case report. AB - A case of central (intraosseous) calcifying odontogenic cyst affecting the mandible is reported. The clinical, radiographic, and histological features are reviewed and discussed. PMID- 8615740 TI - The provision of orthodontic services by general dental practitioners. 2. Factors influencing variation in service provision. AB - Previous work has shown that variations exist amongst general dental practitioners in the volume and type of orthodontic services provided, the type of orthodontic appliances used, and the objectives of the orthodontic treatment. The aims of this survey were to identify practitioner characteristics that account for variations in the level of orthodontic services provided and which distinguish providers and non-providers of orthodontic services. Multiple regression analysis revealed that four practitioner characteristics explained 41 per cent of the variance in the number of orthodontic patients treated. Dentists who treated more orthodontic patients: 1) treated more general practice patients; 2) frequently used multiple sources to keep up to date in orthodontics; 3) perceived their patient base to contain more children; and 4) were likely to have attended a Truitt course. Eleven variables best distinguished providers from non providers of orthodontic treatment; 1, 2 and 3 above had the highest correlation with the discriminant function. The Null Hypothesis that selected characteristics of dentists providing orthodontic services were no different from those of dentists not providing orthodontic services was rejected. The provision of orthodontic services was associated with a higher level of continuing orthodontic education as well as treating more general practice patients, especially children. PMID- 8615741 TI - The stability of facial osteotomies. 4. Maxillary and mandibular (with or without chin) advancement with rigid internal fixation. AB - The short-term (6 weeks postoperative) and long-term (12 months postoperative) horizontal skeletal stability of combined maxillary and mandibular advancement was evaluated by cephalometric analysis of 15 patients. The mean horizontal advancement of the maxilla was 5.84 mm. Six weeks later a mean horizontal relapse of 0.03 mm (0.05%) was identified. The mean horizontal relapse at long-term follow-up was 0.59 mm (10.1%). The mean horizontal advancement of the mandible was 12.35 mm at menton and 12.65 mm at pogonion. At 6 weeks, mean horizontal relapse, respectively at the above landmarks, was 0.11 mm and 0.21 mm (1.3%). The mean horizontal relapse at long-term follow-up was 2.19 mm and 1.98 mm (16.6%) respectively for the same landmarks. Subjectively and objectively, improvements were seen in facial aesthetics and dental occlusion. The results indicate that rigid fixation of osteotomies undertaken to correct 'horizontal facial deficiency' is a surgically predictable and relatively stable procedure when reviewed up to 12 months after surgery. PMID- 8615742 TI - Temporomandibular disorders. 2. Non-surgical treatment. AB - There are many treatment modalities for temporomandibular disorders (TMD), most of which are effective in controlling symptoms, at least in the short term. The non-surgical treatment of temporomandibular disorders continues to be the most effective way of managing over 80 per cent of patients who present with symptoms of temporomandibular pain and dysfunction. In this, the second article in the series, a general overview of the current non-surgical treatment strategies for TMD will be presented. PMID- 8615743 TI - Complete denture problem solving: a survey. AB - An investigation was carried out into the problems experienced by 114 referred patients with complete dentures who were considered to be difficult or to have difficult prosthodontic problems. The commonest problems were those of pain and lack of retention, due mainly to occlusal discrepancies and excessive VDO. Treatment was carried out on an individual basis with a large proportion of dentures being remade. However, a small number was satisfied by counselling alone without procedural treatment. A diagnostic denture technique was used for particularly difficult cases. This technique showed that, in two cases, technical prosthodontic inadequacies could definitively be excluded as the problem. The overall success rate for treatment was 80 per cent. Further studies on a larger patient sample are needed so that specific problems can be linked to cause and outcome in a meaningful manner. PMID- 8615744 TI - Cephalometric analysis of Deutero-Malay Indonesians. AB - A cephalometric study using Downs' analysis was undertaken with lateral cephalometric radiographs for a mixed sample of 50 child, adolescent and young adult Indonesians who presented with Angle Class I occlusions. Significant differences between this study group and the published results from comparative racial types were found for Downs' angle of convexity, Y-axis and incisor inclinations, indicating that Indonesians can be differentiated from other races with similar occlusions by using Downs' analysis. PMID- 8615745 TI - Forces transmitted through EVA mouthguard materials of different types and thickness. AB - Previous studies into sporting mouthguards have been mainly attitudinal or epidemiological. The aim of the present study was to build an impact rig to measure the impact absorbed by mouthguard materials of various thicknesses. The acceleration of the pendulum of the rig was measured and used to calculate the force transmitted to the materials. Impact tests were also performed on three commercially available mouthguard materials. Tests showed that the force transmitted through mouthguard materials was inversely related to the material thickness. Mouthguard construction techniques with ethylene vinyl acetate (EVA) plastics should be monitored to avoid occlusal thinning especially on the incisal edges. Thinning results in reduction in the protection offered by the mouthguard. PMID- 8615747 TI - The first three questions. PMID- 8615746 TI - A survey of the pattern of private general dental practice in Queensland, 1992. AB - In 1987 all non-specialist ADA member private practitioners in Queensland were invited to participate in a survey to determine the number of the various types of permanent restorations placed in one week and the percentage breakdown of the time they devoted to the various areas of dentistry. Results of that survey were published in 1989. The survey with a few minor changes was repeated in 1992. Results show that there has been little change in the pattern of practice over the five years with restorative dentistry still taking a very large proportion of respondents' time and similar materials being used. The dental workforce appears to be relatively stable with low rates of overseas and interstate migration. There is evidence of an increasing number of women dentists in the workforce and this is emphasized by a breakdown of the current undergraduate enrolment. PMID- 8615748 TI - Ectopic third molar in maxillary sinus. PMID- 8615749 TI - Mouthguard protection in sports injuries. PMID- 8615750 TI - Sodium hyaluronate for TMJ disorders. PMID- 8615751 TI - Structure-activity relationship of cytoplasmic 5'-nucleotidase substrate sites. AB - Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), but not the cytosolic AMP preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in vertebrate heart. The published data on substrate specificity involve mainly the naturally occurring nucleoside monophosphates, without a systematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure-activity relationship for c-N-I and c-N-II in detail. The rank order of activity of the test compounds differed substantially between c-N-I and c-N-II. c N-I and c-N-II varied with respect to the following interactions with substrate: (1) hydrogen-bond formation with the substituent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6-position unsubstituted purine ring (more pronounced with c-N-I than with c-N-II). No better substrate than 5'-AMP was found for c-N-I. We propose that c-N-I functions as an AMP-binding protein in the myocardial cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly. PMID- 8615752 TI - Molecular cloning, expression and potential functions of the human proteinase activated receptor-2. AB - We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators. PMID- 8615753 TI - Rat kidney glutathione S-transferase 1 subunits have C-terminal truncations. AB - Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1',3 and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1', 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has a C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal tetrapeptide-deleted form was not observed for rat liver GST 1. PMID- 8615754 TI - Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells. AB - Oxidized cholesterol compounds or oxysterols are thought to be potent membrane destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7beta-hydroxycholesterol (7beta-OH) or 25 hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7beta-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may results from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure. PMID- 8615755 TI - Ability of a beta-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters. AB - The ability of casein in the form of colloidal-sized casein micelles to modulate the phase separation of calcium phosphate during milk secretion is adapted to produce nanometre-sized particles of calcium phosphate stabilized by a casein phosphopeptide (nanoclusters). The nanoclusters were prepared from an undersaturated solution of salts and the peptide by raising the pH homogeneously from about 5.5 to 6.7 with urea plus urease. Chemical analysis and IR spectroscopy showed that they comprise an amorphous dicalcium phosophate bound to the phosphopeptide. Multinuclear NMR spectroscopy of the cluster solutions showed that the small ions and free peptide in the solution were in a state of dynamic exchange with the nanoclusters. The peptide is linked to the calcium phosphate through its sequence of phosphorylated residues, but, in a proportion of adsorbed conformational states, the termini retain the conformational freedom of the unbound peptide. The ability of casein to form nanoclusters in milk suggests a more general mechanism for avoiding pathological calcification and regulating calcium flow in tissues and biological fluids exposed to or containing high concentrations of calcium. PMID- 8615756 TI - Complex regulation of plasminogen activator inhibitor type-1 (PAI-1) gene expression by serum and substrate adhesion. AB - Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily that functions to negatively regulate the plasmin-based pericellular proteolytic cascade, was induced early after exposure of growth-arrested normal rat kidney (NRK) cells to serum containing medium. Increased PAI-1 transcription was rapid (evident within 10 min of serum addition) and involved immediate-early response kinetics. [3H]Thymidine autoradiography was used to map the time frame of PAI-1 expression during a synchronous growth cycle. PAI-1 transcript accumulation peaked in mid-G1 phase (approx. 4-6 h post-stimulation) and declined prior to, or concomitant with, the onset of DNA synthetic phase. Serum increased PAI-1 expression in NRK cells in agarose suspension, as well as monolayer, culture; induction in suspended cells (similar to monolayer culture conditions) also occurred in the presence of cyclohexamide or puromycin. The serum-inductive pathway leading to PAI-1 gene activation is thus functional regardless of adhesive conditions or capacity for de novo protein synthesis. The amplitude of induction and maintenance of expression in later stages of G1, however, were subject to adhesive influences. PAI-1 transcript accumulation at 4 and 8 h post-stimulation in newly adherent cells, moreover, was blocked by puromycin, indicating that both immediate-early and secondary mechanisms regulate PAI-1 mRNA levels during progression of NRK cells through an 'activated' G1 growth phase. PMID- 8615757 TI - Characterization of rat 5alpha-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae. AB - Two isoforms of the rat 5alpha-reductase (5alpha-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other delta4-3-keto steroids (e.g. progesterone and corticoids) into their 5alpha-reduced metabolites, have been cloned. In this study, a convenient and efficient system was developed to overexpress the two isoenzymes in Saccharomyces cerevisiae by using the ubiquitin fusion expression system. Two yeast expression vectors have been prepared, YEpR1 and YEpR2, which code for 5alpha-R type 1 and 5alpha-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5alpha-R type 1 or 5alpha R type 2 cDNAs in frame to the 3' end of the ubiquitin cDNA. The activity of the two isoenzymes produced in yeast was determined in cell lysates at the enzyme pH optima (type 1, pH 7.5; type 2, pH 5.5) and a possible differential intracellular distribution was also evaluated. The kinetic parameters were: type 1, Km 4.6 microM, Vmax.100.6 micrograms/h per mg of protein; type 2, Km 68.6 nM, Vmax. 0.84 micrograms/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5alpha-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5alpha-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000g). The data indicate that yeasts overexpress the two 5alpha-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell. PMID- 8615758 TI - Rapsyn's knobs and holes: eight tetratrico peptide repeats. PMID- 8615759 TI - Capacitative Ca2+ influx and a diffusible influx factor. PMID- 8615761 TI - Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells. AB - The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells. PMID- 8615760 TI - Myc oncogenes: the enigmatic family. AB - The myc family of proto-oncogenes is believed to be involved in the establishment of many types of human malignancy. The members of this family have been shown to function as transcription factors, and through a designated target sequence bring about continued cell-cycle progression, cellular immortalization and blockages to differentiation in many lineages. However, while much of the recent work focusing on the c-myc oncogene has provided some very important advances, it has also brought to light a large amount of conflicting data as to the mechanism of action of the gene product. In this regard, it has now been shown that c-myc is effective in transcriptional repression as well as transcriptional activation and, perhaps more paradoxically, that it has a role in programmed cell death (apoptosis) as well as in processes of cell-cycle progression. In addition, particular interest has surrounded the distinct roles of the two alternative translation products of the c-myc gene, c-Myc 1 and c-Myc 2. The intriguing observation that the ratio of c-Myc 1 to c-Myc 2 increases markedly upon cellular quiescence led to the discovery that the enforced expression of the two proteins individually showed that c-Myc 2 stimulates cell growth, whereas c-Myc 1 appears to be growth suppressing. Clearly, the disparities in the activities of c-Myc, together with the consistent occurrence of mutations of c-myc in human malignancies, means that, although reaching an understanding of the functions of the myc gene family might not be simple, it remains well worthy of pursuit. PMID- 8615762 TI - PC8 [corrected], a new member of the convertase family. AB - A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23-11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8. PMID- 8615763 TI - The human leukotriene A4 hydrolase gene is expressed in two alternatively spliced mRNA forms. AB - We identified a novel short (s-) mRNA of the human leukotriene-A4 (LTA4) hydrolase using sequential reverse transcriptase PCR mapping. The s-mRNA is generated by skipping an 83 bp exon located in the 3' coding region and suggests the expression of an LTA4 hydrolase isoform with a calculated molecular mass of 59 kDa and a distinct C-terminus. Both LTA4 hydrolase mRNAs are constitutively expressed in blood cells, endothelial cells, smooth muscle cells, fibroblasts and tumour cells. The ratios of their mRNA expression levels are cell specific, with relatively high s-form mRNA expression in reticulocytes. Our data strongly suggest that the novel mRNA codes for the structurally related but distinct LTA4 hydrolase isoenzyme that has been postulated. PMID- 8615764 TI - Rapid reduction and removal of HDL- but not LDL-associated cholesteryl ester hydroperoxides by rat liver perfused in situ. AB - To test whether high-density lipoproteins (HDL) could aid in the removal in vivo of potentially atherogenic oxidized lipids, we perfused rat liver in situ with buffer supplemented with isolated human HDL containing small amounts of cholesteryl linoleate hydro(pero)xides [CH18:2-O(O)H]. Perfusion resulted in the rapid removal of Ch18:2-O(O)H from HDL with a half-life (t1/2)of 11.4 min., faster than that of unoxidized cholesteryl linoleate, and dependent of the presence of the liver. In addition, the liver enhanced the reduction of Ch18:2 OOH associated with HDL remaining in the perfusate buffer. Perfusion resulted in the release of a hepatic activity that enhanced the reduction of HDL-associated CH18:2-OOH and was resistant to heat treatment. In contrast with the situation with HDL, low-density lipoprotein (LDL)-associated CH18:2-O(O)H were neither removed nor reduced by perfused rat liver within the time course studied, in support of a possible role for HDL in the detoxification of circulating lipid hydroperoxides in vivo. PMID- 8615765 TI - Top-down control analysis of temperature effect on oxidative phosphorylation. AB - The effects of temperature on the control of respiration rate, phosphorylation rate, proton leakage rate, the protonmotive force and the effective ATP/O ratio were determined in isolated rat liver mitochondria over a range of respiratory conditions by applying top-down elasticity and control analyses. Simultaneous measurements of membrane potential, oxidation and phosphorylation rates were performed under various ATP turnover rates, ranging from state 4 to state 3. Although the activities of the three subsystems decreased with temperature (over 30-fold between 37 and 4 degrees C), the effective ATP/O ratio exhibited a maximum at 25 degrees C, far below the physiological value. Top-down elasticity analysis revealed that maximal membrane potential was maintained over the range of temperature studied, and that the proton leakage rate was considerably reduced at 4 degrees C. These results definitely rule out a possible uncoupling of mitochondria at low temperature. At 4 degrees C, the decrease in ATP/O ratio is explained by the relative decrease in phosphorylation processes revealed by the decrease in depolarization after ADP addition [Diolez and Moreau (1985) Biochim. Biophys. Acta 806, 56-63]. The change in depolarization between 37 and 25 degrees C was too small to explain the decrease in ATP/O ratio. This result is best explained by the changes in the elasticity of proton leakage to membrane potential between 37 and 25 degrees C, leading to a higher leak rate at 37 degrees C for the same value of membrane potential. Top-down control analysis showed that despite the important changes in activities of the three subsystems between 37 and 25 degrees C, the patterns of the control distribution are very similar. However, a different pattern was obtained at 4 degrees C under all phosphorylating conditions. Surprisingly, control by the proton leakage subsystem was almost unchanged, although both control patterns by substrate oxidation and phosphorylation subsystems were affected at 4 degrees C. In comparison with results for 25 and 37 degrees C, at 4 degrees C there was evidence for increased control by the phosphorylation subsystem over both fluxes of oxidation and phosphorylation as well as on the ATP/O ratio when the system is close to state 3. However, the pattern of control coefficients as a function of mitochondrial activity also showed enhanced control exerted by the substrate oxidation subsystem under all intermediate conditions. These results suggest that passive membrane permeability to protons is not involved in the effect of temperature on the control of oxidative phosphorylation. PMID- 8615766 TI - Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control? AB - It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties. PMID- 8615768 TI - Exposure of ligand-binding sites on platelet integrin alpha IIB/beta 3 by phosphorylation of the beta 3 subunit. AB - The exposure of ligand-binding sites for adhesive proteins on platelet integrin alpha IIB/beta 3 (glycoprotein IIB/IIIA) by platelet-activating (PAF) is transient, whereas sites exposed by alpha-thrombin remain accessible. The same difference is seen in the phosphorylation of the beta 3 subunit. Inhibition of protein kinases (1 microM staurosporine) and protein kinase C (10 microM bisindolylmaleimide) closes binding sites exposed by both agonists and induces dephosphorylation of beta 3. Inhibition of Tyr-kinases (20 microM Herbimycin A) has only a slight effect. Inhibition of Ser/Thr-phosphatases (1 microM okadaic acid, 30 s preincubation) changes the transient exposure and beta phosphorylation by PAF into the 'permanent' patterns induced by alpha-thrombin. Inhibition of Tyr phosphatases (100 microM vanadate) has little effect. Preincubation with okadaic acid makes exposed binding sites and phosphorylated beta 3 insensitive to staurosporine, resulting in exposed alpha IIB/beta 3 independent of concurrent phosphorylation/dephosphorylation. The stoichiometry of beta 3 phosphorylation by alpha-thrombin is 0.80+/-0.10. Thus, one of the mechanisms that regulates exposure and closure of ligand-binding sites on the alpha IIb/beta 3 is phosphorylation/dephosphorylation of a Ser/Thr-residue in the beta 3 subunit. PMID- 8615767 TI - Cold-induced reduction in Gi alpha proteins in brown adipose tissue. Effects on the cellular hypersensitization to noradrenaline caused by pertussis-toxin treatment. AB - The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi 1 alpha and Gi 2 alpha, as well as of the beta-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (NA+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold. PMID- 8615770 TI - The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the alpha protons of amino acids. AB - 1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) c catalysed hydrogen-deuterium exchange of the alpha-protons of L- and D-alanine and tryptophan. The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-alpha-glycerol 3-phosphate. In the presence of DL-alpha-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalyzed first-order exchange rates was in the order tryptophan > alanine > glycine. This increase in stereospecificity was largely due to the decrease in the magnitude of the first order exchange rate of the slowly exchanged alpha-proton. A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton of D-alanine. Adding DL-alpha-glycerol 3 phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan. The alpha subunits are shown to increase the exchange rates of the alpha-protons of L alanine and L-tryptophan. We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its alpha-proton can be similar to or larger than that of its alpha-carboxylate group. Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed. PMID- 8615769 TI - Fibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse. AB - Disruption of the murine mdr2 gene leads to the complete absence of biliary phospholipids. We tested the hypothesis that the increase in biliary phospholipid output induced by fibrates is mediated via induction of the hepatic mdr2 gene and its encoded product, the P-glucoprotein canalicular flippase. Increased levels of mdr2 mRNA were observed in the liver of mice treated with different fibrates: ciprofibrate, 660+/-155% (as compared with control group); clofibrate, 611+/-77%; bezafibrate, 410+/-47%; fenofibrate, 310+/-52%; gemfibrozil, 190+/-25% (P <0.05 compared with control group). Induction of expression of the mdr gene family was specific to the mdr2 gene. Two- to three-fold increases in P-glycoprotein immunodetection were evident on the canalicular plasma-membrane domain of clofibrate- and ciprofibrate-treated mice. Biliary phospholipid output increased from 4.2+/-1.2 nmol/min per g of liver in the control group to 8.5+/-0.6, 7.1+/ 2.9 and 5.8+/-2.5 in ciprofibrate-, clofibrate- and bezafibrate-treated mice respectively (P <0.05 compared with control group). Moreover, a significant correlation between biliary phospholipid output and the relative levels of mdr2 mRNA was found (r=0.86; P <0.05). In treated animals, bile flow as well as cholesterol and bile acid outputs remained unchanged. Our findings constitute the first evidence that pharmacological modulation of biliary lipid secretion mediated by fibrates can be related to the overexpression of a specific liver gene product, the mdr2 P-glycoprotein, and are consistent with the hypothesis that the mdr2 P-glycoprotein isoform plays a crucial role in the secretion of biliary phospholipid. PMID- 8615771 TI - Trace metal, acute phase and metabolic response to endotoxin in metallothionein null mice. AB - Accumulation of hepatic zinc via metallothionein (MT) induction during infection/inflammation is postulated to benefit a range of metabolic processes. The metabolic consequences of two doses of endotoxin (LPS) (1 and 5 mg/kg, intraperitoneally) were examined in normal (MT+/+) and MT-null (MT-/-) mice (all results means =/- S.E.M., n=6). At 16 h after 1 mg/kg LPS, hypozincaemia was pronounced in the MT+/+ mice (4.4+/-0.2 microM), concomitant with a 36% increase in hepatic Zn and a > 10-fold increase in hepatic MT. Plasma Zn (16.6+/-0.7 microM) and total hepatic Zn were unchanged in MT -/- mice, confirming the importance of MT in altering plasma and hepatic Zn during inflammation. Plasma iron was lower in LPS-treated MT-/- mice, whereas plasma copper increased to a similar extent in both groups of mice. Plasma fibrinogen more than doubled, and was similar in both groups of mice, which questions the importance of MT in acute phase protein synthesis. Blood and liver glucose concentrations were not significantly different between groups before or after LPS, whereas blood and liver lactate concentrations were significantly lower (31% and 24% respectively) in MT-/- mice after LPS. At 16 h after 5 mg/kg LPS, plasma Zn was decreased even further in MT+/+ mice (2.6+/-0.3 microM), but remained unchanged in MT-/- mice at concentrations significantly above those in 16-h fasted MT-/- mice (15.8+/-0.5 versus 11.3+/-0.3 microM). Total liver Zn was 17% lower than fasting values in MT /- mice, in contrast with 32% higher in MT+/+ mice. Synthesis of MT (in MT+/+ mice) and fibrinogen in all mice was not further enhanced by the higher LPS dose. Blood glucose was significantly decreased by 18% in MT+/+ mice and by 38% in MT-/ mice after 5 mg/kg LPS. There was a marked 44% decrease in liver glucose in MT-/ mice; that in MT+/+ mice was unchanged from fasting levels, implying a deficit in hepatic gluconeogenesis in LPS-treated MT-/- mice. In the absence of any indication of major hepatotoxicity, the results of this study indicate that energy production, and not acute-phase protein synthesis, may be most influenced by Zn supply during endotoxaemia, suggesting that MT has a role in maintaining hepatic and blood glucose in this metabolic setting. PMID- 8615772 TI - Regulation of phosphatidylcholine synthesis in maturing type II cells: increased mRNA stability of CTP:phosphocholine cytidylyltransferase. AB - Pulmonary surfactant phosphatidylcholine synthesis increases in fetal lung type II cells with advancing gestation. This increase is accompanied by an increase in gene and protein expression of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15), which catalyses a regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. In the present study we investigated the role of transcriptional and post-transcriptional mechanisms in the developmental induction of CT mRNA in maturing type II cells. We found that CT mRNA increased 2 fold from days 18 to 21 of fetal rat gestation (term 22 d). This increase in CT mRNA was not accompanied by a developmental increase in CT gene transcription. However, CT mRNA was more stable on day 21 (t1/2 48 h) compared with that on day 18 (t1/2 17 h). Glucocorticoids have been shown to enhance surfactant phosphatidylcholine synthesis in fetal type II cells. Therefore we also examined the effect of maternal glucocorticoid administration to pregnant rats at 19 d of gestation on CT mRNA expression in fetal type II cells isolated 24 h later. Glucocorticoid treatment did not increase type II cell CT mRNA. As reported previously, however, glucocorticoids increased CT activity in the microsomal membrane fraction of fetal type II cells, whereas no differences in cytosolic CT activity were observed. We conclude that the developmental increase in CT mRNA in fetal type II cells is due to a decreased breakdown of the CT transcript and that glucocorticoids regulate fetal type II cell CT activity at a post-translational level. PMID- 8615773 TI - Thrombin stimulates wortmannin-inhibitable phosphoinositide 3-kinase and membrane blebbing in CHRF-288 cells. AB - We have investigated thrombin-stimulated morphological changes and the activation of phosphoinositide 3-kinase (PI 3-K), as manifested by the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (labelled with 32P or myo-[3H]inositol), in CHRF-288 cells, a leukaemic cell line derived from a platelet progenitor cell. We report that these cells, when exposed to thrombin or SFLLRN (the peptide Ser-Phe Leu-Leu-Arg-Asn, a thrombin-receptor ligand) rapidly change shape, forming membrane 'blebs', detectable by differential interference contrast or confocal microscopy, as well as labelled 3-phosphorylated phosphoinositides. The 'blebs' are distinguishable from 'ruffles' or lamellae, since they do not contain phalloidin-detectable actin. Studies with permeabilized cells indicate that PI 3 K is activated synergistically by thrombin+guanosine 5'[gamma-thio]triphosphate. Two forms of PI 3-K, i.e. PI 3-K(gamma) and p85/PI 3-K, regulated by G beta gamma subunits of heterotrimeric G-protein and the small G-protein Rho, respectively, are present in these cells, as is true for platelets. Wortmannin, a known potent and specific inhibitor of PI 3-K activities, inhibits thrombin-stiumlated accumulation of 3-phosphorylated phosphoinositides in a dose-dependent manner (IC50 approximately 10nM), without affecting phospholipase C activation. Pretreatment of CHRF-288 cells with either wortmannin (100 nM) or an unrelated synthetic PI 3-K inhibitor, LY294002 (50 microM), abolishes thrombin-receptor stimulated blebbing. These results suggest that thrombin-stimulated accumulation of 3-phosphorylated phosphoinositide(s) is required for the shape-change response in CHRF-288 cells. PMID- 8615774 TI - Synergistic effects of inositol 1,3,4,5-tetrakisphosphate on inositol 2,4,5 triphosphate-stimulated Ca2+ release do not involve direct interaction of inositol 1,3,4,5-tetrakisphosphate with inositol triphosphate-binding sites. AB - We have previously found that for permeabilized L1210 cells, low micromolar concentrations of Ins(1,3,4,5)P4 added prior to Ins(2,4,5)P3 enhance the effects of suboptimal concentrations of Ins(2,4,5)P3 in causing Ca2+ release from InsP3 sensitive Ca2+ stores [Cullen, Irvine and Dawson (1990) Biochem J. 271, 549-553]. If this was due either to some conversion of added Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by the 3-phosphatase, or to Ins(1,3,4,5)P4 acting as a weak (or partial) agonist on the InsP3 receptor it would be expected that,in the presence of thimerosal to sensitize the InsP3 receptor, the dose-response curve to Ins(1,3,4,5)P4 would be left-shifted by the same extent as that of Ins(1,4,5)P3. This was found not to be the case; the dose-response curve to Ins(1,3,4,5)P4 was not shifted at all by thimerosal. Furthermore, L-Ins(1,3,4,5)P4, which can displace radiolabelled D-Ins(1,3,4,5)P4 but not D-Ins(1,4,5)P3 from their respective high-affinity binding sites, mimicked the effects of D-Ins(1,3,4,5)P4 in enhancing the slow phase of Ins(2,4,5)P3-stimulated Ca2+ release. Ins(1,3,4,5)P4 caused an increase in magnitude of the slow phase of InsP3 stimulated Ca2+ release leaving the magnitude of the fast phase unaltered, in contrast to increasing Ins(2,4,5)P3 concentrations which increased the size of both phases. In addition, Ins(1,3,4,5)P4 decreased the rate constant for the slow phase of Ca2+ release. These findings point strongly to the conclusion that InsP4 is not working directly via the InsP3 receptor but indirectly via an InsP4 receptor. PMID- 8615775 TI - Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing. AB - Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-P04-Hex-Hex-Hex(PO4-ethanolamine)(HexNAc)-Hex N(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a ...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered. PMID- 8615776 TI - Acidic phospholipids inhibit the intramolecular association between the N- and C terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites. AB - Chick vinculin polypeptides expressed in Escherichia coli as glutathione S transferase (GST) fusion proteins have been used to identify the sites involved in the intramolecular association between the 90 kDa N-terminal head and the 30 kDa C-terminal tail region of the vinculin molecule. Fusion proteins spanning vinculin residues 1-258 and 1-398, immobilized on glutathione-agarose beads, were shown to bind a C-terminal vinculin polypeptide spanning residues 881-1066 (liberated from GST by thrombin cleavage). However, the C-terminal polypeptide did not bind to a fusion protein spanning residues 399-881 or to itself. Binding was dependent on residues 167-207 within the N-terminal polypeptide, a sequence also essential for talin binding. Conversely, the 90 kDa head polypeptide was shown to bind to residues 1029-1036 in the tail region of vinculin. The association of the head and tail was inhibited by acidic, but not neutral, phospholipids. Pre-incubation of vinculin with acidic phospholipids exposed the binding site for F-actin and a phosphorylation site for protein kinase C. The phosphorylation site was located in the tail region of the vinculin molecule. These results raise the possibility that acidic phospholipids play a role in regulating the activity of vinculin and therefore the assembly of both cell-cell and cell-matrix adherens-type junctions. PMID- 8615777 TI - Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta glucosidase A. AB - Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms. PMID- 8615778 TI - Human 17 beta-hydroxysteroid dehydrogenase type 1 and type 2 isoenzymes have opposite activities in cultured cells and characteristic cell- and tissue specific expression. AB - 17 beta-Hydroxysteroid dehydrogenase (17HSD) isoenzymes catalyse the interconversion between highly active 17 beta-hydroxy- and low-activity 17-keto steroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize 17HSD activity and the expression of 17HSD type 1 and 2 isoenzymes in several human cell types and tissues. The data indicate that in cultured cells the direction of 17HSD activity is exclusively determined by the expression of these distinct isoenzymes. The intracellular environment could not modulate the direction of the enzyme activities in any of the cell types analysed. 17HSD type 1 acts as a reductase converting oestrone into oestradiol, whereas 17HSD type 2 possesses oxidative activity inactivating oestradiol by converting it into oestrone. The data, furthermore, suggest that of the two 17HSD type 1 mRNAs (1.3 and 2.3 kb), expression of the 1.3 kb mRNA is related to enzyme concentration in all the cell types studied. This mRNA is principally expressed in cells of placental and ovarian origin, but is also present in malignant breast epithelial cells. In contrast, 17HSD type 2 is more widely expressed. It is present in several oestradiol-metabolizing tissues as well as in some target cells of sex steroid action. The opposite reaction directions observed in the cultured cells, together with differences in the distribution of the isoenzymes, suggest that type 1 is involved in oestradiol production in females while type 2 plays a role in the inactivation of this sex steroid in peripheral tissues, both in females and in males. However, some examples exist of simultaneous expression of both enzymes in the same cell type or tissue. PMID- 8615779 TI - Structural and functional analysis of the globular domain IVa of the laminin alpha 1 chain and its impact on an adjacent RGD site. AB - The globular domain IVa (about 250 residues) of the laminin alpha1 chain was obtained in recombinant form from mammalian cell clones. It was prepared either with (alpha1IVa-R) or without (alpha1IVa) an adjacent cell-adhesive RGD site which seems to be masked in laminin-1. The recombinant products could be visualized as globular structures by rotary shadowing, were resistant to trypsin and shared immunological epitopes with laminin-1, indicating folding into a native structure. Sequence analysis of pepsin fragments demonstrated the insertion of the globular domain into an epidermal growth factor-like scaffold which is characteristic of the extracellular laminin domain IV (L4) module. Only little immunological cross-reaction was found, however, with other L4 modules from perlecan and different laminin isoforms. Fragment alpha1IVa-R, but not fragment alpha1IVa, bound to alphaVbeta3 integrin, although to a distinctly lower level than a laminin fragment where the RGD site is fully exposed. The fragments also had no or only little cell attachment activity. This confirmed previous predictions that the globular domain alpha 1IVa masks the RDG site in laminin-1. Domain alpha 1IVa showed, in addition, a weak binding activity for the basement membrane protein fibulin-1. PMID- 8615780 TI - Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells. AB - A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/10(6) cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50=0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 micrograms/10(6) cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC(50)=0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5'-[gamma thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 microM) in permeabilized cells increased PA content from 6.3 micrograms to 48.6 micrograms per 10(6) cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 micrograms. An antagonist of G-proteins, guanosine 5'[beta-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 micrograms/10(6) cells) but blocked the effect of EPO on PA (30.6 micrograms/10(6) cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting the EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction. PMID- 8615781 TI - Molecular species of phospholipids in a murine stem-cell line responsive to erythropoietin. AB - The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (x6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC. PMID- 8615782 TI - Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals. AB - Formation of peroxynitrite by the fast reaction between nitric oxide and superoxide anion may represent a critical control point in cells producing both species, leading to either down-regulation of the physiological effects of superoxide anion and nitric oxide by forming an inert product, nitrate, or to potentiation of their toxic effects by oxidation of nearby molecules by peroxynitrite. (The term peroxynitrite is used to refer to the sum of all possible forms of peroxynitrite anion and peroxynitrous acid unless otherwise specified.) In this report we demonstrate that, in spite of all the antioxidant defences present in human plasma, its interaction with peroxynitrite leads to generation of free radical intermediates such as (i) the ascorbyl radical, detected by direct EPR, (ii) the albumin-thiyl radical, detected by spin-trapping experiments with both N-tert-butyl-alpha-phenylnitrone and 5,5-dimethyl-1 pyrroline N-oxide (DMPO), and (iii) a uric acid-derived free radical, detected as the DMPO radical adduct in plasma whose thiol groups were previously blocked with 5,5-dithiobis-(2-nitrobenzoic acid). The identity of the latter adduct was confirmed by parallel experiments demonstrating that it is not detectable in plasma pretreated with uricase, whereas it is formed in incubations of peroxynitrite with uric acid. Peroxynitrite-mediated oxidations were also followed by oxygen consumption and ascorbate and plasma-thiol depletion. Our results support the view that peroxynitrite-mediated one-electron oxidation of biomolecules may be an important event in its cytotoxic mechanism. In addition, the data have methodological implications by providing support for the use of EPR methodologies for monitoring both free radical reactions and ascorbate concentrations in biological fluids. PMID- 8615783 TI - Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose. AB - Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I-III. Both GSH (IC50 =10 microM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 microM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity. PMID- 8615784 TI - Phosphotyrosine phosphatase associated with band 3 protein in the human erythrocyte membrane. AB - The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by tyrosine kinase. To study the regulation of band 3 phosphorylation, we examined phosphotyrisine phosphatase (PTP) activity in the human erythrocyte. We show that the human erythrocyte membrane contains a band 3 associated neutral PTP which is activated by Mg2+ and inhibited by Mn2+ and vanadate. The PTP is active in the intact cell and in the isolated membrane. A major fraction of the PTP is tightly bound to the membrane and can be extracted from it by Triton X-100; a minor part is associated with Triton X-100-insoluble cytoskeleton. The behaviour of the PTP parallels that of band 3, the major fraction of which is extractable by detergents with a minor fraction being anchored to the cytoskeleton. Moreover, band 3 is co-precipitated when the PTP is immunoprecipitated from solubilized membranes, and PTP is co-precipitated when band 3 is immunoprecipitated. The PTP appears to be related to PTP1B (identified using an antibody to an epitope in its catalytic domain and by molecular mass). The system described here has a unique advantage for PTP research, since it allows the study of the interaction of a PTP with an endogenous physiological substrate that is present in substantial amounts in the cell membrane. The membrane-bound, band 3-associated, PTP may play a role in band 3 function in the erythrocyte and in other cells which have proteins analogous to band 3. PMID- 8615786 TI - Biochemical characterization of mouse brain necdin. AB - Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on microsomal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver staining intensity of the 51 kDa band corresponds to the decrease in the immuno staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits of pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (>200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function. PMID- 8615785 TI - Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation. AB - The activation of cultured Raw 264.7 murine macrophages with interferon gamma and lipopolysaccharide results in the expression of inducible nitric oxide synthase (i_NOS) and the subsequent production of nitric oxide. In the present study, the i-NOS expressed in these activated cells was characterized for possible post translational protein modification by endogenous tyrosine protein kinases. Western-blot analysis using phosphotyrosine antibodies revealed that i-NOS was phosphorylated on tyrosine residues and that this was an early event coinciding with the appearance of newly synthesized i-NOS. A brief exposure of activated cells to vanadate, a tyrosine phosphatase inhibitor, significantly increased the level of i-NOS tyrosine phosphorylation, suggesting that tyrosine phosphatases are dynamically involved in the regulation of this process. Vanadate treatment of activated cells also resulted in a rapid increase in enzyme activity, occurring within 5 min of exposure. Taken together, these results demonstrate that tyrosine kinases and phosphatases are involved in the post-translational modification of i NOS and may potentially play a role in modulating the functional activity of the enzyme in macrophages. PMID- 8615787 TI - Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver. AB - Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated. PMID- 8615788 TI - The suppression of 5-lipoxygenation of arachidonic acid in human polymorphonuclear leucocytes by the 15-lipoxygenase product (15S)-hydroxy (5Z,8Z,11Z,13E)-eicosatetraenoic acid: structure-activity relationship and mechanism of action. AB - (15S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) suppresses in ionophore-A23187-stimulated human polymorphonuclear leucocytes (PMN) the conversion of exogenous arachidonic acid into leukotriene B(4) (LTB4) and (5S) hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). However, contrary to earlier suggestions, 15-HETE is not a genuine 5-lipoxygenase inhibitor under these conditions, but rather suppresses the 5-lipoxygenation of arachidonic acid by switching-over of substrate utilization, as judged from a sizeable formation of labelled (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetr aen oic acid (5,15 diHETE) from 15-[1(-14)C]HETE. Identical results were obtained with human recombinant 5-lipoxygenase. In PMN the formation of 5,15-diHETE is strongly stimulated by either hydroperoxypolyenoic fatty acids or arachidonic acid, suggesting a crucial role of the hydroperoxide tone of the cell. A comparison of a selection of hydroxypolyenoic fatty acids with respect to their capability of suppressing 5-lipoxygenation of arachidonic acid revealed that 15-mono hydroxyeicosanoids throughout exhibit the highest inhibitory potencies, whereas the other HETEs, 5,15-diHETE as well as octadecanoids, are modest or poor inhibitors. The R and S enantiomers of 15-HETE do not differ from each other, excluding a receptor-like binding of the 15-hydroxy group. PMID- 8615789 TI - Time-dependent inhibition of phospholipase C beta-catalysed phosphoinositide hydrolysis: a comparison of different assays. AB - The properties of three different beta-isoforms of phospholipase C (PLC) were analysed using substrate lipids dispersed in phospholipid vesicles, phospholipid detergent mixed micelles and phospholipid monolayers spread at an air-water interface. Phosphatidylinositol 4,5-bisphosphate hydrolysis went virtually to completion in monolayers, but inositol trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by increasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when < or = 10% of PtdIns(4,5)P(2) had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by addition of fresh substrate or fresh enzyme after the slow phase of activity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest that the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipic to PLC in lamellar assays, leading to reduced activity. PMID- 8615790 TI - Cloning of the cDNAs for mast-cell chymases from the jejunum of Mongolian gerbils, Meriones unguiculatus, and their sequence similarities with chymases expressed in the connective-tissue mast cells of mice and rats. AB - By using the combination of reverse-transcription CR and rapid amplification of cDNA ends methods, two distinct cDNAs encoding mast-cell proteases (chymases; MCPs), designated as gMCP-1 and -2, were successfully cloned and sequenced from the jejunum of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. On the basis of a comparison of the deduced amino acid sequences with those of known rodent mast-cell chymases, gMCP-1 was found to be highly similar to mouse mast-cell protease (mMCP)-4 and rat mast-cell protease (rMCP)-1, while gMCP-2 was similar to mMCP-5 and rMCP-3. Alghough mMCP-4 and -5 and rMCP-1 and -3 were restrictedly or mainly expressed in connective-tissue mast cells and serosal mast cells, the gMCP-1 and -2 genes were mainly transcribed in the jejunal mucosa and to a lesser extent in the skin and tongue. Moreover, kinetic study after infection revealed that the amounts of the gMCP-1 and -2 mRNAs in jejunum paralleled well the degree of intestinal mastocytosis. The expression of gMCP-1 and -2 in mucosal mast cells of gerbil jejunum was also confirmed by in situ hybridization. Since a tryptase, another type of MCP, was also expressed in mucosal mast cells of gerbils but not in those of mice and rats, the expression of MPCs in mucosal mast cells of gerbils is different from those of mice and rats. The Mongolian gerbil would be a useful model with which to investigate the physiopathological role of MCPs. PMID- 8615791 TI - Inhibition studies on calf pregastric esterase: the enzyme has no functional thiol group. AB - Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5'-dithiobis(2 nitrobenzoic acid) or 4,4'-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C(12:0). PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated. PMID- 8615792 TI - Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C. AB - During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro. PMID- 8615793 TI - Mutations of recombinant rat liver fatty acid-binding protein at residues 102 and 122 alter its structural integrity and affinity for physiological ligands. AB - Rat liver fatty acid-binding protein (FABP) is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. This property means that the liver protein is functionally different from other FABPs from intestine, muscle and adipose tissue that have a more restricted ligand specificity and stoichiometry. The availability of crystal structures for the latter proteins has highlighted the importance of two arginine residues that are involved in the binding of the fatty acid carboxylate. Only one of these arginine residues, arginine-122, is conserved in liver FABP, whereas the other arginine, at position 102, is replaced by a threonine. In order to gain further insight into the nature of ligand interactions with liver FABP these key residues (102 and 122) have been changed by site-directed mutagenesis. The results with an R122Q mutant highlight the critical role of this arginine in determining ligand affinity, while similar but less dramatic effects were observed with the T102Q mutant. The double mutant T102Q/R122Q was expressed but had lost the ability to bind fluorescent ligands. It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid. It is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein. The portal region is suggested to be more flexible in the mutants (R122Q and to a lesser extent T102Q). The net result is that the ligand specificity of the R122Q mutant changes to that of a protein with enhanced affinity for acyl CoA, lysophospholipids and mono-olein. PMID- 8615794 TI - Cellular processing of the nerve growth factor precursor by the mammalian pro protein convertases. AB - In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6 B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment. PMID- 8615795 TI - Studies on lipids and the activity of Na,K-ATPase in lens fibre cells. AB - Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes. PMID- 8615796 TI - Mechanisms by which the surface expression of the glycosyl-phosphatidylinositol anchored complement regulatory proteins decay-accelerating factor (CD55) and CD59 is lost in human leukaemia cell lines. AB - We have investigated the mechanisms of defects in the glycosyl phosphatidylinositol (GPI)-anchored complement regulatory proteins delay accelerating factor (DAF) and/or CD59 in a panel of human leukaemia cell lines that lack surface expression of these proteins: U937 (DAF+/CD59-), CEM (DAF /CD59+), TALL (DAF-/CD59-) and a substrain of Ramos [Ramos(-)] (DAF-/CD59-). Northern blotting and reverse transcription-PCR revealed that the main cause of the DAF and/or CD59 deficiency is the failure of mRNA expression in most of the cell lines, except in Ramos(-) in which sufficient mRNA for DAF and CD59 was produced. U937, CEM and TALL cells were not defective in GPI anchor formation as assessed by the detection of other GPI-anchored proteins. No gene abnormality corresponding to DAF or CD59 was detected by Southern blotting. Thus the cause of the defects of DAF and/or CD59 in these leukaemia cell lines except for Ramos(-) is virtually undetectable steady-state levels of the relevant mRNA, most likely attributable to lack of transcription in these cell lines. On the other hand, Ramos(-) cells failed to generate a GPI anchor, whereas they normally expressed DAF and CD59 transcripts. The transfection of phosphatidylinositol-glycan class A (PIG-A) cDNA into Ramos(-) cells restored DAF and CD59 expression, indicating that the defective mechanism in GPI anchor formation is similar to that in paroxysmal noctural haemoglobinuria (PNH) cells, i.e. a deficiency of the PIG-A gene product. Thus the mechanisms of the defects of DAF and/or CD59 in human leukaemia cell lines are not uniform, and in most cases are different from that proposed to cause PNH. PMID- 8615797 TI - Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes. AB - Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o phenanthroline was reversed by the addition of zinc ions. Oleate, o phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion. PMID- 8615798 TI - Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding. AB - The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2 cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine specific reagent, also resulted in 50% loss of activity following pseudo-first order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding. PMID- 8615799 TI - Natural-product inhibitors of human DNA ligase I. AB - Enzymatic activity mediated by recombinant human DNA ligase I (hLI), in conjunction with tannin removal procedures, has been applied to a natural-product screen involving approximately 1000 plant extracts and various pure compounds. The primary hLI activity assay involved the measurement of the amount of radiolabelled phosphate in a synthetic nucleic acid hybrid that becomes resistant to alkaline phosphatase as a result of ligation. A bioactivity-guided fractionation scheme resulted in the isolation of ursolic [IC50=100 micrograms/ml (216 microM)] and oleanolic [IC50=100 micrograms/ml (216 microM)] acids from Tricalysia niamniamensis Hiern (Rubiaceae), which demonstrated similar DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid. Protolichesterinic acid [IC50=6 micrograms/ml (20 microM)], swertifrancheside [IC50 = 8 micrograms/ml(11)microM)] and fulvoplumierin [IC50=87 micrograms/ml (357 microM)] represent three additional natural-product structural classes that inhibit hLI. Fagaronine chloride [IC50=10 micrograms/ml (27 micronM] and certain flavonoids are also among the pure natural products that were found to disrupt the activity of the enzyme, consistent with their nucleic acid intercalative properties. Further analyses revealed that some of the hLI-inhibitory compounds interfered with the initial adenylation step of the ligation reaction, indicating a direct interaction with the enzyme protein. However, in all cases, this enzyme inhibitor interaction did not disrupt the DNA relaxation activity mediated by hLI. These results indicate that, although the same enzyme active site may be involved in both enzyme adenylation and DNA relaxation, inhibitors may exert allosteric effects by inducing conformational changes that disrupt only one of these activities. Studies with inhibitors are important for the assignment of specific cellular functions to these enzymes, as well as for their development into clinically useful antitumour agents. PMID- 8615801 TI - A network of specific minor-groove contacts is a common characteristic of paired domain-DNA interactions. AB - Pax proteins are a family of transcription factors conserved during evolution and able to bind specific DNA sequences through a domain called a "paired domain'. The DNA-binding specificity of the Pax-8 paired domain was investigated. Site selection experiments indicate that Pax-8 binds to a consensus sequence similar to those bound by Pax-2 and Pax-5. When consensus sequences of various paired domains are observed in light of recent structural studies describing paired domain-DNA interaction [Xu, Rould, Jun, Desplan and Pabo (1995) Cell 80, 639 650], it appears that base-pairs contacted in the minor groove are conserved, while most of the base-pairs contacted in the major groove are not. Therefore a network of specific minor groove contacts is a common characteristic of paired domain-DNA interactions. The functional importance of such a network was successfully tested by analysing the effect of consensus-based mutations on the Pax-8 binding site of the thyroglobulin promoter. PMID- 8615800 TI - Peptide models for membrane channels. AB - Peptides may be synthesized with sequences corresponding to putative transmembrane domains and/or pore-lining regions that are deduced from the primary structures of ion channel proteins. These can then be incorporated into lipid bilayer membranes for structural and functional studies. In addition to the ability to invoke ion channel activity, critical issues are the secondary structures adopted and the mode of assembly of these short transmembrane peptides in the reconstituted systems. The present review concentrates on results obtained with peptides from ligand-gated and voltage-gated ion channels, as well as proton conducting channels. These are considered within the context of current molecular models and the limited data available on the structure of native ion channels and natural channel-forming peptides. PMID- 8615802 TI - Tilorone-induced lysosomal lesions: the bisbasic character of the drug is essential for its high potency to cause storage of sulphated glycosaminoglycans. AB - The immunomodulatory agent tilorone -2,7-bis-[2-(diethyl-amino)ethoxy]fluoren-9 one- and congeners are potent inducers of lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and cultured fibroblasts of animals and man. All potent inducers of GAG storage hitherto described are bisbasic polycyclic aromatic compounds. They are accumulated in lysosomes and disturb the degradation of GAGs, mainly dermatan sulphate. It has been proposed that the drugs cross-link the polyanionic GAG chains giving rise to undergradable drug-GAG complexes. This hypothesis implies that the bisbasic character of the drug molecules is essential for the side effect in question. In the present study, this was tested by comparing tilorone and its monobasic derivative (MT) with respect to (i) induction of GAG storage in cultured bovine corneal fibroblasts and (ii) physicochemical interactions with GAGs in vitro. The intralysosomal concentration of MT achieved after 1-3 days was of the same order of magnitude as previously shown for tilorone. Nevertheless, under conditions that did not enhance the secretion of a lysosomal enzyme (beta-hexosaminidase, EC 3.2.1.52), the ability of MT to cause storage of [35S]GAGs was significantly lower than that of tilorone. Morphological observations showed that MT was much more potent in causing lysosomal storage of polar lipids than of GAGs. CD spectroscopy with tilorone revealed that the presence of GAGs caused the primarily achiral drug molecules to display CD. This suggested a helical orientation of the tilorone molecules within GAG-drug complexes, and short intermolecular distances which allowed electronic coupling of the aromatic ring systems of adjacent drug molecules. In contrast, MT failed to display any induced optical activity, indicating the absence of highly ordered GAG-drug complexes. In conclusion, the present results show that the substitution of the planar aromatic ring system with two basic side chains is essential for the high potency of tilorone in inducing lysosomal GAG storage. This is paralleled by, and presumably causally related to, strong physicochemical interactions with GAGs. PMID- 8615803 TI - Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity. AB - 1. Propranolol, a beta-blocker, inhibited or stimulated ryanodine binding to both the membrane-bound and purified ryanodine receptor (RyR) depending on the assay conditions. At high NaCl concentrations, propranolol increased the number of ryanodine-binding sites (Bmax) with no effect on the binding affinity. In the presence of 0.2 M NaCl, ryanodine binding was inhibited by propranolol. Half maximal inhibition was obtained at 1.2 mM and complete inhibition at 2 mM propranolol. The inhibitory effect of propranolol obtained at low NaCl concentration was not restored by increasing the NaCl concentration to 1 M. 2. Modulators of the RyR that are known to alter its conformational states, such as adenine nucleotides, Ca2+ concentration and pH, modified the effect of propranolol on ryanodine binding. In the presence of propranolol and at low NaCl concentrations, ryanodine binding was inhibited and showed no Ca(2+)-, pH-, or time-dependence. 3. Propranolol immediately and completely blocked the channel opening of RyR reconstituted into a planar lipid bilayer. Propranolol-modified non-active channel was reactivated to a subconductive state (about 40% of the control conductance) by ATP. 4. Competition experiments between lidocaine (a stimulatory drug) or tetracaine (an inhibitory drug) and propranolol at 0.2 or 1.0 M NaCl, respectively, suggest the existence of different interaction sites for local anaesthetics and propranolol. 5. These results suggest that propranolol interacts directly with the RyR and modifies its ryanodine binding and single channel activities. Propranolol effects are altered by the RyR conformational state, suggesting its possible use as a conformational probe for RyR. PMID- 8615804 TI - Regulation of vesicular pH in liver macrophages and parenchymal cells by ammonia and anisotonicity as assessed by fluorescein isothiocyanate-dextran fluorescence. AB - Short-term-cultivated rat hepatocytes and Kupffer cells were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran, in order to study the effects of aniso-osmotic exposure and NH4Cl on apparent vesicular pH (pHves) by single-cell fluorescence. Following a 2 h loading period with FITC-dextran in normo-osmotic (305 mosmol/l) medium, the apparent pHves was 6.01 +/- 0.05 (n = 39) in parenchymal cells and 4.94 +/- 0.04 (n = 76) in Kupffer cells. Under these conditions pHves in parenchymal cells, but not in Kupffer cells, was sensitive to changes in ambient osmolarity. Inhibition of vacuolar H(+)-ATPase by concanamycin A did not affect the osmosensitivity of pHves in parenchymal cells. However, the effects of anisotonicity on pHves were largely abolished in the presence of 4.4' di-isothiocyanato-stibene-2,2'-disulphonic acid (DIDS) or when extracellular chloride was substituted for gluconate. In neither Kupffer cells, nor liver parenchymal cells did hypo-osmotic cell swelling cause an increase in intracellular Ca2+. With regard to vesicular acidification, the following differences were noted between parenchymal and Kupffer cells. (1) In Kupffer cells endocytosed FITC-dextran reached a strongly acidic compartment with a pH value of approx. 5 within 5 min, whereas it took 4-5 h in parenchymal cells. Modification of pHves by hypo-osmolarity in Kupffer cells was only observed in a short-lived "early' compartment with a pH value of approx. 6. (2) In contrast to pHves in parenchymal cells, pHves in Kupffer cells was very sensitive towards alkalinization by NH4Cl: addition of NH4Cl at 1 or 10 mM increased apparent pHves by 0.80 or 1.46 in Kupffer cells, but only by 0.18 or 0.56 in parenchymal cells. The low ammonia sensitivity of pHves in parenchymal cells was observed not only a the less acidic (pH approx. 6) endocytotic compartment which is reached by FITC dextran within 2 h, but also in the stronger acidic compartment (pH approx. 5) which is reached after 4-5 h. (3) NH4Cl had no effect on the osmosensitivity of pHves in parenchymal cells, whereas in Kupffer cells pHves became sensitive to anisotonicity when NH4Cl was present. Osmosensitivity of pHves in Kupffer cells under these conditions, however, was not affected by genistein, DIDS or colchicine, whereas these compounds abolished the osmosensitivity of pHves in parenchymal cells. It is suggested that regulation of pHves by cell volume in liver parenchymal cells involves changes of vesicular chloride conductance. In addition, there are marked differences between Kupffer and parenchymal cells with respect to vesicular ammonia permeability and the kinetics of endocytotic membrane flow and acidification. PMID- 8615805 TI - The purification and some properties of the Mg(2+)-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae. AB - A purification procedure has been developed for the cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae that yields homogeneous enzyme. The enzyme seems to be a tetramer of identical 58 kDa subunits. The enzyme reaction is strongly stimulated by Mg2+ at low NADP+ concentrations but there is no absolute requirement for bivalent cations. The kinetics of the reaction have been studied in the presence and absence of MgCl2. NADP+ binding studies of the quenching of protein fluorescence in the presence and absence of MgCl2 show that the effect of Mg2+ is to increase the affinity of the enzyme for NADP+ by approx. 100-fold. NADP+ binding causes a slow conformational change in the enzyme and converts the enzyme from the inactive or low-activity form in which it is isolated into the fully active form. This conformational change seems to explain the marked lag-phases seen in enzyme assays. The enzyme is strongly inhibited by disulfiram and pyridoxal 5-phosphate. PMID- 8615806 TI - Phosphorylation of membrane proteins in erythrocytes treated with lead. AB - In immature rat microvessels, endothelial cells and glioma cells, exposure to lead results in an increase in the level of protein kinase C in membranes. In this paper we have extended these studies to human erythrocytes and, in addition, studied the phosphorylation of membrane proteins. A significant increase in the phosphorylation of membrane cytoskeletal proteins of molecular mass 120, 80, 52 and 45 kDa was observed in human erythrocytes treated for 60 min with lead acetate at concentrations greater than 100 nM. These same proteins were phosphorylated when erythrocytes were treated for 10 min with 50 nM phorbol 12 myristate 13-acetate (PMA). Similarly, protein kinase C activity was elevated and an increase in the amount of protein kinase C-alpha was observed in membranes from erythrocytes exposed to concentrations of lead acetate above 100 nM. No changes, however, in the activities of cAMP-dependent protein kinase, protein phosphatases I and IIA or casein kinase were observed. Phosphorylation of these membrane proteins stimulated by lead acetate or by PMA was not observed in erythrocytes depleted of protein kinase C by a 72-h treatment with 500 nM phorbol 12,13-dibutyrate. Finally, no changes in the levels of calcium or diacylglycerol were observed in erythrocytes stimulated with 100 nM lead acetate. These results indicate that, in erythrocytes, lead acetate stimulates the phosphorylation of membrane cytoskeletal proteins by a mechanism dependent on protein kinase C. Since levels of calcium or diacylglycerols did not increase, it appears that lead may activate the enzyme by a direct interaction. PMID- 8615807 TI - Protein tyrosine kinases regulate agonist-stimulated prostacyclin release but not von Willebrand factor secretion from human umbilical vein endothelial cells. AB - The rapid synthesis and release of prostacyclin (PGI2) and the exocytotic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein coupled receptors on endothelium occur via signaling mechanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state of endogenous proteins have been implicated in several cellular processes including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated release of PGI2 and vWF from human umbilical vein endothelial cells (HUVECs) using two chemically and mechanistically dissimilar PTK inhibitors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose dependently attenuated PGI2 release in response to thrombin and histamine (IC50 approx. 20 microM), and to the thrombin-receptor-activating peptide. A more potent inhibition of thrombin- and histamine-induced PGI2 synthesis was observed in cells exposed to ST271. In contrast, neither genistein nor ST271 modulated agonist-drive vWF secretion. At concentrations that abolished PGI2 release, genistein blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI2 generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI2 synthesis by acting at, or distal to, agonist-induced changes in intracellular CA2+ ([Ca2+]i). In fura-2-loaded HUVECs genistein partially reduced the histamine-induced peak [Ca2+]i but had no effect on the thrombin response. Ca(2+)-induced PGI2 release from electrically permeabilized HUVECs was abolished in the presence of ST271 or genistein, but not diadzein. The generation of PGI2 in response to exogenous arachidonic acid was not modulated by genistein or ST271, suggesting that PTK inhibitors do not directly inhibit cyclo-oxygenase activity. Taken together, these results suggest that PTKs regulate PGI2 synthesis and release in HUVECs by modulating, directly or indirectly, a CA(2+)-sensitive step upstream of cyclo-oxygenase. PMID- 8615808 TI - Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2 alpha at the protein kinase C site but not at the protein kinase A-controlled site. AB - Streptozotocin-induced diabetes caused a profound increase in the steady-state level of phosphorylation of the alpha-subunit of the adenylate cyclase inhibitory protein Gi2 in hepatocytes. Unlike hepatocytes from control animals, those from streptozotocin-diabetic animals showed no increase in the phosphorylation of Gi2 alpha in response to a challenge with the protein kinase C activator phorbol myristate acetate. However, a stimulatory effect of 8-bromo-cAMP on Gi2 alpha phosphorylation was evident in hepatocytes from diabetic animals but this was severely reduced compared with that observed in hepatocytes from normal animals. Two-dimensional tryptic phosphopeptide mapping showed that Gi2 alpha in resting hepatocytes from diabetic animals was phosphorylated exclusively at the protein kinase C site (C-site) but no labelling was evident at the protein kinase A regulated site (AN-site). Treatment of hepatocytes from diabetic animals with phorbol myristate acetate did not change this pattern of labelling. In contrast, challenge of hepatocytes from diabetic animals with 8-bromo-cAMP led to the appearance of a new labelled phosphopeptide that was consistent with labelling at the AN-site. Analysis of the C-site and AN-site phosphopeptides from hepatocytes of diabetic animals treated with 8-bromo-cAMP showed that the increase in labelling of Gi2 alpha caused by this ligand could be attributed almost entirely to labelling at the AN-site. Thus streptozotocin diabetes appears to cause enhanced labelling of hepatocyte Gi2 alpha by exclusively increasing phosphorylation at the C-site. It is suggested that the increased labelling at the C-site reflects an augmentation of the protein kinase C signalling system in hepatocytes from streptozotocin-induced diabetic animals. This may have wide spread functional consequences for these cells and may result either from an increased protein kinase C activity and/or a reduction in protein phosphatase 1 and/or 2A activity. PMID- 8615810 TI - Molecular cloning and functional expression of human deoxyhypusine synthase cDNA based on expressed sequence tag information. AB - Deoxyhypusine synthase is an NAD(+)-dependent enzyme that catalyses the formation of a deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from spermidine to the epsilon amino group of a unique lysine residue. We have recently cloned and characterized the Neurospora crassa deoxyhypusine synthase cDNA using a reverse genetics approach. A GenBank search showed that a stretch of the deduced amino acid sequence (96 amino acids) of Neurospora deoxyhypusine synthase matches a short human expressed sequence tag (EST), Z25337, with greater than 70% amino acid identity. Gene-specific primers based on this EST were used together with universal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA library. The 1219 bp and 1078 bp sequences, each containing an open reading frame, encode polypeptides of respectively 368 and 321 amino acids. The short sequence is identical to the long one except that it is missing a stretch of 47 amino acids spanning residues 261-307. The 368-amino-acid sequence of human deoxyhypusine synthase shares a high degree of identity ( > 50%) and similarity ( > 60%) with that of the Neurospora and yeast deoxyhypusine synthases. After cloning into an expression vector, the 368-amino-acid recombinant protein exhibits high deoxyhypusine synthase activity. In contrast, the 321-amino-acid recombinant protein shows no detectable activity. PMID- 8615809 TI - Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure. AB - To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) or hMR (hMR 711-984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone-hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1-944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711-984 region is sufficient for hsp90 MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand. PMID- 8615811 TI - Effects of dexamethasone and transforming growth factor-beta 2 on group II phospholipase A2 mRNA and activity levels in interleukin 1 beta- and forskolin stimulated mesangial cells. AB - The expression of 14 kDa group II phospholipase A2 [also referred to as secretory PLA2 (sPLA2)] is induced in rat glomerular mesangial cells by exposure to inflammatory cytokines and forskolin, a cAMP elevating agent. Previously we have shown that dexamethasone and transforming growth factor-beta 2 (TGF-beta 2) suppress sPLA2 protein synthesis and enzyme activity induced by cytokines and forskolin. The regulation of sPLA2 by pro-inflammatory cytokines suggests that the enzyme may play a role in glomerular inflammatory reactions. In order to understand the regulation of sPLA, in more detail, we investigated whether dexamethasone and TGF-beta 2 also suppress sPLA, mRNA after its induction by either interleukin-1 beta (IL-1 beta) or forskolin. We found that IL-1 beta induced sPLA2 mRNA in rat mesangial cells is not down-regulated by pretreatment of the cells with dexamethasone, even at a concentration of 10 microM, which dramatically decreases sPLA2 protein levels and activity. Metabolic labelling experiments indicated that the decreased sPLA2 levels under these conditions can be explained by inhibition of the rate of sPLA2 synthesis from the elevated mRNA levels. In contrast, the forskolin-induced elevation of sPLA, mRNA is inhibited by dexamethasone in a concentration-dependent manner. Likewise, TGF-beta 2 inhibits the elevation of sPLA, mRNAs induced by either IL-1 beta or forskolin. The decrease in sPLA2 mRNA caused by TGF-beta 2 corresponds with the decrease in sPLA2 enzyme levels and activity. These data suggest that cytokine- and forskolin induced sPLA2, expression is tightly controlled via both transcriptional and post transcriptional mechanisms. Furthermore, we show that pretreatment of mesangial cells with epidermal growth factor prior to stimulation with IL-1 beta or forskolin had no suppressing effect on sPLA2 levels or enzyme activity, as has been reported previously for osteoblasts. PMID- 8615812 TI - Expression of soluble cloned porcine pepsinogen A in Escherichia coli. AB - A system for the production of soluble porcine pepsinogen A (EC 3.4.23.1) was developed by fusing the pepsinogen and thioredoxin genes and then expressing the fused product (Trx-PG) in Escherichia coli. The expressed fusion protein was purified using a combination of ion-exchange and hydrophobic chromatography. Trypsin digestion of the fusion protein yielded pepsinogen which was one residue longer than the intrinsic length. Acidification of either the fusion protein or pepsinogen (tryptic digestion of Trx-PG) yielded recombinant pepsin A (r-pepsin). When compared with commercial porcine pepsin A, r-pepsin had similar milk clotting and proteolytic activities, kinetic parameters and pH dependency. The above results indicate that an expression system was developed which yielded fully active soluble pepsin(ogen) from Escherichia coli. PMID- 8615813 TI - 13C-NMR relation study of heparin-disaccharide interactions with tripeptides GRG and GKG. AB - Heparin is a polydisperse sulphated copolymer consisting mostly of 1-->4 linked glucosamine and uronic acid residues, i.e. 2-deoxy-2-sulphamido-D-glucopyranose 6 sulphate and L-idopyranosyluronic acid 2-sulphate. 13C NMR has been used to study the interactions of heparinase-derived and purified heparin disaccharide with N- and C-terminally-blocked tripeptides GRG and GKG. Titration of the disaccharide with peptide indicates that GRG binds the disaccharide more strongly than does GKG, with interactions in either case being stronger at uronate ring positions. In the presence of GRG, a carboxylate pKa depression suggests electrostatic interactions between the arginine guanidinium group and the uronate carboxylate group. 13C relaxation data have been acquired for all disaccharide and peptide carbons in the presence and absence of GRG and GKG. 13C relaxation rates for the disaccharide are significantly faster in the presence of peptide, especially with GRG. Analysis of these relaxation data has been done in terms of molecular diffusion constants, D [symbol: see text] and D parallel, and an angle alpha between D parallel and a molecular frame defined by the moment of inertia tensor calculated for an internally rigid disaccharide. Disaccharide conformational space in these calculations has been sampled for both uronate half-chair forms (2H1 and 1H2) and over a range of glycosidic bond angles defined by motional order parameters and inter-residue nuclear Overhauser effects (+/- 30 degree from the average). In the absence of peptide, the ratio D [symbol: see text] /D parallel falls between 0.4 and 0.7; therefore molecular diffusion occurs preferentially about D parallel, which runs through both disaccharide rings. In the presence of peptide, D [symbol: see text] /D parallel is decreased, indicating that GRG is oriented along D parallel and proximal to the uronic acid ring. A model for this is shown. PMID- 8615814 TI - Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway. AB - Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor-product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H, U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3 mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We concluded that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell. PMID- 8615815 TI - Characterization of the soluble, secreted form of urinary meprin. AB - A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1 alpha/alpha genotype (high meprin, expressing meprin-alpha and meprin-beta ) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-beta ). Western blotting with antisera specific to the meprin-alpha and the meprin-beta subunits established that the only form of meprin present in urine samples was derived from meprin-alpha. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N terminus of the larger protein band begins with the pro-peptide sequence of the alpha-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X-I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms. PMID- 8615816 TI - Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6. AB - Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, beta cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942-5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833-838]. The pH profiles for wild type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family. PMID- 8615817 TI - The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme. AB - We have investigated the effects of tyrosine nitration (to form the weak acid, 3 nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3 nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme. PMID- 8615818 TI - Functional domains of chlamydial histone H1-like protein. AB - Chlamydial trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1. It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies. However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA-protein and protein-protein interactions. Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, and N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein. In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini. Our results support the role of the carboxyl portion in DNA-protein interaction, a function similar to its eukaryotic counterpart. Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction. Intra- or inter-molecular protein-protein interactions may be necessary to achieve such an effect. PMID- 8615819 TI - Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes. AB - The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6 phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4 positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4. PMID- 8615820 TI - Induction of heat shock proteins and their possible roles in macrophages during activation by macrophage colony-stimulating factor. AB - (1) Treatment of resident peritoneal macrophages for 8 h with macrophage colony stimulating factor (M-CSF) increased release of superoxide anion (O2-) stimulated by phorbol 12-myristate 13-acetate. Gel electrophoresis of pulse-labelled proteins with L-[35S]methionine showed that a number of proteins were induced during activation by M-CSF. Immunoblot analysis with antibody against heat shock protein (HSP) 90, HSP70, or HSP60 demonstrated that M-CSF induced these stress inducible HSPs; the timing of induction and level of each HSP correlated with the increase in O2- production. The activated macrophages acquired resistance to H2O2 induced damage. M-CSF also stimulated the synthesis of a heat shock cognate protein (HSC70); however, the induction occurred at 1 h, when O2- production was not yet augmented, but at which time L-[35S]methionine incorporation into cell proteins was already enhanced. (2) Gel mobility shift assay with oligonucleotide coding for the heat shock element showed that M-CSF activated the heat shock factor within 15 min, and the activation continued for at least 8 h. Northern blot analysis with a cDNA probe for human HSP70 or HSC70 showed that accumulations of HSP70 and HSC70 mRNAs coincided with the inductions of the respective proteins. (3) These results suggest that M-CSF may induce the transcriptional activation of heat shock genes, and that the stress-inducible HSPs as well as HSC70 may play an important role in the activation of macrophages by functioning as molecular chaperones and by protecting the macrophage against the auto-oxidative damage associated with the respiratory burst. PMID- 8615821 TI - Activation of phosphatidylinositol 3-kinase by concanavalin A through dual signaling pathways, G-protein-coupled and phosphotyrosine-related, and an essential role of the G-protein-coupled signals for the lectin-induced respiratory burst in human monocytic THP-1 cells. AB - Stimulation of monocytic THP-1 cells by a lectin, concanavalin A (Con A), resulted in protein-tyrosine phosphorylation and association of some of the thus phosphorylated proteins with the 85 kDa regulatory subunit of PtdIns 3-kinase. Both actions of Con A were not inhibited by wortmannin, a PtdIns 3-kinase inhibitor, or by prior exposure of cells to pertussis toxin which uncouples certain G-proteins from receptors. The binding of PtdIns 3-kinase to the tyrosine phosphorylated proteins increased upon Con A stimulation; there was a marked increase in the enzymic activity in the anti-phosphotyrosine immuno-precipitates from Con A-treated cells. The increase was abolished by wortmannin but not affected by pertussis toxin. The incorporation of 32P into PtdInsP3 also increased during incubation of [32P]P(i)-prelabelled cells with Con A, reflecting activation of whole-cell PtdIns 3-kinase which could not be accounted for solely by the increase in the phosphotyrosine-bound enzyme activity from the following aspects: (1) different concentration dependencies for Con A; and (2) almost total susceptibility of the incorporation to pertussis toxin. This notion appears to be supported by different time courses between increases in PtdInsP3 production and the phosphotyrosine-bound activity. The susceptibility to the toxin may reflect involvement of the toxin-sensitive G-proteins. In contrast, insulin-induced increases in PtdInsP3 production, as well as increases in phosphotyrosine-bound PtdIns 3-kinase activity, were blocked by wortmannin, but never affected by prior exposure of cells to pertussis toxin, excluding a possible involvement of G proteins in the insulin-induced activation. Con-A-induced O2- production was almost inhibited by either pertussis toxin or wortmannin. These results suggest that oligomerization of cell-surface glycoproteins with Con A gives rise to activation of G-protein(s) and certain tyrosine kinase(s), both of which were responsible for PtdIns 3-kinase activation; the G-protein-mediated activation led to the respiratory burst. PMID- 8615822 TI - Diversity of roles of protein kinase C alpha in the proliferation of Swiss 3T3 cells. AB - We examined the role of protein kinase C alpha (PKC alpha ) in the stimulation of DNA synthesis of Swiss 3T3 cells induced by bombesin, platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA). We found that cells in which this kinase had been down-regulated showed a partially abrogated mitogenic response to bombesin. The response to PDGF was unaltered; however, the response to PMA was completely suppressed. The mitogenic effect of maximal doses of bombesin and PMA combined was greater than that of either agent alone, suggesting that bombesin does not fully activate the PKC pathway. Accordingly, bombesin induced PKC alpha translocation from cytosol to membranes was partial, while that observed with PMA was essentially complete. Moreover, exposure to Ro-31-8220, a PKC inhibitor, had significantly greater effects on the response to PMA than on that to bombesin. Our findings point out different roles that PKC alpha may play in diversely activated cells: while, in the case of PMA, stimulation of this kinase may be necessary and sufficient to induce proliferation, it appears to be necessary only for a full response to bombesin, and redundant among the mechanisms triggered by PDGF. PMID- 8615823 TI - Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways. AB - Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3 OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3 kinase is activated by a mechanism involving JAK-2 and IRS-1. PMID- 8615824 TI - Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b. AB - The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2-12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2-5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2-5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu 17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys 18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact. PMID- 8615826 TI - Efficient catalysis by beta-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme. AB - The pH- and temperature-dependence of steady-state kinetic parameters for 6-beta (2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 beta-lactamase, and the viscosity-dependence of K(m)/k(cat) indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4-9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to k(cat). The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation. PMID- 8615825 TI - The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity. AB - The microsomal triacylglycerol transfer protein (MTP), an alpha beta dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The beta subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the alpha subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the alpha and beta subunits were co-expressed, soluble alpha beta dimers were formed with significant triacylglycerol transfer activity. Expression of the alpha subunit with a mutant protein disulphide isomerase polypeptide in which both CGHC- catalytic sites had been inactivated also yielded alpha beta dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the beta subunit in MTP seems to be to keep the alpha subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the beta subunit is not required for this function. PMID- 8615827 TI - Structure of photosystem II in spinach thylakoid membranes: comparison of detergent-solubilized and native complexes by electron microscopy. AB - 1. Electron microscopy of solubilized photosystem II (PSII) complexes and PSII in spinach thylakoid membranes has been carried out and the results have been compared with data obtained from ordered two-dimensional arrays of PSII. Membrane bound PSII is roughly rectangular (17.6 nm x 14.1 nm) with a central stain cavity surrounded by four major lumenal domains. A comparison between the averaged projections of single (non-ordered) particles at 3.8 nm resolution and the Fourier projection maps obtained from ordered arrays (at 2-3 nm resolution) reveals close similarity and excludes the possibility that PSII observed in two dimensional ordered arrays represents an unusual subpopulation. 2. After detergent solubilization, PSII adopts various aggregation states which were analysed by electron microscopy in conjunction with single-particle averaging. Two different types of projection of roughly rectangular shape and of dimensions 30 nm x 17 nm manifesting themselves as tetrameric sandwich structures have been revealed. This conclusion is supported by the presence of at least two axes of 2 fold rotational symmetry running perpendicular to each other and intersecting at the centre of the oligomer. Comparisons of the structures of detergent solubilized and native PSII show that the oligomerization of PSII can be artificially induced by the process of membrane solubilization. PMID- 8615828 TI - Differential regulation of transcription and transcript stability of pro-alpha 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma. AB - Activated fibroblasts were derived from the skin of patients with systemic scleroderma (SSc), used as a model for fibrosis. Such cells are characterized by increased production of collagens and other matrix constituents. Increased collagen and fibronectin production has been correlated with similarly elevated mRNA steady-state levels. In the present study we analysed the contribution of transcriptional activity and post-transcriptional transcript stability to the increases in pro-alpha 1(I) collagen and fibronectin mRNA steady-state levels in activated (scleroderma) fibroblasts. Fibroblasts, when cultured in close contact with a three-dimensional collagenous matrix, down-regulate collagen synthesis. Culture of skin fibroblasts from two patients with SSc in three-dimensional collagen lattices, however, showed 4-fold elevated pro-alpha 1(I) collagen mRNA levels over fibroblasts from healthy donors. Transcription of the COL1A1 gene in SSc fibroblasts was induced 2-3-fold over that in controls in both monolayer and lattice cultures, accounting in part for the elevated steady-state level. A 50% decrease in transcription rate in lattice compared with monolayer culture occurred, as in control cells. In contrast, whereas control cells in lattices responded with decreased (50%) pro-alpha 1(I) collagen mRNA stability, in SSc cells these transcripts were found to be more stable (half-life of 5 h compared with 2 h in control cells). Fibronectin steady-state mRNA levels, in contrast, were not significantly regulated by the three-dimensional environment. In SSc fibroblasts, fibronectin mRNA levels were induced 1.5-4.9-fold over controls. In part, this increase appears to be due to elevated transcription, and an increase in fibronectin transcript stability was also detected. We therefore conclude that activated fibroblasts such as those derived from scleroderma patients utilize transcriptional and posttranscriptional mechanisms to maintain increased collagen and fibronectin production, which contribute to the pathogenesis of the disease. PMID- 8615829 TI - Transcriptional regulation by glucocorticoids of mitochondrial oxidative enzyme genes in the developing rat kidney. AB - Mitochondrial fatty acid beta-oxidation plays a major role in providing the ATP required for reabsorptive processes in the adult rat kidney. However, the molecular mechanisms and signals involved in induction of the enzymes of fatty acid oxidation during development in this and other organs are unknown. We therefore studied the changes in the steady-state levels of mRNA encoding medium chain acyl-CoA dehydrogenase (MCAD), which catalyses the initial step in mitochondrial fatty acid beta-oxidation, in the rat kidney cortex and medulla between postnatal days 10 and 30. Furthermore, we investigated whether the expression of MCAD and of mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid cycle, might be co-ordinately regulated by circulating glucocorticoids in the immature kidney during development. In the cortex, the levels of MCAD mRNA rose 4-fold between day 10 and day 21, and then decreased from day 21 to day 30. In the medulla a postnatal increase in the concentration of MCAD mRNA (8-fold) was observed during the same period. Adrenalectomy prevented the 16-21-day developmental increases in MCAD and mMDH mRNA levels in the cortex and medulla; these could be restored by dexamethasone treatment. A single injection of dexamethasone into 10-day-old rats led to a rise in MCAD and mMDH mRNA levels in the renal cortex due to stimulation of gene transcription, as shown by nuclear run-on assays. Therefore MCAD and mMDH gene expression is tightly regulated at the transcriptional level by developmental changes in circulating glucocorticoid levels. These hormones might thus represent a good candidate as a co-ordinating factor in the expression of nuclear genes encoding mitochondrial enzymes in the kidney during postnatal development. PMID- 8615830 TI - Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP stimulated EAhy 926 endothelial cells. AB - We have investigated the mechanisms that bring about the termination of mitogen activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein synthesis step and by a mechanism that does not involve the inactivation of c-Raf 1 or MAP kinase kinase. PMID- 8615831 TI - Detection of gp91-phox precursor protein in B-cell lines from patients with X linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis. AB - NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22 phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3. PMID- 8615832 TI - Structure of a group C streptococcal protein that binds to fibrinogen, albumin and immunoglobulin G via overlapping modules. AB - Pathogenic streptococci express surface proteins that bind to host serum proteins. A novel multiple-ligand-binding protein has now been identified in a species belonging to serotype C streptococci. This protein binds to fibrinogen, albumin and IgG and was therefore designated FAI protein. The structure of the fai gene has been determined, and deletion analysis and expression of FAI fusion polypeptides revealed that the binding domain for fibrinogen and IgG is located within the nonrepetitive N-terminal half of the protein. A 93-amino acid peptide retained the ability to bind both proteins, whereas a 56-amino acid subpeptide only bound fibrinogen. IgG-binding activity required the complete fibrinogen binding domain and an additional 37 amino acids C-terminal to it, and albumin binding activity was only obtained with a polypeptide reflecting the complete surface-exposed region of FAI protein indicating that the binding sites for each ligand were located on overlapping modules. Signal sequence, C repeat region and C-terminus revealed high homology to group A streptococcal M proteins whereas the N-terminal region containing the fibrinogen/IgG-binding domains is completely different and exhibits no similarity to any other previously characterized protein. Thus FAI protein exhibits a framework structure that might have evolved in group C streptococci via fusion of unrelated sequences, thereby generating an albumin-binding domain in the functional context of multiple-ligand-binding activity. PMID- 8615833 TI - Structure-function relationship of xylanase: fluorimetric analysis of the tryptophan environment. AB - Involvement of one out of three tryptophan residues in the active site of the low molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The work described here aims at: (i) deducing the structure-function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K(SV)) of the solute quenchers. The analysis of K(SV) and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative. PMID- 8615835 TI - Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles. AB - Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (omega(o)) of the system with maximum rate observed at omega(o) 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4 dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56 0.07 and 8.81 0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69 0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40-1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo is discussed. PMID- 8615834 TI - Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum. AB - The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix. PMID- 8615836 TI - Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes. AB - Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague-Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model. PMID- 8615837 TI - Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity. AB - Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, their beta subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-beta subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble alpha subunits in solution. We report here that expression of the alpha subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble alpha-subunit-BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the alpha subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the alpha subunit. Nevertheless the soluble alpha-subunit-BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-beta subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the alpha subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation. PMID- 8615838 TI - Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality. AB - Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine. Both FGF-2 peptides also induced functional modifications of G proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation. PMID- 8615839 TI - One-electron oxidation of ergothioneine and analogues investigated by pulse radiolysis: redox reaction involving ergothioneine and vitamin C. AB - Redox reactions of endogenous and exogenous sulphur-containing compounds are involved in protection against oxidative damage arising from the incidence and/or treatment of many diseases, including cancer. We have investigated, via pulse radiolysis, the one-electron oxidation of ergothioneine, a molecule with antioxidant properties which is detected at millimolar concentrations in certain tissues and fluids subject to oxidative stress, including erythrocytes and plasma. The spectrum of the transient species, assigned to the product of one electron oxidation, observed after reaction of ergothioneine with the oxidizing radicals OH., N3. and CCl3O2. has a maximum absorption at 520 nm and is very similar to that obtained by oxidation of analogous molecules such as 2 mercaptoimidazole, 1-methyl-2-mercaptoimidazole, S-methyl- and S,N-dimethyl ergothioneine. In the presence of vitamin C, the oxidized form of ergothioneine is repaired by a rapid reduction (k = 6.3 x 10(8) M(-1).s(-1)) producing ascorbyl radicals. This co-operative interaction between ergothionine and ascorbate, similar to that previously observed between vitamin E and ascorbate, may contribute to essential biological redox protection. PMID- 8615840 TI - Pentamidine uptake in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes. AB - A transport system for pentamidine in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes has been identified and characterized. Pentamidine is not metabolized by these parasites. Its uptake process is saturable, carrier-mediated and energy-dependent. This drug does not inhibit purine or pyrimidine uptake, whereas it inhibits uptake of several amino acids non-competitively and that of putrescine and spermidine competitively. The results suggest that pentamidine shares polyamine-carrier systems in these parasites. PMID- 8615841 TI - Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes. AB - An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino-(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1 30.4 microM and the Vmax 1.4 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP. PMID- 8615842 TI - Pituitary adenylate cyclase-activating polypeptide induces expression of corticosteroid-binding globulin in cultured fetal hepatocytes: synergy with tri iodothyronine. AB - The purpose of the present study was to determine whether functional receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in cultured rat fetal hepatocytes and eventually play a role in regulating gene expression of corticosteroid-binding globulin (CBG). We found PACAP38 and PACAP27 to elevate cAMP levels in hepatocytes in a dose-dependent manner, with a plateau being achieved at 10 nM and EC50 values of about 0.5-1 nM. PACAP failed to alter the turnover of inositol phosphates, whereas PACAP and VIP stimulated cAMP accumulation in an equipotent manner, suggesting the presence in these cells of type II receptor isoforms. As revealed by measurements of both CBG mRNA levels and concentrations of binding sites, long-term treatment of fetal cells with 10 nM PACAP, although resulting in partial desensitization of peptide-induced cAMP accumulation, caused a significant 3-fold elevation in CBG synthesis. This stimulatory influence of PACAP was mimicked by the cell permeant N6,2'-O dibutyryladenosine 3',5'-phosphate (dbcAMP). Treatment of hepatocytes with tri iodothyronine (T3) enhanced CBG expression and, most interestingly, appeared to synergize with PACAP to elicit a 2-3-fold amplification of CBG synthesis. This study thus provides first evidence for the up-regulation by PACAP and cAMP of CBG expression in fetal hepatocytes and for T3's playing a synergistic role in enhancing PACAP-induced synthesis of the binder. PMID- 8615843 TI - Substrate modulation of aldolase B binding in hepatocytes. AB - The binding properties of hepatic aldolase (B) were determined in digitonin permeabilized rat hepatocytes after the cells had been preincubated with either glycolytic or gluconeogenic substrates. In hepatocytes that had been preincubated in medium containing 5 mM glucose as sole carbohydrate substrate, binding of aldolase to the hepatocyte matrix was maximal at low KCl concentrations (20 mM) or bivalent cation concentrations (1 mM Mg2+) and half-maximal dissociation occurred at 50 mM KCl. Preincubation of hepatocytes (for 10-30 min) with glucose or mannose (10-40 mM), fructose, sorbitol, dihydroxyacetone or glycerol (1-10 mM), caused a leftward shift of the salt dissociation curve (maximum binding at 10 mM KCl; half-maximum dissociation at 35 mM KCl) but did not affect the proportion of bound enzyme at low or high KCl concentrations. Galactose and 2 deoxyglucose had no effect on aldolase binding. Inhibitors of glucokinase (mannoheptulose and glucosamine) suppressed the effects of glucose but not the effects of sorbitol, glycerol or dihydroxyacetone. Glucagon suppressed the effects of glucose, fructose and dihydroxyacetone but not glycerol. Poly(ethylene glycol) (PEG) (2-10%), added to the permeabilization medium, increased aldolase binding and caused a rightward shift in the salt dissociation curve. In the presence of PEG (6-8%), the effects of substrates on aldolase dissociation were shifted to higher salt concentrations (50-100 mM versus 35 mM KCl). The effects of substrates (added to the intact cell) on aldolase binding to the permeabilized cell could be mimicked by addition of the phosphorylated derivatives of these substrates to the permeabilized cell. Of the intermediates tested dihydroxyacetone phosphate and fructose 1,6-bisphosphate were the most effective at dissociating aldolase (A50 values of 20 microM and 40 microM respectively). Other effective intermediates in order of decreasing potency were fructose 1 phosphate, glycerol 3-phosphate, glucose 1,6-bisphosphate/fructose 2,6 bisphosphate. These results show that aldolase B binds to the hepatocyte matrix by a salt-dependent mechanism that is influenced by macromolecular crowding and metabolic intermediates. Maximum binding occurs when hepatocytes are incubated in the absence of glycolytic and gluconeogenic substrates and minimum binding occurs in the presence of substrates that are precursors of either fructose 1,6 bisphosphate or triose phosphates. Since the bound form of aldolase represents a kinetically less active state it is proposed that aldolase binding and dissociation may be a mechanism for buffering the concentrations of metabolic intermediates. PMID- 8615844 TI - Characterization of mitochondria from pig muscle: higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia. AB - Mitochondria were isolated from biopsies of the biceps femoris muscle of Danish landrace pigs. Three groups of animals were compared: (1) normal pigs; (2) pigs that were homozygous with respect to the gene Hal(n)/Hal(n) coding for the porcine malignant hyperthermia syndrome; and (3) heterozygote animals. A newly developed micro-method for preparation and assaying of small quantities of intact mitochondria was employed. With this technique mitochondria from biopsies weighing less than 100 mg were examined with respect to cytochrome content as well as phosphorylating and respiratory activities, including the nonphosphorylating exo-NADH oxidase activity. The mitochondria, prepared in a yield of 48%, showed high respiratory activities with tricarboxylic acid-cycle intermediates and pyruvate, and somewhat lower activity with palmitoyl-carnitine as substrate. The ATP synthase activity was about 1000 micromol ATP/min per g of protein and the maximal respiratory activity approx. 700 micromol of O2/min per g of protein. No differences among the three groups of animals were detected, except for the exo-NADH oxidase activities, which were 43, 78 and 107 micromol of O2/min per g of protein in the groups of normal, heterozygous and homozygous animals respectively. It is concluded that the exo-NADH oxidase activity may be a genetic manifestation of malignant hyperthermia and may play a significant role in the heat production characteristic of the syndrome. PMID- 8615845 TI - Evidence for differences in the post-transcriptional regulation of rat metallothionein isoforms. AB - The expression of metallothionein (MT)-1 and -2 mRNAs in rat liver following administration of Cd or Cu was investigated using specific oligonucleotides. The specificity was confirmed using a competitive prehybridization assay. Cd injection caused a biphasic induction of both isoform mRNAs, whereas Cu induced a sustained, monophasic response. Analysis of polyribosomal RNA showed that, after both Cd and Cu treatments, the recruitment of MT-1 mRNA into polyribosomes paralleled the increase in transcription, but the increase of polyribosomal MT-2 mRNA was less than that of total MT-2 mRNA. This indicates that not all the MT-2 mRNA induced was translated, suggesting that there is translational control of MT 2 mRNA expression, but not of MT-1 mRNA. This hypothesis was supported by the observation that, after Cu treatment, the induction of MT-1 protein was induced to the same extent as MT-1 mRNA, whereas the total MT protein (MT-1 + MT-2) was increased far less (7-fold) than MT-2 mRNA (30-fold). PMID- 8615846 TI - Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca(2+)-ATPase. AB - The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis on the calcium ion-dependent ATPase from plasma membrane (PM-Ca(2+) ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 microM [gamma 32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140% to 84 11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4 0.17 pmol/mg of protein. If added together with [gamma-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 --> E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the k(app) of the transition by 55% to 14 3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its k(app) by 60% to 91 12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca(2+)-ATPase under physiological conditions. PMID- 8615847 TI - Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A. AB - Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS. PMID- 8615848 TI - Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site. AB - The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217. PMID- 8615849 TI - Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase. AB - Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions. PMID- 8615850 TI - Relationships between eicosanoids and cannabinoids. Are eicosanoids cannabimimetic agents?. PMID- 8615851 TI - Acute hepatotoxicity of acetaminophen in rats treated with ethanol plus isopentanol. AB - Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment. PMID- 8615852 TI - Formation of free radicals by gentamicin and iron and evidence for an iron/gentamicin complex. AB - Participation of free radicals in the adverse renal and cochlear side effects of aminoglycoside antibiotics is controversial. We measured the production of free radicals by gentamicin in vitro through the oxidation of arachidonic acid. Gentamicin alone (0.05 to 10 mM) did not cause lipid peroxidation. However, it dramatically promoted radical formation in the presence of iron salts. Peroxidation was maximal at 1 mM gentamicin plus 0.1 mM Fe(II)/Fe(III) (0.05 mM FeSO4 and FeCl3 each). At these iron concentrations, peroxidation was not significant in the absence of gentamicin. Since chelators can enhance iron catalyzed oxidations, this finding suggested that gentamicin-dependent radical formation was based upon iron chelation. This hypothesis was tested by measuring the influence of gentamicin on the oxidation of salicylate by Fe-EDTA complexes, a reaction that is inhibited by competing iron chelators. Gentamicin was a concentration-dependent inhibitor. In contrast, concentrations of gentamicin as high as 50 mM did not interfere with iron-independent salicylate oxidation. These results suggest that gentamicin acts as an iron chelator, and that the iron gentamicin complex is a potent catalyst of free radical formation. PMID- 8615853 TI - Auranofin inhibits the induction of interleukin 1 beta and tumor necrosis factor alpha mRNA in macrophages. AB - Gold compounds are widely used in the treatment of rheumatoid arthritis, but their mechanisms of action remain unclear. We demonstrate here that auranofin (AF) (0.1-3 microM), but neither the hydrophilic gold compounds aurothiomalate (ATM) and aurothioglucose nor methotrexate or D-penicillamine, inhibits the induction of interleukin 1 beta and tumor necrosis factor (TNF) alpha mRNA and protein by either zymosan, lipopolysaccharide (LPS), or various bacteria in mouse macrophages. The auranofin-mediated inhibition of the induction of TNF-alpha mRNA was stronger than that of interleukin (IL) 1 beta mRNA. AF, but not the other drugs, also inhibited zymosan-induced mobilization of arachidonate. The fact that AF inhibited the induction of mRNA for both these proinflammatory cytokines, irrespective of which stimulus was used, may indicate that it affects some common signal transduction step vital to their induction. PMID- 8615854 TI - Effects of decahydroisoquinoline-3-carboxylic acid monohydrate, a novel AMPA receptor antagonist, on glutamate-induced CA2+ responses and neurotoxicity in rat cortical and cerebellar granule neurons. AB - In this study, we examined the effects of a novel water-soluble, putative AMPA receptor antagonist, (-)(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3, 4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY326325), on glutamate-, N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA)-, and kainic acid (KA)-induced elevations of intracellular Ca2+ concentrations ([Ca2+]i) and 45Ca2+ uptake, as well as glutamate agonist-induced neurotoxicity in primary cultures of intact rat cortical and cerebellar granule neurons. In some experiments, the actions of LY326325 were tested in the presence of cyclothiazide, a compound that is known to block glutamate-induced desensitization of AMPA-preferring subtypes of glutamate receptors, thereby largely potentiating the functional effects of AMPA. LY326325 fully blocked the elevations of [Ca2+]i induced by NMDA and non-NMDA glutamate receptor agonists in both cortical and cerebellar granule neurons. The application of increasing concentrations of cyclothiazide was not able to reverse the LY326325-induced blockade of glutamate receptors in cortical neurons. In contrast, the same cyclothiazide treatment fully reversed the blockade produced by the noncompetitive AMPA/KA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8 methylenedioxy-5H-2, 3-benzodiazepine HCl (GYKI 52466). In 45Ca2+ uptake studies. LY325325 inhibited the NMDA-, AMPA-, and KA-induced enhancement of 45Ca2+ uptake in a concentration-dependent fashion in both cortical and cerebellar granule cells. In analogy to the results obtained with [Ca2+]i recordings, cyclothiazide failed to counteract the LY326325-induced blockade of KA-stimulated 45Ca2+ uptake in cerebellar granule neurons, whereas the blockade induced by the noncompetitive AMPA/KA receptor blocking agent GYKI 52466 was fully reversed by cyclothiazide. Because a similar, although not identical pattern of actions was seen following the application of the competitive AMPA/KA receptor antagonist 6-nitro-7 sulphamoyl-benzo(f)quinoxaline-2-3-dione (NBQX), it is suggested that the inhibitory actions of LY326325 are similar to those produced by NBQX but clearly differ from those caused by the noncompetitive AMPA/KA receptor antagonist GYKI 52466. Finally, when the neuroprotective actions of LY326325 on glutamate agonist induced neurotoxicity were examined in cerebellar granule neurons, we found that LY326325 almost completely blocked the neurotoxic actions of NMDA, AMPA, and KA, respectively, whereas it produced only a partial blockade of glutamate-induced neurotoxicity. Taken together, our current results suggest that although LY326325 blocked both nonNMDA and NMDA-induced Ca2+ responses, it still displayed a preferential affinity of nonNMDA receptors as compared to NMDA receptors. However, LY326325 appears to be a less selective AMPA/KA receptor antagonist than NBQX and GYKI52466, respectively. PMID- 8615855 TI - Validation of the (omega-1)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1. AB - The (omega-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes. The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot (r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6 hydroxylation (r = 0.73), 4-nitrophenol hydroxylation (r = 0.84), N nitrosodimethylamine demethylation (r = 0.79) and n-butanol oxidation (r = 0.73). The (omega-1)-hydroxylation of lauric acid was inhibited by ethanol (Ki = 3.5 mM), acetone (IC50 = 10 mM) dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol Ki on the (omega-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (omega-1)-hydroxylation of lauric acid in human liver microsomes, and omega-hydroxylation is mediated by another enzyme. Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (omega-1)-hydroxylation of lauric acid. PMID- 8615856 TI - Na(+)- and Cl(-)-dependent transport of taurine at the blood-brain barrier. AB - The characteristics of carrier-mediated transport of taurine at the blood-brain barrier (BBB) were studied by using primary cultured bovine brain capillary endothelial cells (BCECs), in situ brain perfusion and brain capillary depletion methods in rats. The uptake of [3H]taurine by cultured cells showed that the active transporter functions on both the luminal and antiluminal membranes of BCECs. The kinetic parameters for the saturable transport of taurine were estimated to be: for the luminal uptake, the Michaelis constant, Kt, was 12.1 +/- 0.5 microM, and the maximum uptake rate, Jmax, was 4.32 +/- 0.05 nmol/30 min/mg protein; for the antiluminal uptake, Kt was 13.6 +/- 2.4 microM and Jmax was 2.81 +/- 0.22 nmol/30 min/mg protein. The luminal and antiluminal uptakes of [3H]taurine were each dependent on both Na+ Cl-. Stoichiometric analyses suggest that two Na+ and one Cl- are associated with the luminal uptake of one taurine molecule. beta-Amino acids such as beta-alanine and hypotaurine strongly inhibited the uptake of [3H]taurine, whereas alpha- and gamma-amino acids had little or no effect. Furthermore, by in situ brain perfusion and in vivo brain capillary depletion methods, the carrier-mediated transport found by in vitro experiments was confirmed to function for the translocation of the taurine molecule from the vascular space into the brain. From these results, it was concluded that a Na+ and Cl- gradient-dependent transport (uptake) system for taurine exists in both the luminal and the antiluminal membranes of BCECs. PMID- 8615857 TI - Inhibition of collagen-induced platelet activation by arachidonyl trifluoromethyl ketone. AB - Collagen-induced platelet activation is associated with, and markedly potentiated by, the release of arachidonic acid and its subsequent conversion to thromboxane A2. The precise mechanism of arachidonic acid release is unknown. An inhibitor of isolated cytosolic phospholipase A2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), was used to examine the role that cPLA2 plays in this process. AACOCF3 inhibited platelet aggregation in response to collagen and arachidonic acid but not to thrombin, calcium ionophore, phorbol ester, or a thromboxane mimetic. Thromboxane formation stimulated by thrombin or collagen was inhibited by AACOCF3. However, AACOCF3 did not inhibit collagen-induced [14C]arachidonic acid release. These data are consistent with the inhibitory effects of AACOCF3 on collagen-induced aggregation involving an action on the conversion of arachidonic acid to thromboxane. PMID- 8615858 TI - 2-Arylpropionyl-CoA epimerase: partial peptide sequences and tissue localization. AB - The R-enantiomers of 2-arylpropionic acids (2-APAs) such as ibuprofen (IBU) exhibit the phenomenon of species- and substrate-dependent metabolic chiral inversion. Only R-enantiomers are activated to acyl-CoA-thioesters by an acyl-CoA synthetase via an adenylate intermediate. The acyl-CoA-thioesters are substrates for an epimerase, which is responsible for chiral inversion. A 42 kDa epimerase from the cytosolic fraction of rat livers was isolated and purified to homogeneity. Polyclonal antibodies were raised against the epimerase in rabbits. The anti-epimerase antibodies were used for affinity column chromatography to separate homogeneous protein for amino acid sequence analysis. Sequence data analysis of 3 internal peptide sequences showed 50% and more homology with regions of enzymes involved in fatty acid metabolism. The polyclonal anti epimerase antibodies were used to analyze the tissue distribution of the in guinea pigs and rats by Western blot analysis. Furthermore, the correlation of inversion enzyme activity in various tissues under comparable incubation conditions and cross-reactivity in Western blot analysis was investigated. PMID- 8615859 TI - N6-methyladenosine inhibits murine erythroleukemia cell maturation by blocking methylation of RNA and memory via conversion to S-(N6-methyl) adenosylhomocysteine. AB - We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block commitment of murine erythroleukemia (MEL) cells to terminal erythroid maturation. In this study, we further investigated the mechanism of this blockade. Treatment of MEL cells with N6mAdo inhibited cell growth, prevented accumulation of committed cells, suppressed methylation of total cytoplasmic RNA, and erased the expression of "memory" response, an event that precedes initiation of commitment. Furthermore, N6mAdo increased the level of S adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and altered the SAH/SAM ratio that influences methylation of ribonucleic acid (RNA). Moreover, analysis of the intracellular extracts revealed that N6-mAdo is converted into S-(N6 methyl)-adenosylhomocysteine (N6-SAH) in MEL cells, an active intermediate that affects methylation of RNA. Therefore, we conclude that N6-mAdo prevents induction of MEL cell differentiation by affecting methylation of critical RNA transcripts involved in expression of "memory" and initiation of commitment. It is likely that this inhibition occurs via conversion of N6mAdo into N6-SAH. PMID- 8615860 TI - Differential effects of in vitro peroxidation on peripheral- and central-type benzodiazepine receptors. Protection by diverse antioxidants. AB - The influence of various concentrations of ferrous iron and ascorbate on in vitro peroxidation and drug binding of diverse membrane preparations (cerebral cortex and liver) was studied. Peroxidation was not simply dose-related to ascorbate and ferrous iron, but a complex relationship between iron and ascorbate when added in association was established. Under our conditions 0.01 mM Fe2+ and 0.5 mM ascorbate was the most peroxidative combination for cerebral and liver membranes. Under the same conditions, cerebral membranes were more peroxidated than liver membranes. Considering the consequences of drug binding, peripheral-type benzodiazepine receptors (PBRs) of liver were more affected by peroxidative events than central-type benzodiazepine receptors (CBRs) of the cerebral cortex. The degree of binding disturbance was generally inversely correlated to the degree of peroxidation and this was more significant for liver PBRs than for cerebral CBRs. The liver membrane model was retained for testing in vitro protection by diverse putative antioxidants. Under our conditions desferrioxamine, ethylene diamine tetra acetate (EDTA), trolox, and rutin were good protective antioxidants, whereas phenyl-butyl-nitrone (PBN) and tocopherol were not effective. PMID- 8615861 TI - The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes. AB - The cytotoxic response of Chinese hamster ovary (CHO) cells to challenge with hydrogen peroxide was highly dependent upon the temperature of exposure, being markedly higher at 37 degrees than at 4 degrees C. Increasing intracellular levels of L-histidine prior to challenge with hydrogen peroxide increased the toxicity elicited by the oxidant at both physiologic and ice-bath temperatures. The effect of the amino acid, however, was more pronounced under conditions at 4 degrees C, as compared to 37 degrees C. Indeed, at 4 degrees C the oxidant was nontoxic at submillimolar levels and pre-exposure to L-histidine restored cytotoxicity to levels slightly higher than those observed after treatment at 37 degrees C (in the micromolar range). Pre-exposure to the amino acid increased the production of DNA double-strand breaks (DSBs) elicited by treatment with the oxidant both at 37 degrees and 4 degrees C. A remarkable correlation was found when the level of this lesion was plotted against the cytotoxic response observed using different concentrations of L-histidine or hydrogen peroxide, or treating the cells with the oxidant either at 37 degrees or 4 degrees C, thus suggesting the existence of a cause-effect relationship. The overlapping correlation curves obtained with cells challenged with the oxidant at 4 degrees or 37 degrees C also suggest that similar molecular mechanisms mediate the formation of DNA DSBs under both experimental conditions. Two lines of evidence provide experimental support for this inference: (1) the kinetics of repair of DNA DSBs generated at 37 degrees or 4 degrees C were virtually superimposable; this would suggest that the same repair pathway(s) is/are responsible for the removal of DNA DSBs generated at the two temperatures; and (2) the size distribution of double-stranded DNA fragments produced under the two treatment conditions, resulting in a similar cytotoxic response, was basically identical. This is indicative of remarkable similarities in the topology of chromosomal domains where DSBs are generated. Overall, the results presented in this paper provide further experimental evidence supporting the notion that DNA DSBs are responsible for the L-histidine mediated enhancement of hydrogen peroxide-induced cytotoxicity, and demonstrate that the mechanism whereby the amino acid enhances the ability of hydrogen peroxide to produce DNA double strand breakage and cell killing does not depend on cellular metabolism and/or energy-dependent reactions. PMID- 8615862 TI - Phenytoin covalent binding and embryopathy in mouse embryos co-cultured with maternal hepatocytes from mouse, rat, and rabbit. AB - The anticonvulsant drug phenytoin is teratogenic in a variety of species including humans. Traditional embryo culture studies have employed the addition of 9000 g supernatant (S-9) or microsomal fractions from induced rat or mouse liver as an exogenous bioactivating system to approximate a maternal contribution. However, cellular fractions, unlike cultured intact hepatocytes, may themselves be embryotoxic, and do not reflect the in vivo balance of bioactivation and detoxification. To evaluate in vitro the known in vivo differential species susceptibility to phenytoin teratogenesis, day 9.5 (day of plug = day 1) mouse embryos either were cultured alone for 24 hr or were co cultured with hepatocytes from maternal mice, rats or male rabbits, thereby exposing the embryos to the effects of potential species-specific phenytoin metabolism. In the absence of hepatocytes, phenytoin embryotoxicity was concentration dependent (0, 10, 20 and 60 micrograms/mL), with decreases in embryonic growth, reflected by reduced yolk sac diameter and crown rump length, apparent within the maternal therapeutic range (20 micrograms/mL). Covalent binding of the radiolabeled drug to live embryonic tissue was significantly higher than in control embryos previously killed by fixation, suggesting that the embryo can bioactivate phenytoin to a toxic reactive intermediate. Mouse embryos grew equally well with hepatocytes from all three species, indicating interspecies tissue compatibility. The addition of rat and rabbit hepatocytes, but not mouse hepatocytes, significantly enhanced the phenytoin-induced impairment of mouse embryonic development, as demonstrated by reductions in somite number. The phenytoin-induced impairment of mouse embryonic growth was not enhanced by the addition of rat or rabbit hepatocytes, while mouse hepatocytes conferred protection. The covalent binding of phenytoin to extracellular proteins in the culture medium was not enhanced by the addition of mouse hepatocytes. These results suggest that mouse embryos intrinsically can bioactivate phenytoin to a toxic reactive intermediate, with embryopathic consequences. The protection conferred by maternal mouse hepatocytes suggests a species-specific maternal biochemical balance favouring detoxification that is not shared by rat and rabbit hepatocytes, which enhanced phenytoin embryopathy. Thus, while phenytoin teratogenicity likely involves embryonic bioactivation, maternal determinants may contribute variably to teratologic susceptibility in a species-specific manner. PMID- 8615863 TI - Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3/4. AB - Cytochromes P450 3A3/4 are inordinately important P450 enzymes catalyzing the metabolism of a large variety of clinically useful drugs, steroids, and carcinogens. Two monoclonal antibodies, MAb 3-29-9 and MAb 275-1-2, were prepared to human P450 3A4 from mice immunized with baculovirus-expressed human P450 3A4. MAb 3-29-9 was a powerful inhibitor of the enzymatic activity of P450 3A3/4/5. MAb 3-29-9 inhibited the P450 3A3, 3A4, and 3A5 catalyzed metabolism of substrates of divergent molecular weights, e.g., p-nitroanisole, phenanthrene, diazepam, testosterone, taxol, and cyclosporin. However, MAb 3-29-9 did not give a western blot with P450 3A3 or 3A4. MAb 275-1-2 was non-inhibitory but yielded a strong western blot with P450 3A3 and 3A4 but not with 3A5, and thus distinguished between 3A3/4 and 3A5. The two MAbs did not cross-react with human 2E1, 1A2, 2B6, 2C8, and 2C9; rat 2A1, 3A1/2, 4A1, 4A3, and 2B1; and mouse 1A1 and 1A2. MAb 3-29-9 has been used successfully to measure the quantitative contribution of P450 3A3 and 3A4 to the metabolism of the above-designated substrates in human adult liver. MAb 3-29-9 and MAb 275-1-2 are precise and sensitive reagents for P450 3A studies. PMID- 8615865 TI - Oxygen dependence of hepatocyte susceptibility to mitochondrial respiratory inhibitors. AB - Most zone 3 specific hepatotoxins or their metabolites are mitochondrial toxins, and yet the susceptibility of hepatocytes to respiratory inhibitors at the low O2 concentrations found in zone 3 is not known. Potassium cyanide (CN) and antimycin A (AA) were found to be 5- and 2-fold more cytotoxic at 1% than at 95% O2, respectively. CN also inhibited the respiration of hepatocytes 36% more at 1% O2 than at 95% O2; however, AA inhibited the respiration to the same level at 1% and 95% O2. CN but not AA depleted ATP levels of hepatocytes more extensively at 1% than at 95% O2. The CN-trapping agents dihydroxyacetone, glyceraldehyde, alpha ketoglutarate and pyruvate prevented CN-induced cytotoxicity more effectively at 95% O2 than at 1% O2. In contrast, thiosulfate was less effective in preventing CN toxicity at 95% than at 1% O2. Hepatocyte thiocyanate formation from CN and thiosulfate was much faster at 1% than at 95% O2, suggesting that rhodanese, the mitochondrial enzyme that forms thiocyanate from CN and thiosulfate, is more effective at 1% O2 than at 95% O2. PMID- 8615864 TI - Adenosine modulation of tumor necrosis factor-alpha-induced neutrophil activation. AB - We hypothesized that adenosine, known to be release from inflammatory sites, could lessen the potentially damaging activity of neutrophils (PMN) primed by tumor necrosis factor-alpha (TNF alpha) at sites of infection. We investigated the effect of adenosine on PMN primed with cell-free medium from mononuclear leukocytes (MNL) that had been treated with lipopolysaccharide (LPS) yielding a conditioned medium rich in TNF alpha and on PMN primed with recombinant human TNF alpha (rhTNF alpha). LPS (10 ng/mL) minimally primed PMN, but LPS-MNL-conditioned medium increased PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP) 1242% compared with unprimed PMN. LPS-MNL-conditioned medium contained adenosine (approximately 30 nM). Converting the adenosine in the LPS-MNL-conditioned medium to inosine with adenosine deaminase (ADA) or blocking adenosine binding to PMN with the adenosine receptor antagonist 1,3-dipropyl-8-(phenyl-p-acrylate) xanthine (BW A1433U) resulted in a near doubling of chemiluminescence. The LPS MNL-conditioned medium contained TNF alpha (836 pg/mL; approximately 1 U/mL). Recombinant human TNF alpha (1 U/mL) primed PMN for a 1033% increase in chemiluminescence. Added adenosine decreased rhTNF alpha-primed PMN chemiluminescence (IC50 approximately 100 nM), and adenosine (100 nM) decreased both superoxide and myeloperoxidase release from rhTNF alpha-primed fMLP stimulated PMN. The activity of adenosine was counteracted by ADA and BW A1433U, and the modulating effect of adenosine was on the primed response rather than on priming per se. Thus, physiological concentrations of adenosine reduce the effects of recombinant human TNF alpha and native human TNF alpha (released from LPS-treated MNL) on PMN activity. Endogenous adenosine may preclude or minimize damage to infected tissue by damping the TNF alpha-primed PMN oxidative response. PMID- 8615866 TI - Affinity and dose-dependent digoxin Na+K+ATPase dissociation by monoclonal digoxin-specific antibodies. AB - The effect of three monoclonal digoxin-specific antibodies and of polyclonal Digidot as reference on digoxin dissociation from rat brain Na+K+ATPase microsomes was studied to determine the role of the affinity constant (Ka) and dose of the antibody on the rate of digoxin dissociation from Na+K+ATPase. Stoichiometrical doses of 1C10, 6C9, 9F5 IgG, and Digidot (Ka = 6 10(9), 3.1 10(8), 2.5 10(7), and 8.5 10(9) M-1, respectively) resulted in digoxin dissociation related to Ka. When the IgG:digoxin molar ratio increased from 0.25 to 10, digoxin dissociation from Na+K+ATPase sites also increased according to the Hill equation, allowing comparative parameters among the three antibodies to be determined. 1C10 IgG was 2- and 10-fold more efficacious than 6C9 and 9F5, respectively. This in vitro model appears to be a useful predictive screening assay before in vivo experimentation. PMID- 8615867 TI - Inhibition of choline uptake in syncytial microvillus membrane vesicles of human term placenta. Specificity and nature of interaction. AB - The potency and nature of the inhibitory effect of various cationic drugs on the transport of choline across the placental syncytial microvillus membrane was investigated. Tetraethylammonium, a model substrate for organic cation transport, was a poor inhibitor. Enlarging the degree of alkylation of the quaternary ammonium increased the inhibitory effect, in proportion with increasing lipophilicity. Log concentration vs % control uptake curves showed marked differences in inhibitory potency for the different cationic drugs. Hemicholinium 3 inhibited mediated choline uptake in the micromolar range, whereas atropine and mepiperphenidol were less potent. The H2-receptor antagonists cimetidine, ranitidine, and famotidine inhibited choline uptake in the millimolar ranges. Dixon analysis revealed a competitive nature of inhibition for hemicholinium-3 and atropine (Ki = 40 microM and 1.2 mM, respectively). Cimetidine interacted noncompetitively (Ki = 3.4 mM). Since relatively high concentrations were needed to reach half maximal inhibition, impairment of fetal choline supply due to maternal drug use during pregnancy is not to be expected. PMID- 8615868 TI - Conformational analysis of non-sulfonylurea hypoglycemic agents of the meglitinide family. AB - Non-sulfonylurea hypoglycemic agents of the meglitinide family such as S3075, repaglinide, KAD-1229, and A-4166, were found to display a comparable U-shaped conformation by molecular modelling, with hydrophobic cycles placed at the extremity of each branch and a peptidic bond placed at the bottom of the U. A comparable conformation was observed with the hypoglycemic sulfonylureas glibenclamide and glimepiride. A different conformation with a greater distance between the hydrophobic cycles at the extremity of each branch was found, however, with the biologically inactive enantiomers of A-4166 and repaglinide and the poorly efficient insulinotropic agent meglitinide. The identification of a common conformation of these hypoglycemic agents may help in the design of highly active compounds and provide an imprint of their postulated target receptor on the pancreatic B-cell plasma membrane. PMID- 8615869 TI - Response of [Ah] battery genes to compounds that protect against menadione toxicity. AB - We have studied the response of genes in the dioxin-inducible [Ah] battery to three compounds that protect mouse hepatoma cells (Hepa-1c7c7 wild-type, wt) against menadione toxicity. Pretreatment of wt cells with 25 microM 5,10 dihydroindenol[1,2-b]indole (DHII), 25 microM tert-butylhydroquinone (tBHO) or 10 microM menadione itself, generated substantial protection against toxicity produced by subsequent menadione exposure. The gene response was examined in wt cells, and three mutant lines: CYP1A1 metabolism-deficient (c37 or P1-); nuclear translocation-impaired (c4 or nt-); and AHR-deficient (c2 or r-, containing < 10% of normal functional receptor levels). DHII treatment of wt cells for 12 hr markedly elevated the enzyme activities and mRNA levels of genes in the [Ah] battery: aryl hydrocarbon hydroxylase (Cyp1a1), NAD(P)H:menadione oxidoreductase (Nmol), cytosolic aldehyde dehydrogenase class 3 (Ahd4), and UDP glucuronosyltransferase form 1*06 (Ugt1*06). Treatment of the c4 and c2 cells with DHII failed to induce mRNA levels of the genes, indicating that induction of the [Ah] gene battery by DHII is aromatic hydrocarbon receptor (AHR)-mediated. On the other hand, neither tBHO nor menadione caused increases in CYPlAl mRNA, but tBHQ significantly enhanced the NMO1, AHD4, and UGT1*06 mRNA levels in all three mutant cell lines. In conclusion, we expect one or more putative electrophile response elements (EpRE), previously found in the regulatory regions of the murine Nmol, Ahd4, and ugt1*06 genes, to be functional in responding to phenolic antioxidants. PMID- 8615870 TI - Characterisation of cytosolic phospholipase A2 as mediator of the enhanced arachidonic acid release from dimethyl sulphoxide differentiated U937 cells. AB - Studies were performed to characterise the phospholipase A2 (PLA2) responsible for the greatly increased capacity to release arachidonic acid (AA) of dimethyl sulphoxide (DMSO) differentiated U937 monocytic cells compared to undifferentiated cells (18-fold increase in response to Ca2+ ionophore A23187). Cytosolic PLA2 (cPLA2) activity could be measured in homogenates of differentiated cells, and the highly selective cPLA2 inhibitor arachidonic acid trifluoromethyl ketone reduced A23187 induced [3H]AA release from pre-labelled cells by at least 80%, with an IC50 (12.7 +/- 1.4 microM) not significantly different from that for inhibiting authentic cPLA2 (9.3 +/- 2.0 microM). On the other hand, type II PLA2 activity was not detected in cell homogenates, and [3H]AA release was not inhibited by heparin (1 mg/mL), which binds secreted type II PLA2 and reduces its ability to degrade membrane phospholipids. Stimulation of intact cells with A23187 plus phorbol myristate acetate (PMA) under conditions that released [3H]AA did not increase cPLA2 activity of the cell homogenate, and there was little difference between DMSO differentiated and undifferentiated cells in cPLA2 protein content, cPLA2 specific activity of homogenates, or distribution of cPLA2 between membrane and cytosol in the resting cell. Following stimulation with A23187 plus PMA, no increase in [33P] labelling of cPLA2 immunoprecipitates was seen in cells pre-labelled with [33P] orthophosphate, nor a change in electrophoretic mobility of cPLA2. It was concluded that cPLA2 releases the bulk of AA from stimulated, DMSO differentiated U937 cells. The failure to observe increased cPLA2 specific activity following cellular stimulation could be explained by increased [3H]AA release requiring the activation of only a small proportion of the cell pool of cPLA2 or, alternatively, by increased release reflecting greater Ca(2+)-dependent association of cPLA2 with membrane substrate rather than increased specific activity per se. There was no evidence that any such increased membrane association resulted from cPLA2 phosphorylation. The relative inability of undifferentiated cells to release AA was not due to the absence of cPLA2 or an altered distribution between membrane and cytosol, but suggested the presence of a repressor mechanism that prevents elevated Ca2+ from functionally activating the enzyme intracellularly. PMID- 8615871 TI - Inhibition of complex I by isoquinoline derivatives structurally related to 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). AB - Mitochondrial respiratory failure secondary to complex I inhibition may contribute to the neurodegenerative process underlying nigral cell death in Parkinson's disease (PD). Isoquinoline derivatives structurally related to 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) may be inhibitors of complex I, and have been implicated in the cause of PD as endogenous neurotoxins. To determine the potency and structural requirements of isoquinoline derivatives to inhibit mitochondrial function, we examined the effects of 22 neutral and quaternary compounds from three classes of isoquinoline derivatives (11 isoquinolines, 2 dihydroisoquinolines, and 9 1,2,3,4 tetrahydroisoquinolines) and MPP+ on the enzymes of the respiratory chain in mitochondrial fragments from rat forebrain. With the exception of norsalsolinol and N,n-propylisoquinolinium, all compounds inhibited complex I in a time independent, but concentration-dependent manner, with IC50s ranging from 0.36-22 mM. Several isoquinoline derivatives were more potent inhibitors of complex I than 1-methyl-4-phenylpyridinium ion (MPP+) (IC50 = 4.1 mM), the most active being N-methyl-6-methoxy-1,2,3,4-tetrahydroisoquinoline (IC50 = 0.36 mM) and 6 methoxy-1,2,3,4-tetrahydroisoquinoline (IC50 = 0.38 mM). 1,2,3,4 Tetrahydroisoquinoline was the least potent complex I inhibitor (IC50 approximately 22 mM). At 10 mM, only isoquinoline (23.1%), 6,7 dimethoxyisoquinoline (89.6%), and N-methylsalsolinol (34.8%) inhibited (P < 0.05) complex II-III, but none of the isoquinoline derivatives inhibited complex IV. There were no clear structure-activity relationships among the three classes of isoquinoline derivatives studied, but lipophilicity appears to be important for complex I inhibition. The effects of isoquinoline derivatives on mitochondrial function are similar to those of MPTP/MPP+, so respiratory inhibition may underlie their reported neurotoxicity. PMID- 8615873 TI - Biotransformation of tritiated fentanyl in human liver microsomes. Monitoring metabolism using phenylacetic acid and 2-phenylethanol. AB - Norfentanyl has been identified previously as a urinary metabolite of fentanyl. However, at clinically relevant concentrations, norfentanyl are below the limits of detection. The use of labeled drug in metabolic studies is a standard approach to overcome the limitations imposed by metabolite concentrations that are below detection limits. Unfortunately, the available tritium-labeled fentanyl yields unlabeled norfentanyl following N-dealkylation. Thus, we have developed a technique to monitor the N-dealkylation of fentanyl using the other products of N dealkylation. The biotransformation of fentanyl was studied in human liver microsomes. After incubation with human liver microsomes for 20 min, almost 50% of a 0.03 microM concentration of [3H]-fentanyl was metabolized to the [3H]N dealkylated metabolite phenylacetaldehyde, which was then converted principally to [3H]2-phenylethanol and to a smaller extent to [3H]phenylacetic acid in microsomal incubates. The apparent Km,app and Vmax,app for norfentanyl formation were 82 +/- 21 microM and 4.7 +/- 0.4 nmol product formed/min/nmol cytochrome P450, respectively. Thus, this study defined methodology that can be used to evaluate the metabolism of fentanyl, both in vivo and in vitro, at clinically relevant concentrations. PMID- 8615872 TI - Structure-dependent induction of CYP2B by polychlorinated biphenyl congeners in female Sprague-Dawley rats. AB - The dose-response induction of hepatic microsomal pentoxyresorufin O-dealkylase (PROD) activity by phenobarbital (PB) and several polychlorinated biphenyl (PCB) mixtures and congeners was determined in the immature female Sprague-Dawley rat. At a dose of 75 mg/kg/day of PB for 3 days, the microsomal PROD activity was 2154 pmol/min/mg protein. Aroclors 1260, 1254, 1242, and 1016 did not induce maximal PROD activity at doses up to 500 mg/kg, and only Aroclor 1016 induced > a half maximal response at the 500 mg/kg dose. The relative potencies of eighteen different PCB congeners were also determined, and the structures of these compounds differed with respect to the degree of chlorination (tri- to octochloro) and substitution patterns. The relative potencies of these compounds were estimated by comparing their induced activities at the high dose (150 or 100 mg/kg) with that of PB. The most potent inducers were 2,3,3',4',5,6-hexaCB and 2,2',3,4',5,5',6-heptaCB; at a dose of 150 mg/kg, the PROD activity induced by 2,2'3,4',5,5',6-heptaCB was comparable to that observed for PB. 2,3,3',4',5,6 HexaCB was the most potent inducer, and hepatic PROD activity in rats treated with 150 mg/kg was 4202 pmol/min/mg; this value was higher than that observed for PB at a dose of 75 mg/kg. A second group of congeners including 2,2',3,4,4',5,5' heptaCB, 2,2',4,4',5,5'-hexaCB, 2,2',3,3',4,4',5,5'-octaCB 2,2',4,4'-tetraCB, 2,2',4,5,5'-pentaCB, 2,2',3,4,4',5',6-heptaCB, 2,2',4,4',5-pentaCB and 2,2',3,3',4',5,5',6-octaCB induced PROD activity > or = 1090 pmol/min/mg at the 150 mg/kg dose, and this value was > 50% of the maximal response observed for PB. The remaining compounds, namely 2,4,4'-triCB, 2,2',3,4'-tetraCB, 2,2',5,5' tetraCB, 2,3',4,4',5-pentaCB, 2,3,3',4,4'-pentaCB, 2,2',4,4',5,6'-hexaCB, 2,3,3',4,4',5,5'-heptaCB and 2,2',3,3',4,4,5-heptaCB were all relatively weak inducers of hepatic microsomal PROD activity ( < 450 pmol/min/mg). In parallel experiments, western blot analysis of immunoreactive CYP2B1 and CYP2B2 protein showed that PB, the PCB mixtures, and congeners induced both proteins. Previous studies have identified a cis-acting DNA element that plays a role in regulating CYP2B1/B2 gene expression and binds nuclear trans-acting factor(s) induced by PB. The results of gel electrophoretic mobility shift assays with nuclear extracts showed that both PB and 2,2',3,4',5,5',6-heptaCB induce formation of a common retarded band using a 32P-labeled oligonucleotide corresponding the the cis acting DNA promoter sequence. Both PB and PCBs appear to induce CYP2B1/B2 via a common mechanism. Although the results of this study do not define structure induction (CYP2B1/B2) relationships for PCBs, two compounds, namely 2,3,3',4',5,6 hexaCB and 2,2',3,4',5,5',6-heptaCB, were identified as highly potent inducers. PMID- 8615874 TI - Interaction of ethanol and the organophosphorus insecticide parathion. AB - Phosphorothioate insecticides such as parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate) undergo P450-dependent oxidative desulfuration, leading to both activation and detoxification of these compounds. Consequently, alterations in P450-dependent oxidative desulfuration may affect the acute toxicities of these insecticides. In the present study, pretreatment of mice with 15% ethanol in the drinking water for 6 days antagonized the acute toxicity of parathion, but not its toxic metabolite paraoxon (O,O-diethyl-O-p-nitrophenyl phosphate), suggesting that ethanol affected the oxidative desulfuration of this insecticide. The presence of ethanol within hepatic microsomal incubations did not alter the P450 dependent formation of paraoxon (activation) and p-nitrophenol (detoxification), although p-nitrophenol levels were increased in the presence of ethanol as a result of inhibition of its biotransformation to 4-nitrocatechol by CYP2E1. Ethanol exposure reduced hepatic pyruvate levels, but had no effect on levels of lactate, isocitrate, alpha-ketoglutarate, and malate. Calculation of cytosolic NAD+/NADH and cytosolic NADP+/NADPH redox ratios did not reveal any detectable difference in redox state between control and ethanol-treated mice. Since ethanol did not alter directly the P450-dependent activation or detoxification of parathion, and did not decrease NADPH levels, ethanol's antagonism of the acute toxicity of parathion may result from reduced availability of O2. PMID- 8615875 TI - Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human placental microsomes. AB - The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can be activated metabolically by cytochrome(s) P450 to DNA-damaging agents that result in the formation of tumors in various organs of several animal models. In the present study, 30-min incubations at 37 degrees containing 5 mg/mL pooled human placental microsomes, 36 nmol NNK (including 2 microCi [5-3H]NNK) and a 5 mM concentration of either NADH, NADPH, or both cofactors together resulted in the formation of 11.43 +/- 0.32, 35.40 +/- 4.64, and 44.05 +/- 1.66 pmol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)/mg protein/min (mean +/ SD, N = 3), respectively. Similar experiments using 7, 9, and 11 mM NADH, NADPH, and both cofactors together in equimolar concentrations yielded results that suggest that NADH- and NADPH-dependent reductions of NNK are catalyzed by different enzymes. Computer simulations for the production of NNAL based on various kinetic models corroborated the conclusion drawn from the empirically derived data. In human placental microsomes, the Km,app and Vmax,app for the formation of NNAL were 1021.9 +/- 251.5 microM and 4360.7 +/- 991.7 pmol/mg protein/min, respectively. Inhibition of cytochrome P450-dependent activities by carbon monoxide and dicumarol (100 and 200 microM) resulted in an average increase of NNAL production of 40 and 56%, respectively, suggesting that P450 dependent biotransformation of NNK is occurring in the absence of inhibitors. Similarly, polyclonal goat IgG against rabbit P450 reductase resulted in a 12% increase in the production of NNAL when compared with control values. Thirty micromolar rutin, ethacrynic acid, cibacron blue 3GA, and iodoacetic acid, known inhibitors of certain human carbonyl reductase(s), incubated with placental microsomes containing an equimolar concentration of NNK, did not have a significant effect on the production of NNAL. These results establish that: (1) cytochromes P450 are likely involved in the metabolism of NNK by human placental microsomes, (2) metabolism of NNK to NNAL by human placental microsomes is catalyzed by an NADPH-dependent carbonyl reductase(s) and an NADH-dependent carbonyl reductase(s), and (3) reduction of NNK to NNAL is catalyzed by a placental microsomal carbonyl reductase(s) not previously described. PMID- 8615876 TI - Biochemical modulation of tumor cell energy. IV. Evidence for the contribution of adenosine triphosphate (ATP) depletion to chemotherapeutically-induced tumor regression. AB - DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6 methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis. PMID- 8615877 TI - Inducers of adipose conversion activate transcription promoted by a peroxisome proliferators response element in 3T3-L1 cells. AB - Nonmetabolizable fatty acids are shown here to induce adipose conversion of 3T3 L1 preadipocytes as well as to activate transcription of a reporter plasmid promoted by a peroxisome proliferators response element. This dual activity was also observed using the peroxisome proliferator bezafibrate or the differentiation inducer isobutyl methylxanthine. The data suggest a role for a peroxisome proliferators activated receptor (PPAR) in adipose conversion induced by fatty acids, isobutyl methylxanthine, or xenobiotic amphipathic carboxylates. PMID- 8615878 TI - N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N'-diacetic acid as a new iron chelator with potential medicinal applications against oxidative stress. AB - N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N,-diacetic acid dihydrochloride (OR10141) is a member of a recently described series of "oxidative stress activatable iron chelators." These chelators have a relatively low affinity for iron but can be site-specifically oxidized, in situations mimicking oxidative stress in vitro, into species with strong iron-binding capacity. It is hoped that this local activation process will minimise toxicity compared to strong iron chelators that may interfere with iron metabolism. The present paper describes the results of experiments aimed at characterising oxidative reactions between iron-OR10141 complexes and hydrogen peroxide. Incubation of ascorbate and hydrogen peroxide with the ferric chelate of OR10141 in neutral aqueous solution yields a purple solution with a chromophore at 560 nm, which is consistent with an o-hydroxylation of one of the trimethoxybenzyl rings. Oxidation of OR10141 also takes place, although more slowly, by incubating hydrogen peroxide with ferric OR10141 complex in the absence of reductant. HPLC analysis shows that OR10141 is consumed during the reaction and transformed principally into N-(2-hydroxy 3,4,5-trimethoxybenzyl) N'-(3,4,5-trimethoxybenzyl) ethylenediamine N,N'-diacetic acid. Minor products are also formed, some of which were identified by mass spectrometry. The protective effect of OR10141 in vitro against DNA single strand breaks, protein damage, and lipid peroxidation induced by Fenton chemistry suggests that this compound is able to compete for iron with biological molecules and, thus, that this strategy of protection against oxidative stress is feasible. In addition, preliminary results showing protective effects of OR10141 dimethyl ester against toxicity induced by hydrogen peroxide in cell culture are described. It is concluded that OR10141 and related prodrugs might be useful in vivo in chronic situations involving oxidative stress. PMID- 8615879 TI - Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells. AB - Daunorubicin (DRC) and other anthracyclines are valuable cytotoxic agents in the clinical treatment of certain malignancies. However, as is the case with virtually all anticancer drugs, tumor cell resistance to these agents is one of the major obstacles to successful chemotherapy. In addition to an increased energy-dependent efflux of chemotherapeutic agents, enzymatic drug-inactivating mechanisms also contribute to multidrug resistance of tumor cells. In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significantly lower antineoplastic potency compared to the parent drug. In the present study, we compared two pancreas carcinoma cell lines (a DRC-sensitive parental line and its DRC-resistant subline) with respect to their capacity of DRC inactivation via carbonyl reduction. In addition, we cultured the two cell lines in the presence of increasing sublethal concentrations of DRC. Evidence is presented that DRC treatment itself leads to a concentration-dependent induction of DRC carbonyl reduction in subcellular fractions of both the sensitive and resistant pancreas carcinoma cells, resulting, surprisingly, in different susceptibilities to DRC. The principal difference between the two cell lines becomes most apparent at high-dose DRC supplementation (1 microgram/mL), at which DRC resistant cells exhibited higher inducibility of DRC-inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability. The use of the diagnostic model substrates metyrapone and p-nitrobenzaldehyde reveals that this adaptive enhancement of DRC inactivation can be attributed to the induction of DRC carbonyl reductases different from known aldehyde and carbonyl reductases. In conclusion, these findings suggest that inactivation of anthracyclines by carbonyl reduction is inducible by the substrate itself, a fact that might be considered as one of the enzymatic mechanisms that contribute to the acquired resistance to these drugs. PMID- 8615880 TI - Receptor-independent G protein activation may account for the stimulatory effects of first-generation H1-receptor antagonists in HL-60 cells, basophils, and mast cells. AB - The first-generation histamine H1-receptor antagonists, chlorpheniramine (CPHE) and diphenhydramine (DPH), may activate histamine release from basophils and mast cells. Because CPHE and DPH are cationic-amphiphilic and because several substances with such physicochemical properties activate heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in a receptor independent manner, we asked the question of whether or not H1-receptor antagonists could be G-protein activators as well. In dibutyryl cAMP differentiated HL-60 cells, CPHE and DPH increased cytosolic Ca2+ concentration and azurophilic granule release in pertussis toxin (PTX)-sensitive manners. In HL 60 membranes, PTX-sensitive stimulations of GTPase [E.C. 3.6.1.] and binding of guanosine 5'-[gamma-thio]triphosphate by H1 receptor antagonists were observed. CPHE and DPH also increased GTP hydrolysis by the purified PTX-sensitive G protein, transducin. In all-trans-retinoic acid-differentiated HL-60 cells and rat basophilic leukemia cells (RBL 2H3 cells), H1-receptor antagonists induced, unlike in dibutyryl cAMP-differentiated HL-60 cells, Ca2+ influx without Ca2+ mobilization from intracellular stores. CPHE and DPH also induced serotonin release from RBL 2H3 cells. Our data indicate that first-generation H1-receptor antagonists are receptor-independent G-protein activators and that such a mechanism of action accounts for their stimulatory effects in HL-60 cells, basophils, and mast cells. PMID- 8615881 TI - Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population. AB - KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinol ine-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose dependently increased alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese digestible protein (CDP) of ROS 17/2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1 alpha,25 dihydroxyvitamin D3 (1 alpha,25(OH)(2)D3)-induced increase in osteocalcin synthesized by ROS 17/2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA 098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1 alpha,25(OH)(2)D(3), parathyroid hormone (PTH), and prostaglandin E2 (PGE2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclast-like cells. Coumestrol and 17beta-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17/2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17beta-estradiol on bone tissue is the inhibition of bone resorption. PMID- 8615882 TI - Structure-function relationships of calcium antagonists. Effect on oxidative modification of low density lipoprotein. AB - Human low density lipoprotein (LDL) incubated with active Ca2+ antagonists from three different chemical groups, 1,4-dihydropyridines that are of reduced activity as Ca2+ antagonists, vitamin E, and probucol, was more resistant than control to copper- or human monocyte-induced oxidation, as assessed by thiobarbituric acid reactive substance (TBARS) content, degradation by J774 macrophages, and relative electrophoretic mobility on agarose gel. In the copper induced oxidation system, the drugs tested reduced the TBARS levels of LDL in a concentration-dependent manner. The order of potency was vitamin E > felodipine > 2-chloro analog of nifedipine > nifedipine > amlodipine, nitrendipine, verapamil > diltiazem. In agreement with the results of the TBARS assay, felodipine (25 microM) was also the most effective calcium antagonist in the degradation assay, inducing a significant (P<0.05) 97 +/- 2% reduction in the amount of oxidized [125I]LDL degraded by J774 macrophages compared with nifedipine and its 4-nitro analog, amlodipine, and verapamil. The relative mobility of oxidized LDL on agarose gel was reduced significantly (P<0.05) by felodipine (50 microM) and amlodipine (25 and 50 microM) when compared with control, and was similar to that of native LDL, suggesting an effect of these drugs on the net negative charge of oxidized LDL. In the cell-induced oxidation system, both nifedipine and felodipine (25 microM) induced significant (P<0.05) reductions in the TBARS content of LDL (96 +/- 2 and 65 +/- 9%, respectively) compared with amlodipine, verapamil and the 4-nitro analog of nifedipine. However, in this oxidation system nifedipine was a more effective antioxidant than felodipine. Analysis of the structure-function relationships for the effect of 1,4-dihydropyridines on the oxidative modification of LDL suggests an important role for the 2-substitution of the phenyl ring, and an essential role for the dihydropyridine ring. This study clearly shows that Ca2+ antagonists from different chemical groups have a concentration-dependent effect as antioxidants against LDL oxidation. However, the order of potency of the drug depends on the oxidation system and the assay used to measure the antioxidant effect. Our data suggest that such a protective effect of Ca2+ antagonists against LDL oxidation could play a role in the antiatherosclerotic effect of these drugs. PMID- 8615883 TI - Effects of benzophenanthridine alkaloids on the phosphorylation of an approximately 44 kDa protein present in a mitochondrial fraction of the rat heart. AB - Chelerythrine and sanguinarine, benzophenanthridine alkaloids that are known to have a wide variety of biologic actions including inhibitory activity against the phosphorylation of proteins, were tested for their effects on the phosphorylation of a specific approximately 44 kDa protein present in the mitochondrial fraction of the rat heart. The concentrations required for 50% inhibition were determined to be 90.3 and approximately 200 microM for chelerythrine and sanguinarine, respectively, while the median-effect concentrations were 71 and 98 microM for chelerythrine and 186 microM for sanguinarine. The combination index values, determined from median-effect plots, for the combination of chelerythrine and taurine in a ratio of 1:100 were greater than 1, which indicates that chelerythrine plus taurine is antagonistic. Both chelerythrine and sanguinarine had biphasic (i.e. stimulation and inhibition) effects on the phosphorylation of the approximately 44 kDa protein. It was determined that the biphasic effect for checlerythrine depended upon the time of preincubation at 37 degrees of chelerythrine with the mitochondrial preparation. Preincubation times of 0.5 and 1 min produced 70 and 82% stimulation, while longer preincubation times of 2-22 min resulted in inhibition of the phosphorylation reaction by 40-95%. Dithiothreitol (DTT), a reducing agent, prevented the inhibitory effect of chelerythrine. Glutathione was less effective in protecting the phosphorylation of the approximately 44 kDa protein. It is suggested that the minimum bond of chelerythrine reacts with the thiol group on DTT, thereby preventing chelerythrine from reacting with thiol groups on the kinase responsible for phosphorylating the approximately 44 kDa protein. The inhibitory effects of taurine were only partially eliminated by DTT. PMID- 8615884 TI - Effects of 540C91 [(E)-3-[2-(4'-pyridyl)-vinyl]-1H-indole], an inhibitor of hepatic tryptophan dioxygenase, on brain quinolinic acid in mice. AB - Studies were undertaken to assess the role of the liver in the formation of the neurotoxin quinolinic acid in the brain. A selective and potent inhibitor of hepatic tryptophan 2,3-dioxygenase, 540C91 [(E)-3-[2-(4'-pyridyl)-vinyl]-1H indole], largely prevented the elevation in mouse brain quinolinic acid resulting from parenteral injection of tryptophan (TRP). In contrast, 540C91 did not affect basal levels of the neurotoxin. Following induction of indoleamine dioxygenase with bacterial lipopolysaccharide, 540C91 was less effective in preventing the TRP-induced elevations in quinolinic acid. The data suggest that kynurenines, formed from tryptophan, by the liver and other extrahepatic organs can give rise to brain quinolinic acid. PMID- 8615885 TI - Identification of human liver cytochrome P450 enzymes that metabolize the nonsedating antihistamine loratadine. Formation of descarboethoxyloratadine by CYP3A4 and CYP2D6. AB - [3H]Loratadine was incubated with human liver microsomes to determine which cytochrome P450 (CYP) enzymes are responsible for its oxidative metabolism. Specific enzymes were identified by correlation analysis, by inhibition studies (chemical and immunoinhibition), and by incubation with various cDNA-expressed human P450 enzymes. Descarboethoxyloratadine (DCL) was the major metabolite of loratadine detected following incubation with pooled human liver microsomes. Although DCL can theoretically form by hydrolysis, the conversion of loratadine to DCL by human liver microsomes was not inhibited by the esterase inhibitor phenylmethylsulfonyl fluoride (PMSF), and was dependent on NADPH. A high correlation (r2 = 0.96, N = 10) was noted between the rate of formation of DCL and testosterone 6 beta-hydroxylation, a CYP3A-mediated reaction. With the addition of ketoconazole (CYP3A4 inhibitor) to the incubation mixtures, the residual rate of formation of DCL correlated (r2 = 0.81) with that for dextromethorphan O-demethylation, a CYP2D6 reaction. Rabbit polyclonal antibodies raised against the rat CYP3A1 enzyme (5 mg IgG/nmol P450) and troleandomycin (0.5 microM), a specific inhibitor of CYP3A4, decreased the formation of DCL by 53 and 75%, respectively, when added to 1.42 microM loratadine microsomal incubations. Quinidine (5 microm), a CYP2D6 inhibitor, inhibited the formation of DCL approximately 20% when added to microsomal incubations of loratadine at concentrations of 7-35 microM. Incubation of loratadine with cDNA-expressed CYP3A4 and CYP2D6 microsomes catalysed the formation of DCL with formation rates of 135 and 633 pmol/min/nmol P450, respectively. The results indicated that loratadine was metabolized to DCL primarily by the CYP3A4 and CYP2D6 enzymes in human liver microsomes. In the presence of a CYP3A4 inhibitor, loratadine was metabolized to DCL by the CYP2D6 enzyme. Conformational and electrostatic analysis of loratadine indicated that its structure is consistent with substrate models for the CYP2D6 enzyme. PMID- 8615886 TI - Effect of different chemically modified oligodeoxynucleotides on immune stimulation. AB - Based on previous studies that certain oligonucleotides can stimulate cell proliferation and immunoglobulin production, this study was carried out to establish the relationship between the stimulatory effect and the chemical modification of the oligonucleotide. First, the effects of oligonucleotide and analogs on immune stimulation were studied in vitro using murine splenic lymphocytes. Our results show that cell proliferation and immunoglobulin production (IgG and IgM) depend on the sequence and the chemical modification of the oligonucleotide. Phosphorothioate oligodeoxynucleotides displayed a greater stimulatory effect than partially modified phosphorothioate oligonucleotides. Second, we studied the effects of these chemically modified oligonucleotides after injection in mice. Massive splenomegaly and stimulation of cell proliferation were observed with some phosphorothioate oligonucleotides. These effects were minimized markedly by chimeric and hybrid oligonucleotides. We also demonstrate that in vitro the effects of oligonucleotides on murine lymphocytes were unaffected by T cell depletion, suggesting that oligonucleotides exert their effects mainly on the B cells. PMID- 8615887 TI - Regulation of the stress-like protein adenotin in PC 12 cells by ethanol exposure. AB - The effects of chronic ethanol exposure on the stress-like protein adenotin were investigated using the radioligand [3H]-5'-N-ethylcarboxamidoadenosine ([3H]NECA). A 4-day exposure to 150 mM ethanol increased both the KD and the density of [3H]NECA binding sites. These changes were not due to residual ethanol as the acute addition of ethanol did not alter [3H]NECA binding. Chronic ethanol exposure of A126-1B2-1 cells, which are a mutant PC 12 cell line deficient in protein kinase A (PKA), increased the cellular density of adenotin, but did not affect the KD for the radioligand. Conversely, when PC 12 cells were exposed to 10 microM forskolin for either 2 or 4 days, the cellular density of adenotin was not altered, but the affinity of adenotin for [3H]NECA was reduced significantly. An increase in KD was not observed after a 1-hr exposure of PC 12 cells to forskolin, indicating that the reduction in affinity for the radioligand was not due simply to a PKA-mediated phosphorylation of adenotin. The present study demonstrated that chronic ethanol regulates adenotin through two different mechanisms. The ethanol-induced increase in the density of adenotin does not involve PKA, while the reduction in affinity ap pears to involve a cAMP-dependent mechanism. PMID- 8615888 TI - Cellular fibronectin and von Willebrand factor concentrations in plasma of rats treated with monocrotaline pyrrole. AB - The monocrotaline pyrrole (MCTP)-treated rat is a useful model for the study of certain chronic pulmonary vascular diseases. A single, i.v. administration of a low dose of MCTP causes pneumotoxicity, pulmonary vascular remodeling, sustained increases in pulmonary arterial pressure, and right ventricular hypertrophy in rats. The pulmonary vascular lesions are characterized by endothelial cell alterations, platelet and fibrin microvascular thrombosis, pulmonary edema, and thickening of the intimal and medial layers of the vessel wall. These lesions suggest that some dysfunction of the hemostatic system occurs in the lungs of rats treated with MCTP. We evaluated the concentrations of two adhesion proteins, cellular fibronectin (cFn) and von Willebrand factor (vWF), in the plasma of rats treated with MCTP. We hypothesized that changes in these factors occur along with markers of pneumotoxicity and ventricular hypertrophy and that such changes might contribute to the genesis of the vascular lesions. Enzyme-linked immunosorbent assays were used to measure cFn and vWF concentrations in the plasma of rats after MCTP treatment. Rats treated with a single i.v. injection of 3.5 mg MCTP/kg body weight had delayed and progressive lung injury characterized at 5 days post treatment by increases in the lung-to-body weight ratio and in lactate dehydrogenase activity and protein concentration in cell-free bronchoalveolar lavage fluid (BALF). Values for these markers were further increased at 8 days and reached a plateau thereafter. The number of nucleated cells within the BALF was increased at 8 and 14 days. Right ventricular hypertrophy, an indirect marker of pulmonary hypertension, was evident at 14 days. The cFn concentration was increased in plasma in rats at 8 and 14 days after treatment with MCTP. There was no difference between the vWF concentration in plasma of rats treated with MCTP and those treated with vehicle at any time. We conclude that an increase in plasma cFn concentration occurs prior to the onset of right ventricular hypertrophy and that this change is consistent with a role for cFn in the genesis of vascular remodeling and pulmonary hypertension in the MCTP-treated rat. The lung vascular injury and pulmonary hypertension in this model were not reflected in altered vWF concentration in the plasma. PMID- 8615889 TI - Formation of a highly stable complex between BN 50730 [tetrahydro-4,7,8,10 methyl 1(chloro-2 phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido [4',3'-4,5] thieno [3,2-f] triazolo-1,2,4 [4,3-a] diazepine-1,4] and the platelet-activating factor receptor in rabbit platelet membranes. AB - BN 50730 [tetrahydro-4,7,8,10 methyl-1(chloro-2 phenyl)-6 (methoxy-4 phenyl carbamoyl)-9 pyrido [4',3'-4,5] thieno [3,2-f] triazolo-1,2,4 [4,3-alpha] diazepine-1,4], a novel platelet-activating factor (PAF) receptor antagonist with a hetrazepine structure, decreased the maximal number of binding sites (Bmax) of [3H]PAF in rabbit platelet membranes without altering its dissociation constant. Platelet aggregation induced by 1 microM PAF was prevented by preincubation with 1 microM BN 50730. The washing of the platelets preincubated with BN 50730 failed to revert its inhibitory effects. We conclude that BN 50730 acts as a non competitive antagonist of the PAF receptor, due to the formation of a highly stable drug-receptor complex. PMID- 8615890 TI - Molecular pharmacology of endothelin converting enzymes. AB - A critical processing step in endothelin biosynthesis is the conversion of the intermediate "big endothelin" to its biologically active product catalysed by endothelin converting enzyme (ECE). In this commentary we discuss critically the cellular location, structure, and activity of the isoforms of ECE. The current evidence supporting a metallopeptidase ECE as the physiological regulator of endothelin production is described. Its sensitivity to inhibition by the fungal metabolite phosphoramidon and subsequent cloning of the enzyme indicate it to be a type II integral membrane protein homologous with neural endopeptidase-24.11 (E 24.11), the major neuropeptide-degrading ectoenzyme in brain and other tissues. Unlike E-24.11, however, ECE exists as a disulphide-linked dimer of subunit M(r) 120-130 kDa and is not inhibited by other E-24.11 inhibitors such as thiorphan. Alternative splicing produces two forms of ECE with distinct N-terminal tails. These isoforms of ECE-1 show similar specificity converting big endothelin-1 (ET 1) to ET-1 but big ET-2 and big ET-3 are converted much less efficiently. This suggests that additional forms of ECE remain to be isolated. Immunocytochemical studies indicate a predominant cell-surface location for ECE-1, like E-24.11. This is consistent with the conversion of exogenous big ET-1 when administered in vivo and the inhibition of this event by phosphoramidon. However, mature ET-1 can be detected in intracellular vesicles in endothelial cells, suggesting that some processing occurs in the constitutive secretory pathway. This may be mediated by ECE-2, a recently cloned member of the E-24.11/ECE family which has an acidic pH optimum. Selective inhibitors of ECE may have therapeutic applications in cardiovascular and renal medicine. PMID- 8615891 TI - Non-genomic effects of estrogen and the vessel wall. AB - Estrogen, like other steroids, is now believed to possess rapid membrane effects independent of the classical gene activation pathway of steroid action. The presence of membrane estrogen receptors has been demonstrated in different cell types, but not yet in vascular tissue. In vivo, estrogen administration rapidly promotes acetylcholine-induced vasodilation of the coronary and peripheral vascular beds of postmenopausal women. Estrogen also causes relaxation of precontracted isolated arterial segments and perfused organ preparations, within minutes of administration of the hormone. These rapid vasomotor effects of estrogen may be related to blockade of the cell membrane voltage-dependent calcium channels, resulting in inhibition of extracellular Ca2+ mobilization and flux. Recently, estradiol has been shown to rapidly affect cyclic nucleotide turnover in vascular segments, smooth muscle, and epithelial cell cultures, suggesting the possibility of a "cross-talk" between membrane-mediated events and nuclear receptor activation. PMID- 8615892 TI - Effect of pentoxifylline on the fibrogenic functions of cultured rat liver fat storing cells and myofibroblasts. AB - The effects of pentoxifylline (PTX) an analogue of the methylxanthine theobromine, on basic fibrogenic reactions of cultured fat-storing cells and myofibroblasts (MFB), the cell types most important for the excessive production of extracellular matrix components in fibrosing liver, were studied. The proliferation of MFB (i.e., activated, transdifferentiated fat-storing cells) was more dose-dependently inhibited by pentoxifylline than that of unactivated fat storing cells (ED50 50 microgram/mL). In addition, PTX retarded the transdifferentiation of fat-storing cells into smooth muscle alpha-actin positive MFB, a 50% reduction in actin-positive cells being reached with concentrations of 0.5 mg PTX/mL medium. The transdifferentiation-associated decrease in retinyl palmitate of cultured fat-storing cells was delayed by PTX. The synthesis of [35S] sulfate-labeled glycosaminoglycans (GAG) and total and cellular fibronectin was not significantly reduced by treatment of MFB with PTX up to 1.0 mg/mL. It is concluded that PTX reduces the transdifferentiation of fat-storing cells to MFB and the proliferation of MFB, but leaves the synthesis of extracellular matrix components GAG and fibronectin unaffected. The effect of PTX on the former reactions might account for the reported antifibrogenic properties of this drug in experimental hepatic fibrogenesis. PMID- 8615893 TI - Interaction of hydro- or lipophilic phthalocyanines with cells of different metastatic potential. AB - A highly metastatic (4R) and a nonmetastatic (RE4) transformed rat embryo fibroblast cell line were incubated with lipid-soluble Zn(II)-phthalocyanine (ZnPc) and its water-soluble tetrasulphonated derivative (ZnPcTS) and compared for phthalocyanine uptake. The hydrophobic liposome-delivered ZnPc showed a significantly greater uptake by both cell lines than did ZnPcTS. Moreover, the two phthalocyanines appear to interact with cells according to different pathways, as suggested by the different temperature-dependence of the binding process and the different inhibitory action exerted by selected serum proteins, such as lipoproteins and heavy proteins. Under all experimental conditions, the two cell lines exhibited similar interactions with ZnPc and ZnPcTS, suggesting that heterogeneity of the tumor cell population has a minor influence on the accumulation of photosensitizers. PMID- 8615894 TI - Metabolism of the new immunosuppressor cyclosporin G by human liver cytochromes P450. AB - Cyclosporin G is a new immunosuppressor structurally similar to cyclosporin A. Although this drug is pharmacologically as active as cyclosporin A, it is less toxic, in particular at the kidney level. The aim of this work was to identify the enzyme system(s) involved in the oxidative metabolism of cyclosporin G in man: (1) in a bank of human liver microsomes (n = 22), cyclosporin G oxidase activity correlated significantly with cyclosporin A oxidase activity (P < 0.0001) and with the level of CYP3A4 (P < 0.002), determined by immunoblot; (2) specific inhibitors of CYP3A4, troleandomycin, and ketoconazole, inhibited cyclosporin G oxidase activity by more than 80%; (3) antiCYP3A4 antibodies specifically inhibited this activity by nearly 90%; (4) cyclosporin A was a competitive inhibitor of cyclosporin G oxidase and vice versa; (5). Among a battery of cDNA-expressed CYPs, only CYP3A4 was able to generate detectable amounts of metabolites of cyclosporin G and cyclosporin A with a turnover number close to that calculated from experiments with liver microsomes; (6) in human hepatocytes in culture, pretreatment of cells with rifampicin and phenobarbital, 2 inducers of CYP3A4, produced a great increase in cyclosporin G oxidase activity, while beta-naphthoflavone, an inducer of CYP1As, did not. We conclude that CYP3A4 is the major enzyme involved in the oxidative metabolism of cyclosporin G in human liver. PMID- 8615895 TI - Generation of a free radical from calphostin C by microsomal cytochrome P450 reductase in rat and human liver. AB - Calphostin C (cal C) is a potent and specific protein kinase C inhibitor that is thought to be photoactivated to generate a reactive perylenesemiquinone free radical intermediate. In this electron spin resonance spectrometry study, we show, for the first time, an enzymatic light-independent mechanism of cal C free radical formation. Such a free radical was generated in liver microsomes from rat and humans, and its formation was dependent on the presence of NADPH, functional microsomes, and protein and cal C concentrations. Inhibitor and purified enzyme studies showed that cytochrome P450 reductase is responsible for the cal C free radical observed, which is probably a perylenesemiquinone. PMID- 8615896 TI - Dual effects of SK&F 96365 in human leukemic HL-60 cells. Inhibition of calcium entry and activation of a novel cation influx pathway. AB - The effects of the receptor-mediated Ca2+ entry blocker, SK&F 96365 on thapsigargin (TSG)-induced Ca2+ entry in fura-2-loaded HL-60 cells were studied. After Ca2+ release induced by 30 nM TSG, readmission of Ca2+ resulted in a sustained Ca2+ entry, which could be partially inhibited by 1-3 microM SK&F 96365. Surprisingly, SK&F 96365 at 30-100 microM, instead of causing a stronger inhibition, actually promoted Ca2+ entry. Furthermore, at 16-100 microM, this drug released intracellular Ca2+ on its own and induced Ca2+ entry upon readmission of Ca2+. This SK&F 96365-activated Ca2+ entry pathway was insensitive to nifedipine and, interestingly, accessible to Ni2+ and La3+. However, SK&F 96365 (30 microM) almost completely blocked (basal) Mn2+ entry and only caused 4.4% of the cells to be stained with trypan blue, strongly suggesting that the SK&F 96365-activated cation entry was not due to damage nor to a very nonselective permeabilization of the plasma membrane. These data indicate that low concentrations of SK&F 96365 inhibited Ca2+ entry and higher concentrations activated a novel cation entry pathway. Because these 2 opposing effects overlapped at an intermediate concentration (16 microM), which is within the range commonly used to block Ca2+ entry, cautious use of this Ca2+ antagonist appears to be warranted. PMID- 8615897 TI - Determination of rat brain and plasma levels of the orally active GABAB antagonist 3-amino-propyl-n-butyl-phosphinic acid (CGP 36742) by a new GC/MS method. AB - An involvement of GABAergic neurons has been suggested in the process of memory consolidation based on anatomical evidence and increasing physiological and biochemical data. With the advent of orally active GABAB antagonists, such as CGP 36742, the question of their therapeutic value, for example in Alzheimer's disease, becomes relevant. Therefore, a new GC/MS method was developed to determine the concentration of CGP 36742 (3-amino-propyl-n-butyl phosphinic acid) in various intra- and extracerebral tissues after different routes of application. The compound was chemically derivatised in a two-step process (acylation of the amino group and esterification of the phosphinic acid). The limit of detection of the method was 0.01 microgram/g tissue and 0.0005 microgram/mL plasma. The time-course after i.p. treatment showed peak levels of CGP 36742 between 30 min and 1 hr after injection. After a dose of 100 mg/kg, the concentration in the brain ranged from 1 to 1.4 microgram/g or 6 to 8 microM, assuming that 1 mg tissue equals 1 microL (i.e., below the IC50 of the interaction with GABAB receptors as measured by [3-3H]-aminopropyl-phosphinic acid binding [35 microM]). These results are discussed in light of the psychopharmacological effects (improvement of cognitive performance of rats) of CGP 36742 observed at very low oral doses. PMID- 8615898 TI - Correlation of retention of tumor methylmercaptopurine riboside-5'-phosphate with effectiveness in CD8F1 murine mammary tumor regression. AB - Treatment with a combination (PMA) of (N-phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) induced partial regressions of CD8F1 murine mammary tumors and provided for tumor growth inhibition without regression of Colon 38 tumors. HPLC-nucleotide pool analysis of CD8 mammary tumors obtained at various times after treatment with PMA revealed that MMPR-5'-phosphate, which inhibits de novo purine nucleotide biosynthesis, was constant at levels of approximately 2.5 nmol/mg protein for 72 hr after treatment. In contrast, the MMPR-5'-phosphate levels of C38 tumors decreased from 24-hr levels at 1.5 nmol/mg protein with a half-time of about 24 hr. Treatment of CD8 tumor-bearing mice with iodotubercidin, a potent inhibitor of adenosine/MMPR kinase, at various times after PMA, reversed both the accumulation of high levels of MMPR-5'-phosphate and the number of partial tumor regressions. These data demonstrate that a cycle of MMPR rephosphorylation is active in the CD8 mammary tumor and suggest that this recycling of MMPR is important for the optimal effect of PMA treatment. PMID- 8615899 TI - Correlation of the response to cisplatin of human ovarian cancer cell lines, originating from one tumor but with different sensitivity, with the recovery of DNA adducts. AB - Cis-Diamminedichloroplatinum(II) (CDDP) is used in the treatment of various cancers, with or without ionizing radiation. During treatment, resistance may develop, and cross-resistance can also occur. DNA is the main target for CDDP and ionizing radiation, and we therefore evaluated the correlation between the amount of CDDP-DNA adducts and the cytotoxic activity of CDDP in human ovarian cancer cell lines with different platinum sensitivities. DNA-adduct levels were investigated 18 hr after CDDP exposure in three cell lines originating from the same human ovarian cancer. The least sensitive cells appeared to have the largest amounts of CDDP-DNA adducts, while the most sensitive had higher adduct levels than the parental cells. The proportion of the four adducts measured (i.e., Pt-G, Pt-AG, Pt-GG, and G-Pt-G) was comparable in all cell lines, with a preference for Pt-GG adduct formation (> 50% of the adducts). Intracellular CDDP concentrations were higher in sensitive than in resistant cells, in contrast to the degree of CDDP adduct formation. Data obtained following continuous exposure of CDDP resistant cells to CDDP suggest that DNA repair is partly responsible for resistance to CDDP. We conclude that the amount of CDDP-DNA adduct formation in cancer cells is not a predictor of CDDP cytotoxicity. PMID- 8615900 TI - A checkerboard method to evaluate interactions between drugs. AB - A method to evaluate interactions between biologically active agents is presented. Synergism, zero interaction, and antagonism were easily detected with the three-dimensional approach proposed herein. This method is compatible with a checkerboard design to diagnose the interaction between agents and obviate the need to test their mixtures in a fixed concentration ratio as proposed by Chou and Talalay. Dose-response curves for individual agents were obtained, and experimental data fitted to appropriate equations by nonlinear regression. If zero interaction was present, the predicted effect could be calculated for each combination using the classical isobole equation with any spreadsheet having a command to solve mathematical equations by iteration. This allowed the selection of appropriate concentrations for the combination of two or more agents. Interaction between agents could be assessed in two ways: by comparing experimental with expected effects, if zero interaction is present; or by analyzing the reduction or increase in total dose found as a consequence of the interaction. The applicability of both approaches is discussed and, for purposes of comparison with other methods, examples based on published data are analyzed and commented upon. PMID- 8615901 TI - Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. AB - The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT 29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line. PMID- 8615902 TI - Differential membrane interactions of calcium channel blockers. Implications for antioxidant activity. AB - Lipid peroxidation causes cellular damage during aging and various diseases, including atherosclerosis. Chronic administration of highly lipophilic calcium channel blockers (CCB) may reduce lipid peroxidation as a result of concentration in cell membranes and altering physico-chemical properties of the lipid bilayer. In the study, small angle X-ray scattering was used to examine reconstituted cardiac membrane lipid bilayers in the presence of CCB with various antioxidant activities, including nisoldipine, nifedipine, and diltiazem. Analysis of one dimensional electron density profiles demonstrated that these compounds have different molecular distributions relative to the center of the membrane: diltiazem (+/- 14-22 A), nifedipine (+/- 12-22 A), and nisoldipine (+/- 7-22 A). The overall hydrocarbon core width for control samples was 44 A and was unaffected by the addition of drugs at these concentrations (< 1% by mass). High resolution differential scanning calorimetry indicated that CCB markedly perturbed the thermotropic properties of liposomes, including thermal phase transition temperature and enthalpy, relative to control samples. The effects of these compounds on membrane thermotropic properties correlate with their reported antioxidant activities. These data support the hypothesis that calcium channel blockers have potent physico-chemical interactions with the membrane lipid bilayer, which may underlie their antioxidant activity. PMID- 8615903 TI - Potentiation of mitogen-activated protein kinase by ethanol in embryonic liver cells. AB - Ethanol modulates agonist responses in liver cells, which are the major site of ethanol metabolism. Mitogen-activated protein kinases (MAPKs) are involved in the integration of multiple signaling pathways leading to cellular responses. However, the effect of ethanol on liver MAPK is not known. To this end, we studied the activation of MAPK in a normal mouse embryonic liver cell line (BNLCL2) after acute and chronic exposure to ethanol. Acute exposure to ethanol (0-400 mM) for 1 hr had no effect on either basal or serum- and phorbol-12 myristate-13-acetate (PMA)-stimulated MAPK activity. Chronic exposure to ethanol (0-400 mM) for 24 hr potentiated the stimulation of MAPK by serum, PMA, or thrombin. Maximum potentiation was observed with 200 mM ethanol (2- to 3-fold higher than control cells). Chronic exposure had no significant effect on epidermal growth factor-stimulated MAPK activity. In-gel MAPK assay of cytosolic extracts and of immunoprecipitates obtained with MAPK antibody demonstrated that ethanol potentiated the activation of both p42 and p44 MAPKs. When cells were pretreated with pertussis toxin, the potentiation by ethanol was abolished. It is concluded that ethanol potentiates MAPK in fetal liver cells by a pertussis toxin sensitive G-protein-dependent mechanism. PMID- 8615904 TI - Anandamide- and delta9-tetrahydrocannabinol-evoked arachidonic acid mobilization and blockade by SR141716A [N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4 dichlorophenyl)-4 -methyl-1H-pyrazole-3-carboximide hydrochloride]. AB - The purpose of this study was to investigate whether anandamide induces cannabimimetic responses, mainly mobilization of arachidonic acid, in primary cultures of rat brain cortical astrocytes. Confluent monolayer cultures of astrocytes, prelabeled with [3H]arachidonic acid, were incubated with anandamide or delta9-tetrahydrocannabinol (delta9-THC) in the presence or absence of thimerosal, a fatty acid acyl CoA transferase inhibitor and phenylmethylsulfonyl fluoride, an amidohydrolase inhibitor. Anandamide and delta9-THC induced a time- and concentration-dependent release of arachidonic acid in the presence, but not in the absence, of thimerosal. Anandamide- and delta9-THC-stimulated arachidonic acid release was pertussis toxin-sensitive, indicating a receptor/G-protein involvement. A novel and selective cannabinoid receptor antagonist, SR141716A [N (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole 3-carboximide hydrochloride], blocked the arachidonic acid release, suggesting a cannabinoid receptor-mediated pathway. In astrocytes, the magnitude of anandamide induced arachidonic acid release was equal to that released by equimolar concentrations of delta9-THC. Furthermore, direct assay of amidohydrolase activity indicated that degradation of anandamide into arachidonic acid and ethanolamine was negligible in cortical astrocytes. Our results suggest that anandamide stimulates receptor-mediated release of arachidonic acid, and the receptor may be the cannabinoid receptor. Astrocytes, containing a cannabinoid receptor and lower or negligible amidohydrolase activity, may be an important brain cell model in which to study the cannabimimetic effects of anandamide at a cellular and molecular level. PMID- 8615905 TI - Regulation of mouse liver microsomal esterases by clofibrate and sexual hormones. AB - Carboxylesterase activity was measured using six different substrates in microsomal preparations from female and ovariectomized female mice in order to evaluate the effects of female sex hormones on esterase expression. With three of the substrates (alpha-naphthyl acetate and esters 2 and 3), esterase activity was the same in both groups; however, with the others (rho-nitrophenyl acetate and esters 1 and 4), there was a small increase in activity in ovariectomized females, compared with intact females. Castration of males followed by treatment with testosterone caused only transient increases in activity for four of the substrates (alpha-naphthyl acetate and esters 1, 2, and 3) and no change in activity for the other two (rho-nitrophenyl acetate and ester 4). Treatment of male and female mice with the peroxisome proliferator clofibrate, with or without testosterone, resulted in increased hydrolysis of alpha-naphthyl acetate and rho nitrophenyl acetate, but little change for the other substrates. Clofibrate also induced alpha-naphthyl acetate and rho-nitrophenyl acetate hydrolysis in castrated males, but clofibrate and testosterone administrated together resulted in significant increases of activity with all substrates, which were greater than the additive effects of the two compounds administered separately. These results indicate that clofibrate causes significant alterations in the regulation of esterase activity, whereas sex hormones only cause small changes. However, it would seem that testosterone can synergize the effect of clofibrate in castrated males, resulting in higher levels of activity than with clofibrate alone. Finally, an overall increase in esterase activity might be due to a large increase in the activity of a few esterases or to a small increase in many esterases. Enzyme staining of native polyacrylamide gels reveals that the latter is true, with the majority of esterases present in mouse liver microsomes being induced to a small degree by clofibrate. PMID- 8615906 TI - Scavenging effects of phenylpropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method(95)02255-4. AB - The scavenging activities of six phenylpropanoid glycosides, i.e. leucosceptoside A and martynoside, isolated from Pedicularis alashanica Maxim, verbascoside, and pediculariosides A, M and N isolated from Pedicularis striata Pall ssp. arachnoidea Franch Tsoong, on superoxide anion and hydroxyl radicals have been studied by the spin trapping method. The results demonstrated that the number of phenolic hydroxyl groups in the structures of the phenylpropanoid glycosides is related to their scavenging activities. The scavenging effects of the phenylpropanoid glycosides possessing two o-dihydroxyl groups were stronger than the effects of the compounds possessing two o-hydroxy-methoxy groups. PMID- 8615907 TI - Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo. AB - The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development. PMID- 8615909 TI - Expression of Ca2+/calmodulin-dependent protein kinase types II and IV, and reduced DNA synthesis due to the Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl piperazine) in small cell lung carcinoma. AB - Because changes in intracellular Ca2+ affect progression through the mitotic cell cycle, we investigated the role of Ca2+-binding proteins in regulating cell cycle progression. Evidence was found demonstrating that the activation of Ca2+/calmodulin-dependent protein kinase (CaM kinase) inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells. We also demonstrated that SCLC cells express both CaM kinase type II (CaMKII) and CaM kinase type IV (CaMKIV). Five independent SCLC cell lines expressed proteins reactive with antibody to the CaMKII beta subunit, but none expressed detectable proteins reactive with antibody to the CaMKII alpha subunit. All SCLC cell lines tested expressed both the alpha and beta isoforms of CaMKIV. Immunoprecipitation of CaMKII from SCLC cells yielded multiple proteins that autophosphorylated in the presence of Ca2+ / calmodulin. Autophosphorylation was inhibited by the CaMKII(281-302) peptide, which corresponds to the CaMKII autoinhibitory domain, and by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), a specific CaM kinase antagonist. Influx of Ca2+ through voltage-gated Ca2+ channels stimulated phosphorylation of CaMKII in SCLC cells, and this was inhibited by KN-62. Incubation of SCLC cells of KN-62 potently inhibited DNA synthesis, and slowed progression through S phase. Similar anti-proliferative effects of KN-62 occurred in SK-N-SH human neuroblastoma cells, which express both CaMKII and CaMKIV, and in K562 human chronic myelogenous leukemia cells, which express CaMKII but not CaMKIV. The expression of both CaMKII and CaMKIV by SCLC cells, and the sensitivity of these cells to the anti-proliferative effects of KN-62, suggest a role for CaM kinase in regulating SCLC proliferation. PMID- 8615908 TI - Effects of phenobarbital and interleukin-6 on cytochrome P4502B1 and 2B2 in cultured rat hepatocytes. AB - The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL 6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process. PMID- 8615911 TI - Models for developing integrated curricula. PMID- 8615910 TI - Effect of DNA conformation on cisplatin adduct formation. AB - The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation. PMID- 8615912 TI - Managed care training for internal medicine residents. PMID- 8615913 TI - Incentives for teaching. PMID- 8615914 TI - The Nutrition in Medicine project. PMID- 8615915 TI - Housestaff attitudes about work rounds. PMID- 8615916 TI - Electronic bulletin board for faculty development. PMID- 8615917 TI - Students' attitudes toward alternative health care. PMID- 8615919 TI - Crossing the cultural doctor-patient barrier. PMID- 8615918 TI - Psychiatrists' attitudes toward psychiatry. PMID- 8615920 TI - New opportunities to improve physicians' lifelong learning. PMID- 8615921 TI - Preventing academic medical center from becoming an oxymoron. AB - Many academic medical institutions are facing serious threats to their survival today as changes in the organization and financing of health care delivery and reductions in federal support create damaging pressures. In order for Americans to continue to have the best health care in the world, academic medicine and its crucial contributions of medical education, training, and research must receive adequate support. The author maintains that it is up to the Association of American Medical Colleges and associated organizations to ensure that this message is heard and recognized and to seek an all-payer approach that would support the costs associated with medical education and training and would allow teaching hospitals to compete on a level playing field with non-teaching hospitals. Academic medical institutions must face painful transformations while maintaining their academic missions; the author discusses the nature of these transformations and missions, particularly research, and outlines useful strategies to maintain these missions, such as establishing a centralized approach to curriculum and having clinical and basic science faculty form alliances to address common research problems and secure more research funding. He concludes that many of the structural and other changes that academic medicine's institutions must make may result in true improvements and help maintain the validity of the term academic medical center in the future. PMID- 8615922 TI - Learning to care, for a healthier tomorrow. AB - Despite the fundamental transformations occurring in health care, academic medicine can maintain, and actually enhance, its value to society. But to do so will require a shared vision of where academic medicine should go. The author offers his own vision, one that includes (1) establishing true partnerships between medical schools and the communities they serve; (2) elevating prevention and health maintenance to equal status with diagnosis and treatment; (3) addressing pressing social problems, such as drug abuse, teenage pregnancy, and domestic violence; (4) assuming a leadership role in containing health care costs; (5) harnessing information technology to develop, disseminate, and implement comprehensive clinical information systems, and to provide the public with reliable medical information; (6) fostering racial, ethnic, and gender parity in medicine; (7) carrying out appropriate downsizing and restructuring of the academic medicine enterprise; (8) addressing forcefully the workforce needs for physicians and medical scientists; (9) improving the education of medical students and residents and fostering the conduct of clinical research by creative use of the integrated academic health care systems that teaching hospitals are becoming; (10) developing regional educational consortia to guide important aspects of medical students' and residents' education; (11) building a learning network so that meaningful cross-talk and dissemination of lessons learned can occur among all segments of the academic medicine community; and (12) creating a permanent planning process to guide academic medicine's future. The author concludes that if academic medicine adheres to its tradition of public service and professionalism, its future can be as glorious as its past. PMID- 8615923 TI - Research collaboration between an HMO and an academic medical center: lessons learned. AB - Joint research ventures between health maintenance organizations (HMOs) and academic medical centers (AMCs) can strengthen both organizations from the research and patient care perspectives, by facilitating the exploration of views and cost-effective approaches to diagnoses and treatment. This article describes a research venture between the Fallon Healthcare System, a health maintenance organization (HMO), and the University of Massachusetts Medical Center, an academic medical center (AMC). This research venture, the Worcester Area Trial for Counseling Hyperlipidemia, was a four-year study in which 1,277 participants were recruited from 45 Fallon Clinic physician panels. The study targeted patients with blood cholesterol levels in the upper 25% of the cholesterol distribution. The different priorities and perspectives of the two institutions with regard to health care and research became more apparent as the study progressed. Eventually study personnel needed to re-examine the study objectives and each other's perspectives to accommodate these differences. This learning process showed that research ventures between HMOs and AMCs can prove mutually beneficial. An HMO can gain experience with large government-funded projects. AMCs can study managed care within a controlled setting. However, the differing priorities and perspectives of these institutions' organizational structures require that considerable attention be paid to their representatives' working relationships and perceptions. PMID- 8615924 TI - A strategy for coping with change: an affiliation between a medical school and a managed care health system. AB - Case Western Reserve University School of Medicine (CWRU), a private research focused medical school, and Henry Ford Health System (HFHS), an integrated health system with a preponderance of managed care, have established a formal, broad affiliation that includes substantial commitments that bind the two organizations. Among them are formal full-time faculty appointments at CWRU for qualified professional staff of HFHS, designation of an associate dean for CWRU at HFHS, election of HFHS faculty to key medical school committees such as admission, curriculum, and promotions and tenure, and the commitment of funds to the affiliation by both organizations: a grant from HFHS to CWRU for curriculum development, and investment from CWRU to HFHS. The alliance of two such organizations is made complex by a number of issues. They include differences of institutional cultures as well as traditional issues in academic health centers such as departmental authority over curriculum and faculty appointments, competition for academic preeminence, and competition among hospitals for patients. The affiliation was facilitated by shared commitments to education, agreement on the need to adapt student education to the emerging managed care environment, a shared commitment to health services research, investment in the concept that learners add value to a health care delivery setting, and the desire to develop graduates with knowledge of practice in managed care. The authors conclude that medical schools and integrated managed care health systems gain sufficiently from such an affiliation that the investment of time, effort, and resources is readily justified. PMID- 8615925 TI - A model for distributing teaching funds to faculty. AB - Teaching at medical schools has been supported principally through research and clinical income, but these resources have become more scarce, faculty have begun to look elsewhere for support of student teaching. Yale University School of Medicine has developed a model for distributing a portion of general revenue funds to departments based entirely on their faculty members' commitment to the teaching of undergraduate medical students. This model combines both a qualitative measure (effectiveness) and a quantitative measure (effort) to assess the commitments of faculty to teaching. Initial response has been primarily positive. Both faculty members and chairs have acknowledged that faculty should receive financial support for major teaching responsibilities. Although the amount of funding may not be proportional to the actual effort of faculty members, by apportioning tuition dollars for teaching Yale hopes to send a powerful message about its commitment to its teaching faculty and to its medical students. PMID- 8615926 TI - Medical education in the former Soviet Union. AB - After World War I, medical education in the Soviet Union and medical education in the United States headed in strikingly divergent directions. In keeping with the recommendations of the Flexner report, medical education in the United States became a university-based academic discipline based in the natural sciences. In contrast, the Soviet Union created a series of free-standing medical institutes whose admission, curricular, and pedagogic policies were centrally controlled in strict conformity with political doctrine. Notable features of the Soviet system were narrowly defined professional education; early specialization, beginning in the first year of medical school; and emphasis on empirical clinical training at the expense of scientifically based education. Despite the historical differences between Soviet and American medical education, there are several issues that face present-day medical educators in both the United States and the Soviet successor states. These include an overabundance of specialists, the need to provide equitable professional opportunities for physicians of both sexes, and the need to provide access to medical education for qualified candidates from underrepresented social or ethnic groups or from geographically remote regions. PMID- 8615928 TI - Abraham Flexner Award for Distinguished Service to Medical Education. PMID- 8615927 TI - Academic pediatrics: the view of New York City a century ago. AB - Pediatrics arose as an academic and medical specialty in the United States during the late nineteenth century. This paper documents the first three stages of academic pediatrics in the United States between 1850 and 1950: (1) 1850-1880, a period focused on sanitary reform as a means of reducing infant mortality; (2) 1880-1900, the era where discoveries being made in bacteriology, physiology, and nutrition began to be applied to improving the health of children; and (3) 1900 1930, when the field was characterized by an active health reform movement directed at parents and health care professionals. Three prominent pioneer pediatricians, Drs. Abraham Jacobi, Henry Koplik, and L. Emmett Holt, are profiled as representative practitioners of these eras. PMID- 8615929 TI - David E. Rogers Award. PMID- 8615930 TI - Baxter Award for Distinguished Research in the Biomedical Sciences. PMID- 8615931 TI - AOA Distinguished Teacher Award for Senior Faculty in the Basic Sciences. PMID- 8615932 TI - AOA Distinguished Teacher Award for Senior Faulty in the Clinical Sciences. PMID- 8615933 TI - The mini-med school: bridging the gap. PMID- 8615935 TI - Who should rate candidates in an objective structured clinical examination? AB - PURPOSE: To determine who is the better rater of history taking in an objective structured clinical examination (OSCE): a physician or a standardized patient (SP). METHOD: During the 1991 pilot administration of an OSCE for the Medical Council of Canada's qualifying examination, five history-taking stations were videotaped. Candidates at these stations were scored by three raters: a physician (MD), an SP observer (SPO), and an SP rating from recall (SPR). To determine the validity of each rater's scores, these scores were compared with a "gold standard", which was the average of videotape ratings by three physicians, each scoring independently. Analysis included both correlations with the standard and a repeated-measures analysis of variance (ANOVA) comparing raters' mean scores on each station with mean scores of the gold standard. RESULTS: Ninety-one videotapes were scored by the "gold-standard" physicians. Correlations with the standard showed no clear preference for MD, SPO, or SPR raters. ANOVAs revealed significant differences from the standard on three stations for the SPR, two stations for the SPO, and one stations for the MD. CONCLUSIONS: An MD rater is less likely to differ from a standard established by a consensus of MD ratings than are SP raters rating from recall. If an MD cannot be used, an SP observer is preferable to an SP rating from recall. PMID- 8615936 TI - Prediction of students' USMLE step 2 performances based on premedical credentials related to verbal skills. AB - PURPOSE: To examine the relationship between the objective premedical credentials and performances on Step 2 on the United States Medical Licensing Examination (USMLE) of 480 students in three classes at the Virginia Commonwealth University Medical College of Virginia School of Medicine. The purpose of the study was to seek those selection criteria that might best predict performance on an examination designed to assess problem-solving skills, the essence of clinical medicine. METHOD: Premedical data from two classes (1193, 1994) were analyzed, and a regression equation was used to calculate theoretical USMLE Step 2 scores for the students in the class of 1995, who had not yet taken this examination. The premedical variables were scores on the verbal and math section on the Scholastic Aptitude Test (SAT), scores on the six sections of the pre-1991 Medical College Admission Test (MCAT), grade-point average (GPA) in science courses required of premedical students, and undergraduate major. Once the class of 1995 had taken the USMLE Step 2, the equation was cross validated, and the theoretical and actual scores of the class of 1995 were correlated. RESULTS: The correlation between theoretical and actual scores was r = .443. In the analysis for the classes of 1993 and 1994, the single variables most highly predictive of USMLE Step 2 performance were scores on the verbal section of the SAT (r = .317) and the Skills Analysis: Reading section of the MCAT (r = .331). However, the MCAT scores were excluded from the final regression analysis because of the pre 1991 MCAT cannot be useful in predicting the performances of present medical school applicants. The resulting regression equation (using the SAT verbal section and premedical GPA) was able to account for 21.2% of the variance for the class of 1995. CONCLUSION: The use of the verbal section of the SAT as a predictive factor is unique. It is significant that this variable was strongly related to premedical GPA, suggesting that high verbal aptitude serves one well, even when coping with complex scientific concepts. PMID- 8615934 TI - What does not explain the variation in the direct costs of graduate medical education. AB - BACKGROUND: There is considerable variation in the costs of training residents across hospitals. Previous studies have reported training costs that ranged for $7,500 to $200,000 per resident, with means in the $50,000 to $60,000 range. This paper examines the factors associated with the variation in the direct costs of residency education across hospitals. METHODS: Hospital costs, hospital payment rates, various cost-of-living indices, and hospital characteristics for all hospitals in the United States receiving Medicare funds for residency education in fiscal year 1991 were obtained from various public sources. Bivariate and multivariate analyses were performed to determine whether organizational structure of residency training, specialty mix of residents, quality of training, cost of living in the geographic area, patient mix of the hospital, or other factors could explain some or all of the cost variation. RESULTS: Only a small proportion of the variation in the costs of training residents or payments for residency education could be explained by the factors analyzed. CONCLUSION: Much of the current variation in residency costs and payments is attributable to accounting and not structural or locational factors. Institutions looking for ways to become more efficient could compare costs of training across institutions without sophisticated adjusters. Federal policymakers should consider a national payment rate per resident to replace the current system based on hospital specific costs. This proposal would redistribute dollars across residency programs. The proposal would affect payments for direct medical education in the Medicare program and could form the basis for payment for residency education by all payers. PMID- 8615937 TI - Psychometric test results associated with high achievement in basic science components of a medical curriculum. AB - PURPOSE: To analyze the correlation between scores obtained on the 25 scales and vectors of the California Psychological Inventory (CPI) and academic test results obtained in various subjects by the same students in the first three years of the six-year medical curriculum at Monash University. METHOD: The study participants were 133 students who commenced their medical education in 1990 or 1991 and completed their third year in 1993. The student took the CPI in their first year. The academic test results were classified according to assessment type and content. Pearson product-moment correlation coefficients were then calculated between the CPI scores and the test results. RESULTS: With respect to assessment type, the results for multiple-choice question test (MCQs), practicals, calculations, and to a lesser extent, essays were negatively correlated with some CPI scores that might be notionally desirable in a caring profession. Essay and oral examination results were positively correlated with scales such as Socialization, Self-control, and Work Orientation. Results of oral examinations, unlike those of other types assessments, were positively correlated with the CPI scales of Intellectual Efficiency and Management Potential. Three of the assessment types--MCQs, practicals, and calculations--were highly significantly negatively associated with the Empathy scale. With respect to assessment content, results for cell and tissue studies were negatively correlated with several CPI scales, including Empathy. By complete contrast, the scores from clinical and communication skills assessments were positively correlated with many notionally desirable CPI scales, including Empathy, Responsibility, and Tolerance. CONCLUSION: The results indicate that students with high achievement in many components of the curriculum tend to have personality profiles that seem inappropriate to their chosen careers as physicians. Medical schools espousing humanistic qualities in their educational objectives may be offering a contradictory message to their students by rewarding those with inappropriate personal qualities. PMID- 8615938 TI - Ratings of students' performances in a third-year internal medicine clerkship: a comparison between problem-based and lecture-based curricula. AB - PURPOSE: To compare clinical performances in a third-year medicine clerkship between studies from a problem-based learning (PBL) curriculum and students from a traditional, lecture-based learning (LBL) curriculum. METHOD: The study participants were 88 PBL students and 364 LBL students rotating through a common third-year internal medicine clerkship at the Bowman Gray School of Medicine at Wake Forest University, classes of 1991-1995. Faculty and housestaff assessed the students' performances using four clinical rating scales. The student also completed the medicine student ("shelf") test of the National Board of Medical Examiners (NBME). RESULTS: On average, the PBL students received significantly higher ratings from housestaff and faculty on all four rating scales. No difference in scores on the NBME medicine shelf test was observed. CONCLUSION: The results support the hypothesis that preclinical PBL curricula as found at the Bowman Gray School of Medicine may enhance third-year students' clinical performances. PMID- 8615939 TI - Ventures in education: a pipeline to medical education for minority and economically disadvantaged students. AB - BACKGROUND: Ventures in Education is an independent, nonprofit educational organization established by the Josiah H. Macy, Jr. Foundation to improve the academic achievement of minority and economically disadvantaged students, particularly in science and mathematics. One specific objective has been to increase the number of students who enter schools of the health professions, in particular schools of medicine, which was the focus of this study. METHOD: A search was conducted of the Association of American Medical Colleges' (AAMC's) Student and Applicant Information Management System database, to determine whether any of the 981 graduates in the first five Venture classes (1985 to 1989) of the original five New York City high schools in the program had pursued medical education. RESULTS: The search located 160 of the 981 Ventures graduates, and, of those, 136 had taken the Medical College Admission Test (13.9%), 109 (11.1%) has applied to medical school, 75 (7.6%) had been accepted, and 72 (7.3%) had matriculated into medical school. All of these percentages were significantly higher than the corresponding percentages for the general population. CONCLUSION: The findings have important implications for the AAMC's Project 3000 by 2000, showing that a rigorous academic curriculum with resources for individualized attention can facilitate the entry of minority and economically disadvantage students into medical education, with at least 7.3% of the Ventures graduates entering medical school and nearly 70% of those applying subsequently being accepted. PMID- 8615941 TI - Clinical Aspects of De/Remineralization of Teeth. Proceedings of Models Conference 1994. Rochester, New York, June 11-14, 1994. PMID- 8615940 TI - Determinants of the generalist career intentions of 1995 graduating medical students. AB - Using national databases of the Association of American Medical College, the authors employed logistic regression analysis to show the relative predictive influences of selected demographic, structural, attitudinal, and educational variables on the specialty careers choices of 1995 U.S. medical school graduates. Plans to pursue certification in family practice or an unspecified generalist career could be predicted with moderate success, while choices of general internal medicine and general pediatrics could not. The intentions of the 1995 graduates to pursue generalist specialty, were significantly associated with demographic factors such as female gender, older student age, and rural hometown; early interest in the generalist specialties; attitudes favoring helping people over seeking opportunities for leadership, intellectual challenge, or research; the presence of a department of family medicine in the medical school; and ambulatory care experiences in the third and fourth years. In the multiple regression models used in this study, a number of factors widely touted as important to the cultivation of generalism were not significant predictors of generalist decisions; an institutional mission statement expressly addressing the cultivation of generalist careers; giving admission preferences to applicants who vowed an interest in generalism; public (versus private) school sponsorship; discrete organization units for general internal medical or general pediatrics; the proportion of institutional faculty in the general specialty of medicine and pediatrics; the level of educational debt; the students; clinical experiences in the first and second years of medical school. The authors acknowledge the danger of inferring causal relationships from analyses of this kind, and described how the power of previous associations--e.g., that between a required third-year clerkship in family medicine and graduates' family practice career choices--may be weakened when the independent variable spreads across institutional cultures that at present are less conductive to primary care. The findings of this analysis add to the evidence that generalist career intentions are largely carried on the tide of students' interests and experiences in family medicine and ambulatory primary care. In terms of the predictive values of the input variable in this study, career decisions for the other two generalist specialties--general internal medicine and general pediatrics--were essentially a crapshoot, either because the tactics to promote interest in these fields were ineffective (or confounded), or because the efforts were underdeveloped. Moreover, the statistical models of this study employed quantifiable variables that can be discerned and manipulated to guide the result, whereas medical students tend to identify less tangible elements as more powerful factors influencing their career choices. The results sharpen the strategic focus, but must be combined with those of other, descriptive analysis for a more complete understanding of graduating students' career decisions. PMID- 8615942 TI - The application of in vitro models to research on demineralization and remineralization of the teeth. AB - Progress in in vivo and in situ experimentation has led many researchers to speculate as to the relevance and importance of in vitro testing protocols in caries research. A Medline/Biosis search for the present review revealed well over 300 citations (since 1989) documenting in vitro tests associated with caries research on mineralization and fluoride reactivity. The present survey documents these recent applications of in vitro test methods in both mechanistic and 'profile' caries research. In mechanistic studies, in vitro protocols over the past five years have made possible detailed studies of dynamics occurring in mineral loss and gain from dental tissues and the reaction dynamics associated with fluoride anticaries activity. Similarly, in profile applications, in vitro protocols make possible the inexpensive and rapid--yet sensitive--assessment of F anticaries efficacy within fluoride-active systems, and these tests represent a key component of product activity confirmation. The ability to carry out single variable experiments under highly controlled conditions remains a key advantage in in vitro experimentation, and will likely drive even further utilization, as advances continue in physical-chemical and analytical techniques for substrate analysis in these protocols. Despite their advantages in vitro testing protocols have significant limitations, most particularly related to their inability to simulate the complex biological processes involved in caries. PMID- 8615943 TI - Animal caries models for evaluating fluoride dentifrices. AB - To determine the ability of animal caries models to evaluate the cariostatic potential of fluoride dentifrices, we examined four different coronal caries models. These currently used rat caries models were: Francis' hypomineralized area model, Gaffar's CARA rat model, the Connecticut model, and the Indiana model. Available data indicated that all of these models were capable of detecting significant cariostatic benefits from clinically proven fluoride dentifrices. For the most part, these animal models were also able to demonstrate a response to increasing concentrations of fluoride, particularly if the fluoride was provided in an ionic rather than a complexed form. Data were also presented which indicated the potential for animal caries models to be used to predict the clinical benefits of non-fluoride systems as well as the utility of fluoride systems for the prevention of root-surface caries. It is concluded that animal caries models play a significant role in the development and evaluation of fluoride and other systems for the clinical prevention of dental caries. PMID- 8615944 TI - In situ caries models. AB - By using in situ models, we have the potential to study both fundamental aspects of the caries process as well as more applied research problems such as the effect of food on dental caries and the role of fluoride in caries prevention in human subjects without actually causing caries in the natural dentition. The key experimental parameters that need to be considered in the development of an in situ model are the characteristics of the subject panel, the physical design of the model, the type of hard tissue substrate and the method of assessing mineral status, and the study design and clinical protocol. Each parameter must be carefully considered in relation to the objectives of the research, study design requirements, ethical considerations, impact on clinical relevance, and impact on the control of variation. The major source of variation associated with in situ models should be of biological and not experimental origin. The design and conduct of proper in situ model studies require a clear understanding of the caries process, sound analytical support, and a knowledge of how to work with research subjects to achieve a high level of compliance. Given the complex nature of caries, a combination of hard tissue substrates--including sound, surface softened lesions and subsurface lesions--may be necessary to model all aspects of caries progression and prevention successfully. Internal validation of in situ models using fluoride dose-response controls is considered to be necessary for studies evaluating the efficacy of new fluoride dentifrice formulations. PMID- 8615945 TI - Role of saliva in caries models. AB - The crucial role played by the actions of saliva in controlling the equilibrium between de- and remineralization in a cariogenic environment is demonstrated by the effects on caries incidence of salivary dysfunction and by the distribution of sites of caries predilection to those were salivary effects are restricted. However, of the several properties of saliva which may confer protective effects, it is not certain which are most important. A distinction can be made between static protective effects, which act continuously, and dynamic effects, which act during the time-course of the Stephan curve. Evidence implicates salivary buffering and sugar clearance as important dynamic effects of saliva to prevent demineralization; of these, the buffering of plaque acids may predominate. Enhanced remineralization of white spot lesions may also be regarded as dynamic protective effects of saliva. Fluoride in saliva (from dentifrices, ingesta, etc.) may promote remineralization and (especially fluoride in plaque) inhibit demineralization. The design of experiments using caries models must take into account the static and dynamic effects of saliva. Some models admit a full expression of these effects, while others may exclude them, restricting the range of investigations possible. The possibility is raised that protective effects of saliva and therapeutic agents may act cooperatively. PMID- 8615946 TI - Salivary factors in caries models. AB - Consideration of salivary factors in caries models rarely extends beyond viewing saliva as a sink or diluent for plaque metabolic products, or as a source of buffering, for neutralizing plaque acids. In reality, saliva has a complex chemistry and a wide range of biochemical activities that may significantly affect plaque chemistry and microbiology. Thus, saliva is a major source of microbial nutrients, without which bacterial acid production is diminished. Buffering by salivary bicarbonate, and base production from urea and basic amino acids and peptides, significantly affect Stephan curves. Saliva is supersaturated with respect to basic calcium phosphate salts and contains novel inhibitors of calcium phosphate precipitation, while specific salivary proteins bind calcium. It seems important to consider if this system is reflected in plaque. Saliva, with contributions from serum and bacterial constituents, provides most of the precursors for the acquired enamel pellicle, which acts to slow demineralization during caries attack. Pellicle constituents appear to influence initial bacterial colonization of tooth surfaces and, therefore, may affect the microbial composition of plaque, but their detailed effects on plaque are poorly understood. Microbial adaptations to the anti-bacterial systems also seem important but are poorly investigated. Thus, saliva possesses an array of activities that have substantial actual or potential impact on plaque and, therefore, merit consideration for inclusion in systems intended to model dental caries. PMID- 8615947 TI - The role of microbiology in models of dental caries. AB - Models of dental caries (laboratory, animal, and human in situ models) vary markedly in their microbiological complexity. Laboratory models range from mono cultures of cariogenic species providing an acidic challenge to enamel, to the development of diverse mixed cultures growing on a habitat-simulating medium in an artificial mouth or chemostat. The latter systems are of value in determining either mechanisms of action or cause-and-effect relationships--e.g., between dietary components or antimicrobial agents and the microflora. Laboratory models have also shown that the sensitivity of oral bacteria to inhibitors is markedly reduced when growing in biofilms such as dental plaque. Animal models have proved unequivocally that caries is an infectious and transmissible disease. Their use has enabled comparisons to be made of (a) the cariogenic potential of different bacterial species, (b) the role of the diet, and (c) the effects of potential anti-caries agents. It has been claimed that no caries-protective agent currently in use has failed a rodent test. In situ human models have been designed to permit the development of "natural" plaque on standardized enamel surfaces freely exposed to the human oral environment. The microflora that develops on unadulterated surfaces is similar in composition to that found at comparable sites on vital teeth. Demineralization can be accelerated by the inoculation of additional cariogenic bacteria coupled with either intra- or extra-oral sucrose rinses. The increased realism associated with the transition from laboratory to human in situ models is countered by a reduced ability to control or manipulate the system for experimental purposes. Thus a hierarchy of tests is needed for the study of anti-caries agents, each requiring a varying degree of microbiological complexity. PMID- 8615948 TI - Statistical considerations related to the use of caries model systems for the determination of clinical effectiveness of therapeutic agents. AB - Caries model systems bear great promise for the eventual determination of clinical benefits of therapeutic agents without the time and expense involved in the conduct of a full-fledged clinical trial. However, before such models can routinely be applied for such purposes, there are several issues which must first be resolved, including the consideration of a number of statistical matters. These include the assessment of the validity and reliability of the models to be so used. A procedure presented by Proskin, Chilton, and Kingman provides a quantitative means by which a conclusion concerning clinical effectiveness can be derived on the basis of data obtained through the use of a model system given that certain fairly general underlying conditions prevail. The present paper provides some discussion and recommendations of the author related to statistical matters concerning the use of models, and discusses the derivation and logic of the Proskin, Chilton, and Kingman procedure. PMID- 8615949 TI - The application of in situ models for evaluation of new fluoride-containing systems. AB - Many in situ models have assessed the anticaries potential of fluoride-containing systems (Stookey et al., 1985; Mellberg et al., 1986, 1992a,b; Corpron et al., 1986; Featherstone and Zero, 1992; Ogaard and Rolla, 1992; Stephen et al., 1992). Several models have reportedly been validated according to guidelines proposed by Proskin et all. (1992). The proposed guidelines cover only dentifrices containing sodium fluoride (NaF) or sodium monofluorophosphate (SMFP) as active ingredients. These compounds are the most widely used sources of fluoride in dentifrices, and dose-response clinical standards are available for both. Other fluoride compounds, such as amine fluoride (AmF) and stannous fluoride (SnF2), have also been proven effective in reducing caries (Muehler et al., 1957, 1958; Marthaler, 1968; Lu et al., 1980; Cahen et al., 1982). Profile standards for these fluorides were not included in the proposed guidelines, primarily due to the lack of clinical data necessary to establish a dose response for these ingredients. Criteria for demonstrating the efficacy of these ingredients, along with methods to assess new fluoride compounds, need to be established. In situ models are used to evaluate the anticaries potential of new compounds added to mouthrinses, gums, slow-release devices, etc. (Creanor et al., 1992; Manning and Edgar, 1992; Lamb et al., 1993; Toumba and Curzon, 1993; Wang et al., 1993). Ingredients are often added to dentifrices previously proven effective against caries in order to provide additional benefits of gum health, tartar control, cleaning, etc. Proposals are made regarding the in situ testing of new dentifrices containing clinically proven fluoride compounds other than NaF and SMFP, as well as alternative delivery systems, in order to assist in their evaluation. PMID- 8615950 TI - Role of models in assessing new agents for caries prevention--non-fluoride systems. AB - While fluoride is an effective anti-caries agent, the search for more effective alternative therapies continues. A wide range of non-fluoride anti-caries agents has been postulated, and this paper reviews some of the pre-clinical models that have been utilized in their evaluation and some of the pitfalls that must be avoided. Using data on the potential anti-caries efficacy of phosphopeptides obtained from casein, the caution that must be applied in extrapolating laboratory data to predict clinical performance is discussed. Evaluation strategies that focus on only one potential mode of action (e.g., inhibition of demineralization) may overestimate the true clinical efficacy which may arise from a combination of two or more effects (e.g., inhibition of demineralization and stimulation of remineralization). Although laboratory and in situ data predict anti-caries efficacy for sodium trimetaphosphate in combination with fluoride, this was not found in three-year clinical trials. A possible reason for this, the lack of suitable calibration methods, is discussed. Finally, some comments on the appropriateness of laboratory evaluation strategies are made. PMID- 8615952 TI - Working Group Report 1: Laboratory models for caries (in vitro and animal models). PMID- 8615951 TI - Model investigations of caries inhibition by fluoride-releasing dental materials. AB - Fluoride-releasing dental materials are generally believed to reduce or prevent secondary caries. The evidence for this is largely anecdotal and centers on clinical experience with silicate cements and, more recently, with glass-ionomer cements. Unfortunately, corroborating evidence from controlled clinical trials is inadequate to establish precisely how effective these materials are or under what conditions they might be effective. Even less is known about the clinical effectiveness of newer materials that, often, release less fluoride. In vitro model systems have been used to study the effects of dental materials on de/remineralization of surrounding tooth structure. Fluoride-releasing materials have been shown in these models to reduce demineralization of both enamel and dentin compared with a material that does not release fluoride. This is useful from a mechanistic standpoint, but without clinical "standards" to guide model results, it is not possible to define an acceptable level of fluoride release or the length of time such release is required. A limited number of in situ model studies has been conducted, and some fluoride dose-response information has been obtained. These models are closer to the real situation and perhaps provide the best means to define required levels of fluoride release from materials in the absence of adequate clinical information. PMID- 8615953 TI - Working Group Report 2: In situ caries models, saliva, microbiology, and statistical considerations. PMID- 8615954 TI - Working Group Report 3: Role of models in assessing new agents for caries prevention. PMID- 8615955 TI - Understanding laboratory and clinical research: an overview. PMID- 8615956 TI - University research. PMID- 8615957 TI - The Food and Drug Administration's regulation of dental products. PMID- 8615958 TI - Evaluation programs at the American Dental Association. PMID- 8615959 TI - Product evaluations: a general practitioner's perspective. PMID- 8615960 TI - Comparative attributes for the description of the relative efficacy of therapeutic agents: general concepts and definitions, and application to the American Dental Association guidelines for the comparison of the clinical anticaries efficacy of fluoride dentifrices. AB - Comparative attributes are terms or phrases commonly employed in the vernacular of the biomedical literature to convey the results of comparative studies. Examples are such terms as "equivalent" or "superior." Recently, there has developed an increasing tendency in certain areas of application toward the institution of guidelines which provide both a uniform set of criteria for defining such attributes and a set of requirements which must be satisfied in order to demonstrate that such attributes may hold in a particular situation. The present paper provides a general discussion of comparative attributes, from which the application of the underlying concepts to any particular context might better be understood. Second, it provides an explicit and comprehensive discussion regarding the American Dental Associations's guidelines concerning the attributions of "superiority," "equivalence," and "at least as good as," as they are applied to the comparison of the clinical anticaries efficacy of fluoride dentifrices. PMID- 8615962 TI - Near-total laryngectomy. Patient selection and technical considerations. AB - OBJECTIVES: To investigate the speech and swallowing outcomes of patients undergoing near-total laryngectomy and to determine those perioperative factors that are associated with success. DESIGN AND SETTING: Retrospective analysis of a case series obtained from a hospital-based academic tertiary care center. PARTICIPANTS AND INTERVENTION: Records of all patients who underwent near-total laryngectomy at this institution were reviewed. OUTCOME MEASURES: Wound healing problems, quality of speech, degree of aspiration, and need for shunt revision were recorded. RESULTS: Thirty-nine patients during a 10-year period underwent near-total laryngectomy. Good speech was obtained in 30 (76%). Severe aspiration was a complication in eight patients (21%), necessitating reversal of the shunt in four (10%). Certain technical aspects of this procedure that produce a "hooded" myomucosal shunt were crucial to proper shunt function. Severe aspiration and poor voice outcome were most likely in patients who experienced a postoperative pharyngocutaneous fistula. These fistulas tended to occur at the junction of the pharynx and the upper end of the myomucosal shunt. When this region broke down, the hooding of the shunt was disrupted and its function impaired. CONCLUSIONS: Careful patient selection is crucial to the creation of a functional myomucosal speaking shunt after near-total laryngectomy. In patients at high risk for developing a pharyngocutaneous fistula, where irreversible aspiration through the shunt is then likely, this operation should be avoided and a total laryngectomy with tracheoesophageal puncture considered instead. PMID- 8615961 TI - Is surgery necessary in stage III and stage IV cancer of the head and neck that responds to induction chemotherapy? AB - OBJECTIVE: To study the influence of surgery in patients who achieve a response to induction chemotherapy. DESIGN: Nonrandomized retrospective study. SETTING: Comprehensive cancer center. PATIENTS: The records of all patients with state III and stage IV squamous cell carcinoma of the oral cavity, oropharynx, and hypopharynx were reviewed at the Queensland Radium Institute, South Brisbane, Australia, in the years 1970 through 1990. INTERVENTIONS: There were 314 patients where induction chemotherapy was used as part of a curative treatment. Complete response occurred in 20 patients, partial response in 110 patients, nonresponse in 162 patients, and unknown response in 22 patients. To assess the impact of surgery, the responders were divided into two treatment groups: one group (n = 57) received chemotherapy surgery, and radiotherapy (C/S/XRT), and a second group (n = 73) received chemotherapy and radiotherapy (C/XRT). MAIN OUTCOME MEASURES: Five-year survival, local failure, nodal failure, and distant failure. RESULTS: The 5-year actuarial survival for the chemotherapy responders and nonresponders was 58% and 43%, respectively (P < .05). When analyzed by treatment group, those receiving C/S/XRT had a 65% 5-year survival (95%) confidence intervals [CIs], 53 to 78) and those receiving C/XRT had a 56% 5-year survival (95% CI, 44 to 70). However, when the complete responders were assessed, those receiving C/S/XRT had a 90% 5-year survival (95% CI, 72 to 99) vs a 51% survival (95% CI, 18 to 89) for those treated with C/XRT. For the partial responders, the survival for the groups C/S/XRT and C/XRT were 59% (95% CI, 45 to 79) and 53% (95% CI, 41 to 67), respectively. Chemotherapy responders were less likely to fail locally if they were treated with C/S/XRT than with C/XRT (21% vs 43%, P < .01). CONCLUSION: The results suggest that surgery still has a role to play in patients who achieve a response with chemotherapy, even when the response is complete. PMID- 8615963 TI - The posterolateral neck dissection. Technique and results. AB - OBJECTIVE: To evaluate the effectiveness of the posterolateral neck dissection in providing regional control of metastatic disease to the posterior triangle from head and neck primary tumors as part of a multidisciplinary treatment approach. DESIGN: A case series review of 55 patients treated over a 10-year period form 1982 through 1991 with a minimum of 3 years of follow-up. Factors evaluated included site and histologic type of primary tumors, extent of surgery performed, other therapies provided, pathologic findings, and clinical outcome. SETTING: The University of Texas M.D. Anderson Cancer Center, Houston. PATIENTS: Forty-six male and nine female patients were studied. Three of them had bilateral dissections, for a total of 58 operations. Thirty-five were diagnosed as having melanoma; 10, squamous cell carcinoma, and 10, various other histologic types. INTERVENTION: All patients underwent a posterolateral neck dissection, either alone or as part of a multidisciplinary treatment plan. OUTCOME MEASURES: Factors reviewed were recurrence, either at the primary site or at a regional site, development of distant metastases, and surgical morbidity. RESULTS: Our review showed that, overall, disease was controlled at the site of the primary tumor in 89% of patients (94% of patients with melanoma) and that regional disease was controlled in 93% of patients (89% of patients with melanoma). Surgical morbidity was minimal. CONCLUSION: The "functional" posterolateral neck dissection as practiced at the University of Texas M.D. Anderson Cancer Center is effective surgical therapy that provides control of regional metastatic disease to the posterior neck from head and neck primary tumors. PMID- 8615964 TI - Quality of life and functional status measures in patients with head and neck cancer. AB - OBJECTIVE: To assess the relationship among three validated head and neck specific measures of functional status and a general measure of quality of life in patients with head and neck cancer. DESIGNS: Cross-sectional study using medical chart review, patient interview, and test administration. SETTING: Academic tertiary referral center. PARTICIPANTS: Fifty adults patients 3 months to 6 years after major surgery for head and neck cancer. MAIN OUTCOME MEASURE: Scores from a general measure of quality of life (the Functional Assessment of Cancer Therapy), a subscale specific to head and neck cancer, the University of Washington Quality of Life Questionnaire, and the Performance Status Scale for Head and Neck Cancer Patients. RESULTS: The disease-specific measures of functional status correlate well with one another. However, there were low correlations between the Functional Assessment of Cancer Therapy and the disease specific measures, indicating that general and disease-specific instruments contribute unique information about quality of life. CONCLUSION: A general measure of quality of life augments information obtained by disease-specific instruments by interpreting functional status in the broader scope of the patient's life. PMID- 8615965 TI - Gene therapy for head and neck cancer. Comparing the tumor suppressor gene p53 and a cell cycle regulator WAF1/CIP1 (p21). AB - OBJECTIVE: To compare the efficacy of the tumor suppressor gene wild-type p53 with that of cell-cycle regulator WAF1/CIP1 as single-agent gene therapy for squamous cell carcinoma of the head and neck. EXPERIMENTAL METHODS AND DESIGN: Recombinant cytomegalovirus-promoted adenoviruses containing the wild-type p53 or WAF1/CIP1 (p21) genes were transiently introduced into squamous cell carcinoma of the head and neck cell lines. Standard Western blot analysis was used to determine expression in these cells of the proteins encoded by these genes. A nude mouse xenograft model of squamous cell carcinoma of the head and neck was used to investigate the in vivo efficacy of the repeated gene therapy interventions. RESULTS: Western blot analysis showed marked induction of the WAF1/CIP1 tumor suppressor gene product by both the p21 adenovirus and the wild type p53 adenovirus (as a secondarily transcribed product). In vitro growth curves demonstrated that the wild-type p53 adenovirus significantly inhibited cell growth in these cell lines, whereas direct induction of the p21 gene product did not. Repeated infection with wild-type p53 adenovirus significantly reduced the size of established subcutaneous tumors, whereas infection with a replication defective viral control did not. CONCLUSION: Wild-type p53 adenovirus exhibits substantial in vitro and in vivo tumor suppressor activity in squamous cell carcinoma of the head and neck cell lines. This tumor suppression is not a function of the induced WAF1/CIP1 (p21) transcriptional product. Further studies are required to investigate the potential for induction of apoptosis by gene therapy. PMID- 8615966 TI - Definition of a tumor suppressor gene locus on the short arm of chromosome 3 in squamous cell carcinoma of the head and neck by means of microsatellite markers. AB - BACKGROUND: Tumor suppressor genes are important in the development of head and neck cancer. Using microsatellite markers that map close to the region 3p24-pter, we determined the frequency of allele loss close to this site with a view to narrowing the search for a putative tumor suppressor gene involved in the development of squamous cell carcinoma of the head and neck, which may facilitate future positional cloning techniques. DESIGN: Laboratory-based project with tumor and normal specimens subjected to molecular genetic analysis. Tumor-normal tissue DNA pairs were analyzed for allelic imbalance and microsatellite instability on chromosome 3p in the region 3p24-pter by the polymerase chain reaction and microsatellite markers D3S1304, D3S656, D3S1252, D3S1293, THRB, and D3S1266. SETTING: Molecular genetics and oncology research laboratory. PATIENTS: Paired tumor-normal DNA samples were obtained from 46 patients with tumors of the head and neck. MAIN OUTCOME MEASURES: Detection of loss of heterozygosity and microsatellite instability on chromosome 3 in the region 3p24-p25.1. RESULTS: We found loss of heterozygosity with at least one marker in 48% of informative cases and loss of heterozygosity or microsatellite instability in 57% of informative cases. The minimal region of loss was found in the region bounded by D3S656 and D31293. CONCLUSION: A putative tumor suppressor gene in head and neck cancer lies between D3S656 and D3S1293 in the 3p25.1 region. PMID- 8615967 TI - MYC amplification in squamous cell carcinomas of the head and neck. AB - OBJECTIVES: To establish the frequency of MYC amplification in squamous cell carcinoma (SCC) of the head and neck to evaluate its correlation with clinicopathologic variables that are used in clinical practice. DESIGN: Cohort analytic study. SETTING: University Hospital. PATIENTS: Fifty-nine consecutive patients with SCC of the head and neck. INTERVENTION: Oncologic surgery. MAIN OUTCOME MEASURES: The MYC copy number in tumor samples was estimated with the polymerase chain reaction. The presence or absence of amplification was correlated with the anatomic site, T stage, nodal involvement, pathologic grade, recurrence, distant metastases, and survival. RESULTS: Six SCC specimens (11%) showed MYC amplification. A highly statistical correlation between MYC amplification and T stage was noted (P < .005). Amplification was also significantly correlated with a hypopharyngeal primary site (P < .05). No correlation among amplification status, nodal involvement, pathologic grade, relapse, metastases, and survival was observed. CONCLUSIONS: Amplification of MYC is associated with advanced primary tumors, and it appears to be a late event in the tumorigenesis of SCCS of the head and neck. However, there is no correlation between MYC amplification and prognosis. PMID- 8615968 TI - Preliminary experience with thallous chloride T1 201-labeled single-photon emission computed tomography scanning in head and neck cancer. AB - OBJECTIVES: To test the feasibility of single-photon emission computed tomography (SPECT) scanning with the use of thallous chloride T1 201 in patients with head and neck cancer and to decide its possible applications to improve the diagnosis and staging of head and neck cancer. DESIGN: Findings from SPECT with the use of 4.32 mCi of thallous chloride T1 201 were compared with those from clinical examination, computed tomography (CT), magnetic resonance imaging, ultrasound guided fine-needle aspiration, and histologic studies. Primary sites and neck nodes were separately studied. Accuracy, sensitivity, and specifically were calculated for 19 patients who were being assessed for initial treatment (primary sites) and for 12 neck node dissections in 10 patients. SETTING: The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Amsterdam. PATIENTS: A consecutive sample of 25 patients with head and neck cancers of different sites. The average age of the patients was 60.2 years, and there were 19 men and six women. The sites of the primary lesions were as follows: oropharynx (n = 5), larynx (n = 9), oral cavity (n = 4), nasopharynx (n = 1), hypopharynx (n = 3), and unknown (n = 3). RESULTS: For the primary lesions, SPECT identified 94% of the lesions; SPECT was more accurate in delineating four oropharyngeal lesions and one nasopharyngeal lesion. In 12 neck node dissections, SPECT identified all of the positive lesions and two negative lesions, whereas CT detected two false positive lesions. Single-photon emission computed tomography gave less information about the number of nodes and the anatomy than did CT or magnetic resonance imaging. CONCLUSIONS: Single-photon emission computed tomography with the use of thallous chloride T1 201 appears to be useful in helping to identify occult primary lesions, particularly in the oropharynx. It may assist CT or magnetic resonance imaging in identifying a recurrence of cancer in tissues or in lymph nodes, and in screening for distant metastases. Although no nodes were identified that were not already seen with the use of CT or magnetic resonance imaging, SPECT may help to eliminate the false-positive lesions, and SPECT with the use of thallous chloride T1 201 appears to be a valuable new tool in helping to diagnose and stage head and neck cancer. PMID- 8615969 TI - The role of toluidine blue in assessing margin status after resection of squamous cell carcinomas of the upper aerodigestive tract. AB - OBJECTIVE: To determine the efficacy of toluidine blue in assessing margin status after removal of squamous cell carcinomas of the upper aerodigestive tract. DESIGN: A prospective study of 50 consecutive patients undergoing surgical resection of squamous cell carcinomas of the upper aerodigestive tract was performed during February 1 to December 1, 1993. After tumor resection, toluidine blue was applied directly to the remaining unresected mucosa. The staining characteristics of the mucosa were then compared with those of frozen-section biopsy specimens of the margins and with the permanent histologic findings of the resected tumor specimen. RESULTS: In three cases, toluidine blue identified a positive margin, which was confirmed on frozen and permanent section. In six cases, false-positive staining was noted, which was most frequently related to traumatic handling of the mucosa during the resection. In no case was a positive margin found on histologic staining that failed to stain with toluidine blue. During routine staining of surrounding unresected mucosa, three cases of a second primary tumor that was not seen on routine evaluation before tumor removal were identified with toluidine blue. In one case, a second T1 oral cavity lesion was found, while in the other cases, separate pharyngeal lesions were identified. CONCLUSION: Based on these findings, it appears that toluidine blue improved the ability to assess margin status at the time of resection, and we advocate its use after resection of tumors to the upper aerodigestive tract. PMID- 8615970 TI - Resection margin as a predictor of recurrence at the primary site for T1 and T2 oral cancers. Evaluation of histopathologic variables. AB - OBJECTIVE: To establish whether histopathologic variables other than the pathologist's statement of complete excision predict recurrence of squamous cell carcinoma at the primary site and therefore indicate local postoperative radiotherapy. DESIGN: Retrospective analysis of clinical data and review of slides of resection specimens. SETTING: Tertiary care, hospital-based clinic. PATIENTS: Eighty-two patients who had complete excision only (histologically based) of a T1 or T2 squamous cell carcinoma of the mobile tongue or floor of the mouth but did not receive any form of immediate postoperative radiotherapy. Twenty-nine patients underwent local resection without treatment of the NO neck; in 53 patients a neck dissection was also performed. METHODS: Evaluation of recurrent tumor above the clavicles until 4 years postoperatively and of second and third primaries. Infiltrative depth was evaluated in 73 cases and spidery spread, perineural spread, and angioinvasion in 70 cases. RESULTS: Of the squamous cell carcinoma, 27% were well differentiated and 73% were moderately differentiated; depth of invasion was 5 mm or more in 57%, a spidery growth pattern was present in 51%, there was perineural spread in 16%, and angioinvasion was found in 3%. Recurrences at the primary site, not linked to histopathologic findings, occurred in 4%; 17% of the patients had second primary tumors in the head and neck region, 15% had neck conversions, and 1% had neck recurrence. CONCLUSIONS: When excision of a small squamous cell carcinoma of the mobile tongue or the floor of the mouth is histologically complete, other histopathologic variables are irrelevant in predicting recurrence at the primary site, and local radiotherapy is not indicated, considering the morbidity and high number of second and third primary tumors to be expected that will require future new treatment. PMID- 8615971 TI - Use of a mechanical microvascular anastomotic device in head and neck free tissue transfer. AB - BACKGROUND: The use of mechanical microvascular anastomotic systems for free tissue transfer has previously been reported. Currently, a commercially available coupling device (3M Healthcare, St. Paul, Minn) is widely used for various microvascular free flaps. However, to our knowledge, there are no reports in the literature describing the efficacy of this particular device in regard to free tissue transfer in head and neck reconstruction. OBJECTIVE: To describe the surgical technique, limitations, and guidelines for application of this system for vascular anastomosis in head and neck free tissue transfer. DESIGN: The microvascular anastomotic device was used in 79 head and neck free flaps: radial forearm (n = 28), rectus abdominis (n = 27), fibula (n = 12), lateral thigh (n = 4), iliac crest (n = 3), gracilis (n = 2), jejunum (n = 1), pectoral (n = 1), and lateral arm (n = 1). Follow-up ranged from 6 months to 2.5 years. SETTING: Two major teaching/referral medical centers. PARTICIPANTS: Seventy-six patients ranging in age from 19 to 86 years. INTERVENTION: A total of 105 anastomoses (17 arterial and 88 venous) were performed. OUTCOME MEASURES: Anastomotic times and patency rates were evaluated. RESULTS: the anastomotic times ranged from 8 to 18 minutes for the arteries (average, 10 minutes) and from 4 to 16 minutes for the veins (average, 5 minutes). None of the flaps resulted in venous congestion due to thrombosis at the anastomosis. Two arterial anastomoses resulted in thrombosis, one of which was detected intra-operatively and successfully salvaged with conventional suture anastomosis. CONCLUSIONS: The patency rates with the microvascular anastomotic system appear to compare favorably with those of standard suture techniques. The major advantage is that the time of venous anastomosis is reduced, thereby decreasing the total ischemic time. An additional advantage is the ease with which anastomoses can be performed when the vessels are deep within a wound, where suture placement is difficult. PMID- 8615972 TI - Radiation-induced malignant fibrous histiocytoma in patients with nasopharyngeal carcinoma. AB - OBJECTIVE: To evaluate the prevalence, 15-year cumulative incidence, time interval, and prognosis of radiation-induced malignant fibrous histiocytoma of the head and neck in long-term survivors of nasopharyngeal carcinoma. DESIGN: Cohort. SETTING: Tertiary care hospital. PATIENTS: Eight long-term survivors of nasopharyngeal carcinoma with malignant fibrous histiocytoma in the maxillary sinus or nasal cavity. MAIN OUTCOME MEASUREMENT: Survival of postirradiation malignant fibrous histiocytoma in patients with nasopharyngeal carcinoma. RESULTS: The prevalence of radiation-induced malignant fibrous histiocytoma in long-term survivors of nasopharyngeal carcinoma was 0.38%. The 15-year cumulative incidence was 2.2%. Most tumors occurred in the maxillary sinus and were characterized by spindle-shaped tumor cells with plump nuclei arranged in a whorl or storiform pattern in a fibrous stroma. The mean interval between malignant fibrous histiocytoma and nasopharyngeal carcinoma was 121 months. Local recurrence developed in all cases within 9 months after surgery. Six patients died of disease without distant metastasis within 30 months. Two patients were alive with disease for 20 and 32 months, respectively. CONCLUSIONS: Radiation induced malignant fibrous histiocytoma in the head and neck region in long-term survivors of nasopharyngeal carcinoma is rare. It takes a long time to occur after irradiation and is locally invasive with poor prognosis. PMID- 8615973 TI - Interleukins 2 and 12 activate natural killer cytolytic responses of peripheral blood mononuclear cells from patients with head and neck squamous cell carcinoma. AB - OBJECTIVES: To examine the capacity of interleukin-2 (IL-2) and interleukin-12 (IL-12) to modulate the cytolytic activity of peripheral blood mononuclear cells against squamous cell carcinoma, and to determine whether peripheral blood mononuclear cells from healthy donors respond differently to IL-12 than do peripheral blood mononuclear cells from patients with head and neck squamous cell carcinoma. DESIGN: Case-comparison study of a consecutive sample of patients with head and neck squamous cell carcinoma who were scheduled to undergo surgical excision. PARTICIPANTS: The study included 10 patients with stage III or IV carcinoma matched with 10 volunteer blood donors. INTERVENTION: Isolated peripheral blood mononuclear cells from patients and volunteers were treated with IL-2, 10 U/mL and 100 U/mL; IL-12, 1 U/mL or 10 U/mL; or a combination of IL-2 and IL-12. RESULTS: The combination of IL-2 and IL-12 consistently produced the greatest activation cytolysis than either cytokine alone at all concentrations tested. This increased activity against a squamous cell carcinoma cell line was seen in lymphocytes from volunteers and patients. CONCLUSION: Our findings suggest a new treatment regimen for the patient with head and neck cancer that uses immunomodulation with a combination of cytokines. PMID- 8615974 TI - Neuroectodermal antigens persist in benign and malignant salivary gland tumor cultures. AB - OBJECTIVE: To determine whether a heterogeneous collection of salivary gland tumors shared common antigenic characteristics and growth patterns in tissue culture. DESIGN: Cell cultures were derived from benign and malignant salivary gland neoplasms, cultured conservatively, and serially analyzed for epithelial, myoepithelial, and neuroectodermal antigens. SUBJECTS: Nineteen samples reflecting the spectrum of salivary tumor pathologic characteristics were established in tissue culture. Most were derived from benign pleomorphic adenomas, and several were from carcinomas, including carcinoma ex pleomorphic adenoma, and mucoepidermoid and adenoid cystic carcinoma. RESULTS: All cultures were epithelial as determined by morphologic and antigenic examination, using antibodies for cytokeratin. The phenotype of cells derived from benign tumors, especially the pleomorphic adenomas, resembled those in the original neoplasm. Those from carcinomas were similar, with less differentiated characteristics. Manipulation of growth conditions altered the phenotypes shown in culture. Some cultures contained cells expressing vascular smooth-muscle actin and glial fibrillary acidic protein or nestin. CONCLUSIONS: This model cell system containing proliferative cells from several tumor types is consistent with a stem cell theory of salivary gland tumor origin. Our data were not consistent with the bicellular or multicellular theory. We hypothesize a neuroectodermal origin for this group of apparently heterogeneous tumors. These cultured cells will be valuable for in-depth investigation of the loss of proliferation controls in benign and malignant tumors of the salivary gland. PMID- 8615975 TI - Apoptotic inhibition of head and neck squamous cell carcinoma cells by tumor necrosis factor alpha. AB - OBJECTIVES: To evaluate the effect and mechanism of action of tumor necrosis factor alpha (TNF-alpha) on head and neck squamous cell carcinoma (HNSCC) cell lines. Specifically, to find out whether TNF-alpha induces apoptotic (programmed) cell death. DESIGN: Cytotoxicity and kinetics were evaluated through the use of a methylene blue colorimetric assay. The mechanism of cell death was evaluated through gel electrophoresis of extracted DNA, double-stranded DNA fragmentation, and Hoechst 33342-propidium iodide double staining. TARGET CELLS: Four HNSCC cell lines were investigated: UMSCC-1, UMSCC-8, UMSCC-19, and CAL-27. RESULTS: Tumor necrosis factor alpha at a concentration of 10000 U/mL induced cytotoxic effects in all four cell lines with varying sensitivities. Significant cytotoxic effects required 3 to 4 days of exposure to TNF-alpha. Further experiments demonstrated that TNF-alpha induced double-stranded DNA fragmentation of targets, which preceded the detection of actual cytotoxic effects. Treated HNSCC cell lines had their DNA fragmented into a ladder pattern of endonucleolytic cleavage of multiples of 180 to 200 base pairs, consistent with apoptosis. Propidium iodide Hoechst 33342 double staining confirmed that TNF-alpha-induced nuclear alterations precede permeabilization of the plasma membrane, supporting cell death in these tumor cells by apoptosis. CONCLUSION: Tumor necrosis factor alpha exposure results in variable cytotoxic reactions in HNSCC cell lines, with DNA fragmentation preceding significant cell death. The mechanism of cell death is apoptosis, with typical morphological features. Further studies are needed to better elucidate the role of TNF-alpha in the treatment of in vivo HNSCC. PMID- 8615976 TI - Wound healing. Tensile strength vs healing time for wounds closed under tension. AB - OBJECTIVES: To measure tensile strength of wounds closed with and without tension in rats, delineating the postoperative time interval when tensile strength is equal. To study patterns of growth in tensile strength. DESIGN: Transverse incisions on the backs of control rats were closed with minimal tension. In experimental animals, after excision of skin from the back, wounds were closed with closing tensions in excess of 70 g. Animals were killed at designated healing intervals for tensile-strength testing of wounds. SUBJECTS: One hundred Sprague-Dawley rats were divided into 5 groups days 5, 7, 10, 14, and 21, with equal numbers of control and experimental animals in each group. Wounds from 94 animals were available for breakload testing. INTERVENTION: Closing tensions were measured for transverse incisions on the backs of control animals before closure and after removal of 50 to 60 mm of skin from the backs of experimental animals. RESULTS: Tensile strength was not significantly different on day 5. However, wounds closed under tension showed significantly higher tensile strength on 7, 10, 14, 21. Polynomial regression suggests a cubic relationship between healing time and tensile strength. CONCLUSIONS: Results suggest that tensile strength of wounds closed under tension exceeds that of tensionless wounds as early as day 7 following surgery. This study also illustrates the 2 periods of rapid increase in wound tensile strength that probably coincide with specific stages of wound healing. PMID- 8615977 TI - Imaging quiz case 1. Warthin's tumor (papillary cystadenoma lymphomatosum). PMID- 8615978 TI - Imaging quiz case 2. Arteriovenous malformation (AVM) of the buccal fat pad. PMID- 8615979 TI - Maintaining esthetic restorations--a shared responsibility. PMID- 8615980 TI - Health promotion and disease prevention. PMID- 8615981 TI - Clinical implications of recent advances in salivary research. PMID- 8615982 TI - Role of adjunctive therapy in esthetic management. PMID- 8615983 TI - Protecting esthetic restorations with mouthguards and other appliances. PMID- 8615984 TI - Intraoral esthetic repair of dental restorations. PMID- 8615985 TI - Acceptability of alternatives for conservative restoration of posterior teeth. PMID- 8615986 TI - High tech--the time is now! PMID- 8615987 TI - Ceramic bonded restorations: pathway to natural esthetics. PMID- 8615988 TI - Esthetic considerations in treating periodontal defects associated with bilateral palitoradicular grooves. PMID- 8615989 TI - The porcelain-veneered metal crown with a procelain butt joint: treatment of choice in stress-bearing areas. PMID- 8615990 TI - Evaluating color change following vital tooth bleaching. PMID- 8615991 TI - A lood back at the adhesive resin-bonded cast restoration. PMID- 8615992 TI - Self-esteem and physical attractiveness. PMID- 8615993 TI - Findings of scientific misconduct. PMID- 8615994 TI - Findings of scientific misconduct. PMID- 8615996 TI - [Colorectal cancer: has its diagnosis improved in the last decade?]. AB - OBJECTIVE: To assess whether any shift in pathologic stage, tumor resectability and need of emergency surgery has been observed in colorectal carcinoma patients over the last ten years. EXPERIMENTAL DESIGN: Retrospective study. PATIENTS: We compare 201 patients treated in our Service from January 1981 to December 1983 (Group I) with 306 patients operated from January 1991 to December 1993 (Group II). RESULTS: No change has been noticed in any of the parameters analyzed. CONCLUSIONS: We think that the efforts in earlier detection of CCR should be increased to improve the prognosis of the disease. PMID- 8615995 TI - Colonoscopic findings in patients with lower gastrointestinal bleeding send to a hospital for their study. Value of clinical data in predicting normal or pathological findings. AB - BACKGROUND: Lower gastrointestinal bleeding is a highly frequent clinical problem that may reflect serious pathology in the colon. Colonoscopy is generally accepted as the diagnostic procedure of choice. Decisions as to whether to carry out colonoscopy or not, are not well defined. METHODS: 536 colonoscopies, made to discover the cause of lower gastrointestinal bleeding were analyzed and a final 457 included in the study. All of these patients came to the hospital because they had presented at least in one occasion, one episode of rectal bleeding, and were send by the specialist of the zone, in order to achieve a correct diagnosis of its process. In all cases the following associated symptoms were analyzed: the presence of diarrhea, constipation, abdominal pain and rectal mass on examination. The characteristics of lower gastrointestinal bleeding were analyzed in a subset of 150 consecutive patients. RESULTS: Mean age was 59 +/- 16.9 years. 54.5% were male and 45.5% female. The exploration was normal until the cecum in 146 patients (32%). In the remaining 311, the findings were: polyps (25.1%), diverticular disease (24%), neoplasia (12.6%), inflammatory bowel disease (9.4%), unspecific proctitis (2.4%), ischemic colitis (2.4%), angiodysplasia (1.9%), infectious colitis (1.1%), and miscellaneous (0.7%). An age of less than 40 years and the existence of anal pathology were significantly more frequent among patients with a normal examination (p < 0.001), but with a sensitivity of only 66%. No differences were found among patients with disordered bowel frequency or abdominal pain in relation to the colonoscopic findings. Previous presence of a rectal mass when the examination proved abnormal (p=0.06). Intermittent bleeding and the presence of blood in the stools were more frequent in patients with normal examination (p= 0.07 and p< 0.05, respectively). No significant differences in relation to colour, duration of bleeding, or to whether toilet paper was stained with blood were found. CONCLUSIONS: 1) The more frequent endoscopic findings were polyps and diverticular disease. 2) Clinical data are of little value in predicting a normal examination. 3) Total colonoscopy appears to be the first procedure of choice in all patients with lower gastrointestinal bleeding, irrespective of the clinical data and the presence of anal pathology. PMID- 8615997 TI - [Anal incontinence in patients with rectal neoplasms previous to surgical intervention]. AB - HYPOTHESIS: Patients with rectal carcinoma may have anal continence disorders before the operation, in relation to age. AIM: To evaluate the anorectal function in a consecutive sample of patients with rectal carcinoma before the operation. MATERIAL AND METHODS: 56 consecutive patients with rectal carcinoma were studied and classified into two groups according to anal continence: continent and incontinent. Anorectal function were evaluated in all patients: Perineometry (perineal measurements at rest and during a straining effort), Anal manometry (anal pressures and rectal capacity), Pudendal nerve terminal motor latency. STATISTICAL ANALYSIS: quantitative data: -test (confidence interval), qualitative data: Fischer exact test. RESULTS: Anal continence: continent 41, incontinent 15. All patients with anal incontinence were more than 60 years old (p<0.01). Mean age: continent 61.3 +/- 12.4, incontinent 74.3 +/- 6 (p<0.01, CI 8.02-17.98). Perineal measurement: at rest: continent 2.97 +/- 0.69. incontinent 2.54 +/- 0.56 (p<0.05, CI 0.03-0.83), with straining effort: continent 1.37 +/- 0.86, incontinent 0.81 +/- 0.92 (p < 0.05, CI 0.03 - 1.86). Pudendal latency: continent 1.9 +/- 0.3, incontinent 2.3 +/- 0.5 (p<0.01, CI 0.11-0.69). There was no significant difference in the manometric data. CONCLUSION: Patients with rectal carcinoma have preoperative anal continence alterations, in relation to pelvic disorders and age. PMID- 8615998 TI - [Basal levels of blood pepsinogen I and Helicobacter pylori infection in patients with duodenal ulcer and normal endoscopy]. AB - AIM: To study basal pepsinogen I levels in patients with duodenal ulcer and in subjects with normal endoscopy, depending on Helicobacter pylori status. METHODS: One-hundred and one patients with duodenal ulcer and 74 controls with normal endoscopy were studied. Mean age and gender distribution were: 46 vs 42 years, and 74% vs 43% males, respectively. At endoscopy biopsies from the gastric antrum and body were obtained for histologic (H&E) and microbiologic (Gram and culture) study. Basal levels of serum pepsinogen I were measured (RIA). RESULTS: Among the subjects with a normal endoscopy, those with H. pylori infection had higher pepsinogen I levels (m +/- SD) than non-infected patients (77 +/- 27 vs 62 +/- 28 ng/ml; p < 0.05). Basal levels in duodenal ulcer patients were 107 +/- 38 ng/ml, higher (p < 0.001) than in the group with normal endoscopy (both with and without H. pylori). In multivariate analysis pepsinogen I levels were correlated with H. pylori infection (regression coef.= 17; SE= 8.1), duodenal ulcer (regr. coef.= 22; SE= 5.8) and smoking habit (regr. coef.= 24; SE= 5.2). CONCLUSION: Basal pepsinogen I levels were significantly higher in duodenal ulcer patients than in H. pylori infected subjects with normal endoscopy. The lowest levels corresponded to non-infected patients. Therefore, an additional factor other than H. pylori infection is likely involved in the hyperpepsinogenemia classically reported in duodenal ulcer patients. PMID- 8615999 TI - [Ascitic fluid: benignancy and malignancy parameters]. PMID- 8616001 TI - [Recurrent perforation of of the sigmoid colon associated with Ehlers-Danlos syndrome type IV]. AB - Ehlers-Danlos syndrome type IV is a heritable disease of type III collagen metabolism. Most patients have a defect either in the synthesis or structure of type III procollagen, a finding consistent with the fact that these patients are prone to spontaneous rupture of the aorta and intestines, tissues rich in type III collagen. The diagnosis is suspected in a patient with a combination of clinical manifestations and family history, but it is confirmed only by culture of the patient's skin fibroblasts and the demonstration of a defect in type III collagen metabolism. We present a patient with Ehlers-Danlos type IV syndrome who developed a recurrent colon perforation, and discuss the surgical strategy to prevent recurrences. PMID- 8616000 TI - [Torsion of the great epiploon as cause of acute abdomen]. AB - Two patients with long term evolution of inguinal hernia and acute abdomen caused by torsion of the major omentum are reported. The main physiopathologic mechanisms involved in the torsion of epiploon as well as the clinical data that could enable us to suspect the diagnosis before performing laparotomy, are reviewed. After searching the literature, the low frequency of presentation of the illness is confirmed. Experience shows us that is less than 0.25 per thousand in the last 20 years. PMID- 8616002 TI - [Small cell anaplastic carcinoma of the colon]. AB - Small-cell anaplastic carcinoma of the colon is a very infrequent tumour (less than 1% of all colorectal neoplasms). Thirty-two cases have been described in the international literature up to 1992. A case, in a 54 year-old patient who underwent successful resection is presented. The importance of this tumour is due to its great aggressivity, and its great tendency to produce early hematogenous and lymph node metastases. It implies a bad prognosis and a survival of around 0% at one year. Because of these facts, treatment must include, beside surgical resection, an aggressive systemic protocol. PMID- 8616003 TI - [Droxicam-induced toxic hepatitis]. AB - We present four cases of hepatitis with clinical features indicating a direct link with Droxicam. In all the cases, the presentation was that of acute hepatitis with subsequent resolution, whereas one patient developed autoimmune chronic active hepatitis. A full evaluation including ultrasound, liver biopsy, and serologic markers supported the diagnosis. Due to the fact that the "Direccion General de Farmacia y Productos Sanitarios" has approved the postponement of dispensation of products with Droxicam from February 25, 1995, this drug should not be considered anymore in the future as a potential cause of liver injury (acute or chronic hepatitis). PMID- 8616004 TI - [Papillary tumor of the pancreas diagnosed by fine-needle aspiration]. AB - We report the case of a 48 year-old woman with an epigastric palpable mass identified by abdominal echography and computed tomography as a solid pancreatic tumor. A cytology taken by fine needle aspiration guided by computed tomography was diagnostic of papillary pancreatic tumor, which was confirmed by surgical resection. We emphasize the low frequency and good prognosis of this type of tumor after surgical resection, and the utility of image techniques and fine needle aspiration to obtain a preoperative diagnosis. PMID- 8616005 TI - [Esophageal foreign bodies: endoscopic management]. PMID- 8616006 TI - [Major surgery in geriatric age: are the risk factors quantifiable?]. PMID- 8616007 TI - [2 cases of anisakiasis in the Del Rio Hortega Hospital (Valladolid)]. PMID- 8616008 TI - [Diagnosis of hiatal hernia by radiology and endoscopy]. PMID- 8616009 TI - [Oral video enteroscopy: a prospective study of 30 cases]. AB - The main indication of oral enteroscopy is gastrointestinal bleeding. The literature does not include many references to oral, as opposed to peroperative enteroscopy. OBJECTIVE: To determine the usefulness and the sensitivity of the technique in small bowel diseases in a prospective series of 30 consecutive patients. Included were 3 cases of Crohn's disease, 3 acute gastrointestinal lesions in patients under SNAIDS and a normal gastroscopy, 1 polyp, 3 small bowel tumors and 13 cases of bleeding of unknown origin. RESULTS: Findings included jejunal leiomyomas, tubular adenomas of the jejunum, isolated jejunal erosions, angiodysplasia, mesenteric thrombosis, nodular lymphoid hyperplasia and jejuno ileal isolated Crohn's disease. Tolerance was good without complications. Sensitivity was only 53%, therefore the evaluation of the diagnostic efficacy needs further assessments. PMID- 8616010 TI - Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. PMID- 8616011 TI - Differential effects of the antifolates methotrexate, aminopterin and trimetrexate on murine haemopoietic progenitor cells. AB - We investigated the effects of the antifolates methotrexate (MTX), aminopterin (AMT) and trimetrexate (TMTX) on murine haemopoietic progenitor cells. Colony formation by late progenitors (CFU-C) was inhibited by the antifolates and the addition of leucovorin (LV) to the cultures on day 0 or day 5 wholly or partially reversed the effects of MTX or AMT. The effect of TMTX was only effectively rescued by hypoxanthine plus thymidine (HT). In the high proliferative potential colony forming cell (HPP-CFU-C) assay the antifolates induced formation of smaller colonies, but not a reduction in absolute colony numbers. This effect was reversed by LV and indicated that the antifolates (except high TMTX concentrations) were not toxic to the HPP-CFU-C but reversibly inhibited proliferation of more mature progenitor cells. The direct effects of the drugs on HPP-CFU-C were investigated in limiting dilution assays performed with fractionated bone marrow cells. Untreated cultures contained almost only large colonies, where the addition of antifolates induced a reversible shift towards smaller colonies. The present study indicates that MTX, AMT and low TMTX concentrations are not toxic to HPP-CFU-C but that the drugs induce a developmental arrest in more mature progenitor cell populations. PMID- 8616012 TI - Effects of IL-3 and LPS on transcription factors involved in the regulation of IL 6 mRNA. AB - Recently it has been demonstrated that in vivo application of interleukin-3 (IL 3) is associated with the release of IL-6. This observation suggests that the transcription factors triggered by IL-3 are in great homology with the transcription factors induced by lipopolysaccharide (LPS). The results of the present study with in vitro activated human monocytes demonstrate that IL-3 alone is incapable of inducing IL-6 mRNA, but primes monocytes to enhance the IL-6 mRNA expression when co-stimulated with LPS. The difference in effect between IL-3 and LPS might be related to our observation that IL-3 induces the p5O subunit of the transcription factor nuclear factor-kappa B (NF-Kappa B), whereas LPS appears to induce both the p5O as well as the p65 subunit of NF-kappa B, as demonstrated with RNA studies and electrophoretic mobility shift assays (EMSA). However, no difference was found with regard to the induction of activator protein-1 (AP-1) and NF-IL6 after treatment with IL-3 or LPS alone. Priming with IL-3 followed by LPS stimulation is associated with a reduced expression of NF-kappa B without changing the composition of the complex. In addition, a reduced expression of c fos and c-jun mRNA was noticed, combined with a reduced DNA binding activity of AP-1. However, the expression of NF-IL6 was enhanced when priming with IL-3 followed by LPS. Since AP-1 has been suggested as negative regulator of the IL-6 gene expression, it is conceivable that, after priming with IL-3, the reduced DNA binding activity of AP-1, in conjunction with the increased DNA binding of NF IL6, might result in a synergistic effect on IL-6 mRNA expression, when compared to stimulation with LPS alone. PMID- 8616013 TI - PMA-induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level. AB - We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10(-7)M phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (alpha, beta I, beta II, gamma, delta, epsilon, theta, eta, zeta) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant (P < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC alpha, -beta I, -epsilon, theta and -zeta isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-zeta isoforms. Moreover, PKC-6 delta appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10(-7)M haemin. The possible implications of these findings for a better understanding of the molecular events underlying the process of megakaryocytic differentiation are discussed. PMID- 8616015 TI - Response of myelodysplastic syndrome marrow progenitor cells to stimualtion with cytokine combinations in a stroma-free long-term culture system. AB - The effect of an ex vivo expansion culture system using multiple cytokine combinations was evaluated in 38 cases of myelodysplastic syndrome (MDS) with the aim of overcoming the defective in vitro growth of haemopoietic progenitor cells. A combination of four growth factors (GF) including SCF, IL-3, IL-6 and GM-CSF was identified as the optimal combination for expanding clonogenic progenitor cells in MDS bone marrow liquid cultures. The cultures of 50% of the patients (19/38) responded to GF stimulation (mean CFU-GM fold increase 15.65+/-48 at week 4) and showed morphological features of normal and/or dysplastic myeloid differentiation. In 12/38 cases (31%), complete unresponsiveness to multiple cytokine stimulation was observed; a small number of patients (7/38) showed progressive leukaemic growth along the cultures with the presence of 100% immature blasts at week 4. GM-CSF and c-kit receptors, analysed by immuno histochemistry in 10 patients, were over-expressed in responding patients and either lacking or down-regulated in non-responders. Fluorescence in situ hybridization (FISH) analysis of cultured interphase cells of nine patients (trisomy 8 in eight patients) showed a clear-cut increase in the percentage of cells with three signals in the two responding patients, thus indicating the expansion of a MDS clone. Multiple cytokine liquid cultures seem to be able to override the refractoriness of MDS progenitor cells to GF stimulation in many cases, revealing a heterogeneity which may have prognostic implications and should be considered in ex-vivo and in vivo clinical trials with cytokine combinations. PMID- 8616014 TI - Early suppressive effects of chemotherapy and cytokine treatment on committed versus primitive haemopoietic progenitors in patient bone marrow. AB - These studies investigated the effectiveness of in vivo administration of cytokines in ameliorating potential marrow damage induced by chemotherapy. Breast cancer patients received 5-fluorouracil, leucovorin, doxorubicin and cyclophosphamide (FLAC) followed by either GM-CSF, PIXY321, or no cytokine. Marrow was obtained before and after one or two cycles of FLAC once blood cell counts had recovered. Colony-forming units for granulocytes and macrophages (CFU GM) were used to indicate the effect of therapy on recovery of committed progenitor cells responsible for early blood cell recovery. The frequency and number of CFU-GM in marrow obtained after FLAC + PIXY321 were significantly lower than in marrow obtained after FLAC+GM-CSF or FLAC without cytokine. CD34+ cell numbers were also reduced after FLAC + PIXY321. CFU-GM production in marrow long term cultures (LTC) was used to assess the effect of therapy on primitive progenitors. After 5 weeks the number of CFU-GM in LTC of post-therapy marrow from all three treatment arms was < 15% of the number in pre-therapy LTC. Suppressive effects of FLAC on primitive progenitors were observed even when committed progenitors and CD34+ cells had recovered to pre-therapy levels. These results demonstrate that cytokine treatment did not ameliorate suppressive or toxic effects of FLAC on the functional integrity of the marrow. PMID- 8616016 TI - Kinetics of colony formation by BFU-E grown under different culture conditions in vitro. AB - Colony formation by erythroid burst-forming units (BFU-E) involves a variable number of cell divisions before individual 'subcolonies' begin to appear. Consequently the numbers of subcolonies vary amongst individual bursts. If this observation is interpreted as a reflection of a stochastic process, the number of subcolonies in each individual burst represents the number of divisions by the BFU-E prior to commitment to terminal differentiation. This provides a means for quantitating the probability of erythroid differentiation (pD) and the probability of renewal (1 - pD). In order to determine whether these kinetics of burst formation can be influenced by exogenous factors we used three commercially available media designed for the growth of BFU-E. We found that subcolony numbers per burst ranged from one to 64 and that the cumulative distributions of subcolonies per burst followed a logarithmic curve (r > 0.90). Differences were observed in the distribution of subcolonies per burst when BFU-E were grown in different media (P=0.03; Kruskall-Wallis test). The probability of immediate terminal differentiation (i.e. committment to form a subcolony) was 0.25 for two of the media and 0.7 for the third. The corresponding renewal probabilities were O.75 and O.3. These data indicate that the proliferation kinetics of BFU-E are susceptible to regulation by exogenous factors. PMID- 8616017 TI - Stem cell factor receptor (c-kit, CD117) is expressed on blast cells from most immature types of acute myeloid mallignancies but is also a characteristic of a subset of acute promyelocytic leukaemia. AB - Investigating 208 patients with acute haematological malignancies, we found that stem cell factor receptor (SCFR) was expressed on high numbers of blast cells from the vast majority of patients (93%) with refractory anaemia with excess of blasts in transformation. SCFR was also detected in 62% of AMLs, in which it was directly associated to the expression of CD7, interleukin 6 receptor and CD34, and inversely to that of CD11b and CD14. SCFR-positive cases were preferentially represented in AML-M1 (70%) and in AML-M2 (83%) subsets, whereas only 45% of the remaining samples (M3-M4-M5) exhibited SCFR positively. Interestingly, 50% of cases with acute promyelocytic leukaemia expressed SCFR and this molecule was heterogenously regulated by in vitro treatment with all-trans retinoic acid. PMID- 8616018 TI - Autoantibody against a 58 kD molecule in a patient with neutropenia and NK cell deficiency. AB - A patient with Evans' syndrome is described who developed intermittently an immunocytopenia consisting of autoimmune neutropenia and natural killer (NK) cell deficiency. During this time an autoantibody binding to a 58 kD antigen expressed on granulocytes, activated NK cells and Epstein-Barr virus (EBV) transformed B cells was present. Incubation of activated NK cells and NK cell clones with the autoantibody leads to a significant inhibition of NK activity. Therefore the autoantibody may detect a functionally important activation molecule. PMID- 8616019 TI - Effects of high-dose granulocyte colony-stimulating factor on neutrophil functions. AB - We evaluated the effects of high-dose recombinant human granulocyte colony stimulating factor (rhG-CSF) therapy on N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis and superoxide (O2-) production in neutrophils from four patients with aplastic anaemia. The FMLP-induced chemotaxis and O2- production in the neutrophils of all four patients were normal before the rhG-CSF treatment. After the administration of high-dose rhG-CSF, chemotaxis in agarose was decreased, adherence and O2- production were enhanced in all the patients. An excessive increase of neutrophils with augmented adhesiveness and oxygen radical production may be harmful. Care should be taken in regard to neutrophil toxicity when high-dose G-CSF is used clinically. PMID- 8616021 TI - Atypical chronic myeloid leukaemia, a distinct clinical entity related to the myelodysplastic syndrome? AB - The FAB group has recently published guidelines for distinguishing chronic granulocytic leukaemia (CGL) from chronic myelomonocytic leukaemia (CMML) and atypical chronic myeloid leukaemia (aCML). Whereas CGL is generally recognized to be a distinct entity, there is debate as to whether CMML and aCML are separate disorders or part of a spectrum of myeloproliferative disorders with dysplastic features. Data are presented on 10 cases who developed features of a CML during the course of their disease but who presented with a normal or a low leucocyte count without a monocytosis and were diagnosed as refractory anaemia. This suggests that, at least in some cases, aCML represents an unusual evolution of MDS, and even though these patients have a uniformly poor prognosis it may be premature to regard aCML as a distinct clinical entity. PMID- 8616020 TI - Deletions of the long arm of chromosome 7 in myeloid disorders: loss of band 7q32 implies worst prognosis. AB - Clinical and cytogenetic data were analysed in 54 patients with acute non lymphocytic leukaemias (ANLL) or MDS (myelodysplastic syndromes) and deletion of the long arm of chromosome 7 (7q-), in order to determine if there is a commonly deleted region in 7q and to establish possible correlations between karyotypic features, such as karyotype pattern, karyotype complexity, associated anomalies, and/or the type of deleted segments, and outcome of patients with these disorders. The median follow-up of our patients was 4 months (range 1-89), as was the median survival. In 30% of the cases there was a history of preceding MDS or previous chemotherapy. Clinical and cytogenetic remission was obtained in 7/36 patients treated with chemotherapy (CT). At the time of 7q- detection, three patients previously treated with CT for ANLL were in clinical remission. 5q- was the most recurrent associated abnormality. Complex karyotypes were observed in 68% of the cases. In univariate analysis, statistical differences in survival were observed according to diagnosis (therapy-related and secondary diseases had a worse prognosis than primary disorders), the chromosomal segments deleted (the loss of band 7q32 was of poor prognostic value), the karyotype complexity (patients with single anomalies did better than patients with complex anomalies) and the response to therapy (patients who achieved complete remission had a better survival probability). In multivariate analysis, the loss of band 7q32 was found to be significantly related to very poor prognosis. This finding suggests that band 7q32 may contain critical genes that should be explored at the molecular level. PMID- 8616022 TI - Helper and cytotoxic lymphocyte responses to chronic myeloid leukaemia: implications for adoptive immunotherapy with T cells. AB - We studied 19 patients with chronic myeloid leukaemia (CML) to characterize T cell autologous responses to leukaemia. Third party stimulated alloresponses in mixed lymphocyte reactions were normal in all patients. Using the helper T lymphocyte precursor frequency (HTLPf) assay we demonstrated a low frequency of T helper cells recognizing autologous leukaemia cells from CML blood (1/850 000) and marrow (1/965 000). However, similar frequencies to autologous bone marrow and lymphoid cells were also found in normal individuals. In 11 patients studied, HLA-matched siblings had a higher HTLPf to leukaemia than the patient's autologous response (P < 0.004). Alloresponse in mixed lymphocyte reactions (MLR), and autologous HTLPf to leukaemia, were comparable at all stages of disease progression and time from diagnosis, and independent of treatment given. In order to generate autologous cytotoxic lymphocyte responses to CML, lymphocytes were stimulated with CML cells. Cultures were fed again with CML cells and examined for cytotoxicity after 21 d. Strong lymphokine-activated killer (LAK) cytotoxicity was found against K562 and Daudi cell lines, and to Epstein-Barr virus-transformed allogeneic and autologous lymphoblastoid cell lines. Autologous leukaemia cells were lysed to a lesser extent in only 3/13 patients tested. The findings indicate that immune reactivity in CML is normal but suggest that CML cells are relatively resistant to lysis by cytotoxic T cells. The results do not support the existence of a leukaemia-specific T-cell response in CML. PMID- 8616023 TI - Induction of interferon regulatory factors 2'-5' oligoadenylate synthetase, P68 kinase and RNase L in chronic myelogenous leukaemia cells and its relationship to clinical responsiveness. AB - The genes crucially determining the therapeutic response of chronic myelogenous leukaemia (CML) to interferon-alpha (IFN-alpha) are unknown. Recently, two independent IFN-alpha signalling pathways were identified: the classic pathway mediates induction of 2'-5' oligoadenylate synthetase (2-5 OAS), p68 kinase and IFN regulatory factor-2 (IRF-2), whereas the alternate pathway leads to activation of IFN regulatory factor-1 (IRF-1). We investigated whether deficient or imbalanced expression of components of these two pathways is associated with resistance of CML cells to antiproliferative action of IFN alpha/beta. Constitutive and IFN-induced transcript levels of IFN-dependent genes in mononuclear cells, granulocytes, monocytes, lymphocytes and CD34+ cells of chronic-phase CML and blast crisis patients were assessed by Northern blot techniques and were correlated with subsequent clinical responses to IFN therapy. Our results demonstrated that IFN-alpha or -beta treatment in vitro and in vivo leads to an enhanced expression of IRF-1, IRF-2. RNase L, p68 and 2-5 OAS which was independent of the degree of cellular differentiation and clonal evolution of CML. Neither the magnitude of induction of these genes nor the IRF-1/IRF-2 mRNA balance differed between chronic-phase CML patients responding or failing IFN alpha therapy. These results indicate that failure of IFN-alpha treatment is not due to defects in mRNA induction of the above-mentioned candidate genes for the direct antiproliferative response to IFN type I. PMID- 8616024 TI - Measurement of health-related quality of life in multiple myeloma. Nordic Myeloma Study Group. AB - When a randomized trial (NMSG 4/90) comparing treatment with melphalan/prednisone to melphalan/ prednisone + interferon alpha-2b in newly diagnosed multiple myeloma was initiated in 1990, a quality-of-life assessment was integrated into the study. We used the questionnaire (QLQ-C30) developed by the European Organization of Research and Treatment of Cancer (EORTC) Study Group on Quality of Life. The QLQ-C30 incorporates five functional scales, three symptom scales, a global health and quality-of life scale and some single symptom measures. The questionnaire was completed prior to treatment and after 1, 6, 12, 24, 36 and 48 months. 524 (90.2%) of 581 patients enrolled in the NMSG 4/90 completed the first questionnaire, and 484 (83.3%) completed all questionnaires given to them. All but one of the scales met the minimum criteria of reliability (Cronbach's alpha >/ 0.70). Validity was shown by (1) the ability of the scales to discriminate clearly between patients differing in clinical status as defined by pretreatment W.H.O. performance index and Durie & Salmon stage, and (2) the sensitivity to changes in objective disease status (response and relapse). This is the first report of the measurement of health-related quality of life in a prospective clinical trial in multiple myeloma. The results demonstrate that the QLQ-C30 is a reliable and valid instrument for the measurement of quality of life in these patients. The data will be used for a cost-utility analysis of the results of the NMSG 4/90 trial. PMID- 8616025 TI - Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests. AB - lt could be speculated for patients with myeloma and other lymphoproliferative disorders that peripheral blood stem cells may be preferable to bone marrow for autologous transplantation because they may be less contaminated by neoplastic cells. To test this possibility, the immunoglobulin heavy chain gene rearrangement and limiting dilution polymerase chain reaction were used to sensitively quantify myeloma cells in bone marrow and peripheral blood stem cell collections, taken at a similar time, from eight patients with multiple myeloma. Levels of residual disease in the peripheral blood stem cell harvests were variable and did not reflect the tumour burden in the marrow. Peripheral blood stem cells contained 1.7 to 23700-fold fewer myeloma cells compared with the bone marrow and would have resulted in reinfusion of 0.08 to 59480-fold fewer myeloma cells based on total reinfused CFU-GM and 0.24 to 24700-fold fewer myeloma cells based on total reinfused nucleated cells. Assuming that the proportion of clonogenic myeloma cells is equivalent, peripheral blood stem cells may be better than bone marrow as a source of haemopoietic stem cells for transplantation in multiple myeloma. The clinical followup suggested that patients transplanted with peripheral blood stem cells containing a low number of myeloma cells had better disease control than those transplanted with peripheral blood stem cells containing a high number. PMID- 8616026 TI - Remission maintenance therapy with histamine and interleukin-2 in acute myelogenous leukaemia. AB - Peripheral blasts recovered from patients with acute myelogenous leukaemia (AML) were efficiently lysed by interleukin-2 (IL-2)-activated heterologous natural killer (NK) cells in vitro. The IL-2-induced killing of AML blasts was inhibited by monocytes, recovered from peripheral blood by centrifugal elutriation. Histamine, of concentrations within the micromolar range, abrogated the monocyte induced inhibition of NK-cells; thereby, histamine and IL-2 synergistically induced NK-cell-mediated killing of AML blasts. The effect of histamine was apparently mediated by H2-type histamine receptors (H2R), since the H2R antagonist ranitidine completely blocked the response. Based on these in vitro findings, seven patients with AML in first (n=2), second (n=3) or third (n=2) complete remission (CR) were given home therapy with interleukin-2 (IL-2; O.9 x 10(6) IU x 2 s.c.) and histamine (0.4 - 0.7 mg x 2 s.c.) in cycles of 21 d, separated by 6-week intervals. The patients also received treatment with low dose cytarabine and thioguanine between cycles of histamine/IL-2. Toxicity was moderate and include local reactions to IL-2 at the site of injection and short lasting flush, hypotension, and headache to histamine. The addition of histamine to treatment with IL-2 significantly enhanced the accumulation of CD25+ T cells in peripheral blood as compared to treatment with IL-2 alone (P< 0.003). Five patients remain in complete remission at 9, 18, 21, 24 and 26 months; the two patients in CR3 relapsed after 8 and 33 months, respectively. In the five patients with earlier relapse, the duration of remission after treatment with histamine/IL-2 has in each case exceeded that of previous remissions. We conclude that (i) histamine and IL-2 synergize to kill human AML blasts in vitro, and (ii) histamine/IL-2 is a safe and feasible approach to immunotherapy of AML which merits further investigation. PMID- 8616027 TI - Serum LDH value as a predictor of clinical outcome in acute myelogenous leukaemia of the elderly. AB - 72 patients aged over 60 suffering from acute myelogenous leukaemia (AML) and treated with aggressive chemotherapy were analysed with the aim of finding factors of prognostic relevance. With regard to complete remission achievement, the only variable of statistical significance found was the karyotype at diagnosis. As far as overall survival, in multivariate analysis performed by Cox hazard regression model unfavourable karyotype and serum lactic dehydrogenase (LDH) >400 IU/I were significantly related to an adverse clinical outcome. Finally, serum LDH was the only parameter to significantly influence disease-free survival. We conclude that serum LDH is a main prognostic factor in AML of the elderly and a potentially useful tool for identifying subsets of patients who may obtain a substantial advantage from aggressive chemotherapy. PMID- 8616028 TI - Adult T-cell leukaemia/lymphoma with multiple integrations of human T-cell lymphotropic virus type I proviral DNA: differing clinical features are linked to varied proviral integration. AB - Multiple integrations of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA are occasionally found in tumor cells from patients with adult T-cell leukaemia/lymphoma (ATL). However, the clinical implications of multiple integrations of HTLV-I in ATL have not been well established. We studied 95 patients with ATL to elucidate the relationship between the multiple integrations of HTLV-I and the clinical characteristics. The proviral DNA of HTLV-I was examined by standard Southern blot analysis using the probe of an entire HTLV-I genome and the endonucleases with or without cleavage sites within the provirus. Multiple integrations of HTLV-I were detected in eight patients as extraordinary multiple bands; five patients showed multiple bands of the same intensity, and the remaining three showed multiple bands of differing intensities. The patients were divided into two groups based on these band patterns. One group was considered to exhibit one tumour cell clone carrying multiple copies of the provirus, whereas the other was considered to exhibit multiple tumour cell clones, each carrying one copy of the provirus. The former group of patients manifested a highly aggressive clinical course with frequent peculiar organ infiltrations, including the retina, uvea and muscle, along with the presence of large peripheral leukaemic T cells having flower-like nuclei. The latter group demonstrated an indolent clinical course with skin lesions or small leukaemic T cells having cleaved or lobulated nuclei. These findings suggest that the pattern of multiple HTLV-integrations into the tumor cell(s) has clinical implications in ATL. This may help to explain the heterogeneity of this disease. PMID- 8616029 TI - Elevated serum levels of soluble ICAM-1 in non-Hodgkin's lymphomas correlate with tumour burden, disease activity and other prognostic markers. AB - The serum levels of soluble ICAM-1 (CD54) were significantly elevated in patients with non-Hodgkin's lymphomas (NHL, n=127) and hairy cell leukaemia (HCL, n=15) compared with healthy controls (n=31). In high-grade malignant NHL (n=79) the sICAM-1 levels correlated with the tumour mass as reflected in the Ann Arbor staging system but not with bulky disease. Further, the sICAM-1 levels correlated with disease activity as reflected by the presence of B symptoms and with other known prognostic markers. In particular serum thymidine kinase (sTK). In patients with low-grade malignant NHL (n=48) a trend towards higher serum levels of sICAM 1 was found in patients with advanced stage and B symptoms. In both low and high grade malignant NHL, elevated levels of sICAM-1 were associated with poorer overall and disease-free survival. The present results indicated that sICAM-1 levels have a prognostic power equal to that of other serum markers claimed to be of prognostic value in NHL, namely serum lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), beta-2-microglobulin (beta2m), serum thymidine kinase (sTK), albumin and orosomucoid. The cellular origin and the possible interactions between soluble and surface ICAM-1 and its ligands needs further exploration. PMID- 8616030 TI - Bcl-2 rearrangements with breakpoints in both vcr and mbr in non-Hodgkin's lymphomas and chronic lymphocytic leukaemia. AB - The bcl-2 gene is rearranged in most cases of follicular lymphoma and the breakpoint clusters into two specific regions: mbr and mcr. Rearrangements to immunoglobulin heavy chain genes (IgH) result in a deregulation of the gene and increased transcription of mRNA for the bcl-2 protein. In chronic lymphocytic leukaemia (CLL) expression of bcl-2 protein is increased but rearrangement of the gene can be found only in a minority of cases: commonly a variant translocation with a breakpoint region located 5' of the bcl-2 gene (vcr) with preferential rearrangement to immunoglobulin light chain genes. We have analysed breakpoints in mbr and vcr in malignant cells from 96 patients with B-CLL, 45 with hairy cell leukaemia (HCL) and 41 with high- and low-grade non-Hodgkin's lymphomas (NHL). Vcr rearrangements were observed in nine patients (12%) with B-CLL. Four patients had co-migration of rearranged bcl-2 bands to kappa genes and two patients to IgH. Cytogenetic abnormalities involving 18q21, the site of the bcl-2 gene, was found in two cases only. In several cases with bcl-2 gene rearrangement chromosomal aberrations not including 18q21 were observed. In six patients (two B CLL, one follicular lymphoma, one immunocytoma and two high-grade lymphomas), breakpoints in both vcr and mbr were found. In HCL a rearrangement in the vcr region was found in one case. Bcl-2 protein immunostaining of B-CLL showed intense bcl-2 expression in all cases and no correlation was found between gene rearrangement and protein expression. Our study confirms that breakpoints in the bcl-2 gene commonly cluster to the vcr region in B-CLL, but in most cases over expression of bcl-2 protein has to be explained by other mechanisms than bcl-2 gene rearrangement. We also report that simultaneous breakpoints in mbr and vcr is a recurrent phenomenon in B-CLL and in other high- and low-grade non-Hodgkin's lymphomas. PMID- 8616032 TI - Clinical relevance of residual disease monitoring by polymerase chain reaction in patients with ALL-1/AF-4 positive-acute lymphoblastic leukaemia. AB - In this study we used reverse transcriptase-polymerase chain reaction (RT-PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with All-1/AF-4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults. Eleven patients were treated with high-dose intensive induction and consolidation chemotherapy without bone marrow transplantation and one received conservative treatment due to poor performance status. Three had resistant disease, four relapsed within 12 months after achieving complete remission, and five are in continuous complete remission (CCR) at 32, 39, 52, 53 and 61 months from diagnosis, respectively. The sequential analysis of the ALL 1/AF-4 hybrid transcript showed a persistently negative RT-PCR in the five CCR long-term survivors. The PCR analysis resulted persistently positive in the remaining seven cases, including the four cases who relapsed after the achievement of clinical CR. These data emphasize the clinical relevance of PCR monitoring analysis in t(4;11) ALL patients and should be considered in order to better determine variable post-remission treatment according to risk prediction. PMID- 8616031 TI - Reverse transcriptase/polymerase chain reaction follow-up and minimal residual disease detection in t(1;19)-positive acute lymphoblastic leukaemia. AB - The t(1;19) is the most frequent recurring chromosomal translocation in childhood acute lymphoblastic leukaemia (ALL). In most cases typical chimaeric E21-PBX1 transcripts are expressed as a consequence of this rearrangement, allowing the molecular detection of the t(1;19) at the RNA level. This translocations has been associated with a poor clinical outcome, although intensified chemotherapy has been reported to nullify its adverse prognostic impact. We therefore used reverse transcriptase/polymerase chain reaction (RT-PCR) to detect residual leukaemic cells at successive times during treatment and to monitor the response to chemotherapy in six t(1;19)-positive ALL pediatric patients. Five of these patients rapidly achieved molecular remission and no evidence of minimal residual disease (MRD) was found in the remission bone marrows beyond the third month of treatment. One patient still displayed residual leukaemic cells at the end of therapy, although she has been in continuous complete clinical remission (CCR) for 84 months. However, this patient is peculiar in our series in that two different types of chimaeric E2A-PBX1 transcripts were expressed in her leukaemic cells, only one being detectable in remission. PMID- 8616033 TI - The GIMEMA ALL 0183 trial: analysis of 10-year follow-up. GIMEMA Cooperative Group, Italy. AB - We report the 10-year follow-up of the GIMEMA ALL 0183 trial. From 1983 to 1987, 358 adults with acute lymphoblastic leukaemia (ALL) were entered into this trial, which included a mild induction, an early intensive consolidation, a post consolidation phase randomized in conventional maintenance (arm A) and in more intensive regimen (arm B). CNS prophylaxis did not include CNS irradiation. The overall complete remission (CR) rate was 79.3% (284/358); 212 patient were randomized (110 in arm A and 102 in arm B). The median overall CR duration was 20 months and the median overall survival (OS) 21 months; both curves reach a plateau after 6 years; at 10 years 25% of patients were projected to be in long term remission and survivors. The median disease-free survival (DFS) was 17 months, at 10 years 27% and 28% of patients were DFSs in arm A and in arm B respectively. In multivariate analysis age, WBC count and L2 FAB subtype were found to significantly influence OS and DFS. With regard to our previous report OS appears to linearly correlate with initial WBC count and age (P = 0.0002 and P = 0.042 respectively). 195 (68.7%) patients relapsed (only 25 had isolated CNS). The overall second CR rate was 56.5%; 23 patients underwent transplantation (12 BMT and 11 ABMT). Post-relapse survival was found to be influenced by the duration of first CR. PMID- 8616035 TI - Mutational analysis of the p15 and p16 genes in acute leukaemias. AB - Recently the p16 and related p15 genes have been described as candidate tumour suppressors at chromosome 9p21. These genes have been found to be homozygously deleted at a high frequency in several types of solid tumours and also in acute lymphoid leukaemias. In order to determine whether these genes are more widely involved in haematological malignancies, we have investigated a total of 84 samples that did not have homozygous p16 or p15 deletions from patients with acute lymphoid leukaemia (n=13), acute myeloid leukaemia (n=24) and chronic myeloid leukaemia in blast crisis (n=43) as well as four haemopoietic cell lines. p15 and p16 exon 1 and exon 2 were amplified by polymerase chain reaction (PCR), analysed by single-stranded conformation polymorphism (SSCP) and subsequently by sequencing. Within the p16 gene, a G-->A polymorphism at nucleotide 436 was found in 3/80 (4%) leukaemias and the cell line HL60. This cell line had also a C-->T mutation at nucleotide 232 which causes a premature stop codon. Analysis of the p15 gene revealed a C-->A mutation within the noncoding sequence 27 nucleotides upstream of exon 2 in 10/80 (13%) cases. These data show that inactivation of either the p15 or p16 gene by point mutation is a very uncommon event in acute leukaemias. PMID- 8616034 TI - Secondary acute leukaemias with 11q23 rearrangement: clinical, cytogenetic, FISH and FICTION studies. AB - Three patients with secondary acute leukaemia after treatment with topoisomerase II inhibitor agents are described. Two patients had acute myeloid leukaemia (AML). FAB M5a, one had pro-B-acute lymphoblastic leukaemia (ALL). The interval between initiation of chemotherapy and the onset of secondary acute leukaemia was 19-20 months. 11q23 rearrangements were detected in all cases. They were due to translocations t(11;19) (q23;p13.3), t(11;16)(q23;p13) and t(4;11)(q21;q23), respectively. Fluorescence in situ hybridization (FISH) with Yeast Artificial Chromosome (YAC) probe 13HH4 spanning the ALL-1 gene on 11q23 confirmed that in each case the ALL-1 gene had been disrupted by the translocations. The study underlined the relationship between the development of secondary acute leukaemias with 11q23 rearrangement and previous chemotherapy with topisomerase II inhibitor agents. So far, however, only six adult patients with secondary ALL with t(4;11) after treatment with topoisomerase II inhibitor agents have been reported. All with t(4;11) mostly occurs in infants or young children. Our patient received epirubicin continuously for >19 months. This indicates that both myeloid and lymphoid leukaemias with involvement of the ALL-1 gene can be induced by exogenous agents, especially topoisomerase II inhibitors. Thus they may have a common biological background. This hypothesis was substantiated by means of combined immunophenotyping and FISH (FICTION). In the case of AML M5a with t(11;19), the tumour cells with ALL-1 rearrangement expressed CD34. Moreover, the pro-B-ALL with t(4;11) was CD34 positive. These findings suggest that the cell of origin of secondary AML and ALL with 11q23 rearrangement is an immature haemopoietic progenitor cell. PMID- 8616036 TI - Lack of correlation between ABL-BCR expression and response to interferon-alpha in chronic myeloid leukaemia. AB - Interferon-alpha (IFN-alpha) is useful in the treatment of Philadelphia (Ph) positive chronic myeloid leukaemia (CML). There is, however, a marked heterogeneity among CML patients in relation to their response to IFN-alpha treatment, the reasons for which are unknown. Since the reciprocal ABL-BCR gene is transcriptionally active in only a proportion of CML patients, it has been suggested that response to IFN-alpha may correlate with ABL-BCR expression. In the present study we have tested 209 Ph-positive CML patients for expression of ABL-BCR, BCR-ABL and the normal BCR and ABL genes by reverse transcriptase/polymerase chain reaction (RT/PCR). Whereas BCR-ABL, BCR and ABL transcripts were detected in all the patients, ABL-BCR expression was observed in 59% of the cases. A group of 105 patients within this series was treated with IFN alpha; 33% achieved a complete or major cytogenetic response (< 35% Ph-positive metaphases) and the remaining 67% showed minimal or no response to IFN-alpha. The proportions of patients who were ABL-BCR positive (63%) and ABL-BCR negative (37%) were the same for good responders and poor responders, suggesting that there is no correlation between ABL-BCR expression and cytogenetic response to IFN-alpha in CML. PMID- 8616037 TI - Molecular cloning of the breakpoint of t(11;22) (q23;q11) chromosome translocation in an adult acute myelomonocytic leukaemia. AB - Southern blot analysis with a cDNA probe of MLL indicated that the breakpoint is in a BamHI 8.3 kb fragment which carries the exon 5-11 of MLL gene in DNA from an adult acute myelomonocytic leukaemia with a t(11;22) (q23;q11) translocation. The structural analysis of the rearranged MLL locus demonstrated that the breakpoint is localized between exon 8 and 9 of MLL locus. The normal counterpart fused to the MLL locus was proved to be derived from chromosome 22q11(AF-22) by somatic cell hybrids analysis and FISH. By FISH, AF-22 was localized to the region more centromeric to the BCR gene. PMID- 8616038 TI - Chronic myelomonocytic leukaemia with t(8;9)(p11;q34) in childhood: an example of the 8p11 myeloproliferative disorder? AB - We describe the case of a 10-year-old girl with chronic myelomonocytic leukaemia with the chromosomal translocation t(8;9)(p11;q34), who had developed tonsillar lymphoma as extramedullary involvement at the initial presentation. The cytogenetic study of the cells in both bone marrow and tonsils demonstrated t(8;9)(p11;q34), despite no malignant features in the bone marrow specimens. She developed acute leukaemic transformation 8 months after diagnosis during chemotherapy for lymphoma. Although etoposide reduced the number of blasts, t(8;9)(p11;q34)-bearing cells were not eradicated. Complete remission was obtained following an unrelated bone marrow transplantation. The clinical characteristics of this patient are similar to those of the patients with t(8;9)(p11;q34 or q32) or t(8;13)(p11;q11 or q12) reported previously. The unusual progression of the disease might be associated with the presence of (8;9)(p11;q34), suggesting a part in the 8p11 myeloproliferative syndrome. PMID- 8616039 TI - Spontaneous remission in a patient with therapy-related myelodysplastic syndrome (t-MDS) with monosomy 7. AB - We describe a case of spontaneously reversible refractory anaemia, a subtype of myelodysplastic syndrome (MDS), with monosomy 7 secondary to chronic treatment with azathioprine (AZA) in a young renal transplant recipient. AZA was stopped after that conventional cytogenetics and fluorescence in situ hybridization (FISH) had demonstrated the existence of a monosomy 7 clone, 4 months later, haematological values had considerably improved and the karyotypic examination as well as the FISH analysis were normal. The spontaneous remission of this MDS with monosomy 7, which is usually associated with a particularly poor prognosis, could be due to the recovery of a better immunosurveillance following the withdrawal of AZA. PMID- 8616040 TI - Marrow transplantation for Fanconi anaemia: conditioning with reduced doses of cyclophosphamide without radiation. AB - Nine patients with Fanconi anaemia (FA) were conditioned for HLA-identical sibling bone marrow transplant (BMT) with reduced dose of cyclophosphamide (Cy) without radiation or antithymocyte globulin (ATG). The total dose of Cy was 140 mg/kg (n = 2) or 120 mg/kg (n = 7). The median patient age was 8 years (range 4 19). Graft-versus-host disease (GVHD) prophylaxis was with methotrexate and cyclosporine (n = 8) or cyclosporine alone (n = 1). All patients had sustained engraftment and two developed grade>/= II acute GVHD. Cy toxicity included grade >/= 2 mucositis seen in all evaluable patients and haemorrhagic cystitis in two patients. The Kaplan-Meler survival estimate is 89% with a median follow-up of 285 d (range 56-528). For the purpose of comparison, this report also reviews and updates long-term follow-up data on 32 previously reported FA patients conditioned with 140-200 mg Cy/kg without radiation. The lowest dose of Cy (without radiation or ATG) after which HLA-identical sibling marrow transplant can be successfully performed in FA patients has yet to be determined, but it appears that uniform and sustained engraftment can be achieved with a Cy dose of as low as 120 mg/kg. PMID- 8616041 TI - Resolution of Budd-Chiari syndrome following bone marrow transplantation for paroxysmal nocturnal haemoglobinuria. AB - Thrombosis of the hepatic veins (Budd-Chiari syndrome) is a life-threatening thrombotic complication which can occur in patients with paroxysmal nocturnal haemoglobinuria (PNH). Despite aggressive medical and surgical therapy, mortality from Budd-Chiari syndrome remains high. We report a boy with PNH who developed Budd-Chiari syndrome and underwent syngeneic bone marrow transplantation (BMT). Now, 3 years following BMT, he has had dramatic clinical and radiographic evidence of resolution of the thrombosis. We suggest that BMT for PNH can successfully correct life-threatening thrombosis in patients with PNH. PMID- 8616042 TI - Impaired survival of bone marrow GPIIb/IIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia. AB - Glycoproteic (GP) IIb/IIIa+ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV-1 seropositive thrombocytopenic patients, eight HIV-1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV-1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa+ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa+ cells from HIV-1 seropositive thrombocytopenic patients showed a significant (P < 0.0001) increase of apoptosis with respect to both HIV-1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa+ cells purified from HIV-1 seropositive thrombocytopenic patients was inversely (P < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV-1 disease nor a direct infection with HIV-1 seems to play a primary role in the impaired survival of BMGPIIb/IIIa+ megakaryocytic cells. These findings strengthen the notation that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV-1 related thrombocytopenia. PMID- 8616043 TI - Monoclonal antibody 197 (anti-Fc gamma RI) infusion in a patient with immune thrombocytopenia purpura (ITP) results in down-modulation of Fc gamma RI on circulating monocytes. AB - A 44-year old woman with refractory immune thrombocytopenia purpura was treated with the murine monoclonal antibody 197 in a phase 1 trial. It vitro studies have demonstrated that the monoclonal antibody 197 (subclass IgG2a) binds to two distinct epitopes of Fc gamma RI, with the constant domain binding to the Fc binding portion of the Fc gamma RI and the variable domain binding to a different epitope, resulting in crosslinking and modulation of this receptor. The monoclonal antibody 197 was administered on days 1, 3 and 5 at doses of 0.25 mg/kg, 0.35 mg/kg and 0.45 mg/kg, respectively. The fusions were well tolerated with transient facial flushing, and wheal-and-flare rash during the first infusion, which resolved with a slower infusion rate and the administration of diphenhydramine and acetaminophen. Although a marked clinical improvement did occur with resolution of oral ecchymoses and epistaxis after the first mAb infusion, the initial platelet count of 6 x 10(9)/I did not change appreciable over the 5 d course of monoclonal antibody treatment. Binding of fluorescein labelled monoclonal antibody 197 to peripheral monocytes showed a rapid and persistently decreased mean fluorescein intensity, indicated binding of administered 197 to the monocytes in vivo. Indirect staining for FcgammaRI using fluorescein-labelled goat anti-mouse immunoglobulin was also decreased, suggesting modulation of the receptor. The patient experienced monocytopenia which persisted throughout the 5 d of monoclonal antibody 197 therapy, but reversed following institution of intravenous IgG. These data indicate that intravenous monoclonal antibody 197 induces specific down-modulation of Fc gamma RI expression on monocytes. PMID- 8616044 TI - The role of plasma D-dimers concentration in the exclusion of pulmonary embolism. AB - OBJECTIVE: To determine the role of four ELISA D-dimer assays in the exclusion of pulmonary embolism. DESIGN: Blinded comparison using pulmonary angiography and/or lung scintigraphy as a reference method. SETTING: A secondary and tertiary referral centre. PATIENTS AND METHODS: Consecutive patients with suspected pulmonary embolism underwent lung scintigraphy, followed by angiography if a non diagnostic result was obtained. Comorbid conditions resulting in elevated plasma D-dimer levels were defined a priori. Cut-off levels for 100% sensitivity was determined. A decision-analytic model was used to determine effectiveness and costs in the management pulmonary embolism. MAIN OUTCOME MEASURES: The exclusion efficacy of the various assays at a sensitivity of 100% and cost-effectiveness. RESULTS: A total of 179 patients were included (78 inpatients and 101 outpatients; 74 patients had comorbid conditions). Pulmonary embolism could be adequately excluded in between 8% and 18% of all patients, an in between 3% and 7% and 11% and 27% of inpatients and outpatients, respectively, depending on the assay used. D-dimer assays could exclude pulmonary embolism in <5% of patients with comorbid conditions, whereas this increased to 14-32% in outpatients without comorbid conditions. A cost-effectiveness analysis showed a cost reduction of 10% at a specificity of 30%, largely due to a 28% decrease in angiography requirements. Furthermore, for every 2% decrease to sensitivity, one per 1000 evaluated patients would die as a result of inadequately treated pulmonary embolism. CONCLUSION: D-dimer ELISA assays may have a role in the exclusion of pulmonary embolism in symptomatic outpatients, where the application may reduce angiography by 30% and costs by 10%. PMID- 8616045 TI - Proteolysis of von Willebrand factor is decreased in acute promyelocytic leukaemia by treatment with all-trans-retinoic acid. AB - Plasma von Willebrand factor (VWF) was investigated in five patients with acute promyelocytic leukaemia (APL) before and after administration of the differentiating agent all-trans-retinoic acid (ATRA). The purpose of this study was to see how the proteolytic state associated with APL affects VWF structure and function and whether ATRA reverses any abnormality. At the onset of APL, multimeric analysis of plasma VWF revealed a lack of the largest multimers. After ATRA, there was a progressive correction of the multimeric pattern in all cases, with transient appearance of ultralarge multimers in two cases. Proteolysis was investigated with immunopurified and reduced VWF from each patient's plasma. This was electrophoresed and probed with two monoclonal antibodies that identify the 225 kD native subunit and the three native fragments of 189, 176 and 140 kD and differentiate novel proteolytic fragments produced by different proteinases. At the onset of APL, the 225 kD native subunit was relatively decreased, with the appearance of an array of novel VWF proteolytic fragments, ranging in size from <140 to <225 kD. These novel fragments observed in patients were similar to those produced in vitro by digestion of purified VWF with plasmin or elastase. After ATRA therapy, proteolysis diminished progressively in parallel with the improvement of other haemostatic measurements, but persisted to some extent. We conclude that VWF proteolysis in APL is produced by plasmin and elastase. Changes of VWF structure and function might adversely affect haemostasis in APL. Therefore, improvement of VWF after ATRA administration might explain in part the effectiveness of this drug in reducing haemorrhagic complications. PMID- 8616046 TI - Absence of mutations at the activated protein C cleavage sites of factor VIII in 125 patients with venous thrombosis. AB - Resistance to activated protein C (APC), caused by a mutation at amino acid position Arg506 of the factor V gene, has recently been identified as the most prevalent genetic defect associated with venous thrombosis. Similarly to factor V, mutations at the cleavage sites of factor VIII for APC may occur in patients with venous thrombosis. Here we have analysed 125 consecutive patients with incidental or recurrent venous thromboembolism for the presence of mutations at the cleavage sites for APC at amino acid positions Arg336 and Arg562 of factor VIII. Our findings indicate that mutations at these amino acid positions of factor VIII do not occur in the patient group analysed. PMID- 8616047 TI - Effect of platelet phospholipid exposure on activated protein C resistance: implications for thrombophilia screening. AB - The effect of platelet contamination and freeze-thawing on the activated protein C sensitivity ratio (APCsr) was determined. With increasing platelet count there was a progressive reduction in the ratio. Filtration of samples through a 0.2 microm filter before or after freeze-thawing abolished the development of resistance to the addition of activated protein C indicating that the phenomenon is due to the presence of a particulate factor. Contamination of normal plasma with platelets from a patient with homozygous factor V (FV) deficiency was also associated with the same development of resistance to activated protein C, indicating that the phenomenon was not due to exposure of platelet-derived factor V that might be inaccessible to APC. 82% (96/117) of FVQ506 and 32% (138/430) of FVR506 individuals had APC resistance on analysis of unfiltered plasma. However, 85% (42/50) of FVQ506 individuals had APC resistance on analysis of filtered plasma, whilst only 1/50 FV R506 individuals had APC resistance after filtration. For the purpose of identifying individuals at increased risk of venous thromboembolism due to the presence of the FVQ506 and associated APC resistance a PCR-based genotypic analysis is recommended. PMID- 8616049 TI - The R0Har RH:33 phenotype results from substitution of exon 5 of the RHCE gene by the corresponding exon of the RHD gene. AB - The highly polymorphic Rh (Rhesus) system is encoded by two homologous genes, one encoding the D polypeptide and the other the CcEe polypeptides. Partial D antigens may be caused by gene rearrangements, deletions or point mutations. In this study the molecular basis of R0Har RH:33, a Rh phenotype of low frequency, is described. The R0Har RH:33 phenotype is characterized by partial expression of D, altered expression of e, absence of G and the presence of two antigens of low frequency: Rh33 and FPTT. Southern blot analysis, RHD typing by PCR and sequence analysis of Rh transcripts revealed that the RHD gene is absent in subjects with this phenotype. Apart from the expected RHCE transcripts, a new Rh transcript, RHc(D)(e), was identified in three unrelated individuals expressing R0Har Rh:33. The RHc(D)(e) transcript showed the same sequence as the RHce transcript, with the exception of exon 5, which was substituted by the corresponding exon of the RHD gene. A method for PCR-based genotyping was developed to determine specifically the c(D)(e) haplotype. The c(D)(e) PCR proved to be a reliable alternative method for R0Har RH:33 typing. PMID- 8616048 TI - Elevated plasma levels of vascular cell adhesion molecule-1 (VCAM-1) in diabetic patients with microalbuminuria: a marker of vascular dysfunction and progressive vascular disease. AB - Advanced glycation endproducts (AGEs), which accumulate in diabetic vasculature, result in enhanced expression of endothelial cell-associated vascular cell adhesion molecule-1 (VCAM-1) as well release of a soluble form of VCAM-1 (sVCAM 1) into culture supernatants. We hypothesized that sVCAM-1 in diabetic plasma might reflect early vascular perturbation in diabetic vasculopathy. Diabetic patients with microalbuminuria, a group with a high incidence of vascular complications, had increased plasma levels of sVCAM-1, approximately 1.5-fold greater than diabetic patients without microalbuminuria; P < 0.05. sVCAM-1 may be an indicator of ongoing cellular dysfunction in diabetes, as well as a dynamic surrogate marker for the effectiveness of therapeutic interventions. PMID- 8616051 TI - Final consensus statement of the Royal College of Physicians of Edinburgh Consensus Conference on Autologous Transfusion, 4-6 October 1995. PMID- 8616050 TI - Allogeneic marrow transplantation for aplastic anaemia associated with dyskeratosis congenita. AB - Eight patients with aplastic anaemia associated with dyskeratosis congenita received allogeneic marrow grafts from either HLA-identical siblings (six patients) or HLA-matched unrelated donors (two patients). Patients who received marrow from HLA-identical siblings were conditioned with cyclophosphamide (140 200 mg/kg), with or without antithymocyte globulin. Patients who received unrelated donor marrow were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (1200 cGy). The six patients who survived for >2 weeks following transplant all had haematological evidence of engraftment, and all three patients who survived for at least a year following transplant recovered normal haematological function. Three patients died with respiratory failure and pulmonary fibrosis at 70 d. 8 years and 20 years posttransplant; three patients died during the neutropenic period of invasive fungal infections; one patient died on day 44 of refractory acute graft-versus-host disease; and one patient remains alive 463 d following transplant. The surviving patient recently underwent surgical resection of a Dukes' stage C rectal carcinoma diagnosed 14 months posttransplant. The aplastic anaemia associated with dyskeratosis congenita can be successfully treated by allogeneic bone marrow transplantation; however, this approach does not reverse the other systemic manifestations of the syndrome. The pathogenesis of the intestinal lung disease observed in dyskeratosis congenita patients following marrow transplantation is not understood. PMID- 8616052 TI - t(8:21) Myelodysplasia. PMID- 8616053 TI - Clinical significance of elevated soluble interleukin 6 receptor levels in patients with plasma cell disorders. PMID- 8616054 TI - Fludarabine-related autoimmune haemolytic anaemia in patients with chronic lymphocytic leukaemia. PMID- 8616055 TI - t(2;3) in a case of blastic transformation of essential thrombocythaemia. PMID- 8616056 TI - Hepatitis C virus infection in patients with lymphoproliferative disorders. PMID- 8616057 TI - Acquired haemophilia and its management. PMID- 8616058 TI - rVIIa therapy to secure haemostasis during central line insertion in children with high-responding FVIII inhibitors. AB - The provision of adequate haemostasis in children with haemophilia A (HA) who have developed high-responding inhibitors continues to be a great challenge to haematologists and institutional resources. The most exciting development in the management of acute bleeding in these patients, irrespective of inhibitor titre, has been the use of recombinant activated factor VII (rVIIa). Three severe HA children with high-responding inhibitors underwent four central line insertions (two Hickman catheters and two port-a-caths, one replaced because of infection) with rFVIIa cover to enable continuous FVIII infusions. No adverse haemorrhagic events occurred and immune tolerance therapy (ITT) using high-dose FVIII therapy was initiated and inhibitor eradication was achieved in al three patients at 8, 10 and 7 months. A further patient who had a central line in situ prior to the development of a high-responding inhibitor was also successfully 'tolerized' at 6 months using the same protocol. Interestingly, all four patients had the IVS 22 mutation. PMID- 8616059 TI - A flow cytometric platelet immunofluorescence crossmatch for predicting successful HLA matched platelet transfusions. AB - Platelet crossmatching may provide a useful way of selecting donors for effective platelet transfusions in patients refractory to random donor platelet concentrates due to alloimmunization. We assessed the predictive value of a flow cytometric platelet immunofluorescence crossmatch test for the outcome of HLA matched platelet transfusions in a group of alloimmunized patients. Platelet immunofluorescence (PIFT) crossmatches were performed for 104 HLA-matched platelet transfusions administered to 30 patients. A negative PIFT crossmatch correctly predicted a successful platelet transfusion (1 h post-transfusion platelet recovery >20%) in 56/75 (75%) cases. We also considered non immunological factors that, in combination with alloimmunization, might have contributed to an unsuccessful transfusion result, i.e. fever, septicaemia, splenomegaly, disseminated intravascular coagulation and bleeding. The predictive value of a negative PIFT crossmatch was better when these non-immunological factors were absent [48/59 (81%) correct predictions] than when these factors were present [8/16 (50%) correct predictions] (P=0.01; chi-square test). The effect of ABO incompatibility between donor and recipient on the predictive value of the PIFT crossmatch was also analysed. Positive PIFT crossmatches occurred more frequently in ABO incompatible donor-recipient combinations [in 18/28 (64%) cases] than in ABO-compatible donor-recipient combinations [in 11/76 cases (14%)] (P<0.001, chi-square test). Successful platelet transfusions were observed on 53/76 (70%) occasions in ABO compatible transfusions as compared to 16/28 (57%) in ABO incompatible transfusions. This difference was not statistically significant (P=0.23; chi-square test). Consequently, a negative PIFT crossmatch appeared to be non-predictive for the transfusion outcome in cases of ABO incompatibility between donor and recipient. We conclude that the PIFT crossmatch for platelet donor selection in addition to matching for HLA antigens, is predictive for the outcome of ABO compatible transfusions in alloimmunized recipients and prediction levels are increased when non-immunological causes for platelet refractoriness are absent. PMID- 8616060 TI - Evidence of involvement of cytotoxic antibodies directed against patients's HLA class II produced by transfused donor-derived B cells in post-transfusion graft versus-host disease. AB - Post-transfusion graft-versus-host disease (PTGVHD) is one of the most severe side-effects of blood transfusion. To characterize the effector cells causing this disease, we cloned lymphocytes from a PTGVHD patient's peripheral blood. T cell and B-cell clones were established, the origins of which were proven to be transfused donor lymphocytes. It was found that the B cells produced IgG that mediated complement-dependent cytotoxicity to the cells bearing the patient's HLA class II genotype. Our results suggest, for the first time, the involvement of B cell-produced cytotoxic antibodies directed against patient's HLA class II in the pathogenesis of PTGVHD. PMID- 8616061 TI - Elevated serum interleukin-6 (IL-6) is derived from neoplastic lymphoid cells in patients with B-cell non-Hodgkin's lymphoma: correlation with extent of IL-6 expression and serum concentration. AB - We investigated the expression of interleukin-6 (IL-6) mRNA in tumours from 21 patients with B-cell non-Hodgkin's lymphoma (NHL). Total RNA, extracted from diagnostic tissue specimens, was subjected to semi-quantitative analysis for IL-6 mRNA by the reverse transcription-polymerase chain reaction (RT-PCR). The amount of IL-6 mRNA ws semi-quantified against that in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells. The expression of IL-6 in neoplastic B cells was confirmed by immunohistochemistry. We then looked for correlations between the amount of IL-6 concentrations. The amount of IL-6 mRNA correlated with the circulating IL-6 concentration, suggesting that the malignant cells were the source of IL-6 in these patients with B-cell NHL. Our results suggest that the circulating IL-6 in patients with B-cell NHL is derived from the neoplastic lymphoid cells, and that neoplastic cells may act as modulators of the general status of patients with B-cell NHL. PMID- 8616062 TI - Evidence for a single origin of factor V Leiden. AB - Factor V Leiden is the most common hereditary blood clotting disorder so far identified, with an allele frequency of 4%. The low prevalence of the mutation outside of Europe suggests it occurred as a single event in the European founding population. In this study four polymorphisms have been identified defining different haplotypes in the exon 13 region of the factor V gene. One of these polymorphisms predicts a novel amino acid change, threonine to serine, in the B domain of factor V. Statistical evidence for a single origin of factor V Leiden is provided through the association of the mutation with a single exon 13 haplotype. PMID- 8616063 TI - Diagnosis of APC Resistance during pregnancy. PMID- 8616064 TI - t(2;3)(p23;q26) in a patient with AML M2. PMID- 8616065 TI - G-CSF administration following peripheral blood selected CD34-positive cells autologous transplantation accelerates haematological recovery after myeloablative therapy. PMID- 8616066 TI - Dyskeratosis congenita: an inherited bone marrow failure syndrome. PMID- 8616068 TI - Generation of human natural killer cells from peripheral blood CD34+ cells mobilized by granulocyte colony-stimulating factor. AB - We studied the generation of human natural killer (NK) cells from CD34+ cells that were isolated from peripheral blood stem cells (PBSC) mobilized by granulocyte colony-stimulating factor (g-CSF). The isolated CD34+ cells were cultured in the presence of a combination of interleukin-1 (IL-1alpha), IL-2, and stem cell factor for 5 weeks without marrow stroma. We found that the CD34+ cells isolated from G-CSF-mobilized PBSC (G-CSF/PBSC) could differentiate into a population of NK cells which were CD56+(bright)/CD3- and showed morphologic characteristics of large granular lymphocytes. Immunophenotypic analysis of the NK cells thus generated showed that a small proportion of them expressed CD2, CD8 and CD16 surface markers and approximately half of them coexpressed CD7. This NK population exhibited cytotoxic activity against a NK-sensitive cell line, K562. These observations suggest that CD34+ cells from G-CSF/PBSC contain precursors of NK cells that can differentiate into functional NK cells. PMID- 8616067 TI - Two regulation points for differentiation in the cell cycle of human megakaryocytes. AB - To examine the relationship between cell cycle progression and differentiation in megakaryocytes, variances in the cell cycle during PMA (phorbol 12-myristate-13 acetate)-induced differentiation were analysed in synchronized growing CMK cells. The cells stimulated with PMA in early S phase showed cell cycle arrest in G2 phase. In addition, the cells stimulated with PMA in early G1 phase showed cell cycle arrest in late G1. The expression of gpIIb/IIIa, megakaryocytic differentiation marker, was markedly induced in G2 phase when the cells were stimulated in early S phase, and was also induced in late G1 phase when cells were stimulated in early G1 phase. Therefore, the induction of gpIIb/IIIa expression seems to be associated with cell cycle blockage in both G2 and late G1. Expression of the transcription factor GATA-1 in cells stimulated in early S phase was much higher than that in controls at G2 phase. Furthermore, GATA-1 expression in cells stimulated in early G1 tended to be higher than that in controls at late G1. These periods corresponded to the time that the cell cycle arrest and gpIIb/IIIa expression were induced by PMA. These results suggest that the cell cycle in human megakaryocytic lineage cells may have two regulation points for differentiation. PMID- 8616069 TI - Characterization of peripheral blood progenitor cells (PBPC) mobilized by filgrastim (rHuG-CSF) in normal volunteers: dose-effect relationship for filgrastim with the character of mobilized PBPC. AB - Filgrastim (rHuG-CSF)-mobilized peripheral blood progenitor cells (PBPC) in healthy Japanese volunteers were characterized in detail using two clonal cell culture systems and double-colour flow cytometry to detect multilineage colony forming cells and subsets of CD34+ cells. The kinetics of PBPC during the administration of filgrastim was studied, and possible differences in the character of progenitor cells relative to given doses of filgrastim were investigated. Filgrastim was administered subcutaneously to normal volunteers for 7 d at doses of 100, 200 or 400 microgram/m2 (10 per cohort). Treatment with 100 or 200 microgram/m2 filgrastim was well tolerated; however, the 400 microgram/m2 dose level was not completed because of bone pain and myalgia. The treatment strikingly mobilized various types of progenitor cells, including highly proliferative megakaryocytic colony-forming cells. The number of progenitor cells peaked on days 5 and 6. The fold increase of circulating progenitor cells from the baseline value in the volunteers treated with 200 microgram/m2 filgrastim was more pronounced than in those treated with 100 microgram/m2. Treatment with 200 microgram/m2 also released the less mature progenitor cells (i.e. mixed colony forming cells CD34+/33- cells, and CD34+/HLA-DR-cells) into circulation better than the 100 microgram/m2 dose. These results suggest that daily subcutaneous injection with 200 microgram/m/2 filgrastim for 5 d will effectively mobilize, both qualitatively and quantitatively, PBPC in healthy donors. PMID- 8616070 TI - The regulation of neutrophil phospholipase A2 by granulocyte-macrophage colony stimulating factor and its role in priming superoxide production. AB - Experiments were performed to investigate the relative role of phospholipase A2 (PLA2) in the activation and cytokine-mediated priming of neutrophil superoxide production. PLA2 activity was measured with a radiometric assay which discriminates between PLA2 and the downstream enzyme, 5-lipoxygenase. In cells that had not been primed by prior incubation with granulocyte-macrophage colony stimulating factor (GM-CSF), PLA2 and NADPH oxidase were differentially stimulated by the chemotactic peptide N-formyl-met-leu-phe (FMLP), calcium ionophore, or phorbol ester. In addition, inhibition of PLA2 by mepacrine (0-100 micromol/l) did not concomitantly inhibit FMLP-stimulated superoxide production. These findings suggest that the activity of PLA2 and NADPH oxidase may be uncoupled in the unprimed cell. In cells preincubated with GM-CSF, time- and dose dependent priming of FMLP-stimulated PLA2 responses were observed and inhibition of PLA2 by mepacrine was accompanied by the inhibition of FMLP-stimulated superoxide production down to the level of unprimed cells. The effect of mepacrine was not due to inhibition of FMLP receptor expression. These data suggest that a mepacrine-sensitive PLA2 may have a role in the GM-CSF mediated priming of superoxide production. Using ionophore-stimulated PLA2 activity as a model, we showed that Bordatella pertussis toxin did not inhibit GM-CSF mediated priming, demonstrating that a pertussis-sensitive GTP-binding protein does not mediate signal transduction from the GM-CSF receptor to PLA2. The tyrosin kinase inhibitor, genestein, selectively inhibited GM-CSF primed but not unprimed PLA2 activity, demonstrating that GM-CSF-mediated priming requires tyrosine kinase activity. PMID- 8616071 TI - Serum erythropoietin levels in paroxysmal nocturnal haemoglobinuria: implications for therapy. AB - In order to assess the rationale and possible indications for the use of recombinant erythropoietin in paroxysmal nocturnal haemoglobinuria (PNH), we have measured endogenous erythropoietin (Epo) levels in 18 patients with PNH and in 44 patients with iron deficiency anaemia (IDA). In both groups of patients we found a significant inverse correlation between Epo and haemoglobin (Hb). However, the mean Epo level was significantly higher in the PNH group (385 mU/ml) than in the IDA group (136 mU/ml). The range of Epo levels at any given Hb was greater in the PNH group than in the IDA group. There was a significant positive correlation between Epo and absolute reticulocyte count. Since Epo administration is unlikely to benefit patients with high levels of endogenous Epo, we conclude that in the majority of patients with PNH there is no indication for treatment with Epo. PMID- 8616072 TI - Splenectomy as an adjuvant measure in the treatment of severe aplastic anaemia. AB - The role of splenectomy in aplastic anaemia (AA) is controversial. The hazards of operating on a severely pancytopenic patient, the fear of compromising the patient's immune function, and the improvement of non-surgical treatment have made splenectomy unpopular in this disease. We have evaluated positive and adverse effects of splenectomy in 80 patients with severe aplastic anaemia (SAA) treated with antilymphocyte globulin (ALG) (group A), using 52 nonsplenectomized ALG patients as controls (group B). All patients survived the operation. Nonfatal complications of surgery occurred in 10 (12.5%). Splenectomy induced a significant increase of peripheral blood neutrophils, reticulocytes and platelets within 2 weeks, followed by a continuous increase of all values over the following weeks. 28/132 patients (21%) developed a late clonal disorder of haemopoiesis, paroxysmal nocturnal haemoglobinuria (PNH) or myelodysplastic syndrome (MDS), or both. Their incidence was identical in groups A and B. 13/28 (59%) died, 10/17 (59%) in group A and 3/11 (27%) in group B (not significant (n.s.)). Overall probability of survival at 18 years after ALG was 51+/-6% for group A and 61+/-7% for group B (n.s.). We conclude that splenectomy in AA is safe. It induces an immediate increase of peripheral blood counts and, thereafter, a continuous improvement of haemopoiesis. It does not increase the incidence of late clonal complications but has a borderline effect on mortality from these disorders. Splenectomy should be reconsidered in selective nontransplanted patients who have prolonged transfusion requirements despite otherwise optimal treatment. PMID- 8616074 TI - A case of refractory anaemia with p53 point mutation at codon 249 (AGG to ATG). AB - Although mutations of the p53 tumour-suppressor gene have been observed occasionally in the advanced stages of myelodysplastic syndromes (MDS), they have not been detected in early refractory anaemia (RA) or RA with ring sideroblasts phases. Using single-strand conformation polymorphism analysis and direct sequencing, we searched for p53 mutations in a patient who progressed from RA to overt leukaemia. A p53 mutation at codon 249 (AGG to ATG) was observed in RA, RA with excess of blasts in transformation and overt leukaemia. We describe a case of MDS with p53 mutation at codon 249 detected in the RA phase. PMID- 8616073 TI - Five unknown mutations in the LR pyruvate kinase gene associated with severe hereditary nonspherocytic haemolytic anaemia in France. AB - A survey of PK-deficient patients by molecular biology techniques has been performed in France in 26 unrelated families, in which at least one mutation has been characterized. The patients, of European or North African origin, exhibited approximatively 10% of PK activity. Among the PK-R mutants described, mutation G1529-->A (Arg-509-->Gln) was the most frequent. The strategy followed for the description of PK mutants in France firstly involves determination of this mutation by PCR amplification and restriction enzyme digestion and, secondly, the sequencing of the gene for negative samples. Study of the mutation at residue 509 in 26 unrelated families indicated that 10/52 defective alleles possessed this mutation. Our study described seven different mutations; five of these have not as yet been documented. Two frameshift mutations were found: the deletion of one G base in a repetition of four Gs in position 1231-1234 (PK Mondor), del C-1527 (PK Rouen), and three missense mutations: G382-->C (Ala-114-->Pro) (PK Val-de Marne), C398-->T (Ser-119-->Phe) (PK Beaujon), A1217-->G (Asn-392-->Ser) (PK Paris). Two mutations which were detected have been reported previously: C760-->T (Glu-240-->End) and G1529-->A (Arg-509-->Gln. PMID- 8616075 TI - Plateau phase in multiple myeloma: an analysis of long-term follow-up of 432 patients. Finnish Leukaemia Group. AB - DAta from an 8-year follow-up of 432 myeloma patients were analysed for incidence, duration and prognostic value of plateau phase and factors favouring its achievement. The first-line chemotherapy was melphalan and prednisolone in 121 cases, combination chemotherapy in 311 cases. The survival times were similar despite different response rates. Any response resulted in survival significantly (P<0.001) better than in patients with progressive disease, but the level of response had no influence in this respect. A plateau of at least 3 months was achieved in 81%, at least 6 months in 74%, at least 12 months in 59% and at least 24 months in 33%. Groups with significantly different (P<0.001) survivals were identified: a plateau of <3 months, 3-11 months, 12-23 months, and 24 months or longer, with median survivals of 10, 27, 46 and 81 months, respectively. In the multivariate analysis of pretreatment variables, only haemoglobin (Hb) (P<0.001) and creatinine at 2 months (P<0.01) were significant for a plateau >12 months. After inclusion of chemotherapy data, Hb and time taken to reach the best response were still significant (P=0.002). The predictive power of high Hb and slow response for achieving a plateau of 6 or 12 months was 79%. Accordingly, the criteria for response of treatment for multiple myeloma should include a stable period of at least 3 months; the criteria for a plateau a stable period of at least 12 months in order to have real prognostic significance. PMID- 8616076 TI - Modulation of anti-idiotypic immune response by immunization with the autologous M-component protein in multiple myeloma patients. AB - Multiple myeloma is characterized by a proliferation of clonal B lymphocytes and plasma cells. The idiotypic structure of clonal immunoglobulin (Ig) expressed on the tumour B-cell surface can be regarded as a tumour-specific antigen and, as such, a potential target for anti-idiotypic T and B-cells in an immune regulation of the tumour-cell clone. Active immunization using the autologous mono-clonal Ig as a 'vaccine' was shown to induce tumour-specific immunity in murine B-cell tumours and in human B-cell lymphoma. With the aim to induce or amplify an anti idiotypic response in multiple myeloma, five stage I-III patients were repeatedly immunized with the autologous monoclonal IgG. Induction of idiotype-specific cellular immunity was analysed in vitro by an enzyme-linked immunospot assay (interferon-gamma and interleukin-4 secreting cells). B cells secreting anti idiotypic IgM antibodies were also analysed. An anti-idiotypic T-cell response was amplified 1.9-5-fold in three of the five patients during immunization. The number of B cells secreting anti-idiotypic antibodies also increased in these three patients. In two of the patients induction of idiotype-specific immunity was associated with a gradual decrease of blood CD19+ B cells. The induced T-cell response was eliminated during repeated immunization. Further studies are warranted to optimize the immunization schedule in order to achieve a long lasting T-cell immunity against idiotypic determinants on the tumour clone. A role for immunity in controlling the tumour clone remains to be established. PMID- 8616077 TI - Levels of mdr1 and mrp mRNA in leukaemic cell populations from patients with acute myelocytic leukaemia are heterogenous and inversely correlated to cellular daunorubicin accumulation. AB - Multidrug resistance gene (mdrl) expression is associated with a poor prognosis in acute myelocytic leukaemia (AML). Whether expression of the recently described multidrug resistance-associated gene (mrp) has any prognostic importance in AML is still unclear. The aim of the present study was to investigate the functional role of the mdr1 and mrp mRNA levels in peripheral leukaemic cell populations from patients with AML. Peripheral leukaemic cells from 10 patients with AML were incubated with daunorubicin (DNR). Cellular DNR content was analysed with a fluorescence-activated cell sorter (FACS). From each cell population the 20-25% cells with the lowest and highest DNR content were sorted out, and mdr1 and mrp RNA were quantified in these subpopulations with competitive polymerase chain reaction. The ratio between the mean DNR content in the cell populations with high and low DNR content varied between 1.9 and 6.6. the cell fraction with low DNR content had higher (3.8-40 times)mdr1 mRNA levels in 10/10 patients and higher (1.4-26 times) mrp mRNA levels in 8/10, as compared to the cell fraction with high DNR accumulation. In conclusion, mdr1 and mrp mRNA expressions are heterogenous in leukaemic cell populations from patients with AML. The mdr1 expression, and to some extent mrp expression, is inversely correlated to DNR accumulation in vitro. PMID- 8616078 TI - Molecular detection of t(8;21)/AML1-ETO in AML M1/M2: correlation with cytogenetics, morphology and immunophenotype. AB - The t(8;21) identifies a subgroup of acute myeloid leukaemia (AML) with a relatively good prognosis which may merit different treatment. It is associated predominantly, but not exclusively, with AML M2, and corresponds to rearrangements involving the AML1 and ETO genes. AML1-ETO positive, t(8;21) negative cases are well recognized but their incidence is unknown. In order to determine optimal prospective AML1-ETO RT-PCR screening strategies, we analysed 64 unselected AML M1 and M2 cases and correlated the results with other biological parameters. Molecular screening increased the overall detection rate from 8% to 14%. AML1-ETO was found in 3% (1/32) of AML M1 and 25% (8/32) of M2, including three patients without a classic (8;21) but with chromosome 8 abnormalities. It was more common in younger patients. Correlation with morphology enabled development of a scoring system which detected all nine AML1 ETO-positive cases with a false positive rate of 7% (4/55). Although certain AML1 ETO-positive cases demonstrated characteristic immunological features (CD19 and CD34 expression, CD33 negativity), each of these markers was insufficiently specific to permit prediction in an individual case. We conclude that initial routine prospective molecular screening for AML1-ETO in all AMLs, combined with standardized morphological and immunological analysis, is desirable in order to produce improved prognostic stratification and to determine whether screening can ultimately be restricted to appropriate subgroups. PMID- 8616079 TI - NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas. AB - The chromosomal aberration t(2:5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1+ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin's disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRdelta-chain gene rearranged (Ddelta2-Ddelta3 and Vdelta1-Jdelta1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRdelta chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH. PMID- 8616080 TI - Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice. AB - The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible. PMID- 8616081 TI - The role of intensive therapy and autologous blood and marrow transplantation for chemotherapy-sensitive relapsed and primary refractory non-Hodgkin's lymphoma: identification of major prognostic groups. AB - Patients with intermediate grade non-Hodgkin's lymphoma (NHL) who relapse or fail to achieve a complete remission after anthracycline-containing induction regimens have a poor outcome with conventional-dose salvage treatment. This outcome may be improved with intensive therapy and autologous transplantation (ABMT) but even in patients with proven chemotherapy-sensitive disease, relapse rates of up to 60% are observed. Reliable and powerful prognostic indicators are needed to identify appropriate patients for this expensive procedure and those subjects to whom alternative or additional treatment should be offered. We were interested in testing the hypothesis that tumour burden, and hence remission status immediately prior to transplant, is an important prognostic indicator of survival. We aggressively treated patients with conventional-dose salvage chemotherapy to maximum tumour response, and tested, by multivariate regression analysis, predictors of outcome post-transplant. We studied 81 consecutive patients with intermediate grade and immunoblastic NHL who achieved either a partial (PR) or complete remission (CR) following repetitive cycles of conventional-dose salvage therapy. Intensive therapy consisted of etoposide (60 mg/kg) and intravenous melphalan (160-180 mg/m2) with or without total body irradiation (TBI) followed by infusion of autologous unpurged bone marrow and/or blood cells. The predicted 4-year survival and progression-free survival (PFS) with a median follow-up of 37 months was 58% and 48% (95% confidence interval (CI) 37-55%), respectively. The only factor predictive of outcome was remission status at transplant (P=0.0001). The PFS at 4 years for the CR group was 61% (95% CI 53-75%). In contrast, only 25% (95% CI 11-40%) of patients undergoing autotransplant in PR were progression free at 4 years. We conclude that remission status at transplant after maximum tumour reduction is a powerful prognostic indicator. PMID- 8616082 TI - Analysis of p53 and mdm-2 expression in 18 patients with Sezary syndrome. AB - Sezary syndrome is a leukaemic form of cutaneous T-cell lymphoma which presents with multiple cytogenetic abnormalities and responds poorly to chemotherapy. Because of the importance of the p53 tumour suppressor in maintaining genomic stability and in sensitizing transformed cells to DNA damaging agents, we looked for alternations which may affect p53 functions in 18 patients with Sezary syndrome. Cytogenetic analysis suggested frequent p53 gene inactivation since 6/18 patients had loss of one copy of 17p. However, single-strand conformational polymorphism (SSCP) revealed that p53 gene mutations are relatively rare, occurring in only two of 18 Sezary patients. Neither of these two patients was missing a copy of 17p. Possible abnormalities of p53 pathway function through mdm 2 over-expression were also investigated. Although all 18 patients had normal levels of mdm-2 RNA 4/18 over-expressed mdm-2 protein. One patient with advanced disease and the highest percentage of malignant cells overexpressed mdm-2 protein and possessed a nonsense p53 gene mutation. The five patients with abnormalities of p53 or mdm-2 were found to have significantly highest absolute lymphocyte counts and higher absolute numbers of Sezary cells (P=0-021 and 0.027 respectively). In summary, molecular alternations of 17p and potential p53 pathway abnormalities are a common event in Sezary syndrome and appear to be associated with more advanced disease. PMID- 8616083 TI - Predominant expression of the long isoform of Bcl-x (Bcl-xL) in human lymphomas. AB - Bcl-x is a member of the bcl-2 family of proteins which are characterized by their ability to modulate apoptosis. Alternative splicing results in two distinct bcl-x mRNAs encoding a long isoform, bcl-xL, which acts as a bcl-2 agonist; and a short isoform, bcl-xS, which inhibits bcl-2 effects. The aim of the study was to determine whether bcl-x is expressed in lymphoma tissues and to characterize the respective production of bcl-xs and bcl-xL. We investigated the expression of bcl x mRNA in a series of 50 non-Hodgkin's lymphomas (NHL) and Hodgkin's disease (HD) cases using a RT-PCR method in order to amplify both transcripts simultaneously, and to estimate their relative abundance. The rearrangements of the bcl-2 gene were analysed by RT-PCR expression of the hybrid bcl-2-lgH mRNA. In addition, 20 PCR-positive NHL cases and three HD cases were analysed by immunohistochemistry using bcl-x polyclonal antisera. RT-PCR showed bcl-x expression in 43/45 NHLs and 5/5 HD cases. The bcl-xL transcript was predominant in all positive cases and was associated with variable amounts of bcl-xS. There was no significant correlation between the profile of bcl-xL/bcl-xS expression and the histological and immunological subtyping. Bcl-x immunodetection was positive in the neoplastic cell component in all analysed cases, but the degree of staining was highly variable between cases. Expression of the hybrid bcl-2-IgH gene was detected by RT-PCR in five cases of follicular NHL and in one case of HD, but this group of tumours did not display a particular profile of bcl-xL/bcl-xS expression. We conclude that bcl-x is commonly expressed by malignant cells in various types of malignant lymphomas, with a predominance of the bcl-xL transcript. Since the corresponding bcl-xL isoform can block the cell death machinery and potentialize bcl-2 effects, it may be involved in some pathways of lymphomagenesis. PMID- 8616085 TI - Bone marrow transplantation from partially HLA-matched related donors in adults with leukaemia: the experience at the University Hospital of Essen, Germany. AB - The Seattle group has demonstrated that bone marrow transplantation (BMT) using partially HLA-mismatched related donors is feasible in principal. However, it was unclear whether these results can also be achieved at smaller-sized BMT units. Therefore a matched pair analysis enrolling 52 BMTs from partially HLA-mismatched relatives and 52 control BMTs from HLA-identical siblings was performed at the University Hospital of Essen. Overall survival (OS) and incidence of graft-versus host disease (GVHD) did not differ significantly after study and control BMTs (OS: 52% v 63% 1 year, 46% v 48% 5 years post transplant; acute GVHD: 37% v 35%, chronic GVHD: 67% v 55%). After study BMTs, however, therapy-related mortality (P=0.018) and incidence of graft failure (P=0.002) were increased, whereas relapse rate was reduced (11% v 27%). Two or three mismatches in HVG direction implied the same risk of graft failure as one mismatch. Therefore, (i) OS after BMT from one HLA antigen mismatched relative and from HLA-identical siblings does not differ significantly even when performed at a 'smaller' centre; (ii) two or even three HLA mismatches in host-versus graft (HVG) direction might be acceptable in family BMT for leukaemia. PMID- 8616084 TI - Regulated plasma levels of colony-stimulating factors, interleukin-6 and interleukin-10 in patients with acute leukaemia and non-hodgkin's lymphoma undergoing cytoreductive chemotherapy. AB - Endogenous plasma levels of granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF),IL-6 and IL-10 were measured in a total of 70 patients undergoing cytoreductive chemotherapy for treatment of acute leukaemia or non-Hodgkin's lymphomas. the diagnoses were acute myeloid leukaemia (AML; n = 30), acute lymphoblastic leukaemia (ALL;n=6), non Hodgkin's lymphomas (NHL; n=11) and other malignant haematological disorders including myelodysplastic syndromes (n=23). After chemotherapy, plasma G-CSF was elevated (mean 5.6 ng/ml; range 1.2-10 ng/ml), and was inversely correlated with white blood cell counts (WBC) (r=-0.7, p<0.001). Occurrence of fever (T>38.0 degrees C) during severe myelosuppression (WBC<1x10(9)/1) was associated with an additional increase of G-CSF levels (P<0. (P<0.001). Plasma IL-6 correlated significantly with fever (range <1 to 1100 pg/ml, mean 130 pg/ml; r=0.5, P<0.001) but revealed only a weak association with WBC or platelet counts. In patients treated with recombinant G-CSF (n = 9), an association between IL-6 and fever was still observed after chemotherapy. During the nonfebrile status (total n = 242; AML n = 124), IL-6 levels remained <9 pg/ml in 90% of cases, whereas G-CSF increased with leucopenia (r = -0.72;P<0.001). In contrast, endogenous GM-CSF remained normal and IL-10 showed only a slight increase (21% of samples; maximum 22 pg/ml) in severe leucopenia. In particular, IL-10 levels did not correlate with G-CSF or IL-6 levels. We conclude that systemic release of G-CSF and IL-6 is obviously nit abrogated by cytoreductive chemotherapy in acute leukaemia and NHL may add to the therapeutic efficacy of recombinant cytokines. Also, plasma levels of G-, GM-CSF or IL-6 appear to be regulated by separate mechanisms. PMID- 8616086 TI - Triple immunofluorescence staining for prediction of relapse in childhood precursor B acute lymphoblastic leukaemia. AB - In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+ CD19+ cells and CD20- CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10-28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM sample indicated that only a subpopulation of the CD34+ CD10+ CD19+ and CD34+ CD20- CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted. PMID- 8616087 TI - Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCRdelta gene rearrangements. AB - TCR delta gene recombination patterns were analysed by Southern blot, polymerase chain reaction and nucleotide sequencing in acute myeloid leukaemias, with coexpression of lymphoid antigens (Ly+ AML, n=10) as well as in early T-cell acute lymphoblastic leukaemias with (My+ T-ALL, n=10) and without coexpression of myeloid antigens (My(-) T-ALL, n=9). These 29 acute leukaemias exhibiting TCR delta gene rearrangements were selected from 66 Ly+ AML, 14 My+ T-ALL and 12 My( ) T-ALL cases. Similar recombination patterns, namely D delta 2J delta1 and V delta 1J delta1 gene rearrangements, were observed in Ly+ AML and My+T-ALL. In contrast to V delta2 D delta3 rearrangements in B-cell precursor ALL, these rearrangements require activation of a T-cell-specific TCR delta enhancer. Comparison of My+ T-ALL and Ly+ AML with My(-) T-ALL exhibited a higher incidence of incomplete D delta 2J delta1 rearrangements in My+ T-ALL and Ly+ AML. Since a D delta 2J delta1 rearrangement is an early event in TCR delta recombination, these leukaemias seem to be arrested at an earlier stage of differentiation. Similar patterns of TCR delta rearrangements in My+ T-ALL and Ly+ AML suggest existence of a common myeloid/T-lymphoid progenitor cell. Although weak or missing expression of terminal deoxynucleotidyl transferase (T delta T) was found in 7/10 Ly+ AML cases, no difference was observed in numbers of N-nucleotides inserted in junctional regions when comparing with 3/10 cases exhibiting TdT expression. Since TdT activity is necessary for N-nucleotide addition, this finding suggests down-regulation of T delta T expression after rearrangement took place in these Ly+ AML cases. PMID- 8616089 TI - Cytogenetic remission induced by interferon-alpha in a myeloproliferative disorder with trisomy 8. AB - We report a case of an unusual myeloproliferative disorder with trisomy of chromosome 8 characterized by peripheral blood thrombocythaemia and pronounced bone marrow myelodysplastic features. Treatment with interferon-alpha at intermediate dosages, together with a complete bone marrow and peripheral blood haematology remission, was able to induce a cytogenetic remission, shown by the complete disappearance at the conventional cytogenetic analysis of trisomy 8 abnormality characterizing the neoplastic clone at diagnosis. However, trisomy 8 was still detectable by FISH in a small proportion of cells, showing that profound suppression of, but not complete eradication of, the neoplastic clone had occurred. The ability of interferon-alpha to induce a major cytogenetic remission in a +8, Philadelphia-chromosome negative, myeloproliferative disorder has not previously been documented and may provide further indications for the use of this drug in these disorders. PMID- 8616088 TI - All-trans-retinoic acid (ATRA) responsive skin relapses of acute promyelocytic leukaemia followed by ATRA-induced pseudotumour cerebri. AB - A 30-year-old woman with acute promyelocytic leukaemia (APL) went into complete remission following idarubicin and cytarabine chemotherapy; 18 months later she developed repeated skin relapse, with no bone marrow involvement. DNA and RNA analysis of skin lesions revealed the presence of the PML/RAR alpha hybrid gene, which was not detected at the same time in bone marrow. The skin relapses were successfully treated by all-trans-retinoic acid (ATRA) as single agent over 2 years. However, prolonged administration of ATRA caused pseudotumour cerebri, which disappeared upon drug withdrawal. The absence of the hybrid gene in the bone marrow by RT-PCR analysis led to the patient being autografted. PMID- 8616090 TI - Karyotype conversion in two patients with chronic myeloid leukaemia after busulphan-induced marrow hypoplasia. AB - SUMMARY: We report two patients with chronic myeloid leukaemia (CML) developing hypoplasia and karyotype conversion after conventional busulphan therapy. Initially, the percentage of Ph-positive metaphases in marrow for both patients was 100%, which steadily diminished up to a complete disappearance in case 1 and decreased dramatically in case 2 following hypoplasia. Thereafter Southern blot and RT-PCR assays revealed no abnormalities. Both patients have survived 9 years and remained in good clinical and haematological remission without any treatment until recently. We believe that the high sensitivity to busulphan therapy result in hypoplasia and karyotype conversion, which contributed to prolonged survival. PMID- 8616091 TI - Human herpesvirus-6-associated exanthema in a patient with acute lymphocytic leukaemia. AB - SUMMARY: We report the first case of human herpesvirus-6 (HHV-6) associated exanthema in a patient with acute lymphocytic leukaemia (ALL). We analysed DNA extracted from an exanthematous lesion using the polymerase chain reaction (PCR). DNA was positive for HHV-6 but negative for herpes simplex virus, varicella zoster virus, and cytomegalovirus. Immunohistochemical staining of the skin with monoclonal antibody against (HHV-6 confirmed the infection. The possibility of HHV-6 infection should be considered when an atypical skin rash is seen in patients with ALL. PMID- 8616092 TI - Leukaemic CD5+ B-cell apoptosis: co-incidence of cell death and DNA fragmentation with reduced bcl-2 expression. AB - SUMMARY: Apoptosis and bcl-2 expression were characterized in leukaemic B cells during in vitro culture. Prior to culture, > 85% of cells from each B-CLL patient expressed high levels of bcl-2. Despite this, all leukaemic B-cell populations underwent apoptosis in vitro. Furthermore, bcl-2 was down-regulated such that the B cells displayed a bcl-2high and /or bcl-2low phenotype. However, the overall number of bcl-2-positive cells remained constant. We propose that although enhanced survival of leukaemic B cells in vivo is mediated by the sustained expression of bcl-2, it is apparent that additional mechanisms are capable of overriding the protective effect of bcl-2 when bcl-2 when bcl-2 is expressed at reduced levels. PMID- 8616093 TI - Pathogenicity of IgA and/or IgM antibodies to heparin-PF4 complexes in patients with heparin-induced thrombocytopenia. AB - Antibodies to heparin-PF4 (H-PF4) complexes have been tested and isotyped in 38 patients who developed severe heparin-induced thrombocytopenia (type II HIT). All patients had a platelet count <120x10(9)/1 or a reduction of > 30% of the initial value, occurring at least 5d after the onset of heparin. Thrombocytopenia, which rapidly reversed following the withdrawal of heparin, was associated with thrombosis in nine patients. Although IgG isotypes were found in most cases (n=26), the presence of only IgM and/or IgA was observed in 12 patients, including three cases showing a thrombotic complication. Our results indicate that type II HIT may be induced by IgA and /or IgM anti-H-PF4 antibodies even in the absence of IgG isotypes. This finding demonstrates that platelet Fc receptors (FcgammaRII) are not necessarily involved in the pathogenicity of heparin dependent antibodies and emphasizes the major role of platelet PF4 receptors. The increased expression of the latter following a slight activation by thrombin, and the subsequent binding of IgM and IgA antibodies to H-PF4 on the platelet surface, may directly trigger platelet activation aggregation and thrombosis. Alternatively, thrombocytopenia could be indirectly induced through the mediation of neutrophils, monocytes and lymphocytes which expose receptors for IgA (FcalphaR) or IgM FcmuR). IgM-platelet complexes may also bind and activate complement, leading to platelet activation or destruction. Moreover, the reactivity of the antibodies with glycosaminoglycans-PF4 complexes present on the endothelial surface could also induce endothelial lesions and promote procoagulant activity and predisposition to thrombosis. PMID- 8616095 TI - Physiologic concentrations of arginine vasopressin activate human platelets in vitro. AB - Arginine vasopressin (AVP) is a neurohypophyseal peptide hormone with protean effects. Previous reports had shown that AVP stimulates platelets, but only at concentrations 3-6 logs higher than the normal plasma concentrations in humans. In this study we tested the hypothesis that AVP, at physiologic concentrations, stimulated the expression of an activation-dependent platelet antigen. Platelets obtained from normal volunteers were incubated with increasing concentrations of AVP and the expression of the activation-dependent platelet antigen P-selectin (CD62) was determined by monoclonal antibodies and flow cytometry. There was a concentration-dependent increase in CD62 expression with increasing AVP concentration; at 1 pm AVP, 24.5% (1.3-88.5%) [median (range)] of platelets expressed CD62. The selective vasopressin V1 receptor antagonist d(CH2)(5) Tyr(me)AVP (TM-AVP) completely abolished AVP-stimulated CD62 expression. We conclude that AVP can activate platelets at concentrations found in normal humans, at least in vitro, and that this response is mediated by the platelet V1 receptor. AVP may be a physiologic platelet agonist. PMID- 8616094 TI - Inhibition of platelet adhesion to collagen by monoclonal anti-CD36 antibodies. AB - Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non-denaturing procedure. These antibodies reacted with control platelets, but not Nak(a)-negative platelets which lack CD36, as measured by flow cytometry and immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg2+ -independent conditions but had lit tle effect in the presence of Mg2+. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800s-1), whereas 131.5 was without effect. These are the first anti-CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet-collagen interaction. PMID- 8616096 TI - The impact of prophyactic treatment on children with severe haemophilia. AB - Twenty-seven children with severe haemophilia receiving regular prophylactic factor concentrate were evaluated to examine the overall effectiveness of prophylaxis in modern haemophilia care. The median age at the start of prophylaxis was 6.2 years (range 1.3-15.9 years) and the cumulative length of follow-up was 808 months (mean 30, rang 7-76 months). Nine patients required a central venous catheter for venous access (age range 1.3-5.2 years), eight boys could cannulate themselves and in 10 the parents performed regular venepuncture. The mean dose of concentrate given at the time of study was 31.8 U/kg three times weekly (range 12.5.6 U/kg) or 4900 U/kg/year (range 1900-8200). None developed an inhibitor on prophylaxis, though four had previously had an antibody. The median average annual number of bleeds in the 27 patients prior to prophylaxis was 14.7 (range 3.7-35.4). On prophylaxis this fell to 1.5 (range 0-12.5) (P<0.001) and in the group as a whole the frequency of bleeds diminished in successive years on prophylaxis. All 20 children with evidence of arthropathy improved on prophylaxis and eight had reversal of chronic damage such that their joints appeared normal at the time of study. There were reductions in the need for walking aids, in hospital admissions, and in numbers of school days lost for bleeding episodes. All families feel that prophylaxis has brought about an improvement in quality of life. PMID- 8616097 TI - Development and clinical application of a new ELISA assay to determine plasmin alpha2-antiplasmin complexes in plasma. AB - Plasmin-alpha2-antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine-sepharose and elution with epsilon-aminocaproic acid. This material, characterized by SDS PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified alpha2-antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3.5% (intra-assay) and 10-6% (interassay). A mean value of 573.5+/-131.4 ng/ml was obtained from PAP concentration in a healthy population (n = 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels. PMID- 8616099 TI - Resistance to activated protein C (APCR) in children with venous or arterial thromboembolism. AB - Resistance to activated protein C (APCR), in the majority of cases due to the point mutation Arg 506 Gln of the factor V gene, has emerged as the most important hereditary cause of venous thromboembolism. Using an activated thromboplastin time (aPTT) based method in the presence of APC together with a DNA technique based on the polymerase chain reaction, we investigated 37 children with venous (V: n=19) or arterial (A: n=18) thromboembolism and 196 age-matched healthy controls for the presence of this mutation. In the control group 10 children were detected to be heterozygous for the factor V Leiden mutation, indicating a prevalence of 5.1%. 10/19 children (52%) with venous thrombosis and 7/18 (38%) patients with arterial thromboembolism showed the common factor V gene mutation. Additional inherited coagulation disorders were found in 1/10 (V:10%) and 2/7 (A:28%) APC-resistant patients. Inherited coagulation disorders without APCR were diagnosed in 3/9 (V: 33%) and 2/11 (A:18%) children. Furthermore, we diagnosed exogenous risk factors in 6/10 (V: 60%) and 2/7 (A: 28%) children with thrombosis and APCR. These data are evidence that APCR combined with exogenous reasons may play an important role in the early manifestation of thromboembolism during infancy and childhood. PMID- 8616098 TI - Characterization of a large chromosomal deletion in the PROS1 gene of a patient with protein S deficiency type I using long PCR. AB - A long-PCR-based technique was developed to investigate a possible deletion in the protein S gene, PROS1, in a family with type I protein S (PS) deficiency (pedigree PS62). Long-PCR across introns produced an unexpected 2kb PCR product between exon VII and XII not seen in control individuals, in addition to the expected 2.5 kb exon VII-VIII product. This result suggested that a deletion had occurred between exons VII and XII in the PS-deficient family members. All were heterozygous for the deletion, Sequencing of the cloned 2 kb fragment gave the precise location of the breakpoints within introns 7 and 11. Significant similarity existed in both introns to repetitive sequences, e.g. Alu and Mer12, but no significant similarity was evident between the two introns themselves. The technique of long-PCR is simple and more informative than Southern blotting in detecting and characterizing large intragenic deletions. PMID- 8616100 TI - HELLP syndrome associated with factor V R506Q mutation. AB - The pathogenesis of HELLP (haemolysis, elevated liver enzyme and low platelet count) syndrome, a severe presentation of pre-eclampsia, is still an enigma. Activated protein C resistance resulting from a mutation in coagulation factor V has recently emerged as the leading cause of thrombosis in pregnancy. We report on two patients with HELLP syndrome who were found to be heterozygous for factor V R506Q mutation, leading to activated protein C resistance. These findings suggest that the pathogenesis of HELLP syndrome is associated with a thrombotic process, and point to the potential benefit of anti-thrombotic therapy in this condition. PMID- 8616101 TI - Vascular changes in major and lingual minor salivary glands in primary Sjogren's syndrome. AB - A histological investigation of the vascular changes of three major and lingual minor salivary glands in primary Sjogren's syndrome was carried out on eight autopsied Japanese patients. This study compares vascular lesions in salivary glands between one group of four short-term corticosteroid-treated patients (Cases 1, 3, 4 and 7) and the other group of four long-term corticosteroid treated patients (Cases 2, 5, 6 and 8). We proposed the following five stages for morphogenesis of arteritis; (1) endothelial swelling, (2) thrombosis, (3) fibrinoid degeneration, (4) necrotizing panarteritis and (5) endarteritis obliterans. Endothelial swelling was seen in small-to-large arteries of major salivary glands and the tongue, and this finding was considered as the initial change of vascular lesion. Thrombosis was observed in the small arteries of both organs. Fibrinoid degeneration and necrotizing panarteritis were predominantly localized in small and middle-sized arteries. Endarteritis obliterans was observed in small and large arteries of major and lingual minor salivary glands in primary Sjogren's syndrome. Vascular lesion of this type was common in the four patients who received corticosteroid for more than 12 months. Corticosteroid therapy appears to accelerate the fibrotic change of the vascular wall. Therefore, we suggest that essential vascular lesions of major and lingual minor salivary glands in primary Sjogren's syndrome may include four types (endothelial swelling, thrombosis, fibrinoid degeneration and necrotizing panarteritis), excluding endarteritis obliterans. PMID- 8616102 TI - Hematoxylin staining in quantitative DNA cytometry: an image analysis study. AB - The suitability of commercial and pure aluminum-hematein for quantitative DNA image cytometry was investigated. Cervical smears, breast cancer aspiration biopsies, and rabbit liver tissue imprints were stained with Mayer's and Harris' al-hematein with variable staining times and dye concentrations. Pure and commercial hematoxylin was used. Nucleic acids were removed by enzyme digestion or by HCl-hydrolysis. A standard Feulgen stain served as control. DNA polyacrylamide films were used as staining models. Absorption was measured using a VIDAS image analyzer. DNA in liver cell nuclei was not stained in a stoichiometric dye-DNA ratio. Sequential staining of cervical smears with hematein followed by the Feulgen reaction gave a covariance between 0.77 and 0.88 for IOD. Photometric errors due to unspecific RNA or protein staining were remarkable. Harris' and Mayer's hematein gave comparable results. Pure hematein gave slightly better results than commercial batches. DNA staining in model films was not quantitative with hematein. Al-hematein should therefore not be used for quantitative DNA cytometry. PMID- 8616103 TI - Histometry and the H.O.M.E. concept: an aid to the grading of intra-cervical neoplasia? AB - The view has been expressed that few quantitative methods are of value to the pathologist in purely diagnostic work. Quantitative systems are perceived as too large for the average reporting room, too time consuming to learn, very expensive to buy and quick to become obsolete. Further, the software supplied usually cannot provide fully automated analysis, and user interaction is often tedious. If measurement techniques have little value in diagnosis they may have a role in assessing the prognosis of tumours. High levels of inter- and intra-observer variation in tumour grading have been reported and quantitative methods have been used to reduce this and more emphasis has been placed on the measurement of changes in tissue architecture, which may help to reduce observer variation. This paper describes such a method based on cell sociology, which has been implemented on a quantitative microscope specifically designed for use in the routine diagnostic pathology environment. The results of a preliminary study on grading cervical intraepithelial neoplasia show a significant difference between all groups (P less than 1 x 10(-5)) and a linear trend for the measurement of Area Disorder (P less than 1 x 10(-5)). PMID- 8616104 TI - Flow-cytometric analysis of oxidative and proteolytical activities in tissue associated phagocytes from normal and hypertrophic muscles. AB - The study was conducted by the known tendencies of increased stress susceptibility and metabolic disorders in individuals with hypertrophied muscles due to innate factors or intensive exercise which can induce the overtraining syndrome. Using an animal model, muscle-associated cells from normal (N) and hypertrophic (H) skeletal muscle (m.semitendinosus) were examined in their resting and phorbolmyristate acetate (PMA) stimulated oxidation of dihydrorhodamine (DHR) as well as cathepsin B and L activities. Phagocytes were phenotyped by their casein receptors (CR) and fibroblasts by their surface collagens (I and IV). The portion of CR-cells in single cell suspension was 4-8% and 1-3% in H and N. The CR-cells were enriched by 200 g centrifugation and cultured for 5 days with and without cortisol (C), norepinephrine (NE) and indomethacin (I). NE suppressed dose-dependently CR-expression in N, with increase in H occurring. C, NE and I elevated cathepsin activities only in N. PMA stimulated DHR oxidation in H and N 5- and 2-fold. Only the oxidative rate in N reacted to C, NE and I significantly. The data suggest that the response of muscle-associated cells from hypertrophied and normal muscles to signals released in stress-coping significantly differs. PMID- 8616105 TI - Application of recent stereological tools for unbiased three-dimensional estimation of number and size of nuclei in renal cell carcinoma samples. AB - New stereological methods have appeared in recent years that allow an unbiased and efficient estimation of quantitative characteristics of three-dimensional biological structures. The aim of this study is to apply the new stereological tools on renal tissue and investigate the changes in the number and size of nuclei in renal cell carcinomas. The simple and easy use of the disector, point sampled intercepts and selector methods on kidney biopsies is demonstrated. Using these approaches, the mean number of nuclei per unit reference volume: NV(nucl/tis), the volume-weighted mean nuclear volume: nu V(nucl) and the number weighted mean nuclear volume: nu N(nucl) were estimated in histological sections from 20 renal cell carcinomas. The study was performed on 10 grade 2 and 10 grade 3 renal cell carcinomas previously diagnosed by the pathologist on the basis of nuclear size increase, irregularity and nucleolar prominence. Basically, serial optical kidney sections from a 25-millimicrons thick slice were used for stereological study. The size estimations of the individual particles nu V(nucl) and nu N(nucl) obtained by the 'point-sampled intercepts' and 'selector' methods, respectively, turned out to be the most adequate parameters employed in discriminating between renal carcinoma grades (P less than or = 0.001). PMID- 8616106 TI - Classification of colorectal adenomas with quantitative pathology. Evaluation of morphometry, stereology, mitotic counts and syntactic structure analysis. AB - In search for an objective classification rule for the morphologic changes in colorectal adenomas, the results of nuclear morphometry for assessing geometric characteristics of tumour nuclei, syntactic structure analysis for assessing the arrangement of nuclei in the epithelium, stereological assessment of glandular changes, and area weighted mitotic counts were evaluated in a multivariate analysis. The H&E stained tissue sections of 59 colorectal adenomas were studied, of which 20 showed mild, 20 moderate and 19 severe dysplasia, according to blind duplicate assessments by two pathologists. In a stepwise jackknifed discriminant analysis, the combination of variables was selected that could best discriminate the cases into the previously assessed grade of dysplasia. With the combination of two variables (minimum line length and inner gland surface density), 71% of the cases could be correctly classified according to the previously assessed grade. No cases with mild dysplasia were classified as severe dysplasia and vice versa. However, since the reproduction of subjectively assessed grade was not the ultimate goal, we aimed to discriminate the cases into two groups on the basis of measurements results alone, by means of a cluster analysis. These two groups would merely reflect the cases with major versus minor morphologic changes. The results of the cluster analysis showed that all mild dysplasia cases were allocated into the low grade cluster, and that the moderate dysplasia cases were divided over both the low grade and high grade cluster. Two severe dysplasia cases with borderline morphometric results were allocated into the low grade cluster, and the remaining 16 into the high grade cluster. It is concluded that objective evaluation of dysplasia in colorectal adenomas is possible by a combination of morphometric techniques. The results of the morphometric analyses seem to favor a two-grade classification system rather than a three-grade system. PMID- 8616108 TI - Diagnosis and staging of bronchogenic carcinoma by transtracheal and transbronchial needle aspiration. AB - Transbronchial needle aspiration (TBNA) has been used in diagnosis and staging of bronchogenic carcinoma. However, its true effectiveness seems uncertain and some models of needles are expensive. The aim of this study was to procure new experiences on this method. TBNA was performed in 194 patients with bronchogenic carcinomas. Two models of cheap, re-usable, cytological needles were used. In diagnostic application, TBNA was positive in 34 of 39 (87%) central tumors and in 31 of 45 (69%) peripheral nodules or masses. In 19 patients, TBNA was the only positive sample. In staging application, TBNA was positive in 41 of the 90 cases (46%) in which the spread of the tumor compressed the wall of the airway. When the trachea or bronchus was endoscopically normal at the site of the puncture, TBNA was only positive in 3 of 20 cases (15%). These results suggest that TBNA is effective as a diagnostic tool. However, it appears to be less effective in staging, where the attainment of a good yield with TBNA probably demands a positive computed tomography and the use of a histological needle. PMID- 8616107 TI - K-ras genotypes and prognosis in non-small-cell lung cancer. AB - BACKGROUND: Despite major advances in the treatment of many kinds of cancer over the past 25 years, the overall 5-year survival of non-small-cell lung cancer patients has scarcely improved. Even in stage I which has the best outcome long term survival still falls below 70%. Since intriguing data suggest that the identification of genetic markers might allow prognosis to be assessed case by case. We were prompted to evaluate K-ras gene mutations as a putative prognostic marker in this neoplasm. MATERIALS AND METHODS: We used the polymerase chain reaction (PCR) followed by allele specific oligonucleotide (ASO) hybridization or single-strand conformation polymorphism (SSCP) assays, to detect K-ras mutations in DNA from formalin-fixed, paraffin-embedded tumor samples. K-ras mutations were examined in 192 stage I to IV non-small-cell lung cancer patients. RESULTS: K-ras mutations were detected in 51 of 192 of the cases studied (27%). All K-ras mutations detected by PCR/ASO hybridization were also identified by SSCP. In stage I disease, the median survival was 46 months in those patients whose tumors had no K-ras mutations and 21 months in those with aspartic acid and serine mutations at K-ras codon 12; in patients with stage IIIA disease, median survival time was 16 months in the K-ras negative group and 7 months in the aspartic acid and serine mutation group. No significant differences were observed for the remaining amino acid substitutions of K-ras, nor were they observed at all in more advanced disease. CONCLUSIONS: K-ras gene status has strong prognostic value in patients with stage IIIA non-small-cell lung cancer. The survival curve for patients with stage I and K-ras codon 12 aspartic or serine mutations is close to that of patients with stage IIIA without K-ras mutations. However, a non-small cell lung cancer K-ras genotypic classification should be validated in larger studies. PMID- 8616109 TI - Lung cancer and ipsilateral pleural effusion. AB - Lung cancer presenting with ipsilateral pleural effusion is considered to have a poor prognosis. Thoracoscopy has been invoked as a useful tool for staging those cases before proceeding to thoracotomy, especially in patients with large pleural effusions. In cases where there is only a small effusion or no effusion at all, direct thoracotomy would be the choice, with pleural lavage during the operation and immediate cytology investigation. We performed thoracoscopy in 76 patients with lung cancer and ipsilateral pleural effusion (55 cytologically positive and the remaining negative), and we found only five cases with potentially resectable tumor. They were submitted to thoracotomy and resection could be accomplished in none of them, due to direct mediastinal tumor invasion. On the other hand, we found visceral pleura involvement without effusion in 31 out of 167 patients submitted to thoracotomy for lung cancer in our Institution. The tumor could be resected in all but one of these cases. Talc pleurodesis was performed in all patients who were found to have pleural effusion and non-resectable tumor at thoracoscopy, and we obtained a 68% success rate in cases with no trapped lung, as opposed to 56% in patients with trapped lung (massively covered by fibrin and/or tumor). Pleural fluid glucose and pH are good predictors of the outcome of pleurodesis. PMID- 8616110 TI - The role of induction therapy and surgery for stage IIIA lung cancer. AB - In stage IIIA lung cancer, the role of induction chemotherapy or chemoradiotherapy prior to surgical resection has been studied extensively in patients identified preoperatively as having N2 disease. Both types of induction treatment have resulted in significant response and resection rates. Three trials have randomized patients to this form of treatment versus primary surgery. In all three trials the combined modality therapy has been significantly more effective, resulting in longer median survival times and estimated five year survival times. This new-found optimism for combined modality therapy including surgery is presently being compared to more standard therapy - chemoradiation for patients suffering from this stage of disease. In the future, this type of treatment will be investigated in earlier stage disease, classically treated by surgery, but often yielding less than satisfactory five year cancer free survival times. PMID- 8616111 TI - Molecular genetic tumor markers in the early diagnosis and screening of non-small cell lung cancer. AB - BACKGROUND: Little progress has been made in decreasing lung cancer mortality by applying conventional methods to early diagnosis and screening. Recent advances in molecular oncology, however, have provided tools which may be of use in this area. Many genes involved in controlling cell growth and differentiation are abnormal in lung cancer cells. Such genes include K-ras, p53, rb, myc, her2/neu, and probably one or more tumor suppressor genes on chromosome 3p. The involvement of these genes in lung cancer is reviewed. The K-ras oncogene contains a mutation in codon 12 in many cases of non-small-cell lung cancer, particularly adenocarcinoma, and is thus a potentially useful lung cancer tumor marker. DESIGN; We have developed a highly sensitive, simple assay for ras mutations, and applied it to bronchoalveolar lavage fluid obtained from patients undergoing evaluation for suspected lung cancer. RESULTS: In many cases, the ras assay was more sensitive than routine cytology and histopathology, demonstrating that this is a potentially clinically useful assay. CONCLUSION: Molecular genetic tumor markers, including mutations in ras and other genes, and/or immunohistochemical tumor markers, may provide tools which can be applied to bronchoalveolar lavage fluid or sputum, for use in diagnostic tests and in screening programs. The use of such markers may lead to decreased lung cancer mortality. PMID- 8616112 TI - Issues at the cutting edge in stage III non-small-cell lung cancer. PMID- 8616113 TI - Induction or concomitant chemotherapy and radiotherapy for non-small-cell lung cancer: myth or reality. AB - If radiation therapy was often considered to be a classical treatment for locally advanced non-small-cell lung cancer, the overall results were quite dismal. Failures were related both to local recurrences and distant metastases. During the last year, several studies have clearly suggested that combining radiation with chemotherapy given as an induction program or concurrently has led to some long term survival benefits. This paper reviews briefly the data available. Additional studies are required to better identify the optimal schedule. PMID- 8616115 TI - Chemotherapy in advanced non-small-cell lung cancer. The experience of Italian Cooperative Groups. AB - The clinical experience of Italian Cooperative Groups in the treatment of advanced non-small-cell lung cancer began in the early 1980's with several phase II trials on cisplatin-etoposide (PE) combination. PE was chosen and widely used also in neoadjuvant trials on the basis of several randomized studies showing a favourable safety-efficacy profile of this combination with respect to other chemotherapy regimens. Recently, in order to improve therapeutic results in NSCLC the Italian Oncology Group for Clinical Research compared PE versus mitomycin, ifosfamide and cisplatin (MIC) and mitomycin, vindesine and cisplatin, the three drug regimens most widely used in clinical trials. This trial showed a significant difference in response rate and a survival benefit for MIC and MVP; therefore, we considered MIC or MVP as the reference treatment is advanced NSCLC. PMID- 8616114 TI - Chemotherapy of stage IIIB and IV non-small-cell lung cancer. AB - Currently, lung cancer is a leading cause of death in men with more than half million new cases diagnosed every year. Eighty percent of these tumors are non small-cell carcinoma and 70% of these are unresectable or metastatic at the time of presentation, resulting in dramatically poor survival rates. The increasing number of drugs showing a significant activity against non-small-cell lung cancer and the widespread use of modern cisplatin based regimens offer some hope of progress and suggest that chemotherapy may have a role in treating this disease. A recent meta-analysis has confirmed the modest but significant survival benefit for patients treated with combined chemotherapy both in case of metastatic disease and in addition to radiotherapy, in locally advanced disease. PMID- 8616116 TI - Single agent Taxol, 3-hour infusion, in untreated advanced non-small-cell lung cancer. AB - Currently, only a few chemotherapeutic agents have consistently produced single agent response rates greater than 15% in patients with non-small-cell lung cancer (NSCLC). Taxol has been reported in two phase II studies to have significant activity in NSCLC with response rates of 21% and 24%. Schedule infusion of 24 hours has been used to reduce allergic reactions. The study reported here was a phase II trial of Taxol given by 3-hour intravenous infusions at a 210 mg/m2 dose every three weeks in outpatients setting. It was conducted simultaneously at three centers on chemotherapy-naive patients medicated with unresectable stage III or metastatic NSCLC. Sixty-two patients were initially enrolled; all were premedicated with dexametasone (20 mg), cimetidine (330 mg) and diphenilhydramine (50 mg), given prior to initiation of paclitaxel infusion. Fifty patients were evaluated for toxic effects and 47 for response. Sixteen partial responses (34) and one complete response (2%) were observed, for an overall response rate of 36% (95% confidence internal, 22% to 50%). Taxol was well-tolerated and none of the patients experienced allergic reaction. Granulocytopenia was generally mild. Therapy was interrupted in only two patients because of the development of grade 3 neuropathy. In our experience Taxol is one of the most active cytotoxic drugs targeting non-small-cell lung cancer. PMID- 8616117 TI - Dealing with initial chemotherapy doses: a new basis for treatment optimisation in limited small-cell lung cancer. AB - Treatment of patients with small-cell lung cancer (SCLC) remains disappointing despite initially high complete response rates. The dramatic initial chemosensitivity of tumor cells is rapidly thwarted by the early emergence of chemoresistant clonogenic cells, regardless of front line treatments. Although a dose-response relationship is well established its effect on survival is inconclusive. From 1980 to 1988, 202 patients with limited SCLC were included in four consecutive trials using an alternating schedule of thoracic radiotherapy and chemotherapy. Despite an increase in chemotherapy and/or the total radiation dose, no significant difference was observed between the four trials in terms of response, disease-free or overall survival. However, a retrospective analysis performed on a total of 131 consecutive patients led us to postulate that a moderate increase in the initial dose, i.e. first course, of cisplatin and cyclophosphamide, could improve overall survival. From 1988 to 1991, 105 consecutive patients were included in a large randomized trial to address this question. The difference in treatment options only concerned the initial doses of cisplatin (80 vs. 100 mg/m2) and cyclophosphamide (900 vs. 1200 mg/m2). According to the triangular test used in this study the trial was closed after inclusion of 105 patients, 32 months after the start of the study because, at that time, overall survival was significantly better in the higher-dose group (p = 0.001). This debatable concept of dose-intensity having an impact on survival offers new possibilities for the management of SCLC. The contribution of hematopoietic support may help to validate this concept. PMID- 8616118 TI - Gemcitabine in the treatment of non-small-cell lung cancer. AB - Gemcitabine is a nucleoside analogue with activity in non-small-cell lung cancer (NSCLC). Phase I trials have determined the best tolerated dose of gemcitabine in chemotherapy-naive patients to be 1250 mg/m2 given as a 30-minute infusion weekly x 3 every 4 weeks. The single-agent efficacy of gemcitabine has been assessed in 4 phase II trials (361 patients) at dose of 800-1250 mg/m2/weekly x 3 every 4 weeks. Single-agent response rates (externally verified by Oncology Review Board) were > 20%, with duration of response 7.6-12.7 months and median survival 8-9 months. Dose-limiting toxicity was neutropenia, but this was rare, even at the highest dose levels. In 3 Japanese studies, response rates of 16.3%, 26% and 20.9% were seen in untreated patients. Pooled data from all NSCLC studies shows that responses were seen in stages IIIA, IIIB and IV disease, and were similar in adeno and squamous cell types. Gemcitabine has also been studied in combination with other drugs active in NSCLC. In one study 50 patients were treated with gemcitabine and cisplatin given weekly x 3 every 4 weeks, cisplatin at a dosage of 25-30 mg/m2 and gemcitabine at doses escalating from 1000 mg/m2 in steps of 250 mg/m2 per cycle, 38 of 50 patients have been evaluated to date, with an overall response rate of 31.6%. Dose limiting toxicity was rare, but there was evidence of cumulative haematological toxicity with grade 4 granulocytopenia and thrombocytopenia becoming more frequent with repeated administration. Similar activity was seen when gemcitabine (1000 mg/m2) was combined with monthly cisplatin (60, 75, 100 mg/m2). Other studies have shown that gemcitabine can enhance radiosensitivity in NSCLC and other solid tumour types such as pancreas/breast and colorectal cancer cell lines. PMID- 8616119 TI - Gene therapy for lung cancer. AB - Lung cancer is a leading cause of cancer death and standard chemotherapies are resulting in only marginal improvements in outcome. Experimental approaches involving gene therapy are attractive in this clinical setting. There are two basic types of genes utilized, either those intended to induce immunity or those that are directly tumoricidal. Immunity-inducing genes that have been used in model (and some human) systems include MHC molecules, costimulatory molecules, and cytokines such as IL-2, IL-4, IL-6, GM-CSF. These are intended to induce effective systemic immune responses against tumor antigens which would not otherwise develop. Direct toxic approaches include the reintroduction of tumor suppressor genes or enzymes which convert non-toxic drugs to toxic ones, such as herpes thymidine kinase. As a means for gene delivery, retroviruses are the most common vehicle, although Adenovirus vectors and direct DNA delivery have specific advantages. PMID- 8616121 TI - 1st Andorran Non-Small-Cell Lung Cancer Congress. Andorra, February 22-24, 1995. PMID- 8616120 TI - p53 nuclear immunostaining and gene mutations in non-small-cell lung cancer and their effects on patient survival. AB - BACKGROUND: p53 gene mutations are known to occur in about half of all non-small cell lung cancer (NSCLC) cases. Mutations of the p53 gene usually but not always lead to an increased half life of the p53 protein, and result in a nuclear accumulation of protein which can be detected by immunohistochemistry (IHC). Controversy still exists as to whether the presence of an aberration of the p53 gene or protein is a poor prognostic indicator in patients with NSCLC. PATIENTS AND METHODS: DNA samples and paraffin blocks were obtained from 129 patients of 143 consecutive patients who underwent a pulmonary resection during a 22-month period from July 1991 to April 1993. Mutations of the p53 gene occurring at exons 5-8 were detected by a polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) assay, while the nuclear accumulation of the p53 protein was detected by immunohistochemistry. RESULTS: Of the patients studied, 35% had mutations and 54% showed overexpression, when we defined a positive case as being one in which more than 10% of the tumor cell nuclei were stained. There was a 59.5% concordance between the p53 gene mutations and p53 immunopositivity. p53 immunopositivity in adenocarcinoma and any p53 abnormality (i.e. p53 immunopositivity and/or mutation) in adenocarcinoma were a poor prognostic indicator. However, Cox's proportional hazards model indicated that the stage was the only significant prognostic factor. CONCLUSIONS: p53 immunopositivity and mutations of the p53 gene are frequently seen in NSCLC. They are considered to be mutually related but may sometimes represent a different aspect of p53 abnormality. p53 alteration may be a poor prognostic indicator only in a subset of patients with NSCLC, especially for adenocarcinoma. PMID- 8616122 TI - The magnesium sulphate story. PMID- 8616123 TI - Magnesium sulphate regimens for women with eclampsia: messages from the Collaborative Eclampsia Trial. PMID- 8616124 TI - The modern preventative treatment of recurrent miscarriage. PMID- 8616126 TI - Serum lipids in early pregnancy and risk of pre-eclampsia. AB - OBJECTIVE: To determine whether first and late second trimester serum total and high density lipoprotein cholesterol are associated with blood pressure, uterine artery pulsatility index and pregnancy outcome. DESIGN: A prospective cohort study. Data were analysed using multiple linear and logistic regression analysis. PARTICIPANTS: Three hundred and ninety-three pregnant women requesting chorionic villus sampling because of advanced maternal age (36 years and older). MAIN OUTCOME MEASURES: Serum total cholesterol and high density lipoprotein cholesterol were measured by an automated enzymatic method. Uterine artery flow velocity waveforms were recorded using continuous Doppler ultrasound. Pregnancy outcome was assessed by questionnaire. RESULTS: First trimester serum total cholesterol was significantly associated with the risk of pre-eclampsia, with the adjusted relative risk exceeding 5 for women with serum total cholesterol levels above 6.0 mmol/1 when compared with women with a cholesterol level below 5.0 mmol/1. First trimester serum total cholesterol also showed a significant relationship with diastolic blood pressure (coefficient of linear regression = 0.02 (mmol/1)/mm Hg, 95% CI = 0.01 to 0.03), and the change in both diastolic and systolic blood pressure from the first to the late second trimester was associated with linear changes in serum total cholesterol and high density lipoprotein cholesterol. CONCLUSIONS: These data suggest a relation between serum lipids in early pregnancy and the development of pre-eclampsia. PMID- 8616125 TI - Randomised study assessing the effect of phenytoin and magnesium sulphate on maternal cerebral circulation in eclampsia using transcranial Doppler ultrasound. AB - OBJECTIVE: To assess maternal middle cerebral artery flow velocity patterns as measured by transcranial Doppler ultrasound (TCD) in eclampsia and to investigate the effect of the anticonvulsants magnesium sulphate (MgSO4) and phenytoin on cerebral circulation. DESIGN: Prospective randomised study. SETTING: High care obstetric unit, King Edward VIII Hospital, Durban, South Africa. PARTICIPANTS: Twenty-four eclamptic patients: 13 received MgSO4 and 11 phenytoin. INTERVENTION: Middle cerebral artery flow velocity waveforms were measured using 2 MHz pulsed Doppler ultrasound via the transtemporal approach in eclamptic patients, before and 15 minutes after the loading dose of anticonvulsant. RESULTS: Magnesium sulphate significantly reduced the pulsatility index (P = 0.002) and mean flow velocity (P = 0.02) in the middle cerebral artery, whereas phenytoin failed to produce any statistically significant effect. However, differences between groups were not statistically significant. Systolic and diastolic blood pressures were reduced in both the MgSO4 and phenytoin groups. CONCLUSION: These findings provide firm evidence that MgSO4 relieves cerebral vasospasm, compared with phenytoin, and may therefore be the better drug for the prevention of eclamptic convulsion. PMID- 8616127 TI - Perinatal morbidity in chronic hypertension. AB - OBJECTIVE: To investigate if chronic hypertension in the absence of superimposed pre-eclampsia is associated with increased perinatal morbidity (especially small for gestational age babies and preterm deliveries) when compared to the general obstetric population. DESIGN: A retrospective cohort study. SETTING: A tertiary referral obstetric hospital. PARTICIPANTS: One hundred and fifty-five pregnant women with chronic hypertension who had a diastolic blood pressure of greater than 90 mmHg before 20 weeks or had pre-existing essential hypertension were studied. The study period was January 1 1991 to June 30 1993. MAIN OUTCOME MEASURES: Perinatal related loss rate, birthweight less than the fifth centile (small for gestational age) preterm delivery, placental abruption and development of superimposed pre-eclampsia. RESULTS: Women with chronic hypertension without superimposed pre-eclampsia had an increased rate of small for gestational age babies (10.9%) compared with the general population (4.1%) (odds ratio 2.9 confidence interval 1.6 to 5.0). Women with chronic hypertension without superimposed pre-eclampsia did not have a significant increase in preterm delivery or perinatal loss. Severe hypertension (diastolic blood pressure > or =110) at less than 20 weeks was associated with a trend to an increased risk of small for gestational age babies (odds ratio 3.8 -confidence interval 1.0 to 13.7 ), increased rate of delivery at less than 32 weeks (odds ratio 7.4 -confidence interval 1.9 to 29.5-) and increased rate of superimposed pre-eclampsia (odds ratio 5.2 -confidence interval 1.5 to 17.2-). Women with superimposed pre eclampsia had the greatest perinatal morbidity. CONCLUSIONS: Women with chronic hypertension without pre-eclampsia have an increased risk of delivering a small for gestational age baby. Perinatal morbidity and pre-eclampsia is greatest in women with severe hypertension at less than 20 weeks. Preterm delivery is more common in women with superimposed pre-eclampsia. PMID- 8616128 TI - Selenium deficiency and miscarriage: a possible link? AB - OBJECTIVE: To investigate a possible relationship between a deficiency in serum selenium concentration and first trimester miscarriage. DESIGN: An observational study. SETTING: A teaching hospital in South Wales. PARTICIPANTS AND METHODS: Serum selenium, albumin and total protein concentration were measured in 40 women admitted with first trimester nonrecurrent miscarriage. The results were compared with an equal number of age-matched nonpregnant healthy volunteers, and also from 40 pregnant women attending the antenatal clinic for booking in the first trimester. RESULTS: A reduction in serum selenium normally occurs in the first trimester of pregnancies that progress to term. However, a further statistically highly significant decrease in serum selenium was observed in those women who miscarried. CONCLUSION: Further studies are required to assess the potential benefits of selenium supplements. PMID- 8616129 TI - Fetal pathology in intrauterine death due to parvovirus B19 infection. AB - OBJECTIVES: To study the pathological features of fetuses dying because of parvovirus B19 infection, with particular reference to the presence of hydrops; to assess the usefulness of immunochemistry as a screening method for the detection of parvovirus infection at post-mortem examination. DESIGN: Review of clinical, sonographic, serological and pathological data; immunohistochemical staining of post-mortem tissue. SAMPLE: Cases of intrauterine fetal death occurring during the 18-month period January 1993 to June 1994 inclusive, referred for post-mortem examination to the Pathology Department, Royal Victoria Infirmary, Newcastle upon Tyne. RESULTS: Eleven cases of fetal death due to parvovirus infection were identified. Seven fetuses were less than 18-week size. Three fetuses showed conspicuous hydropic change. One of the 11 cases was detected for the first time by retrospective immunochemical screening. Of cases originating from the Newcastle district, parvovirus infection was responsible for about 10% of all non-malformed fetal deaths occurring between 10 and 24 weeks of gestation referred for pathological examination. CONCLUSIONS: During the period of study, parvovirus infection was a relatively common cause of mid-trimester fetal death. Many fetuses dying because of this infection are not noticeably hydropic, and the possibility of parvovirus infection should be considered in any case of intrauterine fetal death. Immunochemistry can be used to confirm the histopathological diagnosis, and may be of particular help where there is advanced autolysis; immunohistochemical screening may detect occasional cases not initially identified by examination of routinely stained tissue sections. PMID- 8616130 TI - Chlamydial antibodies in women who suffer miscarriage. AB - OBJECTIVE: To ascertain the relation between previous chlamydial infection and miscarriage. DESIGN: A prospective study of 349 women who had a miscarriage. PARTICIPANTS: Women surgically treated for spontaneous pregnancy loss on an outpatient basis at a hospital. SUBJECTS: Three hundred and forty-nine women who miscarried, of whom 91 had at least one previous miscarriage and 33 had had two or more miscarriages previously. Age-matched women who had a normal pregnancy served as controls. INTERVENTIONS: Blood samples were drawn from patients and controls for antibody determination. MAIN OUTCOME MEASURES: Patients and controls were compared regarding the frequency of Chlamydia trachomatis IgG antibodies (titre > or = 1/32). Antibodies to C. pneumoniae were also analysed to study cross reactivity. RESULTS: The frequency of IgG antibodies among the patients was 137/349 (39.3%) which was not statistically different from that among controls (116/349; 33.2%). Even among those who had miscarried previously the antibody frequencies did not differ significantly between patients and controls. The rate of C. trachomatis antibodies was about the same whether or not antibodies to C. pneumoniae were present in both patients and controls. CONCLUSION: No association was found serologically between previous chlamydial infection and miscarriage and no significant cross reactivity between C. pneumoniae and C. trachomatis could be detected. PMID- 8616131 TI - Randomised trial comparing hysterectomy and transcervical endometrial resection: effect on health related quality of life and costs two years after surgery. AB - OBJECTIVE: To compare the impact of endometrial resection and abdominal hysterectomy on a range of health outcomes and health service costs, based on longer term follow up of patients randomised to a clinical trial. DESIGN: A parallel group of randomised control trial. SETTING: The gynaecology department of a teaching hospital. PARTICIPANTS: 196 women requiring surgical treatment for menorrhagia were randomised and received surgery (88 underwent resection and 97 hysterectomy). Longer term follow up was undertaken using a postal questionnaire sent to all 196 women. MAIN OUTCOME MEASURES: Longer term assessment was on the basis of menstrual symptoms, health related quality of life using the Short Form 36 (SF36) and the EuroQol visual analogue scale, patient satisfaction and health service resource cost. RESULTS: Of 196 women who were sent a questionnaire, 155 (79%) responded at an average interval of 2.8 years after initial surgery. All aspects of health outcomes were as good or better in patients randomised to hysterectomy. Among patients randomised to resection, 57% had experienced no improvement in premenstrual symptoms following surgery and 23% had taken time off work due to menstrual problems; among hysterectomy patients, these rates were 23% and 4%, respectively. Women randomised to hysterectomy had better mean scores on seven of the eight dimensions of the SF36 health related quality of life instrument, with the greatest difference being on the pain dimension (P = 0.01). Women randomised to hysterectomy were generally more satisfied with treatment (P = 0.002). By two years after initial surgery, women randomised to resection had a 12% probability of having had a repeat resection and a 16% chance of having had a hysterectomy. As a percentage of the mean total cost associated with women randomised to hysterectomy, the mean total cost of resection was 53% based on four months follow up; this proportion had increased to 71%, based on an average overall follow up of 2.2 years. CONCLUSIONS: These results show that, at an average follow up of 2.8 years among responders to a questionnaire, women randomised to hysterectomy experienced more of an improvement in menstrual symptoms and higher rates of satisfaction with treatment. There is also some evidence of superior health related quality of life amongst hysterectomy patients. However, the health service cost of endometrial resection remains lower than that of hysterectomy. An assessment of the relative cost effectiveness of the two procedures awaits further research. PMID- 8616132 TI - GnRH analogue in assessing chronic pelvic pain in women with residual ovaries. AB - OBJECTIVE: To assess whether suppression of ovarian function by a gonadotrophin releasing hormone (GnRH) analogue could assist in the diagnosis of chronic pelvic pain in women with residual ovaries. DESIGN: Uncontrolled, observational study. SETTING: District general hospital (seven women) and teaching hospital (one woman). SETTING: Eight women with residual ovaries and chronic pelvic pain. INTERVENTIONS: Goserelin 3.6 mg every 28 days was used followed by surgery to remove residual ovaries. MAIN OUTCOME MEASURES: The women's response to goserelin and surgery (12 months or more post-operatively) was assessed clinically. RESULTS. Goserelin was associated with resolution of pelvic pain in the six women who obtained relief of pain with oophorectomy. The only woman who did not respond to goserelin also failed to gain relief with surgery. One woman who responded to goserelin declined surgery. CONCLUSIONS: Suppression of ovarian function by GnRH analogues may allow differentiation of pelvic pain caused by the residual ovary syndrome from other causes. This would enable selection of cases likely to benefit from surgery, avoiding potentially difficult surgery in women who will gain little or no relief of symptoms with surgery. Only eight cases are reported and a randomised controlled trial would be required to determine the place of GnRH agonists in the treatment of the residual ovary syndrome. PMID- 8616133 TI - Obstetric practice and the prevalence of urinary incontinence three months after delivery. AB - OBJECTIVE: To examine the relation between obstetric factors and the prevalence of urinary incontinence three months after delivery. DESIGN: 2134 postal questionnaires sent between August 1989 and June 1991. SETTING: Teaching hospital in Dunedin, New Zealand. SUBJECTS: All women three months postpartum who were resident in the Dunedin area. MAIN OUTCOME MEASURE: Prevalence of urinary incontinence. RESULTS: 1505 questionnaires were returned (70.5% response rate). At three months postpartum 34.3% of women admitted to some degree of urinary incontinence with 3.3% having daily or more frequent leakage. There was a significant reduction in the prevalence of incontinence for women having a caesarean section, in particular in primiparous women with a history of no previous incontinence (prevalence of incontinence following a vaginal delivery 24.5%, following a caesarean section 5.2% P = 0.002). There was little difference between elective caesarean sections and those carried out in the first and second stages of labour. The odds ratios for women having a caesarean section were 0.4 (95% confidence interval (CI) 0.2.-0.7) (all women and all primiparae) and 0.2 (95% CI 0.0-0.6) (primipara with no previous incontinence) in comparison with those having a normal vaginal delivery. The prevalence of incontinence was also significantly lower in women having had two caesarean sections (23.3%; P = 0.05) but similar in those women having three or more caesarean sections (38.9%) in comparison with those women who delivered vaginally (37.7%). Other significant independent odds rations were found for daily antenatal pelvic floor exercises (PFE) (0.6, 95% CI 0.4-0.9), parity > or = 5 (2.2, 95% CI 1.0-4.9) and pre pregnancy body mass index (1.07, 95% CI 1.04-1.10). CONCLUSIONS: Adverse risk factors for urinary incontinence at three months postpartum are vaginal delivery, obesity and multiparity (> or = 5). Caesarean section and daily antenatal PFE appear to be protective, although not completely so. PMID- 8616134 TI - Evaluation of the home pad test in the investigation of female urinary incontinence. AB - OBJECTIVE: To evaluate the use of the home pad test in the management of patients with urinary dysfunction with reference to feasibility, normal data, reproducibility, compliance and accuracy when compared to video urodynamics. DESIGN: A prospective study to determine 1. the accuracy of weighing pads by healthcare workers compared to weighing by patients with a spring balance; 2. the effects of evaporation; 3. the pad weight increase in continent women; 4. the compliance over 194 tests in terms of acceptability and feasibility; 5. the reproducibility in 112 women for the 24 h and 48 h tests, 6. the comparison with video urodynamic studies in 149 patients. SETTING: A London teaching hospital. MAIN OUTCOME MEASURES: Coefficient of variation and mean deviation analysis were employed to determine the accuracy of measurement. Percentage changes in standardised wet pad weights over a period of 8 weeks were used to determine the rate of weight loss due to evaporation. The percentage of patients completing the test satisfactorily was documented. The increase in the weights of all pads worn during the test period was measured in continent volunteers and incontinent patients. Video urodynamic diagnoses were used as the Gold Standard for comparison. RESULTS: Twelve perineal pads were weighed by 15 healthcare workers with a resulting coefficient of variation equal to 1.55% (standard error = 0.09%). The mean deviation between patient-measured pad weights and staff measured weights was 49% (SD = 132%). Pads wetted with saline showed no difference in weight after 1 week and less than 5% change in weight after 8 weeks, with the upper 95% confidence limit of less than 10% loss. The mean pad weight increase over 48 h in continent women was 7.13 g (SD = 4.32 g) giving a 95% upper confidence level of less than 15 g. Compliance was reasonably high with 161 (83%) carrying out the test perfectly. Test-retest analysis of the 24 h and 48 h tests showed a strong relationship with correlation coefficients of 0.90 and 0.94 respectively. The reproducibility was also good, with differences as a percentage of the mean between the first and second test being 6.9% and 1.6% for the 24 h and 48 h tests respectively. There was good concordance when the urodynamic study data was compared with pad weight data in terms of incontinence compared with no incontinence (kappa = 0.65), but a high false positive rate was detected for the pads. When eight of these patients with a false positive pad test had repeat video urodynamic studies, six were found to have detrusor instability on the second study. CONCLUSIONS: The home pad test combined with frequency volume chart documentation is an easy test to perform. It is quite robust and reasonably reproducible. This test should be added to complement the routine urodynamic tests and may have a role in detecting occult detrusor instability. PMID- 8616135 TI - An analysis of recent trends in vacuum extraction and forceps delivery in the United Kingdom. PMID- 8616136 TI - Oscillometric blood pressure measurements in severe pre-eclampsia: validation of the SpaceLabs 90207. PMID- 8616137 TI - Latex allergy as a risk during delivery. PMID- 8616138 TI - Reducing hospital stay after abdominal hysterectomy. PMID- 8616139 TI - Hepatic haematoma related to Glanzmann thrombasthenia in a newborn infant. PMID- 8616140 TI - Chorioamnionitis due to pseudomonas aeruginosa: a complication of prolonged antibiotic therapy for premature rupture of membranes. PMID- 8616141 TI - Cephalic replacement in conjoined twins. PMID- 8616142 TI - Ovarian cancers related to minimal access surgery. PMID- 8616143 TI - The combined oral contraceptive pill: what advice should we give when pills are missed? PMID- 8616144 TI - The combined oral contraceptive pill: what advice should we give when pills are missed? PMID- 8616145 TI - Is third trimester abortion justified? PMID- 8616146 TI - Is third trimester abortion justified? PMID- 8616147 TI - Is third trimester abortion justified? PMID- 8616148 TI - In vitro fertilisation and gamete intrafallopian transfer: an integrative analysis of research, 1987-1992. PMID- 8616149 TI - The prediction of fetal acidosis at birth by computerised analysis of intrapartum cardiotocography. PMID- 8616150 TI - Perineoplasty compared with vestibuloplasty for severe vestibulitis. PMID- 8616151 TI - Complications of pregnancy after infertility treatment: awareness and prevention. PMID- 8616152 TI - The definition of pre-eclampsia. PMID- 8616153 TI - Magnesium sulphate: the time of reckoning. PMID- 8616155 TI - Nucleotide sequence of the PetM gene encoding a 4 kDa subunit of the cytochrome b6f complex from Chlamydomonas reinhardtii. AB - We have determined the nucleotide sequence of the PetM gene from the single celled alga Chlamydomonas reinhardtii. The gene encodes a recently characterized, small protein of the cytochrome b6f complex, and based on this sequence, it is proposed that this protein spans the membrane by a single alpha-helix. Comparison of the nucleotide sequence with the deduced amino acid sequence reveals a 60 amino-acid presequence similar to a stroma-targeting peptide. PMID- 8616154 TI - Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli. AB - Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase. PMID- 8616156 TI - The deduced primary structure of subunit I from cytochrome c oxidase suggests that the genus Polytomella shares a common mitochondrial origin with Chlamydomonas. AB - We cloned and sequenced the mitochondrial gene encoding subunit I of cytochrome c oxidase (coxI) of Polytomella spp., a colorless alga related to Chlamydomonas. The purpose was to explore whether homology between the two species also exists at the level of a mitochondrial enzyme. The gene is 1512 bp long and contains no introns. The translated protein sequence exhibits 73.8% identity with its Chlamydomonas reinhardtii counterpart. The data obtained support the hypothesis that the separation of the colorless alga from the Chlamydomonas lineage was a late event in evolution, that occurred after the endosymbiotic process that gave rise to mitochondria. PMID- 8616157 TI - The cDNA sequence of proton-pumping nicotinamide nucleotide transhydrogenase from man and mouse. AB - cDNA clones for the human and mouse nicotinamide nucleotide transhydrogenases have been isolated and their sequences have been determined. Multiple alignments show that the functional proteins are encoded by single mRNAs. The deduced amino acid sequences are approximately 95% identical for the previously known bovine, and the human and mouse proteins. The major variable region is located in the presequence. It is proposed that all mammalian transhydrogenases have a similar structure. PMID- 8616158 TI - K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles. AB - Respiration-driven Na+ transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K+. Cells from an E. colic mutant deficient in three major K+ transport systems were incapable of accumulating K+ or expelling Na+ unless valinomycin was added. Membrane vesicles from an E. coli mutant from which the genes encoding the two known electrogenic Na+/nH+ antiporters nhaA and nhaB were deleted transported Na+ as well as did vesicles from wild-type cells. Quantitative analysis of Delta psi and Delta pH showed a high driving force for electrogenic Na+/nH+ antiport whether K+ was present or not, although Na+ transport occurred only in its presence. These results suggest that an Na+/nH+ antiporter is not responsible for the Na+ transport. Respiration driven efflux of Na+ from vesicles was found to be accompanied by primary uphill efflux of K+. Also, no respiration-dependent efflux of K+ was observed in the absence of Na+. Such coupling between Na+ and K+ fluxes may be explained by the operation of an Na+, K+/H+ antiporter previously described in E. coli membrane vesicles (Verkhovskay, M.L., Verkhovsky, M.I. and Wikstrom, M. (1995) FEBS Lett. 363, 46-48). Active Na+ transport is abolished when delta mu H+ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na+ transport. Such an effect may be expected if the Na+, K+/H+ antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump. PMID- 8616159 TI - The polar behavior of frog photoreceptors. AB - It was observed that the outer segments of the frog visual rods orient along the direction of an externally applied static electric field. The orientation ability of the rod outer segments seems to be fuelled by the cell energy. The dipolar moment per rod was determined using a model which considers rod outer segments as rigid dipoles interacting with the electric field in a viscous medium. The mean dipolar charge of ROS was determined as being (2.10 +/- 0.17).10(-14)C. PMID- 8616160 TI - Interrelation between mitochondrial respiration, substrate supply and redox ratio in perifused permeabilized rat hepatocytes. AB - A one-step perifusion technique is described for studying the regulation of energy metabolism in intact hepatocytes and in mitochondria of the same cells after their permeabilization by digitonin. Cell count and activities of glutamate dehydrogenase, the latter being used as an indicator of mitochondrial integrity, were found to be nearly unchanged after permeabilization and perifusion for at least 40 min at 37 degrees C. The residual activity of lactate dehydrogenase after permeabilized indicated that permeabilized cells were almost depleted of soluble cytosolic components. The composition of the perifusion medium was chosen so that various metabolic states could be adjusted of both intact and permeabilized hepatocytes without the need to change the perfusion medium. Oxidative phosphorylation of mitochondria within permeabilized hepatocytes remained intact throughout the perifusion as indicated by the response of respiration to the addition of ADP, carboxyatractyloside and uncoupler. The application of the perifusion technique allows us to sample indicator metabolites in the effluent medium like acetoacetate (AcAc) and 3-hydroxybutyrate (HB) for calculating the mitochondrial redox ratios and rates of ketogenesis. In the presence of octanoate and ADP, an improvement of substrate supply by glutamate and malate led to increases in the intramitochondrial HB/AcAc ratio and the respiration rate. Glutamate/malate concentrations of 1 mM resulted in maximal respiration rates, whereas concentrations of 5 mM further enhanced the HB/AcAc ratio. Mitochondria responded to increasing ATP/ADP ratios in the perifusion medium by decreased respiration rates at higher HB/AcAc ratios. By comparing respiration rates and redox ratios of mitochondria in permeabilized cells with those before permeabilization (gluconeogenic conditions of hepatocytes), it is concluded that in the intact cell oxidative phosphorylation is limited with respect to substrate supply as well as by the ATP demand. PMID- 8616161 TI - The coupling of metabolic to secretory events in pancreatic islets. Glucose induced changes in mitochondrial redox state. AB - Mitochondrial NAD+, NADH, NADP+ and NADPH were measured in dispersed pancreatic islet cells incubated in the absence or presence of D-glucose and then exposed for 20 s to 0.5 mg/ml digitonin. The latter treatment resulted in the full release of lactate dehydrogenase without any detectable loss of glutamate dehydrogenase. The permeabilized cells were separated from the incubation medium by centrifugation through an oil layer and their content in pyridine nucleotides measured by a radioisotopic procedure coupled to the classical cycling technique. Relative to basal value, D-glucose, in concentrations of 2.8 and 16.7 mM, caused a concentration-related increase in both the NADH/NAD+ and NADPH/NADP+ ratio. These findings provide the first direct evidence for the induction of a more reduced mitochondrial redox state in glucose-stimulated pancreatic islets. PMID- 8616163 TI - Literature. October-December 1995. PMID- 8616162 TI - Cardiovascular risk in continuous ambulatory peritoneal dialysis patients. PMID- 8616164 TI - Computational formulas for clearance indices in continuous ambulatory peritoneal dialysis. PMID- 8616165 TI - Why are reported relative mortality risks for CAPD and HD so variable? (inadequacies of the Cox proportional hazards model) AB - The relative risks of death for CAPD and HD reported in the literature and at recent meetings most likely reflect case-mix differences and varying percentages of adequately dialyzed patients within the compared populations. A prospective randomized comparison of CAPD and HD patients will be unlikely because of the expense. Therefore, we should attempt to improve the way that both CAPD and HD are practiced. There is no conclusive evidence that the choice of CAPD or HD per se yields a specific modality advantage for survival. PMID- 8616166 TI - Choice of chronic peritoneal dialysis: need for early assessment and education. PMID- 8616167 TI - Is peritoneal dialysis feasible once a large muscular patient becomes anuric? PMID- 8616168 TI - The spectrum of renal osteodystrophy in peritoneal dialysis patients. PMID- 8616169 TI - Elevated lipoprotein(a) and fibrinogen levels [corrected] increase the cardiovascular risk in continuous ambulatory peritoneal dialysis patients. AB - OBJECTIVE: To analyze the relationship between lipoprotein(a) [Lp(a)] and fibrinogen as potential cardiovascular risk factors in patients on continuous ambulatory peritoneal dialysis (CAPD). PATIENTS: A total of 47 uremic patients receiving CAPD, 21 with coronary artery disease (CAD), 26 without CAD. MEASUREMENTS: Lp(a) levels were determined by an immunoradiometric assay. Since Lp(a) serum concentrations vary depending on the size, apoprotein(a) [apo(a)] isoforms were determined (Westernblot). Fibrinogen was quantified according to Clauss. RESULTS: The mean Lp(a) serum concentration was 43 +/- 5 mg/dL (SEM) (median 33 mg/dL) in CAPD patients and 21 +/- 2 mg/dL (8 mg/dL) in controls (p < 0.01). Patients with low molecular weight apo(a) isoforms exhibited substantially elevated Lp(a) levels when compared with patients with high molecular isoforms (p < 0.01). In addition, we found elevated fibrinogen levels in the CAPD patients (538 +/- 61 mg/dL) compared with healthy controls (288 +/- 46 mg/dL). Twenty-one CAPD patients (45%) were suffering from CAD. Patients with CAD had higher Lp(a) levels (54 +/- 5 mg/dL vs 34 +/- 4 mg/dL) as well as higher fibrinogen concentrations (628 +/- 59 mg/dL vs 459 +/- 46 mg/dL). Furthermore, a positive correlation between the fibrinogen levels and the Lp(a) serum concentration was observed (r = 0.45, p = 0.01). CONCLUSION: We suggest that elevated Lp(a) levels are influenced by the allelic variation of the apo(a) isoform. In addition to the typical dyslipidemia found in CAPD patients, high levels of Lp(a) and fibrinogen may contribute to the elevated risk of coronary artery disease and other cardiovascular complications. PMID- 8616170 TI - The role of DEXA bone densitometry in evaluating renal osteodystrophy in continuous ambulatory peritoneal dialysis patients. AB - OBJECTIVE: The aims of this study were to assess the clinical utility of total and regional bone densitometry in a large continuous ambulatory peritoneal dialysis (CAPD) population and to determine the clinical, biochemical, and radiographic variables that best identified osteopenic CAPD patients. DESIGN AND PATIENTS: A cross-sectional study was performed on 45 CAPD patients (19 males, 26 females), comprising the total CAPD population at the Princess Alexandra Hospital. MAIN OUTCOME MEASURES: Total body (TB), anteroposterior lumbar spine (APL), femoral neck (FN), Ward's triangle (WT), and skull bone mineral densities (BMDs) were measured using dual-energy x-ray absorptiometry (DEXA) and then correlated with clinical, biochemical, and radiographic indices of uremic osteodystrophy. RESULTS: BMDs were not significantly different from age- and sex matched reference population data. Considerable regional variation of BMD Z scores were noted between FN (-0.11 +/- 0.23), WT (-0.11 +/- 0.22), and APL (1.22 +/- 0.04) (p = 0.003). APLZ scores were significantly reduced in patients with a previous history of fracture (-1.36 +/- 1.07 vs 0.89 +/- 0.31), bone pain (-0.72 +/- 1.08 vs 1.01 +/- 0.31), or steroid treatment (-0.62 +/- 0.39 vs 1.16 +/- 0.35). Increased BMD Z scores for APL (1.82 +/- 0.57 vs 0.38 +/- 0.29, p < 0.05), FN (0.32 +/- 0.36 vs -0.38 +/- 0.29, p = 0.014), and WT (0.45 +/- 0.38 vs -0.45 +/- 0.26, p < 0.05) were found in patients with radiographic hyperparathyroid bone disease. Both APL BMD Z scores and skull BMDs were weakly correlated with PTH (r = -0.33, p < 0.05 and r = -0.33, p < 0.05, respectively) and with CAPD duration (r = 0.30, p < 0.05 and r = -0.30, p < 0.05). Generally, however, total body and regional BMDs were poorly related to age, renal disease type, dialysis duration, renal failure duration, serum aluminum, calcium, phosphate, alkaline phosphatase, osteocalcin, and parathyroid hormone. CONCLUSIONS: We conclude that the prevalence of osteopenia is not increased in CAPD patients. Clinical and biochemical parameters do not reliably predict BMD measurements, but prior steroids and bone symptoms are major risk factors for important bone loss. Although DEXA can reliably detect osteopenia in different skeletal regions, its usefulness in detecting osteodystrophy is limited by the confounding effects of superimposed hyperparathyroid osteosclerosis, which increases BMD. PMID- 8616171 TI - Creatinine clearance in continuous peritoneal dialysis: dialysis dose required for a minimal acceptable level. AB - OBJECTIVES: To identify the most advantageous formula for estimating creatinine clearance (CCr) and to establish a dose of dialysis that will ensure minimal acceptable levels of creatinine clearance in patients on continuous peritoneal dialysis (CPD). DESIGN: Analysis of all CCr studies performed in CPD patients over 40 months. SETTING: All four dialysis units following CPD patients in one city. One dialysis unit is government-owned, one is university-affiliated, and two are community based. PARTICIPANTS: One hundred and ninety-four patients representing almost the entire CPD population in Albuquerque. INTERVENTIONS: Creatinine and urea clearance studies were performed in 24-hour urine and drained dialysate samples. Creatinine clearance (peritoneal plus urinary) was normalized to either 1.73 m2 body surface area (CCr) or body water estimated by the Watson formulas (KT/VCr). CCr and KT/VCr were either corrected by averaging urinary creatinine and urea clearances or were not corrected. Two dialysis units were designated as the training set (92 patients, 143 clearance studies) and the other two units as the validation set (102 patients, 181 clearance studies). MAIN OUTCOME MEASURES: Minimal acceptable creatinine clearance levels were determined in the training set by computing the creatinine clearance value corresponding to 1.70 weekly KT/V urea by linear regression. Logistic regression models predicting low creatinine clearance were developed in the training set and were tested in the validation set. RESULTS: The following weekly creatinine clearance values corresponded to 1.70 KT/V urea: corrected CCr 52.0 L/1.73 m2, uncorrected CCr 54.4 L/1.73 m2, corrected KT/VCr 1.46, uncorrected KT/VCr 1.53. Logistic regression identified as predictors of low creatinine clearance low daily urine volume (UV) and low daily dialysate drain volume/body water (DV/V) for all four creatinine clearance formulas, plus low/low-average peritoneal solute transport (only for uncorrected CCr) and serum creatinine (for both KT/VCr formulas). In the validation set, the predictive models produced an area under the receiver operating characteristic (ROC) curve between 0.835 and 0.919 indicating very good predictive accuracy. For corrected CCr and anuria, the regression model produced a minimal normalized drain volume (DV/V) value consistent with minimal acceptable CCr equal to 0.305 L/L per 24 hours. This DV/V cutoff detected low corrected CCR in validation set anuric subjects (n = 55) with a sensitivity of 85% and a specificity of 71%. For uncorrected CCR and anuria, DV/V cutoffs were 0.273 L/L per 24 hours (high/high-average peritoneal solute transport) and 0.420 L/L per 24 hours (low/low-average transport). Sensitivity and specificity of these cutoffs in validation set anuric subjects were 87% and 85%, plus 86% and 33%, respectively. CONCLUSIONS: The uncorrected CCr appears to be the most advantageous creatinine clearance formula in CPD, because it allows the use of peritoneal solute transport type in the calculation of the minimal required normalized drain volume. The minimal acceptable uncorrected CCr is 54.4 L/1.73 m2 weekly. To achieve this uncorrected CCr in anuria, the required minimal normalized drain volume is 0.273 L per liter of body water daily if peritoneal solute transport is high or high-average and around 0.420 L per liter of body water daily if peritoneal solute transport is low or low-average. The required total daily drain volume is computed by multiplying the required normalized drain volume by body water. PMID- 8616173 TI - Restoring the therapeutic effects of the patient-physician relationship. PMID- 8616172 TI - Renal transplantation in patients undergoing chronic peritoneal dialysis. AB - OBJECTIVE: To review the outcome of renal transplantation in a group of patients treated with chronic peritoneal dialysis and to compare the results with those of a matched population on hemodialysis. DESIGN: Retrospective study. SETTING: Tertiary, institutional hospital, administering to a population of two million, with 100 patients on peritoneal dialysis. Six hundred and sixty renal transplantations were performed by the end of 1993. PATIENTS: Fifty-six patients on chronic peritoneal dialysis who received 58 cadaveric renal allografts were compared to 58 patients on hemodialysis who received a graft from the same donor (n = 39), or the transplant next to the one performed to the corresponding patient on peritoneal dialysis (n = 19). RESULTS: Patients on peritoneal dialysis showed a lower rate of delayed graft function (24.1 vs 50%, p < 0.05) and a similar incidence of acute rejection than patients on hemodialysis. Also, peritoneal dialysis patients received less supplementary immunosuppression, suffered a lower incidence of late infections (0.93 vs 0.58 episodes/patient), and had a similar incidence of dialysis-related complications (0.25 vs 0.20 episodes/patient). CONCLUSIONS: Patients on peritoneal dialysis do well after renal transplantation. The incidence of some complications, particularly delayed graft function, is lower than in patients on hemodialysis, while the incidence of dialysis-associated complications is similar in both groups. PMID- 8616174 TI - Fusarium peritonitis in a child on peritoneal dialysis: case report and review of the literature. AB - OBJECTIVE: To review various aspects of the management of peritonitis due to Fusarium, a soil mold which infrequently causes infections in humans. DATA SOURCES: A case of Fusarium peritonitis in a child on chronic peritoneal dialysis (PD) is presented. The child developed Fusarium peritonitis 2 weeks after an episode of bacterial peritonitis. His Tenckhoff catheter was removed, and he was maintained on hemodialysis while receiving intravenous amphotericin. Following 2 weeks of treatment with amphotericin, he was successfully returned to PD. A literature review of all previously reported cases of Fusarium peritonitis was then conducted to determine features common to infections caused by Fusarium. Emphasis was also placed on unique characteristics of the organism that may affect patient management, as well as patient characteristics that may increase the risk for infection by Fusarium. RESULTS: Fusarium may cause infection in immunosuppressed individuals, such as cancer patients or patients on chronic PD. The organism has a propensity to attach to foreign bodies such as intravascular and intraperitoneal catheters. Therefore, successful treatment of infections caused by Fusarium may require catheter removal in addition to systemic antifungal therapy. CONCLUSIONS: This report presents the first known case of Fusarium peritonitis in a child. In view of the difficulties posed by this unusual organism, optimal therapy of Fusarium peritonitis should consist of immediate catheter removal and treatment with systemic antifungal drugs. PMID- 8616175 TI - The fate of leached di(2-ethylhexyl)phthalate in patients undergoing CAPD treatment. AB - OBJECTIVES: To evaluate the degree of exposure to and the fate of di(2 ethylhexyl)phthalate (DEHP) and its major derivatives mono(2-ethylhexyl)phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA) in patients undergoing regular continuous ambulatory peritoneal dialysis (CAPD) during a 4-hour dwell period. DESIGN: Prospective, controlled. SETTING: Teaching hospital, Department of Nephrology. PARTICIPANTS: Seven elderly patients on stable CAPD using Fresenius instruments and dialysate and 6 age-matched healthy controls. INTERVENTIONS: During a routinely performed peritoneal equilibration test (PET), blood and dialysate samples were drawn before and 120 and 240 min after the dwell was started. In addition, blood samples were taken from a group of volunteers participating in a pharmacological study. MEASUREMENTS: Quantitative analysis of DEHP and its hydrolysis products was performed by selected ion-monitoring gas chromatography/mass spectrometry, operating the mass spectrometer in a combined positive and negative ion chemical ionization mode. RESULTS: Serum concentrations of DEHP and PA were significantly higher in patients (median: 0.079 microgram/mL, range: 0.032-0.210 microgram/mL; and 0.167 microgram/mL, range: 0.097-0.231 microgram/mL, respectively) than in controls [0.0195 microgram/mL, range: 0.016 0.025 microgram/mL (p = 0.0027) and 0.0120 microgram/mL, range: 0.006-0.034 microgram/mL (p = 0.0026), respectively]. Concentration of MEHP in the fluid of CAPD bags prior to use was four times higher than that of the parent compound. During the first 4 hours of dwell time, the concentrations of MEHP and 2-EH in dialysate consistently decreased from 0.177 (range: 0.137-0.239 microgram/mL) to 0.022 microgram/mL (range: 0.005-0.058 microgram/mL) (p = 0.017), and from 0.087 (range: 0.075-0.097 microgram/mL) to 0.05 microgram/mL (range: 0.023-0.064 microgram/mL) (p = 0.017), respectively, while the concentration of DEHP remained stable. Remarkably high concentrations of PA (0.129 microgram/mL; range: 0.038 0.466 microgram/mL) were found in CAPD bags prior to use, and these concentrations tended to increase during dwell time, without statistical significance, however (0.135 microgram/mL; range: 0.073-0.659 microgram/mL, p = 0.062). CONCLUSIONS: Patients on CAPD are regularly exposed to considerable amounts of phthalic ester derivatives, mainly to MEHP and PA. MEHP seems to be well absorbed by the peritoneal membrane. The long-term effects of this exposure remain to be elucidated. PMID- 8616176 TI - Long-term outcome of diabetic patients receiving peritoneal dialysis. AB - OBJECTIVE: Data from the United States Renal Data Systems (USRDS) suggest that older diabetic patients with end-stage renal disease will have improved survival if they receive hemodialysis versus peritoneal dialysis. Younger diabetic patients have equal survival on either treatment modality. To address more specifically the risk factors for long-term survival of diabetic patients receiving peritoneal dialysis, we analyzed the long-term outcome of 118 diabetics receiving peritoneal dialysis over a decade and compared them to 165 nondiabetic patients. DESIGN: Retrospective analysis utilizing the Cox proportional hazards model to identify risk factors for survival of both diabetic and nondiabetic patients. SETTING: An experienced, single-center, university-based dialysis program. PATIENTS: All patients receiving home peritoneal dialysis for at least one month from 1 January 1981 to 31 December 1990. Diabetics were classified as type I or type II, in addition to age stratification. Most type I diabetic patients used insulin via the intraperitoneal route. MAIN OUTCOME MEASURES: Patient survival and technique survival. RESULTS: The most significant risk factor for diabetic patient survival was diabetes type (relative risk type I to type II 0.14, p < 0.0001). On treatment serum albumin, predialysis blood urea nitrogen and predialysis serum cholesterol were also significant risk factors (p < 0.01). For nondiabetic patients, age, on treatment serum albumin, and current smoking were significant survival risk factors. Survival of patients 55 years or less was not significantly different between diabetic and nondiabetic patients. Survival of patients greater than 55 years was better in nondiabetic patients. CONCLUSION: These findings of a long-term follow-up period suggest a good survival for younger type I diabetic patients receiving peritoneal dialysis. Reasons other than age for the discrepancy in survival of young versus old diabetics receiving peritoneal dialysis should be sought. PMID- 8616177 TI - Treatment modality selection in 150 consecutive patients starting ESRD therapy. AB - OBJECTIVE: The purpose of this study was to assess the reasons for treatment modality selection between hemodialysis (HD) and peritoneal dialysis (PD) in 150 consecutive patients in a single center. DESIGN: This study is a retrospective study using chart review as the data collection method. SETTING: A single tertiary care university teaching hospital. PATIENTS: One hundred and fifty consecutive patients starting end-stage renal disease (ESRD) therapy at the Royal Victoria Hospital in Montreal were assessed. Their treatment modality at 6 weeks after starting dialysis was recorded as their treatment modality. Patients transplanted or who died prior to that 6-week period were excluded. MAIN OUTCOME MEASURES: The treatment modality, that is, either HD or PD, at 6 weeks after the initiation of ESRD was the modality assigned to the patient. RESULTS: One hundred and fifty patients started ESRD therapy of whom 83 went to HD and 67 to PD. Thirty-one patients were directed to HD, including 20 for social reasons, 3 with ostomies, and 6 with unsuitable abdomens. Fourteen patients were directed to PD, including 10 with severe cardiovascular disease, 3 with no vascular access, and one for geographical reasons. Of 31 diabetics who were encouraged to do PD, 17 went to PD and 14 to HD (10 for social reasons, 3 refused PD, and one with an inappropriate abdomen). Seventy-four patients were initially eligible for either PD or HD. Fifty percent (37) went to PD and 50% to HD. Of those going to HD, 15 went to self-care HD, of whom 7 had prior exposure to HD. Eleven were not informed regarding PD. There was no gender preference for PD versus HD. CONCLUSION: We conclude that among informed patients, if given a choice of treatment modality, the majority will choose self-care dialysis including continuous ambulatory peritoneal dialysis (CAPD) or self-care HD. PMID- 8616179 TI - Long-term peritoneal dialysis in the absence of residual renal function. PMID- 8616178 TI - Estimation of total creatinine clearance is unreliable in children on peritoneal dialysis. AB - OBJECTIVE: To test the reliability of creatinine clearance in children on peritoneal dialysis (PD). DESIGN: Longitudinal, case-controlled. SETTING: Routine clinic visits at the pediatric dialysis unit of the Universitatskinderklinik of Vienna. PATIENTS: Eleven children (2-13 years, 10-55 kg) with end-stage renal disease on PD. INTERVENTIONS: Creatinine clearance (CCr) was determined by measuring creatinine excretion (ECr) over 24 hours in both dialysate and urine. Each child had three to five separate measurements of their CCr. At the same time we also calculated the Schwartz formula clearance from the patient's height and serum creatinine, using a modified correlate. MAIN OUTCOME MEASURES: Reliability of CCr was assessed by two approaches. First, we compared each serial measurement with the mean value for each patient and thereby assessed the "intramethodical" variability. Second, we compared each CCr with the simultaneous formula clearance and assessed the "intermethodical" disagreement. RESULTS: Twenty-seven percent of the measurements of CCr were classified as unreliable based on a comparison with the mean value for each patient. Reliability was closely correlated with residual renal function (p < 0.01); only 12% of the measurements in the anuric patients were classified as unreliable (vs 31% in the patients with residual renal function). The simultaneous formula clearance was less variable than the CCr. The formula clearance had a sensitivity of 93% and a specificity of 60% for detecting unreliable values of CCr. CONCLUSION: Estimation of total CCr is unreliable in pediatric patients on PD. A simultaneous formula clearance can be used to detect which values are unreliable. PMID- 8616180 TI - Scanning electron microscopy studies of peritoneal catheter in CAPD peritonitis due to Aspergillus fumigatus. PMID- 8616182 TI - Hemoperitoneum in CAPD patients with hepatic tumors. PMID- 8616181 TI - The influence of dialysate sampling on peritoneal equilibration test results. PMID- 8616183 TI - Molecular diagnosis of tuberculous peritonitis using DNA amplification in a patient with end-stage renal disease. PMID- 8616184 TI - Technique survival and complication rates in a newly started CAPD center (five years of experience) PMID- 8616185 TI - Diurnal blood pressure variation in CAPD patients. PMID- 8616186 TI - Obstructive fibrin clot in the peritoneal catheter. PMID- 8616188 TI - Videolaparoscopy: a new alternative for implantation of peritoneal catheters in ESRD patients with previous abdominal surgeries. PMID- 8616187 TI - The solute removal index: a new name for an old concept. PMID- 8616189 TI - Peritoneal catheter colonization and peritonitis with Aureobasidium pullulans. PMID- 8616190 TI - Emerging infectious diseases: what should Australia do? PMID- 8616191 TI - Climate change in the South Pacific region: priorities for public health research. PMID- 8616192 TI - The control of communicable diseases in Australia. PMID- 8616193 TI - Health at the margins: providing services in remote areas and for marginal populations. PMID- 8616194 TI - Health care policy for aboriginal Australians: the relevance of the American Indian experience. AB - This paper discusses and compares the systems for the delivery of health care services to indigenous peoples in the United States and Australia; both are poor minorities in wealthy countries and many live in remote locations. Three necessary conditions that have shaped the relative success of the Indian Health Service in the United States are relevant to the Australian situation: federal government administration; the separation of the Indian Health Service from other Indian affairs; and the provision of an integrated health service. Ironically, recent policy changes in the United States by the Clinton administration are reducing the federal bureaucracy, and along with it, Indian Health Service funding. In Australia, the states have had responsibility for service delivery to Aboriginal people, there have been no treaties formalising the relationship between indigenous people and the federal government, and Aboriginal health has been switched between different departments while remaining primarily within the Aboriginal affairs (rather than the health) portfolio. Since 1993, there has been pressure to return Aboriginal health to the health portfolio, and in July 1995, funding and administration of Aboriginal health services were moved from the Aboriginal and Torres Strait Islander Commission to the Department of Human Services and Health. PMID- 8616195 TI - Issues in health policy for indigenous peoples in Canada. AB - This paper provides a preliminary report on the work of the Canadian Royal Commission on Aboriginal Peoples. This commission has spent the past four years undertaking a comprehensive review of all matters pertaining to indigenous people in Canada, and will publish a final report in late 1995 or early 1996. The paper provides an overview of health policy issues examined by the commission. The author was employed by the commission in various capacities to contribute to this analysis of indigenous people's health policy concerns. A disproportionate burden of illness has been suffered by indigenous peoples in Canada. Past policy has systematically undermined the capacity of indigenous communities to develop their own health care systems. Current concerns about health problems and services, as expressed by indigenous people at the commission's community hearings, describe a need for a community-controlled and culturally appropriate approach to healing in indigenous communities. The commission's framework for developing an indigenous people's health strategy is intended to ensure that indigenous people have the capacity, the resources and the appropriate political environment in which to implement community healing. Its relevance to the Australian context may best be seen through a careful review of the commission's final report. PMID- 8616196 TI - Evaluating government health and substance abuse programs for indigenous peoples: a comparative review. AB - Most health and substance abuse programs for indigenous peoples in Australia are funded by government. Over the past decade there have been calls for greater accountability in the conduct of these programs. Initial attempts focused on the development of standardised performance indicators, an approach that has been criticised on both political and methodological grounds. Recently, some government agencies have sought to identify culturally appropriate models for the evaluation of programs for indigenous peoples. In a comparative review of the evaluation of indigenous programs in Australia and Canada, conducted for the Western Australian Aboriginal Affairs Department, the authors were not able to identify any generally applicable models. However, this literature review and our own research and experience in working with Aboriginal community organisations have identified some principles that should be an essential part of any attempts to evaluate health and substance abuse programs for indigenous peoples. Underlying these principles is the realisation that evaluation is not a politically or ideologically neutral activity. Theoretical and methodological considerations of the evaluation process must take into account the very real differences between the agendas of indigenous peoples and those who seek to evaluate programs for them. PMID- 8616197 TI - Is there a role for prevention in aboriginal mental health? AB - Primary prevention is consistently identified as a priority in mental health and Aboriginal health planning. In both areas it has proved to be elusive. Aboriginal mental health therefore presents substantial challenges, particularly in remote Australia. In this paper, two brief case histories are used to demonstrate the limits of conventional psychiatric engagement in such settings and explore the dimensions of social disadvantage that compound psychiatric vulnerability. There are difficulties of primary prevention in mental health generally, and access to mental health services for those most at risk, including indigenous Australians, is unacceptably poor. Proceeding to primary prevention in Aboriginal mental health will, of necessity, include rectifying that situation and addressing the social precursors of mental disorders and distress. Such initiatives will require wide and sustained commitment toward long-term goals, with a focus on children and families, and must include effective substance-abuse strategies. PMID- 8616199 TI - Which way kaikai blo umi? Food and nutrition in the Torres Strait. AB - The people of the Torres Strait suffer a disproportionate level of diet-related disease, especially diabetes. The Torres Strait Health Strategy identified the difficulty in obtaining healthy food, particularly fruit and vegetables, as a major problem for people living in the Torres Strait. This study examined traditional plant food supply systems and current local production of fruit and vegetables. The supply system of fruit and vegetables from mainland Australia was also examined. Traditional garden food production was seen to continue (mainly to provide food for ceremonial occasions), but had declined because of easy access to store foods, changes in the physical, social and economic environment, limited access to land and water, and quarantine restrictions on movement of garden produce. Supplies of fruit and vegetables from the Australian mainland were infrequent and the prolonged transit time meant that produce was often in poor condition on arrival and prone to continued rapid deterioration due to limited store-level storage facilities. Demand for fresh produce exceeded supply. PMID- 8616198 TI - Obstetric health services in Far North Queensland: is choice an option? AB - In remote Far North Queensland, evacuation for mandatory hospital births saves lives. However, morbidity and mortality for mothers and infants remain high. Results from a retrospective study of deliveries over a one-year period showed significant differences in obstetric risk and outcomes among rural and urban Aboriginal, Torres Strait Islander and Caucasian women. Choices of birth place for remote residents were severely limited but those women with education and knowledge about how the system worked utilised options alternative to the regional public hospital. The findings from both the retrospective hospital sample and interviews among remote-area residents of Far North Queensland confirm the need to develop community-based perinatal services to reduce cultural and social barriers to clinical care. PMID- 8616200 TI - Contact tracing and sexually transmitted disease among aboriginal men on the Anangu Pitjantjatjara lands. AB - Sexually transmitted diseases (STDs) are a major health problem for Pitjantjatjara Aboriginal people, causing considerable morbidity and being implicated in the potential rapid transmission of human immunodeficiency virus (HIV) infection. This paper presents data in relation to STD contact tracing on the Anangu Pitjantjatjara lands. In-depth interviews were conducted with Aboriginal men and non-Aboriginal clinic staff to ascertain their attitudes to and perceptions of contact tracing. The interviews with Aboriginal men also sought more general information in relation to health-seeking behaviour and knowledge of STDs. While contact tracing has proven an effective method of case detection on the Anangu Pitjantjatjara lands (most named contacts are able to be located and a high proportion are found to be infected), the data suggest that important constraining issues are the reluctance of Aboriginal men to consult with particular health workers because of moiety group considerations, concerns about confidentiality, and the low level of knowledge of STDs, particularly of largely asymptomatic infections such as chlamydia, syphilis and HIV. PMID- 8616201 TI - Fatal attraction: do high-technology treatments for end-stage renal disease benefit aboriginal patients in central Australia? AB - The health problems of Aboriginal Australians, like those of many indigenous peoples, resemble those of the developing world, yet they are dealt with using the tools, techniques, and high-technology medical solutions of first-world health. Such approaches ignore the social components of health and illness, including the need for preventive and educative programs at the primary health care level. The example of endstage renal disease provides a poignant example of the inadequacies of this approach. Central Australian Aboriginal people suffer from a high incidence of kidney disease from numerous causes including non insulin-dependent diabetes mellitus and glomerulonephritis. The high incidence has led to numbers of people developing end-stage renal disease and moving into the Northern Territory-South Australia renal failure program for dialysis and/or transplantation. In requiring patients to leave their lands, communities and families, this program removes people from the religious and social support network that could ensure a reasonable quality of life in their final years, while offering only marginal extensions of those years. Expensive technology programs are of little benefit and of considerable cost to Aboriginal patients and draw attention away from efforts to reduce the exposure of at-risk Aboriginal people to the factors that facilitate the development of end-stage renal disease. PMID- 8616202 TI - Cultural factors in dialysis and renal transplantation among aborigines and Torres Strait Islanders in north Queensland. AB - Australian Aborigines experience end-stage renal disease at 10 times the national average. Although contributing physiological factors have been widely discussed, there has been little research into cultural factors affecting treatment and outcomes. This paper discusses folk and lay understandings of renal physiology and disease aetiology, and social and cultural factors in dialysis and transplantation, in a group of Aboriginal and Torres Strait Island renal transplant recipients. The implications for service delivery include the need for improved and clear information regarding renal disease and treatment and for culturally appropriate and acceptable support systems. Beliefs that continued alcohol consumption and poor nutrition were major reasons for kidney failure and separation from kin and country emerged as significant factors affecting treatment and leading to poor outcomes. PMID- 8616203 TI - Family planning: personal and political perspectives from Choiseul Province, Solomon Islands. AB - Rapid population growth has put family planning on personal and political agendas in the Solomon Islands. With the release of a population policy in 1988, national leaders sanctioned the concept of family planning as a key strategy in reducing the rate of population growth. On a personal level, Solomon Islanders share their government's concern about population problems. There is a shortage of arable land, health services are stretched, and there are limited places in school for children. A study in Choiseul Province, a rural area in Solomon Islands, suggests that people want smaller families but have limited means to control their fertility. Meagre resources and infrastructure, compounded by geography, climate, culture and religion, constrain the development of family planning services. PMID- 8616204 TI - Food intakes of adult Melbourne Chinese. AB - Food intake patterns of 545 adult Melbourne Chinese were studied in 1988 and 1989 using a 220-item food-frequency questionnaire appropriate for Chinese eating practices. Men and women were compared, adjusting for age, time in Australia and education. Men consumed more rice and alcoholic beverages as energy. In women, the energy intake was derived from foods of traditional Chinese types. There were two types of consumption patterns: in the first group were those who acculturated towards an Australian way of eating by replacing some traditional Chinese foods, such as rice, pork, leafy green and cruciferous vegetables, soups and tea, with 'new foods', such as wheat products, red meats and coffee; in the second were those who limited their intake to a handful of traditional Chinese foods as the major source of energy. The educated, the professional and those with an administrative profession, the Australian-born and those with a longer length of stay fitted into the first group, and were more acculturated towards Australia than those born in the People's Republic of China or Vietnam and who migrated at an older age. The first group may benefit from the best of both worlds, but may risk the diseases of an industrialised society. The second group may be trapped at a cultural crossroads and may be unable to make appropriate food choices. Public health efforts in Australia, where one in every five is overseas-born, should provide for nutrition and health education for new and aged migrants of non-European cultural backgrounds. PMID- 8616205 TI - The traditional humoral food habits of pregnant Vietnamese-Australian women and their effect on birth weight. AB - Vietnamese-Australian women have infants with lower birth weight than Australian born women. Traditional humoral food habits, based on ancient Chinese medicine, are more likely to be followed during life changes like pregnancy. These food habits may influence maternal energy intake, weight gain and therefore infant birth weight. This study determined the proportion of pregnant Vietnamese women in southwestern Sydney who practise traditional humoral food habits, and their effect on birth weight. A cohort of 113 pregnant Vietnamese-born women were questioned on smoking status, height, weight, parity, food practices and demographics. Dietary intake was measured in each trimester. Known risk factors and infant birth weights were collected from medical records for participants and nonresponders. Fifty-seven per cent practised the traditional food habits. There were no differences in energy intake or weight gain between the two groups. After adjusting for confounders, birth weight was estimated to be 3257 g (95 per cent confidence interval (CI) 3205 to 3309) and 3272 g (CI 3211 to 3333) for the infants of traditional and nontraditional women respectively. Following humoral food habits does not appear to affect birth weight. Women who choose to follow these traditions should not be discouraged from doing so. PMID- 8616206 TI - Vaccination policies and practices. PMID- 8616207 TI - Australia's role in HIV prevention in the developing world. PMID- 8616208 TI - Cost containment, drugs and community health. PMID- 8616209 TI - A review of simultaneous visual discrimination as a method of training octopuses. AB - I have presented a review and critique of the procedures employed in simultaneous discrimination training experiments using octopuses as subjects. Procedural variables were analyzed statistically for their influence on experimental outcome. The variables most significantly associated with successful discriminations included use of a specific start location for subjects, shock as negative reinforcement, fewer trials per session, more sessions per day, and discriminations based on stimulus brightness. No experiment controlled all potential sources of inadvertent cues, and subjects' performances appeared to be sensitive to exact procedural details. The most common practice diminishing evidence for learning involved reward that coincided with the subject's pre existing preferences. I found no evidence that sub-optimal experimental designs biased experimental outcomes in any significant and systematic way. Although there is sufficient reason for rejecting results of published simultaneous discrimination training experiments, careful conclusive experiments remain to be performed. PMID- 8616210 TI - Axonal regeneration. AB - Axons damaged in a peripheral nerve are often able to regenerate from the site of injury along the degenerate distal segment of the nerve to reform functional synapses. Schwann cells play a central role in this process. However, in the adult mammalian central nervous system, from which Schwann cells are absent, axonal regeneration does not progress to allow functional recovery. This is due to inhibitors of axonal growth produced by both oligodendrocytes and astrocytes and also to the decreased ability of adult neurons to extend axons during regeneration compared to embryonic neurons during development. However once provided with a substrate conducive to axonal growth, such as a peripheral nerve graft, many central neurons are able to regenerate axons over long distances. Over the past year this response has been utilised in experimental models to produce a degree of behavioural recovery. PMID- 8616211 TI - Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits. AB - The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress. PMID- 8616212 TI - Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum. AB - Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5' non-coding leader, the 11 introns, and 3' non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3' non coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3' tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these 'twin' GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed. PMID- 8616214 TI - The phytochrome gene family in tomato includes a novel subfamily. AB - Data presented here define five tomato phytochrome genes (PHY) and indicate the existence of additional PHY in the tomato genome. Portions of each gene, encoding amino acids 203 through 315 in a consensus amino acid sequence, were amplified by polymerase chain reaction. Four of these genes, PHYA, PHYB1, PHYB2 and PHYE, are members of previously identified PHY subfamilies, while the fifth, PHYF, is identified as a member of a new PHY subfamily. PHYA, PHYB1, PHYB2 and PHYE fragments encode amino acid sequences that share 88% to 98% sequence identity with their Arabidopsis counterparts. The PHYF fragment, however, encodes a polypeptide that shares only 65% to 74% sequence identity with previously identified Arabidopsis phytochromes. A phylogenetic analysis suggests that PHYF arose soon after, or perhaps prior to, the origin of angiosperms. This analysis leads to the prediction that PHYF might be widespread among angiosperms, including both monocotyledons and dicotyledons. Each of the five tomato PHY is expressed as a transcript of sufficient size to encode a full-length phytochrome apoprotein. Two PHYF transcripts, 4.4 and 4.7 kb in length, have been detected in 9-day-old light-grown seedlings, consistent with either multiple transcription start sites or differential processing. Analyses of genomic Southern blots hybridized with radiolabelled RNA probes derived from the five tomato PHY, as well as Arabidopsis PHYC, indicate that the tomato genome contains as many as 9 to 13 PHY. The tomato PHY family is apparently not only different from, but also larger than, the PHY family presently described for Arabidopsis. PMID- 8616213 TI - Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum. AB - The phytochrome photoreceptor in the green alga Mesotaenium caldariorum is encoded by a small family of highly related genes. DNA sequence analysis of two of the algal phytochrome genes indicates an atypical gene structure with numerous long introns. The two genes, termed mesphy1a and mesphy1b, encode polypeptides which differ by one amino acid in the region of overlap that was sequenced. RT PCR studies have established the intron-exon junctions of both genes and show that both are expressed. RNA blot analysis indicates a single transcript of ca. 4.1 kb in length. The deduced amino acid sequence of the mesphy1b gene reveals that the photoreceptor consists of 1142 amino acids, with an overall structure similar to other phytochromes. Phylogenetic analyses indicate that the algal phytochrome falls into a distinct subfamily with other lower plant phytochromes. Profile analysis of an internal repeat found within the central hinge region of the phytochrome polypeptide indicates an evolutionary relatedness to the photoactive yellow protein from the purple bacterium Ectothiorhodospira halophila, to several bacterial sensor kinase family members, and to a family of eukaryotic regulatory proteins which includes the period clock (per) and single minded (sim) gene products of Drosophila. Since mutations which alter phytochrome activity cluster within the region delimited by these direct repeats (P.H. Quail et al., Science 268 (1995): 675-680), this conserved motif may play an important role in the signal transducing function of these disparate protein families. PMID- 8616215 TI - A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca(2+)-binding proteins. AB - Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca(2+)-binding sites of Ca(2+)-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca(2+)-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the Ige of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species. PMID- 8616216 TI - Production of biologically active hirudin in plant seeds using oleosin partitioning. AB - A plant oleosin was used as a 'carrier' for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin 'carrier'. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide. PMID- 8616217 TI - The macrophage-specific membrane protein Nramp controlling natural resistance to infections in mice has homologues expressed in the root system of plants. AB - In mice, natural resistance or susceptibility to infection with Mycobacteria, Salmonella, and Leishmania is controlled by a gene named Bcg. Bcg regulates the capacity of macrophages to limit intracellular replication of the ingested parasites, and is believed to regulate a key bactericidal mechanism of this cell. Recently, we have cloned the Bcg gene and shown that it encodes a novel macrophage-specific membrane protein designated Nramp. A routine search of the public databases for sequences homologous to Nramp identified 3 expressed sequence tags (EST) that show strong similarities to the mammalian protein. We report the identification and cloning of a full-length cDNA clone corresponding to a plant homologue (OsNramp1) of mammalian Nramp. Predicted amino acid sequence of the plant protein indicates a remarkable degree of similarity (60% homology) with its mammalian counterpart, including identical number, position, and composition of transmembrane domains, glycosylation signals, and consensus transport motif, suggesting an identical overall secondary structure and membrane organization for the two proteins. This high degree of structural similarity indicates that the two proteins may be functionally related, possibly through a common mechanism of transport. RNA hybridization studies and RT-PCR analyses indicate that OsNramp1 mRNA is expressed primarily in roots and only at very low levels in leaves/stem. DNA hybridization studies indicate that OsNramp1 is not a single gene, but rather forms part of a novel gene family which has several members in all plants tested including cereals such as rice, wheat, and corn, and also in common weed species. The striking degree of conservation between the macrophage-specific mammalian Nramp and its OsNramp1 plant homologue is discussed with respect to possible implications in the metabolism of nitrate in both organisms. PMID- 8616218 TI - The seed lectins of black locust (Robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes. AB - Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust. PMID- 8616219 TI - A non-photosynthetic ferredoxin gene is induced by ethylene in Citrus organs. AB - The sequence and expression of mRNA homologous to a cDNA encoding a non photosynthetic ferredoxin (Fd1) from Citrus fruit was investigated. The non photosynthetic nature of this ferredoxin was deduced from: (1) amino acid sequence alignments showing better scores with non-photosynthetic than with photosynthetic ferredoxins, (2) higher expression in tissues containing plastids other than chloroplast such as petals, young fruits, roots and peel of fully coloured fruits, and (3) the absence of light-dark regulation characteristic of photosynthetic ferredoxins. In a phylogenetic tree constructed with higher-plant ferredoxins, Citrus fruit ferredoxin clustered together with root ferredoxins and separated from the photosynthetic ferredoxins. Non photosynthetic (root and fruit) ferredoxins, but not the photosynthetic ferredoxins, have their closest homologs in cyanobacteria. Analysis of ferredoxin genomic organization suggested that non-photosynthetic ferredoxins exist in Citrus as a small gene family. Expression of Fd1 is developmentally regulated during flower opening and fruit maturation, both processes may be mediated by ethylene in Citrus. Exogenous ethylene application also induced the expression of Fd1 both in flavedo and leaves. The induction on non-photosynthetic ferredoxins could be related with the demand for reducing power in non-green, but biosynthetically active, tissues. PMID- 8616220 TI - Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress. AB - A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and -II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants. PMID- 8616221 TI - Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes. AB - Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5 methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization. PMID- 8616222 TI - The GATA-binding protein CGF-1 is closely related to GT-1. AB - Many light-regulated genes contain a conserved GATA motif in their 5'-upstream region. We have characterized in detail the GATA-binding factor, CGF-1, which bonds within a 73 bp TATA-proximal light/circadian regulatory element in the Arabidopsis cab2 promoter and to two more sites farther upstream. CGF-1 was found to be distinct from other metal-dependent GATA-binding factors, but to have the same sequence requirements for binding and similar physical and chemical properties as GT-1, a factor required for light regulation of the tobacco rbcS-3A gene. CGF-1 was found to be constitutively present in extracts and was shown to be immunologically related to GT-1. The close similarity between CGF-1 and GT-1 suggests that a GT-1-like factor is involved in the phytochrome/circadian regulation of the cab2 gene. CGF-1 and GT-1 were also found to have similar sequence specificities to another constitutively-regulated GATA factor, IBF-2b, which binds the I box region of the tomato nitrate reductase gene. Of three complexes detected using an IBF-2b-specific probe, only one was identical to CGF 1/GT-1. The other two were similar to IBF-2b, demonstrating that CGF-1/GT-1, although very similar, are actually distinct from IBF-2b. These data indicate that more than one factor can bind to the same short sequence and may indicate how constitutively present factors like GT-1 can play a role in light regulation. PMID- 8616223 TI - Repair mechanisms of UV-induced DNA damage in soybean chloroplasts. AB - In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation. PMID- 8616224 TI - Cloning of the amphibolic Calvin cycle/OPPP enzyme D-ribulose-5-phosphate 3 epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects. AB - Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene. PMID- 8616225 TI - The maize GapC4 promoter confers anaerobic reporter gene expression and shows homology to the maize anthocyanin regulatory locus C1. AB - The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) gene family of maize is differentially expressed in response to anaerobic stress. While GapCl and GapC2 are downregulated, GapC3 and GapC4 are anaerobically induced. We have sequenced and analyzed a 3073 bp promoter fragment of GapC4. The promoter confers anaerobic induction of a reporter gene construct in a transient gene expression system in maize. Deletion analysis of the GapC4 promoter revealed a 270 bp long DNA region required for anaerobic induction. This region contains sequence motifs resembling the cis-acting sequences of the anaerobically induced maize Adh1 and Adh2 genes. Furthermore, the 3073 bp GapC4 promoter fragment displays homology to long terminal repeats of maize retrotransposons and to the 3' region of the maize anthocyanin regulatory locus C1. PMID- 8616226 TI - 5'-regulatory region of Agrobacterium tumefaciens T-DNA gene 6b directs organ specific, wound-inducible and auxin-inducible expression in transgenic tobacco. AB - The regulatory activity of a 826 bp DNA fragment located upstream of the pTiBo542 TL-DNA gene 6b coding region was analyzed in transgenic tobacco, using beta glucuronidase (gus) as a reporter gene. The region was shown to drive organ specific, wound- and auxin-inducible expression of the reporter, the effect being dependent on the type and concentration of auxin. PMID- 8616227 TI - The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization. AB - Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals. PMID- 8616229 TI - Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco. AB - The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the beta glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elongation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B + C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene. PMID- 8616228 TI - Impaired splicing of the rps12 transcript in ribosome-deficient plastids. AB - Analysis of RNA maturation in ribosome-deficient plastids of four non-allelic barley mutants revealed an increased accumulation and altered processing of transcripts of the ribosomal protein gene CS12 (rps12) compared to normal chloroplasts. The three exons of rps12 are part of two different polycistronic transcription units. Generation of mature rps12-mRNA involves both cis- and trans splicing. In ribosome-deficient plastids, the cis-intron separating exons 2 and 3 remains entirely unspliced whereas the splicing of the bipartite rps12 trans intron between exon 1 and exon 2 occurs, but at a reduced level. A comparison of the 3' and 5' ends of the two RNAs that are generally assumed to interact during trans-splicing showed a difference in the processing pathways of 3' rps12 transcripts between mutated and normal chloroplasts. Nonetheless, the final products were identical. PMID- 8616230 TI - Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin associated NTPase in pea. AB - A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene. PMID- 8616231 TI - Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. AB - Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais. PMID- 8616232 TI - The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase. AB - The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal 57 residues comprise the chloroplast transit sequence. The Chlamydomonas protochlorophyllide reductase has 66-70% identity (79-82% similarity) to the higher plant enzymes. Transcripts encoding protochlorophyllide reductase are abundant in dark-grown wild-type cells, but absent or at very low levels in cells grown in the light. Similarly, immunoreactive protochlorophyllide reductase protein is also present to a greater extent in dark- versus light-grown wild-type cells. Both pc-1 and pc-1 y-7 cells lack CRlpcr-1 mRNA and the major (36 kDa) immunodetectable form of protochlorophyllide reductase consistent with their inability to photoreduce protochlorophyllide. DNA sequence analysis revealed that the lpcr gene in pc-1 y 7 cells contains a two-nucleotide deletion within the fourth and fifth codons of the protochlorophyllide reductase precursor that causes a shift in the reading frame and results in premature termination of translation. The absence of protochlorophyllide reductase message in pc-1 and pc-1 y-7 cells is likely the consequence of this frameshift mutation in the lpcr gene. Introduction of the CRlpcr-1 gene into pc-1 y-7 cells by nuclear transformation was sufficient to restore the wild-type phenotype. Transformants contained both protochlorophyllide reductase mRNA and immunodetectable enzyme protein. These studies demonstrate that pc-1 was in fact a defect in protochlorophyllide reductase activity and provide the first in vivo molecular evidence that the lpcr gene product is essential for light-dependent protochlorophyllide reduction. PMID- 8616233 TI - Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean. AB - Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KDEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24 kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs. PMID- 8616234 TI - Molecular analysis of a pistil-specific gene expressed in the stigma and cortex of Solanum tuberosum. AB - A gene, sts14, coding for a highly expressed mRNA in pistils of Solanum tuberosum, was isolated. Northern blot and in situ analyses demonstrated that the gene was expressed throughout pistil development in both the stylar cortex and the stigma. The deduced STS14 protein displays similarity to the pathogenesis related PR-1 proteins. A possible function for protection or guidance of the pollen tubes through the pistil is discussed. PMID- 8616235 TI - Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C in synchronized tobacco BY-2 cells. AB - Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5-249) and KatC(207-754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein. PMID- 8616236 TI - Pumpkin hydroxypyruvate reductases with and without a putative C-terminal signal for targeting to microbodies may be produced by alternative splicing. AB - Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A)+ RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing. PMID- 8616237 TI - Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana. AB - The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed. PMID- 8616239 TI - Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants. AB - The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0 degrees C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning. PMID- 8616238 TI - Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens. AB - As ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes such as development and signal transduction. Overexpression of transgenes after genetic transformation is a powerful technique in such studies. To establish a controllable expression system for this experimental approach we expressed a chimeric protein consisting of the Tn1O encoded Tet repressor and the activation domain of Herpes simplex virion protein 16 in the moss Physcomitrella patens. We showed that this protein activates transcription from a suitable target promoter (Top 1O) containing seven operators upstream of a TATA box. In media containing very low levels of tetracycline (1 mg/l), expression levels of a beta-glucuronidase (GUS) reporter gene dropped to <1% of that in the absence of tetracycline. This regulation is due to interference of tetracycline with the DNA binding activity of the Tet repressor portion of the chimeric transcriptional activator. Stable transformants grown for three weeks on tetracycline-containing media showed negligible GUS activity, whereas GUS was expressed strongly within 24 h of transfer to tetracycline-free media. Potent and stringently regulated expression of other, physiologically active genes is thus readily available in the moss system using the convenient ToplO expression system. PMID- 8616240 TI - Transaldolase genes from the cyanobacteria Anabaena variabilis and Synechocystis sp. PCC 6803: comparison with other eubacterial and eukaryotic homologues. AB - We have sequenced and analysed the transaldolase (tal) genes from two cyanobacteria, Anabaena variabilis (ATCC 29413) and Synechocystis sp. PCC 6803, which are filamentous heterocyst-forming and unicellular organisms, respectively. The deduced amino acid sequences of the two cyanobacterial tal genes are 78% identical and are highly homologous to both eubacterial and eukaryotic transaldolases (Escherichia coli, two yeasts, and man) with values ranging from 54 to 60% amino acid identity. In contrast, the transaldolase homologous sequences from the cyanobacterium Nostoc sp. ATCC 29133, from Mycobacterium leprae, and the partial sequence from the higher plant Arabidopsis thaliana have a much lower degree of homology with each other and relative to the sequences mentioned above. These data indicate three different types of transaldolases. PMID- 8616241 TI - A cDNA from pea petals with sequence similarity to pollen allergen, cytokinin induced and genetic tumour-specific genes: identification of a new family of related sequences. AB - A cDNA isolated from pea petals exhibits extensive similarity to pollen allergen genes, a cytokinin-regulated cDNA from soybean suspension cultures, a partial cDNA preferentially expressed in tobacco genetic tumours, four Arabidopsis expressed sequence tags (ESTs) and fifteen rice ESTs. This diverse family of pollen-allergen-likes genes may have a common ancestor or at least share common functional domains. Possession of a putative signal peptide and a presumed extracellular location is a common aspect of this family of sequences. PMID- 8616242 TI - Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA. AB - A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pDl) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5' end of the transcript, the rapid amplification of cDNA end (5'-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis. PMID- 8616243 TI - Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L. AB - Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C4 plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane alpha-helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity purified. The antibody against the recombinant protein cross-reacted with proteins of 31-32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate. PMID- 8616244 TI - Higher-plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement. AB - Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme. We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms. A very high calculated net negative charge of 63 for PGK from H. vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol. We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes. The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria. Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol. Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e. contain more genes of eubacterial origin, than is generally assumed. PMID- 8616247 TI - A guide to naming sequenced plant genes. PMID- 8616246 TI - Two closely related cDNAs encoding starch branching enzyme from Arabidopsis thaliana. AB - Two starch branching enzyme (SBE) cDNAs were identified in an Arabidopsis seedling hypocotyl library using maize Sbe1 and Sbe2 cDNAs as probes. The two cDNAs have diverged 5' and 3' ends, but encode proteins which share 90% identity over an extensive region with 70% identity to maize SBE IIb [12]. Genomic Southern blots suggest that the two cDNAs are the products of single, independent genes, and that additional, more distantly related SBE genes may exist in the Arabidopsis genome. The two cDNAs hybridize to transcripts which show similar expression patterns in Arabidopsis vegetative and reproductive tissues, including seedlings, inflorescence rachis, mature leaves, and flowers. This is the first report of the identification of cDNAs encoding two closely related starch branching enzymes from the same species. PMID- 8616245 TI - Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2' promoter wound-responsive elements. AB - A 318 bp mannopine synthase 2' (mas2') promoter element from the T-DNA of Agrobacterium tumefacians can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from 318 to -213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from -103 to +66 and from -213 to -138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated ( 209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2' TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2' promoter elements in wound-inducible gene expression. PMID- 8616248 TI - Molecular cloning and characterization of a gibberellin-inducible, putative alpha glucosidase gene from barley. AB - A putative alpha-glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A 3 (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal alpha-glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI alpha-amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this alpha-glucosidase gene and two additional sequences that may be related genes or pseudogenes. PMID- 8616249 TI - Random chemical mutagenesis of a specific psbDI region coding for a lumenal loop of the D2 protein of photosystem II in Synechocystis sp. PCC 6803. AB - To identify amino acid residues of the D2 protein that are critical fo r functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautotrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins. PMID- 8616250 TI - Structure and characterization of a putative drought-inducible H1 histone gene. AB - A drought- and abscisic acid (ABA)-inducible gene, His1, was isolated from Lycopersicon pennellii, a drought-resistant relative of cultivated tomato, and the gene structure was defined experimentally. The nucleotide sequence of His1 predicts a protein of 202 amino acid residues, with a significant sequence homology to plant H1 histones. Consensus sequences for both H1 histone-specific promoter elements as well as an ABA-responsive element were identified in the 5' flanking region of His1. Transcripts of this gene accumulate in leaf tissue in response to drought in three tomato species including cultivated tomato (L. esculentum), L. pennellii, and L. chilense, as well as in tobacco. Transcripts for His1 are constitutively expressed in roots; transcript abundances in tomato root tips were equivalent to transcript abundances in more mature regions of the seedling root. The accumulation in leaves of transcripts for His1 preceded visible symptoms of drought stress in the plants. Transcript accumulation was detected in both drought-sensitive and drought-resistant species at similar leaf water potentials, psi W -1.3 to -1.4 MPa. PMID- 8616251 TI - Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway. AB - To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized. The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes. The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool. By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to zeta-carotene. RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm. Other phytoene accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts. RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed. Therefore, endosperm carotenogensis is not regulated by increasing the level of Pds transcripts. PMID- 8616252 TI - Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by corresponding cDNA. AB - ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source. PMID- 8616253 TI - Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene. AB - Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3' ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers. PMID- 8616254 TI - Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase. AB - A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme, the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3' non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (> 60%) but a poor homology is observed when compared to Escherichia coli ICDH (< 20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now. PMID- 8616255 TI - Promoter regions of cysteine endopeptidase genes from legumes confer germination specific expression in transgenic tobacco seeds. AB - Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds. PMID- 8616256 TI - Induction of homologous low temperature and ABA-responsive genes in frost resistant (Solanum commersonii) and frost-sensitive (Solanum tuberosum cv. Bintje) potato species. AB - A DNA fragment corresponding to a low-temperature- and ABA-responsive gene (Scdhn1) was amplified by PCR from genomic DNA of a wild, frost-resistant potato species, Solanum commersonii. A homologous gene (Stdhn1) was identified in Solanum tuberosum cv. Bintje, a frost-sensitive domesticated potato cultivar. The expression of the gene was studied during low temperature and ABA treatments in both Solanum species. The analysis revealed that both low temperature and ABA lead to the accumulation of a 1 kb transcript that corresponded to the PCR fragment. The induction of the gene was relatively rapid and maximum amounts of the transcripts were detected already after 1 day and 7 h of treatment with low temperature and ABA, respectively. Previous results have shown that there is no increase in the amount of endogenous ABA in S. tuberosum during low-temperature treatment, which indicates that two independent signalling pathways lead to the induction of this gene. PMID- 8616257 TI - Sequence analysis of the second largest subunit of tomato RNA polymerase II. AB - We have cloned and sequenced the cDNA encoding the open reading frame of the mRNA of the second largest subunit of RNA polymerase II, or RPB2, of tomato. The mRNA is transcribed from a single-copy gene in the tomato genome and the transcript size of the gene was measured as 4.2 kb by northern analysis. From the deduced amino acid sequence of 1191 residues, a protein of M r 135 000 with an isoelectric point of pH 7.9 was predicted. Alignment of the tomato RPB2 protein sequence with those of the homologous subunits in Arabidopsis, man, Drosophila and yeast showed considerable sequence identity. PMID- 8616258 TI - Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants. AB - In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the beta-glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter. PMID- 8616259 TI - Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants. AB - Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter GUS fusion of A.h.Chi2;1 are described. PMID- 8616260 TI - High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants. AB - Ustilago maydis killer toxins are small polypeptides (7-14 kDa) which kill susceptible cells of closely related fungal species. The KP4 toxin is a single polypeptide subunit with a molecular weight of 11.1 kDa. In this work, a transgenic tobacco plant was constructed which secretes the KP4 toxin at a high level. The KP4 toxin expressed in this transgenic plant was of the same size and specificity as the authentic Ustilago KP4 toxin. The expression level was at least 500 times higher than that of the KP6 toxin expressed in plants. Transgenic crop plants producing the KP4 toxin could be rendered resistant to KP4 susceptible fungal pathogens. PMID- 8616261 TI - Isolation and characterization of the Adh2 5' region from Petunia hybrida. AB - The Adh2 gene from Petunia hybrida has been difficult to clone; exons 1 to 8 were isolated using PCR after unsuccessful screening of three genomic libraries. A combination of inverse and direct PCR strategies has been used to isolate upstream regions of Adh2. Here we report the cloning strategy for the nucleotide sequence of the 5' region of Adh2 from P. hybrida, the locations of the transcriptional start site and putative TATA box, as well as comparative analyses of the upstream regions of petunia Adh2, other Adh genes and other genes induced by hypoxia. PMID- 8616262 TI - Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds. AB - A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root. PMID- 8616264 TI - High-performance liquid chromatography/tandem mass spectrometry identification of salmon calcitonin degradation products in aqueous solution preparations. AB - Degradation impurities have been investigated, by means of high-performance liquid chromatography (HPLC)/ mass spectrometry, in commercial ampoules of salmon calcitonin, aqueous solution, stored for up to 2 years. Oxidation derivatives of the cysteine residues in positions 1 and 7, with cleavage of the disulfide bridge, were observed in all samples. Using our reversed-phase separation, these oxidation derivatives coeluted and presented an HPLC behavior identical to the derivative of salmon calcitonin with the disulfide bridge reduced. In a previous study this last compound was tentatively identified, without confirmation by mass spectrometry, as a major degradation product of salmon calcitonin in aqueous solution. PMID- 8616263 TI - Genomic structure of the rice aldolase isozyme C-1 gene and its regulation through a Ca 2+ -mediated protein kinase-phosphatase pathway. AB - Complementary and genomic DNA clones coding for aldolase C-1, the fourth-type isozyme of aldolase in rice Oryza sativa L., have been characterized. The organization of the gene is quite similar to those encoding rice aldolase C-a and a maize cytoplasmic-type aldolase, in that introns are located in the same position. Amino acid sequences are highly conserved among cytoplasmic aldolases in plants. Expression of the gene in rice callus is activated by a protein phosphatase inhibitor okadaic acid, and is inhibited in the presence of thapsigargin, a reagent which increases calcium influx into the cytoplasm. The inhibition is rescued by the simultaneous addition of protein kinase inhibitor H 7. Thus, it is suggested that expression of the aldolase C-1 gene is regulated through a signal transduction pathway involving a Ca 2+ -mediated protein kinase protein phosphatase system. PMID- 8616265 TI - An electrospray investigation on in vitro glycation of ribonuclease. AB - The in vitro reactions of RNase with different concentrations of glucose or fructose have been studied by means of electrospray mass spectrometry coupled with microcolumn high performance liquid chromatography. The results obtained have shown that, subsequent to the protein glycation, a series of cross-linking products are generated. Their molecular weights demonstrate that severe degradation processes of the proteic substrate takes place after the cross linking process. PMID- 8616266 TI - Electrospray-collision-induced dissociation mass spectrometry of mono-, di- and tri-hydroxylated lipoxygenase products, including leukotrienes of the B-series and lipoxins. AB - Collision-induced dissociation (CID) mass spectra were recorded on underivatized, hydroxylated lipoxygenase products, including S(S)-, 12(S)- and 15(S) hydroxyeicosatetraenoic adds (HETE), leukotriene (LT) B4, LTB5, the LTB4 metabolites 20-OH-LTB4, 20-COOH-LTB4 and 10,11-dihydro-LTB4, the LTB4 isomers 6 trans-LTB4, 6-trans,12-epi-LTB4 and S(S),12(S)-dihydroxyeicosatetraenoic acid (diHETE) as well as S(S),6(R)-diHETE, 14(S), 15(R)-diHETE and lipoxin (LX) A4 and B4. Pseudomolecular [M-H]- parent ions were generated by electrospray ionization. The ions were accelerated to 4 keV, selected by the double focusing sectors Of 2 hybrid tandem mass spectrometer, decelerated to 400 eV and focused into a collision cell containing xenon. The resulting fragment ions were mass analysed by an orthogonal time-of-flight analyser. The CID spectra obtained were structurally informative allowing isomer differentiation. PMID- 8616267 TI - Distonic ion .CH2CH2SCH2+ and the isomeric trimethylene and propylene sulfide radical cations. Assessment of structures and reactivities via decomposition and redox reactions. AB - The distonic radical ion .CH2CH2SCH2+ (1+.), generated by dissociative electron ionization of 1,4-dithiane or 1,4-thioxane, is identified in the gas phase by its collisionally activated dissociation (CAD), neutralization -reionization (+NR+) and charge-reversal (+CR-) mass spectra. The unimolecular chemistry of 1+. is shown to be substantially different from that of the isomeric, ring-closed, trimethylene sulfide ion (2+.). Hence, a substantial isomerization barrier must separate 1+. from the thermodynamically more stable 2+.. Charge permutation (i.e. charge-stripping, +NR+ and +CR-) are far superior, compared to collision-induced fragmentation, for distinguishing 1+. from 2+., mainly because the oxidized (1++ and 2++) and reduced forms (1 and 2 as well as 1-. and 2-.) of these cations have much lower tendencies for isomerization that 1+. and 2+. themselves. The diradical .CH2CH2SCH2. (1), formed by neutralization of 1+., is found to exist as a bound species, requiring appreciable activation energies for both decomposition to CH2CH2 plus SCH2 and ring-closure to 2. The dissociations and redox reactions of the propylene sulfide ion (3+.) are also assessed in this study and clearly indicate that 3+. is a stable C3H6S+. isomer. Further, the C3H6S+. ions from thiane, 1,3-dithiane and 2-methyl-1,3-dithiane are characterized based on their combined CAD, +NR+ and +CR- spectra. The two 1,3-dithianes produce ionized trimethylene sulfide, 2+.. In contrast, thiane gives rise to a C3H6S+. isomer other than 1+.(-3t); the data strongly suggest that this isomer is the 1-propene 1-thiol radical cation, namely CH3CH = CH-SH+.. PMID- 8616268 TI - 1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: presence of an amino terminal helix in solution. AB - 1H, 15N and 13C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007-1010]. In the group I enzyme from porcine pancreas, the amino terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G. H., Kaptein, R. and Verheij, H.M. (1995) Nature Strct. Biol., 2, 402-406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well positioned amino-terminal helix in the absence of substrate. Therefore, there is a clear difference between the conformation group I and group II enzymes in solution. These conformational differences suggest that formation of the amino terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases. PMID- 8616270 TI - Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C') and 3J(H beta,C') coupling constants in desulfovibrio vulgaris flavodoxin. AB - A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C') and 3J(H beta,C') in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95-105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of phi and chi 1 dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant 13C,15N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 3J(HN,C') coupling constants from a single spectrum. PMID- 8616269 TI - High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. AB - An approach to produce 13C- and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40-200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C(6)-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C(6)-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purifies IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI. PMID- 8616272 TI - Suppression of spin diffusion in selected frequency bands of nuclear Overhauser spectra. AB - A variant of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) is described that yields information about cross-relaxation rates between pairs of spins, while the migration of magnetization through several consecutive steps (spin diffusion via neighboring spins) is largely suppressed. This can be achieved by inserting a doubly-selective inversion pulse in a conventional NOESY sequence. PMID- 8616271 TI - Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C /15N-labeled RNA with an HNC-TOCSY-CH experiment. AB - A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in 13C-/15N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs. PMID- 8616274 TI - Respectful treatment of the elderly. PMID- 8616273 TI - Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy. AB - The growth factor receptor-bound protein-2 (Grb-2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly 13C-/15N-enriched protein as well as the protein containing selectively 15N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, 1H alpha, 13C alpha, 13C beta and 13CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423 438]. The core structure of the SH2 domain contains an antiparallel beta-sheet and two alpha-helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment. PMID- 8616275 TI - What we want: qualitative research. Promising frontier for family medicine. PMID- 8616276 TI - Qualitative research. A personal journey. PMID- 8616277 TI - Battling environmental concerns. How physicians and hospitals can work together. PMID- 8616278 TI - Payment equity for prescription drugs. PMID- 8616279 TI - A clinical dilemma. PMID- 8616280 TI - Equality in health promotion. PMID- 8616281 TI - Lead risk during pregnancy. PMID- 8616282 TI - Radiology rounds. The diagnosis is normal pressure hydrocephalus. The treatment is cerebrospinal shunting. PMID- 8616283 TI - [Study on insomnia treatment by family physicians]. AB - OBJECTIVES: To describe treatment of insomnia in general practice and to identify family physicians' training needs in this area. DESIGN: Mail survey using Dillman's total design method. PARTICIPANTS: A sampling of 484 general practitioners in the Quebec City area was done to provide roughly equal representation of six practice settings. The response rate was 65%; 295 of the 315 questionnaires returned were selected for analysis. RESULTS: Most physicians reported treating insomnia with general advice and lifestyle changes; 25% reported prescribing hypnotics frequently; 56% reported they prescribed them occasionally. Although 58% often recommend relaxation techniques, only 8% taught these techniques to their patients. Other cognitive and behavioral approaches are rarely used. Most felt that training in treating insomnia should be offered. CONCLUSION: Cognitive and behavioral approaches are very effective approaches are very effective nonpharmacological treatments for insomnia. General practitioners make little use of these treatments that could be easily integrated into clinical practice. Strategies for increasing their use discussed. PMID- 8616284 TI - Needs for CME in geriatrics. Part 1: Perceptions of patients and community informants. AB - OBJECTIVE: To describe the needs of physicians for continuing education in geriatrics as perceived by patients and community informants. DESIGN: Cross sectional survey by mail and in-person interviews. SETTING: Organizations working with the elderly in the community and patients in a primary care population in Calgary. PARTICIPANTS: Key informants working with the elderly in the community, including managers and providers of physical, psychosocial, educational, or mental health services to the elderly, and the first two geriatric patients visiting physicians after telephone contact from study investigators were surveyed. Twenty-five of 27 key community informants and 32 of 61 geriatric patients responded. MAIN OUTCOME MEASURE: Potential topics for continuing medical education. RESULTS: The 10 most frequently identified topics were communication, time management, attitudes to the elderly, medication, continuity of care, mental health, medical management of complicated cases, knowledge of community resources, health promotion, and compassion. Patients were more concerned than key informants about the process of care. Key informants were concerned about the technical aspects of care. CONCLUSIONS: The process of care as well as technical aspects of care must be addressed in continuing education in geriatrics for physicians. PMID- 8616285 TI - Long hospital stays and need for alternate level of care at discharge. Does family make a difference for elderly patients? AB - OBJECTIVE: To determine whether parental and marital status of elderly patients admitted to acute care affect the likelihood of a need for long hospital stay or alternate level of care (nursing home) at discharge. DESIGN: A 1-year descriptive study was carried out prospectively on elderly hospitalized patients. Marital status and parental status were treated as risk factors for resource use, as were sex, age, admitting service, and diagnosis. SETTING: A 672-bed university hospital. PATIENTS: We studied 495 patients aged 65 years or more sequentially admitted over a 1-year period. Excluded from study were critically ill patients, patients admitted to intensive care, and patients with whom we could not communicate on the day were considered for the study. MAIN OUTCOME MEASURES: Whether acute hospital stay exceed 44 days and need for alternate level of care at discharge. RESULTS: Many (43.4%) of the patients had no spouse and 19.4% had no children; 32.9% stayed 45 days or more and 6.9% required alternate level of care at discharge. Predictive of a long hospital stay were being without children (adjusted RR = 1.85), having a neurologic or psychiatric diagnosis (adjusted RR = 3.39), and having surgery unrelated to reason for admission (adjusted RR = 5.88). Predictive of need for alternate level of care at discharge were increasing age (adjusted RR = 1.08), having no spouse (adjusted RR = 2.59), having no children (adjusted RR = 3.27), and having a neurologic or psychiatric diagnosis (adjusted RR = 7.56). PMID- 8616287 TI - Ottawa ankle rules. AB - The Ottawa ankle rule project demonstrated that more than 95% of patients with ankle injuries had radiographic examinations but that 85% of the films showed no fractures. A group of Ottawa emergency physicians developed two rules to identify clinically important fractures of the malleoli and the midfoot. Use of these rules reduced radiographic examinations by 28% for the ankle and 14% for the foot. PMID- 8616286 TI - Clinical practice guidelines. New-to-practice family physicians' attitudes. AB - OBJECTIVE: To examine the attitudes toward clinical practice guidelines of a group of family physicians who had recently entered practice in Ontario, and to compare them with the attitudes of a group of internists from the United States. DESIGN: Mailed questionnaire survey of all members of a defined cohort. SETTING: Ontario family practices. PARTICIPANTS: Certificants of the College of Family Physicians of Canada who received certification in 1989, 1990, and 1991 and who were practising in Ontario. Of 564-cohort members, 395 (70%) responded. Men (184) and women (211) responded at the same rate. MAIN OUTCOME MEASURES: Levels of agreement with 10 descriptive statements about practice guidelines and analyses of variance of these responses for several physician characteristics. RESULTS: Of respondents in independent practice, 80% were in group practice. Women were more likely to have chosen group practice, in which they were more likely to use practice guidelines than men. Generally favourable attitudes toward guidelines were observed. Physician characteristics occasionally influenced agreement with the descriptors. The pattern of agreement was similar to that noted in the study of American internists, but, in general, Ontario physicians were more supportive. CONCLUSIONS: This group of relatively new-to-practice Ontario family physicians shows little resistance to guidelines and appears to read less threat of external control in them than does the US group. PMID- 8616288 TI - Treating musculoskeletal disease with NSAIDs. Practitioner's guide. AB - Although not a panacea for all painful conditions, nonsteroidal anti-inflammatory drugs (NSAIDs) are the mainstay of treatment for most musculoskeletal disorders in the western world. This article offers a practical guide to NSAIDs based on six clinical issues that strongly affect the outcome of treatment. PMID- 8616289 TI - Diagnosis of deep vein thrombosis. AB - Deep vein thrombosis (DVT), a common disease, can be difficult to diagnose because its clinical features are nonspecific. Venography is the standard test, but other less expensive, easily performed, noninvasive tests are available. At present, duplex ultrasonography is the noninvasive test of choice. PMID- 8616291 TI - Overwork can kill. PMID- 8616290 TI - Patient health questionnaire. Using a patient-focused assessment tool. AB - A patient health questionnaire was developed for use in family practice to help identify lifestyle risk factors, assess health care needs, and better understand patients. This article discusses the questionnaire's development and practical application. PMID- 8616292 TI - Privatising the NHS: dentistry paves the way. PMID- 8616293 TI - Hospital at home. PMID- 8616294 TI - Health and human rights. PMID- 8616295 TI - Travel associated illness. PMID- 8616296 TI - Patenting gene sequences. PMID- 8616297 TI - Threatened surgeon has case reconsidered. PMID- 8616299 TI - Dutch crack down on drugs policy. PMID- 8616298 TI - Judge allows baby to die in peace. PMID- 8616300 TI - Women's prison regime was "squalid and degrading". PMID- 8616301 TI - Doctors launch twins research company. PMID- 8616302 TI - Police investigate suspicious hospital deaths. PMID- 8616303 TI - Effect on lipoprotein profile of replacing butter with margarine in a low fat diet: randomised crossover study with hypercholesterolaemic subjects. AB - OBJECTIVE: To examine the effect on lipid and lipoprotein concentrations when butter or an unsaturated margarine is used for cooking or spreading in a reduced fat diet. DESIGN: Randomised crossover study with two intervention periods of six weeks' duration separated by a five week washout. SETTING: Community setting in New Zealand. SUBJECTS: 49 volunteers with polygenic hypercholesterolaemia and baseline total cholesterol concentration in the range 5.5-7.9 mmol/l. MAIN OUTCOME MEASURES: Concentrations of total and low density lipoprotein, Lp(a) lipoprotein, high density lipoprotein, apolipoprotein B 100, and apolipoprotein A I. RESULTS: Concentrations of low density lipoprotein cholesterol and apolipoprotein B were about 10% lower with margarine than with butter. Lp(a) lipoprotein and high density lipoprotein cholesterol concentrations were similar with the two diets. CONCLUSION: Despite concerns about adverse effects on lipoproteins of trans fatty acids in margarines, the use of unsaturated margarine rather than butter by hypercholesterolaemic people is associated with a lipoprotein profile that would be expected to reduce cardiovascular risk. PMID- 8616304 TI - Clearance of chylomicron remnants in normolipidaemic patients with coronary artery disease: case control study over three years. AB - OBJECTIVE: To test the hypothesis that subjects who clear chylomicron remnants slowly from plasma may be at higher risk of coronary artery disease than indicated by their fasting plasma lipid concentrations. DESIGN: Case control study over three years. SETTING: An 800 bed general municipal hospital. SUBJECTS: 85 normolipidaemic patients with coronary artery disease selected prospectively and matched with 85 normolipidaemic subjects with normal coronary arteries on angiography. INTERVENTIONS: All subjects were given a vitamin A fat loading test which specifically labels intestinal lipoproteins with retinyl palmitate. MAIN OUTCOME MEASURE: Postprandial lipoprotein metabolism. RESULTS: The area below the chylomicron remnant retinyl palmitate curve was significantly increased in the coronary artery disease group as compared with the controls (mean 23.4 (SD 15.0) v 15.3 (8.9) mumol/l.h; 95% confidence interval of difference 4.37 to 11.82). CONCLUSION: Normolipidaemic patients with coronary artery disease had significantly higher concentrations of chylomicron remnants in plasma than normolipidaemic subjects with normal coronary vessels. This may explain the mechanism underlying the susceptibility to atherosclerosis of coronary artery disease patients with normal fasting lipid values. As diet and drugs can ameliorate the accumulation of postprandial lipoproteins in plasma, the concentration of chylomicron remnants should be measured in patients at high risk of coronary artery disease. PMID- 8616306 TI - Commentary: genes for osteoarthritis: interpreting twin data. PMID- 8616305 TI - Genetic influences on osteoarthritis in women: a twin study. AB - OBJECTIVES: To assess the relative contribution of genetic and environmental factors to common forms of osteoarthritis of the hands and knees. DESIGN: Classic twin study with unselected twins who were screened radiologically for osteoarthritis. SUBJECTS: 130 identical and 120 non-identical female twins aged 48-70 recruited from a London based twin register and through a national media campaign. MAIN OUTCOME MEASURES: Similarity in identical compared with non identical twin pairs for radiographic changes at the interphalangeal and first carpometacarpal joints of the hands and the tibiofemoral joint and patellofemoral joint of the knee expressed as intraclass correlations. RESULTS: The intraclass correlations of radiographic osteophytes and narrowing at most sites and the presence of Heberden's nodes and knee pain were higher in the identical pairs. The intraclass correlation of the total radiographic osteoarthritis score in identical pairs (rMZ) was 0.64 (SE 0.05) compared with 0.38 (0.08) in non identical pairs. The proportion of genetic variance of total osteoarthritis score (osteophytes and narrowing) with modelling techniques was estimated at 0.54 (95% confidence interval 0.43 to 0.65) and ranged from 0.39 to 0.65 for different sites and features (p < 0.001) after adjustment for age and weight. CONCLUSIONS: These results demonstrate for the first time a clear genetic effect for radiographic osteoarthritis of the hand and knee in women, with a genetic influence ranging from 39-65%, independent of known environmental or demographic confounders. The results of this study should lead to further work on isolating the gene or genes involved in the pathogenesis of the common disabling disease. PMID- 8616307 TI - Survey of intensive care of severely head injured patients in the United Kingdom. AB - OBJECTIVES: To study practice in intensive care of patients with severe head injury in neurosurgical referral centres in United Kingdom. DESIGN: Structured telephone interview of senior nursing staff in intensive care unit of adult neurosurgical referral centre. SETTING: 39 intensive care units in hospitals that accepted acute head injuries for specialist neurosurgical management, identified from Medical Directory and information from professional bodies. MAIN OUTCOME MEASURES: Details of organisation and administration of intensive care and patterns of monitoring and treatment for patients admitted with severe head injury. RESULTS: Patients were managed in specialist neurosurgical intensive care units in 21 of the centres and in general intensive care units in 18. Their intensive care was coordinated by an anaesthetist in 25 units and by a neurosurgeon in 12. Annual case-load varied between units: 20 received > 100 patients, 12 received 50-100, and seven received 25-49. Monitoring and treatment varied considerably between centres. Invasive arterial pressure monitoring was used routinely in 36 units, but central venous pressure monitoring was routinely used in 24 and intracranial pressure was routinely monitored in only 19. Corticosteroids were used to treat intracranial hypertension in 19 units. Seventeen units routinely aimed for arterial carbon dioxide pressure of 3.3-4.0 kPa, and one unit still used severe hyperventilation to a pressure of < 3.3 kPa. CONCLUSION: The intensive care of patients with acute head injuries varied widely between the centres surveyed. Rationalisation of the intensive care of severe head injury with the production of widely accepted guidelines ought to improve the quality of care. PMID- 8616308 TI - A follow up study of depression in the carers of dementia sufferers. PMID- 8616309 TI - Contrasting epidemiology of aortic aneurysm and peripheral vascular disease in England and Wales. PMID- 8616310 TI - A study of general practitioners' reasons for changing their prescribing behaviour. AB - OBJECTIVES: To explore general practitioners' reasons for recent changes in their prescribing behaviour. DESIGN: Qualitative analysis of semistructured interviews. SETTING: General practice in south east London. SUBJECTS: A heterogeneous sample of 18 general practitioners. RESULTS: Interviewees were able to identify between two and five specific changes that had occurred in their prescribing in the preceding six months. The most frequently mentioned changes related to fluoxetine, angiotensin converting enzyme inhibitors, and the antibiotic treatment of Helicobacter pylori. Three models of change were identified: an accumulation model, in which the volume and authority of evidence were important; a challenge model, in which behaviour change followed a dramatic or conflictual clinical event; and a continuity model, in which change took place against a background of willingness to change, modulated by other factors such as cost pressures and the comprehensible therapeutic action of a drug. Behaviour change was reinforced and sustained by experiences with individual patients. CONCLUSIONS: Multiple factors are involved in general practitioners' decisions to change their prescribing habits. Three models of change can be identified which have important implications for the design and evaluation of interventions aimed at behaviour change. PMID- 8616311 TI - A method of creating a death register for general practice. PMID- 8616312 TI - Evaluating fever in travellers returning from tropical countries. PMID- 8616313 TI - Seabather's eruption--a case of Caribbean itch. PMID- 8616315 TI - ABC of urology. Urinary incontinence and urinary infection. PMID- 8616314 TI - Patients' prerogatives and perceptions of benefit. AB - Patients today demand more information about their treatment. Doctors, however, seem reluctant to cast aside ingrained habits of paternalism, believing they can best interpret therapeutic choices for their patients. Whether doctors can be more objective and effective than patients in interpreting the "probabilities" of medical evidence is open to question. On the other hand, the exercise of choice by patients may itself have a bearing on the probabilities of outcome. Involving patients more in making therapeutic choices is justified if doctors can present options in an unbiased and effective manner and if the process improves the outcome of the care delivered. PMID- 8616316 TI - Dangerous patients with mental illness: increased risks warrant new policies, adequate resources, and appropriate legislation. PMID- 8616317 TI - Commentary: dangerous patients or dangerous diseases? PMID- 8616318 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Gag reflex has no role in ability to swallow. PMID- 8616319 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Speech and language therapists should have participated in study. PMID- 8616320 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Staff find it harder to stop feeding patients with a gastrostomy. PMID- 8616321 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Radiological placement of the gastrostomy tube should be the preferred method. PMID- 8616322 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Mortality associated with nasogastric tube feeding was high. PMID- 8616323 TI - Percutaneous endoscopic gastrostomy feeding after acute dysphagic stroke. Study's methods were inadequate. PMID- 8616324 TI - Girls should be taught at school about importance of folic acid. PMID- 8616325 TI - Health checks and coronary risk. Effect of health checks was underplayed. PMID- 8616326 TI - Serum screening for Down's syndrome. Creates stress for carers and parents. PMID- 8616327 TI - Fertility continues after age 40. PMID- 8616328 TI - Topical acyclovir is beneficial in recurrent herpes labialis. PMID- 8616329 TI - Laboratories should use serum IgM tests to confirm measles. PMID- 8616330 TI - Increased mortality related to asthma among asthmatic patients using major tranquillisers. Underlying ill health is the main risk factor. PMID- 8616331 TI - Training in advanced trauma life support. All aspects of life support should be part of undergraduate curriculum. PMID- 8616332 TI - Training in advanced trauma life support. Go on a course in the United States. PMID- 8616333 TI - Increased mortality related to asthma among asthmatic patients using major tranquillisers. Smoking may increase mortality when patients stop using tranquillisers. PMID- 8616334 TI - Competition continues in the NHS. PMID- 8616335 TI - The Internet. Editorial made extravagant claims. PMID- 8616336 TI - The Internet. Facilities on the Internet may be abused. PMID- 8616337 TI - The Internet. Encryption algorithims are effective in maintaining security. PMID- 8616338 TI - Health Query Language can be used for collecting data from general practices. PMID- 8616339 TI - The Internet. Access is now available in Romania. PMID- 8616340 TI - The Internet. Accessing it is easy. PMID- 8616342 TI - Editing may ruin an obituary. PMID- 8616341 TI - The Internet. MSc in general practice can be done over the Internet. PMID- 8616343 TI - New college of paediatrics and child health is being set up. College must continue to be exclusively for medical practitioners. PMID- 8616344 TI - Rwanda needs visiting teachers and textbooks. PMID- 8616345 TI - Income distribution and mortality: cross sectional ecological study of the Robin Hood index in the United States. AB - OBJECTIVE: To determine the effect of income inequality as measured by the Robin Hood index and the Gini coefficient on all cause and cause specific mortality in the United States. DESIGN: Cross sectional ecological study. SETTING: Households in the United States. MAIN OUTCOME MEASURES: Disease specific mortality, income, household size, poverty, and smoking rates for each state. RESULTS: The Robin Hood index was positively correlated with total mortality adjusted for age (r = 0.54; P < 0.05). This association remained after adjustment for poverty (P < 0.007), where each percentage increase in the index was associated with' an increase in the total mortality of 21.68 deaths per 100,000. Effects of the index were also found for infant mortality (P = 0.013); coronary heart disease (P = 0.004); malignant neoplasms (P = 0.023); and homicide (P < 0.001). Strong associations were also found between the index and causes of death amenable to medical intervention. The Gini coefficient showed very little correlation with any of the causes of death. CONCLUSION: Variations between states in the inequality of income were associated with increased mortality from several causes. The size of the gap between the wealthy and less well off--as distinct from the absolute standard of living enjoyed by the poor--seems to matter in its own right. The findings suggest that policies that deal with the growing inequities in income distribution may have an important impact on the health of the population. PMID- 8616346 TI - Socioeconomic determinants of rates of consultation in general practice based on fourth national morbidity survey of general practices. AB - OBJECTIVE: To identify the socioeconomic determinants of consultation rates in general practice. DESIGN: Analysis of data from the fourth national morbidity survey of general practices (MSGP4) including sociodemographic details of individual patients and small area statistics from the 1991 census. Multilevel modelling techniques were used to take account of both individual patient data and small area statistics to relate socioeconomic and health status factors directly to a measure of general practitioner workload. RESULTS: Higher rates of consultations were found in patients who were classified as permanently sick, unemployed (especially those who became unemployed during the study year), living in rented accommodation, from the Indian subcontinent, living with a spouse or partner (women only), children living with two parents (girls only), and living in urban areas, especially those living relatively near the practice. When characteristics of individual patients are known and controlled for the role of "indices of deprivation" is considerably reduced. The effect of individual sociodemographic characteristics were shown to vary between different areas. CONCLUSIONS: Demographic and socioeconomic factors can act as powerful predictors of consultation patterns. Though it will always be necessary to retain some local planning discretion, the sets of coefficients estimated for individual level factors, area level characteristics, and for practice groupings may be sufficient to provide an indicative level of demand for general medical services. Although the problems in using socioeconomic data from individual patients would be substantial, these results are relevant to the development of a resource allocation formula for general practice. PMID- 8616347 TI - Commentary: the basis of a more rational method of funding primary medical care? PMID- 8616348 TI - Does the variation in the socioeconomic characteristics of an area affect mortality? PMID- 8616349 TI - Effect of inadvertent intradermal administration of high dose percutaneous BCG vaccine. PMID- 8616350 TI - Functional disability and antibody response to influenza vaccine in elderly patients in a Dutch nursing home. PMID- 8616351 TI - Measuring outcomes in primary care: a patient generated measure, MYMOP, compared with the SF-36 health survey. AB - OBJECTIVE: To assess the sensitivity to within person change over time of an outcome measure for practitioners in primary care that is applicable to a wide range of illness. DESIGN: Comparison of a new patient generated instrument, the measure yourself medical outcome profile (MYMOP), with the SF-36 health profile and a five point change score; all scales were completed during the consultation with' practitioners and repeated after four weeks. 103 patients were followed up for 16 weeks and their results charted; seven practitioners were interviewed. SETTING: Established practice of the four NHS general practitioners and four of the private complementary practitioners working in one medical centre. SUBJECTS: Systematic sample of 218 patients from general practice and all 47 patients of complementary practitioners; patients had had symptoms for more than seven days. OUTCOME MEASURES: Standardised response mean and index of responsiveness; view of practitioners. RESULTS: The index of responsiveness, relating to the minimal clinically important difference, was high for MYMOP: 1.4 for the first symptom, 1.33 for activity, and 0.85 for the profile compared with < 0.45 for SF-36. MYMOP's validity was supported by significant correlation between the change score and the change in the MYMOP score and the ability of this instrument to detect more improvement in acute than in chronic conditions. Practitioners found that MYMOP was practical and applicable to all patients with symptoms and that its use increased their awareness of patients' priorities. CONCLUSION: MYMOP shows promise as an outcome measure for primary care and for complementary treatment. It is more sensitive to change than the SF-36 and has the added bonus of improving patient-practitioner communication. PMID- 8616353 TI - All together now: why social deprivation matters to everyone. AB - Inequalities in health in the United Kingdom are widening as a result of economic policy. By focusing on specific diseases, health policy fails to address why less prosperous groups die earlier from most major categories of death. By concentrating on actions which can be taken by individuals and local communities health policy ignores actions which require the support and involvement of society as a whole. Clinicians see the consequences of health and economic policy in their everyday practice and could contribute more effectively to public debate. PMID- 8616354 TI - Grand Rounds--Hammersmith Hospital. A physiology classic revisited after 60 years. PMID- 8616352 TI - Recent advances. Medical genetics. PMID- 8616355 TI - ABC of urology. Urological malignancy--1: Prostate cancer. PMID- 8616356 TI - Adjuvant treatment with tamoxifen. Local research ethics committees should review their approval of prevention trial. PMID- 8616357 TI - Adjuvant treatment with tamoxifen. Side effects may have been overstated. PMID- 8616358 TI - Adjuvant treatment with tamoxifen. Recruitment into studies assessing optimum duration of treatment must continue. PMID- 8616359 TI - Adjuvant treatment with tamoxifen. Clinicians must share knowledge with their patients more readily. PMID- 8616360 TI - Adjuvant treatment with tamoxifen. Multicentre trial should be welcomed. PMID- 8616361 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Scientists who inflame public anxieties must share responsibility for resulting panic. PMID- 8616362 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Doctors and scientists must be able to communicate degree of risk... PMID- 8616363 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Advisory committee's conclusion was based on "absence of any credible alternative". PMID- 8616364 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Government policy has failed. PMID- 8616365 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy... but members of the public make up their own minds about risks. PMID- 8616366 TI - Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Risk to human population is remote. PMID- 8616367 TI - Plasma lactate and blood cyanide in acute cyanide poisoning. Confounding factors should have been considered. PMID- 8616368 TI - Vocational training schemes in south London have been improved. PMID- 8616369 TI - Florence Nightingale's fever. PMID- 8616370 TI - Men's health: don't blame the victims. PMID- 8616371 TI - Ethnic differences in outcome of serum screening for Down's syndrome. Differences in median values may account for differences in false positive rates. PMID- 8616372 TI - Ethnic differences in outcome of serum screening for Down's syndrome. For appreciable ethnic differences in false positive rates, use different cut off points. PMID- 8616373 TI - Ethnic differences in outcome of serum screening for Down's syndrome. Median values and maternal weight specific to the ethnic group should be used. PMID- 8616374 TI - When marginal costs and benefits should be used in screening. PMID- 8616375 TI - Junior doctors' hours. Alternative representative body is needed. PMID- 8616376 TI - Junior doctors' hours. Junior doctors need to have time for their lives outside medicine. PMID- 8616377 TI - Supervised discharge is anachronistic. PMID- 8616379 TI - Potential transmission of BSE via medicinal products. PMID- 8616378 TI - Income inequality and mortality: why are they related? PMID- 8616380 TI - Shortage of organs for transplantation. PMID- 8616381 TI - Landmines: time for an international ban. PMID- 8616383 TI - Belgian doctors' freedom to be guaranteed. PMID- 8616382 TI - The continuing rise in emergency admissions. PMID- 8616384 TI - French doctors may strike against reforms. PMID- 8616385 TI - China tightens up traditional Chinese medicine courses. PMID- 8616386 TI - Irish psychiatric workers criticised. PMID- 8616387 TI - Scottish court will rule on withdrawing treatment. PMID- 8616388 TI - Pill scare linked to rise in abortions. PMID- 8616389 TI - Poverty spreads among children in Britain. PMID- 8616390 TI - France tackles psychotropic drug problem. PMID- 8616391 TI - US could ban patents on medical procedures. PMID- 8616392 TI - Labour targets the chief medical officer. PMID- 8616393 TI - Inequality in income and mortality in the United States: analysis of mortality and potential pathways. AB - OBJECTIVE: To examine the relation between health outcomes and the equality with which income is distributed in the United States. DESIGN: The degree of income inequality, defined as the percentage of total household income received by the less well off 50% of households, and changes in income inequality were calculated for the 50 states in 1980 and 1990. These measures were then examined in relation to all cause mortality adjusted for age for each state, age specific deaths, changes in mortalities, and other health outcomes and potential pathways for 1980, 1990, and 1989-91. MAIN OUTCOME MEASURE: Age adjusted mortality from all causes. RESULTS: There was a significant correlation (r = -0.62 [corrected], P < 0.001) between the percentage of total household income received by the less well off 50% in each state and all cause mortality, unaffected by adjustment for state median incomes. Income inequality was also significantly associated with age specific mortalities and rates of low birth weight, homicide, violent crime, work disability, expenditures on medical care and police protection, smoking, and sedentary activity. Rates of unemployment, imprisonment, recipients of income assistance and food stamps, lack of medical insurance, and educational outcomes were also worse as income inequality increased. Income inequality was also associated with mortality trends, and there was a suggestion of an impact of inequality trends on mortality trends. CONCLUSION: Variations between states in the inequality of the distribution of income are significantly associated with variations between states in a large number of health outcomes and social indicators and with mortality trends. These differences parallel relative investments in human and social capital. Economic policies that influence income and wealth inequality may have an important impact on the health of countries. PMID- 8616394 TI - Inflammation in ischaemic heart disease. PMID- 8616395 TI - Managing peanut allergy. PMID- 8616396 TI - Smoking in public places. PMID- 8616397 TI - Chernobyl 10 years on. PMID- 8616398 TI - Drugs and the fetus. PMID- 8616400 TI - Britain reverses landmine policy. PMID- 8616399 TI - Describing race, ethnicity, and culture in medical research. PMID- 8616401 TI - Mercy killing of mother prompts inquiry. PMID- 8616402 TI - China steps up battle against AIDS. PMID- 8616403 TI - Medical researchers challenge antivivisectionists. PMID- 8616404 TI - Trial begins into victims of CJD growth hormone. PMID- 8616405 TI - US doctors warned about test for breast cancer gene. PMID- 8616406 TI - British GPs take charge of health promotion. PMID- 8616407 TI - New US theory on Gulf war syndrome. PMID- 8616408 TI - Brazilian health workers go on strike. PMID- 8616409 TI - Condom failure is on the increase. PMID- 8616410 TI - Frozen embryos to be stored for longer. PMID- 8616411 TI - German company halts delivery of HIV test kits. PMID- 8616412 TI - C reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. AB - OBJECTIVE: To test the hypothesis that minor chronic insults such as smoking, chronic bronchitis, and two persistent bacterial infections may be associated with increases in C reactive protein concentration within the normal range and that variations in the C reactive protein concentration in turn may be associated with levels of cardiovascular risk factors and chronic coronary heart disease. DESIGN: Population based cross sectional study. SETTING: General practices in Merton, Sutton, and Wandsworth. SUBJECTS: A random sample of 388 men aged 50-69 years from general practice registers. 612 men were invited to attend and 413 attended, of whom 25 non-white men were excluded. The first 303 of the remaining 388 men had full risk factor profiles determined. INTERVENTIONS: Measurements of serum C reactive protein concentrations by in house enzyme linked immunosorbent assay (ELISA); other determinations by standard methods. Coronary heart disease was sought by the Rose angina questionnaire and Minnesota coded electrocardiograms. MAIN OUTCOME MEASURES: Serum C reactive protein concentrations, cardiovascular risk factor levels, and the presence of coronary heart disease. RESULTS: Increasing age, smoking, symptoms of chronic bronchitis, Helicobacter pylori and Chlamydia pneumoniae infections, and body mass index were all associated with raised concentrations of C reactive protein. C Reactive protein concentration was associated with raised serum fibrinogen, sialic acid, total cholesterol, triglyceride, glucose, and apolipoprotein B values. C Reactive protein concentration was negatively associated with high density lipoprotein cholesterol concentration. There was a weaker positive relation with low density lipoprotein cholesterol concentration and no relation with apolipoprotein A I value. C Reactive protein concentration was also strongly associated with coronary heart disease. CONCLUSION: The body's response to inflammation may play an important part in influencing the progression of atherosclerosis. The association of C reactive protein concentration with coronary heart disease needs testing in prospective studies. PMID- 8616413 TI - Psychiatric problems in children with hemiplegia: cross sectional epidemiological survey. AB - OBJECTIVE: To examine the prevalence and predictors of psychiatric problems in children with hemiplegia. DESIGN: Cross sectional questionnaire survey of an epidemiological sample with individual assessments of a representative subgroup. The questionnaire survey was repeated on school age subjects four years later. SUBJECTS: 428 hemiplegic children age 2 1/2-16 years, of whom 149 (aged 6-10 years) were individually assessed. MAIN OUTCOME MEASURES: Psychiatric symptom scores and the occurrence of psychiatric disorder. RESULTS: Psychiatric disorders affected 61% (95% confidence interval 53% to 69%) of subjects as judged by individual assessments and 54% (49% to 59%) and 42% (37% to 47%) as judged from parent and teacher questionnaires, respectively. Few affected children had been in contact with child mental health services. The strongest consistent predictor of psychiatric problems was intelligence quotient (IQ), which was highly correlated with an index of neurological severity; age, sex, and laterality of lesion had little or no predictive power. CONCLUSION: Though most hemiplegic children have considerable emotional or behavioural difficulties, these psychological complications commonly go unrecognised or untreated. Comprehensive health provision for children with chronic neurodevelopmental disorders such as hemiplegia should be psychologically as well as physically oriented. PMID- 8616414 TI - Randomised study of n of 1 trials versus standard practice. AB - OBJECTIVE: To compare outcomes between groups of patients with irreversible chronic airflow limitation given theophylline by n of 1 trials or standard practice. DESIGN: Randomised controlled study of n of 1 trials versus standard practice. SETTING: Tertiary care center outpatient department. SUBJECTS: 31 patients with irreversible chronic airflow limitation who were unsure that theophylline was helpful after an open trial. INTERVENTIONS: n Of 1 trials (single patient randomised multiple crossover comparisons of theophylline against placebo) followed published guidelines. For standard practice patients theophylline was stopped and resumed if their dyspnoea worsened; if their dyspnoea then improved theophylline was continued. For both groups a decision to continue or stop the drug was made within three months of randomisation. MAIN OUTCOME MEASURES: Exercise capacity as measured by six minute walking distance, quality of life as measured by the chronic respiratory disease questionnaire at baseline and six months after randomisation, and proportions of patients taking theophylline at six months. RESULTS: 26 patients completed follow up. 47% fewer n of 1 trial patients than standard practice patients were taking theophylline at six months (5/14 versus 10/12; 95% confidence interval of difference 14% to 80%) without differences in exercise capacity or quality of life. CONCLUSIONS: n Of 1 trials led to less theophylline use without adverse effects on exercise capacity or quality of life in patients with irreversible chronic airflow limitation. These data directly support the presence of a clinically important bias towards unnecessary treatment during open prescription of theophylline for irreversible chronic airflow limitation. Confirmation in a larger study and similar studies for other problems appropriate for n of 1 trials are needed before widespread use of n of 1 trials can be advocated in routine clinical practice. PMID- 8616415 TI - Clinical study of peanut and nut allergy in 62 consecutive patients: new features and associations. AB - OBJECTIVE: To investigate clinical features of acute allergic reactions to peanuts and other nuts. DESIGN: Analysis of data from consecutive patients seen by one doctor over one year in an allergy clinic at a regional referral centre. SUBJECTS: 62 patients aged 11 months to 53 years seen between October 1993 and September 1994. MAIN OUTCOME MEASURES: Type and severity of allergic reactions, age at onset of symptoms, type of nut causing allergy, results of skin prick tests, and incidence of other allergic diseases and associated allergies. RESULTS: Peanuts were the commonest cause of allergy (47) followed by Brazil nut (18), almond (14), and hazelnut (13). Onset of allergic symptoms occurred by the age of 2 years in 33/60 and by the age of 7 in 55/60. Peanuts accounted for all allergies in children sensitised in the first year of life and for 82% (27/33) of allergies in children sensitised by the third year of life. Multiple allergies appeared progressively with age. The commonest symptom was facial angioedema, and the major feature accounting for life threatening reactions was laryngeal oedema. Hypotension was uncommon. Of 55 patients, 53 were atopic--that is, had positive skin results of tests to common inhaled allergens--and all 53 had other allergic disorders (asthma, rhinitis, eczema) due to several inhaled allergens and other foods. CONCLUSIONS: Sensitisation, mainly to peanuts, is occurring in very young children, and multiple peanut/nut allergies appear progressively. Peanut and nut allergy is becoming common and can cause life threatening reactions. The main danger is laryngeal oedema. Young atopic children should avoid peanuts and nuts to prevent the development of this allergy. PMID- 8616416 TI - Glycophorin A biodosimetry in Chernobyl cleanup workers from the Baltic countries. PMID- 8616417 TI - Transformations, means, and confidence intervals. PMID- 8616418 TI - Practicality of recording patient ethnicity in general practice: descriptive intervention study and attitude survey. AB - OBJECTIVE: To assess the feasibility of recording patient ethnicity in primary care using the Office of Population Censuses and Surveys classification. DESIGN: A descriptive intervention study and attitude survey in random samples of adults and primary care staff in randomly selected practices. SETTING: Eight practices in Lincolnshire and seven in Leicester. SUBJECTS AND METHODS: When patients were asked their ethnicity by general practitioners, nurses, or receptionists data were collected for 863 of a possible 880 patients. Of 750 patients sent a questionnaire about their attitudes towards the collection of such data 489 responded. Ninety five primary care staff completed a similar questionnaire. MAIN OUTCOME MEASURES: Time taken to record a patient's ethnicity; attitudes of patients and staff towards such recording, including who should ask, who can respond for others, and whether data can be shared with secondary care. RESULTS: Recording the data took less than a minute for three quarters of patients, but even this would need an average of a week of receptionist time per general practitioner. 72% of patients and 57% of staff agreed that ethnic data could be shared with secondary care, and 73% of patients and 60% of staff felt that the data should probably be collected in general practice. CONCLUSIONS: Ethnicity recording in general practice is feasible and acceptable. Nevertheless, the role of ethnic data in assessing health need in primary care, an adequate recording system, and evidence that recording offers benefits greater than the costs need to be established. PMID- 8616419 TI - An epidemic like any other? Rights and responsibilities in HIV prevention. AB - Throughout the 1980s and into the 1990s, HIV prevention has been closely associated with the protection of individual human rights. Traditional public health measures such as compulsory testing and isolation have largely been rejected as ineffective in public health terms and inappropriate in the context of human rights protection. HIV prevention has been based instead chiefly on elective measures --information, education, counselling, and voluntary testing. In the past five years there have been important clinical, epidemiological, and social developments in the AIDS epidemic. These changes, combined with a growing recognition of possible weaknesses inherent in a strictly voluntarist approach to HIV prevention, may herald a new approach to AIDS control which places more weight on social responsibility in the context of HIV prevention. PMID- 8616420 TI - Addison's disease. PMID- 8616421 TI - Grand rounds--Hammersmith Hospital. Reactive (AA) systemic amyloidosis. A cause of refractory nephrotic syndrome. PMID- 8616422 TI - ABC of Urology. Urological malignancy--II: Urothelial tumours. PMID- 8616423 TI - Ethnicity, race, and culture: guidelines for research, audit, and publication. PMID- 8616424 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Evening primrose oil and magnesium have been shown to be effective. PMID- 8616425 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Good general care may offer as much benefit as cognitive behaviour therapy. PMID- 8616426 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Patients were not representative of all patients with the syndrome. PMID- 8616427 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Cognitive behavior therapy should be compared with placebo treatments. PMID- 8616428 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Essential elements of the treatment must be identified. PMID- 8616429 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Use an interdisciplinary approach. PMID- 8616430 TI - Cognitive behaviour therapy for the chronic fatigue syndrome. Patients' beliefs about their illness were probably not a major factor. PMID- 8616431 TI - Tobacco funding for academics. A public relations disaster. PMID- 8616432 TI - Tobacco funding for academics. Buying respectability. PMID- 8616433 TI - Cheese and Salmonella infection. All milk products should be heat treated. PMID- 8616434 TI - Cheese and Salmonella infection. Cheese lovers should not be condemned to a pasteurised and tasteless product. PMID- 8616435 TI - Cheese and Salmonella infection. Distribution and retail of cheese, not just production, may lead to contamination. PMID- 8616436 TI - Cheese and Salmonella infection. Exceptional cheeses can be made only from raw milk. PMID- 8616437 TI - Prenatal and postnatal prevalence of Turner's syndrome. No scientific evidence for study's conclusions. PMID- 8616438 TI - Prenatal and postnatal prevalence of Turner's syndrome. No reason to doubt standard of prenatal diagnosis. PMID- 8616439 TI - Magnesium is underused in acute atrial fibrillation. PMID- 8616440 TI - Minocycline for acne. Doctors should not change the way they prescribe for acne. PMID- 8616441 TI - Minocycline for acne. Food reduces minocycline's bioavailability. PMID- 8616442 TI - Alternative's to hospital care. Emergency consultation clinic's avert unnecessary admissions. PMID- 8616443 TI - Alternatives to hospital care. Study's results may not apply elsewhere. PMID- 8616444 TI - Nursing shortages. Nationally determined pay could result in high marginal costs. PMID- 8616445 TI - Nursing shortages. Pay increases are most efficient response to labour shortages. PMID- 8616446 TI - Neonatal prevention of iron deficiency. Blood can be transfused from cord clamped at placental end. PMID- 8616447 TI - Finding meaning from loss. PMID- 8616448 TI - Neonatal prevention of iron deficiency. Placental transfusion might reduce prevalence of iron deficiency. PMID- 8616449 TI - Prospects for substance abuse control in South Africa. PMID- 8616450 TI - The 1994 annual lecture of the Society for the Study of Addiction. Commissions (Royal and other) on drug misuse: who needs them? AB - In the context of current calls in the United Kingdom for a Royal Commission on drugs, the political antecedents and consequences of nine major commissions of enquiry into drugs established by governments in the English-speaking world during the last 100 years are described, together with available information on their modus operandi and costs. The different interest groups calling for reform of UK drug laws are described and the broad basis on which a putative Royal Commission might proceed is suggested. Finally, analysis of previous commissions is used to identify factors likely to lead to a new Commission having a substantial influence on government policy on drugs. PMID- 8616451 TI - Ethics and journal publishing: taking the debate forward. The ordeals of editors. PMID- 8616452 TI - Ethics and journal publishing: taking the debate forward. Ethics of refereeing re examined. PMID- 8616453 TI - Ethics and journal publishing: taking the debate forward. The sensible way forward. PMID- 8616454 TI - Ethics and journal publishing: taking the debate forward. Wanted? Standard operating procedures for journals. PMID- 8616455 TI - Ethics and journal publishing: taking the debate forward. A first kick. PMID- 8616456 TI - Ethics and journal publishing: taking the debate forward. A committee for scientific dishonesty--the way forward? PMID- 8616457 TI - Ethics and journal publishing: taking the debate forward. Why it is helpful to know the funding source. PMID- 8616458 TI - Ethics and journal publishing: taking the debate forward. Ethics as everyone's business. PMID- 8616459 TI - Ethics and journal publishing: taking the debate forward. Ethics and the incentive structure. PMID- 8616460 TI - Ethics and journal publishing: taking the debate forward. Evolving standards for authorship. PMID- 8616461 TI - Anterior brain dysfunctioning as a risk factor in alcoholic behaviors. AB - This study assessed the relationship between neuropsychological and electrophysiological functioning and four alcohol-related measures: the Michigan Alcoholism Screening Test (MAST), the age at which the first drink was taken, frequency of drinking to "get high", and frequency of drinking to "get drunk". Ninety-one young adult men with no history of alcohol dependence were recruited. Subjects completed a variety of alcohol-related scales and a battery of neuropsychological tests. Resting EEG activity was also recorded. Stepwise regression analysis found that neuropsychological tests commonly regarded as measuring frontal and/or temporal neocortex functioning predicted the age at which subjects took their first drink and their scores on the MAST. Tests of frontal functioning, along with tests of memory, also predicted the frequency with which subjects reported drinking to "get drunk". Tests of memory also predicted the frequency at which subjects drank to "get high". On two of the alcohol measures, including age at which the first drink was taken and frequency of drinking to "get high", left-frontal slow alpha EEG activity was a significant predictor. These results suggest that markers of anterior brain functioning/dysfunctioning are associated with self-reports of alcohol-related behaviors, and that disturbances in the integrity of the anterior neocortex may be a risk factor in the development of alcohol-related behaviors. PMID- 8616462 TI - The histories of withdrawal convulsions and delirium tremens in 1648 alcohol dependent subjects. AB - A small proportion of alcohol-dependent men and women experience delirium tremens (DTs) and/or convulsions during alcohol withdrawal. While some characteristics of individuals most likely to show these severe sequelae of the abstinence syndrome have been described, it is not clear whether these risk factors operate independently in their association with severe withdrawal. The Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) interview was used to evaluate 1648 alcohol dependent men and women (including 540 women). The background characteristics and drinking histories of the 160 men and 51 women (12.8% of the subjects) who reported ever having had at least one episode of DTs and/or convulsions during withdrawal were compared with the characteristics of the remaining alcohol dependent individuals. Compared to other alcohol-dependent subjects, those with histories of severe withdrawal reported a greater maximum number of drinks in any 24-hour period (40.9 +/- 25.71 versus 24.9 +/- 17.72), more withdrawal episodes (28.2 +/- 33.74 versus 15.9 +/- 26.84), more non medicinal use of sedative-hypnotics (56.4% versus 32.9%) and a greater number of medical problems. Hierarchical logistic regression analysis revealed that the most powerful differences between those with histories of more and less severe withdrawals related to the maximum number of drinks per day and the total number of withdrawal episodes. The remaining variables still added significantly to the relationship to more severe withdrawal. The etiology of DTs and convulsions is complex and involves the interaction of diverse characteristics representing relatively unique domains. It is hoped that these data will help clinicians identify individuals most likely to have experienced severe withdrawal syndromes and will aid researchers attempting to understand more about the etiology of these problems. PMID- 8616463 TI - The AUDIT questionnaire: choosing a cut-off score. Alcohol Use Disorder Identification Test. AB - The Alcohol Use Disorders Identification Test (AUDIT) is a 10-item questionnaire designed by the World Health Organization to screen for hazardous alcohol intake in primary health care settings. In this longitudinal study we examined its performance in predicting alcohol-related harm over the full range of its scores using receiver operating characteristic analyses. Three hundred and thirty ambulatory care patients were interviewed using a detailed assessment schedule which included the AUDIT questions. After 2-3 years, subjects were reviewed and their experience of alcohol-related medical and social harm assessed by interview and perusal of medical records. AUDIT was a good predictor of both alcohol related social and medical problems. Cut-off points of 7-8 maximized discrimination in the prediction of trauma and hypertension. Higher cut-offs (12 and 22) provided better discrimination in the prediction of alcohol-related social problems and of liver disease or gastrointestinal bleeding, but high specificity was offset by reduced sensitivity. We conclude that the recommended cut-off score of eight is a reasonable approximation to the optimal for a variety of endpoints. PMID- 8616464 TI - Management and treatment efficacy of drug and alcohol problems: what do doctors believe? AB - We conducted a survey of the attitudes of postgraduate medical trainees in Australia on the management of drug and alcohol problems and examined the medical practitioner's role in managing drug and alcohol problems, factors influencing prognosis and beliefs about the efficacy of a number of treatment interventions. Of 2461 trainees enrolled in specialty training programmes in internal medicine, psychiatry and general practice 1361 (55%) participated. There was a high level of acceptance of responsibility for management of alcohol and drug problems, with the strongest support observed among psychiatry trainees. However, views of the efficacy of various treatment interventions were less positive. Alcoholics Anonymous was considered to be an approach well supported by the research literature. Dynamic psychotherapy was less well supported, and there was considerable uncertainty about the evidence for brief advice and cognitive behaviour therapies. The opinions expressed on treatment efficacy were in many cases in striking contrast to the research evidence. The implications for future training in drugs and alcohol in specialty programmes are discussed. PMID- 8616465 TI - Determinants of HIV infection and recent risk behaviour among injecting drug users in Berlin by site of recruitment. AB - This study investigated differences in prevalence and determinants of HIV infection, and in recent risk behaviour (previous 6 months) among injecting drug users (IDUs) who are in contact with different types of services for IDUs in Berlin. Participants (n = 557) were recruited from drug-free long-term treatment centres, a storefront agency and a syringe exchange bus. HIV seroprevalence was lowest (3.9%) at the treatment centres, and highest among IDUs at the storefront agency (20.7%). In logistic regression, independent risk factors for HIV infection were duration of injecting drug use, borrowing syringes in prison, sex with HIV-positive partners, and prostitution. Syringe sharing in prison was the most important independent determinant of HIV infection among all three subpopulations of IDUs. Participants entering long-term treatment were most likely, and IDUs at the syringe exchange bus were least likely to have borrowed and passed on syringes in the previous 6 months. In logistic regression, site of recruitment was independently associated with recent borrowing of syringes, but not with condom use. Injection of drugs other than heroin only, and injecting in prisons, were also independent predictors of recent borrowing. The results indicate that IDUs entering treatment form an important target group for health education. There is a need for AIDS prevention measures in prisons. The comparatively low levels of recent injection risk behaviour among IDUs at the syringe exchange bus suggest that this type of intervention may be effective in harm reduction. PMID- 8616466 TI - Psychiatric status and HIV risk reduction among residential drug abuse treatment clients. AB - We investigated the associations of psychiatric symptoms and diagnoses with HIV risk behaviors among 405 clients of two United States residential drug abuse treatment programs at admission and at follow-up. Measures of psychiatric status included the Beck Depression Inventory (BDI), selected diagnoses assessed with the Diagnostic Interview Schedule (DIS-III-R) and the Addiction Severity Index psychiatric composite score (ASI-P). Measures of risk behaviors included: drug injection risk (including sharing and bleaching of needles and syringes), multiple sexual partners and condom use. In multivariate analyses, the BDI at baseline and change in the BDI to follow-up were strongly associated with drug use at follow-up (both injection and non-injection), but not with other risk behaviors. In contrast, psychiatric diagnoses were not statistically associated with risk behaviors at follow-up when baseline behavior was controlled. PMID- 8616467 TI - The Health of the Nation target on syringe sharing: a role for routine surveillance in assessing progress and targeting interventions. AB - The Health of the Nation initiative in the United Kingdom includes a target aimed at reducing the proportion of current injecting drug users who share syringes. The PHLS Collaborative Survey of Salivary Antibodies to HIV and Hepatitis B core in injecting drug users is a comprehensive and national surveillance mechanism which routinely collects data that can be used to monitor progress toward this target. Nineteen per cent of injecting drug users (353/1876) in 1992 and 18% (375/2138) in 1993 shared previously used injecting equipment (difference of 1.3%, 95% Cl -3.7%, 1.1%). Only with further years of data collection will it be possible to tell if this decline represents a real change in behaviour. There was a substantial reduction in the proportion of sharers who received previously used needles and syringes from more than one person, from 45% (138/305) in 1992 to 27% (81/298) in 1993 (fall of 18%, 95% Cl 11%, 26%). This decline could indicate a real reduction in risk behaviour that is not reflected in the target. Monitoring this aspect of sharing could be an important supplementary measure. Women were more likely to have share (adjusted OR = 1.87, 95% Cl 1.53, 2.28) and the likelihood of sharing declined with age (adjusted OR of each 5-year age band = 0.75, 95% Cl 0.72, 0.79). Particular attention should be given to interventions which aim to reduce sharing among women and young people. Clients of agencies at which the main service provided was syringe exchange were less likely to have shared than attenders of other types of agencies (adjusted OR = 0.69, 95% Cl 0.51, 0.93). This suggests that syringe exchange schemes play a role in reducing the transmission of HIV infection. PMID- 8616468 TI - Crack use and injection on the increase among injecting drug users in London. AB - Fears of a crack 'epidemic' in the United Kingdom of the kind experienced in the United States remain unfounded. However, findings from an ongoing serial point HIV prevalence study indicate that the use and injection of crack cocaine among injecting drug users (IDUs) in London is on the increase. Data on patterns of drug use and drug injection were collected over 4 years (1990-93), from IDUs recruited from both drug treatment and community-based settings. All respondents were asked about their drug use in the 6-month period prior to interview. The use of crack cocaine increased significantly from 16% in 1990 to 59% in 1993 and the injection of crack cocaine from 1% to 27%, respectively. The findings suggest that crack cocaine is being injected more regularly, with increases over the 4 year period in those who reported injecting the drug at least once per week. In response this paper considers both the health implications of, and the treatment response to, the increasing use and injection of crack cocaine among IDUs in London. PMID- 8616469 TI - Can outreach work? PMID- 8616470 TI - Life Education's failure to face the facts. PMID- 8616471 TI - Cessation of long-term nicotine gum use. PMID- 8616472 TI - [Langerhans cells in lung pathology]. PMID- 8616473 TI - [Logistic model in diagnosis of lung cancer]. AB - Specificity of the diagnosis of lung cancer (l.c.) based upon clinical and radiological criteria was investigated in patients who died in Hospital of Lung Diseases in Lodz between in 1985 and 1989. Autopsy revealed diagnostic errors in 37% cases. Twenty most common disease features were taken into consideration. Logistic analysis helped to create clinical-mathematical model which allows for interpretation of disease features in two distinct categories: l.c. (non neoplasmatic lung disease). Eight features proved to be significant (changes in RBC, weight loss, chest pain, increased sputum production, haemoptisis, hilar tumor, pleural effusion, peripheral round shadow on chest X-ray). Diagnostic usefulness of the model was checked in prospective study a group of patients who died from properly diagnosed l.c. (499 cases). Our model allowed for proper diagnosis of neoplasmatic cause of the disease with higher probability than morphological methods (including both intravital and autopsy examination). PMID- 8616474 TI - [Comparison of the degree of regression of small cell lung neoplasm in clinical radiologic evaluation with levels of neuron specific enolase (NSE) in serum]. AB - NSE was estimated using Pharmacia test and radioimmunologic method in the serum of 41 SCLC patients before treatment, at the time of maximal tumor response and in some patients also during progression. The level of 12,5 ng/l was regarded as the upper limit of normal. Elevated NSE levels before treatment were found in 12 out of 20 patients with limited disease and in 19 out of 21 patients with extensive disease. Complete remission (CR) was observed only in patients with limited disease and in those in whom NSE serum level was below 50 ng/l. Elevated NSE serum levels decreased in all those in whom partial remission (PR) or CR of tumor was obtained. Decrease of elevated NSE levels was however also observed in some patients with no significant regression of tumor. The decrease of serum NSE level seemed to be a good prognostic factor even in this last group. NSE serum level increased in 7 out 10 patients where progressive disease after PR was found. In 3 patients however NSE level persisted in normal values despite progression. The significance of this fact was discussed. PMID- 8616475 TI - [Evaluation of the value of determining levels of cytokeratin-19 fragments for diagnosis of lung cancer]. AB - Cytokeratins, the intermediate filaments, are expressed by many epithelial cells. Immunohistochemistry revealed the presence of cytokeratin-19 both in bronchial epithelium and in lung cancer cells. The aim of our study was to establish the value of serum cytokeratin-19 estimation by immunoenzymatic assay (Enzymun Cyfra 21-1, Boehringer Mannheim) in the patients (pts) with lung cancer (Ic). 153 pts (104 men, 49 women, median age 50 years) entered this study. The group consisted of 37 pts with benign lung diseases (control group), 56 pts with squamous cell Ic, 37 pts with small cell Ic and 23 with adenocarcinoma. Cut off value was determined at 4 ng/ml, with 96% of specificity. Elevated cytokeratin-19 values were found in 41% of pts with lung cancer (45% of squamous cell Ic, 39% of adenocarcinoma and 35% of small cell Ic). Median cytokeratin-19 values were 2.2 ng/ml in the control group, 3.4 ng/ml in squamous cell Ic, 3.3 ng/ml adenocarcinoma and 2.9 in small cell Ic. Cytokeratin-19 elevation was observed more often in non small cell Ic pts with advanced disease, stage III--46%, stage IV--50% than in early stages (I + II)--34%. In small cell Ic pts the frequency of cytokeratin-19 elevation was 20% in limited disease versus 45% in extensive disease. We conclude that cytokeratin estimation is not valuable in the recognition of histologic type of lung cancer, although elevated levels are seen more often in squamous cell Ic. Cytokeratin-19 estimation may be also helpful in lung cancer staging. PMID- 8616477 TI - [Incidence of beta HCG elevation in serum of patients with small cell lung neoplasms and their prognosis]. AB - HCG-like immunoreactivity has been found in many nontrophoblastic tumors, but the biological behaviour of HCG--producing cells has not been clarified yet. The aim of the study was to estimate the frequency of serum bHCG (sbHCG) elevation in SCLC patients and to assess its possible prognostic role in this type of tumor. 156 SCLC patients entered the study: 93 men, 63 women, median age 58 years. SbHCG activity was measured with immunoenzymassay (Abbott EIA bHCG 15-15) before treatment. ScHCG elevation (5 mIU/ml) was found in 21 of 156 patients (14%). Response to treatment after chemotherapy (CR + PR) was obtained in only 48% of those patients in whom elevated sbHCG was found, in comparison to 73% of response rate observed in the remaining patients. Only 5% of patients with elevated sbHCG survived 2 years, in comparison to 21% of 2-years survivors in the remaining patients. Thus sbHCG elevation in SCLC seems to be a marker of more resistant tumors and of poor prognosis. PMID- 8616476 TI - [Level of cytokeratin-19 in serum of patients with non small cell lung cancer]. AB - The serum levels of cytokeratin-19 were measured in 116 patients--96 with NSCLC and 20 with non-malignant lung diseases. The reference group consisted of 60 healthy volunteers--30 smokers and 30 non-smokers. Significantly elevated Cyfra 21-1 values were observed in NSCLC. There was not a correlation between of cytokeratin-19 serum levels and histologic types of lung carcinoma. Cyfra 21-1 concentrations generally increased with stage of diseases and the highest were in patients with evidence of distant metastases. In NSCLC, the distribution of Cyfra 21-1 varied significantly according to the performance status of NSCLC patients. PMID- 8616478 TI - [Concentration of sialic acid in bronchoalveolar lavage fluid in selected interstitial pulmonary diseases]. AB - Sialic acid is a component of cell membranes and it can take part in immunological processes with lymphocytes and neutrophiles. In the present work the concentration of sialic acid in BALF in selected interstitial pulmonary disease was studied (in sarcoidosis, pulmonary fibrosis, avian fanciers lung) and compared with the control group of healthy persons. The investigations were repeated during the observations. The patients were divided into active and inactive groups. The analysis included the results in 187 patients divided into 9 groups. Sialic acid was measured with colorimetric method with the use of Ehrlich's reagent. The growth of sialic acid concentration in supernatant of BALF was observed in the studied diseases. The increase of sialic acid occurs in inflammatory processes especially those with neutrophiles. The correlation of acid concentration with the percentage or the total number of lymphocytes in BALF was observed. It is specific that the concentration of sialic acid in BALF in patients with avian fanciers lung keeps growing even after the contact with the antigen was discontinued. PMID- 8616479 TI - [Effects of smoking tobacco on exercise tolerance in healthy subjects]. AB - The aim of the study was to investigate the effects of smoking on exercise tolerance in middle aged smokers. Sixty two healthy subjects 55 males and 7 females with mean age 35 +/- 7 years were studied. There were 47 smokers (smoking 21.5 cigarettes per day for a mean of 16 years) and 15 non-smokers (control group). Resting pulmonary function was normal in both groups, however smokers had significantly lower VC as well as MEF 50 and MEF 75. A maximal incremental exercise on cycloergometer using ramp protocol was performed. Ventilatory parameters (breath by breath method) together with transcutaneous oxygen saturation (every minute), heart rate (continuously) and blood pressure (every minute) were recorded. Significant differences in exercise tolerance in studied groups were observed. Smokers tolerated a lower maximal workload for a shorter time and deferred significantly in both maximal oxygen consumption and oxygen consumption at anaerobic threshold. Five persons, all smokers did not reach the anaerobic threshold. In five smokers a decrease in ECG ST segment was observed. No differences were found in breathing reserve, heart reserve or maximal oxygen pulse. It seems that a decreases exercise performance is due to cardiac limitation. PMID- 8616480 TI - [Sloping posture on the unwell side for treatment of pulmonary tuberculosis combined with use of modern intensive antitubercular chemotherapy]. AB - The action of pulmonary tuberculosis treatment with setting the patient in a sloping position on the unwell side has been discussed briefly. The results of this already historical event of the years 1957-1958 have also been presented. It was then that in spite of the imperfection of the antituberculous chemotherapy SM+INH+PAS of that time, the application of the setting in a sloping position made it possible to achieve a high percentage of very good and fast results of pulmonary tuberculosis treatment. The results were three times as good as others till then, considerably sooner than the results of chemotherapy exclusively. The precious experience in setting in a sloping position treatment is worth testing nowadays, merged with the application of intensive chemotherapy RMP+INH+PZA+EMB or SM in cases of neglected or late diagnosis of tuberculosis, or case of tuberculosis refractory to a number medicine refractory mycobacteriosis, but at that time merged with other antituberculous medicines with tested in vitro sensitivity. AIDS patients with tuberculosis infection can also have the setting in a sloping position applied, and since the moment antituberculous medicines are applied, if drug-sensitiveness has been found in inoculations, as drug-fastness frequently occurs in case of patients with AIDS. PMID- 8616481 TI - [New computerized system for clinical evaluation of respiratory regulation]. AB - The foundations for a computer program allowing to monitor "on line" physiological data to clinically evaluate ventilatory control were set up. The new equipment demonstrates important innovations--lowered flow resistance, and decreased dead space, modernization of the blocking steering the computer valve. The new program allowed to decrease the time for analysis from couple of minutes to seconds. The results of ventilatory control studies in 11 healthy volunteers were found in ranges of normal values calculated earlier using an analogue method and are similar to those from literature. PMID- 8616482 TI - [Bronchofibroscopy in removal of foreign bodies from the bronchi in children]. AB - In most cases of bronchial foreign bodies removal in children rigid bronchoscopy is used. In this study three cases of bronchial foreign body removal by bronchofiberoscopy were presented. In some cases that method could be necessary for diagnosing and removal of the foreign body. PMID- 8616483 TI - [A case of lung eosinophilic granuloma (histiocytosis X) without clinical symptoms]. AB - The asymptomatic case of eosinophilic granuloma (Histiocytosis X). 24 years old men with disseminated lung lesions which were present for six years was presented. Histiocytosis X was diagnosed on the basis of the examination of open lung biopsy specimen and high resolution computer tomography. The patient will be observed without treatment as no symptom were present and some regression of lung lesions was noted. PMID- 8616485 TI - [A case of primary chronic lung hemangiopericytoma]. PMID- 8616484 TI - [Treatment of late complications of tuberculous bronchitis with Nd:YAG laser]. PMID- 8616486 TI - [Mediastinal seminoma--diagnostic difficulties]. AB - A case report of a seminoma of extragonadal localization--of the mediastinum--is presented. In specimens obtained during mediastinoscopy sarcoid lesions were detected. Parasternal mediastinotomy allowed to sample larger fragment of the tumor and the final diagnosis was made. PMID- 8616487 TI - [A case of nephropathy with renal insufficiency during the course of pulmonary sarcoidosis]. PMID- 8616488 TI - [Forced oscillation technique in respiratory system studies. I. Theoretical basis, interpretation of results. Equipment]. AB - The paper presents short description of the forced oscillation technique--a noninvasive technique used to examine respiratory mechanics. The setup, interpretation of the results and also advantages and disadvantages of that technique in measurement and interpretation of human respiratory input impedance are described. PMID- 8616489 TI - [Forced oscillation technique in respiratory system studies. II. Clinical interpretation of results in respiratory system diseases]. AB - The paper presents short description of changes in FOT parameters in different respiratory system disease. Clinical interpretation of results and usefulness of FOT are described. PMID- 8616491 TI - Policy. Mass. squelches drive-through deliveries. PMID- 8616490 TI - [Forced oscillation technique in respiratory system studies. III. Compliance and airway resistance in children with lung fibrosis]. AB - In patient with lung fibrosis we need to monitor lung compliance to evaluate the progress of the illness or efficacy of the treatment. This examination is not well tolerated and difficult to perform, especially in children. We compared classic measurements of static compliance of the lung and airway resistance with parameters derived from forced oscillations measurements--compliance and resistance of respiratory system. Correlation coefficients were respectively 0.85 for compliance and 0.70 for resistance. We conclude that it is possible to supplement classic measurements, especially in evaluating the dynamics of illness. PMID- 8616492 TI - Employers. Cut costs now, quality later. PMID- 8616493 TI - Consumers. Phoning in for medical advice. PMID- 8616494 TI - Executive advancement ... College of Managed Care. PMID- 8616495 TI - Physicians ... don't ignore intangibles--especially leadership. PMID- 8616496 TI - Listen to what cutting edge executives have to say about how to succeed with doctors by really trying. AB - As more and more hospitals move to form partnerships with physicians, the executives working to create these new relationships are learning that to really be equal partners takes more than simply installing a senior doctor into a senior executive slot. It takes vision, guts, patience, and commitment. Getting these new ventures to work to everyone's benefit isn't easy, but ultimately it's worth it. Find out what the field's cutting-edge executives have to say about their best strategies to ensure a peaceful--and profitable--partnership. PMID- 8616498 TI - Ben Latimer. Interview by Mark Hagland. PMID- 8616497 TI - There's no place like home. AB - When the school system in tiny Colby, Kans., signed onto a health plan that excluded the only hospital in the entire county, its citizens learned an important lesson. ?If we're not working together,? says the hospital's administrator, ?health plans and medical centers are going to come in here and take business away from us.? Here's what they learned about keeping rural health care rural. PMID- 8616499 TI - Patient-focused. A kinder, gentler ER. PMID- 8616500 TI - Hospital pulse. September 1995. PMID- 8616501 TI - Quality patrol. Having it all--almost. PMID- 8616502 TI - Technology. Finding the right clinical trial--fast. PMID- 8616503 TI - The value of nursing. PMID- 8616504 TI - Some behavioral effects of CNQX AND NBQX, AMPA receptor antagonists. AB - CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and NBQX (2,3-dihydroxy-6-nitro-7 sulfamoyl-benzo[f]quinoxaline), two competitive AMPA (non-NMDA glutamate) receptor antagonists, as well as their interaction with CGP 37849, a competitive NMDA receptor antagonist, were studied in rats and mice. CNQX and NBQX inhibited the locomotor activity of naive rats. No symptoms of behavioral excitation were observed. CGP 37849 induced locomotor hyperactivity which was reduced by CNQX and NBQX. In monoamine-depleted rats (pretreated with reserpine + alpha-methyl-p tyrosine), none of the two quinoxalines nor CGP 37849 antagonized akinesia. The antiakinetic effect of L-DOPA was increased by CGP 37849, but not by CNQX or NBQX. The latter action of CGP 37849 was decreased by CNQX and NBQX. The antiakinetic effect of clonidine was not changed by CNQX. The locomotor hyperactivity induced by apomorphine or cocaine was not modified by CNQX. Neither of the quinoxalines changed the catalepsy induced by haloperidol or spiperone. The fluphenazine catalepsy was slightly decreased by CNQX and increased by NBQX. CNQX and NBQX were inactive in the forced swimming test; CNQX (but not NBQX) increased the CGP 37849-induced reduction of the immobility time. CNQX decreased the muscle tone of hind limbs in naive and monoamine-depleted rats. The obtained results indicate that the AMPA receptor antagonists differ in their neuropharmacological profile from CGP 37849, an NMDA receptor antagonist. There is no positive cooperation (except for the forced swimming test) between NMDA and AMPA receptor antagonists; on the contrary, an antagonistic between them has been observed. PMID- 8616505 TI - Effect of nifedipine on lithium-pilocarpine-induced seizures in the rats. AB - Recent studies have shown that lithium pretreatment of rats potentiates the convulsant effect of pilocarpine. A large body of evidence support the hypothesis that calcium channel antagonists possess antiepileptic properties in various models of epilepsy. This study was designed to investigate effect of calcium channel antagonist, nifedipine on lithium-pilocarpine-induced convulsions in rats. Pretreatment of rats with nifedipine (5 or 10 mg/kg) as well as with a calcium channel agonist BAY k-8644 (2 mg/kg) increased convulsant effect induced by lithium and pilocarpine. The facilitating effect of nifedipine on lithium pilocarpine-induced convulsions was prevented by pretreatment with a cholinergic antagonist atropine. It can be concluded that nifedipine facilitates convulsions in lithium-pilocarpine model of epilepsy and that cholinergic system may be involved in this effect. PMID- 8616506 TI - Estimation of central activity of hydroxyproline--the amino acid characteristic for collagen degradation products. AB - The central activity of hydroxyproline - the amino acid characteristic for collagen degradation products was studied. The levels of urinary and serum hydroxyproline are elevated in many disturbances, among them is a mental deficiency connected with hydroxyprolinemia. Previous studies showed that the mixture of collagen degradation products is biologically active and exerts and effect on central nervous system. In our study hydroxyproline significantly decreased the psychomotor activity of rats in Lat's test, enhanced amphetamine stereotypy in lower doses, and enhanced catalepsy in one of used doses. It prolonged the time of thiopental sleeping. The pentylenetetrazole seizures latency was not significantly prolonged and duration of seizures was shorter after injection (icv) of hydroxyproline. Our investigations show, that hydroxyproline is the active amino acid in the mixture of collagen degradation products and may be responsible for central activity of this mixture. PMID- 8616507 TI - Effects of 5-HT3 receptor antagonists on ethanol-induced hyperlocomotion in mice. AB - We tested how 5-HT3 receptor antagonists, tropisetron (TR) and ondansetron (ON) affect the hyperlocomotion induced by single dose of ethanol EtOH) in mice. EtOH in a dose-dependent manner (0.5-2.0 g/kg ip) increased locomotion of mice. The effect of 2.0 g/kg of EtOH was reduced by ON (0.1 mg/kg and 1.0 mg/kg sc) and, to the lesser extent and only in limited dose range (0.01 mg/kg) by TR. Tropisetron (but not ON) increased concentrations of EtOH in the blood. The data suggest that 5-HT3 receptor is involved in EtOH-induced hyperlocomotion. PMID- 8616509 TI - Some pharmacological properties and cumulative, subchronic and chronic toxicity of 9-methyl-2-[3-(4-phenyl-1-piperazinylpropyl)] -1,2,3,4-tetrahydro-beta carbolin-1-one (B-193). AB - The cumulative, subchronic and chronic toxicity of B-193 were studied on the rats and mice. It was found that this compound exerted weak tendency to cumulation in the body. Only the highest doses of B-193 (70, 40 mg/kg po for 12 weeks) caused the increase of animals mortality. Studies on subchronic and chronic toxicity have demonstrated, that B-193 administrated po or ip for 3 weeks, and po for 12 weeks, in general, neither affects the body weight gain nor the mass and morphology of heart, liver and kidneys, as well as spontaneous locomotor activity of animals. The weak depressant effect of B-193 on peripheral blood morphology was seen only after 3 weeks po or ip treatment with this compound. The moderate effect of B-193 on activity of alanine and aspartate transaminases (A1At and AspAt) and serum protein level found after 3 weeks of treatment, was no longer observed after 12 weeks of treatment. This could indicate that above effects of B 193 are reversible. PMID- 8616508 TI - Role of the serotoninergic system in the regulation of glucocorticoid and mineralocorticoid receptors in the rat hippocampus. AB - Effects of 5-HT receptor agonists (8-OH-DPAT, DOI and mCPP) on the binding parameters of corticosteroid receptors in the hippocampus of adult rats were studied. Glucocorticoid (GR) and mineralocorticoid (MR) receptors were examined by an in vitro [3H]corticosterone binding in cytosol, using the selective GR agonist RU 28362 to discriminate between MR and GR. Treatment with 8-OH-DPAT and mCPP given for 7 but not 1 days increased the density of MR. None of the compounds under investigation influenced the density of GR or the affinity of MR and GR in the rat hippocampus. Our results suggest that, in contrast to the postnatal period, the 5-HT1A and/or 5-HT2C, but not 5-HT2A, receptor is mainly involved in the regulation of MR in adult rats. PMID- 8616510 TI - Cold water stress induced analgesia in unilateral inflammation of the hindpaw in hypertensive and normotensive rats. AB - Analgesia induced by cold water stress (CWS) was tested in the model of monolateral inflammation in spontaneously hypertensive (SHR), renal hypertensive (RHR) and normotensive Wistar (NR) and Wistar Kyoto (WKY) rats. Unilateral hind paw inflammation was induced by Freund's complete adjuvant (FCA). Four days after inoculation all tested rats exhibited profound analgesia following CWS in both inflamed and non-inflamed paws which reached a maximum immediately after CWS and returned to control values within 15 min. The activity of naloxone was tested both by systemic and local injection. Baseline pain thresholds of SHR rats were significantly higher than those of NR, WKY and RHR. Hyperalgesia following systemic intraperitoneal administration of naloxone applied ten minutes before CWS was higher in RHR and WKY than in NR and SHR. When administered directly into the inflamed paw, naloxone did not antagonize CWS-induced analgesia in RHR, in contrast to weak antagonism in NR, while more evident in SHR and WKY. Our results probably reflect biological and genetic variability of intrinsic opioid and inflammatory mechanisms in hypertension. PMID- 8616511 TI - Effect of repeated amitriptyline administration to mice on the T lymphocyte proliferative activity and natural killer cell cytotoxicity. AB - The study examined the effect of repeated amitriptyline administration to mice on the proliferative activity of lymphocytes in response to mitogen stimulation and on the ability of natural killer (NK) cells to evoke lysis of YAC-1 tumor cells in vitro. A relatively short treatment (5 days) produced an increase in the NK activity, but no change in the T cell proliferative response to mitogens. After a 2-week treatment, a transient but significant decrease in the proliferative activity of splenocytes and in the NK activity was observed. Prolonged administration of that drug for more than 3 weeks produced a return to the control level of the NK activity and the T lymphocyte responsiveness to mitogens. The obtained results show that amitriptyline modifies the immune function, and that the observed effect depends crucially on the length of drug administration. PMID- 8616512 TI - Bidirectional modulation of mouse natural killer cell and macrophage cytotoxic activities by enkephalins. AB - Stimulation of a cellular immune function as measured by splenic natural killer (NK) cell activity has been described following systemic treatment with enkephalins. In this study, we evaluated the effects of the central administration of enkephalins on the splenic NK cell activity and macrophage cytotoxicity. Intracerebroventricular injection of leucine-enkephalin (LE) or methionine-enkephalin (ME) induced bidirectional modulation of NK cell and macrophage cytotoxic activities (suppression followed by enhancement). Administration of ME or LE at doses of 100 or 10 micrograms/mouse caused inhibition of NK cell and macrophage activity 2 h after injection. Enhancement of NK cells activity was observed at 96 h and macrophages cytotoxicity at 24 h after icv injection with both enkephalins. Pretreatment with naloxone (20 micrograms/mouse) inhibited the enkephalins-induced augmentation of NK cell and macrophage cytotoxic activity. These findings demonstrate that central actions of enkephalins produce changes in cytotoxicity of NK cells and macrophages. PMID- 8616513 TI - Effects of neuropeptide Y on evoked potentials in the CA1 region and the dentate gyrus of the rat hippocampal slice. AB - The effects of exogenously applied neuropeptide Y (NPY) on evoked field potentials in the CA1 area and dentate gyrus were examined in the in vitro hippocampal slice preparation from rate. The amplitudes of extra-cellularly recorded field potentials from the CA1 region were depressed by NPY (0.1-0.5 microM) in a dose-dependent manner. NPY had only little inhibitory effect in the area dentata. Similar effects were observed when synaptic excitation was recorded in the presence of the GABA(A) receptor antagonist picrotoxin. In the presence of picrotoxin, NPY reduced epileptiform discharges evoked in the disinhibited CA1 pyramidal cells suggesting that NPY may have antiepileptic action in this area. The effect of NPY on epileptiform discharges evoked in granule cells was minor. When NMDA-receptor mediated component of excitatory synaptic transmission was revealed in Mg2+ -free solution, NPY diminished both non-NMDA and NMDA receptor mediated components of the field excitatory synaptic potentials recorded in the CA1 region. Such an effect is line with the reduction of glutamate release at the Schaffer collateral/commissural terminals by NPY. PMID- 8616514 TI - Guanidine derivative U-37883A, inhibits mitochondrial K+ uniport. AB - The activity of potassium uniport in rat liver mitochondria was measured using light scattering technique (mitochondrial swelling). It was shown, that guanidine derivative, U-37883A (4-morpholinecarboxamidine-N-1-adamantyl-N'-cyclohexylhyd rochloride), was able to inhibit A23187 induced potassium uniport activity. The inhibitory effect of U-37883A on mitochondrial swelling gave an IC50 an of 89 +/- microM. No inhibition was observed with U-37883A analog, U-42069D (4 morpholinecarboxamidine), which points to the specificity of U-37883A action. It was also shown, that U-37883A acted on a different binding site in rat liver mitochondria than glibenclamide, inhibitor of the ATP sensitive potassium channel. PMID- 8616515 TI - Possible involvement of D4-like dopamine receptor in regulation of serotonin N acetyltransferase activity in duck retina. AB - The dopamine (DA) receptor regulating serotonin N-acetyltransferase (NAT) activity in duck retina was characterized pharmacologically. Systemic administration to ducks of quinpirole (a D3/D4-DA receptor agonist) and &-OH-DPAT (a D3-DA receptor agonist) suppressed the nighttime NAT activity of the retina in a dose-dependent manner, with quinpirole being two orders of magnitude more potent than 7-OH-DPAT. Bromocriptine (a D2/D3-DA receptor agonist) and SKF 38393 (an agonist of the D1 family DA receptors) were ineffective. The suppressive effect of quinpirole on NAT activity was blocked by spiroperidol and clozapine (antagonists of the D2 family of DA receptors), and not affected by SCH 23390 (an antagonist of the D1 family). The results extend our data on chick retina (which demonstrate an important role of D4-like DA receptor in the control of melatonin biosynthesis) and suggest that DA receptor regulating NAT activity in the duck retina belongs to the D2 family and may represent a D4-like subtype. PMID- 8616516 TI - Specific involvement of striatal D1 and D2 dopamine receptors in the neuroleptic catalepsy in rats. AB - Since highly specific antagonists of D1 (SCH 39166) and D2 (raclopride) dopamine receptors have recently become available, we decided to investigate the role of striatal populations of these receptors in catalepsy - an animal model of neuroleptic-induced parkinsonism in humans. Injections of raclopride (2.5, 5 and 10 micrograms/0.5 microliters) into the ventro-rostral part of the striatum induced a strong, dose-dependent and long-lasting catalepsy. Intrastriatal injections of SCH 39166 (1.5 and 3.6 micrograms/ 0.5 microliters) also evoked a dose-dependent, but short-lasting catalepsy. The present results suggest, that neuroleptic side-effects are specifically dependent on the blockade of D2 and D1 dopamine receptors in the striatum. PMID- 8616517 TI - Immediate and "day-after" effects of morphine on dopamine and serotonin metabolism in various structures of the rat brain. AB - The effects of a single dose of 20 mg/kg ip of morphine on dopaminergic and serotonergic systems in the limbic (cortex, nucleus accumbens) and extrapyramidal (striatum) structures were investigated in rats. The action of morphine was assessed by measuring the regional concentrations of dopamine, serotonin and their metabolites (homovanillic acid- HVA,3,4-dihydroxyphenylacetic acid - DOPAC, 5-hydroxyindoleacetic acid - 5-HIAA) by means of HPLC. We have demonstrated that a single large dose of morphine produces a biphasic change in limbic dopaminergic and serotonergic structures, with an initial activation followed by significant inhibition 24 h later. In contrast, the striatum depression of dopamine level was observed during the initial phase with normalization on the next day. PMID- 8616518 TI - Antidepressant treatment influences cyclic AMP accumulation induced by excitatory amino acids in rat brain. AB - The prolonged imipramine or electroconvulsive shock treatment induced a decrease in ibotenate stimulated increase in cyclic AMP accumulation as well as it inhibited the synergistic interaction between ibotenate and noradrenaline; the glutamate-mediated inhibition of forskolin-stimulated cyclic AMP accumulation was not modified. Our results indicate that certain subtypes of metabotropic glutamate receptor are differently modified by prolonged antidepressant treatment. PMID- 8616519 TI - German Endocrine Society. PMID- 8616520 TI - Primary hyperparathyroidism: genetic heterogeneity suggesting different pathogenesis in sporadic and familial forms of parathyroid hyperplasia and tumors. PMID- 8616521 TI - 11 beta-Hydroxysteroid dehydrogenase: new answers, new questions. PMID- 8616522 TI - Thyrotropin-dependent transforming growth factor beta expression in thyroid gland. PMID- 8616523 TI - New insights into pancreatic beta-cell metabolic signaling in insulin secretion. AB - In recent years, it has become apparent that second messengers and factors other than ATP. metabolically sensitive K+ATP channels and Ca2+ play essential roles in nutrient-induced insulin release. This paper reviews the evidence in support of several new concepts and hypotheses in the field of beta-cell signaling. These include in particular that: a rise in cytosolic Ca2+ is not sufficient to explain the kinetics and extent of secretion induced by glucose; variations in ADP, rather than ATP, regulate beta-cell metabolism and the K+ATP channel; anaplerosis (the replenishment of the citric acid cycle with intermediates) is essential for beta-cell activation: a shift from fatty acid oxidation to esterification is an important event in beta-cell signaling: malonyl-CoA and long chain acyl-CoA esters may act as metabolic coupling factors; glycolytic oscillations underlie, in part, oscillations in electrical activity, cytosolic Ca2+ and insulin release. A metabolic model of fuel sensing that integrates the mode of action of all classes of nutrient secretagogues is proposed. PMID- 8616524 TI - Gender differences in secretory activity of the human somatotropic (growth hormone) axis. PMID- 8616525 TI - Gonads in trouble: follicle-stimulating hormone receptor gene mutation as a cause of inherited streak ovaries. PMID- 8616526 TI - Human kidney 11 beta-hydroxysteroid dehydrogenase: regulation by adrenocorticotropin? AB - In ectopic adrenocorticotropin (ACTH) syndrome (EAS) with higher ACTH levels than in pituitary Cushing's syndrome and during ACTH infusion, the ratio of cortisol to cortisone in plasma and urine is increased, suggesting inhibition of renal 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) by ACTH or by ACTH-dependent steroids. Measuring the conversion of cortisol to cortisone by human kidney slices under different conditions, we tested the possibility of 11 beta-HSD regulation by ACTH and corticosteroids. Slices prepared from unaffected parts of kidneys removed because of renal cell carcinoma were incubated with unlabeled or labeled cortisol, and cortisol and cortisone were quantitated after HPLC separation by UV or radioactive detection. The 11 beta HSD activity was not influenced by incubation with increasing concentrations (10(-12)-10(-9) mol/l) of ACTH (1-24 or 1-39) for 1 h. Among 12 ACTH-dependent steroids tested (10(-9)-10( 6) mol/l), only corticosterone (IC50 = 2 x 10(-7) mol/l), 18-OH-corticosterone and 11 beta-OH-androstenedione showed a significant dose-dependent inhibition of 11 beta-HSD activity. The percentage conversion rate of cortisol to cortisone was concentration dependent over the whole range of cortisol concentrations tested (10(-8) - 10(-5) mol/l. A direct inhibitory effect of ACTH on 11 beta-HSD is, therefore, unlikely. The only steroids inhibiting the conversion of cortisol to cortisone are natural substrates for 11 beta-HSD. Kinetic studies show a saturation of the enzyme at high cortisol concentrations. Thus, the reduced percentage renal cortisol inactivation in EAS seems to be due mainly to overload of the enzyme with endogenous substrates (cortisol, corticosterone and others) rather than to direct inhibition of 11 beta-HSD by ACTH or ACTH-dependent steroids, not being substrates of 11 beta-HSD. PMID- 8616527 TI - Relative value of computed tomography scanning and venous sampling in establishing the cause of primary hyperaldosteronism. AB - The purpose of this study was to evaluate the relative merits of the postural stimulation test, adrenal computed tomography (CT) and venous sampling in the differential diagnosis of patients presenting with primary hyperaldosteronism. The records of 20 patients presenting with primary hyperaldosteronism were reviewed retrospectively. There were 15 patients with a unilateral aldosterone producing adenoma (APA), four patients with idiopathic hyperaldosteronism (IHA) and one patient with primary adrenal hyperplasia (PAH). The postural stimulation test was based on measurements of plasma aldosterone and renin activity at 08.00 h and at noon after 4 h of ambulation. The CT scans of the adrenals were reviewed by a single radiologist. Bilateral venous sampling of adrenal veins was attempted in all patients and blood collected for aldosterone and cortisol assay. Plasma aldosterone concentration increased after 4 h of standing in all cases of hyperplasia but was also demonstrated in 10/15 patients with a surgically-proven APA. If one defines a significant postural rise as being greater than 30%, then 8/15 patients with APA can be considered as being posturally responsive. Computed tomography scanning correctly identified all 15 cases of APA and also classified correctly the remaining five cases of hyperplasia (four cases of IHA and one case of PAH). Venous sampling failed technically in 4/15 cases of APA and in one case of IHA: a total of 5/20 (25%,). A correct diagnosis of APA or IHA was established in all the remaining cases. However, the one case of PAH was treated successfully by adrenalectomy following venous sampling, which suggested a unilateral adrenal lesion: this one result was the only instance where venous sampling altered clinical decision-making. Computed tomography scanning may be used alone to confirm the cause of hyperaldosteronism where postural studies suggest an adrenal adenoma, and such patients may be considered for early surgery. Venous catheterization studies are not necessary routinely. but may still be useful in selected patients, particularly when CT scanning shows no clear lesion. PMID- 8616528 TI - Obvious mRNA and protein expression but absence of mutations of the RET proto oncogene in parathyroid tumors. AB - The study on the expression of the RET proto-oncogene in parathyroid tumors disclosed obvious mRNA expression by the reverse transcription (RT)-polymerase chain reaction (PCR) method and protein expression by Western blotting. To find out whether mutations in the cysteine-rich regions or tyrosine kinase domain of the RET proto-oncogene are etiological for parathyroid tumorigenesis, sporadic parathyroid adenomas and carcinomas, parathyroid tumors from multiple endocrine neoplasia 1, familial isolated hyperparathyroidism or hereditary hyperparathyroidism-jaw tumor syndrome were screened by PCR-single strand conformation polymorphism and PCR restriction fragment length polymorphism. Missense mutations in the cysteine-rich region, or codons 768 or 918 in the tyrosine kinase domain of the RET proto-oncogene, were not detected in any of the examined cases of parathyroid tumors. These results suggest that mutations of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for parathyroid tumors. PMID- 8616529 TI - Increased plasma levels of islet amyloid polypeptide in patients with primary hyperparathyroidism. AB - Amylin, also named islet amyloid polypeptide (IAPP), is a protein that is processed and released from pancreatic beta-cells in parallel with insulin. Islet amyloid polypeptide is currently studied with regard to a role for insulin resistance in non-insulin-dependent diabetes. To elucidate a possible function of IAPP for impaired glucose tolerance in primary hyperparathyroidism (pHPT), we studied plasma IAPP levels during an oral glucose tolerance test (OGTT) in seven pHPT patients before and 8 weeks after surgery and in six healthy subjects. The B glucose level of the patient groups was 4.34 +/- 0.12 mmol/l before and 3.97 +/- 0.16 mmol/l after surgery (NS), while the serum level of insulin was significantly higher before (16.9 +/- 2.8 mlU/l) than after (8.9 +/- 1.9 mlU/l) the operation (p < 0.05), indicating a moderately increased insulin resistance in pHPT. The basal plasma levels of IAPP were significantly higher in pHPT patients before than 8 weeks after surgery (9.71 +/- 1.05 and 4.30 +/- 0.82 pmol/l, respectively: p < 0.01). When compared to the plasma IAPP level of the controls at 1.80 +/- 0.38 pmol/l, pHPT patients had higher IAPP values both before (p < 0.01) and at 8 weeks after (p < 0.05) operation. There was a significant correlation between the serum levels of insulin and plasma levels of IAPP in pHPT patients before (r = 0.87, p < 0.01) as well as 8 weeks after surgery (r = 0.69, p < 0.05). The area under the curve for IAPP during OGTT in pHPT patients was 1872.4 +/- 187.7 pmol.min/l, which is significantly higher than after surgery (1010.8 +/- 93.7 pmol.min/l) (p<0.05) and compared to the area for the controls at 840.3 +/- 49.9 pmol.min/l (p<0.01). In conclusion, pHPT is associated with an increased plasma level of IAPP, correlated to the serum insulin level, but persistently higher than in controls also 8 weeks after surgery. Possibly, increased IAPP levels can have a role for impaired glucose tolerance in pHPT. The hyperparathyroid state might have a specific role for the release of this peptide, otherwise closely connected to insulin secretion PMID- 8616530 TI - Effect of insulin and growth hormone on the synthesis of radiolabelled proteoglycans from cultured human arterial smooth-muscle cells. AB - The present study focuses on the pathogenesis of increased frequency of large vessel disease in diabetes. The diabetic arterial wall displays characteristic alterations of the extracellular matrix secreted by arterial smooth-muscle cells. The effects of insulin and growth hormone on the synthesis of sulphate-labelled proteoglycans and heparan sulphate proteoglycan were studied. Proteoglycans and heparan sulphate proteoglycan were obtained from the medium and the cell layer of cultured human arterial smooth-muscle cells grown in 5% human serum. Heparan sulphate proteoglycan was quantified using ethanol precipitation combined with specific enzyme degradation. Addition of insulin (0.01, 0.05 and 0.10 mU/ml) induced a significant accumulation of 35S-labelled proteoglycans in the cell layer (2p < 0.005 and 2p < 0.001). The relative amount of cell-associated heparan sulphate proteoglycan increased during insulin stimulation (2p < 0.05). Growth hormone stimulation (5.0 and 10.0 ng/ml) caused a significant decrease in the ratio between heparan sulphate proteoglycan and proteoglycan in the cell layer (2p < 0.02 and 2p < 0.01), whereas the distribution of proteoglycans between the cell layer and the medium was unaltered. PMID- 8616531 TI - High frequency of endocrine autoimmunity in patients with sarcoidosis. AB - Autoimmune diseases and sarcoidosis may be related and, especially, the association between sarcoidosis and autoimmune thyroid disease has long been recognized. The frequency and type of endocrine autoimmunity was examined in a series of Swedish patients with sarcoidosis. Of all patients (N = 89) with documented sarcoidosis attending the Department of Pulmonary Medicine between January 1980 and December 1991, 78 patients (44 males and 34 females; median age at the time of the study 48 years. range 22-81 years) were examined at the Department of Endocrinology, Malmo University Hospital, in the present study. Fifteen patients (19.2%) had clinical or serological evidence of endocrine autoimmunity. Two patients had Addison's disease, both with polyglandular autoimmune (PGA) syndrome type II: evidence of thyroid autoimmunity was found in 13 patients, eight with clinical autoimmune thyroid disease (ATD) (two with Graves' disease and six with autoimmune thyroiditis), of whom two had PGA syndrome type III, and five with isolated positive thyroid serology; two patients had insulin-dependent diabetes mellitus and one had premature ovarian failure. The frequencies of Addison's disease, clinical ATD and PGA syndrome type II were significantly higher compared with the frequencies found in the general population. In conclusion, a high frequency of endocrine autoimmunity in patients with sarcoidosis, occurring in about 20% of the cases, was demonstrated. Thyroid autoimmunity and polyglandular autoimmune syndromes occurred most frequently. Complex immunological and genetic mechanisms might explain the association of sarcoidosis and endocrine autoimmune diseases. PMID- 8616532 TI - Prophylactic application of thyrostatic drugs during excessive iodine exposure in euthyroid patients with thyroid autonomy: a randomized study. AB - In a prospective, randomized study we examined the influence of prophylactic short-term thyrostatic therapy on thyroid iodine metabolism in patients with euthyroid autonomy undergoing elective coronary angiography. From a total of 1177 patients, 51 fulfilled the criteria of euthyroid autonomy before coronary angiography (negative thyrotropin-releasing hormone test, 10-min uptake of at least 1.2%, 99mTc and no elevation of free thyroxine and free triiodothyronine indices) and were randomized into three groups: group 1 (N = 17) received 20mg/day of thiamazole and group 2 (N = 17) received 900 mg/day of sodium perchlorate; thyrostatic therapy was begun on the day before angiography and continued for 14 days; group 3 (N = 17) served as controls without treatment. Parameters of thyroid function-free thyroxine (FT4) index and free triiodothyronine (FT3) index, thyrotropin (TSH) and delta-TSH urine iodine excretion and 99mTc uptake were determined before and 30 days after coronary angiography. At the end of the study the mean FT4 index and FT3 index were elevated significantly in the control group compared with baseline values, but were still within the normal range. In contrast, the mean FT4 index and FT3 index remained unchanged in the treated groups. Four mild cases of hyperthyroidism were observed at the end of the study: two cases in the control group and one case in each of the treated groups. Thyrotropin suppression, urine iodine excretion and 99mTc uptake differed significantly between the treated groups and the control group. In the treated groups TSH suppression, urine iodine excretion and 99mTC uptake remained unchanged 30 days after coronary angiography compared with baseline values. In the control group the degree of TSH suppression and the level of urine iodine excretion increased (about twofold) significantly after coronary angiography, whereas 99mTc uptake decreased significantly (ca. 50%). In conclusion, short-term prophylactic thyrostatic therapy seems to have a protective effect against iodine excess in patients with euthyroid autonomy. However, mild hyperthyroidism could not be prevented in some cases. Probably a combination therapy of thiamazole and perchlorate would be more effective. PMID- 8616533 TI - Hypothyroid patients showing shortened responsiveness to oral iodized oil have paradoxically low serum thyroglobulin and low thyroid reserve. Thyroglobulin/thyrotropin ratio as a measure of thyroid damage. AB - In Central Africa, all of northern Zaire is very severely deficient in iodine. A peculiar feature of this endemia is that iodine deficiency and the ensuing thyroid gland stimulation not only leads to goitre formation but also to progressive thyroid involution and to myxoedematous cretinism. An iodine supplementation trial based on oral administration of small doses of iodine was made in 81 schoolchildren. All of them received a small dose of iodine (0.1 ml containing 48 mg) per os and the thyroid status was followed during 4 months. Blood and urine samples were collected at the start of the study, then 2 weeks, 2 months and 4 months after iodine administration. Before iodine supplementation the mean urinary iodine level was 0.18 +/- 0.02 micromol/l, and 10% of the subjects had a urinary iodine level below 0.08 micromol/l. Fifty-two percent of the subjects had a serum thyrotropin (TSH) level above 10 mU/l. All the subjects responded to the administration of iodine. and all of them recovered a euthyroid status. Most of them were still euthyroid at the end of the study. However. within 4 or even 2 months, some subjects (15 % of the total) reverted to hypothyroidism. At the entry of the study these subjects were all hypothyroid and had elevated TSH and paradoxically low serum thyroglobulin (TG) values. In myxoedematous cretins living in the same area, even lower serum TG levels were found. Together with the absence of goitre, a paradoxically low serum TG Suggests a low thyroid reserve, and in the present case a reduced amount of functional thyroid tissue. We show that the serum TG/TSH ratio may be used as a predictive index of thyroid reserve and of positive response to iodine administration. These data further suggest that thyroid damage is not confined to myxoedematous cretins. but is widely distributed in the phenotypically normal population. Widely distributed thyroid damage may render iodine prophylaxis based on oral administration unpredictable. PMID- 8616535 TI - Gonadotropins and gonadotropin receptors during the perimenopause. AB - Twenty-two perimenopausal patients (aged 47-56 years) admitted for elective abdominal hysterectomy and salpingo-oophorectomy were selected to understand better the clinical significance of increasing gonadotropin levels as an indicator of target organ responsiveness. Prior to anesthesia, blood was drawn from the patients for subsequent analyses of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and 17beta-estradiol (E2) levels. Ovarian tissue was obtained during surgery and frozen at -70 degrees C for subsequent analyses for FSH and LH receptor content. The phase of the menstrual cycle of the patients or postmenopause was determined by serum gonadotropin and E2 levels and histological evaluation of the endometrium. Patients with no detectable FSH receptors showed significantly higher serum FSH and LH levels (4.7- and 4.3-fold, respectively) when compared to patients with detectable FSH receptors; FSH receptors were present in 27% of the patients, LH receptors were present in 68% of the patients and a negative correlation was found between serum LH levels and ovarian LH receptors. In postmenopausal patients, neither FSH receptors nor LH receptors were detectable. High serum gonadotropin levels in perimenopausal patients thus suggest the existence of low or undetectable ovarian gonadotropin receptor levels. PMID- 8616534 TI - New approach to the diagnosis of growth hormone deficiency in adults. AB - Pyridostigmine (PD), a muscarinic cholinergic agonist, and arginine (ARG) clearly increase the growth hormone (GH) response to growth hormone-releasing hormone (GHRH) in man. The current study was undertaken to investigate the value and safety of PD + GHRH and ARG + GHRH tests as well as the measurement of serum insulin-like growth factor I (IGF-I) in diagnosing GH deficiency in adults. Fifty four patients considered GH deficient from extensive organic or idiopathic pituitary disease and 326 healthy adults were studied. The IGF-I concentrations were lower than the 3rd percentile of normal values in only 31 of the 54 (57.4%) patients with hypopituitarism. However, the IGF-I levels in hypopituitary patients and in normal subjects overlapped more frequently between 41 and 60 years (50%) and between 61 and 80 years (92.3%) as opposed to between 20 and 40 years (8.6%). In contrast to the IGF-I measurement, the ranges of peak GH responses to PD + GHRH and ARG + GHRH tests were clearly differentiated between the hypopituitary (0.2-6.8 and 0.1-9.5 microg/l, respectively) and normal (17.7 114 and 16.1-119 microg/l, respectively). However, the PD + GHRH test was reliable only in subjects of 20-40 years of age. In conclusion, IGF-I measurement had no value in the diagnosis of GH deficiency in adults aged over 40 years, but is reliable enough when young adults of 20-40 years of age are considered. Both PD + GHRH and ARG + GHRH testing should be considered more reliable biochemical measurements of GH deficiency. In contrast to the PD + GHRH test, the ARG + GHRH test is reliable throughout the adult lifespan and appears to be the most appropriate for patient compliance and safety. PMID- 8616536 TI - Peptide YY does not inhibit glucose-stimulated insulin secretion in humans. AB - Peptide YY (PYY) is localized to gut and pancreatic endocrine cells. it may therefore be involved in the regulation of insulin secretion as a gut hormone as well as an islet local regulator. in laboratory animals, the peptide inhibits stimulated insulin secretion, but its effects in humans are not known. We therefore infused PYY intravenously at a low (1 pmol x kg(-1) x min(-1); N = 4); or a high rate (5 pmol x kg(-1) x min(-1); N = 5) for 120 min in healthy women aged 52 years. After 30 min of infusion, glucose (0.5 g/kg) was injected rapidly. In separate control experiments, saline was infused instead of PYY. We found that PYY did not inhibit the acute insulin response to glucose or affect the glucose elimination rate, and PYY was also without influence on the basal plasma glucose and serum insulin levels during the 30-min infusion before the challenge with glucose. We therefore conclude that intravenous infusion of PYY does not affect glucose-stimulated insulin secretion in man. PMID- 8616537 TI - Aggressive thyroid cancer associated with toxic nodular goitre. AB - Reports of concurrent thyrotoxicosis and thyroid cancer have appeared in the last three decades. While most of the tumours have been clinically inconsequential, it has been suggested that thyroid carcinomas arising in patients with Graves' disease tend to behave aggressively, while those associated with toxic nodular goitre follow a more benign course. We report a contrary clinical experience with four cases of thyrotoxicosis associated with metastatic thyroid cancer, two of which were fatal. All four patients had toxic nodular goitre. Thyroid eye signs were uniformly absent. Two patients had received 131I therapy; none had other history of irradiation to the head or neck. Antimicrosomal and antithyroglobulin antibodies were absent in all four patients. Thyroid-simulating immunoglobulin, which was measured in one patient, was also absent. Histopathological examination of the resected thyroid glands revealed two papillary cancers. one mixed anaplastic/papillary and one anaplastic cancer. All four patients had cervical node involvement and one had pulmonary metastases. Both patients with anaplastic carcinoma succumbed to their disease within 6 months: neither of the patients with papillary cancer had disease recurrence after 2 and 4 years, respectively. The experience reported here of aggressive thyroid cancer associated with toxic nodular goitre may represent coincidence or, alternatively, it may represent the early recognition of a change in the natural history of toxic nodular goitre. PMID- 8616538 TI - Enhanced expression of transforming growth factor beta1 in rat thyroid hyperplasia is thyrotropin induced and time dependent. AB - Forty-three 8-week-old male Wistar rats were studied to evaluate temporal changes of transforming growth factor beta1, (TGF-beta1) mRNA levels in thyroid tissue during pharmacologically induced goiter. Four rats were treated with purified bovine thyrotropin (TSH; Ambinon, 2 mU/day sc) for 7 days before being sacrificed. Thirty-one were treated with propylthiouracil (PTU), added to their drinking water at a concentration of 0.2 g%, and subsequently were sacrificed as follows: five after 1 week (PTU-1): five after 2 weeks (PTU-2); five after 4 weeks (PTU-4); five after 8 weeks (PTU-8); five after 12 weeks (PTU-12). In six rats, after 12 weeks of treatment. PTU was withdrawn for 2 months and subsequently started again in three rats which were sacrificed after 2 weeks (PTU R); the remaining three rats were sacrificed without any further treatment (PTU-R control). Eight rats (control rats) were never treated and served as controls. After sacrifice, blood was drawn for determination of total thyroxine and the thyroid was excised and subdivided into two lobes. Northern analysis for TGF beta1 was performed in one lobe. while histological and immunohistochemical studies were performed in the other lobe. Gene expression of TGF-beta1 was induced in TSH- and PTU-treated rats. In TSH-treated rats TGF-beta1 gene expression was less detectable than in PTU-treated rats, where it became evident after 2 weeks and remained through weeks 4-8. Gene expression of TGF-beta1 wits also seen in PTU-R rats, but not in the control and in the PTU-R control. Immunohistochemical analysis showed a different presence and location for the TGF beta1 protein, which appears to be dependent on the time of exposure to mitogenic stimulus. In conclusion, TGF-beta1 is produced in response to both a direct (TSH by itself) and indirect (TSH induced by PTU-induced hypothyroidism) cellular proliferative stimulus and is not linked to an adaptative phenomenon secondary to hypothyroidism. The immunohistochemical location of TGF-beta1 within the thyrocytes is influenced by mitogen exposure time. A TGF-beta1 immunohistochemical evaluation may be important to define exposure time and activity of goitrogenic stimuli. PMID- 8616539 TI - Influence of oestrous cycle on the pressor recovery following haemorrhage in anaesthetized Brattleboro rats. AB - A sexual dimorphism in the pressor responsiveness to the neurohypophysial hormone vasopressin may be associated with a peripheral interaction between ovarian steroids and the neurohypophysial hormone. Indeed, the ovarian steroids may inhibit the vasopressin-dependent component of the pressor response to haemorrhage. The present study examined the recovery of the arterial blood pressure following it single large (2% v/w) haemorrhage in anaesthetized male Long Evans (LE) rats and females of the same strain during either pro-oestrous or di-oestrous phases of the reproductive cycle. In addition the same recovery process was examined in Brattleboro rats with diabetes insipidus (BDI) lacking circulating vasopressin. All BDI rats had an impaired blood pressure recovery following haemorrhage compared with male rats of the parent LE strain, and this was irrespective of sex or stage of the oestrous cycle. While the blood pressure recovery was more impaired in both groups of BDI female rats than in the males of the same strain during the first 20 min after haemorrhage (both comparisons p < 0.001; ANOVA), there was no difference between the recoveries of the female rats in pro-oestrus or di-oestrus. In contrast a significantly impaired blood pressure recovery was observed in female LE rats at pro-oestrus, when circulating ovarian steroid concentrations are raised, compared with male (p < 0.001: ANOVA) and di oestrous (p < 0.02: ANOVA) rats of the same strain. Heart rate responses to haemorrhage showed strain differences, with LE rats having initial decreased heart rates followed by a recovery process, while the heart rate responses of BDI rats increased immediately. The novel use of the female Brattleboro rat in this study provides evidence for the existence of an important inhibitory interaction between ovarian steroids and vasopressin during the blood pressure recovery phase following haemorrhage, and indicates a possible direct influence of gonadal steroids on the recovery process. PMID- 8616540 TI - Differential subcellular distribution of rat prostatic steroid 5alpha-reductase isozyme activities. AB - The rat prostate, a classical androgen-target tissue, contains both known isozymes of steroid 5alpha-reductase. i.e. type I and type II. So far, the role of the type I isozyme has been proposed as catabolic. The abundant expression of type I 5alpha-reductase in an androgen-target tissue is therefore puzzling. Assessment of the subcellular localization of 5alpha-reductase isozymes in rat prostate might contribute in elucidating their possibly distinct roles. After obtaining crude subcellular fractions by differential centrifugation, both isozyme activities were measured at neutral pH by plotting according to Eadie Scatchard. The observations were extended by assessment of pH-dependent velocity ratios and type II 5alpha-reductase inhibitor sensitivities in these subcellular fractions. The results indicated a preferentially--although not exclusively- nuclear localization for the type I and a predominantly microsomal localization for the type II isozyme activity in the rat prostate. In conclusion, the nuclear localization of the type I isozyme seems not to concur with its proposed catabolic role. PMID- 8616541 TI - Is volume reduction surgery appropriate in the treatment of emphysema? Yes. PMID- 8616542 TI - Is volume reduction surgery appropriate in the treatment of emphysema? No. PMID- 8616543 TI - Health-care technology assessment of surgical procedures: the case of reduction pneumoplasty for emphysema. AB - End-stage chronic obstructive pulmonary disease (COPD) is frequent in the practice of pulmonology. The recent report that reduction pneumoplasty results in lung function improvement in a high proportion of patients with emphysema has raised the question of how effectively the medical profession assesses the efficacy of new surgical procedures. Unlike drugs and devices, the introduction of new surgical procedures is not regulated. Technique changes rapidly for a period of time after new procedures are introduced. Peer review is done mainly by quality-improvement activities in the surgeon's hospital and by publication in peer-reviewed journals. Randomized clinical trials (RCT) are procedurally and ethically difficult to do and few have been done for new surgical procedures. Some trials have been problematic. For example, a controlled cooperative trial of radial keratotomy resulted in unsuccessful antitrust suits brought by physicians and patients alleging that third-party payers would not reimburse them for surgery. RCTs that have been done have tended to be reported long after the procedure has been incorporated into surgical practice. A RCT of reduction pneumoplasty would be impossible at this time because of rapid flux in surgical techniques. There might also be ethical problems because of the substantial improvement in lung function of many patients in the existing case series, a level of improvement seen with no other treatment than lung transplantation. It is proposed that a prospective, cooperative, consecutive-case, observational study (registry) of reduction pneumoplasty for emphysema could greatly speed the acquisition of information about the efficacy of this new procedure. PMID- 8616544 TI - Effects of depletion of cells bearing the interleukin-2 receptor on immunoglobulin production and allergic airway responses in the rat. AB - Lymphocytes, key cells in chronic inflammation, are increased in the airways of asthmatics and have increased expression of the interleukin-2 (IL-2) receptor, a sign of activation. We determined the effects of depleting cells bearing IL-2 receptors on immunoglobulin (Ig) production, airway inflammation, and airway responses after antigen challenge of Brown Norway rats that were sensitized to ovalbumin (OA). Both control and ART-18 (antirat IL-2 receptor) antibodies inhibited plasma specific IgE and the early (ER) and late (LR) airway responses to antigen when given from zero to 14 d after sensitization. When ART-18 was administered from 4 to 14 d after sensitization and compared with control animals, it inhibited OA specific IgE production from Day 21 onward, but it increased total IgE and specific IgG. These changes followed a significant increase in blood CD4+ lymphocytes (%) in ART-18-treated animals 14 d after sensitization. The same protocol of administration did not affect Ig levels at 14 d, but it decreased neutrophil influx into the lungs 8 h after antigen challenge without any effects on the ER and LR. Administration of ART-18 at the time of antigen challenge did not affect the subsequent airway inflammation or the increased responsiveness to methacholine that occurs 32 h after antigen challenge. In summary, depletion of IL-2-receptor-bearing cells affects lymphocyte subsets and immunoglobulin production and it decreases the influx of neutrophils into the lungs 8 h after OA challenge, but it does not significantly inhibit the ER, LR, or increased airway responsiveness after antigen challenge. PMID- 8616545 TI - Inflammatory cellular influx follows capsaicin nasal challenge. AB - Capsaicin is a specific activator of sensory nerve endings. In rodents, mucosal application of capsaicin causes cells to infiltrate the tissue. To examine whether inflammatory-cell influx follows sensory-nerve activation in human airways, we delivered capsaicin (200 microM) nasal spray into the nares of 20 subjects (10 with allergic rhinitis and 10 normal), and measured the total leukocyte content of nasal lavage fluid obtained from 10 min to 4 h after the capsaicin challenge. Vehicle spray (1% EtOH in 0.9% saline) served as a control. Capsaicin challenge caused significant increases from prechallenge leukocyte counts at 10 min (p<0.03), 30 min (p<0.01), and 4 h (p<0.03) after challenge, but not at 1 h after challenge (p = 0.68). Vehicle challenge did not increase leukocyte counts. Differential counts (performed on the 13 of 20 subjects from whom adequate specimens for differential counts were obtained) showed that neutrophils, eosinophils, and mononuclear cells increased at 10 min, 30 min, and 4 h (all p < 0.04), but not at 1 h after capsaicin challenge. Comparing the rhinitic to the normal subjects, we found no significant differences in the cellular response to capsaicin. These data support a nonspecific inflammatory effect of sensory nerve activation in the human nose. Consequently, this work provides evidence that neurogenic inflammation can be induced in the human airway in vivo. PMID- 8616546 TI - Ozone enhances the uptake of mineral particles by tracheobronchial epithelial cells in organ culture. AB - We have previously shown that the basal uptake of mineral particles by tracheobronchial epithelial cells in organ culture is mediated in part by active oxygen species (AOS) and can be greatly augmented by exposure to cigarette smoke, a concentrated source of AOS, and other radicals. We hypothesized that ozone, another generator of AOS in tissues, might have the same effect. To test this hypothesis, tracheal explants were exposed to room air (control) or ozone in varying concentrations from 0.01 to 1.0 ppm for 10 min, and subsequently to a suspension of either amosite asbestos or titanium dioxide (rutile) for 1 h. Explants were then transferred to an air/CO2 incubator for 1 wk to allow particle uptake to occur, and uptake was determined by morphometry. We found that ozone exposure increased the uptake of both asbestos and titanium dioxide in a dose response fashion; this effect appeared at lower exposure levels and was more marked with titanium dioxide than with amosite. The ozone effect could be prevented by addition of catalase but not superoxide dismutase to the particle suspension, or by preincubation of the particles with deferoxamine. These observations indicate that ozone can directly increase uptake of mineral particles by tracheobronchial epithelial cells; this effect occurs with brief exposures at very low ozone levels and appears to be mediated by hydrogen peroxide and possibly by hydroxyl radical. These findings support the general hypothesis that AOS are important mediators of epithelial particle uptake in many different settings. Enhanced uptake may be one of the mechanisms by which ozone impairs particle clearance from the lung and may play a role in the increased morbidity seen in populations with exposure to high levels of both ozone and atmospheric particulates. PMID- 8616547 TI - Long-term effects of home rehabilitation on physical performance in chronic obstructive pulmonary disease. AB - A pilot study was set up to assess the long-term effects of once weekly versus once monthly follow-up of pulmonary rehabilitation after a comprehensive home rehabilitation program on physical performance in patients with chronic obstructive pulmonary disease (COPD) during an 18-mo period. Thirty-six patients with a mean FEV1 of 1.3 +/- 0.4 L (43% pred) were included in the study. Groups A and B (n = 23) visited the physical therapist twice weekly for 3 mo. Thereafter, 11 patients (Group A) had a follow-up of pulmonary rehabilitation once a week, and 12 patients (Group B) had a follow-up once a month. Thirteen patients received no rehabilitation at all (Group C). Long-term home rehabilitation does not appear to improve exercise tolerance; however, on the other hand, there is a deterioration in vital capacity (p < 0.01), walking distance (p < 0.01), and maximal work load (p < 0.05), as shown in the control group. A small improvement in exertional dyspnea (p < 0.01) after 18 mo and inspiratory muscle function (p < 0.05) after 12 mo was shown only in Group A. Because of the insufficient number of patients enrolled in this pilot study, no clear benefit on physical performance of long-term home rehabilitation with either weekly or monthly supervision could be demonstrated. PMID- 8616548 TI - Interaction between cold and hypoxia on pulmonary circulation in COPD. AB - This study was designed to investigate the interaction of mild and localized cold exposure and hypoxia on pulmonary hemodynamics in chronic obstructive pulmonary disease (COPD). Nineteen patients with COPD were studied at sea level and seven at an altitude of 2,640 m. For all patients, pulmonary hemodynamic measurements were performed 10 min after insertion of a catheter in a femoral vein and following 10 min of cold exposure. Cold exposure was restricted to the forehead, and subjects breathed air at ambient temperature. Flow and temperature of air (1.5 L.s(-1), 5 degrees C) to the forehead were chosen to cool down the forehead skin to approximately 20 degrees C without discomfort for the subject. For the seven patients studied at high altitude, the same measurements were also performed after 5 min of oxygen supplementation with and without cold exposure. At sea level, an increase in pulmonary vascular resistance (PVR) during cold exposure was inversely related to the initial PaO2. In six severe hypoxic subjects (PaO2 < 50 mm Hg), PVR increased by 24%. At high altitude, PVR was significantly increased by 15%. After O2 supplementation, cold exposure did not induce an increase in PVR. We concluded that mild and localized cold exposure to the forehead only induced an increase in PVR in COPD patients with severe hypoxia. Moreover, in cold exposure responders, O2 supplementation negated the effect of cold exposure on pulmonary hemodynamics. PMID- 8616549 TI - Do tidal expiratory flow patterns reflect lung mechanics in infants? AB - It has been suggested that during tidal breathing, the time to maximal expiratory flow, as a proportion of total expiratory time (Tme/TE), can be used as an index of airways obstruction. However, the relationship of Tme/TE to lung mechanics in infants is unclear. We examined this relationship in 42 premature infants (21 intubated and 21 not intubated) by comparing direct measures of pulmonary mechanics (lung resistance, dynamic lung compliance, and lung impedance) with Tme/TE measured over the same sample of tidal breathing. Tme/TE was positively correlated with lung compliance, expressed as a percent of the predicted value, in both intubated (r = 0.69, p < 0.005) and nonintubated (r = 0.64, p < 0.02) infants. There was no significant association between Tme/TE and lung resistance, expressed as a percent of the predicted value, in intubated (r = 0.32) and nonintubated (r = 0.23) infants. Tme/TE also showed, in nonintubated infants, a positive association with lung impedance, expressed as a percent of the predicted value, on the basis of the predictive values for compliance and resistance at the infant's unique respiratory rate, but this was significant only because of the influence of compliance on Tme/TE. These findings suggest that, in infants, the relationship between pulmonary mechanics and Tme/TE is complex, with Tme/TE being influenced by the elastic rather than the flow-resistive properties of the lungs. PMID- 8616550 TI - Tidal breathing analysis as a measure of airway obstruction in children three years of age and older. AB - This study was designed to evaluate the value and applicability of tidal breathing pattern analysis to assess airflow obstruction in young children. The time needed to reach maximal tidal expiratory flow (TME) divided by total expiratory time (TE) was measured in 228 healthy children 3 to 11 yr of age, 64 patients with asthma, and 12 children with cystic fibrosis. In 70 patients both TME/TE and forced maximal expiratory flow volume (MEFV) parameters were measured. The mean TME/TE in healthy subjects was 43.0 +/- 7.6%. The within-subject reproducibility was high (repeatability index, 5.3%). In the asthmatic patients the mean TME/TE was significantly lower (30.0 +/- 8.2%, p < 0.001), and it increased to 36.5 +/- 7.9% after bronchodilation (n = 44, p < 0.001). The TME/TE level of the subgroup of patients with asthma and FEV1/FVC > or = 0.80 was lower when compared with age-matched normal subjects (30.9 +/- 8.5, p < 0.0001), but it was in a higher range when compared with asthmatics with FEV1/FVC < 0.80 (25.9 +/ 7.9, p < 0.001). In the cystic fibrosis group the mean TME/TE was 27.4 +/- 10.7% without a significant change after bronchodilation. TME/TE correlated significantly with MEFV parameters. Tidal breathing analysis proved easy to perform in children older than 3 yr of age. The TME/TE ratio may be a reliable and simple indicator for airway obstruction. PMID- 8616551 TI - Recurrent cough in childhood and its relation to asthma. AB - Risk factors for recurrent cough (RC) in childhood, and its relation to asthma were investigated as part of the prospective, longitudinal Tucson Children's Respiratory Study. RC, defined as > or = 2 episodes of cough without a cold in the past year, was assessed by questionnaire in 987 children at age 6. Children having RC without wheeze (n = 154) did not differ from children with neither symptom (n = 610) in serum IgE levels, skin test response, size-corrected forced expiratory flow, or percentage of decline following cold air challenge. In contrast, children with both RC and wheeze (n = 116) had significantly more respiratory illness, more atopy, lower flow at end-tidal expiration (V'maxFRC), and greater declines in lung function following cold air challenge than children with neither symptom. Current parental smoking was a risk for RC without wheeze, whereas male gender, maternal allergy, wheezing lower respiratory tract illness (LRI) in early life, and high IgE were significant risks for RC with wheeze, compared with children having neither symptom. RC early in life resolved in the majority of children, between ages 2-3 yr and age 6, and between age 6 and age 11. High IgE and positive skin prick test were associated with persistence of RC to age 6 among children who wheezed, and markers of allergy were associated with persistence of RC between 6 and 11 yr. These findings suggest that recurrent cough in the absence of wheeze differs in important respects from classic asthma, and using the same label to refer to these distinct syndromes may obscure their diverse pathophysiologies. PMID- 8616552 TI - Familial aggregation of asthma in a South Bavarian population. AB - There is considerable evidence for a significant hereditary component in the pathogenesis of asthma and allergic diseases. The objective of this study was to investigate familial influences unique to the expression of asthma. School children (n = 9,403), 9 to 11 yr of age, were enrolled in a cross-sectional survey in southern Germany. The prevalence of asthma and allergic diseases in parents and children was assessed through parental questionnaires. Atopic sensitization was measured by skin prick tests, and bronchial responsiveness was determined by cold air hyperventilation challenge. The prevalence of asthma alone, i.e., without concomitant hay fever or atopic eczema, increased strongly if nearest of kin suffered from asthma alone (4.7 versus 11.7%, p < 0.0001). A family history of hay fever or atopic dermatitis, excluding asthma, was unrelated to asthma in the offspring (4.7 versus 3.9%). These relations did not change when stratifying for skin prick test reactivity (STR) and bronchial hyperresponsiveness (BHR). STR alone and BHR alone (i.e., without manifestation of disease) were unrelated to a family history of asthma or allergy. The results strongly suggest a separate genetic factor controlling the development of asthma. Skin test positivity and BHR to cold, dry air are unlikely to be the underlying mechanisms through which the inheritance of childhood asthma is transmitted. PMID- 8616553 TI - Concentrations of methacholine for bronchial responsiveness according to symptoms, smoking and immunoglobulin E in a population-based study in Spain. Spanish Group of the European Asthma Study. AB - To assess the simultaneous effects of symptoms related to asthma, smoking, and Immunoglobulin E (IgE) on the concentrations of methacholine at which different individuals fall below 80% the FEV1 values obtained with saline, we used parametric survival methods that fit the dose-response nature of bronchial responsiveness (BR). A general population-based sample of 1,615 adults (aged 20 to 44 yr) from five cities in Spain participating in the European Community Respiratory Health Survey was studied. The total number of subjects whose FEV1 values dropped below 80% was 237 (14.7%). We found that individuals with symptoms related to asthma, whether they were smokers or nonsmokers, responded at lower concentrations of methacholine than individuals without symptoms. The greatest decrease in the concentrations of methacholine at which different percentages of individuals responded was due to symptoms among the nonsmokers (relative percentiles ranging from 0.53 to 0.76, and a 90% confidence band not containing a value one). Among individuals without symptoms, smokers responded at moderately but significantly lower concentrations than nonsmokers, whereas an apparent protective effect of smoking was seen in symptomatic individuals, owing in part to a self-selection bias. The higher the concentration of IgE, the lower were the concentrations of methacholine needed to obtain a given level of BR. Moreover, the magnitude of the decrease in methacholine concentrations leading to a response was constant over all percentages of individuals responding. The analytical methods used in the study permit the incorporation of complexities in the relationships between BR and symptoms, smoking, and IgE, and are generally applicable to situations in which a dose-response relationship occurs. PMID- 8616554 TI - Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness. AB - To investigate genetic factors in asthma and atopy, we sought allelic associations for 12 markers near candidate loci for serum total, immunoglobulin E (IgE), and bronchial hyperresponsiveness (BHR) to inhaled histamine in 131 families comprising 685 individuals, selected randomly without regard to atopy or asthma. Nonparametric linkage and association analyses were performed with the Nonparametric Analysis of Lineage and Association (NOPAR) program, and parametric analyses were performed with the Complex Inheritance with Diathesis and Severity (COMDS) program on an ordered polychotomy of ranked scores. On chromosome 11q, allele 168 at the D11S527 locus was significantly associated with BHR (p<0.0003) but not with log IgE. At the D11S534 locus, allele 235 was significantly associated with log IgE (p = 0.007) but not with BHR. Both D11S527 and D11S534 were too distant from the gene encoding the high-affinity IgE receptor FcepsilonRIbeta to account for the association. At the interleukin-9 (IL-9) locus, the 118 allele showed significant association with serum total IgE (p<0.003) but not with histamine BHR. Parametric tests are more conservative, perhaps because they demand consistency with mendelian inheritance and tight linkage. These findings provide support for the view that both chromosomes 5 and 11 may contain genes relevant to asthma and atopy, a possible candidate being the interleukin-4 (IL-4) gene cluster. Because these associations are extremes in a large number of tests, they require confirmation in other samples. PMID- 8616555 TI - Respiratory symptoms in urban Hispanic and non-Hispanic white women. AB - The prevalence and predictors of wheeze syndromes, including asthma, were examined among 475 non-Hispanic (NH) white and 371 Hispanic pregnant women enrolled in a population-based study in East Boston, Massachusetts. Respiratory symptoms and risk factors were ascertained by questionnaire early in pregnancy. Hispanic and NH white women were of similar age (mean +/- SD, 26 +/- 5 yr), but Hispanics reported less schooling (30 versus 50% completed high school), a lower frequency of household pets (4 versus 47%), and a lower frequency of parental asthma (6 versus 12%). Hispanics smoked significantly less than NH whites did, both in prevalence (8 versus 50%) and number of cigarettes per day among current smokers (12 +/- 9 versus 22 +/- 10; p < 0.0001). Hispanics reported a lower frequency than NH whites did of doctor-diagnosed asthma (6 versus 12%), persistent wheeze (5 versus 19%), and either persistent wheeze or asthma (11 versus 30%). In multivariate analysis, active cigarette smoking and parental history of asthma were associated most strongly with wheeze syndromes. When these two factors as well as educational level, presence of household pets, and height2 adjusted FEV1 were controlled, Hispanics remained at lower risk of asthma (odds ratio [OR] 0.71; 95% confidence interval [CI], 0.37-1.37) and persistent wheeze (OR 0.48; 95% CI, 0.25-0.95) than NH whites. These results suggest that chronic wheeze syndromes are common among young urban women and are associated with both active smoking and a parental history of asthma. Hispanic women from Central and South America living in urban environments in the United States may be at less risk for these conditions than NH white women, in contrast to those from Puerto Rico. PMID- 8616556 TI - Incidence of anaerobes in ventilator-associated pneumonia with use of a protected specimen brush. AB - The role of anaerobic bacteria in ventilator-associated pneumonia (VAP) has been little investigated. In this study we analyzed the incidence of anaerobes in patients with a first episode of bacteriologically documented VAP (> 10(3)CFU/ml), using protected specimen brushes (PSB). We particularly took care to preserve anaerobic conditions during transport and the microbiological procedure. Two groups were considered: group A with anaerobic bacteria recovered from PSB, with or without anaerobes, and group B with aerobic bacteria only. One hundred and thirty patients were included, 30 (23%) in group A, and 100 (77%) in group B. The main anaerobic strains isolated were Prevotella melaninogenica (36%), Fusobacterium nucleatum (17%), and Veillonella parvula (12%). Univariate analysis demonstrated that patients in group A were younger than those in group B (p < 0.05) and their simplified acute physiologic score was higher (p < 0.02). The percentage of patients receiving antibiotics before PSB did not differ significantly between group A (57%) and group B (35%). VAP with anaerobes occurred more often in patients orotracheally intubated than nasotracheally intubated (p < 0.02). Episodes of VAP involving anaerobic bacteria occurred more often in the first five days (early VAP) than after the fifth day (late VAP) (p < 0.05). The 3-mo mortality rate was similar in the two groups, but death occurred earlier in group B (p < 0.01). Multivariate analysis demonstrated that presence of altered level of consciousness (p = 0.0002), higher simplified acute physiologic score (p = 0.003), and admission to the medical ICU (p = 0.02) were the factors independently predisposing to the development of VAP with anaerobes. PMID- 8616557 TI - Primary pulmonary hypertension in HIV infection: an outcome determined by particular HLA class II alleles. AB - Primary pulmonary hypertension (PPH) may have an autoimmune basis that is influenced by host immunogenetics. The pathogenesis of primary pulmonary hypertension in human immunodeficiency virus (HIV) infection is unclear. The objective of this study was to determine whether patients with PPH and HIV infection have distinctive immunogenetic profiles. Ten racially mixed HIV infected patients with PPH were typed for human leukocyte antigen (HLA) class II (DRB1, 3, 4, 5 and DQB1) by DNA-PCR sequence-specific oligonucleotide probes. Results were compared with two control groups: 128 HIV-negative Caucasians and a previously reported group of 97 HIV-positive, racially mixed control subjects. In those with PPH, there was a significantly increased frequency of HLA-DR6 ( DRB1*1301/2 subtypes) and of HLA-DR52 (DRB3*0301 subtype). These findings suggest that HIV-associated PPH reflects a host response to HIV-1 determined by one or more HLA-DR alleles located within the major histocompatibility complex. The same HLA-DR6 subtype found at increased frequency in our patients has previously been associated with the development of a CD8 lymphocytic host response to HIV-1, termed diffuse infiltrative lymphocytosis syndrome (DILS), which resembles autoimmune Sjogren's disease and is associated with prolonged survival. Together, these findings suggest that HIV-positive PPH may represent a clinical outcome that has similarities with that resulting from the immunogenetically determined host response present in DILS. PMID- 8616558 TI - The effect of inspiratory maneuvers on expiratory flow rates in health and asthma: influence of lung elastic recoil. AB - We studied the effect of breath holding and inspiratory speed on airflow during the FVC maneuver in seven healthy subjects and eight patients with asthma. The purpose of the study was to determine whether the effects of inspiratory speed and breath holding on expiratory flow were greater in patients with asthma than in healthy individuals; whether these effects were lessened by inhalation of aerosolized bronchodilator in the patients with asthma; and whether were was a relationship between the lung elastic recoil pressure and the expiratory flow achieved during four different maneuvers. We found that peak expiratory flow rate (PEFR) was significantly lower after both a slow inspiration and a breath hold than after a fast inspiration without a breath hold. In addition, a breath hold was associated with a significantly lower FEV1. The effects of inspiratory speed and breath holding in the patients with asthma were not significantly different from those observed in the healthy subjects. There was a significant relationship between lung elastic recoil pressure at the point of onset of the FVC maneuvers (Pel Blow) and expiratory flow in both healthy and asthmatic subjects. Also, the decrease in Pel Blow with equivalent breath-hold time was greater in asthmatic subjects, which is consistent with an increase in viscoelastic elements in the lung. These findings corroborate previous suggestions that inspiratory speed and the duration of breath holding have significant implications in the performance of spirometry and peak flow measurements, and indicate the importance of standardization of the preceding inspiration when determining FEV1 and PEFR. PMID- 8616559 TI - Standardization of multiple spirometers at widely separated times and places. AB - We designed a system for a multiyear longitudinal study of lung function in 12 widely separated communities, intending to minimize variation in instrument related data. We used multiple rolling-seal spirometer/personal computer systems. Calibrations were checked before, during, and after each day's field testing, using multiple calibration syringes with electronic readouts. The syringes were rotated to obtain data for each syringe-spirometer combination. Before and after each annual field testing season, a laboratory reference spirometer system was calibrated against a water-displacement device and an electronic frequency counter, and then compared against each field spirometer and syringe. Field equipment consistently met American Thoracic Society (ATS) specifications. Variance among spirometers exceeded variance among syringes. A spirometer occasionally changed its volume readout by approximately 1 to 2 %. More rarely, a syringe changed its delivered volume by approximately 1%. Syringes' electronic readouts tracked changes in delivered volume. Syringe readouts were the most stable component of the system, and were more reproducible than the laboratory water-displacement calibration. We conclude that variation in spirometers may limit the reliability of epidemiologic findings, even when these spirometers meet ATS specifications. Frequent calibration checks traceable to an independent standard, and adjustment of individual test results, can reduce measurement error. PMID- 8616560 TI - The effect of the absence of abdominal muscles on pulmonary function and exercise. AB - In order to determine the long-term sequelae of prune belly syndrome (PBS) and whether the absence of abdominal wall musculature impairs exercise performance we studied nine patients 6 to 31 yr of age with PBS. Conventional spirometry, lung volumes, DLCO, and respiratory muscle strength were measured. A progressive 1-min incremental exercise test was performed on a cycle ergometer, and relative abdominal and chest wall displacements were measured by respiratory inductive plethysmography (RIP). Mean values of TLC, FRC, and RV were 94 +/- 12, 88 +/- 13, and 94 +/- 41%, respectively. Mean values of PEFR, FEV1, and FEF25-75 were 83 +/- 24, 92 +/- 23, and 83 +/- 28%, respectively. Maximal expiratory pressures were significantly reduced in seven of nine patients, with marked reduction in four (>3 SD below the mean). Percent predicted maximal VO2 achieved, % maximal work, and % maximal heart rate were 79 +/- 13, 78 +/- 14, and 87 +/- 2%, respectively. All seven subjects with absent abdominal musculature had paradoxical motion of the abdomen during quiet respiration in the erect or sitting position and while exercising. These subjects had synchronous breathing at rest in the supine position. Although the etiology of the relatively low work rates and VO2 achieved was multifactorial, we speculate that the abdominal paradox in these subjects necessitates abnormally large rib cage displacements during exercise, which may be a significant contributing factor to exercise limitation in some of these subjects. PMID- 8616561 TI - The tension-time index of the diaphragm revisited in quadriplegic patients with diaphragm pacing. AB - The fatigue threshold of the human diaphragm, index of its endurance and fatigability, corresponds, during spontaneous breathing, to a tension-time index (TTdi = Pdi/Pdimax x T1/Ttot (i.e., the inspiratory time over the total breath duration) of 0.15 to 0.18. We studied three quadriplegic patients with diaphragm pacing in order to reassess this threshold in patients in whom the contribution of the other respiratory muscles is lacking. Transdiaphragmatic pressure (Pdi) was obtained from the difference between gastric (Pga) and esophageal (Pes) pressures while the electromyograms (EMG) of both hemidiaphragms were recorded with surface electrodes. Four runs at different TTdi were performed on different days in each subject, varying either the Pdi developed per breath, changing the frequency of stimulation, or the T1/Ttot. The time of fatigue was defined when Pdi decreased during the trials by 35% from baseline. No evidence of transmission fatigue (i.e., decline in action potential amplitude) was observed. The TTdi at which fatigue occurred in all the quadriplegic patients was around 0.10 to 0.12, well below the values previously described. After fatigue had occurred, the force recovery during the force-frequency curve was not complete after 2 h at low frequencies, whereas at high frequencies it was fully complete at 30 min. We conclude that when respiration is accomplished only by the diaphragm, without the contribution of the other respiratory muscles, the fatigue threshold is lower than previously reported. PMID- 8616562 TI - The effect of sleep fragmentation on daytime function. AB - Patients with the sleep apnea/hypopnea syndrome suffer from impaired daytime function. This has been attributed to both sleep fragmentation and hypoxemia. To help understand which is casual, we studied the effects of sleep fragmentation alone on daytime function. Sixteen normal subjects were studied on two pairs of two nights. The first night of each pair was for acclimatization, and on the second the subject either slept undisturbed or had sleep fragmented with sound pulses every 2 min. Sound volume and duration was titrated to cause a return to theta or alpha rhythm on the EEG for at least 3 s. Study nights were followed by daytime testing of psychometric function and mood and by a multiple sleep latency test (MSLT) and a maintenance of wakefulness test (MWT). Total sleep time did not differ between study nights (400 +/- 20 SD min undisturbed, 396 +/- 24 min fragmented; p = 0.6). Fragmentation decreased sleep latency on both the MSLT (11 +/- 3, 7 +/- 2 min; p = 0.001) and the MWT (34 +/- 8, 24 +/- 10 min; p<0.001). Energetic arousal (22 +/- 4, 19 +/- 4; p = 0.005) and hedonic tone (29 +/- 4, 27 +/- 4; p = 0.05) decreased after fragmentation. Fragmentation impaired daytime function adjudged by the Trailmaking B (p = 0.05) and PASAT 4-s tests (p<0.03). One night of sleep fragmentation makes normal subjects sleepier during the day, impairs their subjective assessment of mood, and decreases mental flexibility and sustained attention. PMID- 8616563 TI - Influence of treatment on muscle sympathetic nerve activity in sleep apnea. AB - Obstructive sleep apnea (OSA) is a common disorder associated with systemic hypertension, myocardial infarction, stroke, and premature death. Elevated sympathetic tone has been documented previously in OSA and may contribute to the cardiovascular risk. As OSA therapy appears to reduce mortality, we wondered if decreased apnea activity would attenuate the sympathetic hyperactivity of untreated patients. Muscle sympathetic nerve activity (MSNA) was measured during wakefulness via peroneal microneurography in seven patients with documented OSA before and at least 1 mo after compliance-monitored nasal continuous positive airway pressure (CPAP) therapy. Before institution of CPAP therapy, MSNA was high in all patients and decreased after CPAP therapy (baseline versus CPAP: 69.4 +/- 15.3 versus 53.9 +/- 10.5 bursts/min, mean +/- SD; p<0.01). However, the decrease in MSNA was limited to the four patients with the greatest nightly use of CPAP (> or = 4.5 h/night), whereas it remained unchanged in the three patients who were less compliant. There was a direct linear correlation between the decrease in MSNA (bursts/min) and the average hours of CPAP use per night (r = 0.87, p = 0.01). We conclude that in patients with OSA effective reduction in apnea activity with CPAP therapy diminishes the high sympathetic tone present during resting wakefulness. PMID- 8616564 TI - Distinct roles for pneumolysin's cytotoxic and complement activities in the pathogenesis of pneumococcal pneumonia. AB - Pneumolysin, the major Streptococcus pneumoniae cytotoxin, contributes to the early pathogenesis of invasive pneumococcal pneumonia by facilitating intrapulmonary bacterial growth and invasion into the blood. Pneumolysin is a multifunctional toxin, with distinct cytolytic ("hemolytic") and complement activation ("complement") activities that have been mapped to several regions of the molecule. To characterize the specific contributions of pneumolysin's hemolytic and complement properties to the pathogenesis of pneumococcal pneumonia, we compared the in vivo effects of type 2 S. pneumoniae mutant strains, which produce pneumolysins deficient in these activities. The absence of either pneumolysin's hemolytic or complement activities rendered mutant strains less virulent than the wild-type strain during pulmonary infection. Pneumolysin's hemolytic activity correlated with acute lung injury and bacterial growth at 3 and 6 h after endotracheal instillation. In contrast, pneumolysin's complement activity correlated with bacterial growth and bacteremia at 24 h after pulmonary infection. Pneumolysin's complement activity was not associated with the degree of alveolar-capillary injury or recruitment of leukocytes during initial pulmonary infection. However, pneumolysin's complement activity inhibited killing of mutant bacteria in an in vitro complement-dependent neutrophil killing assay. Thus, both pneumolysin's hemolytic and complement activities made specific contributions to the early pathogenesis of pneumococcal pneumonia at different stages of infection and by different mechanisms. PMID- 8616565 TI - Lowering of interstitial fluid pressure will enhance edema in trachea of albumin sensitized rats. AB - Interstitial fluid pressure (Pif) has recently been found to play an important role in edema formation in acute airway inflammation. Because airway inflammation is important in the pathogenesis of asthma, Pif was measured in rat trachea after albumin challenge to rats previously sensitized to chicken egg albumin. In pentobarbital anesthesia (50 mg/kg intraperitoneally) sensitized rats received an intravenous infusion of either saline or albumin, which circulated for 4 min. Circulatory arrest was then induced with saturated KCl intravenously to prevent further edema formation, which will increase Pif and thereby possibly cause an underestimation of an increased negativity of Pif. Pif was measured with sharpened glass capillaries (diameter 3-6 micrometer) connected to a servo controlled counter pressure system. Pif was -1.3 +/- 0.4 mm Hg in controls and 5.8 +/- 0.5 mm Hg in sensitized rats (p < 0.01) after allergen challenge. Airway resistance was measured to verify the occurrence of airway narrowing and increased significantly in sensitized rats after allergen challenge but did not change in controls. The experimental anti-inflammatory drug, alpha-trinositol (D myo-inositol-1,2,6-trisphosphate, 10 mg), given before or after allergen challenge abolished the increased negativity of Pif (p < 0.05), while hydrocortisone (6.25 mg) had no effect. Thus, allergen challenge is associated with a lowering of Pif, which was abolished by alpha-trinositol. PMID- 8616566 TI - Regional variation in iron and iron-binding proteins within the lungs of smokers. AB - The lungs of cigarette smokers are known to contain increased concentrations of extracellular ferritin-bound iron. Reductants present in cigarette smoke may mobilize alveolar ferritin-bound iron, which could then promote oxidative injury to lung cells. Because iron-mediated oxidative injury may be relevant to the pathogenesis of emphysema and lung cancer, which have a predilection for upper lobes, we sought to determine whether concentrations of extracellular iron, ferritin, and transferrin differed in upper and lower lobes of cigarette smokers. Bronchoalveolar lavage (BAL) was performed in the upper and lower lobes of 15 asymptomatic smokers and six healthy nonsmokers. BAL fluid recovered from upper lobes of smokers contained higher concentrations of iron (p < 0.01) and ferritin (p < 0.006) and lower concentrations of transferrin (p < 0.003) compared with the lower lobes. In contrast, BAL fluid recovered from upper and lower lobes of nonsmokers contained much lower concentrations of iron and ferritin, and concentrations were similar in both sites. These findings indicate that, compared with the lower lobes, upper lobes of the lungs of smokers contain higher extracellular concentrations of ferritin-bound iron and decreased concentrations of transferrin. This distribution of lung iron and iron-binding proteins may promote oxidative injury in the upper lobes of smokers. PMID- 8616568 TI - Lymphangioleiomyomatosis: the pathophysiology of diminished exercise capacity. AB - Dyspnea with exertion is nearly always present in patients with pulmonary lymphangioleiomyomatosis, but the mechanisms underlying exercise impairment have not been well defined. Spirometry, lung volumes, lung mechanics, and exercise physiology were performed on a cohort of 16 patients. We determined the relative contribution of airflow limitation, gas exchange abnormalities, and pulmonary vascular abnormalities to the exercise performance achieved. The patients had normal TLC and Vtg, but RV was elevated in 88% of the subjects. A moderate to severe obstructive pattern was present in 69% of the subjects, and the DLCO was reduced, often markedly, in 81% of the subjects. Exercise performance was limited (work load, 68% +/- 6) with abnormalities of ventilatory function and gas exchange present. Strong correlations between overall exercise performance (percent predicted VO2max and maximal work load achieved) and indices of airflow and vascular involvement were present. Poor exercise performance was due primarily to ventilatory limitation. The etiology of this ventilatory limitation appears twofold. First, subjects had a reduced ventilatory ceiling because of airflow limitation. Second, subjects demonstrated an excessive ventilatory response as a result of increased dead-space ventilation thought to be due to disease-associated cystic changes and associated pulmonary vascular dysfunction or destruction. PMID- 8616567 TI - Expression of tumor necrosis factor-receptor superfamily members by lung T lymphocytes in interstitial lung disease. AB - Recently, a novel receptor superfamily has been identified whose members interact with a parallel family of ligands showing homology to tumor necrosis factor (TNF). To investigate the role of these receptor structures in the pulmonary environment, we evaluated the expression of some members of the TNF-receptor (CD27, CD30, CD40, CD95/Fas, CD120a, and CD120b) and TNF-ligand (CD40L, CD70/CD27L, CD30L, and mTNFalpha) superfamilies by bronchoalveolar lavage (BAL) T cells recovered from healthy subjects and patients with interstitial lung disease (ILD). Lung T lymphocytes recovered from control subjects showed a slight expression of CD27 but did not bear CD30, CD40, CD120a, or CD120b antigens. CD27 expression was restricted to normal CD4+ cells. Fas antigen (CD95), which is involved in activation-driven T-cell suicide, and the ligand for CD27 (CD70) were weakly expressed by normal BAL T-cell subpopulations. In patients with sarcoidosis, the majority of pulmonary T lymphocytes were CD4+ cells that expressed low levels of CD27 antigen and an upregulation of CD95 and CD70 molecules. When we characterized lymphocytes accumulating in the lung of patients with HIV infection and hypersensitivity pneumonitis, we demonstrated that T cells accounting for the CD8 alveolitis bore TNF-receptor type 2 (CD120b) at high density and were CD70+ while CD40L, CD30L, or mTNF-alpha expression were not found. The discrete surface expression of the TNF-receptors and TNF-ligands on alveolar T-cell subsets suggests that these molecules play a role in the immune regulatory mechanisms that ultimately lead to the alveolitis in the pulmonary microenvironment of interstitial lung disease. PMID- 8616569 TI - Tuberculosis in HIV-positive patients: cellular response and immune activation in the lung. AB - The host response to Mycobacterium tuberculosis is dependent on the accumulation and activation of cytotoxic and memory CD4+ T cells, resulting in granuloma formation and delayed type hypersensitivity. We characterized the cellular response of radiographically involved lung segments from 17 HIV-positive and 11 HIV-negative patients with acute tuberculosis (TB) using bronchoalveolar lavage (BAL) and compared the response to uninvolved segments, normal control subjects and peripheral blood. In both HIV-positive and HIV-negative patients, radiographically involved segments had significantly increased numbers of total cells per milliliter, percent of neutrophils recovered, and percent of lymphocytes recovered compared with uninvolved segments or normal control subjects, but HIV-positive patients had a lower proportion of lymphocytes in the involved segments than HIV-negative patients with tuberculosis (19 +/- 5% versus 33 +/- 5%; p < 0.05). Lymphocyte subset analysis demonstrated that HIV-positive patients had markedly reduced percentages of CD4+ lymphocytes (CD4+ lymphocytes in HIV-positive TB involved site 25 +/- 6%; HIV-negative TB involved site 73 +/- 2%; p < 0.01) and an increase in the percentage of CD8+ lymphocytes (HIV positive involved site 61 +/- 6% versus HIV negative involved site 19 +/- 3%; p < 0.01). Immunohistochemistry of lung biopsy tissue in five HIV-negative patients showed similar lymphocyte subset profiles as BAL, indicating that BAL reflects cell populations in tissue granulomas. BAL lymphocytes from four HIV-positive and four HIV-negative tuberculosis patients demonstrated immune activation by staining with a murine antibody to TIA-1, a cytoplasmic protein associated with cytotoxicity and apoptosis (HIV positive 48 +/- 6%, HIV negative 31 +/- 7%, normals 11 +/- 5%). Steady state mRNA for gamma-interferon was decreased in four HIV-positive patients when compared with four HIV-negative patients. IL-8 production was comparable in HIV-negative and HIV-positive patients with focal disease but reduced in two patients with miliary tuberculosis. We conclude that HIV-positive patients with+ tuberculosis have a reduced enrichment and activation of immune cells in the lung, and this failure of a CD4+ alveolitis limits an effective immune response. PMID- 8616570 TI - Presentation of AIDS-related pulmonary Kaposi's sarcoma diagnosed by bronchoscopy. AB - Kaposi's sarcoma (KS) is the most common neoplasm in persons infected with the human immunodeficiency virus (HIV). However, information about the presenting features of pulmonary KS is limited. To describe the clinical, laboratory, and radiographic features of pulmonary KS, medical records and chest radiographs of 168 patients with pulmonary KS diagnosed by bronchoscopy during a 7-yr period were reviewed. All of the patients were HIV-seropositive males, of whom 95% identified homosexual or bisexual sex as a risk factor for HIV infection. The median CD4 lymphocyte count was 19 cells/microliter. The most common symptoms were cough, dyspnea, and fever. Patients with a concurrent opportunistic pneumonia had a higher median serum lactate dehydrogenase (LDH) concentration than did those with pulmonary KS alone (p<0.001). The most common chest radiograph findings were bronchial-wall thickening, nodules, Kerley B lines, and pleural effusions. The presence of granular opacities or cystic spaces usually indicated concomitant Pneumocystitis carinii pneumonia (p < 0.001). Twenty-six patients (15.5%, 95% CI = 10.2% to 20.8%) had pulmonary KS in the absence of mucocutaneous involvement. The presentation of pulmonary KS is characterized by symptoms that cannot be distinguished from those of a superimposed infection. An elevated serum LDH concentration or a chest radiograph with granular opacities or cystic spaces should raise the suspicion of concurrent opportunistic pneumonia. The diagnosis of pulmonary KS should be considered in an HIV-infected homosexual or bisexual male with respiratory symptoms even in the absence of mucocutaneous lesions. PMID- 8616571 TI - Safety aspects of local endobronchial allergen challenge in asthmatic patients. AB - Local endobronchial allergen challenge is being increasingly used to investigate the role of allergic inflammation in asthma. However, little information is available about the safety of this procedure and the changes induced in airway physiology. BAL and biopsy were performed at 10 min and at 4 to 6 h, or 24 h after segmental allergen challenge in 49 patients with atopic asthma. Two hours after challenge, FEV1 was reduced from 97.6 +/- 13.9 (mean +/- SD) to 83.4 +/- 21.7% predicted. FEV1 remained reduced at 4 to 6 h (87.7 +/- 20.4%), but it had nearly returned to baseline by 24 h (93.2 +/- 14.0%). When endobronchial challenge was combined with BAL and biopsy, the initial fall in FEV1 was slightly greater (from 101.8 +/- 14.2 to 78.5 +/- 13.6%). Bronchial responsiveness to methacholine was measured in 10 subjects, and it showed a twofold increase 24 h after local challenge and lavage. Significant changes in FEV1 and methacholine PC20 were still detectable 72 h after challenge. Widespread wheezing occurred in 29% of the subjects, but none of the them had to be admitted to hospital. We conclude that local endobronchial allergen challenge, although producing measurable changes in airway physiology, is in general well tolerated and is an acceptable method to investigate airway pathophysiologic processes in patients with mild to moderate asthma. PMID- 8616572 TI - Increased MCP-1, RANTES, and MIP-1alpha in bronchoalveolar lavage fluid of allergic asthmatic patients. AB - Chemokines are cytokines that induce chemotaxis of inflammatory cells. We studied the presence of chemokines in bronchoalveolar lavage fluid (BALF) obtained from nine allergic asthmatic patients and six nonsmoking normal individuals. The cells were pelleted, and ribonucleic acid (RNA) was extracted by using RNAzol B. BALF was assayed for monocyte chemoattractant protein-1 (MCP-1), regulated upon activation in normal T cells, expressed, probably secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin-8 (IL-8) by enzyme linked immunosorbent assay (ELISA). The levels of MCP-1, RANTES, and MIP-1alpha were significantly higher in the asthma patients than in the control subjects (p<0.04). The concentrations of RANTES and MCP-1 correlated with the lymphocyte count in the BAL specimens (r = 0.61 and 0.68, respectively). BALF showed eosinophil chemotactic activity in vitro that was blocked by anti-RANTES and anti MCP-3 antibodies. The total cellular RNA was reverse-transcribed and the complementary deoxyribonucleic acid (cDNA) was amplified with the polymerase chain reaction (PCR) for MCP-1, MCP-3, RANTES, MIP-1alpha, IL-8, and beta-actin. We found that messenger ribonucleic acids (mRNAs) for MCP-1, MCP-3, RANTES, MIP 1alpha, and IL-8 were produced by BAL cells from most asthmatic and normal subjects. We conclude that chemokines are produced in the airways, and that an increased recovery of MCP-1, RANTES, and MIP-1alpha is observed in allergic asthmatic patients. PMID- 8616573 TI - Aerosolized human neutrophil elastase induces airway constriction and hyperresponsiveness with protection by intravenous pretreatment with half-length secretory leukoprotease inhibitor. AB - This study was designed to determine the effects of inhaled human neutrophil elastase (HNE) on airway constriction and airway responsiveness, and to examine the protection by an intravenous recombinant half-length secretory leukoprotease inhibitor, r1/2SLPI in guinea pigs. Aerosol inhalation of HNE (250 microgram/ml, for 3 min) caused a transient but significant airway constriction, in which lung resistance (RL) increased from 194 +/- 18 (mean +/- SEM) to 461 +/- 42 cm H2O/L/s (p < 0.001). Thirty minutes after the end of HNE inhalation, airway responsiveness to intravenous 5-hydroxytryptamine (5-HT) was significantly increased. The provocative dose causing a 200% increase in RL (PD200) was significantly decreased from 10.0 +/- 1.2 to 6.5 +/- 0.8 microgram/kg (p < 0.001). Forty-five minutes after the end of HNE inhalation, total cells in bronchoalveolar lavage fluid (BALF) were significantly increased (p < 0.05). Histologic study of intrapulmonary bronchi demonstrated an acute inflammatory response characterized by damage to the epithelium, airway obstruction by mucus plugs, and recruitment of mononuclear and polymorphonuclear cells to the bronchial epithelium. r1/2SLPI (30 mg/kg) injected 5 min before the initiation of HNE inhalation significantly inhibited the airway constriction (p < 0.05), the airway hyperresponsiveness (p < 0.01), and the increase of cells in BALF (p < 0.05). The present data suggest that HNE plays a role in the induction of airway constriction and airway hyperresponsiveness in various inflammatory lung diseases with pulmonary neutrophil infiltration, such as chronic obstructive pulmonary diseases (COPD) and possibly bronchial asthma. r1/2SLPI may be useful as an antiprotease treatment. PMID- 8616574 TI - Hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium induced by endotoxin. AB - To elucidate the mechanisms of epithelial mucus hypersecretion in upper respiratory airway inflammation, we produced hypertrophic and metaplastic changes of goblet cells in rat nasal respiratory epithelium by intranasal instillation of endotoxin. Significant increase of hypertrophic goblet cells was induced in the septal epithelium transversely sectioned at the level of incisive papilla at 24 h after the intranasal instillation of 0.1 mg of endotoxin. This change was completed after 3 d of endotoxin instillations and recovered by normal epithelium 7 d after the last instillation. Total cell number and the number of basal and ciliated cells counted over 2 mm of basal lamina did not change; however, the number of goblet cells increased and that of nongranulated secretory cells decreased time-dependently after endotoxin instillations. Mitotic rates examined after a 6-h colchicine metaphase blockade were very low at any time point studied, and cell division did not play a major role in this process. These results indicate that endotoxin induces hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium rather than a hyperplastic change, and this metaplasia is produced by direct conversion of nongranulated secretory cells into the goblet cells. Histochemical examination of this epithelium revealed that most of the mucus produced by these goblet cells was sulfomucin. Intraperitoneal injection of antirat neutrophil antiserum or cyclophosphamide depleted circulating blood neutrophils. Endotoxin-induced changes of goblet cells were significantly inhibited in these neutrophil-depleted rats, and intranasal instillation of elastase also induced hypertrophic and metaplastic changes of goblet cells. PMID- 8616575 TI - Polymerase chain reaction detection of Mycobacterium tuberculosis in formalin fixed tissue. AB - Use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue would be of great diagnostic value. However, formaldehyde has been reported to decrease the efficiency of amplification by structurally altering the polynucleotide chain. We sought to determine the ability of the PCR assay to detect M. tuberculosis in formalin fixed, paraffin-embedded tissue in a mouse experimentally infected with the H37Rv strain of M. tuberculosis. Lung tissue from the infected mouse was cultured to determine the number of organisms per gram of tissue. The remaining lung tissue was divided into eight portions, seven of which were fixed in 10% neutral buffered formalin for 24-h intervals over periods lasting from 1 to 7 d, and a control portion was placed in isotonic saline. The tissue samples were then paraffin-embedded, and sections were obtained from each tissue block for PCR analysis. We show that the PCR assay can detect as few as nine organisms in a 5 micron section of tissue, and that up to 7 d of fixation in 10% neutral buffered formalin has a negligible effect on the assay. The PCR assay can detect low numbers of M. tuberculosis organisms in formalin-fixed, paraffin-embedded tissue. PMID- 8616576 TI - Endobronchial ultrasound-guided needle aspiration of mediastinal adenopathy. AB - We conducted a randomized, controlled trial to prospectively confirm that ultrasound-directed transbronchial needle aspiration (USTBNA) results in: (1) improved sensitivity for detecting lymph nodes involved with neoplasm, and (2) a decreased number of aspirates needed to achieve a diagnosis as compared with standard transbronchial needle aspiration (TBNA). The study was conducted in a tertiary medical center on patients undergoing fiberoptic bronchoscopy in the evaluation of enlarged mediastinal lymph nodes. USTBNA or TBNA were followed by rapid, on-site cytopathology examination of the collected specimens. Measurements included the (1) age and sex of the patient, prior diagnosis of cancer, nodal short-axis diameter and node location as determined by computerized tomography (CT), and endobronchial abnormalities at bronchoscopy; (2) number, order, and location of transbronchial aspirates and results of on-site evaluation; (3) results of surgical exploration in patients with negative transbronchial needle aspiration; (4) sensitivity, specificity, and diagnostic accuracy of USTBNA and TBNA; (5) number of aspirates required for successful lymph node aspiration as well as for a diagnosis of cancer for both USTBNA and TBNA; and (6) multiple logistic regression analysis to determine the significance of combinations of clinical predictors and needle aspirate results. Eighty-two bronchoscopic examinations were performed on 80 patients. We found no significant difference between USTBNA and TBNA in sensitivity (82.6% versus 90.5%, respectively), specificity (100% for both), or diagnostic accuracy (86.7% versus 91.7%, respectively). The sensitivity, specificity, and diagnostic accuracy of USTBNA and TBNA were similarly high, regardless of node location (paratracheal or subcarinal). A decrease in the number of aspirates required for lymph node sampling approached statistical significance for all USTBNAs as compared with TBNAs (2.03 +/- 0.19 versus 2.62 +/- 0.25, p = 0.06), but this was not demonstrated for the number required to confirm cancer (1.95 +/- 0.47 versus 2.68 +/- 0.21, p = 0.17). The number of aspirates to successful lymph node aspiration decreased with USTBNA versus TBNA in paratracheal lymph nodes (2.00 +/- 0.20 versus 2.91 +/- 0.34, p = 0.03), but not to a diagnosis of cancer (1.93 +/- 0.25 versus 3.00 +/- 0.58, p = 0.11). No difference was seen in the number of aspirates for subcarinal nodes. The number of TBNA attempts for paratracheal lymph node sampling was inversely correlated with node size (r = 0.48, p = 0.02). No such relation was seen with USTBNA of paratracheal nodes (r = 0.09, p = 0.66), TBNA of subcarinal nodes, or USTBNA of subcarinal nodes. A similar relation was seen between the number of aspirates to a diagnosis of cancer. On multiple logistic regression analysis, a positive transbronchial aspirate was associated only with a larger lymph node and history of prior cancer. We conclude that: (1) in the setting of on-site cytopathology, transbronchial needle aspiration has a high sensitivity, specificity, and diagnostic accuracy in the evaluation of enlarged mediastinal lymph nodes suspected of harboring malignancy; (2) mediastinal anatomy, including vascular structures and lymph nodes, is clearly imaged with endobronchial ultrasonography; (3) a greater short-axis diameter of the mediastinal lymph node and history of a prior malignancy increase the likelihood of a positive transbronchial aspiration; (4) USTBNA exhibits a similarly high diagnostic yield to TBNA in the setting of rapid on-site cytopathology evaluation; (5) USTBNA decreases the number of aspirates required for paratracheal lymph node sampling, which may be particularly useful in sampling smaller paratracheal nodes or at institutions that do not utilize rapid on-site cytopathology evaluation. PMID- 8616577 TI - Balance between alveolar macrophage IL-6 and TGF-beta in lung-transplant recipients. Marseille and Montreal Lung Transplantation Group. AB - Acute inflammation in the lung is characterized by a phase of tissue injury followed by a phase of tissue repair. When the latter is excessive, fibrosis occurs. Alveolar macrophages (AM) can produce cytokines involved in both phases of acute lung inflammation, notably interleukin-6 (IL-6), involved in injury and transforming growth factor-beta (TGF-beta), mediating repair. We hypothesized that AM were activated in both phases, and studied IL-6 and TGF-beta production by AM during complications of lung transplantation, acute rejection (AR), and cytomegalovirus pneumonitis (CMVP). In addition, we analyzed these cytokines in bronchiolitis obliterans (BO), a fibrotic complication of lung transplantation linked to previous AR and CMVP. At the onset of AR and CMVP, IL-6 secretion increased, whereas AM TGF-beta content was increased, but not its secretion. In contrast, with time, IL-6 reached control value whereas TGF-beta secretion rose significantly. In BO, IL-6 was not oversecreted, but TGF-beta increased, notably before functional abnormalities occurred. These results show that during acute complications of lung transplantation, AM display an early activation with oversecretion of IL-6, which is involved in tissue injury, counterbalanced by a late activation in which TGF-beta predominates, mediating tissue repair. The results provide new insights into the pathogenesis of BO, which is linked to acute complications of lung transplantation through this biphasic AM activation. PMID- 8616578 TI - Cytokine production at the site of disease in chronic eosinophilic pneumonitis. AB - Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. We hypothesized that cytokine(s) produced in the disease sites are implicated in the pathophysiology of CEP. We studied peripheral blood and bronchoalveolar lavage fluids (BALF) obtained from two lung segments of a patient with CEP. Seventy times more eosinophils were found in the BALF from an involved lung segment (showing patchy opacification on a chest roentgenogram) than from an uninvolved segment. The eosinophil-active cytokines interleukin-5 (IL-5), IL-6, and IL-10 were strikingly elevated in the BALF from the involved lung segment, whereas no or minimal levels of these cytokines were detectable in the BALF from the uninvolved segment or serum, respectively. Leukocytes in the involved lung segment, but not those in peripheral blood, expressed messenger ribonucleic acid (mRNA) for IL-5, IL-6, and IL-10. In contrast, IL-2, IL-3, IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) were not detected in any sample. These findings suggest that increased production of several cytokines, such as IL-5, IL-6, and IL-10, in the involved lung segment, but not in the uninvolved lung segment or peripheral blood, is a critical pathophysiologic feature of CEP. PMID- 8616579 TI - Interferon-alpha affects the immune response in post-transplant lymphoproliferative disorder. AB - Post-transplant lymphoproliferative disorder (PTLD) is associated with Epstein Barr virus (EBV) and characterized by fever, lymphadenopathy, and graft dysfunction. We describe the clinical course of an EBV seronegative 11-yr-old boy who underwent double lung transplantation and subsequently developed PTLD in the graft. A reduction in immunosuppression and the addition of acyclovir did not result in improvement. Treatment with interferon-alpha (IFN-alpha), however, led to dramatic clinical, radiographic, and histologic improvement. Semiquantitative measurements of cytokine mRNA in his bronchoalveolar lavage cells prior to therapy with IFN-alpha revealed high levels of IL-4 and IL-10 mRNA, which decreased significantly with treatment. We speculate that the beneficial effect of IFN-alpha in the treatment of PTLD is directly related to the inhibition of type 2 helper (Th2-like) T-cells. PMID- 8616580 TI - Testing for human immunodeficiency virus infection in patients with tuberculosis. AB - To investigate the utility of testing all tuberculosis patients for HIV infection in Los Angeles, we prospectively evaluated 183 hospitalized patients with acid fast smear-positive tuberculosis who gave no history of HIV infection. HIV serologic testing was performed, and the presence of risk factors for HIV infection was assessed by interview and review of the medical record. Thirty three patients (18%) were infected with HIV. Based on data obtained by interview, 124 (68%) of 183 patients had at least one risk factor for HIV infection. The most common risk factors were related to heterosexual transmission of HIV (prostitute contact, multiple sex partners, and a history of sexually transmitted disease). These risk factors were frequently not documented in the medical record. Among the 33 patients who were infected with HIV, 30 were identified by interviewing as having significant risk factors. We concluded that most tuberculosis patients in Los Angeles have risk factors for HIV infection and that systematic questioning does not identify risk factors in all HIV-infected tuberculosis patients. These data support current recommendations to screen all tuberculosis patients for HIV infection. PMID- 8616582 TI - A nitric oxide synthase inhibitor impairs memory storage in mice. AB - Posttraining administration of the L-enantiomer of the competitive inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME, 3-100 mg/kg, ip), impaired 48-h retention of a one-trial step-through inhibitory shock avoidance task in male Swiss mice. The effects were dose-dependent and were not observed when the D-enantiomer (D-NAME, 3-100 mg/kg, ip) was injected instead of L-NAME. Retention latencies of mice that had not received a footshock during training were not affected by L-NAME. The memory impairment produced by L-NAME was time-dependent, suggesting an action on memory storage. The effects of L-NAME on memory were overcome by the injection of L-(but not D-)arginine (300 mg/kg, ip) along with the inhibitor. Considered together, these findings suggest that the L-arginine/nitric oxide pathway may be involved in memory storage of an inhibitory avoidance response in mice. PMID- 8616581 TI - Aerosolized cyclosporine in lung recipients with refractory chronic rejection. AB - This study evaluated aerosolized cyclosporine as rescue therapy for lung transplant recipients with unremitting chronic rejection. Nine patients with histologic active obliterative bronchiolitis and progressively worsening airway obstruction refractory to conventional immune suppression received aerosolized cyclosporine. Improvement in rejection histology was seen in seven of nine patients. We compared the changes in the FVC and FEV1 over time using linear regression analysis in these seven histologic responders and nine historical control patients. During the pretreatment period for both the experimental and control groups, the FVC and FEV1 declined at comparable rates. After aerosolized cyclosporine there was stabilization of pulmonary function, whereas in the controls there was continued decline. Cyclosporine blood levels were less than 50 ng/ml 24 h after an aerosolized dose of 300 mg in five patients receiving oral tacrolimus. Nephrotoxicity, hepatotoxicity, and a greater than expected rate of infection was not observed. This study suggests that aerosolized cyclosporine is safe and may be effective therapy for refractory chronic rejection in lung transplant recipients. PMID- 8616583 TI - High level of footshock during inhibitory avoidance training prevents amnesia induced by intranigral injection of GABA antagonists. AB - Disruption of synaptic activity of a number of cerebral structures (e.g., neostriatum, amygdala, and thalamus) produces marked deficits in retention of instrumentally conditioned behaviors. When animals are given a relatively high number of training trials or high intensities of footshock during learning, however, such disruption is considerably less effective. Since there is a close anatomical and functional relationship between the neostriatum and the substantia nigra, it was of interest to determine whether enhanced training with a high level of footshock would prevent the reported amnesic state induced by injections of GABA antagonists into the latter structure. Rats were trained in a one-trial inhibitory task, using 0.2 or 0.4 mA, and then injected with microgram quantities of picrotoxin or bicuculline into the substantia nigra and posterior region of the zona incerta; retention was measured 24 h later. Only those groups that had been injected into the nigra and trained with 0.2 mA showed amnesia. These results support the hypotheses that (a) the normal activity of a set of structures is essential for the development of memory consolidation and (b) after an enhanced learning experience these structures may participate in memory consolidation, but are not necessary for the occurrence of this process. PMID- 8616584 TI - Enhancement of the post-training cholinergic tone antagonizes the impairment of retention induced by a nitric oxide synthase inhibitor in mice. AB - The present experiments examined the role of the central cholinergic system in the memory impairment induced by post-training administration of a nitric oxide synthase (NOS) inhibitor in mice. Male Swiss mice received a one-trial inhibitory avoidance training (0.8 mA, 50 Hz, 1-s footshock) followed immediately by an ip injection of the NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 mg/kg). Retention (cut-off time, 300 s) was tested 48 h after training. The administration of L-NAME results in memory impairment for the inhibitory avoidance task. The effects of L-NAME (100 mg/kg, ip) on retention were reversed in a dose-related manner by the centrally acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, sc) administered 30 min after the NOS inhibitor. Further, L-NAME (100 mg/kg, ip)-induced memory impairment was completely antagonized by the centrally acting muscarinic cholinergic agonist oxotremorine (OTM; 25, 50, or 100 microg/kg, sc) when given 30 min after L-NAME. The peripherally acting anticholinesterase neostigmine (150 microg/kg, sc) did not modify the memory-impairing effects of L-NAME. These findings suggest that the memory impairment following post-training administration of a NOS inhibitor is mediated, at least in part, by a reduction of the activity of central muscarinic cholinergic mechanisms and are consistent with our previous view that nitric oxide may be involved in post-training neural processes underlying the storage of newly acquired information. PMID- 8616585 TI - Alteration of auditory cortex activity with a visual stimulus through conditioning: a 2-deoxyglucose analysis. AB - In two experiments, the 2-deoxyglucose metabolic mapping technique was used to examine the hypothesis that a stimulus of one modality (a light) will begin to activate the sensory cortex of a stimulus of another modality (a tone) with which it has been repeatedly paired. Adult gerbils received repeated presentations of either a light or the light paired with a tone known to affect 2DG labeling patterns in the auditory cortex. Intermittent footshock was included on a pseudo random basis to maintain arousal in the subjects. One day after training, each gerbil was injected with 2DG and either received repeated presentations of the light only or was simply exposed to the training context. Analysis of the auditory cortex revealed no differences in overall metabolic activity of the auditory cortex between the groups. However, in both experiments, the light that was previously paired with the tone changed the relative activity of the cortical subfields compared to the light not previously paired with the tone. Specifically, the results indicate greater activity in the anterior auditory field (AAF-Experiments 1 and 2) and the posterior fields (DPVP-Experiment 2) relative to the primary field AI in response to the light that was previously paired with the tone during training. Gerbils either only placed in the context during the 2DG session or that received unpaired presentations of the light and tone during training did not show this shift in relative labeling between the subfields. Because no differences in overall activity of the auditory cortex were found, we conclude that the shift in relative labeling between the subfields reflects, on average, both an increase in activity of fields AAF and DPVP and a concomitant decrease in AI activity in response to the light stimulus. The results have implications for our understanding both of brain learning mechanisms in general and the potential functions of auditory cortex subfields in particular. PMID- 8616586 TI - Pavlovian conditioning to a diazepam cue with yohimbine as the unconditional stimulus. AB - On multiple occasions, rats were administered diazepam (2.0 mg/kg, ip) followed 30 min thereafter by yohimbine hydrochloride (2.5 or 5. 0 mg/kg) or isotonic saline (forward conditioning groups). Three additional groups (backward conditioning controls) were given equivalent injections, but in reverse order. After eight such pairings, the effects of a single injection of diazepam on motor performance (balancing on a rotating drum) was assessed. Rats that had received either dose of yohimbine during forward conditioning trials maintained their balance longer than the saline controls. After four additional conditioning trials, the animals' activity patterns in a plus-maze screening test for anxiolytics were examined. Placed into the maze after a single test injection of isotonic saline, the behavior of all groups was virtually identical: less than 16% of total entries into or time spent in the four arms of the maze was spent in the two "open" arms (unprotected by surrounding walls). When tested in the maze again, but 35 min after a single injection of diazepam, the groups that had received diazepam but not yohimbine during the conditioning phase exhibited the expected increase in open-arm activity, and equivalent increases were found in backward conditioning groups. However, the group previously conditioned with 2.5 mg/kg of yohimbine following diazepam also showed an increased open-arm activity when tested with diazepam alone, but it was significantly greater than that seen in the control group. In contrast, the group conditioned with 5.0 mg/kg yohimbine following diazepam exhibited no effect of diazepam upon their plus maze activity; consequently, these animals spent less time in the open arms than either of the other groups. Yohimbine alone normally decreases open-arm activity (a putative "anxiogenic" effect) in a linear dose-dependent fashion. The fact that it had a bidirectional conditional effect on the diazepam cue drug demonstrates that a conditional response in drug --> drug conditioning cannot always be predicted on the basis of the behavioral response to the signaled drug. Consideration is given to possible reasons for these effects of diazepam --> yohimbine pairings in terms of the known neuropharmacological properties of yohimbine. PMID- 8616587 TI - Involvement of glutamate receptors, protein kinases, and protein synthesis in memory for visual discrimination in the young chick. AB - Two-day-old chicks were trained to discriminate between edible chick crumbs and arrays of colored beads glued to the floor of their cage. Normal chicks learned this task within a few minutes and retained it for at least 24 h. The role of several biochemical systems known to be required for other forms of early learning in the chick was explored in this task. Antagonists and inhibitors of these systems were used in the doses known to produce amnesia in a related passive avoidance learning model. Drugs were injected intracerebrally just before training, and retention was tested at various times subsequently. The protein synthesis inhibitor anisomycin (240 nmol/chick) was without effect on retention at 30 min posttraining, but chicks were amnestic at 3 and 24 h. The protein kinases inhibitors melittin (1.2 nmol/chick) and 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine hydrochloride (100 nmol/chick) were without effect on retention at 30 min posttraining but were amnestic by 3 h. While these effects are similar to those found for one-trial passive avoidance training, neither the N-methyl-D aspartate receptor antagonists (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (up to 15 nmol/chick) or DL-2-amino-5 phosphonovalerate (1.3 nmol/chick), both of which are amnestic for passive avoidance, nor the non-NMDA-glutamate receptor antagonist 6-cyano-7 nitroquinoxaline-2, 3,-dione (4 nmol/chick) were amnestic for the visual discrimination task. By contrast, the metabotropic glutamate receptor blocker (RS)- alpha-methyl-4-carboxyphenylglycine (300 nmol/chick) injected 5 min pretraining resulted in amnesia at 3 h posttraining. The implications of these findings for the putative "memory consolidation cascade" are discussed. PMID- 8616588 TI - Marked retrograde and anterograde amnesia of a visual discrimination task in rats with selective lesions of the perirhinal cortex. AB - Damage to the temporal cortex (TC), the lateral entorhinal cortex (LEC), or their interconnections has disruptive effects on visual memory. The fiber connections between TC and LEC are relayed in the perirhinal cortex (PC) or in the adjacent white matter of PC. PC seems to make up a particularly important structure for mnemonic processing. The purpose of the present study was to examine whether selective PC lesions might affect retroactive or proactive memory, since TC/LEC transections can cause both retrograde and anterograde amnesia. The results show that both PC and TC/LEC lesions impair retroactive memory to similar degrees (Experiment 1). However, PC lesions yielded a slightly stronger impairment of both acquisition and retention in the proactive paradigm than TC/LEC lesions (Experiment 2). These findings give support to the notion that PC plays an important role in formation of memory. PMID- 8616589 TI - Effects of posttraining administration of glucose on retention of a habituation response in mice: participation of a central cholinergic mechanism. AB - Male Swiss mice were allowed to explore a novel environment, provided by an open field activity chamber, for 10 min. The procedure was repeated twice with a 24-h interval. The difference in the exploratory activity between the first (training) and the second (testing) exposures to the chamber was taken as an index of retention of this habituation task. Posttraining intraperitoneal administration of glucose (10-300 mg/kg) enhanced retention in a dose-related manner, although only the dose of 30 mg/kg of glucose produced significant effects. Thus, the dose response curve adopted an inverted U-shaped form. Glucose (30 mg/kg) given to untrained mice did not modify their exploratory performance when recorded 24 h later. The effects of glucose on retention were time-dependent, suggesting an action on memory storage. The memory-improving actions of glucose were prevented by the simultaneous administration of both the central acting muscarinic cholinergic antagonist atropine (0.5 mg/kg) and by the central acting nicotinic cholinergic antagonist mecamylamine (5 mg/kg). In contrast, neither methylatropine (0.5 mg/kg), a peripherally acting muscarinic receptor blocker, nor hexamethonium (5 mg/kg), a peripherally acting nicotinic receptor blocker, prevented the effects of glucose on retention. Low subeffective doses of glucose (10 mg/kg) and the central anticholinesterase physostigmine (35 microg/kg), but not neostigmine (35 microg/kg), given together, act synergistically and facilitated retention. We suggest that glucose modulates memory storage of one form of learning elicited by stimuli repeatedly presented without reinforcement, probably through an enhancement of brain acetylcholine synthesis and/or its release. PMID- 8616590 TI - Developmental emergence of long-term memory for sensitization in Aplysia. AB - Adult Aplysia exhibit both short-term and long-term memory for sensitization in the gill and siphon withdrawal reflex. Previous developmental studies showed that short-term memory for sensitization emerges late in juvenile development (stage 12; Rankin and Carew, 1988; Wright et al., 1991). In the present study, we examined the development of long-term memory for sensitization. Long-term sensitization of the siphon withdrawal reflex was quantified as an increase in mean response duration observed 20-24 h after receiving a training regime of one or more 90-min sessions of electrical shock to the tail. In the first three experiments we assessed the capacity for long-term sensitization in adults and in juveniles of stages late 12 and early 12. Animals in all three age classes showed long-term sensitization. In a fourth experiment we simultaneously trained and tested both early stage 12 animals and stage 11 animals with identical stimulus parameters that were scaled down to a level appropriate to the smaller stage 11 animals. Under these conditions, the early stage 12 animals demonstrated long term sensitization, while the stage 11 animals still showed no evidence of long term sensitization. These results indicate that long-term sensitization first emerges at early stage 12, which is the same developmental stage in which short term sensitization first emerges. Although these behavioral data do not elucidate underlying mechanisms, the fact that short-term and long-term memory emerge according to the same developmental timetable is consistent with the possibility that these two forms of memory may share at least some common mechanistic features. PMID- 8616591 TI - Retrograde enhancement of human kinesthetic memory by alcohol: consolidation or protection against interference? AB - Alcohol intake is known to impair memory in animals and humans. However, five studies reported that "medium" (0.05 ml/kg body weight) to "high" (1.0 ml/kg body weight) doses of alcohol improved memory when drunk immediately after initial learning of verbal or visual material. It was proposed that alcohol brought about this retroactive facilitation either through enhanced consolidation of memory traces or by protecting against retroactive interference. The present double blind study compared the performances of an alcohol and a placebo group on a kinesthetic memory task before alcohol or placebo intake and at retest 1 h after consumption. A second experiment was identical to the first except that all subjects carried out two trials on a T maze in the hour between testing and retesting. The alcohol group in the first experiment performed significantly better than the placebo group at retest (p < .05) but this was not the case in the second experiment. Alcohol therefore enhanced performance on the kinesthetic memory task in the first experiment but may not have protected against the moderate interference from the T maze in the second. The low levels of alcohol could have had a stimulant effect on trace consolidation, perhaps via raised blood glucose levels. PMID- 8616592 TI - Stria terminalis lesions attenuate memory enhancement produced by intracaudate nucleus injections of oxotremorine. AB - The present study examined the role of the stria terminalis in modulating the memory enhancement produced by posttraining intracaudate nucleus injection of oxotremorine. Male Sprague-Dawley rats with either sham operations or bilateral lesions of the stria terminalis (ST) were trained on a one-trial inhibitory avoidance task and received a unilateral posttraining intracaudate injection of either a buffer vehicle or the cholinergic agonist oxotremorine (0.3 microg/0.5 microl) into a medial region of the caudate nucleus innervated by the ST. Intracaudate injection of oxotremorine improved memory in sham-operated rats. Although ST lesions did not affect retention in rats given intracaudate injections of the buffer vehicle, ST lesions attenuated the memory enhancement produced by posttraining intracaudate injection of oxotremorine. In view of anatomical evidence indicating that amygdalostriatal projections are nonreciprocal, the present findings suggest that amygdala output via the ST is essential for memory enhancement produced by intracaudate injection of oxotremorine. PMID- 8616593 TI - Effects of cerebroventricle administration of acidic fibroblast growth factor on conditioned taste aversion learning in rats. AB - Wistar strain rats were given acidic fibroblast growth factor (aFGF) or denatured aFGF into their cerebroventricle before taste aversion conditioning for saccharin solution. Animals administered with aFGF showed significantly lower aversion threshold for saccharin at the 1st day and preference ratios for saccharin vs distilled water at the 4th, 6th, and 7th day after the conditioning than those administered with denatured aFGF. These results suggests that aFGF in the cerebrospinal fluid facilitates acquisition of the conditioned taste aversion learning. PMID- 8616594 TI - Effects of feeding on conditioned avoidance responses in rats. AB - Possible effects of feeding on learning were studied by comparing learned avoidance rates among three groups of Wistar rats that were given a diet at 1 h (1-h pretest group) and 5 h (5-h pretest group) before and immediately after (post-test group) the conditioned avoidance test. Learned avoidance rates during eight test sessions were higher in the order of the 1-h pretest, post-test, and 5 h pretest groups. This suggests that both pre- and post-test (training) feeding facilitates acquisition of conditioned avoidance learning presumably based on neurohumoral processes influenced by available glucose concentrations. PMID- 8616595 TI - Ethanol withdrawal hyperexcitability in vitro is selectively decreased by a competitive NMDA receptor antagonist. AB - Hippocampal slices were prepared immediately after withdrawal from chronic ethanol in vivo. The decreases in thresholds for production of single and multiple population spikes seen after the ethanol treatment were not evident when CGP39551 was included in the perfusion medium at 20 microM. The decrease in paired pulse potentiation seen during ethanol withdrawal, however, was not prevented by CGP39551. For comparison, the effects of CGP39551, at the same concentration, were examined on the changes in field potentials seen in control slices when the magnesium concentration in the bathing medium was lowered to 250 microM. The decreases in thresholds for multiple population spikes produced by the lowered magnesium were prevented, but not other changes including decreases in single spike thresholds. In addition, this 20 microM concentration of CGP39551 did not prevent epileptiform activity, measured by decreases in thresholds for production of single and multiple population spikes caused by addition of the GABAA antagonist, bicuculline, to control hippocampal slices. PMID- 8616596 TI - Age and the effects of 2-D,L-amino-5-phosphonovalerate in an area of the chick forebrain which is essential for early learning. AB - The intermediate, medial part of the hyperstriatum ventrale (IMHV) is a region of the avian forebrain which is known to be essential for early learning in the domestic chick. The IMHV in an in vitro slice preparation displays two forms of synaptic plasticity. The incidence of both varies with age and is maximal between 3 and 5 days post-hatch. Since NMDA receptors are critical for at least one of these plasticities, we have investigated the relationship between age and the contribution of NMDA receptors to the field response evoked by local, low frequency stimulation and have found that the magnitude of the NMDA-dependent component of the response varies with age peaking between 3 and 5 days post hatch. Spontaneous neural activity, recorded intracellularly, can be completely and reversibly silenced by NMDA receptor blockade and the incidence of spontaneous activity also varies with age, peaking between 3 and 5 days. These results suggest that the IMHV contains NMDA receptors which can be activated near resting membrane potential. Either the efficiency or the numbers of these receptors is maximal at a specific point in development and their peak activity coincides with a peak in synaptic plasticity. These characteristics are similar to those reported for young mammals. PMID- 8616597 TI - Halothane, but not alpha-chloralose, blocks potassium-evoked cortical spreading depression in cats. AB - The effects of two anesthetics, halothane and alpha-chloralose, on induction of spreading depression and on extracellular glutamate elevation after intracortical potassium administration were investigated in artificially ventilated (30% oxygen/70% nitrous oxide) cats. High potassium concentrations were achieved using either direct KCl injections (7 microliters, 150 mM via a micropipette) or microdialysis by supplementing 100, 300 or 500 mM KCl, respectively, for 10 min to the perfusion solution (Ringer's). Changes of the cortical DC potential were recorded adjacent (1-2 mm: electrode DC1) and distant (6-7 mm: electrode DC2) to the injection site. Either under halothane (0.75% in the respiratory gas mixture) or under alpha-chloralose (60 mg/kg i.v.) anesthesia, prolonged negative shifts of the DC potential reflecting the elevated potassium levels after KCl injection were measured near the injection site (electrode DC1). In contrast, spreading depressions (transient short DC deflections) were almost exclusively observed under alpha-chloralose. Spreading depressions recorded with electrode DC1 were superimposed on the prolonged negative DC shifts and they propagated frequently to the more distant site (DC2). Upon KCl administration, dose dependent elevations of extracellular glutamate were measured. These elevations were not significantly altered by the type of anesthesia. Our results suggest that in cats, spreading depression induction is affected by anesthesia, i.e., spreading depression induction is inhibited by halothane as compared to alpha-chloralose. Furthermore, factors other than glutamate or high potassium seem to contribute to spreading depression induction. PMID- 8616598 TI - Concentrations of pituitary adenylate cyclase activating polypeptide (PACAP) in human brain nuclei. AB - Concentrations of pituitary adenylate cyclase activating polypeptide (PACAP) in 79 microdissected human brain regions from adult 40- to 80-year-old adult males were measured by radioimmunoassay. Although PACAP was detectable in all of the brain nuclei investigated, the distribution of the peptide was regionally very heterogeneous. The ratio of the highest to the lowest concentrations measured in brain regions exceeded 160:1. The highest concentrations were found in the dorsal vagal complex, the bed nucleus of the stria terminalis, the median eminence pituitary stalk, and in the periventricular and paraventricular hypothalamic nuclei. They were followed by some hypothalamic (supraoptic and ventromedial), preoptic and brainstem nuclei. High concentrations were also measured in the septum pellucidum, periaqueductal and spinal gray matters, the motor facial, and in the spinal nucleus of the trigeminal nerve. The distribution pattern of PACAP in the human brain was unique; it did not show any similarities to the distributions of other neuropeptides in the central nervous system. PMID- 8616599 TI - The serotonin (5-HT) antagonist methysergide increases neuropeptide Y (NPY) synthesis and secretion in the hypothalamus of the rat. AB - NPY is synthesized in neurons of the hypothalamic arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN), an important site of NPY release. Serotonin (5-HT) has been suggested to induce satiety and 5-HT fibers contact NPY neurons in the ARC and PVN, suggesting that 5-HT could inhibit the ARC-PVN projection. Methysergide is a 5-HT antagonist which stimulates feeding in rats and increases NPY levels in the hypothalamus. To clarify the effects of methysergide on NPY, we examined its effects on NPY synthesis and on NPY secretion in the PVN using push-pull sampling. Hypothalamic NPY mRNA levels were measured in rats (n = 8/group) given either saline or methysergide (10 mg/kg) and killed after 4 h or after 7 days. Food intake was increased by 33% in the acute study and by 9% in the 7-day study (both P < 0.01). NPY mRNA levels were 80% higher in the 7-day study (P < 0.05) and unchanged in the acute study. NPY secretion was measured over a 3-h period after an i.p. injection of methysergide or saline (10 mg/kg, n = 12) with a flow rate of 15 microliters/min. Mean NPY secretion in the methysergide-injected rats was increased by 34% (P < 0.01). We conclude that methysergide induced feeding is associated with increased activity of the NPY neurons in the ARC-PVN projection. This is consistent with our previous findings suggesting that the NPYergic ARC-PVN projection may mediate, at least in part the effects of 5-HT on feeding and energy balance. PMID- 8616600 TI - A comparison of the effects of a sodium channel blocker and an NMDA antagonist upon extracellular glutamate in rat focal cerebral ischemia. AB - Agents such as 619C89 decrease extracellular glutamate concentrations by a primary action at voltage sensitive sodium channels, but NMDA antagonists also have been shown to decrease extracellular glutamate concentration after ischemia. To address the question as to whether 619C89's effect upon extracellular glutamate concentrations is any different than the effect of the NMDA antagonist dextrorphan, 24 rats were given either optimally neuroprotective doses of these drugs or saline prior to middle cerebral artery occlusion. In caudate, the 619C89 treated, but not dextrorphan-treated rats had less microdialysate glutamate than ischemic controls. In cortex, both 619C89- and dextrorphan-treated groups had significantly decreased glutamate compared with ischemic controls. These results support a specific effect of 619C89 upon glutamate release in caudate but not cortex. PMID- 8616601 TI - Alterations of low molecular weight acid phosphatase protein level in Alzheimer's disease. AB - We have previously reported that the activity of low molecular weight (LMW) acid phosphatase, which can remove tyrosine-linked phosphates of epidermal growth factor receptor, was significantly decreased in Alzheimer brains. In the present study, a specific antibody was prepared to analyze the protein level of this enzyme. Western blot analysis indicated that the level of LMW acid phosphatase protein was significantly reduced, whereas the activity of LMW acid phosphatase per enzyme molecule was not changed in Alzheimer brains. These results suggest that the reduction of LMW acid phosphatase activity in Alzheimer brains is due to its decreased protein level in Alzheimer's disease. PMID- 8616602 TI - The effect of transcranial magnetic stimulation of rat brain on behavioral models of depression. AB - Magnetic stimulation of the brain in unanesthetized humans and animals can painlessly induce motor movements and has recently been reported to have antidepressant properties. In behavioral models of depression and electroconvulsive therapy including enhancement of apormorphine-induced stereotypy, reduction of immobility in the Porsolt swim test and increases in seizure threshold for subsequent stimulation, magnetic stimulation of rat brain had effects similar to those of electroconvulsive shock. PMID- 8616603 TI - The AMPA antagonist NBQX provides partial protection of rat cerebellar Purkinje cells after cardiac arrest and resuscitation. AB - Purkinje cell loss in adult rats resuscitated following cardiac arrest is analogous to that seen following human cardiac arrest. Administration of the competitive AMPA antagonist NBQX to rats resuscitated after 10 min duration cardiac arrest rescued 21.5% of the vulnerable Purkinje cell population. These results support the hypothesis that sustained postischemic overexcitation of AMPA receptors may be a driving force in the process of Purkinje cell degeneration. PMID- 8616604 TI - Electrophysiological evidence for oxytocin receptors on sympathetic preganglionic neurones--an in vitro study on the neonatal rat. AB - The action of oxytocin (0.01-1 microM) on sympathetic preganglionic neurones was studied by intracellular recording in slices of neonatal rat thoracic spinal cord. In 85% of the cells superfusion induced a slow tetrodotoxin-insensitive depolarization accompanied by the appearance or increase in frequency of repetitive discharges. Oxytocin also caused some cells to switch from silent neurones to spontaneously active ones. These effects were reversibly blocked by a specific oxytocin antagonist. PMID- 8616605 TI - Exogenous NT-3 mitigates the transganglionic neuropeptide Y response to sciatic nerve injury. AB - Peripheral sciatic nerve transection injury produces a marked increase in immunoreactive NPY in rat lumbar DRG and, as a 'transganglionic' response, in their central axonal projections to the dorsal spinal cord and gracile nuclei. Local application of neurotrophin-3 (NT-3) using implanted silicone chambers applied to the proximal transected sciatic nerve stump significantly diminished the transganglionic NPY response in the gracile nucleus and spinal cord but did not affect the CGRP transganglionic response. Conversely, local application of NGF affected the transganglionic CGRP response to axotomy but did not substantially affect transganglionic gracile NPY upregulation. PMID- 8616606 TI - A paradoxical elevation of brain cyclo(His-Pro) levels in hyperphagic obese Zucker rats. AB - Several studies suggest a role for endogenous cyclo(His-Pro) or CHP in appetite regulation. In the present study, we have examined the regional brain distribution of CHP in hyperphagic obese Zucker rats and their lean littermates. The data show a significant elevation in the levels of CHP in many brain regions, including hypothalamus of the obese rat. Within the hypothalamus, the lateral hypothalamic (LH) nucleus of obese rats had significantly higher levels of CHP when compared to that of the lean littermates. Administration of dehydroepiandrosterone, a steroid hormone known to decrease food intake and body weight gain, to obese rats led to decrease in the levels of CHP in the LH. These data further suggest a role for the endogenous CHP in attenuating food intake. PMID- 8616607 TI - Fast axonal transport rates are unchanged in 6- and 24-month F344 rats. AB - The maximum rate of fast axonal transport in motor axons at 6 and 24 months was measured in F344 rats. Tritiated proline was injected near sciatic motoneurons and rats were killed after 2-5 h. Nerves were processed for liquid scintillation spectroscopy and fast transport rates calculated. The rates, in 6- and 24-month rats, were 373 +/- 12 mm/day and 368 +/- 10 mm/day, respectively. Thus, the maximum fast transport rate is unchanged with age in F344 rats. PMID- 8616608 TI - Potentiation of morphine-induced antinociception in acute spinal rats by the NMDA antagonist dextrorphan. AB - Neurophysiologic and behavioral evidence indicates that excitatory amino acid (EAA) antagonists may provide a new class of selective analgesics for opiate resistant, neuropathic pain syndromes. Therapeutic applications have been limited because of unacceptable side effects of most EAA blockers. However, dextrorphan, a metabolite of the antitussive drug, dextromethorphan, is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) subtype of EAA receptor with few side effects and a moderate analgesic effect in animals with peripheral neuropathy. It may therefore, have clinical benefit, either alone or in combination with opiates, for neuropathic pain. In this study a subeffective dose of dextrorphan (15 mg/kg) was combined with several doses of morphine (1.5, 3.0 and 6.0 mg/kg) and assessed in an animal model of central injury, the tail flick response of the acute spinal rat. At doses which were individually ineffective, the combination of dextrorphan and morphine (15 and 1.5 mg/kg, respectively) produced a significant antinociceptive response. The same dose of dextrorphan also increased the antinociceptive response to 3.0 and 6.0 mg/kg of morphine. Coadministration of low doses of an NMDA antagonist and an opiate, might have clinical benefit for the relief of pain with reduced risk of undesirable side effects. PMID- 8616609 TI - The interaction of clonidine and nitric oxide on feeding behavior in the chicken. AB - Central administration of alpha 2-receptor agonists stimulate food intake in mammalian and avian species. Recently we reported that inhibition of nitric oxide (NO) synthase (NOS) decreased food intake in chickens. In the present study, we investigated whether the increased eating induced by clonidine (Clon), an alpha 2 receptor agonist, is attenuated by NOS inhibition. In the first experiment, four levels (0, 9.4, 18.8 or 37.5 nmol/10 microliters) of Clon were administered into the right lateral ventricle of chickens, and food intake was monitored. Clon increased 30 min-food intake in a dose-dependent manner. In a co-administration study of L-NG-nitro-arginine methyl ester HCl (LNNA), a NOS inhibitor, and Clon, LNNA (0, 1.5, 3.0 or 5.9 mumol) attenuated food intake induced by Clon (37.5 nmol) in a dose-dependent manner. Our results suggest the possibility that NO interacts with adrenergic neurons in the central nervous system to modulate feeding behavior in the chicken. PMID- 8616610 TI - A comparative study of pre-sympathetic and Botzinger neurons in the rostral ventrolateral medulla (RVLM) of the rat. AB - The aim of this study was to investigate the degree of functional and anatomical overlap between two major neuronal subpopulations in the rostral ventrolateral medulla: pre-sympathetic (sympathoexcitatory) neurons, and expiratory neurons of the Botzinger complex. Extracellular recordings were made with dye-filled microelectrodes in pentobarbital anesthetized, paralyzed and artificially ventilated adult Wistar rats. Tests applied included stimulation of baroreceptor afferents, activation of peripheral chemoreceptors and lung stretch receptors, changes in central respiratory drive with hyper- or hypoventilation, nociceptive stimulation, and antidromic stimulation from the T2 segment of the spinal cord or medulla oblongata at obex level. The two groups of neurons showed different patterns of spontaneous activity and generally different responses to these stimuli. The recording positions showed some overlap, but the majority of Botzinger neurons were dorsolateral to pre-sympathetic neurons. There was a large overlap between the location of pre-sympathetic neurons and the lateral part of the C1 adrenergic group, but only a small overlap between these adrenergic neurons and Botzinger neurons. These results indicate that the anatomically adjacent pre-sympathetic and Botzinger expiratory neurons form two functionally distinct neuronal subpopulations. PMID- 8616611 TI - Effects of hyper- and hypoglycemia on blood growth hormone level in free-feeding rats with anterolateral deafferentation of the medial basal hypothalamus. AB - In rats with anterolateral deafferentation of the medial basal hypothalamus, the growth hormone (GH) level in the blood showed irregular and small fluctuations instead of the usual high bursts and low trough level, and the baseline GH level was higher than that in sham-operated rats. Continuous infusion of a glucose solution to operated rats increased the baseline level, GH pulse and pulse amplitude. I.v. bolus injection of the glucose solution resulted in a significant but transient increase in GH level. Insulin-induced hypoglycemia decreased the blood GH level in operated rats more effectively than in sham-operated ones and that was prevented by simultaneous infusion of glucose. Since SS influence on GH secretion had been largely eliminated in rats with anterolateral deafferentation of the medial basal hypothalamus, it is highly unlikely that the effects of hyperglycemia or hypoglycemia on GH secretion were the consequence of altered SS secretion. PMID- 8616612 TI - Sodium and potassium current in neonatal rat carotid body cells following chronic in vivo hypoxia. AB - Chronic hypoxic acclimatization modifies ventilatory reflexes arising from carotid body stimulation. To explore this, the effects of in vivo chronic hypoxia on membrane currents were quantified in chemoreceptive carotid body glomus cells. Pregnant rats were maintained in either normoxia (NORM: inspired oxygen tension 141 mmHg), or hypoxia (CHX: inspired oxygen tension 80 mmHg) from day 3 of gestation, to day 5-10 postpartum. Whole cell patch clamp recordings were then made from the mechanically and enzymatically dissociated carotid body glomus cells of the rat pups (NORM: 41 cells, CHX: 36 cells) and comparisons of means +/ S.E.M. were made with unpaired t-tests. Glomus cells were bright under phase contrast illumination, formed clusters, were histochemically positive for catecholamines and possessed voltage-gated potassium currents that were depressed by acute hypoxia. Acclimatization to chronic hypoxia did not affect rat pup whole body mass (CHX: 12.0 +/- 0.7 g vs. NORM: 11.0 +/- 0.2 g), but it significantly increased blood hematocrit (CHX: 48.7 +/- 0.9% vs. NORM: 37.8 +/- 0.5%, P < 0.05). Sodium current was not uniformly present in glomus cells from either group, but sodium current was observed in a greater proportion of glomus cells isolated from the chronically hypoxic pups (CHX: 72% vs. NORM: 46%, P < 0.05). The mean peak tetrodotoxin-sensitive sodium current evoked by -70 mV to +10 mV depolarizations was greater after hypoxic acclimatization (CHX: -100 +/- 25 pA vs. NORM: -38 +/- 15 pA, P < 0.05), but the sodium current density (pA/pF) was unchanged. In contrast, the mean peak voltage-gated potassium current (pA) evoked by -70 mV to 0 mV depolarizations was unchanged by acclimatization, but the potassium current density (pA/pF) was reduced (P < 0.05). Unchanged sodium current density coupled with decreased potassium current density may make glomus cells more excitable during exposure to chronic in vivo hypoxia. PMID- 8616613 TI - Evaluation of recombinant human basic fibroblast growth factor (rhbFGF) as a cerebroprotective agent using high speed MR imaging. AB - The potential cerebroprotective effects of recombinant human basic fibroblast growth factor (rhbFGF) were evaluated in a feline model of acute cerebral ischemia using high-speed magnetic resonance imaging (MRI) in conjunction with immunohistology. The neuroprotective efficacy of three doses of rhbFGF was evaluated in a unilateral middle cerebral artery (MCA) occlusion/reperfusion model. Ten h following a 2 h period of MCA occlusion in control (vehicle-treated) animals, cerebral perfusion in the ischemic hemisphere was 58 +/- 17% of the contralateral normal hemisphere. Corresponding ischemic/normal perfusion ratios in rhbFGF-treated groups were not significantly different: 54 +/- 16% (14 micrograms/kg/h dose), 40 +/- 19% (42 micrograms/kg/h dose) and 75 +/- 8% (125 micrograms/kg/h dose). Triphenyltetrazolium chloride histopathological assessment demonstrated brain damage in vehicle-treated animals comprising 31 +/- 15% of the hemisphere; in rhbFGF-treated groups injury was not significantly different: 19 +/- 4% (14 micrograms/kg/h rhbFGF), 24 +/- 6% (42 micrograms/kg/h rhbFGF) and 16 +/- 10% (125 micrograms/kg/h rhbFGF). Immunohistochemical analysis of brain sections using glial fibrillary acidic protein (GFAP) revealed that in animals that showed marked perfusion deficits throughout the entire experiment (regardless of treatment), GFAP staining was elevated contralateral to the occlusion and absent ipsilaterally. While some tendency towards protection is found, particularly at higher doses of rhbFGF, it must be concluded that in the chosen stroke model and time interval, the doses used did not significantly improve reperfusion or confer significant cerebroprotective benefit. Non-invasive high-speed MRI was found to be useful for evaluation of putative cerebroprotective agents. PMID- 8616614 TI - Nitric oxide regulation of methamphetamine-induced dopamine release in caudate/putamen. AB - A possible role for NO modulation of dopamine (DA) release in the caudate/putamen (CPU) during methamphetamine (METH) exposure was investigated using in vivo microdialysis in rats. Inclusion of the nitric oxide synthase (NOS) inhibitors NG nitro-L-arginine (NOARG), NG-nitro-L-arginine methyl ester (L-NAME) or D-NAME (less potent inhibitor) in the microdialysis buffer prior to METH minimally affected basal levels of DA, DOPAC or HVA in CPU microdialysate. However, L-NAME and NOARG produced concentration-dependent decreases of up to 64% (100 microM) in CPU DA levels in microdialysate during exposure to four doses of METH (5 mg/kg i.p./2 h), with lesser effects on DOPAC or HVA. Reversal of the NOARG inhibition was produced by inclusion of 500 microM of either L-arginine or L-citrulline in the microdialysate. D-NAME (100 microM) minimally affected levels of DA or metabolites. Paradoxically, inclusion of from 20 to 2 microM of the NOx generators isosorbide dinitrate (ISON) or sodium nitroprusside (SNP) in the microdialysis buffer decreased DA and DOPAC levels in microdialysate during METH exposure. This paradox might result from the concentrations of NOx produced by SNP or ISON being great and not regionally specific resulting in inhibition of DA release and/or synthesis while the NO generated endogenously during METH exposure may have localized and site-specific actions. Alternatively, NOx may inhibit NOS or other enzymes in the NO synthesis pathway, thereby reducing levels of an intermediate (other than NO) which potentiates DA release. In their entirety, our results indicate that NO generation in the CPU may augment the release of DA during METH exposure. PMID- 8616615 TI - Effects of isoflurane concentration on the activity of pontomedullary raphe and medial reticular neurons in the rat. AB - Neurons in the pontomedullary raphe magnus (RM) and adjacent nucleus reticularis paragigantocellularis pars alpha (NRPG alpha) are thought to participate in the modulation of spinal nociceptive transmission. In order to determine whether these cells also contribute to the suppression of nocifensive reflexes produced by general anesthetics, the spontaneous activity of RM/NRPG alpha cells was recorded in rats anesthetized with isoflurane (IF) at several steady state concentrations, corresponding to depths which are below, equal to, or above the threshold for blocking the motor response to noxious stimuli (minimum alveolar concentration, MAC). Neurons were classified by their spontaneous activity patterns and their responses to noxious stimulation as OFF, ON, REGULAR or NEUTRAL cells. After cell classification, unit activity, arterial blood pressure, heart rate, and EEG activity were simultaneously recorded, in the absence of somatic stimulation, for 1 h at each of two or three concentrations of IF. The concentrations tested were low (1.05-1.25%), medium (1.30-1.45%) and high (1.70 1.90%). ON, OFF and some NEUTRAL cells exhibited alternating periods of inactivity and activity when recorded during periods of low and medium anesthetic concentrations. At high steady state anesthetic concentrations, the mean discharge of most OFF, ON and NEUTRAL cells decreased by greater than 25% from their mean discharge rate at the low concentration. REGULAR cells maintained a uniform firing rate at all steady state anesthetic concentrations studied. Since high concentrations of IF do not activate OFF cells, the putative inhibitory output neuron of the RM/NRPG alpha, it is unlikely that the activity of RM/NRPG alpha neurons contributes to the suppression of nocifensive movement by the general anesthetic, IF. PMID- 8616616 TI - Assessment of volumetric sex differences in the song control nuclei HVC and RA in zebra finches by immunocytochemistry for methionine enkephalin and vasoactive intestinal polypeptide. AB - In this study we assessed sex differences in the volume of two vocal control nuclei, the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in zebra finches (Taeniopygia guttata) exposed to various endocrine treatments (neonatal treatment with an aromatase inhibitor and/or an adult treatment with testosterone). The boundaries of these nuclei were defined in sections stained for Nissl substance and compared to alternate sections stained with an immunocytochemical procedure for two neuropeptides, methionine enkephalin and vasoactive intestinal polypeptide. A high density of immunoreactive fibers for methionine enkephalin and vasoactive intestinal polypeptide completely covered the high vocal center in male and female zebra finches and these fibers were not observed in the surrounding neostriatum. With the use of these markers to define the boundary of the high vocal center, it became possible to reconstruct its volume. Positively staining perikarya were not apparent within the boundaries of this nucleus. Immunoreactive fibers for vasoactive intestinal polypeptide and enkephalin also allowed one to define the boundary of the robust nucleus of the archistriatum but they did not fill the entire area of the nucleus as is the case for the high vocal center. When the volumes of both nuclei were reconstructed based on enkephalin or vasoactive intestinal polypeptide immunoreactivity, the presence of a marked sex difference in the volume of these nuclei was confirmed. Neonatal and adult endocrine manipulations did not affect the volumes of these two nuclei measured in Nissl-stained material nor did they affect the volumes as defined based on the peptidergic innervations of the nuclei. Also, the volumetric estimates of these two nuclei derived from sections stained by immunocytochemistry were in good agreement in all groups of birds with the values obtained based on an analysis of the Nissl-stained material. These results illustrate the usefulness of employing a variety of histochemical markers to define a brain area when investigating brain variation and plasticity. PMID- 8616617 TI - TRH in the dorsal motor nucleus of vagus is involved in gastric erosion induced by excitation of raphe pallidus in rats. AB - The influence of excitation of the raphe pallidus neurons on gastric mucosal lesions was examined in urethane-anesthetized rats pretreated with indomethacin (5 mg/kg, i.p., -60 min). Kainic acid (12 ng/30 nl), delivered into the raphe pallidus, increased gastric acid secretion (94.8 +/- 15.9 mumol/60 min) and produced gastric lesions in 2.7 +/- 0.3% of the corpus mucosa. No gastric erosions were observed when kainic acid was injected nearby, but outside, of the raphe pallidus. The gastric lesions induced by microinjection of kainic acid into the raphe pallidus neurons were completely prevented by atropine (0.3 mg/kg, -30 min, s.c.) and bilateral microinjection of TRH antibody # 8964 (1.3 microgram/site) into the dorsal motor nucleus of the vagus (DMN). Microinjection of the TRH antibody into the hypoglossal nucleus or control antibody into the DMN did not modify the mucosal lesions induced by kainic acid into the raphe pallidus. These data suggest that activation of raphe pallidus induced vagal cholinergic mediated gastric erosion through TRH release in the DMN. PMID- 8616618 TI - Tetanus toxin inhibits spontaneous quantal release and cleaves VAMP/synaptobrevin. AB - Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed. PMID- 8616619 TI - Influences of neuronal uptake and D2 autoreceptors on regulation of extracellular dopamine in the core, shell and rostral pole of the rat nucleus accumbens. AB - Fast cyclic voltammetry in rat brain slices containing the nucleus accumbens, was used to examine the regulation of the extracellular concentration of electrically stimulated dopamine overflow in the core, shell and rostral pole. One microM (-) sulpiride, significantly increased dopamine overflow in all 3 regions but only when the duration of stimulation was greater than 500 ms. One microM cocaine, significantly potentiated dopamine overflow in all 3 regions following all patterns of stimulation. In the presence of 1 microM cocaine, superfusion with 1 microM (-)-sulpiride did not result in a further increase in dopamine overflow at any frequency of stimulation in the rostral pole, but significant increases in dopamine overflow were observed after stimulation with 20 pulses at 10 or 20 Hz in the core or shell; the degree of potentiation was greater in the shell than core. Quinpirole inhibited single pulse stimulated dopamine overflow in a concentration dependent manner (maximum inhibition (100%) in all regions) but was significantly less potent in the rostral pole than in the core or shell. Increasing the number of pulses to 2 or 4 pulses at 50 Hz resulted in a shift of the quinpirole dose-response curve to the right in all regions and in the rostral pole, a significant reduction in the maximum inhibition of dopamine overflow to both stimulation parameters. In the shell a significant reduction in maximum inhibition was only seen with 4 pulses at 50 Hz stimulation, whereas in the core there was no change in the maximum inhibitory effect of quinpirole. Neuronal uptake and D2 autoreceptor activity contribute to regulation of the extracellular concentration of dopamine in core, shell and rostral pole. The relative importance of either uptake or autoreceptor control is region and stimulus dependent. PMID- 8616620 TI - The role of GABA in NMDA-dependent long term depression (LTD) of rat medial vestibular nuclei. AB - The role of GABA in NMDA-dependent long term depression (LTD) in the medial vestibular nuclei (MVN) was studied on rat brainstem slices. High frequency stimulation (HFS) of the primary vestibular afferents induces a long lasting reduction of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the MVN. The induction but not the maintenance of this depression was abolished by AP5, a specific blocking agent for glutamate NMDA receptors. The involvement of GABA in mediating the depression was checked by applying the GABAA and GABAB receptor antagonists, bicuculline and saclofen, before and after HFS. Under bicuculline and saclofen perfusion, HFS provoked a slight potentiation of the N2 wave, while the N2 depression clearly emerged after drug wash-out. This indicates that GABA is not involved in inducing the long term effect, but it is necessary for its expression. Similarly, the LTD reversed and a slight potentiation appeared when both drugs were administered after its induction. Most of these effects were due to the bicuculline, suggesting that GABAA receptors contribute to LTD more than GABAB do. According to our results, it is unlikely that the long lasting vestibular depression is the result of a homosynaptic LTD. On the contrary, our findings suggest that the depression is due to an enhancement of the GABA inhibitory effect, caused by an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level. PMID- 8616621 TI - A learned odor evokes an enhanced Fos-like glomerular response in the olfactory bulb of young rats. AB - Young rats exposed to peppermint odor and reinforcing tactile stimulation from postnatal days (PND) 1-18 increase their preference for that odor relative to controls. This early olfactory memory is accompanied by an 80% increase in the density of glomerular-layer cells displaying Fos-like immunoreactivity in response to the learned odor on PND 19. The difference is observed in midlateral portions of the olfactory bulb that align with foci of 2-deoxyglucose (2-DG) uptake in adjacent sections. Trained and control animals are not different in the Fos-like response of juxtaglomerular cells within ventrolateral 2-DG foci. Ratios of midlateral/ventrolateral response differ significantly between trained and control animals and include differences among cells of three staining intensities. These ratios are correlated with ratios of 2-DG uptake (midlateral/ventrolateral foci), which also differ significantly between trained and control rats. Juxtaglomerular cells associated with 2-DG foci also express Egr-1-like immunoreactivity. However, the midlateral Egr-1 response does not differ between trained and control rats. These results show that early memories can be associated with an increased Fos-like response in a primary sensory area of the CNS. They also suggest that only specific regions within the olfactory bulb are modified following the learning of a given odor in early life. PMID- 8616622 TI - Muscle atonia can be induced by carbachol injections into the nucleus pontis oralis in cats anesthetized with alpha-chloralose. AB - Cholinergic excitation of structures in the pontine reticular formation appears to be a key step in the generation of active sleep. For example, muscle atonia which occurs as a result of the postsynaptic inhibition of motoneurons during active sleep is also present after carbachol, a cholinergic agonist, is injected into the nucleus pontis oralis. In the present study, in order to obtain information regarding the mechanisms that generate atonia during active sleep and to provide a paradigm for studying atonia in anesthetized cats, we determined whether cholinergically induced atonia could be generated in an animal that was anesthetized with alpha-chloralose. Cats which were initially anesthetized with alpha-chloralose (40 mg/kg, I.V.) exhibited spikes in the EEG, hippocampus and lateral geniculate nuclei. Muscle atonia occurred after carbachol (200 mM) was injected by microiontophoresis (300-500 nA) into the nucleus pontis oralis; the spikes in the EEG, hippocampus and lateral geniculate nuclei were still present. We believe that the atonia induced by carbachol in alpha-chloralose-anesthetized cats is mediated by the same mechanisms that operate during active sleep in the unanesthetized animal for the following reasons. First, in the same cats when they were not anesthetized with alpha-chloralose, carbachol injections in the identical brainstem sites induced active sleep with its accompanying pattern of muscle atonia. Second, after carbachol was injected into the same sites in alpha chloralose-anesthetized cats, intracellular recordings from lumbar motoneurons revealed that inhibitory postsynaptic potentials were bombarding motoneurons; these inhibitory potentials were similar to those which are present during naturally occurring active sleep. In addition, stimulation of the nucleus reticularis gigantocellularis (NRGc) was found to induce large amplitude depolarizing potentials in lumbar motoneurons in alpha-chloralose-anesthetized cats prior to the administration of carbachol, whereas after its administration, accompanying muscle atonia there were large amplitude hyperpolarizing potentials and a reduction in the amplitude of depolarizing potentials. We therefore conclude that the cholinergically induced processes that initiate and maintain muscle atonia are not blocked by the actions of alpha-chloralose. PMID- 8616623 TI - L-arginine ameliorates cerebral blood flow and metabolism and decreases infarct volume in rats with cerebral ischemia. AB - Effects of L-arginine, 300 mg/kg, i.p., on the regional cerebral blood flow (rCBF), brain metabolism, and infarct volume were examined in spontaneously hypertensive rats subjected to occlusion of both left middle cerebral artery and left common carotid artery. Rats treated with L-arginine had higher rCBF, determined by hydrogen clearance method, in the ischemic core (7 +/- 1 ml/100 g/min, mean +/- S.E.M.) and penumbral regions (16 +/- 2) than did rats treated with saline (5 +/- 0 and 7 +/- 1, respectively). Simultaneously, L-arginine attenuated metabolic derangement in the ischemic tissue at 60 min, i.e. well maintained adenosine triphosphate (ATP) in ischemic region (1.29 +/- 0.07 mmol/kg in L-arginine group vs. 1.05 +/- 0.06 in saline group), and also close to normal levels in ATP (2.61 +/- 0.02 mmol/kg vs. 2.45 +/- 0.05), glucose (2.29 +/- 0.12 mmol/kg vs. 1.80 +/- 0.17) and lactate (1.63 +/- 0.10 mmol/kg vs. 2.24 +/- 0.21) in periischemic region. In another experiment, the effects of L-arginine on rCBF in the subcortical regions and on infarct volume were evaluated. L-arginine, compared with saline, increased rCBF by 8 ml/100 g/min in the ischemic side and reduced infarct volume by 29% at 24 h of ischemia. These findings support that L arginine may be potentially useful for the treatment of acute cerebral ischemia. PMID- 8616624 TI - Pontine and basal forebrain transections disinhibit brown fat thermogenesis in neonatal rats. AB - Bignall, Heggeness and Palmer (1975) were the first to demonstrate increases in metabolic heat production following midpontine transection in neonatal rats. Subsequent work in adult rats has shown that this procedure disinhibits thermogenesis by brown adipose tissue (BAT). Bignall and his colleagues also found that hypothalamic ablation did not result in increased thermogenesis in 5 day-olds, leading them to conclude that thermoregulation depends on more caudal structures at that age. We have also found that midpontine transection disinhibits BAT thermogenesis and, furthermore, have extended that finding to newborn pups. When transections were made in the basal forebrain, however, we also found profound and rapid increases in brown fat thermogenesis. These results suggest the presence of at least two sources of inhibition of BAT thermogenesis in newborn rats: one located in the rostral pons-caudal midbrain and one located in the basal forebrain. PMID- 8616625 TI - GABAergic suppression prevents the appearance and subsequent fatigue of an NMDA receptor-mediated potential in neocortex. AB - We have investigated the regulation of an N-methyl-D-aspartate (NMDA) receptor mediated synaptic potential by gamma-aminobutyric acid (GABA)-mediated inhibition using extracellular and whole-cell voltage clamp recordings in rat auditory cortex in vitro. Single afferent stimulus pulses at low intensity elicited a slow extracellular negativity (Component C) that was mediated by NMDA receptors. At higher intensities, Component C was suppressed by recruitment of GABAergic inhibition. To understand the actions of GABAergic inhibition on Component C, we determined the effects of: (i) paired-pulse stimulation, which depresses GABAergic inhibition; (ii) pharmacological antagonism of GABA receptors; and (iii) afferent stimulation in slices from neonatal rats prior to the development of cortical inhibition. The results indicate that GABAergic inhibition prevents Component C from occurring, thereby preventing its reduction upon repeated stimulation. Whole-cell voltage clamp recordings were used to test the hypothesis that GABAergic suppression occurred by way of membrane hyperpolarization. At hyperpolarized holding potentials no NMDA receptor-mediated synaptic current was elicited, even with paired-pulse stimulation. At depolarized holding potentials a significant NMDA synaptic current was elicited despite the presence of GABAergic synaptic currents. We conclude that membrane hyperpolarization by GABAergic inhibition prevents the appearance and subsequent fatigue of an NMDA receptor mediated synaptic potential. Reduction of inhibition can act as a 'switch' to fully release the NMDA potential as frequently as once every 10-20 s. PMID- 8616626 TI - Repeated spinal cord stimulation decreases the extracellular level of gamma aminobutyric acid in the periaqueductal gray matter of freely moving rats. AB - Most of the previous experimental studies on the antinociceptive effects of electrical spinal cord stimulation (SCS) have focused on short-lasting effects mainly depending on spinal mechanisms. However, patients treated with SCS for chronic pain often report pain relief exceeding the period of stimulation for several hours. The long lasting effect of SCS might not only involve spinal, but also supraspinal mechanisms. A supraspinal region of major importance for the coordination of descending pain inhibition is the periaqueductal grey matter (PAG). The aim of the present microdialysis study, performed in awake freely moving rats, was to investigate if repeated SCS (two 30 min periods separated by a 90 min resting period) alters the extracellular neurotransmitter concentrations in the ventrolateral PAG. In a first series of experiments significantly decreased (-30%; P < 0.05; n = 7) gamma-aminobutyric acid (GABA) levels were detected immediately after the second SCS session. Neither the concentration of serotonin nor that of substance P-like immunoreactivity (SP-LI) was affected by SCS. The decrease of GABA after two SCS sessions was confirmed in a second series of experiments (-30%; P < 0.05; n = 7). No spontaneous decline of GABA was observed in sham-stimulated animals (n = 6). The glutamate concentration was also determined in this latter series of experiments and a significant decrease (-23%; P < 0.05; n = 5) was observed after the second SCS session. As GABA-neurons in the PAG exert a tonic depressive effect on the activity in descending pain inhibitory pathways, a decreased extracellular GABA level in this region, as detected following repeated SCS, might indicate an increased pain inhibition. PMID- 8616627 TI - Sex-related effects on behaviour and beta-endorphin of different intensities of formalin pain in rats. AB - The effects of two intensities of formalin pain on behaviour and beta-Endorphin (beta-EP) concentration in the brain and pituitary were studied in male and female rats. The animals were familiarized with the Hole-Board apparatus for 3 days, and then, after a subcutaneous injection of formalin (50 microliter, 0.1 or 10%) or Sham-injection (Control) in the hindpaw, they were tested in the Hole Board for 60 min. Licking, Flexing and Paw-Jerk of the injected limb were recorded. beta-EP concentration was determined in the hypothalamus (HYP), the periaqueductal gray matter (PAG), the anterior pituitary (AP) and the neurointermediate lobe (NIL). Licking and Flexing durations were greater in females than males only with formalin 10%. Sex differences in beta-EP concentration between the Control groups were found in all tissues except the HYP; beta-EP levels were higher in females in the PAG and NIL, but greater in the AP in males. beta-EP concentration increased in males in the HYP and NIL with formalin 10%; in females, a decrease was found in the HYP with formalin 0.1%. The present results suggest that: (a) there are differences between males and females in the responses to formalin pain, and the nature (pattern and duration) of the sex differences varies according to the pain intensity; (b) there are differences in beta-EP concentration between the two sexes in control animals, and male and female rats also exhibit differences in the modifications of beta-EP in response to formalin-induced pain. PMID- 8616628 TI - Priming of experimental anxiety by repeated subthreshold GABA blockade in the rat amygdala. AB - Blockade of GABAA receptor function in the area of the anterior basolateral amygdala of rats elicits physiological (increases in heart rate and blood pressure) and behavioral changes similar to symptoms of human anxiety states. Repeated subthreshold blockade of GABAA receptors in this region appears to result in a long-term 'priming' of these anxiety-like responses. The present study was conducted to characterize the 'priming' of the heart rate and blood pressure responses and to test if these 'primed' animals would show increases in anxiety responses. Male Wistar rats with arterial catheters placed for physiological measurements were implanted with chronic microinjection cannulae in the anterior basolateral amygdaloid nucleus (BLA) under pentobarbital anesthesia. Repeated daily injections of a subthreshold dose of bicuculline methiodide (GABAA receptor antagonist; BMI) into the BLA elicited 'priming' of physiological responses after 3-5 injections and this response was maintained for at least 6 weeks. The primed animals also showed increased anxiogenic responses to GABAA blockade in the BLA. The 'priming' of anxiety responses were clearly elicited before kindling of seizures as measured by EEG. These results suggest that this 'priming' phenomenon may be similar to kindling and long-term potentiation. This could be one potential mechanism for developing pathological emotional responses, such as chronic, high levels of anxiety. PMID- 8616630 TI - Cholecystokinin modulates mesolimbic dopaminergic influences on male rat copulatory behavior. AB - Much evidence suggests that the neuropeptide cholecystokinin (CCK) functions as a neurotransmitter or neuromodulator in the central nervous system. The CCKa receptor subtype in the nucleus accumbens has been demonstrated to potentiate the behavioral and neurophysiological effects of dopamine. Since the mesolimbic dopamine system participates in the regulation of male rat sexual behavior, the present investigation was undertaken to determine if central CCK modulates this dopaminergic regulation. Electrical stimulation of the ventral tegmental area greatly enhanced several measures of appetitive and consummatory male rat sexual behavior. Administration of a CCKa receptor antagonist to the posteromedian nucleus accumbens reversed the electrically stimulated behavioral enhancement. A CCKb antagonist was without effect. In a second group of animals, administration of either a CCKa or CCKb antagonist to the anterolateral nucleus accumbens reversed the enhancement of consummatory sexual responding produced by electrical stimulation. These results agree with the growing body of evidence supporting different behavioral roles for two distinct CCK systems in the nucleus accumbens. PMID- 8616629 TI - Co-localization of amyloid-associated proteins with amyloid beta in rat soleus muscle in chloroquine-induced myopathy: a possible model for amyloid beta formation in Alzheimer's disease. AB - Chloroquine, a potent lysosomotropic agent, induces myopathy in experimental animals similar to rimmed vacuole (RV) myopathy in humans. The abnormal accumulation of amyloid beta protein (A beta), which is the invariable pathological alterations in the brains affected by Alzheimer's disease (AD), has been demonstrated in denervated soleus muscle fibers in chloroquine-induced myopathy in rats. In AD affected brains, a variety of additional proteins are associated with the extracellular deposition of A beta, which leads to the intracellular accumulation of neurofibrillary tangles and finally to neuronal death. In this study, we demonstrate that amyloid-associated proteins, alpha 1 antichymotrypsin, apolipoprotein E, SP-40,40 and ubiquitin co-localize with A beta in vacuolated muscle fibers in chloroquine-induced myopathy. There are striking similarities in immunopathology between experimental RV myopathy and AD. Chloroquine-induced myopathy in rats provides a suitable model not only to obtain insight into the basic mechanisms underlying RV formation in muscle, but also to understand amyloid precursor protein processing into A beta, and the role of amyloid-associated proteins in terms of the pathogenesis of AD. PMID- 8616631 TI - Histochemical demonstration of nitric oxide synthase-like immunoreactivity in epiplexus cells and choroid epithelia in the lateral ventricles of postnatal rat brain induced by an intracerebral injection of lipopolysaccharide. AB - The present in vivo study showed the expression of nitric oxide synthase-like immunoreactivity in epiplexus cells in the lateral ventricles induced by intracerebral injection of lipopolysaccharide into postnatal rats. Nitric oxide synthase-like immunoreactivity was vigorously expressed in epiplexus cells 1 and 3 days after the lipopolysaccharide injection, but by 7 days post-injection, it became undetectable. The expression of nitric oxide synthase-like immunoreactivity was also observed in some of the choroid epithelial cells. The nitric oxide synthase-like immunoreactivity in these cells appeared to be more intense in the ventricle ipsilateral to the LPS injection than that on the contralateral side. The immunostaining patterns of OX-42 and OX-6 for the detection of complement type 3 receptors and major histocompatibility complex class II antigens respectively paralleled that of anti-nitric oxide synthase, indicating that lipopolysaccharide-induced nitric oxide synthase-like immunoreactivity was primarily in epiplexus cells. Immunoelectron microscopy revealed that the nitric oxide synthase-like immunoprecipitate in epiplexus cells and choroid epithelial cells filled the entire cytoplasm and in some areas associated with the membranes of some of the organelles especially the mitochondria, suggesting that the enzyme is mainly cytosolic. It is speculated that nitric oxide synthase in these cells is involved in the synthesis of nitric oxide. The nitric oxide production, if any, through the enzymatic activity of nitric oxide synthase in epiplexus cells as well as the choroid epithelial cells may be involved in host defense against bacterial endotoxin in the ventricular system of postnatal rat brain. PMID- 8616632 TI - Nerve growth factor alleviates a painful peripheral neuropathy in rats. AB - Nerve growth factor (NGF) reverses some effects of axotomy and prevents toxic neuropathy in adult rodents. We tested the effect of NGF on behavioral hyperalgesia resulting from a chronic constriction injury (CCI) of the sciatic nerve in the rat [5]. CCI rats exhibit thermal hyperalgesia as demonstrated by a reduction of paw withdrawal latency to a noxious thermal stimulus applied to the paw on the side of injury. The mechanical sensitivity of the ipsilateral hindpaw, assessed with von Frey filaments, was also significantly increased. There were no significant changes in nociceptive thresholds on the contralateral side. When NGF was infused directly on the ligated nerve via an osmotic pump (0.5 microgram/microliter/h for 7 days) immediately after the ligation, thermal hyperalgesia was abolished from postoperative days 5 up to at least two weeks. The CCI-induced decrease in mechanical threshold was also abolished by NGF. However, NGF had only a minor effect on the abnormally long response duration, a second measure of mechanical sensitivity, to the mechanical stimulus. Delayed infusion of NGF four days after the ligation failed to block hyperalgesia. Infusion of NGF on the sciatic nerve of rats that had no CCI had no significant effect on paw withdrawal latency. Infusion of anti-NGF antiserum did not enhance hyperalgesia in CCI rats. These results suggest that alterations in neurotrophic factor(s) contribute to the development of behavioral hyperalgesia in an animal model of neuropathy and that NGF may have therapeutic value in the treatment of neuropathic pain in humans. PMID- 8616633 TI - Effects of central administration of arginine-vasopressin on aversive memory retrieval. AB - This study examined whether arginine-vasopressin (A-VP), given before the test would produce an improved retrieval of aversive memory, in the same way as pre exposure to inescapable footshocks, in rats. For this purpose animals conditioned in a T-maze with appetitive (10% sucrose) and aversive (2.0 mA footshock) events were administered (intracerebroventricular) a single dose of 2.5, 5, 10 or 20 ng/rat of A-VP, 20-min before testing. In the retention test conducted with the same training apparatus 72 h after conditioning, the peptide treated rats showed a dose-dependent increase in latencies to enter the previously shocked goalarm, with the absence of such a difference in responding to the non-shocked goalarm. This differential response was not observed in saline treated rats. This effect of peptide on memory retrieval was similar to that seen following inescapable footshock in rats. These results suggest the possible involvement of central vasopressinergic mechanisms in the differential enhancement of memory of helplessness condition. PMID- 8616635 TI - Lidocaine has different effects and potencies on muscle and brain sodium channels. AB - Lidocaine effects were studied at the single channel level on batrachotoxin activated eel electroplax (muscle-derived) and on rat brain sodium channels in planar lipid bilayers to investigate whether these effects were the same on structurally different sodium channels. Lidocaine blocked the open state of brain channels with the same voltage dependence, but with 15-times as high a potency as muscle-derived channels. In brain channels, but not muscle-derived ones, the level of the open channel block showed periods of relief. Lidocaine at microM concentrations stabilized the highest conductance state in both channel types and at mM concentrations stabilized subconductance-like states in electroplax, but not in brain channels. In both channel types, lidocaine increased the lifetime and rate of entry to a long-nonconducting state. Since both channel types were studied under identical lipid and ionic conditions, the observed functional differences in the lidocaine action (effects, potency) must reflect channel structural differences. PMID- 8616634 TI - Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia. AB - Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease. PMID- 8616636 TI - Modulation of adenosine release from rat spinal cord by adenosine deaminase and adenosine kinase inhibitors. AB - Adenosine, a modulator of pain processing in the spinal cord, is metabolized by adenosine kinase and adenosine deaminase. In this study we determined which of these mechanisms is more important for the regulation of endogenous adenosine levels in the rat spinal cord. The effects of the adenosine kinase inhibitors, 5' amino-5'-deoxyadenosine (NH2dAD) and iodotubercidin (IOT), and the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), on adenosine release in a spinal cord superfusion model were studied. DCF markedly increased basal adenosine levels detected in perfusates and was more potent than NH2dAD and IOT in this regard. Coadministration of DCF with NH2dAD produced an enhanced effect compared to the inhibitors alone. NH2dAD, but not DCF, potentiated morphine-evoked adenosine release. These results suggest that adenosine deaminase may be the predominant pathway for adenosine metabolism in this experimental model. PMID- 8616637 TI - Co-localisation of NADPH diaphorase activity and GABA immunoreactivity in local circuit neurones in the medial prefrontal cortex (mPFC) of the rat. AB - This study provides evidence that neurones in the medial prefrontal cortex of the rat (mPFC areas 24b, 25, and 32) containing strong NADPH diaphorase reactivity also contain GABA immunoreactivity. Also demonstrated is the co-localisation of NADPH diaphorase activity with immunoreactivity for the neuronal isoform of nitric oxide synthase (nNOS) in mPFC neurones. Qualitative and quantitative analyses in the light and electron microscopes indicate that strongly NADPH diaphorase reactive cells are a subpopulation of GABAergic local circuit neurones and constitute a very small proportion (0.6-1.1%) of neurones in rat mPFC. These results suggest that NADPH diaphorase reactive cells in rat mPFC can influence neural activity via GABA-mediated and NO-mediated mechanisms. PMID- 8616638 TI - Infarct volume varies with rat strain and vendor in focal cerebral ischemia induced by transcranial middle cerebral artery occlusion. AB - We recently reported that the outcome of focal cerebral ischemia induced by intraluminal middle cerebral artery occlusion may differ depending upon a rat's strain and/or vendor. Sprague-Dawley rats originating from Taconic Laboratories and Charles River Laboratories had infarct volumes several fold larger than Sprague-Dawley rats originating from Simonsen Laboratories. The present study sought to determine whether these differences were restricted to an intraluminal technique or whether strain and vendor dependent differences will also exist in rats subjected to a transcranial method of focal cerebral ischemia. Accordingly, we permanently coagulated the middle cerebral artery via a transcranial approach in Sprague-Dawley rats originating from Simonsen Laboratories and Taconic Laboratories and in Simonsen Laboratories Fischer-344 rats. The cortical infarct volume significantly differed in the following order: Simonsen Laboratories Sprague-Dawley rats < Simonsen Laboratories Fischer-344 rats < Taconic Laboratories Sprague-Dawley rats. The subcortical infarct volume differed statistically in the following order: Simonsen Laboratories Sprague-Dawley rats < Taconic Laboratories Sprague-Dawley rats < Simonsen Laboratories Fischer-344 rats. These results along with our previous findings demonstrate that strain and vendor differences in the outcome of focal cerebral ischemia are independent of the technique applied. PMID- 8616639 TI - N-methyl-D-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats. AB - Intracerebral microdialysis technique was utilized to study the effect of NG nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, on N-methyl-D-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mM D-(-)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mM NG-nitro-L-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats. PMID- 8616640 TI - Variations in vascular practice and the Cochrane Collaboration. PMID- 8616641 TI - Secretion of prostacyclin, tissue plasminogen activator and its inhibitor by cultured adult human endothelial cells grown on different matrices. AB - OBJECTIVES: The aim of this study was to investigate the secretion of prostacyclin (PGI2), tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI- 1) produced by human great saphenous vein endothelial cells (HSVECs) in vitro when seeded to confluency on different matrices. METHODS: The matrices compared were: uncoated plastic, gelatin, collagen type-I gel, fibronectin, fibrin glue, de-endothelialized porcine aorta and ePTFE vascular grafts pre-coated with collagen type-I or human serum. RESULTS: There were no significant differences between basal production of PGI2 on the different matrices. The HSVECs responded with a significant increase in PGI2 secretion after stimulation with thrombin on all matrices, including the ePTFE grafts. A statistically significant higher secretion of tPA secretion was found in supernatants from cells cultured on collagen type-I gel. Interestingly, tPA secretion was lower by cells seeded on both collagen type-I and serum precoated ePTFE as compared to collagen type-1 gel. PAI-1 secretion however was significantly higher on collagen type-1 gel, gelatin, fibrin glue and porcine aorta but not on pre-coated ePTFE grafts. CONCLUSIONS: The findings emphasise the important effect of the matrix on endothelial secretion of PGI2, tPA and PAI-1. Differences in patency after implantation of in vitro endothelialized grafts, may be due to cellular function depending on the types of graft and matrix chosen. PMID- 8616642 TI - Reversal of acute renal failure by kidney revascularisation. AB - OBJECTIVES: To assess whether acute renal failure, due to total or subtotal renal artery occlusion, can be reversed by kidney revascularisation. DESIGN: A retrospective review of surgery for kidney salvage in anuric patients at a University Hospital. METHODS: From 1983 to 1993, eight patients were operated on for occlusive renal artery disease as a cause of acute renal failure, requiring preoperative haemodialysis. On admission the mean serum creatinine was 40 mg/l (354 mumol/dl). The oligoanuria lasted from 12 h to 3 weeks. Renal length of 8 cm or more and visualisation of a patent distal renal artery branches on aortography were arguments that return of renal function could be expected after revascularisation of these non-functioning kidneys. RESULTS: Revascularisation restored immediate urine flow in six cases, with no further need for dialysis in four. Two patients remained oliguric despite successful reperfusion. One of them could be weaned from dialysis after 1 month. Two patients died postoperatively. Five of the eight patients left the hospital with restored renal function. CONCLUSIONS: Patients with acute renal function deterioration due to ischemia of a single or both kidneys can benefit from prompt revascularisation, with significant recovery of renal function in most of them. PMID- 8616643 TI - The role of common carotid artery end-diastolic velocity in near total or total internal carotid artery occlusion. AB - OBJECTIVES: To evaluate the role of the end-diastolic velocity (EDV) in the common carotid artery (CCA) as a marker of internal carotid artery (ICA) occlusion. DESIGN: Validation of retrospective data in a prospective clinical study. METHODS: The EDV in 94 patients with total ICA occlusion and in 24 patients with high grade (95-99%) unilateral ICA stenosis identified on extracranial carotid colour-flow Duplex imaging (CFDI) and arteriography was reviewed, and was retrospectively compared to the EDV of 176 normal individuals. Identification of patients with ICA occlusion was most accurate (99.3%) with an ipsilateral EDV > or = 12 cm/s and a DIFF > or = 10 cm/s (DIFF = contralateral EDV -- ipsilateral EDV). These values were then prospectively applied to all 886 patients (67 with high grade stenosis or occlusion) who underwent CFDI at our institution during 1994. RESULTS: The EDV > or = 12 had a 92% sensitivity, a 99.4% negative predictive value (NPV) and a 85% specificity in distinguishing between occluded and patent ICA's. In combination with a DIFF > or = 10 was 80.4% sensitive and 97.5% specific. The positive predictive value of the EDV > or = 12 in the distinction between 95-99% ICA stenosis and ICA occlusion was 78.3%, and that of the combination was 85.4%. The EDV was rarely zero and 10% of patients with normal or minimally diseased ICA's had an EDV > or = 12 and/or a DIFF > or = 10. CONCLUSIONS: The EDV < or = 12 cm/s is a sensitive marker of ICA occlusion with a high NPV and in combination with the DIFF > or = 10 cm/s, is specific. Nevertheless, EDV parameters are inaccurate in the distinction of 95-99% ICA stenosis from occlusion. Low EDV can be found in a number of patients with minor or no ICA disease, particularly in those with a stroke or silent cerebral infarct. PMID- 8616645 TI - Outcome of unreconstructed chronic critical leg ischaemia. AB - OBJECTIVE: To assess the outcome of unreconstructed chronic critical leg ischaemia with a special reference to the definition of CLI. DESIGN AND SETTING: A retrospective study with 1 year follow-up in an academic referral center (Fourth Department of Surgery, Helsinki University Central Hospital). MATERIAL: 105 consecutive unreconstructed patients with 136 critically ischaemic legs as defined by the European Consensus Document on Chronic Critical Leg Ischaemia. MAIN OUTCOME MEASURES: Major amputations and mortality. RESULTS: 81% of the 136 critically ischaemic legs survival 1 month, 70% three months and 54% one year. Of the 105 patients 93%, 77% and 46% were alive at 1, 3 and 12 months, respectively, whereas survival of patients with nonamputated leg was only 71%, 56% and 28%. Patients with bilateral CLI had a worse prognosis in terms of survival and leg salvage. The leg outcome was not worsened by the presence of diabetes nor by the distal extent of arterial changes. CONCLUSIONS: Although the selection of the present material is likely to cause some bias, unreconstructed CLI seemed to predict a very poor outcome in terms of survival and limb salvage. PMID- 8616644 TI - Infected false aneurysms of the carotid arteries after carotid endarterectomy. AB - OBJECTIVE: To determine the incidence and management of infected false aneurysms following carotid endarterectomy. DESIGN: Case notes of patients undergoing carotid endarterectomy (CEA) between the years of 1980 and 1993 at two major teaching hospitals, or those patients who represented with complications were reviewed. RESULTS: Eight patients were identified with infected false aneurysms, an incidence of 0.625%, in five CEA had been performed at one of the teaching hospitals, whilst in three other cases the primary operation had been done elsewhere. Presentation was a median 19 days following CEA. In five cases the original arteriotomy was closed by direct suture whilst in three a saphenous vein patch was used. Staphylococcal organisms were cultured in all cases. Antibiotics had not been administered at the original operation. Repair with saphenous vein graft from the common to the internal carotid artery had the least complications. CONCLUSION: Infected false aneurysms are a rare complication following CEA, resection of the false aneurysm and reconstruction with autologous saphenous vein is recommended. Ligation alone is associated with a high incidence of stroke. PMID- 8616646 TI - The aortic polytetrafluoroethylene graft: further experience. AB - OBJECTIVES: We analysed our results with the use of aortic polytetrafluoroethylene PTFE grafts over the last 7.5 years. A historical comparison was also made between the results with non-stretch PTFE (NS-PTFE) (1987-91) and stretch PTFE (S-PTFE) grafts (1991-94). MATERIALS: 244 infrarenal aortic replacements or bypasses with PTFE grafts were performed at the University of Iowa Hospitals and Clinics from January 1987 to June 1994. Infrarenal aortic replacement was indicated for aortic aneurysmal disease in 192 patients (elective 151, symptomatic 20, ruptured 21) and bypass for aorto-iliac occlusive disease in 52 patients (disabling claudication 28, limb salvage 24). Patients ranged in age from 37 to 93 years (mean 68 years). There were 161 males and 83 females. Medical risk factors included hypertension (55%), coronary artery disease (31%), COPD (23%), diabetes mellitus (12%) chronic renal failure (9%), and smoking (61%). Aortic replacement or bypass was done with a NS-PTFE graft in 108 patients (44%) and a S-PTFE graft in 136 patients (56%). Postoperative ultrasound (US) scans and/or CT-studies were available in 40 patients with NS-PTFE and 26 patients with S-PTFE grafts. MAIN RESULTS: The 30 day operative mortality was: elective AAA patients (1.3%), symptomatic AAA patients (10%), ruptured AAA patients (48%), limb salvage patients (4.1%) and disabling claudication patients (0%). Graft related complications included five graft limb thromboses (4 NS-PTFE, 1 S-PTFE). Two thromboses occurred perioperatively and the three others at 24, 28 and 30 months postoperatively. Two other graft related complications included a mixed pseudomonas and streptococcus groin infection with a culture negative perigraft fluid collection occurring 3 weeks following surgery (NS-PTFE), and distal aortic anastomotic suture line bleed on the first postoperative day following replacement of a ruptured AAA with a S-PTFE graft. Based on US and/or CT imaging studies, the mean internal diameters of the bodies of 40 NS-PTFE and 26 S-PTFE grafts were 11% and 10% greater than the manufacturer's specified sizes at a mean follow-up duration of 36 and 10 months respectively. CONCLUSIONS: These data reveal that a PTFE graft performs satisfactorily in the aortic position with minimal adverse clinical sequence over a 7.5 year period. Continued long term follow up data will determine the ultimate suitability of aortic PTFE grafts. PMID- 8616647 TI - Vein graft stenosis: incidence and intervention. AB - OBJECTIVES: The incidence of vein graft stenosis ranges from 5%-45%. Reported rates appear to be increasing as technological advances make detection easier. The aim of this study was to review our experiences with regard to the incidence of stenosis in infrainguinal bypass grafts and the outcome of intervention for salvage of failing grafts. DESIGN: Retrospective review of graft surveillance records. SETTING: Vascular Studies Unit, Bristol Royal Infirmary. METHODS: A Duplex-based graft surveillance (GS) programme was used from January 1989 to June 1994 to study 275 primary graft procedures in 250 patients with lower limb ischaemia. Patients were scanned at 1 week, 6 weeks and 3, 6, 9 and 12 months postoperatively. RESULTS: One year cumulative limb salvage, patient survival and primary, primary assisted and secondary patencies were 91%, 83%, 67%, 77% and 84% respectively. Duplex scanning detected 85 vein graft stenoses in 59 patients: an incidence of 21.5%. In addition, 64 potentially graft-threatening inflow (14) and outflow (50) problems were detected in the native vessels of 52 patients from clamp damage or progression of disease (POD). Of the 85 graft stenoses, 40 were treated by balloon angioplasty (PTA) and 20 by surgical intervention and 1 patient's symptoms were treated by chemical sympathectomy. Twenty-four patients were not actively treated. Of the 64 grafts affected by POD, 20 were treated by PTA, 15 by surgery, one with anti-coagulation and 28 had no treatment. Comparing patients with non-treated and treated lesions, the respective 12 month cumulative patencies for patients with graft stenoses were 75% and 87.5% as against 86% and 83% for patients with POD (log rank test 0.1). CONCLUSIONS: These results uphold the perceived benefits of a GS programme, although the evidence from the non treated cases in this series reinforces a need for a large, prospective, randomised trial to confirm the case for GS. PMID- 8616648 TI - A prospective comparison of lower limb colour-coded Duplex scanning with arteriography. AB - OBJECTIVE: To compare the diagnostic value of colour Duplex scanning with arteriography for the detection of arterial disease of the aortoiliac arteries, femoropopliteal arteries and the origins of the tibial vessels. DESIGN: Prospective, semi-blind study. SETTING: Vascular laboratory and radiology department, University Hospital. METHODS: A total of 1658 arterial segments in 148 limbs were studied both by colour Duplex scanning and digital subtraction arteriography. Individual arterial segments were classified on the basis of peak systolic velocity ratios < 2.0, > or = 2.0 or an absent Doppler signal, as 0-49%, 50-99% diameter reduced, or occluded. The same arterial segments were similarly classified on the basis of arteriography and the two modalities were compared using a Kappa (k) analysis. RESULTS: The overall agreement between arteriography and colour-coded Duplex was kappa = 0.74 (95% CI, 0.70-0.78), this indicates substantial agreement. Kappa values (95% CI) from the aortoiliac, femoropopliteal and the origins of the infrapopliteal arteries were kappa = 0.59 (0.49-0.73; moderate agreement), kappa = 0.80 (0.76-0.84; substantial agreement) and kappa = 0.48 (0.35-0.61; moderate agreement) respectively. CONCLUSIONS: We conclude that there is substantial agreement between colour-coded Duplex and arteriography of the lower limbs, and that the ability of colour-coded Duplex to plan and guide lower limb vascular interventions requires investigation. PMID- 8616649 TI - Amputation risk and survival after embolectomy for acute arterial ischaemia. Time trends in a defined Swedish population. AB - OBJECTIVES: To assess the outcome of embolectomy over an 19 year period. METHODS: Time trends in the outcome of acute arterial thrombo-embolectomy of the extremities were analysed in a population-based cohort of 1190 patients operated on between 1965-83. RESULTS: A total of 262 (22%) initial amputations were performed. The limb salvage rates at 5 years postoperatively were lower between 1975-79 (61%) than between 1965-69 (81%). A proportional hazards model revealed a relative hazard (RH) of amputation of 2.2 (95% confidence interval (CI) 1.3-3.3) for 1975-79 compared with 1965-69. Operation at any district hospital entailed a 70% higher risk of amputation (RH 1.7; 95% CI 1.3-2.5) compared with the University hospital. The relative survival rate at 5 years postoperatively decreased towards the end of the study period (33% between 1975-79 compared with 43% between 1965-69). Younger age-groups had a considerably lower risk of death in the University hospital compared with the county and district hospitals. CONCLUSIONS: Contrary to the results in other hospital based reports no improvement in amputation or survival rates since 1965 could be demonstrated in this large series with no patient selection. PMID- 8616650 TI - Screening for abdominal aortic aneurysm: a computer assisted cost-utility analysis. AB - OBJECTIVES: To evaluate the effects of introducing routine ultrasonic screening for the identification and elective surgical treatment of abdominal aortic aneurysms (AAA) at high risk of rupture in the U.K. population of men aged 65-74 years. DESIGN: A computer assisted simulation of an AAA screening programme. The simulation incorporated assumptions gleaned from the literature about the epidemiology of AAA and the costs of screening. In addition, up-to-date costings based on recent Manchester (U.K.) vascular surgery experience are used. SETTING: A dialogue between National Health Service commissioners and providers to explore the feasibility and desirability of introducing AAA screening. CHIEF OUTCOME MEASURE: Cost per quality adjusted life year (QALY) gained. MAIN RESULTS: The absolute cost (circa 1992/3) per QALY gained from screening for and treating aneurysms of > or = 6 cm in diameter of pounds 1500 (benefit not discounted). Offsetting current treatment costs of ruptured aneurysms gives a net additional cost per QALY of pounds 1300. Screening and treating aneurysms of > or = 5 cm leads to a cost per QALY gained exceeding pounds 20000. The findings are robust under sensitivity analysis. CONCLUSIONS: Routine screening for AAAs of size > or = 6 cm compares favourably in terms of cost per QALY gained with services such as breast and cervical cancer screening. PMID- 8616651 TI - Quality of life following percutaneous transluminal angioplasty for claudication. AB - OBJECTIVE: To determine quality of life in claudicants before and after percutaneous transluminal angioplasty (PTA). DESIGN: Prospective study using EuroQuol questionnaire and visual analogue scales. SETTING: District general hospital. MATERIAL: Twenty-nine patients undergoing successful PTA. RESULTS: Before PTA patients showed a significantly lower EuroQuol score compared with the normal population. There was also marked impairment of patients usual activities, mobility and perceived health state. Following successful PTA the EuroQuol score, perceived health state, mobility, usual activities, pain and discomfort and mental state were all significantly improved. CONCLUSIONS: Intermittent claudication impairs quality of life. Following successful PTA there is significant improvement in symptoms and in general health. PMID- 8616652 TI - Ambulatory venous pressure: correlation with skin condition and role in identifying surgically correctible disease. AB - OBJECTIVES: (1) To evaluate the full spectrum of venous skin damage with respect to ambulatory venous pressure. (2) To determine whether the ambulatory venous pressure/tourniquet test can be used to select patients for superficial venous surgery (eg. long or short saphenous stripping). DESIGN: Prospective study. SETTING: Vascular studies unit. MATERIALS AND METHODS: Ambulatory venous pressure was measured in a larger sample of limbs (360) with a wide spectrum of venous disease. In addition the effect of a tourniquet placed below the knee on ambulatory venous pressure and venous refilling time was assessed in 234 limbs. This was compared with Duplex assessment of deep and superficial venous reflux at this site. RESULTS: There was a linear trend towards more severe skin damage with increasing ambulatory venous pressure. Ulceration was associated with more severe calf muscle pump dysfunction (higher ambulatory venous pressure) than were lipodermatosclerosis, eczema or pigmentation. The tourniquet test was not able to distinguish between deep and superficial reflux as determined by Duplex scanning. CONCLUSIONS: Ambulatory venous pressure should be used to quantify venous insufficiency and remains the reference standard test of the venous calf muscle pump. The tourniquet test should not be used to select patients for surgery since it cannot distinguish deep from superficial venous incompetence. Venous reflux is best localised using Duplex ultrasound. PMID- 8616653 TI - Gastric intramucosal pH predicts outcome after surgery for ruptured abdominal aortic aneurysm. AB - OBJECTIVE: The mortality associated with repair of ruptured abdominal aortic aneurysms (RAAA) remains obstinately high and many deaths result from multiple organ failure which is likely to be related to splanchnic ischaemia. The aim of this study is to investigate the importance of splanchnic ischaemia in determining outcome from RAAA by comparing gastric intramucosal pH with other methods of assessing the adequacy of splanchnic oxygenation. DESIGN AND SETTING: Prospective cohort of patients following surgery for RAAA admitted to the Intensive Care Unit of Guy's Hospital, London. OUTCOME MEASURES: Gastric intramucosal pH (pHim) and global haemodynamic, oxygen transport and metabolic variables were measured on admission, at 12 h and at 24 h after admission. Results were compared between survivors and non-survivors and Receiver Operating Characteristic (ROC) curves were constructed to assess the ability of each measurement to predict outcome. RESULTS: The median 24 h APACHE II was 18 and the ICU mortality 45.5%. Gastric pHim was significantly higher in survivors than non survivors at 24 h (7.42 vs. 7.24, p < 0.01). In survivors who had a low intramucosal pH (pHim) on admission there was a significant improvement over the first 24 h (7.26 to 7.40, p < 0.05), whereas in patients who subsequently died, and had a normal pHim on admission, there was a significant fall in pHim (7.35 to 7.16, p < 0.05). ROC curves showed that gastric pHim was the most sensitive measurement for predicting outcome in these patients. CONCLUSIONS: Gastric intramucosal pH is the most reliable indicator of adequacy of tissue oxygenation in patients with RAAA, suggesting that splanchnic ischaemia may have played an important role in determining survival. PMID- 8616654 TI - Anastomotic femoral aneurysms: increase in interval between primary operation and aneurysm formation. AB - OBJECTIVE: Anastomotic pseudoaneurysms continue to be a late complication of vascular surgery, particularly following prosthetic graft procedures. The purpose of this study was to investigate if a previously reported increase in interval between the original operation and the development of pseudoaneurysm was still valid. DESIGN: Retrospective study. MATERIAL AND METHODS: We reviewed the records of 76 patients who presented with 90 femoral aneurysms. The median age was 69 years (range: 39-83). The commonest previous vascular surgery was a aortofemoral bypass in 61 cases. RESULTS: The interval between the original operation and the repair of the pseudoaneurysms was 9 years (range 1 month to 26 years). CONCLUSIONS: This study confirms the previously noted trend of an increasing time to aneurysm formation from 3 years before 1975, 5 years between 1976 and 1980, and 6 years between 1981 and 1990. PMID- 8616655 TI - Neutrophil activation in jugular venous blood during carotid endarterectomy. AB - OBJECTIVES: Neutrophils may play an important role in cerebral ischaemia. We investigated whether neutrophil activation can be detected in cerebral venous blood during the mild cerebral hypoxia and reperfusion that occurs during carotid cross clamping and declamping for endarterectomy. DESIGN, SETTING AND MATERIALS: The ipsilateral jugular bulb was cannulated at operation in 16 patients undergoing carotid endarterectomy. Blood was taken immediately prior to and 30 seconds following internal carotid cross clamping; then immediately prior to, 30 s and 2 m following declamping. Blood was also taken from a peripheral vein in the foot. Intracerebral oxygen saturation (CsO2) was measured continuously by near infrared cerebral spectroscopy. Neutrophil activation was measured by flow cytometric detection of fluorescence to hydrogen peroxide in unstimulated cells and phorbol myristate acetate stimulated cells, and expressed as mean fluorescent intensity (MFI). OUTCOME MEASURES: Neutrophil activation and cerebral oxygenation. MAIN RESULTS: CsO2 fell from 68% (95% Confidence interval 64%-72%) to 63% (59%-68%) following carotid cross clamping (p < 0.05, repeated measures analysis of variance). This recovered slightly during the cross clamp period to 64% but only returned to preclamp levels following declamping (p > 0.05). Neutrophil hydrogen peroxide generation by stimulated neutrophils rose significantly from 0.79 mean fluorescent intensity (0.53-1.19) to 1.46 (0.98 2.20) but no there was no further rise following cross clamp release. There was no significant neutrophil activation in the peripheral samples. CONCLUSIONS: These results indicate that even mild cerebral hypoxia is associated with priming of neutrophils in cerebral venous blood. PMID- 8616656 TI - Early experience with transfemoral endovascular aneurysm management (TEAM) in the treatment of aortic aneurysms. AB - OBJECTIVES: To evaluate the early experience with transfemoral endovascular aortic aneurysm management using the Endovascular Grafting System. DESIGN: Multi centre prospective evaluation of the implantation procedure and early results (median follow-up 153 days). SETTING: Department of Surgery, University Hospital Utrecht, The Netherlands; Department of Surgery, University of Sydney, Australia; University of Leicester School of Medicine, Leicester, U.K., Department of Surgery, Karolinska Hospital, Stockholm, Sweden and Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital; Oxford, U.K. MATERIALS: 31 consecutive patients treated in 13 months. CHIEF OUTCOME MEASUREMENTS: Peri and postoperative morbidity and mortality in accordance with the recommendations of the Ad Hoc Committee on Reporting Standards. MAIN RESULTS: Graft placement was initially successful in all 31 patients. In one patient the endograft had to be replaced by a standard aortic tube graft because of extra graft flow in the aneurysm sac, and complaints of back pain. One patient died from multiple organ failure, 11 days after the operation. In three patients five severe adverse events were recorded. Breaks of the attachment system were encountered in two patients. These failures did not have severe clinical consequences for individual patients. CONCLUSIONS: Transfemoral Endovascular Aneurysm Management is a technically demanding procedure that requires special training in both catheter and surgical techniques. The potential for less operative morbidity when compared to conventional surgery and the prospect of technical improvements in graft and introduction system design will make TEAM an important tool in aneurysm management in the near future. PMID- 8616657 TI - A survey of methods used for cardiac risk assessment prior to major vascular surgery. AB - OBJECTIVES: To assess the frequency with which various methods of cardiac risk assessment are used prior to major vascular surgery and the way in which patients considered to the "high" risk are managed. DESIGN: Questionnaire survey. SETTING: Great Britain and Northern Ireland. MATERIALS: Vascular Surgeons who are current members of the Vascular Surgical Society of Great Britain and Northern Ireland. CHIEF OUTCOME MEASURES: Number of respondents reporting routine or frequent use of particular investigations and methods of management. MAIN RESULTS: Of 246 respondents, 52% had access to a high dependency unit and 77% used intensive therapy units routinely following aortic reconstruction. Some measure of ejection fraction was the most common investigation and was used routinely prior to aortic reconstruction by 35% and often by 33% of respondents, this being more frequent in respondents from teaching hospitals and those carrying out a greater number of reconstructions. Calculated clinical risk indices were rarely used. The identification of high risk patients led to referral to a cardiologist for 90% of respondents and influenced the choice of anaesthetist for 50%. CONCLUSIONS: It is concluded that there is considerable variation in practice, but that those who carry out more vascular surgery are more aggressive in their assessment of cardiac risk prior to reconstruction. PMID- 8616659 TI - Assessment of wall structure and composition of varicose veins with reference to collagen, elastin and smooth muscle content. AB - OBJECTIVES: To compare collagen, elastin and smooth muscle contents of varicose and control long saphenous veins. DESIGN: Collagen, elastin and muscle were estimated stereologically using random sampling and histological staining. MATERIALS: Varicose vein samples were collected from nine patients (mean age 52 years, range 34-64 years) undergoing vein stripping, sample sites being saphenofemoral junction and knee. Control samples were taken from five patients (mean age 58 years, range 38-76 years) presenting for femoral-popliteal bypass at equivalent levels. METHODS: Veins were fixed, sectioned transversely, and stained with Picric Acid Sirius Red. Analysis of samples was performed using point and intersection counting on vertically projected images. RESULTS: Using two way analysis of variance tests, varicose saphenous veins had significantly larger wall areas (p < 0.01) and higher amounts of collagen (p < 0.01). Collagen content and wall area were significantly larger proximally compared to distally in both control and varicose veins (p < 0.05) with a higher content of smooth muscle and elastin in varicose veins proximally compared to distally (p < 0.05). There was no difference in wall thickness or elastin content between the two groups. CONCLUSIONS: This suggests that varicose veins are a dynamic response to venous hypertension and are not thin walled structures as previously thought. PMID- 8616658 TI - Surgical management of brachioaxillary-subclavian vein occlusion. AB - OBJECTIVE: The possibility of using RING PTFE graft as venous bypass to preserve arteriovenous graft function and reduce upper extremity swelling. METHODS: Twenty two patients with stenosis/occlusion of the brachial-axillary-subclavian vein segment in haemodialysis patients (n = 19) and patients with penetration injury (n = 3) who were not candidates for balloon angioplasty were treated with ring PTFE venous bypass in renal patients and jugular to axillary vein transposition for trauma patients and followed for 10-87 months (mean 31) using venography, Doppler analysis and Duplex scanning. RESULTS: There was no death or neurologic deficit resulting from the venous bypass. Resolution of swelling occurred in 8-48 h. 19/22 (86%) of the bypasses and 3/3 transpositions remained patent after a mean follow-up of 31 months (10-87) months. The attrition was due to AV graft occlusion (n = 2) and infection requiring graft removal (n = 1). CONCLUSIONS: Ring PTFE graft is an acceptable venous bypass for brachial-axillary-subclavian stenosis/occlusion to reduce arm swelling and preserve the function of AV grafts in patients with lesions not amendable with balloon angioplasty or thrombolytic therapy. Jugular-axillary transposition is inappropriate for renal patients. PMID- 8616660 TI - Popliteal artery entrapment caused by bony exostosis. PMID- 8616661 TI - Hypothenar hammer syndrome caused by playing tennis. PMID- 8616663 TI - Prostacyclin analogues. PMID- 8616662 TI - Late rupture of femoropopliteal Dacron grafts: a rare complication. PMID- 8616664 TI - Femoral osteochondroma. PMID- 8616665 TI - Pedal aneurysms. PMID- 8616666 TI - Carotid patches. PMID- 8616667 TI - Carotid plaque. PMID- 8616668 TI - Effect of co-fractionation technique in the preparation of palm oil and sal fat based cocoa butter equivalent. AB - Two types of palm oil and sal fat based cocoa butter equivalents, namely fCBE (produced by using co-fractionation method) and mCBE (produced by using conventional method) were prepared. Results showed that the fCBE had triglyceride composition and solidification characteristics closer to the Malaysian cocoa butter than the mCBE produced at the same yield percentage. Increasing acetone washing time had little effect on the fCBE if compared to the effect of increasing palm olein to sal fat blend ratio. Co-fractionation technique increase the compatibility between CBE component triglycerides. Thus, more palm oil can be incorporated in the preparation and the process can be carried out at not low temperature as compared to the conventional method. PMID- 8616669 TI - Dietary nitrite and scavenger antioxidants trace elements. AB - Rats initially weighing 138 +/- 14 g were fed the following diets for 150 days: control (Co), control plus nitrite-bacon-proline 24 mg/kg, 100 g/kg and 10 mg/kg, respectively (NB), NB plus 0.04 micrograms/g selenium (NBSe) and NB plus 0.020 g/kg ascorbic acid (NBC). The NB diet provoked body weight and feeding efficiency enhancement with a reduction in body density increasing serum lactic acid, uric acid and cholesterol levels. The serum selenium decreased by the presence of NB in the diet. The addition of selenium and ascorbic acid to the NB diet prevented the reduction in body density and also affected uric acid and cholesterol levels. It is suggested that the NB diet has adverse effects and that some of the alterations it causes are prevented by the reducing elements selenium and ascorbic acid. PMID- 8616670 TI - Effect of malting time on chemical composition and functional properties of soybean and bambara groundnut flours. AB - Soybean and bambara groundnut were malted for 0, 24, 48, 72, 96 and 120 h, respectively. The malted beans were dried, devegetated and milled into flour. The protein contents of unmalted soybean and bambara groundnut flours which were 48.3 and 18.8% (dry weight), respectively, increased on malting to maximum levels of 56.17 and 28.55% (dry weight) after 2 days before decreasing to 19.51 and 20.92% (dry weight) for soybean and bambara groundnut flours, respectively. The same pattern of change was observed for the fat, soluble sugar and nitrogen contents of the malted bean flours. Approximately 76 and 59% of the phytic acid contents of soybean and bambara groundnut flours were removed, respectively, on malting for 120 h. Malting significantly affected the water and oil absorption capacities as well as the viscosities of both bean flours. The optimum malting time for both beans was found to be between 72 and 96 h. PMID- 8616671 TI - Nutritional quality of green beans (var. Tuf) fermented with starter culture. AB - Green beans (var. Tuf) were blanched at 90 degrees C for 20 min and then fermented in 3.08% salt solution with starter culture. Trypsin inhibitors, vitamins B1 and B2 and protein were determined before and during fermentation as well as after storage for 36 days. Trypsin inhibitors, vitamins B1 and B2 were reduced significantly (P < or = 0.05) during fermentation. A decrease in protein content was observed during the first 2 days of fermentation and thereafter the decrease was not significant (P > or = 0.05). Storage of pasteurised beans at 20 degrees for 36 days had no significant effect on the vitamins and protein. PMID- 8616672 TI - Mineral values of selected plant foods common to southern Burkina Faso and to Niamey, Niger, west Africa. AB - Wild and cultivated fruits, leaves, nuts, seeds, spices and vegetables from southern Burkina Faso and Niamey, Niger, were analysed for their copper, iron, magnesium, manganese and zinc concentrations and compared to imported, exotic reference foods found within the study area. The species analysed covered a broad spectrum of local diet; 33 were wild and 16 were cultivated. The edible wild plants were often the highest in mineral concentrations. Five species analysed, exhibited consistently high mineral values, specifically, Adansonia digitata, Boerhavia diffusa, Cerathoteca sesamoides, Sclerocarya birrea and Xylopia sp. The latter was particularly high in zinc, an observation which suggests that there may be a solid rationale for local traditions which recommended its consumption during pregnancy and lactation. Respondents indicated that during times of drought, wild plants were not consumed in the volume they once were, due to changes of infrastructure and in famine relief programmes. PMID- 8616673 TI - Development by extrusion of soyabari snack sticks: a nutritionally improved soya maize product based on the Nigerian snack (kokoro). AB - A nutritionally improved local snack compared to existing kokoro has been developed by extrusion cooking of different formulations of maize, soybean and condiments such as pepper, onion, salt, palm oil, plantain and banana. The improved snack was named as the 'soyabari snack stick'. The chemical composition of representative extruded products indicates a high level of crude protein, fat, energy, available lysine and improved in vitro digestibility compared to the usual maize-based products. The level of stachyose and raffinose were greatly reduced in the extruded products compared to raw soya. Formulations using various additives yielded products suitable for different consumers' preferences such as hot, sweet, bland, gritty or crispy and acceptable to taste assessors. Soyabari snack sticks were equally acceptable as Bombay mix, a product on the market in London. Sensory analysis showed no significant differences in the two products but the crude fibre content of Bombay mix was higher while the protein was slightly lower than for soyabari sticks. Local ingredients can produce acceptable extrudates. PMID- 8616674 TI - Influence of dietary spices or their active principles on digestive enzymes of small intestinal mucosa in rats. AB - A few common spices or their active principles, were examined for their possible influence on digestive enzymes of intestinal mucosa in experimental rat. The animals were fed the following diets for 8 weeks: control, curcumin (0.5%), capsaicin (15 mg%), piperine (20 mg%), ginger (50 mg%), cumin (1.25%), fenugreek (2%), mustard (250 mg%) and asafoetida (250 mg%). Dietary curcumin, capsaicin, piperine and ginger prominantly enhanced intestinal lipase activity and also the disaccharidases sucrase and maltase. Dietary cumin, fenugreek, mustard and asafoetida brought about decreases in the levels of phosphatases and sucrase. The positive influences of a good number of spices on these terminal enzymes of digestive process could be an additional feature of species that are generally well recognized to stimulate digestion. PMID- 8616676 TI - Blending of palm oil, palm stearin and palm kernel oil in the preparation of table and pastry margarine. AB - Palm oil (PO ; iodin value = 52), palm stearin (POs1; i.v. = 32 and POs2; i.v. = 40) and palm kernel oil (PKO; i.v. = 17) were blended in ternary systems. The blends were then studied for their physical properties such as melting point (m.p.), solid fat content (SFC), and cooling curve. Results showed that palm stearin increased the blends melting point while palm kernel oil reduced it. To produce table margarine with melting point (m.p.) below 40 degrees C, the POs1 should be added at level of < or = 16%, while POs2 at level of < or = 20%. At 10 degrees C, eutectic interaction occur between PO and PKO which reach their maximum at about 60:40 blending ratio. Within the eutectic region, to maintain the SFC at 10 degrees C to be < or = 50%, POs1 may be added at level of < or = 7%, while POs2 at level of < or = 12%. The addition of palm stearin increased the blends solidification Tmin and Tmax values, while PKO reduced them. Blends which contained high amount of palm stearin showed melting point and cooling curves quite similar to that of pastry margarine. PMID- 8616675 TI - Protein quality and urea kinetics in prepubertal Chilean schoolboys. AB - Urea kinetics were measured non-invasively in 12 Chilean schoolboys aged 8-10 years who were receiving one of two diets, either predominantly animal protein or predominantly vegetable protein. Both the diets provided an equivalent level of gross protein, 1.2 g/kg/day. The study diets were given for 10 days to enable adaptation to take place. On the eighth day a single oral dose of 15N15N-urea, 100 mg, was given and the amount of label excreted as 15N15-urea in urine over the subsequent 48 hours was measured. There was little difference in any aspect of urea kinetics between the two diets with urea production (animal, 173 +/- 50 mgN/kg/day; vegetable 179 +/- 53 mgN/kg/day), urea excretion (animal, 86 +/- 19 mgN/kg/day; vegetable, 105 +/- 13 mgN/kg/day), urea nitrogen hydrolysis (animal, 87 +/- 49 mgN/kg/day; vegetable, 74 +/- 42 mgN/kg/day), and the salvaged urea nitrogen derived from hydrolysis which returned to urea formation (animal, 12 +/- 5 mgN/kg/day; vegetable, 17 +/- 9 mgN/kg/day) all being similar. A very high proportion of the salvage nitrogen derived from urea hydrolysis was maintained within the metabolic pool, about 80%, which was equivalent to 0.4 g protein/kg/day. This is the first time urea kinetics have been measured in children of this age and shows that 57% of the ura produced is excreted in urine on average with about 43% of the urea-nitrogen being salvaged for further metabolic interaction. It is concluded that the vegetable based protein diet taken habitually by Chilean children is metabolically equivalent in terms of urea kinetics to a diet based upon animal protein at this level of intake, but that high rates of salvage of urea nitrogen are found on both diets. PMID- 8616677 TI - Estimation of selenium bioavailability from human, cow's, goat and sheep milk by an in vitro method. AB - The trace element selenium (Se) has been recognized to be essential for human health. The dependence of infants on milk as their principal food source, generally low in Se content, makes them more vulnerable to inadequate Se intake. The present study compared the Se availability as estimated by a simulated gastrointestinal digestion procedure, of human milk and some common ruminant milks, namely cow, goat and sheep milk. The Se availability of human milk (11.1%) was significantly higher compared to that of cow (6.8%), goat (6.2%) and sheep milk ( < 2%). Further study suggested that the Se availability may be related to the gastric digestibility of protein. The high Se availability of human milk might be attributed to the high gastric digestibility of human milk protein. It was found that removal of the milk fat fraction increases the Se availability. PMID- 8616678 TI - Availability of calcium for absorption in the small intestine and colon from diets containing available and unavailable carbohydrates: an in vitro assessment. AB - An in vitro method simulating conditions in the small intestine and colon was used to study the effects of various carbohydrates on Ca release from basal diet (BD) containing dairy products. During enzymatic digestion of BD, 28.5 +/- 0.3% of the Ca was released. This was reduced by 3, 22 and 27% by adding bread, psyllium or pectin to BD, respectively (P < 0.05). After enzymatic digestion, the residue from BD was fermented releasing 11.9 +/- 1.2% of the Ca, a value which was significantly less than with pectin (13.7 +/- 0.9%) and greater than with psyllium (4.4 +/- 0.2%) addition. The total Ca release ranged from 26.5 +/- 0.8 to 42.2 +/- 1.0% with bread>BD>pectin>psyllium. Lactulose did not differ significantly from BD. These results suggest that carbohydrates may bind Ca and reduce its availability for absorption in the small intestine. However, if the carbohydrate is fermented, bound Ca may be released for potential absorption in the colon, whereas less fermented carbohydrates may continue to bind Ca in the colon. The in vitro method described may be useful for estimating total Ca availability. However, studies in humans are required to validate these results. PMID- 8616680 TI - [Ultrasonographic aspects of cystic papillary tumor of the pancreas. Apropos of 2 cases]. AB - Cystic papillary pancreatic tumor is an infrequent neoplasm of unknown origin which is presented almost exclusively in young women. It is characterized by an anodyne clinical symptomatology with it frequently treated as an accidental finding. Ultrasonography and computerized axial tomography have a priority role in the study of this entity but the definitive diagnosis is based on the histologic findings. The treatment of choice is surgical removal and although it is a malignant tumor, good evolution is observed with recurrence not being expected. Two new cases of cystic papillary tumor are presented comparing the ultrasonographic appearance of the same with other pancreatic cystic neoplasms. Although the ultrasonographic findings are not characteristic they may be suggestive of papillary tumor. PMID- 8616679 TI - [Endoscopic sclerotherapy is useful in Dieulafoy's disease ]. AB - The clinical histories of 14 patients with digestive hemorrhage due to Dieulafoy disease admitted to the authors' hospital over 74 months were retrospectively analyzed. These cases represent 1.18% of the non varicose upper digestive hemorrhages (CI 95%; 0.57-1.79%). Male predominance (6:1) and advanced age (median: 67.5 years) were observed. The ingestion of potentially gastroerosive drugs, smoking and alcoholism were reported in 57, 57 and 7% of the cases, respectively. Six patients (43%) required more than one endoscopy for diagnosis. The lesion responsible for hemorrhage was found at 6 cm or less from the cardias on 11 occasions (78%). Endoscopic sclerotherapy was performed in the vessel by polidocanol at 2% (1.5-10 ml, median: 6). In 10 cases sclerotherapy was preceded by the injection of adrenaline 1/10,000 (2-11 ml, median: 5). Definitive hemostasis was achieved in 11 patients (78%) (two of these cases required further sclerotherapy); 2 patients required surgery, and one patient (7%) died. Of the 13 surviving patients, none presented relapse after follow up of 2-63 months. We can conclude that Dieulafoy disease is an infrequent but severe cause of digestive hemorrhage, being predominantly found in males over the age of 50 years. Repeated endoscopy is often required to determine diagnosis and treatment. Endoscopic sclerotherapy with polidocanol is effective and safe in this disease although surgery may be required and death may occur. PMID- 8616681 TI - [Recombinant alfa-2 interferon treatment of a patient with chronic hepatitis B and ulcerative colitis]. AB - The case of a patient ulcerative colitis involving an autoimmune base who was treated with recombinant alpha-2 interferon for concomitantly presenting chronic hepatitis B in the replicative phase is reported. With this therapy unreplicative seroconversion of the disease was achieved without modification of the course of the chronic inflammatory intestinal disease (CIID) during treatment or over the posttreatment follow up period. A brief review of the literature was performed concerning the role of autoimmunity in ulcerative colitis, treatment with alpha interferon in chronic hepatitis B and the exacerbation of autoimmune phenomena which may lead to interferon treatment. According to the evolution of this case and the review of the literature, the authors conclude that the existence of CIID does not contra-indicate the use of recombinant alpha-2 interferon in patients with chronic viral hepatitis, although special control of the disease should be carried out during the treatment period. PMID- 8616682 TI - [Ursodeoxycholic acid: an alternative in the treatment of chronic cholestasis]. PMID- 8616683 TI - [Somatostatin and its analogs in the treatment of gastrointestinal and liver diseases]. PMID- 8616684 TI - [Low incidence of hepatitis C virus transmission in stable sexual partners]. PMID- 8616685 TI - [Scrotal edema as initial manifestation of squamous carcinoma of the esophagus]. PMID- 8616686 TI - [Simple pneumomediastinum and upper digestive endoscopy]. PMID- 8616687 TI - [Acute hyperlipemic pancreatitis and pregnancy]. PMID- 8616688 TI - [Abdominal cystic lymphangiomas]. PMID- 8616689 TI - [Leukocytoclastic vasculitis and alcoholic hepatic cirrhosis]. PMID- 8616690 TI - [Spontaneous pneumoperitoneum associated with bronchial carcinoma]. PMID- 8616691 TI - Managed care & conflict of interest. PMID- 8616692 TI - Mother-infant HIV transmission: making the most of what we know. PMID- 8616693 TI - Tissue factor expression in normal and abnormal mammary gland. PMID- 8616694 TI - What is leptin for, and does it act on the brain? PMID- 8616696 TI - Caffeine dependence: an alternative view. PMID- 8616695 TI - Plasmin, fibrin degradation and angiogenesis. PMID- 8616697 TI - Japan settles HIV-tainted blood cases. PMID- 8616698 TI - Buying life insurance from the dying gets riskier. PMID- 8616699 TI - Is new AIDS report just another piece of paper? PMID- 8616700 TI - How much weight should viral load carry? PMID- 8616701 TI - Hippocrates a la mode. PMID- 8616702 TI - There is a renaissance of interest in pallidotomy for Parkinson's disease. PMID- 8616703 TI - Naked DNA: new shots for allergy? PMID- 8616704 TI - Shaking down new epilepsy genes. PMID- 8616705 TI - Food for starving hearts. PMID- 8616706 TI - Genetic studies of manic-depressive illness. PMID- 8616707 TI - The cost of treating Gaucher disease. PMID- 8616708 TI - Cellular proofreading. PMID- 8616709 TI - Genetically modified mice as models of transplant atherosclerosis. PMID- 8616710 TI - Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor type integrins inhibits retinal neovascularization. AB - Retinal neovascularization is a major cause of blindness in such disorders as retinopathy of prematurity, proliferative diabetic retinopathy and senile macular degeneration. Because ligation of vitronectin receptor-type integrins appears to be required for the survival and maturation of newly formed but not quiescent blood vessels in several vascular beds including the retina, blockade of this downstream adhesion receptor system was investigated. In a mouse model of hypoxia induced retinal neovascularization twice daily administration of 1 to 20 mg cyclic alpha v-integrin antagonist peptide per kilogram of body weight reduced capillary proliferation in a dose-dependent fashion--maximum 76%--without obvious side effects. A cyclic control peptide displayed no inhibitory effect on neovascularization. These findings indicate that systemic application of vitronectin receptor antagonists appears to be clinically feasible and is efficient in preventing retinal neovascularization and superior to cytokine blocking strategies. PMID- 8616711 TI - Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. AB - Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress-induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5 (FGF-5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer. PMID- 8616712 TI - Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization. AB - The efficacy of an "allergen-gene immunization" protocol in altering allergic response was examined. Intramuscular injection of rats with a plasmid DNA encoding a house dust mite allergen into the muscle results in its long-term expression and the induction of specific immune responses. Significantly, this approach prevents the induction of immunoglobulin E synthesis, histamine release in bronchoalveolar fluids, and airway hyperresponsiveness in rats challenged with aerosolized allergen. Furthermore, this suppression is persistent and can be transferred into naive rats by CD8+ T cells from gene-immunized rats. These findings suggest that allergen-gene immunization is effective in modulating allergic responses, and may provide a novel therapeutic approach for allergic diseases. PMID- 8616714 TI - Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. AB - Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) offers safe and effective therapy for certain viral infections and could prove useful in the eradication of tumor cells. Whether or not the infused T cells persist for extended periods, retaining their ability to expand in response to antigenic stimulation, is not known. We now report long-term detection of gene-marked Epstein-Barr virus (EBV)-specific CTLs in immunocompromised patients at risk for the development of EBV lymphoproliferative disease. Infusions of CTLs not only restored cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months. Our findings support wider use of antigen specific CTLs in adoptive immunotherapy. PMID- 8616713 TI - Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. AB - The use of replication-defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene-encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses direct against foreign transgene-encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials. PMID- 8616715 TI - Cortisol blockade of progesterone: a possible molecular mechanism involved in the initiation of human labor. AB - In most mammals, labor is heralded by progesterone withdrawal, which is believed to be related to the activation of multiple pathways leading to parturition. In humans, despite no decrease in placental progesterone production, activation of similar pathways preceding labor suggests the presence of an endogenous antiprogestin, which we reasoned might be cortisol, whose secretion from the fetal adrenal rises markedly at the end of human gestation. We report that in primary cultures of human placenta, cortisol is able to compete with the action of progesterone in the regulation of the corticotropin-releasing hormone (CRH) gene. CRH is a peptide highly expressed in human placenta at the end of gestation, which has been suggested to be involved in regulating the timing of parturition. These findings provide a model for functional progesterone withdrawal at the end of human pregnancy, which may be involved in the initiation of labor. PMID- 8616716 TI - Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr Abl positive cells. AB - The bcr-abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML, there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. PMID- 8616717 TI - Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo. AB - Cytosine arabinoside (ara-C) is a cytidine analog that incorporates into replicating DNA and induces lethal DNA strand breaks. Although ara-C is a potent antitumor agent for hematologic malignancies, it has only minimal activity against most solid tumors. The rate-limiting step in intracellular ara-C activation is phosphorylation of the prodrug by deoxycytidine kinase (dCK). The present results demonstrate that both retroviral and adenoviral vector-mediated transduction of the dCK cDNA results in marked sensitization of glioma cells lines to the cytotoxic effects of ara-C in vitro. We also demonstrate that ara-C treatment of established intradermal and intracerebral gliomas transduced with dCK results in significant antitumor effects in vivo. These data suggest that viral vector transduction of the dCK gene followed by treatment with ara-C represents a new chemosensitization strategy for cancer gene therapy. PMID- 8616718 TI - Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. AB - Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies. PMID- 8616719 TI - p53-dependent apoptosis suppresses radiation-induced teratogenesis. AB - About half of human conceptions are estimated not to be implanted in the uterus, resulting in unrecognizable spontaneous abortions, and about 5% of human births have a recognizable malformation. In order to find clues to the mechanisms of malformation and abortion, we compared the incidences of radiation-induced malformations and abortions in p53 null (p53-/-) and wild-type (p53+/+) mice. After X-irradiation with 2 Gy on day 9.5 of gestation, p53-/- mice showed a 70% incidence of anomalies and a 7% incidence of deaths, whereas p53+/+ mice had a 20% incidence of anomalies and a 60% incidence of deaths. Similar results were obtained after irradiation on day 3.5 of gestation. This reciprocal relationship of radiosensitivity to anomalies and to embryonic or fetal lethality supports the notion that embryonic or fetal tissues have a p53-dependent "guardian" of the tissue that aborts cells bearing radiation-induced teratogenic DNA damage. In fact, after X-irradiation, the number of cells with apoptotic DNA fragments was greatly increased in tissues of the p53+/+ fetuses but not in those of the p53-/- fetuses. PMID- 8616720 TI - Inducible nitric oxide synthase gene expression in the brain during systemic inflammation. AB - Inducible nitric oxide synthase (iNOS) is a transcriptionally regulated enzyme that synthesizes nitric oxide from L-arginine that has a key role in the pathophysiology of systemic inflammation and sepsis. Transgenic animals with a null mutation for the iNOS gene are resistant to hypotension and death caused by Escherichia coli lipopolysaccharide (LPS). The regulation of peripheral iNOS has been well studied in sepsis, but little is known about iNOS regulation in the brain during systemic inflammation or sepsis. We know that at baseline there is no detectable iNOS gene expression in the brain, but a detailed neuroanatomical study reveals that early in the course of systemic inflammation there is a profound induction of iNOS messenger RNA in vascular, glial and neuronal structures of the rat brain, accompanied by the production of nitric oxide (NO) metabolites in brain parenchyma and cerebrospinal fluid (CSF). We propose that the spillover of nitrite into the CSF has the potential to be a diagnostic marker for systemic inflammation and sepsis. Pharmacological interventions aimed at regulating iNOS function in the brain might represent a new treatment strategy in sepsis. Brain iNOS may be relevant to the pathophysiology, diagnosis and treatment of systemic inflammation and sepsis. PMID- 8616721 TI - Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. AB - Hematopoietic development is a complex process that involves a large number of growth factors and cytokines. Many cytokines are known to act on more mature, lineage-restricted cells of the hematopoietic system. However, no specific factors have yet been identified that induce the expansion of the most primitive hematopoietic cells without also inducing differentiation. To search for such factors, we isolated novel cell lines from the yolk sac in order to identify genes important in early hematopoietic and endothelial development. This approach led to the discovery of B219, a sequence that is expressed in at least four isoforms in very primitive hematopoietic cell populations and which may represent a novel hemopoietin receptor. The recently published receptor for the obesity (ob) gene product (leptin) is an isoform of B219 with a nearly identical ligand binding domain. B219/obr is expressed in the yolk sac, early fetal liver, enriched hematopoietic stem cells and in a variety of lymphohematopoietic cell lines. B219/obr is also expressed at high levels in adult reproductive organs. B219/obr maps to human chromosome 1p32, a region syntenic with the recently reported location of obr on murine chromosome 4 (ref. 5). PMID- 8616722 TI - Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. AB - The adipocyte hormone, leptin (OB protein), is proposed to be an "adiposity signal" that acts in the brain to lower food intake and adiposity. As plasma leptin levels are elevated in most overweight individuals, obesity may be associated with leptin resistance. To investigate the mechanisms underlying brain leptin uptake and to determine whether reduced uptake may contribute to leptin resistance, we measured immunoreactive leptin levels in plasma and cerebrospinal fluid (CSF) of 53 human subjects. Leptin concentrations in CSF were strongly correlated to the plasma level in a nonlinear manner (r = 0.92; p = 0.0001). Like levels in plasma, CSF leptin levels were correlated to body mass index (r = 0.43; p = 0.001), demonstrating that plasma leptin enters human cerebrospinal fluid in proportion to body adiposity. However, the efficiency of this uptake (measured as the CSF:plasma leptin ratio) was lower among those in the highest as compared with the lowest plasma leptin quintile (5.4-fold difference). We hypothesize that a saturable mechanism mediates CSF leptin transport, and that reduced efficiency of brain leptin delivery among obese individuals with high plasma leptin levels results in apparent leptin resistance. PMID- 8616723 TI - Cystic fibrosis hetero- and homozygosity is associated with inhibition of breast cancer growth. AB - Cystic fibrosis (CF) is the most common lethal recessive genetic disease of the Caucasian population. Although reports of cancer frequency in CF have emphasized an elevated observed-to-expected ratio of 6.5 for digestive tract cancers, these studies also show a significantly decreased observed-to-expected ratio for other malignancies including breast cancer. The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATP channel. We found that heterozygous and homozygous CFTR knockout mice had elevated blood ATP concentrations. Elevated extracellular ATP is known to inhibit tumor growth in vivo and in vitro. Using double mutant mice created by F2 generation crosses of CFTR knockout and nude mice, we observed reduced breast tumor implantability in CFTR homozygous nude animals. Decreased tumor growth rate was observed in both CFTR heterozygous and homozygous nude animals. Extracellular ATP reduced human breast tumor cell growth rate in vitro, and a breast tumor transfected with human CFTR that had high extracellular ATP concentrations in vitro correspondingly had a slower growth rate in vivo. The results suggest that both CFTR heterozygosity and homozygosity suppress breast cancer growth and that elevated extracellular ATP can account for this phenomenon. PMID- 8616724 TI - Magnetic resonance elastography. PMID- 8616725 TI - What have clinical trials taught us about proarrhythmia? AB - Proarrhythmia is defined as the developmental of a new arrhythmia, or the worsening of a preexisting arrhythmia, following the institution of anti arrhythmic therapy. The most important manifestation of proarrhythmia is sudden arrhythmic death. Possible mechanisms of proarrhythmia include early afterdepolarizations, dispersion of repolarization, a conduction-slowing effect that promotes reentry, and the interaction of arrhythmic drugs with ischemia. Recent trials of arrhythmic drugs have focused attention on the increase in mortality due to some of these drugs. In many studies, the effect of placebo has been compared with that of antiarrhythmic drugs on mortality in high-risk patients following myocardial infarction (MI). In most of these trials, anti arrhythmic drugs were associated with an increase in mortality has been most clearly shown with encainide, flecainide, moricizine and d-sotalol. In addition, increased mortality has been suggested in patients treated with antiarrhythmics for atrial fibrillation especially in the presence of structural heart disease. In contrast, several post-MI benefit. This suggests that amiodarone may be safe for the treatment of arrhythmias in the post-MI patient. Further evidence will come from two majors studies (CAMIAT and EMIAT) which should be available by early 1996. PMID- 8616726 TI - An overview of antiarrhythmic drug management of electrical storm. AB - Electrical storm, the most lethal of all cardiac arrhythmias, is defined as incessant or frequently recurrent ventricular fibrillation, or hemodynamically destabilizing ventricular tachycardia. Electrical storm typically develops in older men with coronary artery disease, in the setting of a acute myocardial infarction or following cardiac surgery. Treatment of electrical storm with conventional antiarrhythmic drugs is seldom effective. Amiodarone, an anti arrhythmic agent with established efficacy in the treatment of a variety of atrial and ventricular tachyarrhythmias, is now available as an intravenous formulation in hospitals. Three recent studies have examined the effect of intravenous amiodarone in the management of electrical storm. The studies, performed in critically ill patients, found amiodarone to be effective in the treatment of these serious arrhythmias. Two of the studies compared different dose ranges for intravenous amiodarone, while one study compared amiodarone to intravenous bretylium. The investigators found amiodarone to be as effective as bretylium, and to have significantly fewer adverse effects requiring drug discontinuation. Side effects of intravenous amiodarone included hypotension and bradycardia, both of which were managed by reducing the rate of the infusion. Proarrhythmia, defined as torsade de pointes or new onset ventricular fibrillation, was infrequently seen in the studies. PMID- 8616727 TI - Assessment of arrhythmias after myocardial infarction in the post-CAST era. AB - Prevention of sudden cardiac death after myocardial infarction (MI) has become a continuing challenge for clinical cardiology. Of the many treatment modalities available, some have been shown to save lives while others may even have deleterious effects. Further survival studies exploring the efficacy of prophylactic treatment will be necessary before conclusions for routine clinical practice can be drawn. For this purpose, improvements in the process of risk stratification must be made before these studies are able to demonstrate potential survival benefit. This article will review both the established and the new methods of risk stratification for the post MI patient, with particular attention to antiarrhythmic therapy. PMID- 8616728 TI - New physicians, unlike new mechanics, find pay and location restricted. PMID- 8616729 TI - Diagnosing the ills of our health care system. PMID- 8616730 TI - Error in dosage for Colace docusate sodium in 1996 CPS. PMID- 8616732 TI - Ideas for antismoking messages. PMID- 8616731 TI - Turf not the issue in PEI reform of mental health care. PMID- 8616733 TI - Issues concerning malaria vaccine scientific, not based on racism. PMID- 8616734 TI - A second look at new excimer-laser eye surgery. PMID- 8616735 TI - Developing a Canadian prescribing practices network. Network Development Committee of the Canadian Prescribing Practices Network Project. AB - Expenditure on drug therapy in Canada has been growing at a faster rate than spending on any other aspect of health care. Increasing societal pressure to use scarce resources more efficiently, advances in communication technology and data indicating that there is room for improvement in drug prescribing suggest that the time has come for an organized linkage of the available drug-utilization and health-outcomes data-bases across the country. A national prescribing practices network would assist prescribers, researchers and policymakers to optimize prescribing with respect to both cost effectiveness and health outcomes. The authors outline the main concerns addressed in the 1994 report to the National Pharmaceutical Strategy and present the results of discussions by the Canadian Prescribing Practices Network Project with respect to the potential users and data sources of a national network and the communications technology on which it would rely. PMID- 8616736 TI - Factors determining compliance with screening mammography. AB - OBJECTIVE: To determine factors affecting compliance with screening mammography prescribed by family physicians. DESIGN: Secondary analysis of a nonrandomized trial. SETTING: University-affiliated family medicine clinic in Montreal. PATIENTS: Women aged 50 to 69 years who were given a written prescription for a screening mammography during their visit at the clinic between Oct. 12, 1991, and May 31, 1992, and who had not undergone mammography in the preceding 2 years and had never been treated for breast cancer. Information on the potential factors was obtained through a telephone questionnaire 2 months after the visit. OUTCOME MEASURES: Indicator of compliance presence of result of screening mammography in patient chart, potential factors influencing compliance: age, level of education, marital status, socioeconomic level, smoking status, perceived health status, perceived psychological well-being, risk factors for breast cancer, use of health services including frequency of Papanicolaou test, Health Belief Model variables. RESULTS: Of the 171 eligible women, 113 (66.1%) underwent the prescribed mammography within 2 months after the visit to the clinic, and 149 (87.1%) responded to the questionnaire. The patients' socioeconomic characteristics, perceived health status, health utilization indices and risk factors for breast cancer were not found to be predictors of compliance. The strongest predictor of compliance was the number of previous mammograms. Women who had undergone mammography previously were less likely to be noncompliant than those who had not (odds ratio [OR] 0.11, 95% confidence interval [CI] 0.02 to 0.51; p = 0.005). Women who did not comply were less likely than those who did to believe that a prescription from their physician would convince them to undergo mammography (OR 0.21, 95% CI 0.007 to 0.60; p = 0.004). Other factors associated with noncompliance were the expression of fear of mammography (OR 2.09, 95% CI 1.08 to 4.02; p = 0.03) and the lack of time to take the test (OR 3.07, 95% CI 1.21 to 7.80 p = 0.02). Being a smoker was negatively associated with compliance (OR 0.43; 95% CI 0.22 to 0.86; p = 0.02). The stepwise logistic regression model accounted for 87.5% of the outcome (chi2 for goodness of fit = 164.4; p = 0.0001). CONCLUSION: Family physicians who prescribe screening mammography, even to women who consult for other reasons, are likely to overcome some of the barriers observed in association with population screening rates. However, physician-oriented approaches are not likely to reach the 30% to 40% of reluctant women who appear to hold negative views toward physicians' recommendations. Further study is necessary to determine how better to reach these women. PMID- 8616738 TI - How should we interpret noncompliance with screening mammography? AB - Primary care practitioners have an important role to play in recommending breast cancer screening to patients in the target age group. In this issue of CMAJ (see pages 1335 to 1343) Dr. Marie-Dominique Beaulieu and associates report the results of a program designed to maximize utilization of screening mammography. Only two thirds of eligible women for whom screening mammography was prescribed obtained a mammogram within the 2-month study period. However, when taken in context, this compliance rate is fairly encouraging. There are many possible reasons for noncompliance such as a need for more information or for repeated suggestions. Family physicians should not become disheartened in their efforts to increase the use of screening procedures and may find that collaboration with others in giving consistent messages will help to maximize screening rates within their patient population. PMID- 8616737 TI - Use of the emergency department for nonurgent care during regular business hours. AB - OBJECTIVE: To characterize the patient population seeking care for nonurgent medical problems at an emergency department during regular business hours and to determine why these patients chose the emergency department over alternative care sites. DESIGN: Patient survey (self-administered questionnaire). SETTING: Emergency department at a tertiary care hospital in Montreal. PATIENTS: All ambulatory patients presenting on weekdays between 8 am and 5 pm from Nov. 10 to Dec. 8, 1993, whose condition was determined to be nonurgent. Eligible patients had to be residents of Montreal, who did not have a pre-arranged consultation at the emergency department. Of 202 consecutive eligible patients, 200 agreed to participate. OUTCOME MEASURES: Description of events leading to the visit, including possible attempts by patients to contact their regular physician; patients' knowledge of alternative care options such as provincial CLSGs (centres locaux des services communautaires) and private walk-in clinics. RESULTS: Of the 200 patients 152 (76%) stated that they had not visited an emergency department within the previous month, and only 10 (5%) stated that they were in extreme pain. At least 70% were aware of alternative care options, however, 120 (60%) felt that the emergency department was the best place for them to receive care for their medical problem. In all, 81 patients (40%) were referred to the emergency department; 62 (77%) were referred by a health care professional, 46 (57%) by a physician. CONCLUSION: Most patients are aware of alternatives to the emergency department for care of nonurgent medical problems. Nevertheless, a large number are being referred to the emergency department during regular business hours by health care professionals. This inefficient use of expensive hospital resources requires further investigation. PMID- 8616740 TI - Prevention should be the preferred insurance program for all physicians. AB - Recent fee increases announced to the Canadian Medical Protective Association (CMPA) and the Ontario government's plan to stop its CMPA rebate program for the province's physicians have put the spotlight on medical liability insurance. In this examination of the role played by the CMPA, Ottawa lawyer Karen Capen notes that quality service and attention to physician-patient communication will in most cases ensure a litigation-free professional life. PMID- 8616739 TI - The practitioner, the patient and resistance to change: recent ideas on compliance. AB - Despite the explosion of research into the effect of medical advice on patient behaviour, only about 50% of patients comply with long-term drug regimens. And when it comes to changes in lifestyle, the percentage of patients who comply with medical advice often falls to single figures. Review articles on compliance have traditionally concentrated on factors that make it easier for patients to adhere to medical advice. However, recent articles urge clinicians to be more understanding of the wider implications of compliance in their patients' lives. This article focuses on how clinicians' consulting methods can affect patients' behaviour. Specifically, the authors consider the patient-centred clinical method as well as insights from and consulting techniques pioneered in the addictions field that can help to bring ambivalent patients closer to decisions about change. Instead of seeing resistance to change as rooted entirely in the patient, the authors view it as stemming partly from the way clinicians talk to patients. An advice-giving approach is usually inadequate to motivate people to embark on major lifestyle changes. Instead, the authors propose a negotiation-based framework that harnesses patients' intrinsic motivation to make their own decisions. This approach also promotes clinicians' acceptance of patients' decisions, even if these decisions run counter to current medical wisdom. PMID- 8616741 TI - Cochrane Collaboration helping unravel tangled web woven by international research. AB - Although there are sceptics and critics, the Cochrane Collaboration continues to grow exponentially. With centres in nine countries, its common goal is to create, maintain and disseminate systematic reviews of randomized controlled trials in an effort to bring clinicians the most current, accurate evidence about medical treatments available around the world. The future of the collaboration will depend largely on whether the methodology of their reviews can be shown to be valid and to produce consistently relevant and timely reviews. PMID- 8616743 TI - On the trail of Dr. Fifer. AB - A gift from a patient drew Hope, BC, family physician Gerd Asche irrevocably into the local medical history of the 1858 Fraser River Gold Rush. Because of his interest in Dr. Max William Fifer, Asche undertook research missions in British Columbia, England and the US, converted his computer room to a research and writing centre, and wrote a biography of his predecessor and colleague. He recounts his experience and the growing satisfaction provided by his interest in medical history. PMID- 8616742 TI - A system worth saving. AB - The country will mark Canada Health Day on May 12, so CMAJ asked Dr. Michael Gordon to reflect on Canada's health care system and the changes it has seen and will see. The special day, cosponsored by the Canadian Public Health Association, is designed to highlight "the need for better communication between health professionals and the communities they serve." In this article, Gordon reflects on the dangers facing Canada's medicare system and the need to protect it from the inroads threatened by privatization. PMID- 8616744 TI - Major merger gives London Canada's second-largest teaching hospital. AB - Two of London's three major teaching hospitals joined forces recently to form Canada's second-largest teaching hospital. The merger is considered a harbinger as teaching hospitals strive to maintain current teaching, research and clinical activities in the face of decreasing government spending. PMID- 8616745 TI - Family-friendly HIV and AIDS care the goal at Vancouver's Oak Tree Clinic. AB - A clinic at Vancouver's old Shaughnessy Hospital offers specialized care for women and children infected with HIV. Drs. David Burdge and Jack Forbes, the codirectors, believe the "one-stop-shopping" for services offered by physicians, social workers, nurses, dietitians and pharmacists helps improve quality of life for families living with the consequences of HIV and AIDS. BC has the highest cumulative incidence of AIDS in the country, and demand for the clinic's services has far exceeded expectations. PMID- 8616746 TI - Successive Ontario governments forced to grapple with problem of health care fraud. AB - Albert Shu of Willowdale, Ont., is the 1995 winner of the Amy Chouinard Memorial Essay Contest. Named in memory of long time CMAJ and Canadian Journal of Surgery contributor Amy Chouinard, the competition is intended to stimulate interest in medical and health-related writing among journalism students. The winning essay, written prior to Ontario's 1995 provincial election, examined health care fraud in the province and the impact of the photo health card that was introduced by the New Democrats party government. PMID- 8616747 TI - Local resection of early gastric carcinoma. PMID- 8616748 TI - BRCA1. A new marker in the management of patients with breast cancer? PMID- 8616749 TI - Risk factors for lymph node metastasis from intramucosal gastric carcinoma. AB - BACKGROUND: Although regional lymph node metastasis from intramucosal early gastric carcinoma (EGC) is rare, it is very important to clarify the characteristics of patients having lymph nodal metastases in order to determine appropriate therapy. METHODS: The authors investigated 1196 patients with solitary intramucosal EGC who underwent resection at the National Cancer Center Hospital in Tokyo, with special reference to lymph node metastases. Eight clinicopathologic factors (age, sex, tumor: size, location, macroscopic type, histologic type, histologic ulceration of the tumor, and lymphatic vessel invasion) were investigated by univariate and multivariate analyses for their possible relationship to lymph node metastasis. RESULTS: Lymph node metastases were found in 43 patients (3.5%). Univariate analysis revealed that younger age (< 57 years), macroscopic depressed type, larger tumor size (> or= 30 mm), undifferentiated histologic type, histologic ulceration of the carcinoma, and lymphatic vessel invasion had a significant association with regional lymph node metastasis. Multivariate analysis revealed that lymphatic vessel invasion, histologic ulceration of the tumor, and larger size (> or = 30 mm) were independent risk factors for regional lymph node metastasis. The incidence of lymph node metastasis from intramucosal EGC negative for these 3 risk factors was only 0.36% (1 in 277 patients). CONCLUSIONS: Lymphadenectomy is unnecessary for patients with small intramucosal EGC with neither histologic ulceration of the tumor nor lymphatic vessel invasion because the incidence of regional lymph node metastasis is extremely low in those patients. The therapeutic options for such patients would be local resection or endoscopic resection. PMID- 8616750 TI - Manual dissection of adenocarcinoma of the lower third of the rectum specimens for detection of lymph node metastases smaller than 5 mm. AB - BACKGROUND: The question of whether manual dissection when searching for metastatic lymph nodes from rectal cancer (less than 5 mm) is a reliable method remains controversial. METHODS: We examined 50 consecutive cases of primary adenocarcinoma of the rectum treated with a sphincter-sparing total rectum resection, total mesorectum excision, and coloanal anastomosis. We used a manual method for the detection of lymph nodes. RESULTS: One thousand seven hundred ninety-three lymph nodes were found (mean, 36 per patient). One hundred seventy four contained metastases. Seventy-nine (45.4%) of the affected lymph nodes were less than 5 mm in greatest dimension. The percentage of metastases to small lymph nodes was similar to the percentage reported by Kotanagi (50%), but lower than the report of Herrera (78%), who used a clearing technique to search for regional lymph nodes. CONCLUSIONS: A median 17 months follow-up in these patients demonstrated that metastases in small lymph nodes are important in the accurate staging of rectal tumors and that a manual method of searching for small lymph nodes is reliable. PMID- 8616751 TI - Intraoperative detection of occult colon cancer micrometastases using 125 I radiolabled monoclonal antibody CC49. AB - BACKGROUND: The detection of locally-disseminated disease is one of the principal goals of oncologic surgery. For this study, a hand-held, gamma-detecting probe was used intraoperatively to assess the extent of colorectal carcinoma in patients previously injected with radiolabeled antibody to the TAG-72 antigen (CC49); this technique is known as Radioimmunoguided Surgery (RIGS) (Neoprobe Corporation, Dublin, OH). RIGS-positive areas (i.e. those with increased signal over background) have previously been shown to contain carcinoma in a high proportion of cases. However, some RIGS-positive areas had no tumor detectable by clinical examination or routine histopathologic analysis. This study was undertaken to determine if the presence of occult metastases might account for this disparity. METHODS: A total of 57 regional lymph nodes (LN), resected from 16 patients with primary (9) or recurrent (7) colorectal carcinoma, were studied. The patients were injected with 125I labeled CC49 murine monoclonal antibody approximately 3 weeks prior to surgery. After routine histologic evaluation, the LN were analyzed for occult metastases; paraffin sections were cut at 5 levels (50 micron apart) and were examined by histology (hematoxylin and eosin stain [H & E]) and by immunohistochemistry (IHC) with a cocktail of monoclonal antibodies to cytokeratins. RESULTS: Fifty-seven LN were included in this study; 17 were H & E-positive (i.e., contained tumor by routine histologic examination [overt tumor]), while 40 LN were H & E-negative (i.e., no evidence of tumor after routine histologic examination). Thirty-nine LN were RIGS-positive, but only 14 of these were H & E-positive. Of the 25 RIGS-positive/H & E-negative LN, 10 (40%) demonstrated the presence of occult metastases after serial section/IHC analysis. Thus, a total of 27 LN contained metastatic carcinoma (17 overt, 10 occult); routine histologic analysis was able to identify tumor in only 17 of these 27 LN (63%), while the probe signaled the presence of tumor in 24 of these LN (89%). None of the RIGS-negative/H & E-negative LN were found to have occult metastases (0/15). Specific immunoreactivity with CC49 antibody was observed in 5 of 15 RIGS positive/H & E-negative LN in which no tumor could be identified by any method (histopathology or IHC. CC49 immunoreactivity was not observed in 15 RIGS negative/H & E-negative LN. CONCLUSIONS: The finding of a RIGS-positive LN had a significant association with the presence of tumor cells (P < 0.05). In this study, the RIGS procedure was more sensitive than clinical or histopathologic examination in detecting the regional spread of a tumor. Furthermore, in LN that showed no evidence of tumor by routine histopathologic examination, a positive RIGS reading was significantly associated with the presence of occult LN metastases (P < 0.01). This study is the first to demonstrate the detection of histologically occult tumor by a remote imaging device. RIGS assessment is a highly sensitive method for detecting occult tumor deposits, and may guide therapeutic intervention in patients with colorectal carcinoma. PMID- 8616752 TI - Nonpolypoid adenomas and adenocarcinomas found in background mucosa of surgically resected colons. AB - BACKGROUND: Nonpolypoid (depressed or flat) neoplasias are rarely seen in the colon and rectum. We previously reported their histogenesis and characteristics in patients with familial adenomatous polyposis (FAP), but their development in patients without FAP has not been studied systematically. METHODS: Three hundred specimens of large intestinal mucosa surgically resected from patients with cancers or other diseases, excluding FAP, were examined with a dissecting microscope. The morphologic types, sizes, locations, and frequencies of detectable colorectal neoplasias, and their histologic features, were analyzed. RESULTS: A total of 297 adenomas (240 polypoid, 32 flat, and 25 depressed type) were obtained. Nonpolypoid adenomas were most frequently found in the transverse and descending colon. Almost all depressed adenomas (24 of 25; 96%) were less than 3 mm in greatest dimension and almost all flat adenomas (31 of 32; 96.9%) were less than 3.5 mm in greatest dimension. Three minute, nonpolypoid adenocarcinomas (mean size, 2.6 mm; range, 2.4-2.9 mm) were also detected, two of them already invaded the submucosal layer. CONCLUSIONS: Minute nonpolypoid type adenomas may be present in the background mucosa of patients without FAP. This study suggests that even minute nonpolypoid adenocarcinomas have an increased potential for endophytic growth. PMID- 8616753 TI - Flat adenoma as a precursor of colorectal carcinoma in hereditary nonpolyposis colorectal carcinoma. AB - BACKGROUND: Because flat adenoma shows a higher malignancy rate compared with other types of polyps, it is considered to play an important role in the carcinogenesis of colorectal carcinoma. In the present study, we examined flat adenomas in hereditary nonpolyposis colorectal carcinoma (HNPCC) patients. METHODS: Nine HNPCC patients who presented with flat adenomas were examined. All patients underwent either surgery or endoscopic polypectomy for colorectal carcinoma and/or adenoma. In all patients, annual colonoscopy had been performed once a year throughout the follow-up period after the initial treatment. When colorectal polyps were detected during follow-up colonoscopy, all lesions were endoscopically excised. Clinicopathologic features and microsatellite instability of both malignant lesions and adenomas were examined. RESULTS: Thirteen malignant lesions were detected: seven advanced carcinomas and six early carcinomas. Among 4 early carcinomas with submucosal invasion, 3 lesions (75%) were categorized as superficial type, with a configuration similar to flat adenoma. The frequency of flat adenoma was strikingly high in HNPCC patients in the present study. Among 73 polyps detected, 37 (50.7%) were flat adenomas. Both malignant lesions and flat adenomas had proximal predominance, 61.5% and 59.5%, respectively. Eleven of 15 lesions (73.3%) showed replication error. CONCLUSIONS: These results suggest the importance of flat adenoma as a precursor of colorectal carcinoma in some groups of HNPCC patients. Further study is essential to elucidate the natural history of flat adenomas in HNPCC patients. PMID- 8616754 TI - Phase II study of flutamide in the treatment of hepatocellular carcinoma. AB - BACKGROUND: Hepatocellular carcinoma (HCC) is a male predominant disease and may be an androgen-dependent or androgen-responsive tumor. This Phase 11 study was designed to investigate the clinical activity and toxicity of flutamide in the treatment of patients with advanced HCC. METHODS: Thirty-two patients with measurable advanced HCC were studied. Flutamide, 750 mg per day, was administered orally for 8 weeks. Ten patients died before repeat tumor measurements could be performed. RESULTS: Twenty-two patients were evaluable for response and toxicities. There were no complete responses nor partial responses. Nine of 22 patients (41%) had stable disease and 13 patients (59%) had progressive disease. Serum alpha-fetoprotein was reduced in three patients. The median survival was 10 weeks (range, one to 35 weeks). Toxicities were minimal and tolerable. CONCLUSIONS: Flutamide is not effective in the treatment of advanced HCC. Clinically, HCC may not be an androgen-responsive tumor. Other new methods of treatment of HCC warrants future clinical investigations. PMID- 8616755 TI - Results of extensive surgery for pancreatic carcinoma. AB - BACKGROUND: Since 1973, 210 patients with pancreatic carcinoma have undergone surgery in our clinic, including 144 with carcinoma of the head of the pancreas. Of these 144 patients, macroscopic curative resections were performed on 53 (36.8%). Five patients (9.4%) died within 30 postoperative days, and an additional 3 (5.7%) died within 60 days. The overall median survival was 13 months. Eight of the patients who underwent macroscopic curative resection survived 5 years, giving a 5-year survival rate of 27.4% using the Kaplan-Meier method. The 5-year survival rate was 39.7% after a microscopically curative resection and 0% after a microscopically noncurative resection. METHODS: Outcome was compared based on the extent of pancreatic cancer by constructing survival curves according to the general rules published by the Japan Pancreas Society. RESULTS: There was no statistically significant difference in survival based on tumor size or stage. However, there was a significant difference in the survival of patients with the absence (so) or presence (se) of invasion to the anterior capsule of the pancreas, the absence (rpo) or presence (rpe) of invasion of the retroperitoneal tissue, the absence (ew0) or presence (ew2) of invasion at the surgical margin of resection, and the extent (n0 to n2) of lymph node metastasis. CONCLUSIONS: The results of this study suggest that extended radical pancreatectomy may be indicated for patients with pancreatic carcinoma because standard dissection may fail when the tumor has spread to the retroperitoneum or extrapancreatic nerve plexus. PMID- 8616756 TI - The changes of the stromal elastotic framework in the growth of peripheral lung adenocarcinomas. AB - BACKGROUND: Most peripheral lung adenocarcinomas form a characteristic central fibrosis; however, the mechanism of formation has not yet been clarified. METHODS: The stromal elastosis of 176 patients with peripheral lung adenocarcinomas was studied histologically, morphometrically, radiographically, and clinicopathologically. RESULTS: The tumors were classified into two types, according to the histomorphological pattern of the stromal elastosis: (1) a preserved framework composed of a uniformly thick stroma (Type 1; 20 patients) and (2) a disrupted framework in the central fibrosis in which many air spaces were collagenized or collapsed (Type 2; 156 patients). Around the central fibrosis in the Type 2 tumors, replacement growth took the form of a thin-walled, elastic framework. Image analysis disclosed that the elastotic framework had an increased elastic fiber volume (approximately three- to nine-fold, compared with the normal alveolar wall), especially in Type 1 tumors, related to the contraction of the pre-existing alveolar wall. In Type 2 replacement growth, the amount of elastic fibers was slightly increased, which suggested mild reaction of the pre-existing alveolar wall. Radiographically, Type 1 tumors were characterized by a gradual increase of the density without enlargement, while the Type 2 tumors often enlarged within a short period. Recurrence was seen in 54% of the patients with Type 2 tumors, but in none of those with Type 1 tumors. CONCLUSIONS: In the early development of peripheral adenocarcinoma, there is preservation of the elastotic framework (Type 1) of the stroma due to contraction and thickening of the alveolar walls. As the tumors grow, the elastotic framework is disrupted (Type 2), indicating stromal invasion. Based on the clinicopathologic findings and the outcome, Type 1 tumors may be in situ peripheral lung adenocarcinomas. PMID- 8616757 TI - Fine tuning of epitopic dominance induced by lung cancer on the IgG response to bovine betalactoglobulin: towards a paraneoplastic immune marker. AB - BACKGROUND: Investigating the humoral immune response to mucosal antigens in patients with lung cancer, we have documented a preferential immunoglobulin G (IgG) binding to cryptic epitopes unmasked by the proteolysis of bovine beta lactoglobulin (BLG). In contrast, IgG from healthy controls and patients with chronic bronchitis (COPD) bind preferentially to continuous epitopes presented on both native (n) and denaturated (d) forms of this antigen. The present study further characterized the differences in the epitope profiles recognized on BLG. METHODS: The capacity of individual sera from 65 lung cancer patients, tested before and after cancer removal for the patients with early stage lung carcinoma, 65 healthy controls, and 52 patients with COPD, to prevent the binding of pooled IgG fractions from each population as well as murine monoclonal antibodies (MoAb), specific for BLG, to solid phase bound antigen was evaluated in enzyme linked immunoadsorbent assay using streptavidin-biotin technology. Some of these experiments were also performed with sera from 42 patients diagnosed with other cancers. RESULTS: Compared with control sera and sera from patients with other solid tumors, lung cancer patient sera showed distinct capacities to prevent the binding of murine MoAb as well as human pooled IgG fractions to n- and d-BLG. The inhibition capacities of lung cancer sera changed as soon as five weeks after cancer removal. CONCLUSIONS: The results indicate that the difference in epitope specificity exhibited by lung cancer sera is not restricted to cryptic epitopes, but also affects continuous and discontinuous epitopes, accessible only on the native antigen. A high level of binding discrimination between antibodies from the study populations is also observed at the level of the epitope. This deviation in the epitope specificity of antibodies changes soon after cancer removal, suggesting a tumor-dependent disturbance. Also documented in the Dermatophagoides pteronyssinus model, it opens the way to a new class of paraneoplastic immune markers for this malignancy, with, at first glance, a high specificity level. PMID- 8616758 TI - Atypical adenomatous hyperplasia and adenocarcinoma of the human lung: their heterology in form and analogy in immunohistochemical characteristics. AB - BACKGROUND: In a previous study, morphometry and multivariate cluster analysis was performed on 97 lesions. These consisted of atypical adenomatous hyperplasia (AAH), considered to be an important lesion corresponding to a step of carcinogenesis for adenocarcinoma of the human lung, Clara cell type, and type 2 pneumocyte type adenocarcinomas. Although AAH and the two types of adenocarcinoma were re-classified into three clusters and AAH was defined in clear morphologic terms, the biologic nature of AAH has yet to be clarified. In the present study, immunohistochemical analysis was performed to gain a deeper understanding of the relationship between AAH and the two types of adenocarcinoma, and to compare the results with those obtained by morphometry. METHODS: The 97 lesions analyzed by morphometry were submitted to immunohistochemical analyses using antibodies against surfactant apoprotein A, urine protein 1, carcinoembryonic antigen and cytochrome P-450s (1A1-2, 2B1-2, 2E1). Also examined, as controls, were 17 lesions with adenomatous hyperplasia (AH), a non-neoplastic reactive change of bronchiolo-alveolar cells, 30 areas of normal Clara cells, and 36 areas of normal type 2 pneumocytes. The immunoreactivity was graded by introducing a semi quantitative scoring system. RESULTS: Immunohistochemically, AAHs behaved quite similarly to the lesions classified as Clara cell type or type 2 pneumocyte type adenocarcinomas. For any of the antibodies employed, no significant difference in immunoreactivity was demonstrated among these lesions. CONCLUSIONS: The results suggest, in accordance with our previous morphometry, that AAH is a lesion closely related with Clara cell type and type 2 pneumocyte type adenocarcinomas, probably as their common precursor. However, the two types of adenocarcinomas, despite their characteristic morphologic features, are indistinguishable using the biological indicators applied in this study. PMID- 8616759 TI - Surgical treatment of lung metastases: The European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group study of 255 patients. AB - BACKGROUND: Several reports have shown a prolonged survival after surgical treatment of pulmonary metastases from soft tissue sarcomas. However, it is still unclear which prognostic factors predict a favorable outcome. Series are not comparable and the data are conflicting. Therefore, a multi-institutional study was undertaken to analyze prognostic factors in selecting patients for resection of pulmonary metastases from soft tissue sarcomas. METHODS: This report is a retrospective study of the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group. Two hundred fifty-five patients underwent complete resection of lung metastases from soft tissue sarcomas. Cases with chondrosarcoma and small round cell sarcomas like Ewing sarcoma were excluded. RESULTS: The 3 year and 5 year overall postmetastasectomy survival rates were 54% and 38%, respectively. The disease free postmetastasectomy survival rates were 42% and 35%, respectively. Analysis of prognostic factors for a more favorable outcome revealed disease free intervals of 2.5 years or more, following a resection with microscopically free margins, age less than 40 years, and Grade I and II tumors. These prognostic factors have an independent influence on overall survival, using a multivariate Cox regression model. CONCLUSIONS: Surgical excision of lung metastases from soft tissue sarcomas is well accepted and should be considered as a first line of treatment if preoperative evaluation indicates that complete clearance of the metastases is possible. Further investigation is needed before chemotherapy can be recommended as additional therapy. PMID- 8616760 TI - Prognostic factors in elderly women with metastatic breast cancer treated with tamoxifen: an analysis of patients entered on four prospective clinical trials. AB - BACKGROUND: Information regarding prognostic factors and survival in elderly women with metastatic breast cancer treated with tamoxifen is limited. METHODS: The data from 4 prospective clinical trials were analyzed, including information on 396 postmenopausal women with advanced breast cancer who received tamoxifen as initial therapy for metastatic disease. Emphasis was placed on 184 elderly patients (age greater than 65 years) to characterize the response to therapy, time to progression TTP), overall survival (OS), prognostic factors, and treatment-related toxicity. RESULTS: Among 363 patients with measurable or evaluable disease, the objective response rates were higher in the elderly patients (46% versus 33%, P = 0.06); but age did not achieve significance in a logistic regression analysis (P= 0. 1). The median TTP (10.5 months versus 6.2 months, log rank P = 0.002) and OS (35.7 months versus 28.8 months, log rank P = 0.02) were superior in the elderly cohort. In multivariate analysis, age at diagnosis approached statistical significance (P = 0.055) for TTP but was not significant for OS (P = 0.17). Among elderly patients, disease free interval (DFI) (greater than 5 years), dominant disease site (soft tissue), prior adjuvant chemotherapy, positive estrogen/progesterone receptor (ER/PgR) and performance status (PS) were independent prognostic factors. Hot flashes were common in both younger and older cohorts (25% versus 33%, P = 0.14), while anorexia (14% versus 22%, P = 0.04) and mood changes (2% versus 6%, P = 0.03) were more common in the elderly patients. CONCLUSIONS: There was no indication that elderly women with metastatic breast cancer treated with tamoxifen have a poorer outcome with regard to response rate, TTP or OS; in fact, they appeared to have a slightly better prognosis although this was not significant after adjustment for other prognostic factors. In elderly patients, DFI, PS, positive ER or PGR, and dominant disease site are independent prognostic factors. PMID- 8616761 TI - Levels of 5-hydroxymethyl-2'-deoxyuridine in DNA from blood as a marker of breast cancer. AB - BACKGROUND: Oxidative DNA damage can result from numerous endogenous metabolic processes as well as from exposure to environmental and dietary oxidants. One important type of oxidative DNA damage is the formation of hydroxylated DNA bases. This type of DNA damage may have a role in carcinogenesis. METHODS: We examined the levels of a hydroxylated thymine residue, 5-hydroxy-methyl-2' deoxyuridine, in DNA obtained from the peripheral blood of breast cancer patients and control women. The isolated DNA was analyzed for levels of 5-hydroxymethyl-2' deoxyuridine by gas chromatography with mass spectral detection. RESULTS: The levels of this modified base were significantly higher in 25 breast cancer patients compared with 38 controls, with levels of 0.112 +/- 0.046 in the cancer patients versus 0.083 - 0.025 in the controls, given as pg 5-hydroxymethyl-2' deoxyuridine/ng thymidine, mean +/- standard deviation (P = 0.019). After controlling for various covariates, the adjusted mean levels of oxidative DNA damage were still significantly higher in women with breast cancer relative to controls. CONCLUSIONS: These results indicate that the levels of 5-hydroxymethyl 2'-deoxyuridine in DNA from peripheral nucleated blood may be potentially useful as a marker of breast cancer. PMID- 8616762 TI - Hereditary breast cancer: pathobiology, prognosis, and BRCA1 and BRCA2 gene linkage. AB - BACKGROUND: The purpose of this investigation was to determine if there are pathobiologic differences between BRCA1-related and BRCA2-related hereditary breast cancer (HBC) and non-HBC. METHODS: On the basis of linkage to chromosomes 17q or 13q and/or the presence of ovarian and male breast cancer, HBC families were classified as either "BRCA1-related" (26 families, 90 breast cancer pathology cases) or "Other" (26 families, 85 cases), in which most BRCA2 cases were likely to reside. Cases were compared with 187 predominantly non-HBC cases. Tumors were assessed for histologic type, grade, and ploidy and S-phase fraction by quantitative DNA flow cytometry. Clinical presentation and available follow-up data were obtained. RESULTS: BRCA1-related and Other HBC patients each presented at lower stage (P = 0.003) and earlier age than non-HBC patients (mean, 42.8 years and 47.1 years vs. 62.9 years, P = 0.0001). Compared with non-HBC, invasive BRCA1-related HBC had a lower diploidy rate (13% vs. 35%; P = 0.002), lower mean aneuploid DNA index (1.53 vs. 1.73; P= 0.002), and strikingly higher proliferation rates (mitotic grade 3; odds ratio [OR]= 4.42; P= 0.001; aneuploid mean S-phase fraction 16.5% vs. 9.3%, P < 0.0001). Other HBC patients, including patients in two BRCA2- linked families, had more tubular-lobular group (TLG) carcinomas (OR = 2.56, P = 0.007). All trends were independent of age. A nonsignificant trend toward better crude survival in both HBC groups was age- and stage-dependent. Compared with Other HBC, BRCA1-related HBC patients had fewer recurrences (P = 0.013), a trend toward lower specific death rates, and fared no worse than breast cancer patients at large. Other HBC patients, despite neutral prognostic indicators, fared worse. CONCLUSIONS: BRCA1-related HBCs are more frequently aneuploid and have higher tumor cell proliferation rates compared with Other HBC. Despite these adverse prognostic features, BRCA1-related HBC patients have paradoxically lower recurrence rates than Other HBC patients. The excess of TLG cancers in the "Other" HBC group may be associated with BRCA2 linkage. PMID- 8616763 TI - Quantitative analyses of epidermal growth factor receptors, HER-2/neu oncoprotein and cathepsin D in nonmalignant and malignant uteri. AB - BACKGROUND: Hormone receptors and oncoproteins are receiving increased attention as possible prognostic factors in different carcinomas. Few data are available regarding quantification of their levels of expression in gynecologic malignancies. METHODS: Epidermal growth factor (EGF) receptor specific binding capacities and affinities were measured by ligand binding assay using [125I]EGF in a competition mode with Accufit software (Lundon Software, Inc., Middlefield, OH). HER-2/neu oncoprotein was extracted from membranes and measured using an enzyme-linked immunosorbent assay. Cathepsin D was measured by an immunoradiometric assay using cytosols for steroid receptor analyses. RESULTS: EGF receptors in 23 nonmalignant uteri ranged from undetectable to 50 fmol/mg membrane protein (median, 0), with dissociation constant values of 1.2 x 10(-9) M to 8.5 x 10(-10) M, compared with EGF receptors in 76 endometrial cancers that ranged from undetectable to 7674 fmol/mg (median, 52). HER-2/neu oncoprotein ranged from undetectable to 2.9 HER-2/neu units (HNU)/microg protein (median, 0.6) in 41 nonmalignant uteri and from undetectable to 5.8 HNU/microg protein (median, 2.5) in endometrial cancers (n = 53). Cathepsin D ranged from 5 to 32 pmol/mg cytosol protein (median, 11) in 42 nonmalignant uteri and 18 to 144 pmol/mg protein (median, 42) in 29 endometrial cancers. CONCLUSIONS: Determination of the frequency and levels of EGF receptors, HER-2/neu protein, and cathepsin D in uteri with and without cancer and the availability of reference materials developed in our laboratory, will allow evaluation of their prognostic value in cancers of the uterus. PMID- 8616764 TI - Exogenous sex hormone use, correlates of endogenous hormone levels, and the incidence of histologic types of sarcoma of the uterus. AB - BACKGROUND: We analyzed data from a population-based, multi-center, case-control study to determine whether the occurrence of histologic types of uterine sarcoma is related to exogenous hormone use and/or to two correlates of endogenous estrogens: excess weight and cigarette smoking. METHODS: One hundred sixty-seven women with newly-diagnosed uterine sarcoma (56 leiomyosarcoma, 85 mixed mullerian tumors, and 26 endometrial stromal sarcomas) were interviewed by telephone regarding possible risk factors for these neoplasms, For comparison, 208 women identified at random from the general population of the study areas were interviewed as controls. RESULTS: Use of oral contraceptives was positively associated with the risk of leiomyosarcoma (odds ratios [OR] = 1.7, 95% confidence interval [CI] = 0.7, 4.1), primarily among women who last used these medications 15 or more years prior to diagnosis. Use of noncontraceptive estrogens was directly associated with the risk of mixed mullerian tumors, but only among recent and long-term users of these medications. Women in the highest quantile of body mass index (> or = 27.5 kg/m2) one year prior to diagnosis were at increased risk of each type of uterine sarcoma (leiomyosarcoma, OR = 2.5, 95% CI = 1.1, 5.7; mixed mullerian tumors, OR = 2.9, 95% CI = 1.3, 6.7; stromal sarcoma, OR = 3.5, 95% CI = 1.1, 10.9). Women who had ever smoked cigarettes were at reduced risk of leiomyosarcoma (OR = 0.6, 95% CI = 0.3, 1.1) and stromal sarcoma (OR = 0.5, 95% CI = 0.2, 1.2), but the relationship was not more pronounced among heavy smokers; no association with smoking was observed with mixed mullerian tumors. CONCLUSIONS: Several of these findings parallel those from studies of endometrial carcinoma and may indicate a role for unopposed estrogen in the etiology of histologic types of uterine sarcoma. PMID- 8616765 TI - Involvement of the mitochondrion respiratory chain in the synergy achieved by treatment of human ovarian carcinoma cell lines with both tumor necrosis factor alpha and cis-diamminedichloroplatinum. AB - BACKGROUND: Previous studies have demonstrated that treatment of human tumor cell lines with a combination of cis-diamminedichloroplatinum (CDDP) and tumor necrosis factor-alpha (TNF-alpha) results in additive/synergistic cytotoxic effects and reverses tumor cell resistance to TNF drugs. Free radical intermediates are induced by both TNF-alpha and CDDP; however, the role of free radicals in synergy is not known. This study investigated the effect of two inhibitors on synergy, phenoxan (Phe) and butylated hydroxyanisole (BHA), which inhibit Complex I and Complex I and II of the mitochondrion respiratory chain, respectively. METHODS: Three human ovarian carcinoma cell lines of different sensitivity to TNF-alpha and/or CDDP were selected for the study and consisted of 222, a TNF/CDDP-sensitive line, 222TR (TNF-resistant), a TNF-resistant, CDDP sensitive line, and AD10, a TNF-sensitive, CDDP-resistant line. Cytotoxicity was determined by the microculture tetrazolium dye assay. RESULTS: Synergy in cytotoxicity was achieved in all three lines treated with a combination of TNF alpha and CDDP. Cytotoxicity by either TNF-alpha or CDDP or by both TNF-alpha and CDDP was inhibited in the presence of either Phe or BHA. Pretreatment of tumor cells with either Phe or BHA for up to 4 hours, washed and followed by the addition of the cytotoxic agents (alone or combined), resulted in no inhibitory effect. Pretreatment of the cells with the cytotoxic agent for up to 2 hours, washed and then followed by the addition of Phe, resulted in significant inhibition of cytotoxicity. In contrast to Phe, the addition of BHA as late as 12 hours post pretreatment of the cells with the cytotoxic agent(s) still inhibited cytotoxicity. These results demonstrated that free radicals are involved in cytotoxicity mediated by a single agent, and in synergy with both agents. Further, the results demonstrated that Phe acts at an early stage of the cytotoxic pathway and that BHA acts at both an early and a late stage of the cytotoxic pathway. CONCLUSIONS: These results demonstrated that both TNF-alpha and CDDP rapidly stimulate the induction of free radicals but a lag of several hours was necessary to initiate the irreversible program of cell death. Further, the studies demonstrated that synergy and reversal of drug resistance in ovarian tumor cells by TNF-alpha and CDDP, used in combination, share the same pathway of cytotoxicity as that mediated by TNF-alpha or CDDP used as a single agent. PMID- 8616766 TI - Randomized trial of initial therapy with melphalan versus cisplatin-based combination chemotherapy in patients with advanced ovarian carcinoma: initial and long term results--Eastern Cooperative Oncology Group Study E2878. AB - BACKGROUND: Following surgical debulking, most patients with international Federation of Gynecology and Obstetrics (FIGO) Stage III or IV carcinoma of the ovary receive treatment with combination chemotherapy. However, the optimal postsurgical therapy for ovarian carcinoma remains to be defined. METHODS: To define better the role of initial therapy with a cisplatin-based chemotherapy regimen, the Eastern (Cooperative Oncology Group (ECOG) initiated a randomized, Phase III trial, EST 2878, comparing initial therapy with a single, orally administered alkylating agent, melphalan, versus a complex regimen employing cyclophosphamide, hexamethylmelamine, doxorubicin, and cisplatin (CHAD). Women who failed treatment with melphalan were crossed-over to treatment with CHAD minus the cyclophosphamide (HAD). Study endpoints included response to therapy, time to treatment failure, and overall survival. RESULTS: Between October, 1978, and November, 1980, EST 2878 accrued 253 patients with advanced epithelial carcinoma of the ovary. There were 118 eligible patients initially treated with melphalan and 126 with CHAD. Two patients experienced lethal toxicities, including gastrointestinal hemorrhage (1 patient) and neutropenic sepsis (1 patient), and 22 patients experienced life-threatening toxicities, including hematologic toxicity (21 patients) and anaphylaxis (1 patient). Response to treatment and clinical complete response rates were higher in women receiving CHAD (60% and 38%, respectively) versus melphalan (42% and 21%, respectively) (P = 0.037 and P = 0.024, respectively), but these differences were confined to women older than 50 years of age. Likewise, time to treatment failure was significantly longer in women receiving CHAD (P = 0.014), but the difference was again confined to women older than 50 years of age and to women suboptimally debulked at the time of surgery. Survival did not differ between the two arms (median survivals of 17.5 months with initial melphalan therapy and 19.5 months with CHAD), probably because women treated initially with melphalan received salvage therapy with HAD). Twenty-three patients survived longer than 10 years. Among 18 long term survivors who had retrospective pathologic review, 8 had borderline tumors of the ovary. CONCLUSIONS: In women with advanced ovarian cancer, initial therapy with a cisplatin-based combination chemotherapy regimen resulted in higher clinical complete response rates and longer time to failure compared with initial therapy with a single, oral alkylating agent; however, the benefits of this approach were confined to women older than 50 years of age at diagnosis, and there was no significant difference in survival. PMID- 8616767 TI - Distant metastasis from bilharzial bladder cancer. AB - BACKGROUND: Distant metastasis is rarely described among bilharzial bladder cancer patients. However, with improved 5-year survival rates following adjuvant local therapy, distant metastasis is now reported with increasing frequency. METHODS: Three-hundred-fifty-seven bilharzial bladder cancer patients were treated at the National Cancer Institute in Cairo, Egypt, during the period 1981 1990. They were treated with either cystectomy alone, cystectomy preceded by a short course of preoperative radiotherapy (2000 cGy/5 fractions/1 week), or cystectomy followed by postoperative irradiation (5000 cGy/25 fractions/5 weeks or 3750 cGy/30 fractions/2 weeks). These patients were retrospectively analyzed. RESULTS: The overall 5-year actuarial rate of distant metastasis was 23% (95% confidence interval, 21-25%), which was essentially the same in the 3 therapeutic groups. Both univariate and multivariate analyses revealed that the independent risk factors for distant metastasis were pelvic lymph node involvement (P = 0.005), pathologic stage (P = 0.004), and histopathologic grade (P = 0.05). Histologic type and local pelvic recurrence appeared in the univariate analysis as working risk factors; however, they were proven by multivariate analysis to be dependent on other risk factors. Patients who had none of the independent risk factors had a lower rate of distant metastasis (II%) and a high local control rate (88%). Those who had more than one risk factor had high distant metastasis rate (51%) and low local control rate (41%), regardless of the therapeutic modality used. The identified independent risk factors determined both the distant metastasis and the local control rates. CONCLUSIONS: Unlike previous reports, this rigorous study of distant metastasis in bilharzial bladder cancer revealed an occurrence rate of 23%. This high rate was associated with pelvic lymph node involvement, pathologic stage, and histopathologic grade. Histologic type, local pelvic recurrence, or the addition of pre- or post-operative radiotherapy proved not to be independent risk factors. PMID- 8616768 TI - Physiologic versus neoplastic C-cell hyperplasia of the thyroid: separation of distinct histologic and biologic entities. AB - BACKGROUND: Although hyperplasia of C-cells has been described in association with various pathologic and physiologic conditions, criteria for its diagnosis are poorly defined. Both neoplastic and physiologic C-cell proliferations have been lumped together under the umbrella designation of C-cell hyperplasia (CCH), creating considerable confusion among clinicians and pathologists. METHODS: in order to compare the morphologic and immunohistochemical characteristics of the two major types of CCH, we examined thyroid sections of 17 patients with familial forms of C-cell hyperplasia and/or neoplasia and tissue sections of 19 thyroid glands known to have reactive or physiologic CCH (at least 50 C-cells per one low power field, 100X). Hematoxylin and eosin (H & E) stained sections and immunohistochemical stains for calcitonin were assessed in each case. RESULTS: Physiologic or reactive CCH was not recognized with certainty on H & E stains in any of the cases due to morphologic similarities between C-cells and adjacent follicular cells. Detection of this form of hyperplasia, which was predominantly diffuse, required calcitonin immunostains and quantitative analysis. Conversely, nodular and diffuse neoplastic CCH was easily identified with conventional H & E stains at the periphery of 11/12 (92%) familial medullary thyroid carcinomas (MTC). In the other five cases, neoplastic C-cell hyperplasia was the only pathologic finding on thyroidectomy performed for elevated serum calcitonin levels detected via provocative biochemical screening or identification of the mutated RET proto-oncogene by genetic analysis. The C-cells in this neoplastic form of CCH were large, mildly to moderately atypical, and confined within the basement membrane of thyroid follicles. Moreover, these cells were cytologically indistinguishable from those of invasive MTC cells. CONCLUSIONS: Physiologic and neoplastic CCH are biologically and morphologically distinct entities. The former cannot be recognized with certainty with conventional stains and requires immunohistochemistry and quantitative analysis for diagnosis. The latter consists of mildly to moderately atypical C-cells that can be identified with H & E stained sections. Consequently, the number of C-cells is of no importance for the diagnosis of neoplastic CCH which is considered to be the precursor (medullary carcinoma in situ) of invasive medullary carcinoma. PMID- 8616769 TI - Clonal analysis by polymerase chain reaction of B-cell lymphoma with late relapse: a report of five cases. AB - BACKGROUND: Little is known about the clonal heterogeneity of non-Hodgkin's lymphoma between presentation and relapse, although several such reports have been published on acute lymphoblastic leukemia. METHODS: We examined five patients with B-cell lymphoma who relapsed more than 5 years after initial presentation. Formalin fixed, paraffin embedded tissues were analyzed for clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction (PCR) and sequencing of the PCR products. Four specimens retained the original histologic type, but one showed histologic transformation from diffuse large cell lymphoma to follicular small cleaved cell lymphoma. RESULTS: Although the size of the PCR products looked identical on the gel between presentation and relapse in all patients, only three of the four specimens that retained the original type had identical gene rearrangements at both presentation and relapse. One of these four and the fifth specimen showed novel gene rearrangements. CONCLUSIONS: This study suggests that late relapse lymphoma may present as a new clone. Sequencing of the PCR products is important in the evaluation of clonal heterogeneity. PMID- 8616770 TI - Cancer incidence in the rural community of Tecumseh, Michigan: a pattern of increased lymphopoietic neoplasms. AB - BACKGROUND: The Tecumseh Community Health Study (TCHS), initiated in 1959 at the University of Michigan School of Public Health, has provided a resource for long term prospective studies of the incidence of cancer in the setting of a midwestern rural farming community. METHODS: A survey of total and site specific cancer incidence among 7016 male and female adult residents from 1959-1987 was conducted, and the observed number compared with the expected number, based on the age-sex-race-calendar period-and site-specific cancer incidence rates reported by the Connecticut tumor registry. Based on the results of this survey, a hypothesis was advanced concerning the potential risks of exposures to insecticides and herbicides. This was pursued by analyzing for each county in Michigan the comparative annual number of acres and percentage of acreage treated with agricultural chemicals in 1978 and for the period 1982-1987. Finally, because of the availability of information on lifestyle risk factors that had been collected in the 1960s on all participants, a nested case-control study was implemented. RESULTS: The standardized incidence ratio (SIR), based on the observed number divided by the expected number of invasive cancer cases (all sites combined [excluding nonmelanoma skin cancer]), was not significant in females (SIR, 0.95; 95% confidence interval [CI], 0.86-1.05) nor males (SIR, 0.91; 95% CI, 0.81-1.01). A significant increase was demonstrated for males and females combined in the incidence of lymphopoietic neoplasms, namely non Hodgkin's lymphoma, Hodgkin's disease, and chronic lymphocytic leukemia; the combined SIR was 1.40 (95% CI, 1.03-1.86; P = 0.03). In the Department of Commerce surveys of counties in Michigan, the Tecumseh area (Lenawee County) was ranked highest with respect to the average annual number of acres (240,000 or more) and the percent of acreage (40%) sprayed with herbicides and insecticides. A comparison of temporal trends for cancer incidence since 1973 was reviewed for the Wayne-Oakland-Macomb tricounty area, in which the survey estimated that less than 80,000 acres per year, or less than 8% of acreage, were treated with pesticides. The comparison of the Tecumseh cohort, for all sites combined, was not significantly different from expectation in females (SIR, 1.01; 95% CI, 0.89 1.14), and was decreased by more than 10% in males (SIR, 0.88; 95% CI, 0.77 1.00). However, the SIR for non-Hodgkin's lymphoma in females was significantly elevated (SIR, 1.92; 95% CI, 1.07-3.11, P = 0.02); the trend for increased risks of lymphomas and leukemias was also evident in males. In the nested case-control study, based on risk factor information documented prior to diagnosis, the relative risk of a family history of lymphoma, leukemia, or multiple myeloma was significantly increased among patients with lymphoproliferative neoplasms (odds ratio, 3.81; 95% CI, 1.49-9.79; P = 0.005). CONCLUSIONS: This prospective epidemiologic study conducted in a rural farming community in Michigan has found significant increases in the incidence dence of lymphoproliferative cancers. A plausible hypothesis, consistent with the preliminary findings, is that the reported cancer pattern is an expression of risk resulting from sustained environmental exposures to agricultural chemicals, perhaps in conjunction with familial or genetic factors. PMID- 8616771 TI - Functional adrenal cortical tumors in pediatric patients: a clinicopathologic and immunohistochemical study of a long term follow-up series. AB - BACKGROUND: Controversy exists as to which variable is a reliable predictor of clinical outcome of adrenal cortical tumors in children. METHODS: Twenty patients with adrenal cortical tumors were studied. Tumor weight, histologic features, and percentage of proliferating cell nuclear antigen (PCNA/cyclin) in tumor cells were analyzed to determine the best predictor of clinical outcome. RESULTS: Eleven patients had Cushing's syndrome with virilization and 9 had virilization without Cushing's syndrome. The mean age at diagnosis was 7.1 +/- 5.2 years (range, 0.4-15.6 years). Sixteen patients, with good outcomes have been followed for 10.7 +/- 7.8 years (range, 3-23 years). All but two patients had a tumor weight of less than 100 g (185 g and 800 g, respectively) (mean 47.7 g +/- 46.4 g). Two patients with large tumors (weighing 1000 g and 780 g, respectively) had poor outcomes; 1 died 3 months after surgery with metastasis and the other presented with lung metastasis 18 months after surgery. Histologic features did not correlate with clinical outcome. Overall, PCNA stained cells were 6.96 +/- 8.2% (range, 0-32.5%). PCNA values were significantly lower in tumors of patients with good outcomes (P < 0.002). Within all tumors, we found a weak correlation between tumor weight and PCNA (r = 0.51; P < 0.02), but a better correlation was found between tumor weight and PCNA in patients with Cushing's syndrome (r = 0.70; P < 0.01). Patients with Cushing's syndrome had higher PCNA values than those with virilization syndrome (10.3 +/- 9.6% vs. 2.8 +/- 3.3%; P < 0.03). CONCLUSIONS: Our data show that small tumors (less than 100 g) are associated with good outcome; the two patients with the poorest prognosis had Cushing's syndrome and large tumors (more than 100 g). Histologic features are not adequate predictors of outcome and PCNA may be useful in tumors of patients with Cushing's syndrome, but this parameter should not be used alone. Two patients had virilization syndrome, large tumors (185 g and 800 g, respectively), and good outcomes, which contradicts with the concept that these tumors are usually associated with poor prognosis. PMID- 8616772 TI - Inflammatory myofibroblastic tumor (inflammatory pseudotumor): DNA flow cytometric analysis of nine pediatric cases. AB - BACKGROUND: Inflammatory myofibroblastic tumor or inflammatory pseudotumor is an uncommon lesion reported in various organs and believed to be a non-neoplastic reactive inflammatory condition. The concept of benign lesion has been recently challenged from both clinical demonstration of recurrence and cytogenetic evidence of acquired clonal chromosomal abnormality. Because DNA aneuploidy can be a useful marker for neoplasia, we analyzed nuclear DNA content of these lesions using flow cytometry. METHODS: In this study, inflammatory myofibroblastic tumors from nine children were examined retrospectively by evaluating clinicopathologic features and ploidy. DNA ploidy status was analyzed by flow cytometry in nuclei isolated from paraffin-embedded tumor tissues. RESULTS: Three of the nine patients had local recurrence or distant metastases. Flow cytometric DNA analysis revealed five of the nine cases were diploid and four hyperdiploid (DNA indices 1.14, 1.16, 1.19, and 1.33). All lesions had a low S-phase fraction. Samples from the three subjects with clinical recurrence were all hyperdiploid. CONCLUSIONS: The present data indicate that flow cytometry identifies aneuploidy (hyperdiploidy) in approximately half of the cases of inflammatory myofibroblastic tumors. This feature appears to reflect a more aggressive biologic behavior. In addition to the reported cytogenetic abnormalities, our data suggest that inflammatory myofibroblastic tumor, generally considered a benign reactive inflammatory process, may evolve as a distinct, potentially malignant, lesion. Therefore, flow cytometric DNA analysis is a suitable tool to provide the clinician with both diagnostic and prognostic information and to individuate the most feasible therapeutic approach. PMID- 8616773 TI - Variation in survival of children with cancer within a region of the United Kingdom. AB - BACKGROUND: A population-based study of survival of 678 children, age less than 15 years, diagnosed with cancer in the Southwest region of the United Kingdom showed an improvement in 5-year survival from 53% between 1976 and 1980 to 64% between 1981 and 1985 (P = 0.008). Survival varied significantly among the five counties that make up the region (P = 0.0008); the differences were greatest for central nervous system (CNS) tumors. METHODS: The expertise of local hospitals and entrance into national trials was examined. The region has nine hospitals acting as primary treatment hospitals for children with cancer, two neurosurgical units, and one regional oncology unit. Four of the primary hospitals saw more than six children per year and were designated "large" centers. Five saw fewer than six patients per year and were designated "small" hospitals. RESULTS: There was a significant difference in survival of patients according to the number of patients treated per year per hospital. A hospital that treated fewer than six patients per year was designated "small" and six or more designated "large." At 5 years, patients with CNS tumors experienced a 58% survival rate in large hospitals and a 41% survival rate in small hospitals. (P = 0.03). The rate of entrance into trials for all malignancies did not differ between large (30%) and small (27%) centers. Only 5% of children with CNS tumors were placed on national trials. There was no difference in survival rates for CNS tumors in the two neurosurgical centers. CONCLUSIONS: There were significant differences in survival by county for children with cancer, especially those with CNS tumors. In the first half of the study decade, in hospitals treating fewer than six cases of childhood cancer per year, children with CNS tumors were rarely placed on national protocols and did poorly. PMID- 8616774 TI - The identification of febrile, neutropenic children with neoplastic disease at low risk for bacteremia and complications of sepsis. AB - BACKGROUND: The management of pediatric oncology patients with fever and neutropenia assumes that all patients are at risk for bacteremia, and therefore generally involves hospitalization and broad-spectrum parenteral antibiotics for all patients. The determination of which patients are at low risk for having positive blood cultures and for developing complications related to bacteremia is of potential benefit. METHODS: The records of 161 pediatric patients with neoplastic disease hospitalized for 509 episodes of fever and neutropenia between January 1990 and June 1992 were retrospectively reviewed. Clinical features at initial presentation, clinical and microbiologic documentation of infection, and outcome were analyzed. RESULTS: The only presenting clinical features that correlated with an increased likelihood of having positive blood cultures were chills, hypotension, the requirement for fluid resuscitation (P < 0.001), or a diagnosis of leukemia or lymphoma (P < 0.041). Leukemia patients with relapse admitted for fever and neutropenia were no more likely to have positive blood cultures than those patients in remission. There were ten episodes of fever and neutropenia in which patients were transferred to the intensive care unit (ICU), and two sepsis related deaths. Nine episodes involving ICU management and both deaths were in patients who had persistent fever and an absolute neutrophil count (ANC) of less than 100 after 48 hours of hospitalization (n = 177). Patients with an ANC of less than 100 after 48 hours were twice as likely to have antibiotic changes, 15 times more likely to receive amphotericin B, and were hospitalized twice as long as patients with an ANC of 100 or higher after 48 hours. CONCLUSIONS: Children hospitalized for fever and neutropenia who have persistent fever and an ANC of less than 100 after 48 hours are at high risk for morbidity and are more likely to require antibiotic changes and antifungal therapy. Children who initially lack signs of sepsis, are afebrile, and have an ANC of 100 or higher after 48 hours are at low risk for complications and could be selectively discharged on antimicrobials after a 48-hour period of inpatient hospitalization. PMID- 8616775 TI - A nude mouse Wilms' tumor line (KCMC-WT-1) derived from an aniridia patient with monoalleleic partial deletion of chromosome 11p. AB - BACKGROUND: A candidate tumor suppressor gene, WT-1, is believed to have an important role in the pathogenesis of Wilms' tumor, especially that occurring in patients with congenital aniridia. METHODS: To obtain a stable tumor line to work with, Wilms' tumor tissue was serially transplanted in athymic nude mice. Biopsied Wilms' tumor tissue, derived from an aniridia patient, was transplanted subcutaneously to an athymic nude mouse, and then transplanted serially. Histopathologic and molecular biologic studies were performed on the xenotransplants. RESULTS: The aniridia patient showed partial deletion in one short arm of chromosome 11, which bears the WT-1 gene. The tumor was successfully transplanted in the nude mouse. Although the tumor contained blastemic, organoid, and stromal histologic elements, the organoid element began to decrease after more than 20 passages. Cytogenetic analysis revealed an additional abbreviation of one long arm of chromosome 6. Dot blot analysis showed that the copy number of WT-1 gene was decreased to half the amount in the tumor, in spite of the WT-1 transcript with normal size detected by Northern blotting. CONCLUSIONS: The tumor is expected to bear one WT-1 gene with minute abnormalities as well as one congenitally deleted gene. This tumor line is useful when examining the effect caused by introduction of WT-1 gene to Wilms' tumor in vivo. PMID- 8616776 TI - Metastatic neuroblastoma in children older than one year: prognostic significance of the initial metaiodobenzylguanidine scan and proposal for a scoring system. AB - BACKGROUND: Metaiodobenzylguanidine (mIBG) is a guanethidine analog that has demonstrated a high sensitivity and specificity in detecting bone metastases in about 90% of metastatic neuroblastomas. However, the predictive value of initial mIBG scan in neuroblastoma patients older than 1 year of age regarding response to initial chemotherapy has yet to be ascertained. Therefore, a scoring system for grading the positivity of mIBG scans was devised and applied in a retrospective study in an attempt to determine whether this score had a prognostic value in neuroblastoma patients older than 1 year of age at diagnosis. METHODS: Eighty-six children, older than 1 year of age, with metastatic neuroblastomas were homogeneously treated and had a mIBG scan performed at diagnosis and following the induction regimen to assess bone metastases. Each mIBG scan was assigned a reproducible score and the predictive value of the initial mIBG score was assessed in order to evaluate response to induction regimen. RESULTS: The relative risk of failing to achieve complete remission after four courses of induction therapy was 6.9 times higher in patients who had more than four mIBG spots at diagnosis. A multivariate analysis including the established prognostic factors revealed that the initial mIBG score was the only significant factor (P < 0.001). CONCLUSIONS: The initial mIBG scan is of prognostic significance to predict response to chemotherapy for metastatic neuroblastoma in children older than 1 year of age. A prospective study comparing this initial mIBG score with other recently established prognostic factors is warranted. PMID- 8616777 TI - Predominant 45,X,--Y karyotype in donor cells after allogeneic BMT: cytogenetic and molecular analysis. AB - We describe two women; one (patient 1) with the diagnosis of acute myeloblastic leukemia (AML), the second (patient 2) with myelodysplastic syndrome (MDS). Both patients underwent allogeneic bone marrow transplantation (BMT), from their HLA matched brothers. Cytogenetic analysis after the BMT revealed a chromosomal mosaicism in both patients, with the karyotype 46,XX/45,X with no sign of the Y chromosome. The origin of the clone with monosomy X was determined using cytogenetic analysis including heteromorphism and segregation of DNA polymorphic markers. The results led us to the conclusion that in both patients the origin of the 45,X clone was that of the donors. Patient 1 had MDS-like syndrome after the BMT and was stabilized in the chimeric state; to date she is doing well. Patient II also had MDS. However, in her case, it was her primary disease. The graft in patient II was rejected and she died 6 months after BMT. PMID- 8616778 TI - Molecular rearrangement of the MLL gene in adult acute myeloid leukemia without cytogenetic evidence of 11q23 aberration. AB - Translocations involving chromosome band 11q23 are frequently found in infant acute leukemia and involve rearrangement of the MLL gene. In this study, 29 cases of adult acute myeloid leukemia (AML) were analyzed to determine the frequency of MLL gene rearrangement. Of these, 19 cases were karyotyped and none showed cytogenetic evidence of 11q23 aberration. MLL rearrangements were demonstrable in four cases, giving a frequency of 14% (4/29). Only one of the four cases with MLL rearrangement showed features typical of leukemia with 11q23 aberration; other cases were indistinguishable from those without MLL rearrangement. There was no apparent difference in presentation blast count, remission, and survival duration when cases with or without MLL rearrangement were compared. Clinicopathologic features of adult AML with MLL rearrangements may be heterogeneous. PMID- 8616779 TI - Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis. AB - Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity. PMID- 8616780 TI - Codeletion of the genes for p16INK4, methylthioadenosine phosphorylase, interferon-alpha1, interferon-beta1, and other 9p21 markers in human malignant cell lines. AB - In this study, 27 malignant cell lines, including leukemias, gliomas, and lung and bladder carcinomas were screened for homozygous deletions of the putative tumor suppressor gene p16 (MTS1/CDK4I/CDKN2) and other markers within the chromosome 9p21 region; these include the genes for interferon-alpha1 (IFNA1), interferon-beta1 (IFNB1), methylthioadenosine phosphorylase (MTAP), and two microsatellite markers, D9S171 and D9S169. The purpose of this study was to determine the incidence of codeletion of these markers. Screening for homozygous deletions was carried out using direct polymerase chain reaction of genomic DNA, or, in the case of MTAP, a functional enzyme assay. Of these cell lines, 14 (52%) were found to have homozygous deletions of the p16 gene. Two of the 14 p16 negative cell lines (14%) were found to have homozygous deletions within the p16 domain but but no other 9p21 marker. MTAP was codeleted in 12 of the 14 p16 negative cell lines (86%), whereas IFNA1 was codeleted with p16 in eight of these lines (57%); IFNB1 was codeleted in five (36%) of the p16-deleted cell lines. The D9S171 marker, which may lie greater than 3 cM centromeric to p16, is codeleted in three cell lines (21%); the D9S169 marker, which maps even further toward the centromere, was codeleted in only one cell line (7%). Loss of any 9p21 marker, e.g., MTAP or IFNA1, were invariable predictors of the loss of the p16 gene. In addition, loss of IFNA1 always predicted a loss of MTAP (eight of eight cell lines), although loss of MTAP did not always predict a loss of IFNA1 (four of 12 MTAP-deleted cell lines did not have homozygous deletions of IFNA1). Thus loss of nearby genes occurs in a high percentage of cell lines that bear homozygous deletions of the p16 locus. Codeletion of MTAP or IFN in p16-negative malignant cells is of interest, as loss of these genes may influence the biologic behavior of these cells and render them susceptible to certain therapeutic approaches. PMID- 8616781 TI - A case of therapy-related extramedullary acute promyelocytic leukemia. AB - We report a patient with extramedullary acute promyelocytic leukemia (APL) occurring after radiation therapy for carcinoma of the prostate. To the authors' knowledge, this patient represents the first case of cytogenetically and fluorescence in situ hybridization (FISH) confirmed therapy-related extramedullary APL. In contrast to the majority of previously reported t-APL, this case underwent a very unfavorable course. PMID- 8616783 TI - Translocation (Y;1)(q12;q12) in hematologic malignancies. Report on two new cases, FISH characterization, and review of the literature. AB - Translocation (Y;1)(q12;q12) is a rare cytogenetic anomaly occurring in hematologic disorders thought to affect stem cells. We report here on two new cases, one end-stage myelofibrosis and one chronic myelomonocytic leukemia. The translocation breakpoints were assessed by conventional cytogenetic techniques in both cases and by FISH in the second case. A partial trisomy of the 1q21-qter region could be demonstrated. The data of the literature are reviewed and the possible pathogenetic mechanisms are discussed. PMID- 8616782 TI - Acute leukemia with t(10;11)(p11-p15;q13-q23). AB - We report six patients with acute leukemia characterized by the presence of a t(10;11)(p11-15;q13-q23), either as sole cytogenetic abnormality (three patients) or as part of a complex abnormal karyotype. The morphologic and cytochemical features of four patients were consistent with FAB-M5A, while two patients presented with FAB-L1 characteristics. By immunophenotyping, myeloid leukemia was diagnosed in five patients, including one patient with FAB-L1 leukemia who typed as terminal transferase (TdT)+, CD7 T-cell antigen+ acute myelomonocytic leukemia. Differentiated acute myeloid leukemia (AML) with expression of terminal transferase was found in two of the other cases and monocytic leukemia in two, with co-expression of T-cell antigens in one of them. The second FAB-L1 patient typed as undifferentiated acute lymphocytic leukemia (ALL) expressing myeloid antigens. Serial phenotypic studies in patient 3 during the course of the disease demonstrated a switch from monocytic to lymphoid morphology at the time of first and second relapse, which was paralleled by the appearance of a pre-T ALL immunophenotype with co-expression of the myeloid antigen CD33. These phenotypic changes occurred without apparent alteration in the genotype since t(10;11)(p11.2;q23) remained the only cytogenetic aberration at all stages of the disease. Our observations suggest that the (10;11) variant of 11q aberrations occurs in a bipotential myelomonocytic/T-lymphoid stem cell. PMID- 8616784 TI - Mapping of the translocation breakpoints of primary pleomorphic adenomas and lipomas within a common region of chromosome 12. AB - Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosomal aberrations of 12q14-q15 have indicated that the chromosome 12 breakpoints cluster in a 445-kb region designated ULCR12 (uterine leiomyoma cluster region of the chromosome 12 breakpoints). Here we report the results of FISH studies of five primary pleomorphic adenomas and six primary lipomas and established cell lines of these tumor types characterized by translocations involving the chromosomal segment 12q13-q15. The results reveal that for nearly all tumors and cell lines analyzed, the chromosome 12 breakpoints map within a 350-kb region included in ULCR12, despite the previous cytogenetic assignment of the breakpoints to different bands of that region. In some cases the primary material and additionally analyzed cell lines allowed an even more precise localization of the breakpoints to less than 100 kb. Furthermore, a previously hidden translocation of ULCR12 in one primary tumor could be detected by FISH. PMID- 8616785 TI - Complex cytogenetic rearrangement in a case of unicameral bone cyst. AB - Cytogenetic analysis of a unicameral bone cyst surgically resected in an 11-year old boy revealed a highly complex clonal structural rearrangement involving chromosomes 4, 6, 8, 16, 21, and both 12. These findings reinforce the need for further studies on unicameral bone cysts to verify the frequency and to understand the significance of chromosome anomalies in this type of lesion. PMID- 8616786 TI - Chromosome telomere integrity of human solid neoplasms. AB - Eukaryotic chromosomes contain specialized structures at the termini called telomeres. This region of DNA is required for replication and stability of the chromosome. Telomere reduction can contribute to genetic instability and has been described in certain malignancies (e.g., colon, leukemia, giant cell tumor of bone). To determine whether telomere reduction is a generalized phenomenon in malignancies, the telomere integrity of genomic DNA isolated from tumor cells was determined from 39 individuals with 15 different malignancies categorized as musculoskeletal, epithelial, cranial, or other, and peripheral blood leukocytes from the same patient, when possible, or age-matched controls. Significant telomere reduction occurred randomly across histopathologic groups including giant cell tumor of bone, glioblastoma, colon cancer, and Wilms' tumor while telomere elongation occurred in chordoma. The other remaining 10 malignancies do not show significant differences in telomere lengths compared with controls. PMID- 8616787 TI - Choromosome instabililty and sister chromatid exchange (SCE) studies in patients with carcinoma of cervix uteri. AB - Cytogenetic studies have been carried out using the G-banding technique in peripheral blood lymphocytes of 14 patients with carcinoma of the cervix uteri. Simultaneously, sister chromatid exchange (SCE) was also analyzed in the peripheral blood lymphocytes of these patients, along with those of 20 age matched control subjects. The frequency of aberrant metaphases is significantly higher in patients with carcinoma of the cervix uteri (7.85%) than in the age matched controls (3.35%). A large number of chromosome aberrations in lymphocytes of these patients have also been detected. Sister chromatid exchange (SCE) was also analyzed in lymphocytes of 14 patients with carcinoma of the cervix uteri and 20 age-matched control subjects. The mean SCE frequencies were 9.44 +/- 0.34 (n = 637) and 6.09 +/- 0.24 (n = 900) per metaphase in patients and controls, respectively. The increase of SCE frequency in cancer patients was statistically significant (p < 0.001), but not seen in controls. Our results suggest that patients with carcinoma of the cervix uteri show a degree of chromosomal instability that might be related to a predisposition to neoplasia. PMID- 8616788 TI - Trisomy 21 in solitary fibrous tumor. AB - We found trisomy 21 as the sole chromosome abnormality in a solitary fibrous tumor arising at a nonpleural site. No cytogenetic investigation of solitary fibrous tumors has previously been reported. PMID- 8616789 TI - Near-hexaploid Ph-positive acute myeloid leukemia with major-BCR/ABL transcript. AB - We describe the first case of acute myeloid leukemia (AML) with a Philadelphia (Ph) translocation and a near-hexaploid range chromosome number, whose leukemic cells had the major-BCR/ABL transcript. The genesis of near-hexaploid leukemic cells might be due to endoreduplication of triploid leukemic cells with the Ph, since the relapsed leukemic cells had triploid range chromosomes with double Ph chromosomes. PMID- 8616790 TI - Trisomy 11 with loss of the Y chromosome and trisomy 13 in a case of de novo acute myeloid leukemia. AB - We report a man with de novo acute myeloid leukemia (M4 of the FAB classification) bearing two abnormal clones in the bone marrow cells. The clones showed trisomy 11 with loss of the Y chromosome and trisomy 13, respectively. PMID- 8616791 TI - A case of chromophobe renal cell carcinoma associated with low chromosome number and microsatellite instability. AB - Cytogenetic study in a case of a human chromophobe renal cell carcinoma revealed a hypodiploid chromosome number of 36 with loss of chromosomes 1, 2, 5, 6, 10, 13, 15, 17, 21, and X. The tumor DNA showed microsatellite instability in dinucleotide repeat microsatellite markers. This is the fourth case that has been fully karyotyped and showed a low chromosome number in a chromophobe renal cell carcinoma. Our data in the present study are consistent with those in the literature. It is suggested that human chromophobe renal cell carcinoma may possibly be characterized by tumor cells with low chromosome number or microsatellite instability. PMID- 8616792 TI - t(7;11) and trilineage myelodysplasia in acute myelomonocytic leukemia. AB - A 48-year-old Japanese man was admitted to our hospital because of general fatigue, nasal bleeding, and petechiae on his extremities. He was diagnosed with acute myelomonocytic leukemia with trilineage myelodysplasia (T-MDS). Chromosomal analysis of bone marrow cells revealed t(7;11)(p15;p15), which has been rarely reported but known to be characteristic of Japanese patients. Although t(7;11)(p15;p15) has been reported mainly in acute myelogenous leukemia (AML), it can be occasionally found in so-called stem cell diseases such as chronic myelogenous leukemia or chronic myeloproliferative disorders. Therefore, t(7;11)(p15;p15) might affect trilineage progenitors or stem cells as well as myeloid lineage cells, subsequently resulting in AML with T-MDS, as in our case reported here. PMID- 8616793 TI - Presence of three recurrent chromosomal reaarrangements, t(2;3)(p12;q37), del(8)(q24), and t(14;18), in an acute lymphoblastic leukemia. AB - A complex chromosomal abnormality associating three recurrent rearrangements, t(2;3)((p12;q37), del (8)(q24) and t(14;18)(q32;q21), was detected in a patient with acute lymphoblastic leukemia of the Burkitt type. Southern blot studies showed rearrangements of the MYC, BCL2, and JH genes, thus confirming the cytogenetic data. However, no rearrangement of the LAZ3/BCL6 gene, normally localized on band 3q27, could be detected. The simultaneous presence of three recurrent rearrangements specific for lymphoid malignancies addresses the question of their timing in the malignant process and the prognostic significance of the association of such anomalies. PMID- 8616794 TI - A greater incidence of complex translocations in myeloid leukemias than in lymphomas and lymphoid leukemias associated with IGH rearrangement. AB - We have shown that the incidence of complex translocations is approximately the same in chronic myeloid leukemia, characterized by the t(9;22)(q34;q11), and in acute myeloid leukemias, characterized by the t(15;17)(q22;q11) or t(8;21)(q22;q22). This incidence is almost threefold greater than the incidence of complex translocations in lymphomas and lymphoid leukemias characterized by the t(8;14)(q24;q32) or t(14;18)(q32;q21). The genomic recombination, which gives rise to the translocations in lymphoid cells, results mostly from errors of IGH gene rearrangement. Genomic recombination underlying myeloid leukemias has a different cause, and a clue to this may lie in the greater incidence of complex chromosome rearrangements. PMID- 8616795 TI - Case of acute monocytic leukemia with 47,XY,+X,t(2;10)(q21.1;q26.1) and basophilia. AB - The clinical cytogenetic findings of a patient with acute monocytic leukemia with peripheral and bone marrow basophilia are presented. The cytogenetic analysis of bone marrow cells established a pathologic clone with the following unusual karyotype: 47,XY,+X,t(2;10)(q21.1;q26.1). This chromosome abnormality has not been reported previously in leukemic diseases. PMID- 8616796 TI - Translocation (1;5)(q32;q35) in CD30+ anaplastic large cell non-Hodgkin lymphona of childhood. A case report. AB - The authors studied cytogenetically a case of CD30+ anaplastic large cell non Hodgkin lymphoma previously diagnosed as malignant histiocytosis and detected a translocation involving chromosomes 1 and 5, t(1;5)(q32:q35). After comparing their findings with those from reports in the literature, they comment about the importance of breakpoint q35 on chromosome 5 and point out the importance of associating morphologic, immunoperoxidase, and cytogenetic findings to confirm the diagnosis of this tumor. PMID- 8616797 TI - Translocation (15;17)(q22;q21) in a patient with Klinefelter syndrome. PMID- 8616798 TI - Constitutional trisomy 8 mosaicism and cancer. PMID- 8616799 TI - Partial trisomy of chromosome 4 resulting from a novel t[4;7) translocation in a case of AML M2 with eosinophilia. PMID- 8616800 TI - Trisomy 8 and 20 in desmoid tumors and breast cancer: more than a casual coincidence? PMID- 8616801 TI - Altered CD3 chain and cytokine gene expression in tumor infiltrating T lymphocytes during the development of mesothelioma. AB - The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de activated upon infiltration of the tumor micro-environment. PMID- 8616802 TI - Elevation of internal 6-methyladenine mRNA methyltransferase activity after cellular transformation. AB - A comparison of internal 6-methyladenine mRNA methyltransferase activity in a variety of cell types demonstrated an 8-15-fold increase as a result of cellular transformation. Utilizing adenovirus transformed rat embryo cells, it was found that the increase in methyltransferase activity was concomitant with or occurred rapidly after transformation. An 80-fold increase in activity was observed in the cells isolated from the transformed foci and remained elevated through subsequent passages. The relationship between methyltransferase activity and tumor formation was also investigated. High level expression of the avian ski oncogene in mouse L cells causes a reversion of the transformed phenotype to a non-transformed state, and resulted in a 47% reduction in the specific activity of the methyltransferase as compared with mock transfected cells. PMID- 8616804 TI - Detection of K-ras gene mutations in non-neoplastic lung tissue and lung cancers. AB - Oncogene and tumor suppressor gene mutations are candidate biomarkers for cancer risk assessment and lesion detection. The K-ras oncogene has previously been associated with non-small cell lung cancer (NSCLC), particularly adenocarcinomas in which reported rates of mutation have approached 30-40%. We have analyzed non malignant lung tissue from patients with lung cancer and primary lung cancers for K-ras gene mutations. Mutations were detected in 32% cancers and 29% non malignant lung tissue from patients with cancer. The majority of tumors testing positive were adenocarcinoma of the lung. Normal DNA controls, including peripheral blood lymphocytes and normal lung from non-smokers, were negative. The ability to detect genetic alterations in non-malignant lung tissues is consistent with the concept that genetic alterations are involved in field cancerization of the aerodigestive tract. PMID- 8616803 TI - Loss of human E-cadherin (ECD) correlated with invasiveness of transitional cell cancer in the renal pelvis, ureter and urinary bladder. AB - Loss or decreased expression of E-cadherin (ECD), which forms an epithelial junction complex that includes several other proteins and triggers signal transduction, may contribute to tumor progression. In the present study, we examined 90 transitional cell cancers (TCCs), 47 urinary bladder cancers and 43 ureteral or renal pelvic cancers, as well as TCC and papilloma cell lines to determine whether they express ECD. We classified ECD expression into normo expression (like normal epithelial), decreased and loss of ECD staining on TCCs (urinary bladder, renal pelvic or ureteral). We found that low-stage TCCs expressed normal ECD in 68%, decreased of ECD in 20% and loss of ECD in 12%, whereas high-stage TCCs expressed 29%, 41% and 30% of ECD staining, respectively (P < 0.01). Furthermore, grade 1 TCCs were all estimated to show normo expression, grade 2 TCCS expressed normal ECD in 49%, decreased of ECD in 41% and loss of ECD in 10% grade 3 TCCs classified as 20%, 30% and 50%, respectively (P < 0.01). Staining for cultured cell lines showed positive membranous staining for ECD in a benign papilloma cell line, RT4 and a TCC cell line, HT1376, but not in a TCC cell line, T24. Reverse-transcription polymerase chain reaction showed the presence of ECD and alpha-catenin mRNA in RT4 and HT1376, and only alpha-catenin in T24. Thus, it is more likely that decrease or loss of ECD might contribute to the malignant character of tumor cells and result in tumor progression. PMID- 8616805 TI - Promoting effect of large amounts of vitamin A on cell proliferation of thyroid proliferative lesions induced by simultaneous treatment with thiourea. AB - In order to examine modifying effects of simultaneous treatment with large amounts of vitamin A (VA) and thiourea (TU) on the thyroid tumorigenesis in rats, male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine (2800 mg/kg body weight, single s.c. injection), and starting 1 week later received diet containing 0.1% VA (VA group), drinking water containing 0.2% TU (TU group), 0.2% TU + 0.1% VA (TU + VA group) or tap water/basal diet (control group) for 19 weeks. Serum T3 and T4 in the TU and TU + VA groups were significantly decreased as compared to the control group, while serum TSH levels were remarkably increased. The ratios of T3 and T4 decrease and TSH increase in the TU + VA group were remarkably more pronounced than in the TU group. Thyroid neoplastic lesions were only induced in the TU and TU + VA groups. The multiplicity of intracapsular follicular cell proliferative foci in the TU + VA group was significantly increased as compared to the TU group value. Cell proliferation of hypertrophic and subcapsular follicular cells, as well as in hyperplasias, and neoplasias with adenomatous growth pattern was significantly higher in the combined treatment case than after TU alone. In the liver, centrilobular hypertrophy of hepatocytes was seen in the TU and TU + VA groups, this being especially marked in the latter group. In the combined group case the affected cells were strongly positive for GST-P antibody binding. The results of the present study suggest that cell proliferation of thyroid follicular cell proliferative lesions in rats is enhanced by strong TSH stimulation with simultaneous treatment of TU and large amounts of VA. PMID- 8616806 TI - A dislocated and enlarged proliferative zone in human gastric intestinal metaplasia. AB - There are many published reports suggesting a close relationship between intestinal metaplasia and gastric carcinogenesis, but there are few studies that examine the cellular kinetics of these tissues in humans. Thus, we sought to characterize the proliferative zone of intestinal metaplasia of the human stomach and correlate this with its known malignant potential. We examined the incorporation of bromodeoxyuridine into 228 human endoscopic biopsy specimens from duodenal mucosa (n=35) and non-intestinalized antral mucosa (n=127) as well as antral mucosa with intestinal metaplasia (n = 66). The proliferative zone in specimens with intestinal metaplasia was deeper when compared to non intestinalized antral mucosa, but was more superficial than that of duodenal mucosa. Although the labeling index of intestinalized mucosa was similar to that of non-intestinalized antral mucosa, the size of the proliferative zone was significantly increased in intestinal metaplasia. The dislocation of the proliferative zone with an increase in its size in intestinal metaplasia is considered to be a hallmark of gastric intestinal metaplasia. PMID- 8616807 TI - Antitumour activity of protein A in a mouse skin model of two-stage carcinogenesis. AB - Protein A (PA) is an immunostimulating glycoprotein (mol. wt. 43,000 kDa) obtained from Staphylococcus aureus cowan I. The antitumour property of PA is well documented in the literature in various transplantable tumours of rats and mice. In the present set of investigations, the antitumour property of PA was tested in Swiss albino mice in a two-stage initiation-promotion mouse skin carcinogenesis model. The animals were initiated topically with a single subcarcinogenic dose (52 microgram) of 7,12-dimethylbenzanthracene (DMBA). PA was administered intraperitoneally (1 microgram/animal), twice weekly for 2 weeks. Promotion was performed by twice weekly applications of 12-O- tetradecanoyl phorbol-13-acetate (TPA) at a dose of 5 microgram/animal for 32 weeks. The result showed that the treatment schedule can effectively check the onset of tumorigenesis, the cumulative number of tumours and the average number of tumours per mouse. In the PA administered group, 30% of the animals remained tumour free until the termination of the experiments (i.e. 32 weeks of promotion). Thus the present study proves that protein A can effectively inhibit DMBA initiated and TPA promoted mouse skin carcinogenesis. PMID- 8616808 TI - Inhibition of insulin like growth factor II autocrine growth of Wilms' tumor by suramin in vitro and in vivo. AB - Suramin was found to affect the Wilms' tumor (WT) cell line, W13, by inhibiting in vitro growth (half-maximal inhibitory dose (ID50)=11 microM), insulin like growth factor II (IGF-II) cell binding (ID50 = 10 microM) and IGF-II induced DNA synthesis (ID50 = 8 microM). In addition, suramin inhibited cross-linking of [125I]IGF-II to the type 1 IGF receptor (IGF1R) and type 2 IGF receptor (IGF2R). Disruption of IGF-II/IGF1R interaction appears to be the main mode of action of suramin since the suramin response was abolished in the presence of the IGF1R blocking antibody, alpha IR-3. When administered to athymic mice bearing W13 heterotransplants, suramin suppressed the linear tumor growth rate by 64%. PMID- 8616809 TI - p53 gene mutation in thyroid carcinoma. AB - The pattern of p53 protein expression was examined in 92 cases of thyroid carcinoma. When the cases were divided into two groups with regard to their cytoplasmic staining only or nucleus staining only, the frequency of the nucleus staining group was significantly higher in the poorly differentiated carcinoma (PDC) and undifferentiated carcinoma (UDC) groups (10.5% and 25%) compared with the other groups of histologic subtype (0%). The results suggest positivity in nucleus staining for p53 may be a marker for the biologically worse carcinomas, PDC and UDC, however, tumors showing only cytoplasmic staining of p53 favor a fair prognosis. In this paper, we also elucidate the spectrum of genotypic aberrations of p53 in each histological subtype. Of 92 thyroid tumor samples analyzed, the overall frequency of p53 mutation was 8.5%. The mutations occurred in 4.35% (2/46) ot WDC, 17.2% (5/29) of PDC, and 16.7% (1/6) of oncocytic carcinoma. Two of five PDC cases and one papillary carcinoma revealed point mutations in exon 8 as follows; GTG (val) to CTG (leu) at codon 272 in case 23T, CGA (arg) to CCA (pro) at codon 306 in case of 30T, and CGG (arg) to AGG (arg) at codon 282 in case 28T. All of the p53 mutations detected were represented by single nucleotide changes including two missense and one silent mutation. In contrast to the missense mutations found in PDC, it is interesting to note that the silent mutation was checked in 28T of well differentiated papillary carcinoma. These results represents molecular evidence that p53 gene aberration associated with overexpression of the mutant form of p53 protein plays a crucial role in the biologically aggressive subtypes of thyroid carcinoma, and point mutation only was not sufficient to be a prognostic marker for the biologically aggressive malignancy of thyroid tumors. There was no p53 gene aberration found in four cases of undifferentiated carcinoma (UDC) studied. The results suggest that other unknown factors should be responsible for the aggressiveness in some UDC of thyroid carcinoma except overexpression of p53. PMID- 8616810 TI - Arsenic and chromium enhance transformation of bovine papillomavirus DNA transfected C3H/10T1/2 cells. AB - Tumor promoters such as phorbol esters, teleocidin and okadaic acid increase the numbers of multilayered, transformed foci produced by BPV DNA-transfected C3H/10T1/2 cells. We questioned whether arsenic and chromium, which are known human carcinogens also enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Cr(III) potassium sulfate at 100 microM enhanced transformation by 1.4 fold, but Cr(VI) as potassium chromate did not enhance transformation, although toxicity of potassium chromate may have prevented enhancement of transformation. Sodium arsenite (As(III) at 5 microM and sodium arsenate (As(V)) at 25 microM both enhanced neoplastic transformation by 6-fold. By comparison, in previous studies, sodium orthovanadate (V(IV)) or vanadyl sulfate (V(IV)) at 4 microM enhanced numbers of transformed foci by 25-50-fold. The comparatively strong enhancement of transformation by vanadium and phorbol esters suggests that neoplastic transformation may occur by mechanisms that are common to these compounds including alteration of tyrosine phosphorylation. PMID- 8616811 TI - The lack of an effect by insulin or insulin-like growth factor-1 in attenuating colon-2-mediated cancer cachexia. AB - In several studies, the anabolic hormones insulin-like growth factor-1 (IGF-1) and insulin attenuated several metabolic changes associated with cancer cachexia. In the present study, we evaluated the effect of these hormones on the cachexia associated with colon-26 (C-26) tumor. Healthy age-matched and tumor-bearing mice were treated with two daily doses of IGF-1 (50 micrograms/kg in toto), or insulin (1 U in toto). Determinants of cachexia were body and tumor weight, epididymal fat pad and serum glucose concentrations. Neither IGF-1 nor insulin treatment had a significant effect on the cachectic parameters of C-26-bearing mice. These hormones were biologically active, being capable of inducing weight gain in hypophysectomized mice and hypoglycemia, respectively. Although IGF-1 and insulin have been used to treat cancer-related weight loss, the research presented here suggests that the beneficial effect of these hormones is not universal. PMID- 8616812 TI - A cohort of supporting metabolic enzymes is coinduced with nitric oxide synthase in human tumor cell lines. AB - Although nitric oxide (NO) has cytotoxic activity against certain tumor cell lines, some human tumor cell lines can themselves produce NO by expressing the inducible isoform of NO synthase (iNOS). As rates of cellular NO synthesis play a major role in determining whether NO has cytotoxic or cytoprotective effects at anatomic sites of NO production, identification of cellular processes which regulate rates of NO synthesis by iNOS is important for understanding the role of NO in tumor cell biology. This study demonstrates that argininosuccinate synthetase and GTP-cyclohydrolase-I, which catalyze rate-limiting steps in the synthesis of iNOS substrate (arginine) and cofactor (tetrahydrobiopterin), respectively, are coinduced with iNOS expression in two human tumor cell lines. These results indicate that coinduction of these supporting metabolic pathways helps to maximize cellular NO synthesis by iNOS in human tumor cells, suggesting that these pathways might be useful targets for pharmacologic intervention in NO producing human tumor cells. PMID- 8616813 TI - Inhibition of the proliferation of human neural neoplastic cell lines by cysteamine. AB - Cysteamine (CySH), a thiol compound that crosses the blood-brain barrier, inhibited the proliferation of neural neoplastic cells in vitro. The IC50 of cysteamine with respect to inhibition after 72 h of drug exposure, was approximately 70 microM in the glioma cell line, 2607, and approximately 80 microM in the neuroblastoma cell line, DAOY. Interestingly, the inhibition of proliferation of 2607 cells produced by 72 h treatment with CySH could also be induced with exposure periods as short as 8 h. Another thiol bearing compound, penicillamine methyl ester, also arrested the proliferation of 2607 cells with IC50 approximately 160 microM. Cell cycle analysis revealed that CySH acted to lengthen the cell cycle period of 2607 cells by slowing the passage of cells through S phase and caused the cells to finally arrest in G2/M. In the other cell lines tested, CySH arrested cells in all phases of the cell cycle. These observations suggest that CySH and its congeners may have some utility in the treatment of neoplasia in vivo. PMID- 8616814 TI - Messenger ribonucleic acids for fibroblast growth factors and their receptor in bladder and renal cell carcinoma cell lines. AB - FGF-like growth factors have been detected in the urine of patients with bladder or renal cell carcinoma. FGF-1-like and FGF-2-like proteins have been detected in the urine of patients with bladder carcinoma. However, the expression of FGFs and their receptor in bladder and renal cell carcinoma cells remains limited. We measured the mRNA levels of FGFs and their receptor in these carcinoma cell lines by means of RT-PCR. We detected FGF-8 mRNA expression in murine cell lines of bladder and renal cell carcinomas but not in those of the normal bladder and kidney. Furthermore, FGF-8 mRNA expression was detected in all human bladder and renal cell carcinoma cell lines tested. We also frequently detected FGF-1, FGF-2 and FGF-5 mRNA expression in human bladder and renal cell carcinoma cell lines. These results indicate that FGF-8 is also candidate for marker of these types of carcinoma as well as FGF-1 and FGF-2. PMID- 8616815 TI - Cellular Ras partly mediates gap junction closure by the polyoma virus middle tumor antigen. AB - Endogenous, cellular Ras proteins (c-Ras) mediate the transforming action of the polyoma virus middle Tumor antigen (mT), which is accompanied by elimination of gap junctional, intercellular communication (GJIC). In this report we show that reducing the c-Ras content of murine C3H10T1/2 fibroblasts (10T1/2) through the expression of an anti-sense ras gene, increased GJIC by 60-80% mT totally eliminated GJIC in normal 10T1/2 cells but it reduced GJIC no more than 50% in the c-Ras deficient lines. These results indicate that endogenous c-Ras is at least partly responsible for the mT-induced gap junction closure. PMID- 8616816 TI - Tissue sialic acid and fibronectin levels in human prostatic cancer. AB - We investigated the tissue concentration of sialic acid and fibronectin in patients with prostatic cancer. The mean sialic acid and fibronectin levels in patients with prostatic cancer were 19.02 +/- 6.30 micrograms/mg protein, respectively versus 13.01 +/- 4.53 micrograms/mg protein and 11.77 +/- 6.74 micrograms/mg protein for normal prostatic tissues. Sialic acid and fibronectin levels in cancerous patients were significantly higher than in the control group (P < 0.05). PMID- 8616817 TI - The reliability of the differential polymerase chain reaction compared to restriction fragment length polymorphism for the detection of gene loss in primary tumors. AB - The loss of genetic information from a number of specific regions of the genome has been documented in human tumors in vivo. To estimate the frequency with which IFN beta 1 gene loss occurs in human gliomas, both restriction fragment length polymorphism (RFLP) analysis and polymerase chain reaction (PCR) amplification, using the IFN-gamma gene as a reference, were used and the results obtained with the two methods were compared. The relative intensity of PCR bands (IFN beta 1/IFN gamma) in the gliomas with loss of heterozygosity for the IFN beta 1 was significantly different (P < 0.05) with regard to the gliomas with the IFN beta 1 gene. Consequently, these findings indicate that differential PCR is a reliable and non-radioactive method of demonstrating gene loss in tumors. PMID- 8616818 TI - Simultaneous expression of cadherin-11 in signet-ring cell carcinoma and stromal cells of diffuse-type gastric cancer. AB - We examined the expression of cadherin-11, a type II cadherin, in normal human tissues, cell lines and gastric cancer surgical specimens. Cadherin-11 was expressed widely in adult tissues, except the liver. It was expressed in fibroblast, mesothelial cell lines, and in only two signet ring cell carcinomas out of 16 various cancer cell lines. Cadherin-11 expression was detected in both signet ring cell carcinoma cells and surrounding fibroblasts of surgical specimens by in situ hybridization. These results suggest that cadherin-11 may play a role in the formation of diffuse-type gastric cancer through cancer stromal interactions. PMID- 8616819 TI - Regulation of human Caco-2 intestinal epithelial brush border enzyme activity by cyclic nucleotides. AB - Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways. PMID- 8616820 TI - Failure of cigarette smoke to induce or promote lung cancer in the A/J mouse. AB - A six-month bioassay in A/J mice was conducted to test the hypothesis that chronically inhaled mainstream cigarette smoke would either induce lung cancer or promote lung carcinogenicity induced by the tobacco-specific nitrosamine, 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Groups of 20 female A/J mice were exposed to filtered air (FA) or cigarette smoke (CS), injected with NNK, or exposed to both CS and NNK. At 7 weeks of age, mice were injected once with NNK; 3 days later, they were exposed to CS for 6 h/day, 5 days/week, for 26 weeks at a mean 248 mg total particulate matter/m3 concentration. Animals were sacrificed 5 weeks after exposures ended for gross and histological evaluation of lung lesions. No significant differences in survival between exposure groups was observed. A biologically significant level of CS exposure was achieved as indicated by CS-induced body weight reductions, lung weight increases, and carboxyhemoglobin levels in blood of about 17%. Crude tumor incidences, as determined from gross observation of lung nodules, were similar between the CS exposed and FA groups, and the NNK and CS + NNK groups. Incidences in either of these latter groups were greater than either the CS or FA groups. Furthermore, tumor multiplicity in tumor-bearing animals was not significantly different among any of the three groups (FA, NNK, CS + NNK) in which tumors were observed. Thus, CS exposure neither induced lung tumors nor promoted NNK-induced tumors. Because the CS exposure concentration was probably near the maximally tolerable level, longer exposures should be evaluated to potentially establish a CS-induced model of lung carcinogenesis in the A/J mouse. PMID- 8616821 TI - Microsatellite instability in gastric cancer prone families. AB - We examined for germ-line p53 mutations and microsatellite instability in three gastric cancer patients who had family histories of gastric cancer aggregation. Although no germ-line p53 mutation was detected in these three cases, the replication error (RER) phenotype was observed in two of them. One base deletion in the sequence of ten repeating adenines of the type II transforming growth factor-beta receptor gene was detected in one of these two cases. Furthermore, there were young patients of 50 years and downward in their families. Therefore, it is possible that inherited disorders in mismatch repair systems contribute to high susceptibility to gastric cancers in these families. PMID- 8616822 TI - Responsiveness of hepatocytes from dichloroacetic acid or phenobarbital treated mice to growth factors in primary culture. AB - Hepatocytes isolated from male B6C3F1 mice and maintained in primary culture were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF), acidic fibroblast growth factor (aFGF) alone or in combination with the mitoinhibitory transforming growth factor beta 1 (TGF-beta 1). Groups of mice were exposed to 3.5 g/l dichloroacetic acid (DCA), 0.1% phenobarbital (PB) or the drinking water vehicle for 0, 2, 5, 10, 20, 30, 60, or 90 days. Following a 2 h attachment period, the growth factors with or without TGF-beta 1 were added together with [3H]thymidine. The cells were harvested 48 h later and the incorporation of the labeled thymidine into cellular DNA was determined. Basal DNA synthesis was enhanced following 2 days of PB treatment after which it declined to levels significantly below that in the untreated mice. No early time enhancement of DNA synthesis was measured in the hepatocyte cultures for animals exposed to DCA, but the late time inhibition was also seen. Primary cultures of hepatocytes isolated from control and DCA treated mice exhibited similarly enhanced DNA synthesis in response to eGF, HGF, or aFGF alone or in combination with TGF-beta 1. In contrast, hepatocytes from PB treated animals were refractory to the effects of the growth factors at all time periods. These data suggest that the early depression of cell proliferation we have seen during DCA induced hepatocellular cancer is not due to an impaired ability of hepatocytes to respond to growth factors and that the mechanisms of liver tumorigenesis in the mouse induced by PB and DCA are dissimilar. PMID- 8616824 TI - Human larynx expresses isoforms of the oestrogen receptor. AB - Commercially available enzyme immunoassays (EIAs) were used for oestrogen (ER) and progesterone (PR) receptor determination in the cytosol fraction of 118 human larynx cancer specimens and in the corresponding histologically proven non malignant tissues. Fifty-one ER positive cancerous samples had corresponding non cancerous tissues also expressing the receptor. A high resolution isoelectric focusing (IEF) technique followed by immunoblotting with the H222 anti-ER monoclonal antibody was used to evaluate the presence of ER isoforms in the 51 ER positive human larynx cancer specimens and in their corresponding non-malignant tissues. In both tissues, four ER isoforms were detected, with isoelectric points (pI) similar to those obtained in breast and endometrium carcinomas (6.1, 6.3, 6.6 and 6.8). A significant difference in the expression of ER isoforms between cancerous and non-cancerous tissue was found; precisely, the 94.1% of the ER positive non-malignant specimens co-expressed the four isoforms while they were detected in only the 35.5% of the malignant specimens (P < 0.0001 by Fisher's exact test). In larynx cancer, the concentration values of ER and PR did not correlate, nevertheless tumours co-expressing the four ER isoforms had PR levels significantly higher than those which did not (P = 0.02 by Mann-Whitney Wilcoxon sum rank test). To investigate the possibility that the isoforms of the monomeric 4S form of the ER (those with pI 6.3, 6.6, and 6.8) could dimerise, a cold agarose gel electrophoresis technique was used on IEF-separated ER isoforms. In summary, the evidence shows that all the isoforms are able to form homodimers and that the isoforms at pI 6.3 and 6.8 are able to dimerise with that at pI 6.6 but, under the same experimental conditions, they do not form the 6.3/6.8 heterodimer. It was concluded that: (1) the four isoforms of the ER are co-expressed by the non-malignant human larynx and the cancer loses the capacity to express some of them; (2) the complete complement of ER isoforms (all four) is needed for PR expression; (3) the monomeric 4S isoform with pI 6.6 has the capacity to form homo- and heterodimers, while the remaining two are only able to homodimerise. PMID- 8616823 TI - Overexpression of her-2/neu in human prostate cancer and benign hyperplasia. AB - Overexpression of the neu oncoprotein has been described in several tumor models including breast and prostate cancer. Overexpression of neu has been reported to have prognostic significance in certain tumors but controversy continues regarding the role and frequency of neu overexpression in prostatic cancer. The objectives of the study were twofold. First, to characterize neu expression in prostate cancer in comparison to benign prostatic hyperplasia. Second, to determine whether neu expression correlates with Gleason grade in prostate cancer. Thirty-nine prostate cancers obtained from radical prostatectomy specimens and 10 benign prostatic hyperplasia specimens were included in the study. Specimens were formalin fixed and paraffin-embodied. neu expression was studied by immunohistochemical staining using a monoclonal neu specific AB-3 antibody. All 39 specimens (100%) of prostate cancer showed positive immunostaining of variable degree while 2 (20%) benign prostatic hyperplasia specimens showed positive staining. Thus, neu oncogene is overexpressed in localized prostate cancer compared to benign prostatic hyperplasia. The degree of neu immunostaining did not correlate with Gleason grade and there appeared to be a tendency towards an inverse relationship. The prognostic significance of the varying overexpression is unknown. PMID- 8616825 TI - Rhodamine 123-efflux from hematopoietic subpopulations and leukaemic blast populations marked by PerCP-conjugated monoclonal antibodies. AB - A representative functional assay for determination of drug transporting proteins (e.g. P-glycoprotein) in leukaemic blasts could help to evaluate effects of chemotherapy combined with chemosensitizers. Since subpopulations of normal peripheral blood or bone marrow cells show distinct P-glycoprotein levels, the presence of these cells in leukaemic samples causes a major problem in determination of rhodamine 123 efflux in these types of malignant cells. Additional staining of blasts with specific monoclonal antibodies (marked with FITC (fluorescein) or PE (phycoerythrin) might ensure a selective analysis of a particular subpopulation by flow cytometry, but the emission spectrum of rhodamine 123 interferes with FITC and PE signals and vice versa. This can be avoided by using monoclonal antibodies (mab) conjugated with the newly developed dye PerCP (peridnine chlorophyll protein; Becton/Dickinson), devoid of interfering with the rhodamine 123 fluorescence emission spectrum. Therefore we established an assay for the determination of rhodamine 123 efflux from peripheral blood CD4+, CD8+ or CD56+ subpopulations by detection with PerCP conjugated mab, followed by electronic gating. The problems of varying signal intensities or the need to recompensate during measurement which normally occurred using FITC- or PE-conjugated mab did not emerge by the use of PerCP marked mab. Moreover we could correlate MDR1 gene expression and modulation of rhodamine 123 efflux from the leukaemic blasts by proven P-gp MDR chemosensitizing agents such as SDZ PSC 833, dexverapamil and dexniguldipine. This method gives highly reproducible results of P-gp function in patient samples which should be compared with patient outcome after combined chemotherapy including chemosensitizers. PMID- 8616826 TI - Invasive capacity and regulation of urokinase-type plasminogen activator in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, and a transfectant (S30) stably expressing ER. AB - We assessed the invasive capacities and expression of urokinase-type plasminogen activator (uPA) in the estrogen receptor (ER) negative MDA-MB-231 cell line, and the same cell transfected with an ER expression vector (S30 cells) in response to 17 beta-estradiol (E2; 1 nM) and epidermal growth factor (EGF; 1 ng/ml and 10 ng/ml). The invasive potential of S30 cells was only 50% that of MDA-MB-231 cells and was further reduced by E2. EGF increased the invasiveness of S30 cells, but was unable to reverse the inhibitory effects of E2. Invasion of MDA-MB-231 cells was unaffected by E2 or EGF. EGF increased uPA secretion from both cell lines, as determined by ELISA and zymography, and this correlated with increased expression of uPA mRNA. uPA expression in MDA-MB-231 cells was unaffected by E2; however, S30 cells responded to E2 with downregulation of uPA at both the protein and mRNA levels. PMID- 8616828 TI - Relationship between latitude and melanoma incidence: international evidence. AB - We investigate the relationship between latitude and incidence of melanoma among Whites in different age groups using data from different continents. Relevant data for Whites were obtained for 59 regions around the world. A statistical analysis was carried out using regional dummy variables to eliminate spurious statistical correlation due to clustering. Simple correlation between latitude and incidence of melanoma is strongly negative for almost all age groups. However, once the regional dummies were introduced in the analysis, the relation between latitude and incidence rates disappeared for all age groups but the explanatory power of the regression equation increased substantially. PMID- 8616827 TI - Cytotoxic effects of sinococuline. AB - Three alkaloids, aknadinine, 1-nitroaknadinine and sinococuline, isolated from Stephania sutchuenensis were studied for their effects on a fibroblast cell line, eight tumor cell lines and a rat alveolar macrophage culture. Sinococuline is an effective tumor cell growth inhibitor whereas the toxicity of aknadinine and 1 nitroaknadinine towards all tested cells is low. A dose-dependent decrease in cell viability and in the uptake of tritiated-thymidine, -leucine and -uridine by these cells was observed when they were grown in the presence of sinococuline for 24 h. Exposure to sinococuline in vitro also altered the macrophage function by reducing the production of tumor necrosis factor and reactive nitrogen intermediates. Human leukaemic HL60 cells and mouse fibroblast L929 cells were used to study the underlying mechanism of cytotoxicity and apoptosis seem to be the mode of death induced by sinococuline PMID- 8616829 TI - Reduced levels of cathepsin D associated with tamoxifen resistance and estrogen independence in the ZR-75-1 human breast cancer cell line. AB - The expression and secretion of cathepsin D by ZR-75-1 human breast cancer cells, and tamoxifen-resistant (ZR-75-9a1) and estrogen-independent (ZR-PR-LT) variants was examined by electrophoresis of labeled proteins and Western blotting. Secreted proteins of 160 kDa, 52 kDa and 34 kDa were identified, and in ZR-75-1 cells, they were shown to be estrogen-inducible. Treatment of ZR-75-9a1 cells with 17 beta-estradiol (E2) and the progestin ORG 2058 increased secretion of the 52 kDa protein; ZR-PR-LT cells were unaffected. Western blotting showed that each cell line expressed high levels of the 52 kDa and 34 kDa forms of cathepsin D but that relatively little was being secreted. Each cell line secreted 52 kDa procathepsin D, but 34 kDa mature-cathepsin D was not detected as a secreted protein. ZR-75-1 cells expressed and secreted the highest levels of cathepsin D while ZR-75-9a1 cells expressed and secreted the least. PMID- 8616830 TI - Changes in insulin-like growth factor-I receptor expression and binding protein secretion associated with tamoxifen resistance and estrogen independence in human breast cancer cells in vitro. AB - Conditioned medium from a series of human breast cancer cell lines representing the phenotypes of estrogen dependence ZR-75-1, MCF-7), tamoxifen resistance (Z(+) 75-9a1, LY2) and estrogen independence (ZR-PR-Lt) contained four detectable insulin-like growth factor binding proteins (IGFBPs) of MW 24, 34, 44 and 70 kDa. in the tamoxifen resistant lines secretion of the 24 kDA IGFBP was depressed in comparison to the parent lines whilst secretion of the 44 kDa IGFBP was unaffected. Secretion of this species by ZR-PR-LT cells was reduced. Following incubation in medium containing 1% serum both tamoxifen resistant cell lines secreted higher levels of the 44 kDa IGFBP than the respective parent lines. These changes in IGFBP secretion associated with the development of tamoxifen resistance and estrogen independence were accompanied by changes in the expression of receptors for IGF-1. PMID- 8616831 TI - Barrett's esophagus: metaplastic cells with loss of heterozygosity at the APC gene locus are clonal precursors to invasive adenocarcinoma. AB - Adenocarcinoma in Barrett's esophagus is the second most rapidly increasing cancer in western society. The cause and pathogenesis are unknown. Although histological studies suggest that there is successive progression from metaplasia and dysplasia, with a high risk of subsequent invasive carcinoma, at present there is no direct evidence that metaplastic and dysplastic epithelia are clonal precursors of adenocarcinoma. We selected 12 esophagectomy specimens of Barrett's esophagus patients, which showed a spectrum of normal tissue, metaplasia, dysplasia, and invasive carcinoma in each individual biopsy. We applied the microdissection technique to selectively procure microscopic tissue samples from H&E-stained slides for genetic evaluation using polymorphic markers flanking the APC gene locus. Identical APC gene alterations were found in the dysplastic and adenocarcinoma foci of all informative cases. The same changes were observed even in some metaplastic foci adjacent to dysplasia. Furthermore, clonality analysis of X-chromosome inactivation in female cases verified the same X-chromosome inactivation pattern in carcinoma, dysplasia, and metaplasia adjacent to dysplasia. No APC gene alterations were found in the normal epithelium and metaplasia distant from dysplasia. These data show for the first time that a tissue field of genotypic changes precedes the histopathological phenotypic changes of carcinoma in Barrett's esophagus syndrome. Our findings, in conjunction with the applied tissue microdissection technique, may help identify genotypic changes in patients with Barrett's esophagus before phenotypic changes occur. Therefore, genotyping of Barrett's metaplastic epithelium may supplement the histopathological evaluation of Barrett's esophagus. PMID- 8616832 TI - bcl-xs gene therapy induces apoptosis of human mammary tumors in nude mice. AB - Bcl-xs is a dominant negative repressor of Bcl-2 and Bcl-xL, both of which inhibit apoptosis. We used a replication-deficient adenoviral vector to transiently overexpress Bcl-xs in MCF-7 human breast cancer cells, which overexpress Bcl-xL. Infection with this vector induced apoptosis in vitro. We then determined the effects of intratumoral injection of bcl-xs adenovirus on solid MCF-7 tumors in nude mice. Tumors injected four times with the bcl-xs adenovirus showed a 50% reduction in size. Using terminal transferase-mediated dUTP-digoxigenin nick end labeling, we observed apoptotic cells at sites of bcl xs adenoviral injection. These experiments demonstrate the feasibility of using bcl-xs gene therapy to induce apoptosis in human breast tumors. PMID- 8616833 TI - Lack of tumorigenicity of cholesterol epoxides and estrone-3,4-quinone in the rat mammary gland. AB - The purpose of this study is to test the long-standing hypothesis that endogenous agents found in human breast fluid and in plasma are potential initiators of breast cancer. Therefore, we evaluated the tumorigenicity in the mammary glands of female CD rats of cholestan-5 alpha,6 alpha-epoxy-3 beta-ol (cholesterol-alpha epoxide), cholestan-5 beta,6 beta-epoxy-3 beta-ol (cholesterol-beta-epoxide), and 1,5(10)estradiene-3,14,17-trione (estrone-3,4-quinone). As a positive control, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthren e (BcPDE) was used. Rats were fed a high-fat AIN-76A diet (23.5% corn oil) to mimic the Western dietary composition. Because literature data suggest that the endogenous agents tested in this study are weak electrophiles, the total doses of cholesterol epoxides (12.3 mumol/rat) and of estrone-3,4-quinone (30 mumol/rat)were 10- and 25- fold higher, respectively, than that of BcPDE (1.2 mumol/rat). Each agent was dissolved in DMSO, and one-sixth of the total dose was injected under each of six nipples on the right side. The thoracic glands of the rat were treated at 30 days of age, and those located in the inguinal area were treated on the following day. The experiment was terminated at 44 weeks after treatment. Consistent with our previous study, BcPDE was a strong mammary carcinogen. However, there were no differences between rats treated with DMSO alone or those receiving DMSO containing cholesterol-alpha-epoxide, cholesterol beta-epoxide, or estrone-3,4-quinone. The results of this study clearly indicate, for the first time, that metabolites derived from cholesterol and estrone lack tumorigenic activity in the rat mammary gland, at least under the conditions of the present protocol. PMID- 8616834 TI - Polymorphism at the glutathione S-transferase locus GSTM3: interactions with cytochrome P450 and glutathione S-transferase genotypes as risk factors for multiple cutaneous basal cell carcinoma. AB - The influence of polymorphism in the glutathione S-transferase, GSTM3 gene on susceptibility to cutaneous basal cell carcinoma (BCC) has been investigated. We have reported previously two GSTM3 alleles, GSTM3*A and GSTM3*B, distinguished by a recognition motif for the YY1 transcription factor in GSTM3*B. In this study, immunohistochemistry was used to identify GSTM3 expression in the epidermis of skin samples from 11 controls and 9 patients with BCC. A PCR method was used to identify GSTM3*A and GSTM3*B and thereby the GSTM3 AA, GSTM3 AB, and GSTM3 BB genotypes in 300 controls and 286 Caucasians with 1-35 primary BCCs. Genotypes at GSTM1, GSTT1, and the cytochrome P450 CYP1A1 and CYP2D6 loci were also determined. Frequencies of GSTM3, GSTM1, GSTT1, CYP2D6, and CYP1A1 genotypes in the cases and controls were not different. Dividing the BCC cases into groups of 92 patients with 1 lesion and 194 patients with 2-35 lesions showed that the frequencies of GSTM3 BB (2.6%) and GSTM1 A/B (1.3%) in the group with 2-35 tumors were almost significantly lower than in the group with 1 lesion (7.6%, exact P = 0.0601, chi 2(1) = 3.390; 6.5%, exact P = 0.055, chi 2(1) = 4.946, respectively). Within the cases with 2-35 tumors, a Poisson regression model was used to identify genotypes, characteristics such as skin type, and interactions between genotypes and characteristics associated with increasing numbers of tumors. This showed, after correction for male gender and age, that GSTM3 AA was not associated with risk of increased numbers of tumors, although in combination with skin type 1, GSTM1 null, and CYP1A1 m1m1, the genotype did confer increased risk (P < 0.001, rate ratio, 2.058; P < 0.001, rate ratio, 1.606; P < 0.001, rate ratio, 1.470 respectively). The data suggest that, like other allelic GST, GSTM3 influences cancer risk. As GSTM3 AA was associated with increased tumor numbers, it appears that YY1 acts as an activator of the recognition motif in GSTM3*B. PMID- 8616835 TI - 5-Fluorouracil suppresses nitric oxide biosynthesis in colon carcinoma cells. AB - Nitric oxide is an important cellular mediator that plays a role in regulating cellular proliferation of both normal and tumor cells. In the present study, we characterized nitric oxide production by the human colon adenocarcinoma cell line DLD-1 and examined the effects of 5-fluorouracil (5-FUra), an antimetabolite effective against colon tumors, on nitric oxide production. IFN-gamma was found to be a potent inducer of nitric oxide production in DLD-1 cells. This effect was dependent on L-arginine and blocked by the nitric oxide synthase inhibitors NG monomethyl-L-arginine, nitroarginine, and aminoguanidine. Production of nitric oxide by DLD-1 cells was due to the expression of the inducible (type II) form of nitric oxide synthase. mRNA for the nitric oxide synthase was present in both untreated and IFN-gamma-stimulated cells, as determined by RT-PCR, suggesting that expression of enzyme is regulated posttranscriptionally. Treatment of DLD-1 cells with concentrations of 5-FUra that are not growth inhibitory or cytotoxic strongly inhibited their ability to express nitric oxide synthase and produce nitric oxide in response to IFN-gamma. This effect was not reversed with thymidine, indicating that inhibition of nitric oxide production was due to incorporation of 5-FUra into RNA. However, pretreatment of DLD-1 cells with 5 FUra before stimulation with IFN-gamma also suppressed nitric oxide production. Thus, inhibition of nitric oxide production was not due directly to incorporation of 5-FUra into the mRNA for nitric oxide synthase. Taken together, these data suggest that inhibition of nitric oxide biosynthesis in colon tumor cells by 5 FUra may underlie, at least in part, the efficacy of this antitumor agent. PMID- 8616837 TI - Allelic imbalance on chromosome 13q: evidence for the involvement of BRCA2 and RB1 in sporadic breast cancer. AB - Recently, the breast cancer susceptibility gene BRCA2 has been identified in chromosome 13q, a region that also contains the retinoblastoma gene RB1. To elucidate a possible role of BRCA2 and RB1 in sporadic breast tumorigenesis, allelic imbalance (AI) at 13q loci was examined in 78 primary sporadic breast tumors. AI was found in 52-63% of tumors. Nine tumors showed AI only in the BRCA2 region but not at RB1. Six tumors showed AI at RB1 but not in the BRCA2 region. AI in the BRCA2 region correlated significantly with aneuploidy (P = 0.032) and AI at RB1 with small tumor size (P = 0.025). Our data suggest that BRCA2 and RB1 may be both distinct target loci for AI on chromosome 13 in sporadic breast cancer. PMID- 8616836 TI - Uptake of temozolomide in a rat glioma model in the presence and absence of the angiogenesis inhibitor TNP-470. AB - The angiogenic phenotype is associated with hyperpermeable capillaries. Through treatment with angiogenesis inhibitors capillary permeability may be reduced, and it can be anticipated that cytotoxic agents coadministered may be adversely affected. The current investigation examined this possibility for the combination of TNP-470, an angiogenesis inhibitor, and temozolomide (TMZ), a DNA-alkylating agent with demonstrated activity in brain tumors. TNP-470 (30 mg/kg) was given s.c. on days 6, 8, 10, 12, and 14 following s.c. implantation of rat C6 glioma cells in Sprague-Dawley rats. On the 15th day following tumor implantation, control (no TNP-470) and treated rats received 40 mg/kg of TMZ intraarterially. Prior to dosing, a linear microdialysis probe was placed in the tumor to collect interstitial fluid. Plasma and interstitial fluid samples were collected for 8 h and measured for TMZ by a high-performance liquid chromatography assay. Pharmacokinetic parameters for TMZ were calculated by noncompartmental methods. Total systemic clearance (39.8 +/- 7 versus 44.2 +/- 14 ml min(-1) kg(-1)) and volume of distribution (5.4 +/- 2 versus 5.2 +/- 0.8 L kg(-1)) were not significantly different in control and TNP-470-treated animals. However, the mean TMZ area under the interstitial fluid concentration-time curve was reduced by 25% in the TNP-470-treated group compared to the control (5450 +/- 1892 versus 4120 +/- 1790 micrograms min ml(-1); P < 0.05). It appears that TNP-470 caused this reduction in the tumor uptake of TMZ by its pharmacodynamic action on the tumor vasculature. Since combination regimens using angiogenesis inhibitors and cytotoxic drugs will be needed to determine how such combinations can be used effectively. The current animal model, which utilized tumor microdialysis, can serve as a model to further analyze combination chemotherapy. PMID- 8616838 TI - Apoptotic response to oncogenic stimuli: cooperative and antagonistic interactions between c-myb and the growth suppressor p53. AB - c-myb, a protooncogene prevalently expressed in the hematopoietic tissue, is a transcription factor that contains a DNA-binding domain and an acidic domain and is able to transactivate specific viral and cellular genes. In this report, we show that c-myb can stimulate apoptosis in both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines when coexpressed with p53. Apoptosis is accompanied by increased transactivation of the cell death-associated BAX gene. This effect is c-myb specific, because B-myb is not able to cooperate with p53 in the induction of BAX transcription and apoptosis. Immunoprecipitation studies and gel shift analysis indicate that c-myb does not directly interact with the BAX promoter or the p53 protein but, rather, cooperates through an indirect mechanism. Consistent with the existence of a functional link between c-myb and p53, we also observed that c-myb represses p53-induced activation of the WAF-1 promoter and induces proliferation of SAOS2 cells growth arrested by p53. These results might contribute to the elucidation of the mechanisms underlying p53 dependent pathways of oncogene-induced apoptosis and provide a further example of DNA-binding independent myb activity. PMID- 8616839 TI - Identification of two candidate tumor suppressor genes on chromosome 17p13.3. AB - A second tumor suppressor locus on 17p that is distinct from TP53 has been identified in brain, breast, lung, and ovarian tumors. Using allelic loss mapping and positional cloning methods, we have recently identified two novel genes, which we refer to as OVCA1 and OVCA2, that map to 17p13.3. The two genes are ubiquitously expressed and encode proteins of 443 and 227 amino acids, respectively, with no known functional motifs. Sequence comparison of OVCA1 and OVCA2 revealed extensive sequence identity and similarity to hypothetical proteins from Saccharomyces cerevisiae, Caenorhabditis elegans, and Rattus species. Northern blot analysis reveals that OVCA1 and OVCA2 mRNA were expressed in normal surface epithelial cells of the ovary, but the level of this transcript is significantly reduced or is undetectable in 92% (11/12) of the ovarian tumors and tumor cell lines analyzed. The location, high degree of amino acid conservation, and reduced expression in ovarian tumors and tumor cell lines suggest that decreased expression of these two genes contributes to ovarian tumorigenesis and should be considered candidate tumor suppressor genes. PMID- 8616840 TI - Functional analysis of pRb2/p130 interaction with cyclins. AB - The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the E1A viral oncoprotein were able to rescue the pRb2/p130-mediated G1 growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/p130, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/p130 dramatically increased near the G1-S-phase transition. This suggests that, like the other Rb family members, pRb and p107, the phosphorylation of pRb2/p130 is controlled by the cell cycle machinery and that pRb2/p130 may indeed be another key G1-S-phase regulator. PMID- 8616841 TI - In vivo oxygen consumption rate of DS sarcoma cells on inhibition of DNA synthesis. AB - The effect of inhibiting DNA synthesis on the cellular O2 consumption rate of tumor cells (DS sarcoma) in vivo was analyzed using a photometric technique. Five days after DS-sarcoma ascites was induced in SD rats, animals were treated either with fludarabine (400 mg/kg i.p., 6 h prior to measurements) or lovastatin (3 x 20 mg/kg i.p., 24, 15, and 3 h prior to measurements), drugs that can inhibit tumor cell proliferation. In addition to cellular O2 consumption, the cell cycle distribution and the fraction of DNA-synthesizing cells in the tumor ascites were measured. Both drugs lowered DNA synthesis significantly, an effect that was more pronounced with fludarabine. The cellular O2 consumption rate following lovastatin application was significantly impaired (approximately 33%), whereas fludarabine had practically no effect on the respiration rate of tumor cells. From these data, it is concluded that a reduction in DNA synthesis does not necessarily result in a decrease in the O2 consumption rate of tumor cells in vivo. PMID- 8616842 TI - Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. AB - Angiogenesis is essential for the growth and metastasis of solid tumors. In this study, we examined gene expression of vascular endothelial growth factor (VEGF); its receptor, flt-1; basic fibroblast growth factor; and transforming growth factors (TGFs) alpha and beta in 18 paired cases of human breast carcinomas and the adjacent nonneoplastic tissues. In all of the paired cases, VEGF expression was markedly increased in the carcinomas. In contrast, an insignificant difference was observed in the expression of flt-1, basic fibroblast growth factor, TGF-alpha, and TGF-beta between the malignant breast tissue and the nonneoplastic counterpart. Immunostaining showed variable VEGF positivity of the malignant cells, whereas the nonneoplastic breast epithelial cells were negative. The findings of this study suggest that VEGF is an important angiogenic factor in human breast cancer. PMID- 8616843 TI - Hydroxylforms of p-boronophenylalanine as potential boron carriers on boron neutron capture therapy for malignant brain tumors. AB - Hydroxylforms of boronophenylalanine (BPA) were synthesized by conjugation with a cascade of polyols to decrease the BPA uptake of normal parenchyma without affecting uptake into the tumor. We determined their tumor cell killing effect on boron neutron capture therapy (BNCT) against BPA using the human glioma cell line T98G. The thermal neutron doses yielding the D37 (dose used to inhibit 63% colony formation) values of dl-p-BPA(OH)n were 1.45 x 10(12)nvt (n = 1), 1.33 x 10(12)nvt (n = 2), 3.37 x 10(12)nvt (n = 4), and 1.72 x 10(12)nvt (n = 0). The relative tumor cell killing effect on BNCT of dl-p-BPA(OH)n against dl-p-BPA, which was defined as the ratio of D37-BPA to D37-BPA(OH)n, was 1.18 (n = 1) 1.29 (n = 2), and 0.51 (n = 4). The tumor:normal brain ratio of dl-p-BPA(OH)n in 9L rat brain tumor models was improved 1.2- (n = 1) and 1.4-fold (n = 2) against that of dl-p-BPA without a decrease of its uptake into the tumor. The water solubility of BPA(OH)n increased against BPA, and the toxicity represented as the IC50 value of dl-p-BPA(OH)2 was nearly one half that of dl-p-BPA, being established in our previous works. Hydroxylforms of BPA, especially dl-p BPA(OH)2, might be more suitable boron carriers of BNCT to malignant brain tumors since the radiation injury to the normal parenchyma surrounding the tumor is reduced. PMID- 8616844 TI - Stable transfection of the P-glycoprotein promoter reproduces the endogenous overexpression phenotype: the role of MED-1. AB - Cellular resistance to multiple chemotherapeutic agents is most often due to the overexpression of P-glycoprotein (Pgp). The mechanisms(s) underlying Pgp overexpression had not been determined, due, in part, to a failure to reproduce the overexpression in transient transfection assays. We now report that stable transfection of a Pgp (pgp1) promoter/luciferase construct in the drug-sensitive Chinese hamster cell line DC-3F and its drug-resistant sublines reproduced the overexpression phenotype, with up to 18-fold higher activity observed in the resistant cell lines compared with DC-3F. Moreover, mutation of a pgp1 promoter element, multiple start site element downstream (MED-1), decreased transcription in drug-resistant cells without affecting activity in drug-sensitive cells. This is the first report of a Pgp promoter element differentially regulated in drug resistant cells. Moreover, these data suggest that the regulation of Pgp transcription is modulated by chromatin structure, and that stable transfection may be more suitable for identifying promoter elements important for overexpression in drug-resistant cells. PMID- 8616845 TI - Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. AB - A unique type of rapidly progressive renal fibrosis, designated Chinese herbs nephropathy (CHN), has been described in young Belgian women who had followed a slimming regimen including recently introduced Chinese herbs (Stephania tetrandra and Magnolia officinalis). Aristolochic acid (AA), a known nephrotoxin and carcinogen, was suspected as its causal factor. To substantiate this hypothesis, renal tissue from five patients with CHN and six patients with other renal diseases was analyzed for the presence of AA-derived DNA adducts, a described biomarker of AA exposure associated with its carcinogenic and mutagenic activity. Using the 32P-postlabeling method, a major distinct DNA adduct spot was found in all five cases of CHN and identified by cochromatographic analyses with authentic markers as the deoxyadenosine adduct of AA-I [7-(deoxyadenosin-N6-yl) aristolactam I], the major component of the plant extract AA. This DNA adduct was absent in the six control cases. The 7-(deoxyadenosin-N6-yl)-aristolactam I adduct levels in CHN ranged from 0.7 to 5.3/10(7) nucleotides. Our data demonstrate that AA is implicated in CHN. They suggest a mechanism for the urothelial atypia and cancers observed in this disease and raise the possibility that a DNA mutation is responsible for the kidney-destructive fibrotic process. PMID- 8616846 TI - Wild-type p53 suppresses transcription of the human O6-methylguanine-DNA methyltransferase gene. AB - High-level expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) correlates with cellular resistance to the chloroethylnitrosourea (CENU) class of alkylating agents. Consequently, tumors expressing low levels of MGMT are sensitive to CENU chemotherapy, and any mechanism that can be used to reduce MGMT levels could sensitize resistant tumors. We have demonstrated that transient transfection of wild-type, but not mutant, p53 protein into a p53-null cell line, Saos-2, suppresses MGMT promoter activity in a reporter gene system. In addition, following a 24-h transduction of IMR90 fibroblasts with a wild-type p53-adenoviral vector, endogenous MGMT protein is down-regulated and is no longer detectable 5 days following infection. Because p53 is inducible by ionizing radiation, we propose that existing cancer therapy regimens that combine radiotherapy with CENU chemotherapy may be improved by altering scheduling and allowing enough time between the two therapies for the relatively stable MGMT protein to degrade. PMID- 8616847 TI - Apoptosis-associated derangement of mitochondrial function in cells lacking mitochondrial DNA. AB - U937 cells lacking mitochondrial DNA (rho [symbol: see text] cells) are auxotrophic for uridine and pyruvate, hypersensitive to hypoglycemic conditions, and resistant to antimycin A-induced apoptosis. In spite of their obvious metabolic defects, rho [symbol: see text] cells possess a normal mitochondrial transmembrane potential, as well as near-normal capacity to generate superoxide anion after menadione treatment. Similarly to rho + controls, rho [symbol: see text] cells undergo apoptosis in response to tumor necrosis factor-alpha plus cycloheximide. Detailed comparison of the apoptotic process in rho + and rho [symbol: see text] cells reveals essentially the same sequence of events. In response to tumor necrosis factor/cycloheximide, cells first lose their mitochondrial transmembrane potential (delta psi m) and then manifest late apoptotic alterations, such as generation of reactive oxygen species and DNA fragmentation. Experiments involving isolated mitochondria from rho + and rho [symbol: see text] cells confirm that rho [symbol: see text] mitochondria can be induced to undergo permeability transition, a process thought to account for the pre-apoptotic delta psi m disruption in cells. Like rho + mitochondria, rho [symbol: see text] mitochondria contain a pre-formed soluble factor that is capable of inducing chromatin condensation in isolated nuclei in vitro. This factor is released from mitochondria upon induction of permeability transition by calcium or the specific ligand of the adenine nucleotide translocator atractyloside. In conclusion, it appears that all structures involved in the maintenance and pre-apoptotic disruption of the delta psi m, as well as a mitochondrial apoptotic factor(s), are present in rho [symbol: see text] cells and thus are controlled by the nuclear rather than by the mitochondrial genome. These findings underline the contribution of mitochondria to the apoptotic process. PMID- 8616848 TI - Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal. AB - We have reported previously on the first spontaneously immortalized, nonmalignant human breast epithelial cell line, HMT-3522, which is entirely dependent on exogenous epidermal growth factor (EGF). In passage 118, cells were adapted to grow in medium without EGF and a new growth-transformed subline, HMT-3522/gt-1, was generated and propagated at high growth rate without exogenous EGF (Madsen et al., Cancer Res., 52: 1210-1217, 1992). Here we have used this subline and the continuum of the parent line, HMT-3522/wt, to pose the question whether a relevant change in a physiological parameter of the microenvironment will induce malignant transformation. The two cell lines were cultured under identical conditions with the only exception that EGF was omitted in the medium for gt-1. Initially, wt and gt-1 were identical in terms of karyotype as well as morphology, growth rate, and protein expression as revealed by two-dimensional gel electrophoresis. A highly dramatic shift to phenotype was observed in passage 238 when the gt-1 line became tumorigenic in nude mice. After two mouse-culture passages, the resulting malignantly transformed cell line (HMT-3522/mt-1) was refractory to the growth-modulating effect of EGF and presented an extra copy of a chromosome marker, 7q-, as the only cytogenetic difference from the gt-1. Our results suggest that microenvironmental cues are powerful factors in the induction of malignancy. A major role of EGF receptor in the malignant transformation is emphasized by loss of EGF sensitivity and acquisition of an extra chromosome 7p harboring the EGF receptor gene. We hypothesize that during premalignant hyperplasia, a population of EGF/transforming growth factor alpha autonomous epithelial cells in situ may develop as a consequence of local transforming growth factor alpha deprivation, a condition reflected in the culture model as autonomy after EGF withdrawal. PMID- 8616849 TI - Chromium induces a persistent activation of mitogen-activated protein kinases by a redox-sensitive mechanism in H4 rat hepatoma cells. AB - Chromium is an important industrial metal, an environmental pollutant, and a human carcinogen. To investigate the mechanisms of chromium-induced carcinogenesis, activation of mitogen-activated protein (MAP) kinases ERK1 and ERK2 was examined in rat hepatoma cells following exposure to hexavalent chromium (Cr(VI)). Cr(VI) was found to activate both forms of MAP kinase in a dose- and time-dependent manner. In contrast to the protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate, which induced a transient activation of MAP kinases, Cr(VI) caused persistent activation of these enzymes. Furthermore, unlike phorbol 12-myristate 13-acetate, the ability of chromium to activate MAP kinases was found to be independent of PKC since chromium-induced MAP kinase activation occurred in PKC-depleted cells. Stimulation of ERK1 and ERK2 was associated with the ability of Cr(VI) to increase cellular peroxide levels as determined using the H2O2-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate and flow cytometry. Furthermore, the activation of these kinases by chromium was enhanced in cells treated with the glutathione-depleting agent, L-buthionine-[S,R] sulfoximine, and attenuated in cells pretreated with an agent that elevates cellular levels of glutathione (i.e., N-acetyl-L-cysteine). The ability of chromium to modulate MAP kinase activity in this manner suggests a mechanism of chromium-induced carcinogenesis that involves the persistent stimulation of cellular regulatory pathways. PMID- 8616850 TI - Metabolism and DNA binding of the environmental colon carcinogen 6-nitrochrysene in rats. AB - The environmental contaminant 6-nitrochrysene (6-NC) has been shown to induce adenomas and adenocarcinomas in the colons of rats. The present study aimed at providing a better understanding of mechanisms that are responsible for this effect. Three female CD rats were injected i.p. with [3,4,9,10-3H]6-NC [9 mumol/rat (346 microCi/rat)], and urine and feces were collected daily for 3 days. In the first 24 h, radioactivity corresponding to 1.3% of the dose was excreted in the urine, whereas 23.0% was recovered in the feces. After 3 days, the total excretions in urine and feces were 2.8% and 34.9% of the dose, respectively. Radioactivity measured in various organs 3 days after injection of [3,4,9,10-3H]6-NC amounted to 24.8% of the administered dose. Fecal metabolites were identified, based on comparison of their chromatographic characteristics with those of standards, as trans-1,2-dihydro-1,2-dihydroxy-6-NC, chrysene-5,6 quinone, and 6-aminochrysene (6-AC); the structure of the latter was further confirmed by mass spectrometry and UV spectral analysis. Metabolites identified in the urine were 6-AC, trans-1,2-dihydro-1,2-dihydroxy-6-NC, and trans-9,10 dihydro-9,10-dihydroxy-6-NC in free forms and also as glucuronide and/or sulfate conjugates. The 32P-postlabeling assay was used to determine the metabolic pathways that were leading to DNA adduct formation in the target (colon) and nontarget (liver, lung, and mammary tissues) organs of female CD rats injected with 6-NC under conditions identical to those of the bioassay (total, 14.8 mumol/rat; single i.p. injections on days 1, 8, 15, 22 and 29). Twenty-four h after the last carcinogen administration, the levels of the adduct derived from trans-1,2-dihydro-1,2-dihydroxy-6-AC were higher than those derived from N hydroxy-6-AC in all organs examined; however, the highest levels of DNA adducts were found in the lung and not in the target organ, the colon. Although the role of each adduct in colon carcinogenesis needs to be determined, the results favor the ring oxidation and nitroreduction combination pathway as the primary contributor to the activation of 6-NC as a colon carcinogen in the rat. PMID- 8616851 TI - The expression of gonadotropin-releasing hormone and its receptor in endometrial cancer, and its relevance as an autocrine growth factor. AB - The presence of a direct extra-pituitary action of gonadotropin-releasing hormone (GnRH) via specific receptors in endometrial cancer (EC) has been suggested as an explanation for the therapeutic effect of GnRH analogue (GnRHa) in recurrent disease. We have sought the expression of the GnRH peptide and functional GnRH receptor (GnRH-R) in human tissues and cell lines to investigate the possibility of an autocrine growth regulation mechanism. Using reverse transcription-PCR, differing GnRH mRNA transcripts were detected in two EC cell lines (Ishikawa and HEC-1A), a choriocarcinoma (JEG3) cell line, and tissues from endometrium and placenta. However, secretion of immunoreactive GnRH could be detected by RIA in only 1 of 10 EC tissues in primary culture, and in none of the cell lines. Low levels of GnRH-R mRNA expression were found in the same cells, which were only detectable by reverse transcription-PCR and Southern blotting of the PCR product. In radioligand binding assays using GnRHa goserelin, no pituitary-like, high affinity GnRH binding sites could be found in either EC cell lines or tissues. Low affinity binding (Kd = 1.0 - 3.1 x 10(-7)M) was detected in three of eight (37%) EC tissues. Furthermore, receptor signal transduction measurements carried out in these cells showed no increases in either total inositol phosphate, cyclic AMP production, or cytosolic Ca2+ in response to either GnRH or GnRHa. Finally, no effect of either GnRH or GnRHa on the growth of EC cell lines was detected in vitro, under estrogen-free conditions, assessed by DNA content. Our data suggest that although there is a potential for autocrine activity for GnRH in EC as judged by the presence of mRNA for peptide and receptor, no functional receptor activity could be detected in vitro. Alternative mechanisms should be studied to explain the in vitro action of GnRHa. PMID- 8616853 TI - Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU. AB - O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2' deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues. PMID- 8616852 TI - Fluorescence imaging studies for the disposition of daunorubicin liposomes (DaunoXome) within tumor tissue. AB - Unilamellar liposomes that retain their contents in the systemic circulation can alter the pharmacokinetics of anticancer agents in favorable ways. It has long been recognized that certain liposome compositions may increase the local drug concentration substantially above that achievable with a free drug. We report here that liposomes can alter the in vivo disposition of an entrapped drug not only on a macroscopic but also on a microscopic scale. We show through in vitro studies that intact liposomes composed of distearoylphosphatidylcholine and cholesterol and containing daunorubicin (DaunoXome) are taken up into P1798 tumor cells. These liposomes produce an enhanced cytotoxicity relative to the free drug for incubation times longer than about 8 h. For in vivo studies, we developed and used a noninvasive fluorescence imaging technique to follow the accumulation of liposomal daunorubicin within murine tumors. With this method, we show that the maximum concentration of the available liposomal drug in tumors exceeds that of the free drug, and additionally, liposomal daunorubicin persists at high levels for several days. Total liposome-delivered drug fluorescence from whole tumor extracts peaks at about 8 h. In comparison, the fluorescence intensity of daunorubicin demonstrate persistent high levels of daunorubicin fluorescence within cells and throughout the tumor masses. Free daunorubicin, in contrast, transiently achieves modest levels of fluorescence and rapidly drops to background within a few h. These results indicate distinct mechanisms for the localization of free and liposomal daunorubicin, suggesting that liposmal daunorubicin can provide sustained intracellular levels of the drug within the tumor. PMID- 8616854 TI - Oral administration of anti-doxorubicin monoclonal antibody prevents chemotherapy induced gastrointestinal toxicity in mice. AB - Gastrointestinal mucositis is a common and painful condition that afflicts a proportion of cancer patients receiving chemotherapeutic drugs including anthracyclines, and it has become the dose-limiting toxicity for a number of chemotherapeutic regimens. The murine monoclonal antibody MAD11 recognizes the anthracycline doxorubicin, and systemic administration of this antibody in mice treated with doxorubicin was found previously to prevent the toxic effects of the drug. The purpose of this study was to determine whether gastrointestinal toxicity associated with doxorubicin can be reduced by oral administration of anti-doxorubicin MAD11 in mice. Our experiments show that orally administered MAD11 antibodies: (a) are essentially not absorbed in the blood circulation since less than 0.5% of protein-associated radioactivity was recovered from blood samples; (b) reduce the extent of doxorubicin-induced apoptosis in murine intestinal crypts, as determined by labeling strand breaks with modified nucleotides in an enzymatic reaction; and (c) reduce the body weight loss in mice treated with 12 mg/kg body weight of doxorubicin and decrease the early mortality in mice treated with 16 mg/kg body weight. This type of treatment may be useful in preventing anthracycline-induced gastrointestinal mucositis in cancer patients. PMID- 8616855 TI - Pharmacodynamics of taxol in human head and neck tumors. AB - The pharmacodynamics of taxol in human head and neck squamous cell carcinoma were studied using histocultures of surgical specimens from patients (n = 22). Tumors were treated with taxol for 24 h. The inhibition of DNA synthesis was determined by 48 h cumulative bromodexyuridine (BrdUrd) incorporation. The induction of apoptosis was measured by morphological changes, in situ DNA end labeling, post exonuclease III BrdUrd labeling, and DNA fragmentation. Inhibition of the BrdUrd labeling index (LI) by taxol was incomplete, with 11 tumors showing maximal inhibition (Emax) of 30-50% and the remaining 11 tumors showing and Emax of 50 80%. For both groups, the inhibition approached maximum values at 1 microM taxol concentration; an additional 10-fold increase in drug concentrations did not significantly enhance the inhibition. The taxol concentrations required for a 30% inhibition (IC30) were 4.2 and 0.3 microM for the first and second groups, respectively. The IC30 correlated with the Emax (r2 = 0.39; P < 0.001). Taxol induced apoptosis in all tumors, 11 tumors showed a maximal fraction of apoptotic tumor cells between 3 and 10% and 11 tumors between 13 and 28%, whereas untreated controls showed a maximal apoptotic index of < 1%. For individual tumors, the maximal apoptotic index occurred between 0.1 and 3 microM, and correlated with the BrdUrd LI for the untreated control (r2 = 0.37; P < 0.01). It is interesting that > 95% of apoptotic cells were BrdUrd labeled, whereas not all BrdUrd-labeled cells were apoptotic. To investigate the basis of the variable tumor response to taxol, we determined the expression of multidrug resistance P-glycoprotein (Pgp), p53, and bcl-2 proteins, using immunohistochemical staining and Western blot analysis. Eleven (50%), 10 (45%), and 7 (32%) tumors expressed Pgp, p53, and bcl 2, respectively. Patients with Pgp-positive tumors showed a higher number of affected lymph nodes than those with Pgp-negative tumors (P < 0.05). Compared with moderately and well differentiated tumors, the poorly differentiated tumors expressed p53 and Pgp more frequently and showed a lower maximum inhibition of DNA synthesis and a higher apoptotic fraction after taxol treatment (P < 0.05 in both cases). Pgp expression correlated differently with taxol-induced inhibition of DNA synthesis than with apoptosis; Pgp-positive tumors showed a significantly higher Emax (63%) and IC30 (4.2 microM) but also a higher apoptotic index (17%) than Pgp-negative tumors (Emax 36%; IC30, 0.3 microM; and apoptotic index; 6%; P < 0.05 for all cases). p53 and bcl-2 expression did not correlated with taxol induced inhibition of DNA synthesis or apoptosis. The data indicate that taxol acts through apoptosis and inhibition of proliferation in human head and neck cancer. Pgp overexpression appears to protect cells from the antiproliferative effect of taxol but correlated with a higher apoptosis. PMID- 8616856 TI - Pharmacological and toxicological aspects of new imidazoacridinone antitumor agents. AB - Imidazoacridinones represent a new group of antitumor compounds developed by J. Konopa and coworkers in Gdansk, Poland (W.M. Cholody, J. Med. Chem., 33: 49-52, 1990). The compounds exert activity against a broad spectrum of human tumors in the National Cancer Institute in vitro screening scheme. In this work, the in vitro cytotoxicity, cellular pharmacology, and genotoxic/transforming potential of five selected imidazoacridinones were studied. The compounds were highly cytotoxic (0.01-0.40 microM) to dividing cells, such as Friend erythroleukemia cells (line F4-6), V79 Chinese hamster cells, and exponentially growing C3H/M2 mouse fibroblasts. In contrast, nondividing primary rat hepatocytes and C3H/M2 cells in confluency were less sensitive to the toxicity of the imidazoacridinones. Multidrug-resistant-overexpressing F4-6 cells, 200-fold resistant to doxorubicin, showed only partial resistance (4-10 fold) to the imidazoacridinones. The cellular transport of the fluorescent imidazoacridinones occurred rapidly, and most of the drug fluorescence was localized in the nucleus. Cellular accumulation and retention of two selected imidazoacridinones (C-1310 and C-1311) in sensitive as well as in resistant F4-6 cells were determined with laser-excited flow cytometry. After an incubation with C-1311 and C-1310 for 60 min at 37 degrees C, the cellular accumulation of the less cytotoxic compound C 1310 was greater than that of C-1311, and for both compounds, the fluorescence in the resistant F4-6 cells was one-half of that in the sensitive F4-6 cells. Lowered temperature (4 degrees C) reduced the cellular accumulation for both compounds in the sensitive and in the resistant F4-6 cells and was comparable to the uptake in resistant F4-6 cells. The treatment of the resistant F4-6 cells with the multidrug-resistant modulator verapamil led to an enhanced accumulation of C-1310 and C-1311 by the cells. All five compounds produced a dose-dependent inhibition of [3H]uridine and [14C]thymidine incorporation and, except for C 1336, preferentially inhibited DNA synthesis. The affinity of the imidazoacridinones to DNA is also indicated by an increase of the DNA melting point by 9-11 degrees C. The mutagenic potential of the imidazoacridinones was investigated in the hypoxanthine guanine phosphoribosyl transferase test; the compounds C-1310 and C-1311 were additionally tested in the Salmonella typhimurium-microsome assay. Limited mutagenicity was detected in the hypoxanthine guanine phosphoribosyl transferase test, and in Salmonella typhimurium, mutagenicity was observed only in the strain TA1537. Furthermore, no induction of DNA repair synthesis was observed after treatment of primary hepatocytes with the five imidazoacridinones. The compounds did not transform C3H/M2 fibroblasts. One derivative, C-1336, led to a significant induction of cell differentiation in Friend erythroleukemia cells. The results of this study show that the imidazoacridinones display a strong cytotoxic effect in rapidly dividing cells and only a partial resistance toward multidrug resistant cells; in addition, they showed a limited mutagenic potential in V79 fibroblasts and Salmonella typhimurium and no transforming potential in C3H/M2 cells. The imidazoacridinones are, therefore, an interesting group of new antitumor agents, and further in vivo studies are warranted to explore the usefulness of these compounds for the treatment of human cancer. PMID- 8616857 TI - The bryostatins inhibit growth of B16/F10 melanoma cells in vitro through a protein kinase C-independent mechanism: dissociation of activities using 26-epi bryostatin 1. AB - Bryostatin 1 is a potential cancer chemotherapeutic agent in Phase II clinical trials, with positive responses observed for malignant melanoma, among other tumors. The bryostatins are known to be potent ligands for protein kinase C (PKC), functioning as partial antagonists. In the present study, we explore the mechanism by which the bryostatins inhibit growth to B16/F10 mouse melanoma cells in vitro. Three experimental approaches suggest that the growth inhibition is independent of PKC. First, we characterized in detail the translocation and down regulation of the PKC isozymes alpha, delta, and epsilon in response to phorbol ester and bryostatin 1 in these cells. Although the dose-response curves obtained for the translocation-activation of PKC isozymes showed good correlation with the growth-enhancing activity of phorbol 12-myristate 13-acetate, for no PKC isozyme was there a good correlation with the growth-inhibitory activity of bryostatin 1. Second, inhibition PKC, inhibited the growth of the B16/F10 melanoma cell lines with potency similar to that of bryostatin 1. We confirmed here that 26-epi bryostatin 1 showed 60-fold reduced affinity for PKC and 30-60-fold reduced potency to translocate and downregulate PKC isozymes compared with bryostatin 1. We presumed that the principal toxicity of bryostatin 1 reflects its interaction with PKC, and we would thus predict that epi-bryostatin 1 would be less toxic. Indeed, we found at least 10-fold reduced toxicity of 26-epi-bryostatin 1 in C57BL/6 mice compared with bryostatin 1. We conclude that the growth inhibition of the bryostatins, at least in this system, does not result from interaction with PKC. As exemplified by 26-epi-bryostatin 1, this insight permits the design of analogues with comparable growth inhibition to bryostatin 1 but with reduced toxicity. PMID- 8616858 TI - Nonlinear pharmacokinetics of paclitaxel in mice results from the pharmaceutical vehicle Cremophor EL. AB - Studies in humans and mice have demonstrated a nonlinear pharmacokinetic behavior of paclitaxel. Because of its poor water solubility, the drug is formulated in a mixture of Cremophor EL and ethanol (1:1, v/v; Taxol). We hypothesized that the substantial amounts of concurrently administered Cremophor EL on the disposition of paclitaxel, female FVB mice received paclitazel by i.v. injection at does levels of 2, 10, and 20 mg/kg by appropriate (standard) dilution of the commercially available formulation of paclitaxel (Taxol) with saline. The drug was also given at 2 mg/kg with supplemental Cremophor EL-ethanol to achieve the same amount of vehicle as by standard administration of 10 mg/kg. Furthermore, paclitaxel formulations in Tween 80-ethanol (1:1, v/v) and dimethylacetamide were tested. Plasma samples were collected between 5 min and 48 h, and tissue specimens were sampled at 1, 4, and 8 h after drug administration. Paclitaxel and metabolites were quantified by high-performance liquid chromatography. Cremophor EL levels were determined by a novel high-performance liquid chromatography procedure. For comparative reasons, Cremophor EL was also assayed in plasma samples from three patients receiving a 3-h i.v. infusion of 175 mg/m2 of paclitaxel. A marked nonlinear pharmacokinetic behavior of paclitaxel was observed when the drug was formulated in Cremophor EL-ethanol. The clearance of 2.37 L/h/kg at 2 mg/kg was reduced to 0.33 and 0.15 L/h/kg at 10 and 20 mg/kg, respectively. When 2 mg/kg were given with an amount of Cremophor EL-ethanol matching that of the 10-mg/kg dose level, the clearance was 0.56 L/h/kg. If administered at 10 mg/kg in Tween 80-ethanol or at 2 and 10 mg/kg in dimethylacetamide, the clearances were 2.66, 2.57, and 2.62 L/h/kg, respectively. Despite the fact that much higher plasma levels of paclitaxel are reached when given in the Cremophor EL-ethanol formulation, the tissue levels were essentially similar with all tested drug preparations. The Cremophor EL levels in patients were in the same order of magnitude as those observed in mice after administration of 2 and 10 mg/kg. These data demonstrate that Cremophor EL has a profound effect on the pharmacokinetics of paclitaxel im mice. Because Cremophor EL also contributes substantially to the nonlinear pharmacokinetic behavior of paclitaxel observed in humans. PMID- 8616859 TI - Gene cloning and characterization of Pseudomonas putida L-methionine-alpha deamino-gamma-mercaptomethane-lyase. AB - Methionine dependency has been reported in cancer cell lines and primary tumors. Thus, L-methionine deprivation might have potential value for the treatment of human cancers with a methionine requirement. L-Methionine-alpha-deamino-gamma mercaptomethane-lyase has been reported to decrease plasma methionine levels and to inhibit tumor growth in experimental animals but has not been studied extensively because sufficient homogeneous enzyme was not available. In this study, we cloned the L-methioninase gene from Pseudomonas putida and isolated pure and abundant recombinant enzyme. Both L-methionine and L-cysteine in culture medium were completely degraded by 1 unit/ml purified enzyme. Two hundred and fifty units/kg L-methioninase administered i.v. to mice yielded 0.7 unit/ml of plasma concentration and lowered total plasma sulfur-containing amino acids by more than 75%. Although sensitivity to enzymatic methionine depletion differed among cell lines, leukemia cell lines were generally more sensitive than solid tumor cell lines. The availability of pure recombinant L-methioninase will allow in vivo studies on the antitumor activity and the potential toxicity of enzymatic methionine depletion. PMID- 8616861 TI - Deletions and insertions in the p53 tumor suppressor gene in human cancers: confirmation of the DNA polymerase slippage/misalignment model. AB - We analyzed all published deletions and insertions in the p53 gene to assess the relevance of mutagenesis models. Almost all deletions and insertions can be explained by one or more of the following DNA sequence features: monotonic base runs, adjacent or nonadjacent repeats of short tandem sequences, palindromes, and runs of purines or pyrimidines (homocopolymer runs). Increased length of monotonic runs correlates positively with increased frequency of events. Complex frameshift mutations can be explained by the formation of quasi-palindromes, with mismatch excision and replication using one strand of the palindrome as a template. Deletions and insertions in the p53 tumor suppressor gene may reflect both spontaneous and carcinogen-induced mutagenesis. PMID- 8616860 TI - Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. AB - Radiolabeled antibodies have produced encouraging remissions in patients with chemotherapy-resistant hematological malignancies; however, the selection of therapeutic radionuclides for clinical trials remains controversial. In this study, we compared the internalization, lysosomal targeting, metabolism, and cellular retention of radiolabeled murine and humanized monoclonal antibodies targeting the CD33 antigen (monoclonal antibodies mP67 and hP67, respectively) on myeloid leukemia cell lines (HEL and HL-60) and of anti-carcinoma antibodies (monoclonal antibodies hCTM01 and hA33) targeting breast cancer and colorectal carcinoma cell lines (MCF7 and Colo 205, respectively). Each antibody was labeled with 125I (by the IodoGen method) and with 111In and 90Y using macrocyclic chelation technology. Targeted tumor cells were analyzed for retention and metabolism of radioimmunoconjugates using cellular-radioimmunoassays, Percoll gradient fractionation of cell organelles, SDS-PAGE, and TLC of cell lysates and culture supernatants. Our results suggest that antibodies are routed to lysosomes after endocytosis, where they are proteolytically degraded. [125I]monoiodotyrosine is rapidly excreted from cells after lysosomal catabolism of antibodies radioiodinated by conventional methods, whereas small molecular weight 111In and 90Y catabolites remain trapped in lysosomes. As a consequence of the differential disposition of small molecular weight catabolites, 111In and 90Y conjugates displayed superior retention of radioactivity compared with 125I conjugates when tumor cells were targeted using rapidly internalizing antibody antigen systems (e.g., hP67 with HEL cells and hCTM01 with MCF7 cells). When tumor cells were targeted using antibody-antigen systems exhibiting slow rates of endocytosis (e.g., hP67 on HL-60 cells and hA33 on Colo 205 cells), little differences in cellular retention of radioactivity was observed, regardless of whether 125I, 111In, or 90Y was used. PMID- 8616862 TI - Ploidy and karyotypic alterations associated with early events in the development of hepatocarcinogenesis in transgenic mice harboring c-myc and transforming growth factor alpha transgenes. AB - The cooperation of the c-myc oncogene with the growth factor transforming growth factor (TGF)-alpha in development of liver tumors in transgenic mice has been demonstrated previously. In this study, we analyzed the ploidy and karyotype of c myc, TGF-alpha, parental control, and the double transgenic c-myc/TGF-alpha hepatocytes at 3 weeks of age when the liver is histologically normal and at 10 weeks when the c-myc/TGF-alpha liver is dysplastic and contains basophilic foci. Eighty % of the 10-week hepatocytes were aneuploid, and 32% had chromosomal breakage. Statistically significant breakage was observed in six different chromosomes. Breakage at band A5 and at the border of bands C4/5 of chromosome 1 was observed. Fragile sets on chromosome 4 were most frequent in the middle of the chromosome at bands C2 and C6. Chromosome 6 was fragile at band F2. The region of chromosome 7 at bands B5 and D3 was frequently broken and involved in translocations. Chromosome 12 was broken at bands D1 and D3. The breakage sites on chromosomes 1, 4, 7, and 12 correspond to sites of tumor susceptibility genes in the mouse. Although there was no consistent change in copy number, recurrent translocations between chromosomes 1, 4, 7, 12 and 19 were also observed. These studies demonstrate that the development of dysplasia and basophilic foci in the liver is correlated with aneuploidy and chromosome breakage. The specific fragile sites indicate genetic regions that are altered during early stages of hepatocarcinogenesis. Due to the conservation of genetic linkage groups between mice and humans, the identification of genetic alterations in the mouse during hepatocarcinogenesis may provide critical information about tumor susceptibility genes that are important in the early development of human hepatocellular carcinoma. PMID- 8616863 TI - Allelic loss on chromosome 10 in prostate adenocarcinoma. AB - A total of 83 prostate adenocarcinomas was evaluated for allelic loss on chromosome 10 by analysis of loss of polymorphic microsatellite repeats. Initially, 64 stage B carcinomas were analyzed at 12 loci on chromosome 10. Nine cases showed loss of chromosome 10 sequences, with a fractional allelic loss of 20%. These nine cases were then analyzed at an additional 19 loci to define better the regions of loss. Four areas of loss were identified, including 10p (2 of 64 cases), 10q23.1 (7 of 64 cases), 10q23.3 (4 of 64 cases), and 10q26 (2 of 64 cases). Three loci in these regions, D10S111, D10S185, and D10S192, were then analyzed in 19 advanced (stage C and D) carcinomas. Seven (37%) of 19 advanced carcinomas showed allelic loss at one or more of these loci. A statistically significant increase in the fractional allelic loss at both D10S111 (10p) and D10S185 (10q23.1) was observed. Thus, a complex pattern of loss is seen on chromosome 10 in prostate carcinoma, with regions of loss on 10p and 10q, and such loss occurs at a higher rate in clinically advanced disease. PMID- 8616864 TI - Cooperation between p53-dependent and p53-independent apoptotic pathways in myeloid cells. AB - Apoptosis may involve p53-dependent and -independent pathways. Results presented here suggest a possible cooperation between these two types of pathways. M1/2 is a p53-nonproducer subclone that may undergo either a p53-independent apoptosis following growth factor deprivation or a p53-dependent apoptosis following reconstitution of wild-type p53 expression. The p53-independent apoptosis in these cells is a slow process occurring after a G0-G1 arrest. In contrast, the p53-dependent apoptosis is much more rapid and is characterized by early and late apoptotic phases, taking place in cell arrested at G0-G1 and S phase. The transition from early to late apoptosis correlated with the levels of the p53 protein. Concomitant induction of both apoptotic pathways accelerated cell death and facilitated the transition from early to late apoptotic phase. The interaction between these pathways is further supported by the finding that mutant p53 interferes with p53-independent apoptosis. Thus, although apoptosis can occur via either p53-dependent or -independent pathways, under certain conditions the two pathways may interact with each other. PMID- 8616865 TI - Suppression of the malignant phenotype of human prostate cancer cell line PPC-1 by introduction of normal fragments of human chromosome 10. AB - Numerous studies have detected frequent losses of heterozygosity at polymorphic loci on chromosomal arms 10p and 10q in human prostate cancers. To confirm the presence of tumor suppressor genes in these chromosomal regions, fragments of normal human chromosome 10 tagged with a neomycin resistance gene were transferred into cells from a human prostate cancer cell line. PPC-1, by microcell-mediated chromosome transfer. Two of the six hybrid clones obtained showed decreased tumorigenicity in athymic nude mice and decreased efficiency of colony formation in soft agar compared with PPC-1; the other four retained fully malignant phenotypes. Analysis of polymorphic loci on chromosome 10 in these hybrid clones suggested that a tumor suppressor gene associated with prostate cancer is located within a 17-cM region at distal 10p. PMID- 8616866 TI - Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. AB - The molecular mechanism of cell death due to hypoxia has not been elucidated. Our recent observations that overexpression of the anti-apoptotic proto-oncogene bcl 2 and a bcl-2-related gene, bcl-x, prevents hypoxic cell death suggest that hypoxia induces apoptosis. Using electron microscopy and confocal and nonconfocal fluorescence microscopy, we show here that hypoxia does, in fact, induce both necrosis and apoptosis, and that the proportion of these two modes is highly dependent on the cell type. Overexpression of Bcl-2 or Bcl-Xl blocks hypoxia induced apoptosis in a dose-dependent manner. PMID- 8616867 TI - Heterogeneous mutation of the RET proto-oncogene in subpopulations of medullary thyroid carcinoma. AB - Mutations in the RET proto-oncogene are associated with the pathogenesis of medullary thyroid carcinoma (MTC). In an attempt to understand this process, we examined microdissected subpopulations from MTC and multiple metastases from these tumors. Approximately 80% of sporadic MTC's had at least one subpopulation with the RET codon 918 mutation, which is a mutation previously detected in sporadic MTC as a somatic mutation and in multiple endocrine neoplasia type 2B as a germline mutation. However, the distribution of this mutation was nonhomogeneous, occurring only in subpopulations in most tumors and among subsets of multiple metastases, thus implying that although the codon 918 mutation could be an early event, it is not necessarily an early or essential event in tumorigenesis. This heterogeneity suggests either that the codon 918 mutation can arise as an event in progression within a metastatic clone or within a single tumor, or that MTC can be of polyclonal origin. Of significance, one of two multiple endocrine neoplasia type 2A MTCs carried a somatic mutation at codon 918, in addition to the RET mutation present in the germline. We found no correlation between the presence of other somatic genetic events, such as loss of heterozygosity on chromosome arms 1p and 22q, and RET mutation status in the various subpopulations of MTC. PMID- 8616868 TI - A specific deletion in the breakpoint cluster region of the ALL-1 gene is associated with acute lymphoblastic T-cell leukemias. AB - A variety of chromosomal translocations to the ALL-1 gene are regularly observed in acute leukemias and are thought to play a key role in the leukemogenic process. Chimeric proteins are encoded by the breakpoint regions of the derivative chromosomes have been proposed to be the relevant oncogenic agents. In addition, internal duplications of the ALL-1 gene have been observed in patients with specific acute myeloid leukemias. Thus, it has been hypothesized that oncogenic variants of the ALL-1 protein may be generated by both chimerization and self-fusion, but the critical structural features endowing the altered proteins with their oncogenic potential are still unknown. Here a novel structural alteration of the ALL-1 gene was observed in three patients presenting with acute T-cell leukemia (ALL) without chromosomal translocations or self fusions of the ALL-1 gene. These unrelated patients carried an internal deletion in one of the two alleles of the ALL-1 gene that eliminated parts of introns 7 and 8, together with exon 8. The deletion was found in 3 of 74 ALL patients, but not in acute myeloid leukemias, follicular lymphomas, or peripheral blood leukocytes from healthy donors. One ALL patient showed the deletion at diagnosis but no longer at remission or at 9 months after remission. These findings support the hypothesis that the ALL-1 protein may be converted to an oncogenic variant, not only by chimerization or self-fusion, but also by deletion of sequences coded by exon 8. They further suggest that these three different types of structural alterations of the ALL-1 protein may each cause a distinct disease phenotype. Alternatively spliced mRNA species omitting exon 8 were observed in 14 of 24 ALL patients without detectable macroscopic alterations of the ALL-1 gene and also in peripheral blood leukocytes from healthy donors. PMID- 8616869 TI - The prognostic significance of Bcl-2 and p53 expression in ovarian carcinoma. AB - Advanced ovarian cancer is characterized by poor prognosis and the development of resistance to chemotherapy. We have found that Bcl-2 and p53, two proteins implicated in the control of apoptosis, are differently expressed in the ovarian cell line A2780 and its cisplatin-resistant variant 2780CP, with the resistant line overexpressing both proteins. Transfection of the A2780 cells with a Bcl-2- or p53-expressing plasmid increases resistance to various drugs, including cisplatin, suggesting that Bcl-2 and p53 expression may influence the sensitivity of ovarian cancer cell lines to chemotherapy. Expression of these two proteins in vivo was determined by immunohistochemical staining of ovarian tumor biopsies from 70 patients. We found that Bcl-2 and p53 were expressed in 57 and 61% of specimens examined, respectively. Both p53 and Bcl-2 were found to be independent prognostic indicators of survival in ovarian cancer. Survival was poorer in patients with tumors expressing high levels of p53, whereas expression of Bcl-2 was associated with improved survival. PMID- 8616870 TI - Concentration of vascular endothelial growth factor in the serum and tumor tissue of brain tumor patients. AB - Vascular endothelial growth factor (VEGF) has been investigated as a potent mediator of brain tumor angiogenesis, vascular permeability, and glioma growth. Using a VEGF ELISA, we determined the concentration of VEGF in the sera and tumor extracts of 19 brain tumor patients including glioblastoma, anaplastic astrocytoma, low grade astrocytoma, meningioma, malignant lymphoma, and metastatic brain tumor as well as normal brain. Although VEGF concentration of the serum was not correlated with that of the tissue, VEGF concentrations of glioblastoma cyst fluid were 200-300-fold higher than those of serum in the patients. VEGF concentration in the tumors was significantly correlated with the vascularity measured by counting vessels stained with von Willebrand factor antibody. VEGF protein localized to the cytoplasm of tumor cells and vasculature in gliomas, predominantly in the peripheral microvessel "hot spots" as well as around the necrosis in glioblastomas. VEGF immunopositivities were well reflected with VEGF concentration determined by ELISA. VEGF ELISA demonstrated time dependent increase of the VEGF concentration in the serum-free conditioned medium of various glioma cell lines. The conditioned medium with high VEGF concentration induced endothelial cell migration. These observations suggest that VEGF represents a useful marker and measurable element of glioblastoma angiogenesis. The measurement of VEGF concentration by ELISA in tumor and tumor cyst fluid may allow for the assessment of vascularity in gliomas. PMID- 8616871 TI - Antitumor effects of granulocyte-macrophage colony-stimulating factor production by melanoma cells. AB - The use of immunomodulating gene therapy in the treatment of malignant disease is under intensive investigation. In this study, we examined the potential of melanoma-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) to inhibit melanoma progression in a murine model. The HGH18 murine melanoma cell line was transfected with the murine GM-CSF gene in a SV40 expression vector that resulted in melanoma clones that produced varying amounts of GM-CSF. Syngeneic mice inoculated s.c. with HFH18 parental melanoma cells or HFH18 cells transfected with the GM-CSF gene n the noncoding 3'-5' orientation [HFH18/GM-CSF( ) cells] develop large tumors that reach a mean tumor volume of 3300 mm3 by day 30. In contrast, animals inoculated with two melanoma clones producing high levels of GM-CSF [HFH18/GM-CSF(++) and HFH18/GM-CSF(+ + +)] either completely reject the tumor cells or develop tumors with a mean volume of only 40 mm3. In comparison, animals inoculated with a melanoma clone producing low levels of GM CSF [HFH18/GM-CSF(+)] develop large tumors averaging 2000 mm3, thus demonstrating a dose-response effect of tumor inhibition by melanoma-derived GM-CSF. Additionally, vaccination with irradiated GM-CSF-producing melanoma cells conferred optimal immunogenicity against a subsequent challenge with HFH18 cells. Tissue sections from excised GM-CSF-producing tumor cell inoculation sites but not from HFH18 parental or HFH18/GM-CSF(-) inoculation sites demonstrate a dense inflammatory infiltrate composed of neutrophils, tissue macrophages, and numerous CD4- and CD8-positive lymphocytes but few melanoma cells. Large numbers of dendritic cells and cells expressing the B7-2 costimulatory molecule are detected only within HFH18/GM-CSF(+ + +) melanoma inoculation sites. Our results lend further support to clinical trials of GM-CSF gene therapy in the treatment of advanced malignant melanoma, possibly by the recruitment of dendritic antigen presenting cells. PMID- 8616872 TI - Immunohistochemical staining for markers of future neoplastic progression in the larynx. AB - We performed a retrospective, longitudinal study to determine whether abnormalities in immunohistochemical staining for the epidermal growth factor receptor (EGFR), p53, or cyclin D1 occur before the development of laryngeal carcinoma. Staining was performed on 63 paraffin-embedded biopsies from 18 patients who subsequently developed carcinoma in situ (CIS) or invasive carcinoma of the larynx. These were compared to 71 biopsies from 20 patients who did not develop CIS/cancer (minimum follow-up period, 4 years). Also studied were the 34 biopsies containing CIS and/or carcinoma from those patients who progressed and 22 biopsies obtained concurrently. The two patient groups did not differ significantly in terms of tobacco and alcohol use. Distinct patterns of staining correlated with malignant progression. These included EGFR staining of two thirds or more of the epithelium thickness, a linear basal p53 staining pattern, and strong (3+) staining for cyclin D1 (P < 0.01 for each). These staining patterns also correlated with increasing atypia. In our study population, linear basal staining for p53 and strong staining for cyclin D1 had higher specificity for progression than did EGFR overexpression, which was also seen in association with inflammation and chronic irritation. Marked site-to-site variation was seen in the immunohistochemical staining and in the degree of atypia, suggesting that multiple biopsies are necessary to properly assess risk. These immunohistochemical staining patterns may be clinically useful to predict patients at risk for neoplastic progression. PMID- 8616873 TI - Regulation of the adhesion of a human breast carcinoma cell line to type IV collagen and vitronectin: roles for lipoxygenase and protein kinase C. AB - We have investigated the regulation of adhesion of metastatic human breast carcinoma cells to various protein substrates in the presence or absence of the protein kinase C (PKC) activator, 12-tetradecanoyl phorbol 13-acetate (TPA) or calcium ionophore A23187 (A23187). Both TPA and A23187 dramatically enhanced MDA MB-435 cell adhesion to type IV collagen (collagen IV), vitronectin, and, to some extent, fibronectin and laminin. Adhesion to BSA and polylysine were not affected. TPA and A23187 induced substantial dose-dependent effects that were apparent after 30- and 60-min incubations, respectively, whereas a phorbol ester, which does not activate PKC, had no effect. A23187, but not TPA, induced a release of arachidonic acid (AA) from MDA-MB-435 cells. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, prevented A23187 and exogenous AA, but not TPA, from stimulating cell adhesion to collagen IV. In contrast, the increase in adhesion to vitronectin induced by A23187 and AA was, at best, only partially inhibited by nordihydroguaiaretic acid treatment. Calphostin C, a PKC inhibitor, blocked the stimulation of adhesion by A23187, exogenous AA, and TPA to both collagen IV and vitronectin. Together, these results suggest that calcium mobilization activates the release of AA and its metabolism through a lipoxygenase pathway leading to a rapid increase of MDA-MB-435 cell adhesion to collagen IV, whereas other mechanisms regulate adhesion to vitronectin. Finally, PKC activation, occurring downstream from calcium mobilization or the AA effects, is a key event involved in the regulation of adhesion to both proteins. PMID- 8616874 TI - Alteration of beta-catenin expression in colonic epithelial cells of familial adenomatous polyposis patients. AB - It has been found that beta-catenin, a key regulator of the cadherin-mediated cell adhesion system, forms complexes with adenomatous polyposis coli (APC) tumor suppressor protein, and beta-catenin expression levels are affected by exogenously induced APC protein. The effects of intrinsic APC protein alteration on beta-catenin expression levels and its subcellular localization were examined in colonic epithelia of eight patients with familial adenomatous polyposis. In all eight patients, beta-catenin was immunostained at the membranes of the cell to-cell borders in normal epithelial cells, whereas the nuclei and cytoplasms stained intensely in addition to the membranes in both adenoma and cancer cells. beta-Catenin expression levels in tumor tissues were over three times higher than those in corresponding normal mucosae of all of the three patients, whose resected specimens were available for quantitative immunoblot analysis. In these three patients, mutant truncated APC proteins were detected and shown to have lost the central region, including a known beta-catenin binding domain. beta Catenin was not coimmunoprecipitated with these mutant APC proteins in tumor tissues but was able to be coprecipitated with glutathione S-transferase-fused APC protein containing a beta-catenin binding domain. These results suggest that the absence of wild type APC protein affects the subcellular localization and expression levels of beta-catenin in human tissues. PMID- 8616875 TI - Phorbol ester and cyclic AMP-mediated regulation of the melanoma-associated cell adhesion molecule MUC18/MCAM. AB - MUC18/MCAM is a melanoma-associated cell adhesion molecule that is also occasionally found on carcinomas and other tumor types. On melanomas, MUC18 expression increases with tumor progression and is found on more than 70% of metastatic lesions. To investigate the regulation of MUC18 expression, cell lines of diverse tissue origin were exposed to cytokines, regulators of intracellular cyclic AMP (cAMP), and to phorbol ester. MUC18 expression could not be induced in negative cell lines and could only be modulated by changes in cAMP levels or by exposure to phorbol ester in positive cells. An increase in intracellular cAMP led to an up-regulation in cell surface MUC18 that was maximal at 48 h. Increased MUC18 mRNA levels were observed as soon as 4 h and were 3-fold higher than in control cells by 48 h. Exposure of the cells to phorbol ester reduced MUC18 surface expression to background levels by 24 h. This downregulation was associated with decreased mRNA levels that were apparent at 8 h. By 24 h, steady state levels of MUC18 mRNA had been reduced by 58%. Whereas similar changes in MUC18 surface expression were observed in MUC18-expressing glioma and carcinoma cell lines, melanoma cells were more resistant to the MUC18-modulating effects of cAMP analogues and phorbol ester. These observations suggest that the strong MUC18 expression observed in advanced melanomas may reflect disturbances in the normal regulation of this molecule. PMID- 8616876 TI - K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. AB - Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. A common genetic alteration in lung adenocarcinomas is mutational activation of K-ras. To determine the timing of K-ras activation, we evaluated formalin-fixed and paraffin-embedded tissue samples of 41 AAHs and their paired lung neoplasms from 28 patients for codon 12 point mutations of the K-ras oncogene. K-ras codon 12 mutations were detected using PCR followed by allele-specific oligonucleotide hybridization. Mutations were found in 16 (39%) of the 41 AAHs, 8 (42%) of the 18 adenocarcinomas, and none (0%) of the 5 lung neoplasms that were not adenocarcinomas. Of the 18 patients with both an AAH and a synchronous lung adenocarcinoma, 6 had K-ras mutation in the adenocarcinoma but not in the AAH, 6 had mutations in the AAH but not in the adenocarcinoma, 4 did not harbor mutations in either the AAH or the adenocarcinoma, and 2 had mutations in both their AAH and their synchronous adenocarcinoma. In just 1 of the 18 patients was the same K-ras mutation present in the AAHs and adenocarcinoma of the patient. The detection of independent activating point mutations in a cancer causing gene points to the neoplastic nature of AAH and suggests that glandular neoplasms of the lung arise from a background of field cancerization. PMID- 8616877 TI - Expression of Tn and sialyl-Tn antigens in the neoplastic transformation of uterine cervical epithelial cells. AB - The expression of simple mucin-type carbohydrate antigens, Tn and sialyl-Tn antigens, was evaluated by immunohistochemical staining with monoclonal antibodies in normal squamous epithelium, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma of the uterine cervix. The expression of the Tn antigen detected by HB-Tn1 and B1.1 was found in 17 (20%) and 19 (23%) of the 83 invasive carcinomas, respectively, but was not found in the 36 normal squamous epithelia, 22 severe dysplasias, or 24 carcinomas in situ. The sialyl-Tn antigen was detected by HB-STn1 and TKH-2 in 14 (64%) and 11 (50%) of the 22 severe dysplasias, 13 (54%) and 10 (42%) of the 24 carcinomas in situ and 48 (58%) and 42 (51%) of the 83 invasive carcinomas, respectively, but was completely absent in 36 normal squamous epithelia. Coexpression of the sialyl-Tn antigen was observed in 89% of the cases expressing the Tn antigen. No significant difference was observed between the immunoreactivities of the antigens in the metastatic lymph nodes and primary tumors. No correlation was found between the expression of each antigen and clinical state, histologic type, depth of invasion, parametrial spread, lymphatic and vessel permeation, lymph node metastasis, or 5 year survival rate. The expression of Tn and sialyl-Tn demonstrates a specific change in the neoplastic progression from carcinoma in situ to invasive carcinoma and from normal to dysplasia, respectively, in squamous cell neoplastic lesions of the cervix. Tn and sialyl-Tn antigens may be useful markers for biologic investigation of neoplastic transformation in cervical squamous cell carcinoma. PMID- 8616878 TI - [Tuberculosis semper viva]. AB - At the end of the twentieth century tuberculosis still has the attributes of a multiple, difficult to diagnose, and frequently treacherous disease which is difficult to treat. Due to its high prevalence in the developing countries and due to new medical and sociological elements associated with tuberculosis in economically developed countries with a low prevalence, tuberculosis still remains a paradoxical memento of medical science and practice. We know its etiological agent, treatment and principles of its control and despite this we must assume that it will persist in the human population for at least the period of one human life. PMID- 8616879 TI - [The conditions, indications and contraindications of treatment of diabetes using subcutaneous infusion of insulin]. AB - Adequate treatment of insulin dependent diabetes with insulin, properly implemented and accurately indicated, ensures good control which guarantees a favourable effect on the clinical course of diabetes. The main advantage of treatment is to maintain a stable insulinaemia during the interprandial period and during the night, during the postprandial period its necessary rise to ensure glycogen formation in the liver and peripheral glucose utilization. In some diabetics for certain reasons the usual injection treatment fails and it is thus necessary to know all essential conditions of non-conventional treatment continuous insulin infusions. In the submitted paper the authors emphasize the objective and purpose of insulin treatment by a continuous infusion. Attention is drawn to the most frequent therapeutic errors, to conditions which call for caution, contraindications are defined. PMID- 8616880 TI - [Maternal hyperphenylalaninemia in a population of healty Czech women. 18 years' experience with mass screening, diet therapy and metabolic monitoring]. AB - BACKGROUND: Elevated phenylalanine levels in maternal blood (hyperphenylalaninaemia) during pregnancy damages the developing foetal tissues. Early detection of pregnant women with hyperphenylalaninaemia and adherence to a low phenylalanine diet already before conception and throughout pregnancy can prevent this damage. The objective of the investigation are results achieved screening and strict monitoring of low phenylalanine dietetic treatment in detected pregnant women of the Prague population. METHODS AND RESULTS: 186 350 healthy women of the Prague population were examined by the chromatographic screening test in a venous blood sample during their first visit in a maternity welfare centre and 22 positive cases were detected (incidence 1:8470). In 86% mild, persistent or benign forms of phenylketonuria were involved. Nineteen patients were treated by a low phenylalanine diet and the phenylalanine tolerance was monitored as well as the nitrogen balance, amino acids in serum and urine, protein markers, trace elements, vitamins, lipids, the body mass index-BMI, changes of body weight after introduction of the dietetic treatment and treatment during pregnancy. A significant increase of the phenylalanine tolerance by 20 to 200% was found, mostly in the second half of pregnancy and reduced values of serum and urinary selenium. The decrease of body weight when the diet was introduced and the increment during pregnancy correlated with the BMI value. In the other investigated parameters no significant deviations were found. CONCLUSION: Fifteen healthy children with normal psychomotor development delivered by 12 mothers with hyperphenylalaninaemia provide evidence of the effectiveness of prenatal screening for hyperphenylalaninaemia during pregnancy. PMID- 8616881 TI - [The molecular basis and genetics of diseases associated with disorders of heme biosynthesis]. AB - Haem is a constituent of various haemoproteins which are essential for the function of all living cells. The haem biosynthetic pathway is now well understood and the molecular biology of its function and dysfunction in sideroblastic anemias and porphyrias is currently intensively investigated. Each type of these disorders is the result of a specific reduction in the activity of one of the enzymes of the heme biosynthetic pathway. In this paper a review of the recent progress using molecular probes in the study of sideroblastic anemias and porphyrias is presented. Until now different mutations in genes for haem biosynthesis pathway have been described and other mutations will be identified in the near future and this will provide new tools for the diagnosis of mutated genes carriers. This is particularly important in porphyrias with acute manifestations (acute intermittent porphyria, coproporphyria and variegate porphyria) since the prevention of acute attacks rests on the detection of asymptomatic carriers among members of affected families. PMID- 8616882 TI - [Growth in children with the exudative enteropathy syndrome due to a congenital heart defect--cor triatriatum dextrum]. AB - Retarded growth in a child can be the sign of serious chronic disease. The authors present an account of a six-year-old boy where growth retardation persisted at least from the age of three. During this period his height dropped from the zone between the 25th and 50th percentile into the zone between the 3rd and 10th percentile. From the clinical point of view a large abdomen, loose stools and hypocalcaemia with tetany were striking, as they were moreover refractory to vitamin D2, calcitriol and calcium administration by the oral route. The authors revealed severe hypoproteinaemia, a 150 times increased value of alpha-1-antitrypsin in faeces, and exudative enteropathy syndrome was diagnosed. The cause was venous congestion due to a rare heart disease--cor triatriatum dextrum. The septum in the right atrium was resected. Immediately after surgery the consistency and frequency of stool decreased. Calcaemia and plasma protein levels reached normal levels within two months. A growth spurt of 11 cm/year followed. Fifteen months after operation the patient's height reached almost the 50th percentile. PMID- 8616883 TI - Insulin-dependent diabetes mellitus. PMID- 8616884 TI - Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. PMID- 8616885 TI - Systemic lupus erythematosus. PMID- 8616886 TI - Rheumatoid arthritis. PMID- 8616887 TI - Genetic analysis of autoimmune disease. PMID- 8616888 TI - Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. AB - Using a novel strategy that enables the isolation of previously unknown genes encoding selectable recessive phenotypes, we identified a gene (tsg101) whose homozygous functional disruption produces cell transformation. Antisense RNA from a transactivated promoter introduced randomly into transcribed genes throughout the genome of mouse 3T3 fibroblasts was used to knock out alleles of chromosomal genes adjacent to promoter inserts, generating clones that grew in 0.5% agar and formed metastatic tumors in nude mice. Removal of the transactivator restored normal growth. The protein encoded by tsg101 cDNA encodes a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated previously in tumorigenesis. Overexpression of tsg101 antisense transcripts in naive 3T3 cells resulted in cell transformation and increased stathmin-specific mRNA. PMID- 8616889 TI - Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members. AB - How growth factors influence the fate of multipotent progenitor cells is not well understood. Most hematopoietic growth factors act selectively as survival factors, rather than instructively as lineage determination signals. In the neural crest, neuregulin instructively promotes gliogenesis, but how alternative fates are determined is unclear. We demonstrate that bone morphogenic protein 2 (BMP2) induces the basic-helix-loop-helix protein MASH1 and neurogenesis in neural crest stem cells. In vivo, MASH1+ cells are located near sites of BMP2 mRNA expression. Some smooth muscle differentiation is also observed in BMP2. A related factor, transforming growth factor beta1 (TGFbeta1), exclusively promotes smooth muscle differentiation. Like neuregulin, BMP2 and TGFbeta1 act instructively rather than selectively. The neural crest and hematopoietic systems may therefore utilize growth factors in different ways to generate cellular diversity. PMID- 8616890 TI - Nerve growth factor is an autocrine survival factor for memory B lymphocytes. AB - Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD "virgin" B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes. PMID- 8616891 TI - Direct and long-range action of a DPP morphogen gradient. AB - During development of the Drosophila wing, the decapentaplegic (dpp) gene is expressed in a stripe of cells along the anteroposterior compartment boundary and gives rise to a secreted protein that exerts a long-range organizing influence on both compartments. Using clones of cells that express DPP, or in which DPP receptor activity has been constitutively activated or abolished, we show that DPP acts directly and at long range on responding cells, rather than by proxy through the short-range induction of other signaling molecules. Further, we show that two genes, optomotor-blind and spalt are transcriptionally activated at different distances from DPP-secreting cells and provide evidence that these genes respond to different threshold concentrations of DPP protein. We propose that DPP acts as a gradient morphogen during wing development. PMID- 8616892 TI - The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process. AB - During the cotranslational integration of a nascent protein into the endoplasmic reticulum membrane, the transmembrane (TM) sequence moves out of an aqueous pore formed by Sec61alpha, TRAM, and other proteins and into the nonpolar lipid bilayer. Photocross-linking reveals that this movement involves the sequential passage of the TM domain through three different proteinaceous environments: one adjacent to Sec61alpha and TRAM and two adjacent to TRAM that place different restrictions on TM domain movement. In addition, the TM sequence is not allowed to diffuse into the bilayer from the final TRAM-proximal site until translation terminates. Cotranslational integration is therefore linked to translation and occurs via an ordered multistep pathway at an endoplasmic reticulum site that is multilayered both structurally and functionally. PMID- 8616893 TI - Biogenesis of polytopic membrane proteins: membrane segments assemble within translocation channels prior to membrane integration. AB - The initial steps in the biogenesis of membrane proteins parallel that of secretory proteins. The translocation of membrane proteins, however, must be interrupted prior to the complete traversal of the membrane. This is followed by their folding and integrating into the lipid bilayer. We have previously shown that as each latent transmembrane segment (TMS) in a polytopic membrane protein emerges from the ribosome, it sequentially translocates across the membrane. Here we demonstrate that these translocated TMSs can be extracted from the membrane with urea. This suggests that nascent TMSs do not integrate into the bilayer as they achieve a transmembrane topography. The integration is delayed until after the protein is synthesized and released from the ribosome. Prior to insertion into the bilayer, these TMSs appear to be stabilized by salt-sensitive electrostatic bonds within an aqueous-accessible compartment. PMID- 8616894 TI - Modulation of Neisseria porin (PorB) by cytosolic ATP/GTP of target cells: parallels between pathogen accommodation and mitochondrial endosymbiosis. AB - PorB of the pathogenic Neisseria species belongs to the large family of pore forming proteins (porins) produced by gram-negative bacteria. PorB is exceptional in that it is capable of translocating vectorially into membranes of infected target cells and functions in the infection process. Here we report on an unexpected similarity between Neisserial PorB and mitochondrial porins. Both porin classes interact with purine nucleoside triphosphates, which down-regulate pore size and cause a shift in voltage dependence and ion selectivity. Patch clamp analyses indicate that PorB channel activity is tightly regulated in intact epithelial cells. In light of recent findings on the pivotal role of PorB in virulence and the prevention of phagosome lysosome fusion, these data provide important mechanistic clues on the intracellular pathogen accommodation reminiscent of mitochondrial endosymbiosis. PMID- 8616895 TI - A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. AB - Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus. PMID- 8616896 TI - Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis. AB - Mutations resulting in premature termination codons reduce the corresponding mRNA levels. We describe a cell-free system in which depletion of the mutant immunoglobulin kappa mRNA pool correlates with inefficient splicing and not with RNA decay. Splicing deficiency does not depend on the sequence surrounding the in frame nonsense codon and can be partially corrected by mutating the methionine initiation codon. Despite the apparent link between translation and low mutant mRNA levels, inefficient splicing is not dependent on protein synthesis. Abnormal splicing of mutant immunoglobulin RNA is observed with B-cell but not with HeLa or T-cell extracts. A nonsense mutant beta-globin RNA is normally spliced by B cell extract. We propose that the phenomenon exhibits tissue and gene specificity. PMID- 8616897 TI - Evidence for a new step in telomere maintenance. AB - The strand of telomeric DNA that runs 5'-3' toward a chromosome end is typically G rich. Telomerase-generated G tails are expected at one end of individual DNA molecules. Saccharomyces telomeres acquire TG1-3 tails late in S phase. Moreover, the telomeres of linear plasmids can interact when the TG1-3 tails are present. Molecules that mimic the structures predicted for telomere replication intermediates were generated in vitro. These in vitro generated molecules formed telomere-telomere interactions similar to those on molecules isolated from yeast, but only if both ends that interacted had a TG1-3 tail. Moreover, TG1-3 tails were generated in vivo in cells lacking telomerase. These data suggest a new step in telomere maintenance, cell cycle-regulated degradation of the C1-3A strand, which can generate a potential substrate for telomerase and telomere-binding proteins at every telomere. PMID- 8616898 TI - Three-site synapsis during Mu DNA transposition: a critical intermediate preceding engagement of the active site. AB - The chemical steps of bacteriophage Mu DNA transposition take place within a higher order nucleoprotein structure. We describe a novel intermediate that precedes the previously characterized transpososomes and directly demonstrates the interaction of a distant enhancer element with recombination regions. The transpositional enhancer interacts with the Mu left and right ends to form a three-site synaptic (LER) complex. Under normal reaction conditions, the LER complex is rapidly converted into the more stable Mu transpososomes. However, mutation of the Mu terminal nucleotides results in accumulation of the LER and a failure to form the type 0 transpososome. During the transition from LER to type 0, the Mu DNA termini and the active site of the transposase engage in a catalytically competent conformation. PMID- 8616899 TI - Positional information within the Mu transposase tetramer: catalytic contributions of individual monomers. AB - The strand cleavage and strand transfer reactions of Mu DNA transposition require structural/catalytic contributions from separate polypeptide domains of individual transposase (MuA) monomers within the functional MuA tetramer. Based on catalytic complementation between two inactive MuA variants, we have derived certain rules by which the physical location of a MuA monomer within the transposition complex specifies its role in DNA breakage and transfer. During strand transfer, MuA monomers contributing domain II to the reaction occupy R1 (the subsite proximal to the strand-transferred nucleotide), while those contributing domain IIIalpha occupy R2. The positions of the monomers contributing these two domains appear to be reversed during DNA cleavage. PMID- 8616900 TI - Pharmacokinetics and metabolism of O-(chloroacetyl-carbamoyl) fumagillol (TNP 470, AGM-1470) in rhesus monkeys. AB - The metabolic disposition and pharmacokinetics of TNP-470 were investigated in rhesus monkeys following intravenous administration of 5 mg/kg of [3H]-TNP-470. Rapid and extensive metabolism of parent drug to six metabolites occurred as demonstrated by the absence of unchanged drug in plasma and urine at time points as early as 6 min after administration. Substantial, yet variable, plasma levels of M-IV were detected in all three monkeys with a mean Cmax value of 3.54 microM. Five other metabolites, labeled M-I, M-II, M-III, M-V and M-VI, were also detected in biological fluids of monkeys. M-II, M-V and M-VI exhibited similar kinetic profiles with apparent plasma elimination half-life values of 0.91 +/- 0.37, 2.42 +/- 0.13 and 1.19 +/- 0.29 h respectively. In contrast, M-I, M-III and M-IV exhibited much shorter apparent plasma half-life values of 30 min or less. Urinary recovery within 36 h represented only 19.90 +/- 6.09% of the total administered dose. No radioactivity was detected beyond 36 h and during a 15-day sample collection period, suggesting that nonrenal (biliary) elimination of TNP 470 metabolites is a predominant excretion route in nonhuman primates. This study provides the first detailed in vivo analysis of TNP-470 metabolism and disposition using an animal model highly predictive of humans, consistent with the detection of the same TNP-470 metabolites in human tissues. A detailed understanding of TNP-470 metabolism and disposition is critical to fully elucidate the pharmacodynamic properties of this new anticancer drug as clinical investigations proceed. PMID- 8616901 TI - Efficacy of lonidamine combined with different DNA-damaging agents in the treatment of the MX-1 tumor xenograft. AB - Lonidamine is an antitumor agent with a peculiar mechanism of action, since it differentially impairs the energy metabolism of normal and neoplastic cells. We investigated the effects of lonidamine on the activity of DNA-damaging antitumor agents against the MX-1 human breast carcinoma xenograft. Athymic mice bearing measurable s.c. tumors were treated by a single injection of doxorubicin (i.v.), cyclophosphamide (i.v.), or cisplatin (i.p.) followed by repeated daily injections of lonidamine (i.p. or p.o.). A potentiation of the activity of all these DNA-damaging drugs was achieved when each was given in combination with lonidamine, but for doxorubicin and cyclophosphamide the increase in antitumor activity paralleled the increase in lethal toxicity. In contrast, a therapeutic advantage of the combination was achieved for cisplatin and lonidamine as compared with cisplatin alone. Indeed, 6 mg/kg of cisplatin plus lonidamine cured all tumors, whereas the maximum tolerated dose of cisplatin alone (12 mg/kg) cured only six of eight tumors. In addition, the study indicated that the duration of lonidamine administration after injection of the cytotoxic drug influenced the tumor response and that prolonged treatment resulted in greater efficacy. These results document the ability of lonidamine to modulate the pharmacological activity of DNA-damaging drugs, thus suggesting that lonidamine may be a clinically useful cisplatin modulator. PMID- 8616902 TI - Pharmacokinetics and toxicity of oral and intravenous lonidamine in dogs. AB - Plasma lonidamine concentration and toxicity were investigated in dogs receiving 100, 200, 400, 800, 1200 mg/m2 orally twice daily for 30 days and in dogs receiving single intravenous doses of 200, 400, 800, 1200 mg/m2. Physical or laboratory signs of toxicity were not observed in dogs receiving oral lonidamine, but severe vomiting and signs of acute hepatic and pancreatic toxicity were observed in dogs receiving intravenous doses that exceeded 400 mg/m2. The area under the lonidamine concentration versus time curve (AUC) in dogs receiving 200, 400, and 800 mg/m2 of lonidamine intravenously was a 1.8-, 3.3-, and 8.7-fold higher than in dogs receiving oral lonidamine. This suggests that the bioavailability of oral lonidamine may be limited. However, centrilobular hepatocellular swelling and vacuolation were observed in dogs receiving oral lonidamine. Serum alanine aminotransferase (ALT) activity was increased in dogs receiving intra-venous lonidamine. These findings suggest that lonidamine is hepatotoxic in dogs. However, serum ALT was increased in only 1/4 dogs receiving 400 mg/m2 of lonidamine intravenously and plasma concentration were within the range capable of sensitizing hyperthermia and chemotherapy. Therefore, this dose and route appears to be a viable and controllable method for prospective quantification of lonidamine interaction with systemic chemotherapy and/or hyperthermia. PMID- 8616904 TI - Effect of the exposure dose of etoposide on the cell growth and cell kinetics of human ovarian cancer cells. AB - Although schedule-dependent cytotoxicity of etoposide has been reported, its mechanisms have not been elucidated fully. In this study, we attempted to clarify what concentration, time or exposure dose (ED, concentration of drug x time) of etoposide result in the antitumor effect on human ovarian cancer cells from the standpoint of cell cycle perturbation. The different ED were produced by different drug treatment schedules: 10 microgram/ml x 4 h (ED 40), 1.66 microgram/ml x 24 h (ED 40), 5 microgram/ml x 4 h (ED 20), 0.83 microgram/ml x 24 h (ED 20), 10 microgram/ml x 0.8 h (ED 8), 5 microgram/ml x 1.6 h (ED 8), 2 microgram/ml x 4 h (ED 8), 0.33 microgram/ml x 24 h (ED 8). Cell cycle perturbation on day 5 and the suppression of cell growth were dependent on the drug concentration at the lowest exposure dose (ED 8), but were dependent on ED at the higher EDs (20 or 40). The percentage of cells in the G2/M fraction significantly decreased from day 5 to day 7 in BG-1 cells treated at ED 20 or treated with higher concentrations (10 and 5 microgram/ml) at ED 8, but not in those treated at ED 40 or treated with lower concentrations (2 and 0.33 microgram/ml) at ED 8. Therefore, the cytotoxic mechanism of etoposide may not be explained by simple schedule dependency. PMID- 8616903 TI - A convenient tubulin-based quantitative assay for paclitaxel (Taxol) derivatives more effective in inducing assembly than the parent compound. AB - A room temperature biochemical assay, based on centrifugal removal of tubulin polymer, was developed to permit ready detection of paclitaxel analogs more active than the parent compound and to permit reliable quantification of differences in activity relative to paclitaxel in terms of drug concentration. The assay was validated by comparing paclitaxel to two compounds (docetaxel and 2 debenzoyl-2-meta-azidobenzoylpaclitaxel) known to be more active under multiple reaction conditions. The assay was designed to yield a relatively high EC50 (23 microM) for paclitaxel. This was possible because paclitaxel only weakly induced tubulin assembly at room temperature in 0.4 M glutamate without exogenous GTP. Under these same reaction conditions 50% assembly occurred with 4.7 microM 2 debenzoyl-2-meta-azidobenzoylpaclitaxel and 11 microM docetaxel. These biochemical EC50 values were in agreement with the relative cytotoxicity of the three compounds for human Burkitt lymphoma CA46 cells (IC50 values for paclitaxel, docetaxel, and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel were 40, 10, and 3 nM, respectively). PMID- 8616905 TI - Intrasubject variation in children of ifosfamide pharmacokinetics and metabolism during repeated administration. AB - The aim of this study was to investigate intrasubject variability in ifosfamide (IFO) pharmacokinetics and metabolism which may influence clinical effect, since the pharmacology of this drug is dependent on metabolism. A group of 11 patients (ages 1-16 years) were studied on at least two occasions. IFO, 9 gm-2 was administered as a continuous infusion over 72 h. Plasma and urine samples were collected and concentrations of IFO and its metabolites were determined. Comparisons were made between courses in the same subject, allowing for differences in age and prior IFO treatment. There was a wide variation in drug (twofold) and metabolite (up to tenfold) AUCs between courses in the same patient. Although some patients did show an increase in clearance between courses (up to threefold), there was no significant consistent change in overall pharmacokinetics among the different courses studied in the same patient. There was a significant decrease (up to 63%) in the AUC of the inactive metabolite 3 dechloroethylifosfamide (3-DCI) in later courses compared with the first course studied (P = 0.032, paired t-test). This was matched by an increase in the AUC of the total dechloroethylated metabolites with course (P = 0.015, paired t-test). None of the other metabolites measured showed any consistent change in plasma or urine levels between courses. Overall, the AUC of parent drug correlated with age (r2 = 0.86, P = 0.011), and postinfusion half-life correlated with plasma bilirubin (r2 = 0.89, P = 0.007). This study demonstrated large and seemingly unpredictable intrasubject variability in IFO pharmacokinetics and metabolism during repeated administrations. Investigations relating the clinical effects of IFO to pharmacokinetics and metabolism must take this variation into account. PMID- 8616906 TI - Biotransformation of the platinum drug JM216 following oral administration to cancer patients. AB - This study evaluates the metabolic profile of JM216 [bis(acetato)ammine dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n = 2-4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m2). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt = 5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients' plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis amminedichloro-(cyclohexylamine)platinum(II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis acetatoammine-chloro(cyclohexylalamine)hydroxoplatin um(IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients' plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzymatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(Il) metabolites in plasma that differ significantly from other systemically applied platinum drugs. PMID- 8616907 TI - Analysis of HMG protein binding to DNA modified with the anticancer drug cisplatin. AB - Cisplatin (CDDP) is an effective and widely used cancer chemotherapy drug. High mobility group (HMG) proteins 1 and 2 have been shown to bind with high affinity to CDDP-DNA. In this study we analyzed the interaction of HMG proteins with CDDP DNA. We demonstrate that after binding, HMG proteins can be removed from CDDP-DNA leaving the Pt adducts intact and capable of rebinding HMG proteins. Furthermore, the very HMG proteins that have been removed remain functionally viable and capable of rebinding CDDP-DNA. We also investigated the role that Cys residues play in protein binding. Replacement of Cys 45 or Cys 106 with a Ser residue reduced HMG2 protein binding to CDDP-DNA. These results indicate that Cys residues play a critical role in the high affinity binding of this protein to CDDP-DNA. From these findings, we speculate that the intracellular oxidative environment could affect the redox state of protein thiols in HMG1 and HMG2 and in addition, regulate the ability of these proteins to recognize cis-Pt-DNA adduct formation in tumor cells. PMID- 8616908 TI - Comparison of several antiangiogenic regimens alone and with cytotoxic therapies in the Lewis lung carcinoma. AB - The efficacy of several potential antiangiogenic agents, TNP-470, minocycline, suramin, genistein, interferon delta 4, 14(sulfated)-beta-cyclodextrin and tetrahydrocortisol, alone and in combination with cytotoxic therapies was examined against primary and metastatic Lewis lung carcinoma. The antiangiogenic agents when administered as single agents or in two-agent combinations were only modestly active as antitumor agents. Three antiangiogenic agent combinations, TNP 470/minocycline, TNP-470/14(SO4)beta-CD/THC and minocycline/14(SO4)beta-CD/THC, produced significant increases in tumor growth delay and decreases in the number of lung metastases when administered along with cyclophosphamide compared with cyclophosphamide alone. Two antiangiogenic agent combinations, minocycline/interferon delta 4 and minocycline/14(SO4)beta-CD/THC, produced significant decreases in the number of lung metastases when administered alone with adriamycin compared with adriamycin alone. The antiangiogenic combinations of TNP-470/minocycline, TNP-470/suramin, TNP-470/genistein, TNP-470/interferon delta 4 and TNP-470/l4(SO4)beta-CD/THC, resulted in increased tumor growth delays when administered along with CDDP, BCNU, fractionated radiation or 5 fluorouracil. There was not always a direct correlation between the antiangiogenic regimen that was most beneficial against the primary tumor as compared with disease metastatic to the lungs. These studies establish that a broad range of antiangilogenic therapies can interact in a positive manner with cytotoxic therapies. PMID- 8616909 TI - Lack of in vivo crossresistance with gemcitabine against drug-resistant murine P388 leukemias. AB - Gemcitabine, a novel pyrimidine nucleoside antimetabolite, has shown clinical antitumor activity against several tumors (breast, small-cell and non-small-cell lung, bladder, pancreatic, and ovarian). We have developed a drug-resistance profile for gemcitabine using eight drug-resistant P388 leukemias in order to identify potentially useful guides for patient selection for further clinical trials of gemcitabine and possible noncrossresistant drug combinations with gemcitabine. Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin or etoposide) exhibited no crossresistance to gemcitabine. Leukemias resistant to vincristine (not multidrug resistant), cyclophosphamide, melphalan, cisplatin, and methotrexate were also not crossresistant to gemcitabine. Only the leukemia resistant to 1-beta-D-arabinofuranosylcytosine was crossresistant to gemcitabine. The results suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-beta-D arabinofuranosylcytosine and (2) the lack of crossresistance seen with gemcitabine may contribute to therapeutic synergism when gemcitabine is combined with other agents. PMID- 8616910 TI - Kinetic parameters for reversal of the multidrug pump as measured for drug accumulation and cell killing. AB - We determined the kinetic parameters that describe the effect of 20 different modulators of the multidrug resistance pump on the reversal of cytotoxin accumulation in a resistant strain of P388 leukemia cells (P388/ADR), and on the reversal of cell killing for these cells. When measured by a direct comparison of the amplitude of the pertinent protocol (accumulation or cell killing), the Ki for reversal of accumulation was generally some four or five times larger than that for reduction of cytotoxicity. We showed that this was only an apparent discrepancy, since a full theoretical analysis of the two protocols allowed the intrinsic Ki to be obtained for the two procedures and these computed Ki values were then almost identical. We found that for six of the modulators studied (namely, cyclosporin A, quinidine, dipyridamole, propafenone, mefloquine, tamoxifen) the extent of pump reversal should be better than 90% at tolerated plasma levels culled from the literature. PMID- 8616911 TI - Treatment of metastatic malignant melanoma with cisplatin plus tamoxifen. AB - A phase II study was performed to assess the efficacy and toxicity of the combination of cisplatin (CDDP) and tamoxifen (TAM) in patients with metastatic malignant melanoma (MM). A total of 31 consecutive previously untreated patients with unresectable measurable MM were given 100 mg/m2 CDDP every 21 days and 60 mg TAM every 12 h daily. All courses were given on an outpatient basis. A total of 119 courses of treatment were given. In all, 5 of the 31 patients (16%) had an objective response (95% confidence interval 5.3-34%) and 2 (6%) achieved a clinical complete response. The median duration of response was 7 months. The main side effect was gastrointestinal: 13% of the patients experienced grade 3/4 nausea/vomiting. Hematological or neurological toxicities were mild and rare. In conclusion, the combination CDDP-TAM has limited activity in MM, although its toxicity is tolerable. Our results do not allow us to recommend its use for the treatment of MM. PMID- 8616912 TI - 5-methyltetrahydrofolate or 5-formyltetrahydrofolic acid to modulate 5 fluorouracil's cytotoxic activity in vivo. A phase II study in patients with advanced colon cancer. AB - The purpose of this study was to test the hypothesis that 5 methyltetrahydrofolate (Me-THF), a source of reduced folates alternative to leucovorin, could effectively modulate 5-fluorouracil's (5-FU) cytotoxic activity in patients with advanced colon cancer. A total of 23 patients were enrolled in a phase 11 trial; they received 5-FU as a 30-min infusion at a dose of 370 mg/m2 following a rapid i.v. push of 200 mg/m2 Me-THF, both drugs being given for 5 consecutive days. Cycles were repeated every 4 weeks until disease progression. No patient achieved a complete response. In all, 4 patients showed a partial response (17.4%), 7 developed stable disease (30.4%), and the remaining 12 (52.2%) progressed. Toxicity was acceptable and never exceeded WHO grade III intensity. According to our experience, the MeTHF/5-FU combination does not appear to be an effective treatment for advanced colon cancer. Despite its low toxic profile, in our opinion its wider use should be discouraged. PMID- 8616913 TI - Comparison of the uptake of retinoids 13-cis-retinoic acid and Ro 13-6298 delivered to HL-60 cells by serum albumin or low-density lipoprotein. AB - Retinoids, a class of polyisoprenoids including retinol and retinoic acid, regulate and control diverse physiological functions via their cell differentiating and morphogenic potential. In the present study we showed that the extracellular concentration of retinoid-binding proteins such as albumin limits the amount of retinoid entering the human promyelocytic leukemia cell line HL-60. These cells accumulate 5 -10 times more retinoid when delivered free in solution than when bound to either albumin or low-density lipoprotein (LDL). Moreover. the effect of protein binding is concentration-dependent, with a higher concentration of binding protein corresponding to a lower level of cellular uptake. Furthermore, the uptake of the ester derivative is higher than that of the acidic retinoid. These observations suggest that (a) the cellular uptake of both retinoids occurs via the free form of the ligand in solution, with the free concentration of ligand decreasing as the carrier-protein concentration increases, and (b) according to a passive mechanism, the ester derivative, unionized and lipophilic, enters the cells more easily than does the acidic derivative. PMID- 8616914 TI - Building shelters. Safeguards in public disclosure of outcomes data. PMID- 8616915 TI - Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene. AB - BACKGROUND: Prostacyclin is an important vasoprotective molecule. It inhibits platelet aggregation, monocyte interaction with endothelium, and smooth muscle cell lipid accumulation. Vascular cyclooxygenase-1 (COX-1) is the rate-limiting step in prostacyclin synthesis. The objective of this study was to determine whether adenovirus-mediated transfer of COX-1 could restore COX-1 activity, augment prostacyclin synthesis, and prevent thrombus formation in a porcine carotid angioplasty model. METHODS AND RESULTS: Human COX-1 cDNA driven by a cytomegalovirus promoter was constructed into a replication-defective adenovirus 5 vector by homologous recombination. Recombinant adenovirus without a foreign gene (Ad-RR) and buffer were included as controls. Recombinant Ad-LacZ was used for marking the transfected cells in vivo. In the in vitro experiments, cultured human endothelial cells (ECs) and porcine arterial smooth muscle cells (SMCs) were incubated with Ad-COX-1 for 2 hours and 6-keto-PGF(1 alpha) level and the transgene expression were determined 72 hours after infection. In the in vivo experiments, recombinant adenoviruses were directly instilled into angioplasty injured porcine carotid arteries for 30 minutes. Cyclic flow changes were monitored for 10 days and thrombus formation was examined histologically thereafter. Transgene expression and prostaglandin I2 (PGI2) synthesis by the injured arteries were determined. Cultured ECs infected with Ad-COX-1 produced a fivefold to eightfold increase in PGI2, and the transgene expression in cultured porcine SMCs was demonstrated by Northern analysis. Direct administration of Ad COX-1 at a dose of 3 x 10(10) pfu completely inhibited carotid cyclic flow changes and thrombus formation accompanied by a fourfold increase in PGI2 synthesis by the injured arteries 10 days after infection, whereas Ad-COX-1 at a lower dose, 5 x 10(9) pfu, had no antithrombotic effects when compared with Ad-RR vector and buffer controls. CONCLUSIONS: Adenovirus-mediated transfer of COX-1 to angioplasty-injured carotid arteries was efficacious in augmenting PGI2 synthesis and was associated with an inhibition of thrombosis when a relatively high titer of adenovirus was instilled. PMID- 8616916 TI - Elaboration of type-1 plasminogen activator inhibitor from adipocytes. A potential pathogenetic link between obesity and cardiovascular disease. AB - BACKGROUND: Obesity is known to predispose to attenuated fibrinolysis attributable to increased concentrations in plasma of type-1 plasminogen activator inhibitor (PAI-1), the primary physiological inhibitor of endogenous fibrinolysis. PAI-1 is present in neointimal vascular smooth muscle cells and lipid-laden macrophages. METHODS AND RESULTS: The present study was designed to determine whether PAI-1 expression occurs in adipose tissue as well, thereby potentially contributing to increased cardiovascular risk associated with obesity. 3T3-L1 preadipocytes were differentiated into adipocytes by exposing them to isobutylxanthine (0.5 mmol/L) and dexamethasone (0.25 mumol/L) over 7 days and incubated for 24 hours with transforming growth factor-beta (TGF-beta), known to augment PAI-1 synthesis in several cell types and to be released from platelets when they are activated. TGF-beta increased PAI-1 activity in the conditioned media of the 3T3-L1-derived cells in a concentration-dependent fashion without significantly affecting cell proliferation. Western blotting and immunoprecipitation of 35S-labeled PAI-1 showed that the increased PAI-1 activity paralleled increased PAI-1 protein. Northern blotting showed that increased PAI-1 mRNA preceded increased accumulation of PAI-1 activity and protein in the conditioned media. Furthermore, TGF-beta (10 ng/g body wt) administered in vivo increased PAI-1 activity in mouse plasma and PAI-1 mRNA expression in mouse adipose tissue. CONCLUSIONS: Increased plasma PAI-1 activity in obese human subjects may result from PAI-1 release from an increased mass of adipose tissue, particularly in association with thrombosis and elaboration of TGF-beta from platelet alpha-granules into the circulation. The increased PAI-1 may exacerbate vascular disease by shifting the balance between thrombosis and thrombolysis toward thrombosis and consequently exposing luminal surfaces of vessels to mitogens associated with microthrombi over protracted intervals. PMID- 8616918 TI - High frequency-induced upregulation of human cardiac calcium currents. AB - BACKGROUND: In mammalian heart cells, Ca2+ influx through voltage-gated L-type Ca2+ channels can be upregulated by high rates of stimulation. We have investigated this important adaptive regulation in human cardiomyocytes. METHODS AND RESULTS: Using the whole-cell patch-clamp technique, we found a high frequency-induced upregulation (HFIUR) of the dihydropyridine-sensitive L-type Ca2+ current (ICa) in human cardiomyocytes. ICa was potentiated in a graded manner with increasing rates of stimulation between 0.3 and 5 Hz. Both moderate increase of ICa peak amplitude and marked slowing of current decay contributed to large increases of Ca2+ influx (up to 80%). The maximal potentiation of ICa was reached rapidly after the change in the rate of stimulation (no more than a few seconds). Beta-Adrenergic stimulation of the cells by isoproterenol (1 micromol/L), which is well known to induce a slow (approximately 1 minute) cAMP mediated potentiation of ICa, could enhance (when present) or promote (when absent) the HFIUR of ICa. As a consequence, the increasing effect of isoproterenol on Ca2+ influx through Ca2+ channels was dependent on the rate of stimulation. HFIUR of ICa was altered in patients with ejection fraction lower than 40% and in patients pretreated with Ca2+ antagonists or beta-blockers. CONCLUSIONS: Upregulation of Ca2+ entry through voltage-gated Ca2+ channels by high rates of beating may be involved in the frequency-dependent regulation of contractility (Bowditch "staircase") of the human heart. This process, which is highly sensitive to beta-adrenergic stimulation, may be crucial in adaptation to exercise and stress. PMID- 8616917 TI - Lysis of adult ventricular myocytes by cells infiltrating rejecting murine cardiac allografts. AB - BACKGROUND: Immunologic mechanisms that mediate myocardial cell injury during rejection are not fully understood. We therefore investigated whether cells that infiltrate rejecting cardiac allografts are capable of directly injuring myocytes and whether this injury resembles that produced by cytotoxic T lymphocytes (CTLs) that are generated in a mixed lymphocyte reaction (MLR). METHODS AND RESULTS: Heart-infiltrating cells (HICs) were isolated from murine heterotopic BALB/c cardiac allografts undergoing rejection 6 to 8 days after transplantation into C57BL/6 mice. An in vitro model system of cultured adult murine ventricular myocytes was developed to facilitate investigation of cell-mediated myocyte injury. Isolated adult myocytes were incubated with either HICs or MLR effector cells, and myocyte death was quantified by counting the number of rod-shaped myocytes excluding trypan blue. The frequency of donor-reactive CTLs was similar in the HIC and MLR populations, as assessed by limiting dilution analysis. However, HICs were less efficient at killing donor-strain myocytes than were MLR cells. CTL-mediated cell lysis occurred by 6 hours, whereas myocyte injury produced by HICs was more gradual, with considerable cytotoxicity occurring between 12 and 24 hours. Furthermore, whereas MLR cells lysed only donor-strain myocytes, HIC lysed donor, third-party, and syngeneic myocytes. Treatment of MLR cells and HICs with anti-CD8 antibody plus complement produced a much greater inhibition of MLR cytotoxicity than of HIC cytotoxicity. CONCLUSIONS: These data demonstrate that only a small component of myocyte injury mediated by allograft infiltrating cells can be ascribed to CTLs within the infiltrating cell population. These findings suggest that cell types associated with a delayed-type hypersensitivity response, as well as CTLs, cause myocyte injury during cardiac rejection. PMID- 8616919 TI - The protective dose of the potent GPIIb/IIIa antagonist SC-54701A is reduced when used in combination with aspirin and heparin in a canine model of coronary artery thrombosis. AB - BACKGROUND: Fibrinogen receptor antagonists block the fibrinogen-platelet interaction at the GPIIb/IIIa receptors and inhibit thrombus formation. SC-54701 is the active metabolite of SC-54684A, an orally fibrinogen receptor antagonist. We compared the efficacy of SC-54701A (SCa, hydrochloride salt) with that of aspirin (ASA) or heparin and with combination therapy in a canine model of continuous current injury. METHODS AND RESULTS: Sixty-six dogs were used (6 per treatment). SCa (15-minute loading dose followed by [//] infusion [microgram/kg per minute]: (0.87//0.39=1 X SCa; 0.52//0.23=0.6 X SCa; and 0.425//0.20= 0.5 X SCa), ASA (2.8 mg/kg), heparin (200 U/kg plus 1000 U/h), or saline (0.1 mL/kg) was administered intravenously. Experimental time was 180 minutes of current. Time to occlusion was increased (P < .05) by SCa (T=incidence of thrombosis) (1 X SCa, >180 minutes [T=0]; 0.6 X SCa, 158 +/- 15 minutes [T=2]; 0.5 X SCa, 130 +/- 22 minutes [T=4]), heparin (114 +/- 16 minutes [T=5]), and ASA plus heparin (130 +/- 11 minutes [T=5]) relative to saline (58 +/- 7 minutes [T=6]). Time to occlusion for the SCa treatments was increased compared with ASA (64 +/- 7 minutes [T=6]). When 0.5 X SCa was administered with ASA plus heparin, time to occlusion was >180 minutes [T=0]. SCa provided complete protection at > or = 90% inhibition of ex vivo collagen-induced platelet aggregation. Cyclic flow variations were minimal with SCa or any treatment involving 0.5 X SCa and ASA. CONCLUSIONS: SCa has dose-dependent antithrombotic efficacy and inhibits ex vivo platelet aggregation. ASA, heparin, or saline was ineffective in this model. SCa (0.5X) plus ASA and heparin maximized the antithrombotic effect of this lower dose of SCa. PMID- 8616920 TI - Ranolazine stimulates glucose oxidation in normoxic, ischemic, and reperfused ischemic rat hearts. AB - BACKGROUND: Ranolazine is a novel antianginal agent that may reduce symptoms without affecting hemodynamics and has shown cardiac antiischemic effects in in vivo and in vitro models. In one study it increased active pyruvate dehydrogenase (PDHa). Other agents that increase PDHa and so increase glucose and decrease fatty acid (FA) oxidation are beneficial in ischemic-reperfused hearts. Effects of ranolazine on glucose and palmitate oxidation and glycolysis were assessed in isolated rat hearts. METHODS AND RESULTS: Working hearts were perfused with Krebs Henseleit buffer plus 3% albumin under normoxic conditions and on reperfusion after 30-minute no-flow ischemia and under conditions designed to give either low [low (Ca) (1.25 mmol/L), high [FA] (1.2 mmol/L palmitate; with/without insulin] or high (2.5 mmol/L Ca, 0.4 mmol/L palmitate; with/without pacing) glucose oxidation rates; Langendorff-perfused hearts (high Ca, low FA) were subjected to varying degrees of low-flow ischemia. Glycolysis and glucose oxidation were measured with the use of [5-3H/U-14C]-glucose and FA oxidation with the use of [1 14C]- or [9,10-3H]-palmitate. In working hearts, 10 micromol/L ranolazine significantly increased glucose oxidation 1.5-fold to 3-fold under conditions in which the contribution of glucose to overall ATP production was low (low Ca, high FA, with insulin), high (high Ca, low Fa, with pacing), or intermediate. In some cases, reductions in FA oxidation were seen. No substantial changes in glycolysis were noted with/without ranolazine; rates were approximately 10-fold glucose oxidation rates, suggesting that pyruvate supply was not limiting. Insulin increased basal glucose oxidation and glycolysis but did not alter ranolazine responses. In normoxic Langendorff hearts (high Ca, low FA; 15 mL/min), all basal rates were lower compared with working hearts, but 10 micromol/L ranolazine similarly increased glucose oxidation; ranolazine also significantly increased it during flow reduction to 7, 3, and 0.5 mL/min. Ranolazine did not affect baseline contractile or hemodynamic parameters or O2 use. In reperfused ischemic working hearts, ranolazine significantly improved functional outcome, which was associated with significant increases in glucose oxidation, a reversal of the increased FA oxidation seen in control reperfusions (versus preischemic), and a smaller but significant increase in glycolysis. CONCLUSIONS: Beneficial effects of ranolazine in cardiac ischemia/reperfusion may be due, at least in part, to a stimulation of glucose oxidation and a reduction in FA oxidation, allowing improved ATP/O2 and reduction in the buildup of H+, lactate, and harmful fatty acyl intermediates. PMID- 8616921 TI - Transmural channels can protect ischemic tissue. Assessment of long-term myocardial response to laser- and needle-made channels. AB - BACKGROUND: We previously found that transmural laser channels failed to acutely increase myocardial blood flow. Nevertheless, this method is being used to treat patients with coronary artery disease who are unable to undergo angioplasty or bypass graft surgery and in cases in which previous surgery has failed. To reconcile the lack of an acute increase in blood flow with beneficial effects claimed in patients, our hypothesis was that the channel-making process might, over time, stimulate a protective effect, possibly by the growth of new vessels linking channels to the existing circulation. We tested this hypothesis in rat hearts, which have little native collateral circulation. METHODS AND RESULTS: We made six transmural channels in the left ventricle of each heart using a 400 micrometer-diameter optic fiber coupled to a holmium:yttrium-aluminum-garnet laser or a 400-micrometer-diameter syringe needle. Two months after the channels were made, rats were randomized to either an infarct-size study or analysis of myocardial capillary density. We challenged any induced protective mechanism by acutely occluding the left coronary artery for 90 minutes, followed by 4.5 hours of reperfusion. The artery was then reoccluded, and pigment was injected into the circulation to delineate tissue perfused by the occluded vessel and to detect perfusion via the channels. We used triphenyltetrazolium staining to determine the amount of muscle necrosis and the location of muscle protection. Infarct size in needle-treated hearts was smaller than in controls (15 +/- 6% versus 40 +/- 3% of the left ventricle, P<.01). Infarct size in laser-treated hearts (27 +/- 5%) did not differ significantly from controls; however, all eight laser-treated hearts showed evidence of muscle protection in areas adjacent to channels. We found that the laser-made channels were associated with more fibrosis than the needle-made channels (mean width of fibrosis 430 +/- 50 versus 180 +/- 30 micrometer, P<.0001), and, in tissue remote from channels, fibrosis was increased more in laser-treated hearts (3.6 +/- 0.3%) versus both control (2.5 +/- 0.2%) and needle-treated (2.5 +/- 0.3%) hearts (P<.05). In addition, muscle disarray was seen adjacent to channel-associated fibrosis. We observed injected pigment within fibrosis associated with the channels and in surrounding myocardium. We also found vessels that appeared to be connected to channels; however, there was no overall increase in capillary density. CONCLUSIONS: We were able to protect the heart against coronary artery occlusion by making transmural channels 2 months before occlusion. Channels created by a needle provided greater protection than channels created by a laser, probably because they caused less initial injury. Our results are consistent with the concept that the channels were able to provide blood flow to the tissue directly from the ventricular cavity; however, we cannot rule out the possibility that other mechanisms of protection may be involved. PMID- 8616922 TI - Comparison of sustained antithrombotic effects of inhibitors of thrombin and factor Xa in experimental thrombosis. AB - BACKGROUND: In the pathogenesis of (recurrent) thrombosis, clot-associated thrombin appears to play an important role. Antithrombin III-independent thrombin inhibitors have been shown to neutralize clot-bound thrombin effectively. We compared the sustained antithrombotic effects and the effects on endogenous fibrinolysis of several of these agents with recombinant tick anticoagulant peptide (rTAP), a selective factor Xa inhibitor, and low-molecular-weight heparin (LMWH) in an experimental venous thrombosis model. METHODS AND RESULTS: Rabbits received either recombinant hirudin (rHir), Hirulog-1, CVS#995 (a novel direct inhibitor of thrombin), rTAP, LMWH, or saline. The effect on thrombus growth was assessed by measuring the accretion of 125I-labeled fibrinogen onto preformed nonradioactive thrombi, and the effect on endogenous fibrinolysis was assessed by measuring the decline in radioactivity of preformed 125I-labeled thrombi in rabbit jugular veins. All direct thrombin inhibitors induced a sustained antithrombotic effect compared with either LMWH and rTAP. In addition, CVS#995 also further decreased thrombus size after stopping its infusion, which was due to a significant enhancement of endogenous fibrinolysis. CONCLUSIONS: Direct thrombin inhibition by rHir, Hirulog-1, or CVS #995 induces a sustained antithrombotic effect compared with rTAP and LMWH, which is most likely due to inhibition of clot-bound thrombin. CVS#995 was shown to also enhance the extent of endogenous fibrinolysis to a greater degree compared with rHir and might therefore be an interesting new antithrombotic agent for the treatment of venous and arterial thrombosis. PMID- 8616923 TI - Diabetes exacerbates inflammatory responses to ischemia-reperfusion. AB - BACKGROUND: Diabetes is associated with an increased incidence of ischemic organ damage. The objectives of present study were to compare the leukocyte-endothelial cell adhesive interactions and albumin leakage response of mesenteric venules to ischemia-reperfusion between control rats, rats with streptozotocin-induced diabetes, and rats with hyperglycemia induced by glucose infusion and to define the molecular determinants of the leukocyte accumulation elicited by ischemia reperfusion in diabetic rats. METHODS AND RESULTS: Under baseline conditions, lower venular shear rates and an increased number of rolling leukocytes were noted in diabetic rats, whereas the number of adherent and emigrated leukocytes did not differ from that in control rats. Spontaneous albumin leakage from mesenteric venules was markedly increased in diabetic rats but not in hyperglycemic nondiabetic rats. Ischemia-reperfusion elicited significantly larger increases in leukocyte adhesion and emigration and albumin leakage in diabetic rats. Acute elevation of glucose levels did not modify the microvascular responses to ischemia-reperfusion compared with control rats. Antibodies directed against CD11/CD18, intercellular adhesion molecule-1 (ICAM-1), or P-selectin but not L-selectin significantly decreased the number of adherent and emigrated leukocytes after ischemia-reperfusion in diabetic rats. However, none of the antibodies significantly attenuated the increased albumin leakage response to ischemia-reperfusion in diabetic rats. CONCLUSIONS: These results indicate that diabetes mellitus is associated with exaggerated leukocyte-endothelial cell adhesion and albumin leakage responses to ischemia-reperfusion. The enhanced leukocyte accumulation in response to ischemia-reperfusion is mediated by CD11/CD18-ICAM-1 interactions (firm adhesion) and P-selectin (rolling). The exaggerated albumin leakage response to ischemia-reperfusion in diabetics is not mediated by the recruited inflammatory cells. PMID- 8616924 TI - Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. AB - BACKGROUND: Recordings of outward currents in human ventricular myocytes revealed the presence of a large calcium-insensitive transient outward current. This current has been suggested to contribute significantly to regional electrophysiological heterogeneity in myocardial cells and tissue of several animal species and to cause electrical gradients across the ventricular wall. METHODS AND RESULTS: The patch-clamp technique was used to record action potentials and outward currents in myocytes enzymatically isolated from thin subepicardial and subendocardial layers of human nonfailing and failing left ventricle. In all subepicardial cells studied, a calcium-insensitive transient outward current (Ito1) could be recorded with large density (10.6 +/- 1.08 pA/pF at 40 mV), whereas current density of Ito1 in subendocardial cells was fourfold smaller (2.63 +/- 0.31 pA/pF, P<.0001, nonfailing myocardium). In failing hearts, the density of Ito1 was significantly smaller in subepicardial cells (7.81 +/- 0.53 pA/pF, P=.012) but not different in subendocardial myocytes (2.01 +/- 0.23 pA/pF, P=.25). Rate-dependent reduction of peak Ito1 at a 2-Hz depolarization rate was minimal in subepicardial cells (to 92.3 +/- 1.9%), whereas peak Ito1 in subendothelial myocytes was almost suppressed at 2 Hz (reduction to 13.2 +/- 2.1%, P<.0001). The different rate-dependent reduction of the transient outward current was due to a much slower time course of recovery from inactivation in subendocardial cells. Kinetic data, including action potentials recorded at 35 degree C, allow assessment of the role of the transient outward current for electrical activity and transmural voltage gradients in human left ventricle. CONCLUSIONS: Marked regional differences in density and rate-dependent properties of the transient outward current exist in subendocardial and subepicardial layers in human left ventricular myocardium, causing transmural electrical gradients that are important for normal and pathological electrical behavior of the human heart. The difference in recovery rates of the transient outward current is a distinguishing feature between subepicardial and subendocardial myocytes. PMID- 8616925 TI - Rapid ventricular pacing produces myocardial protection by nonischemic activation of KATP+ channels. AB - BACKGROUND: Rapid ventricular pacing reduces the incidence of ventricular arrhythmias during a subsequent sustained period of ischemia and reperfusion. We investigated whether rapid ventricular pacing also limits myocardial infarction and determined the role of KATP+ channels in the protection afforded by ventricular pacing. METHODS AND RESULTS: Myocardial infarction was produced by a 60-minute coronary artery occlusion in open chest pigs. Infarct size of pigs subjected to 10 minutes of ventricular pacing at 200 beats per minute followed by 15 minutes of normal sinus rhythm before the occlusion (79 +/- 3% of the area at risk, mean +/- SEM) was not different from control infarct size (84 +/- 2%). Thirty-minute pacing followed by 15-minute sinus rhythm resulted in modest reductions in infarct size (71 +/- 2%, P<.05 versus control). Thirty minutes of pacing immediately preceding the occlusion without intervening sinus rhythm resulted in considerable limitation of infarct size (63 +/- 4%, P<.05), which was abolished by pretreatment with the KATP+ channel blocker glibenclamide (78 +/- 4%, P=NS). KATP+ channel activation did not appear to involve ischemia: (1) myocardial endocardial/epicardial blood flow ratio was 1.07 +/- 0.08, (2) phosphocreatine and ATP levels and arterial-coronary venous differences in pH and PCO2 were unchanged, (3) end-systolic segment length did not increase and postsystolic shortening was not observed during pacing, and (4) systolic shortening recovered immediately to baseline levels and coronary reactive hyperemia was absent after cessation of pacing. Administration of glibenclamide after 30 minutes of pacing at the onset of 15 minutes of normal sinus rhythm did not attenuate the protection (73 +/- 3%, P<.05 versus control), suggesting the KATP+ channels did not contribute to the moderate degree of protection that was still present 15 minutes after cessation of pacing. CONCLUSIONS: Rapid ventricular pacing protects the myocardium against infarction via nonischemic KATP+ channel activation. Continued activation of KATP+ channels does not appear mandatory for the protection that is still present 15 minutes after cessation of pacing. PMID- 8616926 TI - Basic fibroblast growth factor restores endothelium-dependent responses after balloon injury of rabbit arteries. AB - BACKGROUND: After experimental angioplasty, partial or complete reendothelialization of the denuded surface occurs; the function of the regenerated endothelium has, however, been shown to be abnormal. Basic fibroblast growth factor (bFGF) is mitogenic for endothelial cells in vitro and in vivo. We investigated whether chronic administration of bFGF in a rabbit model of balloon denudation might not only improve endothelial regrowth but also restore normal physiological responses to endothelium-dependent agonists. METHODS AND RESULTS: Thirty-nine New Zealand White rabbits underwent balloon denudation of the right iliac artery. Twenty rabbits received intravenous administration of bFGF (2.5 micrograms twice a week for 2 weeks). Nineteen rabbits receiving saline injections served as controls. Animals were killed on day 28 for assessment of reendothelialization and neointimal thickening and for analysis of in vitro vasoreactivity. Animals in the bFGF group had a significantly (P<.005) greater degree of reendothelialization than controls (115 +/- 13 versus 55 +/- 6 mm2). Neointimal thickening was similar in the two groups. Four weeks after denudation, endothelium-independent responses did not differ significantly between the two groups. In contrast, the maximal endothelium-dependent acetylcholine-induced relaxation of the bFGF-treated animals (Emax, 40 +/- 7%) was significantly greater than that of the control group (Emax, 11 +/- 9%; P<.05). CONCLUSIONS: Systemic administration of bFGF restores, in large part, the responses of previously denuded arterial segments to endothelium-dependent vasodilators. Angiogenic growth factors may help to reestablish normal endothelial cell function in patients who have undergone angioplasty. PMID- 8616928 TI - Data on giant cell myocarditis sought for new study. PMID- 8616927 TI - Images in cardiovascular medicine. Carcinoid heart disease. PMID- 8616929 TI - Effect of cocaine on left ventricular function. PMID- 8616931 TI - Long-term benefits of thrombolysis. PMID- 8616930 TI - Growth factor therapies for vascular injury and ischemia. PMID- 8616932 TI - Patent foramen ovale and platypnea-orthodeoxia syndrome. PMID- 8616933 TI - Mechanism of ischemic preconditioning. PMID- 8616934 TI - Proliferative response of smooth muscle cells after experimental balloon angioplasty. PMID- 8616935 TI - Arrhythmogenic action of thrombin during myocardial reperfusion via release of inositol 1,4,5-triphosphate. AB - BACKGROUND: Cardiac reperfusion initiates release of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and arrhythmogenesis via norepinephrine stimulation of alpha1 adrenergic receptors. The present study examines arrhythmogenic effects of thrombin-stimulated Ins(1,4,5)P3 release under these conditions. METHODS AND RESULTS: [3H]Ins(1,4,5)P3 release was measured in [3H]inositol-labeled rat hearts by high-performance liquid chromatography. Arrhythmia studies were performed in buffer-perfused rat hearts. Two-minute reperfusion after 20 minutes of global ischemia increased [3H]Ins(1,4,5)P3 from 1123 +/- 77 to 2238 +/- 44 cpm/mg tissue. No increase was observed in catecholamine-depleted hearts (755 +/- 89 cpm/mg). The addition of thrombin (5 IU/mL) or thrombin receptor agonist peptide (TRAP(1-6), 50 micromol/L) restored the reperfusion Ins(1,4,5)P3 response (thrombin, 1518 +/- 68 cpm/mg and TRAP(1-6), 1755 +/- 128 cpm/mg). Ins(1,4,5)P3 release initiated by norepinephrine or thrombin was inhibited by gentamicin (150 micromol/L; 986 +/- 52 and 868 +/- 125 cpm/mg, respectively). The thrombin response was inhibited by the phospholipase C inhibitor U-73122 (5 micromol/L; 394 +/- 59 cpm/mg) but not by its inactive isomer U-73343. The norepinephrine response was not inhibited by U-73122 (2126 +/- 74 cpm/mg). Ventricular tachycardia and ventricular fibrillation were observed in intact hearts but not in hearts from catecholamine-depleted rats (ventricular fibrillation duration, 110 +/- 19 versus 0 +/- 0 seconds). The addition of thrombin or TRAP(1-6) increased arrhythmias in catecholamine-depleted hearts (112 +/- 32 and 89 +/- 28 seconds, respectively). Gentamicin and U-73122 but not U-73343 prevented thrombin induced arrhythmias. Gentamicin inhibited norepinephrine-initiated arrhythmias, but U-73122 was ineffective. CONCLUSIONS: This study demonstrates that the development of reperfusion arrhythmias under these conditions depends on the release of Ins(1,4,5)P3. PMID- 8616936 TI - Outmigration for coronary bypass surgery in an era of public dissemination of clinical outcomes. AB - BACKGROUND: Since 1989, New York State has disseminated comparative information on outcomes of coronary bypass surgery to the public. It has been suggested that this program played a significant role in the 41% decrease in the risk-adjusted mortality rate between 1989 and 1992. We hypothesized that some high-risk patients had migrated out of state for surgery. METHODS AND RESULTS: We reviewed 9442 isolated coronary bypass operations performed from 1989 through 1993 to assess referral patterns of case-mix and outcome. Expected and risk-adjusted mortality rates were computed using logistic regression models derived from the Cleveland Clinic and New York State databases. A mortality comparison was performed using the 1980 to 1988 time period as a historical control. Patients from New York (n=482) had a higher frequency of prior open heart surgery (44.0%) than patients from Ohio (n=6046) (21.5%, P<.001), other states (n=1923) (37.4%, P=.008), and other countries (n=991) (17.3%, P<.001). They were also more likely to be in NYHA functional class III or IV (47.6% versus Ohio 42.7%, P=.037; other states, 41.2%, P=.011; other countries, 34.1%, P=.001). The expected mortality rate was thus higher than among other referral cohorts. The observed 5.2% mortality rate among these patients was significantly greater than the 2.9%, 3.1%, and 1.4% mortality rates observed for patients from Ohio (P=.004), other states (P=.028), and other countries (P<.001). These differences in outcome were not apparent between 1980 and 1988 among referrals from within the United States. CONCLUSIONS: Public dissemination of outcome data may have been associated with increased referral of high-risk patients from New York to an out-of-state regional medical center. PMID- 8616938 TI - Compliance enhancement. A call for multidisciplinary team approaches. PMID- 8616937 TI - Progression of coronary artery disease predicts clinical coronary events. Long term follow-up from the Cholesterol Lowering Atherosclerosis Study. AB - BACKGROUND: Progression of coronary artery disease is assumed to be a surrogate end point for clinical coronary events. Because no single method or measure for a coronary angiographic end point is uniformly accepted as optimal, the utility and validity of surrogate end points for predicting clinical coronary events remain unsettled. METHODS AND RESULTS: The Cholesterol Lowering Atherosclerosis Study randomized 162 nonsmoking, 40- to 59-year-old men with previous coronary artery bypass graft surgery to colestipol/niacin plus diet or placebo plus diet. Atherosclerosis change on 2-year coronary angiograms was evaluated by a consensus panel and by quantitative coronary angiography (average per-subject change in percent diameter stenosis [%S] and minimum lumen diameter [MLD). With all three end points, the benefit of colestipol/niacin treatment on coronary artery atherosclerosis has been reported. Annual follow-up for an average of 7 years (range, 6.3 months to 10 years) has been carried out on all subjects who completed the 2-year angiogram. Clinical coronary events (need for revascularization, nonfatal acute myocardial infarction, and coronary death) have been documented. Risk of clinical coronary events was positively related to coronary lesion progression for all three surrogate end points (P<.05). New lesion formation in bypass grafts (P=.02) and progression of mild/moderate lesions ( < 50%S) were predictive of clinical coronary events (P<.01). Change in MLD contributed significantly to the prediction of clinical coronary events beyond a model with %S alone (P<.05). CONCLUSIONS: In this population of nonsmoking men with previous bypass surgery, both the consensus panel- and quantitative coronary angiography-based end points of coronary artery disease progression predict clinical coronary events. Subjects who demonstrate greater coronary artery lesion progression have an increased risk of future clinical coronary events. Design of shorter, smaller trials of antiatherosclerotic agents is justified. PMID- 8616939 TI - Smoking and cardiac events after venous coronary bypass surgery. A 15-year follow up study. AB - BACKGROUND: The long-term clinical effects of smoking and smoking cessation after venous coronary bypass surgery have not been well established. METHODS AND RESULTS: Four hundred fifteen patients who underwent venous coronary bypass surgery between April 1976 and April 1977 were followed up prospectively for 15 years. Multivariate Cox survival analysis revealed that patients who smoked at the time of surgery had no elevated risks for clinical events compared with nonsmokers. However, smoking behavior at 1 and 5 years after surgery appeared to be an important predictor of clinical events during the subsequent follow-up period. Compared with patients who stopped smoking since surgery, smokers at 1 year after surgery had more than twice the risk for myocardial infarction and reoperation. Patients who were still smoking at 5 years after surgery had even more elevated risks for myocardial infarction and reoperation and a significantly increased risk for angina pectoris compared with patients who stopped smoking since surgery and patients who never smoked. Patients who started to smoke again within 5 years after surgery had increased risks for reoperation and angina pectoris. No differences in outcome were found between patients who stopped smoking since surgery and nonsmokers. CONCLUSIONS: Our results show that smoking cessation after coronary bypass surgery may have important beneficial effects on clinical events during long-term follow-up. PMID- 8616940 TI - Relation between symptom duration before thrombolytic therapy and final myocardial infarct size. AB - BACKGROUND: Myocardial salvage is most likely to occur when thrombolytic therapy is administered within 4 to 6 hours of the onset of symptoms of myocardial infarction. The impact of delays within this early time period on final myocardial infarct size are unknown. The purpose of this study was to quantitate the relation between final myocardial infarct size and duration of symptoms before initiation of thrombolytic therapy in patients treated within 6 hours of symptom onset. METHODS AND RESULTS: The findings from patients in four prospective randomized trials of thrombolytic therapy were combined for analysis. The study population consisted of 432 patients presenting within 6 hours of onset of symptoms of first acute myocardial infarction who met ECG criteria that allowed estimation of myocardial area at risk before treatment with thrombolytic therapy and who had thallium-201 myocardial infarct-size measurements performed several weeks after infarction. ECG analysis revealed no difference in myocardium at risk for infarction as a function of duration of symptoms before initiation of thrombolytic therapy. In contrast, univariate and multivariate analysis showed that final infarct size was highly dependent on duration of symptoms before initiation of therapy. Each 30-minute increase in symptom duration before thrombolytic therapy was associated with an increase in infarct size of 1% of the myocardium. Final infarct size in patients treated 4 to 6 hours after symptom onset was indistinguishable from patients who did not receive thrombolytic therapy. CONCLUSIONS: These findings suggest that for patients treated within 4 to 6 hours of the onset of symptoms, there is a progressive decline in the extent of myocardium salvaged as the duration of symptoms before therapy increases. These results support efforts to minimize the time delay between symptom onset and initiation of reperfusion therapy in all eligible patients. PMID- 8616941 TI - Persistent elevation of plasma insulin levels is associated with increased cardiovascular risk in children and young adults. The Bogalusa Heart Study. AB - BACKGROUND: Hyperinsulinemia has been considered to be a potent cardiovascular risk factor. The present investigation examines persistently elevated fasting insulin levels from childhood to young adulthood and its influence on cardiovascular risk factors. METHODS AND RESULTS: A longitudinal cohort was constructed from two cross-sectional surveys in a community-based population over an 8-year period: 1606 individuals (39% were black) aged 5 to 23 years participated in the first survey. Stability in rankings (persistence) of insulin levels was shown by the presence of significant correlations between year 1 and year 8 values (r=.23 to .36, P<.0001), with a greater magnitude in older subjects. Compared with subjects with levels of insulin consistently in the lowest quartile, those with levels always in the highest quartile showed higher (P<.001) levels of body mass index (+9 kg/m2), triglycerides (+58 mg/dL), LDL cholesterol (+11 mg/dL), VLDL cholesterol (+8 mg/dL), glucose (+9 mg/dL), systolic blood pressure (+7 mm Hg), and diastolic blood pressure (+3 mm Hg); lower (P<.001) levels of HDL cholesterol (-4 mg/dL): and higher (P<.05) prevalence of parental history of diabetes (3.3-fold) and hypertension (1.2 fold). There were 739 young adults aged 20 to 31 years at follow-up. As adults, individuals with consistently elevated insulin versus those with consistently decreased insulin had increased (P<.05) prevalence of obesity (36-fold), hypertension (2.5-fold), and dyslipidemia (3-fold), which was attributed to both baseline insulin and change of insulin from baseline to follow-up. In addition, clustering of these risk factors was stronger (P<.05) in adults with persistent insulin elevation. CONCLUSIONS: Elevated insulin levels persist from childhood through young adulthood, resulting in a clinically relevant adverse cardiovascular risk profile in young adults. PMID- 8616942 TI - Blood pressure in young blacks and whites: relevance of obesity and lifestyle factors in determining differences. The CARDIA Study. Coronary Artery Risk Development in Young Adults. AB - BACKGROUND: Middle-aged black men and women have higher blood pressure, on average, than whites. However, this pattern is inconsistent in children and adolescents. This study explores how differences in lifestyle factors in young adulthood may influence blood pressure patterns in the two races. METHODS AND RESULTS: The Coronary Artery Risk Development in Young Adults (CARDIA) study is an ongoing collaborative investigation of lifestyle and the evolution of cardiovascular disease risk factors in a random sample of young adults ages 18 to 30 years at baseline (1985 to 1986). Data from four examinations over 7 years were analyzed with the use of a method that simultaneously examined cross sectional and longitudinal relationships of lifestyle factors and blood pressure. This study included 1154 black women, 853 black men, 1126 white women, and 1013 white men. Blacks had higher systolic blood pressure and diastolic blood pressure than whites at every examination. Racial differences were much greater in women than in men and increased over time. Within each sex-race group, average diastolic blood pressure over four examinations was positively associated with baseline age, body mass index, and alcohol intake and negatively associated with physical activity, cigarette use, and intake of potassium and protein. Longitudinal change in diastolic blood pressure was positively associated with changes in body mass index and alcohol intake. After adjustment for obesity and other lifestyle factors, black-white diastolic blood pressure differences were reduced substantially: 21% to 75% for men and 49% to 129% for women. Results for systolic blood pressure were similar. CONCLUSIONS: Differences in obesity and other lifestyle factors in young adults largely explain the higher baseline blood pressure and greater increase over time of blacks relative to whites. PMID- 8616943 TI - Energetically optimal left ventricular pressure for the failing human heart. AB - BACKGROUND: An energy-starved failing heart would benefit from more effective transfer of the mechanical energy of ventricular contraction to blood propulsion. However, the energetically optimal loading conditions for the failing heart are difficult to establish. In the present study, we analyzed the optimal left ventricular pressure to achieve maximal mechanical efficiency of the failing heart in humans. METHODS AND RESULTS: We determined the relation between left ventricular pressure-volume area and myocardial oxygen consumption per beat (VO2), stoke work, and mechanical efficiency (stroke work/VO2) in 13 patients with different contractile states. We also calculated the optimal end-systolic pressure that would theoretically maximize mechanical efficiency for a given end diastolic volume and contractility. Left ventricular pressure-volume loops were constructed by plotting the instantaneous left ventricular pressure against the left ventricular volume at baseline and during pressure loading. The contractile properties of the ventricle were defined by the slope of the end-systolic pressure-volume relation. In patients with less compromised ventricular function, the operating end-systolic pressure was close to the optimal pressure, achieving nearly maximal mechanical efficiency. As the heart deteriorated, however, the optimal end-systolic pressure became significantly lower than normal, whereas the actual pressure remained within the normal range. This discrepancy resulted in worsening of ventriculoarterial coupling and decreased mechanical efficiency compared with theoretically maximal efficiency. CONCLUSIONS: Homeostatic mechanisms to maintain arterial blood pressure within the normal range cause the failing heart to deviate from energetically optimal conditions. PMID- 8616945 TI - Prospective 12-year follow-up study of clinical and hemodynamic sequelae after deep vein thrombosis in low-risk patients (Zurich study). AB - BACKGROUND: No prospective study of the long-term sequelae of more than 10 years after acute deep vein thrombosis exists so far. Therefore, 58 low-risk patients with DVT were included in a prospective study to evaluate the natural history of postthrombotic syndrome. METHODS AND RESULTS: Clinical and hemodynamic examinations were performed at the time of admission; after 3, 6, and 12 months; after the 2nd, 3rd, 4th, 5th years; and finally after the 12th year. All patients received heparin initially and oral anticoagulants subsequently. After 12 years, 64% of the patients exhibited normal findings. Mild skin changes were found in 28%, marked trophic changes in 5%, and only one venous ulcer occurred. Regular use of compression stockings was reported by 54% of the patients with multilevel thrombosis. Although mean maximum venous outflow was significantly reduced from the acute event to 12 years later (P<.003) compared with the contralateral leg, a significant (P<.05) improvement was observed 6 months later. Recanalization of calf vein thrombosis was detected by Doppler sonography after 3 months. Sixty four percent of the multilevel thromboses were recanalized completely or in part after 1 year; in 69%, valvular incompetence was found. CONCLUSIONS: In contrast to earlier reports, this prospective study up to 12 years after deep vein thrombosis demonstrates a low incidence of postthrombotic syndrome by administration of oral anticoagulants and regular compression therapy. However, the adverse clinical event rate (mortality 14%) and a recurrence rate of 24% show that the prognosis after deep vein thrombosis does not appear favorable even in low-risk patients. PMID- 8616944 TI - Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. AB - BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5 methyltetrahydrofolate, the major carbon donor in remethylation of homocysteine to methionine. A common MTHFR mutation, an alanine-to-valine substitution, renders the enzyme thermolabile and may cause elevated plasma levels of the amino acid homocysteine. METHODS AND RESULTS: To assess the potential interaction between this mutation and vitamin coenzymes in homocysteine metabolism, we screened 365 individuals from the NHLBI Family Heart Study. Among individuals with lower plasma folate concentrations ( < 15.4 nmol/L), those with the homozygous mutant genotype had total fasting homocysteine levels that were 24% greater (P<.05) than individuals with the normal genotype. A difference between genotypes was not seen among individuals with folate levels > or = 15.4 nmol/L. CONCLUSIONS: Individuals with thermolabile MTHFR may have a higher folate requirement for regulation of plasma homocysteine concentrations; folate supplementation may be necessary to prevent fasting hyperhomocysteinemia in such persons. PMID- 8616947 TI - L-arginine induces nitric oxide-dependent vasodilation in patients with critical limb ischemia. A randomized, controlled study. AB - BACKGROUND: L-Arginine is the precursor of endogenous nitric oxide (NO), which is a potent vasodilator acting via the intracellular second-messenger cGMP. In healthy humans, L-arginine induces peripheral vasodilation and inhibits platelet aggregation due to an increased NO production. Prostaglandin E1 (PGE1) induces peripheral vasodilation via stimulating prostacyclin receptors. METHODS AND RESULTS: We investigated the effects of one intravenous infusion of L-arginine (30 g, 60 minutes) or PGE1 (40 microgram, 60 minutes) versus those of placebo (150 mL 0.9% saline, 60 minutes) on blood pressure, peripheral hemodynamics, and urinary NO3- and cGMP excretion rates in patients with critical limb ischemia (peripheral arterial occlusive disease stages Fontaine III or IV). Blood flow in the femoral artery was significantly increased by L-arginine (+42.3 +/- 7.9%, P<.05) and by PGE1 (+31.0 +/- 10.2%, P<.05) but not by placebo (+4.3 +/- 13.0%, P=NS). Urinary NO3- excretion increased by 131.8 +/- 39.5% after L-arginine (P<.05) but only by 32.3 +/- 17.2% after PGE1 (P=NS). Urinary cGMP excretion increased by 198.7 +/- 84.9% after L-arginine (P<.05) and by 94.2 +/- 58.8% after PGE1 (P=NS). Both urinary index metabolites were unchanged by placebo. CONCLUSIONS: We conclude that intravenous L-arginine induces NO-dependent peripheral vasodilation in patients with critical limb ischemia. These effects are paralleled by increased urinary NO3- and cGMP excretion, indicating an enhanced systemic NO production. Increased urinary NO3- excretion may be a sum effect of NO synthase substrate provision (L-arginine) and increased shear stress (PGE1 and L-arginine). PMID- 8616946 TI - Low-molecular-weight heparinoid orgaran is more effective than aspirin in the prevention of venous thromboembolism after surgery for hip fracture. AB - BACKGROUND: The study objective was to determine the relative efficacy and safety of a low-molecular-weight heparinoid (Orgaran) compared with aspirin for the prevention of postoperative venous thromboembolism in patients undergoing surgery for fractured hips. A double-blind, randomized, controlled trial was used to study 251 consecutive eligible and consenting patients undergoing surgery for hip fracture in seven participating hospitals. METHODS AND RESULTS: Patients received either fixed-dose Orgaran by subcutaneous injection every 12 hours in a dose of 750 anti-Factor Xa units or aspirin 100 mg orally twice daily; both regimens were started 12 to 24 hours after surgery and continued for 14 days or until discharge, if sooner. All patients had postoperative 125I-fibrinogen leg scanning and impedance plethysmography. If the results of one or both tests were positive, then venography was performed. Otherwise, venography was done at day 14, or sooner if the patient was ready for discharge. Pulmonary embolism in symptomatic patients was diagnosed on the basis of a high probability perfusion/ventilation lung scan, a positive angiogram, or a clinically significant embolism detected at autopsy. Evaluable venograms were obtained in 90 of the 125 patients randomly assigned to receive Orgaran and in 87 of the 126 patients assigned to receive aspirin. Venous thromboembolism was detected in 25 (27.8%) patients in the Orgaran group and in 39 (44.3%) patients in the aspirin group. Thus, there was a relative risk reduction of 37% with Orgaran (P=.028; 95% confidence interval, 3.7% to 59.7%). Six (6.8%) of 88 patients in the Orgaran group and 12 (14.3%) of 84 patients in the aspirin group developed proximal deep vein thrombosis or pulmonary embolism, a relative risk reduction of 52% with Orgaran (P=.137; 95% confidence interval, -30.7% to 84.6%). Hemorrhagic complications occurred in 2 (1.6%) patients given Orgaran and 8 (6.4%) patients given aspirin (P=.10). There was one major bleed in the Orgaran group compared with four in the aspirin group. CONCLUSIONS: This study demonstrates that Orgaran is significantly more efficacious than aspirin in preventing postoperative venous thromboembolism in patients undergoing surgery for fractured hips, with no evidence of any increase in hemorrhagic complications. PMID- 8616948 TI - Undetected ventricular fibrillation in transvenous implantable cardioverter defibrillators. Prospective comparison of different lead system-device combinations. AB - BACKGROUND: The purpose of this study was to prospectively analyze redetection problems after unsuccessful shock with different lead systems and devices. METHODS AND RESULTS: We prospectively analyzed detection and redetection characteristics among transvenous implantable cardioverter-defibrillators (ICDs) using standard bipolar and integrated bipolar sensing. Monophasic and biphasic ICDs were included. Subthreshold shocks were intentionally delivered, and redetection of ventricular fibrillation (VF) was assessed before discharge and at 1, 3, 6, and 12 months later. Sensing of VF resulting from antitachycardia pacing and low-energy cardioversion ( < or = 2 J) also was analyzed. Before inclusion in the study, each patient underwent subthreshold shock testing at three different time intervals. Among the 160 ICDs with standard bipolar sensing, 530 VF inductions were analyzed. After the failed shocks, undersensing was more frequent (3% versus 20%, P<.01) but did not remarkably prolong redetection (3.1 +/- 0.8 versus 3.3 +/- 1.1 seconds). Among the 201 ICDs with integrated bipolar sensing, 80 were connected to a CPI device (60 Ventak 1600-Endotak 60 series and 20 PRx II 1715-Endotak 70 series) and 121 to the Ventritex defibrillator (91 Endotak 60 series, 14 TVL systems, and 16 Endotak 70 series). After 252 failed shocks, redetection was prolonged with the CPI system (3.1 +/- 1.4 versus 4.6 +/- 3.6 seconds, P<.05) but did not change after 396 failed shocks with the Ventritex ICD (5.4 +/- 1.9 versus 4.9 +/- 2.2 seconds). This may reflect different nominal settings for detection and redetection. In 9 of 121 patients with Ventritex and 1 of 80 with the CPI ICDs, the devices failed to redetect VF. However, redetection malfunction was never observed in patients with integrated bipolar systems with >6-mm electrode separation. After antitachycardia pacing in 1 patient and a 2-J shock in 1 patient, ventricular tachycardia turned into VF, which was undetected. Both patients used the Endotak 60 series-Cadence combination. None of the patients showing VF undersensing had sudden death at follow-up. Only 3 of the 12 patients with sensing malfunction were on antiarrhythmia drugs at the time of testing. Analysis of endocardial electrograms showed that failure to redetect VF is not associated with a uniform reduction but with a rapid and repetitive change of electrogram amplitude. CONCLUSIONS: Standard bipolar sensing redetects VF more effectively than integrated bipolar sensing. Endocardial electrogram analysis provides insights into the understanding of the mechanism of undersensing, and certain lead-device combinations result in a higher occurrence of VF undersensing. The clinical relevance of this phenomenon remains unknown. PMID- 8616949 TI - Induction of DNA synthesis by a single transient mechanical stimulus of human vascular smooth muscle cells. Role of fibroblast growth factor-2. AB - BACKGROUND: Although mechanical vascular injury leads to smooth muscle cell proliferation that contributes to restenosis after balloon angioplasty, the role of the single transient mechanical stimulation of smooth muscle cells in this process is unknown. METHODS AND RESULTS: To test the hypothesis that a single transient mechanical stimulus can increase DNA synthesis, human vascular smooth muscle cells cultured in a three-dimensional collagen gel system were subjected to transient compression. Transient compression (5-minute duration) of smooth muscle cell-collagen gel cultures in defined serum-free conditions led to delayed increases in [3H]thymidine incorporation. At 12 to 24 hours after compression, there was a 3.3 +/- 0.5-fold (P<.001 versus control) and 3.0 +/- 0.6-fold (P<.002 versus control) increase for 60% and 80% strain, respectively; at 24 to 36 hours after compression, there was a 1.8 +/- 0.5-fold (P<.05 versus control) and 4.3 +/ 0.8-fold (P<.001 versus control) increase. Also, serum-free media conditioned by transiently compressed gel cultures induced DNA synthesis in control, unstimulated smooth muscle cell cultures, suggesting the release of growth factors by transient compression. Although neutralizing antibodies against platelet-derived growth factor did not affect the mechanical induction of DNA synthesis, a neutralizing monoclonal antibody against fibroblast growth factor-2 (FGF-2) decreased this induction by 89% and completely blocked the increase in DNA synthesis caused by media conditioned by transiently compressed gels. Media conditioned by transient compression contained elevated levels of FGF-2 (17 +/- 5 versus 2 +/- 2 pg/mL for control, P<.005) with no increase in lactate dehydrogenase activity, suggesting release of FGF-2 with sublethal cellular injury. CONCLUSIONS: A single transient mechanical stimulus increases DNA synthesis in human vascular smooth muscle cells, in part by autocrine or paracrine FGF-2 release. PMID- 8616950 TI - Monocyte superoxide generation and its IgA-receptor in IgA nephropathy. AB - Recent studies indicated that polymorphonuclear leukocytes (PMNL) were primed to produce superoxide (O2-) in various types of glomerulonephritis with a particular importance in IgA nephropathy (IgAN). In this study, we have examined O2- production and receptor expression for the Fc portion of IgA (Fc alpha R) in monocytes to evaluate their incorporation in IgAN, since infiltration of these cells in the glomeruli are more commonly observed than that of PMNL. Similar to PMNL, monocytes obtained from IgA nephropathy (IgAN) seemed to be primed both non specifically and specifically, as increased O2- generation was observed to N formyl methionyl leucyl phenylalanine (FMLP) and phorbol myristate acetate (PMA), as well as IgA aggregates stimulants, respectively. Monocytes O2- generation to IgA aggregates was comparatively higher in amount than in PMNL, and showed a correlation with the severity of proteinuria in IgAN patients. Flow cytometric assessment showed an increased expression of Fc alpha R on circulating monocytes in IgAN patients which showed a linear correlation with the amount of IgA-induced O2- generation. Comparing with the previous literature on PMNL, inflammation related substances such as cytokines/immune complexes, particularly IgA immune complexes which present in the circulation of IgAN, can prime the phagocytic cells in the circulation for a burst of O2- generation to a second stimulus. The increased expression of Fc alpha R appears to be associated with the increase in priming and the degree of priming can be reflected in the severity of proteinuria/hematuria, although it can not be defined as a cause or consequence of this disease. PMID- 8616951 TI - Glomerular filtration rate and kidney size after six years disease duration in non-insulin-dependent diabetic subjects. AB - The objective of the present study was to estimate how glomerular filtration rate and kidney size change after six years of diabetes in subjects with non-insulin dependent disease. It is a population-based prospective study of a cohort of non insulin diabetic patients (n = 150) diagnosed 1985-1988. The baseline studies utilized a non-diabetic control group, whose basic characteristics were equal to the study group. The setting was a primary health care center in an urban area. Main outcome measures were the glomerular filtration rate and its relation to renal area, mean blood pressure, hemoglobin A1c, serum insulin and cholesterol. Seventeen patients had died and 109 were eligible for evaluation at follow-up. The mean (standard deviation) of the glomerular filtration rate (ml/min/1.73 m2) remained elevated at follow-up, 118 (28), just as it was at baseline, 118 (28) in the diabetic subjects compared to matched non-diabetic subjects, 103 (24) (p = 0.0000). Kidney size (cm2) was larger in diabetic subjects at follow-up, 114 (19) than at baseline, 109 (18) (p = 0.0000) and in non-diabetic subjects 98 (14) (p < 0.0000). This resulted in a decline in glomerular filtration rate per unit renal area in the diabetic subjects at follow-up, 1.0 (0.23) compared to at baseline, 1.09 (0.23) (p = 0.002) and to non-diabetic subjects, 1.07 (0.23). The renal area at baseline was directly and significantly related to the glomerular filtration rate at follow-up (p < 0.001). The relation of baseline serum cholesterol, hemoglobin A 1c and mean arterial blood pressure to the glomerular filtration rate at follow-up was inverse and reached significance in those diabetic subjects having had high filtration rates at baseline but displaying a faster decline than on average i.e. in those patients who were at increased risk of renal insufficiency. We conclude that after the first six years of non-insulin dependent diabetes the glomerular filtration rate remains high. Kidney size increases further from the attained increase at diagnosis and is an important determinant of continuing hyperfiltration. The deleterious effect of serum cholesterol and high blood glucose on the glomerular filtration rate at this early stage of diabetic kidney disease is suggestive. PMID- 8616952 TI - Effects of cyclosporine, azathioprine and prednisone on Kimura's disease and focal segmental glomerulosclerosis in renal transplant patients. AB - Nephrotic syndrome secondary to focal segmental glomerulosclerosis (FSGS) is a common cause of renal failure leading to transplantation. Various immunosuppressive drugs, including cyclosporine, have been used in the treatment of FSGS with varying degrees of success. The patient described herein was afflicted with end stage renal disease secondary to steroid-resistant nephrotic syndrome due to FSGS with coexistent Kimura's disease. After kidney transplantation, the patient experienced a remission of both the Kimura's disease and the nephrotic syndrome in the native kidneys, leading to the resumption of renal function by the native kidneys in spite of severe transplant glomerulopathy. This case suggests that certain cases of FSGS without extensive interstitial disease may benefit from aggressive treatment with a combination of immunosuppressive drugs. PMID- 8616953 TI - Predictive value of IgG subclass levels for infectious complications in renal transplant recipients. AB - Renal allograft recipients are at risk to acquire infectious disease complications primarily after an intensified immunosuppressive therapy. In this study, serum levels of IgG, IgA, IgM and IgG subclasses were compared between transplant patients after different anti-rejection regimen and evaluated as possible predictive markers for infectious implications. Thirty-six renal transplant recipients in the early posttransplantation period were allocated into three subgroups: group 1 consisted of 15 subjects with good primary graft function standing under a basal triple drug immunosuppression; patients of group 2 (n = 9) had a very early acute rejection episode which was treated by high steroid doses and patients of group 3 (n = 12) required antithymocyte globulin (ATG) therapy for acute rejection. Immune parameters were studied 5 weeks after transplantation, when basal triple drug immunosuppression was continued in all patients. Twenty-three age-matched patients under chronic hemodialysis were recruited as a control group. Total IgG serum levels were reduced in transplant patients compared to dialysis subjects, whereas IgA and IgM levels were not altered. IgG subclass analysis revealed, that IgG1 levels were similar in stable transplant compared to chronic hemodialysis patients, but were significantly reduced in rejection patients after steroid pulses or ATG therapy. IgG3 levels were significantly reduced in all transplant recipients compared to dialysis patients without differences between the transplant subgroups. IgG2 and IgG4 subclass levels were similar in all studied subjects. IgG subclass abnormalities were paralleled by a reduction of lymphocyte subsets in rejection patients of groups 2 and 3, which may be the basis for an altered Ig class switching. Seven out of 36 studied transplant patients had severe infections in the early posttransplantation period with four cases of viral infections. Patients with infections had significantly lower IgG1 levels and CD4 positive lymphocyte numbers but similar total IgG levels compared to patients without infections. So IgG1 levels may be of value to predict the occurrence of infectious complications in renal allograft recipients in the early posttransplantation period. PMID- 8616955 TI - Clinical course of hypophosphatemic rickets in 23 adults. AB - Twenty-three adult patients (19 females, 4 males) with x-linked hypophosphatemic rickets (HPR) underwent a retrospective evaluation of the clinical course and a clinical examination by a nephrologist, orthopedic surgeon and dentist. Blood and urine analysis, bone density measurements with QCT and DEXA, ultrasonic examination of the kidneys were performed and the patients were asked to fill in a standardized questionnaire on pain and psychosocial rehabilitation. Mean final height was 152.4 cm +/- 8.5 SD in females and 157.3 cm +/- 8.9 SD in males. Decreased joint mobility was seen in all patients, deviations of the normal leg axis in 18/23 patients in spite of 69 correcting osteotomies in the past. Dental (n = 14) and psychosocial problems were associated with inability to work (n = 8). There was a trend that patients with a very low Tp/GFR had a more severe course of the disease. Early therapy with vitamin D metabolites and phosphate had a beneficial effect on growth, bone density and deformations. Eight patients had nephrocalcinosis due to vitamin D and phosphate therapy and had normal kidney function. Four patients had urinary tract abnormalities. We conclude that patients with HPR should receive continuous interdisciplinary care given by nephrologists, orthopedic surgeons, physiotherapists and dentists not only during childhood but also as adults. PMID- 8616954 TI - The pharmacokinetics of anti-thymocyte globulin (ATG) following intravenous infusion in man. AB - The pharmacokinetics of the distribution and elimination of polyclonal rabbit antithymocyte globulin (ATG) following intravenous infusion was studied in patients who had received renal allografts. ATG concentration was measured using a new enzyme-linked immunoabsorbent assay (ELISA) for the Fc portion of rabbit IgG. Eleven patients received 14 courses of ATG supplied either by Fresenius (F ATG) or Merieux (M-ATG) as a daily infusion of 2-6 mg/kg body weight for a therapeutic course lasting 5-10 days. The washout phase of ATG elimination was analysed over 0-300 days; results were well-fitted by a single exponential decay (r2 > 0.95) giving a mean elimination half-life (t0.5e) of 29.8 days (range 14.3 45.0, n = 9). Data for the first 4 days of treatment were analysed with linear regression to obtain a mean value for the apparent volume of distribution of ATG (Vd) of 0.12 l/kg body weight (range 0.07 to 0.17, n = 5). These results demonstrate that rabbit ATG has a long half-life in human plasma and an apparent volume of distribution of about twice plasma volume. The relationship between the concentration of ATG measured by this Fc receptor assay and its biological activity requires further study. PMID- 8616956 TI - Amino acid loading disrupts whole kidney proximal glomerular-tubular balance in humans. AB - The effect of amino acid infusion on proximal glomerular-tubular balance (PG-TB) was studied in twelve healthy two-kidney volunteers (2K) and eight uninephrectomized subjects (1K) in similar sodium and protein balance, to provide an extended range of values for renal function. Amino acid infusion induced a reversible significant increase in GFR: +23 +/- 4 and +18 +/- 3 ml/min/1.73 m2 in 2K and 1K respectively (p < 0.001), but not in absolute proximal reabsorption (APR, calculated as inulin clearance--lithium clearance): +8 +/- 3 and +6 +/- 3 ml/min/1.73 m2 respectively, NS. Consequently, the fractional increases in APR for both two kidney- and one kidney-subjects (+11 +/- 4 and +14 +/- 6%) were less than the fractional increases in GFR (+20 +/- 3 and 25 +/- 4%), and the effectiveness of PG-TB was significantly decreased to 53.2 and 55.9% (p < 0.001). Urinary sodium excretion also increased by 116 +/- 24 and 95 +/- 29 mumol/min/1.73 m2 in 2K (p < 0.001) and 1K (p < 0.01) respectively. Urinary excretion of guanosine 3',5'-cyclic monophosphate (cGMP) simultaneously increased (p < 0.001) by 223 +/- 77 and 350 +/- 135 pmol/min/1.73 m2 during infusion of amino acids in 1K and 2K respectively. In the whole group of subjects, impairment of the PG-TB was inversely related to the increase in urinary cGMP (p < 0.05). The results demonstrate that amino acid infusion disrupts proximal glomerular-tubular balance in humans due to non-proportional alterations in glomerular filtration rate and proximal reabsorption, and they suggest that a NO dependent mechanism may be involved in the disruption. PMID- 8616957 TI - The acute impact of NaHCO3 in treatment of metabolic acidosis on back-titration of non-bicarbonate buffers: a quantitative analysis. AB - OBJECTIVE: The major non-bicarbonate buffers are intracellular proteins, a detrimental effect of severe acidosis could be their titration with H+. This in turn would lead to their net charge becoming more positive, and possibly, to changes in their shape and function. Since NaHCO3 is a treatment option in patients with severe metabolic acidosis, the purpose of this study was to examine the acute effect of the administration of NaHCO3 on back-titration of non bicarbonate buffers in metabolic acidosis. DESIGN: Prospective, controlled, non randomized laboratory study. SETTING: Research laboratory. SUBJECTS: 21 male Wistar rats. INTERVENTION: Rats were anesthetized, intubated and ventilated. Ventilation was adjusted at the beginning of the experiment to a PCO2 of approximately 30 mmHg, no further adjustments were made thereafter. Acute metabolic acidosis was induced by the infusion of 3.5 mmol of hydrochloric acid over 1 hour. After an equilibration period, 3 groups of seven rats were studied; group I received 0.75 mmol NaHCO3, group II received equimolar NaCl, and group III served as time control. MEASUREMENTS AND MAIN RESULTS: Measurements were made to enable quantitation of how much HCO3 was retained in the ECF and how much was titrated with H+ and was excreted as "acid-base" CO2. Since there are so few H+ present in the ECF in a free or a bound form, and in the absence of an increase in endogenous acid-production, the source of this H+ is from proteins in the ICF. As compared to the NaCl and the time control groups, the administration of NaHCO3 led to significant alkalinization of the ECF, pH rose from 7.22 +/- 0.03 to 7.34 +/- 0.02. Of the 0.75 mmol of NaHCO3 that was administered, 67% or 0.52 +/- 0.08 mmol was retained in ECF. Only a small amount (0.07 +/- 0.09 mmol) of acid-base CO2 was excreted. CONCLUSIONS: The administration of NaHCO3 does not acutely lead to a significant back-titration of non-bicarbonate buffers, especially under conditions of fixed ventilation. PMID- 8616958 TI - Uremic autonomic neuropathy: recovery following bicarbonate hemodialysis. AB - Autonomic function was followed in 8 chronic uremic patients on periodic hemodialysis over a period of almost eight years. The cardiovascular autonomic testing included R-R interval variation test, deep breathing, Valsalva manoeuvre, heart rate and blood pressure responses to standing, sustained handgrip. The patients were investigated on entry into the study and after 18, 56, and 92 months. Six months after the study at time 56 months, they switched from acetate to bicarbonate dialysis. The response to deep breathing test was significantly reduced at time 18 months versus baseline (P = 0.014), but significantly increased at time 92 months versus 56 months (P = 0.042). A significant decrease was found in the systolic blood pressure response to standing between baseline and 18 months (P = 0.014) and in the response to handgrip test between 18 and 56 months (P = 0.014). Multivariate analysis of the autonomic tests by a pattern recognition method (Bayesian analysis) showed that, at the time of entry into the study, two out of eight patients had autonomic damage. At 18 and 56 months, 6/8 patients had autonomic dysfunction. At the last time of investigation, 30 months after the onset of bicarbonate dialysis, all the patients showed a reversal of autonomic damage. Age and duration of dialysis on entry did not affect autonomic function. The present study is the first demonstration that autonomic neuropathy can recover after long-term dialysis. Since chronic hypoxemia is a cause of polyneuropathy, we postulate that: 1) hypoxemia in dialysis patients may have a role in the pathogenesis of uremic polyneuropathy, and particularly of autonomic dysfunction; 2) in patients on bicarbonate dialysis, a greater hemodynamic stability with less hypoxemia may lead to a recovery of autonomic function. PMID- 8616959 TI - Beer potomania: two cases and review of the literature. AB - The association of severe hyponatremia and the ingestion of large quantities of beer, termed beer potomania, has been known for several years. We report two new cases, and review 20 others from the medical literature. These patients usually have a history of binge beer drinking, poor dietary intake, and then present with severe hyponatremia and various mental status changes or seizures. Typical laboratory findings include hyponatremia, hypokalemia, and a very dilute urine. The patients respond quickly to the administration of sodium chloride containing i.v. fluids. We propose that the pivotal pathophysiologic mechanism in beer potomania syndrome is the minimal intake of solute and the hypoosmolality of the beer ingested. This will lead to the inability to excrete sufficient amounts of free water to keep up with the ingestion of large quantities of the hyposmolar beer. Treatment with isotonic sodium chloride results in the rapid clearance of the accumulated excess free water. PMID- 8616960 TI - Urinary activity of N-acetyl-beta-glycosaminidase (NAG) in arterial hypertension. PMID- 8616961 TI - Low Na concentration solution for CAPD. PMID- 8616962 TI - Erythropoietin concentration, body iron and cytokines. PMID- 8616963 TI - Increased prothrombin fragment 1.2 concentrations in hemodialysis patients. PMID- 8616964 TI - Severe secondary hyperparathyroidism: intravenous calcitriol treatment or parathyroidectomy? PMID- 8616965 TI - The diagnosis of preterm labor and the prediction of preterm delivery. AB - Clinical approaches to the diagnosis of PTL and the prediction of PTD are complicated by the absence of a gold standard for the pathogenic process leading to PTD. There is also substantial overlap between the signs and symptoms of PTL and impending PTD, and the normal processes of pregnancies destined to remain uncomplicated (e.g., our inability to convincingly differentiate PTL from Braxton Hicks contractions). Our emphasis on the diagnosis of PTL rather than the pathogenic process preceding PTD also results in failure to detect the 50% of spontaneous PTDs in which uterine contractions follow PPROM. Thus, clinical predictors of incipient PTD including cervical change, uterine contractions, vaginal bleeding, risk scoring schemes, and fetal breathing activity, either have poor sensitivity or specificity, or are accurate only at late stages in the pathogenic process. The most promising approaches to the detection of impending PTD are laboratory indices of the putative pathogenic processes including: maternal serum or plasma CRH, salivary E3, serum collagenase and cervicovaginal cytokines, granulocyte elastase, and FFN levels. However, even if these indices prove sensitive, specific, and early predictors of PTD, they will be useful only if more appropriate therapies are found to treat patients. The latter will depend on addressing the primary causes of chorionic-decidual cell activation (e.g., infection, stress, utero-placental ischemia, hemorrhage, endocrinopathies). PMID- 8616966 TI - Beta-adrenergic agonists. PMID- 8616967 TI - Indomethacin. PMID- 8616968 TI - Magnesium sulfate. AB - Since the first American report on the use of magnesium sulfate tocolysis in 1977, its popularity as a tocolytic agent has increased progressively. Primarily because of its safety and familiarity, magnesium has become the primary tocolytic agent in the majority of U.S. centers. The exact mechanism of action is unknown, and long-term effects on neonates have not been studied. Although randomized studies show similar success compared to other tocolytic agents, no placebo controlled study has shown neonatal improvement with magnesium sulfate tocolysis. This is similar to the studies of beta-sympathomimetic tocolytics and has led some authors (e.g., Higby) to suggest that safe dosages of magnesium sulfate are ineffective in preventing preterm birth and should not be used as a tocolytic agent. Although magnesium sulfate, like other tocolytics, has not fulfilled the initial promise of preventing preterm birth, it does appear if used correctly in a well identified population of patients to at least transiently inhibit preterm labor as well as other tocolytic agents with fewer side effects and fewer contraindications. PMID- 8616969 TI - Calcium channel blockers. PMID- 8616970 TI - Atosiban. AB - The role of oxytocin and oxytocin receptors in preterm labor is perhaps more prominent than once thought. The blockade of oxytocin and/or oxytocin receptors has both a theoretical and clinical aspect. Initial trials with the oxytocin antagonist atosiban have been encouraging with respect to efficacy and side effect profile. Current trials are examining the efficacy and dosing regimens of atosiban. Additional work must be done with this drug. However, if subsequent data demonstrate that atosiban is as effective as ritodrine in obtaining uterine quiescence, on the basis of its milder side effect profile, it should be seriously considered as a tocolytic of choice for the treatment of preterm labor. Moreover, it may have significant appeal for combination use with other tocolytics, given its alternative mechanism of action to betamimetics and magnesium sulfate. PMID- 8616971 TI - Risks and complications of tocolysis. AB - In this article, the author has reviewed available information on maternal, fetal, and neonatal risks and complications of tocolytic therapy. Because no ideal tocolytic agent exists, clinicians must be aware of the potential problems that can be encountered from initiating tocolysis. A variety of tocolytic agents may be effective in delaying preterm delivery. However, whether these agents reduce perinatal morbidity and mortality is debatable. This review emphasizes that serious complications occur from the use of these drugs including maternal, fetal, and neonatal death. Clinicians who use tocolysis, even oral terbutaline, must be aware of the significant side effects associated with these agents. Caution should be exercised not only in their initiation but also in their continuation. Additional research is needed and ongoing to limit the use of tocolytic agents and develop drugs with more efficacy, less risk, fewer complications, and more tolerable means of administration. PMID- 8616972 TI - The clinical use of antenatal corticosteroids. PMID- 8616973 TI - Adjunctive care of preterm labor--the use of antibiotics. PMID- 8616974 TI - Current status of home uterine activity monitoring. PMID- 8616975 TI - Delivery of the premature infant. AB - Significantly preterm delivery should ideally occur in centers with neonatal intensive care units and be attended by pediatric staff. The progress of labor, fetal heart rate patterns, and indications of fetal compromise differ substantially from those seen at term. Steroids and antibiotics are frequently indicated; research is focused on the use of phenobarbital, magnesium sulfate, and thyrotropin-releasing hormone. Selective use of episiotomy may be beneficial. The suggestion that cesarean delivery before the onset of labor reduces intraventricular hemorrhage in very low birth weight infants has not been demonstrated clearly. Vertical uterine incision is frequently needed for cesarean delivery of very low birth weight infants. Cesarean delivery is usually employed for very low birth weight infants in breech presentation or if either fetus in a twin gestation is not in vertex presentation, but is usually not needed for face or compound vertex presentations. Ultrasound examination should be performed to identify major malformations associated with preterm delivery. Serious morbidity is uncommon after 32 weeks' gestation; mortality and disability are concentrated in those with birth weights less than 1,000 grams. In the latter group, the decision of whether to intervene for fetal indications should be considered as soon as it appears delivery is likely. This decision should be based on early pregnancy dating, when available. In the absence of reliable dates, ultrasound measurements of biparietal diameter and femur length are more reliable than estimated fetal weight. PMID- 8616976 TI - New perspectives on the prevention of extreme prematurity. PMID- 8616977 TI - Progestogens, progesterone antagonists, progesterone, and androgens: synthesis, classification, and uses. PMID- 8616978 TI - Desogestrel. AB - Desogestrel is a new, potent progestogen with very low androgenic properties. When used as a contraceptive, it is a strong antiovulatory compound, even at low doses. The clinical efficacy is as good as that of the old progestogens. It has a low incidence of side effects and complications, similar to other progestogens. It may have a role as an anti-androgen in women with hyperandrogenic symptoms in need of adequate oral contraception. PMID- 8616979 TI - Gestodene-containing contraceptives. AB - As GSD is the most potent progestogen used in oral contraceptives, the doses of GSD can be lower than those of other progestogen components. The monophasic (30 micrograms EE + 75 micrograms GSD) and the triphasic formulation (30 micrograms EE + 50 micrograms GSD/40 micrograms EE + 70 micrograms GSD/30 micrograms EE + 100 micrograms GSD) suppress gonadotropin release and ovarian function profoundly and inhibit ovulation reliably. The strong anti-estrogenic and progestogenic effectiveness of GSD is based on the high GSD serum concentrations achieved during daily intake. Because of the weak androgenic properties of GSD, both formulations can be characterized as estrogen-dominant with respect to their hepatic effects. Except for the first cycles, both formulations afford good cycle control, and the rate of side effects is similar to that with comparable low-dose oral contraceptives. The levels of total and free androgens and androgen precursors, as well as of peripheral androgen activity, are significantly reduced, resulting in a reduced incidence of acne. The concentrations of SHBG and other serum-binding globulins are elevated considerably, and thyroid function is almost unaffected. The estrogen-dominant effect on hepatic metabolism of both formulations also is reflected by a significant increase in the levels of triglycerides and VLDL, HDL, and some apolipoproteins, while LDL-CH and total CH remain unchanged. Similar to other low-dose oral contraceptives, the GSD containing preparations cause a slight impairment of glucose tolerance that does not appear to be of clinical relevance. However, a significant increase exists in pro-coagulatory and fibrinolytic activity that leads to a considerable stimulation of fibrin turnover. In predisposed women, this may contribute to an elevated risk of venous and arterial thromboembolic diseases. PMID- 8616980 TI - Norgestimate. PMID- 8616981 TI - The contraceptive use of depot medroxyprogesterone acetate. PMID- 8616982 TI - Implantable levonorgestrel contraception: 4 years of experience with Norplant. PMID- 8616983 TI - The progestin-only pills and the levonorgestrel-releasing IUD: two progestin-only contraceptives. PMID- 8616984 TI - Progestogens in estrogen-replacement therapy. PMID- 8616985 TI - Micronized progesterone: vaginal and oral uses. PMID- 8616986 TI - The rational use of androgens in hormone replacement therapy. PMID- 8616987 TI - Clinical applications of the antiprogestins. PMID- 8616988 TI - Tc-99m sestamibi and In-111 pentetreotide uptake in metastatic papillary thyroid carcinoma. AB - Approximately 25% of metastatic and recurrent differentiated (papillary and follicular) thyroid carcinoma do not concentrate 1-131 Nal. In such cases, diagnosis can be attempted with other radiopharmaceuticals. The authors present a case of recurrent papillary thyroid carcinoma that was well visualized with Tc 99m sestamibi and In-111 pentetreotide; but only poorly visualized by I-131 Nal. PMID- 8616989 TI - Whole-body scintigraphy with radioiodine-131. A comprehensive list of false positives with some examples. AB - Whole-body scintigraphy with radioiodine-131 is an important diagnostic test in the management of patients with differentiated thyroid cancer who have undergone surgical treatment. The scan can demonstrate the presence of residual thyroid or functioning metastases in lymph nodes or distant sites. However, there are a number of potential pitfalls in the interpretation of this scan that could lead to a false-positive diagnosis of cancer. The scintiscans are presented for five patients in whom uptake outside of the thyroid was not due to functioning metastases. Some of these abnormalities are physiologic, such as uptake of iodine in the gastrointestinal tract. A comprehensive list of false-positive results are tabulated. PMID- 8616990 TI - False-positive I-131 images mimicking thyroid cancer metastasis. The nose ring sign. AB - This case report describes an I-131 whole-body scan with false-positive uptake at the site of a nose ring in a patient with follicular thyroid cancer. PMID- 8616991 TI - Radionuclide angiography in evaluation of cold solitary thyroid nodules. Improved diagnostic accuracy using flow and washout analysis. AB - Radionuclide thyroid angiography was performed in 252 patients with hypofunctioning thyroid nodules to evaluate differences in vascular flow and washout patterns in benign and malignant disease. Time activity curves of Tc-99m pertechnetate flow through the cold nodules were generated using region of interest software. Retention ratios of counts in the nodule at 2 minutes after radionuclide administration versus those at peak activity were derived. Patients subsequently underwent surgical excision and histopathologic examination. One hundred forty-four of the 204 benign nodules were avascular with absent radionuclide flow through the nodule. Fifty-six benign nodules were vascular with a prolonged radionuclide washout pattern with retention ratios ranging from 0.62 0.92. Forty-six of the 48 malignant nodules displayed increased perfusion with rapid radionuclide washout with retention ratios ranging from 0.28-0.48. Four benign nodules exhibited a similar flow and washout pattern. Radionuclide thyroid angiography with vascular flow and washout analysis appears to be a useful technique to differentiate between benign and malignant thyroid nodules with a high degree of sensitivity and specificity. PMID- 8616992 TI - The effect of exercise on normal splenic volume measured with SPECT. AB - In a study group of 20 healthy young men, splenic volume was determined with SPECT before and after exercise. A randomly chosen control group of 10 comparable men was studied similarly, but without exercise intervention. The mean splenic volume did not change significantly in the control group (i.e., from 292.9 ml to 282.1 ml [P = 0.75]). The mean splenic volume decreased 60.1 ml from 279.4 to 219.3 ml (21.5%) in the study group and this was highly significant (P = 0.01). Although exercise induced splenic autotransfusion is generally considered to be unimportant in humans, significant splenic contractility was observed with this technique. In the normal individual with a large spleen or with functional splenomegaly, the contractility response may become more important and can now be measured in a quick and easy manner. PMID- 8616993 TI - Relief of common bile duct obstruction during the course of hepatobiliary scintigraphy. AB - Hepatobiliary scintigraphy performed in a patient suspected of having common bile duct obstruction showed persistence of the hepatic parenchymal phase and no bile duct or gallbladder activity during the first hour of imaging. On endoscopic retrograde cholangiopancreatography examination several hours later, an obstructing gallstone was identified in the common bile duct, and the stone was extracted in conjunction with a papillotomy. Delayed scintigraphic images at 6 hours were unchanged from the earlier views, but imaging at 24 hours showed tracer activity in the small bowel and colon along with persistent hepatic parenchymal activity. These results demonstrate that hepatobiliary radiopharmaceuticals remain in an excretable form in the liver in patients with complete common duct obstruction, but that resumption of bile flow and tracer excretion does not occur until a number of hours after relief of the obstruction. PMID- 8616994 TI - Intrahepatic bile duct dilatation and cholestasis in autosomal recessive polycystic kidney disease. Demonstration with hepatobiliary scintigraphy. AB - Fifteen patients with the clinical and radiologic features of autosomal recessive polycystic kidney disease were investigated with radionuclide hepatobiliary scintigraphy. In nine patients (60%), cholestasis and intrahepatic bile duct dilatation were demonstrated. A 10th child had scintigraphic evidence of cholestasis, but the bile ducts were not dilated. The other five children has normal h hepatobiliary scans. The authors conclude that intrahepatic bile duct dilatation with cholestasis (Caroli's disease) is part of the clinical spectrum of autosomal recessive polycystic kidney disease and that hepatobiliary scintigraphy can be of value in determining the extent of hepatobiliary disease in this group of patients. PMID- 8616995 TI - In-111 pentetreotide. Superior imaging agent for gastrinomas. AB - Gastrinomas are usually diagnosed clinically and localized before surgery using various imaging modalities including ultrasound, computed tomography, magnetic resonance imaging, angiography, and venous sampling. The authors present a case in which CT, MRI, and angiography were negative in attempting to localize the gastrinoma, but In-111 pentetreotide localized the lesion, which was proven surgically. PMID- 8616996 TI - Detection of pelvic deep vein thrombosis by subcutaneous radionuclide venography utilizing an acupuncture point. AB - The authors present a case of thrombosis involving the right common iliac vein evaluated with subcutaneous radionuclide venography (SCRNV) after injection at acupuncture points. SCRNV demonstrated interruption of venous flow to the right common femoral vein and right iliac vein with prominent collateral venous drainage into the contralateral deep veins. This technique, which is technically simple and relatively operator-independent, has the potential to become a screen or follow-up test for deep vein thrombosis. PMID- 8616997 TI - Tc-99m tetrofosmin. A new diagnostic tracer for parathyroid imaging. AB - A 42-year old woman with hyperparathyroidism had a CT scan that was suggestive of a small nodular lesion in the left lower neck. Tc-99m sestamibi and Tc-99m tetrofosmin parathyroid imaging were performed 10 minutes and 2 hours after tracer injection. Early imaging with Tc-99m sestamibi demonstrated thyroid and focal uptake in the left lower neck. On delayed imaging, findings suggested a parathyroid adenoma. Imaging with Tc-99m tetrofosmin demonstrated similar findings. The abnormal parathyroid gland was an adenoma. PMID- 8616998 TI - Fortuitous imaging of a primary adrenocortical carcinoma with Tc-99m HDP. AB - The importance of evaluating nonfunctional adrenal masses in the right clinical setting is discussed. A 60-year-old man was initially diagnosed of having a localized lung carcinoma. Metastatic work-up showed an adrenal mass that was not deemed to be related to the lung primary. Although biochemical testing revealed that the adrenal mass was nonfunctional, adrenal scintigraphy was not performed. On resection, the lung neoplasm was shown to be a poorly differentiated adenocarcinoma. Radiologic follow-up of the adrenal finding was recommended. A year later, the patient presented with an abdominal mass that was visualized by bone scintigraphy and, on resection, proved to be adrenocortical carcinoma. In retrospect, the lung mass was a metastasis of an adrenocortical carcinoma. PMID- 8616999 TI - Three-phase bone and Ga-67 scintigraphy in disseminated sporotrichosis. AB - A 27-year-old man, who had been shoveling gravel in southern Texas for 3 years, had a history of papules and nodules in the left lateral wall of the abdomen. The lesions increased in number and severity with spread to other regions of the body. A punch biopsy of the right arm lesion revealed intracellular, round and cigar shaped budding yeast. The cultures grew Sporotrichum schenkii. Three-phase bone imaging and a Ga-67 scan defined the extent of the disease including involvement of the right tibia, left second metacarpal, and the left wrist joints, the latter two of which were not apparent on clinical examination. PMID- 8617000 TI - Scintigraphic findings in a Brodie's abscess. AB - A 9-year-old girl had a 6-month history of left hip pain. Radiographs of the left hip showed a metaphyseal osteolytic lesion with sclerotic borders in the femoral neck. Tc-99m MDP bone imaging and a Ga-67 scan showed focal areas of increased activity in the left femoral neck. These areas of increased uptake corresponded to a lytic area on x-rays, which was due to a Brodie's abscess. The combination of Tc-99m MDP bone and Ga-67 imaging has been widely used in the confirmation of bone infection, increasing the accuracy in the diagnosis of osteomyelitis. However, nuclear scintigraphy has not been previously reported in the confirmation of a Brodie's abscess. PMID- 8617001 TI - Bone SPECT imaging of vertebral hemangioma correlation with MR imaging and symptoms. AB - To evaluate the bone SPECT findings of vertebral hemangioma and their relationship to symptoms and MRI findings, the authors reviewed planar and SPECT bone scans and MR images of 15 vertebral hemangioma in 10 patients. All planar bone scan images demonstrated normal findings, except one, SPECT images demonstrated normal findings in 11 vertebral hemangioma less than 3 cm in diameter. Three of four vertebral hemangioma that were 3 cm or larger showed abnormally increased or decreased uptake on SPECT images. There was a disparity between SPECT imaging and MRI. PMID- 8617002 TI - Lymphoscintigraphy in a case of Merkel cell tumor. AB - The authors report a rare case of Merkel cell tumor with the findings of lymphoscintigraphy. The study was performed to delineate anatomic lymphatic drainage from the primary skin lesion to help surgeons to decide which lymphatic basin(s) should have elective lymph node dissection. The lesion was situated in the midline on the back and at the border zone in the lumbar area from which it could drain superiorly to the axillary lymph nodes(s), inferiorly to the inguinal node(s), or to both directions. Because of this location, prediction of the lymphatic drainage by applying conventional anatomical knowledge from the lymphatic atlas was not easy. However, the lymphoscintigraphy was very helpful and subsequent pathologic examination of surgical specimens confirmed the interpretation of the lymphoscintigraphy. PMID- 8617003 TI - Rupture of abdominal aortic aneurysm demonstrated on Tc-99m RBC gastrointestinal bleeding study. PMID- 8617004 TI - Splenic infarction detected by Tc-99m RBC scintigraphy. PMID- 8617005 TI - Three-phase bone scintigraphy in suppurative tenosynovitis. PMID- 8617006 TI - Bone scintigraphy in a patient with polymyositis. PMID- 8617007 TI - Accumulation of Tc-99m MDP in diaphragm and retroperitoneum of a battered child. PMID- 8617008 TI - Increased symmetric bone uptake during treatment with granulocyte colony stimulating factor and erythropoietin. PMID- 8617009 TI - Hepatobiliary imaging at 18 years after choledochoduodenostomy for biliary atresia. PMID- 8617010 TI - Stone in the cystic duct. Hepatobiliary scintigraphic findings. PMID- 8617011 TI - Discordant uptake of three different agents for liver scintigraphy in a patient with focal nodular hyperplasia. PMID- 8617012 TI - Pulmonary reverse mismatches due to endotracheal tube placement. PMID- 8617013 TI - Lung perfusion imaging in tricuspid atresia after Glenn and Fontan repair. PMID- 8617014 TI - Localization of a bronchopleural fistula in a postpneumonectomy patient using ventilation scintigraphy. PMID- 8617015 TI - Abnormal Tc-99m MDP renal images associated with hemolytic streptococcal sepsis. PMID- 8617016 TI - In-111 labeled leukocyte detection of metastatic renal cell carcinoma. PMID- 8617017 TI - Tc-99m DMSA imaging of crossed fused renal ectopia. PMID- 8617019 TI - Current readings in nuclear medicine. PMID- 8617018 TI - Bladder herniation detected on a bone scan. PMID- 8617020 TI - Perspectives on what's new in plastic surgery. The new millennium. AB - The next millennium will witness the most exciting advances in the field of plastic surgery. Some of these advances are already here today whereas others are just starting to surface. These include soft-tissue and bone reconstruction, tissue engineering, gene therapy, microsurgery, and fetal surgery to name just a few. Fetal wound healing may become a major part of intervention for correction of certain birth defects. Plastic surgery will continue to work with other specialties to solve clinical problems. Innovative techniques and other modalities will continue to rise in plastic surgery. PMID- 8617021 TI - Toe-to-hand transfer. Current concepts, techniques, and research. AB - The current status of toe-to-hand transfer at the Chang Gung Memorial Hospital is presented. Various combinations of toe-to-hand transfers have been used routinely for reconstruction of lost digits as well as parts of the digits distal to the sublimis insertion. Early, one-stage reconstruction is recommended in patients with clean wounds when replantation is not feasible. The decision of which technique to be used in certain patients is highly individualized. Technical refinements have been introduced aimed at simplifying the technique of toe harvesting and improving the function and appearance of the reconstructed digits as well as donor sites. Aggressive motor rehabilitation and sensory re-education are essential for optimal functional recovery. Current research projects involve evaluation of patients using the computerized work simulator, gait analysis, and somatosensory evoked potentials. Morphometric studies of the digital nerves in the fingers and toes have been performed to determine the effect of neurotization upon the degree of sensory recovery. PMID- 8617022 TI - "Microleaps" in the progression of flaps and grafts. AB - No momentous upheavals in the past decade have altered radically the knowledge or application of "flaps and grafts," but "microleaps" in slow and steady increments have ensured a continuum of progress. Fasciocutaneous flaps, owing to an intimate relationship with the superficial nerves, may be neurocutaneous flaps in disguise. Distal-based flaps that recruit orthograde circulation may be made even more reliable for extremity coverage in lieu of complex microsurgical techniques. As success at the recipient site has not become so routine, further outcome improvement has been directed toward enhancing the appearance and residual function at the previously neglected donor site. The future of plastic surgery will continue to parallel advances in the principles of flaps, constrained only by the boundaries of imagination. PMID- 8617024 TI - Gene therapy for plastic and reconstructive surgery. AB - In the future, gene therapy will become the standard treatment for enhancing wound healing and nerve and muscle regeneration and for preventing or treating vessel thrombosis, areas critical to the plastic surgeon and the patient. This review has focused on factors that are either currently used in a clinical situation or have the promise of being developed for clinical use. The role of gene therapy applied to these areas will be to locally place therapeutic genes at the site of injury or surgical repair, which will replace the need to systemically administer therapeutic agents that may have toxic effects, and to express factors in low abundance but at effective and safe levels. This strategy is to produce enough agent to have a therapeutic effect. The use of tissue specific promoters will be beneficial in localizing the production of agents to certain cell types. Gene therapeutic approaches used in plastic surgery may have a significant impact on the health care system by increasing efficiency of treatment and reducing health care costs. The accelerated pace of basic research in gene therapy will result in clinical applications in the near future. PMID- 8617023 TI - Pediatric plastic surgery. AB - Pediatric plastic surgery has seen widespread advances during the past decade. These have been primarily in the biologic understanding of certain disease processes as well as in diagnostic and therapeutic technologies. These advances have contributed to a potential new array of treatment options for congenital deformities and traumatic injuries. This review identifies several of the most significant new trends and treatment concepts in pediatric plastic surgery from anesthesia to wound healing. PMID- 8617025 TI - The role of orbital fat preservation in facial aesthetic surgery. A new concept. AB - Preservation of lower orbital fat in facial rejuvenation is a new concept for preventing the "operated" appearance that may follow fat removal in conventional blepharoplasty. By creating a narrower, more shallow orbit, the surgeon can create a truly youthful eyelid-cheek complex. Orbital fat advanced over the complete inferior orbital rim hides the bony framework that becomes apparent with normal aging. This technique is used routinely in isolated blepharoplasty and with composite rhytidectomy. It accompanies repositioning of the orbicularis oculi muscle, cheek fat, and facial platysma muscle. It is a procedure that can correct postoperative problems caused by excess orbital fat removal, lower eyelid retraction, and contour problems resulting from malar augmentation. The orbital fat is one of the many deep "pieces" of the human face that must be preserved and repositioned for optimal facial rejuvenation. PMID- 8617026 TI - Plastic surgery research. AB - Several areas of plastic surgery research, both in and out of the basic science laboratory, have been highlighted. Progress is being made in our ability to produce biologic structures to replace destroyed or damaged ones in vitro, our ability to manipulate the genetic structure of cells, and our ability to define and potentially manipulate the wound healing process in normal and abnormal healing states. More is also being learned about the biology of nerve repair, the physiologic response to silicone implants, and the mechanism of reperfusion injury. Virtual surgery may become a major part of the planning, performance, and teaching of surgical procedures in the future. Outcomes need to be better defined to prove the value of what plastic surgeons do to increasingly skeptical third parties, and progress is being made in this area as well. These areas represent only a few aspects of what is new in plastic surgery research, but they at least provide a window into the field and demonstrate the increasing sophistication of work being done in all areas. This work, as well as work in other areas, will undoubtedly affect clinical practices in the near future. PMID- 8617027 TI - Plastic surgery's plastics. AB - Alloplastic materials have become an essential part of reconstructing the craniofacial skeleton. This article reviews several of the more commonly used implant materials and summarizes their mechanical properties and use in reconstructive surgery. PMID- 8617028 TI - Lasers in aesthetic surgery. AB - Lasers are now in their third decade of medical application. The use for aesthetic surgery is relative new. Multiple attempts to use lasers in cosmetic surgery, in the past, resulted in less than desirable results. The current generation of lasers now has multiple applications in aesthetic surgery, with much success in tattoo removal, skin resurfacing, and cosmetic procedures. The plastic surgeon, with an augmented armamentarium, is now able to solve old problems and face new challenges. Careful attention to the fine details will allow the surgeon to avoid problems and complications when using the laser for aesthetic surgery. The ease of using the laser, speed of the procedure, patient satisfaction, and the minimal amount of potential complications all encourage the plastic surgeon to utilize the laser for aesthetic surgery. PMID- 8617030 TI - Alpha hydroxy acids in skin care. AB - Although not surgery in the traditional sense, skin care offers the ability to treat patients with a wide range of skin pathology. Skin care is another facet in facial rejuvenation, whose magnitude will increase as the pathophysiology and treatment of photoaging and other skin conditions becomes better understood. Patient satisfaction has been overwhelming for physician-directed skin care, but as the number of products, regimens, and adjuncts increase exponentially, we must emphasize a well-founded, scientific approach to the individual patient's pathology. PMID- 8617029 TI - What's new in aesthetic surgery. AB - This article describes new trends, techniques, and instrumentation in aesthetic surgery. Advances in our understanding of anatomy and the changes that come about with intrinsic and extrinsic factors are discussed. Specifically, anatomic approaches to rhytidectomy, the preservation of lid shape in blepharoplasty, and CO2 facial resurfacing are highlighted. Body contouring surgical techniques, including minimal scar breast reductions, endoscopic-assisted augmentation mammoplasty, and superficial liposuction, are reviewed. PMID- 8617031 TI - Addressing difficult areas in body contouring with emphasis on combined tumescent and syringe techniques. AB - The combined use of the tumescent and syringe techniques is a logical choice in addressing difficult areas in body contouring. Each technique complements the other. The magnification of the deformity by the tumescent technique allows for a more uniform and accurate contour correction. The syringe technique provides precision and accuracy that further contribute to body contouring refinement. The combined use of the tumescent and syringe techniques has allowed greater refinement and precision in achieving satisfactory body contouring and in minimizing complications associated with traditional techniques. PMID- 8617032 TI - The residual cleft lip nasal deformity. An anatomic approach. AB - The cleft lip nasal deformity presents an extremely challenging problem for the reconstructive surgeon. Consistent satisfactory results have been difficult to attain. This article demonstrates that by obtaining sufficient exposure, each abnormal component can be identified and appropriately reconstructed. PMID- 8617033 TI - Bone repair by regeneration. AB - Bone repair by regeneration as we know it continues to undergo changes, with advances approaching that may change our treatment of patients with craniofacial deformities and skeletal defects. Perhaps by the turn of the century, patients born with asymmetric deformities due to lack of growth will be treated early in life by skeletal stretching, and then later in life by skeletal distraction that is followed by use of accelerating factors to assist the healing processes. All of these available modalities are part of the regeneration of new bone formation. The future of such changes is very interesting, and our ability to help our patients will be maximized. We may even look back 25 years from now at bone grafting and find it to be obsolete and crude. It is hoped that with the new modalities being developed, we will not deviate from the use of a bone grafting procedure, which is the workhorse of the craniofacial surgeon. Bone grafting is used by all surgeons working on the craniofacial skeleton despite the problems of unpredictability of healing and an inability to calculate what percentage of the original graft will survive. The transplantation issue will be solved. The problems with donor site morbidity will continue. The use of inorganic bone substitutes will continue to have its limitation, particularly in type II wounds, which we as plastic surgeons see in the craniofacial region. As we redefine our approach to skeletal repair, we may look back and find solutions to some of the major problems we have had. The rapid stretch of soft tissue after facial advancement or structural alteration that is accompanied by a relapse due to the elastic recoil of the soft tissue could be eliminated by gradual distraction. The bone will undergo better functional adaptation when it has a gradual change in structure based on adjustment and molding in a gradual fashion. The problem of donor site morbidity and a prediction formula for bone could be resolved with new bone formation in situ by mineralization of the area under repair. Bone healing enhancers are here to stay and their clinical application will produce a far reaching better final outcome (Fig. 11). PMID- 8617034 TI - Contrast-enhanced MR imaging. PMID- 8617035 TI - Magnetic resonance imaging of the knee: assessment of effectiveness. AB - OBJECTIVES: To quantify how magnetic resonance imaging (MRI) influences clinicians' diagnoses, diagnostic confidence and management plans in patients with knee problems. To investigate whether these changes can bring about an improvement in health. METHODS: This was a prospective observational study on all patients referred to a regional unit for MRI of the knee over a 6-month-period. Data on diagnosis, diagnostic confidence and proposed management before MRI was compared with diagnoses and actual management after MRI. In addition, short form 36 item (SF-36) health survey data was collected at referral and again 6 months later. RESULTS: Three hundred and thirty-two patients were entered into the study. MRI led to previously unsuspected diagnosis in 69 of 269 patients with available data. When MRI confirmed the clinical diagnosis, significant improvements in clinicians' diagnostic confidence were found (P < 0.01 for medical meniscus, P < 0.05 lateral meniscus, P < 0.05 anterior cruciate). MRI led to a change in management in 180 (63%) of 288 patients (where data available). There was a significant shift away from surgical management after MRI (P < 0.01). SF-36 results were available in 206 patients. There was a significant improvement over time in five of the eight SF-36 scales (four at P < 0.001, one at P < 0.01). CONCLUSIONS: Magnetic resonance imaging significantly influences clinicians' diagnoses and management plans. These patients, examined by MRI, also recorded an improvement in health related quality of life. PMID- 8617036 TI - Magnetic resonance imaging of the knee: diagnostic performance studies. AB - OBJECTIVES: To review and reassess the published diagnostic performance statistics for MRI of the menisci and cruciate ligaments. To illustrate the potential sources and effects of bias in the evaluation of this widely accepted diagnostic technique. METHODS: Published evaluations of knee MRI were identified from the literature. Criteria for inclusion in the review were a total sample size > or = 35, arthroscopic correlation of MRI findings and presentation of complete results. Diagnostic performance statistics were then recalculated for each published study. RESULTS: Twenty-two studies were identified with sample sizes between 35 and 1014. The overall sensitivity for MRI of the menisci and cruciates was 0.88 (95% confidence interval 0.86-0.90). The overall specificity was 0.94 (0.93-0.94). Sampling error varied widely amongst studies and was rarely quantified. CONCLUSIONS: Diagnostic performance statistics are widely used. It is still not well appreciated that these are subject to sampling error. Such errors make meaningful comparisons between published studies more difficult. Nevertheless, the results for meniscal and cruciate lesions are consistently high and support the use of MRI for these common problems. The diagnostic performance of other applications of MRI should be subjected to similar critical review. PMID- 8617037 TI - The relative contribution of disc and vertebral morphometry to the angle of kyphosis in asymptomatic subjects. AB - In order to investigate the relative contributions of vertebra and inter vertebral disk to kyphosis, a series of 100 asymptomatic healthy women (age range 39-91 years) were studied to evaluate lateral dorsal appearances and possible related parameters of bone loss. Subjects underwent lateral dorsal spine radiography and single photon absorptiometry of the radius. There was a significant decrease in physical height in relation to age (P < 0.001) and this was directly related to an increasing thoracic kyphosis (P < 0.005). The angle of kyphosis was better related to the average anterior disc height (P < 0.001) than to average anterior vertebral height. The vertebral body ratio, however (anterior/posterior height), was more strongly related to angle of kyphosis than was disk ratio. The results also showed a fall in the proximal and distal radial bone mineral content with age (P < 0.001). These findings suggest that dorsal kyphosis as part of the ageing process may be as closely related to the physical integrity of the disc as to the vertebral body. Hence, therapy for age related bone mineral loss may have limited effect on a kyphotic deformity of the dorsal spine in otherwise asymptomatic patients. PMID- 8617038 TI - The pattern and distribution of calcified mediastinal lymph nodes in sarcoidosis and tuberculosis: a CT study. AB - Following granulomatous involvement, calcification of hilar and mediastinal lymph nodes is common and calcified nodes are frequently identified on computed tomography (CT). The aim of this study was to establish whether there is any difference in the pattern and distribution of such calcifications in tuberculosis (TB) and sarcoidosis. METHODS: The thoracic CT scans of 77 patients with proven TB (n=28) or sarcoidosis (n=49) were studied retrospectively. Lymph nodes were categorized by size and pattern of calcification: diffuse/homogeneous, focal deposits, eggshell, and complete nodal replacement by calcification. The position of hilar or mediastinal calcified nodes was recorded. RESULTS: Nodal calcification was present in 26 fo 49 (53%) sarcoidosis patients and 13 of 28 (46%) TB patients. The mean short axis diameter of calcified nodes was significantly larger in sarcoidosis patients (sarcoidosis 12 mm, TB 7 mm, P < 0.003). A focal pattern of calcification was commoner in sarcoidosis (58% sarcoidosis nodes, 23% TB nodes) and complete calcification in TB (62% TB nodes, 27% sarcoidosis nodes). When hilar node calcification was present it was more likely to be bilateral in sarcoidosis than in TB (65% and 8%, respectively, P < 0.001). CONCLUSION: CT of the mediastinum shows significant differences in distribution and pattern of calcification in lymph nodes in TB and sarcoidosis. Possible explanations for these differences include the route of lymphatic drainage of pulmonary TB and the caseating nature of tuberculous granulomas. PMID- 8617039 TI - Magnetic resonance imaging of pericardial constriction: comparison of cine MR angiography and spin-echo techniques. AB - AIM: Spin-echo (SE) MRI detects pericardial thickening in pericardial constriction but the validity of extrapolating SE criteria to cine MRA imaging has not been tested. Pericardial thickness measured by SE and cine MRA was compared in patients with and without pericardial thickening to determine if the range of pericardial thickness measured by the two techniques is the same. PATIENT AND METHODS: Fourteen patients, investigated for possible pericardial constriction (PC), were compared with 24 subjects without evidence of pericardial disease (controls). Images were acquired using SE and cine MRA. Pericardial thickness was compared with final diagnosis. RESULTS: Pericardial thickening ( > 3.5 mm) by SE detected pericardial constriction: sensitivity = 100% specificity = 96%, kappa = 0.91. Cine MRA had a sensitivity = 86%, specificity = 63%, kappa = 0.33. Maximum differences between SE and cine MRA pericardial thickness ranged from +2.5 mm to -2/7 mm. CONCLUSIONS: Spin-echo identifies pericardial thickening with little overlap between measurements in patients with and without pericardial constriction. Pericardial thickness on cine MRA usually exceeds SE thickness, but with considerable overlap of thickness measurements in patients with and without pericardial constriction. Cine MRA cannot be used alone to diagnose pericardial thickening. PMID- 8617040 TI - Patterns of supradiaphragmatic metastases in testicular germ cell tumours. AB - The imaging and clinical findings of 31 patients with supradiaphragmatic nodal metastases from primary testicular germ cell cancers were reviewed. In 11 patients the primary testicular tumor was a seminoma, and in 20 a non seminomatous germ cell tumour (NSGCT). The patterns of spread of these tumour types were compared. All patients had chest radiographs (CXR): 27 had chest computed tomography (CT). One patient with seminoma and eight with NSGCT (40%) had lung metastases. Mediastinal lymphadenopathy was seen on CT in seven patients with seminoma (64%) and in nine patients with NSGCT (45%). Neck lymphadenopathy was present in 10 of 11 (91%) patients with seminoma, and 13 of 20 (65%) patients with NSGCT. Neck disease co-existed with mediastinal disease in six of 11 (55%) patients with seminoma, but in only two of 20 (10%) patients with NSGCT (P=0.012). This study gives further supporting evidence for the relative importance of haematogenous spread in teratoma, and lymphatic spread in seminoma. The contiguous nature of disease spread from abdomen to chest and neck in seminoma is confirmed. In NSGCT, supradiaphragmatic spread is more random but tends to occur in the paraoesophageal and subcarinal groups. PMID- 8617041 TI - The positive predictive value of mammographic signs: a review of 425 non-palpable breast lesions. AB - The sensitivity of mammography in cancer detection needs to be high but is also important to achieve a high diagnostic specificity to avoid the morbidity associated with unnecessary surgical biopsy. We have reviewed the mammographic features of non-palpable breast lesions to identify factors which may improve the specificity of mammographic interpretation and reduce the number of open surgical biopsies for benign lesions. Four hundred and twenty-five surgical biopsies of non-palpable breast lesions were performed following image-guided localization between January 1987 and April 1994. The mammographic features of these lesions were reviewed and correlated with their histology. Two hundred and twenty-five of the excised lesions were malignant and 200 were benign giving a benign to malignant ratio of 0.88:1 and a positive predictive value (PPV) for malignancy of 53%. Pre-operative fine needle aspiration cytology was performed in 359 cases (84%). The PPV for the various mammographic abnormalities following the assessment process ranged from 4% for well defined masses to 94% for spiculate masses. The PPV for all microcalcifications was 45%. For impalpable lesions it is our policy to recommend surgical excision of all spiculate masses, parenchymal deformities and high risk microcalcifications. Ill defined masses are managed according to fine needle aspiration cytology (FNAC) and/or core biopsy results. Masses which are entirely well-defined are regarded as benign and are not recalled for assessment unless they are new or enlarging. Needle core biopsy has been added to our preoperative assessment of mammographically indeterminate microcalcifications with the aim of reducing the number of benign surgical biopsies. PMID- 8617042 TI - A three centre audit of IVU referrals in patients with asymptomatic microscopic haematuria. AB - There is considerable debate concerning the investigation of patients with asymptomatic microscopic haematuria. Urine dipstick testing is a sensitive screening test but may be positive in some normal individuals. The present consensus is that urine microscopy should be performed to confirm haematuria prior to further investigation. We have performed a retrospective study to establish whether urine microscopy was used in addition to dipstick testing before a request for intravenous urography (IVU) in three centres. IVU request forms from a District General, a Teaching Hospital and a Uroradiological Referral Centre were audited over a 9-month-period. Patients referred with asymptomatic microscopic haematuria were selected. The case notes and urine microscopy results were reviewed. The date of microscopy and its result and the interval between the result and the IVU request were established. One hundred and two cases have been examined, 17 (16.7%) of which were under the age of 40. Only 37 in total had significant haematuria on microscopy and of these, 32 results (31.4% of all cases) were available before the IVU request. In eight patients there was no evidence that urine microscopy had been performed. Fourteen patients had a urinary tract infection. Our findings show that IVUs are often requested on the basis of dipstick testing alone. Only one third of patients had confirmed significant haematuria at the time of IVU request and in some patients infection had not yet been excluded. Although prompt investigation of microscopic haematuria is important, it is essential that the diagnosis is established by microscopy before an IVU is requested. PMID- 8617043 TI - Detection of occult wrist fractures by magnetic resonance imaging. AB - The early detection and management of wrist fractures may avert potential complications and patient morbidity. Five cases are described in which radiographically-occult fractures were clearly demonstrated by magnetic resonance imaging (MRI). These fractures were seen on MRI as a low signal linear band on T1 and T2-weighted images, with surrounding increased signal intensity from bone marrow oedema on T2-weighted images. In one case, intense oedema obscured the fracture line on the initial MR scan. MRI is useful in confirming the presence of a fracture, delineating its exact shape and location, and in the follow-up of these injuries during and after treatment. PMID- 8617044 TI - Osteosarcoma metastatic to the pancreas in young patients. AB - Two rare cases of pancreatic metastases from osteosarcoma are reported. Metastatic spread occurred after treatment with chemotherapy. This unusual phenomenon represents an alternation in the natural history of osteosarcoma with increased long-term survival of patients who are successfully treated with chemotherapy. PMID- 8617045 TI - Case report: myolipoma or liposarcoma--a mistaken identity in the retroperitoneum. PMID- 8617046 TI - Case report: transabdominal ultrasound detection of achalasia. PMID- 8617047 TI - Case report: multiple lytic lesions in HTLV1 associated ATLL in the absence of lymphadenopathy and peripheral blood changes. PMID- 8617048 TI - Case report: bilateral recurrent tension pneumothorax complicating combination chemotherapy for soft tissue sarcoma. PMID- 8617049 TI - Barium enema: do antispasmodics really improve performance and quality? PMID- 8617050 TI - Improving visualization of distal and terminal ileum during the small bowel meal: an evaluation of fluoroscopic manoeuvres. PMID- 8617051 TI - Retrograde radiological retrieval and replacement of double-J ureteric stents. PMID- 8617052 TI - Retrograde radiological retrieval and replacement of double-J ureteric stents. PMID- 8617053 TI - Wessex Regional Radiology Audit: barium enema in colo-rectal carcinoma. PMID- 8617054 TI - Pre-operative cervical spine radiography in rheumatoid arthritis. PMID- 8617055 TI - Chagas' heart disease. PMID- 8617056 TI - Fiberoptic bronchoscopy: enhancing the diagnostic yield. PMID- 8617057 TI - Guidelines in need of guidance. PMID- 8617058 TI - Outcomes of home care for life-supported persons: long-term oxygen and prolonged mechanical ventilation. PMID- 8617059 TI - Vasodilator therapy in acute respiratory failure. PMID- 8617060 TI - Science challenges the dogmas of CPR. PMID- 8617061 TI - Death by syrinx: worse than Ondine's curse? PMID- 8617062 TI - Tough love and the pulmonologist. PMID- 8617063 TI - Diagnostic value of protected BAL in diagnosing pulmonary infections in immunocompromised patients. AB - STUDY OBJECTIVES: To assess the diagnostic utility of protected BAL (P-BAL) in respiratory infections in immunocompromised patients and to examine whether P-BAL alone could substitute the combined use of protected specimen brush (PSB) and BAL in such patients. PATIENTS AND STUDY DESIGN: Thirty-seven immunocompromised patients who underwent PSB, P-BAL, and BAL simultaneously for the diagnosis or exclusion of bacterial or nonbacterial opportunistic respiratory infections were studied prospectively. The P-BAL was performed through the inner catheter of a telescoping plugged catheter with 60 mL of saline solution. MAIN RESULTS: Thirteen (35%) cases of bacterial pneumonia were diagnosed. PSB obtained seven true-positive (TP) results, P-BAL obtained nine, and BAL obtained eight TP. Results of the three techniques were positive and concordant in 6 of the 13 cases. PSB remained free of contamination from oropharyngeal flora in all cases, P-BAL was contaminated twice, and BAL was contaminated in four cases. Opportunistic respiratory infections were diagnosed in 19 patients. P-BAL results were identical to those with BAL in all cases: 18 TP and 1 false-negative. The average volume of P-BAL fluid retrieved was 19 mL, sufficient for all microbiologic and cytologic processings. P-BAL was more time-consuming than both PSB and BAL procedures and was technically more complex. CONCLUSION: P-BAL alone can substitute the combined use of both PSB and BAL in immunocompromised patients and attains a higher sensitivity than PSB in diagnosing bacterial pneumonia. The combined strategy continues to be a good choice, but due to the high incidence of bacterial pneumonia in these patients, a highly efficient diagnostic procedure is required not only for nonbacterial opportunistic respiratory infections but also for bacterial pneumonia. PMID- 8617064 TI - Screening for bronchiectasis. A comparative study between chest radiography and high-resolution CT. AB - STUDY OBJECTIVE: The aim of our study was to investigate whether in the search for bronchiectasis a correlation exists between abnormalities on the chest radiograph and high-resolution CT (HRCT), and if HRCT has an additional value when the chest radiography is normal. SUBJECTS AND METHODS: In a prospective study, chest radiographs were compared with HRCT in 84 patients. Analysis of presence and extent of bronchiectasis were made for each bronchopulmonary lobe. RESULTS: Thirty-seven patients had a normal radiograph, from whom 32 had a normal HRCT. The other 5 had a low HRCT severity score with a mild cylindrical bronchiectasis. From the 47 patients with an abnormal radiograph, 36 had signs of bronchiectasis at HRCT; 11 patients, however, had a normal HRCT. The sensitivity for chest radiography to detect bronchiectasis appeared to be 87.8% with a specificity of 74.4%. We found a significant linear relationship between the severity of bronchiectasis at HRCT and abnormalities as seen on the chest radiograph (r=0.62, p=0.0001). CONCLUSION: A normal chest radiograph almost always excludes relevant bronchiectasis and no further investigation seems necessary. There is a significant linear relationship between the severity of bronchiectasis at HRCT and abnormalities as seen on the chest radiograph. PMID- 8617065 TI - The role of functional respiratory tests in predicting pneumothorax during lung needle biopsy. AB - In 243 of 945 patients having lung percutaneous needle biopsy, we retrospectively evaluated 10 independent variables that potentially influence the incidence of pneumothorax (PNX): age, sex, lesion size, number of needle passes, needle intrapulmonary route, distance of the lesion from the chest wall, FEV1, FVC, residual volume, and total lung capacity percent predicted. The subjects considered (mean age, 61 years; 192 men and 51 women) performed respiratory function tests within 1 year of the procedure. The sample was also subdivided into four functional groups: restricted patients, obstructed ones, obstructed with alveolar hyperinflation patients, and normal subjects. The variables significantly correlated to PNX were the length of the needle intraparenchymal route (p<0.05) and the distance of the lesion from chest wall greater than 0 cm (p<0.01). None of the functional parameters was determinant in predicting PNX occurrence. Bronchial obstruction was not significantly associated with a higher risk of PNX. Nevertheless, when alveolar hyperinflation was associated with bronchial obstruction, the risk increased significantly (odds ratio, 2.38). PMID- 8617066 TI - Transthoracic needle aspiration in patients with severe emphysema. A study of lung transplant candidates. AB - PURPOSE: To describe the risks of transthoracic needle aspiration (TTNA) in a population of patients with severe lung disease: candidates for lung transplantation. MATERIALS AND METHODS: Eight of 190 patients evaluated for lung transplantation underwent TTNA of nine pulmonary nodules (mean diameter, 14 mm; range, 0.8 to 2.2 cm). We evaluated pneumothorax rate, chest tube rate, duration of placement, and pulmonary function test results. RESULTS: All patients had emphysema; two had alpha 1-antitrypsin deficiency. The mean FEV1 of all patients was 0.64 L (22% of predicted; range, 17 to 28%), indicating severe air-flow obstruction. Six patients required a chest tube (50%); three chest tubes were placed emergently on the CT scanner table. Three patients required a second chest tube for persistent air leak. Tubes were in place for 1 to 22 days (mean, 10 days). One patient had chest tubes for 22 days and required intubation. CONCLUSION: TTNA in patients with marked emphysema is complicated by a high incidence of pneumothorax, rapid development of tension pneumothorax and chest tube placement. Since nodules in lung transplant candidates may represent bronchogenic carcinoma, serial CT scans to demonstrate lesion stability or growth, or thoracoscopic resection should be considered as an alternate approach to TTNA to avoid the significant morbidity of the procedure in these patients. PMID- 8617067 TI - Bronchoscopic diagnosis of solitary pulmonary nodules and lung masses in the absence of endobronchial abnormality. AB - To evaluate the individual and additive diagnostic yield(s) of several bronchoscopic sampling techniques for the diagnosis of lung lesions with no corresponding airway abnormalities, consecutive patients with lung nodules or masses were prospectively evaluated between December 1989 and November 1994. A CT of the chest was done in all patients before flexible bronchoscopy (FB). Size, location, and character of the border of the lesion were determined. During FB, using biplane fluoroscopic guidance, the lesion was localized and following sampling techniques were done: brushing, transbronchial lung biopsy (TBLB), and Sofcor transbronchial needle aspiration (STBNA). Bronchial washings (BWs) were collected throughout the procedure. Problems associated with each sampling technique were noted. Forty-nine patients underwent 51 FB. A diagnosis was established by FB in 36 (73%). After a nondiagnostic FB, histologic diagnosis was established in 9 of 13 patients by other methods. A benign or malignant nature of lesion was established in other four patients by clinical follow-up. FB was diagnostic in 32 of 40 (80%) patients with primary lung cancer, in 3 of 6 (50%) patients with benign disease, and in 1 of 3 (33%) patients with metastatic disease to lung. All sampling procedures could be done in 33 of 51 (65%) FBs. Overall diagnostic yields were as follows: BW, 18 of 51 (35%); brush, 25 of 48 (52%); TBLB, 23 of 40 (57%); and STBNA, 19 of 37 (51%). In 12 of 51 (24%) FBs, only one sample was diagnostic. Lesions with sharp borders had a lower combined diagnostic yield, 13 of 24 (54%) compared to lesions with fuzzy borders, 20 of 24 (83%) (p=0.03). Yield of TBLB in lesions with fuzzy borders, 14 of 18 (78%), was higher than the yield for lesions with sharp borders, 6 of 19 (32%) (p=0.005). Size of the lesion in centimeters in patients with a positive FB (4.55+/- 2.35; mean +/-SD) was significantly larger than in patients with a nondiagnostic FB (3.14+/-1.31; mean+/-SD) (p=0.009, t test). Diagnostic yield was directly related to the size of the lesion. For lesions less than 2 cm, yield was 6 of 11 (54%) (p=0.19); for lesions less than 3 cm, yield was 12 of 21 (57%) (p=0.07); and for lesions greater than 3 cm, yield was 24 of 30 (80%). Yield from lesions located in the lower lobe basal segments or the apical segment of upper lobes was lower (11/19, 58%) than that from lesions in other parts of the lung (25/30, 83%) (p=0.05). FB was terminated prior to collecting all samples because of severe bleeding after brushing (n=3) or instability of the patient (n=4). None of the patients required intubation. There were no pneumothoraces. Diagnostic yield of FB depends on the location, size, character of the border of the lesion, and the ability to perform all sampling methods. Brushing, TBLB, and STBNA should be performed in all patients to give the best diagnostic yield. Routine cytologic examination of BW is unnecessary. Methods other than FB should be considered for lesions 2 cm or less in size, especially when they have a sharp border and/or are located in the basal segment of a lower lobe/apical segment of an upper lobe. PMID- 8617068 TI - Silicone stents in the management of benign tracheobronchial stenoses. Tolerance and early results in 63 patients. AB - STUDY OBJECTIVE: To assess tolerance and early results of Dumon silicone stents inserted in patients with nonneoplastic airway obstruction. DESIGN: Tracheobronchial stenting for palliative or curative restoration of airway narrowing has been evaluated in a retrospective study. SETTING: Tertiary-referral teaching hospital. PATIENTS: Between May 1991 and September 1994, 64 patients with a mean age of 52 years had endobronchial silicone stent insertion for benign tracheal stenosis (82% secondary to intubation or tracheostomy injury). Lesions were pure fibrous stenosis in 25 patients and fibroinflammatory stenosis in 38. Prostheses were used for temporary stenting of the airway during 18 months in 48 patients in whom cure was expected and as a procedure for palliation in the remaining 15 patients. INTERVENTIONS: In all cases, the Dumon tracheobronchial stent was implanted with the rigid bronchoscope under general anesthesia. RESULTS: Five patients died (four from unrelated causes); one was due to hypersecretion and airway obstruction at the time of an emergency tracheostomy 20 days after stent insertion. Complications included migration of prostheses in 11 (17.5%) patients, granuloma formation in 4 (6.3%) patients, and airway obstruction due to heavy secretion in 4 (6.3%). In 48 patients who received silicone stents with curative expectations, removal of the device was accomplished in 21 patients. Therapy proved successful in 17 patients with a mean follow-up of 259+/- 173 days and stenosis recurred in 4. In 16 patients, stents still remain for a mean period of 364+/-119 days. In the series of 15 patients in whom silicon stents were implanted for palliation, prostheses were placed permanently in 11 with a mean follow-up of 486+/-260 days. In the remaining four patients with tracheostomy, silicone stents were used after inability to expand the upper limb of the T-tube (two patients) or placed above the tracheostomy stoma to maintain laryngotracheal patency and preserve phonation when a T-tube was poorly tolerated (two patients). CONCLUSIONS: Silicone tracheobronchial stents are effective in the maintenance of airway patency and are associated with good tolerance and infrequent complications that are rarely life-threatening. PMID- 8617069 TI - Energy metabolism of thoracic surgical patients in the early postoperative period. Effect of posture. AB - STUDY OBJECTIVE: To determine the effect of elective thoracic surgery on energy metabolism and gas exchange and to evaluate whether the 30-degree sitting position would affect these variables. DESIGN: Prospective, unblinded, controlled study. SETTING: Surgical ICU in a university hospital. PATIENTS: Twenty-two adult patients undergoing elective pulmonary resection. INTERVENTIONS: Posture change from supine to 30-degree sitting position. MEASUREMENTS AND RESULTS: Oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ), and energy expenditure (EE) were measured by means of computerized indirect calorimetry before and after surgery. Heart rate and respiratory frequency were measured continuously during gas exchange measurement. Blood gases were analyzed with an automated blood gas analyzer. Preoperatively, altering position did not affect energy metabolism, gas exchange, and cardiopulmonary variables. Postoperatively, the measured EE was 116% of the expected value. Mean EE and VO2 values for each position were higher than the preoperative values for the corresponding postures (p<0.05 for each position), while VCO2 increased only in the supine position (p<0.05). Mean percent increases in EE, VO2, and VCO2 were significantly lower in the 30-degree sitting position than in the supine position (EE: 7.9+/-2.7% vs 14.4+/-2.3%; p<0.001; VO2: 9.0+/-3.0% vs 16.4+/- 2.6%; p<0.001; VCO2: 3.2+/-2.1% vs 6.5+/-1.4%: p<0.05). Arterial oxygen tension and all the physiologic indexes of gas exchange for each position were worse than the preoperative values for the corresponding postures (p<0.05 for each position). Mean arterial pressure, heart rate, and respiratory frequency for each position were higher than the preoperative values for the corresponding postures (p<0.05 for each position). No changes in mean values of these variables occurred between the two positions postoperatively. CONCLUSIONS: The early postoperative period of patients undergoing elective thoracic surgery is characterized by a condition of impaired gas exchange and by a hypermetabolic state. Hypermetabolism can be partly mitigated by assuming the 30-degree sitting position. PMID- 8617070 TI - The effect of physical therapy on respiratory complications following cardiac valve surgery. AB - STUDY OBJECTIVE: To determine whether higher personnel intensive chest physical therapy can prevent the atelectasis that routinely follows cardiac valve surgery. DESIGN: Randomized, controlled trial. SETTING: Tertiary care hospital. PATIENTS: Seventy-eight patients undergoing elective cardiac valve surgery between October 1991 and April 1993 were enrolled. INTERVENTIONS: Patients were randomized in an unmasked fashion to receive early mobilization and sustained maximal inflations (lower-intensity treatment) or to receive early mobilization, sustained maximal inflations, and single-handed percussions (higher-intensity treatment.) MEASUREMENTS AND RESULTS: Clinical efficacy was determined by extent of atelectasis, length of ICU stay, total length of hospital stay, and personnel costs. The extent of postoperative atelectasis was similar in both groups on the fifth postoperative day. Postoperative values of FVC and FEV1 were reduced to a similar extent in both groups. Hospital stays and ICU stays were similar regardless of treatment. Physical therapy costs were highest in the higher intensity therapy group. CONCLUSIONS: Postoperative respiratory dysfunction is common but does not usually cause significant morbidity or prolong hospital stay. The routine prescription of high-intensity physical therapy does not improve patient outcomes but does add significantly to patient costs. PMID- 8617071 TI - Sleep deprivation worsens obstructive sleep apnea. Comparison between diurnal and nocturnal polysomnography. AB - Daytime polysomnography (DPG) has been suggested for diagnosis of obstructive sleep apnea syndrome (OSAS), because it is less expensive than whole-night polysomnography investigation (NPG). To ensure sleep during day recordings, patients are often instructed to stay awake the night preceding DPG. This procedure has been validated against NPG, and also against apnea mattress recordings combined with ear oximetry (AMO). Twenty patients with OSAS symptoms were examined with NPG and simultaneous AMO and 2 to 3 weeks later with DPG 3 to 4 h in the morning after 1 nights sleep deprivation. Median apnea-hypopnea index (AHI) of DPG was 37 (95% confidence interval [CI], 19 to 44), significantly higher than median AHI of NPG (14; 95% CI, 12 to 27), whereas median nocturnal oxygen desaturation index (ODI) (11; 95% CI, 9 to 25) did not differ significantly from median AHI of NPG. Sensitivity values for DPG increased from 81 to 100% when the criteria AHI greater than 5, greater than 10, greater than 15, and greater than 20 were used, respectively. Specificity values also increased with the AHI used as cutoff point, from 50% (AHI>5) to 75% (AHI>20). In AMO, there were one false-negative case and four nonclassifiable borderline cases. If these types of simplified tests for OSAS are used for diagnosis, the risk of both false-negative and positive results (DPG) or nonclassifiable borderline cases (AMO) must be considered. Since there was a significant increase in AHI in DPG after sleep deprivation in comparison to conventional NPG, the former procedure should not be used for staging of the disease. These results also stress the importance of advice to OSAS patients concerning regular sleeping habits. PMID- 8617072 TI - Respiratory effort. A factor contributing to sleep propensity in patients with obstructive sleep apnea. AB - To test the hypothesis that respiratory effort during obstructive apneas contributes, together with hypoxemia and sleep fragmentation, to excessive daytime sleepiness, we investigated the relationship between daytime sleepiness and polysomnographic variables in 44 patients with obstructive sleep apnea (OSA). In all patients, daytime sleep propensity was assessed by an 11-item standardized self-questionnaire yielding a sleepiness score and by a modified sleep latency test yielding a mean sleep latency. Respiratory effort during apneas was evaluated by measuring esophageal pressure swings using an esophageal balloon. Within each apneic cycle, we measured the esophageal pressure swings during the first three and the last three occluded efforts during the apnea to define the overall increase, its ratio to apnea duration, and the maximal effort developed during obstruction. In the group of patients as a whole, the sleepiness score was negatively correlated with the mean sleep latency (r=-0.38, p=0.01). The sleepiness score was correlated with the indexes of respiratory effort during apneas (ie, the overall increase, its ratio to apnea duration, and the maximal end-apneic swing in esophageal pressure) and with the apnea+hypopnea index. The mean sleep latency was correlated with all indexes of nocturnal hypoxemia (ie, the mean lowest oxyhemoglobin saturation [SaO2] and the index of apnea associated with a fall in SaO2 below 90% and 80%). We conclude that the degree of respiratory effort during obstructive apneas contributes to self-rated sleep propensity in patients with OSA. PMID- 8617073 TI - Sleep-disordered breathing in men with coronary artery disease. AB - OBJECTIVE: To examine the occurrence of sleep apnea and nocturnal hypoxemia in men with symptomatic coronary artery disease (CAD) and to evaluate the relationship between disordered breathing and coronary artery disease. DESIGN: Case-control study. Cases were randomly selected from men undergoing coronary angiography because of angina pectoris. Controls were age matched and selected from the population registry. Pulse oximetry, oronasal thermistors, body position indicator, and recording of body and respiratory movements were used to quantify desaturations and apneas. SETTING: Norrland University Hospital, a referral center for northern Sweden. SUBJECTS: One hundred forty-two men with angina pectoris and angiographically verified CAD and 50 controls without known heart disease. MAIN OUTCOME MEASURES: The number of arterial oxygen desaturations of 4% or more per hour of sleep, oxygen desaturation index (ODI), and the number of apneas or hypopneas per hour of sleep, apnea-hypopnea index (AHI). RESULTS: Men with CAD had a high occurrence of sleep-disordered breathing measured as ODI of 5 or more, 39% (n=55), or AHI of 10 or more, 37% (n=50), while, the same proportions in controls were 22% (n=11, p<0.05) and 20% (n=10, p<0.05). Mean values of ODI in cases and controls were 6.4 and 2.7, respectively (p<0.001). Multiple logistic regression analysis identified ODI, AHI, body mass index, and hypertension as significant predictors of CAD (p<0.05). CONCLUSION: Sleep- disordered breathing is common in men with CAD. A significant association between sleep apnea with nocturnal hypoxemia and CAD remains after adjustment for age, hypertension, body mass index, diabetes, and smoking. PMID- 8617075 TI - Sleep, breathing, and cephalometrics in older children and young adults. Part II - Response to nasal occlusion. AB - STUDY OBJECTIVES: We postulated that nasal occlusion would provide a challenge enabling us to assess factors predisposing development of sleep apnea in older children/adolescents and young adults. Factors of interest included sex, age, body mass index (BMI), tonsillar hypertrophy, and cephalometric measurements. DESIGN: Sleep and breathing variables were examined and compared for four groups of subjects between one baseline night and one night of nasal occlusion in a sleep research laboratory. SUBJECTS: Healthy, normal boys (n=23, mean age=13.3+/ 2.1 years), girls (n=22, mean age=13.8+/-1.8 years), men (n=23, mean age=22.2+/ 1.5 years), and women (n=24, mean age=22.4+/-1.8 years) were studied. MEASUREMENTS AND RESULTS: The following sleep and sleep-related breathing measures showed significant increases in all four groups from baseline to occlusion: percentage of stage 1, number of transient arousals, transient arousal index, apnea index, respiratory disturbance index (RDI), and mean apnea length. No significant relationships were found between occlusion-night RDI and tonsillar size, cephalometric variables, or BMI, either singly or in combination. CONCLUSIONS: Subjects' responses to nasal occlusion varied: most demonstrated a minimal and clinically insignificant increase in RDI; few showed a marked increase in RDI. Significant increases of sleep fragmentation -- even in the absence of frankly disturbed breathing -- indicate that nasal occlusion may secondarily affect waking function if prolonged over a series of nights. PMID- 8617074 TI - Sleep, breathing, and cephalometrics in older children and young adults. Part I - Normative values. AB - STUDY OBJECTIVES: Aims were (1) to provide normative values for sleep and sleep related breathing variables and physical features (cephalometrics, body mass index [BMI], and tonsillar size) in older children/adolescents and young adults, (2) to describe sex and age group differences, and (3) to evaluate relationships between physical features and sleep-related breathing variables. DESIGN: Standard polysomnographic variables describing sleep and breathing were measured during a single night. Cephalometric measures were obtained from a standing lateral skull radiograph. SUBJECTS: Normal, healthy boys (n=23; mean age=13.3+/-2.1 years), girls (n=22; mean age =13.8+/-1.8 years), men (n=23; mean age=22.2+/-1.5 years), and women (n=24; mean age=22.4+/-1.8 years) with BMI less than 27 were evaluated. RESULTS: Sleep variables showed age group and sex differences consistent with published norms. Slow-wave sleep and rapid eye movement (REM) latency declined with age; transient arousals increased with age. Sleep-related breathing variables showed few changes related to age group or sex; small but statistically significant sex differences were found for arterial oxygen saturation nadir (lower in male subjects) and respiration disturbance index in non-REM sleep (greater in male subjects). Differences in cephalometric measures largely reflected normal growth and expected sex differences. No significant relationships between sleep-related breathing variables and physical findings were observed. CONCLUSIONS: These data provide well-controlled normative values for sleep, breathing, and cephalometrics in a group of normal older children, adolescents, and young adults. The data provide useful reference points for patients of these ages in whom sleep apnea is suspected, particularly since such clinical studies are normally based on first-night polysomnography. Furthermore, these values represent developmentally appropriate grouping of the data. PMID- 8617076 TI - Can sleep and wakefulness be distinguished in children by cardiorespiratory and videotape recordings? AB - Polysomnography, including EEG recording, is the standard method to diagnose obstructive sleep apnea (OSA) in children and adults. Diagnosis of OSA would be considerably simplified if it was shown that sleep could be distinguished from wakefulness without EEG recordings. Therefore, we compared sleep/wakefulness classification using a simplified cardiorespiratory-video (CRV) method with standard polysomnography in 20 children undergoing in-hospital evaluation for OSA. The channels for the simplified montage were chosen because they (1) were suitable for unattended, home recordings, (2) allowed the diagnosis of apneas, hypopneas, desaturation, and movement/arousals, and (3) did not require attachment to the head or face that might disturb the childs sleep. Sleep staging by the two methods was blinded to results of the other method. We evaluated 21,832 30-s epochs--1,092+/-111 (SD) per child. Across 20 subjects, 79.7+/-7.1% of the epochs were sleep. The simplified montage agreed with polysomnographic classification of sleep/wakefulness for 93.8+/-2.5% of the epochs. Of all sleep epochs, 97.7 (96.4, 98.1%) median (interquartile range), were correctly classified; sleep predictive value of the CRV method was 95.2+/-2.8%. Of all epochs classified as wakefulness by polysomnography, 80.1+/-12.8% were correctly classified by the CRV method. The wakeful predictive value was 88.7+/-2.6%. Kappa values averaged 0.8+/-0.1, indicating that agreement between the CRV method and polysomnography did not occur by chance and that the level of agreement was excellent. Thus, sleep can be distinguished from wakefulness in children being evaluated for OSA using a combination of cardiorespiratory and videotape recordings. These results suggest that the CRV method would be useful in a pediatric laboratory setting where EEG recordings are not always possible. They also suggest that overnight, unattended CRV recordings in a childs own home could correctly distinguish sleep from wakefulness. PMID- 8617077 TI - Asthma, employment status, and disability among adults treated by pulmonary and allergy specialists. AB - STUDY OBJECTIVE: Identify risk factors for work disability among adults with asthma treated by pulmonary and allergy specialists. DESIGN: Cross-sectional survey, including retrospective work history data. PARTICIPANTS: Sixty-eight pulmonary and 16 allergy internal medicine subspecialists maintaining a registry of patient visits for asthma; 698 registered patients aged 18 to 50 years, of whom 601 (86%) were studied. MEASURES: Computer-assisted, telephone-administered structured interview assessing asthma severity, perceived general health status, asthma quality of life, demographics, and work history. Complete work disability defined as total work cessation attributed to asthma; partial work disability defined as change in job, duties, or reduction in work hours attributed to asthma. RESULTS: Complete cessation of work due to asthma was reported by 40 (7%; 95% confidence interval [CI], 5 to 9%) and partial work disability by 53 (10%; 95% CI, 7 to 12%) of 550 subjects with a history of labor force participation. Severity of asthma score predicted both complete disability (odds ratio [OR], 7.9; 95% CI, 4.2 to 15 per 10-point increment) and partial disability (OR 2.6; 95% CI, 1.6 to 4.2). Taking illness severity into account, job conditions, occupation, and work exertion carried a combined disability OR of 3.9 (95% CI, 1.7 to 8.6). CONCLUSIONS: Work disability is common among adults with asthma receiving specialist care. Severity of disease is a powerful predictor, but not the sole predictor of disability in this group. Working conditions, including job related exposures, are associated with added disability risk even after taking illness severity into account. PMID- 8617078 TI - Prior diagnosis and treatment of patients with normal results of methacholine challenge and unexplained respiratory symptoms. AB - OBJECTIVE: Previous research indicates that asthma has been underdiagnosed. However, we suspect that recent widespread attention to the underdiagnosis of asthma has led to an overdiagnosis of asthma in some settings. We therefore sought to examine prior diagnosis and treatment of patients referred to our facility and subsequently found to have no objective evidence of variable airflow limitation. DESIGN: Retrospective chart review. SETTING: Hospital-based asthma center. PATIENTS: A referred sample of 263 patients in whom a methacholine challenge (MCC) was conducted after evaluation by our pulmonologists; complete medical histories were available. MAIN OUTCOME MEASURES: Prior respiratory diagnoses, duration of treatment with asthma medications, and diagnosis following assessment by our pulmonologists in 175 patients with a provocative concentration of the substance causing a 20% fall in FEV1 (PC20) greater than 8.0 mg/mL and 88 with a PC20 of 8.0 mg/mL or less. RESULTS: Of those with a PC20 greater than 8 mg/mL, a diagnosis of asthma or possible asthma prior to the challenge study was recorded by their primary care physician in 129 patients (74%). One hundred sixty of 172 patients (88%) with a PC20 greater than 8 mg/mL were diagnosed as not having asthma by our pulmonologists; 109 of 172 patients (62%) had been previously treated with asthma medication(s). The mean duration of asthma treatment was 25.9+/- 56.3 months, and there was no significant difference in the duration of treatment between this group and those who had a PC20 of 8 mg/mL or less. Most of those treated received inhaled beta2-agonists and inhaled corticosteroids. Approximately 61% received two or more classes of medications. CONCLUSIONS: The misdiagnosis of asthma occurs commonly in the referral practice of a tertiary care asthma center. The more frequent use of objective pulmonary function testing in primary practice might reduce the problem of delayed diagnosis and inappropriate therapy for respiratory symptoms. PMID- 8617079 TI - Cumulative dose-response study of non-CFC propellant HFA 134a salbutamol sulfate metered-dose inhaler in patients with asthma. AB - STUDY OBJECTIVE: This study compares the safety and efficacy of HFA 134a salbutamol sulfate (Airomir in the 3M CFC-free system [3M Pharmaceuticals]) and CFC 11/12 salbutamol (Ventolin [Allen & Hanburys]) in a cumulative dose-response (1, 1, 2, 4, 8 inhalations at 30-min intervals) study in asthmatic patients. DESIGN: Randomized, single-blind, two-period cross-over study. PARTICIPANTS: Twenty-four stable mild to moderate asthmatics. MEASUREMENTS AND RESULTS: At all cumulative inhalations, the changes in FEV1 (absolute, percent, and percent predicted) and FVC were equivalent. There was also no significant difference in heart rate, serum potassium level, BP, 12-lead ECG, Holter monitor recordings, or adverse events. Both HFA 134a salbutamol sulfate and CFC 11/12 salbutamol displayed a significant dose-response for FEV1, FEF25-75%, FVC, serum potassium, heart rate, and systolic BP. CONCLUSIONS: HFA 134a salbutamol sulfate and CFC 11/12 salbutamol produced clinically and statistically similar airway responses and side effects. These results indicate that HFA 134a salbutamol sulfate would be a safe and effective substitute for CFC 11/12 salbutamol. PMID- 8617080 TI - Survey of asthma practice among emergency physicians. AB - PURPOSE: The National Asthma Education and Prevention Program (NAEPP) published guidelines for asthma management in 1991. The purpose of this study is to assess the concordance between emergency physicians' practice and the guidelines. DESIGN: Survey mailed to emergency physicians. Non-respondents were mailed a second copy of the survey. PARTICIPANTS: Eight hundred randomly selected active members of the American College of Emergency Physicians. INTERVENTIONS: None. MEASUREMENTS: Participants were asked questions regarding training, current asthma practices, and sources of information on asthma management. RESULTS: Eight hundred questionnaires were mailed, of which 416 (52%) were returned. Sixty-four percent of respondents administer beta-agonists consistent with the NAEPP guidelines. Seventy-five percent of respondents administer corticosteroids in similar accord, while 75% prescribed outpatient corticosteroids in concordance with those recommendations. Forty-seven percent report measuring pretreatment pulmonary function more than half the time and only 38% report checking pulmonary function prior to disposition more than half the time. CONCLUSIONS: Most emergency physicians surveyed use beta-agonists and steroids at least as often as recommended. A minority of emergency physicians reported utilizing pulmonary function testing in the manner recommended by the NAEPP. PMID- 8617081 TI - Bronchogenic carcinoma and coexistent bronchioloalveolar cell adenomas. Assessment of radiologic detection and follow-up in 28 patients. AB - PURPOSE: Bronchioloalveolar cell adenomas (BAAs) have been described in up to 10% of patients with bronchogenic carcinoma. Their prognostic significance is unknown. The purpose of this study was to determine the prognostic implications of finding BAAs coexisting in specimens resected for primary bronchogenic carcinoma and to determine how frequently BAAs can be detected radiologically. METHODS: Follow-up information for a mean of 30 months was obtained on 28 patients with a single primary bronchogenic carcinoma and one or more coexistent BAAs. Preoperative chest radiographs (n=27) and CT scans (n=24) were retrospectively reviewed to assess the number of patients in whom BAAs could be detected radiologically. RESULTS: There was no significant difference between percentage survival of patients with a primary bronchogenic carcinoma and coexistent BAAs when compared with the percentage predicted survival of these patients based on their primary bronchogenic carcinoma alone. BAAs could be detected retrospectively in 1 of 27 (4%) preoperative radiographs and 11 of 24 (46%) CT scans. CONCLUSIONS: On standard preoperative imaging for bronchogenic carcinoma, BAAs were retrospectively detected in more than one third of patients in whom they were detected pathologically. However, the presence of coexistent BAAs with bronchogenic carcinoma does not affect short- and medium-term prognosis. PMID- 8617082 TI - p53 mutations in non-small cell lung carcinomas in Hong Kong. AB - Lung resections from 50 Chinese patients in Hong Kong diagnosed as having non small cell lung carcinoma were examined for the presence of mutations in the p53 gene by polymerase chain reaction single-stranded conformation polymorphism methods and for aberrant protein expression by immunostaining techniques. Eight point mutations in the evolutionarily conserved exon 5 through 8 regions were detected. Abnormal expression of p53 detectable by immunostaining techniques was seen in 23 specimens tested. There was no statistically significant correlation between the detection of p53 aberrations and age, sex, smoking history, histologic type, and tumor stage. Aberrant p53 protein levels detectable by immunostaining were significantly associated with the clinical and nodal staging of the tumors. PMID- 8617083 TI - Significance of hemoptysis following thrombolytic therapy for acute myocardial infarction. AB - PURPOSE: To describe the occurrence, cause, and significance of hemoptysis following thrombolytic therapy for acute myocardial infarction. PATIENTS AND METHODS: We retrospectively reviewed 2,634 patients presenting with acute myocardial infarction who received thrombolytic therapy to determine the incidence of hemoptysis. Chart and radiographic review included the type, dose, and route of thrombolytic therapy. In addition, the onset, duration, and severity of hemoptysis were recorded and correlated with radiographic and bronchoscopic findings. RESULTS: Eleven patients (0.4%) developed hemoptysis following administration of thrombolytic therapy for an acute myocardial infarction. The duration and severity had a wide range, although no patient had significant hemodynamic compromise. The source of hemoptysis was identified in only one patient who had a tongue laceration following cardiopulmonary resuscitation, and blood was seen within the oropharynx and trachea. No definitive cause was identified in all other patients. There was no correlation between the different types or doses of thrombolytic therapy and the duration or severity of hemoptysis. Chest radiographs were nonspecific and demonstrated resolution within 11 days following hemoptysis. CT of the thorax in one patient and bronchoscopy in two patients confirmed chest radiographic findings and in no patient was an underlying pulmonary abnormality identified. CONCLUSIONS: Pulmonary hemorrhage and hemoptysis are unusual complications of thrombolytic therapy in patients with acute myocardial infarction. Although hemoptysis may be the first indicator of an underlying pulmonary abnormality, we found no case in which a significant abnormality was unmasked. This study suggests that follow-up chest radiographs are recommended and further evaluation may be unnecessary if complete resolution is demonstrated. PMID- 8617084 TI - Electrocardiographic and electrophysiologic characteristics of anteroseptal, midseptal, and para-Hisian accessory pathways. Implication for radiofrequency catheter ablation. AB - STUDY OBJECTIVE: To investigate the ECG characteristics, the electrophysiologic properties, and an effective radiofrequency catheter ablation technique in patients with septal accessory pathways. PATIENTS: Forty-six consecutive subjects with septal accessory pathways located in the anteroseptal, midseptal, and para Hisian areas. DESIGN AND INTERVENTIONS: ECGs obtained during sinus rhythm and orthodromic tachycardia, conduction properties obtained from electrophysiologic study, and results of two different ablation techniques were analyzed. MEASUREMENTS AND RESULTS: (1) Twenty-four (52.2%) had manifest preexcitation and 15 (32.6%) had multiple accessory pathways; (2) midseptal pathways could be differentiated from anteroseptal and para-Hisian pathways by a negative delta wave in lead III and a biphasic delta wave in lead aVF during sinus rhythm, and a negative retrograde P wave in two inferior leads during orthodromic tachycardia; (2) midseptal pathways had better antegrade conduction properties and a significantly higher incidence (61.5%) of inducible atrial fibrillation; (4) radiofrequency catheter ablation using lower energy (20+/-6 W) had a comparable effect to ablation using higher energy (36+/-5 W), but without impairment of atrioventricular (AV) node conduction or development of AV block; and (5) during the follow-up period of 26+/-14 months (range, 5 to 54 months), three (6.5%) patients had recurrence. CONCLUSIONS: Midseptal accessory pathways had ECG and electrophysiologic characteristics that were distinctive from those of anteroseptal and para-Hisian pathways. Catheter ablation of these septal pathways using low radiofrequency energy was safe and effective. PMID- 8617085 TI - Predictors of survival in patients receiving domiciliary oxygen therapy or mechanical ventilation. A 10-year analysis of ANTADIR Observatory. AB - STUDY OBJECTIVE: To analyze predictors of survival for patients receiving home long-term oxygen therapy (LTOT) or prolonged mechanical ventilation (PMV) according to the cause of chronic respiratory insufficiency (CRI) and the patients physiologic data. DESIGN: Analysis of a nationwide database (ANTADIR Observatory). SETTING: The national nonprofit network for home treatment of patients with CRI Association Nationale pour le Traitement a Domicile de lInsuffisance Respiratoire Chronique (ANTADIR); founded in France in the 1980s. PATIENTS: There were 26,140 patients receiving LTOT or PMV (noninvasive or via tracheostomy) between January 1, 1984 and January 1, 1993 (chronic bronchitis, 12,043; asthma, 1,755; bronchiectasis, 1,556; emphysema, 551; tuberculosis sequelae, 4,147; kyphoscoliosis, 1,574; neuromuscular diseases, 1,097; pneumoconiosis, 919; and fibrosis, 2,498. MEASUREMENTS AND RESULTS: Survival analysis was performed using the actuarial and the Cox's semiparametric model. The mean survival for patients with chronic bronchitis is 3 years. Survival is slightly better for patients with bronchiectasis and asthma and worse for those with emphysema. Patients with kyphoscoliosis and a neuromuscular disease have the longest survival (8 and 6.5 years, respectively). Patients with CRI due to tuberculosis sequelae experience the same survival as COPD patients (3 years). Prognosis is the worst in patients with pneumoconiosis or fibrosis: 50% of these patients die during the year following the beginning of home treatment. The association of an obstructive lung disease worsens the prognosis of patients with kyphoscoliosis or neuromuscular disease and tends to bring the survival rate of the patients with pneumoconiosis or fibrosis closer to that of COPD patients. In COPD, male sex, older age, lower body mass index (BMI),FEV1 percent predicted,PaO2,and PaCO2 are independent negative prognostic factors. For tuberculous sequelae and kyphoscoliosis, female sex, younger age, a high BMI, PaO2 and PaCO2 (and for kyphoscoliosis a higher FEV1/vital capacity [VC] ratio) are all independent favorable prognostic factors. In pulmonary fibrosis, a lower PaO2 and PaCO2 values, a lower VC percent predicted, and a higher FEV1/VC ratio are negative prognostic factors. CONCLUSIONS: The ANTADIR Observatory allows a unique opportunity to analyze long-term survival of a large population with CRI treated at home. PMID- 8617086 TI - A placebo-controlled trial of prostacyclin in acute respiratory failure in COPD. AB - Although patients with COPD often have elevated pulmonary artery pressures (PAP) and pulmonary vascular resistance (PVR), it is uncertain whether treatment of this pulmonary hypertension is beneficial. We evaluated the extent of pulmonary hypertension in 16 patients with severe COPD complicated by acute respiratory failure and pulmonary hypertension. We assessed the hypothesis that the vasodilator prostacyclin (PGI2) would reduce PVR and improve systemic O2 transport. Patients with a COPD exacerbation requiring mechanical ventilation, and mean PAP greater than 30 mm Hg, were randomized to receive PGI2 or placebo, in addition to conventional therapy. Randomization to PGI2 or placebo therapy occurred 1 to 12 h after intubation, while the patient was mechanically ventilated. An optimal PGI2 dose (2 to 12 ng/kg/min, IV) was established in an initial dose-ranging study and then this dose was infused continuously for 48 h. PGI2 initially reduced PVR, but this effect dissipated within 24 h, indicating the development of tachyphylaxis. Tolerance to the adverse effects of PGI2 (tachycardia, hypotension, flushing, and headache) also developed over time. PGI2 treatment was associated with a significant fall in PaO2 but no increase in systemic oxygen transport. PGI2 proved to be a nonselective vasodilator that caused mild hypoxemia. Despite acute respiratory failure, pulmonary hypertension is mild in patients with severe COPD receiving mechanical ventilation and IV PGI2 is not beneficial in such patients. PMID- 8617087 TI - Dopexamine hydrochloride in septic shock. AB - OBJECTIVE: To test whether dopexamine hydrochloride, by its beta 2-adrenoreceptor and dopaminergic 1 (DA1) and dopaminergic 2 (DA2) agonistic properties, can improve oxygen consumption (VO2) in hyperdynamic patients with septic shock. DESIGN: Prospective, single-cohort study. SETTING: ICU, university hospital. PATIENTS: Twenty-nine postoperative, hemodynamically stabilized, hyperdynamic patients with septic shock. INTERVENTIONS: Short-term application (30 min) of dopexamine hydrochloride at a dose of 2 microgram/kg/min. MEASUREMENTS: Complete hemodynamic profile with O2 transport-related variables at baseline, 30 min after starting the dopexamine infusion, and 30 min after stopping the infusion. MAIN RESULTS: The dopexamine infusion resulted in significant increases in cardiac index (17%) (p<0.001) and O2 delivery (DO2) (16%) (p<0.001). VO2 increased slightly but significantly about 4% (p<0.001) by respiratory gas exchange measurements and 9% (p<0.001) by cardiovascular Fick calculations. The O2 extraction ratio decreased about 8% (0.001). CONCLUSIONS: The addition of dopexamine hydrochloride at a dose of 2 microgram/kg/min resulted in significant increases of DO2 and to a lesser extent VO2. Much of the global DO2 increase was not utilized, because O2 extraction ratio decreased. Direct calorigenic effects of dopexamine and an increase in myocardial VO2 likely account for a large portion of the increase in global VO2. Whether any of the VO2 increase reflects improvement in regions of jeopardized tissue oxygenation remains to be clarified before the definite value of this drug in septic shock can be established. PMID- 8617088 TI - Comparison of wire-guided and nonwire-guided radial artery catheters. AB - OBJECTIVE: To determine if either wire-guided-catheters (WGC) or nonwire-guided catheters (non-WGCs) are associated with a higher rate of successful arterial cannulation overall or when evaluated by a variety of patient and operator characteristics. DESIGN: Prospective clinical trial in a ten-bed adult medical surgical ICU in a 500-bed university hospital. PATIENTS: Adults requiring arterial cannulation for hemodynamic monitoring or frequent blood sampling. A total of 116 attempted arterial cannulations were recorded, and 112 in 67 patients were acceptable for statistical analysis. RESULTS: Overall, no difference in success rates could be demonstrated between WGC and non-WGC. WGCs were associated with a higher success rate in patients with pulses characterized as absent or weak (78% vs 37%; p=0.01). WGCs also had a greater success rate than non-WGCs for more experienced operators, whether defined by seniority (83% vs 44%; p=0.02) or by experience at arterial catheterization (81% vs 48%; p=0.02). CONCLUSION: WGCs were not associated with better success rates overall than were non-WGCs, although there were higher success rates in several subsets of patients and/or operators. Because of their significantly higher costs, WGCs cannot be recommended for routine use for arterial catheterization. In cases where successful catheterization is essential, however, there are circumstances under which they may be the preferred equipment, depending on patient characteristics and operator experience. PMID- 8617089 TI - Selective decontamination of the digestive tract in multiple trauma patients. A prospective double-blind, randomized, placebo-controlled study. AB - STUDY OBJECTIVE: The aims of the study were to evaluate the technique of selective digestion decontamination (SDD) in preventing the development of nosocomial infections in a selected population and to assess the effects on colonization of the oropharynx, nares, and bronchi. A financial assessment was also performed. DESIGN: Prospective, double-blind, randomized placebo-controlled trial using amphotericin B, colistin sulfate (polymixin E), and gentamicin applied to the nares, the oropharynx, and enterally; no parenteral antibiotics were given during the study period. The SDD was applied every 6 h during the study period. SETTING: Multidisciplinary ICU in a university hospital. PATIENTS: A total of 148 trauma patients admitted emergently and intubated within less than 24 h were enrolled. Seventy-two patients who received placebo and 76 treated patients were analyzed on an "intention-to-treat" basis. INTERVENTIONS: Microbiologic surveillance samples of oropharyngeal and bronchial secretions, urine, and any other potentially infected sites were taken at the time of ICU admission and twice weekly thereafter until discharge from the unit. MEASUREMENTS AND RESULTS: With the use of SDD, colonization was significantly reduced in the oropharynx and nares (<0.05) but not in bronchi. However, episodes of bronchopneumonia were significantly reduced (19 in the active group vs 37 in the placebo group; p,0.01). Staphylococcus aureus remained the main potential pathogen causing bronchial colonization and subsequent bronchopneumonia. There was no reduction in the incidence of other infections. Days in the ICU, duration of mechanical ventilation, and mortality rate were unchanged. After the use of SDD, Gram-positive colonization tended to increase and this was mainly due to methicillin-resistant coagulase-negative staphylococci. The total cost of antibiotic therapy ($62,117 [US] in the placebo group and $36,008 in the SDD group) was decreased by 42% with the use of SDD. Clinically important complications of SDD were not encountered. CONCLUSIONS: The use of SDD in this population of trauma patients reduced the incidence of bronchopneumonia and the total charge for antibiotics. Stay in the ICU, mechanical ventilation, and mortality rate were unchanged. Methicillin-resistant coagulase-negative staphylococci were selected by SDD in some patients and the clinical relevance of this colonization needs further evaluation. PMID- 8617090 TI - Cardiopulmonary support mitigates the effects of experimental myocardial ischemia. AB - The effectiveness of cardiopulmonary support (CPS) as a rescue method following failed angioplasty is unknown. The proximal left anterior descending (LAD) was occluded for 20 min in 21 dogs. Group 1 animals (n=15) were given CPS and group 2 animals (n=6) served as controls. During coronary occlusion, animals receiving CPS had increased mean arterial pressure (71+/- 12 vs 58+/-7 mm Hg), decreased left atrial pressure (3+/-3 vs 12+/-3 mm Hg), increased ischemic area blood flow (0.20+/-0.16 vs 0.02+/-0.04 mL/min/g) and myocardial oxygen consumption (0.014+/- 0.008 vs 0.003+/-0.006 mL O2/min/g), decreased remote area myocardial oxygen consumption (0.026+/-0.010 vs 0.091+/-0.047 mL O2/min/g), and an improved myocardial oxygen consumption index (0.60+/-0.33 vs 0.02+/-0.03) when compared with controls (p<0.05). During reperfusion (no CPS), group 1 animals had increased cardiac index (210+/-95 vs 117+/-46 mL/min/kg), renal blood flow (110+/ 38% vs 53+/-45%), ischemic area blood flow (1.13+/-0.40 vs 0.58+/-0.27), and myocardial oxygen consumption (0.066+/-0.015 vs 0.032+/-0.018) when compared with controls (p<0.05). CPS improves oxidative metabolism in selective myocardial segments during coronary occlusion, promotes recovery of the postischemic myocardium, and results in improved peripheral circulation. PMID- 8617091 TI - Increased expression of platelet-derived growth factor A and insulin-like growth factor-I in BAL cells during the development of bleomycin-induced pulmonary fibrosis in mice. AB - Current concepts suggest that macrophages may play a central role in pulmonary fibrosis by virtue of their ability to release a variety of cytokines. In this study, the expression of interleukin (IL)-1 alpha and beta, platelet-derived growth factor (PDGF) A and B, and insulin-like growth factor (IGF) I in BAL cells, which may be involved in fibroblast proliferation, was investigated in murine bleomycin (BLM)-induced pulmonary fibrosis. BAL cells were obtained at 1, 15, and 29 days from Institute for Cancer Research mice after 10 days of intraperitoneal administration of BLM. The relative amounts of cytokine messenger RNA (mRNA) were evaluated by the reverse transcription-polymerase chain reaction method, which simultaneously amplified complementary DNA for cytokines and beta actin as an internal control. The level of IL-1 beta mRNA in BLM-treated mice was increased 4.5-fold compared with that in saline solution-treated (control) mice 1 day after treatment, while no significant differences were observed between the two groups at 15 and 29 days. The mRNAs of PDGF-A and IGF-I in BLM-treated mice were sustained at levels eightfold and threefold to fourfold, respectively, those of controls over 4 weeks. No significant differences were noted in IL-1 alpha and PDGF-B expression between the two groups. We conclude that IL-1 beta released from macrophages may be important in the early phase of inflammatory responses and that PDGF-A and IGF-I may play important roles in the development of BLM induced pulmonary fibrosis. PMID- 8617092 TI - Lung transplantation for cystic fibrosis: special considerations. PMID- 8617093 TI - Spinal cord protection during surgical procedures on the descending thoracic and thoracoabdominal aorta: review of current techniques. PMID- 8617094 TI - Guidelines for the use of nebulizers in the home and at domiciliary sites. Report of a consensus conference. National Association for Medical Direction of Respiratory Care (NAMDRC) Consensus Group. AB - Guidelines for the use of nebulizers outside of the hospital were developed at the request of the Health Care Financing Administration (HCFA) to assist in the preparation of Medicare criteria for reimbursement. The National Association for Medical Direction of Respiratory Care (NAMDRC) convened a consensus conference in Leesburg, Va, with physician representatives from the major medical organizations involved in adult and pediatric respiratory care. Members of the health-care industry also were invited to participate. After review of the pertinent references, members of the faculty were preassigned topics for presentations during the first day of the meeting. Three workshops were organized to address segments of the consensus statement and to develop written reports. Each report was reviewed by the entire group and then finalized. The Consensus Conference recommends that a metered-dose inhaler (MDI) with reservoir chamber is the preferred mode of aerosol therapy for patients outside of the hospital. The circumstances under which a small-volume nebulizer (SVN) may be appropriate are described. The medications that may be administered by SVN are identified with recommendations as to the usual doses to be prescribed. A cost analysis of the various modes of aerosol therapy is presented. These guidelines should be of value to physicians who are prescribing aerosol therapy in the home and also to policy makers who are developing guidelines for reimbursement. PMID- 8617095 TI - Safe load carrying after uncomplicated myocardial infarction: a simple prescriptive method. AB - Measuring maximal handgrip strength at the time of hospital discharge provides a simple method for prescribing load holding and load carrying and patients who have had myocardial infarction. PMID- 8617096 TI - Roentgenogram of the month. Bottle-shaped heart. PMID- 8617097 TI - Clinical conference on management dilemmas. An expanding right upper lobe cavity. PMID- 8617098 TI - Upper airway obstruction in a woman with AIDS-related laryngeal Kaposi's sarcoma. AB - Kaposi's sarcoma rarely causes upper airway obstruction. In the only two previously reported cases, both patients were men who died of hemorrhage shortly after tracheostomy. We describe a 45-year-old woman with AIDS who presented with stridor secondary to Kaposi's sarcoma of the larynx. To our knowledge, this is the first report of this presentation in a woman and the first reported patient with Kaposi's sarcoma to survive tracheostomy. PMID- 8617099 TI - Three cases of dental technician's pneumoconiosis related to cobalt-chromium molybdenum dust exposure. AB - Dental technician's pneumoconiosis (DTP) is a rather recent finding in subjects exposed to the dust generated in dental laboratories producing metal-framed removable partial dentures from alloys based on cobalt, chromium, and molybdenum. This study presents details of the first three Swedish cases of DTP with some emphasis on the diagnostic procedures and the dust exposure. A follow-up of at least 5 years from diagnosis is included. PMID- 8617100 TI - Massive hemoptysis successfully treated by modified bronchoscopic balloon tamponade technique. AB - A 60-year-old man with massive hemoptysis was treated successfully by modified bronchoscopic balloon tamponade technique. Compared with previously reported techniques, the modified technique requires no special catheter, no complicated maneuver, and is able to be applied to more massive bleeding. PMID- 8617101 TI - Extralobar sequestration presenting as massive hemothorax. AB - Pulmonary extralobar sequestration is a rare anomaly, usually diagnosed during the first months of life. A case of extralobar pulmonary sequestration in an adult, manifesting itself as massive hemothorax, is presented. PMID- 8617102 TI - Use of somatostatin analog for localization and treatment of ACTH secreting bronchial carcinoid tumor. AB - A 29-year-old woman presenting with an ectopic adrenocorticotropic hormone syndrome and a nodule of the upper lobe of the left lung was explored by indium 111 (111In) octreotide scintigraphy. This showed a pathologic uptake by the nodule. Treatment with octreotide resulted in the rapid control of hypercortisolism prior to surgery. PMID- 8617103 TI - Pulmonary vein thrombosis and peripheral embolization. AB - A 78-year-old-woman was admitted to the hospital with bilateral femoral arterial occlusion. Her medical history disclosed atrial fibrillation and a left thoracoplasty performed 50 years earlier for treatment of tuberculosis. A transesophageal echocardiogram demonstrated intraluminal thrombus in a left pulmonary vein. The patient recovered after thromboembolectomy. This case documents another uncommon cause of cardiac thromboembolism in which a transesophageal echocardiogram was essential to make the diagnosis. PMID- 8617104 TI - Familial association of primary pulmonary hypertension and a new low-oxygen affinity beta-chain hemoglobinopathy, Hb Washtenaw. AB - A Hungarian-American kindred with familial primary pulmonary hypertension (PPH) and a new, low-oxygen affinity beta-chain variant hemoglobin, Hb Washtenaw, is described. The index case presented with severe PPH and was found to have the abnormal hemoglobin. Two siblings with the abnormal hemoglobin also demonstrated increased pulmonary artery pressures on exercise echocardiography suggestive of early PPH. The occurrence of PPH and the abnormal hemoglobin could be due to genetic or biochemical factors or simply coincidental. A previous study had described a possible association of an abnormal beta-chain variant hemoglobin, Hb Warsaw, and PPH. It was suggested that the putative gene for familial PPH may be located near the beta-globin gene on chromosome 11. The association of PPH and the beta-chain variant hemoglobin in this kindred adds further support to this hypothesis. PMID- 8617105 TI - Tracheoesophageal fistula in a patient with recurrent Hodgkin's disease. A case for hope or despair. AB - Tracheoesophageal fistula (TEF) is a devastating complication of malignancies; however, those associated with Hodgkin's disease (HD) may carry a better prognosis. We present a patient with recurrent HD and TEF. PMID- 8617106 TI - Life support. The debate continues. PMID- 8617107 TI - Life support. The debate continues. PMID- 8617108 TI - Life support. The debate continues. PMID- 8617109 TI - Life support. The debate continues. PMID- 8617110 TI - Get real, Dr. Stobo. PMID- 8617111 TI - Diagnosing pulmonary embolism. Indeed, when will we ever learn? PMID- 8617112 TI - PZA hypersensitivity. PMID- 8617113 TI - Cyfra 21-1 in cavitary lung lesions. PMID- 8617114 TI - Dual therapy versus triple therapy for Helicobacter pylori-associated duodenal ulcers. AB - We compared the ulcer healing effect and eradication of H. pylori by one-week triple therapy of bismuth, metronidazole, and tetracycline with two-week dual therapy of amoxicillin and omeprazole. One hundred twelve patients with confirmed H. pylori infection and duodenal ulcers were recruited in a prospective, randomized, single-blinded trial. Ulcer healing, eradication of H. pylori in the stomach six weeks after randomization and side effect reported by patients during the therapy. Duodenal ulcers were healed in 44 of 49 (89.8%, 95% CI, 81.3-98.3 %) patients receiving triple therapy and in 44 of 53 (83.0%, 95% CI, 72.9-93.1%) patients receiving dual therapy (P=0.32). H. pylori was successfully eradicated in 41 of 49 (83.6%, 95% CI 73.4-94%) patients and in 40 of 53 (75.5%, 95% CI 63.9 87.1%) patients in the triple therapy group and the dual therapy group respectively (P=0.31). Side effects experienced by patients who received triple therapy were significantly more frequent than those who received dual therapy (P=0.0076). In conclusion, a two-week course of omeprazole and amoxicillin achieves a comparable rate of H. pylori and ulcer healing with fewer side effect. PMID- 8617115 TI - High seroprevalence of Helicobacter pylori in diabetes mellitus patients. AB - To assess the prevalence of Helicobacter pylori in diabetes mellitus, a serological test used to detect antibodies to H. Pylori in patients with diabetes mellitus. Within six months, 45 insulin-dependent, 98 non-insulin-dependent, and a control group of 159 outpatients were enrolled in this study. The age adjusted seroprevalence rate of Helicobacter pylori were determined using a commercial anti-Helicobacter pylori IgC and IgA ELISA (Bio-Rad). The prevalence rates increased with age in all age groups until 60-70 years. In diabetic patients, the frequency of Helicobacter pylori infection was higher than control subjects in nearly all age groups, reaching significance in three age categories of NIDDM patients and in one age category in IDDM patients. This higher seroprevalence could not be explained by differences in socioeconomic status or use of antibiotics. PMID- 8617116 TI - Gastric emptying and Helicobacter pylori infection in duodenal ulcer disease. AB - The pathogenetic link between Helicobacter pylori gastritis and duodenal ulcer is still unknown. Fast gastric emptying of liquids might be important in the pathogenesis of gastric metaplasia of the duodenum and duodenal ulcer through an increased exposure of the duodenum to gastric acid. In H. pylori-infected subjects, an abnormal gastric emptying could affect urea breath test results and correlate with histological gastritis. This study was performed to evaluate the gastric emptying of liquids in duodenal ulcer patients with H. pylori infection and the possible relation between the bacterial load, gastric emptying, and urea breath test results. Seventeen duodenal ulcer patients with H. pylori gastritis and 15 healthy volunteers were studied by a [14C]octanoic acid and [13C]urea breath test to evaluate gastric emptying rate and H. pylori status simultaneously. Endoscopy with antral biopsies were performed in all duodenal ulcer patients. Duodenal ulcer patients with H. pylori infection have a normal liquid gastric emptying that is unrelated with histological severity of gastritis. The urea breath test results and the gastric emptying parameters do not correlate with histology. A significant correlation between the gastric emptying and the urea hydrolysis rate is found. It is concluded that H. pylori infection and duodenal ulcer disease is not associated with abnormally fast liquid gastric emptying, and this finding should be taken into account when a casual link between H. pylori infection and duodenal ulcer disease is searched for. The correlation between gastric emptying and urea hydrolysis rate explains why no conclusions on intragastric bacterial load can be drawn from the urea breath test results. PMID- 8617118 TI - Bioavailability and efficacy of omeprazole given orally and by nasogastric tube. AB - We compared the bioavailability and the efficacy of omeprazole provided either as encapsulated enteric-coated granules or as enteric-coated granules delivered via a nasogastric tube in 10 healthy subjects. Omeprazole reduced mean pentagastrin stimulated peak gastric acid secretion by 85.5% +/- 23.7% when delivered orally and by 79.6% +/- 32.1% when delivered by nasogastric tube; the mean plasma omeprazole concentration area under the curve (AUC) was 2.02 +/- 0.79 after oral delivery and 1.74 +/- 1.89 after nasogastric tube delivery. There was no significant difference in these parameters between the two routes of administration, and there was excellent intrasubject correlating between oral and nasogastric percent acid suppression and AUC. There was a close correlation between AUC and percent acid suppression at AUC values below 0.6, and complete acid suppression at AUC values above 0.6, regardless of the delivery route. We conclude that omeprazole delivered as enteric-coated granules via nasogastric tube provides equal bioavailability and gastric acid suppression as omeprazole given orally in its proprietary formulation. PMID- 8617117 TI - Omeprazole causes delay in gastric emptying of digestible meals. AB - We have studied gastric emptying of a solid, realistic meal (800 cal, 15% protein, 45% fat, 40% carbohydrate) in 21 healthy subjects twice, with and without a four-day pretreatment with 40 mg omeprazole. The last dose of the drug was taken 24 hr before the test, to avoid hypothetical nonsecretory side effects of the drug . Gastric emptying was measured by ultrasound of antral diameters. The results show that basal and maximal postprandial antral cross-sectional areas were the same during the two tests. A greater residual distention of the antrum was present throughout the study after the omeprazole treatment, the difference being significant at time 120 and 240. Omeprazole induced a highly significant delay in gastric emptying [control 199.6 (12.6) vs omeprazole 230.9 (12.7) min, mean (1 SEM); P<0.003]. The delay was not due to a prolonged lag phase, but rather to an effect on the slope of the emptying curve. This study shows that in normal subjects omeprazole delays gastric emptying of a digestible solid meal. PMID- 8617119 TI - Effects of cisapride on salivary production in normal subjects. AB - Cisapride improves reflux esophagitis and enhances esophageal acid clearance. To test the effect of cisapride an salivary production, we enrolled 14 healthy volunteers in a double-blind, randomized, placebo-controlled study. Subjects received cisapride, 10mg per os four times a day, or placebo for three days. Saliva, collected during fasted and fed states, was analyzed for volume and buffer capacity. Buffer capacity was expressed as the volume of 0.01 N HCl needed to titrate 1 ml of saliva to pH 6.1. Both volume and buffer capacity significantly increased during the fed state as compared to the fasted on both cisapride and placebo. Cisapride significantly enhanced the postprandial salivary volume and buffer capacity compared to placebo: 29.6 +/- 11.3 ml vs 22.9 +/- 9.5 ml and 1.07 +/- 0.31 vs 0.89 +/- 0.28, respectively (P< 0.0001). Cisapride's enhancement of salivary flow rate and buffer capacity in the fed state may be another mechanism by which it exerts its beneficial effect in patients with reflux esophagitis. PMID- 8617120 TI - Pathways mediating pentagastrin-induced mucosal blood flow response in rat stomachs. AB - The mechanisms of pentagastrin-induced gastric mucosal blood flow (GMBF) response were investigated in anesthetized rats. A rat stomach was mounted on an ex vivo chamber, perfused with saline, and GMBF was measured by a laser Doppler flowmetry simultaneously with acid secretion. Pentagastrin infused intravenously produced a dose-dependent increase of GMBF as well as acid secretion, and its effect reached a maximum at 120 microgram/kg/hr (maximal dose). Pretreatment with omeprazole (60 mg/kg, intraperitoneally) completely inhibited the acid secretory response and the enhancement of GMBF induced by both submaximal (60 micrograms/kg/hr) and maximal doses of pentagastrin. In contrast, the luminal perfusion with glycine (200 mM) to remove luminal H+ almost totally attenuated the increase of GMBF caused by the submaximal dose of pentagastrin, without any effect on acid secretion, but partially suppressed such GMBF responses caused by the maximal dose. Subcutaneous pretreatment with indomethacin, a cyclooxygenase inhibitor, significantly mitigated GMBF response caused by both submaximal and maximal doses of pentagastrin, whereas 8-phenyltheophylline (8-PT), an adenosine antagonist, showed a significant inhibition of GMBF response caused by only the maximal dose. However, the combined administration of 8-PT with glycine perfusion further attenuated GMBF response caused by the maximal dose of pentagastrin, and the additional treatment with indomethacin completely blocked this this GMBF response. We conclude that pentagastrin-induced GMBF responses are mediated by at least two different pathways; one is related to luminal H+ and the other to the parietal cell activity, depending on the dose of pentagastrin. In addition, the latter pathway may be mediated by adenosine, while endogenous prostaglandins may be involved in both pathways. PMID- 8617121 TI - High systemic HCO3(-) and topical 16,16-dimethyl-PGE2 protect gastric mucosa against luminal acid by enhancing its preepithelial buffer capacity. AB - Systemic metabolic alkalosis and topical prostaglandins protect the gastric mucosa against luminal acid. This study investigates whether this protection is mediated by increased epithelial HCO3(-) secretion with resultant alkalization of the pre-epithelial mucus-HCO3(-) buffer layer. surface pH of chambered ex vivo rat gastric epithelium was measured with liquid sensor pH microelectrodes during luminal perfusion of increasing acidities (0, 10, 30, 50, 100 mM HCL). The experimental groups were: (1) control, (2) topical 16,16-dimethyl-PGE2 treatment, (3) high-HCO3(-) metabolic alkalosis, and (4) low HCO3(-) respiratory alkalosis. The gastric mucosa of PGE2-treated and high-HCO3(-) alkalotic rats tolerated significantly better luminal acid than that of controls, but the tolerance of low HCO3(-) alkalotic rats was significantly impaired. There was a significant correlation between arterial HCO3(-) concentration (but not arterial pH) and surface pH (r = 0.81, P < 0.01). This suggests that the gastric mucosa against luminal acid are, at least in part, mediated by enhanced buffer capacity of the pre-epithelial mucus-HCO3(-)layer. PMID- 8617122 TI - Stimulatory effects of sucralfate on secretion and synthesis of mucus by rabbit gastric mucosal cells. Involvement of phospholipase C. AB - We examined the effects of sucralfate on the secretion and synthesis of mucus by cultured rabbit gastric mucosal cells, and the underlying intracellular mechanism. Treatment of mucosal cells with sucralfate (>0.5 mg/ml) for 4 and 8 hr caused a significant increase in the inositol triphosphate (IP3) content in the cells. Neomycin (a phospholipase C inhibitor) at 1 mM markedly inhibited the sucralfate-induced increases in both the IP3 content and mucus secretion and synthesis. Neither 10 nM staurosporine, 1 mM H-7 (protein kinase C inhibitors), nor 5 microM indomethacin (a cyclooxygenase inhibitor) affected the stimulatory effects of sucralfate on mucus secretion and synthesis, but 10 microM TMB-8 (an antagonist of intracellular Ca2+ mobilization)abolished its effects. Taken together, these results demonstrate that sucralfate acts directly an gastric mucosal cells, inducing increases in mucus secretion and synthesis, and that sucralfate causes an increase in the IP3 content, probably through activation of phospholipase C, and the subsequent IP3-elicited Ca2+ mobilization may be involved in the stimulatory effects of sucralfate. PMID- 8617123 TI - Origin of atypical reflux symptoms. A case study showing the importance of reflux composition and posture. AB - In summary we evaluated a 39-year-old man two years after partial esophagectomy and gastroesophageal anastomosis. He had developed recurrent Barrett's esophagus and atypical reflux symptoms. We found free reflux and no antireflux barrier at the hiatus or the esophagogastric anastomosis. Three different reflux techniques performed simultaneously demonstrated that the composition of refluxant varied with posture, explaining the atypical nature of the symptoms. In the left recumbent posture, the refluxate was comprised of acidified liquid and gaseous gastric contents with the patient complaining of heartburn and chest pain. In the right recumbent posture the refluxate was composed of only nonacidic gas, and the patient complained of chest pain without heartburn. We propose that multiple reflux tests performed simultaneously in the setting where a patient experiences his atypical symptoms may help clarify their origin. Furthermore, this case illustrates how posture may dramatically influence refluxant composition. PMID- 8617124 TI - Esophageal dysphagia as the sole symptom in type I Chiari malformation. AB - Chiari malformation, also called Arnold-Chiari deformities, are rare hindbrain herniations that may present in children or adults. The most common symptoms include headache, syncope, disordered eye movement, sensory loss, weakness, and cerebellar features such as ataxia. Dysphagia occurs in 5-15% of patients, although only a few reports describe dysphagia as the only presenting symptom. We report a case of a 27-year-old woman who presented with a three-year history of dysphagia, chest pain, and weight loss. Esophageal manometrics revealed markedly disordered esophageal motility and gastroesophageal reflux. Her symptoms failed to respond to high doses of omeprazole, prokinetics, and eventually surgical fundoplication. The subsequent onset of neurological symptoms led to the diagnosis of Chiari type I malformation. Following posterior craniotomy with decompression, her dysphagia and chest discomfort completely resolved. Repeat esophageal manometrics revealed complete resolution of prior abnormalities. PMID- 8617126 TI - Therapy with omeprazole and clarithromycin increases serum carbamazepine levels in patients with H. pylori gastritis. PMID- 8617125 TI - Achalasia. A possible late cause of postpolio dysphagia. AB - The aim of this paper is to describe a patient with severe postpolio problems who developed achalasia. A 66-year-old patient came to our observation for severe dysphagia. He had suffered from paralytic poliomyelitis at the age of 7 months and had severe residual deficits. At the age of 62 he presented with sudden pain localized in the distribution of the C4 and C5 dermatomes and an inability to abduct the left arm. At the time, he experienced only occasional and mild dysphagia; his esophagus was not dilated and emptied normally. Over the following months his muscular function improved, but dysphagia worsened. We found a megaesophagus with a sigmoid appearance and the manometric features of achalasia. Pneumatic dilatation produced good resolution of dysphagia. A year later manometry showed the reappearance of peristalsis after all wet swallows. In patients with postpolio dysphagia, the possible presence of achalasia must be considered. PMID- 8617127 TI - Nonsteroidal antiinflammatory drug-induced colitis and misoprostol. PMID- 8617128 TI - Increase of swallows before onset of phase III of migrating motor complex in normal human subjects. AB - The aim of our study was to analyze the relation between the deglutitive activity and fasting esophageal and gastric motility in normal subjects. Fifteen healthy subjects (9 males, 6 females) with a mean age of 42 years (range 19-65) were studied. A 24-hr pH-manometric recording was performed using a probe with four solid-state recording sites 10 cm apart, placed so as to record the motor activity of esophageal body, lower esophageal sphincter (LES) or distal part of the esophagus, and gastric antrum. An additional probe with one recording site was placed in the pharynx to evaluate swallowing. A pH electrode was also placed at 5 cm above the sphincter. Data were transferred to a personal computer and analyzed using specific software. Subjects received two meals during the recording session. MMCs were almost exclusively recorded during the nighttime. A mean of 2 +/- 0.94 (SD) MMC per subject was detected with a mean (+/- SD) interval, between each cycle, of 86.11 +/- 34.53 min. During the 30 min preceding gastric phase III, the number of swallows showed an increase that reached statistical significance 5 min before the onset of phase III. A similar pattern was observed for the area under the curve (AUC) of the esophagus and LES. In conclusion deglutition and esophageal motility vary with the MMC, suggesting that the deglutitive activity is part of the interdigestive motility pattern. PMID- 8617129 TI - Influence of blood glucose levels on rat liquid gastric emptying. AB - The glycemic influence on liquid gastric emptying in rats was studied. Diabetic hyperglycemia was induced by streptozotocin-treated rats further received a daily insulin injection ( 2.5 or 10 IU/kg). Immediate hyperglycemia was induced in a separate group of rats by continuous intravenous glucose infusion (44 or 88 mg/kg/min) 10 min before the experiment. Rats were killed 15 min after radiochromium feeding; then the radioactivity of stomach and small intestine were counted to obtain the gastric emptying value. Emptying in diabetic rats was delayed compared with controls (mean +/- SE: 40.9 +/- 2.6% vs. 54.2 +/- 2.8%, P < 0.01). Low-dose insulin treatment reversed the impairment, while high-dose treatment even enhanced emptying. Immediate hyperglycemia induced with two glucose infusions also inhibited gastric emptying. Present results indicate that hyperglycemia elicited with any hyperglycemic model is at least one of the important mechanisms to delay liquid gastric emptying. PMID- 8617130 TI - Resting energy expenditure in patients with alcoholic chronic pancreatitis. AB - The aim of this study was to assess resting energy expenditure in patients with chronic pancreatitis; 33 patients with alcohol-related chronic pancreatitis (group 1: 13 normal weight, group 2: 20 underweight) and 11 undernourished patients without identifiable disease (group 3) were studied. Body composition was determined by bioelectric impedance analysis and fat-free mass was similar among the three groups (76.4 +/- 1.5%, 78.6 +/- 1.3% and 76.8 +/- 2.1% for groups 1, 2, and 3, respectively). The measured resting energy expenditure (REE) was higher than the predicted EE (Harris and Benedict formula and Cunningham's equation) for the underweight patients with chronic pancreatitis (group 2) (P < 0.05), but not for the two other groups. According to Cunningham's equation, 65% of the group 2 patients were hypermetabolic (REE > 110% of predicted EE) versus 23.1% and 20% in groups 1 and 3. When adjusted for fat free mass, REE was significantly (P < 0.01) higher in group 2 (35.0 +/- 0.9 kcal/kg/24 hr) than in the other two groups (30.1 +/- 0.7 kcal/kg/24 hr and 30.8 +/-1.4 kcal/kg/24 hr in groups 1 and 3, respectively). During chronic pancreatitis, weight loss is accompanied by hypermetabolism, which should be taken into consideration during nutritional support. PMID- 8617131 TI - Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis. AB - A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in deltaF508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%. PMID- 8617133 TI - Comparison of post-ERCP biological pancreatitis in chronic alcoholic and control nonalcoholic group. PMID- 8617132 TI - Effect of cisapride on gallbladder emptying and plasma CCK in normal and vagotomized human subjects. AB - Previous studies have provided conflicting results of cisapride on gallbladder emptying in response to a meal. We studied six volunteers and six patients after a truncal vagotomy in a double-blind, placebo-controlled, prospectively randomized study using 10 mg cisapride four times a day for three days. Gallbladder volume was quantitated using ultrasonography, and plasma CCK levels were measured with a sensitive and specific radioimmunoassay using the DINO antibody before and for 90 min after a fatty, mixed meal. Plasma effect was observed on the gallbladder either in normal subjects or vagotomized patients. Paradoxically, residual volume (RV) was increased in the vagotomized patients after treatment with cisapride: RV cisapride 7.1 (4.1-15.9) ml, RV placebo 5.1 (3.8-14) ml, P < 0.05. Further work is required to clarify the mechanisms of action of cisapride on the gallbladder and the sphincter of Oddi. The use of cisapride during litholytic therapy may impair gallbladder emptying and delay stone clearance. PMID- 8617134 TI - Chronic pancreatitis with alpha 1-antitrypsin deficiency: from uncontrolled trypsin activation? PMID- 8617135 TI - Small bowel bacterial overgrowth in patients with alcoholic cirrhosis. AB - A total of 89 patients with alcoholic cirrhosis and 40 healthy subjects were included in a study to assess the prevalence of intestinal bacterial overgrowth and to analyze its relationship with the severity of liver dysfunction, presence of ascites, and development of spontaneous bacterial peritonitis (SBP). Bacterial overgrowth was measured by means of a breath test after ingestion of glucose. Intestinal bacterial overgrowth was documented in 27 (30.3%) of the 89 patients with alcoholic cirrhosis and in none of the healthy subjects. The prevalence of intestinal bacterial overgrowth was significantly higher in cirrhotics with ascites (37.1%) than in those with no evidence of ascites (5.3%) and among patients with Pugh-Child class C (48.3%) than in patients with a class A (13.1%) or B (27%). Twelve (17.1%) of the 70 patients with ascites developed an episode of SBP. The prevalence of spontaneous bacterial peritonitis was significantly higher in patients who had intestinal bacterial overgrowth (30.7%) than in patients who did not (9.09%). We conclude that intestinal bacterial overgrowth occurs in approximately one third of patients with cirrhosis secondary to alcohol, particularly in patients with ascites and advanced liver dysfunction. Moreover, bacterial overgrowth may be a condition favoring infection of the ascitic fluid. PMID- 8617136 TI - Gastrointestinal dysfunction in liver disease and portal hypertension. Gut-liver interactions revisited. PMID- 8617137 TI - Differences in hemostasis among sclerosing agents in endoscopic injection sclerotherapy. AB - Endoscopic injection sclerotherapy is useful in stopping bleeding from esophageal varices. We compared the in vivo effects of sclerosants on thrombogenesis, hemostasis, and endothelial injury. We injected aethoxysclerol (AS) of ethanolamine oleate (EO) into the small veins of the rat intestine. The maximum thrombogenic index with AS was 30.7 and with EO was 9.2. The venous flow stopped sooner with EO than with AS. The thrombi caused by EO were mixed with red blood cells. Heparin pretreatment decreased the thrombogenic index with AS by 96.7%, but not that with EO. The area of the fluorescein-albumin conjugate that permeated from veins with AS was larger than that with EO. The fluorescent intensity with AS was lower than that with EO. We thus conclude that: (1) the size of thrombi is not necessarily proportional to the hemostatic efficacy; (2) change in a patient's coagulation may affect the potential of sclerosants; (3) the excellent hemostatic efficacy of EO is based on localized injury to the endothelium and the involvement of red blood cells aggregation; and (4) in vivo microscopy is useful in determining the rational selection of sclerosants. PMID- 8617138 TI - Peritoneal fibrosis in cirrhotics treated with peritoneovenous shunting for ascites. An autopsy study with clinical correlations. AB - Of 554 cirrhotics autopsied during 1975-1993, 69 had had peritoneovenous shunts. Generalized peritoneal fibrosis with cocoon formation was found in 26 (38%) of those with shunts but in only one of 485 without shunts (P = 0.00002). In 14/26 the fibrosis was asymptomatic, an incidental autopsy finding. Intestinal obstruction in 12/26 (46%), the only symptomatic manifestation, was fatal in five. The etiology of peritoneal fibrosis in shunted patients is unknown. The 26 patients with fibrosis had more prior abdominal operations, complicated abdominal wall hernias, and active biliary tract inflammations; the features differentiated them from the 43 patients without fibrosis. Scores in a 'peritoneal complication index,' that considered multiple risks in the same patients, were significantly higher in those with fibrosis. In addition to these peritoneal injuries or inflammations, the faster ascitic fluid circulation in shunted patients may have increased deposition of fibrin upon the peritoneum. Fibrogenic cytokines, thus spread throughout the abdomen from local sites, may have converted fibrinous adhesions to generalized peritoneal fibrosis. PMID- 8617139 TI - Hepatic encephalopathy after transjugular intrahepatic portosystemic shunt. Incidence and risk factors. AB - Forty-seven consecutive patients were prospectively evaluated to study the incidence of hepatic encephalopathy as well as modifications in the PSE index after TIPS. Various clinical, laboratory, and angiographic parameters were also recorded to identify risk factors for the development of post-TIPS hepatic encephalopathy (HE). Mean follow-up was 17 +/- 7 months. During follow-up, six patients died and one underwent transplantation. All other patients were followed for at least a year. Fifteen patients (32%) experienced 20 acute episodes of precipitated HE (hospitalization was necessary in 10 instances), and five patients (11%) presented a continuous alteration in mental status with frequent spontaneous exacerbation during follow-up. Both precipitated and spontaneous HE occurred more frequently during the first three months of follow-up. Moreover the PSE index was significantly worse than basal values one month after TIPS, thereafter returning to near basal values. HE was successfully treated in all patients but one who required a reduction in the stent/shunt diameter. Increasing age (>65 years) and low portacaval gradient (<10 mm Hg) were predictors of HE after TIPS. A gradual dilation of the stent/shunt should be performed to obtain a portacaval gradient >10 mm Hg to avoid an unacceptable rate of HE after TIPS. PMID- 8617140 TI - Gastric functions in portal hypertension. Role of endothelin. AB - This study investigated the effects of portal hypertension on gastric motor and secretory functions and the role of endothelin in rats. Control; sham-operated; endothelin-A receptor blocker, BQ 485 (1 microgram/kg)-treated; portal hypertensive; and portal hypertension +, endothelin-A receptor blocker-treated rats were subjected to tests of gastric secretory, motor, and mucosal function studies as well as gastric wall polymorphonuclear infiltration. Portal hypertension was induced by partial portal vein ligation. Portal hypertension suppressed gastric acid and total fluid secretion and delayed gastric emptying. An increase in mucosal permeability and no alteration in gastric wall myeloperoxidase activity were observed. The effects of portal hypertension on gastric secretory, motor, and mucosal functions were reversed by treatment with endothelin-A receptor blocker, BQ-485. It is concluded that portal hypertension suppresses the gastric motor and secretory functions and endothelin plays an important role in the pathophysiology of gastric alterations associated with portal hypertension. PMID- 8617141 TI - Lymphangioleiomyomatosis, chylous ascites, and diet. PMID- 8617142 TI - Pyoderma gangrenosum with hepatopancreatic manifestations in a patient with rheumatoid arthritis. AB - Pyoderma gangrenosum is a dermatological disease of unknown origin. We report the case of a 47-year-old woman with cutaneous lesions of pyoderma gangrenosum associated with hepaticopancreatic involvement. We found no other similar cases in the literature. The outcome was favorable with steroid therapy. She was free of symptoms after one year. PMID- 8617143 TI - Incidence and outcome of overt gastrointestinal bleeding in patients undergoing bone marrow transplantation. AB - The purpose of this study was to determine the prevalence, clinical patterns, and outcomes of gastrointestinal bleeding in consecutive patients treated at one bone marrow transplant center. We reviewed the clinical course of 579 consecutive bone marrow transplant recipients who underwent therapy from January 1986 through December 1993. These patients were evaluated for overt gastrointestinal bleeding, defined as hematemesis, melena, hematochezia, or a combination. Overt gastrointestinal bleeding was defined in 43 of 579 patients (7.4%), including 25 men and 18 women undergoing transplantation for hematologic disorders (N = 29) and solid tumors (N = 14). After high-dose cytotoxic chemotherapy, patients were given allogeneic (N = 10) or autologous (N = 33) hematopoietic progenitor cell support obtained from bone marrow, peripheral blood, or both. H2 blockers, sucralfate, or a combination were administered to all patients as prophylactic therapy. Bleeding manifestations included hematemesis(N = 24, melena (N = 8), hematochezia (N = 7), and combinations (N = 4). The median time from bone marrow infusion to the onset of overt gastrointestinal bleeding was 7.5 days (range: 0 45 days). Fourteen patients had evidence of orthostatic hypotension attributable to gastrointestinal bleeding. Esophagogastroduodenoscopy was performed in 26 patients; 18 had diffuse esophagitis and gastritis. Two patients with bleeding ulcers underwent successful electrocautery. Colonoscopy was performed in five patients and revealed a cecal ulcer in one subject, tumor recurrence in one patient, and colitis in another. No patients underwent surgical intervention. Only ine patient died as a result of gastrointestinal bleeding. Overt gastrointestinal bleeding is uncommon in patients undergoing bone marrow transplantation; most episodes are self-limited and do not contribute to overall mortality. Endoscopy is primarily diagnostic as most patients do not have lesions amenable to therapeutic procedures. PMID- 8617144 TI - Prolonged hypergastrinemia does not increase the frequency of colonic neoplasia in patients with Zollinger-Ellison syndrome. AB - Whereas considerable experimental evidence suggests chronic hypergastrinemia can increase the occurrence of colonic neoplasia, the risks in man remain unclear. Zollinger-Ellison syndrome (ZES) is associated with marked plasma elevation of all forms of gastrin and, because of its prolonged course, has been shown to be an excellent model disease to study the effects of chronic hypergastrinemia in man. To determine whether profound chronic hypergastrinemia affects the occurrence of colonic dysplasia and neoplasia, 97 consecutive patients with ZES were studied. All patients underwent colonoscopic examination to the cecum, and the location, size, and type of polyps/tumors were determined. The patients had a mean fasting gastrin level 31 times above normal and a mean disease duration of 10 years; 17/97 (18%) had adenomatous polyps, 67/97 (69%) no adenomatous polyps, and 2/97 (2%) had colonoscopy and/or autopsy studies fo asymptomatic controls. Stratification by age or gender, presence of MEN-I, tumor extent, and duration of degree of hypergastrinemia did not increase prevalence. This study shows that despite prolonged, profound hypergastrinemia, no increased rate of colonic neoplasia (polyps or cancer) was noted. These data suggest that the development of hypergastrinemia secondary to continuous use of H+,K+-ATPase inhibitors for as long as 10 years is unlikely to cause an increased risk of developing colonic neoplasia in man. PMID- 8617145 TI - Increased substance P receptor expression by blood vessels and lymphoid aggregates in Clostridium difficile-induced pseudomembranous colitis. AB - Pseudomembranous colitis is most often caused by toxins secreted by Clostridium difficile following bowel flora overgrowth after antibiotic use. The secretory and inflammatory effects observed in C. difficile toxin A-induced enterocolitis in the rat ileum are inhibited by CP-96,345, a substance P (SP) receptor antagonist. To determine if SP plays a role in the pathogenesis of human pseudomembranous colitis, SP receptor distribution was examined in a toxin A positive specimen of bowel. Quantitative receptor autoradiography was used to examine SP receptors in tissue from a patient who tested positive for C. Difficile toxin. SP receptors were massively increased in small blood vessels and lymphoid aggregates in the pseudomembranous colitis bowel in comparison to control specimens. The SP binding was saturable and exhibited similar affinities for SP and CP-96,345. SP may contribute to the inflammatory response in pseudomembranous colitis via a massive increase in SP receptor antagonists may offer a novel therapeutic intervention for pseudomembranous colitis. PMID- 8617146 TI - Enterocolitis in infantile common variable immunodeficiency. A case report and review of the literature. PMID- 8617147 TI - Pleuropericarditis related to the use of mesalamine. PMID- 8617148 TI - An unusual presentation of intestinal duplication with a literature review. AB - A 52-year-old male presented with urinary symptoms of frequency and hesitation. X rays, ultrasound, and computerized tomography investigations were performed that indicated the diagnosis and position of an enteric duplication cyst. Elective surgery was performed to completely remove the duplication cyst. Histological examination showed that the cyst was lined by stratified squamous, ciliated, and gastric-type epithelium, with a muscularis mucosae and a muscularis Propria. No malignancy or dysplasia was seen. Duplications of the alimentary tract are uncommon congenital abnormalities. They may be multiple and arise at any level from the mouth to the anus. Usually observed early in life, a minority may remain unsuspected until adulthood. The clinical presentations may be vague and diverse depending on their location. These include pain, distention, dysphagia, dyspepsia, and complications involve bleeding, perforation, malignancy, and obstruction of the alimentary tract and vessels. Plain x-rays are of limited use in the diagnosis of duplications but ultrasound findings may be diagnostic, with computerized tomography useful in delineating surrounding structures. Once the diagnosis is established, surgical correction is the treatment of choice, preferably with complete removal. PMID- 8617149 TI - Pain-related and cognitive components of somatosensory evoked potentials following CO2 laser stimulation in man. AB - We recorded cortical potentials evoked by painful CO2 laser stimulation (pain SEP) employing an oddball paradigm in an effort to demonstrate event-related potentials (ERP) associated with pain. In 12 healthy subjects, frequent (standard) pain stimuli (probability 0.8) were delivered to one side of the dorsum of the left hand while rare (target) pain stimuli (probability 0.2) were delivered to the other side of the same hand. Subjects were instructed to perform either a mental count or button press in response to the target stimuli. Two early components (N2 and P2) of the pain SEP demonstrated a Cz maximal distribution, and showed no difference in latency, amplitude or scalp topography between the oddball conditions or between response tasks. In addition, another positive component (P3) following the P2 was recorded maximally at Pz only in response to the target stimuli with a peak latency of 593 msec for the count task and 560 msec for the button press task. Its scalp topography was the same as that for electric and auditory P3. The longer latency of pain P3 can be explained not only by its slower impulse conduction but also by the effects of task difficulty in the oddball paradigm employing the pain stimulus compared with electric and auditory stimulus paradigms. It is concluded that the P3 for the pain modality is mainly related to a cognitive process and corresponds to the P3 of electric and auditory evoked responses, whereas both N2 and P2 are mainly pain-related components. PMID- 8617150 TI - Is the somatosensory N250 related to deviance discrimination or conscious target detection? AB - Effects of attention to, and probability of sudden changes in, repetitive stimuli on somatosensory evoked potentials (SEP) were studied. Low - (30 Hz) and high frequency (140 Hz) vibratory stimuli were delivered in random order to the middle finger of the left hand with different presentation probabilities in different blocks. Also ignore conditions were administered. In the ignore conditions, the probability had no effect on SEPs. However, when the standard stimuli were omitted, the "deviants" elicited small N140 and P300 deflections not observed in response to deviants when standards were also present. In the attention conditions, deviant stimuli (targets) elicited large N250 and P300 deflections which increased in amplitude with a decreased target probability. However, when subjects counted infrequently presented "deviants" alone (standards omitted) the enhanced N140 and the P300 with shortened latency were elicited, but no N250 wave could be found. At the ipsilateral side, a distinct N200 deflection was seen which could be the N250 with a shorter latency because of an easier task (detection instead of discrimination). The results might be interpreted as suggesting that the somatosensory N250 is related to conscious detection of target stimuli. PMID- 8617151 TI - Localization of functional regions of human mesial cortex by somatosensory evoked potential recording and by cortical stimulation. AB - We describe methods of localizing functional regions of the mesial wall, based on 47 patients studied intraoperatively or following chronic implantation of subdural electrodes. Somatosensory evoked potentials were recorded to stimulation of posterior tibial, dorsal pudendal, median, and trigeminal nerves. Bipolar cortical stimulation was performed, and in 4 cases movement-related potentials were recorded. The cingulate and marginal sulci formed the inferior and posterior borders of the sensorimotor areas and the supplementary motor area (SMA). The foot sensory area occupied the posterior paracentral lobule, while the genitalia were represented anterior to the foot sensory area, near the cingulate sulcus. The foot motor area was interior and superior to the sensory areas, but there was overlap in these representations. There was a rough somatotopic organization within the SMA, with the face represented anterior to the hand. However, there was little evidence of the "pre-SMA" region described in monkeys. Complex movements involving more than one extremity were elicited by stimulation of much of the SMA. The region comprising the supplementary sensory area was not clearly identified, but may involve much of the precuneus. Movement-related potentials did not provide additional localizing information, although in some recordings readiness potentials were recorded from the SMA that appeared to be locally generated. PMID- 8617152 TI - Brain-stem auditory evoked potentials (BAEPs) from basal surface of temporal lobe recorded from chronic subdural electrodes. AB - BAEPs were recorded from the basal surface of the temporal lobe by subdural electrodes chronically implanted in 6 patients who were evaluated for surgical management of intractable partial seizures. Near-field recordings were obtained by recording between the subdural electrode closest and most distant to the brain stem. Far-field recordings were obtained by recording between the subdural electrodes and an indifferent electrode over the spinal process of the seventh cervical vertebrae. The recordings were compared with standard ear-vertex recordings. After ipsilateral ear stimulation, the subdural electrode closest to the brain-stem recorded large amplitude waves I and II followed by less well defined waves of longer latencies. Recordings to contralateral stimulation showed no clearly defined waves I and II and a large amplitude wave Vn. Waves III, IV, V, Vn and VI were of opposite polarity after ipsi- and contralateral stimulation. These findings indicate that waves I and II are generated ipsilaterally to the stimulation side, whereas wave Vn has a contralateral origin. Wave Vn may be generated in the brachium of the inferior colliculus, as suggested from dipole configuration studies. This conclusion is consistent with the classical anatomical observations that the supracollicular auditory pathways are predominantly crossed. PMID- 8617153 TI - Brain-stem trigeminal and auditory evoked potentials in multiple sclerosis: physiological insights. AB - Thirty-six patients with multiple sclerosis were evaluated by means of brain-stem trigeminal and auditory evoked potentials. The brain-stem auditory evoked potentials (BAEPs) were abnormal in 26 patients (72.2%). Brain-stem trigeminal evoked potentials (BTEPs) yielded similar results, showing distorted waveforms and/or prolonged latencies in 25 patients (69.4%). As expected, the MRI proved to be the most efficient single test, revealing plaques in 86.4% of the patients evaluated. However, the diagnostic accuracy of MRI was lower than that provided by the combination of the BTEP and BAEP (88.9%). Moreover, in patients having signs of brain-stem involvement, the BTEP, alone and in combination with the BAEP, proved to be more sensitive than the MRI in revealing brain-stem lesions. Correlation between clinical and BTEP findings could be found only in those patients who presented with signs of trigeminal involvement such as trigeminal neuralgia or dysesthesiae. The analysis of the BTEP waveforms showed two distinct types of abnormality-a peripheral type and a central type-suggesting plaques in distinct locations. Both the BTEP and the BAEP demonstrated a correlation with the clinical course of the disease and the condition of the patient at the time of the evaluation. Relapse of the disease was associated with a marked prolongation of the central conduction time in the BTEP and in the BAEP, suggesting the application of such studies to the monitoring of unstable patients in the evaluation of new therapeutic protocols. PMID- 8617154 TI - Evidence of neuronal plasticity within the inferior colliculus after noise exposure: a study of evoked potentials in the rat. AB - Recent investigations have implicated that the central nervous system has a role in the changes that occur in auditory function following acoustic trauma caused by noise exposure. These investigations indicate that the inferior colliculus may be the primary anatomical location in the ascending auditory pathway where noise induced neuronal plasticity occurs, thereby resulting in changes in the neuronal processing of auditory information. In the present investigation, we show that the amplitudes of all peaks in the click-evoked response from the external nucleus of the inferior colliculus decrease during a 30 min exposure to a tone (104 dB sound pressure level (SPL) at 4 kHz and 8 kHz). After tone exposure, the amplitudes of two of the peaks of the response from the external nucleus of the inferior colliculus that reflect the input from more caudal structures slowly returned to baseline levels, whereas the amplitudes of the two peaks reflecting neuronal activity in the inferior colliculus increased above baseline levels and remained at the increased levels for at least 90 min following exposure to the tone. We also show that exposure to a 4 kHz tone at 104 dB SPL causes changes in the neuronal processing of tonebursts in the form of changes in the temporal integration function for one of the peaks of the response from the external nucleus of the inferior colliculus that originates in the inferior colliculus. Before tone exposure the amplitude of this peak decreased with increasing stimulus duration, but after tone exposure the amplitude of this peak was independent of the duration of the toneburst stimulus. We interpret these changes as evidence that noise exposure (tone exposure) causes changes in the excitability of the inferior colliculus that are not seen in more caudal structures, and these changes are probably a result of a change in the balance between inhibition and excitation in the inferior colliculus. PMID- 8617155 TI - Middle and long latency somatosensory evoked potentials after painful laser stimulation in patients with fibromyalgia syndrome. AB - Ten female patients with fibromyalgia syndrome (FS) were investigated with laser evoked potentials (LEPs) after hand stimulations and compared with 10 female pain free and age-matched control patients. FS patients exhibited significantly lower heat pain thresholds than controls (P < 0.05) and had higher amplitudes of LEP components N170 (P < 0.01) and P390 (P < 0.05) in response to intensities of 20 W (beam diameter 5 mm, duration 20 msec, wavelength 10.6 microns). N170 additionally appeared with a broader distribution over bilateral central, vertex and fronto-central leads which contrasted to the control group and studies in healthy subjects where N170 was much more restricted to central and midtemporal positions contralateral to the stimulated hand. Auditory stimuli interspersed between laser impulses that served to announce subjects to rate the perceived pain elicited auditory evoked potentials that were not different between groups indicating no differences of general vigilance level to account for observed LEP effects. P390 amplitude enhancement might indicate greater attention and cognitive processing of nociceptive stimuli in FS subjects. Effects upon N170 rather point to exogenous factors like peripheral and spinal sensitization or reduced cortical or subcortical inhibition of nociception. PMID- 8617156 TI - Source modeling of esophageal evoked potentials. AB - Evoked potentials can be recorded from the scalp after stimulation of the esophagus by balloon distension. The purpose of this study was to estimate the number and localization of sources contributing to the esophageal evoked potential (EEP). The EEP was recorded from 32 scalp electrodes in 5 healthy subjects. Spatio-temporal dipole modeling was performed in the time interval from 185 msec to 525 msec after stimulation (mean values). The EEP was best explained by the combined activity of 1 dipole located relatively high in the midline and 2 lateral dipoles. Given the anatomical projection of esophageal sensory fibers and the location of these dipoles the sources were probably located in the cingulate gyri and insular cortex. There was no evidence that sources in the lower brain stem contributed to the scalp recorded EEP. PMID- 8617157 TI - Somatosensory evoked magnetic fields following stimulation of the lip in humans. AB - The topography of somatosensory evoked magnetic fields (SEFs) following stimulation of the upper and lower lips was investigated in 6 normal subjects. When the lateral side of the upper lip was stimulated, P20m and its counterpart, N20m, were identified in the hemisphere contralateral to the stimulated side. The equivalent current dipoles (ECDs) of N20m-P20m were considered to be located in lip area of the primary sensory cortex (SI). Middle latency deflections (N40m P40m, N60m-P60m, and N80m-P80m) were identified in bilateral hemispheres. Their ECDs were located in the SI in both hemispheres. Long latency deflections (P110m N110m) were recognized in both hemispheres, and their ECDs were located inferior to the SI, in an area considered to be the secondary sensory cortex (SII). When the midline of the lip was stimulated, similar short and middle latency deflections was also identified, but SII deflections (P110m-N110m) were decreased in amplitude. When the lower lip was stimulated, the ECDs of short and middle latency deflections were located at a site in the SI inferior to or near those elicited by upper lip stimulation. The ECDs of P110m-N110m were located in an area of the SII similar to that upon stimulation of the upper lip, but their orientations were different. PMID- 8617158 TI - Digital radiographic images will benefit endodontic services. AB - Future assurances for endodontic services and assessments of traumatized teeth will depend in part on significant improvements to the quality of dental radiographs. Whereas digital images offer many potential sensor, display and archival advantages over film-based radiographs, the marginal image improvements from intraoral sensors primarily benefit patient education. Precisely digitized conventional radiographs are more likely to yield quantum image resolution improvements, although neither dentists nor patients will benefit until cost effective digitizing systems have been developed for the dental office. PMID- 8617159 TI - Apical leakage following root canal dressing with calcium hydroxide. AB - The purpose of this study was to analyse the apical leakage in teeth filled by the lateral condensation technique following medication with calcium hydroxide. One hundred and twenty extracted human teeth were biomechanically prepared by using exclusively reaming motion with files up to #40. Half of the teeth received a calcium hydroxide dressing for 3 days. The medication was removed by irrigation and reaming motion with files #40 up to #70. The teeth were divided in 6 experimental groups, according to the dimension of the utilized instrument. The root canals were filled and posteriorly the teeth were placed into a 2% methylene blue dye solution inside a flask, which was attached to a vacuum pump. Leakage was measured linearly, and the results showed significantly (p<0.01) less leakage in the experimental groups that received calcium hydroxide dressings than in the control groups. The results persisted even after the removal of 300 micrometers of dentin from the root canal dentinal walls. PMID- 8617161 TI - Relationship between file size and stiffness of nickel titanium instruments. AB - The purpose of this study was to evaluate the relationship existing between file size and stiffness for 3 endodontic files made of nickel titanium. Three groups of instruments with different cross-sections were tested: a triangular cross section, a square cross-section and a modified triangular cross-section. The instruments were tested from size 15 to size 40 or 60 according to ANSI/ADA specification no. 28 for binding moment evaluation. There was a statistically significant difference between the 3 groups: the square cross-section K files presented a larger bending moment than the triangular cross-section K files, which presented a larger bending moment than the modified cross-section K files. Like the stainless steel instruments, there was an exponential relationship between file size and bending moment for triangular and square cross-section K files, but a linear relationship between file size and bending moment for the files with a modified triangular cross-section. PMID- 8617160 TI - Sealing ability of potential retrograde root filling materials. AB - The sealing ability of two potential retrograde root filling materials, a light cured glass ionomer cement (Vitrebond) and a reinforced zinc oxide-eugenol cement (Kalzinol) was compared with that of amalgam using three methods of assessment: bacterial leakage, confocal microscopy and Indian ink leakage. the root canals of 80 extracted human single-rooted teeth were prepared. All the teeth were apicected, retrograde cavities were prepared and then divided into four equal groups of 20 teeth. The teeth were sterilised by autoclaving and the retrograde cavities filled with the test materials. In the control group, retrograde cavities in 10 teeth were left unfilled while cavities in another 10 teeth were sealed with cyanoacrylate cement. The teeth were first subjected to a bacterial leakage test using Enterococcus faecalis as a leakage marker. After which, the adaptation of the retrograde root fillings was assessed using a confocal optical microscope. Finally, the teeth were processed for the Indian ink leakage test. Bacterial leakage occurred in more teeth filled with amalgam compared with both Vitrebond and Kalzinol (P<0.001), between which there were no differences. With confocal microscopy, the size of the marginal gap was largest with amalgam and smallest with Vitrebond; all differences were statistically significant (<0.001). Finally, there was more Indian ink penetration with amalgam compared with both Vitrebond and Kalzinol (P<0.001) but there were no differences between Vitrebond and Kalzinol. Although there were individual differences within samples, overall, the three methods of assessment produced similar results. The sealing ability of Vitrebond and Kalzinol was similar and both materials were better than amalgam. PMID- 8617162 TI - Effect of palatal injections on pulpal blood flow in premolars. AB - The effect of palatal infiltration injections using 2% lignocaine hydrochloride with 1:80,000 adrenaline was evaluated for sound maxillary first premolar teeth. The influence of the injections on pulpal blood flow and local anaesthesia was investigated. Ten human volunteers were given the following injections on different days: 1 ml palatal infiltration; 1 ml buccal infiltration; 1 ml palatal and 1 ml buccal infiltration; and 2 ml buccal infiltration. The blood flow was observed with a laser Doppler flowmeter with the probes held against the buccal surfaces of the teeth by a splint. Blood flow was recorded digitally on a computer in machine units. Pulpal anaesthesia was assessed using a monopolar electric pulp tester. All injections significantly reduced blood flow from baseline levels (p<0.05). The 1-ml palatal injection produced a significantly longer period of reduced blood flow (mean 40 min) compared with the other injections (p<0.05). The duration of blood flow reduction was shorter than the duration of pulpal anaesthesia for every injection. Only seven out of 10 palatal injections achieved pulpal anaesthesia. The 2-ml buccal injection and the combined buccal and palatal injections produced significantly longer anaesthesia than the 1-ml buccal injection (p<0.05). PMID- 8617163 TI - Effect of carbamide peroxide and hydrogen peroxide on the surface morphology and zinc oxide levels of IRM fillings. AB - The effect of 10% carbamide peroxide or 10% hydrogen peroxide on the surface morphology and zinc oxide levels of IRM fillings was tested. Ninety IRM samples were treated with either 10% carbamide peroxide, 10% hydrogen peroxide or phosphate buffer which served as control. Treatment consisted of placing the samples in a dry incubator at 37 degrees C for 1, 3 or 7 days. At each time point, the samples were removed from the test solutions, dried and prepared for surface scanning electron microscopy and energy dispersive spectrometric analysis. After 3 days, 10% carbamide peroxide significantly reduced the zinc oxide levels as compared to the 10% hydrogen peroxide group (<0.01) and the controls (p<0.01). 10% hydrogen peroxide reduced the zinc oxide levels similarly to the control. No significant changes in the zinc oxide levels were found between 3 and 7 days in any of the groups tested. Microscopy examination of the carbamide peroxide group revealed granular surface with well defined crystalline areas. In the hydrogen peroxide group, numerous cracks with multiple sun burst like areas were found. At the macroscopic level, the samples of this group appeared cracked and more swollen, as compared to controls and samples treated with carbamide peroxide. In conclusion, both 10% carbamide peroxide and 10% hydrogen peroxide altered the surface morphology and the zinc oxide levels of IRM fillings, but their modes of action differed. PMID- 8617164 TI - Effects of mandibular nerve block on heat- or cold-induced changes in pulpal blood flow in man. AB - Laser Doppler flowmetry (LDF) was used to study the effect of mandibular nerve block, using 3% mepivacain, on heat- or cold-induced changes in pulpal blood flow (PBF) evoked by application of cold or heat to the lingual surfaces of teeth 33 and 43 in nine young subjects. PBF on average showed a 7% increase in response to heat (48 degrees C) and a 20% decrease when exposed to cold (3 degrees C). Neither response was affected by mandibular block. From this we concluded that the effects on PBF of the presently applied heating and cooling of the tooth were solely due to direct thermal influences on the blood vessels, without involvement of vasomotor or sensory nerves. PMID- 8617165 TI - Splints made of wire and composite: an investigation of lateral tooth mobility in vivo. AB - In 103 posttraumatic splints, later tooth mobility was measured with Periotest immediately before and after the routine splint removal. The splints were made of composite resin and an 0.017 X 0.025" orthodontic steel wire. 481 teeth were measured. A statistic evaluation revealed that the immobilisation effect did not exceed normal tooth firmness. Fixation to one neighbouring tooth had less effect than fixation to two. Adjacent tooth gaps reduced the effect. Splint extensions had no influence. With the use of the Periotest device, more than 50% of all teeth with a true mobility of 20 Periotest-units or more were detectable as mobile in spite of the fixed splint. PMID- 8617166 TI - Traumatic crown fractures in permanent incisors with immature roots: a follow-up study. AB - A follow-up study of crown fractured permanent incisors with incomplete root formation was carried out in a group of patients, aged 6-12 years, over a 5-year period in the Dental Clinic of the University of Verona, Italy. The number of injured patients was 55, representing 84 injured incisors. All patients were followed clinically and radiographically using a standardized follow-up protocol. The most common type of trauma was fracture of enamel and dentine without pulpal exposure (80%) and the most common type of treatment was restoration with the acid-etch composite resin technique (46%). Bonding of the crown fragment was performed in 10 instances (12%). At the 5-year-control all teeth with fracture of the enamel had no pulp complications. Four of 67 teeth (6%) with fracture of the enamel and dentine without pulpal involvement showed pulp necrosis and 1 tooth showed pulp obliteration (1.5%). Eight of 14 teeth (57%) with fractures of the enamel and dentine with pulp involvement showed pulp necrosis. Aesthetically 36 of the restored teeth were deemed satisfactory (43%). In 9 teeth the bonded fragment had to be rebonded. 14 teeth were considered unsatisfactorily restored due to wear of the composite (17%). 34 restored teeth had to be retreated because of a new trauma (40%). In one tooth a previous bonded fragment had to be rebonded. These results confirmed that crown fractures without pulp involvement in permanent incisors with incomplete root formation hav a low percentage of pulp complications, while 60% of the teeth with crown fractures with pulp involvement had pulp complications. PMID- 8617167 TI - Use of ultrafast computed tomography in dental surgery: a case report. AB - A case of a transdental fixation planned on the basis of ultrafast computed tomography is reported. This method allows to gain an insight in the osseous structures required for the operative procedure. Ultrafast computed tomography is superior to the conventional radiographs in obtaining accurate information about the vertical bone supply and the extension of the facial osseous sinus wall. A set of axial CT-images with an extension of 3 mm was acquired in the region of the alveolar bone. By means of frontal reconstructions and data processing methods the thickness of the bone layer in transverse direction was accurately determined. The calculated bone thickness allowed to anchor a transdental self tapping titanium screw. PMID- 8617168 TI - Mandibular canine with two roots and three root canals. AB - A rare case of root canal retreatment in a lower canine with two roots and three canals is presented. While the presence of two roots and two canals in one root is not uncommon, the finding of three canals in a lower canine appears not to have been reported previously. PMID- 8617169 TI - Microdialysis study of the neuropharmacokinetics of phenytoin in rat hippocampus and frontal cortex. AB - Acute administration of phenytoin (PHT) is used in the treatment of status epilepticus, yet little is known about the neuropharmacokinetics of PHT in brain extracellular fluid (ECF), the pharmacodynamically relevant compartment. To characterize the neuropharmacokinetics of brain ECF PHT we implanted microdialysis probes in rat hippocampus and frontal cortex and placed a catheter in the internal jugular vein. PHT (50 or 100 mg/kg intraperitoneally, i.p.) was then administered, and microdialysate and serum samples were collected. PHT was rapidly absorbed, with a time to maximum concentration (Tmax) of approximately 20 min for serum concentrations. PHT rapidly entered the brain ECF compartment, with Tmax values similar to those of serum. In brain ECF, PHT concentrations then plateaued for 40-60 min despite decreasing serum concentrations. The area under the brain ECF concentration-time curve (AUC) was higher in hippocampus than frontal cortex. The possible mechanisms for these observations include entry of PHT into specific brain areas both across capillaries and through the cerebrospinal fluid (CSF), extensive binding of PHT in brain white matter, and differing blood flow in different brain regions. PMID- 8617170 TI - Postictal pulmonary edema requires pulmonary vascular pressure increases. AB - The pathogenesis of neurogenic pulmonary edema has been debated for many years. Whether cardiogenic mechanisms and increased pulmonary vascular pressures are primary or even necessary for the production of pulmonary edema has been argued. We used postictal pulmonary edema to study this problem in a sheep model of neurogenic pulmonary edema with bicucullin-induced status epilepticus. Seizure induced increases in pulmonary vascular pressures were averted with a reservoir system to maintain left atrial pressure (LAP) and pulmonary artery pressure (PAP) at preseizure levels. No increase in lung lymph flow occurred during seizures, in contrast to the doubling of lung lymph flow that occurred during seizures when ictal pulmonary vascular hypertension was not blocked. These data support a primary role of pulmonary vascular pressure increases in the production of neurogenic pulmonary edema. PMID- 8617171 TI - Qualitative MRI segmentation in mesial temporal sclerosis: clinical correlations. AB - Magnetic resonance imaging (MRI) is reliable and sensitive in the detection of mesial temporal atrophy in patients with temporal lobe epilepsy. We investigated the MRI patterns of atrophy in 47 patients with histologically confirmed hippocampal sclerosis and correlated the imaging findings to the clinical features and surgical outcome. One hundred percent of patients had hippocampal body atrophy, 70% had hippocampal tail atrophy, 23% had amygdala atrophy, and 10% had focal hippocampal body atrophy without other segmental involvement. Correlative analysis of the segmented MRI findings with other clinical variables, including a previous history of childhood febrile convulsions, showed no significant associations except for younger age of seizure onset associated with hippocampal tail atrophy (p <0.03). No associations between surgical outcome and the patterns of mesial temporal atrophy were detected. Our results demonstrate that variable patterns of atrophy exist in mesial temporal atrophy, but invariably atrophy involves the hippocampal body segment. Segmental MRI analysis in mesial temporal atrophy provides added useful diagnostic information. PMID- 8617172 TI - Hippocampal cell distributions in temporal lobe epilepsy: a comparison between patients with and without an early risk factor. AB - Neuronal cell distributions were measured for anterior and posterior locations in the hippocampi of epilepsy patients who were seizure-free after temporal lobectomy. Patients were divided into two groups, those with an early risk factor, defined as a neurologic insult occurring in the first 4 years of life, and those with no early risk factor. Early-risk patients had lower hilar cell densities, lower granule cell densities, and fewer granule cells per millimeter, a measured related to total granule cell number, than to early risk patients. Moreover, each risk group had different anteroposterior density gradients for granule cells and hilar cells. These differences in cell distribution may arise from different patterns of cell loss of cell migration in the dentate gyrus during development. In the early-risk group, there was also a distinction between patients with a history of febrile convulsions without CNS infection and patients with a history of meningitis or encephalitis. These two subgroups had similar numbers of granule cells, However, the meningitis/encephalitis subgroup exhibited a wider granule cell layer, suggesting that the granule cell layer was more dispersed. Our results support the hypothesis of a predominantly anterior hippocampal insult in temporal lobe epilepsy (TLE). In nonepileptic hippocampus, the ratio of putatively excitatory granule neurons to putatively inhibitory hilar neurons is highest in the anterior hippocampus. This ratio may explain in part why the anterior hippocampus is more prone to cell loss and seizures. PMID- 8617173 TI - Significance of simple partial seizures in temporal lobe epilepsy. AB - We determined how localization of simple partial seizures (SPS) correlated with localization of complex partial seizure (CPS) in scalp/sphenoidal EEG and assessed prognosis after temporal lobe resective surgery in patients with an ictal correlate of SPS in scalp/sphenoidal EEG recordings. EEGs were recorded with the 10-20 system of electrode placement and supplemented with sphenoidal electrodes. Between 1985 and 1992, 183 patients with temporal lobe epilepsy (TLE) reported an aura (SPS) during inpatient monitoring; all were eligible for inclusion in our study. The EEGs during SPS showed ictal changes in 51 patients (28%, 117 SPS). Forty-four patients had unilateral temporal interictal spikes (IIS), and SPS and CPS always arose from the same region. Seven patients had bitemporal interictal spikes; SPS colocalized with CPS in 4 patients (57%), SPS were contralateral to CPS in 2 patients, and 1 patient had bilateral independent CPS but unilateral SPS. SPS accompanied by EEG ictal changes conveyed a favorable prognosis in patients who underwent epilepsy surgery. Scalp/sphenoidal recorded IIS but were less reliable in identifying the location of CPS onset in patients with bitemporal spikes. PMID- 8617174 TI - Implications of seizure termination location in temporal lobe epilepsy. AB - Where propagating symptomatic seizures terminate has not been studied, but might provide insight into mechanisms of seizure termination as well as localization of epileptogenic tissue. We investigated location of seizure termination in 50 refractory temporal lobe epilepsy (TLE) patients who had intracranial EEG recording of spontaneous seizures and subsequent temporal lobe resection with > 1 year follow-up. Only seizures that had onset in the resected temporal lobe were included. Location of the electrical termination for each seizure in each patient was categorized as diffuse, localized to the onset location, or localized elsewhere. The proportion of all seizures in each patient in each category was analyzed with respect to the outcome of surgery. Outcome was classified as seizure-free or persistent seizures. Diffuse seizure termination was noted equally frequently in both outcome groups. However, the 27 patients without seizures postoperatively had a significantly greater proportion of seizures with termination in the onset location (67%) than did the 23 patients with persistent seizures (36%, p < 0.01). The seizure-free patients also had a significantly lower proportion of seizures with localized termination elsewhere than the onset site (13%) than did patients with persistent seizures (45%, p < 0.005). Localization of the site of termination of seizures of focal origin to cortical regions other than the onset is associated with a poorer surgical prognosis. This observation raises the possibility of additional abnormal epileptogenic cortical regions with impaired seizure-terminating capabilities. PMID- 8617175 TI - Will a critical level of hyperventilation-induced hypocapnia always induce an absence seizure? AB - We wished to determine if the degree of hypocapnia correlates with increased frequency of absence seizures and if there is a critical pCO2 at which absence seizures are reliably provoked. Twelve untreated children with newly diagnosed absence epilepsy were continuously monitored by EEG and end-expiratory CO2 recording during quiet respiration and hyperventilation (to absence seizure or exhaustion) while breathing four gas mixtures: (a) room air, (b) 100% O2, (c) 4% CO2 in room air, or (d) 4% CO2 + 96% O2). In quiet respiration, a reduction in number of spike and wave bursts and total seconds of spike and wave was noted in children breathing supplemental CO2 (gases c and d vs. gases a and b), p < 0.05. Supplemental O2 had no effect. Eight subjects had absence seizures elicited with each trial of hyperventilation. All subjects had their own critical pCO2, ranging from 19 to 28 mmHg. Three children had no seizures, two despite hypocapnia to pCO2 of 19 and 21 and 1 who achieved a pCO2 of only 25. In 1, absence seizures were provoked in only six of nine hyperventilation trials to pCO2 of 17-23. In 67% of subjects, absence seizures were reliably provoked by hypocapnia. Critical pCO2 varied among children with absence. Determination of whether variation in sensitivity to hypocapnia may be helpful in determining response to antiepileptic drugs (AEDs) or remission of seizures will require further study. PMID- 8617176 TI - Quantitative comparison of language deficits produced by extraoperative electrical stimulation of Broca's, Wernicke's, and basal temporal language areas. AB - Subdural electrodes were implanted over the language-dominant left lateral convexity in 45 patients undergoing evaluation for epilepsy surgery. In 29 patients, additional electrodes were placed over the left basal temporal cortex. We identified language areas by using intermittent electrical stimuli applied while patients read aloud. Aphasia was classified as expressive or receptive based on additional testing performed when electrical stimulation elicited reading arrest in the absence of direct excitatory or inhibitory motor effects. Using correlated logistic regression, we noted no statistically significant differences among Broca's, and Wernicke's areas and the basal temporal language area (BTLA) regarding the frequency with which electrical stimulation interfered with language. Speech production deficits, however, occurred significantly more frequently in Broca's than in Wernicke's area (p = 0.012). In contrast, language comprehension deficits occurred with equal frequency when Broca's and Wernicke's areas were stimulated. These results suggest that both Broca's and Wernicke's areas play important roles in language comprehension and that the primarily expressive aphasia of patients with lesions of Broca's area results mainly from the predominant participation of Broca's area in language production. PMID- 8617177 TI - Cognitive effects of resecting basal temporal language areas. AB - Electrical stimulation of the basal temporal region of the dominant hemisphere before partial temporal lobectomy for epilepsy sometimes produces temporary interruption of language function, but the significance of removal of this area is unknown. We evaluated the correlation between resection of the basal temporal language areas (BTLA) and certain types of postoperative language deficits. In a population of 25 patients, we mapped the inferolateral temporal lobe with cortical electrical stimulation, verifying the positions of the stimulating electrodes with three-dimensional computed tomography (CT). Eighty percent of the patients developed transient language deficits with stimulation, particularly with tests of confrontation naming and comprehension. BTLA was primarily located in the fusiform gyrus, from 1 to 9 cm from the temporal tip. At testing 6-12 months after operation, patients with BTLA resection performed worse (mean 9% decrease) than those with no BTLA resection (mean 4% improvement) on tests of confrontation naming (p = 0.03). Resection size accounted for less of the variance in decrease in confrontation naming than did resection of the BTLA. Tests of performance I.Q. (PIQ), verbal I.Q. (VIQ), or recognition memory for word and verbal learning showed no significant difference between these groups. Most patients do not have language decrease with removal of basal temporal lobe 5 6 cm from the tip, even with removal of BTLA. Some patients, however, have persistent decrease in naming. PMID- 8617178 TI - Neuroticism in temporal lobe epilepsy: assessment and implications for pre- and postoperative psychosocial adjustment and health-related quality of life. AB - We assessed 77 candidates for epilepsy surgery to determine the association among neuroticism (a dimension of personality characterized by chronic negative emotions and behaviors), psychosocial adjustment as measured by the Washington Psychosocial Seizure Inventory (WPSI), and health-related quality of life (HRQOL) as measured by the Epilepsy Surgery Inventory 55 (ESI-55). Minnesota Multiphasic Personality Inventory 2 (MMPI-2) Neuroticism scale scores were significantly correlated with many domains of patient -perceived psychosocial adjustment and HRQOL regardless of frequency or type of seizures. We then followed 45 of the patients who subsequently underwent epilepsy surgery to determine the influence of neuroticism on postoperative functioning. Two-way analysis of variance (ANOVA) indicated that patients with high preoperative neuroticism had significantly poorer postoperative psychosocial adjustment and HRQOL scores than patients who had low or moderate preoperative neuroticism scores. These results support the validity of the MMPI-2 as a useful measure of neuroticism. Preoperative neuroticism has an important influence on postoperative psychosocial adjustment and HRQOL that is independent of postoperative seizure outcome. Understanding the influence of personality variables, such as neuroticism, on psychosocial functioning both before and after epilepsy surgery is essential in managing intractable seizures. PMID- 8617179 TI - Photic-induced epileptic negative myoclonus: a case report. AB - We present the first documented case of photic-induced epileptic negative myoclonus. A 17-year-old girl had experienced two generalized tonic-clonic seizures (GTCS) while watching television. The only EEG abnormality was a photoparoxysmal response (PPR), which was sometimes accompanied by loss of postural tone in both arms. Valproate was effective in abolishing photosensitivity. Negative myoclonus should be included among the ictal phenomena accompanying PPR. PMID- 8617180 TI - Double pathology in Rasmussen's encephalitis: etiologic considerations. AB - In a 7-year-old girl with epilepsia partialis continua (EPC) involving the left face, arm, and leg for 1 year, serial neuroimaging studies showed progressive, brain atrophy. Because medical treatment was ineffective, she underwent a large fronto-temporal surgical resection. Neuropathological examination showed loss of lamination and dysplastic neurons, gliosis, microglial nodules, and perivascular cuffing. Such "double pathology" (dysgenesia and a chronic inflammatory process) may have implications for the pathophysiology of Rasmussen's syndrome. PMID- 8617181 TI - Gabapentin associated with aggressive behavior in pediatric patients with seizures. AB - Gabapentin (GBP) is a new antiepileptic drug (AED) approved for adjunctive treatment of complex partial seizures with or without seizures secondarily generalization in adults. We report 2 children who received GBP for intractable seizures and who developed intolerable aggressive behavior requiring dose reduction or drug discontinuation. Behavioral changes should be recognized as a possible side effect of GBP, especially in mentally retarded children. PMID- 8617182 TI - Felbamate-induced headache. AB - We prospectively investigated drug-induced headaches (HA) among 60 epileptic patients receiving felbamate (FBM). Twenty patients (33%) experienced HA. HA was pounding in 11 (55%), steady in 9 (45%), moderate or severe in 19 (95%), occurred at least once a week in all patients, and was relieved by nonnarcotic analgesics in 14 (70%). Mean duration on FBM before HA onset was 19 days. HA occurred with higher FBM doses and was relieved in 8 of 13 patients (62%) with FBM dose reduction. FBM was discontinued in most cases because of risks of anemia or hepatitis; not because of HA. Other side effects included insomnia (25%), gastrointestinal symptoms (27%), and agitation or restlessness (23%). HA is a common dose-related complication of FBM, occurs early after initiation of FBM treatment, and is relieved by dose reduction. PMID- 8617183 TI - Report of the International League Against Epilepsy Commission on economic aspects of epilepsy. PMID- 8617184 TI - Diagnosis of food hypersensitivity. PMID- 8617185 TI - The indirect association of lactation with subsequent perimenopausal body weight. AB - OBJECTIVE: We aimed to estimate the relationship of prior breastfeeding to perimenopausal body mass index (BMI) (kg/m2). While most long-term studies of women's body weight after reproductive experience have found a negative relationship between lactation and body weight, most short-term studies found either no impact of lactation on body weight, or decelerated postpartum weight loss among breastfeeding women. DESIGN: We performed a retrospective cohort study. SETTING: The study was done in The Netherlands between 1987 and 1990. SUBJECTS: The 1067 singleton females who were born around the time of the Dutch famine (1 August 1944 through 15 April 1946) at The University of Amsterdam Teaching Hospital were all traced and accounted for. This analysis refers to the 671 who were interviewed and for whom all data elements were available. ANALYSIS: We related parity and number of children breastfed at least 1 week to BMI at age 45 and at the time of marriage, both calculated from respondents: recall of weight and height, by linear multiple regression analysis, adjusting for potentially confounding variables. RESULTS: Among parous women, each additional live birth was associated with a BMI increase of 0.69 kg/m2 (CI = 0.24 to 1.13, P = 0.003), and for each additional child breastfed, the BMI was lower by 0.41 kg m2 (CI = -0.77 to -0.04, P = 0.03) at age 45. However, BMI before any reproductive experience was 0.39 kg/m2 greater (CI = 0.08 to 0.71, P = 0.01) with each additional subsequent birth, and for each additional infant breastfed, 0.34 kg/m2 lower (CI = - 0.60 to - 0.09, P = 0.008). CONCLUSIONS: The negative relationship of prior breastfeeding to perimenopausal weight was probably not causally related to breastfeeding, since the relationship was present before any reproductive experience. The results could also arisen in part because of biased recall of weight; however, the relationship of parity to perimenopausal BMI was far less attenuated by adjusting for prior BMI than was the association with breastfeeding, lending support to an indirect association of breast- feeding with later BMI. PMID- 8617186 TI - Dose-related gastrointestinal response to the ingestion of either isomalt, lactitol or maltitol in milk chocolate. AB - OBJECTIVES: To determine whether there were differences between different polyols (sugar alcohols) in terms of their ability to stimulate intolerance symptoms when consumed in milk chocolate. Also to discover whether symptomatology can be related to the dose of polyol ingested. DESIGN: The study was of a randomised double-blind cross-over design. SUBJECTS: 59 healthy volunteers aged 18-24 years were recruited from the student population of the University of Salford. All subjects successfully completed the trial. INTERVENTIONS: Subjects ingested 100 g milk chocolate containing 40 g bulk sweetner as either sucrose, isomalt, lactitol or maltitol or a mixture (10:30 w/w) of sucrose and isomalt, sucrose and lactitol or sucrose and maltitol. Each bar was taken as breakfast on one day with following products consumed at 1-week intervals. Subjects reported the incidence and severity of the symptoms of flatulence, borborygms, colic, motion frequency and loose stools. RESULTS: The ingestion of 30 g or 40 g lactitol resulted in a significant increase in the incidence and severity of all symptoms examined compared to reactions after the consumption of standard sucrose-containing chocolate (P <0.01). Similarly, 40 g isomalt led to an increased incidence of all symptoms, including mild laxation (P <0.01), but unlike lactitol none was rated as being severe. A reduction in isomalt to 30 g was marked by increased tolerance with evidence of only mild borborygms (P <0.01), mild flatulence, colic, and laxation (P <0.05), with no increase in motion frequency (P <0.35). Ingestion of 40 g maltitol caused less intolerance than 40 g isomalt, with evidence of only flatulence, borborygms and colic (P <0.01), symptoms being rated as only mild. A reduction to 30 g led to a decrease in all symptoms except mild flatulence. Maltitol did not have any laxative effect when ingested at either 30 g (P = 0.32) or 40 g (P = 0.13) per day. CONCLUSIONS: This work has shown that there are significant differences in the reporting of gastrointestinal symptomatology following the consumption of isomalt, lactitol and maltitol incorporated into milk chocolate. However, with all three polyols the incidence and severity of symptomatology was dose dependent. PMID- 8617187 TI - Influence of parboiling and physico-chemical characteristics of rice on the glycaemic index in non-insulin-dependent diabetic subjects. AB - OBJECTIVE: To study the influence of parboiling, amylose content and gelatinisation temperature of rice on the postprandial blood glucose and insulin responses in non-insulin-dependent diabetic (NIDDM) subjects. DESIGN AND SUBJECTS: Twelve NIDDM subjects ingested high (27%) and low amylose content five test meals of 50 g available carbohydrates as white bread, cooked polished rice with high (27%) and low amylose content (12%) with different gelatinisation temperature and as nonparboiled and parboiled. The meals were taken in random order after a 12h fast with approximately 7 days interval. RESULTS: The glycaemic indices (GI) of all rice varieties were lower than that of white bread (P <0.001). Furthermore, GI of parboiled rice with a high amylose content was lower than that of parboiled rice with a low amylose content (50 +/- 7 vs 73 +/- 7, P <0.01). No differences were 47 +/- 4, n.s.), nor between non-parboiled and parboiled rice (50 +/- 7 vs 53 +/- 7, n.s.). Insulin responses to the five test meals were not significantly different in the NIDDM subjects. CONCLUSIONS: In NIDDM subjects the investigated rices were all low glycaemic as compared to white bread,independent of parboiling and physico-chemical characteristics. The mildparboiling process used did not influence GI. The study showed that the amylose content, but not the gelatinisation temperature, may be an useful criterion in selection of low GI rices also after parboiling. PMID- 8617188 TI - In vivo antioxidant effect of green and black tea in man. AB - OBJECTIVE: Evaluation of the vitro antioxidant activity of green and black tea, their in vivo effect on plasma antioxidant potential in man and the effect of milk addition. DESIGN: The antioxidant activity of the tea, with and without milk, was tested in vitro by measuring the length of the peroxyl radical induced lag-phase. The in vivo activity was tested on two groups of five healthy adults. Each group ingested 300 ml of either black or green tea, after overnight fast. The experiment was repeated on a separate day, adding 100 ml whole milk to the tea (ratio 1:4 ). Five subjects acted as controls. The human plasma antioxidant capacity (TRAP) was measured before and 30, 50 and 80 min from the ingestion of tea. RESULTS: Both teas inhibited the in vitro peroxidation in a dose-dependent manner. Green tea was sixfold more potent than black tea. The addition of milk to either tea did not appreciably modify their in vitro antioxidant potential. In vivo, the ingestion of tea produced a significant increase of TRAP (P <0.05), similar in both teas, which peaked at 30-50 min. When tea was consumed with milk, their in vivo activity was totally inhibited. CONCLUSIONS: The paper shows that tea possesses a strong antioxidant activity in vitro which is believed to be exerted by its polyphenols moiety. It also provides compelling evidence that tea has also a potent in vivo activity in man. The promptness of the in vivo response suggests that the absorption of the bioactive components of tea takes place in the upper part of the gastrointestinal system. The inhibition of this effect by milk is thought to be due to the complexation of tea polyphenols by milk proteins. These findings might help to clarify the putative role of dietary poly- phenols in modulating oxidative stress in vivo. PMID- 8617189 TI - Nutrition knowledge and attitudes towards high-fat foods and low-fat alternatives in three generations of women. AB - OBJECTIVES: To assess family resemblance in food habits in three generations of maternally related family members. DESIGN AND SUBJECTS: Ninety-seven adult women, their mothers and grandmothers were asked about nutrition knowledge, attitudes and fat intake. Nutrition knowledge and attitudes were determined by means of a self-administered questionnaire. A food frequency questionnaire was used to assess fat intake. RESULTS: Mean percentage energy derived from fat was 39% for the younger generation, and 40% for their mothers and grandmothers. Generations differed in their nutrition knowledge score (P <0.0001), the grandmothers having a lower nutrition knowledge than the other generations. Correlations of nutrition knowledge scores were 0.30 (95% confidence limits (c.l.) 0.10 and 0.48) between the younger and middle generations, 0.35 (95% c.l. 0.16 and 0.52) between the middle and older generations, and 0.14 (95% c.l. -0.06 and 0.34) between the younger generation and their grandmothers. For attitudes towards high-fat foods and their low-fat alternatives these figures were 0.27 (95% c.l. 0.07 and 0.45), 0.22 (95% c.l. 0.01 and 0.41), and 0.17 (95% c.l. - 0.03 and 0.36), respectively, while for energy percentage of fat intake the correlations were only 0.19 (95% c.l. -0.01 and 0.37), -0.02 (95% c.l. -0.22 and 0.18), and 0.12 (95% c.l. limits 0.08 and 0.31), respectively. Within generations the correlations between attitudes and nutrition knowledge or percentage energy derived from fat were found to be higher in the middle generation than in other generations. No statistically significant correlations were found between nutrition knowledge and percentage energy derived from fat. CONCLUSIONS: From this study it can be concluded that mothers and their adult daughters resemble each other in nutrition knowledge and attitudes. PMID- 8617190 TI - Effects of short-term zinc supplementation on cellular immunity, respiratory symptoms, and growth of malnourished Equadorian children. AB - OBJECTIVE: To assess the effect of zinc supplementation on respiratory tract disease, immunity and growth in malnourished children. DESIGN: A randomized double-blind placebo-controlled trial. SETTING: A day-care center in Quito, Ecuador. SUBJECTS: Fifty children (12-59 months old) recruited by height-for-age and weight-for-age deficit. INTERVENTIONS: Twenty-five children (supplemented, S group) received 10 mg/day of zinc as zinc sulfate, and 25 (nonsupplemented, NS group) received a placebo during 60 days. All were also observed during a 60-day postsupplementation period. Two children of the S group dropped out. Daily the clinical presence of cough, respiratory tract secretions, and fever, was recorded. On days 0,60 and 120, the cutaneous delayed-type hypersensitivity (DTH) to multiple antigens, and anthropometric parameters were assessed. On days 0 and 60 serum zinc levels were also measured. RESULTS: On day 60, DTH was significantly larger (20.8 +/- 7.1 vs 16.1 +/- 9.7 mm), and serum zinc levels were significantly higher (118.6 +/- 47.1 vs 83.1 +/- 24.5 micrograms/dl) in the S group than in the NS group (P <0.05 for each). The incidence of fever [relative risk (RR): 0.30, c.i. = 0.08- 0.95, P =0.02], cough (RR): 0.52, c.i. = 0.32-0.84, P = 0.004) and upper respiratory tract secretions (RR):0.72, c.i. = 0.59-0.88, P = 0.001) was lower in the S group than in the NS group at day 60. At the end of the postsupplementation observation period (day 120), the incidence of fever and upper respiratory tract secretions was the same in both the S and NS groups. The incidence of cough was higher at day 120 in the S group than in the NS group (RR): 2.28, c.i. = 1.37-3.83, P = 0.001). CONCLUSIONS: This study supports a role for zinc in immunity, and immunity to respiratory infections, while pointing out the need for larger studies. PMID- 8617191 TI - Home enteral tube feeding in East Anglia. AB - OBJECTIVE: To establish the profile of patients receiving home enteral tube feeding (HETF) in East Anglia, to assess the adequacy of care, and to consider the causes and implications of differences in the geographic prevalence of HETF. DESIGN: A 1-year prospective study. SETTING: Eight independently funded districts within East Anglia. SUBJECTS: Basic clinical data were available from 234 patients, 191 of which were tube-fed in the same district as that which initiated treatment, with detailed data obtained from 126. OUTCOME MEASURES: Prevalence, complications in relation to the dietetic support available, and standards of care in relation to those set in the reports of the King's Fund and the British Association of Parenteral and Enteral Nutrition. RESULTS: The patients, who were usually elderly subjects or children, had severe disabilities: 40% were unable to walk: 39% were unable to speak; and 20% were housebound. In all cases enteral tube feeding had been initiated in hospital but in this survey we found inadequate training for home care in 23%, inadequate support and follow-up in 20%, inadequate equipment in 41%, uncertainties regarding organisation, and various clinical problems such as feed regurgitation, in 22%, aspiration pneumonia in 13% , which occasionally led to hospitalization (4%), and frequent but usually minor peristomal problems in patients with gastrostomies. There was a direct correlation between the prevalence of HETF (which varied fourfold between districts) and the number of NHS dieticians per head of population; and in inverse correlation with respect to requests by patients for more support (r = 0.97), complaints of blocked tubes (r = 0.82) and the need for hospital help. CONCLUSION: HETF provides an important form of support to a small group of severely disabled patients. There are important differences between districts in the prevalence of HETF which may be related to variation in local expertise and available support staff. The standards of care did not always conform to those set in the King's Fund report and BAPEN report on Enteral and Parenteral Nutrition in the community. PMID- 8617192 TI - Vitamin A deficiency and xerophthalmia in western Yemen. AB - OBJECTIVE: To determine the prevalence of xerophthalmia and the extent of vitamin A deficiency in western Yemen. DESIGN: A stratified cluster sample of children aged 1-5 years with clinical examination for signs of xerophthalmia as well as blood serum survey. SETTING: The 18 districts of western Yemen, of which 10 clusters were chosen at random. SUBJECTS: All children aged 1-5 years resident in the cluster sites (n = 2438). MAIN OUTCOME MEASURES: Clinical signs of xerophthalmia, a history of night blindness, serum retinol levels in a random sample of clinically normal children (n =338) in addition to all children with xerophthalmia. RESULTS: Night blindness was found in 0.5% of the children, Bitot's spots in 1.7%, corneal ulceration in 0.04% and corneal scars in 0.04% Of the subsample, 7.2% (95% confidence interval [c.i.] 4.4-10.0%) had serum retinol values below 10 micrograms/dl; 63.0% (95% c.i. 57.6- 68.4%) had values below 20 micrograms/dl. CONCLUSIONS: Xerophthalmia and vitamin A deficiency are public health problems in western Yemen. PMID- 8617193 TI - Prison deprivation and protein nutritional status of inmates of a developing community prison. AB - OBJECTIVE: This study assesses the protein nutritional status of inmates of a small prison, in the light of a changing serum protein pattern in the environment. DESIGN: Survey of prison inmates. SETTING: A male prison and low social class hospital workers. SUBJECTS: 81 inmates and 62 randomly selected controls, of which 42 of the former and 28 of the latter were further randomly selected. METHODS: Nutritional and confinement history were obtained by a questionnaire, Weights and heights were measured and serum and urine samples collected for protein estimations. RESULTS: The prisoners were confined for 229.1 (SD 210.1) days. 48.1% had BMI < 20 kg/m2 compared to 28.6% of controls (p = 0.04). Their diet was predominantly carbohydrate. Mean total serum protein was 81.5gm/L (6.1) (95% CI = 79.3-83.5) (prisoners); 88.7 gm/L (8.1) (95% CI = 85.7 91.7) (controls). Serum globulin was 44.9 gm/L (6.3) (95% CI = 42.9-46.9) (prisoners); 55.4 gm/L (8.2) (95% CI = 52.3-58.5) (controls) (p <0.001 respectively). More prisoners than controls had severe hypo- albuminaemia (p =0.001). All values were lower in those awaiting trial compared to convicted inmates. Total protein and albumin weakly correlated with duration of confinement (r = -0.23 and -0.15, respectively). CONCLUSION: The study suggest that the prisoners might be undernourished with lower serum proteins than controls, within an average of seven months confinement. The picture appears worse in those awaiting trial and with prolonged duration of confinement. PMID- 8617194 TI - Longitudinal weight changes over four years and associated health factors in 629 men and women aged over 65. AB - OBJECTIVES: (1) To assess changes in body weight longitudinally over 4 years in a representative group of men and women aged over 65 years, living in their own homes. (2) To assess whether initial weight (or weight in proportion to skeletal size) was associated with health 4 years later. (3) To assess whether any changes found in body weight were associated with changes in physical or mental health. (4) To compare the cross-sectional age-related changes in weight found in the initial survey with the longitudinal changes found over 4 years. DESIGN: A large randomised age-stratified survey using a structured questionnaire and measurements of body weight and demispan (for skeletal size), with a 4-year follow-up. SETTING: The survey was conducted in the respondents' own homes. SUBJECTS: 958 subjects age over 65 years, who were recruited from the Nottingham general practitioners' lists, took part in the initial survey; 629 of these subjects completed the second survey 4 years later. RESULTS: (1) The mean 4-year change in body weight was a small but significant loss; in women (n = 385) it was 1.56kg (P <0.001, 95% CI 1.02-2.10) and in men (n = 244) 0.85kg (P = 0.010, 95% CI 0.21-1.49). (2) Initial weight did not predict mortality, new morbidity nor health 4 years later. (3) There were no robust associations between weight change ans either absolute measures of physical health or changes in these measures. (4) The longitudinal change in weight was similar to that predicted by the cross sectional data. CONCLUSIONS: (1) Ageing, in old age, is associated with loss of body weight, but with a large intra-individual variation. (2) Neither initial body weight nor the change, was associated with mortality or morbidity over 4 years in a large representative sample of old people living in their own homes in a food-rich country. PMID- 8617195 TI - The actin binding site of thymosin beta 4 mapped by mutational analysis. AB - We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin. PMID- 8617196 TI - Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle. AB - Fas, also called APO-1, belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits an apoptotic signal within the cell by binding to the Fas ligand. Fas has been implicated in the activation-induced suicide of T cells and cytotoxic T cell activity in the immune system. Non-immune cells such as those in liver, lung and ovary also express Fas, but its role in these cells remains unclear. Ovariectomy has been used to study homeostasis of female reproductive organs, which is regulated by sex hormones. Here we analyzed Fas function in the ovariectomy-induced regression of mouse vaginal epithelial cells. Fas expression was detected in vagina and was elevated after ovariectomy. Fas deficient lpr and lpr(cg) mice did not exhibit ovariectomy-induced regression of vaginal epithelia, whereas uterine regression induced by ovariectomy was not affected in these mice. The vaginas of lpr and lpr(cg) mice were in a persistent estrous stage with cornification of vaginal epithelia, as judged from the cell types in the vaginal fluid. Thus, Fas appears to be involved directly in the regression of vaginal epithelia induced by ovariectomy and during the estrous cycle, suggesting that the physiological role of this receptor extends beyond that exerted on immune cells. This is the first evidence of a role for Fas inducing physiological apoptosis in non-immune cells. PMID- 8617198 TI - The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death. AB - ATP binding cassette (ABC) transporters define a family of proteins with strong structural similarities conserved across evolution and devoted to the translocation of a variety of substrates across cell membranes. A few members of the family are known in mammals, but although all of them are medically relevant proteins, knowledge of their molecular function remains scanty. We report here a morphological and functional study of the recently identified mammalian ABC transporter, ABC1. Its expression during embryonic development correlates spatially and temporally with the areas of programmed cell death. More specifically, ABC1 is expressed in macrophages engaged in the engulfment and clearance of dead cells. Moreover, ABC1 transporter is required for engulfment since the ability of macrophages to ingest apoptotic bodies is severely impaired after antibody-mediated steric blockade of ABC1. A structural homologue of ABC1 has been identified in the Caenorhabditis elegans genome and maps close to the ced-7 locus. Since ced-7 phenotype is precisely defined by an imparied engulfment of cell corpses, it is tempting to surmise that ABC1 might be a mammalian homologue of ced-7. PMID- 8617197 TI - Superoxide anion is a natural inhibitor of FAS-mediated cell death. AB - The cell surface receptor Fas is a major trigger of apoptosis. However, expression of the Fas receptor in many tumor cell types does not correlate with sensitivity to Fas-mediated cell death. Because a prooxidant state is a common feature of tumor cells, we examined the role of intracellular reactive oxygen intermediates in the regulation of Fas-mediated cytotoxicity. Our results show that an oxidative stress induced by increasing the intracellular superoxide anion (O2-) concentration can abrogate Fas-mediated apoptosis in cells which are constitutively sensitive to Fas. Conversely, an O2- concentration decrease is observed to sensitize cells which are naturally resistant to Fas signals. These observations suggest that intracellular O2- may play a key role in regulating cell sensitivity to a potentially lethal signal and provide tumor cells with a natural, inducible mechanism of resistance to Fas-mediated apoptosis. PMID- 8617199 TI - Delineation of the peptide binding site of the human galanin receptor. AB - Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th PMID- 8617200 TI - Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. AB - Transforming growth factor-betas (TGF-betas) are produced by most cells in large latent complexes of TGF-beta and its propeptide (LAP) associated with a binding protein. The latent TGF-beta binding proteins (LTBPs-1, -2 and -3) mediate the secretion and, subsequently, the association of latent TGF-beta complexes with the extracellular matrix (ECM). The association of beta1-LAP with LTBP-1 was characterized at the molecular level with an expression system in mammalian cells, where TGF-beta1 and various fragments of LTBP-1 were co-expressed and secreted with the aid of a signal peptide synthesized to the LTBP-1 constructs. Immunoblotting of the fusion protein complexes indicated that the third 8-Cys repeat of LTBP-1 bound covalently to the LAP region of TGF-beta1. The cysteine required for the association between LTBP-1 and beta1-LAP was mapped to Cys33 of beta1-LAP. The N-terminal region of LTBP-1 consisting of the first 400 amino acids was found to associate covalently with the ECM. The data indicate that an 8 Cys repeat of LTBP is capable of covalent and specific protein-protein interactions. These interactions are mediated by exchanging cysteine disulfide bonds between the core 8-Cys repeat and an optionally associated protein during the secretion. This is, to our knowledge, the first demonstration of an extracellular protein module that is able to exchange cysteine disulfide bonds with heterologous ligand proteins. PMID- 8617201 TI - ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. AB - Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling. PMID- 8617202 TI - The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. AB - Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. PMID- 8617203 TI - Complementation between kinase-defective and activation-defective TGF-beta receptors reveals a novel form of receptor cooperativity essential for signaling. AB - Transforming growth factor-beta (TGF-beta) signals through two transmembrane serine/threonine kinases, T beta R-I and T beta R-II. TGF-beta binds to T beta R II, allowing this receptor to associate with and phosphorylate T beta R-I which then propagates the signal. T beta R-I is phosphorylated within its GS domain, a region immediately preceding the kinase domain. To further understand the function of T beta R-I in this complex, we analyzed T beta R-I-inactivating mutations identified in cell lines that are defective in TGF-beta signaling yet retain ligand binding ability. The three mutations identified here all fall in the kinase domain of T beta R-I. One mutation disrupts the kinase activity of T beta R-I, whereas the other two mutations prevent ligand-induced T beta R-I phosphorylation, and thus activation, by T beta R-II. Unexpectedly, a kinase defective T beta R-I mutant can functionally complement an activation- defective T beta R-I mutant, by rescuing its T beta R-II- dependent phosphorylation. Together with evidence that the ligand-induced receptor complex contains two or more T beta R-I molecules, these results support a model in which the kinase domain of one T beta R-I molecule interacts with the GS domain of another, enabling its phosphorylation and activation by T beta R-II. This cooperative interaction between T beta R-I molecules appears essential for TGF-beta signal transduction. PMID- 8617204 TI - A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. AB - Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed. PMID- 8617205 TI - FluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlA beta activation. AB - The Aspergillus nidulans fluG gene is necessary for the synthesis of a small diffusible factor that is required for the endogenously regulated induction of asexual sporulation that takes place during the development of an air-exposed colony. Previous work established that FluG is present at nearly constant levels throughout the Aspergillus life cycle, leading to the hypothesis that FluG factor is constitutively produced and development initiates after its concentration surpasses a fixed threshold. Here we show that overexpression of fluG can overcome the developmental block normally imposed on vegetative cells in submerged culture and leads to the formation of complex conidiophores that are remarkably similar to wild-tye conidiophores made by air- exposed colonies. This fluG-induced sporulation requires the activities of other early developmental regulatory genes including, flA, flB, flC, flD, flE, and brlA. The requirement for flbA in fluG-induced sporulation is particularly interesting because overexpression of flbA can also induce sporulation in submerged culture and this flbA activity requires fluG. The interdependence of fluG and flbA activities suggests a close relationship between the products of these two genes in controlling conidiophore development. In addition to the endogenous sporulation signal provided by fluG, several environmental factors, including air exposure, carbon or nitrogen stress, and increased osmolarity, can influence developmental activation. We demonstrate that each of these signals requires the brlA beta gene, but not brlA alpha, to initiate conidiophore development. We present a model to account for the complex genetic and environmental controls leading to the activation of brlA beta and sporulation. PMID- 8617206 TI - Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle cell lineages. AB - Targeted inactivation of the myogenic determination genes myf-5 and myoD in mice resulted in moderate (Myf-5) or no muscle phenotypes (MyoD) and double knock-out mutants lacking both genes failed to develop any skeletal muscle. In order to determine the mechanism of this apparent genetic redundancy we investigated the basis of functional overlap between the two genes. Here we demonstrate that Myf-5 and MyoD are not expressed within the same muscle precursor cell, but rather determine different muscle cell lineages arising from independently committed stem cell populations. Selective ablation of Myf-5-expressing muscle precursors from differentiating ES cells does not prevent Myo-D-dependent muscle differentiation. The early muscle progenitor cells which normally express Myf-5 do not develop into later appearing MyoD cells, even when the myf-5 gene has been inactivated. Thus skeletal musculature in vertebrates develops from two separate cell lineages and complementation may occur at the cellular level, but not between different myogenic factor genes within one cell. PMID- 8617208 TI - Functional hierarchy and phenotypic suppression among Drosophila homeotic genes: the labial and empty spiracles genes. AB - The Drosophila homeotic cluster (HOM-C) is made up of eight genes, which specify the identity of cephalic, thoracic and abdominal segments. These genes can be ordered in a hierarchy which correlates with their position along the 5'-3' transcriptional direction. When they are absent, thoracic and abdominal body segments develop the same'ground' pattern, which is thoracic-like but also includes cephalic structures (sclerotic plates). We find that these plates are specified by the homeobox gene empty spiracles (ems) which is not a member of the HOM-C and which is expressed in all body segments. ems mutations, however, only produce defects in anterior head structures and the posterior spiracles. The ems product has the potential to induce sclerotic plates but this potential is suppressed by any of the HOM-C genes, including the labial gene, which we show to be the lowest ranking of the HOM-C hierarchy. This suppression does not occur at the transcriptional or translational level because the ems function is suppressed in cells containing the ems product. Thus, this appears to be the first case of phenotypic suppression operating in normal development. We propose that ems was originally a member of the HOM-C which escaped from the complex and has also acquired new functions during evolution. PMID- 8617207 TI - GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes. AB - The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. PMID- 8617209 TI - Conserved function of anopheles gambiae midgut-specific promoters in the fruitfly. AB - Control of malaria by a methodology that would permit the effective blockage of the Anopheles gambiae midgut wall penetration by Plasmodium parasites requires a detailed understanding of both the physiology of the mosquito's digestion, and of the interactions between the parasite and its host. We have transformed Drosophila melanogaster with several constructs that allow the study of the promoter region of two of the major late trypsin genes of A. gambiae. Using several deletions, we have identified, for both genes, small genomic segments that are sufficient to confer tissue specificity to the promoter in a species that is far away in evolution from the mosquito. This will allow further studies that will enable both the understanding of the blood meal digestion, and may potentially be useful for the design of anti-plasmodial constructs at a later stage. PMID- 8617210 TI - The two PAR leucine zipper proteins, TEF and DBP, display similar circadian and tissue-specific expression, but have different target promoter preferences. AB - The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfection experiments, these two proteins exhibit different activation potentials for two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF-DBP fusion protein, carrying N-terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene as strongly as wild-type DBP. Our results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP. PMID- 8617211 TI - Genetic analysis of glucose regulation in saccharomyces cerevisiae: control of transcription versus mRNA turnover. AB - A major determinant of the steady-state level of the mRNA encoding the iron protein (Ip) subunit of succinate dehydrogenase of yeast is its rate of turnover. This mRNA is significantly more stable in glycerol than in glucose media. Many other genes, for example, SUC2, that are repressed in the presence of glucose are believed to be controlled at the level of transcription. The present study elucidates differences in the regulatory mechanisms by which glucose controls the transcription and turnover of the SUC2 and Ip mRNAs. The signaling pathway for glucose repression at the transcriptional level has been associated with a number of gene products linking glucose uptake with nuclear events. We have investigated whether the same genes are involved in the control of Ip mRNA stability. Phosphorylation of glucose or fructose is critical in triggering the transcript's degradation, but any hexokinase will do. Of the other known genes examined, most, with the exception of REG1, are not involved in determining the differential stability of the Ip transcript. Finally, our results indicate that differential stability on different carbon sources also plays a role in determining the steady state level of the SUC2 mRNA. Thus, glucose repression includes both transcriptional and post-transcriptional mechanisms. PMID- 8617213 TI - Activation and repression of transcription at two different phage phi29 promoters are mediated by interaction of the same residues of regulatory protein p4 with RNA polymerase. AB - Phage phi29 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Arg120 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Arg120, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Arg120 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription. PMID- 8617212 TI - Epstein-Barr virus nuclear antigen 2 is a transcriptional suppressor of the immunoglobulin mu gene: implications for the expression of the translocated c-myc gene in Burkitt's lymphoma cells. AB - A conditional mutant of Epstein-Barr virus nuclear antigen 2 (EBNA2) regulated by estrogen was employed to study the effect of EBNA2 on the cellular phenotype. Activation of EBNA2 in lymphoblastoid cell lines (LCLs) and in B cell lymphoma lines resulted in down-regulation of cell surface IgM and Ig-mu steady-state RNA expression. In LCLs, activation of EBNA2 is required for maintaining proliferation, whereas in Burkitt's lymphoma (BL) cell lines with t(8;14) translocations, activation of EBNA2 induces growth arrest. In these cells, Northern and nuclear run-on analyses revealed rapid simultaneous repression of Ig mu and c-myc transcription as early as 30 min after activation of EBNA2. Since c myc expression is under the control of the Ig heavy chain locus in BL cell lines with a t(8;14) translocation, we propose that Ig-mu and c-myc are down-regulated by EBNA2 through a common mechanism. PMID- 8617214 TI - Preferential binding of an unfolded protein to DsbA. AB - The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. PMID- 8617215 TI - Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria. AB - Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70 dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins. PMID- 8617216 TI - A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. AB - The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state. PMID- 8617217 TI - Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. AB - Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50 kDa (p50) and further distinct 20 and 130 kDa polypeptides were detectable. Class II binding of p50 was characterized in detail. Point mutations within the Ak antigen binding groove destabilized the p50:class II complexes; a mutation outside the groove had no effect. A short segment of p50 was sufficient for association with Ak. The p50 polypeptide was synthesized endogenously, bound to Ak in a pre-Golgi compartment, and was transported to the cell surface in association with Ak. Thus, Ii protects the class II groove from binding endogenous, possibly misfolded polypeptides in the ER. The possibility is discussed that polypeptide binding is an ancestral function of the MHC antigen binding domain. PMID- 8617218 TI - A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa. AB - A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery. PMID- 8617219 TI - Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease. AB - Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease. PMID- 8617220 TI - Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. AB - Muscle wasting is a critical feature of patients afflicted by AIDS or cancer. In a murine model of muscle wasting, tumor necrosis factor alpha (TNF alpha) induces oxidative stress and nitric oxide synthase (NOS) in skeletal muscle, leading to decreased myosin creatinine phosphokinase (MCK) expression and binding activities. The impaired MCK-E box binding activities resulted from abnormal myogenin-Jun-D complexes, and were normalized by the addition of Jun-D, dithiothreitol or Ref-1, a nuclear redox protein. Treatment of skeletal muscle cells with a phorbol ester, a superoxide-generating system, an NO donor or a Jun D antisense oligonucleotide decreased Jun-D activity and transcription from the MCK-E box, which were prevented by antioxidants, a scavenger of reducing equivalents, a NOS inhibitor and/or overexpression of Jun-D. The decreased body weight, muscle wasting and skeletal muscle molecular abnormalities of cachexia were prevented by treatment of TNF alpha mice with the antioxidants D-alpha tocopherol of BW755c, or the NOS inhibitor nitro-L-arginine. PMID- 8617221 TI - The role of the adenovirus protease on virus entry into cells. AB - Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid-stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re-entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry-competent virus but also to disassemble the incoming virus. PMID- 8617222 TI - Crystal structure of the human adenovirus proteinase with its 11 amino acid cofactor. AB - The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy. PMID- 8617223 TI - ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. AB - A cDNA encoding a second zinc transporter (ZnT-2) was isolated from a rat kidney cDNA expression library by complementation of a zinc-sensitive BHK cell line. The protein predicted from the open reading frame of ZnT-2 cDNA has 359 amino acids and initiates with a CTG codon. It resembles ZnT-1 (a plasma membrane protein that stimulates zinc efflux) in overall topology in that it has six membrane spanning domains, a histidine-rich intracellular loop and a long C-terminal tail; however, the overall amino acid identity is only 26%. Unlike ZnT-1, which is in the plasma membrane and lowers cellular zinc by stimulating zinc efflux, ZnT-2 is localized on vesicles and allows the zinc-sensitive BHK cells to accumulate zinc to levels that are much higher than non-transformed cells can tolerate. Zinc was visualized within these vesicles with zinquin, a zinc-specific fluorescent probe. The intracellular compartment that accumulates zinc is acidic as revealed by staining with acridine orange or LysoTracker. Prolonged exposure of cells expressing ZnT-2 to zinc causes an accretion of intracellular vesicles. We suggest that ZnT-2 protects these cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment. PMID- 8617224 TI - Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval. AB - Two new thermosensitive yeast mutants defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2-1 and ret3-1 were defective for ER retrieval, only ret2-1 exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine tagged proteins back to the ER, and the similarity between clathrin and coatomer coats. PMID- 8617226 TI - A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. AB - The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry. PMID- 8617225 TI - Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras. AB - GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction. PMID- 8617227 TI - The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import. AB - Nuclear proteins are targeted through the nuclear pore complex (NPC) in an energy dependent reaction. The import reaction is mediated by nuclear localization sequences (NLS) in the substrate which are recognized by heterodimeric cytoplasmic receptors. hSRP1 alpha is an NLS-binding subunit of the human NLS receptor complex and is complexed in vivo with a second subunit of 97 kDa (p97). We show here that a short amino-terminal domain in hSRP1 alpha is necessary and sufficient for its interaction with p97. This domain is conserved in other SRP1 like proteins and its fusion to a cytoplasmic reporter protein is sufficient to promote complete nuclear import, circumventing the usual requirement for an NLS receptor interaction. The same amino-terminal domain inhibits import of NLS containing proteins when added to an in vitro nuclear transport assay. While full length hSRP alpha is able to leave the nucleus, the amino-terminal domain alone is not sufficient to promote exit. We conclude that hSRP1 alpha functions as an adaptor to tether NLS-containing substrates to the protein import machinery. PMID- 8617228 TI - Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfur clusters on the PsaC protein of photosystem I. AB - The PsaC protein of the Photosystem I (PSI) complex in thylakoid membranes coordinates two [4Fe-4S] clusters, FA and FB. Although it is known that PsaC participates in electron transfer to ferredoxin, the pathway of electrons through this protein is unknown. To elucidate the roles of FA and FB, we created two site directed mutant strains of the cyanobacterium Anabaena variabilis ATCC 29413. In one mutant, cysteine 13, a ligand for FB was replaced by an aspartic acid (C13D); in the other mutant, cysteine 50, a ligand for FA was modified similarly (C50D). Low-temperature electron paramagnetic resonance studies demonstrated that the C50D mutant has a normal FB center and a modified FA center. In contrast, the C13D strain has normal FA, but failed to reveal any signal from FB. Room temperature optical studies showed that C13D has only one functional electron acceptor in PsaC, whereas two such acceptors are functional in the C50D and wild type strains. Although both mutants grow under photoautotrophic conditions, the rate of PSI-mediated electron transfer in C13D under low light levels is about half that of C50D or wild type. These data show that (i) FB is not essential for the assembly of the PsaC protein in PSI and (ii) FB is not absolutely required for electron transfer from the PSI reaction center to ferredoxin. PMID- 8617229 TI - Different modes of proton translocation by sensory rhodopsin I. AB - The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction. PMID- 8617230 TI - Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump. AB - We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model. PMID- 8617231 TI - FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5. AB - The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+( )ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322 355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening. PMID- 8617232 TI - Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin. AB - Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore. PMID- 8617233 TI - A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed. AB - Calcium/calmodulin-dependent protein kinases (CaM kinases) have been reported to be involved in neuroplasticity. We have cloned a new Drosophila CaM kinase gene named caki. We describe the molecular characterization of caki and a behavioral effect of its elimination. The caki gene is extremely large; comparison of the genomic and cDNA sequences reveals that the caki transcription unit is at least 150 kb. The catalytic domain of this new CaM kinase protein shares homology (41%) with type II CaM kinases, while the C-terminal part is divergent. Constitutively expressed Caki protein is enzymatically active since it causes a 3-fold increase in the level of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter in a co-transfusion assay. In situ hybridization shows that during embryogenesis, larval and pupal life, transcription of caki is restricted almost exclusively to the central nervous system. In the adult head, immunohistochemistry reveals Caki protein in the lamina, the neuropil of the medulla, lobula, lobula plate and in the central brain. Mutant caki flies show reduced walking speed in 'Buridan's paradigm'. PMID- 8617234 TI - MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH. AB - MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation. PMID- 8617235 TI - The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. AB - The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo. PMID- 8617236 TI - Lineage commitment of transformed haematopoietic progenitors is determined by the level of PKC activity. AB - Our previous work showed that haematopoietic precursors transformed by the E26 avian leukemia virus undergo multilineage differentiation in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA). Treatment of the cells with high concentrations of PMA (100 nM) favours myelomonocytic differentiation, while lower concentrations (20 nM) induce predominantly eosinophil differentiation. Here we have investigated the role of protein kinase C (PKC) in this process and found that 100 nM, but not 20 nM, PMA dramatically down-regulates total cellular PKC activity, indicating that high PMA concentrations result in less efficient signalling than lower PMA concentrations. Consistent with these findings is the observation that very low PMA concentrations (1 nM), which presumably only moderately activate PKC, induce myeloid differentiation. This suggests the existence of two PKC thresholds which play a role in lineage commitment. To test the model, alpha- and epsilon-PKC isoforms were expressed in E26-transformed progenitors. These cells exhibited myelomonocytic differentiation even in the absence of PMA, while treatment with concentrations of PMA as high as 100 nM led to the differentiation of predominantly eosinophils and failed to downregulate the exogenous PKC. Our results suggest that different levels of PKC activity result in three different phenotypes: (i) no PKC activity maintains the progenitor phenotype; (ii) low PKC activity favours myelomonocytic differentiation; (iii) high PKC favours eosinophil differentiation. PMID- 8617237 TI - Interleukin-2 activation of STAT5 requires the convergent action of tyrosine kinases and a serine/threonine kinase pathway distinct from the Raf1/ERK2 MAP kinase pathway. AB - Interleukin-2 (IL-2) induces DNA binding of STAT5, a member of the family of cytokine-regulated transcription factors termed 'signal transducers and activators of transcription'. IL-2-stimulated STAT5-DNA complexes include two tyrosine phosphoproteins which exhibit distinct mobilities in SDS-PAGE gels. Our studies have shown that IL-2 rapidly induces both tyrosine phosphorylation and serine phosphorylation of STAT5 and that the two STAT5 tyrosine phosphoproteins detected in IL-2-activated cells differ in their levels of phosphorylation on serine residues. The two different phosphoforms of STAT5 have identical in vitro DNA binding specificity and reactivity with tyrosine phosphopeptides, but differ in their cellular localization. As well, the present data indicate that the transcriptional activity of STAT5 is regulated by serine kinases in T lymphocytes. Two previously characterized serine kinases activated by IL-2, MAP kinase/ERK2 and p70 S6 kinase, do not appear to be involved in STAT5 regulation by this cytokine. Accordingly, STAT5 activation in T cells requires the convergent action of tyrosine kinases and a distinct serine/threonine kinase which has not previously been implicated in IL-2 signalling. PMID- 8617238 TI - The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. AB - Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation. PMID- 8617239 TI - The interleukin-7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. AB - The interleukin 7 receptor (IL7R), which contains a unique alpha chain and a gamma chain shared by other cytokine receptors, is indispensable for normal lymphocyte development. The basis for this role is poorly understood. Here we show that the IL7R alpha chain not only causes progenitors to proliferate, but also has a distinct activity in inducing differentiation. First, we identify a single cytoplasmic tyrosine residue in the IL7R alpha chain that is essential for cell cycle entry and proliferation dependent on phosphatidylinositol 3-kinase. We use a mutant alpha chain in which this residue has been altered to reconstitute B lymphopoiesis by retrovirus-mediated gene transfer in cultures of bone marrow from mice deficient in IL7R alpha chain. The mutation abrogates the proliferation of B-lymphocyte progenitors, but reveals a novel function of the alpha chain in promoting immunoglobulin heavy chain gene rearrangement leading to B-cell differentiation. This function is lost (but proliferation sustained) when the cytoplasmic domain of IL7R alpha is replaced by corresponding sequences from the IL2R, despite the similarity on their signalling mechanisms. Thus, the signals which mediate a differentiative function of the IL7R in B lymphopoiesis are specific and distinct from those causing proliferation. PMID- 8617240 TI - Yeast SUB1 is a suppressor of TFIIB mutations and has homology to the human co activator PC4. AB - Activation of transcription in eukaryotes depends upon the interplay between transcriptional activators and general transcription factors. While direct contacts between activators and general factors have been demonstrated in vitro, an additional class of proteins, termed co-activators, is also required of transcriptional activation. Here we describe a yeast protein, SUB1, that was isolated as a suppressor of the cold-sensitive TFIIB R78H mutant. The N-terminal third of SUB1 is highly similar to the mammalian co-activator PC4. We show that increased expression of SUB1 suppresses two alleles of TFIIB (E62G, R78H) specifically and that the deletion of SUB1 is lethal in combination with these same two alleles. We show that SUB1 binds to TFIIB in vitro and that it specifically inhibits the formation of TBP-TFIIB-promoter complexes. Furthermore we show that increasing the copy number of SUB1 stimulates transcriptional activation in vivo. Based on our results and recent observations of others, we propose that SUB1 plays a role in the release of TFIIB from the transcription complex during transcription initiation. PMID- 8617241 TI - A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB. AB - Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B". We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature-sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC-DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA complex. The two-hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B" component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B". An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription. PMID- 8617242 TI - The human papillomavirus type 16 E7 gene product interacts with and trans activates the AP1 family of transcription factors. AB - The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16 induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. PMID- 8617243 TI - DEAF-1, a novel protein that binds an essential region in a Deformed response element. AB - A 120 bp homeotic response element that is regulated specifically by Deformed in Drosophila embryos contains a single binding site for Deformed protein. However, a 24 bp sub-element containing this site does not constitute a Deformed response element. Specific activation requires a second region in the 120 bp element, which presumably contains one or more binding sites for Deformed cofactors. We have isolated a novel protein from Drosophila nuclear extracts which binds specifically to a site in this second region. This protein, which we call DEAF-1 (Deformed epidermal autoregulatory factor-1), contains three conserved domains. One of these includes a cysteine repeat motif that is similar to a motif found in Drosophila Nervy and the human MTG8 proto-oncoprotein, and another matches a region of Drosophila Trithorax. Mutations in the response element designed to improve binding to DEAF-1 in vitro resulted in increased embryonic expression. Conversely, small mutations designed to diminish binding to DEAF-1 resulted in reduced expression of the element. Thus, DEAF-1 is likely to contribute to the functional activity, and perhaps to the homeotic specificity, of this response element. Consistent with this hypothesis, we have discovered DEAF-1 binding sites in other Deformed response elements. PMID- 8617244 TI - C-terminal activating and inhibitory domains determine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8. AB - Pax-5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. In this study we have analysed the structural requirements for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C-terminal serine/threonine/proline-rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from promoter and enhancer positions. This transactivation domain was found to be inactivated by a naturally occurring frameshift mutation in one PAX-5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C-terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in the mammalian Pax-2 and Pax-8, the zebrafish Pax-b as well as the sea urchin Pax-258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C-terminal regulatory module composed of activating and inhibitory sequences. PMID- 8617245 TI - Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase. AB - An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis. PMID- 8617247 TI - Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs. AB - One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation. For type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by assays that monitor restriction in vivo. The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac prophage. In bacteria with elevated levels of EcoKI the co-operative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction. PMID- 8617246 TI - p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. AB - The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene. PMID- 8617248 TI - Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange. AB - Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein. PMID- 8617249 TI - Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. AB - Budding of enveloped viruses has been shown to be driven by interactions between a nucleocapsid and a proteolipid membrane. By contrast, we here describe the assembly of viral envelopes independent of a nucleocapsid. Membrane particles containing coronaviral envelope proteins were assembled in and released from animal cells co-expressing these proteins' genes from transfected plasmids. Of the three viral membrane proteins only two were required for particle formation, the membrane glycoprotein (M) and the small envelope protein (E). The spike (S) protein was dispensable but was incorporated when present. Importantly, the nucleocapsid protein (N) was neither required not taken into the particles when present. The E protein, recently recognized to be a structural protein, was shown to be an integral membrane protein. The envelope vesicles were found by immunogold labelling and electron microscopy to form a homogeneous population of spherical particles indistinguishable from authentic coronavirions in size (approximately 100 nm in diameter) and shape. They were less dense than virions and sedimented slightly slower than virions in sucrose velocity gradients. The nucleocapsid-independent formation of apparently bona fide viral envelopes represents a novel mode of virus assembly. PMID- 8617250 TI - Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c myc dependent transformation and are expressed during neural and epidermal differentiation. PMID- 8617251 TI - Apoptosis: molecular regulation of cell death. AB - The field of apoptosis is unusual in several respects. Firstly, its general importance has been widely recognised only in the past few years and its surprising significance is still being evaluated in a number of areas of biology. Secondly, although apoptosis is now accepted as a critical element in the repertoire of potential cellular responses, the picture of the intra-cellular processes involved is probably still incomplete, not just in its details, but also in the basic outline of the process as a whole. It is therefore a very interesting and active area at present and is likely to progress rapidly in the next two or three years. This review emphasises recent work on the molecular mechanisms of apoptosis and, in particular, on the intracellular interactions which control this process. This latter area is of crucial importance since dysfunction of the normal control machinery is likely to have serious pathological consequences, probably including oncogenesis, autoimmunity and degenerative disease. The genetic analysis of programmed cell death during the development of the nematode Caenorhabditis elegans has proved very useful in identifying important events in the cell death programme. Recently defined genetic connections between C. elegans cell death and mammalian apoptosis have emphasised the value of this system as a model for cell death in mammalian cells, which, inevitably, is more complex. The signals inducing apoptosis are very varied and the same signals can induce differentiation and proliferation in other situations. However, some pathways appear to be of particular significance in the control of cell death; recent analysis of the apoptosis induced through the cell surface Fas receptor has been especially important for immunology. Two gene families are dealt with in particular detail because of their likely importance in apoptosis control. These are, first, the genes encoding the interleukin-1 beta converting enzyme family of cysteine proteases and, second, those related to the proto-oncogene bcl-2. Both of these families are homologous to cell death genes in C. elegans. In mammalian cells the number of members of both families which have been identified is growing rapidly and considerable effort is being directed towards establishing the roles played by each member and the ways in which they interact to regulate apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-myc, or tumour suppressors, e.g. p53, an observation which is consistent with the importance of defective apoptosis in the development of cancer. Viral manipulation of the apoptosis of host cells frequently involves interactions with these cellular proteins. Finally, the biochemistry of the closely controlled cellular self destruction which ensues when the apoptosis programme has been engaged is also very important. The biochemical changes involved in inducing phagocytosis of the apoptotic cell, for example, allow the process to be neatly integrated within the tissues, under physiological conditions. Molecular defects in this area too may have important pathological consequences. PMID- 8617252 TI - Conformational features of a synthetic cyclic peptide corresponding to the complete V3 loop of the RF HIV-1 strain in water and water/trifluoroethanol solutions. AB - The disulfide-bridge-closed cyclic peptide corresponding to the whole V3 loop of the RF HIV-1 strain was examined by proton two-dimensional NMR spectroscopy in water and water/trifluoroethanol solutions. Although most of the peptide is conformationally averaged in water, the NOE data support a beta-turn conformation for the central conservative GPGR region and the presence of nascent helix. Upon addition of trifluoroethanol, helix formation in the C-terminal part becomes apparent. This is confirmed by CD data. NOEs indicative of multiple and transient beta-turns around the Asn6 glycosylation site and NOEs fitting X-ray data on a linear V3 peptide-Fab complex also emerge. The C-terminal helix is shown to have amphipathic character and might thus assist in the infection process. PMID- 8617253 TI - A new mechanism in serine proteases catalysis exhibited by dipeptidyl peptidase IV (DP IV)--Results of PM3 semiempirical thermodynamic studies supported by experimental results. AB - Herein we present results of semiempirical molecular orbital calculations employing the PM3 molecular model. The compounds studied are related to substrates of the serine protease dipeptidyl peptidase IV (DP IV). Our goal was the thermodynamic characterization of the DP IV-enzyme-catalyzed reaction pathway. A new mechanism of serine proteases catalysis is presented. We found that a tetrahedral intermediate can be stabilized by the formation of an oxazolidine ring with the nonscissile P2-P1 peptide bond. In this way, the negative charge of the tetrahedral intermediate around the scissile bond is transferred to the carbonyl oxygen atom of the preceding peptide bond. This negative charge can be compensated by a proton transfer from the positively charged N-terminus to this oxygen atom. It is shown that the positively charged N terminus is the driving force in this particular serine protease mechanism of catalysis. The mechanism is supported by observed secondary hydrogen isotope effects on the C alpha proton for an alanine residue in the P2 position. We suggest a trans-cis isomerisation around the P2-P1 peptide bond in the final step of the acylation and cleavage of the substrates. The results obtained by our theoretical calculations are compared with several experimental findings supporting the suggested mechanism. PMID- 8617254 TI - Biochemical characterization of purified zeta-carotene desaturase from Anabaena PCC 7120 after expression in Escherichia coli. AB - A novel enzyme, zeta-carotene desaturase from the cyanobacterium Anabaena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowed the purification of this enzyme and subsequent enzyme kinetic studies. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion-exchange and hydrophobic interaction chromatography. The correct translational start was confirmed by N-terminal protein sequencing. Substrates for zeta-carotene desaturase apart from zeta-carotene are those carotenes which partially resemble the latter, like neurosporene and beta zeacarotene yielding lycopene and gamma-carotene, respectively as reaction products. Also cis isomers like pro-zeta-carotene were converted to the corresponding products. Km values of 10 microM were determined for both substrates zeta-carotene and neurosporene. The enzyme was inhibited to some extent by the experimental herbicides J852 and LS80707 and also by diphenylamine which is a well-known inhibitor of the bacterial-type phytoene desaturase. PMID- 8617255 TI - Kinetic characterization of the hydrolytic activity of the H+-pyrophosphatase of Rhodospirillum rubrum in membrane-bound and isolated states. AB - Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two pathway reaction scheme in which preformed 1:1 and 1:2 . enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis. PMID- 8617256 TI - Alanine metabolism in isolated human kidney tubules--Use of a mathematical model. AB - To gain insight into the fate of alanine nitrogen and carbon taken up by the human kidney under certain conditions, isolated human kidney cortex tubules were incubated in Krebs-Henseleit medium with L-alanine as substrate. The tubules metabolized alanine at high rates and in a dose-dependent manner. Most of the alanine nitrogen removed was recovered as ammonia and to a lesser extent as glutamate. Glucose, lactate and glutamate were also found to be significant products of alanine carbon metabolism. A simple mathematical model allowing one to calculate flux of alanine carbon through the various metabolic steps involved is proposed and applied to data obtained in experiments in which 5 mM [U-14C]-,[1 14C]-, [2-14C]- and [3-14C]alanine were used as substrates in parallel. About 40% of the alanine carbon removed was recovered as CO2 and oxidation of C1 of alanine accounted for most of the CO2 released from alanine. Calculations reveal that the ATP produced exceeded 3.2-fold the ATP consumed in relation to alanine metabolism. It is concluded that, in human kidney, alanine may serve as an energy supplier and as a precursor of glucose and ammonia. PMID- 8617258 TI - Crystallization and crystal packing of recombinant 3 (or 17) beta-hydroxysteroid dehydrogenase from Comamonas testosteroni ATTC 11996. AB - The enzyme 3 (or 17) beta-hydroxysteroid dehydrogenase from Comamonas testosteroni was crystallized. Crystals, of up to 0.6 mm in their longest dimension and suitable for a crystallographic analysis have been obtained by the vapour diffusion method. They belong to the orthorhombic lattice type and diffract to a maximum resolution of 0.23 nm. A final data set obtained by merging data from three crystals resulted in a completeness of 90% with an Rmerge of 6%. A molecular replacement search carried out by using 3 alpha (or 20 beta) hydroxysteroid dehydrogenase from Streptomyces hydrogenans as a search model allowed us to assign I222 as the correct space group and to propose a model for the crystal packing, with one monomer per asymmetric unit. Thus, the whole unit cell contains two tetramers. The R-factor after rigid body refinement is 48.1%. PMID- 8617257 TI - Gene structure of the murine calcium channel beta3 subunit, cDNA and characterization of alternative splicing and transcription products. AB - The beta3 subunit of high-voltage-gated calcium channels is a peripheral membrane protein that copurifies with neural N-type calcium channels. Murine genomic clones containing the full coding sequence of beta3 were isolated and the exons were mapped and sequenced. The murine calcium channel beta3 subunit is encoded by a unique gene composed of 13 translated exons that spread over approximately 8 kb of genomic sequence. Alternatively spliced transcripts of the beta3 gene were identified and characterized. The primary structure of beta3 is highly conserved between the murine, human, rabbit and rat proteins (98% identity). The intron placement within that primary structure correlates with the previously postulated exon positions in transcripts encoding the members of the calcium channel beta subunit family and confirm a close evolutionary relationship of the beta3, beta1, beta2 and beta4 subunit genes. PMID- 8617259 TI - Photoaffinity labeling of elongation factor-2 with 8-azido derivatives of GTP and ATP. AB - Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido [gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8 azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2. PMID- 8617260 TI - Quinaldine 4-oxidase from Arthrobacter sp. Ru61a, a versatile procaryotic molybdenum-containing hydroxylase active towards N-containing heterocyclic compounds and aromatic aldehydes. AB - Quinaldine 4-oxidase from Arthrobacter sp. Ru61a, an inducible molybdenum containing hydroxylase, was purified to homogeneity by an optimized five-step procedure. Molecular oxygen is proposed as physiological electron acceptor. Electrons are also transferred to artificial electron acceptors with E'o > -8 mV. The molybdo-iron/sulfur flavoprotein regiospecifically attacks its N-heterocyclic substrates: isoquinoline and phthalazine are hydroxylated adjacent to the N heteroatom at Cl, whereas quinaldine, quinoline, cinnoline and quinazoline are hydroxylated at C4. Additionally, the aromatic aldehydes benzaldehyde, salicylaldehyde, vanillin and cinnamaldehyde are oxidized to the corresponding carboxylic acids, whereas short-chain aliphatic aldehydes are not. Quinaldine 4 oxidase is compared to the two molybdenum-containing hydroxylases quinoline 2 oxidoreductase from Pseudomonas putida 86 [Tshisuaka, B., Kappl, R., Huttermann, J. & Lingens, F. (1993) Biochemistry 32, 12928-12934] and isoquinoline 1 oxidoreductase from Pseudomonas diminuta 7 [Lehmann, M., Tshisuaka, B., Fetzner, S., Roger, P. & Lingens, F. (1994) J. Biol. Chem. 269, 11254-11260] with respect to the substrates converted and the electron-acceptor specificities. These dehydrogenases hydroxylate their N-heterocyclic substrates exclusively adjacent to the heteroatom. Whereas the aldehydes tested are scarcely oxidized by quinoline 2-oxidoreductase, isoquinoline 1-oxidoreductase catalyzes the oxidation of the aromatic aldehydes, although being progressively inhibited. Neither quinoline 2-oxidoreductase nor isoquinoline 1-oxidoreductase transfer electrons to oxygen. Otherwise, the spectrum of electron acceptors used by quinoline 2 oxidoreductase and quinaldine 4-oxidase is identical. However, isoquinoline 1 oxidoreductase differs in its electron-acceptor specificity. Quinaldine 4-oxidase is unusual in its substrate and electron-acceptor specificity. This enzyme is able to function as oxidase or dehydrogenase, it oxidizes aldehydes, and it catalyzes the nucleophilic attack of N-containing heterocyclic compounds at two varying positions depending on the substrate. PMID- 8617261 TI - Role of cAMP in the regulation of hepatocytic autophagy. AB - To assess the role of cAMP in the regulation of autophagy, we examined the effects of cAMP analogues and cAMP-elevating agents on freshly isolated rat hepatocytes, using electroinjected [3H]raffinose as an autophagy probe. Glucagon was found to stimulate, inhibit or have no effect on autophagy, depending on the inclusion of metabolites like pyruvate (which caused ATP depletion and autophagy suppression) and amino acids (a complete mixture that antagonized pyruvate) in the incubation medium. Inhibition was also observed with theophylline, a cAMP elevating inhibitor of cyclic nucleotide phosphodiesterases, and with the adenylyl cyclase activator deacetylforskolin. At low concentrations of deacetylforskolin, the inhibition could be abolished by amino acids. N6,2'-O Dibutyryladenosine 3',5'-monophosphate (Bt2-cAMP) strongly inhibited both autophagic sequestration of [3H]raffinose and overall autophagic protein degradation; again, amino acids abolished the autophagy-inhibitory effect of low Bt2-cAMP concentrations. Several other cAMP analogues (8-thiomethyl-cAMP, N6 benzoyl-cAMP, (S)-5,6-dichloro-1-D-ribofuranosylbenzimidazole 3',5' [thio]monophosphate, (S)-8-bromoadenosine 3',5'-[thio]monophosphate) inhibited autophagy as well. The effect of Bt2-cAMP was rapid, dose-dependent, reversible and did not require concomitant protein synthesis. Neither Bt2-cAMP nor deacetylforskolin reduced intracellular ATP levels or cell viability, ruling out inhibition of autophagy by non-specific cytotoxicity. The autophagy-inhibitory effect of Bt2-cAMP could be substantially antagonized (40-50%) by KT-5720, a specific inhibitor of the cAMP-dependent protein kinase A, and by the nonspecific protein kinase inhibitor K-252a. Somewhat surprisingly, KN-62 and KT-5926, allegedly specific inhibitors of Ca2+/calmodulin-dependent protein kinase II and myosin light chain kinase, respectively, were also Bt2-cAMP-antagonistic. These results suggest that cAMP regulates the early, sequestrational step of hepatocytic autophagy by a highly conditional, dual mechanism, inhibition being predominant under most conditions in freshly isolated hepatocytes, whereas stimulation reportedly predominates in vivo. The effect of cAMP is probably mediated by protein kinase A, but other protein kinases would appear to participate in the regulation of autophagic sequestration as well. PMID- 8617262 TI - Identification of a functional cAMP response element in the secretogranin II gene. AB - Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type. PMID- 8617264 TI - Purification and characterization of carbamoyl-phosphate synthetase from the deep sea hyperthermophilic archaebacterium Pyrococcus abyssi. AB - Carbamoyl-phosphate synthetase was purified from the deep-sea hyperthermophilic archaebacterium Pyrococcus abyssi. This enzyme appears to be monomeric and uses ammonium salts as nitrogen donor. Its activity is inhibited by some nucleotides that compete with ATP. In contrast with the carbamoyl-phosphate synthetases investigated so far, this enzyme is very resistant to high temperature. Its low molecular mass (46.6 kDa) and its catalytic properties suggest that the gene coding for this enzyme is a previously postulated ancestor, whose duplication gave the genes coding for carbamoyl-phosphate synthetases and carbamate kinases. PMID- 8617263 TI - Polymerization of 5,6-dihydroxyindole-2-carboxylic acid to melanin by the pmel 17/silver locus protein. AB - Recent advances in melanogenesis have focused on the role of dihydroxyindole-2 carboxylic acid[(HO)2IndCOOH]. For example, it has been shown that formation of (HO)2IndCOOH from dopachrome is catalyzed by dopachrome tautomerase, that the melanogenic protein tyrosinase-related protein (TRP)-1 can oxidize (HO)2IndCOOH to its indole quinone, that (HO)2IndCOOH-melanins can be synthesized chemically, that mammalian melanins are naturally rich in (HO)2IndCOOH subunits, and that (HO)2IndCOOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)2IndCOOH and other precursors become incorporated into melanins in vivo. Accordingly, an activity was partially purified that catalyzed melanin formation with (HO)2IndCOOH as a substrate. Analyses of the (HO)2IndCOOH polymerization factor from Cloudman melanoma cells revealed the following: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N-acetyl glucosamine; it was located predominantly in the melanosomal fraction of cell homogenates; the activity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)2IndCOOH polymerization reaction was inhibited by superoxide dismutase. In addition, the activity was found with the mouse pmel 17/silver locus protein immunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice compared to extracts from Si/Si melanocytes. In summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide-dependent polymerization of (HO)2IndCOOH to melanin in vitro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus. PMID- 8617265 TI - Covalent association of lipopolysaccharide at the hemocyte surface of insects is an initial step for its internalization--Protein-tyrosine phosphorylation requirement. AB - It is well known that lipopolysaccharide (LPS) of Gram-negative bacteria triggers antibacterial responses to mammalian macrophages [Weinstein, S., Gold, M. R. & DeFranco, A. (1991) Proc. Natl Acad. Sci. USA 88, 4148-4152] and insect hemocytes [Charalambidis, N.D., Zervas, C.G., Lambropoulou, M., Katsoris, P.G. & Marmaras, V.J. (1995) Eur J. Cell Biol. 67, 32-41], via protein-tyrosine phosphorylation. In this study we show that insect hemocytes in response to LPS facilitate internalization of LPS (either cell-associated or cell-free). According to our data, the recognition and covalent association of LPS (either cell-associated or cell-free) to the hemocyte surface are essential initial steps for LPS internalization. LPS (Escherichia coli) recognizes membrane effector 47-kDa protein (p47) and then crosslinks to membrane-associated p47 (mp47) via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase, as is the case for cuticular protein-chitin crosslinks during sclerotization [Shaefer, J., Kramer, K.J., Garbow, J.R., Jacob, G.S., Stejskal, E.O., Hopkins, T.L. & Speirs, R.D. (1987) Science 235, 1200-1204]. The covalent association of LPS to the hemocyte surface appears to be a prerequisite for LPS internalization as judged by the resistance of LPS binding to dissociation by proteinase K. In addition, our results show that the effector molecules participating in LPS covalent association at the cell surface and LPS internalization are not involved in LPS-induced activation of hemocytes. However, the fact that genistein, as well as the inhibitors of LPS-dependent secretion, block LPS covalent association at the cell surface and LPS internalization provides a preliminary characterization of an LPS signal-transduction-dependent process which is apparently involved. PMID- 8617266 TI - Identification of a cysteine protease closely related to interleukin-1 beta converting enzyme. AB - The present study describes the identification and molecular cloning of a new member of the interleukin-1 beta-converting enzyme (ICE) family denoted transcript Y (TY). TY is very closely related to both ICE (51% amino acid identity) and a protein named transcript X (TX) (75% amino acid identity) that we recently identified [Faucheu, C., Diu, A., Chan, A.W.E., Blanchet, A.-M., Miossec, C., Herve, F.,Collard-Dutilleul, V., Gu, Y., Aldape, R., Lippke, J., Rocher, C., Su, M.S.-S., Livingston, D.J., Hercend, T. & Lalanne, J.-L. (1995) EMBO J. 14, 1914-1922]. The amino acids that are implicated in both the ICE catalytic site and in the PI aspartate-binding pocket are conserved in TY. Within the ICE gene family, TY belongs to a subfamily of proteins closely related to the prototype ICE protein. Using transfection experiments into mammalian cells, we demonstrate that TY has protease activity on its own precursor and that this activity is dependent on the presence of a cysteine residue at position 245. However, despite the close similarity between TY and ICE active sites, TY fails to process the interleukin-1 beta precursor. In addition, as already observed for ICE and TX, TY is able to induce apoptosis when overexpressed in COS cells. TY therefore represents a new member of the growing family of apoptosis-inducing ICE related cysteine proteases. PMID- 8617267 TI - Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element. AB - Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene. PMID- 8617268 TI - Elongation factor Ts from Thermus thermophilus-- overproduction in Escherichia coli, quaternary structure and interaction with elongation factor Tu. AB - The gene encoding the elongation factor Ts from Thermus thermophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from T. thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins. Purified T. thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys19O by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF-TS, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-TU . EF Ts)2 complex, a ternary EF-TU . EF-Ts2 complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers. PMID- 8617269 TI - Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei. AB - The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle. PMID- 8617270 TI - Use of T7 RNA polymerase in an optimized Escherichia coli coupled in vitro transcription-translation system. Application in regulatory studies and expression of long transcription units. AB - An Escherichia coli coupled in vitro transcription-translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21(DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio between mRNA and ribosomes has to be regulated in the coupled in vitro system. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The coupled in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the coupled in vitro system. PMID- 8617272 TI - Amino acid preferences in the octapeptide subunit of the major histocompatibility complex class I heterotrimer H-2Ld. AB - Major histocompatibility complex class I (MHC-I) molecules are heterotrimers composed of polymorphic alpha-chains, monomorphic beta-chains, and peptides of eight or nine amino acids. The peptides are derived from various intracellularly occurring proteins and are very heterogeneous. They are essential for a stable conformation of the MHC-I protein at physiological temperature. This study presents results from stabilization experiments that were designed to determine the impact of the amino acids in every sequence position of octapeptides on the thermal stability of the mouse MHC-I molecule H2-Ld. OX7 octapeptide libraries with one defined and seven randomized positions were employed as they allow the effects of individual amino acids to be determined. The results confirm the importance of the motif amino acids proline and leucine for positions 2 and 8, respectively, of octapeptides. They are among the most efficient amino acids for these positions. However, with a few exceptions, all amino acids are permitted in all eight sequence positions. Hydrophobic amino acids are generally favored. Charged amino acids, especially aspartic acid and glutamic acid, are disfavored. Stabilization indices were defined as measures for the MHC stabilization power of the amino acids. These indices can serve to predict the efficiency of peptide binding to H-2Ld and can guide the design of T-cell epitopes. PMID- 8617271 TI - Cloning, sequence analysis and overexpression of the rhodanese gene of Azotobacter vinelandii. AB - A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelandii on a 2.3 kb SphI fragment. This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N-terminal sequence of rhodanese purified from A. vinelandii. The sequence of a 1.2-kb region revealed an 813-bp open reading frame that encoded a polypeptide of 271 amino acids, the N-terminal sequence of which was identical to that of A. vinelandii rhodanese. In a search of database entries, eukaryotic rhodaneses and rhodanese-like proteins from bacteria gave the highest scores of identity (27-30%) with the predicted product of the 813-bp open reading frame. A. vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese-like proteins which did not show typical rhodanese activity. Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A. vinelandii rhodanese. The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A. vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication. When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosulfate:cyanide sulfurtransferase activity increased >600 fold in cell-free extracts. A. vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild-type strain with respect to growth on various sulfur sources or nitrogenase activity. Mutants retained 20% of wild type rhodanese thiosulfate:cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A. vinelandii. PMID- 8617274 TI - Formation of pores in Escherichia coli cell membranes by a cecropin isolated from hemolymph of Heliothis virescens larvae. AB - The insect humoral defense system produces antibacterial peptides called cecropins. Cecropins were initially isolated from Hyalophora cecropia pupae and have since been isolated and identified in various insects. In this study, we have isolated and identified a cecropin from Heliothis virescens larvae. Rabbit IgG were raised against synthetic cecropin B. Affinity chromatography with the rabbit anti-(cecropin B) IgG was used to isolate a cecropin from hemolymph of H. virescens larvae. Acid gel electrophoresis followed by a bacterial-overlay analysis showed that Heliothis cecropin is a basic peptide of low molecular mass with bactericidal activity against Escherichia coli K12 D31. Heliothis cecropin is therefore analogous to synthetic cecropin B. One unresolved issue concerning cecropins and other antibiotic peptides is the mode of action by which they kill bacteria. By means of electron microscopy and immunocytochemistry with gold labeled rabbit anti-cecropin IgG, binding of purified and synthetic cecropin to the cell membranes of E. coli K12 D31 cells was observed. Small lesions in the cell membrane were seen that had a diameter of 9.6 nm and internal pore of 4.2 nm. The Heliothis cecropin was found to be a pore-forming molecule that causes lesions in the cell membrane of E. coli K12 D31. The lesions lead to leakage of cytoplasmic contents and death of bacteria. PMID- 8617273 TI - Pressure-tuning the conformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy. AB - A hydrostatic pressure of 1.5 GPa induces changes in the secondary structure of bovine pancreatic trypsin inhibitor (BPTI) as revealed by the analysis of the amide I' band with Fourier-transform infrared (FTIR) spectroscopy in the diamond anvil cell. The features of the secondary structure remain distinct at high pressure suggesting that the protein does not unfold. The fitted percentages of the secondary structure elements during compression and decompression strongly suggest that the pressure-induced changes are reversible. The pressure-induced changes in the tyrosine side chain band are also reversible. The results demonstrate that the infrared technique explores different aspects of the behaviour of proteins in comparison with two published molecular dynamics studies performed up to 1 GPa [Kitchen, D.B., Reed, L.H. & Levy, R.M.(1992) Biochemistry 31, 10083-10093] and 500 MPa [Brunne, R.M. & van Gunsteren, W.F.(1993) FEBS Lett. 323, 215-217]. A possible explanation for the difference is the time scale of the experiments. PMID- 8617275 TI - Expression, lipoylation and structure determination of recombinant pea H-protein in Escherichia coli. AB - A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sativum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form. Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-(1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The three-dimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme. PMID- 8617276 TI - Spinach chloroplast 0-acetylserine (thiol)-lyase exhibits two catalytically non equivalent pyridoxal-5'-phosphate-containing active sites. AB - A synthetic gene encoding the mature spinach- chloroplast O-acetylserine (thiol) lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoacrylate and the pyridoxal 5' phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with the acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the enzyme exhibits two non-equivalent pyridoxal-5'-phosphate containing active sites. PMID- 8617277 TI - Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity. AB - Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity. PMID- 8617278 TI - Primary structure of cyclohydrolase (Mch) from Methanobacterium thermoautotrophicum (strain Marburg) and functional expression of the mch gene in Escherichia coli. AB - The gene mch encoding N5,N10-methenyltetrahydromethanopterin cyclohydrolase (Mch) in Methano-bacterium thermoautotrophicum (strain Marburg) was cloned and sequenced. The gene, 963 bp, was found to be located at the 3' end of a 3.5-kbp BamHI fragment. Upstream of the mch gene two open reading frames were recognized, one encoding for a 25-kDa protein with sequence similarity to deoxyuridylate hydroxymethylase and the other encoding for a 34.6-kDa protein with sequence similarity to cobalamin-independent methionine synthase (MetE). The N-terminal amino acid sequence deduced for the deoxyuridylate hydroxymethylase was identical to that previously published for thymidylate synthase (TysY) from M. thermoautotrophicum. The 3' end of the tysY gene overlapped by 8 bp with the 5' end of the mch gene. Despite this fact, the mch gene appeared to be transcribed monocistronically as evidenced by Northern blot analysis and primer-extension experiments. The mch gene was overexpressed in Escherichia coli yielding an active enzyme of 37 kDa with a specific activity of 30 U/mg cell extract protein. PMID- 8617279 TI - Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5' phosphate-dependent enzyme involved in biotin biosynthesis. AB - The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7-oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over-expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8-amino-7-oxopelargonate formed using either L-[2 2H]alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino-7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L-alanine with the solvent protons. Similarly, 8-amino-7 oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7 oxopelargonate. In addition to these exchange reactions, 8-amino-7-oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino-7-oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch. = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans. = 0.11 h-1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0). The magnitudes of the KIE are consistent with a partially rate limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step. Taken together, all these results show that 8-amino-7 oxopelargonate synthase utilizes mechanism (a). 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of alpha-oxoamines. Based on the fact that all these alpha-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism. PMID- 8617280 TI - A polyferredoxin with eight [4Fe-4S] clusters as a subunit of molybdenum formylmethanofuran dehydrogenase from Methanosarcina barkeri. AB - Formylmethanofuran dehydrogenase (Fmd) from Methanosarcina barkeri is a molybdenum iron-sulfur protein involved in methanogenesis. The enzyme contains approximately 30 mol non-heme iron/mol and 30 mol acid-labile sulfur/mol. We report here the cloning and sequencing of the encoding genes, and that these genes form a transcription unit fmdEFACDB. Evidence is provided that the subunit FmdB harbours the molybdenum-containing active site and may bind one [4Fe-4S] cluster. fmdF encodes a protein with four tandemly repeated bacterial-ferredoxin like domains and is predicted to be a polyferredoxin that could contain as many as 32 iron atoms in eight [4Fe-4S] clusters. The other genes code for proteins without sequence motifs characteristic for iron-sulfur proteins. These findings suggest that most of the iron-sulfur clusters present in the purified formylmethanofuran dehydrogenase are associated with the subunit FmdF. The finding that FmdF forms a tight complex with the other subunits of formylmethanofuran dehydrogenase indicates a function of the polyferredoxin in the reaction catalyzed by the enzyme. fmdE encodes a protein not present in the purified enzyme. All six genes of the fmd operon were expressed in Escherichia coli and yielded proteins of expected molecular masses. A malE-fmdF gene fusion was constructed and expressed in E. coli, making the apoprotein of the polyferredoxin available in preparative amounts. PMID- 8617281 TI - MUC1 glycoforms in breast cancer--cell line T47D as a model for carcinoma associated alterations of 0-glycosylation. AB - A highly immunogenic peptide motif within the tandem repeat domain of MUC1 mucin is assumed to be exposed during development of breast cancer due to altered O glycosylation. To elucidate the structural aspects of these changes, we have isolated and analysed the integrated or secretory MUC1 glycoforms from carcinoma cell lines or solid tumors and from human milk. The buoyant densities measured in CsCl gradients for MUC1 glycoforms from cancer cells revealed heterogeneity of the physicochemical species and a significant reduction of their carbohydrate contents compared to MUC1 from skim milk. Immunoreactivity patterns of MUC1 glycoforms from tumor or T47D cells exhibited a lack of fucosylated Lewis blood group-related antigens and the appearance of core-type antigen sialyl(NeuGl)-TF, Gal beta 1-3(NeuGl alpha 2-6)GalNAc. Structural chemistry of MUC1 oligosaccharides demonstrated that the cancer-associated glycoforms carry mainly sialylated trisaccharides NeuAc alpha 2-3Gal Beta 1-3GalNAc or NeuAc alpha 2 6(Gal beta l-3)GalNAc, exhibit a concomitant decrease in the ratio of GlcNAc/GalNAc, a reduction or disappearance of L-fucose, and a partial substitution of N-acetylneuraminic acid by the N-glycolylated variant. On comparison to the secretory MUC1 in human milk, the glycoforms on human milk fat globule membranes showed apparently identical patterns of O-linked oligosaccharides with a preponderance of neutral polylactosamino-glycans. During serum-free cultivation of T47D cells over 4 weeks, the expression of secretory MUC1 glycoforms was inconsistent based on the decreasing contents of sialic acid and on the concomitant increase of immunodetectable TF antigen. PMID- 8617282 TI - Phytanic acid is a retinoid X receptor ligand. AB - Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9-cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXR alpha, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXR alpha, promotes formation of an RXR alpha/RXR response element complex (as detected by gel retardation), and induces a RXR alpha conformational change similar to that induced by 9-cis retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXR alpha in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism. PMID- 8617283 TI - Expression of active streptolysin O in Escherichia coli as a maltose-binding protein--streptolysin-O fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity. AB - Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N-terminus was separated from the MBP carrier by cleavage with factor Xa. Cleavage with plasmin or trypsin also yielded active, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP-SLO was also hemolytic and cytotoxic to human fibroblasts and keratinocytes. The MBP-SLO fusion protein displayed equal activities to SLO. Sucrose density gradient analyses showed that the fusion protein assembled into polymers, and no difference in structure was discerned compared with polymers formed by native SLO. These studies show that the N-terminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N-terminus of the molecule is probably not inserted into the bilayer. In addition, they provide a simple means for producing mutants for structure/function studies and highly purified SLO for use as a permeabilising reagent in cell biology research. PMID- 8617284 TI - Insulin and phorbol ester stimulate initiation factor eIF-4E phosphorylation by distinct pathways in Chinese hamster ovary cells overexpressing the insulin receptor. AB - We have developed a one-dimensional isoelectric focusing technique to measure changes in the steady-state phosphorylation of the cap-binding initiation factor, eIF-4E. We have used a Chinese hamster ovary cell line transfected with the human insulin receptor (CHO.T cells) to study the regulation of eIF-4E phosphorylation by insulin and other stimuli. Exposure of CHO.T cells to insulin, phorbol ester or serum resulted in a rapid increase (up to twofold) in eIF-4E phosphorylation. As a control, we have also performed experiments with the parental cell line, CHO.K1 cells, in which both serum and phorbol ester, but not nanomolar concentrations of insulin, produce similar changes in eIF-4E phosphorylation. We have used two complementary approaches to study the role of protein kinase C (PKC) in these responses: a highly specific inhibitor of PKC and down-regulation of PKC by prior treatment of the cells with phorbol ester. In CHO.T cells, both approaches indicate that PKC is required for the response to phorbol ester but that insulin and serum each increase eIF-4E phosphorylation by a mechanism(s) independent of this protein kinase. Similarly, PKC is necessary for the effects of phorbol ester, but not of serum, on eIF-4E phosphorylation in CHO.K1 cells. These data indicate that multiple signal transduction mechanisms are involved in the modulation of eIF-4E phosphorylation and the implications of these findings are discussed. PMID- 8617285 TI - Phagocytic activation induces formation of platelet-activating factor in human monocyte-derived macrophages and in macrophage-derived foam cells. Relevance to the inflammatory reaction in atherogenesis. AB - Monocyte-derived macrophages and macrophage-derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3 phosphocholine, PAF-acether, PAF), a proinflammatory phospholipid, in human monocyte-derived macrophages (macrophages) and in cholesterol-loaded macrophage foam cells (foam cells). Adherent human monocyte-derived macrophages were transformed into foam cells upon incubation with acetylated low-density lipoproteins (Ac-LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3 +/- 1.3 microgram/microgram DNA and 0.2 +/- 0.3 microgram/microgram DNA, n = 5, respectively). After phagocytic stimulation with serum-opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5-1.1 pmol PAF/microgram DNA, n = 5, corresponding to 4.0-8.8 pmol PAF/10(6) cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell-associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, approximately 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse-phase liquid chromatography. Addition of exogenous 16:0 lyso-PAF alone triggered PAF formation (0.9-1.7 pmol PAF/microgram DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso-PAF increased PAF formation in an additive manner. Acetyltransferase, the enzyme which acetylates the precursor lyso-PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83 240 pmol PAF formed per min per mg DNA (n = 4); such elevated activity was not increased by OPZ-stimulation. The activity of acetylhydrolase, the PAF-degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded per min per mg DNA (n = 5). Cell-associated acetylhydrolase activity was increased significantly by 40+/-15 % (P < 0.003, n = 5) after 15 - 30 min of activation with OPZ compared with non-stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage derived foam cells upon phagocytic-activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects. PMID- 8617286 TI - Glutamine increases argininosuccinate synthetase mRNA levels in rat hepatocytes. The involvement of cell swelling. AB - Glutamine, one of the most efficient substrates in the urea cycle, was found to induce the accumulation of argininosuccinate synthetase (ASS) mRNA in primary cultured rat hepatocytes. The inducing action of glutamine was obtained at various stages of development and a half-maximal effect was observed at about 3 mM glutamine. This effect was apparent from 6 h and persisted for at least 48 h. NH4Cl addition up to 5 mM did not significantly change the ASS mRNA level. Like glutamine, hypoosmotic medium and aminoisobutyric acid (conditions known to increase cell volume) also increased the ASS mRNA level, which was, in contrast, decreased by hyperosmotic medium. These results demonstrate that the glutamine induced swelling may participate in the observed increase of the ASS mRNA level. This increase in the ASS mRNA level was associated with an increase in ASS activity. PMID- 8617287 TI - Structural organization of the gene encoding the neuroendocrine chaperone 7B2. AB - The neuroendocrine-specific polypeptide 7B2 is a constituent of the regulated secretary pathway. Recently, 7B2 was found to function as a molecular chaperone for prohormone convertase PC2. This report describes the genomic organization of the 7B2 gene which consists of six exons. Exon I corresponds to the 5' untranslated mRNA region, while exons 2 and 3 encode the signal peptide and the amino-terminal half of the 7B2 protein that is distantly related to a subclass of molecular chaperones. The carboxy-terminal half of 7B2, responsible for its inhibitory action on PC2, is encoded by exons 4-6. Primer-extension analysis showed that the human 7B2 gene is transcribed from multiple transcription initiation sites. The human 7B2 gene promoter contains a cAMP-responsive element, an AP-1 site, and several Pit-1/GHF-1-binding domains and heat-shock-element-like sequences but no obvious TATA or CAAT boxes. Of further interest is the finding of two DNA elements which are common to the promoter regions of the 7B2 gene and other genes selectively expressed in neuroendocrine tissues. PMID- 8617288 TI - Stoichiometry, organisation and catalytic function of protein X of the pyruvate dehydrogenase complex from bovine heart. AB - Mammalian pyruvate dehydrogenase complex (PDC) contains a subunit, protein X, which mediates high-affinity binding of dihydrolipoamide dehydrogenase (E3)to the dihydrolipoamide acetyltransferase (E2) core. Precise stoichiometric determinations on bovine heart PDC, by means of two approaches, indicate the presence of 12 mol protein X/mol PDC and 60 mol E2/mol PDC. Studies of the organisation of collagenase-modified PDC by means of covalent cross-linking of N,N'-1,2-phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross-linked products have Mr values corresponding to homodimers of protein X. However, significant formation of higher-Mr aggregates indicates that lipoyl domains of protein X can form an interacting network independent of E2 lipoyl domains. These data suggest that either 12 interacting X monomers or 6 interacting X dimers are involved in the binding of six E3 homodimers to the E2/X core. The presence of 60 E2 subunits/complex also supports proposals for a non integrated external position of protein X. Collagenase-treated PDC possesses residual activity (15 %), indicating that protein-X-linked lipoamide groups can substitute for the lipoyl domains of E2 in overall complex catalysis. Protein-X mediated diacetylation of dihydrolipoamide moieties is also performed by the modified complex which raises the possibility of a unique catalytic function for protein X. PMID- 8617289 TI - The effects of sodium perchlorate on rabbit muscle enolase--Spectral characterization of the monomer. AB - Incubation of rabbit beta beta enolase in NaClO4 (< or = O.3 M) results in a loss of enzymatic activity and striking changes in the second-derivative ultraviolet spectrum of enolase. HPLC gel filtration shows that dissociation of the dimeric enzyme is occurring. We have used molecular modelling, fluorescence and circular dichroic spectroscopy to examine the structural differences between the monomeric and dimeric forms of this protein. In the dimer, the tyrosine residues are in a non-polar environment; upon dissociation, two of them that were at the dimer interface become exposed. This results in large changes in the second-derivative spectrum. Both the tryptophan fluorescence emission spectrum and the aromatic region of the CD spectrum indicate that there are also changes in the environment of other aromatic residues. No perturbations in the peptide bond region of the CD spectrum are observed. We propose that the major structural effect of NaClO4 is to increase the flexibility of the loops connecting the helices and strands of the alpha/beta barrel of enolase. These loops, which contain about half of the aromatic residues, contain some of the residues of the active site and other residues involved in subunit contacts. Increased flexibility of the loops could disrupt both subunit interactions and the structure of the active site. PMID- 8617290 TI - Conformational effects of serine phosphorylation in phospholamban peptides. AB - We have employed one- and two-dimensional 1H-NMR spectroscopy to study the effects of serine phosphorylation on peptide conformations, using cardiac phospholamban as a model system. The non-phosphorylated phospholamban 1-20 peptide has few restraints on the conformations available to it in aqueous solution. Phosphorylation at Ser16 results in greater constraints being placed on the region encompassing Arg14-Thr17, particularly at neutral pH when the phosphate group is in the di-anionic form. These conformational restrictions arise from specific interactions between the side-chain of Arg14 and the phosphate group. While substitution of phosphothreonine at position 16 causes generally similar effects to phosphoserine, aspartic acid has little effect. The results are compared with phosphorylation effects in other systems, including cardiac troponin I. PMID- 8617291 TI - Paramagnetic NMR analysis of the seven-iron ferredoxin from the hyperthermoacidophilic archaeon Desulfurolobus ambivalens reveals structural similarity to other dicluster ferredoxins. AB - The seven-iron ferredoxin from the hyperthermophilic archaeon Desulfurolobus ambivalens has been investigated by one-dimensional and two-dimensional 1H-NMR in its oxidized and dithionite-reduced states. All iron atoms of both the three-iron and the four-iron cluster are bound to cysteine residues whose hyperfine-shifted resonances were characterized. The pattern of these resonances is similar to those from three-iron, four-iron and eight-iron ferredoxins previously described in the literature, but the four-iron cluster has a shift pattern different from that in other seven-iron proteins. A second set of hyperfine-shifted resonances clearly indicates sample heterogeneity, which possibly involves the four-iron cluster. The observation of interresidue NOEs between two different cysteine residues proves the existence of close spatial proximity of the two clusters in D. ambivalens ferredoxin and therefore indicates structural homology to other dicluster ferredoxins. Moreover, this feature is crucial for the sequence specific assignment of the hyperfine-shifted resonances. The C alpha-C beta-S-Fe dihedral angles of the cysteine residues coordinating the four-iron cluster could be estimated, and the electronic structure of the three-iron cluster is discussed. PMID- 8617292 TI - Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides. AB - Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III) specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers. PMID- 8617293 TI - TAP1-deficient mice select a CD8+ T cell repertoire that displays both diversity and peptide specificity. AB - Mice deficient in the gene encoding the transporter associated with antigen processing 1 (TAP1) are defective in providing major histocompatibility complex (MHC) class I molecules with cytosolic peptides. Consequently, these mice express reduced levels of MHC class I glycoproteins on the cell surface, and have reduced numbers of CD8+ T cells in the periphery. In the present study, we have addressed the diversity and specificity of the peripheral CD8+ T cell population in TAP1 -/ mice. CD8+ T cells were polyclonal with regard to T cell receptor (TCR) V beta expression. Overall, V beta usage in TAP1 -/- mice appear to be very similar to that in wild-type mice, with significantly reduced levels of V beta 5.1/5.2 expressing CD8+ T cells as the only clear exception. This polyclonal population of CD8+ T cells readily mounted epitope-specific CTL responses against four out of five well-defined MHC class I-restricted peptides. In contrast to allospecific CTL, peptide-specific CTL from TAP1 -/- mice did not cross-react on cells expressing normal levels of H-2b class I. The present results demonstrate that a polyclonal CD8+ T cell repertoire, displaying both diversity and peptide specificity, is positively selected in mice devoid of a functional peptide transporter. These observations imply that TAP-dependent peptides are not absolutely required for positive selection of a functionally diverse repertoire of CD8+ T cells. PMID- 8617294 TI - Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. AB - Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID). PMID- 8617295 TI - B7-1 synergizes with interleukin-12 in interleukin-2 receptor alpha expression by mouse T helper 1 clones. AB - Expression of interleukin-2 receptor alpha (IL-2R alpha) is critical to induce interleukin (IL)-2-dependent proliferation of T helper (Th)1 clones. The IL-2R alpha expression of Th1 clones is known to be up-regulated by IL-12. Co stimulation via CD28/CTLA-4 is also known to be important for efficient activation of CD4+ T cells. In the present experiments, IL-12-induced enhancement of IL-2R alpha expression of Th1 clones stimulated with B cells as antigen presenting cells (APC) is suppressed by the addition of anti-B7-1. To analyze the mechanism, Th1 clones were stimulated with immobilized anti-CD3 plus IL-12 in the presence or absence of Chinese hamster ovary cells that express mouse B7-1 (B7 1CHO) and the enhancement of IL-2R alpha expression induced by the co-stimulation was analyzed. The results of these experiments indicate that B7-1 synergizes with IL-12 in IL-2R alpha expression of the Th1 clone stimulated with anti-CD3, although B7-1CHO alone did not enhance IL-2R alpha expression of the clones. B7-1 stimulation is not mediated by the enhancement of IL-2 production: B7-1 enhancement of IL-2R alpha expression was FK506 resistant, while the inclusion of FK506 abrogated IL-2 production of the Th1 cells. B7-1 co-stimulation did not stabilize IL-2E alpha mRNA, but did synergize with IL-12 to enhance IL-2R alpha mRNA transcription. PMID- 8617296 TI - Prevention of murine lupus by an I-E alpha chain transgene: protective role of I E alpha chain-derived peptides with a high affinity to I-Ab molecules. AB - The expression of a transgene encoding the I-E alpha chain prevents a lupus-like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the E alpha d transgene-mediated protective effect results from I-E expression or from the generation of I-E alpha chain-derived peptides (E alpha peptide) displaying high affinity for the I-Ab molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus-prone MRL mice; this resulted in three types of (MRL x BXSB)F1 mice, differing in the expression levels of I-E molecules and of E alpha peptides presented by I-Ab molecules. Comparative analysis of these three (MRL x BXSB)F1 mice as well as several BXSB transgenic lines showed that the E alpha d transgene mediated protection paralleled the expression levels of E alpha peptide presented by I-Ab molecules, but not of I-E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I-Ab-restricted T cell-dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell-independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of E alpha peptides prevents, because of their high affinity to the I-A molecules, activation of potential autoreactive T and B cells. PMID- 8617297 TI - CC chemokines induce the generation of killer cells from CD56+ cells. AB - We describe here that members of the CC chemokines exhibit biological activities other than chemotaxis. Macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, monocyte chemoattractant protein-1 and RANTES, but not interleukin (IL)-8, induce the generation of cytolytic cells, designated here as CHAK (CC chemokine activated killer) cells to distinguish them from IL-2-activated (LAK) cells. Like IL-2, CC chemokines can induce the proliferation and activation of killer cells. While incubating CC chemokines with CD4+ or CD8+ cells did not generate CHAK activity, all CC chemokines were capable of inducing CHAK activity upon incubating with CD56+ cells, suggesting that the primary effectors are NK cells. However, the presence of other cell types, such as CD4+ or CD8+, are necessary to induce the proliferation of CD56+ cells. Confirming the involvement of T cell derived factors in inducing the proliferation of these cells, anti-IL-2 and anti interferon-gamma, but not anti-IL-1 beta, anti-tumor necrosis factor-alpha, anti IL-8, or anti-granulocyte/monocyte-colony-stimulating factor inhibited RANTES induced proliferation of nylon wool column-nonadherent cells. Our results may have important clinical applications for the utilization of CHAK cells in the treatment of cancer and immunodeficient patients. PMID- 8617298 TI - Neonatally tolerant rats actively eliminate donor-specific lymphocytes despite persistent chimerism. AB - Rats from the allotype-marked PVG-RT7b and PVG-RT1u-RT7b strains were injected at birth with semi-allogenic F1 bone marrow (BM) cells from athymic nude rats (PVG rnu/rnu x PVG-RT1u-rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor-specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG-RT1u RT7b rats were injected at birth with fully allogeneic PVG-rnu/rnu nude BM cells: as adults, 94% accepted donor-specific skin allografts and 76% of recipients were chimeric. Donor derived CD4 T cells, CD8 T cells and B cells were found in low numbers (less than 2%) in peripheral blood of rats made tolerant by F1 BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor specific haplotype; donor-specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4+CD8- thymocytes or peripheral CD4 T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG-RT1u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi-allogeneic lymphocytes--a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor-derived lymphocytes and putting at risk the very survival of the allogenic BM cells. This has interesting implications for clinical transplantation. PMID- 8617299 TI - CD30 cell expression and abnormal soluble CD30 serum accumulation in Omenn's syndrome: evidence for a T helper 2-mediated condition. AB - Omenn's syndrome (OS) is a severe immunodeficiency, characterized by clinical and laboratory features reminiscent of a T helper type-2 (Th2) response. CD30, a member of the tumor necrosis factor receptor superfamily, has been found to be preferentially expressed by human T cell clones exhibiting a Th2-line profile and function. We investigated whether there are derangement in CD30 expression in tissues, and/or abnormalities in soluble CD30 (sCD30) levels in the serum, or both, of three children with OS and one child with maternal engraftment and Omenn's-like syndrome (OLS). Large proportions of tissue-infiltrating T lymphocytes from all four patients expressed CD30, whereas in control tissues, including peripheral blood, CD30+ T lymphocytes were extremely few or absent. In addition, levels of sCD30 were abnormally increased in all patients' sera. T cell clones were generated from sorted CD30+ and CD30-peripheral blood T cells of the patient with OLS who showed unusually high numbers of circulating CD30+ T lymphocytes. Most CD4+ T cell clones derived from CD30+ cells showed a Th2-like cytokine profile, whereas the majority of clones generated from CD30-T cells were Th1. These findings support the hypothesis that Th2 cells are involved in the pathogenesis of OS. Moreover, they provide evidence that detection of CD30+ T cells in tissues, increased levels of sCD30 in biological fluids, or both, reflect the presence of immune responses characterized by prevalent activation of T cells producing Th2 cytokines. PMID- 8617300 TI - An HLA-B35-restricted epitope modified at an anchor residue results in an antagonist peptide. AB - Peptides associated with HLA-B35 commonly have a proline or occasionally a serine residue in the P2 anchor position of the peptide, with a tyrosine at the C terminus. Based on this motif, we identified an octamer epitope from influenza A matrix protein which is presented by HLA-B35. The requirements for MHC binding and T cell receptor contact have been analyzed using analogs of this peptide with substitutions at positions 1, 2, 4, 7 and 8. The natural epitope contains a serine residue at P2 of the peptide. Substitution of this residue with proline (the favored amino acid in this position in B35-associated peptides) considerably enhances binding to HLA-B35 in the T2-B35 cell line, but the peptide is not recognized by the majority of CTL clones and can antagonize recognition of the index peptide. This suggests that a conservative substitution at the P2 anchor position results in a conformational change in the peptide-MHC surface exposed to the T cell receptor. PMID- 8617301 TI - IgD-receptor up-regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination. AB - Aged individuals (more than 65 years) were classified as antibody (Ab) responders on the basis that they showed increases to more than or = 1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre-immunization samples of blood taken from seven Ab-responders and seven Ab nonresponders were examined for their ability to exhibit up-regulation of IgD receptor (IgD-R) after exposure for 2 h to immobilized cross-linked IgD, as shown by rosetting with IgD-coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD-R up-regulation was greater than 5% in all Ab-responders and less than 4% in all the Ab-nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD-R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894. Injection of influenza vaccine itself also induced IgD-R on PBMC in vivo. The percentage of IgD-R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre-injection levels by day 28 in young subjects and aged Ab responders, but not in Ab-nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab-responders exhibited IgD-R upon immunization in vivo. These findings suggest that Ag injection causes rapid up-regulation of IgD-R by cross-linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD-R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab-responders than in Ab-nonresponders. PMID- 8617302 TI - Biological function and distribution of human interleukin-12 receptor beta chain. AB - We previously described the cloning of a cDNA encoding an interleukin-12 receptor (IL-12R) subunit, designated beta, that bound IL-12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL-12R beta chain, when used in combination, strongly inhibited IL-12-induced proliferation of activated T cells, IL-12-induced secretion of interferon-gamma by resting peripheral blood mononuclear cells (PBMC), and IL-12-mediated lymphokine activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL-2, -4, or -7. Thus, the IL-12R beta chain appears to be an essential component of the functional IL-12R on both T and natural killer (NK) cells. We previously observed that high affinity IL-12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription-polymerase chain reaction analysis showed that IL-12R beta chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non-lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL-12R beta chain (800 2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL-12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL-12R beta chain but, consistent with previous results, did not display detectable IL-12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL-12R beta, but did not bind measurable amounts of IL-12. These results indicate that expression of IL-12R beta is essential, but not sufficient, for expression of functional IL-12R. We speculate that expression of functional, high-affinity IL 12R may require the presence of a second subunit that is more restricted in its expression than IL-12R beta. PMID- 8617303 TI - Selectively impaired development of intestinal T cell receptor gamma delta+ cells and liver CD4+ NK1+ T cell receptor alpha beta+ cells in T cell factor-1 deficient mice. AB - T cell factor-1 (Tcf-1) is a transcription factor that binds to a sequence motif present in several T cell-specific enhancer elements. In Tcf-1-deficient (Tcf-1-/ ) mice, thymocyte development is partially blocked at the transition from the CD4 8+ immature single-positive stage to the CD4+8+ double-positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCR gamma delta+ cells and liver CD4+ NK1.1+TCR alpha beta+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf-1-/- mice. In contrast, thymic and thymus-derived (splenic) TCR gamma delta+ cells are present in normal numbers in Tcf-1-/- mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf-1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCR gamma delta+ cells and liver CD4+ NK1+T cells. PMID- 8617304 TI - Administration of interleukin-10 abolishes innate resistance to Listeria monocytogenes. AB - We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage. PMID- 8617305 TI - Human natural killer cell inhibitory receptors bind to HLA class I molecules. AB - To obtain direct evidence that human natural killer cell (NK) inhibitory receptors physically interact with polymorphic determinants of major histocompatibility complex class I molecules, a receptor termed NK-associated transcript-2 has been produced as a soluble fusion protein with the human IgG1 Fc portion. This soluble receptor binds specifically to HLA-C alleles with serine 77 and asparagine 80 in cell adhesion assays. PMID- 8617306 TI - CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages. AB - We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL 12, but not IL-2, in these cultures by approximately 90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-gamma and granulocyte/macrophage colony stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L. PMID- 8617307 TI - Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP-1. AB - Like many other cytokines and growth factors, interleukin-6 (IL-6) activates p21ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL-6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21ras. In the multiple myeloma cell line LP-1, IL-6 stimulated the tyrosine phosphorylation of Shc in a time- and concentration-dependent manner. This led to the complex formulation of Shc with Grb2, an adaptor protein known to relocate a p21ras-GDP exchange factor. Sos1 (Son-of-sevenless), to the cell membrane. Taken together, these findings suggest that IL-6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL-6 receptor associated non-receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects. PMID- 8617308 TI - A study of complexes of class II invariant chain peptide: major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody. AB - Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP:MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP:MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Li peptide bound to the peptide binding groove. PMID- 8617309 TI - Expression of a mannose/fucose membrane lectin on human dendritic cells. AB - Dendritic cells (CD) are the most efficient antigen presenting cells for T lymphocytes. CD1a+ CD14- CD with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al., J. Exp. Med. 1994. 179: 1109). Human macrophages express a membrane lectin, or sugar-specific receptor, which specifically mediates the binding and endocytosis of mannose- and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens. A similar lectin activity was sought on cultured human DC using flow cytometry and confocal microscopy to detect binding and internalization of fluoresceinated neoglycoproteins [bovine serum albumin (BSA) substituted with sugar residues]. Several neoglycoproteins, especially alpha-L-fucosyl-, alpha-D-mannosyl-, N,N'-di-acetyl-beta-chitobiosyl- and beta-D-glucosyl-BSA, were endocytosed by cultured human CD1a+ DC as well as by CD1a- CD14- cells which were also obtained in the culture. Fuc-BSA and Man-BSA had the same number of binding sites (1.7 x 10(6)/cell) on CD1a+ DC, and bound with an affinity constant close to 10(7) 1/mol. Inhibition experiments indicated that these two neoglycoproteins bound to the same membrane lectin. CD1a+ and CD1a cells were both labeled by an antiserum specific for the human macrophage mannose receptor. The membrane lectin specific for mannose and fucose that is evidenced in these experiments on cultured DC may be similar to the macrophage membrane lectin or may share functional and structural properties with it. PMID- 8617310 TI - The murine homolog of human Ep-CAM, a homotypic adhesion molecule, is expressed by thymocytes and thymic epithelial cells. AB - In this report, we demonstrate that gp40, a molecule previously shown to be expressed by thymic epithelial cell lines in vitro and by thymic epithelial cells in vivo, is the murine homolog of human Ep-CAM, a calcium-independent homotypic adhesion molecule. gp40 is also expressed at low levels by thymocytes and peripheral T cells. In the adult thymus, gp40 expression was inversely related to the state of thymocyte maturation, with the highest levels associated with CD4 CD8- and CD4+CD8+ thymocyte populations. Ultrastructural immunohistochemistry revealed gp40 localization to areas of thymocyte/epithelial contact and demonstrated that gp40 is also expressed by thymic dendritic cells. During fetal development, thymocytes at days 14-16 of gestation expressed high levels of gp40. At later stages, the observed decline in the frequency of gp40+ cells and levels of expression correlated with the emergence of alpha beta+ thymocytes by day 18 of gestation. In short-term cultures, stimulation of unfractionated adult thymocytes with concanavalin A increased gp40 expression, particularly among CD3hi and CD3int thymocyte populations. This demonstration that Ep-CAM, initially considered to be expressed primarily by epithelial cells, is also expressed by thymocytes, T cells and antigen-presenting cells, raises the possibility that Ep CAM may contribute to adhesive interactions between thymocytes and epithelial cells or dendritic cells, either in the context of thymocyte development or peripheral T cell trafficking and function. PMID- 8617311 TI - Membrane exon sequences of the three Xenopus Ig classes explain the evolutionary origin of mammalian isotypes. AB - We have cloned and sequenced the genes corresponding to the membrane exons of the three immunoglobulin (Ig) heavy chain isotypes (mu, upsilon and chi) of Xenopus. Among membrane Ig (mIg) polypeptides, the transmembrane domain are the most highly conserved. The transmembrane and cytoplasmic domains of Xenopus mIgM are similar to the corresponding domains of all known vertebrate mIgM molecules, supporting the idea that amphibian mu gene is homologous, not just analogous, to the mu gene of higher vertebrates. The membrane forms of the two other Ig isotypes mIgX and mIgY exhibit the specific structure found in all Ig membrane exons, but are not homologous with any specific mammalian non-mu Ig isotype; they are most similar to Xenopus mIgM. Based on the conserved transmembrane domains of Xenopus mIgX, mIgY, we suggest that first the upsilon and later the chi genes arose by duplication from the original mu gene. The transmembrane and the 37 amino-acid-long cytoplasmic domains of Xenopus mIgY have conserved residues found in avian mIgY and mammalian mIgG and mIgE, suggesting that the modern isotypes might share a common ancestor with amphibian mIgY. However, while the sequence similarity between the membrane exons of avian mIgY and mammalian mIgG and IgE is striking, the overall similarity with Xenopus mIgY is very low. Thus, the genes giving rise to Xenopus mIgY and those eventually leading to avian mIgY and mammalian mIgG and mIgE must have diverged early in evolution, probably at the level of the primitive amphibians or before. PMID- 8617312 TI - Fas (CD95)/Fas ligand interactions regulate antigen-specific, major histocompatibility complex-restricted T/B cell proliferative responses. AB - The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity. PMID- 8617313 TI - Gamma delta T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ alpha beta T cells. AB - To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, gamma delta T cells and alpha beta T cells expressing one of the two prototypic V beta families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4+ alpha beta T cells expressing either V beta 1 or V beta 2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that alpha beta T cell alloreactivity is determined by CDR3 variability. By themselves, the gamma delta T cells were incapable of inducing GVH reactions. However, host gamma delta T cells were recruited into the donor alpha beta T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that gamma delta T cells may play a secondary role in GVH reactions. PMID- 8617314 TI - Inhibition of I-Ad-, but not Db-restricted peptide-induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice. AB - Thymocytes differentiate by positive and negative selection of immature CD4+ CD8+ T cells. Negative selection occurs by default or by high-affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4+ CD8+ thymocytes, and we have earlier shown that anti-CD3 induced thymic apoptosis can be blocked by RU486 in vivo. We now show that anti CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti-CD3 antibody-mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323 339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I-Ad, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366-374 peptide, recognized in context of Db, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high affinity recognition of endogenous MHC-presented peptides on TEC. PMID- 8617315 TI - Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses. AB - Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9 mer peptide to induce in vivo CD8+ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2Kd; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8+ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4+ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2Kd was demonstrated with the mutant ras4-12(V12) peptide, but not the normal ras4-12(G12) peptide, which specifically inhibited an H-2Kd-restricted, anti-nucleoprotein NP147-155 CTL response in a dose-dependent fashion. An anti-ras CD8+ T cell line was then established from immune splenocytes of BALB/c (H-2d) mice injected with ras4-12 (V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2Kd and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12(Gly-->Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras4-12(V12) peptide was immunogenic for the production of antigen-specific CD8+ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene. PMID- 8617316 TI - The phenotype and fate of the antibody-forming cells of the splenic foci. AB - The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling. PMID- 8617317 TI - Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells. AB - In addition to T cell receptor triggering, activation of T cells requires costimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3 day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAB) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAB against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogenic. as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence of B7-1 expression. PMID- 8617318 TI - P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity. AB - The role of P-selectin in T-lymphocyte accumulation and injury was studied in delayed-type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti-rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte-dependent macrophage influx and injury characteristic of DTH. Up regulation of P-selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti-CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up regulation endothelial cell P-selectin. Treatment with anti-CD4 antibody produced similar results. Blocking P-selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up-regulation of endothelial cell P-selectin was prevented. These data demonstrate that P-selectin is locally up-regulated on endothelial cells in T cell-dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P-selectin in local T cell recruitment and subsequent injury in DTH. PMID- 8617319 TI - CD8/CD4 lineage commitment occurs by an instructional/default process followed by positive selection. AB - In the present study, we investigated the developmental potential of purified populations of transitional CD4inCD8hi and CD4hiCD8in thymocytes that were further defined according to their differentiation stage by their levels of T cell receptor (TCR) expression into TCRlo, TCRin and TCRhi subpopulations. The differentiation potential of each of these subsets was tested in vitro in a single-cell suspension culture assay that showed that CD4inCD8hiTCRhi are precursors of CD8 single-positive cells, whereas CD4hiCD8inTCRin/hi are precursors of both CD4 and CD8 single-positive thymocytes. The analysis of transitional subsets in mutant mice for either beta 2-microglobulin or major histocompatibility complex (MHC) class II further revealed that lineage commitment to the CD8 lineage requires a TCR-MHC class I engagement, presumably at the immature double-positive stage of thymic development, while CD4 commitment does not require an MHC class II-mediated signal, but rather occurs by default. Using the addition of MHC class I- or class II-expressing cells or the addition of total thymocytes to purified sorted transitional precursors for the duration of the cultures in vitro, we identified an additional stage of differentiation for both CD4 and CD8 lineages that requires a positive selection signal. Examination of protein tyrosine phosphorylation of transitional precursors revealed that CD4inCD8hi transitional cells contain a high level of a 70-kDa phosphorylated protein consistent with a role for ZAP70 in the signal transduction during the positive selection of CD8+ cells. PMID- 8617320 TI - Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies. AB - In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds) DNA and/or anti-histone autoantibodies could recognize and influence the fate of cell surface-bound nucleosomes. 125I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of approximately 7nM and a low-affinity site (Kd approximately 400 nM) with a high capacity of 9 x 10(7) sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived ds DNA (180-200 bp) was found to complete for binding of 125I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from 125-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa ds DNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-ds DNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5-10 min which moved toward the perinuclear region by 20-30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the microfilament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-ds DNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis. PMID- 8617321 TI - Anti-parasitic effector mechanisms in human brain tumor cells: role of interferon gamma and tumor necrosis factor-alpha. AB - Toxoplasma gondii, an obligate intracellular parasite, is able to replicate in human brain cells. We recently showed that interferon (IFN)-gamma-activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN-gamma-mediated activation of indolamine 2,3-dioxygenase (IDO), resulting in the degradation of the essential amino acid tryptophan. In contrast, glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN-gamma activation, and IFN-gamma-mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)-alpha alone is unable to activate IDO or to induce toxoplasmostasis in any glioblastoma cell line tested. Interestingly, we found that TNF-alpha and IFN-gamma were synergistic in the activation of IDO in glioblastoma cells 87HG31, 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF-alpha + IFN-gamma activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF-alpha or IFN gamma were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore, it was found that the addition of L-tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF alpha and IFN-gamma may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells. PMID- 8617322 TI - Identification and characterization of a human CD4 silencer. AB - Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage-specific silencing activity to a region of 190 bp. This region contains two protein-binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage-specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein-DNA interaction in CD4 gene regulation. PMID- 8617323 TI - In Th2-biased lymphatic filarial patients, responses to purified protein derivative of Mycobacterium tuberculosis remain Th1. AB - Natural infection with filarial nematode parasites results in immune responses skewed towards T helper (Th)2, while infection with mycobacteria shows many characteristics of a Th1-dominated response. Cytokines typifying Th1, interferon (IFN)-gamma, and Th2, interleukin (IL)-4, were measured following stimulation of peripheral blood mononuclear cells from filarial patients with Brugia malayi adult worm antigen (BmA) and purified protein derivative of Mycobacterium tuberculosis (PPD). In response to PPD, only 1 out of 81 patients produced IL-4, and this at an amount (4.4 pg/ml) just above the detection limit, whereas 59% of patients responded to BmA by releasing IL-4. Conversely, substantial quantities of IFN-gamma were released in response to PPD (geometric mean 37.43 U/ml) compared to low BmA-stimulated IFN-gamma production in the same patients (geometric mean 5.02 U/ml). These results demonstrate that the strong skewing of the cytokine environment towards Th2 in filarial patients in vivo does not influence the predominance of a Th1 type immune response to PPD. PMID- 8617324 TI - Co-localization of beta 2-microglobulin and IgG in human placental syncytiotrophoblasts. AB - The fetal syncytiotrophoblast cells in close contact with maternal blood circulation apparently lack surface expression of HLA molecules, including the HLA light chain beta 2-microglobulin. This is thought to contribute significantly to a successful pregnancy. We find that syncytiotrophoblasts do express beta 2 microglobulin. Beta 2-microglobulin is primarily localized intracellularly in apical granules, and co-localize with human IgG. The origin and function of syncytiotrophoblast beta 2-microglobulin is unknown, but its localization in the syncytiotrophoblasts may implicate beta 2-microglobulin in the transplacental transport of IgG in conjunction with a recently identified class I HLA-like receptor for IgG/Fc. Alternatively, beta 2-microglobulin may associate with a hitherto unidentified class I HLA molecule. PMID- 8617325 TI - Schistosoma mansoni: characterization of an alpha 1-3 fucosyltransferase in adult parasites. AB - We report that extracts of Schistosoma mansoni contain a GDPFuc:Gal beta 1 4GlcNAc (Fuc to GlcNAc) alpha 1-3 fucosyltransferase (alpha 1,3 FT) capable of synthesizing the antigenic determinant known as Lewis x (Le(x), Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-R). When the acceptor lacto-N-neotetraose (LNnT, Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) was incubated with extracts of S. mansoni in the presence of GDPFuc and Mn2+, Fuc was transferred to generate the pentasaccharide lacto-N-fucopentaose III (LNFPIII, Gal beta 1-4[Fuc alpha 1 3]GlcNAc beta 1-3Gal Beta 1-4Glc). The enzyme did not transfer efficiently to the isomeric oligosaccharide lacto-N-tetraose (LNT, Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc). The activity of the schistosome alpha 1,3 FT toward LNnT was dependent upon time, protein and GDPFuc. Interestingly, the schistosome alpha 1,3 FT was also able to transfer Fuc to a sialic acid-containing trisaccharide NeuAc alpha 2-3 Gal beta 1-4 GlcNAc to produce the tetrasaccharide sialyl Lewis x (2,3 sLe(x), NeuAc alpha 2-3 Gal beta 1-4[Fuc 1-3]GlcNAc), although the rate of reaction with the sialylated acceptor was <5% of the rate obtained toward nonsialylated acceptor. The schistosome alpha 1,3 FT was relatively resistant to inhibition by N-ethylmaleimide. The enzymatic properties of the schistosome alpha 1,3 FT resemble those of the human myeloid fucosyltransferase FTIV and not those of other known human fucosyltransferase. PMID- 8617326 TI - Fibronectin-induced intracellular calcium rise in Entamoeba histolytica trophozoites: effect on adhesion and the actin cytoskeleton. AB - The interaction of Entamoeba histolytica trophozoites with fibronectin (FN) promotes adhesion of the protein to the cells and its later degradation by locally released proteases. Binding to FN-covered surfaces induces, in addition, the formation of actin adhesion plates and focal contacts in the amebas. The signaling mechanisms underlying the response to FN are incompletely understood. In this paper we examined the modifications of cytosolic free calcium ([Ca2+]i) induced in the trophozoites by the interaction with FN and their effect on adhesion and the actin cytoskeleton organization. FN produced a sustained rise of [Ca2+]i that could be correlated to the incremented adhesion to FN-covered surfaces. Further increments in [Ca2+]i produced by Ca2+ ionophores A23187 or ionomycin significantly increased the adhesion of trophozoites, whereas depletion of cytoplasmic Ca2+, by treatment with the ionophores in the absence of external Ca2+ or using the chelator BAPTA/AM, blocked it almost completely. To study the role of internal calcium we used the plant lactone thapsigargin, which was found to produce a transient increase of [Ca2+]i but a low stimulatory effect on adhesion and the organization of actin plates. The shifting of soluble actin to the F-actin form and the stabilization of adhesion plates and focal contacts, seen as results, of the FN stimulus, were positively influenced by rises in [Ca2+]i and negatively affected by its decrement. Additional evidence for Ca2+ mediated signaling in the response to FN was provided by the poor adhesion and defective actin plate organization observed in trophozoites treated with calmodulin antagonists. The results presented here suggest that FN action is mainly dependent on the influx of external Ca2+. PMID- 8617327 TI - Leishmania major: infection of human monocytes induces expression of IL-8 and MCAF. AB - Leishmania major, the causative agent of cutaneous leishmaniasis, is an intracellular parasite of monocytic cells. Leishmania lesions are characterized by recruitment of neutrophils, eosinophils, and blood monocytes. To clarify the mechanism of this cellular influx we examined the expression of IL-8 and MCAF, cytokines chemotactic for neutrophils and monocytes, respectively, in human monocytes infected by L. major. Supernatants of monocytes infected with L. major had chemotactic activity for neutrophils and monocytes that was neutralized by anti-IL-8 and anti-MCAF antibodies. Both IL-8 and MCAF mRNAs were induced within 1 hr of infection. Furthermore we showed that infection of monocytes with L. major induces IL-8 and MCAF but not the proinflammatory cytokines IL-1 beta and TNF alpha. These data suggest that the expression of chemokines may contribute to the cellular recruitment in Leishmania lesions. PMID- 8617328 TI - Boophilus microplus: multiple proteolytic activities in the midgut. PMID- 8617329 TI - Leishmania amazonensis: the Asian rhesus macaques (Macaca mulatta) as an experimental model for study of cutaneous leishmaniasis. AB - As a means of assessing the usefulness of the Rhesus macaque (Macaca mulatta) as a nonhuman primate model for studying cutaneous leishmaniasis, monkeys were infected with Leishmania amazonensis. Variation in the level of susceptibility was found; however, animals inoculated with 10(8) promastigotes provided consistent results as indicated by an earlier onset and/or larger size of lesions. Three monkeys, which had recovered from skin lesions, were challenge infected using the same parasite strain/dose; although these animals remained susceptible to homologous infection, lesion size was smaller and healed faster than in the initial infection. The immunologic features during infection were assessed. Levels of IgM and IgG antibodies to promastigote antigens rose during active infection and then declined; immunoblot analyses indicated that numerous leishmanial antigens (predominately >30 kDa) were recognized. Delayed type hypersensitivity (DTH) responses and proliferative responses (PBL) developed during active infection and/or rechallenge. Circulating peripheral T cell subpopulations varied throughout the course of infection. Initially (6-8 weeks p.i.), CD4+ T cells appear to predominate; subsequently (15-21 weeks p.i.), an increase in CD8+ T cells was observed. Pathologic analyses indicated that lesions contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, and formation of tuberculoid-type granulomas. As the progression and resolution of leishmanial infection in rhesus macaques are very similar to those observed in humans, this primate model could be employed for elucidating the mechanisms of protective immunity in cutaneous leishmaniasis. PMID- 8617330 TI - Plasmodium falciparum: naturally occurring rabbit immunoglobulins recognize human band 3 peptide motifs and malaria-infected red cells. AB - The reactivity to overlapping decapeptides based on sequences of human band 3 protein was determined for 24 rabbit serum samples. A high percentage of the sera recognized sequences of amino acid 650-670, as well as several other sequences representing putative exofacial regions of the band 3 protein. When sera were used to stain immunofluorescently human erythrocytes, some of which were infected with P. falciparum, those samples that predominantly reacted with amino acids 650 670 stained P. falciparum-infected red cells, whereas those that reacted with other regions of band 3 stained all erythrocytes. A positive correlation was found between anti-band 3 reactivity and the capacity of a serum to inhibit the cytoadherence of P. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Also, some rabbit sera immunoprecipitated a high-molecular-weight protein from extracts of surface iodinated P. falciparum-infected red cells. PMID- 8617332 TI - Entamoeba histolytica: isoprenylation of p21ras and p21rap in vitro. PMID- 8617331 TI - Plasmodium yoelii: the role of the individual epidermal growth factor-like domains of the merozoite surface protein-1 in protection from malaria. AB - The merozoite surface protein-1 (MSP-1) is a leading candidate for a vaccine targeted at the erythrocytic stages of plasmodial parasite development. Recently, there has been increasing interest in this polypeptide, particularly in the carboxyl-terminal EGF-like domains. We have previously shown that this region from Plasmodium yoelii, when expressed in native configuration, could immunize mice against an otherwise lethal challenge infection. In this model system, protection appears to be predominantly mediated by antibodies. In all rodent immunization studies to date, however, the immunogen has contained both of the postulated EGF-like domains. We report here on the efficacy of immunization with the individual EGF-like domains from P. yoelii in elicitation of a protective host response. Although all animals developed some level of antibody in response to the various immunogens, only those animals immunized with both EGF-like domains produced antibodies which could recognize the native MSP-1 molecule. Antibodies generated against the individual EGF-like domains did cross-react with the double EGF-like domain structure, suggesting that the immunogens had retained elements of native configuration. In addition, only those animals which generated antibodies capable of recognizing native MSP-1 showed any level of protection from challenge infection. These results suggest that determinants unique to the double EGF-like domain structure may be necessary for the generation of antibodies specific for the native configuration of MSP-1 and that these antibodies may play a significant role in protection. PMID- 8617333 TI - Identification of 3-hydroxyproline residues in several proteins of Fasciola hepatica. PMID- 8617334 TI - Crithidia fasciculata: isolation, sequencing, and expression of the hypoxanthine guanine phosphoribosyltransferase gene. PMID- 8617335 TI - The mevalonate pathway in parasitic protozoa and helminths. PMID- 8617336 TI - Trypanosoma congolense: high erythropoietic potential in infected yearling cattle during the acute phase of the anemia. AB - N'Dama (Bos taurus) cattle are known to tolerate trypanosome infections, developing less severe anemia and lower parasitemia, compared to Boran (Bos indicus) cattle. Young calves were also reported to be more resistant to trypanosomiasis than adult cattle. To explore the basis for this resistance, the erythropoietic response was evaluated in four native yearling N'Dama calves and four age-matched Boran calves which developed anemia over a 140- day primary infection with Trypanosoma congolense clone IL 13E3. Similar levels of parasites were detected in the two breeds until 42 days postinfection (dpi). During the same period, a rapid and greater colony-forming units-erythroid response in the bone marrow of yearling Boran calves, compared with N'Dama calves, may explain the unusual absence of breed differences in mean packed cell volumes (PCV). However, this early erythropoietic response was transient and did not result in any rise in PCV from 70 dpi onward. In contrast, in the N'Dama calves, following the erythroid response, the mean PCV was gradually compensated from 56 dpi onward and reached 30% by 126 dpi. This period of PCV recovery coincided with low and intermittent parasitemia and a return of the erythroid progenitor levels to near preinfection values. Elevated levels of erythroid progenitors in the N'Dama calves, occurring 1 week after trypanocidal treatment, returned the PCV to preinfection values. These results suggest that the age of the Bos indicus cattle has an important impact on the early bone marrow response in primary T. congolense infection and confirmed previous suggestions of a high erythropoietic potential in trypanosome-infected young calves. PMID- 8617337 TI - Differential in vitro and in vivo behavior of three strains of Trypanosoma cruzi in the gut and hemolymph of Rhodnius prolixus. AB - A comparison was made of the agglutination and lysis of three strains of Trypanosoma cruzi in gut extracts and hemolymph of Rhodnius prolixus and the results obtained were correlated with the success or failure of the parasite strain to infect the digestive tube or to survive in the hemocel after inoculation. Both T. cruzi strains Dm28c and Cl urine 35 days after feeding with parasites. Concomitantly, both of these strains were agglutinated but not lysed by the crop extracts. In contrast, T. cruzi Y strain parasites rapidly disappeared from the gut and showed no agglutination, but some lysis, in the crop extract. Following inoculation into the hemocel, only the Cl strain survived at high levels and was also the only strain agglutinated significantly in the hemolymph. Both Dm28c and Y strains rapidly disappeared from the hemocel with the former parasite being removed more slowly than the latter, probably due to clearance by the cellular defenses. The rapid clearance of the Y strain was correlated with the presence of a high titer lysin in the hemolymph. Subsequent experiments using FITC-labeled lectins and FACS to probe the carbohydrates on the parasite surfaces showed significant differences between the three strains. Thus, only Dm28c was stained strongly by Arachis hypogea (PNA) lectin, indicating the presence of galactose/N-acetylgalactosamine residues, and Dm28c and Y strains by Phytolacca lectin for N-acetyl glucosamine moieties. Finally, the fact that, in contrast to Dm28c and Y, the Cl strain strongly interacted with Triticum vulgaris (WGA) but not with Phytolacca lectin may be due to the presence of N-acetyl neuraminic acid residues on these organisms. These surface carbohydrate differences may be correlated both to the behavior and agglutination variations between the three strains recorded in this work. PMID- 8617338 TI - Heligmosomoides polygyrus: resistance in inbred, outbred, and selected mice. AB - Mice reared selectively as high- and low- immune responders to reinfection with Heligmosomoides polygyrus showed extremes of resistance and susceptibility after infection when compared to inbred BALB/C, DBA/I, CBA, and SJL/J and outbred Quackenbush (Q) mice. The levels of anti-H. polygyrus total IgG and IgG1, IgG2b, and IgG3 isotype antibodies detected in serum from the seven strains of mice after secondary and the eosinophil leucocyte counts in blood after both primary and secondary infections correlated positively and strongly with the levels of protective immunity developed when evaluated using a resistance index formulated from the infectivity, fecundity, and length of adult worms recovered after infection. PMID- 8617339 TI - A new approach for the identification and the diagnosis of Eimeria media parasite of the rabbit. AB - In this work, we described a new approach for the isolation of a species-specific probe for the Eimeria media parasite of the rabbit based on the use of the random amplified polymorphic DNA (RAPD) technique. A specific fragment of 800 bp of the studied species was isolated after RAPD and then cloned and DIG-radiolabeled. After dot-blotting, we observed that this probe was specific for E. media. Sequencing of the 3' and 5' ends of this probe enabled the determination of two primers that could be used in a PCR reaction. The amplified product of 750 bp was specific E. media. The use of these primers and of our probe allowed the detection of a very small number of oocysts. With a new protocol of DNA purification, 10 purified oocysts were detected by PCR. The efficiency of the amplification was not changed when two species were mixed. The threshold of detection of oocysts in fecal matter was equal to 30. PMID- 8617340 TI - Antibody response in dogs experimentally infected with Leishmania infantum: infection course antigen markers. AB - Five-month-old beagle dogs were experimentally infected with amastigotes of Leishmania infantum and kept for 14 months. Infection course was monitored by clinical examination, serum protein variations, and levels of specific antibodies against Leishmania estimated by indirect immunofluorescence test and Western blotting (WB). Infected animals developed notable changes in serum protein levels reaching maximum protein concentrations 2-3 months postinfection (p.i.) related to the gamma-globulin fraction. Specific antibody titers were in good agreement with the serum protein rise, reaching immunofluorescence values of over 1:800 3 months p.i. Serial Western blotting analysis with L. infantum promastigotes protein showed a strong response against immunodominant antigens of 50-57, 42, and 29 kDa during most of the studied period with immunofluorescence titers of over 1:100 and in addition the response was remarkably homogeneous among the infected dogs. Immunoreactivity patterns displayed time-related variations; the response against 29 and 50-57 kDa was seen very early, followed by the reaction around 42, 76, and 86 kDa. In addition the recognition of peptides around 34-35.4 and 26 kDa was restricted to the acute phase of the experimental infection. Preliminary results obtained in naturally infected dogs seem to support the predictive value of the WB. PMID- 8617341 TI - Plasmodium falciparum: characterization of toxin-associated proteins and identification of a hemoglobin containing parasite cytokine stimulator. AB - Previous studies have indicated the inositol monophosphate (IMP) is a component of the malaria parasite toxin that induces cytokines such as tumour necrosis factor (TNF). To further characterize the toxin we have labeled Plasmodium falciparum in vitro cultures with [14C]inositol or [35S]-methionine and immunoprecipitated the labeled antigens with an antiserum against IMP which blocks malaria parasite-induced TNF production. We detected four proteins associated with IMP when the immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography. To evaluate the capacity of different P. falciparum antigens to induce cytokine production we separated a mixture of exoantigens by SDS-PAGE gels. Antigen fractions of 43-71 kDa and of a low molecular mass of <20 kDa contained the dominant inducers of TNF alpha interleukin 1 alpha, and interleukin 6 production from human mononuclear cells. The low-molecular-mass antigen fraction contained hemoglobin, while no parasite-specific proteins were detectable when tested by immunoblotting. Hemoglobin may act as a carrier for cytokine-inducing malaria parasite toxins. PMID- 8617342 TI - Plasmodium falciparum: differential parasite growth inhibition mediated by antibodies to the antigens Pf332 and Pf155/RESA. AB - Plasmodium falciparum-reactive antibodies can inhibit growth of parasite blood stages in vitro by interfering at different stages of parasite development. Antibodies reactive with repeat sequences in Pf155/ring-infected surface antigen (Pf155/RESA) or in antigen Pf332 inhibit parasite growth as determined by a reduction of newly infected erythrocytes. Antibodies to Pf155/RESA have been implicated in merozoite invasion inhibition. Since it was considered unlikely that antibodies to the late stage antigen Pf332 act on merozoite invasion, we investigated at what developmental stage this growth inhibition takes place. Parasites were cultured in the presence of antibodies to repeat sequences of either Pf332 or Pf155/RESA and were examined with regard to the distribution of different stages and their morphology. As expected, antibodies to both antigens decreased the number of ring stages after reinfection. They did not affect the intraerythrocytic development of ring stages or trophozoites. However, antibodies to Pf332 induced high levels of schizonts displaying an abnormal morphology while antibodies to Pf155/RESA induced considerably lower levels of degenerated schizonts. Thus, Pf332-reactive antibodies seem to interfere with the schizont development by blocking the rupture of mature schizonts or, alternatively, by interfering intraerythrocytically with late stage parasites. PMID- 8617344 TI - Entamoeba histolytica: localization of a 30-kDa cysteine proteinase using a monoclonal antibody. AB - We produced a monoclonal antibody against a major cysteine proteinase of 30kDa from trophozoites of Entamoeba histolytica strain HM1:IMSS. The specificity of the monoclonal antibody was confirmed by specific inhibition of azocasein digestion and by electrophoretic analysis, in the presence of sodium dodecyl sulfate or on a substrate gel, of the antigen precipitated by the antibody. Immunofluorescent staining of trophozoites with the monoclonal antibody revealed heterogeneity in the intensity of whole cell fluorescence and subcellular localization of the stain. The latter was also observed in trophozoites, which were stained by conventional immunohistochemical methods, from experimental liver abscesses in hamsters. Ultrastructural analysis showed antigen distributed mainly in clear amorphous zones in the cytoplasm, which were not limited by a visible membrane. Proteinases are translocated from these compartments to phagocytic vacuoles after trophozoites ingest erythrocytes, suggesting that these regions might be a lysosomal equivalent of this primitive eukaryotic cell. PMID- 8617343 TI - Entamoeba histolytica: involvement of pp125FAK in collagen-induced signal transduction. AB - The interaction of Entamoeba histolytica trophozoites with collagen involves cell adherence, formation, and release of electron dense granules (EDGs) containing collagenase activity leading to the degradation of the bound protein. The binding is thought to be mediated by an "integrin-like" collagen receptor. Since the signal transduction mechanisms triggered by the collagen-trophozoite interaction are unknown, but clearly involve cytoskeletal organization, we decided to explore the role of protein tyrosine phosphorylation in this process. Collagen induces a time-dependent increase in the phosphorylation of several polypeptides migrating around 67 and 110 kDa. One polypeptide of the high-molecular-weight component was identified as a 125-kDa protein with very similar epitopes to the focal treatment was a 42-kDa polypeptide related to the mitogen activated protein kinase (MAPK) family. Our results suggest that tyrosine phosphorylation is involved in collagen signaling in amoebas and that pp125FAK and p42MAPK homologs may play an active role in turning on the genetic program that enables the parasite to invade its host. PMID- 8617345 TI - Diversity of Trypanosoma cruzi stocks and clones derived from Chagas disease patients. II. Isozyme and RFLP characterizations. AB - Isoenzyme and RFLP analyses were carried on freshly isolated Trypanosoma cruzi stocks and subsequent clones derived from patients with chronic Chagas disease. The isoenzymes separated the parasite stocks and clones in two groups: The stock hSLU239 (group I), isolated from a heart disease patient, showed the zymodeme 3 (Z3) profile (M. A. Miles et al., 1977, Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 217-225). The stock mSLU142 (group II), isolated from a digestive disease (megaesophagus) patient, showed the Z2 profile. The parasite clones m1, m2, m3, and m4, derived from mSLU142, and clones h1 and h2, derived from hSLU239, showed isoenzyme profiles similar to those of Z2 and ZA (Miles et al. 1977; J. A. Romanha, 1982, Thesis, Universidade Federal de Minas Gerais). Furthermore, the T. cruzi clones derived from the cardiac disease patient differed from those derived from the megacolon patient in 3 of the 13 enzymes analyzed. RFLP analysis showed polymorphism at the EcoRI and PstI restriction fragments of the DNA sequences coding the glycolytic enzymes ALD, GPI, GAPDH, and PYK and separated the T. cruzi stocks and clones in three groups: I, comprising the stock hSLU239 and clone m4, which was classified as homozygous CC, BB, AA, and AA for the ALD, GPI, PYK, and GAPDH genes, respectively; II, formed by the parasite stock mSLU142 and clones h1 and h2 (derived from hSLU239), which was classified as homozygous AA, AA, CC, and BB for ALD, GPI, PYK, and GAPDH genes, respectively . These findings show that the infection of each Chagas disease patient may be produced by genetically diverse mixed parasite populations. PMID- 8617346 TI - Schistosoma mansoni infection in humans and primates induces cytolytic antibodies to surface Le(x) determinants on myeloid cells. AB - The Lewis x antigen (Le(x); Gal beta 1-4[Fuc alpha 1-3]GlcNac beta1-R), which is present on the surfaces of human cells, is also synthesized by the human helminthic parasite Schistosoma mansoni. We now report that IgM and IgG antibodies to Le(x) antigens are present in the sera of humans and rhesus monkeys infected with S. mansoni, whereas these antibodies are completely absent in uninfected individuals. The sera from infected humans and monkeys mediate specific complement-dependent cytolysis of human promyelocytic leukemic HL-60 cells, which bear surface Le(x) antigen. Furthermore, the major activity in sera from infected individuals toward HL-60 cells is due to anti-Le(x) reactivity. PMID- 8617347 TI - Schistosoma mansoni: the glucose transport protein SGTP4 is present in tegumental multilamellar bodies, discoid bodies, and the surface lipid bilayers. AB - Schistosomes metabolize large quantities of glucose obtained from the host serum by facilitated diffusion through the tegument. Here we have used rabbit antibodies affinity purified against the carboxyl terminus of a facilitated glucose transporter, SGTP4, to localize the antigen in both schistosomula and adults. By ultrastructural immunocytochemical analysis, SGTP4 was localized to both lipid bilayers that cover the tegumental surface of adults and schistosomula. In the inner bilayer of adults, SGTP4 was apparently oriented with the carboxyl terminus on the internal side of the bilayer. SGTP4 was also present in the discoid and multilamellar bodies in adults and the membranous bodies in schistosomula. Finally, the affinity purified antibodies against SGTP4 bound nonspecifically to the head glands and postacetabular glands in schistosomula. The localization of the antigen in the two surface lipid bilayers suggests that SGTP4 may be responsible for transporting glucose from mammalian host serum into the tegument. PMID- 8617348 TI - Thymidine kinase as a selectable marker for studying the biogenesis of glycosomes in Trypanosoma brucei. AB - The import of proteins into the glycosome of T. brucei has been studied as a potential target for antitrypanosomal chemotherapy. We have previously reported on the C-terminal tripeptides, such as SKL and its analogs, which function as targeting signals in the import of proteins into glycosomes. Recently, we tested the herpes simplex virus thymidine kinase gene (tk) as both a potential positive and a negative selectable marker in T. brucei for isolating glycosome-deficient T. brucei mutants in the procyclic form and complementation studies to identify genes involved in glycosomal biogenesis. The transforming vectors that we have constructed contained the hygromycin phosphotransferase gene (hyg) coupled either to the tk gene (ptk) or to the gene encoding the thymidine kinase fused with the glycosomal targeting signal (ptk-SKL) at the C-terminus. Individual constructs were introduced into the T. brucei 427 procyclic cells by electroporation, and the transformants were selected under hygromycin B (50 micrograms/ml) and cloned. The thymidine kinase activity in the crude extracts of transformants was determined. Differential digitonin treatment of the transformants indicated that the tk-SKL protein was apparently localized to the glycosomal fraction, as expected, whereas the tk protein was found in the soluble fraction. [methyl 3H]Thymidine was incorporated into the nucleic acids of the transformant 427/ptk to a level twice as high as that incorporated into 427/ptk-SKL and the wild type. In the presence of 100 microM ganciclovir, the growth of 427/ptk was totally inhibited, whereas growth of 427/ptk-SKL and the wild type was unaffected. When 150 microM trimethoprim was added to the culture medium, growth of 427/ptk-SKL and the wild type was completely arrested while that of 427/ptk remained normal. We have thus established the methodology for both positive and negative selection of potential glycosome-deficient mutants of T. brucei 427/ptk-SKL. PMID- 8617349 TI - Schistosoma mansoni: localization of the SmIMP25 protein in the subtegumental extracellular matrix of schistosomula. PMID- 8617351 TI - The genomic organization of the HSP83 gene locus is conserved in three Leishmania species. PMID- 8617350 TI - Parasite-specific isotype and subclass antibody profiles during acute prepatent human schistosomiasis. PMID- 8617352 TI - Leishmania chagasi: a gene encoding a protein kinase with a catalytic domain structurally related to MAP kinase kinase. AB - The gene for a serine/threonine protein kinase was isolated from Leishmania chagasi. The deduced amino acid sequence encoded by this Leishmania protein kinase (LPK-1) gene possesses all of the 11 conserved subdomains found in most other serine/threonine protein kinase catalytic domains. Sequence alignment with other known protein kinases demonstrates that the 42-kDa LPK-1 is a member of the mitogen-activated protein kinase kinase family involved in the signal transduction pathways in yeast and mammalian cells. The single copy gene for LPK 1 specifies a 3.1-kb mRNA that is expressed in both logarithmic and stationary phase promastigotes with a twofold higher steady-state level in the infective stationary phase promastigotes. PMID- 8617353 TI - Plasmodium falciparum: molecular characterization of multidrug-resistant Cambodian isolates. AB - Clinical resistance to many therapies for malaria is a rapidly evolving problem in most endemic areas, particularly, Southeast Asia. Recent studies have suggested linkages between the mdr-like genes of Plasmodium falciparum and resistance to quinoline containing compounds. Other studies have found an association between allelic polymorphisms in the DHFR gene and antifol resistance in these parasites. The purpose of this study was to further examine these associations in recent isolates from Cambodia. DNA sequences and gene copy number of the pfmdrl and the DHFR-TS gene in 10 Cambodian isolates were analyzed and correlated with the drug sensitivity pattern. No new intragenic alleles were detected in the pfmdrl gene by a full-length DNA sequence analysis of the L 14/Cambodia clone. The allelic variations seen in pfmdrl in these isolates did not correlate with chloroquine resistance as previously reported. The full-length sequence of the DHFR latter findings may be correlated with high-level resistance to the antifolate drugs as has been previously described. None of the Cambodian isolates presented gene amplification in either pfmdrl or DHFR-TS genes. PMID- 8617354 TI - Triplex-mediated cleavage of DNA by 1,10-phenanthroline-linked 2'-O-methyl RNA. AB - We have previously reported that 2'-O-methyl RNAs are efficient probes for duplex DNA. Here we describe the design, synthesis, and DNA cleaving activity of 1,10 phenanthroline (OP)-linked 2'-O-methyl RNA (OP-m). Although a local triple helix was formed, both with OP-m and a control OP-linked DNA at the target sequence of the duplex DNA, the promoter region of the human thrombomodulin gene, the cleavage efficiencies on both strands were not proportional when OP-M was used as a cleavage agent. These results may reflect the structural differences of the respective triple helices and the duplex-triplex junction, formed from the two types of triplex-forming oligonucleotides, 2'-O-methyl RNA and DNA. Since the OP ms were found to work as preferential purine-strand cutters for duplex DNA, they would be useful as unique tools for genome analysis. PMID- 8617355 TI - Crystal structures of human procathepsin B at 3.2 and 3.3 Angstroms resolution reveal an interaction motif between a papain-like cysteine protease and its propeptide. AB - A wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Angstroms resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure. PMID- 8617356 TI - Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPphi14-9 fusion protein. AB - The SRPphi14-9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPphi14-9 form IV crystals belong to the space group P4(1)22/ P4(3)22 with cell parameters a = b = 69.7 Angstroms, c = 95.7 Angstroms, alpha = beta = gamma = 90 degrees. A complete data set to 2.8 Angstroms resolution with an Rsym on intensities of 7.0% was collected on a single flash-frozen crystal. PMID- 8617357 TI - Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9. AB - Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected. PMID- 8617358 TI - The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron-containing pre-tRNAs in wheat germ extract. AB - Southern analysis of Triticum DNA has revealed that nuclear tRNATyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNATyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNATyr family. Four of the sequenced tRNATyr genes code for Triticum tRNA Tyr and two code for tRNA2Tyr. Three genes encode tRNAsTyr which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNATyr genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5' and 3' splice site loops separated by five base pairs can be formed by all pre tRNAs Tyr which are efficiently spliced in the homologous wheat germ extract. PMID- 8617359 TI - Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation. AB - In rod photoreceptor cells, Ca2+-bound recoverin associates with disk membranes and inhibits light-dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+-induced membrane association of recoverin has not been fully evaluated. We found that Ca2+-bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+ recoverin-rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity. PMID- 8617361 TI - Cloning of a potential cytochrome P450 from the archaeon Sulfolobus solfataricus. AB - Abstract A gene, CYP119, for a potential cytochrome P450 has been isolated and sequenced from the extreme acidothermophilic archaeon Sulfolobus solfataricus. The gene predicts a polypeptide of 368 amino acids containing the consensus heme binding sequence Phe-Gly-Xaa-Gly-Xaa-His-Xaa-Cys-Xaa-Gly- Xaa3-Ala-Arg-Xaa-Glu. It most closely resembles the cytochrome P450s found in the bacterium Bacillus subtilis, with which it shares 129 identical amino acid residues (35%). This first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary history of this biologically important enzyme family. PMID- 8617360 TI - Interaction of a human blood group Sd(a-) Tamm-Horsfall glycoprotein with applied lectins. AB - Unlike the human blood group Sd(a+) Tamm-Horsfall glycoprotein (THGP), the Sd(a-) one lacks terminal GalNAcbeta1--> residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a-) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin-a and ricin, all of which recognize the Galbeta1--> 4GlcNAcbeta1--> sequence, although at different strength. The lectin glycan interactions were inhibited by Galbeta1--> 4GlcNAc and Galbeta1--> 4Glc. When the precipitability of Sd(a-) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a-) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin-I (ML-I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a-) THGP showed a strongly diminished affinity for GalNAcbeta1--> active lectins (SBA and WFA) than the Sd(a+) phenotype. PMID- 8617362 TI - Antioxidant activities of carotenes and xanthophylls. AB - The purpose of this study was to assess the relative antioxidant activities of a range of carotenes and xanthophylls through the extent of their abilities to scavenge the ABTS(.+) radical cation. The results show that the relative abilities of the carotenoids to scavenge the ABTS(.+) radical cation are influenced by the presence of functional groups with increasing polarities, such as carbonyl and hydroxyl groups, in the terminal rings, as well as by the number of conjugated double bonds. PMID- 8617363 TI - Cloning and characterisation of the human adenosine A3 receptor gene. AB - We have cloned the gene for the human adenosine A3 receptor and report characterisation of its intron/exon structure and upstream untranslated region. The open reading frame is interrupted by a single intron of approximately 2.2 kb, within the coding sequence for the second cytoplasmic loop. Sequence analysis of the upstream region reveals no TATA box but the transcriptional start site has been mapped to a common nucleotide in three tissues by 5'-RACE and RT-PCR analysis. Northern blotting, 5'-RACE PCR and analysis of upstream sequences, have provided no evidence for the occurrence of further introns in the upstream untranslated sequence or of transcriptional regulation by alternative splicing in this region. PMID- 8617365 TI - The o-diphenol oxidase activity of arthropod hemocyanin. AB - Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport-storage protein. The protein, however, also catalyses with low efficiency the oxidation of o-diphenol to quinone, similarly to tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V(max) exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase. PMID- 8617364 TI - Cell adhesion and integrin binding to recombinant human fibrillin-1. AB - Fibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin alphaVbeta3. Integrins alphaIIbbeta3, alpha5beta1, alpha2beta1 and alpha1beta1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin alphaVbeta3. PMID- 8617366 TI - Structural rearrangements on HIV-1 Tat (32-72) TAR complex formation. AB - Expression of the early genes of the human immunodeficiency virus type-I (HIV-1) genome is under the control of a trans-activator (Tat) protein. HIV-1 Tat action requires binding to TAR (trans-activation responsive element), an RNA sequence located at the 5'-end of all lentiviral mRNAs. We used various spectroscopic methods to investigate conformational changes on HIV-1 TAR binding to the HIV-1 (32-72) Tat peptide BP1. It comprises the RNA binding region and binds specifically to TAR. We conclude from our experiments that the regular A-form of the TAR RNA is slightly distorted towards the B-form when bound to BP1. Thus, the major groove is widened and the binding of BP1 facilitated. BP1 presumably adopts an extended conformation when binding to TAR and may fit well into the TAR major groove. PMID- 8617367 TI - Molecular cloning and characterization of a novel orphan receptor (P2P) expressed in human pancreas that shows high structural homology to the P2U purinoceptor. AB - Here we report the cloning of a gene encoding a new member of the superfamily of G protein-coupled receptors. The gene encodes a protein of 365 amino acids closely resembling two recently cloned nucleotide binding receptors, called P2U and P2Y purinoceptors (71% and 49% sequence identity within the transmembrane domains, respectively). Our studies show that this new putative purinoceptor (designated P2P) is encoded by an intronless single copy gene that is exclusively expressed in pancreas, in contrast to the P2U and the P2Y purinoceptors which are widely distributed throughout the periphery. The identification of a pancreas specific human putative P2 purinoceptor makes it attractive to speculate that the reported actions of ADP/ATP analogues in pancreas on insulin secretion are mediated through this receptor. PMID- 8617368 TI - Vaccinia virus DNA topoisomerase I preferentially removes positive supercoils from DNA. AB - Type I DNA topoisomerases homologous to Escherichia coli topoisomerase I normally only remove negative supercoils from DNA. Topoisomerases I from various eukaryotes share sequence homology and remove both positive and negative supercoils from DNA. Here we report that vaccinia virus topoisomerase I has significant difference in substrate preference from the other homologous type I topoisomerases. Vaccinia virus topoisomerase I shows a definite preference for removal of positive supercoils. In contrast, topoisomerase I from human, wheat germ and Saccharomyces cerevisiae has little preference between positive and negative supercoils. The vaccinia enzyme may have evolved for functions required for optimal viral growth. topoisomerases. PMID- 8617369 TI - Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry. AB - Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of beta-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins. PMID- 8617370 TI - Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells. AB - Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli. PMID- 8617371 TI - M-type K+ current inhibition by a toxin fron the scorpion Buthus eupeus. AB - A number of invertebrate venoms have been tested for effects on M-type K+ currents (IK(M)) in differentiated mouse neuroblastoma X rat glioma NG108-15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited IK(M) by approximately 44% at 50 microgram/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrimidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited IK(M) by approximately 45% with an IC50 of approximately 33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of IK(M) in NG108-15 cells. PMID- 8617372 TI - Genetic analysis of the cytochrome P-45OIIC18 (CYP2C18) gene and a novel member of the CYP2C subfamily. AB - The CYP2C18 gene was investigated in order to characterize its molecular basis in the CYP2C subfamily. A mutation of the CYP2C18 gene was identified at the 5' flanking region of the gene, which could be detected by digestion with DdeI. The allele frequency of the mutant CYP2C18 gene was 21.4%. Genotypes of the polymorphic DdeI site of the CYP2C18 gene were found to be completely consistent with that of the polymorphic CYP2C19 gene (the m1 mutant). The CYP2C18 and CYP2C19 genes were suggested to be linked and located close together on the chromosome. Clones containing the 5'-flanking region of a member of the CYP2C subfamily were obtained from the PCR products from human genomic DNA. The nucleotide sequence of the clone proved to be 90.5% identical to the corresponding region of the CYP2C18 gene. This is very likely to be a novel member of the CYP2C subfamily. PMID- 8617373 TI - Down-regulation of plant V-type H+ -ATPase genes after light-induced inhibition of growth. AB - Cell extension growth in the mesocotyl tip of dark-grown Zea mays L. seedlings is dependent on vacuole enlargement and massive flux of ER and Golgi vesicles. Water flow into the expanding vacuole is driven by ion accumulation, which in turn is energized by the vacuolar H+-ATPase (V-ATPase). The V-ATPase energizes the secondary ion transport into the expanding vacuole. As light exposure leads to a strong inhibition of extension growth, the effect of light on transcript levels for subunits A and c of the V-ATPase was analyzed. Partial homologous cDNAs for subunit A and two isoforms of subunit c were cloned by RT-PCR. In dark-grown seedlings transcript levels for both subunits were much higher in the growing mesocotyl tip than in the fully differentiated mesocotyl tissue. Only in the tip region did light exposure lead to a strong and coordinate down-regulation of both mRNAs whereas in the differentiated mesocotyl only a slight decrease was observed. The results indicate that expression of the 'housekeeping' V-type H+ ATPase is strongly regulated in response to growth rate. PMID- 8617374 TI - cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3'-coding regions. AB - Two cDNA clones encoding stromal (SAP28) and thylakoid-bound (SAP22) ascorbate peroxidase were isolated from a spinach cDNA library constructed by greening cotyledons. The SAP22 and SAP28 contained an open reading frame encoding mature protein of 295 and 345 amino acids with calculated molecular mass of 32239 Da and 37710 Da, respectively, preceded by the common transit peptides of 70 amino acid residues. Interestingly, the N-terminal 364 amino acids of SAP22 were 100% identical with SAP28 except for one C-terminal amino acid residue (Asp-365), and the C-terminal of SAP22, which is the putative transmembrane segment, was 50 amino acids longer than that of SAP28. PMID- 8617375 TI - Ultrastructure of mucous areolae in the pig placenta. AB - The mucous areolae in the pig placenta are characterized by the presence of acid mucopolysaccharides. They have different forms: areas of vascularized trophoblast in early pregnancy, small irregular areolae, vesicles and cysts. The ultrastructure of the mucous areolae in placentae from 18th to 113th day post coitum was investigated. The trophoblast lining the areolae consisted of polarized columnar cells of varied height and width. Only in large cysts that epithelium was extended and flat, only a few micrometers thick. On the free surface of the cells long microvilli formed groups resembling stereocilia. The hyaloplasm of these cells was extremely electron-dense. The apical parts of the cells were filled with mucous granules and exocytosis of their content into the areolar cavity was observed. High synthetic activity of numerous dictyosomes was noted near the nucleus. The basal parts of the cells were divided into lobular processes containing a significant number of elongated mitochondria. In many trophoblastic cells large heterogeneous vacuoles were present. Their content was extruded into areola cavity. The substance filling the mucous areola cavity was heterogenous with some cell debris and whole desquamated cells. The uterine epithelium covering the mucous areolae consisted of high columnar absorptive cells, with the brush border present on their free surface. Apical terminal web contained numerous small coated endocytotic vesicles with a light flocculent content. The filopodia and lamellipodia separated by the intercellular space formed a labyrinth in the basal parts of the cells. The mucous areolae differ from the regular areolae in the ultrastructural and functional features. Possible role of the mucous areolae is discussed. PMID- 8617376 TI - Different isoenzyme patterns of nonspecific esterases and the level of IL6 production as markers of macrophage functions. AB - We examined the isoenzyme patterns of alpha and beta naphtyl acetate esterase and the IL6 production of two macrophage cell lines, which were cloned from a single fusion of macrophage tumor cells and spleen adherent cells. These clones were phenotypically indistinguishable but differ functionally as line 59 presents antigen to Th 1 lymphocytes while line 63 induces suppressor T lymphocytes. Cell extracts of these lines exhibit different isoenzyme patterns of alpha and beta naphtyl acetate esterase at both pH 7.5 and 5.8 but do not differ significantly in the level of produced IL6. Treatment with nitrogranulogen (NG), a derivative of cyclophosphamide, changes the isoenzyme pattern in line 59 and decreases severalfold IL6 production, while in similarly treated line 63 cells isoenzyme pattern remains unaffected but the production of IL6 is significantly increases. We assume that the observed differences between these two cell lines are due to distinct intracellular translation of the membrane signal delivered by NG. The different behaviour of these two parameters can thus be used as a useful tool to further delineate different macrophage subpopulations. We regard it likely that nonspecific esterases may play a role in intracellular processing or trafficking of antigen. PMID- 8617377 TI - Non-isotopic procedure with silver staining of polyacrylamide gels in detection of genomic abnormalities in tumors using microsatellites. AB - Microsatellite-based PCR (polymerase chain reaction) with silver staining of polyacrylamide gels as an alternative approach to detect genomic abnormalities in tumors has been developed. In experiments with a series of primers for microsatellite sequences located on human chromosome 5 we found that a 2- to 4 fold increase or decrease in the ratio of amount of allelic microsatellite sequences can be reliably assessed from the relative intensity of corresponding PCR products. Results of the studies of renal carcinoma cell lines carrying increased or decreased numbers of specific allelic markers on chromosome 5 assessed by cytogenetics, Southern blotting-restriction fragment length polymorphism and microsatellite analyses were consistent. Microsatellite studies performed on samples obtained from formalin-fixed paraffin-embedded archival material allowed detection of genomic abnormalities of chromosome 5 in five of ten primary renal cell carcinomas. PMID- 8617378 TI - Calcium in pollen-pistil interaction in Petunia hybrida Hort. III. Localization of Ca2+ ions and Ca(2+)-ATPase in pollinated pistil. AB - Studies were carried out of CA2+ and CA(2+)-ATPase localization in pollinated (6 and 48 h after pollination) pistils of Petunia hybrida. The results were confronted with CA2+ localization in mature pollen grain and in unpollinated pistil. It has been found that after pollination the number of CA2+ sequestered in the stigmal exudate and in the sporoderm of the pollen grain gets lower. That phenomenon was associated with the appearance of a large number of Sb/Ca precipitates in the submembrane cytoplasm of the germinating pollen. In the vacuolized pollen grain, i.e. grown into a pollen tube, there were only a few precipitates. In the pollen tube, CA2+ were found in the organelles of the tip cytoplasm and in the external pectin cell wall. Studies with the use of 45Ca2+ have revealed that the source of calcium ions incorporated into the pollen tube tip and its pectin wall is the transmitting tract of the style. In the transmitting tract overgrown with pollen tubes, Ca2+ were located in the intercellular matrix and in the transmitting cells. Sb/Ca precipitates occurred in the nuclei, around the secretory vesicles and on the plasmalemma in the transverse walls region. Elevated Ca2+ level was found in degenerating cells (inhibited pollen tubes, transmitting cells, nucellar cells). The progressing degeneration process of the cells of the transmitting tract of the pollinated pistil was associated with a decrease in the activity of plasmalemmal Ca(2+) ATPase. PMID- 8617379 TI - Effect of symplasmic isolation and antigibberellin treatment on morphogenesis in Chara. AB - The effects of plasmolytically induced symplasmic isolation and AMO-1618 treatment on the development of antheridia of Chara vulgaris L. were compared. After 24 h both factors inhibited endoreplication in manubria. In the youngest antheridia the inhibition of 3H-thymidine incorporation following plasmolysis was irreversible while the same effect caused by AMO-1618 disappeared after 3 days. Four-five days after plasmolysis or five days after AMO-1618 treatment antheridia which were at the stage of spermiogenesis had a lower level of DNA in manubria and revealed decreased productivity due to the smaller number of mitotic divisions in antheridial filaments. On the basis of the previous and present data a hypothesis was put forward that in both experiments premature spermiogenesis was caused by a rapid decrease in gibberellin level in the antheridia. PMID- 8617381 TI - Distribution patterns of cytokeratins in epidermis and horny teeth of the adult sea lamprey, Petromyzon marinus. AB - An immunohistochemical characterization of cytokeratins in the skin tissues of the sea lamprey, Petromyzon Marinus was performed using a panel of monoclonal antibodies. Cytokeratins typical for simple epithelia have been detected in the epithelial cells, with a 8/18 pair expression. Granular cells and skein cells showed a labelling of cytokeratins 7, 8, 18 and 19, that is observed also in the non-keratinized layers of the horny teeth. Similar cytokeratins occur in the outermost cell layers of the epidermis; cytokeratin 19 shows a comparatively weaker reaction. These results suggest that the cytokeratin patterns in the above types of cells of adult epidermis are quite different from those in ammocoetes where the expression of cell specific cytokeratins may be correlated with specific programs of epidermal differentiation. PMID- 8617382 TI - Cytokeratin type distribution in the skin and gill epithelia of the Indian freshwater catfish, Heteropneustes fossilis as detected by immunohistochemistry. AB - Based on the general cross-reactivity of the cytokeratins in vertebrates, we describe the immunoreactivity for some mammalian cytokeratins of both the epidermis and gill of H. fossilis. The following monoclonal antibodies, commercially supplied, were tested: K8.13, KL1, AE1 and AE3, which have a wide spectrum of specificity, and LDS-68, M 20, K8.60, KS-B17.2, K4.62, which are more narrowly specific. The reaction of the epithelial cells of the skin to K8.13 was negative in the basal layer, weakly positive in the layers above but strongly positive in some cells of the superficial layer. KL1 was negative in the basal layer, positive in the outer layers. AE1 was strongly positive in the basal layer, negative in the superficial cells. AE3 gave a general but weak reaction in the epithelial cells. K8.60 was negative for the epithelial cells, but reacted positively in the club cells. Club cells also reacted to K8.13 and AE1, and were strongly positive to KL1 and AE3. Goblet mucous cells were negative to all antibodies tested. In the gills, K8.13 labelled cells of both filament and lamellar epithelia. The reaction to AE3 was similar but less intense. KL1 was positive in the basal layer of the filament epithelium but not elsewhere, and K8.60 was negative throughout. AE1 and KS- B17.2 specifically labelled small cells scattered in the filament and lamellar epithelia, which are tentatively identified as neuroendocrine cells. PMID- 8617383 TI - Silver-binding nucleolar organizer regions (AgNORs) in bronchial tumors. AB - Various AgNOR parameters were measured using the computer-aided image analysis in the normal bronchial epithelium (n = 13), in squamous cell carcinoma (n = 25) and in small-cell carcinoma (n = 11). The number and area of AgNOR dots were the highest in squamous cell carcinoma. In small cell carcinoma and in the normal epithelium the values were significantly lower. The number of AgNOR separate locations was higher in tumors in general. The size of single AgNOR dots showed differences between squamous cell carcinoma and small-cell carcinoma. Ratio of AgNOR dot area to nuclear area was similar in various pathological lesions and normal epithelium. There was a positive correlation ( < or = 0.0001) between the number and area of dots, the number of AgNOR separate locations, the area of single AgNOR dots on one hand and nuclear area on the other. PMID- 8617380 TI - Immunohistochemical study of the existence and coexistence of catecholamine synthesizing enzymes and some neuropeptides in perivascular nerve fibres of the main thoraco-cranial arteries in the pig. AB - The presence and pattern of coexistence of catecholamine-synthesizing enzymes and some neuropeptides in nerve fibres supplying thoraco-cranial arteries of the sexually immature gilts were investigated in whole mount preparations. The studied substances included: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (D beta H) (as markers of catecholaminergic nerve fibres), neuropeptide Y (NPY), Leu5-enkephalin (LENK), vasoactive intestinal polipeptide (VIP), calcitonin gene related peptide (CGRP), substance P (SP), galanin (GAL), somatostatin (SOM) and serotonin (5-HT). The arteries were found to be richly supplied by TH/D beta H immunoreactive (TH/D beta H) nerve fibers. Of the neuropeptides studied, NPY (rich innervation), LENK (moderate innervation), VIP (moderate innervation) and CGRP, SP, GAL (only a few nerve fibres) were detected in periarterial nerves. The following patterns of coexistence of the studied substances were found: TH+/D beta H+, TH+/D beta H+/NPY+, TH+/D beta H+/LENK+, TH-/D beta H-/NPY+, TH-/D beta H-/VIP+, TH+/D beta H+/VIP+ (only a few nerve fibres in the cerebral arteries), LENK+/NPY+, LENK-/NPY+, LENK+/NPY-. No SOM and 5-HT-positive structures were observed in the porcine blood vessels. PMID- 8617384 TI - Electron-dense deposits in epididymal cells of rats chronically treated with lead acetate [Pb(II)]. AB - Electron microscopic studies were performed to investigate the influence of chronic lead acetate treatment on morphology of rat epididymis. Dense, lead loaded inclusions were found in the cytoplasm of epididymal principal cells, especially in the caput of epididymis. They were also present, but in smaller amounts, in smooth muscle cells. Usually, the inclusions were located in vacuoles, rarely without any surrounding membrane. Similar lead-containing structures were found in the epididymal lumen. The localization of lead deposits suggests the ability of lead to pass from blood vessels through the epithelial cells of the epididymis to its lumen. It can be therefore postulated, that lead can be eliminated from the male genital tract together with ejaculate. PMID- 8617385 TI - A new approach to the areolar structures in the pig placenta: histochemistry and development of mucous areolae. AB - The pig placenta contains specific structures such as regular and irregular areolae, vesicles and cysts. On the basis of histochemical reactions it can be stated, that irregular areolae, vesicles and cysts differ from the regular areolae. Trophoblast of all the former structures and the substance filling their cavities contain glycosoaminoglycans with sulphuric acid residues (acid mucopolysaccharides). The regular areolae contain PAS-positive glycoproteins and active acid phosphatase. The acid mucopolysaccharides were also observed in the small trophoblast areas with vacuolized cells (TAVC) in the early placenta (18-20 days post coitum). The histochemical features of the investigated structures allow to observe the differentiation and development of TAVC in irregular areolar structures, vesicles and cysts in placentas from the time of implantation until the last day of gestation. Some of these structures fuse with regular areolae forming large irregular areolae described in the literature. The uterine epithelium covering all these structures forms festoon-like folds covered with the brush border. All the structures containing acid mucopolysaccharides can be termed the mucous areolae. PMID- 8617386 TI - Individual model fabrication in maxillofacial radiology. AB - Oral and maxillofacial surgery has long needed a methodology for accurate definition of the third dimension. The introduction of computer-aided tomography in the 1970s provided surgeons with multiple 2-D maps which they themselves had to conceptualize into a third dimension. The later advent of computerized summation of these data made it possible to display a perspective view of the third dimension on a TV monitor. CT, and more recently MRI, with the further analytical refinement afforded by software processing (interactive data presentation, contour detection and summation, hypothetical 3-D construction and interactive visualization) now provide the basic information that is needed for the fabrication of an individual model. Such models can be milled from a variety of materials. More recently, laser-hardened acrylic resins have been shown to be a useful alternative. Both systems are described and their advantages and disadvantages in the planning and performance of oral and maxillofacial surgical procedures are discussed. PMID- 8617387 TI - Does radiographic feature recognition contribute to dentists' diagnosis of pathology? AB - OBJECTIVES: To measure the degree to which dentists can accurately identify radiographic features and use their findings in the diagnosis of three lesions. METHODS: Fourteen radiographic examples were used in this study and five features derived from the literature: border sharpness, border radiopacity, lesion shape, lesion radiopacity and root contact. A 'silver standard' for the presence of the features in the radiographic images of the lesions was established by expert observers; and a diagnostic 'gold standard' based on the histopathology of the lesions was available. Dentists scored each image according to these features and gave their diagnosis. RESULTS: When dentists made different subjective diagnoses for a case, significant differences were found in the extent to which features were reported to be present. The degrees of presence of border sharpness (21%) and border radiopacity (46%) measured using the 'silver standard' were significantly related to the diagnosis of osteosarcoma. CONCLUSIONS: Radiographic information is not used to its full extent and improvement in the recognition of features and the cognitive use of this radiographic information in the diagnostic process could improve diagnostic accuracy. PMID- 8617389 TI - Observer variation in the radiographic assessment of the bone level on the buccal and lingual surfaces of mandibular molars. AB - OBJECTIVES: To assess accuracy and precision of observers' diagnostic performance in locating the buccal/lingual bone level using conventional periapical radiographs. METHODS: Standardized radiographs of molar regions of 10 dried mandibles were obtained. The bone crest on buccal and lingual surfaces of first and second molars was then marked with thin lead foil strips and fresh radiographs taken. A series of increasingly extensive furcation defects was created by sequential bone removal and radiographs with and without markers obtained after each step. The radiographs without indicators were presented to 10 observers on two separate occasions and the most apically located bone level marked on a transparent film. The distance between observers' markings and 'true' bone level on the radiographs was determined. RESULTS: There was wide interobserver variation which increased as the bone loss increased. Lack of consistency was largely attributable to intra-observer variation except when a furcation defect was present. The underestimation of bone loss became more marked as the amount of bone loss increased. CONCLUSIONS: Radiographic determination of bone loss on buccal and lingual surfaces is associated with a considerable lack of accuracy and precision, both of which get worse as bone loss increases. PMID- 8617388 TI - Identification of stroke-prone patients by panoramic and cervical spine radiography. AB - OBJECTIVE: To determine if calcified atherosclerotic lesions in the region of the bifurcation of the common carotid artery (a major cause of stroke) can be identified by conventional panoramic dental radiography. METHODS: All those aged 65 years or older, of 500 consecutive out-patients enrolled in the dental clinic for comprehensive treatment and for whom it was possible to obtain a technically satisfactory panoramic radiograph, were included. The radiographs were evaluated for the presence of radiopacities within the soft tissues of the neck at the level of the lower margin of the third cervical vertebra. An antero-posterior cervical spine radiograph was obtained of those individuals with any such calcifications. Medical histories were obtained from patients with suspected lesions in order to determine the presence of risk factors known to be associated with stroke. RESULTS: Six individuals (4.5% of the final study population) had radiopacities which were confirmed by radiographs of the cervical spine to be calcified atheromas. The medical histories revealed risk factors associated with stroke. CONCLUSION: Panoramic radiographs obtained during the course of routine dental treatment may demonstrate calcified carotid atheromas in patients at risk of stroke. PMID- 8617390 TI - A comparison of six intra-oral X-ray films. AB - OBJECTIVES: To compare objectively and subjectively national-brand dental X-ray films with widely available and less costly brands. METHODS: A range of dental films, including the recently released Kodak Ektaspeed Plus, was purchased from mail-order suppliers at the lowest published price. Objective measurements (film speed, contrast, fog levels, costs, physical characteristics) and subjective evaluations (ease of use, film graininess, overall appearance) were made. RESULTS: Private-label films were obtained at less cost than those of major vendors. Not all film speeds, sizes and wrapping materials were available from individual manufacturers. Objective measures and subjective appraisals showed that Ektaspeed Plus was the fastest film and had good contrast with the clinically useful density ranges. Agfa Dentus was the next fastest film, but it had rather low contrast and was grainier than the other films. The non proprietary brands were difficult to open with primary glove barriers, making effective infection control practices arduous. All films were relatively stable over a 16-week period regardless of storage location. CONCLUSIONS: The major manufacturers offered more choices of film sizes and speeds than did private label suppliers. The films from the major manufacturers, although more expensive, were more conducive to effective infection control practices. Ektaspeed Plus had a performance comparable with or better than the other available films. Based on this preliminary study, it is recommended that this new film is considered as an alternative to D-speed films. PMID- 8617391 TI - Reproducibility of repeat bitewing radiographs determined by measurement of the distance between the amelocemental junction and the alveolar crest: an ex vivo study using human skulls. AB - OBJECTIVE: To assess the reproducibility of repeat bitewing radiographs. METHODS: Five right and left pairs of posterior bitewing radiographs were taken without the use of beam-aiming or film-holding devices in five dry skulls with complete dentitions. The amelocemental junction (ACJ) and alveolar crest (AC) of every interdental site was traced on projected radiographs and the linear distance between the ACF and AC determined. RESULTS: There was close concordance between the ability to read the ACJ on the approximal tooth surface and the AC. The overall readability of interdental sites was 71% although sites at the extremities of radiographs were often unreadable. Kappa analysis of the readability of alveolar bone height from repeat radiographs was 0.52. There were significant differences in the reproducibility of ACJ and AC measurements from repeated radiographs (p<0.001). Intra-examiner variability was significantly less (p<0.001) than that due to repeat radiography with no significant differences between sites. The standard deviation for all surfaces from repeat readings of the same radiographs was 0.12 mm compared with 0.51 mm from repeat radiography. CONCLUSIONS: In this study a freehand technique produced a degree of imprecision in readings of alveolar bone height from successive films of the same site. In clinical terms, a difference of more than 1.4 mm between pairs of serial measurements from the same site would have to occur in order to be certain that the difference was not measurement error but real bone loss. PMID- 8617392 TI - A comparison of three statistics for detecting differences in digitized dental radiographs: a simulation study. AB - OBJECTIVES: Because of methodology-induced structural differences in dental radiographs, determination of change has always depended upon expert interpretation. However, new methods should be able to considerably reduce structured error in digitized subtracted images. Once true change in density is obscured only by random variation in pixel density, statistical methods may be brought to bear on the problem of detecting change. The most appropriate statistic is not obvious, however, since density change can be quantified with respect to both magnitude and dimensional extent. Whereas mean density loss is often intuitively defined as the average density of those pixels losing density (to preclude gaining pixels from offsetting losing pixels), the extent of change may be defined in a variety of ways. In this study, extent was defined as either (a) the total number of pixels losing density, or (b) the size of the largest cluster of losing pixels. The object was to evaluate the comparative statistical power of three possible statistics (based on mean density, number of losing pixels, and size of largest losing cluster) for detecting change. METHODS: In a series of simulations of comparative clinical trials, density was reduced in the centre of 1600-pixel square regions of interest by either one or 10 grey-scale units, and t-tests, based on the three statistics, were then compared for their ability to detect differences. RESULTS: Each of the three statistics was shown to exhibit superior relative power under particular conditions of loss magnitude, loss distribution, and pixel threshold for change. CONCLUSION: Selection of the appropriate statistic for identifying change between radiographs will require further information about the anticipated distribution of density changes for the different disease processes under investigation. PMID- 8617393 TI - Radiographic detectability of periodontal diseases. A comparison of perapical radiography with detailed zonography. AB - OBJECTIVE: To compare the diagnostic accuracy of conventional periapical radiography with detailed zonography using the Scanora system for the detection of periodontal disease. METHODS: We selected for comparison 311 periodontal sites in 165 patients. Five observers independently assessed the periapical radiographs and detailed zonograms for marginal widening of periodontal ligament space, crestal erosion, vertical bone loss, furcation involvement and calculus. RESULTS: ROC analysis revealed no significant differences between the two imaging techniques for either overall or lesion-specific interpretation of periodontal pathology. The sensitivity of periapical radiography was 79%, and of detailed zonography 91% (multiview) and 89% (stereoscopic). Specificities were 82%, 77% and 83%, respectively. CONCLUSION: Detailed zonography performs as well as periapical radiography in the detection of periodontal disease. PMID- 8617394 TI - A dual sensitivity screen system for TMJ image enhancement in cephalometric radiography: sensitometric evaluation. AB - OBJECTIVES: An in vitro evaluation of the image quality of the TMJ OrthoCeph Slimline Cassette System (TOSCS). METHODS: The density response, resolution and clarity of the system were evaluated by use of the characteristic curve, modulation transfer function and root mean square quantum mottle. Relative sharpness was evaluated qualitatively by five observers. RESULTS: Increased density with Trimax 12 gave almost the same contrast as that with Trimax 8 in the diagnostic range, whereas MTF and RMS for Trimax 12 were inferior. CONCLUSIONS: The dual-sensitivity screen system should lead to improved radiographic contrast of the TMJ region in cephalometric radiography. The reduction in sharpness is probably clinically insignificant. PMID- 8617395 TI - An anthropomorphic phantom for periodontal radiography. AB - OBJECTIVE: To construct an anthropomorphic phantom for periodontal radiography. METHODS: The phantom material for the spongiosa was produced by melting polyethylene with calcium hydroxyapatite. Compact bone and teeth were simulated by mixing silicone rubber with calcium hydroxyapatite and calcium carbonate respectively. RESULTS: Radiographs of the phantom show nearly all the structures relevant for periodontal radiography. Measurements of X-ray attenuation and optical density correspond to those of the human mandible. Any defect in the periodontal bone can be reproduced. CONCLUSION: The new phantom will be useful for research and education. PMID- 8617396 TI - Oral presentation of metastatic calcinosis cutis. AB - An 80-year-old man presented with a hard swelling in the lower labial sulcus. Radiological investigation showed a mineralized mass on the labial aspect of an edentulous mandible which on excision, demonstrated the histological features of calcinosis cutis. No other sites seemed to be affected by calcific deposits in this patient, who appears to be the oldest reported case of metastatic calcinosis, and the first intra-oral case, to date. The different forms of calcinosis cutis are highlighted. PMID- 8617397 TI - Thermographic resolution of the prodromal phase of herpes labialis treated with acyclovir. AB - We present a case of recurrent herpes labialis treated with acyclovir. The patient attended within 3 h of the onset of prodrome of the condition, when he appeared clinically normal. Electron infra-fred thermography of the face and lips was undertaken and demonstrated a significant sub-clinical local elevation in temperature of 1.7 degrees C at the site where prodromal symptoms were perceived. Treatment with topical acyclovir cream five times daily was commenced immediately and aborted the developing lesion. Two days after presentation the patient was thermographically normal. To our knowledge this is the first report documenting the abortion of herpes labialis treated with acyclovir where the prodrome was confirmed thermographically. PMID- 8617398 TI - The size of maxillary sinus studied by CT. PMID- 8617399 TI - Chicken thymic nurse cells: an overview. AB - Thymic nurse cells are multicellular complexes located in the subcortical area of the thymus of all avian, mammalian and amphibian species investigated so far. Since their first description in 1980 many studies have been carried out to characterize their morphological and functional properties. The purpose of this review is to summarize recent morphological as well a functional analyses of chicken thymic nurse cells which suggest a role of these cell complexes in T cell selection. PMID- 8617400 TI - In vitro interaction of Perkinsus marinus merozoites with eastern and Pacific oyster hemocytes. AB - This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites. PMID- 8617401 TI - Isolation of a pentraxin-like protein from rainbow trout serum. AB - Serum amyloid P-component (SAP) is a glycoprotein consisting of five or ten noncovalently associated identical subunits of molecular weight 19,000-30,000. Herein we report the isolation and partial characterization of a SAP-like protein from rainbow trout serum. The protein was isolated by calcium-dependent binding to Sepharose followed by ion-exchange and size-exclusion chromatography. Rabbit antibody against human SAP reacted with the trout protein and the NH2-terminal sequence of 16 amino acids showed 60% identity with the first 15 residues of human SAP. SDS-PAGE and endoglycosidase treatment indicated that the trout protein is a glycoprotein in which five or six subunits are linked by disulphide bonds. The subunits have a molecular weight of 37,000 of which approximately 13% is due to carbohydrate. We propose to name the trout protein sulphide linked SAP (SL-SAP). PMID- 8617402 TI - Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity. AB - This study demonstrates for the first time that carp (Cyprinus carpio) neutrophilic granulocytes from the head kidney possess potent spontaneous cytotoxic activity against several human tumor cell lines. Carp head kidney cells isolated at a density of 1.09 g/mL contained more than 90% neutrophilic granulocytes. These cells were round and approximately 10 millimicrons in diameter with reniform or polymorphic nuclei and slightly eosinophilic cytoplasm when stained with Giemsa. Electron microscopy revealed that the cytoplasm contained numerous oval granules, some of which contained a dense rod-shaped core. The neutrophilic granulocytes readily formed conjugates with the human target cells and rapidly killed them. The neutrophilic granulocytes killed human derived target cells better than murine derived target cells. Inhibition of cytotoxicity by catalase suggested that the production of H2O2 is involved as a mediator in the cytotoxic reaction. The size and granularity of the carp effector cells indicate that they are different from the small agranular nonspecific cytotoxic cells (NCC) described in the channel catfish. PMID- 8617403 TI - The in vitro effects of estradiol and cortisol on the function of a long-term goldfish macrophage cell line. AB - We examined the in vitro effects of estradiol and cortisol on the inflammatory function of a macrophage cell line. A dose-dependent inhibition of chemotaxis (0.5 and 0.1 microM, for estradiol and cortisol, respectively) and phagocytosis (1.0 microM for both steroids) were observed after treatment. Estradiol did not affect the nitric oxide production by the macrophage cell line, while cortisol strongly inhibited in vitro nitric oxide production. The steroids did not affect the ability of the macrophages to mount a respiratory burst response as measured by the NBT reduction assay. PMID- 8617404 TI - Monoclonal antibody BLT-1, specific for the bovine homologue of CD5, reacts with the majority of mature T cells, a subpopulation of B cells and stimulates T cell proliferation. AB - Monoclonal antibody (MAb) BLT-1, an IgG2a with kappa light chains, reacted strongly with 21% of bovine thymocytes, weakly with 15% of thymocytes, with a subpopulation of peripheral blood B cells that also expressed CD20 and with peripheral blood T cells. Practically all of the reactive thymocytes were of a large cell subpopulation. By immunoprecipitation, BLT-1 was shown to recognize a membrane molecule with a molecular mass of 67 kDa. In competitive assays for lymphocyte surface binding, BLT-1 and MAb CC-29 (which had been shown previously to react with bovine CD5) blocked one another, indicating that the epitopes recognized were identical or extensively overlapping. In contrast, another CD-5 reactive MAb, CC-17, did not block BLT-1 reactivity with lymphocytes although the reactivity of CC-17 was blocked by BLT-1, suggesting partial overlap of the epitopes or steric hindrance by BLT-1 but not by CC-17. BLT-1 was able to induce proliferation of bovine lymphocytes in culture alone if monocytes were present or in the absence of monocytes synergized with PMA. The results indicate that BLT-1 recognizes an epitope of the bovine homologue of CD5 and that perturbation of the epitope by MAb binding results in signal transduction to bovine lymphocytes. PMID- 8617406 TI - Opioid and anti-opioid peptides. AB - The numerous endogenous opioid peptides (beta-endorphin, enkephalins, dynorphins ... ) and the exogenous opioids (such as morphine) exert their effects through the activation of receptors belonging to four main types, mu, delta, kappa and epsilon. Opioidergic neurones and opioid receptors are largely distributed centrally and peripherally. It is thus not surprising that opioids have numerous pharmacological effects and that endogenous opioids are thought to be involved in the physiological control of various functions, among which nociception is particularly emphasized. Some opioid targets may be components of homeostatic systems tending to reduce the effects of opioids. "Anti-opioid" properties have been attributed to various peptides, especially cholecystokinin (CCK), neuropeptide FF (NPFF) and melanocyte inhibiting factor (MIF)-related peptides. In addition, a particular place should be attributed, paradoxically, to opioid peptides themselves among the anti-opioid peptides. These peptides can oppose some of the acute effects of opioids, and a hyperactivation of anti-opioid peptidergic neurones due to the chronic administration of opioids may be involved in the development of opioid tolerance and/or dependence. In fact, CCK, NPFF and the MIF family of peptides have complex properties and can act as opioid-like as well as anti-opioid peptides. Thus, "opioid modulating peptides" would be a better term to designate these peptides, which probably participate, together with the opioid systems, in multiple feed-back loops for the maintenance of homeostasis. "Opioid modulating peptides" have generally been shown to act through the activation of their own receptors. For example, CCK appears to exert its anti-opioid actions mainly through the activation of CCK-B receptors, whereas its opioid-like effects seem to result from the stimulation of CCK-A receptors. However, the partial agonistic properties at opioid receptors of some MIF-related peptides very likely contribute to their ability to modulate the effects of opioids. CCK- and NPFF-related drugs have potential therapeutic interest as adjuncts to opioids for alleviating pain and/or for the treatment of opioid abuse. PMID- 8617405 TI - An evolutionary conserved target cell antigen along with MHC class I molecules influences susceptibility to murine NK cell lysis. AB - We have previously characterized a novel monoclonal antibody (mAb), termed 18C2, which binds to and inhibits the lysis of target cells by human natural killer (NK) cells. We now show that the anti-target cell mAb 18C2 also recognizes a similar structure on the murine NK sensitive target cell YAC-1, as well as on NK resistant target cells P815 and EL-4, as observed by flow cytometry. Functional studies demonstrated that the mAb 18C2 inhibited the lysis of both NK sensitive YAC-1 target cells, as well as NK resistant target cell lines P815 and EL-4 by freshly-isolated nylon wool nonadherent (NWNA) NK cells, 5-day lymphokine activated killer (LAK) cells and adherent lymphokine activated killer (ALAK) cells. The inhibitory activity of the mAb 18C2 occurred at the target cell level only. Single cell conjugate assays as demonstrated that the structure recognized by the mAb 18C2 was involved in recognition between NK cells and NK target cells, as the mAb inhibited conjugate formation between a variety of effector cells and various target cell lines tested. Further, the role of major histocompatibility complex (MHC) class I antigens in NK cell cytotoxicity was examined. We observed that target cells expressing low levels of MHC class I antigens in association with the novel target cell antigen were more sensitive to NK cell lysis, as compared to cells that co-express higher levels of MHC class I antigen and the target cell antigen. Further, the presence of this antigen across different species suggests this target cell antigen/structure to be highly evolutionarily conserved. PMID- 8617407 TI - Is striatal dopaminergic receptor imbalance responsible for levodopa-induced dyskinesia? AB - Abnormal involuntary movements (dyskinesias) of variable intensity eventually emerge in the majority of Parkinson's disease patients chronically treated with standard oral levodopa. They create social and physical embarrassment and narrow the therapeutic options normally proposed to improve Parkinsonian symptoms. Thus far, indirect clinical and experimental evidence has implicated the potential role of dopamine D1 receptor activation in the generation of dopa dyskinesia. In recent years, our group has tested several dopaminergic agonists of variable half life and selectivity in monkeys rendered parkinsonian following toxic exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These monkeys readily develop dyskinesia when treated with levodopa and provide the best animal model to study this complication. Our results in "drug-naive" and "dyskinesia-primed" MPTP animals suggest that pathological sensitisation of D2 receptor-mediated striatal outflow is necessary and sufficient for the induction of dopa dyskinesia, with perhaps a synergistic contribution from D1 receptors, and that repeated short-lived stimulation is important in the sensitisation process. This model supports the hypothesis that more continuous forms of dopaminomimetic therapy represent the best therapeutic approach for Parkinson's disease and calls for the development of novel D1 agonists for further clinical testing. PMID- 8617408 TI - How do we study autonomic function in humans? PMID- 8617409 TI - Mechanisms of substance P-induced pulmonary oedema in the rabbit: interactions between parasympathetic and excitatory NANC nerves. AB - The pharmacological mechanisms involved in the substance P (SP)-induced pulmonary oedema were studied in isolated perfused rabbit lungs. Substance P induced a dose dependent increase in the capillary filtration coefficient (Kf,c), responsible for oedema. Atropine, hemicholinium-3 and ruthenium red pretreatment partly protected the lungs against SP effects, while tetrodotoxin and hexamethonium did not significantly modify them. (+/-)CP96,345, a NK1 receptor antagonist, completely inhibited the SP-induced increase in the Kf,c. Like SP, acetylcholine (ACh) and capsaicin also increased the Kf,c. Atropine and (+/-)CP96,345 completely blocked the oedema induced by both drugs. Tetrodotoxin and ruthenium red strongly inhibited the response to capsaicin and acetylcholine. It was concluded that SP-induced pulmonary oedema is in part mediated by a stimulating action on cholinergic efferent nerves, with subsequent release of endogenous acetylcholine. Acetylcholine can, in turn, stimulate the release of SP from excitatory non adrenergic, non cholinergic nerves. The effects induced by capsaicin and exogenous acetylcholine, thus endogenous SP, involve tetrodotoxin sensitive mechanisms, while those produced by exogenous SP are tetrodotoxin resistant. PMID- 8617410 TI - Restoration of brain protein synthesis in mature and aged rats by a DA agonist, piribedil. AB - Brain ageing affects numerous cerebral metabolic pathways such as cerebral glucose consumption or protein synthesis rate. The pharmacological effect of a mixed D1-D2 dopaminergic agonist, piribedil, on this last metabolism is reported. Cerebral Protein Synthesis Rate (CPSR) was measured by the [35S]L-methionine autoradiographic procedure in 38 main brain regions of 11 and 26-month-old Wistar rats after a 2-month treatment per os at 9 and 30 mg/kg/day with piribedil. Mean decrease of CPSR was -21% during the 15-month ageing we followed, with important local variations. Mean CPSR increased with the two treatments, +25% in mature and +35% in aged rats. Treatments restored CPSR of aged rats to the exact mature subjects levels in quite all the brain regions. No dose-effect or asymetrical modification was statistically revealed for the two treatments. Metabolic increases involved particularly central brain gray structures, especially some DA targeted brain nuclei concerned with behaviour and learning. This effect argued for a general metabotrophic effect of D1-D2 dopamine stimulation of the brain. The original pattern of local ageing of brain protein synthesis in rat was also incidentally reported. This was the first direct report of a wide and effective metabolic activation of CPSR in the brain during ageing by a curative dopaminergic agonist treatment. PMID- 8617411 TI - Are vasopressin peripheral V1 receptors involved in the development of malignant hypertension and stroke in SHR-SPs? AB - The aim of this study was to determine whether activation of vasopressin (AVP) peripheral V1 receptors is involved in the development of malignant hypertension, stroke, and end-organ damage in stroke-prone spontaneously hypertensive rats (SHR SPs). For this purpose, young salt-loaded SHR-SPs were treated orally daily from their 5th to 34th week of age, by a selective AVP V1 receptor antagonist, SR 49059, used in a dose (30 mg/kg) that achieved complete peripheral V1 receptor blockade. Untreated SHR-SPs served as controls. SR 49059 slightly and transiently (8th to 10th week of age) limited the rise in blood pressure, but thereafter systolic blood pressure values were similar in the two groups of SHR-SPs. Stroke related mortality was not significantly different in SR 49059-treated and in control animals (65% vs 65% at 30 weeks, 65% vs 83% at 34 weeks). SR 49059 did not prevent the increases in fluid intake, diuresis and proteinuria seen in controls. Histological examination of the brain, kidneys and heart revealed that the development of fibrinoid necrosis and arterial thickening was not prevented by SR 49059, nor was that of malignant nephroangiosclerosis and of myocardial infarction and fibrosis. These data strongly suggest that AVP peripheral V1 receptor activation is not involved in the pathological processes that develop in SHR-SPs. PMID- 8617412 TI - Role of the renin-angiotensin system in systemic and regional vascular responses to orthostatic stress in healthy volunteers. AB - The effects of a single oral dose (5 mg) of the angiotensin-converting enzyme (ACE) inhibitor, ramipril, on the systemic and regional vascular responses to simulated orthostatic stress by the lower body negative pressure (LBNP) technique were investigated in eight healthy volunteers, in a double-blind, placebo controlled crossover study. Arterial blood pressure remained unchanged throughout the study. Ramipril increased significantly forearm (venous occlusion plethysmography, + 37% +/- 4% from 1.98 ml.min-1.100 ml-1) and renal (PAH clearance, + 6 +/- 2% from 1.20 1.min-1) blood flows and decreased corresponding vascular resistances, 150 minutes after its administration and before LBNP. It also significantly reduced calculated filtration fraction and inhibited by approximately 86% plasma ACE activity. Lower body negative pressure at -10 and 20 mmHg induced a progressive and significant decrease in central venous pressure and significant increases in forearm, splanchnic (indocyanine green clearance) and total peripheral vascular resistances which were of the same magnitude after ramipril and placebo administrations. Ramipril blunted the LBNP-induced increase in renal vascular resistance observed at -20 mmHg after placebo but a similar increase in glomerular filtration rate (inulin clearance) was observed at LBNP-10 and -20 mmHg after ramipril and placebo. Calculated filtration fraction increased after placebo (LBNP-10 mmHg) and ramipril (LBNP-20 mmHg). Finally, LBNP-induced changes in biological parameters were similar after ramipril and placebo at all levels of LBNP. Thus, ramipril does not interfere with the adaptive forearm and splanchnic vascular responses and preserves renal hemodynamics during orthostatic stress simulated by LBNP in healthy volunteers. PMID- 8617413 TI - Is there a relationship between antipsychotic blood levels and their clinical efficacy? An analysis of studies design and methodology. AB - There are now more than 50 studies concerning neuroleptic blood levels and clinical outcome relationships. Haloperidol, the most studied, is the only antipsychotic permitting some conclusions. A number of authors suggest that the striking lack of agreement between different studies results from heterogeneity of their quality. Here, we have used a scoring system for assessing the quality of those studies. According to this system, none (0/14) of the studies having a score < 0.60 was able to show a therapeutic window, as compared to 53% (10/19) of those having a score > or = 0.60 (p = 0.002, Fisher exact test). Also, the studies able to identify the presence of a therapeutic window during haloperidol treatment were those having sample size > 20 (p = 0.06) and those whose patients were treated with fixed doses (p = 0.02). The diagnosis of schizophrenia in the studies seems not to be an exclusive condition, as compared with those also including schizophreniform and schizoaffective disorders (p = 0.12). Our qualitative analysis of haloperidol blood level publications seem to indicate that an upper limit may exist for haloperidol efficacy; values above this limit seem not to provide any supplementary clinical improvement and may even reduce therapeutic effect. PMID- 8617414 TI - The safety of nitrofurantoin during the first trimester of pregnancy: meta analysis. AB - Asymptomatic bacteriuria is common during pregnancy and may adversely affect both the mother and her fetus. Nitrofurantoin (NF) has been long recognized as an effective agent in both nonpregnant and pregnant women suffering from urinary tract infections. This meta-analysis was conducted in order to evaluate the safety of NF ingested during early pregnancy. Of twenty-two studies, only four met the inclusion criteria and their analysis could not demonstrate any significant correlation between NF ingestion and fetal malformation. The pooled odds ratio was 1.29 with 95% confidence interval 0.25-6.57. Although the number and quality of the studies included are limited, we thought it important to present the existing data. More extensive controlled studies are urgently needed in order to increase the significance of our study. PMID- 8617415 TI - The "optional cross-over design" for randomized controlled trials. AB - In order to facilitate the performance of randomized controlled trials (RCT) in situations where no "hard" endpoints can be identified and the patient's subjective impressions about success of failure of a given treatment are paramount, the "optional cross-over design" is suggested. In this design, patients are randomised to receive either active medication or placebo during phase I. At its end the patient may choose to change to the other treatment arm, if therapy was felt to be unsuccessful (= optional cross-over). In phase II, treatment continues as in phase I except for those patients who have chosen the "optional cross-over". Further cross-over points may ensue depending on the particularities of the situation, however, two such options may be adequate for most studies. At the end of the trial period, the numbers of patients in each treatment arm are counted and evaluated statistically. If an optimally successful remedy is being tested, close to 100% of the study population could finish up in the active treatment arm. If, as in most instances, the remedy is not optimally successful, this percentage will be proportionally less. If an ineffective remedy is being tested, the distribution of the total sample within the two treatment arms approaches 50:50%. The "optional cross-over design" seems suited for RCT in areas where the complex, unmeasurable and subjective experience of the patient are considered to represent adequate endpoints. PMID- 8617416 TI - A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B. AB - To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre S2 region before clearance of HBsAg. After the appearance of circulating anti HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance. PMID- 8617417 TI - Hepatitis B and C coinfections and persistent hepatitis B infections: clinical outcome and liver pathology after transplantation. AB - Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are common complications after orthotopic liver transplantation (OLT), but the liver pathology and clinical outcomes of HBV infection with HCV coinfection have not been thoroughly examined. In this study, we used the polymerase chain reaction (PCR) to detect HBV and HCV in pre- and post-OLT sera of 38 patients and correlated the findings with clinical outcome and liver pathology. Of 13 patients who were HBV and HCV negative before OLT, 9 acquired HBV infection, and 4 developed acquired HBV and HCV coinfections after OLT. Persistent HBV infections were present in 10 patients. Three patients with pre-OLT HBV infections developed persistent HBV and acquired HCV coinfections after OLT; 5 with pre-OLT HCV infections developed acquired HBV and persistent HCV coinfections after OLT, and 7 had persistent HBV and HCV coinfections before and after OLT. Portal/periportal inflammation was the same in all groups; however, lobular inflammation and fibrosis were more severe in patients with persistent HBV infections and in those with acquired HBV and HCV coinfections. Two major histopathological patterns were present in patients with HBV and HCV coinfections, one with predominant features of HCV infection, and the other with those of HBV infection. Patients with post OLT HBV and HCV coinfections had survival rates similar to those with acquired HBV infection, whereas patients with persistent HBV infections experienced more allograft loss caused by chronic hepatitis or fibrosing cytolytic hepatitis, and had a more dire clinical outcome than the others. Although the limited numbers reported in this study prevent a definitive conclusion, our data suggest that in patients with HBV and HCV coinfections, the presence of HCV may improve the clinical outcome as compared with the expected outcome of persistent HBV infection alone. PMID- 8617418 TI - Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus. AB - There have been numerous reports suggesting that human peripheral blood mononuclear cells (PBMCs) can be productively infected with human hepatitis B virus (HBV). We therefore examined whether the PBMCs can be used to establish an in vitro infection system for HBV. Freshly purified PBMCs were incubated with HBV with or without mitogen stimulation. Successful infection was tested using a newly developed PCR method that can differentiate between the relaxed circular (RC) DNA of the virus inoculum and the covalently closed circular (CCC) DNA which is formed only after successful virus entry. This method enables virus uptake to be proven even if the infection is abortive because there is no gene expression because of the lack of liver specific gene expression factors. All attempts to detect CCC DNA after incubation of PBMCs with HBV failed. On the contrary, CCC DNA could easily be detected in infected liver or after in vitro infection of primary human hepatocytes. Because this result appeared to be contradictory to the published data, we analyzed PMBCs isolated from infected patients. We could confirm that HBV DNA and RNA are associated with these cells. However, even after restimulation with mitogens, we could only detect RC DNA. Moreover, we could also demonstrate that viral RNA is present in free virus. Apparently, a certain amount of defective particles do not reverse transcribe the packaged pregenomic RNA. In summary we found no evidence that PMBCs can be infected with HBV and conclude that all previous observations can be explained by adsorbed virus. PMID- 8617419 TI - Hyperinsulinemia in preascitic cirrhosis: effects on systemic and renal hemodynamics, sodium homeostasis, forearm blood flow, and sympathetic nervous activity. AB - Insulin has been shown to be vasodilatory and antinatriuretic and to stimulate sympathetic nervous activity independent of hypoglycemia in healthy normal subjects. It is hypothesized that hyperinsulinemia, which is commonly observed in cirrhosis, may in part be responsible for the systemic vasodilatation, sympathetic activation, and sodium retention in these patients. The aims of this study, in preascitic cirrhotics, were as follows: (1) to document baseline hyperinsulinemia and its effects on sodium handling, forearm and renal circulations, and sympathetic nervous activity; (2) to determine if pharmacological increases in plasma insulin levels would result in an exaggeration of these physiological effects. Seven male, nonobese, well compensated, preascitic cirrhotic patients were studied, after being maintained on a 150 mmol sodium per day diet for 7 days, firstly at baseline level, followed by increasing doses of insulin from 10 to 1,200 mU/m2/min using the euglycemic clamp technique. Systemic and renal hemodynamics, urinary sodium excretion, plasma norepinephrine, and forearm blood flow (FBF) were measured at the end of baseline and each hyperinsulinemic period. Baseline measurements in the cirrhotics, when compared with our laboratory standards obtained from a comparable group of male healthy normals, showed significant hyperinsulinemia (P=.01), associated with significantly higher FBF (P=.02), and glomerular filtration rate (GFR) (P=.02), as well as significantly reduced urinary volume (P=.04) and fractional excretion of sodium (P=.04). Insulin infusions in the cirrhotics produced no further sodium retention, but further forearm vasodilatation occurred at doses > or = 10 mU/m2/min. In contrast, there was no further renal vasodilatation except at very high pharmacological levels of insulin together with an unchanged GFR, natriuresis, and diuresis. Hyperinsulinemia produced no significant effects on the sympathetic nervous activity. In conclusion, these results suggest that hyperinsulinemia may be implicated in the glomerular hyperfiltration and sodium retaining tendency of preascitic cirrhotic patients with glucose intolerance. The ability of the kidneys to escape from the sodium retaining effects may serve as an in-built physiological regulatory mechanism on sodium homeostasis. PMID- 8617420 TI - Fibronectin in human bile fluid for diagnosis of malignant biliary diseases. AB - In a preliminary study, we demonstrated a strong association between the concentration of the glycoprotein fibronectin (FN) in human bile fluid and the presence of malignant biliary diseases. We now present the results of measurements of total FN (tFN) and cellular FN (cFN) within a larger group of 71 patients. Bile fluid was collected during routine endoscopic retrograde cholangiography or by transhepatic puncture, respectively, from patients admitted for examination/treatment of biliary obstruction. Determination of tFN in bile was performed using a previously described time-resolved fluorescence immunoassay (TRFIA). For cFN, a newly developed TRFIA, using a specific monoclonal antibody for the EDA epitope of cFN, was applied. Within the noncarcinoma group of patients (n=50), consistently low concentrations of tFN (median = 5 ng/mL) were found. In most of these cases, the corresponding concentrations of cFN were below the detection limit (2.6 ng/mL) of this assay. Highly significantly elevated concentrations were found for both tFN (median = 1,220 ng/mL) and cFN (median = 243 ng/mL) in the carcinoma group (n = 21) in comparison with the noncarcinoma group (P < or = .01). By adopting cutoff values of 60 ng/mL for tFN and >0 ng/mL for cFN, diagnostic sensitivities for carcinoma of the biliary tract of 0.89 and 0.92, and specificities of 0.96 and 0.98, respectively, were computed. FN in bile fluid is suggested as a sensitive, specific, and easily determined marker for differential diagnosis of malignant and benign diseases of the biliary tract. PMID- 8617421 TI - Interphase cytogenetic studies of human hepatocellular carcinomas by fluorescent in situ hybridization. AB - Although numerous allelic chromosome losses have been reported in hepatocellular carcinomas (HCC), chromosome analysis by cytogenetic methods has rarely been performed in these tumors, unlike other solid malignant tumors. The purpose of the current study was to analyze primary liver tumors by conventional cytogenetic methods and by a new molecular cytogenetic technique, called fluorescent in situ hybridization (FISH), a technique that has been recently proposed to count the number of chromosome copies in interphase nuclei with chromosome centromeric probes. Primary cultures of tumoral cells were prepared to obtain metaphases. Specific chromosomes probes 7, 17, and 20 were used to perform in situ hybridization on isolated intact tumoral cells. Seven cases of primary liver tumors (six cases of HCC and one case of benign focal hepatic nodular hyperplasia) were investigated. A few metaphases were obtained in five of the seven tumors, and in most cases numerical abnormalities were difficult to interpret. In contrast with in situ hybridization, all cases of HCC showed losses and/or gains of chromosomes. Loss of one to three chromosomes occurred in five tumors. A gain of two chromosomes was observed in two of these five tumors. In only one case, a gain of only three chromosomes occurred. In addition, a loss of chromosome 17 was recorded for the benign tumor. These results demonstrate that FISH with specific probes can provide information on chromosome number in the tumoral cells of primary liver tumors even in the absence of analyzable metaphases. This technique opens new possibilities for the investigation of chromosome abnormalities in HCC. PMID- 8617422 TI - Epitope mapping and reactivity of autoantibodies to the E2 component of 2 oxoglutarate dehydrogenase complex in primary biliary cirrhosis using recombinant 2-oxoglutarate dehydrogenase complex. AB - Five different target mitochondrial autoantigens recognized by sera from patients with primary biliary cirrhosis (PBC) have been identified as subunits of the following 2-oxo acid dehydrogenase complexes: the pyruvate dehydrogenase complex (PDC), the branched chain 2-oxo acid dehydrogenase complex (BCOADC), and the 2 oxoglutarate dehydrogenase complex (OGDC). Unlike the E2 subunits of PDC (PDC-E2) and BCOADC (BCOADC-E2), the E2 subunits of OGDC (OGDC-E2) reactivity of PBC sera and the reactive epitope of OGDC-D2 have not hitherto been studied in detail. In this report, we took advantage of a recombinant fusion protein for OGDC-E2 to address these issues. Eighty of 268 (29.9%) PBC patient sera but none of the 45 controls reacted with recombinant OGDC-E2. The recombinant OGDC-E2 was judged to express the immunodominant epitope, because when sera from patients with PBC were preabsorbed with the recombinant fusion protein, such sera were depleted of reactivity against 48 kD OGDC-E2 when probed on beef heart mitochondria (BHM) but retained reactivity toward PDC-E2 and/or BCOADC-E2. Furthermore, affinity purified PBC sera against recombinant OGDC-E2 reacted only with native OGDC-E2 and not with any other enzyme components of the 2-oxo acid dehydrogenase complex. Antimitochondrial autoantibodies (AMA) against OGDC-E2 included immunoglobulin (Ig)G2, IgG3 and IgM and the relative titers were as follows: IgG2 > IgG3 > IgM. Finally, using overlapping recombinant polypeptides, it was determined that a minimum of 81 amino acids (residues 67-147) corresponding to the lipoyl domain of OGDC-E2 are necessary for reactivity, suggesting that a conformational autoepitope is recognized by AMA. These data suggest that each of the 2-oxo acid dehydrogenase enzymes has distinct antigenicity despite their similarities in structure and function. The availability of recombinant OGDC-E2 autoantigen will allow the design of additional studies to further our understanding of the role of mitochondrial autoantigens in the pathogenesis of PBC. PMID- 8617423 TI - Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody. AB - Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem LiALP in the circulation. Several other membrane-bound enzymes, such as gamma glutamyltransferase (gamma-GT), leucine aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP or similar lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments. PMID- 8617424 TI - Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumor. AB - Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. However, the relationship between the vascularity of HCC and the expression of angiogenic factors has not been investigated. In addition, no detailed studies have examined the possible involvement of angiogenic factors in the grade of malignancy of HCC. The aim of this study was to determine which angiogenic factors regulate tumor angiogenesis and contribute to the invasive ability of liver tumors, especially of HCC. Northern blot analysis was used to examine the transcriptional expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and acidic FGF in resected surgical specimens (20 HCC and 9 metastatic liver tumors). Correlations between messenger RNA (mRNA) expression and arteriographic findings, as well as histopathological findings, were evaluated. Immunohistochemistry was performed to identify the localization of cells expressing VEGF in HCC. Higher levels of VEGF mRNA were observed in 12 of 20 HCC and 2 of 9 metastatic liver tumors than in corresponding nontumorous tissues. The degree of VEGF mRNA expression was significantly correlated with the intensity of tumor staining in angiograms (P<.01). On immunohistochemical observation, VEGF protein was intensely detected in HCC cells. Furthermore, basic FGF mRNA was detected in 9 of 20 HCC and was related to the capsular infiltration of cancer cells (P<.05). In contrast, no significant difference was observed in the very low levels of acidic FGF mRNA found in the tumorous and nontumorous portions of the liver. In conclusion, these results suggest that VEGF contributes to angiogenesis of liver tumors, whereas basic FGF may be involved in the invasion of HCC into the surrounding tissues. PMID- 8617425 TI - Fulminant hepatic failure caused by ecarazine hydrochloride (a hydralazine derivative). AB - The cause of fulminant hepatic failure is reported to be unknown in more than half the cases in Japan. We recently reviewed 23 cases of fulminant hepatic failure that had been treated at our hospital. The cause of disease had been regarded as unknown before this study. It was found that seven of these patients had been under ecarazine hydrochloride therapy when they developed fulminant hepatic failure. We examined the reasons why fulminant hepatic failure in these seven patients had not been previously attributed to ecarazine, and found that it could be explained by the following factors: (1) the time from the start of ecarazine therapy to the onset of hepatic failure was long; (2) in all cases, hepatic failure developed more than 10 days after the clinical recognition of hepatitis; and (3) characteristic signs of drug-induced hepatic failure such as a skin rash and positive lymphocytes stimulation test with the drug were absent in all cases. Fulminant hepatic failure in these cases could be characterized by: (1) rapid decrease in serum alanine transaminase (ALT) level after discontinuation of ecarazine, (2) prolonged jaundice despite discontinuation of ecarazine, (3) high incidence of anti-nuclear antibody (ANA) (57%), and (4) histological findings of extensive hepatocellular necrosis ranging from bridging necrosis to massive necrosis. Of the seven patients, four died of fulminant hepatic failure. These four patients had received high doses of ecarazine hydrochloride for prolonged periods. Our data suggest that there may be many cases in which the cause of fulminant hepatic failure or acute hepatitis was not previously determined that can be attributed to long-term drug therapy for chronic diseases. PMID- 8617426 TI - Presence of autonomic neuropathy is a poor prognostic indicator in patients with advanced liver disease. AB - Autonomic neuropathy (AN) is seen in both alcohol-induced and non-alcohol-induced liver disease, and when present is an independent predictor of mortality. We postulated that patients who were awaiting liver transplantation are likely to have a high prevalence of autonomic neuropathy with an associated increase in mortality. To test our hypothesis, we evaluated the presence of autonomic neuropathy using a battery of tests in 33 patients awaiting liver transplantation and prospectively followed them to determine their prognosis. Twenty-two of 33 (67%) patients with liver disease had evidence of autonomic neuropathy; of these, 12 (36%) had evidence of definite and 10 (31%) had early autonomic neuropathy. The prevalence of AN was similar in alcohol-induced and non-alcohol-induced liver disease. Using Child-Pugh classification, 14.3% Child A, 31.3% Child B, and 60% Child C had definite autonomic neuropathy. Six patients died during a median observation period of 10 months, and all had AN. Kaplan-Meier survival analysis showed a significantly higher mortality (P=.05) in patients with AN. On the basis of this observation, we suggest that consideration should be given for early liver transplantation in patients with advanced liver disease and autonomic neuropathy. PMID- 8617427 TI - Identification of bipotential progenitor cells in human liver development. AB - Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks' gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks' gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks' gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks' gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks' gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver. PMID- 8617428 TI - Studies of liver repopulation using the dipeptidyl peptidase IV-deficient rat and other rodent recipients: cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule. AB - The feasibility of liver repopulation with hepatocytes has been shown, although clinical applications demand significant hepatic replacement. To show whether portal vascular bed in large animals could accomodate a greater cell number, we analyzed liver repopulation in syngeneic Fischer 344 rats deficient in dipeptidyl peptidase IV. This system allowed localization of transplanted normal hepatocytes in liver or various ectopic sites, as well as dual studies for analysis of gene expression. Interestingly, the product of a dipeptidyl peptidase IV substrate inactivated bile canalicular adenosine triphosphatase (ATPase) activity in normal but not in dipeptidyl peptidase IV-deficient rats, which allowed localization of dipeptidyl peptidase IV-deficient hepatocytes in normal rat liver for additional reversed transplantation systems. Further studies with genetically marked cells showed that because of the size difference between hepatocytes and portal vein radicles, intrasplenically transplanted cells were distributed in periportal areas (zone 1) in mice, whereas in larger animals (rats or rabbits) cells were also distributed downstream to midlobular (zone 2) or perivenous (zone 3) areas. Transplantation of an escalating number of hepatocytes showed that adult rats tolerated intrasplenic injection of a large cell number in single sessions (up to 1 X 10(8), approximately 10% to 15% of the host hepatocyte mass). Morphometric analysis of recipient livers showed survival of a significantly greater cell number with incorporation in host liver plates. At 4 weeks, transplantation of 2 x 10(7) hepatocytes into adult rats led to a survival of 1.4 +/- 1.0 x 10(6) transplanted cells/cm3 liver, whereas after transplantation of 5 x 10(7) cells or 7.5 x 10(7) cells, the number of surviving transplanted cells in the liver significantly increased to 4.1 +/- 1.4 x 10(6) transplanted cells/cm3 liver (mean, 2.9-fold; P<.003) and 5.5 +/- 1.3 x 10(6) transplanted cells/cm3 liver (mean, 3.9-fold; P<.003), respectively. When cells were injected in greater numbers, transplanted hepatocytes retained normal function and produced more serum albumin or hepatitis B surface antigen in deficient hosts. These data indicate the feasibility in larger animals of significant liver repopulation with hepatocyte transplantation. Use of dipeptidyl peptidase IV-deficient rats should help further analysis of mechanisms in liver repopulation. PMID- 8617429 TI - Ethanol feeding of micropigs alters methionine metabolism and increases hepatocellular apoptosis and proliferation. AB - Chronic alcoholism is associated with increased cancer risk that may be related to ethanol-induced alterations in methionine and deoxynucleotide metabolism. These metabolic relationships were studied in micropigs fed diets for 12 months that contained 40% ethanol or cornstarch control with adequate folate. Ethanol feeding altered methionine metabolism without changing mean terminal liver folate levels. After initial equilibration to diet, ethanol feeding significantly increased monthly serum homocysteine levels while reducing serum methionine levels over the time course of the experiment. After 12 months, hepatic methionine synthase activity and the ratio of S-adenosylmethionine (SAM) to S adenosylhomocysteine (SAH) were significantly reduced in ethanol-fed animals, whereas the ratio of liver deoxyuridine triphosphate (dUTP) to deoxythymidine triphosphate (dTTP) was increased and correlated inversely with methionine synthase activity. These findings were associated with increased frequency of hepatocytes with apoptotic bodies and positivity for proliferating cell nuclear antigen (PCNA) in livers from ethanol-fed minipigs. These studies suggest that chronic ethanol feeding perturbs methionine metabolism by impairment of methionine synthase activity, resulting in deoxynucleoside triphosphate (dNTP) imbalance, increased apoptosis, and regenerative proliferation. These biochemical alterations may provide a promoting environment for carcinogenesis during long term ethanol exposure. PMID- 8617430 TI - The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. AB - The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and have shown that this injury induces the production and release of hepatic-derived tumor necrosis factor alpha (TNF alpha), which mediates, in part, local liver injury following hepatic reperfusion. In the present study, we have extended these previous observations to assess whether an interrelationship exists between TNF-alpha and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein, that may account for some of the pathology of neutrophil-mediated ischemia/reperfusion-induced liver injury. We observed that hepatic ischemia/reperfusion injury leads to: (1) a coincident increase in hepatic neutrophil sequestration, elevated serum alanine aminotransferase (ALT) levels, and hepatic production of epithelial neutrophil activating protein; (2) passive immunization with neutralizing antibodies to TNF-alpha resulted in significant suppression of hepatic-derived epithelial neutrophil activating protein; and (3) neutralization of epithelial neutrophil activating protein by passive immunization significantly attenuated neutrophil sequestration in the liver and serum ALT levels. These findings support the notion that local expression of hepatic epithelial neutrophil activating protein produced in response to TNF alpha is an important mediator of the local neutrophil-dependent hepatic injury associated with hepatic ischemia/reperfusion. PMID- 8617431 TI - Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine. AB - The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. PMID- 8617432 TI - Exaggeration of acute liver damage by hepatic sympathetic nerves and circulating catecholamines in perfused liver of rats treated with D-galactosamine. AB - Effects of electrical stimulation of the hepatic nerves on acute liver damage were examined using isolated rat liver perfused in situ, 24 hours after intraperitoneal injection with D-galactosamine (800 mg/kg). The leakage of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the liver was used as markers of acute liver damage. In perfused livers after treatment with galactosamine, nerve stimulation (20 V, 20 Hz, 2 ms) increased the leakage of LDH and AST about 3-fold over the basal level accompanied by the decrease in flow rate, whereas with control livers the leakage of LDH and AST into the effluent was almost undetectable throughout the perfusion. The rapid increase in the leakage of LDH and AST was observed during nerve stimulation even under conditions where perfusion flow was maintained constant. Such effects of hepatic nerve stimulation on galactosamine-treated livers were mimicked well by infusion of noradrenaline or phenylephrine, and inhibited by the alpha1-antagonist bunazosin. Artificial reduction of perfusion flow alone did not induce the rapid leakage of LDH and AST into the effluent. On the other hand, low concentration (10 nmol/L) of noradrenaline only minimally decreased the flow rate but apparently augmented liver cell damage. The acute liver damage augmented by noradrenaline was dependent on extracellular Ca2+. These results indicate that in the liver, already having been injured slightly, the activation of hepatic sympathetic nerves and circulating catecholamines exaggerates acute liver damage through an action on liver cells, which depends on the influx of extracellular Ca2+. PMID- 8617433 TI - Release of soluble intercellular adhesion molecule 1 into bile and serum in murine endotoxin shock. AB - Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mac-1 (CD11b/CD18) on neutrophils and its counterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule 1 (ICAM-1). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 micrograms/kg Salmonella abortus equi endotoxin alone or in combination with 700 mg/kg galactosamine. In endotoxin-sensitive mice (C3Heb/FeJ), injection of endotoxin did not cause liver injury but induced a time dependent increase of sICAM-1 in serum (300%) and in bile (615%) without affecting bile flow. In galactosamine/endotoxin-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%, respectively. In either case, the increase in sICAM-1 concentrations paralleled the enhanced ICAM-1 expression in the liver. The endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile after endotoxin administration. In contrast, the intravenous injection of murine tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) or IL-1 beta (13-23 micrograms/kg) into endotoxin-resistant mice induced a 225% to 364% increase in serum sICAM-1 and a 370% elevation of the biliary efflux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation during endotoxemia in vivo. The described experimental model can be used to investigate the role of sICAM-1 in the pathophysiology of inflammatory liver disease. PMID- 8617434 TI - Effects of long-term administration of octreotide in portal vein-stenosed rats. AB - The hemodynamic effects of long-term administration of octreotide in portal hypertension has not been established. In addition, whether long-term octreotide treatment prevents the development of portosystemic shunts has not yet been evaluated. Hence, the current study was undertaken to evaluate the effects of long-term administration of octreotide in rats with portal vein stenosis. Immediately after portal vein stenosis or sham operation, rats were given either a long-term octreotide administration of 100 micrograms/kg or a placebo every 12 hours by subcutaneous injection for 14 consecutive days. Systemic hemodynamics and regional blood flows, degree of mesenteric-systemic shunts, and plasma glucagon concentrations were measured after the final dose of octreotide or placebo. A fifth group of portal vein-stenosed rats received hemodynamic and plasma glucagon measurements after 1-day octreotide treatment given at 14 days after surgery. Long-term octreotide treatment modified the hyperdynamic circulation without affecting the degree of mesenteric-systemic shunts, and 1-day octreotide treatment decreased portal tributary blood flow without affecting the portal pressure, systemic hemodynamics, and degree of mesenteric-systemic shunts. Plasma glucagon levels were decreased in portal vein-stenosed rats receiving either long-term or 1-day octreodtide compared with rats receiving placebo. In contrast, chronic octreotide treatment did not affect any of the hemodynamic values or plasma glucagon levels in sham-operated rats. In conclusion, long-term administration of octreotide modified in part the development of portal hypertension and hyperdynamic circulation in portal vein-stenosed rats without affecting the degree of mesenteric-systemic shunts. PMID- 8617435 TI - Inhibition of 5-lipoxygenase promotes the regeneration of the liver after partial hepatectomy in normal and icteric rats. AB - The role of leukotriene (LT) on liver regeneration after hepatectomy is still unknown. LTB4 stagnates in the liver with obstructive jaundice, because LTB4 is excreted in the bile; therefore, LTB4 may have an effect on liver regeneration after hepatectomy with obstructive jaundice. Release of obstructive jaundice and simultaneous 70% hepatectomy was performed in rats to study the effect of 5 lipoxygenase inhibitor (AA-861) on liver regeneration. Group 1 underwent hepatectomy with administration of 0.1 mL dimethyl sulfoxide (DMSO), group 2 underwent hepatectomy with administration of AA-861 (20 mg/kg/d) dissolved in 0.1 mL DMSO, group 3 underwent hepatectomy with administration of AA-861 (40 mg/kg/d) dissolved in 0.1 mL DMSO, group 4 underwent release of obstructive jaundice and hepatectomy with administration of 0.1 mL DMSO, and group 5 underwent relief of obstructive jaundice and hepatectomy with administration of AA-861 (20 mg/kg/d). DMSO or AA-861 was administered 24 hours before, during, and 24 hours after hepatectomy in each group. Whole blood LTB4 and serum alanine aminotransferase (ALT), total bilirubin, and bromodeoxyuridine labeling index (LI) were measured before and after hepatectomy. The LTB4 level increased during obstructive jaundice and after hepatectomy. LTB4 and serum ALT levels were significantly lower after hepatectomy in the rats that were administered AA-861, and a significantly higher LI was observed at 24 hours after hepatectomy in rats receiving AA-861. Inhibition of 5-lipoxygenase promotes liver regeneration and decreases hepatocyte injury after hepatectomy associated with obstructive jaundice. PMID- 8617436 TI - Changes in liver membrane potentials after partial hepatectomy in rats. AB - Changes in potential differences (PD) across hepatocyte membranes after partial hepatectomy may play an important role in hepatic homeostatic mechanisms and regenerative activity. To date, few studies have attempted to describe the extent and duration of such changes. In the present study, we documented PD values immediately before and for a 24-hour period after a partial hepatectomy (PHx) of 70% in healthy adult rats. We also documented PD changes after 30% and 90% PHx and PD values in different lobes of the same liver before and after PHx. Our findings showed that the liver depolarizes promptly (within 5 minutes of PHx) and that changes in PD are sustained for approximately 18 hours before returning to baseline values. We also observed that the magnitude of hepatic depolarization correlates with the extent of PHx. Finally, we did not observe regional differences in PD recordings from various lobes before and after PHx. These findings enhance our understanding of the physiological changes that occur in regenerating livers after PHx in rats. PMID- 8617437 TI - The herbal medicine Inchin-ko-to inhibits liver cell apoptosis induced by transforming growth factor beta 1. AB - Transforming growth factor beta 1 (TGF-beta 1) is reported to play an important role in the induction of liver cell apoptosis. In this report, we demonstrate that TGF-beta 1 induces apoptosis in a rat Morris hepatoma McA-RH8994 cell line and rat primary cultured hepatocytes at similar doses and in a similar manner. Using McA-RH8994 cells, we screened a number of chemical reagents, aqueous extracts of crude drugs, and herbal medicines for their inhibitory activities on TGF-beta 1-induced apoptosis. The results indicate that Artemisiae capillaris spica (ACS) and the ACS-containing herbal medicine Inchin-ko-to, which are used for treatment of various liver disorders, exhibited the most potent anti apoptotic activity. Various chemicals that were reported as inhibitors of apoptosis in other experimental systems showed no evident activity. By contrast, two of nine ACS ingredients we tested, capillin and capillene, showed activity at concentrations of submicrogram per milliliter. The inhibitory effects of Inchin ko-to, capillin and capillene were also confirmed on TGF-beta 1-induced apoptosis of rat primary cultured hepatocytes. Inhibition of undesired apoptosis induced by TGF-beta 1 is expected to be beneficial for the treatment of various inflammatory liver diseases. Our findings therefore suggest the possibility that therapeutic effects of Inchin-ko-to on liver diseases might be associated with its inhibitory activity on TGF-beta 1-induced liver cell apoptosis. PMID- 8617438 TI - alpha2-Macroglobulin is mainly produced by cancer cells and not by hepatocytes in rats with colon carcinoma metastases in liver. AB - Localization and production of alpha2-macroglobulin (alpha2M), a multifunctional binding protein with protease and cytokine scavenging properties, was studied in situ in rat livers containing experimentally induced colon carcinoma metastases by means of immunocytochemistry and in situ hybridization methods. The study was performed to investigate whether alpha2M production by hepatocytes plays a role in the defense against the growth of metastases on the basis of its protease inhibiting capacity. It was found that colon cancer cells in all developmental stages of the metastases contained large amounts of messenger RNA (mRNA) of alpha2M but hardly any alpha2M protein. Cancer cells in culture contained large amounts of both mRNA and protein of alpha2M. In contrast, stromal cells and liver cells did not show positivity for alpha2M mRNA above background levels. The exception was a few layers of hepatocytes around the latest stage of metastases. Hepatocytes contained both alpha2M mRNA and protein only when Kupffer cells were present, indicating that alpha2M mRNA production was induced via Kupffer cells. On the other hand, alpha2M protein was found in high amounts in the sinusoids and stroma of all metastases, irrespective of their developmental stage. Increased levels of alpha2M could not be detected in serum in all but one rat tested (n=8). It is concluded that production of alpha2M by hepatocytes occurs only around the latest developmental stage of metastases and that alpha2M does not play a significant role in the defense against metastatic cancer growth in rat liver. In contrast, cancer cells produce and secrete large amounts of alpha2M, which seems to be linked with their tumorigenicity. We suggest that this alpha2M captures cytokines rather than proteases by complex formation. These complexes were observed using immunocytochemical staining for alpha2M protein indicating that it was captured by either stromal cells, sinusoidal cells, or hepatocytes that are in direct contact with cancer cells, Therefore, changes in serum levels of alpha2M were limited, indicating that these levels do not reflect local production and effects of alpha2M. PMID- 8617439 TI - Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta. AB - The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor beta (TGF-beta)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-beta1. MFB were obtained by subculture of FSC. The TGF-beta concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-beta was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-beta1. The exclusive role of active TGF beta in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-beta receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-beta. PMID- 8617440 TI - Carbon tetrachloride-induced cirrhosis in rats: influence of the acute effects of the toxin on glucose metabolism. AB - In animal models, conflicting results on the effect of cirrhosis on glucose metabolism have been reported. The use of various toxins as well as differences in experimental protocols may be responsible for these controversial data. However, differences may be also be explained by the fact that glucose metabolism has been evaluated following different time intervals after cessation of the toxic injury. Therefore, we have performed intravenous glucose tolerance tests, euglycemic hyperinsulinemic clamps (at 2,6, and 30 mU/kg/min insulin infusion rates), and determination of peripheral tissue glucose metabolic index (by [3H]2 deoxy-glucose injection) in rats treated for 10 weeks with carbon tetrachloride, either 3 days (acute group) or 2 weeks (delayed group) after the last CCl4 dose was administered. Cirrhosis was confirmed by liver histological analysis, and by a 22% (P <.05) decrease in 13C-aminopyrine demethylation. In the acute group, whole-body glucose disposal was decreased at the highest insulin infusion rate only (19.7 +/- 1.2 vs. 23.4 +/- 1.2 mg/kg/min in controls, P <.05). In contrast, results of the delayed group were not different from controls at any insulin infusion rate. Peripheral tissue glucose metabolic index was significantly decreased in all muscles tested in the acute group compared with controls. A significant decrease of glucose utilization was found in some but not all muscles in the delayed group but was less pronounced than in the acute group. In conclusion, this study showed than insulin sensitivity in cirrhotic rats is time dependent with regard to the last CCl4 administration. These results must be taken into account when using this experimental model of liver cirrhosis. PMID- 8617441 TI - Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: the role of glutathione and relevance to hepatic venoocclusive disease. AB - The mechanisms leading to hepatic venoocclusive disease (HVOD) remain largely unknown. Azathioprine and monocrotaline were studied as part of a series of studies looking at a variety of toxins that induce HVOD to find common features that might be of pathogenic significance. In a previous study, dacarbazine showed selective in vitro toxicity to sinusoidal endothelial cells (SEC) compared with hepatocytes and a key role for SEC glutathione (GSH) was demonstrated. Murine SEC and hepatocytes were isolated and studied in culture. Azathioprine and monocrotaline were found to be selectively more toxic to SEC than to hepatocytes. The relative resistance of hepatocytes to azathioprine was due to enhanced GSH defense: hepatocytes exposed to azathioprine maintained intracellular GSH levels better than SEC, particularly when supplemental GSH precursors were added, and hepatocyte resistance was completely overcome by depletion of intracellular GSH. In contrast, monocrotaline toxicity in hepatocytes was largely unaffected by depletion of GSH, which suggests that selectivity of monocrotaline for SEC may be attributable to differences in metabolic activation. Both compounds are detoxified by GSH in SEC, as demonstrated by enhanced toxicity in the presence of buthionine sulfoximine (BSO) and attenuation of toxicity with exogenous GSH. SEC GSH levels were more than 70% to 80% depleted by monocrotaline and azathioprine, respectively, before cell death. Azathioprine and monocrotaline are selectively toxic to SEC; the mechanism of toxicity in the SEC may be caused by profound GSH depletion. PMID- 8617443 TI - Biliary excretion of estradiol-17 beta-glucuronide in the rat. AB - Estradiol-17beta-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol 17beta-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17beta-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 micromol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 micromol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose. PMID- 8617442 TI - Changes in rat liver gene expression induced by thioacetamide: protective role of S-adenosyl-L-methionine by a glutathione-dependent mechanism. AB - Chronic liver damage induced by thioacetamide (TAM) was accompanied by changes in the expression of genes related to growth (beta-actin) and function (albumin and haptoglobin) of the liver. Their messenger RNA (mRNA) levels increased during the first days after TAM administration, but 4 to 7 days after prolonged treatment with this drug, liver gene expression was considerable decreased. TAM-induced changes in albumin and beta-actin mRNA levels were prevented by cotreatment with S-adenosyl-L-methionine (SAM). We have investigated the possible involvement of glutathione in the protective mechanism of SAM. Firstly, we found that TAM treatment in the rat induced changes in liver glutathione disulfide (GSSG) levels, with a concomitant increase in the glutathione reductase enzymatic activity, these changes being abolished when animals were cotreated with TAM and SAM. Secondly, when rats were pretreated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, before thioacetamide administration, the beneficial effect of SAM on liver gene expression was completely abolished. These results were confirmed by assaying the alanine transaminase serum activity, a parameter of liver injury. TAM-treated animals had increases in this serum enzyme, this effect being partially blocked by SAM. However, in BSO-pretreated rats, the protective effect of SAM was impaired. Taking together all these results, we propose a glutathione-dependent mechanism in the SAM protection against TAM hepatotoxicity in the rat. PMID- 8617444 TI - Involvement of calcium in macrophage leukotriene release during experimental cirrhosis. AB - The aim of the present study was to assess the mechanism of 5-lipoxygenase metabolites (LT) secretion by peritoneal macrophages in rats wih CC14 induced cirrhosis. After stimulation with calcium ionophore A23187 or opsonized zymosan, [3H] arachidonic acid labeled macrophages from cirrhotic rats presented a significantly greater secretion of LT than macrophages from healthy controls. In addition, the phorbol ester TPA (protein kinase C activator) increased LT production only in macrophages from cirrhotic animals and not in controls. Although Ca2+ is thought to be involved in 5 lipoxygenase activation, the role of Ca2+ in LT production was studied. The use of a Ca2+-free medium as well as the addition of TMB-8 (an inhibitor of intra-cellular Ca2+ movements and of plasma membrane Ca2+ fluxes) resulted in a fall in LT production greater for macrophages from cirrhotic animals than for controls. The measurement of cytosolic Ca2+ concentration by cytofluorimetry showed that Fluo-3 loaded macrophages from cirrhotic rats had a greater cytosolic CA2+ concentration than macrophages from control animals both in basal conditions and after A23187 stimulation. Study of 45Ca2+ uptake suggest, that extra-cellular Ca2+ is implicated in the elevated cytosolic Ca2+ observed in macrophages from cirrhotic animals as compared to healthy controls. The greater Ca2+ concentration observed in macrophages from cirrhotic rats was not related to a difference in phospholipase C activation because inositol phosphate production did not differ between macrophages from healthy and cirrhotic animals. Taken together these results suggest that as compared to healthy animals, the greater LT production during cirrhosis could be dependent upon a difference in 5-lipoxygenase activation related to a rise in cytosolic Ca2+ concentration independently of inositol phosphates generation. PMID- 8617445 TI - Adaptive response of the enterohepatic circulation of bile acids to extrahepatic cholestasis. AB - Experimental cholestasis induced by ligation of the common bile duct results in morphological and functional changes in the rat hepatocyte. The aim of this study was to evaluate the adaptive response of the transport process involved in the enterohepatic circulation of bile salts to obstructive cholestasis. Male Sprague dawley rats with common bile duct ligation were killed after 48 and 72 hours. Portal and systemic blood and duodenal aspirates were collected. Taurocholate transport was measured in isolated ileal brush border (BBM) and liver basolateral membranes (BLM). Drastic reduction in intraluminal bile salt concentrations in ligated rats accompanied decreases in saturable taurocholate uptake by ileal BBMs. Kinetic analysis indicated that the decrease was attributable to reduction in transporter density and alteration in affinity for the substrate, both of which accentuated with increase in postligation time. In contrast, despite 20 fold higher portal venous bile salt concentrations, taurocholate uptake by hepatic sinusoidal membrane was lower in rats. Kinetic analysis and immunoblots developed using polyclonal antisera to the liver BLM bile acid transporter demonstrated a gradual decrease in transporter density with increase in postligation time accompanied by reduced taurocholate uptake by basolateral membrane. These results further support the concept that the ileal brush border membrane transporter is regulated by the availability of the substrate, whereas regulation of the bile salt transport across the liver BLM is independent of portal venous bile acid concentration. PMID- 8617446 TI - Quantitative image analysis of cytokeratin filament distribution during fetal rat liver development. AB - Recent reports suggest that not only chemical but also mechanical influences from the cellular environment could have profound effects on gene activity and could act on cell differentiation and proliferation. These mechanisms involve a tissue matrix system, which includes extracellular matrix, nuclear matrix, and cytoskeleton. Supposing that the cytoskeleton mediates mechanical transduction from the cell environment to the nucleus, significant differences in the spatial distribution of the cytoskeleton network could reflect variations in environmental signals. Hepatocytes during fetal development provide a useful model to study such variations, because a progressive establishment of intercellular contact is reported. The aim of this work is to discriminate steps in hepatocyte differentiation from fetal to adult livers, using computerized quantitative image analysis of cytokeratin (C8) immunofluorescent localization, visualized by confocal scanning laser microscopy. The filament structure is represented by the gray-scale skeleton of the digital images obtained by specially designed segmentation methods. A set of line features was investigated, including number and length of lines, orientation of lines, and the fractal dimension of the filament network. The features studied showed highly significant differences throughout liver development, with an increase of the total amount of cytokeratin filaments. We could also demonstrate a modification in the structure of the network, being more and more dense, with an increase of connecting points. Moreover, it has been shown that filaments have some orientation in fetal hepatocytes, and that directionality. Thus, significant differences in the pattern of the cytokeratin filament network according to the stages of hepatocyte differentiation have been demonstrated with objective and quantitative methods. PMID- 8617447 TI - Primary biliary cirrhosis clinical research single-topic conference. PMID- 8617448 TI - A novel adenovirus-based system for facilitating retroviral hepatic gene transfer in vivo. PMID- 8617449 TI - Autoimmune hepatitis and/or hepatitis C: how to decide. PMID- 8617450 TI - bcl-2: role in epithelial differentiation and oncogenesis. AB - The precise regulation and maintenance of balance between cell proliferation and cell death in multicellular organisms is critical for tissue homeostasis. bcl-2 initiates a new gene family involved in the regulation of cell death and survival without affecting cell proliferation. Expression of Bcl-2 has been reported in a wide range of hematopoietic cells, nonneoplastic epithelia (both hormone responsive and nonresponsive), and epithelial malignancies. Although the major group of epithelial cells expressing Bcl-2 protein are in the proliferating zones, expression is not directly related to cell proliferation. Bcl-2 is also associated with stem cells committed to differentiation and morphogenesis. The survival advantage provided by Bcl-2 prolongs the life span of epithelial cells with differentiation potential and allows proliferation, differentiation, and morphogenesis to proceed. The gene expression in hormone-responsive organs may contribute to the sustained life of those terminally differentiated epithelial cells and a decrease in Bcl-2 levels leads to cell death by apoptosis. Overexpression of bcl-2 protects epithelial cells from death, but it is neither able to immortalize normal cells, nor to cause tumorigenic transformation of immortalized epithelial cells. Heterogeneous expression of Bcl-2 in epithelial malignancies suggests that the gene is differentially regulated. Furthermore, its expression in association with precancerous lesions suggests a role in the early stage of tumorigenesis. The effects of Bcl-2 expression on sensitivity of epithelial cells to drug, radiation, and hormone therapies vary depending on the typed of tumor. Expression of Bcl-2 is associated with resistance to hormone therapy and recurrence in prostate carcinomas, whereas in lung and breast carcinomas it is associated with a better prognosis. Studies now being performed should clarify the underlying mechanisms of differential gene regulation in different tissues and show the clinical significance of the expression of bcl-2 and other members of the bcl-2 gene family. PMID- 8617451 TI - Telepathology diagnosis by means of digital still images: an international validation study. AB - Telepathology affords the means to provide pathological diagnosis and consultation to remote sites. However, before telepathology can become an acceptable medical tool, it will be vital to determine the diagnostic accuracy of this technology. We report the results of a single-blind study of the accuracy of diagnosis performed using computerized still images obtained from a telepathology workstation used in a French telepathology network. Four pathologists, each working alone, reviewed a total of 200 cases of routine surgical pathology (50 cases each), and performed diagnosis based on both computer CD-ROM still images (CD) and conventional glass slides (GS). Concordance between GS and CD diagnosis, as well as accuracy, were determined. Other factors related to performance were also measured, including diagnostic certainty, reasons for uncertainty, and causes of diagnostic error. Overall, there was good agreement between CS and CD diagnosis. There was 87.5% concordance between CS and CD diagnosis, and comparison to consensus (correct) diagnosis showed accuracy of 95.5% and 88.5% for GS and CD diagnosis, respectively. Marked variability in accuracy of CD diagnosis was observed among the four pathologists, and issues related to image selection and/or quality appeared responsible for 60% of the diagnostic errors. The lack of sufficient images and clinical information were frequently cited as reasons for diagnostic uncertainty, as well as feelings of insufficient expertise. It is likely that the opportunity for interaction with the referring pathologist and the use of subspecialty consultants would likely improve the performance of telepathology. PMID- 8617452 TI - Immunohistochemical evaluation of HER-2/neu expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasms. AB - Although several morphological and molecular genetic studies have implicated various grades of pancreatic duct hyperplasia as precursor lesions to infiltrating pancreatic adenocarcinoma, the identity of preinvasive pancreatic neoplasms remains controversial. In the present study, the authors examined the expression of the epidermal growth factor receptor homologue, HER-2/neu (c-erbB 2), in pancreatic duct lesions adjacent to infiltrating pancreas cancers in a series of 19 cases of pancreatic duct adenocarcinoma. HER-2/neu expression was examined because it has been identified in a proportion of infiltrating pancreas cancers and because it may provide early neoplasms with a growth advantage over adjacent nonneoplastic epithelium. In normal pancreatic ducts and ductules, HER 2/neu expression was absent in all but one case. By contrast, HER-2/neu was expressed in 82% (P = .008 vs normal ) of ducts with flat mucinous hyperplasia, 86% (P = .03 vs normal) of ducts with papillary mucinous hyperplasia without atypia, 92% (P = .001 vs normal) of ducts with atypical papillary mucinous hyperplasia, and all specimens with carcinoma in situ. HER-2/neu expression was observed in 69% (P = .002 vs normal) of the moderately differentiated infiltrating carcinomas and none of the poorly differentiated infiltrating carcinomas. These data establish HER-2/neu as a potential mediator of growth factor-related signal transduction in pancreatic duct lesions, and provide additional support for the hypothesis that lesions formerly regarded as various grades of hyperplasia instead may represent intraepithelial neoplasms with the potential for subsequent invasion and metastasis. PMID- 8617453 TI - Drug-induced immune dysregulation as a cause of atypical cutaneous lymphoid infiltrates: a hypothesis. AB - The authors encountered 22 patients in whom a skin biopsy showed atypical lymphoid hyperplasia and in whom a subsequent drug history showed indigestion of one or more agents before lesional onset. In 13 patients, the biopsy had been performed to rule out a diagnosis of malignant lymphoma, whereas the other nine the clinical impression was that of a drug eruption. Among the more frequently prescribed agents were calcium-channel blockers, angiotensin-converting enzyme (ACE) inhibitors, antidepressants, antihistamines, beta-blockers, benzodiazepines and lipid-lowering agents, all of which are either known to perturb lymphocyte function or have been implicated as a cause of pseudolymphomata. Twelve of the patients were on two or more of these drugs. The effect of drug modulation on the clinical course was assessed. The clinical presentations were as one or more erythematous plaques or multiple infiltrative papules, or as solitary nodules. The patient had been on one or more of the aforementioned drugs from 2 weeks to 5 years before developing the lesions. Resolution of the eruptions occurred in 17 patients within 1 to 32 weeks (mean, 7 weeks) of discontinuing the medication. Five additional patients had complete excision of solitary lesions without recurrence. A history of atopy, autoimmune disease, or previous carcinoma was elicited in five patients. All biopsy specimens showed atypical lymphoid infiltrates, which assumed one or more of the following patterns: mycosis fungoides (MF)-like, a lymphomatoid vascular reaction, lymphocytoma cutis and follicular mucinosis. Based on the histopathology of the biopsied lesions and the clinical course being one of lesional resolution after cessation of drug therapy or excision of a solitary lesion without subsequent recurrence, a diagnosis of drug-associated lymphomatoid hypersensitivity was established in all specimens. A diagnosis of drug-associated pseudolymphoma should be excluded before a diagnosis of cutaneous lymphoma is rendered, and should be considered if the patient is on a drug known to alter lymphocyte function, particularly in the setting of systemic immune dysregulation or multidrug therapy where agent may act synergistically or cumulatively to alter lymphoid function. The authors postulate that the drug may promote an aberrant immune response to an antigen that may be the drug itself or some other stimulus. A skin biopsy may be particularly helpful, as the lesions of drug-associated pseudolymphoma have a morphology distinctive from malignant lymphoma. PMID- 8617455 TI - Can you count on the mitotic index? AB - The mitotic index (MI) is the most commonly use quantitative measure in anatomic pathology but has been widely criticized for supposed "irreproducibility." In this article, the authors show that mitotic figure (MF) counts may be described by a Poisson distribution, give confidence intervals for the MI, and show that the supposed irreproducibility is largely a consequence of the counting techniques. They also show how to obtain the MI to a predetermined level of precision and present an analysis of why mitotic figure counting works, when properly used, despite the inherently low precision of the estimates that are usually obtained. PMID- 8617454 TI - Immunophenotypic and molecular analyses of acquired immune deficiency syndrome related and Epstein-Barr virus-associated lymphomas: a comparative study. AB - Limited information is current available on the molecular and immunophenogenotypic characteristics of CD30-positive anaplastic large cell (ALC) lymphomas occurring in human immunodeficiency virus (HIV)-infected individuals. To address this issue, the authors have undertaken a combined analysis of these lymphomas in a comparison with other Epstein-Barr virus (EBV)-associated tumors in the setting of HIV infection. Twenty-one AIDS-related lymphomas, including five CD30-positive ALC and 11 small noncleaved cell (SNCC) lymphomas, and five Hodgkin's disease (HD) specimens were characterized regarding the immunophenogenotypic features, the frequency and subtype distribution of EBV (as defined by in situ hybridization [ISH], Southern blot, and a polymerase chain reaction [PCR] amplification of the EBV nuclear antigen-2 [EBNA-2] region) antigen expression (latent membrane protein-1 [LMP-1], EBNA-2, and for alterations of the tumor suppressor gene p53. Combined immunophenotypic and immunogenotypic analyses showed a derivation from anomalously matured B cells in four of five CD30-positive ALC lymphomas, whereas SNCC showed features of mature B cells; no evidence of immunoglobulin or TCR gene rearrangement could be obtained in HD cases. Combined ISH and Southern blot analyses revealed that EBV was more strictly associated with HD (five of five) and CD30-positive ALC lymphomas (four of five) than with SNCC lymphomas (four of 11). EBV-positive samples from CD30-positive ALC lymphomas carried type 1 EBV (two of two specimens tested), whereas both EBV subtypes were observed in SNCC lymphomas and HD samples. All three forms of viral latent gene expression were found in the EBV positive CD30-positive ALC lymphomas. SNCC specimens did not express LMP-1 or EBNA-2, whereas HD specimens expressed LMP-1 (four of five tested) but no EBNA-2. Immunostaining for ZEBRA was consistently negative. HHV-6 DNA sequences were detected by PCR in one SNCC of the 19 specimens analyzed. Three out of five CD30 positive ALC lymphoma specimens and six of 10 SNCC showed nuclear staining for p53. No mutation was detected in any of the three CD30-positive Alc lymphoma analyzed, whereas an aberrant SSCP pattern was found in all the four SNCC samples tested. At variance with SNCC lymphomas, AIDS-related B-cell CD30- positive ALC lymphomas are strictly associated with EBV infection and may also express the broad lymphoblastoid cell line-like (LMP-1-positive, EBNA-2-positive) pattern, and lack p53 genetic lesions. Unlike EBV, HHV-6 probably does not represent a relevant factor involved in the pathogenesis of CD30-positive ALC and other HIV related lymphomas. PMID- 8617456 TI - von Hippel-Lindau disease gene deletion detected in microdissected sporadic human colon carcinoma specimens. AB - The progression of human malignancies is thought to involve the inactivation or loss of tumor suppressor genes. Previous studies have suggested that inactivation of tumor suppressor genes on chromosomes 5q, 17p, 18q, and 8p play a role in the development of colorectal carcinoma. However, chromosome 3p at the von Hippel Lindau disease (VHL) gene locus (3p25-26) has not been previously implicated in the development or progression of sporadic colorectal carcinoma. The authors have analyzed VHL gene alterations on chromosome 3p in sporadic human colon carcinomas and adenomas using modified microdissection techniques. These techniques allow for procurement and analysis of selected subpopulations of cells from both paraffin-embedded and frozen human tumor specimens. VHL disease gene deletion was detected by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis in microdissected colon carcinoma specimens. Allelic loss of VHL gene was detected in 7 of 11 (64%) informative patients who underwent colectomy for primary sporadic colon carcinoma. However, no allelic loss of VHL gene was shown in colonic adenomas of eight informative patients. These results indicate that VHL disease gene deletion frequently occurs in sporadic colon carcinoma. Because this deletion was not present in adenomas, VHL gene may play a role in colonic carcinogenesis and represent a relatively late event in colonic neoplasia progression. Additionally, microdissection of tissue sections may be especially useful in detecting allelic loss in PCR-based studies of infiltrating tumors, particularly when the tumor cells represent a relatively small percentage of the total cell population. PMID- 8617457 TI - Recurrent Epstein-Barr virus-associated lesions in organ transplant recipients. AB - Posttransplant lymphoproliferative disorders (PTLD) are related to Epstein-Barr virus (EBV) and range from lymphoid hyperplasias to lymphomas. The authors report 11 transplant recipients with recurrent EBV-associated lesions. Four patients presented with EBV-positive mononucleosis-like lymphadenitis. One had recurrence of a similar lesion and the other three developed polymorphic PTLDs. Matched clonal studies in one patient showed clonal lymphoid and EB viral populations in the recurrent lesion, but not in the initial lesion. Six patients presented with polymorphic PTLDs. Five later developed histologically dissimilar tumors that resembled non-Hodgkin's lymphoma (two B-cell and one T-cell origin), Hodgkin's disease (one patient), or smooth muscle tumor (one patient). Matched clonal studies were available from one patient and showed that the primary and recurrent lesions were clonally distinct. The sixth patient had recurrence of histologically and clonally identical polymorphic PTLD. One patient presented with monomorphic PTLD and developed recurrence of a clonally identical tumor after a 6-month remission. This study shows that a few patients with EBV associated lesions have clinical recurrence, which may be either a relapse of the original process or a new EBV-associated lesion. In some patients, the new lesion appeared to represent a more fully developed malignancy that did the antecedent lesion. PMID- 8617458 TI - Genetic analysis of synchronous mucinous tumors of the ovary and appendix. AB - The coexistence of mucinous ovarian and appendiceal tumors in association with pseudomyxoma peritonei (PP) is well established. However, it has not been determined whether they represent independent or metastatic neoplasms. The authors analyzed microsatellites on chromosome 17q 21.3-22 (nm23), 3p 25-26 (von Hippel Lindau disease [VHL] gene), and 5q 21-22 (D5S346 locus) in 12 synchronous ovarian and appendiceal mucinous lesions. Loss of heterozygosity (LOH) at the nm23 locus has been shown previously in ovarian carcinomas, and genetic alterations at both the 3p and 5q loci have been reported in colorectal carcinomas. The ovarian lesions consisted of nine mucinous tumors of low malignant potential and three invasive adenocarcinomas, and the appendiceal lesions consisted of eight carcinomas without invasion, two invasive carcinomas, and two mucosal hyperplasias. DNA was extracted from microdissected cells obtained from formalin-fixed, paraffin-embedded tissue sections and amplified by polymerase chain reaction. In three specimens, genetic alterations occurred at 17q 21.3-22 in only the ovarian tumors. One of these cases showed LOH on chromosome 5q 21-22 in only the appendiceal tumor. In three other specimens, LOH at the same locus was found in both tumors. Six specimens did not show LOH at any locus. These results suggest that a subset of synchronous mucinos ovarian and appendiceal lesions showing different LOH patterns in both sites most likely represent patients with two separate primary lesions. Another group of specimens with the same allelic loss in both tumors most likely represent patients with a single primary and metastatic spread. Thus, genetic analysis of these lesions may be useful in investigating the origin of histologically similar synchronous tumors. PMID- 8617459 TI - The immunohistochemical discrimination of endometrioid adenocarcinomas. AB - Carcinomas of endometrioid histology frequently arise in the endometrium, ovary, and endocervix and involve the pelvic tissues in women. Adenocarcinomas of psuedoendometrioid morphology developing in the colon also frequently involve the ovary. The authors retrospectively examined 97 adenocarcinomas from the uterus, cervix, ovary, and colon to ascertain whether the site of origin could be determined by using a battery of antibodies with the immunoperoxidase method on formalin-fixed tissue. This study was restricted to tumors with endometrioid morphology. There were 27 endometrial, 16 ovarian, 23 endocervical adenocarcinomas, and 31 psuedoendometrioid colonic adenocarcinomas. The battery of antibodies included vimentin (V), monoclonal carcinoembryonic antigen (mCEA), and monoclonal CEA D-14. V-positive cells were defined by the presence of a crisp paranuclear band of staining, and CEA-positive cells showed irregular or diffuse cytoplasmic staining. V diffusely decorated 22 of 27 (81.4%) of endometrial tumors, 3 of 23 (13%) of endocervical tumors, (rare, focal staining), diffusely stained 5 of 16 (31.3%) of ovarian tumors, and was rare and focal in 2 of 31 (6.4%) of colon tumors. Both CEA antibodies were negative for cytoplasmic staining in both endometrial and ovarian tumors, but decorated from 65.2% (CEA D 14) to 95.6% (monoclonal CEA) of endocervical tumors and from 83.8% (CEA D14) to 90.3% (mCEA) of colonic tumors. The authors conclude that endometrioid adenocarcinomas developing in endometrium and ovary are most often strongly V positive and CEA negative, which greatly aids in distinguishing them from endometrioid or pseudoendometrioid tumors arising in endocervix and colon, which are only rarely, and very focally V and CEA positive. The antibodies do not allow for discrimination between endocervical and colonic tumors. CEA D-14 offered no immunodiagnostic superiority over mCEA. These results support the use of immunohistochemistry is assisting in the distinction of endometrial from endocervical primary sites in curettage specimens and in metastatic sites. PMID- 8617460 TI - Microcalcifications in ductal carcinoma in situ of the breast: histochemical and immunohistochemical study. AB - Thirty cases of ductal carcinoma in situ (DCIS) of the breast, showing histological microcalcifications, were studied to clarify their mechanism of formation. Undecalcified sections revealed three types of calcium precipitates: type I and II granular calcifications (GCs) and laminar calcifications (LCs). In type I GCs the core on which the calcium had deposited was constituted mainly by nuclear debris. Type II GCs were predominantly composed by mucosubstances. LCs were the result of calcium deposits on mucoid or proteinaceous material, arranged in concentric lamellae. LCs and type II GCs were mainly present in well and intermediately differentiated DCIS. Type I GCs were observed in only DCIS with necrosis, frequently being present in intermediately and poorly differentiated DCIS. PMID- 8617461 TI - Immunohistochemical localization of cathepsins D and E in human gastric cancer: a possible correlation with local invasive and metastatic activities of carcinoma cells. AB - The immunohistochemical localization of cathepsins D and E in 44 cases of human gastric carcinoma, using antibodies specific for each enzyme, were investigated. Cathepsin D- and E- positive carcinoma cells were present in all samples. However, the staining intensity varied from cell to cell in the same carcinoma tissue as well as among samples. The most intense immunostaining of both cathepsins was often found in the cells, which were present at the advancing margin of the carcinoma tissues. The incidence of this peculiar localization of intensely stained carcinoma cells significantly correlated with the progression of the carcinoma tissue (D, P < .05; E, P < .01) and with occurrence of the lymph node metastasis (D and E, P < .05). There was no statistical significance between this localization and the histological type (differentiation) of the carcinoma tissues. Cathepsin-positive inflammatory cells infiltrated in and around the carcinoma tissue, and intensely stained inflammatory cells were often located in the stroma at the border of the carcinoma tissue. However, no statistical correlation was noted between the localization of cathepsin-positive inflammatory cells at the border and the stage of progression or the incidence of metastasis. These results indicated that cathepsins D and E in the carcinoma cells located at the advancing margin play an important role in the invasion and subsequent metastasis of human gastric carcinoma. Meanwhile, cathepsin-positive inflammatory cells seem to be less responsible for the biological behavior of carcinoma cells than those in the carcinoma cells themselves. PMID- 8617462 TI - Maternal varicella during pregnancy: correlation of maternal history and fetal outcome with placental histopathology. AB - The authors examined the records from 73 women diagnosed with varicella during pregnancy to correlate placental findings with maternal history and fetal outcome. Fifty-eight of the mothers delivered at the authors' institution, and 19 placentas were available for review. The onset of symptoms of varicella occurred from 27 weeks before delivery to 1 day postpartum. Only on e of the newborns delivered at the authors' institution was diagnosed with probable varicella at birth; the remainder had no unequivocal evidence of infection; however, serological studies were not performed on most of the newborns. The placenta from the newborn with probable varicella showed extensive basal chronic villitis with a lymphohistiocytic infiltrate and occasional multinucleated giant cells. Two other placentas showed rare foci of chronic villitis, and the remainder showed no villitis or other viral-associated changes. Twenty-four (33%) of the 73 women developed varicella pneumonia, and one woman died. Although varicella during pregnancy is associated with high maternal morbidity, fetal disease is uncommon. Most placentas show no virus-associated lesions; however, chronic villitis with multinucleated giant cells in association with a recent history of maternal varicella may be predictive of neonatal infection. PMID- 8617463 TI - Hair granuloma of the prostate. AB - We report the second case of so-called hair granuloma of the prostate in a transurethral resection specimen. We hypothesize that the hair was most likely embedded in bladder/neck/prostate tissue by an earlier perineal prostate needle biopsy. PMID- 8617464 TI - Diffuse bone marrow metastases from glioblastoma multiforme: the role of dural invasion. AB - Two cases of glioblastoma multiforme (GBM) diffusely metastatic to bone marrow are presented; both patients developed back pain, thrombocytopenia, and hemorrhagic diatheses from their diffuse bony disease. In one patient the intracranial and extracranial symptoms were synchronous in their presentation. Both patients had known dural involvement by GBM at the time of craniotomy and dural venous invasion microscopically. Tumor infiltration of dura and dural vessels is a significant mechanism for extracranial spread and should be noted by surgical pathologists in their reports if it is found. Although cases of metastatic GBM have been uncommon in the past, especially without antecedent surgery or ventriculoperitoneal shunt placement, better control of local disease may enhance the possibility of extracranial spread in the future from GBM. The finding of dural invasion by GBM either at surgery or microscopically should prompt oncologists to initiate at least a limited metastatic work-up for this subset of GBM patients. PMID- 8617465 TI - Placental changes in a first trimester missed abortion in maternal systemic lupus erythematosus with antiphospholipid syndrome; a case report and review of the literature. AB - The placental lesions generally attributed to the effects of systemic lupus erythematosus (SLE) on the decidual and placental villi include decidual vasculopathy, placental infarcts and possibly intrauterine growth retardation. The maternal decidual vessels in SLE show a lesion termed "acute atherosis" which is histologically similar to acute vascular rejection in a transplanted kidney. These changes can be so extensive as to result in complete vascular occlusion, decreased placental perfusion and resultant placental villous infarction. In currettings from products of conception in the first trimester, acute atherosis is a rare finding. We describe striking decidual vasculopathy and extensive villous infarction in a case of a first trimester missed abortion in a patient with SLE who had anticardiolipin antibodies (ACL), both immunoglobulin (IgG) and IgM, and lupus anticoagulant (LA) in her serum. To the best of our knowledge, this is the first report of acute atherosis and villous infarction in a first trimester placenta in association with SLE and/or ACL and LA. Pathologists should be aware of these changes and alert clinicians to the possibility of SLE or antiphospholipid and/or anticardiolipin antibodies. PMID- 8617466 TI - Soft tissue oncocytoma. AB - We describe a case of a novel soft tissue neoplasm, composed of a monotonous population of cells. Their cytoplasm was parked with mitochondria, and had no immunohistochemical or ultrastructural evidence of differentiation. The neoplasm, located within the thigh of a 50-year old man, was well-circumscribed but unencapsulated. The patient was clinically free of neoplasm at 1 year follow-up, after complete local excision. We propose the term "soft tissue oncocytoma" for this lesion, because of the similarity of this neoplasm to oncocytomas of other reported sites. PMID- 8617467 TI - Angiotropic large cell lymphoma presenting as primary adrenal insufficiency. AB - Angiotropic large cell lymphoma is reported in two patients who developed severe metabolic acidosis and unexplained hypotension before death. In addition to the usual multiorgan involvement of the disease, the adrenal glands of both patients were symmetrically enlarged. Histologically, the cortical vessels were engorged and filled with neoplastic lymphoid cells of B-cell lineage, whereas the parenchymal cells were compressed and atrophic. Angiotrophic large cell lymphoma seems to be a distinct and unrecognized cause of primary adrenal insufficiency, and should be included in the differential diagnosis of adrenal hormone deficiency. PMID- 8617468 TI - Dysplasia in atypical liver nodes. PMID- 8617469 TI - Metastatic pattern of gastric carcinoma. PMID- 8617470 TI - Static image telepathology in perspective. PMID- 8617471 TI - Clinically silent rejection: what's in a name, anyway? PMID- 8617472 TI - Recommendations for the reporting of resected prostate carcinomas. Association of Directors of Anatomical and Surgical Pathology. PMID- 8617473 TI - The WAF1-mediated p53 growth-suppressor pathway is intact in the coronary arteries of heart transplant recipients. AB - It has been suggested that the interaction of cytomegalovirus (CMV) with the p53 tumor suppressor gene product plays a role in the development of coronary artery restenosis after angioplasty. CMV nucleic acids have been observed in the coronary arteries of allografted hearts, suggesting a possible role for the interaction of CMV with p53 in the development of accelerated graft arteriosclerosis in transplant recipients. Formalin-fixed, paraffin-embedded sections of coronary arteries from 19 transplanted hearts were immunostained for the p53 gene product using Target Unmasking Fluid (TUF)-mediated immunohistochemistry and the anti-p53 antibodies CM1 and DO7. Fresh-frozen sections of coronary arteries were also available from six of the 19 hearts, and these fresh-frozen sections were immunostained for the p53 gene product with the DO7 antibody and for WAF1 using the anti-WAF1 antibody EA10. Focal and weak staining for p53 was observed in smooth muscle and endothelial cells in two of 19 vessels, whereas the remaining 17 did not stain. CMV nucleic acids were previously shown in six of 13 of these hearts by in situ hybridization. The fresh frozen sections of coronary arteries also did not stain for p53, but the smooth muscle cells in these vessels did stain intensely for WAF1. These results suggest three possibilities: (1) CMV-p53 interactions are not important in the development of accelerated graft arteriosclerosis; or (2) there is an interaction, but it is transient and not detectable at the time points examined in this study; or (3) there is an interaction, but binding of CMV to p53 leads to accelerated degradation of p53, as occurs with HPV-E6. The expression of WAF1 further suggests that the WAF1-mediated antiproliferative signal is intact in these vessels. PMID- 8617474 TI - Interphase cytogenetics in mammographically detected breast lesions. AB - Chromosomal aneuploidy in 25 mammographically detected breast lesions (MDBL) were determined on cytological smears using directly labeled pericentromeric probes for chromosomes 7 to 12, 17, 18 and X. The lesions included seven nonproliferative (NP) lesions, seven atypical hyperplasias (AHs), and 11 carcinomas (CAs). No other significant histological findings were identified in the remaining specimens except in two mammographically detected NP lesions, where foci of AH were present in adjacent sections; therefore, these two specimens were included in the AH lesion group (moderately increased risk lesions). Corresponding tissue sections were evaluated, and the results were correlated with fluorescent in situ hybridization (FISH) results. Monosomy was defined as the loss of one signal in > or = 15% of cells, and trisomy or tetrasomy was defined by the presence of three or more signals in > or = 3% of cells. Chromosomal aberrations were detected in 2 of 5 NP, 9 of 9 AH, and 11 of 11 CA groups. The mean number of cells with three or more signals, for all chromosomes, was 1.04 +/- 0.9 in the NP group, 8.5 +/- 9.4 in the AH group, and 20.2 +/- 5.4 in the CAs. A significant statistical difference was noted between the different groups (P = .0001). Chromosomal gain was the most common aberration and involved all chromosomes. The X chromosome was the only individual chromosome with significant differences in NP, AH, and CA groups. Chromosomal loss was observed in five specimens (20%) and involved chromosomes 8, 10, 17, and 18. The authors conclude (1) significant chromosomal aberrations can be detected in AH lesions and in NP epithelium from patients with moderately increased risk lesions; (2) numerical chromosomal aberrations tend to increase with progression of disease; (3) the frequent chromosomal gains/losses involving AH suggest that some AH may display submicroscopic features of malignancy; and (4) combined chromosomal aberrations allow for significant categorization of breast lesions, especially in cytology specimens. PMID- 8617475 TI - Cystic transformation and calcium oxalate deposits in rete testis and efferent ducts in dialysis patients. AB - The histological study of the testes and epididymides obtained from autopsies of 24 men with chronic renal insufficiency revealed bilateral cystic transformation of the rete testis and efferent ducts in patients who underwent hemodialysis or peritoneal dialysis, but not in patients who did not receive this treatment. The lesion was associated with an accumulation of crystalline calcium oxalate deposits in the lumen of the rete testis and efferent ducts, and in the connective tissue adjacent to these excretory ducts. The rete testis epithelium showed columnar transformation with occasional papillary proliferations. Neither atypias or mitoses were observed. In three specimens, fibrosis and giant cell reactions was also present in the rete testis at the level of crystalline deposits. In three specimens, the caput epididymidis was enlarged, and the efferent ducts showed an increase in both tubular diameter and epithelial height, irregular outline, and development of diverticula. The lesions appeared within 30 months after the onset of dialysis. PMID- 8617476 TI - Comparative genomic hybridization: a new tool in cancer pathology. AB - Analysis of genetic abnormalities in tumors is becoming increasingly important in tumor pathology. Techniques available for this purpose include DNA cytometry, tumor cytogenetics (TC), in situ hybridization (ISH), and the microsatellite assay (eg, for the analysis of loss of heterozygosity [LOH]). All of these techniques have certain advantages and disadvantages. The latter make them less suitable for a detailed (eg, DNA cytometry is too crude) and extensive (eg, TC is too laborious, and ISH and LOH too specific) evaluation of the genetic changes throughout the whole genome of tumors in the pathology laboratory. The authors discuss a recently developed molecular cytogenetic technique called comparative genomic hybridization (CGH), which has distinct advantages over the other techniques. Using only a small amount of DNA, CGH can, in a single experiment, provide detailed information on gains and losses of genetic material in a tumor, throughout the whole genome. In short, tumor DNA is labeled with a green fluorochrome, mixed with red labeled normal (diploid) DNA, and hybridized to normal metaphase preparations. The green and red labeled DNAs compete for hybridization to the chromosomes. The green-to-red fluorescence ratio on the chromosomes is a measure of underrepresentation or overrepresentation (loss or gain, respectively) of genetic material of the tumor. CGH has already been applied to tumor cell lines and on fresh or fresh frozen tissues from several types of malignancies, and has revealed chromosomal regions involved in amplifications and deletions that were previously unsuspected. Another important advantage of CGH is its applicability to paraffin-embedded archival material. This application allows analysis of many tumors that are pathologically well characterized and of which the clinical outcome is known. CGH is, therefore, an important new tool in the study of cancer development and perhaps prognosis. PMID- 8617477 TI - Comparison of MIB-1 (Ki-67) antigen and bromodeoxyuridine proliferation indices in meningiomas. AB - Meningiomas from 40 adult patients were labeled immunohistochemically with monoclonal antibodies to bromodeoxyuridine (BUdR) and the Ki-67 antigen, MIB-1. The meningiomas were classified as classical, or benign (n = 31); atypical (n = 4); or malignant (n = 5). Meningeal sarcomas and hemangiopericytomas were excluded. The patient population consisted of 26 women and 14 men, ranging in age from 26 to 75 years. BUdR proliferation indices ranged from 0% to 5.8%, measurements that were expectedly lower than those for MIB-1, which ranged from 1.5% to 19.3%. MIB-1 proliferation indices were not significantly affected regarding steroid pretreatment or age. These results show a good correlation between the BUdR and MIB-1 proliferation markers (rs = 0.72; P < .0001), which supports the use of anti-MIB-1 as an alternative labeling tool to BUdR for the determination of the proliferation index in meningiomas, thus avoiding the administration of a potentially mutagenic drug. PMID- 8617478 TI - Differential expression of the bcl-2 oncogene in human basal cell carcinoma. AB - The bcl-2 protein increases cell longevity and reduces apoptosis. Expression of the bcl-2 oncogene is reported in certain low grade neoplasms including basal cell carcinomas (BCCs). The authors postulated that the indolent variants of BCC, namely, the superficial and circumscribed subtypes, might exhibit greater bcl-2 expression than their aggressive counterparts, and used a monoclonal antibody to identify its protein product in formalin-fixed tissue from 30 BCCs. Expression of bcl-2, observed in 28 to 30 BCCs, was greatest in indolent-growth BCCs and weakest in the aggressive-growth variants (P = .005). Variable bcl-2 expression is seen in BCC and may be integral to both its pathogenesis and its biological behavior. PMID- 8617479 TI - p53 protein overexpression in gallbladder carcinoma and its precursor lesions: an immunohistochemical study. AB - Gallbladder carcinoma is one of the most frequent neoplasms diagnosed in Chile. Although the premalignant lesions have been extensively studied and are well characterized, there is only limited information about the genetic abnormalities that might be important in the pathogenesis of gallbladder carcinoma or that might have prognostic implications. The present study evaluates the immunohistochemical expression of p53 protein in premalignant lesions and invasive carcinoma of the gallbladder, and correlates the p53 expression with histological type, grade of differentiation, and level of invasion of the tumor. The authors studied the immunohistochemical p53 protein overexpression in 52 gallbladder carcinomas, 47 carcinomas in situ (CISs), 34 dysplasias, and 10 specimens with chronic cholecystitis containing normal and metaplastic epithelium. A semiquantitative scoring system was used to assess the p53 reactivity. p53 overexpression was found in 34 of 52 (65.4%) carcinomas, 21 of 47 (44.7%) CISs, and 11 of 34 (32.4%) dysplasias. There were no significant differences in p53 expression in premalignant lesions associated with invasive carcinoma and those that were not. Normal and metaplastic epithelium did not overexpress p53 protein. In adenocarcinomas, no correlation was found between p53 protein overexpression and histological subtype, grade of differentiation, or level of invasion. The high incidence of p53 overexpression in gallbladder carcinoma and its presence in dysplasia, even in specimens without invasive carcinomas, suggests that this abnormality is an important and early event in the pathogenesis of the tumor. The progressively increasing incidence of p53 overexpression observed from premalignant lesions to invasive tumor provides additional support to the view that this is the usual route for the development of infiltrating gallbladder carcinoma. PMID- 8617481 TI - Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms. AB - Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms. PMID- 8617480 TI - Diagnosis of congenital syphilis from placental examination: comparison of histopathology, Steiner stain, and polymerase chain reaction for Treponema pallidum DNA. AB - Congenital syphilis is often a presumptive diagnosis (based on serologies), because confirmation requires identification of Treponema pallidum in fetal/neonatal tissues or in the placenta. Placental histological features associated with congenital syphilis include the triad of enlarged hypercellular villi, proliferative fetal vascular changes, and acute or chronic villitis. The authors blindly evaluated 49 formalin-fixed, paraffin-embedded placentas (38 with positive maternal syphilis serologies; 11 with negative serologies) and compared results of histology, Steiner stain, and polymerase chain reaction (PCR) for T pallidum DNA. Histology was categorized as positive (triad present), suspicious (two thirds of triad present), or negative. Treponemal DNA was detected by amplifying a 189 base pair region of the 47 kd treponemal membrane antigen with 44 cycles of PCR; products were detected by Southern blot. Placentas from the 11 seronegative mothers were all negative by histology, Steiner stain, and PCR. Among the 38 placentas from serologically positive mothers, 4 had positive histology (2 of 4 positive Steiner, 4 of 4 positive PCR); 6 had suggestive histology (0 of 6 positive Steiner; 1 of 6 positive PCR); and, 28 had negative histology (0 of 28 positive Steiner; 1 of 28 positive PCR). PCR identification of treponemal DNA was significantly associated with the triad (P = .0003), proliferative fetal vascular changes (P = .0003), acute villitis (P = .003), chronic villitis (P = .004), and spirochetes on Steiner stain (P = .01). These results (1) confirm a strong association between placental histopathologic features and congenital syphilis; (2) indicate that when such features are present, PCR of placental tissue may confirm the diagnosis of congenital syphilis; and (3) suggest that even when such features are absent, PCR of placental tissue may identify additional cases of histologically unsuspected congenital syphilis. PMID- 8617482 TI - Proliferative activities in conventional chordoma: a clinicopathologic, DNA flow cytometric, and immunohistochemical analysis of 17 specimens with special reference to anaplastic chordoma showing a diffuse proliferation and nuclear atypia. AB - Chordoma shows various degrees of atypia histologically, however, the relationship between the histological features and the biological behavior still remains controversial. The authors subclassified 17 specimens with chordoma into two groups (ie, trabecular type showing a trabecular patterns and solid type mainly consisting of a diffuse proliferation of tumor cells). The histological grading was performed according to the degree of nuclear atypia on a scale of 1 to 3. Using DNA flow cytometric and immunohistochemical techniques, both the proliferative index (% S + G2 + M phase) and the MIB-1 labeling index (LI) of the tumor cells were estimated regarding their proliferative activities. In addition, p53 overexpression was also investigated using immunohistochemical techniques. There were eight (47.1%) specimens of trabecular type and nine (52.9%) of solid type. In nine specimens of solid type, those with higher nuclear atypia (grade 2 or 3) were significantly more frequent (five specimens, 55.6%) than in trabecular type in which all of the eight specimens were grade 1 (P = 0.44). The proliferative index was significantly higher in grade 2 or 3 lesions than in grade 1 lesions (P = .014), and the MIB-1 LI tended to be higher in solid type than in trabecular (P = .088). p53 overexpression was detected in two specimens of solid type, and the MIB-1 LI in these two specimens was significantly higher (P = .037) than that in the specimens without p53 overexpression. It was considered that the preceding anaplastic histological features, including either diffuse proliferation or high grade nuclear atypia, together with p53 overexpression, were thus closely related to the proliferative activities in chordomas. PMID- 8617483 TI - p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias: evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas. AB - Previous studies of vulvar carcinomas have shown two distinct subsets with respect to several clinicopathologic features. In younger women, the tumors are frequently human papillomavirus (HPV) positive, are usually of basaloid or warty histology, and are associated with vulvar intraepithelial neoplasia. In older women, the tumors are usually HPV negative, are typical keratinizing squamous carcinomas, and are associated with squamous hyperplasia--a lesion that has been purported to serve as a precursor to HPV-negative invasive carcinoma. In squamous carcinomas of the cervix, p53 inactivation (through gene mutation or interaction with the HPV E6 oncoprotein) occurs in most cases. Comparatively few studies have assessed p53 mutation and HPV status in vulvar carcinomas, and none has used molecular markers to evaluate squamous hyperplasias as direct precursors of HPV negative invasive cancers. Of 18 invasive squamous carcinomas analyzed, seven (39%) were found to be HPV positive. Four p53 gene mutations were identified--all in HPV-negative tumors. DNA was subsequently prepared from microdissected archival tissues from all four specimens showing p53 gene mutations. DNA was separately isolated from normal squamous epithelium, invasive squamous carcinoma, and associated squamous hyperplasia. In each specimen, the p53 mutation was confirmed in the invasive tumor and absent in both normal and hyperplastic epithelium. To further investigate squamous hyperplasia as a potential precursor of HPV-negative invasive carcinoma, the authors determined the clonality of hyperplastic lesions adjacent to invasive carcinomas with p53 mutation. Clonality analyses were performed using a polymerase chain reaction (PCR)-based assay for X chromosome inactivation. Although all three informative carcinomas tested were monoclonal, corresponding normal epithelia and hyperplastic lesions were polyclonal. These findings underscore the heterogeneity of vulvar cancers with respect to loss of wild type p53 function either by interaction with the HPV E6 oncoprotein or somatic mutation of p53, and suggest that squamous hyperplasias do not serve as direct precursors of HPV-negative squamous carcinomas. PMID- 8617484 TI - Atypical adenomatous hyperplasia (adenosis) of the prostate: development of a Bayesian belief network for its distinction from well-differentiated adenocarcinoma. AB - The diagnosis of atypical adenomatous hyperplasia (AAH) of the prostate and its distinction from well-differentiated prostatic adenocarcinoma with small acinar pattern (PACsmac; Gleason primary grades 1 or 2) are affected by uncertainties that arise from the fact that the knowledge of AAH histopathology is expressed in descriptive linguistic terms, words, and concepts. A Bayesian belief network (BBN) was used to reduce the problem of uncertainty in diagnostic clue assessment, while still considering the dependencies between elements in the reasoning sequence. A shallow network was designed and developed with an open tree topology, consisting of a root node containing two diagnostic alternatives (eg, AAH v PACsmac) and 12 first-level descendant nodes for the diagnostic features. Eight of these nodes were based on cell features, three on the type of gland lumen contents and one on the gland shape. The results obtained with prototypes of relative likelihood ratios showed that belief for the diagnostic alternatives is high and that the network can differentiate AAH from PACsmac with certainty. The features that best contributed to the highest belief were those concerning the nucleolar size, frequency, and location. In particular, after the analysis of five nucleolar features (prominent nucleoli, inconspicuous nucleoli, nucleoli with diameter greater than 2.5 micron, nucleolar margination, and nuclei with multiple nucleoli), the belief for AAH was 1.0, being already close to 1.0 when three were evaluated (the value range is 0.0 to 1.0; the closer to 1.0, the greater the belief). The contribution of the three features concerning the gland lumen contents (mucinous material, corpora amylacea, and crystalloids) was such that the final belief did not exceed 0.8. Results with the group of remaining features (eg, basal cell recognition, gland shape variation, cytoplasm appearance, and nuclear size variation) were slightly better. These features allowed a substantial accumulation of belief that was already greater than 0.9 when three were polled. However, the maximum belief value was never obtained. In conclusion, a BBN for AAH diagnosis offers a descriptive classifier that is readily implemented, and allows the use of linguistic, fuzzy variables, and the accumulation of evidence presented by diagnostic clues. PMID- 8617485 TI - Immunostaining of the p30/32MIC2 antigen and molecular detection of EWS rearrangements for the diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumor. AB - The identification of Ewing's sarcoma (ES) and peripheral neuroectodermal tumor (PNET) among other small round cell tumors (SRCTs) is a critical issue in musculoskeletal pathology because of the lack of clearly distinctive morphological features. In this study, the authors have compared advantages and limits of two procedures that were recently suggested as additional tools for the identification of ES/PNET, the analysis of p30/32MIC2 antigen by immunohistochemistry, and the evaluation of the fusion products of two specific chromosomal aberrations, the t(11;22)(q24;q12) and the t(21;22)(q22;q12), by reverse transcriptase-polymerase chain reaction (RT-PCR). The authors have analyzed the expression of p30/32MIC2 in 28 cell lines and in 90 tumor samples. p30/32MIC2 was highly expressed in ES/PNET but was also present in all the other cell types. The broad spectrum of positivity for p30/32MIC2 in SRCTs of bone was substantially confirmed by the analysis of tissue samples. In the same material, the authors have evaluated the presence of t(11;22) or t(21;22) transcripts (EWS/FLI-1 and EWS/ERG, respectively) by RT-PCR. These transcripts were found in all the cell lines and tissue samples of ES/PNET, but not in other tumors. The authors' results question the use of p30/32MIC2 immunostaining alone for the identification of ES/PNET and suggest the adoption of RT-PCR as an advantageous alternative. Molecular diagnosis of ES/PNET by RT-PCR is highly specific and can be applied to small amounts of tissue. Moreover, RNA extracted from paraffin embedded specimens was shown to be suitable for RT-PCR analysis, thus enabling analysis of archival material. PMID- 8617486 TI - Granulocytic sarcoma of megakaryoblastic differentiation complicating chronic idiopathic myelofibrosis. AB - We report the development of soft tissue granulocytic sarcoma with megakaryoblastic differentiation in a patient with chronic idiopathic myelofibrosis, which has hitherto been undescribed. Although an artifactual signet ring appearance of the blasts was found in the formalin-fixed tissue and caused diagnostic problems, the histological appearance on B-5 fixed material and immunophenotyping confirmed the diagnosis. Cytogenetic analysis showed 47,XY,+der(1)del(1)(?p13) at initial presentation and 47,XY,del(1)(?13),+der(1)del(1)(?p13) at the time of soft tissue blastic transformation, indicating that partial trisomy 1 might be of pathogenetic significance. PMID- 8617487 TI - Histiocytoid cardiomyopathy and sudden death. AB - We present a case of histiocytoid cardiomyopathy resulting in sudden and unexpected death in a 4-month-old infant with Peter's Anomaly and congenital glaucoma. At autopsy, the granular histiocytoid cells that define this entity were found predominantly involving the conduction system, with encasement and partial replacement the His' Bundle. Large aggregates of these cells formed atrioventricular and nodoventricular connections, indicating a possible mechanism for the arrhythmias characteristic of the condition. The striking propensity for involvement of the conduction system in this case lends further support to the view that this disorder represents a developmental anomaly of the Purkinje cell system of the heart. PMID- 8617488 TI - Prothrombotic activation of pulmonary arterial endothelial cells in a patient with tuberculosis. AB - Activation of endothelial cells occurs in response to numerous physiological stimuli and results in the concerted expression of endothelial cell proteins that change the nonthrombogenic intimal surface of a vessel into a thrombogenic surface, with the subsequent development of local thrombosis. For example, both type 1 plasminogen activator inhibitor and tissue factor expression are mediated by endothelial cell stimulation in vitro; however, in contrast to type 1 plasminogen activator inhibitor, it has been difficult to detect tissue factor associated with endothelial cells in vivo. This case study describes the presence of both type 1 plasminogen activator inhibitor and tissue factor antigen associated with pulmonary arterial endothelial cells of a patient exhibiting a mycobacterial infection. The disease was associated with chronic hemoptysis and characterized by extensive tissue destruction and local thrombosis within the pulmonary artery. The data show that conditions occur in vivo in which local thrombosis is associated with increased levels of type 1 plasminogen activator inhibitor and tissue factor. PMID- 8617489 TI - Prostatic tissue in benign cystic ovarian teratomas. AB - Benign prostatic tissue was found in three of 25 benign cystic teratomas of the ovary. Our finding may indicate that prostatic tissue in mature ovarian teratomas is more common than currently reflected in literature. PMID- 8617490 TI - Tendon sheath giant cell tumors. PMID- 8617491 TI - Certification and training in molecular pathology. PMID- 8617492 TI - Susceptibility to insulin-dependent diabetes mellitus maps to a locus (IDDM11) on human chromosome 14q24.3-q31. AB - To locate genes predisposing to insulin-dependent diabetes mellitus (IDDM), an autoimmune disorder resulting from destruction of the insulin-producing pancreatic cells, we are testing linkage of IDDM susceptibility to polymorphic markers across the genome using families with two or more IDDM children. A new susceptibility locus (IDDM11) has been localized to chromosome 14q24.3-q31 by detection of significant linkage to microsatellite D14S67, using both maximum likelihood methods (LODmax = 4.0 at Theta = 0.20) and affected sib pair (ASP) methods (P = 1 x 10(-5)). This represents the strongest reported evidence for linkage to any IDDM locus outside the HLA region. The subset of families in which affected children did not show increased sharing of HLA genes (HLA sharing A mutation at position -1 of the exon 8 splice donor site results in skipping of exon 8 in 97% of the LAL hnRNA originating from this allele, while 3% are spliced correctly, resulting in full-length LAL enzyme. The mutant LAL mRNA codes for a protein lacking amino acids 254 to 277. On the other allele, a G --> T mutation leads to a premature stop codon at Gly245, resulting in inactive LAL enzyme. In addition, the previously identified Leu179 --> Pro mutation is present on this allele, and the LAL mRNA is rendered unstable by the premature stop codon. Analysis of two children with Wolman disease showed that both were homozygous for a G --> A mutation at position +1 of the same splice donor site as for the CESD patient, leading to skipping of exon 8. In contrast to the CESD patient, no correctly spliced mRNA was detectable. We have also expressed a wildtype LAL cDNA and the mutant LAL cDNA from one Wolman patient in Sf9 and H5 insect cells. We demonstrate that the LAL enzyme generated from the wildtype LAL cDNA was active in homogenates from Sf9 and H5 cells, while the enzyme with the internal deletion of 24 amino acids originating from the LAL cDNA of the Wolman patient was not. The combined data provide evidence that the only functionally relevant genetic difference between the Wolman patients and the CESD patient is that the splice defect in Wolman, which affects one of the invariable nucleotides of the splice consensus sequences (position +1), does not permit any correct splicing, whereas the defect observed in CESD (position -1) allows some correct splicing (3% of total LAL mRNA) and therefore the synthesis of functional enzyme. PMID- 8617514 TI - Framework for a physical map of the human 22q13 region using bacterial artificial chromosomes (BACs). AB - Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points. PMID- 8617515 TI - A polymorphic and hypervariable locus in the pseudoautosomal region of the CBA/H mouse sex chromosomes. AB - We have identified a genomic locus (DXYH1) that is polymorphic and hypervariable within the CBA/H colony. Using a panel of C57BL/6 x Mus spretus backcross offspring, it was mapped to the distal end of the X chromosome. Pseudoautosomal inheritance was demonstrated through three generations of CBA/H x CBA/H and CBA/H x C57BL/6 crosses and confirmed through linkage to the Sxr locus in X/Y Sxr x 3H1 crosses. Meiotic recombination frequencies place DXYH1 similar 28% into the pseudoautosomal region from the boundary. The de novo generation of CBA/H variant DXYH1 restriction fragment length polymorphisms during spermatogenesis is suggestive of the germline instability associated with hypermutable human minisatellites. The absence of DXYH1-related sequences in Mus spretus provides DNA sequence evidence to support the observed failure of X-Y pairing during meiosis and consequent hybrid infertility in C57BL/6 x Mus spretus male F1 offspring. PMID- 8617516 TI - 3-Hydroxy-3-methylglutaryl CoA lyase (HL): mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients. AB - 3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3 hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, we found 2 of 33 HL deficient probands to be homozygous for large deletions in the HL gene. PMID- 8617517 TI - Isosexual precocious puberty in a boy with meningomyelocele and cerebral arachnoid cyst. PMID- 8617518 TI - Is INH alone enough for prophylaxis. PMID- 8617519 TI - Palatal palsy in enteric fever. PMID- 8617520 TI - Knowledge, attitude and practice of health workers in immunization. PMID- 8617521 TI - Waugh's syndrome. PMID- 8617522 TI - Chloramphenicol-furazolidone combination in enteric fever. PMID- 8617523 TI - Hereditary pancreatitis with lithiasis in a 7-year-old boy. PMID- 8617524 TI - Congenital segmental dilatation of colon. PMID- 8617525 TI - Megalourethra. PMID- 8617526 TI - Management of the angry parents of severely ill children. PMID- 8617527 TI - A comparison of a 30-cluster survey method used in India and a purposive method in the estimation of immunization coverages in Tamil Nadu. AB - A 30-cluster survey method that is employed for estimating immunization coverages by the Government of India (GOI) was compared with a Purposive method, to investigate whether the likely omission of SC/ST and backward classes in the former would lead to the reporting of higher coverages. The essential difference between the two methods is in the manner in which the first household is selected in the chosen first stage sampling units (villages). With the GOI method, it is often close to the village centre, whereas with the Purposive method it is always in the periphery or in a pocket consisting of SC/ST or backward classes. A concurrent comparison of the two methods in three districts in Tamil Nadu showed no real differences in the coverage with DPT and BCG vaccines. However, the coverage was consistently higher by the GOI method in the case of the Polio vaccine (by 1.5%, 3.1% and 5.3% in the 3 districts), and the Measles vaccine (by 4.8%, 13.3% and 13.9%); the average difference was 3.3% for Polio vaccine (p = 0.08) and 7.3% for Measles vaccine (p = 0.01). PMID- 8617528 TI - Comparative efficacy of furazolidone and nalidixic acid in the empirical treatment of acute invasive diarrhea: randomized clinical trial. AB - Efficacy of furazolidone and nalidixic acid was compared in a randomized trial involving 72 children with acute invasive diarrhea. Thirty six children received furazolidone (7.5 mg/kg/day) and 36 children received nalidixic acid (55 mg/kg/day). Clinical characteristics of the two treatment groups were comparable on admission. Of these, 34 children in furazolidone treated group and 29 children in nalidixic acid treated group completed the full course of treatment and were analyzed finally for clinical efficacy. Clinical cure was observed in 29(85.3%) children treated with furazolidone and 29(100.0%) children treated with nalidixic acid. Nalidixic acid treated group had statistically significantly higher cure rate (p = 0.039) as compared to furazolidone treated group. However, 85% cure rate in furazolidone treated group may be potentially useful for the treatment of acute invasive diarrhea because of decreasing efficacy of nalidixic acid against shigellosis in many countries. PMID- 8617529 TI - A randomized controlled trial of vitamin A supplementation in acute diarrhea. AB - Effect of vitamin A supplementation on duration of diarrhea was evaluated in 108 cases between 6 months to 5 years of age suffering from acute diarrhea of less than 3 days duration and results were compared with equal number of age and sex matched controls having comparable feeding pattern, nutritional and socio economic status and clinical profile who did not receive vitamin A supplementation. There was no significant difference in the mean duration of diarrhea in cases who received vitamin A and the controls. However, on subgroup analysis of the study and control groups a significant (p = 0.009) beneficial effect of vitamin A supplementation was noticed in cases who had a pre-existing vitamin A deficiency with CIC stage 3/5 and above. Even though vitamin A supplementation in malnourished children did not significantly alter the duration of diarrhea, a beneficial effect was observed in children who had CIC state 3 and above in association with malnutrition (p = 0.025). Our results indicate that vitamin A supplementation does not significantly reduce the duration of a diarrheal episode. However, in children with pre-existing vitamin A deficiency particularly those who have associated malnutrition it may have a beneficial effect. PMID- 8617530 TI - Sexually transmitted diseases in children. AB - Fifty eight (16%) of the 362 patients, who reported to Dermatology and STD Department with symptoms pertaining to their genitourinary system were below 14 years of age. Fifty four (93.1%) of these children belonged to families of lower socio-economic strata. All the children were slum dwellers and none of them had studied beyond the third class. Syphilis was seen in 27.6%, gonorrhea in 24.1%, chancroid in 22.4%, candidiasis in 10.3%, condylomata acuminata in 6.9% and herpes genitalis in 6.9% of these children. The probable reasons for the increased prevalence of sexually transmitted diseases in children as ascertained by this study were sexual promiscuity and probably sexual assault. PMID- 8617531 TI - Evaluation of cold chain system in rural areas of Haryana. AB - Evaluation of cold chain system was done in a time bound study during August and September months of 1992 in two districts of Haryana as there were frequent breakdowns of icelined refrigerators during the previous year. The study revealed that defective stabilizers and electricity plugs and sockets were the reason of breakdown in many cases. Temperature maintenance and functioning of deep freezers was satisfactory. Retrospective analysis showed that the polio vaccine samples picked up during 1990-92 were found to be satisfactory by CRI, Kasauli. Use of two ice-pick carrier and thermos flasks was associated with poor temperature maintenance. Seven vaccine carriers out of 25 examined had cracked wall lining. Lids of carriers were also not kept tight during vaccination sessions. Response lag of the health workers and medical officers in case of breakdowns was delayed. A one day refresher course exclusively on cold chain maintenance at community health centre level is recommended. PMID- 8617532 TI - Lung functions in malnourished children aged five to eleven years. AB - Lung functions including FVC, FEV1, PEFR, %FEV1/FVC and Empey Index were measured using P.K. Morgan's pocket spirometer on 122 children of both sexes in the age group of five to eleven years. There were 80 (65.57%) malnourished children according to the Indian Academy of Pediatrics classification. FVC and FEV1 were significantly (p < 0.02) reduced in malnourished children. FVC (r = 0.67, p < 0.001), FEV1 (r = 0.68, p < 0.001) and PEFR (r = 0.53, p < 0.001) showed linear relationship with body surface area in all age groups. Empey index was less than 10 in both healthy and malnourished children. Abnormal respiratory functions should be interpreted with caution in malnourished children. PMID- 8617533 TI - Effecting attitudinal change towards rational drug use. AB - Attitudes of 40 interns towards rational drug use (RDU) were assessed, using a standardized Likert type scale. The assessment was repeated after 4 months to evaluate the effect of usual working conditions of the hospital. After this period, the attitudes had slided towards negative side (p < 0.01). At this point, an intervention in the form of a workshop was provided for half the group while other half served as control. A repeat assessment after another period of 4 months revealed that the attitudes of test group returned towards positive side (p < 0.01) while control group maintained its negative attitudes. PMID- 8617534 TI - Utilization of ICDS scheme in children one to six years of age in a rural block of central India. AB - The evaluation of nutritional and immunization services was undertaken in the rural ICDS block Sanwer (Madhya Pradesh) where the project is functioning from last 3 years. A door to door survey was conducted in 1993 in six Anganwadi areas in ICDS block and five randomly selected matched non ICDS rural area served as controls. There were a total of 709 children in ICDS and 500 in non ICDS block in 1-6 years age group. The difference was not statistically significant for nutritional status in the two blocks, but a remarkably better immunization status (p < 0.005) was observed in non ICDS block. The coverage for DPT (3 doses), and measles vaccination in ICDS block was 79.57% and 45.7%, respectively, while in non ICDS block it was 94.4% and 62.03%, respectively. It seems the ICDS scheme is under utilized by the community and requires immediate attention by the health authorities. PMID- 8617535 TI - Missed opportunities for immunization in children under 2 years attending an urban teaching hospital. AB - A cross-sectional survey was done to assess the missed opportunity for immunization (MOI) in children under two years of age attending Medical Outpatient, Newborn Follow-up Service and Immunization Clinic of Institute of Child Health and to evaluate interventions. Baseline survey phase-I was done and two interventions: (i) education and awareness of immunization among health personnel; and (ii) attaching immunization slip to the outpatient form were done. After each intervention phase-II and phase-III surveys were carried out. The data from the different phases were analyzed for the effect of interventions. The total number of children surveyed were 634; 423 from Medical Outpatients, 108 from Newborn Follow-up Service and 103 from immunization Clinic. MOI was 35.5%, 23.1% and 9.7% in the above health facilities, respectively. After intervention I, the MOI was 24.5% and 12.2% in Medical Outpatient and Newborn Follow-up Service and none in Immunization Clinic. After intervention-II there was an improvement in immunization of 18.4%, 30.4% and 16.0% in the three health facilities mentioned above. MOI was avoided because the medical officers advised immunization in the above children. The difference in the MOI among Medical Outpatient and Immunization Clinic between baseline, phase-I and phase-II were significant (p < 0.001). It is concluded that MOI can be brought down by creating awareness periodically and that attaching an immunization schedule to the outpatient forms is an effective method of reducing MOI. PMID- 8617536 TI - Water electrolyte homeostasis in acute bronchiolitis. AB - Children with acute bronchiolitis frequently require hospitalization and parenteral fluid therapy. Water retention due to impaired renal water excretion has been described in several pulmonary conditions in children. We studied 20 infants (3.6 +/- 2.9 months), hospitalized consecutively for acute bronchiolitis for water and electrolyte changes during the acute stage and compared them to those on recovery. Serum sodium and plasma osmolality, urinary sodium and osmolality were measured in all infants. Ten infants each were assigned alternatively to study body water compartment or renal water handling (water load excretion and free water excretion capacity) on the day of hospitalization and after recovery. Mean ( +/- SD) value of serum sodium of the infants at admission was 132.7 +/- 7.2 mEq/L which increased to 137.1 +/- 5.4 mEq/L on recovery (p < 0.05). Plasma osmolality changed from 284 +/- 14 mOsm/kg at admission to 294 +/- 10 mOsm/kg at recovery (p < 0.05). There was a significant decrease in urinary sodium from 54 +/- 39 mEq/L to 20 +/- 18 mEq/L and urinary osmolality from 415 +/ 213 mOsm/kg to 252 +/- 204 mOsm/kg at admission and at recovery, respectively. All 10 infants showed significant increase in total body water (mean +/- SD; 22.8 +/- 7.5 ml/kg) at admission as compared to that at recovery. The total body water (TBW) excess was mainly in extracellular water compartment (16.3 +/- 3.6 ml/kg). Seven of 10 infants had significant impairment in renal water excretion. Increase in maximum free water clearance of these 7 infants on recovery was 0.69 +/- 0.27 ml/min, i.e., 15 times more than that at admission. It is concluded that bronchiolitis of infancy is characterized by water retention which is caused by impaired renal water excretion. In the management of severe bronchiolitis careful attention to fluid therapy is mandatory; liberal fluid therapy may lead to water intoxication. PMID- 8617537 TI - A multicentre collaborative study of the care of mothers and infants with a comprehensive MCH care package utilizing high risk approach strategy at primary health centres: summary, conclusions and recommendations. PMID- 8617539 TI - Parental awareness and practices in acute diarrhea. PMID- 8617538 TI - Knowledge of Anganwadi workers about growth monitoring in Delhi. PMID- 8617541 TI - Congenital contractural arachnodactyly. PMID- 8617540 TI - Knowledge of college girls on child health. PMID- 8617542 TI - Recurrent Cardiac tamponade: intrapericardial teratoma. PMID- 8617543 TI - Undergraduate pediatric education in India: current concepts. PMID- 8617544 TI - Intrahepatic biliary dilatation with aortoarteritis. PMID- 8617545 TI - Klippel Trenaunay Weber syndrome associated with abdominal hamartoma with undescended testis. PMID- 8617546 TI - Congenital erythropoietic porphyria. PMID- 8617547 TI - Developmental disabilities. PMID- 8617548 TI - Ultrasonography of the brain in preterm infants and its correlation with neurodevelopmental outcome. AB - Two hundred and eighteen preterm neonates had ultrasonography (USG) brain done on third, and/or seventh and fourteenth day of life. Fifty eight (26.3%) had intraventricular/ periventricular hemorrhage, 3 had parenchymal lesions. 46 had Grade I hemorrhage, 9 had Grade II, 2 had Grade III and 1 had Grade IV hemorrhage. Grade III and IV hemorrhages occurred in neonates below 34 weeks gestation. There was an inverse relationship between gestation age and hemorrhage (p = 0.0001). A comparison of incidence of hemorrhage between preterms who were appropriate for gestational age was not significant. Out of the 63 neonates who had serial USGs on the third and seventh day of life, 15 of the 16 bleeds (94%) were detected on the third day itself, indicating it to be a opportune time for doing an USG. USG at term (40 weeks postconceptual age) was done in 99 infants to see if it could correctly predict the neurodevelopmental outcome using the Bayley Scales of Infant Development. Out of these 99 infants, 72 came for the developmental assessment at one year. One neonate who had periventricular leucomalacia with cystic changes on USG at term, was grossly abnormal with cerebral palsy and mental retardation. Six infants showed delayed development with a mean mental development quotient of 79.1 +/- 1.72 at 2 years. The specificity of USG at term for predicting outcome was 89.2% and negative predictive outcome was 90%, indicating that a normal USG at term predicted a good neurodevelopmental outcome. PMID- 8617549 TI - Significance of left ventricular inflow gradients in patients with ventricular septal defect. AB - Hemodynamic data of 167 patients of isolated ventricular septal defect (VSD) was retrospectively analyzed for the presence of left ventricular inflow gradients. End diastolic gradients of > 5 mm Hg between the pulmonary artery wedge pressure and the left ventricular end diastolic pressure were recorded in 40 of these patients. In three of these cases, left atrium was also entered and identical pressure gradients were recorded between the left atrial pressure and the left ventricular end diastolic pressure. Two dimensional and Doppler echocardiographic or operative findings were available in 32 of the 40 patients. No statistical correlation was found between the presence and degree of left ventricular inflow gradients at end diastole and the degree of left to right shunt. Out of a total of 40 patients with left ventricular inflow gradients, gradients of 6-10 mm Hg were present in 24 patients. Echocardiographic or operative findings available in 19 of these did not show any left ventricular inflow obstruction. Enddiastolic gradients of 11-15 mm Hg were present in 14 patients. Echocardiographic or operative findings were available in 11 of these and one of these had congenital mitral stenosis at surgery. End diastolic gradients of more than 15 mm Hg were present in 2 patients and one of these had congenital mitral stenosis at surgery. Thus organic left ventricular inflow obstruction is rare with inflow mitral gradients of upto 15 mm Hg in patients of VSD. PMID- 8617550 TI - Biophysical profile of blood pressure in school children. AB - The study was conducted in an industrial and prosperous city of Punjab to evaluate the biophysical profile of blood pressure (BP) in apparently healthy school children. A total of 2560 children between the ages of 5-15 years were enrolled. Their age, religion, dietary and family history were recorded. Weight and height of all children were measured and body mass index (BMI) calculated. A value of 2.26 or more was taken as obesity. BP measurements were made as per recommendations of the American Heart Association. Systolic as well as diastolic BP increased with age in both sexes, correlation coefficients being 0.59 and 0.6, respectively. A statistically significant linear relationship between BP and weight and height was noted. Children with BMI of > 2.26 had a significantly higher BP (P < 0.01). The mean BP did not vary among different religions. The BP of vegetarians and also non- vegetarians also did not differ. A family history of hypertension was associated significantly with elevated BP (p < 0.01). It is concluded that obesity and a family history of hypertension in children are associated with elevated BP and such children may be at risk for developing hypertension at a later date. They should be followed up and considered for modification of risk factors. PMID- 8617551 TI - Clinical profile of cholera in young children--a hospital based report. AB - Clinical profile of cholera was studied in children attending Diarrhea Training and Treatment Unit from January-December 1993. Out of a total 8714 cases of acute watery diarrhea, 64 children (0.7%) were suspected to have cholera on the basis of acute onset loose water/rice watery stools, high purge rate with or without excessive vomiting and/or severe dehydration. Stool culture was positive for cholera in 33 cases (51.6%). All the isolates were V. cholerae 01 biotype El Tor serotype Ogawa. Sixty four per cent of stool culture positive cases were below 5 years of age. The results assume importance because out of 28 children < 2 years with clinical suspicion of cholera, 11 cases (39.3%) were culture positive for V. cholerae, youngest child being 3 months old. Comparison of various parameters revealed that presence of vomiting > 4 episodes/ day (p < 0.005), frequency of stools >12/24 hours (p <0.002), rice watery stools (p < 0.01) and presence of severe dehydration (p < 0.01) were significant parameters associated with positive stool culture. Beside examination of stool sample by hanging drop method was an excellent diagnostic tool (p < 0.001) with a sensitivity of 51.5%, specificity 100% and positive predictive value of 100%. The isolates of V. cholerae were susceptible to furazolidone, cephelexin, nalidixic acid, norfloxacin and gentamicin. Our observations indicate that cholera is not uncommon in infants and young children. Like children in the older age group, acute onset diarrhea with watery/rice watery stools and high purge rate with or without excessive vomiting and/or rapid development of severe dehydration should arouse suspicion of cholera in younger children also. They should be investigated for cholera even in non-endemic areas and in the absence of cholera outbreaks. PMID- 8617552 TI - Functional and behavioral responses as marker of illness, and outcome in infants under 2 months. AB - Value of abnormal findings on 11 functional and behavioral items were studied for identification of serious illness, presence of bacteremia and prediction of the outcome (recovery or death) in infants upto 2 months of age. All the items were graded on a 3 point scale (0, 1 and 2) in the ascending order of severity. A total of 116 infants who were being evaluated for suspected sepsis were enrolled. The assessment was completed before detailed history and physical examination. Significant associations were observed between presence of serious illness and six items by lambda2-test and/or Pearson's correlation and multiple stepwise regression analysis. These were decreased activity, abnormal quality of cry, presence of pallor, fast breathing, decreased consolability, and consciousness level. The sensitivity of six item model was > 90% and negative predictive value >95%. The negative predictive value of several individual items was also above 90%. Consciousness level was the most important predictor of the outcome followed by poor feeding, hydration, color, consolability and abnormal expression (combined multiple R = 0.51). A total score of 7 or more on above six items had a sensitivity of 80%, and negative predictive value of 97% for death. In conclusion, behavior and functional responses (as mentioned above) were fairly useful in predicting the outcome and/or the severity of the illness. These items may be combined to develop a scale to help in therapeutic decision making. PMID- 8617553 TI - Detection and prevention of childhood disability with the help of Anganwadi workers. AB - OBJECTIVES: To evaluate the role of Anganwadi Workers (AWW) for detection and prevention of disability in children below 6 years of age. DESIGN: Cross sectional and longitudinal follow up. SETTING: Ten Anganwadi Centers in ICDS Urban Project. METHODS: Trained AWWs identified disabilities and instituted preventive measures like immunization and supplementary nutrition. Simultaneous independent verification by pediatricians. Repeat survey after 6 mo of follow-up. RESULTS: Amongst the 1545 children, AWW identified disability in 126 subjects which were verified in 118 cases by pediatricians. The disability rate was 7638 per 100,000 population. Visual, mental, orthopedic, speech and hearing disabilities rates were 4790, 2654, 583, 518 and 453 per 100,000 population, respectively. In the repeat survey, 35 of the 74 children with visual disability (mostly xerophthalmia), 4 of the 9 with orthopedic disability and 3 of the 7 with hearing disability could be managed satisfactorily. CONCLUSIONS: AWW can help in early detection and appropriate management of incipient and preventable childhood disabilities. PMID- 8617554 TI - A comparative clinical trial of albendazole versus metronidazole in children with giardiasis. AB - The adverse effects and treatment failures to some of the currently recommended drugs for giardia infection have given rise to the need for alternative antigiardial agents. In an open, randomized parallel group study, the safety and efficacy of albendazole was compared with metronidazole for the treatment of giardiasis in children. Sixty four children of age ranging from 2-12 years was randomized to receive either albendazole suspension 400 mg daily for 5 days or metronidazole suspension 400 mg daily for 5 days or metronidazole suspension 7.5 mg/Kg thrice daily for 5 days. The mean days required for cure, as evident by absence of cysts and/or trophozoites in the stool specimen, were 3.7 +/- 1.4 and 4.5 +/- 1.1 days, respectively for children on albendazole and metronidazole therapy. Six children on metronidazole therapy developed anorexia 2 to 4 days after the treatment. Albendazole proved as effective as metronidazole in the treatment of giardia infection in children with the added advantage of the absence of anorexia. PMID- 8617556 TI - Therapeutic uses of methyl prednisolone. PMID- 8617555 TI - Why the act? PMID- 8617557 TI - Late onset congenital syphilis. PMID- 8617558 TI - 48 XXXY variant of Klinefelter syndrome. PMID- 8617559 TI - Expression of fragile Xq 27.3 in a patient with clinical features of deletion 9p syndrome. PMID- 8617560 TI - Treatment of severe Salmonella typhimurium infection with ciprofloxacin. PMID- 8617561 TI - Enteric cholecystitis 15 years experience. PMID- 8617562 TI - Pseudohypoaldosteronism in a family with variable presentation. PMID- 8617563 TI - Pancreatoblastoma. PMID- 8617564 TI - Biliary ascites caused by perforation of choledochal cyst. PMID- 8617565 TI - Form fruste choledochal cyst. PMID- 8617566 TI - Acute laryngotracheitis. PMID- 8617567 TI - Anaphylaxis following oral cotrimoxazole. PMID- 8617568 TI - Intestinal parasites in children from middle income families. PMID- 8617569 TI - Acute acalculous cholecystitis in typhoid fever. PMID- 8617570 TI - Spoon feeds--an alternative to bottle feeding. PMID- 8617571 TI - Hepatitis B vaccine. PMID- 8617572 TI - Preclinical pharmacokinetics and antitumor activity of imexon. AB - Imexon is an aziridine compound originally studied for immune-enhancing effects on lymphocytes. The drug was well-tolerated in humans and was shown to be active in a variety of animal tumor models. Recently, imexon has demonstrated antitumor activity in human multiple myeloma cell lines in vitro. The pharmacokinetics of the compound using normal phase HPLC assay were studied in normal mice and in dogs with mast cell tumors. Doses of 100 mg/kg given intraperitoneally produced peak plasma levels over 100 micrograms/ml in mice and the drug was rapidly eliminated with half lives of 8 minutes (alpha phase) and 29 minutes (beta phase). Only 20% of an oral imexon dose was absorbed in the mouse. In dogs, the alpha and beta phase half lives ranged from 18-26 minutes and 91-110 minutes, respectively. Peak levels over 100 micrograms/ml were obtained following intravenous doses of 12.5 mg/kg and 25 mg/kg. Imexon was active in mice bearing either P-388 or L-1210 leukemia, but not in mice with B-16 melanoma. These results suggest that cytotoxic drug concentrations can be obtained in vivo and that imexon is active in lymphoproliferative tumors. PMID- 8617573 TI - Investigation of the comparative effects of 2-chlorodeoxyadenosine on tumor colony forming units in vitro. AB - 2-CdA is a deaminase-resistant purine analogue which has shown clinical activity against various hematological tumors, and is currently undergoing clinical phase II trials. The objectives of our study were to determine the activity of 2-CdA against freshly explanted clonogenic cells from non-hematological human tumors and compare this agent with other clinically useful anticancer agents. We also compared short-term (1 hour) and long-term (21-28 days) exposures. For short-term exposure (1-hour), final concentrations were 0.57, 5.7, 57, and 114 ng/ml. Inhibition of tumor specimens was concentration-dependent: 0.57 ng/ml: 1/51 (2%), 5.7 ng/ml: 4/52 (7%), 57 ng/ml: 11/52 (21%), 114 ng/ml: 27/50 (54%). At concentrations > or = 57 ng/ml, 2-CdA was as active as cisplatin, doxorubicin, 5 fluorouracil, mitomycin-C, vinblastine, and etoposide. For long-term exposures (21-28 days), final concentration of 2-CdA were 0.57, 5.7, and 57 ng/ml. At 0.57 ng/ml, 2-CdA was active in 4/54 (7%) specimens [5.7 ng/ml: 13/54 (24%), 57 ng/ml: 40/54 (74%)]. A head-to-head comparison with short-term exposures demonstrated greater activity if the drug exposure time was extended. Using the strategy for testing other standard agents (in vitro dose of 1/10th achievable peak plasma concentration), one would predict clinical response rates for single agent bolus or short-term administration of 2-CdA to be in the neighborhood of 7%. Longer durations of infusion or multiple doses might increase the response rate to about 24%. If higher peak plasma concentration could be achieved, dose-dependent increases in clinical responses might be achievable. We conclude that 2-CdA is active against clonogenic cells from freshly explanted non-hematological human tumor specimens at high concentrations. PMID- 8617574 TI - Activity of the morpholino anthracycline 3'-deamino-3'-morpholino-13-deoxo-10 hydroxycarminomycin (MX2) against human tumor colony-forming units in vitro. AB - In several preclinical systems, the morpholino anthracycline MX2 has greater antitumor activity than doxorubicin, is less cardiotoxic, and is effective against multidrug resistant cancer cells. We used a human tumor soft-agar cloning assay to test the cytotoxicity of MX2 against single cell suspensions from freshly obtained human tumors. Tumor cells were exposed to MX2 at 0.05 and 0.5 micrograms/ml either for 1 hour (2-1 specimens; 77 [38%] assessable) or continuously (231 specimens; 91 [39%] assessable). Superior antitumor activity was observed with continuous exposure (19% in vitro response at 0.05 micrograms/ml and 69% at 0.5 micrograms/ml) than with 1-hour exposure (1.3% at 0.05 micrograms/ml and 12% at 0.5 micrograms/ml). Activity was seen against all types of cancer tested including renal (91%), melanoma (88%), ovarian (73%), breast (71%) and non-small-cell lung (67%) cancer at a MX2 concentration of 0.5 micrograms/ml after continuous exposure. If appropriate plasma levels can be achieved in patients, MX2 could have significant clinical activity with those tumors. PMID- 8617575 TI - Phase I and pharmacokinetic study of KW-2149 given by 24 hours continuous infusion. AB - KW-2149 is a new mitomycin C (MMC) analog, forming DNA-DNA and DNA-protein crosslinking 20-fold more effectively than MMC. Because of its equal or superior in vitro and in vivo activity compared to MMC, a phase I study was initiated with an intravenous bolus injection every three weeks. This study was interrupted after dose escalation from 5 mg/m2 to 100 mg/m2 because of subacute and dose dependent pulmonary toxicity. Because of the lack of other end-organ toxicity, the moderate hematological toxicity and the observed antitumor effect, a second phase I study was initiated with a 24 hour continuous infusion. The starting dose was 50 mg/m2 and further escalation depended on observed pulmonary toxicity. Four patients were entered into this study and the received in total 17 courses. Toxicity was again mainly restricted to the lungs with one patient suffering grade 2 dyspnoe and another one grade 1 dyspnoe. Three patients had a substantial change in the carbon monoxide (CO) diffusion capacity. Pharmacokinetic data from these patients showed very low plasma levels both for KW-2149, as for both known metabolites M-16 and M-18. This study demonstrates that pulmonary toxicity continues to occur with KW-2149, in spite of the assurance of low plasma levels of both the parent compound and the known metabolites. The interesting activity of this compound has stimulated further in-depth research towards mechanisms of pulmonary toxicity and means of preventing them. PMID- 8617576 TI - Phase II trial of gallium nitrate, amonafide and teniposide in metastatic non small cell lung cancer. An Eastern Cooperative Oncology Group study (E2588). AB - Fifty-five patients with metastatic non-small cell lung cancer (NSCLC) were entered into this phase II randomized study for evaluating three new agents: gallium nitrate, amonafide and teniposide. The patients had to have ECOG performance status 0 or 1, no prior chemotherapy, and adequate hematological, hepatic and renal functions. Forty-seven patients were eligible and evaluable. Fourteen were randomized to receive gallium nitrate, 18 to amonafide and 15 to teniposide. Seventy-four percent of eligible patients were male. The majority of patients (89%) had an ECOG performance status 1. ECOG grade 4 toxicity occurred twice in patients on gallium nitrate, seven times on amonafide and 18 times on teniposide. The cause of death was attributed to amonafide in one patients (from sepsis) and to teniposide in two patients (due to infection and leukopenia). There was no objective response in all the patients entered. The overall survival times ranged from 2 weeks to 156 weeks with a medium of 23 weeks. There were no survival differences among the three treatment arms. We conclude that gallium nitrate, amonafide and teniposide are inactive in metastatic NSCLC and do not warrant any further testing in this disease. PMID- 8617577 TI - Evaluation of merbarone (NSC 336628) in disseminated malignant melanoma. A Southwest Oncology Group study. AB - Merbarone, NSC 336628, is an investigational anticancer drug with activity against experimental animal tumors including melanoma. This paper presents results of a Phase II clinical study of merbarone in patients with biopsy proven stage IV malignant melanoma without prior chemotherapy and with no evidence of CNS involvement. Thirty-five patients with median age 58 (range 27-81), with performance status 0-2 were treated with merbarone 1000 mg/m2/day for five days by intravenous continuous infusion repeated every 3 weeks. All patients (21 males and 14 females) were evaluable for toxicity. Two patients were not evaluable for response having been removed from protocol treatment due to toxicity and received other treatment during the first course of chemotherapy. Among the evaluable patients there was one complete response in a supraclavicular lymph node lasting four months and one partial liver response lasting three months. The remaining thirty-one patients were non-responders. Of these one had a stable disease lasting 21 months. The overall objective response rate was 6% (2/35) with a 95% confidence interval of 1%-19%. Twenty-six of the 35 patients have died. The estimated median survival of the entire group was 9 months with a 95% confidence interval six to eleven months. Renal toxicity was dose-limiting and manifested as increasing serum creatinine (54% of patients), proteinuria (51%) and hematuria (9%). One patient experienced grade 4 creatinine increase, proteinuria and acute renal failure. Other toxicities included nausea (71%), vomiting (51%0, malaise (23%), weakness (20%), alopecia (17%), diarrhea (17), anorexia (14%) transaminase (SGOT, SGPT) increase (14%), constipation (14%), alkaline phosphatase or 5'nucleotidase increase (9%), and fever (9%). Hematologic toxicity (granulocytopenia, leukopenia, and anemia) was generally mild and infrequent (29%, only one patient had grade 4 granulocytopenia). Overall 9 patients (26%) had at least one grade 3 toxicity. We conclude that merbarone at this dose and schedule has detectable but minimal activity in the treatment of metastatic malignant melanoma and given the significant renal toxicity this schedule does not merit further evaluation in this disease. PMID- 8617578 TI - A phase II and pharmacokinetic study of 6S-leucovorin plus 5-fluorouracil in patient with colorectal carcinoma. AB - Leucovorin (LV) is commonly used as a modulator of 5-fluorouracil (5-FU) cytotoxicity. In patient with colon cancer, the addition of LV to 5-FU improves response rates, and in some trials has improved survival in advanced disease and in the adjuvant setting. Leucovorin is generally administered as a racemic mixture, but the isomers differ substantially in pharmacokinetics and biological activity, with 6S-LV the predominant active component. The current study was undertaken to determine the effect of 6R on the pharmacokinetics of 6S-LV, and to characterize the toxicity and antitumor effect of 5-FU when administered with 6S LV to patients with advanced colorectal carcinoma. Thirty patients were treated with weekly 5-FU plus high dose 6S-LV. To determine the effects of 6R-LV on the pharmacokinetics of S6-LV, 20 patients were randomly assigned to receive either 250 mg/m2 6S-LV or 500 mg/m2 6R,S-LV as a 2 hour IV infusion on day -2, and the other preparation on day -1, with pharmacokinetics measured each day. The presence of 6R-LV had no effect on the AUC, Clp, Cmax, or terminal phase t1/2 of 6S-LV. The overall response rate was 40% (C.I. 23-60%). The most frequent toxicities were gastrointestinal. In this small cohort, scheduled and delivered dose intensity was positively associated with response (p = 0.05). These results show that there is no pharmacokinetic advantage to the use of 6S-LV rather than 6R,S-LV as a modulator of 5-FU. PMID- 8617579 TI - Mitomycin C and menadione for the treatment of lung cancer: a phase II trial. AB - A phase II trial of menadione [2.5 gm/m2 as a continuous intravenous (i.v.) infusion over 48 hours] followed by mitomycin C (10-20 mg/m2 i.v. bolus) administered every 4 to 6 weeks was performed in 23 patients with advanced lung cancer. Menadione, a vitamin K analog which lowers intracellular pools of reduced glutathione (GSH), was combined with mitomycin C in an attempt to overcome thiol mediated resistance to alkylating agent chemotherapy. The median age of patients entered on this trial was 62 years; performance status ranged from 60-90%. Two of the 23 patients (9%; 95% confidence interval, 1% to 28%) had objective responses lasting 3.5 months and 13 months respectively, while 4 additional patients developed short unconfirmed responses (lacking follow-up response data to estimate response duration). Median survival for all patients was 5.5 months. Treatment with mitomycin C and menadione was well tolerated except for hematologic toxicity and cardiac events of unclear relationship to the study drugs. Thirty-one percent of treatment courses were complicated by grade 3 or 4 hematologic toxicity including one episode of hemolytic anemia. One patient developed interstitial pneumonitis. Two patients developed a decrease in left ventricular ejection fraction: one patient remained asymptomatic, but the other patient developed congestive heart failure. Although only 9% of patients had confirmed objective responses, 28% (5 of 18) of the patients with non-small cell lung cancer demonstrated biological activity (tumor regression fulfilling the criteria for objective response on a single occasion but 3 patients lacking a follow-up measurement to document response duration) to this combination of mitomycin C and menadione. We conclude that further studies of chemomodulation in non-small cell lung cancer are appropriate. PMID- 8617580 TI - Phase II trial of topotecan in patients with cisplatin-refractory germ cell tumors. AB - Fifteen patients with advanced, cisplatin-refractory germ cell tumors (GCT) were treated on a phase II trial with topotecan. None of the 14 evaluable patients achieved a complete or partial response. Myelosuppression was the major toxicity. The median nadir leukocyte count was 1.75 cells/mm3, neutrophil count was 1.55 cells/mm3, hemoglobin was 8.75 gm/dl, and platelet count was 20,500 cells/mm3. Topotecan is not efficacious in the treatment of cisplatin-refractory GCT. PMID- 8617581 TI - Phase II evaluation of didemnin B in hormonally refractory metastatic prostate cancer. A Southwest Oncology Group study. AB - Didemnin B, a dipsipeptide isolated from the Caribbean tunicate Trididemnum with antitumor and antiviral activity was evaluated in a phase II trial in the treatment of metastatic, hormonally refractory adenocarcinoma of the prostate. Thirteen patients were treated with didemnin B at 3.5 mg/m2 and 20 patients were treated at 6.3 mg/m2 intravenously every 28 days. Response was assessed every 8 weeks. Of 32 evaluable patients there was one partial response for an overall response rate of 3% (95% confidence interval of 0.1-16%). The most common toxicities were nausea, vomiting, and diarrhea. Serious cardiac and pulmonary toxicities were also noted. This drug does not appear to warrant further evaluation in this disease as a single agent. PMID- 8617582 TI - A phase II clinical trial of echinomycin in metastatic soft tissue sarcoma. An Illinois Cancer Center Study. AB - Echinomycin, a cyclic peptide in the family of quinoxaline antibiotics, was evaluated in patient with metastatic, soft tissue sarcoma not previously treated for metastatic disease. The starting dose of echinomycin was 1,200 mcg/m2 administered intravenously, once weekly x 4, followed by a two-week break. The protocol design called for dose escalation on subsequent cycles of therapy, but because of significant toxicity, dose escalation occurred in only 5 of 25 treatment cycles. Severe nausea and vomiting was the most common toxicity. No clinical responses were observed in the 12 evaluable patients. Echinomycin at this dose and schedule is inactive in metastatic soft tissue sarcoma. PMID- 8617584 TI - State-of-the-art instruments for measuring radon/thoron and their progeny in dwellings--a review. AB - Radon and radon progeny measurements are conducted world-wide for the assessment of the radiation dose to workers and the general public. In the last 10 years, a variety of instruments have been developed that utilize different principles of detection for different measurement applications. The various instruments and methods used today depend on whether radon or radon/thoron progeny are being measured, the type of radiation being detected, and also the duration of the measurement. Other important design criteria are applicability, portability, convenience, reliability, and cost considerations. The instruments under consideration use the following detection systems: pulse ionization chambers, electret ionization chambers, scintillation detectors with zinc sulfide ZnS(Ag), alpha particle spectrometers with silicon diodes, surface barrier or diffused junction detectors, registration of nuclear tracks in solid-state materials, and gamma-ray spectometry with NaI(TI) scintillation crystals or germanium lithium (GeLi) semiconductors. Discussed in this paper are the advantages and disadvantages of the various portable instrumentation used for measuring radon, thoron, and their progeny. Also provided is guidance for their application in the field. PMID- 8617583 TI - Phase I trial of diaziquone (AZQ) plus GM-CSF. AB - Diaziquone (AZQ) is a lipid soluble alkylating agent which was designed for increased CNS penetration. Its principle toxicity is myelosuppression. We conducted a phase I trial using AZQ in combination with GM-CSF to determine if the maximal tolerate dose (MTD) of AZQ could be escalated. Using GM-CSF on a standard schedule, we were unable to escalate the previously determined MTD of diaziquone with the use of this colony stimulating factor. PMID- 8617585 TI - A histological kidney study of uranium and non-uranium workers. AB - Kidney tissue sections were obtained at autopsy from seven persons with a history of low level occupational exposure to uranium and histologically compared in a blind study with similar sections obtained from six reference cases. The pathologist was unable to identify the uranium workers or any uranium-specific nephropathy. This suggests that the chronic low level of kidney uranium concentration experienced by these workers, which was an order of magnitude lower than the accepted permissible level of 3 microgram per gram, did not induce any identifiable permanent tissue damage. PMID- 8617586 TI - Dose equivalent near the bone-soft tissue interface from nuclear fragments produced by high-energy protons. AB - During manned space missions, high-energy nucleons of cosmic and solar origin collide with atomic nuclei of the human body and produce a broad linear energy transfer spectrum of secondary particles, called target fragments. These nuclear fragments are often more biologically harmful than the direct ionization of the incident nucleon. That these secondary particles increase tissue absorbed dose in regions adjacent to the bone-soft tissue interface was demonstrated in a previous publication. To assess radiological risks to tissue near the bone-soft tissue interface, a computer transport model for nuclear fragments produced by high energy nucleons was used in this study to calculate integral linear energy transfer spectra and dose equivalents resulting from nuclear collisions of 1-GeV protons transversing bone and red bone marrow. In terms of dose equivalent averaged over trabecular bone marrow, target fragments emitted from interactions in both tissues are predicted to be at least as important as the direct ionization of the primary protons-twice as important, if recently recommended radiation weighting factors and "worst-case" geometry are used. The use of conventional dosimetry (absorbed dose weighted by aa linear energy transfer dependent quality factor) as an appropriate framework for predicting risk from low fluences of high-linear energy transfer target fragments is discussed. PMID- 8617587 TI - Tritium intake by exposure to plastic case watches. AB - In recent years tritium has been used in plastic case watches as permanent light sources on watch dials. To measure the release of tritium through the plastic cases, 82 different waterproofed watches were immersed in a water bath for 24 h, and the tritium concentration of the water was measured. The mean tritium release rate was 24,400 Bq d(-1) ranging from 110-162,000 Bq d(-1). Parallel measurements were made to determine the tritium concentration in the urine of 108 wearers of plastic case watches. The mean tritium concentration in urine was 197 Bq L(-1) up to 1,133 Bq L(-1). The whole body dose resulting from exposure to plastic case is negligibly small but given the pathway of skin absorption, the annual skin dose is 3-4 times higher than the dose limit for the public. Plastic case watches are collector's items and are often kept with other watches in glass cabinets or other containers. Storage of a large number of such watches in one container causes tritium to diffuse through the plastic cases and to contaminate watches that did not contain tritium at first. If the container is more or less airtight, the tritium concentration in the container and the tritium release rate from the watches can reach levels up to 4 MBq d(-1). PMID- 8617588 TI - X-ray beam energy, scatter, and radiation risk in chest radiography. AB - Effective doses for patients undergoing chest radiography were computed utilizing updated weighting factors, published organ doses and measured entrance doses. The effective dose decreases with beam energy (kVp) and reaches a minimum value after 100 kVp, with values when a grid is used (6.90 microSv) being 145% higher at this energy than when no grid is used (2.82 microSv). The uncertainties associated with derivation of the tissue weighting factors are shown not to affect the point at which risk is minimized. Use of the effective dose as the measure of risk does not require special treatment of the remainder tissue as with use of effective dose equivalent. The effective dose required for radiographs of constant optical density was examined to incorporate the behavior of the detector's response to energy and compare results to previous work where exit dose was held constant. The effective dose at 120 kVp with a grid (6.84 microSv) is compared to an estimate of that associated with the current kVp distribution (14.55 microSv). Since image quality is enhanced by the grid, its use in conjunction with a beam energy of 120 kVp could maximize the benefit/risk ratio in chest radiography and should be considered for universal implementation. Such adoption could reduce the population risk compared to current practice. PMID- 8617589 TI - Organ and effective doses in the male phantom ADAM exposed in AP direction to broad unidirectional beams of monoenergetic electrons. AB - Organ and effective doses per unit of fluence are calculated through Monte Carlo simulation of radiation transport (MCNP code, version 4) for the mathematical adult male phantom, ADAM, placed in vacuum and irradiated in the AP direction with broad unidirectional electron beams. The electrons are monoenergetic in the energy range of 0.1 tp 10 MeV. Effective dose per unit of fluence increases with increasing electron energy from 8x10(-14) to 2x10(-10) Sv cm(-2). Corresponding effective dose equivalents are also calculated. At the high end of the energy range considered, the present values agree reasonably well with the results of a series of calculations performed with the MIRD-5 male phantom in the 5 to 46 MeV electron energy range, as published in ICRU Report 43 (1988). The difference between effective dose and effective dose equivalent is relatively small for electron energies between 1.5 and 10 MeV. However, the difference increases rapidly if the electron energy decreases. Below 0.5 MeV, effective dose equivalent is about a factor of 100 less than effective dose. In this low energy range, annual dose limits are reached sooner if effective dose instead of effective dose equivalent is used as parameter of risk assessment. Taking the directional dose equivalent as a substitute for effective dose introduces an unnecessarily large safety factor. PMID- 8617590 TI - Directional detector of gamma ray sources. AB - This work describes the design, development, and characterization of an ionization chamber to detect and locate gamma ray sources with intensities down to 100 mu Gy h(-1). The results show that sources of x ray and gamma radiation in the energy range of 14 to 1,250 keV can be located. The directional sensitivity of this type of monitor is dependent on several parameters, such as photon energy, intensity, and the degree of complexity in terms of the number and type of sources in a contaminated area. An application important to the Army is to identify hotspots in a contaminated area and thereby indicate to personnel safer paths around them. Another possible use for this type of monitor may be in security applications in the prevention of shipping or storing clandestine sources. PMID- 8617591 TI - Evaluation of Monte Carlo simulation of photon counting efficiency for germanium detectors. AB - The reliability of calculating the full-energy peak counting efficiency of germanium detectors by Monte Carlo simulation was evaluated by comparing MCNP-4 code results with measurements over a range of conditions. The measurements were performed for two detectors of 20% and 110% nominal efficiencies with a point source at two distances and with four different volume sources, including a reentrant beaker, placed on the end caps. The radionuclides were National Institute of Standards and Technology standard sources that emit photons between energies of 42.8 and 1,596.4 keV. Each detector was modeled in detail with respect to the dimensions of the detection volume and attenuation material in the can, dead-layer, and crystal holder foil. The measurements of the 42.8-keV photon were used to check dimensions and to change slightly the value of the dead-layer thickness so that the simulation agreed with measurements at this energy. After this adjustment, the average ratio of simulation results to measurements for 13 photon energies above 42.8 keV was between 0.97 and 1.03 for all sets of point and volume source comparisons. Ratios at individual energies were between 0.92 and 1.06 for the point source and between 0.94 and 1.09 for volume sources. The observed differences were consistent with the estimated standard deviations of simulation and of measurement, which ranged from 1 to 6% and typically were 2 to 3% except for larger uncertainties at low energies. On the basis of this comparison, simulation with the MCNP-4 code is equivalent to measurement with photon standards if the detector and source configuration can be modeled within a tenth of a millimeter. PMID- 8617592 TI - Comparative distribution of 241 Am and 239,240 Pu in soils around the Rocky Flats Environmental Technology Site. AB - The distribution and behavior of 241 Am and 239,240 Pu in soils from the buffer zone of the Rocky Flats Environmental Technology Site have been investigated. Concentrations of both radionuclides decreased at similar rates with soil depth. More than 80% of the total inventory of both contaminants was found in the upper 9 cm of the soils with over 50% of the inventory residing in the top 3 cm. Comparison with earlier studies indicate that the plutonium depth profile has not changed significantly over the last 25 y. The inventories of 241 Am and 239,240 Pu decreased with distance from the 903 Pad (a former waste storage site) according to a power function, and the plume extended mainly toward the east. The lateral movement of the two contaminants away from the 903 Pad was not significantly different. The median activity ratio of 241 Am: 239,240 Pu ranged from 17 to 19% and was independent of sampling location and soil depth. This observation provided further evidence that the movement of both contaminants is indistinguishable in the study area. Because of the strong correlation between the two radionuclides, 241 Am concentrations can then be used to infer 239,240 Pu by counting the 241 Am via gamma spectroscopy. PMID- 8617593 TI - Obliquity factors for 60 Co and 4, 10, and 18 MV x rays for concrete, steel, and lead and angles of incidence between 0 and 70 degrees. AB - The attenuation of 60 Co gamma rays and photons of 4, 10, and 18 MV x-ray beams by concrete, steel, and lead has been studied using the Monte Carlo technique for angles of incidence 0, 30, 45, 60, and 70 degrees. Transmission factors have been determined down to <2x10(-5) in all cases. The results show that deviation from the obliquity factor increases with angle but is not significant for angles <45 degrees. At a 70 degree angle of incidence and a transmission factor of 10(-5), the obliquity factor varies between 1.2 and 1.9 for concrete, between 1.4 and 1.7 for steel, and between 1.4 and 1.5 for lead for the range of energies investigated. This amounts to an additional 86 and 50 cm of concrete, 25 and 23 cm of steel, and 8 and 14 cm of lead for 60 Co and 18 MV x rays respectively. The results for 60 Co in concrete and lead are in good agreement with previously published experimental work. Fits to the data using mathematical models allow reconstruction of all data curves to better than 1% on average and 7% in the worst single case. PMID- 8617594 TI - Assessment of radioactive systemic uptakes by deconvolution of individual monitoring results. AB - The methods of interpretation presently available for evaluating individual radioactivity intakes from measured data involve difficulties connected with the adaptation of metabolic models to the situations encountered in practice. These difficulties essentially concern the definition of appropriate parameters for each encountered case, and very often- except for characterized incidents erroneous appreciation of the time course of contamination episodes and of th routes of entry. These considerations led us to develop a simplified method of interpreting monitoring data, by considering separately the data relating to routes of entry and those concerning systemic contamination, i.e., the contamination occurring after the transfer of radionuclides to the blood. An approach to interpreting measurements of systemic contamination is proposed in this study. This method is to calculate, from these measurements, the values for the activity absorbed daily from the routes of entry into the blood using the appropriate retention and excretion functions. a day-to-day follow-up of the absorbed activity becomes possible, thus enabling its real-time evolution to be recorded and easy to consult. A few applications of the method are described, including cases of acute tritium and uranium contamination and of chronic contamination by tritium, uranium, and iodine. The conditions and constraints required to validate the proposed approach are indicated. PMID- 8617595 TI - An in-situ method to measure a soil's undisturbed pore gas radon concentration, diffusion length for radon and air filled porosity. AB - Previous work has shown that work has shown that for soils of insignificant permeability (soils with permeability less than about 10(-12) m2) the important soil parameters for characterizing radon mobility in the soil are the soil's steady-state pore gas random concentration at depth (Cs), the soil's bulk diffusion length for radon (L) and the soil's air filled porosity (pa). Existing methods to measure these parameters have been based wholly or in part on measurements of soil samples taken to a laboratory for analysis. The drawbacks of this approach are twofold: (1) since soil structure can be quite heterogeneous, the sample may not have characteristics indicative of the site as a whole, and (2) since the parameters are dependent on soil structure and the soil structure of the sample may be changed in th e process of acquiring the sample, one may be changing the parameters that one is trying to measure. These problems can be avoided by using a totally in-situ method to measure Cs, L, and pa. This paper describes a totally in-situ method for simultaneously measuring the important soil parameters, based on measurements of the radon concentration as a function of time for the gas in a cavity in the soil. PMID- 8617596 TI - A comparison of values of annual limits on intake presented in ICRP 61 and 10 CFR part 20. AB - The latest (1991) major revision of the U.S. Nuclear Regulatory Commission regulations incorporates internal dose concepts and primary and secondary dose limits adapted from the recommendations of the International Commission on Radiation Protection Publication 30 published in 1979. This work compares values of the Annual Limits on Intake (ALIs) reported in the latest U.S. regulations with those recommended in the more recent, 1990, International Commission on Radiation Protection Publication 61. Overall trends in the updated ALI values compared to those in the regulations, as well as radionuclides exhibiting greater than +/-50% changes in these values, after correcting for differences in the primary dose limits, are reported. PMID- 8617597 TI - Comparison of risk for pre- and post-remediation of uranium mill tailings from vicinity properties in Monticello, Utah. AB - Pre- and post-remedial action dose rates were calculated on 101 Monticello, Utah, properties included in the Monticello Vicinity Property Remedial Action Project. Dose rates were calculated using the RESRAD computer code, which indicated that 98% of the effective dose equivalent was contributed by external gamma radiation and radon emanation. Radium concentrations in pCi g(-1) were averaged for pre- and post-remedial action measurements; point sources were not included in the averages. The volume of the deposit was also used in the dose calculation. In all cases the dose was reduced, and at 77 properties the dose was reduced to 0.30 mSv y(-1) (Department of Energy ALARA recommendation). A paired t-test showed a significant reduction (p<0.05) between the pre- and post-remedial action dose rates, The average cost remedial action, number of persons per household, number of properties remediated, and the reduction of cancer mortalities through remediation resulted in an approximate cost of 11,000,000 per life saved by remediation of mill tailings. PMID- 8617599 TI - Study of radiation doses to personnel in a cardiac catheterization laboratory. AB - Radiation safety guidelines and federal regulations (10 CFR Part 20) require that radiation workers who are likely to receive above a certain percentage of the effective dose equivalent limit be monitored and that exposure be maintained as low as reasonably achievable (ALARA). With few exceptions in our hospital, the badges that exceed the Level 1 ALARA limits were worn by personnel in the Cardiology Division. The objective of this study was to determine the reasons for the high personnel dosimeter readings for staff in the cardiac catheterization laboratory and implement corrective measures to keep exposures ALARA. The physicians were double badged. The fluoroscopic time for each patient examination performed by each physician in the catheterization laboratory was tabulated and compared with the exposure on the dosimeters of the person performing the study. Education and retraining of cardiology staff concerning radiation protection and the use of state-of-the-art equipment are major considerations in reducing worker radiation exposure. PMID- 8617598 TI - Limits of fetal thyroid risk from radioiodine exposure. AB - An incident in which a young women became pregnant soon after being treated with 444 MBq 131 I for Graves disease prompted us to search local records for the occurrence of thyroid abnormalities among people exposed in utero to fallout radioiodine. The data base from the Utah Fallout Study (Kerber et al. 1993) indicated that there had been 480 cohort subjects for whom dose to thyroid from fallout radioiodine had been calculated and who could have received any thyroid dose before birth (2473 subjects had been re-examined in 1985-86 of the 4818 examined in 1965-70). Of these 480 subjects in this category, 403 of them could be located in the 1980's and were examined with abnormalities. Although nodules, thyroiditis, hypothyroidism and goiter were seen among the 375 persons with in utero thyroid doses from fallout radioiodine below 0.42 Gy, no thyroid abnormalities of any occurred in the 4 persons with in utero thyroid doses of 0.5 to 2.6 Gy. In addition, no neoplasia was found in any of the 403 subjects examined about 2 decades after in utero fallout exposure. These limited data do not indicate that the fetal thyroid is more sensitive than the postnatal thyroid by more than about a factor of about 4 when thyroid dose is considered and by not much more than unity when the comparison is based on dose equivalent (x ray vs. radioiodine). PMID- 8617600 TI - Radiocesium contamination at a steel plant in Ireland. AB - Radioactive sources have been inadvertently incorporated into consignments of scrap metal in various locations throughout the world. In 1990, a 3.7 GBq 137 Cs source, due for transfer from a Scottish industrial establishment to one in England, was mistakenly included in a scrap consignment destined for Irish Steel, a metal recycling plant in County Cork in the Republic of Ireland. Unaware of the presence of the source, Irish Steel smelted this consignment in the usual manner. This involved separation of some non-ferrous materials which were then exported, in pellet form, To Pasminco Europe (now Britannia Zinc Ltd.) in Avonmouth, U.K. The presence of 137 Cs contamination in these pellets was detected by the U.K. company in the course of a routine radiation survey. Irish Steel carried out extensive decontamination of its plant, placing the contaminated dust in secure storage. The company has equipped itself with radiation detection devices which monitor incoming scrap. Outgoing products and furnace dust are also monitored on a routine basis. While this incident was of negligible radiological significance as far as personnel were concerned, the financial cost to Irish Steel have been substantial. It highlights the need for surveillance, by national competent authorities, of the movement of radioactive sources from production through use to final disposal. PMID- 8617601 TI - Toxicity indices of radioactive waste. PMID- 8617602 TI - A proposal for development of a multi-wire proportional counter for neutron spectrometry. PMID- 8617603 TI - Updating of the Reference Indian Man data. PMID- 8617604 TI - ICRP equivalent dose calculation of the remainder tissues. PMID- 8617605 TI - Health issues related to the use of hand-held radiotelephones and base transmitters. International Commission on Non-Ionizing Radiation Protection. PMID- 8617606 TI - Requiem or reveille for health services research? PMID- 8617607 TI - Efficiency measurement and the operationalization of hospital production. AB - OBJECTIVE: To discuss the usefulness of efficiency measures as instruments of monitoring and resource allocation by analyzing their invariance to changes in the operationalization of hospital production. STUDY SETTING: Norwegian hospitals over the three-year period 1989-1991. STUDY DESIGN: Efficiency is measured using Data Envelopment Analysis (DEA). The distribution of efficiency and the ranking of hospitals is compared across models using various distribution-free tests. DATA COLLECTION: Input and output data are collected by the Norwegian Central Bureau of Statistics. PRINCIPAL FINDINGS: The distribution of efficiency is found to be unaffected by changes in the specification of hospital output. Both the ranking of hospitals and the scale properties of the technology, however, are found to depend on the choice of output specification. CONCLUSION: Extreme care should be taken before resource allocation is based on DEA-type efficiency measures alone. Both the identification of efficient and inefficient hospitals and the cardinal measure of inefficiency will depend on the specification of output. Since the scale properties of the technology also vary with the specification of output, the search for an optimal hospital size may be futile. PMID- 8617608 TI - Assessment of HCFA's 1992 Medicare hospital information report of mortality following admission for hip arthroplasty. AB - OBJECTIVE: The Health Care Financing Administration (HCFA) produced annually from 1987 through 1994 mortality data information as part of the Medicare Hospital Information Project (MHIP) report. We assessed the validity of these data for hip arthroplasty for one state Medicare population and we analyzed the accuracy of the predictions derived from the Bailey-Makeham mortality model for this procedure. DATA SOURCES AND STUDY SETTING: The study sample consisted of claims and model data from 1,421 Medicare patients who underwent hip arthroplasty at acute care Arkansas hospitals from October 1990 through September 1991. STUDY DESIGN: Patients were stratified into two groups based on reason for surgery (fracture status): reconstruction or fracture management. Patient survival experience was compared between the two groups. The effect of fracture status on the HCFA model's predictive ability was examined empirically and via a simulation study. RESULTS: Our results indicate that hip arthroplasty patients are not uniform with regard to outcome, depending on the reason for the surgery. Patients with fracture had a much higher 30-day mortality rate than those who underwent reconstruction (p < .001). The empirical data and the simulation study suggest that the Bailey-Makeham model underestimates mortality for reconstructive surgery in fracture patients, providing a false benchmark for those institutions that perform hip arthroplasty on predominantly one category of patients. CONCLUSION: Published HCFA data concerning mortality for hip arthroplasty combines two different patient populations into one statistic. Casual examination of these data could result in a false benchmark for analysis of institutional performance. An important implication from this study for policymakers who base decisions on "report cards" or performance measurement reports is that, although they are necessary,generic case-mix, comorbidity, and severity of illness adjustments may not be sufficient to achieve accurate representations of outcomes, and that more disease/procedure--specific adjustments may be needed to avoid inappropriate conclusions. PMID- 8617609 TI - Risk indicators for hospitalization during the last year of life. AB - OBJECTIVE: High levels of hospital expenditures for older people during their last year of life are widely documented. However, evidence of the association between prospectively measured indicators and subsequent hospitalization is sparse. This article investigates the pattern of hospitalization for a sample of Medicare enrollees during their last year of life. DATA SOURCES: Data from the Longitudinal Study of Aging, a national study of persons age 70 and older, are used. Only data on decedents are used. STUDY DESIGN: We determine individual characteristics (including functional status, evidence of disease, living arrangement, and prior hospitalization) shortly before the last year of life. A distinction is made between terminal and nonterminal admissions. National estimates and regression analyses using survey weights are conducted. PRINCIPAL FINDINGS: The likelihood of any use is high regardless of age, functional status, or the presence of major diseases. Although only a few indicators are associated with having a terminal stay, a number of indicators are associated with nonterminal use. Nonterminal stays and total nights hospitalized are positively associated with prior evidence of disease, prior hospitalization, and age, although the probability of nonterminal use decreases with age for persons over 82 years old. The relationship between use and functional status depends on whether persons lived alone, were institutionalized, or had private health insurance. CONCLUSIONS: This study demonstrates that while it is difficult to predict who will be admitted to the hospital at the time of death, a number of characteristics existing before the last year of life are associated with nonterminal hospitalization and total nights hospitalized during the last year of life. PMID- 8617610 TI - Practice setting and physician influences on judgments of colon cancer treatment by community physicians. AB - OBJECTIVE: This article compares judgments about the treatment of Dukes' B2 and C colon cancer made by general surgeons to those of internists and family practitioners. Physician and practice variables were specialty, affiliation with a Community Clinical Oncology Program (CCOP) hospital, time in practice, professional centrality (level of participation in cancer information networks), solo practice, and number of colon cancer patients. DATA COLLECTION METHODS: Data are combined from national probability samples of CCOP- and non-CCOP-affiliated physicians. This study focused on 1,138 internists, family physicians, and general surgeons who participated in decision making for patients diagnosed with Dukes' B2 or C stage colon cancer. Judgments were elicited using brief vignettes. METHODS OF ANALYSIS: Judgments of adjuvant therapy are classified as (a) consistent with the National Institutes of Health Consensus Conference recommendations (experimental for Dukes' B2, accepted for Dukes' C); (b) accepted treatment for both stages; or (c) experimental for both stages. Multinomial logit analyses were used to examine the association of practice setting and physician characteristics to judgments of treatment. RESULTS: Surgeons and CCOP-affiliated physicians were more likely to endorse the NIH consensus conference position. Surgeons, younger physicians, and those in group practice were more likely to approve of chemotherapy for both cancer stages. The most common position (chemotherapy experimental) was more likely from nonsurgeons, solo practitioners, and non-CCOP physicians. CONCLUSION: Physician and practice setting characteristics, including organized structures such as the CCOP, are possible mediating structures that can facilitate dissemination of standards of treatment. PMID- 8617611 TI - Trauma systems and the costs of trauma care. AB - OBJECTIVE: This study examines the cost of providing trauma services in trauma centers organized by publicly administered trauma systems, compared to hospitals not part of a formal trauma system. DATA SOURCES AND STUDY SETTING: Secondary administrative discharge abstracts for a national sample of severely injured trauma patients in 44 trauma centers and 60 matched control hospitals for the year 1987 were used. STUDY DESIGN: Retrospective univariate and multivariate analyses were conducted to examine the impact of formal trauma systems and trauma center designation on the costs of treating trauma patients. Key dependent variables included length of stay, charge per day per patient, and charge per hospital stay. Key impact variables were type of trauma system and level of trauma designation. Control variables included patient, hospital, and community characteristics. DATA COLLECTION/EXTRACTION METHODS: Data were selected for hospitals based on (1) a large national hospital discharge database, the Hospital Cost and Utilization Project, 1980-1987 (HCUP-2) and (2) a special survey of trauma systems and trauma designation undertaken by the Hospital Research and Educational Trust of the American Hospital Association. PRINCIPAL FINDINGS: The results show that publicly designated Level I trauma centers, which are the focal point of most trauma systems, have the highest charge per case, the highest average charge per day, and similar or longer average lengths of stay than other hospitals. These findings persist after controlling for patient injury and health status, and for demographic characteristics and hospital and community characteristics. CONCLUSIONS: Prior research shows that severely injured trauma patients have greater chances of survival when treated in specialized trauma centers. However, findings here should be of concern to the many states developing trauma systems since the high costs of Level I centers support limiting the number of centers designated at this level and/or reconsidering the requirements placed on these centers. PMID- 8617612 TI - The impact of market and organizational characteristics on nursing care facility service innovation: a resource dependency perspective. AB - OBJECTIVE: Using resource dependency theory as a conceptual framework, this study investigates both the organizational and environmental factors associated with an emerging health care service delivery innovation, the provision of specialty care in designated units in nursing care facilities. We consider two types of specialty units, Alzheimer's Disease and subacute care. DATA SOURCES: The Medicare/Medicaid Automated Certification Survey (MMACS) data file was merged with local market area data obtained from the 1992 Area Resource File and with state level regulatory data. STUDY DESIGN: The likelihood of providing Alzheimer's Disease or subacute care in dedicated units was estimated by separate logistic regressions. PRINCIPAL FINDINGS: Results indicate that facilities with fewer Medicare patients are more likely to operate a dedicated Alzheimer's care unit, while facilities located in markets with a large HMO population and greater hospital supply are more likely to operate a subacute care unit. While competition among nursing homes, for the most part, is an incentive to innovate, greater regulatory stringency appears to constrain the development of specialty care units of both types. Finally, organizational characteristics (e.g., size and proprietary status) appear to be important enabling factors influencing the propensity to provide specialty care in dedicated units. CONCLUSIONS: Nursing care facilities are moving toward providing specialty care units partly as a response to a growing demand by resource providers and to maintain a competitive edge in tighter markets. Loosening regulation directed at cost containment would further encourage the development of specialty care but should be preceded by some evaluation of population needs for specialty care and the effectiveness of specialty care units. PMID- 8617613 TI - Single-stranded DNA conformation polymorphism at the Rdl locus in Hypothenemus hampei (Coleoptera: Scolytidae). AB - The homologue of the resistance to dieldrin gene (Rdl) in Drosophila melanogaster was cloned and sequenced in the scolytid beetle Hypothenemus hampei, a coffee pest resistant to cyclodiene insecticides in New Caledonia. The amino acid sequence of the Rdl exon no. 7 protein product in H. hampei was identical to that in D. melanogaster and showed the same amino acid change as that characterizing susceptible vs. resistant D. melanogaster. Samples from natural H. hampei populations (from Asia, the Pacific Islands, Africa and Central America), from reference susceptible (S) and resistant (R) laboratory strains, and from their hybrid progenies, were analysed at the Rdl locus using single-stranded DNA conformation polymorphism on polymerase chain reaction products. The susceptible allele was the only allele present in all samples from natural populations except in the only resistant population known to date (Ponerihouen, New Caledonia). Females and some males obtained at F1 from R x S crosses were heterozygous at the Rdl locus, confirming that this local mate competing species is diplo-diploid. PMID- 8617614 TI - A population genetic analysis of chloroplast DNA in Phacelia. AB - Hierarchical sampling from populations, incipient and recognized varieties within Phacelia dubia and P. maculata has revealed high levels of intraspecific polymorphism in chloroplast DNA. Much of the variation is partitioned between populations as evidenced by population-specific variants at fixation in all three populations of P. dubia var. interior and in both populations of P. maculata. Nine of 16 populations were polymorphic for cpDNA haplotypes. A total of 16 haplotypes was found in a sample of 106 individuals; the most common occurred in eight of the 16 populations and in 31 per cent of the individuals in the entire sample. A phylogenetic analysis revealed four basic plastome types. The two major groups of plastomes were separated by four independent base-pair mutations which suggests an ancient split in the evolution of plastid genomes. Representatives from each major plastome division were found in each of five populations spanning two allopatric varieties of P. dubia. The geographical distribution of haplotypes and lack of evidence for recent admixture argue against migration as a source of the polymorphism. It is more likely that the current taxonomic varieties are descendants of a polymorphic common ancestor. PMID- 8617616 TI - People I have known--from Canfield to Saratoga. PMID- 8617615 TI - The value of tilmicosin in production medicine. PMID- 8617617 TI - What is your diagnosis? Irregular periosteal response, with a radiolucent defect within the distomedial aspect of the patella. PMID- 8617618 TI - What is your neurologic diagnosis? Neospora caninum-induced myositis. PMID- 8617619 TI - ECG of the month. Phase-4 aberrancy in a dog. PMID- 8617620 TI - A survey of veterinarians' knowledge and attitudes about nutrition. PMID- 8617621 TI - Managing for the future. PMID- 8617622 TI - Some confidentiality issues. PMID- 8617625 TI - Femoral head and neck excision in a dog that had previously undergone contralateral hind limb amputation. AB - A German Shepherd Dog that underwent left hind limb amputation at 6 weeks of age because of quadriceps contracture developed arthritis of the remaining coxofemoral joint when it was 6 months old. The dog subsequently underwent femoral head and neck excision, and following rehabilitation that included intensive physical therapy, the dog was able to walk and run without signs of pain or disability. Strength and agility were maintained during a 4.5-year follow up period. This case demonstrates the importance of postoperative management in the successful outcome of femoral head and neck excision in a large dog with only 1 hind limb. PMID- 8617624 TI - Use of a modified surgical approach to the right atrium for retrieval of heartworms in a dog. AB - A 2-year-old 2-kg female Maltese dog was referred for treatment of dirofilariosis and mild caval syndrome characterized by hemolysis and lethargy. Ultrasonography revealed worms within the caudal vena cava, right auricle, right ventricle, and pulmonary artery. Because of the mild clinical signs and small size of the dog, jugular venotomy was not performed, and treatment with sodium caparsolate was instituted. A markedly adverse reaction was noticed on initial injection, characterized by cardiac and respiratory arrest. Further treatment with sodium caparsolate was discontinued. Because of progression of the dog's condition surgical removal of heartworms was elected. A modified surgical approach to the right atrium was performed, using a cannula introduced through a pursestring placed in the wall of the right auricle. This technique allowed almost complete removal of heartworms with minimal blood loss. Postoperative ultrasonography revealed a single heart-worm remaining in the distal portion of the left pulmonary artery, but it was subsequently absorbed. PMID- 8617626 TI - Risk factors for history of previous colic and for chronic, intermittent colic in a population of horses. AB - OBJECTIVE: To identify risk factors for recurrent colic and chronic, intermittent colic in horses. DESIGN: Case control study. ANIMALS: The population included 768 horses examined by veterinarians for emergencies other than colic (control group). PROCEDURE: Horses with colic that had history of colic (n = 232) were compared with those without such history (n = 536), using logistic regression analysis to identify risk factors for history of previous colic and to determine odds ratios (OR) for these associations. Among the 232 horses in the history of colic group, 58 horses that had chronic, intermittent colic were compared with the no history of colic group and the control group to identify factors associated with chronic, intermittent colic, using multiple logistic regression analysis. RESULTS: Among horses with colic, factors significantly associated with history of colic by multiple logistic regression analysis included history of abdominal surgery (OR = 3.1; P < 0.0001), age > 8 years (OR = 1.5; P < 0.0001), feeding of coastal grass hay (OR = 1.34; P 0.012), Arabian bread (OR = 1.28; P = 0.044), and recent change in stabling (OR = 0.76, P = 0.024). Among horses with colic, factors significantly associated with chronic, intermittent colic were history of previous abdominal surgery (OR = 2.2; P = 0.021), age > 8 years (OR = 2.0; P < 0.0001), being a gelding (OR = 1.7 with female as the reference population; P = 0.002), feeding of coastal grass hay (OR = 1.6; P = 0.045), and farm density < 0.5 horses/acre (OR = 1.6; P = 0.003). When the CIC group was compared with the control group, significant risk factors included history of abdominal surgery (OR = 270.7; P < 0.0001), age > 8 years (OR = 2.4; P < 0.0001), recent change in diet (OR = 2.1; P = 0.005), farm density < 0.5 horses/acre (OR = 2.0; P = 0.0001); being a gelding (OR = 1.8, with female as the reference population; P = 0.002), and Arabian breed (OR = 1.6; P = 0.050). CLINICAL IMPLICATIONS: Certain findings of signalment and management factors may identify horses at increased risk of recurrent forms of colic. PMID- 8617623 TI - Behavioral changes associated with suspected complex partial seizures in bull terriers. AB - OBJECTIVES: To identify and treat a range of abnormal behavior, including tail chasing, unprovoked aggression, and extreme irrational fear, in Bull Terriers and to correlate the behavioral signs with electroencephalogram (EEG) or anatomic evidence of abnormal brain geometry or deafness. DESIGN: Prospective clinical study. ANIMALS: 8 affected and 5 unaffected (control) Bull Terriers. PROCEDURE: All dogs were examined neurologically, including use of EEG, brainstem auditory evoked response, and computed tomography or postmortem examination of the brain. In addition, plasma concentrations of zinc, copper, and iron, and the activity of zinc- and copper-dependent enzymes (alkaline phosphatase and ceruplasmin oxidase) were measured in affected and control dogs. RESULTS: An abnormal EEG was found in 7 of 7 affected dogs and in none of the control dogs subjected to this examination. Seven of 8 affected dogs and 2 of 3 controls had various degrees of hydrocephalus. Metal ion and enzyme concentrations were not different between affected and control dogs. Treatment with phenobarbital was effective in 5 of 7 dogs. CLINICAL IMPLICATIONS: Bull Terriers with compulsive tail chasing and extreme affective disorders should be regarded as neurologically disturbed, with partial seizures perhaps underlying their behavior. Treatment with anti convulsants is a logical first step in treatment. PMID- 8617627 TI - Surgical treatment of subchondral cyst-like lesions in the tibia of an adult pony. AB - A 13 year-old pony was evaluated because of right hind limb lameness of acute onset. Radiographs of the right tarsus obtained shortly after the onset of lameness were normal, but results of nuclear scintigraphy were abnormal. Two radiolucent subchondral cyst-like lesions of the distal part of the tibia were seen on radiographs obtained 9 months later. The lesions were surgically decompressed, and the pony was sound 1 year later. It is hypothesized that a traumatic insult created a crack or split in the articular cartilage that allowed subsequent development of the cyst-like lesions. PMID- 8617629 TI - Growth and microbial flora of nonmedicated, segregated, early weaned pigs from a commercial swine operation. AB - OBJECTIVE: To determine whether segregated, early weaned pigs have better growth performance and different microbial flora than those pigs raised on-site. DESIGN: Prospective, observational study. ANIMALS: Pigs from a commercial operation that were known to be infected with several common swine pathogens. PROCEDURE: Pigs (7 to 10 days old) were weaned and segregated from the farm of origin and compared with littermate control pigs (14 to 17 days old) that were weaned and raised on site. Pig weight was measured and microbial flora were isolated at 14-day intervals for 84 days, beginning when the pigs were 7 to 10 days old. RESULTS: At 50 days of age, the segregated, early weaned pigs had a mean weight of 23.7 kg, compared with a mean weight of 12.5 kg for control pigs. Pasteurella multocida was isolated from fewer segregated, early weaned pigs than from controls. Signs of Mycoplasma hyopneumoniae infection were detected in control pigs but not in segregated early weaned pigs. Clinical, serologic, or bacteriologic signs of early postnatal vertical transmission of Actinobacillus pleuropneumoniae were not detected in either group. CLINICAL IMPLICATION: Vertical transmission of M hyopneumoniae was prevented by weaning pigs at 7 to 10 days of age and segregating them off-site, without the use of medication. Although medicated controls were not compared, results from this herd revealed that use of antibiotics is not the most important factor for disease control in segregated, early weaning programs. Minimizing antibiotic use in disease-control protocols reduces costs as well as removes the need for extra-label drugs. PMID- 8617628 TI - A rapid method for determination of blood glucose concentration in cattle. AB - OBJECTIVE: To determine an accurate rapid method for determination of blood glucose concentration in cattle under field conditions. DESIGN: Prospective, randomized, controlled trial. ANIMALS: 62 clinically normal Holstein cattle: 34 cows and 28 calves. PROCEDURE: Glucose concentrations in venous blood samples were measured in duplicate using a rapid, dry-slide chemistry technique for determination of blood glucose concentration and a laboratory-based method for determination of plasma glucose concentration. Analyses of variance were used to determine whether the relationship between results of the 2 methods was affected by the status of the animals (cows vs calves) or the PCV of the blood samples. Simple linear regression was performed to determine the correlation between the 2 methods and the slope, intercept, and residual error variance of the relationship between the methods. RESULTS: There as a significant linear relationship between the 2 methods throughout the range of glucose concentrations. Mean difference between results of the 2 methods (results for laboratory-based method - results for rapid method) was 12.95 mg/dl (SD, 7.20 mg/dl). The PCV did not affect the relationship, and there was no difference between results of the 2 methods for cows versus calves. Correlation between means of the duplicative values determined by use of the 2 methods was high (r = 0.9462). CLINICAL IMPLICATIONS: The good correlation between the 2 procedures and the comparable precision estimates (coefficient of variation, 7.17% for laboratory-based method; coefficient of variation, 10.11% for rapid methods) indicates that using the rapid method to measure blood glucose concentration is valid in cows and calves. PMID- 8617630 TI - Acute recumbency and marginal phosphorus deficiency in dairy cattle. AB - Because of a mixing error at a local feed mill, a diet marginally deficient in phosphorus, compared with recommendation from the National Research Council, was fed to a high-producing dairy herd for 5 months. Two mature cows in early lactation became recumbent. Serum phosphorus concentration in 1 cow was low (1.8 mg/dl), but was not measured in the other cow. Ten other high-producing, first lactation cows in the herd developed severe lameness. Results of analysis of rib bone samples from the recumbent cows were consistent with changes associated with demineralization. Bone ash, calcium, phosphorus, and magnesium concentrations were lower than published ranges for healthy cattle. Serum calcium, phosphorus, and magnesium concentrations in 8 unaffected cows were normal. For 6 unaffected cows, mean serum hydroxyproline concentration was higher during the period that the phosphorus-deficient diet was fed than when an adequate diet was fed. Moderate (15%) restrictions in dietary phosphorus intake, compared with National Research Council recommendations, can possibly result in health problems in high producing dairy cattle. PMID- 8617631 TI - Anesthetic and postanesthetic management of sea turtles. AB - OBJECTIVE: To examine the physiologic effects of inhalation anesthesia in aquatic turtles to improve anesthetic techniques and postanesthetic monitoring. DESIGN: Retrospective case series. ANIMALS: 9 Kemp's ridley sea turtles. PROCEDURE: Isoflurane was used as the general anesthetic during 14 minor surgical procedures. Turtles were orotracheally intubated, and a surgical plane of anesthesia was maintained with 2.7 +/- 0.4% (mean +/- SE) isoflurane. The duration of anesthesia was 131 +/- 12 minutes. Pulse rate, blood pressure, blood gases (PaO2 and PaCO2) and pH, blood lactic acid concentration, and capnography were used to evaluate the physiologic responses of sea turtles to isoflurane. RESULTS: An isoflurane concentration of 3.4 +/- 0.3% provided anesthetic induction in 7 +/- 1 minutes. The mean duration of the recovery phase was 241 +/- 31 minutes. The duration of the recovery phase was not affected by the duration of anesthesia, type of carrier gas, method of ventilatory weaning, or use of selected pharmacologic agents. The recovery phase was characterized by hypoxemia, progressive acidemia, hypercapnia, and lactic acidosis. Awakening in the turtles was preceded by a characteristic tachycardia and tachypnea. All sea turtles recovered from isoflurane anesthesia without apparent adverse effects within 24 hours. CLINICAL IMPLICATIONS: Isoflurane appears to be safe and effective in providing surgical anesthesia in turtles that require a timely return to an aquatic environment. This study should assist veterinarians in predicting the physiologic responses of aquatic turtles to inhalation agents. PMID- 8617633 TI - Observations on veterinary education in Britain. PMID- 8617632 TI - Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals. AB - An Atlantic white-sided dolphin (Lagenorhynchus acutus), a pygmy sperm whale (Kogia breviceps), 2 harbor seals (Phoca vitulina) developed raised, firm, erythematous, cutaneous nodules that were most prominent on their heads, trunks, and on the caudal portions of their bodies. Prior to the onset of the condition, all 4 animals may have been stressed by factors such as being stranded on a beach, being transported long distances, or being relocated locally. Microbial culturing of the lesions on multiple media yielded fungal isolates containing conidia characteristic of Fusarium spp. Hyphae consistent with those of an ascomycete were evident on histologic examination of lesions. In each treated animal, the dermatitis resolved 3 to 4 weeks after completing treatment with ketoconazole. Fusarium spp may be opportunistic invaders of the skin of marine mammals that have decreased immunocompetence or integumentary compromise. PMID- 8617634 TI - Xenotransplantation and food animal practitioners. PMID- 8617635 TI - What is your diagnosis? Right maxillary sinusitis or sinus cyst in a cow. PMID- 8617636 TI - Theriogenology question of the month. Uterine rupture. PMID- 8617637 TI - Cyanosis and intense murmur in a neonatal foal. PMID- 8617638 TI - Use of antimicrobial-impregnated polymethyl methacrylate. PMID- 8617639 TI - Emotional responses of animal shelter workers to euthanasia. PMID- 8617640 TI - The application of simulation to veterinary practice management. PMID- 8617641 TI - Demonstrations of the use of simulation in veterinary practice management. PMID- 8617642 TI - The real job market. PMID- 8617643 TI - Financial evaluation of vaccination and testing alternatives for control of parvovirus-induced reproductive failure in swine. AB - OBJECTIVE: To identify the preferable testing and vaccination strategy for control of porcine parvovirus (PPV) during a 6-month period. DESIGN: Decision tree analysis and computer simulations. SAMPLE POPULATION: Computer modeling of 300-sow farrow-to-finish herd. PROCEDURE: Serologic testing of 30 females to estimate herd PPV prevalence versus not testing any females was the initial decision alternative. On the basis of serologic test results, herds were classified into 1 of 3 PPV-risk categories: low (> or = 80% seropositive females), moderate (40 to < 80% seropositive females), or high (< 40% seropositive females). Vaccinating all females, only gilts, or not vaccinating was the second decision alternative. RESULTS: For initial model assumptions (test sensitivity and specificity = 0.95; test cost = $5/female; vaccination cost = $0.30/dose; vaccination efficacy = 0.95; and foregone gross margin = $10.85/pig), vaccination of all females (with or without serologic testing) was preferable, but the financially preferable option was to omit serologic testing. Most profitable vaccination option varied with foregone gross margin, vaccination cost, and efficacy. For herds in which all sows were known to be immune, vaccinating only gilts was financially preferable, and serologic testing was not warranted. Variation is expected monetary losses was less in vaccination options than with nonvaccination. CLINICAL IMPLICATIONS: For most herds in the United States, serologic screening for PPV prior to selection of a vaccination program is unlikely to be cost-effective, because vaccination is inexpensive ($0.30/dose) and effective (95%). At current profit margins ($10.85/pig), vaccination of all females has the least-risk and is the preferred option. PMID- 8617645 TI - Factors associated with finishing status for dogs competing in a long-distance sled race. AB - OBJECTIVE: To determine whether selected factors were associated with finishing status in a long-distance sled dog race. DESIGN: Prospective, observational study. ANIMALS: 248 dogs participating in the 1994 Iditarod trail sled race that were members of 13 teams that finished the race. Dogs were not selected randomly, but were members of teams that were easily accessible for examination before the race. The proportion of teams that were examined that finished the race (14/17) was similar to the proportion of teams that were not examined that finished the race (36/41). PROCEDURE: Age, sex, body conformation (weight, length, and thoracic width and circumference), cardiac variables (heart rate, natural logarithm of heart period variance, PR interval, QRS duration, QT index, R wave amplitude in leads II and V3, mean electrical axis, presence of cardiac arrhythmias suggestive of myocardial disease), and athletic ranking as assessed by the musher were compared between dogs that finished the race (n = 128) and dogs that did not finish (n = 120). RESULTS: The only factor found to differ significantly (P < 0.05) between finishers and nonfinishers was athletic ranking as assessed by the musher. Athletic rank and QRS duration or QRS duration normalized for body weight were identified by means of logistic regression as variables associated (P < 0.15) with finishing status. There was a significant (P < 0.0001), but weak (R2 = 0.18), linear relationship between race time and mean QRS duration for each team. CLINICAL IMPLICATIONS: Athletic ranking as assessed by the musher was the most important of the studied variables in determining finishing status, whereas age, sex, body conformation, and body weight were unimportant. Duration of the QRS was of minor importance in determining finishing status. PMID- 8617644 TI - Effect of treatment of hyperthyroidism on renal function in cats. AB - OBJECTIVE: To determine whether increases in BUN and serum creatinine (SCr) concentrations, which have been reported to develop after surgical bilateral thyroidectomy in hyperthyroid cats, also develop after treatment of hyperthyroidism with radioactive iodine and methimazole. DESIGN: Prospective, clinical trial. ANIMALS: 58 hyperthyroid cats. PROCEDURE: Urine specific gravity, SCr, BUN, and serum thyroxine (T4) concentrations were determined before and 30 and 90 days after treatment of hyperthyroidism with radioactive iodine, methimazole, or surgical bilateral thyroidectomy. RESULTS: Mean SCr and BUN concentrations determined 30 and 90 days after treatment were significantly higher than those measured before treatment. Mean SCr, BUN, and T4 concentrations were not different among groups before treatment or 30 and 90 days after treatment. CLINICAL IMPLICATIONS: Reduction of serum T4 concentrations after treatment of hyperthyroidism may result in azotemia in older cats with chronic renal disease. Treating azotemic hyperthyroid cats with methimazole until it can be determined whether correction of the hyperthyroid state will exacerbate the azotemia may be prudent. PMID- 8617646 TI - Evaluation of cortical bone damage and axial holding power of nonthreaded and enhanced threaded pins placed with and without drilling of a pilot hole in femurs from canine cadavers. AB - OBJECTIVE: To evaluate the in vitro axial extraction forces necessary to remove pins and to evaluate mechanical trauma resulting from pin insertion, using various types of pins and insertion techniques. DESIGN: Prospective, controlled study. SUBJECTS: Femurs of cadavers of dogs. PROCEDURE: Pins were inserted as follows: 1 nonthreaded pin without drilling of a pilot hole, 1 enhanced threaded pin with drilling of a pilot hole, and 1 enhanced threaded pin without drilling of a pilot hole. After pin insertion, mechanical damage and proper pin insertion was determined by means of radiography. Axial extraction forces were determined for all pins, using a universal testing machine. Mechanical damage was evaluated in 12 additional femurs. After pin insertion, all pins were removed from the bone by use of a low-speed power drill. Samples were sectioned, processed, and evaluated by use of dissecting and scanning electron microscopy. RESULTS: Using radiography, a significant difference was detected in the number of periosteal trans-cortex fractures between the enhanced threaded and nonthreaded pins. Axial extraction force was not significantly different between the enhanced threaded pins, regardless of insertion technique; however, the axial extraction force was significantly greater for enhanced threaded pins, compared with that for nonthreaded pins. Microfractures only were detected on the periosteum of the trans-cortex of enhanced threaded pins by use of scanning electron microscopy. CLINICAL IMPLICATIONS: We cannot recommend a particular insertion technique to decrease mechanical trauma to the bone and to increase axial extraction force needed for removal of enhanced threaded pins from the femur of dogs. PMID- 8617647 TI - Intraoperative use of subtraction angiography and an ultrasonic aspirator to improve identification and isolation of an intrahepatic portosystemic shunt in a dog. AB - Mesenteric portography, using a C-arm fluoroscope equipped with digital subtraction capability, was performed intraoperatively to locate a single, intrahepatic portocaval shunt. An ultrasonic aspirator was used to isolate the shunt, which was ligated completely. Typical portal venous arborization was seen on postligation intraoperative mesenteric portography. Subtraction angiography used intraoperatively and dissection with the ultrasonic aspirator improve the surgeon's ability to localize and isolate intrahepatic portocaval shunts and can reduce surgical time. Film development and patient repositioning and transport, before and after shunt ligation, are not necessary when mesenteric portography is performed intraoperatively, using a C-arm fluoroscope and digital subtraction. Dissection around intrahepatic portocaval shunts is facilitated by the ultrasonic aspirator, which selectively fractures and suctions tissue from around the shunt. Hemorrhage is decreased, because hepatic ductules and small blood vessels are left intact. PMID- 8617648 TI - Photocoagulation of limbal melanoma in dogs and cats: 15 cases (1989-1993). AB - OBJECTIVE: To evaluate immediate clinical effects and long-term results of neodymium:yttrium-aluminum-garnet laser treatment of limbal melanoma in dogs and cats. DESIGN: Retrospective case series. ANIMALS: 13 dogs and 2 cats. RESULTS: At the time of treatment, 9 tumors were progressively enlarging and 4 were static. Recent growth characteristics of 2 tumors were not reported. Total energy applied ranged from 7.5 to 572 J. In all eyes, pigmented tissue shrank after treatment; however, 3 tumors recurred, 1 at 3 months and 2 at 1 year after treatment. CLINICAL IMPLICATIONS: Photocoagulation may be an effective means of treating limbal melanoma in dogs and cats. PMID- 8617649 TI - Large colon resection for treatment of lymphosarcoma in two horses. AB - With the exception of lipoma, neoplasia of the gastrointestinal tract is rare in horses. Lymphosarcoma is the most common neoplasm of the hematopoietic system in horses. In horses with lymphosarcoma of the large colon, clinical signs may include intermittent signs of mild abdominal pain, weight loss, pyrexia, and pelvic flexure impaction caused by impingement of the colonic lumen by the mass. Peritoneal fluid analysis may be normal or have a high total protein concentration. If signs of metastasis are not evident, resection of the large colon affected by the mass may prolong survival. PMID- 8617650 TI - Medical treatment of horses with ileal impactions: 10 cases (1990-1994). AB - OBJECTIVE: To evaluate clinical and laboratory findings for horses treated medically for ileal impactions. DESIGN: Retrospective case series. ANIMALS: 10 horses with primary ileal impaction that were treated successfully with medical treatment alone. PROCEDURE: Medical records were reviewed for all horses with naturally developing ileal impaction seen at our hospital between 1990 and 1994. RESULTS: Transrectal palpation revealed an impaction in the midabdominal area in all horses. Generalized distention of the small intestine was evident in 6 horses, whereas 4 horses were examined early in the course of the condition and did not have intestinal distention. Treatment consisted of intravenous administration of a balanced electrolyte solution, nasogastric intubation and siphonage, and administration of analgesics. Mineral oil was administered after gastric reflux had ceased. Mean time for resolution of ileal impaction was 11.7 hours. CLINICAL IMPLICATIONS: Medical treatment may be a viable alternative for horses that cannot have surgery, provided persistent signs of severe pain or progressive gaseous distention of the small intestine are not features of the condition. Improvement of cardiovascular status, reduction in signs of abdominal pain, decrease in distention of loops of small intestine during repeated transrectal examination, softening of the impaction, and decreases in amounts of gastric reflux were indicative of a response to medical treatment. PMID- 8617651 TI - Forestomach acidosis in six New World camelids. AB - Forestomach acidosis was diagnosed in 2 llamas and 4 alpacas. All were young, group-housed, sexually intact males. Clinical signs included forestomach atony, lethargy, ataxia, diarrhea, and tachycardia. Forestomach distention was observed in only 1 llama. Clinicopathologic abnormalities included low forestomach fluid pH, hyperchloremia, hypokalemia, and metabolic acidosis. Although camelids differ from domestic ruminants in typical management practices and behavioral, anatomic, and physiologic characteristics, they are, nonetheless, susceptible to forestomach acidosis. Gastric fluid analysis was essential for an accurate diagnosis. Four of 6 camelids recovered after PO and IV treatment with alkalinizing agents and fluids, antibiotics, and thiamine. PMID- 8617652 TI - Catheterization of the auricular vein in cattle: 68 cases (1991-1994). AB - OBJECTIVE: To evaluate the use of auricular vein catheters (AVC) in cattle in a clinical setting. DESIGN: Case series. ANIMALS: 57 cattle. PROCEDURE: 68 AVC were placed in cattle for the administration of drugs or rehydration fluids. Catheter size, quantity of fluids administered, duration of administration, drugs administered, duration of catheter maintenance, and problems were recorded. RESULTS: The AVC ranged in size from 20 to 14 gauge, with the latter being the predominate size. A maximum flow rate of 7.7 L/h was achieved, and the flow rate was satisfactory in all but 1 case. The maximum duration of maintenance was > 96 hours. Problems occurred in 29 of 68 (43%) catheterizations; the most frequent problem was occlusion of the catheter, which occurred 16 times (24%). No serious complications occurred. CLINICAL IMPLICATIONS: Auricular vein catheters were a convenient, safe, and low-cost alternative to jugular vein catheters. PMID- 8617653 TI - Influence of energy or protein supplementation during midpregnancy on forage intake of ewes grazing Montana winter range. AB - A 2-yr winter experiment was conducted to determine the influence of either energy or protein supplementation during midpregnancy on fecal output (FO), forage intake, blood metabolite profiles, and BW changes of ewes grazing winter range. Thirty-two Targhee ewes were selected for uniformity in age and BW and assigned randomly to one of four dietary treatments 1) no supplement (NONE); 2) 150 g of barley supplement (BAR); 3) 75 of feather g meal, blood supplement (FM/BM); and 4) 75 g of FM, BM, urea supplement (FM/BU/U). Two 5-d experimental periods were conducted during each winter (January and February). Forage FO (P = 0.9), total FO (P = 0.7), and subsequent forage intake (P < .01) were higher during Yr 1 than during Yr 2. Supplement type did not affect forage DMI when expressed either as grams/day (P = .57) or as a percentage of BW (P = .52). Body weight changes and body condition scores were not affected (P > .10) by year but were affected (P < .01) by treatment; unsupplemented ewes lost more (P < .01) BW and body condition than supplemented ewes. Serum urea N (SUN) concentrations were affected (P < .03) by a year x treatment interaction. Unsupplemented, FM/ BM, and FM/BM/U ewes had higher (P < .10) SUN concentrations during Yr 1 than during Yr 2, averaging 9.8 ml/dL and 7.5 mg/dL, respectively. Barley-supplemented ewes had similar (P > .10) SUN concentrations both years, averaging 7.4 mg/dL. Alternate day supplementation during midpregnancy with energy of protein had no effect on forage DMI of ewes grazing winter range. PMID- 8617654 TI - The effect of ewe body condition at lambing on colostral immunoglobulin G concentration and lamb performance. AB - Body condition was scored at lambing (BCSL) on 101 mature (4 to 7 yr old) Polypay ewes and related to colostral immunoglobulin G (IgG) concentration and lamb performance. Colostrum samples were collected from each ewe within 12 h of lambing and litters of more than two lambs were reduced to two within 2 h of lambing. Colostral IgG concentrations decreased rapidly with time (b = -3.28; R2 = .2132) and linear regression analysis projected that colostral IgG concentrations would diminish to zero milligrams/milliliter by 23 h postpartum. Body condition score at lambing varied from 2.5 to 3.5 and had no effect on colostral IgG concentration, which averaged 79 +/- 5.6 mg/mL, adjusted to the time of parturition. Total birth weights of lambs were higher for the oldest ewes (7 yr old), but this group had the lowest prolificacy. This age group weaned the lowest total lamb weight and number of lambs. Total weight of lambs born was not affected by BCSL. Lamb mortality from birth to weaning was 19.0% and was not affected by BCSL, sex, litter size, or breed of sire, but the older (7 yr old) ewes had greater lamb mortality. Total weight of lamb weaned was not affected by BCSL, although ewes with a BCSL of 3.0 tended ( P = .11) to wean more kilograms of lamb than ewes with a BCSL of 3.5. Ewes bred to Polypay rams weaned more total weight of lamb than those bred to Columbia rams, which was due to increased survival rate to weaning for the Polypay rams. We conclude that, within a range of 2.5 to 3.5, BCSL is not an important factor affecting the colostral IgG concentration, total weight of lamb born, lamb mortality, or total weight of lamb weaned. PMID- 8617655 TI - Value of feather meal in a molasses-based liquid supplement fed to yearling cattle consuming a forage diet. AB - Hydrolyzed feather meal (FM) was compared to other sources of CP to determine its value as a protein source in a molasses-based liquid supplement. Ruminal N escape values (measured in situ) for FM, ring-dried blood meal, cottonseed meal, and soybean meal approximated reported values. Ruminal N escape value for catfish meal was lower than values reported for other fish meals. In two trials (194 d), 84 yearling heifers were allotted to 12 pastures (3 pastures/treatment) and fed one of four supplements (1.7 kg of DM.heifer-1.d-1) containing either molasses and urea; molasses, urea,and FM; molasses, urea, FM, and catfish meal; or molasses, urea, FM, and catfish oil. Heifers were exposed to bulls for 60 d. In two trials, 112 steers were allotted to 16 pastures (4 Pastures/treatment) and fed one of four supplements (1.7 kg of DM.steer-1.d-1) containing molasses and urea; molasses, urea and FM; molasses, urea, FM and ring-dried poultry blood meal; or molasses, urea, and poultry feathers and blood hydrolyzed together. In all trials, cattle fed molasses containing FM had faster gains (P < .05) and heifers had heavier live weights at breeding (P < .05) and higher pregnancy rates (P < .05) than cattle fed molasses and urea. Combining poultry blood with molasses, urea, and FM improved (P < .05) steer gains in one of two trials. Combining catfish meal with molasses, urea, and FM did not improve (P > .05) ADG of pregnancy rate of heifers. Combining catfish oil with molasses, urea, and FM increased (P < .05) ADG in one trial and blood cholesterol and live weight of heifers at breeding in both trials. It is concluded that protein feeds, such as FM, that contain a large portion of ruminally undegradable protein may provide much of the CP in liquid supplements fed to growing cattle consuming moderate quality forage. Adding catfish oil to a liquid supplement may improve ADG and live weight of yearling heifers at breeding. PMID- 8617656 TI - Production responses to various doses and ratios of estradiol benzoate and trenbolone acetate implants in steers and heifers. AB - At each of three locations, 400 steers and an equal number of heifers were randomized to 10 treatment groups. The purpose of the studies was to evaluate the response of feedlot steers and heifers to single implants containing a combination of estradiol benzoate (EB) and trenbolone acetate (TBA) at two different ratios each at three doses. The selected ratios corresponded to 1E2(estradiol-17 beta):5TBA and 1E2:10TBA. The two ratios were each tested at three different EB/TBA doses (1:5 at 20/70, 40/140, and 60 mg/210 mg, 1:10 and 14/100, 28/200, and 42 mg/300 mg). The test groups were compared to those given each of the compounds alone (60 mg of EB or 300 mg of TBA), as well as to groups reimplanted with Synovex S or Synovex H implants and untreated controls. Steers (P < .01) and heifers (P < .05) implanted with the 1:10 E2:TBA implants gained faster and had better feed conversion (FC) than their counterparts given 1:5 E2:TBA over the 140-d trial. The results indicated that both estradiol benzoate and trenbolone acetate contributed to the efficacy of the combination implant. Contour plots of ADG and FC indicate that increasing the amount of EB above approximately 36 and 37 mg does not significantly increase the response of steers. The results of these studies indicate that the 28 EB/200 TBA dose is close to optimal for growth promotion and feed conversion in both heifers and steers. In steers, carcass value was increased (P < .01) in all test groups except the group give TBA only. Despite a slight reduction in marbling score and percentage of Choice carcasses, carcasses of steers treated with either 28 mg of EB/200 mg of TBA or 42 mg of EB/300 mg of TBA were more valuable (P < .05) than carcasses from steers in any of the 1:10 ratio EB/TBA groups. Carcass values for groups reimplanted with Synovex S or Synovex H or implanted with EB alone were not significantly different from those for groups implanted with any dose of the 1:10 EB/TBA ratio. PMID- 8617657 TI - Prediction of carcass and empty body composition of steers by 40K emission detection. AB - Although originally used for prediction of whole body composition (WBC), use of 40K emission detection was later suggested for determination of empty body composition (EBC), Therefore, the present study was conducted to validate existing equations developed to predict WBC and to develop more useful equations to predict EBC or carcass composition (CC) of beef steers. Fourteen crossbred steers were detected for 40K emissions and slaughtered and their chemical composition determined from chemical analyses and total body K determined from 40K emissions. the existing equation for percentage whole body lipid had a slope of .93 and an intercept of -1.62%, indicating a close approximation of percentage of whole body lipid. Percentage of whole body protein was poorly estimated by existing equations. Equations developed for prediction of EBC relied on both BW and predicted K. Coefficients of variation for prediction of empty water or protein were within 5% and those for prediction of empty body lipid were approximately 15%. These results demonstrate that use of predicted K from 40K emission detection enhances the determination of EBC and CC. PMID- 8617658 TI - Effects of calcium soaps of fatty acids on postpartum reproductive activity in beef cows and growth of calves. AB - Beef cows were used to determine the influence of calcium soaps of fatty acids (CSFA) incorporated in a range supplement on postpartum reproductive characteristics and growth of calves. Cows were assigned randomly to receive 0 (C, n = 68) or 125 g/d of CSFA (M, n = 66). Diets were isonitrogenous (23%) and were used during 105 d, beginning at 61 +/- 36 d (range) precalving. Two blood samples were collected monthly (7-d intervals). Weights of calves at 35, 50, and 90 d of age and weaning weight adjusted to 200 d of age were greater in M than in C (46.8 vs 43.8 kg, P < .05; 56.0 vs 50.6 kg, P< .01; 98.8 vs 91.8 kg, P < .01; and 186 vs 173 kg, P < .01, respectively). Body weights at 35 and 50 d postcalving were greater in M than in C cows (334 and 310 kg, P < .01; 329 and 300 kg, P < .01, respectively). A similar tendency was observed in body condition scores in the same postpartum periods (4.1 vs 3.4, P < .01 and 3.6 vs 2.5, P < .01 for M and C, respectively). Concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol, and triglycerides were greater (P < .01) in M than in C cows. Percentage of cycling (progesterone > 1 ng/mL) cows at 30 to 90 d postpartum was 38% in M and 22% in C (P < .02). Percentage of pregnant cows during the first half of the breeding season was greater (P < .02) in M (62.5%) than in C cows (35.5%). We concluded that CSFA incorporated in a range supplement during pre- and postpartum periods improved reproductive efficiency and growth of calves. PMID- 8617659 TI - Calf production by Angus-Hereford and Brahman-Hereford cows on two native rangeland forage systems. AB - Calf birth weights, weaning weights, and preweaning gain of Simmental-sired calves from Angus-Hereford (AH) and Brahman-Hereford (BH) F1 cows grazing native rangeland (NR) or native rangeland-complementary forage (NRCF) systems in the southern Great Plains mixed prairie were evaluated. Calves from AH dams were heavier (P < .001) at birth than calves from BH dams in both forage systems (40 and 37 kg, respectively). However, birth weights of calves from BH dams were 2 kg heavier (P < .001) in the NRCF fall-calving system. Calves in the NRCF system were 122 d older and heavier (P < .001) than the NR calves at weaning (343 and 256 kg, respectively). During the preweaning period, the NR calves gained faster (P < .001) than the NRCF calves. When calves from both systems were evaluated at 200 d of age, NR calves were heavier (P < .001) than NRCF calves. Calves from BH cows were 8 to 16 kg heavier (P < .001) that calves from AH cows at 200 d of age and at weaning. The increased age at weaning associated with the NRCF did not reduce reproductive efficiency. The NRCF system requires less land than a traditional NR system to support a cow-calf pair and seems to be more economically efficient. PMID- 8617660 TI - Application of a computer model to predict optimum slaughter end points for different biological types of feeder cattle. AB - A bioeconomic model was developed to predict slaughter end points of different genotypes of feeder cattle, where profit/rotation and profit/day were maximized. Growth, feed intake, and carcass weight and composition were simulated for 17 biological types of steers. Distribution of carcass weight and proportion in four USDA quality and five USDA yield grades were obtained from predicted carcass weights and composition. Average carcass value for each genotype was calculated from these distributions under four carcass pricing systems that varied from value determined on quality grade alone to value determined on yield grade alone. Under profitable market conditions, rotation length was shorter and carcass weights lighter when the producer's goal was maximum profit/day, compared with maximum profit/rotation. A carcass value system based on yield grade alone resulted in greater profit/rotation and in lighter and leaner carcasses than a system based on quality grade alone. High correlations ( > .97) were obtained between breed profits obtained with different sets of input/output prices and carcass price discount weight ranges. This suggests that breed rankings on the basis of breed profits may not be sensitive to changes in input/output market prices. Steers that were on a grower-stocker system had leaner carcasses, heavier optimum carcass weight, greater profits, and less variation in optimum carcass weights between genotypes than steers that were started on a high-energy finishing diet at weaning. Overall results suggest that breed choices may change with different carcass grading and value systems and postweaning production systems. This model has potential to provide decision support in marketing fed cattle. PMID- 8617661 TI - Influence of grain type, tallow level, and tallow feeding system on feedlot cattle performance. AB - Two experiments were conducted to evaluate the effects of grain type, tallow level, and tallow feeding system on finishing steer performance. Experiment 1 involved 256 yearling steers (359 kg) in a 4 x 2 factorial arrangement of treatments. Steers were assigned randomly to one of four tallow feeding systems: 1) 0% tallow fed throughout the experiment; 2) 4% tallow fed throughout the experiment; 3) 0% tallow fed d 1 through 33 and then 4% tallow fed until slaughter; and 4) 4% tallow fed d 1 through 33 and then 0% tallow fed until slaughter. Tallow treatments were applied to diets containing either dry-rolled corn (DRC) of high-moisture corn (HMC). No fat treatment x grain type interaction (P > .10) was observed. Steers fed 4% tallow throughout the experiment, only during d 1 through 33, or only during d 34 until slaughter were more (P < .10) efficient than steers fed 0% tallow. No differences in DMI or ADG were observed (P > .10). In Exp. 2, 120 large-framed steer calves (286 kg) were blocked by weight and allotted randomly within block to one of three treatments consisting of the addition fo 0, 2, or 4% tallow added d 1 and fed for 197 d. Feed efficiency of calves increased linearly (P < .05) with increasing tallow level. Daily gain was not different (P > .10), but DMI decreased linearly (P < .05) with increasing tallow level. This research indicates that tallow added during or after grain adaptation to DRC- or HMC-based diets fed to yearling steers will result in similar improvement in feed efficiency, and that including up to 4% tallow to diets fed to large-framed calves can significantly improve feed efficiency. PMID- 8617662 TI - Preweaning diet and stall weaning method influences on stress response in foals. AB - The stress response of foals during weaning was examined in terms of a behavioral protocol and the responses of plasma ascorbate, serum cortisol, and the serum cortisol response to an ACTH challenge. The experimental plan was a 2 x 2 factorial of two preweaning diets and two stall weaning methods. Dietary groups included foals raised on pasture supplemented with hay and a pelleted concentrate (PHC) and foals raised on pasture supplemented with hay only (PH). Stall weaning methods included foals placed in stalls singly or in pairs. Sex influences were also examined. The foals exhibited characteristic behavioral and physiological responses to weaning stress. Behavior scores indicated fewer outward signs of stress in single than in paired foals (P = .008) and tended to indicate fewer signs of stress in PHC than in PH foals (P = .15). No differences in plasma ascorbate concentrations were found among treatments. Responses of serum cortisol to an ACTH challenge were lower (representing adrenal depletion arising from stress) in PH than in PHC foals (P = .001) and in paired than in single foals (P = .058). Behavior scores were positively correlated with the response of serum cortisol to ACTH. Both behavioral data and the ACTH response indicated that foals may cope better with weaning when supplemented with concentrate before weaning or when placed singly in stalls rather than in pairs. PMID- 8617663 TI - Feeding wet corn gluten feed to reduce subacute acidosis in cattle. AB - Two experiments were conducted to evaluate the effects of feeding wet corn gluten feed (WCGF) on subacute acidosis in cattle. In Exp. 1, 60 individually fed yearling steers (270 +/- 22 kg BW) were used in a 5 x 2 factorial arrangement of treatments. Steers were assigned to one of five dietary treatments: 1) dry-rolled corn (DRC), 2) 35% WCGF fed d 1 to 132, 3) 86.5% WCGF fed d 1 reduced to 35% WCGF by d 19 and increasing the proportion of DRC, 4) 86.5% WCGF fed d 1 to 132, or 5) 94.5% WCGF fed d 1 to 132. Final diets for Treatments 1 through 4 contained 92% concentrate and 8% alfalfa hay (DM basis). Treatment 5 was a 100% concentrate diet. All diets were fed with or without the addition of escape protein. During d 19 to 24, steers fed WCGF had less (P < .05) DMI variation than steers fed the control diet. Steers fed 86.5 and 94.5% WCGF had lower (P < .05) DMI and ADG than steers fed TReatments 1 through 3, although gain/feed was mot different (P > .10) In Exp. 2, three ruminally fistulated steers (615 +/- 36 kg BW) were used in a repeated 3 x 3 Latin square design. On d 14 of each period, 7.9 kg (DM) of 100% DRC, 50% DRC:50% WCGF, of 100% WCGF was intraruminally dosed as an acidosis challenge. Area within the curve below ruminal pH 6.0 was greater (P < .05) over a 24-h period for steers dosed with 100% DRC than for steers dosed with 50% DRC: 50% WCGF or 100% WCGF. In addition, more (P < .05) ruminal VFA accumulated over 24 h for steers dosed with 100% DRC. These data suggest that feeding WCGF does nor eliminate ruminal acidosis but may reduce the length of time cattle are exposed to the insult. PMID- 8617664 TI - Prediction of genetic values of sires for growth traits of crossbred cattle using a multivariate animal model with heterogeneous variances. AB - The purpose of this study was to evaluate the effect of adjusting for heterogeneous variances across breed groups on prediction of breeding values (PBV) of selected sires and on breed of sire effects. Data on weights at birth (BWT), 200 d (WW), and 365 d (YW) of purebred and crossbred calves from matings of Angus (A), Hereford (H), Polled Hereford, Charolais, Shorthorn, Simmental, Limousin, Maine-Anjou, Chianina, Gelbvieh, Tarentaise, and Salers Bulls to A and H cows were used. Calf performance in H and A dams was treated as a different trait. Models compared included fixed birth year, cow age, and sex classes and crossbreeding effect as a covariate; random direct and maternal genetic and permanent environmental effects were also included, but their variance structure was different. Model I assumed homogeneous variances across breed groups. Model II accounted for heterogeneous variances. Sires were ranked based on PBV from each model, and means of PBV of selected sires were calculated based on Model II. Differences between mean PBV were small for BWT, intermediate for WW, and larger for YW. Differences in PBV of selected sires increased as selection intensity increased, but only for WW and YW. Large differences in mean PBV of selected sires between maternal environments (H vs A) were observed for WW and YW for various sire breeds. Means of PBV of selected sires based on Model II exceeded those based on Model I by 6 to 16 kg of YW for various selection intensities and maternal environments. Estimates of breed of sire effects from Model I or II were similar for BWT and WW, but large differences were found for YW. Results indicate that some additional economic returns may be gained by commercial producers if sires are chosen across breeds based on predicted genetic values computed with models accounting for heterogeneous variances. PMID- 8617665 TI - Direct and maternal genetic responses to selection for weaning or yearling weight or for yearling weight and muscle score in Hereford cattle. AB - An experiment involving crosses among selection and control lines was conducted to partition direct and maternal additive genetic response to 20 yr of selection for 1) weaning weight (WWL), 2) yearling weight (YWL), and 3) an index of yearling weight and muscle score (IXL). Maternal response was estimated from reciprocal crosses among unselected sires and dams of control (CTL) and the selection lines. An Angus line was added to increase the number of reciprocal cross comparisons. Direct responses of WWL, YWL, and IXL linebreds compared with CTL were significant for all traits. Maternal genetic responses were much smaller than direct responses. Direct response in birth weight was largest for YWL, followed by WWL and IXL. Maternal effect of IXL on birth weight was larger and that of WWL and YWL was smaller than CTL. Direct responses in weaning weight did not differ greatly among selection lines; maternal response was greater for IXL than for WWL, which was selected for this trait, and response was negative for YWL. Responses in maternal effects on final weight were much reduced in Hereford crosses because of a negative relation between maternal responses in pre- and postweaning gains, especially in YWL and IXL. However, in Angus crosses, a positive association between pre- and postweaning gains increased maternal responses in final weight. Direct response for postweaning gain was greater in IXL than in YWL of WWL in Hereford crosses. In Angus crosses, YWL had larger direct responses for birth weight, preweaning gain, and postweaning gain than in other lines. The direct response for muscle score from selection in IXL, which was selected for muscle score and yearling weight, was greater than in other lines; maternal response was not important. The greatest gain in final weight was obtained when selection resulted in a favorable change in the total of direct and maternal effects pre- and postweaning, which in this experiment was provided by including a muscle score along with yearling weight as selection criteria. PMID- 8617667 TI - Compensatory growth and carcass quality in growth-restricted and refed beef steers. AB - Beef steers were fed in two phases 1) to determine the relative importance of changes in DMI, gastrointestinal tract fill, energy expenditures, and composition of gain in the compensatory growth phenomenon, 2) to compare the effects of growth restriction due to ad libitum consumption of a low-energy (low concentrate) diet to those of limited intake of a high-energy (high-concentrate) feed, and 3) to examine changes in carcass composition and quality resulting from different types of growth restriction. During the growing phase (237 to 327 kg), steers were fed either a high- (C) of low- (F) concentrate diet. Diet F was available for ad libitum consumption (FA) and diet C was available either for ad libitum consumption (CA) or on a limited basis (CL) to match the live weight gains by the FA group. During the finishing phase (327 to 481 kg), all steers received diet C, either for ad libitum consumption (CA) or restricted (CL) to 70% of the intake by the corresponding CA steers. Backfat thickness was markedly reduced (P < .001) by final feed restriction (7.4 and 6.9 mm for CL-CL and FA-CL respectively), compared with CA-CA (12.6 mm). Backfat also was lower in CL-CA (11.6 mm, P < .10) and FA-CA (9.9 mm, P < .05) than in CA-CA steers. Conversely, marbling scores were similar among groups, except for the FA-CL steers, which had lower marbling scores than FA-CA and CL-CA steers (P < .05). Higher DMI following growth restriction were accompanied by increased rates of live weight (+54 and +27%) and empty body weight (EBW; +57 and +43%) gain for CL-CA and FA-CA steers, respectively, compared with CA-CA steers. Gain:feed (EBW basis) were improved in some restricted/refed groups (+30, +13, and +10%, for Cl-CA, CL-CM respectively CA-CA. Increased DMI played a major role in the compensatory gain response in both CL-CA and FA-CA groups. Maintenance requirement was reduced (-17%) in CL-CA and increased in the FA-CA group (+21%); both changes affected the magnitude of compensatory gain in those animals. In contrast, composition of gain had little or no effect on the compensatory gain response. Programmed feeding can be used to manipulate carcass quality, but low-concentrate feeding during the growing phase may impair overall feedlot performance. PMID- 8617666 TI - Estimation of genetic parameters for litter size in Canadian Yorkshire and Landrace swine with each parity of farrowing treated as a different trait. AB - Genetic variances and covariances for the number of pigs born in total (NOBT), the number of pigs born alive (NOBA), and the number of weaned pigs (NOW) were estimated by REML under an animal model. Data on 30,357 and 42,041 litters born between 1977 and 1992 from Yorkshire and Landrace sows, respectively, were obtained from the Quebec Record of Performance sow productivity program. Data of the first four parities of litter size were used for four different analyses under an animal model: univariate analyses with direct genetic effects only, univariate analyses with maternal and direct genetic effects , seri0s of bivariate analyses with each parity treated as a different trait, and a series of bivariate analyses between NOBT, NOBA, and NOW within each parity. Heritabilities of different parities from univariate analyses under a direct genetic effects model ranged from .10 to .15, .09 to .14, and .06 to .08 for NOBT, NOBA, and NOW, respectively. Estimates of direct heritability from bivariate analyses between parties were consistent with estimates from univariate analyses in Landrace but not in Yorkshire. Genetic correlations between first and secondary parity in Yorkshire were .59, .49, and .17 for NOBT, NOBA, and NOW, and in Landrace were .90, .93, and .81, respectively. Influence of maternal effects on moderate correlations between first and secondary parity in Yorkshire was suggested. Genetic correlations averaged over all parities between NOBA and NOBT or NOW were .97 and .65 in Yorkshire and .97 and .82 in Landrace. A multiple-trait animal model with parities treated as different traits was recommended. PMID- 8617668 TI - Effects of recombinant porcine somatotropin on placental size, fetal growth, and IGF-I and IGF-II concentrations in pigs. AB - The objective of this study was to determine the effects of recombinant porcine somatotropin (rpST) on placental size, fetal growth, and maternal and fetal IGF-I and IGF-II concentrations. Twenty-four pregnant gilts received daily injections of either 1 mL of saline (control) (n = 12) or 5 mg of rpST (n = 12) from d 30 to 43 of gestation. Gilts were slaughtered on d 44 of gestation, reproductive tracts were removed, and fetal weight and length, placental weight, and implantation length were recorded. There was no effect of rpST on fetal or implantation length. Placental weight increased with rpST administration (71.20 +/- 3.52 vs 58.35 +/- 3.41 g; P < .02), as did fetal weight (18.06 +/- .55 vs 16.44 +/- .53 g; rpST vs control, respectively; P < .05). Implantation lengths were partitioned into quartiles to determine the effect of rpST on fetuses with different implantation lengths. The effect of rpST of fetal weight was greater in the first quartile ( < 137.5 mm) than in the fourth quartile ( > 240 mm) (16.04 vs 13.86 g compared with 19.47 vs 18.21 g, respectively). Analysis using a modified Brody curve suggests that the effect of rpST treatment on fetal weight is equivalent to the effect of increasing implantation length by 58.8 mm. Administration of rpST numerically raised IGF-I (P = .07) and IGF-II (P = .12) concentrations in fetal serum. Although maternal serum IGF-I concentrations were similar at d 30, treatment with rpST increased these concentrations over time (77.76, 247.75, 267.85 vs 82.59, 79.59, 77.97 ng/mL on d 30, 37, 43, respectively; P < .001, SE = 14.09). Maternal IGF-II concentrations were also similar at d 30 but decreased over time with rpST treatment (265.78, 219.61, 191.05 vs 285.44, 284.72, 283.05 ng/mL; P < .03, SE = 14.03). Increased maternal IGF-I concentrations may exhibit negative feedback on maternal IGF-II concentrations. The more pronounced effect of rpST on growth in fetuses with shorter implantation lengths suggests that rpST may increase uptake or utilization of nutrients by fetuses. In addition, nutrient transfer across placental membranes may be enhanced by rpST. PMID- 8617669 TI - The effects of in utero exposure of lambs to a beta-adrenergic agonist on prenatal and postnatal muscle growth, carcass cutability, and meat tenderness. AB - The objectives of the present experiment were to examine the effects of in utero exposure to a beta-adrenergic agonist (L644,969) on prenatal and postnatal muscle growth and meat tenderness of lambs. Thirty twin-pregnant Composite IV (1/2 Finnsheep, 1/8 Dorset, 1/8 Rambouillet, 1/8 Targhee, 1/8 Suffolk) ewe lambs were used for this experiment. All ewes were fed an alfalfa hay-corn-based diet throughout gestation and lactation. From d 25 to 95 of gestation, the diet of one half of the ewes contained 2 ppm of L644,969 on an as-fed basis. Treatment did not ( P > .05) affect lamb weights at any point in the growth cycle (birth to 43 kg). Heart weights of neonatal and market lambs were increased ( P < .05) by in utero exposure to L644,969. However, weights of lamb carcass components and weights of individual muscles were not affected by treatment (P > .05). Additionally, treatment did not alter the activities of any of the components of the calpain proteolytic system in neonatal or market lambs. Concomitantly, there was no effect of treatment on myofibril fragmentation index or Warner-Bratzler shear force. Moreover, there was no effect of treatment on muscle fiber type distributions, fiber sizes, or apparent fiber number. It seems that the lack of an effect of treatment on apparent fiber number would explain the lack of an effect on muscle weight. Thus, in utero exposure to L644,969 does not seem to have promise as a method for improving lamb carcass cutability. Other methods of improving the rate and composition of lamb carcass growth while maintaining acceptable meat tenderness must be developed. PMID- 8617670 TI - Maternal and fetal blood levels of glucose, lactate, fructose, and insulin in the conscious pig. AB - To study nutrition and metabolism in the fetal pig, a chronic catheterization method was developed that allows blood sampling in arteries and veins, at both the umbilical and uterine sources, in the conscious, unstressed animal. A catheter was inserted in the fetal aorta through a femoral artery, and another one was introduced in the umbilical vein. A catheter was put in a femoral artery of the sow so that its end was in the abdominal aorta. A fourth catheter was placed in a uterine vein draining the fetoplacental unit studied. This procedure was applied to 18 Large White primiparous sows at 99 d of gestation. Blood samples were drawn simultaneously using the four catheters before a meal at 103 d of pregnancy, and glucose, insulin, lactate, and fructose were determinated. Glycemia was 2.5 times higher in the sow than in the fetus. The extraction coefficient of glucose by the fetus amounted to 14% of the umbilical supply. The insulin level in the fetal pig was very low ( < 5 microU/mL). Lactate and fructose seemed to originate from the placenta. Blood lactate was 2.6 times lower in the sow than in the fetus, and its extraction coefficient by the fetus amounted to 8%. Fructose in the fetal blood was 2.3 times higher than that of glucose. Fructose was not utilized by the pig fetus. The present results obtained in the fetal pig are comparable to the conclusions drawn from studies with other species. PMID- 8617671 TI - Effect of feeding reduced protein, amino acid-supplemented diets on nitrogen and energy balance in grower pigs. AB - Two experiments were conducted to determine the effect of feeding reduced CP, amino acid (AA)-supplemented diets on the nitrogen (N) and energy (E) balance of grower pigs. In Exp. 1, 24 barrows (22.2 kg BW) were fed corn-soybean meal (C SBM) diets containing either 16% CP, 12% CP, or 12% CP supplemented with lysine (LYS), tryptophan (TRP), and threonine (THR). After 6 d of adaptation to the diets and feeding frequency, a 5-d N and E balance trial was conducted. Supplementation of the 12% CP diet with LYS, TRP, and THR improved N retention ( P < .01) but failed to improve N retention to the level attained by pigs fed the 16% CP diet (P < .01). Efficiency of N retention was similar between pigs fed the AA-supplemented 12% CP diet and pigs fed the 16% CP diet (P > .10). Energy retention was increased by AA-supplementation of the 12% CP diet ( P < .10) to a level higher than that of pigs fed the 16% CP diet (P < .01). In Exp. 2, 60 barrows (21.7 kg BW) were fed one of the following diets: 16% CP; 12% CP diet supplemented with indispensable AA (IDAA) to simulate the 16% CP diet; 12% CP supplemented with LYS, TRP, THR, and dispensable AA N (DAAN); 12% CP supplemented with LYS, TRP, and THR; or a 12% CP negative control diet. After 6 d of adaptation to the diets and feeding frequency, a 5-d N and E balance trial was conducted. Nitrogen retention was improved (P < .01) by supplementing the 12% CP diet with LYS, TRP, and THR but remained inferior (P < .01) to that obtained when pigs were fed the other three diets. Pigs fed the 12% CP diet with LYS, TRP, THR, and DAAN supplementation retained less N (P < .07) than pigs fed the 16% CP but retained an amount similar (P > .10) to pigs fed the 12% CP diet with IDAA and DAAN supplementation. Pigs fed the 12% CP diet with LYS, TRP, and THR supplementation exhibited the highest efficiency of N retention (P < .01). Pigs fed the 12% CP diet supplemented with LYS, TRP, THR, and DAAN retained more (P < .01) E than pigs fed the 12% CP diet supplemented with IDAA and DAAN and more (P < .07) E than pigs fed either the 12% CP diet supplemented with LYS, TRP, and THR or the unsupplemented 12% CP diet. Pigs fed the 16% CP diet retained more (P < .06) E than pigs fed the 12% CP diet with IDAA and DAAN supplementation. Although the data show that the efficiency of N retention is greatest when LYS, TRP, and THR are supplemented to the 12% CP diet, maximal N retention is achieved only when the 12% CP diets are supplemented with both the deficient AA and a source of N for dispensable AA synthesis. PMID- 8617672 TI - Effects of glycine and bovine serum albumin on inhibition of propionate metabolism in ovine hepatocytes caused by reduced phenolic monomers. AB - Hepatocytes isolated from sheep were incubated in the presence of reduced phenolics and glycine to determine the effects of these compounds on hepatic propionate metabolism in vitro. 3-Phenyl-propionic (PPA) or t-cinnamic (CA) acids, but not benzoic (BA) or 3-(4-hydroxyphenyl)propionic (4OHPPA) acids, decreased conversion of propionate to glucose at .05 mM in the absence of supplemental glycine. At 1.2 mM, all reduced phenolics decreased conversion of propionate to glucose in the absence of supplemental glycine. Addition of glycine to the incubation medium linearly alleviated the inhibition by BA, PPA, or CA, suggesting that physiological glycine concentrations limited alleviation of inhibition of propionate metabolism. Hippuric acid production increased as glycine concentration increased in the presence of PPA, CA, or 4OHPPA. Bovine serum albumin did not alleviate inhibition of conversion of propionate to glucose caused by BA, PPA, or CA and slightly alleviated inhibition caused by 4OHPPA (.4 mM). Of the reduced phenolics tested, PPA is the most likely to inhibit gluconeogenesis from propionate in ovine liver in vivo. PMID- 8617673 TI - Effects of energy source in the diet on reproductive hormones and insulin during lactation and subsequent estrus in multiparous sows. AB - Two experiments were performed. The first experiment was done to study the effects of dietary energy source on plasma insulin concentration using five gilts in a Latin square design with two diets over two periods. The diets contained either 200 g/kg of cornstarch (Starch) or soybean oil (Fat) as energy sources. Results indicate that insulin response was greater in the Starch-fed than in the Fat-fed gilts. A second experiment was performed in which 18 multiparous sows were fed one of the two experimental diets from farrowing until slaughter at d 35 of subsequent pregnancy. All sows nursed nine pigs. Blood samples were taken from a permanent jugular vein catheter every 12 min during a 12-h period on d 109 +/- 1 of pregnancy, on d 7 +/- 1, 14 +/- 1, and 21 +/- 1 of lactation, and on the day of weaning ( d 22 +/- 1). From 48 h after weaning, blood samples were taken every 4 h until 24 h after ovulation. After that, blood samples were taken at 12-h intervals until d 10 after ovulation. Differences between diets in insulin response were not significant. In Starch-fed sows, LH pulsatility at d 7 of lactation was greater (P < .05), the preovulatory LH surge was greater ( P < .05), and progesterone production was greater (P < .05) from 108 h until 256 h after the LH surge than in the Fat-fed sows. Results indicate that feeding Starch rich diets to multiparous sows compared with Fat-rich diets, on an isocaloric basis, increases LH pulsatility during early lactation, the preovulatory LH surge, and progesterone production after the LH surge. PMID- 8617674 TI - Immunization of prepubertal beef heifers against gonadotropin-releasing hormone: immune, estrus, ovarian, and growth responses. AB - To develop an effective immunization protocol against human serum albumin-Cys-Gly GnRH (HSA-GnRH) conjugate to delay the onset of puberty in heifers, 58 heifers (8 mo of age; mean +/- SE BW = 203 +/- 1 kg) were randomly assigned to each of six treatments: 1) controls, .1 mg of HSA, with diethylaminoethyl (DEAE)-dextran as adjuvant, on d 0 and 28; 2) .1 mg of HSA-GnRH, with DEAE-dextran, on d 0; 3) as 2) and booster on d 28; 4) as 3) but boosters also on d 84, 140, 196, and 252; 5) as 2) but half the conjugate given with DEAE-dextran adjuvant and half with non ulcerative Freund's adjuvant (NUFA), injected in two separate sites; and 6) as 2) but the conjugate given with DEAE-dextran and NUFA, emulsified and injected in two sites. The duration of the experiment was 342 d. Mean plasma GnRH antibody titers (samples every 2 wk) for heifers in Treatments 2 to 6 were 9.4 +/- 1.16, 20.6 +/- 2.21, 43.9 +/- 2.86, 27.9 +/- 2.67, and 44.5 +/- 3.75% binding at a plasma dilution of 1:640. The mean number of times estrus was observed in heifers was less (P < .05; pooled SEM = .53) in Treatments 4 (.2) and 6 (2.4) than in Treatments 1, 2, 3, and 5 (7.8, 7.0, 7.0, and 6.6, respectively). The mean interval to the onset of puberty (the first increase in plasma progesterone > or = .5 ng/mL for > or = 10 d with samples at 3- to 4-d intervals) was greater (P < .05; pooled SEM = 11.6) for heifers in treatments 4 (339 d) and 6 (276 d) than for heifers in Treatments 1, 2, 3, and 5 (164, 159, 165, and 170 d, respectively). Mean ADG of heifers was reduced (P < .05) in treatments 2, 3, 4, and 6 (.71, .72, .68, and .69 kg, respectively) compared with controls (.77). In summary, the multiple booster immunization treatment induced and maintained sufficient anti-GnRH titer to delay puberty for 175 d; a single immunization against GnRH with DEAE and NUFA increased antibody titers enough to delay puberty for 112 d. However, GnRH immunization treatments reduced ADG of heifers in Treatments 2, 3, 4, and 6. PMID- 8617675 TI - Dose response of plasma insulin and glucagon to intravenous n-butyrate infusion in sheep. AB - n-Butyrate (0, 1, 2, 4, 8, 16, 32, and 64 mumol.kg BW-1.min-1) was infused i.v. for 30 min to investigate the physiological effects of n-butyrate on plasma insulin and glucagon responses in sheep. Blood n-butyrate concentrations during n butyrate infusion increased (P < .01) with increasing infusion rates. At the greater infusion rates of n-butyrate, plasma glucose concentrations increased (P < .01) during infusion, and then they decreased (P < .05) to less than the preinfusion values. Plasma insulin concentrations increased (P < .01) dose dependently during n-butyrate infusion at rates of > or = 2 mumol.kg BW-1.min-1. Plasma glucagon concentrations increased (P < .05) during n-butyrate infusion at rates of 32 and 64 mumol.kg BW-1.min-1. It is possible that, in sheep, n-butyrate may have a physiological role in controlling insulin secretion; however, it does not seem to have a role in glucagon secretion. PMID- 8617676 TI - Influence of calf genotype on colostral immunoglobulins in Bos taurus and Bos indicus cows and serum immunoglobulins in their calves. AB - Purebred Bos indicus calves are documented to have lower survival rates than Bos taurus calves. Thus, this study was designed to investigate the possibility that this decreased survival rate may be attributed to dam colostral immunoglobulin (Ig) concentrations and subsequent calf serum Ig concentrations. The specific objective was to determine the effect of breed type of calf on colostrum production, immunoglobulin concentrations in colostrum and calf serum, and availability and absorption efficiency of Ig. Brahman (B) and Angus (A) cattle were reciprocally mated to produce calves of the following types: A x A (n = 8), A x B (n = 9), B x B (n = 11), and B x A (n = 11). At birth, calves were separated from their dams and a blood sample was collected before feeding pooled colostrum (30 mL/kg birth weight) at 1 and 6 h of age. From 6 to 12 h of age, each calf was placed in a box that allowed interaction with the dam but prevented suckling. At 12 h of age, each calf was fed its dam's colostrum and placed with the dam. Additional blood samples were collected at 12, 24, and 48 h after birth. Serum and colostrum samples were analyzed for IgG, IgG1, IgG2, IgM, and IgA using single radial immunodiffusion (RID) assay techniques. The cows were hand-milked after induction of milk letdown with oxytocin at 1 and 12 h after calving. Colostrum volume was recorded, and samples were collected. Brahman cows produced more (P < .001) colostrum at 1 and 12 h than A cows. Total Ig concentrations were obtained by summing IgG, IgG1, IgG2, IgM, and IgA concentrations. Total Ig (P < .02), IgG (P < .005), and IgA (P < .01) concentrations in colostrum were greater in cows producing crossbred calves. Total Ig (P < .006), IgG (P < .02), IgG1 (P < .004), and IgG2 (P < .02) available in colostrum were affected by B x B and A x B breed types of calf. Brahman cows had more Ig available at 1 and 12 h than A cows due to increased production of colostrum. Breed type influenced colostral Ig in cattle. Serum concentrations of total Ig, IgG, IgG1, IgG2, IgM, and IgA in the calf and efficiency of absorption at 6 and 12 h were not affected by breed type, sex of calf, or any interaction. PMID- 8617677 TI - Recombinant bovine somatotropin increases milk yield and calf gain in diverse breeds of beef cattle: associated changes in hormones and indices of metabolism. AB - In Exp. 1, Angus (A, n = 30), Charolais (C, n = 37), and Simmental (S, n = 30) multiparous cows received (s.c.) recombinantly derived bovine somatotropin (bST; sometribove, 500 mg) or vehicle (VEH) at 2-wk intervals from 124 to 228 d postpartum (DPP). Calves were weaned at 228 DPP. Bovine somatotropin increased (P < .01) milk yield and percentage of milk fat similarly in A, C, and S cows. Calf weaning weight was greater (P < .05) in cows treated with bST than in those given VEH. Administration of bST decreased deposition of fat and increased concentrations of IGF-I, insulin, glucose, and nonesterified fatty acids. In Exp. 2, we compared effects of bST initiated before or after the breeding season. Charolais (n = 33) and S (n = 40) cows were administered (at 2-wk intervals) VEH or bST beginning at 28 DPP (B-bST) or bST beginning at 105 DPP (A-bST). Calves were weaned at 243 DPP. Administration of bST before or after the breeding season increased milk yield on DPP 136 and 194; however, yields were greater in A-bST than in B-bST cows. Milk yields were similar in all cows at 236 DPP, corresponding to decreased forage availability. Calf body weight was greater (P < .05) in A-bST than VEH; B-bST calves were similar to VEH. Fat depth was greater in VEH than in bST-treated cows in C but not in S cows. Serum IGF-I was greater in A- and B-bST than in VEH cows. Mean days from calving to serum progesterone > 1 ng/mL and pregnancy rates were similar in VEH, A-, and B-bST cows. Administration of bST increased cow milk yield and subsequent calf weaning weight when initiated after 100 d postpartum. As anticipated, bST increased IGF-I, insulin, glucose, and nonesterified fatty acids. Administration of bST before and during the breeding season did not affect reproductive performance. PMID- 8617678 TI - Evaluation of response to hormonal therapy in prepubertal gilts of different genetic lines. AB - Research was conducted to determine the effect of genetic line on hormonally induced puberty. Two studies were conducted, the first to evaluate estrus response (n = 120 gilts) and the second to evaluate follicular development (n = 24 gilts). Gilts were allotted to treatments in a 2 x 2 factorial arrangement. Gilts from two genetic lines (Age at Puberty x Yorkshire [APY] and Relaxed Selection x Yorkshire [RSY] received either P.G. 600 [symbol: see text] (P) or no injection (C). The percentage in estrus within 5 d after treatment was greater (P < .05) for P than for C gilts. However, no difference was detected for the percentage of P or C gilts that had ovulated within 14 d. For the APY line, P gilts had greater (P < .05) ovulation rates than C gilts. However, for the RSY line, P and C gilts did not differ in ovulation rate. More P gilts than C gilts had follicular cysts (P < .05). At 48 h after onset of estrus, plasma progesterone concentration tended to be greater (P = .13) for P gilts than for C gilts. In Exp. 2, gilts were ovariectomized 18 h after the onset of estrus. Ova were dissected from follicles on one ovary, and follicular fluid was aspirated from the other ovary. No differences were detected for the percentages of ova in various stages of meiosis. For APY gilts, follicular fluid estradiol concentration for P gilts tended to be lower (P = .12) than that for C gilts; however, no differences were detected for RSY gilts. Progesterone concentration in follicular fluid tended (P = .14) to be greater in P gilts than in C gilts. These results indicate that the effect of genetic line on age at puberty should be considered when inducing puberty. PMID- 8617679 TI - The role of insulin-like growth factor I in clenbuterol-stimulated growth in growing lambs. AB - We examined the role of IGF-I in muscle growth stimulated by a beta-adrenergic agonist, clenbuterol. Ewe lambs (90 d old, 20.4 kg mean live weight) were allotted to five groups. A pretreatment control group of five lambs was slaughtered immediately (0 d). The other four groups of six ewes ate freely for 38 or 80 d and were then slaughtered. Half those lambs received clenbuterol (400 micrograms.kg live weight-1.d-1) as a dietary supplement. Blood was collected at intervals from 19 d before supplementation began (0 d) until slaughter. Prerigor muscle samples were sectioned for detection of IGF-I receptors and myofibrillar ATPase activity. Carcass weights were slightly increased by treatment, whereas muscle weights (semimembranosus, gastrocnemius, and biceps femoris) were greatly increased (P < .001), up to 48% at 80 d for semimembranosus. Clenbuterol significantly decreased collagen concentration because myofibrillar proteins were preferentially produced. Collagen solubility was unaffected. Total RNA:total DNA in semimembranosus and gastrocnemius showed transcription was still stimulated between 38 and 80 d. Fiber type area analysis indicated a shift toward glycolytic metabolism, confirmed by iron measurements. However, clenbuterol did not change the portion of muscle occupied by each ATPase class, and the data indicated that type I fibers, though smaller, became relatively more numerous. In spite of significant muscle changes, plasma IGF-I was unaffected by clenbuterol. Similarly, there was no difference in the specific binding of [125I]IGF-I at slaughter between treated and control lambs. However, a response in the first few days of treatment, preceding visible hypertrophy, cannot be excluded. PMID- 8617680 TI - Effect of protein level and source in molasses slurries on the performance of growing cattle fed hay during winter. AB - Four molasses slurries of varying protein level and source were fed in two performance trials (Year 1, 105 d; Year 2, 92 d) to growing cattle (Year 1, 230 kg; Year 2, 247 kg). Treatments were CONTROL (hay only), MOL (molasses-corn meal), MOL-UREA (molasses-urea-corn meal), MOL-SBM (molasses-soybean meal), and MOL-BF (molasses-urea-corn meal blood meal-hydrolyzed feather meal). Animals on all treatments were offered bermudagrass hay (Year 1: 12.8% CP, 50% TDN; Year 2: 12.8% CP, 54% TDN) and a complete mineral mixture free choice. Each treatment was fed to three pens each year with seven animals/pen. Slurries were offered at 2.1 kg/d (DM) and effects on forage intake, ADG, condition score (1 to 9), hip height, and plasma urea nitrogen were monitored. Treatment effects for Years 1 and 2 were analyzed separately due to treatment x year interactions (P < .15) with respect to ADG, hip height change, condition score change, and feed cost of gain. Supplementation increased (P < .001) ADG over CONTROL in Year 1 (.41 vs .06 kg/d) and Year 2 (.69 vs .25 kg/d), increased hip height change by .02 cm/d (P < .001) in Year 1 and by .01 cm/d (P = .012) in Year 2, and decreased (P < .001) loss of body condition in Years 1 and 2. Molasses-urea showed no advantage over MOL in Years 1 and 2. Natural protein (MOL-SBM and MOL-BF) increased ADG by .10 kg/d in Year 1 (P = .001) and by .06 kg/d in Year 2 (P = .077) compared with MOL UREA. Daily gain was improved by MOL- BF by .05 kg/d (P = .109) in Year 1 and by .08 kg/d (P = .063) in Year 2 compared with MOL-SBM. Results indicate that growing cattle fed bermudagrass hay during winter respond positively to energy supplementation in the form of molasses. The addition of animal source protein enhanced this response. PMID- 8617681 TI - Urea ammoniation effects on the feeding value of guineagrass (Panicum maximum) hay. AB - Laboratory, digestion, and growth studies evaluated urea as a source of ammoniation for quality improvement in guineagrass (Panicum maximum) hay. In a laboratory trial, 5.0-kg portions of hay were reconstituted with water to yield final forage moisture concentrations or 25 of 40% and treated with urea at 0, 4, 6, or 8% of the forage DM, with or without urease addition. Main effects of forage moisture or urease addition did not influence (P > .10) CP or NDF concentration or in vitro OM disappearance (IVOMD) of the guineagrass hay. Hay CP concentration and IVOMD increased linearly (P < .01), whereas concentrations of hemicellulose and ADL decreased linearly (P < .05) with increasing urea level. In other experiments, round bales of hay (320 kg) were reconstituted with water to yield final forage moisture concentrations of 25 or 40% and treated with urea at 0, 4, or 6% of the forage DM. The urea solution was applied as a spray onto the cut edges of the bales, or by low pressure (10 psi) injection. Two- and three-way interactions (P < .05) existed among forage moisture concentration, urea application method, and urea level for CP and NDF concentration and IVOMD of the guineagrass hay. Greatest enhancements in these forage quality characteristics were obtained when the urea solution was sprayed onto the hay at the 25% forage moisture concentration. In two digestion and two growth trials, round bales of hay were treated with 0, 4, and 6% urea sprayed onto the hay at the 25% forage moisture level. In each growth trial, 30 St. Croix white hair castrated male sheep (Trial 1:34 +/- 5.5 kg, Trial 2: 17 +/- 3.5 kg) were allotted to six pens of five head each, resulting in two pens per treatment. In the digestion trials, six similar sheep were used in a replicated 3 x 3 Latin square design. In the digestion and growth trials, hay intake increased in a quadratic (P < .05) manner with increasing urea level. Apparent NDF and ADF digestibilities increased linearly (P < .05) with increasing urea level. Linear improvements in ADG (P < .05) and gain/feed (P < .07) were observed with increasing urea level. Urea ammoniation offers potential for improving the feeding value of tropical forages and provides an option for quality forage during the dry season. PMID- 8617682 TI - Influence of moisture content of forage on ruminal functional specific gravity and passage of digesta. AB - Four crossbred beef steers (392 kg BW) fitted with ruminal cannulas were used in a 4 x 4 Latin square experiment with treatments arranged in a 2 x 2 factorial to evaluate effects of forage species (F) and reconstitution (R) on apparent digestibility, ruminal kinetics, and digesta functional specific gravity (FSG). Dietary treatments were alfalfa (Medicago sativa L.) and timothy (Phleum pratense L.) hays (90% DM) and the same hays reconstituted with water to 38% DM. Apparent DM digestibility was unaffected by R or F. Proportion of ruminal digesta with a FSG of .9 to 1.1 or 1.1 to 1.2 decreased linearly (P < .05) after feeding, whereas the proportion with FSG > 1.2 increased linearly (P < .01). Reconstitution decreased (P < .10) the proportion of ruminal digesta with FSG of 1.1 to 1.2 and increased (P < .10) the proportion with a FSG > 1.2 compared with the hay diets. Reconstitution decreased digesta passage rate for timothy forage; no influence on passage rate for alfalfa was detected (F x R, P < .10). Ruminal DM pool was increased by reconstitution (P < .10). Inert particles of 1-mm length passed from the rumen at a faster rate (P < .05) than 3-mm particles. Inert particles with SG of 1.32 passed from the rumen at a faster rate (P < .05) in the reconstituted alfalfa than in the dry alfalfa diet but at a slower rate in the reconstituted timothy than in the dry timothy diet. These results suggest that factors other than SG of the digesta can have a profound effect on passage of forage particles from the rumen. PMID- 8617683 TI - Influence of duodenal slaframine infusion on site of nutrient disappearance from the digestive tract of steers fed a high-concentrate diet. AB - The effect of duodenal slaframine (SF) infusion on site and extent of digestion was determined using four steers equipped with ruminal, duodenal, and ileal cannulas in a 4 x 4 Latin square. A 77% dry-rolled corn diet was provided in 12 equal portions daily at a DMI of 2.26% BW. Slaframine in a .9% saline excipient was infused into the duodenum every 12 h with total daily dose of 0, 30, 60, or 90 micrograms /kg of BW. Slaframine infusion had no effect on ruminal pH, ruminal NH3 N, or solids and liquids passage rate. Slaframine increased (linear, P < .10) total tract OM and starch disappearance and digestibility and tended to increase (linear, P = .14) total tract N digestibility. Ruminal starch disappearance tended to be decreased (quadratic, P = .16) by SF. Small intestinal OM digestibility was increased (linear, P < .10) but starch digestibility in the small intestine was not affected by SF. Increased total tract starch digestibility was caused by increased (quadratic, P < .10) starch fermentation in the large intestine. Ruminal feed N digestibility decreased at the intermediate doses of SF (quadratic, P < .10). Total N digestibility in the small intestine tended to be increased (cubic, P = .13) with 30 and 90 micrograms of SF/kg of BW. Decreased ruminal feed N digestion was compensated for by increased (quadratic, P < .10) small intestinal feed N disappearance for steers treated with intermediate doses of SF. The potential of SF to increase starch digestion in the rumen and small intestine seems to be limited. PMID- 8617684 TI - The effects of ruminal acidosis on volatile fatty acid absorption and plasma activities of pancreatic enzymes in lambs. AB - Twenty crossbred wethers (41.9 +/- 4.0 kg BW), each fitted with a ruminal cannula and a jugular catheter, were used in a completely randomized design to examine the effects of ruminal acidosis on plasma activities of pancreatic enzymes and fractional rates of VFA absorption. Lambs had ad libitum access to a 50% concentrate diet. Acidosis was induced by an intraruminal dose of glucose at 0, 6, 12, or 18 g/kg BW via the ruminal cannula. Ruminal fluid and plasma were collected 0, 4, 8, 12, 18, 24, 36, 48, and 72 h after dosing. Ruminal fluid pH was reduced (linear, P < .001) with increasing ruminal glucose. Total ruminal VFA concentration decreased (linear, P < .01) and D(-)-lactate (linear, P < .01) and L(+)- lactate (linear, P = .07) concentrations increased with increasing ruminal glucose. Activities of amylase and lipase in plasma were not affected by ruminal glucose (P > .10). Ten days after the acidosis insult, rumens were evacuated and contents were replaced with an isotonic Cr:VFA solution to measure ruminal VFA absorption. Ruminal fluid was collected hourly from 0 to 6 h. Fractional rate of acetate absorption was 13% lower for lambs receiving 18 g/kg BW glucose than for control lambs. In addition, fractional liquid passage rate was lower (P < .05) in lambs receiving 18 g/kg BW glucose 6 mo after the insult of acidosis. These data suggest that a short-term, severe insult of acute acidosis does not result in pancreatic tissue damage but may result in reduced ruminal VFA absorption for an extended period of time. PMID- 8617685 TI - Influence of dietary vitamin E on the oxidative stability and quality of pig meat. AB - Oxidation of lipids is a major cause of deterioration in the quality of muscle foods and can directly affect many quality characteristics such as flavor, color, texture, nutritive value, and safety of the food. Lipid oxidation in muscle systems is initiated at the membrane level in the intracellular phospholipid fractions. In the processing of muscle foods, one of the most important questions concerns the methods used to delay the initiation of oxidation and loss of quality. Vitamin E is a major lipid-soluble antioxidant, and one of its primary functions is to maintain and protect biological membranes against lipid peroxidation. Dietary vitamin E supplementation above requirement levels is effective in reducing lipid oxidation. This review focuses on the antioxidant function of vitamin E and how supplementation of the diet of pigs with vitamin E influences the rate of lipid peroxidation, color, water-holding capacity, and cholesterol oxidation in pig meat. PMID- 8617686 TI - A review of dietary vitamin E supplementation for improvement of beef quality. AB - Color is a primary factor used by consumers to judge beef quality, especially freshness. Recent studies indicate that dietary supplementation of vitamin E to beef cattle increases the alpha-tocopherol concentration in muscle and its membranous subcellular fractions. The increased tissue alpha-tocopherol concentration protects not only membranal lipids but also myoglobin from oxidation. This results in delayed onset of discoloration in fresh, ground, and frozen beef and in suppression of lipid rancidity, especially in fresh, ground, and frozen beef and less so in cooked beef. Extension of beef color display life depends on dose level and duration of dietary vitamin E, muscle, and aging period. Cumulative results of experiments conducted to date indicate that beef from animals that receive 500 IU/steer daily of vitamin E for 126 d could assuredly benefit the domestic retail market by extending color display life. Implementation of this technology by the beef industry requires development of a method for rapid determination of alpha-tocopherol concentration in muscle samples collected on the day of harvest. PMID- 8617687 TI - Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrus in cattle: a review. AB - Treatment with GnRH and PGF2 alpha is a practical method for controlling ovarian follicular and luteal functions and increasing the precision of estrus synchronization in cyclic and acyclic postpartum cows and heifers. This method reduces considerably the period of time needed for estrus detection; it synchronizes the estrous cycle of 70 to 80% of the cyclic cows to within a 4-d interval without any detrimental effect on the fertility rate (65 to 85%). Moreover, resumption of ovarian activity and normal fertility in acyclic cows in favored. Administration of GnRH eliminates the large follicles by ovulation or atresia and induces emergence of a new follicular wave within 3 to 4 d after treatment at any stage of the estrous cycle, but it limits further growth of these emerging follicles by increasing atresia. The precision of estrus and the unaltered fertility rate is due to the synchronized selection of a new larger growing follicle, which becomes the ovulatory follicle after PGF(2 alpha)-induced luteolysis 6 d after GnRH treatment. Also, fixed-time AI programs without the need for estrus detection may be possible using a second injection of GnRH in a GnRH-PGF(2 alpha)-GnRH protocol to ovulate the selected follicle at a precise time. We describe a physiological model to explain how the precision of estrus is improved following PGF(2 alpha)-induced luteolysis, via the effect of pretreatment with GnRH on follicular development and luteal functions in cattle. Application of this model to the development of reliable methods of fixed-time insemination is also explored. PMID- 8617688 TI - Review of some aspects of growth and development of feedlot cattle. AB - Growth in animals is defined as accretion of protein, fat and bone. Although growth typically is measured as the change in live weight, nutrient retention is estimated more precisely by measuring empty body weight and composition, whereas production economics are measured ideally through carcass weights and quality. As a percentage of live weight gain, carcass weight gain usually is a much higher percentage during the feedlot phase than during the growing phase of production because dressing percentage (ratio of carcass:live weight) increases with maturation and is greater with concentrate than with roughage diets. At a given fraction of mature body size (maximum body protein mass), body fat percentage seems to be a constant. Mature size may be altered genetically and nutritionally. Protein accretion declines to zero when cattle reach their mature body size (approximately 36% fat in empty body weight in modern cattle) even though mature animals can continue to accrete fat. Although fat accretion can be reduced by limiting the supply of net energy, rate of fat accretion by finishing steers given ad libitum access to high-concentrate diets seems to reach a plateau at approximately 550 g daily. Protein mass, in contrast, increases in proportion to empty body weight. The protein:fat ratio of the carcass can be increased through increasing mature size, by administering hormones or hormonal modifiers, by limiting energy intake during the growing period or finishing period, or by slaughtering cattle at an earlier stage of maturity. Energetically, efficiency of accretion of fat is approximately 1.7 times that of protein. But because more water is stored with deposited protein than with deposited fat, lean tissue gain is four times as efficient as accretion of fat tissue. Conversion of protein to fat is very inefficient, suggesting that excess protein is utilized inefficiently. PMID- 8617689 TI - Changing paradigms in animal agriculture: the role of academia and industry in technology transfer. AB - Challenges abound for academia, industry, and animal agriculture. Universities, especially land-grant universities, are losing their credibility with the public on whom they depend for support. Industries have gone and continue to go through wrenching restructuring, driven by the realities of the marketplace. On the farm and in the classrooms, laboratories, and field research stations of land-grant universities, agriculturalists face a major challenge-society's growing resistance to science and technology. Technology, especially biotechnology, has become suspect in the minds of many people. Solutions to these and other challenges for effective technology transfer in the future will not depend on a single institution, company, or program. Perhaps the most challenging issue is simply how to unite groups and individuals who have been accustomed to having their own separate programs. In the future, "business as usual"; won't work. Academia and industry are being held to new and higher standards of accountability by their clientele (customers). Academia and industry will need to join forces to increase U.S. agriculture's competitiveness in a global environment that demands that the lag time between discovery and adoption of appropriate technology be shortened. PMID- 8617690 TI - Changing paradigms in animal agriculture: addressing animal issues in Washington by a professional society. AB - This article discusses key points that we believe are important as we consider how a professional society in Washington could address animal issues with a changing paradigm as expressed by the public. In summary, our professional perspectives need to change as much as or more than society does. We must understand the advocacy groups and where they are coming from. We have to provide relevant messages to the diverse audiences that play an important role in the public policy process. We have to be clear about our goals and objectives as professionals so as to be more effective as we participate with others in trying to achieve stated goals. We need to understand the broad view of the role of science that exists today, particularly focusing on accountability and relevance of our work to assure the public that their funds are well spent. We need to ensure that all the questions are addressed so that society will maintain trust and respect for the role of science in society. PMID- 8617691 TI - Beef Quality Assurance education via satellite videoconference. AB - A 1.5-h satellite video program was developed for a statewide Beef Quality Assurance (BQA) producer education activity. Twenty-seven downlink sites were identified across the state, and additional known downlink sites included one each in Minnesota, Indiana, and Monterrey, Mexico. The videoconference was formally evaluated by viewers and resource people at each site. Program participants included representatives of the Ohio and National Cattlemen's Associations and two producers currently implementing BQA programs. A toll-free telephone number was available for viewer's questions during the last 15 min of air time and following the video program. A demographic data form and pre- and postconference evaluations were completed by 368 participants. Twenty-seven percent of the respondents were age 56 or older; 76% were age 36 or older. A one to five scale (strongly disagree to strongly agree) was used for evaluation. Beef Quality Assurance was considered by viewers to be an important consideration for the future in pre- (mean = 4.5, SD = .99) and post-evaluations (mean = 4.6, SD = .83). The mean response to the question regarding using more videoconferencing for agriculture programming was 3.9 (SD = .91). Four percent of participants indicated less or no videoconferencing should be used, 24% were neutral, and 72% felt more videoconferencing should be done. Comments were neutral with regard to the length of the conference (mean = 3.5, SD = .86) and the complexity of the subject matter (mean = 3.1, SD = .64). More than 88% of the participants agreed or strongly agreed that they intended to examine their overall production strategies to see where they could improve the quality of beef they produce (mean = 4.3, SD = .85). After the videoconference, 2.6% of respondents still strongly believed that BQA was not an important consideration for the future of the beef industry. The cost of all activities associated with this satellite video program was $13,000. PMID- 8617692 TI - Rapid communication: bovine dinucleotide repeat polymorphisms ABS010, ABS011, ABS012, ABS013, and ABS014. PMID- 8617693 TI - A controlled comparison of light box and head-mounted units in the treatment of seasonal depression. AB - BACKGROUND: Patterns of response to the light box and head-mounted unit (HMUs) in seasonal affective disorder (SAD) appear to differ. The current study employed a "no light" condition to compare the response rates with the light box and HMU against a plausible placebo. METHOD: Forty-three subjects with DSM-III-R nonpsychotic, unipolar major depression, seasonal subtype, were randomly assigned, in a double-blind manner, to receive 2 weeks of active treatment with a light box (N=9) or HMU (N=12) that emitted no visible light, or 2 weeks of placebo treatment with a light box (N=12) or HMU (N=10) that emitted no visible light. Response was defined as a 50% or greater reduction in both the 17-item "typical" score and 8-item "atypical" score on the Structured Interview Guide for the Hamilton Rating Scale for Depression-SAD version (SIGH-SAD). RESULTS: Using ANOVA for repeated measures, with change in total SIGH-SAD score as the dependent measure, we found no significant main effect of light (F=0.20, p=N.S.) or unit (F=0.50, p=N.S.), and no interaction (F=0.21, p=N.S.). Using log-linear analysis, we found no significant difference in response rate between the four cells (likelihood ratio chi-square = 2.1, p=N.S.). Using chi-square analysis, we found no significant difference in response rates between patients who received light (48%) versus patients who received no light (41%; chi-square = 0.2, p=N.S.) or between patients who received the light box (38%) versus HMU (50%; chi-square = 0.62, p=N.S.). CONCLUSION: The failure to detect any significant difference in efficacy between active and placebo treatments calls into question the specificity of light in light therapy for SAD. Methodological limitations, particularly small sample size, are discussed. PMID- 8617694 TI - The use of midazolam with pulse oximetry in the drug-assisted interview. AB - BACKGROUND: The drug-assisted interview appears to be declining in psychiatric practice. This decline may be due in part to complexities in the use of barbiturates, which have a narrow therapeutic index. METHOD: Five patients who had depression and/or posttraumatic stress disorder underwent drug-assisted interviews for which midazolam and pulse oximetry monitoring were used. RESULTS: The midazolam-pulse oximetry technique resulted in consistent and easily monitored levels of conscious sedation. Midazolam was judged comparably effective to amobarbital and easier to use. CONCLUSION: A simple, safe, and effective technique for drug-assisted interviewing for which midazolam and continuous pulse oximetry monitoring are used is described. PMID- 8617695 TI - Response to an open trial of a second SSRI in major depression. AB - BACKGROUND: We evaluated the efficacy of a second serotonin selective reuptake inhibitor (SSRI) in patients who had failed to respond to the first SSRI used. METHOD: Fifty-five patients with major depression who had failed one of the SSRIs for their current depressive episode were included. After failing a trial of one SSRI, they received a second SSRI in an open clinical trial. RESULTS: On the basis of the Clinical Global Impression-Improvement scale, 28 of 55 patients had a marked or complete antidepressant response. CONCLUSION: These data provide preliminary clinical evidence that substituting a second SRI may be a useful clinical alternative in depressed patients who fail to respond to an adequate trial of an SSRI. PMID- 8617696 TI - DSM-IV stereotypic movement disorder: persistence of stereotypies of infancy in intellectually normal adolescents and adults. AB - BACKGROUND: As part of a broader series of studies on unwanted repetitive behaviors, DSM-IV stereotypic movement disorder (SMD) was examined in an intellectually normal population. Repetitive nonfunctional behaviors, or stereotypies, are expressed during early normal development but have not been described in adults without severe psychiatric or intellectual impairment. METHOD: Lifetime and current psychiatric Axis I diagnoses were determined by structured and clinical interviews in subjects who responded to a newspaper advertisement that specifically mentioned rocking and head banging. RESULTS: Of 52 potential subjects who were screened by telephone, 32 had been previously diagnosed with an Axis I psychiatric disorder, which presumably accounted for the repetitive behavior, or were otherwise excluded. Of 20 who were interviewed in person, 12 met DSM-IV criteria for SMD; rocking or thumb sucking was present in 8 of these 12. Four of 8 rockers had a first-degree relative who had a lifetime history of a similar repetitive behavior. A lifetime history of an affective or anxiety disorder was found for 11 of 12 SMD subjects. CONCLUSION: DSM-IV stereotypic movement disorder can be diagnosed in intellectually normal individuals. Although sampling bias was probable, prominent stereotypies in individuals meeting the DSM-IV criteria for stereotypic movement disorder, which are narrower than the DSM-III-R criteria for stereotypy/habit disorder, seem likely to include rocking and thumb sucking. The likelihood of persistence of these behaviors, which are developmentally appropriate in infancy, may be enhanced by comorbidity with anxiety or affective disorders. PMID- 8617697 TI - Clozapine treatment in polydipsia and intermittent hyponatremia. AB - BACKGROUND: Recent case reports indicate that clozapine treatment diminishes excessive diurnal weight gain and alleviates hyponatremia observed in some chronically psychotic patients. We examined the influence of clozapine on sodium metabolism and water regulation across a group of patients with the syndrome of polydipsia and intermittent hyponatremia. METHOD: Eleven patients with treatment resistant DSM-III-R schizophrenia or schizoaffective disorder were studied. Each had a history of repeated diurnal weight gains of greater than 10% with at least one documented bout of hyponatremia in the 6 months before clozapine treatment. We utilized a target weight protocol and serial laboratory measures to compare changes in sodium metabolism and water regulation during 26 weeks of standard antipsychotic medication and 26 weeks of clozapine treatment. RESULTS: Across patients, we found significant improvement in routinely monitored 6 a.m. and 4 p.m. serum sodium, reflecting normalization of sodium metabolism. We also found that the frequency (as reflected by diurnal weight gain), severity (lowest serum sodium), and estimated quantity (calculated urine volume) of polydipsia improved across patients. Improvement in polydipsia and hyponatremia was associated with decreased necessity for monitoring and restrictive interventions, and tended to be associated with psychiatric improvement. CONCLUSION: We found a corrective and stabilizing effect of clozapine on polydipsia and intermittent hyponatremia. Future studies need to examine the relationship of psychiatric improvement and alterations in the regulation of sodium and water physiology to our findings. PMID- 8617699 TI - The use of risperidone in the treatment of bipolar disorder. PMID- 8617698 TI - Causes of haloperidol discontinuation in patients with Tourette's disorder: management and alternatives. AB - BACKGROUND: Neuroleptics are considered the mainstay of treatment in Tourette's disorder, and haloperidol is deemed the treatment of choice by many. Factors such as treatment efficacy and the side effects that appear in response to neuroleptic administration have been implicated in affecting medication compliance. However, a detailed evaluation of these factors has yet to be undertaken in Tourette's disorder. METHOD: Of 51 consecutive referrals to a Tourette's disorder clinic, 48 met DSM-III-R criteria for Tourette's disorder. Of these 48, 28 had previously received neuroleptics. In this set of 28 patients, 24 (16 male, 8 female) had initially received treatment with haloperidol, and they made up the present sample; their ages ranged from 10.4 to 47.9 years (mean = 27.1), and age at onset ranged from 2 to 16 years. Each patient completed an evaluation consisting of a Tourette Syndrome Questionnaire and a clinical interview with the patient and involoved family members. Charts were also reviewed to gather information concerning side effects and other factors that led to haloperidol discontinuation and/or noncompliance. RESULTS: Duration of treatment ranged from 3 days to 14 years (mean = 3.6 years). In this sample, 12.5% (3/24) of the subjects continued medication without interruption (mean +/- SD = 8.4 +/- 5.1 years of medication). Of the 21 patients who discontinued haloperidol, 66.7% (14/21) did so because they experienced intolerable side effects, 9.5% (2/21) because of the fear of experiencing certain side effects, and 14.3% (3/21) because of a combination of these factors. The principal side effects that led to discontinuation included dysphoric reactions, akathisia, nervousness, sedation, dystonic reactions, and cognitive dulling/feeling drugged. CONCLUSION: Careful monitoring of side effects and efficacy is essential to continued compliance with haloperidol. In addition, psychoeducation about potential consequences of medication administration may help promote compliance in those patients who develop fears of possible adverse reactions. PMID- 8617700 TI - Verapamil maintenance therapy in bipolar patients. PMID- 8617701 TI - Diphenhydramine dependence: a need for awareness. PMID- 8617702 TI - Capgras'and Fregoli's syndromes in one family. PMID- 8617703 TI - Use of ECT in treatment of depression in patients with diabetes mellitus. PMID- 8617704 TI - The making of a user friendly MAOI diet. AB - BACKGROUND: Many monoamine oxidase inhibitor (MAOI) diets are considered to be excessively restrictive and founded on poor scientific evidence. We present a safe and practical MAOI diet based on the related clinical and analytic data. METHOD: We used a critical review of the literature and our own tyramine assay results to categorize foods to be restricted absolutely, taken in moderation only, or unrestricted. RESULTS: We recommend that users avoid aged cheese; aged or cured meats (e.g., air-dried sausage); any potentially spoiled meat, poultry, or fish; broad (fava) bean pods; Marmite concentrated yeast extract; sauerkraut; soy sauce and soy bean condiments; and tap beer. Wine and domestic bottled or canned beer are considered safe when consumed in moderation. Other foods not mentioned are considered unrestricted. CONCLUSION: The concerns about perpetuating an overly restrictive MAOI diet include the avoidance by prescribers of a potentially useful treatment option, excessive limitations on lifestyle for patients, and increased risk to patients secondary to noncompliance with the diet. We propose an MAOI diet that has a solid scientific and clinical basis and that is, above all, practical. PMID- 8617706 TI - Absence of a sertraline-mediated effect on digoxin pharmacokinetics and electrocardiographic findings. AB - The effects of oral administration of sertraline on the plasma concentration profile and renal clearance of digoxin were assessed in 20 healthy male subjects in a double-blind, randomized study. METHOD: All subjects first received digoxin 0.5 mg twice daily on Day 1, 0.25 mg twice daily on Day 2, and 0.25 mg daily thereafter. On Day 11, 10 subjects began concomitant sertraline administration with an initial dose of 50 mg/day that was titrated upward over 7 days to 200 mg/day, which was given over the remainder of the study period. The other 10 subjects received concomitant digoxin and placebo for 17 days beginning on Day 11. Trough plasma concentrations of digoxin were monitored daily beginning on Day 7. Blood samples and 24-hour urine collections were used to determine steady state digoxin concentration and renal clearance before, during, and after sertraline coadministration. RESULTS: Sertraline had no effect on digoxin pharmacokinetics, except for a decrease in the time to reach the maximum plasma digoxin concentration (Tmax) compared with placebo (p = .0046), a finding thought to be of limited clinical significance. Side effects of mild-to-moderate severity were reported by 5 of 10 sertraline-treated subjects and by 6 of 10 placebo treated subjects. CONCLUSION: The results of this study suggest that dosing adjustments of digoxin may not be necessary in patients receiving concomitant sertraline administration. PMID- 8617705 TI - Sertraline does not alter the beta-adrenergic blocking activity of atenolol in healthy male volunteers. AB - A double-blind, placebo-controlled, randomized, crossover study was conducted to determine the effect of sertraline on the beta-adrenergic blocking activity of atenolol in 10 healthy male volunteers. METHOD: To assess the existence of any possible pharmacodynamic interaction between sertraline and atenolol, the effect of sertraline and placebo on the dose of intravenous isoproterenol required to increase heart rate by 25 beats per minute (bpm; chronotropic dose25 [CD25]) and the change in heart rate during exercise in atenolol-treated subjects were determined. RESULTS: The mean CD25 of isoproterenol was 2.00 micrograms after administration of placebo plus atenolol 50 mg and 2.03 micrograms after administration of sertralnie 100 micrograms plus atenolol 50 mg. The mean heart rate during exercise testing decreased by 29 bpm after sertraline plus atenolol administration and by 31 bpm after placebo plus atenolol administration. Analysis of variance indicated no statistically significant treatment or sequence effects. Only 1 subject experienced an adverse event--a mild headache after administration of sertraline plus atenolol. No clinically significant electrocardiograph changes were observed after sertraline or placebo administration. CONCLUSION: The results of this study demonstrate that sertraline does not alter the beta-blocking activity of atenolol. PMID- 8617707 TI - Absence of a sertraline-mediated effect on the pharmacokinetics and pharmacodynamics of carbamazepine. AB - A double-blind, randomized, placebo-controlled study was conducted in 14 healthy male volunteers to assess the effects of sertraline on the pharmacokinetics and pharmacodynamics of carbamazepine. METHOD: Subjects received carbamazepine 200 mg once daily for 2 days and every 12 hours thereafter. On Days 16 to 32, subjects also received either sertraline or placebo daily. The dose of sertraline was increased from 50 to 200 mg daily over 7 days; the 200-mg dose was given for 10 days. Samples for pharmacokinetic analyses were obtained on Days 15 and 32; trough plasma concentrations of carbamazepine and its principal metabolite, carbamazepine-10, 11-epoxide (CBZ-E), were determined daily beginning on Day 13. Cognitive function testing was performed on Day 1 before carbamazepine dosing (baseline), Day 15 (carbamazepine alone), and Day 32 (carbamazepine plus sertraline or placebo). RESULTS: There were no significant differences between the sertraline and placebo groups in any of the pharmacokinetic parameters for carbamazepine or CBZ-E. Carbamazepine alone impaired cognitive function. The addition of sertraline did not potentiate these effects. Side effects were reported by 2 subjects in each group, but none were severe. CONCLUSION: These findings indicate that sertraline does not affect the pharmacokinetics of carbamazepine or its principal metabolite and does not potentiate the cognitive effects of carbamazepine. PMID- 8617708 TI - Absence of effect of sertraline on the pharmacokinetics and pharmacodynamics of phenytoin. AB - A double-blind, randomized, placebo-controlled study assessed the effects of sertraline on the pharmacokinetics and pharmacodynamics of phenytoin in 30 healthy male volunteers. METHOD: All subjects received phenytoin throughout the study. The dose of phenytoin was 100 mg three times daily; steady-state trough plasma phenytoin concentrations were determined on Day 6. Concurrent treatment with sertraline (16 subjects) or placebo (13 subjects) was initiated on Day 8 and continued throughout the study in those subjects whose trough plasma phenytoin concentrations were between 5 and 20 micrograms/mL. The dose of sertraline was increased from 50 to 200 mg/day over 7 days; the 200-mg dose was then administered for 10 days. The plasma phenytoin concentration-time profile was determined on Day 7 before the start of sertraline or placebo dosing and at the end of dosing on Day 24. Psychometric testing was done before and after dosing on Days 0, 7, and 24. RESULTS: There were no significant differences between the sertraline group and the placebo group in the pharmacokinetic parameters of phenytoin. In addition, there was no indication that administration of phenytoin alone or concomitant administration of phenytoin and sertraline impaired cognitive function. Treatment-related side effects, primarily headache and nausea, were reported in 8 of 16 sertraline subjects and in 5 of 13 placebo subjects. Two subjects in the sertraline group withdrew because of side effects (rash), and 3 subjects in the placebo group withdrew because of side effects (rash and headache). CONCLUSION: High dosages of setraline did not affect the pharmacokinetics or the pharmacodynamics of phenytoin in ths study performed in healthy volunteers. PMID- 8617709 TI - Reducing the risk of drug-drug interactions: a goal of rational drug development. PMID- 8617710 TI - Absence of effect of sertraline on time-based sensitization of cognitive impairment with haloperidol. AB - This double-blind, randomized, placebo-controlled study evaluated the effects of haloperidol alone and haloperidol plus sertraline on cognitive and psychomotor function in 24 healthy male subjects. METHOD: All subjects received placebo on Day 1 and haloperidol 2 mg on Days 2 and 25. From Days 9 to 25, subjects were randomly assigned to either sertraline (12 subjects) or placebo (12 subjects); the sertraline dose was titrated from 50 to 200 mg/day from Days 9 to 16, and remained at 200 mg/day for the final 10 days of the drug administration period. Cognitive function testing was performed before dosing and over a 24-hour period after dosing on Days 1, 2, and 25. RESULTS: Impairment of cognitive function was observed 6 to 8 hours after administration of haloperidol on Day 2 but was not evident 23 hours after dosing. When single-dose haloperidol was given again 25 days later, greater impairment with earlier onset was noted in several tests in both treatment groups, suggesting enhancement of this effect. There was no indication that sertraline exacerbated the impairment produced by haloperidol since an equivalent effect also occurred in the placebo group. Three subjects (2 on sertraline and 1 on placebo) withdrew from the study because of side effects. Ten subjects in each group reported side effects related to treatment. The side effect profiles of sertraline and of placebo were similar. CONCLUSION: Haloperidol produced a clear profile of cognitive impairment that was not worsened by concomitant sertraline administration. PMID- 8617711 TI - The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells. AB - The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20 carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST lHDAg was found to be an excellent substrate (apparent Km = 0.8 microM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection. PMID- 8617712 TI - Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. AB - Genetic analyses of Caenorhabditis elegans has identified three genes that function in the regulation of nematode cell death. Mammalian homologs of two of these genes, ced-9 and ced-3, have been identified and comprise proteins belonging to the Bcl-2 and ICE families, respectively. To date, it is unclear where the negative regulators, ced-9 and bcl-2, function relative to the death effectors, ced-3 and the mammalian ced-3 homologs, respectively. Here, the molecular order of the cell death pathway is defined. Our results establish that Bcl-2 and Bcl-xL function upstream of two members of the ICE/CED-3 family of cysteine proteases, Yama (CPP32/apopain) and ICE-LAP3 (Mch3). PMID- 8617713 TI - The mercurial insensitive water channel (AQP-4) forms orthogonal arrays in stably transfected Chinese hamster ovary cells. AB - The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P face of MIWC-expressing cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding linear grooves (spacing, approximately 8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays. PMID- 8617714 TI - A unique CD45 glycoform recognized by the serum mannan-binding protein in immature thymocytes. AB - The serum-mannan binding protein (S-MBP) is a calcium-dependent C-type lectin specific for mannose and N-acetylglucosamine. S-MBP is known as a host defense factor involved in innate immunity, where the target ligands for S-MBP should be on the surface of exogenous microorganisms. In this study, we tried to find endogenous ligands for this endogenous lectin. Among the cells tested, only the lymphocytes from thymus of BALB/c mice expressed ligands for S-MBP on their surface, those from bone marrow, spleen, mesenteric lymph nodes and peripheral blood all being negative. Interestingly, among the thymocytes, only the immature thymocytes with the CD4+CD8+CD3low phenotype expressed ligands for S-MBP, and ligands for S-MBP decreased on their maturation. A major cell surface glycoprotein bearing S-MBP ligands was isolated and identified as CD45RO, which is a transmembrane protein with tyrosine phosphatase activity. Deglycosylation experiments with N-glycanase and endoglycosidase H indicated that the S-MBP ligands on thymic CD45 are high mannose type or hybrid type N-linked oligosaccharides. This unique presentation of S-MBP ligands on this special CD45 isoform suggested the possibility that the oligosaccharide portion of CD45 on immature thymocytes is associated with the maturation, development or selection events of thymocytes. PMID- 8617715 TI - Activation of protein kinase A inhibits interferon induction of the Jak/Stat pathway in U266 cells. AB - Activation of early response genes by interferons (IFNs) requires tyrosine phosphorylation of the Stat transcription factors and is mediated by the Jak family of tyrosine kinases. Recent evidence suggests that ERK2 serine/threonine kinase modulates the IFN-stimulated Jak/Stat pathway. In this report we show that in the myeloma cell line U266 protein kinase A specifically interacts with the cytoplasmic domain of the IFNalpha/beta receptor. Treatment of cells with the adenylate cyclase activator forskolin inhibits IFNbeta-, IFNgamma-, and hydrogen peroxide/vanadate-induced formation of complexes that bind to enhancers known to stimulate the expression of IFN-regulated genes. Immunoprecipitations followed by anti-phosphotyrosine immunoblots indicate that tyrosine phosphorylation of the alpha chain of the IFNalpha/beta receptor, Jak1, Tyk2, as well as Stat1 and Stat2 is reduced as a consequence of incubation of cells with forskolin. In contrast, dideoxyforskolin, which fails to activate adenylate cyclase, has no effect on IFN induction of the Jak/Stat pathway. These results indicate a novel regulatory mechanism by which protein kinase A can modulate the Jak/Stat signaling cascade. PMID- 8617716 TI - Trans-repressor BEF-1 phosphorylation. A potential control mechanism for human ApoE gene regulation. AB - Human apolipoprotein E is a plasma lipoprotein that appears to play an important protective role in the development of atherosclerosis. While little is known about the regulation of apoE, recent studies have shown that cytokines repress apoE synthesis both in vivo and in vitro. Furthermore, we have recently shown that the endogenous apoE gene is negatively regulated by the nuclear trans repressor BEF-1 in the human HepG2 cell line. In this study we demonstrate that treatment of HepG2 cells with the cytokine interleukin-1 and interleukin-6 resulted in the induction of an isoform of BEF-1, designated B1. The induction of the B1 isoform could be blocked by the protein kinase inhibitor staurosporine, suggesting that B1 is a phosphorylated form of BEF-1. As further support, the B1 isoform could also be induced by phorbol ester, and subsequently inhibited by staurosporine, implicating a role for protein kinase C-mediated phosphorylation. Quantitation of the levels of the BEF-1 isoforms, and studies in the presence of cyclohexamide, provided evidence for the phosphorylation of an existing intracellular pool of BEF-1, with no change in the total intracellular level. Under conditions that generated increased levels of the B1 isoform, there was a concomitant and proportional decrease in the level of apoE mRNA. The effect did not appear to be the result of improved binding to the apoE regulatory region as the DNA binding affinity of B1 was identical to native BEF-1. Our data suggest that the regulation of apoE by BEF-1 is modulated by differential phosphorylation, possibly through the protein kinase C pathway. PMID- 8617717 TI - Metargidin, a membrane-anchored metalloprotease-disintegrin protein with an RGD integrin binding sequence. AB - Cellular disintegrins are a family of membrane-anchored proteins with structural homology to snake venom metalloproteases and disintegrins. We report here the cDNA cloning and initial biochemical characterization of the first cellular disintegrin protein with an RGD sequence in its disintegrin domain, which we propose to name metargidin (for metalloprotease-RGD-disintegrin protein). The domain organization of metargidin is identical with that of previously reported members of the cellular disintegrin family, comprising (i) a pro- and a metalloprotease domain including a zinc-binding consensus motif, (ii) a disintegrin domain containing the RGD motif, (iii) a cysteine-rich domain, (iv) an epidermal growth factor-like domain, (v) a hydrophobic transmembrane domain, and (vi) a cytoplasmic tail with proline-rich sequences that could act as potential SH3 ligands. Antibodies raised against the cytoplasmic tail of metargidin recognize a glycoprotein of 110 kDa in MDA-MB-468 mammary epithelial carcinoma cells, which can be cell surface-biotinylated, indicating its localization in the plasma membrane. A second protein of 56 kDa co immunoprecipitates with metargidin, suggesting that it is part of a protein complex. These features are consistent with a model in which metargidin is an integrin ligand which, as a transmembrane protein, might function in cell-cell adhesion and/or signaling. PMID- 8617718 TI - Regulation of gene expression by cGMP-dependent protein kinase. Transactivation of the c-fos promoter. AB - The cAMP/cAMP-dependent protein kinase (A-kinase) and Ca2+/calmodulin-dependent protein kinase (Cam-kinase) signal transduction pathways are well known to regulate gene transcription, but this has not been demonstrated directly for the cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway. Here we report that transfection of G-kinase into G-kinase-deficient cells causes activation of the human c-fos promoter in a strictly cGMP-dependent manner. The effect of G-kinase appeared to be mediated by several sequence elements, most notably the serum response element (SRE), the AP-1 binding site (FAP), and the cAMP response element (CRE). The magnitude of G-kinase transactivation of the fos promoter was similar to that of A-kinase, but there were significant differences between G-kinase and A-kinase activation of single enhancer elements and of a chimeric Gal4-CREB transcription factor. Our results indicate that G-kinase transduces signals to the nucleus independently of A-kinase or Ca2+, although it may target some of the same transcription factors as A-kinase and Cam-kinase. PMID- 8617719 TI - Hypotonically induced calcium release from intracellular calcium stores. AB - Osmotic cell swelling induced by hypotonic stress is associated with a rise in intracellular Ca2+ concentration, which is at least partly due to a release of Ca2+ from internal stores. Since osmotic influx of water dilutes the cytoplasmic milieu, we have investigated how nonmitochondrial Ca2+ stores in permeabilized A7r5 cells respond to a reduction in cytoplasmic tonicity. We now present experimental evidence for a direct Ca2+ release from the stores when exposed to a hypotonic medium. The release is graded, but does not occur through the inositol trisphosphate or the ryanodine receptor. Ca2+ seems to be released through the passive leak pathway, and this phenomenon can be partially inhibited by divalent cations in the following order of potency: Ni2+ = Co2+ > Mn2+ > Mg2+ > Ba2+. This release also occurs in intact A7r5 cells. This novel mechanism of hypotonically induced Ca2+ release is therefore an inherent property of the stores, which can occur in the absence of second messengers. Intracellular stores can therefore act as osmosensors. PMID- 8617720 TI - Interleukin-1 alpha activates an NF-kappaB-like factor in osteoclast-like cells. AB - We investigated the NF-kappaB transcription factor in osteoclast-like cells. Osteoclast-like cells were differentiated from mouse bone marrow cells in co culture with mouse calvaria-derived primary osteoblasts in the presence of 1alpha,25-dihydroxyvitamin D3 and prostaglandin E2 in collagen gel-coated dishes. We enriched osteoclast-like cells from the co-cultures by Pronase treatment. When the enriched osteoclast-like cells were treated with phorbol 12-myristate 13 acetate, interleukin-1 (IL-1), calcitonin, or macrophage colony-stimulating factor, only IL-1 activated an NF-kappaB-like factor, which specifically bound to a kappaB motif DNA sequence, as detected by an electrophoretic mobility shift assay. IL-1 also activated NF-kappaB induction in osteoblasts. However, the NF kappaB-like factor induced by IL-1-stimulated osteoclast-like cells is of smaller molecular size than the factor in osteoblasts, as shown by an electrophoretic mobility shift assay. The NF-kappaB activity of osteoclast-like cells was recognized completely by antibodies against the p50 subunit, and only partially by antibodies against the p65 subunit of NF-++kappaB. Antibodies against c-Rel, Rel B, and p52 did not recognize the NF-kappaB-like factor. These results suggest that IL-1 activates an NF-kappaB-like factor in osteoclast-like cells, which contains p50 and p65-related proteins. PMID- 8617721 TI - An unusual dehalogenating peroxidase from the marine terebellid polychaete Amphitrite ornata. AB - The terebellid polychaete Amphitrite ornata produces no detectable volatile halogenated secondary metabolites, but frequently inhabits coastal marine sediments heavily contaminated with anthropogenic or biogenic haloaromatic compounds. This animal contains high levels of two very unusual enzymes, dehalogenating peroxidases. We have purified and partially characterized one of these dehaloperoxidases, DHP I. DHP I is a heme enzyme (Mr = 30,790) composed of two identical subunits (Mr = 15,529) and is very rich in the amino acids aspartic acid (+ asparagine) and glutamic acid (+ glutamine). The enzyme converts trihalogenated phenols, such as 2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for this reaction is 5.0. DHP I is also active against di- and monohalogenated phenols and will oxidize bromo-, chloro-, and fluorophenols. We have identified similar dehaloperoxidase activities in other infaunal polychaetes, including halometabolite-producing species. PMID- 8617722 TI - Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase. AB - RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase. PMID- 8617723 TI - Engineering a novel iron-sulfur cluster into the catalytic subunit of Escherichia coli dimethyl-sulfoxide reductase. AB - Dimethyl-sulfoxide reductase (DmsABC) is a complex [Fe-S] molybdoenzyme that contains four [4Fe-4S] clusters visible by electron paramagnetic resonance (EPR) spectroscopy. The enzyme contains four ferredoxin-like Cys groups in the electron transfer subunit, DmsB, and an additional group of Cys residues in the catalytic subunit, DmsA. Mutagenesis of the second Cys, Cys-38, in the DmsA group to either Ser or Ala promotes assembly of a fifth [Fe-S] cluster into the mutant enzyme. The EPR spectra, the temperature dependences, and the microwave power dependences demonstrate that the new clusters are [3Fe-4S] clusters. The [3Fe-4S] clusters in both of the C38S and C38A mutant enzymes are relatively unstable in redox titrations and have midpoint potentials of approximately 178 and 140 mV. Mutagenesis of the DmsA Cys group to resemble a sequence capable of binding an [4Fe-4S] cluster did not change the cluster type but reduced the amount of the cluster present in this mutant enzyme. This report demonstrates that all four EPR detectable [Fe-S] clusters in the wild-type enzyme are ligated by DmsB. Wild-type DmsA does not ligate an [Fe-S] cluster that is visible by EPR spectroscopy. PMID- 8617724 TI - Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities. AB - Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2 dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively. PMID- 8617725 TI - The plastid-encoded ccsA gene is required for heme attachment to chloroplast c type cytochromes. AB - A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes. PMID- 8617726 TI - In vivo association of v-Abl with Shc mediated by a non-phosphotyrosine-dependent SH2 interaction. AB - A necessary downstream element of Abelson murine leukemia virus (Ab-MLV)-mediated transformation is Ras, which can be activated by the phosphotyrosine-dependent association of Shc with the Grb2-mSos complex. Here we show that Shc is tyrosine phosphorylated and associates with Grb2 in v-Abl-transformed cells, whereas Shc in NIH3T3 cells is phosphorylated solely on serine and is not Grb2-associated. In addition, Shc coprecipitates with P120 v-Abl and P70 v-Abl, which lacks the carboxyl terminus. Surprisingly, a kinase-defective mutant of P120 also binds Shc, demonstrating that Shc/v-Abl association is a phosphotyrosine-independent interaction. Glutathione S-transferase fusion proteins were used to map the interacting domains and showed that Shc from both NIH3T3 and v-Abl-transformed cells binds to the Abl SH2 domain and that P120 v-Abl binds to a region in the amino terminus of Shc. Consistent with these data, a v-Abl mutant encoding only the Gag and SH2 regions was able to bind Shc in vivo. The unique non phosphotyrosine-mediated binding of Shc may allow direct tyrosine phosphorylation of Shc by v-Abl and subsequent activation of the Ras pathway through assembly of a signaling complex with Grb2-mSos. PMID- 8617728 TI - The selenoenzyme phospholipid hydroperoxide glutathione peroxidase controls the activity of the 15-lipoxygenase with complex substrates and preserves the specificity of the oxygenation products. AB - Mammalian 15-lipoxygenases have been suggested to be involved in cell differentiation and atherogenesis because of their capability of oxygenating polyenoic fatty acids esterified to biomembranes and lipoproteins. We investigated the interaction of the lipid-peroxidizing 15-lipoxygenase and the hydroperoxy lipid-reducing phospholipid hydroperoxide glutathione peroxidase during their reaction with biomembranes and lipoproteins and obtained the following results. 1) Lipoxygenase treatment of submitochondrial membranes led to the formation of hydroperoxyphosphatidylethanolamine and hydroperoxyphosphatidylcholine as indicated by high performance liquid chromatography with chemiluminescence detection. In 15-lipoxygenase-treated low density lipoprotein cholesteryl hydroperoxylinoleate was the major oxygenation product. 2) Phospholipid hydroperoxide glutathione peroxidase was capable of reducing the hydroperoxy lipids formed by the 15-lipoxygenase to their corresponding alcohols. 3) Preincubation of low density lipoprotein and submitochondrial membranes with the phospholipid hydroperoxide glutathione peroxidase completely prevented the lipoxygenase reaction. However, addition of exogenous hydroperoxy lipids restored the oxygenase activity. 4) Short-term incubations of the complex substrates with the 15-lipoxygenase led to a specific pattern of oxidation products which was rendered more unspecific at long-term incubation or at high substrate concentrations. If the phosholipid hydroperoxide glutathione peroxidase was present during the reaction, the specific product pattern was preserved. These data indicate that the phospholipid hydroperoxide glutathione peroxidase is capable of reducing hydroperoxy ester lipids formed by a 15-lipoxygenase, and that it may down-regulate the 15-lipoxygenase pathways in mammalian cells. The specificity of 15-lipoxygenase-derived hydroperoxy lipids depends on their immediate reduction to the corresponding alcohols preventing postcatalytic isomerization. PMID- 8617727 TI - A novel site in the muscle creatine kinase enhancer is required for expression in skeletal but not cardiac muscle. AB - Expression of the muscle creatine kinase (MCK) gene in skeletal and heart muscle is controlled in part by a 5' tissue-specific enhancer. In order to identify new regulatory elements, we designed mutations in a previously untested conserved portion of this enhancer. Transfection analysis of these mutations delineated a new control element, named Trex (Transcriptional regulatory element x), which is required for full transcriptional activity of the MCK enhancer in skeletal but not cardiac muscle cells. Gel mobility shift assays demonstrate that myocyte, myoblast, and fibroblast nuclear extracts but not primary cardiomyocyte nuclear extracts contain a trans-acting factor that binds specifically to Trex. The Trex sequence is similar (7/8 bases) to the TEF-1 consensus DNA-binding site involved in regulating other muscle genes. To determine if TEF-1 interacts with Trex, selected TEF-1 binding sites such as GTIIc and M-CAT and two anti-TEF-1 antisera were used in gel shift assays. These experiments strongly suggest that a factor distinct from TEF-1 binds specifically to Trex. Thus it appears that MCK transcription is regulated in skeletal muscles through a Trex-dependent pathway while Trex is not required for MCK expression in heart. This distinction could account partially for the difference in levels of muscle creatine kinase in these tissues. PMID- 8617729 TI - Inhibition of ecto-5'-nucleotidase by nitric oxide donors. Implications in renal epithelial cells. AB - We evaluated, in renal epithelial cells with a proximal tubule phenotype, the effect of nitric oxide (NO) on ecto-5 -nucleotidase (5'-N U), the underlying mechanism and its functional consequence. Sodium nitroprusside (SNP, 1-1000 microM), a NO donor, inhibited 5'-NU activity in a time- and concentration dependent manner. Consequently, NO blunted the inhibition by extracellular cyclic AMP (cAMP, 10-1000 microM) of sodium-phosphate cotransport, a pathway which involves degradation of adenosine monophosphate (AMP) by 5'-NU. SNP-induced inhibition of 5'-NU was not mediated by cyclic GMP, since it was not mimicked by atrial natriuretic peptide, and was reproduced by isosorbide dinitrate and sodium nitrate, two NO donors. SNP and genuine NO decreased the activity of 5'-NU in renal homogenates, and the effect of SNP was potentiated by dithiothreitol and glutathione, but not by nicotinamide adenine dinucleotide. In vivo in rats, kidney ischemia/reperfusion, which activates inducible NO-synthase, inhibited renal 5'-NU. This inhibition was prevented by Nomega-nitro-L-arginine methyl ester, a NO-synthase inhibitor. These results indicate that: (i) NO-related activity inhibited the activity of an ecto-enzyme, 5'-NU, most likely through S nitrosylation of the enzyme; (ii) inhibition of 5'-NU activity by NOx, which can occur in vivo under pathophysiological conditions, affected the extent to which extracellular cAMP inhibited sodium-Pi cotransport. PMID- 8617730 TI - Binding of phosphate, aluminum fluoride, or beryllium fluoride to F-actin inhibits severing by gelsolin. AB - Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO4(2-) binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two Pi binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO4(2-) which is subsequently titrated to H2PO4-. We speculate that beryllium fluoride and aluminum fluoride bind to the HPO4(2-) binding site. The cellular consequences of this model of phosphate release are discussed. PMID- 8617732 TI - Mechanisms leading to and the consequences of altering the normal distribution of ATP(CTP):tRNA nucleotidyltransferase in yeast. AB - CCA1 codes for mitochondrial, cytosolic, and nuclear ATP(CTP):tRNA nucleotidyltransferase. Studies reported here examine the mechanisms leading to and the consequences of altering the distribution of this important tRNA processing enzyme. We show that the majority of Cca1p-I, translated from the first in-frame ATG, is in mitochondria but surprisingly, there is a small contribution to nuclear and cytosolic tRNA processing by this form as well. The majority of Cca1p-II and Cca1p-III, translated from ATG2 and ATG3, respectively, is in the cytosol but both are also located in the nucleus for processing precursors. Altering the cytosolic/nuclear distribution of Cca1p by fusing the SV40 nuclear localization signal to the 5' end of CCA1 causes a growth defect and results in the accumulation of end-shortened tRNAs in the cytosol. These results suggest an important role for Cca1p in the cytosol of eukaryotes, presumably in the repair of 3' CCA termini. These experiments also demonstrate that individual tRNAs are affected differently by reduced cytosolic nucleotidyltransferase and that cells resuming exponential growth are more severely affected than those continuing exponential growth. PMID- 8617733 TI - The role of non-catalytic binding subsites in the endonuclease activity of bovine pancreatic ribonuclease A. AB - Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA. There is much evidence that several subsites, in addition to the main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Pares, X., Nogues, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 abolishes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism. PMID- 8617731 TI - Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf. AB - B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post translational modifications in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B Raf. PMID- 8617734 TI - Mutations at two invariant nucleotides in the 3'-minor domain of Escherichia coli 16 S rRNA affecting translational initiation and initiation factor 3 function. AB - We have investigated the highly conserved GAUCA sequence of small subunit ribosomal RNA. Within this region, the invariant nucleotides G1530 and A1531 of Escherichia coli 16 S rRNA were mutagenized to A1530/G1531. These base changes caused a lethal phenotype when expressed from a high copy number plasmid. In low copy number plasmids, the mutant ribosomes had limited effects when expressed in vivo but caused significant deficiencies in translation in vitro, affecting enzymatic tRNA binding, non-enzymatic tRNA binding, subunit association, and initiation factor 3 (IF3) binding. Mutant 30 S ribosomal subunits showed a 10 fold decrease in affinity for IF3 as compared to wild-type subunits but showed an increased affinity for IF3 when in 70 S ribosomes. Additionally, IF3 did not promote dissociation of 70 S ribosomes, which had mutated subunits as monitored by light-scattering experiments. However, extension inhibition experiments (toeprinting) showed that IF3 retained its ability to discriminate between initiator and elongator tRNAs on mutated subunits. The results indicate that the two functions of IF3, tRNA discrimination and subunit dissociation, are separable and that the invariant nucleotides are important for correct subunit function during initiation. PMID- 8617735 TI - Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor. AB - The WSXWS motif in the extracellular domain defines members of the cytokine receptor family, yet its role in receptor structure and function remains unresolved. To address this question we have generated a panel of 100 mutants within the WSXWS motif of the erythropoietin receptor, which represents all single amino acid substitutions of these five amino acids. All mutants were synthesized at the same level; however, their passage from the endoplasmic reticulum to the Golgi apparatus differed. Because of this, expression of mutant receptors at the cell surface varied more than 300-fold. The tolerance of the tryptophan and serine residues to substitution was quite narrow; as a result, most of these mutants were retained in the endoplasmic reticulum and showed no cell surface expression or reduced cell surface expression. Although many mutants with substitutions at the middle residue of the motif reached the cell surface, it was notable that one mutant, A234E, was processed more efficiently than the wild type receptor and was expressed in elevated numbers at the cell surface. Despite this variation, all mutant receptors that reached the cell surface appeared able to bind erythropoietin and transduce a proliferative signal normally. These results are discussed in terms of a general model for WSXWS function in which the motif contributes to efficient receptor folding. PMID- 8617736 TI - Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites. AB - The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of cleavable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel beta sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites. PMID- 8617737 TI - Biosynthesis of topa quinone cofactor in bacterial amine oxidases. Solvent origin of C-2 oxygen determined by Raman spectroscopy. AB - Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character. PMID- 8617738 TI - Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator. AB - Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia. PMID- 8617740 TI - Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding. AB - Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8 anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate. PMID- 8617739 TI - Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. AB - The partially purified myosin-bound phosphatase had an associated protein kinase that phosphorylated the holoenzyme, primarily on the large (130-kDa) subunit. Phosphorylation of the 130-kDa subunit resulted in inhibition of phosphatase activity. The major site of phosphorylation was threonine 654 of the 130-kDa subunit or threonine 695 of the 133-kDa isoform. Phosphorylation of the large subunit did not dissociate the holoenzyme. Dephosphorylation of the large subunit was achieved by the holoenzyme, and addition of the catalytic subunit of the type 2A enzyme did not increase the rate of dephosphorylation. The associated kinase was inhibited by chelerythrine, with half-maximal inhibition at approximately 5 microM (in 150 microM ATP). The associated kinase phosphorylated two synthetic peptides, one corresponding to the sequence flanking the phosphorylated threonine, i.e. 648-661 of the 130-kDa subunit, and the other to a known protein kinase C substrate, i.e. a modified sequence from the autoinhibitory region of epsilon protein kinase C. The associated kinase was activated by arachidonic and oleic acid and to a lesser extent by myristic acid. The protein kinase that phosphorylated the 130-kDa subunit and resulted in inhibition of myosin phosphatase activity was not identified. PMID- 8617741 TI - 2'-Phospho-cyclic ADP-ribose, a calcium-mobilizing agent derived from NADP. AB - Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD, appears to modulate changes in intracellular free Ca2+ levels by activation of ryanodine sensitive Ca2+ channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP to 2'-phospho-cyclic adenosine diphosphoribose (2'-P-cADPR), cyclized at N-1 of the adenine moiety. An enzyme from canine spleen previously shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase activities also utilized NADP and 2'-P-cADPR as substrates. The apparent Km value for NADP was 1.6 microM compared with 9.9 microM for NAD, and the Vmax with NADP was twice that with NAD, indicating that 2'-P-cADPR is a likely metabolite in mammalian cells. 2'-P-cADPR was as active as cADPR in eliciting Ca2+ release from rat brain microsomes, but was unable to elicit Ca2+ release following conversion to 2'-P-ADPR by the action of canine spleen NAD glycohydrolase. 2'-P-cADPR and 1-D-myo-inositol 1,4,5-trisphosphate (IP3) appear to act by distinct mechanisms as microsomes desensitized to IP3 still released Ca2+ in response to 2'-P-cADPR and vice versa. Also, inhibition of IP3-induced Ca2+ release by heparin had no effect on release by 2'-P-cADPR. Both 2'-P-cADPR and cADPR appear to act by a similar mechanism based on similar kinetics of Ca2+ release, similar dose-response curves, cross-desensitization, and partial inhibition of release by procaine. The results of this study suggest that 2'-P-cADPR may function as a new component of Ca2+ signaling and a possible link between NADP metabolism and Ca2+ homeostasis. PMID- 8617742 TI - Syk, activated by cross-linking the B-cell antigen receptor, localizes to the cytosol where it interacts with and phosphorylates alpha-tubulin on tyrosine. AB - Syk (p72syk) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as alpha-tubulin. alpha-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. alpha-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including alpha tubulin. PMID- 8617743 TI - Regulation of tissue-specific expression of the skeletal muscle ryanodine receptor gene. AB - The ryanodine receptors (RYR) are a family of calcium release channels that are expressed in a variety of tissues. Three genes, i. e. ryr1, ryr2, and ryr3, have been identified coding for a skeletal muscle, cardiac muscle, and brain isoform, respectively. Although, the skeletal muscle isoform (RYR1) was shown to be expressed predominantly in skeletal muscle, expression was also detected in the esophagus and brain. To analyze the transcriptional regulation of the RYR1 gene, we have constructed chimeric genes composed of the upstream region of the RYR1 gene and the bacterial chloramphenicol acetyltransferase (CAT) gene and transiently transfected them into primary cultured porcine myoblasts, myotubes, and fibroblasts. A 443-base pair region upstream from the transcription start site was sufficient to direct CAT activity without tissue specificity. Deletion of a 61-base pair fragment from the 5'-end of the promoter resulted in a marked reduction of CAT activity in all three tissue types. A similar reduction of expression was observed when using a construct with the first intron in antisense orientation upstream from the promoter. In contrast, the first intron in sense orientation enhanced expression only in myotubes, while expression was repressed in fibroblasts and myoblasts. Gel retardation analyses showed DNA binding activity in nuclear extracts for two upstream DNA sequence elements. Our data suggest that (i) RYR1 gene expression is regulated by at least two novel transcription factors (designated RYREF-1 and RYREF-2), and (ii) tissue specificity results from a transcriptional repression in nonmuscle cells mediated by the first intron. PMID- 8617744 TI - Thermodynamic studies of SHC phosphotyrosine interaction domain recognition of the NPXpY motif. AB - The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no significant sequence similarity to the other Tyr(P)-recognizing module, the SH2 domain. We describe the thermodynamic parameters characterizing PI domain binding to various tyrosyl phosphopeptides, using isothermal titration calorimetry. The PI domain forms 1:1 complexes of similar affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of the epidermal growth factor receptor (EGFR) (KD = 28 nm) and an 18 mer (LQGHIIENPQpYFSDACVH) derived from Tyr-490 of Trk (KD = 42 nM). Binding of the EGFR-derived peptide was largely enthalpy-driven at 25 degrees C, while Trk490 peptide binding was entropy-driven. Based on the change in heat capacity upon binding, approximately 700 A2 of nonpolar surface was estimated to be buried upon interaction. Alteration of the Asn or Pro to Ala in the NPXpY motif of the EGFR Tyr-1148 peptide increased the KD of PI domain interactions to 238 and 370 nM, respectively. Alteration of a Leu at position -5 (with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the binding affinity (KD = 580 nM). It is proposed that the PI domain recognizes the beta1 turn that is found in NPXpY containing peptides and also interacts with a larger segment of the peptide than seen for SH2 domains. PMID- 8617745 TI - G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes. AB - Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O (thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function. PMID- 8617746 TI - cAMP-response element-binding protein induces directed DNA bending of the HTLV-I 21-base pair repeat. AB - Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-base pair (bp) repeats in addition to the transactivator protein Tax. Each of the 21-bp repeats contain nucleotide sequences which are homologous to a cAMP response element (CRE) which bind members of the ATF/CREB family of transcription factors. In this study, we investigated whether CREB alone or in the presence of Tax was able to induce DNA structural changes when bound to CRE sites in the HTLV I 21 bp, the cellular somatostatin promoter, or a hybrid CRE construct comprised of both the somatostatin and 21-bp repeat sequences. Circular permutation analysis indicated that CREB was able to induce DNA flexure upon binding to each of these elements. However, phasing analysis, which is a more sensitive method to determine the degree and orientation of directed DNA bending, demonstrated that CREB induced DNA bending of the HTLV-I 21-bp repeat and the hybrid CRE but not the somatostatin CRE. The addition of Tax did not change CREB-mediated bending of the 21-bp repeat or the hybrid CRE although it markedly increased the amount of CREB bound to each of these DNA elements. These results indicate that sequence motifs flanking the CRE in the 21-bp repeat are critical for inducing DNA structural changes and that these changes are likely important in mediating Tax activation of the HTLV-I LTR. PMID- 8617747 TI - Partial G protein activation by fluorescent guanine nucleotide analogs. Evidence for a triphosphate-bound but inactive state. AB - N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation where MANT-guanosine-5'-O-(3 thiotriphosphate) (mGTP gammaS) had the highest fluorescence followed by mGTP and mGDP, respectively (Remmers, A. E., Posner, R., and Neubig, R. R. (1994) J. Biol. Chem. 269, 13771-13778). To directly examine G protein conformations with nucleotide triphosphates bound, we synthesized several nonhydrolyzable MANT labeled guanine nucleotides. The relative maximal fluorescence levels observed upon binding to recombinant myristoylated Goalpha (myrGoalpha) and myrGialpha1 were: mGTPgammaS > MANT-5'-guanylyl-imidodiphosphate > MANT-guanylyl-(beta,gamma methylene)-diphosphonate > MANT-guanosine 5'-O-2-(thio)diphosphate. Using protection against tryptic digestion as a measure of the activated conformation, the ability of the MANT guanine nucleotides to maximally activate myrGo alpha correlated with maximal fluorescence. Biphasic dissociation kinetics were observed for all of the MANT guanine nucleotides. The data were consistent with the following model, [formula: see text] where G protein activation (G*-GXP) is determined by a conformational equilibrium between two triphosphate bound states as well as by the balance between binding and hydrolysis of the nucleotide triphosphate. Compared with myrGialpha1, maximal mGTP fluorescence was only 2 fold higher for the myrGialpha1 Q204L mutant, a mutant with greatly reduced GTPase activity, and only 24% that of mGTPgammaS, indicating that partial activation by mGTP was not just due to hydrolysis of mGTP. These results extend our previous conclusion that GTP analogs do not fully activate G protein. PMID- 8617748 TI - Cloning and expression of the chick anti-Mullerian hormone gene. AB - Mullerian duct regression in male embryos is due to early production by fetal Sertoli cells of anti-Mullerian hormone, a homodimeric protein of the transforming growth factor- beta superfamily. In mammals, both female Mullerian ducts develop into the uterus and Fallopian tubes, whereas in birds, the right oviduct does not develop. To gain insight into sex differentiation in birds, we have cloned the cDNA for chick anti-Mullerian hormone using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of cross-hybridization between mammalian and chick anti-Mullerian hormone DNA. The chick DNA and protein are significantly longer, due to insertions that abolish nucleotide homology, except in the cDNA coding for the C terminal, bioactive part of the protein. Nevertheless, the general structure of the gene, sequenced from the transcription initiation to the polyadenylation site, and the main features of the protein are conserved between the chick and mammals. The chick anti-Mullerian hormone gene is expressed at high levels in Sertoli cells of the embryonic testes and in lower amounts in both ovaries, higher levels being reached on the left side after 10 days of incubation. PMID- 8617749 TI - Activation of heat shock factor by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. AB - Transcriptional activation of heat shock protein genes is a common response to proteotoxic stress. Many drugs and chemicals that form reactive electrophiles modify protein structure by binding covalently to nucleophilic functional groups. Although many of these agents also activate transcription of the inducible member of the hsp70 gene family, it is not clear if covalent modification of cellular proteins per se is sufficient. Iodoacetamide (IDAM) is a prototypical alkylating toxicant that induces hsp70 transcription. However, IDAM-induced cell death is indirectly linked to protein alkylation through depletion of glutathione, induction of oxidative stress, and increased lipid peroxidation. Therefore, we determined if any of these secondary cytotoxic events might lead to activation of hsp70 transcription. IDAM treatment increased hsp70 transcription by activating heat shock transcription factor-1 (HSF1). The addition of antioxidants and iron or calcium chelators prevented cell death but did not prevent hsp70 transcription or HSF1 activation. However, the protein synthesis inhibitor cycloheximide blocked activation of hsp70 by low concentrations of IDAM. Furthermore, the addition of dithiothreitol (DTT) after IDAM removal blocked hsp70 transcription and HSF1 activation without altering IDAM binding. DTT had no effect on activation of HSF1 by hyperthermia. After IDAM treatment, cellular nonprotein and protein thiols had decreased to less than 20 and 70%, respectively, of the value in control cells. DTT treatment in situ prevented the loss of cellular protein thiols and blocked the formation of high molecular weight protein aggregates. Thus, alkylation of proteins is insufficient to activate hsp70 transcription and DNA binding of HSF1. However, cellular thiol-disulfide redox status and formation of disulfide linked aggregates of cellular proteins are linked to HSF1 activation and hsp70 transcriptional activation. PMID- 8617750 TI - HER4-mediated biological and biochemical properties in NIH 3T3 cells. Evidence for HER1-HER4 heterodimers. AB - The EGF receptor family of tyrosine kinase growth factor receptors is expressed in a variety of cell types and has been implicated in the progression of certain human adenocarcinomas. The most recent addition to this family of receptors, HER4, was expressed in NIH 3T3 cells to determine its biological and biochemical characteristics. Cells expressing HER4 were responsive to heregulin beta2 as demonstrated by an increase in HER4 tyrosine phosphorylation and ability to form foci on a cell monolayer. HER4 exhibited in vitro kinase activity and was able to phosphorylate the regulatory subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition studies identified tyrosine 1056 of HER4 as the phosphatidylinositol 3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells conferred responsiveness to EGF with respect to colony formation in soft agar. It was also found that in response to heregulin beta2, endogenous murine HER1 or transfected human HER1 became phosphorylated when HER4 was present. This demonstrates that HER1 and HER4 can exist in a heterodimer complex and likely activate each other by transphosphorylation. PMID- 8617751 TI - Critical amino acids in the transcriptional activation domain of the herpesvirus protein VP16 are solvent-exposed in highly mobile protein segments. An intrinsic fluorescence study. AB - Eukaryotic transcriptional regulatory proteins typically comprise both a DNA binding domain and a regulatory domain. Although the structures of many DNA binding domains have been elucidated, no detailed structures are yet available for transcriptional activation domains. The activation domain of the herpesvirus protein VP16 has been an important model in mutational and biochemical studies. Here, we characterize the VP16 activation domain using time-resolved and steady state fluorescence. Unique intrinsic fluorescent probes were obtained by replacing phenylalanine residues with tryptophan at position 442 or 473 of the activation domain of VP16 (residues 413-490, or subdomains thereof), linked to the DNA binding domain of the yeast protein GAL4. Emission spectra and quenching properties of Trp at either position were characteristic of fully exposed Trp. Time-resolved anisotropy decay measurements suggested that both Trp residues were associated with substantial segmental motion. The Trp residues at either position showed nearly identical fluorescence properties in either the full-length activation domain or relevant subdomains, suggesting that the two subdomains are similarly unstructured and have little effect on each other. As this domain may directly interact with several target proteins, it is likely that a significant structural transition accompanies these interactions. PMID- 8617752 TI - Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors. AB - The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins. PMID- 8617753 TI - Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis. AB - The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion. PMID- 8617754 TI - Activity of the mitochondrial multiple conductance channel is independent of the adenine nucleotide translocator. AB - The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT. PMID- 8617755 TI - Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans. AB - Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH. PMID- 8617756 TI - The maltose transport system of Escherichia coli displays positive cooperativity in ATP hydrolysis. AB - Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis. PMID- 8617757 TI - The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin like enzymes, the 3C-like serine proteases. AB - The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. PMID- 8617758 TI - Human immunodeficiency virus reverse transcriptase. Functional mutants obtained by random mutagenesis coupled with genetic selection in Escherichia coli. AB - We describe catalytically active mutants of HIV RT (human immunodeficiency virus reverse transcriptase) generated by random sequence mutagenesis and selected in Escherichia coli for ability to complement the temperature-sensitive phenotype of a DNA polymerase I (Pol Its) mutant. We targeted amino acids Asp-67 through Arg 78 in HIV RT, which form part of the beta3-beta4 flexible loop and harbor many of the currently known mutations that confer resistance to nucleoside analogs. DNA sequencing of 109 selected mutants that complement the Pol Its phenotype revealed substitutions at all 12 residues targeted, indicating that none of the wild-type amino acids is essential. However, single mutations were not observed at Trp-71, Arg-72, and Arg-78, consistent with evolutionary conservation of these residues among viral RTs and lack of variation at these positions among isolates from patients. The mutations we recovered included most of those associated with drug resistance as well as previously unidentified mutations. Purification and assay of 14 mutant proteins revealed correlation between their DNA-dependent DNA polymerize activity in vitro and ability to complement the Pol Its phenotype. Activity of several mutants was resistant to 3'-azidothymidine triphosphate. We conclude that random sequence mutagenesis coupled with positive genetic selection in E. coli yields large numbers of functional HIV RT mutants. Among these are less active variants which are unlikely to be isolated from HIV-infected individuals and which will be informative of the roles of individual amino acids in the catalytic functions of the enzyme. PMID- 8617759 TI - Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation. AB - The growth promoting activity of the subendothelial extracellular matrix (ECM) is attributed to sequestration of basic fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and its regulated release by heparin-like molecules and heparan sulfate (HS) degrading enzymes. HS is also involved in bFGF receptor binding and activation. The present study focuses on the growth promoting activity and bFGF binding capacity of sulfate-depleted ECM. Corneal endothelial cells (EC) maintained in the presence of chlorate, an inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM containing 10-15% of the sulfate normally present in ECM. Incorporation of sulfate into HS was reduced by more than 90%. Binding of 125I-bFGF to sulfate-depleted ECM was reduced by 50-60% and only about 10% of the ECM-bound bFGF was accessible to release by heparin. Incubation of 125I-bFGF on top of native ECM resulted in dimerization of the ECM bound bFGF, but there was a markedly reduced binding and dimerization of bFGF on sulfate-depleted ECM. ECM produced in the presence of chlorate contained a nearly 10-fold less endogenous bFGF as compared to native ECM and exerted little or no mitogenic activity toward vascular EC and 3T3 fibroblasts. In other studies, we investigated the interaction between chlorate-treated vascular EC and either native or sulfate-depleted ECM. Exogenous heparin stimulated the proliferation of chlorate-treated EC seeded on native ECM, suggesting its interaction with ECM bound bFGF and subsequent presentation to high affinity cell surface receptors. On the other hand, heparin had no effect on chlorate-treated cells seeded in contact with sulfate-depleted ECM or regular tissue culture plastic. Altogether, the present experiments indicate that heparan sulfate proteoglycans associated with the cell surface and ECM act in concert to regulate the bioavailability and growth promoting activity of bFGF. While HS in the subendothelial ECM functions primarily in sequestration of bFGF in the vicinity of responsive cells, HS on cell surfaces is playing a more active role in displacing the ECM-bound bFGF and its subsequent presentation to high affinity signal transducing receptors. PMID- 8617761 TI - The water effect on allosteric regulation of hemoglobin probed in water/glucose and water/glycine solutions. AB - We have previously proposed a role of hydration in the allosteric control of hemoglobin based on the effect of varying concentrations of polyols and polyethers on the human hemoglobin oxygen affinity and on the solution water activity (Colombo, M. F., Rau, D. C., and Parsegian, V. A. (1992) Science 256, 655-659). Here, the original analyses are extended to test the possibility of concomitant solute and water allosteric binding and by introducing the bulk dielectric constant as a variable in our experiments. We present data which indicate that glycine and glucose influence HbA oxygen affinity to the same extent, despite the fact that glycine increases and glucose decreases the bulk dielectric constant of the solution. Furthermore, we derive an equation linking changes in oxygen affinity to changes in differential solute and water binding to test critically the possibility of neutral solute heterotropic binding. Applied to the data, these analyses support our original interpretation that neutral solutes act indirectly on the regulation of allosteric behavior of hemoglobin by varying the chemical potential of water in solution. This leads to a displacement of the equilibrium between Hb conformational states in proportion to their differential hydration. PMID- 8617760 TI - Identification of functional domains in Atp11p. Protein required for assembly of the mitochondrial F1-ATPase in yeast. AB - The Atp11p protein of Saccharomyces cerevisiae is required for proper assembly of the F1 component of the mitochondrial ATP synthase. The mutant atp11 genes were cloned and sequenced from 12 yeast strains, which are respiratory-deficient due to a defect in Atp11p function. Four of the mutations mapped to the mitochondrial targeting domain (amino-terminal 39 amino acids) of Atp11p. All the genetic lesions found in the mature protein sequence were shown to be nonsense mutations. This result is consistent with the idea that Atp11p activity is provided, principally, by the overall structure of a functional domain, and not by specific amino acid residues in a localized active site. Amino-terminal (Edman) sequence analysis of fragments derived from limited proteolysis of purified Atp11p, and in vivo functional characterization of deletion mutants, were employed to locate the position of the active region in the protein. Three domains, separated by proline rich sequences, were identified in the mature protein. The active domain of Atp11p was mapped to the sequence between Phe-120 and Asn-174. The domains proximal (Glu-40 through Ser-109) and distal (Arg-183 through Asn-318) to the active region were found to be important for the protein stability inside mitochondria. PMID- 8617762 TI - Iron regulatory proteins 1 and 2 bind distinct sets of RNA target sequences. AB - Iron regulatory proteins (IRPs) 1 and 2 bind with equally high affinity to iron responsive element (IRE) RNA stem-loops located in mRNA untranslated regions and, thereby, post-transcriptionally regulate several genes of iron metabolism. In this study we define the RNA-binding specificities of mouse IRP-1 and IRP-2. By screening loop mutations of the ferritin H-chain IRE, we show that both IRPs bind well to a large number of IRE-like sequences. More significantly, each IRP was found to recognize a unique subset of IRE-like targets. These IRP-specific groups of IREs are distinct from one another and are characterized by changes in certain paired (IRP-1) or unpaired (IRP-2) loop nucleotides. We further demonstrate the application of such sequences as unique probes to detect and distinguish IRP-1 from IRP-2 in human cells, and observe that the IRPs are regulated similarly by iron and reducing agents in human and rodent cells. Importantly, the ability of each IRP to recognize an exclusive subset of IREs was conserved between species. These findings suggest that IRP-1 and IRP-2 may each regulate unique mRNA targets in vivo, possibly extending their function beyond the regulation of intracellular iron homeostasis. PMID- 8617763 TI - Cloning, expression, and characterization of polyphosphate glucokinase from Mycobacterium tuberculosis. AB - Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using polyphosphate or ATP as the phosphoryl donor. The M. tuberculosis H37Rv gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli. The gene contains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was purified 189-fold to homogeneity and shown to contain dual enzymatic activities, similar to the native enzyme from H37Ra strain. The high G+C content in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycobacterial genes. Two phosphate binding domains conserved in the eukaryotic hexokinase family were identified in the polyphosphate glucokinase sequence, however, "adenosine" and "glucose" binding motifs were not apparent. In addition, a putative polyphosphate binding region is also proposed for the polyphosphate glucokinase enzyme. PMID- 8617764 TI - Cell-type specific recognition of RGD- and non-RGD-containing cell binding domains in fibrillin-1. AB - The fibrillins are large glycoprotein components of 10-nm microfibrils found in the extracellular matrix of most tissues. Microfibrils play a role in elastic fiber assembly and serve to link cells to elastic fibers in the extracellular matrix. To determine whether fibrillin-1 specifically interacts with receptors on cells from fibrillin-rich tissues, we evaluated whether two cell types that produce different types of fibrillin can adhere to purified fibrillin-1 in cell adhesion assays. Our results indicate that both cell types attach and spread on fibrillin-1 and that the RGD sequence in the fourth 8-cysteine motif mediates this interaction. Fibroblast attachment to fibrillin-1 was sensitive to inhibition by antibodies to the alphavbeta3 receptor and by peptides encoding the RGD sequence in fibrillin-1 and the second RGD sequence in fibrillin-2. In contrast, adhesion of auricular chondroblasts to fibrillin-1 was only partially inhibited by these reagents, suggesting that some cell types recognize a second, non-RGD binding site within the fibrillin molecule. These findings confirm and extend ultrastructural studies that suggest a direct interaction between microfibrils and the cell surface and provide a functional explanation for how this association occurs. PMID- 8617765 TI - Compartmental isolation of cholesterol participating in the cytoplasmic cholesteryl ester cycle in Chinese hamster ovary 25-RA cells. AB - Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cholesterol are selectively differentiated with respect to their cellular locations. These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over with a half-time of 7.5 h. When [3H]cholesterol was provided to the cells in serum to trace cellular cholesterol, the specific activities of cellular free and esterified cholesterol (6238 +/- 273 and 5128 +/- 277 cpm/ microg, respectively) failed to equilibrate, indicating that bulk cellular free cholesterol is isolated from that participating in the cholesteryl ester cycle. Using [3H]acetate to trace the fate of endogenously synthesized cholesterol, a failure of equilibration was also observed (specific activities of free and esterified cholesterol = 280 +/- 37 and 458 +/- 8 cpm/ microg, respectively). The lower specific activity of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified. When cells radiolabeled with [3H]acetate were post-incubated in the absence of radiolabel, the specific activity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esterified. PMID- 8617766 TI - Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes. AB - Recombinant c-Jun and c-Fos were ubiquitinylated by the ubiquitin carrier enzymes E214K, E220K, or E232K in the presence of the ubiquitin-activating enzyme, E1. Addition of ubiquitin protein ligase E3 substantially enhanced the E214K-mediated ubiquitinylation of c-Jun and c-Fos. Truncated c-Jun and c-Fos mutant proteins including wbJun and wbFos were also ubiquitinylated under the same conditions, suggesting the sites of ubiquitinylation are located within the dimerization and DNA binding domains of c-Jun and c-Fos. The E3-dependent ubiquitinylation of c Jun was inhibited upon the heterodimerization of c-Jun with c-Fos. Further addition of E220K significantly enhanced ubiquitinylation of c-Jun in the heterodimer suggesting a regulatory role of E220K. Polyubiquitinylated c-Jun, wbFos, and wbJun, but not E220K-ubiquitinylated c-Jun, were readily degraded by the ATP-dependent 26 S multicatalytic proteases. These results suggest that the temporal control of c-Jun and c-Fos may be regulated through the ubiquitinylation pathways, and the ubiquitinylation of c-Jun and c-Fos may in turn be regulated in response to the heterodimerization between them and the cooperation between E220K and E3 mediated polyubiquitinylation. PMID- 8617767 TI - Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants. AB - Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued. PMID- 8617768 TI - The ultrastructure of fibrinogen Caracas II molecules, fibers, and clots. AB - Fibrinogen Caracas II is an abnormal fibrinogen involving the mutation of A alpha serine 434 to N-glycosylated asparagine. Some effects of this mutation on the ultrastructure of fibrinogen Caracas II molecules, fibers, and clots were investigated by electron microscopy. Electron microscopy of rotary shadowed individual molecules indicated that most of the alphaC domains of fibrinogen Caracas II do not interact with each other or with the central domain, in contrast to control fibrinogen. Negatively contrasted Caracas II fibers were thinner and less ordered than control fibers, and many free fiber ends were observed. Scanning electron microscopy of whole clots revealed the presence of large pores bounded by local fiber networks made up of thin fibers. Permeation experiments also indicated that the average pore diameter was larger than that of control clots. The viscoelastic properties of the Caracas II clot, as measured by a torsion pendulum, were similar to those of control clots. Both the normal stiffness and increased permeability of the Caracas II clots are consistent with the observation that subjects with this dysfibrinogenemia are asymptomatic. PMID- 8617769 TI - Modification of an N-terminal regulatory domain of T antigen restores p53-T antigen complex formation in the absence of an essential metal ion cofactor. AB - We have discovered that the ability of the tumor suppressor protein p53 to bind to the viral large T antigen (TAg) oncogene product is regulated by divalent cations. Both proteins were purified from an insect cell line infected with the appropriate baculovirus expression vector. In a two-site capture enzyme-linked immunosorbent assay, complex formation between the purified proteins is strictly dependent on the addition of specific concentrations of divalent metal ions, notably zinc, copper, cadmium, cobalt, manganese, and nickel. In the presence of zinc the pattern of proteolytic fragments obtained when TAg was subjected to proteolysis by endoproteinase Glu-C (V8) was strikingly different, supporting the idea that a conformational change in TAg associated with ion binding is required for it to complex with p53. Monoclonal antibody analysis provides supporting evidence for a conformational change. When TAg was captured onto an enzyme-linked immunosorbent assay plate coated with PAb 419 as opposed to many other anti-TAg antibodies, complex formation was completely independent of the presence of additional divalent cations. Our results suggest that the ability of p53 and TAg to form a stable complex in vitro is dependent upon a regulatory domain residing in the N terminus of TAg, zinc ions or the binding of a specific monoclonal antibody (PAb 419) provoking a conformational change in TAg that facilitates and supports complex formation. PMID- 8617770 TI - FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. AB - CD95 (Fas/APO-1) and tumor necrosis factor receptor-1 (TNFR-1) are related molecules that signal apoptosis. Recently, a number of novel binding proteins have been proposed to mediate the signaling of these death receptors. Here we report that an N-terminal truncation of one of these candidate signal transducers, FADD/MORT1, abrogates CD95-induced apoptosis, ceramide generation, and activation of the cell death protease Yama/CPP32. In addition, this dominant negative derivative of FADD (FADD-DN) blocked TNF-induced apoptosis while not affecting NF- kappaB activation. FADD-DN bound both receptors, and in the case of CD95, it disrupted the assembly of a signaling complex. Taken together, our results functionally establish FADD as the apoptotic trigger of CD95 and TNFR-1. PMID- 8617771 TI - Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase. AB - To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response. PMID- 8617772 TI - p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2. AB - Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185erbB2, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185erbB2 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the approximately 100-kDa ansamycin-binding protein is GRP94. Membrane-associated p185erbB2 exists in a stable complex with GRP94. GA binding to GRP94 disrupts this complex, leading to degradation of pre existing p185erbB2 protein, and resulting in an altered subcellular distribution of newly synthesized p185erbB2. PMID- 8617773 TI - Active site amino acids that participate in the catalytic mechanism of nucleoside 2'-deoxyribosyltransferase. AB - The importance of eight nucleoside 2'-deoxyribosyltransferase residues for catalysis was investigated by site-directed mutagenesis. Each residue was selected because of its proximity to nucleophile Glu-98 or on its potential contribution to intrinsic protein fluorescence. Mutation of Asp-72, Asp-92, Tyr 7, Trp-12, and Met-125 resulted in over a 90% activity loss whereas mutation of Tyr-157, Trp-64, and Trp-127 produced less than a 80% activity loss. The magnitude of the perturbation on catalysis by mutation, however, was dependent on donor substrate. The kcat values for dIno hydrolysis by these mutants were greater than 25% of that for native enzyme. Although mutant and native enzymes bound substrate analogues with comparable affinities, Km values for dIno hydrolysis varied over a 1000-fold range. The pH dependence of Glu-98 esterification by dCyd suggested that amino acids with pK values of 4.2 and 7.5 were relevant for catalysis. The intrinsic protein fluorescence was attributed primarily to Trp-127 (approximately 80%). Pre-steady-state kinetic parameters for deoxyribosylation of mutant enzymes by dCyd, dThd, and dAdo were determined by monitoring changes in enzyme fluorescence. Collectively, results from mutagenesis suggest that, depending upon substrate, either Asp-92 or Asp-72 functions as the general acid catalyst, and that this enzyme undergoes a change in conformation upon Glu-98 deoxyribosylation. PMID- 8617774 TI - Cleavage of oligoribonucleotides by the 2',5'-oligoadenylate- dependent ribonuclease L. AB - RNase L, the 2',5' oligoadenylate-dependent ribonuclease, is one of the enzyme systems important in the cellular response to interferon. When activated in the presence of 2',5'-linked oligoadenylates, RNase L can catalyze the cleavage of synthetic oligoribonucleotides that contain dyad sequences of the forms UU, UA, AU, AA, and UG, but it cannot catalyze the cleavage of an oligoribonucleotide containing only cytosines. The primary site of the cleavage reaction with the substrate C11UUC7 has been defined to be 3' of the UU dyad by labeling either the 5' or the 3' end of the oligoribonucleotide and by examining the reaction products on polyacrylamide sequencing gels. Reaction time courses have been used to determine the kinetic parameters of the cleavage reactions. The effect of the overall length of the oligomeric substrate as well as the sequence of the bases around the position of the cleavage site on the kinetics of the cleavage reaction has been examined. The efficiency with which activated RNase L catalyzes the cleavage of the substrate C11UUC7 is 1.9 x 10(7) m-1 s-1. Because the cleavage of the synthetic oligoribonucleotide can be used to monitor the steady-state kinetics of catalysis by activated RNase L, this method offers an advantage over previous methods of assay for RNase L activity. PMID- 8617775 TI - Amplification of the transketolase gene in desensitization-resistant mutant Y1 mouse adrenocortical tumor cells. AB - As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyclase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity relative to parental Y1 cells and a 28-fold amplification of the transketolase gene as determined by Southern blot hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome. The relationship between overexpression of transketolase and desensitization resistance was evaluated in somatic cell hybrids formed between a desensitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression behaved dominantly, whereas desensitization resistance behaved recessively. These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulatory gene in conjunction with the amplification event. PMID- 8617776 TI - Imaging of intracellular calcium stores in individual permeabilized pancreatic acinar cells. Apparent homogeneous cellular distribution of inositol 1,4,5 trisphosphate-sensitive stores in permeabilized pancreatic acinar cells. AB - Several lines of evidence suggest that the existence of a heterogeneous population of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ stores underlies the polarized agonist-induced rise in cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells (Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677; Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668). To investigate whether the apical pole of acinar cells contains Ca2+ stores which are relatively more sensitive to Ins(1,4,5)P3 than those in basolateral areas, we studied Ca2+ handling by Ca2+ stores in individual streptolysin O (SLO) permeabilized cells using the low affinity Ca2+ indicator Magfura-2 and an in situ imaging technique. The uptake of Ca2+ by intracellular Ca2+ stores was ATP-dependent. A steady-state level was reached within 10 min, and the free Ca2+ concentration inside loaded Ca2+ stores was estimated to be 70 microM. Ins(1,4,5)P3 induced Ca2+ release in a dose-dependent, "quantal" fashion. The kinetics of this release were similar to those reported for suspensions of permeabilized pancreatic acinar cells. Interestingly, the permeabilized acinar cells showed no intercellular variation in Ins(1,4,5)P3 sensitivity. Although SLO treatment is known to result in a considerable loss of cytosolic factors, permeabilization did not result in a redistribution of zymogen granules, as judged by electron microscope analysis. These results suggest that Ins(1,4,5)P3-sensitive Ca2+ stores are unlikely to be redistributed as a result of SLO treatment. The effects of Ins(1,4,5)P3 were therefore subsequently studied at the subcellular level. Detailed analysis demonstrated that no regional differences in Ins(1,4,5)P3 sensitivity exist in this permeabilized cell system. Therefore, we propose that additional cytosolic factors and/or the involvement of ryanodine receptors underlie the polarized pattern of agonist-induced Ca2+ signaling in intact pancreatic acinar cells. PMID- 8617777 TI - Biosynthesis of the unsaturated 14-carbon fatty acids found on the N termini of photoreceptor-specific proteins. AB - In the vertebrate retina, a number of proteins involved in signal transduction are known to be N-terminal acylated with the unusual 14 carbon fatty acids 14:1n 9 and 14:2n-6. We have explored possible pathways for producing these fatty acids in the frog retina by incubation in vitro with candidate precursor fatty acids bearing radiolabels, including [3H]14:0, [3H]18:1n-9, [3H]18:2n-6, and [3H]18:3n 3. Rod outer segments were prepared from the radiolabeled retinas for analysis of protein-linked fatty acids, and total lipids were extracted from the remaining retinal pellet. Following saponification of extracted lipids, fatty acid phenacyl esters were prepared and analyzed by high pressure liquid chromatography (HPLC) with detection by continuous scintillation counting. Transducin, whose alpha subunit (Gt alpha) is known to bear N-terminal acyl chains, was extracted from the rod outer segments and subjected to SDS-polyacrylamide gel electrophoresis and fluorography to detect radiolabeled proteins. Gt alpha was also subjected to methanolysis, and the resulting fatty acyl methyl esters were analyzed by HPLC. The identities of HPLC peaks coinciding with unsaturated species of both phenacyl esters and methyl esters were confirmed by reanalyzing them after catalytic hydrogenation. The results showed that 14:1n-9 can be derived in the retina from 18:1n-9 and 14:2n-6 from 18:2n-6, most likely by two rounds of beta-oxidation, but that neither is produced in detectable amounts from 14:0. Retroconversion of unsaturated 18 carbon fatty acids to the corresponding 14 carbon species showed specificity, in that 18:3n-3 was not converted to 14 carbon fatty acids in detectable amounts. Myristic acid (14:0), 14:1n-9, and 14:2n-6 were all incorporated into Gt alpha. A much less efficient incorporation of 18:1n-9 into Gt alpha was also observed, but no radiolabeling of Gt alpha was observed in retinas incubated with 18:3n-3. Thus, retroconversion by limited beta-oxidation of longer chain unsaturated fatty acids appears to be the most likely metabolic source of the unusual fatty acids found on the N termini of signal transducing proteins in the retina. PMID- 8617778 TI - The three alpha 2-adrenergic receptor subtypes achieve basolateral localization in Madin-Darby canine kidney II cells via different targeting mechanisms. AB - The present studies examined the localization of the alpha2B- and alpha2C adrenergic receptor (AR) subtypes in polarized Madin-Darby canine kidney cells (MDCK II) and the mechanisms by which this is achieved. Previously we demonstrated that the alpha2AAR subtype is directly delivered to lateral subdomain of MDCK II cells. Surface biotinylation strategies demonstrated that the alpha2BAR, like the alpha2AAR, achieves 85-90% basolateral localization at steady-state. However, in contrast to the alpha2AAR, this polarization occurs after initial random insertion of the alpha2BAR into both apical and basolateral surfaces followed by selective retention on the lateral subdomain (t1/2 the apical surface is 15-30 min; t1/2 the basolateral surface is 8-10 h). The alpha2CAR also is enriched on the basolateral surface at steady-state and, like the alpha2AAR, is directly delivered there. Morphological evaluation of the epitope-tagged alpha2AAR, alpha2BAR, and alpha2CAR subtypes by laser confocal microscopy not only corroborated the biochemically-defined basolateral localization of all three alpha2AR subtypes but also revealed that the alpha2CAR uniquely exists in an intracellular compartment(s) as well. Immunofluorescence due to intracellular alpha2CAR partially overlaps that due to calnexin, a marker for endoplasmic reticulum, as well as that due to mannosidase II, a marker for the trans-Golgi network. Taken together, the present findings demonstrate that the alpha2AAR, alpha2BAR, and alpha2CAR subtypes, which possess highly homologous structures and ultimately achieve similar polarization to the lateral surface of MDCK II cells, nonetheless manifest distinct trafficking itineraries. PMID- 8617779 TI - Purification and characterization of a novel stress protein, the 150-kDa oxygen regulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain. AB - As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons. PMID- 8617781 TI - Identification and characterization of a thermostable MutS homolog from Thermus aquaticus. AB - Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family. Here, we describe the isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence homology with MutS homologs from both prokaryotes and eukaryotes. Expression of the T. aquaticus mutS gene in Escherichia coli results in a dominant mutator phenotype. Initial biochemical characterization of the thermostable MutS protein, which was purified to apparent homogeneity, reveals two thermostable activities, an ATP hydrolysis activity in which ATP is hydrolyzed to ADP and Pi and a specific DNA mismatch binding activity with affinities for heteroduplex DNAs containing either an insertion/deletion of one base or a GT mismatch. The ATPase activity exhibits a temperature optimum of approximately 80 degrees C. Heteroduplex DNA binding by the T. aquaticus MutS protein requires Mg2+ and occurs over a broad temperature range from 0 degrees C to at least 70 degrees C. The thermostable MutS protein may be useful for further biochemical and structural studies of mismatch binding and for applications involving mutation detection. PMID- 8617780 TI - Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways. AB - Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway. PMID- 8617782 TI - Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor. AB - The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated. PMID- 8617783 TI - Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins. AB - A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins. PMID- 8617784 TI - Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation. AB - A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235 Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit. PMID- 8617785 TI - Promoter and upstream regulatory activities of the mouse cellular retinoic acid binding protein-I gene. AB - The promoter and its upstream regulatory region of the mouse cellular retinoic acid-binding protein I (crabp-I) gene were examined in transgenic mouse embryos, a mouse embryonal carcinoma cell line P19, and a mouse embryonic fibroblast cell line 3T6. In transgenic mouse embryos, a beta-galactosidase reporter gene under the control of crabp-I promoter and its upstream regulatory region displayed a very specific pattern of expression characteristic of crabp-I gene expression during developmental stages. In tissue culture systems, the minimal promoter of this gene was identified, and regions containing positive and negative regulatory activities were dissected from the upstream 3-kilobase sequence using assays for transient reporter activity. It is concluded that the minimal promoter of the mouse crabp-I gene is located between 120 and 150 base pairs upstream from the transcription initiation site. Several cell type-specific positive and negative regulatory regions for this promoter have been identified. A region encoding a common negative regulatory activity in both P19 and 3T6 cells is also inhibitory to two heterologous promoters, and specific protein-DNA interactions between this DNA fragment and nuclear extracts of P19 and 3T6 are demonstrated by gel retardation experiments. PMID- 8617786 TI - Properties of Ryr3 ryanodine receptor isoform in mammalian brain. AB - Although the RNA for the third isoform (Ryr3) of ryanodine receptor (RyR), a Ca2+ release channel, is detected in specific regions of mammalian brain, little is known about the protein. We investigated Ryr3 in rabbit brain, using an antibody raised against the synthetic peptide corresponding to amino acid sequence 4375 4387 of rabbit Ryr3, the homologue of bullfrog beta-RyR. The antibody which reacted with bullfrog beta-RyR, but not with the other isoforms, Ryr1 or Ryr2, specifically precipitated a single polypeptide from rabbit brain microsomes having a size similar to beta-RyR. Sucrose gradient ultracentrifugation revealed that Ryr3 forms a homotetramer, as true of the other isoforms. Being consistent with the distribution of its RNA, Ryr3 was abundantly expressed in hippocampus, corpus striatum, and diencephalon. Ryr3 demonstrated Ca2+-dependent [3H]ryanodine binding, and caffeine increased its Ca2+ sensitivity. The Ca2+ sensitivity of Ryr3 was also enhanced in a medium containing 1 m NaCl, as observed with beta RyR. [3H]Ryanodine binding gave an estimate of Ryr3 which would be only 2% or less of total RyR in rabbit brain. These results confirm the expression of functional Ryr3 in mammalian brain which is similar to nonmammalian beta-RyR. PMID- 8617787 TI - Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II. AB - Two subunits in RNA polymerase II (e.g. RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g. AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases. We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11. The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1. Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro. Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit. Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II. PMID- 8617788 TI - Cloning and expression of the unique Ca2+-ATPase from Flavobacterium odoratum. AB - The 60-kDa Ca2+-ATPase from Flavobacterium odoratum is kinetically and mechanistically similar to other P-type ATPases, suggesting its use as a model system for structure-function studies of ion transport. A portion of the gene was amplified by polymerase chain reaction of genomic DNA with degenerate oligonucleotide primers, one based on the N-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of P-type ATPases. This gene fragment was used to screen a lambda library of F. odoratum 29979 DNA. Clone "C" is 3.3 kilobases in length and contains one complete and part of a second open reading frame, the first of which encodes a 58-kDa protein containing the exact N-terminal amino acid sequence of the purified protein. We have named this gene cda, for calcium-dependent ATPase. Escherichia coli, transformed with clone C, demonstrates high levels of calcium dependent and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa phosphointermediate, and cross-reacts with antibodies to the purified Ca2+ ATPase. The gene has almost no sequence homology to even the highly conserved regions characteristic of P-type ATPases but does possess significant homology to a protein with alkaline phosphatase activity (PhoD) from Zymomonas mobilis. The putative phosphorylation site is a Walker A (P-loop) ATP binding sequence and is modified relative to P-type ATPases, suggesting that the F. odoratum Ca2+-ATPase may represent an ancestral link between the F- and the P-type ATPases or perhaps a new class of ATPases. PMID- 8617789 TI - Structural determinants in the platelet-derived growth factor alpha-receptor implicated in modulation of chemotaxis. AB - Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals. PMID- 8617790 TI - Suppression of interleukin-1 beta-converting enzyme-mediated cell death by insulin-like growth factor. AB - COS cells are resistant to cell death induced either by interleukin-1beta converting enzyme (*ICE) and ICE homolog (ICH-1L) overexpression or by serum deprivation. COS cells deprived of serum undergo apoptosis after transfection with an ICE expression construct, but not an ICH-1L construct. ICE-mediated apoptosis of COS cells in serum-free medium is suppressed by insulin-like growth factor (IGF)-1 and insulin. Viability of Rat-1 cell line (Rat-1/ICE) expressing low levels of ICE-LacZ fusion protein is lower than those of cell lines expressing either both Bcl-2 and ICE or mutant ICEGly-->Ser during serum deprivation. Enzymatic activation and processing of ICE are observed in cells induced to die by serum deprivation, which are suppressed by IGF-1. IGF-1 or insulin suppresses ICE-mediated cell death without affecting the expression levels of Bcl-2, Bcl-x, or Bax. Taken together, these results indicate that ICE is activated by growth factor deprivation, and IGF-1 is able to suppress ICE mediated cell death through a mechanism independent of the expression of Bcl-2, Bcl-x, or Bax. PMID- 8617791 TI - Roles of active site residues and the NH2-terminal domain in the catalysis and substrate binding of human Cdc25. AB - Human Cdc25 proteins are dual specific protein phosphatases that play important roles in cell cycle regulation. In this study, the catalytic mechanism and substrate binding specificity of human Cdc25A and -B proteins were investigated by site-directed and deletion mutagenesis methods. Mutations of the cysteine or the arginine residues in the active site motif abolished the Cdc25 phosphatase activity. However, the cysteine mutation in both Cdc25A and -B created enzymes that still retain the ability to bind their substrates. This allowed us to test the ability of Cdc25A and -B to bind various cyclin-Cdk complexes in vitro. While Cdc25A Cys --> Ser could interact with cyclin A-Cdk2, cyclin B-Cdc2, and cyclin E Cdk2 strongly, Cdc25B mutant was only found to bind to cyclin A-Cdk2 at significant levels. We also identified Arg452 and Ser449 as two crucial residues that could be directly involved in the molecular interactions between Cdc25 and cyclin-Cdk proteins. Deletion mutagenesis data also indicate that the phosphatase catalytic domains of Cdc25A and -B proteins are located within their carboxyl terminus. PMID- 8617792 TI - In vivo and in vitro iron-replaced zinc finger generates free radicals and causes DNA damage. AB - The estrogen receptor (ER) is a ligand-activated transcription factor whose DNA binding domain (ERDBD) has eight cysteines, which coordinate two zinc atoms, forming two zinc finger-like structures. We demonstrate the capability of iron to replace zinc in zinc finger (hereby referred to as iron finger) both in vivo (using Escherichia coli BL21 (DE3)) and in vitro. Iron has the ability to substitute for zinc in the ERDBD as demonstrated by mobility shift and methylation interference assays of iron finger, which show specific recognition of the estrogen response element. The DNA binding constants for both in vivo and in vitro iron-replaced zinc fingers were similar to that of the native finger. Atomic absorption analysis revealed a ratio of 2:1 iron atoms/mol of ERDBD protein, as found for zinc in the crystal structure of native ERDBD. More importantly, we demonstrate that iron finger in the presence of H2O2 and ascorbate generates highly reactive free radicals, causing a reproducible cleavage pattern to the proximate DNA, the estrogen response element. The deoxyribose method, used to detect free radical species generated, and the resultant cleaved DNA ends, caused by iron finger, suggest that the free radicals generated are hydroxyl radicals. Due to the close proximity of the zinc finger to DNA, we postulate that iron-substituted zinc finger may generate free radicals while bound to genetic regulatory response elements, leading to adverse consequences such as iron-induced toxicity and/or carcinogenesis. PMID- 8617793 TI - Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland specific function. AB - beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis. PMID- 8617794 TI - Examination of the potential functional role of conserved cysteine residues in the hormone binding domain of the human 1,25-dihydroxyvitamin D3 receptor. AB - The significance of conserved cysteines at positions 288, 337, and 369 in the hormone binding domain of the human vitamin D receptor was evaluated by individual site-directed mutagenesis to glycine. Neither nuclear localization nor heterodimerization with retinoid X receptors in binding to the vitamin D responsive element was appreciably affected by altering these cysteines, but vitamin D hormone (1,25-(OH)2D3) activated transcription was compromised significantly in the C288G and C337G mutants. Only the C288G mutant displayed depressed (3-fold) 1,25-(OH)2D3 ligand binding affinity at 4 degrees C, in vitro, although at elevated temperatures (23-37 degrees C), ligand binding was attenuated severely in C288G, moderately in C337G and very mildly in C369G. The degree of impairment of ligand binding at physiologic temperatures correlated with the requirement for increased concentrations of 1,25-(OH)2D3 ligand to maximally stimulate transcriptional activity in co-transfected COS-7 cells. Thus cysteine 288 and, to a lesser extent, cysteine 337 are important for high affinity hormone binding to the vitamin D receptor, which ultimately leads to ligand-dependent transcriptional activation. PMID- 8617795 TI - Constitutive expression of class 3 aldehyde dehydrogenase in cultured rat corneal epithelium. AB - Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5'3' RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner. PMID- 8617796 TI - Cooperativity and segregation of function within the Ig-alpha/beta heterodimer of the B cell antigen receptor complex. AB - The B cell antigen receptor complex contains heterodimers of Ig-alpha and Ig beta. The cytoplasmic tails of each of these chains contain two conserved tyrosines, phosphorylation of which initiates the signal transduction cascades activated by the receptor complex. Although the cytoplasmic domains of Ig-alpha and Ig-beta have been expressed individually and demonstrated to be competent signal transduction units, we postulated that within the context of a heterodimer, Ig-alpha and Ig-beta could have new, complementary or even synergistic functions. Therefore we developed a system to compare the signal transducing capacities of dimers of Ig-alpha/Ig-alpha, Ig-beta/Ig-beta, or Ig alpha/Ig-beta. This was done by fusing the extracellular and transmembrane domains of either human platelet-derived growth factor receptor (PDGFR) alpha or beta to the cytoplasmic tail of either Ig-alpha or Ig-beta. Three cell lines expressing PDGFRbeta/Ig-alpha, PDGFRbeta/Ig-beta, or PDGFRalpha/Ig-beta together with PDGFRbeta/Ig-alpha were established in the murine B cell line A20 IIA1.6. While aggregation of each dimer by itself could induce the tyrosine phosphorylation of cellular substrates, only aggregation of the heterodimer induced the phosphorylation of substrates similar in range and intensity to that induced by the endogenous B cell antigen receptor complex. Interestingly, Ig-beta remarkably enhanced the rapidity (Tmax decreased from 5 to 1 min) and intensity (greater than 10-fold enhancement) of Ig-alpha phosphorylation. Conversely, the phosphorylation of Ig-beta was reduced to undetectable levels when co-aggregated with Ig-alpha. The enhancement of Ig-alpha phosphorylation by Ig-beta correlated with a lowering of the stimulation threshold for tyrosine kinase activation. PMID- 8617797 TI - Identification of a novel protein phosphatase 2A regulatory subunit highly expressed in muscle. AB - Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). We have isolated and characterized three novel cDNAs related to the B' subunit of bovine cardiac PP2A. Two human (B'alpha1 and B'alpha2) and a mouse (B'alpha3) cDNA encode for alternatively spliced variants of the B subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B' subunit. Differences between the deduced sequences of the B alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B' subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B'alpha diverge at a common splice site near the 3'-end of the coding regions. Northern blot and reverse transcription polymerase chain reaction analyses revealed that the B'alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B'alpha proteins readily associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B'alpha was localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein was greatly enhanced by the addition of the C subunit. Expression of the B'alpha subunits in insect Sf9 cells resulted in formation of AC.B'alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B' subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B'alpha suggests that some variants of the B' subunit are involved in the nuclear functions of PP2A. PMID- 8617798 TI - Structure of aquaporin-2 vasopressin water channel. AB - Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct. AQP-2 is selectively permeable to water molecule and is translocated between the apical membrane and subapical endosomes in response to vasopressin. To investigate the localization and structure of the aqueous pathway of the AQP-2 water channel, a series of site-directed mutants was constructed and functionally analyzed. Insertion of N-glycosylation reporter sequence into each hydrophilic loop (HL) indicated that AQP-2 has a six-membrane spanning topology and that insertional mutations in HL-2 or HL-5 do not alter water channel function. Mercury-sensitive site of AQP-2 is located near the second asparagine proline-alanine (NPA) domain at cysteine 181, but not near the first NPA domain. Replacement of HL-3 or HL-4 with the corresponding part of Escherichia coli glycerol facilitator abolished water channel function without changing plasma membrane expression of the channel protein. Introduction of cysteine residues in His-122, Asn-123, Gly-154, Asp-155, or Asn-156 induced partial mercury sensitivity, and point mutations in asparagine 123 significantly altered water permeability. Our results implicate that the structure of AQP-2 is different from models previously proposed for AQP-1 and that HL-3 and HL-4 are closely located to the aqueous pathway. PMID- 8617799 TI - Promoter region of the rat m4 muscarinic acetylcholine receptor gene contains a cell type-specific silencer element. AB - We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5'-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5'-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment. PMID- 8617800 TI - A heterodimeric nuclear protein complex binds two palindromic sequences in the proximal enhancer of the human erbB-2 gene. AB - Increased expression of the protein-tyrosine kinase receptor ErbB-2 occurs frequently in human breast and ovarian cancer and causes transformation in experimental systems. Control of transcription of the erbB-2 gene is an important determinant of receptor expression. Within the human erbB-2 promoter, a 100-base pair (bp) region 5' to the TATA box enhances transcription 200-fold. Two palindromes present in this 100-bp region are important for both positive and negative transcriptional control. A nuclear palindrome binding protein (PBP) has been purified to near homogeneity using ion-exchange, DNA-affinity, and gel filtration chromatography. PBP is a heterodimer consisting of a 69-kDa alpha subunit that binds DNA and a 60-kDa beta subunit that appears to enhance subunit binding. DNase I footprinting and electrophoretic mobility shift assays indicate that PBP binds to the half-site of each palindrome with the core recognition sequence TGGGAG. By DNA binding specificity and lack of immunological cross reactivity, PBP is distinct from NF-kappaB and Ikaros, two proteins with related DNA binding specificities. PBP is proposed to be an important regulator of transcription of the erbB-2 gene. PMID- 8617801 TI - Specific roles of methylcobamide:coenzyme M methyltransferase isozymes in metabolism of methanol and methylamines in Methanosarcina barkeri. AB - An immunochemical approach was employed as a direct test for functional activities of isozymes of methylcobamide:coenzyme M methyltransferase (MT2-M and MT2-A) in the metabolic pathways of methane formation from: methanol, acetate, monomethylamine, dimethylamine, and trimethylamine. Specific removal of the MT2 isozymes from buffer soluble cell extracts of Methanosarcina barkeri was accomplished by use of immobilized, affinity-purified, ovine polyclonal antibodies. Extracts of methanol-grown cells depleted of MT2-M lost entirely the ability to carry out conversion of methanol to 2-(methylthio)ethanesulfonate (methyl-CoM). Methanol:CoM methyl transfer activity was completely restored by addition of purified MT2-M, but no activity was recovered by addition of MT2-A. In contrast, the activity of trimethylamine-grown cell extracts to convert monomethylamine and dimethylamine to methyl-CoM was lost almost entirely by immunosorptive removal of MT2-A. Addition of purified MT2-A, but not MT2-M, to the MT2-A-depleted extract fully reconstituted methyl-CoM formation from both mono- and dimethylamine. Interestingly, in extracts resolved of MT2-A, trimethylamine-dependent methylation of coenzyme M was observed at approximately 20% of the rate of controls not treated with antibody. Furthermore, both isozymes were effective in full restoration of trimethylamine conversion. Tests indicated that neither of the two MT2 isozymes are involved in methane formation from acetate. The results establish that MT2-A plays a specific role in metabolism of methylated amine substrates, whereas, MT2-M functions in methane formation from trimethylamine and methanol. PMID- 8617803 TI - Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism. AB - The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit. PMID- 8617802 TI - N-terminal sequences contained in the Src homology 2 and 3 domains of p120 GTPase activating protein are required for full catalytic activity toward Ras. AB - The p120 GTPase-activating protein (GAP) is a negative regulator of Ras, which has a central role in signal transduction pathways that control cell proliferation. p120 GAP accelerates the conversion of activated Ras-GTP to its inactive form, Ras-GDP, thereby inhibiting mitogenic signaling. To examine potential contributions of p120 N-terminal sequences to regulation of its C terminal catalytic domain, we constructed deletion mutants lacking defined regions, including the variable hydrophobic region as well as the Src homology 2 (SH2) and 3 (SH3) domains. These mutant proteins were expressed in infected Sf9 insect cells from recombinant baculoviruses and assayed in vitro for their ability to stimulate the intrinsic GTPase activity of purified Ras. While deletion of the variable hydrophobic region had no effect on p120 GAP activity, deletion of the entire SH2/SH3/SH2 region severely impaired catalytic activity toward Ras. Deletion of individual SH2 and SH3 domains within this region partially inhibited p120 GAP activity. Moreover, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of the neurofibromin GAP-related domain. These results demonstrate that sequences in the SH2/SH3/SH2 region of p120 GAP are required for full catalytic activity toward Ras. Together with earlier findings that the p120 GAP SH2 domains mediate interactions with several GAP-associated proteins, our results suggest multiple roles for the N-terminal sequences in regulating p120 GAP catalytic activity and mitogenic signaling pathways. In addition, our results raise the possibility that SH2 domain point mutations in p120 GAP detected in some basal cell carcinomas reduce catalytic activity toward Ras and thereby contribute to oncogenesis. PMID- 8617804 TI - The chicken beta-globin gene promoter forms a novel "cinched" tetrahelical structure. AB - We have previously shown that the G-rich sequence G16CG(GGT)2GG in the promoter region of the chicken beta-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029-27035). The K+ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K+-dependent intrastrand "cinched" tetraplex. This structure has several unique features including the incorporation of bases other than guanine into the stem of the tetraplex, interaction between loop bases and bases in the flanking region, and base pairing between bases 3 and 5 of the tetrahelix-forming region to form a molecular "cinch." This finding extends the range of sequences capable of tetraplex formation as well as our appreciation of the conformational complexity of the chicken beta-globin promoter. PMID- 8617806 TI - GLUT1 transmembrane glucose pathway. Affinity labeling with a transportable D glucose diazirine. AB - We synthesized a transportable diazirine derivative of D-glucose,3-deoxy-3,3-azi D-glucopyranose (3-DAG), and studied its interaction with purified human erythrocyte facilitative glucose transporter, GLUT1. 3-DAG was rapidly transported into human erythrocytes and their resealed ghosts in the dark via a mercuric chloride-inhibitable mechanism and with a speed comparable with that of 3-O-methyl-D-glucose (3-OMG). The rate of 3-DAG transport in resealed ghosts was a saturable function of 3-DAG concentration with an apparent Km of 3.2 mM and the Vmax of 3.2 micromol/s/ml. D-Glucose inhibited the 3-DAG flux competitively with an apparent KI of 11 mM. Cytochalasin B inhibited this 3-DAG flux in a dose dependent manner with an estimated KI of 2.4 x 10(-7) M. Cytochalasin E had no effect. These findings clearly establish that 3-DAG is a good substrate of GLUT1. UV irradiation of purified GLUT1 in liposomes in the presence of 3-DAG produced a significant covalent incorporation of 3-DAG into glut1, and 200 mM D-glucose abolished this 3-dag incorporation. Analyses of trypsin and endoproteinase Lys-C digestion of 3-DAG-photolabeled GLUT1 revealed that the cleavage products corresponding to the residues 115 183, 256 300, and 301 451 of the GLUT1 sequence were labeled by 3-DAG, demonstrating that not only the C-terminal half but also the N-terminal half of the transmembrane domain participate in the putative substrate channel formation. 3-DAG may be useful in further identification of the amino acid residues that form the substrate channel of this and other members of the facilitative glucose transporter family. PMID- 8617805 TI - Structural and enzymatic aspects of rhodopsin phosphorylation. AB - Photoactivated rhodopsin (Rho*) is phosphorylated near the C terminus at multiple sites, predominantly at Ser334, Ser338, and Ser343. We systematically examined the sites of phosphorylation upon flash activation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylation at Ser334, Ser338, and Ser343, without changing the ratio between phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (with H7), or reconstitution of protein kinase C with purified ROS membranes. The stoichiometry and the ratio between different phosphorylation sites in purified Rho were also reproduced using RK, purified to apparent homogeneity from ROS or from an insect cell expression system. Thus, we conclude that light-dependent phosphorylation of Rho is mediated primarily by RK. Depalmitoylation of Rho at Cys322 and Cys323 altered the conformation of the C terminus of Rho, as observed by phosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the presence of four conserved amino acids at the C terminus of Rho. The accumulation of phosphorylated Ser334 observed in vivo could result from slower dephosphorylation of this site as compared with dephosphorylation of Ser338 and Ser343. These data provide a molecular mechanism for the site-specific phosphorylation of Rho observed in vivo. PMID- 8617807 TI - Agrin binding to alpha-dystroglycan. Domains of agrin necessary to induce acetylcholine receptor clustering are overlapping but not identical to the alpha dystroglycan-binding region. AB - The synaptic basal membrane protein agrin initiates the aggregation of acetylcholine receptors at the postsynaptic membrane of the developing neuromuscular junction. Recently, alpha-dystroglycan was found to be a major agrin-binding protein on the muscle cell surface and was therefore considered a candidate agrin receptor. Employing different truncation fragments of agrin, we determined regions of the protein involved in binding to alpha-dystroglycan and to heparin, an inhibitor of alpha-dystroglycan binding. Deletion of a 15-kDa fragment from the C terminus of agrin had no effect on its binding to alpha dystroglycan from rabbit muscle membranes, even though this deletion completely abolishes its acetylcholine receptor aggregating activity. Conversely, deletion of a central region does not affect agrin's clustering activity, but reduced its affinity for alpha-dystroglycan. Combination of these two deletions resulted in a fragment of approximately 35 kDa that weakly bound to alpha-dystroglycan, but displayed no clustering activity. All of these fragments bound to heparin with high affinity. Thus, alpha-dystroglycan does not show the binding specificity expected for an agrin receptor. Our data suggest the existence of an additional component on the muscle cell surface that generates the observed ligand specificity. PMID- 8617808 TI - Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo. AB - Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identified regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for interaction with ADA3. Deletions of the region that was required for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex. PMID- 8617809 TI - The regulatory light chains of myosin modulate cross-bridge cycling in skeletal muscle. AB - We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling. PMID- 8617810 TI - All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired. AB - Four transmembrane tyrosine kinases constitute the ErbB receptor family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and ErbB-4. We have measured the endocytic capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. EGF responsive chimeric receptors containing the EGF receptor extracellular domain and different ErbB cytoplasmic domains (EGFR/ErbB) have been employed. The capacity of these growth factor-receptor complexes to mediate 125I-EGF internalization, receptor down-regulation, receptor degradation, and receptor co immunoprecipitation with AP-2 was assayed. In contrast to the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced rapid internalization, down regulation, degradation, and AP-2 association. Also, we have analyzed the heregulin-responsive wild-type ErbB-4 receptor, which does not mediate the rapid internalization of 125I-heregulin, demonstrates no heregulin-regulated receptor degradation, and fails to form association complexes with AP-2. Despite the substantial differences in ligand-induced receptor trafficking between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent capacities to stimulate DNA synthesis in quiescent cells. These results show that the ligand-dependent down-regulation mechanism of the EGF receptor, surprisingly, is not a property of any other known ErbB receptor family member. Since endocytosis is thought to be an attenuation mechanism for growth factor-receptor complexes, these data imply that substantial differences in attenuation mechanisms exist within one family of structurally related receptors. PMID- 8617811 TI - Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins. AB - Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation associated promoters, such as c-fos, which are active during cell division, are turned off. MyoD and myogenin, transcription factors from the basic-helix-loop helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos. On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens. Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes. Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors. We show that myogenin from differentiating myoblasts specifically binds to SRF. In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin. A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast. Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed. Specific binding to SRF was also seen with MyoD. In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested. These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex. PMID- 8617812 TI - Interaction of Shc with adaptor protein adaptins. AB - The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346 355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis. PMID- 8617813 TI - Occurrence of transient multimeric species during the refolding of a monomeric protein. AB - A set of protein fragments from yeast phosphoglycerate kinase were produced by chemical cleavage at a unique cysteinyl residue previously introduced by site directed mutagenesis. Cross-linking experiments showed that the fragments corresponding to incomplete N-terminal domain form stable oligomeric species. Transient oligomeric species were also observed by both cross-linking and light scattering experiments during the folding process of the whole protein. These transient oligomeric species are formed during the fast folding phase and dissociate during the slow folding phase to produce the monomeric active protein. The multimeric species are not required for the protein to fold correctly. Unexpectedly, the distribution of oligomeric species is not dependent on protein concentration during the folding process. A kinetic competition mechanism is proposed as a possible solution to this paradox. These results provide direct evidence that the polypeptide chain can explore nonnative interactions during the folding process. PMID- 8617815 TI - Replacement of the spring clip in the SPEED appliance. PMID- 8617814 TI - Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone. AB - Two beta-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positional identity. Both enzymes hydrolyze the (1,3)-beta-glucan, laminarin, but also hydrolyze (1,3;1,4)-beta-glucan and 4 nitrophenyl beta-D-glucoside. The complete sequence of 602 amino acid residues of the mature beta-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated betaI and betaII, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as beta-glucosidases and although they hydrolyze 4-nitrophenyl beta glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the (1,4)-beta-glucan glucohydrolase type. Both the beta-glucan exohydrolase isoenzyme ExoI and the beta-glucosidase isoenzyme betaII release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain. PMID- 8617816 TI - Pain-free debonding with occlusal rim wax. PMID- 8617817 TI - Managed care in Europe. PMID- 8617818 TI - Sequential slicing of lower deciduous teeth to resolve incisor crowding. PMID- 8617819 TI - Hydrophilic adhesive for bonding to impacted canines. PMID- 8617820 TI - Statically determinate transpalatal arches. PMID- 8617821 TI - Is it ever worthwhile to sue a patient to collect unpaid fees? PMID- 8617823 TI - An informed-consent template. PMID- 8617822 TI - Rigid implant anchorage to close a mandibular first molar extraction site. PMID- 8617824 TI - Selling your practice. PMID- 8617826 TI - Activating the closing-loop archwire. PMID- 8617825 TI - Use of spooled nickel titanium wires as initial archwires. PMID- 8617827 TI - Nonextraction treatment of a high-angle Class II case with a modified Herbst appliance. PMID- 8617828 TI - Attachment of elastomeric thread to a palatally impacted cuspid. PMID- 8617829 TI - Craig Andreiko, DDS, MS, on the Elan and Orthos systems. Interview by Dr. Larry W. White. PMID- 8617830 TI - The 026 practice. PMID- 8617832 TI - Essix technology for the fabrication of temporary anterior bridges. PMID- 8617831 TI - Maxillary second molar extraction treatment. PMID- 8617833 TI - Strategic planning: your map for your future. PMID- 8617834 TI - Indirect-bonded Nance appliance. PMID- 8617835 TI - Headgear and pain. PMID- 8617836 TI - Computer-assisted patient information system. PMID- 8617837 TI - Direct-bonded lingual retainer with a new twist. PMID- 8617838 TI - Force system analysis of V-bend sliding mechanics. PMID- 8617839 TI - Rear delivery system. PMID- 8617840 TI - Use of dividers. PMID- 8617841 TI - Essix thermosealed appliances: various orthodontic uses. PMID- 8617842 TI - A new telescopic maxillary expander. PMID- 8617843 TI - The pseudo-Class I: a newly defined type of malocclusion. PMID- 8617845 TI - A thermal-cured, fluoride-releasing indirect bonding system. PMID- 8617844 TI - Considerations in forming a professional coverage group. PMID- 8617846 TI - The role of the upper first molar in lower incisor crowding. PMID- 8617847 TI - Five steps to successful practice transition. PMID- 8617849 TI - Thermoformed Herbst appliance. PMID- 8617848 TI - Impression technique for patients with limited mandibular opening. PMID- 8617850 TI - Fixed piston appliance for rapid Class II correction. PMID- 8617851 TI - Bonding ceramic brackets with light-cured glass ionomer cements. PMID- 8617852 TI - The K-loop molar distalizing appliance. PMID- 8617853 TI - Bracket placement with the preadjusted appliance. PMID- 8617854 TI - Practice transition concepts. PMID- 8617855 TI - Uprighting fully impacted mandibular second molars. PMID- 8617856 TI - Mandibular protraction appliances for Class II treatment. PMID- 8617857 TI - Surgical-orthodontic treatment of a Class II, division 2 malocclusion. PMID- 8617858 TI - Modified bell-shape bonded lingual retainer. PMID- 8617859 TI - Historic restorations. PMID- 8617860 TI - Mechanical analysis of Class II elastics. PMID- 8617861 TI - Preventive eruption guidance in the 5-to-7-year-old. PMID- 8617862 TI - Removable molar distalization splint. PMID- 8617863 TI - New edgewise bracket with rounded slot and variable ligation. PMID- 8617864 TI - Tolerance to factor VIII inhibitors in hemophilia A patients: a French twist. PMID- 8617865 TI - Progress towards a unifying hypothesis for angiogenesis. PMID- 8617866 TI - Targeted mutagenesis: analysis of phenotype without germ line transmission. AB - The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient, rapid, and complete. Genetic chimeras and mosaics, in which mutant cells are mixed with wild type cells, can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice. PMID- 8617867 TI - Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections. AB - Lipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines. We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-). The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin 1 alpha (IL-1 alpha) plasma concentrations were decreased twofold. LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae. No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice. In contrast, the LPS stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls. This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice. In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection. At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity. PMID- 8617869 TI - Neutralizing antiidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A. AB - Hemophilia A patients producing antibodies towards FVIII are usually treated with infusions of high doses of FVIII in an attempt to "desensitize" them. To examine the mechanisms by which such desensitization operates, sequential plasma samples of two unrelated inhibitor patients were analyzed for anti-FVIII and antiidiotypic antibodies before and during infusions of high doses of FVIII. Anti FVIII antibodies were separated from antiidiotypic antibodies by immunoaffinity chromatography before analysis. We show in the present study that the concentration of anti-FVIII antibodies did not change during a successful desensitization and that antibodies maintained their capacity to inhibit the procoagulant function of FVIII, even though the number of Bethesda units in plasma was reduced to undetectable levels. Using a competition assay with mAbs, we further show that the specificity of human antibodies did not vary significantly during therapy. Finally, we show that the treatment elicited antiidiotypic antibodies, which neutralized the inhibitory capacity of anti-FVIII antibodies. Inhibitor antibodies can therefore not be accurately evaluated in plasma, as their function appears to be neutralized by antiidiotypic antibodies. These findings could have implications for the design of new therapies for hemophilia A patients with inhibitors. PMID- 8617868 TI - Human herpesvirus-6 enhances natural killer cell cytotoxicity via IL-15. AB - The marked tropism of human herpesvirus-6 (HHV-6) for natural killer (NK) cells and T lymphocytes has led us to investigate the effect of HHV-6 on cellular cytotoxicity. We describe here how HHV-6 infection of peripheral blood mononuclear cells (PBMC) leads to upregulation of their NK cell cytotoxicity. The induction of NK cell activity by HHV-6 was abrogated by monoclonal antibodies (mAbs) to IL-15 but not by mAbs to other cytokines (IFN-alpha, IFN-gamma, TNF alpha, TNF-beta, IL-2, IL-12) suggesting that IL-15 secreted in response to viral infection was responsible for the observed effect. Furthermore, NK activation by HHV-6 was blocked with mAb to CD122, as well as by human anti-HHV-6 neutralizing antibodies. Using RT-PCR, we were able to detect IL-15 mRNA upregulation in purified monocyte and NK cell preparations. IL-15 protein synthesis was increased in response to HHV-6. Finally, addition of IL-15 to PBMC cultures was found to severely curtail HHV-6 expression. Taken together, our data suggest that enhanced NK activity in response to viral infection represent a natural anti-viral defense mechanism aimed at rapidly eliminating virus-infected cells. PMID- 8617870 TI - Islet transplantation under the kidney capsule fully corrects the impaired skeletal muscle glucose transport system of streptozocin diabetic rats. AB - Chronic insulin therapy improves but does not restore impaired insulin-mediated muscle glucose uptake in human diabetes or muscle glucose uptake, transport, and transporter translocation in streptozocin diabetic rats. To determine whether this inability is due to inadequate insulin replacement, we studied fasted streptozocin-induced diabetic Lewis rats either untreated or after islet transplantation under the kidney capsule. Plasma glucose was increased in untreated diabetics and normalized by the islet transplantation (110 +/- 5, 452 +/- 9, and 102 +/- 3 mg/dl in controls, untreated diabetics, and transplanted diabetics, respectively). Plasma membrane and intracellular microsomal membrane vesicles were prepared from hindlimb skeletal muscle of basal and maximally insulin-stimulated rats. Islet transplantation normalized plasma membrane carrier mediated glucose transport Vmax, plasma membrane glucose transporter content, and insulin-induced transporter translocation. There were no differences in transporter intrinsic activity (Vmax/Ro) among the three groups. Microsomal membrane GLUT4 content was reduced by 30% in untreated diabetic rats and normal in transplanted diabetics, whereas the insulin-induced changes in microsomal membrane GLUT4 content were quantitatively similar in the three groups. There were no differences in plasma membrane GLUT1 among the groups and between basal and insulin stimulated states. Microsomal membrane GLUT1 content was increased 60% in untreated diabetics and normalized by the transplantation. In conclusion, an adequate insulin delivery in the peripheral circulation, obtained by islet transplantation, fully restores the muscle glucose transport system to normal in streptozocin diabetic rats. PMID- 8617871 TI - Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1 in mice. AB - In a proportion of atopic asthmatics, exposure to a relevant antigen is followed by chronic inflammation in the airways leading to altered airway responsiveness (AR). However, the mechanisms underlying the development of airway hyperresponsiveness still remain unclear. To elucidate the relationship between IgE-mediated reactions and airway hyperresponsiveness, a murine model of passive sensitization and airway challenge with ovalbumin (OVA) was developed using anti OVA IgE and IgG antibodies from murine B cell hybridomas. Passive sensitization by intravenous injection of anti-OVA IgE resulted in immediate cutaneous hypersensitivity and, after airway challenge with OVA on two consecutive days, increased AR in BALB/c and SJL mice. Increased numbers of eosinophils were observed in bronchoalveolar lavage fluid, in cells extracted from the lungs, and in the peribronchial areas of BALB/c mice passively sensitized with IgE and challenged through the airways compared with nonsensitized mice. Eosinophil peroxidase activity was also elevated in lung tissue from these mice. Passive sensitization with anti-OVA IgG1 but not IgG2a or IgG3 was similarly associated with development of skin test reactivity and increased AR after airway challenge, accompanied by an increase in eosinophils in bronchoalveolar lavage fluid. These data suggest that IgE/IgG1-mediated reactions together with local challenge with antigen can result in allergic inflammation resulting in altered airway function. PMID- 8617872 TI - Preferential oxidation of glycogen in isolated working rat heart. AB - We tested the hypothesis that glycogen is preferentially oxidized in isolated working rat heart. This was accomplished by measuring the proportion of glycolytic flux (oxidation plus lactate production) specifically from glycogen which is metabolized to lactate, and comparing it to the same proportion determined concurrently from exogenous glucose during stimulation with epinephrine. After prelabeling of glycogen with either 14C or 3H, a dual isotope technique was used to simultaneously trace the disposition of glycogen and exogenous glucose between oxidative and non-oxidative pathways. Immediately after the addition of epinephrine (1 microM), 40-50% of flux from glucose was directed towards lactate. Glycogen, however, did not contribute to lactate, being almost entirely oxidized. Further, glycogen utilization responded promptly to the abrupt increase in contractile performance with epinephrine, during the lag in stimulation of utilization of exogenous glucose, suggesting that glycogen serves as substrate reservoir to buffer rapid increases in demand. Preferential oxidation of glycogen may serve to ensure efficient generation of ATP from a limited supply of endogenous substrate, or as a mechanism to limit lactate accumulation during rapid glycogenolysis. PMID- 8617873 TI - Autoantibodies to DNA-dependent protein kinase. Probes for the catalytic subunit. AB - DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients. PMID- 8617874 TI - BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells. AB - Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl 2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation. PMID- 8617875 TI - Inhibition of TGF-alpha gene expression by vitamin A in airway epithelium. AB - The autocrine/paracrine growth mechanism has been implicated in the regulation of bronchial epithelial cell proliferation. By inhibiting the expression of the transforming growth factor-alpha (TGF-alpha) gene product, vitamin A is able to suppress the proliferation of tracheobronchial epithelial cells in culture. Similar repressions in TGF-alpha mRNA levels by retinol were observed in airway explant cultures and in a cell line immortalized from normal human bronchial epithelial cells. Both the nuclear run-on transcriptional assay and the transfection study with the chimeric construct of the TGF-alpha promoter and chloramphenicol acetyltransferase reporter gene partly suggest a transcriptional downregulation mechanism of TGF-alpha gene expression by the retinol treatment; however, this inhibition at the transcriptional level cannot account for the total inhibition at the mRNA level. These results suggest that a downregulation of the expression of the TGF-alpha gene at the transcriptional and post transcriptional levels by vitamin A may precede the essential event associated with the homeostasis of normal conducting airway epithelium. PMID- 8617876 TI - Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis. AB - Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1. PMID- 8617877 TI - The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway. AB - Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF. PMID- 8617878 TI - Endothelin(B) receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells. AB - To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET(A) and ET(B) receptor cDNA. In cells overexpressing ET(B), but not ET(A) receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET(B) receptor blocker but was not inhibited by an ET(A) selective receptor blocker. In ET(B)-overexpressing cells, 10(-8) M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in J(Na/H) were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ET(A)-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ET(B) receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Ca-calmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation. PMID- 8617880 TI - Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance. Evidence for different mechanisms and different effects on insulin signaling. AB - Inhibition of insulin receptor signaling by high glucose levels and by TNF-alpha was recently observed in different cell systems. The aim of the present study was to characterize the mechanism of TNF-alpha-induced insulin receptor inhibition and to compare the consequences of TNF-alpha- and hyperglycemia-induced insulin receptor inhibition for signal transduction downstream from the IR. TNF-alpha (0.5-10 nM) and high glucose (25 mM) showed similar rapid kinetics of inhibition (5-10 min, > 50%) of insulin receptor autophosphorylation in NIH3T3 cells overexpressing the human insulin receptor. TNF-alpha effects were completely prevented by the phosphotyrosine phosphatase (PTPase) inhibitors orthovanadate (40 microM) and phenylarsenoxide (35 microM), but they were unaffected by the protein kinase C (PKC) inhibitor H7 (0.1 mM), the phosphatidylinositol-3 kinase inhibitor wortmannin (5 microM), and the thiazolidindione troglitazone (CS045) (2 microgram/ml). In contrast, glucose effects were prevented by PKC inhibitors and CS045 but unaffected by PTPase inhibitors and wortmannin. To assess effects on downstream signaling, tyrosine phosphorylation of the following substrate proteins of the insulin receptor was determined: insulin receptor substrate-1, the coupling protein Shc, focal adhesion kinase (FAK125), and unidentified proteins of 130 kD, 60 kD. Hyperglycemia (25 mM glucose) and TNF-alpha showed analogous (> 50% inhibition) effects on tyrosine phosphorylation of insulin receptor substrate-1, Shc, p60, and p44, whereas opposite effects were observed for tyrosine phosphorylation of FAK125, which is dephosphorylated after insulin stimulation. Whereas TNF-alpha did not prevent insulin-induced dephosphorylation of FAK125, 25 mM glucose blocked this insulin effect completely. In summary, the data suggest that TNF-alpha and high glucose modulate insulin receptor-signaling through different mechanisms: (a) TNF-alpha modulates insulin receptor signals by PTPase activation, whereas glucose acts through activation of PKC. (b) Differences in modulation of the insulin receptor signaling cascade are found with TNF-alpha and high glucose: Hyperglycemia-induced insulin receptor inhibition blocks both insulin receptor-dependent tyrosine phosphorylation and dephosphorylation of insulin receptor substrate proteins. In contrast, TNF-alpha blocks only substrate phosphorylation, and it does not block insulin-induced substrate dephosphorylation. The different effects on FAK125 regulation allow the speculation that long-term cell effects related to FAK125 activity might develop in a different way in hyperglycemia- and TNF-alpha-dependent insulin resistance. PMID- 8617879 TI - Peptone stimulates CCK-releasing peptide secretion by activating intestinal submucosal cholinergic neurons. AB - In this study we tested the hypothesis that peptone in the intestine stimulates the secretion of the CCK-releasing peptide (CCK-RP) which mediates CCK secretion, and examined the enteric neural circuitry responsible for CCK-RP secretion. We used a "donor-recipient" rat intestinal perfusion model to quantify the CCK-RP secreted in response to nutrient stimulation. Infusion of concentrated intestinal perfusate collected from donor rat perfused with 5% peptone caused a 62 +/- 10% increase in protein secretion and an elevation of plasma CCK levels to 6.9 +/- 1.8 pM in the recipient rat. The stimulatory effect of the intestinal washings was abolished when the donor rats were pretreated with atropine or hexamethonium but not with guanethidine or vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride which ablates the myenteric neurons in the donor rats also abolished the stimulatory action of the intestinal washings. Furthermore, treatment of the donor rats with a 5HT3 antagonist and a substance P antagonist also prevented the secretion of CCK-RP. These observations suggest that peptone in the duodenum stimulates serotonin release which activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial CCK-RP containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of CCK-RP may in turn play an important role in the postprandial release of CCK. PMID- 8617881 TI - Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts. AB - Type I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins. Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities. First, they incorporate 3-10-fold less [3H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls. Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [3H] mannose incorporation and the size of LLO. These corrections are not permanent, and the defects immediately reappear when mannose is removed. To explore further the basis of mannose correction, we analyzed the amount of 3H-labeled LLO intermediates. Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10-fold decrease in CDGS cells. Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [3H]mannose appeared to be metabolized normally. Initial velocities of [3H]mannose uptake were two- to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [3H]mannose incorporation in CDGS cells. Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways. Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets. PMID- 8617882 TI - Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells. AB - Recently our laboratory has cloned both the rat canalicular and sinusoidal GSH transporters (RcGshT and RsGshT, respectively; Yi, J., S. Lu, J. Fernandez-Checa, and N. Kaplowitz. 1994. J. Clin. Invest. 93:1841-1845; and 1995. Proc. Natl. Acad. Sci. USA. 92:1495-1499). The current work characterized GSH transport and the expression of these two GSH transporters in various mammalian cell lines. The average cell GSH levels (nmol/10(6) cells) were 25, 22, 32, 13, and 13 in HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. GSH efflux was temperature dependent and averaged 0.018, 0.018, 0.012, 0.007, and 0.019 nmol/10(6) cells/min from HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. Dithiothreitol (DTT), which stimulates rat sinusoidal GSH efflux, stimulated GSH efflux only in HepG2 and HeLa cells which was partially reversed by subsequent cystine treatment. GSH uptake (1 mM plus 35S-GSH) was temperature dependent, linear up to 45 min, and Na+-independent with average rates of 1.12, 0.91, 0.45, and 0.45 nmol/10(6) cells/30 min for HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. BSP-GSH (2mM), which cis-inhibits sinusoidal GSH uptake in rat liver and HepG2 cells, inhibited GSH uptake only in HeLa cells. mRNA and polypeptide of RcGshT are expressed in all cells whereas those of RsGshT are expressed only in HepG2 and HeLa cells. In conclusion, bidirectional GSH transport, mediated by the "canalicular" GSH transporter, is ubiquitous in mammalian cells. Sinusoidal GSH transporter expression is more restricted, being present in HepG2 and HeLa cells. DTT and BSP-GSH affect GSH transport only in cells expressing the sinusoidal transporter confirming their selective action on this transporter. PMID- 8617883 TI - Transendothelial insulin transport is not saturable in vivo. No evidence for a receptor-mediated process. AB - In vitro, insulin transport across endothelial cells has been reported to be saturable, suggesting that the transport process is receptor mediated. In the present study, the transport of insulin across capillary endothelial cells was investigated in vivo. Euglycemic glucose clamps were performed in anesthetized dogs (n = 16) in which insulin was infused to achieve concentrations in the physiological range (1.0 mU/kg per min + 5 mU/kg priming bolus; n = 8) or pharmacologic range (18 mU/kg per min + 325 mU/kg priming bolus; n = 8). Insulin concentrations were measured in plasma and hindlimb lymph derived from interstitial fluid (ISF) surrounding muscle. Basal plasma insulin concentrations were twice the basal ISF insulin concentrations and were not different between the physiologic and pharmacologic infusion groups (plasma/ISF ratio 2.05 +/- 0.22 vs 2.05 +/- 0.23; p = 0.0003). The plasma/ISF gradient was, however, significantly reduced at steady-state pharmacologic insulin concentrations (1.37 +/- 0.25 vs 1.98 +/- 0.21; P = 0.0003). The reduced gradient is opposite to that expected if transendothelial insulin transport were saturable. Insulin transport into muscle ISF tended to increase with pharmacologic compared with physiologic changes in insulin concentration (41% increase; 1.37 +/- 0.18 10(-2) to 1.93 +/- 0.24 10(-2) min-1; P = 0.088), while at the same time insulin clearance out of the muscle ISF compartment was unaltered (2.53 +/- 0.26 10(-2) vs 2.34 +/- 0.28 10(-2) min-1; P = 0.62). Thus, the reduced plasma/ISF gradient at pharmacologic insulin was due to enhanced transendothelial insulin transport rather than changes in ISF insulin clearance. We conclude that insulin transport is not saturable in vivo and thus not receptor mediated. The increase in transport efficiency with saturating insulin is likely due to an increase in diffusionary capacity resulting from capillary dilation or recruitment. PMID- 8617884 TI - Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. AB - Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses. PMID- 8617885 TI - Macrophage/microglial-mediated primary demyelination and motor disease induced by the central nervous system production of interleukin-3 in transgenic mice. AB - Activated macrophage/microglia may mediate tissue injury in a variety of CNS disorders. To examine this, transgenic mice were developed in which the expression of a macrophage/microglia activation cytokine, interleukin-3 (IL-3), was targeted to astrocytes using a murine glial fibrillary acidic protein fusion gene. Transgenic mice with low levels of IL-3 expression developed from 5 mo of age, a progressive motor disorder characterized at onset by impaired rota-rod performance. In symptomatic transgenic mice, multi-focal, plaque-like white matter lesions were present in cerebellum and brain stem. Lesions showed extensive primary demyelination and remyelination in association with the accumulation of large numbers of proliferating and activated foamy macrophage/microglial cells. Many of these cells also contained intracisternal crystalline pole-like inclusions similar to those seen in human patients with multiple sclerosis. Mast cells were also identified while lymphocytes were rarely, if at all present. Thus, chronic CNS production of low levels of IL-3 promotes the recruitment, proliferation and activation of macrophage/microglial cells in white matter regions with consequent primary demyelination and motor disease. This transgenic model exhibits many of the features of human inflammatory demyelinating diseases including multiple sclerosis and HIV leukoencephalopathy. PMID- 8617887 TI - Presence of hypochlorite-modified proteins in human atherosclerotic lesions. AB - Oxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo. PMID- 8617886 TI - Activated platelets signal chemokine synthesis by human monocytes. AB - Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions. PMID- 8617888 TI - Potentiation of the immune response in HIV-1+ individuals. AB - T cells from HIV-1+ individuals have a defect in mounting an antigen specific response. HIV-1 Tat has been implicated as the causative agent of this immunosuppression. We have previously shown that HIV-1 Tat inhibits antigen specific proliferation of normal T cells in vitro by binding to the accessory molecule CD26, a dipeptidase expressed on the surface of activated T cells. We now demonstrate that the defective in vitro recall antigen response in HIV-1 infected individuals can be restored by the addition of soluble CD26, probably by serving as a decoy receptor for HIV-1 Tat. The restored response is comparable to that of an HIV-1- individual, suggesting that early in HIV infection there is a block in the memory cell response, rather than deletion of these cells. PMID- 8617889 TI - Modulation of T cell responses to recall antigens presented by Langerhans cells in HIV-discordant identical twins by anti-interleukin (IL)-10 antibodies and IL 12. AB - Decreased antigen (Ag)-specific T cell (TC) proliferation and IL-2 production are detected in all stages of HIV disease. To determine whether dendritic cell dysfunction and/or abnormal cytokine production contribute to HIV-induced immune dysregulation, we studies TC responses to recall Ags (influenza virus and tetanus toxoid) presented by Langerhans cells (LC) in six pairs of HIV-discordant identical twins, and the modulation of these responses by anti-IL-10 (alphaIL-10) mAbs and IL-12. LC from HIV+ twins induced IL-2 comparable to normal LC in cultures containing TC from uninfected twins. In contrast, IL-2 production was markedly decreased in cultures containing TC from HIV+ twins. IL-12 enhanced Ag specific IL-2 production by TC from two patients with CD4+ counts > 600. In contrast, alphaIL-10 mAbs enhanced IL-2 production in influenza virus-stimulated cultures containing TC from two patients with CD4+ counts < 20. Thus, these findings suggest that immunologic dysfunction of dendritic cells does not contribute to impaired secondary immune responses in HIV+ individuals. Although few patients were studied, partial immune reconstitution in vitro, as demonstrated here, may help to predict those individuals who might benefit from cytokines or antibodies against cytokines as immunotherapy for HIV disease. PMID- 8617890 TI - Blood vessel adaptation to gravity in a semi-arboreal snake. AB - The effects of vasoactive agonists on systemic blood vessels were examined with respect to anatomical location and gravity acclimation in the semi-arboreal snake, Elaphe Obsoleta. Major blood vessels were reactive to putative neurotransmitters, hormones or local factors in vessel specific patterns. Catecholamines, adenosine triphosphate, histamine and high potassium (80 mM) stimulated significantly greater tension per unit vessel mass in posterior than anterior arteries. Anterior vessels were significantly more sensitive to catecholamines than midbody and posterior vessels. Angiotensin II stimulated significantly greater tension in carotid artery than in midbody and posterior dorsal aorta. Arginine vasotocin strongly contracted the left and right aortic arches and anterior dorsal aorta. Veins were strongly contracted by catecholamines, high potassium and angiotensin II, but less so by adenosine triphosphate, arginine vasotocin and histamine. Precontracted vessel were relaxed by acetylcholine and sodium nitroprusside, but not by atrial natriuretic peptide or bradykinin. Chronic exposure of snakes to intermittent hypergravity stress ( + 1.5 Gz at tail) did not affect the majority of vessel responses. These data demonstrate that in vitro tension correlates with that catecholamines, as well as other agonists, are important in mediating vascular responses to gravitational stresses in snakes. PMID- 8617891 TI - Respiratory gas exchange, nitrogenous waste excretion, and fuel usage during starvation in juvenile rainbow trout, Oncorhynchus mykiss. AB - Oxygen consumption, CO2 excretion, and nitrogenous waste excretion (75% ammonia-N and 25% urea-N) were measured daily in 4-g rainbow trout over a 15-day starvation period. Oxygen consumption and CO2 excretion declined while N excretion increased transiently in the mid-part of the starvation period but was unchanged from control levels at the end. Component losses (as percentage of total fuel used) of protein, lipid, and carbohydrate were 66.5, 31.1, and 2.4% respectively, as measured from changes in body weight and body composition, the latter relative to a control group at day 0. Instantaneous fuel use, as calculated from the respiratory quotients and nitrogen quotients, indicated that relative protein use rose during starvation, but contributed at most 24% of the aerobic fuel (as carbon). Lipid metabolism fell from about 68 to 37%, and was largely replaced by carbohydrate metabolism which rose from 20 to 37%. We conclude that the two approaches measure different processes, and that the instantaneous method is preferred for physiological studies. The compositional method is influenced by greater error, and measures the fuels depleted, not necessarily burned, because of possible interconversion and excretion of fuels. PMID- 8617892 TI - Effect of T3 administration on electrophysiological properties of lizard ventricular muscle fibres. AB - We investigated the effects on the electrophysiological properties of ventricular muscle fibres from lizards kept at 20 degrees C of mild and severe hyperthyroidism. The hyperthyroidism was induced by a 4-day treatment with either 0.025 or 1.0 microgram triiodothyronine g-1 body weight, documented by increased serum levels of thyroid hormone. Triiodothyronine treatment did not modify the duration of the action potential recorded in vitro at 25 degrees C from ventricular muscles stimulated at 1 Hz. Recordings at higher temperatures were associated with a faster repolarization phase and a decrease of of action potential duration in both euthyroid and hyperthyroid animals. However, in lizards treated with 1.0 microgram triiodothyronine . g-1 body weight, the 90% repolarization recovery times at 30 and 35 degrees C (95.6 +/- 14.9 ms and 53.0 +/- 6.0 ms, respectively), were significantly shorter than normal (177.6 +/- 29.2 and 107.2 +/ 18.1 ms, respectively). Action potential duration was also dependent on stimulation frequency of the preparations. Increased frequency led to significant decrease of the duration of action potentials recorded at 25 degrees C. In euthyroid preparations the reductions in 90% repolarization recovery time, owing to increase in stimulation frequency to 2.5 and 5 Hz, were 19.3 +/- 1.7 and 35.6 +/- 2.0 ms, respectively. In hyperthyroid preparations, the reductions in the 90% recovery time due to stimulus frequency increases varied from 35.4 +/- 1.9 and 58.1 +/- 2.1 ms at low hormone doses to 38.9 +/- 2.0 and 58.2 +/- 2.1 ms at high hormone doses. As a result of these differences, the action potential durations recorded from the two hyperthyroid preparations at high stimulation rates were shorter than from euthyroid preparations. The results obtained suggest that lizard cardiac tissue is responsive to hormone action at low environmental temperature, but the effects of such action become evident when the temperature and heart rate increase. PMID- 8617893 TI - Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum). AB - The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by angiotensin II analogue Leu8 angiotensin II, with a pA2 value of 7.6. Similarly, the concentration response curve for (Ala-Pro Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA2 value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu8 angiotensin II (10(-8) mol.1(-1)) and DuP 753 (10(-6) mol.1(-1)) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn1-Val5] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu8 angiotensin II. The hydrosmotic response to [Asn1-Val5] angiotensin II(10(-7) mol.1(-1)) was significantly inhibited by DuP 753 (10(-6) and 5 x 10(-6) mol.1(-1)), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10(-6) mol.1(-1) also inhibited the hydrosmotic response to angiotensin III (10(-7) mol.1(-1)). These results suggest that receptors for angiotensin II present isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues. PMID- 8617894 TI - Evolution of digestion of carbohydrates in the separate parts of the digestive tract of the edible snail Helix lucorum (Gastropoda: Pulmonata: Stylommatophora) during a complete 24-hour cycle and the first days of starvation. AB - In the present study we examined carbohydrase activities during a complete 24-h cycle and during the first days of starvation in both adult and juvenile snails. The results indicated the predominant role of the digestive gland in the secretions of the enzymes responsible for degradation of most of the carbohydrates tested. Salivary glands secreted some digestive enzymes but in amounts lower than secreted by digestive gland. Enzymatic activities fluctuated during the first hours of digestion and also after the digestive tract was empty. The relatively high enzymatic activities recorded 24 h after the intake of food and during starvation could be due to the circadian rhythm of this species and/or to the participation of an existing microflora in the digestive tract of Helix lucorum. The double origin (exogenous and endogenous) of some digestive enzymes such as cellulases is discussed. PMID- 8617895 TI - Reducing frailty and falls in older persons: an investigation of Tai Chi and computerized balance training. Atlanta FICSIT Group. Frailty and Injuries: Cooperative Studies of Intervention Techniques. AB - OBJECTIVE: To evaluate the effects of two exercise approaches, Tai Chi (TC) and computerized balance training (BT), on specified primary outcomes (biomedical, functional, and psychosocial indicators of frailty) and secondary outcomes (occurrence of falls). DESIGN: The Atlanta FICSIT (Frailty and Injuries: Cooperative Studies of Intervention Techniques), a prospective, randomized, controlled clinical trial with three arms (TC, BT, and education [ED]. Intervention length was 15 weeks, with primary outcomes measured before and after intervention and at 4-month follow-up. Falls were monitored continuously throughout the study. SETTING: Persons aged 70 and older living in the community. PARTICIPANTS: A total of 200 participants, 162 women and 38 men; mean age was 76.2. MEASUREMENTS: Biomedical (strength, flexibility, cardiovascular endurance, body composition), functional (IADL), and psychosocial well-being (CES-D scale, fear of falling questionnaire, self-perception of present and future health, mastery index, perceived quality of sleep, and intrusiveness) variables. RESULTS: Grip strength declined in all groups, and lower extremity range of motion showed limited but statistically significant changes. Lowered blood pressure before and after a 12-minute walk was seen following TC participation. Fear of falling responses and intrusiveness responses were reduced after the TC intervention compared with the ED group (P = .046 and P = .058, respectively). After adjusting for fall risk factors, TC was found to reduce the risk of multiple falls by 47.5%. CONCLUSIONS: A moderate TC intervention can impact favorably on defined biomedical and psychosocial indices of frailty. This intervention can also have favorable effects upon the occurrence of falls. Tai Chi warrants further study as an exercise treatment to improve the health of older people. PMID- 8617896 TI - Balance and strength training in older adults: intervention gains and Tai Chi maintenance. AB - OBJECTIVE: To determine the effect on balance and strength of 3 months of intensive balance and/or weight training followed by 6 months of low intensity Tai Chi training for maintenance of gains. DESIGN: Randomized control intervention. Four groups in 2 x 2 design: Control, Balance, Strength, Balance + Strength, using blinded testers. SETTING: Exercise and balance laboratory at University of Connecticut Health Center. PARTICIPANTS: Subjects were 110 healthy community dwellers (mean age 80) who were free of dementia, neurological disease, and serious cardiovascular or musculoskeletal conditions. INTERVENTIONS: Short term training (3 months) occurred 3 times/week (45 minutes Balance and Strength, 90 minutes Balance + Strength). Balance training included equilibrium control exercises of firm and foam surfaces and center-of-pressure biofeedback. Strengthening consisted of lower extremity weight-lifting. All subjects than received long-term group Tai Chi instruction (6 months, 1 hour, 1 time/week). MEASUREMENTS: Losses of balance during Sensory Organization Testing (LOB), single stance time (SST), voluntary limits of stability (FBOS), summed isokinetic torque of eight lower extremity movements (ISOK), and usual gait velocity (GVU). RESULTS AND CONCLUSIONS: Balance training meaningfully improved all balance measures by restoring performance to a level analogous to an individual 3 to 10 years younger: LOB = -2.0 +/- 0.3 (adjusted paired differences, P < .005 ANOVA); SST = 7.0 +/- 1.2 sec; and FBOS = 9.0 +/- 2.0% of foot length (P < .05). Strengthening increased ISOK by 1.1 +/- 0.1 Nm kg-1 (P < .005). There was no interaction between balance and strength training. Significant gains persisted after 6 months of Tai Chi, although there was some decrement. PMID- 8617897 TI - Exercise with physically restrained nursing home residents: maximizing benefits of restraint reduction. AB - OBJECTIVE: To evaluate an exercise protocol designed to improve strength and mobility, and to decrease injury risk factors in physically restrained nursing home residents. DESIGN: A randomized controlled trial. PARTICIPANTS: Ninety-seven residents were randomized into either exercise or control groups. Thirty-five exercise and 37 control group residents completed all post-assessments after a 9 week trial. INTERVENTION: Walking or wheelchair movement training was supplemented by rowing exercise three times per week. Practice in behaviors related to safe movement was provided incidental to the exercise. MEASUREMENT: Endurance, speed, and injury risk measures relevant to walking, wheelchair propulsion, and standing were assessed by standardized protocols. Rowing endurance, rowing range of motion, and handgrip strength measures were collected to assess the effect of the rowing component of the exercise protocol. RESULTS: Fifty-four percent of the subjects who provided consent did not complete the protocol because of health status changes, lack of cooperation, or physical limitations that precluded exercise. The subjects who completed the exercise program showed significant improvement on injury risk and measures related to upper body strength (handgrip strength, rowing endurance, wheelchair endurance, and speed). Measures related to lower body strength did not significantly improve. CONCLUSION: Physically restrained residents are very frail, and it is difficult to implement a long-term exercise program with many residents because of the frailty. However, a substantial proportion of residents did cooperate well with the exercise program and showed improvement on measures correlated with decreased injury risk. The exercise program could be easily modified to include more lower body exercise, and the resultant protocol would be an important adjunct to restraint reduction programs. PMID- 8617898 TI - Controlled trial of a geriatric case-finding and liaison service in an emergency department. AB - OBJECTIVE: To evaluate the effects of a program of case-finding and liaison service for older patients visiting the emergency department. DESIGN: Nonrandomized controlled trial with systematically assembled intervention cohort and matched control group. SETTING: An urban teaching hospital. PARTICIPANTS: There were 385 intervention subjects aged 65 years and older and 385 control subjects matched by day of visit, gender, and age within 5 years. INTERVENTIONS: Geriatric medical, dental and social problems were identified in intervention subjects by a geriatric nurse clinician using well validated assessment instruments during a 30-minute evaluation. Recommendations were made to the patient, family, and attending emergency department physician, and attempts were made to arrange appropriate follow-up services. MEASUREMENTS: Frequency with which geriatric problems were identified in intervention subjects; physician, patient, and family compliance with recommendations; and mortality, institutionalization, health status, use of medical and social services, presence of an advanced directive, and quality of life at 3-month follow-up. RESULTS: Sixty-seven percent of patients were dependent in at least one activity of daily living, 82% had at least one geriatric problem identified, and 77% reported at least one unmet dental or social support need. The cost of identifying geriatric and dental/social issues was $5 and $1, respectively, for each problem. Physicians compiled with 61.6% of suggestions, and patients and families complied with 36.6% of recommendations. Mortality and nursing home residence proportions at 3 months were not significantly different (9.3% vs 9.7% and 5.0% vs 2.5% in intervention and control groups, respectively). Intervention subjects reported more difficulty communicating (21% fair or poor ability vs 13%, P = 0.2) than did control subjects. There were strong trends for fewer subsequent visits to emergency departments (0.26 intervention vs 0.39 control, P = .06) and more advance directives in the intervention group (6.7% intervention vs 2.9% control, P = .07). There was no statistically or clinically significant difference in any other health outcome. The number of new dental or social services initiated per patient over the 3-month follow-up was nearly identical (1.7 in the intervention group vs 1.5 in the control). Results in subjects aged 75 years and older and those discharged home from the emergency department were essentially identical to those in the main group. CONCLUSIONS: Numerous previously unrecognized geriatric medical and social problems can be detected in older persons visiting the emergency department. Despite this, an emergency department-based geriatric assessment and management program failed to produce improved outcomes. This suggests that either disease acuity is an overwhelming factor in subsequent outcome or, alternatively, more control over medical and social service delivery during and after the emergency department visit than was demonstrated in this program will be required before successful outcomes can be assured. PMID- 8617899 TI - Prevalence of atrial fibrillation and association of atrial fibrillation with prior and new thromboembolic stroke in older patients. AB - OBJECTIVE: To correlate atrial fibrillation with the incidence of new thromboembolic (TE) stroke in older patients with and without prior TE stroke. DESIGN: In a prospective study of 2101 older patients, electrocardiograms showed that atrial fibrillation was present in 283 patients (13%). At 42-month mean follow-up, atrial fibrillation was associated with the incidence of new TE stroke in patients with and without prior TE stroke. SETTING: A large long-term health care facility where 2101 older patients were studied. PATIENTS: The 2101 patients included 1451 women and 650 men, mean age 81 +/- 8 years (range 60 to 103). MEASUREMENTS AND MAIN RESULTS: Atrial fibrillation was present in 283 of 2101 patients (13%). The mean age was 84 +/- 7 years in patients with atrial fibrillation and 81 +/- 8 years in patients with sinus rhythm (P = .0001). The prevalence of atrial fibrillation was 5% in patients aged 60 to 70 years, 14% in patients aged 71 to 80 years, 13% in patients aged 81 to 90 years, and 22% in patients aged 91 to 103 years (P < .0001). Mean follow-up was 31 +/- 18 months in patients with atrial fibrillation and 44 +/- 27 months in patients with sinus rhythm (P = .0001). Previous TE stroke occurred in 123 of 283 patients (43%) with atrial fibrillation and in 431 of 1818 patients (24%) with sinus rhythm (P < .0001). New TE stroke occurred in 131 of 283 patients (46%) with atrial fibrillation and in 303 of 1818 patients (17%) with sinus rhythm (P < .0001). The log-rank test showed that patients with atrial fibrillation had a significantly higher probability of developing new TE stroke than those with sinus rhythm (P < .0001). The multivariate Cox regression model showed that independent risk factors for new TE stroke were male sex (relative risk = 1.3), prior TE stroke (relative risk = 3.1), and atrial fibrillation (relative risk = 3.3). CONCLUSIONS: Atrial fibrillation, prior TE stroke, and male sex are independent risk factors for the development of new TE stroke in older patients. PMID- 8617900 TI - Left ventricular hypertrophy on electrocardiogram: prognostic implications from a 10-year cohort study of older subjects: a report from the Bronx Longitudinal Aging Study. AB - OBJECTIVE: The objective of this study was to report on the prevalence, incidence and prognosis of left ventricular hypertrophy (LVH) on the electrocardiogram (ECG) in a cohort of ambulatory older men and women. DESIGN: A prospective, longitudinal study of 10 years duration with ECGs obtained at baseline and on an annual basis. SETTING AND PATIENTS: A community-based cohort study consisting of 459 subjects (aged 75-85, mean age 79 years). MEASUREMENTS: Baseline and follow up ECGs were interpreted using the Minnesota Code. Prevalence and incidence of LVH and ECG were determined as well as regression of ECG LVH. Clinical event rates measured were incidence of total mortality, myocardial infarction (MI, fatal and non-fatal), cardiovascular mortality, cardiovascular disease (fatal and non-fatal), stroke (fatal and non-fatal), all-cause dementia, and multi-infarct dementia. Differences in event rates between groups (those subjects with and without LVH) were compared as tests between proportions. A Cox Proportional Hazards Regression Analysis was performed to compare the relative independent predictive values of different competing factors, including age, gender, serum cholesterol, digitalis use, body mass, index, Blessed Dementia Scale, cigarette smoking, LVH at baseline, LVH ar baseline (persisting), new LVH, new LVH (persisting), new LVH (regressed), previous MI by history of ECG, hypertension by history, and cardiomegaly by X-ray (cardiothoracic ratio > or = 50%). RESULTS: At baseline, 9.2% of subjects (n = 42) had LVH on ECG and a mortality rate of 11.7/100 persons years versus 4.9/100 persons years for subjects without baseline LVH (P < .0001), and MI rate of 7.5/100 persons years with LVH versus 2.6/100 persons years without LVH (P < .0001), and a cardiovascular mortality rate of 7.2/100 persons years without LVH versus 2.7/100 person years without LVH. Subjects who developed new LVH on ECG (n = 39) had a mortality rate of 14.4/100 person-years compared with 4.4/100 person-years for those without LVH (P < .0001), a cardiovascular mortality rate of 11.1/100 person years versus 2.0/100 person years without LVH (P < .0001), and an MI rate of 6.1/100 person years versus 2.0/100 person years without LVH (P < .01). Subjects in whom the ECG LVH pattern disappeared over time had fewer cardiovascular mortal and morbid events than those with persistent LVH. According to the regression analyses, persistent LVH from baseline was an independent predictor of MI, overall cardiovascular disease, and total mortality. Newly developing LVH with subsequent regression was an independent predictor of overall cardiovascular disease and death. CONCLUSIONS: An increased prevalence and incidence of LVH on ECG, irrespective cause, is associated with a poor prognosis in very old men and women. Regression of ECG LVH in older people, irrespective of cause, may confer improvement in risk for cardiovascular disease. PMID- 8617901 TI - Age-dependent influence on heart rate variability in salt-sensitive hypertensive subjects. AB - OBJECTIVE: The known association between systemic arterial hypertension in its initial stages and increased sympathetic nervous system drive prompted us to evaluate the influence of age on autonomic nervous system function in subjects with salt-sensitive arterial hypertension. DESIGN: In a randomized study, autonomic nervous system function was assessed by power spectral analysis of heart-rate variability calculated with an autoregressive algorithm in salt sensitive hypertensives and controls at baseline and under sympathetic stress (passive head-up tilt). For 1 week before the study, all subjects kept to a diet supplying 120 mEq sodium. Sodium sensitivity was assessed by measuring and comparing arterial pressures after a 7-day controlled dietary sodium intake of 20 mEq per day after a 7-day period on 220 mEq sodium/day. SETTING: Geriatric division at the I Medical Clinic of the University of Rome "La Sapienza". PARTICIPANTS: Sixty-five patients with salt-sensitive hypertension (age range 19 to 89 years) and 64 age-matched normotensive controls, divided for data comparison into three age-groups: < 44 years; 44 to 64 years; and > or = 65 years. MEASUREMENTS: With an autoregressive algorithm in a power spectral analysis of heart rate variability, we detected four spectral frequency-domains: total power (0.0033 to 0.40 Hz), high-frequency power (0.16 to 0.40 Hz), low frequency power (0.04 to 0.15 HZ) and very-low-frequency power (0.0033 to 0.04 Hz). To determine sodium sensitivity, for 1 week before the study all subjects kept to a diet supplying 120 mEq sodium. Sodium sensitivity was assessed by measuring and comparing arterial pressures after a 7-day controlled dietary intake of 20 mEq per day and after a 7-day period of 220 mEq sodium/day. RESULTS: Results were expressed as natural logarithms of power and normalized units. The hypertensive patients of all ages had significantly lower total power of heart rate variability than the normotensive controls (P < .05). At baseline, the youngest hypertensives had lower natural logarithms and low-frequency normalized units than controls (P < .001). After tilt, only their low-frequency normalized units exceeded those of controls (P < .001). The middle-aged hypertensive group had higher low-frequency normalized units than controls at baseline (P < .05) and after tilt (P < .001). At baseline and after tilt, the oldest hypertensives had lower low-frequency natural logarithms than controls (P < .05) and normalized units equal to those of controls. But the hypertensives of all ages were less able than controls (P < .001) to increase low-frequency power after head-up tilt. In the less than 44-year-old hypertensives, diastolic pressure correlated significantly with low-frequency power of heart rate variability, expressed in normalized units, at baseline (P < .05) and after head-tilt (P < .05). A significant inverse correlation was found between age and the natural logarithm of low-frequency power at baseline (r = -.682, P < .001) and after tilt (r = .800; P < .001). Also, a significant inverse correlation was found to exist in normotensive subjects between the natural logarithm of low-frequency at baseline (r = -.595; P < .001) and after tilt (r = -.391; P < .001). The two regression line coefficients for age correlated significantly (P < .001) with the natural logarithm of low-power frequency after tilt. CONCLUSION: Whereas sodium chloride sensitive hypertension appears to be associated with sympathetic hyperactivity in young and middle-aged subjects, in older people it is not. Sympathetic activity diminishes with age, declining faster in hypertensive subjects. PMID- 8617902 TI - Community-acquired pneumonia in older patients. AB - OBJECTIVES: To document the prevalence of Legionella sp., Mycoplasma Pneumoniae and Influenza A and B in older patients hospitalized for community-acquired pneumonia (CAP) of nursing-home acquired pneumonia (NHAP) and to determine risk factors associated with fatal outcome or prolonged hospital stay. DESIGN: Prospective clinical and serological study. PATIENTS: All patients with CAP of NHAP--confirmed by chest roentgenogram--admitted to a 320-bed acute care geriatric university hospital from May 1, 1988 to August 31, 1989, were included. Serological testing was performed upon admission and after 2 and 4 weeks. Relevant data concerning medical history, clinical examination, and laboratory data were recorded upon admission. Ninety-nine patients (age: 85 +/- 6.3 years, 36 male, 63 female) met inclusion criteria; 20 came from nursing homes, 79 from their homes in the community. MAIN RESULTS: Fourteen patients died during the month after admission. An etiological diagnosis could be established in 22 patients. No cases of Legionella pneumonia and one case of M. pneumoniae were detected. Seven patients had evidence of Influenza pneumonia. Nonsurvivors were more likely to have been admitted from a nursing home and to have a temperature less than 37.5 degrees C and elevated urea nitrogen (BUN). Cyanosis, involvement of upper lobes, elevated white blood cell counts, and higher percentage of band forms were associated statistically with longer treatment. CONCLUSIONS: This study confirms the low prevalence of Legionella sp. and M. Pneumoniae infection in CAP and NHAP in this age group. Risk factors as to outcome and length of treatment may be used as points to identify high risk patients, with special attention to patients coming from nursing homes, and patients with high BUN. PMID- 8617903 TI - Do rehabilitative nursing homes improve the outcomes of care? AB - OBJECTIVES: To compare the differences in outcomes of Medicare patients discharged from hospital to two types of nursing homes, rehabilitative and regular, and to rehabilitative facilities. DESIGN: Criteria for distinguishing rehabilitative nursing homes (RNHs) from ordinary nursing homes (NH), based on staffing criteria, were developed by an expert panel and validated on a national sample of nursing homes. Those criteria that significantly discriminated the two types of NHs were then applied to a sample of nursing homes from a study of the outcomes of care for more than 2500 Medicare patients to classify the nursing homes in which patients were discharged. Actual discharge outcomes were compared with optimal outcomes based of predictive equations for different types of treatment (ordinary NH care, RNH care, and formal rehabilitative care). PARTICIPANTS: Medicare patients with strokes an hip fractures discharged from 52 hospitals in three cities. MEASUREMENTS: A disability scale that weights components of ADL measures was used as the primary outcome indicator. Nursing homes were classified as rehabilitative on the basis of the extent of staffing in rehabilitative areas. RESULTS: Patients discharged to various types of care varied on several parameters. After adjusting for these differences, stroke patients fared better when treated in rehabilitative facilities; there was no substantial benefit for RNH care over NH care. Healthier hip fracture patients who received RNH care fared better, but functional change for sicker hip fracture patients was not different among the three groups. CONCLUSIONS: The study suggests that at least a preliminary distinction among NHs can be made on the basis of staffing patterns and that the benefits of the additional staffing may vary with the problem under consideration. More work is needed to establish just what sorts of patients are most likely to benefit from the higher level of NH care. PMID- 8617904 TI - The change in serum protein concentration in response to the stress of total joint surgery: a comparison of older versus younger patients. AB - OBJECTIVE: To investigate whether the physiological response to surgery-induced stress, as measured by changes in serum secretory proteins, is more profound on older than in younger total joint arthroplasty patients. DESIGN: Retrospective study. SETTING: A 267-bed teaching hospital. PARTICIPANTS: A total of 220 ambulatory patients with normal admission serum albumin levels, of whom 106 were 65 years of age or older (mean age 73.3 +/- 6.2 years) and 114 less than age 65 (mean age 48.8 +/- 12.2 years). METHODS: Serum albumin and transferrin levels obtained at admission an on the fifth and tenth postoperative days were compared in the two age groups. RESULTS: In both age groups, admission serum albumins were significantly higher than on the corresponding postoperative Day 5 levels (40.4 +/- 3.7 g/L vs 25.0 +/- 3.3 g/L, P < .0001 and 39.5 +/- 2.5 g/L vs 23.9 +/- 3.1 g/L, P < .001 in older and younger patients, respectively). The drop in the serum concentration of albumin by postoperative Day 5 in the older patients was not significantly different from that of the younger patients (a drop of 15.6 +/- 3.3 g/L in older vs 15.4 +/- 4.4 g/L for the younger, P = .740). Among the 64 patients who remained in the hospital 10 days subsequent to surgery, the average postoperative Day 10 serum albumin concentration was significantly lower in the older patients when compared with the younger (26.2 vs 29.1 g/L P = .016). Similar results were obtained for serum transferrin. CONCLUSIONS: Subsequent to elective arthroplasty, the magnitude of change in serum albumin and transferrin concentrations is similar in older compared with younger, patients, suggesting that this stress response to surgery is nor age dependent. In contrast, the rate of recovery of the serum protein concentrations to preoperative levels may be slower in the older patients. However, this issue needs to be investigated further. PMID- 8617905 TI - Temporal and regional variability in the surgical treatment of cancer among older people. AB - OBJECTIVES: Numerous studies have documented that older individuals with cancer have been treated less aggressively than younger individuals. We utilized data from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program to assess geographic variation in the effects of age on the surgical treatment of cancer and to examine temporal trends in the treatment of older people between 1973 and 1991. DESIGN: Ecological analyses comparing time trends of treatment in nine regions over a 19-year period. SETTING: Population based data for nine geographic areas of the United States. PARTICIPANTS: Persons with incident cancer of the breast, colon, rectum, lung, ovary, uterus, pancreas, and stomach residing in these regions during the selected years. RESULTS: After age 64, the percentage of patients treated surgically decreased with increasing age for every cancer site studied. Between 1973 and 1991, the likelihood of receiving surgery for cancers of the uterus, colon, rectum, ovary, and breast increased more rapidly among patients ages 65 years and older than among those under age 65. This relative increase occurred consistently across most geographic areas studied. For cancers of the lung, stomach, and pancreas, the effect of age on the likelihood of receiving surgery did not diminish through the period under study.: CONCLUSIONS: Although older people remain less likely to receive surgical therapy than younger persons, for some important cancer sites the gap between the treatment of older and younger individuals narrowed from 1973 through 1991. PMID- 8617906 TI - Patients' willingness to accept life-sustaining treatment when the expected outcome is a diminished mental health state: an exploratory study. AB - OBJECTIVE: To assess patients' willingness to accept life-sustaining treatment when the expected outcome is a diminished mental health state. DESIGN: Structured interviews with a consecutive series of patients. SETTING: A university-based Department of Veterans Affairs Medical Center. PATIENTS: One hundred four male patients (mean age = 65.4 years; range 37-82) seen consecutively in a general medicine clinic. MEASUREMENTS: We assessed the acceptability of temporary ventilatory support of a hypothetical case of severe pneumonia. Patients were asked to consider mental health states involving minor cognitive deficits that varied only in their expected frequency and duration. RESULTS: All patients except one were willing to accept temporary life support in the management of severe pneumonia. Of these 103 patients, 76 patients specified the number of days they would allow themselves to be continued on intubation and ventilatory support. The length of time varied from 1 day to 2 years and was longer when patients reported having had a stroke of reported being in fair of poor health. Fifty patients were willing to accept all diminished mental health states, including the most severe state (F). Patients who reported having been in a medical intensive care unit were more likely to accept intubation in the most severe state (F). CONCLUSIONS: In the case of "severe pneumonia", about half of our older male veterans reported a willingness to accept intubation and ventilatory support even if it resulted in persistent cognitive disability. These results suggest that experience in a medical intensive care unit with the ventilators and experience with strokes may make patients more tolerant of treatments that may result in impaired cognitive function. PMID- 8617907 TI - Effects of exercise on neurobehavioral function in community-dwelling older people more than 75 years of age. AB - OBJECTIVE: We evaluated the effects of exercise on neurobehavioral function in healthy older people more than 75 years of age. DESIGN: A randomized controlled trial with 6-month follow-up was conducted. SETTING: The study was performed in the rural town of Kahoku, Japan, the population of which is considered representative of the older population of Japan. PARTICIPANTS: We studied 42 healthy volunteers (18 men and 24 women; mean age, 79 years (range 75 to 87 years)) who were randomly assigned to one of two groups, exercise or control. INTERVENTION: Subjects assigned to the exercise group were instructed to exercise for 60 minutes twice a week for 6 months. Subjects in the control group were not instructed to engage in an specific exercise regimen. MEASUREMENTS: The following measurements were recorded for both groups at baseline and at 6-month follow-up: (1) Neurobehavioral function as determined by the following tests: Mini-Mental State Exam (MMSE), Hasegawa Dementia Scale Revised (HDSR), Visuospatial Cognitive Performance Test (VCP-test), Button score, Up & Go test, and Functional Reach; and (2) Body mass index and blood pressure. RESULTS: The effects of exercise were shown in the Up & Go test, and Functional Reach (ANOVA with repeated measures). CONCLUSION: This study demonstrates the acceptability and effectiveness of exercise on neurobehavioral function, even in older people more than 75 years of age. PMID- 8617908 TI - Low TSH levels in nursing home residents not taking thyroid hormone. AB - BACKGROUND: Many practitioners perform a thyroid stimulating hormone (TSH) assay as a screening test in older patients. The introduction of sensitive TSH assays with lower normal limits has created a laboratory abnormality that is often of uncertain significance. Mechanisms include autonomous overproduction of thyroid hormone, nonthyroidal illness including medication effects, and hypothalamic/pituitary dysfunction. OBJECTIVE: To characterize the clinical status and course of nursing home residents with low TSH and normal total T4 levels in the absence of treatment with thyroid hormone. DESIGN: Retrospective chart review was performed to determine participants status at the time of low TSH level, with additional recording of follow-up thyroid hormone levels, cardiac rhythm, and mortality. Mortality was compared with that of a control group matched for age and sex. SETTING: A nursing home for veterans and their spouses. MAIN RESULTS: Forty subjects with low TSH and initially normal total T4 were identified. Only three subjects were subsequently diagnosed as hyperthyroid. TSH levels of 18 subjects subsequently normalized, and four additional subjects had low total T3 levels suggesting a nonthyroidal mechanism. Seven subjects died during the first 4 months of follow-up compared with three in a control group (P < .001). Nine of the 40 subjects had a history of or current atrial fibrillation. No new atrial fibrillation was documented during 388 months of EKG follow-up. CONCLUSIONS: Low total T3 levels, TSH normalization, and excess mortality suggest that nonthyroidal illness plays a role in the pathogenesis of low TSH determinants in the nursing home. Autonomous production of thyroid hormone also plays a role. We believe that the term "subclinical hyperthyroidism" should be used only if the clinician believes that autonomous overproduction of thyroid hormone is the cause of a low TSH level. If subsequent research shows correctable adverse consequences associated with subclinical hyperthyroidism from autonomous overproduction of thyroid hormone, a more aggressive diagnostic approach will be needed to define the mechanism of a low TSH level at the time of its discovery. PMID- 8617909 TI - Factors associated with institutionalization of older people in Canada: testing a multifactorial definition of frailty. AB - OBJECTIVES: To test a model of frailty by examining factors associated with institutionalization of older people in Canada; to assess whether diagnostic data provided information about risk beyond that provided by data on functional capacity and demographic variables. METHODS: Cross-sectional study of 1258 institutional subjects and 9113 community-dwelling older adults from the Canadian Study of Health and Aging. RESULTS: Multiple logistic regression analysis showed that female gender, being unmarried, absence of a caregiver, presence of cognitive impairment (including all types of dementia), functional impairment, diabetes mellitus, stroke, and Parkinson's disease were independently associated with being in a long-term care facility. CONCLUSION: Frailty appears to be a multidimensional construct, and not simply a synonym for dependence in Activities of Daily Living. Studies of health outcomes in older people should include diagnostic data as well as demographic information and data on functional capacity. PMID- 8617910 TI - Gender differences in the behavioral manifestations of Alzheimer's disease. AB - OBJECTIVE: To examine the relationship between gender and specific types of behavior problems that occur in patients with Alzheimer's disease. DESIGN: This was an observational study using the Dementia Behavior Disturbance Scale to quantify and define behavioral problems encountered by caregivers. Multiple regressions were used to control for the possible influence of dementia severity as measured by the Mini-Mental State Examination and the duration of dementia. SETTING: Patients were sampled from the outpatient dementia clinics of Roger Williams Hospital and Miriam Hospital in Providence, Rhode Island. PARTICIPANTS: A total of 125 patients with probable Alzheimer's disease, defined by NINCDS ADRDA diagnostic criteria, were included in the study. There were 75 women and 50 men. MAIN OUTCOME MEASURES: Caregivers rated the presence and frequency of 28 different behavior problems from the Dementia Behavior Disturbance Scale. Domains of behavior disturbance were then defined by a factor analysis of the data. RESULTS: Male and female groups were comparable for the demographic variables of age, education, and duration of dementia, as well as severity of depression, degree of cognitive impairment and overall severity of behavior disturbance. Among the six behavior factors that were defined, two were significantly related to gender. One factor, which included apathy and vegetative signs, was related to male gender; a second factor, which included reclusiveness and emotional lability, was related to female gender. CONCLUSIONS: Although overall severity of behavior disturbance in Alzheimer's disease may be related primarily to severity in dementia, significant differences in the types of behaviors manifested exist between males and females with the disease. PMID- 8617911 TI - Prevalence of appropriate and inappropriate indications for use of digoxin in older patients at the time of admission to a nursing home. AB - OBJECTIVE: To investigate the prevalence of digoxin use and appropriate and inappropriate indications for digoxin use in older patients at the time of admission to a nursing home. DESIGN: In a prospective study of 500 consecutive patients aged 60 years of age or older admitted to a nursing home, 96 (19%) patients were receiving digoxin at the time of admission to the nursing home. Appropriate and inappropriate indications for digoxin use were investigated in these 96 patients. SETTING: A large, long-term health care facility where 500 consecutive older patients were studied. PATIENTS: The 500 patients included 344 women and 156 men, mean age 81 +/- 8 years (range 60-100). MEASUREMENTS AND MAIN RESULTS: Ninety-six of the 500 patients (19%) were receiving digoxin at the time of admission to the nursing home. Fifty-one (53%) of the 96 patients receiving digoxin had an appropriate indication for digoxin use, and 45 (47%) had an inappropriate indication for digoxin use. Appropriate indications for digoxin use included atrial fibrillation with or without congestive heart failure (CHF) in 35 patients (36%) and CHF with sinus rhythm and abnormal left ventricular (LV) ejection fraction in 16 patients (17%). Inappropriate indications for digoxin used included CHF with sinus rhythm and normal LV ejection fraction in 18 patients (19%), misdiagnosis of edema or dyspnea as CHF in patients with sinus rhythm and normal LV ejection function in 17 patients (18%), history of possible (undocumented) paroxysmal atrial fibrillation in nine patients (9%), and sinus tachycardia in one patient (1%). Two of the 45 patients (5%) inappropriately treated with digoxin had evidence of digitalis toxicity on their admission electrocardiogram. CONCLUSIONS: The prevalence of digoxin use was 19% in older patients at the time of admission to the nursing home. Almost half of patients (47%) receiving digoxin at the time of admission had an inappropriate indication for digoxin use at that time. PMID- 8617912 TI - Decreased hospital utilization by older adults attributable to a home hospitalization program. AB - OBJECTIVE: To evaluate the cost effectiveness of a short-term home health care program for older people, Home Hospitalization (HH), compared with the alternative of regular ambulatory care with general or geriatric hospitalization as necessary. SETTING: Our HH was initiated in November 1991 to serve Jerusalem residents. Program staff included physicians, nurses, and paramedical professionals. Other medical/hospital services were provided nonselectively by the general medical personnel of the various hospitals in Jerusalem. STUDY DESIGN: Patients over the age of 65 were either referred to the HH program (study group, n = 36,500) or to routine medical care (control group; n = 9000) depending on their Sick Fund assignment. Hospital utilization rates per enrollee were studied prospectively and compared both between the two groups and with hospitalization rates in the year before the initiation of the program. RESULTS: During the first 26 months of operations, the HH program cared for 741 older persons for a total of 37,290 days' care at an average daily costs of $30.06 (1992) and $23.64 (1993). Annual general hospitalization rates per person declined in the study group from 2.80 days in the 1991 baseline period to 2.65 days in 1992 and to 2.54 days in 1993. Hospitalization rates in the control group increased from 2.62 in 1991 to 2.70 days/member and 2.71 days/member in 1992 and 1993, respectively. Annual geriatric hospitalization rates declined considerably in the study group from the 1991 baseline of 1.49 to 1.34 (1992) to 1.33 (1993). The control group experienced a small decrease from 1.64 (1991) to 1.58 (1992) and then a rise to 1.68 days per member in 1993. For the 26-month duration of the program, estimated savings of 20,773 general hospital days ($5.54 million) and 8486 geriatric hospital days ($0.98 million) exceeded its costs ($0.97 million), providing a cost/benefit ratio of 5.7/1. In addition, patient satisfaction was high. CONCLUSION: The HH program provided a cost effective substitute for care in a geriatric or general hospital for Jerusalem's elderly. PMID- 8617913 TI - Get up and move: a call to action for older men and women. PMID- 8617914 TI - The emergency department: a useful site for CGA? PMID- 8617915 TI - Acute medical care in the home. PMID- 8617916 TI - On-site acute care for residents of long-term care facilities. PMID- 8617917 TI - Predictors of mortality in older patients after a stroke. PMID- 8617918 TI - The relevance of genetic testing in late-onset Huntington's disease. PMID- 8617919 TI - Nurse practitioners. PMID- 8617921 TI - The process of dying. PMID- 8617920 TI - Terminal care medical fees for older adults. PMID- 8617922 TI - Acute epiglottitis in a nonagenarian. PMID- 8617923 TI - Raised streptokinase neutralization titers following a single use of topical streptokinase in an older patient. PMID- 8617924 TI - Close relationship between hemostatic factors and acute-phase reaction as normal aging process. PMID- 8617925 TI - A survey of poisoning on older people in Tehran. PMID- 8617926 TI - Septicemia in older people: trends in patients and disease characteristics. PMID- 8617927 TI - Hydroxamate-based metalloprotease inhibitor blocks shedding of L-selectin adhesion molecule from leukocytes: functional consequences for neutrophil aggregation. AB - L-selectin is an adhesion molecule that mediates the recruitment of neutrophils to inflammatory sites and initiates the migration of lymphocytes into the peripheral lymph nodes. In response to cell activation, L-selectin is shed from the cell surface, and altered levels of functional soluble L-selectin are detected in the plasma of patients suffering from numerous inflammatory diseases as well as AIDS. The mechanism that regulates L-selectin shedding is poorly understood. Here we show that a hydroxamate-based metalloprotease inhibitor, N (D,L-[2-(hydroxyaminocarbonyl)- methyl]-4-methylpentano)-L-3-(tert-butyl)-alanyl L-alanine, 2-aminoethyl amide, which blocks leukocyte TNF, TNF receptor, and IL-6 receptor release, also inhibits L-selectin shedding from neutrophils, eosinophils, and lymphocytes. Moreover, we show that such inhibition of L selectin shedding profoundly affects neutrophil aggregation and permits reaggregation in the presence of a heterologous stimulus. PMID- 8617928 TI - A human killer inhibitory receptor specific for HLA-A1,2. AB - Killer inhibitory receptors (KIRs) are transmembrane glycoproteins, expressed on NK cells and a small subset of T cells, that inhibit cell-mediated cytotoxicity upon binding to polymorphic MHC class I determinants on target cells. Although human KIRs specific for HLA-C and HLA-B molecules have been characterized, none have been shown to interact with HLA-A. Here we demonstrate that a member of the KIR cDNA family, designated NKAT4, encodes a 70-kDa receptor specific for HLA-A3. PMID- 8617929 TI - Dendritic cells produce macrophage inflammatory protein-1 gamma, a new member of the CC chemokine family. AB - Langerhans cells (LC) are skin-specific members of the dendritic cell (DC) family. DC are unique among APC for their capacity to activate immunologically naive T cells, but little is known about their chemotactic recruitment of T cells. We now report that LC produce macrophage inflammatory protein-1 gamma (MIP 1 gamma), a newly identified CC chemokine. MIP-1 gamma mRNA was detected in epidermal cells freshly procured from BALB/c mice, and depletion of I-A+ epidermal cells (i.e., LC) abrogated that expression. MIP-1 gamma mRNA was detected in the XS52 LC-like DC line as well as by 4F7+ splenic DC and granulocyte-macrophage CSF-propagated bone marrow DC. XS52 DC culture supernatants contained 9 and 10.5 kDa immunoreactivities with anti-MIP-1 gamma Abs. We observed in Boyden chamber assays that 1) XS52 DC supernatant (added to the lower chambers) induced significant migration by splenic T cells; 2) this migration was blocked by the addition of anti-MIP-1 gamma in the lower chambers or by rMIP-1 gamma in the upper chambers; and 3) comparable migration occurred in both CD4+ and CD8+ T cells and in both activated and nonactivated T cells. We conclude that mouse DC (including LC) have the capacity to elaborate the novel CC chemokine MIP-1 gamma, suggesting the active participation of DC in recruiting T cells before activation. PMID- 8617931 TI - Differential expression of homing molecules on recirculating lymphocytes from sheep gut, peripheral, and lung lymph. AB - The adhesion molecules integrin alpha 4 beta 7 and L-selectin have been hypothesized to help direct selective migration (homing) of lymphocytes to the gut and peripheral lymph nodes, respectively. An important prediction of current models is that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands. To test this prediction, we directly examined the expression of cell adhesion molecules on lymphocytes recirculating through the gut, the periphery, and the lung. Integrin beta 7 was highly expressed on CD4+CD45R- "memory/effector" T cells recirculating through the gut (mesenteric efferent and lower thoracic duct lymph). In contrast, cells recirculating through the periphery (prescapular efferent lymph) and the lung (caudal mediastinal efferent lymph) had much less beta 7 expression. A similar pattern of organ-specific beta 7 expression was seen on B cells. Beta 7 expression corresponded with adhesion to the gut mucosal addressin, MAdCAM-1, in vitro. The peripheral lymph node homing receptor, L selectin, was expressed at higher levels on CD4+CD45R- T cells from peripheral lymph than on cells from gut or lung lymph. These results provide additional strong support for alpha 4 beta 7 and L-selectin involvement in lymphocyte homing to the gut and to peripheral lymph nodes, respectively. Lymphocytes emigrating from the lung expressed low levels of both homing receptors and likely utilize molecules other than alpha 4 beta 7 and L-selectin for migration to the lung and associated lymphoid tissue. PMID- 8617930 TI - Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes. AB - Recent studies have reported that IFN regulatory factor-1 (IRF-1) regulates nitric oxide (NO.) production in murine macrophages (M phi). Since IRF-2 recognizes the same consensus sequence as IRF-1, we postulated that IRF-2 may also regulate NO.. Therefore, the ability of M phi from INF-2 homozygous (IRF-2-/ ) and heterozygous (IRF-2+/-) knockout mice to produce NO. following LPS and/or IFN-gamma stimulation was compared with the responses of IRF-1-/-, IRF-1+/-, and C57BL/6+/+ M phi. IRF-2-/- M phi produced less LPS-induced NO2- than IRF-2+/- or C57BL/6 M phi. LPS and IFN-gamma synergized to increase NO2- production from IRF 2-/- M phi to approximately 50% of IRF-2+/- and C57BL/6 levels. Unexpectedly, IRF 2-/-, IRF-2+/- and C57BL/6 M phi produced comparable levels of inducible NO synthase mRNA in response to treatment with LPS and IFN-gamma. IRF-1-/- M phi produced barely detectable NO2- and low, but detectable, inducible NO. synthase mRNA in response to IFN-gamma and LPS. These results indicate that IRF-1 and IRF 2 differ in their mechanism of NO. regulation and that IRF-2 regulates inducible NO. synthase post-transcriptionally. PMID- 8617932 TI - Ligation of CD40 influences the function of human Ig-secreting B cell hybridomas both positively and negatively. AB - The effect of ligation of CD40 on the proliferation and Ig secretion of a battery of human Ig-secreting hybridomas was examined to determine the regulatory activity of this surface molecule on B cells after initial activation. B cell hybridomas were generated by fusing activated peripheral blood B cells with SPAZ 4, a non-Ig-secreting fusion partner, and were cloned before analysis. All hybridomas expressed CD40 comparably. These hybridomas were stimulated with either recombinant baculovirus-expressed membrane-bound CD40L or a soluble murine CD40L/CD8 construct in the presence or the absence of various cytokines. Concentrations of CD40L that saturated 40 to 100% of CD40 induced initial homotypic aggregation followed by Fas (CD95)-independent apoptosis, with resultant decreases in growth and Ig secretion. Concentrations of CD40L that saturated 15 to 25% of CD40 also stimulated aggregation of all hybridomas. However, proliferation and Ig secretion of 9 of 13 IgM-secreting hybridomas, but none of 14 IgG- or IgA-secreting hybridomas, were enhanced by these concentrations of CD40L. These responses were independent of interactions mediated by the adhesion pair CD1la/CD18-CD54. These results indicate that the impact of CD40 ligation on human Ig-secreting hybridomas varies with the extent of CD40 engagement and depending on whether the hybridoma derived from an activated B cell that had previously undergone switch recombination. PMID- 8617933 TI - Cross-linking of the CD40 ligand on human CD4+ T lymphocytes generates a costimulatory signal that up-regulates IL-4 synthesis. AB - Although there is good evidence that the induction of IL-4 synthesis in CD4+ T lymphocytes is favored by Ag presentation by B cells and not macrophages, the precise molecular signals provided by B cells to T cells that enhance IL-4 synthesis are not clear. To examine this issue, we established an APC-independent system to activate highly purified T cells and induce cytokine synthesis, using immobilized mAbs against several T cell surface molecules, including CD3, CD28, and the CD40 ligand (CD40L). The counter-receptors for all three of these molecules are expressed on B cells, and include CD40, which is expressed primarily on B cells, but also on dendritic cells and thymic epithelium. We found that IL-4 synthesis was greatly enhanced by triggering of CD40L on the T cell surface in conjunction with ligation of CD3/TCR and CD28, whereas ligation of CD3/TCR and CD28 in the absence of CD40L triggering resulted in little or no IL-4 synthesis. CD40L costimulation greatly enhanced IL-4 synthesis both in T cells from normal nonallergic adult subjects as well as in naive T cells from cord blood. Furthermore, we demonstrated that IL-4 synthesis was optimally enhanced when the strength of the CD3/TCR signal was limiting, while IL-4 synthesis was inhibited when CD3/TCR stimulation was maximal. These studies confirm that IL-4 synthesis can be induced in normal T lymphocytes in the absence of exogenous IL 4, and demonstrate that CD40L costimulation is of fundamental importance in regulation of IL-4 production. In addition, these findings provide a mechanism by which B cells preferentially enhance IL-4 synthesis in T cells at low Ag concentrations. PMID- 8617934 TI - A role for c-myc in the regulation of thymocyte differentiation and possibly positive selection. AB - The purpose of thymocyte differentiation is to establish the T cell repertoire and eliminate nonfunctional and autoreactive T cells. In an analysis of potential regulators of this process, we have found that c-myc expression is down-regulated dramatically during early thymocyte differentiation and subsequently up-regulated along with TCR/CD3 in CD4+8+ cells. Transgenic E beta-myc mice that constitutively express c-myc in thymocytes have a larger proportion of thymocytes with high TCR/CD3 and low heat-stable antigen-1 expression than controls, and an increase in the number of transitional cells with a CD4+CD8low phenotype. Mature E beta-myc T cells respond less vigorously than controls to activation through their TCR/CD3 complex, as measured by proliferation and induction of the activation marker CD69. These results are consistent with a hypothesis in which activation of immature T cells through TCR/CD3 induces c-myc up-regulation and drives thymocytes through the initial stage of positive selection. PMID- 8617935 TI - Peptide-based, but not whole protein, vaccines elicit immunity to HER-2/neu, oncogenic self-protein. AB - HER-2/neu, an overexpressed oncogenic protein, has been proposed as a human cancer vaccine target. HER-2/neu is a "self" protein, however, and methods of vaccine strategies that would be effective in immunizing patients to a "self" tumor Ag have not been established. Many of the tumor Ags defined in humans are nonmutated self proteins, e.g., MAGE, and overcoming tolerance may be key in the generation of effective anti-tumor immunity. One theory states that tolerance to self proteins is directed only to dominant epitopes of proteins and not to every portion of the protein. Accordingly, tolerance can be circumvented by immunization to peptide fragments, but not whole protein. The studies outlined here demonstrate rat neu-specific immunity could be elicited in rats by vaccination with immunogenic rat neu peptides, but not by immunization with the intact protein. A rat model was used since rat neu protein is 89% homologous to human HER-2/neu protein and has a similar tissue distribution and level of expression. Rats were immunized with groups of peptides derived from the amino acid sequence of the intracellular domain or extracellular domain of rat neu protein and both groups developed CD4+ T cell immunity and Ab immunity to rat neu peptides and protein. Animals immunized in a similar fashion with intact purified rat neu protein did not develop Ab or T cell immunity to rat neu. Furthermore, rats that developed neu-specific immunity showed no histopathologic evidence of autoimmunity directed against organs expressing basal levels of rat neu protein. These studies suggest an immunization strategy that might be effective in human cancer vaccines targeting self tumor Ag. PMID- 8617936 TI - IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells. AB - IL-12, a potent inducer of IFN-gamma production by T cells and NK cells, has been recently reported to exacerbate an established Th2 response in vivo. However, the effect of IL-12 on Th2-lymphokine production remains unclear. Since IL-10 is a lymphokine associated with Th2 responses which decreases both IL-12-induced IFN gamma production and IL-12 production by macrophages, we have analyzed here, in an APC-free system, the ability of IL-12 to modulate the production of human IL 10 by established Th0, Th1, and Th2 T cell clones (TCC), T cell lines, and purified peripheral blood T cells. IL-12 synergized with anti-CD3 mAb, Con A, or IL-2 in inducing IL-10 production by Th0, Th1, and Th2 TCC and by T cell lines. This effect was dose dependent (from 0.1 to 50 U/ml) and associated with an increase of IL-10 mRNA transcription. As previously reported, IL-12 also enhanced IFN-gamma production by stimulated Th1 and Th0 TCC and, to a lesser extent, IL-4 production by stimulated Th0 and Th2 TCC. These observations were extended to peripheral blood T cells stimulated in the presence of exogenous IL-2. Moreover, using neutralizing anti-IL-2 Ab, we report that endogenous IL-2 produced by stimulated Th0 TCC could in part contribute to the effect of IL-12 on IL-10 and IL-4 production. In conclusion, IL-12 synergizes with IL-2 and other stimuli in inducing IL-4 and IL-10 production by T cells. This property may help to explain why IL-12 does not efficiently down-regulate an established Th2 response. PMID- 8617937 TI - Continuous administration of Il-13 to mice induces extramedullary hemopoiesis and monocytosis. AB - IL-13, a recently identified Th2 cytokine, shares some, but not all, IL-4 functions, including inhibition of monocyte and macrophage activation, stimulation of human B cells, and induction of growth and differentiation of mouse bone marrow cells in vitro. We have now tested the in vivo effects of recombinant mouse IL-13 (rIL-13) from stably transfected, high expressing BW5147 thymoma cells. After purification by anion exchange chromatography, rIL-13 was administered in the peritoneal cavity of BALB/c mice via osmotic pump for 7 days. Spleens from the rIL-13-treated mice were significantly enlarged compared with control spleens due to increased cellularity. In particular, increased numbers of immature erythroblasts and megakaryocytes were observed in splenic sections after rIL-13 treatment. Spleen cells from rIL-13-treated mice showed greatly increased responsiveness in vitro to recombinant forms of mouse IL-3, mouse granulocyte macrophage CSF, or human CSF-1 and, to a lesser extent, to mouse IL-4 or IL-13. Moreover, the rIL-13-treated mice also showed significant increases in CFU-E, CFU C, and erythroid burst colonies in the spleen, further indicating the presence of increased numbers of hemopoietic precursors. Hematologic analyses indicated that rIL-13 treatment induced slight anemia and striking monocytosis. Finally, spleen cells from rIL-13-treated mice produced significantly more IL-6 upon LPS stimulation. Interestingly, the strong Th2 response induced by Nippostrongylus brasiliensis infection was also accompanied by an increase in hemopoietic precursor frequencies in the spleen. Collectively, these data indicate that exogenous rIL-13 induces extramedullary hemopoiesis in mice and suggest that endogenous IL-13 may contribute to replenishment of effector cells during strong Th2 responses. PMID- 8617939 TI - CD2-induced apoptosis in activated human peripheral T cells: a Fas-independent pathway that requires early protein tyrosine phosphorylation. AB - Short-term activated peripheral T lymphocytes are susceptible to apoptotic cell death triggered by CD2 mAbs. The aim of this study was to examine whether the CD2 mediated pathway of apoptosis is linked to the Fas death pathway, as this is the case for CD3/TCR-triggered apoptosis in several models of T cells. Using T lymphocytes from patients harboring Fas gene mutations and displaying a profound defect in Fas signaling of cell death, we show that CD2- (but not CD3-) mediated apoptosis still proceeds normally. In normal activated T cells, CD3-mediated apoptosis is prevented by reagents that block the Fas/Fas-ligand interaction, namely soluble M3 (an antagonistic anti-Fas mAb) and soluble human Fas.Fc, a fusion protein able to bind released Fas-ligand. In contrast, CD2 signaling of apoptosis resists these blocking agents. Neither new protein synthesis nor the activation of calcineurin was required for CD2- and Fas-mediated apoptosis, suggesting that latent cytoplasmic "death" molecules were activated upon stimulation of the cells. In both cases, protein tyrosine kinases were transiently activated, as is exemplified by the autophosphorylation and exokinase activity of p56lck, yielding overlapping yet nonidentical profiles of protein tyrosine phosphorylation. Pretreating the cells with herbimycin A, before the addition of the apoptotic stimuli, almost completely inhibited CD2 transmembrane signaling of apoptosis, but left intact Fas-induced apoptosis. Our data suggest that CD2 is a Fas-independent cell death pathway that might contribute directly to the elimination of T cells expanding during an immune reaction. PMID- 8617938 TI - Kinetically coordinated induction of TAP1 and HLA class I by IFN-gamma: the rapid induction of TAP1 by IFN-gamma is mediated by Stat1 alpha. AB - Transporter associated with Ag processing-1 (TAP1) is induced by IFN-gamma more rapidly than is HLA class I. Kinetic analysis of transfectants reveals that IFN gamma activates the TAP1 promoter more rapidly than the HLA-B7 class I promoter. A gamma-activating sequence (GAS) in the TAP1 promoter is necessary for the rapid induction by IFN-gamma. Two overlapping IFN consensus sequences contribute to the constitutive and TNF-induced expression of TAP1 but are not necessary for the IFN gamma response. Moreover, IFN-gamma activates the GAS-binding protein Stat1 alpha much more rapidly than it induces the IFN consensus sequence-binding protein IRF 1, which mediates the response of the HLA-B7 class I promoter. We conclude that IFN-gamma uses different transcription factors to regulate the sequence of appearance of these interacting gene products. PMID- 8617940 TI - HLA-DQ8 transgenic mice lacking endogenous class II molecules respond to house dust allergens: identification of antigenic epitopes. AB - We have introduced HLA-DQ8 (HLADQB*0302 and HLA-DQA*0301) genes into A beta 0 knockout mice. Transgenic animals were immunized with a whole body extract of Dermatophagoides pteronyssinus (Der p), one of the causative agents of house dust mite allergy. Transgenic mice expressing HLA-DQ8 genes elicited HLA-DQ8 restricted responses driven by CD4+ T cells. Synthetic-overlapping peptides representing a major allergen of house dust mite (Der p 2) were synthesized and used as immunogens. HLA-DQ8+ mice responded to three peptides: 8 (residues 61 80), 11 (residues 91-110), and 13 (residues 111-129). The mice produced IL-2, IL 4, and IL-6 response to Der p challenge, suggesting a mixed Th1/Th2 response. These mice represent a new model for studies of the immune basis of allergy. PMID- 8617941 TI - Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing. AB - MHC class I molecules bind peptides that are translocated from the cytosol into the endoplasmic reticulum by the peptide transporter associated with antigen processing (TAP). Class I heterodimers have been shown to associate with TAP and are released when loaded with peptide. Here, we show the existence of two pools of class I heterodimers, one associated with TAP and one that is free. Whereas the free pool is recognized by the class I-specific Ab W6/32, the TAP-associated pool is not. Analysis of several class I alleles shows binding to TAP with different efficiencies, even at the earliest time points of MHC class I assembly. Most HLA-A and -C alleles tested interacted efficiently with TAP, whereas a considerable number of HLA-B alleles associated very inefficiently or not at all with TAP. This was also observed in cells with nonfunctional TAP. Sequence comparison of the different class I alleles allowed the definition of amino acids in the peptide binding groove that might be involved in TAP association. Binding of peptides to two different pools of class I heterodimers may ensure efficient peptide association in an environment where peptides have a short life span. PMID- 8617942 TI - In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/-) hemopoietic stem cells. AB - The long term marrow-repopulating ability of mouse bone marrow c-Kit+ Sca-1+ Lin(low/-) cells was studied. Injection of as few as 100 c-Kit+ Sca-1+ Lin(low/-) cells could rescue a lethally irradiated recipient mouse and reconstitute both myeloid and lymphoid cells in their peripheral blood for at least 10 mo. When limiting dilution analysis was performed in the presence of host-derived rescue cells, as low as five c-Kit+ Sca-1+ Lin(low/-) cells were shown to be capable of repopulating lymphohemopoietic cells. Subsequently, we examined whether c-Kit+ Sca-1+ Lin(low/-) cells had a capacity for self-renewal in vivo. After transplantation of 500 c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells into lethally irradiated Ly-5 congenic mice, the expansion of cells with the same phenotypes as the injected cells was monitored. By day 28 post-transplantation, more than 50,000 donor type c-Kit+ Sca-1+ Lin(low/-) Ly-5.1+ cells were found in the spleen and over 18,000 cells were found in bone marrow. The expansion in spleen, however, was transient in that by day 60 cells of the donor phenotype were found only in bone marrow. The c-Kit+ Sca-1+ Lin(low/-) LY-5.1+ cells expanded in spleen or bone marrow contained as many high proliferative potential colony forming cells as the originally injected cells. They also contained cells with LTRA, but the frequency appeared to be less compared with naive c-Kit+ Sca-1+ Lin(low/-) cells. These data provide evidence that c-Kit+ Sca-1+ Lin(low/-) cells in bone marrow are capable of multilineage differentiation as well as self renewal and that spleen is a primary site of stem cell expansion after transplantation. PMID- 8617943 TI - Proteolytic cleavage of alpha-actinin by calpain in T cells stimulated with anti CD3 monoclonal antibody. AB - Stimulation of the TCR/CD3 complex on T cells initiates rearrangement of the actin cytoskeleton. The results presented show that a temporal increase in the appearance of filamentous actin begins immediately after stimulation of T cells with immobilized anti-CD3 mAb. The formation of filamentous actin in these stimulated cells reaches a steady state within 30 min after anti-CD3 mAb stimulation. At this time, pseudopod formation is observed and becomes progressively more evident over the next several hours. Experiments were done to investigate the role of the actin cytoskeletal associated proteins, alpha actinin, vinculin, and talin, in the assembly of the actin cytoskeleton in anti CD3 mAb-stimulated T cells. Using immunofluorescence, these three proteins are detected throughout the cytosol in resting T cells. However, after anti-CD3 mAb stimulation of the T cells, these proteins move to one pole of the cell. Electrophoresis followed by immunoblotting of T cell lysates prepared from resting as well as anti-CD3 mAb-stimulated cells revealed that alpha-actinin, but not vinculin or talin, was modified as a consequence of cell activation. Results show that alpha-actinin exists as a 105-kDa subunit in resting T cells, but that anti-CD3 mAb stimulation of T cells leads to the appearance of an 80-kDa lower molecular form of alpha-actinin. Experiments show that this occurs as a consequence of the 105-kDa subunit being proteolytically cleaved by calpain. PMID- 8617945 TI - Il-7 supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal liver progenitor cells. AB - The rearrangement of TCR-beta gene, one of the earliest events in T cell development, consists of two consecutive steps: D-J rearrangement and V-DJ rearrangement. The present study examined the signals supporting D-J beta and V DJ beta rearrangements during early T cell development from progenitor cells that reside in fetal liver. We have found that there is an interval of 1 to 2 days between D-J beta and V-DJ beta rearrangements during the early T cell development from fetal liver progenitor cells in deoxyguanosine-treated thymus lobes. We have also found that IL-7, a cytokine expressed in the subcapsular area of the thymus, can promote D-J beta rearrangement of fetal liver progenitor cells, and that anti IL-7 and anti-IL-7R Abs inhibit the D-J beta rearrangement and further T cell development of fetal liver progenitor cells in the thymus environment. Interestingly, unlike the thymus environment, IL-7 alone was not capable of supporting V-DJ beta rearrangement in the fetal liver cell cultures. These results indicate that D-J beta rearrangement during fetal liver-derived early T cell development is supported in the thymus by IL-7. Furthermore, the present results demonstrate that IL-7, supporting D-J beta rearrangement, does not promote V-DJ beta rearrangement of fetal liver progenitor cells, suggesting that intrathymic molecules promoting V-DJ beta rearrangement are distinct from IL-7 that supports the D-J beta rearrangement. PMID- 8617944 TI - CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C. AB - The human leukemic cell line YT displays spontaneous cytotoxicity against CD80+ and/or CD86+ and ICAM-1+ target cells. In this work, we report that CD28-mediated cytotoxicity of YT involves tyrosine phosphorylation and activation of phosphatidylinositol (PI) 3-kinase, the Tec kinase Itk/Emt, and protein kinase C (PKC). YT mediates lysis of CD80+/CD86+ B lymphoblastoid cell lines and the murine mastocytoma p815 transfected with CD80 or CD86. The lysis was inhibited by two different Pi 3-kinase inhibitors, wortmannin and LY294002. The PKC inhibitors calphostin C and bisindolylmaleimide GF109203X also abolished YT-mediated cytotoxicity. Furthermore, exocytosis of cytolytic effector molecules was also inhibited by PI 3-kinase inhibitors and PKC inhibitors. PMA together with Ionomycin did not induce granule exocytosis or cytotoxicity by YT cells. Treatment of YT cells with PMA for up to 20 h, which depleted PMA-responsive PKC isoforms, had no effect on the CD28-mediated cytotoxicity. This cytotoxicity displayed by PMA-treated YT cells, however, could still be inhibited by Pi 3 kinase inhibitors and PKC inhibitors. Taken together, these results are consistent with a model in which activation of CD28 and LFA-1 induces tyrosine phosphorylation of the CD28 cytoplasmic domain, recruitment and activation of PI 3-kinase, as well as the Tec kinase Itk/Emt, and the activation of PMA nonresponsive PKC isoenzymes. Activation of PI 3-kinase and PMA-nonresponsive PKC isoenzymes is shown to be involved directly in cytolytic granule release by YT cells. PMID- 8617946 TI - Vasoactive intestinal peptide inhibits IL-4 production in murine T cells by a post-transcriptional mechanism. AB - Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes, modulates cytokine expression in T lymphocytes. VIP down-regulates the expression of IL-2 and IL-10 mRNA in T cells stimulated through the TCR associated CD3 complex. In contrast, IL-4 production is inhibited at a post transcriptional level. In this study, we investigate the molecular mechanisms involved in the inhibition of IL-4 production by VIP. At the protein level, the time courses for IL-2 and IL-4 inhibition by VIP are different, with IL-4 being affected approximately 24 h later than IL-2. Northern blots and competitive reverse-transcription PCR analysis confirm the post-transcriptional inhibition of IL-4 production in both murine spleen cells and thymocytes activated through the CD3 complex. VIP does not affect IL-4 secretion, and does not induce a rapid IL-4 reuptake. Exogenous rIL-2 completely reverses the inhibitory effect of VIP, suggesting that VIP inhibits IL-4 production indirectly as a consequence of IL-2 inhibition. Studies regarding the newly synthesized IL-4 protein show that although VIP and exogenous IL-2 do not affect the rate of IL-4 synthesis, VIP reduces significantly the stability of the newly synthesized IL-4 protein, and exogenous IL-2 can restore it to the levels observed in activated cells in the absence of VIP. These results explore the molecular mechanisms involved in the neuroendocrine regulation of cytokine production, and support the idea that neuropeptides released or produced in the local lymphoid microenvironment may participate in the intricate cytokine network controlling local immune responses. PMID- 8617947 TI - Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells. AB - IL-15 is a newly described cytokine produced by monocytes and other non-T cells that utilizes the IL-2R beta- and common gamma-chains, thereby stimulating many NK cell functions previously ascribed to IL-2. Thus, IL-15 may promote NK cell activity during innate immune responses, before the activation of T lymphocytes and subsequent production of IL-2. This study investigated the ability of rIL-15 to substitute for rIL-2 in initiating proliferation of resting human NK cells cocultured with various stimulator cells. NK cell proliferation could not be initiated with rIL-15 as the sole costimulatory cytokine. However, NK cell proliferation was initiated with rIL-15 and either rIL-10 or rIL-12, cytokines also produced by monocytes and other APC and implicated in innate immune responses. Individually, rIL-10, rIL-12, and rIL-15 are effective initiators of NK cell proliferation when combined with submitogenic concentrations of rIL-2, indicating their potential involvement in NK cell proliferation at early stages of an Ag-specific T cell immune response. NK cells proliferating in the different cytokine combinations or optimum concentrations of rIL-2 were indistinguishable in terms of phenotype and cytotoxic activity, but differed in whether they secreted IFN-gamma or IL-5. IFN-gamma was secreted in cultures containing rIL-12, whereas IL-5 secretion was dependent upon interaction of IL-2 with the high affinity IL-2R. These results support the notion that NK cell proliferation occurs at different phases of the immune response with the particular cytokine milieu influencing the repertoire of NK cell-secreted cytokines. PMID- 8617948 TI - Herpesvirus saimiri open reading frame 14, a protein encoded by T lymphotropic herpesvirus, binds to MHC class II molecules and stimulates T cell proliferation. AB - Herpesvirus saimiri (HVS) is an oncogenic, lymphotropic, gamma-herpesvirus that transforms human and simian T cells in vitro and causes lymphomas and leukemias in various species of New World primates. Nucleotide sequence analysis of the HVS genome revealed an open reading frame with 22% amino acid identity to the mouse mammary tumor virus 7 superantigen. In this study, we demonstrate that this open reading frame, HVS14, encodes a heavily glycosylated protein that is secreted. Both the HVS14 present in the supernatant of transfected cells and a chimeric HVS14.Fc fusion protein were found to bind to heterodimeric MHC class II HLA-DR molecules. The supernatant from HVS14-transfected cells induced the proliferation of human PBMC, which could be specifically inhibited by HVS14-specific mAbs. Purified peripheral blood T cells were induced to proliferate in the presence of accessory cells and HVS14-containing supernatant. Whereas the HVS14 protein stimulated T cell proliferation, the HVS14.Fc fusion protein blocked proliferative responses to soluble Ags in vitro. Collectively, these data indicate that HVS14 can function as an immunomodulator that may contribute to the immunopathology of HVS infection. PMID- 8617949 TI - Enhanced development of Th2-like primary CD4 effectors in response to sustained exposure to limited rIL-4 in vivo. AB - Previous studies have established that cytokines regulate development of Th cell subsets from naive CD4 cells, suggesting promising therapeutic potential for cytokines that has not been fully realized, in part due to high dose toxicity. Here we examined effects of sustained delivery of low doses of rIL-4 on development of cytokine-secreting primary CD4 cells in vivo. Diffusion chambers injected with X63.Ag-653 plasmacytoma transfectants that constitutively produce rIL-4 were implanted s.c. in mice 24 to 48 h before immunization with keyhole limpet hemocyanin (KLH). Five days later at the peak of the response, KLH specific CD4 cells from controls, which received chambers containing untransfected plasmacytoma cells, secreted primarily IL-2 and IL-4, with low levels of IFN-gamma and no IL-5 following Ag restimulation. Administration of rIL 4 enhanced IL-2 and IL-4 production by two- to fivefold, with a corresponding decrease in IFN-gamma. As frequencies of KLH-specific precursors that secreted IL 4 were unaltered, exogenous cytokine apparently affected the magnitude of IL-4 secretion by primed CD4 cells. Although IL-4 was undetectable in sera of mice carrying chambers, titers of KLH-specific IgG1 and IgG3 Ab were increased by rIL 4, whereas IgM, IgG2a, and IgG2b levels were unaltered. The results indicate that sustained delivery of low doses of cytokines at sites distant from Ag exposure can selectively potentiate development of CD4 subsets. PMID- 8617950 TI - Unusual uniformity of the N-linked oligosaccharides of HLA-A, -B, and -C glycoproteins. AB - MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells. PMID- 8617951 TI - Tolerance of single, but not multiple, amino acid replacements in antibody VH CDR 2: a means of minimizing B cell wastage from somatic hypermutation? AB - Mutations in the heavy chain complementarity determining region 2 (CDR2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag. A panel of multisite mutants that had lost detectable binding to phosphocholine-containing Ags was previously created by saturation mutagenesis of the CDR2 region of T15. Based on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg52, is intolerant to change, yielding a nonbinder phenotype even with conservative amino acid replacement. Mutation at two other sites, Ala50 and Tyr55, can yield a nonbinder phenotype depending on the amino acid replacement. Single-site mutations of the remaining 14 positions allowed retention of binding ability. Thus, except for positions 50, 52, and 55, multiple mutations must be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure and the interactions of the CDR2 region. Our results imply that introduction of point mutations would not normally delete Ag-binding ability until two or more mutations had accumulated. This would minimize potentially harmful effects of somatic mutation on Ig V region genes and improve the chance of survival for an Ab such as T15, which in its unmutated form is already well suited to bind Ag. PMID- 8617952 TI - Expression and characterization of functionally active recombinant perforin produced in insect cells. AB - A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule. PMID- 8617953 TI - The COI mitochondrial gene encodes a minor histocompatibility antigen presented by H2-M3. AB - We found that (LP x C57BL/6)F1 mice could raise a CTL response against parental C57BL/6 cells. These CTLs recognized a maternally transmitted, H2-M3wt restricted, minor histocompatibility Ag (MiHA) that is widely distributed among many strains of mice and encoded by the COI mitochondrial gene. The wild-type MiHA is the COI N-terminal hexapeptide. Sequencing the 5' end of the COI gene in LP and C57BL/6 mice showed that the LP allele arose by a T-->C transition in the third codon, which caused substitution of threonine for isoleucine. Molecular characterization of this MiHA and the demonstration that it is presented exclusively by H2-M3: 1) support the concept that differential expression of MiHA in MHC-identical animals is caused by polymorphism of the MiHA gene proper; 2) expand our knowledge of the repertoire of self-peptides naturally presented by H2 M3 and show that this MHC class I molecule can present short endogenous peptide ligands; and 3) suggest that mitochondrial DNA mutations that modify the repertoire of H2-M3-associated mitochondrial peptides are representative of mitochondrial DNA mutations in general. PMID- 8617954 TI - Immunogenicity of peptides bound to MHC class I molecules depends on the MHC peptide complex stability. AB - The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines. PMID- 8617955 TI - Recombinant IL-10 and IL-10/Fc treatment down-regulate egg antigen-specific delayed hypersensitivity reactions and egg granuloma formation in schistosomiasis. AB - In infection with the helminth Schistosoma mansoni, parasite eggs elicit a Th cell-mediated hepatic granulomatous inflammation that leads to scarring, portal hypertension, hemorrhage, and death. On the other hand, the cytokine IL-10 has been shown to decrease egg Ag-specific Th cell responses by down-regulating MHC class II as well as B7 costimulatory molecule expression on accessory cells. We now test the effect of IL-10 in vivo on models of egg Ag-specific hypersensitivity as well as on the natural disease. Systemic administration of IL 10 significantly inhibited delayed-type hypersensitivity reactions to egg Ag as well as primary and secondary granuloma formation to eggs embolized in the lung. However, significant inhibition of hepatic granuloma formation associated with the natural infection required the use of an IL-10/Fc fusion protein with a prolonged in vivo half-life. Lymph node cells from IL-10/Fc-treated mice produced less IL-2 and IFN-gamma, and more IL-4 and IL-10 than control cells, suggesting that reduced egg granuloma formation resulted primarily from down-regulation of Th1 responses. These results indicate that suitable administration of exogenous IL-10 can be effective in ameliorating immunopathologic damage associated with schistosomiasis. PMID- 8617956 TI - Salmonella typhimurium aroA- infection in gene-targeted immunodeficient mice: major role of CD4+ TCR-alpha beta cells and IFN-gamma in bacterial clearance independent of intracellular location. AB - Due to the dependency on aromatic precursors, the growth of Salmonella typhimurium aroA- is limited in immunocompetent mice. Here we show that H-21-A beta-/- mice (lacking MHC class II molecules and thus devoid of mature CD4+ TCR alpha beta cells), TCR-beta-/- mice (devoid of TCR-alpha beta cells), and IFN gamma R-/- mice (unresponsive to IFN-gamma) are highly susceptible to S. typhimurium aroA- infection compared with heterozygous controls. In contrast, beta 2m-deficient mice (lacking surface MHC class I and thus devoid of conventional CD8+ T cells) or TCR-delta-/- mice (devoid of TCR-gamma delta cells) were equally as resistant to S. typhimurium aroA- infection as their heterozygous littermates. These findings emphasize the vital role of CD4+ TCR-alpha beta cells and IFN-gamma in resistance against S. typhimurium aroA-. Sublethal inocula of S. typhimurium aroA- led to permanent infection in H-21-A beta-/- mice, suggesting that bacterial starvation is insufficient for sterile clearance in immunocompetent mice and that MHC class II-dependent immune mechanisms are required for pathogen eradication. The TCR-beta-/- mice suffered from salmonellosis more severely than the MHC class II-deficient mutants, suggesting an auxiliary function of CD8+ T cells. Recombinant S. typhimurium aroA-, secreting listeriolysin (Hly) of Listeria monocytogenes, are capable of escaping from the phagosome into the cytosol of the host cell. However, the course of infection of these recombinant S. typhimurium SL7207 Hlys and control strains did not differ in beta 2m-/- mutants. This finding argues against direct correlation of cytosolic location of S. typhimurium SL7207 Hlys with CD8+ T cell dependency of protection. PMID- 8617958 TI - Antibodies to an autostimulatory growth factor (IL-2) or its receptor induce death of leukemogenic cells. AB - We show for the first time that Ab-mediated antagonism of growth factor activity can induce death of all cells in clonal populations surviving and growing by an autostimulatory mechanism. Models of autostimulatory leukemia were generated by transfecting a mouse IL-2-dependent cell line (FD.C/2) with vectors directing production of IL-2 by these cells. One series of clones grew in a density dependent manner in the absence of exogenous IL-2 and produced tumors in syngeneic mice. Although these clones released relatively small amounts of IL-2, their growth and survival was only partially inhibited by Abs to IL-2 or the IL 2R. Another autostimulatory clone was derived, using a different vector, which produced significantly less IL-2. Treatment of cells of this clone with Abs to IL 2 or its receptor resulted in death of all cells. These data demonstrate that Abs that antagonize growth factor action can induce the death of cells transformed by autostimulatory mechanisms. They suggest that while autostimulatory tumors are relatively resistant to Abs that block growth factor action, this is a quantitative phenomenon, and competitive antagonists of growth factor action of sufficiently high affinity may provide effective and specific adjuvants to the treatment of such tumors. PMID- 8617957 TI - CD40-mediated stimulation contributes to lymphocyte proliferation, antibody production, eosinophilia, and mastocytosis during an in vivo type 2 response, but is not required for T cell IL-4 production. AB - CD40/CD40 ligand interactions are required for the development of T cell dependent Ab responses in vivo. The role of these cell surface molecules in contributing to T cell cytokine production and the development of effector populations other than B cells and T cells is, however, less well defined. We have examined the in vivo effects of blocking CD40/CD40 ligand interactions on the type 2 mucosal immune response that follows oral inoculation of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of anti-gp39 (CD40L) mAb (MR1) blocked H. polygyrus-induced elevations in serum IgG1 levels and inhibited elevations in blood eosinophils and mucosal mast cells at day 14 after inoculation. Anti-gp39 mAb markedly inhibited B cell blastogenesis 8 days after H. polygyrus inoculation but did not inhibit elevations in B cell class II MHC expression. Maximal elevations in B7-2 expression required signaling through both CD40 and the IL-4R. Elevations in T cell cytokine gene expression and elevations in the number of IL-4-secreting cells were unaffected by treatment with anti-gp39 mAb, although IL-4 production was inhibited by anti-IL-4R mAb. These results suggest that CD40/CD40L interactions are not required to activate T cells to produce cytokines but are required for the activation and proliferation of other effector cells associated with the type 2 response. PMID- 8617960 TI - Identification and characterization of a C(K/R)TC motif as a common epitope present in all subtypes of hepatitis B surface antigen. AB - The a determinant of hepatitis B surface antigen (HBsAg) is the most critical determinant for both diagnosis and immunoprophylaxis of the hepatitis B virus. We have used synthetic peptides and an anti-a mAb to identify a peptide sequence corresponding to amino acid residues 117 to 128 of HBsAg as an antigenic epitope contributing to the a determinant. Compared to the native protein HBsAg, the cyclic form of the peptide (aa 117-128) is only 20-fold less effective, whereas the linear form of the peptide is 160-fold less effective in the inhibition of mAb binding to HBsAg. Based on these results, we have postulated a previously unidentified disulfide bond between residue Cysl21 and Cysl24. Individual substitution of amino acids in the peptide (aa 117-128) with alanine identified three residues Cys121, Thr123, and Cys124 as the most critical residues for mAb recognition. Substitution of alanine for any one of the three residues caused a substantial loss in binding free energy (greater than 4.5 kcal/mol). Sequence analysis indicated that the C(K/R)TC motif is highly conserved among 100 subtypes and mutants of HBsAg isolates. Collectively, these results show that the cyclic C(K/R)TC motif is an essential part of the a determinant of HBsAg. Synthetic peptides containing the C(K/R)TC motif are potentially useful as alternative hepatitis B vaccines and as diagnostic reagents for the detection of the hepatitis B virus. PMID- 8617959 TI - Resistance of naive mice to murine hepatitis virus strain 3 requires development of a Th1, but not a Th2, response, whereas pre-existing antibody partially protects against primary infection. AB - Murine hepatitis virus strain 3 (MHV-3) produces a strain-dependent spectrum of disease. The development of liver necrosis has been shown to be related to production of a unique macrophage procoagulant activity (PCA), encoded by the gene fgl-2, in susceptible mice. These studies were designed to examine the influence of Th1/Th2 cells on resistance/susceptibility and production of macrophage PCA in resistant (A/J) and susceptible (BALB/cJ) strains of mice following infection with MHV-3. Immunization of A/J mice with MHV-3 induced a Th1 cellular immune response, and one Th1 cell line (3E9.1) protected susceptible mice and inhibited PCA production by macrophages both in vitro and in vivo. In contrast, immunization of BALB/cJ mice with an attenuated variant of MHV-3 derived from passaging MHV-3 in YAC-1 cells resulted in a Th2 response. Transfer of spleen cells and T cell lines from immunized BALB/cJ mice failed to protect naive susceptible syngeneic mice from infection with MHV-3 and augmented macrophage PCA production to MHV-3 in vitro. However, serum from immunized BALB/cJ mice contained high titrated neutralizing Ab that protected naive BALB/cJ animals from lethal primary MHV-3 infection. These results demonstrate that susceptible BALB/cJ mice generate a Th2 response following MHV-3 infection and that these Th2 cells neither inhibit MHV-3-induced macrophage PCA production nor protect naive mice from MHV-3 infection. The results suggest that Ab protects against primary infection but cannot eradicate ongoing infection. Thus, these data define the differential role of Th1/Th2 lymphocytes in primary and secondary MHV-3 infection and emphasize the importance of PCA in the pathogenesis of MHV-3 infection. PMID- 8617962 TI - Liver NK1.1+ CD4+ alpha beta T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. AB - We demonstrate herein evidence that IL-12-activated alpha beta T cells with intermediate TCR (NK1+ TCRint cells) in the liver inhibit metastases in the lung as well as in the liver metastases of i-v. injected tumors. IL-12 administration enhanced NK1 expression of NK1+ TCRint cells (NK1high) and increased CD4 weakly positive (CD4low) TCRint cells, while both CD4+ TCRint cells and double-negative TCRint cells were proportionally diminished. Accordingly, the major parts of NK1high TCRint cells are CD4low cells, and most of these cells are V beta 8+ cells. The cytotoxic assays of IL-12-stimulated hepatic mononuclear cells after treatment with respective Abs and complement in vitro and after sorting revealed that CD4low NK1high TCRint cells are cytotoxic effectors. When IL-12-stimulated hepatic mononuclear cells (but not splenocytes) were transferred into tumor preinjected mice, EL-4 cell metastases in the liver as well as 3LL cell metastases in the lung were inhibited. The antimetastasis of hepatic mononuclear cells transfer was abrogated by the depletion of NK1+ cells, CD3+ cells, or CD4+ cells but not CD8+ cells before transfer. Moreover, transfer of these cells of nude mice into tumor-preinjected mice also inhibited metastases in both organs. Although NK1+ TCRint cells are nearly absent in the hepatic vein blood, a significant proportion of NK1high TCRint cells appeared by IL-12 administration. These results demonstrate that IL-12-stimulated liver NK1high TCRint cells, including extrathymic ones, are major effectors against tumor metastasis and suggest that the cells migrate and inhibit lung metastases. PMID- 8617961 TI - IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines: enhancement by simultaneous B7-1 expression. AB - A number of cytokines and costimulatory molecules involved in immune activation have recently been identified including IL-12, a heterodimeric cytokine that supports the development of cell-mediated immunity, and B7-1, a costimulatory molecule involved in the activation of T lymphocytes. We explored the use of these immunomodulants as molecularly defined adjuvants in the function of recombinant anticancer vaccines using a murine model adenocarcinoma, CT26, transduced with a model Ag, beta-galactosidase (beta-gal). Although IL-12 given alone to mice bearing tumors established for 3 days did not have consistent antitumor activity, a profound therapeutic effect was observed when IL-12 administration was combined with a recombinant vaccinia virus (rVV) encoding beta gal called VJS6. On the basis of the reported synergistic effects of IL-12 and the costimulatory molecule B7-1 (CD80) in vitro, we immunized mice with a double recombinant vaccinia encoding both the model tumor Ag and the costimulatory molecule B7-1, designated B7-1 beta-gal rVV. The adjuvant administration of IL-12 after immunization with this virus significantly enhanced survival in tumor bearing animals. T cell subset depletions demonstrated that the in vivo activity of IL-12 was largely independent of CD4+ T lymphocytes, whereas the in vivo activity of a B7-1 rVV required both CD4+ and CD8+ T cells to elicit maximal therapeutic effect. To our knowledge, this is the first description of B7-1 and IL-12 cooperation in vivo and represents a novel strategy to enhance the efficacy of recombinant anticancer vaccines. PMID- 8617963 TI - MHC class I-dependent presentation of exoerythrocytic antigens to CD8+ T lymphocytes is required for protective immunity against Plasmodium berghei. AB - T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2 microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, although immunization with attenuated SPZ induced production of IL-2, INF-gamma, anti-circumsporozoite protein IgG, and proliferative T cells. The lack of compensatory mechanisms involving non-CD8+ T cells was particularly evident in the failure to adoptively transfer protective immunity with wild-type SPZ-immune splenic T cells. From our data it can be concluded that CD8+ T cells induced during immunization with attenuated SPZ must recognize liver-expressed Ags presented by class I molecules to engage effectively in a response leading to destruction of the malaria parasites. PMID- 8617964 TI - Constitutive expression of B7-1 (CD80) on mouse keratinocytes does not prevent development of chemically induced skin papillomas and carcinomas. AB - Expression of the B7-1 (CD80) costimulatory molecule in a variety of tumor cell lines leads to an enhanced CD8+ T cell response to tumor Ags. We used transgenic mice constitutively expressing B7-1 on keratinocytes (K14/B7-1 line) to determine whether keratinocyte B7-1 expression would inhibit the development of papillomas and carcinomas following two-stage chemical carcinogenesis in skin. FVB inbred mice carrying the K14/B7-1 transgene and controls were initiated with 25 micrograms of 7,12-dimethylbenz[a]anthracene and promoted weekly with 5 micrograms of 12-O-tetradecanoylphorbol-13-acetate for 20 wk. Expression of the B7-1 transgene did not result in statistically significant decreases in the mean number of papillomas or carcinomas compared with controls. The incidence of carcinomas in both transgenic and control mice reached 90% or greater by 60 wk after initiation. Carcinoma cell lines established from the K14/B7-1 mice maintained expression of B7-1 and Kq. These B7-1 expressing carcinomas grew progressively following intradermal injection into syngeneic FVB mice, further demonstrating their inability to evoke protective tumor immunity. These same carcinoma cell lines were rapidly rejected by minor alloantigen-mismatched SWR mice, confirming their susceptibility to immune effector mechanisms. The failure of constitutive B7-1 expression on keratinocytes to prevent the growth of squamous cell papillomas and carcinomas may reflect the limited immunogenicity of tumors arising after initiation-promotion carcinogenesis. Our results in this transgenic model system are further evidence that B7-1 gene therapy alone may not be sufficient to induce protective immunity to some types of tumors. PMID- 8617966 TI - Human skeletal myoblasts spontaneously activate allogeneic complement but are resistant to killing. AB - The complement (C) system has previously been implicated in several diseases of muscle. We here report that human myoblasts or rhabdomyosarcoma cell lines spontaneously activate C through the classical pathway, causing release of anaphylatoxins and coating of myoblasts with opsonic C fragments but without causing cell killing. Survival of myoblasts is a consequence of the abundant expression of the membrane C regulatory molecules MCP and CD59, and neutralization of CD59 renders cells susceptible to C killing. The decay accelerating factor was expressed at a very low level. Myoblasts and rhabdomyosarcoma lines also abundantly express the fluid-phase regulators C1 inhibitor, factor H, C4 binding protein, S-protein, and clusterin and secrete a soluble form of CD59. Expression of membrane and fluid-phase regulators is enhanced by either IFN-gamma or TNF-alpha. Although myoblasts resist C killing, spontaneous activation of C on these cells may have important consequences in inflammatory diseases of muscle where the generation of anaphylactic and opsonic fragments will recruit and activate inflammatory cells. C activation on myoblasts may also have consequences for the use of these cells as vehicles for gene delivery. Inhibition of C using soluble complement receptor I (sCR1) efficiently protected myoblasts from C attack in vitro, and this agent, already being tested in therapy of several C-mediated diseases, might be of value in inflammatory muscle disease and in improving the efficiency of gene delivery. PMID- 8617965 TI - Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts. AB - The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium. PMID- 8617967 TI - Peptides derived from C-reactive protein inhibit neutrophil alveolitis. AB - C-reactive protein (CRP) is the classic acute phase reactant in humans, with serum levels elevated up to 1000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-, LTB4-, IL-8-, and FMLP-stimulated neutrophils in vitro, and rabbits and transgenic mice with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in models of chemotactic factor-induced alveolitis. To evaluate the mechanism of CRP inhibition on chemoattractant-induced neutrophil inflammation in vivo, experiments were performed in mice infused with peptides of human CRP shown to inhibit C5a- and FMLP-stimulated neutrophil chemotaxis in vitro. After direct tracheal instillation of FMLP, mice previously injected via the retro-orbital plexus with CRP peptide 77-82 or 201-206 showed significant reductions (up to 90%) of neutrophils in the bronchoalveolar lavage fluid compared with vehicle treated mice. Both CRP peptides also significantly (up to 55%) inhibited the increase in alveolar total protein levels. Control injections of native rabbit CRP (3 microM) inhibited neutrophil influx by 93% and protein leak by 55% in mice intratracheally instilled with FMLP. Despite similar levels of inhibition, approximately 10-fold more peptide by weight than native CRP was required. These data suggest that CRP degradation products at sites of tissue injury, in particular CRP peptides 77-82 and 201-206, are anti-inflammatory and can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following FMLP-induced inflammation. PMID- 8617968 TI - Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines. AB - Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells. PMID- 8617969 TI - Pathologic leukocyte infiltration of the rabbit aorta confers a vasomotor effect to chemotactic peptides through cyclooxygenase-derived metabolites. AB - We determined the effect of chemotactic peptides FMLP and C5a, postulated to be relatively selective activators of phagocytes, on the thoracic aorta of rabbits subjected to experimental pathologies that allowed infiltration by leukocytes, i.e., dietary atherosclerosis and serum sickness. Aortic ring tissues isolated from hypercholesterolemic rabbits, precontracted or not by phenylephrine, exhibited a rapid and relatively sustained (10 to 20 min) contractile response when challenged by FMLP (10 nM and above); precontracted tissues also responded to C5a (2.5 nM and above). Aortic rings from rabbits with serum sickness (13 days post-BSA injection) exhibited brief contractions that were often followed by a relaxation in phenylephrine-precontracted tissues. In both models, tissues from normal weight-matched animals were not consistently responsive to these peptides. The cyclooxygenase inhibitor indomethacin extensively reduced the contractile effect of either peptide on precontracted aortic rings in both models. Chemotactic peptide-induced increased prostanoid secretion was evident only in the fluid bathing atherosclerotic aortic rings. Morphologic correlations included the demonstration of cells positive for the RAM-11 macrophage marker and the C5a receptors in tissues from rabbits with hypercholesterolemia (numerous clusters of cells) or serum sickness (modest infiltration). Control aortic rings responded to FMLP by a significant contraction if cultured for 2 h in the presence of resident peritoneal cells (84% macrophages), but not in the presence of a high density of PBL (less than 0.5% monocytes). Infiltrating or adherent macrophages in the blood vessel wall confer to some phagocyte activating peptides the role of eicosanoid dependent vasoconstrictor agents. PMID- 8617970 TI - Inhibition of TNF-alpha expression by adenosine: role of A3 adenosine receptors. AB - Adenosine agonists inhibit TNF-alpha production in macrophage and monocytes, but the mechanism is unknown. Therefore, we studied the human macrophage cell line U937 to determine the adenosine receptor subtypes responsible and the intracellular signaling mechanisms involved. The A1/A3 agonist N6-(4-amino-3 iodobenzyl)adenosine (I-ABA) decreased LPS-stimulated TNF-alpha protein production by 79 +/- 5% (p = 0.003). The mechanism was pretranslational, as adenosine receptor stimulation caused a marked decrease in TNF-alpha mRNA. IL-1 beta, IL-6, and IL-8 mRNA were not changed by adenosine agonists. The rank order of agonists as TNF-alpha inhibitors suggested that the A3 receptor might be involved (N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-beta-D-ribofuranosyl] adenosine > 2-chloroadenosine > or = I-ABA > N6 benzyl 5'-N ethylcarboxamidoadenosine (NECA) > NECA > CGS21680 > N6-cyclohexyladenosine), and this was supported by the fact that a mixed A1/A3 antagonist (xanthine amine congener) reversed the effect, whereas A1-specific (1,3-dipropyl-8 cyclopentylxanthine) and A2-specific (3,7-dimethyl-1-propargylxanthine) antagonists did not. Receptor signaling did not involve cAMP or protein kinase A, nor did it alter the activation and binding characteristics of the transcription factor NF-kappa B. However, the composition of the AP-1 transcription complex was altered by I-ABA. These data suggest that stimulation of the A3 adenosine receptor can alter the cytokine milieu by decreasing TNF-alpha. Adenosine agonists or adenosine regulating agents have potential therapeutic uses in acute and chronic inflammatory diseases. PMID- 8617971 TI - Internalization of Kit together with stem cell factor on human fetal liver derived mast cells: new protein and RNA synthesis are required for reappearance of Kit. AB - Kit, the receptor for stem cell factor (SCF) and the product of the c-kit proto oncogene, is expressed on fetal liver-derived mast cell progenitors when cultured with SCF. Decreased levels of Kit on the surface of human fetal liver-derived mast cells after exposure to recombinant human SCF were demonstrated by flow cytometry using the YB5.B8 mAb against Kit. Internalization of Kit along with SCF appears to be the principal means by which Kit is lost from the mast cell surface. Neither the beta 3-integrin CD51/CD61 (alpha v beta 3), nor the beta 1 integrins CD49d,e/CD29 (VLA-4 and -5) appeared to be internalized along with Kit SCF complexes. Reappearance of Kit on day 28 fetal liver-derived mast cells is complete 3 days after exposure of the cells to SCF and is detectable by 2 days. Recovery requires new protein and new RNA synthesis, because Kit did not reappear if cycloheximide or actinomycin D was added to the cells. No substantial change in total Kit mRNA was detected during the resynthesis period, suggesting that post-transcriptional regulation of Kit production is involved. Internalization of Kit in mast cells exposed to soluble SCF may represent a negative regulatory mechanism for this receptor-ligand interaction and down-regulate mast cell properties such as degranulation to SCF. PMID- 8617972 TI - Heat shock mimics glucocorticoid effects on IFN-gamma-induced Fc gamma RI and Ia messenger RNA expression in mouse peritoneal macrophages. AB - Glucocorticoids activate or repress the expression of different genes. In murine macrophages, glucocorticoids exert opposing effects on the IFN-gamma-induced expression of Fc gamma RI and Ia mRNA and cell surface expression; they enhance IFN-gamma-induced Fc gamma RI mRNA and protein expression, yet inhibit IFN-gamma induced Ia mRNA and protein expression. Recently, in transfected cell lines, heat shock (HS) has been shown to promote nuclear accumulation of the glucocorticoid receptor (GR), resulting in potentiation of certain GR-mediated responses. In this study, we compared the effects of HS and dexamethasone (DEX) treatment on the IFN-gamma induction of Fc gamma RI and Ia mRNA in murine primary peritoneal macrophages. Our results show that HS exerted the same opposing effects on these IFN-gamma-responsive genes as DEX at 37 degrees C. The glucocorticoid antagonist RU 486 blocked both DEX and HS-induced enhancement of IFN-gamma induction of Fc gamma RI, suggesting a common GR-mediated mechanism. While RU 486 also reversed DEX-induced repression of Ia mRNA expression, supporting a GR-mediated action, it did not affect HS-mediated repression, raising the possibility of a ligand independent HS response pathway. PMID- 8617973 TI - A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis. AB - The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells. PMID- 8617974 TI - Decreased production of TGF-beta 1 by human alveolar macrophages compared with blood monocytes. AB - As the main immunocytes lining pulmonary alveoli, alveolar macrophages (AM) are critical to the maintenance of immune hemostasis of the lung. This study examined the capacity of AM obtained from healthy individuals in comparison with autologous blood monocytes (MN) to produce transforming growth factor-beta 1 (TGF beta), a pivotal molecule in regulation of immune responses and in promotion of fibrosis. AM produced negligible TGF-beta in response to LPS at both 24 and 72 h of culture. In contrast, LPS induced significant levels of TGF-beta in MN cultures (79.5 +/- 35 pg/ml in AM vs 890 +/- 162 pg/ml in MN, p less than 0.001, at 24 h). AM also produced significantly less TGF-beta than MN in response to phorbol ester and Con A. By northern blot analysis, constitutive expression of TGF-beta mRNA was lower in AM than MN at the time of isolation and after 24 h of culture. Lower expression of steady state TGF-beta message was not due to a more rapid decay of its mRNA in AM. Furthermore, TGF-beta mRNA expression was up regulated by rTGF-beta in MN but was not induced in AM. In contrast to TGF-beta, LPS-stimulated AM produced sixfold higher levels of TGF-alpha at 24 h than MN (p less than 0.01). Production of IL-10 by LPS-stimulated AM was sixfold lower than MN (p less than 0.005) at 24 h of culture, but was comparable with MN at 72 h. Both 10-day cultured monocytes and peritoneal macrophages also had reduced capacity to produce TGF-beta. Therefore, the inability to produce TGF-beta may be a feature of more differentiated mononuclear phagocytes. In health, the reduced expression of TGF-beta by AM and the intact ability to produce TGF-alpha and IL 10 may favor a timely and regulated host response to inhaled pathogens while limiting potentially deleterious inflammatory responses. PMID- 8617975 TI - Fas mediates apoptosis in human monocytes by a reactive oxygen intermediate dependent pathway. AB - Monocyte apoptosis has emerged as a central regulatory event in hemopoiesis and inflammation. Inflammatory cytokines can either promote or prevent monocyte apoptosis. To study the possible role of Fas Ag, a member of the TNF/nerve growth factor receptor family, in monocyte apoptosis, human peripheral blood monocytes activated by IL-1 beta or TNF-alpha were exposed to anti-Fas mAb. Engagement of the Fas Ag resulted in apoptosis of monocytes, as monitored by propidium iodide uptake, decrease in cell size, DNA fragmentation, and characteristic ultrastructural changes. The apoptotic action of Fas was abolished completely by antioxidants such as N-acetylcysteine and glutathione, suggesting a role for reactive oxygen intermediates (ROI) in the death process. Consistent with this observation, Fas stimulation enhanced the fluorescence associated with oxidation of 2',7'-dichlorofluorescein, indicating increased levels of intracellular ROI. Moreover, the exogenous addition of hydrogen peroxide or menadione, an intracellular generator of superoxide anion, was sufficient for the induction of monocyte apoptosis. These data indicate that ROI are key mediators of Fas-induced apoptosis. In contrast to IL-1 beta and TNF-alpha, LPS-treated monocytes were resistant to the apoptotic action of Fas. Under these conditions, LPS did not down-regulate Fas, but inhibited the Fas-dependent elevation of ROI. Therefore, monocytes appear to have a protective mechanism that can interfere directly with the Fas-induced pathway of cell suicide, thereby controlling their destiny. PMID- 8617976 TI - Persistence of dominant T cell clones in synovial tissues during rheumatoid arthritis. AB - In a previous study, we showed that the T cell repertoire is biased in the synovial membrane (SM) compared with peripheral blood during rheumatoid arthritis (RA). The same bias was observed in different joints from the same patient and seems to be the same over time. To discover whether this bias was due to expansion of a clonal subset resulting from activation by conventional Ag(s) or to polygonal stimulation by superantigen(s), we sequenced more than 650 TCRBV-D-J junctional regions from freshly isolated SM and peripheral blood of two DR4-RA patients. From each patient, two SM were obtained on the same day, and a third was obtained later. Several dominant clones were found in SM but not in peripheral blood. Some of them were found only at the first time point in anatomically different SM, the majority persisted over time, and others were detected only at the second time point. Analysis of the complementarity determining region 3 (CDR3) showed a bias in TCRBD and amino acid usage. Valine, encoded by randomly inserted N nucleotides, was used by 45% of dominant clones compared with 18% in the control population (p less than 0.001). In addition, GXXG and TSG motifs were frequently observed in the CDR3 of these dominant clones. These data indicate a dynamic TCR selection process during the perpetuation phase of RA. The dynamic changes of dominant clones also suggest a determinant spreading mechanism during RA. PMID- 8617977 TI - Lack of IL-2 cytokine expression despite Il-2 messenger RNA transcription in tumor-infiltrating lymphocytes in primary human breast carcinoma: selective expression of early activation markers. AB - Tumor-infiltrating lymphocytes (TIL) are found in most human infiltrating ductal breast carcinomas. In studies of other tumors, TIL were capable of activation by IL-2, both in vitro and in vivo, to produce selective tumor cytolysis. Specific TIL-mediated tumor cytolysis in human breast tumors has recently been reported. The large numbers of TIL within human breast cancers imply that an immune response is occurring, since many of these cells express HLA class II as a late activation marker. However, the degree of early activation of the native TIL in breast tumors has not been fully investigated. Early activation markers CD69, CD43, and CD38 together with the IL-2R (CD25) and IL-2 cytokine were examined using mAbs and tissue section immunohistology. In situ hybridization was used to detect IL-2 mRNA (IL-2 mRNA) in parallel with immunohistochemical localization of IL-2. The results revealed the expression of CD69, CD43, and CD38, but markedly low CD25 (IL-2R) and IL-2 protein expression by the TIL. This strongly indicates that the TIL are an activated population of T cells that shows a deficiency in IL 2 protein and IL-2R expression despite adequate levels of IL-2 mRNA. The mechanism for apparent inhibition of IL-2 production and IL-2R expression in the presence of IL-2 mRNA is currently unclear; however, this may explain the relative anergic state of native TIL. PMID- 8617978 TI - Restricted V beta usage by T cells infiltrating rejecting human lung allografts. AB - TCR expression was evaluated in lung transplant patients to determine whether T cells infiltrating rejecting lung allografts employed restricted V beta elements. Serial bronchoalveolar lavage (BAL) specimens were obtained from six lung transplant recipients at approximately 3 wk, 6 wk, and 3 mo post-transplant. T cell lines were established by culturing lavage cells with irradiated donor splenocytes in the presence of low dose IL-2 for 3 wk, and TCR V beta usage was determined by quantitative reverse transcriptase-PCR. Patients were grouped into three categories based on TCR V beta profiles and the clinical status of the allograft. 1) In one patient, BAL-derived T cells expressed heterogeneous V beta repertoires at all time points evaluated. This patient did not experience graft rejection during the 16-mo period of observation, though respiratory infections were diagnosed. 2) In three patients, V beta usage by BAL-derived T cells was restricted during allograft rejection episodes, but was heterogeneous in the absence of rejection and during respiratory infections. In one of these patients, similar V beta repertoires were employed by BAL cells during multiple rejection episodes. 3) In two patients, restricted V beta usage by BAL-derived T cells was observed before and during rejection episodes. Collectively, these data illustrate that human lung allograft rejection, but not pulmonary infection, is associated with T cells expressing a limited number of V beta families. Restricted V beta usage by graft-reactive T cells may allow for the selective elimination of these cells using TCR-specific reagents, thereby promoting allograft-specific tolerance. PMID- 8617979 TI - Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells. AB - A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells. PMID- 8617980 TI - Programmed cell death in peripheral lymphocytes from HIV-infected persons: increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression. AB - We analyzed the potential causes of the increased susceptibility to apoptosis of peripheral lymphocytes from a large cohort of HIV-infected persons that we followed during a 3-yr period. By using quantitative cytofluorometric methods, we demonstrate that all lymphocyte populations were contributing proportionally to the apoptotic population in both groups of donors, but the percentage of T and B cells involved in this cell death process was significantly increased in HIV infected persons. To study the relationship between the increased apoptosis in HIV infection and the activation state of the immune system, we analyzed cell surface expression of activation markers on apoptotic and nonapoptotic cells. We found that in the chronic phase of HIV infection, 50 to 60% of the apoptotic cells exhibited an activated phenotype (they were HLA-DR+, CD38+, CD45RO+, and Fas+), and interestingly, the CD45RO+ subset appeared to be more prone to apoptosis in HIV-positive persons. This study also indicates that the activated phenotype found on apoptotic cells was not a distinctive feature of patients' lymphocytes since it was in similar proportion in apoptotic cells from control lymphocytes. However, a significant correlation was found between the intensity of anti-CD3-induced apoptosis in both CD4 and CD8 T cells from HIV-infected persons and their in vivo expression of CD45RO and HLA-DR molecules. Finally, a significant correlation was found between the intensity of spontaneous or anti CD3-induced apoptosis in total lymphocytes and disease progression; this was confirmed when the CD4 and CD8 T cell subsets were analyzed separately. Altogether these observations indicate that the increased susceptibility to apoptosis of peripheral T cells from HIV-infected persons correlates with disease progression and support the hypothesis that the chronic activation of the immune system occurring throughout HIV infection is the primary mechanism responsible for this cell deletion process. PMID- 8617981 TI - Regulation of cytokine production during development of autoimmune diabetes induced with multiple low doses of streptozotocin. AB - Cytokines have been shown to play an important role in regulating tolerance to islet Ags and provoking destructive islet lesions. However, data from a number of experimental systems have been conflicting, and the role of cytokines produced by T lymphocytes at various stages of diabetes has not been clearly defined. We have studied the production of cytokines in the pancreas during the development of autoimmune diabetes induced in mice by administration of (5) low doses of streptozotocin (STZ) (MDSDM). Diabetes in this model is T lymphocyte dependent. We used techniques of semiquantitative PCR to identify and quantitate cytokines that are produced. We have found that IL-2, IL-4, TNF-alpha, and IFN-gamma are expressed by the time the fourth dose of STZ is given. In the same pancreas, all of these cytokines (including IL-4) may be found. However, expression of IFN gamma, but not IL-4, was limited to intrapancreatic lymphocytes and was not detectable at extrapancreatic lymphoid sites. Moreover, mAbs against IFN-gamma, but not against IL-4 or IL-2, prevent hyperglycemia and insulitis in MDSDM, suggesting that IFN-gamma regulates development of disease. Cells in the pancreases of nondiabetic mice treated with anti-IFN-gamma mAb and STZ show enhanced expression of IL-4, but the prevention of disease is due to blockade of the IFN-gamma itself, and not due to secretion of IL-4, because systemic administration of IL-4 does not prevent MDSDM. Thus, our findings indicate that cytokines produced by Th1 (or T cytolytic 1) and Th2 (or T cytolytic 2) cells are found in the pancreases of mice developing autoimmune diabetes. IFN-gamma is responsible for progression to diabetes, and its production is limited to lymphocytes only at that site. PMID- 8617982 TI - Streptococcal M protein peptide with similarity to myosin induces CD4+ T cell dependent myocarditis in MRL/++ mice and induces partial tolerance against coxsakieviral myocarditis. AB - Immunologic similarities have been demonstrated between Coxsackievirus B3 (CVB3), group A streptococcal M protein, and cardiac myosin. Previous studies have also shown that T lymphocytes obtained from CVB3-infected mice expressing the H-2k MHC haplotype gave an immunodominant proliferative response to the NT4 peptide (GLKTENEGLKTENEGLKTE) of the streptococcal M5 protein. We now show that the NT4 peptide can induce inflammatory heart disease in MRL/++ (H-2k) mice and that induction of anergy to this peptide protects against CVB3-induced myocarditis. MRL/++ mice infected with CVB3 for 7 days or immunized twice at 7-day intervals with the streptococcal NT4 peptide in CFA developed myocarditis. Treatment of the immunized mice with either anti-CD4 or anti-IAk mAbs inhibited cardiac inflammation. Injection of MRL/++ mice with NT4 covalently coupled to syngeneic splenocytes tolerized the animals to this peptide as shown by reduction of the proliferative response. NT4-tolerized mice had significantly reduced myocarditis, although virus titers in the heart were elevated. A control peptide, VP1-10 from the CVB3 capsid protein VP1, did not protect the mice from CVB3-induced myocarditis. The results suggest that immunity to NT4 induced during CVB3 infections is important to the development of cardiac inflammation. PMID- 8617984 TI - Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated. AB - The selective destruction by an autoimmune process of the beta cells in the pancreas is the hallmark of the type 1 insulin-dependent diabetes mellitus. What triggers islet cell-specific autoreactive T and B cells, however, remains unclear. Identification of the targets of the anti-islet cell autoantibodies frequently found in insulin-dependent diabetes mellitus patients and analysis of their sequences should provide some insights into the nature of this disease. We have combined EBV transformation with CD40 activation of peripheral B cells from one patient with insulin-dependent diabetes mellitus to isolate four B cell clones that secrete islet cell-specific autoantibodies, These four human monoclonal autoantibodies are of the IgG1 isotype, and they each recognize a different epitope of the glutamic acid decarboxylase enzyme. Analysis of their variable gene sequences shows that, while clonally unrelated, three of the four human monoclonal autoantibodies use a member of the VH4 family, and two have rearranged the same delta light chain variable gene. The IgG1 isotype of the four autoantibodies as well as the presence of somatic mutations in both heavy and light chain genes provide concrete evidence for their derivation by a T cell dependent immune response. PMID- 8617983 TI - Rapid induction of autoantibodies by endogenous ovarian antigens and activated T cells: implication in autoimmune disease pathogenesis and B cell tolerance. AB - Animals immunized with nuclear antigenic peptides produce autoantibodies to distant antigenic sites and neighboring Ags within a multimolecular complex. This has led to the hypothesis that induction of autoantibodies in systemic autoimmune diseases might be triggered by a T cell epitope. We have investigated the T to B epitope spreading phenomenon based on the murine autoimmune oophoritis model. Mice immunized with a ZP3 T cell peptide spontaneously produced amplified autoantibodies (amAb) against linear ZP3 B cell epitopes outside the peptide immunogen. Each ZP3 B cell peptide, chimerized to a foreign promiscuous T cell epitope, elicited Ab to the peptide within the native ZP3 molecule. Mice with amAb often had no oophoritis; but more importantly, bilateral ovariectomy 1 day before ZP3 T epitope injection inhibited the induction of the amAb response, whereas ovariectomy 2 to 4 days after immunization was not inhibitory. Because endogenous ovarian Ag depletion before detectable ZP3 T cell response (day 5) and oophoritis (day 7) failed to prevent the amAb response, the autoantibodies are likely stimulated by endogenous ZP3 Ags present outside the normal ovaries. AmAb, of only the IgG class, appeared on day 7; this was 2 to 3 days after detectable T cell response, and 5 to 6 days before A response to the T cell peptide immunogen. The rapid, class-switched amAb response indicates that B cells in female mice are not tolerant to self ovarian Ag and they may normally be primed by ZP3. As evidence for their pathogenic potentials, amAb were produced in response to oophoritogenic, nonovarian T cell peptides that mimic ZP3; moreover, an excellent correlation existed between amAb titers and fertility reduction. PMID- 8617985 TI - Blockade of platelet endothelial cell adhesion molecule-1 protects against myocardial ischemia and reperfusion injury in cats. AB - Polymorphonuclear leukocytes have been shown to play an important role in myocardial ischemia (MI) and reperfusion (R) injury. Since blockade of platelet endothelial cell adhesion molecules (PECAM-1) inhibits neutrophil transmigration in vitro and in vivo, the effects of a polygonal Ab directed against PECAM-1 were examined in a feline model of Ml/R. We established cross-reactivity of our anti human PECAM-1 Ab to cat coronary vasculature and neutrophils by immunohistochemistry and flow cytometry. The anti-PECAM-1 Ab markedly blocked leukocyte transmigration into the peritoneal cavity of cats after glycogen induced peritonitis. Then, anti-PECAM-1 Ab (1 mg/kg) was tested to determine whether it attenuates MI/R injury in a well characterized feline model of Ml and R. Anti-PECAM-1 Ab administered 10 min before R significantly inhibited the myocardial necrosis seen 4.5 h post-R compared with that in MI/R cats treated with control isotype rabbit IgG (12 +/- 2 vs 29 +/- 4% of area at risk; p less than 0.01) and significantly attenuated the rise in plasma creatine kinase activity (p less than 0.05). The Ab did not prevent increases in cardiac myeloperoxidase activity within the affected regions and did not significantly inhibit autologous neutrophil adhesion to coronary endothelium after stimulation of either neutrophils (by leukotriene B4) or coronary endothelium (by thrombin) in vitro. These results indicate that in vivo blockade of PECAM-1 significantly attenuates MI/R injury, presumably by inhibiting transendothelial migration of neutrophils. PMID- 8617986 TI - Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy. AB - Gene therapy offers a novel approach to treating human joint diseases such as rheumatoid arthritis. In the present study, we have used the retrovirus, MFG IRAP, to transfer the human IL-1 receptor antagonist protein (IRAP) gene to rabbits' knees and have assessed its impact on inflammatory and chondrodestructive aspects of the acute phase of antigen-induced arthritis in these joints. Surprisingly, intra-articular expression of IRAP was three- to fivefold higher in arthritic knees than in nonarthritic knees, accumulating to levels of over 20 ng/knee in the highest expressing joints. This level of expression produced a marked chondroprotective effect but a milder anti inflammatory one. Both the increased cartilage matrix catabolism and the inhibition of matrix synthesis that occur in antigen-induced arthritis were abrogated in the presence of the IRAP gene; the latter effect was particularly strong. Of the indices of inflammation that were examined, only leukocyte influx into the joint space was inhibited, and this effect declined with time. Concentrations of rabbit IL-1 were reduced by the IRAP gene, suggesting inhibition of an autocrine induction loop. These data demonstrate that the course of arthritic disease in the rabbit knee can be altered by genetic manipulation, thus encouraging the further development of gene treatments for human joint diseases. PMID- 8617987 TI - Human CD4-internal antigen anti-idiotypic monoclonal antibody: induction of a CD4 specific response in humans. AB - We previously showed that anti-idiotypic mAb (mAb2) F16-16D7 (16D7) to the paratope (or paratope-related idiotope) of the anti-CD4 mAb HP2/6 induces anti CD4 Abs in BALB/c mice. In view of the potential ability of 16D7 to induce anti CD4 Ab in humans and the potential benefits of anti-CD4 Abs in the treatment of autoimmune diseases, we evaluated the immunologic response to and assessed the safety of four 2-mg 16D7 s.c. injections in one patient with systemic lupus erythematosus (SLE) and one with rheumatoid arthritis (RA). 16D7 induced anti isotypic and anti-anti-idiotypic Abs (Ab3), which were almost exclusively of the IgG isotype. Ab3 specifically reacted with 16D7 as they inhibited its binding to mAb HP2/6. Although Ab3 did not react with cellular or recombinant CD4 (rCD4), single-cell enzyme-linked immunospot assays of anti-CD4 Ab production revealed many more spot-forming cells in rCD4- and 16D7-coated wells than in wells coated with BSA or 16D7 isotype-matched MK2-23. Spot-forming anti-CD4 Abs were specifically induced by 16D7, since rCD4-dependent spot formation 1) was not observed with PBL from one patient with SLE, one with mixed connective tissue disease, and one with melanoma immunized with MK2-23; and 2) was inhibited by 16D7 and not by MK2-23. Spot-forming anti-CD4 Abs recognize a CD4 epitope identical (or closely related) to that seen by HP2/6, since this specifically inhibited spot formation. A substantial, although transient, CD4+ T cell depletion was only observed in the RA patient. Local and/or general toxicity and laboratory and/or clinical signs indicative of immunodepression or diseases relapse were not observed during an 18-month follow-up. PMID- 8617988 TI - Rheumatoid factor idiotypic and antigenic specificity is strongly influenced by the light chain VJ junction. AB - The aim of this study was to define the structural basis for rheumatoid factor (RF) specificity and for the expression of the RF light chain-associated Ids, 4C9 and 6B6.6, by determining the reactivity of recombined heavy and light chains of Ig derived from monoclonal B cell lines of patients with rheumatoid arthritis and of light chains with site-directed mutations. We found that expression of the 4C9 and 6B6.6 Ids resulted from use of the VkIIIa genes Humkv 328 and Vg, but only in the presence of a permissive VJ junction. Expression of the Ids was independent of heavy chain use for the Humkv328-encoded light chains, but was highly dependent on the associated heavy chain for the Vg-encoded light chains. The RF specificity of the Abs was primarily heavy chain dependent, but the light chain VJ junction was critical in determining the relative avidity of the Abs for Fc. Our study points to the critical contribution of the somatically generated VJ junction to RF autoantibody specificity and to the expression of the two RF associated Ids studied. PMID- 8617989 TI - Can immunomodulatory molecules work topically for psoriasis? PMID- 8617990 TI - Enhanced modulation of keratinocyte motility by transforming growth factor-alpha (TGF-alpha) relative to epidermal growth factor (EGF). AB - Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are high affinity polypeptide ligands for the EGF receptor, which mediates their biologic activities. In this study, we directly compared the actions of both ligands in promoting keratinocyte motility. We found that normal and tumorigenic human keratinocytes responded to activation of the EGF receptor by either EGF or TGF alpha; however, the two ligands did not elicit identical responses with regard to cell locomotion. TGF-alpha was more effective than EGF at promoting colony dispersion (cell scattering), in vitro wound closure, and single-cell migration as assessed by phagokinetic track analysis. In contrast, EGF and TGF-alpha evoked identical profiles for DNA synthesis with regard to concentration dependence and magnitude of response in normal keratinocytes and in a squamous cell carcinoma line. The overall pattern of tyrosine phosphorylation of intracellular substrates was similar when cells were stimulated with either growth factor; however, a limited number of differences in the kinetics or magnitude of protein phosphorylation were detected in subcellular fractions. These findings demonstrate that two growth factors implicated in promoting mitogenesis and locomotion may elicit divergent responses with regard to one biologic activity while retaining similar responses for other activities. This suggests that ligand mediated mitogenic responses may not be tightly coupled to motogenic activity and further illustrates the multifunctional roles of polypeptide growth factors. PMID- 8617991 TI - Hair cycle-dependent changes in skin immune functions: anagen-associated depression of sensitization for contact hypersensitivity in mice. AB - To assess whether hair follicle cycling influences skin immunity, we examined the association between highly synchronized hair follicle cycling and experimental contact hypersensitivity in C57BL/6 mice. Hair cycle synchronization was performed by depilation of hair shafts on the back with telogen skin. Mice were sensitized on the lower back skin with picryl chloride between 0 and 25 d, after anagen induction by depilation, and challenged on the earlobes with picryl chloride 5 d later. The magnitude of contact hypersensitivity was significantly decreased in mice sensitized on day 1, was minimal on day 3 (early anagen), and slowly increased thereafter, reaching level comparable to day 0 on day 25 (telogen). The significantly depressed contact hypersensitivity response in anagen skin was confirmed in mice with spontaneously developed follicles. Lymph node cells taken from mice sensitized with picryl chloride on days 0, 1, and 3 after depilation were cultured in vitro in the presence of syngeneic, haptenized, Langerhans cell-enriched epidermal cells. Marked proliferative responses of lymph node cells to haptenized cells were found in mice not only of day 0, but also of days 1 and 3, suggesting that immune T cells exist even lymph node cells of the low-responsive mice. Flow cytometric analyses demonstrated that the number of intraepidermal Langerhans cells and their functions, including the expression of major histocompatibility complex class II, CD54, and CD86, and mixed epidermal cell lymphocyte reactions, were not changed in skin on days 0,1, and 3. These findings demonstrated that contact hypersensitivity is induced most effectively via skin with telogen hair follicles and that the depressed response in early anagen skin is not simply due to failure in Langerhans cell function or sensitization of T cells. PMID- 8617992 TI - Characterization and subcellular localization of human Pmel 17/silver, a 110-kDa (pre)melanosomal membrane protein associated with 5,6,-dihydroxyindole-2 carboxylic acid (DHICA) converting activity. AB - Pmel 17 is preferentially expressed in pigment cells in a manner suggestive of involvement in melanin biosynthesis. The gene is identical to the silver (si) pigmentation locus in mice. We now produced a recombinant glutathione-S transferase-human Pmel 17 infusion protein and raised polyclonal antibodies against it to confirm the ultrastructural location and presumed site of action predicted by the deduced primary structure of Pmel 17/silver, and to authenticate the specificity of the DHICA converting function as inherent to the silver-locus protein. Full-length Pmel 17 cDNA also produced in insect cells in a baculovirus expression vector to ensure that activity did not originate from a co precipitated protein. Natural hPmel 17 from human melanoma cells has an approximate molecular size of 100 kDa. By immunoperoxidase electron microscopic cytochemistry, the antigen was localized to the limiting membranes of premelanosomes and presumed premelanogenic cytosolic vesicles and, to a minor extent, in the premelanosomal matrix. In an in vitro assay, both the natural and the recombinant Pmel 17 accelerated the conversion of DHICA to melanin. This activity was inhibited by the anti-Pmel 17 polyclonal antibodies, indicating that the acceleration of DHICA conversion by natural protein is genuine and cannot be due to contaminating complexed proteins. We suggest that in situ Pmel 17/silver is a component of a postulated premelanosomal/melanosomal complex of membrane bound melanogenic oxidoreductive enzymes and cofactors, in analogy to the electron transfer chain in mitochondria. PMID- 8617993 TI - E-selectin binds to squamous cell carcinoma and keratinocyte cell lines. AB - E-selectin is an endothelial adhesion molecule that binds carbohydrate epitopes on leukocytes and has been implicated in a potential pathway of tumor metastasis. Keratinocyte cell lines express similar carbohydrate epitopes, one of which, sialyl Lewis X (SL-X) is a ligand for E-selectin and is also expressed by squamous cell carcinomas (SCC) in situ. The functional role of keratinocyte selectin ligands was investigated using a soluble E-selectin chimaeric protein (pE-sel-Ig) containing pig lectin-like and epidermal growth factor-like domains fused to human IgG. After incubation of keratinocyte cell lines (A431 and SVK14) and normal keratinocytes with pE-sel Ig, binding was quantified by flow cytometry. Frozen sections of SCC were overlaid with pE-sel Ig and binding was visualized immunoenzymatically. Immunolabeling was undertaken using monoclonal antibodies (CSLEX-1 and HECA-452), which label E-selectin ligands including sialyl Lewis X. E-selectin bound strongly to A431 and SVK14 cells; the degree of binding paralleled staining intensity with CSLEX-1 antibody. HECA-452 antibody stained A431 cells strongly but SVK14 cells only weakly. Normal keratinocytes and normal epidermis did not express CSLEX-1 or HECA-452 antigens or bind E-selectin. Serial sections of SCC revealed close correlation between fusion protein binding and antibody staining. Antibody pretreatment of tumor sections with CSLEX-1 blocked fusion protein binding, whereas HECA-452 antibody only slightly reduced fusion protein binding. pE-sel Ig pretreated with YT11.1 antibody failed to bind to A431 or SVK14 cells or to SCC. These studies provide functional evidence that SL-X/E-selectin pathways may be important in SCC metastasis and that A431 and SVK14 cells provide a good model to investigate these mechanisms. PMID- 8617994 TI - Wound fluid-derived heparin-binding EGF-like growth factor (HB-EGF) is synergistic with insulin-like growth factor-I for Balb/MK keratinocyte proliferation. AB - Epidermal cell proliferation is required for re-epithelialization during wound repair. Re-epithelialization of partial thickness excisional wounds in pigs is complete by 6 days after injury. The presence of insulin-like growth factor-I (IGF-I) and heparin-binding molecules that are mitogenic for keratinocytes was examined in wound fluid obtained daily from these wounds. Two significant heparin binding growth factor activities for Balb/MK keratinocytes were detected, a major one that was eluted from a heparin affinity column with 1.1 M NaCl and a minor one with 0.5 M NaCl. These activities appeared 1 day after injury, were maximal by 2-3 days later, and disappeared by 6 days after injury. The molecule eluting with 1.1 M NaCl was heparin-binding EGF-like (HB-EGF). The levels of IGF-I in wound fluid were 45-90 ng/ml during the first 3 days following injury, decreased thereafter, and were not detectable 6 days after injury. IGF-I at 100 ng/ml, increased HB-EGF mitogenic activity for Balb/MK keratinocytes by 40-50 fold. We conclude that the synergism between IGF-I and HB-EGF and their relative concentration at the various days after injury may be important variables for regulating re-epithelialization during wound repair. PMID- 8617995 TI - Differential expression of urokinase-type plasminogen activator, its receptor, and inhibitors in mouse skin after exposure to a tumor-promoting phorbol ester. AB - The cellular distribution of mRNAS for urokinase-type plasminogen activator (uPA), its specific receptor (uPAR), and its inhibitors (PAI-1 and -2) in mouse skin was analyzed by in situ hybridization after topical application of the tumor promoter phorbol 12-myristate 13-acetate. In the epidermis, strong signals for uPA and PAI-1 mRNA were detected 24 h after treatment in the basal and suprabasal epidermal keratinocytes in areas with pronounced hyperproliferation and increased terminal differentiation, and in some hair follicle keratinocytes. After 48 h, both uPAR and PAI-2 mRNAs were expressed in the epidermal layers from the suprabasal keratinocytes up to the differentiating cells beneath the cornified layer and in hair follicle keratinocytes. Induction of PAI-2 mRNA was detected in epidermis as early as 3 h after treatment and remained stable for up to 7 days. In the dermis, 5 h after application of phorbol 12-myristate 13-acetate to the skin, uPA mRNA was detected in fibroblast-like cells below and around the skin muscle, and PAI-1 mRNA was detected in stromal cells located above the skin muscle. After longer exposure to phorbol 12-myristate 13-acetate, the PAI-1 mRNA expressing stromal cells were located more superficially, apparently moving toward the epidermal layer. After 9 h, most of the PAI-1 mRNA-positive cells were identified as endothelial cells. Up to 24 h after the application of phorbol 12 myristate 13-acetate, the intensity of the signal for both uPA and PAI-1 increased, followed by a gradual decrease for up to 7 days. These results show that in mouse skin treated with a tumor-promoting phorbol ester, the various components of the plasminogen activation system are expressed by both epithelial and stromal cell types, which in dermis and subcutis are located in different places, depending on the time of exposure to the phorbol ester. Our results suggest that urokinase-mediated extracellular proteolysis has diverse functional roles during the early steps of tumor promotion. PMID- 8617996 TI - Collagenase gene expression in cutis laxa fibroblasts is upregulated by transcriptional activation of the promoter gene through a 12-0-tetradecanoyl phorbol-13-acetate (TPA)-responsive element. AB - Our previous work demonstrated that collagenase mRNA levels are increased in fibroblasts derived from patients with cutis laxa (CL). To pursue the mechanism of the upregulation of collagenase expression, we investigated transcriptional levels of the collagenase gene in CL fibroblasts. Fibroblasts cultured from the skin of three congenital CL patients were studied. Northern blot hybridization revealed 2.8- to 7.3-fold increases in collagenase mRNA levels in CL fibroblasts compared with normal cells. Nuclear run-off experiments demonstrated that the transcription rate of the collagenase gene in nuclei isolated from the same cells was 5.1- to 10.2-fold higher in the CL fibroblasts than in the controls. Transient transfection of a normal collagenase promoter-CAT construct into the cells further showed significantly enhanced transcriptional activity in CL but not in normal fibroblasts. Experiments of transient transfection of deleted or small substituted collagenase promoter-CAT constructs indicated that collagenase transcription in CL fibroblasts was activated the TPA-responsive element site of the collagenase promoter gene. Although the levels of Jun and Fos gene expression did not differ from those observed in normal fibroblasts, AP-1-binding activity, as measured by the ability to bind to an oligonucleotide containing a TPA responsive element, was significantly elevated in CL fibroblasts as compared with normal fibroblasts. These data suggest that collagenase expression is upregulated at the transcriptional level by endogenous activation of DNA binding of AP-1 in CL fibroblasts [corrected]. PMID- 8617998 TI - Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules. AB - Outwardly directed ionic currents have been measured leaving skin wounds in vivo. These currents generate physiologic electric fields of approximately 100 mV/mm, which may function to direct keratinocyte migration toward the healing wound. We investigated whether the substrate on which the keratinocyte migrates modulates the galvanotactic response to an electric migratory signal. Cultured human keratinocytes were plated on different matrices; types I and IV collagen, fibronectin, laminin, and tissue culture plastic. The effect of an applied direct current (DC) electric field on directional migration was monitored by time-lapse video microscopy over a 2-h period. Directionality was quantitated by calculating the cosine of the angle of migration in relation to anodal-cathodal orientation. Migration toward the negative pole was observed on all matrices as compared with controls (no applied field), which displayed random migration. No significant increase in directional response occurred when the field strength was increased by 100 mV/mm (physiologic levels) to 400 mV/mm. The degree of directionality and the average net cell translocation however, varied significantly with the substrate. The greatest cathodal migration in response to a DC electric field was observed with keratinocytes plated on types I and IV collagens and plastic. The directional migratory response was least on a laminin substrate, whereas cells on fibronectin demonstrated a response that was intermediate between those of collagen and laminin. These results suggest that physiologic ionic currents in concert with underlying matrix may influence the rate of reepithelialization of skin wounds. PMID- 8617997 TI - Induction of vitamin D receptor mRNA expression in psoriatic plaques correlates with clinical response to 1,25-dihydroxyvitamin D3. AB - Psoriasis is a skin disorder characterized by hyperproliferation of epidermal keratinocytes. 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and its analogs have been shown to inhibit keratinocyte proliferation in vitro and to be therapeutically effective for the treatment of psoriasis. Some patients with psoriasis, however, do not have a favorable response to 1 alpha,25 (OH)2D3 therapy. To evaluate the differential responsiveness to 1 alpha (OH)2D3 treatment, we examined the expression of vitamin D receptor mRNA in psoriatic lesions by reverse transcription-polymerase chain reaction using glyceraldehyde-3 phosphate dehydrogenase as an internal control. In this double-blind clinical trial, we recruited 18 patients who received topical treatment of 1 alpha,25(OH)2D3 (15 microgram/g Vaseline) or placebo on separated psoriatic lesions for 8 weeks. In patients who showed >90% clinical improvements of their psoriatic lesions with 1 alpha,25(OH)2D3 (n=9), an increase of 130+/-37% in vitamin D receptor mRNA level was observed in 1 alpha,25(OH)2D3-treated lesions when compared with the corresponding placebo controls. There was no increase in vitamin D receptor mRNA level in the lesions treated with this drug in patients who did not respond to the treatment. These data suggest that the antiproliferative activity of 1 alpha,25(OH)2D3 is closely associated with the expression of its cognate receptor. PMID- 8617999 TI - Localization and expression of cornifin-alpha/SPRR1 in mouse epidermis, anagen hair follicles, and skin neoplasms. AB - Recently, cornifin-alpha/SPPR1 has been identified as a putative precursor protein of the cornified cell envelope. In this study, the expression and localization of cornifin-alpha/SPPR1 was examined in untreated and tumor promoter treated mouse skin, hair follicles, and skin neoplasms. Western analysis with antiserum (SQ37A) to a rabbit cornifin-alpha peptide or antiserum (SQ37C) to a human SPRR1 peptide demonstrated a 31-kDa immunoreactive protein in mouse epidermis and Northern analysis revealed the presence of a 1-kb mRNA. Immunohistochemical staining of mouse skin with SQ37A or SQ37C revealed intense and specific staining of the infundibulum, isthmus, and of Henle's layer of the inner root sheath of the lower anagen hair follicle and weak staining of the telogen follicle and the suprabasal layers of the epidermis. Treatment of mouse skin with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) produced a large increase in cornifin-alpha/SPRR1 protein and mRNA. Immunohistochemical localization of cornifin-alpha/SPRR1 in TPA-treated skin indicated that cornifin-alpha/SPRR1 was increased in the suprabasal epidermis but not in the follicle. sn-1,2, didecanoylglycerol, a model lipid second messenger, produced an increase in cornifin-alpha/SPRR1 protein similar to that of TPA, while mirex, a non-phorbol ester-type promoter had no effect. Topical doses of retinoic acid did not repress TPA-induced cornifin-alpha/SPRR1 expression. Papillomas demonstrated a 10- and 100-fold increase in cornifin-alpha/SPRR1 protein and mRNA, and expression was restricted to suprabasal cells. Squamous cell carcinomas exhibited an intermediate level of cornifin-alpha protein, and expression was restricted to keratinized areas. These data indicate: i) cornifin-alpha/SPRR1 is expressed in mouse skin; ii) cornifin-alpha/SPRR1 is localized to specific areas of the anagen hair follicle with weak staining in the telogen follicle and epidermis; iii) epidermal cornifin-alpha/SPRR1 expression is induced by phorbol ester and sn-1,2 didecanoylglycerol but not mirex, and iv) papillomas and squamous cell carcinomas demonstrate a constitutive increase in cornifin-alpha/SPRR1 in differentiated areas of the neoplasms. PMID- 8618001 TI - Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation. AB - RANTES, interleukin-8 (IL-8), and macrophage inflammatory protein-1-alpha (MIP-1 alpha) exhibit different and highly selective chemotactic activity for leukocytes. Resting cultured normal oral and skin keratinocytes produced little if any of these chemokines. Stimulation with 250-1,000 U/ml of tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) induced both cell types to produce RANTES. Protein levels peaked after 48 h and mRNA levels peaked after 8 h of stimulation. Used combination, TNF-alpha, and IFN-gamma synergistically increased mRNA and protein levels. Amounts of 100-1,000 U/ml of TNF-alpha also induced IL-8 production with peak mRNA levels after 4-24 h of stimulation and maximal protein production after 72 h or more. IL-8 production by oral keratinocytes was significantly greater than that by skin keratinocytes. Although IFN-gamma alone did not induce IL-8 production, it enhanced the effect of TNF alpha on both cell types. Stimulation for 24 h with 100-1,000 U/ml of IL-alpha also induced IL-8 production by oral but not skin keratinocytes. No MIP-1 alpha production was detected under the conditions investigated. Keratinocyte production of RANTES and IL-8, under the influence of cytokines such as TNF-alpha or IFN-gamma, provides a mechanism for the selective accumulation of leukocytes into immunoinflammatory diseases of the skin and oral mucosa. Differences in their production may help to explain differences in the presentation of these diseases on the skin and oral mucosa. PMID- 8618000 TI - Neuropeptides induce release of nitric oxide from human dermal microvascular endothelial cells. AB - Nitric oxide is a potent mediator of endothelium-dependent vasodilation, the synthesis of which is catalyzed by the constitutively expressed enzyme endothelial nitric oxide synthase. In this study we have investigated whether human dermal microvascular endothelial cells express endothelial oxide synthase and whether the vasodilator neuropeptides, calcitonin gene-related peptide and substance P, stimulate the release of nitric oxide from these cells. Endothelial nitric oxide synthase was identified by immunohistochemistry in the blood vessels in both the papillary and deep dermis of normal skin, and also in monolayers of human dermal microvascular extracts prepared from both the dermis of normal human skin and human dermal microvascular endothelial cells, a 135-kDa band corresponding to endothelial nitric oxide synthase was identified. Nitric oxide was released from unstimulated human dermal microvascular endothelial cells as assessed by inhibition of platelet aggregation and nitrate formation. Endothelial cell-mediated inhibition of platelet aggregation was blocked by hemoglobin. Calcitonin gene-related peptide, (100 pM to 100 nM) directly inhibited platelet aggregation, and this direct effect was not modulated by microvascular endothelial cells. Substance P (10 nM to 1 muM) and calcitonin gene-related peptide (100 pM to 10 nM) significantly (p<0.05) increased nitrite formation, and this increase was blocked by the competitive nitric oxide synthase antagonist, NG monomethyl-L-arginine. These results demonstrate that endothelial nitric oxide synthase is expressed in the microvascular endothelium of normal human skin and that human dermal microvascular endothelial cells release nitric oxide constitutively and in response to vasodilator neuropeptides. PMID- 8618002 TI - Localization of human T-cell lymphotropic virus-1 tax proviral sequences in skin biopsies of patients with mycosis fungoides by in situ polymerase chain reaction. AB - The histopathologic diagnosis of mycosis fungoides (MF), even when clinical manifestations of the disease seem convincing, is often tenuous. The observation that practically all patients with MF harbor human T cell lymphotropic virus type I (HTLV-I) proviral sequences in their circulating lymphocytes raised the possibility that such viral footprints could be detected in their cutaneous infiltrates. Application of in situ polymerase chain reaction (PCR) to skin biopsies of 11 of 12 patients demonstrated this assumption to be correct. In addition, cells suspected to be keratinocytes were also positive. None of 10 skin biopsies from a variety of sources used as controls, nor 3 lymph node biopsies from patients with B-cell lymphomas, showed any HTLV proviral sequences on in situ PCR. On the basis of these observations, it is concluded that in situ PCR carried out on skin biopsies of patients with presumptive MF may help to established the diagnosis. PMID- 8618003 TI - Proopiomelanocortin gene product regulation in keratinocytes. AB - Proopiomelanocortin (POMC) is the precursor for adrenocorticotropic hormone, melanocyte-stimulating hormones, beta-lipotropic hormone (beta LPH), and beta endorphin. These peptides can function as neurotransmitters, modulate immune responses, and affect melanogenesis. We investigated POMC expression and protein processing in normal human keratinocytes. On Northern blot analysis, the baseline expression of the 1.2-kb POMC transcript was upregulated by ultraviolet radiation (UVR) or by stimulation with interleukin-1 alpha (IL-1 alpha) or phorbol 12 tetradecanoate 13-acetate (TPA). On Western blot analysis, POMC, beta LPH, and beta-endorphin were detected in cell extracts under baseline conditions. beta LPH level increased substantially after UVR, IL-1 alpha, or TPA. Within 36 h after TPA stimulation, beta-endorphin became undetectable in cell extracts, coinciding with an increase of beta-endorphin-immunoreactive protein in the culture medium. Our data establish that keratinocytes synthesize POMC protein as well as its derivatives beta LPH and beta-endorphin, and that this process is modulated by TPA, IL-1A, and UVR. beta LPH and beta-endorphin of keratinocyte origin may thus be involved in melanogenesis and/or immunomodulation in the skin after sun exposure, and their release into the circulation may also have systemic effects. PMID- 8618004 TI - A common insertion mutation in COL7A1 in two Italian families with recessive dystrophic epidermolysis bullosa. AB - Recessive dystrophic epidermis bullosa is ultrastructurally characterized by the absence of anchoring fibrils, and genetic analyses have revealed that recessive dystrophic epidermolysis bullosa results from mutations in the type VII collagen gene (COL7A1). The mutations disclosed thus far are largely family specific, with no evidence for mutational hotspot(s). In this study, we report a recurrent premature termination codon mutation detected in two apparently unrelated Italian families in different regions of the country. This mutation, 497insA in exon 4 of COL7A1, was found in combination with two different premature termination codon mutations in these families. Haplotype analysis suggested a shared genetic background in the allele containing the mutation 497insA, suggesting that this genetic lesion may represent an ancestral mutation within the Italian gene pool. PMID- 8618005 TI - Detection of low-level tumor cells in allergic contact dermatitis induced by mechlorethamine in patients with mycosis fungoides. AB - Two patients with histologically proven mycosis fungoides, a malignancy of phenotypically mature T cells, received a topical challenge with mechlorethamine to areas of clinically uninvolved skin to exclude possible hypersensitivity reactions to this chemotherapeutic agent. In both patients, allergic contact dermatitis (ACD) developed at the sites of the application and resolved completely after withdrawal of mechlorethamine. The lesions were biopsied and analyzed for the presence of clonal T-cell receptor (TCR)-gamma gene rearrangements using two polymerase chain reaction (PCR)-based assays involving denaturing gradient gel electrophoresis (PCR/DGGE) and ribonuclease protection analysis (PCR/RPA). The former method has a clonal detection threshold of 10(-3) 10(-2), while the latter has a sensitivity of 10(-5). In both cases, the ACD lesions were polyclonal by PCR/DGGE. In contrast, PCR/RPA detected tumor-specific TCR-gamma gene rearrangements in these same lesions. This indicates that the ACD lesions contained tumor cells at a density within the 10(-5)-10(-2) range. Analysis of peripheral blood mononuclear cells from both patients failed to detect the malignant clone and showed the same result as blood from four normal individuals. The normal skin from one skin patient also lacked detectable TCR gamma gene rearrangements. These results indicate that mycosis fungoides tumor are present within ACD lesions induced in mycosis fungoides patients and that this phenomenon does not appear to be due to the ubiquitous presence of detectable levels of these tumor cells in the blood or skin. These findings might be explained by nonspecific recruitment of malignant T cells to sites of local inflammation mediated by non-neoplastic antigen-specific T cells. Alternatively, they might be due to the local proliferation of very rare tumor cells in apparently normal skin in response to cytokines generated during the ACD reaction. In either case, the present study offers evidence that the malignant cells in myosis fungoides retain at least some capability of responding in vivo to physiologic stimuli. PMID- 8618006 TI - Expression of full-length desmosomol glycoproteins (desmocollins) is not sufficient to confer strong adhesion on transfected L929 cells. AB - Desmocollins are cadherin-like glycoproteins that are localized in desmosomes. They are thought to play a role in cell adhesion but direct evidence for this is currently unavailable. For this reason we have expressed cDNAs encoding full length bovine desomocollin type 1a and type 1b in mouse fibroblast (L929) cells. This system has previously been used to demonstrate the adhesive properties of E cadherin. E-cadherin-mediated cell-cell adhesion is thought to require interaction of the cytoplasmic domain with the catenins that are expressed in L cells. Because L929 cells do not express cytoplasmic desmosomal components that may be required for desmocollin-mediated adhesion, we constructed a chimeric cDNA encoding the bovine type 1 extracellular domain linked to the mouse E-cadherin transmembrane and cytoplasmic domains. cDNAs were transfected into cells and clones that expressed heterologous protein at the cell surface were isolated. The full-length desmocollins apparently did not interact with any other molecules, but the chimeric protein did bind to endogenous mouse alpha- and beta-catenin. Surprisingly none of the desmocollin-transfected cell lines showed significant adhesive properties under conditions where cells transfected with E-cadherin exhibited strong adhesiveness. We conclude that desmcollin expression alone is not sufficient to confer adhesion on transfected cells and more than one desmosomal component may be required. PMID- 8618007 TI - The dominant T cell clone is present in multiple regressing skin lesions and associated T cell lymphomas of patients with lymphomatoid papulosis. AB - This study was undertaken to determine the clonality of lymphomatoid papulosis (LyP), its clonal relationship to lymphomas, which occur at high frequency in LyP patients, and to define the cell lineage of Reed-Sternberg-like cells in type A lesions of LyP. Punch biopsies of skin of 11 adult patients with LyP were analyzed for morphologic subtype of LyP, surface antigens, and clonal T-cell receptor (TCR) gene rearrangements. Clonal rearrangements were identified by semiquantitative polymerase chain reaction amplification and sequencing of TCR beta chain genes in nine patients and TCR-gamma chain genes in two patients. A single dominant clone was detected in multiple separate LyP lesions, often of different histologies, in nine patients. The same clone was detected in LyP lesions and the anaplastic large cell lymphoma (ALCL) of 2 patients and the mycosis fungoides (MF) of 2 other patients. No dominant clone could be detected in one patient with LyP uncomplicated by lymphoma or in a second patient with LyP and MF. A T-cell lineage was evident for RS-like cells in cell culture and in type A lesions. These results show that multiple regressing skin lesions and associated T cell lymphomas (MF and ALCL) are clonally related in most LyP patients, which suggest that the disease in these patients was initiated by a non random genetic event. PMID- 8618008 TI - Clearing of psoriasis by a novel immunosuppressive macrolide. AB - Accumulating evidence suggests that psoriasis may be a genetically determined immunogenic, inflammatory disorder based on an ongoing autoreactive Th-1 response. Systemic immunosuppressive therapy is highly effective but fraught with longterm side effects. Our research therefore focuses on therapeutic strategies that induce local immunosuppression in the skin by topical, transepidermal delivery of immunosuppressive drugs. SDZ 281-240 is a newly developed macrolide of the ascomycin type. It is immunosuppressive by mechanism of action similar to that of FK506 but has no antiproliferative activity against keratinocytes in vitro. To evaluate whether SDZ 281-240 exhibits antipsoriatic activity when applied topically, we tested 15 patients with severe, recalcitrant psoriasis, using a microplaque assay in randomized, double-blind, placebo-controlled study, comparing the therapeutic efficacy of the macrolide with a potent halogenated corticosteroid and vehicle. All patients showed a significant improvement of psoriatic lesions treated with two concentrations of the macrolide and, as expected, with the corticosteroid but not with placebo. Both concentrations of the macrolide led to clearing of psoriasis after 10 days of treatment and biopsies confirmed a reversal of the histopathological and immunopathological phenotype of psoriasis to that of normal skin. Thus, an immunosuppressive agent that interferes with early T cell activation can be designed to penetrate into psoriatic lesions when applied topically and to be functionally active within the skin to suppress the ongoing psoriatic process. PMID- 8618009 TI - Familial juvenile onset psoriasis is associated with the human leukocyte antigen (HLA) class I side of the extended haplotype Cw6-B57-DRB1*0701-DQA1*0201 DQB1*0303: a population- and family-based study. AB - To further evaluate the nature of the HLA association with psoriasis, HLA haplotypes of 60 patients with type 1 (early onset, positive family history) and 30 patients with type II (late onset, no family history) psoriasis were investigated by polymerase chain reaction sequence-specific oligonucleotide hybridization (HLA class II) and serology (HLA class I). Ethnically matched blood donors (146) served as controls. In type I, but not type II psoriasis, the Caucasian HLA extended haplotype (EH) Cw6-B57-DRB1*0701-DQA1*0201-DQB1*0303 named according to the B allele EH-57.1 was highly significantly overrepresented (p cor= 0.00021). This particular EH was present in 35% of type I psoriatics but only 2% of controls. EH-57.1+ individuals therefore carry a 26 times higher risk of developing type I psoriasis than individuals who are EH-57.1-negative Further analysis of individual HLA alleles revealed that within EH-57.1, HLA class I antigens (Cw6-B57) were associated to a much higher extent with type I psoriasis than the HLA class II alleles (DRB1*0701-DQA1*0201-DQB1* 0303). Pedigree analysis of three multiply affected families over three generations revealed a cosegregation of disease with EH-57.1. These results strongly suggest that a gene for familial psoriasis is associated with the class I side of the extended haplotype Cw6-B57-DRB1*0701-DQA1*0201-DQB1*0303. PMID- 8618010 TI - Ultraviolet B irradiation-enhanced interleukin (IL)-6 production and mRNA expression are mediated by IL-1 alpha in cultured human keratinocytes. AB - Ultraviolet (UV) B radiation may trigger cutaneous inflammatory responses by directly inducing epidermal keratinocytes to elaborate specific cytokines such as interleukin (IL-1) and IL-6. Because IL-1 is a potent inducer of IL-6, one may speculate that the release of IL-6 by keratinocytes after UV exposure is mediated via the release of IL-1 in an autocrine or paracrine manner. We demonstrated that UVB irradiation upregulated IL-1 alpha mRNA at a lower dose (15 mJ/cm2) and then downregulated IL-1 alpha mRNA expression at high doses (30-40 mJ/cm2). The kinetic profile of IL-1alpha mRNA expression showed a biphasic response, with the early increase by 1 h after UV exposure and the secondary increase at 6 h after UV. On the other hand, the expression of IL-6 mRNA was increased with increasing doses of UVB (0-45 m/J/cm2) and showed a single peak at 6 h post UV. These results may indicate that UVB radiation could regulate the expression of IL 1alpha and IL-6 mRNA in keratinocytes by different mechanisms. Our data show that anti-human IL-1alpha antibody inhibits UV-induced IL-6 production and mRNA expression in cultured keratinocytes. The addition of recombinant IL-1alpha to the medium increased IL-6 synthesis and augmented IL-6 production and mRNA expression in cultured human keratinocytes by UVB irradiation. These results support the hypothesis that UVB irradiation-enhanced IL-6 production and mRNA expression may be mediated by IL-1alpha. PMID- 8618011 TI - Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line. AB - Although it is known that sunlight is carcinogenic,few molecular data are available concerning the mutagenic effects of ultraviolet (UV) B (290-320 nm) and UVA (320-400 nm) radiation in human cells. To analyze the biologic effects of UVA and UVB, we irradiated the 293 human cell line, derived from adenovirus transformed human embryonic kidney cells, in which we had stably introduced a shuttle vector harboring the lacZ' bacterial gene as the mutagenesis target. Identical cell survival occurred after UVA doses 700-fold higher than UVB. This comparable to the UVA/UVB ratio that reaches the basal cell layer of the skin after sunlight exposure with UVB sunscreen. The frequency of UVA- and UVB- induced mutations increased with the UV dose as cell survival decreased. At cell survival levels greater than 10%, UVA and UVB induced similar frequencies of mutations in the episomal lacZ gene, whereas for cell survival lower than 10%, UVA induced twice as many mutations as UVB. Sequence analysis of 81 independent lacZ mutants (36 UVA- and 45 UVB-induced) revealed specific characteristics for some UV-induced-mutations, particularly for UVB. Mutations at A/T base pairs were induced more frequently by UVA than by UVB. The UVA-induced mutation spectrum that we have observed in human cells may help help to elucidate the mechanism of skin carcinogenesis. PMID- 8618012 TI - Connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid, and other fibrotic skin disorders. AB - Connective tissue growth factor (CTGF) is a novel peptide that exhibits platelet derived growth factor-like activities and is produced by skin fibroblasts after activation with transforming growth factor-beta. Coordinate expression of transforming growth factor-beta followed by CTGF during wound repair suggests a cascade process for control of tissue regeneration. We recently reported a significant correlation between CTGF mRNA expression and histologic sclerosis in systemic sclerosis. To confirm the relation between CTGF and skin fibrosis, we investigated CTGF gene expression in tissue expression in tissue sections from patients with localized scleroderma, keloid, other sclerotic skin disorders using nonradioactive in situ hybridization. In localized scleroderma, the fibroblasts with positive signals for CTGF mRNA were scattered throughout the sclerotic lesions with no preferential distribution around the inflammatory cells or perivascular regions, whereas the adjacent nonaffected dermis was negative for CTGF mRNA. In keloid tissue, the fibroblasts positive for CTGF mRNA were diffusely distributed, especially in the peripheral expanding lesions. In scar tissue, however, the fibroblasts in the fibrotic lesions showed partially positive signals for CTGF mRNA. In eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture, CTGF mRNA was also expressed partially in the fibroblasts of the fibrotic lesions. Our findings reinforce a correlation between CTGF gene expression and skin sclerosis and support the hypothesis that transforming growth factor-beta plays an important role in the pathogenesis of fibrosis, as it is the only inducer for CTGF identified to date. PMID- 8618013 TI - LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells. AB - This study characterizes a novel basement membrane component that is the target of autoantibodies in patients with linear IgA bullous dermatosis. Tissue surveys showed that this protein localized to the epidermal side of 1 M NaCl split skin and to basement membranes in cornea, oral mucosa, esophagus, intestine, kidney collecting ducts, ureter, bladder, urethra, and thymus, but was absent in lung, blood vessels, skeletal muscle, and nerve. Monoclonal antibody 123, which recognizes this protein, induced dermal-epidermal separation of human skin in situ, and this protein was found, by immunoelectron microscopy, to localize exclusively to anchoring filaments. This protein was secreted as as a 120-kDa peptide from primary cultures of keratinocytes as determined by radioimmunoprecipitation. Monoclonal antibody 123 recognized this protein as a 120-kDa band from conditioned cell culture medium and a 97-kDa band from human skin extracts as shown by immunoblot. Serum from five patients with the autoimmune blistering disorder linear IgA bullous dermatosis specifically recognized bands of 120 and 97 kDa from culture medium and skin extracts, respectively, that were of identical electrophoretic migration to the bands recognized by monoclonal antibody 123. In summary, this study characterizes a novel anchoring filament protein that is the target of linear IgA bullous dermatosis autoantibodies. Because monoclonal antibody 123 induces blistering of human skin, we hypothesize that this protein functions to maintain dermal epidermal cohesion and that autoantibodies in this disease are themselves pathogenic. We propose LAD-1 as the name for this protein. PMID- 8618014 TI - 97-kDa linear IgA bullous dermatosis (LAD) antigen localizes to the lamina lucida of the epidermal basement membrane. AB - Linear IgA bullous dermatosis (LAD) is an autoimmune blistering disease in which IgA autoantibodies develop against the epidermal basement membrane zone. Target antigens of the circulating autoantibodies are thought to be heterogeneous, and their ultrastructural localization has not been fully elucidated. Previous studies with immunoblotting have demonstrated that the 97-kDa autoantigen is detected most frequently in patients' sera and is thought to be a major LAD antigen. Although a recent report suggests that the 97-kDa antigen localized to the hemidesmosomal plaques and the adjacent lamina lucida, discrepancies still exist among previous immunoelectron microscopic findings. To identify the precise localization of the 97-kDa LAD antigen, we used two different low-temperature immunoelectron microscopic techniques. For immunolabeling, we selected five LAD sera that had a high titer of autoantibodies against the 97-kDa LAD antigen. A post-embedding method with cryofixation and freeze substitution failed to immunolabel the 97-kDa LAD antigen. Cryoultramicrotomy with immunoelectron microscopy succeeded in preserving the antigenicity of the 97-kDa LAD antigen. In all cases, the majority of labeling occurred in the lamina lucida beneath the hemidesmosomes. No specific labeling was observed in the hemidesmosomal attachment plaques or the lamina densa or sublamina densa region, including anchoring fibrils. These results indicate that the 97-kDa LAD antigen is a component of the lamina lucida. PMID- 8618015 TI - Retroviral infection with human tyrosinase-related protein-1 (TRP-1) cDNA upregulates tyrosinase activity and melanin synthesis in a TRP-1-deficient melanoma cell line. AB - A human tyrosinase-related protein-1 (TRP-1) cDNA was inserted into the retroviral vector, pBAbe-puro. Sense and anti-sense constructs were identified and transfected, as well as vector-alone, into a retrovirus packaging cell line by a liposome-mediated technique and used in turn to infect a human melanoma line deficient in TRP-1 protein/transcript. Polymerase chain reaction (PCR) amplification of genomic DNA from these infectants, using TRP-1 cDNA-specific primers, demonstrate that PCR products were only identified from the sense- and anti-sense-infected clones, not from the parental cells or vector-alone infectants. Northern analysis demonstrated that TRP-1 sense and antisense infectants produced TRP-1 cDNA-related transcripts. Immunoblotting analysis with TA99 (a monoclonal antibody for TRP-1) demonstrated a single band of normal molecular weight from melanoma cells infected with sense cDNA, not from cells infected with sense cDNA, not from cells infected with anti-sense or vector alone, or from the uninfected-parental melanoma cells. The quantitative and qualitative analysis of melanin in the sense and anti-sense infectant cells demonstrated an increase and decrease in pigmentation, respectively, compared with vector alone. Tyrosine hydroxylase and DOPA oxidase activities of tyrosinase hydroxylase and DOPA oxidase activities of tyrosinase were both increased in sense cDNA infected cells plus unaltered or slightly decreased, respectively, in anti-sense cDNA-infected cells compared with control cells. Immunoblotting analysis with anti-tyrosinase antibody (alpha Ty-SP) demonstrated the amount of tyrosinase was slightly increased in TRP-1 overexpressing cells but slightly decreased in anti-sense infectant cells. We have demonstrated that the expression of exogenous TRP-1 cDNA melanoma cells stimulated the activity of tyrosinase and promoted melanogenesis, indicating that TRP-1 plays a role in regulating tyrosinase activity. PMID- 8618016 TI - Assignment of psoriasin to human chromosomal band 1q21: coordinate overexpression of clustered genes in psoriasis. AB - Psoriasin is an abundant low molecular weight protein in keratinocytes from psoriatic lesions. Because of similarities in gene structure and expression to other genes on human chromosomal band 1q21, we hypothesized that psoriasin might also map to this region. To test this hypothesis, we identified and used a genomic lambda clone (lambda 9.2) as a probe for fluorescent in situ hybridization. lambda 9.2 detected the 1q21 region in 81% of 52 chromosomes 1 examined, although it also hybridized to acrocentric chromosomes. lambda 9.2 DNA yielded polymerase chain reaction amplification of 121-bp sequence colinear with psoriasin cDNA, as did genomic DNA from hybrid cell lines containing all or part of chromosome 1. The psoriasin gene was present on a 380-kb yeast artificial chromosome clone that was previously mapped to 1q21 and shown to contain calcyclin; here it is also shown to contain MRP8 and CaN19. Psoriasin and several other tightly linked 1q21 genes were markedly overexpressed in psoriatic lesions, suggesting a role for these clustered genes in the regulation of epidermal proliferation. PMID- 8618017 TI - Detection of telomerase activity in malignant and nonmalignant skin conditions. AB - Telomeres are the end regions of linear chromosomes, and in normal somatic cells the lengths of telomeres shorten with successive cell divisions. Telomerase, a ribonucleoprotein enzyme, maintains the length of telomeres in immortal and germline cells. Although present in human fetal tissues, shortly after birth telomerase activity is not detectable except in germline cells, hematopoietic cells, and most human primary tumors. In the present study we show telomerase activity to be present in 73 of 77 basal cell carcinomas, 15 of 18 nonmetastatic cutaneous squamous cell carcinomas, and 6 of 7 cutaneous melanomas, contrasting with extremely low levels detected in sun-protected skin. Sun-damaged skin, psoriatic lesional skin, and skin from lesions of poison ivy dermatitis, however, have increased levels of telomerase activity compared to sun-protected skin, although less than that detected in tumor tissue. Because telomerase activity can be found in inflammatory lesions of the skin, this indicates that telomerase activity does not always correlate with the malignant phenotype. In addition, we show that telomerase activity is localized to the epidermis of newborn foreskin, which suggests that telomerase is expressed constitutively by cells in the epidermis. Finding higher levels of telomerase activity in sun-exposed skin compared to nonexposed skin suggests that environmental factors may modulate telomerase activity. PMID- 8618018 TI - Influence of the second COL7A1 mutation in determining the phenotypic severity of recessive dystrophic epidermolysis bullosa. AB - The dystrophic forms of epidermolysis bullosa (DEB) are characterized by fragility of the skin and mucous membranes. The ultrastructural hallmark of DEB is abnormalities in the anchoring fibrils. A recessively inherited variant, the mitis type of DEB (M-RDEB), is characterized by a mild phenotype, including the absence of mutilating scarring of the hands and feet. In this study, we demonstrate that M-RDEB results from the combination of a premature termination codon mutation in one COL7A1 allele, while other mutation consists of different types of genetic lesions. These results define M-RDEB as a distinct clinical entity at the molecular level. PMID- 8618019 TI - A homozygous deletion mutation in the gene encoding the 180-kDa bullous pemphigoid antigen (BPAG2) in a family with generalized atrophic benign epidermolysis bullosa. AB - The 180-kDa bullous pemphigoid antigen (BPAG2) is a candidate gene/protein for mutations in some forms of junctional epidermolysis bullosa. In this study, we searched for mutations in BPAG2 in a large Austrian pedigree with generalized atrophic benign epidermolysis bullosa, a distinct nonlethal form of junctional epidermolysis bullosa, using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis of the polymerase chain reaction products, and direct nucleotide sequencing. We identified a homozygous 2-bp deletion within the coding region of BPAG2 in the affected individuals. This mutation results in a frameshift and downstream stop codons on both alleles, predicting an absence of functional protein. These findings illustrate the molecular basis of the skin fragility in this family and attest to the importance of the 180-kDa bullous pemphigoid antigen in the attachment of the epidermis to the underlying dermoepidermal basement membrane. PMID- 8618020 TI - Compound heterozygosity for nonsense and missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa. AB - Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DNA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5. PMID- 8618021 TI - Genetic basis of Bart's syndrome: a glycine substitution mutation in type VII collagen gene. AB - Bart's syndrome was initially described as a genodermatosis characterized by congenital localized absence of the skin, together with blistering and nail abnormalities. Recent analysis of Bart's original kindred demonstrated ultrastructural abnormalities in the anchoring fibrils and linkage of the inheritance of the disease to the region of chromosome 3 near the type VII collagen gene (COL7A1). We have performed mutation analysis in this family by using electrophoretic heteroduplex analysis followed by direct nucleotide sequencing of DNA. These results disclosed a G-to-A transition within exon 73 of COL7A1, which results in a glycine-to-arginine substitution within the triple helical domain of type VII collagen in affected individuals. In this family, these findings demonstrate that Bart's syndrome is a clinical variant of dominant dystrophic epidermolysis bullosa. PMID- 8618022 TI - A recurrent homozygous nonsense mutation within the LAMA3 gene as a cause of Herlitz junctional epidermolysis bullosa in patients of Pakistani ancestry: evidence for a founder effect. AB - The anchoring filament protein laminin 5 is abnormally expressed in the skin of patients with Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a H-JEB child of first-cousin Pakistani parents, and identified a homozygous C-to-T transition in the LAMA3 gene of laminin 5 resulting in a premature termination codon (CGA-TGA) on both alleles. This mutation, R650X, has been previously reported in two other seemingly unrelated H-JEB individuals of Pakistani ancestry. Although this mutation may represent a mutational hotspot within the LAMA3 gene, haplotype analysis based on a silent intragenic polymorphism (GCC/GCG, alanine 429; GenBank no. L34155), and on three flanking microsatellite polymorphism (D18S45, D18S478, and D18S480), suggests that a common ancestral allele may be present in all three cases. PMID- 8618023 TI - Histamine in human epidermal cells is induced by ultraviolet light injury. AB - Human epidermal cell cultures were examined to determine whether they were capable of histamine release. Results of these studies indicated that keratinocytes contain and release significant amounts of histamine. In the skin of some individuals, histamine content was induced after ultraviolet B light injury, and 40% of subjects demonstrated high basal histamine levels. Mass spectrometric analysis of cell supernatants showed that the histamine was released into the extracellular environment. Such release may contribute to common itching or intensify the inflammatory response in vivo. PMID- 8618024 TI - Absence of down-regulation and translocation of the eta isoform of protein kinase C in normal human keratinocytes. AB - Among 11 isoforms of protein kinase C (PKC), we previously reported that the eta isoform of PKC plays a crucial role in mediating differentiation of keratinocytes. Activation of PKC is associated with its intracellular translocation from the cytoplasm to the plasma membrane, followed by down regulation through proteolytic cleavage of the PKC molecules. In the present study, we demonstrated that the eta isoform of PKC is unique in that it is not translocated nor down-regulated upon stimulation. The level of the eta isoform, assayed by immunoblotting, remained unchanged during the first 12 h and then increased slightly up to 24 h when treated with tumor promoters or activators of PKC in constitutively expressing normal human keratinocytes. The activity of the eta isoform also remained unchanged after the 12-O-tetradecanoyl-phorbol-13 acetate treatment, as judged by binding ATP analog, autophosphorylation, and phosphorylation of an exogenous substrate. The alpha isoform of PKC, however, was rapidly down-regulated and was undetectable by 6 h after the treatment. These observations were further confirmed by immunohistochemical staining of normal human keratinocytes and transiently expressing COS1 cells. In addition, although the alpha isoform rapidly translocated to the plasma membrane, the eta isoform remained in the cytoplasm. PMID- 8618025 TI - Linkage of monilethrix to the trichocyte and epithelial keratin gene cluster on 12q11-q13. AB - Monilethrix is characterized by beaded or moniliform hair, which results from the periodic thinning of the hair shaft. The beaded hair thus produced is subject to excess weathering and premature fracturing at the internodes. Clinically, monilethrix presents with short, fragile, broken hair. The follicular abnormalities range from subtle perifollicular abnormalities range from subtle perifollicular erythema and hyperkeratosis to horny follicular papule formation. At the ultrastructural level, cytolysis and keratin tonofilament clumping (epidermolysis) are seen in the cortical cells of the bulb of the hair follicle. Microsatellite markers flanking the keratin gene clusters at 17q12-q21 and 12q11 q13 were used to perform linkage analysis in a monilethrix pedigree. This study demonstrates linkage of monilethrix in a pedigree to microsatellite DNA loci mapping to the region on chromosome 12 containing the type II keratin cluster. A major group of structural hair proteins, the basic type II trichocyte keratins, map within this epithelial cytokeratin gene cluster. This study implicates a mutation in a trichocyte keratin gene in the pathogenesis of a structural hair disorder. PMID- 8618026 TI - Age should be considered as a risk factor for basal cell carcinoma and DNA repair capacity. PMID- 8618027 TI - What is a caucasian? PMID- 8618028 TI - Specificity of B.C1 for TGK after renaturation prior to transfer of proteins. PMID- 8618029 TI - Dermal mast cell activation by autoantibodies against the high affinity IgE receptor in chronic urticaria. AB - Previous studies identified autoantibodies against the IgE high affinity receptor alpha-chain, Fc epsilon RI alpha, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-Fc epsilon RI alpha autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti-Fc epsilon RI alpha autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4-34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG-mediated. Preincubation of sera or the IgG fraction with a recombinant alpha-chain of Fc epsilon RI inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human Fc epsilon RI alpha antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of Fc epsilon RI, with 0.01-1.0 micrograms/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-Fc epsilon RI alpha autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-Fc epsilon RI alpha autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients. PMID- 8618030 TI - Elevated levels of plasminogen activator inhibitor-1 may account for the altered fibrinolysis by keloid fibroblasts. AB - Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI 1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis. PMID- 8618031 TI - Autoantibodies against desmoplakin I and II define a subset of patients with erythema multiforme major. AB - In a previous report, we described autoantibodies against the desmosomal plaque proteins desmoplakin I and II (dp I and II) in patients with erythema multiforme (EM) major. In the present study we investigated ten EM major and eight EM minor patients for circulating autoantibodies and performed clinical and immunomorphological evaluations. Seven out of ten EM major patients revealed anti dp I and II autoantibodies. Antigens were biochemically characterized by Western blotting and immunoprecipitation of epithelial-cell-derived protein extracts. These autoantibodies bind in vivo to lesional skin/mucosa in a pemphigus-type dotted pattern along the cytoplasmic membranes of keratinocytes. Ultrastructural immunolocalization studies confine in vivo bound autoantibodies to the cytoplasmic desmosomal plaque. Autoantibody binding studies with the sera of such patients demonstrate that the target antigens are not restricted to squamous epithelia but are also expressed in simple and transitional epithelia, on hepatocytes, and on cells of mesenchymal origin, e.g., myocardial cells. Comparing the clinicopathological features of ten patients with EM major, we could not define any discriminating clinical symptoms among patients with or without autoantibodies. Histopathological examination, however, revealed that only patients with EM major and autoantibodies against dp I and II show suprabasal acantholysis in lesional skin and mucous membranes, suggesting a potential role of the humoral immune response in the pathogenesis of this disease. These findings suggest that these autoantibodies define a subset of patients within the clinical spectrum of EM. PMID- 8618032 TI - The metabolism of testosterone by dermal papilla cells cultured from human pubic and axillary hair follicles concurs with hair growth in 5 alpha-reductase deficiency. AB - Androgens regulate the growth of many human hair follicles, but only pubic, axillary, and scalp hair growth occur in men with 5 alpha-reductase deficiency. This suggests that 5 alpha-dihydrotestosterone is the active intracellular androgen in androgen-dependent follicles, except in the axilla and pubis. Since the dermal papilla plays a major regulatory role in hair follicles and may be the site of androgen action, we have investigated androgen metabolism in six primary lines of cultured dermal papilla cells from pubic and axillary hair follicles; previous studies have shown that beard cells take up and metabolize testosterone, retaining and secreting 5 alpha-dihydrotestosterone. After 24 h preincubation in serum-free Eagle's medium 199, 100-mm dishes of confluent cells were incubated for 2 h with 5 nM [1,2,6,7-3H]testosterone. Media were collected and the cells washed with phosphate-buffered saline and extracted with chloroform: methanol (2:1). After the addition of unlabeled and 14C-labeled marker steroids, the extracts were analyzed by a two-step thin-layer chromatography system; steroid identity was confirmed by recrystallization to a constant 3H/14C ratio. Beard and pubic dermal papilla cells were also incubated for 24 h, and the medium was analyzed at various times. The results from pubic and axillary primary cell lines were similar. In both cells and media the major steroid identified was testosterone, but significant amounts of androstenedione were present, indicating 17 beta-hydroxysteroid dehydrogenase activity; androstenedione was also identified within the cells, but a small amount of 5 alpha-dihydrotestosterone was only identified in one pubic cell line. Beard dermal papilla cells secreted large amounts of 5 alpha-dihydrotestosterone into the medium over 24 h in contrast to pubic cells, which produced only very small amounts. The pubic and axillary cell results contrasts with the observations of pronounced 5 alpha dihydrotestosterone in beard cells and confirm that androgen metabolism in cultured dermal papilla cells reflects the parent follicle's ability to respond to androgen in the absence of 5 alpha-reductase type II in vivo. This supports our hypothesis that androgen acts on hair follicles via the dermal papilla and suggests that cultured dermal papilla cells may offer an important model system for studies of androgen action. PMID- 8618033 TI - UVB radiation interrupts cytokine-mediated support of an epidermal-derived dendritic cell line (XS52) by a dual mechanism. AB - We have established long-term dendritic cell lines from the epidermis of newborn mice. These cell lines (XS series) proliferate maximally in response to granulocyte/macrophage-colony stimulating factor, as well as to CSF-1, which is produced by skin-derived NS fibroblast lines and by keratinocytes (albeit in smaller amounts). The purpose of this study was to examine the impact of UVB radiation on CSF-1-mediated interaction of dendritic cells with fibroblasts and keratinocytes. Exposure of NS cells to UVB radiation (unfiltered FS20 sunlamp) decreased CSF-1 production at mRNA and protein levels. Both changes occurred in a dose-dependent fashion, with 50 J/m2 causing a significant reduction. UVB radiation also downregulated CSF-1 mRNA expression by Pam 212 keratinocytes. UVB exposure of XS cells diminished the surface expression of CSF-1 receptors, with 50 J/m2 causing a significant reduction. Thus, UVB radiation interrupts CSF-1 mediated cell-cell interaction by a dual mechanism: downregulating CSF-1 production and abrogating CSF-1 receptor expression. Importantly, granulocyte/macrophage-colony stimulating factor receptor expression by XS cells was also inhibited by UVB radiation, once again, with 50 J/m2 producing significant inhibition. We propose that the resulting CSF-1 deficiency in epidermal microenvironment and unresponsiveness by dendritic cells to relevant growth factors may contribute to UVB-mediated loss of resident epidermal dendritic cells (i.e., Langerhans cells) in skin. PMID- 8618034 TI - Ceramides are transported through the Golgi apparatus in human keratinocytes in vitro. AB - The intercellular lipid sheets of the stratum corneum constitute the epidermal permeability barrier that permits terrestrial life. Although lamellar granules are known to deliver the precursors of the stratum corneum lipids into the intercellular spaces, their site of origin remains unknown. Lamellar granules have characteristics of both secretory granules and lysosomes, which are known to originate from the Golgi apparatus in other cell types. Glucosylceramides, a major component of lamellar granule contents and the precursors of stratum corneum ceramides, have been found to be synthesized primarily in the early compartments of the Golgi apparatus in other cell types. We have investigated the transport and metabolism of a fluorescently labeled ceramide in human keratinocyte cultures using laser-scanning confocal microscopy and lipid analysis. We found that ceramide is metabolized to glucosylceramide and sphingomyelin as it passes through the Golgi apparatus and the metabolites are then delivered to the plasma membrane. Cold temperature, Brefeldin A, and monensin, all known to inhibit transport from the Golgi to the plasma membrane, prevented ceramide metabolites from appearing at the plasma membrane. Because glucosylceramides are one of the most important lipid constituents of lamellar granules, these results support the hypothesis that the Golgi is the origin of lamellar granules. PMID- 8618035 TI - Chronic sun exposure and age are inversely associated with nevi in adult renal transplant recipients. AB - In 126 adult renal transplant recipients who had survived their transplantation for at least 8 years, we determined whether numbers of nevi and the presence of clinically atypical nevi were related to chronic sun exposure. On the basis of a skin examination, three groups were defined: patients with at least one clinically a typical nevus; patients with only clinically normal nevi: and patients without any nevi. The prevalence odds ratio of having any clinically atypical nevi as compared to having only clinically normal nevi was calculated in a logistic model, in relation to gender, skin type, age, sun exposure, and number of keratotic skin lesions present. Similarly, the prevalence odds ratio of having 30 or more nevi compared to fewer than 30 nevi was calculated. We found an inverse association between chronic sun exposure and age with numbers of nevi in adult renal transplant recipients. The presence of clinically atypical nevi was also inversely associated with chronic sun exposure, but this association disappeared after adjustment for age. We did not observe an association of nevi with the number of keratotic skin lesions, nor with humoral immune responses against human papillomavirus and the presence of certain HLA antigens, which are factors associated with nonmelanoma skin cancer in renal transplant recipients. Chronic sun exposure and age appeared to be strong determinants for decreased numbers of nevi in adult renal transplant recipients. Infection with human papillomaviruses does not appear to play an important role. PMID- 8618036 TI - In vivo retinoic acid modulates expression of the class II major histocompatibility complex and function of antigen-presenting macrophages and keratinocytes in ultraviolet-exposed human skin. AB - Because retinoic acid (RA) can alter photoaging of the skin and repeated ultraviolet (UV)-induced immunologic injury may play a role in chronic photoaging, we asked whether RA alters the acute photoimmunologic effects of UV radiation. Two sites from each volunteer were treated with 0.1% RA or vehicle continuously for 24 h before and 24 h after a 4-minimal erythema dose UVB exposure. RA did not function as a sunscreen, as determined by quantitating the increase in redness after 1 minimal erythema dose to vehicle- and RA-pretreated sites (n = 12). By flow cytometric analysis of epidermal cell suspensions harvested 3 d after the UV-EC, RA treatment did not protect CD1+ Langerhans cells from being depleted by UV light and did not modify the number of UV-induced infiltrating CD36+CD11b+CD1-DR+ macrophages. RA treatment did, however, result in a 40% downregulation of human leukocyte antigen (HLA)-DR expression on these infiltrating macrophages (p = 0.016) (n = 11), in conjunction with a decrease in alloantigen-presenting cell activity of RA-treated UV-EC as measured by T-cell proliferations. RA also induced a 72% inhibition of the autologous T suppressor inducer cell proliferation induced by UV-EC (vehicle: 21,813 +/- 7,302 cpm; RA; 5,299 +/-635 cpm) (n = 3). The downregulation could be due to RA-modulated keratinocytes; RA-treated UV-EC keratinocytes depleted of CD1a+ and DR+ antigen presenting cells displayed a greater ability, relative to similarly treated vehicle-EC keratinocytes, to inhibit alloantigen presentation. IN CONCLUSION: (i) in vivo RA treatment did not protect human Langerhans cells from being depleted by UV and did not block infiltration of macrophages into sunburned skin; and (ii) RA did decrease autologous and allogeneic T-cell reactivity induced by macrophage antigen-presenting cells in UV-exposed epidermis, at least in part by downregulating their HLA-DR expression and by upregulating inhibitory signals from UV-irradiated keratinocytes. PMID- 8618037 TI - Interleukin-15 mRNA is expressed by human keratinocytes Langerhans cells, and blood-derived dendritic cells and is downregulated by ultraviolet B radiation. AB - Interleukin (IL)-15 is a recently described cytokine that shares many functional activities with IL-2; however, unlike IL-2, IL-15 is produced by monocytes/macrophages, and not by lymphocytes. In this report, we assessed IL-15 mRNA expression by freshly isolated human epidermal cells, as well as by negatively selected keratinocytes and positively selected Langerhans cells, utilizing reverse transcription and polymerase chain reaction. In addition, cultured keratinocytes, immortalized keratinocytes (HaCaT cells), and dendritic cells expanded from adult peripheral blood in the presence of granulocyte/macrophage-colony stimulating factor and IL-4 were examined for IL-15 transcripts. Using cultured keratinocytes, we also studied the regulation of IL 15 mRNA expression by ultraviolet B radiation in vitro. Freshly isolated keratinocytes, HaCaT cells, and cultured keratinocytes all constitutively expressed IL-15 mRNA, and IL-15 expression was downregulated by ultraviolet B radiation in cultured keratinocytes in a time- and dose-dependent manner. In addition, IL-15 transcripts were constitutively expressed by freshly isolated Langerhans cells. IL-15 produced by keratinocytes, Langerhans cells, and other tissue-specific dendritic cells may be important in attracting and activating antigen-specific Th1 T cells. Furthermore, ultraviolet B-induced downregulation of keratinocyte IL-15 production may contribute to the relative state of immunosuppression induced by sun exposure. PMID- 8618038 TI - The effect of graft-bed irradiation on the healing of rat skin grafts. AB - This study explores the possible side effects on healing skin grafts of irradiation, commonly used intraoperatively following surgical tumor removal. The experimental model involved the delivery of a single 10-Gy dose of electron radiation to the recipient bed of a skin wound, followed by attachment of a full thickness rat skin autograft. Skin graft repair was assessed by light microscopy, immunohistochemistry, and transmission electron microscopy over a 3-week period for grafted and grafted-irradiated groups. Graft-bed irradiation reduced fibrinogen, fibrin, and fibronectin deposition in the wound. It also produced brief changes in the extent of both re-epithelialization and granulation tissue formation, and reduced the diameter of collagen fibrils in the granulation tissue. Despite these changes, the results suggest that graft-bed irradiation only delays the healing process, producing no serious clinical complications at the time points studied. PMID- 8618039 TI - Water increases the fluidity of intercellular membranes of stratum corneum: correlation with water permeability, elastic, and electrical resistance properties. AB - We used the spin label electron spin resonance technique to monitor the hydration effect on the molecular dynamics of lipids at C-5, C-12, and C-16 positions of the alkyl chain. Increase in water content of neonatal rat SC leads to an increase in membrane fluidity, especially in the region near the membrane-water interface. The effect is less pronounced deeper inside the hydrophobic core. The reorientational correlation time at the C-16 position of hydrocarbon chains showed a higher change up to approximately 18% (w/w) of water content. This behavior was accompanied by an exponential decay both in elastic modulus and electrical resistance with water content. On the contrary, the segmental motion at C-5 and C-12 positions of the chain and the permeability constant increased in the range of around 18% w/w) up to the fully hydrated condition (58 +/- 7%). Our results give a better characterization of the fluidity of SC and show that it is the principal parameter involved in the mechanism of the permeability of different compounds through skin. PMID- 8618040 TI - Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice. AB - The epidermis of aged mice displays decreased stratum corneum (SC) lipid content and decreased extracellular bilayers, which result in impaired barrier recovery following the solvent treatment or tape stripping. We assessed the role of altered lipid synthesis as the cause of the abnormal barrier and lipid content in aged epidermis, both under basal conditions and in response to acute barrier perturbations. In aged epidermis ( > or = 18 months), synthesis of one of the three key lipid classes (cholesterol) is decreased under basal conditions, and sterologenesis fails to attain the levels reached in young epidermis following comparable acute perturbations. In contrast, fatty acid and sphingolipid synthesis in aged epidermis increase sufficiently to approach the levels attained in stimulated young epidermis. The abnormalities in sterologenesis in aged epidermis are paralleled by a decrease in activity of its rate-limiting enzyme, 3 hydroxy-3-methylglutaryl-coenzyme A reductase, under basal conditions, and enzyme activity also fails to increase as much as in young epidermis after barrier disruption. That defective lipid generation contributes to the barrier defect is shown directly by the ability of either a cholesterol-containing mixture of SC lipids or cholesterol alone to enhance barrier recovery. Finally, lipid-induced acceleration of barrier recovery in aged epidermis correlates with repletion of the extracellular spaces with normal lamellar structures. Thus, a deficiency in lipid synthesis, particularly in cholesterologenesis, accounts for the barrier abnormality in aged epidermis. PMID- 8618041 TI - Increased concentrations of 3,4-didehydroretinol and retinoic acid-binding protein (CRABPII) in human squamous cell carcinoma and keratoacanthoma but not in basal cell carcinoma of the skin. AB - Retinoids are biologic response modifiers that are present in normal skin and may possibly be perturbed in carcinogenesis. To examine this possibility in human skin, we analyzed vitamin A and cytosolic retinoid binding proteins (cellular retinol binding protein and cellular retinoic acid binding protein [CRABP]) in a total of 38 non-melanoma skin tumors and 25 healthy skin samples using high performance liquid chromatography, radioligand electrophoresis, and reverse transcriptase-polymerase chain reaction. The mean +/- SEM retinol concentration was normal in basal cell carcinoma (0.60 +/- 0.10 microM) and seborrheic keratosis (0.47 +/- 0.07 microM), but increased in keratoacanthoma (1.60 +/- 0.41 microM) and squamous cell carcinoma (1.17 +/- 0.28 microM) (p < 0.05 for both). Also, the concentrations of 3,4-didehydroretinol, a major vitamin A metabolite produced in human skin, were markedly elevated (6-7 times normal) in keratoacanthoma and squamous cell cancer. All types of tumors showed moderately increased levels of cellular retinol binding protein. In addition, keratoacanthoma and squamous cell cancer showed markedly increased levels (6-7 times normal) of CRABPII protein. Transcriptional activity of the CRABPII gene was demonstrated in both normal and neoplastic epidermis, but clear CRABPI mRNA expression was found only in basal cell carcinoma. The data indicate that characteristic perturbations of the vitamin A and retinoid binding protein levels occur in squamous cell-derived skin tumors, but whether these reflect intrinsic errors in retinoid metabolism or are secondary to abnormal cellular differentiation is unknown. PMID- 8618043 TI - The lunula: a magnetic resonance imagining approach to the subnail matrix area. AB - High-resolution sagittal magnetic resonance images depict an oval area in the dermis beneath the nail matrix that gives a particular signal. This study defines the magnetic resonance imaging characteristics of this area and examines its correlation with the lunula. A high-resolution surface gradient coil specially designed for skin imagining was used on a 1.5 T magnetic resonance unit. The subnail matrix (SNM) areas of 12 subjects had a significantly longer T2 relaxation time and a higher enhancement ratio after injection of gadolinium than did the nail bed dermis. The length of the SNM area distal to the free edge of the proximal nail fold was highly correlated with the length of the lunula (R = 0.98) in 30 fingers and 10 toes. The total length of the SNM area was somewhat correlated with the nail thickness (R = 0.86) in 30 fingers. The histology and microvascularization of the subungual tissue in 21 fingers showed that this SNM area had specific features: The area was composed of loose connective tissue without bundles, and the reticular and subdermal vascular networks had large regular meshes in this oval area. The lunula is shown to be linked to a well defined area in the underlying dermis with a specific histology and microvascularization. PMID- 8618042 TI - Lysozyme binds to elastin and protects elastin from elastase-mediated degradation. AB - Lysozyme has been shown to be associated with damaged elastic fibers in many tissues and organs. To better characterize this interaction, binding of lysozyme to elastin was studied using solution-based binding assays. Under physiologic conditions, radio-labeled lysozyme bound specifically to elastin in a time- and concentration-dependent manner. Binding was reversible and was inhibited by unlabeled human and hen lysozyme but not by other proteins. Lysozyme had no elastolytic activity as assessed by a standard tritium-release assay, but, importantly, prevented the proteolytic degradation of elastin by human leukocyte elastase, pancreatic elastase, thermolysin, and Pseudomonas elastase. A striking feature of lysozyme's anti-elastase activity was that it did not function in the classical sense of inhibiting directly the enzymatic activity of the protease. Instead, by binding to elastin, lysozyme prevented the protease from interacting with the elastin substrate in ways that normally favor proteolysis. These results show that lysozyme binds to the elastin component of elastic fibers and that this interaction has important biological consequences for elastic fiber degradation. By preventing degradation of elastin, lysozyme can function as an important natural inhibitor that exerts a protective effect on elastic fibers at sites of tissue injury. PMID- 8618044 TI - Antioxidant nutrients protect against UVB-induced oxidative damage to DNA of mouse keratinocytes in culture. AB - Ultraviolet B radiation (UVB) induces oxidative damage in DNA, resulting in the formation of the adduct 8-hydroxydeoxyguanosine. Previous studies from this laboratory have demonstrated a decrease in antioxidant enzyme defenses after UVB radiation in Skh: HR-1 hairless mice, implicating antioxidant status in protection against oxidative damage. The present study was undertaken to examine mechanisms of UVB damage to DNA and modulation by vitamin C, selenite, or Trolox, a water-soluble vitamin E analog. BALB/c MK-2 mouse keratinocytes were exposed to a dose range of UVB from 4 to 750 mJ/cm2. DNA damage in the form of 80 HdG was measured using high-pressure liquid chromatography coupled with electrochemical and UV absorbance detection. Preincubation of the cells for 2 days with 0.4 or 0.8 microgram/ml ascorbic acid, 10 or 20 micrograms/ml Trolox, and 5 or 12.5 microM selenite resulted in a significant decrease in the number of 8 hydroxydeoxyguanosine adducts per 10(5) deoxyguanines induced by 500 mJ/cm2 UVB. The results indicate a potential role for antioxidant nutrients in protection against UVB damage to skin cells. PMID- 8618045 TI - Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation. AB - The temporo-spatial expression of fibrillin and elastin in skin regenerating from autologous keratinocyte grafts was studied in three burned children. Skin biopsies taken between 5 days and 17 months after grafting were investigated by conventional immunofluorescence, confocal laser scanning, and electron microscopy. Fibrillin, the major component of 10-12nm microfibrils, appeared 5 days after grafting in a band-like fashion similar to collagen VII at the prospective basement membrane, and the formed the characteristic microfibrillar candelabra at the dermo-epidermal junction by fusion of several fine microfibrils to communicating microfibrils projecting downward into the reticular layer of the neodermis. Four to five months after grafting, several communicating microfibrils were connected to a web of horizontally undulating microfibrils of the neodermis which had developed independently. Elastin was first identified in the deeper neodermis 1 month after grafting as granular aggregates and 4 months after grafting on fibrillar structures and surrounding capillaries of the upper neodermis. Association of elastin with microfibrils in the papillary dermis was not detectable before month 17. Our findings suggest that the cutaneous microfibrillar apparatus develops simultaneously at both the dermo-epidermal junction and the reticular dermis and is a prerequisite for elastic fiber formation. In addition, it might be a driving force for the formation of the papilla-rete ridge pattern. PMID- 8618046 TI - Optimization of physiological lipid mixtures for barrier repair. AB - Three stratum corneum lipids, ceramides, cholesterol (CHOL), and free fatty acids (FA), are required for permeability barrier homeostasis. Recent studies have shown that application of one or two of these lipids to perturbed skin delays barrier recovery; only equimolar mixtures allow normal recovery. We asked here whether any physiological lipid mixtures improve barrier repair, as assessed by transepidermal water loss. Whereas an equimolar ratio of ceramides, CHOL, and FA (either the essential fatty acid, linoleic acid, or the nonessential FAs, palmitic or stearic acids) allows normal repair, further acceleration of barrier repair occurs as the ratio of any of these ingredients is increased up to 3-fold. Similar preliminary results were obtained in damaged human skin. Likewise, while acylceramides alone delay barrier recovery, acylceramides: CHOL mixtures within a specific range of molar rations dramatically improve barrier repair. Furthermore, glycosyl ceramides, sphingomyelin, and triglycerides substitute effectively for ceramides and FA, respectively, but neither phospholipids nor cholesterol esters substitute for FA and CHOL, respectively. These studies show the specific requirements of selected stratum corneum lipid mixtures for optimized barrier repair in murine skin, with further validation in human skin. Utilization of physiologic lipids according to these parameters could lead to new forms of topical therapy for dermatoses (e.g., psoriasis, atopic dermatitis, and irritant dermatitis) triggered by abnormal barrier function. PMID- 8618048 TI - Modulation of murine hair follicle function by alterations in ornithine decarboxylase activity. AB - Mice that overexpress a mutated ornithine decarboxylase (ODC) transgene in outer root sheath keratinocytes of the hair follicle were used to study the role of this enzyme in regulating hair follicle structure and function. These transgenic mice have a normal first hair cycle, but lose their hair completely beginning 2-3 wk after birth. Transgene overexpression in follicular keratinocytes is first detected at day 12 after birth, coincident with the development of follicular cysts in the upper portion of the dermis. The onset of keratin 6 expression also begins around day 12; because the promoter/regulatory region of the bovine keratin 6 gene was used to target ODC transgene expression of hair follicle keratinocytes, these data demonstrate the faithful temporal and cell type specific expression of the K6 -driven transgene. The ODC inhibitor 2 difluoromethylornithine could prevent hair loss and partially normalize skin histology if administered before the onset of ODC overexpression. 2 Difluoromethylornithine could also reactivate hair growth in animals with complete hair loss. Our results suggest that ODC is an important regulatory gene for the mouse hair follicle. PMID- 8618049 TI - Interleukin-12 inhibits angiogenesis induced by human tumor cell lines in vivo. AB - Tumor cell-induced angiogenesis, i.e., new blood vessel formation within tumor tissue, is an essential requirement for the growth of solid neoplasms. Interleukin-12 (IL-12) inhibits growth of a variety of experimental tumors in vivo. We tested whether antitumor activity of IL-12 is related to the inhibition of angiogenesis induced by tumor cell lines. Angiogenesis was induced in x-ray immunosuppressed Balb/c mice by intradermal injection of the following human tumor cells: T47D, originating from mammary carcinoma; A431, derived from vulval carcinoma; and Skv, established from bowenoid papulosis, Systemic treatment of the mice with murine IL-12 significantly decreased angiogenesis induced by human tumor cells in a time-and dose-dependent manner. Preincubation of human cells in vitro with IL-12 did not inhibit tumor cell-induced angiogenesis, suggesting that the antiangiogenic capacity of IL-12 is restricted to in vivo conditions. Treatment of the mice with rat antibody against murine interferon-gamma (IFN gamma) resulted in counteracting the antiangiogenic effect of murine IL-12. Furthermore, human IFN-gamma inhibited the angiogenic activity of human tumor cell lines. This indicates that IFN-gamma is a mediator of the antiangiogenic effect of IL-12. The results show that the mechanism of antitumor action of IL-12 may depend not only on the immunostimulatory activity of this cytokine but also on its effect on tumor cell-induced angiogenesis. IL-12 should be considered as a potential candidate for the treatment of angiogenesis-dependent malignancies. PMID- 8618047 TI - Elevation of interleukin 1 receptor antagonist in the stratum corneum of sun exposed and ultraviolet B-irradiated human skin. AB - Keratinocytes produce not only interleukin 1 (IL-1) but also IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1. Because little is known about the presence of IL-1ra in the stratum corneum, we examined the content of IL-1ra in the stratum corneum, especially the balance between IL-1 and IL-1ra. IL 1 alpha and IL-1ra, but not IL-1 beta, were detected in the tape-stripped stratum corneum of healthy volunteers by enzyme-linked immunosorbent assays. IL-1 alpha and IL-1ra were bioactive as determined by thymocyte co-stimulation assay, and their molecular masses were 17 and 20 kDa, respectively, suggesting that the stratum corneum contains active forms of IL-1 alpha and IL-1ra produced by keratinocytes. The stratum corneum of an unexposed area, the inner side of the upper arm. contained more IL-1 alpha than a sun-exposed area, the face. In contrast, the stratum corneum of the sun-exposed area contained a markedly higher amount of IL-1ra than that of the unexposed area. The ratio of IL-1ra to IL-1 alpha was 8 in the unexposed area, and over 100 in the sun-exposed area. Therefore, IL-1 alpha activity was dominant in the unexposed area, and in contrast, IL-1ra activity was dominant in the sun-exposed area. An elevated level of IL-1ra was detected in the stratum corneum of the sun-exposed area independently of age. In the unexposed area, however, IL-1a increased, but IL-1ra decreased, with age, resulting in a significant decline of the ratio of IL-1ra to IL-1a with increasing age. Irradiation of 2 MED of ultraviolet B to the back skin, an unexposed area, resulted in striking elevation of IL-1ra in the stratum corneum in desquamating scales. These data suggest that IL-1ra in the epidermis may be inducible by chronic UV irradiation, although IL-1ra production in the epidermis may decrease with aging in the absence of any stimulus. IL-1ra in the epidermis may play a role in the regulation of IL-1-induced inflammatory responses, and an appropriate balance between IL-1 and IL-1ra may help to maintain homeostasis of the skin. PMID- 8618050 TI - Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers. AB - Although extracellular proteolysis is a prerequisite for normal wound healing, uncontrolled proteolytic tissue destruction appears to be a pathogenic factor in non-healing wounds. The aim of our study was to compare the activities of the serine proteinases of polymorphonuclear origin, elastase and cathepsin G, and the metalloproteinases, gelatinase and collagenase, in chronic leg ulcer exudate (10 patients) and acute wound fluid (6 patients). Serine proteinase activities were low in leg ulcer exudates but very high in some but not all acute wound fluids. Total collagenase activity, measured as activity against type I collagen monitored by SDS-PAGE and densitometry, was higher in chronic leg ulcer exudate than in acute wound fluid and its degree of autoactivation was relatively high. Doxycycline inhibition studies suggested that the collagenase activity in chronic leg ulcer exudate was MMP-1 ("fibroblast-type") and not MMP-8 ("neutrophil type"). Zymographic analysis of the gelatinolytic enzymes in acute wound fluid showed a progressive increase from the day of operation to postoperative day 5, but the degree of activity was lower than in chronic leg ulcer exudate and the low molecular mass activation products were faint. The leg ulcer gelatinase profiles were characterized by high expression of 92/82- and 72/62-kDa duplex bands and by the presence of low molecular mass activation products. Leg ulcer collagenase seems to be derived from mononuclear rather than polymorphonuclear cells, which are known to be involved in acute wound healing. In conclusion, the present study shows that gelatinase and collagenase, but not elastase and cathepsin G are found in chronic leg ulcer exudate. PMID- 8618051 TI - Identification of a 450-kDa human epidermal autoantigen as a new member of the plectin family. AB - The serum from an individual with a subepidermal blistering disease was previously shown to recognized a 450-kDa epidermal autoantigen. The molecular structure of this antigen was investigated by screening a human keratinocyte cDNA library with the patient's serum. One clone, with a 276-bp cDNA insert, that encoded an epitope recognized by the serum was isolated. Rabbit polyclonal antibodies that were prepared against the corresponding fusion protein recognized the 450-kDa epidermal antigen and stained the basal keratinocytes in human epidermis. This clone was used for further screening of the original keratinocyte and HeLa cell cDNA libraries. Two different, but closely related, 0.8- and 2.0-kb cDNAs were isolated, and their deduced amino acid sequences indicated that the encoded proteins belonged to the plectin family. Northern blot analysis of total RNA from human keratinocytes with these cDNA inserts as probes detected RNAs of approximately 12-13 kb. The 0.8-kb cDNA hybridized to polyadenylated RNA species from human skeletal muscle, heart, lung, and kidney, whereas the 2.0-kb cDNA hybridized to transcripts present only in kidney and lung. Southern blot analysis of genomic DNA from the human placenta revealed similar, but not identical, patterns of hybridization with the 0.8- and 2.0-kb cDNAs. Data suggest that the 0.8- and 2.0-kb cDNAs encode two different proteins but are derived from the same gene. PMID- 8618052 TI - Abnormal IL-4 gene expression by atopic dermatitis T lymphocytes is reflected in altered nuclear protein interactions with IL-4 transcriptional regulatory element. AB - Among the atopic disease, atopic dermatitis is characterized by the highest levels of serum IgE and by increased peripheral blood T-cell interleukin-4 (IL-4) production. IL-4 promotes IgE synthesis by B cells and stimulates the growth of IL-4-producing T cells and may contribute to the pathogenesis of this disease. In this study, in situ hybridization established that atopic dermatitis patients have a higher frequency of IL-4-producing peripheral blood T cell when compared to normal subjects. These in vivo-derived T cells were used to examine the signaling requirements of IL-4 production and the nuclear factors that associate with a critical IL-4 transcriptional regulatory element between -88 and -60 relative to the IL-4 transcription initiation site, the activation responsive element. We demonstrate that, as in T-cell lines, proteins belonging to the NF-AT and AP-1 family of transcription factors are present in stimulated cell extracts and specifically associate with the activation responsive element. Dysregulated IL-4 production is reflected in the nuclear proteins that associated this element. Using gel shift assays, we found that 12 of 12 nuclear extracts from stimulated atopic T cells formed the activation-dependent protein-DNA complex, compared to only 2 of 12 normal T-cell extracts. Activation complex formation correlated with the relative level of IL-4 mRNA and protein produced in stimulated T cells, suggesting that abnormal IL-4 gene expression in atopic disease may be linked to alterations in nuclear protein interactions with these promoter elements. PMID- 8618053 TI - Type I oculocutaneous albinism associated with a full-length deletion of the tyrosinase gene. AB - Type I oculocutaneous albinism is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair, and eyes because of deficient activity of tyrosinase (EC 1.14.18.1). Type I oculocutaneous albinism is caused by mutations in the tyrosinase structural gene, TYR; however, no large TYR gene deletions have been identified previously in humans. Here we report a patient with type IB oculocutaneous albinism who is a compound heterozygote for TYR allele containing a mutation that is likely to affect pre-RNA splicing and a paternally inherited allele in which the TYR gene is completely deleted, the first such allele described to date. Aside from the albinism in the proband, his phenotype and that of his normally pigmented father is otherwise normal, suggesting that this TYR deletion does not involve other functionally important contiguous genes. PMID- 8618054 TI - Low frequency of loss of heterozygosity at the nevoid basal cell carcinoma locus and other selected loci in appendageal tumors. AB - Previous studies of loss heterozygosity (LOH) have revealed distinct patterns of allelic loss in some skin tumors. In basal cell carcinomas (BCCs) loss of heterozygosity is virtually restricted to chromosome 9, whereas in squamous cell carcinomas (SCCs) and actinic keratoses loss is more widespread involving chromosomes 3, 9, 13, and 17. Because there are histological similarities between BCCs and some appendageal tumors, and because lines of evidence suggest that BCCs are appendageal in origin, we carried out a limited allelotype in 41 appendageal tumors. The overall frequency of allelic loss was low (4 out of 247 informative loci; 1.6%). LOH was seen in a proliferating trichilemmal cyst (17p), a sebaceous epithelioma (17q), an eccrine porocarcinoma (17q), a trichoepithelioma (9q), and in two basal cell carcinomas showing eccrine or granular cell differentiation that were originally misdiagnosed (9Q). The pattern of loss in this mixed group of appendageal tumors shows differences from both BCCs and SCCs, and further emphasizes the unique genetic profile and behavior of BCCs. The finding of 9q loss in BCCs with eccrine or granular cell differentiation shows that 9q loss occurs in differential histological subtypes of BCCs. PMID- 8618055 TI - Spontaneous melanocytosis in transgenic mice. AB - Clone B is a 2-kb fragment of cloned genomic DNA involved in adipocyte differentiation in vitro. Insertion of this DNA fragment into the genome of a variety of cell lines results in committing the recipient cells to undergo adipocyte differentiation. Construction of transgenic mice with Clone B DNA resulted in an unexpected phenotype--spontaneous melanocytosis. The present study describes the distribution and morphology of melanin-containing lesions in these transgenic mice. Spontaneous melanin-containing dermal lesions appeared on the ears, snout, and perianal regions of transgenic mice by the age of 3-4 months. Multifocal dermal masses rapidly developed into raised lesions, which appeared to spread to adjacent skin. Ultrastructural examination of lymph nodes, spleen, and dermal lesions of these mice revealed membrane-bound melanin with effacement f the organelle structure of severely affected cells. Protein gel electrophoresis revealed elevated activity of tyrosinase in the pinnae, skin, perianal mass, and lymph nodes. This line of transgenic mice may provide a useful model for investigation of the etiology and progression of benign and malignant melanin containing tumors. PMID- 8618056 TI - Suncreens to protect against the immunosuppressive effects of UV radiation (UVR) PMID- 8618057 TI - Constitutional skin color in Caucasians. PMID- 8618058 TI - Compound heterozygosity for nonsense ans missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa. AB - Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of a nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5. PMID- 8618059 TI - KIT expression reveals a population of precursor melanocytes in human skin. AB - Human skin is believed to harbor a reservoir population of precursor melanocytes. It has been difficult to identify these putative cells experimentally, because they lack phenotypic features that define mature melanocytes. We have evaluated expression of the KIT tyrosine kinase receptor, which is critical for melanocyte development, as a possible marker of these cells. Sections of human skin were evaluated with single- and double-immunolabeling techniques. KIT-reactive dendritic cells were identified in the basal layer of the epithelia and were most numerous in the follicular infundibula and the rete ridges. These cells were located on the epithelial side of the basement membrane and lacked expression of cytokeratin and mast cell tryptase. The location of the KIT-reactive cells was distinctly different from that of Langerhans cells (identified with anti-CD1a) or Merkel cells (identified with CAM 5.2). Within the epidermis and upper follicular infundibulum the majority of the KIT-reactive dendritic cells also coexpressed TRP-1, a marker present in differentiated melanocytes. In the deeper follicular regions, the coexpression of TRP-1 in the KIT-reactive cells was absent. Throughout the epidermis and follicle, however, the KIT-reactive cells coexpressed BCL-2, a marker known to be increased in melanocytes. Thus, KIT expression reveals a population of intraepithelial cells that have immunophenotypic characteristics of mature melanocytes within the upper epithelial regions, but lack the differentiated melanocytic phenotype within the deeper follicular regions. We propose that these KIT(+), BCL-2(+), and TRP-1(-) cells constitute a precursor melanocyte reservoir of human skin. PMID- 8618060 TI - Protein-tyrosine phosphatases expressed in mouse epidermal keratinocytes. AB - The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control. PMID- 8618061 TI - Analysis of familial aggregation of atopic eczema and other atopic diseases by ODDS RATIO regression models. AB - In order to determine the relative importance of genetics and the environment on the occurrence of atopic diseases, we investigated the familial aggregation of atopic eczema, allergic rhinitis, and allergic asthma in the relatives of 426 patients with atopic eczema and 628 subjects with no history of eczema (5,136 family members in total). Analyses were performed by regression models for odds ratios (OR) allowing us to estimate OR for the familial aggregation and simultaneously to adjust for other covariates. Three models were analyzed assuming that the OR i) is the same among any two members of a family, ii) depends on different familial constellations, i.e., whether the pairs are siblings, parents, or parent/sibling pairs, and iii) is not the same between the father and the children and between the mother and the children. The OR of familial aggregation for atopic eczema was 2.16 (95% confidence interval (95%-CI) 1.58-2.96) if no distinction was made between the degree of relationship. Further analyses within the members of the family showed a high OR among siblings (OR = 3.86; 95%-CI 2.10-7.09), while the OR between parents and siblings was only 1.90 (95%-CI 1.31-2.97). Only for atopic eczema was the familial aggregation between fathers and siblings (ms: OR = 2.66; fs: OR = 1.29). This can be explained by stronger maternal heritability, shared physical environment of mother and child, or environmental events that affect the fetus in utero. Since for all atopic diseases a stronger correlation was found between siblings than between siblings and parents, our study indicates that environmental factors, especially during childhood, seem to explain the recently observed increased frequencies of atopic diseases. PMID- 8618062 TI - Cutaneous exposure to the superantigen staphylococcal enterotoxin B elicits a T cell-dependent inflammatory response. AB - We analyzed the impact of superantigens secreted by skin-colonizing Staphylococci on the skin and the associated lymphoid tissue following epicutaneous application and intracutaneous injection of small amounts of staphylococcal enterotoxin B (SEB). A single intracutaneous injection of 50 ng of SEB elicited a strong inflammatory response in the skin of BALB/c mice. Three to 6 h later, we observed langerhans cell activation, mast cell degranulation, vasodilation, upregulation of ICAM-1, and induction of VCAM-1 on dermal blood vessels, with vascular adhesion of granulocytes. by 12 to 24 h, cell infiltration of the dermis increased, reaching the epidermis. Among the infiltrating leukocytes, a substantial number of eosinophils was found. After 48 h, the infiltrate was dominated by mononuclear cells. The response to SEB was dose-dependent, and signs of inflammation slowly disappeared over 5 to 7 days. Although the induction of VCAM-1 on dermal blood vessels suggested a role for interleukin-1/tumor necrosis factor-alpha in this reaction, the activation of monocytes/macrophages was not able to substitute for lymphocytes, as severe combined immunodeficiency (SCID) mice (which are lymphocyte-deficient) did not mount an inflammatory skin response to intradermal injection of SEB. The fact that nude mice (T-cell-deficient) also did not mount an inflammatory response to SEB indicated the T-cell dependency of the response. The V beta specificity of the SEB effect was demonstrated by the fact that SJL/J mice, which lack V beta 8+ T cells (the major SEB-reactive T cell population in mice), exhibited much weaker responses. Deletion or tolerization of SEB-reactive V beta T cells was not observed after a single intradermal injection of such minute amounts of SEB. PMID- 8618063 TI - Genes encoding structural proteins of epidermal cornification and S100 calcium binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21. AB - Chromosome 1 reveals in region 1q21 a most remarkable density of genes that fulfill important functions in terminal differentiation of the human epidermis. These genes encode the cornified envelope precursors loricrin, involucrin, and small proline-rich proteins (SPRR1, SPRR2, and SPRR3), the intermediate filament associated proteins profilaggrin and trichohyalin, and several S100A calcium binding proteins. Extending and refining our previous physical map of 1q21 we have now mapped two additional S100A genes as well as the three SPRR subfamilies and resolved the arrangement of involucrin, SPRRs, and loricrin. All genes are linked within 1.9 Mbp of human genomic DNA in the order: S100A10, trichohyalin, profilaggrin, involucrin, SPRR3, SPRR1B, SPRR2A, loricrin, S100A9, S100A9, S100A8, S100A6. Colocalization of genes expressed late during maturation of epidermal cells together with genes encoding calcium-binding proteins is particularly intriguing since calcium levels tightly control the differentiation of epithelial cells and the expression of genes encoding epidermal structural proteins. Accounting for the close functional cooperation among these structurally and evolutionary related genes, we conclude that these loci constitute a gene complex, for which we propose the name epidermal differentiation complex. PMID- 8618064 TI - Hyporesponsiveness in contact hypersensitivity and irritant contact dermatitis in CD4 gene targeted mouse. AB - To determine the role of CD4 molecules in the generation and regulation of contact hypersensitivity (CHS), we treated mice lacking the CD4 gene as a result of targeted disruption with dinitrofluorobenzene to induce CHS. The mutant mice lacking CD4 (CD4(-) mice) showed marked hyporesponsiveness in CHS compared with normal syngeneic C57BL/6 mice (38.3 +/-9.0% of normal at 24 h after the challenge assessed by net ear swelling; p < 0.025). CD4(-) mice had a larger CD4-8- double negative T-cell receptor alpha beta+ cell population in the lymph nodes than did normal mice, and the increase of this cell population was observed in CD4(-) mice after sensitization. Draining lymph node cells from sensitized normal mice restored the responsiveness in CD4(-) mice, but those from sensitized CD4(-) mice were less effective in restoring the CHS response in normal mice. Langerhans cell numbers were normal, and function, as assessed by the ability to present soluble hapten, was not impaired in CD4(-) mice. Skin cytokine profiles demonstrated an increase in interferon-gamma, interleukin-2, and interleukin-4 mRNA levels after challenge in normal mice, whereas this response was blunted in CD4(-) mice. CD4( ) mice also showed hyporesponsiveness in inflammatory reaction to irritant chemicals. These results suggest that the CD4 molecule is required for optimal induction of CHS as well as irritant contact dermatitis and may influence the development of CHS by modulating the cytokine profiles in the skin. PMID- 8618065 TI - [A new avenue of proteoglycan studies: reconstruction of glycosaminoglycan chains using endo-type glycosidases]. PMID- 8618066 TI - [Structure and function of PAS proteins]. PMID- 8618067 TI - [Characteristic of nitric oxide production in macrophages]. PMID- 8618068 TI - [Multiplicity and purification of animal phospholipase D]. PMID- 8618069 TI - [Advanced mass spectrometry in biochemical sciences]. PMID- 8618070 TI - [Type IV collagen: gene expression, supramolecular aggregates, and role in human diseases]. PMID- 8618071 TI - [Metabolic enzymes of neurotoxin: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Parkinson's disease]. PMID- 8618072 TI - [Molecular structure and function of the prostanoid receptors]. PMID- 8618074 TI - [Regulation of gene expression through phosphorylation of transcription factors]. PMID- 8618073 TI - [Galectins: decoder of glyco-code]. PMID- 8618075 TI - [Establishment of immortalized cell lines with differentiation phenotypes from the transgenic mice harboring temperature-sensitive SV40 T-antigen gene]. PMID- 8618076 TI - [Physiological importance of plasmalemmal caveola]. PMID- 8618077 TI - [Protein translocation machinery--how far it has been elucidated by membrane reconstitution approach]. PMID- 8618078 TI - Detection of pulpal circulation in vitro by pulse oximetry. AB - Evaluation of pulse oximetry as a potential method of determining pulp vitality was the subject of this research. An in vitro model of pulpal circulation was fabricated to test the design for a dental pulse oximetry sensor. Blood samples equilibrated with hypoxic gas mixtures were circulated through the model by a peristaltic pump. A pulse was simulated by introduction of gas bubbles into the blood circulation. Pulse oximeter readings for saturation were recorded and compared with blood gas analysis results. Statistical analysis revealed no difference between pulse oximetry and blood gas analysis with a highly significant correlation coefficient. Clinical evaluation of this application is currently in progress. PMID- 8618079 TI - An in vitro evaluation of the sealing ability of Super-EBA cement used as a root canal sealer. AB - Forty-six extracted human teeth, 26 maxillary central incisors, and 20 mandibular incisors were instrumented with flared preparations. The maxillary and mandibular incisors were prepared to #50 and #30 master apical file sizes, respectively. Ten maxillary and mandibular teeth were obturated with a single gutta-percha (GP) cone and Super-EBA cement. Ten maxillary and mandibular incisors were obturated with laterally condensed GP and Roth 801 sealer. Six maxillary incisors were used as controls. Apical leakage of India ink was measured using a stereomicroscope after clearing the teeth. Results showed significantly less leakage in the mandibular incisors filled with GP and Super-EBA (p<0.05). There was no significant difference in leakage when comparing the maxillary central incisors (p>0.05). In this in vitro study, root canal obturation with a single GP cone and Super-EBA showed promise as an effective way to eliminate or reduce apical microleakage. Further studies are needed to test the ability to use this obturation method clinically. PMID- 8618080 TI - Optical detection of hemoglobin in pulpal blood. AB - An in vitro, flow-through optical system was designed to measure hemoglobin (Hb) concentrations in the pulp space. The system included light-emitting diodes and a silicon photodetector positioned on opposing surfaces of human teeth. A syringe pump allowed a controlled flow of blood through the pulp chamber. The Hb concentration was computed as a nonlinear function of transmitted light intensity. Transmitted light intensities were also used as indicators of oxygenation level. Optical measurements correlated with Hb values measured by the conventional cyanmethemoglobin method (r=0.993). The mean percentage error was 5.8%, and the standard error of prediction was 0.77 g/dl for Hb concentrations ranging from 4 to 20 g/dl. Deoxygenated blood exhibited up to 31% lower transmitted intensity. Light transmission through teeth may be useful in the assessment of total Hb and blood oxygenation within the pulp chamber. PMID- 8618081 TI - Histochemical analysis of dental hard tissues following bleaching. AB - The effect of commonly used bleaching materials on the dental hard tissues was tested in extracted human premolars. In each tooth, the apical two-thirds of the root was removed, and the remaining tooth stump was cut longitudinally into two equal segments. The segments were cleaned, dried, and divided into six experimental groups. Each group was treated with one of the following bleaching materials: 30% hydrogen peroxide (HP), 10% carbamide peroxide (CP), sodium perborate (SP), Nu-Smile (NS), Opalescence (Op), and DentlBright (DB). Treatment consisted of immersing the specimens in the respective test material followed by incubation at 37 degrees C for 7 days. The levels of calcium, phosphorus, sulfur, and potassium were measured in the enamel, dentin, and cementum. In the enamel, a significant reduction in the calcium/phosphorus (Ca/P) ratio was found following treatment with HP. In the dentin, a significant reduction in Ca/P ratio was found following treatment with HP,CP, DB, and Op. In the cementum, a significant reduction in the Ca/P ratio was found following treatment with HP, CP, NS, and Op. Changes in sulfur and potassium levels also occurred, but were usually not statistically significant. Significant reduction in sulfur levels occurred only in the cementum following treatment with CP and SP. Sulfur levels increased significantly following treatment with NS. Significant reduction in potassium levels occurred only in the dentin following treatment with CP. It is concluded that bleaching materials may adversely affect the dental hard tissues and should be used with caution. PMID- 8618082 TI - Root canal irrigation with citric acid solution. AB - The purpose of this study was to investigate various properties of citric acid and EDTA solution as decalcifying and cleansing agents in root canal irrigation, and antibacterial effects of citric acid and EDTA solution. Powdered dentin-resin mixtures were used for evaluating the decalcifying effect of citric acid and EDTA solution. Twelve bacterial strains isolated from infected root canals, were used to evaluate the antibacterial effects of citric acid and EDTA solution. Powdered dentin-resin mixture was found to be more soluble in a 0.5, 1, and 2 M citric acid solutions than in a 0.5 M EDTA solution. Citric acid solution showed antibacterial effects on all the bacteria used. PMID- 8618083 TI - Effects of long-term exposure of human periodontal ligament cells to milk and other solutions. AB - Periodontal ligament (PDL) cells cultured from healthy extracted human teeth were exposed to milk, Alcon Opti-Free contact lens solution, K-Mart contact lens solution, saline, and Hank's balanced salt solution. The appearance and rate of loss of the cells from the culture dishes were recorded over time at both room temperature (20 degrees C) and 4 degrees C. The results indicated that saline was superior to either of the contact lens solutions in its ability to maintain the vitality of the PDL cells. Milk at 4 degrees DC provided good short-term viability , but cells did not remain attached after 48 h. At 20 degrees C, however, milk resulted in a 24.4% retention of cells after 72 h. Hank's balanced salt solution was the best storage media, with 46.8% of sells remaining attached after 72 h of exposure. This study supports milk as a good short-term storage medium for maintaining the vitality of PDL cells in vitro. PMID- 8618084 TI - Endodontic applications of guided tissue regeneration in endodontic surgery. AB - There are several possible causes of failure following nonsurgical and surgical endodontic treatments. Many of the causes of these failures can be attributed to the presence of endodontic-periodontic bone loss around roots. The development of guided-tissue regeneration (GTR) procedures in periodontal therapy has let to the successful treatment of some types of periodontal bone loss. These GTR procedures can be adapted for use in endodontic surgery to produce success in cases that previously presented a poor prognosis. this study categorizes all of the different clinical situations in which GTR procedures can be used to treat endodontically related bone loss. Case reports of several categories are presented. PMID- 8618085 TI - An unusual attempt at surgical repair. AB - Gold foil was used in an attempt to repair a periapical surgical site in the lower anterior region. This method of repair was used in the past for surgical closure of persistent oroantral fistulas with some success. In this case, it met with failure. PMID- 8618086 TI - Sealing ability of the vertical condensation with different root canal sealers. AB - This study evaluated the influence of various root canal sealers on the quality of the apical seal of vertically condensed gutta-percha. One hundred and twenty human anterior teeth with single canals were used. After cleaning and shaping to an apical size 30 file, the teeth were divided into 4 equal groups of 30 teeth each and obturated with vertically condensed gutta-percha. In group 1, no sealer was used. In groups 2, 3, and 4, Kerr Pulp Canal Sealer, Roth 801, and AH 26 were used, respectively. Apical microleakage was determined using pressurized fluid filtration measured at different time intervals up to 24 wk. The no-sealer group showed significantly more apical leakage than the other groups. Kerr Pulp Canal Sealer was significantly better than Roth 801 and AH 26 at 24 wk. PMID- 8618087 TI - Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide. AB - Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 microgram/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 microgram/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA but natural human interferon beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS. PMID- 8618088 TI - Evaluation of a pulse oximeter and customized probe for pulp vitality testing. AB - Current routine methods for assessment of pulp vitality rely on stimulation of A delta nerve fibers and give no direct indication of blood flow within the pulp. Recent papers have suggested that pulse oximeters may be used to diagnose pulp vitality by detection of blood flow. In this study, an optimized pulse oximeter probe for teeth was designed, built and tested using the Biox 3740 Oximeter (Ohmeda, Louisville, CO). Following preliminary in vitro tests, the probe was tested clinically. Pulse waveforms from maxillary and mandibular anterior teeth were noted. Simultaneous readings from the subjects' finger were used as controls. Pulse wave readings from the teeth were found to be synchronous with the finger probe, but not consistently. It was easier to maintain continuous readings from mandibular incisors than from maxillary incisors. The average percentage synchronization with the pulse was 28.95% for maxillary incisors and 50.28% for mandibular incisors. This difference was significant (p = 0.05). The overall accuracy of the commercial instrument was disappointing, and in its present form it was not considered to have clinical value. PMID- 8618089 TI - Effect of endodontic instrument handle diameter on operator performance. AB - A phantom head simulator was used by 30 dental students to determine the effect of instrument handle diameter on the time taken to complete a manipulative task. No significant differences were found for handles between 2.5 and 5 mm diameter. Almost one-third of the participants thought that gloves were a hindrance, and it was considered that "glove wrap," where layers of glove roll over one another, might be a problem with small handles. Despite these subjective reports, the students performed no better without gloves when using the 2.5 mm handle. PMID- 8618090 TI - Evaluation of a dentin barrier test by cyctotoxicity testing of various dental cements. AB - In this study, cell reaction after exposure to several dental cements was determined using a dentin barrier test device. The reaction of mouse fibroblasts grown on the "pulpal" side of a bovine dentin disk was determined after exposure to dental cements applied on the "cavity" side of the disk. For pretreatment, dentin disks (500-micrometer-thick) were etched on one side and either sterilized by autoclaving or disinfected with ethanol. It was found for both pretreatment groups that zinc phosphate cement was less toxic than the conventional glass ionomer cements. A light-cured glass ionomer cement and zinc oxide-eugenol were the most toxic materials tested. Disinfecting the dentin slices instead of autoclaving reduced the toxicity of the phosphate cement and the glass ionomer cements tendentiously. Because the strong cytotoxic reaction evoked by zinc oxide eugenol and by the glass ionomer cements is in contrast to in vivo findings, further improvements of the dentin barrier test device will be necessary. PMID- 8618091 TI - Electron microscopic study on interodontoblastic collagen fibrils in amputated canine dental pulp. AB - The purpose of this research was to study the presence and the ultrastructural features of interodontoblastic collagen fibrils and their contribution to the formation of reparative dentin in dog incisors and premolars for a period of 30 days following experimental pulpotomy. On the seventy day after pulp exposure and capping with calcium hydroxide, short cylindrical-shaped cells collected at the coronal end of the vital pulp tissue. Many collagen fibrils were synthesized in the intercellular spaces and in the direction of the long axis of these cells. On the fourteenth day, large bundles of collagen fibrils were observed in the intercellular spaces of young odontoblasts. These bundles spread out in a fan shaped arrangement. On the thirtieth day, the odontoblasts formed a tubular dentin matrix. Small numbers of twisted collagen bundles crossed the distal junctional complex of the odontoblast cell bodies and entered the dentin matrix. PMID- 8618092 TI - Histological findings of human leprosy periapical granulomas. AB - Histological analysis of human leprosy periapical granulomas was conducted to study the histological responses to Mycobacterium leprae. Many Langhans-type giant cells and epithelioid cell tubercles were observed, although M. leprae were not detected in leprosy periapical granulomas. Although numerous T- and B-cells were infiltrated in leprosy periapical granuloma, the T-/B-cell ratio of leprosy is not changed to that of periapical granulomas isolated from normal patients. These findings suggested that human leprosy periapical granulomas develop as a result of immunological responses to M. leprae. PMID- 8618093 TI - An evaluation of the Canal Master, balanced force, and step-back techniques. AB - The step-back (SB), balanced-force (BF) , and Canal Master (CM) instrumentation techniques were compared in 53 mesial canals of mandibular molars using two different instrument types: Flex-R and CM. Canal angulation changes from preoperative to postrotary and postinstrumentation were measured using the Schneider technique (ST) and the long-axis technique (LAT). Instrumentation times were recorded, and then the roots were sectioned at 1, 3, and 5 mm from the apex. Measurements were made of the minimal remaining root structures and canal diameter. Rotary instrumentation resulted in a average loss of curvature of 2.37 degrees, as measured by the ST. The ST showed significant changes in canal angulation between CM (-7.74 degrees) and SB (-5.28 degrees) groups, with p < 0.05. LAT showed significant changes for CM versus BF and SB (p<0.001), with a mean change in angulation of -7.69 degrees, -1.68 degrees, and _0.1 degrees respectively. The BF technique (5.5) was significantly faster than either SB (7.1 min) or CM (8.3 min), with p < 0.05. The remaining mesial-distal root structure at all levels was similar among groups. PMID- 8618094 TI - An in vitro comparison of the Excalibur handpiece and hand instrumentation in curved root canals. AB - Root canal preparation may be the most technique-sensitive and labor-intensive procedure in endodontic therapy. Many automatic handpieces and machine-driven devices have been marketed to expedite this procedure. Excalibur is one such handpiece. The purpose of this study was to compare the Excalibur hand piece with traditional K-files for their effectiveness in shaping curved root canals. A total of 24 standardized canals in extracted human mandibular molars were randomly divided into two groups. They were instrumented either with Excalibur according to the manufacturer's instructions or manually using the step-down technique. The cross-sectional shapes of the root canal at the apical, midroot, and coronal levels were digitized before and after instrumentation. Image analyzer software was used to compare the images. The results showed that the methods removed similar amount of dentin at all three levels. Although the Excalibur handpiece had a slight tendency to straighten the canal more than hand instrumentation, the difference was not significant. Strip-perforation occurred in three specimens in the Excalibur group, but none in the manual group. There was no separation of instrument throughout the experiment. PMID- 8618095 TI - New directions in surgical endodontics; immediate implantation into an extraction site. AB - Endodontic surgical procedures may reveal compromising factors that indicate a modification of the treatment (e.g. tooth extraction, root amputation, etc.). To take advantage of the osseous height and width, as well as the natural tooth angulation, immediate placement of implants after extraction is a reasonable alternative treatment. In this study, 32 titanium alloy implants were inserted immediately after extraction of teeth diagnosed during endodontic surgery as having root fractures, perforations, or endodontic-periodontal complications. After 4 to 6 months of osseointegration, only one implant failed to integrate, and the remaining implants were prosthetically restored. Sixteen months after occlusal loading, bone loss was approximately 1.5 mm for the 31 implants remaining. It seems that the immediate placement of implants following tooth extraction due to endodontic complications is a reliable procedure. PMID- 8618097 TI - Osteoblastoma in the anterior maxilla mimicking periapical pathosis of odontogenic origin. AB - A patient with a 6-yr history of chronic orofacial pain and periapical pathosis in the anterior maxilla presented for evaluation and treatment. Previous root canal therapy had failed to resolve the persistent pain. Further evaluation suggested a non-odontogenic etiology of the patient's symptoms. Exploratory surgery revealed an osseous cavity across the maxillary anterior palatal midline filled with osteoid and early mineralized bone. The tumor was surgically removed in toto. A diagnosis of benign osteoblastoma was made. PMID- 8618096 TI - Blood mercury levels with amalgam retroseals: a longitudinal study. AB - A clinical study was performed to determine if placement of an amalgam retroseal resulted in elevated blood mercury levels. Ten subjects had blood drawn 7 days before and immediately before placement of an amalgam retroseal. Postoperative blood draws occurred at 7 and 30 days. Blood samples were analyzed for mercury content by Cold-Vapor Atomic Absorption Spectrophotometry. No statistically significant increase in blood mercury levels was detected at 7 and 30 days after placement of an amalgam retroseal as compared with preoperative levels (p = 0.97). Findings support the hypothesis that placement of an amalgam retroseal does not result in significant elevations of blood mercury levels. PMID- 8618098 TI - Subpontic hyperostosis. AB - Two cases of subpontic hyperostosis are presented. This condition is an intraoral bony exostosis that has been observed beneath a fixed partial denture. Subpontic hyperostosis seems to be associated exclusively with the posterior mandible, usually the first molar position. The first case occurred in a 64-yr-old Asian male and the second appeared bilaterally in a 79-yr-old female. It has not been described extensively in the literature, with only 33 cases having been reported previously. The etiology of this condition is unknown, with factors such as genetic predisposition, mechanical stress, and inflammation suggested as possible causes. Treatment of this hyperostosis is be surgical excision, and if necessary is usually due to the impingement of the growth on the pontic and the inability of the patient to maintain adequate oral hygiene in the area. PMID- 8618100 TI - Search images: selective attention to specific visual features of prey. AB - In 3 experiments, pigeons (Columba livia) searched a digitized image of a gravel patch on a computer monitor for cryptic grains. Experiment 1 demonstrated that the bird's ability to detect a type of grain improved over successive encounters, and detection of alternative targets was attenuated when a search image was active. Experiment 2 demonstrated search-image effects independently for the 2 grains. Perception was biased to detect wheat grains after wheat encounters and was biased to detect beam grains after bean encounters. Experiment 3 demonstrated that when a search image was activated, selective attention was heightened to the visual features of the grain used to discriminate if from the multicolor gravel background. These results provide strong support for the view that search images are selective attention to specific visual features of cryptic prey. PMID- 8618101 TI - Object representation in the bottlenose dolphin (Tursiops truncatus): integration of visual and echoic information. AB - A dolphin performed a 3-alternative matching-to-sample task in different modality conditions (visual/echoic, both vision and echolocation: visual, vision only; echoic, echolocation only). In Experiment 1, training occurred in the dual modality (visual/echoic) condition. Choice accuracy in tests of all conditions was above chance without further training. In Experiment 2, unfamiliar objects with complementary similarity relations in vision and echolocation were presented in single-modality conditions until accuracy was about 70%. When tested in the visual/echoic condition, accuracy immediately rose (95%), suggesting integration across modalities. In Experiment 3, conditions varied between presentation of sample and alternatives. The dolphin successfully matched familiar objects in the cross-modal conditions. These data suggest that the dolphin has an object-based representational system. PMID- 8618099 TI - Visual search for natural grains in pigeons (Columba livia): search images and selective attention. AB - The experiments reported here were designed to test the suggestion of many researchers that selective attention to visual features of a prey can account for search-image effects. In 3 experiments pigeons ate wheat and vetch grains presented on multicolored and gray gravel trays. In Experiment 1 search-image effects were evident when grains were cryptic but not when they were conspicuous. Experiment 2 demonstrated that search images can be activated when the grains encountered are either cryptic or conspicuous but that search images affect search performance only when the grains are cryptic. Experiment 3 demonstrated that search images are short-term in nature: A 3-min delay between successive encounters with a type of grain disrupted an activated search image. The discussion addresses how these results further develop a model in which search images are viewed as selective attention to visual features of a prey. PMID- 8618102 TI - Averaging temporal duration and spatial position. AB - Pigeons and humans performed on a task in which spatial position and elapsed time redundantly signaled the availability of reward. On each training trial, a landmark moved steadily across a monitor screen. After a fixed amount of time and movement, reward was available for a response. On occasional unrewarded tests, the landmark moved at 0.50, 0.75, 1.00, 1.50, or 2.00 times the training speed. In both pigeons and humans, the central tendency in the response distribution on tests differed across speeds, when measured in terms of both elapsed time and landmark position. Pigeons and humans seem to average a duration of time and a spatial position to find a single criterion time-place corresponding to the expected time-place of reward. PMID- 8618103 TI - The pigeon's recognition of drawings of depth-rotated stimuli. AB - Four experiments used a four-choice discrimination learning paradigm to explore the pigeon's recognition of line drawings of four objects (an airplane, a chair, a desk lamp, and a flashlight) that were rotated in depth. The pigeons reliably generalized discriminative responding to pictorial stimuli over all untrained depth rotations, despite the bird's having been trained at only a single depth orientation. These generalization gradients closely resembled those found in prior research that used other stimulus dimensions. Increasing the number of different vantage points in the training set from one to three broadened the range of generalized testing performance, with wider spacing of the training orientations more effectively broadening generalized responding. Template and geon theories of visual recognition are applied to these empirical results. PMID- 8618104 TI - Selective incorporation of architectural proteins into terminally differentiated molluscan gill cilia. AB - Incubation of excised gills from the bay scallop Aequipecten irradians with 3H leucine demonstrates that many ciliary structural proteins can attain a degree of labeling approaching that previously reported for sea urchin or surf clam embryos undergoing ciliary turnover or regeneration. This labeling is not a consequence of any predominant incorporation into new cilia at the meristematic growth tips of the gill since tissue regions of varying maturity incorporate label into the same proteins at similar levels, with the most mature region having the highest incorporation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analysis of isolated cilia, separated into detergent-soluble membrane/matrix and detergent-insoluble 9+2 axoneme fractions, reveals that 1) tubulin in the membrane/matrix fraction is labeled whereas tubulin in the axoneme is not; 2) no labeled dynein heavy chains are seen in either fraction; 3) the most heavily labeled axonemal components do not appear to any significant extent in the membrane/matrix fraction; and 4) after thermal depolymerization of the microtubules, nearly all labeled proteins reside in the in-soluble ninefold ciliary remnant, the most prominent being tektin A, an integral component of outer doublet microtubules. Further fractionation of the remnant with sarkosyl urea to produce tektin filaments demonstrates two solubility classes of tekin A, only the more soluble of which is labeled. Very similar selective architectural protein labeling patterns have been reported for steady-state cilia of sea urchin embryos, and this may indicate a widespread turnover or exchange mechanism characteristic of cilia heretofore considered static. PMID- 8618106 TI - Unresolved Issues in Beta-Lactam Therapy. Proceedings of the satellite symposium from the 9th Mediterranean Congress of Chemotherapy. Milan, Italy, 13-17 June 1994. PMID- 8618107 TI - Once-a-day cephalosporins: reality or myth? AB - The factors that may influence the pharmacodynamics of antimicrobial agents against microorganisms at the site of infection must be considered before using once-daily antimicrobial chemotherapy in clinical practice. From a pharmacokinetic point of view, we can establish which antibiotic is suitable for once-daily therapy. For aminoglycosides and quinolones, where the bacterial killing is rapid and dose dependent, and there is a post-antibiotic effect (PAE), the pharmacodynamic objective is to maintain the tissue levels way above the minimum inhibitory concentration (MIC) for a short period. This can be achieved by giving single bolus doses at long time intervals. With beta-lactam antibiotics, however, which have a slow time-dependent antibacterial effect and do not display a PAE, the aim is to keep the antibiotic concentration above the MIC for the duration of therapy. PMID- 8618105 TI - Analysis of R59022 actions in Xenopus laevis oocytes. AB - R59022, a diacylglycerol (DAG) kinase inhibitor, stimulated meiotic maturation of Xenopus laevis oocytes when applied extracellularly. The time course of R59022 induced oocyte maturation was proportional to the concentration of R59022 in the low micromolor range, and the 30 microM-induced response was a fast or faster than progesterone-induced maturation. Dose-response analysis yielded an apparent EC50 for R59022-induced oocyte maturation of approximately 15 microM. An increase in total oocyte DAG levels was observed following treatment with 10 microM R59022. Treatment of oocytes with R59022 also resulted in a significant increase in intracellular pH similar to the increase observed with progesterone. When various phosphodiesterase (PDE) inhibitors were tested for their effects on R59022-induced oocyte maturation, papaverine (a potent nonselective inhibitor of PDE) and CI-930 (a selective PDE III inhibitor) were observed to significantly inhibit the R59022-stimulated response. The sensitivity of R59022-induced oocyte maturation to inhibition by papaverine was intermediate between the sensitivities of the IGF-1- or progesterone-induced responses. Treatment of oocytes with R59022 did not significantly affect the level of oocyte PDE activity measured in vivo, suggesting that elevated levels of DAG may parallel observed increases in PDE but do not directly lead to a stimulation of PDE. The t ime course for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation by progesterone rather than IGF-1. Treatment of isolated membranes with R59022 resulted in inhibition of membrane-associated adenyl cyclas e activity that was not mimicked by DAG analogs. Thus, in addition to elevating oocyte levels of DAG, R59022 also has steroid-like actions. PMID- 8618108 TI - Amoxicillin/clavulanic acid vs cefetamet pivoxil in the treatment of acute exacerbation of chronic bronchitis (AECB) in adults. AB - In this open comparative and prospective study 180 adults of either sex were randomised to treatment with either amoxicillin/clavulanic acid (AMC) 500/125mg tid or cefetamet pivoxil (CAT) 500mg bid for 7 days. Demographic data and assessable findings were similar in both groups. Clinical outcomes of 169 assessable patients showed high efficacy of both drugs: 92% with AMC and 96% with CAT. Bacteriological response rates were equivalent in 141 evaluable cases: 84% vs. 89%, respectively. Baseline susceptibility testing (DIN) revealed a notable number of Haemophilus species either intermediately susceptible or resistant to AMC. Gastrointestinal disorders predominated among the adverse events with diarrhea occurring nearly twice as often in the AMC group. CAT is an effective and safe alternative option in the treatment of AECB in adults. The advantage of CAT is its enhanced activity against gram-negative bacteria. It is well tolerated. PMID- 8618109 TI - Cefetamet pivoxil in the treatment of pharyngotonsillitis due to group A beta hemolytic streptococci: preliminary report. AB - Penicillin therapy has considerably reduced the occurrence of serious late complications of streptococcal pharyngotonsillitis. However, treatment failure with this antibiotic is currently reported in up to 30% of the cases. Beta lactamase production by the commensal flora of the tonsils, and poor compliance of patients have been implicated as the main causes of treatment failure. Cefetamet pivoxil is a new oral cephalosporin with a twice daily dosage and striking stability against beta-lactamases. We are conducting a prospective, randomized study in 120 children, comparing the efficacy of cefetamet pivoxil 10 mg/kg bid for 5 and 10 days and phenoxymethyl penicillin 25,000 U/kg tid for 10 days in the treatment of group A beta-hemolytic streptococcus (GABHS) pharyngotonsillitis. Children are enrolled after the positive culture of a throat swab and randomly assigned to one of the three groups. A follow-up check-up is performed at the end of the therapy (clinical check-up) and 3 to 5 days later (bacteriological check-up). The preliminary analysis of 55 cases shows that in 88.9%, 100% and 87.5% of children GABHS was eradicated by 5 days or 10 days of cefetamet pivoxil, or 10 days of penicillin respectively (p = NS). Side effect were mild and transient in cefetamet pivoxil-treated patients, but required cessation of treatment in 2 children treated with penicillin. These results suggest that cefetamet pivoxil therapy for 5 days eradicates GABHS from the throat in streptococcal pharyngotonsillitis as efficiently as cefetamet pivoxil or phenoxymethyl penicillin for 10 days. PMID- 8618111 TI - The beta-lactamase problem: new therapeutic options. AB - As a consequence of their successful use in prophylaxis and therapy, bacterial resistance mediated by beta-lactamases is now widely diffused among beta-lactam antibiotics. Several effective strategies have been suggested in order to overcome this problem. One interesting option is offered by the development of a series of new beta-lactam compounds that possess a very high intrinsic stability to the hydrolytic action of the most common beta-lactamases. Among these molecules the oral third generation cephalosporins represent a significant breakthrough. Cefetamet pivoxil, because of its broad coverage of most gram negative and gram-positive community acquired pathogens, rightly belongs to these new agents. The activity of cefetamet has been confirmed in a survey in Italy involving 4191 isolates. on this collection of strains cefetamet emerged as the most active in vitro compound, followed by cefixime, with all other comparative agents (cefuroxime, cefaclor, cephalexin, cefradoxil, ampicillin, amoxicillin clavulanate, ampicillin-sulbactam, doxycycline, erythromycin and clindamycin) displaying lower eradication rates. According to the data gathered in the Italian survey, cefetamet can be considered the only compound, among those taken into consideration, that might be selected as the drug of choice in the empiric therapy of respiratory and urinary community-acquired infections. In fact, the prevalence of resistance to cefetamet in the most prevalent pathogens occurring in this setting is, at present, sufficiently low to render therapeutic failures, based on this parameter, highly improbable. PMID- 8618110 TI - Betalactam therapy and intestinal flora. AB - Betalactams, mainly when orally administered, may lead to intestinal flora modifications related to their spectrum of activity, rate of absorption and degradation. therefore it is important to investigate the possible influence of recently developed oral cephem derivatives on normal human microflora. We have investigated the impact on normal human intestinal flora in a 10-day course with cefetamet-pivoxil (CET, 500 mg BID) in comparison to cefixime (CFX, 400 mg qD) or cefuroxime axetil (CA, 250 mg BID) in 24 patients suffering from acute exacerbation of chronic bronchitis. Stool specimens were taken before (day 0), at the end (day 10) and 14 days after treatment (day 24) and quali-quantitative microflora composition was determined with a detection limit of 10 CFU/g dry weight. Treatment with CET caused slight and non-significant modifications of normal intestinal flora. On the contrary CFX and CA significantly affect Enterobacteriaceae and clostridia with a concomitant increase in enterococci for CFX. With both CFX and CA there was a new appearance of Salmonella spp. as well as Clostridium difficile in 4 and 2 cases, respectively. Therefore CET seems to affect normal bowel flora minimally in comparison to other oral cephalosporins. This aspect might contribute to the low incidence of GI related side effects in patients treated with CEt for longer than 1 week. PMID- 8618113 TI - Dental radiology quality of care: the dentist makes the difference. PMID- 8618112 TI - Third generation oral cephalosporins: comparative in vitro kinetics. AB - In order to provide additional data on the in vitro antibacterial activity of cefetamet-pivoxil against respiratory pathogens, we determined the bactericidal kinetics of this antibiotic in comparison to cefixime and ceftibuten against H. influenzae, M. catarrhalis, K. pneumoniae, E. coli, S. pneumoniae and S. pyogenes. time-kill studies were performed by using concentrations equal to a x MIC, and 4 x MIC of the antibiotics tested and different inocula (10(5), 10(7) and 10(9) CFU/ml). The strains were incubated in a shaking incubator at 37 degrees C and 1 ml samples were removed at regular intervals for viable count determination. The antibiotics were eliminated by serial ten-fold dilutions in physiological saline before plating. At 4 x MIC of cefetamet and at 10(5) CFU/ml inoculum the results were 99.9% killing or more of H. influenzae and M. catarrhalis at 4 h, and of E. coli and K. pneumoniae within 4 to 6 h, and of E. coli and K. pneumoniae within 4 to 6 h. At the same concentration of MIC and with the same inoculum a 99.9% reduction was achieved against S. pneumoniae within 4 h and against S. pyogenes within 2 h with no regrowth at 24 h in both cases. Similar results were obtained using 10(7) and 10(9) CFU/ml inocula. Cefetamet had efficient killing activity even at lower concentrations. Bactericidal kinetics of cefetamet favorably compare with those of cefixime and ceftibuten. The efficient bactericidal activity of cefetamet-pivoxil indicates a clinical role for this new oral cephalosporin in the treatment of respiratory tract infections. PMID- 8618115 TI - Bilateral radiolucency and radiopacity of the mandible. PMID- 8618114 TI - Infection control in dental radiology. AB - Although exposure to blood is rare in oral and maxillofacial radiology, contact with saliva occurs. Thus the spread of infectious diseases is possible through cross-contamination, and specific infection control protocols and unit dosing of items are needed. This article outlines rationale for implementing state-of-the art infection procedures; and explains federal standards and guidelines with an impact on infection control and occupational safety in dental radiology procedures. PMID- 8618116 TI - Radiopacity in the posterior mandible. PMID- 8618117 TI - Risks from dental radiation in 1995. AB - Risks from dental radiation are primarily to the somatic tissues of exposed persons, although the genetic tissues may receive small amounts of radiation. Maximum permissible doses have been established at various levels depending on the tissue exposed and whether the exposure was occupational or non-occupational: for pregnant workers, the occupational exposure is 1/10 the normal limits. Risks from dental radiation is expressed as the number of extra fatal cancers per million exposed persons, referred to as relative risk. Generally, the relative risk is tenfold greater for the full mouth survey than for a simple panoramic radiograph. Several ways of reducing the risk, to patients as well as healthcare workers, are outlined. PMID- 8618118 TI - Graduate nurse overhires. A cost analysis. AB - Traditionally, the process for filling vacated nursing positions has contributed to a hiatus between the departure of one nurse and the arrival of another who is prepared to assume patient care responsibility. This is due at least partially to limited work force renewal strategies. Given the cost to temporarily fill vacated positions with agency nurses or to pay nurses overtime rates, current methods of replacing departing nurses may no longer be practical in all cases. The authors discuss the strategy of overhiring graduate nurses and compare these costs to more traditional temporary methods of replacing nurses who have resigned. PMID- 8618119 TI - Characterization of an acute inpatient hospice palliative care unit in a U.S. teaching hospital. AB - An existing hospice palliative care inpatient unit was studied from September 1993 to November 1993 to characterize it for two qualities: demographics of admitted patients and family satisfaction after discharge. The purpose of this study was to identify current uses of the unit and determine whether the high level of satisfaction among family members, as perceived by the staff, was accurate. To characterize demographics, 100 consecutive admissions to the unit were assessed prospectively beginning in September 1993. To characterize family satisfaction, a survey was sent to the families of 240 patients who had been cared for on the inpatient unit. Since this study, it has been identified that care on the hospice palliative care unit is provided at a 50% reduction in daily hospital charges. The hospital has benefited from establishing and maintaining an acute care inpatient hospice palliative care unit. Other academic medical centers in the United States should consider a designated unit for symptom management and terminal care as part of their comprehensive range of healthcare services. PMID- 8618120 TI - The Harvard Nursing Research Institute's conference summary. AB - The Harvard Nursing Research Institute organized a national invitational conference on executive nursing leadership in major teaching hospitals and academic health centers. The conference brought together many of the nation's eminent nurse executives and other prominent individuals in healthcare to analyze the unique and complex challenges facing these organizations, expose participants to other perspectives, and articulate specific aims and strategies that might be taken to increase their effectiveness in leading the nursing profession's efforts to shape the ongoing transformation of the healthcare system. PMID- 8618121 TI - Manager caring remains an important consideration. PMID- 8618122 TI - Improving nursing practice, education, and research. AB - Nurse executive leadership in academic health centers is essential to the improvement of nursing education, practice, and research. The author raises questions to highlight the dilemmas in which nursing finds itself at this time of dramatic transformation of health systems. The framework of the "learning organization" is used to examine the way in which leaders are defined in nursing and the way in which they perceive their roles. Comparisons are drawn between educational and practice administrators, and similarities and differences between these two groups are discussed. Integrative faculty practice roles are summarized, and innovations in advanced nursing practice are described briefly. Specific aspects of the research enterprise discussed are collaboration among members of the nursing discipline and examination and evaluation of quality-of care issues. For improvement to occur, the author advocates involved, participatory problem solving by nurse colleagues in education and practice. PMID- 8618123 TI - Integration of emerging designs for the practice and management of nursing. AB - Work redesign and shared governance represent two of the most popular administrative innovations in contemporary nursing. Whereas work redesign creates changes in the content of nurses' jobs, shared governance addresses the organizational context within which nurses are employed. Although the content and context of nurses' work are closely interrelated, many organizations have attended to one of these issues, but failed to give consideration to the other. Building on this background, the state of the art in nursing redesign and restructuring is summarized, and emerging directions in job and organizational design are identified. PMID- 8618124 TI - Noise in the hospital: a quality improvement approach. PMID- 8618125 TI - Computer use for work accomplishment. A comparison between nurse managers and staff nurses. AB - Studies on computers in nursing typically observed nurses' perceptions toward computers. This study examined the computer use of 528 nurses in three urban teaching hospitals, and compared nurse managers' with staff nurses' time spent at the computer and systems frequently used for work accomplishment. Differences were found in their experiences with computerized information systems (CISs) and expectations of CIS use. Although there was striking similarity in the systems used, nurse managers had significantly greater access to computers and technical support. Findings suggest the importance of integrating clinical and managerial tasks if unit work is to be accomplished efficiently and effectively. PMID- 8618126 TI - Newborn readmissions. PMID- 8618127 TI - Managing diverse needs of the technical coworker. PMID- 8618128 TI - Telecommuters. PMID- 8618129 TI - Advanced practice nurses. PMID- 8618130 TI - Nurses go to the home. PMID- 8618131 TI - Diallyl disulfide suppresses the growth of human colon tumor cell xenografts in athymic nude mice. AB - The present studies examined the anti-proliferative effects of diallyl disulfide (DADS) on the growth of human colon tumor cell line, HCT-15, xenografts in 6-wk old female NCr nu/nu mice with an initial body weight of 20-22 g. Intraperitoneal injection of 1 mg DADS thrice weekly reduced tumor volume by 69% (P < 0.05) without apparent ill consequences such as altered growth of the host. Providing this quantity of DADS intragastrically also inhibited growth of the HCT-15 tumor. At equivalent DADS dosages, intraperitoneal treatment was proportionately more effective (P < 0.05) in reducing tumor growth than gastric intubation. Tumor inhibition caused by DADS (0.5 mg thrice weekly) was similar to that occurring with 5-fluorouracil (5-FU) treatment (0.5 mg thrice weekly). Combining DADS and 5 FU was no more effective in inhibiting tumor growth than using either compound alone. However, concurrent DADS treatment significantly (P < 0.05) inhibited the depression in leukocyte counts and spleen weight and prevented the elevated plasma urea caused by 5-FU treatment. These data suggest that DADS, a constituent of garlic oil, reduces the toxicity of 5-FU and is an effective antitumorigenic agent against xenografts resulting from an established human colon tumor cell line. PMID- 8618132 TI - Probiotics, cecal microflora, and aberrant crypts in the rat colon. AB - Our hypothesis was that administration of bifidobacteria, Lactobacillus acidophilus or both to rats will minimize the numbers of aberrant crypts in the distal colon that develop in response to the carcinogen 1,2-dimethylhydrazine (DMH). A series of experiments was designed to test this hypothesis where the treatments used were as follows: skim milk controls (Skim-Basal), skim milk + bifidobacteria (Bifido-Basal), skim milk + fructooligosaccharide (Skim-FOS), and skim milk + bifidobacteria + fructooligosaccharide (Bifido-FOS). In two experiments, bifido-bacteria + FOS administration significantly decreased the number of aberrant crypts that developed, but there was no clear relationship of aberrant crypts to numbers of bifidobacteria or Clostridium perfringens. In the third experiment, the Bifido-FOS treatment led to significantly fewer aberrant crypts and aberrant crypt foci than the Bifido-Basal treatment. The Skim-FOS group had significantly more cecal bifidobacteria than the Skim-Basal group and significantly fewer C. perfringens than the Skim-Basal and Bifido-Basal. In a fourth experiment, L. acidophilus was added as an additional treatment. The number of aberrant crypts was not significantly different among the groups. However, the number of C. perfringens was significantly decreased by the addition of bifidobacteria, L. acidophilus or the combination of the two, whereas the numbers of bifidobacteria and L. acidophilus were not affected by treatment. A significant correlation (R2 = 0.84, P < 0.01) was noted between the body weight of rats at DMH administration and the magnitude of the difference in aberrant crypts between the Skim-Basal rats and the Bifido-FOS rats. The results suggest that there is variability in the effects of bifidobacteria and L. acidophilus administration on both aberrant crypt formation and C. perfringens. PMID- 8618133 TI - Oat, wheat or corn cereal ingestion before exercise alters metabolism in humans. AB - This study was designed to determine metabolic and physical performance responses to ingestion of pre-exercise meals with different macronutrient and fiber profiles. Twelve physically active subjects (6 males and 6 females) were used to investigate the metabolic and physical performance consequences of consuming pre exercise meals consisting of oat, corn, or wheat cereals. A fasting trial served as the control, and all subjects received each treatment in a Latin-square design. Blood samples were drawn before and 85 min after meal ingestion, during 90 min of cycling exercise (60% VO2peak), after a 6.4 km performance ride, and during 60 min of recovery. Expired air samples were collected to determine nutrient utilization. Resting carbohydrate oxidation rates and plasma insulin concentrations after oat ingestion were less than after wheat, and corn and wheat ingestion, respectively (P < 0.05). During exercise, the change in plasma glucose from pre-exercise was greater after consuming wheat and corn compared with oat (P < 0.05), and it was inversely related to pre-exercise plasma insulin concentration (r = -0.55, P = 0.0001). Plasma free fatty acid concentrations were inversely related to plasma lactate concentrations (r = -0.58, P = 0.0001). Free fatty acid concentrations and fat oxidation were greater in fasting trials than all others, but performance ride times did not differ among treatments. Plasma branched-chain amino acid concentrations resembled their respective meal profiles throughout exercise, the performance ride, and recovery. These results indicate that pre-exercise meal composition can influence glucose homeostasis during early exercise and plasma branched-chain amino acid concentrations over a substantial range of metabolic demands. PMID- 8618134 TI - Nutritional quality of a high carbohydrate diet as consumed by children: The Bogalusa Heart Study. AB - To study the nutritional adequacy of a high carbohydrate diet as consumed by children in a 24-h period, a sample of 568 10-y-old children was stratified into four levels of carbohydrate intake: < 45% of total energy (kJ), 45%-50% energy, 50%-55% energy, and >55% energy. Composition of the diet for those consuming >55% energy from carbohydrates was adequate in total energy, with 29% energy from fat, 11 % energy from saturated fatty acid, an average of 88 mg dietary cholesterol/4200 kJ and less than 10% energy from sucrose. Increased carbohydrate intake was attributed to an increased consumption of total sugars, starch and fiber. The percentage of children meeting two thirds or more of the Recommended Dietary Allowances for niacin and zinc was lower in the high carbohydrate intake group than in any of the low carbohydrate intake groups. Children with a high carbohydrate intake consumed more fruits, breads, grains, milk, desserts, candy and non-dairy beverages than those with a lower carbohydrate intake who consumed more meats. To meet current dietary recommendations for increased carbohydrate intake when meal patterns are designed, it is necessary to incorporate adequate amounts of foods from all of the food groups to maximize nutritional quality. PMID- 8618135 TI - Vitamin E deficiency and immune dysfunction in retrovirus-infected C57BL/6 mice are prevented by T-cell receptor peptide treatment. AB - Female C57BL/6 mice were infected with LP-BM5 retrovirus, causing murine acquired immunodeficiency syndrome (AIDS), which is functionally similar to human AIDS. Retrovirus infection inhibited release of T-helper 1 cytokines, stimulated secretion of T-helper 2 cytokines and induced hepatic and cardiac vitamin E deficiency with increased lipid peroxides. We hypothesized that the immune dysfunction caused increased oxidation and loss of vitamin E. Because T-cell receptor (TCR) peptide treatment blocked the excessive stimulation of a T-cell subset by retroviral superantigens, we tested whether maintenance of normal immune function during infection prevented excessive oxidative damage. The TCR peptide treatments with doses > 100 microgram/mouse and administered 2-4 wk postinfection significantly inhibited the retrovirus-induced immune dysfunction, concomitantly reduced tissue oxidative damage and thereby largely maintained vitamin E concentration in the liver and heart. Reducing the dose of peptide or delaying administration until early murine AIDS had developed resulted in severe immune dysfunction that caused elevated tissue lipid peroxidation and loss of vitamin E. The TCR peptide treatment partially maintained production of interleukin-2 (IL-2) and prevented retrovirus-induced elevated production of IL-6 by splenocytes in vitro. In conclusion, TCR peptide treatment during murine retrovirus infection ameliorated immune dysfunction and thus prevented increases in tissue lipid peroxidation and vitamin E loss. T-cell immune dysfunction and its prevention by TCR peptide treatment is important in the therapy of vitamin E deficiency induced by retrovirus infection. PMID- 8618136 TI - Cyclic food restriction alters substrate utilization and abolishes protection from mammary carcinogenesis female rats. AB - We tested the hypothesis that cyclic food restriction abolishes protection against mammary carcinogenesis. Virgin female Sprague Dawley rats (n = 159) were injected intraperitoneally with 25 mg/kg n-methyl-n-nitrosourea at 50 d of age. Eleven days later, rats were given free access to a 24.6 g fat/100 g AIN-76A diet (ad lib-c), fed in two meals (me-c), or fed in two meals restricted in weight by 33% for 1 wk followed by 3 wk of compensatory refeeding (me-r) for 18 wk or 4.5 restriction cycles. Energy and substrate utilization of 15 rats from each group was measured by indirect calorimetry. The me-r rats ate and weighed less (P < 0.0001), had a greater efficiency of food utilization (P < 0.01), and had a 12% higher incidence of mammary cancer (P < 0.0001) than ad lib-c rats after adjusting for the effect of final body weight. Resting metabolic rate was not different among groups, but me-r rats used less glucose during restriction and more glucose and less lipid for energy during body weight recovery than me-c rats (P < 0.0001). Increased energy efficiency and the shift in utilization of glucose and fatty acids followed closely the effects of cyclic food restriction and meal feeding on mammary carcinogenesis. PMID- 8618137 TI - Calcium bioavailability from bovine milk and dairy products in premenopausal women using intrinsic and extrinsic labeling techniques. AB - Stable isotopes were used to compare calcium fractional absorption from intrinsically and extrinsically labeled bovine milk as well as intrinsically labeled dairy product and cheese analogue. Healthy Caucasian women were fed a controlled diet for 4 d during the follicular phase of their menstrual cycle. With breakfast on the third day, participants ingested milk containing 44Ca (intrinsic) and 42CaCl2 (extrinsic) or dairy products containing 44Ca. Total feces were collected for 2 d prior to and 10 d after isotope ingestion. Polyethylene glycol was administered to monitor completeness of fecal collections. Total calcium was determined by atomic absorption spectrophotometry, and isotopic abundance was determined by high resolution fast atom bombardment mass spectrometry. Fractional absorption was determined as the difference between the administered isotopic dose and the quantity of 44Ca or 42Ca excreted in feces. The fractional absorption of calcium from milk was not affected by the method of labeling, lactose content, fermentation or the chemical form of calcium in dairy products or cheese analogue. PMID- 8618138 TI - Oral supplementation with branched-chain amino acids improves transthyretin turnover in rats with carbon tetrachloride-induced liver cirrhosis. AB - The hypothesis that dietary branched-chain amino acid (BCAA) supplementation improves the impaired protein turnover in male Donryu rats with carbon tetrachloride-induced liver cirrhosis was tested. We supplemented cirrhotic rats orally for 2 wk with BCAA solution [26.67 mg BCAA/(100 g body weight . d)], a conventional amino acid mixture [4.25 mg BCAA/(100 g body weight . d)] or saline and fed these three groups the AIN76 basal diet to have similar intakes of total energy and total nitrogen. Normal rats without liver cirrhosis were fed the basal diet similar to the above (noncirrhotic controls). After supplementation, rats were fed intravenous transthyretin (thyroxine-binding prealbumin) doubly labeled with 125I-tyramine-cellobiose and 131I. Kinetic indices including production rate of transthyretin were analyzed from plasma transthyretin disappearance curves. Tissue sites of transthyretin degradation were assayed using a trapped ligand technique by measuring 125I-tyramine-cellobiose levels. The production rate of transthyretin was significantly lower in cirrhotic rats supplemented with saline (mean 25.46 X 10(-3) . h(-1)) compared with noncirrhotic controls (45.08 X 10(-3) . h(-1)) (P < 0.05). This was corrected by supplementing cirrhotic rats with BCAA (37.05 X 10(-3) . h(-1), P < 0.05) but not with conventional amino acid mixture (22.49 X 10(-3) . h(-1)). The accelerated degradation of transthyretin in muscles of cirrhotic rats was improved by BCAA (P < 0.05). In conclusion, dietary supplementation with BCAA improves the impaired transthyretin turnover in rats with liver cirrhosis. PMID- 8618141 TI - Methionine and 2-hydroxy-4-methylthiobutanoic acid are partially converted to nonabsorbed compounds during passage through the small intestine and heat exposure does not affect small intestinal absorption of methionine sources in broiler chicks. AB - Broiler chicks were fed diets supplemented with DL-methionine or DL-2-hydroxy-4 methyl-thiobutanoic acid. At 4 wk of age the chicks were subdivided into thermoneutral (22 degrees C) and heat-exposed (32 degrees C) groups and maintained under these conditions for 48 h. Highly purified 3H-L-methionine (3H-L Met) and 3H-L-2-hydroxy-4-methyl-thiobutanoic acid (3H-L-HMB) were used to evaluate treatment effects on the small intestinal passage of sources of supplemental methionine and on the transport of methionine sources across purified small intestinal brush border vesicles. 3H-L-Met was efficiently absorbed in the upper regions of the small intestine; however, 2.5-3.5% of dietary 3H from birds fed 3H-L-Met remained unabsorbed in the distal small intestine. Dietary 3H (15%) initially associated with 3H-L-HMB was not absorbed during passage down the length of the gut. The HPLC analysis indicated that only 10% of the radiolabeled material remaining in the terminal ileum eluted at the time expected for HMB. Partial breakdown of HMB to nonabsorbed, nonmethionine products during passage down the small intestine may contribute to the difference in biopotency of the two sources of supplemental dietary methionine. Heat exposure did not affect in vivo small intestinal passage or in vitro transport of 3H-L-Met and 3H-L-HMB across small intestinal brush border membrane vesicles. PMID- 8618140 TI - Dietary fiber and short-chain fatty acids affect cell proliferation and protein synthesis in isolated rat colonocytes. AB - Colonic metabolism may be affected by dietary fiber and short-chain fatty acids, the products of fiber fermentation. The aim of this study was to assess the effects of fiber supplementation (150 g/kg diet) on dynamic measurements of metabolism in isolated rat colonic epithelial cells. Additionally, we investigated the effect of in vitro short-chain fatty acid and glutamine concentrations and media osmolarity on oxygen uptake, protein synthesis, cell proliferation and anaplerotic flux. Colonocyte oxygen consumption did not differ due to fiber supplementation or the inclusion of short -chain fatty acids in incubation media. Cell proliferation (3H-thymidine uptake) was increased by fiber consumption (P 0.05). Therefore, capsule afferents continue to behave normally in joints in which the anterior cruciate ligament has been transected. PMID- 8618154 TI - Uniaxial tension inhibits tendon collagen degradation by collagenase in vitro. AB - Tendon structure is governed largely by factors regulating the anabolic and catabolic phases of tenocyte metabolism. Little is known about the mechanisms that regulate the synthesis, activation, and action of metalloproteinases, which are key enzymes in a multifactorial cascade controlling homeostasis of the extracellular matrix. In the present study, we investigated the effect of tension on collagenase-induced degradation of the tendon in vitro by assessing changes in structural and material properties measured during tensile failure tests. Devitalized right-left pairs of rabbit patella-patellar tendon-tibia units were maintained under culture conditions in the presence of 60 U/ml highly purified collagenase for 20 hours. One randomly selected unit from each animal was subjected to a tension that produced a constant 4% elongation or strain (n = 10); the contralateral unit served as a slack comparison (n = 10). In one series of experiments (immediate, n = 5), the tension was applied immediately prior to collagenase exposure. In a second series (delayed, n = 5), it was delayed for 4 hours to allow time for the collagenase to diffuse into the tendon. Additional devitalized and nonincubated units (n = 6) were used as normal controls. Collagenase exposure caused large decreases in stiffness and elongation to failure in slack units. This resulted in greater than 80% reductions in both maximum failure force and energy to failure. In contrast, the loaded unit in both experimental protocols had significantly greater stiffness than control units. In both the immediate and the delayed protocols, the loaded tendons had significantly higher stiffness and failed at significantly higher elongations and maximum forces than the slack tendons. Diffusion studies with and without tension showed the tension did not inhibit diffusion of collagenase into the tendon but did significantly decrease the water content from 64.6 to 57.8%. The data suggest that stresses and strains of the extracellular matrix may modify the kinetics of the bacterial collagenase-collagen interaction. Matrix stress and strain may be an important and overlooked factor that modulates the susceptibility of collagen to proteolytic degradation. PMID- 8618155 TI - Equal effectiveness of electrical and volitional strength training for quadriceps femoris muscles after anterior cruciate ligament surgery. AB - Neuromuscular electrical stimulation and voluntary muscle contraction are two exercise modes widely used in rehabilitation to strengthen skeletal muscle. Since there is no debate as to which mode is most effective, we compared electrical stimulation with voluntary contraction performed at matched intensities following reconstructive surgery of the anterior cruciate ligament. Forty men and women, aged 15-44, were randomly assigned to either an electrical stimulation or a voluntary contraction group. None of the subjects had a previous history of neuromuscular injury. The subjects received treatment for 30 minutes a day, 5 days a week, for 4 weeks. Knee extension torque was monitored during treatment to try to match the absolute muscular tensions (quantified as "activity") achieved during therapy. To match the activity of the subjects in the electrical stimulation group, who were treated at the highest stimulation intensity they could tolerate, the subjects in the voluntary contraction group were paced at progressively increasing intensities corresponding to 15, 25, 35, and 45% of the injured limb's maximum voluntary torque during weeks 1, 2, 3, and 4, respectively. We found no significant difference between the groups in terms of maximum voluntary knee extension torque throughout the study period. In addition, 1 year after surgery, there was still no significant difference between groups with regard to knee extension torque (p > 0.4). These data suggest that neuromuscular electrical stimulation and voluntary muscle contraction treatments, when performed at the same intensity, are equally effective in strengthening skeletal muscle that has been weakened by surgical repair of the anterior cruciate ligament. PMID- 8618156 TI - Reproducibility of isometric trunk extension torque, trunk extensor endurance, and related electromyographic parameters in the context of their clinical applicability. AB - Testing the capacity of the trunk extensor muscles may be useful in the diagnosis of low back pain. In the present study, the reproducibility of measurements of maximum trunk extension force, trunk extension endurance, and related electromyographic parameters was investigated. Intraclass correlations indicated that the reproducibility of maximum force and endurance time was satisfactory. Nevertheless, the smallest difference in these parameters that could be attributed, with 95% confidence, to a change in the condition of a patient was, in general, more than 20%. On the electromyograms, the slopes of amplitude and frequency content appeared to be related to endurance time. The reproducibility of these parameters in terms of the intraclass correlation was again satisfactory; however, the smallest detectable difference generally exceeded 50%. The clinical applicability of the parameters studied is severely limited by a lack of reproducibility. PMID- 8618157 TI - Hamstrings and psoas lengths during normal and crouch gait: implications for muscle-tendon surgery. AB - Crouch gait, one of the most common movement abnormalities among children with cerebral palsy, is characterized by persistent flexion of the knee during the stance phase. Short hamstrings are thought to be the cause of crouch gait; thus, crouch gait is often treated by surgical lengthening of the hamstrings. In this study, a graphics-based model of the lower extremity was used in conjunction with three-dimensional kinematic data obtained from gait analysis to estimate the lengths of the hamstrings and psoas muscles during normal and crouch gaits. Only three of 14 subjects with crouch gait (four of 20 limbs with knee flexion of 20 degrees or more throughout stance) had hamstrings that were shorter than normal by more than 1 SD during walking. Most (80%) of the subjects with crouch gait had hamstrings of normal length or longer, despite persistent knee flexion during stance. This occurred because the excessive knee flexion was typically accompanied by excessive hip flexion throughout the gait cycle. All of the subjects with crouch gait had a psoas that was shorter than normal by more than 1 SD during walking. These results emphasize the need to consider the geometry and kinematics of multiple joints before performing surgical procedures aimed at correcting crouch gait. PMID- 8618158 TI - Restoration of function of the thumb flexor apparatus requires repair of the oblique and one adjacent flexor tendon pulley. AB - Damage to the pulleys of the thumb flexor apparatus may cause bow-stringing of the tendon and affect muscle function. An experiment using the hands and distal forearms of cadavers was designed to determine which damaged pulleys increase excursion length of the flexor tendon with constant tendon and resisting loads. Each specimen was mounted to a loading frame with a dead weight pinned to the tip of the thumb. The thumb flexor tendon was clamped to an actuator that applied a fixed load and measured excursion of the tendon. Ranges of motion of the thumb joint were also measured. The thumb flexor apparatus of each specimen was tested intact first, with the hand in flexed, neutral, and extended positions; then it was tested with progressive sectioning of pulleys from proximal to distal in one group and from distal to proximal in a second group. The length of excursion increased significantly with all pulleys cut but there was no effect on overall range of motion of the thumb. With proximal to distal sectioning, no change in tendon excursion occurred when the flexor retinaculum and the first annular pulleys were cut, until the oblique pulley was sectioned, leaving only the second annular pulley intact (range, 1.17 - 1.31 times that of intact excursion, dependent on position of the hand). With distal to proximal sectioning, tendon excursion was not affected when the second annular and oblique pulleys were cut but did increase when the first annular pulley was sectioned, leaving only the flexor retinaculum intact (range, 1.28 - 1.36 times that of intact excursion). Dependent on the location of damage, therefore, an intact oblique or first annular pulley can maintain normal excursion of the tendon. PMID- 8618159 TI - Mechanical properties of the anterior cruciate ligament chronically relaxed by elevation of the tibial insertion. AB - The effects of stress deprivation on the mechanical properties of the anterior cruciate ligament were studied in a canine model. Fifty-eight mature mongrel dogs were divided into two groups. In the relaxed group (n = 30), the tibial insertion of the anterior cruciate ligament in each right knee was surgically elevated proximally 3 mm from the tibia; the elevation was anatomically reduced in the sham group (n = 28). In order to obtain control data, the left knee in each dog was left untreated. A femur-anterior cruciate ligament-tibia complex was excised from each knee for biomechanical tests at 6 or 12 weeks after surgery. To simplify data analysis, the treat/nontreat ratio (the ratio of the data obtained from the treated knee to that from the nontreated knee) was used. The cross sectional area of the ligament increased significantly in both groups; the area in the relaxed group (average treat/nontreat ratio = 1.37) was significantly larger than that in the sham group (1.16) at 6 weeks but not at 12 weeks. The treat/nontreat ratio of tensile strength in the relaxed group significantly decreased to 0.67 and 0.58 at 6 and 12 weeks, respectively; in the sham group, it significantly decreased to 0.79 at 6 weeks but subsequently increased to 0.87 at 12 weeks. Only at 12 weeks was a significant difference observed between the two groups. This study demonstrated that, in the anterior cruciate ligament, stress deprivation results in a rapid increase in the cross-sectional area, although this effect disappears by 12 weeks, and in a decrease in mechanical strength, although a relatively long period of more than 6 weeks is required for the deterioration. PMID- 8618160 TI - Late changes in bone mineral density of the proximal tibia following total or partial medial meniscectomy. A randomized study. AB - The adaptive bone remodeling in the proximal tibia following medial meniscectomy was measured quantitatively by dual photon absorptiometry. Thirty-three patients who had undergone a meniscectomy (randomized to either total [n=19] or partial [n=14] meniscectomy) performed by open joint surgery approximately 12 years earlier were included in the study. Bone mineral density was measured in the previously injured legs and in the healthy contralateral legs in areas located medially and laterally in the cortical bone of the subchondral plates and below in the trabecular bone of the medial and lateral tibial condyles. The distribution of bone mineral within the proximal tibia showed a characteristic and significant pattern. In the trabecular bone of the healthy contralateral knees, bone mineral density was 15% higher in the medial tibial condyles compared with the values laterally; a total or partial meniscectomy increased this difference to 25%. With regard to the cortical bone of the subchondral plates, the bone mineral density in the healthy knees was 24.8-29.4% higher medially than laterally, whereas after total and partial meniscectomy the differences were, respectively, 37.7 and 41.4%. No significant differences in the distribution of bone mineral density, at either cortical or trabecular measuring sites, were found between totally and partially meniscectomized knees. PMID- 8618161 TI - Localized, tumor-associated osteolysis involves the recruitment and activation of osteoclasts. AB - The cellular and biochemical mechanisms that direct destruction of bone at the site of tumor osteolysis are unknown. In order to understand this process better, a murine model designed for the study of tumor osteolysis was developed and the influence of osteolytic and nonosteolytic tumors on bone was investigated. Tumors developed following femoral intramedullary injection of sarcoma (2472) and melanoma (G3.26) cell lines; however, only tumors from the 2472 cell line caused osteolysis. It was determined that 2472 tumor-induced osteolysis commenced 6 days after the femora had been inoculated with 2472 cells. There were more osteoclasts per millimeter of bone surface in 2472 tumor-bearing limbs (16.7 +/- 5.0) than in sham-injected limbs (3.8 +/- 0.9) (p < 0.015). In addition, an increase in the osteoclast size (area) was detected in 2472 tumor-bearing limbs: 412 +/- 65 micron2 compared with 187 +/- 17 micron2 (p < 0.01). In vitro bone resorption experiments indicated that 2472 tumor cells had a limited ability to destroy bone in comparison with macrophages and osteoclasts. Taken in total, these findings define a model that is useful for the study of tumor osteolysis, and the data from analyses of the model demonstrate that the cellular mechanisms responsible for 2472 tumor-induced osteolysis include both an increase in the number of osteoclasts and activation of mature osteoclasts. PMID- 8618162 TI - Determinants of femoral geometry and structure during adolescent growth. AB - Our goal was to understand developmental determinants of femoral structure during growth and sexual maturation by relating femoral measurements to gender and developmental factors (age, pubertal stage, height, and body mass). The bone mineral content of the femur was measured by dual energy x-ray absorptiometry in 101 healthy Caucasian adolescents and young adults, 9-26 years of age. After some simplifying assumptions had been made, cross-sectional geometric properties of the femoral midshaft were estimated. Two geometry-based structural indicators, the section modulus and whole bone strength index, were calculated to assess the structural characteristics of the femur. Femoral strength, as described by these structural indicators, increased dramatically from childhood through young adulthood. Regressions were performed between these femoral measurements and the developmental factors. Our data show that of age, pubertal stage, body mass, and height, body mass is the strongest predictor of femoral cross-sectional properties, and the correlation of body mass with femoral cross-sectional structure is independent of gender. A model including all four developmental factors and gender did not substantially increase the accuracy of predictions compared with the model with body mass alone. In light of previous research, we hypothesize that body mass is an indicator of in vivo loading and that this in vivo loading influences the cross-sectional growth of the long bones. PMID- 8618163 TI - Role of tumor necrosis factor alpha in particulate-induced bone resorption. AB - The purpose of this study was to determine the role of tumor necrosis factor alpha in bone resorption secondary to mediator release from macrophages exposed to cement particles. The J774 mouse macrophage cell line was exposed to polymethylmethacrylate particles for 24 hours and the resulting conditioned medium was analyzed for prostaglandin E2, tumor necrosis factor alpha, interleukin-1 alpha and beta, and the ability to stimulate release of prostaglandin E2 and 45Ca from radiolabeled mouse calvaria. Macrophage exposure to polymethylmethacrylate particles led to a 9-fold increase in release of tumor necrosis factor alpha (p < 0.01), but did not lead to a significant increase in release of prostaglandin E2, interleukin-1 alpha, or interleukin-1 beta when compared to unexposed cells. Exposure of the macrophages to polymethylmethacrylate particles over a time course from 30 minutes to 96 hours led to an increase in the release of tumor necrosis factor alpha that was initially detected at 30 minutes and was maximum at 48 hours. Incubation of the macrophage-polymethylmethacrylate conditioned medium with rat calvaria significantly increased the release of 45Ca and prostaglandin E2 from the bone. To study the role of release of tumor necrosis factor alpha in bone resorption, the macrophage-polymethylmethacrylate conditioned medium was then preincubated with anti-tumor necrosis factor alpha antibody prior to exposure of the conditioned medium to the calvaria. This preincubation was successful in significantly inhibiting 45Ca release by calvaria (p <0.01) to levels that were not significantly different from the levels of release by unexposed calvaria. Tumor necrosis factor alpha appears to play a critical role in initiating particulate-induced bone resorption. Exposure of macrophages to polymethylmethacrylate particles leads to a significant release of tumor necrosis factor alpha in a time-dependent fashion. This macrophage-polymethylmethacrylate conditioned medium stimulated release of prostaglandin E2 and bone resorption in bone organ culture. The addition of anti-tumor necrosis factor alpha antibody to this in vitro system inhibited the bone resorption stimulated by the macrophage polymethylmethacrylate conditioned medium and partially suppressed the production of prostaglandin E2. The sequence of events in this model for particulate-induced bone resorption appears to be initiated by the production of tumor necrosis factor alpha by the macrophage, followed by production of prostaglandin E2 by cells in bone, and then by bone resorption. PMID- 8618164 TI - Friction and stem stiffness affect dynamic interface motion in total hip replacement. AB - Large cyclic movements between the femoral stem and bone during the first weeks after total hip arthroplasty may hamper bone ingrowth and adversely affect the eventual success of the arthroplasty. Little is known, however, about the magnitude of the motions and its relationship to design and surgical factors. A two-dimensional finite element model of a cementless prosthesis inserted into the proximal femur was constructed to study the effects of two mechanical variables- the stiffness of the implant and the coefficient of friction between bone and implant--on the magnitude of the motions. We investigated the influences of these variables on the subsidence of the prosthesis, the magnitudes of the cyclic motions, and the level of the interface stresses. The presence of friction reduced cyclic motions by about 85% compared with a frictionless interface. Once friction was assumed, varying the coefficient of friction had little effect. The effect of friction on the interface stress state and gross subsidence of the prosthesis was not as great as on cyclic motion. Implant stiffness also affected the magnitudes and distributions of the cyclic motions along the interface. A flexible stem generated motions about three to four times larger proximally than those of a stiff stem, which generated larger motions distally. The influence of stem stiffness on interface stresses and prosthetic subsidence was less than on cyclic motion. The location of the peak shear stresses at the interface around a bonded prosthesis corresponded to the location where cyclic interface motion was maximal for an unbonded prosthesis. However, no direct relationship was found between the magnitudes of peak stresses and the amplitudes of cyclic motions. PMID- 8618165 TI - Differential effects of serum, insulin-like growth factor-I, and fibroblast growth factor-2 on the maintenance of cartilage physical properties during long term culture. AB - The effects of fetal bovine serum, insulin-like growth factor-I, and fibroblast growth factor-2 on the regulation of the functional physical properties of adult bovine cartilage explants during an incubation period of 18-20 days was determined, and the relationship between the measured functional properties of the cartilage and the tissue composition was assessed. Cartilage disks were tested in the uniaxial radially confined configuration by the application of low amplitude oscillatory displacement and measurement of the resultant load and streaming potential. For the control cartilage terminated just after explant, the modulus was 0.39 +/- 0.28 MPa, the open circuit hydraulic permeability was 2.0 +/ 1.0 x 10(-15) m2/(Pa.sec), and the electrokinetic (streaming potential) coefficient was -2.3 +/- 0.6 mV/MPa. Incubation of cartilage in medium supplemented with serum or insulin-like growth factor-I resulted in maintenance of the modulus and electrokinetic coefficient, whereas incubation in basal medium or medium supplemented with fibroblast growth factor-2 led to a marked decrease from control values in the modulus and the amplitude of the electrokinetic coefficient. All of the culture conditions examined resulted in an increase in permeability that was not statistically significant. The variation in the electromechanical properties of all the cartilage samples tested was related to the density of tissue proteoglycan and collagen (hydroxyproline). The modulus was correlated with both the density of tissue proteoglycan (+0.014 MPa/[mg/ml]) and the density of tissue hydroxyproline (+0.008 MPa/[mg/ml]). The electrokinetic coefficient was also correlated with the density of proteoglycan (-0.080 [mV/MPa]/[mg/ml]) and the density of hydroxyproline (+0.064 [mV/MPa]/[mg/ml]). These data indicate that the regulation of chondrocyte matrix metabolism by growth factors can significantly affect the physical properties and function of cartilage. PMID- 8618166 TI - In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure. AB - This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes. PMID- 8618167 TI - Enhanced denaturation of the alpha (II) chains of type-II collagen in normal adult human intervertebral discs compared with femoral articular cartilage. AB - The mechanical strength of connective tissues is dependent on the integrity of their fibrillar collagen frameworks. The objective of the present study was to assess type-II collagen damage (denaturation) in the adult human intervertebral disc compared with articular cartilage, in order to determine whether damage to this molecule may vary in different anatomical sites in the same person. A new immunochemical assay was used to measure the amounts of denatured and total type II collagen in the annulus fibrosus and nucleus pulposus of the L5-S1 disc and in cartilage from the femoral condyles of the same individuals (n = 7). Denaturation of type-II collagen was significantly higher in both the annulus fibrosus and the nucleus pulposus than in articular cartilage. Such increased damage to type-II collagen in the adult disc may have relevance to the more pronounced degenerative changes observed in this tissue compared with articular cartilage. PMID- 8618168 TI - Expression of interleukin-6 in osteoarthritic chondrocytes and effects of fluid induced shear on this expression in normal human chondrocytes in vitro. AB - This study tested the effect of fluid-induced shear on interleukin-6 expression in normal human articular chondrocytes in vitro. As determined by Northern blot analysis, interleukin-6 mRNA expression occurs in chondrocytes from osteoarthritic cartilage but not in normal chondrocytes. Applying fluid-induced shear stress to primary high density cultures of chondrocytes increased interleukin-6 mRNA signal 4-fold at 1 hour and 10 to 15-fold at 48 hours compared with unsheared control cultures. At 48 hours, fluid-induced shear stress increased interleukin-6 protein levels in the culture medium 9 to 10-fold compared with unsheared controls. mRNA signals for interleukin-1alpha, interleukin-1beta, and tumor necrosis factor-alpha in RNA from sheared or control chondrocytes were not detected by Northern blotting. Transforming growth factor beta mRNA signal was detectable but was not affected by shear. In contrast, human lung fibroblasts (WI-38) responded to fluid-induced shear with increased signal for transforming growth factor-beta, but not interleukin-6, mRNA. Both cell types did respond to interleukin-1alpha with increased interleukin-6 mRNA signal. These data demonstrated that distortional forces, such as fluid-induced shear stress, alter interleukin-6 levels in normal chondrocytes in vitro and suggest that increased interleukin-6 expression in osteoarthritic cartilage may result, in part, from alterations in the mechanical loading of the tissue. PMID- 8618170 TI - Effect of alendronate on fracture healing and bone remodeling in dogs. AB - To examine the effect of alendronate (4-amino-1-hydroxybutylidene bisphosphonate) on fracture repair, the drug was given to mature beagle dogs orogastrically at 2 mg/kg/day for 9 weeks preceding fracture. 16 weeks after fracture, or both before and after fracture (25 weeks). A transverse mid-diaphyseal fracture of the right radius was surgically induced and was stabilized by external coaptation splinting. Fracture healing and bone remodeling were evaluated by radiography, gross and histological examination, and bone histomorphometry. The mechanical properties of the fracture callus were determined by a four-point bending test. Radiographs and gross and microscopic examination demonstrated normal bone healing at the fracture site in all dogs. In dogs that received alendronate during the fracture healing period, at 16 weeks the calluses were approximately 2 3 times larger than those in dogs that received a placebo during the healing period. This is consistent with slower callus bone remodeling, an expected pharmacological effect of the compound. Bone histomorphometry demonstrated that treatment with alendronate did not inhibit bone formation or mineralization. Mechanical testing showed that the ultimate load at failure and the flexural rigidity of both the fractured and contralateral intact bone were unaffected by treatment with alendronate. Therefore, in this study, treatment with alendronate before or during fracture healing, or both, resulted in no adverse effects on the union, strength, or mineralization of bone in mature beagle dogs. PMID- 8618169 TI - Formation of osteoclast-like cells is suppressed by low frequency, low intensity electric fields. AB - With use of a solenoid to generate uniform time-varying electric fields, the effect of extremely low frequency electric fields on osteoclast-like cell formation stimulated by 1,25(OH)2D3 was studied in primary murine marrow culture. Recruitment of osteoclast-like cells was assessed by counting multinuclear, tartrate-resistant acid phosphatase positive cells on day 8 of culture. A solenoid was used to impose uniform time-varying electric fields on cells; sham exposures were performed with an identical solenoid with a null net electric field. During the experiments, both solenoids heated interiorly to approximately 1.5 degrees C above ambient incubator temperature. As a result of the heating, cultures in the sham solenoid formed more osteoclast-like cells than those on the incubator shelf (132 +/- 12%). For this reason, cells exposed to the sham solenoid were used for comparison with cultures exposed to the active coil. Marrow cells were plated at 1.4 x 10(6)/cm2 in square chamber dishes and exposed to 60 Hz electric fields at 9.6 muV/cm from days 1 to 8. Field exposure inhibited osteoclast-like cell recruitment by 17 +/- 3% as compared with sham exposure (p < 0.0001). Several variables, including initial cell plating density, addition of prostaglandin E2 to enhance osteoclast-like cell recruitment, and field parameters, were also assessed. In this secondary series, extremely low frequency fields inhibited osteoclast-like cell formation by 24 +/- 4% (p < 0.0001), with their inhibitory effect consistent throughout all variations in protocol. These experiments demonstrate that extremely low intensity, low frequency sinusoidal electric fields suppress the formation of osteoclast-like cells in marrow culture. The in vitro results support in vivo findings that demonstrate that electric fields inhibit the onset of osteopenia and the progression of osteonecrosis; this suggests that extremely low frequency fields may inhibit osteoclast recruitment in vivo. PMID- 8618171 TI - Effect of loading and fracture motions on diaphyseal tibial fractures. AB - A computerized motion sensor was used to record the three-dimensional components of interfragmentary motion during healing in three patients with closed, low energy fractures of the tibial diaphysis treated with functional braces. At the first measurement session 2 weeks after fracture, the patients applied approximately 15 kg to the injured limb. Although this produced 1-4 mm of translation of the fragments, this was recovered when the load was removed. The maximum rotational and angulatory displacements often occurred as the patients rose from the chair with no weight applied to the limb and frequently were reduced as the 15 kg of load was applied. Under load, the maximum axial rotation was 3 degrees and the maximum angular displacement was 1 degree. As with the translations, the initial rotational and angulatory positions of the fragments were recovered when the load was removed and the patient returned to the seated position. At 8 weeks, the patient applied full body weight, producing a maximum interfragmentary translation of 0.5 mm and maximum axial rotation or angulation of 0.5 degrees. Abundant peripheral callus formed in all three fractures, and they healed by 15 weeks through typical gradual consolidation and mineralization of the callus, accompanied by a corresponding reduction in interfragmentary motions. PMID- 8618172 TI - Qualitative and quantitative analysis of orthotopic bone regeneration by marrow. AB - A rat model of a femoral segmental defect was used to specifically test the hypothesis that autogenous marrow has the osteogenic capability to heal a bone defect. The variables analyzed included the ratio of the marrow volume to the defect, implantation of live or dead marrow, and remodeling of established nonunions by implantation of live marrow. The uniqueness of this model allows biomechanical evaluation of the new bone formed by the implant. When live marrow was implanted, woven bone formed at 3 weeks, progressing to early lamellar bone at 6 weeks, with subsequent remodeling for as long as 12 weeks in a volumetric fashion (p < 0.05). Bone marrow, when placed in a fresh femoral defect and given in sufficient amounts, produced a rate of union comparable with that of autologous bone grafts. Mature lamellar bone formed by marrow was evaluated biomechanically; the results were statistically comparable with those of cancellous bone grafts at 12 weeks. Significant bone formation occurred when marrow was percutaneously injected in femoral nonunions, although union and remodeling did not take place in this rat model. Implantation of dead marrow resulted in rare cellular infiltration and minimal bone formation in a manner comparable with that of autogenous cancellous bone grafts. These results indicate that bone marrow can lead to structurally functional bone regeneration in an orthotopic location. PMID- 8618173 TI - Recorticalization after bifocal internal bone transport in the double-plated sheep femur. AB - Distraction osteogenesis and bone remodelling after the end of bone transport were investigated in one femur each of six adult male sheep. A newly designed internal distraction device was used. A custom-made osteosynthesis plate was fixed on the lateral side of the femur, and two transporting plates driven by a transcutaneously inserted screwdriver moved two bone cylinders simultaneously over a 40 mm defect. An additional plate was applied ventrally to stabilize the device. Bone transport was begun 2 weeks postoperatively at 1 mm/day at each transporting plate until they came into contact. New bone formation within the distraction gaps was evaluated by computed tomography scans and was quantified at 4 and 6 months by dual energy x-ray absorptiometry as well as histomorphometry on polyfluorochrome-labelled undecalcified ground sections and microradiographs. At 4 months, all distraction gaps were bridged by abundant newly formed bone trabeculae, which were reduced and condensed to cortex-like layers of new bone at 6 months. Less new bone was always found under the lateral device than in the unplated medical and dorsal segments, but the amount of new bone under the ventral plate was comparable with that in the unplated regions. The results of this pilot study show that distraction osteogenesis can be achieved with an internal device such as this one and that recorticalization and restoration of a medullary canal occur despite the relatively rigid internal stabilization by the plates. PMID- 8618174 TI - Mutations that affect ion channels change the sensitivity of Drosophila melanogaster to volatile anesthetics. AB - We have quantitated the response of D. melanogaster to general anesthetics with a device, the inebriometer, that assays the fly's geotactic and postural behavior. When alleles of several loci that encode or regulate subunits of ion channels were compared with control stocks, several ion channel mutants clearly increased the anesthetic sensitivity of Drosophila. The effects were specific in that: a) for several alleles, genetic tests indicated that the anesthesia phenotype was due to the ion channel mutation and not to extraneous genetic differences between the stocks; b) a given ion channel mutation often affected the response to one anesthetic but not another; and c) the behavior of decapitated flies in the inebriometer indicated that the anesthetic phenotype of several mutants did not merely reflect a global change in the fly's physiology. These results provide support for the idea that ion channels are on the pathway(s) influenced by anesthetics and that different anesthetics use different pathways. They also provide perspective on the behavior of previously isolated mutations (har) that decrease the sensitivity of Drosophila to anesthetics in the inebriometer. PMID- 8618175 TI - 5th European meeting on the neurogenetics of Drosophila. Montpellier, France, 3-6 October, 1994. Abstracts. PMID- 8618176 TI - Hospitalization as a measure of morbidity among very low birth weight infants with chronic lung disease. AB - OBJECTIVE: To examine the spectrum of hospitalization and rehospitalization among very low birth weight (VLBW, <1500 gm) infants with severe chronic lung disease during the first 2 years of life. POPULATION: All 124 VLBW infants admitted to our center from October 1988 to September 1990 who were oxygen and ventilator dependent at 21 days of age. One hundred infants survived to discharge, of whom two subsequently died. The 98 surviving infants are the subject of this report. METHODS: The duration of the neonatal stay, the use of a long-term care facility, and rehospitalizations were recorded to a postnatal age of 24 months. The duration of these hospitalizations and the total duration of hospitalization during the first year of life were correlated with demographic and perinatal risk factors and 20-month outcome. RESULTS: The 98 infants spent a median of 125 days (range, 44 to 365) of their first year hospitalized; the neonatal stay accounted for 85% of this time. Forty-nine of the infants (50%) were rehospitalized in their first year (median stay, 14 days), and 36 (37%) were rehospitalized in their second year (median stay, 7 days). Long-term care facility stay and rehospitalizations accounted for 6% and 9% of the first-year hospitalizations, respectively. A median of 9 days (range, 1 to 365) of the second year of life were spent in hospital. The infants rehospitalized during their first year of life did not differ significantly from those not requiring rehospitalization with regard to maternal demographic descriptors, birth data, severity of chronic lung disease, or measures of 20-month outcome. Both duration of neonatal stay and total hospital stay during the first year were significantly associated with all measures of chronic lung disease severity and with 20-month neurodevelopmental outcome measures, whereas the duration of rehospitalization was associated only with duration of oxygen dependence. CONCLUSION: Among infants with severe chronic lung disease, the total duration of hospitalization during the first year of life provides a better index of morbidity than the number or duration of rehospitalizations alone. PMID- 8618178 TI - Increase in the concentration of transforming growth factor beta-1 in bronchoalveolar lavage fluid before development of chronic lung disease of prematurity. AB - OBJECTIVE: Pulmonary fibrosis is a prominent feature of chronic lung disease of prematurity (CLD). We sought to determine the influence of the potent profibrotic cytokine transforming growth factor beta-1 (TGF-Beta 1) on the development of CLD. METHODS: We determined the concentration of active and total TGF-Beta 1 in bronchoalveolar lavage fluid obtained from 18 infants who subsequently had CLD (mean gestation, 25.7 weeks; birth weight, 816 gm) 15 (29.8 weeks, 1493 gm) who recovered from the respiratory distress syndrome, and 7 (35.1 weeks, 2441 gm) control infants. RESULTS: The concentration of both active and total TGF-Beta 1 was increased in the infants with CLD when compared with the respiratory distress syndrome and control groups. The increase in active and total TGF-Beta 1 was greatest on day 4 of age, when infants who eventually had CLD were compared with those who did not progress to CLD (active TGF-Beta 1, 39.5 vs 4.6 ng/ml; total TGF-Beta 1, 43.8 vs 13.8 ng/ml). In addition, immunocytochemistry studies localized pan-TGF-Beta to alveolar macrophages obtained by bronchoalveolar lavage. CONCLUSIONS: These observations indicate that TGF-Beta 1 may contribute to the fibrotic response that is observed in the lungs of infants who have CLD. PMID- 8618177 TI - Randomized multicenter trial comparing synchronized and conventional intermittent mandatory ventilation in neonates. AB - OBJECTIVE: To compare synchronized intermittent mandatory ventilation (SIMV) and conventional intermittent mandatory ventilation (IMV) in neonates. STUDY DESIGN: Prospective, multicenter, randomized clinical trial. SETTING: Level III neonatal intensive care units at six university or children's hospitals. PATIENTS: Three hundred twenty-seven infants receiving conventional IMV for respiratory distress syndrome, pneumonia, or meconium aspiration pneumonitis were randomly assigned a 7.5 +/- 6 hours of age to either continue with IMV or change to SIMV. Infants assigned to each mode of ventilation had similar birth weight (BW), gestational age, and Apgar scores at birth, and similar oxygenation indexes at randomization. They received similar surfactant therapy and had similar incidence of sepsis, seizures, secondary pneumonia, and necrotizing enterocolitis. In the infants with BW less than 1000 gm, more infants receiving IMV had surgical ligation of their patent ductus arteriosus than did those receiving SIMV (27 vs. 7 %; p = 0.02). ANALYSIS: Data was analyzed overall for all infants and also separately within three BW groups: less than 1000 gm, 1000 to 2000 gm, and more than 2000 gm. The 1000 to 2000 gm BW group was further analyzed in subgroups weighing 1000 to 1499 gm and 1500 to 2000 gm. RESULTS: In all infants, at 1 hour after randomization, the infants receiving SIMV had a lower mean airway pressure than those receiving IMV (8.08 +/- 2.15 vs. 8.63 +/- 2.59; p<0.05), with similar fractions of inspired oxygen and oxygenation indexes. Infants whose BW was 1000 to 2000 gm at 0.5 hour required a lower fraction of inspired oxygen with SIMV than with IMV (0.52 +/- 0.20 vs. 0.62 +/- 0.27; p<0.05) and had better oxygenation at 1 hour, as shown by lower oxygenation indexes with SIMV than with IMV (6.14 +/- 4.17 vs. 9.42 +/- 8.41; p = 0.01). Infants whose BW was 1000 to 2000 gm received a lower number of unit doses of sedative/analgesic drugs per infant during the first 4 days of SIMV than did infants receiving IMV (3.8 +/- 3.4 vs 6.3 +/- 5.5 unit doses; p = 0.02). Infants whose BW was more than 2000 gm had a shorter duration of mechanical ventilation with SIMV than with IMV (median, 72 vs 93 hours; p = 0.02). Three of the forty-six infants receiving IMV but none of the 47 infants receiving SIMV required extracorporeal membrane oxygenation. In the infants with BW less than 1000 gm, fewer infants treated with SIMV required supplemental oxygen at 36 weeks of postconceptional age than did those treated with IMV (47 vs 72%; p<0.05). In 83 infants whose lungs were mechanically ventilated for 14 days or longer, all with BW less than 2000 gm, those treated with SIMV regained their BW earlier than those treated with IMV (median, 21.5 vs 29 days; p<0.01). There were no differences in the rates of death, intraventricular hemorrhage (grades III and IV), air leak, need for pharmacologic paralysis, or need for supplemental oxygen at 28 days. CONCLUSIONS: We found that SIMV was at least as efficacious as conventional IMV, and may have improved certain outcomes in BW-specific groups. PMID- 8618179 TI - Influence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighing 1000 grams or less. AB - OBJECTIVES: To test the hypotheses that (1) infection increases ductal dilatory prostaglandins and inflammatory mediators that may influence the closure of a patent ductus arteriosus (PDA), increasing the incidence of late episodes of PDA (after 7 days) and the rate of closure failures, and (2) the concurrence of PDA and infection increases the risk of chronic lung disease (CLD). METHODS: One hundred fourteen premature infants (birth weight, 500 to 1000 gm) were prospectively assessed for PDA and infection. Serum levels of 6-ketoprostaglandin F1 alpha and tumor necrosis factor alpha were measured routinely in all infants and when PDA or infection was present. Multivariate assessment of risk factors for PDA closure failure and for CLD was done by logistic regression, and expressed as an odds ratio and as 95% confidence intervals. RESULTS: Late PDA episodes were more frequent in infants with infection than in those without infection. A temporally related infection (<5 days between both diagnoses) was associated with an increased risk of PDA closure failure (odds ratio, 19.1 (confidence interval, 4 to 90)). In addition to birth weight and the severity of initial respiratory failure, PDA and infection increased the risk of CLD (odds ratio, 11.7 (confidence interval, 1.7 to 81) for PDA; odds ration, 3.1 (confidence interval, 1 to 11) for infection). Furthermore, when both factors were temporally related, they further increased the risk of CLD (odds ratio, 29.6 (confidence interval, 4.5 to >100)). Infants with infection and those with PDA had higher levels of 6-ketoprostaglandin F1 alpha than did control subjects. Levels of tumor necrosis factor alpha were also elevated in infants with infection and in those with late PDA. CONCLUSIONS: Infection adversely influences PDA outcome by increasing the risk of late ductal reopening and PDA closure failures. Increased levels of prostaglandins and tumor necrosis factor alpha in infants with infection may explain the poor PDA outcome. The concurrence of PDA and infection potentiates their negative effects on the risk of CLD. PMID- 8618180 TI - Deposition pattern of radiolabeled salbutamol inhaled from a metered-dose inhaler by means of a spacer with mask in young children with airway obstruction. AB - BACKGROUND: The exact amount of drug deposited in the respiratory and gastrointestinal tract in children with airway obstruction, when delivered from a metered-dose inhaler (MDI) via a spacer with mask, and its distribution in children with airway obstruction, are unknown. METHODS: We studied 15 children, using salbutamol labeled with technetium 99m. Each patient was imaged with a gamma-camera immediately after one puff of labeled salbutamol was administered via a spacer with mask. Drug deposition was then analyzed to measure the distribution of the labeled spray in the oropharynx, the lungs, the stomach, and the spacer with mask (Aerochamber) itself. RESULTS: Fifteen infants and children (mean age, 21 months (range, 3 months to 5 years); mean weight, 9.3 kg (range, 3.2 to 15 kg)) were studied. Mean aerosol deposition was 1.97% +/- 1.4% in the lungs, 1.28% +/- 0.77% in the oropharynx, and 1.11% +/- 2.4% in the stomach. The remainder was trapped in the spacer. Lung imaging after inhalation from an MDI via a spacer showed widespread deposition of the drug in central and peripheral intrapulmonary airways. In two adult volunteers the deposition after one puff of the same radiolabeled drug, inhaled from an MDI via a spacer with a mouthpiece, was 19% in the lungs and 2% in the stomach. CONCLUSIONS: Infants and toddlers with obstructive lung disease can be reliably and safely treated with inhaled medication administered with an MDI via a spacer with mask. The doses of a drug given from an MDI to infants and toddlers when a spacer with mask is used are not yet well defined but should be higher than the currently recommended doses, perhaps as much as an adult dose. PMID- 8618181 TI - Ultrasound measurement of gastric emptying time in patients with cystic fibrosis and effect of ranitidine on delayed gastric emptying. AB - Intestinal dysmotility is commonly reported in patients with cystic fibrosis (CF); however, gastric motor activity has rarely been investigated. We measured with real-time ultrasonography the antral distention and gastric emptying time of a solid-liquid meal in 29 patients with CF (age range, 5 to 17 years). A significantly prolonged gastric emptying time was present in 26 patients compared with 13 healthy control subjects (age range, 5 to 16 years); an exaggerated antral distention in the fed period was also detected. The patients with CF and delayed gastric emptying were randomly allocated to receive cisapride or ranitidine for 4 weeks. Twelve patients treated with ranitidine and 11 with cisapride completed the trial. There was a marked decrease in gastric emptying time, antral distention, and dyspeptic symptomatic score in patients receiving ranitidine but not in patients treated with cisapride. We conclude that gastric dysmotility is commonly detected in patients with CF and that H2 receptor blockers are more effective than prokinetics in improving dyspeptic symptoms and gastric emptying and distention. PMID- 8618182 TI - Impact of neonatal vitamin A supplementation on infant morbidity and mortality. AB - OBJECTIVE: To determine whether vitamin A supplementation at birth could reduce infant morbidity and mortality. STUDY DESIGN: We conducted a placebo-controlled trial among 2067 Indonesian neonates who received either 52 micromol (50,000 IU) orally administered vitamin A or placebo on the first day of life. Infants were followed up at 1 year to determine the impact of this intervention on infant mortality. A subgroup (n = 470) was also examined at 4 and 6 months of age to examine the impact on morbidity. RESULTS: Vital status was confirmed in 89% of infants in both groups at 1 year. There were 19 deaths in the control group and 7 in the vitamin A group (relative risk = 0.36; 95% confidence interval = 0.16, 0.87). The impact was stronger among boys, infants of normal compared with low birth weight, and those of greater ponderal index. Among infants examined at 4 months of age, the 1-week period prevalence of common morbidities was similar for vitamin A and control infants. However, during this same 4-month period, 73% and 51% more control infants were brought for medical treatment for cough (p = 0.008) and fever (p = 0.063), respectively. CONCLUSIONS: Neonatal vitamin A supplementation may reduce the infant mortality rate and the prevalence of severe respiratory infection among young infants. PMID- 8618183 TI - Low birth weight and morbidity from diarrhea and respiratory infection in northeast Brazil. AB - OBJECTIVE: To compare morbidity and mortality rates of low birth weight (LBW) and appropriate birth weight infants born at term, focusing on diarrheal and respiratory infections. STUDY DESIGN: A cohort of 133 LBW infants (1500 to 2499 gm) and 260 appropriate birth weight infants (3000 to 3499 gm), individually matched by sex and season of birth, were followed for the first 6 months of life. None had congenital anomalies and all were from poor families living in the interior of Pernambuco, northeast Brazil. Data on infant deaths, hospitalizations, and morbidity were collected prospectively through daily home visits (except Sundays) from birth through week 8, then twice weekly for weeks 9 to 26. The effects of birth weight were assessed with a variety of multivariable techniques, controlling for confounders. RESULTS: Of the LBW infants, 56% were wasted (thin), 23% were stunted, and 17% were both wasted and stunted. The LBW infants (median 2380 gm) experienced a sevenfold higher mortality rate and fourfold higher rate of hospitalization than appropriate birth weight infants. Almost all deaths and hospitalizations were in the postneonatal period. The LBW infants also experienced 33% more days with diarrhea and 32% more days with vomiting (p = 0.003 in each case). The prevalences of cough and fever were not significantly different. CONCLUSIONS: Infant deaths, hospitalizations, and diarrheal morbidity are increased in term LBW infants who have only a modest weight deficit. PMID- 8618184 TI - Atypical hemolytic-uremic syndrome: a comparison with postdiarrheal disease. AB - OBJECTIVES: To compare the epidemiologic, laboratory, clinical, and outcome variables of atypical (nondiarrheal) hemolytic-uremic syndrome with those of classic postdiarrheal disease. METHODS: A 24-year retrospective review of 28 episodes of atypical HUS that occurred in 22 children compared with 266 episodes of typical postdiarrheal disease in 265 children treated during the same period. RESULTS: Of the 294 episodes of HUS, 9.5% were atypical (nondiarrheal), and 18% of the patients in the atypical disease group had recurrences. Prodromal features (other than the presence or absence of diarrhea) were similar between the groups. White blood cell count and serum creatinine concentration on admission to the hospital and most abnormal blood urea nitrogen values during hospitalization were significantly lower (p = 0.02) in the patients with atypical HUS. Oliguria, anuria, and the need for dialysis were also less common (p = 0.02) in the atypical disease group. There were no deaths in the subset of patients with atypical disease; 3.4% of the patients in the typical disease group died. Although there were no statistically significant differences in the incidence of end-stage renal disease between the atypical and typical disease groups, two of the four patients with atypical disease who had recurrences also had end-stage renal disease. There were no significant differences in chronic renal sequelae between the groups one or more years after HUS. CONCLUSIONS: In contrast to reports from most other regions, patients with atypical disease in our area of the western United States have milder acute nephropathy and, with the exception of those with recurrence, do not experience worse outcomes. PMID- 8618185 TI - Effect of protein intake on erythropoiesis during erythropoietin treatment of anemia of prematurity. AB - OBJECTIVE: To examine the effect of protein intake on the erythropoietic response of very low birth weight infants to treatment with recombinant human erythropoietin (rHuEPO). STUDY DESIGN: Twenty very low birth weight infants were enrolled in the study and 19 completed the 6 weeks of study. Weekly absolute reticulocyte counts, protein intakes, and growth, as well as selected markers of protein metabolism--prealbumin, albumin, and transferrin--were analyzed. Iron stores were estimated for each infant to exclude iron deficiency as a cause of anemia. The relationship between protein intake and absolute reticulocyte count was evaluated with a linear breakpoint analysis to account for any plateau in the relationship at higher protein intakes. RESULTS: Adequate iron stores were present in all infants, and transferrin concentrations correlated with measured total iron-binding capacity (r = 0.95, p = 0.0001). In the rHuEPO-treated infants, absolute reticulocyte count was significantly associated with protein intake up to 3.1 gm/kg per day and extending to 3.5 gm/kg per day (p = 0.041 to 0.032); beyond this point there was no longer any effect. Moreover, in comparison with the infants who received placebo, the rHuEPO-treated infants had a better daily percent weight gain for a protein intake up to 3.5 gm/kg per day (p = 0.016). CONCLUSIONS: In VLBW infants treated with rHuEPO, higher protein intake up to 3.1 to 3.5 gm/kg per day improved the erythropoietic response, and protein utilization for growth was improved. During treatment with rHuEPO, infants who receive adequate protein to achieve satisfactory growth also receive sufficient protein for erythropoiesis. PMID- 8618186 TI - Pharmacokinetics and effectiveness of recombinant erythropoietin administered to preterm infants by continuous infusion in total parenteral nutrition solution. AB - OBJECTIVES: To compare the pharmacokinetics and effectiveness of continuously administered recombinant erythropoietin (Epo) in total parenteral nutrition (TPN) solution with daily subcutaneously administered Epo. METHODS: Forty preterm infants in the first 72 hours of life were randomly assigned to receive Epo (200 units/kg per day for 10 consecutive days), either subcutaneously (20 infants, 1051 +/- 40 gm, 28.3 +/- 0.4 weeks of gestation; mean +/- SEM), or added daily to their TPN fluids (20 infants, 1028 +/- 36 gm, 27.9 +/- 0.4 weeks of gestation). Both groups received iron supplementation (1 mg/kg per day iron dextran in the TPN solution). Absolute reticulocyte counts and complete blood cell counts with differentials were measured, and transfusions and phlebotomy losses were recorded. Pharmacokinetics were determined in the first 16 infants. RESULTS: In the infants who received Epo subcutaneously, the elimination half-life was 17.6 +/- 4.4 hours on day 3 and 11.2 +/- 1.5 hours on day 10; the volume of distribution was 802 +/- 190 ml/kg on day 3 and 1330 +/- 243 m/kg on day 10. Serum Epo concentrations were higher on day 3 than on day 10 for both groups (subcutaneous: 400 +/- 64 mU/ml vs 177 +/- 29 mU/m, p <0.05; TPN: 395 +/- 64 vs 194 +/- 41 mU/ml, p <0.05). Clearance did not differ between the two groups with regard to route of administration and increased significantly from days 3 to 10 in both groups. Reticulocyte counts were similar in both groups. There were no differences between groups in the number of transfusions given, and the overall decline in hematocrit was similar. No adverse effects of Epo were noted in either group. CONCLUSIONS: Adding Epo to the TPN solution in this population results in similar Epo concentrations, clearance, and effectiveness as subcutaneous dosing. PMID- 8618187 TI - Comparison of five different vaccination schedules with Haemophilus influenzae type b-tetanus toxoid conjugate vaccine. AB - OBJECTIVE: To compare seroresponses to five different vaccination schedules for Haemophilus influenzae type b (Hib)-tetanus toxoid conjugate (PRP-T) vaccine in infants. DESIGN: Four different two-dose schedules were compared, with doses given at 1 and 3 months, 2 and 4 months, 2 and 6 months, or 4 and 6 months of age. One group received three doses at 2, 4, and 6 months of age. The PRP-T vaccine was given in the same syringe with diphtheria-tetanus-pertussis (DTP) vaccine; inactivated polio vaccine (IPV) was given in a separate syringe. Anti Hib polysaccharide antibodies were measured by radioimmunoassay in sera taken before each immunization, 1 month after the second dose, at 7 and at 12 to 24 months of age. SUBJECTS: A total of 196 healthy infants were enrolled between November 1990 and November 1992. RESULTS: After one dose of PRP-T there were no significant differences in geometric mean antibody concentrations (0.09 to 0.13 microgram/ml) or in fold responses among the schedules. The response to the second dose was significantly higher than the response to the first dose given at the same age. The geometric mean antibody concentration was lower in the group vaccinated at 1 and 3 months than in the groups vaccinated at 2 and 4 months, 2 and 6 months, or 4 and 6 months. The three-dose schedule resulted in a significantly higher final antibody concentration than the best two-dose schedule (p <0.001). In most children (64% to 93%), the antibody concentration remained at least 0.15 microgram/ml up to the age of 12 to 24 months. CONCLUSIONS: The Hib conjugate vaccine, PRP-T, administered concomitantly with DTP vaccine and IPV, was immunogenic with schedules starting at 1 to 4 months of age. Two injections of PRP-T vaccine were immunogenic enough to maintain protection up to 12 to 18 months of age. PMID- 8618188 TI - Colonization with antibiotic-resistant Streptococcus pneumoniae in children with sickle cell disease. AB - OBJECTIVE: Because of susceptibility to severe pneumococcal infection, children with sickle cell disease (SCD) routinely receive penicillin prophylaxis. Increasing rates of penicillin resistance have been reported throughout the world. Our objective was to assess the prevalence of nasopharyngeal colonization with Streptococcus pneumoniae and to assess the antimicrobial susceptibility of the organisms in children with SCD. STUDY DESIGN: Nasopharyngeal cultures for S. pneumoniae were obtained from all children with SCD attending clinics in a statewide university-based network. Background colonization rates were determined in children attending day care centers in some of the same locations. All recovered S. pneumoniae organisms were tested for susceptibility to penicillin, and all resistant strains were examined for susceptibility to other antibiotics. RESULTS: Overall nasopharyngeal pneumococcal colonization rates among children with SCD were 12%. Colonization was associated with age less than 2 years (p <0.001) and day care attendance for more than 20 hr/wk (p = 0.00005). More than half of these strains (62%) were resistant to penicillin, 33% having intermediate resistance (minimal inhibitory concentration 0.06 to 1 microgram/ml) and 29%, high level resistance (minimal inhibitory concentration > or = 2.0 microgram/ml). Penicillin resistance was associated with penicillin prophylaxis (p <0.01). Many of these organisms were also resistant to other classes of antibiotics. CONCLUSIONS: Although penicillin prophylaxis and pneumococcal vaccine for patients with SCD have reduced overall nasopharyngeal colonization and disease caused by S. pneumoniae (p <0.001), a higher percentage of colonizing strains are now resistant both to penicillin and to other antimicrobial agents (p <0.01). Newer strategies for preventing disease and early management of suspected pneumococcal infection in these children must be developed. PMID- 8618189 TI - Recognition and management of nonpenetrating cardiac trauma in children. AB - OBJECTIVE: To characterize the evaluation and clinical course of children with nonpenetrating injury to the heart. METHODS: We reviewed the medical records for children admitted to St. Louis Children's Hospital between the years 1987 to 1992 with traumatic cardiac injury. Patients with penetrating trauma were excluded; eight children, ages 4 to 13 years, were the study subjects. Chest x-ray studies, electrocardiograms, and serum creatine kinase values were obtained on admission. Two-dimensional echocardiography was performed when indicated by unexplained hemodynamic instability or abnormal radiographic findings. RESULTS: All children with nonpenetrating cardiac trauma were involved in a motor vehicle accident. The principal cardiac diagnoses were ventricular septal defect (1), mitral regurgitation (1), pericardial effusion (2), contusion (3), and arrhythmia (1). Multisystem injury was present in each case, but cardiac injury was not suspected at the time of admission in seven of the eight patients. The hemodynamic status of four children was compromised 12 to 48 hours after admission; echocardiography was diagnostic in each instance, but the electrocardiogram and creatine kinase values were nonspecific. Two patients eventually required cardiac surgery. CONCLUSIONS: Recognition of blunt cardiac trauma in children may be confounded by associated multisystem injury and the delayed onset of clinical manifestations. Echocardiography is a sensitive diagnostic tool for hemodynamically significant disease, and should be performed promptly when patients have unexplained hypotension or diminished peripheral perfusion. PMID- 8618190 TI - Studies of collagen synthesis and structure in the differentiation of child abuse from osteogenesis imperfecta. AB - OBJECTIVE: To determine whether analysis of collagen synthesized by dermal fibroblasts could identify children with osteogenesis imperfecta (OI) among those suspected to have been abused. METHODS: We reviewed biochemical studies and clinical findings for all children who were referred to us to distinguish OI from abuse during a 4-year period. RESULTS: Cells from 6 of 48 children tested to distinguish OI from abuse had biochemical evidence of OI. In five of the six children with abnormal results on collagen studies, clinical signs of OI in addition to fractures were present on examination by a physician familiar with the condition. In those five cases, the diagnosis of OI was strongly suspected. CONCLUSIONS: OI can be diagnosed by biochemical studies in some cases of suspected abuse, but clinical evaluation by experienced physicians is usually sufficient to do so. When diagnostic uncertainty persists in cases of suspected child abuse, biochemical studies may be a useful adjunct, but routine biopsy for children suspected to have been abused is unwarranted. PMID- 8618192 TI - High frequency of celiac disease in Down syndrome. AB - We screened 115 children with Down syndrome for celiac disease, using antigliadin, antiendomysium, and antireticulin serum antibodies and an intestinal permeability test. Celiac disease was diagnosed in eight children, giving a frequency of 7.0%. We recommend screening for celiac disease in all persons with Down syndrome, with the use of at least the antiendomysium antibody determination. PMID- 8618191 TI - Thyroid function in very low birth weight infants: effects on neonatal hypothyroidism screening. AB - OBJECTIVES: To supply normative data for screening thyroxine (T4) and thyrotropin concentrations correlated with birth weight and age at screening of infants with birth weights ranging from 400 to 5500 gm, and to document the effects of screening of very low birth weight (VLBW) infants, because VLBW infants comprise 0.86% of surviving newborn infants and have very low total T4 concentrations with normal or elevated free T4 concentrations as a result of deficient protein binding of thyroid hormones. STUDY DESIGN: Both retrospective and prospective studies were used. We conducted retrospective analyses of screening of T4 and thyrotropin concentrations in 9,324 term, 18,946 low birth weight, and 3,450 VLBW infants in Massachusetts, and a prospective study of T4 and thyrotropin concentrations in 48 VLBW infants at 2 weeks of age. Forty of the infants also had hormone measurements at 4 weeks, 29 at 8 weeks of age, and 24 had analysis of cord blood samples. RESULTS: Median T4 concentrations for each weight group (in 250 gm increments) increased progressively and significantly up to 2500 gm. Of the surviving VLBW infants, 1.5% had screening T4 concentrations that were unmeasurably low (<3.9 nmol/L (0.3 microgram/dl)). The mean T4 concentration varied with age at screening, increasing from cord blood concentrations to a peak at 1 to 3 days of age and thereafter decreasing to a nadir at about 2 weeks in both low birth weight and VLBW infants. In VLBW infants the mean concentrations return to the level of 1 to 3 days by 4 to 8 weeks of age. The incidence of screening thyrotropin concentrations > or = 40 mU/L correlates inversely with weight. The incidence of early, transient hypothyroidism in VLBW infants defined by this thyrotropin concentration was eight times that in term infants. Two infants had late-onset, transient hypothyroidism at 2 and 7 weeks, respectively. CONCLUSIONS: The normative data related to birth weight and age at screening allow proper interpretation of VLBW results for primary T4 and primary thyrotropin screening programs. Screening of the concentrations of T4 and thyrotropin in VLBW increases the number of secondary measurements of T4 in a primary thyrotropin screening program and the number of secondary thyrotropin measurements in a primary T4 screening program by 6% and 9%, respectively. We recommend screening analyses for VLBW infants in the latter part of the first week of life and again at 2 and 4 to 6 weeks of age. This protocol would increase the number of screening analyses by 1.6%. PMID- 8618193 TI - Isolated rice intolerance: clinical and immunologic characteristics in four infants. AB - We describe four infants with rice intolerance who had no hypersensitivity to other foods. Clinical manifestations included shock, vomiting, and diarrhea. Results of tests for occult blood in stools were positive in all cases; results of all immunologic tests were negative. No symptoms were observed during 6 weeks of a diet free of rice and flour. Successive double-blind challenges caused severe shock in three of the four infants. In addition, we observed histologic alterations in the intestinal mucosa after challenge. PMID- 8618194 TI - Hyponatremia caused by a reset osmostat in a neonate with cleft lip and palate and panhypopituitarism. AB - A neonate with cleft lip and palate and hypopituitarism had persistent hyponatremia despite treatment with hydrocortisone, L-thyroxine, and growth hormone. Serum sodium concentration and urinary osmolality increased and decreased appropriately and concurrently with alterations in sodium and water intake. The ability to regulate serum concentrations of antidiuretic hormone at subnormal serum sodium concentrations indicated a reset osmostat. PMID- 8618195 TI - Transient congenital hypoparathyroidism: resolution and recurrence in chromosome 22q11 deletion. AB - Transient congenital hypoparathyroidism (TCHP), with spontaneous resolution in infancy and subsequent recurrence in childhood, has not been associated with a specific cause. We report three patients with TCHP, initially with severe but transient neonatal hypocalcemia. During childhood, recurrence of hypoparathyroidism and recognition of phenotypic features suggested a diagnosis of velocardiofacial syndrome (VCFS). Features specific for the DiGeorge syndrome, with known clinical and genetic overlap with VCFS, were not present except for hypoparathyroidism. Genetic analysis confirmed chromosome 22q11 deletion in each patient. TCHP may be the earliest specific finding in 22q11 deletion/VCFS subgroup, with other diagnostic features emerging in later childhood. Infants with resolved TCHP need continued observation of parathyroid sufficiency, genetic analysis, and examination for anomalies associated with chromosome 22q11 deletion. PMID- 8618196 TI - The Beck Airway Air Flow Monitor as an aid for evaluation of endotracheal tube placement in neonatal patients. AB - We evaluated the Beck Airway Air Flow Monitor (BAAM), through which airflow makes a whistling sound, for its safety and efficacy in confirming that an endotracheal tube is in the trachea. We studied 46 neonates ranging in weight from 0.6 to 3.7 kg. We found that the BAAM consistently produced the desired whistling sound signaling intratracheal placement of the tube in all infants weighing more than 1.5 kg. No adverse effects or complications were noted. PMID- 8618197 TI - Procalcitonin as a marker for the early diagnosis of neonatal infection. AB - Serum procalcitonin was determined in newborn infants at the time of admission to the pediatrics or obstetrics unit. Increased levels were found in all neonates with bacterial sepsis. Neonates with viral infection, bacterial colonization, or neonatal distress had normal or slightly increased levels. These data suggest that procalcitonin might be of value in diagnosing neonatal sepsis. PMID- 8618198 TI - Comparison of chloral hydrate and midazolam for sedation of neonates for neuroimaging studies. AB - In a crossover study of seven term neonates who had neuroimaging studies, chloral hydrate (75 mg/kg administered orally) was more efficacious (p<0.05) but similar with regard to toxic effects than midazolam (0.2 mg/kg administered intravenously). Thus newer drugs are not necessarily better, and monitoring is essential even after a single oral sedative dose. PMID- 8618199 TI - Steroid-resistant chronic urticaria associated with anti-thyroid microsomal antibodies in a nine-year-old boy. AB - The case of a 9-year-old boy with severe chronic urticaria of 6 months' duration is described. The urticaria was associated with intractable bronchospasm and abdominal cramping and was unresponsive to antihistamines and high doses of corticosteroids. Even though the child was euthyroid, he was treated with thyroid hormone after the presence of anti-thyroid microsomal antibodies was documented. Within 1 month the patient demonstrated full remission. He remained free of symptoms for 9 months after discontinuation of treatment. After a relapse he again responded to thyroid hormone therapy. Children with chronic, intractable urticaria and documented evidence of anti-thyroid microsomal antibodies may benefit from treatment with thyroid hormone. PMID- 8618200 TI - Anemia, blueberry-muffin rash, and hepatomegaly in a newborn infant. PMID- 8618201 TI - Measuring resting energy expenditure in pediatrics. PMID- 8618202 TI - Effect of weight reduction on serum transaminase activities in children with simple obesity. PMID- 8618203 TI - Symptoms and Helicobacter pylori infection in children. PMID- 8618204 TI - Acute pancreatitis in a patient with glutaric acidemia type I. PMID- 8618205 TI - American society and hostility to the goals, methods and ideas which have created our economy and its world-leading medical device industry. PMID- 8618206 TI - Currently practised sterilization methods--some inadvertent consequences. AB - Currently used sterilization techniques such as ethylene oxide, gamma irradiation, and steam sterilization could introduce inadvertent consequences, especially in polymeric materials. These could have far-reaching effects on the biocompatibility of the materials. Some of these consequences are reviewed and a typical example of the effect of steam sterilization on the properties and biocompatibility of polyethylene terephthalate is discussed. PMID- 8618207 TI - Sterilization of chitosan: implications. AB - Chitosan, a biopolysaccharide having structural characteristics similar to glycosaminoglycans, seems to be an ideal non-toxic and bioabsorbable biopolymer. This study highlights the effects of some physical and chemical methods of sterilization on chitosan films, in retaining the original tensile strength besides the efficacy of sterility and degree of hemolysis of the finished films. PMID- 8618208 TI - Development of artificial skin (Template) and influence of different types of sterilization procedures on wound healing pattern in rabbits and guinea pigs. AB - Different types of sterilization procedures have been applied onto artificial skin (Template) developed in our laboratory from polyether urethane, chitosan and polyvinyl alcohol, etc. Studies have been performed to investigate the differences in the wound healing pattern. It appears that quickened wound healing takes place in the rabbit model despite different types of samples and sterilization methods. PMID- 8618209 TI - Controlled release of newer quinolones from biodegradable systems based on poly(lactic acid). AB - The release of newer quinolones (such as pefloxacin, ofloxacin, and ciprofloxacin) from biodegradable poly(D, L lactic acid) has been investigated. The in vitro study showed that drug delivery takes place for about two months and a maximum in concentration was recorded after fifteen days. The release from poly(lactic acid) slabs seemed to give high drug doses that are adequate for the treatment of infections caused by common pathogens. PMID- 8618210 TI - Development of a microporous compliant small bore vascular graft. AB - PURPOSE: To produce biodurable small diameter microporous vascular grafts with self-sealing properties for vascular access, for peripheral vascular and potentially for coronary artery bypass. The prosthesis should retain compliance and pulsatile flow in situ using a unique modus operandi permitting wall compression which accommodates changes in volume. METHOD: We have utilized efficient low temperature coagulation technology to develop a unique range of small diameter microporous vascular grafts using ChronoFlex, a biodurable polycarbonate urethane. RESULTS: Grafts have been subjected to a range of in vitro and in vivo testing, demonstrating excellent physical and mechanical characteristics, self-sealing, maintenance of compliance and pulsatile flow in situ and patency up to twenty-two weeks. PMID- 8618211 TI - Midlife crisis in Chinese men and women. AB - Married adults (N = 1,501) between the ages of 30 and 60 years responded to the 15-item Chinese Midlife Crisis Scale. An examination of the levels of concerns in six midlife age groups showed that although some respondents were dissatisfied with their work and personal achievement, a majority did not indicate dissatisfaction at the crisis level; thus the findings did not lend strong support to the existence of normative midlife crisis. The findings also revealed that although midlife crisis levels were different in the various age groups, there was no clear rise of peak in concerns in any particular age group. Further analyses based on different dimensions of midlife crisis showed that women displayed higher levels of overall midlife concerns and problems and fear of aging than men did. PMID- 8618212 TI - Religiosity, gender, and the double standard. AB - Scottish teenagers (N = 690) participated in a survey concerning the relationship between religiosity, gender, and social judgments of sexual activity. Respondents estimated the number of sexual partners of 20-year old men and women and made evaluative judgments of sexually active men and women on positive and negative dimensions. On both tasks, evidence was obtained for the operation of a double standard. Women were expected to have fewer sexual partners than men, and their sexual activity was judged more negatively on evaluatively negative dimensions. Contrary to findings of previous studies in this area, gender differences in endorsement of the double standard were not found. Only moderate support was found for the view that religiosity contributes to different standards of sexual behavior for men and women, although religiosity had significantly greater influence on judgments made by women than on judgments made by men. PMID- 8618213 TI - Relationship of religion and perceived social support to self-esteem and depression in nursing home residents. AB - This study is an examination of the relationship of religiosity and perceived social support to depression and self-esteem in nursing home residents. Answers to questionnaires administered to 83 nursing home residents indicated that perceived social support from family, public religious activity, and length of stay in the home were related to self-esteem and to depression. Past occupational status was also associated with self-esteem. Health status and having a choice in selecting the nursing home were negatively related to depression. Intrinsic religiosity and the resident's perceived social support from friends were not significantly related to depression or self-esteem. PMID- 8618214 TI - Short-term effects of research participation on college men. AB - College students in a social science core curriculum course were given an option of completing a packet of psychological inventories and demographic questions. The last inventory in the packet, the Lazarus Stress Questionnaire, evaluated the emotional impact of answering the questionnaires. Positive feelings were endorsed significantly more than negative feelings. Further analyses, using the Eysenck Personality Questionnaire and the Life Experiences Survey, revealed characteristics that may predispose participants to positive or negative emotional reactions to participation in research. Results are discussed in terms of self-focus mechanisms and ethical standards in the treatment of students who participate in research. PMID- 8618215 TI - Economic advantage and the cognitive ability of rural children in Zaire. AB - Rural Zairian children (n = 32), 4 to 6 years old, were enrolled in a preschool educational and nutritional enrichment program throughout the school year. As part of the evaluative research for this program, cognitive and motor development of the children was assessed with the American Guidance Service (AGS) Early Childhood Screening Profiles (ECSP; Harrison et al., 1990) battery, adapted to the local Bantu dialect of Kituba. On the ECSP global indicator of cognitive ability, the children in the enrichment program performed significantly better than their counterparts in nearby villages, although the two groups did not differ significantly on motor development or anthropometric indicators of physical development. The results indicate that rural African children with reasonable nutritional status demonstrate significantly improved intellectual development in response to a comprehensive economic enhancement and educational enrichment program for them and their families. PMID- 8618216 TI - Mapping motor neuron activity to overt behavior in the leech. I. Passive biomechanical properties of the body wall. AB - As an initial step in constructing a quantitative biomechanical model of the medicinal leech (Hirudo medicinalis), we determined the passive properties of its body wall over the physiological range of dimensions. The major results of this study were: 1. The ellipsoidal cross section of resting leeches is maintained by tonic muscle activation as well as forces inherent in the structure of the body wall (i.e., residual stress). 2. The forces required for longitudinal and circumferential stretch to maximum physiological dimensions were similar in magnitude. Cutting out pieces of body wall did not affect the passive longitudinal or circumferential properties of body wall away from the edges of the cut. 3. The strain (i.e., the percentage change in dimension of different body segments when subject to the same force was identical, despite differences in muscle cross-sections. 4. Serotonin, a known modulator of tension in leech muscles, affected passive forces at all physiological muscle lengths. This suggests that the longitudinal muscle is responsible for at least part of the passive tension of the body wall. 5. We propose a simple viscoelastic model of the body wall. This model captures the dynamics of the passive responses of the leech body wall to imposed step changes in length. Using steady-state passive tensions predicted by the viscoelastic model we estimate the forces required to maintain the leech at any given length over the physiological range. PMID- 8618217 TI - Behavioral and cellular effects of serotonin on locomotion and male mating posture in Ascaris suum (Nematoda). AB - The site and mode of action of serotonin on locomotion were investigated in the parasitic nematode Ascaris suum. Injection of serotonin into Ascaris immediately caused paralysis in animals that were generating locomotory waveforms. Injected serotonin also increased body length and decreased the number of propagating body waves. Similar injections into the male tail produced a ventral tail curl. Injection of N-acetyl-serotonin had no effect on the generation of locomotory waveforms, but increased the body length and decreased the number of body waves in the waveform. Other biogenic amines were also tested but were much less potent. Serotonin decreased the amplitude of a submaximal acetylcholine-induced muscle contraction and increased the time to attain this contraction. The time course of this effect on the response to ACh was much slower than the action of injected serotonin on locomotory waveforms, suggesting that additional elements are involved in the action of serotonin on locomotory behavior. Serotonin abolished spontaneous slow potentials in VI motor neurons and decreased the frequency of EPSPs in DE2 motor neurons, probably by a pre-synaptic mechanism. In the male tail, serotonin depolarized the male-specific transverse ventral muscle cells, but did not affect either dorsal or ventral longitudinal muscle cells. PMID- 8618218 TI - Synaptic connections of the cuticular stress detectors in crayfish: mono- and polysynaptic reflexes and the entrainment of fictive locomotion in an in vitro preparation. AB - The reflex connections made by Cuticular Stress Detector afferents (CSD1 and CSD2) with motorneurones of the four proximal muscle groups in the 5th walking legs of crayfish (Procambarus clarkii, Pacifastacus leniusculus) have been studied in an in vitro preparation. Reflex responses to mechanical stimulation of the CSDs were studied in single neurones by means of intracellular techniques. Within each motorneurone pool, both excitatory and inhibitory reflex responses occurred, although sometimes no reflex connections were found. When present, they could be classified into 'levation' and 'depression' reflexes, corresponding to negative and positive feedback effects respectively. Each motorneurone receives input from a number of different CSD afferents (mean values between 3.0 and 5.8). Using electrophysiological and pharmacological tests, it was demonstrated that at least 32% of all connections were monosynaptic. In preparations showing fictive locomotion, phasic CSD stimulation was shown to be able to entrain anterior levator and depressor motorneurone activity in 95% of cases. The results thus demonstrate the importance of sensory feedback from the CSDs in shaping the final motor output. PMID- 8618219 TI - A surgical guide for identifying the lateral femoral circumflex vessels during free vascularized fibular transfer for avascular necrosis of the femoral head. AB - Free vascularized fibular transfer for avascular necrosis of the femoral head was developed in an attempt to preserve, rather than replace, the femoral head. In this procedure, the peroneal vessels of the autogenous fibular graft are anastomosed to recipient branches of the lateral femoral circumflex vessels. A problem with this procedure has been the difficulty in locating the lateral femoral circumflex vessels. An anatomic and surgical study was conducted to seek a consistent anatomic landmark for use in isolating these vessels. Ten cadaveric and 29 surgical hip dissections were performed. A falx, or fascial band, was consistently found approximately 10 cm distal to the anterior superior iliac spine; it is attached to the anterior intertrochanteric line of the proximal femur, with additional attachments to the investing fascia of the tensor fascia lata and rectus femoris muscles. Cautious release of this falx from superficial to deep leads to the fat layer surrounding the lateral femoral circumflex vessels. This study has shown that an anatomic landmark exists, which consistently leads to location of the lateral femoral circumflex vessels during a free vascularized fibular transfer. By incorporating the use of this landmark ito the authors' surgical technique, they have significantly reduced surgical dissection time and have provided a safe method for isolating the recipient vessels during this challenging operation. PMID- 8618220 TI - The clinical reliability of vein grafts in free-flap transfer. AB - The use of interposition vein grafts has been associated in the literature with a high complication and flap failure rate. Ninety-three vein grafts in 55 patients over a 46-month period were analyzed to evaluate the clinical reliability of vein grafts in a predominantly trauma patient population (37/55). Fifty-two arterioarterial grafts and 41 veno-venous grafts were performed. A-V loops prior to flap transfer were created in 26 patients. The revision rate was 14.8 percent, with a salvage rate of 75 percent. Flap survival was 96.2 percent, compared to 96.7 percent for a large series of flaps without vein grafts. It can be concluded from the study, that the use of vein grafts is not associated with a higher flap failure rate, when technical pitfalls can be avoided, and close monitoring by an experienced staff is guaranteed. PMID- 8618221 TI - Temporoparietal sandwich technique in acute avulsion injury of the hand. AB - In degloving injuries of the hand, rehabilitation can be delayed due to poor wound healing and partial flap necrosis, if the avulsed tissue is sutured back primarily. By sandwiching a temoroparietal fascial flap between the avulsed flap and the tendons, rehabilitation can be started immediately, even if flap necrosis is present. PMID- 8618222 TI - Penile replantation after self-inflicted complete amputation: case report. AB - A case of penile replantation is reported in which a 37-year-old man suffered a self-inflicted complete penile amputation. Replantation was successfully performed; at 3 months after surgery, a urethrocystogram demonstrated the absence of urethral stricture. Although complete erection has yet to be obtained, no other functional impairments have been encountered at 7 months after replantation. Reconstruction of the venous system is considered to be one of the most important factors in achieving a successful penile replantation. PMID- 8618223 TI - Thrombosis in an ischemia-reperfusion injury flap model: is vessel anastomosis a factor? AB - This study was undertaken to elucidate the possible contribution of vessel anastomosis to the incidence of vessel thrombosis in an ischemia- reperfusion injury flap model in rabbits. Bilateral groin flaps were elevated on isolated vascular pedicles and rendered ischemic for 6 (n = 11), 8 (n = 5), 15.5 (n = 5), or 24 hr (n = 8). After the ischemic episode, an arterial anastomosis was performed on one side, and then perfusion was reestablished on both sides. Although the incidence of thrombosis increased with the interval of ischemia, there was no statistically significant difference in thrombosis rate between the two sides for any of the ischemia intervals studied. The authors conclude that the presence of an arterial anastomosis does not increase the rate of vessel thrombosis in flaps after primary ischemia-reperfusion injury in the rabbit model. PMID- 8618224 TI - Thrombosis and thrombolysis in crushed arteries with or without anastomosis: a new microvascular thrombosis model. AB - This study introduces a new rat thrombosis model in which a 2-mm segment of femoral artery is crushed by an impact load. Ninety-five femoral arteries were divided into five groups. The vessels in Groups I and 2 underwent only crush injuries with a 15-kg and 25-kg load, respectively. Group 3 vessels were not crushed, but did undergo vessel anastomosis by a standard microsurgical technique. The vessels in Groups 4 and 5 were crushed by a 25-kg load, and then divided and anastomosed. During the procedure, the vessel lumina were topically irrigated with saline (Group 4) or heparin solution (Group 5). Thrombosis and thrombolysis were evaluated at set time points up to 56 days after operation. While all vessels in Groups 1 and 3 remained patent, the rate of occlusive thrombus formation in Group 2 significantly (p < 0.001) dropped from 85 percent at day 1 to 11 percent at day 7. The intima and media in Groups 4 and 5 were severely disrupted and often occluded the lumen, Group 5 had a significantly (p < 0.01 to 0.001) lower rate of occlusive thrombus formation (40 to 45 percent) at days 1 and 7 than Group 4 (90 percent). Histology in Groups 2, 4, and 5 at day 1 showed no intimnal and almost no medial tissue left in the crushed area. The adventitia and remaining external elastic lamina were adherent to thrombus in the occluded vessels or covered by fibrin and platelet mesh in the patent vessels. The results documented that spontaneous thrombolysis occurs in thrombosed arteries following crush injury alone, but not in thrombosed arteries after crush injury followed by suture anastomoses. The degree of disruption of the internal elastic lamina and the presence of sutures appear to contribute to occlusive thrombus formation following crush injury and anastomoses. Topical irrigation with heparin solution, at the concentration routinely used clinically, significantly reduces the thrombosis rate at the anastomosis site in crushed vessels, but does not promote thrombolysis. PMID- 8618225 TI - The effect of Beraprost sodium on the survival of subcutaneous transferred jejunum after vascular pedicle interruption in a rat model. AB - Beraprost sodium, a stable PGI2 analog, having antiplatelet aggregation and vasodilating actions, was tested in a rat subcutaneous heterotopic jejunal model for its ability to improve survival after vascular pedicle interruption. Forth Sprague-Dawley rats were divided into four groups: Group 1 (control, ligation of pedicle on postoperative day 5); Group 2 (Beraprost sodium, ligation on day 5); Group 3 (control, ligation on day 7); and Group 4 (Beraprost sodium, ligation on day 7). The resulting viability rates were: Group 1 = 0 percent, Group 2 = 40 percent, Group 3 = 30 percent, Group 4 = 90 percent. These results indicate that the administration of Beraprost sodium facilitates the neovascularization of the transferred intestine and shortens the time required for viability of the transferred tissue, after interruption of the vascular pedicle. PMID- 8618226 TI - Scanning electron microscopy evaluation of endothelial regeneration in 1-mm PTFE prostheses. AB - This study documents the developmental stages of neoendothelium regeneration in 1 mm polytetrafluoroethylene (PTFE) prostheses. Scanning electron microscopy was used to follow the progression of platelet deposition, fibrin layering, and endothelial cell migration along the graft. The endothelial healing process was divided into six stages: platelet aggregation (stage I); fibrin network (stage II); bridging (stage III); progression (stage IVA); transmural migration (stage IVB); intimal closure (stage V); and endothelial thromboresistance (stage VI). PMID- 8618227 TI - Topographic anatomy of the recurrent laryngeal nerve. AB - Anatomic and topographic studies were carried out in 30 recurrent laryngeal nerves (RLN), dissecting the vagus nerve retrogradely to identify corresponding fascicles to the RLN. By obtaining measurements of diameter and length, surgically practical data were obtained, allowing for a possible revision of reconstructive possibilities for nerve injuries to the RLN through dissection and nerve graft. PMID- 8618228 TI - A new vessel occluder for microvascular anastomoses. AB - A vessel occluder has been developed that is easy to use and provides effective, safe, vascular occlusion. The occluder is small and is easily rotatable in the operative field. It consists of a plastic backing and a compliant foam strap that wraps around the blood vessel. The foam absorbs blood and irrigation which keeps the vessels moist during the anastomosis. This vessel occluder possesses the ability to vary the amount of blood flow through the vessel, enabling the surgeon to identify and readily repair anastomotic leaks. This occluder has been used successfully by the authors in 100 anastomoses. PMID- 8618229 TI - Use of a micropunch for arteriotomy in end-to-side anastomosis. AB - A micropunch can mechanically create a precise, clean microarteriotomy that is a simple and perhaps more accurate method, especially in atherosclerotic vessels where plaque or the risk of intimal dissection may sometimes interfere with conventional incision or excision techniques. A 1.5-mm. prototype micropunch was tested in seven Sprague-Dawley rats to create;l an end-to-side femoral vein to artery fistula, with a traditional traction suture method used on the contralateral side as a control. All anastomoses were patent immediately and at 2 weeks, implying no compromise, even in small vessels, by using a micropunch. However, the technique had to be modified, as the 1.5-mm anvil could not be inserted directly into the vessel lumen because of the small size of the rat femoral artery. This indicates a significant limitation of this device for use in vessels with an internal diameter less than that of the chosen anvil size. PMID- 8618230 TI - Antitumor effects of antisense phosphorothioate c-myc oligodeoxynucleotides: a question of mechanism. PMID- 8618231 TI - Biochemical modulation of fluoropyrimidines: is there an optimal (6R,S) leucovorin dose and schedule? PMID- 8618232 TI - The secondary leukemias: challenges and research directions. AB - Acute myelogenous leukemia (AML) arising following exposure to genotoxic agents has been recognized as a distinctive entity for more than 40 years. Secondary, or therapy-related, AML accounts for 10%-20% of all AML cases. This review addresses four overarching areas of investigation focused on secondary AMLs: 1) dissection of the molecular structure of the induced genetic lesions and identification of the functional consequences of these changes, thereby providing clues to the pathogenesis of secondary AML and potentially serving as a basis for innovative therapeutic interventions; 2) identification and characterization of mechanisms of DNA damage and the orderly repair of such damage; 3) identification and application of accurate biomarkers of leukemogenesis for the purpose of risk prediction and quantification, potentially allowing recognition of patients especially susceptible to the leukemogenic effects of chemotherapy (for genetic or acquired reasons) and allowing their treatment for cancer to be modified on the basis of this susceptibility; and 4) design and implementation of longitudinal clinical and genetic monitoring of high-risk populations (i.e., individuals under-going cytotoxic therapies for primary cancers). This review of the literature relating to these areas builds upon these themes and attempts to synthesize these seemingly disparate areas of research so that they can be more effectively utilized together to address the problem of secondary AML. Ultimately, the evaluation of these areas will improve our understanding of de novo leukemia and will serve as a springboard for the development of new concepts of therapy and prevention. PMID- 8618233 TI - Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice. AB - BACKGROUND: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. PURPOSE: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, METHODS: The effects of 15-mer [S]ODNs containing c-myc sense, c myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. RESULTS: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. CONCLUSIONS: treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis. PMID- 8618234 TI - Role of fluoropyrimidine Schedule and (6R,S)leucovorin dose in a preclinical animal model of colorectal carcinoma. AB - BACKGROUND: Fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd), used alone or in combination with other cytotoxic agents, exhibit limited efficacy in the treatment of advanced gastrointestinal cancer. (6R,S)leucovorin (LV), a source of reduced folate cofactor, can modulate (i.e., enhance) the therapeutic efficacy of treatment with these fluoropyrimidines (FPs). The role of FP schedule and lv dose in modulating FP antitumor activity, using clinically relevant drug doses and schedules, has not been fully documented. PURPOSE: We evaluated the antitumor activities and the toxic effects of 5-FU and FdUrd, used either alone or in combination with LV, by following three clinically relevant treatment schedules in rats bearing advanced ward colorectal carcinomas. METHODS: Maximum tolerated doses (MTDs), i.e., doses producing a reversible body weight loss of no more than 20% with no lethality, of 5-FU and FdUrd, either individually or in combination with LV, were used in the following treatment schedules: (I) 4 days of continuous intravenous FP infusion (with or without a daily 2-hour lv infusion); (II) a daily FP intravenous push for 4 days (LV, when given, was administered as a 2 hour infusion, with the FP push given after the first hour of LV treatment); and (III) an FP intravenous push given weekly for 3 weeks (the coadministration of LV and FP was performed as in schedule II). In these studies, LV was given at either a low dose (20 mg/kg [body weight] per day) or a high dose (200 mg/kg per day). The MTDs of 5-FU and FdUrd, with or without LV, were defined in normal rats. Antitumor activities were assessed in animals 12-14 days after they received subcutaneous tumor implants. Toxic effects at the MTD were evaluated in both normal and tumorbearing animals. RESULTS: With schedules I and II, the MTD of 5 FU alone was 35 mg/kg per day; with schedule III, it was 100 mg/kg per week. For FdUrd alone, the MTD was 100 mg/kg per day with schedules I and II and 400 mg/kg per week with schedule III. Coadministration of LV reduced the MTD of both 5-FU and FdUrd by approximately 25%-30%, irrespective of the LV dose used. The dose limiting toxic effects of treatment with 5-FU and FdUrd were diarrhea and/or stomatitis, the relative severity of which depended on the schedule of FP administration. The profile of toxic effects was not altered by LV when used at either dose. FP antitumor activity was modulated by LV in all three treatment schedules, but the greatest effects were seen using schedule III, where more complete tumor regression was seen with high-dose LV than with low dose LV. LV potentiated the antitumor activity of FdUrd to a greater extent than that observed with 5-FU. CONCLUSIONS: In this rat model of colorectal carcinoma, the extent to which FP antitumor activity is modulated by LV depends on the schedule of FP administration and the dose of LV used. PMID- 8618235 TI - Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing gammadelta(+) t-cell receptors. AB - BACKGROUND: The monomeric laminin receptor, a 67-kd high-affinity laminin-binding protein, is expressed by a variety of normal cell types. Overexpression and abnormal surface distribution of this receptor have been demonstrated in tumor cells where it appears to promote tumor invasion and metastasis. Previously, we reported the existence of an association between laminin receptor overexpression by lung cancer cells and the presence of tumor-infiltrating lymphocytes (TILs) bearing gammadelta T-cell receptors. Gammadelta(+) lymphocytes represents a sizable fraction of the TILs in approximately one fourth of lung cancers analyzed thus far. PURPOSE: The aim of this study was to determine whether gammadelta(+) TILs might participate in the immune response against lung cancer through recognition of monomeric laminin receptors expressed by tumor cells. METHODS: Tumor cells from 11 lung cancer specimens exhibiting sizable gammadelta(+) T-cell infiltrates and from 11 other specimens infiltrated predominantly by lymphocytes bearing alphabeta(+) T-cell receptors were analyzed for expression of the monomeric laminin receptor by use of the monoclonal antibody (MAb) MLuC5. Gammadalta TILs and chibeta+ TILs derived from four tumors were each examined for cytotoxic activity toward lung cancer target cells by use of a standard 51Cr release assay and lung tumor cell lines expressing different levels of surface monomeric laminin receptor. The ability of MAbs directed against the laminin receptor (i.e., MLuC5) or against gammadelta T-cell receptors (i.e., TigammaA and A13) as well as laminin peptides known to bind to the laminin receptor to inhibit TIL-mediated target cells lysis was also determined. RESULTS: We confirmed that the association between overexpression of the monomeric laminin receptor by lung tumor cells and the presence of gammadeltadelta+ TILs is statistically significant (two sided P = .003; Fisher's exact test). We also observed a relationship between the levels of laminin receptor expression on cultured lung cancer cells and their susceptibility to specific lysis by gammadelta(+), but not alphabeta+, TILs. This specific cell killing was not T-cell receptor mediated, but it was inhibited by addition of the anti-monomeric laminin receptor MAb MLuC5 and by a synthetic peptide corresponding to amino acids 2091-2108 of the laminin A chain. CONCLUSION AND IMPLICATIONS: Our results indicate gammadelta(+) TILs localized at human lung cancer sites can kill tumor cells in a process that involves interaction with the monomeric laminin receptor. The infiltration of gammadelta(+) TILs at lung tumor sites may represent a first line of defense against cells undergoing malignant transformation. PMID- 8618237 TI - Deletion and translocation involving chromosome 3 (p14) in two tumorigenic Kaposi's sarcoma cell lines. AB - BACKGROUND: Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have shown to have normal diploid characteristics are hyperplastic, and depends on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. PURPOSE: We investigated whether the chromosomal changes that occurred in these KS cell lines were random contribute to the pathogenesis of KS. METHODS: We used the conventional G-banding technique and fluorescence in sti hybridization to identify structural and numerical chromosomal changes in the KS cell lines. RESULTS: We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibits loss of heterozygosity of loci at region 3p14-ter. CONCLUSION: This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. IMPLICATIONS: Future research should focus on the identification cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process. PMID- 8618236 TI - Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue. AB - BACK: The functional organization of polarized epithelia depends mostly on adhesion molecules belonging to the integrin and cadherin families. These molecules either recognize basement membrane components, such as laminins, or form intercellular junctions via homotypic interactions. Such tissue organization is often disrupted upon neoplastic transformation, and the resulting loss of functional polarization and cell cohesion might be a prerequisite for the invasive and metastatic behavior of carcinomas. PURPOSE: We studied modifications on thyroid adhesive mechanisms at various stages of neoplastic progression in terms of adhesion molecule expression, topography, and functional regulation by tyrosine kinases. Starting from this working hypothesis, we sought to identify one or more biological markers that would be suggestive of malignant transformation and poorer prognosis and that could be developed as a reliable indicator(s) in early diagnostic steps. METHODS: The study was carried out on both surgical samples and the corresponding fine-needle aspiration biopsy smears (numbers of specimens collected: 19 adenomas, seven follicular carcinomas, 13 papilary carcinomas, and 39 normal tissues). Immunohistochemistry of tissue sections and smears and immuno-precipitation and western blot analysis of protein extracts were done with a battery of monoclonal and polyclonal antibodies. Northern blotting was performed on RNA extracts from frozen tissue samples and use of an integrin subunit beta4 complementary DNA probe. RESULTS: Our findings can be summarized as follows: 1) In normal thyroid cells, the cooperative role of integrin alpha6beta4 and laminin 5/kalinin in hemidesmosome-mediated adhesion adhesion is missing, and recognition of the basal lamina occurs via integrin alpha3beta1 and laminin 1 and/or 2 (this pattern being maintained in adenomas but altered in carcinomas regardless of their histotype or differentiation grade); 2) only in carcinomas with clinical and/or histologic aggressiveness do neoexpression of integrin subunit beta4 and loss of laminin 2/merosin occur, indicating de novo assembly of integrin alpha6beta4; 3) pericellular redistribution and cytoskeletal disconnection of the E-cadherin-catenin complex occur; and 4) basal E-cadherin tyrosine phosphorylation decreases in carcinomas as compared with that in normal and adenomatous tissue. CONCLUSION: The malignant progression of thyroid tumors involves marked rearrangement of cell-basement membrane and cell-cell adhesion molecules and changes in their cytoskeleton linkage. These rearrangements are also easily and reproducibly detected on fine needle aspiration biopsy smears. IMPLICATIONS: Immunodetection of adhesion molecules in sections and/or fine-needle smears may complement the toolbox of thyroid surgical pathologists; it may expand the possibilities of achieving a correct early diagnosis of thyroid tumors and of gaining some prognostic information on thyroid tumors. PMID- 8618238 TI - Impact of inclusion of subsequent primary cancers on estimates of risks of developing cancer. PMID- 8618239 TI - The tumor enhancement phenomenon: reinterpretation from a Th1/Th2 perspective. PMID- 8618240 TI - Phase II studies: which is worse, false positive or false negative? PMID- 8618241 TI - Are time or intensity factors important to the definition of metastases of unknown origin? PMID- 8618242 TI - Paclitaxel premedication regimens. PMID- 8618243 TI - Re: Helicobacter pylori and atrophic gastritis: importance of the cagA status. PMID- 8618244 TI - Re: How does the MRP/GS-X pump export doxorubicin? PMID- 8618245 TI - Does acetaminophen damage the heart? PMID- 8618246 TI - New ways of looking at an old molecule. PMID- 8618247 TI - APAP-induced heart injury? Maybe yes, maybe no. Next question? PMID- 8618248 TI - Pharmacokinetics of extended relief vs regular release Tylenol in simulated human overdose. AB - BACKGROUND: The purpose of this trial was to compare the pharmacokinetics of the two available acetaminophen dosage forms in simulated human overdose. METHODS: Ten healthy volunteers received acetaminophen, 75 mg/kg orally, either as the regular release or extended relief formulation in a random, crossover fashion. Blood samples were analyzed using a TDx assay and a best fit correlation of data points was determined by PCNONLIN. RESULTS: The area under the curves for extended relief acetaminophen and regular release acetaminophen were 426 mg h/L and 432 mg h/L, respectively (p = 0.768). The mean half times for extended relief acetaminophen and regular release acetaminophen were 4.02 h and 2.56 h, respectively (p < 0.001). The mean maximum serum acetaminophen concentrations were 62.6 mg/L (414.4 mmol/L:) and 94.3 mg/L (624.3 mmol/L) for extended relief acetaminophen and regular release acetaminophen, respectively (p < 0.001) and the mean time to maximum serum acetaminophen concentrations were 0.87 h and 0.75 h for extended relief acetaminophen and regular release acetaminophen, respectively (p = 0.508). CONCLUSIONS: Although the formulations appear to have equal bioavailability, their half-lives and peak concentrations were significantly different. Further study is required to determine whether these differences affect the assessment and management of poisoned patients. PMID- 8618250 TI - Multiple-dose activated charcoal compared to urinary alkalinization for the enhancement of phenobarbital elimination. AB - BACKGROUND: Urinary alkalinization and multiple-dose activated charcoal are modalities advocated for the enhancement of phenobarbital elimination in poisoned patients. However, no studies exist comparing the efficacy of these two means of elimination enhancement. We compared their effects on the pharmacokinetic disposition of intravenously administered phenobarbital. METHODS: Ten healthy volunteers participated in each of three randomly ordered study phases. During each phase, 5 mg of intravenous phenobarbital per kilogram of body weight was administered. During phase I, no interventions were made in attempt to enhance phenobarbital elimination. In phase II, participants underwent 24 hours of urinary alkalinization. Throughout phase III, volunteers received six doses of activated charcoal and two doses of sorbitol over 24 hours. RESULTS: The phenobarbital elimination half-life was 148 hours, 47 hours and 19 hours during the control, alkalinization and charcoal phases, respectively. Statistically significant differences in the elimination of phenobarbital were detected when each of the following phases were compared: I vs II, I vs III and II vs III. CONCLUSIONS: Both urinary alkalinization and multiple doses of activated charcoal are effective for the enhancement of phenobarbital elimination but multiple-dose charcoal was superior to urinary alkalinization in our study population. PMID- 8618249 TI - The use of ondansetron in the treatment of nausea and vomiting associated with acetaminophen poisoning. AB - BACKGROUND: Nausea and vomiting associated with poisoning can complicate treatment and in some cases delay potential antidote administration. Side effect such as lowering the seizure threshold may at times discourage the use of traditional phenothiazine and butyrophenone antiemetics. METHODS: We performed a prospective, single arm, observational study examining the effectiveness of the 5HT3 receptor antagonist ondansetron in the management if nausea and vomiting associated with acetaminophen poisoning. Patients with a history or laboratory evidence of acetaminophen poisoning were eligible for inclusion in the study. Exclusion criteria included age less than 18 or greater than 65, use of other antiemetic therapy within the previous 12 hours, history of preexisting hepatic or hematologic disease, pregnancy, or significant ingestion of other substances. Upon meeting entry criteria, patients were administered 8 mg of intravenous ondansetron. Nausea was graded on a 100 mm scale with number of emetic episodes recorded before and after treatment. RESULTS: Six patients were entered in the study. All patients had nausea and at least one emetic episode prior to ondansetron and prior to administration of N-acetylcysteine. All patients reported relief of nausea after ondansetron. The degree of nausea decreased by an average of 52% at 30 min and 88% at 60 min following ondansetron administration. No significant vital sign changes were recorded in any patient, and there were no complications related to therapy. Three patients were administered N acetylcysteine, and all tolerated this therapy without vomiting after ondansetron. CONCLUSIONS: Ondansetron appears to be a potentially useful adjunct in the management of nausea and vomiting associated with acetaminophen poisoning. PMID- 8618251 TI - Lead intoxication in children with pervasive developmental disorders. AB - OBJECTIVE: To investigate the observation that children with pervasive developmental disorders have later and more prolonged lead exposure and are more likely to be reexposed when compared to lead-poisoned children without pervasive developmental disorders. DESIGN: Retrospective chart review. SETTING: A large, urban lead treatment program. RESULTS: Over a six year period 17 children with pervasive developmental disorders (including autism) were treated. Compared to a randomly selected group of 30 children without pervasive developmental disorders who were treated for plumbism over the sam interval, those with pervasive developmental delay were significantly older at diagnosis (46.5 vs 30.3 months, p = .03) and had a longer period of elevated blood lead levels (39.1 vs 14.1 months, p = .013) during management. Despite close monitoring, state-mandated environmental inspection and prompt lead hazard reduction or alternative housing, 75% of children with pervasive developmental disorders were reexposed to lead during medical management compared with 23% of children without pervasive developmental disorders (p = .001). CONCLUSIONS: 1) lead intoxication among children with pervasive developmental disorders may appear de novo beyond the third year of life and is associated with a high rate of reexposure; 2) the provision of deleaded housing (by current techniques) may not be sufficient to protect these children from repeated lead exposure; 3) these data support recommendations by the Centers for Disease Control that children with developmental delays be closely monitored for the appearance of lead intoxication. This monitoring should continue beyond the third year of life. PMID- 8618252 TI - Circulating testosterone levels in skeletal fluorosis patients. AB - OBJECTIVE: The present study focuses on serum testosterone concentrations in patients with skeletal fluorosis, in order to assess the hormonal status in fluoride toxicity. METHODS: Serum testosterones were compared for patients afflicted with skeletal fluorosis (n = 30) and healthy males consuming water containing less than 1 ppm fluoride (Control 1, n = 26) and a second category of controls (Control 2, n = 16): individuals living in the same house as the patients and consuming same water as patients but not exhibiting clinical manifestations of skeletal fluorosis. RESULTS: Circulating serum testosterones in skeletal fluorosis patients were significantly lower than those of Control 1 at p < 0.01. Testosterone concentrations of Control 2 were also lower than those of Control 1 at p < 0.05 but were higher than those of the patient group. CONCLUSIONS: Decreased testosterone concentrations in skeletal fluorosis patients and in males drinking the same water as the patients but with no clinical manifestations of the disease compared with those of normal, healthy males living in areas nonendemic for fluorosis suggest that fluoride toxicity may cause adverse effects in the reproductive system of males living in fluorosis endemic areas. PMID- 8618253 TI - Coin-operated dry cleaning machines may be responsible for acute tetrachloroethylene poisoning: report of 26 cases including one death. AB - BACKGROUND: Incorrect operations by customers are not uncommon in coin-operated dry cleaning establishments; dry cleaning machines may also be poorly maintained. This may result in retention of large amounts of the cleaning solvent in dry cleaned items. CASE REPORT: A 2-year-old boy was found dead in his bed, with a strong odor of solvent in the room. Toxicological analysis demonstrated tetrachloroethylene poisoning. The solvent had been retained in the double curtains of the bedroom which had been dry cleaned in a coin-operated establishment the same day. A retrospective study at the Paris Poison Center revealed 25 additional cases, all with a favorable outcome. Analysis of the circumstances of these accidents showed that the main causes of tetrachloroethylene retention in clothes are overloading of the machine and dry cleaning of bulky items. However, failure of the dry cleaning machine may also be involved. CONCLUSIONS: To immediately reduce the health risks, consumers were informed both via the mass media and by warnings in coin-operated dry cleaning shops. A second batch of preventive measures is in preparation including modifications of the machines to limit solvent exposure and a specific regulation concerning their inspection and maintenance. PMID- 8618254 TI - Myocardial damage and rhabdomyolysis associated with prolonged hypoxic coma following opiate overdose. AB - CASE REPORT: We report a case of biopsy proven myocardial damage after opiate induced rhabdomyolysis. Myocardial biopsy showed focal lesions formed by small mononuclear inflammatory cells with a few neutrophils, associated with degenerated and necrotic myocardial fibers, interstitial edema and congestion of intrinsic blood vessels. These findings were similar to those seen with other drug overdoses if combined with strenuous muscular effort or hypoxic coma. We hypothesize that myocardial damage is a consequence of intracapillary myohypoxia associated with prolonged hypoxic coma following opiate overdose. PMID- 8618255 TI - Hypoglycemia associated with phenytoin intoxication. AB - BACKGROUND: Diphenylhydantoin, a widely used antiepileptic agent, can alter carbohydrate tolerance, and acute intoxication with diphenylhydantoin can be associated with hyperglycemia. CASE REPORT: We describe a patient who experienced a prolonged episode of hypoglycemia secondary to an acute voluntary intoxication with diphenylhydantoin 20 g and zopiclone 225 mg. This hypoglycemic episode was presumptively attributed to diphenylhydantoin and might be due either to an escape from the inhibitory effects of diphenylhydantoin on insulin secretion or an increased sensitivity of the tissues to insulin. Zopiclone was considered less likely since zopiclone overdoses have been only exceptionally associated with hyperglycemia. PMID- 8618256 TI - Elemental mercury-induced skin granuloma: a case report and review of the literature. AB - BACKGROUND: Injection of elemental mercury is rare and only some 72 cases have been reported in the literature over the period 1923-1995. Direct subcutaneous injection or extravasation of mercury injected into blood vessels can produce local granulomata and abscesses. Unless intravascular mercury injection has occurred, clinical signs of mercury toxicity are usually absent though four cases of systemic toxicity have been reported following isolated subcutaneous injection without evidence of elemental mercury dissemination. CASE REPORT: We report a further case of subcutaneous injection by gunshot of elemental mercury, with subsequent granuloma formation, in a 19-year old man who was admitted with an eight month history of a tender enlarging mass in his left antecubital fossa, while on active military service. Surgical removal of mercury from a presumed mercury-tipped bullet was undertaken but was incomplete and the patient declined further operative intervention as he remained asymptomatic. Chelation therapy was not instituted. Serum and urine mercury concentrations were measured for six years after presentation. CONCLUSIONS: We recommend that cases of subcutaneous metallic mercury injection should be managed by complete surgical excision of the granuloma under X ray control and serial monitoring of blood and urine mercury concentrations. PMID- 8618257 TI - Marijuana and hyperthermia. AB - BACKGROUND: Animal and human laboratory studies suggest marijuana may cause hyperthermia. However, there are no clinical case reports of life-threatening hyperthermia associated with use of marijuana alone. CASE REPORT: We report a patient who developed severe hyperthermia after smoking a marijuana cigarette and jogging on a warm day. He presented with delirium; hot, red, dry skin; and a rectal temperature of 41.7 degrees C. Historical and laboratory data indicated he had used cannabinoids and no other drugs. This is the first report of life threatening hyperthermia temporally associated with use of marijuana alone. PMID- 8618258 TI - Three suicide attempts with moclobemide. AB - OBJECTIVE: To report plasma moclobemide, course and outcome of two cases of overdose with moclobemide alone and one case of combined ingestion of moclobemide and clomipramide. METHODS: Moclobemide identification and quantification was achieved by gas chromatography-mass spectrometry after alkaline extraction. CASE REPORTS: In case 1, plasma moclobemide was 2.8 mg/L with 1.8 mg/L clomipramide; in case 2, 18 mg/L; in case 3 60.9 mg/L and 4.6 mg/L 12 hours later. None of the patients showed serious effects during 24 hours of observation. Plasma moclobemide at 10 to 30 times therapeutic was not associated with major toxic effects. Moclobemide seems to be considerably less toxic than tricyclic antidepressants. PMID- 8618259 TI - Ultra-high dose trimethaphan in an infant with severe hypertension. AB - BACKGROUND: Trimethaphan camsylate is a potent antihypertensive drug used to induce systemic arterial hypotension in patients undergoing major surgery and to treat severe systemic hypertension. The pharmacokinetics and pharmacodynamics of trimethaphan administered in the usual clinical dosages have been previously reported. The effects of trimethaphan when administered in very high doses of 500 1000 times the usual dose have not been reported. CASE REPORT: A case is presented of an infant with severe hypertension who inadvertently received such an overdose of trimethaphan. PMID- 8618260 TI - Fatal iatrogenic iodine toxicity in a nine-week old infant. AB - BACKGROUND: Povidone-iodine has been used since the 1950s for various labelled uses as a topical antiseptic. The toxicity of an excessive dose in internal use is described in this case report. CASE REPORT: A 9-week old infant was treated for colic by a pediatric gastroenterologist with loperamide and the elimination of nonhuman milk. Without improvement he was hospitalized and given an enema of 50 mL of povidone-iodine diluted in 250 mL of a bowel irrigant. The enema was promptly expelled and 50 mL of the described solution was given hourly for three doses by nasogastric tube. The infant was found lifeless three hours after the last dose and resuscitation was unsuccessful. Autopsy showed a corroded and necrotic intestinal tract, serous fluid in body cavities, a blood total iodine of 14,600 micrograms/dL, protein-bound iodine of 3,400 micrograms/dL and inorganic iodine of 11,700 micrograms/dL. PMID- 8618261 TI - Intracardiac injection of T-61, a veterinary euthanasia drug. AB - CASE REPORT: We report a suicidal attempt by intracardiac injection of T-61, a veterinary euthanasia drug containing embutramide, mebezonium and tetracaine in dimethylformamide. The main complications were reversible acute renal failure and pericardial effusion. There was a delayed abnormality of the liver function tests possibly related to the dimethylformamide solvent. A liver biopsy on day 16 showed a normal hepatic architecture with lipid-containing lysosomes and prominent vesicular endoplasmic reticulum noted on electron microscopy. PMID- 8618262 TI - Two very different fatal cases associated with the use of methylenedioxyethylamphetamine (MDEA): Eve as deadly as Adam. PMID- 8618263 TI - Multiple system organ failure, intermediate syndrome, congenital myasthenic syndrome, and anticholinesterase treatment: the linkage is puzzling. PMID- 8618264 TI - Multiple system organ failure: link to intermediate syndrome indirect. PMID- 8618265 TI - Propoxyphene overdose in Chinese subjects. PMID- 8618266 TI - Prolonged paralysis after neuromuscular blockade. PMID- 8618267 TI - This month in Investigative Urology. Commentary on antibody production in response to collagen injection. PMID- 8618268 TI - Delayed graft function in renal transplantation: etiology, management and long term significance. AB - PURPOSE: In cadaveric renal transplantation a period of delayed graft function postoperatively is not uncommon and often associated with a poor outcome. We reviewed the biology of reperfusion injury and delayed graft function in renal transplantation, as well as its prevention, management and long-term effects. MATERIALS AND METHODS: The medical literature covering acute tubular necrosis, delayed graft function in renal transplantation and immunology of ischemia reperfusion injury was reviewed. RESULTS: Delayed graft function is clearly associated with poor allograft survival, and is caused by an interaction of ischemic and immunological factors. Technical and pharmacological maneuvers can improve early function rates. The response to ischemic injury is complex, and may increase graft immunogenicity and promote the chronic proliferative changes seen in chronic allograft nephropathy. CONCLUSIONS: Improvement in early renal function should be a primary goal in renal transplantation to enhance early and long-term results. Basic research into the injury response may yield insights into renal pathophysiology. PMID- 8618269 TI - Clinical and pathological features of hereditary prostate cancer. AB - PURPOSE: We determined whether the clinical and pathological features of hereditary prostate cancer differ from those of sporadic prostate cancer. MATERIALS AND METHODS: We compared the clinical and pathological features of radical prostatectomy specimens from 50 men with and 50 without a family history of prostate cancer who were matched for age and date of surgery. RESULTS: Median serum prostate specific antigen concentration was not significantly different in the 2 groups. Mean Gleason score plus or minus standard deviation in the 50 men with sporadic prostate cancer was 6.2 +/- 1 compared to 5.6 +/- 0.9 in those with hereditary disease (p = 0.008). Of the 50 hereditary and 50 sporadic prostate cancers 35 (70%) and 33 (66%), respectively, were pathologically organ confined (p = 0.69). Median percentage of carcinoma within the gland (determined morphometrically) in men with hereditary disease was 11.4 +/- 8.3 compared to 10.9 +/- 8.9 for those with sporadic cancer (p = 0.63). CONCLUSIONS: In our study population hereditary prostate cancers have significantly lower Gleason scores compared to sporadic carcinomas. Otherwise, there appear to be no substantial clinical or pathological differences. PMID- 8618270 TI - Prospective analysis of the incidence of ipsilateral adrenal metastasis in localized renal cell carcinoma. AB - PURPOSE: The incidence of ipsilateral adrenal metastasis in renal cell carcinoma was evaluated in a prospective fashion to understand more fully the natural history of this disease. MATERIALS AND METHODS: During a 15-month period 128 radical nephrectomies were performed for stages T1 to T3bN0M0 renal cell carcinoma at our institution. Of these specimens 100 adrenal glands were of sufficient integrity to allow for thin sectioning. RESULTS: Among the 100 adrenal glands 4 adrenal lesions were discovered (2 metastatic renal cell carcinomas and 2 benign adrenal lesions). All 4 lesions were identified preoperatively by computerized tomography or magnetic resonance imaging. CONCLUSIONS: Our prospective investigation demonstrates that the rate of ipsilateral adrenal metastasis from renal cell carcinoma is approximately 2%, and suggests that when the ipsilateral adrenal gland is well visualized and of normal integrity on preoperative magnetic resonance imaging or computerized tomography adrenal sparing nephrectomy may be considered a viable treatment option. PMID- 8618272 TI - Effect of colon injury on the management of simultaneous renal trauma. AB - PURPOSE: We determined the effect of simultaneous colon injury on the management of renal trauma. MATERIALS AND METHODS: A retrospective review was done of 62 cases of colon and renal injuries from 1977 to 1994, representing 2.5% of 2,596 renal trauma cases. RESULTS: Renal trauma management was consistent with the grade of renal injury. Renal exploration was performed in 58% of the cases, with nephrectomy performed in 16% of the explorations and only for severely injured kidneys. Urological complications occurred in 16% of the cases but they resulted in loss of only 1 renal unit. CONCLUSIONS: Renal injury and reconstruction should not be treated differently in the face of colon injury, including gross fecal contamination. PMID- 8618271 TI - A prospective study of the intake of vitamins C and B6, and the risk of kidney stones in men. AB - PURPOSE: The association between the intake of vitamins C and B6, and kidney stone formation was examined. MATERIALS AND METHODS: We conducted a prospective study of the relationship between the intake of vitamins C and B6 and the risk of symptomatic kidney stones in a cohort of 45,251 men 40 to 75 years old with no history of kidney calculi. Vitamin intake from foods and supplements was assessed using a semiquantitative food frequency questionnaire completed in 1986. RESULTS: During 6 years of followup 751 incident cases of kidney stones were documented. Neither vitamin C nor vitamin B6 intake was significantly associated with the risk of stone formation. For vitamin C the age-adjusted relative risk for men consuming 1,500 mg. daily or more compared to less than 250 mg. daily was 0.78 (95% confidence interval 0.54 to 1.11). For vitamin B6 the age-adjusted relative risk for men consuming 40 mg. daily or more compared to less than 3 mg. daily was 0.91 (95% confidence interval 0.64 to 1.31). After adjusting for other potential stone risk factors the relative risks did not change significantly. CONCLUSIONS: These data do not support an association between a high daily intake of vitamin C or vitamin B6 and the risk of stone formation, even when consumed in large doses. PMID- 8618273 TI - Laparoscopic live donor nephrectomy: the initial 3 cases. AB - PURPOSE: Successful laparoscopic live donor nephrectomy in 3 patients is described. MATERIALS AND METHODS: The procedures were performed completely laparoscopically and the kidneys were extracted via 8 cm. infraumbilical incisions. RESULTS: In all 3 cases warm ischemic time was less than 5 minutes, and the renal vessels and ureter of the harvested kidneys were of adequate length for routine transplantation. Donors required minimal postoperative parenteral analgesia and were discharged home 1 to 3 days after the procedure. All harvested kidneys were successfully transplanted, and functioned well initially and at hospital discharge. CONCLUSIONS: Laparoscopic live donor nephrectomy may be an alternative surgical modality to conventional open nephrectomy. Advantages include less postoperative pain, shorter hospital stay and convalescence, and a more desirable cosmetic result. Additionally, these advantages may encourage more individuals to consider live donation, resulting in an increase in organ supply. PMID- 8618274 TI - Post-transplant renal artery stenosis: impact of therapy on long-term kidney function and blood pressure control. AB - PURPOSE: We assessed the long-term outcome of different treatment methods for transplant renal artery stenosis. MATERIALS AND METHODS: Outcome data for 23 patients with transplant renal artery stenosis treated during a 16-year period were reviewed and analyzed. RESULTS: There was a higher incidence of renal artery stenosis in cadaveric donor kidneys compared to living donor kidneys (2% versus 0.3%, p < 0.02), and in cadaveric kidneys from pediatric donors less than 5 years old compared to those from adults (13.2% versus 1.3%, p < 0.01). Six patients underwent primary medical treatment for renal artery stenosis, with a successful outcome in 4 (mean followup plus or minus standard error 57 +/- 22 months) and failure in 2. Of the patients 16 were treated with percutaneous transluminal angioplasty, including 12 who were cured or improved with respect to hypertension (followup 44.7 +/- 7.6 months). Five patients underwent surgical revascularization for renal artery stenosis with postoperative improvement of hypertension (followup 18.8 +/- 11.6 months). Overall, 21 of 23 patients (91%) were treated successfully for transplant renal artery stenosis with cure or improvement of associated hypertension. Posttreatment renal function was stable or improved in 18 patients, while renal function deteriorated due to parenchymal disease in 3. CONCLUSIONS: Most patients with transplant renal artery stenosis can be treated successfully. Percutaneous transluminal angioplasty is the initial interventive treatment of choice for high grade renal artery stenosis. Surgical revascularization is indicated if percutaneous transluminal angioplasty cannot be done or is unsuccessful. PMID- 8618275 TI - Primary renal lymphoma: a clinical and radiological study. AB - PURPOSE: We assessed the incidence, clinical and radiological features, and prognosis of patients with renal lymphoma. MATERIALS AND METHODS: We studied 210 patients with symptoms, signs and and radiological findings suggestive of renal cell carcinoma. RESULTS: Final diagnosis in 6 of 210 patients (3%) was primary renal lymphoma. Radiological features were similar to those of renal cell carcinoma. Five of the 6 patients had an International Prognostic Index score of greater than 1. Despite appropriate chemotherapy, only 2 patients remain with complete remission. CONCLUSIONS: Primary renal lymphoma is unusual but not rare. The relatively poor prognosis in our patients could be attributed to the adverse prognostic factors associated with aggressive nodal lymphomas. PMID- 8618276 TI - Disease outcome in patients with low stage renal cell carcinoma treated with nephron sparing or radical surgery. AB - PURPOSE: We investigated the outcome of nephron sparing surgery in patients with low grade and low stage (Robson stage II or less) renal cell carcinoma. MATERIALS AND METHODS: We retrospectively reviewed the records of 185 patients treated with nephron sparing surgery and 209 matched for patient age and sex, and tumor stage and grade who were treated with radical nephrectomy. Kaplan-Meier survival curves were constructed for progression and survival end points. Multivariate analysis was performed to determine the tumor characteristics independently correlated with progression and cancer death. RESULTS: No significant difference was observed with respect to progression-free, crude or cancer specific survival between the nephron sparing surgery and radical nephrectomy groups. Less than 5% of the patients treated with conservative nephron sparing surgery had local recurrence. Tumor size was a strong independent predictor of outcome, whereas Robson stage was not. Patients treated with radical nephrectomy had a significant cancer specific and progression-free survival advantage when controlling for tumor diameter and grade. However, no difference was observed in patients with primary tumor diameters of 4 cm. or less. CONCLUSIONS: Robson staging is inaccurate in predicting tumor behavior. Patients with tumors larger than 4 cm. and a normal contralateral kidney may be best served by radical nephrectomy rather than elective nephron sparing surgery. However, nephron sparing surgery may result in an outcome similar to that of radical nephrectomy for low grade, low stage renal cell carcinomas of 4 cm. or smaller. PMID- 8618277 TI - Venous dissection injuries during laparoscopic urological surgery. AB - PURPOSE: The incidence of major venous dissection injuries during laparoscopic procedures is assessed and recommendations are made for management. MATERIALS AND METHODS: We evaluated our experience with all major intra-abdominal injuries occurring during 274 consecutive laparoscopic procedures performed within a 4 year period. Five patients (1.7%) had a total of 6 major vascular injuries, including gonadal vein avulsion in 1 case, lumbar vein avulsion in 1 and a tear in the inferior vena cava in 4. Two patients sustained inferior vena caval injuries during nephrectomy because of adhesions from previous surgery and 1 of them had 2 venacavotomies. RESULTS: All vascular injuries were venous and 5 of the 6 major vessel injuries were treated successfully endoscopically via intracorporeal suturing techniques. The injury requiring open repair was a gonadal vessel avulsion that occurred during retroperitoneal lymph node dissection early in our laparoscopic experience. Major vessel injuries were more likely to occur during complex laparoscopic procedures in patients who had undergone previous ipsilateral retroperitoneal surgery. CONCLUSIONS: In select situations new techniques can allow for safe endoscopic control and repair of venous injuries during laparoscopic surgery. PMID- 8618278 TI - Prognostic significance of Ki-67 labeling index in urothelial tumors of the renal pelvis and ureter. AB - PURPOSE: We evaluated the prognostic significance of the Ki-67 labeling index in 70 patients with primary urothelial tumors of the renal pelvis and ureter. MATERIALS AND METHODS: Immunohistochemical staining for Ki-67 in archival tumor materials was done by the streptavidin-biotin method. RESULTS: Univariate survival analysis showed that the prognosis of patients with a high Ki-67 labeling index of 21.7 or more was significantly worse than that of patients with an intermediate labeling index of 13.3 to less than 21.7 (p < 0.01) or a low labeling index of less than 13.3 (p < 0.001). Multivariate survival analysis showed that staining for Ki-67 labeling index was significantly correlated with prognosis (p < 0.01). CONCLUSIONS: Analysis of Ki-67 labeling index provides useful prognostic information about patients with primary urothelial tumors of the renal pelvis and ureter. PMID- 8618279 TI - Endoscopic treatment of vesicoureteral reflux in patients with myelodysplasia. AB - PURPOSE: We assessed the usefulness of and indications for endoscopic treatment of vesicoureteral reflux in myelodysplasia patients. MATERIALS AND METHODS: A total of 26 patients treated with intermittent catheterization was divided into 11 (16 ureters) with and 15 without vesicoureteral reflux. In 9 patients (13 ureters) endoscopic correction was performed with 3% atelo-collagen and without anesthesia at the outpatient clinic. In each ureter we obtained the sum of scores for 4 risk factors for upper urinary tract deterioration: bladder compliance less than 10 ml./cm. water, grade 2 to 3 bladder deformity, detrusor-sphincter dyssynergia and urethral closure pressure 50 cm. water or greater. RESULTS: No reflux was demonstrated immediately after the initial collagen injection but cystography 3 to 6 months later showed recurrent reflux in 5 ureters (38%). Repeat injection cured the reflux, with results persisting for an average of 17 months. Mean risk factor score for patients without vesicoureteral reflux was significantly lower than that for patients with reflux. In patients treated with intermittent catheterization and anticholinergic agents the mean score for ureters with an increased or unchanged reflux grade was significantly greater than for those with a decreased grade. CONCLUSIONS: Endoscopic treatment of reflux appears to be safe and useful in patients with myelodysplasia. The treatment is preferable in those with high risk factor scores due to the possibility of increased reflux grade in such patients. PMID- 8618280 TI - Clinical evaluation of cell deoxyribonucleic acid content measured by flow cytometry in bladder cancer. AB - PURPOSE: We evaluated the clinical value of flow cytometry in bladder cancer. MATERIALS AND METHODS: Deoxyribonucleic acid (DNA) content was measured by flow cytometry in 275 untreated patients with bladder tumor followed for 1 to 8 years. Four pathological parameters (stage, grade, observed vascular invasion and associated carcinoma in situ) and 3 flow cytometric parameters (ploidy, number of aneuploid cell lines and DNA index) were defined. RESULTS: Univariate survival analysis showed that every parameter, when considered separately, was a significant prognostic factor (p < 0.0001 in call cases). Multivariate analysis showed that stage (p < 0.0001), DNA index (p < 0.01) and associated carcinoma in situ (p < 0.05) were independent, significant prognostic factors. However, ploidy and DNA index enhanced prognostic information above the traditional stage and grade only in patients with a stage pT1, grade 3 tumor (p < 0.05). Retrospectively, different therapeutic decisions could have been made using DNA content only in 4% of cases. CONCLUSIONS: In patients with bladder cancer DNA content is an independent predictor of survival but its clinical usefulness is limited. PMID- 8618281 TI - The effect of lubricants on viability of bacillus Calmette-Guerin for intravesical immunotherapy against bladder carcinoma. AB - PURPOSE: The viability of bacillus Calmette-Guerin (BCG) is crucial for induction of a local immune response and for effective therapy of recurrent superficial bladder carcinoma. During intravesical instillation of BCG lubricants are administered to assist catheterization, which contain bacteriostatic components that may interfere with the viability of mycobacteria. To verify this assumption, 5 commercially available lubricants were analyzed with regard to inhibition of viable BCG growth. MATERIALS AND METHODS: Five different lubricants and their components were co-incubated with Connaught strain BCG and the resultant growth of BCG was assessed. To prove the significant passage of lubricants into the bladder, fluid was recovered from the bladder after catheterization, analyzed with regard to the bacteriostatic effect and compared to normal urine of different acidity. RESULTS: Significant impairment of BCG viability, dependent on dosage and time of co-incubation, was noted with all lubricants analyzed. Several components, namely lidocaine hydrochloride, glyceryl stearate, propyl-4-hydroxy benzoate and chlorhexidine digluconate, were identified as responsible for this inhibition. Fluid recovered from the bladder after lubricant assisted catheterization also showed an inhibitory effect, indicating significant mixture of the instillate with lubricants. CONCLUSIONS: Generous use of lubricants to assist catheterization during intravesical BCG therapy will result in a clinically significant decrease in the number of intravesically instilled viable mycobacteria. For this reason, during intravesical immunotherapy with BCG only small amounts of lubricants should be used for urethral catheterization, and use of catheters not requiring lubricants should be considered. PMID- 8618282 TI - Primary cisplatin, methotrexate and vinblastine aiming at bladder preservation in invasive bladder cancer: multivariate analysis on prognostic factors. AB - PURPOSE: Although radical cystectomy is the standard therapy for invasive bladder cancer, cisplatin based multi-drug chemotherapy has proved to be effective for advanced transitional cell urothelial carcinoma. The potential for bladder preservation with neoadjuvant chemotherapy is currently under investigation. MATERIALS AND METHODS: A phase 2 protocol is presented for conservative treatment of muscle invasive transitional cell carcinoma of the bladder consisting of primary cisplatin, methotrexate and vinblastine chemotherapy followed by reevaluation for bladder sparing surgery and surveillance. A total of 61 patients completed the protocol with a mean followup of 41.4 months. RESULTS: Initial complete response to chemotherapy associated with tumor stage, size and configuration was noted in 20 patients (33%). Bladder preservation, intended only for the complete response group, was achieved in 16 patients (26%) but only 11 (18%) were alive with the bladder intact at study closure. Disease-free 5-year survival rate was 47% (95% confidence interval 65 to 26%). Tumor stage (p = 0.0007), size (p = 0.0003), response to chemotherapy (p = 0.002), patient age (p = 0.039) and tumor grade (p = 0.048) influenced survival. Multivariate analysis revealed response to chemotherapy (beta = 0.988, p = 0.034) and tumor size (beta = 0.978, p = 0.042) to be the only independent predictors. CONCLUSIONS: Induction of cisplatin, methotrexate and vinblastine chemotherapy is helpful in identifying patients with a greater chance for survival among those with locally advanced bladder cancer. However, a bladder preservation strategy based on this therapy is only of limited success. PMID- 8618283 TI - Five-year followup of a prospective trial of radical cystectomy and neoadjuvant chemotherapy: Nordic Cystectomy Trial I. The Nordic Cooperative Bladder Cancer Study Group. AB - PURPOSE: Chemotherapy is widely used in patients with locally advanced bladder cancer but until now there has been no conclusive evidence that this therapy improves survival. The Nordic Cooperative Bladder Cancer Study Group conducted a randomized phase III study to assess the possible benefit of neoadjuvant chemotherapy in patients with bladder cancer undergoing radical cystectomy after short-term radiotherapy. MATERIALS AND METHODS: Our trial included 325 patients with locally advanced stage T1 grade 3 or stages T2 to T4aNXM0 bladder cancer allocated randomly into a chemotherapy or no chemotherapy group (control). The chemotherapy schedule consisted of 2 cycles of 70 mg./m.2 cisplatin and 30 mg./m.2 doxorubicin with a 3-week interval between the cycles. RESULTS: After 5 years the overall survival rate was 59% in the chemotherapy group and 51% in the control group (p = 0.1). The corresponding cancer specific survival rate was 64 and 54%, respectively. In regard to treatment, no difference was observed for stages T1 and T2 disease, while there was a 15% difference in overall survival for patients with stages T3 to T4a disease (p = 0.03). In a multivariate analysis only chemotherapy and T category emerged as independent prognostic factors. The relative death risk for patients who received chemotherapy was 0.69 (95% confidence interval 0.49 to 0.98) compared to the control group after adjustment for the other tested factors. CONCLUSIONS: Neoadjuvant chemotherapy seems to improve long-term survival after cystectomy in patients with stages T3 to T4a bladder carcinoma, while no survival benefit was found for stages T1 to T2 disease. PMID- 8618285 TI - Invasive bladder cancer--where do we go from here? PMID- 8618284 TI - Reassessment of conservative management for stage T1N0M0 transitional cell carcinoma of the bladder. AB - PURPOSE: We evaluated the outcome of stage T1 transitional cell carcinoma of the bladder treated with local tumor resection and intravesical therapies. MATERIALS AND METHODS: Of 42 patients with stage T1 bladder cancer seen at our clinic during a 10-year period 38 were treated conservatively with local tumor resection, intravesical therapy and long-term followup. Bacillus Calmette-Guerin (BCG) was used as the primary intravesical agent since 1986. RESULTS: Of the 38 patients 15 had initial grade 2 or 2 to 3 tumors, including 9 (60%) who had at least 1 or more local recurrences but without disease progression. The remaining 23 patients had grade 3 or grades 3 to 4 stage T1 tumors, with local recurrence in 17 (74%) and disease progression in 8 (35%). Furthermore, 5 patients (22%) died of the metastasis despite salvage therapies. CONCLUSIONS: For patients with initial grade 2 or grades 2 to 3, stage T1 disease the risk of disease progression is low. Current management with local tumor resection and intravesical BCG is appropriate and should be continued. Patients with high grade, stage T1 disease are at particularly high risk for disease progression and BCG does not seem to decrease this risk effectively. Therefore, immediate cystectomy is appropriate and should be recommended. PMID- 8618286 TI - Use of free grafts in urethral stricture reconstruction. AB - PURPOSE: The indications, contraindications and results of free graft urethroplasty are determined. MATERIALS AND METHODS: A retrospective review was done of 40 consecutive patients who underwent free graft urethroplasty with penile and preputial skin, buccal mucosa and bladder epithelium. RESULTS: Of the 35 patients in whom adequate followup data were available the outcome was successful in 30 (86%). Success was unrelated to donor site, prior intervention or cause of stricture. Failure was attributed to placement of grafts onto the penile urethra and patient age. CONCLUSIONS: For strictures in the bulbar urethra the success rate of free grafts was high. Failures occurred in patients in whom full thickness skin grafts were extended far onto the penile urethra. PMID- 8618287 TI - A new method for laparoscopic access to the space of Retzius during retropubic cystourethropexy. AB - PURPOSE: We assessed the feasibility of a new technique for laparoscopic dissection of the space of Retzius. MATERIALS AND METHODS: In 10 women 40 to 70 years old (median age 45) undergoing laparoscopic retropubic cystourethropexy for stress urinary incontinence hydrodissection was used to create a pneumo subperitoneal space. A suction irrigator probe was inserted into a mid peritoneal incision created with a 5 mm. trocar above the symphysis pubis between the 2 umbilical ligaments. The subperitoneal space was developed and insufflated with carbon dioxide without incising the peritoneum. RESULTS: All procedures were completed laparoscopically without intraoperative or postoperative complications. Operative time for cystourethropexy ranged from 30 to 70 minutes (median 40). Estimated blood loss ranged from less than 50 to 300 ml. (median 100). Patients were discharged from the hospital within 24 to 48 hours. All patients reported satisfactory relief of symptoms at 3 to 6 months of followup. CONCLUSIONS: The new technique is not difficult and may minimize tissue injury. Pneumosubperitoneal pressure provides clear exposure of the space of Retzius with minimal bleeding. PMID- 8618288 TI - The presence of arterial anatomical variations can affect the results of duplex sonographic evaluation of penile vessels in impotent patients. AB - PURPOSE: Since penile arterial communications are present in a significant percentage of impotent patients, we evaluated whether peak systolic cavernous blood velocity after intracavernous prostaglandin E1 injection might be different in patients with and without arterial variants. MATERIALS AND METHODS: Cavernous blood flow was assessed with echo color Doppler ultrasound before and after intracavernous injection of prostaglandin E1 in 63 impotent patients. The penile shaft was accurately evaluated to detect arterial anatomical variants. Clinical erectile response was assessed by visual inspection and palpation. RESULTS: Of 23 patients who obtained a full erection with full rigidity after prostaglandin E1 injection the cavernous peak blood velocities in 11 with penile arterial communications were significantly less than those in 12 without arterial communications. CONCLUSIONS: The generally accepted limit of normal for cavernous peak blood flow obtained after prostaglandin E1 injection (greater than 25 to 30 cm. per second) must be interpreted carefully because lower peak blood velocities may be found in subjects with a full erectile response if arterial communications are present. PMID- 8618289 TI - The value of magnetic resonance imaging in the diagnosis of suspected penile fracture with atypical clinical findings. AB - PURPOSE: We studied the use of magnetic resonance imaging (MRI) in the diagnosis of suspected penile fracture. MATERIALS AND METHODS: Penile fracture diagnosis was based on classic history and typical physical signs in 8 patients who were treated surgically without any further diagnostic procedure. Sonography, cavernosography and MRI were performed in 4 patients with equivocal findings. RESULTS: Only MRI identified rupture of the corpus cavernosum in all 4 cases. CONCLUSIONS: MRI is the most accurate imaging procedure when penile fracture is suspected but clinical findings are unusual. PMID- 8618290 TI - Molecular weights and isoelectric points of sperm antigens relevant to autoimmune infertility in men. AB - PURPOSE: We determined the molecular weights and isoelectric points of antigens in the uncapacitated and capacitated spermatozoa of fertile men binding to the serum immunoglobulin G (IgG) from 8 autoimmune infertile men and 8 fertile nonautoimmune men. MATERIALS AND METHODS: We used double fluorochrome cytotoxicity and immunobead binding assays to determine the sperm antibody status of the study subjects. 2-Dimensional gel electrophoresis and Western blot analysis were used to determine the molecular weights and isoelectric points of sperm antigens binding to serum IgG from these men. Amino acid sequencing of the digested peptides of chosen proteins was accomplished. Immune reactivity to the proteins in autoimmune infertile men was further verified. RESULTS: Serum IgG from fertile men failed to react significantly. Serum IgG from all autoimmune men (100%) showed significant binding to proteins with a molecular weight of 92 kDa. and isoelectric points of 3.5 to 4.0 in the capacitated spermatozoa. Six of 8 infertile men (75%) had serum IgG binding to capacitated sperm antigens with a molecular weight of 18 kDa. and isoelectric points of 4.5 to 5.2. Amino acid sequencing of peptides of the 92 kDa. protein matched complement component 1 (C1) inhibitor, with noted differences in the amino acid sequencing from the latter. The 18 kDa. protein matched calmodulin. We verified that serum IgG from autoimmune infertile men bound with C1 inhibitor and ascertained that the 92 kDa. protein in the spermatozoa was C1 inhibitor-like protein. CONCLUSIONS: Significant antibody responses to C1 inhibitor-like protein and calmodulin were noted in autoimmune men. Both of these proteins may be of testicular origin and these autoimmune responses may be highly relevant to infertility. PMID- 8618291 TI - In vitro fertilization improves pregnancy rates for sperm obtained by rectal probe ejaculation. AB - PURPOSE: We evaluated semen quality and pregnancy rates achieved with sperm obtained by rectal probe ejaculation. MATERIALS AND METHODS: A series of 183 rectal probe ejaculation procedures performed by 1 of us (J. F. E.) on 40 anejaculatory men was reviewed. RESULTS: Motile sperm were recovered from 95% of men undergoing rectal probe ejaculation. Live births were recorded for 15 of 33 couples (45%) via intrauterine insemination (10) or in vitro fertilization (5). Three of the latter 5 pregnancies were achieved with intracytoplasmic sperm injection. CONCLUSIONS: Motile sperm are obtained from most men undergoing rectal probe ejaculation and pregnancy rates obtained with these sperm are improved by in vitro fertilization. PMID- 8618292 TI - Malignant testicular neoplasms in immunosuppressed patients. AB - PURPOSE: Testicular cancers were studied in patients on immunosuppression. MATERIALS AND METHODS: A retrospective analysis was done of testicular cancer in patients on immunosuppression reported on between 1975 and 1995. RESULTS: The management schemes adopted in the immunosuppressed population followed the generally accepted management concepts. No patient with low stage (A to B1) disease died of testis cancer. Only 4 of 66 patients (6.1%) with the acquired immunodeficiency syndrome died of testis cancer compared to 5 of 20 (25%) after transplantation, implying significantly higher cause specific mortality in post transplant patients (Fisher's exact test, p < 0.01). The incidence of adverse effect of therapy did not differ from the expected incidence in other patients. CONCLUSIONS: Patients on immunosuppression with testicular neoplasms should be treated in the standard fashion as indicated by tumor histology and stage of disease, since most will tolerate therapy and benefit from the standard treatment protocols. PMID- 8618293 TI - Teratoma in the orchiectomy specimen and volume of metastasis are predictors of retroperitoneal teratoma in low stage nonseminomatous testis cancer. AB - PURPOSE: We determined whether teratomatous elements in the orchiectomy specimen predict for teratoma in the retroperitoneum in patients who have not received chemotherapy. MATERIALS AND METHODS: We retrospectively reviewed the records of patients with clinical stages A, B and B2 nonseminoma who underwent retroperitoneal lymph node dissection. RESULTS: Teratomatous elements in the orchiectomy specimen predict for retroperitoneal teratoma. CONCLUSIONS: When deciding on treatment for low stage nonseminoma, the presence or absence of teratoma in the orchiectomy specimen should be considered to minimize double therapy. PMID- 8618294 TI - Interstitial radiofrequency therapy of the prostate: results of a pilot study. AB - PURPOSE: We assessed the feasibility of using interstitial radiofrequency therapy for benign prostatic hyperplasia (BPH) in 50 patients. MATERIALS AND METHODS: Therapy was given using a standard diathermy unit as the radiofrequency generator and a disposable needle electrode. The main outcome measure was improvement in symptoms assessed by symptom scores. RESULTS: Patients with bothersome symptoms or acute retention showed clinically significant improvements in symptom score, while those with chronic retention did not. There were no serious complications. CONCLUSIONS: Interstitial radiofrequency therapy may be effective for symptomatic BPH. Further studies with long-term objective data are under way to assess its role in the management of BPH. PMID- 8618295 TI - Transurethral microwave thermotherapy for nonbacterial prostatitis: a randomized double-blind sham controlled study using new prostatitis specific assessment questionnaires. AB - PURPOSE: We investigated the effectiveness and durability of transurethral microwave thermotherapy in the treatment of chronic nonbacterial prostatitis using 2 new prostatitis specific assessments in a randomized, double-blind, sham controlled trial. MATERIALS AND METHODS: Patients with nonbacterial prostatitis were randomly assigned to receive either transurethral microwave thermotherapy or sham therapy. Patients were assessed using a symptom severity index and symptom frequency questionnaire. These 2 new prostatitis symptom assessment tools were validated by applying them to 30 similar patients without prostatitis. All nonresponders received transurethral microwave thermotherapy at 3 months and were reassessed at 6 months. Long-term followup of the responder group averaged 21 months. RESULTS: The symptom severity index and symptom frequency questionnaire were confirmed to be valid for symptom assessment in prostatitis patients. The transurethral microwave thermotherapy group benefited from therapy compared to the sham group. Of the sham group 50% had a favorable response after subsequent transurethral microwave thermotherapy. The 7 responders in the treatment group continued to improve during the subsequent 21 months. CONCLUSIONS: Transurethral microwave thermotherapy appears to be an effective, safe and durable treatment for some patients with nonbacterial prostatitis unresponsive to traditional therapy. PMID- 8618296 TI - The use of a removable stent in patients with prostate cancer and obstruction. AB - PURPOSE: A second generation temporary stent (ProstaCoil*) was inserted into the prostatic urethra of patients with obstruction due to prostate cancer to allow spontaneous voiding during hormonal therapy given to decrease the size of the prostatic mass. MATERIALS AND METHODS: The stent was inserted under fluoroscopic guidance using topical anesthesia in 27 patients (mean age 77 years) who presented with urinary retention due to advanced carcinoma of the prostate. All patients underwent operative or nonoperative hormonal therapy shortly after insertion of the stent. RESULTS: Followup of our patients was 3 to 48 months after stent removal and 15 void spontaneously. In 6 patients the stent was removed 9 to 19 months after insertion due to slow regression of the prostatic mass. Two of these patients required transurethral resection of the prostate and in 3 a new stent was inserted because of recurrent obstruction. two recently treated patients await stent removal and 3 died before removal of the stent. During followup no patient had urinary infection, either with the stent indwelling or after its removal. CONCLUSIONS: Temporary internal stenting of the prostate should be the treatment of choice for relieving obstruction during hormonal therapy given for prostate cancer. PMID- 8618297 TI - Reliability of the International Prostate Symptom Score in the assessment of patients with lower urinary tract symptoms and/or benign prostatic hyperplasia. AB - PURPOSE: The reliability of the International Prostate Symptom Score (I-PSS) was tested in patients with lower urinary tract symptoms and/or benign prostatic hyperplasia. MATERIALS AND METHODS: A total of 71 consecutive men with benign prostatic hyperplasia and/or lower urinary tract symptoms was asked to complete the I-PSS at baseline and 8 weeks later. At the second visit the physician also completed the I-PSS according to the complaints of the patient. Variability between both scores was evaluated by calculation of duplo errors and results were compared to the clinical data. RESULTS: A considerable variability existed between the I-PSS results obtained at baseline and 8 weeks. The duplo error was 4.3. In a regression analysis of I-PSS, including all clinical parameters, only free flow had some predictive value for I-PSS outcomes. CONCLUSIONS: It is important to consider the variability of the I-PSS score when making decisions concerning treatment. PMID- 8618298 TI - Prevalence of urinary symptoms and other urological conditions in Spanish men 50 years old or older. AB - PURPOSE: We determined the prevalence of lower urinary tract symptoms and other urological conditions in Spanish men. MATERIALS AND METHODS: A cross-sectional population survey of 2,002 men 50 years old or older was done using interviewers in the autonomous community of Madrid. The main outcome measures were the self reported International Prostate Symptom Score, and acute retention, incontinence, renal failure, hematuria, urinary infection and bladder stones. RESULTS: The estimated response rate among eligible responders was 68.1%. The prevalence of moderate or severe symptoms was 30.4%, which increased with age. Of men with moderate or severe symptoms 26.5% reported that the symptoms were a medium or great problem and 5.4% reported that the symptoms interfered with daily activities at least sometimes. The prevalence of other urological conditions related to the prostate was 5.1% for acute retention, 1.2% for current use of a catheter, 6.1% for incontinence, 2.4% for renal failure, 2.5% for hematuria, 5.2% for urinary infection and 0.7% for bladder stones. CONCLUSIONS: The prevalence of moderate or severe symptoms is greater than reported in most other studies. Although Spanish men with moderate or severe symptoms report similar levels of bothersomeness, the impact of the symptoms in terms of interference with daily activities and perception of the future is less than that in other countries. PMID- 8618299 TI - The American Urological Association symptom score in the evaluation of men with lower urinary tract symptoms: at 2 years of followup, does it work? AB - PURPOSE: The American Urological Association (AUA) benign prostatic hyperplasia (BPH) guidelines committee established criteria for the diagnosis and treatment of patients with BPH. In a prospective study we determined the usefulness of these guidelines in 145 previously untreated patients with BPH symptoms. MATERIALS AND METHODS: Patients were evaluated initially by AUA symptom score, digital rectal examination, urinalysis, serum creatinine and prostate specific antigen. Based on symptom score, patients with mild symptoms were treated with watchful waiting, while those with moderate and severe symptoms were offered watchful waiting, finasteride alpha-blockers, or laser or transurethral prostatectomy. Minimum followup was 2 years. Patients were offered a change in therapy if they had an intolerable adverse event or no improvement. Analysis included maintenance of therapy at 1 and 2 years, number of office visits and diagnostic tests performed. In addition, all patients were queried regarding which factors influenced their therapeutic choice. RESULTS: Of 37 patients with mild symptoms 31 (81%) remained on watchful waiting at 2 years and 6 advanced to medical therapy. Among 71 patients with moderate symptoms 9 of 15 (60%) remained on watchful waiting, 27 of 36 (75%) remained on alpha-blockers and 12 of 20 (60%) remained on finasteride at 2 years. Of the 37 patients with severe symptoms 1 of 5 (20%) remained on watchful waiting, 1 of 6 (17%) remained on finasteride and 9 of 15 (60%) remained on alpha-blockers, while 3 of 5 (60%) who underwent laser prostatectomy and all 6 (100%) who underwent transurethral prostatectomy received no further treatment. At 2 years 83% of the men who selected either finasteride or alpha-blockers as either the primary or secondary therapeutic choice were still on medications. Most patients with mild (61%) or moderate (51%) symptoms cited adverse events as the predominant concern when selecting therapeutic options. In contrast, efficacy was the overriding concern (70%) in patients with more severe symptoms. CONCLUSIONS: Overall, with these guidelines and the AUA symptom score 110 men (76%) were still on original therapy at 1 year and 99 (68%) at 2 years. Additionally, 31 patients (21%) changed to an alternative, nonoperative therapy. These results suggest that the AUA BPH guidelines provide a rational and balanced approach for evaluation and management of patients with symptomatic BPH. Patients can reasonably expect to remain on the initial therapeutic option for at least 2 years. PMID- 8618300 TI - The American Urological Association symptom index--concerns and confirmation. PMID- 8618301 TI - Physiological variation of serum prostate specific antigen in the 4.0 to 10.0 ng./ml. range in male volunteers. AB - PURPOSE: Because some patients show a surprising variation in serial serum prostate specific antigen (PSA) values, we determined the intra-individual or physiological variation in serum PSA by collecting sera 2 to 3 week apart without any prostatic manipulation. MATERIALS AND METHODS: Because 4.0 to 10.0 ng./ml. PSA is the critical range for decision making, we asked all men with a PSA in this range to return 2 to 3 weeks later for a second measurement. Total serum PSA was determined by the Hybritech Tandem-R, automated Tosoh AIA-600 and Delfia section immunoassays. Free and complexed serum PSA was determined by the Delfia assays. Between assay variation (first blood specimen retested on a separate day with the second blood specimen) was compared to the physiological variation (first versus second blood specimens). RESULTS: Mean coefficient of variation (95% confidence limits) was 10.5% for between assay and 23.5% for physiological evaluations. The preferred analysis of ratio difference variation provided a factor of 0.138 (between assay) and 0.298 (physiological) for 95% confidence limits. Changes in free or complexed PSA were not the cause of physiological variation. CONCLUSIONS: The intra-individual physiological variation is 2 to 3 times the between assay variation for sera drawn 2 to 3 weeks apart with a PSA of 4 to 10 ng./ml. A serum PSA of 4.0 ng./ml. can increase to 5.2 ng./ml. (4.0 x 0.298) and still be within the physiological variability for 95% confidence limits. PMID- 8618302 TI - Prostate specimen reevaluation in patients with organ confined prostate cancer and postoperative biological recurrence. AB - PURPOSE: We evaluated whether detectable levels of prostate specific antigen after radical prostatectomy for stage P2 disease are associated with unconfined cancer overlooked at pathological examination. MATERIALS AND METHODS: Among 129 patients with stages T1 and T2 prostate cancer treated with radical prostatectomy 60 had stage P2 disease. The initial slides from the 7 patients with biological failure were carefully reviewed and, if necessary, the embedded blocks were sectioned every 2 mm. RESULTS: The disease was upstaged histologically from P2 to P3 in 6 of 7 patients by reinspecting the initial slides (3) and examining new slides (3). CONCLUSIONS: A postoperative detectable prostate specific antigen level in cases of stage P2 cancer reflects the presence of unconfined disease that may be overlooked by histopathological examination. PMID- 8618303 TI - Postoperative radiotherapy for stage pT3 carcinoma of the prostate: improved local control. AB - PURPOSE: We determined whether radiotherapy after radical prostatectomy leads to improved results in patients with stage pT3 carcinoma of the prostate. MATERIALS AND METHODS: In a prospective nonrandomized study of 203 patients with clinical stage T2 prostate cancer treated with radical prostatectomy 88 underwent surgery alone, 89 received early postoperative radiotherapy generally because of pathological stage T3 disease and 26 received delayed radiotherapy for local recurrence. The disease was stage pT3N0/X in 135 patients. RESULTS: For patients with pathological stage T3 cancer actuarial local recurrence rates were significantly decreased in the early postoperative radiotherapy group compared to the surgery only group (p = 0.005), while actuarial metastatic rates (p = 0.6) and cause specific survival rates (p = 0.04) were not significantly different. Multivariate analysis for all patients in both groups identified adverse features of increased postoperative prostate specific antigen levels, seminal vesicle involvement, lack of postoperative radiotherapy and positive lymph nodes. Late toxicity was severe (Radiation Therapy Oncology Group grade 3 or 4) in 13 surgery only and 17 early postoperative radiotherapy group patients. Of those who were potent postoperatively the incidence of impotence in the early postoperative radiotherapy group was 89% compared to 59% in the surgery only group (p = 0.003). For patients treated with delayed radiation for clinical local recurrence the actuarial local control rate was 54% after 10 years. CONCLUSIONS: Local radiotherapy appears to improve local control of stage pT3 cancer but has no impact on overall survival. PMID- 8618304 TI - Some aspects of prostate cancer. PMID- 8618305 TI - Repair of rectourinary fistulas using a posterior sagittal transanal transrectal (modified York-Mason) approach: an update. AB - PURPOSE: We report our experience with posterior sagittal, transanal, transrectal repair of rectourinary fistulas. MATERIALS AND METHODS: A total of 16 fistula repairs was done in 15 patients. RESULTS: Of the fistulas 13 occurred after a variety of prostatic procedures, 1 after Y-V plasty and 1 after pelvic trauma (2 repairs were attempted in the latter case). Six patients underwent repair without colostomy. No patient experienced fecal or anal complications and all repairs were successful. CONCLUSIONS: Our surgical approach for repair of rectourinary fistulas is simple, effective, associated with minimal morbidity and cost effective. PMID- 8618306 TI - Reversible clinical outcome after sphincter stent removal. AB - PURPOSE: We determined whether the self-expanding sphincter stent, a potential alternative to conventional external sphincterotomy for the treatment of detrusor external sphincter dyssynergia, causes a permanent effect on the lower urinary tract. MATERIALS AND METHODS: Four spinal cord injured men with voiding symptoms of detrusor external sphincter dyssynergia as noted by complete urological evaluation, including a video urodynamic study, were treated with the self expanding sphincter stent. However, the device was explanted 6 months or longer after insertion in all 4 cases due to stent migration (3) and difficulty with condom catheter urinary drainage (1). RESULTS: All stents were removed completely without damage to the urethra. Mean voiding pressure decreased from 62.5 +/- 39.4 to 20.7 +/- 6.5 cm. water after sphincter stent placement. One year after stent explantation mean voiding pressure remained unchanged from preoperative values of 58.5 +/- 21.5 cm. water. No patient had stress urinary incontinence or endoscopically apparent urethral strictures. CONCLUSIONS: The stent can be removed even after complete epithelialization and an extended interval without damage to external sphincter function or urethral stricture formation. The urinary sphincter stent is an effective, reversible treatment for patients with detrusor external sphincter dyssynergia. PMID- 8618307 TI - Detrusor contractility and compliance characteristics in adult male patients with obstructive and nonobstructive voiding dysfunction. AB - PURPOSE: To understand better the contractility and compliance characteristics of the detrusor in patients with varying degrees of outlet obstruction, we analyzed urodynamic studies in elderly men with obstructive and nonobstructive voiding dysfunction. MATERIALS AND METHODS: All patients were evaluated with video urodynamics, including cystometry, isometric tests, voiding profilometry and post void residual measurement. Bladder compliance, detrusor contractility, detrusor reserve, detrusor instability and the severity of outlet obstruction were determined in each patient. Patients were stratified into 4 groups: urodynamically normal, detrusor instability, outlet obstruction and outlet obstruction with detrusor instability. RESULTS: A significant correlation was found between the maximum isometric contraction pressure and the severity of obstruction in 168 patients. Maximum isometric contraction pressure was significantly greater in patients with than without obstruction, independent of detrusor instability. Although compliance was not significantly different among the groups, the proportion of patients with poor compliance (less than 30 ml./cm. water) was lowest in the normal group. The detrusor reserve was significantly less in patients with chronic retention (post-void residual more than 200 ml.) than in those with lower post-void residuals. CONCLUSIONS: The increase in detrusor contractility with increasing outlet obstruction suggests a compensatory response to obstruction. Furthermore, a decrease in bladder compliance does not appear to be a consistent finding in patients with outlet obstruction, although the proportion of patients with poor compliance is higher in the group with obstruction and/or detrusor instability than in those with normal urodynamic findings. The decrease in detrusor reserve in patients with high post-void residual volumes suggests that the detrusor reserve reflects the degree of detrusor decompensation. PMID- 8618308 TI - Urodynamic evaluation in simultaneous insulin-dependent diabetes mellitus and end stage renal disease. AB - PURPOSE: We evaluated the urodynamic changes produced by insulin-dependent diabetes mellitus with end stage renal disease. MATERIALS AND METHODS: A urodynamic evaluation was performed on 51 young patients (mean age plus or minus standard deviation 35 +/- 6 years) with long-term diabetes mellitus (average 21 +/- 6 years) and end stage renal disease (86% on dialysis). RESULTS: The urodynamic study was abnormal in 84% of the patients. The bladder was hypersensitive in 39% and hyposensitive in 30% of the cases, and maximum vesical capacity was greater than 600 ml. in 33%. An acontractile detrusor was noted in 6% of the patients, while 4% had detrusor hyperreflexia and 35% had bladder outlet obstruction. CONCLUSIONS: A high frequency of vesical alterations was observed, which were modified by association of progressive vesical dysfunction and diabetes mellitus. In diabetes mellitus dialysis protects against detrusor hypocontractility but predisposes the patients to have bladder obstruction. PMID- 8618309 TI - Transcutaneous electrical nerve stimulation and temporary S3 neuromodulation in idiopathic detrusor instability. AB - PURPOSE: We studied the effects of electrical stimulation on idiopathic detrusor instability. MATERIALS AND METHODS: Between January 1993 and December 1994, 30 men and 41 women (mean age plus or minus standard deviation 48 +/- 16 years) underwent transcutaneous electrical nerve stimulation (TENS) of the S2-S3 dermatomes, and 13 men and 22 women (mean age 48 +/- 12 years) underwent S3 neuromodulation. Subjective assessment was performed using a diary and symptom score of 0 to 14. Objective outcome was analyzed with urodynamic studies. RESULTS: Mean duration of TENS was 3 +/- 1 weeks (range 2 to 4). Although there were no major complications 31% of the patients reported local skin irritation. The overall urinary symptom scores improved from 10 +/- 2 (range 5 to 14) before the study to 7 +/- 3 (range 1 to 14) during stimulation. Urodynamic analysis revealed significant (p < 0.05) improvements in total bladder capacity and voided volume, and decreases in the number and frequency of unstable contractions. Mean duration of S3 neuromodulation was 6 +/- 1 days (range 4 to 8 days). Four procedures failed due to electrode displacement in 3 cases and procedure intolerance in 1. Hemorrhage from the puncture site occurred in 1 patient. Overall urinary symptom scores were 10 +/- 3 (range 5 to 14) before the study and 5 +/- 2 (range 2 to 10) during stimulation. Although symptomatic relief was more pronounced with S3 neuromodulation, no statistically significant differences were found regarding urinary symptoms compared to TENS. CONCLUSIONS: In patients with severe detrusor instability refractory to conservative treatments the use of TENS and S3 neuromodulation produced significant changes in urodynamic parameters and presenting symptoms. Our results appear to justify evaluation with neuromodulatory techniques before definitive surgical intervention in these patients. PMID- 8618310 TI - Neuro-urological testing. PMID- 8618311 TI - The value of symptom score, quality of life score, maximal urinary flow rate, residual volume and prostate size for the diagnosis of obstructive benign prostatic hyperplasia: a urodynamic analysis. AB - PURPOSE: We investigated which linear combination of scores for symptoms, quality of life, maximum urinary flow rate, residual volume and prostate size best discriminated men with prostatism who do and do not have obstruction. MATERIALS AND METHODS: Mandatory and recommended tests were performed in 196 men older than 50 years with prostatism. Schafer's obstruction grade was estimated by urodynamic studies. Relative residual volume was defined as residual volume divided by cystometric capacity (times 100%). Correlation coefficients among the different parameters were estimated. Obstruction grade was correlated with linear weighted combinations of the parameters. RESULTS: Of the men 79% appeared to have obstruction. The formula, prostate size (cm.3) -3 x maximum urinary flow rate (ml. per second) + 1/4 of relative residual volume (%), correlated almost maximally with obstruction grade. Including quality of life score or symptom score in this expression had a negative outcome on the correlation. Calculation of this expression resulted in the bladder outlet obstruction number. In more than 50% of the men the bladder outlet obstruction number was greater than -2 and more than 90% had obstruction. In 25% of all men the bladder outlet obstruction number was greater than 13 and more than 95% had obstruction. CONCLUSIONS: Bladder outlet obstruction number may be calculated with an easy to use expression composed of prostate size, maximum urinary flow and relative residual volume. In 50% of the men with prostatism bladder outlet obstruction number will diagnose obstruction with a reliability of more than 90%. PMID- 8618312 TI - Laparoscopic pyelolithotomy for calculus removal in a pelvic kidney. PMID- 8618313 TI - Carbon dioxide and helium insufflation during laparoscopic radical nephrectomy in a patient with severe pulmonary disease. PMID- 8618314 TI - A case of renal cell carcinoma producing granulocyte-macrophage colony stimulating factor. PMID- 8618315 TI - Dramatic response to ifosfamide, mesna and doxorubicin chemotherapy regimen in an adult with clear cell sarcoma of the kidney. PMID- 8618316 TI - Post-transplantation lymphoproliferative disorder in the renal transplant ureter. PMID- 8618318 TI - Spontaneous drainage of an appendiceal abscess into an orthotopic neobladder. PMID- 8618317 TI - Lumbar ureteral shunt: an absolute contraindication for retrograde pyelography. PMID- 8618319 TI - Mixed urinary incontinence secondary to lead poisoning. PMID- 8618320 TI - Parasitic infection of the penis. PMID- 8618322 TI - Re: Improved focusing for extracorporeal shock wave lithotripsy of ureteral calculi. PMID- 8618321 TI - Laparoscopic removal of uterus, seminal vesicle and bilateral ovotestes harboring mature teratoma and carcinoid tumor in an intersex patient. PMID- 8618323 TI - Re: The detection of human papillomavirus deoxyribonucleic acid in intraepithelial, in situ, verrucous and invasive carcinoma of the penis. PMID- 8618324 TI - Re: The detection of human papillomavirus deoxyribonucleic acid in intraepithelial, in situ, verrucous and invasive carcinoma of the penis. PMID- 8618325 TI - Re: Historical review of theories on testicular descent. PMID- 8618326 TI - Re: Testicular torsion in a 59-year-old man. PMID- 8618327 TI - Re: Quality of life: radical prostatectomy versus radiation therapy for prostate cancer. PMID- 8618328 TI - Re: Long-term survival and mortality in prostate cancer treated with noncurative intent. PMID- 8618329 TI - Re: Long-term survival and mortality in prostate cancer treated with noncurative intent. PMID- 8618330 TI - Pediatric kidney: functional outcome after extracorporeal shock wave lithotripsy. AB - PURPOSE: We studied the efficacy, functional outcome and morphological changes following extracorporeal shock wave lithotripsy (ESWL*) of renal calculi in a pediatric population. MATERIALS AND METHODS: We prospectively evaluated 50 patients 2 to 12 years old (mean age 7.64) undergoing ESWL with the Siemens Lithostar Plus. Functional outcome was assessed by comparing total and ipsilateral glomerular filtration rates before and after ESWL. Glomerular filtration rate was measured using Gates' method on a 99mtechnetium-pentetic acid radionuclide renal scan corrected to body surface area times 1.73 m.2. Ultrasound was performed to assess morphological changes after ESWL. RESULTS: The immediate fragmentation rate was 86% and the clearance rate was 82%. At the end of the study 40 patients were evaluable. Immediately after ESWL ultrasonography showed perirenal hematoma in 3 patients, intrarenal hematoma in 2 and subcapsular hematoma in 1. These changes resolved spontaneously at followup. There was no change in total or ipsilateral glomerular filtration rate at a mean followup of 31.7 months. Before and after ESWL total glomerular filtration rates were 86.58 +/- 12.43 and 86.27 +/- 12.82 ml. per minute per 1.73 m.2, respectively. Treated kidney glomerular filtration rate decreased insignificantly from 40.58 +/- 12.61 to 40.41 +/- 12.61 ml. per minute per 1.73 m.2 at 3 months. At the last followup the change in glomerular filtration rate was insignificant. CONCLUSIONS: ESWL is effective in the pediatric population, and it can be safely performed without long-term bio-effects on the function or morphology of the growing kidney. PMID- 8618331 TI - Minimal hydronephrosis in the fetus: clinical significance and implications for management. AB - PURPOSE: We further define the natural history and management of minimal fetal hydronephrosis. MATERIALS AND METHODS: Experience with minimal fetal hydronephrosis (renal pelvic diameter 4 to 10 mm. before 20 weeks of gestation and 5 to 10 mm. from 20 to 24 weeks) was reviewed for 18 months at New England Medical Center. RESULTS: The incidence of minimal fetal hydronephrosis was 2.2% in the 5,900 fetuses screened by ultrasound. In 63% of cases prenatal ultrasound was done more than once, and revealed that dilatation of the urinary tract was stable during gestation in 31 (25%), and became normal in 35 (29%) and worse in 11 (9%). A total of 63 patients (103 renal units) followed for up to 1 year underwent postnatal sonography. Those with abnormalities were screened with a voiding cystourethrogram and renal scan, including 40 with normal postnatal ultrasound and no changes on subsequent evaluation. Of the 23 patients with abnormal findings 4 had functionally significant ureteropelvic junction obstruction and 1 had severe vesicoureteral reflux. A retrospective analysis of the sonograms of patients with minimal fetal hydronephrosis and persistence or progression of hydronephrosis revealed calicectasis, progression of minimal fetal hydronephrosis in utero and/or abnormal renal echogenicity in each fetus. CONCLUSIONS: Our study shows that in a small but significant number of fetuses minimal fetal hydronephrosis will progress, whereas in most it will resolve. PMID- 8618332 TI - Minimizing stomal stenosis in appendicovesicostomy using the modified umbilical stoma. AB - PURPOSE: We describe creation of a modified umbilical stoma as part of continent urinary diversion using appendicovesicostomy. MATERIALS AND METHODS: Umbilical stomas were created using the eversion-inversion principle in 25 patients undergoing appendicovesicostomy. RESULTS: Mean followup was 3 years. Cosmesis of the umbilical stoma was good in all cases. Stomal stenosis required surgical revision in 8% of cases. CONCLUSIONS: We recommend our modified umbilical stomal technique for appendicovesicostomy because it allows good cosmesis without compromising stomal function. PMID- 8618333 TI - Alternative approaches to the prognostic stratification of mild to moderate primary vesicoureteral reflux in children. AB - PURPOSE: We compared the prognostic stratification of primary vesicoureteral reflux by performing staging voiding cystourethrography in all children with a urinary tract infection or only in those with renal scarring on 99mtechnetium dimercapto-succinic acid (DMSA) scintigraphy. MATERIALS AND METHODS: Staging voiding cystourethrography and DMSA scintigraphy were performed in 105 children with a urinary tract infection and reflux persistence was assessed by radionuclide cystography after a 2-year followup. RESULTS: Staging voiding cystourethrography revealed no reflux in 51 children (DMSA positive in 3), grades I to II reflux in 21 (DMSA positive in 6) and grade III reflux in 33 (DMSA positive in 19). On followup radionuclide cystography no new reflux was detected, and it was no longer demonstrated in 23 children (8 with grade III and 15 with grades I to II reflux). The finding of grade III reflux on staging voiding cystourethrography had a 76% positive and a 92% negative value for predicting persistent reflux with an 87% predictive accuracy. Limiting the evaluation of voiding cystourethrography data to the 28 children with a positive DMSA scan the combination of renal scarring and grade III reflux had an 84% positive and an 83% negative predictive value with 83% accuracy. This approach would have prevented 77 children from having to undergo voiding cystourethrography. CONCLUSIONS: Performance of staging voiding cystourethrography exclusively in children with renal scarring on a DMSA scan resulted in predictive accuracy that was close to what was achieved by performing voiding cystourethrography in all children with a urinary tract infection. To be able to limit cystourethrography to a select population could prove to be cost-effective. PMID- 8618334 TI - Does testosterone diffuse down the wolffian duct during sexual differentiation? AB - PURPOSE: Sexual differentiation in gonadal dysgenesis is commonly asymmetrical. In patients with true hermaphroditism there may be an ovary and mullerian duct on 1 side, and a testis and wolffian duct on the other side. Such asymmetry suggests that testicular hormones only act locally at this early stage of sexual differentiation. We tested the hypothesis that testosterone reaches the wolffian duct by transport down the duct rather than by simple diffusion. MATERIALS AND METHODS: Mouse 14-day urogenital ridges were placed in organ culture and microinjected with testosterone-albumin-fluorescein isothiocyanate. RESULTS: At 17 hours fluorescence was found throughout the wolffian duct and by 48 hours it was maximal in the dilated caudal end. CONCLUSIONS: Our results support the hypothesis that androgens may be transported along the wolffian duct. Secretion of testicular hormones into the wolffian duct may maintain hormone levels in the biologically active range. PMID- 8618335 TI - Congenital bilateral absence of the vas deferens and cryptorchidism without cystic fibrosis. PMID- 8618336 TI - Heterotopic gastric tissue in the scrotum. PMID- 8618337 TI - Pelvic clear cell adenocarcinoma in a 14-year-old boy. PMID- 8618338 TI - Evaluation of antibody class in response to bovine collagen treatment in patients with urinary incontinence. AB - PURPOSE: Contigen Bard Collagen Implant (CI), made of highly purified bovine dermal type I collagen (BDC), is used as a bulking agent for the treatment of urinary stress incontinence. The humoral immune response to placement of this material in the urinary sphincter was evaluated. MATERIALS AND METHODS: In a prospective clinical study, patients were treated with CI in the urinary sphincter, and blood was collected at various timepoints following injection. Approximately 28% of the patients treated with BDC demonstrated specific antibodies against bovine type I collagen. Serum samples from 27 patients from this cohort were evaluated. The class specificity of circulating antibodies against bovine collagen was characterized by an indirect enzyme-linked immunosorbent assay. RESULTS: In all patients demonstrating an antibody response to bovine collagen, the predominant immunoglobulin class was IgG, found in 100% of sera samples. Immunoglobulin A was produced in approximately 40% of these patients, and IgM was seen in approximately 0.6%. No specific IgE was detected against bovine collagen in any serum sample. The highest concentrations of IgG and IgA antibody classes were observed 4 to 5 months after the initial treatment with CI. In the multicenter clinical trial, adverse events were reported in approximately 40% of all patients treated with CI (19). There was no correlation found between the production of a specific immunoglobulin class and the onset of any clinical adverse events. CONCLUSIONS: In all sera from patients treated with CI for urinary stress incontinence, antibodies to bovine dermal collagen always were predominantly IgG. Immunoglobulin A was seen in less than half of the sera samples, and IgE was not observed. In addition, no change in the humoral response to CI over time was noted in patients demonstrating presensitization to bovine dermal collagen at the time of initial treatment. Clinical adverse events reported for patients demonstrating pretreatment antibodies against bovine dermal collagen did not differ in type or number when compared with patients having no presensitization. PMID- 8618339 TI - Recording the corpus cavernosum electromyogram: principles and problems. AB - PURPOSE: To apply digital signal acquisition and analyzing techniques to the collection and interpretation of electromyographic data of the cavernous body. MATERIALS AND METHODS: Electromyographic recordings were performed in the cavernous bodies of anesthetized, spontaneously breathing dogs under resting conditions and after intracavernous pharmacostimulation with norepinephrine, angiotensin II, phentolamine/papaverine, diethylether and T61. RESULTS: Resting corpus cavernosum activity was ill-coordinated and provided little information. Signal energy was confined largely to the range below 20 Hz. Pharmacostimulation with norepinephrine or angiotensin increased frequency and amplitude of the potential transients and decreased the random components. Administration of a combination of phentolamine and papaverine made the signals very regular and increased periodicity. Blockade of electrical membrane events with diethylether removed all signal components except for electrical and biological noise. CONCLUSIONS: Our findings indicate that electromyograms from the corpus cavernosum can be recorded even under adverse conditions. Signal properties, however, are such that the application of computer-aided data processing and analysis to the evaluation of these myograms is imperative. PMID- 8618340 TI - NM23 gene expression in human prostatic carcinomas and benign prostatic hyperplasias: altered expression in combined androgen blockaded carcinomas. AB - PURPOSE: To investigate whether NM23 (nm23-H1, nm23-H2), a metastasis suppressor gene family, is a molecular marker indicative of metastatic potential of localized carcinoma of the prostate (CaP). Previously, we found decreased nm23-H2 expression correlated with an increase in stage, and here we have expanded the cohort. MATERIALS AND METHODS: Eighty prostate tissue samples from patients with CaP and benign prostatic hyperplasia (BPH) were examined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for NM23 gene expression. Samples were grouped according to stage, grade and whether the patient received combined androgen blockade (CAB) prior to surgery. RESULTS: We could not confirm our initial results of an inverse correlation of nm23-H2 expression levels with grade. However, two significant results were found after CAB: 1) nm23-H1 expression was reduced (p = 0.003), and 2) nm23-H2 expression across stage and grade was uniformly higher (p = 0.003) than in untreated samples. CONCLUSION: NM23 appears not to be a useful molecular marker of metastatic potential in CaP. The altered gene expression after CAB may relate to a cancer cell subpopulation insensitive to apoptosis induced by hormone withdrawal. PMID- 8618341 TI - In vivo antitumor activity of bropirimine against PAIII and Dunning MAT-LyLu rodent prostate cancers. AB - PURPOSE: To evaluate bropirimine for in vivo activity in rodent prostate cancer. MATERIALS AND METHODS: Subcutaneously injected PAIII and Dunning MAT-LyLu rodent prostate cancer cells caused solid tumors and death in controls. Bropirimine was given on varying schedules at 250 mg./kg. by gavage, and tumor volume and survival were recorded. RESULTS: Bropirimine prevented growth when given on the day of tumor injection and caused 95% of advanced tumors to regress completely in the PAIII model. Bropirimine caused significant growth inhibition and prolongation of survival in the MAT-LyLu model. CONCLUSIONS: Bropirimine has statistically significant in vivo activity against both of these rodent prostate cancer cell lines. PMID- 8618342 TI - Effects of nitric oxide on detrusor relaxation. AB - PURPOSE: Recently we (1994) reported the photo-induced adequate nitric oxide (PIANO) system, in which an NO- or NO2-carrying molecule which has been photoactivated to release NO, could be exploited to investigate the role of NO in various smooth muscle functions. This study was designed to characterize the effect of nitric oxide (NO) exploiting PIANO on rat detrusor relaxation by isometric tension recording and measuring changes in cGMP content. MATERIALS AND METHODS: Exposure to ultraviolet light was used (1 to 60 seconds) to evoke PIANO in the presence of streptozotocin, an NO-carrier, and N omega-nitro-L-arginine (L NOARG), an NO2-carrier. During relaxation the cyclic guanosine monophosphate (cGMP) content was measured by radioimmunoassay. RESULTS: Rat detrusor strips were reversibly relaxed upon NO generation via PIANO. Pyrogallol, an O2 generator, significantly (p < 0.01) diminished PIANO-mediated relaxation. During PIANO-mediated relaxation, the tissue level of cyclic GMP significantly (p < 0.05) increased over that of the control. Furthermore, methylene blue, a guanylate cyclase inhibitor, significantly (p < 0.01) inhibited both the relaxation and the increase of cGMP. CONCLUSION: We concluded that rat detrusor muscle was capable of responding to NO, and these findings might lead to a treatment for bladder instability and detrusor hyperreflexia, by the use of intravesical instillation of NO donors. PMID- 8618343 TI - Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin. AB - PURPOSE: These studies were designed to determine the combined inhibitory effect of a human monoclonal antibody (MAb) and cefsulodin on Pseudomonas aeruginosa renal infection in a neutropenic condition. MATERIALS AND METHODS: Protection against the infection of mice was estimated by survival rate and bacterial numbers in the kidney and blood. Opsonophagocytic assay by human polymorphonuclear neutrophils (PMNs) and fluorescence activated cell sorter (FACS) analysis were also examined. RESULTS: Treatment of infected mice with MAb combined with a suboptimal dose of cefsulodin prevented the mice from developing pyelonephritis and bacteremia and resulted in a significantly higher survival rate than treatment with either MAb or cefsulodin alone (p < 0.01). When bacteria were preexposed to cefsulodin, a significant enhancement in opsonophagocytic killing with MAb was observed. Fluorescence activated cell sorter analysis suggested that the bacteria incubated with 1/4 minimal inhibitory concentration (MIC) of cefsulodin showed greater binding of MAb to bacteria than the control. CONCLUSION: The combination therapy with human antilipopolysaccharide MAb and cefsulodin is useful for P. aeruginosa pyelonephritis in neutropenic hosts. PMID- 8618344 TI - Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder augmentations. AB - PURPOSE: To evaluate small intestinal submucosa (SIS) as a possible bladder augmentation material. MATERIALS AND METHODS: Nineteen male dogs underwent 35 to 45% partial cystectomy with immediate augmentation with SIS grafts. All dogs were evaluated pre- and postoperatively with blood chemistries, urine cultures, intravenous urograms, cystograms and cystometrograms. Postoperatively (1 to 15 months), bladders were examined with routine histology and image analysis. RESULTS: All dogs survived their intended survival period without morbidity. All results were normal. Histologically, all 3 layers (mucosa, smooth muscle, serosa) of the normal bladder showed evidence of regeneration. CONCLUSIONS: Small intestinal submucosa acts as a scaffold for bladder augmentation through regeneration and could be a potential option for bladder reconstruction. PMID- 8618345 TI - Bladder squamous cell carcinomas express psoriasin and externalize it to the urine. AB - PURPOSE: To report a single biomarker, psoriasin (Mr 11.0 kd, pI 6.2), a calcium binding protein which is expressed largely by stratified squamous epithelia and is externalized to the urine of bladder squamous cell carcinoma (SCC) bearing patients. MATERIALS AND METHODS: Protein expression profiles of SCCs obtained immediately after surgery were analyzed by two-dimensional gel electrophoresis and Coomassie blue staining. Protein identity was determined by microsequencing and immunoblotting. Protein expression in cryosections was studied by immunofluorescence. RESULTS: Four patients with SCC were identified from 100 samples of patients with suspected transitional cell carcinoma (TCC). The protein profiles of the 4 SCCs (56-1, grade III, T4; 181-1, grade I, T3; 219-1, grade III, T3 and 239-1, grade not determined, T2-4) resembled that of keratinocytes, suggesting that these cells express an early developmental pattern of gene expression. Besides expressing markers characteristic of keratinizing stratified squamous epithelia, the SCCs exhibited psoriasin, a protein externalized to the medium by keratinocytes. Immunohistochemistry of 3 of the SCCs with psoriasin antibodies showed that the positive cells were confined chiefly to the "squamous pearls." The presence of psoriasin in the urine of the 4 SCC patients was demonstrated by two-dimensional gel immunoblotting. Similar analysis of 43 urines from patients with bladder tumors other than SCC revealed 7 positives, some of which may reflect squamous differentiation. Analysis of the urine of 13 control individuals (12 males matched by age and a 42-year-old female) revealed 2 positives. Immunoblotting of the SCC patients' serum proteins with psoriasin antibodies failed to reveal the protein. CONCLUSION: The results point towards psoriasin, alone or as part of a biomarker profile, as a potential marker for the noninvasive follow-up of patients with SCC. PMID- 8618346 TI - Towards a comprehensive database of proteins from the urine of patients with bladder cancer. AB - PURPOSE: To establish a comprehensive two-dimensional database of proteins from the urine of patients with bladder cancer. MATERIALS AND METHODS: Urines dialized against distilled H2O were freeze-dried and subjected to isoelectrofocusing two dimensional gel electrophoresis. Coomassie brilliant blue stained dry gels were scanned and analyzed with the PDQUEST software. Proteins were identified by one or more of the following techniques: microsequencing (44 proteins), mass spectrometry, comigration and immunoblotting. RESULTS: The urine protein database, which includes all the polypeptides detected in the urines of 50 patients, lists 339 proteins (including variants) of which 124 have been identified. Of these, psoriasin, the psoriasis-associated fatty acid binding protein 5, the gelsolin fragments and prostaglandin D2 synthetase have not been previously described. CONCLUSIONS: The database provides a solid basis for further studies aiming at identifying tumor markers in the urine that may serve as prognostic factors in bladder cancer. PMID- 8618348 TI - A piece of my mind. Friday afternoon. PMID- 8618349 TI - More evidence links NSAID, estrogen use with reduced Alzheimer risk. PMID- 8618350 TI - Politicians, AIDS experts wage budget debate. PMID- 8618347 TI - Developmentally imprinted genes as markers for bladder tumor progression. AB - PURPOSE: Developmentally imprinted genes, such as H19 and insulin-like growth factor-II (IGF-II), play an important role during human embryogenesis and also have been implicated in the pathogenesis of embryonal tumors of childhood. Since H19 is expressed in human fetal bladder, we evaluated 35 bladder carcinomas for H19 expression by in situ hybridization analysis and correlated expression with tumor grade. As a prelude to gene transfer studies to determine if H19 is a bladder tumor oncogene, we also evaluated bladder cell lines for expression of H19, IGF-II, IGF-I and the type I IGF receptor. MATERIALS AND METHODS: H19 expression was evaluated by in situ hybridization analysis in bladder tumor specimens. Northern analysis was used to evaluate the expression of H19, IGF-II, IGF-I and the type I IGF receptor in bladder cell lines. RESULTS: H19 was expressed preferentially in advanced stage tumors: 2 of 12 grade I tumors were H19 positive, whereas 9 of 11 grade II and 7 of 10 grade III tumors expressed H19 (p = 0.004). Additionally, 6 of 6 carcinoma in situ tumors were H19 positive, whereas normal bladder mucosa cells were H19 negative. We found that 3 of 11 cell lines (HT-1376, HT-1197 and 5637) express high levels of H19 mRNA, and each of these cell lines and J82 also express IGF-II. All cell lines examined expressed the type I IGF receptor, whereas there was no detectable IGF-I mRNA. CONCLUSIONS: These data demonstrate that H19 is an oncodevelopmental marker of bladder tumor progression and raise the possibility that H19 may have oncogenic properties in bladder cancer. PMID- 8618351 TI - Can sports-minded kids have too many helmets? PMID- 8618353 TI - ACC features clinical trial presentations. PMID- 8618352 TI - Identifying substance abusers at preschool age. PMID- 8618354 TI - From the Centers for Disease Control and Prevention. Outbreaks of hepatitis B virus infection among hemodialysis patients--California, Nebraska, and Texas, 1994. PMID- 8618355 TI - From the Centers for Disease Control and Prevention. Hantavirus pulmonary syndrome--United States, 1995 and 1996. PMID- 8618356 TI - From the Centers for Disease Control and Prevention. Assessment of testing for and completeness of reporting of vancomycin-resistant enterococci--Connecticut, 1994. PMID- 8618357 TI - Somatization and medicalization. PMID- 8618358 TI - Somatization and medicalization. PMID- 8618359 TI - Somatization and medicalization. PMID- 8618360 TI - Increasing US mortality from infectious diseases. PMID- 8618361 TI - Increasing US mortality from infectious diseases. PMID- 8618362 TI - A 50-year-old woman with spinal stenosis. PMID- 8618363 TI - A 50-year-old woman with spinal stenosis. PMID- 8618364 TI - Very low-fat diets for coronary heart-disease: perhaps, but which one? PMID- 8618365 TI - Triggering myocardial infarction by sexual activity. Low absolute risk and prevention by regular physical exertion. Determinants of Myocardial Infarction Onset Study Investigators. AB - OBJECTIVE: To determine the relative risks of nonfatal myocardial infarction (MI) triggered by sexual activity among the general population and in patients with prior coronary heart disease. DESIGN: Relative risks and effect modification were calculated by the case-crossover method, a new epidemiologic technique designed to quantify the transient change in risk following exposure to a potential disease trigger. SETTING/PARTICIPANTS: A total of 1774 patients with MI were interviewed in 45 hospitals throughout the United States. Data were gathered on potential triggers of MI occurring immediately prior to the event and during the previous year. Results are presented for the 858 patients who were sexually active in the year prior to the MI, with attention to the 273 patients who had coronary artery disease prior to their index MI, and the effect of regular exertion on risk. MAIN OUTCOME MEASURE: The relative risk of nonfatal MI following sexual activity. RESULTS: Of the 858 patients, 79(9%) reported sexual activity in the 24 hours preceding MI, and 27(3%) reported sexual activity in the 2 hours preceding onset of symptoms of MI. The relative risk of MI occurring in the 2 hours after sexual activity was 2.5(95% confidence interval [CI], 1.7-3.7). The relative risk of triggering onset of MI among patients with a history of prior angina (2.1 [95% CI, 0.8-5.8]) or prior MI (2.9 [95% CI, 1.3-6.5]) was not greater than that observed in those without prior cardiac disease. Sexual activity was a likely contributor to the onset of MI in only 0.9% of cases and regular exertion was associated with decreasing risk. CONCLUSIONS: Sexual activity can trigger the onset of MI. However, the relative risk is low, and since the absolute hourly risk of MI is extremely low, the absolute risk increase caused by sexual activity also is extremely low (1 chance in a million for a healthy individual). Moreover, the relative risk is not increased in patients with a prior history of cardiac disease and regular exercise appears to prevent triggering. These findings should be useful for counseling patients and decreasing the fear of sexual activity that often prevents complete rehabilitation from cardiovascular disease. PMID- 8618366 TI - A detailed comparison of physician services for the elderly in the United States and Canada. AB - OBJECTIVE: To assess the relative volume and price of physician services in Canada and the United States. DESIGN: A comparative analysis of 1992 claims data from Canadian provincial ministries of health and from the US Health Care Financing Administration. PATIENTS: All elderly individuals in the 3 largest Canadian provinces, Ontario, Quebec, and British Columbia, and a 1% random sample of US elderly Medicare beneficiaries not enrolled in health maintenance organizations. MAIN OUTCOME MEASURE: The volume of physician services measured in terms of the relative value units used in the Medicare fee schedule to calculate payments, with services disaggregated into clinically meaningful categories. RESULTS: Canadian elderly receive a higher volume of physician services than US elderly. Because the provinces examined paid a much lower price per service, Canada had overall lower expenditures per elderly person than the United States. Canadian elderly received 44% more evaluation and management services, but 25% fewer procedures. Canada has a disproportionately lower volume of procedures for which there is low clinical consensus as to when they are indicated. Such procedures include cataract extractions and knee replacements. CONCLUSION: The lower prices for physician services in Canada permit Canadian elderly to receive a higher volume of evaluation and management services, on the other hand, are constrained by both price and volume. These differences in the volume of physician services may be the result of differences in facility and physician supply. PMID- 8618367 TI - The Ontario trial of active compression-decompression cardiopulmonary resuscitation for in-hospital and prehospital cardiac arrest. AB - OBJECTIVE: To compare the impact of active compression-decompression (ACD) cardiopulmonary resuscitation (CPR) and standard CPR on the outcomes of in hospital and prehospital victims of cardiac arrest. DESIGN: Randomized controlled trial with blinding of allocation using a sealed container. SETTINGS: (1) Emergency departments, wards, and intensive care units of 5 university hospitals and (2) all locations outside hospitals in 2 midsized cities. PATIENTS: A total of 1784 adults who had cardiac arrest. INTERVENTION: Patients received either standard or ACD CPR throughout resuscitation. MAIN OUTCOME MEASURES: Survival for 1 hour and to hospital discharge and the modified Mini-Mental State Examination (MMSE). RESULTS: All characteristics were similar in the standard and ACD CPR groups for the 773 in-hospital patients and the 1011 prehospital patients. For in hospital patients, there were no significant differences between the standard (n = 368) and ACD (n = 405) CPR groups in survival for 1 hour (35.1% vs 34.6%; P = .89), in survival until hospital discharge (11.4% vs 10.4%; P = .64), or in the median MMSE score of survivors (37 in both groups). For patients who collapsed outside the hospital, there were also no significant differences between the standard (n = 510) and ACD (n = 501) CPR groups in survival for 1 hour (16.5% vs 18.2%; P = .48), in survival to hospital discharge (3.7% vs 4.6%; P = .49), or in the median MMSE score of survivors (35 in both groups). Exploration of clinically important subgroups failed to identify any patients who appeared to benefit from ACD CPR. CONCLUSIONS: ACD CPR did not improve survival or neurologic outcomes in any group of patients with cardiac arrest. PMID- 8618368 TI - Energy expenditure with indoor exercise machines. AB - OBJECTIVE: To compare the rates of energy expenditure at given rating of perceived exertion (RPE) levels among 6 different indoor exercise machines. DESIGN: Repeated measures design. PARTICIPANTS: Healthy young-adult volunteers, including 8 men and 5 women. INTERVENTIONS: Subjects underwent a 4-week habituation period to become familiar with the RPE scale and exercise on an Airdyne, a cross-country skiing simulator, a cycle ergometer, a rowing ergometer, a stair stepper, and a treadmill. Following habituation, each subject completed an exercise test with each exercise machine. The exercise test comprised 3 stages of 5 minutes at self-selected work rates corresponding to RPE values of 11 (fairly light), 13 (somewhat hard), and 15 (hard). Oxygen consumption, from which the rate of energy expenditure was calculated, was measured during the last minute of each 5-minute exercise stage. Heart rate was measured during the last minute of each stage of the exercise test, and blood lactate levels were obtained immediately after each exercise stage. MAIN OUTCOME MEASURE: Rate of energy expenditure at specified RPE values. RESULTS: Rates of energy expenditure at a given RPE varied by 1093 kJ/h (261 kcal/h) for the exercise machines. The treadmill induced higher (P < .05) rates of energy expenditure for fixed RPE values than all other exercise machines. The cross-country skiing simulator, rowing ergometer, and stair stepper induced higher (P < .05) rates of energy expenditure than the Airdyne and cycle ergometer. Heart rate varied significantly (P < .01) among exercise machines, with the highest values associated with the treadmill and the stair stepper. Lactate concentration varied significantly (P = .004), with highest values associated with use of the stair stepper and the rowing ergometer. CONCLUSIONS: Under the conditions of the study, the treadmill is the optimal indoor exercise machine for enhancing energy expenditure when perceived exertion is used to establish exercise intensity. PMID- 8618369 TI - Comparison of the effects of over-the-counter famotidine and calcium carbonate antacid on postprandial gastric acid. A randomized controlled trial. AB - OBJECTIVE: To compare an over-the-counter histamine2-receptor antagonist with an antacid as gastric acid reducers. DESIGN: Randomized, double-blind, placebo controlled crossover trial. SETTING: Gastric secretory research laboratory in a Veterans Affairs medical center. PARTICIPANTS: Eighteen healthy volunteers (10 men and 8 women) aged 25 to 62 years with normal gastric acid secretion rates. INTERVENTIONS: The subjects received the histamine2-receptor antagonist famotidine (Pepcid AC, 10 mg), calcium carbonate antacid tablets (Tums, 1000 mg), or placebo medications 1 hour after a test meal. Two identical meals were taken 2.5 and 6.0 hours after the medication was given. MAIN OUTCOME MEASURES: Intragastric pH was maintained at 4.0 by in vivo intragastric titration with 0.3N sodium bicarbonate for 10 hours (1 hour before and 9 hours after medication). Reduction in sodium bicarbonate titrant use in the 2 treatment groups compared with titrant use with placebo was reflective of acid secretion inhibited by a famotidine or acid neutralized by calcium carbonate tablets. RESULTS: When evaluated in increments of 30 minutes, calcium carbonate had a rapid onset of action, neutralizing 6.7 mmol of acid in the first 30 minutes. However, its duration of effect was only 60 minutes. Famotidine had a delayed onset of action compared with antacid, beginning after 90 minutes. However, famotidine had a duration of effect of at least 540 minutes. At its peak effect, 210 minutes after administration, famotidine reduced acid secretion by 7.3 mmol per 30 minutes. CONCLUSIONS: Recommended over-the-counter doses of famotidine and calcium carbonate tablets have different pharmacokinetic profiles when taken in the postprandial period. The antacid has a rapid onset and short duration of action, while the histamine2-receptor antagonist has a delayed onset and a prolonged duration. Their peak potencies are similar. PMID- 8618370 TI - Reestimating date of delivery in multifetal pregnancies. AB - OBJECTIVE: To clarify the optimal estimated date of delivery for multifetal pregnancies. DESIGN, SUBJECTS, AND SETTING: A retrospective study of all 88,936 infants born of multifetal pregnancies and all 6,020,542 infants born of singleton pregnancies that occurred at 26 weeks or more of gestation between 1989 and 1993 in Japan. MAIN OUTCOME MEASURE: Incidence of stillbirth and early neonatal death according to gestational age at delivery. RESULTS: The mean +/- SD duration of pregnancy was 37.0 +/- 2.7 weeks for multifetal pregnancies and 39.6 +/- 1.6 weeks for singleton pregnancies. In multifetal pregnancies, the incidence of stillbirth and of early neonatal death gradually declined until 37 to 38 weeks' gestation and then increased. These parameters in singleton pregnancies declined until 39 weeks' gestation before increasing. The lowest incidence of perinatal death (Stillbirth plus early neonatal death) seen at 38 weeks' gestation in multifetal pregnancies corresponded to that seen at 43 weeks' gestation in singleton pregnancies (10.5 vs 9.7 per 1000 infants). The fist of perinatal death was more than 6 times as high for fetuses of multifetal pregnancies born at 37 weeks or later than for singleton fetuses born at 40 weeks or later (relative risk, 6.6; 95% confidence internal, 6.1 - 7.1). CONCLUSION: Fetuses of multifetal pregnancies are at an increased risk of death after reaching the normative gestational age for singleton pregnancies. Limiting the estimated date of delivery to 37 to 38 weeks may be appropriate in multifetal pregnancies. PMID- 8618371 TI - Users' guides to the medical literature. XI. How to use an article about a clinical utilization review. Evidence-Based Medicine Working Group. PMID- 8618372 TI - A 35-year-old pregnant woman considering maternal serum screening and amniocentesis. PMID- 8618373 TI - Sexual activity triggering myocardial infarction. One less thing to worry about. PMID- 8618374 TI - Health care for older people. A look across a frontier. PMID- 8618375 TI - An experimental model investigation of the opening of a collapsed untethered pulmonary airway. AB - We developed an essentially two-dimensional planar benchtop model of an untethered collapsed airway to investigate the influence of fluid properties (viscosity, mu and surface tension, gamma) and the structural characteristics (effective diameter, D, longitudinal tension, T, and fluid film thickness, H) on airway reopening. This simplified model was used to quantify the relationship between wall deformation and meniscus curvature during reopening. We measured the pressure (P) required to move the meniscus at a constant velocity (U), and found the dimensionless post-startup pressure (PD/gamma) increased monotonically with the capillary number (Ca = microU/gamma). Startup pressures depend on the fluid viscosity and piston acceleration, and may significantly increase reopening pressures. Consistently stable steady-state pressures existed when Ca > 0.5. D was the most dominant structural characteristics, which caused an increase in the post-startup pressure (P) for a decrease in D. An increase in H caused a slight decrease in the reopening pressure, but a spatial variation in H resulted in only a transient increase in pressure. T did not significantly affect the reopening pressure. From our planar two-dimensional experiments an effective yield pressure of 3.69 gamma/D was extrapolated from the steady-state pressures. Based on these results, we predicted airway pressures and reopening times for axisymmetrically collapsed airways under various disease states. These predictions indicate that increasing surface tension (as occurs in Respiratory Distress Syndrome) increases the yield pressure necessary to reopen the airways, and increasing viscosity (as in cystic fibrosis) increases the time to reopen once the yield pressure has been exceeded. PMID- 8618376 TI - The mechanical behavior of a mammalian lung alveolar duct model. AB - A model for the mechanical properties of an alveolar duct is analyzed using the finite element method. Its geometry comprises an assemblage of truncated octahedral alveoli surrounding a longitudinal air duct. The amounts and distributions of elastin and collagen fiber bundles, modeled by separate stress strain laws, are based upon published data for dogs. The surface tension of the air-liquid interface is modeled using an area-dependent relationship. Pressure volume curves are computed that compare well with experimental data for both saline-filled and air-filled lungs. Pressure-volume curves of the separate elastin and collagen fiber contributions are similar in form to the behavior of saline-filled lungs treated with either elastase or collagenase. A comparison with our earlier model, based upon a single alveolus, shows the duct to have a behavior closer to reported experimental data. PMID- 8618377 TI - Axial strain measurements in skeletal muscle at various strain rates. AB - A noncontact optical system using high speed image analysis to measure local tissue deformations and axial strains along skeletal muscle is described. The spatial resolution of the system was 20 pixels/cm and the accuracy was +/- 0.125 mm. In order to minimize the error associated with discrete data used to characterize a continuous strain field, the displacement data were fitted with a third order polynomial and the fitted data differentiated to measure surface strains using a Lagrangian finite strain formulation. The distribution of axial strain along the muscle-tendon unit was nonuniform and rate dependent. Despite a variation in local strain distribution with strain rate, the maximum axial strain, Exx = 0.614 +/- 0.045 mm/mm, was rate insensitive and occurred at the failure site for all tests. The frequency response of the video system (1000 Hz) and the measurement of a continuous strain field along the entire length of the structure improve upon previous noncontact optical systems for measurement of surface strains in soft tissues. PMID- 8618378 TI - Cancellous bone Young's modulus variation within the vertebral body of a ligamentous lumbar spine--application of bone adaptive remodeling concepts. AB - Bone remodeling theory based on strain energy density (SED) as the feedback control variable was used in conjunction with the finite element method to analyze the shape of the vertebral bodies within the ligamentous motion segment. The remodeling theory was once again applied to the altered two motion segments model to predict the Young's modulus distribution of the cancellous bone within the vertebral bodies. A three-dimensional finite element model of the two motion segments ligamentous lumbar spine (L3-5) was developed. Bone remodeling response (external as well as internal) of the motion segments to a uniaxial compressive load of 424.7 N was studied. The external shape of the converged model matched the normal shape of a vertebral body. The internal remodeling resulted in regional cancellous bone Young's moduli (or bone density) distributions similar to those reported in the literature; posterocentral regions of the vertebrae were predicted to have greater values of the elastic modulus than that of the outer regions. The results of the present study suggest that vertebral body assumes an adequate/optimum structure in terms of both its shape and its elastic moduli distribution within the cancellous region in response to the applied load. Extensions of the present model and its clinically relevant applications are discussed. PMID- 8618379 TI - Development and validation of a three-dimensional finite element model of the pelvic bone. AB - Due to both its shape and its structural architecture, the mechanics of the pelvic bone are complex. In Finite Element (FE) models, these aspects have often been (over)simplified, sometimes leading to conclusions which did not bear out in reality. The purpose of this study was to develop a more realistic FE model of the pelvic bone. This not only implies that the model has to be three dimensional, but also that the thickness of the cortical shell and the density distribution of the trabecular bone throughout the pelvic bone have to be incorporated in the model in a realistic way. For this purpose, quantitative measurements were performed on computer tomography scans of several pelvic bones, after which the measured quantities were allocated to each element of the mesh individually. To validate this FE model, two fresh pelvic bones were fitted with strain gages and loaded in a testing machine. Stresses calculated from the strain data of this experiment were compared to the results of a simulation with the developed pelvic FE model. PMID- 8618380 TI - A constitutive relationship for large deformation finite element modeling of brain tissue. AB - Finite element modeling of the finite deformation response of soft tissues presents a formidable challenge. This paper discusses the use of a large strain constitutive relationship suitable for modeling brain tissue as well as other soft biological tissue. Available experimental data on the finite deformation response of brain tissue is used to characterize the constitutive properties. Analytical modeling and finite element simulation of the experiment are performed using the proposed constitutive formulation. The numerical results compare favorably with the experimental data. PMID- 8618381 TI - The application of scanning acoustic microscopy in a bone remodeling study. AB - Scanning acoustic microscopy (SAM) was used in the evaluation of bone remodeling around a cylindrical unicortical defect. SAM is a technique for the nondestructive evaluation of materials, and has only recently been employed as an orthopaedic research tool. The utility of SAM was demonstrated by using it to measure an elastic property known as acoustic impedance. Specifically, the acoustic impedance of bone formed by remodeling around a cylindrical defect was measured. The defects were filled with either a low modulus "void" or rigid inclusion to create various states of stress in the bone in the vicinity of the defect. After six months of implantation of the inclusions in the sheep metatarsal, new bone formation on periosteal and endosteal surfaces about the defect region was observed. These regions of new bone were less stiff and had 18.0 +/- 6.5% lower acoustic impedance than the pre-existing bone in the intracortical region of the metatarsal. There was no difference in the degree of new bone formation about void and rigid inclusions. Both underwent significant adaptational changes in response to the elevated stress about the defect. These changes affected the basic structure of the bone cross-section at the level of the defect and effectively reduced the stress levels about the defect. By using SAM to measure acoustic impedance, it was seen that little internal remodeling occurred in the intracortical region. Hence, the primary mechanism of strain induced bone remodeling observed in this experimental model was surface remodeling. PMID- 8618382 TI - Structural evaluation of ceramic femoral heads: effect of taper friction, support conditions and trunnion compliance. AB - The outcome of a nonlinear finite element stress analysis of ceramic heads for artificial hip joints is presented. The analysis mainly covers the influence of taper friction, support conditions and trunnion modulus of elasticity on the hoop stress distribution at the surface of the head bore. The paper quantifies how much the maximum tensile stress decreases with increasing frictional coefficient, with stiffening of the support and with stiffening of the trunnion material. An appreciable rise of the maximum tensile upon unloading of the head is also shown for the case of cup support. The computational results are found in close correspondence with photoelastic measurements of taper pressures and encourage the use, for preliminary design purposes, of an approximate theoretical model retrieved from the literature. PMID- 8618383 TI - Modeling and simulation of paraplegic ambulation in a reciprocating gait orthosis. AB - We developed a three dimensional, four segment, eight-degree-of-freedom model for the analysis of paraplegic ambulation in a reciprocating gait orthosis (RGO). Model development was guided by experimental analysis of a spinal cord injured individual walking in an RGO with the additional assistance of arm crutches. Body forces and torques required to produce a dynamic simulation of the RGO gait swing phase were found by solving an optimal control problem to track the recorded kinematics and ground reaction forces. We found that high upper body forces are required, not only during swing but probably also during double support to compensate for the deceleration of the body during swing, which is due to the pelvic thrust necessary to swing the leg forward. Other stimulations showed that upper body forces and body deceleration during swing can be reduced substantially by producing a ballistic swing. Functional neuromuscular stimulation of the hip musculature during double support would then be required, however, to establish the initial conditions needed in a ballistic swing. PMID- 8618385 TI - Finite element model study of head impact based on Hybrid III head acceleration: the effects of rotational and translational acceleration. AB - The translational and rotational components of acceleration measured at the center of gravity of a Hybrid III dummy head were used to investigate their individual and combined effects on a two-dimensional finite element model of the human brain. Each component of acceleration generated distinct patterns of deformation. Although translational acceleration is related to pressure and rotational acceleration has a dominant effect on shear deformations, complete acceleration (combination of translation and rotation) yielded the highest values in all stresses and produced a maximum shear stress at the top of the brain. PMID- 8618384 TI - Dynamic models for sideways falls from standing height. AB - Despite our growing understanding of the importance of fall mechanics in the etiology of hip fracture, previous studies have largely ignored the kinematics and dynamics of falls from standing height. Beginning from basic principles, we estimated peak impact force on the greater trochanter in a sideways fall from standing height. Using a one degree-of-freedom impact model, this force is determined by the impact velocity of the hip, the effective mass of that part of the body that is moving prior to impact, and the overall stiffness of the soft tissue overlying the hip. To determine impact velocity and effective mass, three different paradigms of increasing complexity were used: 1) a falling point mass or a rigid bar pivoting at its base; 2) two-link models consisting of a leg segment and a torso; and 3) three-link models including a knee. The total mechanical energy of each model before falling was equated to the total mechanical energy just prior to impact in order to estimate the hip impact velocity. In addition, the configuration of the model just before impact was used to estimate the effective mass. Our model predictions were compared with the results of an earlier experimental study with young subjects falling on a 10-inch thick mattress. Values from literature were used to estimate the soft tissue stiffness. For the models, predicted values for hip impact velocity and effective mass ranged from 2.47 to 4.34 m/s and from 15.9 to 70.0 kg, respectively. Predicted values for the peak force applied to the greater trochanter ranged from 2.90k to 9.99k N. Based on comparisons to the experimental falls, impact velocity and impact force were best predicted by a simple two-link model with the trunk at 45 degrees to the vertical at impact. A three-link model with a quadratic spring incorporated in the knee of the model was the best predictor of effective mass. Using our most accurate model, the peak impact force was 2.90k N for a 5th percentile female and 4.26k N for a 95th percentile female, thereby confirming the widely held perception that "the bigger they are, the harder they fall". PMID- 8618386 TI - Implementation of strain rate as a bone remodeling stimulus. AB - Strain rate is implemented as a stimulus for surface bone remodeling. Using idealized models for trabecular bone structures, the surface remodeling predictions using the strain rate as the stimulus are compared with the predictions using the peak strain magnitude as the stimulus. For a uniaxially loaded cruciform shape, the comparison shows that the two surface remodeling stimuli predict the same final shape under a periodic compressive load, but the two evolutionary paths to final shapes are different. Two biaxially loaded regular grid models of trabecular structure were considered, one a grid of square diamond shaped elements and the other a brick wall patterned grid. For both of these idealized trabecular structures, the comparison shows that the two surface remodeling stimuli predict the same final shape under a periodic compressive load, even from these distinctly different initial grid patterns, and the evolutionary paths to final shapes are quite different. In general the two stimuli do not predict the same remodeling and the conditions under which they do are derived. The models developed are also applied to the data from the animal experiments reported in Goldstein et al. (1991), and it is shown that the strain rate stimulus predicts bone remodeling similar to what was experimentally observed. PMID- 8618387 TI - Relationship between muscle force and stiffness in the whole mammalian muscle: a simulation study. AB - Several types of analyses in biomechanics require estimates of both muscle force and stiffness. Simulations were performed using the two-state cross-bridge Bond Distribution-Moment muscle model of Zahalak (1981), together with other parameters for passive elasticity and tendon compliance, to estimate instantaneous stiffness and to compare these estimates with the wide range of values reported in the literature. While the relatively simple cross-bridge theory appears to approximate the stiffness of skinned muscle fibers, the stiffness of a complete muscle-tendon unit become complex and non-linear due to relative changes in muscle-tendon length and interaction with activation and length dependent passive elastic components. It would appear that the variability in muscle stiffness values reported in the literature can be explained with the D M approach. PMID- 8618388 TI - A model for stress-induced growth in the developing heart. AB - Mechanical loads affect growth and morphogenesis in the developing heart. Using a theoretical model, we studied stress-modulated growth in the embryonic chick ventricle during stages 21-29 (4-6 days of a 21-day incubation period). The model is a thick-walled, compressible, pseudoelastic cylinder, with finite volumetric growth included by letting the rate of change of the local zero-stress configuration depend linearly on the Cauchy stresses. After investigating the fundamental behavior of the model, we used it to study global and local growth in the primitive ventricle due to normal and abnormal cavity pressures. With end diastolic pressure taken as the growth-modulating stimulus, correlating theoretical and available experimental results yielded the coefficients of the growth law, which was assumed to be independent of time and loading conditions. For both normal and elevated pressures, the predicted changes in radius and wall volume during development were similar to experimental measurements. In addition, the residual stress generated by differential growth agreed with experimental data. These results suggest that wall stress may be a biomechanical factor that regulates growth in the embryonic heart. PMID- 8618389 TI - Numerical investigation and prediction of atherogenic sites in branching arteries. AB - Atherosclerosis, a disease of large- and medium-size arteries, is the chief cause of death in the US and most of the western world. It is widely accepted that the focal nature of the disease in arterial bends, junctions, and bifurcations is directly related to locally abnormal hemodynamics, often labeled "disturbed flows." Employing the aorto-celiac junction of rabbits as a representative atherosclerotic model and considering other branching blood vessels with their distinctive input wave forms, it is suggested that the local wall shear stress gradient (WSSG) is the single best indicator of nonuniform flow fields leading to atherogenesis. Alternative predictors of susceptible sites are briefly evaluated. The results discussed include transient velocity vector fields, wall shear stress gradient distributions, and a new dimensionless parameter for the prediction of the probable sites of stenotic developments in branching blood vessels. Some of the possible underlying biological aspects of atherogenesis due to locally significant /WSSG/-magnitudes are briefly discussed. PMID- 8618390 TI - Modeling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells. AB - The arterial media is modeled as a periodic array of cylindrical smooth muscle cells residing in a matrix comprised of proteoglycan and collagen fibers. Using Brinkman's model to describe transmural flow through such a fibrous media, we calculate the effective hydraulic permeability of the media and the wall shear stress on smooth muscle cells. Two interesting results are obtained: first, the wall shear stress on smooth muscle cells is on the order of 1 dyne/cm2, which is the range known to affect endothelial cells in vitro; second, the flow resistance due to smooth muscle cells is not negligible compared to the resistance due to the fiber matrix. PMID- 8618391 TI - Fabrication of vascular replicas from magnetic resonance images. AB - Image processing and Computer Numerical Controlled (CNC) machining techniques have been used to prepare a large-than-life investment cast of an aortic bifurcation from magnetic resonance images of a replica of the vessel. The technique will facilitate experimental studies of vascular fluid dynamics and permit the in vitro reproduction of flows in living subjects. PMID- 8618392 TI - Optimization technique for the calculation of in vitro three-dimensional vertebral motion. AB - A method for the calculation of translations and Eulerian rotations of an orthogonal axis system with respect to a fixed reference is described with application to the measurement of position in a vertebral motion segment. Kinematic equations were derived to compute the three-dimensional motion of a moving vertebra relative to an adjacent fixed body, without the requirement of a direct physical link between the two bodies. For this calculation, the quadratic error of the lengths of six position vectors was minimized to obtain a mathematically optimal estimate of the translations and rotations. Tests with a rigid model resulted in mean maximum overall system errors of 2.8 percent for the measurement of translation (translations less than 3.5 mm) and 6.1 percent for the measurement of rotations (rotations less than 10 deg) limited by transducer accuracy. The mathematical techniques presented for the quantitative description of rigid body motion, based on the measurement of three reference vectors, may be extended to a broad range of kinematic problems. PMID- 8618393 TI - The World Federation of Surgical Oncology. PMID- 8618394 TI - Atraumatic method of intraoperative retrograde transhepatic biliary stent insertion. AB - A significant risk of hepatic injury remains using reported methods of intraoperative retrograde transhepatic stenting (IRTS). Our hypothesis was that we could minimize this risk by: (1) using a pliable sheath to create a stent tract that follows the curve of the biliary tree, (2) decreasing the stent diameter, and (3) avoiding the hepatic hilum. We evaluated the safety of a novel technique of intraoperative stenting employing these three concepts. Twenty-four patients underwent IRTS between 1992 and 1995 at our institution after potentially curative resection (one bypass). Malignant disease was present in 22 of 24 patients. Bile ducts were normal caliber in all patients. There was no operative mortality and 38% operative morbidity, all readily treated. All complications were due to stent dislodgment. There were no deaths. This novel atraumatic method of IRTS has acceptable morbidity and mortality. Complications due to stent insertion are minimal. The technique compares favorably with previous methods of IRTS and offers a viable alternative to the surgeon when a transhepatic stent is required. Anchoring the stent securely to the skin is essential to prevent catheter dislodgement. PMID- 8618395 TI - Combined angioma and glioma (angioglioma). AB - Ten patients in whom tissue proliferation akin to angioglioma occurred within the brain are described; seven of the lesions were supratentorial and three infratentorial. Only 31 accepted instances of such neoplasms have been found in the literature. The combined lesions usually become symptomatic in the second and third decades. In all 10 cases, the angiomatous part of the combined tumors showed characteristic vascular malformation such as severe hyalinization, tortuosity, and some were even calcified. The number of abnormal blood vessels were excessive in all examples. The glial portion consisted of either astrocytoma, oligodendroglioma, or mixtures of these gliomas. Dedifferentiation of the neuroglia combined with neoplastic endothelial proliferation indicates the true neoplastic nature rather than reactive gliosis associated with a vascular anomaly. PMID- 8618396 TI - Management for patients with advanced T4 epidermoid carcinoma of the esophagus. AB - Available data concerning the treatment of patients with advanced T4 esophageal carcinoma are limited. A consecutive series of 42 patients with advanced T4M0 epidermoid carcinoma of the esophagus were studied from June 1987 to July 1992. The aim of this study was to evaluate the efficacy of various therapeutic modalities, and further evaluate the therapeutic options. The various therapeutic modalities included the following: Group I, feeding jejunostomy or endoesophageal intubation, 6 patients; Group II, palliative subtotal esophagectomy only, 8 patients; Group III, bypass procedures without tumor resection, 9 patients; Group IV, nutritional support and then treatment with irradiation (n=8) or concurrent radio-chemotherapy (n=4), 12 patients; Group V, subtotal esophagectomy, followed by aggressive concurrent radiochemotherapy, 7 patients. The total prescribed irradiation dose was 60 Gy (10 Gy/5 fractions/week). A combination regimen of chemotherapy consisted of cisplatin, 5-fluorouracil, and leucovorin (PFL regimen). For the patients undergoing esophagectomy or bypass procedures (n=24), the rates of operative complication and mortality were 45.8% and 25%, respectively. Side effects of adjuvant therapy (n=24) consisted of main airway irritation (100%), mucositis or gastrointestinal symptoms (83.3%), hematologic toxicity (79.2%), esophagitis or gastric ulcer (62.5%), alopecia (37.5%), and pneumonia (20.8%). The mortality due to toxicity of adjuvant therapy was 21.1% (4/19 patients). The mean survival times for each of the different groups was 1.9+/-0.5 months for Group I, 4.8+/-1.6 months for Group II, 5.2+/-1.2 months for Group III, 7.3+/-2.0 months for Group IV, and 20.3+/-2.5 months for Group V, respectively. Compared with patients of Groups I--IV, the Group V patients had a significantly superior one-year survival rate (P<0.01). Our results demonstrated that esophagectomy followed by concurrent irradiation and PFL combination chemotherapy may provide a significant improvement in the quality of life and survival for appropriate patients with advanced T4M0 epidermoid carcinoma of the esophagus. Furthermore, more than one cycle of PFL regimen chemotherapy may result in a better prognosis. During the performance of such an aggressive treatment, the utmost care must be taken with the patient's nutrition and to prevent pulmonary complications. PMID- 8618397 TI - Germ cell testis cancer: 15-year review. AB - Testis cancer affects 2-3 men per every 100,000 in the United States, making it the most common solid tumor of men in the 20-35-year-old age range. Since the average age of active duty military personnel is included in the age range of those at greatest risk for germ cell testis cancer, it is of pertinent clinical importance to physicians who treat these young patients. The National Naval Medical Center has been using cisplatin-based protocols since the time of their introduction. This study reviews the success of treating these patients and examines the treatment failures. PMID- 8618398 TI - Immunohistochemical analysis of the poorly differentiated stomach adenocarcinoma with medullary growth pattern. AB - Poorly differentiated gastric adenocarcinoma with medullary features (poor medullary) is distinguished by a propensity for hepatic metastasis. To classify it antigenically, we compared it to poorly differentiated adenocarcinoma with scirrhous growth pattern (poor scirrhous), well and moderately differentiated adenocarcinoma (differentiated adenocarcinoma), and normal gastric mucosa (foveolar and deep epithelium) using immunohistochemistry with antibodies against CEA, AFP, NSE, and Lewis-type antigens. Lewis(a) antigen was significantly associated with differentiated adenocarcinoma and foveolar epithelium, although Lewis(x) antigen was significantly expressed in poor medullary, poor scirrhous, and deep gland epithelium. From the viewpoint of expression of Lewis(a), there was no significant differentiation between poor medullary and differentiated adenocarcinoma, but it was definite between poor scirrhous and differentiated adenocarcinoma. Therefore, we conclude that in antigenic expression, poor medullary carcinoma is allied with differentiated adenocarcinoma rather than poorly differentiated scirrhous carcinoma. PMID- 8618399 TI - Prospective adjuvant therapy with mitomycin C and carmofur (HCFU) for colorectal cancer, 10-year follow-up: Tokai HCFU Study Group, the first study for colorectal cancer. AB - A joint study was performed by the Tokai HCFU study group, which included 41 institutions to study the usefulness of the concomitant therapy with Mitomycin C (MMC) and Carmofur (HCFU) as a postoperative adjuvant chemotherapy in patients with colorectal cancer who had curative resection. Patients were divided into two groups, Group MMC and Group MMC+HCFU, using the "envelope" method. Among the 172 patients who had the envelope opened, 149 evaluable cases were analyzed for evaluation of the drug. The cumulative 10-year survival rates of Group MMC+HCFU had a statistically significant increase in survival rate compared with Group MMC. In particular, the rate was statistically significant in patients with colorectal cancer who had lymph node invasion. There were no severe side effects due to the adjuvant chemotherapy with MMC+HCFU. Thus the adjuvant chemotherapy with MMC+HCFU is suggested to be a useful and safe postoperative adjuvant chemotherapy. PMID- 8618400 TI - Is there a role for second-look laparotomy in the management of malignant germ cell tumors of the ovary? Experience at Institut Gustave Roussy. AB - The last two decades have seen great improvements in the management of patients with germ-cell tumors of the ovary. The initial treatment approach includes conservative surgery and cisplatin-based chemotherapy in most cases. At completion of chemotherapy, the role of second-look surgery remains questionable. We retrospectively analyzed the long-term outcome (median follow-up, 8 years) of 40 patients who received various chemotherapy regimens after primary surgery and focused on the role of second-look surgery. A second-look laparotomy was performed at completion of chemotherapy in 22 patients. Histological findings were no tumor in 13; mature teratoma in 5; immature teratoma in 1; active disease in 3. Six of the latter nine patients had persistent radiologic abnormalities after chemotherapy. All three patients with active disease had elevated serum tumor markers. Five out of the six patients with residual teratoma lesions had a teratoma component in the primary tumor. According to histological findings at second-look surgery, the number of patients without long-term evidence of disease is 12, 5, 1 and 0, respectively. Eighteen patients were not subjected to second look surgery. One of them had clearly progressive disease and the other 17 experienced a clinical complete response at completion of chemotherapy. All patients but one are alive without evidence of disease. We conclude that second look surgery is not necessary in patients with elevated serum tumor marker levels and in those patients with neither radiologic abnormality nor teratoma element in the primary tumor. However, we recommend a second-look procedure for the small subset of patients with a teratoma component in the primary tumor and persistent radiologic abnormalities along with normal serum tumor markers at the end of chemotherapy. PMID- 8618401 TI - Multimodal therapy for neoplasms arising from a vesical diverticulum. AB - Both radical cystectomy and diverticulectomy for the treatment of neoplasms arising in a vesical diverticulum have resulted in poor survival, mainly secondary to early metastatic spread. In this study, nine patients were treated with a multispecialty approach in hopes of eradicating both local and distant disease. All patients underwent a combination of therapeutic modalities with three patients undergoing pre-operative radiotherapy (RT) and diverticulectomy; diverticulectomy and chemotherapy (three patients); diverticulectomy with chemotherapy and pre-operative RT (two patients); and definitive RT with cisplatin (one patient). With a mean follow-up of 4.0 years (median 3.2), four patients are free of disease, two are dead of other causes, one patient has developed an invasive recurrence, one patient is alive with metastatic disease, and one patient is dead of disease. Five patients (55%) developed local recurrences. Disease-specific survival for the group was 89%. Surgical monotherapy has been ineffective in controlling both local and metastatic disease in patients with diverticular tumors. This study suggests a significant benefit from systemic chemotherapy and RT when combined with surgery for these neoplasms. PMID- 8618402 TI - Retroperitoneal soft tissue sarcomas: a pilot study of intraoperative radiation therapy. AB - This pilot study was conducted to evaluate the feasibility and tolerance of a multimodal therapy of retroperitoneal soft tissue sarcoma (STS), including intraoperative radiation therapy (IORT). Nineteen patients (14 primarily treated patients and 5 treated for a recurrent tumor) were included. Surgery included a complete resection (14), a partial resection (2), and no resection (2). The median IORT dose was 17 Gy. Thirteen patients also received an external radiation therapy (ERT). Nine patients received chemotherapy. There was no postoperative mortality. Immediate postoperative complications occurred in four patients (21%). Delayed complications occurred in six patients, including one lethal iliac artery disruption. With a median follow-up of 17 months, the 2-year disease-free survival rate was 60%, and the 2-year actuarial local control rate was 76%. A multimodality approach of treatment, including IORT and ERT and eventually chemotherapy, appears feasible in patients with retroperitoneal STS. However, the treatment-related morbidity appeared relatively high in this study. PMID- 8618403 TI - Clinicopathological features of multiple primary gastric carcinoma. AB - The clinicopathological features of multiple primary gastric carcinoma in 107 patients who had undergone gastrectomy between 1972 and 1992 were studied and compared with those of single gastric carcinoma in 1,456 patients. The incidence of occurrence of multiple primary gastric carcinoma was 6.8% of patients who had gastrectomy for gastric cancer. Such carcinoma was detected less often in patients <49 years of age. Dominant findings involved an elevated gross appearance, papillary or well-differentiated adenocarcinoma in the histology, and invasion to the depth of mucosa. When multiple primary gastric carcinoma was classified by main and concomitant lesions based on the stage of the disease, concomitant lesions were detected more often in the lower third of the stomach and at the distal site of main lesions located in the upper or middle third of the stomach. These results indicate that the lower third of the stomach and the distal site of the main lesion must be investigated carefully to ensure that incidental concomitant lesions are not overlooked, especially when a patient has the clincopathological features described above. PMID- 8618404 TI - Leiomyoma of the breast. AB - Leiomyoma is the most uncommon benign neoplasm of the breast. We report a case of a middle-aged woman with a palpable breast mass who underwent excisional biopsy. Pathologic examination revealed a leiomyoma. The clinical characteristics, pathologic findings, and proper management of this lesion are discussed. The tumor is thought to arise from the smooth muscle of the endothelium and can be managed similarly to leiomyomas occurring elsewhere. The possible effects of tamoxifen on uterine leiomyomas may be of theoretical concern with breast leiomyomas. The recognition of this entity and an understanding of the management of this rare lesion are necessary by all surgeons who perform breast surgery. PMID- 8618405 TI - Perioperative therapy for locoregional nonsmall-cell lung cancer. AB - Surgical therapy remains the treatment of choice for resectable nonsmall-cell lung cancer (NSCLC). However, the 5-year survival results of surgical therapy is 40-70%, which is far from acceptable. In this report, past results of perioperative therapies were reviewed to identify the future direction of effort in improving the therapy of NSCLC. Two perioperative modes of treatment that may possibly improve postsurgical survival were identified, i.e., neoadjuvant chemotherapy for resectable NSCLC and postoperative specific active immunotherapy. PMID- 8618406 TI - Simple PC-based system for morphometric analysis of synapses. AB - The computer system designed for synaptic morphometry of aksosomatic and aksospine synapses of brain is described in the present paper. It is based on an AT-comparable personal computer equipped with low-cost frame grabber. This hardware configuration allows to input images from any TV source such as TV camera, videorecorder for further processing. The appropriate software was written in Microsoft Quick Basic to measure the main morphometric parameters of axon ending, dendritic spines, total contact, active zone and mitochondria. Number of synaptic vesicles (total and active) were also counted using a mouse as a pointing device. The derivative parameters (vesicle density, mitochondrial density) are then calculated. All measured data are stored in ASCII format, allowing ease in editing and export into other application programs. Statistical evaluation and calculation of histograms is performed by associated program also written in Quick Basic. The advantages and disadvantages of this approach are discussed. PMID- 8618407 TI - A method for 3D reconstruction of neuronal processes using semithin serial sections displayed as a cinematographic sequence. AB - In order to study the organization and distribution of dendrites and axons in the cerebral cortex, we have developed a computer-assisted method for 3D reconstruction of neuronal processes based on serial light microscopic images displayed as a continuous sequence. A series of tangential sections (0.65 micron thick) through rat parietal cortex was aligned, digitized into the computer and then used to build a sequence (stack) of images which was stored to a digital real-time video disk. Apical dendrites located in dendritic bundles in laminae III and IV were traced through the sequence. Two tracing modes were tested: (1) cinematographic mode, in which the image stack was displayed continuously and automatically by the computer at various preset speeds (max. speed: 25 images/s) and (2) stepping mode, in which the interval between each image was varied manually according to the choice of the operator. Coordinates were stored in a database and used to build a 3D reconstruction where apical dendrites were displayed as wires or tubes. Tracing in cinematographic mode was about 3 times faster than tracing in stepping mode. We believe that the former mode exploits the built in 'filtering' capacity of the visual system to perform temporal averaging. PMID- 8618408 TI - A sensitive technique for the detection of the alpha 7 neuronal nicotinic acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and brain. AB - Methyllycaconitine (MLA) is the most potent and selective antagonist of the alpha bungarotoxin sensitive neuronal nicotinic acetylcholine receptor (nAChR). In the present study, an accurate and reproducible technique for the extraction and analysis of MLA from rat plasma and brain is described. This study further sought to determine whether pharmacologically relevant concentrations of MLA could be achieved in brain following peripheral administration. The detection limits for MLA were 0.5 ng/ml for plasma samples and 1.0 ng/g for brain samples. The pharmacokinetic properties of MLA in rat are characterized by a short elimination half-life (19 min) following intravenous (i.v.) administration and poor bioavailability following oral (p.o.) administration. Remarkably, the elimination half-life is significantly longer following p.o. administration (408 min). To assess the extent to which MLA can penetrate into brain, brain and plasma levels of MLA were determined at different time points following intraperitoneal (i.p.) administration of a dose of MLA that produced no observable side effects. Maximal plasma and brain levels were 694 +/- 106 ng/ml and 32 +/- 3 ng/g, respectively. These concentrations are within a range previously reported to selectively block alpha 7 nAChR mediated responses in vitro. Peripherally administered MLA may therefore be a useful tool to further probe the central nervous system functions of the alpha 7 nAChR subunit in vivo. PMID- 8618409 TI - Transneuronal WGA-HRP: can it demonstrate development of ocular dominance patches and effects of monocular deprivation? AB - We have attempted to use intraocular injections of wheat germ agglutinin horseradish peroxidase (WGA-HRP) to label ocular dominance patches in developing layer 4 of cat visual cortex. The cortices of animals killed at 49 days or later showed normal ocular dominance patches similar to those seen in [3H]proline material. Animals killed at 42 days showed some patches, but also showed unsegregated regions. Animals killed younger were difficult to stain and did not have patches. We also examined the ability of the WGA-HRP technique to demonstrate the effects of monocular deprivation (MD). MD for the first 3 months of life produced expansion of the afferents from the nondeprived eye and retraction of the patches from the deprived eye. One week of MD at about 5 weeks of age produced an expansion of the patches innervated by the nondeprived eye, but did not obviously affect the patches innervated by the deprived eye. We conclude that WGA-HRP is useful for examining the effects of long-term MD on ocular dominance patches but not for following the development of segregation. Its advantages over the [3H]proline technique are that it does not require a delay of many weeks before the sections can be examined and is much less expensive. PMID- 8618410 TI - A retrograde double-labeling technique for light microscopy. A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate. AB - A light microscopical, non-fluorescent, retrograde double-labeling technique is described. Cholera toxin B-subunit (CTb) and a conjugate of wheatgerm agglutinin and bovine serum albumin coupled to 10 nm gold particles (gold-lectin) are both excellent retrograde tracers and, when visualized by means of immunohistochemistry and silver intensification, respectively, may be readily identified within the same cell. This light microscopical retrograde double labeling technique is illustrated in rat with experiments designed to investigate the collateralisation (1) of vestibular neurons to the spinal cord and oculomotor complex, (2) of spinal neurons to the left and right lateral reticular nucleus, and (3) of inferior olivary neurons to the uvula of the cerebellum. Advantages over fluorescent double-labeling experiments are found in the fact that the diaminobenzidine reaction product as well as the silver/gold deposits do not fade and can be examined in counterstained sections. Moreover, the injection sites can be kept quite small and may be guided by electrophysiological recording through the injection pipette. PMID- 8618411 TI - Food carrying: a new method for naturalistic studies of spontaneous and forced alternation. AB - Spontaneous alternation has been studied in laboratory tests given to rodents since 1935. Herein is described a simple new technique that allows the animal to perform without intertrial handling. The apparatus has a centered covered 'home base' and two relatively exposed arms. When presented with large food pellets, the rats carry them from the exposed arms to the home base before consuming them. The utility of the method is demonstrated in four exemplar paradigms ranging from two choice tasks to food 'hoarding' and exploratory tasks. The procedure can be modified for forced and other alternation paradigms and allows collection of a variety of psychophysical measures. The strength of the task is that it provides an analogue of natural foraging behavior. PMID- 8618412 TI - Biotin-dextran: fast retrograde tracing of sciatic nerve motoneurons. AB - We present evidence that biotin-dextran (BD) provides good fast retrograde tracing in the rat sciatic nerve. Using BD injected distal to a crush injury of either tibial or common peroneal nerves, spinal cord motoneuron counts after 48 h compare favorably with counts obtained using horseradish peroxidase. Advantages of BD include fine staining of the soma and its branches, increasing the reliability of motoneuron counting, as well as good staining of sciatic nerve axons 30 mm away from the crush site. BD, already demonstrated to be a good anterograde tracer in the central nervous system, is shown to be a good retrograde tracer in a peripheral nervous system model. PMID- 8618413 TI - Monitoring intracellular nitric oxide formation by dichlorofluorescin in neuronal cells. AB - A method for rapid fluorometric assay of intracellular nitric oxide (NO) formation was developed for use in cultured neuronal cells. In a cell-free system 2,7-dichlorofluorescin (DCF), a non-fluorescent species, is oxidized by NO to dichlorofluorescein, a fluorescent compound. Addition of NO to a solution containing DCF increased the fluorescent signal within 10 s and continued to increase slowly over a 10-min period. The intensity of the fluorescence was dependent upon the concentration of NO. In DCF-loaded PC12 cells, addition of NO markedly increased fluorescence (limit of detection = 16 microM NO) and pretreatment with reduced hemoglobin (Hb) inhibited the NO-mediated increase of fluorescence in both the cell-free system and PC12 cells. In PC12 cells loaded with DCF, the NO generator sodium nitroprusside (SNP) produced a rapid increase of fluorescence. To rule out the possibility that reactive oxygen species (ROS) mediated the increased of fluorescence, superoxide dismutase (SOD) and catalase were added to the cuvette. The enzymes did not alter the fluorescence generated after addition of NO to PC12 cells. This assay was used to determine the ability of glutamate to stimulate NO production in cerebellar granule cells. When 10 microM glutamate was added to DCF-loaded cerebellar granule cells, a rapid increase in fluorescence was noted. The fluorescence was blocked approximately 50% after addition of either Hb or SOD, or by pretreatment with NG-nitro-L arginine methyl ester (300 microM), a nitric oxide synthase (NOS) inhibitor. It was concluded that glutamate stimulated intracellular generation of both NO and ROS, and at least 50% of the oxidation of DCF was attributed to intracellular generation of NO. These results demonstrate that oxidation of DCF by NO can be used to measure intracellular generation of NO and by adding either Hb or SOD to the cell system, the extent of oxidation of DCF attributed to NO and ROS can be determined. PMID- 8618414 TI - Combined intracellular stimulation and high speed video motion analysis of motor control neurons in the cockroach. AB - A complete understanding of motor control circuitry requires detailed analysis of the behavior produced by the circuitry as well as the connections between individual neurons. A technique is described for combining high speed video motion analysis of leg movements in the cockroach with electrophysiological techniques such as intracellular stimulation/recording from central neurons and EMG recording from leg muscles. Using a restrained preparation, we have quantified leg movements evoked by intracellular stimulation of individual motor neurons and local interneurons. By incorporating motion analysis into the recording paradigm, the transfer functions from electrical activity to movements can be derived. Because distinct and characteristic responses to single and multiple action potentials are seen in slow, intermediate, and fast motor neurons, it is often possible to identify the motor neuron targets of local interneurons. The ability to analyze movement in any plane is especially useful in situations such as blind neuropilar penetrations, where a more restricted motion transducer arrangement may not be in register with the impaled cell. In addition, it is possible to record and analyze such complex phenomena as coordinated movements in multiple joints produced by local interneurons and reflexes produced by proprioceptive feedback due to activity of one motor neuron. PMID- 8618415 TI - Effects of L-serine on neurons in vitro. AB - We have examined the influence of amino acids on chicken embryonic dorsal root ganglion (DRG) neurons in vitro and have found that serine has a marked impact on the morphology of the developing neurites. In cultures supplemented with L-serine (but not D-serine) at micromolar concentrations (10-200 microM), the length of the neurites was increased by up to 100% and they were seen to develop a more complex branching pattern. These effects of L-serine were found to be concentration-dependent and stereospecific and were observed on several different substrata such as laminin, Ng-CAM and axonin-1. Similar observations were also made in the case of embryonic retinal explants, while the addition of non essential amino acids other than L-serine to DRG neurons was found to have no effect. We conclude that, although belonging to the group of non-essential amino acids and not a recognized neurotransmitter, L-serine is an important factor for the morphological differentiation of neurons in vitro. PMID- 8618416 TI - Repetitive depletion and recovery of intracellular K+ in retinal Muller glial cells during whole-cell voltage-clamp. AB - A procedure was developed allowing repetitive depletion and recovery of K+ from the cell interior of Muller glial cells without patch pipette perfusion. To this end the whole-cell voltage-clamp technique using tight seal pipettes was applied to enzymatically isolated Muller cells of the guinea pig. When K+ was replaced by Cs+ and/or NMDG in the pipette solution voltage-activated outward currents could be generated similar to those found with normal K(+)-containing intracellular solution. This was the result of the distribution of K+ across the cell membrane due to the negative holding potential, i.e., K+ accumulated at the intracellular side of the cell membrane. This distribution should be favored by the low input resistance of Muller cells. K+ could also be removed from the cell interior by depolarizing voltage steps. Simultaneously, K+ influx had to be cut off by using a K(+)-free extracellular solution or blocking K+ channels using Ba2+. This procedure lead to reduction and eventually cessation of the K+ outward currents, indicating extinction of the intracellular K+ pool. The process is reversible, i.e., outward currents can be evoked again by depolarizing voltage steps after renewed application of extracellular K+ and/or removal of Ba2+. Hence, the intracellular K+ pool is filled up again. The described method was applied to demonstrate the dependence of the Muller cell Na+/glutamate transporter on the intracellular K+ concentration. PMID- 8618418 TI - A telemetry study on the chronic effects of microdialysis probe implantation on the activity pattern and temperature rhythm of the rat. AB - The present study describes the effects of implantation of microdialysis probes on temperature and activity rhythms of the rat, measured with a telemetry system. For comparison two widely used types of microdialysis probes were investigated, a transcerebral probe, inserted into the pineal gland and a set of two I-shaped concentric probes, implanted bilateral into the striatum. Starting from 5 days before the operation until 8 days after surgery, activity and temperature recordings were carried out continuously with the help of previously implanted transmitters. In separate experiments the effects of two different types of anaesthesia (chloralhydrate and Hypnorm) were determined. The results show that there is a pronounced effect of surgery on amplitude and rhythmicity of the temperature and activity patterns which is still detectable 6-7 days after operation. Few differences were noticed between the transverse probe and the I shaped probes. Anaesthesia alone induced much smaller changes, most of which had disappeared within 2 or 3 days after the treatment. The duration of action of chloralhydrate is somewhat longer compared to Hypnorm. The conclusion is that when microdialysis is used in behavioural experiments, the effects of the surgical procedure should be taken into account. If these effects are serious, the use of previously implanted guide cannulae might be necessary. PMID- 8618417 TI - AF64A-induced cytotoxicity and changes in choline acetyltransferase activity in the LA-N-2 neuroblastoma cell line are modulated by choline and hemicholinium-3. AB - The cholinergic neurotoxin AF64A (ethylcholine aziridinium) has been used to selectively destroy the cholinergic system. Due to its structural similarity to choline, this compound may be selectively taken up by the cholinergic terminal via the high-affinity choline transport (HAChT) system to produce persistent and selective cholinergic deficits. The mechanism by which it exerts its cholinotoxicity remains to be elucidated. We have examined the effects of AF64A in the human neuroblastoma cell line, LA-N-2, which has an intact sodium-coupled choline uptake system, and is capable of synthesizing acetylcholine (ACh). AF64A (25, 50 and 100 microM) produced dose-dependent increases in cell kill as measured by colony formation assay. The addition of increasing concentrations (10(-5), 10(-4) and 10(-3) M) of choline and hemicholinium-3 (HC-3) protected the cells from the cytotoxic effects of AF64A. At the same doses, AF64A also decreased choline acetyltransferase (ChAT) activity. In the presence of the highest concentration of choline or HC-3 (10(-3) M) which produced complete protection against AF64A's cytotoxicity in the colony formation assay, ChAT activity was restored to control values. These results demonstrate that agents that utilize (i.e., choline) or inhibit (i.e., HC-3) the choline uptake system prevented AF64A-induced cytotoxicity and decreases in ChAT activity, in a manner similar to that which has been observed in chick and rat primary cholinergic cultures in vitro. The LA-N-2 neuroblastoma cell line thus serves well as an in vitro model of the cholinergic neuron and provides a useful system to study the mode of cholinotoxicity induced by AF64A. PMID- 8618419 TI - Producing columnar depositions of neuroanatomical tracer in rat cortex. AB - A new technique was developed to fill columnar regions of cortex with retrograde neuroanatomical tracer. This technique involved (1) standard iontophoresis to eject tracer from a micropipette in concert with (2) displacement of this micropipette radially through the cortex using a computer controlled microdrive. The displacement of the micropipette left an evenly dense deposit of tracer throughout the cortical thickness forming a column of neuroanatomical tracer. The diameter of the columnar injection site was reliably dependent on the iontophoretic current strength, the micropipette displacement parameters, and the nature of the tracer used. This simple technique provided very reproducible results. Several issues related to the interconnections of functionally identified columns of sensory and motor cortex could be addressed with this injection technique. PMID- 8618420 TI - Simultaneous, selective detection of catecholaminergic and indolaminergic signals using cyclic voltammetry with treated micro-sensor. AB - Selective and simultaneous voltammetric analysis of catechols and indoles in vivo and in vitro has until now been feasible only by means of 'slow' scanning methods (scan speed in tens of seconds) such as differential pulse (DPV) and differential normal pulse voltammetry in conjunction with electrically and/or chemically treated carbon-fiber micro-electrodes (mCFE). Faster electrochemical techniques, such as chronoamperometry and cyclic voltammetry (CV), allow more rapid (seconds or fractions of a second) and frequent measurements of these chemicals. However, these methods show poor sensitivity and selectivity in the presence of different electroactive compounds with similar oxidation potentials. In order to analyze whether the lack of sensitivity and selectivity of the fast voltammetric methods results from the rapidity of the measurement or from the use of untreated sensors, the methods of CV (scan speed: 1000 mV/s) and DPV (scan speed: 10 mV/s) have been applied with either untreated or electrically treated mCFE to analyze the in vitro oxidation potential and current values of DA and 5-HT. When associated with untreated mCFE, neither method was able to separate and selectively detect the two compounds dissolved together in an inert vehicle; the voltammogram recorded resulted in a single broad oxidation signal. In contrast, when these techniques were performed with electrically treated mCFE, oxidation signals for DA (peak A) and 5-HT (peak B) were monitored simultaneously at approximately + 65 mV and + 240 mV, with DPV respectively, and at + 120 mV and + 300 mV with CV, respectively. Additionally, CV with treated mCFE on anesthetized rats, simultaneously monitored two striatal signals at approximately + 100 mV and + 300 mV. The oxidation values (Em) and current levels (nA) of these peaks remained stable during control recordings. The current levels were selectively increased by peripheral injection of fluphenazine (DA antagonist) or of 5 hydroxytryptophan (precursor of serotonin). The chemical nature of these two peaks may therefore be considered catecholaminergic and indolaminergic, respectively. Hence, this report provides the first evidence for the feasibility of concomitant in vitro analysis of DA and 5-HT using a rapid scanning method such as CV. In addition, the values of current level (nA) obtained with CV-mCFE for DA and 5-HT are comparable to those monitored with DPV-mCFE, supporting the view that treatment of the sensor is a key point for increasing the selectivity and the sensitivity of these voltammetric techniques. The feasibility of using CV with electrically treated mCFE for fast in vivo analysis of catechol and indole activities is also demonstrated. PMID- 8618421 TI - A Windows software package to record from voltage-clamped Xenopus oocytes. AB - We have written a software package to record, display and analyze membrane currents elicited by neurotransmitter receptors or voltage-activated channels in voltage-clamped Xenopus oocytes. This suite, which consists of 4 applications, runs on IBM-PC compatible microcomputers under Windows 3.1. The recording programs use Direct Memory Access (DMA) to access the analog-digital board. The first program, NicPulse, is aimed at studying voltage-activated channels. It delivers voltage steps to the voltage-clamp and records the resulting membrane current. The second program, NicScope, emulates a dual-trace digital oscilloscope. It operates either in continuous or triggered mode, and is used chiefly to display neurotransmitter-induced responses in oocytes. The third recording program, VRamp, automatically determines the voltage-current relationship of drug-activated responses (I/V curve), by applying a voltage ramp and recording the subsequent clamping current. The last program, NicView, is designed to analyze records taken with NicScope and NicPulse. The present paper will discuss several issues regarding the design of these programs, and will give a brief description of each application. PMID- 8618422 TI - Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage. AB - We have assessed the efficacy of MAP-2 immunohistochemistry as a marker of seizure-related brain damage and its suitability for quantitation of the damage using densitometric and morphometric image analysis. Seizures were produced in rats by administration of 1.5 LD50 soman, an irreversible AChE inhibitor. Our results demonstrate that neuronal damage, assessed using hematoxylin and eosin, and cresyl violet staining, was colocalized on adjacent serial sections with clearly demarcated reductions in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2 immunostaining were found to be exceptionally well suited for quantitation using densitometric and morphometric image analysis. This study represents the first demonstration of seizure-induced excitotoxic alterations in MAP-2. PMID- 8618423 TI - Effects of dextran on hippocampal brain slice water, extracellular space, calcium kinetics and histology. AB - Hippocampal brain slices are valuable models for studying brain function but are compromised by several artifacts, including significant water gain and histologic injury, which occur under certain incubation conditions. Addition of colloid to Krebs-Ringer buffer (K-R) has been shown to eliminate water gain but has not achieved widespread acceptance. We confirm prior observations that dextran and PEG lessen the increase in slice mass during incubation in a dose-dependent manner with no water gain occurring at 4% concentrations. However, we also observe that addition of colloid to standard K-R induces severe neuronal pyknosis. Fortunately, the pyknosis can be eliminated by reduction in buffer osmolarity through adjustment of NaCl, producing markedly improved slice histology in dextran buffer, especially in the CA3 and CA4 regions of the hippocampus which are severely injured when incubated submerged in K-R at 37 degrees C. Extracellular space markers are not affected by either colloid. The volume of distribution for 45Ca is much larger in dextran buffers than in K-R and variability of 45Ca kinetics is also reduced. In the presence of dextran, hypoxia induces significant slice water gain, a relatively selective histologic injury and an alteration of tissue Ca2+ kinetics. Use of dextran buffers may eliminate many troubling brain slice artifacts. PMID- 8618424 TI - A supercharger for single electrode voltage and current clamping. AB - The theory and several circuits for supercharging is described for single micro and patch electrode voltage and current clamping. Supercharging is of use where the charging time of the cell membrane capacity results in the membrane potential change to considerably lag behind the command potential. This membrane capacity charging time is dependent on the series electrode resistance, the membrane resistance and capacity. Thus although amplifier voltage response times may be in microseconds, the membrane potential response to a step command potential often requires several milliseconds. Supercharging overcomes this difficulty by accelerating the membrane capacity charging time by applying a large initial and brief supercharging or 'booster' potential or current, to the command signal. Supercharging can improve membrane potential rise times by more than a hundred and thus bring membrane potentials to a stationary state well under a millisecond. Early changes in membrane properties are thus more readily observed during voltage and current clamping without being masked by capacitive currents. This technique also provides for the measurement of membrane time constants. The circuitry described is a simple add-on to the command signal pathway, is easy to adjust and provides the appropriate supercharging potentials for sequential step command signals of different amplitudes. PMID- 8618425 TI - Embedding, sectioning, immunocytochemical and stereological methods that optimise research on the lesioned adult rat spinal cord. AB - Numerous methods have been developed to embed, section and immunocytochemically label nervous tissue and the method chosen depends upon numerous factors. However, many of these methods have technical drawbacks that make them difficult to use in studies using injured/lesioned tissue. We present here, methods for embedding, sectioning and immunocytochemically labelling lesioned adult spinal cord tissue at the light microscope level. We have developed a novel, gelatine embedding technique for vibratome sectioning which overcomes many of the difficulties encountered with lesioned tissue. Individualised immunocytochemical protocols have also been developed for the antibodies GFAP (to label astrocytes), MBP (to label myelin) and CNP-ase (to label oligodendrocytes). Sequential pre treatment with proteinase-K, methanol and sodium borohydride achieved optimal GFAP localisation. MBP and CNP-ase were optimally localised after sequential pre treatment with proteinase-K (at different concentrations) and sodium borohydride. Methanol pre-treatment resulted in a loss of immunoreactivity for these latter two antibodies. Each protocol achieved full immunocytochemical penetration throughout 40 microns vibratome sections. These techniques enable the new unbiased stereological tools (that is, the Cavalieri and optical disector principles) to be utilised. PMID- 8618426 TI - A single electrode voltage, current- and patch-clamp amplifier with complete stable series resistance compensation. AB - An input headstage for single electrode voltage and current clamping is described which permits selecting desired operational modes during an experiment. These include cell attached or whole cell patch clamping, single micro-electrode voltage or current clamping, potential recording, iontophoresis and voltammetry. Input electrode series resistance with the electrode inserted in a cell or during whole cell patch clamping can be measured at any time. The unique circuitry allows complete compensation of the series resistance with high frequency response. The input series electrode resistance can also vary considerably around an initially set 100% compensation with intrinsic stability. An accelerator 'supercharger' or 'booster circuit' is included which can shorten the rise time of membrane potential to the command potential by a hundredfold. This improves the recording of early ion channel currents. Feedback resistors are selected remotely for the specific experimental need. Signal resolution in patch clamping is that expected from the thermal noise of the feedback resistor and electrode to membrane seal resistance. This headstage circuitry design thus allows interchangeable modes of operation and parameters during an experiment to obtain optimal conditions for signal detection and frequency response. PMID- 8618427 TI - Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus. AB - Quantal content of transmission was estimated for three synaptic systems (crayfish and Drosophila neuromuscular junctions, and rat dentate gyrus neurons) with three different methods of measurement: direct counts of released quanta, amplitude measurements of evoked and spontaneous events, and charge measurements of evoked and spontaneous events. At the crayfish neuromuscular junction, comparison of the three methods showed that estimates from charge measurements were closer to estimates from direct counts, since amplitude measurements were more seriously affected by variable latency in evoked release of quantal units. Thus, charge measurements are better for estimating quantal content when direct counts cannot be made, as in crayfish at high frequency of stimulation or in the dentate gyrus neurons. At the Drosophila neuromuscular junction, there is almost no latency variation of quantal release in realistic physiological solutions, and the methods based upon amplitudes and charge give similar results. Distributions of evoked synaptic quantal events obtained by direct counts at the crayfish neuromuscular junction were compared to statistical distributions obtained by best fits. Binomial distributions with uniform or non-uniform probabilities of release generally provided good fits to the observations. From best fit distributions, the quantal parameters n (number of release sites) and p (their probability of release) can be calculated. We used two algorithms to estimate n and p: one allows for non-uniform probability of release and uses a modified chi square (chi 2) criterion, and the second assumes uniform probability of release and derives parameters from maximum likelihood estimation (MLE). The bootstrap estimate of standard errors is used to determine the accuracy of n and p estimates. PMID- 8618429 TI - An in vitro jaw-nerve preparation for oral sensory study in the rat. AB - We have developed an in vitro jaw-nerve preparation in Wistar albino rats, suitable for the quantitative study of peripheral sensory mechanisms in the oral region. The mandible on one side was excised together with the inferior alveolar nerve and was kept in a modified Krebs-Henseleit solution saturated with O2/CO2 (95:5) gas mixture. Recordings were made from single afferent fibers in the inferior alveolar nerve in response to mechanical stimulation of various oral sites (oral mucosa, periodontal ligament and tooth pulp) by calibrated von Frey hairs. In the case of tooth pulp stimulation, heat and chemical (bradykinin) stimuli were also used. Stable unitary recordings could be made for up to 5 h which was long enough to record the activities evoked by various stimuli applied to the oral area. This paper will describe the procedure for making this preparation together with some responses of single units in the inferior alveolar nerve to mechanical, thermal and chemical stimulations applied to this preparation. PMID- 8618428 TI - Neuromuscular evaluation using rat gait analysis. AB - We have developed a rat gait analysis model to evaluate if ankle angle and other associated gait parameters could consistently define normal peroneal nerve and anterior tibialis muscle function. The second part of the study was designed to determine if such a model would be useful to measure recovery of function after a peroneal nerve crush injury (NCI). A clear plexiglas tunnel was designed for high speed frame videotaping and subsequent computergraphic gait measurement and analysis. Normal gait patterns for ankle angle, back height, step and stride lengths and the stance and swing times were determined in 8 rats. Data analysis demonstrated no significant left/right differences for any of the variables (ANOVA) with the exception of step length. Subsequently, 12 rats with a peroneal NCI were evaluated. All gait parameters evaluated from the injured side were significantly different from the uninjured side after injury except stride length. Ankle angle was the most sensitive outcome variable. Weekly gait analysis provided objective measurements as the ankle angle gradually returned to normal within 3 weeks. The rat gait model is a sensitive and reproducible method for non invasive evaluation of neuromuscular function during nerve recovery after a peroneal crush injury. PMID- 8618431 TI - A rapid correlation method for the analysis of spectro-temporal receptive fields of auditory neurons. AB - The evaluation of spectro-temporal receptive fields in auditory neurons makes use of a correlation technique that needs a high amount of calculation time. We present a method in which 2-ms time bins of post-stimulus time histograms, rather than every action potential, are the basis for correlation with the acoustic spectrum of the stimulus. In a t test the content of each bin is classified as excitatory, inhibitory or responseless. The bin width is adjusted to the temporal resolution of the units as determined in a gap detection analysis. The method presented here saves a substantial amount of analysis time and reveals adequately spectro-temporal receptive fields. PMID- 8618430 TI - Rapid on-line estimation of responses to transcranial magnetic and peripheral nerve electrical stimulation in single human motoneurons. AB - Cross-correlation experiments allow to obtain information about synaptic potentials in human motoneurons. However, recording cross-correlation responses of one motoneuron to transcranial magnetic and electrical peripheral nerve stimulation requires a considerable recording time when both responses are recorded consecutively. In this paper a method is introduced yielding the same information about the responses of a single motoneuron to both types of stimuli while requiring only a fraction of the recording time necessary for a conventional cross-correlation experiment. The main features of the method introduced were: (i) use of the recharging time of the magnetic stimulator for response recording to the electrical stimulus, (ii) use of specific stimulus timing with respect to the motor unit discharges, and (iii) on-line display with statistical testing of the response functions allowing to stop stimulus application, if the responses to both types of stimuli had reached statistical significance. Application of the method is demonstrated with response recording of 70 tibialis anterior motor units from five healthy volunteers to transcranial magnetic and peroneal nerve electrical stimulation. PMID- 8618432 TI - The B cell transcriptional coactivator BOB1/OBF1 gene fuses to the LAZ3/BCL6 gene by t(3;11)(q27;q23.1) chromosomal translocation in a B cell leukemia line (Karpas 231). AB - The LAZ3/BCL6 gene on chromosone 3q27 is recurrently disrupted in B cell non Hodgkin's lymphomas by translocations involving immunoglobulin genes or other chromosone regions. We have cloned the breakpoint region and chromosone derivatives of the t(3;11)(q27;q23.1) translocation, present in a B cell leukemia cell line (Karpas 231), which define a novel 11q23.1 breakpoint site. As a consequence of the translocation, LAZ3 regulatory regions upstream of non-coding exon 2 are replaced by those of BOB1/OBF1, a recently described B cell-specific coactivator of octamer-binding transcription factors. A detailed structural study of the BOB1/OBF1 genomic DNA and of a nearly full-length cDNA revealed particular features in the 3' untranslated region, such as an Alu motif and a polymorphic tetranucleotide microsatellite. Two mutations leading to two potential amino acid changes in the C-terminal region, were also detected in one allele of a lymphoma B cell line, Raji. Due to its cell-specific expression and role as a coactivating transcription factor, chromosomal translocation and/or point mutation of BOB1/OBF1 may contribute to B cell tumorigenesis. PMID- 8618433 TI - Expression of FLT3 receptor and response to FLT3 ligand by leukemic cells. AB - The novel hematopoietic growth factor FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell factor), and FMS. the receptor for M-CSF (macrophage colony- stimulating factor). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (IL-3), PIXY-321 (an IL-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced growth of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, IL-3, IL-6, IL-7, IL-11, IL-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL stimulated leukemia cell growth underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean 20%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic growth factor- dependent cell lines increased their proliferation upon incubation with FL, whereas all growth factor-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M CSF, IL-3, PIXY- 321 or SCF and FL with IL-3 or IL-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic fibroblast growth factor), GM-CSF, IL-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming growth factor-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine. PMID- 8618435 TI - Low-dose melphalan for treatment of high-risk myelodysplastic syndromes. AB - Twenty-one consecutive patients with high-risk myelodysplastic syndromes (MDS) including six with refractory anemia with excess blasts (RAEB) and 15 with RAEB in transformation (RAEBt) were treated with daily oral low-dose melphalan (2 mg/day). Seven patients achieved complete remission (CR), one patient partial response, and four minor response while the remaining eight did not respond. The median age of the patients was 65 (range 56-83 years). The mean total amount of melphalan given was 140+/-19 mg in patients who achieved CR. The median duration of CR was 14.5 months. Serious toxicity was not encountered in any of the cases. Neither marrow suppression nor pancytopenia was observed during the administration of melphalan in patients who achieved CR. The clinical features of CR patients included normal karyotype and hypocellular marrow in biopsied specimen from the lilac bone. These observations suggest that melphalan may exert some differentiation effects on leukemic cells in addition to cytotoxic effects. Our study indicates that daily administration of low-dose melphalan is worth trying in the treatment of elderly patients with high-risk MDS. PMID- 8618434 TI - Causes of initial remission induction failure in patients with acute myeloid leukemia and myelodysplastic syndromes. AB - We analyzed 144 of 475 previously untreated patients with acute myeloid leukemia (AML, n = 87) and 'high-risk' myelodysplastic syndromes (MDS, n = 57) who failed to achieve a complete remission with fludarabine- and high-dose cytarabine containing regimens between 1991 and 1994. The AML and MDS groups were comparable with regard to age, major prognostic indicators, supportive care received, type of antileukemia response and causes of death and were considered together. The causes of death of 118 evaluable patients were compared to a series of 123 AML initial remission induction failures previously reported from our institution. The induction failure rate was significantly lower (30 vs 43% P <0.001). We found a significant decrease in fatal infections (autopsied patients, P = 0.001) and a reduction in bacterial (autopsied and non-autopsied patients, P <0.005) but not fungal (non-autopsied patients, P = 0.93; autopsied patients P = 0.15) infections as causes of induction death. Pulmonary hemorrhage was twice as common in the present report (P < 0.005, with pulmonary hemorrhage almost three times as common as fatal cerebral hemorrhage. A comparison restricted to the AML patients in the present study yielded similar results. We conclude that the mortality profile in AML remission induction has undergone substantial changes at our institution since our last report, probably as a consequence of the introduction of new chemotherapy regimens and supportive care modalities. PMID- 8618436 TI - Clinical and cytologic characteristics of blastic phase in Ph-positive chronic myeloid leukemia treated with alpha-interferon. AB - We report 72 blastic crises (BC), occurring in 238 Ph+ chronic myeloid leukemia (CML) patients treated in chronic phase (CP) with alpha-interferon (IFN) for a median time of 51 months (range 7-96). The 238 patients were grouped by Sokal's risk at diagnosis in low- (LR), intermediate- (IR) and high-risk (HR), and by CP treatment. Group 1: 160 patients (57% LR, 31% IR, 12% HR) given IFN alone in early CP. Group 2: 31 patients (65% LR, 32% IR, 3% HR) given IFN alone in late CP. Group 3: 23 patients (78% LR, 22% IR) given IFN before and after autologous stem cell transplantation (ASCT). Group 4: 24 patients (83% LR, 17% IR) given IFN after ASCT. Of the 72 BC, 52 (72%) were myeloid (My), and 20 (28%) lymphoid (Ly). Overall BC incidence was similar in all CP treatment groups, although with a prevalence of Ly BC in groups 3 + 4 vs groups 1 + 2, (p = NS); the incidence of BC was higher in HR patients (P = NS), but on the whole it was lower than expected on the basis of historical controls. Lymphoid BC was more frequent in LR than in IR + HR patients (P < 0.05), and was more frequent in responders to IFN, than in non-responders (P < 0.05). In conclusion, a subset of patients with low risk at diagnosis, better response to IFN and proneness to evolve into Ly BC can be identified. The role played by IFN in this context remains to be defined. PMID- 8618437 TI - Clinical and morphological features of cases of trisomy 13 in acute non lymphocytic leukemia. AB - Trisomy 13 has been infrequently reported as a primary non-random karyotypic change in myeloid leukemias. To elucidate its clinical significance we examined the clinical and hematological data in nine ANLL patients in whom we found this change, in a series of 175 cytogenetically abnormal ANLL patients. Morphologically, six of the patients were FAB-M1, two were FAB-M4 and one was FAB M5. Bone marrow aspirates contained more than 90% blasts in eight of the patients. By immunophenotype, TdT was present in four of the patients, CD34 was present in four of five patients tested and CD5 was present in one of five patients tested. Blast cells in all patients expressed two or more myeloid surface antigens. These data suggest the proliferation of an immature myeloid cell in these patients. Complete remission was achieved in seven patients; however, remissions were short-lived. Eight patients expired between 1 and 13 months from diagnosis (median survival 5 months). Combining our findings with data in the published literature on trisomy 13 in ANLL, a larger data set consisting of 29 patients was established to determine better the clinical significance of this cytogenetic entity in ANLL. We found that this cytogenetic change has been reported in all subsets of FAB classification excepting M6 and M7. Median age at presentation was 60 years and no association with gender was noted. Median WBC was 29.5 x 10(9)/l, the majority of patients were thrombocytopenic (median platelet count 86 x 10(9)/l) and median survival was 5.2 months. This study associates trisomy 13 with malignant transformation of myeloid progenitor cells. These patients respond well to induction therapy, but relapse occurs quickly and the survival duration is poor. PMID- 8618438 TI - Deletion or lack of expression of CDKN2 (CDK4I/MTS1/INK4A) and MTS2 (INK4B) in acute lymphoblastic leukemia cell lines reflects the phenotype of the uncultured primary leukemia cells. AB - The CDKN2 gene has been recently localized to a chromosomal region found to be deleted in leukemias and solid tumors. CDKN2 encodes a 16 kDa protein product (p16INK4A), which functions as a specific inhibitor or the cyclin-dependent kinases 4 and 6. There have been many reports indicating a higher frequency of deletions of the CDKN2 gene in a variety of tumor cell lines, in comparison to primary tumors. These studies raise the possibility that deletions of CDKN2 may be a rare event in primary tumors, and in fact arise in vitro, during the establishment of permanent cell lines. To address this issue, we determined whether the CDKN2 gene deletions found in acute lymphoblastic leukemia (ALL) cell lines are also detected in the primary leukemia samples. Eleven cell lines were identified which had available frozen primary samples of their original leukemic tissue. Five out of 11 of these cell lines, as well as their primary samples had homozygous CDKN2 deletions. The remaining six cell lines and their primary samples retained at least one copy of the CDKN2 gene. Of the six CDKN2+ cell lines, five expressed CDKN2 mRNA, but only one of these expressed the p16 protein product (as did its primary sample). Our results indicate that CDKN2 deletions present in the studied ALL cell lines arose in the primary leukemic cells, and not during cell line establishment or prolonged in vitro culture. PMID- 8618440 TI - Cloning and characterization of the human tartrate-resistant acid phosphatase (TRAP) gene. AB - The expression and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, have been analyzed extensively in the past. In some diseases, like hairy cell leukemia and Gaucher's disease, cytochemically detected TRAP expression is used as a disease-associated marker. In this paper we describe the isolation of a genomic cosmid clone of the human TRAP gene. Restriction mapping revealed a 22-kb insert and the complete genomic structure of the TRAP gene. A 6 kb HindIII-fragment harboring the entire TRAP gene was subcloned and the 5' flanking region of 3026 bp was sequenced. Analysis of the sequence data showed the presence of potential transcription factor binding sites. Two transcriptional start sites were identified in the untranslated exon 1 at positions -349 and -347 bp relative to the translational start codon. Linked to a luciferase-encoding reporter gene the 5'-flanking region was sufficient to direct transcription in the heterologous cell line BHK-21. Treatment of the transfected cells with different modulators of the intracellular iron content showed that regulation of TRAP expression is dependent on iron. In summary, these data imply a possible functional role of the TRAP gene product either in the storage or the transport of iron. PMID- 8618439 TI - Variable expression of p16 protein in patients with acute myeloid leukemia without gross rearrangements at the DNA level. AB - The p16 protein is an inhibitor of cyclin-dependent kinases (cdk) 4 and 6. Both cdk4 and cdk6 phosphorylate the retinoblastoma protein (pRb) and are thought to be required for cell proliferation. Mutations and homozygous deletions in the p16 gene have been found in a number of cell lines but the incidence of abnormalities in primary tumours is controversial. We have studied the p16 gene in 76 cases of acute myeloid leukemia (AML) and 18 hematologically normal controls by quantitative Southern blotting. No deletions or rearrangements were detected. Twenty-five cases also showed no abnormal band patterns by RT-PCR-SSCP analysis of the coding sequence. We analyzed 60 AML samples for p16 protein expression by Western blot analysis. A reduced level of p16 was observed in six cases, however, none of them showed methylation of the 5'-CpG island. Over-expression of p16 protein was detected in six cases. Studies in cell lines have suggested a feedback loop between p16 and pRb with a futile overproduction of p16 protein in cells containing abnormal pRb. In accordance with these findings, four of the AML cases with high levels of p16 had abnormal pRb (two alteration in band size, two absent). From our data we suggest that gross abnormalities in the p16 gene are rare in AML, but that p16 levels are variable and high levels are associated with pRb abnormalities in a subset of cases. PMID- 8618441 TI - Classification of deletions and identification of cryptic translocations involving 7q by fluorescence in situ hybridization (FISH). AB - Monosomy 7 (-7) and deletion of the long arm of chromosome 7, del(7q), are frequent non-random findings in the myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), particularly associated with therapy-related disease (t MDS and t-AML). The cytogenetic breakpoints of 7q deletions are variable, with both terminal and interstitial deletions reported. It is now believed that most deletions are interstitial, and that the variability in reported breakpoints may be due to the difficulty in determining whether the terminal, pale staining G band is present. It has also been suggested that some reported deletions of 7q may be cryptic translocations. To address these questions, we carried out fluorescence in situ hybridization (FISH) studies on leukaemic cells from a large series of patients using a chromosome 7-specific paint and a 7q telomere-specific probe. Of the 26 cases studied, seven were 'pure' deletions (ie without the involvement of other chromosomes); four were interstitial and two terminal. One further patient had two clones each with a different deletion: one with a terminal del(7)(q22) and the second with an interstitial del(7)(q32-qter). A further nine cases had unbalanced translocations with deletion of 7q terminal sequences. The remaining 10 cases were translocations and complex rearrangements, some involving interstitial deletions of 7q. In two cases in which del(7q) was reported as the sole cytogenetic abnormality by G-banding, FISH revealed cryptic translocations involving 7q. PMID- 8618442 TI - High prevalence of T cell receptor D delta 2(D delta)J delta rearrangement in CD7 positive early T cell acute lymphoblastic leukemia. AB - We have studied the molecular characteristics of the T cell receptor (TcR) genes in 16 patients with CD7+ early T cell acute lymphoblastic leukemia (T-ALL), defined as being positive for CD7 but negative for CD3/4/8, myeloperoxidase (MPO), and CD19/20. Using gene analysis, rearrangement was demonstrated in one patient for immunoglobulin heavy chain (IgH) gene, five for TcR-beta gene, and four for TcR-gamma gene. Fifteen patients (94%) had rearranged band(s) involving the joining region of the TcR-delta chain gene. In nine cases these were the only rearrangements, whereas in six cases TcR-beta and/or TcR-gamma gene rearrangements were found as well. The D delta 2(D delta)J delta 1 rearrangement was demonstrated in 87.5% (14/16) of cases. D delta 2(D delta)J delta 3 was recognized in one patient, D delta 2D delta 3 was found in three, and V(D)DJ using only V delta 2 and V delta 3 was recognized in two patients. We found no V2D delta 3, V3D delta 3, or V1(D)DJ delta rearrangement patterns. Five of nine cases with DDJ delta were positive for cytoplasmic CD3 epsilon(CyCD3 epsilon). Our data suggest that DDJ delta joining occurs at an early stage during T cell differentiation, followed by rearrangements of V delta to the DDJ delta complex. Furthermore, our findings suggest that DDJ delta recombination occurs earlier than expression of CyCD3 epsilon protein products. DDJ delta rearrangements have never been observed in non-T cell malignancies, such as precursor-B-ALL or acute myeloid leukemia. Therefore, detection of DDJ delta rearrangement in the TcR delta locus is a useful tool to establish lineage and clonality of leukemic cells in the most immature stages of T cell development. PMID- 8618443 TI - Internal DNA deletion within the BCL-6 gene on untranslocated chromosome in non Hodgkin's lymphoma with 3q27 abnormality. AB - Chromosomal translocations involving the band 3q27 are recognized as common specific cytogenetic abnormalities in B cell non-Hodgkin's lymphoma (NHL), and the BCL-6 gene, identified on 3q27 was shown to be disrupted by these translocations. Previously, we have reported biallelic BCL-6 rearrangements occurring in some patients with B cell NHL. In the present study, we describe a NHL patient with t(3;22)(q27;q11) translocation. In this patient, biallelic BCL-6 abnormalities were indicated by Southern blot analysis. Further studies revealed that one of the two independent abnormalities was a juxtaposition to the immunoglobulin (Ig) lambda gene associated with chromosomal translocation, whereas the other was an internal DNA deletion of 1.5 kb area on untranslocated chromosome 3. Deletion junctions were located within the first exon and the 5' region of the first intron. The result provides the evidence that, besides chromosomal translocation, submicroscopic local DNA recombination can cause structural alteration of the BCL-6 gene. PMID- 8618444 TI - Clonal cell lineage involvement in myelodysplastic syndromes studied by fluorescence in situ hybridization and morphology. AB - We have used DNA fluorescence in situ hybridization (FISH) in combination with morphology to study cell lineage involvement in peripheral blood and bone marrow smears from 15 patients with myelodysplastic syndromes (MDS) and known numerical chromosomal aberrations. Eleven cases were investigated at diagnosis of MDS, and four at transformation to acute myeloid leukemia (MDS-AML). Using conventional cytogenetics, monosomy 7 was detected in nine cases, monosomy 17 in two, and trisomy 8 in five, either as a single aberration or as part of a complex clone. One case had both monosomy 7 and 17. Three biotinylated DNA probes directed against the pericentromeric region of chromosomes 7, 8 and 17, respectively, were used. Bone marrow smears were first stained with May-Grunewald-Giemsa (MGG) for morphologic evaluation, and regions of interest documented by photography. Later the same regions were targeted after hybridization, which allowed FISH analysis of selected bone marrow cells. In each patient between 267 and 921 cells (mean 453) were studied. In most cases a majority of the non-lymphoid bone marrow cells appeared to be of clonal origin, ie showed either monosomy or trisomy by FISH, while in contrast both lymphocytes and plasma cells generally were disomic, ie displayed two fluorescent spots for each probe. The percentages of clonal bone marrow cells differed greatly between patients, but in a single case there was a good agreement between the extent of granulocytic, monocytic and erythrocytic cell lineage involvement. In relation to the clinical stage of MDS, patients with more advanced disease had a significantly higher mean percentage of bone marrow blasts showing monosomy or trisomy, while disomic, possibly non-clonal cells were more frequent among the earlier forms of MDS. In the four cases of MDS-AML nearly all non-lymphoid bone marrow cells belonged to the abnormal clone. The FISH technique offers information regarding the distribution of clonal and non-clonal bone marrow cells in MDS, which might have prognostic and possibly, therapeutic implications. PMID- 8618445 TI - Familial myeloid leukemia associated with loss of the long arm of chromosome 5. AB - A 24-member kindred is described in which four cases of acute myeloid leukemia (AML), and one case of myelodysplastic syndrome (MDS) occurred over three generations. The proband was diagnosed with AML at age 47; within 6 months, her sister, age 41, was diagnosed with MDS. The proband's father, grandfather and a paternal uncle all died of AML, preceded by a pre-leukemic phase. The five cases had several clinical features in common. In the two sisters and their paternal uncle, cytogenetic analyses of bone marrow cells revealed a common abnormality characterized by loss of the long arm of chromosome 5, del(5q). No constitutional cytogenetic abnormality was detected in mitogen-stimulated peripheral blood lymphocytes from the proband. In addition, there was no history of common environmental or occupational exposure in the family. The occurrence of AML and MDS in three generations of this family most likely resulted from a single gene defect with an autosomal dominant pattern of inheritance. The association with the somatic loss of 5q material in the leukemia cells of affected members suggests that a germline mutation of a leukemia suppressor gene located on 5q might be the primary event responsible for hereditary susceptibility to leukemia in this family. PMID- 8618446 TI - Prognostic significance of peanut agglutinin binding in childhood acute lymphoblastic leukemia. AB - We previously reported the favorable prognosis associated with positive peanut agglutinin (PNA) binding in childhood T cell acute lymphoblastic leukemia (ALL), and hypothesized that this may be related to glucocorticoid sensitivity (Veerman et al. Cancer Res 1985, 45: 1890). The purposes of this prospective study involving 202 children with newly diagnosed ALL were to determine the relationship between PNA binding and (1) immunophenotype; (2) in vitro resistance to prednisolone (PRD) and dexamethasone and other drugs; (3) clinical response to a systemic PRD monotherapy (plus one intrathecal injection with methotrexate); and (4) multidrug chemotherapy. PNA positivity was more frequent in T cell ALL (65% of 43 cases) than in pro-B (0% of seven cases), common (17% of 106 cases) and pre-B (16% of 45 cases) ALL (P < 0.001). PNA binding was not associated with in vitro resistance to PRD or dexamethasone. However, in 38 evaluable T cell ALL patients, nine of 13 PNA-negative cases were clinically poor responders to PRD, while all 25 PNA-positive cases were good responders to PRD clinically (P < 0.0001). The four clinically poor PRD responders with B cell precursor (BCP)-ALL were also PNA negative. Within T cell ALL, PNA-positive patients had a 3.4-fold (95% Cl, 1.1-10.4, P = 0.03) lower relative risk of any event, than PNA-negative patients. Within BCP-ALL, PNA binding was not of prognostic significance. In conclusion, PNA positivity, especially frequent in T cell ALL, is a marker for a subgroup of childhood ALL patients who are very likely to respond well to systemic PRD 'monotherapy'. In addition, PNA positivity is a favorable prognostic factor in T cell ALL. PMID- 8618447 TI - Levels of circulating endothelial adhesion molecules (sE-selectin and sVCAM-1) in adult patients with acute leukemia. AB - The spread of leukemia extends from mobilization of leukemic blast cells from the bone marrow to extramedullary tissue infiltration. Migration of leukemic blasts resembles that of neutrophils and may be regulated by activated endothelial cells via endothelial adhesion molecules such as E-selectin, VCAM-1 and ICAM-1. Plasma levels of soluble adhesion molecules may therefore indicate interaction between leukemic blasts and endothelium, and may be related to leukemic cell mass or subtype of leukemia. In this study, plasma levels of soluble E-selectin, VCAM-1 and ICAM-1 were analyzed in 40 untreated patients with acute leukemia (35 ANLL, five ALL). Plasma concentrations of all three receptors were significantly elevated when compared to healthy controls (P = 0.006, P = 0.0001, and P = 0.0001, respectively) but demonstrated high interindividual variations among leukemic patients. sE-selectin but not sVCAM-1 and sICAM-1 levels correlated with peripheral leukocyte and blast cell counts. Increased levels of either sE-selctin or sVCAM-1 were present in 32 out 40 leukemic patients (80%). FAB subgroups differed in levels of sVCAM-1. The highest levels were measured in acute myelomonocytic and lymphoblastic leukemia, ie in leukemia subtypes with a high incidence of extramedullary blast cell infiltration. PMID- 8618448 TI - Analysis of the platelet-type thrombin receptor in 20 cases of large granular lymphocyte proliferations. AB - The platelet-type thrombin receptor was expressed by large granular lymphocytes (LGLs) in a variety of proliferative diseases. Twenty patients with LGL proliferative disease were examined, including five T cell clones and a variety of polyclonal proliferations, some secondary to rheumatoid arthritis and Felty's syndrome; 17/20 showed high number of CD3+, CD8+, and CD57+ lymphocytes and 9/20 also had high numbers of CD16+ or CD 56+ positive lymphocytes. The thrombin receptor was present on more than 20% of the LGLs in 13/20 patients. The clonal T cell expansions showed the highest receptor expression with greater than 75% cells positive. Regression analysis of all 20 cases showed striking and highly statistically significant positive Spearman rank correlation between the proportion of thrombin receptor and CD57-positive LGLs (r = 0.56, P = 0.009). A negative correlation with CD56 was also found (r = -0.46, P= 0.043). Dual antibody flow cytometry showed the receptor was more often co-expressed with CD57 (64%) than with CD16 (19%) or CD56 (11%). The expression of the platelet-type thrombin receptor by LGLs of this phenotype raises the possibility of a functional role for thrombin in the pathogenesis of LGL proliferative diseases. PMID- 8618449 TI - Two brc-abl junction peptides bind HLA-A3 molecules and allow specific induction of human cytotoxic T lymphocytes. AB - Intracellular processing of the products of the bcr-abl junction region in CML Philadelphia chromosomes would generate novel peptides which, if they are capable of binding to HLA-class I molecules, would be potential targets of a cytotoxic T cell response. The 18 nonamers corresponding to the b2-a2 and b3-a2 fusions and differing from the parental bcr and abl sequences for at least one amino acid have been synthesized and tested for binding with HLA class I alpha chain preparations from HLA-homozygous B lymphoblastoid cell lines. Two peptides derived from the b3-a2 junction bound to HLA-A3 and elicited detectable specific CTL responses in vitro. The binding affinity of one of the two peptides could be increased by appropriate substitutions of the anchor residues with those of the known HLA-A3 anchor motifs. More important, the modified peptide had increased capacity to prime a specific CTL response in vitro. The interaction with HLA-A3 of these two peptides and their substitution derivatives seems to be promising for target trials aimed at eliciting a specific CD8 T cell response against CML. PMID- 8618450 TI - Four subclones with distinct immunoglobulin light chain phenotypes. (kappa+lambda+, kappa+, lambda+ and kappa-lambda-) from acute leukemia. AB - A human acute lymphoblastic leukemia (ALL) cell line, BALM-9, was established from the peripheral blood specimen of a immunoglobin (lg) phenotype, the established BALM-9 cell line expressed both kappa and lambda light (L) chains simultaneously in a range of 30-80%. Two-color flow cytometric analysis demonstrated that there was a distinct population of kappa lambda double positive cells as well as kappa single, lambda single, and double negative populations. Therefore, subclones were obtained from each population by limiting dilution and were designated BALM-9KL (kappa+lambda+), BALM-9K (kappa+lambda-), BALM 9N (kappa lambda-). Western blotting confirmed the results of the immunofluorescence test at the protein level. In BALM-9N, L chains were absent even in the cytoplasm as demonstrated by Western blotting. Evidence that the subclones have the same ancestry was provided both by cytogenetic analysis and by Southern blotting, which revealed the 14q32 chromosomal rearrangement as a common abnormality and the same IgH gene arrangement among the subclones. The existence of a kappa lambda positive B cell population suggests a transient stage of normal B cell maturation. These subclones might represent such a stage and thus provide a useful means of analyzing the mechanism of this double light chain expression. PMID- 8618451 TI - Oral idarubicin pharmacokinetics--correlation of trough level with idarubicin area under curve. AB - Idarubicin is the first anthracycline that can be successfully administered via the oral route and thus may facilitate antineoplastic chemotherapy at an improved quality of life. These perspectives are somewhat hampered by the large variation in bioavailability between individual patients and the obvious requirement to monitor plasma concentration and area-under the curve values (AUC) for an appropriate adjustment of idarubicin dose. In this study we describe the pharmacokinetics of idarubicin and its main metabolite idarubicin in 12 patients after oral application of 20 mg/m2 idarubicin on 3 consecutive days and demonstrate that the 24-h trough levels shows high correlation with AUC and may thus allow a rapid and easy determination of individual drug concentrations and an appropriate dose adjustment. The average terminal half-life was 30.5h for idarubicin and 66.9 h for idarubicinol. The AUC for idarubicin and its main metabolite idarubicinol revealed a substantial interpatient variation with AUC values ranging from 25.7 to 114 ng x h/ml (average 58.1 ng x h/ml) for idarubicin and from 109.4 - 445.2 ng x h/ml (average 287.3 ng x h/ml) for idarubicinol. However, the ratio of idarubicin/idarubicinol differed only two-fold from 1:3.7 to 1:7.7 with an average of 1:5.1. Both idarubicin and idarubicinol concentrations were highly reproducible, however, upon measurements after repeated applications within individual patients. Moreover, idarubicinol and idarubicin AUCs showed a good correlation with r=0.78, indicating that the interindividual variations of idarubicin AUC reflects differences in absorptions rather than metabolism. In order to describe the interindividual bioavailability of idarubicin - represented by AUC - measurement of a single data point with a high correlation with the AUC would be ideal. Our study demonstrates that the 24 h trough level shows such an excellent correlation (r=0.96) with AUC, making it the perfect candidate for fast estimates of the individual bioavailability in a given patient. On this basis, the longitudinal measurement of the 24-h trough level may allow the assessment of the impact of interindividual variations in AUC of clinical outcome and toxicity. PMID- 8618452 TI - Effects of excess GM-CSF levels on hematopoiesis and leukemia development in GM CSF/max 41 double transgenic mice. AB - Double transgenic mice were produced by mating granulocyte-macrophage colony stimulation factor (GM-CSF) transgenic mice with max 41 transgenic mice that exhibit excess granulopoiesis and a predisposition to thymic lymphoma development. Although only two-thirds of the double transgenic mice had elevated circulating GM-CSF levels, double transgenic mice maintained significantly higher blood granulocytes and monocytes and more extreme granulopoietic hypercellularity in the marrow and spleen than max 41 transgenic mice. In double transgenic mice, early death occurred from the GM-CSF transgenic syndrome. Because of these early deaths, the incidence of thymic and generalized lymphomas was artificially lower than in max 41 mice but those lymphomas that did develop occurred earlier than in max 41 mice. While the excess GM-CSF levels in double transgenic mice stimulated increased granulocyte and monocyte formation and peritoneal dendritic cells were excessive, this failed to prevent the spontaneous development of T lymphomas, suggesting that dendritic cell-initiated suppression of tumor development may not be effective with this type of tumor. PMID- 8618453 TI - Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells. AB - To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation. PMID- 8618454 TI - N-ras genes are not mutated in Hodgkin and Reed-Sternberg cells: results from single cell polymerase chain-reaction examinations. AB - Ras mutations play an important role in many human tumors. They usually occur at only three codons (12, 13 and 61) of the three ras gene family members and lead to altered proteins resulting in a constitutively activated downstream signal cascade. We have examined the N-ras gene status in Hodgkin's disease (HD). Little is known about the pathogenetic events leading to malignant phenotype in HD. Since Hodgkin and Reed-Sternberg (H and RD) cells comprise only a minority of the cellular infiltrate in HD-lymph nodes, molecular studies concerning the status of oncogenes have been difficult to perform and have yielded conflicting results. We have established a single cell PCR assay for N-ras analysis and have examined H and RS cells from 12 cases of HD by PCR amplification and direct sequencing. None of the single H and RS cells examined carried N-ras mutations at either codons 12/13 or 61. Therefore, N-ras mutations are not involved in the pathogenesis of HD. PMID- 8618455 TI - Sweet's syndrome involoving the musculoskeletal system during treatment of promyelocytic leukemia with all-trans retinoic acid. AB - Induction therapy of promyelocytic leukemia with all-trans retinoic acid is a standard therapy despite significant side-effects. The most important, the "retinoic acid syndrome", consists of a hyperinflammatory reaction with capillary leakage (edema, pleural, and pericardial effusion), infiltration of myeloid cells into internal organs and systemic signs of inflammation. We describe here two cases of another hyperinflammatory reaction during all-trans retinoic acid therapy, the Sweet's syndrome, consisting of infiltrates of the skin and internal organs by neutrophilic granulocytes. Fever, painful erythematous cutaneous plaques, prominent musculoskeletal involvement (myositis, fasciitis), a sterile pulmonary infiltration and intercurrent proteinuria characterized the clinical course of all-trans retinoic acid-associated Sweet's syndrome. Treatment with glucocorticoids led to resolution of the syndrome within 48 h. Three other cases of all-trans retinoic acid-associated Sweet's syndrome without involvement of internal organs, prominent on our cases, were published previously. Recognition of ATRA-associated Sweet's syndrome is of practical importance. PMID- 8618456 TI - A new variant translocation in acute promyelocytic leukaemia: molecular characterization and clinical correlation. AB - Translocation t(15;17)(q22;q21) is an acquired clonal cytogenetic change present in almost all cases of acute promelocytic leukemia (APL). The molecular genetic basis of the translocation supports its integral role in pathogenesis. We describe a patient with APL in whom the leukaemic clone was characterized by a true variant of the classical t(15;17). The patient whose disease had numerous atypical clinical features, had t(11;17)(q13;121). The chromosome 17 breakpoint was localized to intron 2 of RARA by Southern blotting, and there was no evidence at the molecular level for rearrangement at PML locus. These data, along with previous reports of rare variant translocations in APL, indicate that while dysregulation of RARA by gene fusion may be essential for the APL phenotype, the particular fusion partner may determine clinicopathological aspects, including presentation, response to treatment with all-trans retinoic acid (ATRA), and prognosis. This heterogeneity suggests that the variant fusion partners of RARA in APL encode factors with properties both common to and distinct from those of PML. Investigation of these factors promises to shed light on the complex development pathways involved in the regulation of haematopoiesis. PMID- 8618458 TI - Isochromosome 7q in childhood myelodysplastic syndrome. PMID- 8618457 TI - Complete phenotypic and genotypic lineage switch in a Philadelphia chromosome positive acute lymphoblastic leukemia. PMID- 8618459 TI - Rhodamine 123 efflux in drug resistance assays. PMID- 8618460 TI - Acute Leukemia Forum '95: Advances and Controversies in Induction Therapy and Treatment of Older Adults with AML. Symposium proceedings. San Francisco, California, USA, November 10, 1995. PMID- 8618461 TI - High-dose ara-C in older adults with acute leukemia. AB - The concept of high-dose ara-C (HIDAC) was introduced 15 years ago. Phase I studies established a maximum tolerated dose of 3 g/m2, given every 12 hours for 12 doses. Because dermatologic, gastrointestinal, and central nervous system toxicities were dose-limiting in patients over age 50, a reduction from the maximal dose was suggested. Initial studies with refractory or resistant acute myelogenous leukemia (AML), with or without anthracyclines, demonstrated a significant antileukemic effect of HIDAC, with about 60 percent of patients achieving a complete response. Studies by our group and others have established that older patients can be successfully treated with HIDAC, probably with some added benefit in combining dose-intensive regimens. PMID- 8618462 TI - Differentiating therapy with all-trans retinoic acid in acute myeloid leukemia. AB - The standard treatment of patients with acute myeloid leukemia (AML) has depended on the elimination of the leukemic clone with cytotoxic myeloablation. Differentiation therapy is receiving increasing attention due to the remarkable activity of a vitamin A derivative, all-trans retinoic acid (ATRA), in patients with acute promyelocytic leukemia (APL). The majority of patients treated have achieved complete remission and with rapid resolution of the life-threatening bleeding diathesis. Phase II studies with ATRA in APL indicate certain limitations in the therapeutic use of retinoic acid. Patients achieving complete remission with ATRA alone require postremission chemotherapy, once remission is achieved, to extend remission. The results of differentiating therapy in APL patients has encouraged the use of differentiating therapy in patients with non M3 AML. A number of areas await research, such as the need to develop ways to overcome acquired retinoic resistance and the development of cytochrome P450 inhibitors. Other novel retinoids, such as 9-cis-retinoic acid, may be even more effective than ATRA in APL. Additional important areas of research involve studying combinations of retinoids and other putative differentiating agents, and identifying different selected treatments targeted at specific molecular defects. PMID- 8618463 TI - Postremission therapy of acute myeloid leukemia in older adults. AB - Acute myeloid leukemia (AML) in people over age 60 is characterized by adverse cytogenetic characteristics, prior myelodysplasia, and phenotypic features predictive of poor response to induction chemotherapy and brief leukemia-free survival. Because increased treatment-related toxicity complicates both induction and consolidation chemotherapy, most studies of AML in the elderly focus on induction regimens designed to reduce toxicity. Consolidation usually consists of a repeat cycle of conventional-dose induction chemotherapy. Older patients who achieve complete remission may represent a select population, clinically distinct from patients receiving induction therapy. Regardless of the consolidation regimen, the duration of complete remission is 3 months to 11 months, with long term leukemia-free survival rates of 10 percent to 28 percent. In the UCLA experience, patients over age 60 who received consolidation, chemotherapy and pretreatment characteristics similar to elderly patients undergoing induction. Postremission treatment varied from standard-dose ara-C to high-dose ara-C consolidation followed by autologous stem-cell transplantation. Leukemia-free survival appears to be longer with high-dose ara-C and autologous stem-cell transplantation. Further randomized studies should be conducted to determine the feasibility of and response to postremission treatment. PMID- 8618464 TI - Role of postinduction immunotherapy in acute myeloid leukemia. AB - Because chemotherapy alone does not eliminate clonogenic leukemic cells, disease may recur after induction and consolidation chemotherapy. Studies of patients receiving high-dose chemotherapy supported by bone marrow or blood stem-cell transplantation suggest that immune-mediated effects of transplanted cells help control disease. This antileukemic (graft-versus-leukemia) effect represents several immune reactions. Various approaches to introduce or enhance such immune reactivity in patients with acute myeloid leukemia (AML) are being explored. There is some indication, even in older patients, that immunotherapy is better tolerated than chemotherapy, and efforts are underway to find the most effective treatment for maintaining remission after induction chemotherapy. We have developed a strategy that combines myeloablative chemotherapy with transplantation of autologous stem cells that have been cultured for 1 week in interleukin-2. Low-dose interleukin-2 is also administered for the first week after stem cell infusion. Although all patients developed side effects of fever and fatigue, even older patients tolerated this regimen well. The 3-year disease free survival rate in a high-risk patient group transplanted in early first remission is 41 percent. Several other compounds are being investigated for their ability to enhance immune reactions after induction chemotherapy for AML. Although immunotherapy cannot eliminate overt leukemia and cytoreductive treatment is still needed initially, immunotherapy may find a place in postinduction management of AML to eliminate minimal (residual) disease or control its growth. PMID- 8618465 TI - Treatment of acute myeloid leukemia with antecedent myelodysplastic syndrome. AB - Primary myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are both age-related, with increasing prevalence over age 60. The poor outcome of treatment for elderly AML patients is due both to host-related and intrinsic biologic factors. Some elderly patients have documented MDS for months or years before presenting with AML. Many more, though, have dysplastic morphology in bone marrow and blood cells that suggests an occult preleukemic phase. Cytotoxic chemotherapy in elderly patients has a high morbidity and mortality due to comorbid diseases, diminished tolerance to prolonged pancytopenia, and decreased drug metabolism and excretion; doses, therefore, are often attenuated. The low response rates to conventional remission-induction chemotherapy regimens suggest both intrinsic drug resistance and diminished normal hematopoietic precursors to regenerate following therapy. Less intensive chemotherapy has the potential for less organ toxicity, less hospitalization, and less expense. However, attenuated chemotherapy has the major disadvantage of less antileukemic activity and, as yet, the survival benefit for this approach remains unproven. Nevertheless, low dose ara-C (cytarabine) therapy for 2 to 3 weeks may be beneficial for some patients. The standard response criteria used with acute leukemia to measure disease eradication may not be appropriate for patients with MDS evolving to AML. PMID- 8618466 TI - The role of prognostic factors in acute myeloid leukemia. AB - The Acute Myelogenous Leukemia Cooperative Group in Germany studied the role of different intensities of induction therapy, all followed by similar postremission treatment. Of the 1,034 patients aged 16 to 83, 33 percent were over age 60, 63 percent attained a complete remission, and the overall relapse-free survival rate was 30 percent after 5 years. A significantly higher relapse-free survival was predicted by M3 morphology, a favorable karyotype, including t(8;21), t(15;17), and inv(16), and the absence of dysmyelopoiesis. In contrast, dysmyelopoiesis, high serum lactic dehydrogenase, age over 64, and unfavorable karyotype, including abnormalities of chromosomes 5 or 7 and complex abnormalities, all predicted a low relapse-free survival rate. No comparable impact on relapse-free survival was found from the two randomized different intensities of induction treatment in each age-group. Age, LDH, M3, and karyotype contributed to a prognostic index that identified good, intermediate, and poor prognostic groups. Patients older than age 60 showed significantly less frequent favorable and more frequent unfavorable karyotypes, and received generally less-intensive induction treatment than what younger patients were given. We conclude from this that, unlike some biologic disease characteristics, treatment variables are weak prognostic factors and high age per se may not be an independent factor of overall relapse-free survival. PMID- 8618468 TI - Immunophenotyping and cytogenetics in older adults with acute myeloid leukemia: significance of expression of the multidrug resistance gene-1 (MDR1). AB - One of the best hopes for improving outcome in leukemia involves identification of biologic factors that can predict response and resistance at disease presentation and that can be used to design new treatment regimens that circumvent drug resistance. Recent studies suggest that younger acute myeloid leukemia (AML) patients whose leukemic blasts express the multidrug resistance gene-1 (MR1) have a poor prognosis. These younger MDR1+AML patients are biologically and genetically related more to elderly MDR+ and secondary AML patients than t younger AML patients with MDR1- true de novo AML. Data demonstrate for the first time in a large number of uniformly treated patients whose leukemic blasts were analyzed prior to therapy that MDR1 expression and functional dye/drug efflux are important independent predictors of complete response in AML in the elderly. Unexpectedly, elderly patients with de novo AML who are MDR1- have complete response rates approaching 75 percent, similar to younger AML patients, indicating that they are likely to have good outcomes with induction chemotherapy despite their age. In contrast, elderly MDR1+ de novo AML patients and elderly MDR1+ patients with secondary AML are much less likely to achieve a complete response with current regimens. These data argue for the inclusion of MDR1-modulating agents or drugs that are not MDR1 substrates in the treatment of elderly AML patients. PMID- 8618467 TI - The hematopoietic stem cell in elderly patients with leukemia. AB - Stem cell reserve and the proliferative capacity of pluripotent hematopoietic progenitors as influenced by age are of intrinsic biologic interest and potential therapeutic importance. The latter is especially true in older persons receiving intensive but not marrow-ablative chemotherapy. Studies of stem cell aging using murine models yield conflicting data. Unfortunately, few studies address the effect of aging on human hematopoietic stem cells, largely because there is no true pluripotent stem cell assay. Some work indicates that the proliferative capacity of human hematopoietic stem cells appears to decrease with age but is unclear whether this is of clinical significance. Our preliminary analysis of more than 500 autotransplant candidates by univariate analysis indicates that higher patient age correlates with reduced committed hematopoietic progenitor cell levels but not with delayed engraftment. In the future, the use of combinations of assays that include phenotypic (CD34+thy1+CD38lo) and functional (long-term culture-initiating cell) characteristics may better identify the effects of aging on candidate stem cells. The development of assays that predict delayed hematopoietic recovery after chemotherapy in older adults may help to tailor therapy for specific patients with the hope of reducing morbidity while retaining efficacy. PMID- 8618469 TI - The role of multidrug resistance and its pharmacological modulation in acute myeloid leukemia. AB - Despite more effective treatment for younger patients with acute myeloid leukemia (AML), resistance to conventional antineoplastics has limited such advances in the elderly. Overexpression of the multidrug transporter, P-glycoprotein (Pgp), appears to contribute to treatment failure in de novo AML and has been detected in up to 70 percent of elderly patients. Data also indicate linkage between Pgp and many adverse prognostic features, including cytogenetic pattern, surface phenotype, and evolution from an antecedent hematologic disorder. Pharmacologic inhibitors of Pgp function have been targeted for investigation in elderly AML patients. Non-Pgp mechanisms responsible for multidrug resistance (MDR) phenotypes that are only weakly sensitive to classic Pgp modulators, however, may limit the success of such strategies. Overexpression of the lung-resistance protein (LRP) in AML has also been linked to advanced age, secondary leukemia, and Pgp overexpression. In a study of 66 patients at the Arizona Cancer Center, LRP overexpression was a more important predictor of response to induction therapy for AML than was Pgp. Recent investigations indicate that overexpression of the gene encoding the MDR-related protein (MRP), though rare in de novo AML, may be common in high-risk groups such as relapsed patients and secondary AML. Use of monoclonal antibodies specific for the MRP gene product may further define its prognostic relevance in AML. PMID- 8618470 TI - New chemotherapeutic agents in acute myeloid leukemia. AB - Only two classes of chemotherapeutic agents have shown activity in acute myeloid leukemia (AML): ara-C and topoisomerase II reactive agents. Frontline combinations of these agents produce complete response (CR) rates of 70% and long term event free survival rates of 25%. New agents with different mechanisms of action are being explored. Nucleoside analogs such as chlorodeoxyadenosine (2 CdA) or fludarabine have shown single-agent efficacy and may be synergistic with ara-C. Combination therapy with ara-C and nucleoside analogs have shown promising results both as salvage therapy and in newly diagnosed patients. Combinations of topotecan with ara-C, VP16, and anthracyclines are being pursued, as is testing of other Topo-I inhibitors. Hypomethylating agents (5-azacytidine, decitabine) are showing activity in AML, producing CR rates of 5% to 30% as AML salvage therapy as a single agent, and 40%-60% in combinations. Decitabine may be synergistic with topo I inhibitors, biologic agents, and differentiating agents. Homoharringtonine has modest anti-AML activity, with CR rates of 10% to 30% as salvage therapy. Other classes of agents worthy of continuing investigation are platinum analogs and agents with novel mechanisms of action such as tallimustine. PMID- 8618472 TI - Interpreting data in AML. AB - Steady progress has been made in the treatment of acute myeloid leukemia since the discovery of daunorubicin in the 1960s. Advances in new drug activity have contributed greatly to that and are responsible for curing about 20 percent of patients diagnosed today. Progress in supportive care has been equally important; broad-spectrum antibiotics are now allowing patients to survive long enough to receive an adequate course of chemotherapy, to which most patients respond with a complete remission. More recently, the development of colony-stimulating factors and their safe use during induction therapy in high-risk patients has allowed greater numbers of patients to be adequately treated during the initial phase of therapy. The most significant recent advance in treatment for acute leukemia is the demonstration that certain leukemic syndromes respond dramatically to specific interventions that are substantially less active in other leukemic syndromes. The challenge for the near future is to understand the mechanisms behind these empiric observations and, for the long-term, to learn to use such information to design molecularly targeted therapy for specific leukemic syndromes that are characterized by unique molecular features. Antisense oligonucleotide therapeutic research is one first step in that direction. PMID- 8618471 TI - Use of growth factors during induction therapy for acute myeloid leukemia. AB - The use of colony-stimulating factors (CSFs) in acute myeloid leukemia (AML) remains controversial. Potential uses include shortening the period of neutropenia, inducing leukemic cells into the S-phase of the cell cycle, stem cell protection, inducing differentiation of leukemic cells, interrupting autocrine/paracrine loops, direct inhibition of leukemogenesis, and enhancing antimicrobial function. Data from the nine controlled studies of CSFs that have been reported between 1990 and 1995, with varying patient characteristics and other factors, indicate that growth factors have several uses in AML therapy. The published literature now suggests that the safety of CSFs is no longer a major clinical concern, and significant experience has been gained in reducing the period of neutropenia following induction therapy. Yeast-derived granulocyte macrophage colony-stimulating factor and granulocyte colony-stimulating factor appear to be effective and probably have an important role in the management of older adult patients with AML and for those patients with a significant risk for therapy-related morbidity and mortality. The use of CSFs as priming agents remains experimental; results of further prospective placebo-controlled studies, with laboratory correlates, are awaited. PMID- 8618473 TI - Thrombopoietin: biological and preclinical properties. AB - The characterization and purification of thrombopoietin (TPO) was problematic due to the extremely low levels of protein present in even the richest physiologic sources of the material and to the complex nature of plasma as a starting material for biochemical purification. Although interleukin-3 (IL-3) can initiate megakaryocytic progenitor cell development, it cannot complete this process. TPO is absolutely essential for full theory to clinical trials, and many of the properties predicted by early investigators using semipurified preparations of plasma-derived material have been verified using recombinant protein. TPO increases the size, ploidy, and cell-surface expression of platelet-specific proteins. TPO also stimulates the proliferation of megakaryocytic progenitor cells and augments the erythroid progenitor response to erythropoietin and other early-acting cytokines. TPO levels are inversely related to platelet mass; the recombinant protein stimulates thrombopoiesis and, to a lesser extent, erythropoiesis. Preliminary findings give reason for optimism, but clinical trials will be required to establish the usefulness of this primary regulator of platelet production in hastening hematopoietic recovery in states of natural and iatrogenic marrow failure. PMID- 8618474 TI - Does it matter how remission is achieved in acute leukemia? AB - Patients fail to be cured of acute myeloid leukemia (AML) because they develop absolute or relative resistance to antileukemic drugs such as ara-C (cytarabine) or because they die of complications of bone marrow failure. The Australian Leukemia Study Group (ALSG) has been investigating strategies to improve leukemia control by changing the methods of induction treatment. High-dose ara-C (HIDAC) may overcome ara-C resistance in leukemic blasts, and it has been used as a successful salvage regimen and in postremission therapy. The data from ALSG suggest that a dose-effect relationship exists for ara-C in AML, that the use of HIDAC prolongs remission duration and disease-free survival and that it is tolerable when used as initial induction therapy in patients with de novo AML. A fundamental question still to be answered in ongoing clinical trials is: When should AML treatment be optimally intensified--during the induction phase, immediately after remission, or after 2 to 3 months of preparative regimens, as in some transplantation programs? PMID- 8618475 TI - The World Bank, listening and learning. PMID- 8618476 TI - Gas stoves and respiratory health. PMID- 8618477 TI - Cochlear implantation for children and adults. PMID- 8618478 TI - Negative emotions and coronary heart disease: getting to the heart of the matter. PMID- 8618479 TI - Cholesterol, fibre, and bile acids. PMID- 8618480 TI - Influences of the media: a powerful what? PMID- 8618481 TI - Personality as independent predictor of long-term mortality in patients with coronary heart disease. AB - BACKGROUND: Emotional distress has been related to mortality in patients with coronary heart disease (CHD), but little is known about the role of personality in long-term prognosis. We postulated that type-D personality (the tendency to suppress emotional distress) was a predictor of long-term mortality in CHD, independently of established biomedical risk factors. METHODS: We studied 268 men and 35 women with angiographically documented CHD, aged 31-79 years, who were taking part in an outpatient rehabilitation programme. All patients completed personality questionnaire at entry to the programme. We contacted them 6-10 years later (mean 7-9) to find out survival status. The main endpoint was death from all causes. FINDINGS: At follow-up, 38 patients had died; there were 24 cardiac deaths. The rate of death was higher for type-D patients than for those without type-D (23 [27%]/85 vs 15 [7%]/218; p < 0.00001). The association between type-D personality and mortality was still evident more than 5 years after the coronary event and was found in both men and women. Mortality was also associated with impaired left ventricular function, three-vessel disease, low exercise tolerance, and the lack of thrombolytic therapy after myocardial infarction. When we controlled for these biomedical predictors in multiple logistic regression analysis, the impact of type-D remained significant (odds ratio 4.1 [95% CI 1.9 8.8]; p = 0.0004). In this group of CHD patients, type-D was an independent predictor of both cardiac and non-cardiac mortality. Social alienation and depression were also related to mortality, but did not add to the predictive power of type-D. INTERPRETATION: We found that type-D personality was a significant predictor of long-term mortality in patients with established CHD, independently of biomedical risk factors. Personality traits should be taken into account in the association between emotional distress and mortality in CHD. PMID- 8618482 TI - Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome. AB - BACKGROUND: In laboratory animals, cerebral ischaemia is worsened by hyperthermia and improved by hypothermia. Whether these observations apply to human beings with stroke is unknown. We therefore examined the relation between body temperature on admission with acute stroke and various indices of stroke severity and outcome. METHODS: In a prospective and consecutive study 390 stroke patients were admitted to hospital within 6 h after stroke (median 2.4 h). We determined body temperature on admission, initial stroke severity, infarct size, mortality, and outcome in survivors. Stroke severity was measured on admission, weekly, and at discharge on the Scandinavian Stroke Scale (SSS). Infarct size was determined by computed tomography. Multiple logistic and linear regression outcome analyses included relevant confounders and potential predictors such as age, gender, stroke severity on admission, body temperature, infections, leucocytosis, diabetes, hypertension, atrial fibrillation, ischaemic heart disease, smoking previous stroke, and comorbidity. FINDINGS: Mortality was lower and outcome better in patients with mild hypothermia on admission; both were worse in patients with hyperthermia. Body temperature was independently related to initial stroke severity (p < 0.009), infarct size (p < 0.0001), mortality (p < 0.02), and outcome in survivors (SSS at discharge) (p < 0.003). For each 1 degrees C increase in body temperature the relative risk of poor outcome (death or SSS score on discharge < 30 points) rose by 2.2 (95% CI 1.4-3.5) (p < 0.002). INTERPRETATION: We have shown that, in acute human stroke, an association exists between body temperature and initial stroke severity, infarct size, mortality, and outcome. Only intervention trials of hypothermic treatment can prove whether this relation is causal. PMID- 8618483 TI - Association of respiratory symptoms and lung function in young adults with use of domestic gas appliances. AB - BACKGROUND: There is evidence from some studies that people living in homes with gas stoves and other unvented gas appliances experience more respiratory symptoms than those who use other fuels for cooking and heating, but other studies have found no such association. We have investigated whether the use of gas appliances is associated with an increased risk of respiratory symptoms and whether sensitisation to common environmental allergens modifies any such association. METHODS: A stratified random sample of 15,000 adults aged 20-44 years, living in three towns in East Anglia, UK, were sent a questionnaire on asthma and hayfever. From those who responded, a random sample of 1864 were invited to complete an extended questionnaire that included questions on use of gas appliances, to give blood samples for measurements of total IgE and specific IgE to common allergens, and to undergo tests of respiratory function, 659 women and 500 men agreed to an interview. The association of the use of gas appliances with respiratory symptoms, total IgE, specific IgE, and respiratory function was assessed by logistic and multiple regression models. FINDINGS: Women who reported they mainly used gas for cooking had an increased risk of several asthma-like symptoms during the past 12 months including wheeze (odds ratio 2.07 [95% CI 1.41-3.05]), waking with shortness of breath (2.32 [1.25-4.34]), and asthma attacks (2.60 [1.20 5.6]). Gas cooking increased the risk of symptoms more in women who were atopic than in non-atopic women but the difference did not reach significance (p . 0.05). Women who used a gas stove or had an open gas fire had reduced lung function (forced expiratory volume in 1 s [FEV1]) and increased airways obstruction (FEV1 as a percentage of forced vital capacity) compared with women who did not. These associations were not observed in men. INTERPRETATION: In East Anglia, the use of gas cooking is significantly associated with subjective and objective markers of respiratory morbidity in women but not in men. Women may be more susceptible than men to the products of gas combustion or they may have greater exposure to high concentrations of these products because they cook more frequently than men. PMID- 8618484 TI - Prospective assessment of breastfeeding and breast cancer incidence among 89,887 women. AB - BACKGROUND: The relation between breastfeeding and breast cancer risk has been examined in many studies; some have reported no association, and others a reduced risk, particularly among premenopausal women. In the only prospective cohort study, no association was found. We have assessed prospectively the association between breastfeeding and incidence of breast cancer among 89,887 women in the US Nurses' Health Study. METHODS: In 1986, participants were asked about the number of months they breastfed for all their children combined. Parous women with no history of cancer were included in this analysis. During 6 years of follow-up (513,015 person-years), 1,459 invasive breast cancer cases were diagnosed. FINDINGS: Relative to women who had never breastfed, no significant overall association was found--after adjusting for established risk factors for breast cancer--between a history of having breastfed and subsequent development of breast cancer (relative risk [RR] 0.93, 95% CI 0.83 -1.03). No inverse trend was observed with duration of breastfeeding; women who breastfed for 2 years of longer had a RR of 1.11 (0.90-1.38). Among women who had given birth only once, women who had breastfed their child experienced a lower incidence of breast cancer (RR 0.68, 0.46-1.00). Among premenopausal women, who tended to be near menopause due to the age structure of the cohort, the RR of breast cancer for those who had lactated was 1.16 (0.89-1.50). Premenopausal women who had lactated for 1 year or more had a RR of 1.10 (0.78-1.57). INTERPRETATION: These data suggest that there is no important overall association between breast-feeding and the occurrence of breast cancer. PMID- 8618485 TI - Worsening of pulmonary gas exchange with nitric oxide inhalation in chronic obstructive pulmonary disease. AB - BACKGROUND: Inhalation of nitric oxide (NO) causes selective pulmonary vasodilation and improves arterial oxygenation in acute respiratory distress syndrome. But some patients do not respond or gas exchange worsens when inhaling NO. We hypothesised that this detrimental effect might be related to the reversion of hypoxic vasoconstriction in those patients where this mechanism contributes to ventilation-perfusion (V(A)/Q) matching. METHODS: We studied 13 patients with advanced chronic obstructive pulmonary disease (COPD). We compared their responses to breathing room air, NO at 40 parts per million in air, and 100% O2. Changes in pulmonary haemodynamics, blood gases, and V(A)/Q distributions were assessed. FINDINGS: NO inhalation decreased the mean (SE) pulmonary artery pressure from 25.9 (2.0) to 21.5 (1.7) mm Hg (p = 0.001) and PaO2 from 56 (2) 53 (2) mm Hg (p = 0.014). The decrease in PaO2 resulted from worsening of V(A)/Q distributions, as shown by a greater dispersion of the blood flow distribution (logSD Q) from 1.11 (0.1) to 1.22 (0.1) (p = 0.018). O2 breathing reduced the mean pulmonary arterial pressure to 23.4 (2.1) mm Hg and caused greater V(A)/Q mismatch (logSD Q, 1.49 [0.1]). The intrapulmonary shunt on room air was small (2.7 [0.9]%) and did not change when breathing NO or O2. INTERPRETATION: We conclude that in patients with COPD, in whom hypoxaemia is caused essentially by V(A)/Q imbalance rather than by shunt, inhaled NO can worsen gas exchange because of impaired hypoxic regulation of the matching between ventilation and perfusion. PMID- 8618486 TI - New serotype of Bartonella henselae in endocarditis and cat-scratch disease. AB - BACKGROUND: The fastidious nature of Bartonella henselae is such that demonstration of clinical infection relies mainly on serological or molecular biological methods. Only five isolates have been obtained from patients with cat scratch disease (CSD) and none from endocarditis. METHODS: We isolated B henselae from a CSD patient and, for the first time, from a patient with endocarditis. The isolates were characterised on the basis of morphology, biochemistry, cell-wall fatty-acid analysis, PCR-restriction fragment length polymorphism analysis of the 16-23S intragenic spacer region, and 16S rRNA gene sequences. Characterisation of these isolates indicated them to belong to a new serogroup, which we have called "Marseille", and to a new genotype based on the 16S rRNA gene sequence. The new variant was incorporated into an immuno-fluorescence antibody test (IFAT), which was used to reassess serum samples from 113 CSD patients who were seronegative with the conventional IFAT. FINDINGS: 18 (16%) of these apparently seronegative patients yielded significantly raised titres. 20 CSD patients who were seropositive as judged by the conventional IFAT remained seropositive with the new IFAT. INTERPRETATION: Antigenic variability within the species is one possible reason for inconsistent results in the serological diagnosis of CSD. PMID- 8618487 TI - Oxidative damage to DNA in diabetes mellitus. AB - BACKGROUND: Increased production of reactive oxygen species (ROS) and lipid peroxidation may contribute to vascular complications in diabetes. to test whether DNA is also oxidatively damaged in diabetes, we measured 8 hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative damage of DNA, in mononuclear cells. METHODS: For this laboratory-based study, 12 patients with insulin-dependent diabetes mellitus (IDDM) and 15 patients with non-insulin dependent diabetes mellitus (NIDDM) were matched by age with ten healthy volunteers each. DNA was extracted from mononuclear cells from whole blood. 8 OHdG was assayed by high-pressure liquid chromatography, and ROS were assayed by chemiluminescence. FINDINGS: IDDM and NIDDM patients had significantly higher median concentrations (p , 0.001, U test) of 8-OHdG in their mononuclear cells than their corresponding controls (in fmol/micrograms DNA): 128.2 (interquartile range 96.0-223.2) and 95.2 (64.0-133.5) vs 28.2 (21.7-43.4) and 21.9 (18.0-24.4), respectively. ROS generation by mononuclear cells was also significantly greater (p < 0.01) in diabetic patients than in their controls (in mV): 238.0 (107.0 243.0) and 101.3 (66.0-134.0) vs 69.5 (49.8-91.9) and 56.0 (38.8-62.5), respectively. INTERPRETATION: IDDM and NIDDM patients showed greater oxidative damage to DNA, with increased generation of ROS, than controls. Such changes might contribute to accelerated aging and atherogenesis in diabetes and to the microangiopathic complications of the disease. PMID- 8618488 TI - A man with weight loss, ataxia, and confusion for 3 months. PMID- 8618489 TI - Poetry on rounds: a model for the integration of humanities into residency training. PMID- 8618490 TI - The effect of weapons on health. PMID- 8618491 TI - Making sense. PMID- 8618492 TI - Baboon xenotransplant fails but patient improves. PMID- 8618493 TI - Tweaking T cells with altered antigens. PMID- 8618494 TI - Danish haemophiliacs win in European court. PMID- 8618495 TI - HIV-positive servicemen in USA face discharge. PMID- 8618496 TI - Amaurosis fugax on standing and angle-closure glaucoma with clomipramine. PMID- 8618497 TI - Keloid associated with hypertension. PMID- 8618498 TI - Sheffield risk and treatment table for cholesterol lowering in prevention of coronary heart disease. PMID- 8618499 TI - Sheffield risk and treatment table for cholesterol lowering in prevention of coronary heart disease. PMID- 8618500 TI - Sheffield risk and treatment table for cholesterol lowering in prevention of coronary heart disease. GISSI-Prevenzione Investigators. PMID- 8618502 TI - HIV, confidentiality, gender, and support in rural South Africa. PMID- 8618501 TI - Sheffield risk and treatment table for cholesterol lowering in prevention of coronary heart disease. PMID- 8618503 TI - Intrauterine endoscopic laser surgery for fetal sacrococcygeal teratoma. PMID- 8618504 TI - Serotonin and premenstrual dysphoric disorder. PMID- 8618505 TI - Reversible ageusia associated with losartan. PMID- 8618506 TI - Alopecia side-effect of antituberculosis drugs. PMID- 8618507 TI - Does surge in blood pressure precede or follow stroke? PMID- 8618508 TI - Papular-purpuric gloves and socks syndrome with hepatitis B infection. PMID- 8618509 TI - Upper limb disease in women treated for breast cancer. PMID- 8618510 TI - Beeting (sic) a crimson retreat. PMID- 8618511 TI - Revising the research record. PMID- 8618512 TI - Beeting (sic) a crimson retreat. PMID- 8618513 TI - Is informed consent in neonatal randomised controlled trials ritual? PMID- 8618514 TI - Research in emergency care without consent: new proposed FDA rules. PMID- 8618515 TI - Testing for tuberculosis. PMID- 8618516 TI - Presumed Pott's disease. PMID- 8618517 TI - Evaluation of oral anti-allergic drugs in Japan. PMID- 8618518 TI - Two cadaveric renal transplants in Pakistan from non-heart-beating donors from Maastricht. PMID- 8618519 TI - Acute renal failure. PMID- 8618520 TI - Acute renal failure. PMID- 8618521 TI - Acute renal failure. Madrid Acute Renal Failure Study Group. PMID- 8618522 TI - Mercury poisoning associated with hepatitis-B immunoglobulin. PMID- 8618523 TI - Staphylococcus epidermidis and acute necrotising pancreatitis. PMID- 8618524 TI - Phenotype of Hodgkin and Reed-Sternberg cells. PMID- 8618525 TI - World distribution of factor V Leiden. PMID- 8618526 TI - Pooled plasma derivatives and Creutzfeldt-Jakob disease. PMID- 8618527 TI - Development of the facial recess: implications for cochlear implantation. AB - More attention is being focused on the growth and development of the facial recess because of the use of cochlear implantation in children over 2 years of age and the prospect of using implants in even younger children. The facial recess and the extended facial recess were measured in 123 temporal bones from 73 individuals ranging in age from 8 weeks in utero to 7 years after birth. The goals were to trace the development of the facial recess and to determine whether this area continues to grow into childhood. The facial recess enlarges throughout fetal life with the development of the facial canal and the tympanic annulus. Reichert's cartilage, seen early in utero in the facial recess, gradually resorbs but may persist in the newborn. In full-term infants, the facial recess reaches 3.25 mm at the oval window and 2.62 mm at the round window. The extended facial recess reaches 3.79 mm at the oval window and 3.04 mm at the round window. No statistically significant growth of the facial recess after birth could be demonstrated in this study. The data suggest that the facial recess is probably adult sized at birth and should allow surgical access for cochlear implantation in very young children. PMID- 8618528 TI - [Device for examining the state of bronchial patency using the acoustic method]. AB - An acoustic device was proposed to detect bronchial obstruction by analysing a noise process in the laryngotracheal region at the forced expiration. Clinical trials were under way, which revealed persistent diagnosis signs of bronchial patency impairments associated with changes in the duration and shape of the envelope of a recorded acoustic signal, as well as deviations of its spectral characteristic. PMID- 8618529 TI - [Digital filter for circuit noise suppression in the electrocardiograph]. AB - The paper describes the structure and algorithm of a digital suppression filter for circuit noise at 50 Hz. The filter slightly corrupts an electrocardiographic signal. PMID- 8618530 TI - [Experience in organizing the manufacture of medical equipment at large-scale enterprises]. AB - The Izhevsk Motoplant is one of the largest Russian industrial associations which has successfully mastered and at present serially produces medical articles, including those for examination of the cardiovascular system (EK1T-03M2 and EK1TTsSP-01 electrocardiographs, etc.), apparatuses for maternal and child care (a DLTB -01 set for the diagnosis and treatment of tubal infertility; a complex for resuscitation of the newborn); route emergency and resuscitation cars. The Izhevsk Motoplant actively cooperates with the country's leading designing Research Institute of Medical Instrument Making, Russian Academy of Medical Sciences. PMID- 8618531 TI - [Use of automated information systems Oncoregistry and Specialist-Oncologist in establishing cancer registry of the Leningrad region]. AB - The paper describes the basic features and structure of a cancer register (CR) of the Informational Analytical Service System which can perform a package of interrelated functions to record and analyze an oncological condition, the status of anticancer struggle and the achieved level of specialized care for those who have fallen ill. CR is a fully independent structure of medical application wherein informational provision occupies the dominant position. The basic function of CR is also given in the paper. In Saint Petersburg and in the Leningrad Region there are all necessary conditions for successful CR operation. The basic software support of CR represents the automatic informational systems (AIS): Oncoregister AIS and Specialist-Oncologist AIS. The block diagram of these AISs and their informational communication are presented in this paper. PMID- 8618532 TI - [Doppler echographic criteria in determination of the aerobic threshold and ischemia of the lower limbs]. AB - Doppler echographic criteria were developed to define the anaerobic threshold and to detect lower extremity ischemia. Multi-stage treadmill test provided the criteria involving impairments in the time course of recovery of continuous diastolic blood flow in the lower extremity arteries. The criteria allow the anaerobic threshold to be defined in the range of low and high exercises and the severity of ischemia of the saved and amputated limb to be assessed in arterial occlusions. The diagnostic sensitivity of the criteria is 82-87%, its specificity and predictive value are 95-100%. PMID- 8618533 TI - [ESR-spectroscopy in medical practice and biomedical research]. AB - The authors describe the use of the ESR technique during the past 20 years. There are many examples of the application of the technique in different investigations. The authors propose the new way of training medical students to apply it. PMID- 8618534 TI - [A package of programs of computer-assisted simulation of invasive and noninvasive interventions]. PMID- 8618535 TI - [Use of LKhM-80 chromatograph in the analysis of urinary steroids]. AB - The paper describes a simple way of updating a Russian production-type LKaM-80 chromatograph in order to make a gas chromatographic analysis of urinary steroids for separation by using high performance capillary columns. To connect capillary columns, the units consisting of a gas heaving T-joint, a stream separating chamber, and a releasing restrictor were applied. To achieve satisfactory results, it is necessary to place a column through the tube connecting the union for attachment of the column and the flame-ionization detector so that the end of a capillary should be located at the detector as soon as closer. The updated chromatograph has been successfully used to obtain urinary steroidal profiles which are essential for the differential diagnosis of causes of hyperandrogyny in females, including pregnants. PMID- 8618536 TI - [Specific features of prosthesis of dental defects with ceramic dental bridge construction]. PMID- 8618537 TI - [Covering dental defects with aluminum cast dowel construction and technology of their manufacture]. AB - Clinical and laboratory studies have revealed benefits of aluminum cast constructions. Its cast dowel inserts are used in the system of fixed metal-free dentures made from ceramics (dental bridges, single crowns, etc.). The high efficiency, availability of casting technology for aluminum reduces patients' treatment periods and ensures accurate attachment of dowel insets to the root canal and stump of a tooth. PMID- 8618538 TI - [Specific features of designing a new type of electrocardiograph based on the example of portable EK1TTs02]. PMID- 8618539 TI - [Autonomic device for continuous monitoring and correction of muscle activity: Miotonik-02]. AB - The paper outlines a portable device with self-contained power supply, which records with electrodes applied to the skin and subsequently processes electric myosignals from human muscles. The device is designed for the therapy and rehabilitation of patients with some motor diseases via biological feedback. The paper also considers the block diagram and provides the technical data of the device. PMID- 8618540 TI - [A device for automatic determination, calculation and storage of parameters in cardiointervalography]. AB - Cardiointervalography is a highly informative tool of functional diagnosis and for its performance it is better to apply special devices. This paper outlines a device for automatic determination, calculation and storage of parameters in cardiointervalography and presents its technical data. PMID- 8618541 TI - [The electrode-myocardium interface at the cellular level and prospects of the use of implantable pacemakers]. AB - The interaction of the autonomic nervous system (ANS) and the heart is the subject-matter of new investigations of the interdisciplinary science neurocardiology which represents the newest strategy in cardiac pacing. In this connection the priorities are both the renewal of chronotropy with physiological cardiac pacing of the closed circuit and the prevention and treatment of malignant arrhythmias through ANS modulation. To achieve this requires adequate monitoring the balance of autonomic influences and stimulation and hence neuromodulation of cardiac afferents. Under these conditions, the electrode which is both deflecting and stimulating, and its interface with the body at the cellular level gain prime importance. The physical, electrochemical and physiological properties of the electrode-myocardium interface (boundary) are determined by the structure of the Helmholz bilayer. Translayer electron transport and electrochemical reactions which determine the biological compatibility of the interface may be optimized via the microstructure of the surface of a solid-state component by covering this surface with titanium or iridium nitride with so-called fractal geometry. Experimental and clinical findings have demonstrated that the fractal surface structure ensures extremely low polarization and improvement deflection, hence this deflection of an evoked myocardial cell response monitors a myocardial nervous response as a result of increased chronotropic excitement. The stimulating electrode also monitors the sympathetic activity determined from intracardiac impedance measurements, thus providing a new principle of rhythm adaptation when the pacemaker is an integral part of ANS, recovering normal chronotropy. These benefits of the electrode interface open new avenues for the treatment and prevention of tachyarrhythmias and for the long-term management of the grafted heart to prevent its rejecting processes. PMID- 8618542 TI - Necrotic arachnidism--Pacific Northwest, 1988-1996. AB - Although spider bites are common in many parts of the United States, most domestic spiders are not substantially venomous to man. The best known exceptions are widow spiders (Latrodectus spp., including the black widow L. mactans) and brown spiders (Loxesceles spp., particularly the brown recluse, Lox. reclusa). However, cases of arachnid envenomation from the hobo spider (Tegenaria agrestis) are being reported increasingly in the Pacific Northwest. This report summarizes investigations of three cases of T. agrestis bites among persons in Idaho, Oregon, and Washington; spider bites reported to U.S. poison-control centers during 1994; and emphasizes the need for physicians in the northwestern United States to consider the species as a cause of toxic arachnidism. PMID- 8618543 TI - Lake-associated outbreak of Escherichia coli O157:H7--Illinois, 1995. AB - On July 5, 1995, the Winnebago County Health Department (WCHD) in northern Illinois received a report from the local hospital of five cases of Escherichia coli O157:H7 infection among children who resided in Rockford. Interviews of the children's parents revealed no common food source; however, on June 24-25, they all had visited an Illinois state park with a lake swimming beach. On July 6, the Illinois Department of Public Health (IDPH) closed the swimming beach because of suspected transmission of infection through lake water. WCHD and IDPH investigated the outbreak to assess risk factors for illness and determine the source of infection. This report summarizes the findings of the investigation, which indicate that ingesting contaminated and untreated lake water can result in infection. PMID- 8618544 TI - Electricity-related deaths on lakes--Oklahoma, 1989-1993. AB - Drowning accounts for approximately 4200 deaths each year in the United States. Although electricity is documented infrequently as a cause of such fatalities, contact with electricity can result in death through temporary paralysis and drowning of persons who are swimming or wading, or through electrocution. From June 1989 through September 1993, five persons died on lakes in Oklahoma following contact or suspected contact with electrical currents. Four deaths occurred at two adjoining lakes in northeastern Oklahoma (lake A), and one at a lake in the southern part of the state (lake B). The five deaths occurred among males aged 13-50 years who were either swimming, working on or near docks, or working with electricity when they sustained fatal injuries. This report summarizes the investigation of these deaths by medical examiners (MEs) and the Oklahoma State Department of Health (OSDH). PMID- 8618546 TI - National occupational research agenda. PMID- 8618545 TI - Outbreak of cryptosporidiosis at a day camp--Florida, July-August 1995. AB - On July 27, 1995, the Alachua County Public Health Unit (ACPHU) in central Florida was notified of an outbreak of gastroenteritis among children and counselors at a day camp on the grounds of a public elementary school. This report summarizes the outbreak investigation, which implicated Cryptosporidium parvum as the causative agent and underscores the role of contaminated water as a vehicle for transmission of this organism. PMID- 8618547 TI - Inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) mutagenicity by black and green tea extracts and polyphenols. AB - Solutions of lyophilized preparations of standard black and green tea extracts were made and tested over a range of six concentrations as inhibitors of the mutagenicity caused by the fool mutagen 2-amino-1-methyl-6-phenylimidazo[4,5 b]pyridine (PhIP) in the Salmonella typhimurium TA98 assay containing S9 fraction from rats induced with alpha-naphthoflavone and phenobarbital. Extracts of both black and green tea were equally good inhibitors of mutagenicity. Purified polyphenols were prepared from tea extracts by solvent extraction. The polyphenols of black tea were more potent inhibitors of mutagenicity than the polyphenols of green tea. These findings suggest that black tea may have similar health-promoting properties to those reported previously for green tea. PMID- 8618549 TI - A sensitive color ELISA for detecting polycyclic aromatic hydrocarbon-DNA adducts in human tissues. AB - Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been determined by measurement of DNA adducts in human tissues. Competitive enzyme-linked immunosorbent assays (ELISAs) using antisera recognizing benzo[a]pyrenediol epoxide-modified DNA (BPDE-I-DNA) and color of fluorescence endpoint detection have been used extensively for quantifying PAH-DNA adducts. The fluorescence ELISA (limit of detection 1 adduct/10(8) nucleotides) was previously reported to be more sensitive than the color ELISA (1/10(7)) for measuring PAH adducts (Santella et al. (1988) Carcinogenesis, 9, 1265-1269). However, the fluorescence assay has the disadvantages of greater variation among the replicates and higher background levels than the color assay. Using a newly developed antiserum against BPDE-I-DNA, we have modified the color of ELISA so that it has the same sensitivity as the fluorescence ELISA and requires only 33% of the sample quantity needed for the fluorescence ELISA. The modifications included preincubation of the antiserum with the samples, using microtiter plates with half-size, flat bottom wells, and optimizing the assay conditions. The improved color ELISA was used to analyze DNA samples from human autopsy tissues, including heart, lung, liver, kidney, spleen, pancreas and stomach from smokers and nonsmokers. With the exception of spleen and stomach, all tissues from smokers showed higher PAH-DNA adducts (ranging from 0.3 to 19.0 adducts/10(7) nucleotides) than the tissues from the nonsmokers (0.3 to 3.7 adducts/10(7) nucleotides) in two separate experiments. Among the tissues from smokers, heart showed the highest level of DNA adducts. This study demonstrates that a stable color ELISA with high sensitivity can be useful in assessing human exposure to PAH. PMID- 8618548 TI - Dietary garlic extract in modifying clastogenic effects of inorganic arsenic in mice: two-generation studies. AB - Mice are fed by gavage crude garlic extract (100 mg/kg b.wt.) for 30 consecutive days. One set was administered sodium arsenite (0.1 mg/kg b.wt.) simultaneously. Another set was treated with sodium arsenite only. Mice given distilled water were kept as negative control. Exposed mice from each set were sacrificed and bone marrow preparations examined for chromosomal aberrations and damaged cells. Sodium arsenite is a strong clastogen and the effects were reduced to a significant level by prolonged administration of garlic extract. For F1 studies, exposed male mice were mated with exposed female mice, and the progeny examined. In the progeny, clastogenic effects of sodium arsenite persisted in a lower degree, indicating that the metal is able to cross the transplacental barrier. There was no statistically significant difference between the effect in progeny of parents only given sodium arsenite when given simultaneously for prolonged periods in the parents; however, the effect is meagre in the next generation. PMID- 8618550 TI - Palmitic acid is the major fatty acid responsible for significant anti-N-methyl N'-nitro-N-nitroguanidine (MNNG) activity in yogurt. AB - We describe here the isolation and identification of palmitic acid as being responsible for significant anti-N-methyl-N'-nitro-N-nitroguanidine (MNNG) activity in yogurt. The Ames test (Salmonella typhimurium TA100) was used to direct fractionation of activity. Yogurt was freeze-dried and extracted with acetone to yield a crude extract. The crude extract was purified by normal phase silica gel, Sephadex LH-20, and reversed phase medium pressure liquid chromatographies. The major compound in the active medium pressure liquid chromatographic fractions was determined to be palmitic acid on GC and high pressure liquid chromatography (HPLC) systems, and by nuclear magnetic resonance (NMR) analysis. Other saturated straight chain and methyl branched fatty acids were detected by GC/MS and were later shown to possess anti-MNNG activity. Of the straight chain fatty acids, palmitic acid had the highest anti-MNNG activity. All omega - 1 methyl branched fatty acids tested were more active than their straight chain counterparts. A trace amount of isopalmitic acid (14-methyl pentadecanoic acid), a minor milk lipid, was detected in one of the active fractions, and was later shown to be five times more active than palmitic acid. Isopalmitic acid also inhibited mutagenesis induced 4-nitroquinoline-N-oxide (4NQO), and 7, 12 dimethyl benz[a]anthracene (DMBA), and was found to inhibit the metabolic activation of DMBA. PMID- 8618552 TI - Comparison of standard autonomic tests and power spectral analysis in normal adults. AB - Standard autonomic measures [heart rate response to deep breathing (HR[DB]), systolic blood pressure response to orthostatic load, the 30:15 ratio, and the Valsalva ratio (VR)] and spectral measures of the heart rate (HR) and the arterial blood pressure (ABP) (MF: mid-frequency band at 0.05-0.15 Hz; HF: high frequency band at 0.15-0.33 Hz) were performed in 50 healthy subjects. The supine HR-HF and the tilt ABP-MF were taken as indicators of parasympathetic and sympathetic outflow, respectively. The transfer function magnitude of HR related to the ABP in the mid-frequency band estimated the baroreflex sensitivity. The HR[DB] and the 30:15 ratio were correlated with the parasympathetic spectral measure, and the VR was, surprisingly, only correlated with the sympathetic spectral measure. Significant baroreflex contribution was only evident for the 30:15 ratio. The spectral HR data were highly correlated with their corresponding spectral data of ABP. These results provide insights into autonomic regulation, but further studies on both basic physiological mechanisms of these methods and their clinical value have to be performed before a broad application can be recommended. PMID- 8618551 TI - Quadriceps strength and fatigue assessed by magnetic stimulation of the femoral nerve in man. AB - There is no nonvolitional method of assessing quadriceps strength which both supramaximally activates the muscle and is acceptable to subjects. In 10 normal subjects and 10 patients with suspected muscle weakness we used magnetic stimulation of the femoral nerve to elicit an isometric twitch and measured twitch tension (TwQ), surface electromyogram in addition to the maximum voluntary contraction force (MVC). Supramaximality was achieved in all subjects at a mean of 83% of maximum stimulator output. When supramaximal, TwQ was reproducible (mean coefficient of variation 3.6%, range 0.7-10.9) and correlated well with MVC (r2 = 0.83, P<0.001). In 7 normal subjects we measured TwQ before and after a fatiguing protocol; after 20 min TwQ was a mean of 55% (range 29-77%) of baseline and remained substantially reduced at 90 min. Magnetic femoral nerve stimulation is a painless, supramaximal method of assessing quadriceps strength and fatigue which is likely to be of value in clinical and physiological studies. PMID- 8618553 TI - Rank-ordered regulation of motor units. AB - Myoelectric signals were detected from the tibialis anterior muscle of 5 subjects with a quadrifilar needle electrode while the subjects generated isometric forces that increased linearly with time (10% of maximal voluntary contraction/s) up to maximal voluntary level. Motor unit firing rates were studied as a function of force throughout the full range of muscle force output. The relationship between force and firing rate was found to contain three distinct regions. At recruitment and near maximal force levels, firing rates increased more rapidly with force than in the intermediate region. Furthermore, in the regions with rapid increases, the rate of change of firing rate was correlated to the recruitment threshold, with higher recruitment threshold motor units displaying greater rates of change. In the intermediate region, all motor units had similar rates of change of firing rate. A weak positive correlation was found between initial firing rate and recruitment threshold. Firing rates of motor units at any instant were found to be ordered according to the recruitment order: at any given time in the contraction motor units with lower recruitment thresholds had higher firing rates than units with higher recruitment thresholds. Firing rates of all motor units were observed to converge to the same value at maximal forces. Mechanisms underlying motor unit recruitment and firing rate modulation are discussed in the context of a conceptual model. PMID- 8618554 TI - Heat-shock protein 90 and ubiquitin: developmental regulation during myogenesis. AB - Heat-shock/stress proteins are constitutive and stress-inducible proteins, regulated by a number of factors including developmental processes. The 90-kD heat-shock protein (hsp90) and ubiquitin are up-regulated in regenerating fibers and diseased fibers of Duchenne muscular dystrophy. The aim of the present study was to investigate whether the heat-shock response in regenerating fibers is developmentally regulated or disease-associated. Immunohistochemistry and immunoblot analysis were employed to compare the expression of hsp90 and ubiquitin in normal immature muscle from infants and regenerating fibers in polymyositis and dermatomyositis with the basal expression in normal mature muscle from adults. A significant up-regulation of hsp90 and ubiquitin in regenerating fibers and developing infantile fibers suggests that hsp90 and ubiquitin, during myogenesis, are largely regulated by the activation of developmental mechanisms rather than being primarily disease-related. Modulation of the stress response may promote myogenesis and provide a new therapeutic approach in myopathies. PMID- 8618555 TI - Abolition of sympathetic skin responses following endoscopic thoracic sympathectomy. AB - The recording of sympathetic skin responses (SSRs) is a simple, electrophysiological method to assess sympathetic nerve function. Within the last 10 years, SSRs have mainly been applied to delineate peripheral and central nervous system diseases, although the sympathetic nature of these responses was not fully documented, e.g., by a study of sympathectomy. We therefore recorded SSRs before and after 30 cases of endoscopic thoracic sympathectomy. The main indication was palmar hyperhidrosis, in which we found two types of SSR abnormalities. Most patients exhibited normal SSR waveforms but with increased amplitudes. The other patients exhibited abnormal SSRs which did not occur as single responses but as several consecutive waves. Thoracic sympathectomy always led to significant clinical improvement and to the abolition of ipsilateral palmar SSRs, demonstrating the sympathetic origin of these responses. We suggest that the assessment of sympathetic nerve activity by SSR recordings may be useful in sympathectomy. PMID- 8618556 TI - Broad A band disease: a new benign congenital myopathy. AB - We report a second child with broad A band disease. This child differs from the first in having normal vision and no electrophysiological evidence of a congenital retinal dystrophy. Neurological abnormalities at presentation included diffuse hypotonia, developmental delay, and delayed speech development. Histological and preliminary histochemical evaluation of biopsied thigh muscle showed no abnormality. However, 1-micrometer-thick plastic sections and electron microscopy showed numerous foci of broadened A bands accompanied by loss of distinct I bands. The Z lines in these areas were normal except for a fine waviness. Ultrastructurally, the thick filaments in these lesions appeared misaligned. Immunohistochemical reactions for desmin, vimentin, connectin (titin), and 2B myosin showed normal reactivity. An immunohistochemical reaction for fetal myosin showed sparse reacting fibers, which were unremarkable on adjacent sections stained with hematoxylin and eosin. These findings differ from those of other previously described congenital myopathies. Both of our patients have shown good strength and motor development by 5 years of age, suggesting that this ultrastructural abnormality is essentially benign. PMID- 8618557 TI - Improved methodology for lumbosacral nerve root stimulation. AB - The site of S1-S2 root activation following percutaneous high-voltage electrical (ES) and magnetic stimulation were located by analyzing the variations of the time interval from M to H soleus responses elicited by moving the stimulus point from lumbar to low thoracic levels. ES was effective in activating S1-S2 roots at their origin. However supramaximal motor root stimulation required a dorsoventral montage, the anode being a large, circular surface electrode placed ventrally, midline between the apex of the xiphoid process and the umbilicus. Responses to magnetic stimuli always resulted from the activation of a fraction of the fiber pool, sometimes limited to the low-thresholds afferent component, near its exit from the intervertebral foramina, or even more distally. Normal values for conduction velocity in motor and 1a afferent fibers in the proximal nerve tract are provided. PMID- 8618558 TI - Muscle fiber type correlates with innervation topography in the rat serratus anterior muscle. AB - Previous studies have reported that motoneurons from the sixth spinal nerve (C6) innervate the majority of muscle fibers in the rat serratus anterior (SA) muscle. The seventh spinal nerve (C7) innervates a limited number of SA fibers, increasing caudally. This topographic map is partially reestablished following denervation. In the present study, muscle fibers of the SA were stained with monoclonal antibodies for the muscle-specific fast myosin heavy chain (F-MHC) and slow myosin heavy chain (S-MHC) proteins. We found that the majority of fibers in the SA muscle stained for F-MHC antibody, and the percentage of muscle fibers staining for S-MHC antibody increased caudally. When newborn SA muscles were denervated and then reinnervated by the entire long thoracic (LT) nerve or only the C6 branch to the LT nerve, the reinnervated muscle had the normal proportion of muscle fibers expressing S-MHC protein. However, if the LT nerve was crushed and only C7 motoneurons allowed to reinnervate the SA muscle, a greater percentage of muscle fibers stained for S-MHC antibody than normal. We conclude that there is a correlation between muscle fiber type and innervation topography in the SA muscle of the rat. PMID- 8618559 TI - Transcortical and cervical magnetic stimulation with recording of the diaphragm. AB - Transcortical and cervical magnetic stimulation is a potential method of examining the central inspiratory pathway to phrenic motor neurons. The reliability and accuracy of this technique were studied. We performed magnetic stimulations of the cortex and cervical spinal cord with recording from both hemidiaphragms in 35 normal subjects using two different stimulation coils (90-mm circular coil and 70-mm figure-of-eight coil). Needle electrode recordings and ultrasound real-time documentation in 2 subjects excluded volume-conducted contaminations from adjacent chest wall and abdominal muscles. The effect of diaphragmatic facilitation (stimulation at the end of a deep breath) on latency, and amplitude were compared to the effect of hypothenar muscle facilitation. Normal ranges were established for: latency; central motor conduction time; amplitude; amplitude ratio between between peripheral and both cortical and cervical amplitude; and excitability threshold. The latencies were similar for both coils. The amplitudes were significantly higher, and excitability thresholds significantly lower for the 90-mm circular coil, indicating that this coil is preferable for transcortical diaphragmatic stimulations. The effect of facilitation was greater for hypothenar than diaphragmatic recordings. There was excellent right-left agreement for all measurements. Transcortical and cervical magnetic stimulation with recording from the diaphragm can be used routinely to diagnose and monitor patients with impaired central respiratory drive. PMID- 8618560 TI - Reduced oxidative muscle metabolism in chronic fatigue syndrome. AB - The purpose of this study was to determine if chronic fatigue syndrome (CSF) is characterized by abnormalities in oxidative muscle metabolism. Patients with CFS according to Centers for Disease Control (CDC) criteria (n = 22) were compared to normal sedentary subjects (n = 15). CFS patients were also tested before and 2 days after a maximal treadmill test. Muscle oxidative capacity was measured as the maximal rate of postexercise phosphocreatine (PCr) resynthesis using the ADP model (Vmax) in the calf muscles using 31P magnetic resonance spectroscopy. Vmax was significantly reduced in CFS patients (39.6 +/- 2.8 mmol/L/min, mean +/- SE) compared to controls (53.8 +/- 2.8 mmol/L/min). Two days postexercise there was no change in resting inorganic phosphate (Pi)/PCr or Vmax in the CFS patients (n = 14). In conclusion, oxidative metabolism is reduced in CFS patients compared to sedentary controls. In addition, a single bout of strenuous exercise did not cause a further reduction in oxidative metabolism, or alter resting Pi/PCr ratios. PMID- 8618561 TI - Motor unit estimates obtained using the new "MUESA" method. AB - Motor unit number estimates were obtained from the extensor digitorum brevis and thenar muscles using a new method called MUESA. MUESA is distinguished from other estimation methods in the manner in which it deals with probabilistic motor unit activation, which is more commonly referred to as "alternation." Because of "alternation," incremental increases in the observed muscle potentials often cannot be interpreted in terms of the successive activation of single motor units. In the MUESA method, the nerve is subjected to a number of constant intensity stimulus trains, and the resultant muscle response sequences are decomposed into their constituent motor unit action potentials. In general, if a stimulus train results in the probabilistic activation of n motor units, we can expect to see up to 2n different potentials, with each potential representing a unique combination of active and/or inactive motor units. If all 2n potentials are indeed observed, the decomposition of the observed potential sequence into its constituent motor unit action potentials is very straightforward. For the majority of the cases in which the number of observed potentials is not an integer power of 2, we have developed a novel decomposition method based on the analysis of the relative firing rates of the motor units. PMID- 8618562 TI - Anti-MAG and anti-SGPG antibodies in neuropathy. AB - We compared the binding of human antibodies from patients with neuropathy to the myelin-associated glycoprotein (MAG), to its cross-reactive glycolipid sulfoglucuronyl paragloboside (SGPG), and to sections of peripheral nerve. Titers were correlated with the clinical presentation and results of electrophysiological and pathological studies. Most patients had a predominantly sensory or sensorimotor demyelinating neuropathy and highly elevated antibodies to both MAG and SGPG, but 2 had highly elevated antibodies to MAG alone, and 1 to SGPG alone. Two patients had predominantly motor neuropathy and highly elevated antibodies to SGPG which reacted with MAG by Western blot but not by enzyme linked immunosorbent assay. One patient had amyotrophic lateral sclerosis and antibodies to SGPG but not to MAG. These studies indicate that the neuropathic syndrome associated with anti-MAG or -SGPG antibodies are more heterogeneous than previously suspected, and that although most of the antibodies react with both MAG and SGPG, some may react with MAG or SGPG alone. PMID- 8618563 TI - Focal posterior interosseous neuropathy in the presence of hereditary motor and sensory neuropathy, type I. AB - A 30-year-old male with hereditary motor and sensory neuropathy, type I (HMSN I), presented with asymmetric weakness of finger extension and radial deviation with left wrist extension, previously felt to be a manifestation of the peripheral neuropathy. Nerve conduction studies confirmed HMSN I; however, needle EMG revealed marked, ongoing axonal loss in muscles innervated by the left posterior interosseous nerve (PIN) only. At surgery there was focal fusiform swelling in the PIN at exit from the supinator muscle, compatible with localized hypertrophic neuropathy, which has not been reported before in HMSN I. A concomitant focal mononeuropathy should be considered in cases of hereditary neuropathy with marked asymmetry of weakness. PMID- 8618565 TI - Motor control of the diaphragm in multiple sclerosis. PMID- 8618564 TI - Spastic paraparesis and sensory neuropathy. AB - A 12-year-old developed a slowly progressive spastic gait at the age of 3. A marked loss of pain and temperature sensations led to a mutilating acropathy starting at age 5. Electrodiagnostic studies revealed a symmetric, axonal, predominantly sensory neuropathy, and magnetic resonance imaging ruled out compression of spinal cord. Sural nerve biopsy disclosed a predominant involvement of unmyelinated and a global loss of myelinated fibers, particularly larger ones. Clinical, electrodiagnostic and pathological findings of this case most likely represent an example of the "Cavanagh's variant", an unusual but distinct entity within the hereditary sensory and autonomic neuropathies. PMID- 8618566 TI - The effect of wrist position on the conduction velocity of the ulnar nerve. PMID- 8618567 TI - Spinal cord potentials after transcranial magnetic stimulation during muscle contraction. PMID- 8618568 TI - The cervical radiculopathy screen: optimizing the number of muscles studied. PMID- 8618569 TI - Multifocal motor neuropathy: a source of error in the serial evaluation of conduction block. PMID- 8618570 TI - Restless legs syndrome in patients with polyneuropathy. PMID- 8618571 TI - Lumbrical-interosseous comparison in a distal ulnar nerve lesion. PMID- 8618572 TI - Peripheral myelin protein-22 gene deletion in two unrelated Japanese pedigrees with hereditary neuropathy with liability to pressure palsies. PMID- 8618573 TI - Neuromuscular transmission and acetylcholine receptor antibodies in penicillamine treated Wilson's disease patients. PMID- 8618575 TI - Somatic and autonomic nerve studies in Chagas' disease. PMID- 8618574 TI - HLA antigens typification in two Hispanic patients with thyrotoxic periodic paralysis. PMID- 8618576 TI - Recurrent ulnar neuropathy following burn injury at elbow. PMID- 8618577 TI - Double-peaked potential in the neurophysiological evaluation of carpal tunnel syndrome. PMID- 8618578 TI - Enhancement of "end-plate monophasic waves" during an attack of hypokalemic periodic paralysis. PMID- 8618579 TI - Aerosolized surfactant in adults with sepsis-induced acute respiratory distress syndrome. Exosurf Acute Respiratory Distress Syndrome Sepsis Study Group. AB - BACKGROUND: Patients with acute respiratory distress syndrome (ARDS) have a deficiency of surfactant. Surfactant replacement improves physiologic function in such patients, and preliminary data suggest that it may improve survival. METHODS: We conducted a prospective, multicenter, double-blind, randomized, placebo-controlled trial involving 725 patients with sepsis-induced ARDS. Patients were stratified according to the risk of death at base line (indicated by their score on the Acute Physiological and Chronic Health Evaluation [APACHE III] index) and randomly assigned to receive either continuously administered synthetic surfactant (13.5 mg of dipalmitoylphosphatidylcholine per milliliter, 364 patients) or placebo (o.45 percent saline; 361 patients) in aerosolized form for up to five days. RESULTS: The demographic and physiologic characteristics of the two treatment groups were similar at base line. The mean (+/- SD) age was 50 +/- 17 years in the surfactant group and 53 +/- 18 years in the placebo group, and the mean APACHE III scores at randomization were 70.4 +/- 25 and 70.5 +/- 25, respectively. Hemodynamic measures, measures of oxygenation, duration of mechanical ventilation, and length of stay in intensive care unit did not differ significantly in the two groups. Survival at 30 days was 60 percent for both groups. Survival was similar in the groups when analyzed according to APACHE III score, cause of death, time of onset and severity of ARDS, presence or absence of documented sepsis, underlying disease, whether or not there was a do-not resuscitate order, and medical center. Increased secretions were significantly more frequent in the surfactant group; the rates of other complications were similar in the two groups. CONCLUSIONS: The continuous administration of aerosolized synthetic surfactant to patients with sepsis-induced ARDS had no significant effect on 30-day survival, length of stay in the intensive care unit, duration of mechanical ventilation, or physiologic function. PMID- 8618580 TI - Long-term follow-up of HBeAg-positive patients treated with interferon alfa for chronic hepatitis B. AB - BACKGROUND: In patients with chronic hepatitis B, treatment with interferon alfa and the consequent loss of hepatitis B e antigen (HBeAg) from the blood leads to a reduction in inflammatory activity, but the clinical benefits of this treatment have not been established. We evaluated whether HBeAg seroconversion induced by interferon alfa improves clinical outcome. METHODS: We studied prospectively a cohort of 103 patients treated with interferon alfa for chronic hepatitis B; the mean (+/- SD) follow-up was 50.0 +/- 19.8 months. Fifty-three untreated patients served as controls. RESULTS: After treatment with interferon alfa, 53 of 103 patients no longer had detectable HBeAg or hepatitis B virus DNA, although only 10 patients became seronegative for hepatitis B surface antigen (HBsAg) (Kaplan Meier estimates of cumulative clearance rates at five years, 56.0 percent for HBeAg and 11.6 percent for HBsAg). Of the 53 untreated patients, only 7 spontaneously eliminated HBeAg (28.1 percent at five years), and all remained positive for HBsAg (p < 0.001 for the Comparison with the treated patients, by the proportional-hazards model). During follow-up, 6 of the 103 treated patients died of liver failure, and 2 needed liver transplantation, all 8 were persistently positive for HBeAg. In another eight treated patients, complications of cirrhosis developed; all but one of these patients remained positive for HBeAg. Overall survival and survival without clinical complications were significantly longer in patients who were seronegative for HBeAg after therapy with interferon alfa than in those who remained seropositive (P = 0.004 and P = 0.018, respectively). In a regression analysis, clearance of HBeAg was the strongest predictor of survival. Of the 53 untreated patients, 13 had severe complications (including 4 deaths and 1 need for liver transplantation); all 13 continued to be HBeAg-positive. CONCLUSIONS: In patients with chronic hepatitis B infection, the clearance of HBeAg after treatment with interferon alfa is associated with improved clinical outcomes. PMID- 8618581 TI - Autologous bone marrow transplantation versus intensive consolidation chemotherapy for acute myeloid leukemia in childhood. Pediatric Oncology Group. AB - BACKGROUND: The value of autologous bone marrow transplantation in the treatment of children with acute myeloid leukemia (AML) is unknown. We compared autologous bone marrow transplantation with intensive consolidation chemotherapy as treatments for children with AML in first remission. METHODS: We induced remission with one course of daunorubicin, cytarabine, and thioguanine, followed by one course of high-dose cytarabine (3 g per square meter of body-surface area for six doses). Patients in remission after the second course of induction therapy were eligible for randomization. Between June 1988 and March 1993, 552 of 649 enrolled patients who could be evaluated (85 percent) entered remission. A total of 209 patients were not eligible for randomization; of the remaining 343 patients, 232 were randomly assigned to receive six courses of intensive chemotherapy (117 patients) or autologous transplantation (115 patients). Of the original 649 patients, 189, including 21 with Down's syndrome, were nonrandomly assigned to receive intensive chemotherapy. RESULTS: The rates of event-free survival and overall survival for the entire group at three years were 34 +/- 2.5 percent and 42 +/- 2.6 percent, respectively. For patients who were randomly assigned to one of the two treatment groups, the mean (+/- SE) rates of event free survival three years after randomization were not significantly different in the two groups when examined by intention-to-treat analysis: 36 +/- 5.8 percent for the intensive-chemotherapy group as compared with 38 +/- 6.4 percent for the autologous-transplantation group; and the relative risk of treatment failure for the chemotherapy group as compared with the autologous-transplantation group was 0.81 (P = 0.20 by the log rank test; 95 percent confidence interval, 0.58 to 1.12). Overall survival at three years followed a similar pattern. There was a lower relapse rate (31 percent vs. 58 percent, P < 0.001) but a higher rate of treatment-related mortality (15 percent vs. 2.7 percent, P = 0.005) in the group treated with autologous transplantation than in the intensive-chemotherapy group. The event-free survival at three years for the nonrandomized intensive chemotherapy group was 39 +/- 5.1 percent, and for a contemporaneous group of patients each of whom received a histocompatible bone marrow transplant from a sibling, it was 52 +/- 8.0 percent. CONCLUSIONS: Treatment of children with AML in first remission with either autologous bone marrow transplantation or intensive chemotherapy prolongs event-free survival equally. PMID- 8618582 TI - Famotidine for the prevention of gastric and duodenal ulcers caused by nonsteroidal antiinflammatory drugs. AB - BACKGROUND: Acid suppression with famotidine, a histamine H2-receptor antagonist, provides protection against gastric injury in normal subjects receiving short courses of aspirin or naproxen. The efficacy of famotidine in preventing peptic ulcers in patients receiving long-term therapy with nonsteroidal antiinflammatory drugs (NSAIDs) is not known. METHODS: We studied the efficacy of two doses of famotidine (20 mg and 40 mg, each given orally twice daily), as compared with placebo, in preventing peptic ulcers in 285 patients without peptic ulcers who were receiving long-term NSAID therapy for rheumatoid arthritis (82 percent) or osteoarthritis (18 percent). The patients were evaluated clinically and by endoscopy at base line and after 4, 12, and 24 weeks of treatment. The evaluators were unaware of the treatment assignment. The primary end point was the cumulative incidence of gastric or duodenal ulceration at 24 weeks. RESULTS: The cumulative incidence of gastric ulcers was 20 percent in the placebo group, 13 percent in the group of patients receiving 20 mg of famotidine twice daily (P = 0.24 for the comparison with placebo), and 8 percent in the group receiving 40 mg of famotidine twice daily (P = 0.03 for the comparison with placebo). The proportion of patients in whom duodenal ulcers developed was significantly lower with both doses of famotidine than with placebo (13 percent in the placebo group, 4 percent in the low-dose famotidine group [P = 0.04], and 2 percent in the high dose famotidine group [P = 0.01]). Both doses of famotidine were well tolerated. CONCLUSIONS: Treatment with high-dose famotidine significantly reduces the cumulative incidence of both gastric and duodenal ulcers in patients with arthritis receiving long-term NSAID therapy. PMID- 8618583 TI - Images in clinical medicine. Dysmorphic urinary erythrocytes. PMID- 8618584 TI - Does increased access to primary care reduce hospital readmissions? Veterans Affairs Cooperative Study Group on Primary Care and Hospital Readmission. AB - BACKGROUND: For chronically ill patients, readmission to the hospital can be frequent and costly. We studied the effect of an intervention designed to increase access to primary care after discharge from the hospital, with the goals of reducing readmissions and emergency department visits and increasing patients' quality of life and satisfaction with care. METHODS: In a multicenter randomized, controlled trial at nine Veterans Affairs Medical Centers, we randomly assigned 1396 veterans hospitalized with diabetes, chronic obstructive pulmonary disease, or congestive heart failure to receive either usual care or an intensive primary care intervention. The intervention involved close follow-up by a nurse and a primary care physician, beginning before discharge and continuing for the next six months. RESULTS: The patients were severely ill. Half of those with congestive heart failure (504 patients) had disease in New York Heart Association class III or IV; 30 percent of those with diabetes (751 patients) had end-organ damage; and a quarter of those with chronic obstructive pulmonary disease (583 patients) required home oxygen treatment or oral corticosteroids. The patients had extremely poor quality-of-life scores. Although they received more intensive primary care than the controls, the patients in the intervention group had significantly higher rates of readmission (0.19 vs 0.14 per month, P = 0.005) and more days of rehospitalization (10.2 vs 8.8, P = 0.041). The patients in the intervention group were more satisfied with their care (P < 0.001), but there was no difference between the study groups in quality-of-life scores, which remained very low (P = 0.53). CONCLUSIONS: For veterans discharged from Veterans Affairs hospitals, the primary care intervention we studied increased rather than decreased the rate of rehospitalization, although patients in the intervention group were more satisfied with their care. PMID- 8618585 TI - Acute renal failure. PMID- 8618586 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 17-1996. A 48-year-old man with the acquired immunodeficiency syndrome, abdominal pain, and bloody diarrhea. PMID- 8618587 TI - The acute respiratory distress syndrome. PMID- 8618588 TI - Chronic viral hepatitis--benefits of current therapies. PMID- 8618589 TI - Questions about the value of early intervention. PMID- 8618590 TI - Clinical problem-solving: a broken heart. PMID- 8618591 TI - Clinical problem-solving: a broken heart. PMID- 8618592 TI - Clinical problem-solving: a broken heart. PMID- 8618593 TI - Clinical problem-solving: a broken heart. PMID- 8618594 TI - Cardiac pacing. PMID- 8618595 TI - Intravenous epoprostenol for primary pulmonary hypertension. PMID- 8618596 TI - Intravenous epoprostenol for primary pulmonary hypertension. PMID- 8618597 TI - Streptococcal skin infections. PMID- 8618598 TI - Hepatocellular carcinoma (case 2-1996) PMID- 8618599 TI - Hepatocellular carcinoma (case 2-1996) PMID- 8618600 TI - Lack of sustained efficacy of interferon in patients with chronic hepatitis C. PMID- 8618601 TI - More on headphone neuralgia. PMID- 8618602 TI - Infection with hepatitis GB virus C in patients on maintenance hemodialysis. AB - BACKGROUND: A recently discovered non-A-E hepatitis virus has been designated hepatitis GB virus C (HGBV-C), but little is known about its mode of transmission and its clinical manifestations. We studied 519 patients on maintenance hemodialysis to determine whether they were infected with HGBV-C. METHODS: HGBV-C RNA was identified in serum by a reverse-transcription-polymerase-chain-reaction assay with nested primers deduced from a non-structural region. A nucleotide sequence of 100 bp in the nonstructural region was determined on HGBV-C clones. RESULTS: HGBV-C RNA was detected on 3.1 percent of the patients on hemodialysis (16 of 519), as compared with 0.9 percent of healthy blood donors (4 of 448, P<0.03). None of the 16 patients had evidence of active liver disease, although 7 were also infected with hepatitis C virus. Eight patients with HGBV-C infection were followed for 7 to 16 years. In two patients the virus was present at the start of hemodialysis. One had a history of transfusion, and HGBV-C persisted over a period of 16 years; the other became free of HGBV-C after 10 years. In five patients, HGBV-C RNA was first detected 3 to 20 weeks after blood transfusion and persisted for up to 13 years. One patient with no history of transfusion was infected with an HGBV-C variant with the same sequence as in two of the patients with post-transfusion HGBV-C infections. CONCLUSIONS: Patients on maintenance hemodialysis are at increased risk for HGBV-C infection. This virus produces persistent infections, which may be transmitted by transfusions but may also be transmitted by other means. PMID- 8618603 TI - Oral ganciclovir for the prevention of cytomegalovirus disease in persons with AIDS. Roche Cooperative Oral Ganciclovir Study Group. AB - BACKGROUND: In the advanced stages of the acquired immunodeficiency syndrome (AIDS), cytomegalovirus (CMV) disease, particularly vision-damaging retinitis due to CMV is common. We evaluated prophylactic treatment with orally administered ganciclovir as a way to prevent CMV disease. METHODS: We conducted a prospective, randomized, double-blind, placebo-controlled study of CMV infected persons with AIDS with either CD4+ lymphocyte counts of < or = 50 per cubic millimeter or counts of < or = 100 per cubic millimeter in those with a history of an AIDS defining opportunistic infection. Patients were randomly assigned, in a 2:1 ratio, to receive either oral ganciclovir (1000 mg three times daily) or placebo. RESULTS: The study was stopped after a median 367 days of follow-up. In an intention-to-treat analysis, the twelve month cumulative rates of confirmed CMV disease were 26 percent in the placebo group (n = 239) and 14 percent in the ganciclovir group (n = 486), representing an overall reduction in risk of 49 percent in the ganciclovir group (P < 0.001). The incidence of CMV retinitis after 12 months was 24 percent in the placebo group and 12 percent in the ganciclovir group (P < 0.0001). The prevalence of CMV-positive urine cultures at base line was 42 percent; after two months it was 43 percent in the placebo group and 10 percent in the ganciclovir group (P < 0.0001). The one year mortality rate was 26 percent in the placebo group and 21 percent in the ganciclovir group (P = 0.14). Therapy with granulocyte colony stimulating factor was more frequent in the ganciclovir group (24 percent) than in the placebo group (9 percent). CONCLUSIONS: In persons with advanced AIDS, phophylactic oral ganciclovir significantly reduces the risk of CMV disease. PMID- 8618604 TI - Prediction of the need for intensive care in patients who come to emergency departments with acute chest pain. AB - BACKGROUND: Patients who come to the emergency department with chest pain are a heterogeneous group. Some have ischemic heart disease that may lead to serious complications, whereas others have minor disorders. We performed a study to identify clinical factors that predict which patients will have complications requiring intensive care. METHODS: We first studied 10,682 patients with acute chest pain at seven hospitals between 1984 and 1986 (derivation set) to identify potential clinical predictors of the development of major complications. We then validated these predictors in a separate set of 4676 patients at one hospital between 1990 and 1994 (validation set). RESULTS: In the derivation set of patients, we identified the following set of clinical features, which, if present in the emergency department, were associated with an increased risk of complications: ST-segment elevation or Q waves on the electrocardiogram thought to indicate acute myocardial infarction, other electrocardiographic changes indicating myocardial ischemia, low systolic blood pressure, pulmonary rales above the bases, or an exacerbation of known ischemic heart disease. On the basis of these criteria, the patients in the validation set were stratified into four groups, with the risk of major complications in the first 12 hours ranging from 0.15 to 8 percent. After 12 hours, the probability of a major complication could be updated on the basis of whether the patient had already had a complication of major severity, a complication of intermediate severity, or a myocardial infarction (independent relative risks, 18.9, 7.7 and 4.0, respectively, as compared with patients without prior complications or myocardial infarction). CONCLUSIONS: The risk of major complications in patients with acute chest pain can be estimated on the basis of the clinical presentation and new clinical observations made during the hospital course. These estimates of risk help in making rational decisions about the appropriate level of medical care for patients with acute chest pain. PMID- 8618605 TI - Role of ploidy, chromosome 1p, and Schwann cells in the maturation of neuroblastoma. AB - BACKGROUND: Neuroblastoma is a heterogeneous disease, with manifestations ranging from spontaneous regression to lethal spread. Sometimes the tumor spontaneously differentiates toward a benign ganglioneuroma (maturing neuroblastoma). The prognosis is frequently related to ploidy, deletions in the short arm of chromosome 1, and amplifications of the N-myc oncogene. Maturing neuroblastomas consist of both neuronal cells and Schwann cells. We investigated the genetic composition of both cell types in maturing neuroblastomas, to determine the relation between genetic abnormalities and maturation. METHODS: We studied 20 maturing and mature neuroblastomas by in situ hybridization to count the chromosomes and evaluate possible deletions in the short arm of chromosome 1 in neuronal and Schwann cells. The DNA content of the cells was measured by flow cytometry. RESULTS: Neuroblastic and ganglionic cells showed aberrations in the number of chromosomes. In situ hybridization and flow cytometry demonstrated near trip-loidy in 18 of 19 tumors and pentaploidy in the remaining tumor. The Schwann cells in all 20 neuroblastomas contained normal numbers of chromosomes. In 18 tumors studied, there were no chromosome 1 deletions in either type of cell. CONCLUSIONS: The Schwann cells in maturing neuroblastomas differ genetically from the neuronal cells. The normal number of chromosomes in Schwann cells and the abnormal number in neuroblastic ganglionic cells suggests that Schwann cells are a reactive population of normal cells that invade the neuroblastoma. Near-trip loidy of neuroblastoma cells and intact chromosome 1 are presumably genetic prerequisites for spontaneous organoid maturation, because we found no diploidy or chromosome 1 depletions in the neuronal cells of spontaneously maturing neuroblastomas. PMID- 8618606 TI - Images in clinical medicine. Hypopyon. PMID- 8618607 TI - Shattuck Lecture--evaluating the health risks of breast implants: the interplay of medical science, the law, and public opinion. PMID- 8618608 TI - Hip fracture. PMID- 8618609 TI - Adhesion molecules--Part 1. PMID- 8618611 TI - The cloning and clinical implications of HGV and HGBV-C. PMID- 8618610 TI - Clinical problem-solving. A stressful interaction. PMID- 8618612 TI - Schwann cells as antineuroblastoma agents. PMID- 8618614 TI - Management of gout. PMID- 8618613 TI - AIDS prevention--sexual ethics and responsibility. PMID- 8618615 TI - Management of gout. PMID- 8618616 TI - Serum leptin in normal-weight and obese humans. PMID- 8618617 TI - Myocardial ischemia detected by ambulatory monitoring after myocardial infarction. PMID- 8618618 TI - Myocardial ischemia detected by ambulatory monitoring after myocardial infarction. PMID- 8618619 TI - Trandolapril in patients with left ventricular dysfunction after myocardial infarction. PMID- 8618620 TI - Acellular pertussis vaccines. PMID- 8618621 TI - Acellular pertussis vaccines. PMID- 8618622 TI - Case 4-1996: paralysis due to schistosomiasis. PMID- 8618623 TI - Case 4-1996: paralysis due to schistosomiasis. PMID- 8618624 TI - Hepatitis GB virus C in patients on hemodialysis. PMID- 8618625 TI - [Listeria meningitis in adults]. PMID- 8618626 TI - [Emerging pathogens]. PMID- 8618627 TI - [Hepatitis C, awakening of a giant]. AB - Cloning of the hepatitis C virus (HCV) was reported in 1989. By now, the entire viral genome has been sequence. It consists of a single-stranded positive RNA, with relationship to the Flaviviridae. The envelope region shows considerable variability. 6 major genotypes have been described. HCV is transmitted via the parenteral route, mainly blood, rarely by sexual contact. Hepatitis C occurs worldwide and is found in 0.01 to 1.5% of blood donors. The immune response is unable to clear the virus in 80% of infected subjects, probably because of the hypervariability. In the acute phase the hepatitis has only mild symptoms and the chronic hepatitis usually also runs a mild course. After many years liver cirrhosis may develop in 20% of cases; in these subjects there is a high incidence of hepatocellular carcinoma. The diagnosis can be made by detection of anti-HCV antibodies in the blood and an immunoblot confirmation test. The viral genome can be detected by the HCV-RNA (PCR) test. Immunisation against hepatitis C is not possible yet. Therapy with interferon results in an initial response in 45% and a sustained response in < 20% of the patients. Interferon therapy reduces the incidence of hepatocellular carcinoma. PMID- 8618628 TI - [Human herpes viruses type 6 and 7; causative agents of, among others, exanthema subitum]. AB - A serendipitous discovery during early AIDS investigations was human herpes virus type 6 (HHV-6). Two years later (1988) it was shown that HHV-6 and later on also HHV-7 are the causes of exanthema subitum, a childhood disease with previously unknown causation. HHV-6 and HHV-7 are the main cause of febrile seizures. It is assumed that 90% of children are infected before they are three years old. The viruses are also found in adults; HHV-6 may cause mononucleosis and hepatitis. HHV-6 and HHV-7 infect CD4+ cells and may influence the course of HIV infection. In AIDS patients HHV-6 and cytomegalovirus are often isolated together from the lungs, possibly because they activate each other. Another possibility is that the circumstances in the lungs are favourable to both. HHV-6 and HHV-7 infection may be serologically diagnosed. There is little experience with antiviral treatment. PMID- 8618629 TI - [Yersinia infections]. AB - The genus Yersinia contains three pathogenic species: Y. pestis, Y. enterocolitica and Y. pseudotuberculosis. All pathogenic strains contain a 70 kb plasmid coding for a number of virulence factors, of which outer membrane proteins including an adhesin are the most important. In 65% of the patients the infection is self-limiting, but in the others a chronic local inflammation develops. Persistence of the infection has been associated with the immune status and the HLA-B27 antigen. Y. enterocolitica leads to abdominal complaints in young children that usually subside spontaneously. The infection is more serious with increasing age, and in people older than 30 years may involve a septicaemic form with multiple abscesses in various organs, or a lymphadenopathic form with generalized lymphadenitis mimicking a haematologic malignancy. Yersinia can be easily isolated during the acute phase by culture; during the chronic and persistent phase it is not cultivable anymore. Then serology should be performed, anti-Yop serology being the most reliable. Yersinia is susceptible to most antibiotics, except penicillins. Local penetration of the antibiotic may be a problem, however. First-choice antibiotics are cotrimoxazole, tetracyclines, chloramphenicol and fluoroquinolones. Systemic extra-mesenteric infections should always be treated with antibiotics. PMID- 8618630 TI - [Enterococci; increase in infection rate and antibiotic resistance]. AB - Enterococci (mainly Enterococcus faecalis and E. faecium) are increasingly isolated as causative agents of infections, notably in hospitalised patients. Most infections are urinary tract infections, septicaemia and endocarditis. Dutch figures indicate that 10.8% of all bacteria isolated in seven regional microbiological laboratories were enterococci; the samples came not only from hospital infections. Of the isolates 34% were from faecal samples, 14% from urinary tract samples and 14% from genital organ samples. Antibiotic resistance is an increasing problem. Enterococci are moderately resistant to the antibiotics commonly used to treat Gram positive infections. The infections may be treated with (high dose) penicillin and (or) in combination with an aminoglycoside or with a glycopeptide. Unrestricted use of antibiotics increases the risk of resistance. Recently enterococci have been isolated which are resistant to ampicillin, gentamycin and vancomycin: such infections are virtually untreatable. PMID- 8618632 TI - [Methicillin-resistant Staphylococcus epidermidis]. AB - Staphylococcus epidermidis has long been overlooked as a pathogen. Modern medicine gives this bacterium ample opportunities to display its pathogenic properties, notably as a colonizer of medical devices (catheters, implants), on which it grows, protected by an exopolysaccharide ('slime') layer. In addition, more and more methicillin-resistant strains are isolated. In Dutch intensive care units S. epidermidis is responsible for 20% of all infections, approximately one third of these isolates are methicillin resistant. As most people have S. epidermidis on their skin, spread and cross infection are unavoidable; still a judicious antibiotic use help to keep the frequency of resistance as low as possible. PMID- 8618631 TI - [Infections with verocytotoxin-producing Escherichia coli and hemolytic-uremic syndrome]. AB - Infections with verocytotoxin-producing Escherichia coli and (VTEC), and especially with serotype O157, are the main cause of haemolytic-uraemic syndrome (HUS) in children in the Netherlands. 8% of the patients infected develop HUS; the incidence is 2/105/year in children under 5 years. The infection may be asymptomatic, but may also lead to mild to haemorrhagic diarrhoea and to haemorrhagic colitis. Up to 10% of the patients die in the acute phase of the disease and in up to another 10% the renal damage does not resolve completely. In 1993 bovine meat samples examined by polymerase chain reaction revealed VTEC in 16% of the cases; however none of the isolated serotypes was known to be pathogenic in humans. Epidemiological investigations are being carried out in the cattle population. Verocytotoxins are exotoxins that bind to surface receptors on cells after which part of the toxin is internalized where it inhibits protein synthesis. The functional receptor is glycosphingolipid globotriaosylceramide, a molecule normally only expressed in the renal glomeruli of children under three years of age. PMID- 8618633 TI - [Problems of resistance in Streptococcus pneumoniae]. AB - Penicillin-resistant pneumococci are an increasing worldwide problem that until now has spared the Netherlands and a small number of other European countries. In countries with resistant pneumococci empirical therapy of otitis media and of meningitis is difficult. Special antibiotics such as clindamycin and ceftriaxone may be needed or combinations of vancomycin and high doses of cephalosporin. In spite of antibiotic resistance pneumococcal pneumonia can be treated successfully with high doses of benzylpenicillin or cephalosporin. In case of an infection outside the central nervous system, it is not clear what level of minimal inhibitory concentration indicates that therapy will fail, probably > 4 mg/l. Even in regions with much resistance such levels are rare. It is fortunate that antibiotic resistance only occurs in a limited number of pneumococcal strains, which in addition happen to be included in antipneumococcal vaccines. PMID- 8618634 TI - [Cat-scratch disease and other infections caused by Bartonella species]. AB - Bartonella henselae, the causative agent of cat-scratch disease, was identified recently by DNA amplification techniques. Several other Bartonellae (most of which were called Rochalimaea before) cause disease in humans: B. bacilliformis (Carrion's disease), B. elizabethae (endocarditis) and B. quintana (bacillary angiomatosis and peliosis, chronic bacteraemia and endocarditis, trench fever). B. henselae is transmitted to humans by scratch or bite of a bacteraemic, but asymptomatic, cat, which event may be followed by regional lymphadenitis (classical cat-scratch disease), bacillary angiomatosis or peliosis of liver and spleen (in immune compromised, e.g. HIV-infected individuals) or chronic bacteraemia and endocarditis (in elderly individuals). The incidence in the Netherlands of cat-scratch disease is > 2/100,000/year. If a Bartonella infection is suspected, specific immuno-assays and polymerase chain reaction assay may be applied for diagnosis. Culture of the organism is difficult. Macrolides and tetracyclines have been shown to be effective in treatment of disseminated infections. The natural (self-limiting) course of regional lymphadenitis however is not affected by antibiotic treatment. PMID- 8618635 TI - [The changing pattern of Candida infections: different species and increased resistance]. AB - The incidence of invasive infections with Candida has strongly increased during the last few decades. Candida is now one of the commonest hospital pathogens. Many infections are nowadays caused by other species than C. albicans, notably C. tropicalis, C. krusei and C. glabrata. Particularly in HIV seropositive patients the yeast may develop resistance to fungistatic drugs. The altered species frequency and the resistance appear to be caused by the use of fungistatic agents of the imidazole group. To counter these developments the choice of antifungal therapeutics should be made very carefully. PMID- 8618636 TI - [Malaria and drug resistance]. AB - Drug resistance is a major problem in malaria. The resistance mechanism remains unresolved but contributing factors are probably heavy drug use, parasite selection, cross resistance and genetic influences of drugs. Plasmodium ovale en P. malariae are sensitive to the current antimalarial drugs. P. vivax has some chloroquine resistant strains, notably on Papua New Guinea, Irian Jaya and other islands in the Pacific. The geographical distribution of P. falciparum strains resistant to proguanil and pyrimethamine is not well known. Chloroquine-resistant strains are found in South East Asia, the Amazon region (almost 100% resistance in both regions) and in Africa south of the Sahara (resistance not everywhere 100%). Sulfadoxine-pyrimethamine is not an effective treatment in South East Asia and the Amazon region; it is useful in tropical Africa. Mefloquine resistance is a problem mainly confined to Thailand. There is cross resistance between halofantrine and mefloquine. Decreased sensitivity to quinine was reported from Thailand, but it remains an effective drug, notably when given in combination with tetracycline or doxycycline. In cases of severe or complicated malaria intravenous quinine is still the preferred therapy. Resistance to artemisinine has not yet been reported. Pharmaceutical companies show little interest in antimalarial drug development, which in view of the increasing drug resistance is a matter of great concern. PMID- 8618637 TI - [3 emerging protozoal infections in The Netherlands: Cyclospora, Dientamoeba, and Microspora infections]. AB - Increased international travelling, an increased number of patients with immunosuppression caused by HIV infection, and renewed interest in known but little studied microorganisms, resulted in a more frequent finding of certain medically important parasites. Three emerging protozoal infection, Cyclospora cayetanensis, Dientamoeba fragilis and Microspora (Enterocytozoon bieneusi and Encephalitizoon) are causative agents of diarrhoea. Encephalitozoon infections are also associated with hepatitis, hepatoconjunctivitis and nephritis. C. cayetanensis infection was diagnosed in 28 patients in the years 1992-1995 in the Academic Medical Centre in Amsterdam, half of these patients returning from a visit to Indonesia. D. fragilis has a prevalence in the Netherlands of 8% among patients with diarrhoea lasting longer than one week referred for parasitological investigation. The prevalence of E. bieneusi in HIV positive patients with diarrhoea was 8-10% in the Academic Medical Centre in the last five years. Infection with Encephalitozoon appears to be endemic in the Netherlands. PMID- 8618638 TI - [In search of Frederik van Eeden's character]. PMID- 8618639 TI - [Diagnosis and treatment of gout: not always simple]. PMID- 8618640 TI - [Recommendations for toxicologic studies in sudden, unexpected death]. PMID- 8618641 TI - [Signals for cellular selection for life and death by a new family of cytokines]. PMID- 8618642 TI - [Gout: current aspects of etiology, diagnosis and therapy]. PMID- 8618644 TI - [Epileptic insults, cerebral infarction and rhabdomyolysis as complications of amphetamine use]. AB - In a 16-year-old boy with acute generalised epileptic convulsions, cerebral infarction and rhabdomyolysis were diagnosed. The urine was positive for amphetamine. Until that moment the patient had denied using drugs. He recovered and was discharged after nine days. Recreational use of ecstasy (methylenedioxymethamphetamine) and other amphetamine derivatives is gaining popularity worldwide. This drug abuse is rarely reported spontaneously. PMID- 8618643 TI - [Regular vitamin A supplements are safe for pregnant women who consume few liver products]. AB - OBJECTIVE: To determine how much vitamin A is consumed through liver and liver products by non-pregnant and pregnant women aged 16-50 years, and to determine the implications for the use of multivitamin products. DESIGN: Secondary analysis on data from representative database Dutch National Food Consumption Survey. METHOD: Data were obtained from a Dutch National Food Consumption Survey (1992, method published earlier) regarding 1725 non-pregnant and 58 pregnant women aged 16-50 years who did or did not consume liver and (or) liver products. RESULTS: Average daily vitamin A intake (two consecutive days), was 850 retinol equivalents (RE) for non-pregnant and 990 RE for pregnant women, respectively (recommended daily allowances are 800 RE and 1000 RE). Average intakes of those not eating liver or liver products were 540 RE and 720 RE per day. In about 70% and 50% of the women respectively the intake was below the minimal requirement of 600 RE per day. The use of a vitamin A supplement providing 1200 RE per day among the non-liver users would in none of the cases have resulted in intakes higher than the threshold level of 7500 RE for teratogenic risks. Occasionally in 2-3% of the women, not using liver or liver products, maximum intake would exceed 3000 RE per day (the upper safe limit of intake according to the Dutch Health Council/Nutrition Council Committee). However, women using liver or liver products would be at risk of having too high intakes, above the threshold level of 7500 RE, irrespective of the use of vitamin supplements. CONCLUSION: Regular vitamin A supplements may be safely used by pregnant women who consume little or no liver or liver products. PMID- 8618645 TI - [Reversible tooth discoloration during oral use of antibiotics]. AB - From January 1991 until June 1995. 25 cases were reported to the Netherlands Pharmacovigilance Foundation LAREB of yellow to brown tooth discoloration following the oral use of medication; 21 cases (84%) involved antibiotics, of which 14 were amoxicillin. 17/21 patients were children, with ages ranging from 1 to 10 years. All children used liquid formulations (suspension or solutabs). Discoloration was reversible in all cases, but had a protracted course in some. Presumably a pigment precipitated on (and not in) the teeth, but the nature of the pigment was uncertain. PMID- 8618646 TI - [Complaints and claims about obstetric care; expert testimony from one expert reported over 10 years (1984-1995)]. PMID- 8618647 TI - [Is 'Cuba-therapy'useful in retinitis pigmentosa?]. PMID- 8618648 TI - Is selective reporting of well-designed clinical research unethical as well as unscientific? PMID- 8618649 TI - [Rhesus blood group]. PMID- 8618650 TI - Problems with myofascial pain syndrome and fibromyalgia syndrome. PMID- 8618651 TI - The neurologist and the dying patient. PMID- 8618652 TI - Acute illness myopathy. PMID- 8618653 TI - The cost of strokes: Two views. PMID- 8618654 TI - Do the facts and figures warrant a 10-fold increase in the performance of carotid endarterectomy on asymptomatic patients? AB - The detailed results of the Asymptomatic Carotid Atherosclerosis Study (ACAS) have been published. Electrifying reports in the media suggested that 53% fewer strokes would occur if individuals with 60% or greater stenosis were submitted to endarterectomy. The burning question is whether the evidence from this trial, and those preceding it, is sufficiently compelling to persuade any or all individuals with carotid stenosis, but free of any hemisphere and retinal symptoms, to have carotid endarterectomy. Based on a variety of population samplings, it is reasonable to estimate that approximately two million people are living in North America and Europe with asymptomatic lesions comparable with those studied in the ACAS. PMID- 8618655 TI - Clinical neuromythology XV. Feinting science: Neurocardiogenic syncope and collateral vasovagal confusion. PMID- 8618656 TI - Practice parameters for the diagnosis of patients with nervous system Lyme borreliosis (Lyme disease). Quality Standards Subcommittee of the American Academy of Neurology. PMID- 8618658 TI - Fatigue in multiple sclerosis compared with chronic fatigue syndrome: A quantitative assessment. AB - Fatigue, a common complaint among patients with multiple sclerosis (MS), is poorly characterized. We developed a computerized method that quantitatively measures fatigue, and defined a fatigue index (FI), which is the ratio between the integral of muscle strength decay over time and maximal voluntary contraction. Thirty patients (mean age, 37.4 +/- 10.3 years) were examined - 20 patients with pyramidal tract involvement and 10 patients with involvement of other neurological systems. We evaluated 10 patients during relapse and 3 months afterwards, and compared their results with those of four patients with chronic fatigue syndrome (CFS) and 13 age-matched health subjects. The FI was significantly higher in the MS patients as compared with the CFS patients and normal controls: 34.2 +/- 6.4% versus 27.5 +/- 1.0% and 23.6 +/- 6.8%, p < 0.05. Within the MS group, the FI correlated with the presence of pyramidal signs- 43.5% compared with 33% in patients without pyramidal signs, p < 0.01. In MS patients, fatigue worsened during a relapse affecting the pyramidal tract, but not during a relapse in other systems. These results demonstrate that fatigue can be quantitatively measured in MS patients, and that pyramidal dysfunction leads to increased fatigability. PMID- 8618657 TI - Depression and multiple sclerosis. AB - The objective of the present study were (1) to ascertain the lifetime risk of a depression in a representative group of multiple sclerosis (MS) patients, (2) to assess the morbidity risks for depression among first-degree relatives of these MS patients, and (3) to compare these familial risks for first-degree relatives of MS patients with those for first-degree relatives of a primary depression population, i.e., depression but no MS. We psychiatrically evaluated 221 MS patients (index cases) using a structured clinical interview for the DSM-III-R and calculated the rate and lifetime risk of depression for these index cases using the product limit estimate of survival function. We obtained psychiatric histories for all first-degree relatives of index cases, and we calculated morbidity risks for depression for these relatives using the maximum likelihood approach and compared the risks using the likelihood ratio tests. Index cases had a 50.3% lifetime risk of depression. Morbidity risks for depression among first degree relatives of index cases were decidedly lower when compared with morbidity risks among first-degree relatives of the reference population. Although there appears to be a very high rate of depression among MS patients, the data for their first-degree relatives do not support a clear genetic basis for this depression, or at least the same genetic basis that probably operates within families when depression occurs in the absence of MS. PMID- 8618659 TI - Skin lesion with a fatal neurologic outcome (Degos' disease) AB - Degos' disease, a rare multisystem vasculopathy of unknown etiology, only occasionally involves the nervous system. We report the Mayo Clinic experience of the neurologic features of Degos' disease in a series of 15 patients. All 15 patients had the typical skin lesions of Degos' disease, confirmed by skin biopsy. Ten patients developed neurologic manifestations including fatal hemorrhagic or ischemic strokes (n=5), disabling polyradiculoneuropathy (n=1), and nonspecific neurologic symptoms without objective findings (n=4). Results of laboratory tests varied but none were pathognomonic of the disease. Long-term follow-up revealed death in six patients; nine patients were nearly asymptomatic. Immunosuppressive and antiplatelet agents were not of benefit. CNS infarcts and hemorrhages with intravascular thrombi, but without evidence of vasculitis, were characterized features at autopsy. PMID- 8618660 TI - Risk of dementia among relatives of Alzheimer's disease patients in the MIRAGE study: What is in store for the oldest old? AB - Despite recent advances in the molecular genetics of Alzheimer's disease (AD), several fundamental questions concerning risk of illness are unresolved, namely, if Mendelian factors account for the incidence of the disease, and if AD is an inevitable consequence of the aging process. This study was designed to address these issues and other aspects of familial aggregation of the disorder. A consecutive sample of 1,694 patients who met criteria for a diagnosis of probable or definite AD were ascertained in 13 centers participating in the Multi Institutional Research in Alzheimer Genetic Epidemiology (MIRAGE) project. Lifetime risk and age at onset of AD among various strata of 12,971 first-degree relatives was estimated using survival analysis procedures. The lifetime risk of AD in first-degree relatives was 39.0% +/- 2.1% by age 96 years. Age-specific risk of AD declined after age 90 and the data set included 61 apparently unaffected persons who survived to age 96 without becoming demented. Female relatives had a higher risk of AD than male relatives at all ages. By age 80, children of conjugal AD couples had a cumulative risk of 54%, 1.5 times greater than the sum of the risks to children having affected mothers or fathers, and nearly 5 times greater than the risk to children having normal parents. Children of affected fathers had a cumulative risk that was 1.4 times the corresponding risk to children of affected mothers. Risk assessment in early-onset and late onset families, using various strategies for determining the age cut-off, yielded contradictory results. These data suggest the following: (1) the lifetime risk among relatives does not support a simple autosomal dominant inheritance pattern of disease; (2) women are innately more susceptible to AD than men; (3) the proportion of hereditary cases may be higher in men than women; (4) distinction between early- onset and late-onset forms of AD has little meaning in the absence of a biological marker; (5) the risk of AD decreases after age 90; and (6) AD therefore may not be an inevitable concomitant of the aging process, a conclusion that has profound implications for basic and applied AD research. The age- and sex-specific lifetime risks derived from this study are sufficiently robust to be a reliable source of information for counseling relatives of AD patients. PMID- 8618661 TI - Are malnutrition and stress risk factors for accelerated cognitive decline? A prisoner of war study. AB - We set out to test the hypothesis that severe malnutrition and stress experienced by prisoners of war (POWs) are associated with cognitive deficits later in life. We assessed 101 former Australian POWs of the Japanese and 108 veteran control subjects using a battery of neuropsychological tests, a depression scale, a clinical examination for dementia, and CT. We divided the POWs into high weight loss (>35%) and low weight loss groups (<35%). We found no significant differences in cognitive performance between the POWs and control subjects or between high and low weight loss groups on any of the tests or in the prevalence of dementia. Scores on the depression scale showed that the former POWs had more depressive symptoms than the control subjects a decade previous, but the difference had diminished over time. This study does not support the hypothesis that malnutrition is a risk factor for accelerated cognitive decline nor the theory that severe stress can lead to hippocampal neuronal loss and cognitive deficits. Cognitive deficits in earlier studies of former POWs may have been associated with concurrent depression. PMID- 8618662 TI - The consortium to establish a registry for Alzheimer's disease (CERAD). Part XIV: Demographic and clinical predictors of survival in patients with Alzheimer's disease. AB - We made follow-up observations on 1,036 Consortium to Establish a Registry for Alzheimer's Disease (CERAD) patients with Alzheimer's disease (AD) enrolled in 21 university medical centers in the United States. Evaluations were scheduled annually for as long as 7 years; at the time of analysis, there were 332 deaths. The median duration of survival from time of entry into CERAD was 5.9 years (95% CI; 5.6 to 6.4 years). Factors independently affecting survival were sex, age, and severity of dementia as measured by the Clinical Dementia Rating scale and the Blessed Scale for activities of daily living. The median survival after entry was 5.7 years for men, compared with 7.2 years for women. For men age 70, 75, and 80 years, median survival times were 6.5, 5.5, and 4.4 years, values notably less than those for the general population. Neither race, education, nor marital status significantly affect survival. This large nationwide study confirms the fact that AD is associated with shorter survival, particularly in men, subjects age 70 or older, patients with greater impairment in daily activities of living, and those with more severe dementia. PMID- 8618663 TI - Prediction of probable Alzheimer's disease in memory-impaired patients: A prospective longitudinal study. AB - We determined whether a battery of neuropsychological tests could predict who would develop Alzheimer's disease (AD) in a group of 123 memory-impaired nondemented patients. Patients were followed longitudinally for 2 years with a research battery of neuropsychological tests. After 2 years, 29 developed probable AD, and 94 did not develop dementia. We used logistic regression analyses to examine the classification accuracy of subjects' performance at entry to the study on the research battery. The logistic regression model was significant with an accuracy of 89%, sensitivity of 76%, and specificity of 94%. Two tests contributed significantly to this model: the delayed recall from the Rey Auditory Verbal Learning Test and the Mental Control subtest of the Wechsler Memory Scale. These two tests alone produced the same accuracy, sensitivity, and specificity as the larger model. These results demonstrate that probable AD can be predicted with a high degree of accuracy and with a relatively brief battery of neuropsychological tests. PMID- 8618664 TI - Toward a neurologic model of competency: Cognitive predictors of capacity to consent in Alzheimer's disease using three different legal standards. AB - OBJECTIVE: To identify cognitive predictors of competency performance and status in Alzheimer's disease (AD) using three differentially stringent legal standards for capacity to consent. DESIGN: Univariate and multivariate analyses of independent neuropsychological test measures with three dependent measures of competency to consent to treatment. SETTING: University medical center. SUBJECTS: 15 normal older controls and 29 patients with probably AD (15 mild and 14 moderate). MAIN OUTCOME MEASURES: Subjects were administered a batter of neuropsychological measures theoretically linked to competency function, as well as two clinical vignettes testing capacity to consent to medical treatment under five legal standards (LSs). The present study focused on three differentially stringent LSs: the capacity simply to "evidence a treatment of choice" (LS1), which is a minimal standard; the capacity to "appreciate the consequences" of a treatment of choice (LS3), a moderately stringent standard; and the capacity to "understand the treatment situation and choices" (LS5), the most stringent standard. Control subject and AD patient neuropsychological test scores were correlated with scores on the three LSs. The resulting univariate correlates were than analyzed using stepwise regression and discriminant function to identify key multivariate predictors of competency performance and status under each LS. RESULTS: No neuropsychological measures predicted control group performance on the LSs. For the AD group, a measure of simple auditory comprehension predicted LS1 performance (r(2)=0.44, p < 0.0001), a word fluency measure predicted LS3 performance (r(2)=0.58, p < 0.0001), and measures of conceptualization and confrontation naming together predicted LS5 performance (r(2)=0.81, p < 0.0001). Under discriminant function analysis, confrontation naming was the best single predictor of LS1 competency status for all subjects, correctly classifying 96% of cases (42/44). Measures of visumotor tracking and confrontation naming were the best single predictors, respectively, of competency status under LS3 (91% [39/43]) and LS5 (98% [43/44]). CONCLUSIONS: Multiple cognitive functions are associated with loss of competency in AD. Deficits in conceptualization, semantic memory, and probably verbal recall are associated with the declining capacity of mild AD patients to understand a treatment situation and choices (LS5); executive dysfunction with the declining capacity of mild to moderate AD patients to identify the consequences of treatment choice (LS3); and receptive aphasia and severe dysnomia with the declining capacity of advanced AD patients to evidence a simple treatment choice (LS1). The results offer insight into the relationship between different legal thresholds of competency and the progressive cognitive changes characteristic of AD, and represent an initial step toward a neurologic model of competency. PMID- 8618666 TI - Hippocampal volumes in Alzheimer's disease, Parkinson's disease with and without dementia, and in vascular dementia: An MRI study. AB - Hippocampal atrophy detected by volumetric MRI is a sensitive feature of early Alzheimer's disease (AD), but there are no studies evaluating hippocampal atrophy by MR volumetry in other dementing diseases. We therefore compared hippocampal volumes in a total of 113 subjects: 50 patients with mild to moderate AD, 9 patients with vascular dementia (VaD), 12 patients with idiopathic Parkinson's disease (PD) without dementia, 8 patients with PD and dementia (PDD), and 34 elderly control subjects. Thin, coronal, contiguous images were obtained by a 1.5 T MR imager. All patient groups had significantly smaller volumes of the hippocampus compared with the control group. In the PDD group, the absolute volumes were even smaller than in the AD group. In the PD group, the volumes were diminished to a lesser but significant extent. The volumes in the VaD group varied: of nine patients, two had no atrophy, three had unilateral, and four had bilateral atrophy. We postulate that hippocampal atrophy does not seem to be a specific phenomenon of dementia in AD but also occurs in VaD and PDD, and even in PD when no dementia is present. However, coexistence of AD pathology in our PD and VaD patients cannot be ruled out. Further studies with access to neuropathologic data are needed. PMID- 8618665 TI - Apolipoprotein E epsilon4 association with dementia in a population-based study: The Framingham study. AB - Apolipoprotein E type 4 allele (apoE epsilon4) is associated with Alzheimer's disease (AD) in the late-onset familial form and in sporadic cases, but the age associated risk in a randomly sampled elderly population is not established. We examined the association of apoE epsilon4 with AD and other dementias (mainly multi-infarct or dementia following stroke) in 1,030 persons aged 71 to 100 years in the population-based Framingham Study cohort. Kaplan-Meier survival analysis revealed that 55% of the apoE epsilon4/epsilon4 homozygotes developed AD by age 80, whereas 27% of apoE epsilon3/epsilon4 heterozygotes developed AD by age 85, and 9% of those without a 4 allele developed AD by age 85 years. In comparison with persons without a 4 allele, the risk ration for AD was 3.7 (95% CI = 1.9 to 7.5) for apoE epsilon3/epsilon4 heterozygotes and 30.1 (95% CI = 10.7 to 84.4) for apoE epsilon4 homozygotes. ApoE epsilon2 (2/2, 2/3, or 2/4 genotypes) was associated with an absence of AD. One-half (n=21) of the 43 AD patients were either homozygous or heterozygous for apoE epsilon4. We found evidence for an association of apoE epsilon4 with other dementia, primarily multi-infarct dementia and stroke. The risk ratio was 2.3 (95% CI = 0.9 to 6.1) for non-AD dementias among persons with apoE epsilon3/epsilon4. Although the apoE epsilon4 allele is a potent risk factor for AD and may be associated with other forms of dementia, most apoE epsilon4 carriers do not develop dementia, and about one-half of AD is not apoE epsilon4 associated. The low positive predictive value of this marker (0.10) suggest that use of apoE genotyping as a screening test for AD is not supported. PMID- 8618667 TI - Temporal pattern of cognitive decline and incontinence is different in Alzheimer's disease and diffuse Lewy body disease. AB - Incontinence is a hallmark of dementia, but little is known about its inception in different types of dementing disease. We recorded the dates of onset of dementia and of urinary incontinence in 73 demented patients followed for 5.6 +/- 2.5 years. The pathologic diagnosis was Alzheimer's disease (AD) in 29 cases, diffuse Lewy body disease (DLBD) in 11 cases, AD with Lewy bodies (AD+LB) in 13 cases, and AD with vascular lesions (AD+VL) in 20 cases. The onset of urinary incontinence was significantly earlier in DLBD cases (3.2 +/- 1.4 years after dementia onset) than in AD (5.9 +/- 2.5), AD+LB (5.8 +/- 2.4), and AD+VL (6.5 +/- 2.3) (p < 0.01). At the onset of bladder incontinence, the mean score in the Extended Dementia Scale was significantly higher (i.e., cognition was better) in DLBD cases (109.3 +/- 70.8) than in AD (21.3 +/- 40.4), AD+LB (45.6 +/- 45.1), and AD+VL (39.2 +/- 54.9) cases (p < 0.01). Urinary incontinence is associated with severe cognitive decline in pure AD but usually precedes severe mental failure in DLBD cases. This temporal pattern of cognitive decline and incontinence could be useful in differentiating these two dementing illnesses. PMID- 8618668 TI - Focal temporal lobe dysfunction in probable Alzheimer's disease predicts a slow rate of cognitive decline. AB - The memory disorder in Alzheimer's disease (AD) can be described as having two components: one primarily a defect in secondary memory and the other a defect in executive processes. We compared and contrasted the pattern of neuropsychological impairment in AD patients as a function of their memory and executive deficits. A K-Means cluster analysis identified four groups of patients. All four groups had impaired episodic and semantic memory and three had progressively more severe impairments in executive functions. The fourth group had normal executive functions; this group (N=32), described as having a "temporal lobe" pattern of impairment, had a significantly slower rate of progression of their dementia, with visual-construction skills virtually spared. These data demonstrate the existence of a subgroup of AD patients with a consistent pattern of impairment who progress more slowly than other patients over the course of 2 years and who maintain some specific cognitive abilities. This suggests that the mechanism of their disease may be different. PMID- 8618669 TI - Compensatory reallocation of brain resources supporting verbal episodic memory in Alzheimer's disease. AB - Conscious recall of past events that have specific temporal and spatial contexts, termed episodic memory, is mediated by a system of interrelated brain regions. In Alzheimer's disease (AD) this system breaks down, resulting in an inability to recall events from the immediate past. Using subtraction techniques with PET acquired images of regional cerebral blood flow, we demonstrate that AD patients show a greater activation of regions of cerebral cortex normally involved in auditory-verbal memory, as well as activation of cortical areas not activated by normal elderly subjects. These results provide clear evidence of functional plasticity in the AD patient's brain even if those changes do not result in normal memory function, and provide insights into the mechanism by which the AD brain attempts to compensate for neurodegeneration. PMID- 8618670 TI - Age and education correction of Mini-Mental State Examination for English and Spanish-speaking elderly. AB - Previous research has shown that the Mini-Mental State Examination (MMS) is biased as a measure of cognitive impairment in minority and low-education patients. The purpose of this study was to (1) develop a statistical correction for effects of age and education and (2) test the efficacy of the statistically adjusted MMS (MMSAdj) as a screening test for dementia using different ethnic groups and education levels. We used a population-base community survey sample (n=590) composed of 46.6% Hispanics and 53.4% non-Hispanics to derive the statistical correction, defined as:MMSAdj = Raw MMS - (0.471 X [Education-12]) + (0.131 X [Age-70]). Ethnicity and language of test administration were not significantly related to MMSAdj in the community survey sample, but the raw MMS was strongly influenced by these factors. We used an independent sample (n=2,983) of patients evaluated through the California Alzheimer's Disease Diagnostic and Treatment Centers to test the diagnostic accuracy of the MMS and the MMSAdj across low- and high-education groups and across whites, Hispanics, and blacks. Results showed greater stability of sensitivity and specificity across education levels and ethnic groups for the MMSAdj than for the raw MMS and suggest that the MMSAdj is a preferable measure of cognitive impairment for low- education and minority individuals. PMID- 8618672 TI - Neuropsychological functioning in cortical-basal ganglionic degeneration: Differentiation from Alzheimer's disease. AB - Patients with cortical-basal ganglionic degeneration (CBGD) display prominent rigidity and apraxia, exhibit an asymmetric onset of symptoms, and may show other symptoms including abnormal saccadic eye movements, the "alien limb" sign, limb dystonia, and myoclonus. We compared the neuropsychological test performances of 21 CBGD patients with 21 Alzheimer's disease (AD) patients displaying no extrapyramidal symptoms and with 12 ADA patients who did show such symptoms. Groups were matched for age, educational level, and overall severity of dementia. Since the cognitive deficit was mild in most CBGD patients, most AD patients included in this study were also only mildly demented. The CBGD patients performed significantly better than the AD patients on test of immediate and delayed recall of verbal material; whereas the AD patients (with or without extrapyramidal symptoms) performed better on tests of praxis, finger tapping speed, and motor programming. The CBGD and AD groups all displayed prominent deficits on tests of sustained attention/mental control and verbal fluency, and exhibited mild deficits on confrontation naming. The CBGD patients endorsed significantly more depressive symptoms on the Geriatric Depression Scale. PMID- 8618671 TI - Cerebral amyloid deposition and diffuse plaques in "normal" aging: Evidence for presymptomatic and very mild Alzheimer's disease. AB - The presence of senile plaques in the neocortex of apparently nondemented elderly persons often is accepted as part of "normal" aging. Alternatively, because cerebral deposition of beta-amyloid may be a key mechanism in the development of Alzheimer's disease (AD), the presence of beta-amyloid-containing plaques may represent very early AD. To examine the relationships of cognitively normal aging, very mild dementia of the Alzheimer type, and the presence of neocortical senile plaques, we performed clinicopathologic correlation in 21 longitudinally studied healthy elderly subjects (84.5 +/- 6.6 years old at death). Nine subjects had strikingly high plaque densities in the neocortex; two of these subjects died of head injury before which there was no evidence of cognitive impairment. The other seven subjects with high plaque densities had clinical evidence for very mild cognitive impairment (Clinical Dementia Rating score of 0.5) at some time during their course and mildly impaired psychometric performance at last assessment before death. The remaining 12 subjects had no clinical or psychometric impairment and had few or no neocortical AD lesions. These results suggest that senile plaques may not be part of normal aging but instead represent presymptomatic or unrecognized early symptomatic AD. The high density of senile plaques (predominately of the diffuse subtype) in the cortex of subjects just at the threshold of detectable dementia is consistent with the hypothesis that beta amyloid deposition is an initial pathogenetic event in the development of AD. PMID- 8618673 TI - Dementia and adult-onset unprovoked seizures. AB - OBJECTIVE: We tested the hypothesis that dementia increases the risk of unprovoked seizure among adults. Partial- and generalized-onset seizures were considered together and separately. Additionally, we explored whether the increased risk was restricted to Alzheimer's disease (AD), as previously shown. DESIGN: Subjects in this population-based case-control study were 145 incident cases of first unprovoked seizure (without prior stroke, CNS infection, brain tumor, head trauma, mental retardation, or cerebral palsy) aged 55 years or older and 290 controls matched to cases on age, gender, and duration of medical follow up. Using the records-linkage system of the Rochester Epidemiology Project, we obtained, for both cases and matched controls, information on dementia prior to onset of unprovoked seizure. Subjects were classified as having dementia if they met ad hoc criteria equivalent to those in the DSM-III. AD was distinguished from other dementias. RESULTS: Both a diagnosis of AD and a diagnosis of other dementia were associated with at least a six-fold increased risk of unprovoked seizure when controlling for age, sex, and length of medical follow-up in Rochester. There was no difference in risk when comparing generalized-onset seizures with partial-onset seizures. CONCLUSIONS: In the absence of other prior neurologic insult, both AD and other dementias increase the risk of generalized- and partial-onset unprovoked seizures. PMID- 8618674 TI - Muscle is electrically inexcitable in acute quadriplegic myopathy. AB - We directly stimulated muscle in three patients with acute quadriplegic myopathy to determine whether paralyzed muscle in this syndrome is electrically excitable. Two of the patients had been treated with neuromuscular blocking agents and corticosteroids, and one patient had been treated with corticosteroids alone. We found that paralyzed muscle is electrically inexcitable in affected patients. Muscle regained electrical excitability over weeks to months. The recovery of muscle excitability paralleled the clinical recovery of patients, suggesting that paralysis in this syndrome is secondary to electrical inexcitability of muscle membrane. PMID- 8618675 TI - Primary or working memory in frontal lobe epilepsy: An 18FDG-PET study of dysfunctional zones. AB - INTRODUCTION: We previously demonstrated that patients with frontal lobe epilepsy show deficits on a visual working memory paradigm and that this paradigm produces increased 18FDG uptake in the dorsolateral prefrontal cortex (DPFC), premotor cortex, angular and supramarginal gyri, basal forebrain, and ventral frontal poles of normal subjects when compared with a control task. We hypothesized that subjects with frontal lobe epilepsy would have impaired frontal activation during this task. METHODS: One resting and two activated images were obtained with 18FDG PET in 15 subjects and 14 controls. One was a delayed (DMS) and one an immediate (IMS) match to sample paradigm. Discriminant and factor analyses were used to analyze the data, supplemented by selected t tests. RESULTS: No differences in glucose uptake were found between the DMS and IMS in the epilepsy subjects, in distinct contrast to controls. A comparison between controls and epilepsy subjects showed differences both ipsilateral and contralateral to the epileptic focus in the frontal regions involved in the task, with small changes in nonfrontal, task-related regions as well. The task itself brought out or highly exaggerated differences seen at rest. There was weak evidence that other frontal and temporal regions were attempting to compensate for the DPFC deficit. CONCLUSION: A unilateral epileptic focus is capable of suppressing function along a large task-related circuit ipsilateral and contralateral to the focus. Peripheral cortical regions compensate poorly for the area of dysfunction. PMID- 8618676 TI - A positron emission tomography study of primary orthostatic tremor. AB - Primary orthostatic tremor (OT), a clinical syndrome in which a rapid (14 to 16 Hz), regular lower limb tremor causes unsteadiness on standing, may be associated with a postural upper limb tremor of similar frequency. We used H2 15O PET to analyze the abnormal pattern of cerebral activation associated with the postural upper limb tremor in four patients with primary OT. Patients had regional cerebral bloodflow (rCBF) measured during involuntary tremor while maintaining a posture with their outstretched right upper limb and again at rest. Tremor was associated with abnormal bilateral cerebellar and contralateral lentiform and thalamic activation. These findings were evident on group analysis of pooled PET data after transformation into standard stereotactic space and in single subjects when PET images were coregistered with structural MRI of the brain. At rest, cerebellar blood flow was significantly increased bilaterally in OT when compared with age- and sex-matched controls. We have previously demonstrated similar abnormal bilateral cerebellar activation in essential and writing tremors and conclude that abnormal bilateral overactivity of cerebellar connections is a common feature of tremulous disorders. PMID- 8618677 TI - Epileptic negative myoclonus: An EEG-single-photon emission CT study indicating involvement of premotor cortex. AB - We report a combined EEG-single-photon emission CT (SPECT) study on a patient with epileptic negative myoclonus (ENM). Clinically, the ENM was characterized by brief repetitive lapses in postural tone of the right upper extremity when the arms were held outstretched, whereas no movement effect was observed during rest. Ictal EEG showed repetitive left frontal spikes with a maximum at electrodes EC1 and F1. EMG silent periods lasting from 100 to 200 ms followed the onset of the EEG transients by a latency of 20 to 40 ms. The N20 component of median nerve somatosensory evoked potentials-representing a biological marker of the location of central fissure-showed a phase reversal between electrodes P3 and C1 and thus was located considerably posterior to the spike maximum. We obtained accurate anatomic reference of cerebral blood flow changes visible on SPECT by a special coregistration technique of MRI and SPECT. SPECT performed during ENM showed a marked regional hyperperfusion in the left middle frontal gyrus and a less pronounced increase in tracer uptake in the left supramarginal gyrus. Our results suggest that ENM is generated by epileptic activity in the premotor area in the middle frontal gyrus corresponding to Brodmann's area 6. PMID- 8618678 TI - Fatal insomnia in a case of familial Creutzfeldt-Jakob disease with the codon 200(Lys) mutation. AB - Fatal familial insomnia (FFI) has been exclusively associated with a pathogenic mutation at codon 178 in the PRNP gene coupled with methionine (Met) at codon 129. We now describe a subject with familial Creutzfeldt-Jakob disease, heterozygous for the pathogenic lysine (Lys) mutation at codon 200 and homozygous for Met at codon 129 of the PRNP gene, who was affected by severe insomnia. At autopsy the patient had significant involvement of the thalamus, as previously described in subjects affected by FFI with the codon 178 mutation. This case demonstrates the wide variability of the clinical expressions in patients with the codon 200 mutation, that may include insomnia and thalamic pathology. PMID- 8618679 TI - A prion disease with a novel 96-base pair insertional mutation in the prion protein gene. AB - There are coding mutations in the prion protein gene in familial Creutzfeldt Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, and other phenotypes that make up the inherited prion diseases. Insertional mutations consisting of two, five, six, seven, eight, and nine additional octapeptide repeat elements are seen in the inherited prion diseases and usually present as atypical dementias with considerable intrafamilial phenotypic variability. A four-octarepeat insertion was reported previously in an individual without neurodegenerative disease who died of hepatic cirrhosis. Here we report a novel four-octarepeat insertional mutation in a case with classical clinical, electroencephalographic and histopathologic features of CJD with the unusual finding of pronounced prion protein immunoreactivity of the molecular layer of the cerebellum. PMID- 8618680 TI - Physiological studies of spinal inhibitory pathways in patients with hereditary hyperekplexia. AB - Because hereditary hyperekplexia results from a defect in the glycine receptor, we studied in five patients several spinal inhibitory pathways that are thought to use either glycine or gamma-aminobutyric acid as a neurotransmitter. Three patients had a mutation in the alpha1 subunit of the glycine receptor, whereas two sisters with the same clinical syndrome did not have this mutation. Compared with normal subjects, reciprocal inhibition between flexor and extensor muscles of the forearm was diminished during the first period of inhibition and preserved during the second period of inhibition in all three patients tested. Facilitation after the early period of inhibition was prominent. Recurrent inhibition of the soleus H reflex was normal in four patients, as was inhibition of the H reflex produced by Achilles' tendon vibration. There was no significant difference in nonreciprocal (Ib) inhibition between patients and normal individuals, The findings suggest that disynaptic reciprocal inhibition in humans is mediated through glycinergic interneurons, but that recurrent inhibition may have a contribution from nonglycinergic mechanisms. PMID- 8618681 TI - Oculopharyngeal muscular dystrophy in two unrelated Japanese families. AB - The occurrence of oculopharyngeal muscular dystrophy (OPMD) in Orientals is uncertain. We identified two unrelated Japanese families, including 30 affected individuals (14 men, 16 women, mean age 58 years) of OPMD through four generations, with complete penetrance. Their major clinical manifestations were late-onset bilateral ptosis and dysphagia. Histologic studies of slightly affected muscles reveal mild myogenic changes, occasional rimmed vacuoles, and small angulated fibers. By contrast, the severely involved cricopharyngeal muscle showed marked loss of fibers and massive proliferation of connective tissue. Ultrastructural studies of four different biopsied muscles disclosed subsarcolemmal intranuclear tubulofilamentous inclusions, identical to those of non-Japanese OPMD patients. PMID- 8618682 TI - Genetic localization of Bethlem myopathy. AB - Bethlem myopathy is a rare autosomal dominant myopathy characterized by slowly progressive limb-girdle muscular atrophy and weakness, and contractures of multiple joints. To identify the genetic localization we used highly polymorphic microsatellite markers in a genome-wide search in six Dutch families. After excluding genetic linkage with 52 markers distributed evenly over the autosomes, significant linkage was present with the 21q22.3 locus PFKL (two-point lod score of Zmax = 6.86 at theta = 0.03). There was no indication of genetic heterogeneity. The pattern of recombinations observed with adjacent markers indicated a localization distal to PFKL. Recombination of a marker within the collagen 6a1 gene (COL6A1) excluded this apparent candidate gene in one of the Bethlem myopathy families. The disease gene is most likely located in the region between COL6A1 and the telomere of chromosome 21q. PMID- 8618683 TI - Brain choline-containing compounds are elevated in HIV-positive patients before the onset of AIDS dementia complex: A proton magnetic resonance spectroscopic study. AB - The CNS is frequently involved in human immunodeficiency virus (HIV) infection. In recent studies using proton magnetic resonance spectroscopy, investigators found a significant reduction in N-acetyl aspartate, a metabolic marker of neurons, in late stages of dementia. To further understand the relationship between proton magnetic resonance spectroscopy changes and clinical disease and dementia, we compared 20 HIV-infected patients presenting at varying stages of acquired immunodeficiency syndrome (AIDS) dementia complex and infection to 10 age-matched controls. We found a significant reduction in N-acetyl aspartate/creatine only in patients who had advanced dementia and CD4 counts less that 200/microliter. By contrast, a significant elevation in compounds containing choline was present in patients in the early stages of HIV infection of who had CD4 counts greater than 200/microliter, in patients with normal MRI scans, and in all AIDS dementia complex groups, including subjects with no or minimal cognitive impairment. An elevated choline level also occurred in later stages of HIV infection (CD4 < 200/microliter). Our results suggest that an increase in choline occurs before N-acetyl aspartate decrements, MRI abnormalities, and the onset of dementia, and may therefore provide a useful marker for early detection of brain injury associated with HIV infection. PMID- 8618684 TI - Analysis of the KSP repeat of the neurofilament heavy subunit in familiar amyotrophic lateral sclerosis. AB - We examined the neurofilament heavy subunit (NEFH) as a candidate gene for familial amyotrophic lateral sclerosis. We screened the KSP repeat region of the NEFH gene in 117 unrelated individuals who inherited familial amyotrophic lateral sclerosis as an autosomal trait but who do not have the mutation in the SOD1 locus, and we found no variants in any individual. We conclude that the motor neuron degeneration observed in non-SOD1 familial amyotrophic lateral sclerosis is not due to mutations in the KSP repeat of the NEFH gene. PMID- 8618686 TI - Levodopa and deprenyl treatment effects on peripheral indices of oxidant stress in Parkinson's disease. AB - The oxidant stress theory of Parkinson's disease (PD) hypothesizes that levodopa treatment may be potentially harmful and this is supported by studies demonstrating levodopa toxicity to cultured dopaminergic neurons. These in vitro experiments, however, lack the physiologic protective mechanisms present in vivo. Oxyradical damage to cell membranes liberates malondialdehyde, which we measured in the serum of 27 PD patients just before and after levodopa (with carbidopa) administration. We also measured plasma products of the two routes by which levodopa potentially generated oxyradicals: (1) 5-S-cysteinyl-dopa (derived from levodopa autoxidation), and (2) 3,4-dihydroxyphenylacetic acid (DOPAC), produced by monoamine oxidase (MAO) metabolism of dopamine. Following levodopa/carbidopa administration, both of these plasma products were markedly increased; however, the mean serum malondialdehyde concentration was unchanged and remained similar to the normal control group (N=15) value. Chronic treatment with the MAO-B inhibitor, deprenyl (N=16), was not associated with any differences in serum malondialdehyde or plasma 5-S-cysteinyl-dopa concentrations compared with those not treated with deprenyl (N=11). The post-levodopa rise of plasma DOPAC was only slightly attenuated with deprenyl therapy, consistent with a predominant MAO-A effect in the circulation and peripheral organs. Thus, in contrast to in vitro studies, we did not detect evidence of oxidative damage in the circulation following levodopa administration, despite marked increase in the products of dopamine oxidative metabolism. PMID- 8618685 TI - Decrease of the D3 dopamine receptor mRNA expression in lymphocytes from patients with Parkinson's disease. AB - We investigated the dopamine receptor (DAR) mRNA expression in peripheral blood lymphocytes from 45 patients with Parkinson's disease (PD) and 21 age-matched controls using the quantitative reverse transcription and polymerase chain reaction method. Beta-actin mRNA was used as an internal control to evaluate the relative expression level of the DAR mRNA. There was a statistically significant decrease of the D3 dopamine receptor (D3R) mRNA expression in PD patients compared with that in controls. There was no change in expression of the D5 dopamine reception mRNA in PD patients. A further binding study showed reduction of the D3R binding sites in PD lymphocytes. The decrease of the D3R mRNA expression correlated with the degree of clinical severity in PD patients. PMID- 8618687 TI - Consequences of nigrostriatal denervation on the gamma-aminobutyric acidic neurons of substantia nigra pars reticulata and superior colliculus in parkinsonian syndromes. AB - To examine the effects of nigrostriatal denervation on the substantia nigra pars reticulata (SNpr), one of the main outputs of the basal ganglia, we used quantitative in situ hybridization to analyze the messenger RNA coding for Mr 67,000 glutamic acid decarboxylase (GAD67 mRNA) in the SNpr neurons from patients with Parkinson's disease (PD), monkeys rendered parkinsonian by 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), and their respective controls. In MPTP intoxicated monkeys, the expression of GAD67 mRNA was increased in the SNpr neurons, and the increase was reversed by L-dopa treatment. There were no differences in the level of GAD67 mRNA between PD patients who had been treated with L-dopa and control subjects. Combined with the previously reported increased expression of GAD67 mRNA in the internal segment of the pallidum of MPTP intoxicated monkeys, these data suggest that the gamma-aminobutyric acid (GABAergic) activity of the output system of the basal ganglia is globally increased by nigrostriatal denervation. We also analyzed the level of GAD67 mRNA expression in the superior colliculus, a structure that receives the inhibitory influence of the GABAergic neurons of the SNpr and that is involved in eye movement control. GAD67 mRNA expression was reduced in both MPTP-intoxicated monkeys, whether or not they received L-dopa therapy, and PD patients, compared to their respective controls. This decrease may result from the hyperactivity of the inhibitory nigrotectal pathway, but also from other influences since it was not corrected by L-dopa therapy. These changes may account for the slight ocular motor and visuospatial cognitive impairment occurring in PD, even after L-dopa therapy. PMID- 8618688 TI - Congenital muscular dystrophy syndromes distinguished by alkaline and acid phosphatase, merosin, and dystrophin staining. AB - Congenital muscular dystrophy syndromes are characterized by congenital weakness, contractures, and dystrophic features on muscle biopsy. However, these syndromes are often difficult to diagnose precisely because their clinical and pathologic characteristics are not specific and resemble changes in other myopathies. We examined muscle biopsies from 20 children with a congenital muscular dystrophy syndrome. Disease controls with dystrophies or other myopathies (n=19) and normal individuals (n=15) were studied for comparison. In each biopsy we determined (1) numbers of muscle fibers with alkaline phosphatase (AlkP) staining, (2) numbers of acid phosphatase-(AcP) positive cells, (3) dystrophin levels by immunocytochemistry, and (4) the distribution of merosin and laminin-A staining. A ratio of AcP:AlkP staining was calculated for each biopsy. In nine patients with congenital muscular dystrophy (younger than 4 years of age) with normal dystrophin, the AcP:AlkP ratio was low (0.09 +/- 0.03). In contrast, in Duchenne muscular dystrophy, the AcP:AlkP ratio was 15 times higher (1.6 +/- 0.04, p=0.001). The three children with congetial muscular dystrophy syndromes and reduced dystrophin and one child with facioscapulohumeral dystrophy had AcP:AlkP ratios in the range of Duchenne muscular dystrophy patients (2.4 +/- 1.4). Low Ac:AlkP ratios were related to relative absence of AcP-positive cells. Merosin staining was absent in 5 of the 17 congenital muscular dystrophy biopsies tested. None of the 5 children with merosin-negative but all 12 with merosin-positive stains walked (p=0.0002). We conclude that a pattern of few AcP-positive cells in the setting of numerous AlkP staining muscle fibers has specificity for congenital muscular dystrophy syndromes and provides histopathologic support for the diagnosis. Reduced merosin in muscle predicts more severe weakness and long term disability. PMID- 8618689 TI - Congenital muscular dystrophy: Clinical and pathologic study of 50 patients with the classical (Occidental) merosin-positive form. AB - We studied 50 patients with the merosin-positive form of congenital muscular dystrophy (MP-CMD) clinically and pathologically. The frequency of MP-CMD in our laboratory was approximately one-half that of the Fukuyama type and one-sixth that of Duchenne muscular dystrophy. The early signs of MP-CMD included decreased fetal movement during pregnancy (14%) and poor suck (42%), floppiness (30%), and respiratory difficulty (16%) in early infancy. Eighty-six percent of the patients had delayed motor development. Ninety-two percent of the patients followed beyond age 4 years had learned to walk. The disease was relatively slowly progressive, except in six patients who rapidly lost ambulation. Almost all patients had normal IQ, except four who were mildly to moderately retarded. Of the patients examined by cranial CT/MRI, 24% showed cerebral atrophy and 11% had areas of white matter lucency. Muscle biopsy results in those younger than 5 years showed mild dystrophic changes consisting of variation in fiber size and scattered necrotic and regenerating fibers. In older children, there were additional chronic dystrophic changes, including fiber splitting (32%), moth-eaten appearance (32%), marked fatty replacement (46%), and abnormal fiber type distribution (59%). The manifestations of MP-CMD were generally milder and more slowly progressive than those of the Fukuyama type and merosin-negative form of congenital muscular dystrophy. PMID- 8618690 TI - Acute type II myofiber atrophy in critical illness. AB - Two patients with acute onset of profound weakness and loss of muscle bulk during a critical illness had unusual histopathological changes of type II myofibers. Both patients had respiratory failure and prolonged neuromuscular blockade. High dose steroids were used in only one case and atracurium in the other. Muscle biopsies at days 30 to 32 demonstrated widespread atrophy, basophilic cytoplasm, and vesicular nuclei, features suggestive of regeneration involving virtually all type II myofibers and sparing type I myofibers. This may be another variant or critical illness myopathy or a variation of the pathology during the course of illness. PMID- 8618691 TI - Chronic inflammatory demyelinating polyneuropathy associated with malignant melanoma. AB - We report three patients who developed chronic inflammatory demyelinating polyneuropathy (CIDP) in association with malignant melanoma. In two cases, melanoma was discovered during the initial evaluation for neuropathy. Two patients also had vitiligo, an antibody-mediated disorder that may complicate melanoma. Melanoma cells and Schwann cells are both of neuroectodermal cell origin, with shared surface antigens. Shared immunoreactivity may account for the association between melanoma and CIDP, as with vitiligo. PMID- 8618692 TI - Recovery from coma caused by primary CNS mantle cell lymphoma presenting as encephalitis. AB - We report a 74-year-old woman with progressive cognitial deterioration and changes in personality. She had no clinical signs of an inflammatory CNS process, but brain CT and MRI scans and cytologic examination of the CSF were initially indicative of encephalitis and ventriculitis. Antiviral and antibacterial therapy had no effect on the course of symptoms, and patient became comatose. We established the diagnosis of a primary CNS mantle cell lymphoma (PCNSL) and began corticosteroids. Within a few days the patient became alert and was able to walk again. Nonenhancing and non-space-occupying PCNSLs are rare but must be considered in the differential diagnosis of coma and encephalitis. Comatose PCNSL patients without radiographic evidence for herniation can be successfully treated with corticosteroids even if the EEG has a burst suppression pattern. PMID- 8618693 TI - Cerebral infarction associated with Kearns-Sayre syndrome-related cardiomyopathy. AB - We present the clinical and neuroradiologic findings of a 31-year-old man with Kearns-Sayre syndrome- related dilated cardiomyopathy who experienced a left middle cerebral artery territory stroke, thought to be due to cardiogenic embolism. The rate of clinically apparent cardiomyopathy in Kearns-Sayre patients can be expected to increase as their survival is prolonged by the use of cardiac pacemaker devices. Under these circumstances, stroke caused by cardiogenic embolism, which is presently rare, may become more common. PMID- 8618694 TI - Late-onset generalized disorder of peroxisomes. AB - We diagnosed a unique peroxisomal disorder in a 32-year-old man with profound mental retardation, mild facial dysmorphism, retinal pigmentary degeneration, seizures, and sensorineural deafness. Although plasma very-long-chain fatty acid profile suggested X-linked adrenoleukodystrophy, marked reduction in fibroblast lignoceric acid oxidation and the presence of cytosolic catalase were consistent with Zellweger syndrome (ZS). Unlike ZS, functional peroxisomes were present as indicated by the density of peroxisomes (1.175 gm/ml) similar to peroxisomes from control cells and by partial deficiencies of fibroblast phytanic acid oxidation and dihydroxyacetone phosphate acyltransferase activity. These findings indicate that this patient has a previously undescribed group 3 peroxisomal disorder (multiple peroxisomal enzyme deficiencies with preserved peroxisomes). PMID- 8618695 TI - Leukoencephalopathy associated with cobalamin deficiency. AB - Patients with cobalamin deficiency may experience cognitive impairment or neuropsychiatric symptoms. Although abnormalities of central myelin are the presumed cause of these manifestations, there is a paucity of reports of white matter lesions as shown on neuroimaging studies, and the effects of cobalamin replacement on these lesions are not known. We report a man with subacute cognitive impairment associated with cobalamin deficiency and remarkable confluent white matter abnormalities on MRI, confirmed by biopsy. With cobalamin replacement, both his cognitive deficits and imaging abnormalities partially resolved. This case indicates that leukoencephalopathy, in the absence of anemia or myelopathy, should be added to the spectrum of disorders associated with cobalamin deficiency. Early detection and treatment may be associated with a greater potential for recovery. PMID- 8618697 TI - Cerebrospinal fluid concentrations of soluble tumor necrosis factor receptor in bacterial and aseptic meningitis. AB - Soluble tumor necrosis factor receptor (sTNF-R) is the natural homeostatic regulator of the action of TNF-alpha. The level of sTNF-R reflects the true biologic activity of TNF-alpha. We investigated whether sTNF-R increases in CSF during patients' acute stage of bacterial and aseptic meningitis by measuring p60 sTNF-R in CSF by a sandwich enzyme immunoassay. The concentrations of sTNF-R were significantly higher in the CSF of children with bacterial meningitis than in the CSF of those with aseptic meningitis or of control subjects. The patients with bacterial meningitis who died or had severe neurologic sequelae had higher sTNF-R levels than those who survived. Our findings suggest that the sTNF-R level in CSF during acute bacterial meningitis is important for predicting neurologic sequelae. PMID- 8618696 TI - Hereditary spastic paraplegia linked to chromosome 15q: Analysis of candidate genes. AB - We previously reported an extended kindred with autosomal dominant uncomplicated hereditary spastic paraplegia (HSP) and found close linkage between the disorder and microsatellite polymorphisms on chromosome 15q. Multipoint linkage analysis reached a maximum LOD score (10.16) between D15S128 and D15S156, a region that includes genes encoding alpha5 and beta3 subunits of GABAA receptor. Theoretically, abnormal GABA-mediated neurotransmission could produce spasticity and possibly other changes of HSP. We used genetic linkage analysis to evaluate these two HSP candidate genes and observed obligate recombinants for polymorphisms immediately adjacent to (or within untranslated regions of) genes encoding alpha5 and beta3 GABAA receptor subunits. Although these genes are linked tightly to the HSP locus, our findings conclusively exclude these genes from being responsible for HSP in this kindred. PMID- 8618698 TI - Epilepsy surgery in the setting of periventricular leukomalacia and focal cortical dysplasia. AB - We report an infant who had successful epilepsy surgery for intractable infantile spasms in the setting of bilateral periventricular leukomalacia and remote germinal matrix hemorrhage. Although MRI gave evidence of a diffuse cerebral insult and EEG showed hypsarrhythmia, focal epileptogenicity was suggested by previous partial seizures with onset over the right temporoparietal-occipital region and PET hypometabolism in that same area. Right temporoparietal and lateral occipital resection at 15 months resulted in seizure freedom and dramatic developmental progress at 1-year follow-up. Histopathologic examination of resected tissue showed cortical dysplasia, possibly due to the same insult that also resulted in the bilateral periventricular leukomalacia. From an etiologic perspective, this is one of very few reported cases strongly implicating acquired focal cortical dysplasia in response to a prenatal insult. From a clinical perspective, the case illustrates that the spectrum of potential surgical candidacy in infants may be broader than usually suspected. PMID- 8618699 TI - Ventral tegmental area injury and frontal lobe disorder. AB - A patient developed acute behavioral changes implicating frontal-executive dysfunction. His clinical signs suggested mesencephalic injury, and a cranial MRI scan showed an abnormality restricted to a small region of the ventral midbrain. We suggest that the patient's behavioral disorder originated from disruption of the ventral tegmental area or it projections, structures that influence frontal brain processes via the mesocortical dopamine tract. PMID- 8618700 TI - Cerebral angiography complications link cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy to familial hemiplegic migraine. PMID- 8618701 TI - Improvement of both tardive dystonia and akathisia after botulinum toxin injection. PMID- 8618702 TI - Development of general weakness in a patient with amyotrophic lateral sclerosis after focal botulinum toxin injection. PMID- 8618703 TI - Severe and prolonged dysphagia complicating botulinum toxin A injections for dystonia in Machado-Joseph disease. PMID- 8618704 TI - Machado-Joseph disease: A proposal of spastic paraplegic subtype. PMID- 8618705 TI - Medullary lesions and unusual bilateral paroxysmal dystonia in multiple sclerosis. PMID- 8618706 TI - Chronic demyelinating polyneuropathy: Improvement after sepsis. PMID- 8618707 TI - Chronic progressive monomelic sensory neuropathy. PMID- 8618708 TI - Abnormality of nerve conduction in diabetic neuropathy: Is there a functional relationship with the carnitine system? PMID- 8618709 TI - Choreoathetotic movements: A possible side effect of gabapentin. PMID- 8618710 TI - Exacerbation of seizures in Lennox-Gastaut syndrome by gabapentin. PMID- 8618711 TI - Choreiform syndrome associated with fluoxetine treatment in a patient with deficient CYP2D6 activity. PMID- 8618712 TI - Inpatient costs of specific cerebrovascular events at five academic medical centers. AB - We estimated the hospital costs for patients with different cerebrovascular events and applied patient and administrative variables to explain the variance of the cost estimates with particular attention to the relationship between patient age and cost. The study sample was drawn from an administrative data set of all hospital discharges from five academic medical centers for the 1992 calendar year. Using International Classification of Diseases (ICD-9-CM) primary diagnosis codes, cases were classified into cerebrovascular subgroups: subarachnoid hemorrhage (SAH), intracerebral hemorrhage (ICH), ischemic cerebral infarction (ICI), and transient ischemic attack (TIA). The ICD-9-driven data file was supplemented with billing data containing inpatient charges reported in UB-82 format. Costs were imputed by applying Medicare charge-to-cost ratios and regional wage adjustments to the billing data. We estimated relationships between inpatient costs and a number of demographic and administrative variables. A statistically significant difference was found between cerebrovascular subgroups for both the mean cost per discharge (p<0.01) and the mean cost of an inpatient day (p<0.01). The mean cost per discharge for each subgroup was as follows: SAH, $39,994 (n=218); ICH, $21,535 (n=258); ICI, $9,882 (n=908); TIA, $4,653 (n=303). Likewise, the mean cost per inpatient day was as follows: SAH, $2,215; ICH, $1,396; ICI, $1,036; TIA, $1,117. Length of stay as a measure of resource use was strongly predictive of inpatient cost, explaining 72 to 82% of the variation in cost. Demographic variables (i.e., age, gender, race, insurance status), however, revealed virtually no predictive power, accounting for less than 10% of the variance in each of the four subgroups. There are substantial differences in the patient-level cost of hospital services for stroke-related events. After controlling for the type of cerebrovascular event, basic demographic variables and insurance status (including Medicare) contribute little to the total cost of inpatient care. More important factor include stroke severity, social factors, and clinical practice variations. PMID- 8618713 TI - Utilization of acute care services in the year before and after first stroke: A population-based study. AB - There is a need for accurate population-based data on the utilization of medical resources after stroke. The present study used the Rochester Stroke Registry to identify all Rochester, Minnesota residents with confirmed first stroke (hospitalized and nonhospitalized) during the period of 1987 to 1989 (n=292). Events were categorized by type of stroke and assigned Rankin severity. Inpatient and outpatient acute care activity for the 12 months before and after stroke for each individual were obtained from billing tapes provided by Mayo Clinic, Olmstead Medical Group, and affiliated hospitals. These providers account for >95% of acute care received by Rochester residents. The results showed that despite high poststroke mortality, total charges in the year after stroke were 3.4 times those for the previous year. Although greater than 50% of utilization in the year poststroke occurred within the first 30 days, mean monthly charges for acute care remained significantly above prestroke levels for up to 5 months after the event. Poststroke charges per person-day of follow-up were significantly higher for individuals who were hospitalized for the event, who had subarachnoid hemorrhage, whose stroke occurred after admission to the hospital for another reason, and who died within 7 days. Significantly lower poststroke charges were evident for persons with mild cerebral infarctions and persons whose stroke occurred in a nursing home. Neither prestroke utilization, age category, nor sex were predictive of poststroke charges. The unique population-based data presented here have important implications for efforts toward stroke prevention, intervention, and cost containment. PMID- 8618714 TI - Palliative care in neurology. The American Academy of Neurology Ethics and Humanities Subcommittee. PMID- 8618715 TI - Assessment: Clinical autonomic testing report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. PMID- 8618716 TI - Practice parameters for the diagnosis of patients with nervous system Lyme borreliosis (Lyme disease). PMID- 8618717 TI - Editorials in scientific publications. PMID- 8618718 TI - Animal radical right. PMID- 8618719 TI - Animal radical right. PMID- 8618720 TI - Animal radical right. PMID- 8618721 TI - Animal radical right. PMID- 8618722 TI - Chiropractic complications. PMID- 8618723 TI - Chiropractic complications. PMID- 8618725 TI - Chiropractic complications. PMID- 8618724 TI - Chiropractic complications. PMID- 8618726 TI - Chiropractic complications. PMID- 8618727 TI - Chiropractic complications. PMID- 8618728 TI - Expert witness. PMID- 8618729 TI - Expert witness. PMID- 8618730 TI - Expert witness. PMID- 8618731 TI - Expert witness. PMID- 8618732 TI - Expert witness. PMID- 8618733 TI - Radiation damage. PMID- 8618735 TI - Gene therapy for cancer. PMID- 8618734 TI - Apolipoprotein-epsilon4 and head trauma: Synergistic or additive risks? PMID- 8618736 TI - The use of general practice services by 18 year olds in New Zealand. AB - AIMS: To ascertain the opinions of 861 eighteen year olds about their use and opinion of health services. METHODS: A computer generated questionnaire delivered to 440 males and 421 females at or around their eighteenth birthday. RESULTS: General practice was the overwhelmingly predominant provider of health services to this group with 69% of males and 86% of females having consulted. Satisfaction with the service given by general practitioners was high, as it was with other less common providers. Female users were significantly more likely than males to have problems of embarrassment with the doctor or a worry that their parents might be told of the consultation. While only 4% of females preferred to discuss their health problems with a male doctor, 88% had a male doctor. For 50% of the females, choice of doctor depends on the nature of the problem for which they consult. CONCLUSIONS: General practice is seen by these adolescents as providing a highly satisfactory service and the general practitioner is by far the most commonly consulted health professional. There is, however, no doubt that more choice in the gender of the doctor would improve satisfaction with the service. The study also demonstrates that having the same doctor for all problems is not necessarily acceptable to adolescent female patients. PMID- 8618737 TI - Asthma management at Christchurch Hospital: compliance with guidelines. AB - AIM: To audit compliance with guidelines for the assessment and management of adult patients admitted to Christchurch Hospital with acute asthma. METHODS: An asthma admission form and management guidelines, based on international consensus statements, were designed for use by resident staff at Christchurch Hospital. Compliance with these guidelines was audited during the winter of 1994 by means of retrospective case record review. RESULTS: One hundred and forty three admissions were screened. The form was used in 99 patients (69%), of which 97 had records available for audit. Sixty two patients were admitted under general medical services and 35 under respiratory specialist services. The median age was 34 years (range 14-84) and 77% were female. The history including interval status was adequately documented in over 95% of cases. Peak flow rate was recorded on admission in 93 patients (96%) and spirometry in 62 (64%). During the acute phase of treatment 528 items were prescribed, of which 382 (72%) were appropriate according to the guidelines. The major area (55%) of nonguideline prescribing was the use of nebulised ipratropium in addition to salbutamol for mild or moderate asthma. Written evidence of asthma education was present in 42 (43%). In 34 patients (35%) there was specific reference to the introduction of an asthma action plan. Of the 33 smokers only 17 appeared to have been given smoking cessation advice. Discharge prescribing complied with the guidelines in 71%. The most common variation from the guidelines for discharge therapy related to the manner of prednisone dose reduction. The readmission rate at 1 month was 11%. CONCLUSIONS: The introduction of an asthma admission form enhanced the quality of clinical data gathering by junior staff. Compliance with management guidelines was adequate. Specific sections pertaining to the use of chest radiographs, arterial blood gases and the prescribing of ipratropium and prednisone will be reviewed in updated guidelines. PMID- 8618738 TI - Older adults with lifelong intellectual handicap in New Zealand: prevalence, disabilities and implications for regional health authorities. AB - AIM: To find and describe the total population of persons in New Zealand born before 1940 with lifelong intellectual disability and to provide health and services data for regional health authorities. METHOD: An intensive community based case finding process identified 197 (19%) people not known to the two major service producers, the public hospitals and New Zealand Society for the Intellectually Handicapped (IHC). Prime caregivers provided information about personal characteristics, health status, and services utilisation and needs. RESULTS: One thousand and sixty-three people with lifelong intellectual disability were identified and surveyed. Forty two percent lived in public hospitals, 7% with family, 13% in rest homes and 38% in community based facilities. The national prevalence was 1.43 with wide regional variation. Prevalence in Southern RHA was 2.34, in Central 1.76, in Midlands 0.70 and in North Health 0.87. The age range was 51 to 88, with 56% aged between 51 and 60 years, 32% between 61 and 70 and 12% over the age of 71 years. Twenty three percent had no major condition, disability or disease, 28% had some unspecified neurological impairment, 4% cerebral palsy, 13% Down's syndrome, 17% epilepsy, 21% musculoskeletal impairment, 14% cardiovascular disease and 25% a psychiatric diagnosis. A significantly higher number of physical disabilities was found in people living in hospitals, including 15 of the 17 blind people and 41 of the 53 people with mobility problems. There was wide variation among RHA areas in demand for therapeutic services, community and social support services. CONCLUSION: Past policies and practices of hospitalization have impacted on the present distribution of the study group. The services system must now accommodate regional differences and characteristics of this population and new demands to achieve goals of community integration. PMID- 8618739 TI - Older adults and healthy lifestyle issues: results of a community study. AB - AIM: To discover sources of information about, levels of understanding of, and degrees of commitment to a healthy lifestyle on the part of elderly people in the community as a preliminary to mounting health promotion and education initiatives. METHOD: In 1992 500 people aged 60 and over were surveyed anonymously using a 33 item questionnaire. This sought information on a wide range of health and lifestyle issues in older age. The material in this paper refers to only four of the questions asked, namely those relating to sources of, knowledge of, and interest in information on health and ageing. RESULTS: Doctors were perceived to be the most important sources of health information (89% of respondents rating them as "very" or "moderately" important). Relatives/friends and books/magazines were the next most important sources (56 and 55% respectively). High levels of misinformation about lifestyle issues were revealed. Whilst 85+% of respondents answered correctly that smoking was deleterious and strong social ties advantageous to good health in old age there was confusion about causes of osteoporosis, use of vitamins, likelihood of developing dementia and even the importance of exercise. This has implications for the content of health promotion programmes. Contrary to previously published research, we found no correlation between educational or socioeconomic status or gender, and knowledge about health issues and healthy lifestyles. CONCLUSION: Studies of this type clearly have the ability to provide a range of information which ought to be available to those responsible for the planning of health promotion and education initiatives for older adults. PMID- 8618740 TI - Breast cancer follow up; how much is enough? PMID- 8618741 TI - The cost of a medical education: a student perspective. PMID- 8618743 TI - Sexual activity between doctors and patients. PMID- 8618744 TI - Eating fruit and hypertriglyceridaemia. PMID- 8618742 TI - Sexual activity between doctors and patients. PMID- 8618745 TI - Intrapapillary and peripapillary hemorrhage with incomplete PVD. PMID- 8618746 TI - Diagnosis of the conjunctival biopsy in OCP. PMID- 8618747 TI - Blastomycosis of the eyelid. PMID- 8618748 TI - Vertical strabismus after local anesthesia. PMID- 8618749 TI - Retinal light damage and eye surgery. PMID- 8618750 TI - The "poor man's" corneal shield and retinal light protector. PMID- 8618751 TI - The importance of "quality of care". PMID- 8618752 TI - Prognostic factors for retinal vein occlusion: prospective study of 175 cases. AB - BACKGROUND: The prognosis of retinal vein occlusion is highly unpredictable because nonischemic types may convert into ischemic types within the first months. This study was designed to identify epidemiologic characteristics of the different types of retinal vein occlusion, their visual outcome, and their prognostic factors. METHODS: The authors analyzed prospectively the data from patients who have had retinal vein occlusion with complete medical and biologic examination, including fluorescein angiography, and a 1-year follow-up. RESULTS: One hundred seventy-five retinal vein occlusion eyes consisted of 120 central retinal vein occlusions (CRVO), 7 hemicentral occlusions, and 48 branch occlusions. In initially nonischemic CRVO eyes, retinal ischemia developed in 54%. The study of prognostic factors in the CRVO group showed that older age, male sex, and the number of risk factors (systemic vascular risk factors and glaucoma) were correlated with a poor visual outcome and with the development of retinal ischemia, as well as baseline visual acuity, initial extent of retinal ischemia, and rheologic findings (hematocrit, fibrinogen, and erythrocyte aggregation levels). Logistic regression underlined the prognostic role of sex, the number of risk factors, erythrocyte aggregation, and initial clinical features. Persistent macular edema was shown to be associated with hyperlipidemia and cardiovascular history, and inversely correlated to glaucoma. CONCLUSION: Because clinical characteristics of CRVO may worsen, the authors' results provide a basis to predict visual outcome by taking into account epidemiologic and rheologic findings. A careful follow-up of these patients is recommended. PMID- 8618753 TI - Full panretinal photocoagulation and early vitrectomy improve prognosis of florid diabetic retinopathy. AB - BACKGROUND: Florid diabetic retinopathy (FDR) is a rare form of proliferative diabetic retinopathy (PDR) that is characterized by a bilateral rapidly progressive, very severe ischemic retinopathy. Florid diabetic retinopathy was reported to carry a high risk of blindness. This study was conducted to determine whether visual prognosis of FDR can be improved by appropriate photocoagulation and surgical management. METHODS: The authors retrospectively studied 20 patients (40 eyes) who were treated from October 1978 to February 1994. Systemic risk factors, visual acuity, complete ocular examination, and fundus findings, as well as fluorescein angiography, were analyzed with respect to photocoagulation and surgical management. Mean follow-up was 3.6 years. RESULTS: All patients had poorly controlled type I diabetes (mean duration, 13.5 years), which often was associated with systemic complications. Mean initial visual acuity was equal to or better than 20/40 in 32 eyes (80%). During the course of the study, high-risk PDR was observed in 38 eyes (95%) and vitreous hemorrhage occurred in 26 eyes (65%). Extensive full subconfluent panretinal photocoagulation was performed completely in 37 eyes (92.5%). Vitrectomy was necessary in 15 eyes (37.5%). Macular edema was present in 30 eyes (75%). Major complications included retinal detachment that required surgery (2 eyes, 5%) and neovascular glaucoma (2 eyes, 5%). However, final visual acuity was equal to or better than 20/40 in 23 eyes (57.5%) and less than 5/200 in only 4 eyes (10%). CONCLUSION: These results suggest that aggressive treatment of FDR with extensive panretinal photocoagulation and early vitrectomy, when necessary, may result in a much better prognosis than has been reported previously. PMID- 8618754 TI - Endophthalmitis in patients with retained lens fragments after phacoemulsification. AB - PURPOSE: To review the treatment and outcomes of patients presenting with concurrent endophthalmitis and retained lens fragments after phacoemulsification. METHODS: A retrospective chart review was conducted on patients presenting with culture-proven endophthalmitis and retained lens fragments after phacoemulsification between 1990 and 1994. RESULTS: Five patients were identified with culture-proven endophthalmitis and retained lens fragments after phacoemulsification. In all patients, coagulase-negative staphylococci were cultured from the vitreous fluid. One patient also had positive cultures for Proteus mirabilis and Escherichia coli. The interval between cataract surgery and treatment ranged from 5 days to 6 months. Echography was beneficial in showing retained lens fragments in five of five patients when media opacities obscured the view of the fundus. Four patients had vitrectomy and removal of retained lens fragments during their initial treatment. The fifth patient was treated with intravitreal antibiotics alone and continued to have marked inflammation, eventually requiring vitrectomy for removal of the retained lens fragments. A final visual acuity of 20/400 or better was achieved in four of the five patients. CONCLUSIONS: Patients may present with endophthalmitis in the setting of retained lens fragments after phacoemulsification. In such cases, the preferred initial management may be pars plana vitrectomy, removal of retained lens fragments, and injection of intraocular antibiotics. In eyes with endophthalmitis and opaque media, echography is a useful screening modality. PMID- 8618755 TI - Subfoveal neovascular membrane removal in patients with traumatic choroidal rupture. AB - PURPOSE: To describe the clinical outcomes of patients undergoing pars plana vitrectomy to remove subretinal neovascular membranes caused by traumatic choroidal ruptures. METHODS: Three patients with traumatic choroidal rupture in whom subfoveal choroidal neovascularization developed underwent pars plana vitrectomy with surgical excision of the neovascular membrane. Surgical specimens were examined histopathologically in two patients. RESULTS: The choroidal neovascularization was removed completely in each patient. Visual results were excellent with visual acuities improving to 20/30 or better in each patient. Recurrence of choroidal neovascularization has not been observed. Fibrovascular membranes with reactive retinal pigment epithelium were observed in two specimens examined histopathologically. CONCLUSION: Surgical removal of subretinal neovascular membranes emanating from traumatic choroidal ruptures produced an excellent visual outcome in three patients studied. The neovascular membranes, which were removed with minor disturbance to the underlying pigment epithelium, have similar characteristics to those obtained from patients with ocular histoplasmosis syndrome. PMID- 8618756 TI - Submacular neovascular membrane and focal granulomatous inflammation. AB - BACKGROUND: Subretinal choroidal neovascular membranes in persons younger than 55 years old are commonly idiopathic or associated with the ocular histoplasmosis syndrome. There have been a few reports describing the histopathologic features of these membranes. Studies have shown that idiopathic membranes have the same morphologic features as membranes in age-related macular degeneration except for the absence of basal laminar deposits. METHOD: The authors studied a clinicopathologic case of a macular lesion associated with two peripheral hypopigmented spots in a healthy 30-year-old woman. RESULTS: The clinical and fluorescein angiographic findings in this patient were characteristic of submacular neovascular membranes, except that the edge of the lesion remained distinct in the late phase of the fluorescein angiogram. Results of histopathologic examination of the surgically excised membrane showed a well circumscribed granuloma containing some eosinophils. Attenuated vascular spaces were present within the hard tubercle. Special stains for micro-organisms were negative. The patient had no evidence of a systemic inflammatory disease. CONCLUSIONS: A visible edge despite intense staining in the late phases of a fluorescein angiogram may suggest the possibility of subretinal granulomatous inflammation in a lesion that otherwise appears like a neovascular membrane. The clinical distinction between this pattern of subretinal neovascularization and a typical idiopathic membrane may be important because subretinal granulomatous inflammation could respond to treatment with systemic corticosteroids. PMID- 8618757 TI - Autologous platelet concentrate as an adjunct in macular hole healing: a pilot study. AB - PURPOSE: A pilot study was undertaken to assess the efficacy of autologous platelets in macular hole healing. PATIENTS AND METHODS: Eight eyes of eight patients with stage 3 or 4 macular holes, two of which had failed to heal after previous vitrectomy and gas tamponade, were included. The procedure consisted of pars plana vitrectomy with removal of posterior cortical vitreous, stripping of associated epimacular membranes, 15% perfluoroethane-air tamponade, and instillation of autologous platelet concentrate onto the posterior pole. Strict postoperative facedown positioning was observed for 12 days. Postoperative evaluation included visual acuity measurement, biomicroscopic macular appearance and scanning laser ophthalmoscope examination. The follow-up period ranged from 3 to 13 months (mean, 7 months). RESULTS: Of eight eyes, flattening of the surrounding retina and closure of the hole were achieved in seven (87.5%). Visual acuity improved two lines or more in four eyes (50%) Four eyes (50%) reached a postoperative visual acuity of 20/50 or more. Increased nuclear sclerosis was observed in six eyes (75%), and retinal detachment occurred in two eyes (25%). CONCLUSIONS: Autologous platelet concentrate administered peroperatively in full thickness macular holes seems to be a safe and effective adjunct to vitrectomy with removal of posterior hyaloid and gas tamponade. A larger multicenter randomized prospective study is underway to verify these encouraging results before advocating the use of autologous platelets in macular hole surgery. PMID- 8618758 TI - Visual acuity of eyes after vitrectomy for retinopathy of prematurity: follow-up at 5 1/2 years. The Cryotherapy for Retinopathy of Prematurity Cooperative Group. AB - PURPOSE: To provide long-term follow-up on the structural status and visual function at 5 1/2 years of age for 128 eyes of 98 infants who participated in the multicenter randomized clinical trial of cryotherapy for retinopathy of prematurity in whom total retinal detachment developed from retinopathy of prematurity (ROP) by the 3-month study examination. Fifty-four patients had lensectomy-vitrectomy procedures in one or both eyes before 1 year of age (n=72 eyes), and 44 patients did not (n=56 eyes). METHODS: When the children were 5 1/2 years of age, an eye examination was performed and residua of ROP was assessed. Recognition acuity (Early Treatment of Diabetic Retinopathy Study chart) and grating visual acuity (Teller acuity card procedure) assessments were undertaken by testers who were masked to the status of each of the child's eyes. RESULTS: At least partial retinal attachment was present at 5 1/2 years in 21% compared with 28% at 1 year of age (not significant). All except one of the eyes tested at 5 1/2 years had vision limited to light perception or no light perception, regardless of whether a vitrectomy had been performed. One eye that underwent vitrectomy had minimal pattern vision. The two eyes that were reported previously to have minimal pattern vision at 1 year of age were blind at the longer-term follow-up. CONCLUSIONS: The poor visual outcome after a lensectomy-vitrectomy procedure for retinal detachment due to ROP demands that emphasis be placed on prevention of retinal detachment in premature infants. PMID- 8618759 TI - Visual loss in cytomegalovirus retinitis caused by cystoid macular edema in patients without the acquired immune deficiency syndrome. AB - PURPOSE: Although serous macular exudation has been described in patients with the acquired immune deficiency syndrome (AIDS) with active cytomegalovirus (CMV) retinitis, cystoid macular edema (CME) is not encountered in this clinical setting. In contrast to these findings, we describe vision loss due to CME occurring in immunosuppressed patients without AIDS treated for CMV retinitis. METHODS: Three patients (four eyes) with systemic immunodeficiency presenting with vision loss underwent ophthalmologic examination, including fundus photography and fluorescein angiography. Systemic evaluation was performed to establish the etiology of immunodeficiency and to rule out human immunodeficiency virus infection. Patients were treated with topical corticosteroid and nonsteroidal anti-inflammatory medications for CME. RESULTS: All patients had severe generalized immune deficiency, related either to drug-induced immunosuppression or primary immunodeficiency. Laboratory studies confirmed the presence of systemic CMV infection. Affected eyes had mild reduction of central vision (range, 20/40 to 20/60). Three of four affected eyes had resolving CMV retinitis outside the posterior pole with mild panuveitis. These eyes showed CME on clinical examination and fluorescein angiography. The CMV lesions regressed after reduction of immunosuppressive agents or after systemic antiviral treatment. Response of CME to topical anti-inflammatory medication was variable. CONCLUSIONS: Cystoid macular edema can occur in the setting of resolving CMV retinitis in patients with immunodeficiency other than AIDS. This entity is distinct from serous macular exudation, which can occur in patients with AIDS with active CMV retinitis involving the posterior pole. The disparity between patients with and without AIDS in the development of CME may be important in understanding the pathogenesis of CME. PMID- 8618760 TI - Enlarged blind spots in chorioretinal inflammatory disorders. AB - PURPOSE: This study was undertaken to better characterize patients with multifocal choroiditis and panuveitis (MCP), punctate inner choroidopathy (PIC), multiple evanescent white dot syndrome (MEWDS), and diffuse subretinal fibrosis syndrome. The specific aim was to determine whether these disorders were different entities or part of a spectrum of diseases with similar features. METHODS: Seventy-nine patients were included in this study. Most of the patients have been followed up prospectively since July 1980 with some found retrospectively. RESULTS: Forty-one patients had MCP, 16 had PIC, 6 had diffuse subretinal fibrosis syndrome, and 16 had MEWDS. Patients with MCP had visual loss and visual field defects caused directly by visible lesions or recurrent inflammation around old lesions. In particular, clustering of lesions around the optic nerve and nasal periphery was seen in patients with MCP and appeared to be related to visual field loss. Patients with PIC also had enlarged blind spot and other field defects explained by fundus lesions. Patients with PIC and MCP did not have recurrent lesions on extended follow-up. Patients with diffuse subretinal fibrosis syndrome represented a subset of patients characterized with lesions in the posterior pole, sever scarring, and visual loss. Patients with MEWDS had the least inflammation with symmetrically distributed lesions. Minimal permanent chorioretinal scarring was seen in patients with MEWDS. Visual field defects improved in most patients with MEWDS and PIC, whereas most patients with MCP and diffuse subretinal fibrosis syndrome did not improve. CONCLUSIONS: Although enlarged blind spots are a feature of all four disorders, other clinical, angiographic, and electroretinographic evidence suggest that these are different entities. PMID- 8618761 TI - The use of botulinum A toxin to ameliorate facial kinetic frown lines. AB - PURPOSE: External photography and subjective response were used to evaluate the use of botulinum A toxin to diminish glabellar kinetic folds. METHODS: Eleven patients with glabellar folds and midline forehead wrinkling received one to four injections of 0.1 ml of 100 U/1 ml botulinum A toxin. The injections were given into the procerus or corrugator muscles or both. The number of injections corresponded to the wrinkle lines in each patient. The patients were examined and photographed just before the injections and at 7 to 10 days after the injections. Treatment efficacy was judged by photographic evaluation and by the patient's subjective evaluation of the effect of the treatment. RESULTS: Photographic evaluation showed objective improvement in the glabellar wrinkling in 6 of 11 patients in relaxed facial position and in all 11 patients during contraction of the periocular mulscles. Ten of the 11 patients reported satisfaction with their cosmetic results and indicated that they would choose to have the procedure done again. CONCLUSIONS: The results of this study suggest that botulinum A toxin is a safe and effective treatment for glabellar folds. PMID- 8618762 TI - Silicone frontalis slings for the correction of blepharoptosis: indications and efficacy. AB - PURPOSE: To determine the efficacy of silicone rod frontalis sling ptosis repair in selected patients. METHODS: The authors retrospectively studied 35 consecutive patients who underwent silicone sling ptosis repairs in 6 lids at the University of California, San Francisco. RESULTS: Preoperative diagnoses included congenital ptosis causing developmental delay or possible amblyopia in children younger than 3 years of age, chronic progressive external ophthalmoplegia, third-nerve palsy, myasthenia gravis, and ocular restriction secondary to glaucoma filtering valves. With a mean follow-up of 22 months, good-to excellent final lid height was achieved in all 61 lids. Recurrence of the ptosis occurred in four lids (7%), requiring replacement of the silicone rod in two lids and revision of the original sling in two lids to reach the final lid height. Chronic exposure keratopathy without corneal infection occurred postoperatively in 9 (15%) of 61 eyes, all in patients with an inadequate or absent Bell phenomenon. Chronic corneal problems did not develop in any of the children. Extrusion of the sling with or without infection occurred in three foreheads (5%) in two patients younger than 15 years of age. CONCLUSION: Silicone rod is an effective material for use in frontalis suspension in treating severe ptosis with poor levator function. Children younger than 3 years of age with congenital ptosis and developmental delay or possible amblyopia can undergo silicone frontalis suspension to achieve good visual results. The elasticity and ease of adjustment of the silicone rod are ideal characteristics for a suspensory material used to correct severe ptosis associated with a minimal or absent Bell phenomenon, such as in chronic progressive external ophthalmoplegia, myasthenia gravis, or third nerve palsy. PMID- 8618763 TI - Scleritis and mucosal-associated lymphoid tissue lymphoma: a new masquerade syndrome. AB - PURPOSE: To present a new masquerade syndrome showing features of mucosal associated lymphoid tissue (MALT) lymphoma associated with choroidal white dots and scleritis. Differentials including systemic lymphoma, central nervous system lymphoma, and etiologies of white-dot syndromes and scleritis are discussed. PATIENTS AND METHODS: A 42-year-old man who had decreased vision and ocular redness of his right eye for 4 years had a biopsy-proven diffuse anterior and posterior scleritis associated with intense circumferential perilimbal chemosis and ipsilateral yellow-white choroidal dots. A new conjunctival biopsy was performed because of unresponsiveness to high-dose systemic steroid and cyclophosphamide therapy. Immunostains for lymphocyte markers were preformed. RESULTS: A morphologically and immunohistochemically typical, monotypical mu kappa immunoglobulin light chain secreting B-cell MALT-lymphoma was diagnosed. Eighteen months after completion of radiotherapy, the patient recovered completely, except for the choroidal dots, which remained unchanged. CONCLUSION: When scleritis, even histologically proven, fails to respond to immunosuppressive therapy, a new biopsy is mandatory to rule out a misdiagnosed MALT lymphoma. PMID- 8618764 TI - Pretreatment with topical diclofenac sodium to decrease postoperative inflammation. AB - PURPOSE: Anti-inflammatory medications are traditionally administered to the eye only postoperatively for control of inflammation. Because the presumed mechanism of action of nonsteroidal anti-inflammatory drugs (NSAIDs) is to inhibit the formation of prostaglandin mediators of inflammation, the author studied the effect that pretreatment with an NSAID had on postoperative inflammation. METHODS: In a prospective study, 60 patients scheduled for phacoemulsification with silicone lens implantation were randomly assigned to receive either (1) pretreatment with one drop of diclofenac sodium 0.1% four times per day for the 3 days before surgery plus one drop of diclofenac sodium 0.1% every 15 minutes for four doses, beginning 1 hour before surgery along with the dilating drops; (2) no pretreatment, but one drop of diclofenac sodium 0.1% every 15 minutes for four doses beginning 1 hour before surgery along with the dilating drops; or (3) no diclofenac sodium drops at all before surgery. No anti-inflammatory medications were given either during or immediately after surgery, and all patients were begun on a regimen of one drop of diclofenac sodium 0.1% to the operated eye four times per day after being examined on the first postoperative day. Postoperative inflammation was measured on the first postoperative day and at 1 week with a laser cell and flare meter. RESULTS: On the first postoperative day, the mean flare score for group A was 25.59 photons/millisecond; for group B, 29.69 photons/millisecond; and for group C, 33.07 photons/millisecond. The difference between groups A and C was statistically significant. The differences between groups A and B and between groups B and C were not statistically significant. There was no statistically significant difference for cell scores at 1 day or for cell or flare scores at 1 week. CONCLUSIONS: The pretreatment with an NSAID before cataract surgery can reduce the amount of initial postoperative inflammation. PMID- 8618765 TI - Various glaucomatous optic nerve appearances: clinical correlations. AB - PURPOSE: To study the prevalence of risk factors for glaucoma as well as the pattern of visual field defects and their progression in patients with open angle glaucoma with different and distinct optic nerve appearances. METHODS: One thousand seven hundred eleven optic disc stereo photographs of patients with glaucoma and ocular hypertension and of those suspected of having glaucoma were reviewed to identify pure examples of discs with four different optic disc appearances: focal ischemic discs, myopic glaucomatous discs, senile sclerotic discs, and generalized enlargement of the optic cup discs. The clinical charts of the selected patients were reviewed, with emphasis on the presence of predetermined ocular and systemic risk factors. Their automated visual fields also were analyzed. RESULTS: Thiry-four patients with focal ischemic discs, 38 with myopic glaucomatous discs, 22 with senile sclerotic discs, and 23 with generalized enlargement of the optic cup discs were selected. Patients with myopic glaucoma and generalized enlargement of the optic cup discs were significantly younger than patients with focal ischemic and senile sclerotic discs. There were more women in the focal ischemic group. Patients with senile sclerotic discs had a significantly higher prevalence of ischemic heart disease; they also had a higher prevalence of systemic hypertension, which did not reach statistical significance. Migraine was 2.5 times more frequent in the focal ischemic group than in the other groups. Intraocular pressure was significantly higher in the generalized enlargement group. The pattern of visual field defect in the four groups also was distinctly different. CONCLUSIONS: Patients with different disc appearances, selected only from their disc photographs, showed differences in their demographic characteristics, prevalence of certain systemic risk factors, intraocular pressure levels, and the pattern of their visual field damage. These findings suggest that these various disc appearances probably represent different populations of patients with glaucoma with, possibly, different pathogenic mechanisms. PMID- 8618766 TI - Bleb-related endophthalmitis after trabeculectomy with mitomycin C. AB - PURPOSE: To determine whether filtering blebs resulting from adjunctive use of mitomycin C (MMC) leads to an increased risk of endophthalmitis. METHODS: The authors retrospectively reviewed the records of 232 consecutive trabeculectomies performed at the W. K. Kellogg Eye Center with adjunctive use of MMC from May 1990 through June 1993. Data obtained from the records included patient age, sex, race, type of glaucoma, site of filtration surgery, concentration and duration of exposure to MMC, presence of early or late bleb leakage, and the occurrence of endophthalmitis. RESULTS: Three patients were lost to follow-up less than 1 month after surgery. A total of 229 eyes of 192 patients (11 women and 82 men) were included in the study. Mean follow-up of patients remaining free of infection was 18.5 +/- 10.8 months (range, 1-44 months). The overall incidence of bleb-related endophthalmitis was 2.6%. Endophthalmitis developed in 8% of patients (4 or 50) in whom an inferior approach was used and in 1.1% (2 or 179) in whom a superior approach was used (P = 0.02, Fisher's exact test). The estimated odds ratio for the development of endophthalmitis after trabeculectomy with adjunctive MMC for inferior versus superior filtration sites was 7.7. CONCLUSION: Short-term follow up of trabeculectomies performed with adjunctive use of MMC demonstrates an overall incidence of endophthalmitis comparable to filtrationprocedures performed with 5-fluorouracil or without antifibrotic agents. However, inferior trabeculectomy performed with adjunctive MMC carries a significantly increased risk of bleb-related endophthalmitis compared with filters performed superiorly. PMID- 8618767 TI - Detection of incident field loss using the glaucoma hemifield test. AB - PURPOSE: To examine different definitions of incident visual field loss among patients with elevated intraocular pressure and varying numbers of abnormal glaucoma hemifield test results over an average of 6 years of follow-up. METHODS: A cohort of patients with annual C-30-2 Humphrey visual fields were followed for a minimum of 5 years. Three different definitions of field loss were compared: 1, 2, or 3 consecutive annual abnormal glaucoma hemifield test results. RESULTS: Of 253 subjects, 506 eyes were followed for 5 to 9 years. If incident field loss was defined as one or more normal fields followed by one abnormal glaucoma hemifield test result, the incidence of field loss was 63.6 per 1000 person-years of follow up. For two or three consecutive abnormal glaucoma hemifield test results, the rates were 27.6 and 19.2 per 1000 person-years of follow-up, respectively. Among patients with field loss in one eye at the start of the study, the incidence of field loss in the fellow eye using 1, 2, or 3 consecutive abnormal fields as the definition of incident field loss was 60.9, 55.5, and 25.5 per 1000 person-years of follow-up, respectively. Three years after incident field loss, 31.9% (1 abnormal test result), 76.5% (2 abnormal test results), and 88.5% (3 abnormal test results) of eyes with incident field loss had an abnormal hemifield test result. For eyes with one, two, and three consecutive abnormal glaucoma hemifield test results at the start of the study, 59.2%, 83.6%, and 89.1%, respectively, had an abnormal field 3 years later. CONCLUSIONS: One abnormal glaucoma hemifield test result is not a consistent criterion for defining incident field loss. The use of two or three consecutive abnormal fields to define incident field loss makes it more likely that subsequent test results will be abnormal. PMID- 8618768 TI - Tear flow and evaporation in patients with and without dry eye. AB - PURPOSE: To evaluate the steady-state tear flow and evaporation from the ocular surface of patients with and without dry eye. METHODS: Two groups of patients, 21 with dry eye and 34 without dry eye, with similar age distributions were selected by criteria based on tear osmolarity, Schirmer test, meibomian gland loss, and dry eye symptoms and were compared for tear flow, tear volume, percent turnover, and surface evaporation. RESULTS: Tear flow averaged 0.10 +/- 0.08 microliters/minute in patients with dry eye versus 0.15 +/- 0.12 microliters/minute in patients without dry eye (P = 0.002). Tear volume averaged 2.13+/- 1.3 microliters in patients with dry eye versus 2.23 +/- 2.5 microliters in patients without dry eye (P = not significant) and tear turnover averaged 5.3 +/- 2.9% in patients with dry eye versus 8.2 +/- 4.3% in patients without dry eye (P = 0.019). Evaporation averaged 25 +/- 35 X 10(-7) g/cm(2)/second in patients with dry eye versus 13 +/- 6 X 10(-7) g/cm(2)/second in patients without dry eye (P = 0.003). CONCLUSIONS: Measured tear flow was significantly lower than previously determined in patients with and without dry eye. Evaporation was increased in patients with dry eye and accounted for the majority of the tear loss in patients with dry eye. Normal tear osmolarity can be maintained, even with low tear flow, if evaporation is kept within the normal range. PMID- 8618769 TI - Longitudinal tear study after cyclosporine in kidney transplant recipients. AB - PURPOSE: To determine Schirmer wetting (tear flow) in patients before and after kidney transplantation where systemic cyclosporine was used as an immunosuppressive. METHODS: Patients with end-stage renal disease who received living donor or cadaveric kidney transplants and simultaneous systemic cyclosporine were recruited. A 4-minute Schirmer test with topical anesthetic was performed in both eyes of each person before cyclosporine was initiated and three times after transplantation. The tear flow values from both eyes were averaged before analysis. RESULTS: Tear flow increased significantly from precyclosporine levels of 19.4 +/- 1.5 mm/4 minutes (mean +/- standard error of the mean, n = 7; readings at 1 to 3 days before cyclosporine was begun) to 28.4 +/- 2.4 mm/4 minutes at 1 to 2 months after cyclosporine (P < 0.01), 26.4 +/- 2.0 mm/4 minutes at 3 to 5 months (P < 0.05), and 24.4 +/- 2.1 mm/4 minutes at 9 to 18 months (P < 0.05) after initiation of cyclosporine therapy. No relation existed between tear flow and systemic cyclosporine concentration. CONCLUSION: Tear flow was significantly enhanced by systemic cyclosporine when given to renal allograft recipients as an immunosuppressive. The increased flow rate was sustained over the maximum follow-up of 18 months and indicates an unexpected, beneficial side effect of cyclosporine, even in the absence of a deficit in baseline tear production. PMID- 8618771 TI - Extensive detachment of Descemet membrane after holmium laser sclerostomy. AB - PURPOSE: To describe a corneal complication and its surgical repair after holmium laser sclerostomy. METHOD: A 63-year-old woman had extensive detachment of Descemet membrane 3 months after holmium laser sclerostomy. The authors describe the case history of a detached Descemet membrane secondary to holmium laser sclerostomy and its subsequent repair. RESULTS: The patient achieved a visual acuity of 20/20 in the involved eye after descemetopexy with sodium hyaluronate and air combined with suturing. CONCLUSIONS: Detachment of the Descemet membrane should be recognized as a potential complication of holmium laser sclerostomy. Suturing should be considered as a method of repair if there is not spontaneous reattachment of the Descemet membrane. PMID- 8618770 TI - Intraoperative application of topical mitomycin C for pterygium surgery. AB - BACKGROUND: Postoperative recurrence of pterygium occurs in many patients. The authors studied the recurrence rate of pterygium after administration of a single intraoperative dosage of topcial mitomycin C at the completion of pterygium excision. METHODS AND PATIENTS: Eighty-one patients underwent excision of the pterygium, leaving the sclera bare. The first 60 patients were randomized into two treatment groups of 30 patients each. Their remaining 21 patients were offered mitomycin C. Group 1 included 49 patients (30 randomized and 19 of the remaining 21 patients) who received an intraoperative application of 0.02% (0.2 mg/ml) mitomycin C for 5 minutes, and group 2 included 32 patients (30 randomized and 2 of the remaining 21 patients) who received NaCl 0.9% instead of mitomycin C. Patients were followed from 12 to 28 months in a masked manner. RESULTS: The pterygium recurred in 2 (4%) of 49 patients in group 1 and in 15 (46.7%) of the 32 patients in group 2 (P = 0.0001). A delay of epithelialization for 5 and 10 weeks occurred in two patients in group 2 and granuloma manifested in one patient in group 1. CONCLUSION: This study indicates that intraoperative administration of a single dosage of 0.02% mitomycin C is an effective treatment for prevention of recurrence of pterygium. PMID- 8618772 TI - Surgery of the hereditary subluxated lens in children. AB - PURPOSE: To describe the criteria and techniques for the surgery of hereditary subluxated lens in children and analyze the visual and surgical outcome after an extended period of follow-up. METHODS: From 1982 to 1994, 65 children who had subluxation of their lens were followed in the authors' clinic. Thirty-seven children (59 eyes) underwent surgery (27 eyes had Marfan syndrome; 23 eyes had essential dislocation; and 9 eyes had a diagnosis of homocystinuria, aniridia, microphthalmia, or Weill-Marchesani syndrome). The indications for surgery were best-corrected visual acuity of less than 20/70, forward sub-luxation of the lens to the anterior chamber, monocular diplopia, or rapidly progressing posterior subluxation of the lens. All 54 eyes with subluxation of the crystalline lens behind the iris underwent pars plana lensectomy combined with anterior vitrectomy. In five eyes with total forward dislocation of the lens in the anterior chamber, a limbal approach for removal of the lens and protruding vitreous was used. RESULTS: Postoperative follow-up ranged from 12 to 144 months (average, 55 months). After surgery, 522 eyes (88%) of the entire group achieved an improvement (2 lines or more on the Snellen chart) in best-corrected visual acuity. Seven eyes (12%) did not show improvement and remained with the same visual acuity as before the surgery. Retinal detachment was detected in one eye 2 years after surgery and was the only major postoperative complications observed in these patients. CONCLUSION: These results and the unique continuous and long term follow-up on our patients demonstrate that lensectomy of ectopia lentis combined with anterior vitrectomy using a closed-system technique is a beneficial and relatively safe procedure. PMID- 8618773 TI - Detection of Epstein-Barr virus genome in ocular tissues. AB - BACKGROUND: Epstein-Barr virus (EBV) is a ubiquitous mucosal pathogen with a propensity for lifelong, asymptomatic persistence. Because of reported association between EBV and ocular inflammatory disorders, we tested ocular tissues from normal eyes for presence of the EBV genome. METHODS: Ten freshly harvested cadaveric human eyes were dissected into limbal cornea, central cornea, aqueous humor, iris, vitreous humor, and optic nerve. Total cellular DNA preparations were screened for DNA sequences specific to EBV's large internal repeat region. After Southern transfer, polymerase chain reaction products were detected by a 32P-labeled oligonucleotide probe specific to amplified sequences internal to the polymerase chain reaction primers. RESULTS: Seven of ten eyes from deceased donors yielded a polymerase chain reaction product, indicating presence of EBV genome. In all, 12 (20%) of 60 cadaveric ocular samples contained EBV DNA. Only the optic nerve was consistently negative for EBV DNA. CONCLUSIONS: Detection of EBV DNA in cadaveric ocular tissues indicates a broad anatomic distribution of this persistent mucosal pathogen. The frequency with which EBV was found at apparently normal ocular sites raises the possibility for viral involvement in disease states, but emphasizes the need for specific criteria to implicate EBV in ocular pathology. PMID- 8618774 TI - Therapeutic decisions involving disparate clinical outcomes: patient preference survey for treatment of central retinal artery occlusion. AB - BACKGROUND: Major therapeutic decisions are made by patients with information and guidance provided by their physicians. The values patients place on different outcomes and the risks they are willing to accept are important factors in making these decisions. New beneficial therapies associated with potentially serious complications are now available for some blinding diseases. The authors aim to determine the maximum amount of risk of stroke and death persons would accept to recover vision. METHODS: Standardized survey of adults with normal vision. RESULTS: Thirty-nine percent and 37% of surveyed adults would accept some risk of stroke and death, respectively, to triple the chances of recovering 20/100 visual acuity in one eye when binocular. More than 80% of persons would accept these risks if they were monocular. Maximum risk scores were significantly higher in the monocular case scenarios than in binocular case scenarios. Medical students and eye physicians were more likely to accept risk than persons with high school or university educational backgrounds. CONCLUSIONS: The value persons place on vision when weighed against the risk of stroke or death varies considerably. More persons are willing to accept life-threatening risks if they are monocular. The reasons physicians and medical students are more likely to accept serious risks to improve vision than nonphysicians is unclear. Further studies are needed to determine how physicians' values effect the patient decision-making processes. PMID- 8618775 TI - Effects of cytomegalovirus infection on growth factor production in endothelial cells and fibroblasts. AB - To determine whether cytomegalovirus (CMV) infection alters growth factor production from endothelial cells (EC) or fibroblasts, we infected human umbilical vein EC with CMV VHL/E, a strain of CMV with affinity for human EC, and we infected human foreskin fibroblasts with CMV AD169. CMV caused cytopathic effect and positive CMV staining by immunofluorescence within 5 d, effects not seen in cells infected with UV-irradiated CMV or in uninfected (control) cells. The supernatants from the EC were assayed for platelet-derived growth factor (PDGF)-like protein using a radioreceptor inhibition assay, and EC and fibroblasts were assayed for basic fibroblast growth factor (bFGF) by Western blot analysis. There were no significant differences in PDGF production between groups of EC: CMV-infected EC, 13.5 +/- 2.6; UV-irradiated infected EC, 12.1 +/- 3.6; control EC, 12.9 +/- 1.7 fmol/10(6) EC (mean +/- SD, n = 10, p = NS). There were also no significant differences in bFGF production between CMV-infected EC, UV-irradiated infected EC, and control EC as evidenced by similar intensity of migration of bFGF as a single band at approximately 18 kD (n = 5). In contrast, CMV infection of fibroblasts induced a shift in production of bFGF to higher molecular weight forms migrating at 24 and 26 kD molecular mass. alpha-Interferon failed to alter bFGF production. We conclude that CMV VHL/E infection of EC does not directly alter PDGF or bFGF production from EC. However, CMV infection of cultured human fibroblasts qualitatively alters bFGF by inducing a shift to higher molecular weight forms. PMID- 8618776 TI - Adrenocortical and behavioral predictors of immune responses to starting school. AB - Associations between major psychologic stressors and immune function have been documented in previous research, but few studies have investigated immune changes attending minor, normative stressors. This study examined adrenocortical and behavioral predictors of immune responses to starting kindergarten in 39 five year-old children, who completed laboratory visits for venipunctures 1 wk before (time 1) and 1 wk after (time 2) school entry. At time 1, children were also immunized with pneumococcal vaccine. Immune responses were measured as change scores for T (CD4+ and CD8+) cells, B (CD19+) cells, lymphoproliferative responses to pokeweed mitogen (PWM), and type-specific pneumococcal antibody responses (ABR). Adrenocortical response was assessed as the change in salivary cortisol level, and behavioral difficulty with school adjustment was scored using parental ratings of behavior problems, stress due to changes in routines, and degree of adaptive challenge. Salivary cortisol rose after kindergarten entry (means = 0.39 +/- 0.28 to 0.49 +/- 0.36 micrograms/dL, p = 0.03) and was unrelated to behavioral difficulties. CD4+ cells increased in number, whereas PWM declined, and CD19+ cells showed a borderline increase. Change in salivary cortisol was positively associated with change in CD19+ (delta CD19+) and inversely related to ABR. Scores for behavioral difficulty were inversely associated with delta CD4+ and delta CD19+. These data suggest that: 1) school entry is a stressor capable of evoking elevations in cortisol and behavior problems, accompanied by shifts in functional and enumerative measures of immune status; and 2) children with greater adrenocortical reactivity have increases in B cell numbers and less effective B cell-mediated antibody production, whereas children with more behavioral difficulties show declines in all T and B cell subsets. PMID- 8618777 TI - Changes in hepatic nitrogen balance in plasma concentrations of amino acids and hormones and in cell volume after overnight fasting in perinatal and adult rat. AB - Important regulatory factors of intrahepatic protein synthesis and proteolysis are amino acids, glucagon, insulin, and cell volume. We have investigated the changes in these factors with development and after an overnight fast and evaluated their contribution to changes in the hepatic nitrogen balance in vivo. In the fed state, glucagon levels were highest in suckling animals and gradually declined in older rats, whereas the concentration of insulin increased during development. The amino acid concentrations in liver and plasma declined during the suckling period to levels that in vitro are highly permissive for induction of autophagic proteolysis. In all age groups investigated, fasting was associated with a drop in hepatic protein content, together with a marked decrease in hepatocellular volume and insulin concentrations. On the other hand, glucagon concentrations and the concentration of many amino acids in plasma and liver responded to fasting with a pronounced decrease in perinatal and suckling animals, but this response had become blunted at weaning and had disappeared in adult animals. These findings suggest that insulin and/or hepatocellular volume are more likely candidates as short-term physiologic regulators of the hepatic nitrogen balance than are glucagon or amino acids. In glucose-supplemented fetuses, high levels of insulin could not compensate for a decreased hepatocellular volume in averting a catabolic state, suggesting that cell volume is the more important factor. Although our study cannot discriminate between the effects of fasting on protein synthesis and degradation, our findings show unequivocally that, for a rapid growth of the liver, suckling animals have to be fed around-the-clock. PMID- 8618778 TI - Effect on placental transfer of exogenous lipid administered to the pregnant rabbit. AB - The transfer of lipids across the placenta was measured after infusion of an emulsion of triacylglycerol and phospholipid (intralipid) into 10 anesthetized rabbits. Maternal and umbilical venous and arterial samples were collected at 10 min intervals. All samples were analyzed for concentration and fatty acid composition of FFA, triacylglycerol, and phospholipid fractions. At the end of the infusion period of intralipid, there was a significant increase in the maternal concentrations of total triacylglycerol (p < 0.01) and of total phospholipid (p = 0.01) but not of total FFA (p > 0.05). Maternal plasma triacylglycerol and phospholipid altered in composition to match that of the infused Intralipid by the end of the infusion. Despite the significant rise in maternal triacylglycerol and phospholipid concentrations, the umbilical vein artery difference for these lipid fractions remained unchanged and very low. In contrast, the umbilical vein-artery difference for FFA (p < 0.02) rose gradually throughout the experiments. There was no significant change in the fatty acid composition of the maternal FFA, but the fatty acid composition of the umbilical vein-artery difference for FFA changed to reflect the composition of Intralipid. These experiments show that exogenous triacylglycerol and phospholipid dramatically alter the lipid fractions presented to the placenta in the maternal plasma but do not cross the placenta intact. However, the composition of the FFA crossing the placenta is modulated to become more similar to that of the exogenous lipid. PMID- 8618779 TI - 1995 John Howland Award presentation to Floyd W. Denny, Jr. PMID- 8618780 TI - Disease, war, and biology: languages for medicine--and pediatrics. PMID- 8618781 TI - Society for Pediatric Research presidential address 1995: marrying our medicine to biology. PMID- 8618782 TI - Receptor cell biology: receptor-mediated endocytosis. AB - Receptor-mediated endocytosis (RME) provides one major pathway for the trafficking of extracellular molecules into the cell. This involves the binding of a ligand to a specific cell surface receptor, clustering of the ligand receptor complexes in coated pits, invagination and pinching off of the coated pits to form coated vesicles, and delivery of coated vesicles to discrete membrane-limited cytoplasmic sorting organelles, endosomes. Within these endosomes, ligands and receptors are each targeted to their appropriate cellular destination (e.g., lysosome, cytoplasm, opposite cell surface). The cell and molecular biologic basis for such a tightly regulated process is now beginning to be understood and is reviewed herein. PMID- 8618783 TI - Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues. AB - Replication-deficient adenoviruses have been used to transfer various genes of interest to mammalian tissues in vivo. Effective gene therapy for inborn genetic defects presenting with significant morbidity and mortality at birth will require correction of the defect prenatally. To test the hypothesis that intra amniotically administered adenovirus transfers gene expression to fetal tissues, replication-deficient human type 5 adenovirus carrying the lacZ gene which encodes nuclear-targeted bacterial beta-galactosidase (Av1LacZ4) was instilled into the amniotic cavity of fetal sheep (10(10) to 1.5 x 10(11) pfu) and fetal mice (10(9) pfu) at 0.8 term gestation. Amniotic membranes and gastrointestinal and respiratory tract tissues were harvested after 3 d, bacterial beta galactosidase activity was determined by 5-bromo-4-chloro-3-indoyl-beta-D galactopyranoside (X-gal) enzyme-histochemistry, and tissue integrity was assessed in sections stained with hematoxylin and eosin. Bacterial beta galactosidase activity was abundant in amniotic membranes and present in lower levels in esophagus, stomach, and small intestine as well as in conducting airways and pulmonary alveoli. To determine whether gene transfer by intraamniotic injection of adenovirus was dose-dependent, Av1Luc1, an adenoviral vector carrying the gene for luciferase (10(5)-10(9) pfu), was injected intraamniotically into fetal mice at 0.8 term gestation. Luciferase activity measured after 3 d in tissue homogenates of Av1Luc1-treated fetal mice revealed a linear dose response in amniotic membranes and gastrointestinal and respiratory tract organs. Intraamniotic administration of an adenoviral gene vector leads to expression of the transferred gene in amniotic membranes as well as in fetal gastrointestinal and respiratory tract tissues in a dose-dependent manner. PMID- 8618784 TI - Immunolocalization of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF-receptor in normal and injured developing human lung. AB - The family of growth factors that includes epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are thought to play a role in the regulation of fetal lung development and epithelial repair after injury. To further elucidate the potential role of these growth factors and their receptor in normal human lung development and in response to injury, their distribution was determined by immunohistochemistry in normal fetal lung, as well as both normal and injured postnatal human lung. We studied 14 specimens of human lung tissue: from three fetuses, four normal infants, two preterm infants with hyaline membrane disease, and five infants with late bronchopulmonary dysplasia (BPD). EGF, TGF-alpha, and EGF receptor (EGF-R) colocalized in airway epithelium in normal fetal and in postnatal human lung. They were also colocalized in scattered alveolar epithelial cells in postnatal lung. Large numbers of alveolar macrophages immunostained for EGF, TGF-alpha, and EGF-R in lungs with late stages of BPD. The colocalization of these growth factors suggests parallel expression of EGF family members. Moreover, the colocalization of these growth factors with their receptor in developing lung suggests that they may act through an autocrine mechanism. The prominent expression of these growth factors in alveolar macrophages in BPD suggests they may be involved with the pathogenesis of this disease. PMID- 8618785 TI - Comparison of pulmonary neutrophils in the adult and neonatal rat after hyperoxia. AB - The mechanisms of the increased tolerance to hyperoxia of neonatal animals of many species is incompletely understood. To investigate the etiology of this difference we compared neutrophil entry into the lungs of neonatal and adult rats after hyperoxic exposure. Adult rats were studied after exposure to > or = 98% O2 for 60 h and neonatal rats after 3 and 7 d. Neonatal survival was prolonged compared with that reported for adult rats (77% after 7 d of exposure). In adult rats, there were significant increases in pulmonary neutrophils after 60 h of O2 exposure. In neonatal rats, these changes were not evident after 72 h of exposure, but pulmonary neutrophils increased after 7 d of hyperoxia. Before mortality, pulmonary neutrophils were distributed differently in the age groups. After 7 d of O2 exposure in the neonates, total neutrophil counts in lung tissue (21.92 +/- 7.29 per cm2 grid) and lung myeloperoxidase (0.085 +/- 0.02 U/mg protein) remained significantly lower than in adults after 60 h of O2 exposure (41.44 +/- 9.08 per cm2 grid and 0.411 +/- 0.085 U/mg protein, respectively). However, in histologic specimens, O2-exposed neonates had higher percentages of neutrophils free in the alveolar air space than did adults, corresponding to a trend toward higher neutrophil counts in bronchoalveolar lavage fluid in the neonates. It appears that, in addition to delay in neutrophil influx into the lung, neonatal rats have lowered retention of neutrophils to the alveolar tissue. PMID- 8618786 TI - Regulation of CTP:phosphocholine cytidylyltransferase by cytosolic lipids in rat type II pneumocytes during development. AB - CTP:phosphocholine cytidylyltransferase (CT) catalyses a rate regulatory step in the de novo synthesis of surfactant phosphatidylcholine (PC). We have previously shown that CT activity increases during late gestation in alveolar type II cells, and that this increase is most pronounced in microsomes. As it is known that CT is activated by lipids, we investigated the lipid activation of CT in fetal type II cells during late gestation. The degree of activation of cytosolic CT by PC/oleic acid (OA) (1:1 molar ratio) vesicles was gestation-dependent (a 3-fold stimulation on d 18 and a 1.5-fold stimulation on d 21). In contrast, microsomal CT activation by PC/OA vesicles (1.5-fold) remained constant with advancing gestation. Lipids extracted from microsomes of fetal type II cells of different gestational ages (d 18-21) did not differ in their ability to activate either cytosolic CT of d 18 or 21 fetal type II cells, purified CT from adult lung, or delipidated purified CT. In contrast, lipids extracted from cytosol of fetal type II cells of different gestational ages (d 18 and 21) differed in their ability to activate either delipidated cytosolic CT of fetal type II cells, or delipidated purified CT from adult lung. Day 21 cytosolic lipids activated CT more than d 18 cytosolic lipids. Both cytosolic and purified CT, when delipidated by acetone/butanol extraction, showed reduced activities. Several lipids were tested for their ability to activate cytosolic CT. Acidic phospholipids and the mixture of PC/OA (1:1) were the strongest stimulators of cytosolic CT activity. We conclude that cytosolic but not microsomal lipids are involved in the developmental activation of cytosolic CT in fetal type II cells at late gestation. PMID- 8618787 TI - Dexamethasone enhances surfactant protein gene expression in streptozotocin induced immature rat lungs. AB - Because surfactant protein (SP) mRNA levels in rat fetuses are increased by maternal dexamethasone (dex) treatment and decreased in streptozotocin-induced diabetic (STZ-DB) pregnancy, we investigated the in vivo effects of dex on SP gene expression in STZ-DB pregnancy. The mRNA levels of SP (SP-A, SP-B, SP-C) were assessed in d 18 and 20 fetuses by Northern blot analysis, and nuclear run on assays were performed with lung nuclei from d 20 fetuses (term = 22 d). Our findings indicate: 1) dex causes a greater increase in SP-A and SP-B mRNA levels in d 18 (12-16-fold) compared with day 20 (4-6-fold) fetuses (p < 0.05) in normal and STZ-DB pregnancy; 2) a 2-3-fold increase in SP-C mRNA levels was observed in response to dex in d 18 and 20 fetuses; 3) the increase in transcription of SP-A and SP-B in d 20 fetuses after dex is 68 and 60%, respectively, of the increase in their mRNA levels whereas in STZ-DB, the decrease in transcription compared with mRNA levels is 3.67-fold for SP-A and 2.42 fold SP-B; and 4) changes in SP-C transcription in either in vivo model, dex-treated or STZ-DB, correspond well with changes in mRNA levels. Together, these findings indicate that dex can enhance SP expression in STZ-DB immature lungs and support differential regulation of fetal SP genes in the models studied. PMID- 8618788 TI - Intratracheal pulmonary ventilation versus conventional mechanical ventilation in a rabbit model of surfactant deficiency. AB - Intratracheal pulmonary ventilation (ITPV) enhances the clearance of CO2 from dead space and lungs by a bias flow of gas administered in the distal trachea. ITPV flow is continuously administered through a separate catheter placed within an endotracheal tube (ETT). After exiting from catheter's tip in the distal trachea, the flow of gas is redirected outward away from the lungs. We hypothesized that, compared with conventional mechanical ventilation (CMV), ITPV may increase minute CO2 clearance (VCO2), reduce the partial pressure of CO2 dioxide in arterial gas (PaCO2), and reduce distal tracheal peak inspiratory pressure (dPIP). We induced surfactant deficiency in 15 adult rabbits by lung lavage with 10 mL/kg normal saline. Animals were ventilated through a double lumen 4.0 ETT, inserted through a tracheotomy incision. dPIP, distal positive end expiratory pressure, and distal mean airway pressure were monitored, and the mean exhaled CO2 concentration was measured. For ventilator rates (respiratory rate) of 30, 45, and 70 breaths/min, the study included two phases: phase I compared CO2 clearance and PaCO2 between ITPV and CMV using similar ventilatory pressures; phase II evaluated the effectiveness of ITPV in reducing dPIP and tidal volume (Vt), compared with CMV, while maintaining eucapnea. When comparing ITPV and CMV, the following results (mean +/- SD) were achieved at respiratory rate of 30, 45, and 70 breaths/min, respectively. Phase I ITPV resulted in mean percent reduction of PaCO2 by 31.4 +/- 10%, 37.1 +/- 9.7% and 38.3 +/- 9%; mean percent increase in VCO2 by 61.3 +/- 29%, 56 +/- 23, and 98 +/- 40%, compared with CMV. Phase II ITPV resulted in mean percent reduction of dPIP by 35.5 +/- 14%, 38 +/- 10.8%, and 37.2 +/- 13.7%, and mean percent reduction in Vt by 34.7 +/- 12.9%, 36.4 +/- 15%, and 52.7 +/- 10.7%, compared with CMV. The changes in PaCO2, VCO2 (phase I), and dPIP and Vt (phase II) were all significantly more than 25% (p < 0.05). Oxygenation and pH were not significantly different between ITPV and CMV. We conclude that, in a surfactant deficiency rabbit model, ITPV is an efficient mode of assisted ventilation that increases CO2 clearance and reduces ventilator pressures required for adequate ventilation. We speculate that ITPV can minimize lung barotrauma associated with mechanical ventilation. PMID- 8618789 TI - Aspirin versus indomethacin treatment of patent ductus arteriosus in preterm infants with respiratory distress syndrome. AB - Indomethacin (Indo) is commonly used for treatment of patent ductus arteriosus (PDA) but has renal failure as a main side effect. Aspirin (ASA) is an alternative, but there are no controlled trials on its efficacy. We randomly assigned 75 premature infants suffering from respiratory distress syndrome (RDS) (mean gestational age: 29.6 +/- 2.5 wk, mean birth weight: 1295 +/- 464 g) (+/- SD) and on artificial ventilation at the start of the study (mean: 3.4 d of life), to either Indo (3 x 0.2 mg/kg/12 h) or ASA (4 x 15 mg/kg/6 h). PDA and degree of shunting were evaluated by echocardio-Doppler; side effects were carefully recorded. PDA closed in 35/38 patients from the Indo group (92%) and in 16/37 patients from the ASA group (43%) (p < 0.0001). Nineteen patients needed further treatment with Indo or surgery (17 in the ASA group and 2 in the Indo group). The only side effect observed was a decrease of uresis in the Indo group during 4 d post treatment (p < 0.01). Closing of PDA was positively correlated with gestational age, but not with time of starting Indo/ASA or grade of shunting. We conclude that ASA is not as effective in closing PDA as Indo, but has no adverse effect on uresis. PMID- 8618790 TI - Inotropic effect of increasing concentration of Ca2+ in the fetal rat heart with retinoic acid-induced malformations. AB - Cardiac malformations (pulmonary trunk stenosis, ventricular septal defect, and double outlet right ventricle) were induced by the administration of two doses of retinoic acid (RA) to Wistar rats on d 13 of pregnancy. Contractile performance of the isolated perfused rat heart and its inotropic response to Ca2+ (0.6-10.0 mmol.L-1) was studied in 20-d-old fetuses. The body weight of RA-exposed fetuses was significantly lower compared with controls. RA negatively influenced the contractile parameters of the fetal rat heart. The most pronounced effect was, except at a Ca2+ concentration of 2.5 mmol.L-1, observed at developed force at all other concentrations. Simultaneously, the sensitivity to Ca2+, expressed as the Ca2+ concentration at which 30% of maximum was attained, ws significantly lower in RA-exposed hearts. This implies that the malformed heart is more dependent on the extracellular sources of Ca2+. PMID- 8618791 TI - Differential gene expression and regulation of renal angiotensin II receptor subtypes (AT1 and AT2) during fetal life in sheep. AB - Previous studies have shown that angiotensin II subtype 2 (AT2) receptors appear early during renal embryonic development. Factors involved in the regulation of AT2 receptors during renal development, however, have not been investigated. The present study was designed 1) to characterize the ontogeny of renal AT2 gene expression during the last half of gestation in fetal sheep and newborn lambs, 2) to compare changes in AT1 and AT2 gene expression during renal development, 3) to determine the influence of AII in modulating renal AT1 and AT2 gene expression during fetal life, and 4) to characterize the role of cortisol in modulating renal AT2 gene expression during the last trimester of gestation in fetal sheep. To perform these studies, we first isolated and cloned a polymerase chain reaction product that has 92 and 90% homology with the cDNA encoding the human and rat AT2 receptors, respectively. Using this sheep AT2 cDNA probe, we demonstrated that the sheep AT2 gene was encoded in a single locus. In addition, we showed that renal AT2 mRNA expression was high early during fetal life (60-90 d gestation) and decreased rapidly thereafter. In contrast, the expression of renal AT1 receptor gene was low at 60-d gestation and increased during the last trimester of gestation. We found that a continuous i.v. infusion (1 mL/h) of AII (9.5 mM/n) for 24 h, which raised plasma AII levels from 84 +/- 9 pg/mL to 210 +/ 21 pg/mL, decreased the expression of both renal AT1 and AT2 genes in third trimester fetal sheep. On the other hand, we observed that cortisol, known to decrease AT1 gene expression in the fetus, had no effect on AT2 gene expression. In summary, this study demonstrates that AII, but not glucocorticoids, contributes to the regulation of renal AT2 gene expression during development and that there is differential regulation of AT1 and AT2 receptors. PMID- 8618792 TI - Limb reduction defects after prenatal inhibition of nitric oxide synthase in rats. AB - To determine the influence of nitric oxide (NO) on vascular tone during fetal development, timed pregnant rats received the NO synthase inhibitor NG-nitro-L arginine methyl ester for consecutive 4, 7, or 14 d before parturition (postorganogenesis). Offspring demonstrated limb reduction defects (incidence, 53%) involving either or both hindlimbs, whereas forelimbs were uniformly spared. Defects were dose-dependent but independent of the duration of administration occurring with equal frequency in 4-, 7-, and 14-d treatment groups. Histologic analysis revealed features characteristic of vascular disruption with hemorrhagic necrosis and loss of structure. The defects were prevented by concurrent maternal administration of L-arginine or the NO donors S-nitroso-N-acetyl-penicillamine and sodium nitroprusside. Defects were not seen after prenatal treatment with aminoguanidine. To study basal and agonist-mediated NO release, newborn femoral and brachial arteries were cannulated with a glass micropipette under constant pressure, and changes in intraluminal diameter (micrometers) were measured in response to acetylcholine and the NO synthase inhibitor N omega-nitro-L-arginine. Newborn femoral and brachial vessels demonstrated a dramatic (59%) decrease in resting diameter compared with adult vessels (16%). These findings suggest that basal NO release is upregulated during fetal development concurrent with the processes that increase maternal NO release. The data also suggest that up regulation of NO release occurs throughout the fetal systemic circulation and is not restricted to hindlimbs. This is the first study to demonstrate inhibition of NO release in the pathogenesis of limb reduction defects. PMID- 8618793 TI - Selective destruction of nitric oxide synthase neurons with quisqualate reduces damage after hypoxia-ischemia in the neonatal rat. AB - The vulnerability of the developing CNS to hypoxia-ischemia (H-I) differs from that of the mature brain and is due in part to release of nitric oxide (NO) from parenchymal neurons. If NO is important in the generation of excitotoxic injury after H-I in the developing CNS, then selective destruction of the neuronal nitric oxide synthase (nNOS) cells before H-I should lessen the injury seen after the insult. Using low dose quisqualic acid (QA) injected into neonatal (postnatal d 7) parietal cortex, the nNOS neurons were eliminated while sparing other neuronal and glial populations as ascertained by NADPH diaphorase histochemistry, nNOS immunocytochemistry, and Nissl counterstain. Animals subjected to focal ischemia followed by global hypoxia 24 h after the intracortical injection of QA had more viable cortex remaining than vehicle-injected animals (83.4 +/- 4.3% versus 62.7 +/- 8.3%) and lower injury severity represented by less neuronal loss and gliosis. Intracortical injections of QA without H-I resulted in minimal cell loss at the injection site with elimination of nNOS neurons throughout the parietal cortex. Microglial and astrocytic proliferation was seen in areas damaged by H-I 3 wk after injury and clearly marked infarcted areas. Prevention or elimination of NO production from nNOS cells can prevent much of the delayed neuronal necrosis seen after H-I in the developing CNS. PMID- 8618794 TI - Quantitative relationship between brain temperature and energy utilization rate measured in vivo using 31P and 1H magnetic resonance spectroscopy. AB - In neonatal and adult animals, modest reduction in brain temperature (2-3 degrees C) during ischemia and hypoxia-ischemia provides partial or complete neuroprotection. One potential mechanism for this effect is a decrease in brain energy utilization rate with consequent preservation of brain ATP, as occurs with profound hypothermia. To determine the extent to which modest hypothermia is associated with a decrease in brain energy utilization rate, in vivo 31P and 1H magnetic resonance spectroscopy (MRS) was used to measure the rate of change in brain concentration of phosphocreatine, nucleoside triphosphate, and lactate after complete ischemia induced by cardiac arrest in 11 piglets (8-16 d). Pre ischemia metabolite concentrations and MRS-determined rate constants were used to calculate the initial flux of high energy phosphate equivalents (d[approximately P]/dt, brain energy utilization rate). Baseline physiologic and MRS measurements were obtained at 38.2 degrees C and repeated after brain temperature was adjusted between 28 and 41 degrees C. This was followed by measurement of d[approximately P]/dt during complete ischemia at 1-2 degrees C increments within this temperature range. Adjusting brain temperature did not alter any systemic variable except for heart rate which directly correlated with brain temperature (r = 0.95, p < 0.001). Before ischemia brain temperature inversely correlated with phosphocreatine (r = -0.89, p < 0.001), and reflected changes in the phosphocreatine-ATP equilibrium, because brain temperature inversely correlated with intracellular pH (r = -0.77, p = 0.005). Brain temperature and d[approximately P]/dt were directly correlated and described by a linear relationship (slope = 0.61, intercept = -12, r = 0.92, p < 0.001). A reduction in brain temperature from normothermic values of 38.2 degrees C was associated with a decline in d[approximately P]/dt of 5.3% per 1 degree C, and therefore decreases in d[approximately P]/dt during modest hypothermia represent a potential mechanism contributing to neuroprotection. PMID- 8618795 TI - Effect of theophylline on metabolic and ventilatory response to hypoxemia during quiet sleep in piglets. AB - Theophylline, a drug frequently used in newborns, stimulates respiration and increases the metabolic rate in a sustained fashion; hypoxemia, on the other hand, decreases metabolic rate and increases ventilation slightly and, at times, only transiently. This study looked at the effect of theophylline on the metabolic and ventilatory response to hypoxemia in piglets. We studied two groups of piglets during normoxia and hypoxemia: first during a baseline period; and second, after the infusion of either theophylline or a placebo. All studies were done in quiet sleep, 2 d after instrumentation was performed to place vascular catheters and electroencephalographic electrodes. O2 consumption (VO2) and CO2 production (VCO2) were measured in a metabolic chamber, and alveolar ventilation (VA) was then derived from VCO2 and PaCO2. We found that theophylline did not abolish the small decrease in oxygen consumption brought about by hypoxemia. Nor did theophylline augment the ventilatory response to hypoxemia. In fact, the percent change in alveolar ventilation decreased slightly: going from 17 +/- 8% during the baseline period to 9 +/- 6% (p < 0.005) after theophylline administration. We found a significant increase in respiratory exchange ratio (R) in response to hypoxemia (from 0.87 +/- 0.05 to 0.97 +/- 0.04, p < 0.001). However, after the administration of theophylline, additional exposure to hypoxemia did not result in a change in R. In summary, our results show that, in sleeping newborn piglets, theophylline does not abolish the decrease in oxygen consumption observed in response to hypoxemia; nor does it enhance the ventilatory response to a moderate degree of hypoxemia. PMID- 8618796 TI - Glottal patency during experimental seizures in piglets. AB - Tracheostomized cats and piglets demonstrate respiratory stimulation during experimental seizures, whereas airway intact piglets demonstrate hypoventilation and increased subglottic resistances. The purpose of this study was to characterize the role of the vocal folds in contributing to these increased subglottic resistances during experimental seizures. A controlled animal study was performed in six anesthetized, spontaneously breathing, hyperoxic piglets who had subglottic pressure and airflow measured. A fiberoptic video scope directed into the cephalad trachea recorded subglottic images of the vocal folds. Glottal area patency (GAP) was evaluated at inspiratory onset (Io), peak inspiratory pressure (Ip), and expiratory onset (Eo) for four to five consecutive breaths under baseline control, ictal, interictal, and anticonvulsant conditions. Seizures were induced with i.v. pentylenetetrazol or bicuculline. Normalized GAP was greatest at Ip under all conditions, except anticonvulsant. During ictal periods, piglets demonstrated significant reductions in mean GAP throughout the respiratory cycle (Io, 98%; Ip, 78%; Eo, 98%), compared with baseline (p < 0.001, repeat measures analysis of variance). During ictal discharges, hypoventilation and glottal obstruction resulted in significant reductions in mean arterial pH ( 0.35) and PaO2 (-39 kPa) and elevations in PaCO2 (+8.1 kPa), compared with baseline conditions. During interictal conditions mean GAP at Ip was increased, whereas at Eo (-66%) GAP was reduced, compared with control. These data demonstrate that the vocal folds are tonically adducted throughout the respiratory cycle during ictal phases and have increased expiratory adduction during interictal phases of seizures induced with standard i.v. convulsants in hyperoxic piglets. PMID- 8618797 TI - Effect of drugs on chemoreceptor responsiveness in fetal sheep. AB - This study was designed to examine the effects of the drugs ketamine, morphine, pentobarbital, and propranolol on fetal chemoreceptor responsiveness. Eleven fetal lambs (gestational age 125-133 d) were chronically instrumented with a catheter in a hindlimb artery and vein and a forelimb artery; a carotid arterial oximeter catheter was placed in six of these fetuses. An inflatable cuff occluder was placed around the maternal hypogastric artery. Acute fetal hypoxemia was induced repeatedly by reducing uterine blood flow. Fetal heart rate, arterial pressure, and carotid arterial oxygen saturation were monitored continuously before and after administering ketamine, morphine, pentobarbital, or propranolol to the fetus. The ratio delta heart rate/delta O2 saturation has been shown previously to be a reproducible index of chemoreflex response. The differences in baseline values and changes with drugs were compared by multiple regression analysis coded by effects. Chemoreflex response was markedly attenuated by ketamine and morphine but not by pentobarbital or propranolol. Because the cardiovascular response to hypoxemia is blunted by some drugs, caution should be exercised in interpreting heart rate responses to hypoxemia in the fetus when these drugs have been administered to the mother. PMID- 8618798 TI - Cerebral responses to maternal cocaine injection in immature fetal sheep. AB - Previous studies in near-term sheep have shown that maternal cocaine injection causes acute fetal cerebral vasodilation along with transient hypoxemia and hypertension. Preterm sheep fetuses have lower cerebral O2 consumption (CMRO2) and their cerebrovascular responses to hypoxemia are attenuated compared with near-term fetuses. We therefore tested the hypothesis that fetal cerebrovascular responses to maternal cocaine injection may also differ earlier in gestation. We studied nine immature fetal sheep at 0.65 gestation using the same experimental protocol we used in previous studies in near-term sheep. Fetal studies were done in utero, 2 d after vascular catheter placement. We measured cerebral blood flow (CBF) using microspheres, arterial and sagittal sinus O2 content, and cocaine concentrations. We calculated cerebrovascular resistance (CVR) as mean arterial blood pressure divided by CBF. Measurements were made before and 2, 5, and 15 min after a 2 mg/kg maternal cocaine injection. At 2 min, fetal Cao2 decreased (18 +/ 6%, mean +/- SEM), and there was cerebral vasoconstriction (CVR increased by 22 +/- 5%). At 5 min, CBF increased (19 +/- 9%), but because blood pressure increased also, CVR returned to baseline, and therefore there was no vasodilation compared with baseline. Furthermore, at 5 min there was a 22 +/- 6% decrease in Cao2 and a 21 +/- 6% increase in mean arterial blood pressure. There were no changes in CMRo2 throughout the study, but at 2 min, cerebral O2 delivery decreased. Differences in cerebrovascular responses to maternal cocaine injection earlier in gestation may be due to differences in vascular development and/or to developmental differences in responses to cocaine, cocaine metabolites, and/or to hypoxemia. PMID- 8618799 TI - Bilirubin induces a calcium-dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase II activity in vitro. AB - Excessive bilirubin levels in newborn infants result in long-term neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate excitotoxicity. Because neonatal bilirubin exposure results in neuronal loss in developing brain systems, we tested whether bilirubin exposure would induce an immediate inhibition of CaM activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase II activity (IC50 = 16.78 microM). At concentrations above 50 microM, bilirubin exposure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activity (p < 0.001, t test, n = 10). Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 +/- 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 +/- 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase II activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase II activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which bilirubin exposure results in delayed neuronal cell death in developing brain. PMID- 8618800 TI - Serum osteocalcin and bone and liver alkaline phosphatase isoforms in healthy children and adolescents. AB - We report reference values for osteocalcin and bone and liver alkaline phosphatase (ALP) isoforms in serum from 114 healthy children and adolescents, age 7-16 y. The bone and liver ALP isoforms were measured by high performance liquid chromatography (HPLC). Six ALP isoforms were separated and quantified in all samples: one bone/intestinal (B/I), two bone (B1 and B2), and three liver ALP isoforms (L1, L2, and L3). It is especially important to make distinctions between ages and gender during puberty in the interpretation of biochemical markers of bone. We observed that girls reached adult levels for osteocalcin, total ALP, and bone ALP isoforms, B/I, B1, and B2, earlier than boys. Girls in the oldest age group (15-16 y) and in the pubertal stage groups IV-V, reached higher levels than boys for the calculated ratio B1/B2 due to a more rapid decline of B2 compared with B1. This observation might indicate that B2, to a higher extent than the B1 isoform, represent a more mature expression of osteoblast differentiation during matrix mineralization in growing bone. Serial measurements of these new bone ALP isoforms could provide new insights into bone and mineral metabolism. PMID- 8618801 TI - Isolated growth hormone deficiency: testing the little mouse hypothesis in man and exclusion of mutations within the extracellular domain of the growth hormone releasing hormone receptor. AB - The phenotypic characteristics of isolated growth hormone deficiency (IGHD) type IB in humans, such as autosomal recessive inheritance, time of onset of growth retardation, diminished secretion of growth hormone (GH) and IGF-I, proportional reduction in weight and size, and delay in sexual maturation, has much in common with the phenotype of the homozygous little/little (lit/lit) mouse. Sequencing of the GH releasing hormone (GHRH) receptor in lit/lit mice has shown a single nucleotide substitution within the extracellular peptide binding domain at codon 60 that changed aspartic acid to glycine. Therefore, the GHRH receptor is a reasonable candidate gene for causing IGHD in humans. DNA from 65 unrelated healthy Caucasians of normal stature and 65 children with IGHD type IB of whom 12 did not respond to exogenous treatment with GHRH were studied. Restriction endonuclease analysis, linkage studies, and polymerase chain reaction amplification and sequencing of the whole extracellular domain including the first three membrane spanning domains of the GHRH receptor gene were performed. None of the analyses revealed any structural abnormalities in these patients with IGHD. This suggests that a lit/lit mouse equivalent is an unlikely explanation for the majority of children with IGHD. Although gross structural abnormalities in the whole gene have been ruled out in this study, mutations in the carboxyl terminus are still possible, and, therefore, the remaining part of the gene needs to be sequenced. PMID- 8618802 TI - Isolated central hypothyroidism in short stature. AB - Mild hypothyroidism can be difficult to diagnose, particularly when it is caused by deficiency of TSH or TSH-releasing hormone (TRH). Such central hypothyroidism occurs most often in association with growth hormone deficiency (GHD). Isolated central hypothyroidism has been considered rare. The TSH surge test is the most sensitive test currently available for confirming the diagnosis. Results are abnormal in many cases where response to TRH is normal. Short children referred between 1986 and 1994 for evaluation (n = 235, 74 girls; height 2 or more SD below mean for age; no history of radiation therapy) were selected for study. TSH samples were obtained hourly for study of TSH surge (1400-1800 h and 2200-0400 h). Growth hormone (GH) samples were obtained every 20 min (2000-0800 h). GH stimulation tests included arginine, insulin, and L-dopa. Thirty-nine children were found to have GHD (peak GH < or = 7 micrograms/L) and 15 had mild primary hypothyroidism (TSH 5-15 mU/L). Of the remaining 181 children with apparent idiopathic short stature (ISS), 30 had a blunted TSH surge. All 30 had free thyroxine (FT4) in the lowest third of the normal range, consistent with values seen in documented central hypothyroidism. They were 13% of the total group, 16% of the ISS children, and 32% of the ISS children with FT4 in the lowest third of normal. (In comparison, diagnosis of GHD was made in 16% of the total group.) In conclusion, use of a direct FT4 assay to screen short children for possible subtle hypothyroidism (along with other appropriate testing) identifies a subgroup with a one in three possibility of an easily treated cause--isolated central hypothyroidism. PMID- 8618803 TI - Longitudinal change of sonographic ovarian aspects and endocrine parameters in irregular cycles of adolescence. AB - We longitudinally studied clinical endocrine and ultrasound parameters of the ovaries in 73 healthy adolescents having persistent menstrual irregularities. After the first examination, they were reexamined after a variable period ranging from 2 to 7 y. During the first examination, three basic features of the ovaries were observed: homogeneous (36%), multifollicular (23%), and polycystic (41%). Polycystic ovaries were most frequent, and they generally exceeded the normal adult range. During the last examination, in the entire group of irregular adolescents, homogeneous ovaries decreased (-14%), polycystic ovaries increased (+18%), and a further higher number of subjects exceeded the normal adult range (+10%). The subjects with enlarged ovaries had the highest values of LH, testosterone, and androstenedione. Fourteen subjects out of 46 (30%), with normal ovarian volume in the first examination, registered an ovarian enlargement in the last examination, exceeding the normal range. Moreover, a change from the homogeneous or multifollicular structure to the polycystic one was observed. Twenty-one subjects out of 27 (78%) with enlarged ovaries in the first examination confirmed the high ovarian volume and the unchanged structure in the last examination, whereas six subjects (22%) showed ovaries within the normal adult range; the polycystic structure was substantially confirmed. These results indicate the following. 1) Homogeneous, multifollicular, and polycystic ovaries can usually be found in the postmenarcheal period. 2) Enlarged ovaries, polycystic structure, hyperandrogenemia, and high LH values are strongly linked, and they are frequent in irregular cycles even in the absence of signs of hyperandrogenism. These characteristics may all persist or in various aggregations become a permanent feature. 3) Only a few subjects may lose ovarian enlargement and show a change in the polycystic structure; however, they frequently maintain hyperandrogenemia. 4) During the postmenarcheal period, normal ovarian characteristics may suddenly change, and the ovaries may take on a polycystic structure and increase in volume. Moreover, some endocrine parameters may reach pathologic levels. PMID- 8618804 TI - Biochemical mediators of meningeal inflammatory response to group B streptococcus in the newborn piglet model. AB - The meningeal inflammatory response to a heat-killed mutant unencapsulated strain of type III group B Streptococcus (GBS) was studied in a newborn piglet model. GBS (10(9) colony-forming unit equivalents) or saline (control) was inoculated intraventricularly. Serial cerebrospinal fluid measurements were done at baseline and over the course of the next 24 h for cytochemical changes and production of tumor necrosis factor (TNF) and prostaglandins. In separate experiments, we defined the time course of early changes during the first 6 h and dose response relationship over a range of inocula 10(6) to 10(9) colony-forming unit equivalents. The intraventricular inoculation of the heat-killed unencapsulated GBS induced marked leukocytosis and increased protein by 6 h. These changes were preceded by a several hundredfold increase in TNF (maximum at 2 h) and prostaglandins (maximum at 2-4 h). The early and sharp rise in TNF suggests its pivotal role in initiating the inflammatory cascade. The magnitude of the inflammatory response increased with increasing bacterial dose over the range studied. To study the effect of encapsulation of GBS in the induction of meningeal inflammation, we compared the response to the unencapsulated mutant strain with that to the encapsulated parent strain. The encapsulated strain produced much smaller inflammatory changes, and only with high doses of bacteria. The GBS cell wall appeared to be the primary bacterial product triggering inflammation. Intraventricular injection of the heat-killed unencapsulated GBS with exposed cell wall can serve as a valid model for studying neonatal meningitis. PMID- 8618805 TI - Comparison of M and T type antigen testing to field inversion gel electrophoresis in the differentiation of strains of group A streptococcus. AB - Recent clusters of patients with acute rheumatic fever and invasive group A Streptococcus (GAS) have stimulated renewed interest in the epidemiology of streptococcal infections. We compared conventional serotyping for M and T antigens and serum opacity factor with field inversion gel electrophoresis (FIGE) for distinguishing among GAS. Fifteen pairs of throat isolates obtained from children positive for GAS before and after therapy were evaluated by conventional serotyping and by FIGE after SmaI digestion. Ten of the 15 pairs were identical by serotyping. FIGE correctly identified the 10 concordant and 5 discordant pairs. Individual clones were identified within each M type tested, including analysis performed on additional isolates of M1 and M3 obtained from the Centers for Disease Control and Prevention. This preliminary experience suggests that FIGE can successfully determine whether serial isolates from a given patient represent persistence of one strain or acquisition of a new strain of GAS and that this method might provide an alternative typing system for GAS. PMID- 8618806 TI - Platelets and neutrophils from healthy term neonates exhibit increased levels of immunoglobulins. AB - Immune-mediated thrombocytopenia and neutropenia are not uncommon problems in the newborn period. Such cytopenias have been associated with increased levels of IgG, IgM, or both, in the serum, documented by indirect assay, and/or on the cell surface, documented by direct assay, and decreased cell survival. However, interpretation of measurement of platelet- or neutrophil-associated antibodies is problematic due to the lack of data from healthy neonates. In this study, both direct and indirect neutrophil- and platelet-associated IgG and IgM were measured in cord samples from 44 healthy, term neonates. These infants had increased amounts of direct platelet-associated IgG and direct neutrophil-associated IgM and IgG compared with adult controls. Serum samples from these healthy newborns manifested a significantly decreased level of IgM binding to target platelets compared with serum from healthy adult controls. There was not a significant difference in direct platelet-associated IgM, or indirect Ig to neutrophils or platelets. Complete blood counts drawn at 24 h of age were within normal limits in 34/35 infants studied. Moreover, there was not a statistical difference in platelet or neutrophil Ig studies between the newborns of multiparous and primigravida mothers. The physiologic consequences of the increased amounts of these Ig to the survival and function of platelets and neutrophils in neonates is unclear. However, these values must be considered for proper interpretation of platelet and neutrophil Ig measurements in newborns with cytopenias. PMID- 8618808 TI - [Stress echocardiography in the diagnosis and assessment of ischemic heart disease]. PMID- 8618809 TI - [Prognostic value of nocturnal pulse-oximetry in patients with chronic obstructive pulmonary disease treated with oxygen at home]. AB - The aim of this study was to investigate if results of overnight pulse-oximetry (OPO) predict survival in COPD patients undergoing long-term therapy (LTOT). 97 COPD patients (74 M and 23 F) qualified for LTOT from 1986 to 1992 were studied. When qualifying for LTOT patients were mean age 59 years old, had severe airway obstruction (mean FEV10,9L), hypoxaemia (mean PaO2 53 mmHg) and hypercapnia (mean PaCO2 48 mmHg). Two OPO were performed in each patient: the first on atmospheric air and the second while breathing oxygen. All patients were observed for at least 2 years or until death. Fifty five patients died during the observation time. All deaths were related to the primary disease. Survival analysis by Cox formula showed the prognostic value of mean, initial and minimal arterial oxygen saturation while breathing air and of initial oxygen saturation while breathing oxygen. Also classical factors, such as age or FEV1, had the prognostic value in this group of patients. IN CONCLUSION: results of overnight oxygen saturation affect prognosis in COPD patients despite long-term domiciliary oxygen treatment. PMID- 8618807 TI - Anaphylactic reactions to bee-sting challenges in allergic children are not modified by endogenous catecholamines. AB - To investigate the role of basal catecholamine levels and the response of the adrenergic system to expected bee stings, plasma catecholamines were measured before and 1 and 2 min after bee-sting challenges. Twenty-one children (aged 4-15 y) with bee-sting allergies were selected for sequential challenges to establish the need for venom immunotherapy. The time interval between the challenges varied from 2 to 6 wk. Epinephrine, norepinephrine, and dopamine plasma levels were measured using a simultaneous single-isotope radioenzymatic assay. On the first challenge, 33% of the children experienced a normal local reaction, 29% a large local reaction, and 38% a systemic reaction. On the second challenge in 18 out of 21 subjects, 67% experienced a normal normal local reaction, 22% a large local reaction, and 11% a systemic reaction. Epinephrine and norepinephrine plasma levels increased significantly on the first and second challenges. Dopamine plasma levels showed a significant increase on the first challenge only. Plasma catecholamine levels after the second challenge revealed a significant positive correlation between epinephrine increases measured 1 and 2 min after the challenge and the concomitant sting reaction. Basal epinephrine, norepinephrine, and dopamine plasma levels did not differ significantly between patients who experienced different types of sting reactions. Based on our data, we conclude that clinical reactions to in-hospital insect-sting challenges are not affected by early increases in plasma catecholamine levels produced by the expected stress situation. PMID- 8618810 TI - [Does multiple use of a cuprophan dialyser influence levels of chromium and cadmium during hemodialysis in patients with chronic kidney failure?]. AB - 42 patients between 21 and 63 years of age with chronic renal failure (CRF) treated with haemodialysis and 16 healthy subjects between 19 and 72 years of age were observed. All patients were divided in two groups: 15 patients treated with haemodialysis with cuprophan dialyser used first time and 27 patients treated with haemodialysis with cuprophan dialyser used more than three times. The blood samples in haemodialyzed patients were withdrawn four times: just before dialysis, during haemodialysis (after one hour of dialysis at both ends of the dialyzer) and after haemodialysis. Chromium and cadmium concentration in all examined groups, in dialysis fluid and in demineralized water were measured by flame atomic absorption spectrophotometry. During haemodialysis a decrease of serum chromium concentration and an increase of serum cadmium concentration in two groups of haemodialyzed patients were observed. No significant differences in serum chromium and cadmium concentration before dialysis, during dialysis and after dialysis were observed between two groups of haemodialyzed patients. CONCLUSION: The multiple use of cuprophan dialser did not influence significantly serum chromium and cadmium concentration in haemodialyzed patients. PMID- 8618811 TI - [Influence of long-term treatment with human recombinant erythropoietin on secretion of hormones regulating carbohydrate metabolism in hemodialyzed patients with chronic uremia]. AB - The study aimed to assess the influence of long-term rhuEPO treatment on secretion of pancreatic hormones (insulin, glucagon). A total of 27 haemodialyzed and 9 healthy subjects were examined. Nine patients with uraemic anaemia were treated with rhuEPO for 12 months (EPO group) while another nine patients did not receive rhu-EPO (non-EPO group), but were monitored biochemically and clinically as patients of the EPO group. The third group (HD) comprised nine haemodialyzed patients with a haematocrit value of > 30%, without rhu-EPO therapy. In all subjects plasma levels of glucose, insulin (IRI) and glucagon (Glu) were estimated before and after administration of the test meal. Patients of the EPO and non-EPO group were examined before and after 6 and 12 months of rhu-EPO treatment (EPO group) or clinical monitoring (non-EPO group) respectively, while only one test was performed in patients of the HD group and healthy subjects. During the observation period fasting glicaemia did not change. Six months of rhu EPO therapy was followed by an increase of fasting insulinaemia and decrease of basal plasma level of glucagon. At that time point rhu-EPO therapy also increased the response of IRI and Glu to the test meal and the insulin/glucose index. After 12 months of rhu-EPO therapy basal insulinaemia and insulin/glucose index returned to the pretreatment value while plasma level of glucagon and the response to the test meal were lower than pretreatment one. Our results suggest that rhu-EPO treatment exerts a profound effect on carbohydrate metabolism and secretion of IRI. Glu, which seems to be dependent upon duration of rhu-EPO therapy and not only, or exclusively to improvement of the haematological status. PMID- 8618812 TI - [Humoral immune response to tissue injury, characterized by a rise in serum immunoglobulin E]. AB - We have observed that acute myocardial infarction is associated with the immunological response characterized a transient rise of serum immunoglobulin E (IgE). We wondered whether this reaction was specific for myocardial infarction or whether it reflected a more generalized phenomenon, perhaps triggered by tissue injury. We, therefore, made a large prospective study on patients undergoing various surgical procedures. These were the patients undergoing coronary artery bypass graft, who did (n = 39) or did not (n = 42) develop perioperative myocardial infarction, patients subjected to thoracic operations (n = 33) patients having cholecystectomy (n = 17) or repair of the inguinal hernia (n = 18) and 30 healthy volunteers forming the control group. Blood samples were drawn before the operation and then after the operation at 8, 16, 24, 48, 72, 120, 168 and 216 hours. In all samples, concentrations of serum immunoglobulins E were determined on blinded specimens by an automated microparticle enzyme immunoassay while immunoglobulins G, A and M were determined using nephelometry. In all groups studied, except the control group, serum IgE began to rise shortly after the operations reached a peak by the fifth day, and then gradually declined. This behaviour of IgE serum levels was in striking contrast to that of the remaining serum immunoglobulins G, A and M which showed a rapid fall after surgical interventions, followed by a gradual return to the initial values. The stimulation of hypothalamus-adrenal axis and release of glucocorticosteroids, interacting with de novo synthesized interleukin-6, could explain this newly observed phenomenon of the immunoglobulin E response to the tissue injury. Behaviour of serum IgE, as described by us, bears much resemblance to that of acute phase proteins. In conclusion, we hypothesize that immunoglobulin E may act in human organism as an acute phase protein. PMID- 8618813 TI - [Influence of treatment using recombinant tumor necrosis factor alpha (hrec TNF alpha) on circadian rhythm of cortisol secretion in patients with advanced neoplastic disease]. AB - Tumor Necrosis Factor alpha is one of cytokines administered in immunotherapy of advanced neoplastic disease. Its action is exerted through immune endocrine and central nervous systems. The purpose of this study was to assess the influence of hrec TNF alpha administration in patients with advanced neoplasms on the level of cortisol in serum and its circadian rhythm. Serum cortisol was determined several times a day before and just after hrec TNF alpha first immunotherapeutic cycle. The results of the investigation were analysed with the Cosinor test. Serum concentration of hormone was determined using radioimmunoassay method. The results of this study showed the decreasing influence of hrec TNF alpha administration on cortisol secretion by adrenals with no disturbances in that circadian rhythm. IN CONCLUSION: It might be possible that some side effects of hrec TNF alpha administration are connected with the decreasing cortisol level. PMID- 8618814 TI - [Evaluation of the antihypertensive efficacy of urapidil in the treatment of hypertension emergencies]. AB - Hypertensive crisis is defined as an acute elevation of the blood pressure involving the risk of life. Agents used to the treatment of hypertensive emergencies should lower the blood pressure under control and produce minimal adverse effect. The aim of this study was to evaluate the antihypertensive efficacy of urapidil i.v. in hypertensive emergencies. Twenty three patients (pts) with the hypertensive crisis in association with ischaemic heart disease and/or acute left ventricular failure were studied. Urapidil was given intravenously in the emergent treatment to the group of 23 pts in the mean dose of 50 mg. Systolic (RRs) diastolic blood pressure (RRd) and heart rate were measured within 4 hours after the drug administration. In this group of pts the significant decrease in RRs and RRd after 2 min. of administration of urapidil was observed and the maximum effect (p < 0.05) occurred within 40 min. The heart rate decreased by 8% and was significantly different (p < 0 > 05) at the maximum point of the drug action. Urapidil administered in 25-75 mg i.v. appeared an effective antihypertensive agent in more than 90% of patients with hypertensive emergencies. What was striking, no reflex tachycardia was observed after i.v. administration of urapidil despite its antihypertensive action. PMID- 8618815 TI - [Thrombophilia in a family with resistance to activated protein C and protein S deficiency]. AB - Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in minority of cases. Herein, we present a family study of 42 years old woman with recurrent deep vein thrombosis which occurred first time four years ago during pregnancy, in subclavian vein, in relation to cardiac stimulator implantation because of atrio-ventricular III(0) block. Her laboratory investigation demonstrated normal APTT time, prothrombin time, platelet number, antithrombin III and protein C activity. Plasma antiphospholipid antibodies contents was within the normal range. The result of activated protein C(APC) resistance test was abnormal (R=1.64). Family study revealed similar degree of APC-resistance defect in her DVT symptomatic mother and two healthy young daughters (R=1.73 and 1.54 respectively). Additionally, a slightly reduced total protein S plasma concentration was found in the patient and her two children. The influence of a slightly reduced protein S level on the results of APC-resistance was excluded by evaluation of normalized activated protein C sensitivity ratio (nAPC-SR) as described de Ronde and Bertina. PMID- 8618816 TI - [Adrenal cortex carcinoma--progress in diagnosis and therapy]. PMID- 8618817 TI - [Pathogenetic aspects of infective endocarditis]. PMID- 8618818 TI - [Sarcoidosis-lymphoma syndrome in a woman with acromegaly]. AB - The occurrence of lymphoid malignancy tissue, most often Hodgkin or non-Hodgkin lymphoma, after several years of the diagnosis of sarcoidosis has been named sarcoidosis-lymphoma syndrome. The pathogenesis of this syndrome is probably connected with dysregulation of T lymphocytes. A woman with acromegaly with non Hodgkin lymphoma diagnosed six years after sarcoidosis is presented in this paper. The CVP chemotherapy helped to establish 9-years, lasting remission still. PMID- 8618819 TI - [Syndrome X--current level of knowledge]. PMID- 8618820 TI - The currents of life: the terminal electron-transfer complex of respiration. PMID- 8618821 TI - Biochemistry meets genetics in the holoenzyme. PMID- 8618822 TI - Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center. AB - Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins. PMID- 8618823 TI - Truncated WT1 mutants alter the subnuclear localization of the wild-type protein. AB - WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures. PMID- 8618824 TI - The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin. AB - In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction. PMID- 8618825 TI - Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells. AB - There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor. PMID- 8618826 TI - Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation. AB - We have constructed simian virus 40 minireplicons containing uniquely placed cis,syn-thymine dimers (T <> T) for the analysis of leading- and lagging-strand bypass replication. Assaying for replication in a human cell-free extract through the analysis of full-size labeled product molecules and restriction fragments spanning the T <> T site resulted in the following findings: (i) The primary site of synthesis blockage with T <> T in either the leading or lagging strand was one nucleotide before the lesion. (ii) Replicative bypass of T <> T was detected in both leading and lagging strands. The efficiency of synthesis past T <> T was 22% for leading-strand T <> T and 13% for lagging-strand T <> T. (iii) The lagging strand T <> T resulted in blocked retrograde synthesis with the replication fork proceeding past the lesion, resulting in daughter molecules containing small gaps (form II' DNA). (iv) With T <> T in the leading-strand template, both the leading and lagging strands were blocked, representing a stalled replication fork. Uncoupling of the concerted synthesis of the two strands of the replication fork was observed, resulting in preferential elongation of the undamaged lagging strand. These data support a model of selective reinitiation downstream from the lesion on lagging strands due to Okazaki synthesis, with no reinitiation close to the damage site on leading strands [Meneghini, R. & Hanawalt, P.C. (1976) Biochim. Biophys. Acta 425, 428-437]. PMID- 8618828 TI - Two alternative processing pathways for a preprohormone: a bioactive form of secretin. AB - An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors. PMID- 8618827 TI - Improved biodistribution, tumor targeting, and reduced immunogenicity in mice with a gamma 4 variant of Campath-1H. AB - Radiolabeled antibodies have shown promise for the treatment of lymphoma and for solid tumor targeting. Campath-1H is a humanized monoclonal antibody that reacts with the CD52 antigen present on human lymphoid and myeloid cells. Campath-1H is a gamma1 (G1) isotype that induces lymphopenia via an Fc-mediated mechanism(s). Isotype switches were engineered, and the resulting antibodies were expressed in NS0 mouse myeloma cells and biosynthetically radiolabeled with [35S]methionine. The forms included G1, G4, and a G4 variant that contained alanine substitutions at (EU numbering) Leu-235, Gly-237, and Glu-318. All isotypes bound antigen equivalently as assessed by target cell binding in vitro. The G4 variant had a greatly reduced capacity to interact with Fc receptor by virtue of reduced binding to THP-1 human myeloid cells and by a 1000-fold increase in EC50 to intermediate antibody-dependent cellular cytotoxicity. The pharmacokinetics of the isotypes were compared in CD-1 (nu/nu) mice bearing an experimental antigen expressing tumor. The plasma half-life and tumor uptake were increased for the G4 variant. The G4 variant showed significantly less spleen, liver, and bone uptake but similar uptake in the lung, kidney, and stomach and lower tissue-to-blood ratios. Immunogenicity was assessed after repeated monthly administrations of unlabeled antibody in BALB/c mice. A 50% reduction in the incidence of anti globulin response was observed for the G4 variant. These properties suggest that antibodies with reduced Fc receptor interaction merit additional study as potential targeting vehicles relative to other isotypes for radioimmunotherapy or situations where diminished normal tissue binding contributes to efficacy. PMID- 8618829 TI - The law of mass action governs antigen-stimulated cytolytic activity of CD8+ cytotoxic T lymphocytes. AB - An analysis of the initial antigen-recognition step in the destruction of target cells by CD8+ cytolytic T lymphocytes (CTLs) shows that a relationship in the form of the law of mass action can be used to describe interactions between antigen-specific receptors on T cells (TCRs) and their natural ligands on target cells (peptide-major histocompatibility protein complexes, termed pepMHC complexes), even though these reactants are confined to their respective cell membranes. For a designated level of lysis and receptor affinities below about 5 X 10(6) M-1, the product of the required number of pepMHC complexes per target cell ("epitope density") and TCR affinity for pepMHC complexes is constant; therefore, over this range TCR affinities can be predicted from epitope densities (or vice versa). At higher receptor affinities ("affinity ceiling") the epitope density required for half-maximal lysis reaches a lower limit of less than 10 complexes per target cell. PMID- 8618830 TI - Targeting p53 as a general tumor antigen. AB - A major barrier to the design of immunotherapeutics and vaccines for cancer is the idiosyncratic nature of many tumor antigens and the possibility that T cells may be tolerant of broadly distributed antigens. We have devised an experimental strategy that exploits species differences in protein sequences to circumvent tolerance of high-affinity T cells. HLA transgenic mice were used to obtain cytotoxic T lymphocytes specific for peptides from the human p53 tumor-suppressor molecule presented in association with HLA-A2.1. Although such p53-specific cytotoxic T cells did not recognize nontransformed human cells, they were able to lyse a wide variety of human tumor cells lines, thus confirming the existence of broadly distributed determinants that may serve as targets for immunotherapy. PMID- 8618831 TI - Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. AB - Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens. PMID- 8618832 TI - Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C. AB - The adult skeletal muscle Na+ channel mu1 possesses a highly conserved segment between subunit domains III and IV containing a consensus protein kinase C (PKC) phosphorylation site that, in the neuronal isoform, acts as a master control for "convergent" regulation by PKC and cAMP-dependent protein kinase. It lacks an approximately 200-aa segment between domains I and II though to modulate channel gating. We here demonstrate that mu1 is regulated by PKC (but not cAMP-dependent protein kinase) in a manner distinct from that observed for the neuronal isoforms, suggesting that under the same conditions muscle excitation could be uncoupled from motor neuron input. Maximal phosphorylation by PKC, in the presence of phosphatase inhibitors, reduced peak Na+ currents by approximately 90% by decreasing the maximal conductance, caused a -15 mV shift in the midpoint of steady-state inactivation, and caused a slight speeding of inactivation. Surprisingly, these effects were not affected by mutation of the conserved serine (serine-1321) in the interdomain III-IV loop. the pattern of current suppression and gating modification by PKC resembles the response of muscle Na+ channels to inhibitory factors present in the serum and cerebrospinal fluid of patients with Guillain-Barre syndrome, multiple sclerosis, and idiopathic demyelinating polyradiculoneuritis. PMID- 8618833 TI - Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique. AB - Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned. PMID- 8618834 TI - Synthesis and coupling reactions of alpha,alpha-dialkylated amino acids with nucleobase side chains. AB - Several di- and tripeptides containing protected purine (adenine) and pyrimidine (thymine) residues on their side chains were synthesized. The parent amino acids alpha, alpha-dialkylated in a symmetrical manner. An effective coupling procedure was developed for these sterically hindered amino acids: the fluoren-9 ylmethyloxycarbonyl-protected amino acid was dehydrated to its oxazolinone form, which was coupled in good yields with amino esters in hot tetrachloroethane. PMID- 8618835 TI - Adaptive mutation sequences reproduced by mismatch repair deficiency. AB - Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution. PMID- 8618836 TI - The protein-folding activity of chaperonins correlates with the symmetric GroEL14(GroES7)2 heterooligomer. AB - Chaperonins GroEL and GroES form, in the presence of ATP, two types of heterooligomers in solution: an asymmetric GroEL14GroES7 "bullet"-shaped particle and a symmetric GroEL14(GroES7)2 "football"-shaped particle. Under limiting concentrations of ATP or GroES, excess ADP, or in the presence of 5'-adenylyl imidodiphosphate, a correlation is seen between protein folding and the amount of symmetric GroEL14(GroES7)2 particles in a chaperonin solution, as detected by electron microscopy or by chemical crosslinking. Kinetic analysis suggests that protein folding is more efficient when carried out by a chaperonin solution populated with a majority of symmetric GroEL14(GroES7)2 particles than by a majority of asymmetric GroEL14GroES7 particles. The symmetric heterooligomer behaves as a highly efficient intermediate of the chaperonin protein folding cycle in vitro. PMID- 8618837 TI - Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA. AB - Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125 bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle. PMID- 8618838 TI - DNA profile match probabilities in a subdivided population: when can subdivision be ignored? AB - Li and Chakravarti [Li, C.C. & Chakravarti, A. (1994) Hum. Hered. 44, 100-109] compared the probability (MO) of a random match between the two DNA profiles of a pair of individuals drawn from a random-mating population to the probability (MF) of the match between a pair of random individuals drawn from a subdivided population. The level of heterogeneity in this subdivided population is measured by the parameter F, where there is no subdivision when F = 0 and increasing values of F indicate increasing subdivisions. Li and Chakravarti concluded that it is conservative to use the match probability MO, which is derived under the assumption that the two individuals are drawn from a homogeneous random-mating population without subdivision. However, MO may not be always greater than MF, even for biologically reasonable values of F. We explore here those mathematical conditions under which MO is less than MF, and we find that MO is not conservative mainly when there is an allele with a much higher frequency than all the other alleles. When empirical data for both variable number of tandem repeat (VNTR) and short tandem repeat (STR) systems are evaluated, we find that in the majority of cases MO represents a conservative probability of a match, and so the subdivision of human populations may usually be ignored for a random match, although not, of course, for relatives. Loci for which MO is not conservative should be avoided for forensic inference. PMID- 8618839 TI - Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis. AB - In most plants amino acids represent the major transport form for organic nitrogen. A sensitive selection system in yeast mutants has allowed identification of a previously unidentified amino acid transporter in Arabidopsis. AAT1 encodes a hydrophobic membrane protein with 14 membrane spanning regions and shares homologies with the ecotropic murine leukemia virus receptor, a bifunctional protein serving also as a cationic amino acid transporter in mammals. When expressed in yeast, AAT1 mediates high-affinity transport of basic amino acids, but to a lower extent also recognizes acidic and neutral amino acids. AAT1-mediated histidine transport is sensitive to protonophores and occurs against a concentration gradient, indicating that AAT1 may function as a proton symporter. AAT1 is specifically expressed in major veins of leaves and roots and in various floral tissues--i.e., and developing seeds. PMID- 8618840 TI - Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis. AB - Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) disease states, including the animal model of multiple sclerosis (MS) and experimental allergic encephalomyelitis. We have examined post-mortem brain tissues collected from patients previously diagnosed with MS, as well as tissues collected from the brains of patients dying without neuropathies. Both Northern blot analysis and reverse transcriptase (RT)-driven in situ PCR (RT-in situ PCR) studies demonstrated that inducible NO synthase (iNOS) mRNA was present in the brain tissues from MS patients but was absent in equivalent tissues from normal controls. We have also performed experiments identifying the cell type responsible for iNOS expression by RT-in situ PCR in combination with immunohistochemistry. Concomitantly, we analyzed the tissues for the presence of the NO reaction product nitrotyrosine to demonstrate the presence of a protein nitrosylation adduct. We report here that iNOS mRNA was detectable in the brains of 100% of the CNS tissues from seven MS patients examined but in none of the three normal brains. RT-in situ PCR experiments also demonstrated the presence of iNOS mRNA in the cytoplasm of cells that also expressed the ligand recognized by the Ricinus communis agglutinin 1 (RCA-1), a monocyte/macrophage lineage marker. Additionally, specific labeling of cells was observed when brain tissues from MS patients were exposed to antisera reactive with nitrotyrosine residues but was significantly less plentiful in brain tissue from patients without CNS disease. These results demonstrate that iNOS, one of the enzymes responsible for the production of NO, is expressed at significant levels in the brains of patients with MS and may contribute to the pathology associated with the disease. PMID- 8618841 TI - Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation. AB - To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix. PMID- 8618842 TI - Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends. AB - An assay that allows measurement of absolute induction frequencies for DNA double strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes. PMID- 8618843 TI - Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. AB - Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr encoded signal transduction system for which the octapeptide is the ligand. PMID- 8618844 TI - Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro. AB - Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence. PMID- 8618845 TI - Retrotransposon insertion induces an isozyme of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster. AB - The insertion of the blood retrotransposon into the untranslated region of exon 7 of the sn-glycerol-3-phosphate dehydrogenase-encoding gene (Gpdh) in Drosophila melanogaster induces a GPDH isozyme-GPDH-4-and alters the pattern of expression of the three normal isozymes-GPDH-1 to GPDH-3. The process of transcript terminus formation inside the retrotransposon insertion reduces the level of the Gpdh transcript that contains exon 8 and increases the level of the transcript that contains exons 1-7. The induced GPDH-4 isozyme is a translation product of the three transcripts that contain fragments of the blood retrotransposon. The mechanism of mutagenesis by the blood insertion is postulated to involve the pause or termination of transcription within the blood sequence, which in turn is caused by the interference of a DNA-binding protein with the RNA polymerase. Thus, we show the formation of a new functional GPDH protein by the insertion of a transposable element and discuss the evolutionary significance of this phenomenon. PMID- 8618846 TI - T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination. AB - UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts. PMID- 8618848 TI - Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor alpha, and human immunodeficiency virus infection. AB - We tested the hypothesis that increases in tumor necrosis factor alpha (TNF alpha) induced by human immunodeficiency virus (HIV) are associated with the increases in slow-wave sleep seen in early HIV infection and the decrease with sleep fragmentation seen in advanced HIV infection. Nocturnal sleep disturbances and associated fatigue contribute to the disability of HIV infection. TNF-alpha causes fatigue in clinical use and promotes slow-wave sleep in animal models. With slow progress toward a vaccine and weak effects from current therapies, efforts are directed toward extending productive life of HIV-infected individuals and shortening the duration of disability in terminal illness. We describe previously unrecognized nocturnal cyclic variations in plasma levels of TNF-alpha in all subjects. In 6 of 10 subjects (1 control subject, 3 HIV-seropositive patients with CD4+ cell number > 400 cells per microliters, and 2 HIV-positive patients with CD4+ cell number < 400 cells per microliters), these fluctuations in TNF-alpha were coupled to the known rhythm of electroencephalogram delta amplitude (square root of power) during sleep. This coupling was not present in 3 HIV-positive subjects with CD4+ cell number < 400 cells per microliters and 1 control subject. In 5 HIV subjects with abnormally low CD4+ cell counts ( < 400 cells per microliters), the number of days since seroconversion correlated significantly with low correlation between TNF-alpha and delta amplitude. We conclude that a previously unrecognized normal, physiological coupling exists between TNF-alpha and delta amplitude during sleep and that the lessened likelihood of this coupling in progressive HIV infection may be important in understanding fatigue-related symptoms and disabilities. PMID- 8618847 TI - Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors. AB - The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. PMID- 8618849 TI - Liver colonization competence governs colon cancer metastasis. AB - Tumors that metastasize do so to preferred target organs. To explain this apparent specificity, Paget, > 100 years ago, formulated his seed and soil hypothesis; i.e., the cells from a given tumor would "seed'' only favorable "soil'' offered by certain groups. The hypothesis implies that cancer cells must find a suitable "soil'' in a target organ--i.e., one that supports colonization- for metastasis to occur. We demonstrate in this report that ability of human colon cancer cells to colonize liver tissue governs whether a particular colon cancer is metastatic. In the model used in this study, human colon tumors are transplanted into the nude mouse colon as intact tissue blocks by surgical orthotopic implantation. These implanted tumors closely simulate the metastatic behavior of the original human patient tumor and are clearly metastatic or nonmetastatic to the liver. Both classes of tumors were equally invasive locally into tissues and blood vessels. However, the cells from each class of tumor behave very differently when directly injected into nude mouse livers. Only cells from metastasizing tumors are competent to colonize after direct intrahepatic injection. Also, tissue blocks from metastatic tumors af fixed directly to the liver resulted in colonization, whereas no colonization resulted from nonmetastatic tumor tissue blocks even though some growth occurred within the tissue block itself. Thus, local invasion (injection) and even adhesion to the metastatic target organ (blocks) are not sufficient for metastasis. The results suggest that the ability to colonize the liver is the governing step in the metastasis of human colon cancer. PMID- 8618850 TI - Palmitoylation of the GluR6 kainate receptor. AB - The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor. PMID- 8618851 TI - A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus. AB - Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules. PMID- 8618852 TI - Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission. AB - To prevent mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, it is important to identify its determinants. Because HIV-1 RNA levels can be reduced by antiviral therapy, we examined the role of maternal plasma HIV-1 RNA level in mother-to-child transmission. We used quantitative competitive PCR to measure HIV-RNA in 30 infected pregnant women and then followed their infants prospectively; 27% of the women transmitted HIV-1 to their infants and maternal plasma HIV-1 RNA level correlated strikingly with transmission. Eight of the 10 women with the highest HIV-1 RNA levels at delivery (190,400-1,664,100 copies per ml of plasma) transmitted, while none of the 20 women with lower levels (500-155,800 copies per ml) did (P = 0.0002). Statistical analysis of the distribution of HIV-1 RNA loads in these 30 women projected a threshold for mother-to-child transmission in a larger population; the probability of a woman with a viral RNA level of < or = 100,000 copies per ml not transmitting is predicted to be 97%. Examination of serial HIV-1 RNA levels during pregnancy showed that viral load was stable in women who did not initiate or change antiviral therapy. These data identify maternal plasma HIV-1-RNA level as a major determinant of mother-to-child transmission and suggest that quantitation of HIV-1 RNA may predict the risk of transmission. PMID- 8618853 TI - Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene. AB - In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6. PMID- 8618854 TI - Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta. AB - Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors. PMID- 8618856 TI - Active site topology and reaction mechanism of GTP cyclohydrolase I. AB - GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135. PMID- 8618855 TI - Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles. AB - Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease. PMID- 8618857 TI - Selective tumor kill of cerebral glioma by photodynamic therapy using a boronated porphyrin photosensitizer. AB - The prognosis for patients with the high-grade cerebral glioma glioblastoma multiforme is poor. The median survival for primary tumors is < 12 months, with most recurring at the site of the original tumor, indicating that a more aggressive local therapy is required to eradicate the unresectable "nests" of tumor cells invading into adjacent brain. Two adjuvant therapies with the potential to destroy these cells are porphyrin-sensitized photodynamic therapy (PDT) and boron-sensitized boron neutron capture therapy (BNCT). The ability of a boronated porphyrin, 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX tetrakiscarborane carboxylate ester (BOPP), to act as a photosensitizing agent was investigated in vitro with the C6 rat glioma cell line and in vivo with C6 cells grown as an intracerebral tumor after implantation into Wistar rats. These studies determined the doses of BOPP and light required to achieve maximal cell kill in vitro and selective tumor kill in vivo. The data show that BOPP is more dose effective in vivo by a factor of 10 than the current clinically used photosensitizer hematoporphyrin derivative and suggest that BOPP may have potential as a dual PDT/BNCT sensitizer. PMID- 8618858 TI - Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda. AB - The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively. PMID- 8618859 TI - How are close residues of protein structures distributed in primary sequence? AB - Structurally neighboring residues are categorized according to their separation in the primary sequence as proximal (1-4 positions apart) and otherwise distal, which in turn is divided into near (5-20 positions), far (21-50 positions), very far ( > 50 positions), and interchain (from different chains of the same structure). These categories describe the linear distance histogram (LDH) for three-dimensional neighboring residue types. Among the main results are the following: (i) nearest-neighbor hydrophobic residues tend to be increasingly distally separated in the linear sequence, thus most often connecting distinct secondary structure units. (ii) The LDHs of oppositely charged nearest-neighbors emphasize proximal positions with a subsidiary maximum for very far positions. (iii) Cysteine-cysteine structural interactions rarely involve proximal positions. (iv) The greatest numbers of interchain specific nearest-neighbors in protein structures are composed of oppositely charged residues. (v) The largest fraction of side-chain neighboring residues from beta-strands involves near positions, emphasizing associations between consecutive strands. (vi) Exposed residue pairs are predominantly located in proximal linear positions, while buried residue pairs principally correspond to far or very far distal positions. The results are principally invariant to protein sizes, amino acid usages, linear distance normalizations, and over- and underrepresentations among nearest neighbor types. Interpretations and hypotheses concerning the LDHs, particularly those of hydrophobic and charged pairings, are discussed with respect to protein stability and functionality. The pronounced occurrence of oppositely charged interchain contacts is consistent with many observations on protein complexes where multichain stabilization is facilitated by electrostatic interactions. PMID- 8618860 TI - Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development. AB - Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP 4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm. PMID- 8618861 TI - Regulation of the cyclin E gene by transcription factor E2F1. AB - A variety of results point to the transcription factor E2F as a critical determinant of the G1/S-phase transition during the cell cycle in mammalian cells, serving to activate the transcription of a group of genes that encode proteins necessary for DNA replication. In addition, E2F activity appears to be directly regulated by the action of retinoblastoma protein (RB) and RB-related proteins and indirectly regulated through the action of G1 cyclins and associated kinases. We now show that the accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the cyclin E and cyclin D1 promoters, both promoters are activated by E2F gene products, and at least for cyclin E, the E2F sites contribute to cell cycle-dependent control. Most important, the endogenous cyclin E gene is activated following expression of the E2F1 product encoded by a recombinant adenovirus vector. These results suggest the involvement of E2F1 and cyclin E in an autoregulatory loop that governs the accumulation of critical activities affecting the progression of cells through G1. PMID- 8618862 TI - Phospholipase D signaling is essential for meiosis. AB - Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation. PMID- 8618863 TI - Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution. AB - Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain. PMID- 8618865 TI - A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC). AB - Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state. PMID- 8618864 TI - Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia. AB - The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell. PMID- 8618866 TI - Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages. AB - The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170 kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate. PMID- 8618867 TI - Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3. AB - The inheritance of much early-onset Alzheimer disease (AD) has been linked to a dominant-acting locus on chromosome 14. Recently, the gene likely responsible for this genetic linkage has been identified and termed AD3. Five mutations have been found in AD3 that segregate with the disease phenotype in seven AD families and are not present in unaffected individuals. Here we report the existence of a gene encoding a seven transmembrane domain protein very similar to that encoded by AD3 in structure and sequence. This gene is located on chromosome 1, is expressed in a variety of tissues, including brain, and is predicted to harbor mutations causing nonchromosome 14 familial AD. The presence of several S/TPXX DNA binding motifs in both the AD3 protein and the AD3-like protein /AD4 protein suggests a possible role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. Ways in which mutations in either gene could lead to AD are discussed. PMID- 8618868 TI - d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. AB - d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation. PMID- 8618869 TI - Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds. AB - To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution. PMID- 8618870 TI - A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation. AB - The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable. HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer. The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding. These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide. They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state. Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA. PMID- 8618871 TI - Identification of an inducible surface molecule specific to fusing macrophages. AB - Multinucleated giant cells and osteoclasts arise through the fusion of mononuclear phagocyte precursors. To elucidate the mechanism by which cells of monocytic lineage fuse and differentiate into giant cells and osteoclasts, we hypothesized that, as with other cell fusion events, specific surface molecules mediate the adhesion/fusion process. It has been observed that macrophages can be induced to fuse with one another in response to specific stimuli or when placed in a specific microenvironment. The formation of giant cells is primarily associated with chronic inflammatory reactions and tumors, while osteoclasts differentiate on bone which they resorb. The fact that, under normal conditions, macrophages and monocytes fail to fuse in regions and tissues where they are present in large numbers suggests the regulated and transient expression of potential fusion molecules. To identify such a fusion-associated molecule, we established a macrophage fusion assay and generated monoclonal antibodies (mAbs) that alter the fusion of macrophages in vitro. We selected four mAbs that each had the ability to block the fusion but not the aggregation of macrophages in vitro. All four antibodies recognize surface proteins of 150 kDa. The expression of the antigens recognized by all four mAbs is restricted to macrophages that have been induced to fuse in vitro and in vivo and is inducible, transient, and regulated, as neither nonfusing macrophages nor macrophages fused in vitro express these antigens. These results support the hypothesis that macrophage fusion is mediated by specific fusion/adhesion molecules and also provide a means to study the molecular mechanisms of macrophage fusion. PMID- 8618872 TI - Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated. AB - Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance. PMID- 8618873 TI - Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands. AB - We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule. PMID- 8618874 TI - Purification, cDNA cloning, functional expression, and characterization of a 26 kDa endogenous mammalian carboxypeptidase inhibitor. AB - The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223 amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation. PMID- 8618875 TI - Osmotic regulation of cytokine synthesis in vitro. AB - These studies were undertaken to investigate the therapeutic mechanism of saturated solutions of KI, used to treat infectious and inflammatory diseases. The addition of 12-50 mM KI to cultured human peripheral blood mononuclear cells resulted in 319-395 mosM final solute concentration and induced interleukin (IL) 8 synthesis. Maximal IL-8 production was seen when 40 mM salt was added (375 mosM) and was equal to IL-8 induced by endotoxin or IL-1 alpha. However, there was no induction of IL-1 alpha, IL-1 beta, or tumor necrosis factor to account for the synthesis of IL-8; the effect of KI was not due to contaminating endotoxins. Hyperosmolar NaCl also induced IL-8 and increased steady-state levels of IL-8 mRNA similar to those induced by IL-1 alpha. IL-8 gene expression was elevated for 96 hr in peripheral blood mononuclear cells incubated with hyperosmolar NaCl. In human THP-1 macrophagic cells, osmotic stimulation with KI, NaI, or NaCl also induced IL-8 production. IL-1 signal transduction includes the phosphorylation of the p38 mitogen-activated protein kinase that is observed following osmotic stress. Using specific blockade of this kinase, a dose-response inhibition of hyperosmolar NaCl-induced IL-8 synthesis was observed, similar to that in cells stimulated with IL-1. Thus, these studies suggest that IL-1 and osmotic shock utilize the same mitogen-activated protein kinase for signal transduction and IL-8 synthesis. PMID- 8618876 TI - Identification of a cytoplasmic, phorbol ester-inducible isoform of protein tyrosine phosphatase epsilon. AB - The protein-tyrosine phosphatase epsilon (PTP epsilon) is a transmembranal, receptor-type protein that possesses two phosphatase catalytic domains characteristic of transmembranal phosphatases. Here we demonstrate the existence of a nontransmembranal isoform of PTP epsilon, PTP epsilon-cytoplasmic. PTP epsilon-cytoplasmic and the transmembranal isoform of PTP epsilon have separate, nonoverlapping expression patterns. Further, the data clearly indicate that control of which of the two isoforms is to be expressed is initiated at the transcriptional level, suggesting that they have distinct physiological roles. PTP epsilon-cytoplasmic mRNA is the product of a delayed early response gene in NIH 3T3 fibroblasts, and its transcription is regulated through a pathway that requires protein kinase C. The human homologue of PTP epsilon-cytoplasmic has also been cloned and is strongly up-regulated in the early stages of phorbol 12 tetradecanoate 13-acetate-induced differentiation of HL-60 cells. Sequence analysis indicates and cellular fractionation experiments confirm that this isoform is a cytoplasmic molecule. PTP epsilon-cytoplasmic is therefore the initial example to our knowledge of a nontransmembranal protein-tyrosine phosphatase that contains two tandem of catalytic domains. PMID- 8618877 TI - The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion. AB - Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand. PMID- 8618878 TI - Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens. AB - The virulence (vir) genes of Agrobacterium tumefaciens are induced by low molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains. In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains. On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains. The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3' methoxy groups, such as acetovanillone. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid. Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation. PMID- 8618879 TI - Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed. AB - When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, the beta galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the beta-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro. Aside from very slow mutants that were not sufficiently processive to transcribe the lacZ gene, the lower the polymerase speed, the higher the beta-galactosidase yield per transcript. In particular, a mutant which was 2.7-fold slower than the wild-type enzyme yielded 3.4- to 4.6-fold more beta-galactosidase per transcript. These differences in yield vanished in the presence of the rne-50 mutation and therefore reflect the unequal sensitivity of the transcripts to RNase E. We propose that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymerase, the longer the lag between the synthesis of this site(s) and its shielding by ribosomes, and the lower the transcript stability. PMID- 8618880 TI - The sigma factor sigma s affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5. AB - Pseudomonas fluorescens Pf-5, a rhizosphere-inhabiting bacterium that suppresses several soilborne pathogens of plants, produces the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol. A gene necessary for pyrrolnitrin production by Pf-5 was identified as rpoS, which encodes the stationary-phase sigma factor sigma s. Several pleiotropic effects of an rpoS mutation in Escherichia coli also were observed in an RpoS- mutant of Pf-5. These included sensitivities of stationary-phase cells to stresses imposed by hydrogen peroxide or high salt concentration. A plasmid containing the cloned wild-type rpoS gene restored pyrrolnitrin production and stress tolerance to the RpoS- mutant of Pf 5. The RpoS- mutant overproduced pyoluteorin and 2,4-diacetyl-phloroglucinol, two antibiotics that inhibit growth of the phytopathogenic fungus Pythium ultimum, and was superior to the wild type in suppression of seedling damping-off of cucumber caused by Pythium ultimum. When inoculated onto cucumber seed at high cell densities, the RpoS- mutant did not survive as well as the wild-type strain on surfaces of developing seedlings. Other stationary-phase-specific phenotypes of Pf-5, such as the production of cyanide and extracellular protease(s) were expressed by the RpoS- mutant, suggesting that sigma s is only one of the sigma factors required for the transcription of genes in stationary-phase cells of P. fluorescens. These results indicate that a sigma factor encoded by rpoS influences antibiotic production, biological control activity, and survival of P. fluorescens on plant surfaces. PMID- 8618882 TI - Functional antagonism between the retinoic acid receptor and the viral transactivator BZLF1 is mediated by protein-protein interactions. AB - The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) family of transcription factors. Like several other members of the bZip family, transcriptional activity of BZLF1 is modulated by retinoic acid receptors (RARs). We present evidence that the RAR alpha and BZLF1 can reciprocally repress each other's transcriptional activation by a newly discovered mechanism. Analysis of RAR alpha mutants in transfection studies reveals that the DNA binding domain is sufficient for inhibition of BZLF1 activity. Analysis of BZLF1 mutants indicates that both the coiled-coil dimerization domain and a region containing the transcriptional activation domain of BZLF1 are required for transrepression. Coimmunoprecipitation experiments demonstrate physical interactions between RAR alpha and BZLF1 in vivo. Furthermore, glutathione S-transferase-pulldown assays reveal that these protein protein interactions are mediated by the coiled-coil dimerization domain of BZLF1 and the DNA binding domain of RAR alpha. While RAR alpha is unable to recognize BZLF1 binding sites, the RAR alpha can be tethered to the DNA by forming a heteromeric complex with BZLF1 bound to DNA. Tethering RARs via protein-protein interactions onto promoter DNA suggest a mechanism through which RARs might gain additional levels of transcriptional regulation. PMID- 8618881 TI - Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease. AB - Apolipoprotein E (apoE) is critical in the modulation of cholesterol and phospholipid transport between cells of different types. Human apoE is a polymorphic protein with three common alleles, APO epsilon 2, APO epsilon 3, and APO epsilon 4. ApoE4 is associated with sporadic and late-onset familial Alzheimer disease (AD). Gene dose was shown to have an effect on risk of developing AD, age of onset, accumulation of senile plaques in the brain, and reduction of choline acetyltransferase (ChAT) activity in the hippocampus of AD subjects. To characterize the possible impact of the apoE4 allele on cholinergic markers in AD, we examined the effect of apoE4 allele copy number on pre- and postsynaptic markers of cholinergic activity. ApoE4 allele copy number showed an inverse relationship with residual brain ChAT activity and nicotinic receptor binding sites in both the hippocampal formation and the temporal cortex of AD subjects. AD cases lacking the apoE4 allele showed ChAT activities close or within age-matched normal control values. The effect of the apoE4 allele on cholinomimetic drug responsiveness was assessed next in a group (n = 40) of AD patients who completed a double-blind, 30-week clinical trial of the cholinesterase inhibitor tacrine. Results showed that > 80% of apoE4-negative AD patients showed marked improvement after 30 weeks as measured by the AD assessment scale (ADAS), whereas 60% of apoE4 carriers had ADAS scores that were worse compared to baseline. These results strongly support the concept that apoE4 plays a crucial role in the cholinergic dysfunction associated with AD and may be a prognostic indicator of poor response to therapy with acetylcholinesterase inhibitors in AD patients. PMID- 8618883 TI - Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities. AB - Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits. PMID- 8618884 TI - Ceramide formation during heat shock: a potential mediator of alpha B-crystallin transcription. AB - Ceramide has been identified as a potential second messenger that may mediate cell differentiation and apoptosis after exposure to hormonal agonists such as 1 alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, or gamma-interferon. The secondary cellular events that follow ceramide generation remain undefined. We report that in NIH WT-3T3 cells, ceramide induces an enhancement of gene transcription of alpha B-crystallin, a small heat shock protein. The levels of alpha B-crystallin, as measured by Northern blot and immunoblot analyses, were increased by the addition of an exogenous short-chain ceramide, N acetylsphingosine, or by increasing endogenous intracellular ceramide by inhibition of glucosylceramide synthase. Similar effects were not seen in the expression of the closely related gene, Hsp25. To ascertain whether ceramide mediated gene transcription was a feature of the heat shock response, cell ceramide was measured in heat shocked cells and observed to be elevated 2-fold immediately upon the return of cells to 37 degrees C. Thus ceramide formed after heat shock treatment of 3T3 cells may mediate the transcription events associated with the cell stress response. PMID- 8618885 TI - A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. AB - The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell. PMID- 8618886 TI - Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius. AB - Some intron-containing rRNA genes of archaea encode homing-type endonucleases, which facilitate intron insertion at homologous sites in intron- alleles. These archaeal rRNA genes, in contrast to their eukaryotic counterparts, are present in single copies per cell, which precludes intron homing within one cell. However, given the highly conserved nature of the sequences flanking the intron, homing may occur in intron- rRNA genes of other archaeal cells. To test whether this occurs, the intron-containing 23S rRNA gene of the archaeal hyperthermophile Desulfurococcus mobilis, carried on nonreplicating bacterial vectors, was electroporated into an intron- culture of Sulfolobus acidocaldarius. PCR experiments demonstrated that the intron underwent homing and spread through the culture. By using a double drug-resistant mutant of S. acidocaldarius, it was shown that spreading resulted partly from a selective advantage of intron+ cells and partly from intercellular mobility of the intron and homing. PMID- 8618887 TI - Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness. AB - A murine model for antigen-induced bronchial hyperreactivity (BHR) and airway eosinophilia, two hallmarks of asthma, was developed using ovalbumin-immunized mice, which produce large amounts of IgE (named BP2, "Bons Producteurs 2," for High Line of Selection 2). A single intranasal ovalbumin challenge failed to modify the bronchial responses, despite the intense eosinophil recruitment into the bronchoalveolar lavage fluid and airways. When mice were challenged twice a day for 2 days or once a day for 10 days, BHR in response to i.v. 5 hydroxytryptamine or to inhaled methacholine was induced in BP2 mice but not in BALB/c mice. Histological examination showed that eosinophils reached the respiratory epithelium after multiple ovalbumin challenges in BP2 mice but remained in the bronchial submucosa in BALB/c mice. Total IgE titers in serum were augmented significantly with immunization in both strains, but much more so in BP2 mice. Interleukin 5 (IL-5) titers in serum and bronchoalveolar lavage fluid of BP2 mice were augmented by the antigenic provocation, and a specific anti-IL5 neutralizing antibody suppressed altogether airway eosinophilia and BHR, indicating a participation of IL-5 in its development. Our results indicate that the recruitment of eosinophils to the airways alone does not induce BHR in mice and that the selective effect on BP2 mice is related to their increased IgE titers associated with antigen-driven eosinophil migration to the epithelium, following formation and secretion of IL-5. PMID- 8618888 TI - A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme. AB - The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II) induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease. PMID- 8618890 TI - Intravenous cocaine, morphine, and amphetamine preferentially increase extracellular dopamine in the "shell" as compared with the "core" of the rat nucleus accumbens. AB - The nucleus accumbens is considered a critical target of the action of drugs of abuse. In this nucleus a "shell" and a "core" have been distinguished on the basis of anatomical and histochemical criteria. The present study investigated the effect in freely moving rats of intravenous cocaine, amphetamine, and morphine on extracellular dopamine concentrations in the nucleus accumbens shell and core by means of microdialysis with vertically implanted concentric probes. Doses selected were in the range of those known to sustain drug self administration in rats. Morphine, at 0.2 and 0.4 mg/kg, and cocaine, at 0.5 mg/kg, increased extracellular dopamine selectivity in the shell. Higher doses of cocaine (1.0 mg/kg) and the lowest dose of amphetamine tested (0.125 mg/kg) increased extracellular dopamine both in the shell and in the core, but the effect was significantly more pronounced in the shell compared with the core. Only the highest dose of amphetamine (0.250 mg/kg) increased extracellular dopamine in the shell and in the core to a similar extent. The present results provide in vivo neurochemical evidence for a functional compartmentation within the nucleus accumbens and for a preferential effect of psychostimulants and morphine in the shell of the nucleus accumbens at doses known to sustain intravenous drug self-administration. PMID- 8618889 TI - Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli. AB - As shown in the accompanying paper, the magnetic dipolar interaction between site directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed. PMID- 8618891 TI - UGA suppression by a mutant RNA of the large ribosomal subunit. AB - A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon. PMID- 8618892 TI - Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor. AB - The estrogen receptor (ER), a 66-kDa protein that mediates the actions of estrogens in estrogen-responsive tissues, is a member of a large superfamily of nuclear hormone receptors that function as ligand-activated transcription factors. ER shares a conserved structural and functional organization with other members of this superfamily, including two transcriptional activation functions (AFs), one located in its amino-terminal region (AF-1) and the second located in its carboxyl-terminal, ligand-binding region (AF-2). In most promoter contexts, synergism between AF-1 and AF-2 is required for full ER activity. In these studies, we demonstrate a functional interaction of the two AF-containing regions of ER, when expressed as separate polypeptides in mammalian cells, in response to 17 beta-estradiol (E2) and antiestrogen binding. The interaction was transcriptionally productive only in response to E2, and was eliminated by point or deletion mutations that destroy AF-1 or AF-2 activity or E2 binding. Our results suggest a definitive mechanistic role for E2 in the activity of ER- namely, to alter receptor conformation to promote an association of the amino- and carboxyl-terminal regions, leading to transcriptional synergism between AF-1 and AF-2. The productive re assembly of two portions of ER expressed in cells as separate polypeptides demonstrates the evolutionarily conserved modular structural and functional organization of the nuclear hormone receptors. The ligand-dependent interaction of the two AF-containing regions of ER allows for the assembly of a complete activation function from two distinct regions within the same protein, providing a mechanism for hormonally regulated transcription. PMID- 8618893 TI - Mammalian phospholipase D: activation by ammonium sulfate and nucleotides. AB - Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and this dramatic action of ammonium sulfate is not simply due to such high ionic strength. Addition of ATP but not of nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-imido]diphosphate further enhanced the PLD activation approximately equal to 2- to 3-fold. This enhancement by ATP needed cytosol, implying a role of protein phosphorylation. A survey of PLD activity in rat tissues revealed that, unlike in previous observations reported thus far, PLD was most abundant in membrane fractions of kidney, spleen, and liver in this order, and the enzymatic activity in brain and lung was low. PMID- 8618894 TI - Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor. AB - A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect. PMID- 8618895 TI - Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis. AB - Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28 fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site. PMID- 8618896 TI - Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region. AB - A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation. PMID- 8618897 TI - Differentiation of immortal cells inhibits telomerase activity. AB - Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5' TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity. PMID- 8618898 TI - Liver-specific expression of the human factor VII gene. AB - Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays. PMID- 8618899 TI - Isolation and characterization of a pseudoautosomal region-specific genetic marker in C57BL/6 mice using genomic representational difference analysis. AB - Representational difference analysis was used to identify strain-specific differences in the pseudoautosomal region (PAR) of mouse X and Y chromosomes. One second generation (C57BL/6 x Mus spretus) x Mus spretus interspecific backcross male carrying the C57BL/6 (B6) PAR was used for tester DNA. DNA from five backcross males from the same generation that were M. spretus-type for the PAR was pooled for the driver. A cloned probe designated B6-38 was recovered that is B6-specific in Southern analysis. Analysis of genomic DNA from several inbred strains of laboratory mice and diverse Mus species and subspecies identified a characteristic Pst I pattern of fragment sizes that is present only in the C57BL family of strains. Hybridization was observed with sequences in DBA/2J and to a limited extent with Mus musculus (PWK strain) and Mus castaneus DNA. No hybridization was observed in DNA of different Mus species, M. spretus, M. hortulanus, and M. caroli. Genetic analyses of B6-38 was conducted using C57BL congenic males that carry M. spretus alleles for distal X chromosome loci and the PAR and outcrosses of heterozygous congenic females with M. spretus. These analyses demonstrated that the B6-38 sequences were inherited with both the X and Y chromosome. B6-38 sequences were genetically mapped as a locus within the PAR using two interspecific backcrosses. The locus defined by B6-38 is designated DXYRp1. Preliminary analyses of recombination between the distal X chromosome gene amelogenin (Amg) and the PAR loci for either TelXY or sex chromosome association (Sxa) suggest that the locus DXYRp1 maps to the distal portion of the PAR. PMID- 8618900 TI - Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases. AB - We previously characterized a methionine aminopeptidase (EC 3.4.11.18; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppressor of the slow-growth phenotype of the map1 null strain. The DNA sequence of MAP2 encodes a protein of 421 amino acids that shows 22% identity with the sequence of yeast Met-AP1. Surprisingly, comparison with sequences in the GenBank data base showed that the product of MAP2 has even greater homology (55% identity) with rat p67, which was characterized as an initiation factor 2 associated protein but not yet shown to have Met-AP activity. Transformants of map1 null cells expressing MAP2 in a high-copy-number plasmid contained 3- to 12 fold increases in Met-AP activity on different peptide substrates. The epitope tagged suppressor gene product was purified by immunoaffinity chromatography and shown to contain Met-AP activity. To evaluate the physiological significance of Met-AP2, the MAP2 gene was deleted from wild-type and map1 null yeast strains. The map2 null strain, like the map1 null strain, is viable but with a slower growth rate. The map1, map2 double-null strains are nonviable. Thus, removal of N terminal methionine is an essential function in yeast, as in prokaryotes, but yeast require two methionine aminopeptidases to provide the essential function which can only be partially provided by Met-AP1 or Met-AP2 alone. PMID- 8618901 TI - Identification of signals required for the insertion of heterologous genome segments into the reovirus genome. AB - In cells simultaneously infected with any two of the three reovirus serotypes ST1, ST2, and ST3, up to 15% of the yields are intertypic reassortants that contain all possible combinations of parental genome segments. We have now found that not all genome segments in reassortants are wild type. In reassortants that possess more ST1 than ST3 genome segments, all ST1 genome segments appear to be wild type, but the incoming ST3 genome segments possess mutations that make them more similar to the ST1 genome segments that they replace. In reassortants resulting from crosses of the more distantly related ST3 and ST2 viruses that possess a majority of ST3 genome segments, all incoming ST2 genome segments are wild type, but the ST3 S4 genome segment possesses two mutations, G74 to A and G624 to A, that function as acceptance signals. Recognition of these signals has far-reaching implications for the construction of reoviruses with novel properties and functions. PMID- 8618902 TI - Genetic heterogeneity in isogenic homozygous clonal zebrafish. AB - The C32 isogenic homozygous diploid (IHD) strain of the zebrafish (Danio rerio) was found to be polyallelic at a malate dehydrogenase locus (sMdh-A). A variant allele is thought to have arisen via mutation within the past 10 bisexual generations that have maintained the strain since its last gynogenetic cloning event; this unique allele now predominates at the sMdh-A locus. The estimated mutation rate in this species is sufficiently high that long-term genetic homogeneity of its IHD clones cannot be assumed. Researchers using such bisexually maintained clones should be aware that they are not necessarily using genetically uniform subjects. Genetic uniformity of cloned IHD zebrafish will be maximized if experimental subjects are obtained soon after a cloning event. PMID- 8618903 TI - Brain localization for arbitrary stimulus categories: a simple account based on Hebbian learning. AB - A central theme of cognitive neuroscience is that different parts of the brain perform different functions. Recent evidence from neuropsychology suggests that even the processing of arbitrary stimulus categories that are defined solely by cultural conventions (e.g., letters versus digits) can become spatially segregated in the cerebral cortex. How could the processing of stimulus categories that are not innate and that have no inherent structural differences become segregated? We propose that the temporal clustering of stimuli from a given category interacts with Hebbian learning to lead to functional localization. Neural network simulations bear out this hypothesis. PMID- 8618904 TI - Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method. AB - Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV infected infants and 63 seroreverting infants, born with passively acquired anti HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03). PMID- 8618906 TI - Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia. AB - The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7 biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels. PMID- 8618905 TI - The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c. AB - Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags. PMID- 8618907 TI - Stimulation of protective CD8+ T lymphocytes by vaccination with nonliving bacteria. AB - Infectious diseases caused by intracellular microbes are responsible for major health problems, and satisfactory control will ultimately depend on efficient vaccination strategies. The general assumption is that activation of protective immune responses against intracellular microbes dominated by CD8+ T cells are achieved only by live vaccines. In contrast, we here demonstrate stimulation of protective immunity in mice against the intracellular pathogen Listeria monocytogenes by vaccination with heat-killed listeriae. Vaccine-induced immunity comprised cytolytic and interferon gamma-producing CD8+ T lymphocytes. CD8+ T cells from vaccinated donor mice transferred protection against listeriosis. Moreover, vaccination with heat-killed listeriae induced production in CD4+ T cell-deficient, H2-A beta gene-disrupted mutant mice. We conclude that antigens from killed listeriae are introduced into the major histocompatibility complex class I pathway and thus are recognized by CD8+ T cells. The practicability of killed vaccines against human infectious diseases therefore should be reevaluated. PMID- 8618908 TI - In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones. AB - Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus. PMID- 8618909 TI - Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte activated hepatocytes. AB - Using transgenic mice that replicate the hepatitis B virus (HBV) genome, we recently demonstrated that class I-restricted, hepatitis B surface antigen specific cytotoxic T lymphocytes (CTLs) can noncytolytically eliminate HBV pregenomic and envelope RNA transcripts from the hepatocyte. We now demonstrate that the steady-state content of these viral transcripts is profoundly reduced in the nucleus and cytoplasm of CTL-activated hepatocytes, but their transcription rates are only slightly reduced. Additionally, we demonstrate that transcripts covering the HBV X coding region are resistant to downregulation by the CTL. These results imply the existence of CTL-inducible hepatocellular factors that interact with a discrete element(s) between nucleotides 3157 and 1239 within the viral pregenomic and envelope transcripts and mediate their degradation, thus converting the hepatocyte from a passive victim to an active participant in the host response to HBV infection. PMID- 8618910 TI - Synthesis and assembly of self-complementary calix[4]arenes. AB - A calix[4]arene was designed to reversibly dimerize and form an egg-shaped enclosure. Adhesive interactions in the assembly were provided by four self associating ureas, which form a cyclic array containing 16 hydrogen bonds. The synthesis was completed in four steps from the previously described O,O',O",O"' tetrabenzylcalix[4]arene. Evidence for dimerization of the calixarene tetraurea was provided by H NMR, mass spectrometry, and the observation of encapsulated molecules. The resulting cavity was of sufficient size to capture guests such as ethyl benzene and p-xylene. PMID- 8618911 TI - Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands. AB - Two dodecapeptides belonging to distinct classes of Src homology 3 (SH3) ligands and selected from biased phage display libraries were used to investigate interactions between a specificity pocket in the Src SH3 domain and ligant residues flanking the proline-rich core. The solution structures of c-Src SH3 complexed with these peptides were solved by NMR. In addition to proline-rich, polyproline type II helix-forming core, the class I and II ligands each possesses a flanking sequence that occupies a large pocket between the RT and n-Src loops of the SH3 domain. Structural and mutational analyses illustrate how the two classes of SH3 ligands exploit a specificity pocket on the receptor differently to increase binding affinity and specificity. PMID- 8618912 TI - Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells. AB - The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies. PMID- 8618913 TI - Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus. AB - The immunoglobulin kappa gene locus encodes 95% of the light chains of murine antibody molecules and is thought to contain up to 300 variable (V kappa)-region genes generally considered to comprise 20 families. To delineate the locus we have isolated 29 yeast artificial chromosome genomic clones that form two contigs, span > 3.5 megabases, and contain two known non-immunoglobulin kappa markers. Using PCR primers specific for 19 V kappa gene families and Southern analysis, we have refined the genetically defined order of these V kappa gene families. Of these, V kappa 2 maps at least 3.0 Mb from the joining (J kappa) region and appears to be the most distal V kappa gene segment. PMID- 8618915 TI - Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors. AB - When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8 cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors. PMID- 8618914 TI - Vitronectin is not essential for normal mammalian development and fertility. AB - Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components. PMID- 8618916 TI - Studies on mu and delta opioid receptor selectivity utilizing chimeric and site mutagenized receptors. AB - Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding. PMID- 8618917 TI - Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase. AB - The mechanism by which the endogenous vasodilator adenosine causes ATP-sensitive potassium (KATP) channels in arterial smooth muscle to open was investigated by the whole-cell patch-clamp technique. Adenosine induced voltage-independent, potassium-selective currents, which were inhibited by glibenclamide, a blocker of KATP currents. Glibenclamide-sensitive currents were also activated by the selective adenosine A2-receptor agonist 2-p-(2-carboxethyl)-phenethylamino-5'-N- ethylcarboxamidoadenosine hydrochloride (CGS-21680), whereas 2-chloro-N6 cyclopentyladenosine (CCPA), a selective adenosine A1-receptor agonist, failed to induce potassium currents. Glibenclamide-sensitive currents induced by adenosine and CGS-21680 were largely reduced by blockers of the cAMP-dependent protein kinase (Rp-cAMP[S], H-89, protein kinase A inhibitor peptide). Therefore, we conclude that adenosine can activate KATP currents in arterial smooth muscle through the following pathway: (i) Adenosine stimulates A2 receptors, which activates adenylyl cyclase; (ii) the resulting increase intracellular cAMP stimulates protein kinase A, which, probably through a phosphorylation step, opens KATP channels. PMID- 8618918 TI - Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators. AB - Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation. PMID- 8618919 TI - Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. AB - Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines. PMID- 8618921 TI - Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung. AB - Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. PMID- 8618920 TI - Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments. AB - Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15. PMID- 8618922 TI - Inhibition of a plant virus infection by analogs of melittin. AB - An approach that enables identification of specific synthetic peptide inhibitors of plant viral infection is reported. Synthetic analogs of melittin that have sequence and structural similarities to an essential domain of tobacco mosaic virus coat protein were found to possess highly specific antiviral activity. This approach involves modification of residues located at positions analogous to those that are critical for virus assembly. The degree of inhibition found correlates well with sequence similarities between the viral capsid protein and the melittin analogs studied as well as with the induced conformational changes that result upon interaction of the peptides and ribonucleic acid. PMID- 8618923 TI - Learning about categories in the absence of memory. AB - A fundamental question about memory and cognition concerns how information is acquired about categories and concepts as the result of encounters with specific instances. We describe a profoundly amnesic patient (E.P.) who cannot learn and remember specific instances--i.e., he has no detectable declarative memory. Yet after inspecting a series of 40 training stimuli, he was normal at classifying novel stimuli according to whether they did or did not belong to the same category as the training stimuli. In contrast, he was unable to recognize a single stimulus after it was presented 40 times in succession. These findings demonstrate that the ability to classify novel items, after experience with other items in the same category, is a separate and parallel memory function of the brain, independent of the limbic and diencephalic structures essential for remembering individual stimulus items (declarative memory). Category-level knowledge can be acquired implicitly by cumulating information from multiple training examples in the absence of detectable conscious memory for the examples themselves. PMID- 8618924 TI - Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. AB - cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man. PMID- 8618926 TI - Peptide synthesis using unprotected peptides through orthogonal coupling methods. AB - We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency. PMID- 8618925 TI - Steroid receptor heterodimerization demonstrated in vitro and in vivo. AB - The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the intracellular receptor superfamily that bind as homodimers to the same hormone response elements (HREs). Physiological evidence suggests that MR and GR interact with each other in cells that express both receptors, implying that they might directly interact in the regulation of transcription initiation. Indeed, we have found that coexpressed MR and GR interact functionally at the transcriptional level and furthermore that they interact physically through heterodimer formation at a shared HRE in vitro and in vivo. We suggest from these findings that heterodimerization may play an important role in steroid receptor transcriptional regulation. PMID- 8618927 TI - UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression. AB - The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes. PMID- 8618928 TI - Intron phase correlations and the evolution of the intron/exon structure of genes. AB - Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns. Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets. We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution. Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions. This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes. PMID- 8618929 TI - Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse. AB - This study examines the question of whether apolipoprotein E (apoE) alters steady state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse. PMID- 8618930 TI - Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L. AB - An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides. PMID- 8618931 TI - Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA. AB - A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes. PMID- 8618933 TI - Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B cell lymphoma. AB - The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression. PMID- 8618932 TI - Characterization of the promoter region of the mouse Xist gene. AB - The mouse Xist gene is expressed exclusively from the inactive X chromosome and may be implicated in initiating X inactivation. To better understand the mechanisms underlying the control of Xist expression, we investigated the upstream regulatory region of the mouse Xist promoter. A 1.2-kb upstream region of the Xist gene was sequenced and promoter activity was studied by chloramphenicol acetyltransferase (CAT) assays after transfection in murine XX and XY cell lines. The region analyzed (-1157 to +917 showed no in vitro sex specific promoter activity. However, a minimal constitutional promoter was assigned to a region from -81 to +1, and a cis element from -41 to -15 regulates promoter activity. We showed that a nuclear factor binds to an element located at -30 to -25 (TTAAAG). A second sequence at -41 to -15 does not act as an enhancer and is unable to confer transcriptional activity to the Xist gene on its own. A third region from -82 to -41 is needed for correct expression. Deletion of the segment -441 to -231 is associated with an increase in CAT activity and may represent a silencer element. PMID- 8618935 TI - Skeletal muscle as a myocardial substitute. AB - During the last several years, there has been intense worldwide interest concerning the use of skeletal muscle as a form of cardiac assistance. For over 10,000 people in the United States diagnosed each year with irreversible heart failure, the 1-year mortality approaches 50%. This comes despite recent advances in medical therapy, heart transplantation, and the artificial heart program. Because of the limitations of these treatments in terms of effectiveness, cost, and availability, we have used a different approach for cardiac augmentation. Skeletal muscle is shaped into the form of a pumping chamber and then used to aid the function of the failing myocardium. Another approach is cardiomyoplasty, where the latissimus dorsi muscle is wrapped around the heart and stimulated to contract in synchrony with the patient's failing myocardium. More than 500 patients have undergone cardiomyoplasty worldwide. These two areas of investigation represent the principle methods for skeletal muscle cardiac assistance. PMID- 8618934 TI - Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers. AB - The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity. PMID- 8618936 TI - Origin and implications of bovine spongiform encephalopathy. AB - All spongiform encephalopathies in animals, including humans, are slow developing infectious diseases. The current working theory links the origin of bovine spongiform encephalopathy (BSE) to the feeding of cattle with meat and bone meal prepared from scrapie-infected sheep remains. Recycling of cattle meat and bones (MBM) essentially resulted in the selection of a single strain from the "wild type", a mixture of 20 strains. The BSE agent is easily transmitted through ingestion, with some evidence of vertical transmission. Paradoxically, cattle have selected a major new strain which appears to be more virulent than an unselected strain found in scrapie sheep. The same strain of BSE agent is implicated in the occurrence of spongiform encephalopathy in domestic cats, tiger, and some exotic species of ruminants in zoos. The properties of BSE and its spread into cattle are still disputed. Since our understanding of the disease and its transmissibility in humans must await observations that will be made over some years to come, it is important to keep a reasonable perspective and ensure that any speculative comment is consistent with fact. In risk assessment in such circumstances, it is tempting give too much credence to persuasive parallels when direct relevant information is not available. On the other hand, it would also not be wise to assume that the disease will die by itself and will have no effect on humans. PMID- 8618937 TI - A selenium supplement associated or not with vitamin E delays early renal lesions in experimental diabetes in rats. AB - Seventy rats were separated into five groups: one group of 12 was used as a control and received a purified diet, and four groups of streptozotocin-induced diabetic rats, totalling 58, were fed the same diet without or with selenium (Se) supplementation. Of the noncontrol rats, 14 were without supplementation (Group D), 14 were fed a Se-rich yeast diet (i.e., selenion) (Group DSel), 14 received selenomethionine (Group DSm), and 16 received selenomethionine + tocopherol acetate (Group DSmE). Supplementation with Se in all groups was 0.99 micromole/100g of diet and with tocopherol acetate was 0.145 micromole/100 g. All diabetic rats were mildly balanced by insulin. After 24 weeks of diet, plasma glucose tended to decrease in diabetic Se-supplemented groups DSmE > DSm > DSel versus Group D. In DSm and DSmE groups, plasma lipid peroxides also decreased compared with Group D, but this decrease reached significance only for DSmE (P < 0.01 for both TBARS and conjugated dienes). Plasma triglycerides also decreased in DSm and DSmE groups versus Group D (P < 0.01; P < 0.05, respectively). At the same time, Se increased significantly in kidneys of Groups DSel and DSm versus D and more weakly in Group DSmE, but in this case was associated with a large increase of vitamin E. These beneficial effects of selenium supplement and more so of selenium combined with vitamin E were associated with a protection of kidneys in diabetic rats which found expression in a significant correction of renal hyperfiltration (P < 0.05) and in a diminution of the number and severity of glomerular lesions (P < 0.0005). PMID- 8618938 TI - Endogenous basal nitric oxide production does not control myocardial oxygen consumption or function. AB - Previous studies from our laboratory have shown that an extrinsic nitric oxide (NO) donor (i.e., nitroprusside) caused vasodilatation and negative inotropy by activating guanylate cyclase and increasing myocardial cyclic GMP. We tested the hypothesis that endogenous myocardial NO production would limit myocardial oxygen consumption and function in vivo. We used the NO synthase inhibitors N(G)-nitro-L arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA) in nine open-chest anesthetized mongrel dogs. Either L-NAME (6 mg/kg) or L-NMMA (3 mg/kg) were infused into the left anterior descending coronary artery (LAD). The circumflex (CFX) coronary artery region served as a control. Regional segment work was calculated as the integrated product of local force (miniature transducer) and segment shortening (ultrasonic crystals). Local myocardial O2 consumption was determined using an ultrasonic LAD flow probe and local arterial venous O2 content difference (oximetry). Cyclic GMP levels were obtained via a radioimmunoassay. Both L-NAME and L-NMMA caused a local decrease in coronary blood flow (LAD flow: 80 +/- 8 to 69 +/- 7 ml/min/100 g [means +/- SEM]) and increased O2 extraction (9.1 +/- 0.6 to 10.2 +/- 0.7 ml O2/100 ml). However, this led to no change in local O2 consumption. LAD segment force was not altered (12.1 +/- 0.7 to 11.6 +/- 0.9 g), nor was the percent shortening changed (10.8 +/- 1.8% to 10.0 +/- 1.4%) by L-NAME or L-NMMA, leading to no net change in segment work. Myocardial cyclic GMP levels were not different in a comparison between the LAD (1.7 +/- 0.4 pmoles/g) and control (1.7 +/- 0.2) regions with either L-NAME or L NMMA. We conclude that blockade of endogenous NO production with L-NAME and L NMMA is sufficient to cause vasoconstriction in the heart of anesthetized dogs. However, this dose did not lead to alteration in local myocardial function, O2 consumption, or cyclic GMP levels. PMID- 8618939 TI - Relation of ornithine decarboxylase and tyrosine kinase activity in the jejunal mucosa in vivo. AB - Our aim was to study the relationship between jejunal mucosal activity of ornithine decarboxylase and tyrosine kinase during proliferation in adolescent rats in vivo. Their relationship in the proliferating intestinal mucosa under in vivo conditions has not been reported before. From the results of in vitro studies, it was speculated that tyrosine kinase activity modulated ornithine decarboxylase activity during colonic mucosal proliferation (Majumdar AP. Am J Physiol 259:G626-G630, 1990). Jejunal mucosal hyperplasia was induced by Type 1 diabetes and suppressed in both control and diabetic rats by administration of difluoromethylornithine. Jejunal mucosal weight and enzyme activity were determined after 3, 6, and 10 days, and tyrosine-specific phosphorylated proteins after 10 days of induction of diabetes. Difluoromethylornithine suppressed jejunal mucosal proliferation and tyrosine kinase activity after the 6- and 10- day study periods. After the 3-day study period although jejunal mucosal growth was suppressed, tyrosine kinase activity was not. Activity of tyrosine kinase and ornithine decarboxylase were highly significantly correlated at all time periods in both control and diabetic rats. Tyrosine-specific phosphorylated proteins of 34, 54, 80, and 200 kDa proteins were observed in jejunal mucosa of both control and diabetic rats. In the difluoromethylornithine-treated rats, phosphorylation of the above proteins was negligible while the phosphorylation of a 14-kDa protein was prominent. We speculate that in vivo ornithine decarboxylase activity may be modulating tyrosine kinase activity and that phosphorylation of a 14-kDa protein was associated with suppressed mucosal growth in difluoromethylornithine treated rats. PMID- 8618941 TI - Role of neuromedin B in the in vitro thyrotropin release in response to thyrotropin-releasing hormone from anterior pituitaries of eu-, hypo-, and hyperthyroid rats. AB - A role of neuromedin B (NB), a bombesin-like peptide, as an inhibitory paracrine/autocrine regulator of thyrotropin secretion has been suggested. We previously reported (10) that basal thyroid-stimulating hormone (TSH) release in vitro was decreased by NB and increased in the presence of a highly potent antiserum against NB (aNB). In these experiments, we studied the effects of NB (10(-11) - 10(-7) M) and antiserum against NB (aNB, 1:2000 dilution) on basal TSH release and the response to thyrotropin-releasing hormone (TRH) (0.5 x 10(-8) M) from incubated anterior pituitaries from eu-, hypo-, and hyperthyroid rats. As expected, in euthyroid rats NB decreased basal and TRH-stimulated TSH release, but only at the highest concentration tested (10(-7) M). Incubation of the pituitaries from euthyroid rats with the antiserum against NB increased basal TSH release above that from glands of normal rabbit serum-incubated controls, as anticipated based on the concept that NB inhibits TSH release from the pituitary glands of euthyroid animals. The antiserum did not augment the response to TRH, suggesting that NB released in this situation, although suppressing basal release, had no effort on the stimulated release induced by TRH. Glands from hypothyroid rats had a slightly lower basal TSH release and decreased response to TRH than glands from euthyroid rats. They responded with a decrease in basal TSH release at a much lower concentration of NB (10(-9) M) than pituitaries from euthyroid animals. Surprisingly, pituitaries from hypothyroid rats showed a paradoxical increased release of TSH in response to the lowest concentration of NB (10(-11) M), which decreased with increasing concentrations and was not distinguishable from control release in the presence of TRH at the highest concentration of NB (10(-7) M). We hypothesize that the increased responsiveness to the inhibition of basal TSH release by NB in the hypothyroid pituitaries may be related to an upregulation of NB receptors in this situation, in which the release of NB is diminished because of loss of feedback via thyroid hormones. The view that NB secretion was reduced in the hypothyroid situation was supported by the fact that there was no change in TSH release or the response to TRH following treatment with aNB in these animals. Remarkably, in the glands from the hyperthyroid rats, although basal TSH secretion was significantly lower than that from euthyroid pituitaries and response to TRH was also decreased, NB (10(-11) 10(-7) M) instead of decreasing TSH release augmented it significantly. Also, the response to TRH was significantly augmented but only at the lowest concentration of NB tested (10(-11 M). That NB was probably being secreted in vitro from the hyperthyroid pituitaries was indicated by an increased basal TSH release as well as a higher TSH medium concentration after TRH in the presence of the aNB. These results support the concept that the glands from the hyperthyroid animals secrete more NB because of positive feedback of thyroid hormones directly on the thyrotropes to increase NB synthesis and release which downregulates NB receptors on the gland. This downregulation of receptors in some manner reverses the inhibitory action of NB on basal and TRH-stimulated TSH release. In conclusion, the results provide further evidence for an important role of NB as an autocrine regulator of TSH release, which is modulated by increased release of NB induced by thyroid hormones. PMID- 8618940 TI - Effects of amiodarone-induced phospholipidosis on pulmonary host defense functions in rats. AB - The effect of the induction of pulmonary phospholipidosis by amiodarone on selected pulmonary host defense functions was studied in male Fischer-344 rats. One week of daily amiodarone treatment resulted in a 4.5-fold increase in total phospholipid in alveolar macrophages recovered from the lungs by bronchoalveolar lavage. The presence of the phospholipidosis had no effect on the phagocytosis of heat-killed yeast cells, the induction of luminol-dependent chemiluminescence, or the spontaneous release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), or spontaneous and LPS-stimulated release of IL-1 by alveolar macrophages in vitro. In contrast, the LPS-stimulated release of IL-6 and TNF alpha by phospholipidotic alveolar macrophages was enhanced compared with control cells. The pulmonary clearance of Listeria monocytogenes following intratracheal administration of the bacteria was not affected by the phospholipidotic condition. It appears that, in the context of the functions studied, the induction of pulmonary phospholipidosis by amiodarone does not impair pulmonary host defense processes in rats, and may actually be associated with the augmentation of some activities. PMID- 8618942 TI - Antioxidant capacity in different tissues of young and old rats. AB - The antioxidant capacity in heart, liver, lung, and kidney was studied in young (6 months) and old (22 months) male Fischer 344 rats, using the oxygen radical absorbance capacity (ORAC) assay system with two different reactive oxygen species (ROS) generators. The results indicated that liver in old rats had significantly lower peroxyl radical absorbance capacity (ORACROO), units/g wet wt), but higher hydroxyl radical absorbance capacity (ORACOH), units/mg protein) than in young rats. The decreased liver ORACROO in the old rats was mainly due to the loss of cytosol protein, while the increased liver ORACOH in the old rats was a result of an increased resistance of cytosol proteins to the attack of ROS. This conclusion was further supported by the finding that the contribution of nonprotein fraction of liver cytosol to the ORACOH of the cytosol decreased with age. No effect of age was found on either ORACROO or ORACOH in other tissues. The antioxidant capacity for both ORACROO and ORACOH, was usually high in liver and kidney but low in lung and heart. PMID- 8618943 TI - Potentiation of nitric oxide-mediated vasorelaxation by xanthine oxidase inhibitors. AB - Nitric oxide (NO), now almost synonymous with endothelium-derived relaxing factor (EDRF), reacts with superoxide anion radical (O2-) and forms a potentially toxic molecular species, peroxynitrite (ONOO-). Because xanthine oxidase (XO) seems to be a major O2- -producing enzyme in the vascular system, it is important to clarify the mechanism of XO regulation of NO/EDRF. We first characterized the inhibition of XO in vitro by three types of pyrazolopyrimidine derivatives. Kinetic studies indicated that 4-amino-6-hydroxpyrazolo[3,4-d]pyrimidine (AHPP) and allopurinol competitively inhibited the conversion of xanthine to uric acid catalyzed by XO, with apparent Ki values of 0.17 +/- 0.02 and 0.50 +/- 0.03 micro M respectively; alloxanthine inhibited this conversion in a noncompetitive manner with an apparent Ki value of 3.54 +/- 1.12 microM. O2- generation in the xanthine/XO system assayed by lucigenin-dependent chemiluminescence was suppressed most strongly by AHPP in a dose-dependent fashion; allopurinol itself appears to reduce the enzyme by transfer of an electron to O2, thus generating O(2-). AHPP significantly augmented EDRF-mediated relaxation of aortic rings from both rabbits and spontaneously hypertensive rats (SHR) in a dose-dependent manner, whereas allopurinol did not affect the relaxation and only marginal potentiation of the vasorelaxation was observed with alloxanthine. Finally, iv injection of AHPP (50.4 mg/kg; 100 micromol/300 g rat) reduced the blood pressure of SHR rats to 70% of the initial pressure; this pressure is almost the blood pressure of normal rats. Allopurinol (100 micromol/300 g rat; iv) showed transient decrease in blood pressure and moderate reduction of hypertension of SHR (10%) was observed with iv injection of alloxanthine (100 mumol/300 g rat). On the basis of these results, it seems that XO regulates EDRF/NO via production of O2-. PMID- 8618944 TI - Thermoregulation with age: restoration of beta(3)-adrenergic responsiveness in brown adipose tissue by cold exposure. AB - The beta(3)-adrenergic-stimulated thermogenic response in brown adipose tissue (BAT) is impaired in senescent rats, whereas cold-induced thermogenesis is not. To determine if cold exposure can restore beta(3)-adrenergic receptor responsiveness in senescent rats, we examined BAT mitochondrial GDP binding in young and old rats, and UCP mRNA levels in young rats following stimulation by the beta(3)-adrenergic agonist CGP-12177 with and without prior cold exposure. F 344 male rats were maintained at thermoneutrality or exposed to 8 degrees C for 48 hr, followed by a 24-hr period of rewarming before administration of 0.75 mg/kg CGP-12177 or vehicle solution. During the rewarming period, GDP binding remained elevated but UCP mRNA levels with a half-life of 11 hr returned to levels observed in the thermoneutral controls. In young rats, both cold exposure and administration of the beta(3)-adrenergic agonist to thermoneutral controls increased GDP binding 2-fold and UCP mRNA levels 4-fold. However, in cold-exposed young rats, there was no further increase with beta(3)-agonist treatment. In senescent control rats, CGP-12177 did not increase GDP binding, but cold exposure did. However, in cold-exposed old rats, the beta(3)-agonist was now able to increase GDP binding. The induction of UCP mRNA by CGP-12177 was also investigated and found to be 25% less in senescent compared with young rats. These observations indicate that cold exposure restores the impaired beta(3) adrenergic signal transduction in BAT from senescent rats. One possibility is that cold exposure induces the synthesis of one or more components in the beta(3) adrenergic pathway in senescent rats. PMID- 8618945 TI - Copper intestinal absorption in the rat: effect of free fatty acids and triglycerides. AB - The absorption of some minerals has been shown to be affected by the presence of unhydrolyzed dietary triglycerides and free fatty acids generated from their partial hydrolysis. Since copper (Cu) can form poorly soluble soaps with long chain fatty acids, we examined whether the uptake of Cu from the intestinal lumen is altered by the presence of fatty acids and triglycerides using an in vivo jejunal perfusion procedure. Long-chain fatty acids palmitate and stearate at 1.0 mM reduced Cu absorption rates compared with infusates without either fatty acid or triglycerides (means +/- SEM, controls: 104.4 +/- 8.8 pmole/min x cm vs palmitate: 12.5 +/- 17.6, P < 0.01; stearate:37.2 +/- 25.6, P < 0.05). Medium chain free fatty acids had no effect on Cu absorption (caprylate: 90.6 +/- 14.9, not significant; caproate: 69.5 +/- 14.2, not significant). Similarly, neither an emulsion of medium chain nor long-chain triglycerides at a total 1.0 or 2.5 mM concentration altered Cu absorption. The inhibitory effect of palmitate and stearate on Cu absorption was accompanied by a reduction in lumen-to-mucosa water influx (controls: 5.33 +/- 0.26 microl/min x cm vs palmitate: 3.20 +/- 0.70, P < 0.01; stearate: 3.36 +/- 0.52, P < 0.01). The data are consistent with a potential impairment of Cu intestinal absorption by long-chain free fatty acids which may accumulate in the jejunum following excessive fat intake and/or lipid malabsorption. PMID- 8618946 TI - How do long-chain free fatty acids cross cell membranes? PMID- 8618947 TI - Protein-mediated facilitated uptake processes for fatty acids, bilirubin, and other amphipathic compounds. PMID- 8618949 TI - Progress in cloning embryos from domesticated livestock. PMID- 8618948 TI - Eukaryotic polypeptide elongation system and its sensitivity to the inhibitory substances of plant origin. AB - The structural and functional characteristics of the elongation system (ribosomes and elongation factors) are presented. The immunochemical and diagnostic meaning of the ribosome investigations is considered. Evidence of the participation of ribosomes in the first step of protein glycosylation is presented. The heterogeneous elongation factor eEF-1, isolated from Guerin epithelioma, can be separated into three fractions: one of them functionally corresponds to EF-1 alpha, the second on to EF-1 beta gamma, and the third is an unidentified, active aggregate named EF-1B, which contains the subunit forms EF-1 alpha and EF-1 beta gamma, and other polypeptides showing protein kinase activity. The aggregate EF 1B can be autophosphorylated, while the subunit forms EF-1 alpha and EF-1 beta gamma can neither become autophosphorylated nor phosphorylate other polypeptides. The subunit form EF-beta gamma consists from two polypeptides of 32 and 51 kDa, corresponding to other eukaryotic beta and gamma polypeptides, respectively. EF-1 beta gamma is thermostable and protects against thermal inactivation of EF-1 alpha in the EF-1 alpha-EF-1 beta gamma complex. Pure eEF-2 preparations isolated from normal and neoplastic tissues show different structural features. The existence of eEF-2 in multiple forms, differing in molecular mass, have been found. The eEF-2 with molecular weight of about 100 kDa can be phosphorylated, while eEF-2 of about 65 kDa was not phosphorylated by protein kinase eEF-2. The phosphorylated eEF-2 lost its activity, and this effect was reversed by dephosphorylation. The eEF-2 (65 kDa) was isolated from the active polyribosomes, and it may directly participate in the translocation step of the peptide elongation. It was noted that the components of elongation system can be inhibited, in separate steps, by the substances isolated from various sources of plant origin. Alkaloids emetine and cepheline, cardiac remedy digoxin, saponin glycoside, and its aglycon directly inactivated ribosomes. Quercetin inhibited eEF-1 activity by directly influencing its subunit form EF-1 alpha. eEF-2 was shown to be a target site of the inhibitory action of the glycoside isolated from Melissa officinalis leaves. PMID- 8618950 TI - Utilization of the bone/liver alkaline phosphatase activity ratio in blood plasma as an indicator of ascorbate deficiency in salmonid fish. AB - The goal of this study was to test the hypothesis that the ratio of liver to bone alkaline phosphatase in blood plasma reflects the ascorbate status in scurvy prone teleost fish (rainbow trout [Oncorhynchus mykiss]). The studies focused on finding a method for distinguishing bone alkaline phosphatase present in blood plasma from other alkaline phosphatase isoforms. We tested temperature optima and thermostability of liver, kidney, gill cartilage, and intestinal alkaline phosphatases. We did not observe differences among liver, bone, and kidney enzymes with respect to temperature optima and thermostability. We partially purified alkaline phosphatase from juvenile rainbow trout vertebrae and liver using n-butanol solubilization and ammonium sulfate fractionation. We found a difference between bone alkaline phosphatase, which precipitated in 0%-20% ammonium sulfate saturation, and liver enzyme, which required 40%-50% ammonium sulfate saturation to precipitation. We conducted a series of urea inactivation studies on partially purified enzymes from liver and vertebrae. Urea differentially inhibited the enzymes with t 1/2 = 1.1 and 0.4 min, for bone and liver, respectively. Subsequently, we subjected blood plasma alkaline phosphatase to urea inhibition, and using regression analysis we calculated the ratio of liver to bone alkaline phosphatase. We found that thus obtained ratios of bone enzyme in blood plasma correlated with liver ascorbate concentration. Bone alkaline phosphatase declined in ascorbate deficiency 10-fold, whereas low ascorbate status resulted in a 3.5-fold decrease. In order to draw a general conclusion on the linearity of the response of blood plasma/bone alkaline phosphatase as an indicator of ascorbate deficiency in fish, further studies must include analysis of individual fish followed in the process of developing avitaminosis. PMID- 8618951 TI - Fatty acids enter cells by simple diffusion. PMID- 8618952 TI - Vasoactive intestinal peptide stimulates prolactin mRNA expression in turkey pituitary cells: effects of dopaminergic drugs. AB - It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased with 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quinpirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture. PMID- 8618953 TI - Elimination of EL-4 and L1210 murine tumors by 1,3-bis (2-chloroethyl)-1 nitrosourea requires an intact immune response. AB - The chemotherapeutic agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) is commonly used to treat several types of human cancers. Recent investigations have suggested that elimination of tumor by BCNU is dependent on more than the cytotoxic activity of the drug. We have extended those findings by showing that cyclosporin A (CS) can inhibit the BCNU-mediated rejection of EL-4 or L1210 tumors in mice. It was shown that mice could be cured of EL-4 or L1210 ascites tumors with a single intraperitoneal injection of BCNU. When CS, an inhibitor of the activation of T lymphocytes, was administered to mice that had received either EL-4 or L1210 tumor and were treated with BCNU, nearly all the mice died by Day 60. When CS was administered to BCNU-treated mice starting at 1, 2 or 3 weeks after tumor injection, inhibition of the BCNU therapy did not occur. Finally, the ability of animals that had been cured of tumor by the BCNU therapy to reject a lethal challenge dose of homologous tumor was shown to be CS insensitive. These results suggest that the BCNU-mediated elimination of tumor from mice requires a functional immune response in addition to the cytotoxic activity of this chemotherapeutic agent. PMID- 8618954 TI - Differential effects of diabetes and glomerulonephritis on glomerular basement membrane composition. AB - The hallmark of renal diseases involving the glomerulus is the presence of proteinuria. While the routes of pathogenesis of proteinuria have not been established, alterations in the barrier function of the glomerular basement membrane (GBM) have been implicated. We evaluated the effect of streptozotocin diabetes and passive Heymann nephritis (PHN) over time on the macromolecular composition of rat GBM to determine if changes in composition correlate with proteinuria. Six to twelve rats from each group (control, diabetic, and PHN) were sacrificed 1, 5, 28, 56, or 84 days after induction of disease. Identical amounts of GBM were subjected to a sequential extraction procedure, and type IV collagen, entactin, laminin, fibronectin, and anionic charge content were quantitated in the extracts. Type IV collagen and entactin content did not change with time or disease. Both laminin and fibronectin contents increased with time in GBM in all groups, but this increase was significantly greater in diabetic GBM. A significant decrease in anionic charge content of GBM coincided with the onset of albuminuria at Day 28 in diabetes, but no change was seen in PHN. In diabetic rats, the increase in laminin content over control preceded the onset of albuminuria, while the increase in fibronectin was not apparent until after albuminuria was present. In PHN, no differences in type IV collagen, entactin, laminin, fibronectin, or anionic charge content of GBM were found compared with control, even though profound albuminuria was evident from Day 5 through 84. Thus, while alterations in laminin and fibronectin content may contribute to the loss of glomerular permselectivity in streptozotocin diabetes, such changes apparently are not involved in PHN. PMID- 8618955 TI - Adhesion proteins increase cellular attachment, follicle-stimulating hormone receptors, and progesterone production in cultured porcine granulosa cells. AB - We sought to determine the influence of different constituents of the extracellular matrix on porcine granulosa cell function by assessing cellular attachment, cellular morphology, follicle-stimulating hormone (FSH) receptors, and progesterone production. Cells from immature porcine ovarian follicles were cultured for up to 6 days in serum-free medium containing porcine FSH (pFSH, 10 ng/ml) in culture dishes either uncoated or coated with one of the following adhesion proteins: gelatin (1 mg/cm2), fibronectin (1 microgram/cm2), laminin (1 microgram/cm2), type I collagen (10 micrograms/cm2), or type IV collagen (7.8 micrograms/cm2). Fibronectin, laminin, type I collagen, and type IV collagen increased cellular attachment significantly (P < 0.05). All adhesion proteins except gelatin influenced cellular morphology. Cells cultured on laminin or type IV collagen formed dense clusters of rounded cells. Cells cultured in dishes coated with each adhesion protein except gelatin had higher 125I-pFSH binding per cell than cells cultured in uncoated dishes, with increases of 7- to 12-fold over control (P < 0.05). All adhesion proteins increased progesterone production, ranging from 10- to 50-fold over control (P < 0.05). In summary, not only did adhesion proteins increase attachment to the dishes but they also increased FSH receptors and differentiated function (progesterone production) of granulosa cells from immature porcine ovarian follicles. PMID- 8618956 TI - Efficacy of the class III antiarrhythmic agent azimilide in rodent models of ventricular arrhythmia. AB - Azimilide exhibited antiarrhythmic activity in several rodent models of ventricular arrhythmias. In the mouse chloroform model, azimilide provided limited efficacy by the i.p. route (50% at 100 mg/kg versus 20% by vehicle), and no efficacy by the oral route (300 mg/kg). In a rat model in which arrhythmias are induced by ligation and reperfusion of the left descending coronary artery (CALR model), azimilide provided dose-dependent (1-18 mg/kg) efficacy by the intravenous route. The estimated dose that suppressed ventricular fibrillation (VF) was 5.0 mg/kg i.v. At 18 mg/kg i.v. azimilide also partially suppressed ventricular tachyarrhythmia (VT) and extrasystoles (VES). Rats dosed orally (100 mg/kg) were fully protected from VF. In isolated guinea pig hearts exposed to 1 microM ouabain, azimilide at 10 microM prevented the VT and VF seen in 69% and 23%, respectively, of control hearts. In anesthetized guinea pigs, azimilide at 10 and 30 mg/kg i.v. increased the dose of ouabain required to induce VES. While sematilide, dofetilide, and E-4031 significantly increased sensitivity to the arrhythmogenic actions of ouabain (by lowering the dose that caused VF), azimilide did not. Azimilide's antiarrhythmic profile in these rodent models differs from that of other class III agents, since azimilide had less efficacy in the mouse chloroform model, could suppress VT and VES as well as VF in the CALR rat model, and protected from or did not aggravate cardiac glycoside-induced arrhythmias in guinea pigs. These results demonstrating the antiarrhythmic efficacy of azimilide in the intact animal suggest that the compound has a different profile than other class III agents. PMID- 8618958 TI - Speaking rate effects on stops produced by Spanish and English monolinguals and Spanish/English bilinguals. AB - Four groups of 10 subjects each (English and Spanish monolinguals, and two groups of Spanish/English bilinguals) produced Spanish or English sentences at speaking rates designated 'normal', 'slow', and 'fast'. Voice onset time (VOT) was measured in word-initial tokens of /p/ and /t/ found in sentence-initial, medial, and -final words. The four groups produced comparable changes in sentence duration across the three rates. The speaking rate changes exerted less effect on the VOT in stops spoken by the Spanish than the English monolinguals. Moreover, whereas English monolinguals produced /p,t/ (with shorter VOT at a fast than at a normal rate, many Spanish monolinguals showed a trend in the opposite direction. As expected, all 10 early bilinguals produced English stops with VOT values that were similar to the English monolinguals'. They also showed speaking rate effects on VOT that were similar to those observed for the English monolinguals. The late bilinguals, who had begun learning English as adults, showed smaller effects of speaking rate on VOT than did the English monolinguals. Their mean VOT values for English stops spanned a wide range of values. Just 3 of the late bilinguals produced English stops with VOT values that fell within the range of values observed for the English monolinguals. PMID- 8618957 TI - Age of learning affects rate-dependent processing of stops in a second language. AB - The aim of this study was to assess the effect of speaking rate changes on the perception of English stop consonants by four groups of subjects: English and Spanish monolinguals, 'early' Spanish/English bilinguals who learned English in childhood, and 'late' bilinguals who learned English in adulthood. Subjects identified, and then later rated for goodness as exemplars of the English /p/ category, the members of two voice onset time (VOT) continua. The English monolinguals identified a well-defined range of VOT stimuli as English /p/, and stimuli with longer VOT values as 'exaggerated' instances of English /p/. Their goodness ratings increased as VOT increased, then showed a systematic decrease as VOT began to exceed values typical for English /p/. The English monolinguals' goodness ratings also varied systematically as a function of speaking rate, which was simulated in the two continua by varying syllable duration. The Spanish monolinguals, on the other hand, failed to consistently identify any of the stimuli as English /p/. Although speaking rate influenced their goodness ratings, the Spanish monolinguals' rate effects differed significantly from the English monolinguals'. The early bilinguals resembled the English monolinguals, and differed from the Spanish monolinguals to a greater extent than did the late Spanish/English bilinguals. This was taken as support for the hypothesis that early bilinguals are more likely than are late bilinguals to establish new phonetic categories for stop consonants in a second language. PMID- 8618959 TI - Physiology and biochemistry of Drosophila learning mutants. AB - Single gene mutants of Drosophila that are defective in learning/memory processes have increased substantially our understanding of the physiology, biochemistry, and anatomy underlying conditioned behaviors. Drosophila learning mutants can be separated into two general classes, those with structural defects in the brain and those without (conditioning mutants) any obvious brain alterations. From studies of brain structural mutants, two neuroanatomic areas have merged as important for normal conditioned behavior: the mushroom bodies and the central complex. Biochemical and molecular genetic studies of the conditioning mutants have implicated numerous types of molecules in learning, but the adenosine 3',5' cyclic monophosphate (cAMP) second messenger pathway has emerged as especially important. Five different genes in this pathway, amnesiac (a product similar to adenylate cyclase activating peptides), dunce (cAMP phosophodiesterase), rutabaga (adenylyl cyclase), DCO (protein kinase A), and dCREB2 (cAMP-response element binding protein), have proven important for normal learning. The products of many of these learning mutants are enriched in mushroom bodies, which highlight the importance of mushroom bodies for normal learning and the cAMP second messenger cascade for the physiology of mushroom body cells in their role(s) underlying learning. Physiological studies of the mutants have demonstrated that plastic properties of synaptic transmission, including facilitation and posttetanic potentiation, are abnormal in the mutants. An appendix describing the currently used paradigms to test Drosophila behavior is included. PMID- 8618960 TI - Degeneration and regeneration of axons in the lesioned spinal cord. AB - For many decades, the inability of lesioned central neurons to regrow was accepted almost as a "law of nature", and on the clinical level, spinal cord and brain lesions were seen as being irreversible. Today we are starting to understand the mechanisms of neuronal regeneration in the central nervous system and its presence in the periphery. There is now a rapid expansion in this field of neuroscience. Developmental neurobiology has produced tools and concepts that start to show their impact on regeneration research. This is particularly true for the availability of antibodies and factors and for the rapidly growing cellular and molecular understanding of crucial aspects of neurite growth, guidance, target finding, and synapse stabilization. New cell biological concepts on the mechanisms of neuron survival and death and on the interaction of inflammatory cells with the central nervous system also find their way into the field of spinal cord and brain lesions and have, indeed, led already to new therapeutic approaches. This review briefly summarizes the current knowledge on the mechanisms involved in degeneration and tissue loss and in axonal regeneration subsequent to spinal cord lesions, particularly in mammals and humans. PMID- 8618961 TI - Molecular diversity of myofibrillar proteins: gene regulation and functional significance. AB - Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers. PMID- 8618962 TI - Paracrine regulation of the renal microcirculation. AB - There has been an explosive growth of interest in the multiple interacting paracrine systems that influence renal microvascular function. This review first discusses the membrane activation mechanisms for renal vascular control. Evidence is provided that there are differential activating mechanisms regulating pre- and postglomerular arteriolar vascular smooth muscle cells. The next section deals with the critical role of the endothelium in the control of renal vascular function and covers the recent findings related to the role of nitric oxide and other endothelial-derived factors. This section is followed by an analysis of the roles of vasoactive paracrine systems that have their origin from adjoining tubular structures. The interplay of signals between the epithelial cells and the vascular network to provide feedback regulation of renal hemodynamics is developed. Because of their well-recognized contributions to the regulation of renal microvascular function, three major paracrine systems are discussed in separate sections. Recent findings related to the role of intrarenally formed angiotensin II and the prominence of the AT1 receptors are described. The possible contribution of purinergic compounds is then discussed. Recognition of the emerging role of extracellular ATP operating via P2 receptors as well as the more recognized functions of the P1 receptors provides fertile ground for further studies. In the next section, the family of vasoactive arachidonic acid metabolites is described. Possibilities for a myriad of interacting functions operating both directly on vascular smooth muscle cells and indirectly via influences on endothelial and epithelial cells are discussed. Particular attention is given to the more recent developments related to hemodynamic actions of the cytochrome P-450 metabolites. The final section discusses unique mechanisms that may be responsible for differential regulation of medullary blood flow by locally formed paracrine agents. Several sections provide perspectives on the complex interactions among the multiple mechanisms responsible for paracrine regulation of the renal microcirculation. This plurality of regulatory interactions highlights the need for experimental strategies that include integrative approaches that allow manifestation of indirect as well as direct influences of these paracrine systems on renal microvascular function. PMID- 8618963 TI - Malignant hyperthermia: excitation-contraction coupling, Ca2+ release channel, and cell Ca2+ regulation defects. AB - Malignant hyperthermia (MH) is a disorder of skeletal muscle in which certain anesthetic agents trigger a sustained elevation in myoplasmic Ca2+ concentration that activates metabolic and contractile activity. This review focuses on the biochemical and physiological alterations in the skeletal muscle of MH susceptible (MHS) pigs and humans that appear responsible for this inherited disorder. In porcine MH, these studies identified the skeletal muscle sarcoplasmic reticulum Ca2+ release channel gene (RYR1) as the site of the defect. A mutation in this protein results in altered excitation-contraction coupling and secondary changes in porcine muscle structure and function. Although RYR1 mutations have been reported in many MHS human families, there is also significant genetic heterogeneity, and much less is known as to the underlying mechanism responsible for altered human myoplasmic Ca2+ regulation. The effects of caffeine and anesthetic agents on MHS and normal muscle are also discussed to better understand the basis for the in vitro clinical test for this disorder and mechanisms responsible for the initiation and maintenance of MH episodes in susceptible individuals. Finally, we examine the possiblity of a defect in Ca2+ regulation in tissues other than skeletal muscle. Current understanding of the molecular basis of MH elegantly illustrates the successful integration of knowledge obtained from all fields of biological and clinical science. PMID- 8618964 TI - Transcriptional control of osteoblast growth and differentiation. AB - Osteoblast differentiation is a multistep series of events modulated by an integrated cascade of gene expression that initially supports proliferation and the sequential expression of genes associated with the biosynthesis, organization, and mineralization of the bone extracellular matrix. Transcriptional control defines regulatory events operative both developmentally and for support of bone tissue-specific properties. This review focuses on components of transcriptional regulation that function in growth control during osteoblast proliferation and those that postproliferatively contribute to maturation of the bone phenotype. Emphasis is on transcription of the cell cycle regulated histone gene and the bone-specific osteocalcin gene as paradigms for genes with promoter elements exhibiting responsiveness to a broad spectrum of physiological regulatory signals. Additionally, the potential contributions provided by the three-dimensional organization of the histone and osteocalcin gene promoters to integration of regulatory activities at multiple, independent, and overlapping regulatory domains are explored. PMID- 8618965 TI - The histomorphologic changes in vascularized bone transfer and their interrelationship with the recipient sites: a 1-year study. AB - In 13 New Zealand White rabbits with a mean age of 6 months, vascularized bone transfers incorporated as paired auricular anterior myo-osseous flaps were harvested; they were placed in either an inlay or an onlay position relative to the zygomatic arch. The onlay bone transfers were placed either in full contact or in partial contact with the zygomatic arch. The animals were sacrificed 1 year after transfer. At 1 year, the inlay transfer simulated the adjacent zygoma in width and thickness. Onlay full contact transfers maintained significant aug mentation in thickness of the zygoma, while the onlay partial contact transfers did not; the thickness of the augmented zygoma in the onlay full contact subgroup was significantly greater than that in the onlay partial contact transfers. The onlay partial contact grafts had remodeled into the zygoma in bone contact, where the orientation of mismatched osteons within the bone transfers had transformed to match that of the native zygoma. In areas of bone contact between the onlay and the host bone, full-thickness conversion from a cortical to a trabecular architecture had occurred in both the transfer and host bones. These findings have numerous implications regarding mechanisms that could be exploited clinically to optimize the survival of a bone transfer; they also raise questions regarding alteration of the recipient bed after placement of an onlay bone transfer. PMID- 8618967 TI - Where have all the old patients gone? PMID- 8618966 TI - Internal mastopexy following explantation. AB - Controversy over the theoretical complications from silicone gel breast prostheses has led many patients to pursue explantation or removal of the implants. Consequently, the breast is often misshapened. An internal mastopexy can be performed in conjunction with explantation, requiring little additional operative time and no additional scars. Although limited by tissue availability, the procedure can minimize ptosis and improve projection. On the basis of our experience with 27 patients, we feel that the internal mastopexy following explantation is a worthwhile consideration for selected patients. PMID- 8618968 TI - Great toe-to-hand transfer nourished by arterial inflow through the venous system. AB - Revascularization of tissues through their venous system is currently used in vascularized venous flaps and in replantation of some fingertips. A toe-to-hand transfer that suffered prolonged and unexplainable arterial spasm, unrelenting to the usual therapeutic measures, was revascularized through its venous system. Tissue perfusion in the toe began 24 hours after the vascular repairs were through, because arterial flow was hampered by the venous valves in the toe. Tissue perfusion was poor initially but became stable 72 hours postoperatively, and the toe survived. The only complications were epidermolysis and pseudoarthrosis. We consider this technique for tissue revascularization as a suitable salvage method in cases where all other therapeutic measures fail. PMID- 8618969 TI - Exploration of intracranial structures endoscopically through minimal craniotomies. AB - This study brings to light the potential use of intracranial endoscopic techniques that can be combined with osteotomies and bone distraction to improve precision and reduce complications in craniofacial surgery. It also anticipates the widespread use of endoscopic techniques to reduce the morbidity and mortality of plastic surgical and neurosurgical intracranial surgery. PMID- 8618970 TI - Dermabrasion using an ultrasonic surgical aspirator. AB - We used an ultrasonic surgical aspirator on the epidermal surface to perform dermabrasion instead of the conventional motor-driven grinder. It was determined on histologic examination that it is possible to fragment the epidermis with greater selectively using the ultrasonic surgical aspirator. Abrasion also can be performed safely on spotty lesions and intricate, problematic regions with the ultrasonic surgical aspirator. We feel that the ultrasonic surgical aspirator is a promising device for use in dermabrasion. PMID- 8618971 TI - Brow lift by flap transposition in the glabrous brow area. AB - In brow lift, if a direct approach is utilized, the suture line medially is placed in a normal frontalis crease, and then the lateral part of the suture line, by lateral transfer of a small superiorly based flap, is caused to fall into a natural "laugh line" or "crow's feet" crease, three improvements result: 1. Greater control and predictability of shaping and position than with other techniques; 2. A more normal appearance, including better lateral lift and a much more inconspicuous scar than with supraciliary direct lift; 3. A longer-lasting result. In approximately 150 patients, scar appearance has been very gratifying indeed, with only 3 limited segmental scar revisions and 2 secondary midforehead elliptical excisions. It is believed that the principle exhibited in the tightening of the upper suture line by flap transfer, referred to by analogy as the worm principle, accounts for much of the improvement, especially in durability. This principle is seen as an aid in thinking through the mechanics of several other plastic surgical situations as well. PMID- 8618972 TI - Nipple reconstruction with a new local tissue flap. AB - We present a one-staged local cutaneous-fat flap for nipple reconstruction. It has proven reliable in producing a geometrically correct nipple without complication. It represents a modification of existing cutaneous or dermal-fat flaps with theoretical and apparent benefits inherent in its design. PMID- 8618973 TI - Tendon-graft harvest using endoscopic guidance. AB - Traditional methods for harvesting tendon grafts using direct visualization commonly require multiple minute incisions or possibly a single incision if blind dissection by means of a tendon stripper is acceptable. Endoscope-assisted tendon graft retrieval also can be limited to a solitary incision but, simultaneously because of the advantage of continuous visualization, permits a more precise and safer dissection as regards important contiguous anatomic structures. Upper extremity tendons can be readily manipulated by this technique, but at least with currently available equipment the more rigid deep fascia of the lower extremity so far has made such an adaptation cumbersome. The value of the surgical endoscope as an adjunct not only for tendon-graft harvest but perhaps someday for minimally invasive tendon transfer or tenolysis is encouraging. PMID- 8618974 TI - Primary closure of the donor site for the Littler neurovascular island flap transfer. AB - After successful transfer of the Littler neurovascular island flap, patients may be troubled by persistent problems at the donor site. We describe a technique that allows primary closure of this site, which has significantly improved appearance and sensation of the donor finger. PMID- 8618975 TI - Tissue expansion: a method to preserve bone length and joints following traumatic amputations of the leg--a follow-up of five legs amputated at different levels. AB - Tissue expansion of adjacent intact skin and subcutaneous tissue has in five legs provided high-quality soft-tissue coverage of the distal end in legs amputated at different levels without further shortening of the bone. The sensitivity of slowly expanded flaps is temporarily affected but eventually seems to return to normal. Expanded flaps have endured both sitting and the use of prostheses for 4 to 5 years. In one case the technique made it possible to save the knee joint. Besides being a functional reconstruction, the appearance of the reconstructed parts much improved. A technique of overexpansion and double flap coverage of the bone utilizing deepithelialization is described. PMID- 8618976 TI - An unusual complication following intralesional triamcinolone injections. PMID- 8618977 TI - ePTFE (Gore-Tex) facial augmentation. PMID- 8618978 TI - Flexion deformity of the PIP joints due to tuberculous arthritis: 7-year follow up. PMID- 8618979 TI - Abdominal closure after TRAM flaps. PMID- 8618980 TI - The platysma aponeurosis. PMID- 8618981 TI - Dealing with a distorted ear in a face lift. PMID- 8618982 TI - Endoscopic retrieval of packing sponge from wound. PMID- 8618983 TI - Body dysmorphic disorder and the plastic surgeon. PMID- 8618984 TI - Penis reconstruction with an island composite flap of the deep inferior epigastric vascular pedicle. PMID- 8618985 TI - Autologous blood use in reduction mammaplasty. PMID- 8618986 TI - Microvascular anatomy of the galeal and temporoparietal fascia. PMID- 8618987 TI - Breast reconstruction with myocutaneous flaps in previously irradiated patients. PMID- 8618988 TI - Does patient-controlled analgesia lead to delayed diagnosis of lower limb compartment syndrome? PMID- 8618989 TI - A new dressing for skin graft with hydrocolloid and staples. PMID- 8618990 TI - A prospective study of the relationship between strabismus and head posture in patients with frontal plagiocephaly. AB - A prospective study was performed on 16 unoperated patients with frontal plagiocephaly to characterize the relationship between strabismus and abnormal head posture. Serial eye examinations were performed preoperatively and for 24 to 46 months following fronto-orbital advancement. In 14 patients (88 percent), preoperative clinical examination and CT scan indicated coronal plagiocephaly (synostotic); unicoronal synostosis was documented during fronto-orbital advancement in all these patients. Ten patients had abnormal head posture on preoperative examination, 9 of whom had strabismus at some time during the study. These 9 patients all had unicoronal synostosis with ipsilateral strabismus and a contralateral head tilt. All had eye muscle findings consistent with superior oblique paresis, although in 2 patients these signs first developed following fronto-orbital advancement. Strabismus resolved spontaneously in 2 patients between 2 and 8 months following fronto-orbital advancement; the remaining 7 patients underwent extraocular muscle surgery following fronto-orbital advancement, with early resolution of strabismus in all cases. The head tilt resolved or improved significantly in all 9 patients following resolution of the strabismus. Two patients had recurrent superior oblique paresis following surgical correction, necessitating secondary strabismus surgery. The present study indicates that extraocular muscle dysfunction is the major cause of abnormal head posture in patients with coronal plagiocephaly and emphasizes the need for long-term ophthalmologic surveillance in these patients. PMID- 8618991 TI - Correction of ocular dystopia. AB - The purpose of this study was to examine results with elective surgical correction of enophthalmos. The study was a retrospective assessment in a university-based referral practice. A consecutive sample of 10 patients who developed ocular dystopia following orbital trauma was examined. The main outcome measures were a subjective evaluation by patients and objective measurements of patients' eye position. The intervention was three-dimensional orbital reconstruction with titanium plates. It is concluded that satisfactory correction of enophthalmos and ocular dystopia can be achieved with elective surgery using titanium plates. In addition, intraoperative measurements of eye position in three planes increases the precision of surgery. PMID- 8618992 TI - Delayed in utero repair of surgically created fetal cleft lip and palate. PMID- 8618993 TI - The use of magnetic resonance angiography prior to pharyngeal flap surgery in patients with velocardiofacial syndrome. AB - Twenty consecutive patients with velocardiofacial syndrome underwent magnetic resonance angiography (MRA) to determine if abnormalities of the neck arteries would contraindicate pharyngeal flap surgery. All 20 patients were found to have anomalies to the carotid arteries, vertebral arteries, medially placed internal carotids, low carotid bifurcations, and tortuous or kinked internal carotids. The internal carotids were found to be almost directly under the mucous membrane of the pharynx in two patients. In these two patients, the arteries were close to the pharyngeal midline at the base of the first cervical vertebra and might easily be severed during the raising of a pharyngeal flap. Hypoplastic vertebral arteries also were found. One patient had an extra neck vessel. The anomalies of the internal carotids did not have a strong correlation with endoscopically observed pulsations in the position affected the location of the internal carotids did not have a strong posterior pharyngeal wall. It also was found that head position affected the location of the internal carotid arteries when they were located close to the pharyngeal mucous membrane. The information provided in the MRA studies allowed assessment of the arterial anomalies in relation to the flap donor site so that the patients in the sample who underwent pharyngeal flap surgery using a short superiorly based flap had no major bleeding complications. PMID- 8618994 TI - Alteration in facial sensibility in adolescents following sagittal split and chin osteotomies of the mandible. AB - Static two-point discrimination, pressure, and vibratory threshold values were measured bilaterally at standard coordinates in the area of the face innervated by the mental nerve in 115 adolescents (230 nerves). The patients were divided into four groups: normal adolescents who had not undergone any orthognathic surgery (group I controls, n = 134 nerves, mean age 18 years, SD = 3), those 1 year after undergoing bilateral sagittal split osteotomies of the mandible (group II, n = 14 nerves, mean age 19 years, SD = 2), those 1 year after undergoing an osteoplastic genioplasty (group III, n = 40, mean age 19 years, SD = 3), and those 1 year after undergoing a combination of bilateral sagittal split osteotomies and an osteoplastic genioplasty (group IV, n = 42 nerves, mean age 19 years, SD = 3). Subjective residual numbness at the 1 year postoperative interval was reported by 2 of 7 patients in group II, 2 of 20 patients in group III, and 14 of 21 patients (67 percent) in group IV. Long-term subjective numbness involved only the chin skin in 16 of 18 patients experiencing residual numbness and was perceived as problematic in the remaining 2 (group IV) patients whose subjective numbness also was measured objectively in the chin, lower lip (mucosa and skin), and gingiva. Objectively, the mean threshold values of the three sensory modalities tested were higher in group IV patients than in the remaining groups at all coordinates tested, but significant differences (p < 0.05) were found only between the mean two-point discrimination of group IV patients and the control group in the region of the chin skin. The high percentage of patients documented to have subjective and objective sensory disturbance after undergoing a combination of sagittal split osteotomies of the mandible and an osteoplastic genioplasty (group IV) may be explained by the "double crush syndrome." PMID- 8618995 TI - The deep temporal lift: a multiplanar, lateral brow, temporal, and upper face lift. AB - An extended brow upper facelift technique employing a multiplanar, temporal approach is described. Fresh cadaver dissections are utilized to demonstrate the anatomic basis of the approach. A complete mobilization of the orbital portion of the orbicularis oculi muscle away from the orbital rim is described. Release of the malar orbicularis (zygomatic cutaneous ligament) above the zygomaticus major muscle and over the origin of the masseter muscle is emphasized. Indications and patient selection are reviewed and the clinical benefit to the brow, lateral canthus, lower lid, and malar orbicularis cheek pad is demonstrated in different patient groups. PMID- 8618996 TI - The silicone columellar strut. AB - The placement of a silicone columellar strut serves to correct columellar retraction, to improve the nasolabial angle, and to increase tip projection by elevating the medical crura of the lower lateral cartilages. The potential complications of extrusion, infection, and migration are minimal if the implant is placed in a separate watertight compartment with a tension-free closure and no impingement of the nasal dome. PMID- 8618997 TI - Implantable cardioverter-defibrillator: another device to cover. AB - The implantable cardioverter-defibrillator is a mechanical device developed to manage patients with life-threatening arrhythmias when pharmacologic control has failed or produced unacceptable side effects. It is a significant amount of foreign material with a generator pack (volume 113 to 145 cc, weight 197 to 235 gm) and two or three leads and patches that are inserted into or placed on the heart. Although it has worked very well in preventing premature death, there have been complications associated with the device itself. The most significant of these has been exposure and/or infection. We present three patients who have experienced this problem. Improved coverage has been accomplished by burying the implant beneath the rectus abdominis muscle in situations where skin and subcutaneous tissue alone have proved inadequate. By dividing one or two tendinous inscriptions and the anterior limb of the internal oblique fascia, a musculofascial pocket is created to contain the generator and lead wires. This provided satisfactory coverage in two of our three patients. The single failure resulted from external trauma to the abdominal wall. PMID- 8618999 TI - Periareolar mammaplasty: double skin technique with application of polyglactine or mixed mesh. AB - This paper presents the author's experience of 7 years in 254 patients operated on using a personal mammaplasty technique with a periareolar approach. The technique is based on the principle that the skin alone does not prevent early ptosis. This paper proposes the repositioning of all breast connective structures, treating the glandular set separately from the cutaneous lining, which is doubled, applying a circular flap of dermis with the central pedicle to the areolar region. For this purpose, the following structures have been employed, which, once repositioned and together, will act as a support network: (1) anterior pectoral fascia, (2) Cooper's intramammary ligament, (3) periareolar dermal flap, employed as skin inner lining. (4) application of absorbable polyglactine 910 or mixed mesh (with polyester) as a sandwich between both layers of skin, and (5) external cutaneous lining that composes the assembly of double skin. PMID- 8618998 TI - Breast reduction under intravenous sedation: a review of 50 cases. AB - Breast reduction is a surgical procedure most commonly performed on an inpatient basis under general anesthesia. In the current climate of health care reform, we must evaluate such procedures to determine if there are alternate, less expensive, but equally safe means to perform them. Our purpose is to present our experience with 50 bilateral breast reductions performed under local anesthesia with intravenous sedation between October of 1991 and October of 1994. We have excluded bilateral reductions under 500 gm total, unilateral reductions, mastopexies, and gynecomastia procedures. Patients were sedated with intravenous Versed and fentanyl and a local solution consisting of marcaine, lidocaine, and 1:2000,000 epinephrine. Intercostal blocks were not used routinely. Monitoring and sedation were performed by nonanesthesia personnel in 49 patients. There were no complications relating to the sedation or to the local solution. All reductions were performed by the inferior pedicle technique. The average patient age was 28.0 years (20 to 67 years). The total breast tissue resected was 1372 gm (516 to 2948 gm), with 33 patients having resections greater than 1000 gm. Operative times averaged 3 hours (115 to 275 minutes). Forty-nine of the 50 patients tolerated the procedure with little or no recall. Twenty-eight patients were discharged on the same day as admission. One patient recalled some significant discomfort during parts of the procedure. All stated that they would again have the procedure performed under local anesthesia with intravenous sedation. Our conclusions are as follows: (1) Breast reduction can be performed safely and comfortably under local anesthesia with intravenous sedation. (2) Patients should be chosen on their acceptability as intravenous sedation candidates and not with regard to the amount of breast tissue removed. (3) There will be a subset of patients who can be discharged on the same day. PMID- 8619000 TI - Is there liability with chemotherapy following immediate breast construction? AB - Review of 46 consecutive patients undergoing immediate breast reconstruction with tissue expanders and subsequent implant placement was conducted to assess the potential liability of adjuvant chemotherapy. Twenty-three patients required chemotherapy, while 23 did not. Critical comparison of complications and outcome of the two groups revealed no significant differences. Within the parameters of the study (avoidance of expansion or surgery during the period of chemotherapy), there appeared to be no disadvantage posed to the immediate reconstruction patient by adjuvant chemotherapy. We feel that this option can continue to be offered despite the anticipation of probable chemotherapy. PMID- 8619001 TI - Obstetrical brachial plexus palsy: results following neurolysis of conducting neuromas-in-continuity. AB - Sixteen infants with conducting neuromas-in-continuity at primary brachial plexus exploration underwent microsurgical neurolysis of their lesions. For each patient, the immediate preoperative scores for individual joint movements were compared with scores at the last examination. In the Erb's palsy group (n = 9), significant improvement was seen in shoulder movements, elbow flexion, supination, and wrist extension (paired t test, p < 0.05). Clinically useful improvements in function was seen at the shoulder and elbow (Fisher's exact test, p < 0.05). In the total palsy group (n = 7), significant improvement in shoulder abduction, shoulder adduction, elbow flexion, and extension of the wrist, fingers, and thumb was seen (paired t test, p < 0.05), but there was no significant improvement in the proportion of patients with useful functional outcomes. Neurolysis in Erb's palsy improves both muscle grade and the functional ability of patients. Neurolysis does not provide useful functional recovery in patients with total plexus palsy. PMID- 8619002 TI - Consideration of a thin flap as an entity and clinical applications of the thin anterolateral thigh flap. AB - A defatted (thinned) anterolateral thigh flap was designed to reconstruct skin defects requiring thin flap coverage. We used this flap as a free flap for five cases of skin defects, and the outcomes of the reconstructions were all successful. The vascular pedicle of this flap, the cutaneous perforator of the lateral circumflex femoral artery, is about 8 cm long and 2 mm in diameter, and it is ideal for microvascular anastomosis. Thinning is performed in about 3 to 4 mm of thickness almost uniformly except for the vascular pedicle. It was ascertained as one of the useful donor sites of the free thin flap. The virtue of the thin anterolateral thigh flap is its uniform thinness compared with other thin flaps reported previously--the thin groin flap and the thin rectus abdominis musculocutaneous flap. We considered thin flaps as an entity, and they are classified into three types. PMID- 8619003 TI - A reappraisal of axial and nonaxial lower leg fascial flaps: an anatomic study in human cadavers. AB - The aim of this study was to determine the sources of blood supply to the deep fascia of the lower leg and therefore to identify potential new sites where axial and nonaxial flaps might be raised. The deep fascia was harvested from 18 embalmed cadaver lower legs. The blood supply to the deep fascia was examined with the aid of a dissecting microscope. Four clinically important sources of supply to the deep fascia were identified. These were (1) axial and nonaxial fasciocutaneous perforators, (2) nonaxial musculocutaneous perforators, (3) axial cutaneous branches of the sural arteries, and (4) an axial fascial branch from the saphenous artery (present in 75 percent of the dissections). This study has highlighted the potential for utilizing not only the fasciocutaneous system and sural system of vessels but also the fascial branch of the saphenous artery in the design of lower leg axial fascial flaps. Nonaxial fascial island flaps may be raised utilizing not only the fasciocutaneous system of vessels but also the musculocutaneous system of vessels. PMID- 8619005 TI - Effects of Antidepressants and antipsychotics on the 5HT2 receptor-mediated signal transducing system in human platelets. AB - The IC50 values of the antidepressants amoxapine, mianserin, desipramine, clomipramine and imipramine, and the antipsychotics spiroperidol, chlorpromazine, levomepromazine and haloperidol for 3H-ketanserin binding, 5HT-induced intracellular Ca2+ increase, 5HT-induced shape change and 3H-paroxetine binding in human platelets were measured and the correlations of each parameter were examined. Results were as follows. Both the antidepressants and the antipsychotics had inhibitory effects on 3H-ketanserin binding, 5HT-induced intracellular Ca2+ increase and 5HT-induced shape change and there were significant positive correlations between the IC50 values of these three parameters. On the other hand, there were no significant correlations between the IC50 values in 3H-paroxetine binding and those in the other three parameters. These results suggest that 3H-ketanserin binding, 5HT-induced intracellular Ca2+ increase and 5HT-induced shape change are useful and reliable tools for the assessment of the effects of the antidepressants and the antipsychotics on the 5HT2 receptor-mediated signal transducing system in human platelets. PMID- 8619004 TI - Schizophrenia and psychostimulant abuse: a review and re-analysis of clinical evidence. AB - The authors selected articles from those published between 1975 and 1994 that specifically documented psychostimulant abuse in patients determined to be schizophrenic according to recent and relatively uniform diagnostic criteria. These articles indicated that the incidence of psychostimulant abuse in schizophrenics is 2-5 times higher than that of the general public. Additionally, unlike the decline in stimulant use seen in older adults in the general population, high rates of abuse appeared to be maintained in schizophrenics. Although the incidence of abuse in this group was high, comparisons of abuse rates generated by self report with those obtained by urinalysis indicated that the frequency of abuse is being underestimated by 15-21%. Potential factors contributing to stimulant abuse in schizophrenics, including the disease process, and the influence of chronic neuroleptic medication, were evaluated. Results indicated that the incidence of psychostimulant abuse was neither a common property of psychiatric patients, nor exclusive to schizophrenics, but appeared to be related to chronic treatment with neuroleptic drugs. Symptom severity was generally similar in schizophrenic abusers and non-abusers, which also suggested a degree of independence from the disease process. In a majority of the studies surveyed, abuse of stimulants followed disease onset. It was also found that stimulant abuse was associated with marked increases in hospitalization in this patient group, including those known to be neuroleptic medication compliant. Possible explanations for the initiation and maintenance of psychostimulant abuse in schizophrenics are discussed in relation to clinical and preclinical evidence on drug addiction. PMID- 8619007 TI - A comparative study of the psychological effects of DN-2327, a partial benzodiazepine agonist, and alprazolam. AB - The effects of single oral doses of DN-2327 (DN, 2 mg or 3 mg), a newly developed partial benzodiazepine receptor agonist, and alprazolam (APZ, 0.8 mg), a full receptor agonist, on psychomotor function and short-term memory were assessed using three psychometric tests: letter cancellation, visual vigilance and Sternberg's memory scanning task. Twelve healthy male volunteers participated in this study. Randomized, double-blind, cross-over test sessions were conducted at 2-week intervals. Both 3 mg DN and 0.8 mg APZ increased the time to completion of the letter cancellation task at 3 h after administration, but neither had any effect on accuracy of response. In the visual vigilance task, which required relatively intense concentration and continuous attention, both the number of errors and reaction times to correct responses significantly increased from 1.5 to 3.5 h after administration of 3 mg DN and at 3.5 h after administration with 0.8 mg APZ. DN at 2 mg also significantly increased the number of errors from 1.5 to 3.5 h after administration, but it did not affect reaction times. In the memory scanning task, 3 mg DN, but not 2 mg DN or APZ, significantly increased overall reaction times at 2 h after administration. These performance deficits paralleled the time-course changes in serum concentrations of both drugs and appeared to be associated with the hypnotic-sedative effects of the drugs tested. These findings did not support those of previous preclinical studies of DN, indicating superiority of DN over conventional full benzodiazepine agonists/anxiolytics in terms of adverse behavioral consequences. PMID- 8619006 TI - Methamphetamine facilitates ethanol-induced depressions in cerebellar Purkinje neurons of prazocin- or DSP4-treated rats. AB - Methamphetamine (MA) and ethanol (EtOH) are two commonly abused drugs. Previous behavioral studies indicated that MA may synergistically alter EtOH responses. In the present study, we found that local application of MA did not potentiate ethanol-induced depressions of the spontaneous activity of Purkinje neurons in urethane-anesthetized rats. We and others previously found that, in cerebellar Purkinje neurons, EtOH and gamma-amino-butyric acid (GABA)-mediated depressions can be enhanced by norepinephrine (NE) acting via beta-adrenergic receptors while these responses are decreased by activation of alpha-adrenergic receptors. In the present experiment, after blocking alpha-adrenergic receptors with prazocin, MA significantly enhanced EtOH responses in most of neurons studied. It has been reported that MA may directly and indirectly enhance alpha-adrenergic and beta adrenergic receptor-mediated responses. The present study may suggest that MA can negatively modulate (antagonize) the depressant effects of ethanol via the alpha adrenergic receptor, which oppose the positive modulatory mechanism (potentiation of EtOH depression) via actions of the beta-adrenergic receptors. We found that lesioning NE neurons with N-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride (DSP4), a selective noradrenergic neurotoxin, enhance the MA-facilitated ethanol responses, suggesting that this action of MA may not require NE. Since it has been reported that MA increases serotonin (5-HT) and catecholamine release from their nerve terminals, MA may potentiate EtOH depressions by facilitating the release of NE and 5-HT. Taken together, our data suggested that MA may modulate EtOH responses via catecholaminergic and serotonergic mechanisms in cerebellar Purkinje neurons. PMID- 8619008 TI - Diazepam prevents progression of kindled alcohol withdrawal behaviour. AB - The purpose of the present experiment was to study the "kindling" hypothesis of alcohol withdrawal stating that exposure to repeated episodes of alcohol withdrawal results in an increased severity of subsequent withdrawal reactions. Two groups of male Wistar rats were subjected to 13 episodes of 2 days severe alcohol intoxication and 5 days alcohol withdrawal. Animals in the control group (n = 80) developed clinical withdrawal signs following each intoxication episode. In the diazepam group (n = 80) the withdrawal reactions during episodes 1-9 were blocked by intraperitoneal diazepam administration (0-30 mg/kg) 8, 11 and 15 h into withdrawal. During episode 10-13 diazepam treatment was terminated and convulsive withdrawal behaviour was observed 9-15 h after last alcohol dose. The probability of seizure activity during these four withdrawal episodes was calculated as 0.239 and 0.066 in the control and the diazepam groups, respectively. Based on Monte Carlo simulation techniques, this difference was found to be statistically significant (P < 0.05). No differences in the non convulsive alcohol withdrawal symptoms tremor, hyperactivity and rigidity were detected between controls and diazepam animals after diazepam treatment. It was concluded that the increased convulsive behaviour in the control group was caused by cumulated kindling-like cerebral alterations during the previous repeated alcohol withdrawal phases. PMID- 8619009 TI - GM1 ganglioside administration partially counteracts the morphological changes associated with haloperidol treatment within the dorsal striatum of the rat. AB - Haloperidol, a typical antipsychotic drug, causes an increase in the mean percentage of synapses within the situation containing a discontinuous, or perforated, postsynaptic density (PSD) following 1 month of treatment (Meshul et al. 1994). This effect is not observed with the atypical antipsychotic drug, clozapine, following subchronic administration (Meshul et al. 1992a). This morphological change is also associated with an increase in the density of dopamine D2 receptors. The synapses containing the perforated PSD are asymmetrical and the nerve terminals contain the neurotransmitter, glutamate, as demonstrated by immunocytochemistry. We have also shown that subchronic treatment with haloperidol (0.5 mg/kg per day, 30 days) results in a decrease in the density of glutamate immunoreactivity within asymmetric nerve terminals associated with perforated and non-perforated PSDs (Meshul and Tan 1994). This could be due to an increase in glutamate release, perhaps due to activation of corticostriatal synapses. Agnati et el. (1983a) reported that administration of GM1 ganglioside blocks the increase in dopamine D2 receptors following haloperidol treatment. GM1 has also been shown to attenuate the release of glutamate (Nicoletti et al. 1989). In order to determine if similar treatment with ganglioside could block the haloperidol-induced ultrastructural changes notes above, rats were co-administered GM1 (10 mg/kg per day) and haloperidol (0.5 mg/kg per day) for 30 days. We report that GM1 blocked the haloperidol induced increase in striatal asymmetric synapses containing a perforated PSD, but had no effect on the increase in dopamine D2 receptors or the decrease in nerve terminal glutamate immunoreactivity. GM1, either alone or co-administered with haloperidol, also caused a small, but significant, increase in the density of all asymmetric synapses within the striatum. It is possible that the effect of GM1 in attenuating the haloperidol-induced change in glutamate synapses with perforated PSDs is primarily postsynaptic, since GM1 did not block the change in density of glutamate immunoreactivity within asymmetric nerve terminals. PMID- 8619011 TI - Effects of transderman nicotine on mood and sleep in nonsmoking major depressed patients. AB - The role of nicotine as an indirect cholinergic agent in sleep has been studied in normal subjects. There are no studies of its effects on sleep in depressed patients. Nicotine transdermal patches (17.5 mg), were studied in eight depressed patients (DSM-III-R) and eight normal volunteers. Subjects wore placebo and nicotine patches for 24 h. Depressed patients showed increased REM sleep without changes in other sleep variables. They also showed a short term improvement of mood. Normal volunteers had sleep fragmentation, and reduction of REM sleep time. No major side effects were reported in either group. PMID- 8619010 TI - Prior morphine exposure enhances ibogaine antagonism of morphine-induced locomotor stimulation. AB - Ibogaine is currently being investigated for its potential use as an anti addictive agent. In the present study we sought to determine whether prior morphine exposure influences the ability of ibogaine to inhibit morphine-induced locomotor stimulation. Female Sprague-Dawley rats were pretreated once a day for 1-4 days with morphine (5, 10, 20 or 30 mg/kg, i.p.) or saline and then received ibogaine (40 mg/kg, i.p.) 5 h after the last morphine pretreatment dose. Compared to rats pretreated with saline, rats pretreated with morphine (10, 20 or 30 mg/kg, i.p.) before ibogaine (40 mg/kg, i.p.) showed a significant reduction in morphine-induced (5 mg/kg, i.p.) locomotor stimulation when tested 29 h after ibogaine administration. Furthermore, this effect was apparent over a range of ibogaine (5-60 mg/kg, i.p.) and morphine test (2.5-5 mg/kg, i.p.) dosages. Doses of ibogaine (5 and 10 mg/kg, i.p.) which alone were inactive inhibited morphine induced locomotor activity when rats had been pretreated with morphine. These results, showing that morphine pre-exposure affects ibogaine activity, suggest that variable histories of opioid exposure might account for individual differences in the efficacy of ibogaine to inhibit opioid addiction. PMID- 8619012 TI - Dopamine agonists facilitate footshock-elicited locomotion in rats, and suppress level-press responding for food. AB - Several dopamine agonists (apomorphine, quinpirole, 7-OH-DPAT, and U-91356A) suppressed locomotor activities of rats exploring a Y-maze, presumably through activation of dopamine autoreceptors. If brief electric shocks were applied to the grid floor during exploration, locomotion was unchanged in control rats, but the locomotor suppression from the dopamine agonists was converted to a profound stimulation. This locomotor stimulation was completely antagonized by pretreatment with sulpiride. SKF 38393 and clonidine produced no locomotor stimulation in the shock environment. To test whether the locomotor stimulant effect from dopamine agonists generalized to a food-reinforced behavior, rats were trained to lever-press for food according to a multiple (VI-10", VI-40") schedule. The above compounds only suppressed responding with no stimulation, and the suppressant effect on food-reinforced behavior was also blocked by sulpiride. It is concluded that the behavioral inhibitory effect from dopamine autoreceptor activation can be readily overcome by exteroceptive stimulation, which uncovers a powerful motor stimulant effect. This stimulant effect, however, did not generalize to lever-press responding for food. PMID- 8619013 TI - Vesamicol, an acetylcholine uptake blocker in presynaptic vesicles, suppresses rapid eye movement (REM) sleep in the rat. AB - Vesamicol inhibits acetylcholine uptake in presynaptic vesicles and reduce its release. The present study was performed in order to test the effects of this drug in a cholinergic related function as rapid eye movement (REM) sleep. Wistar male rats were implanted for sleep recordings. In addition, a stainless steel cannula was implanted into the left lateral ventricle for intracerebroventricular (ICV) injections. In experiment 1, a dose-response curve was performed. Saline or vesamicol (20, 40, 80 and 100 micrograms) were injected. Following the ICV injections, animals' sleep was recorded for 8 h. In experiment 2, after adaptation and baseline recordings, animals received 50 micrograms vesamicol ICV at 1000 hours. every 24 h for 2 consecutive days. After each injection an 8-h sleep recording session was performed. Two subsequent recovery recordings were allowed. Results obtained in experiment 1 showed a dose-response reduction of REM sleep with significant values at 80 micrograms and 100 micrograms of vesamicol. The main findings in experiment 2 were a reduction in REM sleep time and an increase in REM sleep latency. On the recovery days, a dramatic rebound of REM sleep was observed. Vesamicol behaved as an anticholinergic drug. It produced a reduction in REM sleep time and a rebound of this sleep stage after its withdrawal. PMID- 8619014 TI - Temperature, food intake, and locomotor activity effects of a 5-HT3 receptor agonist and two 5-HT3 receptor antagonists in rats. AB - This study investigated three physiologic functions known to be modulated by serotonin-temperature, food intake and locomotor activity - using the 5-HT3 receptor agonist, m-chlorophenylbiguanide (m-CPBG), and two 5-HT3 antagonists, MDL-72222 and ondansetron. m-CPBG produced dose-dependent elevations in rectal temperature. MDL-72222, which had no effects on temperature when given alone, significantly attenuated m-CPBG-induced hyperthermia. Food intake in food deprived rats was reduced during the first hour by the highest dose of m-CPBG. Food intake was also dose-dependently reduced by MDL-72222; m-CPBG plus MDL-72222 led to greater reductions in food intake. Food intake in freely fed rats was unaffected by m-CPBG or MDL-72222. Locomotor activity was unaffected by m-CPBG, but was dose-dependently reduced by MDL-72222, an effect which may have contributed to its hypophagic effects. Ondansetron, used in ten-fold lower doses than MDL-72222, was inactive in all of these paradigms. These data: (1) provide some evidence for 5-HT3 receptor-mediated changes in temperature; (2) are in agreement with two prior studies which reported locomotor activity reductions following 5-HT3 antagonists; but (3) do not support an important role for 5-HT3 receptors in the regulation of food intake in rats. PMID- 8619015 TI - Pharmacokinetic and pharmacodynamic responses to caffeine in poor and normal sleepers. AB - Pharmacokinetic and pharmacodynamic responses to caffeine (2.5 mg/kg) were compared between ten healthy self-rated poor sleepers and ten normal sleepers. Sleep pattern assessed by the Pittsburgh Sleep Quality Index (PSQI). There was no significant difference in mean estimated daily caffeine consumption between the groups. The poor sleepers had significantly higher scores for neuroticism on the Eysenck Personality Questionnaire (EPQ) and anxiety on the Hospital Anxiety Depression (HAD) scale, compared with normal sleepers. Caffeine pharmacokinetics were assessed by measurement of saliva caffeine concentrations. Poor sleepers showed significantly greater variability in caffeine Cmax, clearance had half life, compared to normal sleepers. Pharmacodynamic measures included heart rate, blood pressure, visual analogue scales for concentration, vigilance and relaxation, psychomotor performance [Digit Symbol Substitution Test (DSST) and tapping rate (TR)] and EEG activity [Contingent negative variation (CNV), auditory evoked potential and power spectral analysis]. Prior to caffeine administration, poor sleepers compared to normal sleepers had faster heart rates, lower ratings for concentration and relaxation, poorer performance on the DSST, greater CNV magnitude, faster peak alpha frequency and lower delta, theta and beta power. These differences persisted after caffeine ingestion and overall differences between the groups on these measures were significant (P < 0.01 .001). Post-dose, but not pre-dose, scores for vigilance and TR were significantly lower overall in poor compared with normal sleepers. Despite the baseline differences between poor and normal sleepers, the changes following caffeine administration were similar in direction and magnitude in both groups. PMID- 8619016 TI - Melatonin phase advances circadian rhythm. AB - We studied the effect of acute (1 day) and subacute (7 days) treatment with melatonin (0.5 mg) on the endogenous rhythms of melatonin secretion in 12 healthy male volunteers, using a placebo-controlled, double-blind, cross-over design. Melatonin given at 1700 h for 7 days significantly advanced the onset of endogenous melatonin secretion, while a single dose was without effect. These data are consistent with the hypothesis that melatonin plays a role in the organisation of circadian rhythms in humans and suggest that appropriately timed melatonin administration may provide a means of altering the timing of circadian cycles. PMID- 8619017 TI - Fenton chemistry: an introduction. AB - In 1876, Fenton described a colored product obtained on mixing tartaric acid with hydrogen peroxide and a low concentration of a ferrous salt. Full papers in 1894 and 1896 showed the product was dihydroxymaleic acid. Haber, Weiss and Willstatter proposed in 1932-1934 the involvement of free hydroxyl radicals in the iron(II)/hydrogen peroxide system, and Baxendale and colleagues around 1950 suggested that superoxide reduces the iron(III) formed on reaction, explaining the catalytic nature of the metal. Since Fridovich and colleagues discovered the importance of superoxide dismutase in 1968, numerous studies have sought to explain the deleterious effects of cellular oxidative stress in terms of superoxide-driven Fenton chemistry. There remain questions concerning the involvement of free hydroxyl radicals or reactions of metal/oxo intermediates. However, these outstanding questions may obscure a wider appreciation of the importance of Fenton chemistry involving hypohalous acids rather than hydrogen peroxide as the oxidant. PMID- 8619018 TI - Catalytic metals, ascorbate and free radicals: combinations to avoid. AB - Trace levels of transition metals can participate in the metal-catalyzed Haber Weiss reaction (superoxide-driven Fenton reaction) as well as catalyze the oxidation of ascorbate. Generally ascorbate is thought of as an excellent reducing agent; it is able to serve as a donor antioxidant in free radical mediated oxidation processes. However, as a reducing agent it is also able to reduce redox-active metals such as copper and iron, thereby increasing the pro oxidant chemistry of these metals. Thus ascorbate can serve as both a pro-oxidant and an antioxidant. In general, at low ascorbate concentrations, ascorbate is prone to be a pro-oxidant, and at high concentrations, it will tend to be an antioxidant. Hence there is a crossover effect. We propose that the "position" of this crossover effect is a function of the catalytic metal concentration. In this presentation, we discuss: (1) the role of catalytic metals in free radical mediated oxidations; (2) ascorbate as both a pro-oxidant and an antioxidant; (3) catalytic metal catalysis of ascorbate oxidation; (4) use of ascorbate to determine adventitious catalytic metal concentrations; (5) use of ascorbate radical as a marker of oxidative stress; and (6) use of ascorbate and iron as free radical pro-oxidants in photodynamic therapy of cancer. PMID- 8619019 TI - Role of Fenton chemistry in thiol-induced toxicity and apoptosis. AB - Under certain conditions, many radioprotective thiols can be toxic, causing loss of colony-forming ability in cultured mammalian cells in a biphasic fashion whereby the thiols are not toxic at high or low concentrations of the drug, but cause decreased clonogenicity at intermediate (0.2-1.0 mM) drug levels. This symposium paper summarizes our studies using dithiothreitol (DTT) as a model thiol to demonstrate the role of Fenton chemistry in thiol toxicity. The toxicity of DTT in V79 cells has several characteristics: it is dependent on the medium used during exposure of cells to the drug; the toxicity is decreased or prevented by addition of catalase exogenously, but superoxide dismutase has no effect; the toxicity is increased by addition of copper, either free or derived from ceruloplasmin in serum; and the toxicity can be modified intracellularly by altering glucose availability or pentose cycle activity. Thus the data are consistent with a mechanism whereby DTT oxidation produces H2O2 in a reaction catalyzed by metals, predominantly copper, followed by reaction of H2O2 in a metal-catalyzed Fenton reaction to produce the ultimate toxic species, .OH. Studies comparing 12 thiols have shown that the magnitude of cell killing and pattern of dependence on thiol concentration vary among the different agents, with the toxicity depending on the interplay between the rates of two reactions: thiol oxidation and the reaction between the thiol and the H2O2 produced during the thiol oxidation. The addition of other metals, e.g. Zn2+, and metal chelators, e.g. EDTA, can also alter DTT toxicity by altering the rates of thiol oxidation or the Fenton reaction. Recent studies have shown that in certain cell lines thiols can also cause apoptosis in a biphasic pattern, with little apoptosis at low or high drug concentrations but greatly increased apoptosis levels at intermediate (approximately 3 mM) thiol concentrations. There appears to be a good correlation between those thiols that cause loss of clonogenicity and those that induce apoptosis, suggesting similar mechanisms may be involved in both end points. However, thiol-induced apoptosis is not prevented by addition of exogenous catalase. These observations are discussed in relation to the possible role of Fenton chemistry in induction of apoptosis by thiols. PMID- 8619020 TI - Role of guanosine triphosphate in ferric ion-linked Fenton chemistry. AB - We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3 carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules. PMID- 8619021 TI - Radiobiology of ultrasoft X rays. V. Modification of cell inactivation by dimethyl sulfoxide. AB - The effects of the radioprotector dimethyl sulfoxide (DMSO) were investigated for carbon-K (0.28 keV) and aluminum-K (1.47 keV) X rays compared with 60Co gamma rays for inactivation of mouse C3H 10T1/2 cells. The protection factor for 2 M DMSO was found to be 2.8 for both of the ultrasoft X-ray energies, which is not significantly different from the protection factor of 2.6 found for gamma rays. The results indicate that the proportion of scavengeable lethal damage from gamma and X rays does not depend on the proportion of the total energy that is deposited by low-energy electrons of relatively high ionization density. PMID- 8619022 TI - Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells. AB - Research to determine the effects of defined numbers of alpha particles on individual mammalian cells is helpful in understanding risks associated with exposure to radon. This paper reports the first biological data generated using the single-particle/single-cell irradiation system developed at Pacific Northwest Laboratory. Using this apparatus, CHO-K1 cells were exposed to controlled numbers of 3.2 MeV alpha particles, and biological responses of individual cells to these irradiations were quantified. Chromosomal damage, measured by the induction of micronuclei, was evaluated after no, one, two, three or five particle traversals. Exposures of up to five alpha particles had no influence on the total numbers of cells recovered for scoring. With increased numbers of alpha particles there was a decrease in the ratio of binucleated to mononucleated cells of 3.5%/hit, suggesting that alpha particles induced dose-dependent mitotic delay. A linear hit-response relationship was observed for micronucleus induction: Micronuclei/binucleated cell = 0.013 +/- 0.036 + (0.08 +/- 0.013) x D, where D is the number of particles. When the estimated dose per alpha-particle traversal was related to the frequency of induced micronuclei, the amount of chromosomal damage per unit dose was found to be similar to that resulting from exposures to alpha particles from other types of sources. Approximately 72% of the cells exposed to five alpha particles yield no micronuclei, suggesting the potential for differential sensitivity in the cell population. Additional studies are needed to control biological variables such as stage of the cell cycle and physical parameters to ensure that each cell scored received the same number of nuclear traversals. PMID- 8619023 TI - Micronucleus induction in human lymphocytes: comparative effects of X rays, alpha particles, beta particles and neutrons and implications for biological dosimetry. AB - The cytokinesis-block micronucleus assay in peripheral blood lymphocytes has the potential for being a simple and rapid method of biological dosimetry. This technique has been used to study the induction of micronuclei in the blood from 12 donors after exposure to a range of radiations with track-averaged LET values ranging from 0.26 to 44 keV microns -1. Data based on the average response of the 12 individuals for 250 kVp X rays were found to agree well with results published previously from other laboratories using similar techniques. Low dose-limiting RBE values relative to 250 kVp X rays for the radiations studied were found to be 0.50 for strontium/yttrium-90 beta particles, 6.9 for 20-23 keV microns -1 alpha particles and 17 for 24 keV neutrons. The pattern of the variation of individual radiosensitivity was found to be complex and dependent on dose, and the evaluation of individual radiosensitivity based on the response at one dose only can be misleading. It is concluded that, although the cytokinesis-block micronucleus assay in blood lymphocytes is a radiobiologically appropriate technique to use for biological dosimetry, its practical implementation may be limited by a need to perform individual pre-exposure calibrations. PMID- 8619024 TI - The kinetics of repair of sublethal radiation-induced damage in pig skin: studies with multiple interfraction intervals. AB - The kinetics of the repair of radiation-induced sublethal damage (SLD) was studied for the epidermis of the pig. A total of either 7 or 14 interfraction intervals with incomplete repair was achieved by giving 28 fractions either as 7 x 2 fractions/day plus a top-up dose of 17 Gy (half tolerance) or as 14 x 2 fractions/day. The dose per fraction ranged from 1.96-4.82 Gy. A total of 9 intervals ranging from 0.17 h up to 8 h between fractions was used. The incidence of moist desquamation, as an estimate of acute epidermal response, was used as an end point to establish dose-effect relationships. The data were analyzed using either the incomplete repair model of Thames, assuming mono-exponential repair kinetics, or a modified version of the incomplete repair model, assuming bi exponential repair of sublethal damage. Both methods of analysis allowed for the longer overnight interval between fractions. Analysis assuming mono-exponential repair gave a T1/2 of 0.74 h for the combined data, although there was a trend toward a longer half-time when only the longer interfraction intervals ( > 1.0 h) were used in the analysis. A further analysis using the modified version of an incomplete repair model gave a fast and a slow component of repair with significantly different half-times of 0.09 and 4.5 h, respectively. Varying the number of incomplete repair intervals by replacing half the number of fractions with a single half-tolerance top-up dose did not modify the kinetics of repair significantly, in terms of either the repair half-times or the proportion of repair associated with a fast and slow component. Reanalysis of data published previously for 3 and 4 fractions using the modified incomplete repair model again resulted in two components of repair, represented by the significantly different half-times of 0.17 and 3.0 h. These values were similar to those obtained from the multiple-fraction experiment. These data clearly demonstrate that an acutely responding tissue is associated with a long T1/2 for the repair of SLD which is independent of the dose per fraction. For accelerated fractionation schedules in the clinic, using multiple fractions per day, these results suggest a need to control the intervals between fractions carefully and when appropriate to reduce the total dose to avoid serious normal-tissue complications. PMID- 8619025 TI - Mortality from leukemia after irradiation in infancy for skin hemangioma. AB - From 1920 through 1959, 14,624 infants were exposed to ionizing radiation for skin hemangioma at Radiumhemmet. They were all less than 18 months old (mean 6 months) at the time of first exposure. The irradiated hemangiomas were located all over the body. The weighted bone marrow dose was on average 0.13 Gy (range < 0.01-4.6 Gy). During the period 1920-1986, 20 deaths from leukemia (11 childhood and 9 adult) were observed in the cohort compared with 17 expected during the same period. There were no significant associations between childhood leukemia and radiation dose. Despite the relatively large number of infants studied, the low average dose to bone marrow limited the possibility of detecting a small radiation risk as might be predicted from other radiation studies. PMID- 8619026 TI - Influence of fractionation and fluence rate in photodynamic therapy with Photofrin or mTHPC. AB - Various schedules of fractionated photodynamic therapy (PDT), delivered at two different light fluence rates, were investigated in the RIF1 tumor model in an attempt to minimize the development of hypoxia during PDT and thereby improve tumor response relative to single treatments. The photosensitizers Photofrin and meta-tetrahydroxyphenylchlorin (mTHPC) were used in combination with either interstitial or superficial illumination. For both methods of illumination, equal volumetric light doses gave similar tumor responses, as measured by tumor regrowth times and number of cures. Fractionation of superficial illumination did not generally improve tumor response compared with a single illumination with the same total light dose. The only fractionated schedules which demonstrated a trend for increased cure were six fractions of superficial illumination given with short (1 h) dark periods between illuminations. Using both photosensitizers, an increase in tumor regrowth time occurred when tumors were illuminated interstitially with continuous light at a linear diffuser output of 50 mW compared with 100 mW per cm diffuser length. Discontinuous illumination with alternating light and dark periods of 30 s improved the tumor response further for mTHPC-mediated PDT at a fluence rate of 100 mW cm(-1). No improvement in response was seen by discontinuous interstitial illumination after Photofrin mediated PDT. These results demonstrate that lower fluence rates and/or fractionating the light dose delivered can improve the response of the RIF1 tumor to PDT but that the choice of dark intervals between fractions is critical. PMID- 8619027 TI - Measurement of differences in pO2 in response to perfluorocarbon/carbogen in FSa and NFSa murine fibrosarcomas with low-frequency electron paramagnetic resonance oximetry. AB - We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response. PMID- 8619028 TI - Protein-protein interactions between the Escherichia coli single-stranded DNA binding protein and exonuclease I. AB - It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria. PMID- 8619030 TI - DNA breakage upon K-shell excitation of phosphorus as a model for direct effects in radiation biology. AB - Single-strand breaks (SSBs) and double-strand breaks (DSBs) induced in DNA under phosphorus K-shell resonant absorption have been studied using supercoiled plasmids. The kinetics of the production of SSBs and DSBs exhibits a linear and a quadratic dependence, respectively, on photon fluence. Cross sections and quantum yields have been measured. The resonant photoexcitation of the phosphorus atoms was found to increase the DSB/SSB ratio compared to the off-resonance excitation. This enhancement factor can be related to the measured enhancement of the rate of cellular death and gene mutation in yeast under similar experimental conditions reported previously in the literature. Such resonant excitation of a specific atom belonging to DNA turns out to be an elegant method to investigate pure direct effects. PMID- 8619029 TI - Contrasting mechanisms of the myeloprotective effects of interleukin-1 against ionizing radiation and cytotoxic 5-fluorouracil. AB - Pretreatment with a single dose of interleukin-1 (IL-1) counteracts the myelosuppressive effects of radiation. In contrast, multiple doses are required to protect against several cytoablative drugs, suggesting different mechanisms. We examined the possibility that myeloprotection is due to IL-1-induced cycling of primitive progenitor cells. First, we evaluated the effect of the time between administration of IL-1 and 5-fluorouracil (5-FU), which kills cycling cells but spares quiescent early progenitors, on their interaction. Pretreatment with a single dose of IL-1 resulted in the death of mice treated with 5-FU provided IL-1 was given 18 h, but not 4 or 48 h, prior to administration of sublethal doses of 5-FU. Second, evaluation of primitive hematopoietic progenitor cells, 13-day spleen colony-forming units (CFU-S) and CFU with high proliferative potential revealed that treatment with 5-FU 18 h after administration of IL-1 results in reduction of CFU-S by 98% and of CFU with high proliferative potential by 65%, but only a 7 and 10% reduction, respectively, at 48 h. Third, in contrast to protection from death by pretreatment with a single dose of IL-1 at 24 h, two injections of IL-1 at 72 and 24 h before irradiation abrogated such protection. Similarly, the toxicity of 5-FU to progenitor cells was reduced when two injections of IL-1 were administered 48 h apart. This correlates with the time of up-regulation in the bone marrow cells of TGF-beta. These findings suggest that, depending on the schedule of treatment, administration of IL-1 may result in cycling of primitive progenitors, to protect against radiation, and may cause inhibition of cycling to protect against chemotherapeutic drugs. PMID- 8619031 TI - A statistical approach for analyzing clonogenic survival data. AB - The assay of colony-forming efficiency is a mainstay in the measurement of cell response in vitro to many physical and chemical agents. Currently, data on colony forming efficiency can be calculated in a variety of ways. Authors rarely describe in detail the methods used to determine the extent of biological variation within experiments. The use of standard methods of data analysis and presentation would improve interpretation of data and facilitate comparison between laboratories. Here we propose such a method. Binomial and Poisson probability theory were used to increase the accuracy of the estimate of the surviving fraction and to create an objective criterion for determining whether data obtained from serial dilutions of cell numbers used in the assay of colony forming efficiency should be excluded or included for further analysis. The variability inherent in the calculation of surviving fraction was determined by using Fieller's theorem, a special statistical application for assessing ratios of estimates, to determine the 95% confidence interval. All calculations were done on a simple and commercially available spreadsheet program. PMID- 8619033 TI - In memoriam Roy Charles Thompson (1920-1995). PMID- 8619032 TI - Radiation-induced formation of a crosslink between base moieties of deoxyguanosine and thymidine in deoxygenated solutions of d(CpGpTpA). AB - A new type of tandem base lesion has been observed in d(CpGpTpA) X-irradiated in aqueous solution. The lesion is attributed to the formation of a covalent bond between the C8 carbon atom of guanine and the methyl carbon atom of thymine. This tandem base lesion is formed in the absence of oxygen. It is the main product produced by ionizing radiation under these conditions. PMID- 8619034 TI - Comments on "Effects of low doses and low dose rates of external ionizing radiation: cancer mortality among nuclear industry workers in three countries" by E. Cardis et al. (Radiat. Res. 142, 117-132, 1995) PMID- 8619035 TI - Comments on the paper by Cardis et al. (Radiat. Res. 142, 117-132, 1995) PMID- 8619036 TI - Comments on "A critical analysis of the use of radiation inactivation to measure the mass of protein" by Lidzey et al. (Radiat. Res. 143, 181-186, 1995) PMID- 8619037 TI - The use of alanine/boron mixtures in neutron dosimetry. PMID- 8619038 TI - Current research suggests a change in irrigation therapy. PMID- 8619039 TI - Case #10. Neurilemoma. PMID- 8619040 TI - The pursuit of professionalism. PMID- 8619041 TI - Select protective gear carefully. PMID- 8619042 TI - Personal panache--and protection, too. PMID- 8619043 TI - RDH discovers new technique through in-office research. PMID- 8619044 TI - Pack instruments with care to ensure proper sterility. PMID- 8619045 TI - Clinical depression: a debilitating disease. PMID- 8619046 TI - Passport. PMID- 8619048 TI - The basics of research. PMID- 8619047 TI - All sterilization methods have their pros and cons. PMID- 8619050 TI - 10 steps to improve your professional image. PMID- 8619049 TI - Case #11. Gingival cyst. PMID- 8619051 TI - Will pocket depths be future indicators of heart disease? PMID- 8619052 TI - You can wait and watch or you can take action. PMID- 8619053 TI - New ways to fight perio infection subgingivally are making headway. PMID- 8619054 TI - HIV drugs. PMID- 8619055 TI - The right touch. PMID- 8619056 TI - Keeping close tabs on process best way to ensure sterility. PMID- 8619057 TI - Case #8. Granular cell tumor. PMID- 8619058 TI - Is a vaccine for perio disease on the horizon? PMID- 8619059 TI - Helping the homeless. PMID- 8619060 TI - Office design a key factor in maintaining infection control. PMID- 8619061 TI - New designs on infection control. PMID- 8619062 TI - Designing women. PMID- 8619063 TI - Anticoagulants. AB - Treatment plans for patients taking anticoagulants can become complicated. Anticoagulants predispose a patient to bleeding problems. Many drugs used in dentistry cannot be taken concomitantly with these medications. PMID- 8619064 TI - Make the commitment to meet the needs of our special patients. PMID- 8619065 TI - Interproximal brush, stick, may work better than floss. PMID- 8619066 TI - Case #9. Inflammatory fibrous hyperplasia. PMID- 8619067 TI - It takes two. PMID- 8619068 TI - Clean instruments properly to achieve optimum sterility. PMID- 8619069 TI - An ounce of prevention... PMID- 8619070 TI - Anticonvulsants. PMID- 8619071 TI - FDA recommends taking patients off Felbatol. PMID- 8619072 TI - Sweden may show us the way to our professional future. PMID- 8619073 TI - Basic science is changing the way we practice dental hygiene. PMID- 8619074 TI - Case #1. Acute necrotizing ulcerative gingivitis. PMID- 8619075 TI - The road to excellence. PMID- 8619076 TI - What desktop publishing can do for you--and your patients. PMID- 8619077 TI - Six ways to learn to love your job again. PMID- 8619078 TI - Have faith in the validity of biological monitoring. PMID- 8619080 TI - Case No. 2. Osteoma. PMID- 8619079 TI - Antidepressants. PMID- 8619081 TI - Two-week recall is helpful to specific patients' needs. PMID- 8619082 TI - Proper storage of sterile instruments is final step. PMID- 8619083 TI - The ABCs of a classroom visit. PMID- 8619084 TI - Bright smiles, bright futures. PMID- 8619086 TI - Designing ideas. PMID- 8619085 TI - Keep it under locks. PMID- 8619087 TI - Limited exposure. PMID- 8619088 TI - Snuff it out. PMID- 8619089 TI - How to be a good hygienist in a really bad dental office. PMID- 8619090 TI - Epidemiology of rheumatoid arthritis. AB - Rheumatoid arthritis is a relatively common disorder that affects men and women at the prime of their lives. Only 30% of the causes of rheumatoid arthritis can be attributed to genetic factors; the rest remain unexplained. Descriptive and analytic epidemiologic methods may lead to a better understanding of the causative, precipitating, and modulatory factors in rheumatoid arthritis. PMID- 8619091 TI - Rheumatoid arthritis. Outcome measures. AB - Rheumatologists have been pioneers in the development and use of clinical measures for outcome assessment. The Lansbury Index (1958) and the Empire Rheumatism Gold Trial (1960) used sophisticated double-blind pseudo-placebo controlled trial designs and standardized prespecified clinical outcome measures to establish the clinical usefulness of a drug whose benefit did not become evident until it was administered for several months. Since these studies, other studies have establish the clinical and statistical groundwork for rheumatoid arthritis outcome measures. In 1980, the Health Assessment Questionnaire and the Arthritis Impact Measurement Scales were added. Future development of paradigms for the decision process in the clinical management of individual rheumatoid arthritis patients will no doubt incorporate standard outcome measures to provide the data upon which management decisions can be based. PMID- 8619092 TI - Assessment of long-term outcomes of rheumatoid arthritis. How choices of measures and study designs may lead to apparently different conclusions. AB - In this article, previous review articles concerning long-term outcomes of rheumatoid arthritis are extended. A summary is provided of evidence that impressions concerning the long-term natural history and results of therapy in rheumatoid arthritis are strongly influenced by the types of measures and study designs used to assess patient status and outcomes. PMID- 8619093 TI - T cells in rheumatoid arthritis. Paradigms and facts. AB - Rheumatoid arthritis is characterized by a strong HLA-DRB1 association and a histologic picture consistent with an antigen recognition event by tissue infiltrating T cells. Basic immunology has seen major progress in the understanding of the T-cell receptor-MHC-antigen interaction; however, the role of T cells and disease-associated HLA-DRB1 alleles in rheumatoid arthritis remains elusive. Recent studies on the genetics of the HLA-DRB1 association and the diversity of the repertoire of synovial T cells, and treatment studies with T cell depleting antibodies, have suggested that the model of T cells recognizing an arthritogenic antigen in association with a HLA-DR molecule is too simplistic. The findings are more consistent with a regulatory role of T cells. Patients with rheumatoid arthritis have a unique T-cell repertoire that not only reflects the influence of disease-associated HLA-DRB1 alleles but also is greatly skewed by the clonal expansion of few CD4+ and CD8+ T-cell specificities. Understanding these repertoire changes appears to be promising not only in permitting understanding of the pathogenesis of this disease but also in designing T-cell targeted treatment strategies. PMID- 8619094 TI - Oncogenes in rheumatoid arthritis. AB - The evolving knowledge of the actions and interactions of (proto)oncogenes in cancer has deeply influenced the understanding of other nonmalignant diseases. In RA, the longstanding pathohistologic evidence of transformed-appearing synovial cells at the site of bone and cartilage attachment and joint destruction can now be explained in terms of alterations of cell regulation, cell cycle, and apoptotically triggered cell death. The detection of upregulated oncogenes and their gene products at these sites supported the hypothesis of an aberrant synovial cell type invading the joint. Interestingly, there are hints that this transformation of synovial cells may require more than one activated oncogene. A model was introduced by Carson and Ribero in 1993. In this model, a primary stimulus affects the cell and leads to the enhanced transcription of an oncogene (i.e., c-myc). A second stimulus activates other oncogenes and determines if this cell (i.e., a synovial fibroblast) proliferates (marked by the presence of bcl-2 mRNA) or undergoes apoptosis (marked by fas mRNA and Fas expression at the cell surface). This co-upregulation might explain why some investigators could not detect a significant upregulation of oncogenes in cultured synovial fibroblasts devoid of their normal milieu. Based on the results of the specific activity of Fas and perforin and recent data from our laboratory, we have modified Carson's model to include these data. As there exists an established retroviral model in which the tax sequence of the HTLV retrovirus initiates central oncogene transcription similar to those activated in RA, the retroviral particles, which do not resemble any other known retrovirus but are detectable in the synovial fluid, might well be an important stimulus in the pathogenesis of RA. To simplify the puzzling events of oncogene interactions in RA, we have summarized the data and propose that an oncogene network acts as a pathogenic mechanism in the synoviocytes of the rheumatoid joint. Similar to the "cytokine network" regulating the T-cell-dependent pathway, the "oncogene network" is presumably the major T-cell-independent pathway in RA (Fig. 4). PMID- 8619095 TI - The neutrophil in rheumatoid arthritis. AB - The destructive capacity of the neutrophil has long been appreciated, and the presence of extraordinary numbers of neutrophils in the synovial fluid of patients with RA supports a role for these cells in the pathogenesis of joint destruction. In this article, we reviewed the current state of knowledge of neutrophil function in the inflammatory response, and emphasized the subjects of neutrophil/endothelial adhesion and the role of chemoattractants and cytokines in neutrophil mobilization. We also discussed the mechanisms of action of neutrophil destruction of cartilage and the interplay of signals between the neutrophil and the chondrocyte. The capacity of many of the drugs used to treat RA to interfere with one or several of these processes underscores the importance of the neutrophil in RA and suggests that future therapeutic strategies could target neutrophil activation within the synovial space. PMID- 8619096 TI - Role of adhesion molecules in the pathogenesis of rheumatoid arthritis. AB - The pathogenesis of rheumatoid arthritis centers on as yet unknown initiating events in the synovium that result in synovial vessel proliferation, and upregulation of endothelial cell ligands for leukocyte adhesion molecules. Ligation of adhesion molecules on synovial microenvironment cells and immune cells probably regulates synovial and immune cell inflammatory cytokine production. Interruption of adhesion molecule function and interruption of inflammatory cytokine production are promising new sites of therapeutic inhibition of synovial inflammation. PMID- 8619097 TI - Eicosanoids in rheumatoid arthritis. AB - Eicosanoids are potent mediators in the cellular microenvironment. Eicosanoids have different effects depending on tissue or organ, the polyunsaturated fatty acid content of the diet of the individual, and the net effect of local microenvironmental factors--as eicosanoids, cytokines, and hormones modulate each others' effects through a complex, multilevel network of interactions. In general, eicosanoids have significant net proinflammatory effects. In RA, the net proinflammatory effects of the prostanoids is underscored by the effectiveness of the cyclooxygenase antagonists (NSAIDs), and recent data indicate a proinflammatory effect of the leukotrienes. Changes in the dietary polyunsaturated fatty acid composition to increased intake of marine n-3 fatty acids and/or dihomogamma-linolenic acid may favorably modulate eicosanoid synthesis towards less inflammatory or antiinflammatory eicosanoids and may ameliorate disease activity in RA. Recent advances in the biochemistry and molecular biology of the eicosanoid receptors and the synthetic pathways of eicosanoids will provide opportunities for advances in the therapeutics for RA, including selective cyclooxygenase (PGHS-2) antagonists, selective eicosanoid receptor antagonists and agonists, and selective inhibitors of PGH2 isomerases and enzymes of the 5-lipoxygenase pathway. PMID- 8619098 TI - Marine and botanical lipids as immunomodulatory and therapeutic agents in the treatment of rheumatoid arthritis. AB - Fish and plant seed fatty acids have antiinflammatory and immunomodulating properties that make them of potential use in the treatment of rheumatoid arthritis. A better understanding of their effects on the immune system ultimately may lead to the development of more benign therapy for rheumatoid arthritis patients. PMID- 8619099 TI - Investigational agents for rheumatoid arthritis. AB - Agents ranging from simple analgesics to antiinflammatory drugs to powerful immunomodulators have been used for the treatment of rheumatoid arthritis with varying success. Despite the availability of agents that are believed to be "second line" or "disease modifying," many patients either do not respond adequately to available agents or must discontinue their use because of intolerable or dangerous adverse reactions. For this reason, researchers continue to search for more efficacious and less toxic agents for patients with rheumatoid arthritis. This article describes pharmaceutical agents currently under investigation for use in rheumatoid arthritis, including the antiinflammatory agents, zileuton and tenidap, and the immunosuppressive agents, leflunomide, mycophenolic acid (RS-61443), tacrolimus (FK-506), sirolimus (rapamycin), amiprilose (therafectin), cladribine (2- chlorodeoxyadenosine), and azaribine. PMID- 8619100 TI - Biologic interventions in rheumatoid arthritis. AB - An increasing understanding of the immunopathogenesis of rheumatoid arthritis and advances in biotechnology have lead to the therapeutic application of immune specific interventions. The complexity of this disorder has generated numerous investigations using a wide range of biologic interventions. This article presents the current and potential targets for such biopharmaceutical agents and discusses their utility in rheumatoid arthritis. PMID- 8619101 TI - Antibiotic therapy for rheumatoid arthritis. Scientific and anecdotal appraisals. AB - Minocycline is arguably the most interesting new drug for rheumatoid arthritis since the development of methotrexate. Tetracycline compounds have long been used by rheumatologists who were considered mavericks by their peers, and recent controlled studies have demonstrated their antirheumatic activity. The reason that minocycline works is unclear, and their niche in the treatment of rheumatoid arthritis remains to be established. Nonetheless, it is clear that some patients with rheumatoid arthritis respond favorably to this form of treatment. PMID- 8619102 TI - Cyclosporine in rheumatoid arthritis. AB - Rheumatoid arthritis is a chronic inflammatory process of unknown etiology that leads to significant morbidity and accelerated mortality in the 10 to 15% of patients with severe proliferative and erosive synovitis that is unresponsive to conventional therapies. T cells play a key role in the initiation and perpetuation of the process. Based on its immune action, cyclosporine has been used in rheumatoid arthritis in a variety of worldwide clinical trials. This article presents the results of several pivotal studies and outlines the development of cyclosporine in the treatment of rheumatoid arthritis. PMID- 8619103 TI - The changing face of therapy for rheumatoid arthritis. AB - This article reviews past and present prescribing patterns for patients with rheumatoid arthritis and reveals some striking differences over time. For many practitioners, methotrexate has become the centerpiece around which other therapies are built; however, we are still learning a great deal about this drug. In this article, some recent advances are described. The prospects for further therapeutic development are discussed, with a focus on the present state-of-the art combination chemotherapeutic regimens. Consideration is given to ways in which new approaches might facilitate clinical research in this severely underfunded area. PMID- 8619104 TI - Validity of the recording of ischaemic heart disease and chronic obstructive pulmonary disease in the Saskatchewan health care datafiles. AB - The internal validity of the recording of information about ischaemic heart disease (IHD) and chronic obstructive pulmonary disease (COPD) in the administrative health care datafiles of the Canadian province of Saskatchewan is investigated. Comparisons between hospital data and medical charts for acute myocardial infarction and chronic airways obstruction patients showed excellent diagnostic agreement: 97 per cent and 94 per cent, respectively. Appropriate physician service claims were identified for 89 per cent of hospitalizations for IHD and COPD and exact concordance between diagnoses in the two datafiles varied between 15 per cent for acute/sub-acute IHD and 80 per cent for asthma; including any physician diagnosis within the same broad category (IHD or COPD) increased concordance to 79-94 per cent for IHD and 64-88 per cent for COPD. Contextual information related to the hospitalizations was clinically and epidemiologically realistic. PMID- 8619106 TI - A personal view of some controversies in allocating treatment to patients in clinical trials. AB - A non-exhaustive but nevertheless wide-ranging if biased and personal view of various matters affecting the allocation of treatments to patients in controlled clinical trials will be undertaken from the standpoint of a statistician working in drug development. The following topics are considered: the use of ?placebos' in the run-in phase; the use of crossover trials; selection of patients; various matters concerning centres; two extreme alternatives to randomization, and various matters linking blinding and allocation. PMID- 8619105 TI - A program to implement a search method for identification of clinical subgroups. AB - A procedure for identifying subgroups that are homogeneous with respect to an outcome variable is described. The method, search partition analysis (SPAN), is formulated in terms of a numeric outcome variable y and a set of predictors, explanatory variables or risk factors x = x1,x2,...,x p. The objective is to split observations into two groups by a binary partition, specified using Boolean expressions of the predictors, x, such that y is as homogeneous as possible in the resultant groups; uniformly ?low' in one and uniformly ?high' in the other. Subgroups within each of the two groups can be identified from the Boolean expressions. SPAN implements a search for the ?best' partition from among a class of regular Boolean expressions. Features of the method are described, including how to measure partition homogeneity, complexity penalizing, search strategies and subgroup definition and representation. The approach is illustrated with analyses of predictors of low birth weight and predictors of impaired glucose tolerance for screening purposes. PMID- 8619107 TI - Effect of non-random missing data mechanisms in clinical trials. AB - A simple form of non-ignorable missing data mechanisms based on two parameters is used to characterize the amount of missing data and the severity of non randomness in clinical trials. Based on the formulation, the effect of non randomly missing data on simple analyses which ignore the missing data is studied for binary and normally distributed response variables. In general, the effect of the non-randomly missing data on the bias and the power increases with the severity of non-randomness. The bias can be positive or negative and the power can be less than or greater than when the data are missing at random. The results of the analysis, ignoring the missing data, can be seriously flawed if the non randomness is severe, even when only a small proportion of the sample is missing. The problem is more pronounced in the case of normally distributed response variables with unequal variances. PMID- 8619108 TI - Bayesian approaches to random-effects meta-analysis: a comparative study. AB - Current methods for meta-analysis still leave a number of unresolved issues, such as the choice between fixed- and random-effects models, the choice of population distribution in a random-effects analysis, the treatment of small studies and extreme results, and incorporation of study-specific covariates. We describe how a full Bayesian analysis can deal with these and other issues in a natural way, illustrated by a recent published example that displays a number of problems. Such analyses are now generally available using the BUGS implementation of Markov chain Monte Carlo numerical integration techniques. Appropriate proper prior distributions are derived, and sensitivity analysis to a variety of prior assumptions carried out. Current methods are briefly summarized and compared to the full Bayes analysis. PMID- 8619109 TI - Non-parametric methods for analysing recurrent complications of varying severity. AB - Non-parametric methods have recently been proposed to take into account the recurrent nature of complications in clinical trials, by estimating the prevalence Q(t), defined as the probability of relapse-free patients being in a complication state at time t. Recoveries are also considered. These methods have been adapted in this paper to take into consideration not only the recurrent nature of complications but also their severity through the interpretation of average weight functions. Results of a randomized trial comparing late complications following two alternative doses of pre-operative internal brachytherapy in patients with localized stage cervix carcinoma are used to illustrate the methods. PMID- 8619110 TI - Non-parametric estimation of the post-lead-time survival distribution of screen detected cancer cases. AB - The goal of screening programmes for cancer is early detection and treatment with a consequent reduction in mortality from the disease. Screening programmes need to assess the true benefit of screening, that is, the length of time of extension of survival beyond the time of advancement of diagnosis (lead-time). This paper presents a non-parametric method to estimate the survival function of the post lead-time survival (or extra survival time) of screen-detected cancer cases based on the observed total life time, namely, the sum of the lead-time and the extra survival time. We apply the method to the well-known data set of the HIP (Health Insurance Plan of Greater New York) breast cancer screening study. We make comparisons with the survival of other groups of cancer cases not detected by screening such as interval cases, cases among individuals who refused screening, and randomized control cases. As compared with Walter and Stitt's model, in which they made parametric assumptions for the extra survival time, our non-parametric method provides a better fit to HIP data in the sense that our estimator for the total survival time has a smaller sum of squares of residuals. PMID- 8619111 TI - Interval censored survival data and multistate compartmental models in the analysis of first appearance of Plasmodium falciparum parasites in infants. AB - A statistical model for interval censored data is described. Assuming a piecewise constant incidence enables us to analyse very unbalanced data in a generalized linear model. The distribution of age at first appearance of P. falciparum parasites in infants in Liberia has been estimated. A new graphical method for presentation of test results on all children was developed. In an illness--death model it is described how the proportion of undetected and detected malaria parasitemias depends on parasite rates and testing frequency. The incidence of detectable malaria parasitemia was 0.14 per month in infants under 4 months of age, and 0.60 per month in children over 4 months (p < 0.001). The congenital resistance to malaria in African infants living in a highly endemic area had largely disappeared by the age of 4 months; before this age children were partly protected. PMID- 8619112 TI - [Wilhelm Conrad Roentgen--century of X ray discovery]. PMID- 8619113 TI - [Food irradiation]. AB - A worldwide standard on food irradiation was adopted in 1983 by Codex Alimentarius Commission of the Joint Food Standard Programme of the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO). As a result, 41 countries have approved the use of irradiation for treating one or more food items and the number is increasing. Generally, irradiation is used to: food loses, food spoilage, disinfestation, safety and hygiene. The number of countries which use irradiation for processing food for commercial purposes has been increasing steadily from 19 in 1987 to 33 today. In the frames of the national programme on the application of irradiation for food preservation and hygienization an experimental plant for electron beam processing has been established in Institute of Nuclear Chemistry and Technology. The plant is equipped with a small research accelerator Pilot (19MeV, 1 kW) and an industrial unit Elektronika (10MeV, 10 kW). On the basis of the research there were performed at different scientific institutions in Poland, health authorities have issued permission for irradiation for: spices, garlic, onions, mushrooms, potatoes, dry mushrooms and vegetables. PMID- 8619114 TI - [Identification of foods preserved by radiation]. AB - Analytical methods suitable for the detection of irradiated foods are reviewed. The detection methods are classified as physical, chemical, microbiological and biological, respectively. Reliability, robustness, sensitivity, accuracy and simplicity of each method are discussed. PMID- 8619115 TI - [Content estimation of nitrates and nitrites in vegetables from the province of Lodz]. AB - The contents of nitrates and nitrites in selected vegetables reaped in summer and autumn in 1993 were investigated. The samples of vegetables were collected directly from the producers from area of Lodz and from outside of the city. Nitrates were reduced on a cadmium column to nitrites, where-upon they were determined colorimetrically using sulfanilic acid and N-1 Naphthylethylenediamine. It has been found that the contents of nitrates in tested vegetables are mostly higher than the allowable values published in the Ordinance of Minister of Health and Social Welfare from 8th of October 1993. The average contents of nitrates and nitrites in most of tested vegetables were higher in the vegetables from the outskirts of the city than in ones from outside of the city in the province. PMID- 8619116 TI - [Studies on the contents of nitrates and nitrites in selected fresh and heat processed vegetables]. AB - The levels of nitrates and nitrites were determined in fresh vegetables and the same products subjected to culinary processing such as boiling. Nitrates were reduced on a cadmium column to nitrites, where upon they were determined colorimetrically using sulfanilic acid and N-1-naphthyl-ethylenediamine. Thermal processing of these vegetables reduced the level of nitrates by about 50% and the nitrites loss reached even 100%. PMID- 8619117 TI - [Study of the level of aromatic amines in food products]. AB - The aim of this study was to adapt the method of total aromatic amines determination in dyes for food purposes and application in food industry. The level of total aromatic amines, expressed as aniline, was found in the range of 2.6 to 4.0 x 10-(6) g/kg in selected food products--coffee, walnuts, hazlenuts, peanuts. The proposed method is simple, easy to perform and reliable, therefore may be recommended to use in food industry laboratories. PMID- 8619119 TI - [Use of HPLC technique for determination of aspartame and acesulfam-K in processed fruit products]. AB - A liquid chromatographic method for the determination of the intense sweeteners- aspartame and acesulfam-K in fruit and vegetable nectars was described. Samples were extracted with water, then clarified with Carrez solutions. An aliquot of the extract was analyzed on C-18 reverse-phase column with UV detection. Mean recoveries ranged from 95.9 to 101.8%. The method is suitable for routine determinations of both sweeteners. PMID- 8619118 TI - [Effect of selected additives on the quality of the Wegirka Zwykla brand of frozen plums]. AB - The aim of the work was to determine the effect of sugar, pectin formulation, and L-ascorbic acid on selected physico-chemical properties and organoleptic quality of frozen Wegierka Zwykla plums. Sugar at a dose of 10 g per 100 g of fruit in a loose form or as 60% syrup was used separately or with an addition of pectins or 0.5% L-ascorbic acid. A 1.5% solution of pectins at a dose of 10 g per 100 g of fruit was used separately or with an addition of 0.5% l-ascorbic acid. Plums were cut into halves, stoned, treated with an additives, placed in plastic containers, and frozen at -30 degrees C. The temperature of -20 degrees C at which frozen products were stored, was obtained after 90 min. The evaluation was carried out at the stage of row material and of frozen fruit directly after freezing and after 12-month storage. In the case of vitamin C and anthocyanins analyses were conducted at 3-month intervals. Fresh fruit contained 18.4% of dry weight, 11.3% of total sugars, 0.81% of acids as malic acid, 12.7 mg/100 g of vitamin C, and 17.9 mg/100 g of anthocyanins. The addition of additives contributed to variability of frozen fruit with respect to the level of dry matter and sugars, and brought about a slight dilution of acids, vitamin C, and anthocyanins. Freezing did not significantly change the content of vitamin C and anthocyanins. On the other hand, one-year storage of the product frozen without the additives brought about a decrease in the content of vitamin C to 3.6 mg/100 g and of anthocyanins to 1.9 mg per 100 g of fruit. In the samples with the additives the respective values were 4.6-13.5 mg and 3.2-6.7 mg. After 12-month storage the organoleptic quality of frozen products without the antioxidants was 3.82 and of those with the antioxidants added 4.04-4.76 in a 5-score scale. From the analysed variants of additives used in freezing plums for dessert sugar syrup with an addition of L-ascorbic acid may be recommended on account of a good preservation of components, high organoleptic value, and also simple way of application. A solution of pectins with L-ascorbic acid can be used in preparing frozen fruit for people not tolerating sugar in their diet. PMID- 8619120 TI - [Evaluation of the combined effect of cupric chloride and sodium nitrite on selected biochemical parameters in rat plasma (subchronic exposure)]. AB - The study was performed on 4 groups of male Wistar rats, receiving p.o. through 3 months every day: 1). Sodium nitrite in dose 30 mg/kg b.w. x day (0.2 LD50); 2). Cupric chloride in dose 4.67 mg/kg b.w. x day (0.03 LD50); 3 ). Cupric chloride and sodium nitrite in amounts as above, and 4). Control group--received dest. water. The activity of alanine aminotransferase (AlAT), aspartate aminotransferase (AspAT), gamma-glutamyltransferase (GGTP-ase) and creatinine and urea level in blood plasma were determined 24 hours after the last application of compounds. There was showed, that every day rats' intoxication with sodium nitrate during 90 days caused the significant increase of gamma glutamyltransferase activity and decrease of urea level in the blood plasma. Subchronic exposure to copper and copper with sodium nitrate causes no effect on biochemical parameters were studied. PMID- 8619121 TI - [Genotoxic properties of 5-nitrofuran compounds]. AB - 5-nitrofurans are a large group of nitro-compounds and worldwide as human and veterinary drugs, food additives or preservatives. However, many adverse effects of these compounds and among mutagenic as well as carcinogenic activities create doubts about safety of their use. Present paper deals with various mechanisms of genotoxic action of 5-nitrofurans. Main metabolic pathways leading to the formation of their active metabolites are discussed. PMID- 8619122 TI - [The effect of nitrofurazone and furazolidone on induction of cytochrome P-450 in the CYPIA test]. AB - The double CYPIA-TEST (cytochrome P-450) induction assay) was used to discriminate between the forms of cytochrome P-450 (the 3-methylcholantrene type (3-MC) or phenobarbital type (PB)) induced by nitrofurans; nitrofurazone and furazolidone. The test consisted of the studies by the technique of Ames of the ability of the S9 preparation obtained from livers of rats induced by nitrofurans to cause the metabolic activation of ethidium bromide (EtBr)--for induction of 3 MC type or cyclophosphamide (CPA) for PB type of cytochrome P-450. The mutagenicity of activated EtBr and CPA was checked with TA98 and TA100 of S. typhimurium respectively. It was found that only furazolidone at cumulative dose 3 x 80 mg/kg of body weight induce the 3-MC-type of cytochrome P-450. PMID- 8619123 TI - [Evaluation of the chorioallantoic membrane in the chick embryo to test the irritation potential of chemical and cosmetic products]. AB - A large number of new chemicals and cosmetics are introduced every year and it is necessary to find a reliable method for the detection of potential irritants. Hitherto used, for this purpose, in vivo rabbit eye test (Draize's test) should be substituted by alternatives because of ethical and legal aspects. One of the most promising method predicting the irritant potential is test performed on the chorioallantoic membrane of the chick embryo (CAM-test) [5]. This membrane is a complete tissue including arteries, capillaries and veins and is technically easy to study. The aim of the study was to check reliability and sensitivity of the test. The principles of the method are simple. The test substance (pure, dissolved or suspended) is applied to the CAM at a volume of 200 microliters. The blood vessels and the remaining parts of the membrane are examined and scored for irritant effects (hyperaemia, lysis or coagulation) after 0.5, 2 and 5 minutes treatment. The numerical, time-dependent score (Table III) are summed to give a single numerical value indicating the irritation potential of the tested substance (Table II). The final assessment is based on the mean value from four experiments. A summary of the results of CAM irritation testing of some of chemicals and cosmetics is given in Tables IV, V and VI. Table VII shows results of assessment of irritation potential of various cosmetics tested by two methods: CAM and Draize eye tests. It was found that CAM test is a rapid and very sensitive method which can give an information on the potential of irritation of chemical substances and cosmetics. PMID- 8619124 TI - [Evaluation of "the ever changing" educational program]. AB - The educational program "The Always Changing Program" was performed in 1500 primary schools in 21 major towns in different voievodships and concerned 200 thousands children from 5 and 6 classes. The studies aimed at the evaluation of its usefulness for health education in primary schools as well as of feedback reaction from pupils. 1406 schools and 1425 school children who completed this program participated in the study. The results showed that both schools and coordinators from the Sanitary Epidemiological Stations are well prepared to introduce this program. The approaches applied by schools were in accordance with the intentions of the program. The program was positively evaluated by school authorities as far as its contents and additional tools in which it was equipped are concerned. The need for the continuation of this program was expressed together with the proposals concerning its extension including films. The opinions expressed by school children allow to conclude that the program is useful and that this kind of education is expected to be continued in schools. It was found different feedback reaction depending on sex and social status. This creates the need for more specific approaches for defined groups of children. The evaluation of the influence of this program on specific knowledge and behavioral patterns in children are planned. PMID- 8619125 TI - [Evaluation of school and afterschool activities of public and nonpublic secondary school students]. AB - The studies were performed on 825 school children (512 girls and 313 boys) from 1st and 2nd classes of secondary schools in som voievodship capital cities. 406 school children were from public, and 419 from non-public schools. The questionnaire prepared in the Institute for Children and Youngsters Institute in Berlin was used in this study. The evaluation of collected responses made possible to state the following conclusions: The organization of school and out school activities in public and non public schools was incorrect in several aspects ie. incorrect from the hygienic point of view organization of classes during day, too early beginning of the classes, too late ending of the classes in some week days, too long time needed to complete homework, and too late return to home after completing out school activities. The difficulties in homework completing were stated by school children from both public and non public schools. In 52% cases the parents helped in homework and 12% of children reported private lessons as an additional help in homework. The analysis of responses concerning frame of mind of school children showed better situation of pupils from non public schools. Only 15% of non public school children expressed reluctancy towards schools, as compared to 21% from public schools. The relationships between pupils and teachers did not worsened during consecutive years in non public schools, as opposite to public schools where the worsening of these relationships during the consecutive years was evident. PMID- 8619126 TI - Analysis of the heavy metal-responsive transcription factor MTF-1 from human and mouse. AB - Heavy metal-induced transcription in mammalian cells is conferred by the metal responsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-1 and is lost when human MTF-1 is fused to the general activation domain of herpesvirus VP16. Wild type and truncation mutants of MTF-1 fused to VP16 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility. PMID- 8619127 TI - Efficient modification of the APRT gene by FLP/FRT site-specific targeting. AB - The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5 X 10(-5), which were 6-22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination. PMID- 8619128 TI - Breakpoints and junctional regions of intragenic deletions in the HPRT gene in human T-Cells. AB - DNA sequences of the deletion breakpoints of 24 human T-lymphocyte hprt gene mutations are reported. These independent deletions ranged in size from 18 to 15655 base pairs. Seven of the 21 in vivo mutations arose in normal adults, three in normal children, eight in radioimmunotherapy patients and three in platinum chemotherapy patients. One in vitro mutation was isolated after 93cGy radon exposure and two after 300cGy gamma radiation. The breakpoints were found to be non-random and a cluster of small deletions in exon 6 is reported. Ten of the mutations had 2-5bp direct repeats at the breakpoints. There was no excess of "deletion-associated" motifs over that expected by chance. Some breakpoints do occur at consensus topoisomerase II cleavage sites and the centromeric end of a Donehower sequence occurs exactly at a telomeric breakpoint. Three mutants had breakpoints at hairpins expected by the model of Glickman and Ripley. PMID- 8619130 TI - Three region-specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24. AB - Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70 94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33. PMID- 8619129 TI - The XIST locus replicates late on the active X, and earlier on the inactive X based on FISH DNA replication analysis of somatic cell hybrids. AB - We have recently reported results of DNA replication analysis of three X-linked loci (FRAXA, F8C and XIST) on the X chromosomes in male and female fibroblasts using fluorescence in situ hybridization (FISH) (1). Although our findings that XIST replicates later on the active X than on the inactive X are similar to those of Boggs & Chinault (2) based on a FISH assay in female lymphoblasts, they are the opposite of observations recently reported by Hansen et al. (3) using a different technique. Because our conclusions about the inactive X were deduced from the behavior of the active X in male cells, we reexamined the time when these loci replicate on the human inactive X chromosome isolated from its homolog in somatic cell hybrids. We also studied the same chromosome as an active X in related hybrids. The results provide direct evidence that the expressed XIST locus on the inactive X replicates earlier than its repressed homolog on the active X and earlier than the FRAXA locus which is repressed on this chromosome. The silent XIST locus on the active X replicates late along with F8C which is also not transcribed in these cells. Possible reasons for the different results obtained by Hansen et al. (3) are discussed. PMID- 8619131 TI - Localization of the mouse lissencephaly-1 gene to mouse chromosome 11B3, in close proximity to D11Mit65. AB - Lissencephaly is a human brain malformation manifested by a smooth cerebral surface and severe mental retardation. Some of the patients have been shown to have deletions in chromosome 17p13.3, and recently, LIS-1 has been proposed to be the disease-associated gene. We have now mapped the mouse homolog of LIS-1 to mouse chromosome 11B3 by using fluorescence in situ hybridization to metaphase chromosomes. The analysis of yeast artificial chromosome clones placed Lis-1 in close proximity to the microsatellite marker D11Mit65. PMID- 8619132 TI - Localization of the human homolog of the yeast cell division control 27 gene (CDC27) proximal to ITGB3 on human chromosome 17q21.3. AB - The human homolog of the Saccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene, BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of human CDC27 on human chromosomes 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of human CDC27 to the same yeast artificial chromosomes (YACs) positive for ITGB3. The estimated distance between ITGB3 and human CDC27 is less than 600 kb. PMID- 8619133 TI - Gene expression in response to retinoic acid in novel human chromosome 21 monochromosomal cell hybrids. AB - To access a wide a variety of expressed sequence from human chromosome 21 we have placed this chromosome into undifferentiated P19 mouse embryonic carcinoma cells. Cell lines resulting from these experiments have a range of morphologies and a wide variety of karyotypes. We have studied the retinoic acid response of five cell lines, compared to P19 cells, by observing three markers of retinoic acid induced P19 differentiation--cell morphology, RAR alpha and Wnt1 transcription. We see an 'early' retinoic acid response effect, however this response breaks down by the time the 'late' gene. Wnt1 would be transcribed in P19 cells. A highly responsive cell line will be useful for cloning expressed sequences from human chromosome 21 which are produced by early genes in retinoic acid inducible pathways, such as those involved in neurogenesis. PMID- 8619134 TI - Essential drug list ready to go. PMID- 8619135 TI - Determining the prevalence of Alzheimer's disease in elderly South Africans. PMID- 8619136 TI - Regional health developments - highlights from the 45th WHO African Regional Committee meeting. PMID- 8619137 TI - Therapy with parasiticidal drugs in cysticercal encephalitis--is it indicated? PMID- 8619138 TI - Restructuring rural health. PMID- 8619139 TI - Health effects of passive smoking in adolescent children. AB - OBJECTIVE AND DESIGN: To study the effects of passive smoking on health in adolescent schoolchildren by questionnaire, spirometry and laboratory investigations. SETTING: Two schools in the Vanderbijlpark area. PARTICIPANTS: Seven hundred and twenty-six high-school children of average age 16 years. OUTCOME MEASURES: Lung function, serological abnormality or historical (i.e. questionnaire) evidence of ill health. RESULTS: The prevalence of respiratory illness before and after 2 years, respiratory symptoms, earache over the past year, low birth weight and learning difficulties were found to be significantly increased in the children exposed to parenteral smoke in the home, especially those exposed to maternal smoking. Spirometric and laboratory parameters, however, were not affected by passive smoking. PMID- 8619140 TI - Accumulating experience in a child abuse clinic. AB - OBJECTIVE: To examine the patient profile encountered in the first year of operation of the Child Abuse and Neglect Clinic of the Transvaal Memorial Institute. DESIGN: Record review of all cases presenting to the Clinic from May 1988 to April 1989. RESULTS: Females comprised just over 80% of the 227 patients. Sexual abuse was the presenting complaint in 89.8%. Most were young, 7% under 3 and 55% under 10 years of age. Almost one-third of the boys and 5.0% of the girls had chronic signs of anal abuse. Of the girls 56% had signs of chronic and 10% signs of acute vaginal abuse. Where the certainty of sexual abuse was high, 60% of the girls and 45% of the boys had suffered penetrative abuse. The perpetrators were almost invariably known to the child; biological family members accounted for 38% of perpetrators, and if all relations are included (biological, step and 'common law'), family members were the perpetrators in 66% of cases. Strangers were the perpetrators in only 7% of our cases. The majority of perpetrators were male. Behaviour problems were recorded in 73% of cases. Many different problems were noted; the most common were school problems (21%), masturbation (19%), 'clingy' behaviour (12%), and withdrawal and depression (11.5%). CONCLUSIONS: Certainty of diagnosis should be specified. We use four categories: proven, highly suspected, unproven but still suspected, and no abuse. For sexual abuse we also differentiate between penetrative, non-penetrative, 'type uncertain' and no abuse. Training of other health personnel in child abuse management is now a priority in our setting. PMID- 8619141 TI - Diagnostic efficiency of the disposable Monotest in detecting tuberculosis infection by setting objective-specific cut-off points for positivity. AB - The disposable multiple-puncture tuberculin test device, Monotest, was recently introduced in South Africa for tuberculin test screening of tuberculosis infections. Three studies were carried out to compare the intradermal Mantoux test with the Monotest. In the first study, conducted on confirmed hospitalised tuberculosis patients, 307 subjects underwent a 2 TU RT 23 (Statens Seruminstitut, Copenhagen) Mantoux test, 155 a 5 TU test of the same antigen, and 111 a Monotest. In the second study, another group of 98 confirmed tuberculosis patients was double-tested with 5 TU RT 23 by Mantoux test and Monotest. In the first study, 100% of 5 TU Mantoux tests resulted in indurations > or = 5 mm, 97% of 2 TU Mantoux indurations were > or = 10mm, and 96% of Monotests produced indurations > or = 2 mm (manufacturer's recommended cut-off point). In the second study, 99% of the 5 TU Mantoux tests measured > or = 5 mm, and 100% of Monotest indurations were > or = 2 mm. In both studies, raising the cut-off point for the Monotest to > or = 4 mm produced sensitivities of 95% and and 100% respectively. All these tests may therefore be regarded as highly sensitive. In a third study, 58 healthy schoolchildren were double tested with 2 TU RT 23 and with Monotest. Results indicated that if the positivity cut-off point of the Monotest is set a 8 mm instead of at 2 mm, sensitivity suffers slightly, decreasing from 100% to 90%, while specificity increases considerably from 8% to well over 80%. Receiver operating characteristic analysis indicated the high likelihood that the Monotest as well as the Mantoux test could discriminate between infected and uninfected subjects. For both tests the Wilcoxon statistic exceeded 0.8. The Monotest is a useful alternative to the Mantoux test. It is recommended that if tuberculosis is suspected, reaction > or = 4 mm be interpreted as positive. For screening purposes, a cut-off point of 8 mm seems to distinguish best between the infected and the uninfected individual. PMID- 8619142 TI - Early bactericidal activity of ethambutol, pyrazinamide and the fixed combination of isoniazid, rifampicin and pyrazinamide (Rifater) in patients with pulmonary tuberculosis. AB - The early bactericidal activity (EBA) of ethambutol, pyrazinamide and the fixed combination of isoniazid, rifampicin and pyrazinamide (Rifater: Mer National) was evaluated in patients with pulmonary tuberculosis who were sputum-positive on microscopy for acid-fast bacilli. Twenty-eight patients (mean age 33 years and weight 51 kg on average; range 40-59 kg) were studied. The fall in viable counts of Mycobacterium tuberculosis in sputum collections during the 2 days following the start of treatment was estimated from counts of colony-forming units (CFUs) of M. tuberculosis per ml of sputum cultured on selective 7H10 agar medium. The EBA for ethambutol determined in 9 patients was 0.245 +/- 0.046, log10 CFU/ml sputum/day, that for pyrazinamide was 0.003 +/- 0.014 log10 CFU/ml sputum/day and that for Rifater 0.558 +/- 0.054 log10 CFU/ml sputum/day. The results obtained are similar to those reported in a previous study of the first 2 days of treatment, but in smaller numbers of patients, and confirm the moderate EBA of ethambutol while pyrazinamide is again shown to have very little EBA. Rifater has a marked EBA which may be due mainly to the action of isoniazid. This methodology may be valuable in the rapid evaluation of the bactericidal activity of new antituberculosis agents and the comparison of different dose sizes of agents of the same class. PMID- 8619143 TI - Effect of an immunisation campaign in Natal and KwaZulu on vaccinaton coverage rates, 1990-1991. AB - In 1990 the Department of National Health and Population Development of South Africa launched a nationwide immunisation campaign targeted mainly at measles. In order to measure the effect of the campaign on vaccination coverage rates for children, pre- and post- campaign vaccination coverage surveys were undertaken using a modified Expanded Programme for Immunisation technique, stratified for race and urban/rural residence. The results in KwaZulu-Natal showed no significant increase in measles vaccination coverage for any race rates after the campaign (as documented by Road-to-Health cards). There was a decrease in coverage of the black population. However, when a history of measles vaccination was accepted, the results showed an increase in coverage. The results call into question the effectiveness of immunisation campaigns as a strategy for raising vaccination coverage levels, as well as their having a sustained impact on the incidence of measles. Alternative strategies, such as the strengthening and expansion of existing primary health care services, should be considered. PMID- 8619144 TI - The lipid and lipoprotein profile of the urban black South Africa population of the Cape Peninsula - the BRISK study. AB - OBJECTIVE: To determine the lipid and lipoprotein profile of the urban black South African population of the Cape Peninsula. DESIGN: Cross-sectional design. SETTING: The seven black residential areas of the Cape Peninsula. PARTICIPANTS: A stratified proportional sample was drawn from the study area. The sample consisted of 422 men and 544 women aged between 15 and 64 years. OUTCOME MEASURES: Lipid and lipoprotein levels. Risk levels for coronary heart disease (CHD). RESULTS: The total cholesterol (TC level) was low compared with other South African groups studied. Men had a mean TC of 3.98 mmol/l and women 4.15 mmol/l. Low-density lipoprotein cholesterol (LDLC) values for men (2.03 mmol/l) were lower than those for women (2.30 mmol/l). Men (1.35 mmol/l) and women (1.37 mmol/l) had similar high-density lipoprotein cholesterol (HDLC) levels. Both sexes had a prevalence of protective HDL/TC ratios above 30% for all age groups. High HDL3C levels and low HDL2C levels were found in both men and women. Apolipoprotein A and B followed the trends of HDLC and LDLC and showed no difference between the sexes. The plasma triglyceride (TG) levels increased with age in both sexes. Men displayed higher TG levels than women in all age groups. Seventeen per cent of men and 26% of women had a moderate-to-high risk for CHD, given their TC levels. Other lipid-related risk factors indicated low risk for CHD. PMID- 8619145 TI - A re-evaluation of isotope screening for skeletal metastases in node-negative breast cancer. AB - OBJECTIVE: To determine the accuracy and cost-effectiveness of skeletal scintigraphy in women with early, node-negative (T1-2N0M0) breast cancer. DESIGN: Retrospective, where scintigraphic prediction of metastases was compared with the criterion standard of radiological confirmation during a follow-up of 5 - 10 years. SETTING: Tertiary referral breast clinic at Groote Schuur Hospital. PATIENTS: Six hundred and seventy-three women with clinical T1-2N0M0 breast cancer who had skeletal scintigraphy between 1974 and 1987, and who had been followed up for more than 5 years. INTERVENTIONS: Initial skeletal scintigraphy, annual follow-up with radiological examination of symptomatic areas. MAIN OUTCOME MEASURES: Correlation of the sites indicated by scintigraphy with the initial presence or later development of metastases at 1 - 10 years, and the cost. RESULTS: Five hundred and sixty-one (83.4%) scans were normal, 35 (5.2%) indicated benign processes, and 77 (11.4%) were suggestive or diagnostic of metastatic disease, with radiological confirmation in 3 (initial detection rate 3/673, 0.44%; accuracy rate 3/77, 3.9%). Of the remaining 74 abnormal scans without radiological confirmation of metastases, 62 has a focus at a single site, and 45 were of low intensity and equivocal, with no apparent explanation. The cumulative sensitivity for predicting site of metastases at 1 year was 33% (3/9) and the positive predictive value 4.0% (3/75). At 10 years the sensitivity was 5.0% (3/60) and the positive predictive value 5.0% (3/65). The total cost of screening was calculated to be R323 460.00, suggesting that the cost for each patient in whom metastases were detected was R64 629.00. CONCLUSION: While scintigraphy may be of value in symptomatic or more advanced disease, screening of node-negative women had a minimal detection rate, was expensive and cannot be supported. PMID- 8619146 TI - Evidence of undue lead exposure in Cape Town before the advent of leaded petrol. AB - Lead concentrations were determined in the exhumed teeth of 28 people who lived in the Cape Town area before the combustion of leaded petrol (i.e. before 1922). The lead content of circumpulpal dentine was analysed by graphite-furnace atomic absorption spectrometry. The mean lead level in the dentine of primary teeth (N = 6) was 109 micrograms/g, while that in secondary teeth (N = 22) was 315 micrograms/g. The current lead levels in circumpulpal dentine of Cape Town residents are reported to be 74 micrograms/g and 16 micrograms/g for primary and secondary teeth respectively. It was found that lead pollution of the human body during the period 1812-1922 in the Cape Town area was substantially higher than at present. We conclude that the main reasons for this were the widespread use of lead piping and soldering of water tanks, which resulted in a higher incidence of lead poisoning than that attributable to leaded petrol. PMID- 8619147 TI - Computerised cardiotocography in a high-risk unit in a developing country--its influence on inter-observer variation and duration of recording. AB - OBJECTIVE: To determine the role of computer-assisted cardiotocography in an obstetric special care unit and its influence on inter-observer variation in interpretation, proposed management and monitoring time. DESIGN: A prospective comparative study. SETTING: The obstetric special care unit, Tygerberg Hospital, W. Cape. STUDY POPULATION: A group of 10 registrars in obstetrics who have had experience in the interpretation of both standard and computer-assisted cardiotocographs. MAIN OUTCOME MEASURES: The influence of method of cardiotocograph recording on inter-observer variation in respect of suggested management of the patient, as well as the observer's opinion of the duration of the recording. RESULTS: Variation in suggested management decreased significantly after assessment of the computer reports, compared with the standard cardiotocographs. While delivery was regarded to be indicated in 3.5% of patients and an immediate repeat of the cardiotocograph in a further 10%, no such action was proposed after evaluation of the computer reports of the same recordings. Thirty-four per cent of tracings were considered to have been too long and 12.5% too short. However, suggested management in 40% of the latter cases seemed inappropriate for tracings regarded as of too short a duration. CONCLUSION: While computer-assisted cardiotocographs significantly decrease inter-observer variation in the proposed management of patients, its cost-effectiveness in an obstetric special care unit in a developing country should be validated, as it might increase monitoring time. PMID- 8619148 TI - High frequency of the median artery of the forearm in South African newborns and infants. AB - In a sample of 60 neonates and infants from black communities in the Johannesburg area, the median artery of the forearm was found in 50% of individuals (11.7% in one forearm only, 38.3% in both forearms). The frequency per forearm was 44.2%, much higher than that found in any previous study, even among adults from the same community (27.1% per forearm). The artery occurs bilaterally significantly more often than it does in one antimere only. There are no differences in its frequency between sexes or between antimeres. The artery provides an additional route of blood supply to the forearm that should be kept in mind by hand surgeons. It can also be harvested for vascular grafts. PMID- 8619149 TI - The MASA and the doctor in full-time public service. PMID- 8619150 TI - HIV in northern KwaZulu-Natal. PMID- 8619151 TI - QT-interval prolongation with Ecstasy. PMID- 8619152 TI - Airborne micro-organisms in a health care environment. PMID- 8619154 TI - HIV positivity--disclosure and counselling. PMID- 8619153 TI - Drugs in sport. PMID- 8619155 TI - Prenatal screening for Down syndrome and neural tube defects. PMID- 8619156 TI - Alternative medicine--a doctor's perspective. PMID- 8619157 TI - Alternative medicine--a doctor's perspective. PMID- 8619159 TI - Private clinics and hospitals--superior service, scary bills. PMID- 8619158 TI - Alternative medicine--a doctor's perspective. PMID- 8619160 TI - Epidemiology of pleural effusions. PMID- 8619162 TI - A MASA for the future. PMID- 8619161 TI - Flushes, night sweats and palpitations need to be treated seriously. PMID- 8619164 TI - Tobacco and the institutionalized mentally retarded: usage choices and ethical considerations. AB - Patterns of tobacco use were observed among 749 people diagnosed with mental retardation residing in a state-operated facility. Specifically, individual preference for tobacco products and frequency of use were documented. Subjects were observed using several types of tobacco products: cigarettes, cigars, chewing tobacco, snuff, and cigarette butts. Approximately 7% (n = 52) used at least one form of tobacco; ten individuals used more than one tobacco product. Interestingly, 20.5% of the individuals diagnosed with mild or moderate mental retardation (n = 122) consumed tobacco products, a pattern of behavior that closely approximates that of the general population (20-24%). Ethical considerations pertaining to the availability of tobacco products to this special population are presented. PMID- 8619163 TI - Tuberculosis risk in the hospital dental practice. AB - Tuberculosis has re-emerged as a serious public health concern. Multidrug resistant strains and an increase in the number of high-risk groups are posing a difficult problem for health care providers. The risk of TB transmission in hospital dental practices is potentially increasing. A 20-question survey was mailed to the membership of the American Association of Hospital Dentists, addressing various issues relating to tuberculosis. One hundred thirty-two surveys were analyzed. Twelve per cent of respondents reported at least one TB skin test conversion by a dental provider within the past year at their institution. Five respondents reported one dental provider contracting TB through patient contact. Oral TB was reported in 21 cases. Over 34% reported that active TB patients are not isolated to negative-pressure isolation rooms, 45% reported that patients are allowed to frequent public areas, and only 59% believed that drug compliance monitoring was adequate. Over 86% support TB screening in the Hospital Dental Practice. It was concluded that Hospital Dental Practice personnel may be at increased risk for exposure to TB. Dental providers must exercise strict TB prevention and employ meticulous referral and follow-up procedures for high-risk patients. PMID- 8619165 TI - Pemphigus vulgaris--the potential for error: a case report. AB - The importance of correlation, referral, and collaboration between dermatology, or any other medical department, and the oral health profession is reported through a case presentation. A case of pemphigus vulgaris diagnosed from a tongue biopsy and confirmed by immunofluorescence is reported. The patient was treated for actinic keratosis of the scalp for 16 months due to inadequate biopsy material. The diagnostic value of proper biopsy material is stressed. The clinical manifestations leading to a misdiagnosis between actinic keratosis and pemphigus vulgaris are discussed. PMID- 8619166 TI - Oral health care of the mentally retarded and other persons with disabilities in the Nordic countries: present situation and plans for the future. AB - In the Nordic countries, the general goal of policies for persons with disabilities is their integration into society. At the same time, the social and economic situation has worsened, and, as a consequence the structures of the welfare state seem gradually to break down. This paper describes the present situation and plans for the future of oral health care for the mentally retarded and others with disabilities in the changing society in the Nordic countries. PMID- 8619167 TI - Hypoxemia due to inserting a bite block in severely handicapped patients. AB - In severely handicapped patients with severe cerebral palsy, stridor can be caused by a bite block in the mouth. So that dental treatment can be performed safely for these patients, the effect of a bite block on the respiratory function of 30 severely handicapped patients was investigated with two pulse oximeters. Of the 30 subjects examined, 96.7% showed SpO2 values above 95% in the resting state, and 26.7% showed SpO2 values under 94% after insertion of a small or middle-sized bite block. Patients who did not show SpO2 decrease while using a small bite block did have deterioration of SpO2 values when a middle-sized bite block was used. According to analysis by AIC, patients whose weight was less tha 20 kg, who were shorter than 130 cm, and who were more severely handicapped tended to have a deterioration of breathing function when using a bite block PMID- 8619168 TI - Tartar-control toothpaste as a possible contributory factor in the onset of superficial mucocele: a case report. AB - Superficial mucoceles are small, clear vesicles that occur on clinically non inflamed mucosa and are often misdiagnosed as vesiculobullous disorders. Soft palate, retromolar pads, and posterior buccal mucosa are common sites of involvement. The lesions are more common in women than in men and need no treatment. This paper describes a 71-year-old female who demonstrated some of the pitfalls in diagnosis and a possible contributory role of a tartar-control toothpaste in the onset of the lesions. PMID- 8619169 TI - Ethical issues encountered by dentists in the care of institutionalized elders. AB - Increasing numbers of institutionalized elders have very poor oral health. It has been suggested that ethical problems may influence dentists who attempt to provide oral care for these people, but little attention has been given to research in this area. A qualitative interview method was used to investigate the the views and experiences of dentists working with institutionalized elders. Particular attention was given to the ethical difficulties encountered and how the dentists resolved them. Ten dentists experienced in long-term care were interviewed individually by means of open-ended questions. Thematic analysis identified ethical problems focused on the difficulty of identifying the wishes of patients or predicting the outcome of treatment. The participants reported few difficulties in making clinical decisions in this setting. However, analysis revealed that the ethical perspectives of the dentists varied substantially. Variation was notable particularly in their preference for ideal or practical treatment and in their preference for autonomy of beneficence. PMID- 8619170 TI - Multiple endocrine neoplasia syndrome, type III: review and case report. AB - Successful treatment of the multiple endocrine neoplasia type III (MEN III) syndrome requires early diagnosis. It is highly possible that the patient's dentist may be the first practitioner with the opportunity to diagnose this potentially fatal syndrome. Additionally, patients with this syndrome having a pheochromocytoma and needing invasive dental treatment pose a life-threatening dental management risk. This article presents a review of the MEN II syndrome and a case report on the surgical management of a MEN III patient with a pheochromocytoma. PMID- 8619171 TI - Facial developmental vascular anomalies. AB - Developmental vascular anomalies of the head and neck may give rise to profound esthetic problems that are a challenge to treat. These vascular anomalies may also be of significance in dentistry, particularly where extractions have to be carried out. This brief report describes a number of children who presented with different variations of these anomalies. PMID- 8619172 TI - Perceived barriers to oral health care among the homebound. AB - Questionnaires and oral examinations were completed on 50 clients of a social service agency which provides home-based services to functionally dependent elderly. Nearly 61% of all respondents classified their oral health as frail/poor, and 82% reported a perceived need for some oral health care. When asked if they considered themselves homebound, 60% reported being homebound from 1-10 years (mean = 4.7 years). Two or more home services were received by 80% of the homebound group compared with just 45% of the not-homebound group. Paying for dental care, transportation difficulties, and poor health were the most frequently identified barriers that limited access to oral health care. PMID- 8619173 TI - A comparison of opinions from parents of disabled and non-disabled children on behavior management techniques used in dentistry. AB - The purposes of this study were to compare the acceptance of pediatric dental behavioral management techniques by 40 parents of children with disabilities with that of 40 parents whose children were not disabled and to determine the effect of prior information on the level of acceptance for both groups of parents. An instrument containing a demographic questionnaire and using a visual analog scale asked parents to indicate acceptance level of hand-over mouth, sedation, restraint using Papoose board, and general anesthesia for either a check up/cleaning, dental filling, or treatment of a toothache. One half of each parent group received a written description and rationale for the behavior management technique prior to rating acceptance, and the other half did not. Although differences were found between parents of the disabled and non-disabled and between those informed and not informed, only one technique and procedure (restraint for check-up/cleaning) was significantly different for acceptability (p < or = 0.05), and that was between uninformed parents of non-disabled children and informed parents of disabled children. We conclude that having a disabled child or receiving a prior rational for pediatric behavior management techniques was not significantly related to differences in acceptance of the techniques for the procedures described. PMID- 8619174 TI - The measurement of mandibular cortical bone height in osteoporotic vs. non osteoporotic postmenopausal women. AB - The asymptomatic progression of osteoporosis, in conjunction with the possibility of catastrophic disability, makes this disorder a major public health priority. Various body sites, including the mandible, have shown susceptibility to decreasing bone density. In 1986, Benson et al. proposed a radiomorphometric technique called the Panoramic Mandibular Index (PMI) as an inexpensive noninvasive dental technique for osteoporosis screening, although no osteoporotic subjects were included in their study. The purpose of our study was to determine whether osteoporotic postmenopausal women would show a decrease in mandibular cortical bone height, as measured by the PMI index, when compared with nonosteoporotic postmenopausal women. Seventy-two Caucasian females (33 cases/39 controls), aged 54-71 years old, were selected through records and screening via a dual-energy x-ray absorptiometry scan (LUNAR-DEXA). ANOVA analysis indicated no differences in the mean PMI between case and control groups (0.37 +/- 0.15 and 0.38 +/- 0.13, respectively; p = 0.69). Other techniques, such as computer digitized radiography should be explored to test the validity of the PMI. PMID- 8619175 TI - A retrospective review of a service to provide comprehensive dental care under general anesthesia. AB - Day-stay general anesthesia is indicated for a number of dental reasons, not least for those patients who are unable to accept routine dental care. Since 1979, the Dental Hospital of the University of Newcastle upon Tyne, UK, has provided a weekly day-stay service for the dental care of such patients. Reviews of this service were undertaken in 1983 and again in 1993. The latter study investigated the provision of care for 265 patients and compared this with that for the 96 patients reviewed in the earlier study. This paper presents the results of the review and underlines the need for a very aggressive approach to preventive dental care for patients treated by this modality. PMID- 8619176 TI - Measurement in surgical clinical research. PMID- 8619177 TI - Management of vagus nerve injury afer carotid endarterectomy. AB - BACKGROUND: Inadvertent injury to the vagus nerve or its branches during carotid endarterectomy can result in adductor vocal cord paralysis (hoarseness) and cricopharyngeal dysfunction (dysphagia) with aspiration, known as "double trouble." This study describes our experience in the management of this complication in cases where conservative treatment failed. METHODS: All patients were examined by a vascular surgeon, a head and neck surgeon, and a speech therapist. Their examinations included comprehensive speech evaluation, video stroboscopy, video fluoroscopy, and methylene blue testing for aspiration. All patients underwent Teflon injections to medialize the paralyzed vocal cord and a cricopharyngeal myotomy to restore swallowing and alleviate aspiration. RESULTS: Fourteen patients, eight men and six women, were treated. The duration of dysfunction was 24 weeks in two patients, 6 weeks in four patients, 4 weeks in three patients, and 1 week in five patients. Five patients had severe dysfunction (defined as difficulty in swallowing both solid and liquid foods with more than 20% aspiration), seven patients had moderate dysfunction (defined as difficulty swallowing solid food with aspiration of less than 20%), and two patients had mild dysfunction (defined as difficulty in swallowing solids but with no aspirations). After the Teflon injections and myotomy, 13 of 14 patients had satisfactory outcomes, including normal voice and swallowing. CONCLUSIONS: Vagus nerve injury from a carotid endarterectomy can be a debilitating complication. Prevention, early recognition, and prompt correction of these injuries are important in the management of this complication. PMID- 8619178 TI - A critical analysis of cerebral computed tomography scanning before elective carotid endarterectomy and its correlation to carotid stenosis. AB - BACKGROUND: Cerebral computed tomography (CT) scanning has been suggested to play a role in the management of patients before carotid endarterectomy (CEA). This prospective study analyzes the value of CT scanning before elective CEA and the correlation of CT findings to significant carotid stenosis. METHODS: This study includes 131 consecutive patients considered for CEA during a 2-year period. All patients underwent carotid duplex ultrasonography, carotid arteriography, and CT scanning. RESULTS: Eighty patients (61%) had transient ischemic attacks or prior strokes, and 51 (39%) had nonhemispheric symptoms or were asymptomatic. The CT scan was abnormal in 36 (27%) patients; however, no brain tumors or abnormalities to affect clinical management were revealed. Ninety-two CEAs were performed on 87 patients. Twenty-nine (32%) in the operative group had abnormal CT scans, but these did not influence operative decisions. On the basis of this rate of 0% of patients with CT findings to change surgical management in 92 cases, a maximum true rate of occurrence of up to 5% could be detected with alpha equals 0.05 by sampling a population of this size. Four patients (4%) had postoperative cerebral vascular accidents, and all of these had normal preoperative scans. Patients with 50% or more carotid stenosis on arteriogram were significantly more likely to have abnormal CT scans than patients with less than 50% stenosis (20% versus 7%, p = 0.0034). As carotid stenosis became more significant, the frequency of abnormal CT scans increased (p < 0.01). The cost of CT scanning was $66,089.50 in this study. CONCLUSIONS: Significant carotid stenosis was associated with a higher frequency of abnormal CT scans; however, routine preoperative CT scanning was unnecessary before elective CEA. PMID- 8619179 TI - Clinical features of small hepatocellular carcinomas as assessed by histologic grades. AB - BACKGROUND: Ninety-seven patients with small hepatocellular carcinomas (HCCs) measuring 3 cm or less in size and three patients with adenomatous hyperplasia who underwent radical hepatic resection were examined in this study. METHODS: The lesions were classified into four groups according to the following histologic grading criteria: group A, adenomatous hyperplasia (n = 3); group B, early HCC (n = 6); group C, well-differentiated HCC (wHCC) (n = 32); and group D, moderately or poorly differentiated HCC (n = 59). The involvement factors that seemed to be important or to characterize the progression of HCC and the survival rates were compared among the four histologic groups. RESULTS: The frequency of patients with tumors larger than 2.0 cm in size and that of patients with 200 or more ng/ml serum alpha-fetoprotein increased with the progression of histologic malignancy. Tumor staining on the angiogram, capsular formation, and extranodular invasion were never seen in groups A and B, but they began to appear in group C and increased remarkably in group D. The 5-year survival rates of the patients in groups B, C, and D were 100%, 60%, and 27%, respectively, and statistically significant differences were seen among them. In comparative evaluation of the group C patients the lesions that showed no tumor staining had no capsule, and those that had no capsule had no extranodular invasion. The 5-year survival rate of patients with wHCC without extranodular invasion (81%) was significantly higher than that of patients with extranodular invasion (35%) (p < 0.05). CONCLUSIONS: It may be recommended to provide the category of wHCC without extranodular invasion for pathologic classification of clinically early HCC (i.e., HCC of high curability). PMID- 8619180 TI - Biliopancreatic diversion for obesity at eighteen years. AB - BACKGROUND: Surgical attempts to treat obesity began because of the discouraging results of conservative medical treatment, which successfully achieved initial weight loss but failed to maintain it. Gastric restrictive procedures, currently the most popular surgical methods for obesity therapy, have proved to be effective in initiating weight loss, but some concerns regarding their long-term efficacy in weight maintenance have arisen. METHODS: Of a total of 1968 obese patients who underwent biliopancreatic diversion since 1976, the last consecutive 1217 underwent the "ad hoc stomach" type of diversion with a 200 cm alimentary limb, a 50 cm common limb, and a gastric volume varying between 200 and 500 ml. Mean age was 37 years old (11 to 69 years), and mean excess weight was 117%. Maximum follow-up was 115 months with nearly 100% participation. RESULTS: In the last half of the series, operative mortality was 0.4% with no general complications and with early surgical complications of wound dehiscence and infection (total, 1.2%) and late complications of incisional hernia (8.7%) and intestinal obstruction (1.2%). Mean percent loss initial excess weight (IEW) at 2, 4, 6, and 8 years was 78 +/- 16, 75 +/- 16, 78 +/- 18, and 77 +/- 16 in the patients with IEW up to 120% and 74 +/- 12, 73 +/- 13, 73 +/- 12, and 72 +/- 10 in those with IEW more than 120%. A group of 40 patients who underwent the original "half-half" biliopancreatic diversion maintained a mean 70% reduction of IEW during a 15-year follow-up period. Specific late complications included anemia (less than 5%), stomal ulcer (2.8%), protein malnutrition (7% with 1.7% requiring surgical revision by common limb elongation or by restoration). Clinical problems from bone demineralization were minimal in the short term and almost absent in the long term. CONCLUSIONS: Biliopancreatic diversion is a very effective procedure but is potentially dangerous if used incorrectly. PMID- 8619181 TI - Prevention of spinal cord injury after transient aortic clamping with tissue factor pathway inhibitor. AB - BACKGROUND: Lower limb paralysis that occurs in 11% of patients after treatment of thoracic and thoracoabdominal aortic aneurysms is unpredictable and at present not preventable. The proposed cause for the neurologic changes is believed to be spinal cord ischemia combined with ischemia/reperfusion injury. Recombinant tissue factor pathway inhibitor (rTFPI), a multivalent Kunitz-type inhibitor that binds to tissue factor-VIIa complex, was evaluated. METHODS: The effectiveness of rTFPI as an agent to limit spinal cord ischemia/reperfusion injury was studied in a rabbit spinal cord made ischemic for 20 minutes. rTFPI or phosphate-buffered saline solution (control) was given in randomized blinded fashion at the onset and conclusion of ischemia. Animals underwent neurologic evaluation at 24 hours in a blinded fashion with a modified Tarlov Scale to rate the lower limb paralysis (score of 4 = normal function, score of 0 = complete paralysis). RESULTS: Seventy-five percent of the TFPI-treated animals had Tarlov scores of 3 to 4, whereas only 29% of the animals treated with phosphate-buffered saline solution had such scores (p < 0.0014). Spinal cord histologic findings correlated with the neurologic findings. CONCLUSIONS: We believe that TFPI has unique inhibitory properties that make it an effective agent in limiting postoperative paraplegia associated with spinal ischemia. PMID- 8619183 TI - Pancreatic juice output after pancreatoduodenectomy in relation to pancreatic consistency, duct size, and leakage. AB - BACKGROUND: A soft pancreas with a main pancreatic duct (MPD) with normal diameter has been considered a high risk for pancreatic anastomotic leakage because of a relatively high output of pancreatic juice, but data are lacking. METHODS: An attempt was made to assess the relationship between the consistency of the pancreas, MPD diameter, pancreatic juice output, and pancreatic leakage after partial pancreatoduodenectomy. The pancreatic parenchyma was classified as of soft, intermediate, and hard consistency in 70 consecutive patients undergoing operation (groups 1, 2, and 3, respectively) by one surgeon. The MPD diameter was determined by means of endoscopic pancreatography or abdominal ultrasonography. Pancreatic juice output was measured for 21 days after operation by using a catheter inserted into the MPD. Anastomotic leakage was identified radiologically by using contrast medium. RESULTS: The mean (SD) pancreatic juice output during a period of 10 days (postoperative days 5 to 14) was 1554 (1073) ml in group 1 (n = 29), 1513 (1060) ml in group 2 (n = 13), and 187 (220)ml in group 3 (n = 28) (groups 1 and 2 versus group 3, p < 0.0001). The MPD diameter was 3.0 (1.6) mm in group 1, 5.9 (2.5) mm in group 2, and 6.6 (2.6) mm in group 3 (group 1 versus groups 2 and 3, p = 0.0001). Anastomotic leaks occurred in four (14%) patients in group 1, three (23%) in group 2, and none in group 3 (p < 0.05). CONCLUSIONS: Patients with a pancreatic parenchyma with an intermediate or normal consistency produced more pancreatic juice and had a higher leak rate. PMID- 8619182 TI - Clinical assessment compared with cyst fluid analysis in the differential diagnosis of cystic lesions in the pancreas. AB - BACKGROUND: In adults 80% to 90% of cystic lesions in the pancreas are pseudocysts and the remainder are mostly neoplastic cysts. To choose optimal treatment for an individual patient, exact nonoperative diagnosis would be preferable. This study was done to assess the value of cyst fluid analysis, compared with clinical and radiologic findings, in the differential diagnosis of pancreatic cystic lesions. METHODS: Twenty-two patients with a cystic lesion in the pancreas underwent operation, cyst wall biopsy, and aspiration of cyst fluid. Carcinoembryonic antigen (CEA), CA 19-9, pancreatitis-associated protein (PAP), and total protein concentration, amylase activity, and cytologic findings were studied. Final diagnosis was pseudocyst in 14 patients, serous cystadenoma in two, mucinous cystadenoma in two, and mucinous cystadenocarcinoma in four patients. RESULTS: Clinical and radiologic judgment correctly differentiated pseudocysts and neoplastic cysts. Cyst fluid aspiration did not succeed in two patients with mucinous cystadenocarcinomas because of the high fluid viscosity. Cyst fluid amylase activity was high (greater than 16,000 IU/ml) in all but one pseudocyst and low (less than 83 IU/ml) in all but one neoplastic cyst. CEA level was lower in pseudocysts than in neoplastic cysts, but with an overlapping value between the groups. Mean CA 19-9 concentration was higher in pseudocysts than in neoplastic cysts, but with wide overlap between the groups. Pancreatitis associated protein and total protein concentration and cystic fluid cytologic findings did not differ between various types of cysts. CONCLUSIONS: Clinical judgment including careful history and radiologic studies seems to be the most reliable method of differentiating neoplastic pancreatic cysts from pseudocysts. Amylase and CEA levels give suggestive information, but cyst fluid analysis may be misleading in an individual patient. PMID- 8619184 TI - Reflex sympathetic dystrophy: does sympathetic dysfunction originate from peripheral neuropathy? AB - BACKGROUND: Sympathetic dysfunction in reflex sympathetic dystrophy (RSD) has been purported to consist of an afferently-induced increase in efferent sympathetic nerve impulses (somato-sympathetic reflex) and/or denervation-induced supersensitivity to catecholamines. In addition, both the central and peripheral nervous systems have been claimed to be involved. It was the aim of this study to obtain more insights into these underlying mechanisms. METHODS: In the affected extremeties of 42 patients with RSD we investigated as indirect measures of sympathetic (dys)function: (1) skin blood flow and the vasoconstrictive response to dependency of skin microvessels by means of laser Doppler flowmetry (distal to the site of trauma), (2) relative distention of the brachial artery and changes in relative distention consequent to a cold pressor test by means of ultrasonic vessel wall tracking (proximal to the site of trauma), and (3) arterial blood pressures by means of the Finapres technique. Both provocation tests induce a sympathetically mediated response. Patients were divided into three categories according to their perception of skin temperature in their injured limb (stage I, stationary warmth sensation; stage II, intermittent warmth and cold sensation; or stage III, stationary cold sensation). RESULTS: Distal to the site of trauma, when compared with controls, skin blood flow was increased at stage I and decreased at stages II and III, whereas the vasoconstrictive response to dependency was impaired at all three stages. Proximally, when compared with controls, relative distention of the brachial artery and its response to the cold pressor test were decreased at all three stages. No differences were observed in pulse pressure between patient groups and controls. CONCLUSIONS: These results suggest that sympathetic dysfunction in extremities of patients with RSD distal to the site of trauma consists of hypersensitivity to catecholamines at stages II and III as a result of autonomic denervation at stage I, whereas proximal to the site of trauma sympathetic nerve impulses may be increased at all three stages. PMID- 8619185 TI - Does the choice of material influence early morbidity in patients undergoing carotid patch angioplasty? AB - BACKGROUND: This study was undertaken to determine whether the choice of material influences the early morbidity of patients undergoing carotid patch angioplasty. METHODS: Before undergoing carotid endarterectomy, 190 patients were randomized to receive 207 patch closures with either Dacron (USCI Sauvage knitted velour) or saphenous vein harvested from the thigh. RESULTS: One hundred seven Dacron and 100 vein patch angioplasties were performed. No significant difference was seen between the two groups in patient age, sex preoperative risk factors, or indication for operation (p > 0.25 for each variable). Among the patients undergoing Dacron patch angioplasty three strokes (two temporary and one permanent), seven episodes of bleeding requiring reoperation, and two neck wound infections requiring rehospitalization occurred. The final 32 patients with Dacron patch closures had their anticoagulation reversed and had no bleeding complications. Complications inpatients undergoing vein patch closure included one fatal perioperative stroke, two episodes of bleeding requiring reoperation including one patch rupture, and three groin infections requiring hospitalization. No significant difference was seen between the two groups in the rate of perioperative stroke (p = 0.62), episodes of bleeding (p = 0.17), or infection (p = >0.67). CONCLUSIONS: Carotid patch angioplasty can be performed with an acceptably low complication rate with either Dacron or vein, and the choice of patch material does not clinically affect patient morbidity. However, reversal of anticoagulation is recommended to minimize bleeding complications in patients undergoing Dacron patch angioplasty. PMID- 8619186 TI - Acadesine and lipopolysaccharide-evoked pulmonary dysfunction after resuscitation from traumatic shock. AB - BACKGROUND: We have reported that the purine precursor acadesine (AICAR) improved the microcirculation, repleted adenosine triphosphate, and attenuated local and lung neutrophil infiltration after intestinal reperfusion and that it quickly improved systemic hemodynamics after resuscitation from hemorrhagic shock. This study evaluated the therapeutic potential of AICAR after fluid resuscitated trauma. METHODS: Anesthetized (fentanyl) mongrel pigs were subjected to tissue injury plus hemorrhage and randomized to receive resuscitation fluids comprised of shed blood plus either lactated Ringer's solution (LR) or AICAR (1 or 10 mg/kg bolus + 0.5 mg/kg/min x 30 min). Thereafter either LR or AICAR (1 or 10 mg/kg) was administered at 12-hour intervals for 72 hours. In a smaller series (n = 7) a single bolus (0.5 mg/kg) of the adenosine deaminase inhibitor deoxycoformycin was administered at the time of resuscitation. After 72 hours, and endotoxin challenge (0.5 microgram/kg, lipopolysaccharide [LPS]) was administered. RESULTS: At 1 mg/kg (n = 9), AICAR had no obvious effect versus LR (n = 31). At 10 mg/kg AICAR (n = 11), the fluid required to stabilize hemodynamics after trauma was higher (66 +/- 5 versus 52 +/- 3 ml/kg/hr, p = 0.014), but there were fewer deaths 3 days after trauma versus LR (0 of 11 versus 4 of 31, p = 0.210), fewer deaths within 5 hours after LPS administration (3 of 11 versus 16 of 27, p = 0.074), and a longer survival time after LPS administration (4.5 +/- 0.3 versus 3.9 +/- 0.2 hr, p = 0.054). Deoxycoformycin had similar salutary effects on survival after LPS administration. LPS increased protein permeability of pulmonary capillaries, increased peak inspiratory pressures on constant tidal volume, increased dead space ventilation, and caused progressive arterial desaturation on 0.65 FiO2 (all p < 0.05). This pulmonary dysfunction was associated with a compensatory increase in cardiac output, decrease in systemic vascular resistance, increase in O2 consumption, and rise in plasma cortisol level (all p < 0.05). All these changes were blunted or eliminated with 10 mg/kg AICAR. Hematocrit and systemic pressures were maintained relatively constant after LPS administration with fluid resuscitation, but less was required with AICAR versus LR (40 +/- 8 versus 83 +/- 14 ml/kg/hr, p = 0.023). AICAR caused a concentration-related reduction in CD18 expression on LPS-stimulated neutrophils in vitro, but there was no effect versus LR on circulating leukocyte counts in vivo and no effect of AICAR on LPS-stimulated production of tumor necrosis factor in vitro or in vivo. CONCLUSIONS: 1. AICAR reduced the pulmonary dysfunction associated with posttrauma endotoxemia but had no effect on circulating leukocytes, so its mechanism could be related to an adenosine-mediated improvement in peripheral perfusion or O2 use. 2. AICAR is a generic compound that is safe and apparently efficacious in human beings, so AICAR prophylaxis could be cost-effectively administered to trauma patients. PMID- 8619187 TI - Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody mediated immunity commensurate to the trauma. AB - BACKGROUND: Measuring serum cytokines, pituitary hormones, or acute phase proteins during or after surgery is not an optimal method for quantifying the impact of surgical procedures. In an effort to assess surgical stress by means of the immune response, we focused on changes in cell-mediated and antibody-mediated immunity as illustrated by the type 1/type 2 T-helper (Th1/Th2) cell balance. The sensitivity of this approach was evaluated by comparing laparoscopic and conventional cholecystectomy (LCE, CCE). METHODS: In a pragmatic prospective study 43 patients with symptomatic cholelithiasis were operated on either by LCE (n = 25) or CCe (n = 18). Blood sampling was done 24 hours before surgery, immediately before incision, and 2, 24, and 48 hours after surgery. Cell surface markers and cytokine production were used to characterize the Th1/Th2 balance and were measured by means of flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: Activation of Th2 cells evokes the production and secretion of interleukin-4 (IL-4), which up-regulates the expression of immunoglobulin E receptors (Fo epsilon RII, CD23) on B cells. Phytohemagglutinin-induced IL-4 production in freshly isolated peripheral blood mononuclear cells from patients increased more after CCE than LCE (IL-4, +41% versus +17%; p < 0.05). Also the expression of CD23 on B cells was higher after CCE than LCE (+146% versus +63%; P < 0.01). CD30, a membrane molecule that belongs to the tumor necrosis factor receptor superfamily and probably is an important indicator of Th2 activity, was more evaluated on T cells from patients who underwent CCE. The Th1 response, characterized by phytohemagglutinin-induced IFN-gamma secretion in peripheral blood mononuclear cells and up-regulation of human leukocyte antigen-DR expression on monocytes, was lower after CCE than after LCE. CONCLUSIONS: This study shows that surgical stress induces a shift in the Th1/Th2 balance toward Th2, suggesting that cell-mediated immunity is down-regulated and antibody mediated immunity is up-regulated after surgery. The evaluation of this shift may be clinically meaningful and help quantify even less invasive surgical procedures. When comparing CCE and LCE in this not strictly randomized study, we found LCE to be the less stressful procedure. PMID- 8619188 TI - An animal model for measurement of protein metabolism in the skin. AB - BACKGROUND: A suitable in vivo approach with which to quantify skin protein metabolism has not been found, and consequently no information exists relating protein synthesis to protein breakdown in the fasted state. METHODS: Stable isotope-labeled phenylalanine was infused into five fasted rabbits, and amino acid and protein kinetics were calculated from a three-compartment model with the rabbit ear used as an arteriovenous balance mode. Results were compared with the more traditional but limited direct incorporation technique to measure synthesis. RESULTS: The model-derived skin protein synthesis rate was 0.34%/hr +/- 0.04%/hr, which was almost identical to the value of 0.30%/hr +/- 0.01%/hr determined by the direct incorporation method. The model-derived skin protein breakdown rate was only slightly higher than the synthesis rate, meaning protein balance was generally maintained in the fasted state because of the efficient reuse of amino acids from protein breakdown. CONCLUSIONS: The newly described rabbit ear model is a reliable approach to the determination of the amino acid and protein kinetics in the skin in vivo. Efficient reuse of amino acids released from proteolysis explains the maintenance of skin integrity during brief fasting. PMID- 8619189 TI - Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer. AB - BACKGROUND: Angiogenesis is a prerequisite for tumor growth and metastasis. Tumor angiogenesis may be mediated by several angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha, and basic fibroblast growth factor. METHODS: Differential mRNA expressions of VEGF, PDGF (A chain), transforming growth factor alpha and basic fibroblast growth factor in 32 primary invasive breast tumors were examined by reverse transcriptase-polymerase chain reaction. We analyzed relationships between mRNA expressions of these angiogenic factors and the degree of angiogenesis, tumor size, and metastasis. Quantification of angiogenesis was achieved by the immunohistochemical staining of endothelial cells with antibody to CD31. RESULTS: VEGF and PDGF-A mRNAs were expressed more frequently in breast tumors than in nontumor breast tissues, whereas no difference was found in expression frequency of either transforming growth factor-alpha or basic fibroblast growth factor mRNA. Vascular counts in tumors correlated with each expression frequency of VEGF and PDGF-A mRNA. PDGF-A mRNA was expressed more frequently in tumors with lymph node metastasis than in those without metastasis. CONCLUSIONS: Expression of VEGF and PDGF mRNAs detected by reverse transcriptase polymerase chain reaction in breast tumors correlates with tumor-related characteristics of angiogenesis and metastatic potential. Analysis of these mRNAs by reverse transcriptase-polymerase chain reaction may be useful for assessing the biologic behavior of a breast tumor before surgical treatment. PMID- 8619190 TI - Fluid resuscitation with deferoxamine hetastarch complex attenuates the lung and systemic response to smoke inhalation. AB - BACKGROUND: We determined the effect of infusing the iron chelator deferoxamine complexed to hetastarch on the degree of lung dysfunction and systemic abnormalities produced by a severe smoke exposure. METHODS: Adult sheep were given a smoke exposure under anesthesia that produced a peak carboxyhemoglobin between 40% and 45%. Twenty-eight sheep were studied; eight were given smoke alone and resuscitated with sufficient lactated Ringer's solution to maintain baseline hemodynamics. Seven sheep were given a bolus plus 1 ml/kg/hr of a 10% deferoxamine-hetastarch solution for resuscitation; five were given hetastarch alone. The response was compared with eight controls during a period of 24 hours. RESULTS: Smoke alone and smoke with hetastarch resulted in a shunt fraction of greater than 25% and a 50% decrease in compliance, severe airway inflammation, mucosal slough, atelectasis, and some alveolar edema. Increased lipid peroxides measured as malondialdehyde were present in airway fluid. In addition, oxygen consumption increased by 100% early after injury, net 24-hour positive fluid balance was almost 3 L, and a significant increase occurred in liver lipid peroxidation. The group given deferoxamine had a significantly attenuated lung response, with only modest airway damage lung dysfunction, and minimal systemic changes including a net positive fluid balance of just over 1L and no liver lipid peroxidation. CONCLUSIONS: An iron chelator deferoxamine complexed to hetastarch, given after a severe smoke exposure, significantly attenuates the airway and the systemic inflammatory (oxidant) injury, indicating free iron release and subsequent increased oxidant activity to be a major etiologic factor. PMID- 8619192 TI - Early postoperative small bowel obstruction caused by spilled gallstones during laparoscopic cholecystectomy. PMID- 8619191 TI - Metastatic renal cell carcinoma to ampulla of Vater: an unusual lesion amenable to surgical resection. PMID- 8619193 TI - Upper extremity arterial thrombosis associated with immunohemolytic anemia. PMID- 8619194 TI - Vein aneurysms of the lower extremities. PMID- 8619195 TI - Neoplastic seeding in the gallbladder fossa after laparoscopic cholecystectomy. PMID- 8619196 TI - Liver transplant recipients and enteral feeding. PMID- 8619198 TI - Effect of lymph node dissection on the prognosis in patients with node-negative early gastric cancer. PMID- 8619199 TI - Percutaneous technique of hepatic venous isolation and charcoal hemoperfusion with a dual-balloon vena cava catheter. PMID- 8619197 TI - Implantation metastasis of esophageal carcinoma to the surgical wound after radical esophagectomy. PMID- 8619200 TI - Randomized controlled trials in surgery: Issues and problems. PMID- 8619201 TI - Late outcome of amputees with premature atherosclerosis. AB - BACKGROUND: Peripheral atherosclerosis in young adults has been associated with a high amputation rate. Our purpose was to examine the natural history of amputees with premature atherosclerosis. METHODS: We compared 50 consecutive young patients undergoing dysvascular amputation who were 49 years of age or younger (mean age +/- SEM, 43 +/- .7 years) with 75 consecutive men and women ranging in age from 60 to 75 years (mean age, 67 +/- .6 years) who were undergoing major amputations for atherosclerosis during the same period. RESULTS: Before undergoing amputation 46 (92%) patients in the study group underwent a mean of 3 +/- 0.3 vascular operations, and 49 (65%) older patients in the control group underwent a mean of 2 +/- 0.2 vascular operations (p = 0.003). The mean time from onset of symptoms to first amputation was not different in the study group versus the control group. Of those patients surviving until discharge, 20 (45%) patients in the study group and 21 (31%) patients in the control group became community or household ambulators, whereas 24 (55%) patients in the study group and 47 (69%) patients in the control group were confined to wheelchairs or were bedridden (p = 0.17). Seventeen (74%) patients in the study group who did not require contralateral amputations became community ambulators, as did 18 (38%) members of the control group (p = 0.01). Twenty (40%) patients in the study group and 39 (52%) patients in the control group died during the study period. The mean age at death was 48 +/- 1 years for the study group and 69 +/- .8 years for the control group (p < 0.001). Cumulative 5-year survival (62% patients in study group, 47% patients in control group) was not different between the two groups (p = 0.41, Kaplan-Meier). CONCLUSIONS: Compared with older counterparts, amputees with premature atherosclerosis have a higher number of failed bypasses before undergoing amputation and die at a younger age. However, both groups have a similar cumulative survival after amputation. Fewer than one half of young patients undergoing dysvascular amputation ultimately achieve ambulation, suggesting that major amputations are a harbinger of long-term disability and dependency in these patients. Because young patients had a higher potential for rehabilitation after unilateral amputation, these patients should be monitored closely for development of ischemia in the contralateral limb. PMID- 8619202 TI - Increased mast cell infiltration in varicose veins of the lower limbs: a possible role in the development of varices. AB - BACKGROUND: This study shows increased infiltration of mast cells in the walls of varicose veins in the lower limbs as an explanation of the pathogenesis of varix formation. METHODS: Great saphenous veins exhibiting varicosity were histologically examined after vein stripping surgery, and the numbers of mast cells in the varicose lesions were estimated in 20 high-power fields (x400). Normal-looking regions of the veins were referred to as controls, and normal saphenous veins were prepared during coronary artery bypass grafting and designated baseline controls. RESULTS: The varicose lesions showed a greater extent of mast cell infiltration (15.0 +/- 8.4 cells; mean +/- standard deviation), whereas control veins (5.9 +/- 4.0) and baseline control veins (4.4 +/-2.9) had a smaller number of mast cells. CONCLUSIONS: The study suggests that increased mast cell infiltration contributes to the development of varicose veins. PMID- 8619203 TI - Preoperative intrahepatic segmental cholangitis in patients with advanced carcinoma involving the hepatic hilus. AB - BACKGROUND: Major hepatic resection for biliary tract carcinoma continues to be a risky operation. In this study we examined the influence of preoperative intrahepatic segmental cholangitis on posthepatectomy mortality. METHODS: We analyzed retrospectively the clinical features of 118 patients who underwent liver resection including more than two segments for biliary tract carcinoma involving the hepatic hilus. RESULTS: Intrahepatic segmental cholangitis was encountered before operation in 22 cases. The morbidity and mortality rates for these patients were significantly higher than those of patients without preoperative cholangitis. Selective percutaneous transhepatic biliary drainage was performed before operation in 11 patients for segmental cholangitis. The morbidity rate of patients after hepatectomy was significantly lower than that of patients treated without percutaneous transhepatic biliary drainage. CONCLUSIONS: The presence of preoperative intrahepatic segmental cholangitis is a major prognostic factor in the outcome of major hepatic resection for biliary carcinoma. Selective percutaneous transhepatic biliary drainage for preoperative intrahepatic segmental cholangitis plays an important role in reducing complications after major hepatic resection. PMID- 8619204 TI - Clinical significance of serum hepatocyte growth factor in orthotopic liver transplantation. AB - BACKGROUND: Hepatocyte growth factor (HGF) plays a key role in the regulation of liver regeneration after hepatocyte damage. Changes in HGF production reflect the status of the regeneration process. METHODS: Serum concentrations of HGF and energy substrates were measured during and after liver transplantation in 30 recipients. RESULTS: In the patients with compromised grafts (group A) HGF concentrations were persistently high after reperfusion, whereas in the patients with well-functioning grafts (group B), HGF concentrations decreased rapidly and remained low 4 hours after reperfusion. The patients in group A who died had persistently high concentrations of HGF. The surviving patients with reversible primary graft dysfunction in group A exhibited low concentrations 48 hours after reperfusion. The decrease in HGF concentration preceded the decrease in aspartate aminotransferase concentration. The metabolic parameters that reflect carbohydrate metabolism by the graft paralleled the changes in HGF. CONCLUSIONS: HGF may be more sensitive and specific in predicting early graft function than prothrombin time, ratio, aspartate aminotransferase, or arterial ketone body ratio. The determination of HGF levels after liver transplantation may yield valuable information for evaluating early graft function and making an early decision to repeat a graft procedure in an acutely ill patient. PMID- 8619205 TI - Detection of liver metastases from colorectal carcinoma: is there a place for routine computed tomography arteriography? AB - BACKGROUND: A prospective evaluation of the liver by preoperative ultrasonography, conventional computed tomography (CT), and continuous CT angiography (CCTA) was performed in 60 patients with primary or secondary colorectal carcinoma. METHODS: The standards of reference were palpation of the liver and intraoperative ultrasonography. The imaging techniques were assessed independently of each other. RESULTS: In 37 patients 105 liver metastases were identified; 23 patients had no metastases. CCTA and a high sensitivity of 94% (99 lesions identified) in contrast to ultrasonography (48%) and conventional CT (52%). The superiority of CCTA was also manifest in lesions less than 1 cm in diameter. However, the high sensitivity was accompanied by a high false-positive rate, particularly because of variations in the perfusion of normal liver parenchyma. Overall, CCTA had the highest accuracy (74%) compared with ultrasonography and CT (both 57%). The data indicate that preoperative ultrasonography and conventional CT have low sensitivity in the detection of liver metastases. CONCLUSIONS: Although CCTA seems to be superior to other preoperative imaging techniques, the too low specificity will hamper its routine application in patients with hepatic metastases from colorectal carcinoma. PMID- 8619206 TI - Surgical treatment strategy for patients with stage IV hepatocellular carcinoma. AB - BACKGROUND: This study was conducted to identify the prognostic indicators for patients with stage IV hepatocellular carcinoma (HCC), as well as to clarify the strategy of surgical treatment for those patients. METHODS: Forty-six patients with stage IV HCC were included in this study. Prognostic factors were univariately and multivariately analyzed. Furthermore, the significance of intraoperative treatment for residual tumors was investigated in patients with an absolute noncurative operation. RESULTS: The poor prognostic factors were as follows: host factors, Child's classification of B and C and immunosuppressive acidic protein level of greater than 400 micrograms/ml; tumor factors, tumor diameter of greater than 5 cm, poorly differentiated histologic features, positive portal vein invasion, and intrahepatic metastases involving more than three segments; others, an absolute noncurative operation and no preoperative treatment. Tumor diameter of more than 5 cm was then suggested to be an independent prognostic indicator. Survival of patients with stage IV-A HCC who underwent a curative operation was similar to that of those with stages III HCC: Furthermore, the survival of patients with Stage IV-A who had an absolute noncurative operation but underwent either intraoperative microwave coagulation or ethanol injection to the residual HCCs was not statistically different from that of those with a curative operation. CONCLUSIONS: Therefore for stage IV-A HCC surgical treatment is considered to be both useful and the first choice of treatment when all the tumors in the liver can be removed or when the residual tumors can be treated during operation by either microwave coagulation or ethanol injection as a result of an incomplete removal of the tumors. PMID- 8619207 TI - Perisurgical erythropoietin application in anemic patients with colorectal cancer: A double-blind randomized study. AB - BACKGROUND: Blood transfusions are associated with higher postoperative morbidity and tumor recurrence rates in colorectal cancer surgery, To reduce the need for transfusions in patients with tumor-induced anemia who are not suitable for autologous blood donation, it was tested whether perisurgical erythropoietin application would be able to stimulate hematopoiesis adequately. METHODS: In a double-blind randomized study 150 IU/kg body weight erythropoietin was given subcutaneously every 2 days beginning 10 days before operation and continuing until postoperative day 2. Twenty patients were randomized into the erythropoietin group with three observed dropouts and 10 patients into the placebo group. RESULTS: In the erythropoietin group two episodes of hypertension and one deep venous thrombosis were observed. Preoperative hemoglobin response in the erythropoietin group (p = 0.069) was paralleled by a highly significant reticulocyte increase (p = 0.0004). However, frequency of blood transfusion was not different between both study groups (erythropoietin, 1.82 +/- 0.80 units/ patient; placebo, 1.80 +/- 0.97 units/patient). If iron availability was analyzed, a strong correlation between ferritin blood levels and transferrin iron saturation with hemoglobin response was observed in regression analysis (p < 0.001). CONCLUSIONS: These results indicate that hematopoiesis in anemic patients with colorectal cancer can be stimulated by erythropoietin; however, clinical efficacy is to be expected only in selected patients with high iron availability, which calls for further studies combining erythropoietin and parenteral iron application. PMID- 8619208 TI - Effects of isotonic saline solution resuscitation on blood coagulation in uncontrolled hemorrhage. AB - BACKGROUND: It has been suggested that fluid resuscitation before surgical control of hemorrhage may lead to increased bleeding because of the elevated blood pressures and clotting factor dilution. This study was designed to assess the effects of isotonic saline solution resuscitation on blood coagulation during uncontrolled hemorrhage. METHODS: Twenty-four female Sprague-Dawley rats were randomized into four groups with different resuscitation regimens: group A, no resuscitation; group B, 40 ml/kg in 4 minutes; group C, 80 ml/kg in 4 minutes; and group D, 80 ml/kg in 1 minute. Baseline blood samples were collected just before a sharp resection of 75% of the tail to initiate the hemorrhage; 15 minutes later the resuscitation began. Additional blood samples were obtained at 60 minutes after resection. The blood was analyzed for platelets, fibrinogen, prothrombin time, and activated partial thromboplastin time. RESULTS: The largest differences between time 0 and 60 minutes were observed in group D with platelets decreasing 43.36% +/- 7.86%, fibrinogen decreasing 57.10% +/- 16.88%, and prothrombin time increasing from an average 16.5 to 19.2 seconds. These differences was statistiacally significant (p <0.05) with the Student's test. CONCLUSIONS: The results suggested that even though the volume of resuscitation fluid did not appear to affect clotting time when compared with that of nonresuscitated animals, the rate of extremely large volume infusions may play an important role in the cessation of bleeding and consequently in the management of uncontrolled hemorrhagic shock. PMID- 8619209 TI - Combined laparoscopic and endoscopic approach in patients with cholelithiasis and choledocholithiasis. AB - BACKGROUND: The Optimal management of common bile duct stones in patients undergoing laparoscopic cholecystectomy remains controversial. METHODS: A prospective study was conducted in 145 of the 481 patients who had a preoperative endoscopic retrograde cholangiogram before their laparoscopic cholecystectomy. RESULTS: Endoscopic retrograde cholangiogram was successful in 138 patients (95%), and common duct calculi were found in 72 (50%) of them. Endoscopic sphincterotomy with ductal clearance was achieved in 62 of 67 patients during a mean of 1.4 sessions (range, 1 to 5). Five (3.4%) patients had complications after endoscopic intervention, all of which resolved uneventfully . Fourteen patients underwent laparoscopic common duct exploration, five had failed endoscopic extraction, five had their common duct stones left intentionally for laparoscopic intervention, and, in addition, four of the seven patients who had a failed endoscopic retrograde cholangiogram had stones identified by intraoperative cholangiogram. Ten of these 14 patients underwent a successful laparoscopic common duct exploration. Laparoscopic cholecystectomy was successfully completed in 134 of the 145 patients, and none had major intraoperative or postoperative complications. The mean postoperative stay was 2.7 days for those patients who underwent a successful laparoscopic procedure. The overall mean number of admissions for completing the treatment was 2.3. CONCLUSIONS: Combined laparoscopic and endoscopic approach is a viable option for patients with gallstones and choledocholithiasis. PMID- 8619210 TI - Living-unrelated renal transplantation provides comparable results to living related renal transplantation: a 12-year single-center experience. AB - BACKGROUND: The increasing success of renal transplantation is paralleled by the increased size of the waiting list. Efforts to increase the donor pool have included the use of living-unrelated kidney donors (LURDs). METHODS: During a 12 year period our center performed 309 transplantation from living donors; 279 patients received living-related donor (LRD) transplants, and 30 patients received LURD transplants. During the same period 543 patients received cadaveric renal donor transplants. A total of 86.7% of LURD transplants were spousal transplants. A total of 29% of the patients who received LRDs were human leukocyte antigen-identical with their donors and 53% were haploidentical, versus 0 human leukocyte antigen-identical or haploidentical in the LURD group. RESULTS: Twenty-seven (90%) Of 30 LURD recipients are alive, as are 240 (86%) of 279 LRD recipients. Mean current creatinine is 1.6 mg/dl for the LURD group and 1.7 mg/dl for the LRD group Kaplan-Meier 1- and 5-year graft survival was 94.9% and 82.9% for the LRD group, 93.1% and 85.9% for the LURD group (p = not significant), and 84.6% and 70.7% for the cadaveric renal donor group (p < 0.05). CONCLUSIONS: LURD patient and graft survival is comparable to LRD transplants despite inferior human leukocyte antigen matching. LURD transplant survival is superior to that of cadaveric renal donor transplants. LURDs are an excellent but underused source of organs for renal transplant recipients. PMID- 8619211 TI - Analysis of Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder after lung transplantation. AB - BACKGROUND: Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorder (PTLD) is a serious complication of lung transplantation. Besides immunosuppression the risk factors for PTLD development are largely unknown. METHODS: The incidence of PTLD was ascertained in a lung transplant population consisting of 45 patients. Nine patients (20%) experienced PTLD. The clinical, histologic, and human leukocyte antigen (HLA) data were collected on all patients. The incidence of EBV infection in lymphoid tissue taken at the time of engraftment was studied by using EBV in situ hybridization. RESULTS: All patients with PTLD had polymorphous lymphoproliferations, seven of which were polymorphous B-cell hyperplasias and two of which were polymorphous B cell lymphomas. EBV was identified in all lesions. All patients with polymorphous B-cell hyperplasias had clinically unsuspected disease, five of which were identified at autopsy. The two polymorphous B-cell lymphoma lesions were monoclonal and regressed with immunosuppression reduction. EBV in situ hybridization on donor or recipient lymph nodes obtained at engraftment from the 45 transplant recipients showed no difference in the number of EBV positive cells in patients with and without PTLD. Cyclosporine and PTLD and azathioprine dosages and cyclosporine levels were similar between patients with and without PTLD. PTLD was seen in patients with high cumulative doses of antilymphocyte globulin. Analysis of HLA status showed a predominance of HLA A2 and DR7 in the donors of the patients with PTLD, whereas donor HLA B7 was more common in patients without PTLD> CONCLUSIONS: Detailed studies are necessary to further elucidate the risk factors for PTLD development in the lung transplant population. PMID- 8619212 TI - Early outcome after open versus extraperitoneal endoscopic tension-free hernioplasty: a randomized clinical trial. AB - BACKGROUND: The use of minimal access surgery for repair of groin hernias is controversial. The aim of this study was to compare endoscopic tension-free hernia repair with open tension-free hernia repair within a randomized clinical trial. METHODS: One hundred twenty patients were randomized by four surgeons during a 1-year period. Early outcome measures were then analyzed by intention to treat. RESULTS: Median postoperative pain scores (63 [interquartile range (IQR), 23 to 81] versus 35 [IQR, 17 to 62]; p = 0.004) and analgesia requirements (2.5 [IQR, 2 to 4] doses verus 2.0 [IQR, 1 to 3] doses; p = 0.0008) were significantly less for patients undergoing endoscopic hernia repair. Hospital stay (1 [IQR, 0 to 1] day versus 2 [IQR, 1 to 2] days; p < 0.0001) was also significantly reduced for the endoscopic group. Wound complications occurred significantly more frequently in the open group. No difference in pulmonary function or metabolic response to trauma (interleukin-6, C-reactive protein, glucose, albumin) was observed between the groups. CONCLUSIONS: This study shows significant short-term advantages for endoscopic tension-free repair over open tension-free repair. However, larger studies with a longer follow-up period are required to establish the relative merits of both procedures in the management of patients with groin hernias. PMID- 8619213 TI - My dear Paul: letters from Harvey Cushing. PMID- 8619214 TI - Overexpression of p53 protein and DNA content are important biologic prognostic factors for thyroid cancer. AB - BACKGROUND: Many factors gave been reported to be of prognostic importance for thyroid cancer. Biologic aggressiveness may influence postoperative recurrences and the prognosis of thyroid cancer. Immunohistochemical staining for the p53 protein and DNA content are novel factors that suggest biologic aggressiveness. METHODS: Retrospective study of the survival rate after operation of differentiated thyroid cancer was undertaken at Osaka Police Hospital. Age, gender operative method, extent of lymph node dissection, use of radioiodine, primary or recurrent tumor, tumor size and invasion, lymph node involvement, presence of distant metastases, DNA ploidy, percentage of S phase and G2M phase fractions, positive staining for the p53 protein, and histologic type and subtype were evaluated as possible prognostic factors by univariate and multivariate analyses of survival. RESULTS: Positive staining for the p53 protein was related to postoperative local recurrence, and DNA ploidy was related to distant metastatic recurrence. Univariate analysis suggested that age, tumor size and invasion, lymph node involvement, presence of distant metastases, percentage of S phase fraction, histologic subtype, DNA ploidy, and positive staining for the p53 protein were significant prognostic factors. Multivariate analysis suggested that positive staining of the protein and DNA ploidy were independent prognostic factors for overall survival. CONCLUSIONS: Both positive staining for the p53 protein and DNA ploidy, which suggest biologic aggressiveness, are independent prognostic factors for overall survival of patients with thyroid cancer, Examination of these biologic factors may provide new information regarding postoperative recurrences and the prognosis of thyroid cancer. PMID- 8619215 TI - Pancreatic regeneration (reg) gene expression in a rat model of islet hyperplasia. AB - BACKGROUND: After pancreatectomy, regeneration of acinar and islet elements occurs. Recent data from a model of islet hyperplasia in the hamster suggested that induction of a local pancreatic factor stimulates islet formation. We postulate that the reg gene may be this factor. METHODS: We studied reg expression during induction of islet growth by using a similar model in the rat. Rats underwent surgical wrapping of the splenic lobe of the pancreas or sham operation. RESULTS: At 2 days ductular proliferation and immunohistochemical evidence of insulin within ductular epithelia were evident in the wrapped lobe. By 14 and 56 days islet number per square millimeter increased 63% and 43%, respectively. Reg mRNA levels, measured by Northern blot analysis with a rat reg cDNA probe (n = 5), increased 300% at 2 days in the wrapped lobe and decreased to that of unwrapped controls by 14 days. In situ hybridization showed localization of reg to the acinar cells. In unwrapped gastric lobes of animals who underwent surgical wrapping of the splenic lobe, no change in islet number per square millimeter or reg gene expression was noted. CONCLUSIONS: Surgical wrapping of the pancreatic splenic lobe induces local reg gene expression that is temporally associated with duct cell hyperplasia. This is followed by islet formation within the wrapped lobe. Reg may play a role in the induction of new islets from ductular precursors and in maintenance of normal islet function. PMID- 8619216 TI - Therapeutic efficacy of an antineutrophil monoclonal antibody, Urge-8, against acute necrotizing pancreatitis in rats. AB - BACKGROUND: Although they have critical roles in the defense mechanism against invading microorganisms, neutrophils may facilitate exacerbation of critical conditions associated with acute necrotizing pancreatitis by a discharge of granule contents into the organ tissues. Because of this constitution of neutrophils, the therapeutic efficacy of Urge-8, a mouse monoclonal antibody to neutrophils, on the survival of rats with experimentally induced acute necrotizing pancreatitis was investigated in vivo. METHODS: Acute pancreatitis was induced by injection of trypsin mixed with sodium chenodeoxycholic acid into pancreatic ducts. Urge-8 was infused intravenously 30 minutes after pancreatitis was induced, a series of vital signs was taken, and plasma amylase level was estimated. RESULTS: Hemorrhagic necrosis of the pancreas and intraabdominal bleeding were observed 1 hour after the pancreatitis-inducing drugs were injected, and death occurred at 240.9 +/- 24.6 minutes (mean +/- SD)in the control group. With Urge-8 administration, however, the survival time was significantly prolonged to 395.2 +/- 64.4 minutes (p = 6.0 x 10-(10) versus control). Failure in the vital signs (mean arterial pressure, heart rate, and body temperature) was significantly ameliorated by injection of Urge-8. The plasma amylase level was elevated after pancreatitis was induced and peaked at 3 hours (4915 +/- 1966 IU/L in mean +/- SD). This level was suppressed during the first 3 hours by injection of Urge-8 (3372 +/- 1223 IU/L); however, the amylase level increased thereafter, becoming comparable with the peak in the control group, and then death occurred. Arterial blood gas and plasma electrolyte analyses showed that pH, base excess, and plasma potassium levels in the group treated with monoclonal antibody were significantly improved. CONCLUSIONS: It was suggested that neutrophils play some critical role in the exacerbation of acute necrotizing pancreatitis and its related symptoms. Although not capable of preventing death in our model, treatment with the antineutrophil monoclonal antibody Urge-8 after the onset of acute pancreatitis prolonged the survival time significantly. PMID- 8619217 TI - Retrograde right hepatic trisegmentectomy. PMID- 8619218 TI - Surgery for massive pulmonary embolism and cavoatrial renal carcinoma. PMID- 8619219 TI - Hepatolithiasis with situs inversus: first case report. PMID- 8619220 TI - Intraoperative localization of metastatic colorectal carcinoma. PMID- 8619222 TI - [Fish diseases]. PMID- 8619221 TI - [Testicular tumors in dogs: a literature review]. AB - The incidence of testicular tumours in dogs is higher than in other species. The main three types are: Sertoli cell tumour, seminoma, and Leydig cell tumour. Metastases are rare. Sertoli cell tumours, and to a lesser extent Leydig cell tumours, are often associated with feminization, which occurs in 19% and 5% of cases, respectively. Seminomas are rarely associated with feminization. Feminization seems to be the result of an excessive oestrogen production by the tumour. In severe cases this may lead to bone marrow depression. Atrophy of the contralateral testis is a common finding. It is not clear whether this is a result of feminization or of age because most tumours occur in older dogs. By investigating the morphology of the testis, and the endocrinological and fertility status of the dog this phenomena is hopefully going to be explained. Extra attention is given to the pathogenesis of feminization. PMID- 8619223 TI - [Hygienic blood collection]. PMID- 8619224 TI - [Study of pregnancy and pseudopregnancy in goats]. PMID- 8619225 TI - [Listeria in goats]. PMID- 8619226 TI - [Cow pregnancy yes or no?]. PMID- 8619227 TI - [Anesthesia in cesarean section in dogs]. PMID- 8619228 TI - Possible mechanism of induction of benign prostatic hyperplasia by estradiol and dihydrotestosterone in dogs. AB - A mechanism of induction of canine benign prostatic hyperplasia (BPH) by androgen stimulated growth of prostatic cells exposed to estrogen-induced toxicity. Estrogen is thought to be activated by redox cycling of catechol metabolites, a mechanism of generation of active radicals. Mongrel dogs, 4 animals/group, were sham-operated (controls) or were treated for 60 days with implants of 5 alpha dihydrotestosterone and/or estradiol-17 beta. The activities of drug and hormone metabolizing enzymes (aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O deethylase, and estradiol-17 beta-2- and -4-hydroxylase) in prostate were significantly elevated by chronic treatment with either estradiol-17 beta or 5 alpha-dihydrotestosterone plus estradiol-17 beta, but not in kidney or liver which are not targets of hormone-induced hyperplasia. Activities of the detoxifying enzymes catalase and glutathione peroxidase I in prostate were increased by estradiol-17 beta treatment, whereas in kidney or liver they were not affected or changed to a lesser degree than observed in prostate. Free radial induced damage (carbonyl content) of proteins was observed in prostate of dogs treated with either estradiol-17 beta or 5 alpha-dihydrotestosterone plus estradiol-17 beta and in liver protein by treatment with either 5 alpha dihydrotestosterone or 5 alpha-dihydrotestosterone plus estradiol-17 beta. It was concluded that the patterns of estradiol-17 beta- or 5 alpha-dihydrotestosterone plus estradiol-17 beta-induced increases in activities of catechol estrogen synthase and of other drug metabolizing enzymes were consistent with the postulated free radical generation by redox cycling of catechol estrogen specifically in the prostate. The increases in activities of detoxifying enzymes may represent an (albeit insufficient) response to prostatic free radical damage to protein in prostate is consistent with a postulated induction of BPH via injury by estrogen metabolites followed by 5 alpha-dihydrotestosterone-stimulated growth of altered prostatic cells. PMID- 8619229 TI - Comparative effects of Cd2+ and Cd-metallothionein on cultured kidney tubule cells. AB - The effects of Ca2(+) and Cd-metallothionein on two cultured cells with proximal tubule characteristics, mouse kidney cortical cells and pig kidney LLC-PK1 cells, have been compared. Cd2+ inhibits Na(+)-glucose cotransport in LLC-PK1 cells and in the process decreases the number of binding sites for [3H]phlorizin, a competitive inhibitor of glucose for the Na(+)-glucose cotransporter. During 24 hr incubation and over a range of concentrations in the two cell types, only Cd2+ inhibited Na(+)-glucose cotransport even when approximately equal concentrations of intracellular Cd resulted from these treatments. Indeed, at low concentrations of Cd-metallothionein in mouse cells, transporter activity was elevated. Extension of incubations to 72 hr in mouse cells led to increased Cd uptake and reduction in cell density with both sources of Cd but only a progressive decline in Na(+)-glucose cotransport activity with Cd2+. Zn-metallothionein was without effect under comparable conditions. Both forms of Cd were accumulated by these cells, with the large majority of the metal ion localizing in metallothionein as a Cd, Zn-protein in LLC-PK1 cells. Under equal exposure conditions, the net uptake of Cd from Cd-metallothionein in the two cell types. It is evident that the mechanisms of toxicity of Cd2+ and Cd-metallothionein as well as their modes of uptake differ in these two cell types. PMID- 8619230 TI - Metallothionein-I and -II knock-out mice are sensitive to cadmium-induced liver mRNA expression of c-jun and p53. AB - Zinc pretreatment has been shown in vitro (rat myoblasts) to induce metallothionein (MT) and inhibit cadmium (Cd)-induced protooncogenes c-myc and c jun mRNA levels. therefore, the purpose of this study was to determine whether the mRNA expression of the protooncogene c-jun as well as the tumor suppressor gene p53 is increased by Cd in the intact animal and, more specifically, in the target organ for Cd toxicity, the liver. Additionally, modulation of the expression of these genes was investigated in the absence of MT. The effect of CdCl2 on the mRNA levels of c-jun and p53 was studied in livers of C57BL/6J (control) and MT-null mice by Northern- and slot-blot analyses. The mRNA for c jun and p53 were increased by Cd in a dose-dependent fashion. In the control mice, Cd induced c-jun mRNA (5-fold) at 3 and 12 hr and p53 mRNA (1.8- to 2-fold) at 6 and 12 hr. Compared to controls, the MT-null mice were more sensitive to the Cd-induced gene expression. The magnitude of the inductions was more pronounced and the elevated mRNA levels of c-jun and p53 were seen at lower doses of Cd (10mumol/kg in MT-null mice vs 40 mmol/kg in control mice). In conclusion, these data demonstrate that Cd induces mRNA expression of the protooncogene c-jun and tumor suppressor gene p53 in liver, and that MT modulates this effect. PMID- 8619231 TI - Effects of superoxide dismutase and U74389G on acute trimethyltin-induced cochlear dysfunction. AB - Reactive oxygen species (ROS) generation has been implicated in the ototoxicity induced by several pharmaceutical agents. The present study examined the efficacy of the ROS scavenger polyethylene glycol-conjugated superoxide dismutase (PEG.SOD), and a 21-aminosteroid (U74389G), in ameliorating the acute cochlear dysfunction induced by the environmental ototoxicant trimethyltin chloride (TMT). Thirty pigmented guinea pigs were divided into six experimental conditions, as defined by preexposure and exposure administrations: saline-saline, PEG.SOD saline, U74389G-saline, saline-TMT, and U74389G-TMT. Electrophysiological potentials (compound action potential (CAP) and cochlear microphonic (CM) were recorded for 10 acoustic frequencies, and then a preexposure agent (U74389G, 4.5mg/kg; PEG.SOD, 10,000 U/kg; or 0.9% saline) was administered i.v. Thirty minutes later, potentials were retested, and an exposure agent (0.9% saline or TMT, 0.5 mg/kg) was administered i.p. CM and CAP were retested at 30, 60, and 90 min postexposure time points, in order to assess changes in auditory responsiveness. Mean changes in 1.0-microV root mean square CM isopotential values did not differ significantly in any group from those of saline-saline controls. In the saline-TMT group, CAP thresholds increased significantly at all test frequencies relative to those of the saline-saline control group. While mean CAP threshold shifts in the U74389G-TMT group were as great as those in the saline-TMT group, such values in the PEG.SOD-TMT group were significantly lower. These results suggest that ROS formation may be involved in acute TMT-induced CAP disruption, for which PEG.SOD, but not U74389G, provides partial protection. PMID- 8619232 TI - Cellular uptake of trivalent arsenite and pentavalent arsenate in KB cells cultured in phosphate-free medium. AB - Trivalent arsenite (As(III)) and pentavalent arsenate (As(V)) have been shown to have differential uptake mechanisms. In regular RPMI 1640 medium, As(III) was about 40-fold more toxic to KB oral epidermoid carcinoma cells. However, the cytotoxicity and intracellular accumulation of As(V) were dramatically enhanced, equalling those of As(III) when cells were grown in phosphate-free RPMI medium, As(V) uptake was dose-dependently inhibited by phosphate, mersalyl acid (a membrane sulfhydryl agent), and energy poisons, such as sodium azide and potassium cyanide. These results suggest that As(V) and phosphate share a common transport system. In contrast, As(III) uptake was not affected by the above agents. However, the initial uptake rates of As(III) were linearly correlated with its extracellular concentrations, suggesting that As(III) uptake is probably accomplished through simple diffusion. Our results also show that As(III) and As(V) are excreted from KB cells at a comparable rate, and at least half of As(V) is reduced to the more toxic As(III) prior to excretion into the medium. Therefore, the toxicity of As(V) may in part result from its reduction to As(III). PMID- 8619233 TI - Construction of a physiologically based pharmacokinetic model for 2,4 dichlorophenoxyacetic acid dosimetry in the developing rabbit brain. AB - A physiologically based pharmacokinetic (PBPK) model that describes the kinetics of organic anions by using 2,4-dichlorophenoxyacetic (2,4-D) as a representative compound was constructed for the developing rabbit brain at near-term pregnancy (Gestation Day 30). The model consisted of brain, body, and venous and arterial compartments for the mother which were linked to the fetus by a placenta. Maternal brain compartments in the model were brain plasma, cerebrospinal fluid (CSF), and brain tissue including hypothalamus, caudate nucleus, hippocampus, forebrain, brainstem, and cerebellum. The fetus consisted of brain, body, amniotic fluid, and venous and arterial compartments. the maternal body had both a central and a deep compartment; the fetal body had only one compartment. Maternal blood flow to the fetus was modeled as blood flowing to the placenta, where it was equilibrated before it reached the fetus. The brain uptake was membrane-limited by the blood-brain barrier, with saturable clearance from the CSF into the venous blood by the choroid plexus in both fetus and mother. The model was used to compare concentrations of 2,4-D in maternal and fetal brain, maternal and fetal plasma, and amniotic fluid over time with experimental data from pregnant rabbits given 2,4-D intravenously (1, 10, or 40 mg/kg). The model adequately simulated the 2-hr time course of 2,4-D concentrations in both mother and fetus. With continued development, this generic PBPK model should be a useful tool for evaluating the safety of organic acid neurotoxicants in the developing brain. PMID- 8619234 TI - Distribution and retention of cadmium in metallothionein I and II null mice. AB - Cadmium (Cd) is known to have a long biological half-life in the body, possibly due to its binding to metallothionein (MT). This study was designed to determine the role of MT in the tissue distribution and retention of Cd using MT-I and -II null (MT-null) mice. Mice were given 109CdCl(2) (15 mumol/kg, 25 microCi/kg, i.p.), and radioactivity was quantified in 14 major organs at 2 hr, 1, 2, 3, 7 and 15 days thereafter. The lack of MT in MT-null mice 2 hr after Cd administration (74% vs 72% of the dose, respectively). However, the elimination of Cd was much faster in MT-null mice than in control mice. In control mice, approximately 40% of Cd administered was found in liver 24 hr after administration, and the majority was bound to MT. In contrast, only 20% of Cd was found in liver of MT-null mice, which was not bound to MT. Cd concentrations in kidney, pancreas, and spleen were also lower in MT-null than in control mice 1 week after administration. No apparent difference in Cd retention in other organs was noted between control and MT-null mice over the 15-day period. Cd concentration in kidney continued to increase with time in control but not in MT null mice, indicating that an important source of Cd in the kidney is the uptake of CdMT. In conclusion, the present data indicate that MT does not play a role in the initial distribution of Cd to tissues, but does play a major role in the elimination of Cd, especially from liver, kidney, and pancreas. These data support the conclusion that the persistence of Cd in the body is at least partially due to Cd binding to MT in tissues. PMID- 8619235 TI - Alterations in rat brain thyroid hormone status following pre- and postnatal exposure to polychlorinated biphenyls (Aroclor 1254). AB - The effect of daily oral maternal exposure to 0, 5, or 25 mg/kg body wt of a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) on Days 10 to 16 of gestation on plasma and brain thyroid hormone concentrations and peripheral thyroid hormone concentrations and peripheral thyroid hormone metabolism were examined in fetal and weanling rats. Plasma thyroid hormone levels and hepatic microsomal thyroid hormone glucuronidation were also examined in pregnant rats and the adult offspring. Plasma and brain levels of PCBs and hydroxylated PCB metabolites were analyzed in fetal, weanling, and adult offspring. Maternal exposure to Aroclor 1254 significantly decreased fetal (Gestation Day 20) and neonatal (Postnatal Day 4) plasma total thyroxine (T4) and free T4 levels in a dose-dependent manner. Effects of maternal Aroclor 1254 exposure on plasma total and free T4 concentrations were less pronounced in offspring at 21 days of age and absent 90 days after birth. Plasma concentrations of thyroid-stimulating hormone were unaltered in fetuses, neonates, weanling rats, and adult offspring following maternal treatment with Aroclor 1254. the concentration of T4 was severely depressed in the forebrain and cerebellum of fetal rats on Day 20 of gestation following maternal Aroclor 1254 exposure. Brain triiodothyronine (T3) concentrations in the Aroclor-exposed fetuses were significantly decreased relative to control values only in the low-dose group. On Day 21 postpartum T4 concentrations were significantly decreased in the forebrains of female weanling rats from the 25 mg Aroclor 1254/kg dose group, and no reductions were observed in forebrain T3 concentrations in male or female neonates. The deiodination of T4 to T3 was significantly increased in fetal forebrain homogenates by both PCB treatments. In female weanling brain homogenates the deiodination of T4 to T3 was significantly decreased in the low-dose group and unaltered in the high-dose group. No alterations in brain thyroid hormone metabolism were observed in forebrain homogenates from adult offspring exposed pre- and postnatally to Aroclor 1254. Hepatic microsomal T4 glucuronidation was significantly decreased in fetal microsomes following perinatal PCB exposure and significantly increased in weanling hepatic microsomes in a dose-dependent manner. An accumulation of mainly one PCB metabolite, 2,3,3',4',5-pentachloro-4-biphenylol was observed in fetal plasma and forebrain on Gestation Day 20 and in neonatal and weanling rat plasma on Postnatal Days 4, 21, and 90. The plasma level of 2,3,3',4',5 pentachloro-4-biphenylol was higher than that of the persistent PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl in the control and PCB-exposed offspring up to Postnatal Day 21, and even after 90 days, the 2,3,3',4',5-pentachloro-4 biphenylol was present in amounts approximately equal to those of CB 153. Although PCB levels were relatively high in the weanling rat forebrain, no hydroxylated PCB metabolites were detected. On Day 90 postpartum, plasma levels of PCBs and 2,3,3',4',5-pentachloro-4-biphenylol were still elevated in the offspring of PCB-treated dams relative to controls. These results suggest that the accumulation of hydroxylated PCB metabolites in fetal plasma can reduce fetal plasma T4 levels and accordingly fetal brain T4 levels. However, in late gestational fetuses, the induction of brain type II thyroxine 5'-deiodinase activity compensates for decreases in brain T4 levels, so that brain T3 levels are maintained. PMID- 8619236 TI - Pulmonary activation and toxicity of cyclopentadienyl manganese tricarbonyl. AB - Cyclopentadienyl manganese tricarbonyl (CMT) produces acute pulmonary injury following cytochrome P450 mixed-function oxidase (CYP450) activation. The current studies were designed to characterize the role of hepatic and/or pulmonary CMT activation and the subsequent pneumotoxicity of this compound following subcutaneous injection in the male Sprague-Dawley rat. Both pulmonary and hepatic tissues were capable of CYP450-dependent CMT metabolism in vitro. Phenobarbital pretreatment, which induced hepatic but not pulmonary CMT metabolism, protected against CMT-depended pneumotoxicity suggesting escape of an active CMT metabolite from the liver is not responsible for the pneumotoxic response. Animals were also pretreated with either m-xylene or 3-methylindole, each of which reduce CMT metabolism in the lung but not in the liver. These pretreatments also reduced CMT dependent pulmonary damage. Protection against toxicity by two compounds that inhibit pulmonary but not hepatic CMT metabolism provides strong evidence that CMT-induced pneumotoxicity is due to the activation of CMT within the lungs. Histopathological studies revealed that CMT induced an alveolar injury without apparent damage to the bronchiolar airways. Based on this pattern of injury, studies were performed with freshly isolate alveolar type II (ATII) cells as these cells are thought to contain significant CYP450 activity. However, CMT metabolism was not detectable in ATII cells in vitro. Although CMT was cytotoxic to ATII cells in vitro, this response was not inhibited by metyrapone indicating CYP450 activation was not involved in the in vitro phenomenon. Together these data suggest in situ activation of CMT is necessary for the alveolar toxicity of this compound; however, activation does not occur in ATII cells. PMID- 8619237 TI - In vivo and in vitro studies of perchloroethylene metabolism for physiologically based pharmacokinetic modeling in rats, mice, and humans. AB - In vivo experiments in rats and mice and in vitro experiments in rats, mice, and humans have been used to develop and validate a "2nd generation" physiologically based pharmacokinetic (PBPK) model for perchloroethylene (PERC). The refined PBPK model should be useful in the preparation of carcinogenic risk assessment based on amounts of PERC metabolites formed in the livers of rodents and humans according to procedures developed by EPA. A sensitivity analysis of the PBPK model revealed that the most significant uncertainties in this process (other than the choice of the appropriate dose/response model based on mechanism of action of PERC) were in the techniques used to estimate rates of PERC metabolism in humans. In vitro studies with human tissues reported help define what some have called the range of "equally reasonable alternatives" for estimating human risk. PMID- 8619238 TI - Glutathione conjugation of 1,2:3,4- diepoxybutane in human liver and rat and mouse liver and lung in vitro. AB - 1,3-Butadiene (BD) has been classified as a probable human carcinogen based on sufficient evidence of a carcinogenic response in B6C3F1 mice and Sprague-Dawley rats and limited human evidence of carcinogenicity. Mice are much more susceptible to BD-induced carcinogenicity than rats. Previous in vitro studies revealed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. BDE was also readily quantitated in blood and tissues of mice exposed to BD but could not be detected in rats similarly exposed. These findings suggest a key role for BDE in BD-induced carcinogenicity. The purpose of this study was to characterize the glutathione (GSH) conjugation of BDE by liver and lung cytosol from B6C3F1 mice and Sprague-Dawley rats and human liver cytosol from six different individuals in vitro. BDE and glycine-[2-3H]GSH were incubated, at pH 7.4, with cytosol. 13C NMR and mass spectral analysis indicated formation of two isomeric conjugates, S-(1-(hydroxy-methyl)-2,3-epoxypropyl)glutathione and S-(2 hydroxy-3,4-epoxy--butyl)glutathione, which were rapidly hydrolyzed in cytosol to the corresponding trihydroxy conjugates. Total conjugates were quantitated by HPLC. Conjugation of BDE with GSH followed Michaelis-Menten kinetics in human as well as rat and mouse cytosolic fractions. The conjugation rates in mouse and rat liver cytosol were similar (Vmax 162 +/- 16 and 186 +/- 37 nmol/mg protein/min, respectively) and an order of magnitude higher than in human liver cytosol (Vmax 6.4 +/- 1.9 nmol/mg protein/min). the apparent KM values were lower in human (2.1 +/- 1.4 mM) than mouse (6.4 +/- 1.6 mM) or rat (24 +/- 6 mM) liver. Mouse lung cytosol (Vmax 38.5 +/- 2.5 nmol/mg protein/min, KM 1.70 +/- 0.37mM) is also more efficient in GSH conjugation than rat lung cytosol (Vmax 17.1 +/- 3.0 nmol/mg protein/min, KM +/- 1.7 mM). These results suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in hepatic or pulmonary GSH conjugation of BDE. Also, considering the low oxidation rates of BD to BMO and of BMO to BDE in humans as compared to mice, the relatively low capacity of GSH conjugation of BDE in human liver will not necessarily lead to increased BDE blood levels in humans potentially exposed to BD. PMID- 8619239 TI - Neurotoxic convulsions induced by histamine H2 receptor antagonists in mice. AB - Convulsive potency was evaluated to investigate the mechanism of neurotoxic convulsion induced by histamine H2 receptor antagonists (H2 blockers). Four H2 blockers, cimetidine (721-1236 nmol), ranitidine (477-954 nmol), famotidine (7.4 44 nmol), and nizatidine (226-603 nmol) were administered intracerebrally (i.c.) to mice. Dose dependency of clonic and/or tonic convulsion was observed, and the ED50 values of convulsive occurrence for cimetidine, ranitidine, famotidine, and nizatidine were 997, 662, 23.4, and 404 nmol, respectively. Intraperitoneal pretreatment of muscimol, aminooxy acetic acid, diazepam, (+/-)2-amino-7 phosphonoheptanoic acid (APH), or (+)MK801 suppressed the tonic convulsion after i.c. administration of ranitidine, but had no effect on clonic convulsion. Furthermore, the convulsive threshold concentration in the brain determined by constant rate infusion of ranitidine was not affected by the pretreatment of muscimol, diazepam, APH, and MK801. Ed50 values for convulsive occurrence after i.c. administration of four H2 blockers correlated well with the EC50 values for gastric acid secretion inhibition. The convulsive threshold concentrations of cimetidine and ranitidine in the brain were 11 and 2.5 microM, respectively, which were similar to the dissociation constants determined from the inhibition of gastric acid output in mice. From these results, tonic convulsion induced by H2 blockers can be suppressed by GABAergic or glutamatergic anticonvulsants, while clonic convulsion induced by H2 blockers may be associated with the blockade of H2 receptor in the brain and not be directly associated with the GABA and glutamate-mediated neurotransmission. PMID- 8619240 TI - 3-Aminobenzamide: effects on cytochrome P450-dependent metabolism of chemicals and on the toxicity of dichlobenil in the olfactory mucosa. AB - Treatment with 3-aminobenzamide, known as an inhibitor of poly(ADP ribose)polymerease, decreased the toxicity and covalent binding of the herbicide dichlobenil (2,6-dichlorobenzonitrile; 12 mg/kg; i.p.) in the mouse olfactory mucosa. In vitro studies showed that 3-aminobenzamide markedly reduced the NADPH dependent covalent binding of [14C]dichlobenil and the hydroxylation of p nitrophenol which have previously been suggested to be mediated by a common form of cytochrome P450 (P450) in rat olfactory microsomes (Eriksson and Brittebo, Chem.-Biol. Interact. 94,183-196, 1995). Furthermore, 3-aminobenzamide markedly reduced the P450-dependent metabolic activation of [3H]NNK (4-(N-methyl-N nitrosamino)-1-(3-pyridyl)-1-butanone) in rat olfactory microsomes and slightly decreased the P450 2B1-dependent pentoxyresorufindealkylase activity in liver microsomes of phenobarbital-treated rats. The present results suggest that 3 aminobenzamide is also an inhibitor of P450 and that the lack of toxicity of dichlobenil in the olfactory mucosa of 3-aminobenzamide-treated mice is related to a decreased metabolic activation of dichlobenil at this site. Further experiments showed that there was no evidence for a binding of [14C]dichlobenil metabolites to calf thymus DNA or a formation of mutagenic dichlobenil metabolites in Ames' Salmonella assay when dichlobenil was incubated in the presence of homogenates of the olfactory mucosa. Finally, analysis of proteins from olfactory microsomes incubated with [14C]dichlobenil using SDS PAGE/fluorography revealed a binding of metabolites to all major proteins. Addition of glutathione or the P450-inhibitor metyrapone prevented the binding, suggesting the formation of relatively stable electrophilic products which can leave the activating enzyme and then unselectively bind to the major olfactory microsomal proteins. PMID- 8619241 TI - Bioavailability of cadmium from shellfish and mixed diet in women. AB - Dietary intake and uptake of cadmium (Cd) were studied in nonsmoking women, 20-50 years of age, consuming a mixed diet low in shellfish (N = 34) or with shellfish once a week or more (N = 17). Duplicate diets were collected during 4 consecutive days for the determination of Cd content. The women kept detailed dietary records, and the intake of energy and various nutrients was calculated. The shellfish diets (median 22.3 micrograms Cd/day) contained twice as much Cd as the mixed diets (median 10.5 micrograms Cd/day; p < 0.0001). Cadmium in feces corresponded to 100 and 99% of that in duplicates of shellfish diets and mixed diets, respectively, indicating a low average absorption of the dietary Cd. In spite of the differences in the daily intake of Cd, there was no statistically significant difference in the concentrations of Cd in blood (B-Cd, shellfish group 0.25 micrograms/liter, mixed diet group 0.23 micrograms/liter) or urine (U Cd, 0.10 micrograms Cd/liter in both groups). This indicates a lower absorption of Cd in the shellfish group than in the mixed diet group or a difference in the kinetics. A higher gastrointestinal absorption of Cd in the mixed diet group could partly be explained by lower body iron stores as measured by the concentrations of serum ferritin (S-fer, median 18 micrograms/liter, compared to 31 micrograms/liter in the shellfish group). In the mixed diet group, S-fer was negatively correlated with B-Cd and the main determining for B-Cd besides U-Cd in the multiple regression analysis, indicating an increased absorption of Cd at low body iron stores. When women with S-fer exceeding 20 micrograms/liter were compared, the higher dietary intake of Cd in the shellfish group compared to the mixed diet group (24 versus 10 micrograms/day) resulted in higher B-Cd (0.26 versus 0.16 micrograms/liter), although not in proportion to the difference in Cd intake. Thus, there seems to be differences in the bioavailability and/or kinetics of dietary Cd related to the type of diet. This is, to our knowledge, the first study where the influence of various types of diets and nutritional factors on the intake and uptake of cadmium in human subjects has been studied. PMID- 8619242 TI - Toxicokinetics of lead in lactating and nonlactating mice. AB - Toxicokinetics of lead in lactating and nonlactating mice were studied after a single intravenous injection of 0.05 mg of lead (2.5 mCi 203Pb)/kg. Lead concentrations in blood, plasma, and milk were measured for 10 days following dosing. The volume of distribution based on plasma lead was more than two times larger in lactating than in nonlactating mice, 133 and 58 liter/kg, respectively. Plasma lead clearance in lactating mice was 4.25 liter/hr/kg compared with 1.07 liter/hr/kg in nonlactating mice. However, no such pronounced difference in blood lead clearance was found between the two groups, indicating that this parameter does not reveal the kinetic characteristics during lactation. Milk was found to be an additional route of excretion for lead. About 1/3 of the administered dose of lead was excreted in milk. Accordingly, milk clearance contributed to 1/3 of the total plasma clearance in the mice. The relationships of lead in plasma to lead in whole blood as well as lead in milk to lead in whole blood were nonlinear, with a relatively higher increase in plasma and in milk lead levels at higher blood lead levels. This nonlinearity may be explained by a reduced uptake of lead in erythrocytes as the lead binding sites of these cells become saturated. In lactating mice, the maximum binding capacity of lead in red blood cells was even lower than in nonlactating mice. Similar nonlinear relationship have have also been found in human studies although at much higher levels of lead in blood. The milk:plasma concentration ratio for lead was found to be between 50 and 100 after 24 hr, demonstrating a much more efficient excretion of lead into milk than what is known from human studies. Differences in the milk composition may be one explanation for the species differences in milk excretion of lead. The present study shows that physiological changes during lactation alter the pharmacokinetics of lead in mice. PMID- 8619243 TI - Differential effects of nonhydroxylated flavonoids as inducers of cytochrome P450 1A and 2B isozymes in rat liver. AB - Flavanone, flavone, and tangeretin differentially affected the activities of cytochrome P540 1A and 2B isozymes in rat liver. Flavone and, to a lesser extent, tangeretin, increased activities of ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, and pentoxyresorufin O-dealkylase (PROD), whereas flavanone mainly enhanced PROD activity. Immunoblot analysis indicated that flavone and tangeretin increased cytochrome P450 1A1, 1A2, and 2B1,2 forms, whereas flavanone only enhanced the cytochrome P450 2B isozymes. Northern blot study showed that flavone and tangeretin increased the level of the cytochrome P450 1A2 mRNAs. The concentration of the other mRNAs were slightly or not affected by flavonoids. These results suggest that the induction of P450 1A2 by flavone and tangeretin might involve a transcriptional and/or post transcriptional mechanism. PMID- 8619244 TI - Molybdate impairs glycosaminoglycan sulfation in rat cartilage. AB - The mechanism by which molybdate produces joint and bone deformities is not known. However, we recently reported that molybdate decreases the sulfation of acetaminophen in rat liver. Therefore, the purpose of the present study was to determine the effect of molybdate on the sulfation of glycosaminoglycans (GAGS) and whether manganese-induced joint and bone deformities might be due to this mechanism. Molybdate (15 mmol/kg, po) did not alter total GAG content in patella and articular cartilage of rats assayed 24 hr after administration. Nevertheless, [35S]sulfate uptake into both patella and articular cartilage was decreased dramatically (70 and 50%, respectively) by molybdate. Molybdate did not affect GAG chain elongation, as there was no effect of molybdate on [3H]glucosamine uptake into patella cartilage. These results indicate that molybdate impairs sulfation of GAGs. In an attempt to determine the mechanism of the molybdate induced decrease in GAG sulfation, the effect of molybdate and sulfate on the incorporation of sulfate into GAGs was examined in vitro using monolayer chondrocyte cultures. Molybdate reduced the sulfation of GAGs in chondrocytes in vitro, but only at concentrations higher than the blood concentration of molybdate given in vivo that decreased GAG sulfation. Molybdate given in vivo decreases plasma sulfate to levels, which when used in vitro, decreases GAG sulfation. Therefore, molybdate treatment decreases sulfate availability and the sulfation of glycosaminoglycans. PMID- 8619245 TI - Reproductive toxicity and growth effects in rats exposed to lead at different periods during development. AB - The reproductive toxicity and growth effects of developmental lead exposure were assessed using a rat model in which 0.6% (w/v) lead acetate was administered in the drinking water ad libitum. Three series of experiments were conducted in which lead exposure was initiated beginning in utero, prepubertally, or postpubertally. Lead effects were measured on reproductive physiology and endocrinology, sexually dimorphic hepatic testosterone hydroxylation, and growth rates in both male and female animals. In male animals secondary sex organ weights were significantly decreased only in animals exposed prepubertally. In addition, serum testosterone levels were significantly suppressed, most severely in animals exposed from in utero (in the in utero group). Little effect was observed in adult female rats. However, in female animals exposed prepubertally, delayed vaginal opening and disrupted estrus cycling was observed. More severe reproductive disruption was accompanied by suppression of circulating estradiol in the in utero group. Effects on circulating sex steroids were accompanied by variable effects on circulating luteinizing hormone (LH) levels, pituitary LH, and pituitary LH beta mRNA, suggesting a dual site of lead action: (a) at the level of the hypothalamic pituitary unit, and (b) directly at the level of gonadal steroid biosynthesis. Prepubertal growth in both sexes was suppressed 25% in the in utero group. However, pubertal growth rates were significantly suppressed only in male animals and postpubertal growth was not significantly different from controls in any of the experiments, despite continued exposure to high lead levels in the drinking water. In addition, at age 85 days, male specific hepatic hydroxylation of testosterone at positions 2 alpha and 16 alpha, which is catalyzed by a cytochrome P450 isozyme CYP 2C11, itself regulated by sexually dimorphic growth hormone secretion, was unaffected. This suggests that the growth effects of lead are possibly due to a delay in the development of sex specific pituitary growth hormone secretion patterns rather than a persistent developmental defect. Thus, the reproductive and growth effects of lead are complex and sex-dependent, and appear to involve multiple sites on the hypothalamic-pituitary-gonadal axis. PMID- 8619246 TI - Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black. AB - Chronic inhalation of carbon black can produce carcinomas in rat lungs. At present the mechanisms underlying the rat lung tumor response to carbon black are unknown, although a significant role for inflammation and cell proliferation has been postulated. To investigate the processes which may contribute to development of rat lung tumors after carbon black exposure, we characterized the effects of subchronic inhalation of carbon black by rats on mutagenesis in alveolar epithelial cells, pulmonary inflammation, inflammatory cytokine/growth factor expression, and lung histopathology. Briefly, rats were exposed for 6 hr/day, 5 days/week for up to 13 weeks to 1.1, 7.1, and 52.8 mg/m3 carbon black and the effects on the lung were characterized after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery. Endpoints characterized after carbon black exposure included mutation in the hprt gene of alveolar epithelial cells, changes in bronchoalveolar lavage fluid markers of lung injury and inflammation, expression of mRNA for the chemokines, MIP-2 and MCP-1, and lung histopathology. Lung burdens of carbon black were also determined. After 13 weeks of exposure to 1.1, 7.1, and 52.8 mg/m3 carbon black, lung burdens were 354, 1826, and 7861 micrograms carbon black, respectively. The lung clearance of carbon black appeared impaired after exposure to 7.1 and 52.8 mg/m3 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the group exposed to 52.8 mg/m3. No increase in hprt mutation frequency was observed for epithelial cells obtained from rats exposed to 1.1 mg/m3 carbon black. The observation that genotoxic effects (i.e., mutations) on alveolar epithelial cells occurred only after carbon black exposures which resulted in significant inflammation and epithelial hyperplasia supports the hypothesis that inflammatory cell-derives oxidants and increased cell proliferation play a role in the pathogenesis of rat lung tumors in response to carbon black. PMID- 8619247 TI - Wood-derived estrogens: studies in vitro with breast cancer cell lines and in vivo in trout. AB - The wood-derived compound, beta-sitosterol (purity > 90%), was shown to be estrogenic in fish. It induced the expression of the vitellogenin gene in the liver of juvenile and methyltestosterone-treated rainbow trout. Structural similarities to beta-sitosterol notwithstanding, cholesterol, citrostadienol, beta-sitostanol, and 5-androstene-3 beta,17 beta-diol, an estrogenic member of the androstenic steroid group, were inactive. An abietic acid mixture (37% abietic acid, 6% dehydroabietic acid, and a remainder of unknown compounds) showed slight hormonal activity in feed, but it was completely inactive when given intraperitoneally in implants. The estrogenic component of the abietic acid preparation was not identified. In addition, to beta-sitosterol and abietic acid, several other wood-derived compounds including betulin, isorhapontigenin, isorhapontin, and pinosylvin were estrogenic in breast cancer cells (MCF-7 or T 47D). However, betulin and pinosylvin, available in sufficient amounts for in vivo testing, did not induce the expression of the vitellogenin gene. Differences in the primary sequences of human and fish estrogen receptors (hormone as well as DNA-binding regions) or uptake and metabolism of the compounds may explain the discrepancy between the two estrogen bioassays. Wood-derived compounds such as beta-sitosterol, present in pulp and paper mill effluents, may account for the weak estrogenicity of debarking effluent seen at the vitellogenin expression bioassay. PMID- 8619248 TI - Oleander toxicity: an examination of human and animal toxic exposures. AB - The oleander is an attractive and hardy shrub that thrives in tropical and subtropical regions. The common pink oleander, Nerium oleander, and the yellow oleander, Thevetia peruviana, are the principle oleander representatives of the family Apocynaceae. Oleanders contain within their tissues cardenolides that are capable of exerting positive inotropic effects on the hearts of animals and humans. The cardiotonic properties of oleanders have been exploited therapeutically and as an instrument of suicide since antiquity. The basis for the physiological action of the oleander cardenolides is similar to that of the classic digitalis glycosides, i.e. inhibition of plasmalemma Na+,K+ ATPase. Differences in toxicity and extracardiac effects exist between the oleander and digitalis cardenolides, however. Toxic exposures of humans and wildlife to oleander cardenolides occur with regularity throughout geographic regions where these plants grow. The human mortality associated with oleander ingestion is generally very low, even in cases of intentional consumption (suicide attempts). Experimental animal models have been successfully utilized to evaluate various treatment protocols designed to manage toxic oleander exposures. The data reviewed here indicate that small children and domestic livestock are at increased risk of oleander poisoning. Both experimental and established therapeutic measures involved in detoxification are discussed. PMID- 8619249 TI - Nephrotoxicity of inorganic mercury co-administrated with L-cysteine. AB - In the present study, we tested the hypothesis that co-administration of low nephrotoxic doses of inorganic mercury (Hg++) with L-cysteine (in a 1:2 mol ratio of inorganic mercury to L-cysteine), alters significantly the nephropathy induced by inorganic mercury. In the first experiment, the effect of co-administering L cysteine on the nephropathy induced by a 1.8 or 2.0 micromol/kg dose of inorganic mercury was evaluated in rats 24 h after the administration of inorganic mercury. According to histopathological assessment of sections of kidney and evaluation of the urinary excretion of lactate dehydrogenase, total protein and inorganic mercury (which were used as indices of renal injury), the severity of renal injury in rats co-administered the L-cysteine with the inorganic mercury was significantly greater than that in corresponding rats injected with only inorganic mercury. In a second experiment, the disposition of mercury was evaluated 1 h after the administration of 1.8 micromol inorganic mercury/kg with or without 3.6 micromol L-cysteine/kg. The renal accumulation of mercury, specifically in the cortex and outer stripe of the outer medulla, was significantly greater the rats co-administered the inorganic mercury and L cysteine than in the rats given only inorganic mercury. In addition, the content of mercury in the blood and liver was significantly lower, and the fraction of mercury in the blood present in the plasma was significantly greater, in the rats co-administered inorganic mercury and L-cysteine than in the rats given only inorganic mercury. On the basis of the findings from this study, the nephropathy induced by low nephrotoxic doses of inorganic mercury is made more severe when the inorganic mercury is co-administered in a 1:2 mol ratio with L-cysteine. Moreover, it appears that the enhanced severity in the nephropathy induced by the co-administration of inorganic mercury and L-cysteine is linked to an increase in the tubular uptake of mercury in the cortex and outer stripe of the outer medulla. PMID- 8619250 TI - Immunotoxicity of a reconstituted polynuclear aromatic hydrocarbon mixture in B6C3F1 mice. AB - Previous studies on the immunotoxicity of a complex mixture of polynuclear aromatic hydrocarbon (PAH) by-products from a manufactured gas plant indicated possible synergistic interactions which were investigated by determining the immunosuppressive effects of a reconstituted PAH mixture in female B6C3F1 mice challenged with TNP-haptenated sheep red blood cells (SRBCs) (T-cell-dependent) or trinitrophenyl-lipopolysaccharide (TNP-LPS) (T-cell-independent) antigens. The reconstituted PAH mixture contained the following 17 congeners: 2-rings (indan, naphthalene, 1- and 2-methylnaphthalene), 3-rings (acenaphthylene, acenaphthene, dibenzofuran, fluorene, phenanthrene and anthracene), and > or = 4-rings (pyrene, fluoranthene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene), and resembled mixtures identified as by products from manufactured gas plants. The reconstituted mixture and the 2-, 3- and > or = 4-ring PAH fractions all caused a dose-dependent decrease in the splenic plaque-forming cell (PFC) response to SRBCs or TNP-LPS, and their ED50 values for the four treatment groups were 86, 354, 145, and 23 or 163, 439, 637 and 31 mg/kg, respectively. The corresponding ED50 values for decreased serum anti-TNP IgM levels for these same mixtures were (TNP-haptenated SRBCs, T-cell dependent) 144, 231, 42 and 27 units, respectively, and (TNP-LPS, T-cell independent) 161, 406, 312 and 69 units, respectively. The suppression of anti TNP IgM titers was similar to the suppression of the PFC response and shows that antigen-specific immunoglobulin titer can be used as a biomarker of PAH exposure. A direct comparison of the immunotoxic responses of the reconstituted PAH mixture and the corresponding dose of the > or = 4-ring PAHs indicated that the latter fraction was primarily responsible for the activity of the reconstituted mixture. PMID- 8619251 TI - Toxic effect of valproic acid on tryptophan binding to rat hepatic nuclei. AB - This study evaluated whether valproic acid, a branched-chain fatty acid which has been used in the treatment of seizures, would influence the binding Of L tryptophan to rat hepatic nuclei. Previous studies have indicated that binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. In this study, we investigated whether valproic acid, which under certain conditions is heptatoxic, would influence L-tryptophan binding to rat hepatic nuclei as assayed by in vitro L-(5-3H)tryptophan binding. Our results indicate that the addition of valproic acid to hepatic nuclei or nuclear envelopes in vitro has little influence on their L-(5-3H)tryptophan binding. On the other hand, when valproic acid (80 mg/100 g body weight) is tube-fed 2 h before killing, the isolated nuclei show decreased specific L-tryptophan binding (total binding minus non-specific binding using unlabeled L-tryptophan (10(-4)M), at 2000-fold excess) compared with controls. Other fatty acids (oleic, palmitic or linoleic acid at 10(-4)M) when added with excess, unlabeled L-tryptophan (10( 4)M) in vitro to hepatic nuclei revealed some (but less than with valproic acid) decreased specific binding compared with controls. At high doses, valproic acid (80 mg/100 g body weight) appears to decrease tryptophan-induced stimulation of hepatic protein synthesis, probably in a hepatotoxic manner. PMID- 8619252 TI - Sodium 2,3-dimercapto-1-propanesulfonate (DMPS) treatment does not redistribute lead or mercury to the brain of rat. AB - Since there has been concern about whether any of the chelating agents used therapeutically might cause an initial redistribution of heavy metals to the brain and since the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (Dimaval, DMPS) has been used to treat heavy metal intoxication in humans, the hypothesis that DMPS does not redistribute and increase lead or mercuric ions in the brains of rats was tested. Lead acetate at a concentration of 50 mg/l was made available in the drinking water of rats for 86 days. Other rats received intraperitoneal injections of 0.50 mg Hg/kg (as mercuric chloride) each day for 5 days a week for a total of 32 or 41 days. Animals were divided into groups and given, i.p., either 0.27 mmol DMPS/kg body weight or saline, each day for 1, 2, 3 or 4 days. Lead or mercury concentrations of the brain were determined after each group received DMPS for the different number of days. DMPS treatment did not result in any initial increase of lead or mercuric ions in the brain. The mercury content of the kidney decreased. The results of these experiments demonstrated that lead or mercuric ions were not redistributed to or increased in the brains of rats during the initial days of DMPS treatment. PMID- 8619253 TI - Sensitization to 2,4-dinitrochlorobenzene: influence of vehicle on absorption and lymph node activation. AB - Effective skin sensitization is dependent upon immune activation of lymph nodes draining the site of exposure. The influence of vehicle formulation on the vigour of lymph node cell proliferative responses to 2,4-dinitrochlorobenzene (DNCB) has been examined. Mice (BALB/c strain) were exposed topically to 0.5% DNCB dissolved in either acetone or propylene glycol (PG). A significantly greater lymph node cell proliferative response was induced by DNCB in acetone. The observed differences were not attributable to variations in the numbers of immunostimulatory dendritic cells accumulating in the draining nodes following sensitization. In parallel studies, the absorption and cutaneous disposition of DNCB dissolved in acetone or PG were measured in vitro using static diffusion cells and full thickness mouse skin. Although flux of DNCB through the skin was comparable with both vehicles over 24 h, the absorption of the allergen during the first 4 h of exposure was significantly faster when acetone was used as the vehicle. Localization of DNCB demonstrated that much less of the chemical allergen was present in the skin at 4 h when applied in PG vehicle. However, there were no measurable vehicle effects on skin disposition of DNCB at 24 h. These data indicate that the sensitization potential of DNCB is influenced significantly by the nature of the vehicle used, possibly due to consequential effects on chemical absorption and disposition. The studies described in this paper reveal that the application vehicle may have a significant influence on the ability of DNCB to induce immune activation of draining lymph nodes and hence skin sensitization and that this may in turn be associated with important changes in the absorption and/or disposition of the chemical within the skin. PMID- 8619254 TI - Repeated high dose oral exposure or continuous subcutaneous infusion of 2 methoxyacetic acid does not suppress humoral immunity in the mouse. AB - 2-Methoxyethanol (ME) has been shown to be immunosuppressive in rats but not mice, with oxidation of ME to 2-methoxyacetic acid (MAA) being a prerequisite for immunosuppression. MAA is more rapidly cleared by mice than rats, consequently this study was designed to determine if increasing the bioavailability of MAA in mice might play a role in this species difference. Female B6C3F1 mice were given MAA by oral multiple daily high doses or by continuous subcutaneous infusion via mini-osmotic pumps. Humoral immunity was evaluated in MAA-exposed mice using the plaque-forming cell (PFC) response to either sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). Female F344 rats were also used to compare the effects of multiple daily MAA exposure on these humoral immune responses. Rats and mice were dosed orally twice a day for 4 days by gavage with MAA at dosages ranging from 40-320 mg/kg/day and 240-1920 mg/kg/day, respectively. All animals were immunized on the first day of dosing and body and lymphoid organ weights and PFC responses to SRBC or TNP-LPS were evaluated 4 days later. While body weights in rats were unaffected, thymus weights were reduced at all dosages of MAA and spleen weights were reduced at 160 or 320 mg/kg/day. PFC responses to SRBC and TNP-LPS were suppressed in rats at dosages of 160 and 320 mg/kg/day. In contrast, thymus weights of mice were reduced only at 960 mg/kg/day or greater, with no effect on spleen or body weights. Furthermore, neither the PFC response to SRBC nor the response to TNP-LPS was suppressed in mice exposed to any oral dosage of MAA. In the continuous infusion study, mice were subcutaneously implanted with mini-osmotic pumps containing MAA which was delivered at 840 mg/kg/day over a 7-day period. Continuous exposure to MAA via mini-osmotic pumps did not suppress the PFC response to either SRBC or TNP-LPS, but rather significantly enhanced the response to TNP-LPS. These results indicate that mice are insensitive to MAA even at the high dosages given as a bolus or continuously over 1 week. The data further support earlier work, which suggested that the observed difference between rats and mice for MAA-induced immunosuppression appears to be unrelated to the availability of MAA to target lymphoid tissue in these rodent species. PMID- 8619255 TI - Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney. AB - The organization of the metallothionein (MT) gene family has been demonstrated to be much more complex in humans than in the mouse, and possibly rodents in general. For humans, the MTs are encoded by a family of genes located at 16q13 representing 10 functional and 7 non-functional MT isoforms. In the present study, the 5' and 3' untranslated region sequences of the highly conserved, functional MT genes were utilized to generate primer pairs for the analysis of isoform-specific MT mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR). Human kidneys from 13 weeks gestation through adulthood were examined for the expression of MT protein and mRNA. Immunohistochemical analysis demonstrated MT immunoreactivity to be confined exclusively to the proximal tubules of the adult and developing kidney. For all MT-positive cells, MT was localized in the cytoplasm and nuclear localization was variable. There was no correlation between nuclear staining and stage of development. Of the 10 MT genes examined (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3, and MT 4), mRNAs representing the MT-1E, MT-1F, MT-1X, and MT-2A genes were consistently expressed in all samples regardless of gestational age. There was no indication of a 'fetal form' of MT analogous to that noted to occur in human liver. Messenger RNA for the MT-1A gene was detected in 2 of 6 renal samples without correlation to gestational age. In no instance was mRNA for the MT-1B, MT-1G, MT 1H, MT-3 or MT-4 genes detected. These studies detail the initial determination of MT gene expression in the human renal system and provide the PCR primers for testing and determination of MT gene expression in other organ systems. PMID- 8619256 TI - Correlations of the induction of microsomal epoxide hydrolase activity with phase II drug conjugating enzyme activities in rat liver. AB - Within the selective induction of phase II enzymes following treatment with dipyridyls or N-heterocyclic analogs of phenanthrene, strong correlations (r > or = 0.70) are observed between the increase of microsomal epoxide hydrolase (mEH) activity and UDP-glucuronosyltransferase (UGT) activities towards 4-nitrophenol, 1-naphthol and morphine. The present study investigates whether this correlation is maintained with inducing agents known to also increase phase I enzyme activities. Rats were treated with beta-naphthoflavone, isosafrole, phenobarbital, ethanol, dexamethasone and clofibric acid regimens in which P450 isozyme induction could be confirmed. Comparisons between the responses of mEH, UGT and glutathione S-transferase (GST) activities were made. mEH activity was increased by beta-naphthoflavone, isosafrole, phenobarbital and clofibric acid. The elevation in mEH activity by these agents showed modest but significant correlations with GST activities toward all the substrates monitored (r values range between 0.49 and 0.65) and a strong correlation with UGT activity towards only one substrate, morphine (r = 0.70). This study suggests that induction of mEH activity correlates with the increases in select phase II enzyme activities whether it is accompanied by P450 induction or not. PMID- 8619257 TI - Comparative study of the metabolism of triphenyltin in hamsters and rats after a single oral treatment with triphenyltin chloride. AB - Our previous work has shown that triphenyltin compound induces the diabetogenic effects, such as hyperglycemia and hypertriglyceridemia, on hamsters, but not on rats. In the present study, it is examined whether the species differences in the metabolic fate of triphenyltin exist for susceptibility between hamsters and rats. Triphenyltin chloride was orally dosed to hamsters and rats, and triphenyltin and its metabolites, mono- and diphenyltin, and inorganic tin, in liver, kidney, pancreas, brain, and blood were determined by gas chromatography periodically for 96 h after the treatment. Triphenyltin levels in the tissues of both species were almost maxima within 24 h after treatment. Although there were relatively high levels of triphenyltin in the tissues of hamsters dosed with triphenyltin chloride, compared with those in the rats, the proportion of metabolites to triphenyltin were lower than those in the rats. In particular, hamsters were more susceptible than rats to the pancreatic accumulations of triphenyltin and good correlation exists between the tin concentrations in the pancreas and the plasma glucose levels in triphenyltin-treated hamsters. These findings suggest that the dearylation of absorbed triphenyltin in hamsters is slower than that in rats and that triphenyltin-induced hyperglycemic action depends upon the amount of tin compounds absorbed into the pancreas. Furthermore, most of the tin compounds in the brains of both species were triphenyltin. This result shows that the metabolism of triphenyltin in the brains of both species was different from that in other tissues. PMID- 8619258 TI - Oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates. AB - The reaction of human erythrocyte acetylcholinesterase (AChE) with a set of structurally related phosphoramidates was studied in order to investigate the properties of phosphorylated enzyme and the effects of 4 oximes PAM-2, TMB-4, HI 6 and BDB-106 on the reactivation of inhibited AChE. Second-order rate constant of the phosphorylation reaction of the compounds towards the active site of AChE range between 5.0 x 10(2) and 4.9 x 10(6) M-1min-1 and their inhibitory power (I50) was from 7.3 x 10(-5) to 5.7 x 10(-9) M for 20 min incubation at 37 degrees C. The oximes used were weak reactivators of inhibited AChE except for (C4H9O)(NH2)P(O)DCP (DCP, -O-2,5-dichlorphenyl group) and (C6H13O)(NH2)P(O)SCH3 where we have obtained good reactivation. Imidazole oxime BDB-106 proved to be a potent reactivator of tabun-inhibited AChE. PMID- 8619259 TI - Anesthetic activity of monoketones in mice: relationship to hydrophobicity and in vivo effects on Na+/K+ -ATPase activity and membrane fluidity. AB - The in vivo anesthetic activity of monoketones in mice was examined in relation to their hydrophobicity and to the in vivo effects on Na+/K+ -adenosine triphosphatase (Na+/K+ -ATPase) activity and membrane fluidity. Anesthetic potency (AD50) of monoketones was determined; AD50 implys the dose required to anesthetize 50% of the animals from the treated group. The n-octanol/water partition coefficient (P) was used as an index of hydrophobicity. Membrane fluidity was determined by using 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-(4 trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH as fluorescence probes. Log (1/AD50) was the parabolic function of log P, log ((1/AD50) = 0.167(log P)2 + 0.698 log P - 1.365, and the log P that corresponds to the minimum AD50 was estimated to be 2.09. Brain synaptosomes were prepared from mice that were considered anesthetized with each of the 4 monoketones (1.5-fold AD50), methyl n-propyl, methyl n-amyl, methyl 3-methylhexyl and methyl n-octyl ketone. The Na+/K+ -ATPase activity was inhibited by methyl n-propyl ketone alone, membrane DPH fluidity was decreased by each of the 4 monoketones, and membrane TMA-DPH fluidity was decreased by methyl n-propylketone alone. These results suggest an involvement of the decreased DPH fluidity in monoketone-induced anesthesia. PMID- 8619260 TI - Induction of renin release from isolated glomeruli by inorganic mercury(II). AB - Mercury(II) ions are known to accumulate in the kidney and their effect upon the renin-angiotensin system has also been described. The question, however, whether mercury(II) also exerts direct effect on the juxtaglomerular cells (JGC) to induce renin release remained to be answered. Suspension of isolated glomeruli was used to measure the mercury(II)-induced renin release in vitro. The glomeruli were isolated from female BALBc mice. HgCl2 was found to be capable of inducing renin release directly from JGC. The effect is concentration-dependent (P < 0.05, r = 0.914 and P < 0.01, r = 0.982, with and without Neutral Red vital staining) and becomes apparent already at a mercury(II) ion concentration as low as 1 microM. The renin-releasing effect of the mercury ion is to be inhibited by dithiothreitol (DTT) (renin activity 20.37 vs. 2.60 ng/ml.h in supernatant) as well as the elevated osmotic concentration of the incubating bath medium (20.37 vs. 6.84 ng/ml.h). This suggests that certain membrane sulfhydryl groups are implicated in the process on the one hand, and it is also in accordance with the known sensitivity of the renin granules to osmotic pressure on the other hand. Light and electron micrographs also demonstrate the direct, effective role of Hg(II) in the renin release process. Therefore, it is assumed that apart from its influence on tubulo-glomerular feedback a direct way of action of mercury(II) on renin release must also be taken into account. PMID- 8619261 TI - Effect of recombinant human hemoglobin on human bone marrow progenitor cells: protection and reversal of 3'-azido-3'-deoxythymidine-induced toxicity. AB - Long-term therapy of AIDS patients with 3'-azido-3'-deoxythymidine (AZT) is limited by hematopoietic toxicity. While the mechanism(s) of this toxicity remain elusive, various strategies are being developed to reduce these toxic effects including combination therapy with non-myelotoxic anti-human immunodeficiency virus (HIV) drugs and/or administration of protective or rescue agents, such as cytokines and growth factors. Using a physiologically relevant human CD34+ bone marrow cell liquid culture system, a crosslinked human recombinant hemoglobin (rHb), currently in Phase II clinical trials, was investigated for effects on hematopoiesis and for its potential in protecting or reversing AZT-induced hematopoietic toxicity. These investigations demonstrated that 0.01, 0.1, or 1 microM human rHb did not affect the proliferation of erythroid or myeloid lineage cells. A concentration of 1 microM rHb partially protected erythroid lineage cells from an inhibition of proliferation induced by 0.1 and 1 microM AZT. Inhibition of proliferation of cells previously exposed to AZT was not reversed at this concentration. These data suggest that human rHb may be of benefit in reducing the toxic effects of AZT in the bone marrow of AIDS patients. PMID- 8619262 TI - Serum biochemical changes associated with cystic ovarian degeneration in pigs after atrazine treatment. AB - Biochemical and histopathological parameters of the ovarian function were observed to assess the toxic effect of low dose of atrazine, an s-triazine herbicide, in female pigs (gilts) undergoing intensive breeding. Female pigs (cross-bred between Swedish and German landrace) received 2 mg atrazine kg-1 body wt. in feed daily during 19 days of the oestrous cycle. The last treatment day corresponded to day -3 of the onset of the next expected oestrus. Blood samples were collected 3 times daily at 3-h intervals on the first 5 post-treatment days. Serum 17 beta-oestradiol (17 beta-E) and progesterone (P) concentrations were determined. A significantly higher (P < 0.05 and P < 0.001, respectively) serum 17 beta-E concentration was recorded 48 and 24 h before the onset of the next expected oestrus in atrazine-treated pigs, as compared to intact pigs. The onset of the next expected oestrus failed to occur, but no other adverse clinical reactions associated with the treatment were recorded. Histopathological examination of the ovaries of treated pigs indicated multiple ovarian follicular cysts and persistence of corpus luteum. Biochemical and histopathological findings showed that subacute exposure of female pigs to low dose of atrazine prolonged their oestrous cycle. PMID- 8619263 TI - Modern veterinary blood banking practices and their applications in companion animal practice. AB - There are two main reasons for blood component therapy: the need for an increase in oxygen-carrying capacity and improved hemostasis. Additionally, blood component therapy is used to increase plasma protein concentrations. Component therapy entails separating whole blood into its cellular and plasma components and administering the appropriate blood component based on the patient's needs. General considerations such as compatibility, defined expectations of benefits, and topics including specific disease screening, preparation of blood components, preparation of blood products for administration, and typing and crossmatching in relation to blood component therapy and its application in companion animal practice are discussed. PMID- 8619264 TI - Blood components. Collection, processing, and storage. AB - Veterinary blood component preparation methods are based on human protocol with veterinary modification. This article covers the practical aspects of donor selection, blood collection, and the various options for separation of whole blood into components. Obligations are met to ensure high quality products yet allowances are made where possible to account for problems that are unique to veterinary medicine. PMID- 8619265 TI - Selection of anticoagulant-preservatives for canine and feline blood storage. AB - Blood or blood component transfusions have become a well recognized, lifesaving form of therapy in veterinary medicine. Blood used for small animal transfusions may be collected and prepared with a variety of anticoagulants, anticoagulant preservatives, or additive solutions. Selection of the most appropriate of these collection or storage solutions requires a knowledge of their formulations and of the shelf-lives previously established for storage of canine or feline red blood cells. Other factors that should be considered in the selection process are based on the specific transfusion needs of a clinic and its patients, including whether the blood will be used fresh or stored, the length of storage time desired, and whether components will be prepared. New products and techniques for blood storage continue to be developed, offering exciting new possibilities for the future practice of veterinary transfusion medicine. PMID- 8619266 TI - General principles of small animal blood component administration. AB - Administration of blood components instead of whole blood has become the most important means of transfusion support in dogs and in some settings in cats as well. Component administration entails the transfer of biological material and carries an inherent risk of disease transmission and adverse reactions. This article reviews the indications, pretransfusion considerations, and follow up in small animal patients receiving blood component therapy. PMID- 8619267 TI - Autotransfusion in the emergency patient. AB - As the need for blood products increases in the veterinary emergency setting, the technique of autologous transfusion is being re-explored. Analogous transfusions provide an immediate source of blood with known compatibility to the patient. With the advent of passive canister collection systems, the technique is becoming simplified and affordable for the veterinary community. Although complications such as coagulopathies and microemboli have been reported with the procedure, attention to patient selection, technique, and patient monitoring following transfusion greatly minimizes potential drawbacks. PMID- 8619268 TI - Feline transfusion medicine. Blood types and their clinical importance. AB - Anemia is the most common indications for a blood transfusion in cats and fresh whole blood is typically administered. Recent experimental studies and clinical reports have elucidated the importance of feline blood types in transfusion medicine. The AB blood group system, the only recognized feline blood group system, consists of three blood types: type A is most common, type B is frequently found in certain breeds, and type AB occurs rarely. The blood type frequently varies geographically among domestic shorthair cats and between breeds. Because of the presence of naturally occurring alloantibodies, only AB matched transfusions are effective and safe. Blood typing is now readily available and incompatibilities are easily recognized with blood crossmatching tests. Owing to the general presence of strong anti-A alloantibodies in type B cats, type A blood given to a type B cat results in life-threatening acute hemolytic transfusion reactions. Immediate withdrawal of a transfusion and supportive care may save a patient. Other transfusion reactions, unrelated to AB mismatch, cause only mild and transient signs. Peculiarities of feline blood banking also are reviewed. PMID- 8619269 TI - Canine blood groups and their importance in veterinary transfusion medicine. AB - Over 13 canine blood groups have been described. Eight DEA types are recognized as international standards. Typing sera produced by canine alloimmunization exists for six DEA types: 1.1, 1.2, 3, 4, 5, and 7. Naturally occurring antibody is found against DEA 3, 5, and 7. DEA 1.1 and 1.2 antibody-antigen interactions result in acute hemolytic transfusion reactions. DEA 3, 5, and 7 antibody-antigen interaction in vivo results in permanent red blood cell sequestration and loss in 3 to 5 days. DEA 4 antibody-antigen interactions produce no effect on red blood cell survival in vivo. A dog possessing DEA 4 and no other antigen is considered a "universal" donors. Veterinary transfusion medicine has advanced beyond uncrossmatched, untyped red blood cell transfusion. Whenever possible, transfusion should be between typed and crossmatched individuals. "Universal" donors and crossmatch should be utilized when typing of the recipient is not feasible. Canine blood typing is routinely performed in service laboratories across North America. In-clinic assays are not available for all canine blood group antigens. Recent production of monoclonal antibodies will lead to biochemical definition of the canine blood groups DEA 1.1 and 3. Additional efforts to define the erythrocytes on a molecular level are underway. Advances efforts in this areal will allow for more rapid and uniform testing of the canine red blood cell. Future exploration of DEA type and disease association is needed. A known association exists between DEA 1.1 and neonatal isoerythrolysis. Further screening of the dog population for DEA type may yield markers for autoimmune and neoplastic disease. PMID- 8619270 TI - Canine transfusion reactions and their management. AB - There is a wide range of mechanisms by which transfusion reactions may occur. These reactions typically are categorized as immune- or nonimmune-mediated and also as to whether they are acute or delayed in nature. The type and severity of clinical signs vary according to the specific reaction present. Many reactions can be prevented with the use of standard and appropriate transfusion medicine procedures. These methods include careful collection and storage of blood products, adequate screening and blood typing of donor dogs, crossmatching donor and recipient blood, use of component therapy, correct administration of blood products, and the use of pretransfusion prophylaxis when appropriate. Because many reactions are dose dependent, careful monitoring of transfusions cannot be overemphasized. Rapid recognition of a transfusion reaction and immediate discontinuation of the transfusion, along with appropriate supportive therapy, is essential for the successful treatment of transfusion reactions. A summary of transfusion reactions including clinical signs, diagnosis, and basic treatment protocols is given in Table 4. When used appropriately, transfusion of blood products can be a highly beneficial, low-risk form of therapy. PMID- 8619271 TI - Transfusion therapy in emergency and critical care medicine. AB - The administration of blood or its components can provide the life-saving element for a critically ill small animal patient. Shock from acute massive hemorrhage produces catastrophic cardiovascular changes requiring rapid, accurate resuscitation techniques for survival. Bleeding that occurs more slowly allows for the opportunity to discover and treat the inciting cause and to administer specific blood component. PMID- 8619272 TI - Use of blood and blood components in canine and feline patients with hemostatic disorders. AB - Therapy with blood and blood products related to hemostatic disorders in small animal practice is reviewed. Administration of platelet rich plasma and platelet concentrates in thrombocytopenia or thrombopathia is discussed. Vascular purpuras, vasculitis, and vascular inherited defects are also considered. Inherited coagulation disorders are summarized and the therapeutic choices in treating these disorders are also proposed. In addition, acquired coagulation disorders are briefly reviewed. PMID- 8619273 TI - The role of blood component therapy in the management of canine and feline patients with cancer. AB - Blood component therapy may be required for a variety of pathologic reasons in the cancer patient. The tumor itself not only affects various cell lines, but treatment with chemotherapeutic agents can induce cytopenias. In addition, chronic and acute hemorrhage can be caused by tumor erosion and rupture or by paraneoplastic coagulopathies. Component replacement will depend on the underlying hematologic deficiencies and should be performed in conjunction with treating the cause of the deficiency. PMID- 8619275 TI - Hematopoietic growth factors: frontiers for cure. AB - Hematopoietic growth factors are cytokines that regulate the growth, development, and function of hematopoetic lineages. These hematopoietic growth factors have great potential for improving the quality of life and for reducing toxicity. This article reviews some of the most clinically relevant hematopoietic growth factors and what is known about their use in veterinary medicine. In particular, the following hematopoietic growth factors are discussed: erythropoietin, stem-cell factor, interleukin-3, interleukin-5, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor. PMID- 8619274 TI - Blood substitutes: oxygen-carrying acellular fluids. AB - Blood substitutes are acellular fluids that can transport and deliver oxygen to the tissues. The blood substitutes most likely to be available and effective for veterinary use are the hemoglobin-based oxygen carriers (HBOCs) containing modified human, mammalian, or recombinant hemoglobin. HBOCs can be stored at room temperature and do not require typing or crossmatching. Chemical modifications of hemoglobin affect its physiologic and pharmacologic properties and facilitate oxygen transport. Although many of the metabolic and toxicologic consequences of HBOC infusion are not fully understood, an approved product will likely be available in the near future. PMID- 8619276 TI - Gonadotrophin releasing hormone treatment of Holstein cows with follicular cysts monitored by skim milk progesterone determination. AB - Progesterone was assayed in skim milk fraction of 38 cystic cows, 26 (68.4%) of which had basal ( < 0.5 ng/ml) progesterone concentrations and were diagnosed as having follicular cysts. These cows were allotted at random to one of 3 treatments: (1) a sham injection of sterile water in 7 cows as controls; (2) a single injection (1 mg) of GnRH analog in 8 cows; (3) double injections (1 mg) of GnRH analog at 7-day interval in 11 cows. According to rectal palpation confirmed by skim milk progesterone determination, the double injection group responded well (P < 0.05) compared with the single injection and control groups (81.8% vs 62.5 vs 42.9%). The interval calving to 1st insemination, interval treatment to conception and interval calving to conception were significantly (P < 0.01) shorter in the double injection group than in the other 2 groups. Also, the conception rate to first insemination was significantly (P < 0.05) higher (63.6% vs 37.5% vs 42.9%). The data provide evidence for reduction in infertility and reproductive disorders in cystic cows given double injections of GnRH. PMID- 8619278 TI - [Identification of aerobic and facultatively anaerobic sporulating bacteria isolated during the primary milk collection]. AB - Aerobic and/or facultatively anaerobic sporulating Gram-positive bacteria of the genus Bacillus influence nutritive and sensory properties of pasteurized milk by their proteolytic and lipolytic activity. Since particularly raw milk is a source of pasteurized milk contamination by spore-producing bacteria, our investigations were focused on identification of bacilli from operations of milk agricultural primary production. The species B. licheniformis and B. cereus (Crielly et al., 1994) are the most frequently isolated ones in the process of milk production. While B. licheniformis as well as B. subtilis and B. pumilus are mesophilic species, B. cereus is rather psychrotrophic, and as to their health impacts they cause diseases from foods (Griffiths, 1990; Christiansson, 1992; Becker et al., 1994). Sixty-six strains were isolated and identified from operations of milk agricultural primary production (Tab. I). B. licheniformis occurrence (58 strains) was most frequent in the set of samples, followed by B. subtilis (5 strains), B. pumilus (one strain) and B. cereus (one strain), i.e. these are species classified to morphological group I (oval, cell-nonswelling spores). Only one strain Paenibacillus amylolyticus (formerly Bacillus amylolyticus) was isolated from morphological group II (oval, cell-swelling spores). Species representation of isolated strains is in agreement with literacy data (Phillips and Griffiths, 1986; Sutherland and Murdock, 1994; Crielly et al., 1994;). Our results did not confirm the seasonal occurrence (winter-summer) of mesophilic and psychrotrophic bacilli species as reported in literature (McKinnon and Pettipher, 1983; Sutherland and Murdock, 1994; Crielly et al., 1994). Biochemical and physiological characteristics of 66 isolates (Tab. I) are in agreement with literary data (Gordon et al., 1973; Parry et al., 1983; Priest et al., Ash et al., 1993). Strong proteolytic, amylolytic or lipolytic activities of the tested strains could influence the nutritive value of milk as a raw material. Taking into account the dominant representation of bacilli of morphological group I in raw and pasteurized milk (Sutherland and Murdock, 1994; Crielly et al., 1994) their review with basic phenotypic characteristics is shown in Tab. II. As follows from our results mesophilic species from so called "B. subtilis group" (96.9%) were isolated from agricultural primary production of milk most frequently. This is the reason why in addition to B. cereus it is also necessary to identify these species: seven tests shown in Tab III are recommended for their rapid differentiation. PMID- 8619277 TI - [The effect of copper on immune responses in guinea pigs with experimental ascariasis]. AB - A subchronic effect of copper on selected immunological parameters was studied in guinea pigs with experimental ascariosis (Ascaris suum). CuSO4 given orally for 28 days moderately increased T and B cells in the animal lymphoid organs. CH50 and AH50 complement levels remained unchanged during CuSO4 administration. A subsequent infection of subchronically intoxicated guinea pigs caused a repeated insignificant stimulation of B cell values while the percentage of T cells was not increased. On the contrary, the complement levels increased significantly in intoxicated infected animals, namely CH50 levels from day 5 to day 15 and AH50 levels from day 1 to day 7 post infection. No statistically significant differences were observed in comparison with infected non-intoxicated guinea pigs. The lean intensity of infection evoked by A. suum larvae migrating in the lungs of intoxicated animals was 29% lower than in control. PMID- 8619279 TI - [The effect of acidification on Salmonella enteritidis in a defined medium]. AB - Effects of five acids on three highly virulent strains of Salmonella enteritidis in a defined medium were investigated at various inoculum sizes. The following minimal inhibition concentrations (MIC) were found for the acids: citric and tartaric-0.2%, pH 4.0 and 3.8, respectively; acetic and propionic-0.1%, pH 4.5 and 4.6, respectively; hydrochloric-0.05%, pH 3.6. Bactericidal effect was evident in the organic acids at the concentration 0.4% within the pH range 3.3 3.9, and in the hydrochloric acid at the concentration 0.025%, pH 2.5 (Fig. 2, Tab. I). As to the intensity of inhibition at MIC for Salmonella suspensions with cell densities 7 to 8 logs per ml, the acids were arranged into the following ascending order: citric < tartaric < propionic < acetic < hydrochloric. The respective order for the cell densities 6 to 7 log per ml was: propionic < tartaric < hydrochloric < citric < acetic. The mean difference between the initial and the final count of salmonellae was -1.3 to -1.8 log and -2.5 to -4.6 log for the weakest and the strongest acids, respectively (Tab. II). Differences not only between the acids, but also between the strains, depending on inoculum sizes, were found at the nearest-to-MIC lower concentrations (subMIC). The weakest effects at subMIC were found in propionic (0.05%; pH 5.0) and citric (0.1%; pH 4.5) acids. Tartaric acid (0.1%; pH 4.5) did not stop the growth of salmonellae at cell densities 1.7 to 2.6 log per ml, but, unlike the former two, inhibited strain II (food isolate) and strain III (egg isolate) at cell densities 0.5 and 0.6 log per ml, respectively. The strains survived the acidification with acetic acid at cell densities 4.6 to 4.9 log per ml, but the densities decreased by 1.3 to 1.7 log. Hydrochloric acid (0.025%; pH 4.6) inhibited strains II and III already at 2.3 to 3.0 log per ml, whereas strain I (human carrier isolate) was relatively acid-fast even at 1.7 log per ml. Limiting factors in the acid inhibition of salmonellae include not only the type, concentration and dissociation rate of the acid, but also the amount of the exposed Salmonella cells. PMID- 8619280 TI - Evaluation of canine lymphocyte blastogenesis prior and after in vitro suppression by dog demodicosis serum using ethidium bromide fluorescence assay. AB - The mitogen induced blastogenic response of lymphocytes from normal dogs and dogs with generalized demodicosis (GD) was measured by the ethidium bromide (EB) fluorescence assay. Serum from GD dogs significantly suppressed the in vitro reactivity to Con A of peripheral blood lymphocytes (PBL) from normal dogs and GD dogs, however with a different percentage of suppression 40.6 and 81.2%, respectively. As a result, the degree of lymphocyte blastogenesis suppression in GD dogs did not parallel the immunosuppressive potency of their serum (Tab. IV). The data indicate that PBL obtained from GD dogs did not respond to Con A as well in the presence of serum from normal dogs as did PBL from normal dogs (Tab. IV). In one third of examined GD dogs a similar situation was described also by Hirsh et al. (1975). The basis for this cellular modified response is unknown. It does not appear that the age or the chronicity of the disease are related to this observation. Further studies are necessary to elucidate this relation. The GD dogs showed not only a significant depression of the lymphocyte response to Con A but also enhancement of the ability of unstimulated cells to proliferate was also observed (Tab. IV). Similar observation was reported by others (Barriga et al., 1992). The meaning of this is not clear at present. This finding is discussed in the light of proposed different effects of the parasite or the host's reactivity to the parasite on different subsets of lymphocytes. No significant difference of PBL responsiveness to Con A between healthy dogs with respect to the age (Tab. III) and the time of examination (compare results in Tabs. I and IV) was observed. Autologous serum showed a better responsiveness of normal canine lymphocytes to Con A than fetal calf serum (FCS). It is suggested that the use of FCS might lead to an erroneous judgement (Tab. I). Both lectins, Con A and PHA induced cell proliferation of healthy dogs in very similar amount (Tab. II). Our results indicated that EB fluorescence assay is a useful method for detection a respondence of canine lymphocyte blastogenesis. PMID- 8619281 TI - The equine enteric nervous system--neuron characterization and distribution in adults and juveniles. AB - A study of myenteric and submucosal plexuses was undertaken in the jejunum and ileum of horses and ponies in which no clinical or pathological evidence of intestinal abnormality was apparent. Complete transverse sections of the intestine, stained by a modified haematoxylin and eosin method, were examined using up to 20 sequential sections per animal. Information was gathered from adult, juvenile and fetal equidae. In adults, the longitudinal muscle layers were thinner than the circular muscle layers and the ileum had thicker layers compared to the jejunum. In adults, the submucosal plexus had more neurons per section than the myenteric plexus by mean ratios of 1:3 in the jejunum and 1:1.9 in the ileum. In juveniles, the ratios were respectively 1:1.8 and 1:1.5 and in the fetus 1:2.5 and 1:1.3. The three-dimensional distribution of neurons in both plexuses varied from animal to animal and no consistent pattern was observed. Groups of neurons contained between one and 42 cells per section examined and their length in a cranio-caudal direction varied from 10 to over 100 microns. There were few statistical differences observed between the cranial, middle and caudal portions of either the jejunum or the ileum when neuron groups or neuron numbers per section were examined in 10 adult animals. PMID- 8619282 TI - Detection of Clostridium chauvoei in formalin-fixed, paraffin-embedded tissues of sheep by the peroxidase-antiperoxidase (PAP) technique. AB - A peroxidase-antiperoxidase (PAP) technique was used to detect Clostridium chauvoei in tissue sections from sheep inoculated intramuscularly with a pure culture of this microorganism. Samples of various tissues were taken for bacteriology, histopathology and immunohistochemistry. A primary antiserum against C. chauvoei for use in the PAP technique was produced in rabbits. Formalin-fixed, paraffin-embedded sections of muscle samples were positively and specifically stained by the PAP technique. The results were consistent with those obtained by bacteriology, but the PAP test was simpler, quicker and less expensive than the bacteriological procedures. The use of the PAP technique would be appropriate for detecting clostridial infections without the constraints of conventional identification methods, especially where laboratory conditions for anaerobic procedures are not readily available. PMID- 8619283 TI - Production and characterization of rabbit anti-idiotypic antibodies directed against a murine monoclonal anti-B. abortus antibody. AB - The protective anti-beta abortus monoclonal antibody ISS/32 (Ab1) was used as an immunogen to induce anti-idiotypic antibodies (Ab2) in rabbits. The purpose was to produce and characterize anti-idiotypic antibodies that share conformational similarity with the corresponding bacterial epitope recognized by Ab1. The rabbit anti-IdAb so induced was isolated and affinity-purified. Its specificity for the paratope of Ab1 was determined by evaluating its ability to compete with B. abortus for binding to Ab1 in a competitive ELISA assay. The anti-idiotypic ISS/32 antibodies were able to compete with B. abortus for binding to Ab1 in a dose-dependent manner. Hence, the data indicated that the rabbit anti-Id ISS/32 antibodies reacted with or near the antigen-binding site of Ab1. PMID- 8619285 TI - Serum biochemical values and mineral element contents of tissues in yaks. AB - Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium. PMID- 8619284 TI - Preliminary findings of altered follicular activity in Holstein cows with coagulation factor XI deficiency. AB - Factor XI (F XI) deficiency is an autosomal recessive coagulopathy found in Holstein cattle. Affected animals have a 50% greater prevalence of repeat breeding. Therefore, several parameters describing ovarian function were studied. Daily blood sampling revealed that progesterone concentrations were slower to decline from a peak at day 16 (p < 0.01) to values less than 3 nmol/L in F XI deficient cows (5.14 +/- 0.69 days (mean +/- SD) versus 4.05 +/- 0.63 days in control animals), resulting in an oestrous cycle length of 24.7 +/- 2.1 days compared to 22.9 +/- 3.0 days, respectively. This was not due to an alteration in the availability of prostaglandin F2 alpha (PGF2 alpha) or oxytocin (OT) involved in luteolysis. No significant differences (p > 0.05) were seen between normal (n = 7) and F XI-deficient (n = 7) cows in the peak values or the area under the curve for the pulse in 13,14-dihydro-15-keto PGF2 alpha in response to OT challenge or in the parameters describing the pulse of ovarian OT secretion after PGF2 alpha injection (n = 7 for each) between days 12 and 14. Ovulatory follicular development was assessed by ultrasound monitoring and plasma 17 beta oestradiol values at 8-h intervals after a luteolytic injection of cloprostenol (n = 6 for each). Follicular diameter was smaller (p < 0.05) and accompanied by lower peak oestradiol values near the time of ovulation in F XI-deficient cows. The results suggest that the oestrous cycle in F XI-deficient cows is characterized by a slower process of luteolysis that may be associated with smaller follicular development. PMID- 8619286 TI - The estimated long-term impact of tsetse control on the size of the population of cattle in the Didessa Valley, western Ethiopia . AB - The long-term impact of tsetse control on cattle population size in the Didessa Valley, western Ethiopia, was analysed using an age-structured population model. A prior analytical assessment revealed that the risk of cattle dying in the tsetse-unprotected villages ranged from 4 to 9 times higher than in the tsetse protected village. Model results show that during a period of 10 years the cattle population in the tsetse-protected village of Meti is likely to increase from 167 to 583 animals, while that in the adjacent tsetse-unprotected village of Gale remains almost constant. Model simulations also predict that improving the survival rate of calves in the tsetse-unprotected villages of Taikiltu and Temoloko (which presently have calf mortality rates of up to 35% would bring a substantial increase in their cattle population. PMID- 8619287 TI - The prevalence and epidemiology of Anoplocephala perfoliata infection in Norway. AB - The caecum and the adjacent 30 cm lengths of ileum and large colon of 201 horses from two different regions of Norway (Ostlandet and Trondelag) were examined for the presence of Anoplocephala perfoliata. In all, 20% of the horses were infested with the cestode (27% in Ostlandet and 7% in Trondelag). The mean number of worms in infected horses from the two regions was 18 and 6, respectively. Information was obtained on the age, sex, breed, type of pasture and anthelmintic treatment after the grazing season for 183 of the 20 horses. The degree of infestation with A. perfoliata was not influenced by age, breed or sex. The odds ratio (confidence limits) for using permanent pastures that had been grazed by horses for at least the 5 last years in succession was 8.8 (3.2-24.4). There was a significantly higher prevalence of A. perfoliata infestation in Ostlandet (odds ration 3.1 (1.1 9.1). The relatively low prevalence of A. perfoliata infestation in Norwegian horses compared to the reported prevalences in other countries and the differences in the prevalence between Ostlandet and Trondelag are discussed. Gross pathological examination identified lesions such as mucosal thickening, hyperaemia and/or erosions. These lesions were found in all of the infected horses, compared with only 5% of the non-infected horses. Examination of faecal samples from 26 of the infected horses detected cestode eggs in only three cases. It was concluded that faecal examination is an inadequate method for the diagnosis of A. perfoliata infestation. PMID- 8619289 TI - Afferent activity in the superior spermatic nerve of lambs--the effects of application of rubber castration rings. AB - Electrophysiological techniques were used to record afferent activity in the superior spermatic nerves of young lambs under general anaesthesia. Receptive fields were identified in the pampiniform plexus and the deep tissue of the testis in response to mechanical stimulation. Application of a standard rubber castration ring to the scrotal neck evoked vigorous afferent activity, including some from formerly silent units with receptive fields particularly in the pampiniform plexus. Some of this multi-unit discharge adapted rapidly within 10 s of the application of the ring and was followed by a discharge pattern which decayed exponentially over 90 min. The rate of decay of this discharge showed more than one exponent (time constant) with inflections at approximately 90 s and 16 min. After the application of the castration ring, quantitatively controlled scrotal compression continued to excite receptors, though a declining frequency was recorded over the period of observation. It was concluded that: (a) rubber castration rings initiated afferent activity which persisted for periods in excess of 90 min, a time course which is similar to the behavioural and humoral changes in the conscious animal; (b) both standard and small rubber castration rings were ineffective in rapidly producing neuronal pressure block of the slowly conducting afferent fibres in the superior spermatic nerve; (c) intra-testicular injection of local anaesthetic rapidly blocked afferent fibres running in the superior spermatic nerve. PMID- 8619288 TI - The distribution of Parascaris equorum eggs in the soil profile of bare paddocks in some Norwegian studs. AB - Fifteen bare paddocks, which consisted of soil only and were located on 12 different studs, were examined for their content of Parascaris equorum eggs in the upper 15 cm of the soil profile. The paddocks were classified into three different groups according to the type of soil: clayey soil (A), morainic soil (B) and gravel or gravel-like sand (C). Soil profiles were collected down to a depth of 15 cm and were divided into three layers: 0-5 cm (D1), 5-10 cm (D2) and 10-15 (D3). The eggs in each layer were counted and identified as infective or noninfective eggs. The paddocks in group C, which had good drainage conditions, had significantly lower numbers of eggs in the whole profile and in D1 and D2 than the paddocks in groups A or B. Furthermore, there was a significantly higher proportion of the total egg count present in D3 in the group C paddocks. This may have been due to a higher degree of passive transportation of eggs down in the profile in the gravel or gravel-like sand. Even though there was a significantly higher proportion of infective eggs in the soil from the group C paddocks, the lower total numbers of eggs resulted in a lower total number of infective eggs in those paddocks. The study showed that the soil type was an important factor in determining the content of P. equorum eggs in the upper layer of the soil profile in bare paddocks and consequently for the potential infestation of horses with P. equorum. PMID- 8619290 TI - The effects of three models of airway disease on tidal breathing flow-volume loops of thoroughbred horses. AB - The effects of histamine and methacholine aerosols and of a fixed inspiratory resistance on tidal breathing flow-volume loops (TBFVL) were investigated using 18 unsedated, standing, healthy thoroughbred horses. The data were first analysed using traditional flow-volume loop indices and then reduced using standardized factor scoring coefficients obtained in a previous study in this laboratory using similar experimental techniques. On the basis of resting TBFVL analysis, the degree of pulmonary dysfunction caused by inhalation of histamine and methacholine aerosols with concentrations of 10 and 2 mg/ml, respectively, was similar. The fixed resistance also caused significant changes in the resting spirogram and TBFVL indices, suggesting that this model may prove valuable for further studies involving upper respiratory tract (URT) conditions. Administration of histamine and methacholine aerosols resulted in significant changes in all factor scores, although most of the observed changes were due to the effects of these aerosols on the respiratory rate. These findings re emphasize the importance of the effects of respiratory rate on pulmonary mechanics. Application of the resistance resulted in significant changes in factor score 3, the 'inspiratory' factor, which lends support to the validity of this model for URT conditions. The close agreement between the factor scores obtained under controlled conditions in this study and in a previous study in this laboratory confirms that the factor analysis used for both of these studies provides an adequate means of reducing TBFVL data obtained from thoroughbred horses. The large intra- and inter-individual variation observed both with the indices of TBFVL and with the factor scores limits the potential of these variables for detecting individual animals with obstructive airway disease. Re evaluation of these indices under the stress of exercise may reduce the variability observed in these data and may increase the magnitude of differences between different animals, providing a means of detecting individual animals with subclinical obstructive airway conditions. PMID- 8619291 TI - Small intestine and small colon neuropathy in equine dysautonomia (grass sickness). AB - The number of neurons in the coeliacomesenteric ganglia and the myenteric and submucosal plexuses of the jejunum, ileum and small colon, and the pathological changes induced in them, were studied in various types of equine dysautonomia. In all forms of dysautonomia, severe and extensive neuron loss and damage occurred in the ileum. In acute and subacute dysautonomia, jejunal neuron loss and damage were severe, but in chronic cases significantly less loss or damage occurred. The damage followed the same pattern in the small colon but it was always less obvious than in the jejunum. The distribution of the damage was uniform within a segment of the intestine. In fatal cases of dysautonomia, the clinical severity and duration of illness seems, in most instances, to be related to the amount of neuronal disruption occurring in the jejunum. Severe disruption results in acute/subacute dysautonomia, while milder damage leads to the chronic form. No case of dysautonomia was encountered in which enteric neuron loss and damage occurred without significant neuronal disruption also occurring in the coeliacomesenteric ganglia. Heal neuronal damage and loss are not invariably worse than that in the jejunum, and the possible reasons for this, together with the relationship between neuronal damage and possible causes of dysautonomia, are discussed. PMID- 8619293 TI - [The mechanism of anti-insulin effect of apoprotein B]. AB - The existence of common epitope in insulin molecule and apolipoprotein B one was shown by means of enzyme immunoassay. Similar epitopes were revealed in apoB containing lipoproteins (VLDL and LDL) by dot-immunobinding and immunoelectroblotting. Epitopes locate on the surface of lipoproteins and are not screened by lipids. The existence of common epitopes is the reason of competition between insulin and LDL. The experiments with isolated perfused rat hindquarter demonstrate that insulin increased the glucose uptake by muscle and LDL decreased it. Simultaneous perfusion with LDL and insulin leads to reduced insulin action due to completion for insulin receptors. After the recovery of S-S bounds apoB forms some proteins, containing common epitopes with insulin. The presence of such proteins was shown in lipoprotein-free serum. Apoprotein B and its derivates seem to be insulin antagonists with marked antiinsulin action. PMID- 8619292 TI - Pathogenicity of quail's inclusion body hepatitis virus (avian adenovirus-1) for Japanese quails and broiler chicks. AB - Quail (Coturnix coturnix japonica) and broiler (Gallus domesticus) chicks were inoculated experimentally with IBH virus (avian adenovirus-1) derived from quails to determine its pathogenicity. Quail chicks were inoculated by the intraperitoneal route at 3, 4, 5, 6 or 7 weeks of age. Lesions were encountered most frequently in the liver, kidneys and lungs. These included pale, swollen and mottled liver, swollen nephrotic kidneys, and congested and pneumonic lungs. The lesions were severe in birds inoculated at 5 weeks of age. Large basophilic intranuclear inclusion bodies were seen in hepatocytes and occasionally in the renal epithelium. The results showed that this isolate is pathogenic for quails above 3 weeks of age. Broiler chicks were inoculated at 4 weeks of age by the intraperitoneal route. The lesions produced in these chicks were similar to those of adenovirus-induced inclusion body hepatitis. Viral antigen was also demonstrated by dot-ELISA in suspension of liver tissue from both quail and broiler chicks. PMID- 8619294 TI - [Tocopherol level and lipid peroxidation in Wistar rat tissues during adaptation to cold]. AB - The content of lipid peroxidation (LPO) products and tocopherol was studied in the plasma, adrenals, brown fat, heart, thymus, liver tissue, microsomes and mitochondria of Wistar rats in various periods of cold acclimation (+4 degrees C during 2, 5, 24 hours and 5, 10, 15, and 49 days). Within the first 24 hours of cold exposure, there were periods of low levels of LPO products which, as their following elevation, did not coincide in time both for various tissues and for subcellular structures. The liver was used as an example to show that the activity of glutathione reductase and glutathione peroxidase enhanced. The rats' adaptation to moderately low temperatures occurred with the stabilized LPO processes and elevated body's tissue tocopherol levels: in the first hours of exposure by mobilizing its endogenous stores, on long-term exposure by enhancing its utilization from the diet. PMID- 8619295 TI - [Effect of free radicals on proliferation and metabolism of chondrocytes and on sensitivity of a cellular monolayer to proteases]. AB - Oxygen radicals (OR) were demonstrated to suppress the proliferation of chondrocytes and to decrease the synthesis of collagen by these cells. The concomitant action of OR and proteases (collagenase or trypsin) sharply increased the detachment of cells and destruction of the intercellular matrix at very low enzymatic concentrations (10-100-fold dilution of routine doses for cultural techniques) as compared with the control. PMID- 8619297 TI - [Effect of sodium and magnesium ions on development of a "calcium paradox" in cardiac muscle]. AB - The isolated rat heart was used to examine the impact of ion concentrations of sodium ions in the Ca-free medium on the development of cardiomyocytic damage after the former sodium ion concentration had been restored in the solution (the calcium paradox). Lowering a concentration of sodium ions in the Ca-free medium was found to enhance the development of the calcium paradox if the perfusion media contained no magnesium ions. Adding magnesium (0.125-1.2 mM) throughout the experiment reversed the action of the decreased sodium medium and contributed to the preservation of high concentrations of energy-rich compounds, to that of mitochondrial conjugation of oxidation and phosphorylation, to more than 4-fold cardiac release of myoglobin. An artificially elevated sodium ion concentration (as high as 170-220 mM) was effective in preventing cardiac energy impairments irrespective of the magnesium ion concentration. The positive action of the unchanged concentration of sodium ions during the calcium paradox was completely reversed by strophanthin. The findings are in a good agreement with the so-called sodium hypothesis of calcium paradox development. PMID- 8619298 TI - [Isolation of carnosine synthetase from animal and human muscles]. AB - Carsinosine synthetase was comparatively estimated in the homogenates of chicken sternal muscle and human skeletal and heart muscles. Partially purified enzyme was obtained from chicken muscle, showing its activity of 60 nmoles carnosine/mg per min. The levels of carnosine synthesized was measured by three different methods: column chromatography on Dowen 50 x 8 resin, and thin-layer chromatography on Fixion or Silufol plates. The Silufol chromatography method was found to be sensitive and useful for measurement of the enzyme in abnormal tissues. Carnosine synthetase activity in chicken sternal muscle was compared with that published in literature. Enzymatic activities in the human skeletal and heart muscle were found to be different: carnosine synthetase from skeletal muscle was 10 times higher than that from the myocardium. PMID- 8619296 TI - [Dysfunctions of the cellular enzyme apparatus of organs and tissues in surgical diseases]. PMID- 8619299 TI - [Lipid composition and phospholipase A2 activity in rat placental cell membranes in hypoxic states]. AB - When in the hypoxic pressure chamber and under high-altitude conditions, rats showed changes in the lipid profile of plasma membranes and in the activity of placental phospholipase A2. More profound changes occurred in the placental membranes of the rats staying under hypoxic conditions during placentation. Placental plasma membranes in the animals exposed to hypoxia in the second gestational half were more stable, their lipid composition was less altered. PMID- 8619301 TI - [On vitamin B1 metabolism in avitaminosis and its correction with thiamine and taurine]. AB - The levels of phosphate esters and the activities of thiamine biotransformation enzymes in the blood and tissues of albino rats were studied during oxythiamine induced B1 deficiency and after metabolic correction with thiamine and taurine. Among thiamine phosphates, the most informative indicators of thiamine deficiency were shown to be triphosphate esters and free thiamine diphosphate. The biosynthetic enzymes thiamine kinase and thiamine diphosphate kinase played a decisive role in maintaining the initial rate and in recovering the physiologically active forms of vitamin B1. The activation of hydrolytic enzymes of thiamine phosphate esters occurred by producing abundant free thiamine diphosphate and thiamine triphosphate. Within the first hours, taurine favoured the acceleration of phosphoester biosynthesis and, accumulating in the tissues, inhibited vitamin phosphorylation reactions. PMID- 8619302 TI - [Approaches to assessing dynamics of permeability and duration of thiaminase I action, administered parenterally to animals]. AB - Two approaches and direct isotopic assay were employed to examine the time course of permeability and duration of action of tissue thiaminase I injected parenterally to albino mice. The injected enzyme was found to be detectable in renal and hepatic tissues 3 hours later. The action of the enzyme dermally injected in a dose of 1.2 IU/kg in the same tissues lasted 4 days. Experimental findings led to the conclusion that the thiamine degradation enzyme may be used in experimental biology in order to estimate the duration of its action and the time course of gross molecular permeability in the tissue of test animals. PMID- 8619300 TI - [Change in lipid peroxidation depending on hormonal reactions during lengthy electric stimulation of the rabbit hypothalamic dorsomedial nucleus]. AB - A model of chronic emotional stress (ES) induced by electrostimulation of the hypothalamic dorsomedial nucleus in 69 rabbits was used to examine the relationship of blood hormonal changes and lipid peroxidation (LPO) activity in blood and myocardial cells. The elevated concentrations of stress hormones at the initial stages of chronic ES (the first 2 series) caused LPO with high activities of antioxidative enzymes (AOEs). The subsequent stages of chronic ES (from 60 to 120 days) were associated with the decreased role of major stress hormones (adrenocorticotropic hormone, cortisol, catecholamines) and the increased significance of parathyroid hormone and active renin (angiotensin I). A significant direct correlation was found between the blood level of these hormones and the rate of LPO in the myocardium and blood. At the same time the activity of AOEs progressively decreased and all rabbits exhibited myocardial cell necrotic foci. PMID- 8619303 TI - [Participation of a diazepam-binding inhibitor in the interaction between NAD and GABA-benzodiazepine receptor complex in synaptic membranes]. AB - Diazepam-binding inhibitor isolated from synaptic membranes exerts a pronounced inhibitory effect on the specific benzodiazepine-receptor binding of 3H flunitrazepam and simultaneously leads to an increase of synaptosomal uptake of 14C-GAMA. At the same time the inhibitor also depresses the specific binding of 14C-NAD by synaptic membranes, but displaying a greater effect. In both cases the inhibition was competent. Whether the isolated neuropeptide may act as an intermediary in the interaction with the reception system of NAD with GABA benzodiazepine-receptor complex. PMID- 8619304 TI - [Intensification of alcohol motivation in CBA mice by means of transferring syngeneic splenocytes or blood serum]. AB - CBA mice were subjected to forced alcoholization. After ethanol withdrawal, adoptive transfer of splenocytes, sera, and macrophages was made in intact recipient mice. Splenocytic transfer enhanced alcohol consumption when they had an option to have water or 15% ethanol solution during 7 days. Serum transfer increased ethanol consumption during 24 hours. Macrophageal transfer failed to affect its consumption. PMID- 8619305 TI - [Effect of lipophilic derivatives of a divinyl ether-maleic anhydride copolymer on immunocompetent cells]. AB - A series of lipophilic derivatives of the copolymer divinyl ether with maleic anhydride were synthesized and tested for their effects on immunocompetent cells. Increases in the length of a hydrophobic moiety and its quantity in the polymer chain of the agent were demonstrated to substantially modify the factors of animals' humoral and cell-mediated immunity. This phenomena is well associated with the examined superficial qualities of solutions of the agents and their mycelium forming capacity. PMID- 8619306 TI - [Reactions between protease inhibitors in blood plasma and collagen]. AB - The interaction of three macroglobulins and three serpines, as well as inter alpha-trypsin inhibitor (ITI) with alpha 1- and alpha 2-chains of collagen I which were immobilized on nitrocellulose. All seven proteinase inhibitors were shown to have a certain affinity for collagen. The binding of alpha 2 macroglobulin and gestation-associated protein A with collagen chains is largely determined by the conformational state of these macrophages. alpha 2-Antiplasmin, proteinase alpha 1-inhibitor, antithrombin III and ITI with collagen yield complexes that are resistant to urea, sodium dodecylsulfate, and Trilon B. PMID- 8619307 TI - [Platelet glycosaminoglycans in paraproteinemic hemoblastoses]. AB - The content and composition of platelet glycosaminoglycans (GAG) were studied in 24 patients with paraproteinemic hemoblastoses (22 patients with myelomic disease and 2 with Waldenstrom's disease). According to the GAG levels, the patients were divided into 2 groups: 1) 6 patients with its higher levels (181 +/- 8 micrograms of uronic acids per 100 mg of acetone-dried cells) and 2) 18 patients with its close-to-normal levels (129 +/- 4 micrograms per 100 mg of the cells). Marked manifestations of hemorrhagic diathesis were found in 5 patients in Group 1 and in 1 in Group 2. Chondroitin sulfate was demonstrated to prevalent in the platelets of patients with paraproteinemic hemoblastoses, as in those of healthy persons. There was GAG electrophoretic profile depletion due to the reduced number of minor components of non- and low-sulfated GAG in 14 patients. It is suggested that the changes detected in the content and composition of GAG may contribute to platelet dysfunction in these diseases. PMID- 8619308 TI - [Criteria of supply of vitamins B1, B2, and B6 in children with insulin-dependent diabetes mellitus]. AB - By mathematically analysing the curves of urinary excretion of vitamins, their plasma and erythrocytic concentrations or of TDP-effect, by constructing and mathematically interpreting the variation curves of distribution of a given plasma concentration of riboflavin and pyridoxal phosphate for 10-14-old-year children suffering from insulin-dependent diabetes mellitus after supplementation of vitamin, as a criterion of normal requirement for vitamin B2, the authors are prone to recommend the concentration of riboflavin over 10 micrograms/ml in plasma and over 96 micrograms/ml in erythrocytes, the hourly excretion of more than 27 micrograms. It has been ascertained that the criteria for the optimal body's requirements for vitamins in diabetes mellitus children do not differ from those in healthy age-matched children. Thus, the value of TDP-effect is less than 1.25, the concentration of pyridoxal phosphate is over 8.4 micrograms/ml plasma, the excretion values of thiamine and 4-pyridoxic acid are 13.5 and 64.0 micrograms/h, respectively. PMID- 8619309 TI - [Characteristics of extracellular superoxide dismutase]. PMID- 8619310 TI - Ribosomal frameshifting in yeast viruses. AB - Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting. PMID- 8619312 TI - Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources. AB - Expression of the peroxisome-deficient (Per-) phenotype by per mutants Hansenula of polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6ts and per14-11ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D-alanine (for both mutants) or methylamine (for per14-11ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno)cytochemical analyses indicated that during growth of cells on methanol in the presence of either D-alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol. PMID- 8619313 TI - GSG1, a yeast gene required for sporulation. AB - We have identified a gene, GSG1 (general sporulation gene 1), required for sporulation in Saccharomyces cerevisiae. Diploids homozygous for a disruption of GSG1 fail to sporulate. The gene has an open reading frame of 2094 bp, encoding a polypeptide with an expected size of 77 kDa. GSG1 is expressed mitotically in both a and alpha haploids, and both mitotically and meiotically in diploids. The message level of GSG1 increases approximately two-fold after 4-6 h of sporulation. gsg1 mutants enter pre-meiotic DNA synthesis later than wild-type diploids. Mutant diploids are not rescued by spo13. These results suggest that GSG1 has a role late in meiosis following DNA replication. PMID- 8619311 TI - UVS112--A gene involved in excision repair of yeast. AB - In this study we show that the previously described uvs112 (uvs12) mutation blocks one of the steps of the excision repair pathway. The properties of this mutation permit the assignment of the UVS112 gene to the RAD3 epistasis group. It was established that the uvs112 mutation caused a 2.5-fold reduction in the number of recombinants produced by conversion and also significantly increased the frequency of mitotic crossing-over in interplasmid recombination. Tetrad analysis placed the UVS112 gene on the left arm of chromosome IX, approximately 20 cM from HIS5. The analysis of mitotic recombination revealed that UVS112 lies between HIS6 and HIS5, and is an allele of the RAD25 gene. PMID- 8619314 TI - A double flow cytometric tag allows tracking of the dynamics of cell cycle progression of newborn Saccharomyces cerevisiae cells during balanced exponential growth. AB - Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell. In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations. PMID- 8619315 TI - Gcs1, a gene encoding gamma-glutamylcysteine synthetase in the fission yeast Schizosaccharomyces pombe. AB - By complementation of a mutant resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) we have identified the gcs1 gene, encoding a putative gamma glutamylcysteine synthetase. The gene is possibly interrupted by two introns and has 49% identical and 80% similar amino acids compared with the homologous protein from rat. In comparison with the Saccharomyces cerevisiae homologue it possesses 41% identical and 74% similar amino acids. PMID- 8619316 TI - The sequence of 24.3 kb from chromosome X reveals five complete open reading frames, all of which correspond to new genes, and a tandem insertion of a Ty1 transposon. AB - We have determined the complete nucleotide sequence of a 24.3 kb segment from chromosome X carried by the cosmid pEJ103. The sequence encodes five complete open reading frames (ORFs), none of which correspond to previously described genes; however, four of these ORFs display interesting similarities with sequences present in the databanks. The sequence also contains a tandem insertion of a Ty1 element. An investigation of the Ty1 polymorphism in other strains has revealed that the original insertion occurred within an ORF. Finally, the structure of the Ty1 repeat suggests a mechanism by which it may have been generated. PMID- 8619318 TI - A 43.5 kb segment of yeast chromosome XIV, which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames. AB - A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA(phe) and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unidentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected. PMID- 8619317 TI - The sequence of an 11.1 kb fragment on the left arm of Saccharomyces cerevisiae chromosome VII reveals six open reading frames including NSP49, KEM1 and four putative new genes. AB - We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins. PMID- 8619319 TI - The RAD58 (XRS4) gene: map position on the right arm of chromosome XIII. PMID- 8619320 TI - Current awareness on yeast. PMID- 8619321 TI - The NO synthase inhibitor L-NNA depresses neurohypophysial vasopressin but not its precursor amidating enzymes in salt-loaded rats. AB - The arginine vasopressin (AVP) producing hypothalamo-neurohypophysial system also has high activities of NO-synthase. Vasopressin production and secretion is drastically upregulated during salt intake and the NO-producing enzyme may be involved. We have studied the influence of the NO-synthase inhibitor NG-nitro-L arginine (L-NNA) on neurohypophysial and hypothalamic AVP and its amidating enzymes in salt-loaded and control rats as well as on stimulated AVP release in vitro in such rats. Rats were given 2% NaCl solution as the only fluid for 4 days and then returned to tap water. The specific amount of AVP (microgram (mg protein)-1) and the activities of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), components of its amidating processing enzyme in the supraoptic (SON) and paraventricular nuclear (PVN) regions, did not change during the salt-loading or the following recovery period. In contrast, the AVP and PHM and PAL in the neurohypophysis fell drastically during the salt loading. After that, PHM and PAL increased even more rapidly than AVP, the latter reaching control levels in about 10 days. Salt loading did not change the protein content of the neurohypophysis. When salt loading was performed after administration of L-NNA, the neurohypophysial AVP at the end of the salt loading and 3 days later was lower than in rats not receiving L-NNA, whereas PHM and PAL were not affected. Fractional AVP release from isolated neurohypophyses of salt loaded rats treated with L-NNA and stimulated with K+ was similar to that found in non-treated rats. It is suggested that L-NNA may affect translation or precursor processing of AVP. PMID- 8619322 TI - Attenuation of low pH-, but not capsaicin- or PGI2-evoked CGRP-release by endothelium removal using saponin. AB - The aim of the present study was to evaluate the role of the endothelium in low pH-, capsaicin-, and prostacyclin (PGI2)-evoked release of calcitonin gene related peptide (CGRP)-like immunoreactivity (-LI) from C-fibre afferents in the isolated, perfused guinea-pig heart. CGRP-LI release, and formation of the stable PGI2-metabolite 6-keto-PGF1 alpha, in response to moderate acidosis (pH 7, 6, but not 5) were significantly reduced after removal of endothelium using saponin (50 micrograms mL-1) perfusion. In contrast, the release of CGRP-LI evoked by capsaicin (10(-7) M) or PGI2 (10(-5) M) remained unchanged after removal of the endothelium. Saponin treatment did not influence the vasodilator action of CGRP, whereas the vasodilation evoked by substance P (SP) was abolished. It is concluded that CGRP release evoked by low pH, but not that evoked by capsaicin or exogenous PGI2, is partly endothelium dependent. Our data suggest that endothelially produced PGI2 is involved in low pH-evoked release of CGRP from capsaicin sensitive nerves in the heart. PMID- 8619323 TI - Regional differences in endothelium-dependent relaxation in the rat: contribution of nitric oxide and nitric oxide-independent mechanisms. AB - Relaxant effects of acetylcholine (ACh), histamine, calcitonin gene-related peptide (CGRP) and the calcium ionophore A23187 were examined in rat femoral (phi approximately 0.2 mm), mesenteric (0.2 mm), intrarenal (0.2 mm) and hepatic (0.3 mm) arteries, and aorta (2 mm). Acetylcholine elicited an endothelium-dependent relaxation in all arteries. Histamine induced an endothelium-dependent relaxation in aorta, and mesenteric and intrarenal arteries, whereas a partly endothelium dependent and mainly endothelium-independent relaxation was observed in hepatic and femoral arteries, respectively. In hepatic, mesenteric and intrarenal arteries, CGRP induced an endothelium-independent relaxation, whereas either small or no relaxation was obtained in aorta and femoral arteries respectively. A23187 induced an endothelium-dependent relaxation in the aorta and hepatic artery, whereas A23187 had no relaxant effect in femoral, mesenteric and intrarenal arteries. N omega-nitro-L-arginine (L-NOARG, 0.3 mM) reduced the maximum ACh-induced relaxation (in the presence of 10 microM indomethacin) by 66% in the aorta, and abolished the relaxation in femoral and intrarenal arteries. A marked L-NOARG/indomethacin-resistant relaxation was obtained in mesenteric and hepatic arteries. Levcromakalim induced a concentration-dependent and almost complete relaxation in all arteries. When contracted by a 60 mM K+ solution, all arteries responded to ACh with a relaxation that was abolished by L-NOARG. These results demonstrate marked regional differences with regard to the vascular effects of ACh, histamine, CGRP and A23187. Whereas nitric oxide appears to mediate endothelium-dependent relaxation regardless of the vascular region, an L NOARG/indomethacin-resistant relaxation, presumably mediated by an endothelium deprived hyperpolarizing factor, was observed only in mesenteric and hepatic arteries, and aorta. PMID- 8619324 TI - The high frequency component of heart rate variability reflects cardiac parasympathetic modulation rather than parasympathetic 'tone'. AB - This study was designed to evaluate the effect of modulating cardiac parasympathetic input on the high frequency component of heart rate variability. We stimulated the right vagus nerve with three different stimulation patterns in anaesthetized, vagotomized and spinal anaesthetized dogs. We kept the mean stimulation frequency constant; controlled the amplitude of modulation with programmed stimulation patterns, and analysed the resulting heart rate variability by power spectral analysis. Constant frequency vagal stimulation increased the cardiac interval, but did not change heart rate variability markedly. There was a slight increase, from 11 +/- 2 to 27 +/- 11 ms2, in the high frequency component. However, when the instantaneous stimulation frequency oscillated between 4 and 17 Hz during 5 s period, we could produce a marked heart rate variation, with 91 +/- 9% of the variation corresponding to the frequency of the modulation (0.20 Hz). The high frequency component was 12932 +/- 7701 ms2. With an increased magnitude of modulation, i.e. the difference between minimum and maximum instantaneous frequency, the high frequency component increased to 32711 +/- 17943 ms2. Thus, the high frequency component of heart rate variability reflects the magnitude of fluctuation in the cardiac parasympathetic input rather than parasympathetic 'tone'. PMID- 8619325 TI - Septal lesions block sympathetic activation following frontal cortex stimulation in the rat. AB - The rates of oxygen consumption, colonic and interscapular brown adipose tissue temperature, and discharge of sympathetic nerves innervating the same tissue were recorded before and after orbital frontal cortex stimulation in three groups of rats. These groups consisted of animals with electrolytic lesions of the nucleus medialis septi, with lesions of nucleus lateralis septi or with sham lesions. The values of all the variables considered were similar during the pre-stimulation time in the three groups of rats. There was an increase in all the parameters in sham-lesioned animals after electrical stimulation, while no changes were found in the two groups of injured rats. The results showed that the frontal cortex is involved in the control of thermogenesis through the activation of the sympathetic nervous system. Medial and lateral septal nuclei, in spite of their neurochemical and pharmacological differences, share a common role in the pathway from the frontal cortex to the hypothalamic area and the amygdala, which in turn drive the sympathetic nervous system. PMID- 8619326 TI - Phosphate excretion during 24 h of hypoxia in conscious rats. AB - The objective of this study was to investigate renal phosphate excretion during 24 h of hypoxia in conscious rats fed by total parenteral nutrition. Wistar rats weighing 190 g were exposed to hypoxia (inspired oxygen fraction = 0.10) or normoxia (inspired oxygen fraction = 0.21) for 24 h in a normobaric chamber. Renal clearance and hormonal studies were performed. The results showed a greater fractional excretion of phosphate (5.37 +/- 0.07%, P < 0.05) and hypophosphataemia (7.40 +/- 0.12 mg dL-1, P < 0.01) in hypoxic rats (n = 10) than in normoxic rats (n = 13; 3.50 +/- 0.37% and 8.02 +/- 0.16 mg dL-1, respectively). In addition, during hypoxia there was a significant decrease in the excretion of urinary adenosine 3',5'-cyclic monophosphate per glomerular filtrate (2.97 +/- 1.27 nmol dL-1, P < 0.005), a parameter of the renal action of parathyroid hormone, and a stable level of serum parathyroid hormone (10.2 +/- 2.6 ng mL-1) (cf. normoxia: 8.57 +/- 0.70 nmol dL-1 and 8.0 +/- 1.7 ng mL-1, respectively). However, creatinine clearance and the renal adenosine triphosphate level, both of which affect adenosine 3',5'-cyclic monophosphate excretion, were not different between the two groups. These data suggest that exposure of conscious rats to 24 h of hypoxia causes renal hyporesponsiveness to physiological levels of parathyroid hormone, which is manifested as a decrease in adenosine 3',5'-cyclic monophosphate excretion. Phosphaturia is not a direct net effect of hypoxia and secondary hypocapnia on renal phosphate transport, which is known to be regulated by parathyroid hormone through adenosine 3',5'-cyclic monophosphate. PMID- 8619327 TI - Effects of exercise-training on hypoxia and angiotensin II-induced pulmonary vasoconstrictions. AB - The present study was undertaken to determine whether exercise-training for 6 weeks would inhibit pulmonary vasoconstriction induced by hypoxia in isolated, blood-perfused rat lungs. Hypoxic pulmonary vasoconstriction (HPV) and angiotensin II (AII)-induced pulmonary vasoconstriction were significantly less in the exercise-trained (ET) group than in the control (cont) group with all challenges. Normoxic pulmonary arterial and capillary pressures in the ET group were significantly lower than those in the cont group and capillary pressor response to hypoxia was less in the ET group than in the cont group. In conclusion, it appears that HPV and AII-induced vasoconstrictions can be reduced by exercise-training, because it would seem that exercise-training repeated responses to increased shear-stress resulting from elevated blood flow in pulmonary vessels. PMID- 8619329 TI - Theophylline increases coronary vascular tone in humans: evidence for a role of endogenous adenosine in flow regulation. AB - To elucidate the role of adenosine in coronary vasoregulation, we studied the effects of adenosine antagonism (by theophylline) on coronary blood flow at different levels of adenosine formation (stimulated by hypoxia and exercise). Six healthy subjects were studied. Coronary sinus (CS) blood flow (thermodilution) and cardiac oxygen extraction [(A-CS)O2D] were determined while breathing room air at rest, and 12% oxygen, both at rest and during light exercise, on two occasions. One of the experiments was performed during infusion of theophylline. The basal CS flow was 118 (67-168) mL min-1 (mean and 95% confidence interval), and the (A-CS)O2D was 125 (111-142) mL L-1. Inhalation of 12% O2 decreased the arterial haemoglobin oxygen saturation to 83 (80-86)% at rest and to 77 (73-81)% during exercise. CS flow increased to 167 (93-214) and 261 (179-343) mL min-1, respectively, and (A-CS)O2D decreased to 102 (85-119) and 94 (77-111) mL L-1, respectively. Theophylline, at a dose lacking effects on myocardial work, markedly attenuated the coronary flow response to exogenous adenosine, and decreased CS flow to 89 (58-119), 120 (79-161) and 190 (162-218) mL min-1 at normoxic rest, hypoxic rest and hypoxic exercise, respectively. The overall decrease amounted to 23% (P < 0.05). The calculated coronary vascular conductance also decreased by 23% (P < 0.05) and (A-CS)O2D increased by 15% (P < 0.001). In conclusion, the data support the hypothesis that endogenous adenosine is involved in regulation of human coronary tone. PMID- 8619328 TI - Flecainide blocks the stimulatory effect of veratridine on slowly adapting pulmonary stretch receptors in anaesthetized rabbits without changing lung mechanics. AB - We examined the responses of slowly adapting pulmonary stretch receptors (PSRs), total lung resistance (RL) and dynamic lung compliance (Cdyn) to administered veratridine before and after pretreatment with atropine or flecainide in anaesthetized, artificially ventilated rabbits with bilateral vagotomy. Administration of veratridine (10 and 30 micrograms kg-1) caused vigorous stimulation of PSRs, resulting in a tonic discharge of receptors during both inflation and deflation, but did not significantly alter either RL or Cdyn. The veratridine-induced PSR stimulation became more prominent, as the dose of this alkaloid was increased. Pretreatment with atropine (1 or 2 mg kg-1) had no significant effect on the excitatory response of PSRs to veratridine. The veratridine-induced PSR stimulation was inhibited by treatment with flecainide (1, 2 and 3 mg kg-1), a sodium channel blocker, and this inhibition was dose dependent. These results suggest that activation of PSRs following veratridine administration probably related to the increased influx of sodium ions into the receptive terminals but does not depend upon bronchoconstriction. PMID- 8619330 TI - Lymphocyte redistribution in connection with physical activity in the rat. AB - Previous studies have demonstrated numerous immunobiological changes in connection with exercise. A decrease in peripheral blood mononuclear white cells (PBMC) 2 h after intense exercise has been shown. This lymphocytopenia in humans after exercise is thought to be of great importance regarding the morbidity to viral infection. We constructed an animal experimental set-up, previously published, to investigate the exercise-induced lymphocyte redistribution. The experimental set-up allowed us to draw blood from catheters implanted in the right carotid artery in rats. PBMC were isolated and labelled with In111 and reinfused before the exercise run on a treadmill to exhaustion. The runner and control rats were killed and dissection performed 1 h after the exercise. Tissue samples were weighed and measured in a gamma counter. Furthermore, blind microscopic examinations of selected tissues were performed to study a hypothesized accumulation of blood mononuclear cells in relation to muscle fibre lesions. We found that the total number of PBMC in the running rats was decreased (P = 0.018) and granulocytes increased, 1 h after the exercise (P = 0.028). Similar findings in humans in connection with physical activity have been observed. The percentage of total injected counts per minute per gram tissue (% c.p.m. g-1) showed significantly lower values in the liver and kidney from runners than from controls (P = 0.032 and P = 0.028). These findings might be the result of a visceral hypoflow in connection with exercise. Furthermore, a tendency to decreased % c.p.m. g-1 in the lungs were seen in the exercised rats (P = 0.083) indicating a possible redistribution from the lungs during the run. Light microscopy demonstrated an accumulation of PBMC around muscle fibre lesions, but there was no significant difference between runners and controls. Furthermore, no significant difference in % c.p.m. g-1 was found between working muscle groups in runner and control rats. In conclusion, the demonstration of the redistribution of PBMC from the liver and kidney in the exercised rats and the absence of any significant accumulation of PBMC in working muscles or other organs, do not explain the lymphocytopenia demonstrated here. PMID- 8619331 TI - Bioassay of gastrin using the isolated vascularly perfused rat stomach. A new, simplified and sensitive method. AB - Radioimmunoassays are sensitive and specific methods for measurement of the concentrations of regulatory peptides. However, aspects of physiological, pathophysiological and pharmacological research require knowledge about the biological activity which does not necessarily vary concomitantly with immunological activity. The present work describes a simplified bioassay for gastrin based on the gastric histamine releasing properties of this peptide, using an isolated vascularly perfused rat stomach preparation with a crystalline perfusate and a specific radioimmunoassay for histamine. The establishment of a dose-response curve is described, as well as the utilization of the bioassay on sera from patients with hypergastrinaemia. The method is sensitive for gastrin in the low (4 pmol L-1) picomolar range. PMID- 8619332 TI - Endogenous NPY acting on the Y1 receptor accounts for the long-lasting part of the sympathetic contraction in guinea-pig vena cava: evidence using SR 120107A. PMID- 8619333 TI - Cardiovascular effects of the microsphere suspending agent, Tween 80, in pigs. PMID- 8619334 TI - Effects of swimming training on the lysosomal enzyme system in brown adipose tissue of rats: an analogy between swimming exercise and cold acclimation. PMID- 8619335 TI - Positive or negative symptoms--which are more appropriate as diagnostic criteria for schizophrenia? AB - For over a decade there has been a consensus that the diagnosis of schizophrenia should rest upon the presence of positive symptoms. Recently it has been suggested to give negative symptoms, which have played a prominent role in research, more diagnostic importance again. This study investigated the usefulness of that suggestion. In a sample of 489 inpatients covering the whole range of psychiatric diagnoses, the frequencies and prevalences of positive and negative symptoms were determined. Analyses of variance were calculated to assess the diagnostic validity of the different classes of symptoms. The study demonstrates that positive symptoms are of much higher diagnostic value than negative symptoms. A change of diagnostic procedures giving more importance to negative symptoms is discouraged. PMID- 8619336 TI - A prospective three-year follow-up study of borderline personality disorder inpatients. AB - Prospective long-term follow-up studies on patients with borderline personality disorder (BPD) have been uncommon. Clinical data suggest that their treatment is highly demanding and that short-term results are sometimes limited. In this study, changes in symptoms and social management were monitored during a hospitalization period of 91 days (mean, range 21-296 days) and during a 3-year follow-up period in 62 patients admitted during 1989 to an open ward specializing in the psychotherapeutic treatment of BPD. The patients were thoroughly evaluated, using various rating scales, at the beginning and at the end of the index admission and after the follow-up period. Forty-two patients (70%) participated in the follow-up evaluation. Most patients suffered from overt anxiety and depressive symptoms at the beginning of hospitalization, and these declined significantly during hospital treatment. At the end of the follow-up period, depressive and anxiety symptoms were at the same level as on discharge, as assessed by the Beck Depression Inventory and Hamilton Depression Rating Scale. Although treatment response was otherwise maintained, the patients often showed suicidal behavior. During the follow-up period the sample clearly differentiated in two groups: those continually fit for work (33%) and those chronically incapable of working (46%). PMID- 8619337 TI - Adolescent suicide, depression and family dysfunction. AB - This study investigated associations between adolescents' perceptions of their family dynamics (McMaster Family Assessment Device, FAD) and depression, suicide thoughts and attempts. High school students (mean age 15.2 years) completed self report questionnaires including the Beck Depression Inventory (BDI), the FAD, questions about suicidal thoughts, plans and attempts, deliberate self harm, and selected life experiences. Univariate analysis showed that family dysfunction measured on the FAD is associated with thinking and planning suicide, deliberate self harm, suicide attempts, as well as severe depression (BDI > or = 22). Despite this, stepwise regression indicated that family dysfunction influences suicide behaviors indirectly through other variables such as depression. The FAD is recommended as a useful addition to questionnaires seeking to identify vulnerability to both depression and adolescent attempted suicide in early detection studies. PMID- 8619338 TI - Attempted suicide predicts suicide risk in mood disorders. AB - Suicide risk was studied in a sample of 346 mood disorder inpatients, 92 of whom were admitted after a current suicide attempt. The overall suicide mortality after a mean observation period of 6 years was 8%. The potential of attempted suicide to predict suicide risk in hospitalized patients with mood disorders was studied by survival analysis after subgrouping on the basis of whether a current suicide attempt had occurred or not. The suicide risk the first year after attempting suicide was 12% (11/92), compared with 2% (4/254) in the mood disorder subgroup with no current suicide attempt. The long-range suicide risk after a current suicide attempt in depression was 15% (14/92) as compared with 5% (13/254) among those without a current suicide attempt. It is concluded that a current suicide attempt in mood disorder inpatients predicts suicide risk particularly within the first year and should be taken very seriously. PMID- 8619340 TI - The prescription of psychotropic drugs in primary health care. AB - We followed the prescription of psychotropic drugs by primary health care physicians over a 3-year period. The material consisted of 1000 randomly selected adult primary health care patients. At least one psychotropic drug was prescribed to one third (n = 307) of the sample during the follow-up period. The most commonly used drug category was that of benzodiazepines, which was prescribed to 24% of the sample. An antidepressive was prescribed to 8%, a neuroleptic to 2% and "other psychotropic drugs" to 10% of the sample. The most important predictor for prescribing psychotropic drugs was recognition of mental problems at the initial survey, but age and marital status also had an association with the prescription. No gender difference was found after controlling for mental problems. Psychotropic medication was also prescribed to 13% of the patients who had no sign of mental disorder. PMID- 8619339 TI - Social phobia: the clinical efficacy and tolerability of the monoamine oxidase -A and serotonin uptake inhibitor brofaromine. A double-blind placebo-controlled study. AB - Seventy-seven patients with a primary diagnosis of social phobia (DSM-III-R) were randomized to treatment with the reversible and selective monoamine oxidase type A inhibitor brofaromine (n = 37) or placebo (n = 40) for 12 weeks in a double blind trial. A fixed dose of 150 mg/day or a matching placebo was given after a 2 week dose titration phase. Patients with additional diagnoses of simple phobia, generalized anxiety disorder, dysthymia or major depressive disorder currently in remission were accepted. Patients with other Axis I mental disorders were excluded. In the brofaromine group, 78% of the patients scored much or very much improved on the Clinical Global Impression scale compared with 23% in the placebo group. The anxiety and avoidance scores on the Liebowitz Social Anxiety Scale (LSAS) were significantly reduced in favor of brofaromine. The clinical effects were not significantly correlated with the plasma concentration of brofaromine. After 12 weeks the brofaromine group scored significantly lower than the placebo group on a core depression part of the Montgomery-Asberg Depression Rating Scale. After 12 weeks of treatment the brofaromine group had significantly higher total scores on the LSAS than an age- and gender-matched group of healthy controls. The brofaromine group improved further during 9-month follow-up treatment period, whereas 60% of the placebo responders who continued long-term treatment relapsed. The most common side effects in the brofaromine group were sleep disturbances, dry mouth and nausea. PMID- 8619341 TI - Clinical evidence for genomic imprinting in bipolar I disorder. AB - The phenotypic indicators of the genomic imprinting model were applied to clinical psychopathology data on 100 bipolar (BP) I probands and their families. The paternal transmission was associated with a significantly younger age of onset of the BP illness in probands and a higher rate of affective disorders in first- and second-degree relatives. The effect of the sex of the transmitting parent on age of onset in probands decreased but remained significant when controlling for the effect of the probands' age at investigation. Probands' sex had no significant influence on their age of onset. The severity of the BP illness in probands in terms of number of illness episodes and annual frequency was not influenced by the sex of the transmitting parent. PMID- 8619342 TI - Family environment predictors of outcome in schizophrenic patients in Spain: a nine-month follow-up study. AB - This study investigates the effects of perceived family environment on clinical outcome among patients in Spain who suffer from schizophrenia. Forty-five consecutively admitted DSM-III-R schizophrenic patients were assessed monthly with the Brief Psychiatric Rating Scale during a 9-month period. Patients and parents rated the family environment through the Family Environment Scale (FES). FES factors were considered separately for each family member, since parents' and patient's perceptions of the family environment were weakly correlated. Stepwise multiple regression analysis showed that patients' perceptions of family control and intellectual-cultural orientation predicted rehospitalization. Patients' and mothers' ratings of family control and fathers' scores of conflict and moral religious emphasis predicted psychotic relapse. However, fathers' scores of family cohesion predicted higher negative symptoms. Prior admissions, age of onset and use of depot medication tended to predict outcome in conjunction with the family variables. PMID- 8619343 TI - A nationwide representative sample of treatment-seeking alcoholics: a study of psychiatric comorbidity. AB - In order to elucidate the psychiatric comorbidity of patients in alcohol and other substance use disorder treatment we examined a representative sample of such patients in Iceland (249 men and 102 women). Over 70% of pure alcoholics and over 90% of polysubstance users had comorbid diagnoses, a prevalence higher than in the Epidemiological Catchment Area study in the United States, but similar to clinical studies from North America. The most prevalent disorders were: affective (33%), anxiety (65%), antisocial personality disorder (28%) and psychosexual dysfunction (20%). Pure alcoholics and polysubstance users in studies on psychiatric comorbidity should be separated. Anxiety and affective disorders influence treatment seeking. Findings concerning the impact of psychiatric comorbidity on course should be comparable between North America and Europe. PMID- 8619344 TI - Is treatment in groups a useful alternative for psychiatry in low-income countries? An evaluation of a psychiatric outpatient unit in Nicaragua. AB - Centro de Atencion Psicosocial in Leon, Nicaragua is a psychiatric outpatient unit that has developed a group-oriented model of working, in which 80% of all visits are in groups: first-admission groups, insight-oriented group psychotherapy, psycho-educative, family groups and relatives groups. The aim of the present study was to analyze patient characteristics and make a preliminary study of improvement, compliance and patient satisfaction in a 1-year perspective. One hundred consecutive visits were assessed, 44 of them first admissions. They were assessed according to all axes of DSM-III-R plus the Structural Clinical Interview for DSM-III Disorders. A 1-year follow up was conducted on 39 of 41 selected patients within the major diagnostic groups. One of 4 patients had a psychotic disorder where schizophrenia dominated. Among nonpsychotics major depression, anxiety and adjustment disorders were most frequent. Personality disorders were common (80%) among nonpsychotic patients, paranoid, obsessive-compulsive, passive-aggressive and masochistic personality disorders dominating. The illiteracy rate was 10%, but 50% had high school or university background. Severity of mental disorders and functional level did not differ between educational levels. There was a strong male dominance in all diagnostic, socioeconomic and educational level strata and few old patients. Improvement in functional level was clinically and statistically significant in all groups, and more than two thirds were very satisfied with the group treatment offered. PMID- 8619347 TI - Xenetix--a milestone in diagnostic imaging. PMID- 8619346 TI - Manic depressive (bipolar) disorder. PMID- 8619345 TI - Mental well-being in people with non-insulin-dependent diabetes. AB - We assessed the prevalence of minor mental disorder and depression in elderly patients with non-insulin-dependent (Type 2) diabetes mellitus as compared with control subjects and evaluated the associates of impaired mental well-being in diabetic patients. The study consists of a community-based group of patients (n = 82) with a 10 years' known duration of disease and nondiabetic control subjects (n = 115). In addition to clinical and laboratory examinations, self-rating questionnaires assessing minor mental disorder (General Health Questionnaire, GHQ) and depression (Zung Self-rating Depression scale) were completed by the patients and control subjects. The mean scores of GHQ and Zung scores tended to be higher in diabetic than in control subjects, but the frequency of case subjects was not different between the diabetic (GHQ: 40%; Zung: 11%) and nondiabetic groups (GHQ: 36%; Zung: 7%). These findings were explained by more severe symptoms in diabetic case subjects as compared to nondiabetic case subjects. From the various parameters studied, the presence of symptomatic neuropathy was most strongly associated with depression and minor mental disorder in diabetic subjects. The results suggest that the impact of Type 2 diabetes per se on minor mental disorder or depression in elderly subjects is not overwhelming. However, a subgroup of diabetic patients seems to have markedly impaired mental well-being, and the treatment of its underlying factors may improve overall treatment compliance. PMID- 8619348 TI - Toxicologic profile of iobitridol, a new nonionic low-osmolality contrast medium. AB - PURPOSE: The toxicologic profile of iobitridol, a new nonionic low-osomolality contrast medium, was evaluated in compliance with the current regulatory requirements in Europe, the USA and Canada. MATERIAL AND METHODS: The toxicity of iobitridol was tested following acute or repeated i.v. administration in several different species (mouse, rat, dog); single oral administration in the mouse and intracisternal injection in the rat. Furthermore, teratogenicity and mutagenicity were evaluated in the rat and rabbit. Local perivenous toxicity was assessed in the rabbit. RESULTS: The acute toxicity of iobitridol in the mouse is equivalent to that of iohexol, a reference product tested under the same conditions. Chronic administration (daily injections i.v. injection over 4 weeks) in the rat and dog did not demonstrate any particular toxicity for iobitridol. It should be noted that, unlike iohexol, iobitridol did not provoke any vacuolization of the renal tubular cells in the rat following repeated injections. Furthermore, this contrast agent did not show any teratogenic or mutagenic potential. The typical local inflammatory signs observed following perivenous injection in the rabbit were low in intensity and reversible. CONCLUSION: The toxicologic profile of iobitridol appears to be favorable and does not show any particular risk for clinical use under the usual indications of water soluble iodinated contrast agents. PMID- 8619349 TI - Pharmacokinetic profile of iobitridol. AB - Iobitridol (Xenetix) is a new triiodinated monomer, nonionic, low-osmolality contrast agent. Animal pharmacokinetic studies (rats, rabbits, dogs) and in vitro studies show that it is a marker of extracellular fluid, i.e. it is distributed in the interstitial space, it does not penetrate into cells, it does not cross the healthy blood-brain barrier (BBB), it does not bind to proteins, and it is eliminated by glomerular filtration without secretion or reabsorption. These characteristics determine its use as an urography and angiography contrast agent as well as the precautions necessary to be taken, which are classical for contrast agents of this classification, related to administration in pathophysiologic populations (patients with renal failure, pregnant women or nursing mothers, neonates, etc. ) PMID- 8619350 TI - Intravascular contrast agents. Current problems and future solutions-- a review. PMID- 8619351 TI - Pharmacologic profile of iobitridol, a nonionic iodinated contrast medium. AB - The present article combines and summarizes the preclinic studies carried out in vitro and in vivo to determine the pharmacologic and biochemical profile of iobitridol, a new nonionic iodinated low-osmolality contrast medium (CM). The effects of this product on the main hemodynamic, bronchopulmonary, neurologic, renal, blood chemistry and electrophysiologic parameters and RBC morphology were studied in detail in comparison with CM in the same chemical category or with reference substances of the same osmolality. The in vivo studies were performed under conditions resembling clinical use. Iobitridol showed an excellent pharmacologic and biochemical profile, which was identical or superior to that of other products in its category. PMID- 8619352 TI - Time course of biodistribution and changes in density following administration of iobitridol in rabbits. A comparative study vs iohexol. AB - PURPOSE: A new nonionic low-osmolality contrast medium, iobitridol (Xenetix 350) was compared with iohexol (Omnipaque) after i.v. injection in anesthetized rabbits to assess efficacy in CT examinations and biodistribution. MATERIAL AND METHODS: The densities in test tubes and the pharmacogenetics and biodistribution of iobitridol 350 and iohexol were compared in rabbits. CT of the brain, liver, the abdominal aorta and the kidneys was performed before and after injection of the contrast media. RESULTS: In aqueous medium, iobitridol absorbed roentgen rays in a manner exactly identical to that of iohexol. Within 15 min following injection of iohexol and iobitridol at a dose of 300 mg I/kg, both contrast agents resulted in aortic enhancement which decreased with time. An increased attenuation of the liver also occurred, decreasing with time. There was no significant enhancement in the brain but enhancement was found in the renal pelvocalyceal cavities 10 min postinjection. No significant difference was found between the 2 contrast agents under the study conditions. CONCLUSION: As could be expected from its behavior as a tracer of extracellular fluid, iobitridol resulted in significant changes in the signal, corresponding to its vascular, hepatic and renal pharmacokinetics in rabbits. PMID- 8619353 TI - Efficacy and safety of iobitridol versus iohexol for contrast-enhanced CT of the head. AB - PURPOSE: The efficacy and adverse reactions of iobitridol versus iohexol in contrast-enhanced CT (CECT) of the head were investigated. MATERIAL AND METHODS: This double-blind randomized parallel study compared the clinical and biological safety of iobitridol and iohexol (350 mgI/ml), administered intravenously (1 ml/kg) to 276 patients undergoing CECT of the head. Vital signs were assessed just before after injection, and 24 hours after. A biologic examination was performed before and after injection. Both groups were comparable at inclusion. RESULTS AND CONCLUSION: The within-group variation for vital signs after injection and 24 hours after was not clinically relevant and no significant difference was evident between treatment groups. The incidence of adverse events was similar in both groups (11.0% for iobitridol and 7.1 % for iohexol), consisting most often of a sensation of warmth. Biologic parameters remained stable and did not differ significantly between groups. The quality of imaging was rated good or excellent in 70% of cases and a very high diagnostic discrimination was achieved (98%). Iobitridol compared favorably with iohexol for head CECT. PMID- 8619354 TI - Iobitridol 300 compared to iopromide 300--a double-blind randomized phase-III study of clinical tolerance in total body CT. AB - PURPOSE, MATERIAL AND METHODS: The clinical safety of iobitriodol 300 mg I/ml and iopromide 300 mg I/ml were compared in a randomized double blind phase-III study conducted on 60 patients undergoing abdominal CT for a variety of indications. Each examination was rated as diagnostic or nondiagnostic and the image quality was noted. Nature, onset, intensity as well as outcome of each adverse reaction were recorded. RESULTS: There was no significant difference in imaging quality and side effects between the contrast media. In this study, both iobitridol and iopromide provided excellent image quality and a low rate of side effects. CONCLUSION: Iobitridol is a safe and effective nonionic contrast agent for contrast-enhanced body CT. PMID- 8619355 TI - Clinical tolerance and diagnostic efficacy of iobitridol in contrast-enhanced CT in children. AB - PURPOSE: The purpose of this double-blind, randomized parallel trial was to evaluate and compare the clinical safety and the diagnostic efficacy of the new nonionic triiodinated contrast agent iobitridol (300 mg I/ml) with those of iohexol (300 mg I/ml) in CT examinations in children. MATERIAL AND METHODS: Eighty children of either sex were included in the study. Of those, 40 patients received iobitridol, 40 iohexol. Mean volume injected i.v. was 1.8 ml/kg b.w. Adverse reactions occurring during 24 hours after the injection of the contrast agent were recorded. RESULTS: Image quality was judged good or excellent in all study examinations and all were diagnostically informative. There was no significant difference in clinical safety between the 2 groups and only adverse reactions of mild or moderate intensity were reported in both groups. CONCLUSION: Iobitridol appears to be a safe, well tolerated and effective contrast agent, when used for brain and body CT in children. PMID- 8619356 TI - Clinical safety and efficacy of iobitridol in urography. AB - PURPOSE: Evaluation of imaging quality and safety of iobitridol 300 compared to iohexol 300 in urography. MATERIALS AND METHODS: 180 patients were included in an urography multicenter study (3 centers, 60 patients in each). RESULTS: There was no significant difference in either the imaging quality or the clinical safety between the 2 contrast media groups. CONCLUSION: Iobitridol is a safe and efficient contrast agent in urography. PMID- 8619357 TI - Clinical tolerance and diagnostic efficacy of iobitridol 300 in lower limb angiography. AB - PURPOSE: The diagnostic efficacy and the clinical tolerance of iobitridol 300 was evaluated in 68 patients receiving intraarterial injection in 2 monocentric comparative randomized double-blind trials with iopromide 300 (30 patients) and iopamidol 300 (40 patients) as reference. RESULTS; There is no significant difference in quality of opacification or in diagnostic efficacy between the products in each trial. No major adverse reactions occurred after the injection of iobitridol 300. Excluding a heat sensation, 5 of the patients who received iobitridol had minor reactions consisting of nausea with vomiting and pain during the injection. Five patients in the reference group also experienced adverse events. CONCLUSION: Iobitridol is well tolerated after intraarterial administration and it has the same diagnostic quality as iopromide and iopamidol. PMID- 8619358 TI - Safety and efficacy of the new iodinated nonionic low-osmolality contrast medium Iobitridol (Xenetix) in coronary and ventricular angiography. AB - PURPOSE: The aim of this phase-III clinical trial was prospectively to evaluate the clinical safety and diagnostic efficacy as well as the effects on laboratory and electrocardiographic parameters of new iodinated, nonionic, low-osmolality contrast medium, iobitridol(Xenetix 350) during coronary angiography in adults in comparison to an iodinated, nonionic, low-osmolality reference product, iohexol (Omnipaque). MATERIALS AND METHODS: This 2-center, comparative, randomized, double-blind trial involved 90 patients, 46 receiving iobitridol and 44 iohexol. Clinical safety was evaluated by recording the adverse events observed during investigation and by the patient's assessments. Electrocardiographic effects on laboratory parameters were evaluated as well as diagnostic efficacy. RESULTS: The age, sex, presence of risk factors and clinical picture ( unstable angina, postinfarction) were not significantly different between the 2 treatment groups. The incidence of significant coronary lesions (stenosis 50%) was also not significantly different between the 2 treatment groups. Clinical safety was good in both groups. Four patients experienced an adverse event, 2 in each group. In the iobitridol group, one patient had an episode of hypertension, followed by hypertension and prolonged chest pain was observed and one patient had sinus tachycardia. In the iohexel group, chest pain was observed in one patient and nausea in another. ECG safety was good in both groups. Transient excitability disorders were observed in 10 and 6 patients, respectively. Moderate conduction abnormalities were noted in one patient in each group and repolarization abnormalities in 10 and 8 cases, respectively. Safety in laboratory parameters was good with no significant changes in either. Diagnostic efficacy was good to excellent in all patients. CONCLUSION: Diagnostic efficacy and safety of iobitridol 350 in terms of effects on clinical, laboratory and electrocardiographic parameters were comparable to those of the nonionic reference product. PMID- 8619359 TI - Stabilization of the hydrophilic sphere of iobitridol, a new nonionic iodinated contrast agent. AB - The chemotoxicity of iodinated contrast agents is essentially related to the spatial accessibilty of lipophilic and polarizable iodine atoms. This accessibility was examined in iobitridol by HPLC and 1H- and 13C-NMR spectroscopy,taking into account both the static distribution of the hydrophilic groups around the triiodinated benzene ring and in particular the dynamic modulation of this distribution. The aim of this latter parameter is to prevent distortion of the hydrophilic sphere of nonionic agents when faced with a hydrophobic environment. Iobitridol is characterized by 2 tertiary carbamoyl substituents whose high rotation barriers (deltaG*353=27.6 kcal for E/Z-rotation and deltaG*345=17.3 kcal for syn/anti-rotation) stabilize the hydrophilic sphere. The 3rd dihydroxylated anilide substituent does not undergo SMILES rearrangement and provides iobitridol with its even hydrophilic distribution. Iobitridol presents remarkable chemical solubility (>140% m/v) and stability. The iobitridol molecule was specifically designed with the aim of stabilizing the hydrophilic sphere around the triiodinated benzene ring, therefore permanently masking access to the iodine atoms. This new concept represents a further step forward towards the synthesis of new iodinated contrast agents which should be totally inert vis a-vis biological membranes and proteins. PMID- 8619360 TI - Safety and efficacy of Xenetix, a new iodinated contrast agent, in pediatric angiocardiography. AB - PURPOSE: The aim of this work was to study the safety and efficacy of iobitridol 350 in pediatric angiocardiography compared to iopamidol 370. MATERIAL AND METHODS: 40 children participated in each test group. Each examination was rated as diagnostic or nondiagnostic and the image quality was noted. Nature,onset,intensity as well as outcome of each adverse reaction was reported. RESULTS: There was no significant difference in either image quality or clinical safety between the 2 groups. CONCLUSION: Iobitridol is a safe and effective contrast agent for pediatric angiocardiography. PMID- 8619361 TI - Clinical experience with iobitridol 250-300 in digital subtraction angiography. Double-blind randomized studies vs. iopromide and iohexol. AB - Iobitridol was clinically tested in DSA against iopromide (i.v. DSA) or iohexol (i.a. DSA) in 139 patients. Seven patients participated in the i.v. DSA trial and 60 in the i.a. DSA study. Each examination was rated as diagnostic or not and the image quality was noted. Nature, onset, intensity and outcome of each adverse reaction were recorded. There was no significant difference in imaging quality and side effect occurrence between the 2 groups. We conclude that iobitridol is a safe and efficient contrast medium, which can be recommended for DSA examinations. PMID- 8619362 TI - Iobitridol 250 vs iohexol 240 in phlebography of th lower limbs. A double-blind clinical trial. AB - Iobitridol 250 (Xenetix 250) was compared with iohexol 240 (Omnipaque 240) in phlebography of the lower limbs by means of a multicenter, randomized double blind trial in 70 patients. No significant difference was demonstrated between the 2 groups in terms of diagnostic efficacy, image quality or clinical safety. Only minor reactions were recorded in the 2 groups, essentially consisting of mild or moderate heat sensations. The only insufficiently informative examinations were not due to the contrast agent. Iobitridol 250 therefore appears to be a well-tolerated contrast medium, suitable for use in phlebography of the lower limbs. PMID- 8619363 TI - Bone fragility in transgenic mice expressing a mutated gene for type I procollagen (COL1A1) parallels the age-dependent phenotype of human osteogenesis imperfecta. AB - An inbred strain of transgenic mice that expressed a mutated gene for type I procollagen and that developed spontaneous fractures was used to study the effects of age on the phenotype of fragile bones. The mutated gene has been shown to cause depletion of type I collagen in the transgenic mice because it generated shortened pro alpha 1(I) chains that bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the endogenous mouse COL1A1 gene. For this study, femurs from transgenic mice ranging in age from 0.5-24 months were examined. The results demonstrated that the level of expression of the transgene was independent of age. Femurs from the transgenic mice were more fragile than controls at 0.5 and 1.5 months, they were biomechanically normal at 6 months, and then they were more fragile at 24 months. The normal biomechanical properties of the bones from the transgenic mice at 6 months were accompanied by periosteal thickening of the bones together with an increase in the collagen content that was not associated with a proportional increase in mineral content. The results indicated that the effects of age, mechanical stress, and hormonal action produced a biological compensation for the mutated gene by either increasing collagen synthesis of bone, decreasing collagen degradation, or both. The biological compensation was apparently lost by 24 months when the outer diameters of the femurs were again less than in controls, the cortical thickness was about the same as in controls, and both the collagen and mineral contents were less than controls. The results demonstrated that bone fragility in the transgenic mice paralleled the age-dependent phenotype of human osteogenesis imperfecta. Therefore the transgenic mice appeared to be useful models for osteogenesis imperfecta. They also may be useful models for some forms of osteoporosis. PMID- 8619364 TI - Effects of recombinant insulin-like growth factor-I and growth hormone on bone turnover in elderly women. AB - We evaluated the effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone (GH) on calciotropic hormones and bone turnover markers in 16 healthy elderly women 71.9 +/- 1.3 years of age (mean +/- SEM). Subjects consumed a fixed diet providing 1000 mg of calcium and 0.9 g/kg of protein for 10 days before starting baseline 24-h urine and blood collections. Specimens were collected for 6 consecutive days before initiating subcutaneous injections of GH (25 micrograms/kg/day, n = 5) and IGF-I at 60 micrograms/kg b.i.d. (high-dose, n = 5) or at 15 micrograms/kg b.i.d. (low-dose, n = 6) for 28 days. Resorption markers included urine hydroxyproline (OHP), total pyridinolines (PYD), and N telopeptide; formation markers include osteocalcin, skeletal alkaline phosphatase (sALP), and type I procollagen carboxy-terminal extension peptide (CICP). For each subject, baseline daily turnover markers varied substantially (DV = 16-22%). With GH and high-dose IGF-I, resorption and formation markers increased progressively to maximum levels at day 21. For GH, the increase in day 21 PYD, N telopeptide, osteocalcin, and CICP was 143, 111, 53, and 81%, respectively (p < 0.96-0.02). For high-dose IGF-I, these increases were 108, 81, 77, and 111% (p < 0.02-0.002). However, with low-dose IGF-I no change was observed in resorption markers while osteocalcin and CICP increased progressively (day 21, % increases = 88 +/- 51, 36 +/- 14). Twenty-four hour urine collections during the last days of baseline and of study drug were taken as six 4 h aliquots. When deoxyPYD was measured on these samples in the low-dose IGF-I group, a significant increase was observed only on the 0800-1200 h aliquot. Serum phosphorus concentrations increased with GH (21.2 +/- 3.3%) and high-dose IGF-I (8.8 +/- 3.6%) by day 21 but actually decreased by day 28 (-9.7 +/- 2.7, p < 0.02) with low-dose IGF-I. Urinary phosphorus excretion decreased with high-dose IGF-I only. Twenty-four hour calcium excretion increased with all treatments. These results indicate that both GH and high-dose IGF-I activate remodeling osteons. By contrast, low-dose IGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption and may therefore provide a useful means to increase bone mass. The results also suggest some of the GH action on renal phosphorus handling represents a direct action of GH on the nephron which does not involve the intermediacy of IGF-I. Finally, even under controlled conditions bone turnover markers exhibit substantial daily variation so that a very large treatment effect will be required for these markers to have clinical utility. PMID- 8619365 TI - The effects of recombinant human insulin-like growth factor (rhIGF)-1 and rhIGF 1/IGF binding protein-3 administration on rat osteopenia induced by ovariectomy with concomitant bilateral sciatic neurectomy. AB - We compared the effect of administration of recombinant human insulin-like growth factor-1 (rhIGF-1) alone or the rhIGF-1/IGF binding protein-3 (IGFBP-3) complex on osteopenia in rats. Female Sprague-Dawley rats (8 months old) underwent combined ovariectomy and bilateral sciatic neurectomy (OVX-NX) or sham operation only. After 2 months, the OVX-NX animals were injected subcutaneously with rhIGF 1 alone or with rhIGF-1A IGFBP-3 equimolar complex for 4 weeks. The IGF-1 contents and dose were 0.3 mg/kg of body weight (BW) three times/week, 3 mg/kg of BW once/week, or 3 mg/kg of BW three times /week. At the end of the experiment, the 4th and 5th lumbar vertebrae (L4, L5) and the proximal tibiae were removed after tetracycline labeling, and histomorphometrical analyses were performed on undecalcified sections using Villanueva's staining. The cancellous bone volume at L5 significantly increased by thickening of the trabecular width in rats treated with the complex. However, the increase in the values at the proximal tibia was not significant. The bone formation rates (BFR/BS) in the lumbar vertebrae of rats treated with the complex three times a week at doses of 0.3 mg/kg of BW and 3 mg/kg of BW were both significantly increased but the parameter increase was less marked with the dose of 3 mg/kg of BW once/week. The BFR/BS did not increase significantly in animals treated with IGF-1 alone. These findings clearly demonstrated that the effect of systemically administered rhIGF-1 on bone formation was markedly potentiated when combined with IGFBP-3 in estrogen deficiency combined with reduced activity. The action of IGF-1 was less potent on the bone in paralyzed limbs. The action of rhIGF-1/IGFBP-3 on trabecular bone appeared to depend not only on the dose but also on the frequency of administration and the parts of the skeleton in rats. PMID- 8619366 TI - Short-term treatment with growth hormone stimulates osteoblastic and osteoclastic activity in osteopenic postmenopausal women: a dose response study. AB - To investigate the potential use of growth hormone (GH) in Activate-Depress-Free Repeat treatment of postmenopausal osteoporosis, we measured changes in serum levels of biochemical markers of bone turnover, insulin-like growth factor-I (IGF I), calciotropic hormones, and bone mineral density in 40 postmenopausal women with osteopenia (ages 52-73 years) in response to 7 days of treatment with either placebo or GH (0.05, 0.10, or 0.20 IU/kg/day) administered subcutaneously in the evening. GH treatment increased serum osteocalcin (p < 0.01) and C-terminal type I procollagen propeptide (p < 0.01) and also serum levels of type-I collagen telopeptide (p < 0.001), fasting urinary hydroxyproline/creatinine (p < 0.05), pyridinoline/creatinine (p < 0.05), and deoxypyridinoline/creatinine (p < 0.01) in a dose-dependent fashion. Even the lowest dose of GH tested induced a significant increase in these parameters; however, the effects were transient lasting only 1-2 weeks. In the highest dose group, however, a somewhat prolonged effect (30 days) on serum osteocalcin was observed. Furthermore, GH increased serum levels of IGF-I, insulin, and tri-iodothyronin. No effect on serum 1,25 dihydroxyvitamin D3 or parathyroid hormone could be demonstrated. Adverse effects were mainly related to fluid retention. They were clearly dose-dependent and rapidly reversible. In conclusion, short-term GH treatment stimulates bone formation and bone resorption in postmenopausal women with osteopenia. PMID- 8619368 TI - Circadian variation in urinary excretion of bone collagen cross-links. AB - Bone resorption can be evaluated by measuring the urinary excretion of collagen type I cross-linked N telopeptides (NTx). Since it is difficult to obtain (and verify) 24 h urine collections from patients, untimed spot urines are more practical. Such measurements, however, need correction for urine dilution and potentially may vary with collection time since a circadian rhythm in bone metabolism has been reported. This study examined cross-link excretion in urine voids serially collected during a 24 h period from subjects living their normal daily routine (as opposed to a controlled hospital setting). This mimics the situation for walk-in patients visiting a clinician and providing a spot urine. A total of 35 dentists (20 males, 15 females) collected all urine voids separately over a 24 h period. Urines were analyzed for creatinine and NTx. The effects of time of day on the excretion rates of these metabolites (in nmol/h) and on the cross-link:creatinine ratio were assessed. A circadian rhythm was evident in the excretion rate of creatinine with a peak in the late afternoon (18% higher than the 24 h mean, p = 0.0004). The NTx excretion rate peaked in the morning (9% higher than the 24 h mean) but this latter rhythm was not statistically significant (p = 0.31). The NTx:creatinine ratio fell during the day from a high (122% of the 24 h mean) in the early morning to a low in the early evening. This rhythm in the NTx:creatinine ratio in untimed spot urines was statistically significant (p < 0.0001). In conclusion, the NTx:creatinine ratio in spot urines from adult outpatient subjects showed a significant circadian rhythm. Variations in creatinine excretion were the primary cause. Time of day should, therefore, be taken into account when comparing test results of spot urines with normal ranges or with other samples from the same subject. PMID- 8619367 TI - Parathyroid hormone-related protein is an autocrine modulator of rabbit proximal tubule cell growth. AB - Parathyroid hormone-related protein (PTHrP), a likely mediator for humoral hypercalcemia of malignancy, is also synthesized in various normal tissues. In the kidney, PTHrP, mainly detected in proximal and distal tubules, has been shown to stimulate proliferation of rat mesangial cells in culture. Experiments were carried out to investigate the possible mitogenic effect of PTHrP in cultures of rabbit proximal tubule cells (PTC). Immunocytochemical analysis, using antihuman (h)PTHrP antibodies to (38-64) and (107-111) epitopes in the PTHrP molecule, showed strong cytoplasmic staining in PTC and proximal tubule-like LLC-PK1 cells. PTC secreted immunoreactive PTHrP (54.8 +/- 7.0 fmol/10(6) cells) into the culture medium. Human PTHrP(1-141) stimulated proliferation in subconfluent cultures of these cells dose-dependently. This effect was similar to that induced by [Tyr34]hPTHrP(1-34) amide (hPTHrP[1-34]), hPTHrP(1-86), and bovine (b)PTH(1 34), while hPTHrP(38-64) amide, hPTHrP9107-111) amide, and hPTHrP(107-139) amide were ineffective. Addition of anti-hPTHrP neutralizing antibodies to (1-34), (38 64), and (107-111) epitopes of PTHrP decreased PTC growth. The mitogenic effect of these agonists was abolished in confluent PTC. In contrast, [Nle8,18, Tyr34]bPTH(3-34)amide (bPTH[3-34]) increased DNA synthesis in either subconfluent or confluent PTC. In LLC-PK1 cells, which also secreted PTHrP and are devoid of PTH receptors, none of these peptides affected proliferation. Forskolin (10 microM) or H-8 (2 microM), a protein kinase A inhibitor, did not affect basal or hPTHrP(1-34)-stimulated DNA synthesis, respectively, in subconfluent PTC. On the other hand, 10 nM staurosporine and 100 nM calphostin C, protein kinase C (PKC) inhibitors, blunted the effects of hPTHrP(1-34) or bPTH(3-34) on DNA synthesis in these cells. These studies suggest that PTHrP may function as an autocrine factor in the regulation of proximal tubule cell growth by a PKC-mediated mechanism. PMID- 8619369 TI - Regulation of gap junction intercellular communication by pH in MC3T3-E1 osteoblastic cells. AB - Gap junction intercellular communication (GJIC) may be related to coordinating the function of osteoblasts during bone mineralization. Since an alkaline pH supports mineral deposition while an acidic pH promotes mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pHo) Functional GJIC was assessed by fluorescent dye transfer after microinjection, and connexin protein abundance was examined by immunoprecipitation and immunoblotting in MC3T3-E1 cells, a model of osteoblast-like cells. The percent of cells coupled by GJIC was found to be 40.7% (24 of 59 injected cells) at pH 6.9, 72.2% (26 of 36) at pH 7.2, and 92.8% (26 of 28) at pH 7.6. A decrease in GJIC was detectable by 30-60 minutes of exposure to a pHo of 6.9. Decreased gap junction communication was also found in cells after 3, 8, and 24 h of incubation in a bicarbonate-CO2 system at an ambient pH of 6.9. Connexin protein abundance experiments showed that at after exposure to a pH of 6.9 for 2.75 h, the specific band(s) at 41-43 kD were fainter compared with these same band(s) at pH 7.2 and 7.6. There was no significant difference in band densities at pH 7.2 and 7.6. Determination of intracellular pH (pHi) showed that it was similar to pHo after 2.75 h of incubation at each ambient pH. When pHi was clamped at 6.9 or 7.2, there was a time-dependent decrease in the gap junction coupling frequency at a pHi of 6.9 when pHo was 7.2. Steady-state mRNA levels were decreased at pHo 6.9 but were unchanged at either pHo 7.2 or 7.6. Our conclusions are that (1) longer incubations ( > or = 2.75 h) at low pHo decrease GJIC which in part may be due to a decrease in connexin protein abundance perhaps as a result of a decrease in connexin steady-state mRNA expression; (2) GJIC inhibition or augmentation found at low and high pHo, respectively, suggests that gating of the GJ channel by pH may also occur; (3) pho-induced alterations in GJIC in the MC3T3-E1 osteoblastic model are related to concomitant changes in pHi. PMID- 8619370 TI - Predictors of hip fractures in elderly men. AB - To assess the influence on the risk of hip fractures in men of medical conditions associated with secondary osteoporosis or with an increased likelihood of falling, we conducted a population-based nested case-control study among the 232 Rochester, Minnesota, men with an initial hip fracture due to moderate trauma in 1965-1989 and an equal number of age-matched control men from the general population. Information on selected medical and surgical conditions and certain behavioral risk factors prior to fracture (or comparable index date for controls) was obtained from inpatient and outpatient medical records in the community that averaged over 36 years in duration. After adjusting for age, obesity, and inactivity, disorders linked with secondary osteoporosis were associated with a 2 fold increase in the risk of hip fracture in men (odds ratio [OR] 2.3; 95% confidence interval [CI] 1.3-4.3), while conditions linked with an increased risk of falling were associated with almost a 7-fold increase in risk (OR 6.9; 95% CI 3.3-14.8). These factors together appeared to account for about 72% of the hip fractures in men. Increased attention must be paid to these conditions which, in aggregate, are very common in elderly men and lead to a substantial increase in the risk of hip fracture with its devastating sequelae of death, disability and cost. PMID- 8619371 TI - Measurement of femoral geometry in type I and type II osteoporosis: differences in hip axis length consistent with heterogeneity in the pathogenesis of osteoporotic fractures. AB - The epidemiologic patterns of vertebral and femoral fractures are sufficiently different to suggest that they represent distinct disorders (type I versus type II osteoporosis) although osteopenia is common in both. To determine whether differences in femoral geometry, one of the main determinants of bone quality, might contribute to the heterogeneity in osteoporotic fractures, we obtained dual energy X-ray absorptiometry scans on 210 women age 60 or older, including 105 type I fracture cases, 30 type II patients, and 75 controls. Hip axis length, measured on the scan printout, was significantly increased (p < 0.01) in hip fracture patients compared with women with postmenopausal osteoporosis, whereas femoral neck density (BMD) was equal in both groups. The best discrimination between both fracture types was obtained by a logistic regression model based on age and axis length. Adding BMD to the model did not improve the discriminative power (p = 0.67). These data provide further evidence that geometric characteristics may be implicated in hip fracture risk. Furthermore, these findings suggest that an increase in hip axis length may predispose osteopenic subjects to a femoral localization of fragility fractures, consistent with the postulated heterogeneity in the pathogenesis of osteoporotic fractures. PMID- 8619372 TI - Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi. AB - The organic matrix of renal calculi has long been considered to influence the crystal growth that occurs in these pathological mineral deposits. Recent advances in characterizing individual organic moieties from mineralized tissues in general and the combined use of antibodies raised against these molecules with different immunocytochemical approaches have allowed their precise distribution to be visualized in a variety of normal and pathological mineralized tissues. The present ultrastructural study reports on the epithelial expression and extracellular localization of several noncollagenous proteins in rat and human kidney stones using high-resolution colloidal-gold immunocytochemistry. To this end, we have examined in an ethylene glycol-induced calcium oxalate model of urolithiasis in the rat, and in human kidney stones, the distribution of certain noncollagenous and plasma proteins known to accumulate in bone and other mineralized tissues that include osteopontin, osteocalcin, bone sialoprotein, albumin, and alpha 2HS-glycoprotein. Of these proteins, osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein) were prominent constituents of the calcium oxalate-associated crystal "ghosts" found in the nuclei, lamellae, and striations of the organic matrix of lumenal renal calculi in the rat and of small crystal ghosts found within epithelial cells. Immunocytochemical labeling for both proteins of the content of secretory granules in tubular epithelial cells from treated rats, together with labeling of a similarly textured organic material in the tubular lumen, provides evidence for cosecretion of osteopontin and osteocalcin by epithelial cells, their transit through the urinary filtrate, and ultimately their incorporation into growing renal calculi. In normal rat kidney, osteopontin was localized to the Golgi apparatus of thin loop of Henle cells. In human calcium oxalate monohydrate stones, osteopontin was similarly detected in the lamellae and striations of the organic matrix. Based on these data, it is proposed that during urolithiasis, secretion of osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein), and the subsequent incorporation of these proteins into kidney stone matrix, may influence the nucleation, growth processes, aggregation, and/or tubular adhesion of renal calculi in mammalian kidneys. PMID- 8619373 TI - Radial and ulnar cortical thickness of the second metacarpal. AB - Differential bone mass at various skeletal sites, which may be due to mechanical stress exerted by the muscles attached to the bone, has been demonstrated for athletes who exert one limb more than the other. The question arises as to whether this bilateral asymmetry extends to the two sides of the same bone with different muscular attachments. The objectives of this study were to ascertain whether the radial and ulnar sides of the second metacarpal have similar cortical thickness and determine if bone mass decreases equally with age on the radial and ulnar sides. Hand-wrist radiographs were obtained from 201 male and 191 female Caucasian participants of the Baltimore Longitudinal Study of Aging. Differences between radial and ulnar cortical thickness within age groups were tested with Student's t-test and between age groups using analysis of variance. RAdial cortical thickness of the second metacarpal was found to be 11-12% greater in men and 10-12% greater in women than ulnar cortical thickness in both the left and right hands. Age-related changes in radial cortical thickness were evident in both sexes. In men, radial cortex decreased linearly from age 40 to 89. For women, there was a sharp decline in radial thickness from age 50 to age 60. Ulnar cortical thickness declined from age 50 to 60 for women only. Muscle attachment along the radial length of the second metacarpal may influence the accumulation of bone mass on the radial side at younger ages while muscle disuse may precipitate the loss of bone preferentially from the radial side. PMID- 8619375 TI - Soft tissue body composition: familial resemblance and independent influences on bone mineral density. AB - Familial resemblance in fat and lean soft tissue and the influence of these body composition measures on bone mineral density (BMD) were determined in 162 adult members of 42 families. Whole body fat and lean mass and BMD of the spine and femoral neck were measured by dual-energy X-ray absorptiometry and BMD of the heel and radius by single-photon absorptiometry. Significant correlations of lean tissue were observed between mothers and daughters (r = 0.35, p < 0.05), fathers and sons (r = 0.36, p < 0.05), and daughters and sons (r - 0.42, p < 0.01) after adjustment for age, height, fat mass, physical activity, and parity (among women). Fat tissue was not significantly correlated among any family member pairs. Lean tissue, but not fat, was positively associated with BMD in one or more family member subgroups at all skeletal sites. These findings suggest a hereditary component to muscle mass. Previously documented familial similarities in bone density may be related in part to the influence of lean mass on bone density. PMID- 8619374 TI - PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats. AB - Using in situ hybridization, we correlated the expression of mRNA for the parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor with bone formation and resorption in undecalcified serial sections of bones from growing rats. In addition we investigated the presence of biologically active receptors in the same locations using an in vivo autoradiographic technique. In the ulnae of growing rats, there are well defined zones of cortical bone formation and resorption. These contribute to the modeling drifts by which the bone achieves its adult shape. Forming surfaces incorporate fluorochrome labels, are lined with osteoid, and have a layer of cuboidal osteoblasts that have a high alkaline phosphatase activity. Resorbing surfaces have no fluorochrome incorporation, no osteoid, and are lined with resorbing cells with high tartrate resistant acid phosphatase (TRAP) activity. PTH/PTHrP receptor mRNA was expressed predominantly on forming but not on resorbing bone surfaces and colocalized with sites of binding of radiolabeled PTH after intravenous injection. PTH/PTHrP mRNA expression on osteocytes was inconclusive but radiolabeled PTH bound to a proportion of osteocytes in all regions of the cortex although binding was not specifically related to areas of bone formation or resorption. These results suggest that in growing animals the actions of PTH or PTHrP are connected more with bone formation than resorption. Such a role may be linked to the ability of PTH to induce bone formation in adults but does not explain the actions of the hormone in regulating resorption. Binding of PTH to osteocytes increases the evidence for a physiological role for these cells. PMID- 8619376 TI - Comparative effects of 1,25-dihydroxyvitamin D3 and EB 1089 on mouse renal and intestinal 25-hydroxyvitamin D3-24-hydroxylase. AB - EB 1089 is a vitamin D analog that is less potent than 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in its calcemic action but more potent in its antiproliferative action. We characterized the interaction of 1,25(OH)2D3 and EB 1089 with renal 25 hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, and compared the effects of 1,25(OH)2D3 and EB 1089 on induction of 24-hydroxylase mRNA in mouse kidney and intestine. 1,25(OH)2D3 and EB 1089 were competitive inhibitors of 24-hydroxylase activity. However, the Ki for 1,25(OH)2D3 (5.2 +/- 2.5 nM) was significantly lower than that for EB 1089 (286 +/- 59 nM). In the kidney, the time course and extent of 24-hydroxylase mRNA induction, relative to 18S rRNA, was similar for 1,25(OH)2D3 and EB 1089 with a peak response at approximately equal to 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24-hydroxylase mRNA, relative to 18S rRNA, was approximately 50% lower for EB 1089 than for 1,25(OH)2D3 at 3 h (p < 0.05) and 6 h (p < 0.05) while at 16 h 24-hydroxylase mRNA was no longer detectable. Moreover, while both 1,25(OH)2D3 and EB 10898 elicited a similar dose dependent induction of 24-hydroxylase mRNA in the kidney (EC50 = 0.4 +/- 0.13 and 0.3 +/- 0.08 ng/g for EB 1089 and 1,25(OH)2D3, respectively), the EC50 for EB 1089 (6.6 +/- 1.7 ng/g) was significantly higher than that for 1,25(OH)2D3 (0.9 +/- 0.32 ng/g) in the intestine (p < 0.01). EB 1089 was also less effective than 1,25(OH)2D3 in the induction of intestinal but not renal calbindin-D9k mRNA. To determine the mechanism for tissue-specific differences in potency, we determined the binding affinity of 1,25(OH)2D3 and EB 1089 for the vitamin D receptor. In the kidney, Kd values for 1,25(OH)2D3 (0.40 +/- 0.95 nM) and EB 1089 (0.48 +/- 0.04 nM) were not different. However, in the intestine, the Kd for EB 1089 (1.43 +/- 0.19 nM) was significantly higher than that for 1,25(OH)2D3 (0.85 +/- 0.06 nM; p < 0.05). Our results demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25(OH)2D3 for renal 24-hydroxylase, suggesting that it is more resistant to catabolism by the C-24 oxidation pathway; and (ii) EB 1089 and 1,25(OH)2D3 exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor. PMID- 8619377 TI - End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes. AB - The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate for apoptosis. Dual parameteric flow cytometric contour plots of Hoechst and 7-amino-actinomycin D fluorescence showed that abut 8% of cells in the plate were apoptotic. Most of these cells were in hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death. PMID- 8619378 TI - Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts. AB - A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix. PMID- 8619379 TI - Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro. AB - A chemically defined serum-free medium, which supports the development of bones and fibrous tissues of rat calvaria from nonmineralized mesenchymal precursor tissues, was employed to investigate tissue interactions between the dura matter and overlying tissues. Fetal calvarial rudiments from stages prior to bone and suture morphogenesis (fetal days 19 and 20) and neonatal calvarial rudiments with formed sutures (day 1) were cultured with and without associated dura mater. Removal of calvaria for in vitro culture allowed the examination of suture morphogenesis in the absence of tensional forces exerted on the sutures via fiber tracts in the dura mater originating in the cranial base. Ossification of frontal and parietal bones proceeded in a fashion comparable to development in vivo, but the cranial (coronal) sutures--primary sites for subsequent skull growth--were obliterated by osseous tissue union in the absence of dura mater. Bony fusion did not occur when rudiments were cocultured with dura mater on the opposite sides of 0.45 microns polycarbonate transwell filters, suggesting that the influence of dura mater on sutural obliteration was mediated by soluble factors rather than cell-cell or cell-matrix interactions. These results indicate that cell signaling mechanisms rather than biomechanical tensional forces are required for morphogenesis of the calvaria. PMID- 8619380 TI - Effects of aluminum on bone surface ion composition. AB - Aluminum induces net calcium efflux from cultured bone. To determine whether aluminum alters the bone surface ion composition in a manner consistent with predominantly cell-mediated resorption, a combination of cell-mediated resorption and physicochemical dissolution or physicochemical dissolution alone, we utilized an analytic high-resolution scanning ion microprobe with secondary ion mass spectroscopy to determine the effects of aluminum on bone surface ion composition. We cultured neonatal mouse calvariae with or without aluminum (10( 7) M) for 24 h and determined the relative ion concentrations of 23Na, 27Al, 39K, and 40Ca on the bone surface and eroded subsurface. Control calvariae have a surface (depth approximately 6 nm) that is rich in Na and K compared with Ca(Na/Ca) = 24.4 + 1.4, mean + 95% confidence limit of counts per second of detected secondary ions, K+Ca = 13.2 + 0.9). Aluminum is incorporated into the bone and causes a depletion of surface Na and K relative to Ca (Na/Ca = 9.6 + 0.7, K/Ca = 4.9 + 0.4; each p < 0.001 versus control). After erosion (depth approximately 50 nm), control calvariae have more Na and K than Ca (Na/Ca = 16.0 + 0.1, K/Ca = 7.5 + 0.1); aluminum again depleted Na and K relative to Ca (Na/Ca = 4.1 + 0.1 K/Ca = 1.9 + 0.1; each p < 0.001 versus control). Aluminum produced a greater net efflux of Ca (362 +/- 53, mean +/- SE, nmol/bone/24 h) than control (60 +/- 30, p < 0.001). With aluminum, the fall in the ratios of both Na/Ca and K/Ca coupled with net Ca release from bone indicates that aluminium induces a greater efflux of Na and K than Ca from the bone surface and is consistent with an aluminum-induced removal of the bone surface. This alteration in surface ion concentration and calcium efflux is consistent with that observed when calcium is lost from bone through a combination of cell-mediated resorption and physicochemical dissolution. PMID- 8619382 TI - Equivalent deficits in bone mass of the vertebral body and posterior processes in women with vertebral fractures: implications regarding the pathogenesis of spinal osteoporosis. AB - Reduced bone mass of the spine in women with vertebral fractures is attributed to excessive trabecular bone loss from the vertebral body. However, the measurement obtained by posteroanterior (PA) scanning includes the posterior processes and the vertebral body, each comprising about 50% of the total vertebral mass. Thus, the deficit in bone mass by PA scanning may be due to deficits in one or both of these structures. We asked two questions: (1) In healthy women, is the age related diminution in bone mass of the vertebral body greater than the diminution at the posterior processes? (2) In women with vertebral fractures, is the deficit in bone mass at the vertebral body, the fracture site in spinal osteoporosis, greater than at the posterior processes? Bone mass of the posterior processes and vertebral body of the third lumbar vertebra was measured by lateral scanning using dual-energy X-ray absorptiometry (DXA). Compared with 27 premenopausal women, deficits in 27 postmenopausal women at the posterior processes and vertebral body, respectively, were 35.9 +/- 3.7 and 25.2 +/- 4.1% (p < 0.05); t score, -1.5 +/- 0.2 and -1.1 +/- SD (p = 0.09). Compared with the postmenopausal (age-matched) women, deficits in 21 women with vertebral fractures at the posterior processes and vertebral body, respectively, were 22.6 +/- 4.9 and 24.5 +/- 8.3% (p = NS); Z score, -0.8 +/- 0.2 and -0.8 +/- 0.3 (p = NS). In vivo the bone mass of the vertebral body as a percentage of the whole vertebra was 45.7 +/ 0.1 in premenopausal women, 48.9 +/ 1.9 in postmenopausal women, 51.5 +/- 1.1 in women with low bone mass but no fractures, 52.7 +/- 2.4 in women with vertebral fractures, and 51.9 +/- 2.5% in vitro, based on autopsy specimens from 19 postmenopausal women aged 65 - 95 years. The lower spinal bone density measured using PA scanning in women with spine fractures may not be due to excessive or disproportionate trabecular bone loss from the vertebral body because comparable deficits are found at the posterior processes. Whether these deficits are due to reduced peak bone mass, trabecular bone loss, cortical bone loss, or varying combinations of these mechanisms remains to be established. PMID- 8619381 TI - Technical considerations of dual-energy X-ray absorptiometry-based bone mineral measurements for pediatric studies. AB - Dual X-ray absorptiometry (DXA) measurements have been shown to provide useful information on bone mineral status in young pediatric subjects. The purpose of this study was to challenge this system under various conditions to determine the clinical and experimental parameters that may be encountered which could interfere with DXA-based bone mineral content (BMC) and bone mineral density (BMD) measurements. Variations in data acquisition, including the covering of step phantom (external calibration standard) with a cotton blanket or partial exclusion of step phantom in the scan field, tissue freezing, or the presence of small nonmetallic objects, did not significantly alter DXA BMC or BMD measurements. By contrast, the presence of movement artifact, radiographic contrast media, and nonmetallic orthopedic casts significantly interfered with DXA BMC and BMC measurements. Variability in operator-dependent analysis of DXA scans occurred with regional analysis of whole body scans for DXA BMC and BMD measurements (average coefficient of variation was 2.9% and 1%, respectively, depending on the region analyzed) but did not affect the total (whole body) result. A minor adjustment in the manual delineation of the step phantom during data analysis may result in almost a 30% difference in DXA BMC and BMD. We conclude that movement artifact, radiographic contrast media, nonmetallic or orthopedic cast, and variations in operator-dependent data analysis may interfere with DXA BMC and BMD measurement in young pediatric subjects. Therefore, appropriate care should be taken to reduce or eliminate such interference. PMID- 8619383 TI - Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures. AB - Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-GP to inorganic phosphate (P(i)) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al3+)on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3 300 muM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above. Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization. PMID- 8619384 TI - Segregation analysis and variance components analysis of bone mineral density in healthy families. AB - Bone mineral density (BMD) was measured in 1992-93 in 129 nuclear families, including 258 parents and 183 children, and was analyzed for familial resemblance factors. BMD measurements were adjusted on weight and age. Segregation analysis rejected the monogenic hypothesis and exhibited a strong polygenic component. Variance components analysis was then used to estimate the parameters of a multivariate normal model including an additive polygenic component, a common environment factor, and a residual specific to each individual. The genetic component was independent of sex and age. The common environmental factor was not significant. The variance of the residual specific factor appeared to be a quadratic function of age, reaching its minimum value at 26.4 years. Consequently, the maximum value for heritability (ratio of genetic variance to total variance) is observed at this age (h2 = 0.84). According to this model, the correlation between two relatives is a function of the ages of each individual in the pair. PMID- 8619385 TI - [Multiple primary malignant neoplasms associated with genitourinary cancer]. AB - Between October 1980 and December 1994, we treated 392 patients with malignant neoplasms associated with genitourinary organs. We made a statistical study on 42 patients (10.6%) with multiple malignant neoplasms. The average age of 42 patients was 72.2 years and 83% of the patients were male. Malignant neoplasms originating from bladder, prostate or kidney were observed in 19 cases (35%), 19 cases (35%) or 10 cases (11%), respectively. The incidence of prostatic cancer was higher than that in other single primary malignant neoplasms associated with genitourinary organs. The other organs having malignant neoplasms accompanying genitourinary organs were the stomach (39%), lungs (12%), esophagus (9%), and pancreas (9%). Only 16 patients (35%) had synchronous multiple malignant neoplasms. However, 35 cases (75%) including these cases had second primary malignant neoplasms within 5 years of the first. In conclusion, the incidence of multiple malignant neoplasms with genitourinary cancer was as high as 10.6% and the prognosis of these patients was poor. These findings suggest the necessity of careful follow-up on patients with genitourinary cancer. PMID- 8619386 TI - [Characteristic clinical features of pyelonephritis caused by Enterococcus faecalis]. AB - A clinical study was performed in 13 cases of refractory Enterococcus faecalis pyelonephritis that were detected in the Department of Urology, Hiratsuka Municipal Hospital, from April 1975 to March 1995. The characteristic features were that pyelonephritis was commonly seen in females, and clinical symptoms (low grade intermittent fever, low back pain, general malaise, etc.) were continuously refractory. Bacteriuria or polymicrobial infections were often found, and bacterial count was often as high as 10(2)-10(4)/ml. Nevertheless we considered that E. faecalis caused of refractory pyelonephritis must not have week adherence and pathogenesis to the kidney, owing to the clinical symptoms and the basic subjects. Furthermore this infection was difficult to cure completely with antimicrobials having activity against E. faecalis, and long-term treatment was needed. Therefore, we recommend that treatment for refractory E. faecalis pyelonephritis be carefully selected according to clinical symptoms and the bacterial density of this strain. PMID- 8619387 TI - [Clinical evaluation of intra-operative radiotherapy combined with subtotal cystectomy for invasive bladder carcinoma]. AB - From 1981 to 1994, intra-operative radiotherapy after subtotal cystectomy was performed on 22 patients with invasive bladder carcinoma on whom radical cystectomy could not be recommended because of old age or condition. All the patients received 25 to 30 Gy of radiotherapy focused on trigonum and internal urethral orifice after subtotal cystectomy with uretero-cutaneostomy. Of 22 patients, 15 patients died. Five patients died of bladder cancer, one died of gastric cancer, one died of rectal cancer and the others died of pneumonia, heart failure, sepsis and senility. The five-year survival rate was 41% and the cause specific five-year survival rate was 75%. Local recurrence was seen only in one patients, who received second intra-operative radiotherapy and recovered well in complete remission. We believe that intra-operative radiotherapy after subtotal cystectomy is useful for patients with invasive bladder carcinoma on whom radical cystectomy could not be recommended because of old age or condition. PMID- 8619388 TI - [Relationship between pretreatment serum levels of prostate specific antigen and bone metastasis in prostate cancer]. AB - We retrospectively reviewed pretreatment levels of serum prostate specific antigen (PSA) in 138 newly diagnosed, untreated patients with prostate adenocarcinoma to evaluate the usefulness of serum PSA levels to predict results of radionuclide bone scans. All of the 24 patients with serum PSA levels above 200 ng/ml showed positive bone scans whereas those with serum PSA levels below 10 ng/ml included only three cases (3/51, 5.1%) positive for bone metastasis. All of the three had poorly differentiated adenocarcinoma of the prostate. In conclusion, bone scans may not be necessary in the cases with serum PSA levels less than 10 ng/ml except for those with poorly differentiated adenocarcinoma. PMID- 8619390 TI - [Results of transurethral resection of prostate plus incision (TUR-P+I) for benign prostatic hypertrophy]. AB - The results of 14 patients treated by TUR-P+I and 15 patients by TUR-P between September 1991 and August 1993 were reviewed to evaluate the effects of TUR-P+I. Tur-P+I is a combined technique of channelling TUR-P and transurethral incision of the bladder neck and the prostate. After receiving modest TUR of the adenoma, the bladder neck and anatomical capsule of the prostate was incised by electroresectoscope at 6 o'clock position from the bladder neck toward to verumontanum. Before operation, the maximum floor rate, average flow rate, and residual urine volume were measured, which were respectively 9.9 +/- 5.6 ml/s (M+SEM), 4.4 +/- 2.3 ml/s, and 130 +/- 80 ml in TUR-p+I group, and 11.6 +/- 2.9 ml/s, 4.3 +/- 1.8 ml/s, and 60 +/- 60 ml (p < 0.01) in TUR-P group. The operation time and resected tissue weights were similar in both groups; 68 +/- 16 min and 11 +/- 5.2 g in TUR-P+I group, and 72 +/- 25 min and 11 +/- 6.5 g in TUR-P group. The post-operative maximum flow rate, average flow rate and residual urine volume were respectively improved to 19.0 +/- 5.7 ml/s, 9.3 +/- 3.7 ml/s, and 20 +/- 20 ml in TUR-P+I group, and 14.6 +/- 6.1 ml/s (p = 0.057), 6.6 +/- 2.6 ml/s (p < 0.05), and 30 +/- 30 ml (not significant) in the TUR-P group. Neither significant blood loss nor complications were experienced in either procedure. It is suggested that TUR-P+I could be a safe and effective alternative of TUR-P. Longer follow-up and the prospective study are required to establish the value of the current combined technique. PMID- 8619389 TI - [Combination therapy with estrogen and UFT in newly diagnosed prostatic cancer (poorly differentiated, stage D2)]. AB - To determine whether long-term oral administration of UFT, a combination of 5 fluorouracil and uracil, in addition to conventional estrogen therapy improved the response and survival of the patients with advanced stage D2 prostate adenocarcinoma, a randomized prospective study was performed with either estrogen alone (Honvan 200 mg/day or presexol 1 mg/day: group A) or estrogen plus UFT (400 mg/day:group B). This study comprises 34 newly diagnosed patients with poorly differentiated prostatic adenocarcinoma (18 patients in group A and 16 in group B). Survival from all causes of death or cancer-specific death were compared using Kaplan-Meier actual methods among the patients separated by histological composition of tumors analyzed WHO histologic patterns, score of extent of disease (EOD), and with or without normalization of serum PSA or PAP levels after treatment. Although combination therapy with UFT against overall survival was effective without statistical significance, better survival in this group than the patients treatment with estrogen alone assessed among the patients whose tumor contained more than 70% of medullary and/or column-cord histological components. The survivals among the patients with EOD score 3 and whose serum PSA or PAP levels did not lead to decrease within normal levels after treatment were also better in group B than in group A. These findings suggest the validity of the combination therapy with UFT in addition to estrogen against highly advanced prostatic cancer patients whose tumor composed of abundant non-hormone-refractor histological components. PMID- 8619391 TI - [Treatment of scrotal hydrocele using fibrin adhesive]. AB - For the purpose of adhesion of the tunica vaginalis in scrotal hydrocele, we used fibrin adhesive, Bolheal. Between September 1993 and December 1994, 13 patients with scrotal hydrocele received occlusion therapies using Bolhead. The therapy was effective in 1 of the 6 patients (17%) administered 2.4 mg of fibrinogen and 750 units of thrombin into the vaginal cavity after aspiration of fluids, and in 5 of the 7 patients (71%) administered 240 mg of fibrinogen and 7.5 units of thrombin. Therefore, fibrin adhesive may be useful in the treatment of scrotal hydrocele. PMID- 8619392 TI - Effects of luteinizing hormone-releasing hormone (LH-RH) analogue on the function of the isolated rabbit corpus cavernosum. AB - An adverse effect of the administration of luteinizing hormone-releasing hormone (LH-RH) analogue is impotence. The effects of LH-RH analogue injection on the function of the rabbit corpus cavernosum were investigated. Eighteen male rabbits were divided into three groups, i.e., LH-RH analogue injection, castration, and sham-operated control groups. After measurement of serum testosterone, the LH-RH analogue (1.5 mg/kg leuprolide acetate) was injected once in the LH-RH group, castration in the castrated group, and sham surgery in the control group. Four weeks later, the rabbits were maintained in the same circumstance and serum testosterone was measured once a week. Four weeks after preparation, all rabbits were used for in vitro experiments. At one week the serum testosterone level of the LH-RH and castrated groups decreased significantly from that in the sham operated control group, which was sustained for the next 3 weeks. Although contractile strength of the corporal tissue taken from the castrated group was weakened in response to phenylephrine and KCl, corporal contractile strength of the LH-RH group was not. Under precontraction with 200 microM phenylephrine relaxation of the corpus cavernosum in response to field stimulation and sodium nitroprusside significantly decreased in both the LH-RH and castrated groups. However, there were no differences in the maximal relaxations induced by ATP and bethanechol between the three groups. In conclusion, the LH-RH analogue impaired the relaxation of the corpus cavernosum induced by field stimulation and sodium nitroprusside as much as castration did. Although the corporal contraction to phenylephrine and KCl was decreased by castration, it was not altered by the LH RH analogue. PMID- 8619393 TI - [Chyluria treated with stripping of the renal pedicle: a case report]. AB - A 54-year-old male was referred to our outpatient clinic because of milky urine. He had lived in Kumamoto, an area of endemic filariasis. There were no microfilaria found in his blood smear. Lymphangiography showed dilation of the retroperitoneal lymphatics and bilateral renal refluxes. The thoracic duct was obvious. The initial treatment consisted of irrigating the bilateral renal pelvis with silver nitrate solution, but was not successful and recurrent acute urinary retention developed. Therefore, we performed stripping and ligation of the bilateral renal lymphatics. Chyluria disappeared immediately after surgery. The etiology and various treatments for chyluria are discussed. PMID- 8619394 TI - [Femoral testis: report of three cases]. AB - Three patients were referred to our clinic with the chief complaint of emptiness of the right scrotal contents. Every physical examination in these patients revealed a little-finger-tip sized mass corresponding to the normal testis at the right side of the femoral region. Right orchiopexy by the dartos-pouch technique was successfully performed. On every surgical exploration of these patients, the gubernaculum was fixed to the femoral skin and the testis was found to be normal in size. Our cases are the 2nd, 3rd and 4th cases of femoral testis, reported in the Japanese literature. PMID- 8619395 TI - [Leiomyosarcoma of the scrotum: a case report]. AB - A case of leiomyosarcoma of the scrotum is reported. A 44-year-old man referred to our hospital with the complaint of swelling of the left scrotal contents. The lesion was about 3 cm in diameter, isolated from testis, epididymis and spermatic cord. Ultrasonography revealed hypoechoic and slightly heterogenic area in the lesion. Surgical resection was easily done. Histological examination showed leiomyosarcoma. Adjuvant therapy was not performed. Three years after the resection, he is alive without any sign of recurrence or metastasis. PMID- 8619396 TI - [Accessory scrotum with lipoma: a case report]. AB - A newborn male infant was referred to our clinic because of anomalies of external genitalia. The infant was born at term after a normal pregnancy and delivery, and was appropriate for gestational age. A tumor mass covered with scrotum-like skin on its tip was noticed in the right side of perineum between the scrotum and anus. The raphe, which ran along the midline at the penis and the normal scrotum, circumscribed the right side of this structure. Both testes had descended into the scrotum. The condition was associated with incomplete penoscrotal transposition. There was no other urological anomaly including lower urinary tract. Histological findings of the tumor indicated perineal lipoma, and the scrotum-like portion was diagnosed as an accessory scrotum. Thirty-one report cases of the accessory scrotum including our own were reviewed and discussed. PMID- 8619397 TI - Bedside measurement of factor VIII:C activity in individuals with hemophilia A. AB - Factor VIII replacement therapy for patients with hemophilia A is conventionally monitored using a plasma-based factor VIII:C assay (a modified activated partial thromboplastin time [APTT] test). The plasma factor VIII assay requires the preparation of plasma from citrated whole blood and measurement of the clotting times of mixtures of patient plasma, factor VIII-deficient substrate, and APTT reagent. Results are not routinely available in less than 1.5 hr, reducing the clinical value of the laboratory data regarding the ability to immediately adjust patient therapy. Results from the whole blood factor VIII assay, performed on a portable coagulation analyzer and using test tubes prefilled with the necessary APTT and factor VIII-deficient reagents, are available within 5-7 min. This immediate determination of the factor VIII:C level from citrated whole blood provides the opportunity to greatly reduce turnaround time and improve the efficacy of factor VIII replacement therapy. Based on clotting time, factor VIII:C activity is read from a standard curve. A clinical evaluation of this whole blood test was performed in two hemophilia centers. A high degree of correlation was seen (r=0.813, n=220) between the whole blood values obtained and conventional laboratory results. This level of correlation was superior to that obtained when comparing two different plasma-based systems(r=0.753, n=23). Factor VIII:C activity levels measured using the whole blood assay system were similar,irrespective of the test operator (laboratory technologist, nurse clinician, or patient). This study indicates that the whole blood factor VIII assay provides results comparable to those of conventional plasma-based assays, but in a more rapid and efficient manner. It provides an opportunity to reduce unnecessary patient consumption of replacement preparations, hence reducing the cost of hemophilia A maintenance and prophylaxis regimens, and to reduce overall patient exposure to human blood products. PMID- 8619398 TI - Determination of plasma soluble fibrin using a new ELISA method in patients with disseminated intravascular coagulation. AB - We measured plasma levels of soluble fibrin (SF) in 98 patients suspected of having disseminated intravascular coagulation (DIC) using a newly developed enzyme-linked immunosorbent assay (ELISA) and investigated the correlations between SF determinations and measurements of other hemostatic molecular markers to determine the diagnostic usefulness of determinations of SF. Patients were classified into four groups according to their clinical and laboratory findings: overt DIC (n =33), subclinical DIC (n =23) hypercoagulability (n =22), and non DIC (n =20). SF levels were significantly higher in patients with overt DIC compared with the other three groups and were significantly higher in the subclinical DIC and hypercoagulability groups compared with the non-DIC patients. SF levels increased significantly with each increase in the clinical stage. Although levels of thrombin-antithrombin III complex (TAT), prothrombin fragment 1 + 2 (PF 1+2), cross-linked fibrin degradation products (XDP), and plasmin antiplasmin complex (PAP) were significantly increased in patients with overt DIC compared with non-DIC patients, the values of these hemostatic molecular markers did not consistently show an increase in association with advances in the disease stage. Plasma levels of SF in patients with overt DIC showed a positive correlation with levels of TAT, XDP,and FDP(E), but not with PF1+2 and PAP. Analysis of receiver-operating characteristic curves showed that the sensitivity and specificity of SF were similar to those of XDP for diagnosis of DIC. The sensitivity and specificity of SF for diagnosis of overt DIC were both above 90% when the cut-off value was set at 65 mu g/ml.plasma levels of SF were also increased in patients with extravascular fibrin formation without DIC. Our findings suggest that measurement of plasma levels of SF by this ELISA method is useful for the diagnosis of DIC and the evaluation of the patient's clinical status. PMID- 8619399 TI - Clinical correlates among 49 families with hemophilia A and factor VIII gene inversions. AB - Inversions between a gene A copy within intron 22 of the factor VIII gene and additional copies outside the factor VIII gene were found in 49 families with hemophilia A. Inversion patterns were that of recombination with a distal gene A copy in 34, a proximal copy in 14, and a third (variant) copy in one. Baseline factor VIII clotting levels were <1% of normal in 43 and 1% in 6. No inversion was detected in 61 other families whose affected members had < or = 1% activity levels nor in 42 families with moderately severe hemophilia A and 2-5% baseline levels. Both high titer and low level alloantibody inhibitors were found in patients with of without an inversion. Of 13 high titer inhibitors, 8 were persistent and 1 of these patients had an inversion. Of 5 that responded to daily factor VIII infusions, 4 were in patients with gene inversions. Of the 49 families with an inversion, the occurrence of hemophilia was isolated in 30 and the mother was a carrier in the 25 in which additional family members were informative. In three of these families with isolated occurrence, the maternal grandmother was a carrier whereas in three others a de novo mutation occurred in the maternal grandfather's factor VIII gene. Screening for gene inversions in patients with severe (or "borderline" severe) hemophilia A provides a direct marker of the mutation in 45% of families. It is useful even if there is no living affected member and in predicting the likely severity of an infant in which there are no reliable baseline clotting activities, including 70% of families with isolated occurrences of hemophilia A. PMID- 8619400 TI - Effects of humic acid on the viability and coagulant properties of human umbilical vein endothelial cells. AB - We have previously shown that humic acid (well-water humic acid, HA, and synthetic humic acid, SHA) enhances cell surface expression of tissue factor (TF). Here we report that incubation of human umbilical vein endothelial cells (HUVEC) for 2 hr with HA or SHA cause a rapid rise in TF mRNA levels, as shown by Northern blot analysis. To understand the cytotoxic and fibrinolytic effects of HA and SHA on cultured HUVEC, the cells treated with varying concentrations of HA and SHA for various periods of time. Both HA and SHA (10-200 micrograms/ml) inhibited the viability of subconfluent HUVEC, cultured in the presence or absence of 20% FBS (Fetal Bovine serum) in the culture medium, in a dose dependent manner. Both HA and SHA induced surface changes in the HUVEC as revealed by scanning electron micrography (SEM). However, protocatechuic acid, the monomer of SHA, did not significantly inhibit cell growth, and showed a cytotoxic effect only at 200 micrograms/ml. Furthermore both HA and SHA stimulated HUVEC to produce plasminogen activator inhibitor (PAI-1) and tissue plasminogen activator (t-PA) in a dose and time dependent fashion; the amount of PAI-1 produced was found to exceed that of t-PA. The monomer of SHA did not have this stimulatory effect. These results distinctly suggest that in addition to the inhibition of viability HA is involved in TF induction and PAI-1 synthesis in HUVEC and these may be some of the plausible mechanisms underlying the thrombotic disorders in Blackfoot disease. PMID- 8619401 TI - Sickle cell acute chest syndrome associated with parvovirus B19 infection: case series and review. AB - Acute Chest Syndrome (ACS) continues to be a major source of morbidity and mortality among patients with sickle cell disease. It is characterized by the presence of pleuritic chest pain, fever, raises on lung auscultation, and pulmonary infiltrates on chest x-ray [Castro et all: Blood 84:643-649]. The pathophysiology of this disorder remains poorly understood leading to the descriptive term "Acute Chest Syndrome" designated by Charache et al. [Arch Intern Med 139:67-69, 1979]. Typical bacterial pathogens are seldom isolated in adults, although they play a significant role in the pathogenesis of this entity in children. Until recently, the technology to accurately study viral infection as a precipitating cause of ACS has been unavailable. Parvovirus B19 is being increasingly recognized as an important human pathogen, and has been established as the cause of transient "aplastic crisis" in patients with sickle cell diseases [Saarien et al: Blood 67:-11411-11417, 1986; Young: Sem Hematol 25:159-172, 1988]. We present three patients with hemoglobin SC variant of sickle cell disease who developed ACS in association with acute parvovirus B19 infection, one of which died of respiratory failure. Parvovirus B19 infection was established by polymerase chain reaction for parvovirus B19 DNA, and the presence of parvovirus B19 specific IgM antibodies. These cases suggest that parvovirus B19 may be associated with more than self-limited illness in patients with sickle cell disease, and that this ubiquitous virus may merit further study as a precipitating cause of ACS. PMID- 8619402 TI - High density sickle cell erythrocyte core membrane skeletons demonstrate slow temperature dependent dissociation. AB - We have previously demonstrated that slow dissociation of HDSS membrane skeletons in high ionic strength Triton X-100 buffer was related to a posttranslational modification in beta-actin, in which a disulfide bridge was formed between cysteine 284 and cysteine 373[Shartava et al: J Cell Bio 128:805, 1995]. These previous dissociation assays were limited to two homozygous (SS) sickle cell patients and a single temperature (37 degrees C). In the current work, we have expanded the SS subjects to 9 and have carried out dissociation assays at 0, 24, 30, 34, and 37 degrees C. At 0 degrees C there was limited dissociation of spectrin and actin from normal(AA), low density sickle cell(LDSS), and high density sickle cell (HDSS) core skeleton up to 24 hr. The first order rate constants for dissociation of spectrin, at 0 degrees C, was 0.030-0.035 x 10-4 sec-1 for AA,LDSS, and HDSS core skeletons. However at 24, 30, 34, and 37 degrees C the rate of dissociation of spectrin from HDSS core skeletons was significantly slower than the rate of dissociation from AA core skeletons. Having determined the first order rate constants for spectrin dissociation at these specified temperatures, we then asked whether dithiothreitol (DTT) would hasten the dissociation of core skeletons. The presence of DTT caused the rate of dissociation of the HDSS membrane skeleton to become statistically indistinguishable from the rate of dissociation of AA membrane skeletons. This is consistent with the suggestion that reversible thiol oxidation is responsible for the slow dissociation of the HDSS membrane skeleton. PMID- 8619403 TI - Partial repression of human gamma-globin genes by LCR element HS3 when linked to beta-globin genes and LCR element HS2 in MEL cells. AB - Clues for overcoming fetal (gamma-) globin gene repression in adult human erythroid cells may come from understanding why repression of isolated gamma globin genes has not previously been achieved in the adult erythroid environment of mouse erythroleukemia cells (MEL). Repression of human gamma-globin genes has been demonstrated in MEL cells when transferred as part of the entire beta-globin gene cluster packaged in chromatin. Major differences in these approaches are prior packaging into chromatin and the presence of additional sequences, notably from the locus control region (LCR). In this report we focus on the contribution to gamma-globin gene repression that multiple elements of the LCR may have. We first show preferential activation of beta-globin genes over gamma-globin genes in MEL cells when linked to each other and to LCR sequences containing the core elements of DNase I hypersensitive sites 4, 3, and 2. Removal of the HS4 element had no effect, however, removal of the 225 bp HS3 core element resulted in a five fold increase in gamma-globin gene expression. The enhancer 3' to the A gamma globin gene also had no apparent effect on gamma-globin gene expression. These results provide first evidence of gamma-globin gene repression involving the core region of HS3 in the presence of the core region of HS2 and a beta-globin gene. A mechanism for repression involving sequestration of the gamma-promoter away from the strong enhancer activity of HS2 is proposed. PMID- 8619405 TI - Coagulation abnormalities in patients with Gaucher's disease: effect of therapy. AB - Various coagulation defects have been associated with Gaucher's disease, including factor IX deficiency and acquired von Willebrand's disease (VWD). We performed repeated coagulation assays in 9 patients with Gaucher's disease over a period of 2 years. The prothrombin time (PT) and fibrinogen levels were normal in 8 of 9 patients, while the partial thromboplastin time (PTT) was abnormal in 5 of 9; all mixing PTT tests showed correction. Factor IX was normal repeatedly in the 7 of 7 patients tested. In contrast, factor XI was decreased in 3 of 9 patients assayed. Anticardiolipin (ACL) IgM was normal in all patients. ACL IgG was highly variable; levels were abnormal at least once in 6 of 8 patients, but were also normal at least once in 7 of 8 patients. Factor VIII was also quite variable: levels were decreased at least once in 4 of 9 patients, and normal at least once in 8 of 9 patients. Von Willebrand factor antigen (VWF Ag) studies were normal in 7 of 8 patients, but VWF activity was decreased at least once in 4 of 8 patients. In some patients, these problems could be overcome by specimen dilution. In ony 1 patient was VWF Ag decreased; this patient had a factor VIIIC level of 13% , and VWF activity of 18.7%. Coagulation assays performed before and after alglucerase administration failed to demonstrate any significant improvement in these assays, and neither was there a consistent improvement over the duration of therapy. We suggest that previously reported decreases in factor IX and VWF may be secondary to the interfering presence of increased cerebroside levels. Caution must be used in the interpretation of clotting assays in the patient with Gaucher's disease. PMID- 8619404 TI - Analysis of PIG-A gene in a patient who developed reciprocal translocation of chromosome 12 and paroxysmal nocturnal hemoglobinuria during follow-up of aplastic anemia. AB - The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow-up of AA. All metaphases in CD59-deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59-deficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single-base deletion mutation in the PIG-A gene of this patient's peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation. PMID- 8619406 TI - Aplastic anemia as the sole presentation of systemic lupus erythematosus. PMID- 8619407 TI - Haplotype analysis of the Mexican frameshift Cd 11 (-T) and -28 A->C beta thalassemia alleles. AB - The origins of the -28 A->C and frameshift Cd 11 - T(Fs CD 11-T)alleles were investigated by beta-globin cluster haplotype analysis. These alleles were found in a Mexican mestizo family with beta-thalassemia (beta-thal). The -28 A->C mutation was described previously in Kurdish Jews linked to the most common haplotype in the world(+----++),the same haplotype observed in this Mexican family. Therefore, it is not possible to assess a new origin of the -28 A->C mutation in our population. The Fs Cd 11 -T allele, not reported to date in any other populations, was linked to the -++--+-haplotype (sixth in frequency in the world). This haplotype has not been reported in association with any beta-thal mutant, suggesting a Mexican origin for the Cd 11 -T mutation. PMID- 8619408 TI - Improvement of erythropoiesis during treatment with deferiprone in a patient with myelofibrosis and transfusional hemosiderosis. PMID- 8619409 TI - Granulocytic sarcoma of the uterus complicating myelodysplastic syndrome. PMID- 8619410 TI - Chronic consumption coagulopathy and popliteal aneurysm. PMID- 8619411 TI - Persistent polyclonal B lymphocytosis. PMID- 8619412 TI - Thrombin-antithrombin III and prothrombin fragment 1.2 levels in early respiratory distress syndrome. PMID- 8619413 TI - Treatment and prophylaxis of hypermenorrhea with leuprorelin in premenopausal women affected by acute leukemia at diagnosis. PMID- 8619414 TI - Waldenstrom's macroglobulinemia transformed into immunoblastic lymphoma presenting with malignant ascites. PMID- 8619415 TI - Acute leg ischaemia as a presentation of hyperleukocytosis syndrome in acute myeloid leukaemia. PMID- 8619416 TI - Simultaneous occurrence of lupus anticoagulant, factor VIII inhibitor and localized pemphigoid. PMID- 8619417 TI - Successful treatment of acute promyelocytic leukemia in a pregnant Jehovah's Witness with all-trans retinoic acid, rhG-CSF, and erythropoietin. PMID- 8619418 TI - Symptomatic porphyria cutanea tarda and B-immunoblastic lymphoma: is there an association? PMID- 8619419 TI - Langerhans cell (eosinophilic) granulomatosis. A clinicopathologic study encompassing 50 years. AB - We summarize our experience with 238 cases of Langerhans cell granulomatosis (LCG), 198 of whom were followed for a median period of 10.5 years. Our patients did well unless overtreated, and no deaths were attributed to the disorder itself. The disease may appear in unifocal or multifocal form, and treatment is based on this fact. Virtually all patients recovered completely except for occasional residual orthopedic problems or residual diabetes insipidus. Several of the patients underwent subsequent pregnancies without difficulty. The granulomas primarily occur in bone, but lung, skin, and lymph nodal involvement is not uncommon. Involvement of thyroid, thymus, and other sites is rare. The hallmark of the disease is the accumulation of Langerhans cells (LCs). We review the pathology of LCG by histology, electron microscopy, and immunolabeling. LCs originally were identified in squamous epithelium, but these cells are part of the widespread system of dendritic cells. The latter cells, which arise from CD34+ progenitors, are specialized and efficient antigen-presenting cells for T cell-mediated immunity. In LCG, however, the major associated cells are not T cells, but mature eosinophils: hence the original name eosinophilic granuloma. Confusion about terminology has been based upon the scanty and rather crude pathology reports in the original literature. The term histiocytosis X was meant to cover a spectrum of three diseases--eosinophilic granuloma, Hand-Schuller Christian disease (HSC), and Letterer-Siwe disease (LS)--but HSC and LS have no basis in pathology and hence the terms are meaningless. The term HSC has become a synonym for multifocal eosinophilic granuloma (LCG). The term LS has been used in reporting a number of benign, malignant, or unknown conditions. We prefer the term LCG to avoid confusion with the term histiocytosis X because there is evidence that the LC is not a member of the mononuclear phagocyte system and hence not a tissue macrophage, and because the use of the term "histiocyte" has become a convenience in much of the literature when reporting incompletely understood diseases. PMID- 8619420 TI - Atypical adenomatous hyperplasia and bronchoalveolar lung carcinoma. Analysis by morphometry and the expressions of p53 and carcinoembryonic antigen. AB - Atypical adenomatous hyperplasia (AAH) of the lung is a putative precursor of bronchoalveolar carcinoma (BAC). To define the steps in its development and to clarify at which stage critical cellular events occur, we studied 65 lesions of AAH, early BAC, and overt BAC by morphometric analysis and immunohistochemical evaluation of expression of p53 protein and carcinoembryonic antigen (CEA). Both the nuclear area and lesion size increased from AAH to early BAC and to overt BAC; the standardized variation of nuclear area was smallest in overt BAC. Discriminant analysis using these morphometric parameters revealed high accuracy rates for the respective categories. Analysis of distribution of lung lesions in terms of nuclear area and lesion size yielded effective, potentially diagnostic cutoff values for distinction between AAH and early BAC. Both p53 and CEA expression tended to increase with the advance of atypia grade. In particular, high-level p53 expression was strongly correlated with overt BAC. These findings indicate that our classification of lung lesions is reproducible and thus useful for analyzing the development of BAC. Furthermore, some kinds of p53 gene abnormalities that are correlated with high-level p53 expression likely play an important role in the progression of early to overt BAC. PMID- 8619421 TI - Renal allograft involvement by Epstein-Barr virus associated post-transplant lymphoproliferative disease. AB - This study describes nine cases of post-transplant lymphoproliferative disease (PTLD) presenting as renal allograft dysfunction. Onset of symptoms was 34 to 265 days post-transplant, typically (in six of nine cases) after refractory rejection treated with OKT3. Diagnosis was made by histopathologic examination of needle biopsy (three of nine cases) or allograft nephrectomy (six of nine cases) specimens. Disease was confined to the allograft in three patients. The morphology was polymorphic in eight cases and monomorphic in one case. Five cases showed monotypic kappa or lambda light chain expression. Expansile lymphoid infiltrates, serpiginous necrosis, nuclear atypia, and presence of Epstein-Barr virus RNA helped to distinguish PTLD from severe rejection. Tubular damage and venulitis was common in PTLD lesions, but arterial involvement was not prominent. Infiltration of the ureter, hilar adipose tissue, and nerve twigs was frequent in nephrectomy specimens. Reduction of immunosuppression led to resolution of PTLD in two of three cases diagnosed by needle biopsy, but severe acute rejection led to graft loss in one case; the third case progressed to fatal multisystem disease. Among cases diagnosed at nephrectomy, two of six patients died of disseminated PTLD and one of six died of sepsis. The five surviving patients are alive 41 to 99 months after initial diagnosis without evidence of recurrent PTLD. PMID- 8619422 TI - Lymphoid changes of the nasopharyngeal and palatine tonsils that are indicative of human immunodeficiency virus infection. A clinicopathologic study of 12 cases. AB - We report 12 cases in which the histomorphologic changes of the nasopharyngeal tonsils (adenoids) or palatine tonsils suggest infection with the human immunodeficiency virus (HIV). The patients included 10 men and two women, aged 20 to 42 years (median, 33 years). The clinical presentation included airway obstruction, pharyngitis, fever, and a tonsillar or adenoidal mass lesion. Histologic evaluation of the excised adenoids or tonsils in 10 of the cases demonstrated a spectrum of changes including florid follicular hyperplasia, follicle lysis, attenuated mantle zone, and the presence of multinucleated giant cells (MGC). The latter characteristically localized adjacent to the surface or tonsillar crypt epithelium. Two of the 12 cases showed marked lymphoid depletion with absent germinal centers, plasmacytosis, and stromal vascular proliferation. Immunohistochemical evaluation for HIV p24 core protein showed reactivity in 10 of 12 cases localized to follicular dendritic cell network (FDC), the MGC, scattered interfollicular lymphoid cells, and cells identified within the surface or crypt epithelium. Localization of viral RNA by in situ hybridization paralleled the HIV p24 immunohistochemical findings. Additional significant findings included the presence of both CD-68 and S-100 protein in the MGC and the presence of S-100 protein in dendritic cells. Other than HIV, no microorganisms were identified. At the time of presentation, eight patients were not known to be a risk for HIV infection, nor were they known to be HIV infected or suffering from AIDS. In these patients, HIV infection was suspected on the basis of the histologic changes seen in the resected tonsillar and adenoidal tissue. Serologic evaluation (by enzyme-linked immunosorbent assay), confirmed the presence of HIV infection. Our findings suggest the possibility of HIV dissemination through the upper aero-digestive tract mucosa via target cells, such as intraepithelial dendritic cells, submucosal macrophages, and T-lymphocytes. Subsequent presentation of viral antigens to the tonsillar and adenoidal lymphoid tissues results in enlargement of these structures that clinically may simulate a neoplastic proliferation but causes histomorphologic changes that are highly suspicious for HIV infection even in asymptomatic HIV-positive patients. PMID- 8619423 TI - Carcinoma-like signet-ring cells in gastric mucosa-associated lymphoid tissue (MALT) lymphoma. AB - We noticed the presence of epithelial signet-ring cells (SRCs) in a proportion of primary gastric B-cell lymphomas, and in some endoscopic biopsies we found it difficult to decide whether they represented an associated carcinoma. To evaluate the frequency and nature of this phenomenon, we reviewed 108 stomachs resected for primary lymphoma, including 70 mucosa-associated lymphoid tissue (MALT) and 38 non-MALT lymphomas. We found SRCs, either isolated or grouped in clusters, in 26 of 70 MALT lymphomas. The SRCs were always localized in the superficial portion of the lamina propria and associated exclusively with lymphomatous areas. Isolated and scarce SRCs were also found in four of 22 cases of polyclonal atypical lymphoid hyperplasia. Our data suggests that SRCs occurring in gastric MALT lymphomas represent a particular type of LEL in which the foveolar cells disaggregated by the lymphomatous infiltration acquire a globoid, signet-ring appearance. These "foveolar" LELs are found in 37% of MALT lymphomas and are usually associated with the more classic and constant "neck" LELs, which are localized between the foveolae and mucopeptic glands. An awareness of the existence of the foveolar LEL may help avoid overdiagnosis of SRC carcinoma on gastric endoscopic biopsies. PMID- 8619424 TI - Signet-ring cells associated with pseudomembranous colitis. AB - After being treated for pneumonia with antibiotics, an 82-year-old man with a history of colonic carcinoma developed pseudomembranous colitis with toxic megacolon. In addition to the classic features of pseudomembranous colitis, the colon displayed signet-ring cells confined to the surface epithelium and crypts. A misdiagnosis of recurrent carcinoma was avoided after a review of the history and the endoscopic findings and by noting the location of the signet-ring cells. PMID- 8619425 TI - Strongyloides stercoralis eosinophilic granulomatous enterocolitis. AB - Six patients suffering from an unusual form of colitis produced by Strongyloides stercoralis hyperinfection are described. In contrast to the usual Strongyloides hyperinfection syndrome, in which small intestinal and pulmonary manifestations are seen in patients with some forms of immunodeficiency, the patients described here presented with only a characteristic transmural eosinophilic granulomatous inflammation affecting mostly the colonic wall and clinically mimicking ulcerative colitis or Crohn's disease. This Strongyloides eosinophilic granulomatous enterocolitis apparently results from a florid inflammatory response by eosinophils, histiocytes, and giant cells with formation of granulomas that destroy the larvae entering the colon. This morphologic picture differs from that of the well-described hyperinfection syndrome, in which the bulk of the larvae pass through the colonic wall to complete the life cycle, with only a few larvae destroyed in the colon. The probable pathophysiologic mechanism of this unusual manifestation of hyperinfection is discussed based on the anatomic and clinical observations of patients who presented at different stages in the evolution of their condition and whose length of follow-up varied. PMID- 8619427 TI - The morphologic spectrum of non-Hodgkin's lymphomas with BCL1/cyclin D1 gene rearrangements. AB - BCL1/PRAD1 gene rearrangements involving the cyclin D1 gene are a feature of about 70% of centrocytic/mantle-cell lymphomas (CC/MCL) but are identified in only a small proportion of other B-cell non-Hodgkin's lymphomas. Of 37 lymphomas found to have BCL1/cyclin D1 (PRAD1, CCND1) gene rearrangements, 30 fit the morphologic and immunophenotypic criteria for typical CC/MCL. Seven cases with morphologic features atypical for CC/MCL were identified. CD5+ monoclonal B cells were documented in all these cases. Six cases were subsequently stained for cyclin D1 protein, and all showed nuclear positivity. Five cases had variably sized foci of cells with moderately abundant pale cytoplasm resembling parafollicular/monocytoid B cells, marginal zone cells, hairy cells, or even proliferation centers. Transformed-appearing cells were also present in some lymphomas. In one case, striking follicular colonization created a markedly nodular growth pattern mimicking a follicular lymphoma. A sixth case had a marked predominance of small, round lymphocytes at some sites, mimicking a small lymphocytic lymphoma. Five of these six cases also had areas more typical of CC/MCL. The seventh case was a CD5-positive splenic marginal zone-like lymphoma (SMZL) with plasmacytic differentiation and circulating villous lymphocytes consistent with a splenic lymphoma with villous lymphocytes (SLVL). These cases illustrate the morphologic spectrum of small B-cell lymphoid neoplasms that have BCL1/cyclin D1 gene rearrangements and overexpression of cyclin D1. Despite the BCL1 translocation and cyclin D1 overexpression, the splenic lymphoma with plasmacytic differentiation was definitely not a CC/MCL and fit the clinicopathologic entity of SMZL/SLVL. The other six cases are best considered CC/MCL variants based on a combined morphologic, immunophenotypic, and genotypic evaluation. Genotypic or immunophenotypic studies to identify cyclin D1 rearrangements and overexpression, although not pathognomonic, are useful in recognizing these variant CC/MCL cases, which can mimic almost any of the other well-described but more indolent low-grade B-cell lymphomas and leukemias. Some of the variant CC/MCL cases had features in common with the CD5+ cyclin D1+ SMZL/SLVL, suggesting a possible relationship between these two otherwise distinct entities. PMID- 8619426 TI - Splenic marginal zone lymphoma. A distinct B-cell neoplasm. AB - The splenic marginal zone is a morphologically and perhaps immunologically distinct B-cell compartment. Lymphomas arising from cells of the splenic marginal zone are rare. Here we describe the morphologic, immunologic, and clinical features of 14 cases. Patient age ranged from 35 to 79 years (median, 68 years) with a male-to-female ratio of 1:1.8. The spleen was uniformly enlarged (median, 1,540 g; range, 388-3,845 g) in all patients, the neoplastic infiltrate had a nodular pattern in three cases, nodular and diffuse in seven cases, and diffuse in four cases. The neoplastic cells had small to medium-sized nuclei with round, oval, or slightly indented contours, small eosinophilic nucleoli, and a moderate amount of pale cytoplasm. Extrasplenic involvement was present in 12 patients. Lymph nodes often had a vaguely nodular pattern and preservation of sinuses; bone marrow was infiltrated focally (seven cases) or diffusely (one case). Five patients had hepatic involvement. Ultrastructurally, neoplastic cells differed from other small B cells and resembled normal marginal zone cells by having long, serpentine rough endoplasmic reticulum profiles. All lymphomas marked as B cells and light chain restriction was demonstrated in 12 cases. Bcl-2 protein expression was present in all cases. Most cases (70%) were negative for DBA.44 (CD72). Plasmacytic differentiation was present in three cases. In conclusion, splenic marginal zone lymphoma is a B-cell neoplasm with distinctive clinical, morphologic, immunologic, and ultrastructural characteristics. PMID- 8619429 TI - Pagetoid melanocytosis. PMID- 8619428 TI - Hepatosplenic gamma delta T-cell lymphoma. PMID- 8619430 TI - Pulmonary lymphoma in an immunocompromised host. PMID- 8619431 TI - Effect of the sickle cell trait status of gametocyte carriers of Plasmodium falciparum on infectivity to anophelines. AB - Insect-reared Anopheles gambiae were experimentally fed with the blood of naturally infected human volunteers carrying gametocytes of Plasmodium falciparum. Infection of at least one mosquito was successful in 86 experiments. For these gametocyte carriers, the hemoglobin types studied were AA (normal, n = 77), AS (heterozygous sickle cell, n = 8), and SS (homozygous sickle cell, n = 1). The mean of the percentages of infected mosquitoes by gametocyte carriers of AS hemoglobin was almost double that of carriers of AA: 30.4% versus 17.5%. The genetic protection in humans conferred by the beta(s) gene in its heterozygous form seems to be associated with an increasing effect on P. falciparum transmission from humans to mosquitoes. The epidemiologic and evolutionary aspects of this finding are discussed. PMID- 8619433 TI - Defecation habits of cats and dogs and contamination by Toxocara eggs in public park sandpits. AB - The defecation habits of cats and dogs in three sandpits in urban public parks were observed by camcorder. Cats were the main cause of fecal contamination of these sandpits. Most (80%) feline defecations occurred at night between 6:00 pm and 6:00 am. Each of the sandpits was used habitually as a defecation site by 4 24 cats, but these cats seemed to defecate elsewhere, as well. Fecal deposits within the sandpits were evenly distributed and did not tend to be concentrated in one area, suggesting that the cats avoided previously deposited feces when choosing a place to defecate. One sandpit was strongly contaminated and two were weakly contaminated with Toxocara eggs. Because sandpits are widely used as play areas for young children, effective sanitation measures should be implemented to prevent the contamination of sandpits by Toxocara eggs. PMID- 8619432 TI - Detection of Plasmodium vivax and Plasmodium falciparum circumsporozoite antigen in anopheline mosquitoes collected in southern Thailand. AB - During a 13-month study on the ecology of malaria vectors in five villages in southern Thailand, Anopheles specimens collected on human-bait, bovid-bait, and in light traps were tested for the presence of Plasmodium vivax and P. falciparum circumsporozoite antigen by enzyme-linked immunosorbent assay. Plasmodium vivax antigen was detected in seven specimens and P. falciparum in 21 specimens, together representing 0.4% of the 7,938 specimens tested. In one village, Palao U, circumsporozoite antigen was detected in 16 (0.7%) of the 2,196 specimens tested. In this village, combined rates of infection with P. falciparum and P. vivax were 0.6% for An. minimus, 1.1% for An. sawadwongporni, and 1.5% for An. maculatus. Circumsporozoite antigen was also detected in An. dirus, An. nivipes, An. barbirostris group, and An. hyrcanus group specimens. Combined P. falciparum and P. vivax entomologic inoculation rates in the wet season (March-October) were 0.05 for An. minimus, An. maculatus, and An. dirus, but 0 for An. sawadwongporni. Rates were higher in the dry season (November-February): 0.26 for An. minimus, 0.13 for An. maculatus, 0.13 for An. sawadwongporni, and 0 for An. dirus. The vectorial capacity, calculated based on human biting rate and rate of survival, of An. minimus during the dry season was more than two-fold higher than that of An. maculatus, the species with the second highest vectorial capacity. PMID- 8619434 TI - Occurrence of hantavirus within the rodent population of northeastern California and Nevada. AB - These studies were initiated to determine the prevalence and hosts of hantaviruses within the rodent population of Nevada and northeastern California. A total of 1,867 rodents were collected, sexed, weighed, identified, and tested by enzyme-linked immunosorbent assay for the presence of antibody against hantavirus nucleocapsid. The primary hosts for hantaviruses in this region were found within the family Muridae (Peromyscus maniculatus. Reithrodontomys megalotis. and Microtus montanus). Studies over time of animals within a defined geographic area indicated that animals with hantavirus antibody are not distributed uniformly over the rodent population in a specific area but were found in foci spanning a distance of only several hundred meters. The antibody prevalence in a given geographic area remained relatively constant with repeated sampling of between 0% and 30%. These data support the hypothesis that rodents within the family Muridae are the primary reservoir for hantaviruses, and the primary risk to biologists for exposure to hantavirus is by contact with members of this family. PMID- 8619435 TI - Short report: polymerase chain reaction detection of hepatitis E virus in north African fecal samples. AB - Epidemics of enterically-transmitted non-A, non-B hepatitis were described in 1983-1984 involving French soldiers in Chad and in 1979-1980 in residents of Algeria. Hepatitis E virus (HEV) was subsequently implicated by serology. In this study, the presence of HEV in patient stool specimens from both outbreaks and from sporadic cases in residents of Chad (1994) was documented. This virus was detected in fecal suspensions by antibody capture of the virus and reverse transcriptase-polymerase chain reaction amplification of the viral RNA in the 3' end of open reading frame 2. Two of five epidemic cases from Chad (1983-1984) were positive, as well as one of five sporadic cases from Chad (1994), and two of three epidemic cases from Algeria (1979-1980). Of these 13 patients, 12 had detectable anti-HEV IgG in their serum. These results confirmed that HEV was the cause of hepatitis in at least five of these 13 patients. PMID- 8619436 TI - Vector competence of Egyptian mosquitoes for Rift Valley fever virus. AB - Reintroduction of Rift Valley fever (RVF) into Egypt in 1993 raised concerns about the potential for Egyptian mosquitoes to transmit the virus. We evaluated the ability of Aedes caspius, Culex pipiens, Cx. antennatus, Cx. perexiguus, Cx. poicilipes, and Anopheles pharoensis collected in the Aswan area and Cx. pipiens collected in the Nile Delta to transmit RVF virus. All mosquito species tested were susceptible to RVF virus infection, with An. pharoensis and Ae. caspius being the most sensitive to infection. However, none of 12 An. pharoensis, including 10 with a disseminated infection, transmitted RVF virus by bite. In contrast, nearly all Cx. pipiens (87%, n = 15) and Cx. perexiguus (90%, n = 10) with a disseminated infection transmitted virus. Overall transmission rates for mosquitoes exposed to hamsters with a viremia > or = 10(7) plaque-forming units/ml were Ae. caspius, 20% (n = 5); Cx. pipiens, 7% (n = 102); Cx. antennatus, 7% (n = 30); Cx. perexiguus, 11% (n = 9); and An. pharoensis, 0% (n = 7). Based on abundance, susceptibility to infection, ability to transmit virus, and feeding behavior, Ae. caspius appeared to be the most efficient vector of the Egyptian mosquitoes evaluated. While less susceptible than Ae. caspius, Cx. pipiens, Cx. antennatus, and Cx. perexiguus were also potential vectors during this RVF outbreak in Egypt. PMID- 8619437 TI - The effectiveness of annual versus biennial mass chemotherapy in reducing morbidity due to schistosomiasis: a prospective study in Gezira-Managil, Sudan. AB - The most serious complication of schistosomiasis is periportal fibrosis, which affects a large number of subjects in endemic areas. Population-based chemotherapy remains the most effective way of controlling this disease. In an attempt to find the best way to deliver chemotherapy to the endemic population, we compared the impact of repeated annual versus biennial mass chemotherapy on morbidity due to schistosomiasis in two villages in Gezira, Sudan. One village was given five rounds of mass chemotherapy annually in the years 1990-1994 while the other village was given three rounds of mass chemotherapy biennially from 1988 to 1994. Before treatment, these villages had similar intensity of infection and prevalence. One round of either annual or biennial treatment reduced the intensity of infection, but not prevalence or morbidity. After two rounds of annual chemotherapy, infection rates, bloody diarrhea, and fibrosis in those 20 years of age and less were significantly reduced. Two rounds of biennial chemotherapy had a similar effect on rates and bloody diarrhea; however, fibrosis was reduced only after the third round of biennial chemotherapy. Prevalence of hepatosplenomegaly increased after both treatment regimens. Reinfection was most prominent in those 5-14 years of age. These findings support the general notion that repeated chemotherapy may be needed in areas of high transmission of schistosomiasis. We recommend two rounds of annual mass chemotherapy to significantly reduce infection and morbidity. PMID- 8619438 TI - Antigenic components of excretory-secretory products of adult Fasciola hepatica recognized in human infections. AB - The antigenic components of excretory-secretory products (ESP) of adult worms of Fasciola hepatica were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using sera from 20 patients infected with F. hepatica. Sera from 184 other parasitic infections and 20 healthy volunteers were also analyzed. It was found that the ESP were composed of more than 11 polypeptides; five components detected in fascioliasis sera had molecular weights of 12.4, 16.4, 19.4, 25, and 27 kilodaltons (kD). Only the 25- and 27-kD components were recognized by all 20 fascioliasis sera. Using the ESP as antigen, it was possible to perform an enzyme-linked immunosorbent assay with a sensitivity of 95% and a specificity of 97%. Sera from other parasitic infections had antibodies to antigenic components with apparent molecular weights of 37, 38.4, 52, 63, 73, 87, 109, and 116 kD that were also found in sera from fascioliasis patients. These findings suggested that the 25- and 27-kD antigenic components may be sensitive and specific for the diagnosis of human fascioliasis. PMID- 8619439 TI - Circulating anodic antigen for detection of Schistosoma mansoni infection in Egyptian patients. AB - We evaluated the ability of circulating anodic antigen (CAA) to identify infection with Schistosoma mansoni in a prospective cohort study of 257 Egyptian men, 147 with infection diagnosed by repeated Kato thick smears, and 110 without detectable infection. The CAA levels were obtained and the stool examinations were performed two weeks and one, two, four, and six months after praziquantel therapy for infected men. A CAA enzyme-linked immunosorbent assay was repeated twice on subjects who were otherwise negative for schistosomiasis. Circulating anodic antigen was detected in 117 cases, with an overall test sensitivity before treatment of 0.8. Sensitivity was related to the intensity of infection, ranging from 1.00 with > 400 eggs per gram (epg) of feces to 0.60 for those with < 100 epg. After praziquantel therapy, the level of the antigen was significantly reduced. Specificity was excellent before treatment (1.00, 95% confidence interval = 0.97-1.0), but it decreased to 0.98 four months after treatment. Likelihood ratios were significant for all titers > or = 4. We conclude that CAA has moderate sensitivity and excellent specificity when used to identify infection with schistosomiasis, as well as to monitor the results of therapy after at least one month after treatment. PMID- 8619440 TI - Acute-phase proteins and serologic profiles of chagasic children from an endemic area in Bolivia. AB - Chagas' disease represents a major public health problem in Latin America. In endemic areas, it is important to detect acute and even asymptomatic infections in children so that specific therapy can be started immediately. We studied 203 sera from children from the region of Cochabamba, Bolivia. A high percentage of seropositive individuals was found in the three villages studies. Levels of alpha 2 macroglobulin (A2M) and C-reactive protein (CRP) increased in a significant number of children with acute Chagas' disease. The combined analysis of serologic and biochemical parameters can define the different stages of acute infection by Trypanosoma cruzi: 1) an early stage, with an increase only in specific immunoglobulin M (IgM) levels; 2) intermediate stages, with high specific IgM and IgG levels and/or high anti-galactose (anti-Gal) levels and increased A2M and/or CRP levels; and 3) a late acute stage, with low IgM levels but high A2M, CRP, anti-Gal, and specific IgG levels. The detection of high IgG levels alone is indicative of the chronic/indeterminate stage of Chagas' disease. We also show serologic differences between seropositive asymptomatic villagers and symptomatic patients undergoing medical care; asymptomatic cases presented higher levels of A2M and lower levels of specific antibodies. PMID- 8619441 TI - Short report: development of a new diagnostic method for Plasmodium falciparum infection using a reverse transcriptase-polymerase chain reaction. AB - We describe here a reverse transcriptase-polymerase chain reaction method for the detection of malaria parasites. Ten in vitro-cultured isolates of Plasmodium falciparum and 16 specimens from patients infected with P. falciparum were used to examine the specificity and sensitivity of the test. The sensitivity of the test was 0.3 parasites per microliter of blood. Specificity was determined by matching the sequences of the specimens' DNA to published sequences of 18S ribosomal RNA genes in the species-specific region. The test proved to be very sensitive and specific for the detection of P. falciparum infection. PMID- 8619442 TI - Serum neopterin, interleukin-4, and interleukin-6 concentrations in cerebral malaria patients and the effect of iron chelation therapy. AB - To determine if iron chelation therapy alters immune responses in children with cerebral malaria, we retrospectively measured mean serum levels of neopterin, interleukin-4 (IL-4), and IL-6 in children who received desferrioxamine B or placebo for three days in addition to quinine-based therapy. Mean levels of neopterin, IL-4, and IL-6 were elevated above the expected normal range on admission. Neopterin correlated significantly with the degree of anemia, IL-4 with the duration of fever prior to admission, and IL-6 with parasite density. Serial measurements of cytokines and neopterin were performed over four days in 39 children, 21 randomized to receive desferrioxamine B and 18 to receive placebo. Mean concentrations of neopterin did not change significantly in either group while levels of IL-4 increased significantly in the placebo group (P = 0.04) but remained unchanged in the desferrioxamine B group. Interleukin-6 concentrations decreased markedly in both groups (P < 0.025). Stable IL-4 levels in children given desferrioxamine B may represent an inhibition of the T helper lymphocyte-2 (TH-2) response resulting from a strengthened TH-1 response associated with iron chelation therapy. Any effect of iron chelation on immunity in the setting of severe malaria will have to be confirmed in future prospective investigations. PMID- 8619443 TI - Ultrastructural aspects of Plasmodium falciparum-infected erythrocyte adherence to endothelial cells of Saimiri brain microvasculature. AB - We have recently shown that some squirrel monkeys (Saimiri sciureus) develop cerebral malaria when experimentally infected with asexual blood stage forms of different Plasmodium falciparum isolates. Since cerebral malaria is neither an inconsistent nor predictable event, several clones of endothelial cells isolated from the squirrel monkey brain microvasculature have been developed. Infected red blood cell (IRBC) adherence involved the knobs and direct membrane interactions through pseudopodes and microvilli on the Saimiri brain endothelial cell (SBEC) surface, similar to that observed with both brain microvascular endothelial cells from a patient who died of cerebral malaria and the rhesus monkey/P. coatneyi cerebral malaria model. The involvement of pseudopodes and microvilli increase the endothelial cell surface for the attachment of IRBCs; however, they are already present before the SBECs are exposed to IRBCs. With some SBEC phenotypes, embedding of IRBCs into the cytoplasma membrane of the endothelial cell was observed, resulting in an extremely close apposition of both SBEC and IRBC membranes during the adherence process. Once IRBCs are adherent, particularly for the embedding type, heterocellular communication-like structures between the cells become apparent. The upregulation of CD36 and intercellular adhesion molecule-1 by soluble recombinant (sr)-tumor necrosis factor-alpha or sr interferon-gamma did not modify the IRBC interactions with SBECs at the ultrastructural level. The study shows further that the observed differences of IRBC adherence are due to unidentified phenotypic differences of SBECs rather than to a parasite isolate or particular endothelial cell receptor-associated phenomenon. Exploring P. falciparum IRBC cytoadherence in the squirrel monkey using a homologous physiologic target cell model in vitro should be useful for the evaluation of vaccine strategies and drugs to prevent human cerebral malaria. PMID- 8619444 TI - Biological behavior of Leishmania amazonensis isolated from humans with cutaneous, mucosal, or visceral leishmaniasis in BALB/C mice. AB - Leishmania amazonensis causes a wide spectrum of disease in humans. In this study, we evaluated BALB/c mice infected with five strains of L. amazonensis isolated from patients with either cutaneous, mucosal, or visceral leishmaniasis. Mice infected with cutaneous and mucosal isolates developed ulcerating footpad lesions with parasite-loaded macrophages and extensive tissue destruction. Skin metastases, early dissemination of parasites to the spleen, and high anti Leishmania antibody levels were also noted. Mice infected with L. amazonensis strains isolated from patients with visceral disease had a controlled infection, with small footpad lesions with mononuclear cell infiltration, few infected macrophages, and granuloma formation. They had no skin metastases, delayed dissemination of the parasite to the spleen, lower levels of IgG and higher levels of IgG2a against L. amazonensis. These findings demonstrate an unexpected resistance of BALB/c mice to the infection with L. amazonensis isolated from patients with visceral leishmaniasis. This resistance seems to be due to differences in these parasites that may be related to the altered course of the disease in humans and in isogenic BALB/c mice. PMID- 8619445 TI - Rapid detection of pyrimethamine susceptibility of Plasmodium falciparum by restriction endonuclease digestion of the dihydrofolate reductase gene. AB - A rapid and simple method to detect pyrimethamine susceptibility of Plasmodium falciparum by analyzing DNA from whole blood is presented. Samples from cultured isolates and from patients infected with P. falciparum were spotted onto filter paper disks, dried, and stored for subsequent analyses. After extracting the P. falciparum DNA using Chelex-100 ion-chelating resin (Bio-Rad, Richmond, CA), the polymerase chain reaction (PCR) was used to amplify the dihydrofolate reductase (dhfr) gene. The PCR product of 727 basepairs was digested with the Alu I restriction endonuclease to detect whether the isolates were sensitive or resistant to the antimalarial drugs pyrimethamine and cycloguanil. This reaction endonuclease digests only DNA from pyrimethamine-sensitive parasites because the recognition locus of Alu I is changed by mutations giving rise to pyrimethamine and cycloguanil resistance. This method is simple and sensitive and could therefore bu used to study the epidemiology of pyrimethamine resistant in P. falciparum. The DHFR gene of pyrimethamine-resistance clones from Vietnamese patients showed three amino acid changes that were previously found in pyrimethamine-resistant isolates. Two other clones, T9/94 and T9/96, originally isolated from a single malaria patient from Thailand, had different DHFR gene sequences. The nucleotide sequence of the DHFR gene from T9/96 was identical to the wild-type DHFR sequence, whereas T9/94 possessed amino acid substitutions at positions 16 and 108 that have been described in cycloguanil-resistant parasites. PMID- 8619446 TI - Simultaneous measurement of proguanil and its metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography, and its preliminary application in relation to genetically determined S-mephenytoin 4' hydroxylation status. AB - A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers. PMID- 8619447 TI - Randomized controlled trial of artesunate plus tetracycline versus standard treatment (quinine plus tetracycline) for uncomplicated Plasmodium falciparum malaria in Brazil. AB - A triple-blind, randomized, clinical trial was undertaken in a Brazilian Amazon region to compare the effectiveness of oral artesunate (seven days, total dose = 0.75 g) plus tetracycline (seven days, total dose = 10.5 g) (AT) and oral quinine (three days, total dose = 6 g) plus tetracycline (seven days, total dose = 10.5 g) (QT) against uncomplicated Plasmodium falciparum malaria. Effectiveness was assessed by cure rates (World Health Organization [WHO]) and parasite clearance at day 2. Patients were randomized, 88 to each group. The groups had similar baseline clinical characteristics. The incidence of side effects was much higher in the QT group (82%) than in the AT group (50%) (P < 0.001). Cure rates were similar: 80% in the AT group and 77% in the QT group (P = 0.68). Parasitemia (by day 2) cleared faster in the AT group than in the QT group (98.5% versus 47.6%, respectively; P < 0.001). These results indicate that the combination of artesunate plus tetracycline is effective in the treatment of uncomplicated falciparum malaria and may provide a useful alternative to other treatment regimens. PMID- 8619448 TI - Short report: severe hiccups secondary to doxycycline-induced esophagitis during treatment of malaria. AB - A 51-year-old man who was treated with quinine and doxycycline for Plasmodium falciparum malaria acquired in West Africa developed hiccups soon after his first dose of antimalarial therapy. Endoscopic examination performed when his hiccups became intractable showed an esophageal erosion and ulcer most likely due to doxycycline. The patient's symptoms resolved on treatment with omeprazole and sucralfate. PMID- 8619449 TI - Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand. AB - One hundred one adult patients with acute uncomplicated falciparum malaria were treated with pyronaridine. All patients were admitted to the Bangkok Hospital for Tropical Diseases for 28 days to exclude reinfection. Sixty-nine patients (Group I) received pyronaridine 1,200 mg over a three-day period and 32 patients (Group II) received 1,800 mg pyronaridine over a five-day period. Cure rates for the two groups were 63% (38 of 60) for Group I and 88% (23 of 26) for Group II (P<0.05). No RII or RIII type response was seen. Mean fever and parasite clearance times were not significantly different in the two groups. The drug was well-tolerated. In vitro drug sensitivity tests of the paired parasite isolates obtained prior to treatment and after recrudescence indicated that the Plasmodium falciparum isolates of the successfully treated patients had a lower mean concentration for 50% inhibition of growth (IC50) and a much narrower range of the individual IC50 values (15.69 +or- 3.82 ng per ml (mean +or- SD)) as compared with those from the recrudescence cases (22.98 +or- 12.05 ng per ml). Nevertheless, there was no evidence of an increase of the IC50 and IC95 values after recrudescence. The results of the study show that pyronaridine alone at a total dose of 1,800 mg given over five days is well-tolerated in patients suffering from acute uncomplicated malaria and has evident activity against multidrug-resistant falciparum malaria. However, it cannot be recommended for use in Thailand as long as the recrudescence rate is as high as 12%. Further studies of its combinations with other antimalarial drugs are needed. PMID- 8619450 TI - Comparative clinical trial of artesunate followed by mefloquine in the treatment of acute uncomplicated falciparum malaria: two- and three-day regimens. AB - The difficulties in treating drug-resistant falciparum malaria in Thailand are compounded by the necessity of giving antimalarials over long periods of time. The resultant decrease in patient compliance not only lowers cure rates but also predisposes to the further spread of drug resistance. We compared the efficacy of two sequential treatment regimens given over two and three days in 111 patients with acute uncomplicated falciparum malaria. Sixty-seven patients received two 400-mg doses of artesunate (total dose = 800 mg) followed by two doses of mefloquine (750 mg given immediately and 500 mg 12 hr later; total dose = 1,250 mg) in Group 1. Forty-four patients (Group II) received four 200-mg doses of artesunate (total dose = 800 mg) given 12 hr apart followed by a mefloquine regimen similar to that for Group I. All patients were admitted to hospital in Bangkok for 28 days to preclude reinfection. Ninety-six patients completed the study. Cure rates for the two groups were 84% (49 of 58) for Group I and 100% (38 of 38) for Group II. The mean parasite clearance time and fever clearance time were significantly shorter in Group II (P < 0.02). There were no serious adverse reactions. All nine of the treatment failures in Group I were of the RI types. The results indicate that the sequential treatment with artesunate followed by mefloquine given over three days is effective and well-tolerated in patients with acute, uncomplicated falciparum malaria and suitable as an alternative treatment for multidrug-resistant falciparum malaria. PMID- 8619451 TI - Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. AB - The functional role of bacteria in the midgut of adult mosquitoes is unknown. In this study, we examined the population dynamics of midgut bacteria of laboratory reared Anopheles stephensi, An. gambiae, and An. albimanus. Mosquito midguts were dissected under sterile conditions and examined for the presence of bacteria using standard microbiologic techniques. Ninety percent and 73% (n = 30) of newly emerged An. gambiae and An. stephensi, respectively, harbored bacteria. In contrast, only 17% (n = 23) of An. albimanus harbored any bacteria. The bacterial population increased 11-40-fold in the presence of a blood meal, but then decreased to pre-blood meal levels in 3-5 days. Pseudomonas cepacia, Enterobacter agglomerans, and Flavobacterium spp. were found in all three anopheline species. Midgut bacteria were acquired both transtadially and through the sugar meal. Transtadial transmission was demonstrated by successfully passaging Escherichia coli HS5 from the larval to the adult stage. However, midgut bacteria were acquired more efficiently through the sugar meal than through transtadial passage. An increase in midgut bacterial counts after mosquitoes were exposed to a bacteria/sugar suspension significantly reduced oocyst infection rates and densities in Plasmodium falciparum-infected mosquito cohorts. Since bacteria occur naturally in wild mosquitoes, it may be possible to modify anopheline vector competence using introduced or indigenous bacteria. PMID- 8619452 TI - The midgut bacterial flora of wild Aedes triseriatus, Culex pipiens, and Psorophora columbiae mosquitoes. AB - The midgut bacterial flora of wild-caught Aedes triseriatus, Culex pipiens, and Psorophora columbiae mosquitoes was investigated. Dissected midgut contents were examined using quantitative aerobic bacterial cultures. Individual colonies (n = 134) were subcultured and identified to species. Midgut bacterial counts changed dramatically during mosquito development. A 280-1,100-fold decrease in the bacterial population occurred between the larval stage and pupal emergence, whereas a subsequent 70-16,000-fold increase occurred after blood-feeding. Bacterial identifications revealed a complex flora with up to nine genera identified during any stage of development. Species most frequently isolated were Serratia marcescens, Klebsiella ozonae, Pseudomonas aeruginosa, and Enterobacter agglomerans. The presence of genetically well-characterized bacteria in the midgut flora of mosquitoes may provide a means of expressing novel genetic products in vector species. PMID- 8619453 TI - Surgical incision and (trauma): the verdict is...guilty! PMID- 8619454 TI - Serial blood lactate levels can predict the development of multiple organ failure following septic shock. AB - BACKGROUND: Despite successful initial resuscitation, septic shock frequently evolves into multiple system organ failure (MSOF) and death. Since blood lactate levels can reflect the degree of cellular derangements, we examined the relation between serial blood lactate levels and the development of MSOF, or mortality, in patients with septic shock. PATIENTS AND METHODS: In 87 patients with a first episode of septic shock, we measured initial lactate (at onset of septic shock), final lactate (before recovery or death), "lactime" (time during which blood lactate was > 2.0 mmol/L, and the area under the curve (AUC) for abnormal values (above 2.0 mmol/L). These measurements were correlated with survival and organ failure and scored for four systems (ie, respiratory, renal, hepatic, and coagulation), adding to a maximal score of 8. RESULTS: Thirty-three (38%) patients survived. Of the 54 (62%) nonsurvivors, the 13 patients who died during the first 24 hours of septic shock had higher initial blood lactate levels than those who died later (mean +/- standard deviation 9.6 +/- 5.3 mmol/L versus 5.6 +/- 3.7 mmol/L, P< 0.05). The 74 patients who survived the first 24 hours of shock, were studied in more detail. On presentation, survivors had a significantly higher mean arterial pressure (76 +/- 12 mm Hg versus 63 +/- 20 mm Hg, P < 0.001) and arterial pH (7.40 +/- 0.07 versus 7.37 +/- 0.09, P< 0.05) than nonsurvivors. Although the differences in initial blood lactate levels between survivors and nonsurvivors did not reach statistical significance (4.7 +/- 2.5 mmol/L versus 5.6 +/- 3.7 mmol/L), only the survivors had a significant decrease during the first 24 hours of septic shock. The survivors had a significantly lower lactime and AUC than the nonsurvivors. The duration of lactic acidosis was the best predictor of survival (multiple regression analysis, R2 = 0.266, P <0.001), followed by age, heart rate, and mean arterial pressure. Patients with lower organ failure scores had lower initial blood lactate, lactime, and AUC. The duration of lactic acidosis was the only significant predictor of organ failure. CONCLUSIONS: In patients with septic shock, serial determinations of blood lactate levels are good predictors of the development of MSOF an death. In this respect, the duration of lactic acidosis is more important than the initial lactate value. Although a number of factors may contribute to hyperlactatemia, these observations are compatible with a direct role of prolonged tissue hypoxia in the development of complications following septic shock. PMID- 8619455 TI - Water-soluble contrast material has no therapeutic effect on postoperative small bowel obstruction: results of a prospective, randomized clinical trial. AB - BACKGROUND: Hyperosmotic water-soluble contrast materials have been fo und to be helpful diagnostic tools in postoperative small-bowel obstruction (POSBO); however, their therapeutic value remains controversial. PATIENTS AND METHODS: A prospective, randomized clinical study was conducted to examine the use of meglumine ioxitalamate as a supplement to the standard conservative treatment of POSBO. Patients with POSBO (n = 50) suitable for a conservative approach were randomized to receive standard conservative treatment with (n = 25) or without (n = 25) the addition of 100 mL of meglumine ioxitalamate via the nasogastric tube (patients with diffuse carcinomatosis and early POSBO were excluded). Both groups were compared for resolution of obstruction, need for surgical relief of obstruction, and complications. RESULTS: Seven (14%) patients required surgery: 3 in the contrast material group and 4 in the control group (P = not significant [NA]. Resolution of symptoms was achieved in nonsurgical patients within an average of 25.7 hours in the contrast material group and 28.7 hours in the control group (P = NS). There was no mortality in this study. In 2 (4%) patients (1 in each group), strangulated bowel was found during surgery, but only the 1 (2%) patient in the contrast material group required bowel resection. No difference was found in the length of hospital stay or rate of complications. There were no complications that could be attributed to the use of the contrast material itself. CONCLUSIONS: Although water-soluble contrast material is a safe and useful diagnostic tool, it offers no advantage as a supplement to the usual conservative treatment of POSBO. PMID- 8619456 TI - Recovery patterns of liver function after complete and partial surgical biliary decompression. AB - BACKGROUND: Recovery patterns of liver function after surgical drainage of obstructing biliary system have not been studied properly, particularly after a single-lobe biliary decompression where atrophic-hypertrophic changes of the liver may affect the recovery of liver functions. PATIENTS AND METHODS: Thirty patients with malignant obstructive jaundice had their liver functions evaluated biochemically both 1 week and 1 day preoperatively, and at 4 points postoperatively: 3 days, 1 week, 3 weeks, and 6 weeks. Half of them underwent complete biliary drainage procedures, whereas the remaining half had partial drainage (decompression of the left lobe only by means of segment III duct enteric bypass). RESULTS: For those with complete drainage, serum alkaline phosphatase (AP) and gamma glutamyl transpeptidase (GGT) were 40% to 50% of preoperative levels 3 days after surgery (P<0.005), and were about twice the norm at 6 weeks. Their serum total and direct bilirubins (TB, DB) were approximately 60% reduced 1 week after the drainage (P<0.05). For partial drainage, serum AP and GGT decreased by 50% at 1 week (P<0.05), but were still very high 6 weeks after the drainage. The TB and DB decreased significantly 1 week postoperatively, and were three times the norm at 6 weeks. Serum albumin decreased sharply at 3 days and returned to normal levels 3 weeks after either complete or partial biliary drainage. Aminotransferase enzymes responded differently between the two groups. The levels dramatically declined one week after complete drainage and were slightly higher than normal thereafter. Following partial drainage, the enzyme levels were unchanged throughout the study period. CONCLUSION: Complete biliary drainage can nearly normalize the liver functions by 6 weeks, and biliary drainage of one lobe of the liver can effectively recover the liver functions. PMID- 8619457 TI - Serum Amylase elevation following hepatic resection in patients with chronic liver disease. AB - BACKGROUND: Factors liable to cause hyperamylasemia after hepatectomy were studied retrospectively in 140 patient with chronic liver disease. METHODS: The pringle maneuver was performed in 113 patients (Pringle group), the hemihepatic vascular occlusion technique in 21 (hemihepatic group), and no vascular occlusion in 6 (no-occlusion group). RESULTS: In the Pringle group, postoperative serum amylase levels were elevated significantly in comparison with the preoperative levels, but were not elevated in hemihepatic and no-occlusions groups. In the Pringle group, there were 4 patients whose postoperative serum amylase levels exceeded 3.5 times the upper limit of the normal range together with serum pancreatic isoamylase or lipase elevation or both. When compared with the other 109 patients, these 4 patients had a significantly longer vascular occlusion time (51 +/-3 minutes versus 94 +/- 8 minutes P<0.005). One of them developed pancreatitis and died from hepatic failure. CONCLUSION: Prolongation of portal congestion carries a potential risk of serum amylase elevation and pancreatitis after hepatectomy in patients with underlying liver disease. PMID- 8619458 TI - Extrapelvic endometriosis: diagnosis and treatment. AB - BACKGROUND: Young women with nondescript abdominal pain can be difficult to diagnose. Although extrapelvic endometriosis is infrequent, we have treated 7 patients over the past 3 years with endometriosis in the abdominal wall, inguinal canal, or surgical incisions as the etiology of their symptoms. PATIENTS AND METHODS: We reviewed the medical records of patients whose final pathology report confirmed a diagnosis of extrapelvic endometriosis. Seven women who were treated at the University of Rochester Strong Memorial Hospital from May 1, 1991 through April 30, 1994 were identified. RESULTS: All patients were premenopausal with no history of pelvic endometriosis. In 4 patients, symptoms were cyclical. Surgical excision was initially curative in 5 patients. Two women required reexcision. The diagnosis of endometriosis was established at exploration by gross appearance and by frozen section. CONCLUSIONS: Endometriosis should be included in the differential diagnosis of a symptomatic mass in a celiotomy scar, the abdominal wall, or the inguinal canal. Principles of management include obtaining an accurate diagnosis and performing an adequate excision to prevent recurrence. PMID- 8619459 TI - The effect of anesthesia type on needle localization breast biopsy: another point of view. AB - BACKGROUND: Efforts directed at early detection of breast cancer have resulted in an increased incidence of nonpalpable mammographic lesions that warrant excisional biopsy. The most common localization method is by use of the hook wire technique, or needle localization biopsy. Although much has been written about the localization technique, the impact of the method of anesthesia on the accuracy of the biopsy and especially on the completeness of the excision has not been clarified. PATIENTS AND METHODS: We studied 450 needle localization breast biopsies to determine whether the type of anesthesia (local versus general) influenced the accuracy and completeness of the biopsy. We compared 153 biopsies performed under local anesthesia to 297 done under general anesthesia. RESULTS: The use of local versus general anesthesia did not affect accuracy; however, it did determine the inability to achieve clean margins (27.6% versus 7.3%, respectively, P <0.02). It was more difficult to excise completely specimens located deeper than 3 cm in the breast, when the localizing needle travelled more than 3 cm, and when the lesions were of the microcalcification mammographic pattern. CONCLUSIONS: For lesions mammographically suspicious for malignancy, mainly those located deeper than 3 cm, general anesthesia is preferred. PMID- 8619460 TI - Acute diverticulitis of the right colon. AB - BACKGROUND: Acute diverticulitis of the right colon is a rare condition with a higher incidence in Oriental populations than in Occidental populations. PATIENTS AND METHODS: A retrospective review was conducted between 1982 and 1993 on 22 surgically treated Chinese patients (14 men, 8 women; mean age 47 years) with documented right colon diverticulitis. RESULTS: Most patients presented with right lower quadrant pain and local peritoneal signs. Acute appendicitis was the preoperative diagnosis in 82% (18/22) of the patients. Only one diagnosis subsequently proved correct. The pathology was easily recognized in 4 patients during surgery, while examination of the resected specimen confirmed the intraoperative suspicion in 13 patients. The right colon was resected and an ileocolonic anastomosis performed in 21 patients; the remaining patient underwent diverticulectomy and drainage of a pericolic abscess. There was no postoperative mortality, and 4 patients developed wound infection. CONCLUSIONS: Our results showed that acute diverticulitis of the right colon was encountered once in 180 cases of acute appendicitis. Local resection or colectomy with primary anastomosis without bowel preparation is the procedure of choice. PMID- 8619461 TI - Treatment of avascular ulcers with cytokine-induced tissue generation and skin grafting. AB - BACKGROUND: Recombinant platelet-derived growth factor (rPDGF-BB) stimulates migration and proliferation of fibroblasts and smooth muscle cells and induces the rapid development of granulation tissue. This study investigated the use of rPDGF-BB and skin grafting to heal avascular ulcers using a rabbit model. We further investigated the effect of a hyaluronic acid carrier and a vascular pedicle on rPDGF-induced tissue generation in this model. METHODS: Large avascular ulcers were created on both ears of New Zealand white rabbits. One ulcer was treated with topical rPDGF-BB, the other with control buffer. After 5 or 7 days, the amount of granulation tissue migration from the wound margin was measured, and the wounds were skin grafted. In another group of ulcers, rPDGF-BB treatment was combined with a hyaluronic acid disk or an intact central axial ear artery and vein. RESULTS: Whereas control wounds generated 1.00 +/- 0.27 mm and 1.75 +/- 0.25 mm of granulated tissue migration from the wound margin by days 5 and 7, respectively, rPDGF-BB treatment increased the amount of granulation tissue migration to 1.88 +/- 0.23 mm and 3.00 +/- 0.86 mm by days 5 and 7 after treatment, respectively (P <0.05 at both time points). Hyaluronic acid disks stimulated 2.50 +/- 0.22 mm of granulation tissue migration by day 7 in controls; when rPDGF-BB was added to the carrier, the migration increased to 4.50 +/- 0.29 mm from the wound margin (P<0.05). Granulation tissue migration was further increased with an intact vascular pedicle: 5.75 +/- 0.48 mm in controls versus 7.75 +/- 0.25 mm with rPDGF-BB treatment (P<0.01). Skin grafts applied to the treated ulcers failed to survive in all groups except those with intact vascular pedicles. CONCLUSIONS. This study demonstrates that rPDGF-BB can increase the amount of granulation tissue generated over an avascular wound. This tissue generation is enhanced by both a hyaluronic acid carrier and a vascular pedicle. A vascular pedicle was required for skin graft survival. This study supports the role of hyaluronic acid in rPDGF-BB induced tissue generation and the requirement of a direct blood supply for functional cytokine-induced tissue generation. PMID- 8619462 TI - Thrombolysis theraphy in patients with femoropopliteal synthetic graft occulsions. AB - BACKGROUND: The aim of this study was to evaluate the efficiency of thrombolysis in the presence of an occluded femoropopliteal synthetic graft. PATIENTS AND METHODS: Over a 3-year period, 46 occluded femoropopliteral grafts were treated with urokinase and reconstruction. The cases were divided into three groups: group 1 (n=25), complete thrombolysis followed by reconstruction or angioplasty or both; group 2 (n=5), complete thrombolysis alone; and group 3 (n=16), failure of thrombolysis requiring reconstruction or leading to amputation. Patients were completely observed after treatment for more than 1 year. RESULTS: There are no fatal complications among patients with thrombolytic therapy. In group 1, the 3 year patency rates were 12% and the 3-year limb salvage rates were 77%. In group 2, the 3-year patency rates and the limb salvage rates were 20% and 80%, respectively. The group 3 patency rates and the limb salvage were 8% and 40%, respectively. The best results were achieved in patients who had thrombolysis followed by reconstruction (group 1) and in those who had thrombolysis alone (group 2). limb salvage was poor in patients with failure of lytic therapy regardless of the reconstruction (P<0.01). CONCLUSION: The use of intra-arterial urokinase followed by secondary vascular reconstructive procedures was studied. The patient with synthetic graft occlusion still has a reasonably favorable prognosis for long-term limb salvage when thrombolysis is successful. PMID- 8619463 TI - Prospective study of safety of lower extremity phlebography with nonionic contrast medium. AB - BACKGROUND: Lower extremity deep venous thrombosis (DVT) following hig h osmolar ionic contrast phlebography has been reported to vary between 9% to 31%. The purpose of this study was to determine the incidence of minor and major adverse reactions and postphlebographic DVT when using nonionic contrast (iopamidol). PATIENTS AND METHODS: One hundred fifty-seven patients with clinically suspected DVT were studied prospectively. One hundred eleven patients had prephlebography duplex ultrasound, and 102 patients were examined in the vascular laboratory for delayed side effects 1 week after phlebography. The presence of phlebography induced DVT was assessed using color duplex ultrasound. The mean amount of contrast used 102 ml. RESULTS: Minor adverse reasons, including nausea, local pain, and dizziness, occurred in 11 (7%) of 157 patients; however, no major complications or postphlebographic DVT was found in the 102 patients who underwent postphlebography duplex ultrasound. Phlebography and pre- and postphlebography duplex ultrasound showed no acute DVT in 70 patients. The maximum hypothetical true rate of major complications (ie, postphlebography DVT) that would result in no detectable events in a population of 102 patients with follow-up (for a probability of P <0.05) is 2.9. CONCLUSION: lower extremity phlebography using nonionic contrast material is safe, with no incidence of postphlebography DVT in our series. Its utilization should be encouraged if duplex ultrasound is not available. PMID- 8619465 TI - Conversion of failed or complicated vertical banded gastroplasty to gastric bypass in morbid obesity. AB - BACKGROUND: Previous studies have documented a significantly better we weight loss for gastric bypass (GBP) than for vertical banded gastroplasty (VGB). Additional problems associated with VBG include intractable vomiting or gastroesophageal (GE) reflux, intragastric migration of the polypropylene band, staple line disruption, or inadequate weight loss due to excessive ingestion of high-calorie liquid or soft carbohydrates. PATIENTS AND METHODS: Fifty-eight morbidly obese patients underwent conversion from VBG to GBP for either weight loss failure (15) or complications of VBG (43), including 2 who where referred with anastomotic leaks and peritonitis, 3 with band erosion, 15 with staple line disruption, and 23 with stomal stenosis, of whom 6 had severe GE reflux, with a Barrett's esophagus in 1. RESULTS: Percentage of excess weight loss in the 53 patients followed up for at least 1 year after conversion increased from 36% +/- 24% to 67% +/- 18%, and in the 15 "sweets eaters" from 20% +/- 19% to 70% +/- 19% (both P <0.001), was equal to weight loss after primary GBP, and was reasonably constant over 8 years in those patients who could be contacted for follow-up, although average follow-up after 5 years was only 45% All patients had resolution of GE reflux symptoms immediately after surgery and for at least 1 year or at last contact. Complications of conversion included 2 anastomotic leaks with major wound infections (1 in a referred patient requiring emergency subtotal gastrectomy following a VBG leak), 3 staple line disruptions (2 subclinical), 3 small-bowel obstructions, and 20 marginal ulcers or stomal stenoses (all responded to endoscopic balloon dilation or acid reduction therapy). Hemoglobin, calcium, and vitamin B12 levels remained within normal levels with prophylactic supplementation in patients who returned for follow-up evaluation. CONCLUSIONS: These data support the efficacy of conversion to GBP in morbidly obese patients with a failed or complicated VBG. PMID- 8619464 TI - Long-term subjective functional outcome of surgery plus postoperative radiotheraphy for advanced stage oral cavity and oropharyngeal carcinoma. AB - BACKGROUND: Although long-term cures have been achieved for locally advanced squamous cell carcinomas of the head and neck, there is a paucity of information available regarding patients' perspectives of their functional outcome. PATIENTS AND METHODS: Thirty-five long-term survivors free of disease following surgery and postoperative radiotherapy for advanced cancers of the oral cavity and oropharynx were sent questionnaires to evaluate their long-term functional outcome after therapy. The questionnaires included a subjective performance status scale that assessed the patient perceived (1) ability to eat in public, (2) understandability of speech, and (3) normalcy of diet. Twenty-nine of 35 patients participated in this function assessment and are the subjects of this report. RESULTS: The mean function scores for all patients were as follows: 72 for eating in public, 69 for understandability of speech, and 58 for normalcy of diet. Functional results were further analyzed by T stage and anatomic subsite. Inferior results were noted with increasing T stage. A two-way analysis of variance showed that this difference was significant even after adjusting for the effect of anatomic subsite (P = 0.0002, P = 0.018, and P = 0.0018 for the three outcome variables). In addition, patients with base of tongue lesions had a worse functional outcome for both early T state (T1/T2) and advanced T stage (T3/T4) when compared to other subsites. This difference averaged across T stage was statistically significant for understandability of speech (P = 0.0019) and normalcy of diet (P = 0.013), but was not significant for eating in public (P = 0.16). CONCLUSIONS: This performance status scale was found to be a useful tool for functional assessment following definitive therapy for advanced stage head and neck carcinomas. These subjective functional scores deteriorated with increasing T stage. In addition, functional scores for oral tongue, floor of mouth, and tonsillar primaries were superior to those for base of tongue lesions. These functional outcome scores are consistent with the extent of surgery required for the base of tongue subsite and are in direct relation to the patients' T stage in this study population. PMID- 8619466 TI - Postoperative complications requiring relaparotomies after 700 gastretomies performed for gastric cancer. AB - BACKGROUND: Prevention of fatal postoperative complications and improved management of patients with complications are important means of increased survival in gastric cancer patients. PATIENTS AND METHODS: A study of 700 patients undergoing gastrectomy was performed to examine factors that contributed to a high rate of postoperative complications. RESULTS: Of 700 patients undergoing gastrectomy for adenocarcinoma, 40 (5.7%) underwent reexploration because of serious complications. The frequency of the relaparotomies varied from 2.1% and 4.4% after regular subtotal and total gastrectomies, respectively, to 20% and 30.4% after palliative and conventional total gastrectomies, respectively. The complications that required reexploration most frequently were anastomotic leakage and incompetence of sutures (11, 27.5%), intra-abdominal abscesses (8, 20%), and pancreatic necrosis (7, 17.5%). A combination of preventive measures allowed the attainment of low rates of esophagojejunal anastomotic leakage (0.8%). CONCLUSION: We believe that the decision to perform an urgent reexploration, based on clinical findings, should generally be made by a group of experienced surgeons (not only the primary surgeon). Timely relaparotomy prevented death in 37.5% of the patients with serious acute postoperative complications. PMID- 8619467 TI - Anti-TNFalpha therapy improves survival and ameliorates the pathophysiologic sequelae in acute pancreatitis in the rat. AB - BACKGROUND: Elevated levels of tumor necrosis factor-alpha (TNFalpha) have been measured in a lethal model acute pancreatitis (AP) and may contribute to the pathophysiologic sequelae of the disease. METHODS: To determine the significance of anti-TNFalpha therapy on survival and disease manifestations in a clinically relevant model of AP, a rat model was developed using a retrograde pancreatic ductal infusion of bile. Animals were randomized to no treatment (n = 30) or treatment with anti-TNFalpha antibody 15 minutes prior to induction of AP (n = 30). Five treated and 5 untreated rats were killed at various time periods up to 72 hours to provide temporal characterization of TNFalpha activity in AP. RESULTS: A burst Of TNFalpha activity in the serum of untreated pancreatitis animals between 1 and 3 hours after induction of the disease is prevented by pretreatment with anti-TNFalpha antibody. CONCLUSIONS: These findings provide a plausible mechanism for the improvement in biochemical and histologic parameters as well as in overall survival in an experimental model of acute pancreatitis in the rat. PMID- 8619468 TI - The learning curve for totally extraperitoneal laparoscopic inguinal hernia repair. AB - BACKGROUND: Several laparoscopic techniques have been introduced to re pair inguinal hernia, the newest and most promising being a totally extraperitoneal approach. Nevertheless, the surgeon may encounter several complications and technical difficulties associated with the transition from the conventional anterior operation. METHODS: In late 1993 and 1994, 120 patients were operated on for inguinal hernia using the totally extraperitoneal approach by four laparoscopic surgeons inexperienced in this new technique in a secondary referral setting. Their learning curve was assessed through operation time, perioperative and postoperative complications, and technical difficulties. RESULTS: Median operative time decreased significantly (P = 0.0003) when going through the learning curve. During the initial part of the learning curve, conversion to another technique was necessary in 10 (8%) cases, and in 6 of these cases, conversion was needed for a peritoneal tear (relative risk for conversion if peritoneal tear was present: 4.0; 95% confidence interval 1.2 to 13.1, P = 0.025). The median operative time for Nyhus type IIIb and IVb hernias was significantly longer than for other types (70 versus 55 minutes, P = 0.003). Median postoperative stay was 2 days (range 0 to 7). There were 10 recurrences within 6 months due to technical or judgement errors. CONCLUSIONS: For surgeons, the learning curve for totally extraperitoneal laparoscopic hernia repair can be overcome; however, the presence of an experienced surgeon during the procedure is vital, as this may prevent unnecessary recurrences. PMID- 8619469 TI - Extended lateral segmentectomy using intraoperative ultrasound to obtain a partial liver graft. AB - The important features of extended lateral segmentectomy to obtain a partial liver graft comprising the left lateral segment and the left half of the medial segment are described with special reference to anatomical variation of the hepatic venous system. Ramification patterns of the hepatic vein tributaries around the juncture of the major hepatic veins with the inferior vena cava are delineated before starting liver resection, using intraoperative ultrasound. The left medial vein draining the left part of the medial segment is recognized close to the confluence of the middle and left hepatic veins. This tributary flows into the left hepatic vein in the majority of cases, but sometimes into the middle hepatic vein. The liver transection line is established in order to obtain the graft, including the drainage area of the left medial vein. Intraoperative ultrasound is indispensable for identifying the left medial vein in extended lateral segmentectomy. PMID- 8619470 TI - Laparoscopic Billroth II distal subtotal gastrectomy with gastric stump suspension for gastric malignancies. AB - BACKGROUND: Laparoscopy has played an ill-defined role as a diagnostic tool for the staging of gastric and other intra-abdominal malignancies for a long time. The widespread use of the laparoscopic approach for the treatment of some benign abdominal diseases, such as biliary lithiasis and gastroesophageal reflux disease, has encouraged the authors toward its use in the treatment of malignant gastric neoplasms, both for palliation and for curative surgery. METHODS: A five puncture technique for laparoscopic distal subtotal gastrectomy, omentectomy, division of the left gastric artery at its origin, and D1 lymph node dissection has been developed by the authors, and is fully depicted and discussed. Reconstruction of digestive continuity is achieved through a posterior transmesocolic side-to-side stapled gastrojejunostomy, facilitated by an original method of suspension of the gastric stump to the anterior abdominal wall. RESULTS: In a preliminary series of 10 cases, this technique was demonstrated to be safe, showing no mortality, and having morbidity rates comparable to those of open surgery. CONCLUSION: The operation is effective, with a mean number of resected nodes comparable to that usually achieved in open surgery, and no cases of conversion to laparotomy. PMID- 8619471 TI - Pelvic exenteration and its modifications. AB - Since it was first reported in 1948, pelvic exenteration has been used in the treatment of advanced pelvic cancers. The original procedure has been modified in an attempt to preserve urinary or fecal continence. A literature review was performed on selected series of total pelvic exenterations and modified pelvic exenterations in order to assess and discuss the different types of pelvic exenterations and the indications, contraindications, morbidity, mortality, and results of these procedures. According to the series reviewed, morbidity after pelvic exenteration ranges between 32% and 84%, postoperative mortality ranges from 0% to 14%, and and 5-year survival varies from 23% to 68% These numbers indicate that total pelvic exenteration and its modifications are a complex group of surgical procedures with significant early and late postoperative morbidity and mortality. While the authors do feel that these findings indicate that pelvic exenteration should only be undertaken by experienced surgeons at specialized centers, the authors caution that, about all, their findings indicate that the potential curability of a patient with adjacent organ involvement should not be compromised by doing less than an en bloc resection. PMID- 8619472 TI - A gas chromatographic analysis of fecal short-chain fatty acids, using the direct injection method. AB - A simple, reproducible, and rapid gas chromatographic method for short-chain fatty acid determination in human feces was developed. It involves direct injection of fecal supernatants into the gas chromatograph, without any pretreatment. Contamination of the gas chromatographic column with nonvolatile fecal material was prevented by the use of a glass liner in the injector. This liner, which acted as a precolumn, was stoppered with a glass wool plug at the lower end of the liner. Injection was performed against the glass wall of the liner, ensuring an immediate contact of the injected sample with the hot glass wall. More than 100 injections of fecal supernatants could be carried out before the liner had to be replaced by a new one. Peak tailing and ghosting was prevented by the use of formic acid in the fecal samples. The method gave sharp peaks with baseline separation for all the fatty acids. PMID- 8619473 TI - Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose. AB - Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose. This compound has a biotin as one functional group and a nitrilotriacetic acid as the other. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot. PMID- 8619474 TI - A "double adaptor" method for improved shotgun library construction. AB - The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation. PMID- 8619475 TI - Formation of N-substituted 2-iminothiolanes when amino groups in proteins and peptides are modified by 2-iminothiolane. AB - The reagent 2-iminothiolane (2-IT) is used to introduce thiol groups into proteins and peptides by reactions of their amino groups. In this study, we report that the thiol adduct initially formed by the reaction of an amine with 2 IT (a 4-mercaptobutyramidine) is unstable and decays by a first-order process to a nonthiol product (an N-substituted 2-iminothiolane) with the loss of ammonia. The thiol adducts derived from amines of low pKa values (approximately 8; e.g., alpha-amino groups in peptides) decay more rapidly than those derived from amines of high pKa values ( similar 9.5; e.g., benzylamine, ethanolamine, lysine residues in proteins), with half-lives at pH 8 ranging from 0.3 to 3 h at 23 degrees C, and from 1 to 44 h at 0 degrees C. In the case of reactions of peptides with 2-IT, the substituents at the alpha-carbon also influence the decay of the initial thiol adducts. The decay of the initial thiol adduct to an N substituted 2-iminothiolane was confirmed for the reaction between benzylamine and 2-IT by the isolation of N-benzyl-2-iminothiolane and its characterization by elemental analysis and mass spectrometry. The decay of the initial 4 mercaptobutyramidine is prevented if the thiol group is capped, e. g., in the form of a disulfide group, or if the solution is acidified (pH 3 to 4). Immediate capping of the thiol is, therefore, recommended when using 2-IT in the formation of bioconjugates. For amines of high pKa, the N-substituted 2-iminothiolane product can be cleaved by hydroxylamine, resulting initially in a thiol which then decays to N-hydroxy-2-iminothiolane regenerating the original amine. For amines of low pKa, the N-substituted 2-iminothiolane product can be hydrolyzed at pH 5 to generate a stable thiol with an amide functionality (an N-substituted 4 mercaptobutyramide). PMID- 8619477 TI - A comparison of measured and calculated single- and double-stranded oligodeoxynucleotide extinction coefficients. AB - The hyperchromicity resulting from exhaustive hydrolysis of several single stranded and double-stranded oligodeoxynucleotides (ODN) with snake venom phosphodiesterase or snake venom phosphodiesterase/DNase I was used to measure the ODN's extinction coefficient at 260 nm. These extinction coefficients were compared with those obtained using nonempirical approximations. For the oligodeoxynucleotides investigated, the nonempirical approximations yielded extinction coefficients within 20% of the measured values. PMID- 8619476 TI - Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase. AB - We now report a solid-phase assay for CMPNeuAc: Galbeta1-3/4GlcNAc-R alpha-2,3 sialyltransferase (alpha2,3ST) that is nonradioactive and allows specific identification of the sialylated product. An acceptor glycoprotein, desialylated fetuin, is immobilized on a microtiter plate. The transfer of sialic acid from CMPNeuAc generates the product NeuAc alpha2-3Gal beta1-4GlcNAc-R that is specifically bound by biotinylated Maackia amurensis leukoagglutinin (MAL). The binding of biotinylated MAL is measured with either an absorbance-based reagent (streptavidin conjugated to alkaline phosphatase) or a light-based reagent (streptavidin conjugated to the bioluminescent protein aequorin, Aqualite). The rat liver alpha2,3ST was used to optimize the assay. The formation of product is linear with respect to time and dependence on the amounts of CMPNeuAc, enzyme, and acceptor coated on the plates. As little as 0.2 microU of enzyme can be measured using the streptavidin-aequorin reagent. The assay is useful with crude tissue extracts, as demonstrated by the determination of the alpha2,3ST activity in human serum and in microsomes of HL-60 and Chinese hamster ovary cells. PMID- 8619478 TI - N,N'-bis[3,3'-(dimethylamino)propylamine]-3,4,9, 10-perylenetetracarboxylic diimide, a dicationic perylene dye for rapid precipitation and quantitation of trace amounts of DNA. AB - A novel dicationic dye with a polycyclic aromatic perylene core and flexible cationic side chains- N,N'-bis[3, 3'-(dimethylamino)propylamine]-3,4,9,10 perylenetetracarboxylic diimide-termed "DAPER," was synthesized and characterized. The dye appears to exist in a highly stacked form in aqueous solution. DAPER precipitates extremely low concentrations of DNA very rapidly, efficiently, and with a stoichiometry of one tightly bound dye per DNA base pair, corresponding to a neutral complex. Precipitation may occur due to side-by-side association between the polyanionic DNA helix and polycationic dye stacks. DNA precipitation by DAPER is less sensitive to DNA concentration and length, and prevailing salt concentrations, than precipitation with ethanol or propanol. DAPER can be quantitatively extracted from DNA into a standard phenol:chloroform mixture under slightly alkaline conditions. The recovered DNA is suitable for treatment with enzymes typically used in DNA sequencing procedures. The amount of DNA precipitated is accurately determined by visible absorption or fluorescence spectroscopic analyses of the phenol:chloroform extracts. Several samples of DNA can be precipitated, recovered, and quantitated in about 1 h using standard microscale procedures and equipment. The unique qualities of DAPER provide the basis for a very sensitive, rapid, and versatile method for simultaneous precipitation and quantitation of microgram and submicrogram amounts of DNA. PMID- 8619479 TI - Nitric oxide production by the rat insulinoma cell line, RINm5F, Is specific for IL-1: a spectrophotometric IL-1 bioassay. AB - Cytokines inhibit glucose-induced insulin secretion from pancreatic beta-cells by stimulating the expression of nitric oxide synthase and the increased production of nitric oxide (NO). We have found that the rat insulinoma cell line, RINm5F, responds specifically and linearly to murine and human interleukin-1beta (IL 1beta) and IL-1alpha in the range of 0.1 to 1 unit/ml to produce nitric oxide. Other cytokines, including IL-2, IL-4, IL-6, IL-9, IL-11, IL-15, tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide fail to stimulate nitric oxide formation by RINm5F cells either alone or in combination. In addition, these cytokines do not significantly potentiate or attenuate the IL-1 response. This unprecedented specificity to IL-1 has been further developed as a sensitive and specific assay for IL-1 bioactivity. Quantitation by this new bioassay of human IL-1beta and IL-1 released from activated murine peritoneal macrophages showed a close correlation with the quantitation of IL-1 by enzyme immunoassay (ELISA). This new bioassay, which is specific, nonradioactive and inexpensive, represents a significant improvement over current bioassays for IL-1. PMID- 8619480 TI - Quantitative competitive polymerase chain reaction: analysis of amplified products of the HIV-1 gag gene by capillary electrophoresis with laser-induced fluorescence detection. AB - A quantitative competitive polymerase chain reaction (PCR) assay for the detection and quantification of HIV-1 has been developed using capillary electrophoresis. Separation of the PCR products is carried out in coated capillaries filled with replaceable linear polyacrylamide, and detection is performed using laser-induced fluorescence. The quantitative capabilities of this assay are described. We show results from analysis of a noninfectious plasmid encoding the HIV genome and integrated proviral DNA. Potential applications of this assay are discussed. PMID- 8619481 TI - Calibration of oxygen-dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. AB - Oxygen-dependent quenching of phosphorescence is a function of the frequency of collision between the phosphor and molecular oxygen and of the efficiency of energy transfer during these collisions. Thus, quenching is dependent on the rate of diffusion of the phosphor and its molecular environment. For measurements in biological samples, the Pd-porphyrin is bound to serum albumin, and this provides a uniform microenvironment for the phosphor which is relatively unaffected by changes in the pH and ionic composition of the medium. Calibration of the phosphor is of particular value because it is absolute, i.e., the calibration is valid independent of the laboratory and the time of measurement. This paper reports the calibration constants determined for Pd-meso-tetra (4-carboxyphenyl) porphine, as measured by two independent methods: by stoichiometric titration of the oxygen with ascorbate in the presence of ascorbate oxidase and by comparison with a high-accuracy oxygen electrode. The measurements were carried out in a specially designed thermostatted vessel in which the oxygen electrode and phosphorescence lifetime measurements of oxygen were made simultaneously. The calibration constants for the oxygen-dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine were determined as a function of albumin concentration, ionic strength in medium, pH, and temperature. PMID- 8619482 TI - Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids. AB - Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96 well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification. PMID- 8619483 TI - Preparation of colloidal gold particles of various sizes using sodium borohydride and sodium cyanoborohydride. PMID- 8619484 TI - A method to radioiodinate hyaluronic acid and its use as a probe to detect hyaluronic acid-binding proteins. PMID- 8619485 TI - Enhancement and stabilization of dithizone vital staining for zinc in rat organs using adduct formation. PMID- 8619486 TI - Catalytic hydrogenation of leukotriene B4 enhances sensitivity and specificity of gas chromatography-tandem mass spectrometry techniques and enables simultaneous analysis with cysteinyl leukotrienes in biological fluids. PMID- 8619488 TI - Detection of "lost" plasmids from Escherichia coli using excess ampicillin. PMID- 8619487 TI - Differential whole-cell extract preparation and electrophoretic mobility shift assay to evaluate the effect of tyrosine phosphatases on DNA binding activity of transcription factors. PMID- 8619489 TI - S1 nuclease protection assay using streptavidin dynabeads-purified single stranded DNA. PMID- 8619490 TI - A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. PMID- 8619491 TI - Beta-lactoglobulin B: a proposed standard for the study of reversible self association reactions in the analytical ultracentrifuge? AB - Bovine beta-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and pH 2.0-3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer-dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Omega function all returned identical parameters. PMID- 8619492 TI - Gas chromatography-mass spectrometry analysis of vitamin E and its oxidation products. AB - To facilitate studies of vitamin E ( alpha-tocopherol) antioxidant actions in tissues, we have developed a stable isotope dilution capillary gas chromatography mass spectrometry assay for alpha-tocopherol and its three principal oxidation products, alpha-tocopherolquinone, 5,6-epoxy-alpha-tocopherolquinone, 2, 3-epoxy alpha-tocopherolquinone, and for alpha-tocopherolhydroquinone, a reduction product of alpha-tocopherolquinone. Deuterium-labeled internal standards (5, 7 [2H3-methyl]-alpha-tocopherol, 2,6-[2H3-methyl]- alpha-tocopherolquinone, 2,6 [2H3-methyl]-5,6-epoxy- alpha-tocopherolquinone, 2,6-[2H3-methyl]-2,3-epoxy- alpha-tocopherolquinone, and 2-[2H3-methyl]- alpha-tocopherolhydroquinone are added to cell or tissue homogenates. The products then are extracted and converted to O-trimethylsilyl derivatives. Products are analyzed by capillary gas chromatography with on-column injection and detected by selected ion monitoring of characteristic fragment ions in electron ionization mode. Standard curves were linear from 25 fmol to 2 pmol for products and from 25 fmol to 4 pmol for alpha tocopherol. The use of 2H6- and 2H3-internal standards for alpha tocopherolquinone and alpha-tocopherolhydroquinone permits simultaneous analysis of both products despite possible redox interconversion during sample workup. alpha-Tocopherol oxidized in microsomes treated with azo-bis(amidinopropane HCl) was quantitatively accounted for as the epoxyquinones, alpha-tocopherolquinone, and alpha-tocopherolhydroquinone. However, over half of the oxidation products were present in microsomes as acid-labile tocopherone precursors. This method permits comprehensive assessment of vitamin E status in tissues and quantitative studies of vitamin E antioxidant actions and turnover. PMID- 8619493 TI - A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride membrane after thin-layer chromatography blotting and in situ mass spectrometric analysis of the ligands. AB - A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride (PVDF) membrane has been developed. Glycosphingolipids were separated on a high-performance thin-layer chromatography (HPTLC) plate and transferred onto a PVDF membrane by TLC blotting [Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221, 312-316]. The PVDF membrane was blocked with phosphate-buffered saline containing 4% casein and 0.1% methionine and overlaid with 35S-labeled Escherichia coli possessing K99 fimbriae (E. coli K99) at 37 degrees C for more than 2 h. Binding of the 35S-labeled E. coli K99 was detected with a bioimaging analyzer. Radioactivities were located on the bands corresponding to N-glycolylneuraminic acid containing glycosphingolipids such as sialylparagloboside, GM2, and GM3 with hydroxy fatty acid in ceramide moiety, and a weak binding was detected on the band of N-acetylneuraminylparagloboside. Furthermore, an in situ mass spectrometric analysis of the ligand glycosphingolipids on the membrane was demonstrated. The present method has several advantages compared with the overlay binding assay on the HPTLC plate as follows: (i) the method is simple and rapid; (ii) the membrane is easy to handle; (iii) binding is clear with low background; (iv) a small amount of [35S]methionine is required; and (v) sensitive ligand characterization can be done by in situ mass spectrometric analysis. PMID- 8619494 TI - Analysis of total lipid extracts from human liver by 13C and 1H nuclear magnetic resonance spectroscopy. AB - Lipid extracts of liver tissue from normal donors and from patients with acute liver failure were analyzed by 13C NMR and 1H NMR at 9.4 T. The spectra allowed estimation of (i) the free fatty acid to total fatty acid chains ratio, (ii) the polyunsaturated to monounsaturated lipids ratio, (iii) the glycerophospholipids to triacylglycerols ratio, (iv) the total cholesterol to total fatty acids molar ratio, (v) the acylcholesterol to cholesterol ratio, (vi) the phosphatidylcholine to phosphatidylethanolamine molar ratio, and (vii) the unsaturation ratio of the fatty acid chains. The values obtained for normal livers agree with literature data on lipid composition obtained by other techniques. The two pathologic livers differ from the normal in the composition of some lipids and show a higher free fatty acid to total fatty acid chains ratio and a lower polyunsaturated to monounsaturated lipid ratio, a lower acylcholesterol to cholesterol ratio, and a lower phosphatidylcholine to phosphatidylethanolamine ratio, whereas the ratio of total cholesterol to fatty acids and the unsaturation ratio remained unchanged. The detailed assignment of the 13C NMR spectra of lipid extracts from normal and pathologic human liver may contribute to direct future research programs on 13C NMR spatially localized spectroscopy of the liver in vivo. PMID- 8619495 TI - Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity. AB - A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a. PMID- 8619496 TI - The use of t-butyl hydroperoxide as a probe for methionine oxidation in proteins. AB - The susceptibility of native recombinant interferon gamma (rIFN-gamma, Actimmune) and recombinant tissue-type plasminogen activator (rt-PA, Activase) to methionine oxidation when treated with the oxidizing agent t-butyl hydroperoxide (TBHP) was investigated. The results showed that two of the five methionine residues in rIFN gamma were susceptible to oxidation by TBHP, while three of the five methionines in rt-PA were found to be oxidizable. The oxidized methionine residues were found to be in the sulfoxide [Met(O)] form, and no other residue(s) appeared to be modified during the TBHP treatment. These results also showed that during treatment of a native protein with TBHP only the exposed methionine residues were oxidized. The biological activity of both molecules were unaffected by the treatment with TBHP. A comparative study between TBHP and hydrogen peroxide (H2O2) demonstrated that H2O2 was also a methionine-specific oxidizer. However, this study also showed that H2O2 was not able to distinguish between exposed and buried methionine residues, as significant portions of all five methionine residues in native rIFN-gamma were oxidized by treatment with H2O2. TBHP should be useful for identifying surface methionine residues in a protein of unknown structure and a valuable reagent for methionine oxidation in pharmaceutical stability studies. PMID- 8619497 TI - Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form. AB - A fundamental step in the pathogenesis of spongiform encephalopathies (prion diseases) is the conversion of the cellular isoform of prion protein (PrPC) into the infectious form (scrapie isoform, PrPSc), apparently by a conformational mechanism. Comparison of the native secondary and tertiary structures of both proteins is essential to elucidate the molecular basis of this transformation. To obtain sufficient quantities of native-like PrPC, we have developed a semipreparative method to purify PrPC from hamster brains. PrPC was solubilized from purified synaptosomal and microsomal membranes by the nonionic detergent n octyl- beta-glucopyranoside; the soluble fraction was loaded at pH 7.5 onto a semipreparative cation-exchange TSK-SP-5PW (HPLC) column. The fractions eluted by linear NaCl gradient and enriched for PrPC were sequentially purified using an immobilized ion-affinity HPLC column charged by Co2+, followed by wheat germ agglutinin (WGA)-affinity HPLC or size-exclusion HPLC (SE-HPLC) using a TSK G3000SW column. More than 95% purity was achieved after SE-HPLC as estimated by quantitative densitometry of the silver-stained SDS-PAGE gel; the recovery of total brain PrPC was >/=8%. The purified PrPC was a monomer with an intact N terminus, and with a Stoke's radius of 26 A, corresponding to that expected from the molecular weight for a native protein. The presence of the native-like conformation was further verified by peptide mapping after limited trypsin proteolysis, and by the apparent unfolding in guanidine hydrochloride, as detected by SE-HPLC. PMID- 8619498 TI - High-performance liquid chromatographic assay for tryptase based on the hydrolysis of dansyl-vasoactive intestinal peptide. AB - A rapid, sensitive, semiautomated method to measure the activity of mast cell derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin. PMID- 8619499 TI - A modified Coomassie blue staining of proteins in polyacrylamide gels with Bismark brown R. AB - A rapid and sensitive protein staining method employing a mixed dye technique has been developed. Solutions of Coomassie brilliant blue R-250 (CB, 0.2%) and Bismark brown R (BBR, 0.05%) were mixed in the volume ratio of 1:0.75 for staining (final molar ratio, 4.5:1). In this staining, BBR inhibits the binding of CB to gel matrix by forming ion-pairing complex with excess CB; therefore, it reduces staining/destaining times and also enhances the staining effect of CB on protein band. As a result, the mixed dye staining enables complete staining/destaining within 20/25 min and detection of up to 25 ng of bovine serum albumin. PMID- 8619500 TI - Amination of polystyrene microwells: application to the covalent grafting of DNA probes for hybridization assays. AB - Amine functionalization of polystyrene microwells for covalent binding of DNA is described. Polystyrene support was first carboxylated by permanganate oxidation in diluted sulfuric acid. These functions were activated with water-soluble carbodiimide and grafted with N-methyl-1,3-propane diamine to introduce a free secondary amino group on the support. The samples were characterized by X-ray photoelectron spectroscopy and radiochemical assay. The conditions for covalent linkage of secondary amine on polystyrene microwells were optimized. Functionalized supports were used for covalent binding of a DNA capture probe for the detection of human cytomegalovirus in a sandwich hybridization assay. Sensitivity of the assay compared very well with a commercially available surface, Covalink-NH, microwell plate obtained by electromagnetic irradiation. PMID- 8619501 TI - Spectrophotometric assay using streptavidin-alkaline phosphatase conjugate for studies on the proteolysis of polymer bead-bound peptides. AB - A simple method for indirectly measuring the concentration of biologically available peptides on the surface of polymer beads synthesized for the peptide or nonpeptide library is developed. To identify very small differences in the amount of cleaved peptides after alpha-chymotrypsin treatment, the so-called "shaving" process, a system of biotin and streptavidin-alkaline phosphatase conjugate was used. To this conjugate, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (NBT) are coupled consecutively, to carry out alkaline phosphatase reaction and color reaction, respectively. The proteolytic action of alpha chymotrypsin on surface peptides located on polymer beads was detected and cleaved peptides were quantified spectrophotometrically by analyzing unreacted NBT. Using this method, the enzymatic "shaving" process of surface peptides on polymer beads can be optimized by examining the effects of the morphology of polymer beads and the length of spacers on how easily enzymes such as alpha chymotrypsin can access the surface of polymer beads. Quantitative understanding of molecular interactions on the bead surface will give us very useful information in designing polymer bead and bead-bound oligopeptide structures for the peptide or nonpeptide library. PMID- 8619502 TI - Isolation of acute-phase proteins from plasma for determination of fractional synthesis rates by a stable isotope tracer technique. AB - Because of the unavailability of convenient tracer methods, there is no information on the kinetic changes responsible for the increased or decreased pool sizes of the acute-phase proteins (APPs) during the metabolic response to trauma and infections. We have developed a stable isotope tracer method to measure the synthesis rates of the APPs transferrin, haptoglobin, and alpha1 antitrypsin. The proteins were isolated from plasma by either direct or sequential immunoprecipitation and purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. With the exception of haptoglobin, the major band of each protein and its standard ran to a position corresponding to its molecular mass. The major band of haptoglobin and its standard ran to a position corresponding to a molecular mass of slightly less than 44 kDa, which corresponds to the haptoglobin beta chain, molecular mass 42.5 kDa. To measure the fractional synthesis rates (FSRs) of these proteins, three children were infused with 2H3 leucine for 8 h, and the amount of 2H3-leucine incorporated into the proteins was measured by gas chromatography-mass spectrometry. The plateau isotopic enrichment of very low density lipoprotein-apoB-100-bound leucine was used to estimate the isotopic enrichment of the hepatic protein synthetic precursor pool. FSRs (%/day) of the proteins were haptoglobin, 50 +/- 8; transferrin, 19 +/- 1.1; and "alpha"1 antitrypsin, 10 +/- 1. 5. PMID- 8619503 TI - Prior myocardial infarction and prognostic outcome in patients with unstable angina in a postdischarge follow-up. AB - The authors investigated how a previous myocardial infarction (MI) affects the prognosis of unstable angina pectoris in patients with maintained or slightly reduced left ventricular performance. From January 1991 to August 1993, 131 patients hospitalized with the diagnosis of Braunwald's class II-III unstable angina and ejection fraction > 40% were included. The enrolled patients were divided into two groups: (1) group I: unstable angina with prior MI (n = 70, 49 men, 21 women, aged between fifty-one and eighty years, mean: 65.7 +/- 8.5 years, Braunwald's class III: 71.4%), (2) group II: unstable angina with previous infarction (n = 61, 31 men, 30 women, aged between forty-nine and eighty, mean: 66.3 +/- 7.9 years, Braunwald's class III: 83.6%). The follow-up varied between six and twenty-four months. The frequency of major cardiovascular events (deaths, MI, reinfarction, heart failure, and recurrent unstable angina) and the number of revascularization procedures (percutaneous transluminal coronary angioplasty [PTCA] and coronary artery bypass grafting [CABG]) established during follow-up were evaluated. Hospitalization was 10.1 +/- 2.9 days in group I and 8.6 +/- 2.6 days in group II (P < 0.01). The duration of the follow-up was comparable between the two groups. Based upon predischarge noninvasive evaluation, patients in both groups were selected to undergo coronary and ventricular angiography: 38 of 70 (55.7%) in group I and 39 of 61 (62.3%) in group II; among them, 52.9% in group I and 24.6% in group II (P < 0.05) were submitted to coronary revascularization, while the others received medical treatments: 33 of 70 in group I and 46 of 61 in group II (P < 0.05). In the subset of patients submitted to angiography, the severity of coronary disease did not differ between the groups, and group I showed a statistically lower ejection fraction than group II (P < 0.005). The frequency of major cardiovascular events demonstrated a mortality rate of 2.9% in group I and 1.6% in group II. Acute MI/reinfarction accounted for 2.9% of the cases in group I and 3.3% in group II. Heart failure was present in 2.9% of group I. Recurrence of unstable angina was diagnosed in 11.4% of group I and 6.5% of group II. CABG and PTCA were performed, respectively in 7.1% and 5.7% in group I and in 6.6% and 4.9% in group II. During follow-up 75.7% of patients in group I and 80.3% in group II were asymptomatic. No significant differences in the frequency of cardiovascular events were reported between the two groups. As result of more aggressive therapeutic approaches following the detection of residual ischemia in patients with prior infarction, the authors conclude that the prognosis of unstable angina in the group with previous infarction does not seem to differ from that of unstable angina in the absence of prior necrosis in patients whose left ventricular function is maintained or slightly decreased. PMID- 8619504 TI - TcPo2 measurement reproducibility during stress in stage II obliterative arterial disease. AB - Distal transcutaneous oxygen pressure measurement (TcPo2) is a noninvasive method of evaluating tissular hypoxemia in peripheral arterial disease. The poststress area of hypoxemia is a usefull technique for globally quantifying different parameters represented by TcPo2 curves during exercise. Although its use is increasingly widespread, the reproducibility of this method is poorly documented. TcPo2 was monitored three times at twenty-four hour intervals in 5 patients with stage II obliterative arterial disease during a treadmill walking test. In order to get uniform measurement conditions, each patient remained lying and then stood until TcPo2 became stable. The stress duration was calculated so that the pain step could not be reached. TcPo2 curves were digitized and a specific image analyzer was used to make replicate measurements. The area under the curve was computed, the horizontal axis determining the mean TcPo2 value at rest, the vertical axis representing the end of the exercise period. The corresponding areas under the curves ranged from 34 to 2212 mm2 (573.60; SD 826). Significant correlation coefficients were obtained among replicate measurements (first-second day, first-third day). However, owing to the wide range of area values, the authors decided to compute and use the coefficient of variation (STD/mean), since it was more representative of reproducibility. The mean of its value for 5 patients was 21%. Observation of the examination conditions resulted in several findings, especially the ability of certain patients to adapt their efforts to the exercise. These results indicate that TcPo2 poststress area measurements are reproducible, but the conditions of the exercise have to be rigorously defined and may still be improved. PMID- 8619505 TI - Acute exposure to carbon monoxide does not affect plasma lipids, lipoproteins, and apolipoproteins. AB - STUDY OBJECTIVES: To examine the effects of acute exposure to carbon monoxide and hypoxia on plasma lipids, lipoproteins, and apolipoproteins. DESIGN: Random-order assignment to blinded, inhaled exposures of carbon monoxide and hypoxia. SETTING: Research laboratory of ambulatory subjects. SUBJECTS: 10 elderly, male nonsmokers with chronic stable angina. INTERVENTION: Random-order two-hour inhaled exposure to clean air at sea level, carbon monoxide at sea level, carbon monoxide at high altitude, and clean air at high altitude. MEASUREMENTS: Fasting plasma lipids, lipoproteins, and apolipoproteins before and after exposures. RESULTS: No differences were noted between fasting plasma lipid, lipoprotein, or apolipoprotein levels before and after exposures. CONCLUSION: Acute exposure to carbon monoxide and high altitude does not affect fasting plasma lipid, lipoprotein, or apolipoprotein levels. PMID- 8619506 TI - Granule-laden perivascular cells observed in rabbits with experimental cerebral atherosclerosis. AB - Granule-laden perivascular cells distributed around the fine vessels in brain of hypertensive rabbits fed a cholesterol diet were examined by electron microscopy. Perivenule granule-laden cells contained secondary lysosomes, residual bodies, Golgi vesicles, fusion vesicles, and vacuoles. In periarteriole granule-laden cells, secondary lysosomes and vacuoles were not prominent. Findings indicated that the granule-laden cell may be a histiocyte that appears during the vascular reaction to hypertension and hypercholesterolemia. This vascular reaction may occur more strongly in the veins than the arteries. PMID- 8619507 TI - Training physicians and health care providers to accurately read coronary arteriograms. A training program. AB - Patterns in visual interpretation of coronary arteriograms (CAs) frequently cause incorrect assessment of percent diameter stenosis (%DS). These errors result in overestimating the results of angioplasty as well as of the number of arteries significantly affected by coronary artery (CAD) disease. METHODS: Forty-one physicians, nurses, and students participated in the standardization of 45 Kodachromes (39 arteries, 6 phantoms) and 5 photographic reproductions. Eleven of the 41 participated in a three-part training program designed to eliminate errors and improve accuracy of interpreting %DS from CAs. RESULTS: Improvement in reading %DS was seen in 69% of CAs with statistical (P < or = 0.05) improvement in one third of these cases, whose narrowings ranged from 4% to 84%DS. Variability of reporting was reduced in 26% of the cases. Skewing, representing an overestimation of "severe" disease and underestimation of "less severe" disease, was reduced with statistical improvement (P < or = 0.05) in reported %DS noted after training. Similar improvement was seen with phantoms but not in photographic images where the arterial edges were outlined. CONCLUSION: The outcomes of clinical management, invasive and interventional (mechanical, thrombolytic) procedures, as well as research studies depend in part upon the accuracy of reading %DS from CAs. Most studies to date have completed using extremely unreliable estimates of %DS with resultant problems in data interpretation. The use of this standardized training program has led to significant improvement in accurately assessing CAD. PMID- 8619508 TI - A simple, noninvasive method to investigate vascular characteristics in children. AB - Palpation of pulses is an ancient clinical technique. In modern medical terms, arterial pulse analysis permits the evaluation of vascular characteristics, the important parameters for study of the properties of vessels. However, the various parameters used to describe the vascular characteristics have been conventionally deriving from expensive, invasive, and complicated methods and involving complex computations. The authors designed, therefore, a simple, noninvasive system to evaluate vascular characteristics. The system consisted of two units of pressure transducer for simultaneous recording of two peripheral arterial pulses, an analog signal processor, an A/D converter, and a personal computer. The vascular characteristics were analyzed by use of the Windkessel model and cross correlation function. The analysis program was designed by members of this team. Through this system, systemic arterial compliance and pulse wave velocity can be obtained. Thirty-five children (11 normal children and 24 children with various stable, mild congenital heart diseases) were enrolled for data analysis. For these 35 children without hypertension or other clinically apparent arterial disease, the pulse wave velocity in the upper limb as between 3.7 and 16.8 m x sec-1, with a mean +/- standard deviation of 9.3 +/- 3.3 m x sec-1. The arterial compliance among the 11 normal children was between 0.72 and 1.72 mL x mmHg-1 x m 2 with a mean of 1.15 mL x mmHg-1 x m-2; both were consistent with the previously reported values obtained from complex methods. This system provides data comparable with those obtained from invasive methods. In the past, the arterial characteristics could not be extensively studied owing to the invasive nature of the conventional methods. This new, simple, convenient noninvasive system will thus provide a convenient method for clinical use in evaluating the vascular characteristics of patients, especially of children. PMID- 8619509 TI - Dipyridamole technetium-99m Sestamibi imaging in the diagnosis of syndrome X. AB - In a middle-aged woman with anginal chest pain and a normal-appearing angiogram, dypiridamole technetium-99m Sestamibi scintigraphy, a noninvasive method, provided the diagnosis of syndrome X and was used in follow-up to monitor the course of disease. PMID- 8619510 TI - Morphologic changes in pulmonary vasculature with arteriographic correlation. AB - During fetal life the pulmonary circulation is one of high resistance. Soon after birth with the onset of breathing and thereafter, sequential structural changes in pulmonary vasculature take place. Several authors have reported differently on this structural remodeling. This study describes the changes in the pulmonary vasculature occurring following birth. Twenty-five cases submitted to autopsy with ages ranging from newborns up to fourteen years were studied. Postmortem arteriography was undertaken in most of the cases and arterial morphology was quantitated by histomorphometry. It was found that the thick-walled pulmonary arteries of the newborn show a reduction in medial thickness over a period of one month, the reduction being most rapid in the first three days. After one month there is no significant change in medial thickness until fourteen years of age. PMID- 8619512 TI - Uhl's anomaly. A case report. PMID- 8619513 TI - Computed tomography and magnetic resonance findings in long-standing patent ductus. Case reports. AB - Two adult patients with patent ductus arteriosus (PDA), resulting in Eisenmenger's syndrome, were evaluated by computed tomography and Magnetic Resonance imaging of the chest, which clearly demonstrated the patent arterial duct as well as the morphologic features of long-standing pulmonary arterial hypertension. Massively dilatated, calcified pulmonary arteries were seen, and the arterial duct was identified as a vascular channel between the distal aortic arch and the main pulmonary artery. PMID- 8619511 TI - Massive cerebral infarction: a potential fatal complication of plaque fissuring of lipid-rich atheromas of the carotid artery. Case reports. AB - The authors report 2 patients who died of massive cerebral infarction secondary to plaque fissuring of and hemorrhage into their carotid atheromas, resulting in overlying thrombosis and total occlustion of the internal carotid arteries. In both cases, the atheromas caused more than 75% reduction in luminal size of the carotid artery. In addition, they were rich in lipid content, which accounted for more than 40% of the cross-sectional area of the atheromatous plaques. It is postulated that high-grade stenotic carotid atheromatous plaques with a high lipid content are at an increased risk of giving rise to subsequent neurologic complications, including massive cerebral infarction, as exemplified by the present 2 reported cases. PMID- 8619514 TI - Unusual presentation of Takayasu arteritis with cardiac involvement and imitation of juvenile arteriosclerosis. A case report. AB - A twenty-five-year-old Caucasian man with Takayasu arteritis, who was formerly diagnosed as suffering from premature arteriosclerosis, is described. Necropsy disclosed involvement of the entire aorta and its major branches, the pulmonary arteries, the coronary arteries, the intramyocardial arteries, and the heart valves, a combination hitherto not described. Literature concerning heart involvement in Takayasu arteritis is reviewed, and the differential diagnosis is discussed. PMID- 8619515 TI - Heparin-induced thrombocytopenia with multiple cerebral infarctions simulating thrombotic thrombocytopenic purpura. A case report. AB - The authors describe a patient with stroke, treated with heparin for unstable angina, whose clinical features mimicked those of thrombotic thrombocytopenic purpura (TTP). His condition eventually proved to be caused by heparin-induced thrombocytopenia (HIT), complicated by thrombosis (HITT). The absence of microangiopathic hemolytic anemia should question the diagnosis in a presumed TTP patient. Early diagnosis of HITT is possible since recently two highly sensitive and specific tests have become available. Heparin treatment has to be stopped immediately if HITT is diagnosed. First-choice antithrombotic treatment in HITT patients is danaparoid. PMID- 8619516 TI - A rare case of arteriosclerosis obliterans without prominent risk factors complicated by idiopathic thrombocytopenic purpura. A case report. AB - An eighty-four-year-old man was admitted to the hospital because of pain at rest in the lower extremities. On physical examination, trophic changes of the skin and petechiae in the limbs were observed. Computed tomographic scan of the abdomen showed focal renal infarctions and calcification of the descending aorta. Moreover, radionuclide imaging of the arterial system revealed complete obstructions of the two right iliac arteries and the left external iliac artery, where collateral flows were observed. Laboratory examination showed a severe thrombocytopenia caused by immunoglobulin G (IgG)-type autoantibody against platelets. He was diagnosed as having arteriosclerosis obliterans complicated by idiopathic thrombocytopenic purpura, although no known risk factors promoting atherosclerosis other than age were evident. In such a case with hemorrhagic diathesis, a hemorheologic agent and the vasodilator prostaglandin could confer advantages in relieving and controlling the ischemic leg pain without hemorrhagic complications. Moreover, small doses of the initial prednisolone therapy for ITP might also be recommended to avoid thrombus formations in the atherosclerotic lesions. PMID- 8619517 TI - Multiple organ manifestations in thromboangiitis obliterans (Buerger's disease). A case report. AB - Thromboangiitis obliterans (TAO) occurs almost exclusively in young male smokers. Its involvement of the small and medium-sized arteries and veins leads to ischemic complaints and/or changes in the extremities. The possibility of organ involvement is a matter of controversy. The authors report a case of TAO with multiple organ involvement, including myocardial, splenic, and cerebral infarctions; pulmonary embolisms; and probable intestinal ischemia during a twenty-three-year course. PMID- 8619518 TI - Autoimmune ataxic neuropathies (sensory ganglionopathies): are glycolipids the responsible autoantigens? PMID- 8619519 TI - Exercise, drug treatment, and the optimal care of multiple sclerosis patients. PMID- 8619520 TI - Experimental sensory neuropathy induced by sensitization with ganglioside GD1b. AB - Three of six rabbits immunized with purified GD1b developed ataxic sensory neuropathy. They laid on the floor with their limbs splayed out, and their movements were awkward; but muscle power, tonus, and superficial sensation appeared to be intact. Sciatic nerve motor conduction studies were normal. Axonal degeneration was present in the dorsal column of the spinal cord, in the dorsal roots, and in the sciatic nerve. Some of the nerve cell bodies in the dorsal root ganglia had degenerated and disappeared. No demyelinative lesions or mononuclear cell infiltrations were seen in those regions. No pathological changes were present in the other three immunized rabbits that showed no clinical symptoms. Control rabbits inoculated only with adjuvants showed neither clinical symptoms nor pathological changes. Anti-GD1b antibody was raised in the sera from all six rabbits immunized with GD1b. The monoclonal anti-GD1b antibody GGR12 immunostained about one-half the rabbit primary sensory neurons. Sensitization with GD1b, therefore, may cause ataxic sensory neuropathy in rabbits due to antibody-mediated damage to the primary sensory neurons. PMID- 8619521 TI - Impact of aerobic training on fitness and quality of life in multiple sclerosis. AB - Fifty-four multiple sclerosis (MS) patients were randomly assigned to exercise (EX) or nonexercise (NEX) groups. Before and after 15 weeks of aerobic training, aspects of fitness including maximal aerobic capacity (VO2max), isometric strength, body composition, and blood lipids were measured. Daily activities, mood, fatigue, and disease status were measured by the Profile of Mood States (POMS), Sickness Impact Profile (SIP), Fatigue Severity Scale (FSS), and neurological examination. Training consisted of 3 x 40-minute sessions per week of combined arm and leg ergometry. Expanded Disability Status Scale (EDSS) scores were unchanged, except for improved bowel and bladder function in the EX group. Compared with baseline, the EX group demonstrated significant increases in VO2max, upper and lower extremity strength, and significant decreases in skinfolds, triglyceride, and very-low-density lipoprotein (VLDL). For the EX group, POMS depression and anger scores were significantly reduced at weeks 5 and 10, and fatigue was reduced at week 10. The EX group improved significantly on all components of the physical dimension of the SIP and showed significant improvements for social interaction, emotional behavior, home management, total SIP score, and recreation and past times. No changes were observed for EX or NEX groups on the FSS. Exercise training resulted in improved fitness and had a positive impact on factors related to quality of life. PMID- 8619522 TI - Relations of genetic and environmental factors in the etiology of epilepsy. AB - We assessed the relations of genetic and environmental factors in the etiology of epilepsy. The study population comprised 9,705 first-degree relatives of 1,951 adults with epilepsy ascertained from voluntary organizations. We calculated standardized morbidity ratios for specific etiologies of epilepsy in the relatives of probands with the same etiologies, using population incidence rates from Rochester, MN, as the reference. Relatives of probands with idiopathic/cryptogenic epilepsy had increased risk for idiopathic/cryptogenic epilepsy and for epilepsy associated with neurological deficit presumed present at birth (cerebral palsy or mental retardation) but not for symptomatic epilepsy associated with postnatal central nervous system insults. Relatives of probands with neurodeficits had increased risks for idiopathic/cryptogenic epilepsy. Risk for epilepsy was not increased among relatives of probands with postnatal symptomatic epilepsy. The degree of increased risk of idiopathic/cryptogenic epilepsy in relatives of probands with idiopathic/cryptogenic epilepsy diminished with increasing age of the relatives; risk was not increased at age 35 or older. These findings support the possibility of shared genetic susceptibility to epilepsy and cerebral palsy, and suggest that the genetic contributions to postnatal symptomatic epilepsy are minimal. PMID- 8619523 TI - Regional metabolic correlates of surgical outcome following unilateral pallidotomy for Parkinson's disease. AB - Stereotaxic ventral pallidotomy has been employed in the symptomatic treatment of patients with advanced Parkinson's disease (PD). To understand the pathophysiology of clinical outcome following this procedure, we studied 10 PD patients (5 men and 5 women; mean age 60.0 +/- 6.1 years; mean Hoehn and Yahr stage 3.8 +/- 1.0) with quantitative 18F-fluorodeoxyglucose (FDG) and positron emission tomography (PET). All patients were scanned preoperatively; 8 of 10 patients were rescanned 6 to 8 months following surgery. Clinical performance was assessed off medications before and after surgery using standardized timed motor tasks. We found that preoperative lentiform metabolism correlated significantly with improvement in contralateral motor tasks at 1 week, 3 months, and 6 months following unilateral pallidotomy (p<0.03). Postoperatively, significant metabolic increases were noted in the primary motor cortex, lateral premotor cortex, and dorsolateral prefrontal cortex (p<0.01) of the hemisphere that underwent surgery. Improvement in contralateral limb motor performance correlated significantly with surgical declines in thalamic metabolism (p<0.01) and increases in lateral frontal metabolism (p<0.05). Principal components analysis disclosed a significant covariance pattern characterized by postoperative declines in ipsilateral lentiform and thalamic metabolism associated with bilateral increase in supplementary motor control metabolism. Subject scores for this pattern correlated significantly with improvements in both contralateral and ipsilateral limb performance (p<0.005). These results suggest that pallidotomy reduced the preoperative overaction of the inhibitory pallidothalamic projection. Clinical improvement may be associated with modulations in regional brain metabolism occurring remote from the lesion site. PMID- 8619524 TI - Thalamic metbolism and corticospinal tract integrity determine motor recovery in stroke. AB - We studied the role of remote metabolic depressions and pyramidal tract involvement regarding motor recovery following a first hemiparetic ischemic stroke. In 23 patients the regional cerebral glucose metabolism (rCMRGlu) was measured with positron emission tomography and the location and spatial extent of the stroke lesions were assessed by magnetic resonance imaging. Motor impairment during the acute and chronic stages (4 weeks after stroke) was determined by a motor score and recordings of magnetic evoked motor potentials. Twelve patients recovered significantly, whereas 11 patients retained a disabling hemiparesis. In contrast to patients with good motor recovery, rCMRGlu was severely depressed in the thalamus on the lesion side in patients with poor motor recovery. This patient group also showed more severe damage to the pyramidal tract on magnetic resonance images and a more pronounced reduction of the magnetic evoked motor potential amplitude. Neither the size of the stroke lesions nor the spatial extent of the lesional and remote rCMRGlu depressions outside the thalamus correlated with the thalamic hypometabolism and the improvement of the motor score. We conclude that preservation both of parts of the pyramidal tract and of the thalamic circuitry is a major determinant for the quality of hand motor recovery following acute brain ischemia in the adult. PMID- 8619525 TI - Hypersomnia following paramedian thalamic stroke: a report of 12 patients. AB - Paramedian thalamic stroke (PTS) is a cause of organic hypersomnia, which in the absence of systematic sleep-wake studies has been attributed to disruption of ascending activating impulses and considered a "dearoused" state. However, an increasing mount of data suggests a role of the thalamus in sleep regulation and raises the possibility that a sleep disturbance contributes to hypersomnia in PTS. We evaluated 12 patients with magnetic resonance imaging-proven isolated PTS and hypersomnia with 10 to >20 hours of sleep behavior per day. Nocturnal polysomnographic findings paralleled the severity of hypersomnia. All subjects had increased stage 1 NREM sleep, reduced stage 2 NREM sleep, and reduced numbers of sleep spindles. In patients with severe hypersomnia, slow-wave (stages 3-4) NREM sleep was often reduced, but there were no major REM sleep alterations. Daytime sleep behavior was associated mostly with stage 1 sleep by electroencephalogram; there was no correlation between hypersomnia and results of nap tests. We conclude that hypersomnia following PTS is accompanied by deficient arousal during the day and insufficient spindling and slow-wave sleep production at night. These observations support the hypothesis of a dual role of the paramedian thalamus as "final common pathway' for both maintenance of wakefulness and promotion of NREM sleep. PMID- 8619526 TI - Glial differentiation predicts poor clinical outcome in primitive neuroectodermal brain tumors. AB - Primitive neuroectodermal tumors (PNETs) of the central nervous system, including medulloblastomas (PNET/MB), are the most common malignant brain tumor of childhood. These tumors often express proteins characteristic of glial differentiation (glial fibrillary acidic protein, GFAP), neuronal differentiation (neurofilament proteins, NFPs), and/or photoreceptor differentiation (retinal-S antigen). To identify biological factors of prognostic significance in PNETs, the expression of glial, neuronal, or photoreceptor antigens was evaluated in the tumor specimens of 86 patients with PNETs by immunohistochemistry after microwave antigen enhancement. Patterns of differentiation were then compared with patient relapse-free survival. Multivariate analysis of PNET immunohistochemistry and clinical variables indicated GFAP expression conferred a 6.7-fold greater risk of relapse than tumors that did not express GFAP or NFPs. Increased risk of relapse was directly related to the amount of GFAP expression. Tumors exhibiting clumps or sheets of GFAP-staining cells were associated with a 3.0-fold increased risk of relapse compared with tumors that did not express GFAP, irrespective of immunohistochemical evidence of other differentiation, while scattered GFAP staining was not associated with increased risk of relapse. These findings indicate that expression of GFAP in PNETs has prognostic power comparable with the most significant clinical factors currently used to predict clinical outcome. PMID- 8619527 TI - Spinocerebellar ataxia 3 and Machado-Joseph disease: clinical, molecular, and neuropathological features. AB - Patients with spinocerebellar ataxia 3 (SCA3) and Machado-Joseph disease (MJD) carry an expanded CAG repeat in the MJD1 gene. One hundred twenty families of different geographic origin with autosomal dominant cerebellar ataxia (ADCA) type I were tested. Thirty-four families (126 patients) carried an expanded CAG repeat. The expanded and the normal allele did not overlap and the repeat was unstable during transmission, with variation in the size of the CAG length ranging from -8 to +5 and a mean expansion of 0.86 repeats without differences according to the parental sex. There was a combined effect of the number of CAG repeats of the expanded and normal allele on the age at onset, which accounted for 70% of its variability. The length of the CAG repeat influenced the frequency of clinical signs associated with cerebellar ataxia, such as abnormal tendon reflexes or decreased vibration sense, whereas the interindividual variation of supranuclear ophthalmoplegia, sphincter and swallowing difficulties, and amyotrophy was mostly determined by different disease durations. We compared the clinical profile of 91 SCA3/MJD patients with 51 SCA1 and 32 SCA2 patients. There were striking differences between the SCA3/MJD and SCA2 but not with SCA1 groups of patients. Despite their clinical similarities, distinct neuropathological features were observed in 2 SCA3/MJD and 2 SCA1 patients. PMID- 8619528 TI - Unstable triplet repeat and phenotypic variability of spinocerebellar ataxia type 1. AB - A Siberian kindred with spinocerebellar ataxia genetically linked to the SCA1 locus on chromosome 6p has been screened for the CAG triplet expansion within the coding region of the SCA1 gene. The kindred includes 1,484 individuals, 225 affected and 656 at risk, making this collection the largest spinocerebellar ataxia type 1 (SCA1) pedigree known. Each of the studied 78 SCA1 patients carried an expanded allele containing a stretch of 39 to 72 uninterrupted CAG repeats. Normal alleles had 25 to 37 trinucleotide repeats. Expanded alleles containing 40 to 55 repeats were found in 26 at-risk relatives. The number of CAG repeats in the mutated allele was inversely correlated with age at disease onset. Cerebellar deficiency was present in each patient and its severity was moderately affected by the number of CAG repeats. In contrast, the associated signs, dysphagia, diffuse skeletal muscle atrophy with fasciculations, and tongue atrophy were absent or mild in patients with low CAG repeat numbers, but severely complicated the course of illness in patients with a larger number of repeat units. One female mutation carrier was asymptomatic at age 66, more than 2 standard deviations beyond the average age of risk, suggesting incomplete penetrance. In 2 symptomatic individuals who had an expanded number of CAG repeats on both chromosomes, age at onset, rate of progression, and clinical manifestation corresponded to the size of the larger allele. PMID- 8619529 TI - Chromosome 12-linked autosomal dominant scapuloperoneal muscular dystrophy. AB - Scapuloperoneal syndromes are characterized by their distribution of muscle weakness and wasting. The reported pattern of inheritance has been variable. Both neurogenic and myopathic forms of autosomally dominantly inherited scapuloperoneal syndrome have been described. It has been suggested that these are variants of other neuromuscular diseases. We examined 44 members from a family with 14 members affected with a scapuloperoneal syndrome. Physiological and histological analysis implied that this condition is predominantly myopathic. Linkage analysis was done to confirm the genetic etiology of the disease in this family and to evaluate the possibility that it is a allelic variant of other neuromuscular diseases. Genetic analysis demonstrated linkage of the disease to chromosome 12, which makes it genetically distinct from other loci known to cause neuromuscular disease. Muscle fibers with hyaline desmin-containing cytoplasmic inclusions in combination with focal myopathic changes may be a disease-specific morphological marker of the disease. PMID- 8619531 TI - Vestibular dysfunction in chronic inflammatory demyelinating polyneuropathy. AB - Chronic inflammatory demyelinating polyneuropathy (CIDP) has occasionally been associated with clinical or laboratory evidence (magnetic resonance imaging,[MRI], visual evoked response, and brainstem auditory evoked response [BAER] of cranial neuropathy. In most cases, the relationship of cranial nerve involvement to CIDP remains unclear. A 45-year-old woman noted foot numbness, limb weakness, gait and postural instability, and oscillopsia. An IgG kappa monoclonal gammopathy of undetermined significance was found. Bilateral vestibulopathy was documented by clinical examination, bithermal calorics, rotary chair testing, BAERs, and dynamic posturography. MRI with gadolinium demonstrated enhancement of cranial nerve VIII bilaterally. Over the next 6 years, the patients's relapsing and remitting course of CIDP and vestibulopathy was assessed by quantitative muscle and vestibular function testing (clinically and neurophysiologically), and dynamic visual acuity. There was a striking synchronization between her CIDP and vestibulopathy with respect to clinical course including relapses and responses to immune therapy. The response to therapy, and evidence derived from clinical and laboratory investigations, suggest that the vestibular dysfunction was immune mediated. PMID- 8619530 TI - Upregulation of Bcl-2 protein in the myasthenic thymus. AB - We examined the expression of Fas antigen and Bcl-2 protein in thymic tissue surgically resected from 10 patients with myasthenia gravis, using immunocytochemical techniques. Histologically, thymic tissues from 7 myasthenia gravis patients showed hyperplasia, while 3 other patients had thymomas. In hyperplastic thymic tissue, immunoreactivity for Fas antigen was observed mainly in the network of medullary epithelial cells. In contrast, expression of Fas antigen was rare in the cortex. Fas antigen was also detected to some degree in thymoma tissue from 3 patients. Bcl-2 protein was highly expressed in the medullary thymocytes in the hyperplastic thymic tissue, whereas its staining was quite low in myasthenia gravis thymomas. The number of Bcl-2-positive thymocytes in the medulla was significantly greater in the hyperplastic myasthenia gravis thymic tissue than in the control thymic tissue. These findings suggest that Bcl 2 protein may be upregulated in the myasthenia gravis thymus and that this phenomenon may be related to impaired apoptotic cell death of autoreactive thymocytes in myasthenia gravis. PMID- 8619532 TI - Gamma knife pallidotomy in advanced Parkinson's disease. AB - Posteroventral pallidotomy as a treatment for Parkinson's disease (PD) has been the subject of increasing interest. We treated 4 nondemented patients with advanced PD, 2 with severe bradykinesia and a declining response to medication, and 2 with marked clinical fluctuations. All patients received 180 Gy delivered in one sitting to the right posteroventral pallidum site, used by Laitinen and colleagues, adjusted as needed, to avoid the optic tract. Only 1 patient changed significantly. Dyskinesia completely resolved on the side contralateral to the lesion in this patient. This same patient also became transiently demented and psychotic. The other 3 patients suffered no clearly identifiable beneficial or harmful effects. Follow-up magnetic resonance imaging scans of the brain at 1 year revealed lesions exactly where targeted although of unequal sizes. Our negative experience forces us to conclude that either larger volumes of tissue must be ablated, that physiologic monitoring is required for placing a lesion, that our subjects were poor candidates for the procedure, or that surgical ablation and radiation cause tissue damage of different types with different results. PMID- 8619534 TI - T-cell-mediated ganglionitis associated with acute sensory neuronopathy. AB - A 67-year-old man presented with acute painful sensory loss, areflexia, ataxia, urinary retention, and severe constipation and became unable to walk within 2 weeks. He died suddenly 5 weeks after the onset of symptoms. Autopsy revealed widespread inflammation of sensory and autonomic ganglia with immunocytochemical evidence of a CD8+ T cell-mediated cytotoxic attack against ganglion neurons. This observation suggests a novel pathogenetic mechanism of immune-mediated human ganglion cell damage comparable to mechanisms operating in polymyositis. PMID- 8619533 TI - Anti-GD1a ganglioside antibodies in peripheral motor syndromes. AB - High titers of anti-GD1a antibodies have been found in patients with Guillain Barre syndrome or motor neuropathy. To determine the possible diagnostic relevance of these antibodies, we measured serum anti-GD1a IgG and IgM antibodies by enzyme-linked immunosorbent assay in 195 patients with different motor syndromes and in 335 control subjects. Moderately high antibody titers (1/1,280 1/5,120) were occasionally found in patients with chronic inflammatory demyelinating polyneuropathy (5%), multifocal motor neuropathy (18%), lower motor neuron disease (3.8%), or amyotrophic lateral sclerosis (1.8%) and in immunological control subjects (1.2%), while titers of 1/20,480 or higher were only found in 2 patients with Guillain-Barre syndrome (IgG in both) and 2 with motor neuropathy and IgM lambda monoclonal gammopathy improving with immunotherapy. In both motor neuropathy patients and the Guillain-Barre syndrome patient who were retested during recovery, anti-GD1a titers decreased concomitantly with clinical improvement. High anti-GD1a antibody titers may be found in several motor syndromes but only markedly increased anti-GD1a titers are strictly associated with potentially treatable dysimmune neuropathies. PMID- 8619535 TI - High apolipoprotein E epsilon 4 allele frequency in age-related memory decline. AB - Many studies have demonstrated a strong association between the presence of one or two epsilon 4 alleles and Alzheimer's disease (AD), although few data are available on the apolipoprotein E (APOE) epsilon 4 frequencies at the preclinical stages of AD. Thus, with a view to determining whether APOE genotyping could be useful in the early detection of AD, we determined the Apoe allele frequencies in patients with memory complaints without dementia (age-related memory decline, ARMD). We found an APOE epsilon 4 allele frequency of 0.315 in the ARMD group, similar to 0.293 in the AD group, in contrast to 0.057 in the control group. Significant differences (t=-2.91, df=25, p=0.008) were found between the Alzheimer's Disease Assessment Scale (ADAS) total scores in the ARMD patients with at least one epsilon 4 allele (mean=24.2) compared with the ARMD patients without the epsilon 4 allele (mean=14.7). Our results suggest that the patients with memory complaints, a high ADAS score, and the presence of one or two APOE-4 alleles could be at high risk for developing AD. Thus, we propose that genotyping in conjunction with the ADAS scale may prove useful as diagnostic markers of AD in the presymptomatic stages. PMID- 8619536 TI - Re: Pharmacokinetics and pharmacodynamics of rHCNTF in rodents. PMID- 8619537 TI - Detrimental effect of nitric oxide inhibition in experimental bacterial meningitis. PMID- 8619538 TI - Pharmacological pallidotomy with glutamate antagonists. PMID- 8619539 TI - Polycystic ovarian syndrome in women with epilepsy: epileptic or iatrogenic? PMID- 8619540 TI - The response to levodopa in Parkinson's disease: imposing pharmacological law and order. AB - The seemingly unpredictable response to levodopa in patients with Parkinson's disease can be understood as an interaction between several distinct pharmacological effects of levodopa. The most important are a short-duration response with a half-life of minutes to hours and a long-duration response with a half-life of days, superimposed on diurnal motor variation. A negative response characterized by brief worsening before and after the short-duration response and dyskinesia accentuate the short-duration response. These various responses are modified by disease progression and long-term levodopa therapy. Pharmacodynamic modeling of the short-duration response indicates that with time, the response becomes less graded and small changes in levodopa concentrations can produce big changes in response. In this setting, unpredictability arises from the variation in absorption and distribution of levodopa. PMID- 8619541 TI - Levodopa-induced dyskinesias improved by a glutamate antagonist in Parkinsonian monkeys. AB - Antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor have been reported to potentiate the antiparkinsonian action of levodopa and reverse levodopa-induced motor fluctuations in animal models of Parkinson's disease. To evaluate the effect of NMDA receptor blockade on dyskinesias complicating the response to long-term levodopa therapy, we studied the selective antagonist LY235959 in six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. Drugs were administered subcutaneously, LY235959 at doses of 0.5, 1.0, 3.0, and 5.0 mg/kg and levodopa/benserazide at doses that produced moderate dyskinesias while almost totally reversing parkinsonian signs. Compared with vehicle control injections, LY235959 (3.0 mg/kg) abolished oral dyskinesias and diminished choreic dyskinesias by 68% (p < 0.01). Lower doses had smaller effects, although still significant, on oral dyskinesias (55% reduction at 1.0 mg/kg, p < 0.05). The highest LY235959 dose (5.0 mg/kg) prolonged oral dyskinesia suppression, but tended to increase dystonia severity. LY235959 had no effect on motor function when given alone and did not reduce the antiparkinsonian response to levodopa. These findings suggest that NMDA receptor blockade may ameliorate the dyskinetic complications of long-term levodopa therapy, without diminishing the beneficial effects on parkinsonian signs. PMID- 8619542 TI - Obesity and endocrine disorders in women taking valproate for epilepsy. AB - We recently reported the frequent occurrence of polycystic ovaries and hyperandrogenism in women taking valproate for epilepsy, especially when the medication was started before the age of 20 years. In the present study we evaluated the association of obesity and hyperinsulinemia with valproate-related polycystic ovaries and hyperandrogenism in women with epilepsy. Sixty-five women participated in the study. Twenty-two received valproate monotherapy and 43 received carbamazepine monotherapy. In addition to clinical examination, vaginal ultrasonography was performed to determine ovarian size, and the concentrations of serum sex hormones, insulin, insulin-like growth factor 1, and the insulin like growth factor-binding proteins 1 and 3 (IGFBP-1 and IGFBP-3) were measured. Fifty-nine percent of the women on valproate were obese, and in a retrospective analysis an indisputable weight gain (mean, 21 kg; range, 8-49 kg) was found in 50% of the women taking valproate. Fourteen (64%) of the women on valproate had polycystic ovaries, hyperandrogenism, or both. These women were obese, and in addition to elevated serum androgen levels, they had high concentrations of fasting serum insulin and low levels of serum insulin-like growth factor-binding protein 1. Valproate therapy for epilepsy is associated with weight gain during treatment in approximately 50% of women patients. The weight gain can be progressive, and is associated with hyperinsulinemia and low serum levels of insulin-like growth factor-binding protein 1, which may lead to hyperandrogenism and polycystic ovaries. PMID- 8619543 TI - Brain thiamine, its phosphate esters, and its metabolizing enzymes in Alzheimer's disease. AB - Clinical data suggest that high-dose thiamine (vitamin B1) may have a mild beneficial effect in some patients with Alzheimer's disease (AD). Since this action could be related to a brain thiamine deficiency, we measured directly levels of free (nonphosphorylated) thiamine and its phosphate esters, thiamine monophosphate and thiamine diphosphate (TDP), and activities of three TDP metabolizing enzymes (thiamine pyrophosphokinase, thiamine diphosphatase, and thiamine triphosphatase) in autopsied cerebral cortex of 18 patients with AD and 20 matched controls. In the AD group, mean levels of free thiamine and its monophosphate ester were normal, whereas levels of TDP were significantly reduced by 18 to 21% in all three cortical brain areas examined. Activities of the TDP metabolizing enzymes were normal in the AD group, suggesting that decreased TDP is not due to altered levels of these enzymes. The TDP decrease could be explained by a cerebral cortical deficiency in AD of ATP, which is needed for TDP synthesis. Although the magnitude of the TDP reduction is slight, a chronic subclinical TDP deficiency could contribute to impaired brain function in AD and might provide the basis for the modest improvement by thiamine in cognitive status of some patients with AD. PMID- 8619544 TI - Brain protein and alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease. AB - To determine whether the reduction in brain alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease (AD) is associated with an abnormality in one of its three constituent enzyme subunits, we measured protein levels of alpha ketoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), in postmortem brain of 29 patients with AD (mean age, 73 years; age range of onset, 50-78 years) and 29 control subjects. In the AD group protein levels of all three subunits were significantly reduced by 23 to 41% in the temporal cortex, whereas in the parietal cortex (El: -28%; E3: 32%) and hippocampus (E3: -33%) significant changes were limited to El and E3. alpha-Ketoglutarate dehydrogenase complex activities were more markedly reduced (by 46-68%) and did not correlate with protein levels, suggesting that decreased enzyme activity cannot be primarily explained by loss of alpha-ketoglutarate dehydrogenase complex protein. We did not find two E2 immunoreactive forms in the brain of any patient, as has been reported in fibroblasts of patients with very early-onset chromosome 14-linked AD. We conclude that brain protein and activity levels of alpha-ketoglutarate dehydrogenase complex are reduced in patients with AD who have onset after 50 years and suggest that these changes, which are also observed in other human brain disorders, may represent a nonspecific consequence of different neurodegenerative processes. Nevertheless, reduced levels of this rate-limiting enzyme of the Krebs cycle could contribute to the brain neurodegenerative mechanisms of AD. PMID- 8619545 TI - Paramyotonia congenita: the R1448P Na+ channel mutation in adult human skeletal muscle. AB - Twitch force and Na+ currents were investigated in a muscle biopsy specimen from a patient with paramyotonia congenita carrying the dominant Arg-1448-Pro mutation in the skeletal muscle sodium channel. Cooling of the muscle fibers caused sustained membrane depolarization that resulted in reduced twitch force. Membrane repolarization, produced by a K+ channel opener, partly prevented and antagonized the drop in twitch force. Patch-clamp recordings on sarcolemmal blebs revealed a distinctly slower Na+ current decay on paramyotonia congenita muscle compared to control muscle. In addition, patches with mutant Na+ channels showed a significantly higher frequency of steady-state openings, which increased with cooling. Activation of mutant channels was not affected, whereas the steady-state inactivation curve was shifted by -5 mV and showed less voltage dependence. We suggest that the weakness of cooled muscle can be explained by a combination of the increased steady-state Na+ current and the left-shifted inactivation curve. PMID- 8619546 TI - GTP-cyclohydrolase I gene mutations in hereditary progressive amd dopa-responsive dystonia. AB - Recently, mutations of the GTP-cyclohydrolase I (GTP-CH I) gene, which catalyzes the first step in the tetrahydrobiopterin (BH4) biosynthesis, were discovered in Japanese patients with hereditary progressive dystonia/dopa-responsive dystonia (HPD/DRD). However, it has not been confirmed that non-Japanese patients also contain mutations in the same gene, or whether these mutations are specific to HPD/DRD. In this study, two novel nonsense mutations in exon I of the GTP-CH I gene and a new mutation at the splice acceptor site of intron I were identified in an autopsied case of English-Irish descent and 2 Japanese patients with HPD/DRD. In the latter, cerebrospinal fluid (CSF) neopterin levels (which may reflect the GTP-CH I activity in the brain) were reduced to 18% and 37% of controls. A therapeutic trial of oral BH4 was ineffective, however, in a genetically proven patient. In contrast, no mutations in any exons of the GTP-CH I gene were found in 2 patients with early-onset parkinsonism with dystonia (EOP D) who developed dopa-responsive parkinsonism and dystonia at 6 and 8 years old, respectively. Neopterin levels in CSF were well preserved in 6 EOP-D patients. These data suggest that, among patients of different racial backgrounds, the pathogenesis of HPD/DRD, unlike EOP-D, involves partial reduction of the brain GTP-CH I activity consequent to mutations in the GTP-CH I gene. Measurement of CSF neopterin concentration may be useful for the differential diagnosis between HPD/DRD and EOP-D. PMID- 8619547 TI - Intercellular adhesion molecule-1-deficient mice are less susceptible to cerebral ischemia-reperfusion injury. AB - Neutrophil emigration is mediated by adhesion proteins that are highly expressed on the endothelial surface during inflammatory processes in the brain. Intercellular adhesion molecule-1 (ICAM-1) is an inducible adhesion molecule that binds to leukocyte integrins and facilitates neutrophil adhesion and transendothelial migration. To study the role of ICAM-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in ICAM-1-deficient mice generated by gene targeting in embryonic stem cells. Transient focal ischemia was induced by occluding the left middle cerebral artery for 3 hours followed by a 21- or 45-hour reperfusion period. When compared with their wild-type littermates, ICAM-1-deficient mice were less susceptible to cerebral injury as demonstrated by a 5.6- or 7.8-fold reduction in infarction volume, respectively. These data support the premise that neutrophil adhesion in ischemic areas may be deleterious and that ICAM-1 deficiency reduces neurological damage after transient focal cerebral ischemia. PMID- 8619548 TI - Immune attack on the Schwann cell surface in acute inflammatory demyelinating polyneuropathy. AB - The localization, mode of action, and roles of complement in the Guillain-Barre syndrome have been controversial. We used high-resolution immunocytochemistry to localize complement activation products in early stages of the acute inflammatory demyelinating polyneuropathy (AIDP) pattern of Guillain-Barre syndrome. Three AIDP subjects who were autopsied had had symptoms for 3 to 9 days at the time of death. Immunocytochemistry was performed on etched, epoxy resin-embedded sections, and the next thin section was compared by electron microscopy (thick/thin sections). Many fibers had a rim of the complement activation marker C3d and the terminal complement complex neoantigen C5b-9 along the outer surface of the Schwann cells. Ultrastructural analysis of these C3d-positive fibers showed mild vesicular changes of the outermost myelin lamellae. Vesicular degeneration was seen before the invasion of macrophages into the myelin, and was the predominant change in the subject with symptoms for 3 days. C3d staining was not found on myelin membranes. The results suggest that at least some forms of AIDP are complement mediated. We speculate that complement is activated by antibody bound to epitopes on the outer surface of the Schwann cell and that the resulting complement activation initiates the vesiculation of myelin. PMID- 8619549 TI - A newly recognized autosomal dominant limb girdle muscular dystrophy with cardiac involvement. AB - Sixty-five members of three families with limb girdle muscular dystrophy (LGMD) underwent neurological, cardiological, and ancillary investigations. Thirty-five individuals were diagnosed as having slowly progressive autosomal dominant LGMD. Symmetrical weakness started in the proximal lower limb muscles, and gradually upper limb muscles also became affected. Early contractures of the spine were absent. Contractures of elbows and Achilles tendons were either minimal or late. Serum creatine kinase activity was normal to moderately elevated. Electromyogram and muscle biopsy were consistent with a mild muscular dystrophy. Cardiological abnormalities, found in more than one-half the patients, included dysrhythmias and atrioventricular (AV) conduction disturbances presenting as bradycardia, syncopal attacks necessitating pacemaker implantation, and sudden cardiac death. There was a significant relation between the severity of AV conduction disturbances and age. In nearly all patients, neuromuscular symptomatology preceded cardiological involvement. The early recognition of this previously not described, autosomal dominant LGMD with life-threatening cardiac involvement offers an opportunity for therapeutic intervention. PMID- 8619550 TI - Infantile spasms: III. Prognostic implications of bitemporal hypometabolism on positron emission tomography. AB - Positron emission tomography (PET) of brain glucose utilization is highly sensitive in detecting focal cortical abnormalities in patients with infantile spasms even when the computed tomographic (CT) and magnetic resonance imaging (MRI) scans are normal. Of 110 infants with spasms evaluated for potential surgical intervention during an 8-year period, we encountered 18 infants (7 males, 11 females; age range, 10 mo to 5 yr) with a common metabolic pattern on positron emission tomography (PET) consisting of bilateral hypometabolism in the temporal lobes. CT and MRI scans did not reveal any focal abnormalities in the 18 infants. Video-electroencephalographic monitoring indicated either bilateral or multifocal epileptogenicity, or failed to show any epileptic focus, so that none of the 18 infants were considered candidates for resective surgery. These patients were then enrolled in a prospective study aimed at determining long-term outcome in the presence of bilateral temporal PET hypometabolism. Analysis of outcome in 14 of the 18 subjects (follow-up period, 10 mo to 10 yr 5 mo; mean, 3 yr 11 mo +/- 2 yr 4 mo [SD]) revealed the following: (1) all had severe developmental delay and had failed to gain significant milestones; (2) language development had been minimal or absent; (3) 10 of the 14 met the DSM-IV criteria for autistic disorder. Our findings indicate that patients with infantile spasms and bitemporal glucose hypometabolism on PET comprise a relatively homogeneous group and are typically not candidates for cortical resection. The long-term outcome of these infants is particularly poor and the majority are autistic. PMID- 8619552 TI - Antiamphiphysin antibodies with small-cell lung carcinoma and paraneoplastic encephalomyelitis. AB - Paraneoplastic encephalomyelitis developed as the presenting feature of small cell lung carcinoma in 3 patients. Two patients with paraneoplastic encephalomyelitis manifested predominantly as subacute sensory neuronopathy did not improve after prednisone treatment and chemotherapy. The third patient had severe axial and limb rigidity and myoclonus, which partially improved after chemotherapy and treatment with intravenous immunoglobulin and prednisone. Serum from each patient immunocytochemically stained the neuropil and to a lesser degree the neuronal cytoplasm in human cerebral and cerebellar cortex. On immunoblots of human neuronal extracts, each patient's serum contained high-titer IgG antibodies reacting with a protein band of apparent molecular mass 125 kd. This autoantibody pattern is indistinguishable from antibodies recently identified in several women with breast carcinoma and stiff-man syndrome. Screening of a human brain complementary DNA expression library with patient serum yielded clones whose sequence is identical to that of the synaptic vesicle related protein amphiphysin. Reverse transcriptase-polymerase chain reaction demonstrated expression of amphiphysin in 8 of 10 small-cell lung carcinomas and in 5 of 14 breast carcinomas. These observations highlight the clinical and serological heterogeneity of paraneoplastic central nervous system disorders: Patients with a given clinical syndrome may have different antineuronal antibodies, and patients with a given autoantibody specificity have differing clinical presentations. PMID- 8619551 TI - The effect of ethanol on alcohol-responsive essential tremor: a positron emission tomography study. AB - We used H2 15O positron emission tomography (PET) to investigate the effect of ethyl alcohol on regional cerebral blood flow in 6 patients with alcohol responsive essential tremor and 6 age-matched control subjects. The patients were scanned while at rest and during involuntary postural tremor of the extended right arm. Normal control subjects were scanned at rest and during passive wrist oscillation of the right arm at tremor frequency. Regional cerebral blood flow associated with these conditions was measured before and after oral administration of 2 to 3 units of alcohol. The mean blood alcohol level was 35.3 +/- 20.0 mg/dl in the patient group and caused marked suppression of tremor; it was 33.9 +/- 12.9 mg/dl in the control group. Similar to previous PET studies on essential tremor patients, tremor compared with rest was associated with bilateral cerebellar activation including the cerebellar vermis. This pattern of activation differed from passive wrist oscillation where ipsilateral cerebellar activation was observed. Ethanol ingestion led to bilateral decreases of cerebellar blood flow in both tremor patients and normal subjects, and this was associated with suppression of tremor in the patients. Alcohol-associated increases of regional cerebral blood flow were observed in the inferior olivary nuclei in the patients but not in the control subjects. We conclude that alcohol induced suppression of essential tremor is mediated via a reduction of cerebellar synaptic overactivity resulting in increased afferent input to the inferior olivary nuclei. PMID- 8619553 TI - Acute paresis of extraocular muscles associated with IgG anti-GQ1b antibody. AB - There have been several case reports of acute ophthalmoparesis without ataxia subsequent to infection or immunization. The nosological position and therapy for acute ophthalmoparesis have yet to be established. Sera from patients are reported to have IgG anti-GQ1b antibody, which is frequently found in sera from patients with Fisher's syndrome. To establish the etiology of acute ophthalmoparesis, I tested sera from 8 patients with acute ophthalmoparesis for anti-GQ1b antibody. High IgG anti-GQ1b antibody titers were present in sera from patients in the acute phase of the illness. I describe the successful treatment with plasmapheresis and intravenous immunoglobulin. Some cases of acute ophthalmoparesis following infection or immunization may be categorized as an auto-immune disease related to Fisher's syndrome. PMID- 8619554 TI - A novel point mutation in the McLeod syndrome gene in neuroacanthocytosis. AB - McLeod syndrome is an X-linked recessive disorder, characterized by neuromuscular and hematopoietic dysfunction. Two cases of McLeod syndrome were reported in a family with neuroacanthocytosis and, remarkably, 1 of them was female. Direct sequence analysis of the McLeod gene in 12 members of the family revealed a novel point mutation in exon 2 that creates a frameshift and results in premature termination of translation. There was marked skewing of X inactivation in the severely affected female. PMID- 8619555 TI - Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex. AB - Glutamate transport is critical for synaptic inactivation of glutamate and prevention of excitotoxicity. The following four glutamate transporters have been identified in human brain: EAAT1, EAAT2, EAAT3, and EAAT4. Deficient glutamate transport has been identified in the motor cortex and the spinal cord of tissue from amyotrophic lateral sclerosis (ALS) patients. The defect appears to be due to a selective loss of the astroglial specific glutamate transporter protein EAAT2. In these studies we sought to extend our understanding of glutamate transporters in ALS by examining the mRNA for each transporter subtype in ALS motor cortex. All tissue was matched for age and postmortem delay. There was no quantitative change in mRNA for EAAT1, EAAT2, or EAAT3 in ALS motor cortex, even in patients with a large loss of EAAT2 protein (95% decrease compared with control) and decreased tissue glutamate transport (73% decrease compared with control). These studies suggest that the dramatic abnormalities in EAAT2 may be due to translational or post-translational processes. PMID- 8619557 TI - High frequency of apolipoprotein E epsilon 2 in patients with cerebral hemorrhage due to cerebral amyloid angiopathy. PMID- 8619556 TI - Expression of Hel-N1 and Hel-N2 in small-cell lung carcinoma. AB - Hel-N1 and HuD are neuron-specific RNA-binding proteins that are antigenic targets of anti-Hu antibodies. Although expression of Hu antigens is most commonly seen in small-cell lung carcinoma, their exact identity (e.g., Hel-NI, Hel-N2, HuD, and HuC cannot be distinguished by immunological methods. Analysis of messenger RNA expression is needed for this distinction. Here we demonstrate that Hel-NI and Hel-N2 are expressed in small-cell lung carcinoma using reverse transcription-polymerase chain reaction. PMID- 8619558 TI - Lack of an association between apolipoprotein E epsilon 4 and cerebral amyloid angiopathy in elderly Japanese. PMID- 8619559 TI - Methotrexate in the treatment of multiple sclerosis. PMID- 8619560 TI - Oligodendroglial development in fetal cerebrum. PMID- 8619561 TI - Management of meningitis caused by penicillin-resistant Streptococcus pneumoniae. PMID- 8619562 TI - Pharmacokinetics of MDL 63,246, a new semisynthetic glycopeptide antibiotic, in the rat. AB - Following intravenous administration in the rat, the concentration of MDL 63,246 in plasma was high and long-lasting. Concentrations fell with an apparent three exponential decay. MDL 63,246 was distributed in a high volume and was cleared quite slowly. The pharmacokinetics of MDL 63,246 in the rat appear to be dose proportional in the dose range of 20 to 50 mg/kg of body weight. When administered subcutaneously, MDL 63,246 was slowly absorbed from the injection site, and the extent of availability was good, being 70.1%. MDL 63,246 was eliminated slowly by both renal and fecal excretion. MDL 63,246 is rapidly and extensively distributed into the tissues. At 0.5 h after drug administration, radioactivity was detected in all organs examined. At 24 h after administration, the total concentrations of radioactivity still increased in some organs which represent a slowly equilibrating compartment, but only the kidneys and liver showed a higher total concentration of radioactivity than plasma. Between 24 and 192 h after treatment, total concentrations of radioactivity decreased very slowly, and finally, apart from brain, all tissues showed higher concentrations than plasma, indicating a very high affinity of MDL 63,246 for tissues. The ratio of the concentration of radioactivity in blood to that in plasma ratio was 0.58, indicating that MDL 63,246 does not diffuse into erythrocytes and that binding to the erythrocyte membrane does not occur. All of these findings appear to correlate with the excellent in vitro and in vivo activities of the compound, suggesting that MDL 63,246 could be therapeutically efficacious at lower dosages and longer treatment intervals than those currently used for vancomycin and teicoplanin. PMID- 8619563 TI - In vivo therapeutic efficacies of PD 138312 and PD 140248, two novel fluoronaphthyridines with outstanding gram-positive potency. AB - PD 138312 and PD 140248 are novel broad-spectrum 7-pyrrolidinyl fluoronaphthyridines with a cyclopropyl or a difluorophenyl substitution at the 1 positions, respectively. They have been demonstrated to have excellent in vitro activity against gram-positive organisms. These compounds were evaluated for their in vivo potencies against acute systemic infections in mice and in a mouse pneumococcal pneumonia model. They were very effective by both the oral and subcutaneous routes of administration. Most remarkable were their comparative median protective values against methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. In general, these compounds were 28- to 100-fold more active than ciprofloxacin against these clinically significant organisms when the drugs were given orally and 10- to 38-fold more active when the drugs were given parenterally. Average ratios of drug concentrations in mice after drug administration by the oral route to that after administration by the subcutaneous route indicate 34 to 44% greater bioavailabilities of PD 138312 and PD 140248 compared with that of ciprofloxacin. In a multidose pneumococcal mouse pneumonia model these new quinolones were extremely effective, with median curative doses of 2 to 2.8 mg/kg of body weight per dose. Ciprofloxacin was ineffective (median curative dose, >100 mg/kg per dose) in this model. Comparative pharmacokinetic studies in mice revealed a relative superiority of PD 140248. Peak levels of PD 140248 in blood after the administration of a single oral 50-mg/kg dose were twice those of PD 138312 and ciprofloxacin, with PD 140248 having a substantially longer half-life. These results indicate that PD 138312 and PD 140248 have excellent therapeutic potential against clinically important gram-positive pathogens when the drugs are administered both orally and parenterally. PMID- 8619564 TI - Growth inhibition of Ureaplasma urealyticum by the proton pump inhibitor lansoprazole: direct attribution to inhibition by lansoprazole of urease activity and urea-induced ATP synthesis in U. urealyticum. AB - The proton pump inhibitors (PPIs) omeprazole and lansoprazole and the acid activated analog of lansoprazole AG-2000, which potently inhibit the urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), also inhibited the urease activities of cell-free extracts as well as intact cells of Ureaplasma urealyticum. The 50% inhibitory concentrations were between 1 and 25 microM. These compounds also inhibited the ATP synthesis induced by urea in ureaplasma cells. The 50% inhibitory concentrations for ATP synthesis were close to those for urease activity, but they were lower than those of urease inhibitors, such as acetohydroxamic acid, hydroxyurea, and thiourea. In addition, one of the metabolites of lansoprazole found in human urine, M-VI, also inhibited ureaplasmal urease activity and the ATP synthesis induced by urea at almost the same concentrations as those of lansoprazole. The inhibition of PPIs against ureaplasma urease was very similar to those against H. pylori urease, suggesting that the inhibitory mechanism against these ureases was due to the blockage of the SH residues on the cysteine of the enzyme. Omeprazole, lansoprazole, AG-2000, and M-VI inhibited the growth of U. urealyticum. Since ureaplasma urease is thought to be involved in the pathogenicity of this organism in the urogenital tract, PPIs and their analogs may be useful as chemotherapeutic agents against diseases caused by U. urealyticum. PMID- 8619565 TI - In vitro activities of cefotaxime, ceftriaxone, ceftazidime, cefpirome, and penicillin against Streptococcus pneumoniae isolates. AB - The in vitro activities of four extended-spectrum cephalosporins and benzyl penicillin were evaluated against 698 clinical isolates of Streptococcus pneumoniae, including 130 (19%) penicillin-intermediate and 84 (12%) penicillin resistant strains. Cefotaxime and ceftriaxone were essentially identical in their antipneumococcal activities: both were active against penicillin-susceptible strains and most penicillin-intermediate strains. Cefpirome was twice as potent as cefotaxime and ceftriaxone against penicillin-resistant strains. Ceftazidime was 8- to 16-fold less active than cefotaxime and ceftriaxone against S. pneumoniae in vitro, and thus, its spectrum included only penicillin-susceptible strains. PMID- 8619566 TI - Fluconazole treatment of Candida albicans infection in mice: does in vitro susceptibility predict in vivo response? AB - A series of fluconazole-susceptible and-fluconazole resistant Candida albicans fungal isolates were used to infect mice intravenously. Mice were treated with varying doses of fluconazole beginning one day after infection. For all of the 6 fluconazole-susceptible isolates, fluconazole was highly effective at <0.25 mg/kg of body weight twice daily. By contrast, fluconazole was less effective in at least 6 of 10 fluconazole-resistant isolates and was ineffective at > or = 40 mg/kg twice daily in 4 fluconazole-resistant isolates. Although the correlation is not precise, in vitro susceptibility testing of C. albicans can predict in vivo response to fluconazole. PMID- 8619567 TI - Pharmacokinetics and metabolism of 14C-isepamicin in humans following intravenous administration. AB - Twelve healthy adult male volunteers received 1 g (base equivalent) of 14C isepamicin (131 microCi) as an intravenous bolus over 5 min. The areas under the plasma concentration-time curves at infinity for isepamicin (196 micrograms*h/ml) and total radioactivity (164 micrograms*h/ml) were similar, indicating no biotransformation of isepamicin. The disappearance of isepamicin from plasma followed a triexponential decline, with half-lives of 0.17, 2.12, and 34 h for the alpha, beta, and gamma phases, respectively. However, the contribution of the gamma phase to the total area under the concentration-time curve was only 2.6%. There were no detectable metabolites in plasma and urine, confirming that isepamicin was not biotransformed. The cumulative levels of isepamicin and total radioactivity excretion in urine from 0 to 120 h were 97.3 and 92.1% of the dose, respectively, indicating that the drug was excreted mainly as unchanged isepamicin in urine. PMID- 8619568 TI - Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus. AB - The gene encoding M2, the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomyces cerevisiae. By using single and multicopy plasmids containing GAL promoter-M2 fusions, a correlation was observed between plasmid copy number and growth in medium inducing M2 expression. Cells expressing M2 from multicopy plasmids have reduced growth rates, suggesting that high levels of M2 are toxic to growth. The addition of amantadine, a compound known to block the ion channel activity of certain M2 alleles, restores the growth rates to wild-type levels in cells expressing an amantadine-susceptible allele of M2 but not an amantadine-resistant allele of M2, suggesting that M2 expression in S. cerevisiae results in the formation of functional M2 ion channels. Measurements of extracellular acidification by microphysiometry suggest that proton efflux in M2-expressing cells is altered and that the addition of amantadine permits the reestablishment of the proton gradient. The growth impairment phenotype resulting from M2 expression was used to develop a high-capacity screening assay which identified a novel inhibitor possessing an antiviral profile similar to that of amantadine. PMID- 8619570 TI - Antipneumocystis activity of 17C91, a prodrug of atovaquone. AB - The prophylactic efficacy of 17C91, a carbamate prodrug of atovaquone (ATQ), was investigated in a severe combined immunodeficient mouse model of Pneumocystis carinii pneumonia (PCP). At an oral dosage equivalent to 100 mg of ATQ per kg of body weight per day, 17C91 protected 9 of 10 mice from PCP and had a prophylactic efficacy comparable to that of co-trimoxazole (at 250 mg of sulfamethoxazole plus 50 mg of trimethoprim per kg per day orally). The intensity of P. carinii infection (infection score) of mice treated with 17C91 correlated with the concentration of ATQ in the plasma, with clearance of the infection associated with plasma ATQ levels of >35 micrograms/ml. 17C91 given orally provided enhanced levels of ATQ in the plasma compared with the conventional ATQ formulation. Additional studies reported in this paper demonstrate that the prophylactic activity of 17C91 against PCP in severe combined immunodeficient mice is comparable to that of a new oral microparticulate formulation of ATQ. PMID- 8619569 TI - In vivo efficacy of trovafloxacin (CP-99,219), a new quinolone with extended activities against gram-positive pathogens, Streptococcus pneumoniae, and Bacteroides fragilis. AB - The interesting in vitro antimicrobial activity and pharmacokinetics of the new quinolone trovafloxacin (CP-99,219) warranted further studies to determine its in vivo efficacy in models of infectious disease. The significance of the pharmacokinetic and in vitro antimicrobial profiles of trovafloxacin was shown through efficacy in a series of animal infection models by employing primarily oral therapy. Against acute infections, trovafloxacin was consistently more effective than temafloxacin, ciprofloxacin, and ofloxacin against Streptococcus pneumoniae and other gram-positive pathogens while maintaining activity comparable to that of ciprofloxacin against gram-negative organisms. In a model of murine pneumonia, trovafloxacin was more efficacious than temafloxacin, while ciprofloxacin failed against S. pneumoniae (50% protective doses, 2.1, 29.5, and >100 mg/kg, respectively). In addition to its inherent in vitro potency advantage against S. pneumoniae, these data were supported by a pharmacokinetic study that showed levels of trovafloxacin in pulmonary tissue of S. pneumoniae-infected CF1 mice to be considerably greater than those of temafloxacin and ciprofloxacin (twice the maximum drug concentration in serum; two to three times the half-life, and three to six times the area under the concentration-time curve). Against localized mixed anaerobic infections, trovafloxacin was the only agent to effectively reduce the numbers of recoverable CFU of Bacteroides fragilis ( >1,000-fold), Staphylococcus aureus (1,000-fold), and Escherichia coli ( >100 fold) compared with ciprofloxacin, vancomycin, metronidazole, clindamycin, cefoxitin, and ceftriaxone. The in vitro and in vivo antimicrobial activities of trovafloxacin and its pharmacokinetics in laboratory animals provide support for the ongoing and planned human phase II and III clinical trials. PMID- 8619571 TI - In vitro activities of antimicrobial combinations against Stenotrophomonas (Xanthomonas) maltophilia. AB - Stenotrophomonas (Xanthomonas) maltophilia is inherently resistant to multiple antimicrobial agents. In order to investigate the in vitro potential of combinations of antimicrobial agents, we obtained 230 epidemiologically unrelated clinical isolates from seven hospitals across Canada and from Northwestern Memorial Hospital in Chicago. Ticarcillin-clavulanate combined with ciprofloxacin or trimethoprim-sulfamethoxazole were assayed for synergy against 31 ticarcillin resistant strains of S. maltophilia by using microtiter checkerboard panels and against 20 strains by using time-kill methodology. The combination of ciprofloxacin with ceftazidime was also evaluated by time-kill studies. Ticarcillin-clavulanate plus trimethoprim-sulfamethoxazole demonstrated synergy by checkerboard panels, with fractional inhibitory concentration indices ranging from 0.033 to 0.49, and by time-kill studies for all 20 strains tested. Synergy between ticarcillin-clavulanate plus ciprofloxacin was found by the checkerboard method for 24 of 31 strains (77%), with fractional inhibitory concentration indices ranging from 0.188 to 0.75. A correlation between synergy by the checkerboard method and the reference time-kill study method was not observed for ticarcillin-clavulanate plus ciprofloxacin, with results for 3 of 10 strains being nonconcordant. Synergy with both ticarcillin-clavulanate plus ciprofloxacin and ceftazidime plus ciprofloxacin by the time-kill method was found to correlate with ciprofloxacin MICs of <32 micrograms/ml and zone diameters of >15 mm on Mueller-Hinton agar. Evaluation of these combinations in vivo may be warranted. PMID- 8619572 TI - Safety and efficacy of long-term use of rimantadine for prophylaxis of type A influenza in nursing homes. AB - The safety and efficacy of rimantadine for long-term prophylaxis of influenza A (H3N2) infection were evaluated among elderly residents in 10 nursing homes. Within each nursing home, participating residents were randomly assigned to receive placebo or rimantadine at 100 or 200 mg/day. Residents were evaluated daily for symptoms and significant health events as possible side effects, as well as for influenza-like illness. The study medications were administered to 328 residents for up to 8 weeks, with no statistically significant differences in the frequencies of gastrointestinal or central nervous system symptoms between the groups. However, residents in the active medication groups were more likely to withdraw from the study and to experience various health events including death; some but not all of these differences were statistically significant. Efficacy evaluations were carried out on the 68 vaccinated residents in the two nursing homes with demonstrated influenza virus activity. Rimantadine appeared to provide an additional protective effect beyond vaccination in reducing the risk of clinical and laboratory-confirmed influenza-like illness; however, the efficacy estimates were never statistically significant. The efficacies of the 100- and 200-mg/day dosages were generally similar. When data for the 100- and 200-mg/day dosage groups were combined and compared with data for the group receiving placebo, the efficacy of rimantadine in reducing the risk of clinical influenza-like illness was estimated to be 58 percent (P = 0.079). The results suggest the relative safety and clinical efficacy of using rimantadine for influenza prophylaxis among vaccinated elderly individuals and support the recommendation for a dosage reduction to 100 mg/day in this population. PMID- 8619573 TI - SC-52151, a novel inhibitor of the human immunodeficiency virus protease. AB - SC-52151 is a potent, selective, tight-binding human immunodeficiency virus (HIV) protease inhibitor containing the novel (R)-(hydroxyethyl) urea isostere. The mean 50% effective concentration for lymphotropic, monocytotropic strains and field isolates of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus is 26 ng/ml (43 nM). The combination of SC-52151 and nucleoside reverse transcriptase inhibitors synergistically inhibited HIV-1 replication without additive toxicity. An extended postantiviral effect correlates with inhibition of gag and gag-pol polyprotein processing. SC-52151 is highly protein bound ( >90%) in human plasma, and the level of partitioning into erythrocytes is low. Physiological concentrations of alpha-1-acid glycoprotein, but not albumin, substantially affect the antiviral potency of SC-52151. The oral bioavailability of [14C]SC-52151 is 17% when it is administered as an elixir to the rat, dog, or monkey. Oxidation of the t-butyl moiety is the major route of biotransformation, and elimination is mainly by biliary excretion. No toxicologically significant effects have been observed in animals. Pharmacokinetic and metabolism studies in multiple animal species predict 20 to 30% systemic bioavailability, an elimination half-life of 1 to 2 h, and a volume of distribution of greater than 3 liters/kg in humans. PMID- 8619574 TI - High-throughput screen for detecting antimycobacterial agents. AB - A simple, robust assay system which can be used to screen for inhibitors of mycobacterial growth has been developed. A strain of the rapidly growing saprophyte Mycobacterium aurum is used as the test organism. Inhibition of its growth is highly predictive of activity against Mycobacterium tuberculosis, which cannot itself be used in screening because of its growth characteristics and highly infectious nature. The viability of M. aurum in the presence of a test sample is monitored by measuring the uptake of radiolabelled uracil into the cells. In a microtiter plate format, the screen has the potential for testing several thousand samples per day. PMID- 8619575 TI - Decreased activity of erythromycin against Streptococcus pyogenes in Taiwan. AB - A total of 78 clinical isolates of Streptococcus pyogenes were collected from January 1992 through December 1993 from patients in southern Taiwan. The in vitro activities of 10 antimicrobial agents were determined by the agar dilution method. Penicillin, cephalothin, cefotaxime, vancomycin, and ofloxacin were shown to be active against S. pyogenes isolates, with MICs at which 90% of isolates are inhibited (MIC90s) being < or = 0.03, < or = 0.13, < or = 0.13, < or = 0.13, and < or = 0.25 microgram/ml, respectively. Erythromycin and azithromycin both had poor activities (MIC50s, 16 and >128 micrograms/ml, respectively; MIC90s, >128 and >128 micrograms/ml, respectively). The activities of tetracycline, clindamycin, and chloramphenicol against a significant number of these isolates were also limited. As the MICs of clindamycin and chloramphenicol for the isolates increased, the MICs of the two macrolides also increased. Clindamycin, chloramphenicol, and the two macrolides were less potent against isolates recovered form throat swab samples than against those from blood or other sources. Isolates of the T12 and T1 serotypes accounted for 53.8% of all isolates. The majority (87.5%) of the isolates recovered from throat swab samples were of the T12 serotype, whereas 19.2% of the isolates recovered from blood were of the T12 serotype. In contrast, 66.7% of the isolates of the T1 serotype were derived from blood but none were derived from throat swab samples. Of the 33 T12 serotype isolates, erythromycin MICs for 78.8% of the isolates were >128 micrograms/ml. Because of the poor activities of erythromycin and azithromycin against S. pyogenes isolates from patients in southern Taiwan, these drugs should no longer be considered the drugs of choice for the management of group A streptococcal infections among patients who live in this area. PMID- 8619576 TI - In vitro activities of 22 beta-lactam antibiotics against penicillin-resistant and penicillin-susceptible viridans group streptococci isolated from blood. AB - A total of 410 strains of viridans group streptococci isolated consecutively from blood were tested by the microdilution method for in vitro susceptibility to 22 beta-lactam antibiotics. One hundred thirty-eight strains (33.6%) were resistant to penicillin with a MIC range of 0.25 to 8 micrograms/ml. MICs of all beta lactam agents tested were higher for penicillin-resistant strains than for susceptible strains. These antibiotics were classified into three groups according to their in vitro activities (MICs at which 50 and 90% of the isolates are inhibited). Beta-Lactams of the first group (these included imipenem, cefpirome, FK-037, cefditoren, cefotaxime, ceftriaxone, and cefepime) showed activities higher than or similar to that of penicillin against penicillin resistant viridans group streptococci. However, 80% of highly penicillin resistant Streptococcus mitis organisms required cefotaxime and ceftriaxone MICs of > or = 2 micrograms/ml (range, 2 to 16 micrograms/ml). Beta-Lactams of the second group (cefpodoxime, ampicillin, amoxicillin-clavulanate, piperacillin, and cefuroxime) showed lower activities than penicillin. Finally, antibiotics of the third group (cephalothin, oxacillin, ceftazidime, cefixime, cefaclor, cefetamet, cefadroxil, cephalexin, and ceftibuten) showed poor in vitro activities. Therefore, some of the beta-lactam agents included in the first group could be an acceptable alternative in the treatment of serious infections due to strains highly resistant to penicillin, although clinical experience is needed. PMID- 8619577 TI - Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem. AB - In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 micrograms/ml), 20 of which were also resistant to imipenem (mean MIC, >16 micrograms/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25- to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 microgram/ml and 0.5 microgram/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms. PMID- 8619578 TI - Preclinical evaluation of HBY 097, a new nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 replication. AB - HBY 097 [(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4 dihydroquinoxaline-2(1H)-thione] was selected from a series of quinoxalines as a nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (NNRTI). HBY 097 was shown to be a highly potent inhibitor of HIV-1 induced cell killing and HIV-1 replication in a variety of human cell lines as well as in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against a variety of clinical isolates of HIV-1 including different HIV-1 subtypes and viruses resistant to 3'-deoxy-3'-azidothymidine. Mutant reverse transcriptases which arise as a consequence of treatment with other nonnucleoside inhibitors of HIV-1 reverse transcriptase were still inhibited by HBY 097 at relatively low concentrations. An HIV-1MN variant resistant to inhibition by HBY 097 displayed in the reverse transcriptase gene a mutation causing a substitution at position 190 of a glutamic acid for a glycine residue (G190 --> E), which is characteristic for quinoxaline derivatives. The drug was demonstrated to possess a favorable toxicity profile and to show good oral bioavailability in both mice and dogs. As a consequence of its outstanding properties, HBY 097 was selected for further development and is at present undergoing clinical trials. PMID- 8619579 TI - Effect of a bacterial lipopolysaccharide on biliary excretion of a beta-lactam antibiotic, cefoperazone, in rats. AB - Klebsiella pneumoniae O3 lipopolysaccharide (LPS) has been found to dramatically modify the pharmacokinetics of the beta-lactam antibiotic cefazolin in rats. This study investigated the effect of LPS on the biliary excretion of the beta-lactam antibiotic cefoperazone (CPZ) in rats. CPZ is known to be actively secreted into the bile by a carrier-mediated transport system. LPS (250 micrograms/kg of body weight) was infused for 20 to 30 min 2 h before an intravenous administration of CPZ (20 mg/kg). The pharmacokinetic parameters of CPZ were estimated by a noncompartment model. LPS induced a significant decrease in the systemic clearance (by approximately 50%) and an increase in the mean residence time of CPZ. Significant decreases were also seen in the bile flow rate and in the biliary recovery of unchanged CPZ in the LPS-treated rats. LPS tended to increase the proportion of urinary excretion of CPZ. LPS significantly decreased the biliary clearance (by approximately 55%) and renal clearance (by approximately 35%) of CPZ. However, no changes in the volume of distribution at steady state for CPZ were observed between the treatment groups. Our findings suggest that LPS induces changes in the pharmacokinetics of CPZ as a result of changes occurring in the biliary secretory system. PMID- 8619580 TI - Modulation of biofilms of Pseudomonas aeruginosa by quinolones. AB - The interaction between four fluoroquinolones (ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin) and biofilms of Pseudomonas aeruginosa in wells of microtiter plates and on segments of vascular catheters were studied in an in vitro model of vascular catheter colonization. Subinhibitory concentrations (one half, one-fourth, and one-eight of the MIC) of the fluoroquinolones reduced the adherence of P. aeruginosa to 30 to 33, 44 to 47, and 61 to 67% of that of controls, respectively. The addition of high concentrations of the fluoroquinolones (12.5 and 400 micrograms/ml) to preformed biofilms (grown for 48 h at 37 degrees C) decreased the adherence of P. aeruginosa to 69 to 77 and 39 to 60% of that of controls, respectively. In an in vitro model of vascular catheter colonization, subinhibitory concentrations (one-half, one-fourth, and one-eight of the MIC) of fluoroquinolones reduced the number of adherent bacteria to 21 to 23, 40 to 46, and 55 to 70% of that of the controls, respectively. Scanning electron microscopy demonstrated a significant reduction in glycocalyx formation and adherent bacteria in the presence of pefloxacin at one-half to one-eight of the MIC. Vascular catheter segments precolonized with P. aeruginosa for 24 h and exposed to the fluoroquinolones at 4 to 25 times the MIC (50 micrograms/ml) for 2 h showed <5% growth of adherent cells compared with controls. No adherent organisms were cultured in the presence of 8 to 50 times the MIC (100 micrograms/ml). Scanning electron microscopy studies of preformed biofilms exposed to pefloxacin verified the results obtained by culture. These data show that subinhibitory concentrations of ciprofloxacin, norfloxacin, pefloxacin, and ofloxacin inhibit the adherence of P. aeruginosa to plastic surfaces and vascular catheters. Clinically achievable concentrations of fluoroquinolones (50 to 100 micrograms/ml) were able to eradicate preformed biofilms on vascular catheters. PMID- 8619582 TI - Mutations in katG gene sequences in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis are rare. AB - In this study, a battery of oligonucleotides was directed toward the katG gene and PCR-single-stranded conformation polymorphism (SSCP) analysis was used to search for katG gene deviations in clinical isolates of Mycobacterium tuberculosis from different geographical regions. Since a complete deletion of the katG gene was not found, it is suggested that deletion is not a major mechanism of isoniazid (isonicotinic acid hydrazide; INH) resistance in these isolates. However, 7 of 39 isolates (4 of 25 from South Africa and 3 of 14 from other geographical regions) showed mobility shifts by SSCP analysis, suggesting aberrations in the katG gene. Direct sequence analysis confirmed that the mobility shifts were due to Thr-275-->Ala (Thr275Ala), Arg409Ala, Arg463Leu, and Asp695Ala mutations and a 12-bp deletion in the 5' region of the katG gene. Mutations at codons 275, 463, and 695 created altered restriction sites for HhaI, MspI, and HaeIII, respectively, and sequence results, supported by restriction fragment length polymorphism analysis, suggested that the PCR-SSCP procedure is a good indicator of mutations in PCR-amplified fragments. Identical mutations at codons 463 and 275 were found in isolates from different geographical regions. This may suggest a common evolutionary event, but one of the control isolates (susceptible to INH [3%; n = 30]) also had a mutation at codon 463. The results suggest that variations in the katG coding gene sequences of INH-resistant isolates of M. tuberculosis are infrequent and that defects in other regions of the M. tuberculosis genome are of equal or greater importance in contributing to the acquisition of resistance to INH. PMID- 8619581 TI - Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta lactamase isolated from Escherichia coli. AB - Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A beta-lactamase. Detailed comparison of Toho-1 with other beta-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity. PMID- 8619583 TI - Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States. AB - We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same. PMID- 8619584 TI - Vancomycin or vancomycin plus netilmicin for methicillin- and gentamicin resistant Staphylococcus aureus aortic valve experimental endocarditis. AB - Using a rabbit model of aortic valve endocarditis, we studied the efficacy of vancomycin alone or in combination with netilmicin and/or rifampin against a methicillin- and gentamicin-resistant strain of Staphylococcus aureus (MGRSA). Antibiotics were given for 6 to 12 days, as follows: vancomycin (15 mg/kg of body weight every 12 h [BID] intravenously), vancomycin plus netilmicin (2.5 mg/kg BID intramuscularly), vancomycin plus rifampin (10 mg/kg BID intramuscularly), and vancomycin plus netilmicin plus rifampin at the same routes, dosages, and schedules mentioned above. Netilmicin was given to two additional groups at a higher dosage (6 mg/kg every 24 h intramuscularly) alone or in combination with vancomycin (15 mg/kg BID intravenously) for 12 days. All regimens resulted in undetectable bacterial counts in a significant proportion of vegetations (except netilmicin alone) or reduced the bacterial counts in the vegetations compared with the counts in the untreated controls (P<0.01 to P<0.001). No resistance to rifampin or netilmicin developed during therapy. It is concluded that in the treatment of experimental aortic valve endocarditis caused by MGRSA (i) vancomycin as monotherapy is as efficacious as the triple combination, (ii) the addition of netilmicin (once daily or BID) to vancomycin does not improve the efficacy of the latter antibiotic, even in the presence of rifampin, and (iii) a 12-day course in more effective than a 6-day one, but not at a statistically significant level. PMID- 8619585 TI - In vitro and in vivo activities of the benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis. AB - The in vitro and in vivo activities of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium tuberculosis were studied. The MIC at which 50% of the isolates are inhibited (MIC50) and the MIC90 of KRM for 30 fresh isolates of M. tuberculosis measured by the BACTEC 460 TB System were 0.016 and 2 micrograms/ml, respectively. These values were much lower than those for rifampin (RMP), which were 4 and >128 micrograms/ml, respectively, and considerably lower than those for rifabutin (RBT), which were 0.125 and 8 micrograms/ml, respectively. A correlational analysis of the MICs of these drugs for the clinical isolates revealed the presence of cross-resistance of the organisms to KRM and either RMP or RBT although the MICs of KRM were distributed over a much lower range than were those of the other two drugs. KRM and RMP at concentrations of 1 to 10 micrograms/ml almost completely inhibited the bacterial growth of RMP sensitive strains (H37Rv, Kurono, and Fujii) of M. tuberculosis phagocytosed in macrophage-derived J774.1 cells. KRM was more active than RMP in inhibiting the growth of the RMP-resistant (MIC = 8 micrograms/ml) Kurata strain but failed to show such an effect against the RMP-resistant (MIC >128 micrograms/ml) Watanabe stain. When KRM was given to M. tuberculosis-infected mice at dosages of 5 to 20 mg/kg of body weight by gavage, one daily six times per week from day 1 after infection, it was much more efficacious than RMP against infections induced in mice by the RMP-sensitive Kurono strain, as measured by a reduction of rates of mortality, a reduction of the frequency and extent of gross lung lesions, histopathological changes in lung tissues, and a decrease in the bacterial loads in the lungs and spleens of infected mice. KRM also displayed significant therapeutic efficacy against infection induced by the RMP-resistant Kurata strain, while neither KRM nor RMP was efficacious against infection by the RMP resistant Watanabe strain. In the case of infection with the Kurono strain, the efficacy of the drugs in prolonging the time of survival was in the order KRM, RBT, RMP. KRM was much more efficacious than RMP, when given at 1- to 4-week intervals. These findings suggest that KRM may be useful for the clinical treatment of tuberculosis contracted through RMP-sensitive strains, even when it is administered at long intervals. PMID- 8619586 TI - Metabolic pathways for activation of the antiviral agent 9-(2 phosphonylmethoxyethyl)adenine in human lymphoid cells. AB - 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), the acyclic phosphonate analog of adenine monophosphate, is a promising antiviral drug with activity against herpesviruses, Epstein-Barr virus, and retroviruses, including the human immunodeficiency virus. In order to be active, it must be converted to the diphosphate derivative, the putative inhibitor of viral DNA polymerases. The metabolic pathway responsible for activation of PMEA is unclear. The metabolism of PMEA was investigated in human T-lymphoid cells (CEMss) and a PMEA-resistant subline (CEMss(r-1)) with a partial deficiency in adenylate kinase activity. Experiments with [3H]PMEA showed that extracts of CEMss phosphorylated PMEA to its mono- and diphosphate in the presence of ATP as the phosphate donor. No other nucleotides or 5-phosphoribosyl pyrophosphate displayed appreciable activity as a phosphate donor. Subcellular fractionation experiments showed that CEMss cells contained two nucleotide kinase activities, one in mitochondria and one in the cytosol, which phosphorylated PMEA. The PMEA-resistant CEMss mutant proved to have a deficiency in the mitochondrial adenylate kinase activity, indicating that this enzyme was important in the phosphorylation of PMEA. Other effective antiviral purine phosphonate derivatives of PMEA showed a profile of phosphorylating activity similar to that of PMEA. By comparison, phosphorylation of the pyrimidine analog (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine proceeded by an enzyme present in the cytosol. We conclude from these studies that adenylate kinase which has been localized in the intermembrane space of mitochondria is the major route for PMEA phosphorylation in CEMss cells but that another hitherto unidentified enzyme(s) present in the cytosol may contribute to the anabolism of the phosphonates. PMID- 8619587 TI - Population pharmacokinetics of stavudine (d4T) in patients with AIDS or advanced AIDS-related complex. AB - The population pharmacokinetics and bioavailability of oral stavudine (d4T; 2',3' dideoxy-3'-deoxythymidine) was determined in 81 patients with AIDS or AIDS related complex (ARC) enrolled in phase I and phase I/II dose-ranging trials. Each patient underwent inpatient pharmacokinetic studies following administration of the first oral stavudine dose; 59 patients were restudied after chronic therapy for an average of 19 days. Thirty-three of these patients also received a single intravenous stavudine dose prior to starting an oral regimen. A two compartment model with first-order absorption and elimination was used as the structural pharmacokinetic model. A basic model provided the following population parameter estimates (interpatient variability expressed in parentheses as percent coefficient of variation): clearance/bioavailability = 30.9 (24.5%) liters/h; volume of distribution/bioavailability = 8.42 (not modeled) liters; volume of distribution at steady state/bioavailability = 68.9 (105%) liters; intercompartmental clearance/bioavailability = 12.4 (26%) liters/h; and first order absorption rate constant = 1.32 (78.9%) liters/h. In the subset of 33 patients receiving both intravenous and oral doses, the bioavailability of stavudine was estimated to be 99.1% (18.5%). Total body weight, stage of disease (AIDS versus ARC), and an oral stavudine dose of > or = 200 mg were found to have a statistically significant but a clinically marginal effect on the estimate of the oral clearance of stavudine. This analysis shows the high degree of bioavailability of stavudine in patients with AIDS and ARC and the relatively low degree of interpatient variability in oral drug clearance compared with those of other nucleosides. Population pharmacokinetic analysis is a useful tool for assessing the combined effects of several patient variables on the pharmacokinetic properties of drugs in human immunodeficiency virus-infected patients. PMID- 8619588 TI - Treatment of Mycobacterium haemophilum infection in a murine model with clarithromycin, rifabutin, and ciprofloxacin. AB - An animal model of disseminated Mycobacterium haemophilum infection was utilized to compare treatment with azithromycin, ciprofloxacin, rifabutin, and the combination of clarithromycin with rifabutin. Following subcutaneous challenge with M. haemophilum, local and disseminated infection occurred only in immunosuppressed mice. For disseminated infection, ciprofloxacin was relatively ineffective therapy. Clarithromycin and rifabutin alone significantly reduced the tissue burden in the spleen after 4 weeks of therapy. Combination therapy with rifabutin and clarithromycin was superior to 4 weeks of treatment with the individual agents. When immunosuppressed mice were treated for 20 weeks with the combination of rifabutin and clarithromycin, the tissue burden remained reduced in the spleen at 1 month following the completion of therapy. Combined rifabutin and clarithromycin provide effective treatment for M. haemophilum in this model. PMID- 8619589 TI - Antimycobacterial activity of a new rifamycin derivative, 3-(4 cinnamylpiperazinyl iminomethyl) rifamycin SV (T9). AB - The antimycobacterial activities of a new rifampin (RIF) derivative, 3-(4 cinnamylpiperazinyl iminomethyl) rifamycin SV (To), against 20 susceptible and multidrug-resistant strains of Mycobacterium tuberculosis and 20 Mycobacterium avium complex (MAC) strains were investigated. The radiometric MICs of T9 for M. tuberculosis were significantly lower than those of RIF. The MICs of T9 and RIF at which 90% of the RIF-susceptible strains were inhibited were < or = 0.25 and < or = 0.5 micrograms/ml, respectively. Interestingly, T9 had lower MICs against some RIF-resistant M. tuberculosis strains. T9 had better activity against MAC strains, and the MIC at which 90% of the MAC strains were inhibited was < or = 0.125 micrograms/ml, and that of RIF was < or = 2.0 micrograms/ml. T9 also showed high in vitro bactericidal and intracellular activities which were significantly superior to those of RIF against both M. tuberculosis, and MAC strains. More importantly, T9 showed excellent in vivo activity against M. tuberculosis H37Rv compared with that of RIF in both the lungs and spleens of C57BL/6 mice, indicating the potential therapeutic value of T9 in the treatment of mycobacterial infections. PMID- 8619590 TI - Antimicrobial activity of CS-940, a new trifluorinated quinolone. AB - The antimicrobial activity of CS-940, a new trifluorinated quinolone drug, was tested against 761 clinical isolates. CS-940 activity against members of the family Enterobacteriaceae was most similar to that of ciprofloxacin and ofloxacin, with a large range of MICs inhibiting 90% of isolates tested (MIC90S) of 0.015 to 16 micrograms/ml (median MIC90, 0.06 micrograms/ml). CS-940 had greater activity than ciprofloxacin or ofloxacin when they were tested against Acinetobacter spp. (MIC90S, 0.03 micrograms/ml) and Stenotrophomonas (Xanthomonas) maltophilia (MIC90S, 2 micrograms/ml). CS-940 demonstrated a high degree of potency against Haemophilus influenzae, Moraxella catarrhalis, and Neisseria spp. (MIC90S, < or = 0.06 micrograms/ml). CS-940 was two- to eightfold more active than ciprofloxacin or ofloxacin against oxacillin-susceptible Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and coagulase-negative Staphylococcus spp. CS-940 was also very active against Streptococcus spp. and enterococci, for which MIC90S were < or = 2 micrograms/ml; for Enterococcus faecium, however, the MIC90 was 4 micrograms/ml. CS-940 was generally less active than a comparison investigational fluoroquinolone, clinafloxacin. This compound appears promising by in vitro test analysis and warrants further in vivo trials. PMID- 8619591 TI - In vitro antibacterial activity and beta-lactamase stability of a new carbapenem, BO-2727. AB - The in vitro activity of BO-2727, a new carbapenem, was compared with those of meropenem, biapenem, imipenem, and ceftazidime. BO-2727 was four- or eightfold more active than the other carbapenems against methicillin-resistant staphylococci and Pseudomonas aeruginosa strains, including imipenem- and ceftazidime-resistant bacteria. BO-2727 was quite stable to penicillinases, cephalosporinases, and oxyiminocephalosporinases, but not to metallo-beta lactamase. Time-kill studies against Staphylococcus aureus Smith, Escherichia coli ML4707, and P. aeruginosa GN11189 showed that BO-2727 has potent bactericidal activity at concentrations greater than the MIC. PMID- 8619592 TI - Potent and specific inhibition of human immunodeficiency virus type 1 replication by 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-Y1 N,N-dialkylcarbamate derivatives. AB - 4-(2,6-Dichlorophenyl)-1,2,5-thiadiazol-3-yl N,N-dialkylcarbamate (TDA) derivatives were found to be highly potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in a variety of cell cultures. The most potent congener of TDA derivatives, RD4-2024, inhibited HIV-1 replication by 50% at concentrations of 12.5 and 4.8 nM in MT-4 cells and peripheral blood mononuclear cells, respectively. These concentrations were more than 2,000- and 30,000-fold lower than its 50% cytotoxic concentrations, respectively. Although the TDA derivatives were active against 3'-azido-3' deoxythymidine-resistant HIV-1, no antiviral activities were observed against HIV 2 and nonnucleoside reverse transcriptase inhibitor-resistant mutants of HIV-1. The TDA derivatives inhibited recombinant HIV-1 reverse transcriptase activity, depending on the template-primer used for the assay. However, they did not interact with HIV-2 reverse transcriptase. Thus, the TDA derivatives belong to the family of nonnucleoside reverse transcriptase inhibitors. Because of their potent anti-HIV-1 activities in vitro and their low levels of toxicity in mice, the TDA derivatives deserve further evaluation as candidate drugs for the treatment of patients with AIDS. PMID- 8619593 TI - In vitro interactions between different beta-lactam antibiotics and fosfomycin against bloodstream isolates of enterococci. AB - The effects of 16 different beta-lactam-fosfomycin combinations against 50 bloodstream enterococci were compared by a disk diffusion technique. Cefotaxime exhibited the best interaction. By checkerboard studies, the cefotaxime fosfomycin combination provided a synergistic bacteriostatic effect against 45 of the 50 isolates (MIC of cefotaxime at which 90% of the isolates were inhibited, >2,048 micrograms/ml; MIC of fosfomycin at which 90% of the isolates were inhibited, 128 micrograms/ml; mean of fractional inhibitory concentration indexes, 0.195). By killing curves, cefotaxime (at 64 micrograms/ml) combined with fosfomycin (at > or = 64 micrograms/ml) was bactericidal against 6 of 10 strains tested. PMID- 8619594 TI - NP-06: a novel anti-human immunodeficiency virus polypeptide produced by a Streptomyces species. AB - From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 microM when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively, NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion. PMID- 8619595 TI - Pharmacokinetics of cefpirome in pediatric patients. AB - Cefpirome is a new investigational cephalosporin. We designed a study to determine the pharmacokinetics and tolerance of cefpirome in pediatric patients. A single dose of cefpirome was administered intravenously over 15 min to 18 patients (age 0.5 to 18 years). The doses were 10 mg/kg of body weight for five patients, 25 mg/kg of body weight for seven patients, and 50 mg/kg of body weight for six patients. Blood samples were collected at 0, 0.25, 0.5, 1, 3, 5, and 8 h after the dose, and cefpirome was measured by a high-performance liquid chromatography method. The maximum concentration in serum ranged from about 53.6 to 454 micrograms/ml after doses of 10 to 50 mg/kg. The total body clearance, apparent volume of distribution, and elimination half-life were 2.15 +/- 0.70 ml/min/kg, 0.32 +/- 0.32 liter/kg, and 1.8 +/- 1.3 h, respectively. No significant adverse effects were attributed to cefpirome. These data may be useful in conducting efficacy and safety studies of cefpirome in pediatric patients. PMID- 8619596 TI - Population differences in ganciclovir clearance as determined by nonlinear mixed effects modelling. AB - We examined the pharmacokinetics of ganciclovir in different populations of cytomegalovirus (CMV)-infected patients through the use of nonlinear mixed effects modelling. As expected, patient weight and estimated creatinine clearance were shown to be important covariates in the serum ganciclovir clearance. Unexpectedly, major differences in ganciclovir clearance between different populations of patients were found. Human immunodeficiency virus (HIV)-infected patients with CMV retinitis cleared ganciclovir 41% faster than HIV-infected patients only shedding CMV into the urine. Solid-organ transplant patients had a serum clearance one-fourth that of HIV-infected patients, even with correction for creatinine clearance. These findings require prospective validation and may have important implications for ganciclovir dosing in different populations of CMV-infected patients. PMID- 8619597 TI - Short-term topical therapy of experimental tinea pedis in guinea pigs with lanoconazole, a new imidazole antimycotic agent. AB - The therapeutic efficacy of short-term treatment with a 1% cream of lanoconazole, a new imidazole antimycotic agent, in comparison with that of a 1% cream of terbinafine was evaluated in the guinea pig model of tinea pedis. Each agent was topically applied once a day for 3 or 7 consecutive days, starting on day 10 postinfection, and a culture study was conducted on day 5 after the last treatment with each agent. The 1% cream of lanoconazole was as highly effective as the 1% cream of terbinafine in terms of eradicating the fungi from the infected feet. PMID- 8619598 TI - Postantibiotic effects and Burkholderia (Pseudomonas) pseudomallei: evaluation of current treatment. AB - The postantibiotic effects (PAEs) OF 16 antibiotics or antibiotic combinations currently used against Burkholderia (Pseudomonas) pseudomallei were determined. The beta-lactams had zero PAEs, while the carbapenems and ciprofloxacin induced median PAEs of 1 and 5 h, respectively. These results emphasize the need to maintain antibiotic levels above inhibitory concentrations in blood throughout the dosing interval in cases of severe melioidosis. PMID- 8619599 TI - Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli. AB - The DNA fragment (3.3 kb) containing the erythromycin resistance determinant was cloned from Escherichia coli Tf481A and sequenced. Deletion and complementation analyses indicated that the expression of high-level resistance to erythromycin requires two genes, mphA and mrx, which encode macrolide 2'-phosphotransferase I and an unidentified hydrophobic protein, respectively. PMID- 8619600 TI - Anti-Mastigina activities of eight contact lens solutions. AB - The effects of eight contact lens solutions on a Mastigina sp., which was associated with the infected eye of a patient, were studied. The solutions which killed the organism promptly were those which are used for gas-permeable and hard contact lenses. Some solutions for soft contact lenses were more effective than others. PMID- 8619602 TI - Relapse of Toxoplasma encephalitis and susceptibility to pyrimethamine: lack of evidence of treatment-induced resistance. PMID- 8619601 TI - Emergence of in vitro resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Japan. AB - To investigate emerging fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Japan, we compared the in vitro antimicrobial susceptibilities of 79 gonococcal isolates from 1992 through 1993 to 14 fluoroquinolones and 14 other antibiotics with those of 27 isolates from between 1981 and 1984. The MICs at which 90% of the isolates were inhibited by nine fluroquinolones, including norfloxacin, enoxacin, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, fleroxacin, levofloxacin, and sparfloxacin, for isolates from 1992 to 1993 were 8 or 16-fold higher than those for isolates from 1981 to 1984. Furthermore, the MICs at which 90% of the isolates were inhibited by five fluroquinolones, including OPC-17116, T-3761, DU-6859a, AM-1155, and Q-35, that have recently been synthesized but have not yet been introduced for clinical use in Japan for isolates from 1992 to 1993 were also 2- to 16-fold higher than those for isolates from 1981 to 1984. The gonococcal isolates from 1992 to 1993 showed no significant decreases in susceptibility to beta-lactams, tetracyclines, macrolides, and spectinomycin, compared with those for isolates from 1981 to 1984. Our data indicate that the incidence of gonococcal strains with decreased susceptibilities to fluoroquinolones is increasing in Japan. PMID- 8619603 TI - Type 1 phosphatase inhibitors reduce the restoration of guanine nucleotide exchange activity of eukaryotic initiation factor 2B inhibited reticulocyte lysates rescued by hemin. AB - In heme-deficient reticulocyte lysates, the alpha-subunit of eukaryotic initiation factor-2 (eIF-2alpha) is phosphorylated due to the activation of the heme-regulated eIF-2alpha kinase (HRI). Phosphorylation of eIF-2alpha impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF 2alpha kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2alpha and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin and puromycin, which do not affect the eIF-2alpha phosphorylation. Also, the functional eIF-2B activity that is available in hemin supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates. PMID- 8619604 TI - The effect of cornea proteoglycans on liposome peroxidation. AB - Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress. PMID- 8619605 TI - Relation between the activities reducing disulfides and the protection against membrane permeability transition in rat liver mitochondria. AB - The oxidation of some mitochondrial sulfhydryl groups acts as an inducer of the mitochondrial membrane permeability transition. This membrane damage can be reversed by regeneration of the sulfhydryl groups. Hence the mitochondrial activities reducing disulfides are especially important for the defense against oxidative injury. The ability of isolated rat liver mitochondria to reduce disulfides was examined by the reduction of 5,5'-dithiobis-(2 nitro-benzoic acid) (DTNB), the reaction catalyzed by thioredoxin reductase and glutathione reductase. The incubation of mitochondria with DTNB induced their swelling which was prevented by EGTA, cyclosporin A, Mg2+, and pyrophosphate. The rate of DTNB reduction by mitochondria was compared with the changes in their volume and in the total content of their sulfhydryl groups. The reduction of DTNB was stimulated by cation chelators, both unphysiological and physiological, and was suppressed by up to 65% during mitochondrial swelling. In mitochondria treated with tert-butylhydroperoxide the total content of mitochondrial sulfhydryl groups decreased in the correlation with the suppression of DTNB reduction and with mitochondrial swelling. The content of nonprotein sulfhydryl groups was significantly lowered under all conditions applied. However, the protein sulfhydryl groups were preserved in the presence of EGTA or Mg2+ when the rate of DTNB reduction was relatively high and this was associated with the strong inhibition of mitochondrial swelling. It is suggested that during oxidative stress mitochondrial activities reducing disulfides are involved in the maintenance of sulfhydryl groups in mitochondria thus protecting their membranes against permeabilization. PMID- 8619606 TI - Aluminum facilitation of iron-mediated lipid peroxidation is dependent on substrate, pH and aluminum and iron concentrations. AB - It has been suggested that aluminum (Al) plays a role in neurological disorders. The mechanism of its neurotoxicity has not been established. Brain lipid peroxidation (LP) contributes to neurodegeneration. There have been conflicting reports concerning the Al effect on LP. In the present study, LP of three Folch Fractions from bovine brain and five pure phospholipids was determined in the presence of varying concentrations of iron (Fe) and Al at pH 5.5 and 7.4. Lipid peroxidation was measured as thiobarbituric acid reactive substances. Iron initiated LP, whereas Al did not. However, Al significantly facilitated Fe mediated LP of bovine brain Folch Fractions I and III, bovine brain-derived phosphatidylserine, and egg yolk phosphatidylcholine. Bovine brain phosphatidylserine was the most susceptible substrate among the lipids tested. Aluminum facilitation of LP was Al and Fe concentration dependent. The peroxidation was greater at pH 5.5 than 7.4. There was no significant Al effect with Folch Fraction V, bovine brain-derived phosphatidylethanolamine, phosphatidylcholine, or sphingomyelin. This study confirmed the ability of Al to facilitate Fe-mediated LP and identified the substrates, pH, and Al concentrations favoring the peroxidation. A potential mechanism for Al facilitation of Fe-mediated LP is proposed. PMID- 8619607 TI - Peroxidase-mediated bromination of unsaturated fatty acids to form bromohydrins. AB - Eosinophil peroxidase and myeloperoxidase (MPO) catalyze the oxidation of bromide by hydrogen peroxide to produce hypobromous acid (HOBr). Hypochlorous acid, which is also generated by MPO, reacts with unsaturated fatty acids to form chlorohydrins. In this study the equivalent reaction of HOBr, produced from MPO, bromide, and hydrogen peroxide, with oleic (18:1), linoleic (18:2), and arachidonic (20:4) acids has been investigated. Thin-layer chromatography detected one major product of higher polarity than the unmodified fatty acids and additional more polar products with the polyunsaturated fatty acids. Similar results were observed with N-bromosuccinimide-derived HOBr. Gas chromatography mass spectrometry (GC-MS) and electrospray MS identified the major products of 18:1 as the isomeric 9,10-bromohydrins based on retention time and mass spectrometric isotope and fragmentation patterns. The products of 18:2 and 20:4 were too unstable for analysis by GC-MS. Electrospray MS identified the mono- and bisbromohydrins formed from 18:2 and 20:4 based on mass/charge ratios of the molecular ions and the presence of bromine isotope patterns. Other oxidation products not containing bromine, such as dihydroxy derivatives, were detected as well. Fatty acid bromohydrins could contribute to the antimicrobial activity and inflammatory tissue damage by eosinophils and neutrophils, and could potentially be useful specific markers for HOBr production in vivo. PMID- 8619608 TI - Suppression of endothelin-1 production in cultured human umbilical vein endothelial cells by heparin fractions separated by strong anion exchange chromatography. AB - Heparin has been shown to lower the production/secretion of the vasoconstrictive peptide endothelin-1. Endothelin-1 production is stimulated by thrombin, and it has been proposed that heparin binds to the anion-binding exosite of thrombin, preventing it from stimulating endothelin-1 production. To further test this proposal, heparin was fractionated by strong anion exchange chromatography (QAE Sephadex A-25) into four fractions. These fractions had anticoagulant activities that increased linearly with charge, as defined by the median salt concentration needed for elution from the column. The fractions also differed in the total number of sulfates per mole of heparin, which was dependent on the molecular mass of the fractions rather than charge density. The fractions were found to significantly differ fron each other in their ability to suppress endothelin-1 production. The fraction eluting from the ion exchange column at the highest salt concentration had the greatest suppressive effect. Addition of sodium or potassium chloride to the media interfered with the ET-1 suppressive effect of unfractionated heparin, whereas lithium chloride had no effect. These data show that charge interactions between heparin and thrombin may be important in regulating the production of endothelin-1 and in regulating other thrombin dependent functions. PMID- 8619610 TI - Functional significance of the Cu,ZnSOD in Escherichia coli. AB - Diethyl dithiocarbamate (DDC) was used to inhibit the copper- and zinc- containing superoxide dismutase (Cu,ZnSOD) of Escherichia coli in order to expose its physiological function. DDC inhibited the aerobic growth of a sodA sodB mutant much more than it did the growth of a SOD-replete parental strain and this inhibitory effect was oxygen-dependent. The SOD mimic MnTMPyP markedly diminished the growth inhibitory effect of DDC. Transfer of the sodA sodB strain from anaerobic to aerobic conditions induced fumarase C, which is a member of the soxRS regulon. DDC augmented this induction. These results indicate that the Cu,ZnSOD provides a defense against oxidative stress, which is more important to the sodA sodB mutant than to its SOD-replete parental strain. PMID- 8619609 TI - Photocatalytic and free radical interactions of the heterocyclic N-oxide resazurin with NADH, GSH, and Dopa. AB - Electron donating free radicals NAD(.), (.)CO2(-), MV(.)+, and e(aq)-, generated by pulse radiolysis, reduce resazurin (RNO) with rate constants of 1.9 x 10(9), 2.8 x 10(9), 4.8 x 10(9), and 2.3 x 10(10) M(-1) s(-1), respectively, neutral solution. The semireduced dye (RN(.)-O- disproportionates slowly to RN (resorufin) and RNO. There was little evidence that RN(.)-O- behaves as an oxidizing species capable of initiating chain reactions, for instance via oxidation of NADH to NAD(.). The oxidizing radicals GS(.), (.)OH, and N3(.) interact with RNO via complex consecutive processes, probably by addition elimination reactions. Stable products generated upon oxidation of RNO by N3(.) exhibit a red-shifted absorption, but GS(.) and (.)OH also cause partial reduction to RN. Neither O2(.)- nor dopa semiquinone nor tyrosine phenoxyl radicals appear to interact with RNO. Radicals formed by reaction of (.)OH with (Gly)3 reduce RNO to RN with stoichiometry near two (gamma-radiolysis), and there is evidence (pulse radiolysis) for direct slow O-atom transfer from RNO to these species. Resazurin is highly photosensitive under anaerobic conditions in presence of H-atom donors like NADH, GSH, or dopa. Under aerobic conditions RNO becomes an efficient catalyst of red light induced photooxidation of these donors; the RN(.)-O- intermediate, formed in the photooxidative process, is apparently recycled to RNO by O2, and by other electron acceptors. Our results suggest that RNO can behave as a photoactive, free radical generating xenobiotic compound. PMID- 8619611 TI - Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli. AB - cDNAs for human cytochrome P450 2E1 and rat NADPH-cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins in Escherichia coli was monitored by immunoblotting, spectroscopy, and catalytic assays. The cDNAs were separated on the coexpression plasmid by 22 nucleotides, with the P450 cDNA preceding the reductase cDNA. P450 content in solubilized cell membranes, whether expressed alone or coexpressed with P450 reductase, was approximately 0.11 nmol/mg of protein, and approximately 0.8 nmol could be obtained per liter of culture. Reductase content was five- to sixfold greater than P450 content when coexpressed, but severalfold less than that obtained when expressed without the upstream P450 cDNA, indicating differences in both stability and translatability between the two proteins. Solubilized membranes from cells expressing both proteins catalyzed aniline hydroxylation, p-nitrophenol hydroxylation, and N-nitrosodimethylamine demethylation at rates equivalent to those obtained by combining P450 and reductase preparations; addition of purified reductase to these membranes did not augment the activity. However, in contrast to results obtained with P450 2E1 expressed in other heterologous systems, addition of rabbit liver cytochrome b5 to preparations catalyzing p-nitrophenol or N-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substrates in vivo could not be demonstrated. Nonetheless, the ability to coexpress P450 and reductase in E. coli so as to generate a functional monooxygenase system in vitro enhances the utility of this organism for the expression and characterization of cloned P450 isoforms. PMID- 8619612 TI - Relevance of lactate dehydrogenase activity to the control of oxidative glycolysis in pancreatic islet B-cells. AB - The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase was also much lower than that of the cytosolic NAD linked glycerophosphate dehydrogenase . A comparable hierarchy in the activity of these enzymes was observed in non-B islet cells. These findings reinforce the view that the preferential stimulation of oxidative glycolysis observed in insulin-producing cells, when exposed to high concentrations of D-glucose, is attributable to a Ca2+-induced activation of the mitochondrial FAD-linked glycerophosphate dehydrogenase, rather than to saturation of the catalytic activity of lactate dehydrogenase. PMID- 8619613 TI - Catabolism of isobutyrate by colonocytes. AB - Isolated colonocytes have more capacity for the oxidation of isobutyrate and alpha-ketoisovalerate than isolated enterocytes. Both enterocytes and colonocytes express high levels of 3-hydroxyisobutyryl-CoA hydrolase, an enzyme activity important in maintaining low intracellular concentrations of methacrylyl-CoA, a common, potentially toxic intermediate in the catabolic pathways of these compounds. In spite of comparable 3-hydroxyisobutyryl-CoA hydrolase activities in both cell types, and much greater amounts of 3-hydroxyisobutyrate dehydrogenase in colonocytes than in enterocytes, only the colonocytes produced 3 hydroxyisobutyrate as an endproduct of alpha-ketoisovalerate and isobutyrate catabolism. Butyrate very effectively inhibits isobutyrate catabolism by colonocytes, most likely by competitively inhibiting activation of isobutyrate to its CoA ester. Oleate also inhibits isobutyrate catabolism, but at a site more distal than butyrate. Starvation of rats for 72 h decreased the capacity of colonocytes for butyrate but not isobutyrate catabolism. We conclude that isobutyrate could function as a carbon source for energy and anapleurosis in colonocytes under conditions of defective butyrate oxidation or low butyrate availability. PMID- 8619614 TI - Phenobarbital-induced activation of CYP2H1 and 5-aminolevulinate synthase genes in chick embryo hepatocytes is blocked by an inhibitor of protein phosphorylation. AB - The phenobarbital-induced activation of cytochrome P4502H1 (CYP2H1) and 5 aminolevulinate synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital induced activation of CYP2H1 and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of the CYP2H1 gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5' flanking region of the CYP2H1 gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of the CYP2H1 gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-O-tetradecanoyl-phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-amino-purine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process. PMID- 8619615 TI - Kinetics of the formation of p-670 and of the decay of compound III of horseradish peroxidase. AB - The kinetics of the decay of compound III of horseradish peroxidase formed from compound II and excess hydrogen peroxide and of the formation of p-670 from this preparation were studied as a function of pH at 25 degrees C and constant ionic strength of 0.11. Compound III undergoes an irreversible formation of p-670 and a slow, spontaneous decay to ferriperoxidase via compound II and the rate of compound III decay is less than that of p-670 formation at all pH values. Both reactions follow pseudo first order kinetics at all pH values. The observed rate constant for compound III decay decreases with increase in pH with a minimum at pH 8.0. Also the observed rate constant for p-670 formation increases with increase in pH with maximum also at pH 8.0. The slow decay of our preparation of compound III and its unique formation of p-670 as compared to oxyperoxidase prepared from ferrous horseradish peroxidase and molecular oxygen (J.B. Wittenberg, R. W. Noble, B.A. Wittenberg, E. Antonini, M. Brunori, and J. Wyman (1967) J. Biol. Chem. 242, 625-34) are discussed in terms of the presence of excess hydrogen peroxide in our preparation. Ferriperoxidase and p-670are formed from compound III in two parallel reactions. PMID- 8619616 TI - Calcium dependent association of surfactant protein A with pulmonary surfactant: application to simple surfactant protein A purification. AB - Surfactant protein A (SP-A) is an abundant lipoprotein component of pulmonary surfactant that plays multiple roles in surfactant homeostasis within the lung. A simple and rapid purification procedure for SP-A is described. Purified surfactant is washed by centrifugation with Ca2+ containing buffer to remove residual soluble proteins. Following the Ca2+ buffer wash, the surfactant pellet is washed in buffer containing EGTA and Mg2+ which releases the bound SP-A in almost pure form. Subsequent chromatography of the SP-A on Sephacryl S-500 yields homogeneous preparations of the protein. The SP-A purified using this procedure requires no exposure to either detergents or organic solvents to remove lipid. SP A prepared by this new method inhibits lipid secretion from alveolar type II cells as effectively as SP-A prepared by other methods. In addition, the SP-A depleted surfactant produced in the first step of this procedure is capable of binding exogenous SP-A in a time dependent, saturable and Ca2+ dependent manner. PMID- 8619617 TI - Conditions to study nitric oxide generation by polymorphonuclear cells from an inflammatory exudate in rats. AB - Superoxide and nitric oxide release by leukocytes has been usually performed after exposure to a particular stimulus. We measured the generation of superoxide and nitric oxide by cells isolated from an inflammatory exudate of rats in either the absence or the presence of a variety of stimuli. Nonstimulated leukocytes generated superoxide radical (1.2 nmol x 10(6) cells(-1)) and nitric oxide (3.8 nmol x 10(6) cells(-1)) after 2 h incubation. When cells were incubated with lipopolysaccharides, opsonized zymosan or phorbol 12-myristate 13-acetate, superoxide level increased while nitric oxide decreased. Phorbol 12-myristate 13 acetate (100 ng/ml) induced a decrease of 0.88 nmol x 10(6) cells(-1) compared with nonstimulated cells, and incubation with N-iminoethyl-L-ornithine increased superoxide production by 0.81 nmol x 10(6) cells(-1). These results provide clear evidence that cells from an inflammatory exudate which are already triggered are able to generate a considerable amount of nitric oxide and in less proportion superoxide, that the measure of nitric oxide must be performed without a further stimulus, and that both molecules react in an equimolar proportions to give peroxynitrite anion. PMID- 8619618 TI - Maltitol and maltobionate act differently on maltose- and maltooligosaccharide hydrolysis by human small intestinal glucoamylase-maltase indicating two different enzyme binding modes. AB - The hydrolysis of maltose and maltotriose at the same catalytic site of glucoamylase-maltase has been demonstrated. Maltitol acts as a competitive inhibitor, Ki = 69 (+/-10) mM, of the maltose hydrolysis and as a noncompetitive inhibitor of the hydrolysis of maltotriose, Ki = Kii = 29 (+/-4) mM, and maltotetraose, Ki = Kii = 30 (+/-3) mM. Maltobionate was not hydrolyzed by the enzyme and did not influence the maltose hydrolysis. In contrast, in hydrolysis of maltooligosaccharides it acts as an uncompetitive inhibitor. For the hydrolysis of maltotriose, Kii = 25(+/-8) mM, and maltotetraose, Kii = 30(+/-4) mM was found. According to a characteristic of this rare inhibition pattern a simultaneous decrease of the apparent Km and the apparent Vmax of maltooligosaccharide hydrolysis with increasing maltobionate concentrations was observed. We were able to discriminate two different binding modes for glucoamylase-maltase. Maltitol binds to the free enzyme (maltose binding mode) as well as to the maltooligosaccharide-enzyme complex, whereas maltobionate binds only to the oligosaccharide-enzyme complex (oligosaccharide binding mode). This could be shown by the different inhibition behaviors of maltitol and maltobionate depending on the substrate: maltose or maltooligosaccharides. PMID- 8619619 TI - Complete primary structure of the subunits of heterodimeric phospholipase A2 from Vipera a. zinnikeri venom. AB - Molecular weight determination of dimeric phospholipase A2 from Vipera aspis zinnikeri venom (PLA2-I) was performed with electrospray ionization mass spectrometry (ESI-MS). PLA2-I consists of an acidic and a basic subunit (subunit A and B), which bind noncovalently and dissociate under highly acidic conditions. The protonated molecular ions of subunit A and B were measured to be 13,655.9 and 13,842.6, respectively. The complete amino acid sequence was also determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion. Both subunit A and B consist of 122 amino acid residues and contain 7 disulfide bonds. The theoretical molecular mass calculated from the primary structure completely agree with the ESI-MS data. THe sequential homology between subunit A and B was 63.9%; however, subunit A lacks enzymatic and biological activities that are characteristic for phospholipase A2. Although the amino acid residues essential for calcium binding (Tyr28, Gly30, Gly32, and Asp49) and catalysis (Asp92) were preserved, replacement of functionally important residue (His48) for catalysis with a Gln was found in subunit A. In addition, substitution of acidic amino acid residues for basic ones and hydrophilic residues for hydrophobic ones were observed in subunit A. Presumably, these changes in the primary structure of subunit A resulted in the loss of enzymatic activity and an increase in the binding ability with subunit B. PMID- 8619620 TI - Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4. AB - The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation. Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile 114--> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity. PMID- 8619621 TI - Oxidation of cytosolic NADH via complex I of heart mitochondria. AB - The exogenous NADH dehydrogenase of heart mitochondria is increasingly reported to mediate cardiomyopathies following adriamycin treatment or reperfusion of ischemic hearts. A great number of studies on the biochemistry and pathobiology exists which indirectly support the existence of this dehydrogenase. Our studies exclude both the rotenone-insensitive NADH dehydrogenase of the outer membrane and the endogenous NADH dehydrogenase of damaged mitochondria as being responsible for external NADH consumption. Reducing equivalents from external NADH were demonstrated to enter complex I of the respiratory chain from the cytosolic phase. Our data support earlier reports on the physical association of the exogenous NADH dehydrogenase with the inner mitochondrial membrane excluding oxidation of external NADH via an enzyme of artefactual origin. PMID- 8619622 TI - Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. AB - Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells. Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials. Recently we expressed the recombinant alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column. Purified (r)alpha-GAL, migrating as a single band of 41 kDa on a SDS-PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (K(m) =0.363 mM and V(max) = 46.9 U/mg). Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substrate p nitrophenol-alpha-D-galactopyranoside. Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface. In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies. Thus, with a simple procedure for over expression and purification of (r)alpha-GAL from P. pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells. PMID- 8619623 TI - Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. AB - We observed that protein (bovine serum albumin) carbonyl content increases upon hypochlorite oxidation, and this increase is inhibited in a concentration dependent manner in the presence of hypochlorite scavengers. Based on this observation, we tested whether some known hypochlorite scavengers (lipoic acid, cysteine, and glutathione) and some other antioxidants (uric acid, ascorbic acid, alpha-tocopherol, and probucol) could prevent protein carbonyl formation. N acetylcysteine, dihydrolipoic acid, cysteine, and glutathione (reduced form, GSH), which otherwise could not be tested in a previously reported 5-thio-2 nitrobenzoic acid test system, were successfully evaluated in our assay. The hypochlorite scavenging capacity of different compounds, compared by determining the IC50 (concentration which produces 50% inhibition), showed that the compounds tested have the following potency: dihydrolipoic acid > GSH, N-acetylcysteine > cysteine > S-methyl glutathione > lipoic acid, ascorbic acid > cystine, GSSG, and uric acid. No scavenging ability was observed for either alpha-tocopherol or probucol. PMID- 8619624 TI - Acceleration of peroxynitrite oxidations by carbon dioxide. AB - Stopped-flow kinetic studies of the isomerization of peroxynitrite to give nitrate have been performed in carbonate-enriched buffers using pH jump and carbonic anhydrase as probes. The data are consistent with the reaction of CO2 and the peroxynitrite anion rapidly forming an unstable nitrosoperoxy-carbonate anion adduct, O=N-OOCO2- (1). The CO2 catalysis of the isomerization of peroxynitrite is not accompanied by the formation of nitrite, hydrogen peroxide, or other hydroperoxidic material like peroxycarbonate. The reaction proceeds via the transient formation of an oxidant or oxidants that is (are) capable of promoting electrophilic nitration reactions. We propose that O=N-OOCO2- rearranges to give a nitrocarbonate anion, O2N-OCO2- (2) which in turn, may serve as the proximal oxidant in biological systems that produce peroxynitrite. At least four different mechanistic classes of reactions that have been ascribed to peroxynitrite can be envisioned to involve 2: (a) hydrolysis to nitrate, (b) one electron or (c) two-electron oxidations, and (d) electrophilic nitration. Given the fast reaction of peroxynitrite with carbon dioxide and the ubiquitous presence of the latter, the role of CO2 cannot be neglected in complex peroxynitrite reactions in vitro and in vivo. PMID- 8619625 TI - Alterations in phospholipid bilayers caused by oxyethylenated nonylphenol surfactants. AB - The interactions of oxyethylenated nonylphenols [ethylene oxide units (EO) averaging between 5 and 30] with phosphatidylcholine liposomes were investigated. Three parameters were regarded as corresponding to the effective surfactant/lipid molar ratios at which the surfactant system (a) resulted in the transient liposome reorganization for the release of 50% of 5(6)-carboxyfluorescein (CF) trapped in the liposomes, R(50%CF); (b) saturated the liposomes, R(SAT); and (c) led to a complete liposome solubilization, R(SOL). From these parameters the surfactant partition coefficients K(50%CF), K(SAT), and K(SOL) were determined. Permeability alterations were determined as a change in CF released from the interior of vesicles and solubilization as a decrease in the static light scattering of liposome suspensions. R(SAT) and R(SOL) showed the lowest values in the critical micellar concentration (CMC) range between 0.040 and 0.087 mM and 0.050 and 0.075 mM, respectively [hydrophilic-lipophilic balance (HLB) number ranging between 13 and 16 and 14 and 15], which corresponded approximately to the nonylphenols with 10--20 and 15 EO units, respectively. However, K(50%CF), K(SAT), and K(SOL) drastically decreased as the number of EO units increased, especially in the EO range between 5 and 15. Thus, the highest surfactant affinity with bilayers (maximum K values) and its maximum ability to alter these structures (minimum R values) were governed by the CMC surfactants and their HLB numbers and, consequently, by the EO units present in the surfactant structure. The free surfactant concentrations were lower than their CMCs at sublytic level, whereas they remained similar to these values during bilayer saturation and solubilization. The nonylphenols investigated showed higher activity versus liposomes than that reported for Triton X-100, the oxyethylenated nonylphenol with an average of 10 ethylene oxide units showing low toxicity in biological tests. PMID- 8619626 TI - Purification and characterization of salivary kallikrein from an insectivore (Scalopus aquaticus): substrate specificities, immunoreactivity, and kinetic analyses. AB - We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pI of 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro Phe-Arg, and Val-Leu-Arg-7-amino-4-methylcoumarin, gave K(m) values for mole kallikrein of 3.3, 46.1, and 2.8 microM, respectively, and specificity constants, kcat/K(m), of 3818, 165, and 8714 s-1 pM-1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family. S. aquaticus is recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research. PMID- 8619627 TI - Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): dose- and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A. AB - Glucocorticoids are being found to influence expression of cytochrome P450 (CYP) genes in multiple subfamilies in mammals (J.S. Sidhu, and C.J. Omiecinski (1995) Pharmacogenetics 5, 24--36). In the present study we investigated CYP1A and CYP3A expression in the fish Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1) after coadministration of CYP1A and CYP3A inducers, including glucocorticoids. A putative CYP3A protein is expressed in PLHC-1 cells but its content was not altered by exposure of cultures to the prototypical mammalian CYP3A inducers dexamethasone (DEX), pregnenolone-16 alpha-carbonitrile (PCN), or rifampicin (RIF). However, when coadministered with 3,3', 4,4' tetrachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), DEX but not PCN or RIF caused increases in the degree of CYP1A induction by these aryl hydrocarbon receptor (AHR) agonists. This increase was seen both in CYP1A protein content and rates of ethoxyresorufin-O-deethylase (EROD) activity. DEX alone caused no induction of CYP1A, indicating that the enhancement of CYP1A induction caused by DEX + AHR agonists was not an additive effect but rather a potentiation. The dose of DEX required for maximal potentiation was three orders of magnitude greater at 48 h than the dose required at 24 h. Moreover, the degree of potentiation of CYP1A induction was much greater at the lower doses than at the highest doses of TCDD. There was up to 20-fold potentiation of EROD induction in cultures exposed to 0.1 nM TCDD. Two other glucocorticoid receptor (GR) agonists, cortisol and prednisone, also produced a strong potentiation of CYP1A induction, but other mammalian CYP3A inducers that are not GR agonists, such as the anti-glucocorticoid PCN, the anti-mineralocorticoid spironolactone, or the macrolide antibiotics RIF and troleandomycin, did not potentiate the CYP1A induction in PLHC-1 cells. Addition of the mammalian GR antagonists PCN or RU 38486 reduced the DEX-mediated potentiation of CYP1A induction, whereas spironolactone had no effect on the potentiation. RU 38486 also potentiated the induction of EROD activity by the TCDD, which suggests that RU 38486 acts as a partial GR agonist in PLHC-1 cells. These results suggest that potentiation of CYP1A induction in this nonmammalian cell line proceeds by a classical GR mediated pathway, independently of the expression of CYP3A. However, the complex interaction between doses of both GR and AHR agonists and duration of exposure, suggests that additional processes influence this potentiation. The unusually strong potentiation at lower doses of TCDD may make PLHC-1 cells particularly suitable in exploring further the consequences of this potentiation. PMID- 8619628 TI - Filaggrin linker segment peptide and cystatin alpha are parts of a complex of the cornified envelope of epidermis. AB - We showed that filaggrin linker segment peptide (FLSP) is a glutamine-rich substrate of epidermal transglutaminase (TGase), conjugating enzymatically with a phosphorylated cystatin alpha (P-cystatin alpha) which is a lysine-rich substrate of the enzyme. This finding suggested that FLSP would be a component of cornified envelope of the epidermis as well as P-cystatin alpha. Here, we investigated the in vivo location of FLSP. An antibody against the peptide conjugated with keyhole limpet hemocyanin (FLSP/KLH) reacted specifically with FLSP on immunoblots. Immunofluorescence histochemistry located specific staining with this antibody on keratohyalin granules and the cell membrane region of the stratum corneum. Specific staining was not detected when the antiserum was first absorbed by FLSP. Preembedding immunoelectron microscopic analysis showed that anti-FLSP/KLH antibody labeled with gold particles reacted with cornified envelope prepared from newborn rat stratum corneum. The high molecular weight protein enzymatically synthesized from phosphorylated cystatin alpha and FLSP by TGase reacted with both anti-FLSP/KLH antibody and anti-P-cystatin alpha antibody on Western blotting. These findings suggest that the FLSP-cystatin alpha conjugate is a component of the cornified envelope of the epidermis in rats. PMID- 8619629 TI - Long loop residues 33-58 in the human glycoprotein hormone common alpha subunit contain structural components for subunit heterodimerization and human follitropin-receptor binding. AB - The family of human glycoprotein hormones that includes follitropin (FSH) are heterodimeric proteins, each composed of single alpha and beta subunits that are non-covalently linked but tightly associated. Previous studies by this laboratory, which used a synthetic peptide approach, suggested that residues 51 58 of the long loop of FSH alpha (aa 33-58) were the minimal alpha-subunit contact area between the subunits. Since carbohydrate at N52 is important for receptor activation but not for receptor binding, a link between receptor activation and heterodimer assembly was established. To address this issue, four composite alanine substitution mutants, 37YPTPL41/37APAPA41, 46TML48/46AAA48, 49VQK51/49AAA51, and 55SES57/55AAA57, were constructed by site-specific mutagenesis and expressed in insect cells. With the exception of the TML mutant, all alpha-subunit forms were produced at a level similar to that of the wild-type alpha subunit (10 micromilligram(s)). The TML mutant was not secreted. When coexpressed with the human FSH (hFSH) beta subunit the 49VQK51/49AAA51 mutant and wild-type hFSH were expressed at similar levels (1-3 micromilligram(s)). In contrast, the 55SES57/65AAA57 mutation evidenced barely detectable levels of heterodimeric hFSH, and 37YPTPL41/37APAPA41 was not detectable as heterodimer, measured in a capture enzyme-linked immunosorbent assay format that detects only heterodimeric hormone. The 49VQK51/49AAA51 mutant was devoid of receptor-binding activity, suggesting that these residues are a key alpha-subunit determinant for follitropin-receptor interaction. The 55SES57/55AAA57 mutant, though scarcely made, retained receptor-binding activity comparable to the wild-type hormone. This work demonstrates for the first time a receptor-binding region in the FSH alpha subunit, within sequence 49VQK51. Residues within 55SES57 and 37YPTPL41 are involved in subunit assembly. Homology modeling of FSH, based on the human chorionic gonadotropin crystal structure, revealed that the FSH receptor-binding site is composed of residues from both subunits assembled through subunit association. PMID- 8619630 TI - Functional organization of mammalian hexokinases: both N- and C-terminal halves of the rat type II isozyme possess catalytic sites. AB - Previous work has shown that catalytic function is associated exclusively with the C-terminal half of the Type I isozyme of mammalian hexokinase. In contrast, we now demonstrate that both halves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalytically important Ser residue to Ala at analogous positions in either the N- or the C-terminal halves (S155A or S603A, respectively) of the rat Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mutated enzyme (S155A/S603A). Catalytic activity was retained in a chimeric hexokinase comprising the N-terminal half of Type II hexokinase and catalytically inactive (by site-directed mutation) C-terminal half of the Type I isozyme. The N and C-terminal catalytic sites of Type II hexokinase are similar in V(max) and K(m) (approximately equal to 130 microM) for glucose; however, the N-terminal site has a lower (0.45 vs 1.1 mM) K(m) for ATP, is slightly more sensitive to inhibition by the product analog 1,5-anhydroglucitol-6-P, and is much more sensitive to inhibition by P(i). It is suggested that the Type II isozyme most closely resembles the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the precursor for the contemporary Type I, Type II, and Type III mammalian isozymes. Subsequent evolutionary changes could then have led to functional differentiation of the N- and C-terminal halves, as seen with the Type I (and possibly the Type III) isozyme. PMID- 8619631 TI - Reaction of phosphatidylcholine hydroperoxide in human plasma: the role of peroxidase and lecithin:cholesterol acyltransferase. AB - In order to elucidate the reason why phosphatidylcholine hydroperoxide is unstable in human plasma, 1-palmitoyl-2-linoleoylphosphatidylcholine hydroperoxide (PLPC-OOH) was incubated aerobically in human plasma at 37 degrees C, and its decomposition products were measured. The major product was the corresponding alcohol (PLPC-OH) and this reduction probably occurred by an enzymatic process since no acceleration in ascorbate depletion and no significant decrease in other plasma antioxidants were observed upon addition of PLPC-OOH. Cholesteryl linoleate hydroperoxide and its alcohol (Ch18:2-OH) were also detected as minor products. Similarly, 1-stearoyl-2 arachidonoylphosphatidylcholine hydroperoxide gave its alcohol (SAPC-OH) as a major product and cholesteryl arachidonate hydroperoxide and its hydroxide (Ch20:4=OH) as minor products. These oxidized cholesteryl esters are likely to be produced by the action of lecithin:cholesterol acyltransferase (LCAT) present in high-density lipoprotein (HDL) since (a) incubation of PLPC-OH and SAPC-OH in human plasma gave Ch18:2-OH and Ch20:4-OH, respectively, (b) isolated human HDL converted PLPC-OH to Ch18:2 OH and SAPC-OH to Ch20:4-OH while isolated human low density lipoprotein was inactive for this conversion, and (c) formation of oxidized cholesteryl esters in plasma and HDL was inhibited by the LCAT inhibitor 5,5'-dithiobis(2-nitrobenzoic acid). A possible beneficial role of LCAT for converting phosphatdylcholine hydroperoxide to cholesteryl ester hydroperoxide is also discussed. PMID- 8619632 TI - cDNA and protein sequence of a major form of P450, CYP2L, in the hepatopancreas of the spiny lobster, Panulirus argus. AB - A P450 fraction was previously isolated from spiny lobster hepatopancreas microsomes and shown in reconstitution experiments to be efficient in catalyzing the monooxygenation of benzphetamine, aminopyrine, benzo(a)pyrene, progesterone, and testosterone. In this study, N-terminal sequence information up to residue 39 was obtained from this P450 and used to design degenerate primers for screening a cDNA library constructed from hepatopancreas mRNA. Clones were obtained that contained part of the coding region of a P450 protein. Further exact primers were designed that permitted the isolation of clones containing coding information for other parts of the P450 sequence, as well as a clone that coded for the complete P450 protein sequence. The open reading frame of the complete coding region corresponded to a protein of 492 amino acids. The deduced amino acid sequence of this P450 was about 36% similar to individual mammalian P450s in the 2 family and did not show strong matches with other proteins in the data base. Based on sequence and the previously determined function, this spiny lobster P450 was assigned by the P450 nomenclature committee to a new P450 subfamily, CYP2L. This is the first description of a P450 primary sequence from a marine crustacean species and the first assignment of an invertebrate P450 into the 2 family. PMID- 8619633 TI - Oxidative stress response in iron-induced renal carcinogenesis: acute nephrotoxicity mediates the enhanced expression of glutathione S-transferase Yp isozyme. AB - An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we investigated the free radical-induced oxidative stress response in this carcinogenesis model, focusing on the expression of glutathione S-transferases (GSTs) which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells. A single intraperitoneal Fe-NTA treatment (15 mg Fe/kg body weight) induced a rapid oxidative stress, which was monitored by the accumulation of lipid peroxidation products and the loss of sulfhydryl contents in the kidneys, resulting in a 30% reduction of GST activity 1 h after an Fe-NTA treatment. The enzyme activity returned to the control level after 16 h. The immunoblot analysis of GST isozymes demonstrated that the level of alpha-class GSTs (GST-Ya and GST-Yc) and pi-class GST (GST-Yp), major GST isozymes constitutively produced in the kidney, decreased immediately within 1 h of the Fe-NTA treatment. The onset of the recovery of GST Yp protein levels was detected 3 h after the Fe-NTA treatment. The enhanced production of GST-Yp in gene expression was evident in the drastic elevation of mRNA levels and these increases coincided with a substantial rise in the GST activity and protein levels. The alpha-class GSTs were not inducible by treatment with Fe-NTA. The immunohistochemical analysis demonstrated that the expression of GST-Yp was strongly induced in the regenerating proximal tubular cells. A steady accumulation of GST-Yp protein was observed in the subacute toxicity experiments with multiple injections of Fe-NTA. These results suggest that the enhanced expression of GST-Yp is important in mediating cell repairs or increasing the resistance to subsequent injury. PMID- 8619634 TI - Characterization of the activated form of the aryl hydrocarbon receptor in the nucleus of HeLa cells in the absence of exogenous ligand. AB - The aryl hydrocarbon receptor (AhR) is known to mediate 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)-induced toxic effects. Immunocytochemical studies revealed that AhR in HeLa cells is localized throughout the cell. Upon TCDD treatment most of the cytoplasmic receptor is translocated into the nucleus in a time-dependent manner. A significant amount of AhR was found to be tightly associated with the nuclear fraction of untreated HeLa cells. The level of receptor in the nuclear fraction was approximately 16% of the total cellular receptor pool. Further characterization of AhR heterocomplex from the HeLa nuclear fraction by sucrose density gradient analysis revealed that the AhR was present in the 6 S form, and that the nuclear AhR could be coimmunoprecipitated using anti-Arnt mAb. The ability of the AhR to specifically interact with dioxin responsive elements (DRE) was demonstrated utilizing wild-type and two mutant DREs in gel shift assays. These results would suggest that, in HeLa cells, the AhR-Arnt heterodimer is associated with the nuclear fraction under normal culture conditions. Therefore, HeLa cells can be used as a model system to study the biochemical and molecular function of the Ah receptor and the process that leads to activation of the AhR in the absence of exogenous ligand. PMID- 8619635 TI - Characterization of products from the reactions of N-acetyldopamine quinone with N-acetylhistidine. AB - When insects harden or sclerotize their exoskeletons, quinones of N-acetylated catecholamines such as N-acetyldopamine (NADA) undergo nucleophilic addition reactions with amino acids such as histidine in cuticular proteins. To determine the products that might form when this type of reaction occurs during cuticle sclerotization, the reactions between electrochemically prepared NADA quinone and N-acetylistidine (NAcH), a protein model nucleophile, have been investigated at pH 7. Two major products, 6-[N-(N-acetylhistidyl)]-N-acetyldopamine and 2-[N-(N acetylhistidyl)]-N-acetyldopamine, were purified by semipreparative reversed phase liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy. The relative molar ratio of the C(6) mono addition adduct to the C(2) mono-addition adduct is 87:13. UV/vis spectroscopic analysis shows that both products have an absorption maximum at 284 nm. Cyclic voltammetry shows that these adducts are oxidized less readily than NADA. PMID- 8619636 TI - The exit from G(0) into the cell cycle requires and is controlled by sarco(endo)plasmic reticulum Ca2+ pump. AB - The intracellular calcium pump sarco(endo)plasmic reticulum Ca2+ (SERCA) is responsible for the formation of the Ca2+ gradient across the endoplasmic reticulum membrane, and this gradient is used to generate the Ca2- signal during agonist-stimulated cell growth. In the present study, the role of SERCA in both cell cycle and growth control was investigated using cultured rat aortic endothelial cells (RAEC). Using a novel DNA transfection approach, cell lines were established that showed varying degree of SERCA activity through the down regulation of the endogenous SERCA gene (B. F. Liu, X. Xu, R. Fridman, S. Muallem, and T. H. Kuo, J. Biol. Chem. 271, 1--9, 1996). Cell proliferation studies indicated that the lower SERCA expressing cells exhibited a slower growth pattern without altering the doubling time which was similar for both parental and transfected RAEC lines. G1 to S phase transition was prolonged with a smaller proportion of cells entering DNA synthesis as indicated by thymidine incorporation assay. Comparison of transfected cell lines indicated a tight coupling of SERCA activity and the length of the G1 period. Down-regulation of SERCA gene expression was accompanied by increased mRNA levels of p21 (WAF1/CIP1), a universal cell cycle inhibitor. The delay in G1 to S progression also coincided with the up-regulation of p53 mRNA and underphosphorylation of the retinoblastoma protein. This study suggests that Ca2+ metabolism in the agonist mobilizable pool controls the cell cycle through the regulation of genes operating in the critical G1 to S checkpoint. PMID- 8619638 TI - Developmental changes in the phospho(enol)pyruvate carboxykinase gene expression in small intestine and liver of suckling rats. AB - The cytosolic enzyme phospho(enol)pyruvate carboxykinase (PEPCK) is markedly expressed in the intestinal mucosa of suckling rats. The expression is located in the small intestine, but there is no expression in stomach, colon, or cecum. The expression changes with age. The mRNA levels at birth are very low, increase after the first lactation, reach maximum levels between 3 and 9 days after birth, and then decrease smoothly. At weaning, when animals begin to feed on a solid chow diet, the expression falls to adult levels, which are hardly detectable. Mother's milk may influence the intestinal expression, since in rats weaned at Day 18, 3 days before normal weaning, the mRNA levels decreased dramatically. mRNA levels for PEPCK in liver present a rather different developmental pattern from that of intestine, remaining high at weaning and in adult rats. On the ninth day after birth, the mRNA levels are the same in intestine and liver. PMID- 8619637 TI - The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols. AB - Sterol biosynthesis requires the removal of the 14 alpha-methyl group from lanosterol in animals and fungi and from obtusifoliol in plants. This reaction is catalyzed by a microsomal cytochrome P450, the sterol 14 alpha-demethylase (P450(14DM), which is the only P450 described so far to be expressed in different phyla. A cDNA encoding human P450(14DM) was isolated from a liver cDNA library using a partial rat lanosterol 14 alpha-demethylase cDNA probe. The deduced amino acid sequence is 93% and 38--42% identical to rat and fungal P450(14DM), respectively. Expression of the human CYP51 cDNA in Escherichia coli showed that the cDNA encodes an enzyme having lanosterol 14 alpha-demethylase activity. Northern blot analysis showed that CYP51 mRNA is ubiquitously expressed with highest levels in testis, ovary, adrenal, prostate, liver, kidney, and lung. Many genes involved in cholesterol homeostasis are regulated by cholesterol or its metabolites. In the case of CYP51, cholesterol deprivation led to a 2.6- to 3.8 fold induction of mRNA levels in human adrenocortical H295R cells and this effect was suppressed by the addition of 25-hydroxycholesterol. In human hepatoma HepG2 cells, no effect of cholesterol deprivation was observed; however, the levels of CYP51 mRNA were reduced 4- to 6-fold by the addition of 25-hydroxycholesterol. Thus, like several other genes in the cholesterol biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, HMG CoA reductase, squalene synthase, and farnesyl diphosphate synthase, the expression of the human CYP51 is suppressed by oxysterols. PMID- 8619639 TI - Proteasome activator PA28 and its interaction with 20 S proteasomes. AB - An activator of the 20 S proteasome has been purified to apparent homogeneity from rabbit erythrocytes, liver, and skeletal muscle. The activator displays an M(r) of about 200,000 upon sizing chromatography and, as judged by gel electrophoresis under denaturing conditions, is composed of two species of subunit of about equal abundance and with M(r) of 31 and 29 kDa. Upon isoelectric focusing, the activator is resolved into two major bands with pI values in the range of pH 5.1 and 5.5, corresponding to the two subunits. Limited proteolytic cleavage with trypsin results, for each subunit, in a distinct fragmentation pattern, indicating that in the rabbit, the native activator molecule occurs either as two homomultimers or as heteromultimers. The activator shows no hydrolytic activity by itself. However, when combined with proteasomes, it enhances, in a dose-related manner, the distinct peptidase activities of the proteinase. The activation process requires binding of the activator protein to the proteinase. This association, however, is reversible with recovery of active proteinase and activator protein. In vitro experiments suggest that, in vivo, the activator is bound to 20 S proteasomes rather than occurring as the free molecule. PMID- 8619640 TI - Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes. AB - Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH cytochrome P450 reductase. PMID- 8619641 TI - Discrimination between the four tryptophan residues of MM-creatine kinase on the basis of the effect of N-bromosuccinimide on activity and spectral properties. AB - Rabbit muscle cytosolic creatine kinase (MM-CK) has been treated with N bromosuccinimide, a reagent known to oxidize selectively the indole moiety of tryptophan residues of proteins in acidic conditions. Inactivation of the enzyme is achieved by modification of one residue per monomer. NBS treatment decreases the ultraviolet absorbance at 280 nm and the intrinsic fluorescence of the protein. From these data it can be deduced that the quantum yields of the four tryptophan residues of each monomer are different due to the more or less hydrophobic environment of each of them and that at least two of them are sufficiently close to Cys 282 to allow fluorescence energy transfer to an extrinsic fluorophore bound to this residue. The accessibility to iodide of the tryptophans has been evaluated during guanidinium chloride denaturation. These data allowed us to acquire a new insight into the environment, the contribution to intrinsic fluorescence and the role in enzymatic activity and fluorescence resonance energy transfer of the tryptophan residues of CK and to tentatively assign a position in the sequence to each of them. PMID- 8619642 TI - -Indications and complications of ambulatory surgery in a general practice operating room-. PMID- 8619643 TI - -Epidemiology of breast cancer-. PMID- 8619644 TI - -Results of ongoing breast cancer prevention studies-. PMID- 8619645 TI - -Complications of hysteroscopy and laparoscopy from the anesthesiologic viewpoint . PMID- 8619646 TI - -Tamoxifen and the endometrium: monitoring and evaluation of endometrial changes . PMID- 8619647 TI - -Optimizing patient information-. PMID- 8619648 TI - -Possibilities and limits of imaging for evaluating breast implants-. PMID- 8619649 TI - -"Is Switzerland an area for trying new non-homologous medical products?"-. PMID- 8619650 TI - -Breast implants: prevention and therapy of early and late complications. New products and alternatives. PMID- 8619651 TI - The transverse rectus abdominis muscle flap for breast reconstruction. PMID- 8619652 TI - -Toxoplasmosis in pregnancy: arguments in favor of systematic screening in Switzerland-. PMID- 8619653 TI - -Screening for toxoplasmosis in pregnancy-. PMID- 8619655 TI - -Are blood transfusions still necessary?-. PMID- 8619654 TI - -Increased AFP values-. PMID- 8619656 TI - -Amniocentesis or chorionic villi sampling--when and why?-. PMID- 8619657 TI - -Hormone substitution in postmenopause-. PMID- 8619658 TI - -Psychosocial aspects of hormone substitution in postmenopause-. PMID- 8619659 TI - -Complications. Surgical endoscopy versus gynecologic minimal invasive surgery-. PMID- 8619660 TI - -Neonatal asphyxia as the cause of brain damage in children?-. AB - The prevalence of cerebral palsy (CP) has increased over the last 15 years in most countries. This is explained by an improved survival of very low birth weight prematures. In term infants birth asphyxia is of minor significance as a cause for CP. In only 10% of all CP cases following delivery at term, birth asphyxia must be discussed as a possible cause. In premature deliveries events during the perinatal period are of greater significance for the later development of a CP. Only severe forms of oxygen deficit, leading to tissue damage in the brain and other organs with clinical symptoms during the first days of life, are of significance for the long term prognosis. Even in the presence of severe birth asphyxia the causal relationship with a psychomotor handicap is not proven, since brain damage may have developed during pregnancy before the onset of labour and may be the cause of birth asphyxia. Brain damage and birth asphyxia may be the result of a common pathology of pregnancy. PMID- 8619661 TI - -Prenatal diagnosis of brain damage in the fetus-. PMID- 8619662 TI - -Diagnosis of fetal hypoxia by monitoring during labor-. PMID- 8619663 TI - -Gynecologic endoscopic surgery. What are the limits?-. PMID- 8619664 TI - -Perinatal asphyxia of the neonate at term-. PMID- 8619665 TI - -Brain damage in infancy: mostly not intrapartum. On the etiology of mental and neurologic handicaps-. PMID- 8619666 TI - -Axillary lymph node excision: for what reasons and indications-. PMID- 8619667 TI - -Breast neoplasms: axillary lymph node excision--at what price-. PMID- 8619668 TI - -Adjuvant systemic therapy of breast carcinoma-. PMID- 8619669 TI - Primary cardiac lymphoma. AB - A case of primary cardiac non-Hodgkin's B cell lymphoma is described in a patient presenting with obstructive right heart failure. Unlike the majority of cases, in this case the tumor was diagnosed ante mortem. THe patient's history combined with the aggressive use of noninvasive echocardiography are helpful in diagnosing this rare lesion. PMID- 8619670 TI - Primary cardiac lymphoma: echocardiographic characterization and successful resection. AB - We present a case of primary cardiac lymphoma in a patient with dyspnea and hypoxemia. Transesophageal echocardiography reveals a large right atrial mass and an atrial septal aneurysm with right-to-left shunting through a patent foramen ovale. The patient underwent resection and atrial reconstruction. Pathology was a B cell lymphoma with diffuse large cell histology. There was no evidence of extracardiac involvement, and the patient is well 3 months postoperatively with a normal transthoracic echocardiogram. PMID- 8619671 TI - Extramedullary hematopoiesis simulating posterior mediastinal tumors. AB - We report the case of a patient with intrathoracic extramedullary hematopoiesis presenting as a posterior mediastinal tumor in response to chronic hemolytic anemia. Noninvasive studies including chest roentgenograms, computed tomographic scans, magnetic resonance images, and nuclear scans can establish the diagnosis in most cases. In equivocal cases, transthoracic needle biopsy and open biopsy should be considered. Surgical resection is recommended only for patients with symptoms of compression. PMID- 8619672 TI - Pulmonary autograft in ventricular septal defect-aortic insufficiency complex. AB - Pulmonary autograft aortic root replacement in a child with the ventricular septal defect-aortic insufficiency complex is described. It offers all the advantages of the autograft, avoids closure of the ventricular septal defect, and prevents the use of prosthetic material. PMID- 8619673 TI - Surgical treatment of multiple coronary aneurysms in an elderly man. AB - We report an unusual case involving a 70-year-old man with multiple coronary aneurysms, including a 5-cm aneurysm of the right coronary artery. The precise size was determined with magnetic resonance imaging, although the aneurysms were diagnosed initially on angiography. Multiple coronary aneurysms are rare in adults, The cause may have been atherosclerosis, syphilis, or collagen vascular disease, all of which cause weakening of the media. The patient underwent successful operation, including coronary artery bypass grafting. PMID- 8619674 TI - Maze procedure and anomalous coronary artery repair. AB - A 60-year-old woman has anomalous origin of the left coronary artery from the pulmonary artery, mitral regurgitation with left ventricular dysfunction, and atrial fibrillation. We performed mitral valve annuloplasty, maze procedure, and intrapulmonary tunnel repair of anomalous origin of the left coronary artery. The patient regained normal sinus rhythm and showed improved left ventricular function and no mitral regurgitation. PMID- 8619675 TI - Dissection of an allograft ascending aorta after aortic root replacement. AB - THe case of a 57-year-old man with dissection of an allograft ascending aorta 2 months after aortic root replacement is presented. Most likely traumatic in origin, this unusual complication was managed by Dacron graft replacement of the ascending aorta using hypothermia and circulatory arrest. PMID- 8619676 TI - Blood cyst of the tricuspid valve. AB - Blood-filled cysts of the heart valves are commonly reported at postmortem examination of infants but are rarely seen in older children and adults. These cysts appear to be benign lesions and should be removed only if they cause problems. We present the case of a patient with a tricuspid valve blood cyst that was surgically removed and then discuss the differential diagnosis and management of these cysts. When a cardiac mass has features suggestive of a blood cyst, radical resection should not be performed unless histologic confirmation of tumor is obtained. PMID- 8619677 TI - Atrial cannulation for left ventricular assistance: superiority of the dome approach. AB - Successful left ventricular support is dependent on adequate inflow drainage. We describe atrial cannulation using the dome of the left atrium, which in our experience has resulted in excellent inflow drainage. PMID- 8619678 TI - Surgical atrial septostomy without cardiopulmonary bypass. AB - Surgical atrial septostomy using a special atriotomy knife without cardiopulmonary bypass in patients with obstruction of the left-sided atrioventricular valve and complex cardiac anomalies is described. This procedure is effective, safe, and economical for patients in the acute stage after intracardiac repair, and available for patients with a closed fossa ovalis. PMID- 8619679 TI - Exposing the circumflex coronary artery: the heartflip technique. AB - A method to expose the circumflex coronary artery in its course in the atrioventricular groove is introduced. No special equipment or assistance is required. This method also can be applied to expose the obtuse marginal branches of the circumflex coronary artery. Adverse effects have not been observed. PMID- 8619680 TI - Simplified technique for hemi-arch replacement during open distal anastomosis: the "calla" method. AB - During open distal anastomosis for type A dissecting aneurysm, the beveled end of the graft was rolled back like a bract of the Calla flower and inserted into the aortic lumen. The inverted graft was anastomosed using forehand continuous sutures. After completion of the distal anastomosis, the inverted graft was pulled out and then the proximal anastomosis was completed. This "Calla flower" deformation facilitates the hemostatic distal anastomosis in hemi-arch replacement during open distal anastomosis. PMID- 8619681 TI - Mitral valve injury after blunt chest trauma. AB - Isolated mitral valve injury after blunt chest trauma is a very rare event. This disruption, causing sudden and severe mitral regurgitation, will rapidly lead to congestive heart failure and death unless operatively corrected. A high index of suspicion coupled with appropriate diagnostic tests will provide the diagnosis and allow operative correction. We report a patient who survived this injury and review all previous reports of blunt traumatic disruption of the mitral valve. PMID- 8619682 TI - Postoperative mediastinitis: classification and management. AB - Although the incidence of mediastinal wound infection in patients undergoing median sternotomy for cardiopulmonary bypass is less than 1%, its associated morbidity, mortality, and "cost" remain unacceptably high. There is considerable lack of consensus regarding the ideal operative treatment of complicated median sternotomy wounds. The aim of this article is to review the current preventive, diagnostic, and therapeutic techniques offered to patients with mediastinitis. We also propose a new classification for postoperative mediastinitis. Data from the English-language literature suggest that the type of mediastinitis and direct assessment of the mediastinum under general anesthesia are the main determinants of the nature of subsequent operative treatment. Wound debridement and removal of foreign materials are essential steps of whatever procedures are applied. Closed mediastinal irrigation can be successful in type I mediastinitis, whereas major reconstructive operation is probably the treatment of choice for patients with mediastinitis types II to V. Refinement of the current diagnostic tools and further evaluation of the benefits of primary sternal fixation in combination with a reconstructive procedure in mediastinitis types I to III could improve the outcome of this dreaded complication. PMID- 8619683 TI - Diagnosis and management of pulmonary embolism. PMID- 8619684 TI - Eosinophil granulocyte activation and heparin-coated cardiopulmonary bypass. PMID- 8619685 TI - Topical aprotinin in cardiac operations: a note of caution. PMID- 8619687 TI - Primary cardiac valve tumors. PMID- 8619686 TI - Troponin-T in cardiac surgery. PMID- 8619688 TI - Defibrillator patch constriction. PMID- 8619689 TI - Coronary bypass in women. PMID- 8619690 TI - Retrograde cerebral perfusion. PMID- 8619691 TI - Deep hypothermic circulatory arrest: a need for caution. PMID- 8619692 TI - Monitoring of systemic anticoagulation during cardiopulmonary bypass. PMID- 8619693 TI - Neurologic sequelae of deep hypothermic circulatory arrest in cardiac transplant infants. AB - BACKGROUND: Considerable controversy exists experimentally and clinically regarding adverse neurologic effects that may follow deep hypothermic circulatory arrest. Moreover, the techniques of DHCA have never been standardized. METHODS: We prospectively studies the neurodevelopmental outcome in 38 infants undergoing cardiac transplantation using DHCA before the age of 4 months (mean age, 37.0 days). Neurodevelopmental outcome in the 22 boys and 16 girls was tested up to 2.5 years after transplantation using Bayley scale of infant development. Bayley scores were compared with the rate of core cooling and the length of DHCA in all patients. Deep hypothermic circulatory arrest was accomplished using an asanguineous prime resulting in hematocrits of 5% +/- 5% and ionized Ca2+, 0.4 +/ 0.1 mmol/L. No surface precooling was used, but the head was packed in ice. Mean cooling time was 14.0 +/- 3.5 minutes, resulting in rectal temperatures of 18 degrees +/- 2.5 degrees C. Duration of DHCA ranged from 42 to 70 minutes (mean duration, 56.0 +/- 6.6 minutes). RESULTS: Postoperatively, the mean Bayley psychomotor development index was 91 (range, 50 to 130) and mental development index was 88 (range, 50 to 130). No relationship was found between either the rate of cooling or the duration of DHCA and Bayley scores (r = 0.227 and r = 0.322, respectively). CONCLUSIONS: These data suggest that neither the rate of cooling nor DHCA times between 42 and 70 minutes using profoundly low hematocrits and low ionized calcium levels has any measurable effect on neurologic outcome up to 2.5 years postoperatively. It is possible that adverse neurologic outcomes from DHCA reflect particular methods of achieving DHCA. PMID- 8619694 TI - Coronary operations in patients with spinal cord injury. AB - BACKGROUND: The risk of heart disease in patients with spinal cord injury is similar to that in the general population. The physiologic derangements raise special problems in patients with SCI having coronary operations. METHODS: From January 1980 to May 1995, we performed coronary artery bypass procedures on 20 patients with SCI; 4 were tetraplegic and the remainder were paraplegic. The indication for operation was angina: unstable (13), exertional (4), or postinfarctional (3). Bowel and bladder care was given immediately before operation; operating room tables were double padded and a pelvic wrap was used to protect the back. Electric wheelchairs were used for early mobilization. RESULTS: Vasomotor instability from cardiopulmonary bypass was not present in patients with SCI. Pharmacologic support was required in the operating room by 4 patients for low vascular resistance, but in only one case in the intensive care unit. One patient required ventilation support for more than 24 hours. All patients were able to cough effectively. No thoracic wound complications occurred. There were three operative deaths, all in patients with multiple risk factors. The acute hospital stay averaged 9.3 days; patients were then transferred to an SCI unit for rehabilitation, were upper-extremity weight bearing was restricted for 2 to 4 weeks. CONCLUSIONS: Patients should not be denied coronary artery bypass procedures because of an SCI, but their special needs must be managed properly. PMID- 8619695 TI - Response of kaolin ACT to heparin: evaluation with an automated assay and higher heparin doses. AB - BACKGROUND: Because previous reports suggest that the linear relationship between celite activated clotting time (ACT) values and heparin sodium is disrupted if values exceed 500 to 600 seconds, this study was designed to evaluate the relationship of kaolin activated clotting time (ACT) values to high in vitro heparin concentrations. In addition, the relationship of kaolin ACT to heparin concentration as determined manually was compared with that obtained with an automated heparin dose response assay. METHODS: Blood specimens were obtained prior to and after heparin administration from 41 cardiac surgical patients requiring cardiopulmonary bypass in this institutional human studies committee approved study. Five ACT instruments were used to evaluate the response of kaolin ACT to manually added heparin at two anticoagulation levels: low range (ACT values of less than 500 seconds) and high range (ACT values of 500 seconds or greater). Specimens were also used to measure kaolin ACT values at three heparin concentrations with an automated heparin dose response assay (HDR) using a Hepcon instrument. RESULTS: A greater response of kaolin ACT to heparin was seen with high-range ACT values than low-range ACT values as illustrated by greater (p = 0.002) mean slope values (low range, 99 +/- 30 s/U/ mL; high range, 128 +/- 50 s/U/ml). Good correlations were obtained between heparin concentration and either low- or high-range ACT values as demonstrated by mean correlation coefficients (low range, 0.992; high range 0.982). The response of low-range kaolin ACT values to heparin was greater than that obtained with the automated heparin dose response assay as illustrated by greater (p = 0.005) mean slope values (low range, 99 +/- 30 s/U/mL; HDR, 82 +/- 21 s/U/mL). Good correlations were observed for the relationship between heparin and ACT values obtained with the HDR assay (r = 0.998). CONCLUSIONS: A variable response of kaolin ACT to heparin among patients was demonstrated in our study, especially when ACT values exceeded 500 seconds. We found that the response of kaolin ACT to higher heparin concentrations was acceptable for clinical monitoring based on good correlations obtained in individual patients. The HDR assay generally overestimates a patient's heparin requirements; most likely, this is due to a lower response of kaolin ACT to heparin concentration that is reflected in this assay. Because and exceptional correlation can be obtained between kaolin ACT values and heparin concentration using the assay, this automated assay can identify heparin resistant patients who may need further treatment. PMID- 8619696 TI - Surgical repair of transposition of the great arteries in neonates with persistent pulmonary hypertension. AB - BACKGROUND: Pulmonary hypertension due to persistent fetal circulation is rarely associated with transposition of the great arteries and intact ventricular septum. Previous attempts at management of affected neonates using prostaglandin E1 and balloon atrial septotomy followed by surgical repair have been largely unsuccessful. METHODS: Between September 1992 and April 1995, 45 neonates underwent repair of transposition of the great arteries with the arterial switch operation. Two patients (4%) with transposition of the great arteries and intact ventricular septum presented with profound reversed differential desaturation and right-to-left shunting at the level of the ductus arteriosus after balloon atrial septotomy. A diagnosis of persistent pulmonary hypertension was established and both neonates entered an experimental management protocol using inhaled nitric oxide and rapid arterial switch operation. RESULTS: Preoperative hemodynamic stabilization was achieved in 1 patient using 40 parts per million of inhaled nitric oxide, whereas the other required in addition extracorporeal membrane oxygenation for severe biventricular dysfunction. Both underwent successful surgical repair 4 to 5 days after admission, but received postoperatively 1 week of inhaled nitric oxide therapy for persistent pulmonary hypertension. Follow-up echocardiography at 3 months showed good biventricular function and normal geometry of the ventricular septum, suggesting low pulmonary artery pressure, in both. CONCLUSIONS: A management protocol using inhaled nitric oxide and extracorporeal membrane oxygenation followed by the arterial switch operation was successfully used in neonates with transposition of the great arteries, intact ventricular septum, and persistent pulmonary hypertension. Wider use of preoperative and postoperative inhaled nitric oxide may improve the surgical outcome of this difficult subset of patients. PMID- 8619697 TI - Long-term results with the Medtronic-Hall valvular prosthesis. AB - BACKGROUND: Although more than 170,000 Medtronic-Hall mechanical valvular prostheses have been inserted world-wide, long-term results are available on only a small percent of those valves inserted. METHODS: A prospective data registry of all Medtronic-Hall cardiac prostheses inserted by one surgeon was used to identify 460 valves inserted during 391 operations from 1983 to 1994: single aortic (n = 210), single mitral (n = 115), or double aortic and mitral (n = 66) replacements, including three tricuspid valve replacements. Follow-up was sought five times in 10 years and was available for 280 (99%) of 283 survivors with only an isolated aortic or mitral Medtronic-Hall valve followed up for at least 1 year (1,246 patient years). RESULTS: Hospital mortality was 4.6% (18 patients). Of 40 late deaths, eight were valve-related (0.6% per patient-year). The linearized rates of complications for aortic and mitral valve replacements (percent per patient-year) were, respectively: structural deterioration, 0 and 0; nonstructural dysfunction, 0.1 and 2.1; thromboembolism, 1.3 and 2.1; thrombosis, 0 and 0.2; anticoagulant-related bleeding, 1.7 and 1.9; and prosthetic valve endocarditis, 0.6 and 1.0. Actuarial freedom from reoperation at 10 years was 97% for aortic and 88% for mitral valves. CONCLUSIONS: The Medtronic-Hall mechanical valvular prosthesis has excellent durability and acceptably low rates of valve related complications and remains my mechanical prosthetic valve of choice for both aortic and mitral valve replacements. PMID- 8619698 TI - Incidence of vocal fold paralysis in infants undergoing ligation of patent ductus arteriosus. AB - BACKGROUND: Left-sided, iatrogenic vocal fold paralysis (IVFP) secondary to recurrent laryngeal nerve injury is a potential complication of ligation of patent ductus arteriosus (PDA). This study investigates specific risk factors associated with IVFP. METHODS: A retrospective chart review was performed for all infants 12 months of age or younger who underwent operative PDA closure at the University of Iowa from January 1, 1991, to January 1, 1994. RESULTS: Six cases of IVFP were diagnosed in 68 infants who underwent PDA ligation using clips (52.9%), suture ligatures (41.2%) or both (5.9%). Compared with infants without postoperative IVFP, infants with IVFP were smaller at birth (0.9 versus 2.3 kg; p < 0.001) and more premature (gestational age, 26.3 versus 33.8 weeks; p < 0.001), and were smaller (1.1 versus 3.4 kg; p < 0.001) and younger (31.9 versus 88.4 days; p < 0.001) at operation. Weight gain from birth to operation was significant only in infants without postoperative IVFP (p < 0.05). Although the overall incidence of IVFP in all infants undergoing PDA closure was 8.8%, five of the six cases (83.3%) of IVFP occurred in extremely low birth weight infants, ie, those weighing 1 kg or less at birth. Among the cohort of extremely low birth weight babies undergoing operation, the incidence of IVFP was 22.7%. Iatrogenic vocal fold paralysis was associated only with the use of surgical clips; however, because clips were used in 90.9% of the premature infants requiring PDA ligation, it was not possible to establish whether suture ligature is a safer technique. CONCLUSIONS: This study demonstrates that the single major risk factor for IVFP after ligation of PDA is birth weight less than 1 kg. PMID- 8619699 TI - The gastrointestinal tract: an underestimated organ as demonstrated in an experimental LVAD pig model. AB - BACKGROUND: Although hemodynamic stability and renal function are important and are monitored closely in patients with implanted left ventricular assist devices (LVAD), the gastrointestinal tract may be underestimated in the early postoperative period with regard to adequate perfusion. We investigated renal, intestinal, and whole body metabolic changes in response to variations in LVAD flow and inspired oxygen concentration (FiO2). METHODS: Left ventricular assist devices were implanted in 10 adult pigs (weight, 55 +/- 1.76 kg). Renal vein (RV), superior mesenteric vein (SMV), and pulmonary artery (PA) blood oxygen saturation and lactate concentration were measured and used as tissue perfusion markers. These measurements were made at baseline and after changes in LVAD flow or FiO2. RESULTS: Oxygen saturation in the PA, SMV, and RV decreased significantly after a reduction in LVAD flow (P < 0.05), with a greater reduction in the SMV than in the PA and RV (p < 0.05 at LVAD flow 3.5L/min; p < 0.01 at LVAD flow 2.0 and 1.0 L/min). The lactate concentration in the PA and SMV increased significantly (p < 0.01) with decreased flow, with a greater increase in the SMV than in the PA (p< 0.05), whereas it remained unchanged in the RV. Oxygen saturation in the PA, SMV, and RV decreased significantly after a reduction in FiO2 (p < 0.05). Lactate concentration in the PA, SMV, and RV increased significantly at FiO2 of 0.10 (p < 0.05). Lactate concentration in the PA and SMV was significantly higher than that in the RV at Fi)2 of 0.10 (p < 0.01). CONCLUSIONS: The results show that the gastrointestinal tract is at high risk during low perfusion or low FiO2, whereas the kidneys' metabolic function appears to be less disturbed. In clinical practice, this emphasizes the need to ensure adequate blood flow and respiratory function, especially after extubation, in patients with implanted LVAD. This might avoid intestinal ischemia and subsequent endotoxemia. Gastrointestinal tonometry may help in the assessment of intestinal perfusion. PMID- 8619700 TI - Double-horned or caplike right ventricle: diagnosis and operative treatment. AB - BACKGROUND: Three patients reported here and 4 from the literature serve as background for the state-of-art diagnostic and operative considerations for an unusual congenital cardiac malformation: double-horned or caplike right ventricle. METHODS: This is a retrospective analysis of cardiac catheterization, cineangiography, and two-dimensional echocardiography findings, as well as palliative and corrective operations in 3 previously unreported patients. Four patients from the literature are reviewed. RESULTS: Characteristic morphologic features recognizable by invasive and noninvasive imaging distinguish double horned right ventricle from complex malformations such as criss-cross hearts, superior-inferior ventricles, and univentricular hearts with a small outflow chamber. CONCLUSION: Double-horned or caplike right ventricle is a congenital malformation characterized by an unusual ventricular morphology, which may be the result of incomplete development of the right ventricle. The two-horned appearance may be secondary to an absence of the apical trabeculated compartment, with the left ventricle wedged between the two horns. It is invariably associated with double right ventricular outlet. Surgical experience so far suggest that most patients with typical double-horned right ventricle should be considered for anatomic surgical correction. PMID- 8619701 TI - Effects of short-term supplementation with coenzyme Q10 on myocardial protection during cardiac operations. AB - BACKGROUND: Coenzyme Q10 (CoQ10) is a naturally occurring vitamin-like substance that may have a beneficial role in ischemia-reperfusion injury. Coenzyme Q10 administered either as an additive to cardioplegia or as long-term preoperative oral supplementation has been reported to ameliorate myocardial injury after cardiac operations. METHODS: To determine whether short-term supplementation with large doses of CoQ10 (600 mg in divided doses 12 hours before operation) was effective in myocardial protection, 20 patients with well-preserved left ventricular function (ejection fraction greater than 0.50) undergoing elective coronary revascularization were enrolled in a prospective, double-blind, placebo controlled randomized trial. Serial concentrations of CoQ10, myoglobin, creatine kinase MD fraction, and cardiac troponin T were measured preoperatively and 1, 6, 24, 72, and 120 hours postoperatively. Efficacy of myocardial protection was also assessed by clinical outcome and serial changes in electrocardiographic indices. RESULTS: The patient groups were similar with respect to preoperative and intraoperative characteristics. There was no significant difference in the preoperative plasma levels of CoQ10. These levels fell significantly in both groups after operation, although the magnitude of the decrease was less in the CoQ10-supplemented group (43% versus 60%). In both groups, there were significant postoperative increases in myoglobin, creatine kinase MB fraction, and cardiac troponin T. The magnitude of increases in cardiac troponin T was greater in the CoQ10-supplemented group, reaching marginal overall statistical significance (p = 0.06). CONCLUSIONS: Short-term supplementation with large doses of CoQ10 does not lead to improved myocardial protection in patients undergoing coronary revascularization with well-preserved ventricular function and relatively short ischemic times. PMID- 8619702 TI - Intermittent aortic cross-clamping and cold crystalloid cardioplegia for low-risk coronary patients. AB - BACKGROUND: Blood cardioplegic strategies have been shown to increase myocardial oxygen uptake, replenish depleted energy stores, and improve myocardial function and survival in the high-risk subset of patients. However, the superiority of these techniques over intermittent aortic cross-clamping and crystalloid cardioplegia in low-risk patients is still controversial. METHODS: This study consisted of two parts. In the first part, we assessed the results of a recent cohort of 399 consecutive low-risk patients undergoing their first coronary artery bypass grafting between 1993 and 1995 using cold crystalloid cardioplegia (n = 128) and intermittent aortic cross-clamping (n = 271). In the second part of the study, 40 consecutive low-risk patients undergoing elective first time coronary artery bypass grafting were randomly divided into two equal groups. One group received cold crystalloid cardioplegia and the other group had myocardial management with intermittent aortic cross-clamping. The two groups were compared with respect to hemodynamic, biochemical and ultrastructural changes. RESULTS: The overall mortality rate, the perioperative myocardial in the need for intraaortic balloon pumps, and the need for inotropic agents were 0.25%, 1.5%, 1%, and 5.8%, respectively. No significant differences were observed between the groups with respect to these clinically defined end points. CONCLUSIONS: Both intermittent aortic cross-clamping and cold crystalloid cardioplegia techniques may be used safely in low-risk patients undergoing first-time coronary artery bypass grafting. PMID- 8619703 TI - Coarctation repair using end-to-side anastomosis of descending aorta to proximal aortic arch. AB - BACKGROUND: Recurrent aortic coarctation after primary operative repair in the neonate and small infant is seen most commonly within the first year of life. Inadequate removal of ductal tissue, failure to address hypoplasia of the aortic arch, and suture line tension have been cited as important factors in early recurrence. METHODS: To address these issues, we have used a technique of coarctation resection and extended anastomosis of the descending aorta to the undersurface of the aortic arch. THe salient features of this approach include extensive mobilization of the aortic arch and neck vessels, careful trimming of all ductal tissue, ligation of the isthmus just beyond the left subclavian artery, and end-to-side anastomosis of the descending aorta to a separate incision in the undersurface of the aortic arch proximal to all tubular hypoplasia. Between July 1992 and January 1995, 19 consecutive neonates (median age, 13 days) and 4 consecutive infants under 3 months of age (median age, 69 days) with a mean peak systolic upper to lower extremity resting gradient of 27.9 +/- 16.9 mm Hg underwent repair of aortic coarctation and tubular hypoplasia of the arch. Other procedures performed at the time of repair included ligation of a patent ductus arteriosus (n = 19), pulmonary artery banding (n = 3), and closure of ventricular septal or atrial septal defect (n = 3). RESULTS: There were no perioperative deaths. Early postoperative complication included a recurrent laryngeal nerve injury and a transient focal tonic clonic seizure. There was one late death, after a subsequent intracardiac surgical procedure, at a median follow-up of 16 months (range, 1 to 29 months). Twenty-one of 22 late survivors were free of recurrent aortic coarctation by echocardiography findings and clinical examination, with a median upper to lower extremity gradient of 0 mm Hg. Reintervention for recurrent aortic coarctation was not required in any survivor. CONCLUSIONS: The technique described herein completely removes all potentially abnormal tissue from the aorta, including ductal tissue and all tubular hypoplastic tissue proximal to the coarctation site. PMID- 8619705 TI - Stab wounds of the innominate artery. AB - BACKGROUND: Innominate artery stab wounds are rarely encountered, and the optimal management of this injury is different from that of blunt innominate injury in that permanent bypass shunting should not be necessary. METHODS: The records of 19 patients with stab wounds of the innominate artery who were treated by our department from January 1982 to June 1995 were reviewed. RESULTS: Eighteen patients (95%) sustained zone 1 neck stabs, with a similar proportion having only a single stab wound. Seventeen (89%) of the 18 patients having chest roentgenograms had mediastinal widening. Thirteen patients (68%) were hemodynamically stable at admission; the remainder were unstable (26%) or moribund (5%). Fourteen patients (74%) underwent angiography, with no false negative studies for arterial injury. Associated injuries to thoracic viscera occurred in 4 patients (21%). All injuries were repaired with either direct suture (18 of 19) or prosthetic interposition grafting (1 of 19). One patient required cardiopulmonary bypass to repair complex injuries. The overall mortality rate was 5% (1 of 19), and complications occurred in 2 patients (11%). CONCLUSIONS: Innominate artery stab wounds can be managed successfully without permanent bypass shunting and with a low mortality rate. PMID- 8619704 TI - Usefulness of pulsatile bidirectional cavopulmonary shunt in high-risk Fontan patients. AB - BACKGROUND: A bidirectional cavopulmonary shunt has been performed for the high risk Fontan patient. It is well known that in the presence of the bidirectional cavopulmonary shunt alone to secure pulmonary blood flow, the central pulmonary artery size decreases over time. We have performed pulsatile bidirectional cavopulmonary shunt (PBCPS), keeping pulmonary blood flow from the ventricle through the stenotic pulmonary valve, or a Blalock-Taussig shunt in patients who do not meet the criteria for the Fontan operation. METHODS: Eleven patients who underwent PBCPS between 1989 and 1993 were reviewed. We compared the results of cardiac catheterization immediately before PBCPS and during the postoperative observation period (310 +/- 257 days). RESULTS: Pulmonary blood flow and arterial oxygen saturation increased significantly after PBCPS (p = 0.01). Pulmonary artery area index showed a tendency to increase (p = 0.11). The mean number of risk factors for the Fontan procedure decreased significantly for 1.8 +/- 1.1 to 0.7 +/- 0.8 after PBCPS (p < 0.05). Overall, 5 of the 11 patients (45.5%) met the criteria for the Fontan procedure, and a fenestrated Fontan procedure was carried out in 4 of them. CONCLUSIONS: The PBCPS is useful for high-risk Fontan patients not only in the staged Fontan operation, but also as definitive palliation. PMID- 8619706 TI - Intraoperative TEE assessment of ventricular septal defect with aortic regurgitation. AB - BACKGROUND: It is desirable to repair but not replace the aortic valve in patients with ventricular septal defect and acquired aortic regurgitation. Precise definition of the valvar pathology with monitoring of its repair perioperatively would enhance the surgical management of this condition. METHODS: Fourteen consecutive patients (age, 10.6 +/- 6 years; weight 29.7 +/- 5.7 kg) who underwent repair of ventricular septal defect with aortic regurgitation were studied by intraoperative transesophageal echocardiography. The severity of prolapse of each of the individual aortic cusps and its adjacent sinus was assessed and the valvar regurgitation quantified by Doppler-derived regurgitant indices. The echocardiographic and surgical findings were correlated and the preoperative and postoperative echocardiographic data were compared to assess the effectiveness of operation. RESULTS: Eight subarterial and six perimembranous defects were located accurately and their sizes (11.8 +/- 3.0 mm) correlated well (r = 0.80) with the surgical measurements. Transesophageal echocardiography detected prolapse of the aortic valve and its sinus in all 14 patients. The severity of the prolapse was severe in 10, moderate in 4, and mild in 5 leaflets. One the basis of these findings, together with the Doppler-derived mean regurgitant indices, exploration of the valve and valvuloplasty were executed appropriately in 12 of 14 patients. In all 14 patients, transesophageal echocardiography after bypass revealed no further cuspal prolapse and significant reduction of the mean regurgitant index (0.55 +/- 0.23 to 0.17 +/- 0.15, p < 0.0001). Residual ventricular septal defect was detected in 5 patients and the only patient with significant shunting who required reexploration was identified correctly. CONCLUSIONS: Intraoperative transesophageal echocardiography can assess effectively the surgical repair of ventricular septal defect with aortic regurgitation and provide information that directs and alters surgical plans to the benefit of patients. PMID- 8619707 TI - Does blood flow through holmium:YAG transmyocardial laser channels? AB - BACKGROUND: Early reports indicate that transmyocardial laser revascularization improves symptoms in patients with refractory angina. However, there is little experimental evidence of whether blood flow through channels is the mechanism of action. METHODS: Endocardial channels were made in the distribution of the left anterior descending coronary artery in canine hearts (n = 5) using a holmium:yttrium-aluminum garnet laser. Hearts were excised acutely while perfused in a retrograde fashion from a second dog so that the aortic valve always remained closed. The proximal left anterior descending coronary artery was ligated. To measure direct transmyocardial blood flow, colored microspheres were injected into the left ventricular chamber. RESULTS: The number of spheres per gram of tissue in the channel region was significantly higher than in the control region (low load, 302.5 +/- 169.0 versus 41.8 +/- 59.4; high load, 208.4 +/- 138.3 versus 5.8 +/- 11.7; both, p < 0.05). However, the estimated regional blood flow through the channels was extremely low (<0.01 mL/g/min. In the chronic setting (n = 4) (2-week survival), no flow as detected through the channels, and the endocardial entry points were closed. CONCLUSIONS: Transmyocardial blood flow does not appear to occur through channels made with the holmium:yttrium-aluminum garnet laser. It remains to be determined whether this is the case with other types of lasers. PMID- 8619708 TI - "Legs" technique for management of widely separated coronary arteries during ascending aortic repair. AB - BACKGROUND: Widely separated coronary arteries with significantly diseased tissue continues to challenge surgeons repairing ascending aortic aneurysms. METHODS: Occasional troublesome leaks around coronary ostial anastomoses and Cabrol graft thrombosis prompted a change of our operative management of this condition. Collagen-impregnated 8-mm "legs" grafts are used to connect the coronary arteries to the composite graft. Ten patients, aged 14 to 70 years, underwent the operation. RESULTS: The first patient is 15 years after the operation and is symptom free. One patient died of an arrhythmia 1 month after discharge. Eight patients are living and well 11/2 to 4 years postoperatively. CONCLUSIONS: Advantages of direct interposition (legs) grafts are as follows: the coronary arteries are separately perfused and the risk of catastrophic thrombosis from a longer high-volume graft is eliminated. Problems with coronary ostial mobilization are avoided. The technique allows full visualization and hemostatic suture line testing with cardioplegia before aortic declamping. Space constraints with reoperations are easily managed, whereas other techniques may result in graft compression on refilling of the heart and termination of bypass. The technique is carried out with ease and reproducibility, and the availability of new graft material has made it our treatment of choice for ascending aortic composite graft replacement. PMID- 8619709 TI - Mortality and neurologic morbidity after repair of traumatic aortic disruption. AB - BACKGROUND: Traumatic disruption of the thoracic aorta frequently results in death before operative repair. The determinants of mortality after repair, however, are uncertain. In addition, intraoperative strategies for reducing the incidence of spinal cord injury remain controversial. METHODS: The records of 45 consecutive patients undergoing repair of traumatic disruption of the thoracic aorta at a single institution during a 9-year period were reviewed in a retrospective fashion. Patient age ranged from 15 to 81 years (mean age, 33.9 years). Twenty-two patients (49%) had multiple associated injuries, and 8 (18%) had isolated aortic injuries. Nine patients (20%) experienced preoperative hypotension (systolic blood pressure of less than 90 mm Hg). Repair was performed with partial bypass in 22 patients, a heparinized shunt in 2, and no distal perfusion (clamp and sew technique) in 21. RESULTS: Nine patient (20%) died after operation. Multivariate logistic regression analysis of preoperative and intraoperative variables identified advancing age and preoperative hypotension as independent predictors of operative death. The presence of associated injuries was not an independent predictor of operative death. All 4 patients with injuries proximal to the aortic isthmus died. Ten patients were excluded from analysis of spinal cord injury either because of preoperative neurologic deficit or because of death before postoperative evaluation. Six (17%) of the remaining 35 patients had development of paraplegia: 5 of the 15 patients having the clamp and sew technique, 1 of the 2 with a shunt, and 0 of the 18 patients with bypass (p < 0.05, clamp and sew versus bypass). In the clamp and sew group, patients in whom paraplegia developed had significantly longer aortic clamp times than those without neurologic injury (40.6 +/- 4.4 minutes versus 28.7 +/- 2.9 minutes, respectively; p < 0.05). CONCLUSIONS: Advancing age, preoperative hypotension, and perhaps injury location are important determinants of death after repair of traumatic disruption of the thoracic aorta. Adjunctive perfusion with partial bypass should be used during repair to reduce the incidence of spinal cord injury. PMID- 8619710 TI - Coronary sinus ostial atresia with persistent left superior vena cava. AB - BACKGROUND: Atresia of the coronary sinus orifice with a persistent left superior vena cava is an intrinsically benign cardiac anomaly with important surgical implications. METHODS: THe medical records of 5 patients with atresia of the coronary sinus orifice with a persistent left superior vena cava were reviewed retrospectively, and a computer search of the world literature describing this cardiac malformation was undertaken. RESULTS: The 5 patients ranged in age from 9 months to 5 years. In 2, the diagnosis was made preoperatively by angiocardiography, and in 3, the abnormality was found incidentally at the time of cardiotomy for repair of associated congenital heart disease. Four of the 5 patients underwent repair of associated cardiac lesions. During operation, care was taken to avoid disruption of left superior vena cava flow to prevent coronary venous obstruction. All patients survived and are doing well at follow-up. CONCLUSIONS: Atresia of the coronary sinus orifice with persistent left superior vena cava is, in itself, a benign anomaly without physiologic consequence. However, the recognition of this lesion during repair of associated cardiac lesions is of vital importance to the cardiac surgeon. Interruption of this sole route of coronary venous drainage can potentially lead to myocardial ischemia and necrosis. PMID- 8619711 TI - Pathologic aspects of polytetrafluoroethylene sutures in human heart. AB - BACKGROUND: Polytetrafluoroethylene (PTFE) sutures have been widely used as a mitral chord substitute. We present the cases of 4 patients who underwent mitral valve repair with chordal replacement by PTFE sutures and these required another operation. This gave us the chance to examine the PTFE sutures. METHODS: Structural analysis of the PTFE sutures was performed 26 to 378 days postoperatively. The specimens were examined grossly, microscopically, and by scanning or transmission electron microscopy or both. RESULTS: The PTFE suture in 1 patient was found to be completely covered with endothelial cells 154 days postoperatively. There was no calcification, and the flexibility and pliability of the PTFE sutures was preserved. Even though the PTFE sutures seemed uncovered on visual inspection, there was a thin lining of collagen and fibrin on the surface. Endothelial cells were seen in areas that looked clear in one specimen 26 days postoperatively. CONCLUSIONS: We think that the new layer of collagen could be promising in terms of durability and that the endothelial layer wil resemble normal tissue in its anticoagulant properties. PMID- 8619712 TI - Arterial impedance in patients during intraaortic balloon counterpulsation. AB - BACKGROUND: Symptomatic improvement of a patient's hemodynamic condition during intraaortic balloon counterpulsation (IABC) is considered to result largely from a reduction in afterload. Afterload can be accurately quantified by arterial input impedance measurements. Here we report the effect of IABC on arterial impedance in humans. METHODS: To characterize the effects of IABC on arterial input impedance, impedance measurements were obtained using aortic annulus Doppler flow and pressure from the aortic balloon catheter. Impedance spectra were compared between the cardiac cycles preceding and following the cycle with IABC in 25 patients. RESULTS: Intraaortic balloon counterpulsation increased stroke volume (23%; p = 0.001), reduced myocardial oxygen demand (11%; p = 0.02), and decreased the aortic pressure at the onset of systole (16%; p = 0.001). There was also a decrease in systemic vascular resistance (24%; p = 0.001), characteristic arterial impedance (21%; p = 0.002), and pulse wave reflection (20%; p = 0.006). Linear regression analysis showed that an increase in stroke volume was predicted only by the decrease in systemic vascular resistance (r = 0.81; p = 0.001). CONCLUSIONS: The reduction in systemic vascular resistance appeared to be the major mechanism by which IABC improved cardiac pumping efficiency. This effect may result from the passive distention of the peripheral vascular bed due to the propagation of the balloon-augmented diastolic pressure through the arterial system. PMID- 8619713 TI - Mitral valve remodeling: long-term results with posterior pericardial annuloplasty. AB - BACKGROUND: We studied the long-term results of a technique of mitral annuloplasty using autologous pericardium. METHODS: Between June 1989 and December 1994, 113 mitral valvuloplasties were performed for myxomatous degenerative disease. Repair of isolated anterior leaflet prolapse was performed in 26 patients (23%), posterior leaflet prolapse in 38 (33.6%), and prolapse of both leaflets in 49 (43.4%). Posterior pericardial annuloplasty was performed in all patients. In 20 patients, the pericardial graft was marked with metal clips for postoperative cinefluoroscopic assessment of annulus motion. RESULTS: The operative mortality rate was 2.7% (3/113). One patient died of myocardial infarction and 2 of low cardiac output syndrome. One patient required replacement of the mitral valve 2 days after operation because of dehiscence of the annular plication. Follow-up (average length, 32.41 +/- 20.09 months; range 1 to 71 months) was 97% complete and revealed good clinical and functional results: 95 patients (84.1%) were in New York Heart Association class I and had no regurgitation or only mild residual regurgitation. Postoperative transmitral flow indices were almost normal (mitral valve area = 3.7 +/- 0.4 cm2; peak flow velocity = 1.06 +/- 0.2 m/s). Only 3 patients had reoperation within 3 years (actuarial 5-year reoperation-free rate, 89.7%) and event-free survival at 5 years was 91%. In patients with metal clips marking autologous pericardium, planimetry of the area derived by fluoroscopic examination showed systolic narrowing of annulus size (8.5% +/- 6.4%; p < 0.01) and a slight systolic fall in the anteroposterior diameter of the annulus contour (5.9% +/- 3.8%; p < 0.01). CONCLUSIONS: Posterior pericardial annuloplasty seems to be a safe, effective and easily performed technique and a more physiologic correction that preserves mitral annulus motion. PMID- 8619714 TI - Intraoperative autotransfusion reduces blood loss after cardiopulmonary bypass. AB - BACKGROUND: A combination of several techniques is necessary to minimize the transfusion requirements for open heart operations. The benefit of plasmapheresis remains in doubt because of smaller and less effective platelets obtained with this technique. Therefore, we evaluated the effects of whole blood intraoperative autotransfusion as part of a blood conservation protocol. METHODS: One hundred patients undergoing coronary artery bypass graft operations were randomized to an autotransfusion group (group A) or control group (group C). Group A patients had a 10 mL/kg of whole blood removed before cardiopulmonary bypass; they had retransfusion at the termination of cardiopulmonary bypass and heparin reversal. Both groups had intraoperative cell saving and autotransfusion of shed mediastinal blood postoperatively. The indications for blood transfusion were standardized, and the physicians ordering blood products were blinded to the study. RESULTS: Compared with the control group, patients in the autotransfusion group had a 28% reduction of chest tube drainage at 8 hours and a 45% reduction in the total homologous blood units transfused. CONCLUSIONS: Autotransfusion during cardiopulmonary bypass provides benefit in addition to other techniques in reducing blood loss and the need for blood products in the postoperative period. PMID- 8619715 TI - Circulating endothelin in cardiac operations: influence of blood pressure and endotoxin. AB - BACKGROUND: Endothelin is involved in the control of cardiovascular and renal functions and acts as a neuromodulator. METHODS: In a prospective study among 15 male patients who underwent coronary artery bypass grafting, we investigated the release pattern and possible stimuli of circulating endothelin. RESULTS: We detected a steep increase in endothelin concentrations after the onset of cardiopulmonary bypass (CPB), and a second minor increase during CPB. The steep increase in endothelin concentrations correlated with the change in arterial pressures at the onset of CPB (r = -0.57; p < 0.03). The slow increase in endothelin concentrations during CPB, however, correlated with mean endotoxin levels during and after CPB (r = 0.60; p < 0.02). CONCLUSIONS: The change in arterial pressure at the onset of CPB seems to induce a steep and fast increase in circulating endothelin level, which is probably mediated through the baroreceptors. The slow increase in endothelin level during CPB is associated with increased circulating endotoxin concentration. It may be that either endothelin-mediated vasoconstriction induces endotoxin transmigration from the intestine or endotoxin stimulates secretion from endothelial cells. PMID- 8619716 TI - Skeletonized and pedicled internal thoracic artery grafts: effect on free flow during bypass. AB - BACKGROUND: The skeletonization technique of the internal thoracic artery (ITA) is used as a dissection technique for myocardial revascularization procedures. This study compared free flow between skeletonized ITA grafts and ITA pedicled grafts. METHODS: The ITA pedicled grafts were sprayed and wrapped in sponges soaked in dilute papaverine solution in 14 patients and prepared with intraluminal papaverine injection in 18 patients. For 23 other patients, the ITA was skeletonized. We measured the first free flow from the distal ITA early after the start of cardiopulmonary bypass and the second free flow just before the ITA was grafted to the left anterior descending artery. RESULTS: The first flow was greater in the skeletonized ITAs than in the ITA pedicled grafts with topical application of papaverine alone (38.9 +/- 15.8 versus 18.0 +/- 6.8 mL/min; p < 0.001). For the second flow, the pedicle grafts with intraluminal papaverine injection and the skeletonized ITAs showed greater flow rate than the pedicled grafts with topical application of papaverine (67.4 +/- 25.5 and 59.7 +/- 22.5 versus 38.1 +/- 13.1 mL/min; p < 0.005 and p < 0.05, respectively), but there was no significant difference between the former two groups (p = 0.53). CONCLUSIONS: Skeletonization of the ITA is as efficient a strategy to increase the flow as intraluminal papaverine injection for the ITA pedicled graft. When the ITA is harvested in a skeletonized fashion, arterial spasm and reduced early flow can be avoided, even without intraluminal injection of papaverine. PMID- 8619717 TI - Internal thoracic artery graft function during exercise assessed by transthoracic Doppler echography. AB - BACKGROUND: Noninvasive quantitative assessment of internal thoracic artery (ITA) graft function at rest and during exercise is important in patients who have undergone coronary artery bypass grafting. METHODS: Blood flow in the ITA graft was measured using transthoracic color Doppler echography before and after operation in 50 patients who underwent coronary artery grafting using an ITA to the left anterior descending artery. The patients were divided into three groups according to the degree of coronary stenosis and previous anterior myocardial infarction: Group 1 included 12 patients with severe (90% or more) coronary stenosis accompanied by anterior infarction. Group 2 included 26 patients with severe coronary stenosis without anterior infarction. Group 3 included 12 patients with moderate (75% or less) coronary stenosis without anterior infarction. Transthoracic echographic images of the ITA were obtained through the first intercostal space using a 7.5-MHz probe, and the diameter and cross sectional area of the ITA were measured on B-mode imaging. Systolic, diastolic, and mean blood flow velocity and volume were measured by the Doppler method. RESULTS: Internal thoracic artery diameter increased significantly from 2.2 mm to 2.4 mm after operation. The ITA flow patterns in both flow velocity and volume changed from systolic-dominant to diastolic-dominant after operation. Postoperative ITA graft flow was 82 +/- 24 mL/min, 53 +/- 30 mL/min, and 31 +/- 15 mL/min (p < 0.01, group 1 versus 3; p < 0.05, group 1 versus 2) and percent diastolic fraction of ITA flow was 72%, 68%, and 62% (not significant) in groups 1, 2, and 3, respectively. Compared with intraoperative ITA flow, which was measured using an ultrasound transit-time flowmeter, postoperative ITA graft flow was increased in group 1, but not changed in group 2 or 3. The ITA graft flow was measured before and after exercise in 19 patients and was compared with ITA flow in 10 normal control subjects. The ITA graft flow increased significantly with exercise in all patients in the three groups. Percent diastolic fraction of ITA flow increased significantly with exercise in patients with severe coronary stenosis (groups 1 and 2), but decreased significantly in patients with moderate stenosis (group 3). CONCLUSIONS: Changes in native coronary artery and ITA graft may be predicted by assessing ITA flow pattern during exercise. Transthoracic color Doppler echography is a clinically useful noninvasive method of assessing ITA graft function at rest and during exercise. PMID- 8619718 TI - Cardiopulmonary bypass in patients with heparin-induced thrombocytopenia using Org 10172. AB - BACKGROUND: In patients with heparin-induced thrombocytopenia undergoing cardiac operations, anticoagulation with heparin should be avoided. The low-molecular weight glycosaminoglycan Orgaran has been used as an alternative, but the overall experience is limited. METHODS: Two patients with heparin-induced thrombocytopenia underwent cardiopulmonary bypass using Orgaran for anticoagulation. A 30-year-old woman suffered from emboli to her brain through a secondary atrial septal defect, a 14-year-old boy from ischemia of his left leg due to recurrent embolism originating from the mitral valve. In both cases, cardiopulmonary bypass was performed in a routine manner, except for using low dose Orgaran instead of heparin. Anticoagulation was monitored during cardiopulmonary bypass by measuring Orgaran plasma levels and activated clotting time. RESULTS: No thromboembolic or bleeding complications occurred during and after atrial septal defect repair and mitral valve replacement, respectively. In the former case, thrombotic material from the inferior vena cava was removed during hypothermic circulatory arrest within the same procedure. Activated clotting time did not correlate with plasma levels of Orgaran. CONCLUSIONS: Orgaran might be a useful alternative for anticoagulation during extracorporeal circulation. Adequate dosages and measurement of plasma levels are recommended for its use in cardiopulmonary bypass. PMID- 8619719 TI - Is the preconditioning response conserved in senescent myocardium? AB - BACKGROUND: Senescent myocardium differs from adult myocardium at both functional and cellular levels. To adjudicate the efficacy of ischemic preconditioning as an alternative or adjuvant myoprotective strategy a reproducible, age-independent, intact laboratory model is necessary. METHODS: Adult (0.5 to 1.0 years) and senescent (5.7 to 8.0 years) sheep underwent 60 minutes of normothermic regional ischemia with 150 minutes of reperfusion. Group II (adult-ischemic preconditioning) and group IV (aged-ischemic preconditioning) underwent preconditioning with three 5-minute episodes of normothermic regional ischemia. Group I (adult-control) and group III (aged-control) were not preconditioned. RESULTS: Risk size and infarct size weights were delineated by monastryl blue pigment infusion and buffered tetrazolium solution. Ischemic preconditioning was evidenced by an infarct size reduction of 54% for adult sheep and 47% for senescent sheep (p < 0.01 versus age-matched controls; p = not significant for adult versus senescent). CONCLUSIONS: The data suggest that the cellular pathways involved with the preconditioning response are well preserved in senescent myocardium and support the utility of the ovine heart model to investigate the clinical relevance of ischemic preconditioning for the increasingly aged population presently undergoing cardiac operations. PMID- 8619720 TI - Cerebral oxygenation measured by near-infrared spectroscopy: comparison with jugular bulb oximetry. AB - BACKGROUND: Near-infrared spectroscopy is a potential tool for measuring adequacy of cerebral oxygenation during cardiac operations. The cerebral microcirculation is predominantly venous (by volume) and therefore regional cerebral oxygenation measured by near-infrared spectroscopy should reflect jugular bulb venous saturations. METHODS: We compared simultaneous regional cerebral oxygenation and jugular bulb venous saturation measurements in 40 children (median age, 4.5 years; range 2 weeks to 14.5 years) in the cardiac catheter laboratory (n = 29) and during cardiac operations (n = 11). RESULTS: For all patients combined the correlation between regional cerebral oxygenation and jugular bulb venous saturation was 0.69 (p < 0.0001) and was similar for the two groups. For individual children undergoing cardiac operations excellent correlations were obtained (r = 0.78 to 0.96; median, 0.91). However, at low values of jugular bulb venous saturation, regional cerebral oxygenation tended to run high, whereas the converse was true for high values of jugular bulb venous saturation. CONCLUSIONS: These findings suggest that near-infrared spectroscopy may be a useful tool for assessing intravascular cerebral oxygenation during pediatric cardiac operations. Prospective studies of neurologic outcome will be required to establish the value of this technique for assessing the adequacy of cerebral protection. PMID- 8619721 TI - Aortic aneurysms at the site of the repair of coarctation of the aorta: a review of 48 patients. AB - BACKGROUND: Study of the long-term results from 1 to 24 years after coarctation of the aorta repair in 891 patients showed that in 48 (5.4%, Mean age, 30.9 +/- 1.1 years) aneurysms had developed at the site of repair. Aneurysms arose in 43 (89.6%) of the patients in whom repair was done with help of synthetic patch aortoplasty, in 4 (8.3%) of the patients after coarctectomy with "end-to-end" anastomosis, and in 1 patient (2.1%) after coarctectomy with a prosthetic graft replacement. METHODS: Reoperation included aneurysm resection, which was performed in 30 patients (62.5%), followed by prosthetic graft replacement (n = 19), synthetic patch aortoplasty (n = 6), aneurysmorrhaphy (n = 3), or prosthetic bypass graft (n = 2). RESULTS: Four (13.8%) patients died after reoperation. All 18 patients who were not reoperated on died of a ruptured aortic aneurysm 7 to 15 years after repair of coarctation of the aorta. CONCLUSIONS: With the aim to prevent aneurysm development at the site of coarctation of the aorta repair, severe limitation of indications for synthetic patch aortoplasty is necessary. It can be used only in adult patients with a not too big narrowing. Patients after primary correction of coarctation of the aorta must avoid strenuous physical activity. Chest roentgenography should be performed in these patients each year, and each year they have to be seen by a cardiac surgeon. Suspicion of aneurysm development demands hospitalization, aortography, and reoperation. Preference is given to prosthetic graft replacement using an approach through the left fourth intercostal space with distal circulatory support by means of temporary bypass shunting. Infected aneurysms can be primarily bypassed through the right anterior thoracotomy with the creation of permanent bypass with the help of a vascular graft between the ascending and descending thoracic aorta. PMID- 8619722 TI - Transposition of the great arteries complicated by tricuspid valve incompetence. AB - BACKGROUND: Tricuspid valve insufficiency secondary to structural anomalies of the valve itself or to an iatrogenic complication of the Rashkind procedure is very rarely associated with transposition of the the great arteries. This condition represents an interesting perioperative challenge. Rapid restoration of the tricuspid valve to a low-pressure system by arterial switch operation associated with tricuspid repair should theoretically improve the outcome in terms of myocardial and valve function. METHODS: Thirteen of 839 patients who underwent an arterial switch operation for various forms of transposition of the great arteries presented with moderate to severe tricuspid insufficiency. Three of them had a ventricular septum defect. Nine experienced severe cardiac failure with profound hypoxemia. Ventilatory support was necessary in 7, 6 had renal or hepatic dysfunction, and 5 had coagulation disorders. Inotropic support was started preoperatively in 8 patients. RESULTS: Tricuspid lesions were as follows: primary annular dilatation and lack of coaptation at the commissural level (n = 1), straddling tricuspid valve (n = 1) redundant tricuspid valve tissue leading to left ventricular outflow tract obstruction (n = 1), small cleft of the septal leaflet (n = 1), and dysplastic valve tissue with juxtacommissural regurgitation (n = 1). In 8 patients, the cause of the tricuspid valve insufficiency was most probably an iatrogenic lesion, with rupture of the papillary muscle (n = 2), rupture of the chordae (n = 1), or tear of the anterior leaflet (n = 5), whereas no clear cause could be found in 1 patient. Repair consisted of the arterial switch operation associated with tricuspid valve repair in 10 patients. In 2 patients with only discrete anomaly and in 1 without a clear cause of tricuspid regurgitation, no valve repair was performed. Three patients had their ventricular septal defect closed. There were only one early and one late death, both not related to the tricuspid lesions. Late postoperative (mean, 6.5 years) evaluation revealed normal left ventricular function in 10, with no tricuspid incompetence in 7 and trivial tricuspid insufficiency in 3. CONCLUSIONS: Restoration of an incompetent tricuspid valve in a low-pressure system by the arterial switch operation combined with valve repair provides good ventricular and valvar results. Preoperative management and appropriate timing of operation seem to be of utmost importance. PMID- 8619723 TI - Selective lung ventilation during thoracoscopy: effects of insufflation on hemodynamics. AB - BACKGROUND: Positive-pressure insufflation during thoracoscopy has been advocated by some authors to facilitate exposure of the intrathoracic structures by expediting collapse of the lung. We hypothesized that insufflation during thoracoscopy may result in hemodynamic compromise despite selective lung ventilation. METHODS: After placement of invasive monitoring lines, six adult swine underwent selective lung ventilation and thoracoscopy. Baseline measurements of hemodynamic indices were taken before selective lung ventilation. The right lung then was collapsed; data were obtained at insufflation pressures up to 10 mm Hg and were compared with baseline values using Student's t test. RESULTS: Cardiac index, mean arterial pressure, and left ventricular stroke work index decreased, whereas pulmonary artery and central venous pressures increased (p < 0.05) at insufflation pressures of 5 mm Hg and greater. CONCLUSIONS: Positive-pressure insufflation during thoracoscopy resulted in significant hemodynamic compromise despite the use of selective lung ventilation. Conversion to thoracotomy may be an alternative if positive-pressure insufflation is necessary to perform the thoracoscopic procedure. PMID- 8619724 TI - Lidocaine reduces reperfusion injury and neutrophil migration in canine lung allografts. AB - BACKGROUND: Depletion of neutrophils (PMNs) and inhibition of PMN endothelial adhesion ameliorate post-ischemic lung reperfusion injury. Lidocaine reduces PMN adhesion to endothelial surfaces in vivo, and inhibits upregulation of PMN CD11b/CD18 (Mac-1) in vitro. We evaluated the effect of lidocaine on reperfusion injury, PMN adhesion, and PMN migration in preserved lung allografts. METHODS: Donor lungs were flushed with modified Euro-Collins solution (4 degrees C) after prostaglandin E1 administration (250 micrograms), inflated with 550 mL (inspired oxygen fraction = 1.0), and stored for 24 hours at 1 degree C. Left lung allotransplantation was performed in 13 mongrel dogs. Immediately after reperfusion the recipient right pulmonary artery and bronchus were ligated to permit assessment of allograft function during a 6-hour postreperfusion period. Allograft gas exchange (every 15 minutes) and hemodynamics (every 60 minutes) were assessed. Peripheral blood PMN CD11b expression was determined by flow cytometry. After sacrifice allograft bronchoalveolar lavage fluid PMN count and allograft tissue myeloperoxidase activity were measured. Two groups were studied: In group I (n = 8) lidocaine hydrochloride was added to the donor flush (20 mg/L) solution. In addition lidocaine was given to the recipient at the time of thoracotomy (intravenous bolus of 4 mg/kg) followed by a continuous infusion of 4 mg/kg/h during implantation and the assessment period. Three dogs that did not reach effective lidocaine blood levels at the time of reperfusion (3 to 4 micrograms/mL) were excluded from analysis. Group II animals (n = 5) received no lidocaine. RESULTS: Gas exchange in group I was superior throughout the assessment period (p < 0.05). Bronchoalveolar lavage fluid PMN count in group I was reduced (0.36 x 10(6)PMN/mL versus 6.2 x 10(6) PML/mL; p < 0.03). Group I allograft myeloperoxidase activity was 0.17 U/mg/min compared with 0.28 U/mg/min in group II (p < 0.01). In lidocaine-treated animals PMN CD11b expression was maintained at basal levels 2 hours after reperfusion, compared with group II, in which upregulation of CD11b was observed. Lower lobe wet/dry ratio was not different in the two groups. CONCLUSIONS: Our observations indicate that lidocaine reduces reperfusion injury and inhibits PMN adhesion and subsequent migration to the lung allograft. PMID- 8619725 TI - Inhalation of nitric oxide after lung transplantation. AB - BACKGROUND: Pulmonary hypertension is a postoperative complication that may adversely affect the outcome of lung transplantation. The effect of nitric oxide (NO) inhalation on pulmonary hemodynamic indices after lung transplantation was studied and compared with findings in control pigs. METHODS: Varying concentrations of NO were inhaled by 5 pigs after left lung transplantation and right pneumonectomy and by 5 controls after right pneumonectomy at an inspired oxygen fraction of 0.21 and 0.5. Hemodynamic data were recorded continuously, and fast circulatory courses were analyzed. RESULTS: Inhalation of NO reduced pulmonary vascular resistance and mean pulmonary arterial pressure in all pigs, but the decrease was pronounced and dose dependent only at an inspired oxygen fraction of 0.21 in the pigs that had transplantation. These were the only pigs that became hypoxic. With the termination of NO, there was a dose-independent rebound pulmonary vasoconstriction in the controls, especially at an inspired oxygen fraction of 0.21, but not in the pigs that had transplantation. This response was transient and could be blunted with a higher inspired oxygen fraction. CONCLUSION: Inhalation of NO reduced pulmonary vascular resistance in the transplanted lung and may be useful in the treatment of pulmonary hypertension after lung transplantation. The rebound pulmonary vasoconstriction with the termination of NO inhalation stresses the need to be aware of this effect and to wean NO carefully in clinical situations. This study showed oxygen dependency, which has to be taken into consideration in dose-response studies involving NO inhalation. PMID- 8619726 TI - ET-Kyoto solution for 48-hour canine lung preservation. AB - BACKGROUND: ET-Kyoto (ET-K) solution, proven safe for 20-hour lung preservation, was modified to achieve longer preservation: ET-K2 solution with more buffer capacity and ET-K3 with less potassium. METHODS: Lungs were preserved with one of the three solutions (with prostaglandin E1 at 4 degrees C for 48 hours (n = 5 for each). Left lung transplantation was performed and evaluated for 6 hours. RESULTS: Each solution became acidic after preservation (p < 0.01), though the change was lowest in the ET-K2 solution. All animals in the ET-K and ET-K3 groups survived for 6 hours after reperfusion, but only 1 survived in the ET-K2 group (p < 0.05). In all groups, partial pressure of oxygen in arterial blood decreased gradually after reperfusion. Pulmonary vascular resistance after reperfusion was significantly lower in the ET-K group than in the ET-K3 group (p < 0.01). Scanning electron microscopic examination showed that endothelial cell swelling and disruption were milder in the ET-K group (with the solution containing potassium of 44 mEq/L) than in the ET-K3 group. CONCLUSION: Lung preservation can be achieved for 48 hours in ET-K and ET-K3 solutions. Enhancement of buffer capacity provides no advantage. Potassium at 44 mEq/L does not cause deterioration of endothelial cells. PMID- 8619727 TI - Isolated lung perfusion with doxorubicin reduces cardiac and host toxicities associated with systemic administration. AB - BACKGROUND: For patients with malignant neoplasms metastatic to lung, systemic chemotherapy in doses high enough to achieve significant survival improvement is often limited by host toxicity. Isolated single-lung perfusion offers the advantage of delivering high-dose organ-specific chemotherapy while minimizing systemic toxicity. We compared the cardiac and systemic toxicities associated with intravenous administration versus isolated single-lung perfusion with doxorubicin. METHODS: Thirty-three male Fischer 344 rats weighing 275 to 300 g were randomized into three groups: normal control rats (n = 11), intravenous doxorubicin (7/mg/kg) (n = 11), and isolated left lung perfusion with 320 micrograms doxorubicin/mL (n = 11). Animals undergoing isolated single-lung perfusion were anesthetized with pentobarbital, intubated, and ventilated, and then had left thoracotomy with cannulation of the pulmonary artery and a pulmonary venotomy; pulmonary artery and vein were clamped proximally. Animals were perfused for 10 minutes at a rate of 0.5 mL/min, followed by a 5 minute rinse with buffered hespan solution. Arteriotomy and venotomy were repaired and circulation was restored. Daily weights were recorded. On day 24, cardiac output was determined in all groups by injection of radiolabeled chromium 51 microspheres. RESULTS: Animals treated with 7 mg/kg intravenous doxorubicin had a significant weight loss as compared with those treated with isolated lung perfusion (209.2 +/- 29.9 g versus 302.3 +/- 10.1 g; p < 0.01). Animals treated with isolated single-lung perfusion, after recovering from surgical stress, resumed normal growth pattern. Significant cardiac toxicities were seen in intravenously treated animals; cardiac index (27.4 +/- 6.9 versus 39.4 +/1 6.3 mL/min/100g body weight and heart weights (0.56 +/- 0.04 versus 0.88 +/- 0.09 g) were reduced in the intravenously treated group as compared with the group treated with isolated single-lung perfusion. In addition, severe hematologic toxicities are associated with intravenous doxorubicin administration. CONCLUSIONS: Intravenous administration of doxorubicin is associated with severe host toxicities, which include weight loss, decreased cardiac function, and hematologic toxicity. Isolated lung perfusion with high-dose doxorubicin is well tolerated and is associated with minimal host toxicity. PMID- 8619728 TI - Low-potassium solution for lung preservation in the setting of high-flow reperfusion. AB - BACKGROUND: We previously demonstrated that standard preservation using Euro Collins solution impairs lung function in the setting of high-flow reperfusion because of potassium-induced vasoconstriction. Preservation strategies for single lung transplantation are an important factor in patients with pulmonary hypertension. This study investigates the hypothesis that low-potassium preservation solution will improve function of lungs subjected to high-flow reperfusion. METHODS: Twenty-one New Zealand white rabbit lungs were harvested and studied on an isolated, blood-perfused model of lung function after 4 hours of cold ischemia at 4 degrees C. Control lungs were preserved with 50 mL/kg of cold saline solution flush (group I). Experimental lungs were preserved with low potassium solution (group II) or Euro-Collins solution (group III) at similar temperatures and volumes. RESULTS: The pulmonary arteriovenous oxygen gradient at the end of the 30-minute high-flow reperfusion period was significantly higher in group II compared with group III (121.3 +/- 19.2 mm Hg versus 31.1 +/- 4.2 mm Hg; p < 0.001). The pulmonary vascular resistance was significantly lower in group II than in group III (46.3 +/- 1.8 x 10(3) dynes x s x cm(-5) versus 79.8 +/- 8.4 x 10(3) dynes x s x cm(-5); p < 0.01. The percent decrease in dynamic airway compliance in group III was significantly greater than in group I and II (-51.0% +/- 13.3% versus -10.2% +/- 3.4% and -11.2% +/- 2.8%, respectively; p < 0.001). Similarly, the wet to dry ratio of the lungs in group III was significantly greater than in groups I and II (13.9 +/- 2.3 versus 5.9 +/- 0.2 and 6.0 +/- 0.4, respectively; p < 0.001). CONCLUSIONS: These data demonstrate that a low potassium preservation solution yields improved lung function after high-flow reperfusion in an ex vivo rabbit lung model. Lung preservation should be aimed at the clinical setting. PMID- 8619730 TI - Primary repair of complete sternal cleft with pectoralis major muscle flaps. AB - We report the successful coverage of a complete sternal cleft with exposed pericardium using bilateral pectoralis major muscle flaps in a newborn. The patient was transferred to our institution with nearly complete sternal aplasia, rectus diastasis, and only dessicated pericardium covering the visible heart. Complete closure without compromise of cardiac function was achieved at 2 days of age with bilateral pectoralis major muscle flaps and fasciocutaneous advancement flaps. PMID- 8619729 TI - Prospective analysis of pneumonectomy: risk factors for major morbidity and cardiac dysrhythmias. AB - BACKGROUND: Data were acquired prospectively on 136 consecutive patients undergoing pneumonectomy for cancer from 1988 to 1993, to define factors that increase the risk of major morbidity and postoperative cardiac dysrhythmias. METHODS: There were 81 patients (60%) with non-small cell lung cancer (standard pneumonectomy) and 55 patients (40%) with malignant pleural mesothelioma (extrapleural pneumonectomy). RESULTS: Four perioperative deaths occurred (3%) with no identifiable associated risk factors. Twenty-three patients (17%) had a major complication with an increase in the median length of stay from 7 to 11 days (p < 0.01). Age greater than 65 years, right-sided procedures, and dysrhythmias were associated with an increased risk of a major complication (p < 0.05). Thirty-two patients (24%) had supraventricular dysrhythmias, which occurred on postoperative days 1 to 2 (n = 8), 3 to 4 (n = 13), 5 to 6 (n = 6), and 7 to 12 (n = 5). The median length of stay increased from 8 to 11 days with dysrhythmias (p < 0.05). Factors associated with an increased risk of dysrhythmias included age greater than 65 years, intrapericardial or extrapleural pneumonectomy, right-sided procedure, and any major complication. CONCLUSIONS: Pneumonectomy can be performed safely in selected patients with cancer. Supraventricular dysrhythmia was the most common complication noted with a peak incidence at 3 to 4 days after resection. PMID- 8619731 TI - Successful repair of a massive coronary arteriovenous fistula in a 68-year-old man. AB - Successful ligation of a massive coronary arteriovenous fistula in a 68-year-old man is reported. The defect, which extended from the left coronary artery to the coronary sinus, had been diagnosed 17 years earlier. At the time of repair, the patient's age and fistula related complications made him a relatively high-risk operative candidate. In light of this case, operative approaches for repairing coronary arteriovenous fistulas are discussed. PMID- 8619732 TI - Origin of the left coronary artery from the right pulmonary artery. AB - We report the successful surgical treatment of a 12-year-old boy with a rare type of Bland-White-Garland syndrome with mitral regurgitation, in which an anomalous left coronary artery arose from the middle portion of the right pulmonary artery, employing the direct translocation of the left coronary artery and mitral valvuloplasty. PMID- 8619734 TI - Anterior pulmonary translocation without conduit for the repair of truncus arteriosus. AB - A technique with autologous tissue for the correction of type III truncus arteriosus is described. The truncal root was excised as a cylinder that incorporated pulmonary arteries and that was translocated anterior to the ascending aorta. The proximal section of the cylinder was closed and the pulmonary tract was reconstructed with anastomosis of a widely opened distal section to the right ventricle. Autologous pericardium was sutured to the entire surface of the pulmonary tract. PMID- 8619733 TI - Right ventricular obstruction in aortic dissection: a mechanism of hemodynamic collapse. AB - The main and right pulmonary arteries may be compressed by the false lumen of a type I aortic dissection. We report a 73-year-old women with a dissection of the aorta in whom echocardiographic examination revealed acute pulmonary arterial compression causing right ventricular failure and hemodynamic collapse. PMID- 8619735 TI - Traumatic tricuspid regurgitation with cyanosis: diagnosis by transesophageal echocardiography. AB - This case report demonstrates the utility of transesophageal echocardiography in the rapid diagnosis of cardiac injury from blunt thoracic trauma. Initial transesophageal echocardiography identified a flail tricuspid valve leaflet and regurgitation in a patient with jugular venous distention and hemodynamic instability. Progressive hypoxemia prompted repeat transesophageal echocardiography with contrast enhancement, which revealed opening of the foramen ovale and a right-to-left interatrial shunt. Operative repair of the lesion was lifesaving. PMID- 8619736 TI - Interference with anticoagulation monitoring by procainamide-induced lupus anticoagulant. AB - A patient scheduled for coronary revascularization was discovered to have elevated partial thromboplastin and activated clotting times. Preoperative testing revealed a lupus anticoagulant, probably secondary to long-term procainamide therapy. The resultant inability to use conventional anticoagulation monitoring for cardiopulmonary bypass was solved by direct measurement of heparin concentration. Operation and recovery were uneventful, and the patient was treated with long-term warfarin anticoagulation for this hypercoagulable state. PMID- 8619737 TI - Successful repair of a right ventricular rupture at the atrioventricular groove. AB - We report a patient who presented with a delayed spontaneous right ventricular rupture at the anterior atrioventricular groove after open heart operation. Successful surgical repair consisted of reestablishing anterior atrioventricular groove continuity by pericardial patch placement on the arrested heart. We discuss the risk factors that could initiate the primary tear and contribute to the extension of this type of right ventricular rupture. PMID- 8619738 TI - Aortopulmonary fenestration with pulmonary artery septation for single ventricle and subaortic obstruction. AB - A neonate with functional single ventricle and severe subaortic obstruction received a palliative procedure that involved a side-to-side anastomosis of the aorta and pulmonary artery, placement of a patch in the main pulmonary artery to divide the systemic and pulmonary circulations, and creation of a small opening in the patch to provide pulmonary blood flow. The patient recently underwent a bidirectional Glenn procedure at 10 months of age. This procedure obviates the need for a modified Blalock-Taussig shunt and may provide a more reliable source of blood to promote growth of both pulmonary arteries. PMID- 8619739 TI - Proficiency testing program for Lyme disease. PMID- 8619740 TI - Calcific bodies may mimic yeasts in histologic sections of pulmonary nodules. PMID- 8619741 TI - Lipid-laden alveolar macrophages as an indicator of aspiration pneumonia. PMID- 8619742 TI - On the need for more expertise in death investigation (and a National Office of Death Investigation Affairs?) PMID- 8619743 TI - Bedside glucose monitoring. Comparison of performance as studied by the College of American Pathologists Q-Probes program. AB - OBJECTIVE: To measure changes in practice characteristics and accuracy of bedside glucose monitoring between studies performed in 1991 and 1994. DESIGN: Participants in a 1991 and a 1994 bedside glucose monitoring study of the College of American Pathologists Q-Probes program were compared using data collected by a questionnaire for 15 quality variables and data from paired specimens for accuracy. Accuracy goals of bedside testing were those defined by the American Diabetes Association as within 10% of the laboratory value. Accuracy was estimated using a daily paired comparison of patient specimens for 30 days, one bedside glucose instrument, and individuals who commonly performed these measurements. SETTING: Institutions subscribing to the College of American Pathologists' voluntary quality improvement program, Q-Probes. PARTICIPANTS: In 1991 and 1994, 605 and 544 institutions collected data about performance characteristics, whereas 171 and 242 institutions participated in the accuracy components, respectively. MAIN OUTCOME MEASURE: Changes in accuracy between 1991 and 1994. RESULTS: When compared, improvement occurred for 12 of 15 quality variables. However, no significant changes were found for the percentage of values within stated accuracy goals. CONCLUSIONS: Although the process of bedside glucose testing improved between the 1991 and 1994 studies, the testing accuracy remained unchanged. PMID- 8619744 TI - Bedside glucose monitoring quality control practices. A College of American Pathologists Q-Probes study of program quality control documentation, program characteristics, and accuracy performance in 544 institutions. AB - OBJECTIVE: To investigate the adequacy of bedside glucose monitoring (BGM) quality control documentation and monitoring, characterize program structure and organization, and identify characteristics associated with the ability to produce accurate results. DESIGN AND SETTING: College of American Pathologists Q-Probes laboratory quality improvement study in 544 institutions. MAIN OUTCOME MEASURES: Percent compliance with quality control (QC) documentation, appropriate corrective action, and frequency of inappropriate patient testing, and the percentage of BGM results within +/-10% and +/-15% of a corresponding clinical laboratory glucose result. RESULTS: Five hundred forty-four institutions reviewed a total of 19543 individual QC paper documents from 2543 separate BGM instruments. Ninety percent of QC determinations that should have been performed and noted on these documents were recorded; of those performed, 2.8% of QC results were outside of the acceptable range. Thirty-two percent of the out-of range QC results had no record of corrective action. There were 527 reported instances of one or more patients being tested when there was no record of corrective action for out-of-range QC results. There were 20665 undocumented potential QC events, with 2053 instances of one or more patients being tested when there was no documentation. Two hundred forty-two institutions submitted 6653 paired BGM and clinical laboratory results for comparison. Approximately 56% of BGM results were within +/-10%, and 74% were within +/-15% of the corresponding clinical laboratory result. Factors associated with better accuracy performance are discussed. CONCLUSIONS: There is a need for improving compliance with QC documentation, improving appropriate corrective action follow-through, decreasing the frequency of inappropriate patient testing, and improving BGM accuracy performance. We provide recommendations for improvement. PMID- 8619745 TI - Quality assurance of autopsy permit form information, timeliness of performance, and issuance of preliminary report. A College of American Pathologists Q-Probes study of 5434 autopsies from 452 institutions. AB - OBJECTIVE: To develop a multi-institutional reference database of autopsy practices and performance for quality improvement purposes. DESIGN: In 1992, participants in the Q-Probes quality improvement program of the College of American Pathologists each prospectively evaluated consecutive autopsies performed over a 6-month period, up to a maximum of 20 autopsies per institution. SETTING: Hospital-based autopsies, excluding forensic cases and stillborn infants. PARTICIPANTS: Four hundred forty-nine North American institutions and three Australian laboratories. MAIN OUTCOME MEASURES: Completeness of information contained on autopsy permit forms, timeliness of autopsy performance between patients' deaths and autopsy prosections, and turnaround time of preliminary autopsy reports. RESULTS: In the aggregate database of 5434 autopsy cases, 7 of 11 selected data items were consistently present on autopsy permit forms in 80% of the participating institutions. The median percentage of autopsies in which permission was given for an unrestricted (complete) autopsy was 71%. The following median time intervals were obtained: time of the patient's death to time the autopsy permission was received, 5 hours, 23 minutes; time the autopsy permission was received to time the prosection was started, 3 hours, 30 minutes; and time of the patient's death to time the prosection was started, 14 hours, 52 minutes. Differences were observed in some time intervals when the participating institutions were grouped by reported demographic characteristics. Preliminary reports were completed in 2 days or less in 80.9% of the autopsies. CONCLUSIONS: Through this multi-institutional study, we have documented a consistent core of autopsy permit form information requested and a wide range of time intervals elapsed between the patients' deaths and autopsy performance. We have also established that the majority of participating institutions meet the College of American Pathologists' laboratory accreditation standard of providing a documented preliminary report of the gross pathologic diagnoses submitted to the attending physicians and institutional record within 2 working days following autopsy completion. PMID- 8619746 TI - The clinical usefulness of the preoperative bleeding time. AB - OBJECTIVES: To determine the clinical utilization of the Simplate bleeding time test as a preoperative screen, to examine the clinical utilization of the bleeding time test by multiple surgical services, and to correlate the indicators of bleeding risk (bleeding history, thrombocytopenia, prolonged prothrombin time/activated partial thromboplastin time, increased creatinine, and medications known to interfere with platelet function) with the bleeding time and the occurrence of clinically significant perioperative bleeding. DESIGN: Retrospective data analysis. SETTING: A large tertiary-care hospital. PATIENTS: One hundred sixty-seven consecutive surgical patients tested for preoperative bleeding time. MAIN OUTCOME MEASURES: The occurrence of clinically significant perioperative bleeding and the positive and negative predictive value of the preoperative screening bleeding time test. RESULTS: Patients with a positive bleeding history were more likely to have an abnormal bleeding time (P = .04), but there was no statistically significant association between patients with an abnormal bleeding time and the other indicators of bleeding risk examined or the occurrence of clinically significant perioperative bleeding. The positive predictive value of the preoperative bleeding time was 5%, and the negative predictive value was 95%. CONCLUSIONS: Screening for preoperative bleeding time is not a reliable test for assessing the risk of clinically significant perioperative bleeding and should not be used for this purpose. PMID- 8619747 TI - Utility of a rapid polymerase chain reaction panel for the detection of molecular changes in B-cell lymphoma. AB - OBJECTIVE: To evaluate the utility of a polymerase chain reaction (PCR)-based, B cell-related molecular panel in the diagnostic workup of hematologic specimens. DESIGN, SETTING, AND PATIENTS: A PCR panel was applied to 89 specimens from 87 patients, including 55 cases (57 specimens) of known monoclonal B-cell lymphoma, 10 cases of Hodgkin's disease, 2 cases of T-cell lymphoma, and 20 specimens of polyclonal reactive lymphoid tissues. The panel comprised a seminested PCR procedure for immunoglobulin heavy-chain (IgH) gene rearrangement and unnested PCR detection of t(14;18) and t(11;14). RESULTS: Immunoglobulin heavy-chain was detected in 63%, evidence of t(14;18) in 35%, and evidence of t(11;14) in 3.5% of all the B-cell lymphoma cases. Seventy-seven percent of all cases demonstrated IgH- and/or bcl-2-associated translocations using these primers. The IgH primers alone detected clonality in 82% (28/34) of the nonfollicular lymphomas and 35% (8/23) of the follicular lymphomas, with no false positives in the non-B-cell lymphoma specimens. The addition of two primer sets for the detection of both IgH and bcl-2/JH significantly improved the detection of molecular abnormalities in the follicular lymphoma group from 35% to 70% (16/23). However, no change was noted in the overall detection rate for the nonfollicular lymphoma group. Adding primers for bcl-1/JH did not change the overall detection rate for either group. CONCLUSIONS: Seminested PCR for IgH detected monoclonality in the majority of the nonfollicular lymphomas and is a valuable diagnostic tool in this group. The combination of different primer sets for the detection of IgH gene rearrangement and bcl-2/JH is most desirable for follicular lymphomas. PMID- 8619748 TI - Histologic, microbiologic, and clinical correlates of the diagnosis of sarcoidosis by transbronchial biopsy. AB - OBJECTIVE: To determine the frequency of positive microbiologic cultures in patients with epithelioid granulomas and negative histochemical stains for microorganisms in transbronchial biopsy specimens. Secondary objectives were to compare the histologic features of sarcoidosis with those of infectious granulomas and to assess the reliability of histology in establishing the diagnosis of sarcoidosis. DESIGN: Retrospective study. Specific histologic features of transbronchial biopsy specimens were correlated with clinical and microbiologic data, final diagnosis, and an estimate of the probability, on admission, that the patient had sarcoidosis. SETTING: A large, urban, tertiary care, university-affiliated hospital. PATIENTS: Ninety-two adult patients in whom epithelioid granulomas, negative for microorganisms on Ziehl-Neelsen and Gomori methemaine silver stain, were found in transbronchial biopsy specimens. Patients were identified through a search of surgical pathology files from 1975 to 1987. RESULTS: Ten patients (10.9%) had mycobacterial or fungal granulomas, while 82 had sarcoidosis. In all patients with a high clinical probability of sarcoidosis, the diagnosis was confirmed. Transbronchial biopsy specimens from patients with infectious granulomas had fewer granulomas (2.0 +/- 1.7 (SD) versus 7.1 +/- 6.6; P<.01), which involved a smaller proportion of lung tissue per case (9.5 +/- 10.0% versus 26.6 +/- 24.0%; P<.01). Sarcoid granulomas often exhibited Schaumann bodies (69.5% versus 10%; P<.01). Necrosis tended to predominate in infectious granulomas (19.5 versus 40%; not significant). CONCLUSIONS: Numerous granulomas, Schaumann bodies, and a high clinical probability of sarcoidosis are significantly associated with that diagnosis. Necrosis does not exclude sarcoidosis. Clinicopathologic assessment of transbronchial biopsy specimens is useful in predicting the final diagnosis of sarcoidosis but does not obviate the need for microbiologic cultures, which were positive in 10.9% of patients in this study. PMID- 8619749 TI - Ganglion cell-containing tumors of the pituitary gland. AB - The ganglion cell-containing tumors of the pituitary are rare lesions of undetermined histogenesis and nosology. A review of the literature revealed 42 such tumors, including the 3 cases described below. On the basis of this review, the tumors were divided into two histologic groups, one consisting of both adenomatous and gangliocytic elements (32 cases), and the other of the gangliocytic component only (10 cases). The first group of tumors were more common in females (23 of 32 cases) and were often active endocrinologically (28 of 32 cases), and acromegaly was the most common manifestation (19 cases). The second group was also more common in females (7 of 10 cases) but was less frequently active endocrinologically (3 of 19 cases). We review histologic and immunocytochemical findings in these tumors and discuss their histogenesis. We propose that the term mixed pituitary adenoma-gangliocytoma be used for the first group and gangliocytoma for the second. The two groups should be kept separate until their histogenesis is better understood. PMID- 8619750 TI - Expression of p53 in conjunctival melanocytic nevi. An immunohistochemical study. AB - The objective of this study was to evaluate conjunctival nevi for p53 gene mutations. We studied 11 conjunctival nevi by immunohistochemistry with the DO7 monoclonal p53 antibody as well as the cell proliferation marker, MIB1. Of the 11 cases, 2 were negative, 2 had less than 1%, and 7 had more than 2% p53 immunopositive nuclei with no direct correlation with MIB1 positivity. Our results suggest altered expression of p53 in conjunctival nevi. PMID- 8619751 TI - Kikuchi's histiocytic necrotizing lymphadenitis associated with ruptured silicone breast implant. AB - OBJECTIVE: In this report we explore the relationship between Kikuchi's necrotizing lymphadenitis (Kikuchi-Fujimoto disease, KD) and a leaky silicone breast implant. PATIENT: The simultaneous occurrence of KD and silicone lymphadenopathy in an axillary lymph node of a patient with a leaking silicone breast implant is reported. Since both KD and silicone implants have been implicated in autoimmune diseases, including systemic lupus erythematosus, serologic tests for antinuclear antibodies and rheumatoid factor were performed. RESULTS: Axillary lymph nodes showed both silicone lymphadenopathy, as well as classic morphologic and immunophenotypic features of KD. Screening tests for systemic autoimmune disorders were within normal range, suggesting that the unusual Kikuchi's-like immune reaction in one axillary lymph node was localized. The patient has no evidence of progressive immunologic disorders 3 years later. Subsequent lymph node biopsies showed silicone adenopathy with no evidence of KD. CONCLUSIONS: Our findings indicate that silicone compounds may be associated with transient abnormal immune reactions and lend further support to the hypothesis that KD represents an exuberant T-cell-mediated immune response to a variety of nonspecific stimuli. PMID- 8619752 TI - A case of composite Hodgkin's disease and chronic lymphocytic leukemia in bone marrow. Lack of Epstein-Barr virus. AB - We report Hodgkin's disease arising in a 68-year-old patient with a history of chronic lymphocytic leukemia for 8 years. The patient presented with a 4-month history of weakness, loss of appetite, and a 15-pound weight loss. A bone marrow biopsy showed two distinct histologic types of lymphoma: chronic lymphocytic leukemia and Hodgkin's disease. Immunohistochemical studies showed that chronic lymphocytic leukemia cells were composed of kappa-light chain-restricted monoclonal B cells. The Reed-Sternberg cells expressed CD15. Epstein-Barr virus RNA was not identified in either the Reed-Sternberg cells or cells of chronic lymphocytic leukemia by in situ hybridization. To our knowledge, this is the second reported case of composite Hodgkin's disease and chronic lymphocytic leukemia involving the bone marrow. PMID- 8619753 TI - Testicular extramedullary myeloid cell tumor in a patient with myelodysplastic syndrome. AB - We report herein a case of extramedullary myeloid tumor arising bilaterally in the testes of a 66-year-old man, who had previously been diagnosed with myelodysplastic syndrome. Light microscopy of the testicular neoplasm demonstrated a tumor composed of large, slightly polygonal cells with pale blue to weakly eosinophilic cytoplasm. The tumor cells were immunoreactive for CD45, myeloperoxidase, lysozyme, CD43, and MB2. Many of the cells also expressed chloroacetate esterase. Peripheral blood and bone marrow findings were consistent with chronic myelomonocytic leukemia (FAB-CMML), particularly in the most recent material, which showed clear cellular dysplasia and an increase in the percentage of blasts in the bone marrow (15% to 20% of all nucleated cells). This case of extramedullary myeloid tumor is unusual in view of the patient's age and the testicular location. It emphasizes the importance of including extramedullary myeloid tumor in the differential diagnosis of histologically undifferentiated large-cell tumors, as well as a need to use a broad panel of immunohistochemical stains in such cases. PMID- 8619754 TI - Multiple primary neoplasms. Ovarian carcinoid tumor, mucinous cystadenoma of low malignant potential tumor of left ovary, and adenocarcinoma of the colon. AB - We describe a case of primary left ovarian carcinoid tumor with metastases to the right paraovarian tissue, left fallopian tube, the right lung, omentum, cul-de sac, pericolonic fat, and, most likely, metastasis to the contralateral ovary, as well as a simultaneous left ovarian mucinous cystadenoma of low malignant potential and a well-differentiated colonic adenocarcinoma. Primary ovarian carcinoids are almost always unilateral. Metastases from such tumors to the lung and adrenal gland are very rare. To our knowledge, no such combination of all the above tumors has been reported. PMID- 8619755 TI - Papillary carcinoma of the thyroid with mucoepidermoid differentiation. AB - A case of papillary carcinoma of the thyroid with mucoepidermoid differentiation is reported. There have been different hypotheses of the histogenesis of this tumor, one of which attributes the origin of the tumor to the ultimobranchial body, mainly because of the presence of neuroendocrine markers. In our case, no neuroendocrine immunohistochemical markers were demonstrated, but a progressive transition between follicular cells and mucinous cells with gradual loss of thyroglobulin immunoreactivity and acquisition of polyclonal carcinoembryonic antigen reactivity was noted. Therefore, we propose that mucoepidermoid carcinoma may be a simple metaplastic transformation of a papillary carcinoma, because the thyroid glandular epithelium, which is of endodermal origin, is capable of differentiating easily into squamous, mucus-secreting, or even polypeptide secreting epithelium. PMID- 8619756 TI - Multiple cutaneous plexiform schwannomas. Report of a case and review of the literature with particular reference to the association with types 1 and 2 neurofibromatosis and schwannomatosis. AB - Plexiform schwannomas are relatively rare, benign peripheral nerve sheath tumors, which usually arise in either the dermis or subcutaneous tissue, although rare cases originate in skeletal muscle or other deep somatic soft tissue sites. These tumors may occur singly or as multiple lesions and may be localized to one anatomic site or diffusely distributed. Rare cases have been associated with "schwannomatosis" as well as type 1 neurofibromatosis (von Recklinghausen's disease). We report an unusual case of multiple cutaneous plexiform schwannomas associated with bilateral acoustic neuromas as well as other intracranial and intraspinal neoplasms. In addition, we examine the relationship between the various forms of cutaneous schwannoma, particularly the plexiform variant, and both types 1 and 2 neurofibromatosis; we also examine several purported cases of schwannomatosis. PMID- 8619757 TI - Lymphoplasmacytic aortitis and acute aortic dissection. An uncommon association. AB - A 43-year-old white man with a history of cigarette smoking, hypertension, nephrolithiasis, and cervical degenerative arthritis was hospitalized for sudden onset severe, substernal, and pleuritic chest pain with epigastric radiation. Despite evaluation, the cause remained unclear and the patient expired on hospital day 5. Autopsy revealed acute Stanford type A aortic dissection, hemopericardium, and hemothorax. Grossly, the aorta and its branches, including uninvolved medium-sized arteries, displayed extreme mural fragility. Microscopic examination showed a primary lymphoplasmacytic aortitis-periaortitis without giant cells. Rents within the tunica media, medial-adventitial inflammation, and elastic fiber disruption were limited to sites of gross aortic dissection. Muscular arteries showed patchy, chronic arteritis-periarteritis without giant cell infiltrate or aneurysm formation. This case documents an unusual association of primary lymphoplasmacytic aortitis and aortic dissection. PMID- 8619758 TI - Steroid cell tumor of the broad ligament arising in an accessory ovary. AB - OBJECTIVE: To determine the histogenesis of an unusual steroid cell tumor that occurred in the broad ligament. PATIENT AND METHODS: The tumor occurred in a 29 year-old woman who had evidence of virilization. The preoperative testosterone level was greater than 700 ng/dL, but it returned to normal after surgery. She was living and well without evidence of recurrent tumor 1 year after operation. The tumor was examined using light and electron microscopy and immunohistochemical stains. RESULTS: The tumor was composed of polygonal cells with prominent eosinophilic cytoplasm. No significant nuclear atypia or mitotic activity was identified; however, the tumor was large, and necrosis and hemorrhage were identified on gross and microscopic examination. A graafian follicle and a primordial follicle were present at the periphery of the tumor within the connective tissue stroma in one section, providing evidence for the presence of an accessory ovary. A separate normal ovary was present on the same side. The tumor mass was connected to the surface of the eutopic ovary by thin membranous tissue. CONCLUSION: To our knowledge, we report the first case in the modern literature of an extraovarian steroid cell tumor arising in the broad ligament, and we present evidence supporting its origin from an accessory ovary. PMID- 8619759 TI - Personal computer-based 3-dimensional ultrasound biomicroscopy of the anterior segment. AB - OBJECTIVE: To develop a practical, inexpensive system for 3-dimensional ultrasound biomicroscopic imaging of the anterior segment with a commercially available high-frequency ultrasound imager and a personal computer. METHODS: Sequential, high-frequency, ultrasound biomicroscopic images of the anterior segment were obtained with a motorized scanning control arm designed in our imaging laboratory. Images were acquired by a personal computer-based video capture device. Ultrasound slice data were then reconstructed as 3-dimensional volumetric images by a personal computer and commercially available software. RESULTS: Four 3-dimensional visualization formats were developed to enhance the clinical utility of high-frequency ultrasound. Rotational animation sequences were created that detailed the extent and anatomy of a filtering bleb, intraocular lens subluxation, focal angle closure from an iridociliary cyst, intraocular foreign bodies, and an iris tumor. CONCLUSIONS: Three-dimensional, high-frequency ultrasound of the anterior segment enhances our ability to visualize spatial relationships between adjacent anatomic structures. The low cost and ease of use of this system make widespread clinical application practical. PMID- 8619761 TI - The association of postoperative subjective visual function with acuity, glare, and contrast sensitivity in patients with early cataract. AB - BACKGROUND: It has been shown in our previous studies that early cataracts affect vision in ways that can be measured by objective means and that this objective impairment in visual acuity, glare, and contrast sensitivity can be successfully reversed by cataract surgery. OBJECTIVE: To evaluate the association of subjective visual function with objective measures of acuity, glare, and contrast sensitivity in patients who were symptomatic from early cataract. METHODS: We administered a task-oriented questionnaire prior to and 4 months after cataract surgery to patients who were symptomatic from early cataract (median preoperative ETDRS [Early Treatment Diabetic Retinopathy Study] visual acuity of 20/40 [range, 20/20 to 20/80]); ETDRS visual acuity, disability glare, and contrast sensitivity were also measured at those times. RESULTS: Uncomplicated cataract surgery resulted in resolution or improvement of subjective symptoms for the great majority of subjects, and in a few subjects new symptoms developed or current symptoms worsened. We found a positive association between postoperative improvement in subjective visual function (as measured by the questionnaire) and postoperative improvement in objective visual function (as measured by visual acuity and contrast sensitivity). We also found that the greater the degree of preoperative impairment in objective visual function (as measured by visual acuity and contrast sensitivity), the greater the postoperative improvement in subjective visual function (as measured by the questionnaire). No such association was found for our disability glare test. CONCLUSIONS: Cataract surgery for symptomatic individuals with mild impairment in visual acuity does relieve visual symptoms, and preoperative measurement of contrast sensitivity can help determine who with early cataract is most likely to report subjective improvement in vision. PMID- 8619760 TI - Triple vs nonsimultaneous procedures in Fuchs' dystrophy and cataract. AB - OBJECTIVES: To determine the outcome and refractive status after triple and nonsimultaneous procedures for Fuchs' endothelial dystrophy and cataract. DESIGN: Records of 236 patients with Fuchs' endothelial dystrophy who were examined during 1988 were reviewed retrospectively. SUBJECTS: Group 1 consisted of 93 patients who had triple procedures (penetrating keratoplasty, extracapsular cataract extraction, and posterior chamber intraocular lens implantation); group 2 consisted of 32 patients who had nonsimultaneous procedures (penetrating keratoplasty followed by extracapsular cataract extraction and posterior chamber intraocular lens implantation). Variables in the first eye that had surgery for each patient were compared between the groups by means of unpaired t tests and Fisher's exact test. RESULTS: Mean follow-up after undergoing transplantation was 6 years in group 1 and 8 years in group 2. Clear grafts were obtained in 89 (96%) of eyes of group 1 and in 29 (91%) of eyes in group 2 (P = .37). A best-corrected visual acuity of 20/40 or better was achieved in 60 (65%) of eyes in group 1 and in 21 (66%) of eyes in group 2. Refractive errors within 2 diopters of emmetropia were found in 37 (42%) of eyes in group 1 and in 15 (48%) of eyes in group 2 (P = .51). Mean refractive cylinder was 3.9 diopters in group 1 and 4.1 diopters in group 2 (P = .67). CONCLUSIONS: There was no statistically significant difference in the outcome and refractive status after triple and nonsimultaneous procedures. To avoid increased cost and delay in visual rehabilitation, we recommend a triple procedure for patients with Fuchs' endothelial dystrophy and visually significant cataracts. PMID- 8619762 TI - The stability of perfluoro-N-octane during vitreoretinal procedures. AB - OBJECTIVES: To examine the propensity for intraoperative procedures, such as endolaser, to generate polar impurities in perfluorocarbon liquids, either by degradation of the compound or by dissolution of materials contacting the liquid, given the value of these liquids as adjuncts to vitreoretinal procedures and the importance of using pure and inert liquid. METHODS: Perfluoro-N-octane liquid recovered from patients after vitreoretinal procedures was analyzed by gas chromatography, nuclear magnetic resonance, ultraviolet spectroscopy, and a cell proliferation assay. Similar analyses were performed on pure and impure perfluoro N-octane exposed in vitro to superclinical energy levels of argon and YAG laser, endodiathermy, and endoillumination. RESULTS: No change in chemical structure and only minor (parts per million) increases in dissolved contaminants were observed. The perfluoro-N-octane liquid retained its inertness as indicated by the inability of fibroblasts to attach and proliferate on its surface. CONCLUSION: The structure and biologic inactivity of perfluoro-N-octane are unaffected by vitreoretinal surgical manipulations. PMID- 8619763 TI - Risk factors for central retinal vein occlusion. The Eye Disease Case-Control Study Group. AB - OBJECTIVE: To identify possible risk factors for central retinal vein occlusion (CRVO). DESIGN: Between May 1, 1986, and December 31, 1990, 258 patients with CRVO and 1142 controls were identified at five clinical centers. Data were obtained through interviews, clinical examinations, and laboratory analyses of blood specimens. RESULTS: An increased risk of CRVO was found in persons with systemic hypertension, diabetes mellitus, and open-angle glaucoma. Risk of CRVO decreased with increasing levels of physical activity and increasing levels of alcohol consumption. In women, risk of occlusion decreased with use of postmenopausal estrogens and increased with higher erythrocyte sedimentation rates. Cardiovascular disease, electrocardiographic abnormalities, history of treatment of diabetes mellitus, higher blood glucose levels, lower albumin globulin ratios, and higher alpha-globulin levels were associated with increased risk only for ischemic CRVO. Systemic hypertension was associated with increased risk for ischemic and nonischemic CRVO, but odds ratios were greater for the ischemic type. CONCLUSIONS: Our results suggest a cardiovascular risk profile for persons with CRVO, in particular, patients with the ischemic type. The findings reinforce recommendations to diagnose and treat systemic hypertension, advise patients to increase physical exercise, and consider use of exogenous estrogens in postmenopausal women. PMID- 8619764 TI - The expanding clinical spectrum of unilateral acute idiopathic maculopathy. AB - OBJECTIVE: To report on new features of unilateral acute idiopathic maculopathy (UAIM). PATIENTS: We have evaluated an additional 17 patients with UAIM since 1991. This is a report of new features of the maculopathy noted in seven patients from this new series. RESULTS: New clinical findings in UAIM included eccentric macular lesions, subretinal exudation, papillitis, and bilaterality. The occurrence of UAIM in association with pregnancy and human immunodeficiency virus was also observed. CONCLUSIONS: The description of these newly reported features broadens our understanding of the nature of UAIM. With recognition of the expanded clinical spectrum of this disorder, a more confident approach to diagnosis and management may be achieved. PMID- 8619765 TI - Evaluation of patients with retinitis pigmentosa receiving electric stimulation, ozonated blood, and ocular surgery in Cuba. AB - OBJECTIVE: To evaluate the effect of intervention with electric stimulation, autotransfused ozonated blood, and ocular surgery, performed in Cuba, on the course of the common forms of retinitis pigmentosa. DESIGN: Ocular evaluations over 6 to 8 months before and after intervention in Cuba. SETTING: Evaluations performed at a US clinical research facility. PATIENTS: Ten adult patients aged 25 to 67 years with retinitis pigmentosa. MAIN OUTCOME MEASURES: Visual acuity, visual field area, and electroretinogram (ERG) amplitude. RESULTS: No significant change in visual acuity or visual field area was observed on average between preintervention and postintervention values over a 6- to 8-month interval. Mean 30-Hz cone ERG amplitude declined by 15.5% between preintervention and postintervention values (P = .006). When data on change in visual field area from 1 statistically significant outlier were excluded from the analysis, a significant decline of 12.9% in mean visual field area was observed (P = .025). CONCLUSIONS: These data support the conclusion that the intervention offered in Cuba provides no benefit to patients with retinitis pigmentosa as measured by visual acuity, visual field area, and ERG. The magnitudes of the mean declines observed in ERG amplitude and visual field area over a 6- to 8-month interval, relative to those reported in previous studies, raise the possibility that this intervention may worsen the course of the disease. PMID- 8619766 TI - Visual field loss following vitreous surgery. AB - OBJECTIVE: To assess possible causes of visual field loss following vitreous surgery. DESIGN: Charts of 8 patients prospectively identified, who developed visual field loss following vitreous surgery, were reviewed to characterize this newly recognized syndrome and assess possible causes. RESULTS: Two patients had preexisting chronic open-angle glaucoma and 1 had ocular hypertension. Indications for surgery included 4 eyes with macular holes, 1 eye with epiretinal membrane, 2 eyes with rhegmatogenous retinal detachment, and 1 eye with retinal detachment and giant retinal tear. All patients received retrobulbar anesthesia. Seven of 8 patients had fluid/gas exchange with installation of long-acting bubbles. In 1 patient with a macular hole, a small hemorrhage was noted along a vessel coming off the nerve superotemporally while attempting to engage the posterior cortical vitreous intraoperatively. This patient developed an inferior visual field defect. No intraocular pressure (IOP) measurements greater than 26 mm Hg were recorded in any eye perioperatively. Visual field defects included 4 eyes with inferotemporal defects, 2 eyes with inferior altitudinal defects, 1 eye with a cecocentral scotoma, and 1 eye with a superonasal defect. Only 1 patient had worsened visual acuity. A relative afferent pupillary defect was observed in 4 eyes and disc pallor in 5 eyes. CONCLUSIONS: Central or peripheral visual field loss can now be recognized as a possible complication of vitreous surgery. In some cases, a relative afferent pupillary defect and optic disc pallor are present, suggesting that the optic nerve is the site of injury. Possible mechanisms include ischemia due to elevated IOP or fluctuations in IOP, optic nerve damage from retrobulbar injection, direct intraoperative mechanical trauma to the optic nerve, indirect injury from vigorous suction near the optic nerve leading to shearing of peripapillary axons or vessels, or a combination of these. Certain optic nerves may be more susceptible to injury because of preexisting compromise from glaucoma or vascular disease. PMID- 8619767 TI - 24-hour blood pressure monitoring in patients with anterior ischemic optic neuropathy. AB - OBJECTIVE: To define parameters of ambulatory diurnal blood pressure in patients who had experienced anterior ischemic optic neuropathy (AION) in a case controlled study. PARTICIPANTS AND METHODS: Twenty-four patients with AION and 24 control subjects who were matched for age, gender, medical diagnoses, and medications underwent ambulatory automated blood pressure monitoring for 24 hours. RESULTS: The overall diurnal pattern of blood pressure appeared to be normal in all subjects, showing lower blood pressures at night than during the day, an overnight nadir, and an ascending blood pressure curve in the morning to reach daytime levels. Also, patients with AION did not differ from control subjects with respect to the nighttime diastolic nadir or daytime peak systolic blood pressure. However, during the daytime, patients with AION had lower mean systolic and diastolic blood pressures than did matched control subjects. The widest difference between their blood pressure curves occurred after awakening in the morning, when patients with AION had a less steep and more irregular rise of blood pressure. Patients who had signs of vertebrobasilar insufficiency in addition to AION had lower mean diastolic blood pressure during both daytime and nighttime and a lower minimum daytime diastolic blood pressure. CONCLUSIONS: On ambulatory measurements of diurnal blood pressure, patients with AION consistently had a lower mean blood pressure than did control subjects and a lag in the usual rise in blood pressure in the morning to meet increasing daytime demands for perfusion. Chronic hypoperfusion of small end-arterial vessels that supply the optic nerve head may predispose to AION, and may be caused by relative hypotension owing to overtreated hypertension or to abnormal vascular autoregulation. Internists should be asked to monitor blood pressure carefully when treating hypertensive patients who are at risk for AION, to avoid hypotension, especially on awakening in the morning. PMID- 8619768 TI - Effect of adhered bacteria on the binding of Acanthamoeba to hydrogel lenses. AB - OBJECTIVES: To determine the effect of Pseudomonas aeruginosa and Staphylococcus epidermidis on the binding of Acanthamoeba species to hydrogel lenses. METHODS: Cells of amebae and bacteria were incubated with different types of hydrogel lenses. Densities of amebae that were bound to the lenses after rinsing were determined from direct counts with a cell detachment procedure and from scintillation counts of cells, which were radiolabeled with tritiated leucine. RESULTS: With both methods, amebae showed significantly increased binding to hydrogel lenses with attached P aeruginosa. The numbers of amebae that were retained on lenses with attached S epidermidis were not significantly different from those that were retained on lenses without bacteria. The binding of amebae to unworn hydrogel lenses, in contrast to the irreversible adherence of P aeruginosa, was tenuous. CONCLUSIONS: The binding of Acanthamoeba species to unworn hydrogel lenses was tenuous and appeared to be related to water content, surface tensions, and ionic charge. The presence of adhered P aeruginosa on the hydrogel lenses facilitated the binding of Acanthamoeba species. The cocontamination of lens systems with bacteria (eg, P aeruginosa) may be a prime factor in the development of amebic keratitis. PMID- 8619769 TI - The effects of corticosteroids of adenoviral replication. AB - OBJECTIVE: To evaluate the effects of Pred Forte (prednisolone acetate; Allergan Pharmaceutical, Irvine, Calif) on the replication of different adenoviral serotypes in vitro and in the adenovirus type 5/New Zealand rabbit ocular model. METHODS: The 50% inhibitory doses of Pred Forte and its components were determined for common ocular serotypes. The effects of continuous topical treatment with Pred Forte for 18 days were evaluated (eg, conjunctivitis, subepithelial immune infiltrates, and serial ocular viral titers) in the adenovirus 5/New Zealand rabbit ocular model. RESULTS: Pred Forte and prednisolone acetate inhibited adenoviruses 1, 5, 8, and 19 in vitro. In vivo, 1% Pred Forte significantly reduced conjunctivitis (P = .04) and subepithelial infiltrates (P = .02), but enhanced viral replication (P = .01) on days 9 to 21 and increased the duration of viral shedding (P < .001). CONCLUSIONS: Despite demonstrated anti-inflammatory and anti-immune effects, prolonged treatment of acute adenoviral infections with topical Pred Forte is not recommended because of the enhanced risks of viral transmission and community epidemics. PMID- 8619770 TI - Aging effects on accommodation and outflow facility responses to pilocarpine in humans. AB - OBJECTIVE: To determine the relationships among age, outflow facility, and refractive and facility responses to pilocarpine in humans. METHODS: Refraction, intraocular pressure, and outflow facility were determined in 30 normal volunteers aged 20 to 75 years, by coincidence refractometry, applanation tonometry, and Schiotz tonography, respectively, before and 1 hour after a 30 microL drop of 2% or 6% pilocarpine. Simple regression of baseline facility, postpilocarpine facility, and facility change, on age and refractive change singly and jointly, was performed. Stepwise regression models and graphic conditioning plots were used to determine, for each facility variable, its relationship to age or refractive change specifically. RESULTS: Baseline outflow facility and maximum pilocarpine-induced refractive change (ie, accommodation) declined with age, but the decrease in intraocular pressure and the facility response to pilocarpine did not. After adjusting for age, for baseline facility, there was no further relationship to 6% pilocarpine-induced accommodation, and a slight residual relationship to 2% pilocarpine-induced accommodation. After adjusting for both 2% or 6% pilocarpine-induced accommodation, the relationship to age was still significant. The facility increase after 2% or 6% pilocarpine did not depend on age and/or accommodative amplitude. CONCLUSIONS: In humans, as previously described in rhesus monkeys, an age-related loss of ciliary muscle mobility may compromise the basal function of the trabecular meshwork. However, unlike monkeys, humans exhibit no loss of the intraocular pressure or outflow facility response to pilocarpine with age. PMID- 8619771 TI - Referral patterns of uveitis in a tertiary eye care center. AB - OBJECTIVE: To analyze the referral patterns and diagnosis of uveitis during the past decade in a large tertiary eye center. DESIGN: The records of 1237 patients with uveitis referred to the Immunology Service of the Massachusetts Eye and Ear Infirmary from 1982 to 1992 were classified and analyzed. Data regarding sex, race, nationality, referral site, ages at presentation and onset of uveitis, ocular involvement, clinical characteristics, ocular condition, and systemic disease associations were obtained. RESULTS: The mean age at onset of uveitis was 37.2 years; the male-to-female ratio was 1:1.4. Most patients were white (85.8%), born in the United States (83.1%), and referred from within New England (84.7%). Anterior uveitis was most common (51.6%), followed by posterior uveitis (19.4%), panuveitis (16.0%), and intermediate uveitis (13.0%). Chronic (58.3%), nongranulomatous (77.7%), and noninfectious (83.1%) were the most frequent types of uveitis. The most common entities included idiopathic (34.9%), seronegative spondyloarthropathies (10.4%), sarcoidosis (9.6%), juvenile rheumatoid arthritis (5.6%), systemic lupus erythematosus (4.8%), Behcet's disease (2.5%), and the acquired immunodeficiency syndrome (2.4%). CONCLUSION: The appearance of new uveitic entities, such as the acute retinal necrosis syndrome, multifocal choroiditis and panuveitis, birdshot retinochoroidopathy, and acquired immunodeficiency syndrome-related uveitis, and the reemergence of the classic infectious causes of uveitis, tuberculosis and syphilis, have changed the way we approach the diagnosis and management of posterior and panuveitis at the Massachusetts Eye and Ear Infirmary. PMID- 8619772 TI - Practice revenue and cost distribution for ophthalmology, 1988 to 1993. AB - As opthalmologists need to better manage their practices, information regarding distribution of practice costs becomes more relevant. In this study, we compare revenues and costs from published sources to determine changes over time and across surveys. We also evaluate the reliability and validity of these statistics. Data were obtained from the Health Care Financing Administration (for 1988), the American Medical Association (for 1988, 1990, 1992, and 1993), and the Medical Group Management Association (for 1988, 1990, 1992, and 1993) and were compared across years and surveys. We found large differences among the surveys in both dollar amounts and percentages of total revenue for some of the reported cost categories. Analysis of the data over time showed less of a decline in physician earnings than expected, although there were large increases in the category "other costs." We found considerable divergence among the statistical results. Opthalmologists, public policymakers, and managed care organizations must exercise great caution in interpreting such data and in applying their findings to individual ophthalmic practices and practitioners. PMID- 8619773 TI - Uveitis and the Tower of Babel. PMID- 8619774 TI - The Cuban experience. False hope for a cure for retinitis pigmentosa. PMID- 8619775 TI - Malignant melanoma of the optic nerve. AB - A 67-year-old man was diagnosed as having a melanocytoma of the optic disc in the left eye. Observation during a 5-year period showed no change in the lesion. At age 72 years, he had abrupt visual loss to no light perception in the affected left eye. Clinical examination disclosed little enlargement of the papillary tumor but ultrasonographic evidence of optic nerve infiltration. Precontrast magnetic resonance imaging studies disclosed a hyperintense infiltrative lesion in the enlarged left optic nerve. Enhancement features of the lesion excluded a hemorrhagic process. The eye was removed with a 22.5-mm segment of optic nerve. Histopathologic examination showed a large, necrotic, mixed-cell malignant melanoma confined to the optic nerve. No choroidal involvement or viable melanocytoma cells were documented. This case stresses that it may be difficult to differentiate a melanocytoma from a primary malignant melanoma of the optic nerve. PMID- 8619776 TI - Histology of fetal eyes with oculocutaneous albinism. AB - The diagnosis of tyrosinase-negative oculocutaneous albinism (OCA) was made in a 19-week-old fetus by skin biopsy. Because the parents had an 11-year-old son with tyrosinase-negative OCA, they requested that the fetus be aborted at the 20th week of gestation. A histological analysis of the eyes was performed. Throughout the retina, the ganglion cell layer was separated from the inner neuroblastic layer by the inner plexiform layer. However, the number of ganglion cells was decreased and the nerve fiber layer was immature. Bipolar and horizontal cells had begun to segregate into the inner nuclear layer. Rods and cones were identifiable in the posterior, but not peripheral, retina. Cones were more numerous in the center of the retina, and no rod-free area was identifiable. In addition, the ciliary body (epithelial folds, blood vessels in the mesodermal connective tissue core, and ciliary muscle) was less developed than in a normal fetus. Melanosomes in the retinal pigment epithelium only contained filaments without melanization and were therefore classified as stage I or II melanosomes. However, the ciliary epithelium also contained some stage III melanosomes with melanin adherent to the filaments. PMID- 8619777 TI - Six-year follow-up of an idiopathic retinal vasoproliferative tumor. PMID- 8619778 TI - Candida endophthalmitis in Job syndrome. PMID- 8619779 TI - Presumed orbital hemangioma associated with the blue rubber bleb nevus syndrome. PMID- 8619780 TI - Adjustable suture technique for levator recession. AB - OBJECTIVE: To determine whether an adjustable suture technique is clinically useful in levator recession surgery. DESIGN: Consecutive clinical series. SETTING: Inpatient hospital and ambulatory surgical center. PARTICIPANTS: Ten patients who were undergoing levator recession surgical procedures for correction of eyelid retraction constituted the group of subjects of this study. OUTCOME MEASURES: Outcome measures included margin-reflex distance and palpebral fissure measurements. RESULTS: Postoperative margin-reflex distance and palpebral fissure measurements were within 0.5 mm of the desired eyelid position in 10 or 14 procedures and within 1 mm of the desired position in 12 of 14 procedures. CONCLUSION: Adjustable sutures may be a useful adjunct in levator recession surgery. PMID- 8619781 TI - Ocular inflammation secondary to a punctal foreign body. PMID- 8619782 TI - Bacterial ulcer 3 days after excimer laser phototherapeutic keratectomy. PMID- 8619783 TI - Comparison of methods for detecting keratoconus using videokeratography. PMID- 8619784 TI - Efficacy of ofloxacin vs cefazolin and tobramycin in the therapy for bacterial keratitis. PMID- 8619785 TI - Management of overfiltering and leaking blebs with autologous blood injection. PMID- 8619786 TI - A catheter to deliver retrobulbar medication. PMID- 8619787 TI - Importance of increasing public awareness regarding glaucoma. PMID- 8619788 TI - Nitric oxide stimulates the phosphorylation of rap1b in human platelets and acts synergistically with iloprost. AB - Phosphorylation of rap 1b in human platelets correlates with both an upward shift of the protein on sodium dodecyl sulfate polyacrylamide gels and the translocation of the phosphorylated protein to the cytosolic fraction of platelets. We reported that this phenomenon occurs in platelets in response to agents that stimulate adenylate cyclase and thereby activate the cyclic AMP dependent protein kinase. We now have evidence that phosphorylation of rap1b in platelets is also induced by nitric oxide generating compounds through stimulation of guanylate cyclase and activation of the cyclic GMP-dependent protein kinase. We observed time-dependent phosphorylation of rap1b and dose dependent inhibition of collagen-stimulated aggregation in washed platelets incubated with S-nitroso serum albumin. In the presence of a combination of iloprost and 3-morpholinosydnonimine, when both PKA and PKG are activated, phosphorylation of rap1b increased synergistically to a level three times higher than the sum of their individual actions. PMID- 8619789 TI - Apoptosis in human hepatoma cell line induced by 4,5-didehydro geranylgeranoic acid (acyclic retinoid) via down-regulation of transforming growth factor-alpha. AB - Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression. PMID- 8619791 TI - Glu-416 of beta-galactosidase (Escherichia coli) is a Mg2+ ligand and beta galactosidases with substitutions for Glu-416 are inactivated, rather than activated, by MG2+. AB - Glu-416 of beta-galactosidase (E. coli) was replaced with Gln and Val using site directed mutagenesis. The substituted enzymes displayed a greatly decreased sensitivity to Mg2+. Equilibrium dialysis studies indicated that wild-type beta galactosidase bound Mg2+ tightly, whereas E416V-beta-galactosidase did not. In addition, the pH profile of E416V-beta-galactosidase was unaffected by the presence or absence of 1 mM Mg2+. Surprisingly, both substituted enzymes were inactivated, rather than activated, by Mg2+ but high amounts of Mg2+ were needed (1 mM). E416Q-beta-galactosidase was unstable when stored in the presence of Mg2+. The substituted enzymes displayed a dramatically lowered affinity for the synthetic substrate, ONPG, and for IPTG (a substrate analog inhibitor) in both the presence and the absence of Mg2+. PMID- 8619790 TI - Identification of 6H1 as a P2Y purinoceptor: P2Y5. AB - We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Y purinoceptor. This identification is based on first on the degree of sequence identity shared with recently cloned members of the P2Y receptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATP alpha S specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2 purinoceptors, with ATP being the most active (K (i)), 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP alpha, beta-methylene ATP, UTP, thus confirming 6H1 as a member of the growing family of P2Y purinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5. PMID- 8619792 TI - cDNA sequence analysis and expression of cardiotoxins from Taiwan Cobra. AB - The cDNAs encoding cardiotoxins I, III and N were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription polymerase chain reaction. A high degree of sequence homology was observed with the three cardiotoxins. The cardiotoxins were subcloned in the expression vector pET 20b(+) and transformed in BL 21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli and purified by reversed phase high performance liquid chromatography. The purified recombinant cardiotoxin III showed an immunoreactivity with anti-cardiotoxin III antibodies as revealed by immunoblot analysis. PMID- 8619793 TI - Inverse relation of autocrine motility factor receptor and E-cadherin expression following MDCK epithelial cell transformation. AB - Autocrine motility factor interacts with its cell surface receptor (AMF-R) to stimulate tumor cell motility. To study AMF-R expression following transformation of polarized epithelial MDCK cells, we have used the invasive Moloney sarcoma virus transformed MDCK (MSV-MDCK) cell population. Decreased E-cadherin expression of the transformed MSV-MDCK clones is associated with both increased cellular motility and increased AMF-R expression. Increased AMF-R expression is due to MSV transformation as differentially motile MSV-MDCK clones, which either retain low E-cadherin, exhibit equivalent high levels of AMF-R. Loss of the polarized epithelial phenotype and increased cellular motility following transformation of MDCK cells is thus associated with a shift from a high E cadherin/low AMF-R to a low E-cadherin/low AMF-R phenotype. PMID- 8619794 TI - Nitric oxide-donor compounds inhibit lipoxygenase activity. AB - The nitric oxide (N0-releasing agents sodium nitroprusside (SNP) and S-nitroso-N acetylpenicillamine (SNAP) inhibit dioxygenase activity of lipoxygenase in human platelets and human CHP100 neuroblastoma cells, leading the latter to necrosis. The effect of both NO-donors on the dioxygenase reaction was investigated by using soybean lipoxygenase type II (LOX-2) as a model for the mammalian enzyme. SNP and SNAP were competitive inhibitors of LOX-2, with inhibition constants of 525 microM and 710 microM, respectively. Both compounds inactivated LOX-2 by reducing the catalytic iron to the inactive Fe(II) form and counteracted the H2O2 mediated activation of the LOX-2 catalyzed dioxygenase reaction. Similarly, the co-oxidative and per-oxidative activities of LOX-2 were also inhibited by the NO releasing agents. These findings suggest that the biological role played by NO can be mediated, at least in part, by the inactivation of lipoxygenase, a key enzyme for the arachidonic acid metabolism in human cells. PMID- 8619795 TI - DNA-helicase activity from sea urchin mitochondria. AB - As a step toward the characterization of the main components of mitochondrial DNA replication apparatus in sea urchin, we report the identification of a DNA helicase activity in Paracentrotus lividus mitochondria. The activity was detected in a protein fraction obtained by fractionating on DEAE-Sephacel a lysate of gradient purified mitochondria from paracentrotus lividus eggs. The mitochondrial helicase unwound, in the presence of ATP and Mg++, a 39-base oligonucleotide annealed to single-stranded M13mp18 (+) DNA. Its direction of movement is 3' to 5' with respect to the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by the Escherichia coli rep helicase and by the helicase from bovine brain mitochondria. These features suggest that the sea urchin mitochondrial helicase could function in enabling the polymerization of the H-strand during mitochondrial DNA replication. PMID- 8619796 TI - Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system. AB - A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein. PMID- 8619797 TI - Human CFTR gene sequences in regions flanking exon 10: a simple repeat sequence polymorphism in intron 9. AB - A 2,908-bp segment of genomic DNA containing exon 10 and flanking intron regions of the human cystic fibrosis transmembrane conductance regulator gene was sequenced. A 30-bp sequence discrepancy and three missing nucleotides were detected when compared to a previously published 831-bp sequence. In the 30-bp region of sequence discrepancy, only a primer based on the new sequence information presented in this study gave products from polymerase chain reaction amplification of cellular DNA and a plasmid DNA encompassing the exon 10 region of CFTR. A 4-bp (TAAA) simple repeat sequence was also identified in intron 9 region. This repeat is dimorphic with nine (TAAA)9 or eleven (TAAA)11 copies on different chromosomes. Eleven repeats were exclusively associated with chromosomes carrying the delta508 mutation. Both 9 and 11 repeats were detected in non-delta508 chromosomes. PMID- 8619798 TI - Verification of the role of PCP 4-monooxygenase in chlorine elimination from pentachlorophenol by Flavobacterium sp. strain ATCC 39723. AB - The bacterial enzyme PCP 4-monooxygenase from Flavobacterium sp. strain ATCC 39723 catalyzes the oxygenolytic removal of the first chlorine from pentachlorophenol. PCP 4-monooxygenase is an FAD binding, NADPH requiring oxygenase, with similar functional domains as other bacterial flavoprotein monooxygenases specific for phenolic substrates. However, the definitive proof for the singular role of an oxygenolytic elimination of the primary chlorine from pentachlorophenol by Flavobacterium sp. has awaited the development of a genetic system whereby targeted mutagenesis via allelic exchange could be carried out with the corresponding gene from PCP 4-monooxygenase, pcpB. We report the development of a genetic system for Flavobacterium sp. strain ATCC 39723, and its application in targeted mutagenesis of the pcpB allele for elimination of PCP 4 monooxygenase activity. PMID- 8619799 TI - The mineralocorticoid hormone receptor and action in the eye. AB - Immunoblotting with a polyclonal antibody, directed against the mineralocorticoid receptor protein purified from rat kidney in presence of the receptor-specific ligand RU 26752, labeled a single 98-102 kDa band in soluble extracts from bovine retina and from cultured bovine retinal pigment epithelial cells, identical to the receptor in several other tissues from the rat. The antibody also immunoprecipitated the receptor-3H-RU 26752 complex in bovine retinal extract. The growth of the isolated pigment epithelial cells was inhibited by RU 26752 and ZK91587, two ligands specific to the mineralocorticoid receptor. Successive passages in culture led to the disappearance of immunoreactivity in Western blots, concurrently with the refractoriness of the cells to growth inhibition by the two antagonists. On sections of the human eye, mineralocorticoid receptor specific immunofluorescence was observed in retinal cone cells, pigment epithelium, epithelium of ciliary body, iris, cornea and lens. To our knowledge, this is the first ever demonstration of the mineralocorticoid receptor in ocular tissues. PMID- 8619800 TI - Effect of serine O-glycosylation on cis-trans proline isomerization. AB - In order to examine the ability of an O-glycosylated serine residue preceding proline to stabilize a cis amide conformation in a fashion similar to that observed with aromatic amino acid residues, we prepared a series of glycosylated analogs of a small linear peptide which we have previous reported to contain a cis conformation of an amide bond between tyrosine and proline. The glycopeptides were prepared by incorporating glycosylated N alpha- (fluoren-9-yl) methoxycarbonyl (Fmoc) amino acids into a standard solid phase peptide synthesis protocol. The peptides and glycopeptides were analyzed using 2-dimensional NMR spectroscopy. Unlike the case where the residue preceding proline was tyrosine, no signals corresponding to a cis proline conformation were detected in the spectra of the two glycopeptides containing serine O-glycosylated with either beta-linked N-acetyl glucosamine or alpha-linked N-acetyl galactosamine in the position preceding proline. PMID- 8619802 TI - Identification of the lysosomal membrane glycoprotein Lamp-1 as a receptor for type-1-fimbriated (mannose-specific) Escherichia coli. AB - The presence of several glycosylated sites with high-mannose oligosaccharides on the lysosome-associated membrane glycoproteins (Lamps)_ combined with the fact that neutrophil Lamps are present in mobilizable organelles inspired us to investigate their ability to bind type-1 fimbriated (mannose-binding) Escherichia coli and subsequently define a potential function for the Lamps in human neutrophils. Bacterial binding to Lamps purified from chronic myeloic leukemia cells was investigated by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transfer to a blotting membrane and overlay with type-1-fimbriated bacteria. The overlays were developed by growth. The bacteria bound readily to Lamp-1 while there was almost no binding to Lamp-2. Hence, we state that a possible function for neutrophil Lamp-1 is bacterial binding. PMID- 8619801 TI - Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C. AB - Exposing normal human breast epithelia (HBE) cells, which were growth arrested by a 3-day culture in EGF-deprived medium, to the microtubule disrupting agent, demecolcin (N-deacetyl-N-methyl-colchicine), for 2 hr significantly stimulated the initiation of DNA synthesis 22 hr later. The demecolcin-induced DNA synthesis was not accompanied by activation of the EGF receptor and it was inhibited by calphostin C, an inhibitor of protein kinase C (PKC), and U-73122, an inhibitor of phospholipase C (PLC). Contrary to this, the EGF-induced DNA synthesis was inhibited by tyrphostin A25, a specific inhibitor of the EGF receptor tyrosine kinase, and calphostin C. The results suggested that the involvement of PLC and PKC in the demecolcin-induced signal transduction pathway leads to the initiation of DNA synthesis. PMID- 8619803 TI - Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity. AB - PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27 30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95 110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity. PMID- 8619804 TI - cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog. AB - In Drosophila, it has been shown that protein kinase A and hedgehog have antagonistic actions during the formation of imaginal disks. In vertebrate skin, sonic hedgehog is expressed specifically in the feather bud epithelia. using an in vitro explant culture model we showed that dibutyryl cAMP, a protein kinase A (PKA) activator, suppresses the expression of Sonic hedgehog, (Shh) and continuous feather growth. The results suggest that Shh and PKA also have antagonistic action during vertebrate skin morphogenesis. PMID- 8619805 TI - Mixed dimers formed by crosslinking of native and glycated proteins in the absence of free sugar. AB - After a hyperglycaemic episode, glycated proteins remain in the body until removed by protein turnover. We have shown that in the absence of free sugar, such proteins can crosslink to native proteins, forming mixed dimers. They can also induce native proteins to crosslink into homodimers, presumably by release of a soluble crosslinking agent. Similar reactions in vivo could be responsible for the deposition of serum proteins in diabetic kidney, nerve and other tissues. Exposure to glycating sugar for brief periods, or a low concentration, still produced glycated protein capable of crosslinking to other proteins under sugar free conditions. These crosslinks are nonfluorescent, unlike the advanced glycation endproducts usually observed. PMID- 8619806 TI - The solution structure of a psoralen cross-linked DNA duplex by NMR and relaxation matrix refinement. AB - The three dimensional structure of the DNA oligomer d(5'GGGTACCC-3')2 cross linked with 4'-aminomethyl-4, 5', 8-trimethylpsoralen(AMT) has been determined by two-dimensional NMR and a relaxation matrix refinement method. NMR data and structural calculation establish that the cross-linking of the psoralen in the B DNA duplex retains Watson-Crick type hydrogen bonding throughout the duplex, although the thymine residue which is cross-linked with psoralen forms a weaker hydrogen bond. The psoralen cross-linking in DNA duplex induces significant change of the local DNA structure, but has no important effect on overall DNA helix. Our observation places an upper limit of 10 degrees on overall DNA bending by psoralen cross-linking, which is consistent with the result of HMT-octamer cross link [Spielmann, H. P., Dwyer, T. J., Sastry, S. S., Hearst, J. E., and Wemmer, D. E., (1965) Proc. Natl. Acad. Sci. USA 92, 2345-2349] and gel electrophoretic study [Haran, T. E. and Crothers, D. E. (1988) Biochemistry 27, 6967-6971]. PMID- 8619807 TI - Pretreatment with H2O2 decreases the Ca2+ sensitivity of the exocytosis of glutamate in cerebrocortical synaptosomes. AB - The treatment of cerebrocortical synaptosomes with low concentrations of H2O2 induces a long-lasting inhibition of the Ca2+ -dependent release of glutamate induced by KCl or ionomycin, without interfering with the cytosolic calcium and without damaging the synaptosomes (Zoccarato, F., Valente, M., and Alexandre, A. (1995) J. Neurochem. 64, 2552-2558). We report now that the inhibition exerted by H2O2 decreases (from 50 +/- 9% to 25 +/- 11%) if exocytosis is triggered by high (80 mM) rather than by low (30 mM) KCl. Similarly the inhibition decreases when glutamate release is triggered by high rather than by low ionomycin. The decreased inhibition by H2O2 on increasing KCl is accompanied by an increase of [Ca2+]i. We conclude that the treatment with H2O2 decreases the CA2+ sensitivity of the synaptosomal exocytotic apparatus. PMID- 8619808 TI - NMR-based, molecular dynamics- and random walk molecular mechanics-supported study of conformational aspects of a carbohydrate ligand (Gal beta 1-2Gal beta 1 R) for an animal galectin in the free and in the bound state. AB - The binding of a carbohydrate to a lectin may affect the conformation of the ligand. To address this question for the galectin from chicken liver, the conformation of Gal beta 1-2Gal beta 1-R was analyzed in the free and in the galectin-bound state with 2D-ROESY- and 1D- as well as 2D-transferred NOE experiments. A computer-assisted analysis of spatial parameters of the ligand by molecular dynamics (MD) and random walk molecular mechanics (RAMM) calculations, taking different dielectric constraints from epsilon = 1 to epsilon = 80 and various force fields into account, were instrumental to define the energetic minima of the free state. NMR-derived interresidual distance constraints enabled a conformational mapping. The two overlapping interresidual distance constraints obtained from transferred-NOE experiments of the galectin-ligand complex clearly support the notion that the conformation of the disaccharide in the bound state is at least very close to its global energy minimum state in solution. PMID- 8619809 TI - Low M(r) phosphotyrosine protein phosphatase interacts with the PDGF receptor directly via its catalytic site. AB - Many proteins bind to the activated platelet derived growth factor receptor (PDGF R) either directly or by means of adapter molecules. Up to now all these proteins were shown to transmit and amplify the signal started with PDGF-R stimulation. In a recent study our group had demonstrated that low M(r) phosphotyrosine protein phosphatase (LMW-PTP) specifically interacts with PDGF-R in NIH3T3 cells. In the present study we have attempted to clarify the modality of interaction, both in vivo and in vitro, of these two proteins, using a catalytically inactive LMW-PTP mutant. Our results indicate that LMW-PTP and PDGF-R interact directly, without the necessity of any adapter protein. This interaction leads to PDGF-R dephosphorylation and, presumably, interrupts one or more of the mitogenic pathways that originate from receptor activation. PMID- 8619810 TI - Reversible red-ox reactions of the diiron site in the mouse ribonucleotide reductase R2 protein. AB - The red-ox reactions of the dinuclear iron center of mouse R2 protein upon interaction with different reductants (dithionite alone and with mediators) and oxidants (PES, methylene blue, hydrogen peroxide and para-benzoquinone) have been studied by EPR and optical spectroscopy. The obtained results indicate that the transitions between Fe(III)Fe(III), Fe(II)Fe(III) and Fe(II)Fe(II) states of the dinuclear iron center are reversible and the mu-oxo-bridge may be formed upon oxidation by non-oxygen oxidants. In contrast to the case for the E. coli R2 protein, dithionite alone reduces the tyrosyl radical and diiron center in mouse R2 protein. PMID- 8619811 TI - The sigma receptor ligand, reduced haloperidol, induces apoptosis and increases intracellular-free calcium levels [Ca2+]i in colon and mammary adenocarcinoma cells. AB - The sigma receptor ligand reduced haloperidol (50 and 100 microM), potently inhibited cell proliferation, and induced apoptosis in WIDr colon and MCF-7 adenocarcinoma cell lines. Apoptosis was confirmed after drug treatment of the cells by the presence of nuclear fragmentation after staining of the cells with Hoechst 33258 and cellular DNA fragmentation ELISA and by condensation of the heterochromatin using transmission electron microscopy. However, internucleosomal DNA cleavage was not detected using gel electrophoresis. Reduced haloperidol (100 microM) increased the intracellular free calcium levels [Ca2+]i in both cell lines, which was independent of extracellular calcium, suggesting that the rise in [Ca2+]i was from intracellular stores and that an increase in [Ca2+]i may act as a "trigger" for apoptosis in these cell lines. PMID- 8619812 TI - Completion of the amino acid sequence of the C-terminal half of the porcine estradiol receptor by Edman degradation: reconfirmation of the absence of O linked sugars and phosphates. AB - The peptide A569-Y582 of the porcine estradiol receptor containing the missing sequence T570-M581 (1) was isolated and sequenced. The 4 seryl- and 2 threonyl PTH amino acids were recovered in normal yields, excluding their posttranslational modification and reconfirming the absence of O-glycosylation and O-phosphorylation in H267-I595. PMID- 8619813 TI - Cloning and characterization of a ribosomal P protein from Taenia solium, the aetiological agent of human cysticercosis. AB - A cDNA encoding the complete open reading frame of the Taenia solium acidic ribosomal phosphoprotein P2 has been cloned. The 417 bp cDNA (arpP2) was isolated from a T. solium cDNA expression library by PCR using P protein specific pimers. The ORF encodes a protein of 121 amino acids exhibiting 40-55% identity to mammalian, fungal, insect, and parasite P2 proteins. The inferred molecular mass of the protein is 12,592 Daltons, similar to that reported for other ribosomal P2 proteins. After subcloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and was shown to have a molecular mass of 15 kDa which is equivalent to the expected size of the full length recombinant fusion protein, comprising the 12.5 kDa arpP2 plus an additional 2 kDa for the N-terminal fusion peptide incorporating the six histidine residues required for purification. PMID- 8619815 TI - Glycated Cu,Zn-superoxide dismutase in rat lenses: evidence for the presence of fragmentation in vivo. AB - Cu,Zn-superoxide dismutase (Cu,Zn-SOD) exists in tissues of rats as both glycated and non-glycated forms when separated by boronate acid column chromatography. Glycated Cu.Zn-SOD is most abundant in rat lenses compared to other tissues. In normal rats lens levels of glycated Cu.Zn-SOD showed a gradual increase with age, whereas in diabetic rats substantial increases were observed. Immunoblotting analyses, using anti-hexitol lysine IgG, indicated that glycated Cu.Zn-SOD contains Amadori products. Moreover, Cu.Zn-SOD in lenses was site-specifically fragmented probably because of glycation. This the first report of a fragmented protein, such as Cu,Zn-SOD, occurring in vivo. PMID- 8619814 TI - C-terminal fragments of alpha- and beta-tubulin form amyloid fibrils in vitro and associate with amyloid deposits of familial cerebral amyloid angiopathy, British type. AB - Familial amyloidosis, British type, is an autosomal dominant disease characterized by progressive dementia, spastic paralysis and ataxia. The identity of the accumulating amyloid is not known, thus preventing the definitive classification of the disease. Biochemical methods were used to characterize the nature of the amyloid deposits from the brain tissue of one individual who died with this disease. The purified tissue material was subjected to trypsin digestion and subsequent N-terminal sequence analysis. Major tryptic fragments yielded the sequences VGINYQPPTVVPGGDLAK, FDLMYAK, GLTVPEL and GYLTVAAVFR, which are all tryptic fragments of the C-termini of human tubulin subunits alpha and beta. Synthetic peptides based on the sequences of these fragments formed amyloid fibrils in vitro fitting the characteristic definition of amyloid. These findings suggest that the C-terminal fragments of both alpha- and beta-tubulin are closely associated to the amyloid deposits of familial amyloidosis, British type. PMID- 8619816 TI - Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis. AB - A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome. PMID- 8619818 TI - Enhanced expression of beta-adrenergic receptor kinase 1 in the hearts of cardiomyopathic Syrian hamsters, BIO53.58. AB - We cloned an entire encoding sequence of beta-adrenergic receptor kinase 1 (beta ARK1) cDNA from the hearts of Syrian hamsters through reverse transcription and subsequent polymerase chain reaction. The cloned cDNA contained 2067 nucleotides coding 689 amino acids. The sequence had 95% homology to rat beta ARK1 and 90% homology to human homologue. Cardiomyopathic Syrian hamster, BIO53.58, has been used as a model animal of congestive heart failure. M-mode echocardiography confirmed that left ventricular contractility of 20-week-old BIO53.58 was markedly reduced. The expression of beta ARK1 mRNA in the hearts of BIO53.58 was significantly increased compared to control hamsters, F1b, suggesting that the enhanced beta ARK1 expression is acting as a negative feedback mechanism in order to maintain intracellular homeostasis against accelerated stimulation by catecholamines via phosphorylation of beta-adrenergic receptor. PMID- 8619817 TI - The expression of truncated MK in human tumors. AB - Midkine (MK) is a heparin binding growth/differentiation factor different from fibroblast growth factors (FGFs), and is largely composed of two domains which are found by a folded polypeptide chain interconnected by disulfide bridges. Polymerase chain reaction revealed the presence of a short MK mRNA in 7 among 12 human tumor cells which expressed MK mRNA. All of 4 pancreatic carcinoma cell lines expressed the short species in addition to the full size mRNA. The short mRNA lacked an exon and resulted in the lack of a more N-terminally located domain. The truncated form of MK was found also in some surgically removed specimens of human tumors, but not in noncancerous tissue. PMID- 8619819 TI - VLA-5-mediated interaction with fibronectin induces cytokine production by human chondrocytes. AB - Adhesion molecules of the integrin family, including very late activation antigens (VLA), have been implicated in various cellular functions. In this study, we investigated the contribution of integrin-mediated interaction with ECM proteins to the cytokine gene expression in human chondrocytes. Human articular chondrocytes expressed VLA-1, -2, -3 and -5 on the cell surface, and could adhere to various ECM proteins, especially to fibronectin (FN). Furthermore, the production of GM-CSF and IL-6 was potently induced by culturing chondrocytes on immobilized FN. This stimulative effect of FN was completely inhibited by an anti integrin alpha 5 chain mAb, as well as by anti-integrin beta 1 chain mAbs. These results indicate an important role of the VLA-5-mediated interaction with FN in regulating inflammatory cytokine production by human articular chondrocytes. PMID- 8619820 TI - Nested-polymerase chain reaction for the detection of Helicobacter pylori infection with novel primers designed by sequence analysis of urease A gene in clinically isolated bacterial strains. AB - We have established a highly sensitive semi-nested PCR assay for the detection of H. pylori infection using gastric juice samples, which can be aspirated with disposable nasogastric tubes. The primers targeting H. pylori urease A gene were designed based on the sequence conservation analysis of sixteen H. pylori strains isolated from Japanese patients. The efficacy of the PCR assay, designated as the URA-PCR, was confirmed by in vitro and in vivo assessments. Its sensitivity was 97.5% in the gastric juice samples aspirated from forty patients with proven H. pylori infection, and was significantly higher than that obtained with previously described PCR assays. PMID- 8619821 TI - A model molecule of the hydrogen-bonded chain in the active site of bacteriorhodopsin. AB - We synthesized a 2-N-methylaminoethyl-tetramethylquanidine amide of Kemp's triacid (CP2). Furthermore, we added to CP2 tetrabutylammonium 4-tert butylphenolate. The pKa value of 4-tert-butylphenol is comparable to that of tyrosine. We studied by FT-IR spectroscopy CP2 and the complex of CP2 with 4-tert butylphenolate. This complex is a model for the hydrogen-bonded chain in the active centre of the bacteriorhodopsin molecule. An intense continuum in the FT IR spectra demonstrates that this hydrogen-bonded chain shows large proton polarizability due to collective proton motion. As earlier demonstrated also Schiff base carboxylic acid bonds show large proton polarizability. Thus, in all these hydrogen bonds protons can easily be shifted by collective proton motion due to changes of the local electrical fields and by changes of specific interactions arising from conformational changes. PMID- 8619822 TI - Isolation of a genomic DNA fragment having negative vitamin D response element. AB - The vitamin D receptor (VDR) binds to the vitamin D response element (VDRE) in the promoter region of target genes and acts as a ligand-dependent transcriptional regulator. In order to identify novel VDREs and new genes that are regulated by the active form of vitamin D [1,25-(OH)2D3], rat genomic DNA fragments bound by VDR were isolated. One of these fragments, designated as VBF5 was transcribed and the transcript was down-regulated by 1,25-(OH)2D3. These data strongly indicate that VBF5 may contain a VDRE regulating negatively an unidentified gene expression. PMID- 8619823 TI - Partial amino acid sequence of a novel 40-kDa human aortic protein, with vitronectin-like, fibrinogen-like, and calcium binding domains: aortic aneurysm associated protein-40 (AAAP-40) [human MAGP-3, proposed]. AB - A microfibrillar protein (40 kDa) purified from the adventitia of the human abdominal aorta is immunoreactive with IgG harvested from the wall of abdominal aortic aneurysms. We have partially sequenced this protein and found that it has fibrinogen alpha-, beta-, and gamma-like domains, a vitronectin-like domain, and a possible site for binding calcium. Because of homologies with other microfibril associated glycoproteins and because it is the third member of the family to be characterized in man, we suggest the name MAGP-3. PMID- 8619824 TI - Constitutive expression of hepatocyte growth factor may maintain the sheet construction of gastric epithelial cells through facilitating actin-myosin contractile system. AB - Previously, we have demonstrated that hepatocyte growth factor (HGF) plays an important role in the repair process of gastric ulcer, showing that HGF expression is specifically increased at gastric ulcer edge. In the present study, we demonstrated the constitutive expression of HGF mRNA in normal mucosa, which is as much as 0.1-1.0 attomole/micrograms total RNA. In order to evaluate the hypothesis that HGF might have some role in maintenance of gastric mucosa or prevention of injury initiation, we developed an in vitro model using rabbit gastric epithelial cell in primary culture. 1% ethanol destroys the cell to cell contact to disrupt the monolayer sheet of the cells without causing any damage to cell viability, indicating that irritants may initiate the mucosal injury. HGF remarkably prevented the disruption induced by the ethanol without eliciting proliferation or migration. This action of HGF was suppressed by actin selective inhibitor, cytochalasin B, indicating that it was mediated by an actin-myosin contractile system. In conclusion, constitutively expressed HGF may prevent the initiation of gastric epithelial disruption, which is dependent on some sort if mobile action of the cells. PMID- 8619826 TI - Conversion of acetylcholinesterase hydrophilic tetramers into amphiphilic dimers and monomers. AB - Exposure of purified hydrophilic tetramers of acetylcholinesterase (AChE) from fetal bovine serum to various guanidinium chloride (Gdn) concentrations led to inactive tetramers (2 M Gdn) and dimers (6 M Gdn). The native tetramers were almost fully monomerized by reduction, a minor fraction of the released monomers remaining active. Sedimentation analysis and hydrophobic chromatography showed that the modified tetramers, dimers and monomers had amphiphilic properties. Intrinsic fluorescence spectra and binding of the amphiphilic probe, 1-anilino-8 naphthalene sulfonate (ANS), revealed that AchE subunit in the modified tetramers were in a 'molten globule' structure, the dimers in a denatured stated, and the inactive monomers in a 'native-like' structure. These data show that AChE subunits possess a flexible conformation, which may be important for generating a full set of molecular forms. In addition, the behavior of the active monomers with amphiphiles may explain the interactions of type II AChE forms with membranes. PMID- 8619825 TI - Identification of a promoter region in the rat prion protein gene. AB - We have demonstrated the presence of a rat prion protein (RaPrP) gene promoter upstream of multiple initiation sites. A 0.1-kb fragment upstream of the 5' untranslated region contains specific DNA motifs characteristic of promoter elements including an AP-1 binding site, an inverted CCAAT motif and three inverted Sp-1 binding sites. This fragment directs transcription of a luciferase reporter gene in pheochromocytoma cells (PC12) and rat glioma cells (C6), suggesting that it contains the promoter for the RaPrP gene. To more precisely localize the transcription regulatory elements in this region, a series of 5' deletion mutants were generated. Deletion analysis showed that an inverted CCAAt and adjoining Sp-1 binding sequences may play an important role in transcription of the RaPrP gene. PMID- 8619827 TI - Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rat. AB - Adrenomedullin (AM) is a vasorelaxant peptide that was recently isolated from human pheochromocytoma. In contrast to human (h) AM, which has vasodepressor activity, a synthetic N-terminal fragment of hAM, hAM-(1-25)-NH2 showed vasopressor activity in the anesthetized rat. The N-terminal peptides hAM-1-31) NH2, hAM-(1-25)-OH, hAM-(1-21)-NH2, acetyl-hAM-(16-21)-NH2, and acetyl-hAM-(16 36)-OH all showed vasopressor activities. The potency of hAM-(1-21)-NH2, acetyl hAM-(16-21)-NH2 was greater than that of hAM-(1-25)-NH2. Pretreatment with phenoxybenzamine, guanethidine, or reserpine attenuated vasopressor activities of these peptides. These data suggested that vasopressor activity of N-terminal fragment of hAM is due to a stimulation of endogenous catecholamine release. PMID- 8619828 TI - High-yield synthesis of N-acetyllactosamine by regioselective transglycosylation. AB - The synthesis of N-acetyllactosamine (D-Galp beta 1-4D-GlcpNAc) with very low contamination of its isomer N-acetyllactosamine (D-Galp beta 1-6D-GlcpNAc) was obtained by use of regioselective transglycosylation activity of beta galactosidase from Bacillus circulans using lactose as the donor of D-Galp and D GlcpNAc as the acceptor. The reaction was conducted at 15 degrees C and at pH 5.0. The incubation time was considerably reduced and the yield improved 100% with respect to the best results so far described in the literature. PMID- 8619829 TI - The expression and characterization of five recombinant murine alpha 1-protease inhibitor proteins. AB - The Mus musculus alpha 1-protease inhibitor gene cluster encodes five highly related proteins. The most significant amino acid polymorphisms lie within the reactive-site loop which is important in determining serpin substrate specificity. All five genes are transcribed in M. musculus adult liver and presumably secreted into plasma. In an attempt to characterize their protein products all five cDNAs were expressed in recombinant mammalian cells and the protease inhibition activity of each determined. Only two of the proteins were efficient inhibitors of neutrophil elastase, the major physiological target of the sole human alpha 1-protease inhibitor (antitrypsin). Four of the proteins were active against chymotrypsin, while no substrate could be identified for the fifth. PMID- 8619830 TI - Suppression of the neoplastic phenotype by replacement of the Tsc2 gene in Eker rat renal carcinoma cells. AB - The hereditary renal carcinoma (RC) in the rat, originally reported by R. Eker in 1954, is an excellent example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. Recently, we have identified a germline mutation of the sclerosis (Tsc2) gene in the Eker rat (Nature Genetics 9, 70-74, 1995), suggested to be a novel tumor suppressor gene, fitting Knudson's two-hit hypothesis. In this study, the effect of wild-type Tsc2 gene expression in Eker RC cells was tested using a tetracycline-responsive promoter system. Transfection and expression of a exogenous Tsc2 gene affected both cell morphology and growth rate. This demonstration of suppression of the neoplastic phenotype provides direct evidence for an essential role of the Tsc2 gene in tumorigenesis. PMID- 8619831 TI - Oxidative stress response in iron-induced acute nephrotoxicity: enhanced expression of heat shock protein 90. AB - Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we determined the proteins preferentially produced in response to the Fe-NTA-induced oxidative injury. A single intraperitoneal Fe-NTA treatment led to the enhanced production of a number of proteins with molecular masses of 85-95 kDa. These included heat shock protein 90 (HSP90) as determined by immunoprecipitation. The enhanced production of HSP90 was prominent in the renal tubular cells. Steady accumulation of HSP90 was observed in the subacute toxicity experiments with multiple injections of Fe-NTA, suggesting that the enhanced production of HSP90 is important in increasing resistance to subsequent injury caused by the Fe-NTA induced oxidative stress. PMID- 8619832 TI - Topoisomerase II inhibition differentially modulates Caco-2 intestinal epithelial cell phenotype. AB - Caco-2 intestinal epithelial cells differentiate spontaneously after confluence when contact inhibition slows proliferation. We hypothesized that such reversible differentiation might be dependent on DNA synthesis and repair. We studied the effects of the topoisomerase II inhibitor etoposide on Caco-2 proliferation and on the differentiation markers alkaline phosphatase and dipeptidyl dipeptidase specific activity, as well as cell motility. Etoposide (0.3-10 microM) dose dependently inhibited proliferation and alkaline phosphatase activity. However, etoposide (0.7-3 microM dose-dependently stimulated dipeptidyl dipeptidase activity. Above this concentration, dipeptidyl dipeptidase was also inhibited. Similar effects on enzyme activity were observed when proliferation was blocked with mitomycin C. Etoposide (1-10 microM) also dose-dependently inhibited cell motility. The selective stimulation of dipeptidyl dipeptidase activity by etoposide may offer a clue to the regulation of intestinal brush border enzyme expression at the molecular level. PMID- 8619833 TI - The novel regulatory mechanism of Fas system-mediated apoptosis in mesangial cells: implication to mesangial proliferative glomerulonephritis. AB - Fas Ag is a cell surface molecule that transduces the signal for apoptosis. Since mesangial cells (MC) play important roles in regulating glomerulonephritis, we investigated regulatory mechanisms of Fas system in MC. Fas Ag was expressed on MC from normal mice. This Fas Ag expression was down-regulated by inducing proliferation with platelet-derived growth factor or 18% fetal bovine serum, but was reversed when cycloheximide was added to the culture. Anti-Fas Ab alone did not induce apoptosis in MC, but MC became susceptible to apoptosis induced by anti-Fas Ab is actinomycin D or cycloheximide was added. Noteworthy findings are that mRNA of Fas ligand was expressed in MC. Taken together, MC appears to control the proliferation by regulating Fas system-mediated apoptosis, at least in part, through an autoregulatory mechanism with Fas Ag Fas ligand expressed on their own MC. PMID- 8619835 TI - DNA-dependent protein kinase inhibitor (OK-1035) suppresses p21 expression in HCT116 cells containing wild-type p53 induced by adriamycin. AB - The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK. PMID- 8619834 TI - Regulation of the nuclear factor of activated T cells in stably transfected Jurkat cell clones. AB - Two Jurkat cell clones have been stably transfected with a reporter vector for the nuclear factor of activated T cells (NFAT). Upon stimulation, they express high levels of secreted heat stable placental alkaline phosphatase. With these clones, we demonstrated that NFAT activation induced by phorbol 12-myristate 13 acetate and ionomycin was inhibited by both cyclosporin A (CsA) (IC50 = 8 nM) and FK506 (IC50 = 160 pM), presumably by inhibition of calcineurin activity. Selective phosphatase inhibitors for protein phosphatase 1 (PP1) and 2A (PP2A) that do not inhibit calcineurin, such as okadaic acid and calyculin A, also inhibited NFAT activation with IC50s of 87 nM and 4 nM, respectively, suggesting that okadaic acid and related inhibitors may block NFAT activation through the inhibition of PP1, instead of PP2A. NFAT activation was also inhibited by agents that increase cAMP concentrations such as dibutyryl cAMP, forskolin and prostaglandin E2. These stable Jurkat cell clones provide a convenient and sensitive tool to study NFAT regulation. PMID- 8619836 TI - Regulation of jun expression and activation of AP-1 activity by erythropoietin. AB - Erythropoietin (EPO) is a molecule which regulates hemoglobin gene expression during erythroid proliferation and differentiation. However, the mechanism by which it regulates gene expression is poorly understood. Secondly, unlike other hematopoietic factors which induce expression of early response genes, it is unknown whether EPO is capable of inducing the expression and activation of early response genes. In this study, evidence is presented that EPO induces the expression of early response genes c-jun, junD and c-fos mRNA, stimulates jun protein synthesis and induces activation of 5 x AP-1/CAT activity in hyman erythroleukemia K562 cells. Also, EPO appears to regulate jun expression at transcriptional, post transcriptional and translational levels. These observations suggest that jun and fos expression may be relevant in the mechanism of growth control by EPO. PMID- 8619837 TI - Sequence analysis of four acidic beta-crystallin subunits of amphibian lenses: phylogenetic comparison between beta- and gamma-crystallins. AB - beta-Crystallins composed of the most heterogeneous group of subunit chains among the three major crystallin families of vertebrates, i.e. alpha-, beta- and gamma crystallins, are less well understood at the structural and functional levels than the other two. They comprise a multigene family with at least three basic (betaB1-3) and four acidic (betaA1-4) subunit polypeptides. In order to facilitate the determination of the primary sequences of all these ubiquitous crystallin subunits present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses. We report here a protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta-crystallin acidic subunit polypeptides by polymerase chain reaction (PCR). Four complete full-length reading frames with two each of 597 and 648 base pairs, which cover four deduced protein sequences of 198 (betaA1-1 and betaA1-2) and 215 (betaA3-1 and betaA3-2) amino acids including the universal initiating methionine, were revealed by nucleotide sequencing. They show about 96 98% sequence similarity among themselves and 76-80%, 80-83% to the homologous betaA1/A3 crystallins of bovine and human species respectively, revealing the close structural relationship among acidic subunits of all beta-crystallins even from remotely related species. In this study a phylogenetic comparison based on amino-acid sequences of various betaA1/A3 crystallins plus the major basic beta crystallin (betaBp) and gamma-crystallin from different vertebrate species is made using a combination of distance matrix and approximate parsimony methods, which correctly groups these betaA crystallin chains together as one family distinct from basic beta-crystallins and gamma-crystallin and further corroborates the supposition that beta- and gamma-crystallins form a superfamily with a common ancestry. PMID- 8619838 TI - Involvement of a disulfide bridge in catalytic activity of camel lens xi crystallin. AB - Chemical modification studies were performed to elucidate the role of cysteine residues in the catalytic activity of camel lens xi-crystallin. 5,5'-dithiobis (2 nitrobenzoic acid) (DTNB) titration of the camel lens xi-crystallin revealed that it contained 3 SH-groups and a disulfide bridge per subunit. One of the three SH groups was readily accessible, whereas the other two were buried. Inactivation of xi-crystallin by DTNB was caused by a modification of one cysteine residue per subunit. The resulting DTNB-modified enzyme was reactivated by dithiothreitol (DTT) or KCN. The inactivation was partially protected by NADPH, whereas 9,10 phenanthrenequinone (PQ) enhanced the modification of the enzyme. These results indicate that the SH- group being modified is located at/near the NADPH binding site which is not essential for catalysis. Incubation of the enzyme with DTT led to a substantial loss of the enzyme activity. However, DTT inhibition was prevented completely by preincubation of enzyme with PQ but not by NADPH indicating that an essential disulfide-bridge is present at the substrate binding site of xi-crystallin. PMID- 8619839 TI - Development of radioimmunoassay for human leptin. AB - Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma leptin-LI concentration in nonobese subjects (17.6 10 mV) upon the addition of either immunosuppressor to the cells. Our findings revealed that the voltage-dependent potassium current in human peripheral blood lymphocytes is inhibited by Cyclosporin A and other immunosuppressors, resulting in a depolarized membrane potential. PMID- 8619843 TI - Heme oxygenase-1 gene expression by a glutathione depletor, phorone, mediated through AP-1 activation in rats. AB - This study shows that induction of heme oxygenase-1 (HO-1) gene expression by a glutathione (GSH) depletor, phorone, is inhibited by cycloheximide pretreatment and involves changes in c-jun, not c-fos, mRNA. Buthionine sulfoximine (BSO) enhanced markedly both c-jun and HO-1 gene expression evoked by phorone. Phorone dramatically increased AP-1 binding activity, which was blocked by unlabeled AP-1 oligonucleotide and abolished by anit-Jun antibodies, but not anti-Fos antibodies. In addition, pretreatment with dexamethasone, an inhibitor of AP-1 DNA binding, inhibited phorone-mediated HO-1 mRNA induction. These findings suggest that HO-1 induction by phorone is likely to involve in the activation of AP-1 (Jun/Jun) binding, which could be associating with GSH depletion. PMID- 8619844 TI - Spectroscopic characterization of bendazac and benzydamine: possible photochemical modes of action. AB - The involvement of near-UV light in cataract development suggests that potential anti-cataract drugs may display unusual spectroscopic properties. As bendazac impedes certain effects associated with lens opacification, we have characterized the singlet and triplet states of bendazac and its analog, benzydamine, by fluorescence and phosphorescence methods. These compounds have much shorter triplet state lifetimes compared to the triplet state lifetimes observed in proteins. Our results raise the possibility that the photoprotective action of these compounds may result from their ability to dissipate energy through the triplet state. We propose alternative modes for the photoprotective actions of these compounds. PMID- 8619845 TI - The reactions catalyzed by the inducible bifunctional enzyme of rat liver peroxisomes cannot lead to the formation of bile acids. AB - The ability of the bifunctional 2-enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase from rat liver peroxisomes to metabolize (24E)-3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA, a presumed intermediate during the beta-oxidative degradation of the steroid side chain in the formation of cholic acid, was investigated. The bifunctional enzyme efficiently hydrated the above compound specifically to (24S,25S)-3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta cholestanoyl-CoA, but the dehydrogenase component of the enzyme was virtually inactive toward this product. In contrast, the bifunctional enzyme efficiently catalyzed the dehydrogenation of the (24S,25R) diastereomer of the above hydroxy intermediate to two products whose uv absorbance and chemical properties were consistent with those of alpha-methyl-beta-ketoacyl-CoAs. These results suggest that the bifunctional enzyme is not sufficient for the formation of a 24-keto intermediate in bile acid biosynthesis. PMID- 8619846 TI - Purification and chemical characterization of a potent inhibitor of the Na-K-Cl cotransport system in rat urine. AB - A potent inhibitor of the Na-K-Cl cotransport system was purified from urines of salt-loaded rats. Mass spectroscopy revealed a molecular mass of 242 Da. Nuclear magnetic resonance showed a spectrum identical to that of 3,4-dihydro-3-(4 hydroxyphenyl)-2H-1-benzopyran-7-ol (an "estrogen-like" isoflavonoid: equol). This compound inhibited cotransport fluxes at similar concentrations (IC50=16-24 microM) as furosemide (IC50 approximately 10 microM). Cotransport inhibitory activity of urines from rats drinking tap water was fully explained by urinary equol concentrations (approximately 27 microM, measured by high-performance liquid chromatography). Slat-loading increased urinary equol excretion, but not sufficiently high to fully explain the very important increase in cotransport inhibitory potency. We conclude that: (i) under basal conditions urinary equol can regulate Na-K-Cl cotransport activity in the kidney and (ii) salt-loading should evoke the appearance of other cotransport inhibitors. PMID- 8619847 TI - cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). PMID- 8619848 TI - Effects of unilateral cryptorchidism on the expression of gonadotropin receptor mRNA. AB - Expression patterns of mRNAs of FSH and LH receptors were examined by Northern blot in adult rat testes after induction of unilateral cryptorchidism. At 4 weeks after the operation, signals of Northern blot were markedly increased in both receptor mRNAs in the abdominal testes as compared to those in the scrotal testes. In addition, there was a remarkable change in size pattern of LH receptor transcripts; the scrotal testes of cryptorchid rats and the intact testes of control rats expressed the transcripts of 7.0, 4.3, 2.5, and 1.8 kb, while the abdominal testes hardly expressed the transcript of 1.8 kb. This finding suggests that the expression of LH receptor mRNA is regulated locally within testis. PMID- 8619849 TI - Effect of nonenzymatic histone acetylation on chromatin high-order folding. AB - In the present study we have made attempts to estimate the effect of histone acetylation on the folding of the chromatin fibril into its high-order structures. Histones were modified directly in isolated nuclei using acetyl adenylate as an acetyl donor. High-order folding of acetylated chromatin was analyzed by titration with increasing amounts of ethidium bromide. We have shown that chromatin with non-enzymatically acetylated histones exhibits a less folded conformation when compared to intact chromatin. We discuss the molecular bases of this phenomenon and propose a novel generalized model for regulation of chromatin high-order folding. PMID- 8619850 TI - Studies of human and rat blood under oxidative stress: changes in plasma thiol level, antioxidant enzyme activity, protein carbonyl content, and fluidity of erythrocyte membrane. AB - Previously by selection and inbreeding of Wistar rats susceptible or resistant to the cataractogenic effect of galactose the S and R rat strains were developed as a model of oxidative stress. It was earlier found S rats have elevated level of OH-radical generation and enhanced lipid peroxidation compared with that for R rats. Our data show that superoxide dismutase and catalase activities in the blood of S rats are twice lower than corresponding values of R rats. Increased level of disulfides and protein carbonyl groups was obtained in the blood plasma of S-rats. It was found that fluidity of erythrocyte membrane measured by photobleaching method is definitely higher for S rats compared with that for S rats. Similar effect of decreased catalase activity and increased disulfide content has been observed for kyphoscoliosis patients. PMID- 8619851 TI - Molecular cloning and functional expression of the cytochrome P450 11B hydroxylase of the guinea pig. PMID- 8619852 TI - Mechanical effects on the kinetics of the HIV proteinase deactivation. AB - The proteinase from HIV undergoes rapid and irreversible deactivation caused by mild mechanical stirring. Both the free enzyme and the ternary Michaelis complex disappear in two separate first-order processes, with half-times of 3.0 and 0.8 minutes, respectively. Ignoring these deactivation steps distorts the results of kinetic analyses. PMID- 8619853 TI - High rates of substrate hydroxylation by human cytochrome P450 3A4 in reconstituted membranous vesicles: influence of membrane charge. AB - CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higher Vmax for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the Km. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane. PMID- 8619854 TI - The gene of the protease inhibitor SKALP/elafin is a member of the REST gene family. AB - Members of the REST gene family characteristically have a transcription unit consisting of three exons. The first and the last exon are conserved among members, while the second exon--encoding almost all of the mature protein- differs considerably. The so far known REST genes are highly, and almost exclusively, expressed in the seminal vesicles. By sequence analysis we have now identified the gene for the protease inhibitor SKALP/elafin as a new member of the REST gene family. The protein is expressed in the epidermis and serves, like the product of several REST genes, as substrate for transglutaminase. We have also found what seems to be a locus encompassing both transglutaminases and REST genes centered around the region q12 on the human chromosome 20, raising the question whether the enzymes and substrates have evolved in parallel. PMID- 8619855 TI - cDNA sequence analysis and expression of the a chain of beta-bungarotoxin from Bungarus multicinctus (Taiwan banded krait). AB - The cDNA encoding the A chain of beta-bungarotoxin (beta-Bgt) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequence encoding the A chain revealed that the determined one was different from the known A chains (A1, A2, A3 and A4). Nevertheless, the amino acid sequence and cDNA sequence of the new A chain (A5) was highly homologous with those of other A chains. The A5 chain was subcloned into the expression vector pT7-7 and transformed into BL21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli, and the refolded A chain was purified by reversed phase high performance liquid chromatography. The purified recombinant A chain exhibited an about 16% phospholipase activity of beta-Bgt. These results strongly suggest that the A chain is an active subunit responsible for the phospholipase activity of beta Bgt. PMID- 8619856 TI - Structural characterization of the human soluble epoxide hydrolase gene (EPHX2). AB - The structural organization of the human gene encoding soluble epoxide hydrolase (EPHX2) was determined. Cosmid clones containing the EPHX2 gene were identified by hybridization of a human genomic library with PCR amplified cDNA to the mouse or human soluble epoxide hydrolase as probes. The gene consists of 19 exons and is approximately 45 kb of which more than 15 kb were sequenced. The coding sequence corresponds to 555 amino acid residues, i.e., one additional residue to those in the reported cDNA sequence. The sized of the introns were defined by sequencing or PCR analysis and they contained various repetitive structure elements as expected. PMID- 8619857 TI - Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain. AB - The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the death-mediating protease ced-3. The mammalian homolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1beta converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor alpha, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor alpha-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death. PMID- 8619858 TI - Basement membrane regulates gene expression in HEC1B(L) endometrial adenocarcinoma cells. AB - The morphology and differentiation of human endometrial adenocarcinoma cells HEC1B(L) are influenced by cell-matrix interactions. Culturing of HEC1B(L) cells on an extracellular matrix (ECM) induced the formation of a three-dimensionally arranged, highly ordered branching network of HEC1B(L) cells. In an effort to identify biologically important genes that are involved in this in vitro differentiation process of endometrial tumor cells we applied the method of "differential display." We isolated two DNA fragments, which were differentially regulated by ECM. One of these fragments, MESR4/13, was downregulated by ECM and contains a TG rich sequence. The second fragment, MES13/15, demonstrates high sequence similarity to MESR4/13 except for the TG-rich region and is induced by ECM. Probably these genes are potent markers to study the molecular basis of the process of in vitro differentiation of endometrial adenocarcinoma cells. PMID- 8619859 TI - Persistent activation of CDK4 during neuronal differentiation of rat pheochromocytoma PC12 cells. AB - It has been shown that suppression of cyclin-dependent kinase 4 (CDK4) expression or its kinase activity is a critical event for the differentiation of hematopoietic and myoblastic cells. To investigate the role of CDK4 during NGF mediated neuronal differentiation of PC12 cells, we examined the changes in the expression and kinase activity of CDK4. Unexpectedly, both the expression of CDK4 and its kinase activity remained high throughout 10 days of exposure to NGF. Furthermore, constitutive overexpression of the human CDK4 did not inhibit the differentiation. These results suggest that suppression of CDK4 is not required for the NGF-induced neuronal differentiation of PC12 cells. PMID- 8619860 TI - Human CAAF1 gene--molecular cloning, gene structure, and chromosome mapping. AB - We have isolated and characterized a cDNA and the gene of the human homologue of CAAF1, a novel member of the S100 calcium-binding protein family. The 276-bp open reading frame encoded a 92-amino acid polypeptide with a predicted molecular mass of 10,575 Da. The deduced amino acid sequence of human CAAF1 showed 66% homology to bovine CAAF1. The human CAAF1 gene consisted of three exons, with the two EF hand motifs of the CAAF1 protein separately encoded by exons 2 and 3. This gene was expressed at a high level in polymorphonuclear leukocytes and at an intermediate level in esophageal mucosa. The tissue distribution of CAAF1 mRNA was different from that of other S100 proteins. Direct R-banding fluorescence in situ hybridization revealed that the human CAAF1 gene was mapped to chromosome 1q21.2-q22, where most of the S100 genes form a cluster. PMID- 8619861 TI - cDNA sequence analysis and expression of the expression of the ribosomal protein S24 during oogenesis and embryonic development of the sea urchin Paracentrotus lividus. AB - A gene for the Paracentrotus lividus ribosomal protein S24, called P1-S24, has been isolated and sequenced. Ribosomal protein P1-S24 consists 130 amino acids and has a molecular weight of 14869 Da. Sequence analysis shows a high percentage (90%) identity with the corresponding gene of Strongylocentrotus purpuratus. Hybridization of the cDNA to digested sperm DNA suggests that P1-S24 is represented in no more than two copies. Studies of the temporal expression of P1 S24 gene indicate a good correlation between this and the expression of the rRNA genes both during oogenesis and embryonic development. PMID- 8619862 TI - Tetrahydropterins interfere with the G protein pathway in Dictyostelium discoideum. AB - The cellular slime mold Dictyostelium discoideum produces tetrahydrodictyopterin, the D-threo-isomer of tetrahydrobiopterin. During cyclic AMP coordinated aggregation, the G protein linked signalling pathway is involved in the regulation of the initial enzyme GTP cyclohydrolase I [M. Gutlich et al., Biochem. J. 314, 95-101 (1996)]. We now find that cyclic AMP stimulated binding of GTPgammaS to the membrane fraction is inhibited by tetrahydrodictyopterin and tetrahydrobiopterin. Inhibition was a function of GTPgammaS concentration and the analysis of the kinetic data pointed to a competitive type of inhibition. The inhibition of G protein activation was accompanied by a loss of adenylyl cyclase activation. Because the GTPgammaS- and G protein-dependent reduction of receptor affinity for cyclic AMP was also attenuated, we conclude that tetrahydrodictyopterin and tetrahydrobiopterin interfere with activation of G proteins by inhibiting GDP-GTP exchange. PMID- 8619863 TI - The C-terminal RNLL sequence of the plasma retinol-binding protein is not responsible for its intracellular retention. AB - An in vitro model system using COS cells that transiently express human plasma retinol binding protein has been set up in which we are able to mimic the retinol dependent secretion of this protein observed in hepatocytes. In the absence of its ligand, plasma retinol binding protein is retained in the endoplasmic reticulum. It contains a C-terminal sequence, RNLL, that could function as a cryptic KDEL motif and thus be responsible for its retention in the endoplasmic reticulum. The model system has been used to test a mutant lacking these four last amino acids for retention and retinol induced secretion. The results obtained show that although plasma retinol binding protein is retained in the endoplasmic reticulum, the RNLL sequence does not seem to be responsible for its retention. PMID- 8619864 TI - Molecular cloning of cDNA and analysis of expression of the gene for alpha glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L. AB - Previously, we identified and purified an alpha-glucosidase with a molecular mass of 70 kDa from a homogenate of the hypopharyngeal gland of the older worker bee (forager bee) Apis mellifera L. (T. Kubo et al, J. Biochem., 1996, 119, 291-295). Here, we isolated and sequenced a cDNA for the alpha-glucosidase. The cDNA encoded a protein consisting of 650 amino acids, which had high sequence identity with fruit fly and mosquito possible maltase gene products. RT-PCR showed that the gene was expressed specifically in the hypopharyngeal gland of the forager bee. PMID- 8619865 TI - In vivo trapping of nitric oxide in the brain of neonatal rats treated with the HIV-1 envelope protein gp 120: protective effects of alpha-phenyl-tert butylnitrone. AB - AIDS dementia complex is a neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction. The HIV-1 envelope glycoprotein gp 120 has been implicated in the development of AIDS dementia. This protein has been shown to be neurotoxic and to cause learning impairment and retardation of the development of complex motor behavior in rat neonates. Nitric oxide has been implicated in gp 120-induced neurotoxicity. In the present study, we report for the first time in vivo evidence for the formation of nitric oxide in the CNS as a result of multiple subcutaneous injections of gp 120 to neonatal rats. Nitric oxide was trapped in the brain of neonatal rats by N-methyl-D-glucamine dithiocarbamate-Fe and the nitric oxide content measured by electron paramagnetic resonance spectroscopy. The nitrone-based spin trap alpha-phenyl-tert butylnitrone at 50 mg/kg was found to prevent gp 120-mediated nitric oxide formation and to also protect against gp 120-induced behavioral impairment. PMID- 8619866 TI - Increased circulating hepatocyte growth factor in the early stage of acute myocardial infarction. AB - We measured serum hepatocyte growth factor (HGF) in patients with acute myocardial infarction, angina pectoris, and other heart diseases. In patients with acute myocardial infarction, blood was collected at the time of admission. Serum HGF was elevated within 3 hours in 8 of 10 patients (80%) with acute myocardial infarction after onset of chest pain (9.4 +/- 3.2 ng/mL, mean +/- SEM, values in normal subjects <0.39 ng/mL). Mean value of serum HGF was 11.0 +/-2.6 ng.mL (n=11) in patients who admitted to the hospital between 6 and 9 hours and 13.1 +/- 5.7 ng.mL between 12 and 24 hours after onset. Elevated HGF levels were significantly more frequent than those of creatine kinase within 3 hours, and elevated levels correlated well with those of serum creatine kinase at 6-9 hours after onset of acute myocardial infarction. No increase in serum HGF value was found in patients with angina pectoris or other heart diseases. Thus, measurement of HGF is a sensitive method for early diagnosis of acute myocardial infarction. PMID- 8619868 TI - Characterization of differentially expressed genes following brief cardiac ischemia. AB - For the study of ischemia-related adaptations we employed DDRT-PCR on mRNA obtained from heart regions undergoing brief (10') occlusions/reperfusions. We found and sequenced 52 differentially expressed clones that we further characterized using Northern analysis with RNA from a wide variety of tissues. We selected only clones (1) with a new sequence, (2) that showed a Northern signal, and (3) that exhibited organ-specific (heart, muscle) or (4) situation-specific (ischemia, non-specific stress) expression. We found two new heart-specific transcripts and three new stress-inducible genes. The transcripts with known sequences showed an expression pattern typical for repair processes after ischemia-reperfusion (ubiquitin, ATPases). About 20% of clones were truly differentially expressed. PMID- 8619867 TI - The interferon-inducible growth-inhibitory p202 protein: DNA binding properties and identification of a DNA binding domain. AB - p202 is an interferon-inducible protein whose expression in transfected cells inhibits proliferation. p202 binds to the retinoblastoma tumor suppressor protein in vitro and in vivo and the transcription factors AP-1 c-Fos and c-Jun, NF kappaB p50 and p65, and inhibits the transcriptional activity of these factors in vivo. Here we report that p202 nonspecifically binds to double-stranded DNA and to single-stranded DNA in vitro. Analysis with recombinant p202 revealed that DNA binding activity is intrinsic to p202. A C-terminal deletion mutant of p202 exhibited DNA-binding properties, indicating that the C-terminus is dispensable for DNA binding. We also found that underphosphorylated p202 efficiently binds to DNA. Our data suggest that DNA binding activity of p202 may contribute to its functions. PMID- 8619869 TI - Two transcripts with different sizes derived from a rice homeobox gene, OSH1. AB - A rice homeobox gene, OSH1, contains two functionally independent promoters which generate a larger transcript and a smaller transcript. In Arabidopsis, each promoter can drive the expression of a reporter gene in a different manner, indicating that the expression of different sized transcripts is independently regulated by each promoter. Over-expression of the larger transcript in transformed plants caused altered morphologies (Matsuoka et al., Plant Cell, 1993, 5, 1039-1048); in contrast, over-expression of the smaller transcript did not cause any morphological changes. The results suggest that the product of the smaller transcript fails to alter the expression of its target gene(s) in the transformants, while that of the larger transcript is capable of altering the expression of its target gene(s). PMID- 8619870 TI - Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel. AB - This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed. PMID- 8619871 TI - Stereospecific effects of R-lipoic acid on buthionine sulfoximine-induced cataract formation in newborn rats. AB - This study revealed a marked stereospecificity in the prevention of buthionine sulfoximine-induced cataract, and in the protection of lens antioxidants, in newborn rats by alpha-lipoate, R- and racemic alpha-lipoate decreased cataract formation from 100% (buthionine sulfoximine only) to 55% (buthionine sulfoximine + R-alpha-lipoic acid) and 40% (buthionine sulfoximine + rac-alpha-lipoic acid) (p<0.05 compared to buthionine sulfoximine only). S-alpha-lipoic acid had no effect on cataract formation induced by buthionine sulfoximine. The lens antioxidants glutathione, ascorbate, and vitamin E were depleted to 45, 62, and 23% of control levels, respectively, by buthionine sulfoximine treatment, but were maintained at 84-97% of control levels when R-alpha-lipoic acid or rac-alpha lipoic acid were administered with buthionine sulfoximine; S-alpha-lipoic acid administration had no protective effect on lens antioxidants. When enantiomers of alpha-lipoic acid were administered to animals, R-alpha-lipoic acid was taken up by lens and reached concentrations 2- to 7-fold greater than those of S-alpha lipoic acid, with rac-alpha-lipoic acid reaching levels midway between the R isomer and racemic form. Reduced lipoic acid, dihydrolipoic acid, reached the highest levels in lens of the rac-alpha-lipoic acid-treated animals and the lowest levels in S-alpha-lipoic acid-treated animals. These results indicate that the protective effects of alpha-lipoic acid against buthionine sulfoximine induced cataract are probably due to its protective effects on lens antioxidants, and that the stereospecificity exhibited is due to selective uptake and reduction of R-alpha-lipoic acid by lens cells. PMID- 8619872 TI - mRNA differential display reveals Krox-20 as a neural plasticity-regulated gene in the rat hippocampus. AB - The prolonged maintenance of hippocampal long-term potentiation depends on de novo protein and RNA synthesis, indicating an involvement of altered gene expression in long-lasting plastic changes in synaptic efficacy. We have used an mRNA differential display technique to identify a set of genes that are induced by neural activity in the rat hippocampus. Sixteen independent cDNAs were isolated whose mRNA level was markedly modulated by convulsive seizure. One of these encodes Krox-20, a zinc finger DNA binding protein. High frequency tetanic stimulation of perforant pathway, which elicited a persistent long-term potentiation (>10 h), rapidly induced expression of krox-20 mRNA in the hippocampus of urethane-anesthetized rat. The increase in krox-20 mRNA was transient and NMDA receptor-dependent. These results suggest a role for krox-20 in the maintenance of long-term potentiation. PMID- 8619873 TI - Translational regulation in vivo of the Drosophila melanogaster mRNA encoding succinate dehydrogenase iron protein via iron responsive elements. AB - Some mRNA encoding proteins related to iron metabolism contain a specific stem loop structure--iron responsive element (IRE)-in the 5' UTR. Binding of the iron regulatory protein (IRP) to the IRE, in response to decreases in cellular iron levels, leads to a block in translation of these mRNAs. We here describe the drosophila melanogaster succinate dehydrogenase iron protein (SDH-IP) as a fourth example of an mRNA species being translationally regulated by an IRE, based on iron dependent regulation of SDH-IP translation in vivo by immunoprecipitations and northern blotting in drosophila cell lines. Addition of hemin to the drosophila cells lead to fragmentation of the SDH-IP, which might suggest additional mode to specifically down-regulate the expression of this protein and Krebs cycle function. PMID- 8619874 TI - beta-Estradiol inhibits Na+-P(i) cotransport across renal brush border membranes from ovarectomized rats. AB - Estrogens play a major role in mineral homeostasis, however it remains unclear whether they exert regulatory action upon reabsorption of phosphate (P(i)) in proximal tubules of the kidney. We investigated the effect of beta-estradiol E2 injected to thyroparathyroidectomized and ovariectomized rats upon Na+ cotransport of P(i) and other solutes across renal brush border membrane (BBM). In BBM from kidneys of E2-treated rats the capacity for Na+-P(i) cotransport was considerably suppressed (delta% - 42; P<0.01) whereas Na+-cotransports of L proline D-glucose and SO4 across the same BBM did not differ from controls. We surmise that E2 inhibits selectively Na+-P(i) cotransport by direct interaction with E2 receptors in proximal tubular cells. These results indicate the existence of the inhibitory effect of estrogens upon renal proximal tubular Na+-P(i) cotransport and, by extention, proximal P(i) reabsorption. We suggest that this modulatory action of E2 plays a role in pathophysiology of mineral metabolism due to estrogen deficiency and should be considered when estrogens are used for pharmacotherapy of postmenopausal osteoporosis and some types of cancer. PMID- 8619875 TI - Lack of involvement of protein kinase A phosphorylation in voltage-dependent facilitation of the activity of human cardiac L-type calcium channels. AB - Phosphorylation by protein kinase A is thought to be involved in voltage dependent facilitation of calcium channels. Here we have shown that the subunit complex of a cloned human cardiac calcium channel, expressed in Xenopus oocytes, responds to voltage-dependent facilitation by an approximately 50% increase of the calcium channel peak current. The removal of all protein kinase A consensus sequences by site-directed mutagenesis decreased but did not eliminate the response to prepulse facilitation. Moreover, Rp-cAMP-S, an inhibitor of protein kinase A, could not prevent facilitation of the wild-type calcium channel currents. Similarly, AMP-PNP a nonhydrolyzable analog of ATP, while significantly decreasing the whole-cell current amplitude, failed to reduce the response to double-pulse facilitation. Therefore, we conclude that the voltage-dependent facilitation of cloned calcium channel currents is not due to enhancement of phosphorylation, but probably to some type of voltage-induced conformational change in the channel. PMID- 8619876 TI - Host-parasite interaction in human onchocerciasis: identification and sequence analysis of a novel human calgranulin. AB - A novel S100 ca2+-binding protein, termed calgranulin-related protein (CGRP), was purified to homogeneity from extracts of adult Onchocerca volvulus, a human tissue-dwelling parasite. Its complete amino acid sequence was determined using microanalytical techniques. The primary structure of CGRP consists of 91 residues and displays identity with the recently reported partial sequence of an S100 protein present in human neutrophils. The human origin of CGRP is supported by the occurrence in O. volvulus extracts of additional human neutrophil proteins, including migration inhibitory factor-related protein 8 and defensins. The results suggest that these proteins interact with the worm surface following their release by activated neutrophils in the course of inflammatory reactions caused by O. volvulus infection. PMID- 8619877 TI - Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris. AB - A 1.8-kb NsiI-StuI fragment containing the recA gene of Xanthomonas campestris pv. campestris was cloned by a PCR-based approach and complementation of Escherichia coli HB 101. Sequence analysis of this fragment revealed an ORF (orf343) of 1,032 bp able to encode a protein of 343 amino acids with a calculated MW of 37,021 Da, a size similar to the values detected by in vitro system and Western blotting. It showed 69.6% identity to the E. coli RecA in amino acid sequence. Amino acid residues of the E coli RecA associated with functional activities are conserved in this Xc17 RecA. The recA mutant, L1, constructed by gene replacement, was sensitive to ultraviolet irradiation and methyl methanesulfonate, and deficient in homologous recombination. PMID- 8619878 TI - Detection of CYP3A5 allelic variant: a candidate for the polymorphic expression of the protein? AB - In liver samples from 19 Caucasian subjects, the CYP3A5 protein was detected in 74% of individuals (14/19), while the messenger was shown to be expressed in 100% of individuals, assessed by the RT-PCR method. In order to characterise the putative mutation(s) in the messenger, accounting for the absence of protein accumulation, the full coding region of the CYP3A5 cDNA was sequenced for two unrelated individuals, one expressing the protein at a high level and one being defective. A point mutation in exon 11 at position 1280 (C-->A) was found to cosegregate with the absence of protein accumulation in 2 of the 5 defective individuals. This mutation produces a change in the amino acid at position 398 (Thr-->Asn). Whether this mutation affects the stability of the protein or whether it is in linkage desequilibrium with other mutation(s) in the non-coding region of the messenger is not known. The C-->A change at 1280 generates a Tsp509 I site which can be used for routine evaluation of the frequency of this mutation in population studies. PMID- 8619879 TI - Soluble 5'-nucleotidase from thyroid gland partial purification and properties. AB - A soluble 5'-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The Km values were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax:IMP>CMP>AMP>UMP.Mg2+ was necessary for the enzyme activity. PMID- 8619880 TI - Site-directed mutagenesis of a novel serine arylesterase from Vibrio mimicus identifies residues essential for catalysis. AB - Site-directed mutagenesis (SDM) of an arylesterase (the arylesterase) from Vibrio mimicus revealed that residues S29, H153, and D96 constituted a catalytic triad. The use of a serine residue for ester hydrolysis by the arylesterase proves that the enzyme is a novel serine arylesterase. SDM also showed that D28 was necessary for the esterase activity; to our knowledge it is the first time that a residue immediately preceding the active-site serine in esterases was shown biochemically to possess such a property. The results further suggest that D28 plays a role in substrate-binding. Residue 31 was firmly shown to participate in the binding of N acetyl-D, L-phenylalanine beta-naphthyl ester (NAPNE), an artificial substrate for chymotrypsin. The S31G enzyme showed a 4 fold decrease in the Km for NAPNE over that of wild type enzyme, proving residue 31 is important for substrate specificity. A mechanism for binding and catalysis of esters by the arylesterase is proposed, which includes the unique role of S31 for aromatic (hydrophobic) acyl-binding. The biochemical properties of the arylesterase suggest that the enzyme stands out as a member of a distinct subfamily within a recently proposed, lipolytic enzyme family. PMID- 8619881 TI - Bioassay of endothelium-derived hyperpolarizing factor. AB - Endothelium-derived hyperpolarizing factor (EDHF) mediates vasodilatation in certain blood vessels, together with prostacyclin and NO. However, its chemical nature is not known. A perfusion-superfusion cascade was developed to confirm the diffusible nature of EDHF. Canine carotid arteries with endothelium were used as donors of vasoactive substances, whereas rings of coronary artery without endothelium were used as detectors. Inhibitors of NO synthesis and cyclooxygenase were present throughout, to avoid interference from NO and prostanoids. Measurements of membrane potential and isometric tension, in coronary arteries without endothelium (used as detectors), demonstrated the release of EDHF from the carotid arteries, following treatment with 8-methoxypsoralen, bradykinin and thimerosal. The K+-channel blocker tetraethylammonium inhibited the action od EDHF in the detectors. Thus, these results demonstrate that endothelial cells release a diffusible activator of K+-channels in vascular smooth muscle. PMID- 8619882 TI - Selective pharmacological inhibition of distinct nitric oxide synthase isoforms. AB - Nitric oxide (NO) is produced in physiological and pathophysiological conditions by three distinct isoforms of NO synthase (NOS): endothelial NOS (ecNOS), inducible NOS (iNOS), and brain NOS (bNOS). Selective inhibition of iNOS may be beneficial in various forms of shock and inflammation, whereas inhibition of bNOS may protect against neuroinjury. This article surveys the enzymatic mechanism of NO production, lists the strategies and pharmacological tools for selective inhibition of distinct NOS isoforms, and considers the side-effects of the various approaches. Selective inhibition of NOS isoforms is achieved by: (a) targeting the differential co-factor (calmodulin or tetrahydrobiopterin) requirement of various NOS isoforms, and NOS; (b) targeting the differential substrate requirements of cells expressing various isoforms of NOS (L-arginine uptake blockers or arginase); (c) the use of pharmacological agents that are selectively taken up by cells expressing various isoforms of NOS (7 nitroindazole); or (d) developing pharmacological NOS inhibitors with isoform specificity. The amino acid-based NOS inhibitor, NG-nitro-L-arginine, shows a preference for ecNOS and bNOS over iNOS, whereas L-N6-(1-iminoethyl)lysine is selective for iNOS over bNOS. Certain non-amino acid-based small molecules, such as aminoguanidine and certain S-alkylated isothioureas, also express selectivity towards iNOS and have anti-inflammatory and anti-shock properties. 7 nitroindazole, a bNOS-selective inhibitor, protects in central nervous system injury. Clearly, there are a number of distinct approaches that are worthy of further research efforts in order to achieve even more selective targeting of various NOS isoforms PMID- 8619883 TI - Inhibition of metmyoglobin/H2O2-dependent low density lipoprotein lipid peroxidation by naturally occurring phenolic acids. AB - The ferrylmyoglobin <==> metmyoglobin redox transitions promoted by hydrogen peroxide and dietary phenolic acids and their potential role in the oxidation of LDL were studied. The use of parinaric acid incorporated in LDL as a probe for radicals (detected by fluorescence quenching of the probe) revealed an oxidative stress inside LDL shortly ( < 1 min) after addition of hydrogen peroxide to metmyoglobin in the aqueous phase outside the particle, reflecting an efficient access of the oxidant to LDL lipids. However, the propagation step of peroxidation only occurs after a lag phase, as detected by the kinetics of oxygen consumption. Triton X-100 decreases but does not suppress the lag phase of oxidation. Addition of metmyoglobin (without peroxide) to LDL was not followed by significant oxidation during the time of the experiment, unless Triton X-100 was present in the medium. When dietary phenolic acids were present in the medium before peroxide addition, an inhibition of parinaric acid fluorescence quenching and oxygen consumption was recorded as a function of concentration and substitution pattern on the phenol ring of the phenolic acids. This was associated with a conversion of ferrylmyoglobin to metmyoglobin. The results indicate that the naturally occurring phenolic acids prevent ferrylmyoglobin dependent LDL oxidation in a way strongly dependent on the substitution pattern on the phenol ring. Among the phenolic compounds studied, the o-dihydroxy derivatives of cinnamic and benzoic acids (caffeic, chlorogenic, and protocatechuic acids), in a molar ratio of 1 to metmyoglobin, efficiently blocked LDL oxidation initiated by ferrylmyoglobin. Replacement of one OH group from catecholic structure with an H (p-coumaric acid) or methoxy group (ferulic acid) decreased the antioxidant activity. Also, the catechol structure fused in heterocyclic rings with adjacent carbonyl groups (ellagic acid) resulted in decreased antioxidant activity. These observations correlate with the efficiency of phenolic acids to reduce ferrylmyoglobin to metmyoglobin. Therefore, the protection of LDL against oxidation is assigned to the reduction of the oxoferryl moiety of the hemoprotein to the ferric form. Additionally, it is suggested that an access constraint of oxidants plays a minor role in the ferrylmyoglobin induced oxidation against LDL. PMID- 8619884 TI - Expression of xenobiotic-metabolizing cytochrome P450 forms in human full-term placenta. AB - The expression of individual xenobiotic-metabolizing cytochrome P450 (CYP) genes in human placenta was studied at the mRNA level by reverse transcriptase polymerase chain reaction (RT-PCR). mRNAs of CYP1A1, CYP2E1, CYP2F1, CYP3A3/4, CYP3A5, and CYP4B1 were detected by RT-PCR, and CYP1A2, CYP2A6/7, CYP2B6/7, CYp2C8-19, CYP2D6, and CYp3A7 were not detected. Several enzyme activity assays and immunoblasts were used to further characterize expression of forms producing detectable mRNA transcripts. The catalytic activities of 7-ethoxycoumarin O deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were substantially increased in response to maternal cigarette smoking, and paralleled the amount of CYP1A1 mRNA and protein. Aromatase activities were slightly lower in placentas exposed to cigarette smoke compared with nonexposed placentas. These data show that several xenobiotic-metabolizing CYP genes are expressed in human placenta at a low level. The significant of such low-level expression is unknown, but it may have local physiological or toxic consequences. PMID- 8619886 TI - Insulinomimetic effects of myricetin on lipogenesis and glucose transport in rat adipocytes but not glucose transport translocation. AB - Myricetin is a naturally occurring flavonol that is commonly found in tea, berries, fruits, and medicinal plants. It mimics insulin in stimulating lipogenesis and glucose transport in rat adipocytes in vitro. It was found to stimulate lipogenesis in rat adipocytes and enhance the stimulatory effect of insulin. The EC50 was estimated to be about 65 microM. Myricetin did not have any effect on insulin receptor autophosphorylation nor on the tyrosine kinase activity of the receptor. However, myricetin stimulated both D-glucose and D-3-O methylglucose uptake in rat adipocytes. The Vmax of glucose transport was increased, but the Km did not change significantly. Immunoblot analysis of Glut4 in rat adipocyte plasma membrane showed that the stimulation of glucose transport was not a consequence of glucose transporter translocation. Instead, the stimulation in glucose uptake probably was due to a change in the intrinsic activity of the glucose transporter possibly caused by alterations in membrane fluidity or transporter-lipid interactions as a result of the insertion of myricetin into the membrane bilayer. Thus, myricetin may have therapeutic potential in the management of non-insulin-dependent diabetes mellitus by stimulating glucose uptake without the presence of fully functional insulin receptor. PMID- 8619885 TI - RP 64477: a potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase with low systemic bioavailability. AB - RP 64477 (N-butyl-3-(p-decyloxybenzamido)-4-(methylthio)benzamide) has been shown to be a potent inhibitor of the cholesterol esterifying enzyme Acyl-coenzyme A:cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) in intestinal, hepatic, adrenal, and arterial tissue preparations obtained from a range of animal species. Drug concentrations producing 50% inhibition of enzyme activity (IC50 values) ranged from 14-283 nM. Inhibition by RP 64477 in a rabbit intestinal enzyme preparation was shown to be non-competitive with respect to the substrate oleoyl-CoA. In whole cell assays using human intestinal (CaCo-2), hepatic HepG2) and monocytic (THP-1) cell lines, RP 64477 inhibited ACAT activity with IC50s of 113, 503, and 180 nM, respectively. RP 64477 (0.03% w/w by diet) reduced significantly cholesterol absorption in cholesterol/cholic acid-fed rats from 94+/- 8% to 65 +/- 4%. In cholesterol-fed rabbits, cholesterol absorption was reduced from 72 +/- 5% to 50 +/-5% and 44 +/- 5% at dose levels of 10 and 30 mg kg-1 b.i.d., respectively. Plasma cholesterol levels were reduced dose dependently in both cholesterol/cholic-acid-fed rats and cholesterol-fed rabbits. Neither cholesterol absorption nor plasma cholesterol levels were reduced significantly in animals maintained on standard laboratory diets. Pharmacokinetic studies indicated that RP 64477 were very poorly absorbed following oral administration to rats. Plasma levels of drug were < 2 ng mL-1 following a dose of 2000 mg kg-1 p.o.. When radiolabelled RP 64477 was administered orally, limited absorption was indicated by the overwhelming elimination of radioactivity in the faces (96.4% of administered material) coupled with low renal clearance (0.6% of dose) and biliary excretion (0.05% of dose). In conclusion, this work shows that RP 64477 is a potent inhibitor of ACAT obtained from a range of animal species and man. Inhibition of cholesterol absorption and hypocholesterolaemic activity has been demonstrated in rats and rabbits maintained on diets supplemented with cholesterol. Pharmacokinetic studies indicate low systemic exposure to RP 64477 as a result of limited absorption of this drug. PMID- 8619887 TI - Identification of two oligodeoxyribonucleotide binding proteins on plasma membranes of human cell lines. AB - Two oligodeoxyribonucleotide (oligodN) binding proteins of approximately 100-110 kDa were identified in the plasma membranes of human HL-60, HepG2, H1, and KB cells by a photolabeling technique. Solubilization of cellular membranes with a nonionic detergent did not interfere with the binding of these two proteins to oligodNs, and both proteins were susceptible to serine protease action. The binding affinities of these two proteins to oligodNs were found to be similar; Scatchard plot analysis revealed the Kd for phosphodiester (PO) 21-mer oligodeoxycytidine to be 60 nM and binding sites numbered approximately 1.2 x 10(6)/cell for HepG2 cells. Both phosphorothioate (PS) and PO oligodNs could bind to these two proteins with the binding affinity for PS oligodNs being much stronger than that for PO oligodNs. The binding to oligodNs was affected by the ionic strength of the reaction. Dextran sulfate, tRNA, and double-stranded DNA inhibited the binding of oligodNs, whereas ATP, ADP, AMP, and TTP had no effect. Given their high affinity for oligodNs, these membranes proteins may play an important role in the action of oligodNs. PMID- 8619888 TI - Purification and characterization of an Ah receptor binding factor in chromatin. AB - Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We previously reported the binding of the Ah receptor, partially purified form rabbit liver, to receptor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromatin proteins (CP) were isolated by absorption of chromatin to hydroxylapatite followed by sequential extraction with 3 M NaCl and 1-8 M guanidine hydrochloride (GdnHCl). In the present study, we continued the purification of the CP5 fraction, which exhibited AhRBF activity. The proteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor binding, a portion of each Cl Sepharose 6B fraction was reconstituted to rabbit double-stranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter binding assay. Two protein fractions with a molecular weight in the range of 10,000-14,000 demonstrated high affinity binding to the Ah receptor. The binding of AhRBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be > 90% specific for [3H]TCDD-R. Further purification was achieved by preparative ADS-PAGE, and AhRBF activity was attributed to two fractions with molecular weights between 12,000 and 10,000. A kDa protein with AhRBF activity was found to have an isoelectric point (pI) of > or = 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyanogen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a variety of systems, this is the first report of a specific interaction of AhR with histone H4. PMID- 8619889 TI - Deltamethrin-induced thymus atrophy in male Balb/c mice. AB - The action of deltamethrin, a potent type II synthetic pyrethroid insecticide, on the thymus of the Balb/c mouse was studied in vivo and in vitro. We found that deltamethrin produced atrophy in the thymus in a dose- and time-dependent fashion. The lowest effective dose was found to be 6 mg/kg, 24 hr after a single intraperitoneal treatment. Treated animals did not recover during the time-course of the experiment (365 days after treatment); however, deltamethrin did not affect the body weight of the treated animals during the course of the study. To determine if deltamethrin-induced [Ca2+]i signaling could lead to thymic atrophy via programmed cell death, mice were treated with 25 mg deltamethrin/kg for 24 hr or the isolated thymocyte suspension was treated with 50 microM deltamethrin. A significant stimulation of inositol 1,4,5-triphosphate (IP3) and inositol 1,4 diphosphate (IP2) production was found after 24 hr of deltamethrin-1R (active isomer) treatment. An inactive stereoisomer of deltamethrin (i.e. 1S) did not cause a significant rise in the production of 1P3 and 1P2. In addition, deltamethrin-1R induced a transient increase of [Ca2+]i mobilization in the thymocyte suspension after 10 min of in vitro treatment, and substantially reduced the rate of calcium-calmodulin (Ca/CaM)-dependent protein dephosphorylation in in vivo treated animals (25 mg deltamethrin/kg for 24 hr). The in vivo effects of deltamethrin treatment demonstrated induction of DNA fragmentation and cell death in thymocytes. Moreover, using a histochemical approach, it was evident that deltamethrin at 25 mg/kg was able to produce cell death in the thymus of treated animals 72 hr after treatment. In the present work, we found that cell death was apoptotic in nature as noted first by the inhibition of deltamethrin-induced cell death by aurintricarboxylic acid, an inhibitor of apoptosis, and second, by internucleosomal DNA fragmentation, a hallmark of apoptosis, produced by deltamethrin in treated animals as well in thymocyte suspensions. In addition, the involvement of the Ca/CaM-dependent protein phosphorylation-dephosphorylation cascade in the induction of apoptosis by deltamethrin was supported by the protective role of the calmodulin inhibitor trifluoperazine against the apoptotic effect of deltamethrin on thymocyte suspension. Our results suggest that deltamethrin induced thymus atrophy and altered the Ca/CaM-dependent protein kinase-phosphatase cascade, which might induce programmed cell death. PMID- 8619890 TI - Expression of I2-imidazoline sites in rat prostate. Effect of castration and aging. AB - Clonidine, idazoxan, and related imidazoline adrenergic drugs bind to non adrenergic sites in brain and several peripheral tissues. These sites, termed imidazoline receptors, appear to exist in two major subclasses, I1 sites labeled by clonidine and I2 sites labeled by idazoxan. In this study, we investigated whether rat prostate expresses imidazoline receptors and, if so, whether their expression can be regulated by circulating testosterone. Studies in rat ventral prostate membrane revealed that [3H]idazoxan, but not [3H]p-aminoclonidine, bound to non-adrenergic sites. The binding of [3H]idazoxan was saturable (Bmax: 941 +/- 105 fmol/mg protein) and high affinity (KD: 16.4 +/- 2.3 nM). The rank order of the inhibition of binding by imidazoline ligands was cirazoline > clonidine > UK 14,304 > guanabenz, indicating an I2 subclass of imidazoline receptors. Bilateral orchiectomy increased the number of binding sites (Bmax) for [3H]idazoxan without changing the affinity (KD). Testosterone replacement, while completely restoring the plasma testosterone levels, only partially reversed the increase in Bmax. In contrast, the binding of [3H]idazoxan to prostate membranes of rats in different age groups (4, 7, and 16 months) revealed a progressive decrease in the Bmax without any change in KD. We conclude that the rat prostate expresses the I2 subclass of imidazoline receptors and that the expression is regulated by circulating testosterone. PMID- 8619891 TI - Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides. AB - Multidrug resistance protein (MRP) is a member of the ATP-binding cassette superfamily of transport proteins which has been demonstrated to cause multidrug resistance when transfected into previously sensitive cells. Sixteen eicosomeric oligonucleotides complementary to different regions along the entire length of the MRP mRNA reduced MRP mRNA and protein levels in drug-resistant small cell lung cancer cells that highly overexpress this protein. In MRP-transfected HeLa cells that express intermediate levels of MRP, one oligonucleotide, ISIS 7597, targeted to the coding region of the MRP mRNA, decreased the levels of MRP mRNA to < 10% of control levels in a concentration-dependent manner. This effect was rapid but transient with a return to control levels of MRP mRNA 72 hr after treatment. A double treatment with ISIS 7597 produced a sustained inhibition, resulting in a greater than 90% reduction in MRP mRNA for 72 hr and a comparable decrease in protein levels. Increased sensitivity to doxorubicin was observed under these conditions. Northern blotting analyses using two DNA probes corresponding to sequences 5' and 3' of the ISIS 7597 target sequence, respectively, revealed the presence of low levels of two smaller sized RNA fragments as expected from an RNase H-mediated mechanism of action of the antisense oligonucleotide. These studies indicate that a specific reduction in MRP expression can be achieved with antisense oligonucleotides, a finding that has potential implications for the treatment of drug-resistant tumors. PMID- 8619892 TI - Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-DI-1- pyrrolidinyl-7H-pyrrolo[2,3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger. AB - Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 microM ferrous sulfate and 200 microM EDTA, Fe2+/EDTA) resulted inhibition of the Na+/K+ -pump ATPases was also associated with membrane protein crosslinking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 microM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U 89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U 89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes. PMID- 8619893 TI - Correlation of altered tyrosine phosphorylation with methotrexate resistance in a cisplatin-resistant subline of L1210 cells. AB - Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistant in L1210/DDP cells. PMID- 8619894 TI - Identification of bilirubin UDP-GTs in the human alimentary tract in accordance with the gut as a putative metabolic organ. AB - The initial identification of traditionally hepatic enzymes expressed in the gut has led to the hypothesis that the gut may function as a metabolic organ. The UDP glucuronosyltransferases (UDP-GTs) play an important role as phase II metabolizing enzymes. Previously members of this family have been identified in the gut by non-isoform specific immunoreactivity, and a small amount of bilirubin glucuronosyltransferase activity was detected in the colon. Recent reports of gut transplantation to reverse the metabolic defect in Gunn rats raised further interest in the expression and distribution of human bilirubin (UDP-GTs (HUG Br 1 and HUG Br 2) in the human alimentary tract. The availability of molecular genetic probes for HUG Br 1 and HUG Br 2 permits the screening of the alimentary tract for the presence of isoform specific message. RNA samples extracted from pinch biopsy specimens of buccal mucosa, esophagus, stomach body, antrum, duodenum, and colon were analyzed for expression of HUG Br 1 and HUG Br 2. HUG Br 1 hybridization was detected in duodenum > colon, whereas HUG Br2 hybridization was detected in duodenum > esophagus > colon. Immunoreactivity data confirmed the presence of HUG Br 1 protein at low levels in the duodenum, whereas the less abundant HUG Br 2 protein was below the limits of detection of isoform specific anti-peptide antibodies. Bilirubin specific reactivity was demonstrated in duodenal samples but not antrum samples, consistent with the molecular genetic data. The presence of functional bilirubin UDP-GT isoforms in human alimentary tract supports the notion that the gut may function as a metabolic organ and may have diagnostic and therapeutic implications for disorders of bilirubin metabolism. PMID- 8619895 TI - Cytotoxic effects of a doxorubicin-transferrin conjugate in multidrug-resistant KB cells. AB - Cancer chemotherapy is often limited by the emergence of multidrug-resistant tumor cells. Multidrug resistance (MDR) can be caused by amplification of the MDR genes and overexpression of the P-glycoprotein, which is capable of lowering intracellular drug concentrations. A doxorubicin-transferrin conjugate has been synthesized and exerts its cytotoxic effects through a transmembrane mechanism. We have examined the cytotoxicity of this conjugate and compared it with doxorubicin in sensitive (KB-3-1) and in multidrug-resistant KB cell lines (KB-8 5, KB-C1, and KB-V1). In the clonogenic assay, doxorubicin exhibited IC50 concentrations of 0.03 and 0.12 microM in the sensitive (KB-3-1) and resistant (KB-8-5) cell lines, respectively, whereas, doxorubicin-transferrin conjugate was more effective with IC50 concentrations of 0.006 and 0.028 microM, respectively. In highly multidrug-resistant KB-C1 and KB-V1 cells, doxorubicin up to 1 microM did not cause any cytotoxic effects, while the doxorubicin-transferrin conjugate inhibited colony formation of these cells with IC50 levels of 0.2 and 0.025 microM, respectively. These results demonstrate that doxorubicin-transferrin is effective against multidrug-resistant tumor cells. PMID- 8619896 TI - Independent induction of morphological transformation of CHO cells by receptor activated cyclic AMP synthesis or by receptor-operated calcium influx. AB - Morphological transformation of Chinese hamster ovary (CHO) cells can be induced by exogenous addition of cyclic AMP (cAMP) or through the stimulation of G protein-coupled receptors ectopically expressed in these cells. The morphological transformation has been shown to represent a phenotypic suppression of CHO cell tumorigenic potential. Studies were undertaken to determine which receptor activated signal transduction pathway initiates the progression from a tumorigenic to a non-tumorigenic phenotype. Stimulation of CHO cells expressing the dopamine D1 receptor (CHOD1) with a D1 selective agonist, SKF38393, resulted in an increase in cAMP accumulation which correlated with morphologic transformation. SKF38393 had no effect on intracellular calcium levels, arguing against a requirement for phospholipase C or calcium mobilization in the D1 stimulated morphology change. In contrast, stimulation of muscarinic m5 (CHOm5) or vasopressin V1a (CHOV1a) receptors expressed in CHO cells with carbachol or arginine vasopressin (AVP), respectively, did not result in an increase in intracellular calcium and a morphology change. The time course of carbachol stimulated calcium influx correlated with the time course of morphological transformation, but not with carbachol-stimulated cAMP or inositol, 1,4,5 trisphosphate (IP3) accumulation. Furthermore, no increase in cAMP accumulation was observed in AVP-stimulated CHOV1a cells, suggesting a cAMP-independent stimulation of the transformation process. Carbachol-stimulated CHO cells expressing the m2 muscarinic receptor (CHOm2) failed to undergo a morphological transformation, yet released IP3. Therefore, phospholipase C-mediated signal transduction is not sufficient for the morphological transformation of CHO cells. It appears that receptor-stimulated morphologic transformation of CHO cells can be induced via two independent signaling pathways, mediated by adenylate cyclase or receptor-operated calcium channels. PMID- 8619897 TI - Development of an in situ toxicity assay system using recombinant baculoviruses. AB - A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8 diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern. PMID- 8619898 TI - Transfer of PCBs via lactation simultaneously induces the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf in neonates. AB - At the first day of lactation, maternal rats were injected with a single i.p. dose of 100 or 250 mg/kg body weight of a mixture of polychlorinated biphenyls (Aroclor 1254). This treatment caused significant increases in both material and neonatal hepatic cytochrome P-450, cytochrome b5, and cytochrome-c-(P-450) reductase. Transfer of PCBs via lactation resulted in significant increases in hepatic enzyme activities catalysed by neonatal CYP1A1, CYP1A2, CYP2B1, CYP3A1, and CYP2E1 using a variety of substrates. In contrast, the metabolism of dimethylnitrosamine and aminopyrine was only marginally (up to 2-fold) increased in maternal animals four days post treatment. Further measurements showed significant increases in maternal and neonatal epoxide hydrolase, glutathione-S transferase, and UDP-glucuronyl transferase activities, thus suggesting a coordinated response for an induction of CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP2E1, CYP3A1, and CYP4A1 in both maternal and neonatal CYP2C6, and at the higher dose the expression of neonatal CYP2E1 was significantly reduced. Northern blot analysis provided further evidence for significant increases in maternal and neonatal hepatic CYP1A1, CYP1A2, CYP2B1, and CYP2E1 mRNA, but reduced amounts of CYP2C7 and CYP4A1 mRNA. Additional Northern blot hybridization experiments may suggest an increased expression of the protooncogenes c-Ha-ras and c-raf in the mother and the neonate upon treatment of maternal rats with Aroclor 1254. Lactation itself may result in an increased expression of the latter protooncogenes, but the mRNA of the protooncogenes c-erb A and c-erb B was not detected in any of the tissues examined. PMID- 8619899 TI - Effects of recombinant drug-specific single chain antibody Fv fragment on [3H] desipramine distribution in rats. AB - Tricyclic antidepressant overdose can be reversed in rats by drug-specific antibody Fab fragments, but the required Fab dose may itself by toxic. We studied the potential use of a smaller, recombinant desipramine (DMI)-specific single chain Fv fragment (B9-sFv) for this purpose. Anesthetized rats received a tracer (subtoxic) dose of [3H]-DMI followed in 15 min by B9-IgG, B9-Fab, B9-sFv (0.1 mumol of binding sites) or BSA. Each of the active treatments produced a rapid and substantial increase in the serum radiolabel concentration, whereas BSA did not (P < 0.001). The increase in serum radiolabel concentration 1 min after treatment was 13.3-fold with B9-IgG, 10.0-fold with B9-Fab and 7.3-fold with B9 sFv. Serum antibody concentrations were also highest after B9-IgG and lower with B9-Fab or B9-sFv. The 24-hr urinary excretion of radiolabel did not differ among groups, but was extensive even in the BSA group and probably represented the excretion of DMI metabolites. B9-sFv concentrations in urine or buffer at 37 degrees declined by >90% over 24 hr, but this fragment was much more stable in serum, retaining 70% of its activity after 96 hr. These data demonstrate that B9 sFv can alter markedly the distribution of [3H]-DMI in vivo. The rapidity of this effect, and its magnitude in comparison with Fab fragment or IgG, suggest that further study of B9-sFv as a treatment of DMI overdose is warranted. PMID- 8619900 TI - Constitutive activation of chimeric m2/m5 muscarinic receptors and delineation of G-protein coupling selectivity domains. AB - To derive structure/function relationships for muscarinic receptor/G-protein coupling, the m2 and m5 muscarinic receptors and a series of m2/m5 chimeras were tested for agonist binding and functional responses in a cellular proliferation/transformation assay. m5, which mediates stimulation of phosphatidylinositol turnover, displayed robust activity in the proliferation assay, whereas m2, which mediates inhibition of adenylyl cyclase, was inactive in the proliferation assay. Chimeras that contained m2 sequences in the i2 or i3 loops had impaired activity or were inactive, respectively. Chimeras that contained m2 segments reaching from the N-terminus to TM2, or from TM6 to the C terminus, had enhanced activity relative to m5, and a chimera with both of these elements was constitutively activated. PMID- 8619901 TI - Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure. AB - An expression plasmid for mammalian cells (CLDN10B) has been modified to add nucleotides encoding hexahistidine and the FLAG peptide (H/F) to cDNAs. The new mammalian expression plasmid has been named pDoubleTrouble (pDT). The plasmid and a recombinant baculovirus were used to produce native-and H/F-human A1 and A2A adenosine receptors, optimally expressed in CHO-K1 and Sf9 cells, respectively. Binding to recombinant H/F-A1 receptors (Bmax = 30 pmol/mg protein) was characterized using [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and 125I-N6 aminobenzyladenosine (125I-ABA). Binding to H/F-A2A receptors (Bmax = 48 pmol/mg protein) was characterized using [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) and [3H]2-[4-(2-carboxyethyl)phenethylamino]-NECA ([3H]CGS21680). By comparison to native receptors, the addition of H/F to the amino termini of these receptors had no effect on the binding affinities cyclic AMP accumulation in intact cells was not affected by the H/F extension. Anti-FLAG and Ni-nitrilotriacetic acid affinity chromatography resulted in high yield ( >50% overall recovery) of nearly homogeneous deglycosylation with N-glycosidase F. We anticipate that pDT will be generally useful for facilitating the purification in high yield of recombinant receptors and other proteins by single or sequential affinity chromatography steps. PMID- 8619902 TI - Cephalosporin and carbacephem nephrotoxicity. Roles of tubular cell uptake and acylating potential. AB - Three beta-lactams, desacetylcephaloglycin, ampicillin, and loracarbef, were studied to test a hypothesis derived from retrospective analysis of previously studied cephalosporins: that beta-lactam nephrotoxicity develops in approximate proportion to tubular cell antibiotic concentrations and lactam ring reactivities. Concentrations of each beta-lactam (and insulin) in rabbit renal cortex and serum were measured at the end of 0.5-hr infusions of 100 mg antibiotic/kg body weight and 0.5 to 0.67 hr later. Total cortical AUCs (total areas under the curve of concentration and time in renal cortex) and transported cortical AUCs (total minus insulin-space beta lactam) were calculated from these measurements. Reactivities, determined by the rate constants of lactam-ring opening at pH 10, were taken from the literature. Nephrotoxicity was quantified by grades of proximal tubular cell necrosis and by serum creatinine concentrations 2 days after infusion of 100-1500 mg/kg of the antibiotics. Desacetylcephaloglycin was slightly less nephrotoxic than cephaloglycin; the AUCs reactivities, and toxicities of these two cephalosporins fit the proposed model, particularly when allowance is made for hepatic and renal deacetylation of cephaloglycin. The very low AUCs, limited reactivity, and absence of nephrotoxicity of ampicillin also fit the model. Loracarbef had a transported AUC less than three times, and reactivity one-thirtieth, those of cefaclor, respectively. Although only at 1500 mg/kg, loracarbef was significantly more nephrotic than cefaclor. If the relativity of loracarbef with its targeted bacterial proteins, which is essentially the same as that of cefaclor, is considered instead of the base hydrolysis rate constant, than loracarbef also fits the model. By the same analysis, the comparatively high in vitro stability of other carbacephems, although pharmaceutically convenient, may not limit their nephrotoxicity. PMID- 8619903 TI - [3H]WIN 35,428 [2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane] binding to rat brain membranes. Comparing dopamine cell body areas with nerve terminal regions. AB - Potential differences between somatodendritic acid and axonal dopamine transporters were examined by comparing the binding constants of [3H]WIN 35, 428 [2 beta-carbomethoxy- 3 beta-(4-fluorophenyl)tropane] binding to membranes prepared from the rat ventral mesencephalon, containing A9 and A10 dopamine cell bodies, and from the nucleus accumbens. Saturation analysis of [3H]WIN 35,428 binding, in the presence of compounds to occlude norepinephrine and serotonin transporters, was performed by both the "unlabeled" method (varying unlabeled ligand) and "labeled" method (varying radioligand). The density of binding was substantially lower in the ventral mesencephalon than in the nucleus accumbens, but the binding affinity was only slightly different. Likewise, the differences between the two regions in the inhibitory potency of cocaine and GBR 12909 [1-(2 di(4-fluorophenyl)-methoxy-ethyl)4-(3-phenylpropyl)piperazine] were not substantial. The results suggest that somatodendritic and axonal dopamine transporters in the ventral mesencephalon and nucleus accumbens are not very different as far as their binding domains for uptake blockers such as cocaine and GBR 12909 are concerned. PMID- 8619904 TI - Suppression of plasma estradiol and progesterone concentrations by buthionine sulfoximine in female rats. AB - Glutathione (GSH) is an important factor involved in the resistance of tumor cells to anticancer agents. Buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, effectively decreases cellular GSH concentrations both in vitro and in vivo. Depletion of GSH by BSO sensitizes a variety of cancer cells to chemotherapeutic agents. Therefore, BSO has been on clinical trial as an anticancer adjuvant. For this purpose, it is important to understand the effect of BSO treatment not only on the sensitivity of tumor cells to anticancer agents, but also on the metabolism and function of normal tissues. The present study was undertaken to determine the effect of BSO treatment on GSH concentrations in the blood, liver, and ovary, and changes in concentrations of ovarian hormones and other important components in plasma. Female Sprague-Dawley rats, 90 days of age, were treated with 2.0 mmol/kg BSO in saline by intraperitoneal injection, twice daily for 7 days. This treatment depressed GSH concentrations in the blood, liver and ovary by 95, 75, and 85%, respectively. Several blood components were measured. These included red blood cells, hemoglobin, ceruloplasmin, hematocrit, mean corpuscular volume and hemoglobin concentration, alkaline phosphatase, urea nitrogen, creatine and creatinine, glucose, cholesterol, triglycerides, triiodothyronine (T3), thyroxine (T4), and hormones including estradiol, progesterone, and prolactin. BSO treatment significantly (P < 0.05) elevated and lowered plasma concentrations of ceruloplasmin and urea nitrogen, respectively, More importantly, plasma concentrations of estradiol and progesterone were decreased markedly (P < 0.05) in the BSO-treated animals. The hormonal results suggest that investigations on the role of BSO-induced GSH depletion in the treatment of malignancies both with and without hormone dependence in women should be undertaken. PMID- 8619905 TI - Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: effects of the Swedish Alzheimer's disease APP 670/671 mutation on binding levels. AB - We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [3H] quinuclidinyl benzilate ([3H]QNB) and the M2/M4 selective antagonist [3H] (+/-)-5,11-dihydro-11-([(2-[(di propylamino)methyl]-1- piperidinyl]ethyl)amino]carbonyl)-6H-pyrido(2,3 b)(1,4)benzodiazepine-6- one ([3H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [3H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine > himbacine > methoctramine > (+/-)-p-fluoro-hexahydro-sila difenidol hydrochloride > pirenzepine > muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25-35% lower levels of muscarinic receptors labelled with [3H]QNB, [3H]N-methyl scopolamine and [3H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing. PMID- 8619906 TI - Chronic treatment with the uncompetitive NMDA receptor antagonist memantine influences the polyamine and glycine binding sites of the NMDA receptor complex in aged rats. AB - Receptor binding studies on rat cortical membranes were used to characterize the NMDA receptor in aged rats (22 months) treated for 20 months with a memantine containing diet delivering 30 mg/kg/day in comparison to aged and young/adult rats treated with control-diet. Spatial memory impairing effects of (+)-MK-801 (0.16 mg/kg) in the radial maze was not altered within the course of memantine treatment (up to 16 months). However, chronic memantine-treatment significantly increased the number of [3H]MK-801 binding sites and the affinity of [3H]glycine. A non-significant trend to such changes was also seen in aged-control rats. Glycine-dependent [3H]MK-801 binding (functional binding under non-equilibrium conditions at a fixed L-glutamate concentration) revealed that a decreased ability of glycine to stimulate channel opening in aged rats was partially attenuated by the long-term memantine treatment. Furthermore, an increased ability of spermidine to enhance [3H]MK-801 binding in aged-control rats was even more pronounced in the aged memantine-treated group. Together these findings may indicate that changes in functional receptor-channel properties during the process of aging occur prior to a detectable loss of binding sites and that memantine enhances an endogenous compensatory mechanism triggered by glutamatergic hypofunction which is suggested to take place in aging. PMID- 8619907 TI - Effect of coadministration of glutamate receptor antagonists and dopaminergic agonists on locomotion in monoamine-depleted rats. AB - Combinations of dopaminergic agonists with glutamate receptor antagonists have been suggested to be a possible alternative treatment of Parkinson's disease. To gain further insights into this possibility, the antagonist of the competitive AMPA-type glutamate receptor NBQX and the ion-channel blocker of the NMDA glutamate receptor (+)-MK-801 in combination with the dopamine D1 receptor agonists: SKF 38393, SKF 82958 and dihydrexidine; the dopamine D2 receptor agonist bromocriptine and the dopamine-precursor L-DOPA were tested in rats pretreated with reserpine and alpha-methyl-p-tyrosine. MK-801 on its own induced locomotor behaviour and potentiated the antiakinetic effects of dihydrexidine and L-DOPA but not of the other dopamine agonists tested. NBQX neither on its own nor coadministered with the dopamine agonists tested had an antiakinetic effect. These results indicate that agents, blocking the ion-channel of the NMDA receptor, might be useful adjuvants to some but not all dopaminomimetics in therapy of Parkinson's disease. The same does not seem to be true for the AMPA antagonist NBQX. PMID- 8619908 TI - Determinants of neuronal firing pattern in the guinea-pig subthalamic nucleus: an in vivo and in vitro comparison. AB - To ascertain the extent to which neuronal firing pattern in the subthalamic nucleus (STN) is determined by afferent inputs, a comparison was made between STN neurons recorded in vivo and in vitro (a largely denervated preparation). In vivo, the majority of cells exhibited an irregular firing pattern, although some showed evidence of burst firing. In contrast, all cells had a regular firing pattern in vitro. Electrical stimulation of the striatopallidal complex in vivo induced a short latency inhibition in STN neurons, followed by a burst of spikes. These effects could be reproduced in vitro; hyperpolarising pulses gave rist to a slow depolarising potential upon termination, which was accompanied by a burst of action potentials. Hence, the evidence suggests that afferents play an important role in determining the firing pattern of STN neurons. However, the cells also possess intrinsic membrane properties which allow inputs to trigger either single spikes or bursts. PMID- 8619909 TI - Differential effects of three dopamine receptor agonists in MPTP-treated monkeys. AB - The behavioral effects of cabergoline, pergolide and bromocriptine were investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned parkinsonian cynomolgus monkeys with attention to the induction of hyperactivity, as evidenced by irritability, excitability and aggressiveness. All three drugs improved the parkinsonism in a dose-dependent fashion following a single injection. Among the three dopamine (DA) receptor agonists used, the antiparkinsonian effect of pergolide was the strongest and had an immediate effect, while cabergoline showed the longest duration of the antiparkinsonian effect and was least potent in inducing hyperactivity. PMID- 8619910 TI - The neural mechanisms and progressive nature of symptoms of Parkinson's disease- based on clinical, neurophysiological and morphological studies. AB - The neural mechanism of parkinsonian motor symptoms, i.e., rigidity, tremor and akinesia, which are the result of nigrostriatal dopamine deficiency, is interpreted from long-term observations on the effect of surgical and pharmacological treatment of the disease in relation to the neuropathological findings within the substantia nigra zona compacta (SNc). Rigidity, tremor and secondary akinesia start first with degeneration of the ventral tier of the SNc followed by spread of the pathology to the dorsal tier, which may produce primary akinesia. Later, locus ceruleus pathology will be added. Spread of pathology is extremely slow in the juvenile or early onset parkinsonism (JP) compared with that in Parkinson's disease (PD). This spreading of pathology from one functional system to another might be one of the key factors responsible for the progressive worsening of the disease, which is different in speed between JP and PD. PMID- 8619911 TI - Stereological estimation of nuclear mean volume in invasive meningiomas. AB - A stereological estimation of nuclear mean volume in bone and brain invasive meningiomas was made. For comparison the nuclear mean volume of benign meningiomas was estimated. The aim was to investigate whether this method could discriminate between these groups. We found that the nuclear mean volume in the bone and brain invasive meningiomas was larger than in the benign tumors. The difference was significant and moreover it was seen that there was no overlap between the two groups. In the bone invasive meningiomas the nuclear mean volume appeared to be larger inside than outside the bone. No significant difference in nuclear mean volume was found between brain and bone invasive meningiomas. The results demonstrate that invasive meningiomas differ from benign meningiomas by an objective stereological estimation of nuclear mean volume (p<0.00005). PMID- 8619912 TI - In vitro susceptibility of 124 Xanthomonas maltophilia (Stenotrophomonas maltophilia) isolates: comparison of the agar dilution method with the E-test and two agar diffusion methods. AB - The in vitro susceptibility of 124 Xanthomonas maltophilia isolates was tested by four methods: Agar dilution (reference method), E-test, a disk diffusion and a tablet diffusion method. Trimethoprim-sulfamethoxazole had the highest activity against X. maltophilia, followed by a combination of aztreonam-clavulanic acid at different ratios, the ratio 1:1 being the most active with a susceptibility rate of 85% as compared to 2% for aztreonam alone. Addition of the beta-lactamase inhibitor tazobactam to piperacillin enhanced the rate of susceptible isolates from 31% to 53%, Relatively few isolates were susceptible to ciprofloxacin (27%) and gentamicin (9%). Generally, the disk diffusion method had a considerably higher frequency of "very major" discrepancies when compared with the agar dilution method than with the other methods. The susceptibility of X. maltophilia to trimethoprim-sulfamethoxazole and ciprofloxacin could reliably be determined by all the diffusion methods tested, but otherwise the agar dilution method is to be preferred. A standardized and reliable diffusion method for susceptibility testing of X. maltophilia remains to be found. Trimethoprim-sulfamethoxazole must be considered the drug of choice in the treatment of severe X. maltophilia infections. The combination aztreonam-clavulanic acid is promising, but must be proved in a clinical setting. PMID- 8619913 TI - A comparison of the immunogenicity of the native and denatured forms of a protein. AB - The effect of heat denaturation on the physicochemical and immunological properties of a model protein, ovalbumin, and its formaldehyde/lysine-treated form was investigated. Polyacrylamide gel electrophoresis and gel filtration showed that heat denaturation converted ovalbumin to high Mr polymers, whereas formaldehyde/lysine-treated ovalbumin remained monomeric with only a small proportion forming oligomers. NMR analysis demonstrated that non-denatured structures could easily be differentiated from the denatured structures. Intraperitoneal immunization of rabbits and mice showed that both native and denatured forms of ovalbumin induced an immune response, but denatured forms of ovalbumin were found to be less immunogenic and to have a lower epitope density than native ovalbumin. Analysis of the antisera in crossed immunoelectrophoresis showed that they were specific for either native or denatured forms of ovalbumin. These findings were further investigated by ELISA and immunoaffinity chromatography, and the high specificity and low cross-reactivity was confirmed. We conclude that the immunogenic epitopes on denatured ovalbumin are different from those on ovalbumin, and that these epitopes reflect a continuum of denatured conformations. PMID- 8619914 TI - No correlation between CD73 expression and ecto-5'-nucleotidase activity on blood mononuclear cells in vitro. Evidence of CD73 (ecto-5'-nucleotidase) on blood mononuclear cells with distinct antigenic properties. AB - Monoclonal antibodies against ecto-5'-nucleotidase (ecto-5'-NT) have been clustered to CD73. The aim of the present study was to elucidate whether specific ecto-5'-NT activity on blood mononuclear cells (BMC) was correlated with CD73 expression measured by flow cytometry. During culture of CD73-negative lymphocytes the percentage of cells with ecto-5'-NT activity increased without there being a comparable increase in CD73 expression. After 2 days' culture, 9% (3-16, median and range given) demonstrated ecto-5'-NT activity, whereas CD73 positive cells comprised only 1.6% (0.0-3.0). These results show the existence of lymphocytes with ecto-5'-NT activity but without expression of CD73. The ecto-5' NT activity increased significantly (p<0.02) during culture of unseparated BMC, whereas the number of anti-CD73 binding sites did not change. As a consequence. the number of anti-CD73 binding sites per U ecto-5'-NT activity decreased during culture. Exposure to interleukin-4 or prostaglandin E2 changed the enzymatic activity but not the number of anti-CD73 binding sites. Treatment of BMC with phosphatidylinositol-specific phospholipase-C released 57% (51%-75%) of the ecto 5'-NT activity into the supernatants, without a detectable decrease in CD73 expression. The fact that ecto-5'-NT was removed from the supernatant after precipitation with anti-CD73 showed that the released ecto-5'-NT activity was due to enzymatic activity of CD73 molecules. The results of this study indicate that CD73 exists on BMC in isoforms with distinct capacities to bind mouse monoclonal anti-CD73. PMID- 8619915 TI - Dynamics of bacterial translocation in acute liver injury induced by D galactosamine in rat. AB - Bacterial translocation may play a role in acute liver injuries as high rates of infectious and septic complications are observed in these clinical conditions. Increased passage of endotoxin and translocating bacteria not only potentiates the extent of liver injury, but may also play a determining role in its final outcome. In this paper the incidence of bacteria] translocation in acute liver injury in rats is evaluated with other important pathological changes observed at different time points after liver injury induced by intraperitoneal administration of D-galactosamine. The bacterial translocation to the blood and other extraintestinal sites starts 3 h after induction of liver injury and is not found to be related to light microscopic changes in the liver or ileal or cecal mucosa, detectable levels of endotoxin in the portal blood, or DNA changes in the small and large intestinal mucosa, but corresponds to the release of liver enzymes in the serum. PMID- 8619916 TI - Conservative hydrophobic interdomain contacts of IFN-gamma remain in P17 matrix protein of HIV-1. AB - A constructed scheme of the surface layers containing helices C, D, and E' of various polypeptide chains which participate in the interdomain contacts in IFN gamma demonstrated two sites of localization of the conservative hydrophobic amino acids. An analogous scheme of the interaction of helices B, C, and D in the p17 matrix protein of HIV-1 showed that the majority of the hydrophobic positions are similar. These data confirm the structural similarity between p17 and IFN gamma. PMID- 8619917 TI - Systemic treatment in the rat with epidermal growth factor causes polycystic growth of the ovaries. AB - BACKGROUND: It has previously been suggested that epidermal growth factor (EGF) plays a role in the function of the ovary. We administered systemic EGF to assess the influence of EGF receptor stimulation on the morphology of the ovaries. METHODS: Forty-eight female Wistar rats were allocated to five groups receiving EGF treatment (150 microgram/kg/day) for 0 (controls), 1, 2, 3 and 4 weeks. All rats were exactly 8 weeks at the start of the experiment and 12 weeks at sacrifice. The EGF was administered in the weeks prior to sacrifice. At sacrifice, the perfusion-fixed ovaries were removed and weighed, and the volumes of tissue components were quantified using stereology. RESULTS: EGF administration increased the total weight of the ovaries from 129 +/- 18 mg in the controls to 158 +/- 29 mg (p<0.05) after one week. In subsequent weeks the total weight increased to 230 +/- 73 mg (p<0.001). The weight gain after one week of treatment was exclusively due to a fourfold increase in follicular cyst volume (p<0.01). In subsequent weeks the cyst volume was increased up to eightfold. After 2, 3 and 4 weeks of treatment the quantity of luteinizing cells was likewise increased by 70% (p<0.01). CONCLUSION: EGF administration causes the follicular cells to produce cysts and increases the quantity of luteinizing cells. PMID- 8619918 TI - Polyomavirus-infected oligodendrocytes and macrophages within astrocytes in progressive multifocal leukoencephalopathy (PML). AB - Polyomavirus-infected oligodendrocytes and in some instances virus-carrying macrophages were demonstrated within the cytoplasm of astrocytes by double immunostaining of brain lesions in a case of PML following bone marrow transplantation. The infected oligodendrocytes and CD68+ cells were usually partially or completely invested by the cytoplasm of reactive and infected or noninfected giant pleomorphic astrocytes. By electron microscopy (EM), internalized infected oligodendrocytes were shown to release polyomavirus particles possibly infectious to the astrocytes. These observations seem to indicate that astrocytes in PML can become infected by direct contact with internalized infected oligodendrocytes and possibly also by virus-loaded macrophages. Correspondingly, astrocyte/oligodendrocyte/macrophage cell-to-cell interactions appear to play an important role in the pathogenesis of PML lesions. PMID- 8619919 TI - Predictors of joint damage in rheumatoid arthritis. AB - Rheumatoid arthritis (RA) is the dominant form of destructive chronic arthritis with the potential to cause substantial disability and permanent functional impairment. The final extent and progression rate with time, however, varies markedly. In order to study effects of intervention and to support early aggressive and atoxic therapy in selected cases, predictive disease markers are needed. Recent advances regarding joint tissue composition and pathophysiology have defined a number of biological marker candidates which need to be explored for possible prognostic information. Some markers are characteristic for RA, such as rheumatoid factors and certain autoantibodies, which although they are more prevalent among patients with aggressive disease are not sensitive as predictors in early disease. Genetic susceptibility markers have been claimed to be good predictors of persisting arthritis in early synovitis clinics, but their role as severity markers in established disease is limited. Unspecific markers of inflammation, notably ESR or CRP when persistently elevated, are useful to monitor disease course and newer markers need to document their superiority over these. Another group of markers are attractive because of enriched or exclusive occurrence in joint tissue, and altered metabolism in joint disease. Thus, collagen type III propeptides, hyaluronates, and neopterin originating in the synovium could be useful, and, in particular, hyaluronate levels indeed do provide some predictive information. Highly tissue-specific cartilage metabolites include aggrecan fragments, collagen II fragments, cartilage oligomeric matrix protein (COMP) and the extraarticular cartilage matrix protein (CMP). When used alone or in combination in early disease some information can be obtained which may in the future facilitate prognostication. Bone metabolism can be monitored and there are different markers for synthesis and resorption. Meanwhile, whilst the new markers are essential research tools, their routine clinical usefulness remains to be proven. PMID- 8619920 TI - Diffuse filiform polyposis of the colon in Crohn's disease. Case report. AB - A case of giant diffuse filiform polyposis of the entire colon with pathological features of Crohn's disease in a 30-year-old man is presented. The most important aspects and difficulties with differential diagnosis of filiform polyposis are discussed. PMID- 8619921 TI - Combined acinic cell mucoepidermoid carcinoma of the parotid gland. Report of a case with immunohistochemical study. AB - A case where mucoepidermoid and acinic cell carcinoma occurred simultaneously in the left parotid gland of a 67-year-old man is presented. Histologically, the neoplasm contained diagnostic areas of these tumors close to each other within the same tumor mass. A complete immunohistochemical study was performed and confirmed the presence of both components. To the best of our knowledge no similar case has previously been reported. PMID- 8619922 TI - Macrophage inflammatory protein-1 alpha mediated growth inhibition in a haemopoietic stem cell line is associated with inositol 1,4,5 triphosphate generation. AB - Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells. PMID- 8619923 TI - The Hox-2.4 gene is not involved in the generation of IL-3 dependent multipotent FDCP-mix cell lines. AB - The establishment of IL-3-dependent multipotent progenitor cell lines from Hox 2.4-expressing bone marrow cells suggests that homeobox genes may contribute to immortalization of early myeloid cells. A survey of 20 independently derived multipotent IL-3-dependent cell lines established from either src-virus-infected long-term bone marrow cultures (FDCP-mix) or Multi-CSF-virus (M3MuV)-infected bone marrow revealed that Hox-2.4 was not expressed in any of these cell lines. In addition DNA rearrangements were not observed. We conclude that activation of Hox-2.4 is not an obligatory event in the immortalization of early myeloid cells. PMID- 8619924 TI - Stimulation of thrombopoiesis in mice by fibroblast growth factor 9. AB - Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9 treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes. PMID- 8619925 TI - PDGF-BB triggered cytoplasmic calcium responses in cells with endogenous or stably transfected PDGF beta-receptors. AB - Platelet-derived growth factor-BB (PDGF-BB) triggered signal transduction was investigated in human foreskin fibroblasts with endogenous PDGF beta-receptors, and porcine aortic endothelial (PAE) cells with stably transfected PDGF beta receptors. Immunoprecipitation and immunoblotting showed that PDGF induced dose dependent autophosphorylation of PDGF beta-receptor, and the PLC-gamma associates with autophosphorylated PDGF beta-receptors and becomes phosphorylated. Activation of PLC-gamma is known to induce fluctuations of the concentration of cytoplasmic calcium ([Ca2+]i). Microfluorometry and digital imaging were employed for measurements of the concentration of [Ca2+]i. In both cell types the growth factor induced four types of [Ca2+]i responses; no rise, a small and sluggish monophasic rise, a biphasic rise with an initial transient peak followed by a sustain elevation, and finally regular oscillations. The frequencies and amplitudes of the oscillatory responses were independent of agonist concentration after stimulation with PDGF-BB. Latency, the period from application of stimulus to the first [Ca2+]i peak, was reduced at higher concentrations of agonist. Also, the proportion of responding cells increased with higher concentrations of ligand. Oscillations of [Ca2+]i were elicited at submaximal concentrations of agonist. In PAE cells PDGF-BB triggered a single [Ca2+]i peak in absence of external Ca2+. Ligand-induced oscillations and sustained increases of [Ca2+]i were counteracted by the inorganic Ca2+ channel blocker Ce3+. These results show that similar types of [Ca2+]i responses occur in different cell types independently of whether the PDGF beta-receptors are expressed endogeneously or after transfection. Potentially, the different [Ca2+]i responses have distinct physiological consequences. PMID- 8619926 TI - Localisation of bradykinin-like immunoreactivity and modulation of bradykinin evoked phospholipase D activity by 17 beta-oestradiol in human endometrium. AB - Bradykinin may act as a promoter of endometrial regeneration. Bradykinin-like immunoreactivity was detected immunocytochemically in the glandular epithelium and stroma of human endometrium. The staining was localized around the stroma and especially in the cells undergoing mitosis. Relatively weak staining was seen in the stromal cells of secretory endometrium, which was predominantly localised around the basal vacuoles of endometrial glands. During the late secretory phase, the intensity of staining was diminished throughout the endometrium: the glandular epithelium showed weak staining and stroma appeared negative. As phosphatidate, the product of phospholipase D pathway, may mediate cell proliferation, the effect of 17 beta-oestradiol on bradykinin-evoked phospholipase D activity assayed as accumulation of [3H]phosphatidylbutanol ([3H]PtdBut) was examined in [3H]myristic acid-labelled primary cultures of human endometrial stromal cells. Bradykinin induced a rapid accumulation of [3H]PtdBut in a time-dependent manner, indicating phospholipase D activation. Pretreatment of stromal cells with 17 beta-oestradiol enhanced the bradykinin-evoked phospholipase D activity. These results suggest that bradykinin-like immunoreactivity is strongly associated with proliferative stromal cells undergoing mitosis, a process that may be mediated by phospholipase D activation as the magnitude of this enzyme's activation in vitro appears to be regulated by 17 beta-oestradiol. PMID- 8619927 TI - The interdependent modulation of hyaluronan synthesis by TGF-beta 1 and extracellular matrix: consequences for the control of cell migration. AB - The principal objective of this communication has been to determine the manner in which two tissue culture substrata (plastic dishes and type I collagen gels) modulate the response of adult skin fibroblasts to TGF-beta 1 with respect to hyaluronan (HA) synthesis. Our results indicate that (a) fibroblasts cultured on collagen gels synthesised more HA compared to cells plated at the same density on plastic dishes, (b) this up-regulation in total HA synthesis by collagen-cultured cells was accompanied by an increase in the relative proportion of high molecular mass species of newly synthesised HA, and (c) the specific effect of TGF-beta 1 on HA synthesis was dependent upon the substratum: i.e. TGF-beta 1 inhibited HA synthesis by subconfluent fibroblasts cultured on both substrata, had no apparent effect on confluent cells cultured on collagen gels, and stimulated HA synthesis by confluent cells cultured on plastic dishes. The TGF beta-stimulated of HA synthesis by confluent fibroblasts cultured on plastic dishes persisted when these cells were transferred to collagen gels in the absence of further TGF-beta 1: interestingly, a second exposure of these plastic pre-incubated cells to TGF beta 1 whilst growing on collagen resulted in a down-regulation in HA synthesis. Confluent fibroblasts pre-incubated with TGF-beta 1 for 24 h on plastic dishes (i.e. under conditions which stimulate HA synthesis) also displayed an HA dependent stimulation in cell migration when subsequently plated onto collagen gels in the absence of further cytokine. PMID- 8619928 TI - Identification of fibroblast growth factor 9 (FGF9) as a high affinity, heparin dependent ligand for FGF receptors 3 and 2 but not for FGF receptors 1 and 4. AB - Fibroblast growth factors (FGF) are multifunctional, heparin binding polypeptides that share structural similarity, but differ in their target cell specificity and expression pattern. Here we describe the cloning and expression of the mouse homologue of FGF9, and the use of a panel of soluble FGF receptors and genetically engineered cells to study its receptor binding specificity. FGF9 is found to bind with high affinity (kd: 0.25 nM) to FGFR3, for which a specific ligand has not yet been identified. FGF9 can also bind, albeit with a lower affinity, to FGFR2 but does not bind FGFR1 or FGFR4. There is no significant binding to either FGFR3 or FGFR2, expressed either as soluble receptors or in heparin sulfate deficient cells, in the absence of heparin. Moreover, receptor binding of FGF9 requires heparin in a manner specific to the receptor type. In conclusion FGF9 presents a unique case of ligand-receptor specificity and fulfills the criteria as a high affinity, heparin-dependent ligand for FGFR3. PMID- 8619930 TI - Genetic predisposition to breast cancer. PMID- 8619931 TI - Hypertrophic pulmonary osteoarthropathy: is there a role for radiotherapy to symptomatic sites? Case report and literature review. PMID- 8619929 TI - Colocalisation of vascular endothelial growth factor and its Flt-1 receptor in human placenta. AB - Vascular endothelial growth factor (VEGF) is an angiogenic protein which acts on both endothelial and trophoblast cells. In first trimester placenta, VEGF immunoreactive protein was detected in cytotrophoblast shell suggesting a role in the regulation of cytotrophoblast growth and differentiation as they also expressed VEGF receptor (flt-1) protein. VEGF and flt-1 immunoreactive proteins were expressed in Hofbauer cells within the villous mesenchyme, macrophages and in maternal decidual cells while weak VEGF immunoreactive protein was seen in syncytiotrophoblast surrounding the placental villi in first and second trimester placentae. At term, there was relatively weak VEGF and flt-1 immunostaining in the syncytiotrophoblast while intense VEGF immunostaining was seen in the Hofbauer and maternal decidual cells. Extravillous trophoblast showed immunostaining for flt-1 but no staining for VEGF. Both amnion and chorion expressed strong VEGF immunoreactivity throughout gestation. Smooth muscle cells surrounding the vein and arteries of the umbilical cord showed weak VEGF immunoreactivity while no immunoreactivity was localised in endothelial cells. VEGF stimulated parathyroid hormone-related protein (PTHrP) release (mean (+/- SD): basal, 0.96 +/- 0.03; 10 ng/ml VEGF165, 2.07 +/- 0.18 and 20 ng/ml VEGF165, 2.43 +/- 0.18 pmol/l/well of PTHrP1-86) in condition medium from immortalised first trimester trophoblast cell line. These results suggest that VEGF in addition to acting as an autocrine mitogen for trophoblast proliferation may also function as a paracrine mediator of vascular tone by releasing vasorelaxants from trophoblasts. PMID- 8619932 TI - Bilateral occipital bone infarction probably due to disseminated zygomycosis in a patient with lymphoma. PMID- 8619933 TI - Disease extent and response to chemotherapy in non-small cell lung cancer. PMID- 8619934 TI - ERRP-29 and ER staining in uterine lipoma and lipoleiomyoma. PMID- 8619935 TI - Oestrogen receptor analysis of paraffin sections and cytosol samples of primary breast cancer in relation to outcome after adjuvant tamoxifen treatment. The South Sweden Breast Cancer Group. AB - The aim of the present study was to compare oestrogen receptor (ER) analysis results obtained in cytosols of frozen breast cancer tissue (using biochemical assay) with those obtained in paraffin-embedded tissue (using immunoperoxidase staining with monoclonal antibodies (DAKO-ER, 1D5), and an ER positivity cut-off level of >10% stained nuclei). In 86% (84/98) of the samples the same ER status (28 negative and 56 positive) was obtained with both procedures. In eight cases, the paraffin section was ER positive but the corresponding cytosol sample ER negative, whereas six cases showed the opposite pattern. The ER positive subgroup manifested better outcome after adjuvant treatment than the ER negative subgroup (p = 0.003 (cytosol), and p = 0.004 (paraffin)). As compared with the percentage of stained nuclei, staining intensity yielded no additional information. Although the results of ER analysis of paraffin-embedded material seem promising, it is too early to prefer it to frozen tissue, though this would be useful when no frozen tissue is available. PMID- 8619936 TI - Use of a polyclonal antibody for the determination of the prognostic value of c erbB-2 protein over-expression in human breast cancer. AB - A rabbit-specific polyclonal antibody was obtained raised to a synthetic peptide corresponding to the 1238-1255 C-terminal predicted sequence of the c-erbB-2 protein. This antibody was used in an immunohistochemical procedure to detect the c-erbB-2 protein on a series of 88 paraffin-embedded human breast carcinomas. In 14/88 cases (16%) the c-erbB-2 protein was found to be overexpressed (immunohistochemical score > 1) with a good concordance with the previously determined mRNA level (79/88 cases: 90%). Prognostic significance of c-erbB-2 protein overexpression as detected by immunohistochemistry was tested by the log rank test. The relative risk of relapse is higher for patients with an immunohistochemical score > 1 (p = 0.00002). In a multivariate analysis of the c erbB-2 immunohistochemical score was the only powerful parameter (p < 1 x 10(-3). In conclusion, this antibody seems to be a valuable tool in estimating the c-erbB 2 protein regarded in our series as a parameter able to identify a subgroup of operable breast cancer patients with a high risk of relapse. PMID- 8619938 TI - Measurement and practical aspects of quality of life in breast cancer. AB - There are many aspects of quality of life which are non-intuitive and need to be studied to get information for the improvement of cancer treatment. There is general agreement that quality of life should be measured as an important end point in clinical trials, especially when palliative treatment is intended. The present paper summarizes the main aspects of measurement of quality of life as well as the results of quality of life studies concerning breast cancer. Many practical problems in our health care system clearly undermine the quality of life of cancer patients. The Finnish Hospital League ran a project to identify factors affecting quality of life in the treatment of breast cancer. Four important aspects are discussed, i.e. individual treatment choices, communication, psychological support and continuity of care. Most of the findings are likely to be generalizable within a Nordic cultural context, although Finland still devotes fewer resources to psychosocial support than do other Nordic countries. PMID- 8619937 TI - Social support and immune status during and after chemotherapy for breast cancer. AB - Social support and immune status were assessed in women treated with adjuvant chemotherapy for breast cancer. Perception of enhanced attachment was associated with an increased number of white blood cell levels three months after, but not during, chemotherapy. After treatment, patients with high attachment ratings had higher numbers and proportions of granulocytes, and lower proportions of lymphocytes and monocytes. It is concluded that the support experienced by a cancer patient can be associated with counts and proportions of leukocytes, but that this effect, if present during chemotherapy, is overridden by the biological factor that affects the haematopoetic process. PMID- 8619939 TI - High dose rate intracavitary brachytherapy in the treatment of nasopharyngeal carcinoma. AB - A retrospective study on 61 patients, with local persistent or recurrent nasopharyngeal carcinoma (NPC), treated during 1990-1992 with high dose rate intracavitary brachytherapy alone or combined with external irradiation, is presented. All 39 patients with persistent disease were treated solely with brachytherapy. The actuarial 3-year local failure-free survival (LFFS) rates of the persistent and recurrent groups were 82% and 45% respectively. The corresponding disease specific survival rates were 82% and 62%. Fifteen patients with recurrence received the combined modality treatment and their 3-year LFFS rate was 65%. Three out of 7 patients treated by brachytherapy could be controlled locally. The total dose given to the floor of sphenoid was an important predictor of local control. Of the 23 patients with persistent disease treated with < 17.5 Gy to this area, 6 failed locally as opposed to none of the 16 patients receiving a higher dose (p = 0.031). For those with recurrence treated by the combined modality, none of the 7 patients given >/= 57.5 Gy recurred while 5 local failures were observed among those receiving a smaller dose (p = 0.041). The general implications of these results for the treatment of NPC recurrence are discussed. PMID- 8619940 TI - Prediction of complications in gamma knife radiosurgery of arteriovenous malformation. AB - The incidence of complications following radiosurgical treatment of arteriovenous malformations (AVM) is presented. A simple relationship exists between average dose and risk of complications, and on this basis a model is presented that gives a qualitatively correct description of this relationship. The parameters of the model have been determined using a clinical material of 862 AVM treatments to give a quantitatively correct description of the risk of complications. The dose response curve is described by a double-exponential function. An accurate description of the dose-response curve at high dose levels is shown to be very important in radiosurgery. PMID- 8619941 TI - Sustained-release metoclopramide plus methylprednisolone versus placebo plus methylprednisolone as antiemetic prophylaxis during non-cisplatin chemotherapy. A randomized double-blind cross-over trial. AB - In a randomized double-blind cross-over trial, sustained-release metoclopramide (S) plus methylprednisolone (M) was compared with placebo (P) plus methylprednisolone as antiemetic prophylaxis during two cycles of non-cisplatin chemotherapy. S was administered as 60 mg every 12 h commencing on the evening before chemotherapy up to total of 300 mg metoclopramide in 2.5 days. Evaluation of nausea and vomiting was done by self-assessment schemes and visual analog scales. Fifty patients were included and 36 fulfilled both cycles. Mild nausea and vomiting were experienced by 81% and 83% in the S + M and P + M groups, respectively, while 42% and 39% showed complete control of nausea and vomiting during the first day of treatment. Moderate-dose S did not add to the antiemetic efficacy of M in non-cisplatin chemotherapeutic regimens. PMID- 8619942 TI - Use of tetranectin, CA-125 and CASA to predict residual tumor and survival at second- and third-look operations for ovarian cancer. AB - Tetranectin (TN), CA-125 and CASA were measured in serum prior to 63 second-look and 5 third-look operations for ovarian cancer. Patients with residual tumor had significantly lower levels of TN and higher levels of CASA and CA-125 compared with tumor-free patients. The predictive values PVPos = 100% and PVNeg = 50.9% were found for TN at 9.3 mg/l. For CASA, a predictive value PVPos = 100% was found at 10 U/ml with a corresponding PVNeg = 52.7%. At the cut-off 35 U/ml for CA-125, the PVPos was 100% and the PVNeg = 53.6%. By combining the markers, PVNeg increased to 61.7% with a PVPos on 100%. Significantly differences in survival were found by lifetable analysis between patients tested as positive and negative respectively for any of the markers. Using multivariate Cox analyses, it was found that every marker had an independent prognostic function for survival. PMID- 8619943 TI - Combination chemotherapy with a five-drug regimen for invasive thymoma. AB - Nine patients with stage III and stage IV thymoma were treated with cisplatin, vincristine, lomustine, cyclophosphamide and prednisolone. Two patients (22%) obtained remission, and four patients (44%) showed no change for 11 to 31 months. It was found that this five-drug regimen did not improve the results obtained by other chemotherapy modalities for thymoma. PMID- 8619944 TI - Palliation of dysphagia in patients with malignant esophageal strictures. Comparison of results of radiotherapy, chemotherapy and esophageal stent treatment. AB - Dysphagia is the earliest and the most common symptom of malignant disease in the esophagus. The palliative effects on dysphagia of radiotherapy (RT) and chemotherapy (CT) were evaluated retrospectively and compared with the effect of the self-expanding stent, evaluated in the prospective study. After completion of treatment, 78 (56%) of 140 patients treated with RT; 31 (49%) of 63 patients treated with CT; and 53 (81%) of 66 patients treated with stent insertion were free from dysphagia. Stent treatment has a good and prompt effect on dysphagia and can be recommended for palliation of patients with malignant esophageal strictures. PMID- 8619946 TI - Influence of fraction size on the development of late radiation enteropathy. An experimental study in the rat. AB - The use of large fraction sizes in radiotherapy may be associated with an increased risk of complications from late responding normal tissues. However, in the intestine, chronic injury may develop either as primary late effect or secondary to disruption of mucosal integrity as so-called consequential injury. Mucosal damage is relatively less sensitive to changes in fraction size than late reacting, slowly proliferating cells. The relationship between fraction size and chronic radiation enteropathy in a given situation may thus depend on which of the two mechanisms that predominates. Most previous studies of the influence of fraction size on radiation injury are confounded by differences in treatment time. The present study was therefore designed to assess subacute and chronic radiation enteropathy after three different fractionation regimens where fraction size was the only experimental variable. A total of 96 male Sprague-Dawley rats were orchiectomized and a functionally intact loop of small intestine was transposed into the left scrotum. These 'scrotal hernias' containing intestine were subsequently exposed to 50.4 Gy localized fractionated irradiation over 18 days with either 2.8 Gy every 24 h, 4.2 Gy every 36 h, or 5.6 Gy every 48 h. Control animals were sham irradiated. The animals were observed for development of intestinal complications (intestinal obstruction or enterocutaneous fistula formation) up to 6 months after irradiation. Histologic damage was assessed in groups of animals at 2 weeks (subacute injury) and 26 weeks (chronic injury), using a previously validated radiation injury score (RIS). RIS increased significantly with increasing fraction size at both observation times. However, the increase was more pronounced at 26 weeks than at 2 weeks. Increased chronic injury was characterized by increased incidence and severity of mucosal ulceration, serosal thickening, vascular sclerosis and intestinal wall fibrosis. We conclude that increasing fraction size increases both subacute and, even more markedly, chronic injury in the intestine. With the fractionation regimens used here, the chronic radiation enteropathy develops as a combined consequential and primary late effect, but the primary mechanism predominates. PMID- 8619945 TI - Relationship between intestinal fibrosis and histopathologic and morphometric changes in consequential and late radiation enteropathy. AB - Intestinal fibrosis is a marked feature of late radiation enteropathy. This study assessed the time dose fractionation relationships of radiation-induced fibrosis in order to elucidate possible pathogenetic mechanisms. In 290 male Sprague Dawley rats, a loop of small bowel was transposed to the left side of the scrotum. Three weeks later, the transposed segment was irradiated with either single dose or various fractionated regimens. The animals were observed for radiation-induced intestinal complications and killed in groups, 2 and 26 weeks after completion of irradiation. A semiquantitative histopathologic radiation injury score, morphometry of the submucosa, submucosal arterioles, intestinal surface area, and relative collagen content were used as endpoints. Fibrosis, measured by collagen assay and radiation injury score, increased with total dose, increasing fraction size and reduction in overall treatment time. This paralleled the results of morphometric assessment of mucosal surface area. Differences in vascular morphometry were only statistically significant in response to changes in total dose and fraction size and not with changes in overall treatment time. We conclude that fibrosis increases with increasing observation time, increasing fraction size, increasing total dose, and reduction of interfraction interval. Consequential injury, occurring as a result of disruption of mucosal integrity, seems to be an important mechanism for development of intestinal fibrosis. In contrast, vascular injury is relatively independent of this mechanism. PMID- 8619947 TI - Primary invasive breast carcinoma in Oslo 1980-1989. Incidence and delay. AB - A retrospective review was performed on 2704 consecutive patients in Oslo in whom histologically or cytologically confirmed primary invasive breast carcinoma had been diagnosed between 1980 and 1989. The age-adjusted incidence rates were significantly higher in the city of Oslo compared with those of the whole country and remained unchanged during the study period. The percentage of patients who could be treated radically remained unchanged. Among the patients with radical treatment the distribution of pT category and stage was similar during the first and last years. The median delay (from onset of symptoms to start of treatment) of two months remained unchanged during the decade and was not related to patient's age, histological grade or tumour localization in the quadrants of the breast. With increasing duration of delay the number of patients not suitable for radical treatment increased. When considering all 2704 patients, the radicality of treatment, the patient's age and delay were correlated with tumour-specific survival and remained independent factors of tumour-specific survival in the multivariate analysis. The unchanged distribution of pT category and stage in the radically treated patients during the decade surveyed is most probably related to the lack of screening mammography in Norway. PMID- 8619948 TI - Antitumor and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma in vivo. AB - The antitumor and radiosensitizing effects of withaferin A (WA), a steroidal lactone from Withania somnifera, was studied on Ehrlich ascites carcinoma in vivo. The acute LD50(14) for WA in Swiss mice was approximately 80 mg/kg. Twenty four hours after i.p. inoculation of 10(6) tumor cells, WA was injected i.p. at different dose fractions (5 or 7.5 mg/kg x 8, 10 mg/kg x 5, 20 or 30 mg/kg x 2) with or without abdominal gamma irradiation (RT, 75. Gy) after the first drug dose. Increase in life span and tumor-free survival were studied up to 120 days. The drug inhibited tumor growth and increased survival, which was dependent on the WA dose per fraction rather than the total dose. Combination of RT with all the drug schedules increased tumor cure and tumor-free survival, the best effect seen after 2 fractions of 30 mg/kg each. In another experiment WA was given as 2 (40 mg/kg x 2), 3 (30 mg/kg x 3) or 4 (20 mg/kg x 4) fractions at 5, 7 or 10 days after tumor inoculation with or without RT after the first drug dose. At 7 and 10 days after inoculation the drug was effective only at 40 mg/kg x 2, but with RT 30 mg/kg x 3 produced an equal effect (20% survival) on 7 day old tumors. PMID- 8619949 TI - S1 nuclease: immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site. AB - A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site. PMID- 8619950 TI - Searching for pharmacophoric patterns in databases of three-dimensional chemical structures. AB - This paper provides an overview of the research that has been carried out in Sheffield over the last decade into searching techniques for databases of three dimensional (3D) chemical structures. A 3D structure or query pattern is represented by a labelled graph, in which the nodes and the edges of the graph are used to represent atoms and the associated inter-atomic distances, respectively. The presence of a pharmacophore in each of the structures in a database can then be tested by means of a subgraph isomorphism algorithm, the computational requirements of which are minimized by the use of an initial screening procedure that eliminates the majority of the structures from the subgraph-isomorphism search. Analogous graph-based representation and searching methods can also be used with flexible 3D structures: in this case, the edges of the graphs represent inter-atomic distance ranges and a final conformational search needs to be carried out for those molecules that match the query pharmacophore in the subgraph-isomorphism search. The paper also reviews related work on the automatic identification of pharmacophoric patterns and on 3D similarity searching. PMID- 8619951 TI - Blocking of CD4 cell receptors for the human immunodeficiency virus type 1 (HIV 1) by chemically modified bovine milk proteins: potential for AIDS prophylaxis. AB - The chemical transformation of synthetic combinatorial libraries to increase the diversity of compounds of medicinal interest was reported recently. Chemical modification of natural products represents a complementary approach to accomplish this aim. Modification of lysines by aromatic acid anhydrides, preferentially by 3-hydroxyphthalic and trimellitic anhydrides and trimellitic anhydride chloride, converted commonly available proteins (human and bovine serum albumin and casein) into potent inhibitors of (i) binding between the HIV-1 gp 120 envelope glycoprotein and the CD4 cell receptor, probably owing to their binding to CD4, and (ii) infection by HIV-1. Modified bovine milk proteins are also potent HIV-1 inhibitors and may have potential for anti-HIV-1 prophylaxis. PMID- 8619952 TI - Conservation of water molecules in an antibody-antigen interaction. AB - The solvation of the antibody-antigen Fv D1.3-lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3-lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-anti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody-lysozyme complexes. The antibody-lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3-lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody-antigen interaction the solvent molecules fill many interface cavities. Comparison with x ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Up to 23 additional water molecules are also found in the antibody-antigen interface, however these waters do not bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody- antigen interface with the formation of the complex. While the current crystal models of the D1.3-lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well- ordered interface waters contribute a net gain of 10 hydrogen bonds to complex stability. PMID- 8619953 TI - Control of axis formation in Xenopus by the NF-kappa B-I kappa B system. AB - We describe the isolation and analysis in Xenopus of Xrel2, a novel member of the NF-kappa B/Rel protein family that remains to be described in other vertebrates. We show that Xrel2 is expressed throughout development but with higher levels in pre-gastrula embryos. Like other NF-kappa B/Rel proteins, Xrel2 protein is able to bind DNA at a kappa B-Motif. Ectopic expression of Xrel2 disrupts normal morphogenesis at the early gastrula stages suggesting that the NF-kappa B/Rel family have developmental functions at stages earlier than previously thought. We also show that the Xrel2 over-expression phenotype can be rescued by co expression of I kappa B-alpha and that ectopic expression of I kappa B-alpha or I kappa B-gamma alone has no effect on development. Finally, we show that Xrel2 does not divert animal caps from an ectodermal to a mesodermal cell fate. Overall, these results suggest that the NF-kappa B/Rel family does have key functions in early vertebrate development, however, there is not a simple conservation of the Drosophila dorsal pathway. PMID- 8619954 TI - Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish. AB - We isolated two zebrafish sequences containing a homeobox related to orthodenticle (otd), a gene expressed in the developing head of Drosophila. One of these is clearly homologous to Otx1, a homeobox gene previously reported to be expressed in the developing rostral brain of the mouse. We termed this zebrafish gene otx1. The second gene is not as closely related to Otx1 and is equally divergent from Otx2, a second homeobox gene expressed in the developing rostral brain of the mouse. We termed it otx3, even if a corresponding murine Otx3 gene has not been reported yet. Both genes are expressed in early-gastrula zebrafish embryos in the involuting presumptive anterior mesendoderm. With the extension of the body axis, the expression domain of both genes extends to neuroectodermal regions fated to become fore- and mid-brain. From this stage the expression domains of the two genes differ slightly from each other but both cover the rostral brain with a sharp posterior boundary coinciding with that between midbrain and hind-brain. This late expression closely corresponds to that of the murine Otx1 gene, whereas the earliest expression of both zebrafish otx genes is different from that of Otx1 and reminiscent of that of Otx2 in the mouse. In this light, the zebrafish otx1 and otx3 genes appear to share some expression features of both murine Otx1 and Otx2. It will be of considerable interest to study the specific role of the various genes of the otx family in the development of the zebrafish brain regions. The peculiar spatio-temporal pattern of these genes during early zebrafish gastrulation suggests a role of this gene family in interactions between anterior mesendoderm and neuroectoderm. PMID- 8619955 TI - eFGF is expressed in the dorsal midline of Xenopus laevis. AB - A detailed study of the expression pattern of embryonic fibroblast growth factor (eFGF) during early Xenopus development has been undertaken using whole-mount DIG in situ hybridization. We show that the zygotic expression of eFGF is activated in the mesoderm of the early gastrula and is first visualized as a ring around the blastopore, with significantly higher levels of expression on the dorsal side of the embryo. As gastrulation proceeds, eFGF transcripts become increasingly abundant in the dorsal blastopore lip. In the early neurula eFGF expression can be detected in the extreme posterior of the embryo around the closed blastopore and in the cells of the notochord. This latter result is significant and represents the first report of a Xenopus FGF that is expressed in the notochord. In addition, we show that during gastrula and neurula stages, expression of eFGF closely follows the expression of the Xenopus brachyury (Xbra) gene. During later development eFGF expression is localized to the tail-bud region and a stripe at the mid-brain/hind-brain junction. These data provide further evidence that FGFs play an important role in regulating the expression of brachyury in the developing mesoderm. PMID- 8619956 TI - Posterior localization of the Drosophila Gi alpha protein during early embryogenesis requires a subset of the posterior group genes. AB - Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures bicoid, swallow, and exuperantia did not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations which eliminate posterior structures, cappuccino, spire, staufen, mago nashi, valois, and oskar, prevented the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes lower in the putative hierarchy vasa, tudor nanos, and pumilio did not affect Gi alpha redistribution. From these results we conclude that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen nanos. PMID- 8619957 TI - Characterization of cDNAs encoding two chick retinoic acid receptor alpha isoforms and distribution of retinoic acid receptor alpha, beta and gamma transcripts during chick skin development. AB - The amino acid sequence of the retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) can be divided into six functional domains (A-F), different isoforms arising from the presence of different A domains by differential splicing. In order to address the respective roles of the different RARs during skin morphogenesis in birds, cDNAs encoding two chick RAR alpha isoforms (alpha1 and alpha2) have been isolated. While the A1 and B-F domains of the RAR alpha are highly conserved across species, the chick A2 domain contains 50% specific amino acids. The three RAR alpha, beta and gamma genes display specific patterns of expression during chick skin morphogenesis. As in mouse, RAR alpha and gamma transcripts are present in both the dermis and epidermis during the first stages of skin appendage formation. Furthermore, Northern blot analysis suggests that different RAR alpha and gamma isoforms could be successively required during feather formation. The RAR gamma gene, continuously expressed in the epidermal cells in both chick and mouse, is thus likely to play a similar role in skin development in these two species. However, RAR alpha transcripts, only transiently detected during mouse skin development, still accumulate in epidermis during the later stages of chick skin differentiation. Furthermore, RAR beta transcripts, never detected during normal development in mouse skin, are actually present at the early stages of chick skin morphogenesis. Thus, our results suggest that the role of the three RAR in skin development has not been strictly conserved in the different classes of vertebrates. PMID- 8619958 TI - Developmentally regulated chromatin acetylation and histone H1(0) accumulation. AB - There exists a close relationship between core histone acetylation and the induced expression of the histone H1(0) gene. We took advantage of this fact to evaluate the influence of chromatin hyperacetylation on the developmentally regulated expression of this specific gene. In this study, the in situ immunodetection approach has been used to analyze both the acetylated histone H4 isoforms and histone H1(0) accumulation during early Xenopus laevis development. We have chosen two stages of development, gastrula stage, when H1(0) is not expressed and not inducible by butyrate treatment, and stage 27 when H1(0) is not expressed but is inducible by butyrate. At stage 27 of development, the early induced accumulation of histone H1(0) under butyrate treatment, occurs mainly in tissues that express the protein normally during later development. These experiments suggest that histone acetylation may be part of a pathway which, in a specific set of cells, keeps H1(0) and probably a series of specific genes, competent for transcription, but cell-specific factors are involved in the induced expression of these genes. PMID- 8619959 TI - Differential expression of the full-length and secreted truncated forms of EGF receptor during formation of dental tissues. AB - The developmental regulation of various receptor forms may be a key-element in the local fine tuning of growth factor effects. The present study focuses on the tissue- and stage-specificity of the alternative splicing of EGF receptor transcripts in the rat incisor. In situ hybridization, as well as light- and electron-microscopic immunolocalization were performed with a set of tools which enable us to discriminate the full-length and secreted truncated forms of EGF receptor. Our data show that, apart from a transient expression in differentiating odontoblasts, EGF receptor expression was predominantly observed in the dental epithelium. In the crown, the expression of the full-length EGF receptor was maximal during preameloblast proliferation and differentiation, decreased in differentiated ameloblasts, and remained low throughout enamel secretion. On the other hand, maturation stage ameloblasts, which regulate the final mineralization of enamel, express high levels of the full-length EGF receptor. In contrast with ameloblasts, epithelial supra-ameloblastic cells, which are not directly involved in the deposition of enamel matrix, showed an alternating predominance of the secreted truncated form during the secretion stage, and the full-length form during the maturation stage. The presence of the secreted truncated EGF receptor form was supported by the electron microscopic detection of extracellular aggregates of immunoreactive EGF receptor. Finally, Northern-blotting of enamel organ samples confirmed the presence of transcripts corresponding to mRNAs of both EGF receptor forms. During root formation, a decreasing gradient of full-length EGF receptor form expression was observed from the apical loop to the disrupting zone in root epithelium. The secreted truncated EGF receptor form was essentially detected in epithelial cells of the disrupting zone of root epithelium. During crown formation, the secreted truncated EGF receptor form, which appears to be synthesized by epithelial supra-ameloblastic cells and secreted toward ameloblasts, may competitively bind EGF receptor ligands and modify activation of the full-length EGF receptor. PMID- 8619960 TI - Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors. AB - An excess of retinoic acid (RA) in the mouse embryo in utero produces hypochondrogenesis and severe limb bone deformities. Since one of the RA receptors--RAR-beta 2, is specifically induced in the limb bud cells upon treatment of embryos with teratogenic doses of RA, we investigated if this receptor played a role in teratogenesis by regulating the process of chondrogenesis. In micromass cultures of mouse limb bud mesenchymal cells, we found that a downregulation of RAR-beta 2 as well as several other RAR isoforms by supplementation of the culture medium with specific oligodeoxynucleotides stimulated chondrogenesis: cartilage nodule number, sulfated proteoglycans, and synthesis of collagen type IIB were all enhanced in a dose-dependent manner. However, only the antisense RAR-beta 2 probe efficiently prevented the strong inhibitory effects of exogenous RA on chondrogenesis in these cells. The data suggest that the RAR-RA complexes play a role in position-dependent patterning of the limb skeleton in normal development and that, in particular, RAR-beta 2 serves to prevent the mesenchymal cells from expressing their chondrogenic bias. Our results further strengthen the argument that RA-dependent elevation in RAR beta 2 levels plays a unique role in RA-induced teratogenesis. PMID- 8619961 TI - Lung fluid restriction affects growth but not airway branching of embryonic rat lung. AB - During the later stages of fetal life lung growth and development is dependent upon a variety of factors, including a normal amount of liquid within the lung's lumen. To investigate whether embryonic lung epithelium secretes fluid and whether lung liquid is essential for proper embryonic airway lung growth and branching, we incubated 12-day rat lung primordia (term=22 days) in submersion culture in serum-free medium for 48 h in room air (21% O2/5% CO2). Under these conditions, lung growth and branching proceeded but at a slower rate when compared to growth and branching in vivo. Neither addition of serum nor incubation in a fetal O2 concentration (=3% O2) changed the growth rate or the degree of branching in vitro. The luminal area of the explant increased progressively with time in culture. Inhibitors of active Cl- secretion (200 microM bumetanide and 1 mM furosemide) significantly reduced the lumen size compared with control. A similar effect was noted with lung explants of 13-15 days of gestation. Branching morphogenesis was not impaired by lung fluid reduction. Reduction of luminal liquid significantly increased DNA synthesis of 12-day embryonic lung explants, but this effect of bumetanide and furosemide on DNA synthesis was reversed when 13-15 day lung explants were used. These data suggest that embryonic lung epithelium secretes fluid and that the secretion is chloride dependent. Lung fluid is involved in controlling lung growth but not branching of the embryonic rat lung. PMID- 8619962 TI - Nuclear remodelling and early development in cryopreserved, porcine primordial germ cells following nuclear transfer into in vitro-matured oocytes. AB - Nuclear transfer was conducted in the pig using, as karyoplasts, primordial germ cells which had been cryopreserved. The cytoplasts were presumptive S- or MII phase, in vitro-matured oocytes, which had been enucleated mechanically. Enucleation was effective in 94.3% of cases. Karyoplasts were introduced into the perivitelline space, in close contact with the cytoplasts, and the complexes fused by electrical stimulation and activation. Activation was successful in 82 88% of nonmanipulated, pulsed oocytes, and in 55% of germ cell-oocyte complexes. The reconstituted embryos were examined for nuclear remodelling and cleavage in vitro. Nuclear swelling was more prominent when MII-phase cytoplasts, rather than S-phase, cytoplasts, were used. After 24 h in culture, the cleavage rate was not significantly different whether blastomeres or primordial germ cells were used as karyoplasts, and whether MII-phase or S-phase cytoplasts were used. However, after 72 h in culture, the developmental rate was higher when MII-phase cytoplasts (75%) were used for the recipients of blastomeres compared with S phase cytoplasts (38.5%, p < 0.05). Similar tendencies were observed with germ cell nuclear transfer when inositol was used as medium for electrofusion (60% vs 27.8%, p < 0.05). Furthermore, when MII-phase cytoplasts were used, the nuclear transferred embryos derived from blastomeres developed at a significantly higher rate than from primordial germ cells (37.5%, p < 0.05). We conclude that cryopreserved primordial germ cells are competent to undergo nuclear remodelling and cleavage during 72 h or incubation in vitro to the 4-cell stage, following nuclear transfer to enucleated, activated (S-) or MII-phase oocytes. This experimental system may help to elucidate events in the early development of pig embryo following nuclear transfer using germ cells as karyoplasts. PMID- 8619964 TI - Expression of membrane targeted aequorin in Xenopus laevis oocytes. AB - We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved. PMID- 8619963 TI - Cell mixing during the early development of mouse aggregation chimera. AB - Two different inbred strain combinations of mouse aggregation chimeras - C3H/HeN (H-2k) x C57BL/6N (H-2b) and C3H/HeN x BALB/cA (H-2d) were used for cell mixing analysis at two points in time - 24 h after aggregation (just prior to transplantation into foster mothers) and 7.5 days post coitum (p.c.). The cell proportion of two H-2 haplotypes at the blastocyst stage was studied using a fluorescence-labeled monoclonal antibody recognizing a C3H-specific alloantigen - CSA (C3H strain-specific antigen) and laser scanning confocal microscopy. The 7.5 day-old chimeras were sectioned and subsequently processed by sensitive biotinylated antibody - avidin peroxidase immunohistochemical technique. Our results showed that 24 h after aggregation (blastocyst stage), there was equal cell mixing and no mouse strain used in the present study was dominant at this time. In 7.5-day-old C3H/HeN x BALB/cA chimeras, cells of both genotypes were intermingled, but the C3H/HeN strain was dominant in all cases. In contrast, the combination C3H/HeN x C57BL/6N clearly showed reduced numbers of C3H/HeN cells (CSA-positive) in 83% of the chimeras evaluated. Generally, CSA positive cells were found only in randomly distributed small distinct areas representing less than 20% of embryonal cells. Surprisingly, the extraembryonal ectoplacental cone was uniformly CSA positive in some C3H/HeN x C57BL/6N chimeras. Furthermore, in 36% of normally implanted chimeras of both strain combinations progressive degeneration was observed. We suggest that the cell mixing pattern as well as the absolute number of cells derived from each strain in the aggregation chimera can be affected by specific immune interactions involving H-2 haplotype combinations of the allogeneic fetus and the fully immunocompetent host organism, at later points in development. PMID- 8619965 TI - ENDO A cytokeratin expression in the inner cell mass of parthenogenetic mouse embryos. AB - During the preimplantation period of development, the first cellular polarization and diversification of the mouse embryo occurs. This process starts at the eight cell stage and is directly driven by the cytoskeleton. Cell polarization finally leads to the first embryonic epithelium, the trophectoderm, characterized by the presence of cytokeratins. It has not been described whether genomic imprinting, an epigenetic modification of certain genes depending on the parent-of-origin, affects preimplantation development. However, implantation is one of the steps in which an exceptionally high mortality rate is observed in mouse parthenogenetic embryos, a phenomenon that may be influenced by a deficiency in trophectoderm differentiation. To assess this possibility we analyzed the expression of various cytoskeletal proteins in late preimplanted embryos. No differences were observed in the expression of microtubules and microfilaments, but surprisingly, the undifferentiated cells of the parthenogenetic inner cell mass showed distinct cytokeratin staining. This anomalous cytoskeleton expression may be considered as one of the earliest manifestation described to date of the effect of genomic imprinting in development. PMID- 8619966 TI - Differential spatiotemporal expression of E- and P-cadherin during mouse tooth development. AB - Changes in E- and P-cadherin (E- and P-CD) expression during embryonic mouse first molar development were analyzed by immunohistochemistry. During the induction and morphogenesis stages (bud, cap and early bell stages), E-CD was expressed in the cells of the invaginating epithelial tooth bud and in the cells of the outer enamel epithelium, stellate reticulum and stratum intermedium, suggesting a role for this molecule in the maintenance of enamel organ architecture. On the other hand, P-CD was strongly expressed in the inner enamel epithelium suggesting its participation in the processes of mesenchymal induction. during the cytodifferentiation stage (late bell stage), E-CD was expressed in polarizing preameloblasts, but cadherin expression was restricted to the basal and apical poles of differentiated secretory ameloblasts, where the zonula adherens type of cell-cell junctions is located. The present study demonstrates for the first time the spatiotemporal expression of cadherins during tooth development and suggests differential and specific roles for E-CD and P-CD during the morphogenesis and cytodifferentiation processes of this organ. PMID- 8619967 TI - A cephalometric evaluation of the dental and facial-skeletal effects using the Bionator with stepwise protrusive activations. AB - A study was conducted to ascertain dental effects of step-wise mandibular advancement with Bionator therapy. A sample of 24 girls in the age group of 9 to 12 years having Class II Division 1 malocclusion was well matched for age, severity of malocclusion and craniofacial morphology. The sample was divided into three groups. Eight girls underwent step-wise mandibular advancement in three stages, while eight were treated with single step advancement. Eight girls acted as control who did not undergo any treatment. The total treatment/observation period was 9 months. The cephalometric analysis revealed that the progressive mandibular advancement enhances favorable facio-skeletal changes, more so in the mandible. The sagittal correction was predominantly of skeletal type with step wise advancement, while with single step advancement it was due to both skeletal and dental changes. PMID- 8619968 TI - Antibacterial activity of fluoride release sealants on mutans streptococci. AB - This study was conducted to study the antibacterial activity of sealants on mutans streptococci. The antibacterial effect of fluoride release sealants was estimated by using agar plates infected with several strains of S. mutans and S. sobrinus. Both bacterial groups were sensitive to sealant antibacterial activity, there was not statistical difference in the inhibition between both groups. The Teethmate-F was the only active material in this study and showed more than four times fluoride release than FluoroShield. The inhibition activity was associated with sealant fluoride release after the setting of this material. PMID- 8619969 TI - Apical microleakage of primary teeth root canal filling materials by clearing technique. AB - The purpose of this study was to evaluate apical leakage of primary teeth root canals sealed with four different root canal filling materials by clearing technique. Dye penetration ratios of all groups were measured by light microscopy. The mean dye penetration was determined in the group of ZOE to be 0.57 mm. This ratio was 1.05 mm in the group of ZOE + glutaraldehyde (GA). In the groups of Kri I and Ca(OH)2, dye penetrations were 0.47 mm, 0.56 mm respectively. The highest apical leakage value was evaluated in the glutaraldehyde + ZOE group (p<0.05), whereas, no statistical difference was found between the other groups. PMID- 8619970 TI - Recall intervals: effect on treatment needs: a retrospective study. AB - The purpose of this retrospective study was to examine the effect of recall intervals on the incidence of dental caries. Data were collected from patient records of a private pediatric dental practice. Variables examined were time of the recall interval, age, race and sex of the patient, and whether the patient lived in a fluoridated area. There were 207 patients who qualified for the study. Of the 207 patients in the study, 173 did not have any teeth with dental caries at the recall visit. A significant difference between increased caries activity and recall interval was not found in this study. There was no significant difference found between the explanatory variables and caries activity. PMID- 8619971 TI - Recall intervals: effect on treatment needs of the handicapped patient: a retrospective study. AB - The purpose of the retrospective study was to examine the effect of recall intervals on incidence of dental caries in handicapped patients. Data was collected from patient records of a private pediatric dental practice. Variables examined were time of the recall interval, age, race and sex of the patient, handicap, and whether the patient lived in a fluoridated area or not. Approximately six hundred charts were reviewed which resulted in 83 patients that qualified for the study. Of the 83 patients in the study, 57 did not have dental caries at the recall visit. The relationship between increased caries activity and recall interval was not significant. However, a trend indicating an increased chance of developing caries after a twelve month recall interval was detected. PMID- 8619972 TI - Electromyographic and ultrasonographic observations of masseter and anterior temporalis muscles in children. AB - Muscle thickness and activity of masseter and anterior temporalis muscles in children with Class I molar occlusion having normal anterior relation, anterior deep-bite and anterior open-bite were measured using ultrasonographic and electromyographic techniques. The results demonstrated an increased muscle thickness in the deep-bite followed by open-bite subjects. Muscle activity was high in the same groups. A significant correlation between thickness and activity was noted only in the anterior temporalis muscle of subjects with normal anterior relation. Activity index indicated that masseter contributed to the higher activity in the clenching effort. PMID- 8619973 TI - Mandibular position and head posture as a function of eye dominance. AB - A recent study conducted at Tufts University, School of Dental Medicine has confirmed the relationship between head posture, mandibular position and eye dominance. The purpose of this study is to assess the temporary eye dominance of children and the change relative to head posture and mandibular position. Twenty children were included in this study after the completion of the ophthalmological exclusion screening. All subjects had the sign of eye dominance screened with eye dominance tests. The mandibular position and the 3-dimensional posture of head were recorded. The eye patches were applied over the dominant eye for one hour to achieve the effect of temporary eye dominance change for 10 children. Ten children had the same procedures except for the eye patch procedure. The reexaminations were performed at the end of one hour visit for both groups. Mandibular midline data revealed no change in both control and experimental group. Statistically significant difference in the transverse plane of head posture was observed by means of Student's t test in experimental group. The mean transverse measurement difference was 14.7 degrees, with a standard deviation of 6.4265 degrees. The T value was 5.66, with a probability of p=0.0003 at DF=9. The differences of positions were not statistically significant (P=0.05) on horizontal and vertical planes. The transverse head postural change has been demonstrated by means of temporary eye dominance change. This study highlights several areas: the importance of clinical intervention in young children, the difference on transverse dimensional change, and the importance of role of pediatric dentistry among interdisciplinary cooperation. Further investigation and early clinical intervention is encouraged. PMID- 8619974 TI - Immediate esthetic treatment of an avulsed/replanted permanent incisor with extensive root resorption: a case report. AB - This article describes the treatment of a 10 year-old patient with complete root resorption of the maxillary permanent incisor following avulsion and replantation 4 years earlier. The remaining natural crown was used to immediately solve the esthetic problem created by its extraction. The crown was attached with an orthodontic wire and composite resin to adjacent teeth. This temporary treatment does not elicit any damage to adjacent teeth, and can be easily removed. Therefore, it does not affect any plans that might be considered in the future as the permanent treatment. PMID- 8619975 TI - Unmonitored apexification of wide open apex in nonvital, immature incisor: a case report. AB - Apexification is the accepted procedure to form an apical stop in nonvital teeth with incomplete root formation. A case is presented in which apexification with calcium hydroxide was performed on erupting maxillary central incisor in 7-year old female child with immature root (half root) formation and wide open apex. Although it was an induced procedure, but unmonitored. Patient did not return until 18 months after his first visit. Treatment then concluded with gutta percha obturation against firm "cap shaped" apical stop. PMID- 8619976 TI - Eruption anomalies of the maxillary permanent cuspids in children with cleft lip and/or palate. AB - Eruption anomalies of the maxillary permanent cuspid on the cleft side was analyzed in a sample of 77 children with mono- and bi-lateral cleft lip/palate. The main findings were: 1) The permanent cuspid showed an initial mesial position in relation to the root of its primary predecessor in a relatively high number of cases. Nearly all those cuspids that showed an anomalous position (mesial or distal) in relation to the corresponding primary teeth at the time of the first observation erupted in malposition (palatally in the cleft area, mesially or distally to the primary cuspids). 2) In most cases presenting a mesially positioned permanent cuspid at the time of first observation, the permanent later incisor was congenitally missing, microdontic or in a mesial position in relation to the cleft. 3) Distally positioned permanent cuspids were always associated with a supernumerary lateral incisor. 4) A congenitally missing lateral incisor could represent a predisposing factor to a mesial position of the permanent cuspid. An early radiographic evaluation of the position of the permanent cuspid was suggested especially in those children presenting a congenitally missing lateral incisor. PMID- 8619977 TI - Unusual nasal foreign body detected on routine dental radiography: case report. AB - This report describes an unusual nasal foreign body in a 4-year-old boy discovered incidentally in a routine dental radiograph. A small piece of rubber eraser was lodged in the right nasal cavity, causing unilateral nasal obstruction and discharge, sneezing, snoring and breathing difficulty. In this case, discovery of unknown intranasal object was diagnostic for a condition suspected of pathological origin. Dentists may play a significant role in the diagnosis of intranasal foreign objects in children, through careful clinical examination and interpretation of dental radiographs. Early diagnosis is emphasized in order to avoid complications. PMID- 8619978 TI - Teething and acute graft vs. host disease: a clinical observation. AB - A 5-month-old girl, treated with bone marrow transplantation for severe combined immunodeficiency, developed two episodes of severe acute graft vs. host disease (GVHD) while teething. A dramatic improvement in GVHD was noted after the completion of tooth eruption. It is suggested that the release of interleukin-1, associated with the traumatized gingival tissue, is the triggering mediator for the GVHD. PMID- 8619979 TI - Generalized microdontia and associated anomalies: a clinical, genetic, radiologic and dermatoglyphic study. AB - The clinical, genetic, radiological and dermatoglyphic findings of a case showing generalized microdontia associated with an extra maxillary central incisor, hypoplastic maxilla, prognathic mandible, wide-set of the ears, hooked nose, astigmatism, camptodactyly, flexion contractures of the distal interphalangeal joints of the fingers, thinning of the fingers towards the distal end of the palm, and complete webbing of the IVth and Vth toes (syndactyly type III) and short stature were presented. PMID- 8619980 TI - Hajdu-Cheney syndrome: case report. AB - A case of Hajdu-Cheney syndrome with acro-osteolysis of the left hand, bizarrely shaped skull, early eruption of permanent teeth and ventricular septal defect is presented. Biochemical investigations revealed high levels of acid phosphatase. The child was treated routinely and the lower primary central incisors were extracted due to early lingual eruption of the permanent central incisors. PMID- 8619981 TI - Supernumerary and congenitally absent teeth: a literature review. AB - This review article examines supernumerary teeth as to prevalence, location, clinical and radiographic appearance, etiology, complications and management of supernumerary teeth; congenital absence of teeth as to prevalence and location, clinical features and complications, etiology, treatment considerations, and coexistance of supernumeraries and congenital absence of teeth. PMID- 8619982 TI - Delayed tooth formation in children exposed to tobacco smoke. AB - Panoramic radiographs of 203 Caucasoid children between the ages of seven and ten years were examined for an evaluation of dental development. Four groups were studied: a control group in which neither parent had smoked during the pregnancy concerned, a group exposed to tobacco smoke from the mother only, a group exposed to smoke from the father only, and finally a group exposed to tobacco smoke from both parents. In each case, the dental age (determined according to the method of Moorrees) was contrasted to the chronological age of the subject. Overall results showed that there were no differences in dental ages related to gender. Maximum differences between chronological and dental ages were found in children subjected to cigarette smoke from both parents. Here, a 35% reduction in dental maturation was noted. Most affected teeth were the maxillary second premolars, while the central incisors were the least affected. PMID- 8619983 TI - X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains. AB - Low- and wide-angle X-ray diffraction were used to determine the structural organization of lipids isolated from the stratum corneum extracellular matrix that forms the major water permeability barrier in mammalian epidermis. Hydrated pig skin ceramides gave a single low-angle reflection of about 62 angstroms and a wide-angle-reflection at 4.15 angstroms. The addition of either cholesterol or fatty acid, the other major lipid components of the skin stratum corneum extracellular matrix, modified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separation, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:cholesterol:palmitic acid (similar to that found in stratum corneum) produced a diffraction pattern with a single sharp wide-angle reflection at 4.10 angstroms and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 angstroms. The repeat period and intensity distribution of the low-angle data were similar to those found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725-3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 183-192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 angstrom repeat period decreased only about 3 angstroms as water was removed by incubation in low relative humidity atmospheres. The 130 angstrom repeat period depended on the presence of a particular ceramide, N-(omega-acyloxy)-acylsphingosine, which is found only in the epidermis. In contrast, 2:1:1 mixtures of brain ceramide:cholesterol:palmitic acid gave reflections of 56 and 34 angstroms. These results indicate that a structure with dimensions similar to those of the lamellar repeating unit found in skin stratum corneum does not depend on the presence of protein but does depend on the presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid. PMID- 8619984 TI - Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin. AB - Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1 228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin. PMID- 8619985 TI - Ca2+-binding stoichiometry of calbindin D28k as assessed by spectroscopic analyses of synthetic peptide fragments. AB - Calbindin D28k is an intracellular Ca2+-binding protein noted for its abundance and specific distribution in mammalian brain and sensory neurons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative sites, previous studies have been unsuccessful in definitively establishing the stoichiometry of Ca2+ binding. We describe a synthetic approach to identify the number of Ca2+-binding sites in which 6 33-residue peptides, designated EF1-EF6, corresponding to the 6 EF-hand sequences of calbindin D28k, were made. The response of each peptide to Ca2+ addition was assessed by 1H NMR spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis. The Ca2+ binding by CD experiments was performed at two peptide concentrations, 20 and 200 microM, and the NMR studies at peptide concentrations ranging from 20 to 100 microM. The CD and 1H NMR data show that five of the six peptides bind Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other four functional sites. In contrast, EF2 does not appear to bind Ca2+ under any of the spectroscopic conditions tested. The data suggest that at least five of the six putative sites in the native protein bind Ca2+, although their relative affinities cannot be deduced from studies of the isolated peptides. PMID- 8619986 TI - Mutational analysis of the role of hydrophobic residues in the 338-348 helix on actin in actomyosin interactions. AB - Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin. Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin. In addition, the Vmax of the actin activated S1 ATPase was reduced 4-fold with no change in the Km. In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase. The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s). Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments. However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s). The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system. The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments. PMID- 8619987 TI - Hydrophobic alkyl headgroups strongly promote membrane curvature and violate the headgroup volume correlation due to "headgroup" insertion. AB - The ability of lipid aggregates to form planar bilayers, rather than highly curved micellar or inverted structures, is dependent on the relative geometries of the headgroup and hydrocarbon regions. The headgroup volume approach to lipid structure provided a quantitative link between a lipid's headgroup size and its ability to promote curved, inverted hexagonal (H(II)) structures in a phosphatidylethanolamine (PtdEtn) matrix [Lee et al. (1993) Biophys. J. 65, 1429 1432]. Phosphatidylalkanols (PtdAlks) are shown here to promote curvature with a potency that far exceeds and a chain length dependence contrary to the expectations of the headgroup volume approach, suggestive of an atypical alkyl "headgroup" conformation. A homologous series of 3-substituted triacylglycerols (TAGs), for which 3-acyl "headgroup" insertion is established, exhibits a chain length dependence similar to the PtdAlks, evidence that the deviation is of common origin. The potency of the TAGs to promote curvature is unprecedented, and the onset of saturation, which parallels the dramatic promotion of curvature, occurs at mole fractions as low as 0.0025. The potency of the PtdAlks or TAGs to promote curvature exceeds that of all mammalian phospholipids examined. Thermodynamic analysis implicates the enthalpic curvature stress imparted upon the membrane matrix as the dominant energetic factor. The imparted stress ranges from -930 J mol(-1) for phosphatidylcholine to +7.5 kJ mol(-1) for 3-palmitoyl TAG. The results affirm the geometric considerations of membrane structure and indicate that alkyl headgroups tend to insert into the bilayer and increase the enthalpic curvature stress within the membrane. PMID- 8619988 TI - Kinetics of PAPS translocase: evidence for an antiport mechanism. AB - In order to gain an understanding of the mechanisms involved in the transfer of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the cytosol where it is synthesized to the Golgi lumen where it serves as the universal sulfate donor for sulfate ester formation in higher organisms, we have undertaken a kinetic characterization of the PAPS translocase from rat liver Golgi. Analyzing the PAS translocase activity in both intact Golgi vesicles and in a reconstituted liposome system, we have determined a number of physical and kinetic parameters. Strong competitive inhibition in zero-trans uptake experiments only with beta methylene PAPS and adenosine 3',5'-biphosphate (PAP) suggest the transporter is highly specific for the 3'-phosphate. The demonstration of trans acceleration as observed by stimulation of transport activity under exchange conditions suggests that the translocase is a carrier with distinct binding sites accessible from both faces of the membrane. The behavior of the PAPS translocase in the presence of equilibrium concentrations of PAP supports the function of an antiport mechanism. Thus the translocase is characterized by its kinetic properties as a specific transporter of PAPS which acts through an antiport mechanism with PAP as the returning ligand. This characterization of the transport activity has proved instrumental in the identification of an approximate 230 kDa Golgi membrane protein as the PAPS translocase protein [Ozeran, J.D., Westley, J., & Schwartz, N.B. (1996) Biochemistry 35, 3695-3703 (accompanying paper)]. PMID- 8619989 TI - Identification and partial purification of PAPS translocase. AB - Sulfation of all macromolecules in higher organisms requires the high-energy donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is synthesized via the sequential actions of two cytoplasmic enzymes, ATP sulfurylase and APS kinase, and then must be transferred across the Golgi membrane for utilization by lumenal sulfotransferases. Following the kinetic characterization of the PAPS translocase as a specific transporter that act through an antiport mechanism with PAP as the returning ligand [Ozeran, J.D., Westley, J., Schwartz, N.B. (1996) Biochemistry 35, 3685-3694 (accompanying paper)], the present study describes the identification and physical characterization of the PAPS translocase from rat liver Golgi membranes. The following evidence suggests the PAPS translocase is a membrane spanning protein of approximately 230 kDa: isolation by affinity chromatography on beta-methylene PAPS matrices of a 230 kDa Golgi membrane protein concomitant with PAPS translocase activity; demonstration that the 230 kDa protein possesses the only PAPS binding site accessible to the cytoplasmic face of intact Golgi membranes, while several other PAPS binding proteins are labeled in solubilized membrane preparations; reduction in size of the 230 kDa membrane protein and loss of PAPS translocase activity following protease treatment; estimation via hydrodynamic analysis of a molecular size of the membrane protein associated with PAPS translocase activity; and correlation of beta-methylene PAPS binding and labeling of the 230 kDa Golgi protein with PAPS translocase activity in artificial liposomes. These and the accompanying data have permitted the identification of the first of a potentially large class of Golgi membrane nucleotide-metabolite transporters. PMID- 8619990 TI - Phosphorylation of beta III-tubulin. AB - There is considerable evidence that mammalian beta-tubulin is phosphorylated. Specifically, of the seven beta isotypes, the phosphorylated one is beta III, the isotype found almost entirely in neurons. The phosphate is added at a serine and perhaps a tyrosine near the C-terminus. All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of beta III is unknown. Nor is it known if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments. In addition, the role of tubulin phosphorylation is unknown. We have purified the alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers from bovine brain and have determined their phosphate content chemically. We have found that alpha-tubulin is not phosphorylated and neither are the beta II or beta IV isotypes. However, beta III is phosphorylated with a stoichiometry of about 1.52 mol/mol. We have found that the phosphate on beta III is resistant to a wide variety of phosphatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule associated protein 2 (MAP 2). However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins. Our results suggest the possibility that beta III phosphorylation may play a role in regulating microtubule assembly in vivo. PMID- 8619991 TI - Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2. AB - Ser-228 has been shown to be essential for the catalytic activity of the human cytosolic phospholipase A2 (cPLA2). However, its involvement in catalysis has not yet been demonstrated. Using site-directed mutagenesis, active-site directed irreversible inhibitors, and the novel fluorogenic substrate 7-hydroxycoumarinyl gamma-linolenate, evidence is presented to show that the hydroxyl group of Ser 228 is the catalytic nucleophile of cPLA2. Replacement of Ser-228 by Ala, Cys, or Thr resulted in the inability of these mutants to mediate calcium ionophore induced PGE2 production in COS-7 cells cotransfected with the cPLA2 mutants and cyclooxygenase-1. Cell lysates from these transfected cells also had undetectable levels of cPLA2 phospholipid hydrolyase activity as did the affinity column purified S228A and S228C cPLA2 mutants overexpressed in insect cells. The loss in activity was not due to the inability of the mutant enzymes to translocate to the substrate lipid interface since the purified S228C cPLA2 mutant, like the wild type, translocated to the phospholipid membrane in the presence of calcium as judged by fluorescence energy transfer. However, when an activated substrate, 7 hydroxycoumarinyl gamma-linolenate (pKa approximately 7.8 for its leaving group) was used as substrate, there was a significant level of 7-hydroxycoumarin esterase (7-HCEase) activity (about 1% of wild type) associated with the purified S228CC cPLA2 mutant. The S228A cPLA2 mutant was catalytically inactive. Contrary to wild type cPLA2, the 7-HCEase activity of the thio-cPLA2 was not titrated by the irreversible active-site-directed inhibitor methyl arachidonyl fluorophosphonate, but rather titrated by one equivalent of arachidonyl bromomethyl ketone, an arachidonyl binding site directed sulfhydryl reagent. These results are compatible with the hydroxyl of Ser-228 being the catalytic nucleophile of cPLA2 and that cysteine can replace serine as the nucleophile, resulting ina thiol-cPLA2 with significantly reduced catalytic power. PMID- 8619992 TI - Major groove recognition elements in the middle of the T7 RNA polymerase promoter. AB - T7 RNA polymerase recognizes a relatively small promoter extending only 17 base pairs upstream from the start site for transcription. A model for this recognition suggests that the enzyme interacts with the major groove of duplex DNA in the region centered at position -9 [Muller, D.K., et al. (1989) Biochemistry 28, 3306-3313], and recent kinetic analyses of promoters containing base analogs at positions -10 and -11 have provided support for this model [Schick, C., & Martin, C.T. (1993) Biochemistry 32, 4275-4280; Schick, C., & Martin, C.T. (1995) Biochemistry 34, 666-672]. In the current work, we extend this analysis across the proposed major groove, identifying specific base functional group contacts at positions -9 through -5. Specifically, the 6 carbonyl of guanine at positions -9 and -7, the 6-amino group of adenine at position -8, the 5-methyl group of thymine at position -6 and the 2-amino group of guanine at position -5 are identified as primary contacts. The results strongly support the model for duplex recognition in this region of the promoter and suggest that recognition continues along one face of the helix beyond the major groove and into the adjoining minor groove at position -5, where helix melting begins. PMID- 8619993 TI - dNTP binding site in rat DNA polymerase beta revealed by controlled proteolysis and azido photoprobe cross-linking. AB - Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263 280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol. PMID- 8619995 TI - Kinetics of the interaction between DNA and the type IC restriction enzyme EcoR124II. AB - Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restriction modification enzyme from Salmonella typhimurium, with DNA. The DNA is immobilized on the surface of an optical waveguide, and the enzyme is introduced in bulk solution flowing over the DNA under controlled hydrodynamic conditions. The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecules per base pair determined to a high accuracy. Dissociation of the protein was measured by switching flowing protein to protein-free buffer. Binding to two different kinds of DNA, with and without the specific sequence recognized by EcoR124II, was investigated. Protein binding and dissociation ("nonspecific" binding), quantified by association and dissociation rate coefficients ka and kd, were the same for both types, but the DNA carrying the recognition site showed an additional process, "irreversible" association (i.e. dissociation was not observed on the time scale of the experiments) of the protein, quantified by a rate coefficient ks. Some inferences regarding the mechanism of base pair searching are made from the measured ka, kd, and ks values. PMID- 8619994 TI - Interactions between RNA polymerase and the positive and negative regulators of transcription at the Escherichia coli gal operon. AB - The simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I "footprint" titration analysis, and structurally, by the use of hydroxyl radical (.OH) and 5-phenylphenanthroline (5OPP) "footprinting." In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a small energetic preference for P1. The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter. This cooperativity is inhibited by the binding of GalR to the upstream operator, OE, but does not result in the repartitioning of RNAP between the P1 and P2 promoters. These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to OE represses transcription by inhibiting the CAP-RNAP cooperativity. It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to OE prevents these contacts from occurring. Changes in nuclease reactivity at the internal operator OI (centered at +53.5) take place upon RNAP binding. These changes are dependent on the DNA sequence present at OI and on the presence or absence of CAP. They are independent of the helical phasing between the promoters and OI and of the distance between them. These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites. PMID- 8619996 TI - Conformational switches involved in orchestrating the successive steps of group I RNA splicing. AB - Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omegaG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing. PMID- 8619997 TI - An amphiphilic lipid-binding domain influences the topology of a signal-anchor sequence in the mitochondrial outer membrane. AB - Mas70p is targeted and inserted into the mitochondrial outer membrane in the N(in)-C(cyto) orientation, via an NH2-terminal signal-anchor sequence. The signal anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10), followed by the predicted transmembrane segment (amino acids 11-29). Substitution of the NH2-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in the membrane (N(cyto)-C(in)). This substitution resulted in an increase in the net positive charge of the hydrophilic region, from +4 to +8. In contrast to the endoplasmic reticulum and the bacterial inner membrane, where the net positive charge is an important determinant in conferring protein topology in the lipid bilayer, we show here that the reversal of the Mas70p signal-anchor was not due to differences in the number and positions of basic amino acids in the hydrophilic domain. However, a reduction in the hydrophobic moment of predicted amphiphilic helices containing an arginine, obtained by converting the apolar amino acids flanking the arginine to polar residues, caused the otherwise N(cyto) C(in) signal-anchor to re-adopt the original N(in)-C(cyto) orientation of Mas70p. The reduced hydrophobic moment at the NH2-terminus significantly reduced the ability of this domain to bind to synthetic liposomes whose lipid composition reflected that of the outer membrane. These results identify amphiphilicity as an important determinant in causing retention of the NH2-terminus of a mitochondrial signal-anchor on the cytosolic side of the outer membrane. In addition to potential interactions between this domain and cytosolic-exposed components of the import machinery, this retention may result as well from interaction of the NH2-terminus with the surrounding membrane surface. PMID- 8619998 TI - Amphiphilicity determines binding properties of three mitochondrial presequences to lipid surfaces. AB - The interactions of three peptides, which correspond to presequences that direct mitochondrial protein import, with model membrane systems were characterized using NMR, fluorescence, and circular dichroism spectroscopies. The positively charged peptides adopted an ordered secondary structure only when the negatively charged phospholipid, cardiolipin, was present in small unilamellar vesicles. Conversely, the peptides adopted an ordered secondary structure in the presence of micelles formed from both formally neutral and negatively charged detergents. The peptides had the same relative affinity for micelles and small unilamellar vesicles containing 20% cardiolipin. Amide proton exchange rates showed that the region of the helical structure which had the greatest hydrophobic moment interacted most readily with micelles. Therefore, it appears that a major determinant of binding to lipid surfaces is the ability of the peptide to attain the correct orientation of hydrophobic and hydrophilic groups. For the three peptides studied, affinity also correlated with the length of the helix, but not with hydrophobic surface area. In each case, the interacting segment of the peptide was toward the C-terminal end of the helix. Previous work has allowed us to postulate that the N-terminus of the presequence is vital for import [Wang, Y., & Weiner, H. (1993) J. Biol. Chem. 268, 4759-4765] and the C-terminal end is essential for membrane interaction [Karslake, C., Piotto, M., Pak, Y. K., Weiner, H., & Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878]. On the basis of the data that are now available, it appears that the interaction with membrane surfaces may depend on the location of an amphiphilic region of the sequence that is near but not necessarily at the C-terminus. PMID- 8619999 TI - Phosphorylation of C8 and C9 subunits of the multicatalytic proteinase by casein kinase II and identification of the C8 phosphorylation sites by direct mutagenesis. AB - Two 29 kDa subunits of the multicatalytic proteinase (proteasome) complex, the C8 and C9 components, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). The major phosphate acceptor is the C8 subunit being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by Glu-C endoprotease digestion from the in vivo 29 kDa labeled subunit and from the in vitro phosphorylation of the recombinant C8 subunit with CKII are identical, suggesting that CKII is likely responsible for the in vivo phosphorylation of the C8 subunit. The in vitro stoichiometry of phosphorylation of the proteasome complex and the recombinant C9 and C8 subunits by CKII is 2 2.5, 0.2, and 2 mol of phosphate per mole, respectively. Several C8 protein constructs allow the location of the CKII phosphorylation sites to be the COOH terminal portion of the protein, and direct mutational analyses show that Ser-243 and Ser-250 are the residues of the C8 subunit phosphorylated by CKII. The in vitro phosphorylation of the proteasome by CKII does not affect its proteolytic activity (on proteins or fluorogenic synthetic peptides), therefore suggesting its involvement in the interaction of the proteasome with other cellular proteins, i.e. in the formation of the 26S complex and/or in the interaction with the nuclear translocation machinery. PMID- 8620000 TI - Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids. AB - Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant. PMID- 8620001 TI - Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family. AB - Cleavage and subsequent release of the extracellular domains of receptor protein tyrosine phosphatases (RPTP) occur at high cell density and may have an important role in regulating their activity. Because cleavage of RPTP occurs at a target motif (RXK/RR) recognized by a family of subtilisin/kexin-like endoproteases, we postulated that members of the subtilisin family may have an important role in this cleavage. We show in this report that the membrane-associated RPTPmu--both in its full 200-kDa form and as a 100-kDa cleavage product--is upregulated 4- and 7-fold, respectively, as human umbilical vein endothelial cells (HUVEC) approach confluence. To determine whether RPTPmu cleavage depended on PC5 (a subtilisin/kexin like endoprotease present in endothelial cells), we transfected COS cells with expression plasmids coding for RPTPmu and PC5 or the closely related protease PACE4. PC5, but not PACE4, cleaved RPTPmu, and RPTPmu cleavage was absent in COS cells transfected with an expression plasmid encoding a mutant PC5 whose active-site serine had been mutated to alanine. We also performed RNA blot analysis to determine whether PC5 expression was affected by confluence in HUVEC. PC5 mRNA levels were upregulated by more than 30-fold when confluence in HUVEC increased from 25% to 100%. These results indicate that PC5 may have an important role in mediating the cleavage of RPTPmu in response to contact inhibition in HUVEC. PMID- 8620002 TI - A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. AB - Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. Alpha-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5'-monophosphate (OMP) in the presence of divalent cations, preferably Mg2+. OMP is thereafter converted to uridine 5'-monophosphate by OMP decarboxylase. We have determined the 2.4 angstrom structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer. We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase.OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis. Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S. typhimurium OPRTase.OMP and E. coli OPRTase are found in the substrate binding regions: the 5'-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73). PMID- 8620003 TI - Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process. AB - Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein. A possible mechanism for branched intermediate formation is an N O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate. This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp. GB-D. Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified. In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products. Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction. These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing. PMID- 8620004 TI - Individual subunits of heterodimers comprised of retinoic acid and retinoid X receptors interact with their ligands independently. AB - The retinoid X receptor (RXR) is a member of a family of transcription factors, known as hormone nuclear receptors, that mediate the effects of hydrophobic hormones on gene transcription. RXR, which is activated by 9-cis-retinoic acid (9cRA), can modulate several signaling pathways by virtue of its ability to form heterodimers with other members of the receptor family, as, for example, the retinoic acid receptor (RAR). The roles of the individual receptors within heterodimers are not clear as yet. It was recently reported that heterodimerization inhibits transcriptional activation by RXR, an effect that was attributed to an inability of RXR within heterodimers to bind its ligand. This inhibition was reported to depend on the association of heterodimers with cognate DNA and on the level of saturation of the RAR subunit within the heterodimers. In the present work, the ligand-binding characteristics of RXR and RAR individually and within heterodimers were examined by fluorescence-based methods. The results indicate that heterodimerization with RAR does ot alter the ligand-binding capacity of RXR nor the rate of dissociation of the ligand from this receptor. The ligand-binding capacity of RXR also was not affected by association of heterodimers with cognate DNA nor by the level of saturation of the RAR subunit. The data indicate further that the affinity of RAR for 9cRA is considerably higher as compared to RXR and that this differential affinity is retained within RAR-RXR heterodimers. Thus, binding of ligands by subunits within RAR-RXR heterodimers proceeds independently. PMID- 8620005 TI - Membrane stiffness and channel function. AB - Alterations in the stiffness of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein function. Gramicidin channels can be used as molecular force transducers to measure such changes in bilayer stiffness. As an application, we show that N-type calcium channel inactivation is shifted reversibly toward negative potentials by synthetic detergents that decrease bilayer stiffness. Cholesterol, which increases bilayer stiffness, shifts channel inactivation toward positive potentials. The voltage activation of the calcium channels is unaffected by the changes in stiffness. Changes in bilayer stiffness can be predicted from the molecular shapes of membrane-active compounds, which suggests a basis for the pharmacological effects of such compounds. PMID- 8620006 TI - Lipid chain dynamics and molecular location of diacylglycerol in hydrated binary mixtures with phosphatidylcholine: spin label ESR studies. AB - The lipid chain motions in hydrated binary mixtures of dimyristoylglycerol (DMG) with dimyristoylphosphatidylcholine (DMPC) have been studied by using ESR spectroscopy of analogues of both components that are spin-labeled at one of eight different positions along the sn-2 chain. The phase diagram of the binary mixtures divides into three separate regions along the composition axis, corresponding to the formation of isothermally melting compounds, with DMPC/DMG stoichiometries of approximately 1:1 and 1:2 mol/mol in the gel phase [Heimburg, T., Wurz, U., & Marsh, D. (1992) Biophys. J. 63, 1369-1378]. In the first region (up to 50 mol % DMG), comparison of the chain flexibility profiles, and the chain profiles of the polarity-dependent isotropic hyperfine coupling constant, of the two different spin-labeled components indicates that DMG is incorporated in the fluid lipid bilayer in a manner similar to that of the host DMPC but is situated approximately two CH2 groups deeper into the hydrophobic interior. At lower contents of DMG, the chain packing is increased by the addition of DMG, whereas at higher DMG contents the lipid chain order decreases rapidly, on reaching the inverted hexagonal phase of the second region of the phase diagram. In the second region of the phase diagram (50-67 mol % DMG), the DMG fits better into the fluid inverted hexagonal phase than into the fluid lamellar phase of the first region and is located only approximately one CH2 group deeper than the corresponding DMPC. In these first two regions of the phase diagram, the ESR spectra of both spin-labeled components display an axial anisotropy that evidences the increasing angular amplitude of motion with position down the chain that is characteristic of liquid crystalline fluid phases. In the third region of the phase diagram (above 67 mol % DMG), the fluid phase consists of isotropically tumbling DMG molecules in which the DMPC molecules are incorporated as inverted micelles as indicated by the residual anisotropic motion of the spin-labeled phosphatidylcholine analogues. PMID- 8620007 TI - Zinc stimulates Mg2+-dependent 3'-processing activity of human immunodeficiency virus type 1 integrase in vitro. AB - Human immunodeficiency virus type 1 integrase (HIV-1 IN) catalyzes both 3'-donor processing and strand transfer reactions. Previous studies have determined that the N-terminal region, a putative zinc finger, is capable of binding Zn2+. The function of zinc coordination to this domain, however, is still unknown. In this report, we present evidence that Mg2+-dependent 3'-donor processing by HIV-1 IN is enhanced by the addition of Zn2+ in vitro. This activity is inhibited in the presence of the chelator 1,10-phenanthroline (OP). In addition, the Mg2+ dependent 3'-donor processing activity is more sensitive to the concentration of IN than is the Mn2+-dependent activity. A combination of dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG) was found to further activate the Mg2+ dependent 3'-donor processing activity while diminishing the Mn2+-dependent activity. These results suggest factors such as substrate-length, concentration of IN, Zn2+ coordination, and protein-protein interactions are important for efficient and specific donor processing activity with Mg2+ in vitro. PMID- 8620008 TI - DNA unwinding induced by zinc finger protein binding. AB - Zinc finger domains of the Cys2His2 type are found in a large number of eukaryotic proteins. Various proteins containing these domains have been shown to bind specifically to DNA, RNA, and DNA-RNA hybrids. Structural studies of zinc finger protein-DNA complexes have revealed that the DNA molecules are underwound relative to canonical B-form. It has not been clear if zinc finger proteins recognize preexisting underwound conformations of DNA or if they induce such conformations upon binding. We report that the DNA binding domains of Sp1 and several designed zinc finger proteins unwind DNA upon binding. The extent of unwinding is consistent with that observed in zinc finger protein-DNA cocrystal structures. These DNA deformations may be important in determining overall binding affinities as well as influencing binding site preferences. Furthermore, changes in DNA conformation upon zinc finger protein binding may affect protein protein interactions important for transcriptional regulation and other activities of zinc finger proteins. PMID- 8620009 TI - Effects of acetyl CoA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase. AB - The approach to steady-state for the formation of the enzyme-carboxybiotin complex obeys first-order kinetics, with the proportion of the total enzyme present as the enzyme-carboxybiotin complex in the steady-state being about 60%. The approach to steady-state for ATP cleavage also obeys first-order kinetics. The apparent first-order rate constants for the approach to steady-state, in the presence and absence of acetyl CoA, respectively, are 6.6 and 0.028 s(-1) for ATP cleavage and 6.1 and 0.028 s(-1) for enzyme-carboxybiotin formation. The similarities of the values of the rate constants for the two reactions indicates that there is a common rate-limiting step. The large enhancement of these rate constants in the presence of acetyl CoA suggests that a major effect of acetyl CoA in the reaction is to enhance the rate of the step in which the putative carboxyphosphate complex is formed and in which ATP is cleaved. In addition, in the presence of acetyl CoA, the formation of the enzyme-carboxybiotin complex is much more tightly coupled to ATP cleavage in the presence of acetyl CoA than in its absence. Modeling studies were performed, and reaction schemes are proposed which give simulations similar to the experimental data. In the reaction schemes, the carboxyphosphate intermediate is able to undergo abortive decomposition without carboxylating biotin. The rate of this abortive reaction is greatly reduced in the presence of acetyl CoA. PMID- 8620010 TI - High-pressure denaturation of staphylococcal nuclease proline-to-glycine substitution mutants. AB - Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42. PMID- 8620011 TI - Affinity of fatty acid for (r)rat intestinal fatty acid binding protein:further examination. AB - The enhancement of the fluorescence quantum yield of 1,8 anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a "reference" dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained "corrected" titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than wer the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatty acids. PMID- 8620012 TI - Factor H co-purifies with thrombospondin isolated from platelet secretate. AB - Thrombospondin is a trimeric glycoprotein that has several known functions, including roles in platelet aggregation, phagocytosis and an inhibitor of angiogenesis. Typically the molecule is isolated from platelet secretate by heparin affinity followed by sizing chromatography. In this study, purity is analysed by 7.5% SDS-PAGE under reducing conditions when thrombospondin monomers run as a band at around 180 kDa. Under nonreducing conditions of 7.5% SDS-PAGE, thrombospondin does not penetrate beyond the stacking gel; however, under these conditions a major contaminating band can be seen which, upon reduction, merges into the thrombospondin band. Further purification of this contaminating protein was achieved by DEAE chromatography and it was identified as Factor H by peptide sequencing and immunoblotting. Factor H function was demonstrated by the ability of the protein to function as a cofactor in the Factor-I-mediated cleavage of C3b. Since Factor H has several known functions, such contamination could confound functional studies of thrombospondin thus purified and a pre-elution step of the heparin affinity column is recommended. PMID- 8620013 TI - The hydration of proteins in solutions by self-diffusion coefficients. NMR study. AB - The hydration of the globular (lysozyme, albumin) and fibrillar (fibrinogen) proteins in solution has been determined from the measurements of the self diffusion coefficient by NMR pulsed gradient method. It has been concluded that the concentration dependencies of the self-diffusion coefficient of water molecules in protein solution are controlled by two additive factors: obstruction effect correlated with the protein dimension and direct hydration effect proportional to the number of the adsorption sites on the protein surface. PMID- 8620014 TI - Substrate specificity and transglucosylation catalyzed by cycad beta-glucosidase. AB - Initial rates of transglucosylation with diglucosides and diglucose azoxyglycosides as acceptor by cycad beta-glucosidase were tentatively obtained. The formation of beta-1,3 glucosidic linkage was predominant, except for neocycasin A (beta-laminaribioside of methylazoxymethanol, MAM) as an acceptor. With neocycasin A as an acceptor, beta-1,4 and beta-1,6 glucosidic linkages were formed but beta-1,3 linkage was not. Whereas with laminaribiose as acceptor, laminaritriose and triose with beta-1,6 linkage were formed, but triose with beta 1,4 linkage was not. On the other hand, with other diglucoses and neocycasin B (beta-gentiobioside of MAM) as acceptor, all the linkages formed were beta-1,3 glucosidic. The aglycone of azoxyglycosides, MAM, affected the kind of linkages formed in the trisaccharides. When initial rates of the linkage formation of the transglucosylation at 100 mM acceptor were compared with the hydrolysis rates obtained by Lineweaver-Burk plot, the order of formation rates of the di- and tri glucosides by transglucosylation was the same as obtained for the hydrolysis parameter, kcat/Km. Km values for various substrates could be grouped according to the kind of the linkages (beta-1,3, beta-1,4, and beta-1,6) first split by the enzyme. PMID- 8620015 TI - Characteristics of phosphatidic acid-containing lipoproteins which selectively inhibit bitter taste: high affinity to frog tongue surface and hydrophobic model membranes. AB - In previous studies (Katsuragi and Kurihara (1993) Nature 365,213--214; Katsuragi et al. (1995) Pharm. Res. 12,658--662) we showed that a lipoprotein composed of phosphatidic acid (PA) and beta-lactoglobulin (LG) selectively suppressed the taste responses to bitter substances without affecting those to other taste stimuli in the frog and man, while complexes composed of other lipids except for phosphatidylserine and LG had little inhibitory activity. In the present study, we found that the lipoproteins having inhibitory activity are adsorbed on the frog tongue surface, while those having no inhibitory activity are not adsorbed. We also examined adsorption of the lipoproteins on model lipid membranes coated on a quartz-crystal microbalance by measuring changes in its frequency. The lipoproteins having inhibitory activity were well adsorbed on the hydrophobic lipid membranes, while the lipoproteins having no inhibitory activity were little adsorbed on the membranes. It seems that receptor sites for bitter substances on the taste cell membranes are hydrophobic and those for other taste stimuli such as salts, acids and sugars are hydrophilic. Hence, the binding of PA-LG to hydrophobic sites of the receptor membranes will lead to selective inhibition of bitterness. PMID- 8620016 TI - Conversion of metmyoglobin to NO myoglobin in the presence of nitrite and reductants. AB - Formation of NO myoglobin through the reaction of horse heart metmyoglobin with NADH in the presence of nitrite was observed optically at pH 5.5. Superoxide generation during the reaction was demonstrated using the ESR spin trap, 5,5 dimethyl-1-pyrroline-1-oxide. A weak optical spectrum corresponding to oxymyoglobin appeared transiently and the spectrum of NO myoglobin then developed. The conversion to NO myoglobin was eliminated in the presence of catalase, SOD or 5,5-dimethyl-1-pyrroline-1-oxide. The kinetics of NADH oxidation and oxygen consumption catalyzed by myoglobin showed an initial lag phase, indicating a chain reaction. When the oxygen was exhausted, the NO form emerged. The duration of the lag phase was prolonged by an increase in the concentration of catalase, SOD or 5,5-dimethyl-1-pyrroline-1-oxide, whereas it disappeared in the presence of H2O2. The spectral change from metmyoglobin to NO myoglobin was also observed under anaerobic conditions though the rate was slower than that obtained under aerobic conditions, while the spectral change was accelerated in the presence of H2O2. Nitric oxide (NO) was derived through the reaction of nitrite with NADH. The formation of NO myoglobin from metmyoglobin is explained in terms of the NADH-oxidase reaction catalyzed by myoglobin. Ascorbate and GSH also serve as reductants though NO myoglobin was formed slowly. PMID- 8620017 TI - Preferential transfer to truncated oligosaccharides to the first sequon of yeast exoglucanase in Saccharomyces cerevisiae alg3 cells. AB - In addition to the exoglucanases (Exg) secreted into the culture medium by wild type cells, ExgIa and ExgIb, which have oligosaccharides attached to both potential N-glycosylation sites, Saccharomyces cerevisiae alg3 mutant secreted substantial amounts (35--44%) of underglycosylated and unglycosylated forms. Quantification of these forms indicated that no more than 78% of the available N sites were occupied. About 50% of the transferred oligosaccharides were endo H sensitive, indicating that the lipid-linked precursor had completed its synthesis to Glc3-Man9-GlcNAc2. The other 50% remained endo H-resistant and, accordingly, it should be derived from the precursor oligosaccharide Man5-GlcNAc2 synthesized by this mutant. A closer analysis of forms that have received two oligosaccharides (ExgIb) showed that the first sequon was enriched in truncated residues, whereas the second one was enriched in regular counterparts. Similarly, analysis of the individual underglycosylated glycoforms indicated that 38% of the oligosaccharides attached to the second site were regular. This percentage dropped to 20% for glycoforms carrying the oligosaccharide in the first sequon. The preferential transfer of truncated oligosaccharides to the first glycosylation site seems to be a consequence of (1) the low percentage of truncated lipid linked oligosaccharides that receives the glucotriose unit, and (2) the effect of the glucotriose unit on the selection of N-sites to be glycosylated. PMID- 8620018 TI - Regulation of the supply of cytosolic oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. I. The effect of covalent modification on PEPC activity, pH response, and kinetic properties. AB - The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by covalent modification is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Extracts for studies on in vivo PEPC phosphorylation were prepared from barley leaf protoplasts by rapid filtration, fractionating the cell within less than 1 s. Measurements of in vitro PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. The relative PEPC phosphorylation state increased upon illumination and decreased upon redarkening under photorespiratory and non-photorespiratory conditions. PEPC activity measured in the presence of malate (3 mM) under photorespiratory conditions showed the same response indicating that a light induced increase in PEPC activity and decrease in malate sensitivity is caused by an increased phosphorylation level of the PEPC protein. PEPC activity was pH dependent. At the physiological cytosolic pH, activity was suboptimal, but most sensitive towards malate inhibition and glucose 6-phosphate stimulation. The presence of malate increased the sensitivity of PEPC activity towards pH changes. The response of PEPC activity to changing pH was not affected by changes in the activation state of the enzyme. The Km (phosphoenolpyruvate, PEP) is about 1 mM. Upon illumination the Km (PEP) decrease significantly. Vmax was unaffected by the light treatment. The presence of physiological concentrations of glucose 6 phosphate decreased Km (PEP) 5- to 10-fold and increased Vmax by about 35%. The effect of glucose 6-phosphate was strongest (up to 7-fold) at subsaturating PEP concentrations stimulating PEPC activity to nearly maximal rates. The results show that an increase in PEPC phosphorylation state causes an increase in PEPC activity as well as in substrate affinity leading to an increased production of OAA in the light. PMID- 8620019 TI - Regulation of the supply of oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. II. Effects of metabolites on PEPC activity at different activation states of the protein. AB - The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by metabolites is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6 bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2 Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose 6-phosphate, fructose 6 phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P(i) stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6 phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P(i) activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C3 plants. PMID- 8620020 TI - Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex. AB - Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl D-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample cell and activated by lipopolysaccharide and gamma-interferon at 37 degrees C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample-cell, and the time-evolution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method, NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages. PMID- 8620021 TI - Comparative 1H-NMR studies on the physical state of water in soft contact lens and mouse lens. AB - The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%. PMID- 8620022 TI - Kinetic studies of the oscillatory dynamics in the peroxidase-oxidase reaction catalyzed by four different peroxidases. AB - Oscillatory kinetics in the peroxidase-oxidase reaction catalyzed by structurally different peroxidases were investigated using NADH as a substrate. For horseradish peroxidase, lactoperoxidase, and soybean peroxidase the oscillatory waveforms of their dominating enzyme intermediates, ferric peroxidase and compound III, are similar. Coprinus peroxidase, on the other hand, has ferrous peroxidase and compound III as the dominating intermediates. The oscillatory waveform of its compound III differs from the waveforms of compound III of the three other peroxidases. Also, the phase plot of the signal for compound III versus the oxygen concentration for Coprinus peroxidase differs from the corresponding phase plots obtained using other peroxidases. A detailed model of the reaction mechanism is proposed, which is able to simulate these different kinds of behaviour. Substituting NADH with dihydroxyfumaric acid as a substrate, oscillations in the oxygen concentration were observed for about 1.5 h when a concentrated solution of this substrate was continuously fed to a solution containing horseradish peroxidase. This is the first demonstration of sustained oscillations with this substrate. PMID- 8620023 TI - Effects of free fatty acids on the binding of bovine and human serum albumin with steroid hormones. AB - Recent studies have shown that, in addition to free steroid hormones, those bound to albumin in plasma may also be available to tissues. In this report, the effects of free fatty acids (FFA) on the binding of steroids to albumin were compared for the cases of bovine serum albumin (BSA) and human serum albumin (HSA). The apparent association constant, Ka, was estimated from the changes in the equilibrium partition coefficient of steroids between the aqueous/hexane phases caused by the addition of albumin to the aqueous phase. In the case of BSA, Ka for progesterone and testosterone increased upon binding of FFA (myristic, palmitic and stearic acid) to BSA and the maximum value of Ka for these steroids could be attained by 3--4 mol of FFA bound per mol BSA. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA. On the other hand, the affinities of ethynylestradiol to both BSA and HSA were unaffected below 4 mol of FFA binding per mol. PMID- 8620025 TI - Complexation of spermine and spermidine by myo-inositol 1,4,5-tris(phosphate) and related compounds: biological significance. AB - D myo-inositol 1,4,5-tris(phosphate) (Ins(1,4,5)P3) displays a multicoordination site arrangement that allows strong interactions with polycationic species such as the naturally occurring polyamines spermine and spermidine. In the present work, the complexation of these polyamines by Ins(1,4,5)P3 and related compounds was quantitatively investigated. The study was performed in a 0.1 M tetramethylammonium p-toluenesulfonate (Me4NOTs) solution at 25 degrees C. For purpose of comparison, the complexation of the polyamine-ATP systems were also considered in the same experimental conditions. 31P-NMR experiments showed for Ins(1,4,5)P3 and its analogues, the formation of complexes of a 1:1 stoichiometry. As expected, the most stable complexes are formed between the most charged partners. In addition, the basicity of the phosphate groups seems to govern the stability of the complexes. If both ATP and Ins(1,4,5)P3 are present at the same concentration, the latter interacts preferably with the polyamines. Ins(1,4,5)P3-spermine complex formation provides a possible simple explanation for the inhibition by spermine of Ins(1,4,5)P3-induced Ca2+ release. Spermine will undoubtedly compete with metallic cations such as Ca2+ in the intracellular medium and consequently, may play a regulatory role in the signal transduction mediated by Ins(1,4,5)P3. PMID- 8620026 TI - Structure of a D-protein gene and amino-acid sequences of the highly repetitive D proteins secreted by the accessory glands of the mealworm beetle. AB - The D-group proteins form the major component of the proteinaceous secretion of the tubular accessory glands of the yellow mealworm beetle, Tenebrio molitor. In a previous paper, we reported the sequence of two D-protein cDNAs and their inferred translation products. Both proteins contain three highly repetitive domains (A, A' and B). In this paper, we present the cDNA-inferred sequences of 8 more D-proteins, none of which contains an A' domain. We also present the structure of a D-protein gene. Southern analysis suggests that genes coding for an A' domain are relatively rare. Genes with a total of 7 or 8 (A + B domain) repeats seem most common. PMID- 8620024 TI - Oscillations in the peroxidase-oxidase reaction: a comparison of different peroxidases. AB - The nonlinear behavior of the peroxidase-oxidase reaction was studied using structurally different peroxidases. For the first time sustained oscillations with peroxidases other than horseradish peroxidase in a single-enzyme system were observed. All peroxidases that showed significant oxidase activity were able to generate sustained oscillations. When adjusting the overall reaction rate, either of the two modifiers 2,4-dichlorophenol or Methylene blue could be omitted from the reaction. Due to the observation of different enzyme intermediates when using different peroxidases, we conclude that the mechanisms responsible for oscillatory kinetics may vary from one peroxidase to the other. PMID- 8620027 TI - Change in the N-terminal domain conformation of annexin I that correlates with liposome aggregation is impaired by Ser-27 to Glu mutation that mimics phosphorylation. AB - Annexin I is a member of the annexin family of calcium-dependent membrane binding proteins. The core domain of these proteins is similar in all annexins but the N terminal domain is specific for each member. This domain is thought to regulate annexin function through phosphorylation. In annexin I, Ser-27 is one of the amino acids that can be phosphorylated by protein kinase C. Phosphorylations are thought to regulate some annexin I functions by increasing calcium requirement. Two mutants were prepared in this study: S27E and S27A proteins. The first mimics phosphorylation while the second prevents phosphorylation at residue 27. Wild type annexin I and S27A mutant protein showed the same calcium dependence for phospholipid vesicles aggregation, while S27E mutant protein showed a higher calcium requirement and a low maximal extent of aggregation. By contrast, liposome binding and self-association required identical calcium concentrations for the wild-type and the two mutant proteins. To examine whether the regulation observed is due to modification of the N-terminal structure, we investigated conformational changes by using two approaches. Firstly we analysed proteinase sensibility. Limited proteolysis of the N-terminal tail showed similar patterns for the three proteins. Using drastic conditions of proteolysis, we observed strong resistance of the core domain to digestion in the presence of calcium. Secondly, since Ser-27 is located on the N-terminal domain that contains a tryptophan located at position 12, the fluorescence of this residue was analysed during Ca2+-binding of wild-type annexin I and S27E mutant protein. The results demonstrated that Ca2+ induces a slight change in the Trp environment of wild type annexin I, corresponding to a burying of the residue. No changes in fluorescence features were observed with S27E mutant protein. The results obtained show that phosphorylation of the N-terminal tail plays a regulatory role in phospholipid vesicle aggregation, which is based on a mechanism distinct from protein self-association. This phosphorylation induces a conformational change in the tail probably related to aggregation property. PMID- 8620028 TI - Binding of 2-hydroxy-5-nitrobenzyl alcohol to rat alpha class glutathione S transferases; evidence for binding at tryptophan 21. AB - 2-Hydroxy-5-nitrobenzyl alcohol (HNB) was prepared from dimethyl(2-hydroxyl-5 nitrobenzyl)sulfonium bromide (HNBB). HNB binds to glutathione S-transferases (GSTs) 1-2 and 2-2 with moderate affinity at a site separate from 1-anilino-8 naphthalenesulfonate (ANS). Intrinsic fluorescence due to Trp-21 is strongly quenched by HNB binding but there is no effect on catalytic activity. There appear to be two HNB binding sites per dimer in each GST isoenzyme. We suggest that HNB binds directly at Trp-21 of each subunit and that previously reported quenching of intrinsic fluorescence in these proteins upon ligand binding may be due to indirect structural effects rather than direct binding at this residue. PMID- 8620030 TI - Investigation of the interaction of m-calpain with phospholipids: calpain phospholipid interactions. AB - Phosphatidyl inositol, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidic acid and sphingomyelin were all found to be effective at reducing the Ca2+ requirement for m-calpain autolysis. In the absence of phospholipid, pig kidney m-calpain required 1.4 mM Ca2+ for 50% autolysis under the assay conditions used. Phospholipids caused a reduction in this Ca2+ requirement to a value between 0.45 mM Ca2+ for phosphatidyl glycerol and 1.1 mM Ca2+ for phosphatidyl ethanolamine. Previous studies (Crawford, C., Brown, N.R. and Willis, A.C. (1990) Biochem. J. 265, 575-579) have shown that the most probable site for phospholipid interaction in calpain is the N-terminal region between residues 39 to 62 of the small subunit of calpain (G17TAMRILGG). In this study we examine the possible role of this G17TAMRILGG region. Three synthetic peptides corresponding to parts of this sequence were used to examine the phospholipid binding sequence. Analysis of the phospholipid vesicle binding properties of these peptides suggested that both the TAMRIL and polyglycine sequences were required for binding to phosphatidyl inositol vesicles. PMID- 8620029 TI - Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae. AB - The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3 Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD+, PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M-1 min-1. A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M-1 min-1. Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402. PMID- 8620031 TI - Effect of the ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system. AB - A sensitive assay which examines the effects of ATP level on the overall activity of a cell-free translation system in a protein synthesis is described. The translational activity of cell-free system was measured in terms of a rate of protein synthesis directed by the 'test' template. The test template encodes a photoluminescent protein, obelin accumulation was determined from the kinetic curves of obelin. The rate of obelin mRNA translation. Time-dependent nucleotide level measurements were conducted throughout the translation processes. It has been shown that the rate of translation decreases exponentially with the decrease of the ATP level. This fall in the overall translation rate is due in part to the mRNA becoming inactive in the translation process. This is not caused by degradation, this mRNA can be restored for translation in a fresh cell-free system by phenol treatment. The reported results provide evidence that the level of ATP unambiguously determines the translational activity of the system. PMID- 8620032 TI - RAP kinase, a new enzyme phosphorylating the acidic P proteins from Saccharomyces cerevisiae. AB - A new protein kinase, showing a high specificity for the ribosomal acidic P proteins (RAP kinase) has been purified and characterized from Saccharomyces cerevisiae extracts. Purification was carried out by four chromatographic steps, including DEAE-cellulose, phosphocellulose, heparin-Sepharose and P protein Sepharose. The purified enzyme preparation contains only one polypeptide of around 55 kDa as determined by SDS gel electrophoresis and gradient centrifugation. RAP kinase is different from all previous well-characterized kinases and does not show cross-reaction with antibodies to the 71 kDa 60S ribosomal subunit-specific kinase PK60 previously reported. The enzyme uses ATP as a better phosphate donor and is less sensitive to heparin than casein kinase II but is moderately affected by salt. Among the different substrates tested, ribosomal acidic proteins are preferentially modified by RAP kinase, which phosphorylates only serine residues in the four P proteins as well as the related ribosomal protein P0. Casein is phosphorylated at a much lower level. All the data indicate that RAP kinase might be the enzyme responsible for the phosphorylation of the P proteins, and in this way may also participate in a possible translational regulatory mechanism. PMID- 8620033 TI - Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa. AB - Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M 1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS. PMID- 8620034 TI - Refolding of riboflavin carrier protein as probed by biochemical and immunological parameters. AB - The unfolding of the chicken egg white riboflavin carrier protein by disulfide reduction with dithiothreitol led to aggregation with concomitant loss of ligand binding characteristics and the capacity to interact with six monoclonal antibodies directed against surface-exposed discontinuous epitopes. The reduced protein could, however, bind to a monoclonal antibody recognizing sequential epitope. Under optimal conditions of protein refolding, the vitamin carrier protein regained its folded structure with high efficiency with simultaneous complete restoration of hydrophobic flavin binding site as well as the epitopic conformations exposed at the surface in a manner comparable to its native form. PMID- 8620035 TI - Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2 nitrophenyl) ethyl, a photoreversible thiol label. AB - 1-Nitro-2-phenylethene (beta-nitrostyrene, 1) which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817 818), was demonstrated in this work to be an irreversible inhibitor of beta galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2 Nitroethene 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of beta-galactosidase at pH 8.3 with o nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 x 10(-4) M) ranged from 1.67 x 10(4) S-1 after 30 min of preincubation to <0.07 x 10(4) S-1 after 180 min preincubation. For compound 2 (9.5 x 10(-5) M) Kcat values ranged from 2.70 x 10(4) S-1 following 30 min preincubation to 1.15 x 10(4) S-1 after 180 min of preincubation; the changes in Km(app), however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity. PMID- 8620036 TI - Conformational analysis of pentapeptide sequences matching a proposed recognition motif for lysosomal degradation. AB - A selective pathway for the degradation of specific long-lived cytosolic proteins is activated in response to starvation in vivo or to serum withdrawal from cultured cells. It involves recognition of a targeting motif by a member of the hsp70 family. A 5-residue targeting motif has been proposed on the basis of sequence comparisons. We investigate whether there is any structural basis for this motif being the true recognition signal. We examine the conformations of four motif peptides in proteins that are either known to be serum regulated or are from related vertebrate species, and two equivalent peptides in bacterial proteins that closely resemble other regulated proteins. Our studies show that all the motif sequences are located near the ends of surface helices with one or more of the residues buried in the structure, yet it is known that members of the hsp70 family tend to interact with extended peptide chains. Furthermore, recognition by these proteins generally requires a specific ordering of key residues, yet the motif implies a largely order-independent sequence characterized by residue type only. We conclude that the proposed motif is unlikely to be the true targeting signal for lysosomal degradation unless additional factors apply. PMID- 8620037 TI - Preparation and characterization of a C-terminal fragment of pregnancy zone protein corresponding to the receptor-binding peptide from human alpha 2 macroglobulin. AB - Digestion of the pregnancy zone protein with papain at pH 4.5 yields an 18 kDa C terminal fragment. This fragment consists of the 145 C-terminal amino-acid residues cleaved at Asn-1288 Ile and is homologous to the C-terminal receptor binding fragment of human alpha 2-macroglobulin obtained by cleavage with papain. The fragment contains an intrachain disulfide bond between 1308Cys and 1423Cys corresponding to that between 1304Cys and 1419Cys in alpha 2-macroglobulin. An oligosaccharide chain, is present in the C-terminal fragment of pregnancy zone protein as in human alpha 2-macroglobulin. The PZP C-terminal fragment was demonstrated to bind to the LRP/alpha 2M-receptor. Both the pregnancy zone protein and alpha 2-macroglobulin fragments bind three mAb's (alpha 1:1, R35, and 7H11D6) generated against alpha 2-macroglobulin. The mAb 7H11D6 was generated against the alpha 2-macroglobulin-proteinase complex (Isaacs, I.J., Steiner, J.P., Roche, P.A., Pizzo, S.V. and Strickland, D.K. (1988) J. Biol. Chem. 263, 6709-6714) and the binding of this to the C-terminal fragments of both pregnancy zone protein and alpha 2-macroglobulin indicates that both proteins use the same receptor recognition site for binding to the LRP/alpha 2M-receptor. PMID- 8620038 TI - Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. AB - The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation. PMID- 8620040 TI - Characterization and N-terminal sequencing of a calcium binding protein from the calcareous concretion organic matrix of the terrestrial crustacean Orchestia cavimana. AB - We extracted proteins from the organic matrix of calcareous concretions, which represents the calcium storage form in a terrestrial crustacean. Electrophoretic analyses of water-soluble organic-matrix proteinaceous components revealed 11 polypeptides, 6 of which are probably glycosylated. Among the unglycosylated proteins, we characterized a 23 kDa polypeptide, with an isoelectric point of 5.5, which is able to bind calcium. Its N-terminal sequence is rich in acidic amino acids (essentially aspartic acid). All these characteristics suggest its involvement in the calcium precipitation process within the successive layers of the organic matrix. PMID- 8620039 TI - Stereo-selective affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5-imidazolyl)propionic acid. AB - The role of configuration for the affinity labelling of sheep liver sorbitol dehydrogenase by chloro-substituted analogues of 2-bromo-3-(5 imidazolyl)propionate (BrImPpOH) has been studied. A saturation kinetics mechanism applies which includes formation of a reversible complex with the enzyme prior to alkylation of Cys-43. The pseudo first-order inactivation rate constant, k2, and the dissociation constant for the reversible enzyme-affinity label complex. KEI, were determined at pH 7.4 and 23.5 degrees C. The stereo isomers of each affinity label exhibit different kinetic characteristics but, unlike with horse liver alcohol dehydrogenase, the discrimination between them is not absolute. For the different affinity labels, k2 varies with 2-chloro-3-(5 imidazolyl)methylpropionate (Me-ClImPpOH) > 2-chloro-3-(5-imidazolyl)propionate (ClImPpOH) > 2-chloro-3-(5-imidazolyl)propanol (ClImPOH), consistent with their order of inherent reactivity, and the specificity constant k2/KEI varies with (S) Me-ClImPpOH > (S)-ClImPpOH > (S)-ClImPpOH > (R)-Me-ClImPpOH > (R)-ClImPpOH. Models of the affinity labels were built into the active site of the predicted subunit structure of the enzyme by using a computer-controlled display system. In each binary complex, the imidazole moiety of the affinity label was liganded to the catalytic zinc atom, and the angle Scys-C alpha-Cl was linear, in accordance with an SN2 mechanism. Both enantiomers of each label could form plausible complexes with the enzyme model, in agreement with the kinetic data. The enantiomeric selectivity, rather than absolute specificity, of the reaction appears due to the anion-binding site in sorbitol dehydrogenase being less developed than in horse liver alcohol dehydrogenase. PMID- 8620042 TI - [The review Nutricion Hospitalaria included in Index Medicus and Medline]. PMID- 8620041 TI - Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue. AB - P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300-380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 --> Lys-81 and Asp-82 --> Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue. PMID- 8620043 TI - [Complications of the use of central venous catheters]. PMID- 8620044 TI - [Energy expenditures, hypothermia and unusual energy substrates]. PMID- 8620045 TI - [Metabolic monitoring of heart surgery patients during the immediate postoperative period]. AB - The situation of aggression is accompanied by metabolic alterations with an important neuro-endocrinal context. Extracorporeal surgery is an especially aggressive situation which involves both surgery and the added metabolic state of hypothermia. In 48 patients subjected to extracorporeal circulation with hypothermia, the metabolic parameters of Oxygen Consumption (VO2), Production of carbon dioxide (VCO2), Respiratory Quotient (RQ) and Energy Expenditure (GE) were studied. It was observed that the patients could be divided into two subgroups, depending on their metabolic response. Studying the RQ parameter, it was seen that this divided patients into two subgroups depending on their posterior evolution. Patients in whom RQ did not exceed 0.8 showed more favourable evolution than those in whom RQ was higher than 0.8. The correct metabolic response was regarded as a parameter indicative of the evolution, directly correlated to the hemodynamic parameters studied. In one patient, creatine phosphate was infused at a dosage of 30 mg/kg for 60 min, and an improvement was noted in GE and Cardiac Index. It was concluded that the intravenous intake of creatine phosphate in patients undergoing extracorporeal surgery improved both metabolic parameters and the myocardiac function. PMID- 8620046 TI - [A comparative study of hepatic cholestasis after infusion of long chain triglycerides and a mixture of medium and long chain triglycerides]. AB - A randomized, double blind prospective study made on surgical patients who required parenteral nutrition during a 10-day period, with complete fasting. The patients were required to show a normal hepatic function measured by gamma-GT, alkaline phosphatase (FA), normal bilirubin and ALT. The evolution of the cholestasis parameters was observed on days 0, 1, 3, 8 and 10. An increase in gamma-GT was observed in the groups. This was much greater in the group with LCT (p<0.005) on the tenth day than in the MCT/LCT group. FA increased only in the LCT group, and was statistically significant (p<0.001) on the tenth day compared with the MCT/LCT group. PMID- 8620047 TI - [Variability of anthropometric parameters]. AB - Presentation of a comparative study of four ideal weight tables applied to the same population, in order to evaluate the differences or similarities between the same. Significant differences (p<0.001) were found between the four tables studied (WHO, USA, Barcelona, Tenerife) when classifying a patient into one of the three groups defined (malnutrition, normal and obese). The maximum differences found were between the Barcelona and Tenerife tables, which classified 13,7% and 24.05% respectively as cases of malnutrition and 35.9% and 16.89% respectively as obese. PMID- 8620048 TI - [Deep venous thrombosis of the upper limb. A prospective study of the central venous catheter as an etiologic factor and clinical and subclinical incidence of pulmonary thromboembolism]. AB - The thrombogenicity presented in different types of endovenous catheters and their anomalies are the cause of the development of pulmonary thromboembolism (PTE) in some patients, secondary to deep venous thrombosis (DVT) of the upper limbs. Presentation of a study made on the incidence of PTE in patients with prior history of DVT of the upper limbs. Of the 30 cases of DVT of the upper limbs studied, 20 were directly attributed to catheters. 18 were attached to a central catheter and the other 2 one or two peripheral catheters. 0,32% of DVT of the upper limbs secondary to a central catheter was calculated. Five of the 20 DVT patients (25%) had symptomatic or sub-clinical DVT. Emphasis was placed on the importance of DVT and its intrinsically serious nature and the need for studies on this condition, since it is possible for the patient not to develop the complete symptoms of DVT at the onset, which led to death in one patient. We recommend the establishing of strict norms with regard to the indications for inserting the central catheter and the choice of the correct material, aseptic and non-traumatic insertion, radiological control (essential) of the position of the catheter and its tip, establishing of a protocol for the correct maintenance and a device for controlling thrombotic complications in upper limbs, to ensure rapid treatment and a rapid check of the possibility of DVT by pulmonary gammagraphy during the first 24-48 hours. PMID- 8620049 TI - [Incidence of malnutrition in a surgery department]. AB - Presentation of a description of the initial results obtained in nutritional evaluation of patients admitted for elective surgery during a 6-month period. A total of 212 patients were studied, recording malnutrition in 25%. This was severe in half of the cases. As a result of the malnutrition, a significant reduction was observed in the number of positive responses to the Multitest and the total count of lymphocytes. PMID- 8620050 TI - [Effects of branched-chain amino acids (BCAA) in complete hypocaloric nutrition of surgical patients with neoplasms of the digestive tract]. AB - One of the generic indications of parenteral peripheral malnutrition is the immediate postoperative period following surgery of the digestive tract. In the series presented, fifty patients with digestive neoplasia of different locations were studied, with slight or moderate malnutrition upon admittance. After the operation, feeding was done using hypocaloric parenteral solutions for the first seven days of the postop. period. In the qualitative intake of nitrogen in this type of nutrition, branched chain amino acids are important due to their beneficial effects on metabolism, and for this reason two complete hypocaloric solutions containing 45% and 15.5% of these amino acids were administered to each respective group under study. To justify the effect of improving the proteic synthesis attributed to these amino acids, the levels of rapid turnover proteins, transferrin, prealbumin and retinol-binding protein were determined. PMID- 8620051 TI - [Complications of intravenous therapy. Influence of parenteral nutrition, central venous pressure and antibiotics]. AB - With regard to the controversy on whether the use of central venous catheters for the perfusion of other solutions as well as parenteral nutrition means an increase in the risk of sepsis caused by the catheter, a prospective study was made on 313 central intravenous catheters placed in patients admitted to the Intensive Medicine Unit of La Paz General Hospital over a one-year period. Collection of data on both catheters and patients was done following a protocol applied in all cases. From the results obtained, it can be concluded that in critical patients in whom a single central venous catheter has been placed, this can be used for the infusion of other liquids, apart from parenteral nutrition, without involving an increased risk of sepsis caused by the catheter. This pathology seems to bear more relation to the amount of time the catheter remains in place. PMID- 8620052 TI - Nitric oxide regulates peroxisomal enzyme activities. AB - We have previously shown that peroxisomes are involved in the production and detoxification of reactive oxygen species and that peroxisomal functions are damaged by such oxygen species. Since nitric oxide is not only a cellular messenger, but also a free radical, it would be interesting to detect a connection between nitric oxide levels and peroxisomal enzyme activities. To determine if nitric oxide has an effect on the activities of peroxisomal functions and whether this effect is based solely on its chemical properties as reactive oxygen species or its action as a second messenger, effectors of the cellular nitric oxide level were applied to a cell model (human skin fibroblasts in culture) or directly to the enzymatic assays or both. If applied to the monolayer at non-cytotoxic concentrations, N-nitro-L-arginine methyl ester hydrochloride, an inhibitor of nitric oxide synthase (EC 1.14.13.39), increased catalase (EC 1.11.1.6) activity by more than 10% and decreased the activity of the peroxisomal fatty acid oxidation system by more than 10%. The effect was concentration-dependent. L-Arginine had the contrary effect. Combinations of L arginine and N-nitro-L-arginine methyl ester hydrochloride compensated one another. If applied directly to the assays, S-nitroso-N-acetylpenicillamine and sodium nitroprusside inhibited catalase activity in a concentration-dependent manner. Sodium nitro-prusside had no effect on the peroxisomal beta-oxidation system unless cells were pretreated with N-nitro-L-arginine methyl ester overnight (50% inhibition). The results show a differential effect for the application of nitric oxide-effectors on fibroblast monolayers, cell suspensions and under assay conditions. Depending on the conditions of the incubation, nitric oxide applied to the cell monolayer at low doses acts as a second messenger in cells rather than as reactive oxygen species. Under assay conditions the effect of nitric oxide is more likely that of a reactive oxygen species because it inhibits all measured enzyme activities. PMID- 8620053 TI - Differential induction of peroxisomal enzymes by hypolipidaemics in human (HepG2) and rat (MH1C1) hepatoma cell lines. AB - Human (HepG2) and rat (MH1C1) hepatoblastoma cells were incubated with different concentrations of the hypolipidaemics cetaben, clofibrate and thyroxine. The enzymatic activities of catalase, peroxisomal bifunctional enzyme, succinate dehydrogenase, and 3-oxoacyl-CoA thiolase were measured. In order to determine the point of regulation of the enzymatic activities Northern and Slot blot experiments with probes for peroxisomal bifunctional enzyme, catalase and fatty acyl CoA oxidase were performed on total RNA. Catalase activity was enhanced in HepG2 cells treated with 3 mmol/l clofibric acid to 135% of control and the mRNA value to 2.6 fold, whereas in cetaben treated cells the enhancement (up to 119% of control) was less pronounced. In MH1C1 cells catalase activity was not changed by any of the drugs. The activity of the peroxisomal bifunctional enzyme was not affected in HepG2 cells by clofibric acid and cetaben, whereas the mRNA level was elevated to 2.3 fold by 10 micromol/l cetaben. At high concentrations of cetaben all enzyme activities were decreased in both cell lines due to its high cytotoxicity. Our data show that, due to the differences in the genomic organisation, the regulation of the enzyme activities is different in human and rat, but the results from the human and rat hepatoblastoma cells correlate with the findings in whole man and rat, so that a human in vitro system is more suitable for pharmacological tests. These results suggest that the human hepatoma cell line HepG2 may be a useful model system for studies of the influence of hypolipidaemics on the peroxisomal enzyme system. PMID- 8620054 TI - Iron binding capacity of trimidox (3,4,5-trihydroxybenzamidoxime), a new inhibitor of the enzyme ribonucleotide reductase. AB - Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Trimidox, a newly synthesized compound, inhibits this enzyme and has in vitro and in vivo antitumour activity. As trimidox was able to upregulate the expression of the transferrin receptor in HL-60 human promyelocytic leukaemia cells, we have now investigated the capability of trimidox to interfere with iron metabolism. We show by photometric and polarographic methods that trimidox is able for form an iron complex. However, its cytotoxic action cannot be circumvented by addition of iron-saturated transferrin or iron-ammonium citrate, indicating that the iron complexing capacity is not responsible for the mechanism of action of this compound. When HL-60, K562 or L1210 leukaemia cells were incubated with the trimidox-iron complex itself, we could observe increases of the 50% growth inhibitory capacity of the complex in comparison with trimidox alone. We conclude that trimidox is able to form an iron complex, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Further studies will have to elucidate the molecular mechanism of action of this new and promising anticancer agent. PMID- 8620055 TI - Non-transferrin iron uptake by HeLa cells cultured in serum-free media with different iron sources. AB - HeLa cells cultured in defined serum-free media supplied with iron wither in the form of diferric transferrin (transferrin-dependent cells), ferric citrate at 500 micromol/l (high-iron dependent cells) or ferric citrate at 5 micromol/l (low iron dependent cells) accumulate iron from ferric citrate in different ways. The uptake rate in transferrin-dependent cells is always much lower in the other two lines. In all three, the uptake rate rises almost linearly with the concentration of iron up to 10 micromol/l. In high-iron dependent cells, the uptake of radiolabelled iron is suppressed by a 100-fold excess of the iron complex, whereas this same excess stimulates iron uptake in the other two lines. The same concentrations of pure citrate completely inhibit iron uptake by all three types of cell. Only high-iron dependent cells take up citrate at measurable and reproducible rates. These rates are independent on the presence of iron, and the uptake is inhibited by an unlabelled surplus. The pH-dependence of iron uptake in high-iron dependent cells is also different from that of the other cells. Low iron dependent cells transferred to medium containing 500 micromol/l iron show increased uptake rates within 3 to 7 h, and after overnight maintenance in this medium they acquire the uptake characteristics of high-iron dependent cells. The special characteristics of iron uptake by high-iron dependent cells are paralleled by low binding activity of iron-regulatory protein to iron-responsive elements of RNA. We conclude that low-iron dependent cells maintain their iron supply from the culture medium by unspecific uptake of oligomeric complexes, while cells in media with a high content of low-molecular weight iron induce a specific uptake system which might have a protective function. PMID- 8620057 TI - Time-resolved immunofluorometric assay for the quantification of lipoprotein(a) in serum. AB - Although two recent studies have failed to reveal lipoprotein(a) (LP(a)) serum concentrations > 300 mg/l to be an independent risk factor for early onset of atherosclerosis, Lp(a) serum concentrations are frequently measured to evaluate the additional risk of coronary heart disease. We describe a time-resolved immunofluorometric assay (TRIFMA) for quantifying Lp(a) levels in humans serum using commercially available reagents, which is rapid, robust and simple to perform. The two-site immunometric assay was based on microtitre plates as solid phase coated with a polycloncal anti Lp(a) antibody. The liquid-phase antibody was labelled with biotin and detected by europium labelled streptavidin in the DELFIA 1232 fluorometer. The measuring range was 2-1600 mg/l. The intra-assay imprecision was < 7% (CV), the inter-assay imprecision < 12% (CV). No interference was detected with plasminogen concentration up to 2.2 g/l. There was an acceptable correlation with a commercially available enzyme immunoassay (r = 0.95) and with electroimmunodiffusion (r = 0.85) on 100 routine serum samples measured. The assay appeared to detect different Lp(a) isoforms as dilution curves were parallel for B/F, S2 and S4 isoforms. PMID- 8620056 TI - Apolipoprotein E and complement C3 polymorphism and their role in the response to gemfibrozil and low fat low cholesterol therapy. AB - Three different allelic variants of apolipoprotein E determine, in concert with other gene products, the levels of plasma lipoproteins. Recently, cleavage products of the complement C3 molecule have also been implicated in determining plasma triacylglycerol concentrations. This study presents data of an ongoing study to dissect the role of the apolipoprotein E gene locus in the response to low fat/low cholesterol diet combined with gemfibrozil treatment. In addition, for the first time, the significance of C3 allelic variants to such hypolipidaemic therapy response was analysed. To this end data from 81 obese hyperlipoproteinaemic patients (Fredrickson type II/A and B and type IV and V) confirmed the usefulness of the combined gemfibrozil/diet treatment and unveiled apolipoprotein E allele group specific therapy responses. The mean changes of lipid properties due to combined treatment was 15% for total cholesterol, 48% for triacylglycerols and 28% for atherogenic index. Division into hyperlipidaemia types according to Fredrickson and subgrouping into E2, E3 and E4 groups (apolipoprotein E2/2 and 2/3, apolipoprotein E3/3 and apolipoprotein E4/2 and 4/3 phenotype groups respectively) exposed pronounced differences from these mean changes, suggesting substantial influence of apolipoprotein E variants on this therapy. We observed triacylglycerol reductions of from 17% in type IIA apolipoprotein E3 group patients up to 78% in the type IV and V-apolipoprotein E2 group. Thus it might be concluded the apolipoprotein E genotyping aides therapy success prediction. Although, low sample number in some subgroups obscures significance in this pilot study, significant therapy success emerges for the E3 and E4 group in type IV and V hyperlipidaemia and type IIB-apolipoprotein E3 homozygous patients can be predicted to respond better than apolipoprotein E2 carriers. Finally, we present evidence that positive changes of lipid properties are also determined by the "fast" complement C3 allel (C3-F). Patients with complement factor C3-FS pattern respond better to treatment than patients with C3 SS configuration. In summary these data endorse the genotyping of apolipoprotein E alleles to predict maximal success of "fibrate" treatment. In addition they argue strongly for further assessment of the involvement of complement C3 allelic variations in lipid homeostasis. PMID- 8620058 TI - The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival. AB - The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions. This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions. These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell. We studied the various chemical and biochemical aspects involved in the MTT assay. Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay. In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival. PMID- 8620059 TI - Serum class I and II alcohol dehydrogenase activity during the course of viral hepatitis. AB - We examined the activities of class I and II alcohol dehydrogenase isoenzymes in the sera of patients with viral hepatitis using the fluorogenic substrates 4 methoxy-1-naphthaldehyde for class I and 6-methoxy-2-naphthaldehyde for class II. It was found that serum activities of class I and II alcohol dehydrogenase isoenzymes over the course of five weeks of hospitalisation were higher than those of controls. The greatest increase in activities was found at the onset of disease, exceeding the mean control value by about 30 fold for class I and 4 fold for class II. These activities were lower than that of aminotransferase but higher than those for lactate dehydrogenase, alkaline phosphatase and gamma glutamyltransferase. Thereafter, the activity of alcohol dehydrogenase isoenzymes gradually decreased, but did not reach the values of the control groups in the last period of the study. Activities of class I and II alcohol dehydrogenase isoenzymes correlated well with those of alanine and aspartate aminotransferase and lactate dehydrogenase in the first weeks of illness. These results clearly demonstrate that especially the activity of class I alcohol dehydrogenase isoenzyme measured by a fluorimetric method can be a useful marker of liver cell damage in the course of viral hepatitis. PMID- 8620060 TI - Predictive value of urinary neopterin in patients with lung cancer. AB - Concentrations of neopterin, which is produced by human monocytes/macrophages when stimulated by gamma-interferon, were measured in urine specimens from 72 patients with lung cancer at diagnosis. Other routine clinical and laboratory variables were concomitantly determined. Neither neopterin nor any other laboratory variable studied showed a significant correlation with clinical indicators of the disease (morphologic type, tumour stage, grading, lymph node status, presence of distant metastases). The cancer patients were followed up for up to 10 years, and the abilities of all variable to predict fatal outcome were assessed. In univariate survival analyses, all clinical indicators except morphologic type (P = 0.86) were significant predictors of survival (P < 0.002), but of all the laboratory variables studies, only neopterin was significantly predictive (P = 0.0013). By multivariate survival analysis, a combination of four variables was found to jointly predict survival: lymph node status (P = 0.003), multivariate model), tumour stage (P = 0.0006), grading (P = 0.0047) and neopterin (P = 0.0047). The data suggest that certain aspects of immune activation may have adverse consequences for the prognosis of patients with lung cancer. PMID- 8620061 TI - Peripheral blood monocyte counting: towards a new reference method. AB - Flow cytometric enumeration of monocytes stained with fluorescence-labelled monoclonal antibodies has been proposed as a possible reference method for monocyte counting. We compared precision and accuracy of monocyte counting of the Coulter STKS, the Cobas Argos 5 Diff, the 800-cell manual differential, and the Coulter Epics Profile II flow cytometer using double-staining with fluorescence labelled monoclonal antibodies (CD45-FITC and CD14-PE). Precision: STKS, Argos and Profile II achieved a precision analogous to a 3423-, 1298-, and 11089-cell differential, respectively, confirming the superiority of automated methods. Accuracy (136 normal and abnormal samples): Correlation of automated methods with the manual differential was good (STKS: r = 0.934, Argos 5 Diff: r = 0.808, Profile II: r = 0.924; Spearman's rank correlation coefficient). The mean relative STKS monocyte result was 0.52 +/- 1.63% (mean +/- SD) higher than the manual differential, whereas the Argos 5 Diff results were 1.22 +/- 2.51% lower (p < 0.001). Profile II results showed a small bias against the manual differential (-0.18 +/- 1.44%, p < 0.05). Analysing 135 healthy adult subjects on the Profile II, males were found to have a higher mean monocyte count (relative count: 6.95 +/- 1.43% vs. 5.86 +/- 0.98%; absolute count: 0.48 +/- 0.15 x 10(9)/l vs. 0.39 +/- 0.11 x 10(9)/l, p < 0.001) and a higher and wider normal range than females (relative count: 4.97 to 9.78% vs. 4.26 to 7.81%, absolute count: 0.30 to 0.84 x 10(9)/l vs. 0.25 to 0.65 x 10(9)/l). Flow cytometry based on fluorescence labelled monoclonal antibodies for monocyte enumeration seems an efficient tool to evaluate the monocyte counting performance of haematology analysers and an ideal successor to the manual differential as reference method for monocyte counting. PMID- 8620062 TI - System identification of the low-dose kinetics of p-aminohippuric acid. AB - The renal clearance of p-aminohippuric acid, due to tubular secretion in addition to glomerular filtration, can only be determined by kinetic experiments. Maximal information can be gained from observed temporal marker concentration profiles by fitting dynamic mathematical models of the processes involved, such as absorption, distribution, and elimination, to the kinetic data. Thereby the values of the system constants, such as fractional elimination or fractional distribution rates, and their accuracy measures are determined by methods which are based firstly on measured time-dependent data elicited in an individual test object by perturbing inputs and secondly, on mathematical formulations of prior knowledge of the underlying physiological system. Such methods of model adaptation are called system identification. In this context a computer-based method of system identification and error estimation for the system constants of two-compartment models matched a dynamic concentration profiles of p aminohippuric acid is presented. The method is used of single-injection experiments to demonstrate that such a technique is able to correctly estimate the clearance of p-aminohippuric acid if sufficiently long experimental protocols are chosen, and to ascertain the sufficient length of a protocol for an individual subject. The renal clearance of p-aminohippuric acid is known to exhibit concentration-dependence generally, but to achieve its maximal value when low doses are applied. The present study deals with the low-dose kinetics of p aminohippuric acid. PMID- 8620063 TI - Creatinine and surveys: an assessment. AB - We analysed the results of surveys on creatinine held in The Netherlands during the years 1992, 1993 and 1994. Assay results of 113 samples were reviewed: 88 human sera and 25 samples of animal origin. The results of 5 creatinine assays, 4 based on the Jaffe reaction and 1 enzymatic procedure, are discussed. The enzymatic assay showed by far the best performance, while some of the Jaffe methods differed considerably. All results were evaluated by reference to a HPLC based selected method for creatinine. Our study shows the need for caution when applying survey performance criteria for creatinine. PMID- 8620064 TI - Usefulness of reference materials in calibration of enzyme activities. AB - alpha-Amylase, alkaline phosphatase and gamma-glutamyltransferase were studied in a multicentre evaluation. Analyses were performed on different patient samples. Each enzyme was assayed in two different laboratories at both 30 and 37 degrees C, with widely used reagent kits and with the IFCC reference method (if in existence). Results differed considerably according to the measurement procedure. Data also showed that it was not possible to employ a constant conversion factor for one enzyme and different techniques between 30 and 37 degrees C. Calibration with three reference materials extensively improved the intermethod consistency for most of the tested measurement procedures. It was possible to transfer accuracy from the method used for the certification of the reference material to routine procedures, by using the reference material as calibrator. Temperature did not seem to be a crucial variable for the implement of the enzyme calibrator approach. PMID- 8620065 TI - Biochemical and enzymological study of lactate dehydrogenase isoenzymes from commercial quality control sera and several animal tissue sources. AB - We assayed the isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in commercial quality control sea and several animal tissue extracts, using electrophoresis. We compared the Km values and activation energies of the isoenzymes, in order to find suitable animal tissue sources with a similar isoenzyme profile to that of human serum lactate dehydrogenase. Some of the control sera contained only one isoenzyme fraction corresponding to porcine heart isoenzyme-1 or chicken heart isoenzyme-1, which showed essentially no changes of enzyme activity as a function of pyruvate substrate concentration. Other control sera, which contained isoenzymes from human red cells haemolysates, or from animal tissue extracts with a human serum matrix, showed significant changes of enzyme activity was a function of substrate concentration, and showed different Km values and activation energies from those of human serum. Of the serum and tissue samples from several animal sources, rat heart and kidney extracts showed the greatest similarity to human serum, with respect to the electrophoretic pattern and the Km, pH optimum and activation energy of lactate dehydrogenase isoenzymes. PMID- 8620066 TI - Cardiac troponin I release correlates with myocardial infarction size. AB - Cardiac troponin I, creatine kinase, and creatine kinase MB activity were tested in serial blood samples from 15 patients with first-time Q wave acute myocardial infarction (2 anterior and 13 inferior wall infarctions). All patients received intravenous thrombolytic therapy. Cardiac troponin I and creatine kinase MB activity were compared with scintigraphic estimates of myocardial scar (single photon emission computed tomography [SPECT] with 99mTechnetium-isonitrile [Tc sestamibi]) on late images at rest about 5 weeks after myocardial infarction. Scintigraphic defect sizes ranged from 3.2 to 41.2% (median: 27.3%) of left ventricle. Cardiac troponin I increased and peaked in parallel with creatine kinase MB activity, and the peak values correlated with each other (r = 0.76, p = 0.002). Troponin I stayed increased for several days longer than creatine kinase and creatine kinase MB activity. It could be detected at least until the 4th day after admission. Significant correlation coefficients were found between 99mTc isonitrile defect sizes and areas under cardiac troponin I curves (r = 0.53, p = 0.042) and between 99mTc-isonitrile defect sizes and cumulative creatine kinase MB activity release (r = 0.64, p = 0.01). Animal studies have already shown a very close correlation between histologic infarct size and SPECT 99mTc-isonitrile defect size. Therefore, our results indicate that cardiac troponin I release in patients with acute myocardial infarction is also correlated with infarct size. PMID- 8620068 TI - "BKK system"--an initiative for efficient quality assessment in diagnostic laboratories of countries of the former Soviet Union. PMID- 8620067 TI - Effect of the pH and the importance of the internal standard on the measurement of the urinary catecholamines by high-performance liquid chromatography. AB - Clinical manifestations of phaeochromocytoma are not always sufficient for its early diagnosis. It is therefore necessary to confirm hypersecretion of catecholamines. Current methods for the measurement of catecholamines are based on their oxidative properties, and the majority of the laboratories often use HPLC methods for catecholamine testing. However, the extraction procedures used for the biogenic amines differ. We use a method of ion-exchange chromatography which is performed at pH 6.5. In order to avoid the spontaneous oxidation of the catecholamines, the urine samples has to be collected on HCl, which gives a pH of approximately 2. Occasionally, the acidified urine sample has a pH less than 1 requiring the addition of NaOH to reach a pH of 6.5, necessary for the adsorption of catecholamines on cation exchanger resins. This phenomenon produces a decrease in the peak areas but the use of an internal standard allows the final results to be corrected. PMID- 8620069 TI - Evaluation of AutoDELFIA and access automated immunoassay systems for third generation assays for thyrotropin. AB - We evaluated the technical performance of two new commercial automated immunoassay systems of third-generation assays for thyrotropin. The interassay CV was 2.8% for AutoDELFIA and 3.25% for Access at thyrotropin concentrations of approximately 1.3 mIU/l. The lower detection limits of the assays were 0.023 mIU/l for AutoDELFIA and 0.0096 mIU/l for Access, and the functional sensitivity for a CV of 20% was 0.027 mIU/l and 0.028 mIU/l, respectively. Sample to sample carry over was negligible (0.0016% for AutoDELFIA and 0.005% for Access). The range of linearity was acceptable for Access (102-115%) but not for low thyrotropin concentrations in AutoDELFIA (143% for a thyrotropin value of 0.48 mIU/l). Correlation between AutoDELFIA and Access was adequate (r = 0.999). We conclude that both automated immunoassays offer good reliability, practicability and performance characteristics. PMID- 8620071 TI - General obstetrics in 1995: the successful application of the most conventional clinical concepts to the evaluation of the most recent innovations. PMID- 8620070 TI - Evaluation of the DPC IMMULITE random access immunoassay analyser. AB - The automated immunoassay analyser, IMMULITE, developed by DPC, was evaluated. IMMULITE is an automated system that allows random access in combination with immediate and continuous access. In this study we evaluated the IMMULITE on four panels of analytes: thyroid, fertility, tumour and "non-routine" markers. We observed good within-rum reproducibility (ranging from 2.3-15.9% CV, for low controls, to 2.7-8.7% CV for high controls) as well as between-day imprecision (ranging from 3.7-24.6% CV for low controls, to 2.5-11.8% cv for high controls). The analytical sensitivity of the assays ranged generally from very sensitive to acceptable. The dilution curves for all assays were nearly linear i.e. the maximum deviation of the observed from the expected recovery was 8%. Correlation between IMMULITE and other assays (AxSYM, IMx, TDx, AIA-1200, DPC-c. a. c., Medgenix) varied from r = 0.931 to r = 0.994, except for lutropin and parathyrin with coefficients of correlation of r = 0.594 and r = 0.591. The slopes of the regression lines ranged from 0.745 to 1.327, except for parathyrin where a slope of 2.389 was found. Inter-laboratory correlation was very good between the two locations (Sittard and Apeldoorn) and varied from r = 0.984 to r = 0.999; slopes of the regression lines varied from 0.924 to 1.086. We conclude that the DPC IMMULITE system is suitable for testing routine analytes in random access mode as well as for testing "non-routine" analytes on an automated immunoassay analyser. PMID- 8620072 TI - Antenatal Care. AB - Recently published papers on antenatal screening show that a policy of routine cervical examination does not lead to a reduction in preterm birth, perhaps because the results of the cervical examination do not influence the decisions umbilical Doppler ultrasonography shows that the systematic use of this method for high-risk pregnant women reduces the odds of perinatal mortality by 38%. Studies on antenatal screening for Group B Streptococcus followed by intrapartum treatment show that the antenatal screening can be effective in reducing the rate of infection. PMID- 8620073 TI - Maternal adaptation to pregnancy. AB - Pregnancy is a time when significant maternal cardiovascular and hemodynamic changes occur. Such alterations may include blood volume, heart rate, stroke volume, cardiac output, and systemic vascular resistance. These physiologic changes are usually well tolerated by the pregnant patient, and they must be recognized and understood by the physician to distinguish them from what is abnormal. Changes begin early in gestation and continue as pregnancy advances, and are totally reversible after delivery. This review summarizes updates on cervical, hemodynamic, cardiovascular and endocrinologic changes during pregnancy. PMID- 8620074 TI - The antiphospholipid syndrome. AB - The antiphospholipid syndrome in obstetrics represents a striking model of maternofetal pathology related to systemic and intraplacental thrombosis. Two pro coagulant antibodies are now well known: lupus anticoagulant and anticardiolipin antibody. Therapy is preventive and efficient. It is based on a cautious choice from three drugs: aspirin, heparin, and corticosteroids. PMID- 8620075 TI - Induction of labor. AB - Several mechanical and biochemical methods have been used to induce labor throughout modern obstetrics. We review and compare mechanical and medical methods of labor induction, along with several new uterotonic agents such as mifepristone (RU 486) and misoprostol, which can be conveniently administered orally and managed in the outpatient clinic. PMID- 8620076 TI - Management of labor and labor complications. AB - Obstetricians assume a dual role in the provision of health care for both mother and fetus during labor. Not only do they function as consultants for medical and surgical problems peculiar to labor, but they also assume a more broad-based role in the prevention of labor complications to achieve the best possible health maintenance for both patients. This includes the provision of continuous health care throughout pregnancy, delivery, and postpartum management. A summary of the common problems of management of labor and labor complications is provided, including fetal monitoring during labor, augmentation of labor, vaginal delivery after cesarean section, epidural analgesia and its effects on delivery, and fecal incontinence after delivery. PMID- 8620077 TI - Normal puerperium and breast-feeding. AB - Mothers who have just given birth need support from medical staff and also from their home surroundings. The type of support given to mothers and their new babies varies in different countries and cultures, but should be equally adequate and beneficial. Breast-feeding, as the very best method of early nutrition of the newborn, is the central event of puerperium. Its importance, and other practical approaches during this period are discussed and reviewed. PMID- 8620078 TI - Postpartum haemorrhage. AB - Most cases of postpartum haemorrhage are caused by uterine atony, maternal soft tissue trauma, retained placenta or its parts, and obstetric coagulopathy. The factors most significantly associated with haemorrhage include advanced maternal age, prolonged labour, pre-eclampsia, obesity of mother, multiple pregnancy, a birth weight of more than 4000g, and previous postpartum haemorrhage. It seems that multiparity itself is only a weakly associated factor. The prophylactic use of oxytocic drugs (oxytocin or its combination with ergometrine at the third stage of labour is always recommended for decreasing the bleeding. Prostaglandins should be used as a second line treatment if uterine atony cannot be abolished by uterine massage and oxytocin infusion. In the surgical management, the role of hypogastric artery ligation is decreasing. The stepwise uterine devascularization may be a reasonable method in the most severe uncontrollable postpartum bleeding. The uterine tamponade with gauze or specific tubes may also be a useful alternative in some cases. Selective arterial embolization is a promising new method that seems to have success in controlling the heavy postpartum bleeding unresponsive to more usual measures. However, the value of this method should be evaluated in bigger series. PMID- 8620079 TI - Prenatal care: an update and future trends. AB - Prenatal care gained universal acceptance during this century, but its efficacy and impact has been questioned widely. Many studies have linked inadequate, or lack of, prenatal care with adverse pregnancy outcomes; however others have failed to confirm the association. This article reviews salient aspects of standard prenatal care, and presents future trends in this field. PMID- 8620080 TI - Fetal assessment in low-risk pregnancy. AB - The value of directed management based on contemporary fetal assessment techniques in the high-risk pregnancy is undisputed. Whether such benefits are evident in the low-risk pregnancy is now an issue of considerable clinical and economic importance. Recent literature suggests that extension of fetal surveillance to the low-risk pregnancy yields significant improvement in pregnancy outcome. PMID- 8620081 TI - Multifetal pregnancies. AB - The incidence of multifetal pregnancies has been increasing steadily. Fetal complications such as prematurity, discordant growth, intrauterine fetal demise, twin-twin transfusion, and congenital anomalies account for a large share of perinatal morbidity and mortality. Maternal complications from multiple gestations are also significant. Recent trends in antepartum management such as multifetal pregnancy reduction, specialized prenatal care, and well-timed delivery have improved outcomes. Intrapartum management, including the possibility of asynchronous birth and critical assessment of mode of delivery, has also led to better outcomes. This review provides a brief summary of each of these topics with, a particular focus on the recent literature. PMID- 8620082 TI - Ectopic pregnancy. AB - The clinical diagnosis of ectopic pregnancy is well established. Serial human chorionic gonadotropin titers and transvaginal ultrasound have a high detection rate. In addition, serum progesterone levels are an indicator for eligibility to a medical protocol. The treatment is now increasingly conservative, with alternatives to laparoscopic treatments available. An unruptured ectopic pregnancy allows nonsurgical management, but there is an urgent need for better tests to detect tubal rupture. PMID- 8620083 TI - The evaluation of accelerated fetal growth. AB - The timely recognition of fetal macrosomia may reduce the complications associated with vaginal delivery of a macrosomic fetus. Today, the most frequently used tool for identification of fetal macrosomia is ultrasound. Although many different calculations have been applied, the most commonly used is the estimation of fetal weight. Generally, the detection rate of fetal macrosomia is 33-82%, with a specificity of 70-100%, a positive predictive value of 40-83%, and negative predictive value of 66-92%. Adding amniotic fluid volume, cheek-to cheek diameter or fetal subcutaneous tissue: femur length ratio may improve the accuracy of the diagnosis. Other promising diagnostic tools include the echo planar imaging and the neural network. Despite the progress that has been achieved since the use of Nagele's rule, our ability to fetal macrosomia remains limited. PMID- 8620084 TI - Abnormal fetal presentations. AB - Current trends in the obstetrical management of abnormal fetal presentations have contributed to the sharp rise in the overall use of cesarean section for delivery. Recent literature questions this need. Many investigators are re evaluating this shift from more traditional approaches. PMID- 8620085 TI - Obstructed labor and shoulder dystocia. AB - Dystocia of labor has become one of the leading indications for operative delivery during the past few years. Dystocia of the first stage of labor complicates 8-11% of all vertex delivery, and in the second stage of delivery it is at least as common. Dystocia may result in part from three factors: uterine activity, the fetus, and the pelvis. In each case of abnormal labor, assessment should be made according to those criteria. Shoulder dystocia is an infrequent, unanticipated, and unpredictable nightmare for the obstetrician. Although it is difficult to predict shoulder dystocia, effort should be made to prevent it. Tight glucose control in the management of diabetic patients will reduce the incidence of macrosomic fetuses. Cesarean section should be considered for diabetic women carrying fetuses with estimated fetal weight of greater than 4250g and for non-diabetic women carrying fetuses with estimated fetal weight of greater than 4500g. In all cases good clinical judgement can reduce the rate of shoulder dystocia. However, in some cases it remains a problem for the obstetrician and because it occurs so rarely, the care provider may have limited skills to manage this condition. PMID- 8620086 TI - Ultrasound features of normal early pregnancy development. AB - Recent advances in ultrasound imaging have improved the assessment of early pregnancy development. The gestational sac can be detected within the uterine cavity as early as 4 weeks and 3 days after the last menstrual period. High resolution images of early embryos have enabled ultrasound studies of normal brain, abdominal wall or limb development. The definition of standard developmental morphological features may open the possibility of screening for structural defects early in the first trimester of pregnancy. PMID- 8620087 TI - Anti-neutrophil cytoplasmic antibodies in generalized autoimmune diseases. AB - Anti-neutrophil cytoplasmic antibodies (ANCA) are a system of autoantibodies which are strongly associated with the primary systemic vasculitides (PSV). cANCA, as detected by indirect immunofluorescence, are mostly reactive to proteinase 3 (PR3) and bear high sensitivity and specificity for Wegener's granulomatosis. pANCA have varied subspecificities and clinical associations. The most important subspecificity of pANCA is anti-myeloperoxidase (MPO), which is strongly associated with microscopic polyaniitis and pauci-immune crescentic glomerulonephritis. pANCA also occur at low to moderate frequency in other PSV, collagen vascular disease, inflammatory bowel disease and autoimmune liver disease. In systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), pANCA of varied subspecificity have been found, including anti-MPO at low rate and low titer, while cANCA and anti-PR3 were generally absent. Consequently, anti PR3 (PR3-ANCA) and anti-MPO (MPO-ANCA) at moderate and high titer are distinguishing features of the PSV and, in an appropriate clinical setting, argue strongly against the presence of SLE or RA. Since no significant clinical association has been found for other pANCA subspecificities, cANCA, PR3-ANCA and MPO-ANCA remain the critical elements of ANCA testing in the clinical diagnosis of generalized autoimmune diseases. PMID- 8620088 TI - Eosinophils in allergy: role in disease, degranulation, and cytokines. AB - For over 100 years, the eosinophil has been associated with allergic disease. At present, eosinophils appear to be associated pathologically with asthma, atopic dermatitis, allergic rhinitis, eosinophilic gastroenteritis, and certain eye diseases. The effector functions of eosinophils appear to be derived primarily from release of lipid mediators and proteins, including cytokines and granule proteins. Eosinophil degranulation results in the release of several cytotoxic cationic granule proteins. Furthermore, release of cytokines by eosinophils and other cells involved in inflammation amplifies and regulates localized immune responses. Altogether, the eosinophil's capacity to release and be influenced by a variety of mediators, including the granule proteins and cytokines, implicates this cell in the pathology of inflammation and in the perpetuation of the inflammatory response. PMID- 8620089 TI - Molecular analysis of HLA-DPB1 alleles in idiopathic systemic sclerosis patients and uranium miners with systemic sclerosis. AB - According to clinical mainifestation and autoantibody pattern [anti-Scl-70, anti centromere antibodies (ACAs)], systemic sclerosis is a connective tissue disease with heterogenous subgroups. PCR-sequence-specific-oligonucleotide typing was used to study the genetic association of HLA-DPB1 alleles in 54 patients with idiopathic systemic sclerosis, 26 uranium miners with systemic sclerosis and 70 unrelated healthy control subjects. Systemic sclerosis patients with and without former employment in mines were divided into two subgroups according to their scleroderma-typical autoantibody specificities--anti-Scl-70 positive and ACA positive--and third subgroup comprising the rest. Statistical analysis revealed a significantly increased frequency of DPB1*1301(p=0.0001, corrected p=0.011) in idiopathic anti-Scl-70-positive systemic sclerosis cases when compared with unexposed controls. In the same group, we observed an enhanced frequency of DPB1*0601 and *1701 alleles. Since these three alleles carry the information for a glutamic acid residue in position 69 of DPB1, we tested the association of this residue with anti-Scl-70 expression. A strong association between anti-Scl-70 positivity in idiopathic systemic sclerosis patients and amino acid residue 69 of DPB1 was observed when compared with anti-Scl-70-negative idiopathic systemic sclerosis patients (p=0.0009) or unrelated controls (p=0.0007). ACA expression was not associated with the presence of any DPB1 allele tested. The data show that anti-Scl-70 expression in idiopathic systemic sclerosis patients is linked with DPB1*1301 whereas anti-Scl-70-positive miners do not show such a DPB1 association. Futhermore, the data indicate that glutamate 69 of DPB1 might be involved in the susceptibility to idiopathic anti-Scl-70 expression. PMID- 8620090 TI - Ovalbumin-liposome conjugate induces IgG but not IgE antibody production. AB - Antibody response after immunization with surface-coupled ovalbumin (OVA) of liposomes was investigated in mice. OVA was coupled to the surface of liposome via amino groups using glutaraldehyde. OVA-liposome conjugate induced a significant anti-OVA IgG antibody production in mice. However, no IgE antibody production specific for OVA was observed. Immunization with OVA-liposome induced IgE-specific unresponsiveness even after the subsequent challenge with OVA adsorbed with with aluminium hydroxide (OVA-alum), which induces a high level of IgE antibody production. Furthermore, following the primary immunization with OVA alum, a secondary challenge with OVA-liposome boosted anti-OVA IgG but not anti OVA IgE antibody production. These results show the potential of the antigen liposome conjugate for the development of a vaccine with the least allergic reaction and also for the application of immunotherapy. PMID- 8620091 TI - Prolactin-induced immunoglobulin and autoantibody production by peripheral blood mononuclear cells from systemic lupus erythematosus and normal individuals. AB - Prolactin (PRL) has been shown to have immunoregulatory effects on a variety of immune responses. Its effect on B cell immune responses is suggested by in vitro data demonstrating a direct effect on B cell activation and differentiation, and also in vivo data demonstrating a biphasic stimulation of antibody production to sheep red blood cells. In addition, it has been shown both in animal models and patients with hyperprolactinemia that PRL may influence the presence of certain autoantibodies. The objective of this work was to study the effect of PRL on the induction of immunoglobulins, and anti-DNA and rheumatoid factor (RF) autoantibody production from peripheral blood mononuclear cells (PBMCs) from normal individuals and systemic lupus erythematosus (SLE) patients. Six female SLE patients and 10 normal individuals (5 females and 5 males) were studied. Ficoll-Hypaque-isolated PBMCs (1x10(6) cells/ml) with high concentrations of PRL (10(-4)-10(-8)M) and pokeweed mitogen (PWM) diluted to 1:400. An ELISA assay was used for immunoglobulins, RF and anti-dsDNA antibodies. PRL stimulated IgG and IgM production in a biphasic manner in normal PBMCs. Enhanced synthesis was observed at 10(-6) M, and a stimulatory effect was again observed at higher doses of PRL (10(-4))M. In contrast, only a mild stimulatory effect was observed in IgG synthesis by SLE PBMCs. These changes in Ig synthesis, however, did not reach statistical significance. PRL also induced IgG and IgM anti-dsDNA antibodies by both normal and SLE lymphocytes, but no differences were observed when compared to PWM stimulation. PRL induced IgM RF synthesis by normal lymphocytes but had no effect on SLE PBMCs. This study demonstrates that PRL induced immunoglobulin synthesis by normal and to a lesser degree by SLE lymphocytes, and also induced anti-dsDNA antibody by normal and SLE PBMCs, and IgM RF by normal PBMCs. However, the exact mechanism(s) of PRL action on the immune response awaits elucidation. PMID- 8620093 TI - Association of immunological changes with clinical efficacy in atopic eczema patients treated with traditional Chinese herbal therapy (Zemaphyte). AB - The efficacy of the Chinese herbal therapy (Zemaphyte) has been well established as a treatment for atopic eczema (AE) in clinical trials. The purpose of this study was to probe the immunological changes that occurred when patients were treated with the herbs for a period of 8 weeks. This treatment decreased serum IgE complexes (p less than 0.05) but did not affect total serum IgE or CD23 expression on peripheral blood monocytes. Peripheral blood mononuclear cells from patients before and after treatment were cultured overnight with interleukin 4 and the ability of this cytokine to induce CD23 on monocytes from treated patients was found to be significantly diminished (p less than 0.01). Soluble interleukin 2 receptor and soluble vascular cell adhesion molecule were both raised in the serum of AE patients compared to control individuals. Both these parameters were decreased following treatment (p less than 0.05). All these changes coincided with improvement in erythema and surface damage scores. There was no alteration in soluble intracellular adhesion molecule or soluble CD23. The results of these investigations would suggest that this herbal treatment has the ability to target various immunological parameters which may be involved in the pathogenesis of AE. PMID- 8620092 TI - Functional disturbance of naive T lymphocytes in very high IgE producers: depletion of interleukin-4-induced interleukin-4-producing cells. AB - We examined the capacity of T cells from normal individuals and allergic patients with very high IgE to differentiate into interleukin-4(IL-4)-producing cells in vitro. T cells incubated with anti-CD3 monoclonal antibody plus IL-4 or plus anti IL-4 antibody in the presence of antigen-presenting cells for 7 days were restimulated and their capacity to express IL-4 mRNA was examined by RT-PCR. In T cells from normal individuals, there was a marked increase in the expression of IL-4 mRNA following the addition of IL-4. After fractionation of normal T cells into naive T (CD45RA+) and memory T (CD45RO+) cells, induction of the increase of IL-4 mRNA was restricted to the naive T cell population. In contrast, in T cells from allergic patients, the stimulation of whole or naive T cells with anti-CD 3 monoclonal antibody in the presence of IL-4 induced much less IL-4 mRNA. These findings suggest the presence of a functional abnormality in IL-4-dependent development of IL-4-producing T cells in the peripheral-blood naive T cells from allergic patients. PMID- 8620094 TI - Immune responses to a Nippostrongylus brasiliensis infection in athymic and euthymic rats: surface expression of IgE receptors, IgE occupancy and secretory ability of mast cells. AB - The significance of T cells in an allergy-related immune response was investigated. Athymic, nude (Lewis rnu/rnu) rats lacking T lymphocytes and euthymic (Lewis+/+) rats were infected with the nematode Nippostrongylus brasiliensis. The density and occupancy of IgE receptors on peritoneal mast cells were quantified using a cytofluorometric method. The secretory ability of the mast cells as a function of anti-IgE challenge was evaluated in terms of histamine releasability in vitro. It was found that the IgE receptors were markedly up-regulated and the IgE occupancy increased on the mast cells of both athymic and euthymic rats during the parasite infection. However, at its peak, IgE occupancy was significantly higher in euthymic rats than in athymic ones. To our knowledge, this is the first proof of the possibility of a T-cell-independent IgE response in vivo. The IgE-mediated histamine releasability of mast cells was significantly enhanced 1 week after the infection in euthymic rats but not in athymic ones. These results thus indicate that the IgE immune response can occur in the absence of, but is augmented by, T cells. They also suggest that the concept of the IgE response should be widened to comprise not only increased IgE production but also an up-regulation of Ig receptors and an increase in IgE occupancy on mast cells, as well as an increase in the secretory ability of these cells. The latter is T cell dependent, although it is not directly related to the density of IgE-receptor complexes on the mast cells but is more likely due to a stimulation of the post-receptor signal transduction mechanism. PMID- 8620095 TI - Extracellular guanosine 3',5'-cyclic monophosphate and disodium cromoglycate share a similar spectrum of activity in the inhibition of histamine release from isolated mast cells and basophils. AB - In comparison to adenosine 3',5'-cyclic monophosphate (cAMP), surprisingly little is known regarding the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the functional modulation of mast cells and basophils. In the present study, the ability of cGMP, cAMP, and a range of related compounds, to inhibit immunologically induced histamine release from these cells was investigated and compared to the anti-asthmatic drug disodium cromoglycate (DSCG). Exogenously applied cGMP produced a potent inhibition of histamine release from rat peritoneal mast cells, but cAMP had a negligible effect. The attenuation noted with cGMP was markedly reduced if cells were pretreated with the nucleotide before stimulation. Similar results were obtained with DSCG. The inhibitory and tachyphylactic effects noted with cGMP were mimicked by direct derivatives of the compound but not by a range of other cyclic or guanosine nucleotides. The time courses of the induced tachyphylaxis seen with cGMP and DSCG were similar, and cross-tachyphylaxis between the two compounds was observed. In addition, both cGMP and DSCG showed a comparable spectrum of activity against mast cells isolated from the mouse, guinea pig, and human. The parallel effects of the two agents suggests that they may inhibit mediator release from mast cells through similar mechanisms. PMID- 8620096 TI - Epidermal urocanic acid and suppression of contact hypersensitivity by ultraviolet radiation in Monodelphis domestica. AB - A single specific epidermal photoreceptor for the immunosuppressive action of UV radiation has not been defined, although separate evidence is accruing in favour of each of two candidates, trans-urocanic acid and DNA. In Monodelphis domestica, specific photoreactivation repair of UV radiation-induced pyrimidine dimers has been shown to abrogate the suppression of contact hypersensitivity (CHS), thus suggesting that DNA is the target for this impairment. However, the both haired and hairless mice, immunosuppressive effects of UV radiation have been reproduced by the exogenous administration of the UV photoproduct of urocanic acid, cis urocanic acid. We show here that the epidermis of M. domestica contains urocanic acid, that UV irradiation of the shaved dorsal skin has resulted in an increase in epidermal cis-urocanic acid and that the topical application of a cis-urocanic acid-containing lotion significantly depressed the capacity of Monodelphis to respond to contact sensitisers, in a manner analogous to these responses in the hairless mouse. Therefore in Monodelphis, suppression of CHS by UV irradiation appears to involve both urocanic acid photo-isomerisation and epidermal DNA damage. PMID- 8620098 TI - Compound 48/80-induced conjunctivitis in the mouse: kinetics, susceptibility, and mechanism. AB - A mouse model of conjunctivitis has been developed by topical application of compound 48/80 (C48/80), an agent that triggers mast cell degranulation. We examined the responsiveness of C57BL/6, C3H/HeN, and ASW/J mouse strains to C48/80 stimulation, and of a mutant strain with mast cell depletion (WBB6F1/J and its sham control). Conjunctivae were collected and examined histopathologically at 15 min and 1,6,24,48 and 72 h after topical C48/80 administration. Conjunctival inflammation developed in all strains, although the severity varied. The conjunctivitis was characterized clinically by irritation, discharge, erythema, and chemosis. Pathology showed conjunctival infiltration with neutrophils, macrophages, CD4+ T lymphocytes, and a few eosinophils. Degranulation of mast cells and evacuation of goblet cells were also observed. Late-phase inflammatory reactions peaked 6-24 h after C48/80 administration and resolved by 48-72 h. WBB6F1/J mice had much less inflammation than their sham controls. In conclusion, topical C48/80 induced a conjunctival inflammatory response similar to allergen-induced conjunctivitis. The depletion of mast cells significantly reduced the inflammation. This model which consistently mimics the clinical signs and histopathological processes of allergic conjunctivitis in humans, is practical and reliable for the evaluation of new anti-allergic medications and for the investigation of conjunctival cellular responses in the allergic inflammatory cascade. PMID- 8620097 TI - Cetirizine reduces ICAM-I on epithelial cells during nasal minimal persistent inflammation in asymptomatic children with mite-allergic asthma. AB - It has been recently demonstrated that individuals who suffer from mite allergy present mucosal inflammation even when asymptomatic. This situation is characterized by infiltration of inflammatory cells (eosinophils and neutrophils) and by ICAM-I expression on epithelial cells. It has been called 'minimal persistent inflammation' (MPI) for its relationship with natural exposure to allergen, which is continuous in the case of mite allergy. ICAM-I (or CD54) expression on epithelial cells is relevant for several reasons: (a) healthy individuals and patients with pollen allergy out of the pollen season do not express this molecule; (b) ICAM-I is the natural ligand of LFA-1 (an integrin expressed on granulocytes), and (c) ICAM-I is also receptor for rhinoviruses. It is well known that viral infections precede asthmatic attacks; consequently, this correlation is more frequent in cases of mite allergy. Cetirizine is an antiallergic drug that can reduce both inflammatory infiltrate and ICAM-I expression induced by allergen-specific conjunctival challenge. The aim of this study was to evaluate the effect of cetirizine on MPI in 20 children (5-14 years old) with mite allergy. All the children suffered from mild asthma and 9 also had rhinitis (they had been asymptomatic, and thus not treated, for 2 months). The study was double-blind, placebo controlled and randomized and children took Cetirizine or placebo for 15 days. At the beginning and end of the study, nasal scrapings were performed to evaluate inflammatory cell infiltration (eosinophils and neutrophils) and ICAM-I expression on epithelial cells. Cetirizine-treated children showed a significant reduction (or even total absence) of ICAM-I expression on epithelial cells (p less than 0.002) and a reduction trend in inflammatory cell counts compared with placebo. In conclusion, Cetirizine might be envisaged as fruitful for the prolonged treatment of allergic children, including during clinical latency, to prevent possible relapse or rhinovirus infections. PMID- 8620100 TI - Participation of platelets in protection against larval Taenia taeniaeformis infection in mice. AB - The participation of platelets in the protection against larval Taenia taeniaeformis was studied. CB-17 SCID mice, susceptible to T. taeniaeformis, were protected against a challenge infection with T. taeniaeformis by the passive transfer of platelets from T. taeniaeformis-infected normal CB-17 mice, resistant to T. taeniaeformis. PMID- 8620099 TI - Catecholamine and beta-adrenoceptor influences on airway reactivity to antigen in guinea pigs. AB - The aim of the present study was to analyze the increased airway reactivity to antigen induced by beta-adrenoceptor blockade, adrenalectomy or medullectomy and to assess the contribution of circulating catecholamines to the increased reactivity. In anesthetized guinea pigs sensitized to ovalbumin (OA), administration of OA produced a dose-related bronchoconstriction characterized by threshold increases in airway insufflation pressure at 0.1 mg/kg i.v. and a near maximal increase by 0.3 mg/kg i.v. Pretreatment with R(+) propranolol (0.5 mg/kg i.v.) 5 min prior to antigen did not significantly alter airway responses to antigen when compared to vehicle-treated animals. However, pretreatment with 0.5 mg/kg i.v. S(-) propranolol, racemic propranolol or nadolol markedly enhanced (10 to 15-fold) the airway response to the low-dose antigen. In addition, in guinea pigs which had been adrenalectomized, the reactivity to low-dose antigen was enhanced to a similar extent as that of beta-antagonist-treated animals when compared to sham-operated animals. Baseline plasma concentrations of epinephrine were significantly higher in sham-operate guinea pigs (1,494 +/- 223 ng/ml) when compared to adrenalectomized animals (412 +/- 44 ng/ml). Upon antigen exposure, epinephrine levels rose 5-fold (6,859 +/- 1,308 ng/ml) from baseline in sham operated guinea pigs and were not significantly changed in adrenalectomized animals (848 +/- 208 ng/ml). Specific airway conductance measurements in conscious guinea pigs revealed that animals which had been medullectomized 2 weeks previously responded to lower provocative concentrations of aerosol OA (0.05-0.5%) than corresponding sham-operated animals. Airway reactivity to inhaled acetylcholine (0.1-1%) was similar in medullectomized and sham guinea pigs. Plasma concentrations of epinephrine were significantly lower in medullectomized guinea pigs (327 +/- 88 ng/ml) when compared to sham-operated animals (832 +/- 162 ng/ml). The results of the present study indicate that beta adrenoceptor antagonism or changes in circulating epinephrine levels markedly alter the response to antigen in sensitized guinea pigs. PMID- 8620101 TI - Incomplete aspirin desensitization in an aspirin-sensitive asthmatic. AB - Aspirin desensitization is a valuable treatment for aspirin-sensitive sinusitis. We present a case where long-term desensitization failed. While undergoing desensitization, our patient had prolonged severe asthmatic reactions and therefore received high intravenous doses of prednisone. We hypothesize that high steroid doses administered at the time of desensitization may have raised the threshold of intolerance to a point where the administered aspirin doses were tolerated. Consequently, symptoms of intolerance subsided during the procedure. Subsequent tapering down of the daily prednisone dose caused a re-emergence of the symptoms of intolerance, apparently due to a decrease in the intolerance threshold. PMID- 8620102 TI - Monitoring of pesticide residues in human milk, soil, water, and food samples collected from Kafr El-Zayat Governorate. AB - Pesticide residues in human milk and environmental samples from Kafr El-Zayat Governorate in Egypt were analyzed. This governorate is located near one of the biggest pesticide factories in Egypt. Organochlorine and organophosphorus pesticides were monitored, including those that have been prohibited from use in Egypt. Human milk samples (31 samples) from Kafr El-Zayat were compared with 11 samples collected from Cairo. Data were compared with results from studies performed in 1987 and 1990. The present study showed that aldrin and dieldrin, heptachlor and heptachlor epoxide, and endrin residues have been eliminated from human milk. Estimated daily intakes (EDIs) of DDT complex and gamma-HCH by breast fed infants in Kafr El-Zayat were 85.96 and 3.1% of the respective acceptable daily intakes (ADIs). beta-HCH residues showed an increasing pattern, especially in human milk samples from Cairo. DDT complex and HCH isomers in orange, spinach, lettuce, potatoes, and clover samples ranged from undetectable to very low concentrations. Higher levels of DDT and HCH were detected, but aldrin, dieldrin, endrin, and the heptachlors were not detected in food of animal origin. Residues in fish samples were below maximum residue limits established by some developed countries. Those in animal milk samples approached the extraneous residue limits of the Codex Committee on Pesticide Residues. HCH residues in soil were negligible, but DDT residues in soil were somewhat higher. Among water samples, groundwater samples had the highest residues of HCHs and DDTs, followed by Nile River water and then tap water. However, the organochlorine pesticide residues were found at concentrations below the maximum allowable limits set by the World Health Organization for drinking water. Among 12 organophosphorus pesticides monitored as parent compounds, dimethoate, malathion, methamidophos, and chlorpyrifos residues were detected in low concentrations in soil samples from a pesticide factory. No organophosphorus pesticide residues were found in plant samples, except for very low residues of dimethoate in an orange sample. Water samples were devoid of organophosphorus residues as parent compounds. PMID- 8620103 TI - Determination of combined residues of metalaxyl and 2,6-dimethylaniline metabolites in urine by gas chromatography/mass spectrometry. AB - A gas chromatographic/mass spectrometric (GC/MS) method for determination of combined residues of the fungicide metalaxyl and its metabolites in urine containing the 2,6-dimethylaniline moiety is described. The method is a modification of a method of Balasubramanian and Perez for analysis of metalaxyl in vegetables. Noted modifications include replacement of steam extraction with extraction by methylene chloride and use of electron impact ionization GC/MS in the selected-ion mode. The method is linear over the range of 0.1-5 micrograms 2,6-dimethylaniline/g urine and has a detection limit of 0.025 microgram/g. PMID- 8620104 TI - Effects of sodium sulfite on recovery and composition of detergent fiber and lignin. AB - Use of sodium sulfite to reduce nitrogenous contamination in fiber analysis was evaluated. The effects of sodium sulfite on analytical accuracy and precision were examined for amylase-treated neutral detergent fiber (aNDF), sequentially determined acid detergent fiber (sADF), and acid detergent lignin (sADL) in animal feeds. In one experiment, 0.5 g sodium sulfite was added per sample during neutral detergent (ND) extraction. The treatment consistently reduced aNDF, sADF, and sADL values of 180 alfalfa samples and improved precision (decreased within sample variance of replicated analyses). The greatest effect was on precision of sADL analysis, with within-sample variance reduced by more than 50%. In a second experiment, 24 animal feeds were analyzed for a aNDF, sADF, and sADL with and without addition of 0.5 g sodium sulfite per sample during ND extraction. Nitrogen contents of the recovered fiber fractions were determined. Sodium sulfite reduced fiber and lignin values and decreased nitrogen concentration in residues. Within-sample variance was lower in all analyses. In a third experiment, 23 animal feeds were analyzed for aNDF with sodium sulfite at 0, 0.25, 0.5, and 1.0 g per sample. Average aNDF of feeds was reduced by each additional increment of sodium sulfite; however, 1.0 g sodium sulfite resulted in only a slight reduction in aNDF compared with 0.5 g. Therefore, 0.5 g sodium sulfite per sample should be added to samples prior to aNDF analysis. PMID- 8620105 TI - History of the Food and Drug Administration's Total Diet Study (Part II), 1987 1993. AB - The Total Diet Studies conducted by the U.S. Food and Drug Administration (FDA) provide yearly information on levels of pesticide residues, contaminants, and nutrients in the food supply and diets of specific age-sex groups. They also identify trends and changes in the levels of these substances in the food supply and in diets over time. Results are useful in making policy decisions regarding the safety of the food supply, food additives, pesticide use, nutrient fortification, and food labeling. This paper provides information on studies performed by FDA from 1987 to 1993. PMID- 8620106 TI - Collection and analysis of butyltin compounds in air at nanogram-per-cubic-meter levels. AB - An analytical method for determining butyltin chlorides in air at low nanogram per-cubic-meter levels was developed. Butyltin chlorides investigated were mono-n butyltin trichloride (MBTC), di-n-butyltin dichloride (DBTC), and tri-n-butyltin chloride (TBTC). These tin chlorides were trapped on cartridges packed with Porapak-N, eluted with methylene chloride containing 0.3% HCl, hydridized with sodium borohydride, and analyzed by capillary gas chromatography with flame photometric detection. On the basis of a 20 m3 sample, a detection limit of 0.05 ng/m3 can be achieved. PMID- 8620107 TI - Estimated content percentages of volatile liquids and fat extractables in ready to-eat foods. AB - Content percentages of volatile liquids and fat extractables in 340 samples of ready-to-eat foods were determined gravimetrically. Volatile liquids were determined by drying samples in a microwave oven with a self-contained balance; results were printed out automatically. Fat extractables were extracted from the samples with mixed ethers; extracts were dried and weighed manually. The samples, 191 nonfat and 149 fatty (containing ca 2% or more fat) foods, represent about 5000 different food items and include infant and toddler, ethnic, fast, and imported items. Samples were initially prepared for screening of essential and toxic elements and chemical contamination by chopping and mixing into homogenous composites. Content determinations were then made on separate portions from each composite. Content results were put into a database for evaluation. Overall, mean results from both determinations agree with published data for moisture and fat contents of similar food items. Coefficients of variation, however, were lower for determination of volatile liquids than for that of fat extractables. PMID- 8620108 TI - Simultaneous determination of multiple tetracycline residues in milk by metal chelate affinity chromatography: collaborative study. AB - To meet federal and state regulatory needs, a liquid chromatographic (LC) method with ultraviolet (UV) detection was developed for determination of 7 tetracyclines at 30 ng/ml in milk. Raw milk samples are defatted, acidified, and centrifuged to remove proteins, and tetracyclines are specifically absorbed from the milk by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns. Tetracyclines are removed from these columns with EDTA-containing buffer, and extracts are further cleaned by ultrafiltration. Finally, extracts are concentrated and analyzed simultaneously by using on-line concentration. This method was validated in a collaborative study that involved 11 laboratories, including the authors' laboratory. Each laboratory was asked to prepare and analyze known control and fortified milk samples, as well as 18 coded blind samples. Eight laboratories completed all analyses. Average interlaboratory recoveries for the known fortified samples ranged from 59% (methacycline at 15 ng/ml) to 78% (oxytetracycline at 60 ng/ml). Average recovery for each of 7 residues at 30 ng/ml were between 60 and 110%, meeting single-residue guidelines for accuracy set by the U.S. Food and Drug Administration. Reproducibility relative standard deviation (RSDR) for the known fortified samples varied from 11 to 39%, with 6 of 7 residues at the 30 ng/ml level having RSDR values at or below 20%. Seven of 8 laboratories correctly identified blind control milk samples and all 28 residues present in blind samples. The metal chelate affinity-LC method for determination of multiple tetracycline residues in milk has been adopted first action by AOAC INTERNATIONAL. PMID- 8620109 TI - Wiley Award Address. Evolution of methods for the detection of Salmonella in foods. PMID- 8620110 TI - Assay of biogenic amines involved in fish decomposition. AB - A liquid chromatographic (LC) method is described for quantitative determination of putrefaction amines: putrescine, cadaverine, histamine, spermidine, and spermine. These amines are extracted from fish by grinding with perchloric acid at -20 degrees C. The amines are reacted with dansyl chloride at alkaline pH, dried under a stream of nitrogen, placed in an automatic injector at -20 degrees C, and separated in a Kromasil C18 reversed-phase column at 25 degrees C. A new gradient elutes the amines in 22 min and regenerates the column in 30 min. Regression lines are obtained with high coefficients of correlation. This method is faster and more reproducible than other methods and shows that the quality index is a reliable, albeit species-specific, criterion of fish decomposition. PMID- 8620111 TI - Determination of D-malic acid in apple juice by liquid chromatography: collaborative study. AB - Eleven laboratories collaboratively studied a liquid chromatographic (LC) method for determination of D-malic acid in apple juice. The mobile phase consisted of mM L-valine and 8 mM copper acetate adjusted to pH 5.5 with NaOH. The UV detector was set at 330 nm, and a single reversed-phase LC column was used. Seven paired samples containing various amounts of D-malic acid ranging from 0 to 188 mg/100 mL of 12 Brix pasteurized apple juice were tested by each collaborator. Repeatability and reproducibility coefficients of variation ranged from 1.0 to 3.5% and 7.7 to 11.7%, respectively, within the range of 26 to 188 mg D-malic acid/100 mL of 12 Brix apple juice. The collaborative study results demonstrated that the method could quantitate the economic adulteration of apple juice with DL malic acid at lower levels than those reported with previous methods. The LC method for determination of D-malic acid in apple juice has been adopted first action by AOAC INTERNATIONAL. PMID- 8620112 TI - Determination of dietary fiber and carbohydrate composition of foods by a modified version of the Englyst method. AB - A modified version of the Englyst procedure for determination of dietary fiber and carbohydrate fractions of foods is described. The method is based on hydrolysis of separated fractions and capillary gas chromatographic (GC) analysis of derivatized monomer sugars, UV photometry of uronic acids, gravimetric determination of residue, and analysis of nitrogen content of the residue by using a C, H, N elemental analyzer. Modifications of fractionation, derivatization, and GC procedures are discussed. Rigorous sample preparation is required for reproducibility of 200 mg test portions. Contaminants interfering with chromatography must be minimized. Results for reference samples (wheat bran and Buerre Bosc pears) and white and whole meal bread agree with literature values. PMID- 8620113 TI - Interpretation of combined 2H SNIF/NMR and 13C SIRA/MS analyses of fruit juices to detect added sugar. AB - The site-specific natural isotopic fractionation studied by nuclear magnetic resonance (SNIF/NMR) method measures site-specific isotope contents in a variety of organic compounds by deuterium nuclear magnetic resonance spectroscopy. This technique, together with SIRA/MS (stable isotope ratio analysis/mass spectrometry) provides a powerful tool for food authentication and characterization. By using the ethanol resulting from sugar fermentation as a molecular probe, SNIF/NMR (deuterium) and SIRA/MS (13C) have been used together for authentication of fruit juices. The influence of deuterium content of the fermentation water on the isotopic parameters is shown and a means for normalizing the results is proposed. A large number of authentic juices have been analyzed to define the variation of isotopic ratios in natural juices. On the basis of these data, a set of rules was designed to enable interpretation of isotopic parameters in terms of possible adulteration of fruit juices by sugar addition. Results of analyses of Florida orange juice are presented. Orange juice samples from Brazil and Israel are included as 2 extreme cases. Assignment limits for a sample of orange juice of unknown origin also are given. These assignment limits are also provided for apple and grapefruit juices. PMID- 8620114 TI - Vitamin D in infant formula and enteral products by liquid chromatography: collaborative study. AB - Results from a collaborative study of a new liquid chromatographic (LC) method for determination of vitamin D in infant formulas and enteral products are presented. Each of 15 laboratories was provided with 11 blind duplicate samples covering a range of approximately 200-500 International Units/quart (normal dilution), a system suitability sample, and the U.S. Pharmacopeia ergo- and cholecalciferol standards. Product types included liquid and powder forms of milk (whey and casein), soy, and hydrolyzed protein-based infant formulas and enteral products. The method includes a single liquid-liquid extraction following saponification, solid-phase extraction, and then concentration by evaporation. An isocratic, nonaqueous, chromatographic system with reversed-phase, zero endcapped C18 column, and UV detector set at 265 nm are used. Statistical evaluation of data from participating laboratories show the average reproducibility and repeatability of the method across all samples to be excellent, with RSDR and RSDr values of 13.48 and 9.44, respectively, after elimination of outliers. The LC method for determination of vitamin D in infant formulas and enteral products has been adopted by AOAC INTERNATIONAL. PMID- 8620115 TI - Determination of shell content in palm kernel cake. AB - A method for determining shell in palm kernel cake (PKC) is described. This simple and rapid method requires little pretreatment compared with the method currently used in PKC trade, in which the sample undergoes defatting, acid and alkali digestion, and washing, before a chloroform-alcohol solution is used to separate the shells. In the proposed method, only defatting the sample is required. The shells are separated by the density difference between the shell and PKC in a potassium iodide solution. Recoveries of at least 93% were obtained, and the correlation coefficient between the actual shell content and the determined shell content was 0.999, with gradients of 0.97 and 0.98 for fine and coarse shell, respectively. PMID- 8620116 TI - Determination of chlorobiphenyls in seal blubber, marine sediment, and fish: interlaboratory study. AB - Between 1988 and 1994, the International Council for the Exploration of the Sea, the Intergovernmental Oceanographic Commission, and the Oslo and Paris Commissions organized a stepwise interlaboratory study for determination of chlorobiphenyls (CBs) in marine media. The final parts of this study, in which 53 laboratories from 14 countries participated, focused on long-term precision, cleanup, and extraction. Calibration was controlled continuously by analysis of 10 CBs in an unknown solution. Participants were requested to analyze 3 CBs in a certified reference material fish oil (6 times); 10 CBs in cleaned and uncleaned marine sediment and seal blubber extracts; and 10 CBs in seal blubber oil, dried marine sediment, and wet, lean fish muscle tissue. The long-term precision study showed that, compared with earlier exercises in which only duplicate analyses were required, repeatability increased about 1.5-fold compared with reproducibility. The mean standard error for reproducibility of determination of 10 CBs in standard solutions improved from 1.22 to 1.15. The standard error improved from 1.36 to 1.25 (without CBs 28 and 31) for seal blubber oil and from 1.36 to 1.22 for dried marine sediment. In seal blubber oil and dried marine sediment, the major CBs 118, 138, 153, and 180 can now be determined by the group of participating laboratories with a reproducibility of 1.5 (about 50%). No significant differences were found between results for cleaned-up and uncleaned extracts. No acceptable results could be obtained for determination of CBs in lean fish muscle tissue. Biplots of principal component analyses are extremely helpful in evaluating the data generated by this type of study. PMID- 8620117 TI - Monitoring of pesticide residues in fresh vegetables, fruits, and other selected food items in Belgium, 1991-1993. AB - To estimate the exposure of the Belgian population to food contaminated with pesticide residues and to determine what pesticides people are actually consuming, a total diet study-individual approach-was performed. Fourteen kinds of fruits, 22 kinds of vegetables, and 7 other food items (coffee, drinking water, rice, tea, wine, bran, and wheat flour) were selected as major representatives of the Belgian diet. During the 2 years of study (April 1991 March 1993), about 3,698 samples were analyzed and 21 163 analyses were performed. The first part of this study demonstrates that no residues are found in 31.3% of leafy vegetables, 72.3% of other vegetables, 51.4% of fruits, and 67.2% of other samples. In particular cases, some critical situations still exist, especially for leafy vegetables. Also, contamination of foreign samples is not easy to determine, because origins are not always traceable. PMID- 8620118 TI - The coming of age of band ligation for oesophageal varices. PMID- 8620119 TI - The paperless general practice. PMID- 8620120 TI - Persistent tachypnoea in neonates. PMID- 8620121 TI - Blood donation--altruism or profit? PMID- 8620122 TI - Judge rules in favour of "do not resuscitate". PMID- 8620123 TI - Scottish court gives right to die. PMID- 8620124 TI - Dutch GP on murder charge over euthanasia claim. PMID- 8620125 TI - Bavaria threatens to reduce abortion access. PMID- 8620126 TI - Has mortality from melanoma stopped rising in Australia? Analysis of trends between 1931 and 1994. AB - OBJECTIVE: To describe recent trends in mortality from melanoma in Australia. DESIGN: An analysis of trends in age standardised and age and sex specific mortalities by year of death and median year of birth (cohort). SETTING: Australia. SUBJECTS: All deaths from melanoma registered in Australia between 1931 and 1994. RESULTS: Melanoma mortality rose steadily from 1931 to 1985. From 1959 the annual rate of increase was 6.3% in men and 2.9% in women, resulting in mortalities of 4.82 and 2.51 per 100,000 person years in 1985 and 1989, respectively. Mortalities for both sexes seem to have plateaued from June 1985 onwards. In 1990-4 the rate rose by 3.7% in men to 5.00 per 100,000 and in women it fell by 5.2% to 2.38 per 100,000. The non-significant increase after 1985 in mortality in men was restricted to those aged over 70 years of age, whereas the fall in rates in women was mostly in those aged under 55 years. This pattern was generally reflected in the state trends, though with some variation: rates for women in Queensland had peaked in the late 1970s; while rates for men in New South Wales continued to rise in 1990-4, placing them above those for Queensland. Examination of mortalities specific for age, period, and cohort for Australia as a whole showed several salient features. Rates in men rose steeply in cohorts born before about 1930; were stable in cohorts born between 1930 and 1950; and fell in more recent cohorts. Rates in women showed similar changes but about five years earlier. CONCLUSION: Melanoma mortality in Australia peaked in about 1985 and has now plateaued. On the basis of trends in cohorts it can be expected to fall in coming years. PMID- 8620127 TI - Incidence and thickness of primary tumours and survival of patients with cutaneous malignant melanoma in relation to socioeconomic status. AB - OBJECTIVE: To study incidence of and survival from cutaneous malignant melanoma in relation to socioeconomic status. DESIGN: Application of Carstairs deprivation score to all malignant melanoma patients diagnosed in a geographically defined area over a 15 year period. SETTING: West of Scotland (area population 2,716,900). SUBJECTS: 3142 patients first diagnosed with malignant melanoma in the period 1979-93. INTERVENTIONS: Surgical excision of primary malignant melanoma with additional treatment as appropriate and follow up until December 1994. MAIN OUTCOME MEASURES: Malignant melanoma incidence, primary tumour thickness and five year survival by socioeconomic status. RESULTS: From 1979 to 1993, the age standardised incidence rate for cutaneous malignant melanoma was 9.1/100,000 for the most affluent men and 2.4/100,000 for the least affluent men and 16.1/100,000 and 5.0/100,000 respectively for most and least affluent women (P < 0.001 for trend in both). The incidence increased steadily over time in both sexes in all socioeconomic groups. Good prognosis tumours ( < 1.5 mm thick) were most common in the most affluent men and women, and over the study period the proportion of such tumours increased most in the intermediate affluence group (both sexes) and in the least affluent women. Five year disease free survival from melanoma for the sexes combined was 81% for most affluent, 77% for intermediate, and 73% for least affluent groups. Even after adjustment for known prognostic factors of tumour thickness, ulceration, age, and body site of primary melanoma, the more affluent the group, the better the survival. CONCLUSION: Although the incidence of cutaneous malignant melanoma is higher among more affluent people, the prognosis is better in this group than for less affluent individuals. Early diagnosis campaigns should be targeted particularly to less affluent men and primary prevention campaigns should emphasise the greater risk in more affluent women. PMID- 8620128 TI - Association between incidence of non-Hodgkin's lymphoma and solar ultraviolet radiation in England and Wales. AB - OBJECTIVES: To examine whether the incidence of non-Hodgkin's lymphoma in different areas of England and Wales is associated with levels of solar ultraviolet radiation. DESIGN: Geographically based study examining the association between incidence of non-Hodgkin's lymphoma and estimated levels of solar ultraviolet radiation, controlling for social class and employment in agriculture. SETTING: 59 counties in England and Wales. SUBJECTS: All registered cases of non-Hodgkin's lymphoma during the period 1968-85. MAIN OUTCOME MEASURE: Age and sex adjusted odds ratio for non-Hodgkin's lymphoma in each county. RESULTS: Incidence of non-Hodgkin's lymphoma was significantly associated with solar ultraviolet radiation levels (P < 0.001), even after social class and employment in agriculture were controlled for (P = 0.004). In a comparison of counties in the highest and lowest quarters of solar ultraviolet radiation, the relative risk of non-Hodgkin's lymphoma was 1.27 (95% confidence interval 1.24 to 1.29), rising to 1.34 (1.32 to 1.37) after adjustment for social class and employment in agriculture. CONCLUSIONS: The incidence of non-Hodgkin's lymphoma in different areas of England and Wales is positively associated with levels of solar ultraviolet radiation. These results are consistent with the hypothesis that exposure to solar ultraviolet radiation increases the risk of non-Hodgkin's lymphoma. PMID- 8620129 TI - Blood money: blood donors' attitudes to changes in the New Zealand blood transfusion service. PMID- 8620130 TI - Low triiodothyronine concentration in preterm infants and subsequent intelligence quotient (IQ) at 8 year follow up. PMID- 8620131 TI - Current practice and complications of temporary transvenous cardiac pacing. PMID- 8620133 TI - Short acting dihydropyridine (vasodilating) calcium channel blockers for hypertension: is there a risk? PMID- 8620132 TI - Randomised controlled trial of general practitioner versus usual medical care in an urban accident and emergency department: process, outcome, and comparative cost. AB - OBJECTIVE: To see whether care provided by general practitioners to non-emergency patients in an accident and emergency department differs significantly from care by usual accident and emergency staff in terms of process, outcome, and comparative cost. DESIGN: A randomised controlled trial. SETTING: A busy inner city hospital's accident and emergency department which employed three local general practitioners on a sessional basis. PATIENTS: All new attenders categorised by the triage system as "semiurgent" or "delay acceptable." 66% of all attenders were eligible for inclusion. MAIN OUTCOME MEASURES: Numbers of patients undergoing investigation, referral, or prescription; types of disposal; consultation satisfaction scores; reattendance to accident and emergency department within 30 days of index visit; health status at one month; comparative cost differences. RESULTS: 4684 patients participated. For semiurgent patients, by comparison with usual accident and emergency staff, general practitioners investigated fewer patients (relative difference 20%; 95% confidence interval 16% to 25%), referred to other hospital services less often (39%; 28% to 47%), admitted fewer patients (45%; 32% to 56%), and prescribed more often (41%; 30% to 54%). A similar trend was found for patients categorised as delay acceptable and (in a separate analysis) by presenting complaint category. 393 (17%) patients who had been seen by general practitioner staff reattended the department within 30 days of the index visit; 418 patients (18%) seen by accident and emergency staff similarly reattended, 435 patients (72% of those eligible) completed the consultation satisfaction questionnaire and 258 (59% of those eligible) provided health status information one month after consultation. There were no differences between patients managed by general practitioners and those managed by usual staff regarding consultation satisfaction questionnaire scores or health status. For all patients seen by general practitioners during the study, estimated marginal and total savings were Ir1427 pounds and Ir117,005 pounds respectively. CONCLUSION: General practitioners working as an integral part of an accident and emergency department manage non-emergency accident and emergency attenders safely and use fewer resources than do usual accident and emergency staff. PMID- 8620134 TI - ABC of urology. Urological malignancy--III: Renal and testicular carcinoma. PMID- 8620135 TI - Pulmonary hypoplasia presenting as persistent tachypnoea in the first few months of life. PMID- 8620136 TI - Management of HIV infected health care workers: lessons from three cases. AB - Three cases in which doctors in Glasgow were diagnosed as having HIV infection were all handled differently in relation to telling patients and the media. In the first patients were not told because the doctor had been doing administrative work and there was thought to be no risk to patients; although the media did report the case, it accepted the assurances given. In the second case, where a doctor had done many jobs in different specialties and places, the media identified the doctor before most patients had been informed: most calls to the helpline subsequently set up by the health authority were from patients who had not been treated by this doctor. This episode, however, allowed the incident team to be prepared for the next case, enabling the helpline to be established swiftly. In this case the doctor voluntarily identified himself, and this served to allay public fears and reduce the number of inappropriate calls to the helpline. PMID- 8620137 TI - The use of transformation when comparing two means. PMID- 8620138 TI - Specialist surgeons and survival in breast cancer. Breast cancer is a medical, not a surgical, disease. PMID- 8620139 TI - Specialist surgeons and survival in breast cancer. Large differences in survival are not explained. PMID- 8620140 TI - Specialist surgeons and survival in breast cancer. A multidisciplinary approach is needed. PMID- 8620141 TI - Specialist surgeons and survival in breast cancer. Case selection bias affected results. PMID- 8620142 TI - Specialist surgeons and survival in breast cancer. More centralisation of services is not needed. PMID- 8620143 TI - Specialist surgeons and survival in breast cancer. Protocols are important. PMID- 8620144 TI - Health services must develop services to reduce crime and violence. PMID- 8620145 TI - Minimum standards should be set for near patient testing. PMID- 8620146 TI - Psychosis in Afro-Caribbean people. Further data should have obtained. PMID- 8620147 TI - Psychosis in Afro-Caribbean people. "Afro-Caribbean" could have been of Chinese, Indian, European, or African extraction. PMID- 8620148 TI - Performance of neonatal screening programme must be monitored. PMID- 8620149 TI - Postoperative deep vein thrombosis and surgery for varicose veins. PMID- 8620150 TI - Postoperative pulmonary complications. Pain relief improves respiratory function. PMID- 8620151 TI - Postoperative pulmonary complications. Adequate pain relief is also necessary. PMID- 8620152 TI - Postoperative pulmonary complications. Obesity, pain, and sedation are important. PMID- 8620154 TI - False localising signs in the spinal cord. PMID- 8620153 TI - Postoperative pulmonary complications. Laparoscopic surgery leads to better postoperative pulmonary function. PMID- 8620155 TI - Future of tropical medicine. PMID- 8620156 TI - Value of ECGs in identifying heart failure due to left ventricular systolic dysfunction. Echocardiography is still necessary. PMID- 8620157 TI - Value of ECGs in identifying heart failure due to left ventricular systolic dysfunction. Courses on interpreting ECGs would improve general practitioners' skills. PMID- 8620158 TI - Value of ECGs in identifying heart failure due to left ventricular systolic dysfunction. ECGs are valuable in hospital as well as general practice. PMID- 8620159 TI - Value of ECGs in identifying heart failure due to left ventricular systolic dysfunction. Likelihood ratios should have been given. PMID- 8620160 TI - Learning from primary care in developing countries. PMID- 8620161 TI - Methadone maintenance reduces injecting in prison. PMID- 8620162 TI - Training in substance misuse for GPs. Courses are available in Lothian. PMID- 8620163 TI - Training in substance misuse for GPs. Services need to be adequately resourced. PMID- 8620164 TI - Code is needed to protect inpatients from press publicity. PMID- 8620165 TI - Television programme distorted dangers of diagnostic radiology. PMID- 8620166 TI - [Diagnosis of allergy to venom of Hymenoptera]. PMID- 8620167 TI - [Evaluation of allergic inflammation parameters in bronchoalveolar lavage in patients with asthma]. AB - The study was set up to evaluate the changes in broncho-alveolar lavage cell count in asthmatics after bronchial provocation. Similarities and differences between atopic (A) and nonatopic (N) asthma were given a special concern. Twenty mild asthmatics--10 A and 10 N. There was no difference in BAL eosinophila between these groups before provocation. After provocation more eosinophils were found in A group (p = 0.06). Neutrophils were more numerous in N on both occasions--0 = 0.00003 and p = 0.006 respectively). Lymphocytes showed a similar pattern--p = 0.03, p = 0.003. A positive correlation between BAL eosinophilia and bronchial hyperreactivity was shown in both groups. Inflammation, including inflammatory cells plays a major role in bronchial asthma pathophysiology. It seems to be more expressed in nonatopic patients. PMID- 8620169 TI - [Spirometric investigation and spiro-ergometric test in evaluation of exercise tolerance in patients suffering from aluminosis and silicosis]. AB - The main purpose of our research was to estimate the ventilation and physical efficiency in 6 patients suffering from aluminosis and silicosis. Chest X-ray examinations, statical spirometric investigations and spiroergometric tests (TSE) were performed. Oxygen levels achieved in this group were equal or even higher than medium values achieved by healthy people. Ability to physical effort can be estimated only using TSE. PMID- 8620168 TI - [Activation of T lymphocytes and severity of atopic bronchial asthma in children]. AB - Recent studies point out to the role of activated T lymphocytes in the pathogenesis of bronchial asthma. We analyzed (1) T subpopulations and (2) expression of the cell surface activation markers (the light chain of interleukin 2 receptor (IL-2R) and HLA-DR antigen) in the peripheral blood of 36 children with stable atopic asthma and 10 non-atopic control subjects. Flow cytometry and double phenotyping with monoclonal antibodies conjugated with fluorescein and phycoerythrin were used. The results of these studies were correlated to the degree of asthma severity. We found no differences between the two evaluated subgroups (severe asthma--AC and moderate asthma--AU) in the percentage of T cells with CD3 and CD4 antigen. In the severe asthma subgroup there were the following differences as compared with those from control subjects: decrease of the percentage of CD8+ lymphocytes (p < 0.01) and the increase of activated cells expressing IL-2R (p < 0.001) and HLA-DR (p < 0.001). Differences in the expression of activation markers were more pronounced in the CD4+ subpopulation (CD4+/IL-2R, p < 0.001 and CD4+/HLA-DR+, p < 0.001), while they were less visible, but still significant, in the CD8+ cells (CD8+/IL-2R+, p < 0.01 and CD8+/HLA-DR+, p < 0.05). When comparing the results in severe asthma subgroup with those in moderate asthma, significant elevation was found in CD4+/IL-2R+ (p < 0.03), CD4+/HLA-DR+ (p < 0.05) and CD8+/IL-2R+ (p < 0.03) double positive cells. These observations may indicate a role of activated T lymphocytes, CD4+ and CD8+, in the pathogenesis of atopic bronchial asthma in children and existence of prolonged activation factors in chronic severe asthma. PMID- 8620170 TI - [Symptoms of atopy in persons exposed to chronic immunosuppression of polycyclic aromatic hydrocarbons]. AB - The frequency of the atopy symptoms was estimated in 126 coke oven workers chronically exposed to polycyclic aromatic hydrocarbons (PAHs). The assessment was based on questionnaire, point skin tests with the allergens of dust, feathers, mould grass as well as on the measurements of total blood serum IgE concentration. The control group was consisted of 75 men, workers of cold rolling mill division where the environmental conditions were much better. It was observed that positive questionnaire data and positive skin tests were significantly less frequent in men exposed to PAHs. The men serum IgE values were not statistically different in both group workers although in coke oven workers the tendency to higher IgE values was observed. It is rather suggested that more useful method might be the measurement of specific serum IgE. PMID- 8620172 TI - [Assessment of skin reactivity in patients with bronchial asthma undergoing non specific immunotherapy]. PMID- 8620171 TI - [Use of thermography methods for evaluating skin prick tests. I. Background temperature of area where skin prick tests are performed. Optimal color scale of temperature differences registered by liquid crystals and conditions of their use]. AB - The aim of the study was to estimate the value in visualization of temperature gradients in assessing results of skin prick tests. The study group consisted of 10 patients suspected of having allergic diseases in whom skin prick tests were carried out. They were performed typically using the Bencard allergens kits. A positive control was performed using different solutions of histamine (10 mg/ml, 1 mg/ml, 0.1 mg/ml). The traditional method comprising of measurement of erythema, and whed was compared with visual thermography and open liquid crystal themography. The background temperature of typical areas chosen for skin prick tests were compared under condition of thermographic evaluation. The skin of the upper region of the back was chosen for uniform temperature background. Basing on the analysis of the background temperature and in areas of high reaction to histamine and allergens the temperature and in areas of high reaction to histamine and allergens the temperature gradient was calculated to be 2.2 degrees -2.5 degrees C. In order to visualize this gradient in composition of liquid crystals was made of a working range of 34.3 degrees--36.3 degrees C. Criteria for a correct thermographic evaluation of skin prick tests were established after studying the effect of manipulation of the area examined, local lowering of the temperature, use of cold solutions of allergens. Further studies are planned in order to assess the practical use of this thermographic analytical method. PMID- 8620173 TI - [Selected cell-mediated immunologic parameters in patients with intrinsic asthma treated with long-term corticotherapy]. AB - Total lymphocytes number, proportions of T lymphocyte subset, nonspecific suppressor activity induced by Con A and T-lymphocytes ability for IL-2 production were assessed. Group of 46 patients with intrinsic asthma (24 females and 22 males) including 20 asthmatics treated during 5-7 years with glucocorticosteroids were studied. Control group consisted of 12 healthy persons. Statistically significant reduction of total T cells, T-suppressor and T-helper subpopulations, ability for IL-2 production and nonspecific suppressor activity was found. PMID- 8620174 TI - [Efficacy of flunisolide in treatment of patients with bronchial asthma]. AB - In 30 patients with bronchial asthma an influence of flunisolid (Bronilide) on spirometric values, bronchial reactivity and pathological signs was evaluated. No changing in spirometric values and bronchial reactivity and decrease complaints during the treatment with higher doses of Bronilide were noticed. No significant side effects were observed. PMID- 8620175 TI - [Assessment of respiratory function in patients with chronic obstructive pulmonary diseases treated with budesonide]. AB - The aim of the study was to carry out assessment of the respiratory function of patients with COPD undergoing a three month (600 microgram) treatment with an inhaled steroid--Budesonide. This was a double blind study carried out on 57 patients. During treatment with Budesonide a clinical improvement was seen. Respiratory function parameters did not alter throughout the 3 month treatment period, although bronchial reactivity diminished. Side effects were seen only in 3 patients--in all it was dysphonia. It seems that Budesonide can be added to other treatment protocols in patients with exacerbations of chronic bronchitis. PMID- 8620177 TI - [The influence of misoprostol on post-aspirin bronchoconstriction in patients with aspirin sensitive asthma]. AB - It is believed that aspirin (ASA) and other nonsteroidal antiinflammatory drugs elicit dysponea in ASA sensitive asthmatics by blocking the cyclooxygenase. It is unclear whether this bronchospasm is due to shunting of arachidonic acid into the lipooxygenase pathway or removal of cyclooxygenase product which prevent bronchospasm. Diminished tissue concentration of PGE may cause bronchoconstriction. PGE play also modulatory function to mast call decreasing the release of mediators of anaphylaxis. There are some evidences concerning the mast cell degranulation in postaspirin reaction in ASA sensitive asthmatics. The authors investigated the influence of synthetic analogue of PGE1--misoprostol (Cototec, Searle) on the postaspirin bronchoconstriction in seven ASA sensitive asthmatics aged 33-62. Aspirin threshold doses ranged from 10 to 150 mg. Postaspirin bronchoconstriction begun usually within 1-2 hrs after digestion of ASA and 200 micrograms were additionally given 2 h later. Seven days later misoprostol (400 micrograms) was administered together with previously determined dose of ASA. One the other day the bronchodilating effect of misoprostol alone was examined. In all but one patients we observed the protective influence of misoprostol on ASA induced bronchoconstriction. Max. fall in FEV1 in % after ASA in each of the patients was 40, 25, 24, 33, 47 and 54, and after ASA with misoprostol, respectively 10, 9, 4, (+8), 10, (+2) and 45. Misoprostol given together with ASA attenuated aspirin-induced bronchoconstriction reaching statistical significance at 3 and 3.5 h, and also diminished extrapulmonary symptoms. The authors discuss the possible mechanism of protective influence of misoprostol. PMID- 8620178 TI - [A case of aspergilloma in a adenocarcinoma cavity in a patient with a history of pulmonary tuberculosis]. PMID- 8620176 TI - [A trial of ipratropium bromide dose optimalization in patients with atopic asthma]. AB - The effect of ipratropium bromide inhalation in cumulated doses upon the change of VC, FEV1 and MEF50 was evaluated in 10 patients with atopic bronchial asthma. It was two days examination. On the first day measurements were obtained immediately after inhalation of 2 doses (40 mcg) ipratropium bromide which were repeated in 30 minutes intervals. On the second day measurements were repited after placebo inhalation used in the same intervals. Examinations were finished inhalation of 40 mcg of fenoterol. The study confirms the full effectiveness of high doses of ipratropium bromide in patients with atopic asthma. Improve in spirometric parameters were observed after 40 mcg of ipratropium but the statistically significant differences were found in cumulated doses between 80 and 160 mcg ipratropium bromide without side effects. After fenoterol inhalation further improvement was observed. PMID- 8620179 TI - [Treatment of a patient with obstructive sleep apnea syndrome superimposed on chronic obstructive pulmonary disease]. AB - History of a middle aged obese male, presenting with severe obstructive sleep apnea (OSA) and chronic obstructive pulmonary disease (COPD) is described. Provisionally patient was started on CPAP and long-term domiciliary oxygen therapy (LTOT). OSA was successfully treated by surgical repair of nasal patency and partial uvulectomy. There was also remarkable improvement in ventilatory indices after steroid therapy. There was no further need for CPAP and LTOT. PMID- 8620180 TI - [Problems with genetic studies of atopy]. PMID- 8620181 TI - [The role of viral respiratory tract infections on pathogenesis of bronchial asthma. I. Clinical and experimental investigations]. PMID- 8620182 TI - [Role of viral respiratory tract infections on the pathogenesis of bronchial asthma. II. Effect on the immune system]. PMID- 8620183 TI - [Adhesion molecules in bronchial asthma]. PMID- 8620184 TI - Chiral inversion of fenoprofen in horses and dogs: an in vivo-in vitro study. AB - Fenoprofen (FPF) is a chiral non-steroid antiinflammatory drug, marketed as a racemic mixture of its R(-) and S(+) enantiomers. Its stereoselective disposition in humans and animals is due to a chiral inversion converting R(-)FPF into S(+)FPF. The first step of this reaction, which produces an acyl-CoA thioester, is catalysed by an acyl-CoA ligase. A stereospecific high performance liquid chromatography assay was used to study the disposition of FPF enantiomers in four geldings and three male beagle dogs, following intravenous doses of racemic FPF (1 mg/kg in horses), R(-)FPF (0.5 mg/kg in horses, 1 mg/kg in dogs), and S(+)FPF (0.5 mg/kg in horses, 1 mg/kg in dogs). A unidirectional stereoinversion of the R(-) enantiomer into its optical antipode (38% in horses, 90% in dogs) was demonstrated. This explained the clear enantioselective behaviour of FPF in both species. Acyl-CoA ligase activity (Km = 473.2 +/- 92.5 microM; Vmax = 23 +/- 3.3 nmol/min/mg) has also been quantified in vitro on equine hepatic microsomes, using a high performance liquid chromatography method to measure thioester formation. The present study showed that, in horses and dogs, as previously demonstrated in rats and sheep, the R(-)FPF clearance was better correlated with ligase activity than with inversion rate. A highly significant linear relationship was demonstrated between these variables. PMID- 8620185 TI - [Bactericidal effect of colistin on Escherichia coli. Model and simulation or the pharmacokinetic-pharmacodynamic relation for prediction of efficacy in veterinary antibiotic therapy]. AB - Pharmacodynamics studies consider three main parameters: the size of the bacterial population, the concentration of the antibiotic and the duration of its action. The pharmacodynamic characteristics of colistin were studied in vitro with Escherichia coli. The bacterial kinetics were fitted using differential equations. The mathematical model gave qualitative and quantitative information about the characteristics of the antibiotic-bacteria association. Above all, when linked to a pharmacokinetic model, the model permitted the prediction of the drug's efficiency. Simulations of various dosage levels in which the administration route, dose size, or interval between doses varied, permitted a more rational optimization than a prediction of efficacy based on the time taken to achieve antibiotic plasmatic concentrations above the minimal inhibitory concentration. Pharmacokinetic/pharmacodynamic modeling seems to be an interesting possibility for determining antibiotic dosing levels during the preclinical phase. PMID- 8620186 TI - Antagonism of lactic acid bacteria towards Staphylococcus aureus and Escherichia coli on agar plates and in milk. AB - The antagonistic effect of lactic acid bacteria (LAB, including Lactobacillus acidophilus, L. bulgaricus, L. casei and Streptococcus thermophilus) on Staphylococcus aureus and Escherichia coli was evaluated on MRS agar with the deferred and cross-streaking techniques, and in milk with the plate counting method. All LAB were repressive to S aureus and E coli on the agar medium. However, their suppressive activity was significantly reduced when the agar medium was buffered to pH 7.2. In normal milk, L acidophilus strains A and B, S thermophilus and its combinations with L acidophilus A and L bulgaricus 6032 were inhibitory to S aureus, while in mastitic milk, only S thermophilus and its combinations showed inhibition. L acidophilus A and L bulgaricus 34104 were repressive to E coli growth in normal milk. S thermophilus and its combinations were inhibitory to E coli in both the normal and mastitic milk samples. These results indicate that the antagonistic activity of LAB on pathogenic bacteria varied with the type of media in which the tests were done, and that testing of in vitro antagonism in milk would be more informative than that in artificial media for in vivo tests concerning the possible roles of competitive microbiological ecology in mastitis control. PMID- 8620187 TI - Reconstitution of mastitic milk by adding blood plasma and leukocytes into low cell count milk. AB - Milk from inflamed quarters is high in somatic cells, proteolytic activity and lysosomal enzyme activity. Addition of homologous blood plasma and leukocytes into low cell count milk did not increase the plasmin activity, total proteolytic activity or N-acetyl-beta-D-glucosaminidase (NAGase) as anticipated, even if the reconstituted milk samples were stimulated by endotoxin or opsonized zymosan. Addition of urokinase activated the latent proteolytic system in plasma supplemented milk. The reactive sulfhydryls in milk samples with different treatments decreased significantly after incubation, indicating the presence of an oxidative mechanism in milk (eg, sulfhydryl oxidase). The high background resazurin reduction of fresh milk might be due, in part, to the cellular activity of somatic cells. This study indicates that a more complex system including inflammatory mediators and complex cellular interactions is required for expression of plasmin-activator activity and for full activation of the proteolytic and lysosomal enzymes in mastitic milk. PMID- 8620188 TI - The neuraminidase of Newcastle disease virus inhibited in the elution-inhibition reaction. AB - The neuraminidase (NA) on strain 575 of Newcastle disease virus (NDV) was inhibited with elution-inhibition (EI) antibodies causing permanent agglutination patterns of red blood cells (RBC). Detection of EI antibody was unaffected by passage through sephadex, centrifugation or plaque purification of NDV. The haemagglutination-inhibition (HI) and EI reactions as well as the NA inhibition test all showed an increase in titre as immunization progressed. Fluorescent stained IgG on RBC from the EI reaction appeared as foci but as a halo if NDV was disrupted with ether suggesting NDV aggregation. The haemagglutinin (HA) on "sensitized' RBC from the EI reaction was not neutralized and agglutinated newly added RBC. The NA cleaved fetuin at 49 to 88% of the maximum values while bound to RBC by EI antibody. Added NA substrates failed to block the EI reaction. The order of inactivation at 53 or 56 degrees C but not 50 degrees C was: the putative EI antibody determinant > NA > HA. The term elution inhibition is suggested for the antibody responsible for the EI reaction. PMID- 8620189 TI - In vivo replication of African swine fever virus (Malawi '83) in neutrophils. AB - The presence of virus replication centers in neutrophils from pigs inoculated with a highly virulent strain of African swine fever virus is described for the first time in vivo. Virus antigens were observed from 3 days post-inoculation (dpi) onwards by means of an immunohistochemical technique. At this time (3 dpi), transmission electron microscopy studies revealed the presence of large amounts of neutrophils in the vascular lumens. At 5 and 7 dpi, neutrophils with phagosomes frequently contained virus particles. In addition, within the cytoplasm of some mature and immature neutrophils, both viral particles and virus replication centers were observed at 5 and 7 dpi. PMID- 8620190 TI - [Hemodynamics of right circulation in ewes: normal values]. AB - The right cardiac pressure was measured on 24 anesthetized adult ewes (Halothane) with a Swan Ganz catheter. After a review of the catheterization technique, the results (mean +/- standard deviation) in mmHg were: right auricle 17 +/- 5, right ventricle 30 +/- 6 (systolic) and 12 +/- 6 (telediastolic), pulmonary artery 29 +/- 6 (systolic), 13 +/- 6 (diastolic) and 20 +/- 6 (mean), capillary pressure 17 +/- 7. We observed slight variations in the pressure curve morphology compared to those found for man and the pressures were 5-10 mmHg higher than what is observed in man and slightly higher than those observed in dogs. These results also demonstrate a great variation between animals. PMID- 8620191 TI - Pulmonary intravascular macrophages in deer. AB - Pulmonary intravascular macrophages (PIMs) have been found in the septal capillaries of deer lungs. Lung samples from adult deer were fixed in 2.5% glutaraldehyde, and then routinely processed for electron microscopy. The main features of the PIMs were the presence of tubular invaginations in the membrane (micropinocytosis vermiformis), phagosomes, and junctions with endothelial cells. A mean of 4.4 of these junctions was recorded per cell. They comprised segments ranging from 67 to 289 nm in length, where the plasma membranes were separated by spaces from 10 to 25 nm wide. In these areas the cytoplasm underlying the membranes showed evidence of increased electron density. When PIMs were compared with alveolar macrophages, it could be seen that although the PIMs were more numerous (more than twice), they were also smaller than the alveolar macrophages (47.625 versus 101.260 microns2 respectively. PMID- 8620192 TI - The development of tissue lesions in the snail Lymnaea glabra exposed to a sublethal dose of molluscicide. AB - Histological examinations were undertaken in adult Lymnaea glabra to determine whether tissue lesions develop in snails that survived exposure to a molluscicidal agent and thus impair survival or reproduction capacities of remaining snails. The snails were exposed for 4 days to sublethal doses of niclosamide (0.21 mg/L), 3,4-dichloro-2-benzamido-5-nitrothiazole (0.13 mg/L), or 3,5-dichloro-2-benzamido-5-nitrothiazole (0.15 mg/L) at 20 degrees C. After exposure, the surviving snails were maintained under normal conditions (oxygenated water) for a further 21 days. The niclosamide group revealed epithelial necrosis in the digestive glands and the gonads. This was followed by reconstitution from day 12 or 19. The same sequence of tissue lesions also occurred in the kidney, however, a second phase of epithelial necrosis developed in the reconstituted epithelium after day 19. In the two other groups, tissue lesions of the three viscera developed in very similar manner, regardless of the molluscicide chosen for these experiments. Although the molluscicidal doses were sublethal in these snails, 12-19 days at 20 degrees C were required to reconstitute the visceral epithelium. Snails that survived the molluscicidal agent were thus impaired for one week or more in normal conditions. PMID- 8620193 TI - [Histopathological analysis of bovine livers infested by Dicrocoelium dendriticum]. AB - The object of this study was to determine the evolution of the lesions provoked by dicrocoeliasis in hepatic tissue, as a function of the quantity of parasites. Of the 274 bovine livers taken from the slaughterhouse and autopsied for Dicrocoelium dendriticum infection, 191 demonstrated hepatic lesions that revealed a proliferation of bile ducts, modifications of the bile ducts and hepatic fibrosis lesions. The modifications of the surfaces of the bile ducts and the hepatic fibrosis lesions increased with the level of infestation from 0 to 300 D dendriticum. Above these values, 301 to 600 flukes, a decrease was observed. PMID- 8620194 TI - Policy. Health and lack of wealth. PMID- 8620196 TI - Technology. New life for old equipment. PMID- 8620195 TI - Payment ... so-called health reforms actually cost more and delivered less. PMID- 8620197 TI - Quality watch ... Quality Measurement Advisory Service. PMID- 8620198 TI - Employers. Helping workers get healthy. PMID- 8620199 TI - To buy or not to buy, that is the question. AB - Though people will always need hospital care, it isn't clear if managed care companies will always need to own hospitals. In fact, some executives now say that they can get the care they need at the price they want without the organization itself holding the deed. But others are cringing at the thought of For Sale signs sprouting up on hospital lawns across America. Who wins--and who loses--when managed care companies decide that owning their own hospitals just isn't worth it anymore? PMID- 8620200 TI - A virtual dilemma. AB - When a group of doctors is faced with a managed care company claiming that the executives of a "virtual" network may lose a contract because they're holding out for too much money, is that a sign that such a firm isn't the answer to hospital physician integration? PMID- 8620202 TI - Employers. Workers' health is retailer's business. PMID- 8620201 TI - The FDA just says yes. AB - Will a new and, some say, improved FDA improve the conditions, financial and otherwise, of patient care? Many argue that the acceleration of the FDA approval process comes at a high price. As new drugs hit the market faster--and with less proof of their long-term benefits--health care costs are going up, up, up, and society is facing painful rationing decisions. PMID- 8620203 TI - Outreach. Aural support for cancer sufferers. PMID- 8620204 TI - Top 10 list. Ready for a grayer America? PMID- 8620205 TI - Insurance issues. Arizona voters to decide Medicaid expansion. PMID- 8620206 TI - Direct contracting. Riskier business. PMID- 8620207 TI - Managed care ... size isn't everything. PMID- 8620208 TI - Physicians. Public Citizen names names. PMID- 8620209 TI - Fewer gripes from Medicare HMO patients. PMID- 8620210 TI - Consumers. Surfing the Web for a doctor. PMID- 8620211 TI - Columbia's got the (Ohio) blues. A company known for its drive and audacity pulls off another big deal. PMID- 8620212 TI - Under scrutiny. As public anxiety grows over health care horror stories, consumers are starting to fight back. Guess who's winning. AB - "We're getting dozens of calls every day from people who are frustrated and fed up," says one health care consumer rights advocate. The scenario is familiar: first come the horror stories, then trailblazing, media-engaging lawsuits, and finally the public learning curve starts to accelerate. Then the heat gets turned up on the government to act. That's where we're at right now. Where will we be tomorrow? PMID- 8620213 TI - Cast me not off in the time of age. PMID- 8620214 TI - From sea to shining sea. PMID- 8620215 TI - Make no little plans. Community health planning is an antidote to a world in which the strong survive and the weak usually don't. But in an era of scaling back, does it have a future? PMID- 8620216 TI - Clinical trials. Forward motion on medical advances. PMID- 8620217 TI - Billing. Surgery at discount prices. PMID- 8620218 TI - Pharmaceuticals. Rural drugstore's bitter pill. PMID- 8620219 TI - Outreach. Pregnant teens get the "Baby Blues". PMID- 8620220 TI - Urgent care. Emergency measures to lure patients. PMID- 8620222 TI - HospitalPulse ... December 1995. PMID- 8620221 TI - Policy. HCFA's new take on physician incentives. PMID- 8620223 TI - Trust and chaos. PMID- 8620224 TI - The safety of acellular pertussis vaccine vs whole-cell pertussis vaccine. A postmarketing assessment. AB - OBJECTIVE: To determine the impact of the introduction of acellular pertussis vaccine for the fourth and fifth doses of the diphtheria and tetanus toxoids and pertussis vaccine series in children on rates of reported vaccine-associated adverse events in the United States. DESIGN: Analysis of postmarketing vaccine adverse event data from the Vaccine Adverse Event Reporting System during the years 1991 to 1993. POPULATION STUDIED: Approximately 27 million doses of diphtheria and tetanus toxoids and pertussis vaccine and 5 million doses of diphtheria and tetanus toxoids and acellular pertussis vaccine were distributed from 1991 to 1993 to children 15 months to 7 years of age. MAIN OUTCOME MEASURES: Rates of reported fever, seizures, and hospitalizations after pertussis vaccination. RESULTS: Rates of reported adverse events per 100,000 vaccinations were significantly lower after administration of diphtheria and tetanus toxoids and acellular pertussis vaccine than diphtheria and tetanus toxoids and pertussis vaccine for the following outcomes: all reports, 2.9 vs 9.8; fever, 1.9 vs 7.5; seizures, 0.5 vs 1.7; and hospitalizations, 0.2 vs 0.9. CONCLUSIONS: These results confirm that minor adverse events are less frequent after administration of the acellular pertussis vaccine. In addition, these data suggested that seizures and hospitalizations associated with pertussis vaccination are less frequent after administration of the acellular pertussis vaccine in age groups for which it is now recommended. PMID- 8620225 TI - Nothing to whoop about. PMID- 8620226 TI - Efficacy of topical anesthesia in children. AB - OBJECTIVES: To compare the efficacy of three formulations of a topical anesthetic solution composed of various concentrations of tetracaine hydrochloride, adrenaline (epinephrine), and cocaine hydrochloride (TAC), and to compare the cost of the topical anesthetic solutions with the cost of lidocaine infiltration. DESIGN: Randomized, double-blind clinical trial. SETTING: Urban pediatric emergency department. PARTICIPANTS: One hundred fifty-six children 3 to 18 years of age and older requiring topical anesthesia for suturing of lacerations. INTERVENTION: Children received 3 mL of one of the following study solutions: TAC 1 consisting of 0.5% tetracaine, 1:2000 dilution of adrenaline, 11.8% cocaine; TAC 2 that contained 1% tetracaine, 1:2000 dilution of adrenaline, 4% cocaine; or TAC 3 made up of 1% tetracaine and 4% cocaine, without adrenaline. MEASUREMENTS OR MAIN RESULTS: Patients were randomized to group 1 (n = 49), group 2 (n = 49), or group 3 (n = 58), and received TAC 1, TAC 2, or TAC 3, respectively. Patients in the three study groups were similar for age, gender, anatomic location and length of the laceration, and history of sutures or use of topical anesthesia. Based on the physician assessment of achievement of complete, partial, or no anesthesia, solutions containing 11.8% cocaine (TAC 1) and 4% cocaine with adrenaline (TAC 2) were more likely to produce complete anesthesia than the solution with 4% cocaine without adrenaline (TAC 3) (P < .001, chi 2). This difference was only noted when the laceration involved the face or scalp. A second dose of the TAC 3 solution was more often required to produce anesthesia when compared with the other two study drugs (P < .003, chi 2). The final cost to produce 3 mL of the study drugs, including the vials, was $16.39 for TAC 1, $8.67 for TAC 2, and $8.41 for TAC 3. CONCLUSIONS: The application of a TAC solution containing 4% cocaine is as effective as a TAC solution containing 11.8% cocaine. Use of the 4% solution decreases the cost of the agent. Adrenaline is a necessary ingredient in the anesthetic solution. PMID- 8620227 TI - Increased drug use among old-for-grade adolescents. AB - OBJECTIVE: To determine whether students older than most other students at their grade level ("old for grade") are more likely to report engaging in alcohol, tobacco, and drug-related behaviors. DESIGN: Cross-sectional analyses of the Centers for Disease Control and Prevention Youth Risk Behavior Survey. SETTING: Monroe County, New York. PARTICIPANTS: A total of 1396 high school students from selected classrooms; 68 classrooms randomly selected within schools with the number of students per school proportionally selected from the 28 schools in the county. MAIN OUTCOME MEASURE: Rates of drug-related behaviors by age-for-grade status. RESULTS: Thirty-six percent of adolescents surveyed were old for grade. Adjusting for multiple potential confounders, old-for-grade high school students were more likely to report being regular smokers, chewing tobacco, drinking alcoholic beverages, driving in a car with someone who had been drinking, using alcohol or other drugs before last sexual intercourse, using cocaine in the past month, ever using crack, and using injected or other illicit drugs. CONCLUSIONS: Old-for-grade status is a potentially important marker for drug-related behaviors in adolescents. The antecedents of adolescent risk-taking behavior may begin before the teen years, and prevention of school failure or interventions targeted toward old-for-grade children could affect their propensity to experiment with or use drugs during adolescence. PMID- 8620228 TI - Primary care involvement among hospitalized children. AB - OBJECTIVE: To examine relations between characteristics of a child's usual source of primary care and involvement of that source before and during hospitalization. DESIGN: Medical record review of pediatric hospitalizations. SETTING: All hospitals in Boston, Mass; New Haven, Conn; and Rochester, NY admitting children during the calendar years 1988 through 1990. PATIENTS: The study included 1875 randomly selected pediatric hospitalizations for five diagnostic groups (i.e., asthma and other lower respiratory tract disease, abdominal pain [including appendicitis], meningitis [bacterial and viral], toxic ingestions, and head injury). Hospital records selected were limited to children aged between 1 month and 12 years and residing in the three study communities. OUTCOME MEASURES: Whether the primary care source examined the child before admission to the hospital, referred the child to the emergency department, or served as the in hospital attending physician. RESULTS: Of the medical charts reviewed, 85.7% identified primary care sources. Children in Rochester had higher rates of medical visits before admission (P < .04), referrals (P < .001), and in-hospital care provided by the primary care physician (P < .001, chi 2) than children in Boston and New Haven. Patterns of primary care involvement also varied by source of care within cities, after controlling for income and severity of illness. Compared with children from Rochester community-based private practices, children in Boston receiving care from health centers, hospitals, or community-based private practices generally had 25% to 50% lower likelihood of positive findings on all primary care involvement measures. Children in New Haven receiving care from community-based private or hospital-based practices also had lower rates, but involvement rates were not higher when they received care from health centers. Other children in Rochester and children receiving care from health maintenance organizations in all cities demonstrated almost no significant differences compared with data from Rochester community practices. CONCLUSION: The source of primary care is associated with patterns of prehospital and hospital care among hospitalized children, although specific associations vary by city. PMID- 8620229 TI - Diffusion of innovative approaches to managing hypoplastic left heart syndrome. AB - OBJECTIVE: To study the perceptions of outcome and the diffusion into practice of innovative approaches such as palliative surgery and heart transplantation to treat hypoplastic left heart syndrome. DESIGN: A forced-choice questionnaire was sent to 108 US neonatology section heads. Responses were analyzed using Wilcoxon matched pairs, chi 2 analysis, and multivariant logistic regression. RESULTS: Ninety-three questionnaires (86%) were returned. All respondents discussed palliative surgery or transplantation or both with parents; 71 (76%) of 93 also discussed comfort care. Nineteen (24%) of 80 recommended comfort care only, 51 (64%) of 80 recommended surgery only, and 10 (12%) of 80 recommended both. Of the 61 respondents recommending one or both surgical procedures, palliative surgery was recommended by 44 and transplantation by 33. Respondents perceived that transplantation was associated with a lower 1-year mortality than palliative surgery (P < .001) and with a better quality of life (P < .001). CONCLUSIONS: Palliative surgery and transplantation are widely used to treat hypoplastic left heart syndrome. The continued availability of comfort care suggests that these surgical procedures are still in transition from experimental technology to standard of care. PMID- 8620231 TI - A new filter paper method to measure capillary blood lead level in children. AB - OBJECTIVE: To develop and evaluate a new filter paper method to determine capillary blood lead levels accurately in children. DESIGN: Paired comparison of lead levels determined in capillary whole blood dried on filter paper with lead levels in venous whole blood samples determined by a reference method. SETTING: Children's Hospital of Michigan clinics, Detroit. PATIENTS: One hundred children aged 9 months to 6 years. INTERVENTIONS: Lead concentrations determined in capillary whole blood samples dried on filter paper were compared with concentrations measured in paired venous whole blood samples by a reference method. MAIN OUTCOME MEASURES: Comparability of the two lead assay methods was assessed with the concordance coefficient. The sensitivity, specificity, and positive predictivity of the capillary filter paper method relative to the reference method were determined at three intervention decision concentrations of blood lead defined by the Centers for Disease Control and Prevention. RESULTS: There was high agreement between the two assay methods, with a concordance coefficient of O.96. The capillary filter paper assay had a sensitivity of 90% and specificity of 90% for differentiating blood lead levels of 0.48 mumol/L (10 micrograms/dL) or more. Blood lead levels of 0.72 mumol/L (15 micrograms/dL) or more and 0.96 mumol/L (20 micrograms/dL) or more were identified with 98% and 94% sensitivity and 98% and 99% specificity, respectively. Positive predictivity was 93%, 98%, and 97%, respectively, at the three blood lead concentration decision points. CONCLUSION: The capillary filter paper method for blood lead analysis described herein provides a convenient, sensitive, accurate, and inexpensive method to examine children for elevated blood lead levels. PMID- 8620230 TI - Prenatal and perinatal risk and protective factors for neonatal intracranial hemorrhage. National Institute of Child Health and Human Development Neonatal Research Network. AB - OBJECTIVE: To identify prenatal and perinatal risk and protective factors for grade III and IV intracranial hemorrhage (ICH) in 4795 singleton infants (weight, < or = 1500 g). METHOD: Prenatal and perinatal risk and protective factors for ICH were examined initially by univariate analysis and adjusted for year of birth, followed by multivariate logistic regression analysis that adjusted simultaneously for the effects of year of birth and prenatal and perinatal characteristics. SETTING: Seven tertiary care neonatal-perinatal centers. RESULTS: By univariate analysis, African-American race, prenatal care, older maternal age, hypertension or preeclampsia, antenatal steroid administration, cesarean section delivery, increasing birth weight, increasing gestational age, and female gender of the infant were protective prenatal or perinatal factors. Antepartum hemorrhage, the presence of labor, and breech presentation were perinatal factors that were associated with an increased risk of ICH. By using staged logistic regression, a model of combined prenatal and perinatal characteristics that influenced grade III and IV ICH was developed. Significant protective factors against ICH included a complete course of antenatal steroid therapy, African-American maternal race, female gender of the infant, hypertension or preeclampsia with no antepartum hemorrhage, increasing gestational age, and increasing birth weight. CONCLUSION: Antenatal steroid administration is a therapeutic intervention that is associated with a decreased risk for neonatal grade III and IV ICH. PMID- 8620232 TI - Evidence for selective health maintenance organization enrollment among children and adolescents covered by Medicaid. AB - OBJECTIVE: To determine whether children and adolescents are selectively enrolled in health maintenance organizations (HMOs) based on age, gender, diagnosis, or prior utilization. DESIGN: Case-control study. New HMO enrollees were compared with a control population of non-HMO enrollees. SETTING: Medicaid claims data and HMO participation records for the Medicaid and Aid to Families of Dependent Children sector in Baltimore, Md. RESULTS: Controlling for age, significant differences in prior health care utilization as measured by Medicaid expenditures and hospital days were noted. Children enrolling in HMOs had significantly lower prior utilization than children from the control population as measured by dollar expenditures and hospital days. Young children enrolling in HMOs were only half as likely to have prior claims for asthma. Conversely, adolescents enrolling in HMOs had significantly higher prior utilization than adolescents from the control population. The difference among adolescents was due to a higher birth rate among new HMO enrollees in that age bracket. CONCLUSIONS: Voluntary HMO enrollment of children covered by Medicaid and Aid to Families of Dependent Children sector was subject to selection biases that may be economically favorable to the HMOs and may undermine the cost-containment goals of prepaid health care for Medicaid participants. Voluntary capitated systems where fee-for-service remains a significant alternative must monitor for these selection biases that are not allowed for in the adjustments to capitation rates. PMID- 8620233 TI - Who needs an immunization in a pediatric subspecialty clinic? AB - BACKGROUND: The Standards for Pediatric Immunization Practices recommends that subspecialty clinics screen children's immunization status and ensure the receipt of needed immunizations. OBJECTIVES: To determine the proportion of children presenting to a pediatric subspecialty clinic in whom immunization status can be assessed, and which of those assessed are due an immunization (eligible to receive an immunization on the day of clinic visit). DESIGN: Standardized survey of 196 patients or accompanying children presenting to a pediatric cardiology clinic. Need for immunizations was determined by the Advisory Committee on Immunization Practices recommendations. RESULTS: The reason for visit included 58% return (enrolled in the clinic), 25% initial, and 17% accompanying another patient. Usual immunization provider included 51% health department, 42% primary care physician, and 7% military. We could assess the immunization status of 79 (40%) of 196, and 19 (24%) of these 79 were due an immunization. Logistic regression analysis revealed that children enrolled in the clinic were more likely to be due for immunization than those presenting for initial visits (38% vs 8%; adjusted odds ratio, 7.42; 95% confidence interval, 1.43 to 38.55). CONCLUSIONS: We could not assess the immunization status of most children presenting to this pediatric clinic. Patients enrolled in the clinic were at increased risk for being due immunization. Having a primary care physician as a provider of immunizations did not ensure the receipt of immunizations. Pediatric subspecialists should assess the immunization status of their patients and make sure that they receive needed immunizations. PMID- 8620234 TI - Anti-inflammatory therapy reduces wheezing after bronchiolitis. AB - OBJECTIVE: To evaluate whether early anti-inflammatory therapy with nebulized cromolyn sodium or budesonide reduces wheezing after bronchiolitis. DESIGN AND SETTING: A randomized, controlled study in a university hospital that provides primary hospital care for all pediatric patients in a defined area. PATIENTS: One hundred consecutive infants younger than 24 months treated in the hospital for acute bronchiolitis. INTERVENTIONS: Thirty-four patients received cromolyn sodium, 20 mg four times a day for 8 weeks and 20 mg three times a day for 8 weeks, and 34 patients received budesonide, 500 micrograms twice a day for 8 weeks and 250 micrograms twice a day for 8 weeks, by a foot pump with a face mask; 32 patients in the control group received no therapy. MAIN OUTCOME MEASURES: Numbers of physician-diagnosed wheezing episodes, hospital admissions for bronchial obstructions, and symptomatic days recorded by the parents. RESULTS: Children in the cromolyn sodium (19%) and budesonide (16%) groups had significantly fewer physician-diagnosed wheezing episodes than those in the control group (47%) during the second 8-week period (P < .05). A significant reduction in hospital admissions for bronchial obstructions was seen in the budesonide group and in the children with atopy in both treatment groups (P < .05). The children with atopy had significantly more subsequent wheezing episodes and hospital admissions than those without atopy (P < .05). The numbers of symptomatic days did not differ significantly among the three groups. CONCLUSIONS: Early anti-inflammatory therapy with nebulized cromolyn sodium or budesonide reduces the number of wheezing episodes and hospital admissions after bronchiolitis. Children with atopy are at high risk of subsequent wheezing episodes, and they particularly benefit from anti-inflammatory therapy. PMID- 8620235 TI - Tympanic temperature asymmetry and stress behavior in rhesus macaques and children. AB - OBJECTIVES: To examine left-to-right tympanic membrane temperature asymmetries and their possible association with biobehavioral stress responses in rhesus macaques and children. SUBJECTS AND DESIGN: Infrared tympanic membrane thermometry was completed bilaterally in 19 two-year-old rhesus macaques and 18 eight-year-old children in a cross-sectional, laboratory-based study. Unidirectional temperature gradients were calculated as the mean of two left sided measurements minus the mean of two right-sided measurements. Biobehavioral stress responses were assessed in monkeys as agitated motor activity and adrenocortical activation after separation from the social group, and in children as parent-reported resilience to psychological stress and child behavior problems. RESULTS: Significant asymmetry was detected in tympanic membrane temperatures in both monkey and child samples, with left-sided temperatures measuring slightly but significantly higher than those from the right tympanic membrane. Higher-magnitude left-to-right temperature gradients were associated with stress-related locomotion in macaques and with lower resilience and more behavior problems in children. CONCLUSIONS: There are small but reliable asymmetries in the tympanic membrane temperatures of young human and nonhuman primates. Tympanic membrane temperature gradients reflect important individual differences in biologically derived responses to psychological stressors. PMID- 8620236 TI - Educational interventions to alter pediatric emergency department utilization patterns. AB - OBJECTIVE: To test the hypothesis that educating parents about use of their primary care provider and providing information about common pediatric illnesses will reduce visits to the pediatric emergency department (PED). DESIGN: Prospective, randomized, controlled trial conducted from September 1, 1993, to October 31, 1994. SETTING: Pediatric emergency department of an urban university hospital. PARTICIPANTS: Parents of 130 patients seen in the PED for minor illness. INTERVENTIONS: Subjects were randomized to intervention or control groups. Parents in both groups were interviewed about their child's health and use of health care services. The intervention group received education on pediatric health care issues; the control group received usual PED discharge instructions. Use of the PED by all subjects was tracked for 6 months by telephone follow-up and medical record review. MAIN OUTCOME MEASURES: Differences between the two groups in total number of return visits to the PED and return visits to the PED for minor illness. RESULTS: Sixty-seven (97%) of the 69 patients in the intervention group and 56 (92%) of the 61 patients in the control group identified a primary care provider. At 6-month follow-up, 21 patients (30%) from the intervention group and 16 (26%) from the control group had returned to the PED (P = .68, chi 2). Seventeen (81%) of intervention group returnees to the PED had minor illness, as did 11 (69%) of control group returnees. CONCLUSIONS: A one-time educational intervention in the PED does not alter long-term emergency department utilization habits. More extensive education and greater availability of primary care providers may be needed to decrease use of the PED for minor illness. PMID- 8620237 TI - Residents on the transport team. Balancing service and education. AB - OBJECTIVES: To describe an educational program for pediatric house staff who participate in interhospital transport and to present an evaluation of the educational program. DESIGN: Educational program evaluation that used multiple confidential surveys of participating pediatric house staff. SETTING: The interhospital transport team of a large, urban pediatric hospital. PARTICIPANTS: Twenty-six pediatric second-year residents who participated in required rotations with the transport service. INTERVENTION: The institution of an educational program designed specifically for the clinical transport rotation. RESULTS: Before their service with the transport team, residents have doubts about their clinical skills, fund of knowledge, and ability to practice independently in a mobile environment. These doubts decrease as the residents participate in the educational program during their transport clinical service. All residents perceive service on the transport rotation to be of educational benefit with regard to communications or cognitive skills. Alternatively, transport service provides little opportunity for residents to improve technical skills. Among the various skills that residents could obtain during transport service, improvement in clinical judgement was most commonly cited by residents who performed more than 10 transports. Among the specific curricular components in the educational program, interactive teaching methods were judged to be more valuable than written materials. CONCLUSIONS: Serving as a physician on interhospital transports can be an important educational opportunity for pediatric house staff when that experience is concentrated in a designated rotation and combined with a focused educational program. Contrary to expectations, there was little opportunity for residents to develop technical skills on transport. Therefore, educators should focus on the development of communications and cognitive skills. Interhospital transport programs that debate the use of residents as members of the service should consider the potential educational benefit of the clinical service to house staff. PMID- 8620238 TI - Henry Koplik, MD, the Good Samaritan Dispensary of New York City, and the description of Koplik's spots. PMID- 8620239 TI - Radiological case of the month. Sequential magnetic resonance imaging of a falx interdural hematoma. PMID- 8620240 TI - Picture of the month. Rubeola (measles). PMID- 8620241 TI - Pathological case of the month. Congenital dermatofibrosarcoma protuberans. PMID- 8620242 TI - Benign intracranial hypertension in infants with cystic fibrosis. PMID- 8620243 TI - Concerns of secondary fever in Streptococcus pneumoniae meningitis in an era of increasing antibiotic resistance. PMID- 8620244 TI - Cystic fibrosis in Asian Indians. PMID- 8620245 TI - Elevated environmental lead levels in a day care setting. PMID- 8620246 TI - A matter of trust: mandatory HIV testing. PMID- 8620247 TI - Adolescent sexuality: looking beyond treatments. PMID- 8620248 TI - The use of methylphenidate in Michigan. PMID- 8620249 TI - Childhood immunizations in the emergency department. PMID- 8620250 TI - Bloody insensitive. PMID- 8620251 TI - Early neurologic outcome after open heart surgery on young infants. PMID- 8620252 TI - A pigmented paraspinal plaque in an infant. PMID- 8620253 TI - More good news for family medicine. PMID- 8620254 TI - Symptoms and complications of adult diabetic patients in a family practice. AB - OBJECTIVES: To determine the frequency of common symptoms and complications amoung adult patients with either non-insulin-dependent or insulin-dependent diabetes mellitus and to examine associations of these problems with vascular disease risk factors. DESIGN: Cross-sectional determination of the prevalence of symptoms, complications, and risk factors among adult patients with diabetes, and a case-control design of a subsample of those case patients with non-insulin dependent diabetes mellitus who were age-, race-, and sex- matched 2:1 to adult control patients with hypertension. SETTING: A large family practice ambulatory care unit in which the patients were demographically representative of the Piedmont region of North Carolina. PATIENTS: Three hundred thirty-six adults participated; of these participants, 223 case patients had non-insulin-dependent diabetes mellitus and 23 case patients had insulin-dependent diabetes mellitus. An additional 90 control patients were selected who had hypertension but did not have diabetes. MAIN OUTCOME MEASURES: Frequencies and odds ratios of symptoms and complications that were categorized by diabetic type, another chronic disease (eg hypertension), or vascular risk factors (eg, the duration of diabetes, levels of glycosylated hemoglobin, fasting blood glucose, total cholesterol, and high density lipoprotein cholesterol, or mean arterial blood pressures). METHODS: Standardized medical histories, physical examinations and anthropometric and serum chemistry studies were done. Microalbuminuria was measured by unrinary albumin excretion ratios. RESULTS: Results: Symptoms of polydipsia, fatigability or cold intolerance, palpitations, dyspnea, orthostasis, indigestion, frequent urination, male impotence, blurred vision, paresthesias, forgetfulness occurred in more that one third of the adult diabetic patients as did complications of hypertension, microalbuminuria, peripheral neuropathy, and hypercholesterolemia. Those patients with non-insulin-dependent diabetes mellitus were more likely than those with insulin-dependent diabetes mellitus to have recent symptoms of polydipsia, chest pains, shortness of breath, orthostasis, light-headedness, and vaginal discharge. They were also more likely to have hypertension and hypercholesterolemia, but less likely to have dipstick proteinuria. Patients with non-insulin-dependent diabetes mellitus were more likely than those with hypertension alone to have a variety of general, cardiovascular, and neurologic symptoms and more likely to have peripheral vascular disease, neuropathy, retinopathy, and proteinuria. Regardless of the diabetic type, nearly half of the patients had microalbuminuria. The presence of symptoms was often associated with the duration of diabetes and poor glycemic control as were complications, but complications were often also associated with dyslipidemias and an elevated mean arterial pressure. CONCLUSIONS: Previous studies have documented such a wide variety of symptoms and complications, but these studies have been based on those patients who attended specialized referral settings. Our findings show that these problems are surprisingly common among adult diabetic patients who are cared for in a primary care setting. The fact that nearly half of the patients had microalbuminuria suggests that the onset of significant vascular complications had already begun in most of these patients. The occurrence of symptoms of depression, anxiety, panic, and forgetfulness were unexpectedly common and may have adversely affected the ability of diabetic patients to comply with the intended therapy. PMID- 8620255 TI - An intervention for preventing alcohol use among inner-city middle school students. AB - OBJECTIVE: To examine the effectiveness of a brief, school-based intervention for preventing alcohol use. DESIGN AND SETTING: Randomized, control trial assigning inner-city public school students to an intervention program or a comparison program. PARTICIPANTS: Sixth, seventh, and eighth grade students in Jacksonville, Fla (N=104). INTERVENTIONS: Students assigned to the intervention program were given a self-instructional module and corresponding audiotape, a health consultation with a physician or nurse, and a follow-up consultation with a trained peer health model. MAIN OUTCOME MEASURES: Alcohol consumption during the month after the intervention and students' assessments of the interventions were measured. RESULTS: Students' t tests showed participants were more satisfied with physician or nurse consultations than with peer consultations or the self instructional module and audiotapes (P=.05). Analysis of covariance tests showed significant main effects for 30-day quantity of alcohol use (F=5.15, P=.02), with intervention students reporting less alcohol consumption at follow-up than comparison students, and for 30-day frequency of alcohol use (F=5.92,P=.01) with intervention students again showing less frequent use at follow-up. CONCLUSIONS: A multicomponent, school-based intervention using print and audiotape media, brief physician or nurse consultations, and follow-up peer contacts holds promise in altering short-term alcohol use and selected behavioral factors among inner city youth. PMID- 8620256 TI - Breaking the silence. Battered women's perspectives on medical care. AB - OBJECTIVE: To determine the barriers to identification and management of domestic violence from the battered women's perspective. DESIGN: Qualitative research methods using semistructured focus groups. SETTING: Urban and suburban community based organizations serving women and their families in the San Francisco Bay (Calif) area. PARTICIPANTS: Fifty-one women with histories of domestic violence comprised eight focus groups divided as follows: two groups of Latino (n=14), two groups of white (n=14), Asian (n=14), and two groups of African-American (n=9) women. RESULTS: Participants from all ethnic groups identified major factors that affect identification and management of battered women in the health care setting. Factors that interfere with patient disclosure included threats of violence from the partner, embarrassment, adherence to gender roles, concerns about police involvement and lack of trust in the health care provider. One factor that predisposed a woman to seek help from providers was a need for the providers to exhibit compassion, awareness, and respect for the patient's need to make the final decisions about her situation. Most participants said that providers should take the initiative to ask directly about domestic violence, establish a supportive patient-provider relationship, and refer battered women to available community resources. The major institutional barriers to using the health care system included the high cost of medical care and long waiting periods. CONCLUSIONS: Many battered women experience social, institutional, and provider barriers to obtaining help from the health care system for problems related to domestic violence. Providers as well as institutions can overcome these barriers through an understanding of the social context of domestic violence and the victim's needs. Identification may be improved through a trusting patient-provider relationship and by direct questioning about domestic violence. PMID- 8620257 TI - Common upper-extremity injuries. AB - In the daily practice of family medicine, injuries to the upper extremity are frequently encountered. Most of these injuries can by easily treated by the primary care physician who has an understanding of the joint anatomy and treatment principles. Some injuries, however, may appear relatively minor, yet require prompt referral for surgical care. We reviewed the pathoanatomy, historical and physical examination findings, and treatment of the more common injuries to the hand, wrist, elbow, and shoulder. PMID- 8620259 TI - Postural vertigo. Quick relief from the postural vertigo component of vestibular diseases. AB - Patients who had disorders of the vestibular system with a component of benign postural vertigo as a symptom were studied, using an examination table suitable for the canalith (otolith) repositioning maneuver as described by Epply, followed by lack of recumbency for 48 hours. The patients regularly had resolution or decreased intensity of symptoms, as did those described by Epply. A repeated positioning maneuver may be needed in some of the patients. The application of a vibrator, as previously described has not been found to be essential. PMID- 8620258 TI - Subungual hemorrhages. A primary manifestation of diabetes mellitus. AB - Subungual hemorrhages may be observed in a variety of systemic diseases and in some otherwise healthy persons. We describe three male patients who were referred because of toenail hemorrhages. Analytical investigations showed hyperglycemia in all the patients. Toenail bilateral hemorrhages were the first manifestation of previously undiagnosed type II diabetes mellitus in all cases. Ophthalmologic examination showed signs of background diabetic retinopathy in all three patients. The existence of retinopathy suggests that subungual hemorrhages might be due to microvascular involvement and could herald the presence of diabetes mellitus. PMID- 8620260 TI - Maximizing the referral of older women for screening mammography. AB - The primary care physician is in a crucial position to facilitate mammography referral of women older than 50 years. Physician underestimation of the importance of the physician role or overestimation of patient resistance can result in lost opportunities for referral. Research is summarized on the impact of physician encouragement on use of mammography and on sources of reluctance to get mammography as reported by patients. Common patients' concerns are discussed. Mammography referral can usually be accomplished successfully by raising the issue and briefly addressing patients' concerns in regard to breast cancer and screening mammography. PMID- 8620261 TI - Combined methotrexate and misoprostol for early induced abortion. PMID- 8620262 TI - Combined methotrexate and misoprostol for early induced abortion. PMID- 8620263 TI - Combined methotrexate and misoprostol for early induced abortion. PMID- 8620264 TI - Combined methotrexate and misoprostol for early induced abortion. PMID- 8620265 TI - Medical decision making and perceived socioeconomic class. AB - OBJECTIVE: To examine the effects that a physician's knowledge of a patient's socioeconomic status or profession has on clinical decision making in an outpatient setting. METHODS: We mailed a survey to all 336 members of the Academy of Medicine of Cleveland who are general internists, family practitioners, or general practitioners. Physicians were randomized before the initial mailing to receive one of the two questionnaires. Physicians in group A were given two simulated clinical scenarios in which the socioeconomic status or profession of the patient was identified, followed by management options. Physicians in group B were given the same scenarios without any suggestion of the patient's profession or social standing. Outcomes that reflect decision making, intensiveness of evaluation, and treatment course were compared for the two groups. Data were analyzed using t tests and chi 2. After Bonferroni correction, a P < or = .01 was considered significant. RESULTS: The response rate was 60%. For clinical scenario 1, no difference was noted in the number of tests ordered. However, physicians in group A responding to clinical scenario 2 ordered more total tests than did physicians in group B (P = .03), including complete blood cell counts (45% vs 29%, P = .01). In both scenarios, earlier follow-up visits were scheduled by physicians in group A compared with those in group B (P = .01). CONCLUSION: Our results do not support findings by other investigators that more tests are ordered when physicians perceive their patients to be of higher socioeconomic status. Intensity of follow-up, however, was greater when physicians believed the patient was in a more prominent profession or was of higher socioeconomic status. PMID- 8620266 TI - The clinical value of computerized information services. A review of 98 randomized clinical trials. AB - OBJECTIVE: To review all randomized clinical trials addressing the efficacy of clinical information systems and to determine the clinical settings, types of interventions, and effects studied. DATA SOURCES: Extensive and systematic MEDLINE searches were conducted using a combination of medical subject headings (MeSH) and textword terms to collect trial reports. Manual searches of books and monographs as well as informal contacts were also used. STUDY SELECTION: The eligibility criteria were (1) randomized controlled clinical trial, (2) computerized information intervention in the study group, and (3) effect measured on the process or outcome of care. DATA EXTRACTION: Two research assistants independently abstracted from the selected reports the following structured information: trial sites, computerized interventions, effect variables, and outcomes. Three investigators evaluated the combined list of trial features for setting, intervention, and effect. The statistical analysis included an evaluation of agreement in developing classifications and an analysis of the ratio of positive trial outcomes. DATA SYNTHESIS: Most information services were tested in outpatient care (82%), particularly in primary care (66%). The information intervention targeted the provider in 64% of the trials. The effect was primarily measured for the process of care (76%). Provider prompt/reminder, computer-assisted treatment planner, interactive patient education/therapy, and patient prompt/reminder were significantly successful interventions (sign test, P < .05). CONCLUSIONS: Randomized clinical trials confirm that four generic information interventions are active ingredients of computer systems and can make a significant difference in family medicine (physician and patient reminders, treatment planner, and patient education). To manage care and improve quality, primary care computer systems should incorporate these effective information services. PMID- 8620267 TI - Episodes of care for abdominal pain in a primary care practice. AB - OBJECTIVES: To explore the usefulness of episodes of care in describing the clinical epidemiology of abdominal pain in the primary care setting and to develop methods to analyze clinician decision-making strategies during abdominal pain episodes. DESIGN: Complete episodes of care for nonpregnant adults with nonspecific abdominal pain from an established episode-based clinical information system were supplemented and validated by medical record review. Utilization decisions during episodes were quantified by summing the costs of all visits, services, tests, and referrals ordered or performed by the clinician. A decision model was used to analyze significant influences on utilization decisions. SETTING: An established faculty practice site of a Midwestern academic family practice department. SUBJECTS: Two hundred ten nonpregnant adults who had nonspecific abdominal pain. MAIN OUTCOME MEASURES: Utilization and costs generated during the episode of care. RESULTS: The average abdominal pain episode required 1.32 visits and cost $123.36. In more than half of all episodes (51%), a specific diagnosis was not reached. The most common specific diagnoses were gastritis and gastroesophageal reflux disease (5% each). Bivariate analyses showed that two variables, clinician uncertainty about diagnosis and a nonspecific diagnosis, were significantly associated with episode cost. Patient age, gender, comorbidity, and the presence or absence of specific clinical findings were not associated with episode cost. Stepwise regression modeling resulted in a two-factor model. Clinician uncertainty and complexity explained only 9% of the variance in episode cost. CONCLUSIONS: Episodes of abdominal pain most often remained undiagnosed. The decision model did not predict episode cost. Utilization decisions did not seem to be driven by commonly cited clinical risk factors, but by diagnostic uncertainty or individualized decision rules. PMID- 8620268 TI - Abdominal pain. What happens in primary care? PMID- 8620269 TI - Current concepts. Photoprotection. AB - Photoprotection encompasses all methods to prevent UV radiation (UVR) damage to the skin, including sunscreens, clothing, seeking shade, and duration and time of the day spent outdoors under UVR. As scientific research validates short- and long-term detrimental effects of UVR, physicians and the public must become increasingly aware of these problems to avoid them. Photoaging is defined. Choice of sunscreens, their Food and Drug Administration labeling, and future sunscreen products are reviewed. Hazards of UVR on the skin include acute sunburn, photocarcinogenesis, immunologic suppression, and photoaging. Distinguishing between UV-A and UV-B damage to the skin is discussed. Education of physicians and their patients is crucial to reduce future photodamage to our population, especially with a reduction of the ozone layer and with patients having more free time. The complete skin examination is emphasized as a method to detect photodamaged skin and give patients insight to provide themselves with future photoprotection. A summary of advice for patients is provided for physicians to give to their patients. PMID- 8620271 TI - Ampicillin-specific rashes. AB - Ampicillin is one of the most common drugs to elicit a rash, with an overall incidence of 3% to 8%. "Ampicillin-specific" rashes are thought to be nonhypersensitivity reactions and cause maculopapular erythema with minimal irritation or pruritus. If the rash is indeed an ampicillin-specific one, then discontinuation of ampicillin is not mandatory, and subsequent use of ampicillin or other beta-lactam antibiotics is tolerated. On the other hand, true hypersensitivity reactions with urticarial and anaphylactic properties demand prompt discontinuation of the drug and warrant supportive care. Unfortunately, there is no immediate definitive scientific method to differentiate between the two. PMID- 8620270 TI - Adherence to single daily dose of aspirin in a chemoprevention trial. An evaluation of self-report and microelectronic monitoring. AB - A consecutive sample of 64 healthy adults (33 female and 31 male) were recruited at the University of Michigan Medical Center, Ann Arbor. Data were available for analysis on 57 subjects. The participants were asked to take a single daily dose of aspirin ranging from 0 to 640 mg. Adherence to the daily aspirin ingestion was measured by self-report and the Medication Event Monitoring System (MEMS, Aprex Corp, Fremont, Calif); adherence rate for the study population was 35%. The adherence rates for all dosing errors between self-report and Medication Event Monitoring System were significantly different (P = .002). There was no significant gender difference in adherence rates. Adherence to regular aspirin ingestion was poor in healthy, paid subjects despite explicit, written and verbal instructions. Patient self-report alone is not a reliable measure of adherence. PMID- 8620272 TI - Preliminary experience with split liver transplantation. AB - BACKGROUND: The purpose of split liver transplantation is to alleviate the organ shortage for patients with end-stage liver disease. The procedure, however, has not gained wide acceptance. This is related not only to the complexity of the procedure but also to poorer results and the complications reported to be associated with the technique. STUDY DESIGN: We report 12 split liver transplantation procedures, seven in children and five in adults. Selection criteria were the same as those for whole-size liver transplantation. Patient and graft survival as well as complications were analyzed. Results were analyzed by Wilcoxon life tables. RESULTS: Patient and graft survival rates are 91.6 and 75 percent, respectively. One patient died at 2.5 months after transplantation because of lymphoproliferative disease. Another had acute vanishing bile duct syndrome and required retransplantation at 1.5 months. One patient had retransplantation because of hepatic artery thrombosis. Bile leaks occurred in two patients and hemothorax in one patient. CONCLUSIONS: Our results indicate that split liver transplantation has become a more acceptable method of hepatic transplantation and should be encouraged. Several guidelines can enhance success rates. PMID- 8620273 TI - Type I gastric ulcer treated by parietal cell vagotomy and mucosal ulcerectomy. AB - BACKGROUND: Type I gastric ulcers occur at the gastric incisura and do not coexist with duodenal or pyloric ulcers. Antrectomy and Billroth I anastomosis are the most frequent operations used for treatment of patients with this lesion. STUDY DESIGN: Postoperative results, including recurrence, were evaluated in 48 patients with a Type I gastric ulcer who were treated by parietal cell vagotomy and mucosal excision of the ulcer and had a mean follow-up of eight years. RESULTS: There was no operative mortality and no major operative complications occurred. The patients have had follow-up examination for a mean of eight years. All but four patients were in Visick I and II categories when last examined. Four patients were in category IV because they required a second gastric operation. The cumulative probability of recurrent ulcer rate calculated by life table analysis was 6.5 plus or minus 9.5 (standard error of the mean) percent at nine years. CONCLUSIONS: Parietal cell vagotomy and ulcerectomy is an excellent operation for patients with Type I gastric ulcers and provides an alternative to antrectomy for patients with this lesion. PMID- 8620275 TI - Meropenem versus tobramycin with clindamycin in the antibiotic management of patients with advanced appendicitis. AB - BACKGROUND: Meropenem (MP), a new carbapenem antibiotic, has excellent antimicrobial activity against the enteric flora commonly encountered in acute appendicitis. Although similar to imipenem, it may have clinical advantages. STUDY DESIGN: We compared patients with advanced appendicitis (gangrenous or perforated) treated with 1,000 mg MP every eight hours with those given the combination of tobramycin 5 mg/kg/day at eight hour intervals and clindamycin 900 mg every eight hours. Both treatments were given intravenously. Patients were randomized to either group of the double-blind study. RESULTS: Of 129 evaluable cases, 63 received MP and 66 received both tobramycin and clindamycin (T/C). The two groups were similar in age, sex, and severity of disease. The mean number of days of postoperative fever (MP = 3.1 +/- 1.7 SD compared to T/C = 4.4 +/- 2.2 SD, p < or = 0.01), days of antibiotic therapy (MP = 6.1 +/- 1.6 SD compared to T/C = 7.3 +/- 2.2 SD, p = 0.01), and therefore hospital stay (MP = 8.0 +/- 3.5 SD compared to T/C = 9.4 +/- 2.6 SD, p < 0.01) were significantly better for patients treated with MP. No difference was found between the numbers of failures in each group (MP = 5 compared to T/C = 6). CONCLUSIONS: This study demonstrates a small but significant reduction (approximately one day) in post-operative fever, duration of antibiotic treatment, and hospital stay for patients treated with MP compared to those treated with T/C. PMID- 8620274 TI - A review of 240 patients undergoing distraction osteogenesis for congenital post traumatic or postinfective lower limb length discrepancy. AB - BACKGROUND: We reviewed 281 lower limb lengthenings in 240 patients treated at three centers for congenital, post-traumatic, or postinfective limb length discrepancy (LLD) in the period 1984 to 1992. STUDY DESIGN: A retrospective review was done of hospital charts and radiographs of patients who had completed lengthening at least 12 months prior to the beginning of the study. The variables studied included patients' age at operation, the bone segment lengthened, whether a corticotomy or an osteotomy had been done, the amount of lengthening planned and achieved, the level(s) of corticotomy or osteotomy, the external fixator used, and the difficulties encountered. RESULTS: Limb length discrepancy was reduced within 2 cm (1.6 percent or lower) of the normal contralateral leg in 249 (89 percent) lengthenings in 208 patients. The average time spent with the fixator in situ was 186.4 days (range, 94 to 617 days), with an average healing index of 35.3 days per cm (range, 26 to 43 days per cm). Femoral osteotomies or corticotomies healed faster than tibial osteotomies or corticotomies. The healing index in post-traumatic and postinfective LLD was significantly lower than in congenital conditions. The younger patients showed a significantly decreased time to bone healing and fewer complications. CONCLUSIONS: A significantly greater number of difficulties were found in patients whose lengthening exceeded 18 percent of the original length of bone. Bifocal lengthenings healed significantly faster than single level ones. The three external fixators used were equally effective for lengthenings of less than 20 percent. The Ilizarov and the Monticelli-Spinelli circular fixators were associated with a significantly decreased number of complications when lengthenings above 20 percent were performed. PMID- 8620277 TI - Management of recurrent and perivascular femoral hernias by giant prosthetic reinforcement of the visceral sac. AB - BACKGROUND: Classical hernioplasties have been used to manage primary femoral hernias for over a century. In women, infrainguinal repair of the parietal defect is simple and successful. In men, femoral hernias are frequently associated with inguinal hernias and, therefore, a Cooper's ligament repair is indicated. For recurrent femoral hernias, however, the classical hernioplasties are often inadequate just as they are for the repair of recurrent inguinal hernias and a prosthetic repair is indicated. Giant prosthetic reinforcement of the visceral sac (GPRVS) is the descriptive name of a properitoneal groin hernioplasty with a large piece of Mersilene. The repair focuses on retaining the peritoneum rather than repairing the parietal defect and is efficient, anatomic, sutureless, and tension-free. It is the only repair that reliably eliminates all hernias of the groin, including perivascular femoral hernias. STUDY DESIGN: In this study, GPRVS by way of an abdominal incision was used to treat recurrent and perivascular femoral hernias. Also included are a description of and experiences with a new technique of unilateral GPRVS performed through an infrainguinal approach. RESULTS: The data reveal no recurrences in 69 problem femoral hernias of which 15 were primary (two perivascular) and 54 recurrent (four perivascular). CONCLUSIONS: Giant prosthetic reinforcement of the visceral sac performed transabdominally or by way of the newly described infrainguinal method is a useful and reliable method to treat primary, recurrent and perivascular femoral hernias. PMID- 8620276 TI - Routine preoperative infusion cholangiography versus intraoperative cholangiography at elective cholecystectomy: a prospective study in 995 patients. AB - BACKGROUND: There has been a resurgence of interest in recent years in preoperative infusion cholangiography (PIC). The role of routine PIC compared to routine intraoperative cholangiography (IOC) has not been clearly defined. STUDY DESIGN: In our department between 1985 and 1991, 1,042 of 1,576 consecutive patients with biliary calculous disease had elective cholecystectomy: 694 patients were prospectively scheduled for PIC, and 348 patients were randomly allocated to IOC. The patients in the PIC and IOC groups were similar with regard to age, history of biliopancreatic complications, and laboratory findings. The cost of PIC in Sweden is nearly five times greater than the cost of IOC. RESULTS: Satisfactory opacification of the biliary system was obtained in 90.1 and 96.8 percent of patients who underwent PIC and IOC, respectively. Preoperative infusion cholangiography required support by IOC in 19.5 percent of patients. There were no statistically significant differences between the PIC and IOC groups with regard to the incidence (7 percent in both groups) of or positive predictive value (68 and 80 percent, respectively) for bile duct stones, rate of retained stones (6 and 20 percent, respectively), intraoperative (5.6 and 6.3 percent, respectively) or postoperative (13.3 and 15.9 percent, respectively) morbidity, or incidence of bile duct anomalies (0.9 and 0.3 percent, respectively). Median operative time was longer in patients with (95 minutes) compared to those without (75 minutes) IOC (p < 0.001). More postoperative complications occurred after bile duct exploration (26 of 75 patients) compared to cholecystectomy alone (114 of 917 patients, p < 0.001). The 30-day mortality was zero. Minor bile duct injuries occurred in two patients (0.2 percent) at cholecystectomy, (one with and one without bile duct exploration). In no patient was the cholangiographic finding of a biliary anomaly crucial for the safe execution of cholecystectomy. CONCLUSIONS: In our study, PIC and IOC were comparable, but routine use of either method did not promote the safety of cholecystectomy and thus their routine use is not warranted. The shorter operative time and preoperative identification of common bile duct (CBD) stones provided by PIC might favor this examination when applied selectively in patients with increased risk of having CBD stones. However, this potential advantage is offset by the need for PIC to be supported by IOC in approximately 20 percent of patients. Also, the cost of PIC is greater than the cost of IOC. PMID- 8620278 TI - Evaluation of tissue trauma after laparoscopic and abdominal hysterectomy: measurements of neutrophil activation and release of interleukin-6, cortisol, and C-reactive protein. AB - BACKGROUND: Trauma and major surgery stimulate a cascade of events that mediate the inflammatory response. The aim of our study was to determine whether or not hysterectomy leads to release of cytokines, cortisol, and C-reactive protein (CRP), activation of neutrophils, and activation of the complement cascade. A further aim was to compare laparoscopic and abdominal hysterectomy with regard to the same parameters. STUDY DESIGN: Twenty-four consecutive patients were randomized to either abdominal (n = 12) or laparoscopic hysterectomy (n = 12). Blood samples were drawn preoperatively, intraoperatively, and then at one minute, 24 hours, and seven days postoperatively. Interleukin-6 (IL-6) levels were used to evaluate cytokine release, cortisol and CRP to evaluate the inflammatory response, and polymorphonuclear (PMN) elastase to detect neutrophil activation. To evaluate complement activation, the terminal C5b-9 complement complex (TCC) was determined. RESULTS: Interleukin-6 concentrations were significantly elevated one minute and 24 hours postoperatively in both groups. Independent of the surgical technique or operative time, the highest IL-6 concentration was reached four hours after beginning the operation. Cortisol levels were significantly elevated during and after the operation in both groups. C-reactive peptide levels were significantly elevated in both groups 24 hours and seven days after the operation. Polymorphonuclear elastase was elevated 24 hours postoperatively in both groups. There were no signs of complement activation during the operative period or postoperatively in either patient group. CONCLUSIONS: Our results indicate serious tissue trauma during both laparoscopic and abdominal hysterectomy. The extent of surgical trauma did not differ between the two operative methods. PMID- 8620279 TI - Good Samaritan surgeon wrongly accused of contributing to President Lincoln's death: an experimental study of the President's fatal wound. AB - BACKGROUND: When President Abraham Lincoln was shot in the back of the head at Ford's Theater in Washington, D.C., on April 14, 1865, he was immediately rendered unconscious and apneic. Doctor Charles A. Leale, an Army surgeon, who had special training in the care of brain injuries, rushed to Lincoln's assistance. When Doctor Leale probed the wound in Lincoln's thickened scalp, feeling for the bullet, he dislodged a blood clot, and Lincoln began to breathe again. However, Lincoln progressively deteriorated and died at 7:22 AM on April 15, 1865. During the postmortem examination of Lincoln's body, numerous secondary missiles of bone and metal were found in the track of pultaceous brain tissue, extending completely through the brain to the front of the skull. In February 1995, an article in a popular magazine alleged that Doctor Leale had caused further (fatal) damage to Lincoln's brain by thrusting his finger into the brain through the bullet hole. The article alleged (wrongly) that most bullet wounds of the brain incurred in Civil War times were not fatal. STUDY DESIGN: The following study demonstrates that it is impossible to introduce even the tip of the little finger through a hole in the skull resulting from a .41-caliber bullet fired from a derringer. In our study, a .41-caliber derringer was used to fire bullets into numerous fresh skulls; the bullet holes all had razor-sharp edges and were much too small to accommodate a fingertip. RESULTS: Thus, the allegation that President Lincoln's brain was damaged further because Doctor Leale thrust his finger through the bullet hole into the brain parenchyma is not valid. In this study, experimental data are presented to demonstrate the foregoing point. CONCLUSIONS: The wound made by John Wilkes Booth's derringer ball in Lincoln's brain was devastating; it was clearly the cause of his death. Good Samaritan surgeon Leale has been falsely accused of contributing to Lincoln's death. PMID- 8620280 TI - Split liver transplantation: can it fulfill its potential? PMID- 8620281 TI - Treatment of type I gastric ulcer. PMID- 8620282 TI - Flexible self-retaining blades to facilitate "mini-incision" surgery. PMID- 8620283 TI - Loop enterostomy in newborns with necrotizing enterocolitis. PMID- 8620284 TI - Stapled laparoscopic splenectomy: initial experience. PMID- 8620285 TI - Malpractice litigation involving patients with carcinoma of the breast. PMID- 8620286 TI - Method for image-based measurement of the reversible and irreversible contribution to the transverse-relaxation rate. AB - A multislice NMR imaging pulse sequence capable of measuring both the reversible and irreversible contribution to the transverse-relaxation rate (R'2 and R2) in a single scan is described. The method, termed GESFIDE (gradient-echo sampling of FID and echo) is based on sampling the descending and ascending portions of a Hahn echo with a train of gradient echoes. R2 and R'2 are computed by exploiting the differential evolution of the transverse magnetization before and after the phase-reversal pulse. Salient features of the method are its insensitivity to RF pulse imperfections and its high precision and efficiency. Applications examined involve the characterization of magnetically inhomogeneous tissues and biomaterials such as trabecular bone marrow in the skeleton and brain iron. PMID- 8620287 TI - Three-dimensional solid-state NMR correlation experiment with 1H homonuclear spin exchange. PMID- 8620288 TI - Correcting NOESY cross-peak intensities for partial relaxation effects enabling accurate distance information. PMID- 8620289 TI - Measurement of microwave-induced heating of mammary tumors in animal models using cobalt NMR. PMID- 8620290 TI - DOSY-NOESY: diffusion-ordered NOESY. PMID- 8620291 TI - Undergraduate education in rheumatology. PMID- 8620292 TI - Sjogren's syndrome revisited: autoimmune epithelitis. PMID- 8620294 TI - Bone resorption by tartrate-resistant acid phosphatase-positive multinuclear cells isolated from rheumatoid synovium. AB - Inflammatory reactions in rheumatoid arthritis (RA) often cause severe joint destruction. However, the mechanism of bone destruction is still a matter of controversy. To determine whether multinuclear cells found in the rheumatoid synovium can resorb bone, isolated synovial cells were assessed for tartrate resistant acid phosphatase (TRAP) staining and the ability to resorb bone in a dentine resorption assay. TRAP-positive multinuclear cells were found in six out of 10 samples. These six samples showed resorption pit formation on dentine slices. The other four samples did not form resorption pits. The results of this study demonstrate that TRAP-positive multinuclear cells isolated from the rheumatoid synovium form resorption pits on dentine slices. Our results suggest that inflamed synovial cells in rheumatoid joints might participate in bone destruction. PMID- 8620293 TI - Nitric oxide production in cells derived from the human joint. AB - We have investigated the ability of cells derived from the human joint to generate nitric oxide (NO). Synovial fibroblasts, articular chondrocytes and osteoblasts were cultured from tissues of patients undergoing hip replacement surgery, and synovial fluid leucocytes were obtained from patients undergoing joint aspiration. There was little spontaneous generation of NO by any of the cells after culture, but synovial fibroblasts, articular chondrocytes and osteoblasts all produced large quantities of NO in response to a cytokine mix of interleukin (IL)-1 beta + tumour necrosis factor alpha (TNF alpha) + interferon (IFN gamma). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of mRNA transcripts for the inducible isoform of NO synthase in cytokine-stimulated but not in unstimulated cells. In contrast, leucocytes from synovial fluid did not produce NO either spontaneously or after cytokine stimulation, and mRNA for inducible NO synthase (iNOS) was not detected in these cells even by nested PCR. There were significant differences in the regulation of NO production between chondrocytes and other cells. Only chondrocytes generated NO in response to IL-1 beta or TNF alpha alone, whereas synovial fibroblasts and osteoblasts required the presence of at least two cytokines to generate NO. Dexamethasone (10(-6)M) had a small but significant inhibitory effect on NO production by chondrocytes, synovial fibroblasts and osteoblasts. Our results indicate that several cells within the human joint have the potential to generate NO in the presence of an appropriate pro-inflammatory cytokine stimulus, while leucocytes in synovial fluid are not a significant source of NO. The data support suggestions that NO is produced within the inflamed joint in diseases such as rheumatoid arthritis. PMID- 8620295 TI - Adhesion of rheumatoid lymphocytes to mucosal endothelium: the gut revisited. AB - By a study of the adhesion of rheumatoid mononuclear cells, we have sought to clarify the homing properties and origins of cells likely to be involved in the pathogenesis of this disease. The adhesion of mononuclear cells from patients with rheumatoid arthritis (RA) was enumerated by an in vitro adherence assay using frozen sections of endothelium-containing gut lamina propria (EGLP) from porcine small intestine. Preliminary studies verified the involvement of known adhesion molecules by inhibition assays using monoclonal antibodies Meca-367, Mel 14 and Hermes-3. Twenty-five paired samples of peripheral blood (PB) and synovial fluid (SF) were studied, plus six from synovial membrane (SM) and eight from patients with other diseases. There was a significantly greater degree of adherence to EGLP by SF cells than PB (mean adherence 266 +/- 22 cells/mm(2), compared to 136 +/- 13 cells/mm(2), respectively, the majority of which were CD8+ cells; P=0.02, Mann-Whitney U-test for 25 paired samples). The results of the monoclonal antibody inhibition assays were in keeping with the involvement of homing receptors to gut endothelium in our assay system. Synovial fluid lymphocytes from RA patients exhibited adhesion properties more in keeping with lymphocytes of mucosal than of lymph node origin. Synovial membrance lymphocytes, by contrast, showed poor adherence to endothelium-containing lamina propria. The gut, as an immune lymphoid organ, may thus play a contributory role in this disease, possibly through the pathological seeding of cells into the synovial space. PMID- 8620296 TI - Active systemic lupus erythematosus is associated with the recruitment of naive/resting T cells. AB - The aim of this study was to determine whether active systemic lupus erythematosus (SLE) is associated with recruitment of resting CD45RA+ T cells or reactivation of CD45RO+ memory T cells. Three-colour immunofluorescence was used to determine CD45 isoform expression by CD4+ T cells from 28 patients with SLE. Newly recruited and highly differentiated primed T cells were distinguished by their CD45RB expression. The pattern of CD45 isoform expression varied directly with time since the onset of symptoms in patients with active SLE. Shortly after symptoms appeared, most cells were CD45RA+ resting cells or CD45RO(dull)RB(bright) early primed cells. However, over the course of active disease, patients accumulated CD45RO(bright)RB(dull) cells which represent an advanced state of differentiation. The switch from an early to late primed phenotype correlated significantly with time since the onset of symptoms. The recruitment of resting T cells in active SLE, rather than the simple reactivation of existing memory clones, has implications for understanding the pathology of this disease and for treating it. PMID- 8620297 TI - CAMPATH-1H in rheumatoid arthritis--an intravenous dose-ranging study. AB - Forty-one patients with active and refractory rheumatoid arthritis(RA) received a total of 100, 250 or 400 mg of CAMPATH-1H (CAMPATH is a trademark of Glaxo Wellcome group companies, registered in the US Patent and Trademark Office) over 5 or 10 days in an open, uncontrolled study. Following therapy, patients were monitored for adverse effects and disease activity for 6 months. Therapy was associated with prolonged peripheral blood lymphopenia in all dosing cohorts. During the month immediately following therapy, lymphopenia was most profound in the 400 mg cohorts. The first dose of monoclonal antibody (Mab) was associated with a 'flu'-like syndrome, more pronounced at higher initial doses. One patient developed haemolytic-uraemic syndrome. There were a number of dose-related infections during the early post-treatment period and one fatal opportunistic infection which followed additional immunosuppressive therapy. Antiglobulin responses developed in 9 of 31 patients tested. The majority of patients showed symptomatic improvement following therapy and 20% of patients maintained a 50% Paulus response at 6 months, all of whom were in the 250 or 400 mg cohorts. CAMPATH-1H appears to be an effective treatment for RA. Allowing for the small number of patients treated, infections were more common with higher doses, although this was not true for adverse events overall, and therapeutic responses were more sustained at higher dosing levels. The broad specificity of CAMPATH-1H may be appropriate for the immunotherapy of RA and future studies should aim to define a dose with an optimal therapeutic ratio. PMID- 8620298 TI - Arthritis associated with monoclonal gammapathy: clinical characteristics. AB - We report nine cases of arthritis associated with a monoclonal gammapathy. Joint involvement was noted simultaneously or after the diagnosis of monoclonal gammapathy was made. The cases had oligoarthritis or polyarthritis mimicking rheumatoid arthritis. However, rheumatoid factor was absent in all patients, and distal interphalangeal joints were involved in two cases and sacroiliitis in one. The plasma cell dyscrasia was a multiple myeloma in two cases and monoclonal gammapathy of undetermined significance in the other patients. The light chain isotype was kappa in eight of our patients. A type I cryoglobulinaemia was associated in four cases; it was detected in the synovial fluid of two of them. We suggest that the occurrence of paraproteinaemia with chronic arthritis is more than a chance association. Moreover, a monoclonal gammapathy should be searched for in patients presenting with atypical seronegative arthritis. PMID- 8620299 TI - Trabecular and cortical bone loss in systemic lupus erythematosus. AB - We measured lumbar spine, hip (total and sub-regions) and total body bone mineral densities (BMDs) by dual-energy X-ray absorptiometry (DXA) in 47 premenopausal female patients suffering from systemic lupus erythematosus (SLE). As compared to health controls, SLE patients had lower BMDs at all trabecular and cortical sites. Comparison of BMDs between patients ever and never treated with glucocorticoids indicated that patients who had ever received glucocorticoids had a significantly lower lumbar spine BMD compared to those who never did. Moreover, bone loss in patients ever treated with glucocorticoids was commensurate with their cumulated oral glucocorticoid intake. Interestingly, patients never treated with glucocorticoids had a lower hip BMD compared to controls, thereby suggesting that the disease per se might induce some bone loss. Taken together, SLE patients suffer from a significant trabecular and cortical bone loss indicative of an increased risk of future fracture. PMID- 8620300 TI - SLICC/ACR Damage Index is valid, and renal and pulmonary organ scores are predictors of severe outcome in patients with systemic lupus erythematosus. AB - We investigated the Systemic Lupus International Collaborative Clinics/American College of Rheumatology (SLICC/ACR) Damage Index as a predictor of severe outcome and an indicator of morbidity in different ethnic groups, and in regard to its validity. We retrospectively studied disease course within 10 yr of diagnosis in an inception cohort of 80 patients with systemic lupus erythematosus (SLE). The mean renal damage score (DS) at 1 yr after diagnosis was a significant predictor of endstage renal failure and the mean pulmonary DS at 1 yr significantly predicted death within 10 yr of diagnosis. Compared to Caucasians, Afro Caribbeans and Asians had significantly higher mean total DS at 5 and 10 yr, and higher mean renal DS at 10 yr. At 5 yr, the mean renal DS in Afro-Caribbeans and the mean neuropsychiatric DS in Asians were significantly higher than in Caucasians. The rate of endstage renal failure in Caucasians was significantly lower than in the other ethnic groups. Our results confirm the validity of the SLICC/ACR Damage Index. PMID- 8620301 TI - Serum cholesterol and risk of rheumatoid arthritis in a cohort of 52 800 men and women. AB - Recent epidemiological studies have suggested that joint risk factors occur for rheumatoid arthritis (RA) and coronary heart disease. We studied serum cholesterol concentration for its association with the incidence or RA in 28 362 men and 24 444 women free from arthritis at baseline. During a mean follow-up of 21 yr, 161 men and 351 women developed RA. Of these incident cases, 119 men and 229 women were rheumatoid factor (RF) positive. The serum cholesterol concentration was directly proportional to the risk of RF-positive RA among women and RF-negative RA among men; the age-adjusted relative risks (95% confidence intervals) per S.D. (1.4 mM/l) of the cholesterol distribution were 1.20 (1.05 1.38) and 1.56 (1.15-2.10), respectively. No association was observed, however, for RF-negative RA among women or RF-positive RA among men. The results suggest that a still unknown factor closely associated with serum cholesterol may be involved in the aetiology of RA, but complex interactions with sex and RF status seem to occur. PMID- 8620302 TI - Rheumatic disorders of the eye and the various structures involved. PMID- 8620303 TI - Rheumatology and medical education in Great Britain. AB - Rheumatology has been relatively under-represented in UK medical school curricula to date. The incidence of rheumatic disease in the community is not reflected by the amount of time spent on it in undergraduate medicine. In addition, the emphasis in medical colleges is on the less common conditions like systemic lupus erythematosus and vasculitis, rather than the commoner treatment of sore shoulders and backs. This article reviews the current changes in the philosophy of medical education in the UK and the response of the General Medical Council of Great Britain towards updating curricula. It explains some of the new teaching and assessment methods being increasingly used in today's medical colleges, and encourages rheumatologists to become actively involved in teaching and curricular reform. PMID- 8620304 TI - Quality of life measures. AB - Quality of life measures have become increasingly popular as outcome measures despite the lack of consensus on a definition of quality of life. This review describes the most frequently used measures, and discusses the conceptual and measurement issues surrounding quality of life measurement. Finally, it tries to place quality of life in the World Health Organization's model of disease impact. PMID- 8620305 TI - Hyper-IgM syndrome associated with rheumatoid arthritis: report of RA in a patient with primary impaired CD40 pathway. AB - We studied a patient who had a typical seronegative rheumatoid arthritis (RA) and an immunodeficiency with hyper-IgM (HIM syndrome). CD40L was normally expressed by activated T cells, but CD40-mediated signal transduction was defective in B cells, preventing heavy chain switching (CD40L+ type of the HIM syndrome). These data suggest that a typical RA can develop in at least some patients with dysfunction of the CD40 pathway, i.e. in the absence of a normal co-operation between T and B cells. Accordingly, the blockade of CD40L-CD40 interactions, which has been proposed as a treatment of RA, might not be adapted to all patients. PMID- 8620306 TI - Iliopsoas bursitis-an unusual presentation of metastatic bone disease. AB - Diagnostic uncertainty concerning the nature of an enlarging inguinal mass in an elderly male with a short history of hip pain was resolved by a combination of ultrasound and magnetic resonance imaging (MRI). Subsequent investigations showed that the enlarged iliopsoas bursa, which contained a number of atypical cells, was an unusual presenting feature of a destructive metastatic lesion in the right hip. PMID- 8620307 TI - Thyrotoxicosis and renal tubular acidosis presenting as hypokalaemic paralysis. AB - A 34-yr-old Chinese woman presented with hypokalaemic periodic paralysis. She had a goitre and was biochemically thyrotoxic. However, she also had urinary potassium loss with a metabolic acidosis and reduced ability to acidify her urine. The co-existence of distal renal tubular acidosis (RTA) was confirmed. There was no evidence of xerostomia or xerophthalmia, although anti-Ro antibody and rheumatoid factor were positive. Paralytic attacks did not recur after the thyrotoxicosis was controlled with radioactive iodine. Possible pathogenic mechanisms for the association of these disorders are discussed. Female patients presenting with thyrotoxic periodic paralysis (TPP) should be thoroughly investigated for possible additional precipitating factors in view of the strong male predominance of TPP, particularly when there are atypical metabolic features. PMID- 8620308 TI - Clubbing in human immunodeficiency virus infection. AB - We describe two patients who developed clubbing shortly after HIV seroconversion. None of them had the full syndrome of hypertropic osteoarthropathy. Other causes of clubbing are unlikely. We suggest that clubbing may belong to the spectrum of arthropathies associated with HIV infection. PMID- 8620310 TI - Osteitis condensans ilii. PMID- 8620309 TI - Successful treatment of Reiter's syndrome in a patient with AIDS with methotrexate and corticosteroids. PMID- 8620311 TI - Chronic polyarthritis and graft-versus-host disease. PMID- 8620312 TI - Pancreatic function in systemic sclerosis. PMID- 8620313 TI - Interference by IgM rheumatoid factor on ELISA for antibodies to mycobacterial 65 kDa heat shock protein in rheumatoid arthritis. PMID- 8620314 TI - Measuring outcome in rheumatoid arthritis--which measures are suitable for routine clinical use? PMID- 8620315 TI - [The dipeptide L-alanyl-L-glutamine--its preparation and therapeutic use]. AB - L-glutamine, representing one of the principal sources of nitrogen for the organism, can be used in infusion solutions employed in clinical nutrition in the form of the dipeptide L-alanyl-L-glutamine. It can be prepared with the use of biotechnological methods, viz. the enzymic synthesis of the peptide, and fermentation preparation of L-glutamine and the proteases catalyzing the formation of the peptidic bond. Suitable producers of amino acids and enzymes are microorganisms (both bacteria and fungi). The procedures used in the preparation of the components as well as the dipeptide itself are a combination of biotransformations and fermentations. PMID- 8620317 TI - [Pharmaceutical care abroad and in the Czech Republic]. AB - The position of the pharmaceutical service in the society of future generation will depend on the quality of pharmaceutical care. Its importance has followed from the requirements of the patients themselves. By accepting his new role, the pharmacist is able to influence the relationship of the patient to the drug in all situations when man encounters the drug: in the course of hospitalization, during the transition from hospital to domestic care, in domestic treatment, in dispensation of drugs supplied on medical prescription, and in the purchases of over-the-counter drugs. In order to cope with these tasks, the pharmacist must cooperate more intensively with a wider team of health workers of the primary and secondary care. There will be necessary changes in the hitherto style of work of pharmacies and both in the graduate and further education of pharmacists. The aim is a safe and rational use of drugs, prevention of wasting, and education of people to take the responsibility for their health. PMID- 8620316 TI - [Comparison of the therapeutic effectiveness of selected cholinesterase reactivators with atropine in acute fosdrine poisoning in mice]. AB - In experiments on male mice, the effect of the cholinesterase reactivators obidoxime, methoxime and HI-6 in combination with atropine sulfate on the acute intoxication with the organophosphorous insecticide fosdrine was tested in dependence on the period of administration of drugs after intoxication and on the dose of oxime by influencing the LD50 value in 48-hour survival of experimental animals. It has been demonstrated that the rate of the therapeutic intervention is a much more important factor influencing the effect of oximes than the dose of oximes. A shortening of the period of drug administration from 2 minutes to 30 seconds substantially increases the effects of all three oximes. A comparison of the effects of all three reactivators has shown that the oxime HI-6 is significantly more effective than the remaining two reactivators in the case of therapy of intoxication 30 seconds after the application of the noxa. In the therapy of intoxication 2 minutes after the exposure of experimental animals to fosdrine, the effect of the antidotal therapy was relatively low regardless of the selected oxime. PMID- 8620318 TI - [HPLC analysis of non-steroidal antirheumatic agents in biological material. I. Salicylates]. AB - The present paper surveys both published HPLC methods estimating salicylates in biological materials and an HPLC method for estimation of salicylic acid and its metabolites in the samples of whole blood, isolated erythrocytes and plasma. For liquid-liquid extraction, salicylates were analyzed on reverse phases Separon SGX C18 with the mobile phase methanol-water (pH 2,5) and detected at 236 nm. The elaborated method was used in a pharmacokinetic study of salicylates on rabbits. PMID- 8620320 TI - [Work presented at the Conference on Vasculitides, organized by the Slovak Immunology Society, the Slovak Angiology Society, the Slovak Allergology and Clinical Immunology Society and the Rheumatic Disease Research Institute. Piestany, 11-12 May 1995]. PMID- 8620319 TI - [Molecular and cellular mechanisms in inflammatory reactions]. AB - Inflammation is a complex stereotypical reaction of the body expressing the response to damage of its cells and/or vascularized tissues. It is the body's reaction to invasion by an infectious agent, antigen challenge or even just physical, chemical, or traumatic damage. The development of inflammatory reactions is controlled by a number of cellular and molecular components. Leukocytes (namely neutrophils and macrophages) belong to the key inflammatory cells. Their accumulation in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells within the microcirculation. The nature and magnitude of the adhesive interactions that take place within postcapillary venules are determined by a variety of factors, of which the contribution of different adhesion molecules (selectins, integrins, members of immunoglobulin superfamily) to leukocyte rolling, adherence, and emigration in venules is discussed. The main purpose of inflammation seems to be to bring fluids, proteins and cells from the blood into the damaged tissues for the elimination of injuring agent and triggering the healing and repairing processes. This is under the control of inflammation mediators which include vasoactive substances, proinflammatory and antiinflammatory cytokines, chemokines, acute phase reactants, bioactive lipids (prostanoids, platelet activating factor--PAF) and products of the plasma enzyme systems (complement, the coagulation clothing, kinin and fibrinolytic pathways) which are shortly reviewed. Several neuroendocrine hormones, neuropeptides, neurotransmitters and mainly glucocorticoids also play an important role of endogenous regulators of any inflammatory process. (Tab. 4, Fig. 1, Ref. 52.). PMID- 8620321 TI - Antineutrophil cytoplasmic autoantibodies (ANCA) associated vasculitides- immunologic and clinical aspects. AB - The author reviews the opinions on vasculitis pathogenesis which is based on the direct noxa impact, active immunity process against the specific components of the vascular tissue and/or passive impact of the inflammatory process upon vessels. The detection is especially based on two most important ANCA tests, i.e. indirect immunofluorescence and ELISA. The author includes also the sensitivity and specificity of these examinations and their predictive value in progressive glomerulonephritis, Wegener's granulomatosis and other diseases. (Tab. 1, Ref. 20.). PMID- 8620322 TI - [Adhesion molecules and cytokines in vasculitides]. AB - Renal vasculitis syndromes include particular characteristic changes in concentrations of some cytokines in plasma or urine. Preliminary results suggest that the systemic lupus erythematodes with affliction of the kidneys is specifically concomitted by the increase in IL-8, both in plasma and urine. ANCA positive renal vasculitis syndromes appear to coincide with a typical increase in the synthesis of interleukin-6 in the kidneys. We suggest that the monitoring of individual cytokine levels in plasma and urine will enable to study in greater detail the immunopathogenesis of renal vasculitis syndromes and the extent of local production of cytokines which may cause further progression of renal lesions. (Fig. 4, Tab. 1, Ref. 10.). PMID- 8620323 TI - [Diagnostic and pathogenic significance of antineutrophil cytoplasmic antibodies in vasculitides]. AB - OBJECTIVES: The study represents a complex report on diagnostic and pathogenic significance of antineutrophil cytoplasmic autoantibodies (ANCA). Our report is founded on the results of our clinical and experimental studies. METHODS: ANCA was examined by indirect immunofluorescence, or ELISA-anti MPO. The data for evaluation of diagnostic ANCA associations were gained by means of questionnaires. The experimental part involved examination of the impact of sera containing ANCA and monoclonal antibodies (MoAb) against proteinase 3 and control MoaB specific for neutrophils. RESULTS: Within the course of 1 year we have examined 1010 samples, ANCA was found in 65 patients: 26 cases of systemic vasculitis, 12 cases of other autoimmune diseases, 9 cases of isolated glomerulonephritis, 7 cases of inflammatory intestinal diseases, 6 cases of ophthalmic inflammatory diseases, and 6 cases of other diseases. Vasculitis of small vessels found in our group of patients are concomitted by ANCA positivity in 90% (Wegener's granulomatosis 15/16, Churg--Strauss's syndrome 3/5, MPA 9/9), vasculitis of medium vessels 12% (classical polyarteritis nodosa 1/2, Kawasaki disease 0/2, Henoch--Schonlein's purpura 0/4, Takayasu's arteritis 1/4). The ANCA containing sera inhibit the candidacidal activity by 65% in average. THe inhibitory impact is yielded by anti-proteinase MoAb, in contrast to the control MoAb. CONCLUSION: c-ANCA in high concentration is specific for active form of Wegener's granulomatosis, other types of ANCA in low titres are not of diagnostic value for vasculitis. ANCA is concomitted especially by small vessels vasculitis. The proved inhibition of microbicidal PMN activity by ANCA can participate in some clinical signs of immunodeficiencies in vasculitis. (Tab. 5, Ref. 18.). PMID- 8620324 TI - [Classification and pathogenesis of vasculitides]. AB - Vasculitis is a heterogeneous group of diseases which is characteristic by inflammatory cellular infiltration of the vascular wall often being concomitted by fibrinoid necrosis. The pre-condition for stating the diagnosis is the primary localization of inflammation in the vascular wall. Affliction of vessels can be focal or segmental. Focal vasculitis afflicts merely a part of the vessel's wall perimeter, and aneurysms may develop. Segmental vasculitis refers to a lesion of the entire perimeter of the vascular wall which often leads to stenosis or occlusion of the vessel. Non-specific histologic findings of perivascular infiltrates cannot be considered as evidence of vasculitis. The authors present the classification of vasculitis, its development and relation to etiopathogenesis of the disease, including histopathology and clinical symptoms. (Fig. 1, Tab. 9, Ref. 43.). PMID- 8620325 TI - [Vasculitides of the coronary arteries]. AB - It is generally accepted that myocardial ischemia, and its extreme consequence, acute myocardial infarction, can result from transient or permanent disproportion between myocardial oxygen demand and coronary artery blood supply. Insufficient coronary artery blood supply may have many reasons. The aim of the study is to point to the clinical features of the coronary vasculitides as well as to the diagnostic and therapeutic possibilities. Coronary artery involvement in infectious angiitis, in Takayasu's arteritis, in granulomatous giant cell arteritis, in thromboangiitis obliterans, in polyarteritis nodosa, in Wegener's granulomatosis and in Churg--Strauss syndrome is discussed. The diagnosis of coronary vasculitis must be supposed in every patient with primary or secondary vasculitis in whom chest pain or cardiac failure appear. In young patients with clinical, electrocardiographic or laboratory signs of coronary artery disease, especially in absence of risk factors for atherosclerosis, the diagnosis of coronary vasculitis must be considered in differential diagnosis. (Fig. 4, Tab. 1, Ref. 32.). PMID- 8620326 TI - [Angiography in the diagnosis of vasculitides]. AB - A review of angiographic findings in vasculitides classified according to Lie is introduced. Angiography is an important investigation in angiitis that affects large, medium and visualizible small vessels. Its importance resides in 1. diagnosis, 2. selection of adequate therapeutic intervention. This work includes the description of angiographic findings in Takayasu's arteritis and Buerger's disease. (Fig. 4, Ref. 26.). PMID- 8620327 TI - [Cutaneous symptoms in vasculitides]. AB - Vasculitis has a very variable dermal manifestation. It includes purpura, pustules, bullae, ulcers, nodosits, pomphi etc. Dermal manifestation can represent the initial signs in systemic vasculitis and therefore its early clinical and histopathologic evaluation represents a presupposition for the determination of the subsequent examination route. The study gives information on general morphogenesis, principles of the correctly performed probatory excision, and clinical and histopathologic patterns of individual vasculitis types. (Tab. 2, Ref. 8.). PMID- 8620328 TI - [General therapeutic approaches in systemic vasculitides]. AB - The paper presents general therapeutic principles applied in systemic vasculitis. Several factors may help to contain the clinical activity, such as the location and extent of the inflammatory process affecting the vascular system, the ultimate narrowing of the vascular lumen with subsequent ischemia of the affected tissue and organ. Treatment of vasculitis involves besides glucocorticoids not only cytotoxic drugs (cyclophosphamide, chlorambucil, methotrexate), and immunomodulatory therapeutic agents with immunosuppressive action (cyclosporin A) but also other immunomodulatory drugs, as e.g. dialyzed homogenate of leukocytes (DHL), pentoxyphylline, hydrolytic enzymes, and monoclonal antibodies. The authors emphasize the importance of a complex approach in the management of systemic vasculitis. (Tab. 1, Ref. 36.). PMID- 8620329 TI - [Systemic enzyme therapy in diseases of the vascular system]. AB - The treatment of autoimmune and immune complex diseases of the vascular bed consists--similarly as of immunopathologic processes of other systems--in the use of risky immunosuppressive agents and antiinflammatory as well as symptomatic therapy. In the article the author informs about the possibility to use in these indications (and in addition also in other angiologic diseases) the systemic enzyme therapy, residing in the oral application of high-dosed combinations of several animal and plant proteolytic enzymes. About four tens years of positive medical empirical experience have been supported by a concentrated sophisticated research and approximately 150 clinical studies according to GCP. These revealed in most autoimmune and immune complex diseases a surprisingly high effectiveness and complete harmlessness of the enzyme therapy. After the short introduction mentioning the important indications for enzyme therapy in the field of clinical immunology, the major attention is paid to the results of enzymotherapy in angiology. Strong evidence indicates that enzymotherapy ameliorates the disturbed composition and properties of blood and vessel walls, acts preventively as well as therapeutically in thromboses, thromboflebitides and consequences of venous insufficiency; it seems be prospective in afflictions of arterial bed, including vasculitides and glomerulonephritides, also. The key feature of enzymotherapy is the immunomodulatory activity. There exists a strong evidence for the favourable modulation of pathogenic autoantibodies, inhibition of the neogenesis of immune complexes and cleavage of their deposits, normalization of the T cell system, network of cytokines, adhesion molecules and inflammatory cascades. Besides the direct peptidolytic and proteolytic effects of hydrolases, the indirect effects realized in the course of interaction between the resorbed enzymes and their natural "partners"--antiproteases (mainly alfa-2-macroglobulin)--have become a topic of intensive research. The author feels, that systemic enzyme therapy should become a regular component of the treatment of immunopathologic processes in general and of angiologic diseases specially. (Ref. 42.). PMID- 8620330 TI - [Surgical aspects of Buerger's disease]. AB - BACKGROUND: The Buerger's disease (BD) is known for more than 100 years. It inflicts especially young men, smokers, at the age of about 40 and manifests itself by ischemia in the periphery of limbs which cannot be explained by precedent injury, embolism, diabetes or hyperlipemia. The disease inflicts the medium or small peripheral arteries of extremities. The occurrence of BD is most frequent in the Mediterranean and East-Asian countries. The therapy is so far symptomatic and is unsuccessful in cases when the patient is reluctant to cease smoking. AIM: The study is aimed at ascertainment of the results of surgical therapy in 14 patients with BD hospitalized in the Surgical Clinic FN in Plzen during the period from 1986 to 1995. METHODS: The average age of patients was 39.6 +/- 1.2 y. The ratio males/females was 13:1. All patients were smokers. 4 patients had a trophic defect in the periphery of the upper and 11 of the lower limbs. 9 (64.3%) patients displayed the Raynod's phenomenon and seven (50%) had thrombophlebitis. All patients were treated conservatively (vasodilators, anticoagulants, antiaggregants, corticoids, prostacyclins, nifedipine). Sympatectomy (lumbal, upper thorax) was performed in 10 (71.4%), and profundoplasty in 2 patients (14.3%). RESULTS: Conservative therapy, sympatectomy and revascularization methods failed to have a long-term effect. Repeated hospitalization of the patients was necessary, namely 7 times in average, and limbs were amputated in high percentage of patients (50%). However in cases when patients ceased smoking, the disease withdrew or became stabilized. CONCLUSION: Despite the fact that BD has become less frequent in our population, it still disables young groups of patients. The therapy is so far symptomatic and the successfulness of therapy strongly depends on the patient's willingness to cooperate (cessation of smoking) and as such it is often unsuccessful. The surgical therapy, so far, fails to have a long-term effect and high percentage of limb amputation is necessary. (Ref. 11.). PMID- 8620331 TI - Autocrine induction of DNA synthesis by mechanical injury of cultured smooth muscle cells. Potential role of FGF and PDGF. AB - To determine whether replication of arterial smooth muscle cells (SMCs) in response to mechanical injury would occur in the absence of serum and other cells, we created an in vitro model in which confluent, growth-arrested cultures of rat SMCs were injured by gentle pressure of a soft plastic tube and then kept in serum-free medium for up to 4 days. Replication of SMCs in and around the injury, as measured by tritiated thymidine incorporation, was noted within 24 hours and peaked at 48 hours after injury, whereas noninjured cells remained quiescent. An increased expression of platelet-derived growth factor (PDGF) A mRNA, noted 6 hours after injury, was followed by an increased PDGF AA immunoreactivity in SMCs in and around the zone of injury at 24 and 48 hours after injury. A PDGF A chain antisense oligonucleotide inhibited 87.0 +/- 4.0% (P < .005) of SMC replication in the injury zone, whereas the corresponding sense oligonucleotide reduced SMC replication by only 37.2%. An antibody to fibroblast growth factor (FGF) almost completely inhibited SMC replication in the injured zone, whereas an antibody to PDGF AA was without effect. Incubation of SMCs with FGF increased PDGF A mRNA levels in SMCs, and 5 mumol/L PDGF A antisense oligonucleotides reduced FGF-induced SMC replication by 62%. Taken together, these results demonstrate that injured rat SMCs in culture release FGF that activates DNA synthesis of neighboring SMCs both by a direct mechanism and by stimulating the production of PDGF AA. PMID- 8620333 TI - Effects of diet and exercise on qualitative and quantitative measures of LDL and its susceptibility to oxidation. AB - The purpose of this study was to investigate the effects of an intensive diet and exercise program on the quantity and quality of LDL as well as its susceptibility to in vitro oxidation. The diet was low in fat (< 10% kcal) and cholesterol (< 100 mg/d), while high in complex, unrefined carbohydrates (> 70% kcal) and fiber (35 g/1000 kcal). The study was composed of 80 participants in a 3-week residential program where food was provided ad libitum and there was daily aerobic exercise, primarily walking. In each subject, preparticipation and postparticipation fasting blood samples were drawn and LDL was isolated via density gradient ultracentrifugation. LDL particle diameter was determined by gradient gel electrophoresis of serum (n = 23). Isolated LDL was either separated into 6 subfractions by saline gradient equilibrium ultracentrifugation (n = 26) or subjected to in vitro copper oxidation (n = 32). Significant reductions (P < .01) in serum levels of cholesterol (20%). LDL-cholesterol (20%), HDL-cholesterol (17%), triglycerides (26%), and glucose (16%) as well as in body weight (4%) were noted for the total population. The mean particle diameter of the LDL increased (24.2 +/- 0.2 to 25.1 +/- 0.14 nm, P < .01) and was correlated with the reduction in serum triglycerides (r = .58, P < .01). Six of 22 subjects changed in LDL phenotype from B (< or = 25.5 nm) to A (> 25.5 nm). The percentage of LDL cholesterol carried in the more dense subfractions fell significantly, while that carried by the less dense fractions increased. Initial oxidation levels fell (21%), while the lag time before copper-induced oxidation increased (13%). Reductions were observed in both the rate of oxidation (16%) and peak oxidation (20%). All of these changes should result in a dramatic reduction in the risk for atherosclerosis and its clinical sequelae. PMID- 8620332 TI - Active oxygen species and lysophosphatidylcholine are involved in oxidized low density lipoprotein activation of smooth muscle cell DNA synthesis. AB - It has recently been shown that oxidative modification of LDL enhances the mitogenic effect of LDL on smooth muscle cell (SMC) DNA synthesis. However, because of its complex chemical structure, the mitogenic components have not been well characterized. Exposure of LDL to the oxidant Cu2+ is followed by a rapid accumulation of peroxides that peaks after 8 to 12 hours and a conversion of the phospholipid phosphatidylcholine into lysophosphatidylcholine that continues for up to 48 hours. Most of the mitogenic activity is formed during the first 4 hours of oxidation. Both superoxide dismutase and catalase effectively inhibit the mitogenic activity of oxidized LDL, suggesting involvement of reactive oxygen intermediates. In the presence of 1% serum, low concentrations of hydrogen peroxide activated SMC DNA synthesis in a dose-dependent manner, with a maximal effect at a concentration of 200 mumol/L, whereas higher concentrations were inhibitory. Lysophosphatidylcholine also enhanced SMC DNA synthesis, with a maximal stimulation at a concentration of 10 mumol/L. Oxysterols, which also accumulate in oxidized LDL, effectively inhibited DNA synthesis. These results demonstrate that oxidation of LDL is associated with formation of several substances affecting the growth of SMCs. Among these substances, low levels of reactive oxygen intermediates and lysophosphatidylcholine stimulate DNA synthesis, whereas at a higher concentration they, as well as oxysterols, are inhibitory. PMID- 8620334 TI - Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans. AB - We have recently demonstrated that cultured human alveolar macrophages efficiently convert cholesterol into excretable 27-oxygenated products. We show here that increasing the intracellular concentration of cholesterol by a factor of 10 leads to about a twofold increase in the excretion of 27-oxygenated products from cultured macrophages. Inhibition of the sterol 27-hydroxylase caused a significant intracellular accumulation of cholesterol. A direct comparison was made between flux of cholesterol and 27-oxygenated products from macrophages preloaded with [4-14C]cholesterol. Under the specific conditions employed with fetal calf serum in the culture medium, the flux of 27-oxygenated products was about 10% of that of cholesterol. Since the sterol 27-hydroxylase, which converts cholesterol to 27-oxygenated products, is present in many cell types, we suggest that 27-oxygenation is a general mechanism for removal of intracellular cholesterol. To evaluate this hypothesis, we measured the net uptake by the human liver of circulating 27-oxygenated products, which was found to be about 20 mg/24 h. This uptake corresponds to approximately 4% of the bile acid production, assuming quantitative conversion into bile acids. It is concluded that the 27-hydroxylase pathway is of significance for elimination of extrahepatic cholesterol. PMID- 8620335 TI - Ethanol-induced redistribution of cholesteryl ester transfer protein (CETP) between lipoproteins. AB - Since alcohol drinking reduces the concentration and activity of plasma cholesteryl ester transfer protein (CETP), we investigated the effects of alcohol on its synthesis and secretion by perfusing rabbit livers for 4 hours in the absence or presence of ethanol. The quantity of CETP mRNA in the perfused livers did not differ between the control and ethanol (25 mmol/L or 50 mmol/L) perfusions. CETP activity was determined by incubating [3H]cholesteryl ester labeled human LDL and unlabeled human HDL with the perfusion medium after removing the endogenous VLDL (secreted by the perfused liver) by ultracentrifugation. CETP activity in the perfusion medium increased at a linear rate that was not affected by ethanol. When the VLDL was removed by precipitation with polyethylene glycol or a heparin-Sepharose column instead of ultracentrifugation, practically no CETP activity was detected in the ethanol perfusions, whereas these procedures did not affect CETP activity in the control perfusions. Inhibition of ethanol oxidation by 4-methylpyrazole resulted in CETP activity similar to that of the controls. We conclude that ethanol does not affect the synthesis or secretion of CETP, but its oxidation may alter the distribution of CETP in lipoproteins. CETP seems to be present in VLDL as well as in HDL, and since VLDL is more rapidly catabolized with HDL, this may explain the low plasma CETP concentration associated with alcohol consumption. PMID- 8620336 TI - Isolation and characterization of human antioxidized LDL autoantibodies. AB - Autoantibodies to oxidized LDL have been reported in normal subjects and in patients with arteriosclerosis, but their possible pathogenic role is not yet well defined. One important problem is the existence of contradictory data reported by different groups concerning the associations between antioxidized LDL autoantibodies and the presence or progression of arteriosclerotic lesions. Such contradictions led us to decide to isolate and characterize antioxidized LDL antibodies by affinity chromatography with the use of oxidized LDL cross-linked to Sepharose. Antioxidized LDL antibodies were isolated from selected serum samples obtained from eight subjects. Seven of them (six patients and one control subject) had high levels of antioxidized LDL antibody during screening. The other subject, a healthy volunteer, had a low level of antibody. All purified antibodies contained IgG (of subclasses 1 and 3) as the predominant isotype and were primarily specific for oxidized LDL but showed some cross-reactivity with malondialdehyde-modified LDL and native LDL. Two of the purified antibodies cross reacted with cardiolipin. We determined average dissociation constants for the antioxidized LDL antibodies purified from five individuals, which varied between 2.4 x 10(-7) and 7.5 x 10(-7) mol/L, whereas the average dissociation constant of rabbit hyperimmune anti-LDL antibody was determined to be 2.7 x 10(-8) mol/L. In conclusion, we have purified human autoantibodies reactive with oxidized LDL that appear to be predominantly of moderate-to-low affinity and of variable cross reactivity. The predominance of IgG1 and IgG3 antibodies is significant from the standpoint of potential pathogenicity, since these two subclasses activate the classic complement pathway system and have the highest binding affinities for Fc gamma receptors on phagocytic cells. PMID- 8620337 TI - Association of localized Ca2+ gradients with redistribution of glycoprotein IIb IIIa and F-actin in activated human blood platelets. AB - We monitored the intracellular distribution of ionized free Ca2+ concentration ([Ca2+]i) in individual human platelets by digital imaging fluorescence microscopy with fura 2 during platelet activation induced by surface contact or a soluble platelet agonist (thrombin). Contact of platelets with glass resulted in pseudopod formation and spreading, accompanied by a nonuniform rise in [Ca2+]i. The rise in [Ca2+]i was maximal during pseudopod formation. Locally elevated [Ca2+]i was frequently found in pseudopodia and at the edge and core of spread platelets. This pattern was faithfully duplicated by the local pattern of distribution of the cytoskeletal components F-actin, gelsolin, and surface glycoproteins (GP) IIb-IIIa but not by calmodulin. Platelets stimulated by thrombin also showed an inhomogeneous rise in [Ca2+]i, which was well correlated with the staining of F-actin and GPIIb-IIIa. Cytochalasin D, an inhibitor of actin polymerization, inhibited the inhomogeneous increase or redistribution of F actin and GPIIb-IIIa but did not inhibit the rise in mean [Ca2+]i. These observations suggest that a localized change in [Ca2+]i may be associated with cytoskeletal reorganization and redistribution of GPIIb-IIIa in activated platelets. PMID- 8620338 TI - Effects of colestipol alone and in combination with simvastatin on apolipoprotein B metabolism. AB - The effects of colestipol therapy alone (20 g/d) or combined with simvastatin (20 mg/d) were examined in a group of eight male patients with primary moderate hypercholesterolemia (total cholesterol > or = 6.5 mmol/L [> or = 250 mg/dL]) who had undergone coronary artery bypass grafting more than 3 months previously. Colestipol therapy decreased total cholesterol by 14% (P < .001) and LDL cholesterol (LDL-C) by 23% (P < .001), while dual therapy decreased total cholesterol by 38% and LDL-C by 52% (both P < .001 versus baseline). No significant changes were observed in plasma triglyceride, VLDL cholesterol, or HDL cholesterol levels. VLDL subfraction turnovers were conducted at baseline and again on each regimen. ApoB kinetic parameters derived from a multicompartmental model suggested that colestipol therapy resulted in an expansion of the total VLDL apoB pool (36%, P < .05) that was largely due to a fall in the clearance rate of VLDL1 apoB (49%), while the LDL apoB pool decreased 23% as a result of diminished direct LDL input. The model used also revealed that addition of simvastatin to the resin therapy caused increases in the fractional transfer rates of VLDL2 to IDL and IDL to LDL together with a 37% increment in the LDL apoB fractional catabolic rate. Compared with baseline, combined therapy generated falls in both IDL (35%, P = .01) and LDL (37%, P < .04) apoB pools due to enhanced clearance of IDL (214%, P < .03) and reduced total input of LDL (39%, P < .003). PMID- 8620339 TI - Higher serum bilirubin is associated with decreased risk for early familial coronary artery disease. AB - Mildly increased serum bilirubin has recently been suggested as a protective factor, possibly reducing the risk of coronary artery disease (CAD) by acting as an antioxidant. We tested this hypothesis by examining serum bilirubin concentrations and other coronary risk factors in 120 men and 41 women with early familial CAD and 155 control subjects. At screening, both cases and control subjects were 38 to 68 years old. Early familial CAD patients had experienced myocardial infarction, coronary artery bypass grafting, or coronary angioplasty by age 55 years for men and 65 for women and had another sibling similarly affected. The average total serum bilirubin concentration was 8.9 +/- 6.1 mumol/L in cases and 12.4 +/- 8.1 mumol/L in control subjects (P = .0001 for difference). In univariate analysis stratified by sex, serum bilirubin was strongly and inversely related to CAD risk, with relative odds of 0.4 to 0.1 (relative to the lowest quintile, P = .04 to .00001) in both men and women as bilirubin increased into the upper two quintiles. Multiple logistic regression analysis was performed including age, sex, smoking, body mass index, diabetes, hypertension, plasma measured LDL cholesterol, HDL cholesterol, triglycerides, and serum bilirubin as potential risk factors. Bilirubin entered as an independent protective factor with an odds ratio of 0.25 (P = .0015) for an increase of 17 mumol/L (1 mg/dL). The standardized logistic regression coefficient for bilirubin was -.33 compared with -.34 for HDL, suggesting that the protective effect of bilirubin on CAD risk in the population is comparable to that of HDL cholesterol. A history of cigarette smoking was associated with significantly lower serum bilirubin concentration and appeared to attenuate the protective effect of bilirubin. PMID- 8620340 TI - In hypercholesterolemia, lower peripheral monocyte count is unique among the major predictors of atherosclerosis. AB - Many studies have shown that enhanced monocyte adherence is an important factor in the initiation of atherosclerosis. Because the relationships between circulating monocyte count and atherosclerosis or its major predictors have received little attention, we conducted this study with the aim of clarifying these relationships. The study included 409 men and women who underwent a carotid artery duplex study and white blood cell analysis (Sysmex Cell Counter) during a 2-day health check at our hospital in 1994. We found no correlation between preexisting carotid atherosclerosis and monocyte count. After adjustment for age and sex, hypercholesterolemia, among the major predictors of atherosclerosis, showed a unique correlation with both lower monocyte count and percentage (P < .001, P < .0001, respectively), whereas smoking was correlated with a higher monocyte count (P < .001). There was a slight but nonsignificant increase in monocyte count in hypertension, diabetes, and hypertriglyceridemia. Our results imply that: (1) hypercholesterolemia has a strong, peripheral monocyte-reducing effect, probably due to direct enhancement of monocyte adhesion to the endothelium, which subsequently initiates the atherosclerotic process, and (2) the mechanisms of other predictor(s)-induced atherosclerosis may be quite different from that of hypercholesterolemia. Another possible explanation for the inverse correlation between monocyte count and serum cholesterol level is that decreased monocyte levels might lead to hypercholesterolemia because of decreased uptake of cholesterol from the plasma by less monocyte-derived macrophages. The reasons why preexisting carotid atherosclerosis did not correlate with monocyte count are also discussed. PMID- 8620341 TI - Plasma fibrinogen and coronary heart disease in elderly Japanese-American men. AB - Clinical and epidemiological studies consistently indicate that elevations in plasma fibrinogen concentration are associated with the presence and development of coronary heart disease (CHD). These elevations are strongly correlated with smoking behavior and may play a significant role in mediating a relation of smoking to CHD. This cross-sectional survey of 3571 elderly Japanese-American men, aged 71 through 93 years, represents survivors of the Honolulu Heart Program cohort. Active smokers are almost twice as likely to be represented in the highest quintile of the fibrinogen distribution compared with the lowest quintile (9.8% versus 5.3%, respectively). The highest prevalence of CHD (34%) was noted in past and current smokers who were in the highest quintile of fibrinogen. The age-adjusted relative odds of prevalent CHD comparing the average fibrinogen levels in the first and fifth quintiles were 1.36 (95% confidence interval, 1.13 to 1.64). After adjustment for smoking status, blood pressure, total and HDL cholesterol, diabetes status, hematocrit, and white cell count, the association between fibrinogen and CHD was changed slightly and remained statistically significant (P < .05). These findings in an elderly cohort of Japanese-American men are consistent with previous studies among middle-aged adults demonstrating fibrinogen to be associated with indicators of clinical CHD and CHD risk factors. Because of the cross-sectional nature of this study, it is not possible to distinguish whether the observed relation of fibrinogen to prevalent CHD is causal or whether it represents a marker of active and progressive disease. PMID- 8620342 TI - Characteristics of the insulin resistance syndrome in a Japanese population. The Jichi Medical School Cohort Study. AB - We investigated the relationships between hyperinsulinemia (a major indicator of the insulin resistance syndrome), blood pressure, dyslipidemia, and coagulation factors in 2606 community-dwelling Japanese individuals as part of the Jichi Medical School Cohort Study. An age-related decrease of the fasting insulin level was found in men but not in women. Body mass index, systolic and diastolic blood pressure, triglyceride and fasting glucose levels, and factor VII activity all increased in both sexes as the insulin level became higher, while the HDL cholesterol level decreased. In addition, total cholesterol and LDL cholesterol levels increased as the insulin level became higher and lipoprotein(a) levels decreased in the men. Fibrinogen levels were not related to the insulin level in either sex. Multiple logistic regression analysis revealed that fasting insulin levels were positively correlated with body mass index and fasting glucose and factor VII activity levels, whereas they were negatively correlated with HDL cholesterol in both sexes. In addition, fasting insulin levels were positively correlated with LDL cholesterol levels in men and with triglyceride levels in women. Our results indicate that hyperinsulinemia is associated with high factor VII activity in a general Japanese population as well as with high blood pressure and dyslipidemia. The accumulation of these cardiovascular risk factors in hyperinsulinemic subjects appears to contribute to cardiovascular events in the Japanese as well as in westerners. PMID- 8620343 TI - Vital exhaustion, anger expression, and pituitary and adrenocortical hormones. Implications for the insulin resistance syndrome. AB - This study was undertaken to examine whether there are psychological factors that can incline an individual toward coronary heart disease and that can in turn identify a pattern of pituitary and adrenocortical responses that is associated with the Insulin Resistance Syndrome (IRS). The study was performed with 69 normotensive and 21 unmedicated borderline hypertensive men (age range, 30 to 55 years). Type A behavior, hostility (defined as cynicism, pessimism, and paranoia), vital exhaustion, and anger expressions were the behavioral variables studied. Among these, only the vital exhaustion-anger-out factor identified the neuroendocrine pattern that predicted the IRS. This neuroendocrine pattern consisted primarily of an adrenal responsiveness to ACTH and secondarily of a high mean basal cortisol-to-mean basal ACTH ratio. The contribution of this last variable was, however, slightly questionable. Instead of the traditional coronary prone factors, ie, type A behavior and hostility, the findings emphasize the significance of vital exhaustion and emotional distress. The findings have been discussed in terms of defeat reaction, hypocortisolemia, and visceral obesity. PMID- 8620344 TI - Genetic analysis of the IRS. Pleiotropic effects of genes influencing insulin levels on lipoprotein and obesity measures. AB - Insulin resistance is part of a metabolic syndrome that also includes non-insulin dependent diabetes mellitus, dyslipidemia, obesity, and hypertension. It has been hypothesized that insulin resistance represents the primary physiological defect underlying this syndrome. Since insulin resistance is at least partially genetically determined, we hypothesized that genes influencing insulin resistance would have pleiotropic effects on a number of other traits, including triglyceride (TG) and HDL cholesterol levels, body mass index (BMI) and body fat distribution, and blood pressure levels. To investigate this hypothesis, we analyzed data obtained from individuals in 41 families enrolled in the San Antonio Family Heart Study. Statistical methods that take advantage of the relatedness among individuals were used to differentiate between genetic and nongenetic (ie, environmental) contributions to phenotypic variation between traits. Serum levels of fasting and 2-hour insulin (measured in 767 and 743 nondiabetic family members, respectively) were used as a measure of insulin resistance. The genetic correlations were high between insulin levels (both fasting and 2-hour) and each of the following: BMI, HDL level, waist-to-hip ratio, and subscapular-to-triceps ratio, indicating that the same gene, or set of genes, influences each pair of traits. In contrast, the genetic correlations of insulin levels with systolic and diastolic blood pressures were low. We have previously shown that a single diallelic locus accounts for 31% of the phenotypic variation in 2-hour insulin levels in this population. We conducted a bivariate segregation analysis to see if the common genetic effects on insulin and these other traits could be attributable to this single locus. These results indicated a significant effect of the 2-hour insulin locus on fasting insulin levels (P = .02) and BMI (P = .05), with the "high" insulin allele associated with higher levels of fasting insulin but lower levels of BMI. There was no detectable effect of this locus on HDL level, TG level, subscapular-to-triceps ratio, or blood pressure. Overall, these results suggest that a common set of genes influencing insulin levels also influences other insulin resistance syndrome-related traits, although for the most part this pleiotropy is not attributable to the 2-hour insulin level major locus. PMID- 8620345 TI - Triiodothyronine exerts a major pleiotropic effect on reverse cholesterol transport phenotypes. AB - The thyroid hormone triiodothyronine (T3) is known to be a potent mediator of APOA1 gene expression. With the use of multivariate quantitative genetic analysis, we have assessed the magnitude of shared effects of T3 on plasma concentrations of apolipoprotein AI (apo AI) and three related phenotypes: HDL-C, apo AII, and LpAI (which is a concentration of apo AI that contains HDL particles). Maximum likelihood techniques were used to simultaneously estimate mean effects and variance components in large, extended Mexican American families living in San Antonio, Tex. We found that T3 accounted for 16%, 23%, 21%, and 37% of the additive genetic variance in HDL-C, apo AI, apo AII, and LpAI, respectively, while explaining virtually none of the random environmental variance in these phenotypes. T3 also has a pronounced effect on the pairwise genetic correlations among the four phenotypes: After the pleiotropic effects of T3 concentrations are controlled for, the genetic correlations are reduced by 6% in the case of HDL-C and apo AI and 97% for apo AII and LpAI. Thus, genes that influence T3 have a significant effect on HDL-C, apo AI, apo AII, and LpAI and also on the correlations among these phenotypes. PMID- 8620346 TI - Two novel molecular defects in the LCAT gene are associated with fish eye disease. AB - A 53-year-old man with a severely reduced HDL cholesterol level, dense corneal opacities, normal renal function, and premature coronary artery disease was investigated together with 16 members of his family. The proband was diagnosed with fish eye disease. As in previously reported patients with fish eye disease, the endogenous plasma cholesterol esterification rate was near normal, yet lecithin:cholesterol acyltransferase (LCAT) activity was almost absent when measured with exogenous HDL analogues used as substrate. Direct sequencing of the LCAT gene revealed two novel missense mutations in exon 1 and exon 4, resulting in the substitution of Pro10 with Gln (P10Q) and Arg135 with Gln (R135Q), respectively. Both missense mutations were located on different alleles. Genetic analysis by polymerase chain reaction revealed 4 carriers of the P10Q and 3 carriers of the R135Q defect. Functional assessment of both missense mutations revealed that when exogenous HDL analogues were used as substrate, the specific activity of rLCAT p10Q was 18% of wild type (WT); however, when LDL was used as substrate, the activity was 146% of WT. By contrast, rLCATR135Q was inactive against both substrates. Thus, we conclude that the LCATR135D mutation is causative for complete LCAT deficiency and that the clinical phenotype of fish eye disease seen in this patient is due to the Pro10 mutation. The presence of premature coronary artery disease in the absence of other risk factors in this new case of fish eye disease raises questions regarding the risk of atherosclerosis, which has previously been reported to be nonexistent. PMID- 8620347 TI - Deletion polymorphism in the angiotensin-converting enzyme gene in patients with a history of ischemic stroke. AB - We evaluated the genotypes of the angiotensin-converting enzyme (ACE) gene in 101 subjects with and 109 subjects without a history of ischemic stroke. All were attending a metabolic ward. The two groups were compared for major risk factors for ischemic events. Genotypes were determined by polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene. Deletion polymorphism of the ACE gene (DD genotype) was shown to be more common in subjects with a history of stroke than in those without (relative risk, 1.76; confidence intervals, 1.02 to 3.05). A positive family history for ischemic complications of atherosclerosis was also more common in subjects with documented events (relative risk, 1.99; confidence intervals, 1.10 to 3.59). DD genotype and a positive family history were strong independent discriminators of cerebral ischemia. Plasma levels of tissue-type plasminogen activator (TPA) and plasminogen activator inhibitor-1 help identify subjects with a history of cerebral ischemic episodes. When such fibrinolytic variables were included in the analysis, the DD genotype still strongly and independently discriminated subjects with a stroke history and significantly interacted with TPA levels > 10 ng/mL in such identification. We conclude that in subjects attending a metabolic ward, homozygosity for a deletion polymorphism of the ACE gene consistently discriminates subjects with a stroke history. Interaction with TPA improves such identification. PMID- 8620348 TI - Relation of intima-media thickness to atherosclerotic plaques in carotid arteries. The Vascular Aging (EVA) Study. AB - This study examined the relation between arterial wall thickness and local atherosclerosis in the carotid arteries (CAs) and their specific risk factors. B mode ultrasonography of the CAs was performed in a cohort of 516 men and 756 women aged 59 to 71 years who had been recruited for the European Vascular Aging Study. Ultrasound examination included measurement of intima-media thickness of the common CA (CCA) and the sites of plaque in the internal CA and bifurcations. Significant associations between increases in CCA intima-media thickness and both the presence and severity of atherosclerotic plaque were found in men and women. Examination of specific risk factors for increases in CCA intima-media thickness in the presence of plaque showed that, after adjustment for sex, both ultrasound measurements were independently related to age, body mass index, hypertension, and ever smoking (versus never smoking). Diabetes and current smoking were associated with intima-media thickness only, whereas hypercholesterolemia was related to plaque only. However, when subjects who were taking lipid-lowering drugs were excluded, lipoproteins and apolipoproteins were more consistently related to intima-media thickness than to plaque. In subjects free from any antihypertensive treatment, both intima-media thickness and plaques were independently associated with systolic blood pressure. After adjustment for sex and other risk factors, the odds ratio for having at least one plaque associated with a 0.10-mm increase in CCA intima-media thickness was 1.18 (95% confidence interval, 1.05 to 1.32). In this relatively aged population, increases in intima media thickness as measured in the CCAs were clearly related to locally detected atherosclerosis and known risk factors for atherosclerosis. Longitudinal studies are needed to clarify the role of arterial wall thickening in the atherosclerotic process. PMID- 8620349 TI - Effect of age on the pattern of short-term albumin uptake by the rabbit aortic wall near intercostal branch ostia. AB - Lipid deposition occurs more frequently downstream than upstream of branches in immature human aorta but the opposite pattern is seen in mature vessels. These distributions may reflect variation in the uptake of plasma macromolecules by the aortic wall. We have recently shown that the quasi-steady state uptake of albumin is greater downstream than upstream of branches in immature rabbit aortas and that the opposite pattern occurs in mature animals. Additionally, there is a sharp drop in the mean uptake shortly after weaning. In the present study, the mechanisms underlying these phenomena were investigated by examining the short term uptake of albumin and its distribution across the wall. Albumin was labeled with a fluorescent dye and introduced into the circulation of conscious New Zealand White rabbits. Thoracic aortas were fixed in situ 10 minutes later and were sectioned through the center of intercostal ostia. Fluorescence from sections was measured by using digital imaging fluorescence microscopy and was converted to tracer concentrations after appropriate autofluorescence levels had been subtracted. In animals aged 45 days, more tracer was detected in the wall downstream than upstream of branches; the difference between regions was > 100% of the mean value. This percentage halved and the mean uptake decreased almost threefold by 75 days. In mature animals, the mean value remained at the 75-day level but the converse distribution was seen; 22% more tracer was detected upstream than downstream. These trends were insensitive to the depth of the intimal-medial layer examined. In each region, the maximum tracer concentration occurred close to the luminal surface but not always within the first 2.9-microns thick layer of the wall. Maxima were similar in magnitude to those observed at quasi-steady state, but the fall with increasing distance into the wall was much sharper. In many cases concentrations remained constant over most of the media, and rises toward the adventitial boundary were rarely seen. Uptake after 10 minutes predominantly reflects the rate at which tracer enters the wall. The concentration profiles were consistent with most of the tracer having entered from the luminal surface and with the involvement of convective transport. The trends observed with age closely paralleled those occurring at quasi-steady state. Consequently, the latter are also likely to be determined by changes in the resistance of the wall to macromolecule influx. PMID- 8620350 TI - Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly. AB - Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid associating properties of apo A-I, the first step is to understand the lipid associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220 241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation. PMID- 8620351 TI - Poland and United States Collaborative Study on Cardiovascular Epidemiology. A comparison of HDL cholesterol and its subfractions in populations covered by the United States Atherosclerosis Risk in Communities Study and the Pol-MONICA Project. AB - HDL cholesterol (HDL-C) levels are inversely related to coronary heart disease (CHD) risk, and HDL-C distributions vary among countries. Poland is one of the few developed countries in which CHD rates are increasing at the same time that US rates have been falling, but whether these differences are explained by differences in risk factors such as HDL-C has not been determined. To examine this possibility, levels of HDL-C and its subfractions were compared in US and Polish urban and rural men and women aged 45 to 64 years. Age-adjusted HDL-C means were 0.20 mmol/L higher in urban Polish men and 0.37 mmol/L higher in rural Polish men than in their US counterparts (P < .0001); means in urban Polish women were 0.06 mmol/L higher (P < .05) and in rural Polish women 0.09 mmol/L higher (P < .001) than in their US counterparts. Adjustment for age, education, alcohol intake, smoking, BMI, heart rate, and menopause status (in women) had little effect on differences. Means of HDL2 and HDL3 levels showed similar between country differences, although differences were minimal for HDL2 in urban men and women, and HDL3 means did not differ between rural women. BMI was inversely related to HDL-C and both subfractions in all gender-country-site strata (P < .001), and alcohol was directly related to HDL-C (P < .001) in all strata except Polish women. Cigarette smoking was negatively related to HDL-C and both subfractions in all US samples except HDL2 in urban men, whereas in Polish samples, significant associations were found only in urban women for HDL-C and in rural and urban women for HDL3. Age, heart rate, and education showed inconsistent or no association with HDL-C and its subfractions in either country. This profile of HDL-C and its subfractions in Polish samples contrasts sharply with the opposite trend in CHD mortality rates, which suggests either that other risk factors may account for the trends or that the relationship between HDL-C and CHD may differ between the two countries. PMID- 8620352 TI - Gender, marital closeness, and depressive symptoms in elderly couples. AB - Data from the Established Populations for the Epidemiologic Study of the Elderly (EPESE) in New Haven include independent interviews with husbands and wives in 317 community-dwelling older couples. Drawing on these data, we (a) describe the prevalence of three aspects of marital closeness: having a confidant, perceived emotional support, and reciprocity between spouses' reports of marital closeness; (b) evaluate their associations with depressive symptoms according to both a respondent's own and his or her spouse's reports; and (c) examine gender similarities and differences in the prevalence and the associations of the closeness variables. Both husbands and wives responded more strongly to their spouse's than to their own. Different dynamics operate, with husbands having fewest depressive symptoms when they have emotionally independent wives, and wives having low levels when they feel important emotionally to their husbands. Dyadic closeness was associated with fewer symptoms in wives and more symptoms in husbands. PMID- 8620353 TI - Amount of care given and caregiving satisfaction: a latent growth curve analysis. AB - We examined the wear-and-tear hypothesis using data from 4 annual interviews with 130 (128 White) middle-aged daughters caring for their physically impaired, elderly mothers. We formulated a latent growth curve model hypothesizing that increases in the amount of care given by daughters caused a decrease in caregiving satisfaction, independent of caregiving duration. We found considerable individual variability and change in both caregiving satisfaction and the amount of care given in univariate latent growth curve analyses. Contrary to the wear-and-tear hypothesis, a multivariate latent growth curve analysis revealed duration of caregiving had no effect on either initial caregiving satisfaction or change in satisfaction. An elaborated wear-and-tear model was supported, however. The mechanism for decline in satisfaction is an increase in the amount of care given. PMID- 8620354 TI - Age differences in postural stability are increased by additional cognitive demands. AB - We report an investigation of postural stability in two groups of volunteers (mean ages of 57 and 77). Participants were required to stand on a force platform while performing five cognitive tasks: (1) random digit generation, (2) Brooks' spatial memory, (3) backward digit recall, (4) silently counting from 1-100, and (5) counting backward in threes (aloud). There was also a control condition in which there was no cognitive task. Postural stability was adversely affected by age in all conditions. Moreover, the difference between the two age groups was significantly greater when performing tasks 2 and 3, in comparison with the age difference in the control condition. Regression analyses revealed that the effect of age on postural stability while performing these particular tasks remained significant even after the following measures were included in the regression: postural stability in the control condition, cognitive performance, intelligence, and speed. We suggest that age differences in postural stability are increased by cognitive tasks requiring use of the visuo-spatial sketchpad component of working memory. PMID- 8620355 TI - Predicting age-related differences in visual information processing using a two stage queuing model. AB - Recent work on age-related differences in some types of visual information processing has qualitatively stated that younger adults are able to develop parallel processing capability, while older adults remain serial processors. A mathematical model based on queuing theory was used to quantitatively predict and parameterize age-related differences in the perceptual encoding and central decision-making aspects of a multiple-frame search task. Statistical results indicated main effects for frame duration, display load, age group, and session of practice. Comparison of the full model and a restricted model indicated an efficient contribution of the encoding speed parameter. The best-fitting parameter set indicated that (1) younger participants processed task information with a two-channel parallel system, while older participants were serial processors; and (2) perceptual encoding had a large impact on age-related differences in task performance. Results are discussed with implications for human factors design principles. PMID- 8620357 TI - Advancing caregiver research: weighing efficacy and feasibility of interventions. PMID- 8620356 TI - Where in an ordered sequence of variables do independent age-related effects occur? AB - A series of regression analyses was conducted to determine which variables in an ordered sequence had significant age-related effects after control of the immediately preceding variable in the sequence. Independent age-related effects in these types of analyses are particularly interesting because they represent age-related influences that are not mediated through earlier variables. A total of 56 analyses are reported with ordered variables representing: (a) successive trials or sessions in learning; (b) progressively more intervening events during the retention interval of a memory task; (c) successively longer stimulus presentation durations; and (d) increased processing complexity. In most of the analyses a very large proportion of the age-related effects on later variables was found to be mediated through earlier variables in the sequence. PMID- 8620358 TI - Differentials in active life expectancy in the older population of the United States. AB - This study clarifies the process by which mortality and disability interact to determine differences in active life expectancy by age, sex, race, and education for the U.S. population 70 years of age and over. The analysis is performed using data from the Longitudinal Study of Aging and multistate life tables constructed using the results of hazard models. Women spend more years than men both active and inactive at every age; however, the proportion of life that is expected to be active is smaller for women. These differences are largely due to mortality differences favoring women. Persons with less than a high school education have shorter total and active life expectancies but similar expected lengths of inactive life compared to those with more than a high school education. There are no significant race differences in total life expectancy for race-education groups of the older population; but Blacks have lower expected active life than non-Blacks because of worse functioning. PMID- 8620359 TI - The moderating influence of service use on negative caregiving consequences. AB - This investigation adapts the social support conceptual framework to examine the moderating influence of community service use by impaired older persons on the negative consequences of caregiving for informal helpers. The model is modified by defining services as a type of social support that can counteract the stress associated with various care recipient impairments. Results from multivariate analyses of data from 401 caregivers suggest that certain services for care recipients can reduce the adverse effects of certain impairments on informal caregivers. The use of health care service by care recipients who are more disabled is related to lower levels of caregiver depression, health deterioration, and social isolation. Personal care service use consistently offsets the negative effects of care recipients' behavioral problems. Additionally, the use of household service when care recipients exhibit behavioral problems is associated with lower levels of caregiver depression. PMID- 8620360 TI - Trends in retirement age in the United States, 1955-1993, by sex and race. AB - To meet the need for a labor-force-based time series on the average age at retirement differentiated by sex and race, net labor force withdrawal rates (NWR) are calculated on a cohort basis for periods of time. The NWR are used to calculate the medium age of exit from the labor force. From the late 1950s through the late 1980s, declines of about 3-4 years occurred among Black and White women and men. These results are very similar to those obtained from Social Security data. The decline slowed during the 1970s and leveled off during the 1980s and early 1990s. The findings are a valuable addition to the meager knowledge available now about the retirement behavior of women and Blacks. The labor force and the Social Security time series of the average age of retirement are useful, especially in combination, to private and public planners and policymakers. PMID- 8620361 TI - Profiling plans for retirement. AB - Actual decision making for retirement is largely inaccessible to investigation, yet research can focus on plans as a window into the preretirement process. This article proposes a construct that profiles five generic types of retirement plans, including plans to retire completely, change jobs, never retire, and uncertainty about retirement. The heuristic value of the construct lies in its recognition of the heterogeneity of retirement intentions. The five plan types were operationalized among workers aged 51-61 in the 1992 Health and Retirement Study. Convergent validity was demonstrated by comparisons to analogous survey questions. Construct validity was shown by predictable relationships between intentions and elements of workers' opportunity structure. The retirement-plans construct can serve as the foundation for a taxonomy of specific retirement plans (e.g., about timing, employment), to organize research on stability and change in retirement intentions, and characterize the path dependence of eventual retirement behavior. PMID- 8620362 TI - Health and social precursors of later life retirement-community migration. AB - This study uses the developmental migration paradigm developed by Litwak and Longino (1987) as a framework to investigate the health and social determinants of late-life elderly mobility to retirement communities. Data from four waves of the Longitudinal Study of Aging are used to predict the likelihood of moving from the general community to two types of retirement communities--those with group meals and those without group meals. Multinomial logistic regression reveals that the likelihood of migrating to both types of retirement communities increases as disability advances to moderate levels, but declines as disability becomes severe. Migration to retirement communities is also more likely among elderly persons who live alone and among those whose children do not live nearby. The results suggest that retirement community migration in middle and late old age is motivated not only by social and amenity considerations, but also by the need for assisted living arising from physical frailty. Implications for developmental migration theory and long-term care policy are discussed. PMID- 8620363 TI - The meaning of older adults' health appraisals: congruence with health status and determinant of mortality. AB - This study explored open-ended responses regarding attributions underlying health appraisals made by older adults, resulting in five categories (physical health, attitudinal/behavioral, externally focused, health transcendence, nonreflective). The older the respondents, the less likely they were to focus on physical aspects of their health. Health optimists were the most likely to make attitudinal/behavioral or health transcendent attributions, while poor-health realists were most likely to mention physical health aspects and least likely to make attitudinal or behavioral attributions. While poor-health realists were at the highest risk of dying within a three-year period, health optimists were significantly less likely to die than poor-health realists, in spite of sharing similar health status. Respondents who were unable to identify underlying attributions were significantly more likely to die than were those identifying any other attribution. In conclusion, health attributions provide unique insight into the complex relationship between older adults' health appraisals, health status, and mortality. PMID- 8620364 TI - The renal bone diseases in children treated with dialysis. AB - Renal osteodystrophy represents a spectrum from high- to low-turnover bone lesions. The specific pattern, however, may change during selected therapeutic interventions. As in the past, osteitis fibrosa remains the most frequent histologic lesion in pediatric patients on dialysis, although recently the prevalence of low-turnover bone lesions without aluminum toxicity has been increasing in the pediatric population. This may be a consequence of aggressive calcitriol and calcium therapy. The different factors involved in the development of secondary hyperparathyroidism include hyperphosphatemia, hypocalcemia, altered vitamin D synthesis, impairments in parathyroid hormone (PTH) secretion and metabolism, and, recently, possible downregulation of renal PTH/PTH-rP messenger RNA receptor. New developments in molecular biology have demonstrated the relationship between vitamin D and PTH. The use of high-dose pulse intravenous, intraperitoneal, and oral calcitriol therapy has significantly decreased serum PTH levels and retarded the progression of osteitis fibrosa. These therapeutic interventions, however, may have led to the development of adynamic bone lesions. The impact of adynamic bone lesions in the young and growing skeleton remains to be determined. PMID- 8620365 TI - Recombinant human erythropoietin therapy in children on dialysis. AB - The addition of recombinant human erythropoietin (rHuEPO) to the therapeutic regimen for children with chronic renal failure (CRF) is one of the most important improvements in care in the last 20 years. Anemia had played an important role in the morbidity of chronic dialysis treatment. Before the availability of rHuEPO, repeated erythrocyte transfusions provided incomplete treatment and had significant long-term sequelae. Recombinant erythropoietin treatment resulted in the amelioration of anemia and marked reduction in transfusions. Additional benefits of the correction of anemia with rHuEPO include improvements in exercise tolerance and regression of ventricular hypertrophy. Many rHuEPO-treated patients have had subjective increases in appetite, but there has been no consistent improvement in dietary intake or anthropometric measures. Correction of anemia with rHuEPO has not been shown to improve the growth of children with CRF receiving dialysis. The most significant adverse effects of rHuEPO are the development of iron deficiency and the exacerbation or development de novo of hypertension. RHuEPO treatment has been shown to treat the anemia of CRF in children safely and effectively. In most cases, putative inhibitors of erythropoiesis and blood loss can be overcome. Many of the symptoms previously ascribed to "uremia" have improved with correction of anemia. The full implications of treatment of anemia with rHuEPO will be clearer when the health outcomes for children who never become severely anemic or require transfusions are more completely studied. PMID- 8620366 TI - The team approach to the management of children on chronic peritoneal dialysis. AB - The diagnosis of chronic renal failure has a profound and lasting impact on a child and family, with the potential for impairment of the child's physical, mental, and social development. To achieve an ideal outcome, the peritoneal dialysis (PD) team must focus on preparing the child and family to perform home dialysis, prescribe the dialysis regimen most compatible with the patient's lifestyle and clearance requirements, ensure optimal nutrition, and facilitate psychosocial adaptation to PD. Close follow-up is essential for early detection, prevention, and treatment of potential complications of dialysis. A multidisciplinary team approach encompassing nursing, medicine, nutrition, and social work best suits the needs of the child and family. PMID- 8620367 TI - Recombinant human growth hormone for children with renal failure. AB - The pathogenesis of growth retardation in children with chronic renal failure (CRF) is clearly multifactorial. A major breakthrough in the understanding of the pathogenesis of uremic growth failure was achieved only recently by a more detailed analysis of the growth hormone (GH)/insulinlike growth factor (IGF) axis. Uremia is characterized by an insensitivity to the somatotropic action of GH. The mechanisms that account for this insensitivity include reduced hepatic GH receptor expression, decreased production of IGF-I, and inhibition of IGF bioactivity by increased binding of IGFs to their specific binding proteins. Recombinant human growth hormone (rhGH) in supraphysiological doses is able to overcome the partial GH resistance and to stimulate longitudinal growth under both experimental and clinical conditions. One possible mechanism of action of rhGH in uremia is the restoration of circulating IGF bioactivity, which results from the differential regulatory effect of rhGH on circulating IGF-I and IGFBP-3 concentrations. RhGH has proven to be an effective, safe, and well-tolerated new treatment modality for growth-retarded children at all stages of CRF. There is strong evidence that final height will increase in these children. Other than a modest chronic stimulation of insulin secretion, no frequent side effects have been observed; in particular, no acceleration in loss of residual renal function has been seen in children treated before the onset of end-stage renal failure. In children after transplantation, rhGH is also effective, but the potential risk of interference with graft function is not yet sufficiently defined. PMID- 8620368 TI - Dialysis and renal transplantation in infants with irreversible renal failure. AB - Historically, infants with irreversible renal failure fared poorly, and aggressive medical intervention was considered futile. Although the care of this population clearly remains a challenge, technical advances and clinical experience have now made dialysis and transplantation reasonable and successful therapeutic options. This report provides a discussion of practical guidelines and patient care issues particular to the infant with end-stage renal disease. Topics addressed include nutritional requirements, neurodevelopmental abnormalities, and the possible contribution of alterations of the immune system to patient morbidity. Specific technical considerations for the performance of peritoneal dialysis, hemodialysis, and transplantation in the very small infant are also presented. PMID- 8620369 TI - Progress in renal transplantation for children. AB - Renal transplantation continues to be the goal of therapy for children with end stage renal disease. Patient age, primary renal disease, psychosocial status, living versus cadaver donor allograft, optimal immunosuppressive therapy, urologic status, and maximization of growth and development must be considered in determining the optimal time for transplantation. Immunizations should be up to date, and the immune status of both the donor and the recipient with regard to Epstein-Barr virus, cytomegalovirus, human immunodeficiency virus, and hepatitis A, B, and C must be known. Prednisone, imuran, cyclosporine, and T cell antibodies remain the mainstay of immunosuppression. However, new therapies, such as FK-506, rapamycin, mofetil, brequinar, leflunomide, and human leukocyte antigen-derived peptides, are under investigation for use in transplantation. Complications, including infection, rejection, and malignancy, continue to be problematic in pediatric renal transplantation. Although patient and graft survival has improved over time, outcomes in pediatric renal transplantation continue to lag behind those in young adults. PMID- 8620370 TI - Urinary tract reconstruction in children undergoing renal transplantation. AB - In children, congenital urinary tract anomalies contribute to end-stage renal disease in 20% to 30% of cases. As more and more children with myelomeningocele, posterior urethral valves, prune belly syndrome, and other serious congenital anomalies of the urinary tract survive early infancy, more of these patients will be in need of renal transplantation. Of these, a significant proportion will have persisting abnormal anatomic and physiological characteristics of the urinary tract requiring reconstructive surgery before transplantation. Before undertaking these procedures, urologic evaluation should be performed in all such children. Comprehensive evaluation includes a careful history and physical examination with radiological imaging of the urinary tract (renal ultrasonography and voiding cystourethrography). In selected instances, further evaluation of bladder function and urethral anatomy may require urodynamic evaluation or cystourethroscopy. The goal of these investigations is to ensure that the bladder will hold urine at a low intravesical pressure during the storage phase and that it can be evacuated with certainty. This presentation focuses on the pretransplantation evaluation and the various possible urinary tract reconstructions that may be performed in children destined for renal transplantation. PMID- 8620371 TI - Working with noncompliant and abusive dialysis patients: practical strategies based on ethics and the law. AB - The patient population in dialysis facilities today reflects common societal problems such as human immunodeficiency virus infection, illicit drug use, distrust of and disrespect for authority, and a propensity toward violence. An increase in calls from dialysis units for guidance in dealing with noncompliant and abusive patients prompted ESRD Network 5 to examine this problem and develop an educational program, "Working with Noncompliant and Abusive Patients." This article provides an overview of the ESRD Network 5 study of the ethical, legal, psychosocial, and administrative aspects of this problem, presents practical strategies for working with such patients, and demonstrates the application of these strategies in three cases. It emphasizes the importance for dialysis units of four elements in the successful treatment of such patients: instruction for all levels of dialysis staff; a team approach; written policies; and patient education at the time of admission about these policies, including the consequences of verbal and physical abuse and the circumstances under which patients will be discharged from the dialysis unit. PMID- 8620372 TI - From the patient's desk. Twenty-seven years and counting. PMID- 8620373 TI - How to create presentations for case presentations, staff meetings, and lectures. PMID- 8620374 TI - Clinical changes in the gingiva as a result of at-home bleaching. AB - An investigative study was performed to evaluate changes in gingival health with the application of a 10% carbamide peroxide bleaching gel using exposure times of 2 and 7 hours (overnight). The presence or absence of gingival inflammation was recorded using the Loe and Silness Gingival Index at intervals over a 28-day period. The results showed no statistical significance between the preoperative gingival index scores and those recorded at any point during the study, regardless of exposure time. PMID- 8620375 TI - Management of oral pain in patients with malignant diseases. AB - Painful oral complications are common in patients undergoing treatment for malignant diseases. Correct diagnosis of painful oral lesions, as well as identification of etiological factors and appropriate therapy, are imperative for this group of patients. Oral mucosites, infections, and graft-versus-host disease and their respective current and prospective methods of management are reviewed. PMID- 8620376 TI - Maxillofacial prosthetic rehabilitation combined with plastic and reconstructive surgery. PMID- 8620377 TI - Carrier interpretation of benefits vs provider fees: Part 2. PMID- 8620378 TI - Pin-retained restorations for severely worn posterior teeth. AB - Restoring severely worn posterior teeth with cast restorations presents a challenge to the dentist. The shortened clinical crowns have insufficient axial wall length for adequate retention and resistance form. Pins, along with other preparation retentive forms, can avoid periodontal support loss created by lengthening procedures. This article describes pin preparation of severely worn mandibular premolars and molar restoration by pin-retained gold castings. These and other teeth are used for retention of a mandibular removable partial denture. PMID- 8620379 TI - Film factors relevant to color reproduction requirements for use in the dental office. PMID- 8620380 TI - The boy with the golden tooth. PMID- 8620381 TI - An experimental technique to repair cracked teeth using calcium phosphate, melted by a laser beam: an in vitro evaluation. AB - Using a neodymium: yttrium-aluminum-garnet laser beam to seal vertical root fracture lines with tricalcium phosphate paste represents an alternative treatment for cracked teeth with noted clinical results. This article describes a study of the permeability of molten crystals of hydroxyapatite in the dentin of a cracked root after crack lines have been filled with a preparation of tricalcium phosphate melted by a neodymium: yttrium-aluminum-garnet laser beam. The morphology of the sealed cracks was analyzed under a scanning electron microscope that showed a deep fusion of tricalcium phosphate along crack lines. PMID- 8620382 TI - HIV-positive health-care professionals: perspectives and recommendations. PMID- 8620383 TI - Multiple-diastema porcelain laminate veneers: a case study. AB - There is some controversy about whether or not tooth preparation is needed before placing porcelain laminate veneers. Without adequate preparation, the added surface contour may increase the risk of periodontal disease. When properly executed, veneers for cosmetic dentistry should be reversible. Many teeth that could have been saved with the use of a conservative laminate veneer are destroyed by full crown reduction. The case presented in this article illustrates the preparation of six maxillary anteriors with with pronounced spaces between them for laminate veneer placement. PMID- 8620384 TI - The future of solo dental practice: another perspective. PMID- 8620385 TI - Use and misuse of disinfectants. PMID- 8620386 TI - Noncarious dental "abfraction" lesions in an aging population. AB - A new classification for noncarious dental lesions has evolved from the dental literature. The name given to these lesions, dental "abfractions," is a theory propounding tooth fatigue, flexure, and deformation through biomechanical loading of tooth structure, primarily at the cervical regions of the dentition. These lesions are typically wedge shaped with sharp line angles, but occlusal abfractions have been observed as circular invaginations. Dental abfractions can occur alone and are sometimes associated with toothbrush abrasion and erosion from endogenous or exogenous acids. Treatment consists of the application of composite resin or glass-ionomer cement restorations and/or the discontinuance of the etiology of these lesions. If esthetics are not a primary concern of the patient and the tooth is not structurally compromised, many of these lesions can be observed, provided that the patient is informed that bruxism or malocclusion problems exist. PMID- 8620387 TI - Insurance reimbursement matters. PMID- 8620389 TI - A computer-prepared employee manual for the 21st century. PMID- 8620388 TI - Xerostomia: recognition and management of hypofunction of the salivary glands. AB - Unfortunately, xerostomia manifests itself in the presence of multiple chronic and acute disease states. Hyposalivation can not only persist as a side effect related to the medical management and concomitant drug use in the treatment of the disease, but it may functionally impair our patients and affect their overall health and quality of life. It is the intent of this article to assist the clinician in the recognition of those disease entities that may induce the side effect of xerostomia. In addition, protocols for the management of decreased salivary flow for each of these conditions are reviewed. PMID- 8620390 TI - Obstructive sleep apnea: a mandibular positioning device for treatment and diagnosis of an obstruction site. AB - Obstructive sleep apnea (OSA) is a common condition with serious health and social consequences for millions of people. If untreated, it has significant effects on mortality, particularly by contributing to the rate of hypertension, stroke, and cardiac pathology. Standard therapy involves the use of a nasal continuous positive airway pressure device during sleep. This keeps the upper airway from collapsing, avoiding the cause of apneas and hypopneas. The inconvenience and side effects of the device make compliance with its use less than ideal. There are several surgical techniques that have been developed to correct OSA. These techniques have had variable success rates and are accompanied by some morbidity and mortality. Intraoral devices that position the mandible forward show promise for increasing the hypopharyngeal airway. A technique for physiologically determining an effective jaw relationship is described, as is a design for a device that maximizes nighttime retention. Radiographic analyses in the upright and supine positions are used to determine adequate airway improvement. The intraoral device can be used as a diagnostic tool to select patients who will most likely benefit from surgery. PMID- 8620391 TI - A 10-year literature review of a split-shanked threaded post. AB - A review of 10 years of literature on prefabricated threaded and passive post systems is presented along with recent literature supporting the validity of the split-shanked threaded post design, Flexi-Post. It is important to note that a split-shanked threaded post is altogether different from a solid-shanked threaded post. The research discussed in this article confirms the high retention of the Flexi-Post and dispels the notion that high retention must be accompanied by high stress. PMID- 8620392 TI - The clinician who helped introduce osseointegration to periodontists. PMID- 8620393 TI - Treatment planning and site development for the implant-assisted periodontal reconstruction. AB - Implants can be used to recreate an edentulous dentition that is esthetic, comfortable, and functional by augmenting the total surface area of load carrying abutments. Traditionally, implants were placed in areas of the mouth where adequate amounts of bone were present. Today, clinicians are strategically planning and developing implant sites to optimize oral health. This article will describe methods of treatment planning and site development for the reconstruction of the implant-assisted periodontal restoration. PMID- 8620394 TI - Presurgical treatment planning for the anterior single-tooth implant restoration. AB - The anterior single-tooth implant restoration may be one of the most demanding procedures in dentistry. The edentulous area, implant anatomy, and implant component anatomy are just some of the factors that should be evaluated. Bone dimension, restorative dimension, and their relationship must also be considered. This article will review presurgical treatment planning for the anterior single tooth implant restoration. PMID- 8620395 TI - The implant periapical lesion: etiology, prevention, and treatment. AB - A classification of implant periapical lesions that separates them into inactive and infected has been suggested. The inactive form is likely to be an apical scar, resulting from a residual bone cavity created by placing an implant that was shorter than the prepared drill site. The infected focus probably occurs when an implant apex is placed in proximity to an existing infection or when a contaminated implant is placed. Bone necrosis caused by overheating during preparation may also be a causative factor. Suggested preventions of implant periapical lesions include careful management of contaminants and heat generation during implant surgery. Treatment varies according to the type of lesion. The inactive type is observed and monitored. The infected type requires surgical intervention, elimination of the infection, and an implant apical resection or implant removal depending on the extent of the infection and the stability of the implant. PMID- 8620396 TI - Considerations for single-unit esthetic implant restorations. AB - Single-tooth replacement in contemporary dentistry is often accomplished with an osseointegrated implant-supported restoration. To accomplish this in an esthetically acceptable manner requires a thorough course of treatment, including diagnosis, treatment planning, surgery and restorative and preventive care. This article discusses surgical considerations that are applicable before implant placement, as well as at the time of stage I and II surgery. Furthermore, the interrelationship between the tissues and the restoration is addressed. Preservation of the result by preventive-care protocol is shown to be essential. PMID- 8620397 TI - Primary care meets managed care--who is the gatekeeper? PMID- 8620398 TI - Restoration-driven implant placement with restoration-generated site development. AB - In any restoration or natural tooth, the surrounding soft-tissue profile plays an integral role in the final esthetics of a case. Similarly in implant restorations, it is no longer sufficient to merely attach a prosthetic device to the underlying fixture, but for optimal esthetics it has become essential for the implant site to be reconstituted in a three-dimensional approach. This invariably involves redevelopment or replacement of lost hard tissue and redevelopment of the correct soft-tissue profile, so that the implant can be placed in the desired position as determined by the restoration, while the soft-tissue profiles are in turn generated by the actual form and contours of the prosthetic device. This article addresses an approach to implant site development. PMID- 8620399 TI - Techniques for ideal implant placement in the mandibular first molar position. AB - Implant restoration of the mandibular first molar can be particularly challenging because of the heavy occlusal force exerted on this tooth. Bone quantity and quality, as well as fixture characteristics, play important roles in the long term success of any implant. In this article, techniques for the ideal placement of an implant in the lower first molar site are discussed for both abundant and moderate bone quantity and for bone quality that is either moderately or minimally dense. Placement depth and the benefit of using a commercially pure grade 4 titanium fixture for mandibular first molar restorations are also discussed. In cases where adequate supporting bone exists, wide-diameter implants can offer an advantage by anchoring the implant into more dense bone that is concentrated toward the outer edges of the underlying bone. These implants also provide greater surface area, which reduces the amount of force directed to the underlying bone and may increase the long-term prognosis of the implant. PMID- 8620400 TI - Diagnosis and evaluation of complications and failures associated with osseointegrated implants. AB - Osseointegrated implants fail for a number of reasons. Failures should be classified based on when in the sequence of therapy they occur. The two stages of therapy to consider when analyzing the causes of failure are the periods before and after loading the implant. Causes for failure during these time periods are discussed, as well as ways to avoid them. PMID- 8620401 TI - Have we found the "definitive cancer biomarker"? The diagnostic and therapeutic implications of human chorionic gonadotropin-beta expression as a key to malignancy. PMID- 8620402 TI - A 10-year experience with Japanese-type radical lymph node dissection for gastric cancer outside of Japan. AB - BACKGROUND: The prognosis for surgically treated gastric cancer remains poor in most Western countries compared with reports from Japanese investigators during the past 3 decades. METHODS: A radical surgical procedure principally to extended lymphadenectomy as defined by the Japanese Research Society for Gastric Cancer was performed prospectively from January 1984 to June 30, 1994 for 512 patients with gastric cancer, 345 of whom were treated with potentially curative surgery. Clinical, histopathologic, and surgical factors were examined for their influence on long term survival by univariate and multivariate analyses. RESULTS: Five- and 10-year survival rates for all patients were 40.5% and 34.3%, respectively, and for patients who underwent tumor resection were 45.7% and 38.6%, respectively. For patients who underwent curative surgery, 5- and 10-year adjusted survival rates were 57.7% and 44.3%, respectively, with a median survival of 96 months. Postoperative hospital mortality was 6.8%:4.9% for R-0 resected patients, 9% for R-1 and R-2 resected patients, and 13.4% for those with palliative procedures. Multivariate analysis using the Cox model identified age older than 65 years, prior total gastrectomy, an increasing number of positive lymph nodes, a high pathologic N classification, male sex, a high pT classification, and low preoperative hemoglobin level as detrimental factors with an independent influence on survival. CONCLUSION: Radical lymphadenectomy in this 10-year Austrian study yielded survival rates similar to those in Japanese investigations without sacrificing low postoperative mortality. In particular, the relatively high overall survival rates seemed to reaffirm the value of radical lymph node dissection with wide resection margins. PMID- 8620403 TI - A clinicopathologic study of 233 cases with special reference to evaluation with the MIB-1 index. AB - BACKGROUND: Few articles have analyzed the prognostic data from a large series of primary gastric lymphoma classified according to the concept of mucosa-associated lymphoid tissue (MALT). METHODS: The resected specimens from 233 patients with primary gastric lymphoma were investigated retrospectively, including immunostaining with MIB-1 (Ki-67). RESULTS: Histologically, 70 (30%) of the cases were low grade B-cell lymphoma of MALT, 27 (12%) low grade B-cell lymphoma of MALT with a focal high grade component, 100 (43%) high grade B-cell lymphoma of MALT, 15 (6%) other B-cell lymphomas, 14 (6%) T-cell lymphomas, and 7 (3%) undetermined. Macroscopically, 96 (41%) were superficial-spreading type, 100 (43%) mass-forming, 14 (6%) diffuse-infiltrating, and 23 (10%) unclassified. The MIB-1 index correlated with phenotype, histologic grade, stage, depth of invasion, and macroscopic type. A significantly better survival was noted for young patients, and those with tumors of a B-cell phenotype, histologic low grade, macroscopic superficial-spreading type, low stage, low depth of invasion, and low MIB-1 index. No significantly different survival rates were found between the patients who underwent gastric resection alone and those who also received additional chemotherapy. By Cox multivariate analysis, independent prognosticators included B-cell phenotype, low stage, and macroscopic superficial spreading type. CONCLUSIONS: In addition to stage, phenotype and macroscopic type are also important prognostic indicators of primary gastric lymphoma. Immunostaining with MIB-1 had limited independent value for predicting prognosis. PMID- 8620404 TI - How tumor stage affects surgeons' surveillance strategies after colon cancer surgery. AB - BACKGROUND: The factors that influence decision making among surgeons are not well understood. This study sought to evaluate how the tumor stage of patients subjected to potentially curative surgery for colon cancer affects the follow-up strategies used by practicing surgeons. METHODS: Hypothetical patient profiles and a detailed questionnaire based on these profiles were mailed to 2733 members of two major surgical societies. The effect of TNM Stage on the surveillance strategies chosen by the respondents was analyzed. RESULTS: Seven of the nine most commonly used surveillance modalities all were performed significantly more frequently with increasing TNM Stage. This effect persisted through 5 years of follow-up. The other two modalities (computed tomography and bone scan) were performed too infrequently for meaningful analysis. CONCLUSIONS: Surgeons performing surveillance after potentially curative surgery for otherwise healthy patients with colon cancer modify their strategies according to the patient's TNM Stage. These data should help in the design of prospective trials related to this topic. PMID- 8620405 TI - Preclinical and clinical evaluation of 5-fluorouracil biochemical modulation with folinic acid and hydroxyurea for patients with colorectal carcinoma. AB - BACKGROUND: Approximately 140,000 new cases of colorectal carcinoma will be diagnosed in 1995 in the United States, and more than one-third of these patients will die from progressive disease. Despite the modest improvement in response rate with chemotherapy, little improvement in patient survival has been noted. Consequently, the evaluation of new agents, modalities, and combinations is needed. METHODS: Two cell lines, HCT 116 and COLO 320 HSR, were treated with various concentrations of 5-fluorouracil (5-FU), folinic acid (FA), and hydroxyurea (HU). Subsequently, 41 patients with advanced, measurable metastatic colorectal carcinoma were enrolled in the study. Patients were treated with oral doses of HU (500 mg) every 8 hours on Days 1 and 2, 5-FU (400-500 mg/m2) intravenously Day 2 and FA (100 mg/m2) intravenously on Day 2 of every week for 6 consecutive weeks, followed by a 2-week rest period. All patients were evaluable for toxicity, and 40 were evaluable for response. RESULTS: In both cell lines, the combination of 5-FU/FA/HU consistently produced the best cytotoxic effect. Clinically, the maximum tolerated dose of 5-FU was established at a level of 500 mg/m2 (450 mg/m2 for patients older than 70 years of age). Ten patients experienced Grade 3 or 4 toxicity, consisting mainly of diarrhea. Eleven of 40 evaluable patients responded (three complete responses, eight partial responses), with a median survival of 12+ months and time to progression of 8.5+ months. CONCLUSION: The biochemical modulation of 5-FU with FA and HU were significantly effective in treating patients with metastatic colorectal carcinoma. Overall, this regimen was well tolerated with only moderate toxicity. Further studies incorporating intravenous HU as well as a randomized Phase III study of 5 FU/FA/HU versus 5-FU/FA are recommended. PMID- 8620406 TI - Aggressive primary hepatic lymphoma in Chinese patients. Presentation, pathologic features, and outcome. AB - BACKGROUND: Primary non-Hodgkin's lymphoma of the liver is rare. In this study, the presentation, pathologic features, and outcome of seven Chinese patients with primary hepatic lymphoma are described. METHODS: From 1984 to 1994, the clinical records of 14 Chinese patients with non-Hodgkin's lymphoma and histologically proven liver involvement were reviewed. Seven (four males, three females; median age, 54 years) were considered to have primary hepatic lymphoma. Histologic and immunohistochemical studies were performed on paraffin embedded liver tissue. RESULTS: "B" symptoms including fever (86%) and weight loss (57%) were the most striking presenting features. Hepatomegaly was present in all patients, splenomegaly in three (43%), and thrombocytopenia in six (86%). Only one patient was hepatitis B surface antigen-seropositive. None had preexisting liver disease. Histologic subtypes, though heterogeneous, were mostly unfavorable and consisted of diffuse large cell lymphoma (two patients), small lymphocytic lymphoma (one patient), lymphoblastic lymphoma (one case), mantle cell lymphoma (one patient), anaplastic large cell Ki-1 lymphoma (one patient), and hepatosplenic T-cell lymphoma (one patient). Three patients expressed B-cell and 2 expressed T-cell phenotypes. Six patients received cytotoxic chemotherapy. One had resection and one had splenectomy, but none achieved complete remission, and only one remained alive as of this writing. The median survival was 3.7 months (range, 8 days to 47.7 months). CONCLUSION: Chinese patients with primary non-Hodgkin's lymphoma of the liver have prominent "B" symptoms, disease with a highly aggressive course, a poor response to local and systemic treatment, and short survival. Hepatitis B virus infection is not a major etiologic factor for these patients. PMID- 8620408 TI - Hepatitis C virus genotypes and development of hepatocellular carcinoma. AB - BACKGROUND: Among several genotypes of hepatitis C virus (HCV), genotypes 1b (or II) and 2a (or III) are predominant in Japan. Although it has been shown that the efficacy of interferon treatment of patients with chronic hepatitis C varies with the HCV genotype, the relationship between HCV genotype and the development of hepatocellular carcinoma (HCC) has not been well described. METHODS: The genotypes and serum levels were determined for HCV-RNA in 72 patients with HCC and 131 patients without HCC, all of whom were positive for second-generation HCV antibody and HCV-RNA. In addition, clinical data from 34 patients with HCC who each had a history of blood transfusion were analyzed. RESULTS: Fifty-seven (79.2%) of 72 patients with HCC had genotype 1b HCV, whereas 101 (77.1%) of 131 patients without HCC had genotype 1b, indicating that there was no significant difference in the prevalence of genotype 1b HCV between the patients with and without HCC. Furthermore, comparison of patients with HCC with genotype 1b HCV with those with genotype 2a who had a history of blood transfusion did not differ significantly in the number of years from blood transfusion to diagnosis of HCC. Levels of HCV-RNA were not significantly different among patients with liver diseases of various stages. CONCLUSIONS: Hepatocellular carcinoma develops in patients with either genotype 2a HCV or genotype 1b HCV. A difference in genotype is not likely to be responsible for the difference in development of HCC. PMID- 8620407 TI - Epstein-Barr virus-related localized hepatic lymphoproliferative disorders after liver transplantation. AB - BACKGROUND: Localized hepatic post-transplant lymphoproliferative disease is uncommon. In such cases, lymphocyte Epstein-Barr virus (EBV) infection may promote an intrahepatic B-lymphocyte monoclonal expansion. METHODS: From 1990 to 1991, 149 patients underwent liver transplantation for various liver failures. Immunosuppressive therapy was azathioprine, cyclosporine-A, and methylprednisolone. Rejection episodes were treated by methylprednisolone bolus injection with or without OKT3 therapy. Three patients (2%), aged 38, 50, and 47 years, developed lymphoproliferative disease localized in the transplanted livers within 5 months of liver transplantation (a patient had been immunosuppressed for 3 years before the lymphoproliferative disease occurred within the third allografted liver). Diagnoses were obtained by fine needle aspiration. In situ hybridizations were performed with the kappa/lambda mRNA-kit FITC DAKO (DAKO Corporation, Carpenteria, CA) and the early mRNA-EBER oligonucleotide FITC DAKO: RESULTS: Lymphoproliferative diseases were all classified as diffuse polymorphic large cell lymphomas in the working formulation and considered as lymphoproliferative disorders with polymorphic large cells in the Frizzera classification. All large cells were CD20-positive, CD45-positive and CD45RO negative. In situ mRNA light chain hybridization demonstrated monoclonality in two cases. In all three cases, EBV mRNA was detected in large cells by early mRNA EBV (EBER) in situ hybridization. Patients were treated with doxorubicin, cyclophosphamide, vincristine, and VM26. Two patients maintained a complete remission 3 years after six cycles of chemotherapy, whereas one died of an early opportunistic infection. CONCLUSION: Epstein-Barr virus may play a special role in the pathogenesis of lymphoproliferative disorders that develop in patients who have undergone liver transplantation. PMID- 8620409 TI - Phase I/II trial of dexverapamil, epirubicin, and granulocyte-macrophage-colony stimulating factor in patients with advanced pancreatic adenocarcinoma. AB - BACKGROUND: The purpose of this study was to determine the maximum tolerated dose (MTD) of a cytotoxic regimen consisting of the second-generation chemosensitizer dexverapamil (DVPM), high dose epirubicin, and recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) in pancreatic carcinoma. PATIENTS AND METHODS: Twenty-eight previously untreated patients with locally advanced or metastatic adenocarcinoma of the pancreas were studied. Treatment consisted of oral DVPM at a dose of 1000-1200 mg/day for 3 days, epirubicin administered as an intravenous bolus injection on Day 2 with an initial dose of 90 mg/m2, and a dose of GM-CSF of 400 micrograms administered subcutaneously from Day 5s through 14. Epirubicin dose escalation levels were 90, 105, 120 and 135 mg/m2. Consecutive cohorts of four to eight patients were planned at each dose level. Treatment cycles were repeated every 3 weeks. RESULTS: Hematologic toxicity, specifically granulocytopenia, constituted the dose-limiting toxicity with an MTD of 120 mg/m2 for epirubicin. Despite routine supportive therapy with GM-CSF, four, two, and five patients experienced Grade 4 granulocytopenia during their first two treatment courses at levels 105, 120, and 135 mg/m2, respectively. Grade 4 granulocytopenia was observed in two, three, and one additional patients during subsequent courses with these levels. Nonhematologic toxicity was uncommon, generally modest, and did not correlate clearly with the anthracycline dose. Dexverapamil-related cardiovascular symptoms occurred frequently, but they never resulted in serious toxicity requiring active medical intervention or permanent discontinuation of therapy. Nine of 28 patients achieved partial responses to this therapy. Stable disease was observed in nine patients, and tumor progress occurred in 10. CONCLUSION: The MTD of epirubicin for this regimen with DVPM and GM-CSF was 120 mg/m2 every 3 weeks. Though it remains uncertain whether the encouraging response activity observed in this disease-oriented Phase I study was, in fact, due to successful modulation of multidrug resistance, these results suggest that this regimen is likely to be an effective and tolerable treatment strategy for patients with pancreatic cancer, which should be evaluated further. PMID- 8620410 TI - Radioimmunoassay for plasma glutathione S-transferase-pi and its clinical application in gastrointestinal cancer. AB - BACKGROUND: Plasma acidic glutathione S-transferase (GST-pi) concentrations in 135 patients with gastrointestinal cancer were measured by an improved radioimmunoassay (RIA). METHODS: Blood was collected into a silicified glass tube containing a specific anticoagulant. The plasma, which was separated rapidly by centrifugation with adequate force, underwent RIA procedures (double antibody and polyethylene glycol separation method). RESULTS: The improved RIA for plasma GST pi was specific and sensitive. Plasma GST-pi in 75% of patients with esophageal cancer, 64.44% of those with gastric cancer, 74.41% of those with colon cancer, 85.19% of those with hepatocellular carcinoma, and 60.00% of those with pancreatic cancer was elevated higher than 13.99 micrograms/l (mean + 1.96 standard deviation of normal controls). Plasma GST-pi levels in patients with gastric cancer with Stages I-II, III and IV disease increased in proper order. Plasma GST-pi concentrations in 57% of patients with colon cancer decreased to the normal range 14 days after surgery. Plasma GST-pi levels of patients with recurrent colon cancer were higher than of those with primary colon cancer. CONCLUSION: The procedure for specimen collection must be critical. Plasma GST-pi may be useful in diagnosing gastrointestinal cancer and in monitoring its clinical course. PMID- 8620411 TI - Alpha-1-antichymotrypsin expression in lung adenocarcinoma and its possible association with tumor progression. AB - BACKGROUND: Alpha-1-antichymotrypsin (ACT) is a serine protease inhibitor, expression of which has been shown in various tumor types, but its biologic and clinical implications in tumor tissues are obscure. The authors examined ACT expression in lung adenocarcinoma to determine its clinicopathologic and prognostic significance. METHODS: First, reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers specific for ACT and Western blotting and immunohistochemical methods using anti-ACT antibodies were performed in several lung adenocarcinoma cell lines. Secondly, ACT expression in clinical materials was examined immunohistochemically. RESULTS: By RT-PCR, Western blotting, and immunohistochemical methods, ACT synthesis was confirmed in several lung adenocarcinoma cell lines. Seventy-five (52%) of 170 surgically resected lung adenocarcinomas showed positive staining for ACT mainly in the cytoplasm, and the incidence of ACT expression was significantly higher in advanced T classification tumors (P = 0.009) or large sized tumors (P = 0.004). Tumors with a higher rate of mitosis were significantly positive for ACT expression (P = 0.023). Patients with ACT-positive adenocarcinoma had a shorter disease free survival (DFS) and a poor prognosis compared with those who were ACT-negative, most significantly among Stage I tumors (DFS, P = 0.003; overall survival, P = 0.006). In a multivariate analysis, the P value of ACT expression status in Stage I tumors was marginally significant (overall survival; P = 0.058). CONCLUSIONS: These results using cell lines suggest the potential productivity of ACT by lung adenocarcinoma cells. The data in clinical materials, combined with results of the previous report that ACT in breast cancer acts as a minor growth factor-like substance, suggest that ACT expression in lung adenocarcinoma also may be associated closely with tumor progression and especially with tumor growth. Alpha 1-antichymotrypsin expression status in Stage I tumors may be a potential independent prognostic factor. PMID- 8620412 TI - Malignancy-related pericardial effusion. 127 cases from the Roswell Park Cancer Institute. AB - BACKGROUND: Malignancy-related pericardial effusions may represent a terminal event in patients with therapeutically unresponsive disease. However, select patients with malignancies sensitive to available therapies may achieve significant improvement in palliation and long term survival with prompt recognition and appropriate intervention. METHODS: From 1968 to 1994, 150 invasive procedures were performed for the treatment or diagnosis of pericardial effusion in 127 patients with underlying malignancies. These cases were reviewed retrospectively to best identify the clinical features, appropriate diagnostic workup, and optimal therapy for this complication of malignancy. RESULTS: Dyspnea (81%) and an abnormal pulsus paradoxus (32%) were the most common symptoms. Echocardiography had a 96% diagnostic accuracy. Cytology and pericardial biopsy had sensitivities of 90% and 56%, respectively. Fifty-five percent of all effusions were malignant comprising 71% of adenocarcinomas of the lung, breast, esophagus, and unknown primary site. In 57 patients, a malignant effusion could not be determined, and no definitive etiology could be established for 74% of these effusions. Radiation-induced, infectious, and hemorrhagic pericarditis each were identified in fewer than 5% of cases. CONCLUSIONS: Subxyphoid pericardiotomy proved to be a safe and effective intervention that successfully relieved pericardial effusions in 99% of cases with recurrence and reoperation rates of 9% and 7%, respectively. Survival most closely was related to the extent of disease and its inherent chemo-/radiosensitivity, with 72% of the patients who survived longer than 1 year having breast cancer, leukemia, or lymphoma. PMID- 8620413 TI - Surgical indications for Ewing's sarcoma of the pelvis. AB - BACKGROUND: Despite advances in adjuvant therapy, Ewing's sarcoma of the pelvis remains an anatomic site with a poor prognosis. This study evaluate the role of surgery in the management of patients with pelvic Ewing's sarcoma who also received conventional radiation therapy and chemotherapy. METHODS: From May 1978 to February 1994, 19 patients with Stage IIB Ewing's sarcoma of the pelvis were treated at the UCLA Medical Center (Los Angeles, CA). There were eight lesions of the ilium, two of the sacrum, and nine involving two adjoining regions of pelvis. All patients received conventional medical management. The 19 patients were divided into two groups according to treatment modality. A group of 12 patients (Group A) had surgical resection, and their results were compared with those of another group of 7 patients (Group B) who did not have surgery. RESULTS: The 5 year cumulative survival (Kaplan-Meier method) was 39% for all patients, 51% for Group A, and 18% for Group B. The 3-year cumulative survival was 59% for all patients, 72% for Group A, and 36% for Group B. Although the survival rate of Group A seemed better than that of Group B, the difference was not statistically significant (P = 0.093, log rank method). This study also suggested that, regardless of treatment modality, the outcome of patients with lesions involving two adjoining pelvic bones was poorer than that of those with a single lesion. In Group A, the 3-year cumulative survival rate for patients with single bone lesions (n = 8) was 86% and for patients with lesions involving two adjoining pelvic bones (n = 4) was 50% (P = 0.045, log rank method). Furthermore, the statistical analysis of the combined data of the single pelvic bone lesions in UCLA and that of Mayo Clinic series (n = 16 for surgery group and n = 15 for nonsurgery group) confirmed the better results for the surgical patients, which was consistent with the results from the Mayo Clinic with an even greater significance (P < 0.002). CONCLUSION: This study demonstrates that surgery plus chemotherapy and radiation therapy is helpful for treating patients with pelvic Ewing's sarcoma so long as the tumor is limited to a single pelvic bone. PMID- 8620414 TI - High grade soft tissue sarcoma of the flexor fossae. Size rather than compartmental status determine prognosis. AB - BACKGROUND: High grade soft tissue sarcoma arising in the popliteal space, axilla, and antecubital fossae (flexor fossae tumors) have by convention been classified as extracompartmental tumors by the accepted staging and grading criteria of the Musculoskeletal Tumor Society (MSTS). Advances in neoadjuvant chemotherapy and radiation therapy have made surgical resection more feasible. The hypothesis to be tested is that compartmental status may not be of prognostic significance if the tumor is adjusted for size, histologic grade, and distant metastasis after undergoing adjuvant chemotherapy and radiation. METHODS: From June 1976 to December 1992, 22 patients with high grade soft tissue sarcomas of the flexor fossae (Group A) were treated at UCLA Medical Center. The histologic subtypes were liposarcoma (five), synovial cell sarcoma (eight), malignant fibrous histiocytoma (four), leiomyosarcoma (two), angiosarcoma (two), and rhabdomyosarcoma (one). The popliteal fossa was the location in 11, the axilla in 10, and the antecubital fossa in 1. Wide resection was attempted in all patients after preoperative chemotherapy and radiation therapy. Amputation was performed in 5 patients because of repeated or extensive recurrent tumor. A group of 77 patients (Group B) with high grade soft tissue sarcoma located within an extremity compartment were chosen to test the hypothesis that survival of patients with tumors in the flexor fossae is equal to that of patients with intracompartmental tumors of similar size and grade if both are given adjuvant therapy. This group was chosen so that histologic subtype, size, sex, and location would be similar in the two groups. The authors selected thigh and calf tumors for comparison with popliteal fossa tumors and periscapular, deltoid, and arm tumors for comparison with axilla and antecubital fossae tumors. All of these patients had similar treatment and follow-up protocols. The median follow-up of survivors in Group A was 104 months and for patients in Group B was 79 months. RESULTS: The 5-year cumulative survival rate (Kaplan-Meier method) of patients in Group A was 76%, and 67% for those in Group B. The difference was not significant. Three patients in Group A (14%) and 17 (22%) in Group B had local tumor recurrence. Eight patients in Group A (36%) and 27 (35%) in Group B had lung metastases. Age, sex, histologic subtype, and surgical margins did not affect survival outcomes, lung metastasis, and local recurrence. However, patients with larger tumors (maximum dimension > or = 8 cm or cross-sectional area > or = 40 cm2) had significantly poorer survival, more metastases, and local recurrences. CONCLUSION: Flexor fossae sarcomas do not have a poorer prognosis than extremity intracompartmental tumors when adjusted for size, distant metastasis, and histologic grade when they are treated with adjuvant radiation therapy, chemotherapy, and surgery. PMID- 8620415 TI - The expression of insulin-like growth factor and its binding protein mRNA in the endometrium of postmenopausal patients with breast cancer receiving tamoxifen. AB - BACKGROUND: Insulin-like growth factor I (IGF-I) is known to mediate estrogen effect in the uterus, whereas IGF binding proteins (IGFBPs) modulate the biologic effects of IGF-I. Tamoxifen may act via the IGF-IGFBP system in the postmenopausal endometrium. METHODS: Endometrial samples were collected from 16 postmenopausal women with breast cancer of whom 9 received tamoxifen and the remaining 7 received no hormonal treatment. The expression of messenger RNA for IGF-I, IGF-II, and the IGFBPs 1-6 was studied using dot blot and Northern blot techniques. RESULTS: Expression of mRNA for IGF-I, IGFBP-2, -3, -4, and -6 was present in endometrial specimens. The expression of IGF-I mRNA was similar in the tamoxifen-treated and control patients, whereas mRNA expression for IGF-II was not detected. The expression of IGFBP-2 and -4 mRNA predominated in the endometrium of patients who received tamoxifen. CONCLUSIONS: These findings demonstrate that the IGF-IGFBP system is present in the postmenopausal endometrium and may be modulated by tamoxifen. PMID- 8620416 TI - T lymphocytes infiltrating advanced grades of cervical neoplasia. CD8-positive cells are recruited to invasion. AB - BACKGROUND: Impaired cellular immunity appears to be a risk factor for progression of cervical neoplasia, but the immunobiology of neoplastic progression is poorly understood. The objective of this study was to characterize the subpopulations of T lymphocytes that infiltrate various grades of cervical neoplasia including metaplasia to invasive cancer in immunocompetent women. METHOD: In 65 patients with a spectrum of cervical disease ranging from normal cytology to carcinoma, the relative proportions of total T lymphocytes and CD4- or CD8-expressing (helper or cytotoxic) T lymphocyte subsets were determined by immunohistochemistry. RESULTS: When the invasive carcinoma stromal infiltrate was compared with the infiltrate of preinvasive lesions, the numbers of total T cells and the CD8-positive subset increased significantly in the invasive cancers (P < 0.005). Although immunocyte infiltrates were highly concentrated in focal clusters beneath the preinvasive squamous lesions, the CD8-positive immunocytes diffusely infiltrated the invading tumor. CONCLUSIONS: The CD8-positive T cell infiltrate far exceeded the CD4-positive cells in the invasive, but not in the preinvasive lesions, a finding that suggests that CD8 cells are recruited preferentially to cervical lesions with progression to invasion. PMID- 8620417 TI - Risks of cancer among members of families in the Gilda Radner Familial Ovarian Cancer Registry. AB - BACKGROUND: Increasing scientific and public interest in hereditary cancer syndromes has created a need for estimates of lifetime cancer risks among members of families with such syndromes. METHODS: Data from the Gilda Radner Familial Ovarian Cancer Registry were used to evaluate risk for cancers of the breast, cervix, uterus, colorectum, and prostate in members of 143 families containing three or more reported cases of ovarian cancer among first- or second-degree relatives. These risks were compared with those that were expected based on general population rates obtained from the Connecticut Tumor Registry. RESULTS: Overall, family members' risk of cancer at any nonovarian site was 1.5 times that of the general population (P < 0.001). Among female members, risk for cancer of the breast was 2.5 times that of the general population. Risk for cancer of the uterus was 5 times that of the general population and increased with increasing number of first-degree relatives with ovarian cancer. Among male family members having three or more first-degree relatives with ovarian cancer, prostate cancer risk was 4.5 times that of the general population. No excess risks were observed for cancer of the colorectum. CONCLUSIONS: These data support previous reports of coaggregation of cancer of the breast, uterus and ovary, and suggest coaggregation between cancer of the ovary and prostate. Differences in cancer risk profiles observed in these families with multiple ovarian cancer and in carriers of the gene BRCA1 suggest that hereditary ovarian cancer is genetically heterogeneous. PMID- 8620418 TI - Leiomyosarcoma of the prostate. Report of 23 cases. AB - BACKGROUND: Leiomyosarcoma of the prostate is a rare neoplasm that accounts for less than 0.1% of prostate malignancies. Previous reports of this neoplasm consisted of single case studies or small series, often combined with cases of rhabdomyosarcoma. The relationship of prognosis with histologic and immunohistochemical findings has not, to the authors' knowledge, been described in a large series of cases, and the efficacy of various treatments is uncertain. METHODS: The authors undertook a clinicopathologic study of all cases of prostate leiomyosarcoma observed at their institution from 1929 to 1994. Twenty-three cases were retrieved from the files of the Department of Pathology, Mayo Clinic (Rochester, MN), and clinical follow-up was available for 14. Immunohistochemical studies, including actin, desmin, S-100 protein, keratin, and vimentin were performed for 18 cases. RESULTS: Patients ranged in age from 41 to 78 years, with a mean of 61 years. Presenting symptoms included urinary obstruction (100%), perineal pain (25%), burning on ejaculation (7%), and weight loss (7%). The neoplasms ranged from 3.3 to 21 cm (mean, 9 cm) in greatest dimension and were often associated with necrosis. Seven tumors were Grade 2, 10 Grade 3 and 6 Grade 4 (Broders' grading system; scale, 1-4). Mitotic figure counts varied from 2 to 24 per 10 high power fields. Fifteen of 15 (100%) cases were immunoreactive for vimentin, 10 of 16 (63%) were immunoreactive for actin, and 3 of 15 (20%) were weakly reactive for desmin. Keratin expression was observed in 4 of 15 cases (27%), and S-100 protein was negative in all cases. Treatment varied, and usually included a combination of radiation therapy, chemotherapy, and radical prostatectomy or cystoprostatecomy. Follow-up ranged from 2 to 72 months, with a mean of 19 months. Ten patients died from tumor 3 to 72 months (mean, 22 months) after diagnosis. Four patients were alive, including three with residual tumor and one without evidence of tumor at 2, 4, 30, and 4.5 months, respectively. Local recurrence occurred in 10 of 11 patients, including 5 who had gross residual tumor present after surgery. Metastases developed up to 40 months after surgery (mean, 10.3 months), and most frequently involved the lungs. CONCLUSIONS: These findings indicate that prostate leiomyosarcoma has a varied histologic appearance ranging from spindled cell neoplasm reminiscent of smooth muscle to pleomorphic sarcoma. Epithelioid features may be present. Most tumors are immunoreactive with antibodies to vimentin and actin, and reactivity with antikeratin antibodies does not exclude the diagnosis of leiomyosarcoma. Prostate leiomyosarcoma has a poor prognosis, although the length of survival is variable. Radical surgery was the treatment of choice in the current series, but complete excision was difficult in most cases and did not result in cure. PMID- 8620419 TI - The antiandrogen withdrawal syndrome. Experience in a large cohort of unselected patients with advanced prostate cancer. AB - BACKGROUND: Flutamide withdrawal has been reported to be therapeutically efficacious for patients with hormone-refractory prostate cancer, with a reported prostate specific antigen (PSA) response rate of 29%. METHODS: to evaluate the results of flutamide withdrawal in a large group of unselected patients, the medical records of 107 consecutive patients with metastatic prostate cancer who developed progressive disease while receiving flutamide therapy were reviewed retrospectively. Flutamide withdrawal was undertaken at the time of disease progression. RESULTS: Eighty-two patients were evaluable. Of these, three had a > 80% fall in PSA value, and another nine had a > 50% decrease, for a response proportion of 14.6% (95% confidence interval 7.8%-24.2%). The median response duration was 3.5 months (range, 1-12+ months). Eight of patients treated with combined androgen blockade at the time of diagnosis of metastatic disease had a response (14%), whereas 4/25 responses (16%) were noted in patients in whom flutamide was added later, at the time of first progression. When patients who responded were compared with patients who did not respond, there was not a significant difference in age, pretreatment PSA level, type of gonadal androgen deprivation, or the likelihood of prior combined androgen blockade versus late addition of flutamide. The duration of prior therapy with flutamide was longer in patients who responded (21.5 vs. 12.0 months). CONCLUSIONS: These findings confirm the flutamide withdrawal phenomenon in a large group of unselected patients, although its frequency is not as high as previously reported. In contrast to earlier reports, whether patients have had initial hormonal therapy with combined androgen blockade or monotherapy does not appear to be predictive of the likelihood of response to antiandrogen withdrawal. PMID- 8620420 TI - Serum acute phase reactants and prognosis in renal cell carcinoma. AB - BACKGROUND: Inflammatory parameters as acute phase proteins commonly are elevated in patients with renal cell carcinoma. Some of these acute phase reactants have been proposed to influence survival. METHODS: In 170 patients with renal cell carcinoma, the authors studied six acute phase reactant parameters (erythrocyte sedimentation rate [ESR], C-reactive protein, haptoglobin, ferritin, orosomucoid, and alpha 1-antitrypsin) that were compared with stage and grade. RESULTS: The acute phase reactants correlated well with each other and with stage and grade. All acute phase reactants separately were found to be significant prognostic factors of survival using the log rank test. However, when a multivariate Cox analysis was performed, only stage, grade, and ESR were identified as independent prognostic factors, whereas the other factors were not. CONCLUSIONS: The study suggests that all acute phase reactants separately were found to be significant univariate prognostic factors, but in a multivariate analysis, ESR was the only independent prognostic parameter for survival. PMID- 8620421 TI - The role of nuclear morphometry for predicting disease outcome in patients with localized renal cell carcinoma. AB - BACKGROUND: More than one-third of patients with localized renal cell carcinoma (RCC) will have disease progression after nephrectomy. Present histopathologic variables cannot accurately predict the outcome of individual patients. METHODS: Nuclear morphometry was performed by an image analyzer on histologic sections from 39 specimens of pathologic T1 and T2 classification RCC. All patients underwent radical nephrectomy and were followed for a mean of 7.6 years. A univariate analysis and then a multivariate stepwise regression method were used to correlate results with patients' outcome. RESULTS: The best predictors of disease free interval were mean nuclear elongation factor (MNEF) (P = 0.023), mean nuclear regularity factor (MNRF) (P = 0.034), and mean nuclear area (MNA) (N = 0.038). Univariate analysis identified a significant correlation between patient survival and MNEF (P = 0.009), MNRF (P = 0.020) and MNA (P = 0.023). Combination of MNEF and MNA was even more strongly associated with survival (P = 0.0013). Multivariate analysis revealed that MNA (P = 0.044) and MNEF (P = 0.045) correlated independently with survival. CONCLUSION: These results suggest that nuclear morphometry provides objective independent prognostic information for patients with localized RCC. PMID- 8620422 TI - Cancer occurrence among dyestuff workers exposed to aromatic amines. A long term follow-up study. AB - BACKGROUND: Although the occupational exposure to some aromatic amines is recognized to cause bladder carcinoma, the long term effect of such exposure on the risk for disease, including other malignant tumors, remains unknown. METHODS: A total of 442 dyestuff workers exposed to one or more substances including benzidine (BZ), beta-naphthylamine (beta-N), alpha-naphthylamine (alpha-N), and dianisidine were followed completely until December 1992 (average time since first exposure, 39.4 years). Besides the underlying cause of death, the incidence of urothelial carcinoma was determined by periodic urologic screenings. RESULTS: Analyses of site-specific cancer mortality revealed a remarkable increased risk for bladder carcinoma for those engaged in BZ manufacture (standardized mortality ratio [SMR] = 63.6), BZ use (SMR = 27.0) and beta N manufacture (SMR = 48.4), but not for those who were exposed to alpha-N. The increased risk of cancer mortality for other organs was not significant for any exposure classes. The crude incidence rate per 1000 person-years of bladder carcinoma was estimated to be 8.7 for those engaged in BZ manufacture, 2.9 in BZ use, 7.7 in beta-N manufacture and 1.0 in beta-N use. Regardless of the class or type of exposure, the adjusted incidence rate of urothelial carcinoma increased with the duration of exposure. The adjusted incidence rate for BZ manufacture remained high (3.8-12.8) during the entire observation period, whereas that for BZ use increased from 0.0 to 4.4 as the time since first exposure increased from less than 10 years to 30+ years. CONCLUSIONS: Occupational exposure to either BZ or beta-N demonstrated an extremely strong and prolonged effect on workers' risk for urothelial carcinoma, particularly for bladder carcinoma, but not for malignant neoplasms of other organs. PMID- 8620423 TI - Spinal metastases from solid tumors. Analysis of factors affecting survival. AB - BACKGROUND: Factors affecting survival were determined for 109 patients with thoracic spine metastases and cord compression. Lung, prostate, and breast were the most common primary sites (78%). All patients had surgical decompression of the spinal cord, and 99% received radiotherapy. METHODS: Survival was determined based on anatomic site of primary carcinoma, preoperative neurologic deficit, extent of disease, number of vertebral bodies involved, tumor location (site of cord compression), and age. The respective compounding weight of the negative prognostic factors also was analyzed in terms of survival. RESULTS: The overall median survival was 10 months. Patients preoperatively ambulatory survived statistically significantly longer than nonambulatory patients or those with sphincter incontinence (P = 3.469 x 10(-6)). Patients with renal cell carcinoma survived the longest, followed by those with breast, prostate, lung, and colon cancer. Patients with breast cancer survived statistically longer than those with lung cancer (P = 0.039). Patients with one vertebral body involved survived statistically significantly longer than patients with multiple vertebral level involvement (P = 0.027). Extent of disease, age, and tumor location did not significantly influence survival. In patients with vertebral column disease, the presence of two or more poor prognostic indicators (leg strength 0/5-3/5, lung or colon cancer, multiple vertebral body involvement), had a compounding adverse effect on survival. CONCLUSIONS: For patients with spinal metastases and cord compression, the factors found to affect survival include preoperative neurological status, anatomic site of primary carcinoma, and number of vertebral bodies involved. Patients with vertebral column disease and two or more of the poor prognostic indicators have a short life expectancy, and, therefore, radical surgery is not recommended because the benefits may not be substantial. PMID- 8620424 TI - Diagnostic accuracy of ultrasound and computed tomography in the staging of Hodgkin's disease. Verification by laparotomy in 100 cases. AB - BACKGROUND: A staging laparotomy still is considered the gold standard to detect occult abdominal involvement in Hodgkin's disease. Computed tomography and ultrasound are routinely available for diagnostic imaging. To the authors' knowledge, the exact contribution of ultrasound for the staging of Hodgkin's disease has not been reported in a large series of patients before this study. METHODS: The diagnostic accuracy of abdominal ultrasound was compared with that of computed tomography and laparotomy in 100 patients with biopsy-proven Hodgkin's disease. Liver, spleen, paraaortic, and iliac lymph nodes were evaluated separately. RESULTS: Seventeen patients had a higher disease stage after surgery (17%). Considering only patients without known abdominal disease (supradiaphragmatic involvement), 14/79 (18%) had a positive staging laparotomy. Ultrasound had superior sensitivity for detecting splenic involvement with Hodgkin's disease (sensitivity, 63% compared with 37% for computed tomography). The specificity of both methods for detecting splenic disease was identical (99% vs. 96%). Inhomogeneities of structure or small nodular infiltrates were detected preferentially by ultrasound. Hepatic involvement also was visualized better by ultrasound than by computed tomography. Lymph nodes at the splenic hilus were recognized by both methods with identical sensitivity (64% vs. 62%). Paraaortic and iliac lymph nodes were recognized with greater sensitivity by computed tomography than by ultrasound (sensitivity, 93% and 100% vs. 77% and 67%, respectively). CONCLUSIONS: These results indicate that ultrasound and computed tomography each have their weaknesses and strengths and therefore should be combined, if possible. Ultrasound is the fastest and least invasive method and has particular accuracy for detecting splenic involvement, whereas computed tomography is more accurate in detecting involvement of paraaortic or iliac lymph node. If cost is important in the staging of Hodgkin's disease and if computed tomography is considered the standard, patients whose results are negative by computed tomography should be examined by ultrasound, focusing on splenic texture and size. Even in the era of combined modality treatment, surgical staging may be necessary to detect occult abdominal disease in a certain number of cases. PMID- 8620425 TI - Human chorionic gonadotropin-beta subunit gene expression in cultured human fetal and cancer cells of different types and origins. AB - BACKGROUND: The authors' previous investigations using living cultured human cancer cells and cells isolated from cancer tissues, analytical flow cytometry, and monoclonal antibodies directed to epitopes located in five different sites of the human chorionic gonadotropin (hCG) molecule, identified the presence of membrane-associated hCG, its subunits and fragments, by cells from all cancers, irrespective of type and origin, indicating that the expression of these sialoglycoproteins is a common phenotypic characteristic of cancer. Although benign neoplasms do not express these compounds, cultured human embryonic and fetal cells also express the same materials. To corroborate these findings, five fetal cell lines and 28 cancer cell lines were randomly selected from those previously studied, to determine the presence of translatable levels of hCG-beta (hCG beta) mRNA. METHODS: All cell lines were grown under identical conditions. Determination of hCG beta mRNA was made by extracting the total RNA from the cells, followed by synthesis of cDNA with RNase H- reverse transcriptase and polymerase chain reaction amplification using specific hCG beta-luteinizing hormone-beta (hLH beta) primers. The presence of amplified hCG beta cDNA was corroborated by hybridization of the product with an hCG beta-specific oligonucleotide and Southern blot analyses of the hybridization products. Gestational choriocarcinoma cells and HeLa adenocarcinoma of cervical cells, known producers of biologically active hCG, were positive control subjects, and human pituitary cells were used as negative control subjects. RESULTS: The results showed single and multiple hCG beta gene activation by the fetal cells and the different types of cancer, indicating that at any given time, there is the possibility of activation of as many as four genes of the six genes of the hCG beta-hLH beta gene cluster, even though alternative gene splicing cannot be ruled out. CONCLUSIONS: In addition to the authors' previous findings, the results of these studies support the concept that cancer is a problem of development and differentiation, and, to the authors' knowledge, prove definitively for the first time that synthesis and expression of hCG, its subunits, and its fragments, is a common biochemical denominator of cancer, providing the scientific basis for studies of its prevention and/or control by active and/or passive immunization against these sialoglycoproteins. PMID- 8620426 TI - A 15-year experience with chemotherapy of patients with paraganglioma. AB - BACKGROUND: Paraganglioma is a rare tumor thought to arise from the neuroectodermally derived paraganglionic cells that are dispersed widely along the autonomic ganglia. They can metastasize to bones, lymph nodes, and lungs and occasionally present with spinal cord compression. Up to 60% of retroperitoneal paragangliomas can be functional, with symptoms and signs of norepinephrine overproduction. METHOD: A 15-year experience with chemotherapy of patients with paraganglioma was reviewed. The patient population was identified through a search of the data-base maintained by the Departments of Melanoma-Sarcoma Medical Oncology and Pathology at the University of Texas M.D. Anderson Cancer Center (Houston, TX). RESULTS: Thirteen of 84 patients with histologically confirmed diagnosis of paraganglioma were treated with chemotherapy. The median age was 42 years (range, 25-67 years); there were eight males and five females. Primary sites included retroperitoneum (seven patients), head and neck (two patients), pelvis, bladder, mediastinum, and paravertebral (one patient each). Twelve patients received chemotherapy for metastatic disease, and 1 had an unresectable mediastinal primary tumor. Eleven patients received cyclophosphamide, doxorubicin, and DTIC/dacarbazine (CyADIC)/cyclophosphamide, vincristine, doxorubicin and DTIC/dacarbazine (CyVADIC), and 2 received doxorubicin and dacarbazine (ADIC) at standard doses for a median of 4 cycles (range, 2-12 cycles). Six of 13 patients achieved an objective partial remission (response rate = 46%, 95% confidence interval = 19-73%); 6 other patients had stable disease, and one developed progressive disease. At the time of last follow-up, eight patients were alive with disease, four died, and one patient was alive with no evidence of disease. The median follow-up from diagnosis was 45 months (range, 12-300 months). CONCLUSION: Cyclophosphamide, doxorubicin, dacarbazine chemotherapy is active in the treatment of patients with paraganglioma. PMID- 8620427 TI - Epstein-Barr virus-associated leiomyosarcomas in liver transplantation recipients. Origin from either donor or recipient tissue. AB - BACKGROUND: Leiomyosarcoma, a mesenchymal malignancy with smooth muscle differentiation, is extremely rare in children. Immunosuppression, due to either antirejection medication in organ transplantation recipients or human immunodeficiency virus infection (HIV), appears to constitute a predisposition. METHODS: Two cases of leiomyosarcoma in pediatric liver transplantation recipients were investigated and compared clinically with respect to site of origin and course of the disease and pathologically by routine histology and electron microscopy, by forensic DNA methodology for origin from donor or recipient tissue, and by EBER-1 in situ hybridization for evidence of latent Epstein-Barr virus (EBV) infection. RESULTS: A 9-year-old male developed a high grade, poorly differentiated leiomyosarcoma in his allografted liver 2 years after transplantation, and despite antineoplastic chemotherapy, he died of metastatic disease. The genotype of his tumor indicated an origin from allografted tissue. A 12-year-old female had a low grade retroperitoneal leiomyosarcoma involving the superior mesenteric vein. After resection, she remained disease free without chemotherapy. The genotype of her tumor indicated an origin from native tissue. In both tumors, latent EBV infection was documented. CONCLUSIONS: Neoplastic smooth muscle proliferation in immunosuppressed liver transplantation recipients is analogous to the more common posttransplantation lymphoproliferative disorder in involving transformation of either engrafted donor tissue or recipient tissue elsewhere in the body, in displaying a wide spectrum of histologic differentiation, grade and clinical behavior, and in exhibiting evidence of latent EBV infection. PMID- 8620428 TI - p53 mutations in gastric cancers from Taiwan. AB - Mutations of the p53 gene were investigated in 80 surgical specimens of primary gastric cancer by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis. Mutations were detected in 18 tumors (22.5%) and localized to exons 5, 7 and 8. Mutations did not follow a random distribution among different subtypes, but instead clustered in the group of papillary adenocarcinomas, in which 7/12 (58.3%) cases were mutated. Positivity for p53 mutation was significantly higher in intestinal-type (37.5%) than in diffuse-type carcinomas (12.5%). These results suggest that gene alterations of p53 are not rare and may participate in the carcinogenesis of intestinal-type carcinomas of the stomach. Twenty of 21 p53 mutations were represented by single nucleotide changes, mostly missense mutations (19 events) and one nonsense mutation. Transversional mutations constitute the majority of p53 mutations (65%) and only 20% of mutations show G:C to A:T transitions. It is possible that the etiologies of gastric cancer in different geographical areas are different. PMID- 8620429 TI - Effect of glycosylation of a synthetic MUC1 mucin-core-related peptide on recognition by anti-mucin antibodies. AB - Human epithelial mucins are heterogeneously glycosylated proteins associated with breast and ovarian cancer. Several peptide-reactive anti-mucin MUC1 monoclonal antibodies are used in experimental and diagnostic assays but it is not known how glycosylation of the mucin influences antibody recognition. In this report we show that increasing glycosylation of a synthetic 25-amino acid fragment of the MUC1 core protein with N-acetylgalactosamine (GalNAc) elicits different responses in its recognition by two anti-MUC1 antibodies, C595 and HMFG1. We propose that increasing glycosylation of the synthetic mucin fragment produces an alteration in the structure of the epitope which enhances binding in C595, but not in HMFG1. PMID- 8620430 TI - Effect of the length of alkyl chain on the cytochrome P450 dependent metabolism of N-diakylnitrosamines. AB - Liver microsomes from control and treated rats (P4501A, 2B, 2E1-induced) metabolize at variable metabolic rates eight N-nitroso-di-n-alkylamines, including five symmetrical (N-nitroso-dimethyl, -diethyl, -dipropyl, -dibutyl and -diamyl-amines) and four asymmetrical (N-nitrosomethylethyl, methylpropyl, methylbutyl, and methylamyl-amines), into aldehydes. Thus, the longer the alkyl chain of symmetrical N-nitrosamines, the smaller was the metabolic rate of the corresponding aldehyde formation. The chain length of the alkyl group of N nitroso-methylalkylamines modified the oxidation of the alkyl moiety: the oxidation by CYP2E1 decreased as the n-alkyl chain length increased and conversely for the oxidation by CYP1A and CYP2B. Finally, the longer the n-alkyl chain length of asymmetrical N-nitrosamines, the greater was the oxidation of methyl groups. PMID- 8620431 TI - Isolation of spindle-shaped cell populations from primary cultures of Kaposi's sarcoma of different stage. AB - Cell lines derived both from sporadic and epidemic KS biopsies show similar characteristics: a mixture of mesenchymal and vascular markers as well as production of factors which recruit endothelial cells in vitro and induce neoangiogenesis in vivo. Most established KS spindle cell strains are derived from patch or plaque stage KS lesions, which are easily collected during routine biopsies. Here we have characterized KS-derived spindle cell lines obtained from the four different stages typical of KS progression: angiomatous macula, patch, plaque and nodular KS to show if the similar features of our KS cell lines are linked to a particular stage of progression or to an in vitro selection/differentiation during KS cell culture. These four KS cell lines have shown the same pattern of characterization as the previous established KS cell lines, apart from an early selection of the spindle cell population we have also observed an easy inducible phenotypic differentiation through a myofibroblastic spindle cell type simply plating cells on gelatin-coated flasks. These data confirm the hypothesis of spindle cell selection in culture and the possible differentiation of these mesenchymal cells. PMID- 8620432 TI - Alterations in lymphocyte subsets in blood may predict resectability in carcinoma of cardia or oesophagus. AB - Impaired immune responses in patients with carcinoma of cardia or oesophagus have previously been reported. However, we do not know whether resectability correlates with specific immunological variables. Immunological assessment was performed in 35 such cancer patients including measurement of total T cells (CD3+) and T cell subsets (CD4+ and CD8+), NK cells (CD16+) and B cells (CD19+) in blood. In vitro lymphocyte responses to phytohemagglutinin (PHA) separated from peripheral blood were quantitated. The numbers in peripheral blood of both total T cells (CD3+) and B lymphocytes (CD19+) were significantly lower in the inoperable patients compared to resected patients (P < 0.01). The number of NK cells (CD16+) was, however, not significantly lower in the inoperable patients compared to the patients operated for cure. Lymphocyte responses to PHA in vitro were similar in resectable and non-resectable patients, but significantly lower in inoperable patients compared to the controls (P < 0.01). In conclusion, resectability in carcinoma of cardia or oesophagus is associated with changes in both T (CD3+) and B (CD19+) cell subsets. PMID- 8620434 TI - Activity of creatine kinase MB-isoenzyme in rat serum after heart irradiation and/or farmorubicin (4'-epidoxorubicin) treatment. AB - Activity of creatine kinase (CK, EC 2.7.3.2) and its CK-MB isoenzyme were evaluated in rat serum after external irradiation of the heart with 20 Gy given as A single dose or 4 x 5 Gy and also after treating the animals with farmorubicin 10 mg/kg in a single dose of 4 x 2.5 mg/kg. Fractionated irradiation or repeated injection of farmorubicin induced high five-fold transient increment of CK-MB isoenzyme activity in rat serum. Combined treatment (4 x 5 Gy heart irradiation, week 1, after 2 days pause 4 x 2.5 mg/kg farmorubicin, week 2) showed increase of MB isoenzyme activity in serum roughly comparable with that measured in animals treated with farmorubicin alone. The behaviour of MB isoenzyme activity after single doses of radiation 1 x 20 Gy or farmorubicin 1 x 10 mg/kg was dissimilar to that after repeated doses and was generally lower. Specially, a high single dose of farmorubicin led at first to suppression of CK MB isoenzyme activity and then to an increase, however, slower than after fractionated treatment. Generally, elevation of CK-MB isoenzyme activity in rat serum after heart irradiation and farmorubicin injection (scheme independent) was many times higher than the total CK activity increment. Results suggest that estimation of CK-MB isoenzyme activity in serum is more sensitive and definitive evidence of early cardiac damage exerted by gamma-rays and/or farmorubicin than total CK activity. PMID- 8620433 TI - Rosemary extract and carnosol stimulate rat liver glutathione-S-transferase and quinone reductase activities. AB - The effects of dietary intake and intraperitoneal (i.p.) administration of an extract of the spice rosemary and of the rosemary constituent carnosol on the liver activities of glutathione-S-transferase (GST) and NAD(P)H-quinone reductase (QR) in the female rat were evaluated. Rosemary extract at concentrations from 0.25 to 1.0% (by wt.) in the diet resulted in a significant 3.5- to 4.5-fold increase in liver GST and a 3.3- to 4.0-fold increase in liver QR activities compared to controls. Carnosol supplemented in the diet at levels from 0.01 to 1.0% did not enhance GST activity. When rosemary extract and carnosol were administered i.p. there was a significant increase in liver GST and QR activities. The injection of rosemary extract (200 mg/kg) was associated with 1.5 fold and 3.2-fold increases in GST and QR activities, respectively, compared to controls. The injection of carnosol at doses from 100 to 400 mg/kg was associated with 1.6- to 1.9-fold increases in GST activity and 3.1- to 4.8-fold increases in QR activity, compared to controls. These data indicate that rosemary extract in the diet or injected i.p. and carnosol administered i.p. are effective enhancers of the in vivo activity of liver GST and QR in the female rat. PMID- 8620435 TI - The prognostic importance of fibronectin and sialic acid levels in human pituitary adenomas. AB - In this study, fibronectin and sialic acid levels were determined in human pituitary adenomas. The mean fibronectin and sialic acid levels for human pituitary adenomas were found to be 31.64 +/- 15.82 microgram/mg protein and 21.90 +/- 9.82 microgram/mg protein, respectively, versus 6.30 +/- 2.96 microgram/mg protein and 9.88 +/- 2.81 microgram/mg protein for the normal brain tissues. Fibronectin and sialic acid levels were significantly higher (P < 0.001) in human pituitary adenomas than the normal brain tissues. In human infiltrative and non-infiltrative pituitary adenomas, the mean fibronectin and sialic acid levels were found to be 40.87 +/- 15.90 microgram/mg protein, 27.59 +/- 11.10 microgram/mg protein and 22.40 +/- 9.51 microgram/mg protein, 16.21 +/- 3.20 microgram/mg protein, respectively. Fibronectin and sialic acid levels were slightly elevated (P < 0.05) in human infiltrative pituitary adenomas compared with non-infiltrative adenomas. PMID- 8620436 TI - Formation of etheno and oxoethyl adducts in liver DNA from rats exposed subchronically to urethane in drinking water and ethanol. AB - Exposure of Fisher-344 male rats to 10 000 ppm of urethane in drinking water for up to 90 days or in 5% ethanol for up to 14 days caused the formation of 7-[2' oxoethyl]guanine (OEG) and 1,N(6)-ethenoadenine (epsilon A) in liver DNA. Mild acid DNA hydrolysates were analyzed by high-performance liquid chromatography with photodiode array detection and fluorometry. The identification of OEG and epsilon A was confirmed by coelution with the authentic standards. Forty and 67% of rats showed OEG and epsilon A adducts at 2 and 90 days of treatment with urethane in drinking water, respectively. In comparison, only 0 and 10% of rats showed adducts at 2 and 14 days of treatment with urethane in 5% ethanol, respectively. Neither OEG nor epsilon A was observed in control rats receiving water or 5% ethanol. Although these data are still preliminary, they appear to suggest that ethanol may inhibit formation of DNA adducts by urethane. Studies designed to produce more conclusive information about the role of ethanol in modifying DNA damage induced by urethane in vivo are in progress. PMID- 8620437 TI - Specific sequences of fibronectin activate the protein kinase C signal transduction pathway in invasive bladder cancer. AB - The mechanism of human bladder cancer cell invasion is not clear, but it appears that extracellular matrix components, such as fibronectin, may be involved. To investigate the role of fibronectin in tumor cell invasion and progression, we used an in vitro invasion assay to define the motility stimulating fragment of fibronectin for invasive human bladder cancer T24 cells. Using a modified Boyden chamber assay and purified fragments of fibronectin, we demonstrated that both the 120 kDa chymotrypsin generated fragment of fibronectin (containing the cell attachment RGD motif and additional sequences towards the carboxyl-terminal heparin binding domain), as well as the trypsin generated 60 kDa fragment of fibronectin (containing the carboxyl-terminal heparin binding domain and additional sequences towards the cell attachment RGD motif), were able to stimulate the migration of invasive human bladder cancer T24 cells. Control fragments containing only the amino-terminal gelatin binding region of fibronectin did not stimulate the motility of the human bladder cancer T24 cells. To determine the molecular mechanism in which these fragments may stimulate the migration of the T24 cells, we assayed for intracellular signal transduction pathway protein kinase C (PKC). We demonstrated that both the 120 kDa and the 60 kDa fragments were able to stimulate the activation of protein kinase C. Non motility stimulating fragments of fibronectin were not able to activate protein kinase C. We conclude that the PKC signal transduction pathway may be involved in matrix mediated motility, and suggest that the inhibition of such pathway(s) may alter the malignant phenotype of human bladder cancer. PMID- 8620438 TI - A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer. AB - In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet. PMID- 8620439 TI - Exogenous gamma-linolenic acid alters hormone stimulated cyclic AMP levels in U937 cells. AB - Polyunsaturated fatty acids are selectively cytotoxic in culture. Incorporation of these fatty acids leads to profound changes in membrane fatty acid composition which in turn may alter the activity of transmembrane receptor/effector systems. In U937 cells, hormone stimulated production of cyclic AMP can be reduced by 30% following incubation with gamma-linolenic acid (18:3n-6). It is suggested that beta-adrenoreceptor number, subtype and adenylyl cyclase stimulation may be regulated by alterations in membrane fatty acid composition as a result of changes in the levels of polyunsaturated fatty acids and alterations in eicosanoid production. PMID- 8620440 TI - Combined effects of 13-cis-retinoic acid, tamoxifen and interferon on the growth of human breast cancer cells. AB - We studied the effect of 13-cis-retinoic acid (13-cRA) alone and in combination with interferons (IFNs) and tamoxifen (TAM) in two established human breast cancer cell lines: the estrogen-sensitive CG-5 and the estrogen-insensitive MDA MB-453 cells. 13-cRA (10(-9)-10(-5) M) significantly reduced the growth of both cell lines in a dose-dependent fashion, after 3 and 6 days of treatment. When the retinoid (10(-9)-10(-5) M) was combined with natural beta-IFN (100-1000 IU/ml) for 6 days, we observed a growth inhibition more pronounced than that produced by each of the two single agents in both CG-5 and MDA-MB-453 cells. Only in the former model was the inhibitory effect synergistic at all the drug concentrations used. Association of 13-cRA (10(-9)-10(-5) M) and recombinant alpha2a-IFN (100 1000 IU/ml) or TAM (10(-7)-10(-6) M) did not determine an additive or synergistic effect on the growth of CG-5 cells. PMID- 8620441 TI - Quantitation of the nucleophosmin/B23-translocation using imaging analysis. AB - We have previously detected by immunofluorescent assay that the cellular localization of nucleophosmin/B23 (NPM) shifts from the nucleolus to the nucleoplasm (NPM-translocation) after exposure of cells to multiple agents. In order to improve the quantification of the NPM-translocation, we have developed a digital imaging technique. Human Lo leukemia cells, MCF-7 breast carcinoma cells, and fresh human leukemia cells were exposed to anthracyclines or actinomycin D for 4 h. The degree of NPM-translocation was determined and presented as the localization index (LI). Control cells had a LI of about 10, which indicates that the majority of NPM was localized in nucleoli. The LI for drug-treated cells decreased in a dosage- and time-dependent manner. The effect of two classes of anthracycline (daunomycin and aclacinomycin A) and different types of intercalators (daunomycin and actinomycin D) had additive effects on induction of NPM-translocation. The imaging procedure was easily applied to fresh leukemia cells, thus providing useful information regarding drug effects on cancer cells. PMID- 8620442 TI - Effect of free fatty acids on two-stage skin carcinogenesis in mice. AB - The effect of n-3 and n-6 free fatty acids on two stage skin carcinogenesis in mice was studied. Stearic, linoleic, arachidonic, and eicosapentaenoic acids inhibited both initiation and promotion stages of skin carcinogenesis. Although no direct correlation between lipid peroxidation and papilloma formation was observed, a trend towards an increase in the formation of lipid peroxides with the inhibitory effect of fatty acids on papilloma development was noted. In general, the fatty acids applied were incorporated mainly into the phospholipid and free fatty acid pools. All the fatty acids tested, except eicosapentaenoic and docosahexaenoic acids, inhibited the binding of benzo[a]pyrene (BP) to DNA. On the other hand, BP and croton oil-induced skin cell proliferation was not influenced by any of the fatty acids used. In conclusion, the results of the present study suggest that inhibition of papilloma formation by free fatty acids is complex and can be attributed to a limited extent only to their ability to inhibit BP binding to DNA, to block cell proliferation and enhance the lipid peroxidation process. PMID- 8620443 TI - Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract. AB - The in vitro inhibitory effect of Beta vulgaris (beet) root extract on Epstein Barr virus early antigen (EBV-EA) induction using Raji cells revealed a high order of activity compared to capsanthin, cranberry, red onion skin and short and long red bell peppers. An in vivo anti-tumor promoting activity evaluation against the mice skin and lung bioassays also revealed a significant tumor inhibitory effect. The combined findings suggest that beetroot ingestion can be one of the useful means to prevent cancer. PMID- 8620444 TI - Expression of the c-jun, jun-B, ets-2 and liver regeneration factor-1 (LRF-1) genes during promotion and progression of rat liver carcinogenesis in the resistant hepatocyte model. AB - During promotion in the RH-model, the mRNA expression of c-jun and LRF-1 was 2- to 8-fold elevated in both initiated and uninitiated rats receiving 2-AAF. The increase was more pronounced in male than in female rats, and GH treatment of male rats down-regulated the expression towards the level in females. The level in uninitiated 2-AAF-treated livers was as high as in isolated early nodules. jun B also showed 3- to 8-fold increased expression, but without sex differences. An increased nuclear transcription of the LRF-1 and jun-B genes but not of c-jun was observed. During progression, LRF-1 and ets-2 showed a 2- to 3-fold higher expression in persistent nodules and hepatocellular carcinomas than in the corresponding surrounding liver tissues, whereas the expression of the jun genes was 3- to 4-fold increased both in lesions and in surrounding livers when compared to age-matched control rats. In conclusion, while the changes during promotion might not be connected with control of early focal growth, the increased levels of LRF-1 and ets-2 in advanced lesions might indicate that these genes could contribute to the growth advantage for persistent nodules during progression. PMID- 8620445 TI - Medium-term bioassay for the hepatocarcinogenicity of hexachlorobenzene. AB - Hexachlorobenzene (HCB) is an important environmental contaminant derived mainly from industrial and agricultural sources. It is carcinogenic in mice, rats and hamsters. It has now been studied in a medium-term bioassay for carcinogenicity based on the induction of preneoplastic lesions in the liver. We report here that the bioassay can rapidly detect carcinogenic doses of HCB and that there is a clear dose-response relationship. At the lowest dose of HCB administered, the incidence of preneoplastic lesions in the liver was no different from that in controls. PMID- 8620446 TI - Effect of tamoxifen on intraperitoneal N-nitroso-N-methylurea induced tumors. AB - The effect of tamoxifen (TAM) was evaluated on a mammary tumor model induced in Sprague-Dawley rats by intraperitoneal administration of three N-nitroso-N methylurea (NMU) doses. Animals received TAM (1 mg/kg per day) from 10 days before the first NMU dose up to 140 days later. Thereafter, treatment was discontinued and the observation period was extended 60 days longer. Mean overall latency period, tumor number per rat and tumor incidence were recorded. Significant differences between treated and control batches were observed in tumor number per rat (1.8 +/- 1.1 versus 5.2 +/- 1.6; P < 0.05) and in tumor incidence (50% versus 100%; P < 0.05), respectively. No significant difference in latency period between both batches was recorded. All lesions induced in the control batch were malignant, whereas only 45% of those induced in TAM-treated animals were malignant and the remaining 55% were preneoplastic. At 60 days after treatment discontinuance, tumor incidence increased to 90% and also tumor number per rat increased to 4.6 +/- 1.5. TAM effect was also evaluated in rats with NMU induced tumors by treatment with 1 mg/kg per day during 60 days starting when tumors reached a 1.5-cm diameter. Regression to less than 80% of initial size in 49% of the tumors was observed, while in ovariectomized rats, 33% of tumors regressed. Estrogen receptor content, ER (fmol/mg protein) and Kd (nM) in control tumors were: 56 +/- 10 and 0.5 +/- 0.1. In tumors of TAM-treated animals, ER was less than 5 fmol/mg protein. Findings demonstrate that TAM significantly decreased the appearance of tumors induced in rats by i.p. injection of NMU and when TAM treatment was initiated after tumor induction, some tumors failed to respond to hormonal manipulation. Differential tumor growth response after TAM or oophorectomy in each tumor indicates that in the same rat it is possible to distinguish hormone-dependent and hormone-autonomous tumor populations. Hormonal regulation of tumor growth can be under intrinsic control, regardless of the hormonal status of the whole organism. PMID- 8620447 TI - Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro. AB - Extracts of saffron (Crocus sativus L.) have been reported to inhibit cell growth of human tumor cells. In order to study the cytotoxic effect of the characteristic compounds of saffron spice, we have isolated crocin, crocetin, picrocrocin and safranal. Doses inducing 50% cell growth inhibition (LD50) on HeLa cells were 2.3 mg/ml for an ethanolic extract of saffron dry stigmas, 3 mM for crocin, 0.8 mM for safranal and 3 mM for picrocrocin. Crocetin did not show cytotoxic effect. Cells treated with crocin exhibited wide cytoplasmic vacuole like areas, reduced cytoplasm, cell shrinkage and pyknotic nuclei, suggesting apoptosis induction. Considering its water-solubility and high inhibitory growth effect, crocin is the more promising saffron compound to be assayed as a cancer therapeutic agent. PMID- 8620448 TI - Identification of antimutagenic substances in an extract of edible red alga, Porphyra tenera (Asakusa-nori) AB - Recently, a relatively strong antimutagenic activity has been detected in the extract of Porphyra tenera (Asakusa-nori in Japanese) which showed a suppressive effect on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002 (Okai et al. (1994) Cancer Lett., 87, 25-32). In the present paper, we analyzed the active principles for the antimutagenic activity in an extract of Porphyra tenera and detected three color spots on a silica gel TLC plate which indicated very similar Rf values and absorbance spectra of standard pigments such as beta-carotene, chlorophyll a and lutein. The seaweed pigments recovered from preparative silica gel TLC corresponding to beta-carotene, chlorophyll a and lutein exhibited significant suppressive activities against mutagen-induced umu C gene expression and combined treatment with these pigments showed an additive effect compared with single treatment with each pigment. Furthermore, the standard pigments prepared from other biological sources also exhibited similar anti-mutagenic activities. The significance of this finding is discussed from the protective role of seaweed pigments against mutagenesis probably associated with carcinogenesis. PMID- 8620449 TI - Cytotoxic effects of 1 alpha,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line. AB - 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3) has recently been reported to exert a toxic effect on both rat and human glioma cell lines. However the potential clinical use of 1 alpha,25(OH)2D3 in the treatment of glioma is impaired by its potent hypercalcemic effects. We have therefore investigated the effects on glioma cell growth of several vitamin D3 analogues which have previously been shown to be less calcemic in vivo than 1 alpha,25(OH)2D3. The present study shows that several analogues are able to induce, in vitro, the death of rat glioma cells (C6.9). The compound KH 1060 appears to be the most effective in the induction of cell death, while MC 1288 and CB 1093 are as potent as 1 alpha,25(OH)2D3. EB 1089 was somewhat less effective than 1 alpha,25(OH)2D3 and MC 903, which is currently used in the treatment of psoriasis, has only a weak activity on C6.9 cells. The effective doses used are around 10(-9) M for 1 alpha,25(OH)2D3 and 10(-10) M for KH 1060. Interestingly, the toxic effect exerted by 1 alpha,25(OH)2D3 and its analogues is accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene. These findings, together with the fact that the therapies currently available for glioma are only palliative, suggest that 1 alpha,25(OH)2D3 analogues such as KH 1060, EB 1089 or CB 1093, alone or in combination with other therapeutic approaches, could be of potential interest in the treatment of brain glial tumors. PMID- 8620450 TI - Expression of a specific mouse germ cell nuclear antigen (GCNA1) by early embryonic testicular teratoma cells in 129/Sv-Sl/+ mice. AB - Spontaneous testicular teratomas which develop at a high rate in 129/Sv-Sl/+ mice are thought to be derived from germ cells. The teratomas present initially as groups of atypical germ-like cells within seminiferous cords of the 15.5 days post coitum (dpc) embryonic testes. These pluripotent teratoma stem cells are capable of differentiating into many kinds of tissues in adult mice. In this immunohistochemical study, we have examined the testes of 129/Sv-Sl/+ mice to determine whether the teratoma cells which developed in these gonads retain the nuclear antigen GCNA1. GCNA1 is a 110 kDa mouse Germ Cell Nuclear Antigen recognized by a rat monoclonal antibody 10D9G11. GCNA1 is expressed in mouse germ cells after they migrate into the genital ridge (11.5 dpc), throughout embryonic development until postnatally germ cells arrive at the diplotene/dictyate stage of the first meiotic division, when it is no longer expressed. Early foci (16.5 dpc) of teratoma stem cells in 129/Sv-Sl/+ mice strongly express GCNA1, but down regulate GCNA1 expression by 19.5 dpc. The loss of GCNA1 expression from teratoma stem cells late in embryonic development is in contrast to embryonic gonocytes which retain GCNA1 expression throughout fetal development. All postnatal undifferentiated and differentiated teratoma cells did not appear to express GCNA1. The expression of the germ cell specific nuclear antigen GCNA1 in early teratoma stem cells further demonstrated that the testicular teratomas originate from early germ cells. The stronger reaction of monoclonal antibody 10D9G11 to GCNA1 within early teratoma cells compared to normal germ cells makes GCNA1 useful in identifying early embryonic tumor foci. PMID- 8620451 TI - Involvement of methionine in the synthesis of certain membrane-associated nucleotide sugars by human amnion (WISH) cells. AB - A plasma membrane preparation from human amnion (WISH) cells contained uridine diphosphate sugars and methyl-uridine diphosphate (mUDP) sugars. The synthesis of mUDP-glucose, mUDP-mannose, and mUDP-fucose by the membrane preparation occurred when supplemented with uridine-5'-diphosphate-glucose and S-adenosyl-L methionine. It is suggested that this newly recognized route for fucose biosynthesis might be employed by certain transformed cells, and may partly account for the methionine dependence of certain human tumors. Additionally, it is suggested that, in colon cancer, a deficiency of folic acid and methionine might affect mUDP-sugar biosynthesis rather than the methylation of DNA. PMID- 8620452 TI - The later administration of progesterone more rapidly activates dormant mouse mammary tumor cells initiated by 3'-methyl-4-dimethylaminoazobenzene. AB - Implantation of progesterone at 1 month of age induced the development of mammary tumors in female C57BL/6 x DS-F1 mice that had been treated with 3'-methyl-4 dimethylaminoazobenzene (3'-Me-DAB) neonatally, and that had undergone ovariectomy and received implants of estradiol-17beta (E2) pellets at 1 month of age, and the incidence of mammary tumors became 100% at 15 months of age. On the other hand, no mammary tumors developed in these mice with implants of E2 pellets alone. Implantation of progesterone alone also induced no mammary tumors in mice that had been treated with 3'-Me-DAB neonatally, and had undergone ovariectomy at 1 month of age. Implantation of progesterone at 4, 6, 8, and 10 months of age also caused the prompt development of mammary tumors as implantation of progesterone at 1 month of age. When ages at which the incidence became 50% were estimated on curves of the incidences, these ages on implantation of progesterone at 1, 4, 6, 8, and 10 were about 11, 13, 14, 14, and 14 months of age. These results suggest that progesterone together with estrogen promotes the development of mammary tumors induced by 3'-Me-DAB, and that the later progesterone is administered, the more rapidly it activates dormant mammary tumor cells initiated by 3'-Me-DAB. PMID- 8620453 TI - Immortalization of human adult prostatic adenocarcinoma cells by human papilloma virus HPV16 and -18 DNA. AB - Primary prostate epithelial and prostate adenocarcinoma cells cultured in serum free medium grew for up to 10 passages before senescence. Cells from prostate adenocarcinoma of a 55-year-old patient without lymph node involvement were transfected with plasmids containing recombinant human papilloma virus HPV16 or HPV18 DNA and the selectable neomycin-resistance gene. After G-418 selection, cells underwent crisis, and surviving cells infected with retroviruses encoding the HPV18 E6/E7 genes (HPV-PAC1), transfected with a head-to-tail dimer of the complete HPV16 genome (HPV-PAC2), or transfected with HPV18 E6/E7 early genes (HPV-PAC3) were established. HPV-PAC1 and HPV-PAC2 cultures appeared morphologically similar to primary cultures even after 40 passages. However, HPV PAC2 cultures had a clonal morphology. All lines were positive for cytokeratin 18, had acquired vimentin expression, and contained either HPV16 or HPV18 sequences integrated into host DNA. None was tumorigenic in nude mice or formed colonies in soft agar. These cells did not secrete prostate specific antigen nor respond to androgen although tamoxifen inhibited the growth of the cells. Immunohistochemistry showed no evidence of p53 overexpression. Further characterization of these cell lines and examination of their response to chemotherapeutic agents may provide relevant information for the study of hormone independent PC. PMID- 8620454 TI - G --> A mutation of ras genes and infrequent p53 gene mutation in rat transplantable thyroid carcinoma lines from tumors induced in vivo by N-bis(2 hydroxypropyl)nitrosamine. AB - In a comparative study, we have investigated mutational activation of three kinds of ras genes and the p53 gene (exons 5-8) in 19 rat transplantable thyroid carcinoma lines derived from in vivo tumors induced by DHPN. Mutations were identified using single-strand conformation polymorphism and DNA sequencing analysis, and activated ras oncogenes were detected in 6 lines (31%). These all had mutations in Ki-ras codons at 12 or 63, and one of them also possessed a Ha ras mutation at codon 12 as a double mutation. Three mutations of Ki-ras at codon 12 involved the second nucleotide and two the first position, the other being found at the first nucleotide in codon 63. Base alterations of p53 gene were found in two lines. One had an insertion of 1 base (thymine) between codon 206 and codon 207. Historically, it was diagnosed as a poorly differentiated papillary carcinoma (scirrhous pattern). In the another case, there was no amino acid change although one base substitution occurred at codon 283 (GAG --> GAA) of exon 8. These results indicate that, in the ras family, DHPN induces Ki-ras gene activation preferentially and that p53 mutation may be infrequent in thyroid carcinogenesis in rats, our data thus corresponding well with the previous reports that an inactivated p53 gene only plays a major role in human undifferentiated thyroid carcinomas. PMID- 8620455 TI - Intratumor administration of fusogenic liposomes containing fragment A of diphtheria toxin suppresses tumor growth. AB - Previously, we reported that experimental i.p. administration of fusogenic liposomes containing fragment A of diphtheria toxin (DTA) completely regressed ascites tumors without any severe side effects. In this study, we examined the therapeutic effects of intratumor injection of fusogenic liposomes using ddY mice implanted with Sarcoma-180 (S-180) cells intradermally. Intratumor injections of fusogenic liposomes containing DTA significantly inhibited the tumor growth as assessed by the relative mean tumor volume, and by the survival time of mice. No therapeutic effects were observed when simple liposomes containing DTA or empty fusogenic liposomes were administered. Using [3H]inulin encapsulated in fusogenic liposomes as a marker, we demonstrated that fusogenic liposomes delivered their contents into the solid tumor cells about 15 times more efficiently than simple liposomes. These results suggest that intratumor administration of fusogenic liposomes containing DTA is a highly effective approach to the local treatment of solid tumors. PMID- 8620456 TI - The antitumor phospholipid analog, hexadecylphosphocholine, activates cellular phospholipase D. AB - Hexadecylphosphocholine (HePC), a glycerol-free phospholipid analog, belongs to a new class of drugs that demonstrate selective anticancer activity. The mechanisms underlying the anticancer activity are unclear. To investigate possible signal transduction relationships we examined the influence of HePC on cellular phospholipid metabolism. When HePC was added to cultured human breast fibroblasts (CCD-986-SK cells) that had been radiolabeled with fatty acid, phosphatidylethanol (PEt, the transphosphatidylation product of phospholipase D (PLD)) formation was stimulated as early as 5 min after addition. In cells labeled with [3H]choline, HePC treatment caused release of choline-containing metabolites to the culture medium, concurrent with PEt formation. HePC also elicited formation of diacylglycerol (DG) which, after 30 min increased 3.5-fold over control. As little is known regarding HePC and PLD, attention was directed towards studies on PC metabolism by PLD. PEt formation was shown to be optimal at 20-50 microM HePC, and structure-activity studies showed HePC to be more potent than either lyso-phosphatidylcholine or 1-hexadecyl-2-O-methyl-rac-glycero-3 phosphocholine for PLD activation. PLD activity induced by HePC was totally inhibited by cellular pretreatment with phorbol dibutyrate, and 59% diminished by pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor. Our results demonstrate for the first time that HePC activates PLD, and suggest that PKC participates in this response. The relationship of PLD to the anticancer properties of HePC may be clinically relevant to drug actions. PMID- 8620457 TI - Promotion of colon carcinogenesis through increasing lipid peroxidation induced in rats by a high cholesterol diet. AB - To examine the influence of hypercholesteremia on 1,2-dimethylhydrazine (DMH) induced rat colon cancer, Sprague-Dawley rats received dietary cholesterol (CH, 0 2%) and cholic acid (CA, 0.25%) with or without DMH (20 mg/kg, s.c. injection) for 18 weeks. The rats receiving dietary cholesterol and cholic acid all significantly increased total serum cholesterol and lipids but only a high cholesterol diet (2% CH plus 0.25% CA) decreased the activity of glutathione peroxidase (GSH-Px) and increased the formation of peroxides in the colon (P < 0.01). The rats that received the combination of DMH and high cholesterol diet enhanced these effects. At the end of the experiment, the diet group administered DMH and high cholesterol (2% CH plus 0.25% CA) developed colon adenoma at 50% of incidence in pathological examination, but no colon adenoma formed in the rats treated with high cholesterol alone. It is supposed that a non-carcinogenic agent like cholesterol may potentiate the carcinogenicity of DMH in rats via an increase of lipid peroxidation and decrease in the activity of peroxidase in the target organ. PMID- 8620458 TI - Tamoxifen antagonizes proliferation and invasion of estrogen receptor-negative metastatic follicular thyroid cancer cells via protein kinase C. AB - Tamoxifen inhibits invasion and growth of estrogen-receptor negative follicular thyroid cancer (FTC) cells in vitro and in vivo. To study the mechanisms involved, we documented the effects of tamoxifen and staurosporine on three metastatic FTC-cell lines. TPA (10 ng/ml) enhanced invasion and growth of FTC by 15% (P < 0.02). Tamoxifen (1.5 micromol/l) inhibited invasion of FTC133 by 36% (FTC236 30%; FTC238 32%; P < 0.01). TPA reversed the tamoxifen-mediated inhibition of invasion by 35% in FTC133 and 30% in FTC238 (P < 0.02). Staurosporine (10 ng/ml) inhibited invasion and growth of all FTC. At 0.1-1 ng/ml it enhanced the inhibitory effects of tamoxifen, but did not further inhibit invasion or growth at higher concentrations. We conclude that the antiproliferative and antiinvasive effects of tamoxifen on follicular thyroid cancer cells are at least partly mediated by an inhibition of protein kinase C. PMID- 8620459 TI - Title aggregation patterns of argyrophilic nucleolar organizer regions induced by 5-fluorouracil in the nuclei of MCF-7 human breast cancer cells. AB - The effects of tamoxifen and 5-fluorouracil (5-FU) on the patterns of argyrophilic nucleolar organizer regions (AgNORs) in MCF7 human breast cancer cells were studied. Tamoxifen and 5-FU both inhibited the growth of MCF-7 cells by 18% by day 3 of culture, but each had different effects on the AgNORs. Whereas no significant changes were induced by tamoxifen, effects on the AgNORs of MCF-7 cells by 5-FU were dramatic: 5-FU treatment changed the pattern of AgNORs, reducing the number of satellites by aggregation, typically to a single aggregation around nucleoli in a sphenoidal fashion. We named these morphological changes: fluorouracil induced AgNOR aggregations (FAA). Following treatment with 500 ng/ml 5-FU, FAA developed rapidly. AgNORs forming two or three aggregates in 24% (6 h), 24% (12 h), 40% (24 h) and 34% (48 h) of cells, compared to a control rate of 14%. Single large aggregate was rarely found in untreated cultures but after 6, 12, 24 and 48 h treatment with 500 ng/ml 5-FU, AgNORs had formed a single aggregate in 6, 8, 16 and 22% of cells, respectively. FAA were observed at a concentration of 100 ng/ml 5-FU; 48 h treatment resulted in cells in which two or three aggregates were increased by 24% and single aggregate by 16%. These large single aggregates were larger than nucleoli stained by Papanicolau staining. PMID- 8620460 TI - Synergistic inhibitory effects of interferon-alpha and 5-fluorouracil in meningioma cells in vitro. AB - We have investigated the effects of interferon-alpha (IFN-alpha) and 5 fluorouracil (5-FU) on meningioma cells in two different culture systems, evaluated by the uptake of radiolabelled methionine. With both IFN-alpha and 5-FU an inhibitory effect on the uptake of radiolabelled methionine by the meningioma cells was demonstrated, and we found a synergistic inhibitory effect with a combination of IFN-alpha and 5-FU. To obtain a maximal inhibition of cell metabolism without causing cell toxicity, we were able to decrease the dose of 5 FU by simultaneously adding IFN-alpha. Our results suggest that a combined treatment of IFN-alpha and 5-FU may be a successful alternative for patients with inoperable meningiomas. A novel in vitro positron emission tomography technique was used for the study of metabolic changes in tumour cells caused by drug treatment, which is complementary to conventional cell culture techniques. PMID- 8620461 TI - Analysis of nm23-H1 expression in breast cancer. Correlation with p53 expression and clinicopathologic findings. AB - Metastasis is the most frequent cause of death in patients with breast cancer. The nm23-H1 and p53 genes have been involved in the development of breast cancer metastasis. We have analyzed the correlation between the expression of nm23 protein and several established clinicopathologic factors. Our results show that the antimetastatic role of nm23-H1 is not related to the cell proliferative status or tumor grade and that it is not associated with the expression of p53. We also demonstrate a strong inverse relationship between the expression of nm23 H1 protein, lymph node metastasis and vascular invasion. These data support the antimetastatic role of the nm23-H1 gene and suggest that nm23-H1 and p53 genes may be involved in different steps of the metastatic process. PMID- 8620462 TI - Inhibition of aromatase activity and growth suppression by 4-methoxy-4-androstene 3,17-dione in an androgen sensitive human prostatic carcinoma cell line. AB - Aromatization of testosterone to estradiol was investigated in a human prostatic carcinoma cell line, LNCaP. A saturable, dose and time-dependent aromatization of testosterone was observed. Kinetic parameters, Km (201 nM) and V(max) (0.76 pmol/h) per mg) and also the inhibition constants (Ki) for various aromatase inhibitors were calculated from standard Lineweaver-Burke plots. The steroidal aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OHA) and its derivative, 4-methoxy-4-androstene-3,17-dione (4-OMA), inhibited aromatization of testosterone in a competitive pattern of inhibition. The derivative 4-OMA is the stronger inhibitor of the two, with an apparent Ki of 1.12 microM, whereas the apparent Ki of 4-OHA is 3.28 microM. Long term incubation with 4-OMA suppressed proliferative activity of LNCaP cells in the presence of physiological levels of testosterone (10(-10) M to 10(-7) M). In contrast, 4-OHA was a growth promoter. These results suggest a potential role for aromatase in hormone responsive prostate cancer. PMID- 8620463 TI - Identification of an RB-responsive region in the 5' untranslated region of the RB gene. AB - To investigate the ability of the retinoblastoma gene product (RB protein, pRB) to regulate its own expression, cotransfection assays using human RB promoter luciferase fusion plasmids and a human pRB expression plasmid were employed. In B104, a rat neuroblastoma cell line, pRB stimulated luciferase activity about 2 fold from the wild-type promoter, and about 4-fold from a mutant promoter with a mutation in the retinoblastoma binding factor 1 (RBF-1) site. The RB-responsive region was mapped to a novel 44 bp sequence in the 5' untranslated region in both wild-type and mutant promoters. When apparent stimulation of luciferase activity by pRB was observed, the luciferase mRNA level did not increase, suggesting that through this 44 bp region, pRB could post-transcriptionally regulate its own expression. PMID- 8620464 TI - Inhibition of human colon cancer by intermittent opioid receptor blockade with naltrexone. AB - Nude mice inoculated with human colon cancer (HT-29) and receiving 0.1 mg/kg naltrexone (NTX) beginning immediately after tumor cell injection exhibited a marked retardation in tumorigenicity. This dosage of NTX, which blocked opioid receptors for 6-8 h/day, resulted in a delay of 2.4-fold in tumor appearance compared to control subjects. At the time (10 days) when all control mice had tumors, 80% of the mice in the 0.1 mg/kg NTX group had no signs of neoplasia. Binding capacity, but not affinity, of [3H][Met5]-enkephalin was reduced 85% of control levels in tumor tissue from mice of the 0.1 NTX group. Plasma, but not tumor tissue levels of [Met5]-enkephalin were elevated (2.5-fold) in contrast to control values. These results suggest that daily intermittent opioid receptor blockade with NTX provokes the interaction of opioids and receptors in the interval following drug availability, with opioids serving to inhibit tumorigenicity of human colon cancer. PMID- 8620465 TI - Use of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes. AB - With a view towards the design of systems capable of combining the use of chemotherapy and photodynamic therapy in the treatment of cancer and other disorders, it has been proposed that photosensitized erythrocytes might be employed as carriers/vehicles for agents such as cancer chemotherapeutics. In studying the light dependent release of entrapped agents from such a system, the efficacy of light induced release is usually studied by measuring release of an entrapped component into centrifugation supernatants following photoactivation. It has hitherto been extremely difficult to examine what occurs upon immediate irradiation at the microscopic level in real-time. In this study we demonstrate that, using real-time confocal laser scanning microscopy, it is possible to directly observe immediate short-term events occurring during direct irradiation with the visualizing beam. Following irradiation of photosensitized erythrocytes with the visualizing beam form the confocal scanning system, it was noticed that some from of cell-disruptive event occurred. In this study we demonstrate a dose dependent response between this relatively immediate, light induced disruptive event with respect to both irradiation exposure and photosensitizer concentration. We suggest that this system may provide a novel means of observing, at a microscopic level, events occurring in real-time during photodynamic therapy. PMID- 8620467 TI - Co-localization of suramin and serum albumin in lysosomes of suramin-treated human colon cancer cells. AB - Suramin is a polysulfonated compound currently under investigation for the treatment of various types of cancer. Pharmacokinetic studies from clinical trials in humans have shown that most of the circulating drug is associated with serum albumin. The objective of the present study was to investigate the intracellular localization of suramin and serum albumin in human colon cancer cells (HT-29-D4) upon suramin treatment. For this purpose, combined gold labeling and autoradiographic methods were performed on HT-29-D4 cells grown in serum free medium containing both [3H]suramin and colloidal gold-albumin. These morphological experiments demonstrated for the first time that suramin and serum albumin were co-localized in the same cellular compartment (i.e. the lysosomal system) of the suramin-treated HT-29-D4 cells. The albumin-directed targeting of suramin in lysosomes may allow the drug to inhibit the activity of several lysosomal hydrolases, resulting in a lysosomal storage disorder. PMID- 8620466 TI - Molecular non-genetic biomarkers related to Fenarimol cocarcinogenesis: organ- and sex-specific CYP induction in rat. AB - Selective biochemical markers of effect have been used to evaluate some non genotoxic cocarcinogenic properties of Fenarimol. Several CYP-dependent reactions have been monitored in liver, kidney and lung microsomes of male and female Sprague-Dawely rats treated (i.p.) with 200 or 400 mg/kg body wt dose of this pesticide. Highly specific substrates were used as probes of various isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences in the inductive response by Fenarimol, has been recorded in this investigation, the kidney (mainly male) being more responsive when compared to other tissues. A 6.6-fold increase in the 2B1-like activity, probed by dealkylation of pentoxyresorufin was observed in the liver at a higher dose. On the contrary, a marked induction of CYP1A1 mediated ethoxyresorufin O-deethylase activity, ranging from 20- to 35-fold in female and male, respectively, was observed in the kidney at a lower dose tested. In the lung, at a higher dose, the p-nitrophenol hydroxylase activity (2E1) was enhanced up to 3.5-fold in male animals, whereas the 3A-like activity, probed by the N demethylation of aminopyrine, was induced up to 2.6-fold in females. A weak, although significant reduction of CYP2B1 isoforms in lung was also recorded. Taken together, these data corroborated by means of Western immunoblotting analysis (using rabbit polyclonal antibodies anti-CYP 2B1/2, 1A1, 2E1, and 3A1/2) indicate a possible cotoxic, comutagenic cocancerogenic and promoting potential of this fungicide. PMID- 8620468 TI - Tumorigenesis disrupts hormonal regulation of tenascin expression in regressing Dunning R 3327 H prostate carcinoma. AB - We recently reported that androgen ablation either by orchiectomy or antiandrogen treatment resulted in the expression of the extracellular matrix glycoprotein tenascin in the regressing rat prostate. With the study presented here we investigated whether tenascin is expressed in the Dunning R 3327 H tumor and if orchiectomy and antiandrogen treatment affect tenascin expression. Experimentally, male rats were inoculated s.c. with pieces of Dunning tumor into the hind limb of both sides. Three months after inoculation rats were either orchiectomized or received a daily dose of 3 mg of cyproterone acetate or flutamide. Following a treatment period of 13 weeks, orchiectomy reduced tumor area by more than 60% compared to untreated controls. Cyproterone acetate and flutamide reduced tumor area significantly up to 30%. The amount and intensity of tenascin immunoreactivity appeared to be independent of the hormonal treatment and rather correlated to the content of tumor stroma. Those tumors with small, densely packed glandular ducts possessing almost no stromal tissue stained weakly for tenascin, whereas those tumors with larger ducts and significant stroma stained intensely. Staining intensity was particularly high at these sites where tumors infiltrated neighboring tissues, in proximity of infiltrating blood cells and close to necrotic areas. In summary, our results demonstrate a specific pattern of tenascin expression in Dunning R 3327 H rat prostate carcinomas, which appear to be independent of the hormonal treatment. We therefore conclude that tumorigenesis disrupts hormonal regulation of tenascin expression which we detected in the normal prostate gland after treatment with antiandrogens. PMID- 8620470 TI - Deregulated c-myc expression is insufficient for emergence of the tumorigenic phenotype in malignancy-suppressed intratypic T-cell lymphoma hybrids: an in vitro model for multistep lymphomagenesis. AB - The T-cell lymphoma cell line YACUT was fused with the Mls-1a antigen-responsive non-tumorigenic T-cell line G4 to construct growth-arrested hybrids which could be induced to proliferate in the presence of Mls-1a antigen. Prolonged growth of the hybrids by repeated antigenic stimulation resulted in the emergence of cells with transformed phenotype, which was accompanied by a reversion of c-myc expression to the levels of the YACUT lymphoma parent and an increase in the number of YACUT-derived chromosome 15 carrying the rearranged pvt-1 gene. Despite these two changes, early passage transformed hybrids as well as proliferation suppressed hybrids were non-tumorigenic in vivo. The fact that only late passage transformed hybrids produced tumors in vivo indicated that additional genetic and/or epigenetic alterations are required for the emergence of hybrid lines with tumorigenic phenotype. Thus, this experimental system offers tangible possibilities for delineation of the three distinct phenotypes which could be exploited for the investigation of the multistep process of lymphomagenesis. PMID- 8620469 TI - Granulocyte-macrophage colony-stimulating factor accelerates growth of Lewis lung carcinoma in mice. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) has not been found to exert any influence on the proliferation of Lewis lung carcinoma (LLC) cells in vitro. Nevertheless, when administered intraperitoneally, GM-CSF accelerated the growth of subcutaneously growing LLC in mice. PMID- 8620471 TI - Human renal cell carcinogenesis is accompanied by a coordinate loss of the tissue specific transcription factors HNF4 alpha and HNF1 alpha. AB - Human renal cell carcinogenesis is usually accompanied by dedifferentiation processes including the loss of expression of tissue specifically expressed genes. Based on the hypothesis that these dedifferentiation processes might be attributed to a functional change in tissue specific transcription factors, we have analyzed the expression and function of the tissue specific transcription factor HNF4 alpha in human renal cell carcinomas. By Western blot analysis and gel retardation assay using HNF4 alpha specific antibodies, we observed that in most cases the amount as well as the binding activity of HNF4 is reduced in the tumor samples compared to the corresponding normal tissues. Furthermore, we found a clear correlation between the HNF4 alpha binding activity and the amount of another transcription factor (HNF1 alpha), which is thought to be transcriptionally activated by HNF4 alpha. We therefore speculate that disruption of the HNF4 alpha/HNF1 alpha pathway of kidney specific gene expression might be an important molecular mechanism in renal cell carcinogenesis. PMID- 8620472 TI - Alveolar stem cells in canine bronchial carcinogenesis. AB - Alveolar type II cells are not present in normal epithelium of canine segmental bronchi but after carcinogen exposure they do occur in intra-epithelial lesions with all degrees of atypia and in invasive lesions with different glandular growth patterns. Immunohistochemistry for proliferation markers (PCNA; Ki-67) strongly suggest that such novel type II cells are pluripotential stem cells in canine bronchial carcinogenesis. Very likely, bronchial carcinogenesis is subject to an oncofetal mechanism of differentiation: bronchial epithelial retrodifferentiation followed by novel differentiation of alveolar tumor stem cells. PMID- 8620473 TI - Metastasis-related cell functions in primary and metastatic tumor cells of AKR lymphoma. AB - The metastatic phenotype is of extreme complexity. To complete all the stages of metastasis, the tumor cell must possess a whole series of functional abilities. Multiple biological markers are therefore needed to achieve a deeper understanding of the metastatic phenotype. In the present study we compared primary (PT) to metastatic tumor (MT) cells of two AKR lymphoma variants with respect to several cellular functions relevant to various steps of tumor dissemination. The MT cells of the TAU-44 variant had a higher capacity than the PT cells to attach to endothelial monolayers and ECM, exhibited a more elevated motility and a higher capacity to grow in the spleen as a metastatic target organ. However, the TAU-44-MT cells had a lower ability to grow in the kidney than the PT cells. The TAU-33-MT cells had a higher ability to attach to endothelial cells and to grow in both spleen and kidney but were less motile compared to PT cells. Metastatic cells showed, on the whole, higher ability to perform in most, but not all, stage-specific models than primary tumor cells. PMID- 8620474 TI - Induction of 8-hydroxy-2'-deoxyguanosine in CHO-K1 cells exposed to phenyl hydroquinone, a metabolite of ortho-phenylphenol. AB - The induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative DNA modification, was investigated in CHO-K1 cells exposed to phenyl-hydroquinone (PHQ), a major metabolite of ortho-phenylphenol (OPP), an antimicrobial. Addition of PHQ at a concentration of 50 microM to CHO cell suspensions (10(6) cells/ml) induced slight elevation of intracellular 8-OHdG levels. Pretreatment of CHO cells with 3-amino-1,2,4-triazole (AT, 20 mM) enhanced PHQ-induced 8-OHdG formation which was accompanied by cell death. Pretreatment of CHO-K1 cells with AT (20 mM) and deferoxamine (DeFe, 20 mM) inhibited the formation of 8-OHdG as well as cell death caused by PHQ. Neither AT nor DeFe affected cell viability or the formation of 8-OHdG in untreated CHO cells during the incubation period. The loss of cellular glutathione induced by the addition of PHQ alone was enhanced by the pretreatment of CHO cells with AT or AT plus DeFe. When PHQ was added to AT pretreated cell suspensions, the concentration of PHQ decreased with time. This decrease was accompanied by the formation of phenyl-benzoquinone (PBQ). These results suggest that the reactive oxygen species derived from autoxidation of PHQ which converts to PBQ via phenyl-semiquinone elicit DNA damage in CHO cells, especially when the activity of cellular catalase is inhibited. PMID- 8620475 TI - Radiosensitizing effect of cisplatin in prostate cancer cell lines. AB - The radiosensitizing effect of platinum compounds has been demonstrated in a number of tumors. In prostate cancer, clinical and preclinical data concerning an eventual efficacy of the concept of radiosensitization are lacking. In the present study cisplatin and carboplatin have been used as a model to explore radiosensitization in in vitro prostate cancer cell lines. Human (DU-145) and rat (R3327-MATLyLu) prostate tumor cells were irradiated with doses ranging from 0 to 8 Gy in the presence of various concentrations of either cisplatin or carboplatin. For the evaluation of the combined effect of the two treatment modalities, a simple model is presented. Supra-additive treatment effects of combinations of platinum drugs with radiotherapy, both at clinically achievable doses, were shown on the basis of surviving fractions of tumor cells and proved to be significant. These data strongly suggest that radiotherapy may be effectively combined with radiosensitizers such as platinum drugs in prostate cancer therapy, to yield synergism in treatment efficacy. PMID- 8620476 TI - Expression of P-glycoprotein, encoded by MDR 1 gene, a metabolically active efflux pump in murine mast cells. AB - Mast cell line (MC/9) derived from normal murine liver was examined for the expression of P-glycoprotein (P-gp) at the level of protein with C-219 and JSB-1 monoclonal antibodies, using flow cytometry and Western blot, and at the level of mRNA by the reverse transcriptase-polymerase chain reaction. The function of P-gp was analyzed by the accumulation and efflux of rhodamine 123 (Rh123) in the presence or absence of cyclosporin A (CSA) and a non-immunosuppressive analog of CSA (CSA-1). P-gp both at the protein and mRNA levels was expressed in mast cells. Intracellular accumulation of Rh123, in the presence of CSA and CSA-1 was significantly greater than in their absence. Furthermore, both CSA and CSA-1 inhibited Rh123 efflux from mast cells. These data suggest the presence of a functionally active P-gp in mast cells. A possible physiologic role for P-gp in the secretion of certain mediators/cytokines from mast cells is suggested. PMID- 8620477 TI - Establishment of a rat thyroid carcinoma cell line in vitro demonstrating high DNA synthesis in response to insulin-like growth factor I. AB - We previously established transplantable rat thyroid carcinoma cell lines in vivo from primary thyroid tumors induced by N-bis-(2-hydroxypropyl)nitrosamine (DHPN). In the present study, an insulin-like growth factor I (IGF-I)-responsive cell line (TRTC-G1-C-A4) in culture was derived from one (well differentiated papillary type) of these carcinoma cell lines G1. TRTC-G1-C-A4 cells were found to exhibit specific saturable binding of IGF-I with a Kd of 1.16 nM at approximately 43.6 fmol/10(5) cells. Inclusion of IGF-I (10 and 50 ng/ml) in the culture medium resulted in a significant increase of [3H]thymidine incorporation and marked cell proliferation. IGF-II (10 ng/ml) and insulin (1 microgram) produced no such effects. The molecular weight of IGF-I receptors on the cell membrane was determined by Western blotting analysis, a single band of binding proteins with a molecular weight of 125 kDa being evident under non-reducing conditions. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the TRTC-G1-C-A4 cells contained IGF-I receptor mRNA with a sequence corresponding to that determined from rat uterus. These results demonstrate that the IGF-I receptor can be expressed in a thyroid carcinoma with an important contribution to cell growth. PMID- 8620478 TI - Antitumor effect induced by the expression of granulocyte macrophage-colony stimulating factor gene in murine colon carcinoma cells. AB - Murine colon carcinoma cells which secrete several kinds of cytokine after retroviral transduction with corresponding genes, were examined for their antitumor effects in syngeneic mice. The mice inoculated with granulocyte macrophage-colony stimulating factor (GM-CSF) producer cells showed not only prolonged survival but also reduced tumorigenicity. The antitumor effect caused by the expression of interleukin-4 was less than that of GM-CSF, and interleukin 6 producer cells did not show any effects on the survival of the host animals. Histological examination of the GM-CSF-producing tumor revealed predominant infiltration of neutrophils and necrotic change of the tumor. The present study indicates the feasibility of cancer gene therapy with the expression of GM-CSF gene in tumor cells. PMID- 8620479 TI - Involvement of B lymphocytes in the growth inhibition of human pulmonary melanoma metastases in athymic nu/nu mice by an antibody-lymphotoxin fusion protein. AB - Antibody-cytokine fusion proteins can target biologically active cytokines to various tumor sites, achieving local concentrations sufficient to induce host immune responses leading to tumor elimination. Here, we demonstrate the therapeutic efficacy of a tumor-specific antibody-lymphotoxin fusion protein (ch225-LT) on xenografted pulmonary metastases of human melanoma. In vitro studies indicated a direct cytotoxic effect of such construacts on melanoma cells via the induction of apoptosis, as demonstrated by cell cycle analysis and DNA fragmentation. However, ch225-LT lacked any therapeutic effect in immune deficient C.B17 scid/beige and scid/scid mice, indicating the insufficiency of this direct mechanism in vivo. In contrast, in athymic nu/nu mice, ch225-LT completely inhibited outgrowth of the xenografted tumor. This therapeutic effect was accompanied by infiltrations of CD45+, Mac-1+, and asialo-GM1+ cells into the tumor; B220+ cells were present in the surrounding tissue and the periphery of the tumor. The functional role of asialo-GM2+ cells was confirmed by in vivo depletion studies. Our data indicate that an antibody-lymphotoxin fusion protein effectively inhibits the growth of disseminated melanoma metastases by mechanisms that function in the absence of mature T cells, but require B, NK, and other asialo-GM1+ cells. PMID- 8620480 TI - Involvement of CPP32/Yama(-like) proteases in Fas-mediated apoptosis. AB - Fas (Apo-1/CD95) belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits apoptotic signals by binding to its ligand. Interleukin-1beta-converting enzyme (ICE), which shows substantial homology to the product of the cell death gene, ced-3, of Caenorhabditis elegans, is reported to be involved in Fas-mediated apoptosis. Using two human carcinoma-derived cell lines with undetectable levels of ICE, we found that an agonistic antihuman Fas antibody induces the activation of CPP32/Yama(-like) proteases that are ICE( like) protease family members, and that a tetrapeptide inhibitor of CPP32/Yama protease, DEVD-CHO, inhibits the Fas-mediated activation of the proteases, Fas mediated apoptosis, and CPP32/Yama(-like) proteolytic activities in vitro. Fas mediated apoptosis is inhibited by the CPP32/Yama inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO, suggesting a dominant role for the CPP32/Yama(-like) proteases and not ICE itself in Fas-mediated apoptosis of the human carcinoma cell lines. PMID- 8620481 TI - Design of a novel bicistronic expression vector with demonstration of a p16INK4 induced G(1)-S block(1). AB - Traditional eukaryotic gene expression systems have many shortcomings that make them unsuitable for the analysis of cytotoxic and growth-arrest genes. An intestinal alkaline phosphatase bicistronic expression vector was specifically designed to facilitate such studies. Intestinal alkaline phosphatase serves as a marker of cells that have been transfected and, therefore, must also be co expressing the gene under study. Using flow cytometry, a trivariate analysis was performed on p16INK4-transfected U87 glioblastoma cells. An average G1-S block of 77.5% was demonstrated compared to controls despite a 1% transfection efficiency. This vector has universal applications, including: (a) analysis of cytotoxic and growth-inhibitory genes in transient assays; (b) 100% enrichement in gene expression studies, especially in low transfection efficiency experiments; and (c) facilitation of the study of cell cycle kinetics. PMID- 8620482 TI - Exceptional chemopreventive activity of low-dose dehydroepiandrosterone in the rat mammary gland. AB - To determine if the chemopreventive activity of dehydroepiandrosterone (DHEA) in the rat mammary gland can be dissociated from its toxicity, two studies were conducted in which low doses of DHEA were administered alone and in combination with other agents to rats treated with N-methyl-N-nitrosourea. Beginning 1 week prior to administration of 35 mg N-methyl-N-nitrosourea per kg body weight, groups of 20 female Sprague-Dawley rates were fed AIN-76A diet supplemented with DHEA alone (800 or 400 mg/kg diet), DHEA + tamoxifen (80 or 40 microgram/kg diet), DHEA + carbenoxolone (3500 or 1750 mg/kg diet), or DHEA + tamoxifen + carbenoxolone. When administered alone at either 800 or 400 mg/kg diet, DHEA reduced mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence in all DHEA combination regimens was also < or = 5%. The dose levels of DHEA used induced no toxicity or alteration in body weight gain. These results indicate that dietary supplementation with low doses of DHEA has chemopreventive efficacy greater than or equal to that of endocrine ablation. This protection may be mediated by the induction of differentiation in the mammary parenchyma. PMID- 8620483 TI - The farnesyltransferase inhibitor FTI-277 radiosensitizes H-ras-transformed rat embryo fibroblasts. AB - Many tumor cells have a greater resistance to ionizing radiation than their normal counterparts, suggesting that the development of drugs that can reduce that radioresistance would potentiate the efficacy of radiation therapy. Because activated H-ras expression has been shown to markedly increase radiation resistance in some transformed cells, the inactivation of H-ras would then be predicted to radiosensitize these tumor cells, while leaving normal cells unaffected. H-ras depends for activity upon farnesylation, which can be blocked by farnesylation inhibitors, including the compound FTI-277. In keeping with this prediction, inhibition of H-ras processing using FTI-277 resulted in higher levels of apoptosis after irradiation and increased radiosensitivity in H-ras transformed rat embryo cells but did not affect control cells. These experiments suggest that farnesylation inhibitors may prove clinically useful as radiosensitizers of tumors that depend on ras function. PMID- 8620484 TI - Alternative splicing and differential expression of DT-diaphorase transcripts in human colon tumors and in peripheral mononuclear cells in response to mitomycin C treatment. AB - The two-electron bioreductive enzyme DT-diaphorase catalyzes the metabolism of quinones. The existence of several distinct sizes of DT-diaphorase mRNA transcripts has been observed in human tissues. One of these, an alternatively spliced mRNA that lacks exon 4, has been recently found to be expressed at levels comparable to those of the full-length mRNA. The protein encoded by the mRNA lacking exon 4 has minimal catalytic activity, consistent with the elimination of the quinone-binding site coded for by this exon. We have pursued a number of approaches to examine the significance of this splice variant. We identified a similar truncated transcript in a human HepG2 cDNA library. To determine the frequency of expression of this form of DT-diaphorase in the general population, we examined mRNA obtained from the peripheral mononuclear cells of 16 patients and found substantial interindividual variability in the patterns of transcript expression. Following treatment of these 16 patients with 20 mg/m2 mitomycin C (MMC), the induction of DT-diaphorase transcripts was demonstrated. In most patients, expression of the variant transcript (lacking exon 4) remained constant, while that of the full-length mRNA was elevated. The extent of induction also showed interindividual variability. In one patient, while both transcripts were present at baseline, expression of the variant transcript disappeared almost completely after MMC treatment. To analyze these events under more controlled conditions, we examined the effects of MMC treatment on two human colon tumor cell lines. MMC treatment induced expression of the full-length mRNA but did not influence the abundance of the variant transcript. We then performed single-strand conformational polymorphism analysis of genomic DNA from the 16 patients to investigate the potential role of cis-acting factors in the variable splicing responses. Two patients demonstrated sequence differences in the region spanning exon 4, but in neither was the change in a region critical to splicing regulation. These data demonstrate that the expression of DT-diaphorase in hyman cells is polymorphic, and that the levels of individual transcripts can be regulated by exogenous factors. The findings support a role for alternative splicing in the control of DT-diaphorase gene expression. PMID- 8620485 TI - Combination gene therapy for oral cancer in a murine model. AB - Combination therapy involving adenovirus-mediated transfer of the genes for herpes thymidine kinase (tk) and murine interleukin 2 (mIL-2) was used to treat head and neck cancer in C3H/HeJ mice. Tumors were generated by transcutaneous injection of 5 X 10(5) murine squamous carcinoma cells into the floor of the mouth of these syngeneic mice. After 1 week, recombinant adenoviral vectors containing both therapeutic and control genes in various combinations were injected directly into the established tumors, and subsequently all mice were administered ganciclovir twice daily (25 mg/kg) for 6 days. Animals receiving either tk alone or tk + mIL-2 demonstrated significant tumor regression compared to mIL-2 alone or control vector-treated mice (P < 0.008). Mice receiving both tk + mIL-2, however, also demonstrated a significantly greater regression of tumors compared to those treated with tk alone (P<0.008), indicating a synergistic effect of the combination gene therapy. This synergism was confirmed in survival studies because tk + mIL-2 treated mice showed increased survivals (P=0.0002). Clinical and microscopic exam of regional surrounding tissues and distant organs showed no evidence of cytotoxicity for representative animals in each experimental group. These results suggest that combination tk and mIL-2 gene therapy may provide a powerful new modality for the treatment of head and neck cancer. PMID- 8620486 TI - Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region. AB - The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene. PMID- 8620487 TI - Cloning and expression analysis of human bleomycin hydrolase, a cysteine proteinase involved in chemotherapy resistance. AB - A cDNA encoding human bleomycin hydrolase, a member of the cysteine proteinase family of proteins, has been cloned from a human brain cDNA library. The isolated cDNA contains an open reading frame coding for a polypeptide of 456 amino acids that contains all of the structural features characteristic of cysteine proteinases, including the cysteine, histidine, and asparagine residues that are essential for the catalytic properties of these enzymes. The deduced amino acid sequence for human bleomycin hydrolase shows 92, 40, and about 35% of identities with those determined for rabbit bleomycin hydrolase, yeast bleomycin hydrolase, and bacterial aminopeptidase C, respectively. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that human bleomycin hydrolase is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in norman cellular protein degradation and turnover. Preliminary expression analysis of bleomycin hydrolase in different human tumors showed increased expression of the enzyme in a series of head and neck carcinomas when compared with paired adjacent normal mucosa. We also observed a variable degree of bleomycin hydrolase expression in different types of lymphoma, with low or undetectable levels in Hodgkin's disease samples and higher levels in Burkitt's lymphomas. These results are consistent with a proposed role for human bleomycin hydrolase in resistance of some tumor to bleomycin chemotherapy. PMID- 8620488 TI - Correlation of KAI1/CD82 gene expression with good prognosis in patients with non small cell lung cancer. AB - As part of our evaluation of members of the transmembrane 4 super-family as possible prognostic predictors, we performed a retrospective study on the expression of the recently identified KAI1 gene by tumors of the lung. This gene, which is identical to CD82, suppresses tumor metastasis of prostate cancer, and its decreased expression may be involved in malignant progression. We used reverse transcription-PCR to analyze tumor tissues from 151 lung cancer patients; 74 tumors were stage I, 17 were stage II, and 60 were stage III. Our results indicate that while 35 patients had tumors in which the KAI1/CD82 gene was conserved (positive), 116 patients had tumors with reduced gene expression (negative). The overall survival rate of patients with KAI1/CD82-positive tumors was significantly higher than that of patients with KAI1/CD82-negative tumors (77.4% versus 38.5%; P=0.002). Furthermore, the overall survival rate of patients with KAI1/CD82-positive adenocarcinoma was also much higher than that of individuals whose adenocarcinoma had reduced KAI1/CD82 expression (73.4% versus 27.1%;P=0.009). Multivariate analysis with the Cox regression model indicated that KAII/CD82 positivity correlated best with the overall survival rate, except for lymph node status. Our data suggest that high KAII/CD82 gene expression by tumors of the lung may be associated with a good prognosis. These findings complement our earlier studies on MRP-1/CD9, another member of the transmembrane 4 superfamily, whose reduced expression in non-small cell lung cancer appears to be a factor of poor prognosis. This set of observations suggests that assessment of the expression status of KAI1/CD82 and MRP-1/CD9 by tumors may provide prognostic information on the clinical behavior of lung cancer. PMID- 8620489 TI - Analysis of the major histocompatibility complex class I antigen presentation machinery in normal and malignant renal cells: evidence for deficiencies associated with transformation and progression. AB - In some human tumors, reduced or defective MHC class I surface expression has been attributed to functional deficiencies of the genes of the antigen-processing machinery, the proteasome subunits low molecular weight (LMP)-2 and LMP-7, as well as the peptide transporters associated with antigen processing (TAP)-1 and TAP-2. Using normal epithelial kidney cells (MZ1851NN) and renal cell carcinoma cell lines established from the primary tumor (MZ1851RC) and a lymph node metastasis (MZ1851LN) of the same patient, we investigated whether the modulation of MHC class I antigens, TAP and LMP molecules, occurs during transformation and subsequent progression. The mRNA and protein expression of MHC class I heavy and light chain TAP and LMP was strongly reduced in MZ1851RC when compared to the corresponding normal kidney cells MZ1851NN, and this suppression was even more pronounced in the metastatic cell line MZ1851LN. In addition, the activity of the TAP molecules, as measured by peptide translocation assays, was also markedly diminished in MZ1851RC compared to MZ1851NN cells and was further down-regulated in cells of the metastatic lesion. MHC class I surface expression was enhanced by either culturing MZ1851RC and MZ1851LN cells at 26 degrees C instead of 37 degrees C or by incubation of both cell lines with class I-specific binding peptides, whereas MHC class I surface expression of MZ1851NN cells was not affected under these culture conditions. IFN-alpha and in particular IFN-gamma treatment enhances the steady-state mRNA and/or protein levels of TAP, LMP, and MHC class I genes of MZ1851 cell lines but had no additional effect on the stability of MCH class I surface expression. These data indicate that malignant transformation and subsequent in vivo selection of renal tubular cells can lead to the recovery of carcinoma cells that show stable expression of an immune escape phenotype. Deficiencies associated with this phenotype involve all levels of the MHC class I-restricted antigen presentation machinery, are at least partially reversible by IFN treatment, and are even more pronounced in cells that had acquired metastatic potential. PMID- 8620490 TI - Overexpression of the insulin-like growth factor-1 receptor and autocrine stimulation in human cervical cancer cells. AB - We characterized mechanisms of growth control involving insulin-like growth factor-1 (IGF-1), IGF-2, and IGF-1 receptor (IGF-1R) by investigating their expression in human cervical cancer cell lines, primary cervical tumor cell cultures, and normal ectocervical epithelial cells maintained in short-term culture. By reverse transcription followed by PCR, IGF-1 mRNA was not detected in any of the cell lines, whereas IGF-2 mRNA transcripts were detected in all of them. Using the RNase protection assay, low levels of IGF-2 mRNA were also detected in all of the cervical cancer cell lines, primary cervical tumor cell cultures, and normal ectocervical cultures tested, but no IGF-1 transcripts were detected. Scatchard analysis revealed 3- and 5-fold increases in IGF-1R expression by the primary cervical cancer cell cultures and cervical cancer cell lines, respectively, compared with the normal ectocervical cells. In proliferation assays, epidermal growth factor (EGF) consistently enhanced cervical cancer cell growth, but an antisense oligonucleotide to IGF-2 uniformly inhibited the EGF-induced mitogenic effect. These studies suggest that autocrine production of IGF-2 and overexpression of the IGF-1R are important components controlling the proliferation of cervical carcinoma cells, and that autocrine IGF 2 production in cervical cancer cells may participate in the mitogenic signaling of EGF. PMID- 8620491 TI - Complete exon structure of the ALL1 gene. AB - The ALL1 gene is found rearranged in approximately 10% of acute lymphoblastic leukemias and in over 5% of acute myeloid leukemias. The gene undergoes fusion with either a variety of partner genes located on different chromosomes or with itself. To further characterize the role of the ALL1 gene in the leukemogenic process, and possibly in solid malignancies, we defined its complete genomic structure. The gene, which spans a region on chromosome band 11q23 approximately 90 kb in length, consists of 36 exons, ranging in size from 65 bp to 4249 bp. The determination of intronic sequences flanking the exon boundaries will allow the determination of whether point mutations may be responsible for inactivation of the gene in solid tumors showing loss of heterozygosity at region 11q23. PMID- 8620492 TI - Uncoupling of M-phase kinase activation from the completion of S-phase by heat shock. AB - Chronic exposure of asynchronous HeLa cell cultures to 41.5 degrees C leads to an accumulation of cells in the S-phase, spontaneous premature chromosome condensation, and loss of clonogenicity (M.A. Mackey, S. L. Anolik, and J. L. Roti Roti. Cancer Res., 52: 1101-1106, 1992). In this report, we show that increases in histone H1 kinase activity during 41.5 degrees C exposure occur coincidentally with the appearance of premature chromosome condensation. Furthermore, this kinase activity is shown to be associated with M-phase kinase complexes containing cyclin B1. These increases in the activity of M-phase kinase were found to occur concomitantly with an elevation in cyclin B1 mRNA and an accumulation of cyclin B1 protein. Because cyclin B1 transcription begins in the S-phase, it is probable that the heat-induced delay in the S-phase allows the accumulation of abnormally high cyclin B1 levels. Elevated cyclin B1 levels could then account for the observed abrogation of the cell cycle checkpoint, which usually assures that mitosis does not proceed until DNA replication is complete. This involvement of M-phase kinase in heat-induced cytotoxicity demonstrates the importance of the coordinate regulation of the processes of DNA replication and entry into mitosis. PMID- 8620493 TI - Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. AB - Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4-kb tumor specific Int3 RNA transcript that encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development were curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene, which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter, the activity of which, unlike that of the MMTV long terminal repeat, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In nonbreeding WAP/Int3 females, mammary tumor incidence also reached 100%, but only after 70 weeks. The WAP/Int3 mammary tumors were highly malignant, and most tumor-bearing females, irrespective of breeding history, developed metastatic lung lesions. These results suggest that WAP promotor-targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into nontransgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated. PMID- 8620494 TI - Mechanism of oxidative DNA damage induced by delta-aminolevulinic acid in the presence of copper ion. AB - Delta-Aminolevulinic acid (ALA) is a heme precursor accumulated in lead poisoning and acute intermittent porphyria. ALA-induced DNA damage in the presence of metal ions was investigated with a DNA sequencing technique and a high-performance liquid chromatograph equipped with an electrochemical detector. ALA caused damage to DNA fragments obtained from c-Ha-ras proto-oncogene in the presence of Cu(II), but only slightly in the presence of Fe(II). ALA + Cu(II) induced piperidine labile sites at thymine residues, especially in the 5'-GTC-3' and 5'-CTG-3' sequences of double-stranded DNA. Catalase and bathocuproine inhibited DNA damage induced by ALA + Cu(II). Typical .OH scavengers did not inhibit DNA damage, suggesting that active species other than .OH play a more important role in DNA damage. 8-Hydroxy-2'-deoxyguanosine formation by ALA increased with ALA concentration in the presence of Cu(II). Electron spin resonance studies using alpha-(1-oxy-4-pyridyl)-N-tert-butylnitrone as spin trap showed that carbon centered radicals were generated during Cu(II)-catalyzed autoxidation of ALA. The major pathway of ALA autoxidation consists for the formation of 4,5-dioxovaleric acid and NH(4)+. Formation of a pyrazine derivative through ALA autocondensation was also observed. Concomitantly, O2- and H2O2 were generated during the Cu(II) catalyzed ALA autoxidation. These results indicate that H2O2 reacts with Cu(I) to form a crypto-OH radical, such as the Cu(I)-peroxide complex, causing DNA damage. The possible mechanism for metal-dependent DNA damage by ALA is discussed in relation to the carcinogenicity of lead compounds and the increased frequency of liver cancer in acute intermittent porphyria. PMID- 8620495 TI - Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells. AB - The hormones derived from vitamin A and related synthetic ligands (retinoids) are important regulators of differentiation and development and have been shown to be therapeutically useful in the treatment of cervical cancer. All-trans-retinoic acid exerts its effects by activation of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers. These heterodimers bind to the retinoic acid response elements of target genes to regulate gene expression. RXR ligands act through RXR homodimers to regulate gene expression. In the present study, we describe the effects of RAR- and RXR-specific ligands on the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) production and cell proliferation in human ectocervical epithelial (ECE) cell lines. Treatment of ECE16-1 cells with a RAR-specific ligand (TTNPB) or a ligand that interacts with both RAR and RXR receptors (9-cis-retinoic acid) increases IGFBP-3 levels and suppresses cell proliferation. In contrast, RXR-specific ligands (AGN191701, SR11217, and SR11237) do not regulate proliferation and slightly suppress the IGFBP-3 level. Cotreatment with increasing concentrations (0.01-1000nm) of RXR specific ligand antagonizes the growth suppressive and IGFB-3-increasing effects of 1000 nM TTNPB. Similar results are observed in two other ECE cell lines, ECE16 D1 and ECE16-D2. These results indicate that RXR-specific ligands can antagonize RAR responses in these cell lines and suggest that a RAR-specific retinoid may be superior to one with mixed RAR/RXR binding activity for inhibiting cervical cancer cell proliferation. Moreover, the antagonism of RAR-dependent responses by RXR-specific ligands is consistent with a squelching model in which the RXR specific ligand drives formation of RXR/RXR homodimers at the expense of the more active RAR/RXR heterodimers. PMID- 8620496 TI - A new sensitive method for determination of intracellular 1-beta-D arabinofuranosylcytosine 5'-triphosphate content in human materials in vivo. AB - A new sensitive method for the measurement of 1-beta-D-arabinofuranosyl-CTP (ara CTP), an intracellular active metabolite of 1-beta-D-arabinofuranosylcytosine (ara-C), in human materials in vivo has been established. An acid-soluble fraction containing ara-CTP was extracted from blastic cells by ara-C treatment with trichloroacetic acid (final concentration, 0.3 M) neutralized with an equal volume of cold freon containing 0.5 M tri-n-octylamine. The ara-CTP fraction was separated from the acid -soluble fraction by high-performance liquid chromatography (TSK gel diethylaminoethyl-2 SW column) eluted with 0.05 M phosphate buffer (pH 6.9) and 20% acetonitrile. ara-CTP was lyophilized, dephosphorylated to ara-C by incubation with 10 units alkaline phosphatase for 12 h at 55 degrees C, and measured by RIA using anti-ara-C serum. Recovery through the whole procedure was 92%. In the human chronic myelogenous leukemia cell line K562, the intracellular ara-CTP levels produced when the cells were incubated with ara-c were assayed as above, and they showed a linear increase depending on Ara-C concentrations from 0.01 to 10 microns, demonstrating a very close correlation with the labeled ara CTP levels yielded by cells on incubation with radiolabeled ara-C (r2 = 0.99). The detection limit was 0.1 pmol/5 x 10(6) cells, and a sample amount of only 5 x 10(6) cells was enough for each assay. In the clinical applications, our method proved capable of detecting a wide concentration range of ara-CTP produced when patients were treated with ara-C or its derivatives from very low to intermediate doses. No radiolabeled drug was necessary. The method was very useful for in vivo pharmacodynamic studies of ara C therapy. PMID- 8620498 TI - Production of a novel monoclonal antibody, JT-95, which can detect antigen of thyroid carcinoma. AB - Monoclonal antibody (MAb) JT-95 was produced by immunization of mice with membrane fractions of a human thyroid carcinoma. Immuno-histochemical staining has demonstrated that the antigen recognized by JT-95 is strongly expressed in 95 (95%) of 100 cases of papillary carcinomas and in 3 (75%) of 4 cases of follicular carcinomas. In benign diseases of the thyroid gland, MAb JT-95 reacted with 0 (0%) of 39 adenomas, 1 (4%) of 21 adenomatous goiters, 0 (0%) of 8 hyperthyroidism specimens, and 3 (38%) of 8 chronic thyroiditis specimens. The antigen detected by MAb JT-95 has an apparent Mr 250,000 in thyroid carcinomas. Moreover, circulating antigen in thyroid carcinoma patients was detected by MAb JT-95 in an ELISA and in Western blotting. The circulating antigen has a Mr 105,000. MAb JT-95 conjugated with (131) I was administrated to nude mice bearing a human thyroid carcinoma. JT-95 131I accumulation at the transplanted tumor was visualized by autoradiography with 2.68-14.75-fold higher levels detected at the xenograft compared to that for normal organs. Based on these data, MAb JT-95 may be useful in the diagnosis detection and therapy of thyroid carcinoma. PMID- 8620497 TI - Factors influencing the pharmacokinetics, dosimetry, and diagnostic accuracy of radioimmunodetection and radioimmunotherapy of carcinoembryonic antigen expressing tumors. AB - The aim of this study was to examine factors that may influence the pharmacokinetics, diagnostic accuracy, and dosimetry in radioimmunodetection and radioimmunotherapy with anti-carcinoembryonic antigen (CEA) monoclonal antibodies (mAbs). Data from 275 patients with CEA expressing tumors were analyzed retrospectively. Of these, 69 patients devoid of human antimouse antibody (i.e., 31 colorectal, 9 lung, 7 breast, 4 ovarian, 6 pancreatic, 9 medullary thyroid, 1 gallbladder, and 1 salivary gland cancer, and 1 primary tumor of unknown origin) underwent a low-protein-dose diagnostic study (0.3-2.6 mg of protein; 6.8-28.8 mCi 131I-labeled IgG or fragments), followed within 4 weeks by a high-protein dose therapy injection (4.0-27.5 mg of protein; 29.8-238.9 mCi). The anti-CEA antibodies NP-4 (Ka=10(8)M-1) and MN-14 (ka=10(9)M-1) were used. Plasma clearance, the molecular composition of radioactivity in the plasma, and the cumulated activity in organs and tumors were determined. Radiation doses were derived from the Medical Internal Radiation Dose scheme. At a low-protein dose and over a similar range of plasma CEA, a significantly higher percentage of MN 14 than of NP-4 was complexed with circulating CEA, consistent with its higher affinity. Complexation was reduced with increasing protein doses. However, the targeting sensitivity was not affected. Profound differences were found in the clearance of the antibody between different types of cancer. Colorectal cancer patients cleared the antibody significantly faster from blood (T1/2=17.6+/-12.6 versus 44.2 +/- 23.7 h) and whole body (t1/2= 53.2 +/- 30.1 versus 114.6+/-59.7 h) than all other tumor types (P <0.001). Consequently, significantly lower red marrow (2.1 +/- 1.0 cGy/mCi versus 4.3 +/- 1.6 cGy/mCi) and whole-body doses (0.5 +/- 0.3 cGy/mCi versus 1.0 +/- 0.4 cGy/mCi) were seen in colorectal cancer patients as compared with other tumor types (P < 0.001). This clearance is probably due to hepatic metabolism of the immune complexes. Clearance rates were especially high in patients with colorectal cancer having large liver metastases and elevated liver enzymes (rapid hepatic clearance with liberation of free I-). In contrast, a disease-stage and plasma CEA-matched cohort of colorectal cancer patients, examined with the 131 I-labeled anti-colon-specific antigen p mAb Mu-9, showed normal murine IgG pharmacokinetics (n=22;3 of them compared intraindividually to MN-14). Only in colorectal cancer patients did complexes between mAb and CEA tend to clear rapidly, whereas Mu-9 had normal kinetics in these patients. This suggests that different CEA-expressing cancer types may produce heterogeneous CEA molecules and that the variability in mAb clearance is due to varying clearance rates of these different circulating CEA subspecies. Disease-related alterations in antibody metabolism are unlikely, given that only anti-CEA antibodies exhibit this phenomenon. PMID- 8620499 TI - Loss of sst2 somatostatin receptor gene expression in human pancreatic and colorectal cancer. AB - Five somatostatin receptor subtypes (sst1 to sst5) have been cloned. We demonstrated previously that sst2 and sst5 mediate the antiproliferative effect of the somatostatin analogues octreotide and vapreotide. Using reverse transcription-PCR, we investigated gene expression of the five receptors in 47 human normal and cancerous tissues or cell lines from pancreatic and colorectal origin. mRNAs of somatostatin receptor subtypes were detected in 98% of samples, with more than two mRNA subtypes being expressed in 55% of cases. sst1, sst4, and sst5 were heterogeneously expressed in both normal and cancerous tissues; sst3 was rarely or not expressed. sst2 was present in normal pancreatic tissues but was absent in exocrine pancreatic carcinomas and their metastases. sst2 mRNAs were detected in normal colon, sporadic polyadenomas, and 50% of Dukes' stage B and 20% of Dukes' stage C carcinomas but were undetectable in Dukes' stage D carcinomas, hepatic metastases, and adenomas from familial adenomatous polyposis. The loss of sst2 expression could represent a growth advantage in these tumors and provide an explanation for the lack of therapeutic effect of somatostatin analogues in such adenocarcinomas. A subtyping of somatostatin receptors should be carried out before considering a somatostatin analogue treatment in patients with colorectal or pancreatic cancer. PMID- 8620500 TI - Intensified antitumor immunity by a cancer vaccine that produces granulocyte macrophage colony-stimulating factor plus interleukin 4. AB - Vaccination with irradiated tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF tumor vaccine) induces a potent systemic antitumor immunity. To develop a protocol for cancer therapy to further augment the host immune response, we examined the effects of the GM-CSF tumor vaccines simultaneously producing additional cytokines. We prepared cancer vaccines expressing double cytokines by sequential recombinant retrovirus mediated genetic transductions. We then used a murine intracerebral tumor model in which the GM-CSF tumor vaccine was less effective in immunopotentiation and evaluated tumor vaccines producing various cytokines in conjunction with GM-CSF. The cytokine combination of GM-CSF and interleukin 4 induced more potent antitumor immunity than GM-CSF alone. An in vivo depletion test showed that CD4+, CD8+, and asialoGM1+ cells were required for the optimum function of the GM-CSF plus interleukin 4 tumor vaccine. Histological examinations revealed infiltration of inflammatory cells at the site of tumor cell challenge as well as at the site of vaccination, indicating the induction of a systemic antitumor immune response which reached the central nervous system. Our findings suggest the feasibility of applying the intensified vaccination strategy to treat human cancers including malignant brain tumors. PMID- 8620501 TI - Hypersensitivity of human testicular tumors to etoposide-induced apoptosis is associated with functional p53 and a high Bax:Bcl-2 ratio. AB - Metastatic testicular cancers are curable, whereas bladder cancers and most other solid tumors are not. Cell lines derived from human testicular (GH, GCT27, and 833K) and bladder (RT4, RT112, and HT1376) tumors retain this differential chemosensitivity in vitro. We have investigated the hypothesis that differential sensitivity to chemotherapy is related to differences in the threshold of susceptibility to undergoing apoptosis. Sensitivity to etoposide was not directly related to the frequency of DNA strand breaks. DNA damage was on average 2-fold greater in the testicular than the bladder tumor cell lines; in contrast, the testicular tumor lines were 15-fold more sensitive to etoposide cytotoxicity than the bladder tumor lines (IC90 values of 19 +/- 6 versus 293 +/- 180 microM, respectively). Using equidamaging (550 rad equivalents) etoposide treatments, the percentage of cells that underwent drug-induced apoptosis was on average higher in the testicular tumor cell lines than the bladder tumor cell lines. The testicular tumor lines have two characteristics that could confer sensitivity to drug-induced apoptosis. First, they have functional p53: the product of the p53 dependent gene waf-1 was increased after etoposide treatment. Second, the testicular tumor lines expressed relatively high levels of the apoptosis promoting protein Bax, but there was no expression of the suppressor of apoptosis Bcl-2. In contrast, only one of the three bladder cell lines (RT4) had functional p53, and all of the bladder lines had readily detectable levels of Bcl-2 and low levels of Bax. In the testicular cell lines, increases in p53 and p53 transactivated genes were associated with apoptosis but not arrest in G1. In contrast, in the bladder cell line (RT4), increases in p53 and Waf-1 were associated with both arrest in G1 and apoptosis. The differences in the ratio of Bax:Bcl-2 could contribute to the differential sensitivity of the two tumor types. However, in contrast to earlier reports, the ratio of Bax and Bel-2 was not perturbed by DNA damage. PMID- 8620502 TI - Interaction of ionizing radiation with paclitaxel (Taxol) and docetaxel (Taxotere) in HeLa and SQ20B cells. AB - Altered gamma-ray response by brief (1 h), concomitant exposure to paclitaxel (Taxol) or docetaxel (Taxotere) was investigated in growing HeLa and SQ20B human tumor cells in vitro. For both cell lines, both taxoids were able to reduce or enhance radiation cell killing, depending on the drug concentration. Large reduction of radiosensitivity (up to 3.3-fold reduction relative to radiation alone) was observed in HeLa cells over a wide range of drug concentrations, extending to 1.5- (paclitaxel) or 3.3- fold (docetaxel) the IC50s determined for drug alone. This antagonistic effect was also observed with SQ20B cells. It disappeared for drug concentrations exceeding 0.9 (SQ20B), 1.6 (HeLa; paclitaxel), and 3.4 (HeLa; docetaxel) IC50 equivalents, above which a drug dose dependent, supra-additive radiation-drug interaction was observed. Reduction of radiation susceptibility in the low-drug dose range also held for mid-G1 synchronized HeLa cells, i.e., in the cell cycle compartment characterized as the most resistant one to docetaxel (C. Hennequin et al., Br. J. Cancer, 71: 1194 1198, 1995). In the case of SQ20B cells, the cytotoxicity of either drug or radiation alone was primarily dependent on the state of growth, with quiescent (G(0)) cells showing increased radiosensitivity and reduced drug toxicity compared to the growing fraction. The effect of taxoids (1-h contact) was finally investigated in sequential treatment as a function of the time elapsed between radiation and exposure to drugs. In HeLa cells, the postirradiation time dependence of the response to combined treatment was biphasic. The radioprotecting potential of either taxoid disappeared in approximately 1.5 h following radiation. At longer postirradiation delays, radiation-induced redistribution in the cell cycle appeared to be the major determinant of HeLa cell survival, in relation to the differential cell cycle phase specificity of each drug. Pronounced paclitaxel recovery versus increased sensitivity to docetaxel occurred over 8 h after irradiation. SQ20B cells showed monophasic radiation recovery with both drugs over the same time range. PMID- 8620503 TI - Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. AB - c-Raf-1 (Raf-1) is a central component of signal transduction pathways stimulated by various growth factors, protein kinase C, and other protein kinases. Raf-1 activation is thought to be initiated at the plasma membrane after its recruitment by Ras. Raf-1 activation is associated primarily with proliferation and cell survival, but it has also been implicated in apoptosis. Raf-1 has also been shown to form complexes with both R-Ras and Bcl-2, raising the possibility that this component of cellular Raf-1 plays a role in apoptosis. Recently, taxol was reported to induce Bcl-2 phosphorylation and inactivation. We have previously demonstrated Raf-1 activation following taxol in MCF7 cells. We now present evidence that taxol fails to stimulate either apoptosis or phosphorylation of Bel 2 in the absence of Raf-1. Moreover, Raf-1 activation by taxol coincided with Bel 2 phosphorylation, showing similar dose and time dependence. Thus, our data support a role for a distinct subcellular component of Raf-1, which is taxol but not phorbol myristate acetate sensitive, in mediating an apoptotic pathway involving Bc1-2. PMID- 8620504 TI - Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin: heterogeneous localization and reversibility. AB - DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino) methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug stimulated DNA cleavage in the chromatin of living cells. PMID- 8620505 TI - Evaluation of efficient chemoembolization mixtures by magnetic resonance imaging therapy monitoring: an experimental study on the VX2 tumor in the rabbit liver. AB - To find effective chemoembolization mixtures, we tested combinations of carboplatin with the embolizates Spherex and Gelfoam in comparison to a therapy with NaCl-solution, a treatment with the cytostatic drug only, and a therapy with each of the embolizates alone. The experiments were carried out using as a model the VX2 tumor in the liver of male chinchilla rabbits (five for each group). Carboplatin was revealed by the 3-(4,5-dimethylthiazole-2)-yl-2,5 diphenyltetrazolium bromide test to be a potent cytostatic drug for VX2 rabbit tumor cells. We used magnetic resonance imaging to examine the tumor volume and signal intensity enhancement up to 15 min after Gd-DTPA administration within the tumor and liver before and after the different therapies. These parameters allowed us to evaluate tumor growth and vitality as well as liver injury for the different therapy types. The results found by magnetic resonance imaging corresponded very well to those obtained by histological analysis of the tumors. The chemoembolization therapies were significantly more efficient than the other therapies, as indicated by the reduction of signal intensity enhancement after contrast agent administration within the tumor and by the histologically determined necrotic fraction after therapy. In addition, we found a significant decrease of the tumor volume and no significant live injury for a therapy with Carboplat and Gelfoam. PMID- 8620506 TI - Changes in cellular components of spleen and lymph node cells and the effector cells responsible for Meth A tumor eradication induced by zinostatin stimalamer. AB - We reported previously that pretreatment with zinostatin stimalamer (ZSS) eradicated Meth A tumors in BALB/c mice. We herein investigated cellular components of spleen and lymph node cells of Meth A-bearing ZSS-pretreated mice by flow cytometry; the antitumor effector cells by in vivo depletion of T cells, NK cells, or macrophages; and host-mediated antitumor activity associated with ZSS treatment after tumor transplantation. ZSS given on day-3 transiently decreased the number of spleen cells. The percentage of T cells increased, but B cells and macrophages decreased. B cells decreased in inguinal lymph nodes in Meth A-bearing ZSS-pretreated mice, but increased in Meth A-bearing control mice. In vivo depletion experiments using antibodies or carrageenan showed that antitumor effector cells for tumor eradication are Thy1.2+/Lyt2.2+ and that at least a part of them are asialo GM1+. Thy1.2+/Lyt2.2+/asialoGM1- cells are important in generation of the antitumor activity of ZSS; however, L3T4+ T cells are also involved in initiation of tumor eradication. The result of ZSS treatment after tumor transplantation suggests that ZSS might exhibit antitumor activity by augementating host-mediated antitumor resistance, as well as its intrinsic cytocidal activity. PMID- 8620507 TI - Resistance of AIDS-associated Kaposi's sarcoma cells to Fas-mediated apoptosis. AB - Escape of tumor cells from apoptotic-mediated stimuli results in tumor cell survival and resistance to cytotoxic mechanisms. Kaposi's sarcoma (KS) is the most common malignancy associated with AIDS, although its pathogenesis is not known. It is clinically important to determine whether AIDS-KS cells are resistant to apoptosis via the Fas system. Three isolates of AIDS-KS cells were studied. Although all KS cells express Fas on the cell surface, these cells were resistant to cytotoxic anti-Fas antibody (IgM, CH-11). Treatment of AIDS-KS cells with actinomycin D sensitized the tumor cells to anti-Fas cytotoxicity and apoptosis. Apoptosis was assessed by morphological changes and DNA fragmentation analysis. Three possible mechanisms related to AIDS-KS cells, resistance to anti Fas cytotoxicity were examined. First, synthesis and secretion of soluble Fas by the tumor cells can neutralize antibody-induced cytotoxicity. However, none of the three types of KS cells expressed soluble Fas mRNA as determined by reverse transcription (RT)-PCR. Second, the expression of the proto-oncogene bcl-2 can protect cells from apoptotic signals. Analysis of bcl-2 mRNA by RT-PCR revealed that all three AIDS-KS cells express very low levels of bcl-2 mRNA. Third, the Fas-associated phosphatase-1 (FAP-1) is an antiapoptotic molecule reported to interact with Fas and can block transduction of the apoptotic signal. RT-PCR analysis revealed that all three types of AIDS-KS cells express high levels of FAP-1 mRNA, and treatment of KS cells with actinomycin D reduced the levels of FAP-1 mRNA significantly. These findings demonstrate that AIDS-KS cells are resistant to Fas-mediated apoptosis and suggest that FAP-1 may be involved in the acquisition of resistance of AIDS-KS to anti-Fas antibody-mediated apoptosis. PMID- 8620509 TI - Aberrant hypermethylation at the bcl-2 locus at 18q21 in human lung cancers. AB - Accumulating evidence suggests that altered DNA methylation may play a role in the oncogenesis of human neoplasms, including lung cancer. The presence of aberrant hypermthylations at 3p, 9p, 11p, ad 17p, which are known to be hot spots for allele loss in lung cancers, is suggested to be a reflection of the existence of tumor suppressor genes in these chromosomal regions. In the present study, we investigated the methylation status of the Rb locus at 13q14 as well as that of the bcl-2 locus at 18q21 in 134 lung cancer specimens, representing all major histological subtypes. As a result, 18q21 was identified to be the fifth chromosomal region affected by frequent tumor-specific aberrant hypermethylation in lung cancers. The occurrence of aberrant hypermethylation at the bcl-2 locus at 18q21 was restricted to non-small cell lung cancers, and among non-small cell lung cancers, such epigenetic aberrations were observed most frequently in adenocarcinomas without any association with bcl-2 expression. Interestingly, allelic loss at the bcl-2 locus was also seen in 40% (7 of 17 informative cases) of adenocarcinomas; this frequency was also the highest among values for the various histological subtypes of lung cancers. These results suggest that aberrant hypermethylation at the bcl-2 locus may be a reflection of a putative tumor suppressor gene residing at 18q21, and aberrant hypermethylation might play a role in its inactivation. In contrast, altered methylation status of the Rb locus appears to be quite rare in lung cancers, if present at all. PMID- 8620508 TI - Differential sensitivity of human and mouse alkyltransferase to O6-benzylguanine using a transgenic model. AB - O6-benzlguanine (O6-bG) potentiates nitrosourea cytotoxicity of human tumor xenografts in nude mice by inactivating O6-alkylguanine-DNA alkyltransferase (AGT). Recent reports dispute whether murine AGT, in cell-free systems, is less sensitive to O6-bG than the human AGT protein, raising the possibility that efficacy seen in the mouse host may not predict the therapeutic index observed in clinical trials. To establish whether mouse and human AGT have different sensitivity to O(6)-bG, we evaluated in vitro and in vivo models of O(6) methylguanine-DNA methyltransferase gene (MGMT) expression in the same genetic background. The 50% inhibitory concentration of O6-bG for inactivation of mouse AGT was >10-fold higher than for the human protein in MGMT-transfected Chinese hamster ovary (CHO) cells. A dose of O6-bG, which inactivated human AGT, markedly sensitized human MGMT-transfected CHO cells to 1,3-bis (2-chloroethyl)-1 nitrosourea (BCNU), whereas mouse MGMT-transfected CHO cells were much more resistant. O6-bG inactivation of AGT in vivo was studied in livers of human MGMT transgenic mice expressing both human and mouse AGT. After a single dose of O6-bG i.p., the 50% inhibitory concentration of AGT was higher for mouse than for human AGT. To reconcile our finding with those of others, we sequenced the mouse MGMT cDNA and found that mutation of amino acid residue Leu180 was associated with O6 bG resistance. These studies provide strong evidence that inactivation of AGT both in vivo and in vitro by O6-bG is species selective and impacts O6-bG mediated enhancement of BCNU toxicity. This may influence the therapeutic index of O6-bG-BCNU combinations observed in human tumor xenograft-bearing mice. PMID- 8620510 TI - Distal nephron renal tumors: microsatellite allelotype. AB - Tumors of varying malignant potential arise from the complex epithelial lining of the nephron. Although the molecular characteristics of renal clear cell carcinomas, which arise from the proximal tubule, have been studied, little is known about tumors that develop from other parts of the renal tubular system. To elucidate common molecular lesions that may contribute to the development or progression of nonproximal tubule renal tumors, we performed a detailed microsatellite allelotype of lesions thought to arise from the renal collecting duct. Eighteen collecting duct carcinomas (CDCs) and 13 renal oncocytomas were studied using highly informative microsatellite markers on all autosomal arms. Loss of heterozygosity (LOH) was identified on multiple chromosomal arms in CDCs and renal oncocytomas. Microsatellite analysis revealed LOH of 1q in 57% of informative CDCs. LOH was also observed on arms 6p (45%), 8p (40%), and 21q (40%). In renal oncocytomas, LOH of 1q occurred in approximately 30% of tumors, but 1p LOH was observed in 57% of informative cases analyzed. High levels of LOH were also observed on arms 8p, 14q, 19q, and 21q in the oncocytomas studied. Loss of chromosomal arm 3p was infrequent in both tumor types. Our results suggest that the molecular events that contribute to the development of distal nephron tumors are distinct from those associated with the etiology of proximal tubule renal cancers. PMID- 8620511 TI - Human colorectal carcinomas express high levels of high mobility group HMGI(Y) proteins. AB - A correlation has previously been demonstrated between the presence of the three HMGI proteins (HMGI, HMGY, and HMGI-C) and the expression of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells; this being subsequently extended to experimental thyroid, lung, prostate, mammary, and skin carcinomas. Recently, we have demonstrated that expression of HMGI and HMGY proteins, coded for by the HMGI(Y) gene, is associated with the malignant phenotype of human thyroid neoplasias. Here, we show that HMGI(Y) gene expression is present both at the RNA and protein level in human colorectal carcinoma cell lines and tissues examined in this study. Conversely, no HMGI(Y) proteins were detected in normal intestinal mucosa. Therefore, these results suggest an involvement of HMGI and HMGY proteins overexpression in colorectal tumorigenesis. PMID- 8620512 TI - Autocrine motility factor signals integrin-mediated metastatic melanoma cell adhesion and invasion. AB - The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C. PMID- 8620513 TI - CD11a-CD18 and CD102 interactions mediate human myeloma cell growth arrest induced by CD40 stimulation. AB - We have recently demonstrated that the CD40 molecule was expressed on both normal human plasma cells and most malignant plasma cells, i.e., myeloma cells. Thus, we have investigated its putative role in the proliferation of myeloma cells. We report that 7 of 15 myeloma cell lines were CD40+ but only one, XG2, presented a high level of CD40 expression. We show that the CD40 stimulation by anti-CD40 monoclonal antibodies (mAbs) of the interleukin 6-dependent myeloma cell line XG2 induced a total inhibition of its proliferation. This inhibition was also observed when cells were either cultured in the "CD40 system," where the anti CD40 mAb has been immobilized on fibroblasts expressing Fc receptors or in the presence of a soluble chimeric CD40 ligand molecule. This inhibition of proliferation was neither accompanied by differentiation nor apoptosis. Triggering CD40 induced an homotypic aggregation of XG2 cells, and the inhibition of proliferation was totally prevented by a blocking anti-CD18 mAb. Although the CD11a-CD18 ligands, i.e., CD50, CD54, and CD102, were all expressed on XG2 cells, only a blocking anti-CD102 mAb inhibited the CD40-induced growth arrest. Our data demonstrate that CD40 triggering on XG2 cells induced a myeloma cell growth arrest mediated by lymphocyte function-associated antigen 1 and intercellular adhesion molecule 2 interactions. PMID- 8620514 TI - Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability. AB - We have tested the hypothesis that endometrial precancers persist in uteri of patients with endometrial carcinoma and are monoclonal. Twenty-two hysterectomies with both well-differentiated endometrial adenocarcinoma and adjacent (normal or abnormal) noncancerous endometrium underwent successful clonal analysis using a PCR assay for nonrandom X chromosome inactivation. Monoclonal lesions included endometrial carcinoma, endometrial polyps, and atypical endometrial hyperplasias, whereas normal and anovulatory endometrium were polyclonal. Comparison of the specific X chromosome copy preferentially inactivated by the matched monoclonal cancers and associated monoclonal lesions allowed us to exclude polyps, but not endometrial hyperplasias, as potential precancers. The repetitive genetic marker (HUMARA) for X inactivation was altered in some cancers, permitting identification of microsatellite instability (RER+). Two patients with RER+ cancers also had adjacent RER+ hyperplasias. The seven monoclonal and two RER+ hyperplasias had focal or diffuse cytological atypia, a feature previously associated with risk for endometrial cancer. We conclude that: (a) putative endometrial precancers and cancers share a monoclonal growth pattern; (b) cancers with microsatellite instability may acquire this feature as precancers; and (c) monoclonal endometrial precancers have the morphology of hyperplasias, which vary in the extent of cytological atypia and degree of architectural complexity. PMID- 8620515 TI - Somatostatin and vasoactive intestinal peptide receptors in human mesenchymal tumors: in vitro identification. AB - Somatostatin and vasoactive intestinal peptide (VIP) have been shown to be of diagnostic and therapeutic interest in several types of human epithelial tumors expressing the respective receptor. The present study evaluates the presence of somatostatin and VIP receptors in 64 primary or metastatic human mesenchymal tumors. In vitro receptor autoradiography on cryostat sections was performed using 125I-labeled [Tyr3]-octreotide as well as 125I-labeled [Leu8,D-Trp22,Try25] somatostatin-28 as radioligands for somatostatin receptors and 125I-labeled VIP as radioligand for VIP receptors. Somatostatin receptors were identified in bone and vascular/perivascular tumors (3 of 3 osteosarcomas, 1 of 1 giant cell tumor, 2 of 2 angiosarcomas, and 4 of 4 hemangiopericytomas), in 2 of 2 synovial sarcomas, in 2 of 5 histiocytomas, and in several muscle cell tumors (1 of 2 leiomyomas, 2 of 4 leiomyosarcomas, and 3 of 5 rhabdomyosarcomas) but were absent in 4 liposarcomas, 3 mesotheliomas, 3 chondrosarcomas, 10 Ewing sarcomas, 11 schwannomas, and 5 Wilms' tumors. VIP receptors were identified in 3 of 3 differentiated liposarcomas, 2 of 2 angiosarcomas, 4 of 4 hemangiopericytomas, 2 of 2 synovial sarcomas, 3 of 3 mesotheliomas, 5 of 5 Wilms tumors, as well as in 2 of 5 histiocytomas, 1 of 2 leiomyomas, 2 of 4 leiomyosarcomas, 3 of 3 intermediately differentiated rhabdomyosarcomas, and 1 of 3 osteosarcomas but not in chondrosarcomas, Ewing sarcomas, schwannomas, or undifferentiated rhabdomyosarcomas. The receptors were located on neoplastic cells. The somatostatin receptors were of high affinity and of high specificity for biologically active somatostatin analogues with high affinity for somatostatin-14 and somatostatin-28 as well as for octreotide, thus representing the sst2 subtype; in a few cases of tumors having somatostatin receptors with low affinity for octreotide, in situ hybridization techniques identified preferentially sst1 mRNA. These data suggest that human mesenchymal tumors may be targets for somatostatin and/or VIP receptor in vivo imaging; they may also be potential targets for somatostatin or VIP analogue therapy. PMID- 8620516 TI - Disialosyl galactosylgloboside as an adhesion molecule expressed on renal cell carcinoma and its relationship to metastatic potential. AB - Aberrant glycosylation expressed in specific types of human cancer may define stage, direction, and fate of tumor progression. Well-studied examples are expression of sialosyl-Lewis(x) or sialosyl-Lewis(a) in colorectal carcinoma and histo-blood group A and H/Le(y) in lung cancer. In renal cell carcinoma (RCC), expression of sialosyl-Lewis(x) has no correlation with metastatic potential. Clinicopathological studies have revealed that the degree of expression of disialosyl galactosylgloboside (DSGG) and monosialosyl galactosylgloboside is correlated with metastatic potential (to lung and lymph nodes) of RCC and inversely correlated with patient survival. In the present study, we compared the adhesion of RCC lines to sections of various tissues measured by Stamper-Woodruff assay and other similar assays under dynamic flow conditions. Of the eight RCC lines tested, only TOS-1 (which expresses DSGG) bound strongly to lung tissue sections. TOS-1 did not bind to sections of liver, kidney, or lymph nodes. In the same eight RCC lines, we also compared expression of DSGG and monosialosyl galactosylgloboside (reflected by reactivity with RM1 and RM2), overall ganglioside patterns, and correlation with lung tissue-binding ability. Under both static and dynamic flow conditions, the binding of TOS-1 cells to lung alveolar tissue was correlated with their DSGG expression, i.e., the binding was inhibited by RM2 but not by RM1. This binding was also inhibited by sialidase but not by EDTA (i.e., it was CA 2+ independent). The other seven cell lines (TOS-2, TOS-M, SMKT-R1, -R2, -R3, and -R4, and ACHN), which do not express DSGG, showed much weaker adhesion to lung tissue. None of the eight cell lines showed E- or P selectin-dependent adhesion. These results suggest the existence of a yet uncharacterized sialoadhesive receptor++ that specifically recognizes DSGG. This receptor could be the binding target in RCC metastasis to lung. PMID- 8620517 TI - Migration of brain tumor cells on extracellular matrix proteins in vitro correlates with tumor type and grade and involves alphaV and beta1 integrins. AB - An important contributor to the malignancy of brain tumors is their ability to infiltrate the brain. Extracellular matrix molecules and cell adhesion molecules on cell surfaces play key roles in cell migration. In the present study, we used reaggregates of dissociated cells from freshly excised human brain tumors to analyze the migration of cells from human brain tumors of different types and grades on many different adhesion proteins adsorbed to glass substrates. Proteins were chosen based on their presence in normal or neoplastic nervous tissue, and included the extra-cellular matrix molecules fibronectin, collagens, fibrinogen, laminin, tenascin-C, thrombospondin, and the neuron-glia cell adhesion molecule, Ng-CAM. Cells from astrocytomas (n = 24) migrated on a variety of substrates, in contrast to cells from primitive neuroectodermal tumors cells (n=6), which only migrated well on laminin, fibronectin, or type IV collagen but not on the other substrates. Typically, migrating cells from astrocytomas of all grades had long, slender processes, were usually bipolar, and their cell bodies did not spread well on any substrate. Although there was variability in the migration of cells from astrocytomas of the same grade, cells from high-grade astrocytomas tended to migrate more extensively (42.3 +/- 4.7 micrometers/16 h: n = 16) than cells from lower grade astrocytomas (28.9 +/- 3.9 micrometers/16 h; P = 0.07; n = 8); the most striking differences were observed for collagen substrates, on which cells from lower grade astrocytomas migrated at very low levels (7.6 +/- 2 .6 micrometers/16 h) and cells from high-grade astrocytomas at higher levels (24.4 +/- 5.2 micrometers;P = 0.01). In contrast to primary cells from glioblastomas (n = 13), glioblastoma cell lines (n = 10) consistently spread on various substrates and migrated at high levels (69.5 +/- 7.6 versus 46.4 +/-5.7 micrometers/16 h; P = 0.03), in particular, on collagens (108.4 +/- 20.2 versus 28.0 +/- 6.1 micrometers/16 h; P= 0.001). Specific monoclonal antibodies to alphaV and beta1 integrin monomers completely inhibited the migration of astrocytoma cells on most substrates, suggesting that alphaV and beta1 integrins play a crucial role in brain tumor infiltration. These studies also suggest that although a large number of extracellular matrix molecules may promote tumor cell migration, disrupting the function of only a few tumor cell receptors may be critical for tumor infiltration in the brain. PMID- 8620518 TI - Bone sialoprotein peptides are potent inhibitors of breast cancer cell adhesion to bone. AB - Bone and bone marrow are important sites of metastasis formation in breast cancer. Extracellular matrix proteins with attachment properties are generally believed to play a key role in tumorigenesis and metastasis formation. We have investigated whether mammary carcinoma cells (MDA-MB-231) can recognize constructs of the fairly bone-specific human bone sialoprotein, which encompass the RGD sequence (EPRGD-NYR). Exogenously added bone sialoprotein peptides with this amino acid sequence in their backbone structure, but not the more common fibronectin-derived GRGDS peptide, strongly inhibited breast cancer cell adhesion to extracellular bone matrix at micromolar concentrations. Most cyclic derivatives with the EPRGDNYR sequence were more effective inhibitors of tumor cell adhesion to bone than their linear equivalents. Furthermore, changes in the RGD-tripeptide of the backbone structure of the constructs, removal of the NYR flanking sequence, or a different tertiary cyclic structure significantly decreased their inhibitory potencies. In addition, the RGE-analogue EPRGENYR was capable of inhibiting breast cancer cell adhesion to bone, albeit to a lesser extent. We conclude therefore, that the inhibitory potency of the bone sialoprotein-derived peptides on breast cancer cell adhesion to bone is not solely due to a properly positioned RGD-motif alone but is also determined by its flanking regions, together with the tertiary structure of the EPRGDNYR peptide. Synthetic cyclic constructs with the EPRGDNYR sequence may, therefore, be potentially useful as antiadhesive agents for cancer cells to bone in vivo. PMID- 8620519 TI - In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425. AB - In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of 125I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments. PMID- 8620520 TI - Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells. AB - The development of cellular resistance to immunotoxins has been demonstrated in a variety of models and can involve a number of mechanisms. For the present study, an immunotoxin was utilized composed of an anti-melanoma antibody ZME-018 recognizing a 240-kDa surface glycoprotein (gp 240) and the plant toxin gelonin. Human melanoma cells (A375-M) were grown in the presence of increasing amounts of ZME-gelonin and a clonal variant (A-375-ZR) was developed that was 100-fold resistant to ZME-gelonin compared to parental cells. Scatchard analysis showed that the A375-M parental cells had 260 X 10(3) ZME-gelonin-binding sites/cell with relatively low affinity (5 nM). In contrast, resistant A375-ZR cells demonstrated a reduced number of low-affinity sites (160 x 10(3)/cel1), but showed a small number (47 x 10(3)) of higher-affinity sites (0.8 nM). Internalization rates and degradation rates of 125I-labeled ZME-gelonin were identical in both the parental and resistant cells. A375-ZR cells were found to be more resistant to vincristine and doxorubicin than were parental cells. Both cell lines were almost equally sensitive to native gelonin, 5-fluorouracil (5 FU), cisplatin. melphalan, carmustine, interferon gamma (IFNgamma) and IFNalpha. In addition. both cell lines were equally sensitive to another gelonin antibody conjugate that binds to cell-surface, GD2 (antibody 14G2A). However, resistant cells were twice as sensitive to the cytotoxic effects of etoposide than were parental cells. Finally, a variety of agents were tested in combination with ZME gelonin against A375-ZR cells in an attempt to identify agents to augment immunotoxin cytotoxic effects against resistant cells. The agents 5-FU, cisplatin, IFNgamma, IFNalpha, and etoposide were the most effective in augmenting the cytotoxicity of ZME-gelonin against resistant cells. These studies suggest that development of resistance to one immunotoxin does not cause development of cross-resistance to other gelonin immunotoxins. Further, specific biological response modifiers and chemotherapeutic agents may be effective in augmenting the effectiveness of immunotoxins and specifically targeting or reducing the emergence of immunotoxin-resistant cells. PMID- 8620521 TI - Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2. AB - Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300,000 IU hnIL-2/day (8.2 +/- 10.2 compared to 1.1 +/- 1; P<0.001). The inhalation of 150,000 IU also induced increases in the index (AI = 2.3 +/- 1.9), however, without reaching statistical significance. In addition at 300000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4 +/- 2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r(s) = -0.5; r(s) = -0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. PMID- 8620522 TI - Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma. AB - The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50-60 microg), low dose intratumoral administration of OK-432 (3-12 KE) was performed. Thereafter, 2 x 10(5) JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum alpha-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity. PMID- 8620523 TI - The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine. AB - The identification and cloning of several tumour antigens together with an improvement in the understanding of the mechanisms involved in antigen presentation and immune recognition has opened up the possibility of using active specific immunotherapy as a treatment for certain cancers. This review discusses the tumour-associated MUC1 gene product of the polymorphic epithelial mucin (PEM), as a potential target molecule for cancer treatment. PEM is both over expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule. Furthermore, immune responses specific for PEM have been detected in cancer patients. Both syngeneic and transgenic murine model systems have been developed in order to compare the efficacy and toxicity of various PEM-based immunogens in tumour rejection studies, and to further improve the understanding of antigen presentation and the mechanisms underlying tumour rejection. Such models also allow the examination of MUC1-based immunogens as a treatment for existing tumours. Clinical trials in progress using immunogens based on the MUC1 gene product are briefly discussed. PMID- 8620524 TI - Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A. AB - A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions x human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733 2. Eighty-two of the 83 patients treated with mmAb 17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab2). Auto antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti anti-idiotypic antibodies (ab3), were induced by the mAb17-1A therapy. Patients with detectable ab3 after treatment had significantly higher ab2 levels than those not developing ab3. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab2 as well as induction of anti-anti-idiotypic antibodies (ab3) compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab3) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab3 (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. PMID- 8620525 TI - Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2. AB - A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the C gamma1 gene of chl4.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumour load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. PMID- 8620526 TI - Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion. AB - The immunological properties of tumour-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3 positive cells were specifically separated from these cell fractions with anti CD3 magnetic beads. These pure CD3-positive PBL (CD3+PBL) and TIL (CD3+TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1alpha (IL-1 alpha), IL-1 beta, IL-2, interferon gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3+TIL and CD3+PBL were compared in each patient similar values for IL-1 alpha, IL-1 beta, IFNgamma and TNFalpha were found. However CD3+TIL produced significantly lower levels of IL-2 than CD3+PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3+TIL as compared to the CD3+PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3+TIL and CD3+PBL derived from patients with renal cell carcinomas. PMID- 8620527 TI - Interferon gamma (IFNgamma) gene transfer of an EMT6 tumor that is poorly responsive to IFNgamma stimulation: increase in tumor immunogenicity is accompanied by induction of a mouse class II transactivator and class II MHC. AB - Abstract Interferon gamma (IFNgamma) is an important cytokine with immunomodulatory properties that include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. In this study a retroviral vector was used to introduce the IFNgamma gene into EMT6 tumor cells to assess the effect of IFNgamma gene expression on tumor immunogenicity. Transfectants were selected in G418-containing tissue-culture medium and were determined to express the inserted IFNgamma gene by reverse transcriptase/polymerase chain reaction. Flow-cytometric analysis revealed that parental unmodified EMT6 cells constitutively expressed only class I MHC and were poorly responsive to exogenous IFNgamma stimulation, whereas class II MHC was induced in IFNgamma-transfected cells. The induction of class II MHC in IFNgamma transfected cells correlated with the expression of a mouse class II transactivator gene that was dormant in unmodified or mock-transfected cells. In addition, IFNgamma-gene-transfected tumor cells were found to secrete up to 17 ng IFN (equivalent to 75 units/10(6) cells) by enzyme-linked immunosorbent assay (ELISA). Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was significantly inhibited in immunocompetent mice. Rechallenge of animals that rejected an IFNgamma-transfected EMT6 clone (EMT6 B17) with parental EMT6 cells resulted in tumor rejection, suggesting that IFNgamma-transfected EMT6 cells were able to induce long-term immunity. Mixing experiments using gene-transfected and unmodified tumor cells demonstrated that 10% of IFNgamma-transfected cells in the population was sufficient to protect mice against subsequent challenge with tumorigenic EMT6 cells. These studies demonstrate that the immunogenicity of tumor cells that are poorly responsive to exogenous IFNgamma can be enhanced by inserting and expressing the IFNgamma transgene. These findings also suggest a role for class II MHC in reducing tumorigenicity of the EMT6 tumor and inducing long-term tumor immunity. PMID- 8620528 TI - Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. PMID- 8620529 TI - The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. AB - V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins was previously shown to demand only a single signal sequence. Here, we established conditions where 12- and 23-spacer signal sequences are both necessary for cleavage. Coupled cutting at both sites requires only the RAG1 and RAG2 proteins, but depends on the metal ion. In Mn2+, a single signal sequence supports efficient double strand cleavage, but cutting in Mg2+ requires two signal sequences and is best with the canonical 12/23 pair. Thus, the RAG proteins determine both aspects of the specificity of V(D)J recombination, the recognition of a single signal sequence and the correct 12/23 coupling in a pair of signals. PMID- 8620531 TI - The crystal structure of the DNA-binding domain of yeast RAP1 in complex with telomeric DNA. AB - Telomeres, the nucleoprotein complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability. In the yeast S. cerevisiae, telomeric DNA is bound in a sequence-specific manner by RAP1, a multifunctional protein also involved in transcriptional regulation. Here we report the crystal structure of the DNA-binding domain of RAP1 in complex with telomeric DNA site at 2.25 A resolution. The protein contains two similar domains that bind DNA in a tandem orientation, recognizing a tandemly repeated DNA sequence. The domains are structurally related to the homeodomain and the proto-oncogene Myb, but show novel features in their DNA-binding mode. A structured linker between the domains and a long C-terminal tail contribute to the binding specificity. This structure provides insight into the recognition of the conserved telomeric DNA sequences by a protein. PMID- 8620530 TI - RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. AB - A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent AO site in the 5' ETS and cleavage in the 3' ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5' ETS RNA at AO site in vitro, in the absence of snoRNA or other factors. A synthetic 3' ETS substrate is specifically cleaved at a site 21 nt downstream of the 3' end 28S rRNA. These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage. PMID- 8620532 TI - Mammalian SH2-containing protein tyrosine phosphatases. PMID- 8620533 TI - The FHIT gene 3p14.2 is abnormal in lung cancer. AB - To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis. PMID- 8620534 TI - Role of the INK4a locus in tumor suppression and cell mortality. AB - The cell cycle inhibitor p16INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19ARF and the adjacent gene encoding p15I1NK4b, both negative regulators of cell proliferation. We report the phenotype of mice carrying a targeted deletion of the INK4a locus that eliminates both p16INK4a and p19ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate rapidly and have a high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-ras into INK4a-deficient fibroblasts can result in neoplastic transformation. These findings directly demonstrate that the INK4a locus functions to suppress neoplastic growth. PMID- 8620535 TI - The yeast CDC16 and CDC27 genes restrict DNA replication to once per cell cycle. AB - CDC16 and CDC27 were identified as genes in S. cerevisiae necessary to limit DNA replication to once per cell cycle. A screen for mutants that overreplicated their DNA uncovered new conditional alleles that cause accumulation of up to 8C DNA. DNA overreplication involves all chromosomes and does not require passage through mitosis or another START. It occurs within a single cell cycle and can cause arrest at the MEC1 checkpoint. Remarkably, Clb2-Cdc28 activity remains elevated in the overreplicating cells. These observations distinguish CDC16 and CDC27 from other mutants that accumulate extra DNA after completing an aberrent mitosis, or skipping mitosis altogether, and entering a second, inappropriate G1 and S phase. CDC16 and CDC27 may contribute to replication control by targeted proteolysis of an S phase initiator. PMID- 8620536 TI - MafB is an interaction partner and repressor of Ets-1 that inhibits erythroid differentiation. AB - Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner. MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine-zipper domain. Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits Ets-1-mediated transactivation of the transferrin receptor, which is known to be essential for erythroid differentiation. Accordingly, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits differentiation without affecting cell proliferation. These results highlight the importance of inhibitory interactions between transcription factors in regulating lineage-specific gene expression. PMID- 8620537 TI - Regulatory circuits controlling photosynthesis gene expression. PMID- 8620538 TI - Integrin activation by R-ras. AB - Expression of a constitutively active R-ras converted two cell lines that grow in suspension into highly adherent cells. There was little change in cell surface expression of integrins, but attachment to surfaces coated with the integrin ligands was greatly enhanced. Cells transfected with activated R-ras bound integrin ligands from solution with higher affinities and assembled severalfold more fibronectin matrix than control transfectants. Introduction of a dominant negative R-ras into adherent cells reduced the adhesiveness of the cells, indicating that endogenous R-ras can control the ligand-binding activity of integrins. These results provide a mechanism for the modulation of integrin ligand-binding activity as well as novel function for R-ras. PMID- 8620539 TI - Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation. AB - E. coli preprotein translocase comprises SecA and SecY/E/G complex. SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion. SecG renders the translocase highly efficient. An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite the exposure of this region to the inside of membrane vesicles in the absence of preprotein translocation. When preprotein translocation was started with ATP and then blocked by the inhibition of ATP hydrolysis, the C-terminal region was exposed to the outside of membrane vesicles. Another region of SecG showed a change in membrane sidedness upon preprotein translocation, indicating that SecG undergoes topology inversion. This topology inversion was tightly coupled to the SecG function and linked with the insertion-deinsertion cycle of SecA. PMID- 8620540 TI - Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. AB - S. cerevisiae inherits its vacuole by projecting vacuole-derived membrane vesicles and tubules into the bud, where they fuse to establish the daughter vacuole. This homotypic fusion event can be assayed in vitro. It requires Sec17p and Sec18p, the homologs of the mammalian alpha-SNAP and NSF, which cooperate in multiple steps of membrane trafficking. We now report that Sec17p, Sec18p, and ATP are only needed for an early stage of the reaction that results in Sec17p release. Sec17p and Sec18p actions precede, and are needed for, the step employing the Ras-like GTPase Ypt7p. Sec18p-driven release of Sec17p can even precede vacuole docking, as it can occur prior to mixing of vacuoles and is insensitive to vacuole concentration. Sec17p and Sec18p thus may function in a predocking stage of the reaction, rather than in bilayer fusion per se. PMID- 8620541 TI - Molecular glue: kinase anchoring and scaffold proteins. PMID- 8620542 TI - Araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. AB - In Drosophila imaginal wing discs, the achaete-scute (ac-sc) proneural genes and rhomboid (veinlet) are expressed in highly resolved patterns that prefigure the positions of sensory organs and wing veins, respectively. It is thought that these patterns are generated by a combination of factors (a prepattern) regulating these genes. We provide evidence for the existence of this prepattern by identifying two of its factors, Araucan and Caupolican. They are members of a novel family of homeoproteins, with homologs in vertebrates. Araucan and Caupolican, present in domains of the imaginal discs larger than those expressing ac-sc and rhomboid, are necessary for expression of these genes in the overlapping domains. Araucan and Caupolican appear to be positive, direct regulators of ac-sc. PMID- 8620544 TI - Immunoglobulin production induced by CD57+ GC-derived helper T cells in vitro requires addition of exogenous IL-2. AB - Germinal centers (GC) are well-defined areas in lymphoid organs were B cells proliferate and differentiate in response to T-cell-dependent antigens. The GC comprises B cells, follicular dendritic cells, tangible body macrophages, and a low number of CD4+ T cells. A large portion of these T cells expresses CD57. We have examined the ability of the CD4+ CD57+ GC T cells to become activated and to take part in B cell activation processes. These T cells coexpress CD45RO, CD69, CD28, and upon mitogenic stimulation CD25. The cell population was found neither to contain nor to be able to produce any specific mRNA for IL-2, IL-4, and IFN gamma upon activation. Levels of mRNA encoding CD40 ligand was also undetectable under similar conditions. Furthermore, in contrast to ordinary CD4+ T cells, this population expressing CD57 was unable to induce B cells to Ig production in the presence of pokeweed mitogen or SEA unless IL-2 was added to the cultures. However, despite their apparent lack of function CD4+ CD57+ GC T cells were found to rescue GC B cells from cell death in vitro to the same extent as CD4+ CD57+ Th cells. The phenotypical and functional differences found between these Th cells and regular Th-cells suggest that they either represent a T cell subset with distinct properties within the GC yet to be determined or that they represent T cells, late in the immune response, having lost most of their original functions and capabilities. PMID- 8620543 TI - A comparison of proliferative response to IL-7 and expression of IL-7 receptors in intermediate TCR cells of the liver, spleen, and thymus. AB - It is well established that IL-7 supports the earliest differentiation of both T and B cells in fetal and adult life. On the other hand, mature lymphocyte subsets tend to decrease the response to IL-7 in case of T and B cells. In a recent study, NK1.1+ T cells in the thymus are also found to efficiently respond to IL-7 and express IL-7 receptors (IL-7R). This population is generated through an alternative intrathymic pathway. A similar population, namely, T cells with intermediate levels of TCR (i.e., int TCR cells) are known to be generated through extrathymic pathways. In this respect, the proliferative response to IL-7 and the expression of IL-7R in int TCR cells of various organs were compared. Whole liver MNC and isolated int TCR cells from the liver were found to proliferate in response to IL-7. Moreover, a considerable population of int TCR cells in both the liver and thymus were found to carry a higher density of IL-7R on the surfaces than high TCR cells. More precisely, the intensity of IL-7R on int TCR cells in the thymus was the highest but those on int TCR cells in other organs were slightly lower (i.e., int TCR cells in the thymus greater than int TCR cells in the liver greater than high TCR cells). Taken together with the result of expression of IL-7 mRNA by hepatocytes and thymic tissues, it is concluded that IL-7 is one of the most important growth factors for int TCR cells both in the liver and thymus. PMID- 8620545 TI - Positive selection by thymic nurse cells requires IL-1 beta and is associated with an increased Bcl-2 expression. AB - A temperature-sensitive line of thymic nurse cells (tsTNC-1) that maintains the ability to selectively internalize immature alpha beta TCRloCD4+CD8+ thymocytes in vitro was used in long-term coincubation experiments to determine nurse cell function during the process of MHC restriction. The thymocyte subset released from its association with TNCs contained both viable and apoptotic cells. The cells that remained within intracytoplasmic vacuoles died through the process of programmed cell death. Surviving or rescued thymocytes in the released population displayed an increase in Bcl-2 protein expression. The rescue activity of TNCs was drastically reduced with the addition of antibodies against either class I or class II MHC antigens to cocultures. A subset of the TNC-rescued population matured from the alpha beta TCRloCD69- phenotype to alpha beta TCRhiCD(69+) expressing cells only when IL-1 beta was added to cocultures. These results suggest that TNC rescue of early double-positive thymocytes from apoptosis is associated with an interaction between the TCR and the MHC and the onset of Bcl-2 expression. Maturation of thymocytes within the TNC-rescued population requires the costimulatory effects of IL-1 beta. PMID- 8620547 TI - Altered CD45 expression in malignant B-1 cells. AB - CD45 is an important surface glycoprotein which has an intrinsic tyrosine phosphatase activity and has been implicated in cell proliferation, signaling, and differentiation and is associated with the B cell receptor during signaling. In this manuscript, the role of CD45 expression in the development of B-1 malignancies in NZB mice, which serve as a model for human diseases such as chronic lymphocytic leukemia, was investigated. B-1 cells spontaneously hyperproliferate and form a clonal hyperdiploid malignant population in aging NZB mice. Phenotypic analysis indicates that the NZB malignant B-1 cells are bright for IgM, but have reduced levels of CD45 relative to normal, nonmalignant B cells (both B-1 and B-2) and are characterized by dull or negative expression of the CD45 isoform B220/6B2 normally found on all B cells. Malignant B-1 cells demonstrated decreased RNA levels of CD45 relative to IgM expression, while nonmalignant B-2 cells showed similar levels of RNA expression for both CD45 and IgM. As CD45 exists in several isoforms and B cells express the highest molecular weight isoform (B220), malignant B-1 cells were further analyzed with respect to their isoform usage. Although, at the RNA level malignant B-1 cells showed the presence of the of the B220 form of CD45, western blot analysis of B220 protein suggested a posttranslational glycosylation defect in the CD45/B220 expression recognized by the mAb 6B2. F1 recipients of premalignant NZB B-1 cells which had been sorted for IgMhi, B220/6B2negative cells developed hyperdiploid malignant donor B-1 clones earlier than did recipients of NZB B-1 cells which were bright for B220/6B2. However, all the malignant B-1 clones of NZB origin which developed in recipients of both transfer populations were B220/6B2 negative. This indicated that abnormal expression of CD45 may be prerequisite for long-term growth and malignant transformation. Thus alterations in CD45 may result in abnormal functioning of the malignant B-1 cells which may further affect the proliferation of, or signaling within, these cells. PMID- 8620546 TI - Age-related decreases in IL-2 production by human T cells are associated with impaired activation of nuclear transcriptional factors AP-1 and NF-AT. AB - Although transcriptional factors AP-1 and nuclear factor of activated T cells (NF AT) are important for the normal induction of IL-2, it is unknown if the age related decline in IL-2 production by activated human T cells may be associated with aberrancies in transcriptional regulatory proteins. In the current studies, IL-2 production by T cells from elderly (mean 78 years) and young (mean 37 years) humans was measured in cultures stimulated with PHA, PHA plus PMA, crosslinked anti-CD3 mAB OKT3 plus PMA, or PMA plus ionomycin. Substantial decreases of IL-2 production were observed for cell cultures from 7 of 12 elderly individuals in response to the different stimuli, whereas the levels of IL-2 produced by stimulated T cells from other elderly individuals were equivalent to those observed for stimulated T cells of young subjects. Analyses of nuclear extracts by electrophoretic DNA mobility shift assays showed that decreased IL-2 production by stimulated T cells of elderly individuals was closely associated with impairments in the activation of both AP-1 and NF-AT. By contrast, T cells from elderly subjects with normal levels of IL-2 production exhibited normal activation of AP-1 and NF-AT. In addition, the results of competition experiments analyzing the normal components of NF-AT showed that the age-related reductions in stimulus-dependent NF-AT complexes corresponded to the slow migrating complexes that were composed of c-Fos/c-Jun AP-1. The resting and stimulated levels of NF kappa B were reduced in T cells from certain elderly individuals; however, alterations of NF kappa B did not correlate with changes in IL-2 expression. Thus, these results show that age-related impairments in the activation of AP-1 and NF-AT are closely associated with decreased expression of IL-2 and further suggest that aberrancies in the signaling pathways important for the induction of transcriptionally active c-Fos/c-Jun AP-1 may contribute to the impaired activation of NF-AT. PMID- 8620548 TI - Differential activation of cell-mediated immune functions by encapsulated and surface-linked liposomal antigens. AB - Liposomes act as powerful adjuvants if physically associated with a protein antigen. Their effect on the immune response, however, varies with the nature of this linkage, surface-linked and encapsulated antigens having different properties. Cytometric analysis and cytokine measurements indicate that this difference may be due to the differential activation of T lymphocyte populations. Surface-linked antigen appears to preferentially stimulate CD4+ T cells to proliferate and mature into a typical Th1 phenotype; this is indicated by a positive shift in the CD4+/CD8+ ratio of sensitized splenocytes, a massive production of interferon-gamma, and the absence of interleukin-4 secretion. In contrast, encapsulated antigen, while stimulating spleen cell proliferation, does not significantly affect the CD4+/CD8+ ratio and induces only low levels of interferon-gamma production in the absence of interleukin-4 secretion. These results suggest that CD4+ and CD8+ populations are both expanded in response to encapsulated antigen but that neither typical Th1 nor Th2 phenotypes are induced. High-resolution immunocytochemical investigations show that this differential activation of T cell populations may be related to a different intracellular trafficking of antigens into professional antigen-presenting cells. Whereas surface-linked antigen remains predominantly in endosomal compartments where it may be associated with major histocompatibility (MHC) class II products for presentation to CD4+ T cells, encapsulated antigen escapes into the cytosol, reaching the MHC class I pathway for presentation to CD8+ T cells. The results therefore suggest that both liposomal antigens stimulate cell-mediated immunity albeit differently. This behavioral difference may be of practical importance in the design of adjuvants for the preferential potentiation of specific cytotoxic effector functions. PMID- 8620549 TI - Role of interferon-gamma against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): involvement of the parasite's secretory phospholipase A2. AB - We examined the role of IFN gamma in protection against Toxoplasma gondii in the monocytoid cell line THP1. The addition of IFN gamma to cultured infected THP1 cells reduced the number of parasitized cells without altering intracellular multiplication during the first 24 hr. This reduction was potentiated by bacterial lipopolysaccharide (LPS). We also examined the role of an enzyme important for T. gondii cellular invasion, secretory phospholipase-A2 (sPLA2) and its relation with IFN gamma-induced protection. Treatment of cells or parasites with a specific inhibitor of sPLA2 significantly reduced the number of infected cells at 6 hr. The addition of exogenous sPLA2 from Naja naja venom did not interfere with the protective effect of IFN gamma and conferred protection when used alone. PLA2 activity was measured in supernatants of parasites maintained in the presence of IFN gamma, and the results suggested that IFN gamma opposes cell invasion by T. gondii by suppressing parasite production of PLA2. PMID- 8620551 TI - T cell receptor V beta repertoire in the thymus and spleen of mice infected with Trypanosoma cruzi. AB - The T cell receptor (TCR) V beta repertoire was studied in BALB/c, CBA/HJ, and CBA/J mice experimentally infected with Trypanosoma cruzi. The percentage of expression of 14 V beta chains of the variable domain of the TCR in the thymus and spleen was evaluated. In the thymus of acutely infected with BALB/c and CBA/HJ mice there was an increase in the expression of positively selected V beta families. These changes in the V beta chains usage in the thymus paralleled the enrichment of CD4+ and CD8+ single-positive T cells. During the acute infection, several changes were observed in the peripheral expression of V beta families, such as of V beta 6 in BALB/c (a 36% increase in CD8+ T cells of the corresponding levels of V beta), of V beta 8 in CBA/HJ (a 37% decrease in CD8+ cells), and of V beta chains 8 and 14 in CBA/J mice (V beta 14+CD4+ cells increased 19%, and V beta 8 expression decreased 19 and 33% in CD4+ and CD8+ cells, respectively). In chronically infected BALB/c and CBA/HJ mice, no change in the V beta families was observed, neither in the thymus nor in the spleen. In acutely infected mice, the alterations of the peripheral expression of positively selected V beta families could be due to the stimulation by T. cruzi antigens and/or cytokines; the homeostatic mechanism/s that maintains the selection of the TCR V beta repertoire did not seem to be severely affected during the infections. PMID- 8620550 TI - Fusion of a signal sequence to the interleukin-1 beta gene directs the protein from cytoplasmic accumulation to extracellular release. AB - Interleukin (IL)-1 differs from most other cytokines by the lack of a signal sequence, which results in the retention of the immature proform intracellularly (i.c.). Several cell types have the capacity to produce IL-1, but release has been shown to be restricted predominantly to monocytes/macrophages and associated with apoptosis of the producer cell. These features have limited the studies on IL-1 in early T cell-APC interactions. To develop a model for studying the biological effects of IL-1 beta release during long-lasting immune responses, we have established cells transfected with IL-1 beta cDNA constructs. To construct a hybrid gene for IL-1 beta release, the signal sequence from the related IL-1 receptor antagonist was fused to the gene encoding the 17-kDa mature form of IL-1 beta. A murine fibroblast cell line was transduced with retroviral technique and analyzed for the expression of human IL-1 beta, with or without a signal sequence (ssIL-1 beta and IL-1 beta, respectively). The fibroblasts transduced with either IL-1 beta or ssIL-1 beta expressed similar levels of human IL-1 beta mRNA. High levels of IL-1 bioactivity were recorded in freeze-thaw extracts from cells expressing the IL-1 beta protein i.c., and in supernatants of ssIL-1 beta transduced cells, which indicates that the initial formation of a proform of IL-1 beta is not required for correct folding of the protein. Treatment of ssIL-1 beta transduced cells with Brefeldin A (BFA), an inhibitor of protein transport in the endoplasmatic reticulum, induced accumulation of the protein i.c. BFA treatment did not affect IL-1 beta-transduced cells, while lipopolysaccharide-activated human monocytes increased the secretion of IL-1 beta. Cytoplasmic staining of single cells demonstrated that expression of the ssIL-1 beta gene directed the protein to a perinuclear Golgi-like compartment, whereas cells transduced with IL 1 beta cDNA showed a diffuse cytoplasmic distribution pattern. Secretion of IL-1 beta from human monocytes was under certain conditions accompanied by cell death. In contrast, in the fibroblast cell line transduced to secrete IL-1 beta, no accompanying cell death could be detected. Gene targeting of IL-1 to the secretory or cytoplasmic pathway may be useful for elucidating the role of IL-1 in T cell-APC interactions, avoiding cell death of the producer cells. PMID- 8620552 TI - Expression of CD80 enhances immunogenicity of cervical carcinoma cells in vitro. AB - Although cervical carcinoma cells may express the human papillomavirus oncoproteins E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. This failure may be due to a lack of expression of costimulatory molecules, such as CD80 (B7.1). To augment the immunogenicity of cervical carcinoma cells, we transfected human papillomavirus (HPV)-transformed cell lines, CaSki and HeLa, with the CD80 expression vector pBJ. Alloantigens on the tumor cells were used for the stimulation of peripheral blood lymphocytes (PBLs). Cocultivation of PBLs and tumor cells resulted in proliferation of CD4+ and CD8+ T lymphocyte subsets. CD80-expressing tumor cells induced proliferation of allogeneic PBLs two-to sixfold compared to control cell lines. Cocultivation of allogeneic PBLs with CD80-positive tumor cells for 3 weeks gave rise to cytotoxic T cells capable of lysing untransfected parental tumor cell lines. Our results demonstrate an immunostimulatory effect of CD80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine. PMID- 8620553 TI - Recipient polyclonal B cell activation and immunoglobulin production induced by priming with a retroviral superantigen. AB - The superantigen vSAG-7 (or MIs 1a) is a membrane glycoprotein encoded by the endogenous retrovirus mouse mammary tumor virus 7 (MMTV-7) and is highly stimulatory for V beta 6/CD4+ T cells. Priming of adult MMTV-7-negative mice with vSAG-7-expressing cells initially results in the activation of the peripheral V beta 6/CD4+ T cell compartment and is followed by T cell tolerance to the superantigen. During the course of tolerance induction the number of recipient B lymphocytes increases in the lymph nodes, but not the spleen, of vSAG-7-primed recipients. These B cells also express increased levels of class II MHC and present passively acquired superantigen. In this study we asked if these effects on the host B cell compartment are followed by the production of immunoglobulin. Priming of MMTV-7-negative BALB/c or CB.17 mice with vSAG-7-expressing cells from DBA/2 mice induced increases of both IgM and IgG2a in the serum. Use of Igh congenic CB.17 (IgMb) mice as recipients of the vSAG-7-presenting cells from DBA/2 (IgMa) donors indicated that the IgM and IgG produced were entirely of host origin. Priming with vSAG-7 also amplified (four- to fivefold) the antibody producing cell response induced to a suboptimal dose of sheep RBC. Priming with purified B cells from vSAG-7 donors resulted in recipient V beta 6/CD4+ T cell activation and increased numbers of recipient B cells in the lymph nodes, but did not induce immunoglobulin production. In contrast, priming with purified CD8+ T cells resulted in increased quantities of serum IgM but not vSAG-7-reactive T cell activation or increased numbers of recipient B cells in the lymph nodes. These results indicate that the T cell activation and increased B cell number with upregulated class II MHC expression initially observed following vSAG-7 priming of adult MMTV-7-negative recipients are not linked to the induction of the recipient-derived immunoglobulin production. PMID- 8620554 TI - Signaling pathways for antigen receptor-mediated induction of transcription factor CREB in B lymphocytes. AB - We previously reported that cross-linking surface immunoglobulin (sIg) leads to induction of the transcription factor CREB in B lymphocytes through phosphorylation at Ser133, despite the lack of an increase in cAMP. Further, cAMP raising agents fail to induce CREB Ser133 phosphorylation and CRE-dependent gene expression in these cells, which differs sharply from the situation in PC12 rat pheochromocytoma cells where CREB responds to elevation of cAMP through the activity of protein kinase A. In this study, we characterized the signal transduction pathways leading from sIg engagement to CREB activation. By using specific inhibitors for protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and protein kinase A (PKA), we found that anti-Ig induced CREB Ser133 phosphorylation depends on PKC, but does not require activation of PKA or CaM kinase II. The differential responsiveness of CREB to forskolin in PC12 cells and BAL-17 B cells may relate to the more marked elevation of cAMP in the former as opposed to the latter; however, high concentrations of dbcAMP which should readily enter B cells and artificially increase cAMP levels still failed to induce CREB Ser133 phosphorylation, even in conjunction with a phosphodiesterase inhibitor. Taken together, the cAMP/PKA pathway does not appear to be as active a contributor to CREB phosphorylation in B lymphocytes as in PC12 cells, and does not appear to be involved in sIg induced, PKC-dependent, CREB activation. PMID- 8620555 TI - Differential effects of opioids on the proliferation of a macrophage cell line, Bac 1.2F5. AB - Chronic treatment of mice with morphine selectively abrogates the terminal differentiation of committed bone marrow progenitor cells to form macrophage colony forming units. To understand the molecular mechanisms involved in morphine mediated suppression of myeloid cell differentiation, we investigated the use of a macrophage cell line, Bac 1.2 F5. In vitro proliferation of this cell line is dependent on the exogenous supply of macrophage colony stimulating factor. Treatment of Bac 1.2F5 cells in vitro with morphine showed a dose-dependent inhibition of proliferation which was associated with morphological changes. Characterization of the binding site revealed that the binding site for morphine on these cells is different from the classical opioid receptors described in the brain. In addition to the putative novel class of morphine receptors, Bac 1.2F5 cells also expressed the delta opioid receptors as determined by RT-PCR analyses. These studies show that Bac 1.2F5 cells are suitable for the molecular characterization of opioid effects on the proliferation and differentiation of myeloid progenitor cells. PMID- 8620557 TI - Involvement of two distinct killing mechanisms in bystander target cell lysis induced by a cytotoxic T lymphocyte clone. AB - Cytotoxic T lymphocyte (CTL) can lyse target cells by mainly two different mechanisms including granule exocytosis- and Fas-based cytotoxicity. In addition, CTL also lyse bystander target cells. In this study, we examined whether these two different killing mechanisms also cause bystander target cell lysis. EL-4 cells, representing bystander target cells, were incubated with CD8+ CTL clone B7B7 and antigen-treated P815 cells. EGTA-MgCl2 inhibited bystander lysis and reduced the release of BLT esterase. In addition, lysis of Fas-negative bystander cells (thymocytes from C57BL/6J-lpr/lpr not expressing Fas antigen) was also detected. CTL preactivated with antigen-treated P815 cells maintained a killing activity on bystander cells in the presence of cycloheximide (CHX). Furthermore, the presence of anti-Fas mAb inhibited the lysis of EL-4 cells by CTL stimulated with anti-TCR mAb. Thus, our results suggest that granule exocytosis- and Fas mediated signal pathways cause the lysis of bystander target cells. PMID- 8620556 TI - Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection. AB - Respiratory virus infections are a serious health challenge. While models exist to study immune mechanisms of the respiratory tract, they have not allowed analysis of the interaction of the lower respiratory tract with other components of the mucosal immune system. This study demonstrates that reovirus 1/Lang, an effective gut mucosal immunogen, also provides a useful model of respiratory mucosal infection. Intra-nasal infection of Balb/c mice resulted in severe viral bronchopneumonia. Major components of the cellular inflammatory response in the lung interstitium and alveolar spaces were CD8 lymphocytes. Lung lymphocyte populations exhibited lysis of reovirus-infected, but not uninfected target cells after in vitro culture. The GCT antigen, a germinal center B-cell and CD8 T-cell marker, was present on 21-60% of the inflammatory lymphocytes. A novel population of GCT-expressing CD4+ lymphocytes unique to reovirus-stimulated lung alveolar and interstitial lymphocyte populations was identified. PMID- 8620559 TI - Differential basal protein tyrosine phosphorylation in natural killer (NK) and T cells: a biochemical correlate of lymphoid functional activity. AB - Despite the similarities between natural killer (NK) and T cells, these lymphocytes have dramatically different functional phenotypes. To identify potential biochemical parameters that correlate with the "primed" NK phenotype, we have investigated protein tyrosine phosphorylation in NK and T cells. Examination of tyrosyl phosphorylation in NK cells showed that they have higher levels of phosphorylation than resting T cells. Consistent with this, the concentrations of the tyrosine kinase inhibitor, herbimycin A, required to inhibit FcR-mediated Ca2+ flux in NK cells were much higher than those required for inhibition of T cell receptor-mediated Ca2+ mobilization. Differences in phosphorylation were not due to purification artifact lymphocyte src-family kinase, p56lck or the protein tyrosine phosphatase CD45. Thus, we have identified high basal tyrosyl phosphorylation as a striking biochemical feature of NK cells that correlates with the unique functions of this subset. PMID- 8620558 TI - Selective induction of CD11a,b,c/CD18 and CD54 expression at the cell surface of human leukocytes by muramyl peptides. AB - Muramyl dipeptide (MDP), murametide, and murabutide which belong to the family of the immunoadjuvant muramyl dipeptides were applied directly to fresh human whole blood and the expression of some surface markers involved in cell adherence in distinct leukocyte populations was investigated. CD11a,b,c/CD18, CD54, CD49d were selected for their involvement in cell adherence, and transferrin receptor (CD71) and low-affinity IgE receptor (CD23) were selected as markers for activated cells. Whereas CD11a was increased only on monocytes, CD11b, CD11c, and CD18 were strongly enhanced on monocytes and polymorphonuclear cells (PMNs) after treatment with MDPs. This increase in membrane expression of integrins, such as CD11b, was not associated with mRNA synthesis, suggesting a mobilization of the CD11b,c/CD18 intracellular pools present in these cells. In contrast, treatment with MDP, murametide, or murabutide enhanced ICAM-1 (CD54) expression on monocyte and PMN cell surface in association with ICAM-1 mRNA synthesis. No variation of CD49d expression was detected on leukocyte surface after incubation with MDPs. Transferrin receptor (CD71) expression and low-affinity receptor for IgE (CD23) expression were increased on monocyte only after incubation with LPS used as positive control. Moreover, no observable change in the selected markers was detected on lymphocyte after MDPs or LPS treatment. These results indicate that MDPs seem to act preferentially on monocytes and PMNs in increasing the level of molecules involved in cellular adhesion process, either in provoking the expression of preformed molecules or in inducing their synthesis. This contributes to understanding the mechanism of the activities of muramyl peptides on specific and nonspecific immunity. PMID- 8620560 TI - Characterization of cadherins expressed by murine thymocytes. AB - Thymocytes develop in close apposition to the stromal cells of the thymus. The ontogeny of thymocytes is dependent on intimate interactions between these cells and the stromal cells. The molecular mechanisms involved in regulating thymocyte stromal cell interactions remain to be clearly defined. In this study, we utilized a polymerase chain reaction strategy to identify members of the cadherin family of cell adhesion molecules that are expressed by CD4+ CD8+ thymocytes, the major cell type in the thymus. One classical cadherin (E-cadherin), three atypical cadherins (OB-cadherin) K-cadherin, and cadherin-8), and two novel cadherins (T1-cadherin and T2-cadherin) were found to be expressed by the CD4+ CD8+ thymocytes. The discovery that these cells display multiple cadherins opens a new area of investigation concerning the adhesive mechanisms involved in modulating thymocyte-stromal cell interactions. We speculate that cadherins will prove to play an essential role in the ontogeny of thymocytes. PMID- 8620561 TI - Methylene blue plus light-induced lipid peroxidation in rat liver microsomes: inhibition by nicotinamide (vitamin B3) and other antioxidants. AB - Methylene blue plus visible light, in the presence of oxygen, induced lipid peroxidation in rat liver microsomes, as assessed by the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides and the loss of membrane-bound enzymes. Peroxidation was enhanced by deuteration of the buffer and inhibited by scavengers of singlet oxygen (1O2) and superoxide (O2.-). The damage induced seemed to be mainly due to Type II involving 1O2 and to a lesser extent Type I reactions with O2.- and hydroxyl radical (.OH) as intermediates. Nicotinamide or vitamin B3, an endogenous metabolite occurring at high concentrations in tissues, had a relatively high rate constant of 1.8 x 108 M-1 s-1 with 102 and had a significant inhibitory effect on lipid peroxidation induced by photosensitization. This effect was both time- and concentration dependent, high inhibition being associated with millimolar concentrations. Chemically related endogenous compounds like tryptophan and isonicotinic acid also had significant inhibitory properties. Similar protective effects were observed with natural antioxidants such as beta-carotene, canthaxanthin, lipoic acid, glutathione, alpha-tocopherol and to a lesser extent ascorbic acid. Nicotinamide was a more effective antioxidant than ascorbic acid. It also showed a similar inhibitory effect against NADPH-ADP-FE3(+)-induced lipid peroxidation. Our results suggest that nicotinamide had significant ability to protect against photosensitization-induced cytotoxicity and cell damage and that it may do so by its ability to react with 102 and other reactive oxygen species. PMID- 8620562 TI - Reaction of epichlorohydrin with 2'-deoxynucleosides: characterization of adducts. AB - Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance and thus has the potential for human exposure in the workplace. It has been shown to be genotoxic in several systems and is a compound capable of reacting with biological nucleophiles. This study details the products formed from the reaction of ECH with 2'-deoxynucleosides at pH 7 and 37 degrees C for 6 h. Reaction with 2'-deoxyguanosine yielded 7-(3-chloro-2-hydroxypropyl) guanine (7-CHP-Gua) resulting from alkylation at N-7 of 2'-deoxyguanosine followed by depurination. Two unusual adducts were also partially characterized which resulted from further reaction of 7-CHP-Gua with another molecule of ECH to yield 1,7-bis(3-chloro-2 hydroxypropyl)guanine (1,7-bis-CHP-Gua) which could then cyclize with the exocyclic amino group to yield 1,N2-(2-hydroxypropano)-7-(3-chloro-2 hydroxypropyl) guanine (1,N2-HP-7-CHP-Gua). Reaction with 2'-deoxyadenosine gave only one product, namely 1,N6-(2-hydroxypropano)-2'-deoxyadenosine (1,N6-HP dAdo). The reaction of 2'-deoxythymidine with ECH also yielded one product which was identified as 3-(3-chloro-2-hydroxypropyl)-2'-deoxythymidine (3-CHP-dThd). A 3-(3-chloro-2-hydroxypropyl)-2'-deoxyuridine (3-CHP-dUrd) product was isolated from the reaction of ECH with 2'-deoxycytidine. This product most likely resulted from the deamination of an initially formed 3-(3-chloro-2-hydroxypropyl) -2' deoxycytidine (3-CHP-dCyd), a phenomenon which we have previously reported to occur during the reaction of 2'-deoxycytidine with other aliphatic epoxides. Evidence is also presented that 3-CHP-dUrd is converted to 3-(2,3 dihydroxypropyl)-2'deoxyuridine (3-DHP-dUrd) under physiological conditions, with a half-life of 213 h. Reaction of ECH with calf thymus DNA (pH 7.0, 37 degrees C, 3 h) resulted in the formation of 7-CHP-Gua (200 nmol/mg DNA. PMID- 8620563 TI - Interaction between the substrate and the high-valent-iron-oxo porphyrin cofactor as a possible factor influencing the regioselectivity of cytochrome P450 catalysed aromatic ring hydroxylation of 3-fluoro(methyl)anilines. AB - In the present study the in vitro and in vivo aromatic ring hydroxylation of a series of amino and/or methyl containing fluorobenzenes, i.e. 3 fluoro(methyl)anilines, was investigated and compared to the calculated density distribution of the reactive frontier pi-electrons of the aromatic substrate. This was done (1) to study to what extent the regioselectivity of the aromatic ring hydroxylation of the 3-fluoro(methyl)anilines could be predicted on the basis of the calculated chemical reactivity, as was previously observed for a series of fluorinated benzenes and monofluoroanilines, and (2) to investigate which factors contribute to possible deviations from the predictions on the basis of the calculated chemical reactivity. Results obtained show that the in vitro and in vivo aromatic ring hydroxylation of the series of 3-fluoro(methyl)anilines correlates qualitatively with the calculated frontier orbital density distribution for electrophilic attack by the cytochrome P450(FeO)3+ species. These results indicate that the HOMO/HOMO-1 frontier orbital densities, i.e. the chemical reactivity of the carbon centres for an electrophilic attack, predict the preferential as well as the non-reactive sites for cytochrome P450 catalysed aromatic ring hydroxylation of the tested model compounds. The absolute values, however, deviated in a systematic way; C4 para hydroxylation being observed to a higher extent than expected on the basis of chemical reactivity and C2/C6 ortho hydroxylation being observed to a lower extent than expected. Additional experiments were performed using different microsomal preparations and microperoxidase-8. The latter is a mini-heme protein of eight amino acids without a substrate binding site. In incubations of the model compounds with different types of microsomal preparations, as well as with MP-8 and purified reconstructed cytochrome P4502B1, similar systematic deviations between the predicted and observed regioselectivity of aromatic hydroxylation were observed. These results show that the regioselectivity of aromatic ring hydroxylation of the 3 fluoro(methyl)anilines cannot be predominantly ascribed to an interaction between the substrate and the substrate binding site of the cytochromes P450 dictating a specific stereoselective positioning of the substrate in the active site. More likely, the systematic deviations between the observed and predicted regioselectivity of hydroxylation of the tested model substrates should be ascribed to an (orienting) interaction between the substrate and the activated cytochrome P450(FeO)3+ cofactor. PMID- 8620564 TI - Mono- and dimethylation of arsenic in rat liver cytosol in vitro. AB - Production of methylarsonate and dimethylarsinate from radiolabelled [73 As]arsenite and [73 As]arsenate was examined in an assay system that contained cytosol prepared from a 20% homogenate (w/v) of livers from 8- 10-week-old male Fischer 344 rats. After a 60-min incubation at 37 degrees C with added S adenosylmethionine and glutathione, up to 50% of carrier-free [73As]arsenite and about 15% of carrier-free [73As]arsenate were methylated. Incubation of cytosol at 100% degrees C for 1 min before addition to the assay system completely abolished methylation of arsenite. Production of methylarsonate increased in proportion to the arsenite concentration in the assay system; however, 50 microM arsenite inhibited production of dimethylarsinate. Methylarsonate production from carrier-free [73-As]arsenite was not dependent on addition of exogenous S adenosylmethionine to the assay system. Addition of 0.1 mM S-adenosylmethionine maximized dimethylarsinate production. Addition of 0.1 or 1.0 mM S adenosylhomocysteine decreased methylation of arsenite, especially dimethylarsinate production. Omission of glutathione from the assay system nearly abolished the methylation of arsenite. Addition of exogenous glutathione to the assay system (up to 20 mM) decreased protein binding of arsenic and increased the production of methylarsonate and dimethylarsinate. The effects of sodium selenite, mercuric chloride, EDTA, p-anisic acid and 2,3-dichloro-alpha methylbenzylamine on the methylation of arsenite were determined. Addition of 10 microM selenite to the assay system nearly abolished the formation of either methylated species. Addition of 1 or 10 microM mercuric chloride inhibited dimethylarsinate production in a concentration-dependent manner but had little effect on methylarsonate yield. Addition of 10 mM EDTA to the assay system inhibited formation of both methylated metabolites, suggesting that an endogenous divalent cation might be involved in enzymatic methylation of arsenic. Neither p anisic acid, an inhibitor of cytosolic methyltransferases, nor 2,3-dichloro-alpha methylbenzylamine, an inhibitor of microsomal methyltransferases, inhibited the conversion of inorganic arsenic to mono- or dimethylated metabolites. PMID- 8620565 TI - Decreased glutathione peroxidase activity in mice in response to nafenopin is caused by changes in selenium metabolism. AB - The activity of selenium-dependent glutathione peroxidase is known to be reduced in the liver of both rats and mice after exposure to nafenopin, as well as other peroxisome proliferators. The mechanism for this down-regulation is not known, but might involve changes in incorporation of selenium into selenoproteins. In this paper we show that both incorporation of selenium into selenoproteins and the level of selenium in liver is reduced in mice treated with nafenopin. The activity of selenium dependent glutathione peroxidase (GPx), as well as incorporation of selenium into its 23 kD subunit were found to be decreased. Contrary to what might have been expected, the decreased GPx activity was detected concomitantly with a slight increase in mRNA levels after 10 days of treatment, while a small decrease in mRNA levels was detected in treated animals after 26 weeks, together with the decrease in GPx-activity. Incorporation of selenium into liver fatty acid binding protein (L-FABP) was also decreased, even though large increases in protein and mRNA levels were detected. Taken together these data suggest that the decrease in GPx-activity in response to nafenopin is due to post-transcriptional mechanisms, involving changes in selenium metabolism. PMID- 8620566 TI - Liver endogenous antioxidant defenses in mice fed AIN-76A diet and infected with murine AIDS. AB - The effects of murine AIDS infection on endogenous antioxidant defenses in mice fed the AIN-76A liquid diet were investigated. C57BL/6 female mice were divided into 2 groups: one group was injected interperitoneally with LP-BM5 murine retrovirus (MAIDS) stock, and the other group served as the non-infected control. Two weeks after the infection, the mice were killed and livers were excised for biochemical analysis of the antioxidant defenses. Liver reduced glutathione (GSH) levels and activities of both cytosolic superoxide dismutase (SOD) and mitochondrial SOD were significantly depressed by MAIDS infection. Activities of glutathione reductase (GR) selenium (Se)-dependent glutathione peroxidase (GPx), catalase and glutathione-S-transferase (GST) toward 1-chloro-2,4-dinitrobenzene (CDNB) were not affected by MAIDS infection. A previous study by this laboratory using the Lieber-DeCarli (L-D) all purpose liquid diet caused a decline in total SOD activity and GPx activity, but not GSH levels. The results suggest that MAIDS infection depresses liver antioxidant defenses; however, MAIDS infection of mice fed the AID-76A liquid diet depresses different liver antioxidant defense parameters when compared to those of the mice fed the L-D all purpose liquid diet. PMID- 8620567 TI - Alpha-naphthylisothiocyanate-induced elevation of serum bile acids: lack of causative effect on bile acid transport. AB - In recent years chemicals including chlorinated solvents have been found to interfere with the transport of bile acids (BA) by hepatocytes, which probably accounts for the raised serum bile acids (SBA) after exposure. However, the known cholestatic agent, alpha-naphthylisothiocyanate (ANIT) has never been fully examined for its effect on these processes. Accordingly, the direct effects in vitro and the effects of in vivo treatment on bile acid transport have been investigated in this study. Direct addition of ANIT (5-100 microns) to hepatocytes isolated from untreated rats did not result in any change in uptake or efflux of taurocholic acid (TC), one of the most obviously elevated SBA in ANIT-treated rats. Additionally, accumulation of TC over an extended incubation period was not affected by ANIT. In vivo treatment with ANIT (50 mumol/kg i.p. on each of 3 consecutive days) resulted in a marked elevation of total serum bile acids (TSBA) and a slight increase in the activity of serum alkaline phosphatase (ALP) and a very mild hyperbilirubinemia, while other markers of liver injury were unaltered. In hepatocytes isolated from these rats, Km and Vmax for uptake and V0 for efflux were no different between ANIT and vehicle-treated animals. In conclusion, ANIT showed no effects on transport of BA on in vitro exposure or after treatment in vivo where SBA were clearly elevated. The lack of effects of ANIT on transport of bile acids is consistent with other postulated mechanisms of action. Furthermore, this indicates that the effects noted with solvents are not necessarily replicated by substances known to cause histopathological cholestasis. PMID- 8620568 TI - Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia. AB - Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15 oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines. The structures of these compounds were determined by analysis of their spectroscopic data. The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition. The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol. The adduct with beta mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself. The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677. The effects of 13 methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells. Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed. The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific. No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15 oxozoapatlin. These data indicate that the cytotoxic activity of 13-methoxy-15 oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression. PMID- 8620569 TI - Increases in tissue levels of ubiquinone in association with peroxisome proliferation. AB - Rats were treated with various peroxisome proliferators and concomitant changes in ubiquinone levels were monitored. In addition to clofibrate and di(2 ethylhexyl)phthalate, acetylsalicylic acid, 2-ethylhexanoic acid, thyroxine and dehydroepiandrosterone were used as proliferators. Administration of these compounds increased the contents of ubiquinone in liver and, to some extent, in kidney and muscle. No change in corresponding valued for heart or brain were observed. The treatments did not influence cholesterol levels, but increased the amounts of dolichol in the liver to various extents. Treatment of rats with the catalase inhibitor aminotriazole increased the ubiquinone levels in kidney, heart and muscle but not in liver. Comparison of peroxisomal fatty acid beta-oxidation with ubiquinone amounts in liver homogenates after treatment with a number of peroxisome proliferators demonstrated a direct correlation between these two parameters. Subcellular fractionation of liver after peroxisome proliferation revealed that the ubiquinone level was increased in mitochondria and lysosomes which are the main compartments for this lipid, but an increase was also observed in both peroxisomes and microsomes. The increase in hepatic ubiquinone after treatment with various types of proliferators was related to the decrease in blood cholesterol level. These results show that the volume of the peroxisomal compartment and the ubiquinone content in animal tissues are interrelated. PMID- 8620570 TI - Catalysis of nitro-aci tautomerism of the genotoxicant 2-nitropropane by cytosol from rodent and human liver. AB - 2-Nitropropane (2-NP) is a genotoxicant and hepatocarcinogen in rodents. Conversion to propane 2-nitronate (P2N), the anion of the tautomeric aci form of 2-NP, seems to be a pivotal part of the mechanism by which 2-NP causes its toxicity. We tested the hypothesis that the tautomeric equilibrium is influenced by enzymes in the liver, the target organ of 2-NP toxicity. Rat or mouse hepatocytes were incubated with 2-NP, P2N or the 2-NP isotopomer 2-deutero 2 nitropropane (2H-2-NP), which equilibrates with P2N much more slowly than 2-NP. Tautomers were analyzed by HPLC. The rates of conversion of 2-NP to P2N expressed as nmol P2N x (10(6) cells/ml)-1 x min-1 were 4.0 and 4.2 in the presence of hepatocytes from rats or mice, respectively, and 2.6 in the absence of cells. Production of 2-NP to P2N expressed as nmol 2-NP x (10(6) cells/ml)-1 x min-1 was increased from 6.1 in the absence of cells to 11.9 or 9.9 in the presence of hepatocytes from rats or mice, respectively. The rate of formation of P2N from 2H 2-NP as compared to 2-NP was characterised by a primary isotope effect of 3.4 and 3.8 in hepatocytes from rats and mice, respectively, contrasting with a value of 9.6 measured in medium omitting cells. When 2-NP was incubated with subfractions of rodent or human liver homogenate, production of P2N by cytosol was between 7.3 (mouse liver) and 28.1 times (human liver) higher than that observed in microsomes. Similarly generation of 2-NP from P2N by cytosol exceeded that in microsomes by a factor of two. Tautomerism in heat-activated cytosol, mitochondria or microsomes was not different from that in buffer only. The results suggest that the nitro-aci tautomerism of secondary nitroalkanes is catalysed by a hepatic enzyme which resides predominantly in the cytosol and may thus contribute to the generation of the toxic species via which 2-NP exerts its toxicity. PMID- 8620571 TI - Redox behaviour of nifuroxazide: generation of the one-electron reduction product. AB - The electrochemical properties of nifuroxazide have been investigated in aqueous and aqueous-DMF mixed solvents. In aqueous media, a single, irreversible four electron reduction occurs to give the hydroxylamine derivative. In mixed media, a reversible one-electron reduction to form a nitro radical anion takes place. Cyclic voltammetric studies show that the anion radical product is stable, although the nitro radical anion intermediate shows a tendency to undergo further chemical reactions. A comparison with the voltammetric behaviour of other nitrofurans such as nifurtimox, nitrofurazone and furazolidone is made. The electrochemically-obtained parameters are correlated with the in vivo studies of oxygen consumption on Trypanosoma cruzi cell suspensions. PMID- 8620572 TI - Effect of exposure to four organic solvents on hepatic cytochrome P450 isozymes in rat. AB - Changes of cytochrome P450 isozymes in livers of rats after exposure to four solvents at 4000 ppm for 6 h, were studied by enzyme assays and immunochemical detection using antibodies to cytochrome P450 isozymes. Toluene, benzene and trichloroethylene (TRI) exposure resulted in a significant increase in the activities of nitrosodimethylamine demethylase (152%, 134% and 118%) and 7 pentoxyresorufin O-depentylase (14-, 5- and 2.5-fold), respectively. 1,1,1 Trichloroethane (TCE) showed little effect on the activities of the enzymes. Anti CYP2E1 and anti-CYP2B1/2 inhibitable activity of toluene side-chain oxidase was significantly enhanced in toluene-, benzene- and TRI-treated rats. Anti-CYP2C11 inhibitable activity was greatly reduced as compared with control. The change in CYP2E1 and CYP2C11 was confirmed by the increase and decrease in the activities inhibited by 4-methylpyrazole and cimetidine, respectively. Western blot analysis revealed that the increase in peak area of bands recognized by anti-CYP2E1 was consistent with toluene inhibition results. CYP2B1/2 was not detectable in control rats, but it was strongly induced by toluene, followed by benzene and TRI. Some increases in the peak areas of bands recognized by anti-CYP2A1 and CYP 4A1 were also observed in the three solvents exposed rat microsomes. Little immunoreactivity was found with anti-CYP1A1 in all microsomes, and no obvious change in peak area of bands recognized by anti-CYP3A and anti-CYP2C13 was observed. TCE exposure showed little effect on these bands. The formation of phenol and hydroquinone from benzene was enhanced to different degree by toluene, benzene and TRI. The hydroxylation of testosterone at 6 beta and 7 alpha was increased by benzene, and benzene and TRI, respectively. However, the metabolism at 16 alpha and 2 alpha was profoundly suppressed by the solvents except TCE. These results showed that the four solvents have different effects on specific cytochrome P450 isozymes and on the metabolism of both endogenous and exogenous substances. PMID- 8620573 TI - Xenobiotic biotransforming enzymes in the central nervous system: an isoform of flavin-containing monooxygenase (FMO4) is expressed in rabbit brain. AB - The flavin-containing monooxygenase (FMO, EC 1.14.13.8) is involved in the metabolism of a number of important xenobiotics including many which affect the central nervous system (CNS). Recently, reports in the literature concerning the amount, activity, location, and isozyme characteristics of this enzyme in the brain have presented conflicting evidence. In order to resolve some of the controversy surrounding FMO in the brain, a highly sensitive method for the detection of flavin-containing monooxygenase (FMO) mRNA in whole brain was employed. A poorly conserved region of FMO transcripts was used to design five sets of oligonucleotide primers. Each primer set was specific for one of the five currently known isoforms of FMO. Four and five isoforms, respectively, are expressed in rabbit liver and kidney, as determined by reverse transcription polymerase chain reaction. However, only one set of primers amplified a specific rabbit brain cDNA fragment. The sequence of the amplification produced affirmed its identity as a segment of FMO4 cDNA. Thus, the FMO of rabbit brain may consist of a single, as yet uncharacterized isozyme and, contrary to several recent reports, is likely to be expressed at low levels. PMID- 8620574 TI - Oxazolidonylethyl adducts to hemoglobin and DNA following nornitrogen mustard exposure. AB - Formation of adducts to hemoglobin (Hb) and DNA of nornitrogen mustard (NNM) was studied with the aim of developing a method for monitoring exposure to NNM. Adducts to N-terminal valines in Hb were studied by the N-alkyl Edman method using pentafluorophenyl isothiocyanate (PFPITC) as the derivatizing reagent. In preliminary studies five major Hb adducts were shown to be formed in reaction of NNM with red cell hemolysate in vitro. Following treatment with PFPITC three of these were found to be pentafluorophenylthiohydantoins (PFPTHs) of N-alkylated valines and the fourth probably originates from NNM esters in which PFPITC had reacted with the nitrogen of N-chloroethylaminoethyl. A PFPTH was found to originate from N-2-(3-oxazolidonyl)ethylvaline, Val-OZ. Val-OZ is formed in reaction, with ring closure to oxazolidone, of CO2 with the 2-chloroethylamino group in the primary valine-N adduct. Besides a few other adducts, Val-OZ was also observed in mouse Hb following injection of NNM, and also after injection of cyclophosphamide. Following reaction in vitro of NNM with DNA, three major adducts to guanine-N-7 were observed; one of them, 7-(N'-(2-chloroethyl)-2 aminoethyl]-guanine (NNMCl), was converted by carbonate to 7-(2-3 oxazolidonyl)ethyl]guanine (Gua-OZ). In mice treated with NNM, Gua-Oz was the only DNA adduct observed. Val-Oz is a chemically stable Hb adduct, potentially useful for monitoring exposures to NNM and cyclophosphamide. PMID- 8620575 TI - In vivo potentiation of 1,2-dibromoethane hepatotoxicity by ethanol through inactivation of glutathione-s-transferase. AB - In the rat, a single ethanol (EtOH) pretreatment (2.5 g/kg b.w., per os) was able to strongly enhance the cytotoxicity of 1,2-dibromoethane (DBE)(87 mg/kg b.w., per os). The principal metabolic routes of DBE involve both oxidative and conjugative transformations. Microsomal cytochrome P450 content and dimethyl nitrosamine demethylase activity were not changed, while a significant loss of cytosolic total GSH-transferase was observed in rats killed 6 h after EtOH pretreatment. Pretreatment with methylpyrazole, an inhibitor of alcohol dehydrogenase prevented the effects provoked by ethanol. The major EtOH metabolite, acetaldehyde. seemed thus to play a fundamental role in the mechanism responsible for the potentiation of DBE toxicity mediated by EtOH. To further support this hypothesis, disulfiram (75 mg/kg b.w.), an inhibitor of aldehyde dehydrogenase, was given i.p. to rats. When DBE was administered to disulfiram- and EtOH-pretreated rats, a marked increase of liver cytolysis was shown and cytosolic GSH-transferase activity was further inhibited if compared to that induced by EtOH treatment alone. The results are consistent with the hypothesis that EtOH given to rats increases DBE liver toxicity because its major metabolite, acetaldehyde, reduces the DBE conjugates to GSH transferase, with consequent shift of DBE metabolism to the oxidative route and accumulation of reactive oxidative intermediates no longer effectively conjugated with GSH. PMID- 8620577 TI - Metabolism of benz[alpha]anthracene by human bone marrow in vitro. AB - The metabolism of polycyclic aromatic hydrocarbons by bone marrow, mononuclear cells from normal donors and leukaemia patients in remission has been investigated. When benz[alpha]anthracene (BA) was included with marrow under cell culture conditions, it was converted to materials which were resolved into three peaks by normal phase HPLC, and which had the chromatographic characteristics of BA-dihydrodiols. Formation of hydroxymethyl-or dihydrodiol-derivatives of 7, 12 dimethylbenz[alpha]anthracene were not detected under the same conditions. The BA metabolites were identified as BA-5,6-dihydrodiol, BA-10,11-dihydrodiol and BA 8,9-dihydrodiol. The identification was based upon chromatographic properties of the metabolites during normal and reverse phase chromatography and on UV spectral and fluorometric characterization. It was not possible to detect the formation of BA-3,4-dihydrodiol since this dihydrodiol co-elutes with BA-8,9-dihydrodiol and BA-10,11-dihydrodiol during normal phase and reverse phase chromatography, respectively. the UV spectra of BA-3,4-dihydrodiol does not have features which enable it to be readily identified in the presence of these other compounds. Formation of the dihydrodiol-metabolites was dependent on cell number and temperature. Two general cytochrome P450 inhibitors, carbon monoxide and piperonyl butoxide, blocked the formation of metabolites but the cyclooxygenase inhibitor, indomethacin had no effect. Large variations were observed in the capacity of marrow from different individuals to form benz[alpha]anthracene dihydrodiols but, in each sample where dihydrodiols were formed, the relative amount of each metabolite was BA-8,9-dihydrodiol >> BA-5,6-dihydrodiol > BA-10,11 dihydrodiol. Factors which may contribute to this variation, including disease status, genetic and environmental agents, are considered. PMID- 8620576 TI - 5-Methylcytosine attack by hydroxyl free radicals and during carbon tetrachloride promoted liver microsomal lipid peroxidation: structure of reaction products. AB - We recently reported that trichloromethyl and trichloromethylperoxyl radicals attack 5-methylcytosine (5MC) to give several products derived from hydroxylation, deamination or halogenation reactions. Hydroxyl radicals and lipid peroxidation (LP) are more frequently involved in deleterious pathological or toxicological processes than those CCl4 derived radicals and thus we considered it of interest to test whether they also alter 5MC. We observed that OH radicals generated by 0.1 mM Fe2+/2.5 mM H202 at 25 degrees C for 1 h led to the production of 5-hydroxymethylcytosine (5MHC). When OH generation was performed with UV light (254 nm, 3400 muWatt/cm2) and 2mM H202 during 4 min at 25 degrees C the following products were observed: 5-hydroxy-5-methylhydantoin, 5 hydroxyhydantoin, 5MHC, thymine glycol (two isomers) and 5-hydroxymethyl-6 hydroxycytosine. When 5MC was exposed to liver microsomal suspensions in the presence of NADPH generating system and carbon tetrachloride during 1 h at 37 degrees C and under air, the formation of only 5HMC was observed. Detection and identification of all reaction products was done by GC/MS analysis of trimethylsilyl derivatives of the bases. If similar reactions occurred in DNA, these results might be of relevance to gene control, differentiation and carcinogenesis. PMID- 8620578 TI - Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats. AB - The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2 BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2 DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes. PMID- 8620579 TI - Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase. AB - Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns. PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[alpha]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz[alpha]anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo[alpha]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals. ortho-Quinones that reduced cell viability produced O2-. and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only. PAH ortho quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA. PMID- 8620580 TI - Effect of structurally diverse peroxisome proliferators on rat hepatic sulfotransferase. AB - Exposure to perfluorocarboxylic acids, pthalate esters, and some hypolipidemic agents results in the proliferation of peroxisomes in the rodent liver. The structural diversity of these compounds suggests mechanistic diversity in their toxicity as well. To establish reliable biomarkers of peroxisome proliferation (PP) in compounds with distinct chemical toxicities, this study investigated the effect of in vivo exposure to perfluoro-n-octanoic acid, perfluoro-n-decanoic acid, di(2-ethylhexyl)phthalate (DEHP) and clofibrate on two-dimensional electrophoretic protein patterns of rat hepatic sulfotransferases, ST1A1, ST1C1 and ST2A1. After exposure to peroxisome proliferative doses, both ST1A1 and ST1C1 abundance in whole liver homogenates was significantly reduced, but only as a result of perfluorocarboxylic and exposure. The well-established PPs, DEHP and clofibrate had no effect on sulfotransferase expression whatsoever. The observed down-regulation of these STs is significant with respect to their normal detoxication activities and its potential correlation to carcinogenesis warrants further study. The present investigation supports previous studies that demonstrate the unique features of perfluorocarboxylic acid toxicity, relative to classic peroxisome proliferators and endorses the continued use of 2D protein mapping of Sts and other proteins as biomarkers of chemical toxicity. PMID- 8620581 TI - Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products. AB - The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme tyrosinase. Tyrosinase was used to oxidize eugenol. No inhibition was observed in the absence of tyrosinase. The rate of irreversible inhibition of GSTs was highest in mouse cytosol, and lowest in rat cytosol. In addition, the irreversible inhibition of human and rat GSTs by eugenol was studied using purified isoenzymes of man and rat. The human GST isoenzymes A1-1, M1a-1a and P1-1 and the rat GST isoenzymes 1-1, 2-2, 3-3, 4-4 and 7-7 were irreversibly inhibited by eugenol in the presence of tyrosinase. In this respect human GST P1-1 and rat GST 7-7 were by far the most sensitive enzymes; human GST A2-2 was not inhibited. Indications were found that human GST P1-1 may be inhibited via three mechanisms: in addition to the well documentated nucleophilic addition of quinones and oxidation of cysteine residues, a covalent subunit cross-linking was also observed. The reversible inhibition of human and rat GST by eugenol, eugenol methyl ether, isoeugenol methyl ether, 2-allylphenol and 4-propylphenol was also studied using purified isoenzymes. The reversible inhibition of human and rat GSTs, using 1-chloro-2,4-dinitrobenzene as substrate, was expressed as I25. All compounds caused moderate reversible inhibition (I25 ranged from 0.2 to 5.4 mM for human GSTs and from 0.4 to 4.9 mM for rat GSTs). In rat, eugenol methyl ether was the strongest inhibitor. In human, the overall inhibiting capacities of eugenol, eugenol methyl ether, isoeugenol methyl ether and 4-propyl phenol were more or less similar; 2-allylphenol was the poorest inhibitor. PMID- 8620582 TI - 32P-postlabelling of bulky human DNA adducts enriched by different methods including immunoaffinity chromatography. AB - DNA adducts in lymphocytes and granulocytes of men exposed occupationally and environmentally to high concentrations of aromatic compounds in air were measured by the 32P-postlabelling method. Adducts in the same samples were characterized using nuclease P1 enrichment, butanol extraction and immunoaffinity purification with an antiserum raised against benzo[alpha]pyrene diol epoxide (BPDE). Only part of the adducts found in human samples were extracted by butanol. It also seemed, that only a small part of them belonged to the group of polycyclic aromatic hydrocarbons (PAHs) recognised by the antibody. Relative content of hydrophobic adducts and those with a structure similar to PAHs was higher in winter samples (when exposure to aromatic chemicals in air was higher) in comparison to samples collected in summer. PMID- 8620583 TI - Disinfectant effects of "Taisalite" and "Taifresh Ace" containing Irgasan DP300 against MRSA and HIV. AB - Sterilization and disinfection in dental clinics and laboratories are important in controlling disease vectors such as methicillin-resistant Staphylococcus aureus (MRSA) and human immunodeficiency virus (HIV). We determined the inactivation of disinfectant effects of "Taisalite" and "Taifresh Ace" containing Irgasan DP300 on MRSA, Streptococcus pyogenes, Candida albicans, and HIV. Exposure for 10 seconds to 50% or 5% Taisalite solution inactivated HIV completely. HIV was also completely inactivated by 10 minutes of exposure to 50% or 5% of Taifresh Ace solution. Taisalite possessed strong bactericidal activity against S. pyogenes and MRSA. C. albicans was resistant to 10 to 30 second exposures to Taisalite. MRSA and C. albicans were killed completely by exposure to 90% Taifresh Ace solution for 10 minutes. S. pyogenes was not highly sensitive to Taifresh Ace. The present study showed that Irgasan DP300 containing Taisalite is an excellent disinfectant against HIV and MRSA and that Taifresh Ace is a useful detergent against these micro-organisms. PMID- 8620584 TI - Site-specific CPITN score and periodontal disease activity assessed by the paper BANA assay. AB - The purpose of this study was to examine the association between site-specific periodontal conditions assessed by CPITN and BANA test results. A total of 161 Japanese company employees aged 18-59 were examined for their periodontal conditions at mesio-buccal and mid-lingual sites of the maxillary right and mandibular left first molars using CPITN. Code 2 (calculus) was divided into Code 2+ and Code 2- by the presence or absence of bleeding. Plaque samples were collected from the examined sites with a scaler and placed on BANA reagent cards. The results indicated a close relationship between site-specific CPITN Codes and the BANA test results except at sites designated Code 2-. The proportion of sites with strong-positive reactions were 19.0% for Code 0, 21.4% for Code 2-, 29.9% for Code 1, 37.0% for Code 2+ and 50.0% for Code 3. These data indicated that the teeth given higher CPITN Codes tend to have higher risks for periodontal disease. From these results, it was concluded that the site-specific CPITN score is a good indicator of periodontal disease activity and that a higher priority for treatment should be given to the sites with pocket formation (Code 3 and Code 4) and/or gingival bleeding (Code 1 and Code 2+). PMID- 8620585 TI - Functioning survival rate of the fixture and superstructure of osseointegrated implants (first report). AB - Osseointegrated implant bridges (OIB) have shown excellent progressing in the majority of cases. Since 1983, OIBs has been used to treat about 110 cases in Tokyo Dental College Hospital with more than 500 fixtures. The survival rate of OIB, especially the functioning survival rate of these fixtures, is described, herein. The survival rate of the superstructure of OIB has been almost 100% in both maxilla and mandible. However, the functioning survival rate of OIB fixtures was 88% in maxillary cases and 96% in mandibular cases at less than 1 year. Very few fixtures had to be removed after more than 1 year in either maxilla or the mandible. It appears that the functioning survival rate of OIB fixture is generally decided during the period just between connecting these components and 1 year. A few fixtures may need to be removed later. PMID- 8620586 TI - An experimental study of osteogenesis by autografted dental pulp, periodontal ligament, and bone marrow in vivo. AB - Osteogenic activity of autografted dental pulp, periodontal ligament, and bone marrow of rat in vivo was investigated. Immunolocalization of ALPase in situ was also studied. One month after the transplantation, osteodentin was formed in all the dental pulp transplants (100%), bone or cementum like tissues were created in 20% of periodontal ligament transplants, and bone like tissues were in 20% of bone marrow transplants. After two months, osteodentin was produced in all the dental pulp transplants (100%) and bone like tissue were in 50% of both periodontal ligament and bone marrow transplants. Immunohistochemically, positive reactions to ALPase in situ were detected in cells just below the odontoblast layers in dental pulp, surface layers of alveolar bone in periodontal ligament, and endosteal membrane of bone marrow space. From these results, it was suggested that the cells of these three kinds of tissue can be termed osteogenic fibroblasts in vivo. PMID- 8620587 TI - Oral focal acantholytic dyskeratosis induced in sialoadenectomized rats painted orally with the carcinogen 4-nitroquinoline N-oxide. AB - This study examines the effects of xerostomia on the development of mucosal changes proceeding carcinoma in rats painted orally with 4-nitroquinoline N-oxide (4NQO). Sialoadenectomy enhanced the incidence of focal acantholytic dyskeratosis (FAD) on the palate and tongue of rats painted on the palate with 0.5% 4NQO three times weekly. At one month, no changes were observed. After two months of painting with 4NQO, sialoadenectomized rats, but not controls, had FAD. After three months, FAD was seen in both test and control groups, but the incidence of FAD was higher in the test group and seen more on the palate than on the tongue of xerostomic animals. Neither test nor control animals developed carcinoma within this study period. PMID- 8620588 TI - Bacteriological diagnosis of periodontal disease. AB - Dental plaque bacteria cause virtually all the forms of inflammatory periodontal disease. Periodontitis is caused by the specific periodontopathic bacteria, which induce destruction of connective tissue attachment and adjacent alveolar bone. Examinations to identify the infections by Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Bacteroides forsythus, Fusobacterium nucleatum, Eikenella corrodens and Treponema denticola have recently become essential in diagnosis of periodontal disease. Bacterial examination permits (1) identification of the causative bacteria, (2) assessment of disease activity and (3) monitoring of the effective of periodontal treatments. The author describes details of accurate and rapid methods for detecting periodontopathogens. Transmission of periodontopathic bacteria as clarified by bacterial examination is also discussed in this review. The pathogenic potential of specific bacteria varies among patients and periodontally healthy individuals and can be controlled by host defense mechanisms such as immune responses. The roles of immune responses against periodontopathic bacteria in balance shifts of periodontal disease processes are therefore also discussed in this review. PMID- 8620589 TI - Absorbed doses modified by exposure settings with rotational panoramic radiography. AB - Absorbed doses to parotid gland, cervical vertebrae and thyroid gland were measured with various exposure settings of the tube voltage and tube current during use of rotational panoramic radiographs , Veraview (J. Morita Corp., Kyoto, Japan) and PM 2002 CC (Planmeca Oy, Helsinki, Finland) using Rando phantom and thermoluminescence dosimeters. The magnitude of each absorbed dose was greatest in the parotid gland, followed in order by the cervical vertebrae and thyroid gland and was proportional to tube current. The increase of the thyroid gland absorbed dose with increasing tube voltage was steeper than were the increases in the absorbed doses in either the parotid gland or the cervical vertebrae. PMID- 8620590 TI - Absorbed dose reduced by sliced exposure using sector selector system with rotational panoramic radiography. AB - Sliced exposures which restrict the exposed area and adjust the region of diagnosis are possible using a sector selector system which is installed in the rotational panoramic radiograph, PM 2002 CC (Planmeca Oy, Helsinki, Finland). The effects of altering the horizontal and vertical sector selector system on thyroid gland dose were measured using Rando Phantom and thermoluminescence dosimeters. The horizontal sector selector system clarified that the most of the thyroid gland dose was delivered from exposure of the mandibular region. The vertical sector selector system clarified that scattered x-rays which originated from primary x-rays curved at the incisor and canine, premolar and molar, and ramus regions contributed equally to the thyroid gland dose. PMID- 8620591 TI - Distribution of organized sensory nerve endings in the human periodontal ligament. AB - The purpose of the present study was to clarify the distribution pattern of organized nerve endings in human-periodontal ligament. The materials investigated were obtained from the first premolar teeth. The fresh periodontal ligament of the extracted tooth was stained using the methylene blue vital staining method. Then the whole-mount preparations of the periodontal ligament were observed by light microscope. Three kinds of receptors (encapsulate corpuscles, bush-like endings and free-nerve endings) were located in the ligament. The ratio of total number of encapsulated corpuscles to bush-like endings was one to three. The density of the encapsulated corpuscles ranged from 0.2/mm2 to 1.2/mm2 and the bush-like endings ranged from 1.3/mm2 to 3.2/mm2. The most organized nerve endings were distributed at the middle one third in the ligament. The distribution of bush-like endings, which differed from that of encapsulated corpuscles, might contribute to their response thresholds and static properties. PMID- 8620592 TI - Oral health status related to subgingival bacterial flora and sex hormones in saliva during pregnancy. AB - The purpose of this study was to investigate oral health status in relation to subgingival microflora and sex hormone concentrations in saliva during pregnancy. Oral health examinations were performed on 39 subjects: of 19 pregnant women (mean age 28.5), and 8 women in the fifth post-partum month (mean age 27.1), and 12 non-pregnant women (mean age 22.9). Periodontal conditions of bleeding on probing, redness, swelling, and probing depth were examined. Subgingival microbial compositions were examined with several selective media. Concentrations of estradiol and progesterone in saliva were determined during pregnancy and the first post-partum month. From the third to the fifth month of pregnancy, the number of gingival sites where probing caused bleeding increased concomitantly with increasing percentages of Prevotella intermedia. The number of gingival sites with redness and swelling increased in the fourth and seventh months of pregnancy and decreased in the last month of pregnancy to the level of the second month. The gingival inflammatory signs, however, tended to increase in the first post-partum month. The probing depth of gingiva gradually increased during pregnancy and the first post-partum month. Percentage of P. intermedia increased in the fourth month of pregnancy with increasing of hormones in saliva. The concentrations of hormones in saliva attained peaks in the ninth month of pregnancy. Thereafter, proportion of P. intermedia decreased. As a further step, these pregnant women were divided into three groups on the bases of percentages and detectable rates of P. intermedia. Gingival inflammation was more extensive in the high group subjects in which more than 15% P. intermedia in total CFU was detected; its severity also tended to increase. PMID- 8620593 TI - alpha 1-adrenergic receptor subtypes. Molecular structure, function, and signaling. PMID- 8620594 TI - Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. AB - Signalling pathways determining the shear stress-induced production of NO from endothelial cells in situ were investigated using a bioassay system in which shear stress was increased by inducing vasoconstriction in an endothelium-intact donor segment (rabbit iliac artery) while maintaining a constant luminal perfusion rate. Shear stress-induced NO production, as assessed by changes in the tone of a preconstricted endothelium-denuded detector ring, was biphasic and consisted of an initial transient (20- to 25-minute) Ca(2+)-dependent phase followed by a Ca(2+)-independent plateau phase, which was maintained as long as the donor segment remained constricted. Stretching the donor segments to their in vivo length abolished the initial phase without affecting the plateau phase of NO release. Inhibition of the Na(+)-H+ exchanger using HOE 694 elicited an intracellular acidification which attenuated shear stress-induced NO production. The specific protein kinase C inhibitor, Ro 31-8220, was without effect, whereas the unspecific inhibitors, staurosporine and calphostin C, abolished the shear stress-induced production of NO. Erbstatin A, a tyrosine kinase inhibitor, attenuated the shear stress-induced tyrosine phosphorylation of specific cellular proteins and abrogated the associated NO production. In summary, these data indicate that shear stress activates the NO synthase at basal levels of [Ca2+]i via a mechanotransduction cascade that involves tyrosine phosphorylation and can be modulated by changes in pHi. The apparent fundamental alteration of the endothelial NO synthase under shear stress that renders its maintained activation independent of an increase in [Ca2+]i is probably the consequence of a change in the enzyme microenvironment. PMID- 8620595 TI - Salicylate or aspirin inhibits the induction of the inducible nitric oxide synthase in rat cardiac fibroblasts. AB - To determine if fibroblasts are a source of NO inflammatory myocardial diseases, we have studied the effect of cytokines on the inducible NO synthase (iNOS) in neonatal cardiac fibroblasts and tested whether nonsteroidal anti-inflammatory drugs can diminish the induction of iNOS. In primary cultures, interferon gamma (IFN), interleukin-1 beta (IL-1), or tumor necrosis factor-alpha (TNF) separately did not stimulate nitrite production, whereas IFN combined with IL-1 or TNF synergistically induced iNOS, both at the level of steady state mRNA and nitrite accumulation. Steady state mRNA levels for iNOS were obvious as early as 3 hours after the addition of IFN + TNF and remained elevated for at least 72 hours. Sodium salicylate inhibited cytokine-induced nitrite accumulation in a time- and dose-dependent manner (IC50, 750 mumol/L). The inhibition was reversible and occurred when salicylate was added either before or after cytokine induction. Aspirin (1 mmol/L) also inhibited nitrite production, whereas indomethacin (25 mumol/L) or acetaminophen (100 mumol/L) did not. TNF, either alone or combined with IFN, significantly stimulated prostaglandin E2, which was inhibited by either salicylate (4 mmol/L) or indomethacin (25 mumol/L). Salicylate, when given either before or after IFN + TNF, reduced mRNA levels of iNOS induced by cytokines. Salicylate did not affect iNOS enzymatic activity when added to the cytosolic lysate, although it was able to reduce enzymatic activity to 32% of induced levels when given to intact cells. These studies implicate cardiac fibroblasts as a source of NO in inflammatory cardiac diseases and suggest a possible therapeutic role for salicylate and aspirin in diminishing the steady state levels of iNOS mRNA. PMID- 8620596 TI - Inhibition of inducible nitric oxide synthase prevents myocardial and systemic vascular barrier dysfunction during early cardiac allograft rejection. AB - NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature. PMID- 8620597 TI - Lysophosphatidylcholine promotes P-selectin expression in platelets and endothelial cells. Possible involvement of protein kinase C activation and its inhibition by nitric oxide donors. AB - Lysophosphatidylcholine (LysoPC), an atherogenic lysophospholipid contained in oxidized low-density lipoprotein (LDL), has been shown to stimulate protein kinase C (PKC). Since PKC activators are suggested to elicit rapid P-selectin expression in platelets and endothelial cells, we examined whether LysoPc promotes P-selectin expression in platelets and P-selectin-mediated leukocyte adherence to endothelial cells via a mechanism involving PKC activation. LysoPc, but not phosphatidylcholine (PC), which is a major phospholipid component in native LDL, significantly upregulated P-selectin on cat platelets by flow cytometric analysis. This P-selectin upregulation by LysoPC was significantly attenuated by two PKC inhibitors, 7-hydroxystaurosporine (UCN-01) and N,N,N trimethylsphingosine, and by two NO donors, CAS1609 and sodium nitroprusside. Submicellar concentrations of LysoPc significantly activated PKC in platelets, and this was inhibited by either UCN-01 or CAS1609. LysoPC, but not PC, significantly increased adherence of autologous cat polymorphonuclear leukocytes to coronary vascular endothelium, which was also markedly attenuated by UCN-01 and by CAS1609. LysoPC induced P-selectin expression on the surface of cat coronary vascular endothelium as assessed by immunohistochemical analysis. These results suggest that LysoPC, an atherogenic lysophospholipid contained in oxidized LDL, rapidly induces P-selectin expression in both platelets and endothelial cells at least partially via PKC activation. Furthermore, NO generating agents may inhibit P-selectin upregulation by LysoPC. Since P-selectin may play an important role in initiating atherosclerosis, our data provide further insight into the mechanism of early stages of atherogenesis and of NO mediated inhibition of atherosclerosis. PMID- 8620598 TI - Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. AB - During fetal development, a specialized vessel the ductus arteriosus, shunts blood from the pulmonary artery to the aorta, thus bypassing the lungs. The ductus differs primarily from the great vessels in that it is a muscular rather than an elastic artery, and the etiology of this differential development remains controversial. We present evidence that retinoic acid (RA) may contribute to the unique muscle phenotype of the ductus arteriosus. Using a transgenic mouse carrying an RA response element-lacZ transgene that expresses beta-galactosidase (beta-gal) in response to endogenous RA signals during embryonic and fetal development, we observe a strong beta-gal signal in the ductus arteriosus. By immunofluorescence, this signal colocalizes with the expression of the adult specific smooth muscle myosin heavy chain isoform, SM2. The beta-gal signal is present throughout fetal development and persists in the neonate until the ductus arteriosus is completely closed. beta-Gal-positive cells are first detected by immunofluorescence at 13.5 days postcoitum (dpc) in the mesenchyme surrounding the ductus. By 15.5 dpc, very intense beta-gal staining localizes to the ductus arteriosus but is absent or minimal in the pulmonary trunk and aortic arch; by 17.5 dpc, the smooth muscle layers of the tunica media in the ductus arteriosus exhibit positive beta-gal staining. Immunostaining with antibodies against smooth muscle myosins shows that, while SM1 is expressed in all embryonic vessels, SM2 is precociously expressed in the ductus arteriosus. Furthermore, SM2 expression can be detected in the ductus as early as 15.5 dpc. In the neonate, the beta-gal signal persists in the smooth muscle layer of the ductus and immunostaining colocalizes with SM2 expression. These data suggest that RA may play a role in inducing and maintaining smooth muscle differentiation in the developing ductus arteriosus and may promote precocious expression of the adult vascular phenotype. PMID- 8620599 TI - Developmental remodeling of the internal elastic lamina of rabbit arteries: effect of blood flow. AB - We examined remodeling of the internal elastic lamina (IEL) of rabbit arteries from 3 to 23 weeks of age. The IELs were fenestrated at all ages; however, the sizes of the fenestrae increased dramatically during postnatal development. Mean areas occupied by the individual fenestrae of the carotid artery IEL increased from 11.3 +/- 0.7 microns2 in 3-week-old rabbits to 61.2 +/- 5.5 microns2 in adult rabbits. The estimated number of fenestrae per vessel also increased greatly, from 2.68 x 10(5) to 9.27 x 10(5); however, the increased number of fenestrae did not keep pace with growth of the artery, since fenestrae per square millimeter decreased by 26%. Large increases in the size of fenestrae were also observed in the renal and iliac arteries, although greater decreases in fenestrae per square millimeter occurred with age (70% in iliac arteries). Morphological assessments suggested that enlarging fenestrae frequently fuse with neighbors. By contrast with other arteries, the IEL of the abdominal aorta was not a continuous fenestrated sheet in young animals, perhaps reflecting the extensive remodeling that this vessel undergoes in the postnatal period. We decreased common carotid blood flow by 70% in 5 rabbits at 10 weeks of age by ligating the ipsilateral external carotid artery, and we approximately doubled blood flow in 5 others at the same age, by contralateral common carotid ligation. At 15 weeks of age, fenestrae in the artery carrying increased flow were 39% larger than fenestrae in the control artery, whereas fenestrae were 53.5% smaller after 70% decreases in flow (P < .05). We conclude that flow-dependent enlargement of fenestrae contributes to developmental remodeling of the IEL. Remodeling of the IEL may also have important implications for transport of materials and cell-cell communication between the intima and media. PMID- 8620600 TI - Identification of protein kinase C isoforms in rat mesenteric small arteries and their possible role in agonist-induced contraction. AB - We have identified immunologically the protein kinase C (PKC) isoforms present in rat mesenteric small arteries, defined their distribution between particulate and soluble fractions, and studied their involvement in phorbol ester-induced contraction. Our analysis revealed the presence of the CA(2+)-dependent PKCs (alpha and gamma), Ca(2+)-independent PKCs (delta and epsilon), and the atypical isoform (zeta). PKCbeta could not be detected, whereas PKCgamma is likely to be of neural origin. All isoforms exhibited different distributions. PKCalpha, PKCepsilon, and PKCzeta were found in both particulate and soluble fractions. In contrast, PKCdelta was mainly in the particulate fraction, and PKCgamma was in the soluble fraction. Phorbol esters, which activate PKC and cause smooth muscle contraction, downregulated only the alpha and delta isoforms. This was associated with a parallel loss of contractile response to phorbol ester. The force developed to submaximal concentrations of noradrenaline was decreased after phorbol dibutyrate pretreatment, although the sensitivity and maximal response were unchanged. Phorbol ester pretreatment did not affect the contractile response to vasopressin. The sensitivity to non-receptor-mediated contraction, caused by k+ in the presence of prazosin, was slightly reduced by 4 alpha- and 4 beta-phorbol ester pretreatment. Maximal tension in response to this agonist was not affected. We conclude that PKCalpha and/or PKCdelta is necessary for phorbol ester-mediated contraction but is not essential for noradrenaline-, vasopressin-, or k(+)-induced contraction, demonstrating differences in the mechanisms involved in the contractile response between these agents. PMID- 8620601 TI - Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2. AB - [Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd approximately 2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is approximately 5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 approximately 0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2-loaded A7r5 cells. Frequency of CA2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(beta-mercapto-beta,beta,-pentamethylenepropionic acid),2-0 methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca(2+)-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 mumol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 mumol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca(2+)-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis. PMID- 8620602 TI - Binding of cytosolic proteins to myofibrils in ischemic rat hearts. AB - Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility. PMID- 8620603 TI - Passive load and angiotensin II evoke differential responses of gene expression and protein synthesis in cardiac myocytes. AB - This study introduced an improved model of loaded adult cardiocytes to address a proposed requirement for angiotensin II (Ang II) in the transduction pathway between load on the cardiac myocyte and its early anabolic responses of gene expression and acceleration of protein synthesis. The isolated cardiocytes were subjected to passive load by step increments of stretch and responded with proportional acceleration of protein synthesis in both adult and neonatal cardiocytes; this response was unaltered by 1 mumol/L [Sar1, Ile8]Ang II, an antagonist peptide to Ang II. Ang II from 1 nmol/L to 10 mumol/L did not increase protein synthesis after 4 hours in adult cardiocytes nor at 100 nmol/L in neonatal cardiocytes. However, 100 nmol/L Ang II did increase [3H]phenylalanine incorporation into neonatal cardiocyte protein over a 24-hour period by 10%, whereas passive load increased [3H]phenylalanine incorporation into protein by 30%, which was not blocked by [Sar1, Ile8]Ang II. Thus, the anabolic effect of load does not require ANG II to increase either 4-hour protein synthesis in both adult and neonatal cardiocytes or 24-hour [3H]phenylalanine incorporation into protein in neonatal cardiocytes. The genetic response of the cardiocyte to load was examined by assessing c-fos and Na+-Ca2+ exchanger mRNA levels, because there are rapidly expressed at the onset of cardiac pressure overload. The c-fos mRNA was increased fourfold within 1 hour after 100 nmol/L Ang II treatment of either adult or neonatal cardiocytes. This c-fos induction was blocked by [Sar1, Ile8]Ang II. One hour after loading of adult cardiocytes, induction of c-fos expression was increased threefold; this was also blocked by [Sar1, Ile8]Ang II. Thus, load-induced c-fos expression was Ang II dependent in adult cardiocytes. In contrast, exchanger mRNA levels were increased threefold 1 hour after loading of adult cardiocytes, but this increased expression was not blocked by [Sar1, Ile8]Ang II. For additional comparison, c-fos expression was induced by Ang II and phorbol myristate acetate, which did not induce exchanger expression; conversely, exchanger expression was induced by veratridine, which did not increase c-fos expression. Thus, separate c-fos and exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na+-Ca2+ exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na+-Ca2+ exchanger gene expression in cardiocytes; however, load induced c-fos expression is Ang II dependent. PMID- 8620604 TI - Phospholamban gene dosage effects in the mammalian heart. AB - Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in phospholamban levels, hearts from wild type, phospholamban-heterozygous, and phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The phospholamban heterozygous mice expressed reduced cardiac phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative phospholamban levels among wild-type, phospholamban-heterozygous, and phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction. PMID- 8620605 TI - Modification of viral myocarditis in mice by interleukin-6. AB - Inflammatory cytokines play a key role in the myocardial injury produced by viral myocarditis. Although interleukin-6 (IL-6) reportedly possesses antiviral properties, its effect in viral myocarditis is unclear. To investigate the role of IL-6 in viral myocarditis induced by encephalomyocarditis virus (EMCV) in mice, we evaluated (1) the survival rate following IL-6 administration, (2) the viral titer in the heart, (3) viral replication in the heart by in situ hybridization, (4) histopathological changes using immunohistochemical staining, (5) neutralizing antibody against EMCV, (6) circulating interferon and tumor necrosis factor-alpha (TNF-alpha), (7) viral suppression in vitro by IL-6, and (8) natural killer (NK)-cell activity. Eight-week-old C3H/HeJ mice were injected intraperitoneally with EMCV (day 0) and were also injected subcutaneously twice daily for 4 consecutive days with 10 micrograms/0.1 mL of human IL-6 on day -4 (group A), day 0 (group B), or day +4 (group D) for 4 days. As a control, 0.1 mL PBS instead of IL-6 was injected on day 0 for 4 days (group C). Certain mice were killed on day 4. The myocardial virus titers, viral replication in situ, and NK cell activity in the spleen were determined. Decreased viral titer and viral replication in the heart reduced the titer of circulating TNF-alpha, and lower NK cell activity was observed in group B versus group C (control group). The titer of neutralizing antibodies against EMCV was significantly (P < .05) increased in group B compared with group C. The remaining mice were killed on days 10 and 30 after infection. The ratio of heart weight (HW) to body weight (BW) and myocardial injury in group B were reduced versus group C on days 10 and 30. The HW of group B on day 30 did not differ from the normal control group. The ratio of splenic weight to BW and the ratio of thymic weight to BW of group B increased on day 10, with expanded follicles observed in the spleen and enlargement of the medulla observed in the thymus. Immunohistochemical study revealed an increased percentage of macrophages in the heart and spleen of group B. In summary, IL-6 reduces myocardial damage in mice with viral myocarditis. Modification of immune responses together with reduction in viral replication appears to be the mechanism of the IL-6 effect. Although IL-6 is likely important in the process of viral antigen presentation, early activation of immune responses and attenuation of viral replication appear most significant, as reflected in the limited time window during which IL-6 is effective in myocarditis. PMID- 8620606 TI - Variability of spontaneous Ca2+ release between different rat ventricular myocytes is correlated with Na(+)-Ca2+ exchange and [Na+]i. AB - We have studied the factors responsible for the variation of the frequency of "waves" caused by spontaneous Ca2+ release in rat ventricular myocytes. The experiments were performed in isolated myocytes using the fluorescent indicators Indo-1 (to measure [Ca2+]i) and SBFI (to measure [Na+]i). After electrical stimulation (either with action potentials or voltage-clamp pulses), some cells showed spontaneous Ca2+ release. The frequency of this release, where present, was variable. The Ca2+ content of the sarcoplasmic reticulum (SR) was measured by applying caffeine (10 mmol/L). The resulting increase of [Ca2+]i activated the electrogenic Na(+)-Ca2+ exchange, and the integral of this current was used to estimate the Ca2+ content of the SR. The SR Ca2+ content was significantly higher in cells that oscillated at high rates ( > 10 . min-1) than in those that were quiescent. The rate of removal of Ca2+ from the cytoplasm by non-SR mechanisms was measured by adding caffeine (10 mmol/L) and measuring the rate constant of decay of the resulting increase of [Ca2+]i. Cells that had a high rate constant of decay of [Ca2+]i had a low frequency of oscillations. Measurements of [Na+]i showed a positive correlation between the frequency of spontaneous SR Ca2+ release and [Na+]i. After cessation of stimulation, there was a gradual decrease of [Na+]i, which was correlated with a parallel decrease of the frequency of oscillation rate. We conclude that the variability of frequency of spontaneous SR Ca2+ release is due to variations of the rate of Ca2+ removal from the cell, which are probably due to Na(+)-Ca2+ exchange. The variability of Na(+)- Ca2+ exchange rate, in turn, is likely to result from variations of [Na+]i. PMID- 8620607 TI - Histamine H3-receptor-mediated inhibition of calcitonin gene-related peptide release from cardiac C fibers. A regulatory negative-feedback loop. AB - Antidromic stimulation of cardiac sensory C fibers releases calcitonin gene related peptide (CGRP), which increases heart rate, contractility, and coronary flow. C-fiber endings are closely associated with mast cells, and CGRP may release mast-cell histamine. Because prejunctional histamine H3-receptors inhibit transmitter release from autonomic nerves, we tested the hypothesis that H3 receptors modulate CGRP release in the heart. CGRP released by bradykinin in the electrically paced guinea pig left atrium and by capsaicin in the spontaneously beating isolated heart caused marked positive inotropic and chronotropic effects, respectively. Capsaicin significantly enhanced the overflow of CGRP (fivefold) and histamine (twofold) into the coronary effluent. All of these effects were prevented by prior chemical destruction of C fibers in vivo. The H3-receptor agonist imetit attenuated the inotropic response to bradykinin by 50%. Imetit also decreased the capsaicin-induced tachycardia and the increase in CGRP overflow by 50%. Imetit, however, did not modify the response to exogenous CGRP. The effects of imetit were blocked by the H3-receptor antagonist thioperamide. Notably, thioperamide by itself potentiated the capsaicin-evoked increases in heart rate and CGRP overflow (by 25% and 50%, respectively). Thus, our findings identify a negative-feedback loop, whereby CGRP releases histamine from cardiac mast cells and histamine in turn inhibits CGRP releases by activating H3 receptors on C-fiber terminals. Because CGRP release is augmented in pathophysiological conditions, such as septic shock, heart failure, and acute myocardial infarction, modulation of CGRP release may be clinically relevant. PMID- 8620608 TI - The alpha3 isoform protein of the Na+, K(+)-ATPase is associated with the sites of cardiac and neuromuscular impulse transmission. AB - The alpha (catalytic) subunit of the Na+ pump (Na+, K(+)-ATPase) has three isoforms; alpha1 is ubiquitous, skeletal muscle expresses predominantly alpha2, and alpha3 has been localized to specific types of neurons and, possibly, to axonal processes. The alpha3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na+ pump alpha subunit and labeled alpha-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that alpha3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed alpha3 immunoreactive protein, as assessed by two isoform specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that alpha3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na+,K(+)-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle. PMID- 8620609 TI - Altered ventricular and myocyte response to angiotensin II in pacing-induced heart failure. AB - Alterations in the cardiac response to angiotensin II (Ang II) may contribute to the functional impairment in tachycardia-induced heart failure (congestive heart failure [CHF]). Accordingly, we studied the response to Ang II in eight conscious instrumented dogs before and after inducing CHF. Left ventricular (LV) performance was assessed by measuring LV pressure and LV volume. Isolated myocyte function was evaluated using computer-assessed videomicroscopy. In conscious animals before CHF, Ang II produced a load-dependent slowing of the time constant of LV relaxation (tau) and did not depress intact LV contractile function. After CHF, although Ang II produced a similar increase in LV systolic pressure, the increases in LV diastolic pressure and time constant tau were much greater, and contractile performance was depressed. These changes persisted when the elevation of end-systolic pressure was prevented by nitroprusside. Similar changes were also present after autonomic blockade. In isolated myocytes, before CHF, Ang II (10(-6) mol/L) produced a slight positive inotropic effect. In contrast, after CHF, Ang II produced a negative inotropic effect and slowed the rate of relengthening. The effects in the intact LV and myocytes were reversed by an Ang II AT1 receptor blocker (losartan). We conclude that pacing-induced CHF alters the LV and myocyte response to Ang II, so that Ang II produces direct depressions in intact LV contraction, relaxation, and filling and exacerbates myocyte contractile dysfunction. These effects are mediated through the activation of AT1 receptors. PMID- 8620610 TI - Decreased energy reserve in an animal model of dilated cardiomyopathy. Relationship to contractile performance. AB - An animal model was used to test the hypothesis that in heart failure the decrease in the ability to resynthesize ATP through the creatine kinase (CK) reaction (which we call energy reserve) contributes to the inability of the heart to maintain its normal function and contractile reserve. One-week-old turkey poults were fed furazolidone for 14 days to induce dilated cardiomyopathy. Isolated Langendorff-perfused hearts from these myopathic animals showed a 73% decrease in baseline isovolumic contractile performance. Neither increasing [Ca2+]o nor electrical pacing rate increased isovolumic contractile performance. Measured by 31P nuclear magnetic resonance magnetization transfer and chemical assay, ATP concentration was decreased by 23%, phosphocreatine concentration by 42%, CK enzyme activity by 34%, and the pseudo first-order rate constant for the CK reaction by 50%. Measured CK reaction velocity decreased by 71%. The reduced ability to increase cardiac performance in response to increasing [Ca2+]o in hearts with lower CK reaction velocity was reproduced in part by feeding a separate group of turkey poults beta-guanidino-propionic acid to specifically reduce CK reaction velocity by decreasing guanidino substrate concentration. These hearts had normal baseline performance but blunted contractile reserve. These observations provide further support for the hypothesis that a decrease in energy reserve via the CK system contributes to reduced cardiac function in the failing heart. PMID- 8620611 TI - Adrenergic modulation of ultrarapid delayed rectifier K+ current in human atrial myocytes. AB - The ultrarapid delayed rectifier K+ current (IKur) in human atrial cells appears to correspond to Kv1.5 cloned channels and to play an important role in human atrial repolarization. Kv1.5 channels have consensus sites for phosphorylation by protein kinase A and C, suggesting possible modulation by adrenergic stimulation. The present study was designed to assess the adrenergic regulation of IKur in human atrial myocytes. Isoproterenol increased IKur in a concentration-dependent manner, with significant effects at concentrations as low as 10 nmol/L. The effects of isoproterenol were reversible by washout or by the addition of propranolol (1 mumol/L). Isoproterenol's effects were mimicked by the direct adenylate cyclase stimulator, forskolin, and by the membrane-permeable form of cAMP, 8-bromo cAMP. Isoproterenol had no effect on IKur when the protein kinase A inhibitor peptide, PKI(6-22)amide, was included in the pipette solution; in a separate set of experiments in which isoproterenol alone increased IKur by 45 +/- 9% relative to control, subsequent superfusion with isoproterenol in the presence of the protein kinase inhibitor H-7 failed to alter IKur. In contrast to isoproterenol, phenylephrine (in the presence of propranolol to block beta adrenegic effects) induced a concentration-dependent inhibition of IKur, with significant effects observed at concentrations as low as 10 mumol/L. The inhibitory actions of phenylephrine were reversed by the addition of prazosin and prevented by coadministration with a highly selective inhibitor of protein kinase C, bisindolylmaleimide. These results indicate that beta-adrenergic stimulation enhances, whereas alpha-adrenergic stimulation inhibits, IKur and suggest that these actions are mediated by protein kinase A and protein kinase C, respectively. The modulation of IKur by adrenergic influences is a potentially novel control mechanism for human atrial repolarization and arrhythmias. PMID- 8620612 TI - Multiple mechanisms of Na+ channel--linked long-QT syndrome. AB - Inheritable long-QT syndrome (LQTS) is a disease in which delayed ventricular repolarization leads to cardiac arrhythmias and the possibility of sudden death. In the chromosome 3-linked disease, one mutation of the cardiac Na+ channel gene results in a deletion of residues 1505 to 1507 (Delta KPQ), and two mutation result in substitutions (N1325S and R1644H). We compared all three mutant-channel phenotypes by heterologous expression in Xenopus oocytes. Each produced a late phase of inactivation-resistant, mexiletine- and tetrodotoxin-sensitive whole cell currents, but the underlying mechanisms were different at the single-channel level. N1325S and R1644H showed dispersed reopenings after the initial transient, whereas Delta KPQ showed both dispersed reopenings and long-lasting bursts. Thus, two distinct biophysical defects underlie the in vitro phenotype of persistent current in Na+ channel-linked LQTS, and the additive effects of both are responsible for making the Delta KPQ phenotype the most severe. PMID- 8620613 TI - Nitric oxide synthase activity in guinea pig ventricular myocytes is not involved in muscarinic inhibition of cAMP-regulated ion channels. AB - It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of beta-adrenergically stimulated ion channels in cardiac sinoatrial node cells (J Gen Physiol. 1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl currents. Myocytes isolated from guinea pig hearts stained positively for NADPH diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the R(-)-isoproterenol bitartrate (Iso)-activated Cl- current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl- current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl- currents could not be mimicked by the NO donors sodium nitroprusside,3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl- current could not be blocked by the NOS inhibitor NG-monomethyl-L-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of beta-adrenergically regulated Cl- channels in these cells. PMID- 8620614 TI - Unitary Cl- channels activated by cytoplasmic Ca2+ in canine ventricular myocytes. AB - Recent whole-cell studies have shown that Ca(2+)-activated Cl- currents contribute to the Ca(2+)-dependent 4-aminopyridine-insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl(-)-sensitive currents are activated by Ca2+ release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl- to identify 4 aminopyridine-resistant unitary Ca(2+)-activated Cl- channels, Ca2+ applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl- selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca2+ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca2+ (ranging from pCa > or = 6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NPo, where N is the number of channels opened and Po is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NPo on cytoplasmic Ca2+ yielded an apparent Kd of 150.2 mumol/L. At pCa 3, an average channel density of approximately equal to 3 microns-2 was estimated. Calculations based on these estimates of cytoplasmic Ca2+ sensitivity and channel current amplitude and density suggest that these small-conductance Cl- channels contribute significant whole-cell membrane current in response to changes in intracellular Ca2+ within the physiological range. We suggest that these small-conductance Ca(2+)-activated Cl- channels underlie the transient Ca(2+)-activated 4 aminopyridine-insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias. PMID- 8620615 TI - NO flow helps clear murky waters? PMID- 8620616 TI - Selection of ligands for polyclonal antibodies from random peptide libraries: potential identification of (auto)antigens that may trigger B and T cell responses in autoimmune diseases. AB - The development of random peptide libraries has increased our possibility for analyzing the structural features involved in binding events. Recently, reports have appeared in which these libraries have been successfully used to investigate binding properties of homogeneous proteins such as monoclonal antibodies. However, a more general application of peptide libraries would be the use of polyclonal sera or fluids from patients with autoimmune diseases in biopanning experiments. This would subsequently allow the identification of (auto)antigen leads responsible for the initiation and/or perpetuation of the immune response in these patients. Moreover, the strategy allows the structural characterization of autoantibody specificities in body fluids that have been produced in vivo without the introduction of bias due to preferential B cell growth under in vitro conditions. The application of this novel strategy for selection of antibody ligands for polyclonal sera as well as to study the nature of immune responses to defined proteins will be discussed with emphasis on the development of peptide reagents for diagnostic and vaccine use. PMID- 8620617 TI - B-cell proliferation and differentiation in common variable immunodeficiency patients produced by an antisense oligomer to the rev gene of HIV-1. AB - The immunostimulatory activity of a phosphorothioate oligodeoxynucleotide (27 mer) that is antisense to the rev gene of HIV-1 was studied on normal human lymphocytes and on cells from patients with common variable immunodeficiency (CVI). For peripheral blood mononuclear cells from nine normal individuals, the proliferation index (16.8 +/- 12.5) after anti-rev oligomer exposure was proportional to the percentage of peripheral B-cells (r = 0.76, P = 0.02). In five experiments, enriched B- or T-cell populations had proliferation indices of 47.2 +/- 32.9 and 2.4 +/- 1.9, respectively. The addition of T-cells to anti-rev oligomer treated B-cells had no effect (proliferation index = 47.5 +/- 38.1). After anti-rev oligomer stimulation, autoradiography, and counterstaining for B- and T-cell markers, all detectable [3H]thymidine uptake was by CD19-positive cells. Eight of the 14 CVI patients had a proliferation index and secreted levels of IgM and IgG comparable to cells from normal individuals. In contrast to normal cells, the direct correlation between proliferation of peripheral blood mononuclear cells and the percentage of peripheral B-cells was weak in samples from 13 CVI patients (r = 0.4, P = 0.2). These findings indicate that peripheral blood B-cells from about half of CVI patients proliferate and produce immunoglobulin after exposure to anti-rev oligomer. These data demonstrate that under the appropriate circumstances, B-cells of some CVI patients can proliferate and differentiate normally. PMID- 8620618 TI - In vitro inhibition by intravenous immunoglobulin of human T cell-dependent B cell differentiation induced by staphylococcal superantigens. AB - Treatment with intravenous Ig (IVIG) is efficacious not only in humoral immunodeficiency diseases but in several nonimmunodeficiency disorders as well. Since microbial superantigens (SAg) have been postulated to play a role in promoting in vivo pathogenic autoantibody production and since IVIG preparations are rich in anti-SAg antibodies, we tested whether IVIG could inhibit in vitro SAg-driven human T cell-dependent B cell differentiation. We demonstrate that IVIG inhibits such B cell differentiation by at least three different mechanisms. Early addition of IVIG inhibits B cell differentiation not only in SAg-stimulated PBMC cultures but in anti-CD3- and pokeweed mitogen (PWM)-stimulated cultures as well, pointing to a SAg-nonspecific inhibitory effect. However, anti-SAg antibodies contained in IVIG can also effect SAg-specific inhibition, since polyclonal rabbit anti-SAg antisera added early to peripheral blood mononuclear cell (PBMC) cultures inhibit neither anti-CD3- nor PWM-driven B cell differentiation and inhibit B cell differentiation triggered only by the specific SAg against which the individual antiserum was raised. Finally, late addition of IVIG at a time at which B cells have already committed to terminal differentiation inhibits SAg-driven, but not anti-CD3- or PWM-driven, generation of Ig-secreting cells (IgSC). This late inhibition is associated with enhanced SAg-dependent cytolytic activity against Raji cell targets which is dramatic in PBMC cultures but is often not detectable in T + B cell cultures. Reconstitution of T + B cell cultures with natural killer cells restores the enhancing capacity of IVIG on SAg-dependent cytolytic activity as well as the late inhibitory effects of IVIG on IgSC generation. Understanding the multiple mechanisms through which IVIG can inhibit SAg-driven B cell differentiation may offer a rational basis for determining which patients are likely to favorably respond to IVIG administration. PMID- 8620619 TI - Existence and failure of T-cell homeostasis prior to AIDS onset in HIV-infected injection drug users. AB - Prior studies, based on populations of homosexual men, have shown that during HIV infection, levels of total circulating T-cells (CD3+ lymphocytes) remain constant for long periods of time after seroconversion. This suggested homeostatic phenomenon was observed to break down about 18 months prior to AIDS diagnosis with a quick loss of T-cells. The objective of this study was to determine whether (a) total T-cells are maintained at a constant level for long periods of time among HIV-infected injection drug users (IDUs) and (b) total T-cells decline before AIDS onset in this risk group and, if so, by how long. The design and setting was prospective follow-up, with semiannual clinic visits, of 646 HIV infected IDUs who participate in the ALIVE study (Baltimore, MD). Among AIDS cases, T-cell levels remained quite stable at about 1500 cells/microl up to approximately 24 months prior to AIDS. However, a steep decline in CD3+ cell levels began approximately 24 months prior to AIDS diagnosis and was -17.5% per 6 months in the last 18 months before AIDS. Among seropositive IDUs without AIDS, a gradual decline of less than -4% per 6 months was observed. These trends remained virtually unchanged after accounting for current injection drug use, smoking, and HIV-related medications. IDUs, like homosexual men, exhibited T-cell homeostasis following HIV infection, as well as failure of this homeostasis about 2 years before AIDS. Although the mechanisms for the maintenance and later failure of the homeostasis of T-lymphocytes are not well understood, the observation has a potentially important prognostic value as well as biological interest. PMID- 8620621 TI - Tenfold increased incidence of spontaneous multiple myeloma in long-term immunosuppressed aging C57BL/KaLwRij mice. AB - Persons undergoing maintenance immunosuppressive treatment (MIST) were shown to be at increased risk for the development of early malignancies, often of cells of the immune system. Very little is known about the late effects of MIST. Some clinical studies indicated an age-related increase in the incidence of plasma cell disorders, in particular in that of multiple myeloma (MM). In the present study the influence of MIST on the development of monoclonal B-cell proliferative disorders, monoclonal gammopathies (MG), was studied in an animal model, the C57BL/KaLwRij mouse. This strain is known for its susceptibility to develop with aging MG similar to those in humans. Two widely used treatment protocols (azathioprine/prednisolone and Cyclosporin A/prednisolone) were tested in young and adult mice. Both regiments were shown to increase 10-fold the incidence of spontaneous multiple myeloma. Unexpectedly, the same high incidence of MM and in addition the development of a life-shortening lymphoblastic lymphoma were found in a high frequency in the control group that received Cremophor EL only, i.e., the solvent of Cyclosporin A. Repeated experiments with another lot of Cremophor showed a 6-fold increased frequency of NM but no lymphoblastic lymphoma. With respect to the life-span and the incidence of hemopoietic neoplasms the least harmful drugs for MIST appeared to be azathioprine/prednisolone. The results of the experiments in this C57BL/KaLwRij mouse model give a warning for increased incidence of MM in susceptible aging individuals and address a question whether Cremophor EL is a safe solvent for Cyclosporin A. PMID- 8620620 TI - The mechanism of autoantibody formation to cartilage in rheumatoid arthritis: possible cross-reaction of antibodies to dietary collagens with autologous type II collagen. AB - In order to study the mechanism of autoantibody formation to type II collagen in rheumatoid arthritis (RA), IgG and IgA antibodies in sera from 259 RA patients and 285 non-RA controls were evaluated for their specificity as to collagen type (I and II) and species (chick, bovine, and porcine) using an improved enzyme linked immunosorbent assay. IgG and IgA anti-type II collagen antibodies were commonly found in both RA (IgG, 41%; and IgA, 45%) and non-RA (IgG, 36%; and IgA, 31%) sera. Both IgG and IgA collagen antibodies were highly reactive with one or more heterologous type II or type I collagen; however, approximately 35% of IgG and 50% of IgA antibody-positive sera from both RA patients and non-RA controls cross-reacted with human type II collagen (HII) to some degree. However, no antibodies specific to HII were observed in either RA or control sera. In individual patient sera, IgG and IgA antibodies had identical collagen-type and species specificities. Importantly, IgG anti-HII antibodies purified from RA sera by affinity chromatography reacted equally with human, chick and bovine type II collagens, suggesting reactivity with conserved epitopes shared by all three species. In contrast, purified IgG anti-HII antibodies from non-RA control sera commonly lacked reactivity with one or the other of the heterologous type II collagens, suggesting reactivity limited to epitopes shared by HII and only one of the heterologous type II collagens. These data suggest that dietary collagens could elicit circulating IgG and IgA anti-collagen antibodies that cross-react with autologous type II collagen. Also the epitope specificity of IgG autoantibodies may be relevant to the pathogenesis of RA. PMID- 8620622 TI - Changes within T cell receptor V beta subsets in infants following measles vaccination. AB - Measles produces immune suppression which contributes to an increased susceptibility to other infections. Recently, high titered measles vaccines have been linked to increased long-term mortality among some female recipients. Because the mechanisms by which wild-type or attenuated live-vaccine strains of measles virus alter subsequent immune responses are not fully understood, this prompted an examination of the changes within the peripheral blood T cell receptor V beta repertoire following measles immunization. Twenty-four 6- and 9 month-old infants were studied at 2 weeks and 3 months following immunization by semiquantitative reverse transcription-polymerase chain reaction. There was a significant increase in V beta 2 expression (P less than 0.05), and a decrease in the V beta 4 subset (P less than 0.03) 2 weeks following vaccination with subsequent return to baselines at 3 months in vaccine recipients who seroconverted. These data suggest that measles virus may affect immune responses in part by altering the T cell receptor repertoire. PMID- 8620623 TI - Intrathymic injection of polynucleosomes delays autoantibody production in BXSB mice. AB - T-cell dependent autoimmunization with nucleosomes appears to be an early event in the induction of lupus anti-chromatin antibodies. We investigated this phenomenon by injecting H1-stripped chromatin polynucleosomes into the thymuses of BXSB male lupus-prone mice. In comparison to uninjected controls, the production of IgG antichromatin, anti-native DNA, and anti-denatured DNA were significantly reduced among the injected animals for a period of 8 to 10 weeks. Peripheral T-cells from intrathymic (i.t.)-treated animals showed decreased proliferative responses to polynucleosomes compared to those from uninjected controls. Treatment did not affect T-cell antigen receptor V beta profiles, excluding the possibility that results were due to superantigen-imposed deletions. In situ staining using the TUNEL method demonstrated that generation and phagocytosis of apoptotic material in thymuses of unmanipulated BXSB mice were similar to normal controls. These findings show that polynucleosomes likely comprise the antigens for helper T-cell engagement and induction of lupus associated anti-chromatin antibodies. Bypassing the underlying defect of T-cell tolerance for polynucleosomal antigens among BXSB mice, by i.t. administration of exogenous polynucleosomes, results in abrogation of autoantibody production. PMID- 8620624 TI - Sera of patients with rheumatoid arthritis contain antibodies to recombinant human T-lymphotrophic virus type I/II envelope glycoprotein p21. AB - A possible retroviral etiology for rheumatoid arthritis (RA) has been raised by results of recent studies. Therefore, we examined sera of patients with RA, including those with coexisting Felty's syndrome or leukemia of large granular lymphocytes, for the presence of antibodies to retroviral proteins of human T lymphotrophic virus type I and type II (HTLV-I/II). Reactivity to recombinant HTLV-I envelope protein rgp21 alone was the primary pattern observed. Twenty-five percent of RA sera, 28% of Felty's syndrome sera, and 30% of large granular lymphocyte leukemia/RA sera reacted with rgp21, each significantly more than the 8% of normal sera (P less than 0.01). Removing rheumatoid factor did not abolish reactivity with rgp21 in any of six RA sera tested. Immunoreactivity to the authentic viral protein was confirmed by using purified rgp21 that was cleaved by CNBr to remove the bacterial fusion peptide, or by blocking sera with a synthetic peptide corresponding to the fusion peptide. Only one serum, from a patient with RA, showed definite evidence for prior infection with prototypic HTLV-II. These data indicate that 25% of RA sera have IgG antibodies to recombinant HTLV-I envelope protein rgp21, which is highly homologous to envelope protein gp21 of HTLV-II. These findings provide potentially novel clues regarding the pathogenesis of RA. PMID- 8620625 TI - Similarity of expression of activation markers and CD28 on gamma delta and alpha beta-receptor T cells in HIV infection. AB - During human immunodeficiency virus (HIV) infection, phenotypic analysis of circulating gamma delta+ T cells showed a downregulation of the CD28 surface antigen, as recently demonstrated for CD4+ and CD8+ alpha beta+ T cells. The downregulation of the CD28 molecule predominated on CD8+ gamma delta+ T cells. Moreover, an increased expression of CD38 and/or HLA-DR molecules was found on gamma delta T cells as reported for alpha beta T cells indicating that all categories of circulating T lymphocytes share similar phenotypic abnormalities in HIV-infected patients. These unique changes in the different T-cell subsets might be induced by sustained activation of the immune system or by the rapid turnover of T cells and argue for a global dysregulation of T lymphocytes during HIV infection. PMID- 8620626 TI - Increase in lymphocyte subsets following treatment of HIV-associated neutropenia with granulocyte colony-stimulating factor. AB - Recombinant human granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in patients with cancer and HIV disease. Since lymphocyte counts have been reported to increase with G-CSF therapy, we studied the effect of G-CSF on lymphocyte subsets in HIV-infected patients. Six patients with HIV associated neutropenia were treated with G-CSF and had significant increases in white blood cell counts. G-CSF induced a significant rise in total lymphocytes, total T-cells, CD8 T-cells, and natural killer cells. A smaller but statistically significant increase in CD4 T-cells and cytotoxic CD8 T-cells was also noted. We conclude that G-CSF has the ability to raise lymphocyte subset levels in patients with HIV disease. The potential immunologic benefit of G-CSF therapy merits further investigation. PMID- 8620627 TI - Inhibition of spontaneous immunoglobulin production by ganglioside GM2 in human B cells. AB - The effects of gangliosides on spontaneous immunoglobulin (Ig) production in human B cells were studied. Of the various gangliosides tested, including GM1, GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b, only GM2 inhibited Ig production, but not thymidine uptake, in human B cell lines. Moreover, the GM2-induced inhibition was blocked by anti-GM2 mAb, but not by control IgM. Of various cytokines, IL-10 and TNF-alpha each partially counteracted the GM2-induced inhibition, and addition of both IL-10 and TNF-alpha completely counteracted the inhibition. On the other hand, anti-IL-10 mAb plus anti-TNF-alpha mAb inhibited spontaneous Ig production. GM2 inhibited endogenous production of IL-10 and TNF-alpha without affecting the binding of IL-10 and TNF-alpha in B cell lines. GM2 also specifically inhibited spontaneous production of Ig, IL-10, and TNF-alpha in in vivo activated B cells obtained from normal donors. This inhibition was blocked by anti-GM2 mAb and was counteracted specifically by IL-10 plus TNF-alpha. Collectively, GM2 may inhibit spontaneous Ig production by inhibiting endogenous production of IL-10 and TNF-alpha in B cells. PMID- 8620628 TI - A "nouvelle vague" of animal models: transgenic and knockout mice. PMID- 8620629 TI - Animal models of human disease. Transgenic and knockout models of autoimmunity: Building a better disease? AB - The development of transgenic and knockout technologies has driven an explosion in new animal models of disease. These engineered diseases are a departure from previous animal models in that pathological syndromes are created from a priori assumptions about how disease pathogenesis could develop. Such models have been useful in providing new information on the functions of effector molecules such as cytokines, and in following the behavior of lymphocytes in vivo. However, the contrived nature of the systems might generate false information, unless validated by careful reference to human disease and spontaneous disease in other animal models. PMID- 8620631 TI - The immune response: the afferent arm. AB - The key to understanding afferent immunity is the mechanism of activation of T lymphocytes by specialized antigen presenting cells, which bind antigenic peptide to Class II major histocompatibility molecules, and stimulate T cells via Signal 1 (antigen) and Signal 2 (costimulation). The best studied costimulatory pathway is the interaction of B7-1 or B7-2 ligand molecules on antigen presenting cells with CD28 or CTLA-4 receptors on T cells. T cell signaling occurs through the T cell receptor-CD3 complex and is augmented by cosignaling via CD4, CD8, and CD45. The activation of T cells to alloantigen occurs by either a direct pathway of recognition of allogenic major histocompatibility molecules (with or without an associated endogenous peptide), or by an indirect pathway of recognition of processed donor alloantigens via recipient antigen presenting cells. Afferent immunity on the musculoskeletal system is of special interest because of the absence of viable donor antigen presenting cells in processed grafts that makes them susceptible to the indirect pathway of alloantigen recognition. PMID- 8620630 TI - Immunologic responses in human recipients of osseous and osteochondral allografts. AB - A multiinstitutional study was carried out to evaluate immunologic responses for human recipients of massive frozen (-80 degrees C) osseous and osteochondral allografts. Allografts were used to reconstruct skeletal defects associated with a variety of traumatic degenerative and neoplastic disorders. Serum samples were obtained before surgery and from 1 month to 4 years after surgery. Sera were tested by microcytotoxicity against T cells from 60 donors for human leukocyte antigen Class I antibodies and against beta 2-microglobulin treated B cells from 40 donors for human leukocyte antigen Class II antibodies. Panels were selected to represent the majority of known human leukocyte antigen specificities. Of the 84 cases evaluated, 62 (74%) received blood transfusions and 28 of 44 (64%) female recipients had been previously pregnant. Sensitization before transplant was shown in 33 of 84 (39%) patients. After grafting, 49 of 84 (58%) recipients showed evidence of sensitization to Class I antigens and 46 of 84 (55%) recipients showed evidence to sensitization to Class II antigens. Overall sensitization was 67%. PMID- 8620632 TI - Human leukocyte antigen matching, radiographic score, and histologic findings in massive frozen bone allografts. AB - Forty-six patients receiving frozen bone allografts, preoperatively tissue typed for human leukocyte antigen and ABO antigens, were radiographically evaluated according to the Musculoskeletal Tumor Society scoring system at a mean followup of 55 months. Patients who matched for 1 or 2 Class I human leukocyte antigens with the donor scored higher than patients totally mismatched, but differences were not significant. Matching for Class II human leukocyte antigen and ABO antigens seemed not to influence radiographic outcome of allografts. In sixteen patients histologic specimens were obtained. Five of 16 patients who showed histologic parameters of an immune response scored significantly lower than those who did not. Processed frozen bone allografts, because of their lack of viable donor cells, most likely trigger an indirect pathway of alloantigen recognition in the recipient. This type of recognition may generate in the recipient either a chronic type of rejection or an immunologic state of tolerance to grafted antigens that cannot be measured with human leukocyte antigen blood tests. This may explain difficulties in correlating human leukocyte antigen mismatches between the donor and recipient with frozen bone allograft performances. PMID- 8620633 TI - Effects of cyclosporin A on bone turnover and on resorption of demineralized bone matrix. AB - The effects of the immunosuppressive drug Cyclosporin A on orthotopic bone turnover and on the resorption of demineralized bone matrix were studied. In the first experiment, rats were labeled with 3H-proline and 45Calcium and treated with Cyclosporin A 2 mg/kg/day or placebo for 2 weeks. Cyclosporin A treatment resulted in an early transient increase in matrix and mineral turnover in the metaphyseal region of the tibia. Furthermore, Cyclosporin A increased mineral turnover in the diaphyseal part of the tibia, but the matrix turnover was unaffected. No osteopenia was seen after 2 weeks. In the second experiment, prelabeled (3H-proline) demineralized bone matrix from rats and rabbits was implanted in the abdominal walls of growing rats that were treated with Cyclosporin A or placebo. After 4 weeks there was no difference in the remaining activity in the implants from Cyclosporin A or placebo treated rats. Cyclosporin A treatment increased mineral content in demineralized allogenic bone matrix implants by 1/3. In the demineralized xenogenic bone matrix implants, mineral content was 4 times higher in the cyclosporin A treated implants. PMID- 8620634 TI - Carpal tunnel release. Correlations with preoperative symptomatology. AB - Fifty patients (54 hands) who underwent carpal tunnel release for carpal tunnel syndrome were evaluated to determine the relationship between the prominence of specific clinical symptoms and the early results of carpal tunnel release. Patients were evaluated preoperatively, 3 weeks after surgery, and 3 months after surgery by questionnaire, physical examination, and Semmes-Weinstein monofilament pressure testing. The symptoms evaluated included hand/wrist/forearm pain, night pain/paresthesias, intermittent paresthesias, hand clumsiness, hand weakness, constant numbness, and difficulty with work related tasks. All symptoms showed significant improvements at 3 months after surgery. Overall symptom reduction at 3 months after surgery was 49% +/- 73%. Overall satisfaction at 3 months after surgery was 7.8 +/- 2.8 (0 to 10 scale). the severity of preoperative subjective hand weakness was significantly associated with surgery and with less improvement of function at 3 months after surgery and with less satisfaction with overall symptom relief at 3 months after surgery. Although subjective outcomes in this study were markedly improved after carpal tunnel release regardless of preoperative symptomatology, patients with more preoperative night symptoms and intermittent paresthesias and less preoperative hand/wrist pain, numbness, weakness, clumsiness, and difficulty with work related tasks were the most satisfied with their surgery. PMID- 8620635 TI - Spondylolisthesis with sciatica. Magnetic resonance findings and chemonucleolysis. AB - Fifteen patients with bilateral lumbar isthmic spondylolisthesis and unilateral sciatica were examined with magnetic resonance imaging. All patients had a disc protrusion at the level of the spondylolisthesis. Nine patients had a central herniated disc that caused dural sac deformation; in 6 of these patients there was extension to the disc tissue into the foramen. In 5 patients there was no clear dural sac deformation, but there was a foraminal disc protrusion that caused nerve root compression. In 1 patient there was a hernia lateral to the foramen. In none of the patients was there evidence of compression by bony elements. No abnormalities on adjacent levels were found. All 15 patients were treated with chemonucleolysis. There were no complications. A followup study was done after 19 months (range, 10-42 months). The result was rated as good or excellent in 10 of 12 patients with a spondylolisthesis of L5 and in 1 of 3 patients with a spondylolisthesis of L4. Magnetic resonance imaging showed a decrease in dural sac deformation in 4 patients, no clear decrease in 3, and a slight increase in 2. There were no distinct foraminal changes in 9 of 11 patients. PMID- 8620636 TI - Preoperative nutritional status and outcome of elective total hip replacement. AB - Preoperative malnutrition increases the morbidity rate and length of hospitalization for various types of surgical patients. However, among patients who undergo elective total hip replacement, it is unclear how preoperative nutritional data can be used to detect a high risk subgroup. The purpose of this study was to identify preoperative nutritional factors that could be used to define a subgroup of patients who have undergone elective total hip replacement who are at high risk for poor post-operative outcome. Preoperative nutritional factors were evaluated in 89 consecutive patients who underwent elective total hip replacement. An inverse relationship was found between serum albumin and length of stay. Patients with an albumin level less than 3.9 were twice as likely to require prolonged hospitalization ( > 15 days) when compared with those in whom the albumin level was 3.9 or greater. Complications were related to the preoperative orthopaedic diagnosis of avascular necrosis of the hip. A subgroup of the patients undergoing elective total hip replacement who are at risk for prolonged recovery can be identified preoperatively by using a serum albumin level of less than 3.9 g/dL. The traditional normal range for albumin may be inappropriate for these patients. PMID- 8620637 TI - Long term bone remodeling around the Charnley femoral prostheses. AB - Femoral bone remodeling after total hip replacement was studied by following patients who received 326 Charnley femoral prostheses for 10 to 20 years (mean, 13.3 years). The radiographic state of the bone remodeling was visually assessed and measured with a digitizer. Demineralization that started proximally and then progressed distally caused cortical thinning, which correlated with widening of the intramedullary canal, not with changes that developed in the periosteal width, and occurred in the medial femoral neck, around the proximal half of the stem, and around the distal half in 87%, 33%, and 10%, respectively. Cortical thinning around the distal half of the stem was always accompanied by proximal thinning, and extensive cortical thinning (both proximal and distal) correlated with both lower clinical scores and radiologic loosening of the femoral prosthesis. A low canal flare index of Noble, a large canal width, and a patient age of 60 years or more were risk factors for extensive cortical thinning. Accelerated polyethylene wear was related to resorption of the medial femoral neck but not to cortical thinning or radiological loosening. Cortical thickening occurred only around the distal half of the stem in 29%. These findings establish a basis for the performance of cemented femoral prostheses, and allow comparison of bone remodeling when evaluating other femoral prostheses. PMID- 8620638 TI - The effect of preoperative exercise on total knee replacement outcomes. AB - This study compared the effects of preoperative physical therapy of general cardiovascular conditioning exercises with the routine procedure of no preoperative physical therapy on patients undergoing primary total knee replacement. Thirty patients were randomly assigned to 1 of 3 groups. Group 1 was the control group. Group 2 participated in a physical therapy program designed to strengthen the upper and lower limbs and improve knee range of motion. Group 3 participated in a cardiovascular conditioning program, consisting of arm ergometry, cycle ergometry, aquatic exercises, and aerobic activity. All patients were evaluated preoperatively and postoperatively using the Hospital for Special Surgery Knee Rating, the Arthritis Impact Measurement Scale, and the Quality of Well Being instrument. Both experimental groups tolerated their respective exercise protocols extremely well. All 3 groups showed significant improvement postoperatively as measured by the Hospital for Special Surgery Knee Rating, the Arthritis Impact Measurement Scale and the Quality of Well Being measurement scales. However, neither type of preoperative exercise added to the degree of improvement after surgery at any of the postoperative evaluations. PMID- 8620639 TI - Component design affecting patellofemoral complications after total knee arthroplasty. AB - Three hundred one primary cemented total knee arthroplasties were performed in 289 patients. Two different prostheses were used; 148 knees received the Miller Galante I prosthesis and 153 knees received the Press Fit Condylar prosthesis. Minimum followup was 2 years. The groups were similar in all parameters both preoperatively and postoperatively, with the exception of the patellofemoral complication rate. Knees that were implanted with the Miller-Galante I prosthesis experienced a complication rate of 10.1%, while those with the Press Fit Condylar prosthesis experienced a complication rate of 0.7%. The distinct difference in the patellofemoral complication rate may be due to the differences in design of the femoral component. Features that may have contributed to increased patellofemoral morbidity included a short, narrow anterior flange; a shallow patellar groove; and an abrupt anterior to distal transition with a smaller radius of curvature. Because subtle design difference can have a profound effect on clinical outcome, long term evaluation of new designs should be completed before widespread use. The clinician also should be aware of the desirable design features when choosing a component. PMID- 8620640 TI - Nonoperative treatment of plantar interdigital neuroma with a single corticosteroid injection. AB - Patients who received a single corticosteroid injection for treatment of third webspace plantar interdigital neuroma were studied retrospectively. Forty-three patients (51 feet) were available for followup study (followup mean, 4 years; range, 2 to 6 years). Mean age of patients was 53 years. Pain initially was relieved in 36 patients (41 feet [80%]). Twenty-four feet (47%) ultimately required surgical excision, while most of the remaining 27 feet (53%), which had not been treated surgically, were the source of residual symptoms in patients. A single corticosteroid injection cannot be recommended as a cure for symptoms of third webspace neuroma, but it can be offered as a temporizing measure or as nonoperative treatment. A single corticosteroid injection does not preclude a successful surgical result. PMID- 8620641 TI - Surgery versus functional treatment in ankle ligament tears. A prospective study. AB - A prospective study was performed to compare surgical treatment (primary repair plus early controlled mobilization) to functional treatment (early controlled mobilization alone) in severe (Grade III) lateral ligament injuries of the ankle. Thirty surgically treated patients were compared with 30 age, height, weight, gender, and sporting activity matched, functionally treated similar patients. In both treatment groups, all but 1 patient had a stable ankle at 9 months. Compared with the functional group, the range of motion of the ankle joint was restricted in the surgical treatment group at 6 weeks but did not normalize during the followup. The functional group showed no restrictions. A specific scoring scale developed for subjective and functional followup evaluation of in injured ankle also was used as an outcome criterion. Nine months after the injury, excellent or good scores were achieved in 87% of the functionally treated patients and 60% of the surgically treated patients, respectively. The results of this study indicated that early mobilization gives better results than surgery plus mobilization in the treatment of the complete tears of the lateral ligaments of the ankle. PMID- 8620642 TI - Humeral shaft nonunion treated by a Seidel interlocking nail with a supplementary staple. AB - Thirty-two adult patients with humeral shaft nonunions were treated with Seidel interlocking nails, and 6 of these 32 patients required an added staple. The indication for inserting a staple was rotational instability, despite use of a distal spreading screw. All 6 patients with nonunions were followed for at least 1 year (median, 1.5 years), and all experienced a solid union. The union period was a median of 5 months, with a range of 3 to 7 months. No complications were noted. The author believes that all humeral shaft nonunions may be treated by a Seidel interlocking nail with or without a staple supplementation and cancellous bone graft. The technique is simple, and its results are satisfactory. Predrilling the cortices of both fragments is key to successful insertion of a staple. PMID- 8620643 TI - Open tibial fractures treated with the Ex-fi-re external fixation system. AB - Fifty open tibial fractures were treated with the Ex-fi-re external fixation system from 1987 to 1994. According to the Gustilo and Anderson classification, there were 12 Grade I, 14 Grade II, 10 Grade IIIA, 13 Grade IIIB, and 1 Grade IIIC injuries. Eight fractures were segmental. The average patient patient age was 39 years (range, 16-85 years). With the reduction unit of the system, displaced tibial fractures could be reduced by the functions of the device in contrast to the manual reductions needed with other unilateral devices. Compression could be applied even to oblique fractures. The reductions were performed by this unit in all cases and were classified as exact in 28 of 41 cases. Exact reduction was defined as a reduction in which there was no more than 2 mm of translational displacement. The 8 segmental fractures were not classified according to reduction. Forty-three fractures healed with no secondary procedure. Three secondary bone graftings, 4 fibulotomies, and 3 renamed intramedullary fixations were performed in 6 patients. Thirty-two skin grafts were performed. There was 1 fracture site infection, and 1 curettage and 1 sequestrectomy were performed after union. At 1 year followup there were no signs of infection. Median time to union was 20 weeks, and median time to full unprotected weightbearing was 22 weeks. Fractures with an exact reduction had a median time of union of 19 weeks, compared with a median of 31 weeks in reductions with greater than 2 mm translational displacement. The exact reduction and translational compression applied in oblique fractures appeared to contribute to early consolidation. PMID- 8620645 TI - Percutaneous pin fixation of chronic slipped capital femoral epiphysis. AB - Eighteen consecutive patients with 24 chronic-slipped capital femoral epiphyses were treated with percutaneous Knowles pin fixation. The average followup was 41 months (range, 21 to 64 months). All patients experienced complete closure of the growth plate within 12 months. There was no evidence of avascular necrosis, chondrolysis, slip progression, pin penetration, hardware failure, or intertrochanteric femoral fracture. Treatment of chronic slipped percutaneous pin is a safe and effective procedure. PMID- 8620644 TI - Treatment of a bone defect of the forearm by bone transport. A case report. AB - The technique of bone transport has been used in the lower extremities to treat acute and chronic bone defect. It has not been applied to the upper extremities. An 8.0-cm defect of the ulna was treated with this technique, using a unilateral bone transport system. At completion of the transport, bone graft was brought to the docking site. A 4.0-cm radius defect was treated with free fibula graft and intramedullary nailing. Treatment was completed with removal of the external fixator at 10 months. Complication was limited to transient superficial pin site infection. PMID- 8620646 TI - Arthrographic evaluation of developmental dysplasia of the hip. Outcome prediction. AB - Arthrograms of 35 hips in 33 children less than 2 years of age with typical development dysplasia of the hip were reviewed. After arthrograms were repeated for 11 hips 6 weeks following the initial test, results were classified into 6 types based on medial pooling ratio and morphology of the acetabular limbus. Using modified Severin's criteria for outcome evaluation, 7 of the 11 hips had been upgraded in type. All hips that were classified as Type I by arthrogram had Severin I results. The relation of arthrographic type and radiographic results was statistically significant. Immediate open reduction is recommended in hips classified as Type VI at first arthrogram or Type III and above at repeat arthrogram. PMID- 8620647 TI - Prosthetic reconstruction for periacetabular malignant tumors. AB - This article reports the reconstruction method and functional results of a newly designed tumor hip prosthesis with a constrained joint mechanism which was used after treatment for 13 primary and 5 metastatic periacetabular malignant bone tumors. The overall 5-year survival rate for patients with primary tumors (n=13) was 50%. The local recurrence rate was 30%. More than 90% of the patients whose hips were reconstructed with the constrained tumor hip prosthesis experienced pain relief and were able to walk with a cane. No patients had greater than 3 cm shortening of the involved limb. Infection was the most serious complication. This prosthesis restored iliofemoral stability after surgical resection of periacetabular malignant tumors. PMID- 8620648 TI - Use of preoperative autologous blood donations and erythropoietin for treatment of giant cell tumor of the ischium. AB - A 24-year-old man with an osteolytic lesion of the ischium was referred to the authors' institution. Computed tomography and magnetic resonance imaging studies showed that the lesion extended to and involved the subchondral bone of the acetabulum. Histologic examination of the biopsy specimen revealed giant cell tumor of bone. Following the biopsy, autologous blood was collected 4 times with recombinant human erythropoietin treatment definitive surgery was performed. Three weeks after the biopsy, the lesion was curetted and bone cementation was performed. The total blood loss during surgery was 3100 ml, which was replaced successfully with stored autologous blood without the need for homologous blood transfusion. The authors believe that without the erythropoietin treatment, autologous blood could have been collected only 3 times instead of 4 times, and the patient would have needed homologous blood. PMID- 8620650 TI - Glomus tumor of the musculotendinous junction of the rotator cuff. A case report. AB - A rare case of glomus tumor of the rotator cuff is presented. A 35-year-old man had a 20-year history of left shoulder pain. Through physical examination, muscular atrophy in the supraspinatus and infraspinatus muscles and restriction of shoulder motion was seen. Magnetic resonance imaging showed an abnormal oval shadow in the supraspinatus musculotendinous junction, and postcontrast computed tomographic scanning showed a small calcification focus in the tumor. Surgical excision of the tumor and rotator cuff reconstruction using Debeyre's procedure were done. Microscopic examination of the tumor showed a typical glomus tumor. It was difficult to diagnose glomus tumor in this patient before surgical intervention. Retrospective consideration indicated that the history of the present illness, computed tomographic scanning, and magnetic resonance imaging were critical for differential diagnosis. PMID- 8620649 TI - The immune response: the efferent arm. AB - Once naive T cells encounter antigen, they become primed effector cells. The scope of effector functions mediated by these cells defines the efferent arm of the immune response. The change from naive to primed effector cell is known as adaptive immunity and takes 2 forms: cell mediated, in which T cells mediate effector function, and humoral, in which antibodies are the effector molecules. There are 3 types of effector T cells: inflammatory CD4 T cells, which activate macrophages; helper CD4 T cells, which help B lymphocytes produce antibody; and cytotoxic CD8 T cells, which kill their target cells. The interaction of primed effector cells with their targets results in phenotypic changes in the cells and the secretion of cytokines. These cytokines may be secreted by the primed effector T cell, the target cell, or both. Cytokines function in either autocrine (secreted and used by the same cell) or paracrine (secreted by 1 cell and used by a different cell) circuits and have marked regulatory effects on cells in both the immune and skeletal systems. Many of these cytokines, which were once thought to be products exclusively of immune cells, are now known to be produced by cells of the skeletal system. Both the specific and nonspecific components of the immune response have profound effects on remodeling of the musculoskeletal system during normal and pathologic states. PMID- 8620651 TI - Multifocal Ewing's sarcoma and hypercalcemia. A case report. AB - An unusual case of Ewing's sarcoma in which the earliest sign of disease was the development of hypercalcemia is described. Radiologic examinations showed extensive osteolysis involving all of the trunk, and the proximal femora and humeri. An ill defined destruction of the left fibula was also noted. No definite primary site was found. The lungs and other visceral organs were free of metastasis. To the best of the author's knowledge, no comparable case has been reported in the literature. PMID- 8620652 TI - Development of in vitro biocompatibility assays for surgical material. AB - This work reports the development of a test to evaluate the biologic effects of implant material used in orthopaedics and traumatology based on the examination of inflammation and allergic reactions at the cellular level. The variation in arachidonic acid metabolite production by murine peritoneal macrophage cultures was studied using different powders of implant material. Macrophage activation by zymosan served as a control. Mouse peritoneal macrophages were labeled with 14C arachidonic acid, and the synthesis of cyclooxygenase products (6-keto prostaglandin F1 alpha; prostaglandins F2 alpha, E2, D2; and thromboxane B2) and lipoxygenase products (hydroxyeicosatetraenoic acids) was analyzed and quantified by chromatography. Results obtained through these assays support the reported clinical data that chrome and nickel increase the production of hydroxyeicosatetraenoic acids by mouse peritoneal macrophages. HXPATRI, titan oxide, and monoclinic zircon also increase the production of hydroxyeicosatetraenoic acids in contrast to other powders tested (alumina, HXPBL, chrome cobalt alloy, stainless steel 316L, titan, quadratic zircon), which have little effect on the production of arachidonic acid metabolites by the lipoxygenase pathway. It is concluded that determination of arachidonic acid metabolite production by murine peritoneal macrophage cultures is appropriate for evaluating implant material. PMID- 8620653 TI - Rabbit articular cartilage defects treated with autologous cultured chondrocytes. AB - Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery. PMID- 8620654 TI - Scapular pain and swelling in a 60-year-old man with Paget's disease. PMID- 8620655 TI - Retroviruses and bone diseases. AB - In 1980, retroviruses were shown to be pathogenic to humans, and experimentation on animals involving retroviruses as causal agents of tumors and degenerative diseases of bone, brain, and lung gained interest. Osteopetrosis, which can be either inherited in rodents or retrovirally induced in cats, is exemplary. Because of replication cycle, retroviruses can be propagated not only as infectious agents but also as cellular genes. If a retroviral infection occurs in germ line cells, the viral genes, which must integrate in the host's DNA, can be passed on to the progeny and inherited as Mendelian characteristics. Therefore, a retroviral etiology could account for diseases that present either as sporadic (infectious) or familial (inherited), although they may be similar in their clinical manifestations. This approach led to the finding of 2 new human retroviruses: 1 in a patient who had sporadic benign osteopetrosis, and the other in a patient who had sporadic paraarticular osteoma. In both patients, the retrovirus was isolated from mononuclear blood cells, not from bone cells, because of the links between bone and the immune system. A systematic search for retroviruses in patients who have sporadic bone disease, which also may appear as inherited disease, has yet to be performed. Patients with sporadic disease could be managed by antiretroviral agents such as Zidovudin. PMID- 8620656 TI - Human leukocyte antigen system and the immune response to it. AB - Immunologic rejection of tissue grafts follows recognition of donor alloantigens; either those resulting from ABO incompatibility of those encoded by the human major histocompatibility complex, HLA. Alloantigens encoded by HLA are present on membrane proteins that are expressed constitutively by tissues or whose expression can be induced by cytokines released during inflammation. Genes of the HLA complex are highly polymorphic resulting in variations in amino acid sequence that shape the peptide binding pocket of HLA molecules and define the complementary structure that interacts with the T lymphocyte receptor for antigen. Variants of HLA proteins expressed by the allografts that are not expressed by the recipient can stimulate the immune response to the allograft resulting in rejection both by humoral antibody and through attack by T lymphocytes. Class II HLA antigens on donor cells can stimulate these responses directly by contact with recipient T cells. However, rejection also may result when HLA antigens are released from the graft, processed to peptides, and presented to recipient T cells by cells expressing recipient HLA Class II molecules. Rejection can be avoided by preventing activation of T lymphocytes, by minimizing differences in HLA proteins between recipient and donor or by avoiding preexisting responses to donor HLA antigens. PMID- 8620658 TI - A biological analysis of individuality. PMID- 8620657 TI - Immunologic mechanisms in common rheumatologic diseases. AB - Rheumatoid arthritis and seronegative spondyloarthropathies are rheumatologic diseases that likely are caused by inflammatory reactions occurring in genetically predisposed individuals mounting an immune response to the antigen. Understanding the immunopathology of these diseases provides insight into their etiology, pathogenesis, and a rationale for therapies targeting immune component interactions. Although the antigen in rheumatoid arthritis is not known, several bacterial antigens have been associated with seronegative spondyloarthropathies. These antigens result in an interaction between the human leukocyte antigen-B27 restricted CD8 positive T lymphocytes and the antigen presenting cell, producing an inflammatory response. Rheumatoid factors are autoantibodies directed against the fragment crystallizable portion of the immunoglobulin G. Rheumatoid factor immunoglobulin G immune complexes contribute to the inflammatory events in the rheumatoid joint, and may play an important role in antigen presentation. A novel antigen capture enzyme linked immunosorbent assay was developed that mimicked B cell surface expressed rheumatoid factor. Conversely, a direct binding enzyme linked immunosorbent assay mimicked secreted rheumatoid factor. Comparison of rheumatoid binding enzyme linked immunosorbent assays showed that the physical state of rheumatoid factor can affect binding characteristics. The state of glycosylation of immunoglobulin G may contribute to its antigenic structure. These physical characteristics may be important in rheumatoid factor's pathogenic role in rheumatoid arthritis. PMID- 8620659 TI - Lymphocyte in vitro response to human giant cell tumors. AB - Peripheral blood lymphocytes and tumor cells were obtained from 31 patients with giant cell tumors of bone and cocultured in vitro in a mixed lymphocyte tumor cell assay. The lymphocyte proliferative response was measured by incorporation of 3H thymidine. Also, the patients' lymphocytes were tested for proliferative reactivity to phytohemagglutinin and allogenic lymphocytes to evaluate nontumor immunologic competence. Mixed lymphocyte tumor cell assays showed higher lymphocyte stimulation in patients with Stage I as compared with Stages II and III giant cell tumors. The proliferative response was blocked partially when the patients' sera was used to supplement the cultures. Lymphocytes from patients with a recurring tumor showed lower responses, but the differences with primary tumors were not significant. This evidence suggests that there is an immune response to giant cell tumor antigens and that this response might be related to the aggressiveness of the tumor. PMID- 8620661 TI - Immune response to synthetic materials. Sensitization of patients receiving orthopaedic implants. AB - Metallic orthopaedic devices are composed of elements that are known to be skin sensitizers in the general population. There is concern about the possibility of sensitivity reactions in patients bearing these implants. Blood samples were drawn from 22 patients having primary total joint replacement and who had no known prior metal allergies or exposure. Repeat blood samples were drawn 3 months to 1 year later. All preoperative blood samples showed no immune reactions against titanium, cobalt, chromium, or nickel ion solutions in a leukocyte migration inhibition test. Thirty two percent (7 of 22) of the patients developed sensitivity to at least 1 of the antigens, but only 5 percent (1 of 22) developed a severe reaction. Review of the literature and these studies has indicated that such reactions can occur. However, the incidence seems to be very low. PMID- 8620660 TI - The immune response to implant materials in humans. AB - The etiology of aseptic loosening of prosthetic joint replacement components is unclear. Implant materials have been considered biologically inert, but recently studies indicate that inflammatory reactions directed against the implanted materials may contribute to aseptic loosening. Data suggesting a progression from a simple inflammatory reaction to complex immune responses against the biomaterials are reviewed. The cellular responses to particles of polymethylmethacrylate, ultrahigh molecular weight polyethylene, and alloys of cobalt-chromium and titanium were assayed in vitro to determine cell proliferation in patients with underlying diagnoses of osteoarthrosis, rheumatoid arthritis, and avascular necrosis who had joint replacement. Control populations were provided by patients with similar diagnoses who were preoperative surgical candidates. The underlying diagnoses did not seem to influence responses to particle stimulation. Elevated responses to both acrylic and cobalt-chromium were observed in patients with aseptically loosened prostheses. These findings suggest that the development of a cellular response to particulate debris may be significant in the pathogenesis of aseptic loosening. PMID- 8620662 TI - Immune response to nonspecific and altered tissue antigens in soft tissue allografts. AB - Soft tissue allografts have many uses in orthopaedic surgery, including knee ligament reconstruction, hand tendon surgery, shoulder instability, and rotator cuff reconstruction. The predictable biologic incorporation of soft tissue allografts without rejection or fear of disease transmission continues to be a goal of basic science researchers. A review of the current knowledge if the immune system response to donor specific, nonspecific, and altered tissue antigens in soft tissue or tendon allografts is presented. An in vitro study was done in an attempt to decrease immunogenicity of a frozen bone-ligament graft by adding irrigation with Betadine scrub solution and hydrogen peroxide to the conventional storage process of freezing. Although the irrigation with cytotoxic agents would undoubtedly further decrease immunogenicity, it also decreased stiffness and maximum load by 15%. Whether this decreased strength and stiffness would compromise the incorporation and long term success of soft tissue allografts would need to be studied by in vitro experiments. PMID- 8620663 TI - The humoral response to vascular and nonvascular allografts of bone. AB - The cytotoxic donor specific antibody response after vascularized and nonvascularized bone allograft implantation was assessed in rats and dogs. Nonvascularized segmental femoral grafts were studied in rats; nonvascularized fresh and cryopreserved massive osteochondral allografts were studied in dogs; and vascularized and nonvascularized fibular allografts were studied in dogs. The major histocompatibility complex antigens of all animals were defined. All grafts were stabilized by internal fixation and the antibody response was measured in a 51chromium release microcytotoxicity assay using donor lymphocytes as target cells. In all cases, donor specific antibody responses were elicited by major histocompatibility complex mismatched grafts. The response was directed primarily at Class I specificities although there was likely and antiClass II response as well. Among fully mismatched grafts, antidonor antibody was detectable earlier in animals receiving vascularized grafts (1 week after surgery) than in animals receiving nonvascularized grafts (3 weeks after surgery). Massive grafts elicited a sustained response whereas relatively smaller grafts, such as the fibula did not. The antidonor antigen antibody response was transient and less frequent in animals receiving frozen grafts. The clinical implications of these data are unclear. Although some improvement of clinical outcome has been observed with grafts matched for major histocompatibility complex antigens, the potential benefits of tissue antigen matching or modulation of the host immune response remain unresolved. PMID- 8620664 TI - Immune response inhibition by irrigating subchondral bone with cytotoxic agents. AB - Attempts have been made (in the recent past) to inhibit the immune response to fresh osteoarticular (shell) allografts because the occurrence and the magnitude of this response is considerably greater and more harmful than that seen after frozen bone and soft tissue allografts. To decrease in immunogenicity of these fresh grafts, the subchondral bone of rat distal femur allografts was irrigated with Betadine scrub solution (n = 10) or Triton-X (n = 11) before transplantation (Study 1). The Triton-X significantly reduced the immunogenicity of the grafts, but the Betadine scrub solution had no effect. A similar experiment with Triton-X was done in sheep where trochlear knee autografts (n = 3) were compared with unirrigated allografts (n = 3) and allografts receiving irrigation with Triton-X (n = 3) (Study 2). All 3 Triton-X irrigated allografts had no immune response, and showed much improved grafts compared with the control allografts (where an immune response developed in 2 of 3). Neither of the 2 allograft groups were as good as the autografts. These techniques may prove useful for inhibiting the recipient immune responses to fresh osteoarticular allografts in humans requiring partial joint reconstruction. PMID- 8620665 TI - Determination of a new 5-lipoxygenase inhibitor, zileuton, and its inactive N dehydroxylated metabolite in plasma by high performance liquid chromatography. AB - A rapid and sensitive assay was developed for the measurement of plasma concentrations of zileuton racemate, a potent inhibitor of 5-lipoxygenase. Zileuton and its inactive N-dehydroxylated metabolite were extracted from human, monkey, and rat plasma by use of a solid-phase extraction column (Analytichem Bond Elut). The compounds were then separated by reverse-phase high performance liquid chromatography (HPLC) on a Supelcosil LC-18 column and quantified on the basis of ultraviolet absorption at 260nm relative to an internal standard. The extraction recovery of zileuton, as determined by HPLC assay, was 77.9 +/- 1.7%. Recovery of the metabolite was 85.8 +/- 0.7%. Calibration curves for both compounds were linear over the zileuton concentration range 0.01 to 10.0 mg/L (correlation coefficients > 0.987), while the intra- and interassay coefficients of variation were < 15.6%. In practice, > 97% of blinded daily spiked control samples for zileuton and > 90% of those for the metabolite were within 10% of their target concentrations. PMID- 8620666 TI - Pharmacokinetic interactions between zileuton and prednisone. AB - A randomized double-blind placebo-controlled crossover study evaluated the effects of zileuton 600mg 4 time daily on the pharmacokinetics of prednisolone after a single 400mg oral dose of prednisone. the effects of the single prednisone dose on the steady-state pharmacokinetics of zileuton were also evaluated. Multiple doses of zileuton had no significant effects on mean peak plasma concentration (Cmax), time to Cmax(tmax), or area under the plasma concentration-time curve from 0 to infinity (AUC0-infinity) values for prednisolone after oral administration of prednisone 40mg. A slight but statistically significant increase in the mean half-life (t1/2) of prednisolone was detected with zileuton + prednisone administration compared with prednisone + placebo (from 2.8 to 2.9 hours); however, this change was of no clinical relevance. Mean Cmax values of zileuton after coadministration with prednisone were similar to those of zileuton alone. While the single 40mg dose of prednisone resulted in a slight but statistically significant decrease in the mean zileuton AUC value from 0 to 6 hours (AUC0-6) [from 23 to 20 mg/L/h] and a reduction in tMAX (from 2.3 to 1.7 hours), these results were not considered to be clinically significant. Therefore, it is considered that zileuton and prednisone may be coadministered with minimal risk of a clinically significant pharmacokinetic interaction. PMID- 8620667 TI - The pharmacokinetic and pharmacodynamic interactions between the 5-lipoxygenase inhibitor zileuton and the cyclo-oxygenase inhibitor naproxen in human volunteers. AB - The potential pharmacokinetic and pharmacodynamic interactions between zileuton, a 5-lipoxygenase inhibitor, and naproxen, a nonsteroidal anti-inflammatory drug that acts as a cyclo-oxygenase inhibitor, have been investigated in 24 healthy volunteers. Coadministration of these 2 drugs had no effect upon the plasma concentration-time curves of either zileuton (800mg) or naproxen (500mg) when compared with each drug administered alone. Both naproxen plasma concentrations during the elimination phase and area under the plasma concentration-time curve values were statistically significantly raised upon coadministration with zileuton, when compared with naproxen alone. However, these differences in these 2 values were sufficiently small to be of no clinical significance. There is no evidence that the combination of zileuton and naproxen had an effect on leukotriene B4 levels that was different from the inhibitory effect of zileuton alone, or had an effect on serum thromboxane B2 levels that was different from the effect of naproxen alone. Moreover, inhibition of the 5-lipoxygenase pathway by zileuton did not appear to aggravate the gastrointestinal adverse events commonly associated with naproxen administration. It is concluded that zileuton and naproxen may be coadministered with minimal risk of a clinically significant interaction. PMID- 8620668 TI - Pharmacokinetics and pharmacodynamics of zileuton after oral administration of single and multiple dose regimens of zileuton 600mg in healthy volunteers. AB - The pharmacokinetics and pharmacodynamics of zileuton were determined after oral administration of single dose (600mg) and multiple dose regimens [600mg every 8 hours (q8h regimen) and 600mg every 6 hours (q6h regimen)] in 12 healthy male subjects aged 18 to 50 years. Steady-state park plasma concentration (Cmax), time to Cmax, apparent total plasma clearance, and apparent terminal phase volume of distribution values after the q8h and q6h regimens were 3.07 +/- 1.13 and 4.37 +/ 1.02 mg/L, 1.5 +/- 0.9 and 1.5 +/- 0.9 hours, 793 +/- 233 and 579 +/- 162 ml/min (47.6 and 34.7 L/h), and 179 +/- 126 and 115 +/- 29L, respectively (mean +/- SD). Trough zileuton plasma concentrations (Cmin) immediately before the morning dose were higher than Cmin immediately before the afternoon dose, suggesting a diurnal variation in the pharmacokinetics of zileuton. Accumulation of zileuton occurred with more frequent dose administration, although there was no unexpected accumulation of the parent drug or the N-dehydroxyzileuton metabolite. The q6h regimen of zileuton 600mg was superior to the q8h regimen in maintaining trough plasma concentrations of zileuton above 1.5 mg/L, corresponding to approximately 70 to 80% inhibition of leukotriene B4 biosynthesis. PMID- 8620669 TI - In vitro plasma protein binding of zileuton and its N-dehydroxylated metabolite. AB - An ultrafiltration technique or equilibrium dialysis has been used to study the in vitro human plasma protein binding of racemic zileuton, its individual enantiomers, and its pharmacologically inactive metabolite N-dehydroxyzileuton. The plasma protein binding of zileuton and N-dehydroxyzileuton over the concentration range of 0.1 to 100 mg/L averaged 93.1 +/- 0.22 and 92.0 +/- 0.12%, respectively. However, there appeared to be a stereoselective effect, with the R(+) enantiomer of zileuton demonstrating greater binding to plasma proteins than the S(-) enantiomer (96 vs 88%, respectively). Zileuton was bound to both human serum albumin (40 g/L) and alpha 1-acid glycoprotein (1 g/L), although binding affinity to albumin was approximately 3-fold greater. Displacement interactions of zileuton with warfarin, salicylate, theophylline, naproxen, ibuprofen, prednisone, and terfenadine were minimal. The blood to plasma concentration ratio for zileuton and N-dehydroxyzileuton ranged from 0.65 to 0.68, indicating that these compounds were mainly distributed in the plasma. Thus, zileuton is approximately 93% bound to plasma proteins at expected therapeutic concentrations in vitro, and this figure is largely unaffected by several commonly prescribed agents with which the drug may be coadministered. PMID- 8620670 TI - The pharmacokinetics of zileuton in healthy young and elderly volunteers. AB - The effects of age and gender on the single and multiple dose pharmacokinetics of zileuton have been examined in a phase I nonblinded study. A total of 27 healthy volunteers were evaluable, 9 in the young group (age range 20 to 40 years; 5 males and 4 females) and 18 in the elderly group (range 65 to 81 years; 9 males and 9 females). A single oral dose of zileuton 600mg was given to all volunteers on day 1 of the study and at 6-hour intervals from days 3 to 7. Analysis of variance showed slight but significant decreases in the mean apparent clearance of total and free drug in the healthy elderly population after a single zileuton dose, but no significant age-related differences after multiple 6-hourly doses. Similarly, zileuton peak and trough plasma concentrations, and values for half life, volume of distribution and protein binding were not significantly affected by age after either a single dose or multiple administration. Moreover, gender effects on the pharmacokinetics were also absent after correction for bodyweight differences. From the results of the present study, it is concluded that there is no pharmacokinetic basis for alteration of zileuton dosage schedules in elderly patients. PMID- 8620671 TI - The effect of mild or moderate hepatic impairment (cirrhosis) on the pharmacokinetics of zileuton. AB - The pharmacokinetics of zileuton and its R(+) and S(-) glucuronide metabolites were determined after single and multiple (400mg every 8 hours) oral dose administration in healthy subjects (n = 5) and patients with mild or moderate hepatic impairment (cirrhosis; n = 8). The clearance of total zileuton (unbound plus bound to plasma proteins) in patients with hepatic impairment (approximately 350 ml/min) was approximately half than in healthy subjects (approximately 670 ml/min), with similar values in patients with mild or moderate cirrhosis. However, the clearance of unbound zileuton in patients with moderate hepatic impairment was nearly half that in patients with mild hepatic impairment, and one quarter that in healthy subjects. On the basis of these findings, it may be necessary to reduce the dose in patients with impaired hepatic function to maintain levels similar to those in healthy subjects. PMID- 8620672 TI - The effect of food on the pharmacokinetics of zileuton. AB - The present study was undertaken to assess the effect of food on the pharmacokinetic parameters of zileuton. In a nonblinded crossover study, 18 healthy male volunteers who had fasted overnight were randomised to receive a single oral dose of zileuton 600mg in the presence or absence of food consisting of a standardised breakfast on the following morning. The mean zileuton peak plasma concentration (Cmax) increased significantly by 27% after food intake, while the mean area under the plasma concentration versus time curve increased by only 1.4%, a difference that was not statistically significant. The mean time to Cmax was unaffected by the presence of food, as were the other pharmacokinetic parameters assessed. Overall, the results suggest that food has a relatively small effect on the rate of zileuton absorption compared with the fasting state, while the bioavailability of the drug appears to be unaffected. Thus, it is concluded that it is appropriate to administer zileuton with or without food. PMID- 8620673 TI - Pharmacodynamic and stereoselective pharmacokinetic interactions between zileuton and warfarin in humans. AB - A double-blind parallel randomised study was conducted to assess the effects of multiple oral doses of zileuton (600mg every 6 hours) or matching placebo on the steady-state pharmacokinetics and pharmacodynamics of warfarin titrated to a prothrombin time of 14 to 18 seconds in 24 healthy adult male volunteers. Serial blood samples were collected for assessment of prothrombin times and R- and S warfarin plasma concentrations. Coadministration of zileuton and warfarin had no effect on S-warfarin pharmacokinetics but statistically significantly increased mean R-warfarin plasma concentrations and decreased mean R-warfarin total oral plasma clearance compared with warfarin alone (by 15%). This stereoselective interaction was accompanied by an increase in mean morning (predose) and evening (12-hour postdose) prothrombin times from 17.5 to 19.8 seconds and 17.1 to 19.1 seconds, respectively; the corresponding changes in the placebo group were from 18.1 to 18.8 seconds and 17.3 to 17.5 seconds. Thus, multiple dose administration of zileuton appears to significantly alter steady-state R-warfarin pharmacokinetics and pharmacodynamics. Careful monitoring of prothrombin times with appropriate dose titration of warfarin is recommended with concurrent therapy of zileuton and warfarin. PMID- 8620674 TI - Effect of zileuton on theophylline pharmacokinetics. AB - In controlled trials involving asthma patients, zileuton - a selective 5 lipoxygenase inhibitor - has significantly improved pulmonary function and reduced symptoms. Since theophylline is frequently prescribed for asthma, we designed a placebo-controlled randomised crossover trial to examine the influence of zileuton on theophylline pharmacokinetics. 16 healthy adult males were given theophylline (Slo-Phyllin) 200mg 4 times daily for 5 days and either zileuton 800mg twice daily or a matching placebo. After a 15-day washout period, theophylline was resumed and the other study drugs reversed. During coadministration with zileuton, mean peak theophylline levels rose from 12.14 to 20.99 mg/L (p < 0.001), while the apparent plasma clearance dropped from 3.74 to 1.91 L/h (p < 0.001). The time to the peak theophylline concentration was delayed by 0.5 hours and the half-life was significantly prolonged by 1.5 hours. 14 volunteers reported 44 mild or moderately severe adverse events, possibly or probably related to coadministration of zileuton, and 8 volunteers reported 8 such events with placebo coadministration. Three volunteers receiving theophylline plus zileuton withdrew from the trial prematurely. Thus, a pharmacokinetic interaction that may produce theophylline toxicity exists between zileuton and theophylline. Accordingly, theophylline dosages in patients receiving zileuton should be adjusted to maintain levels within the therapeutic range. Upon initiation of zileuton, the typical asthma patient may require dosage reductions of one-half, and monitoring of plasma theophylline concentrations is recommended. PMID- 8620675 TI - Lack of pharmacokinetic interaction between zileuton and phenytoin in humans. AB - A randomised double-blind crossover study was undertaken in 20 healthy adult male volunteers to assess the effects of multiple oral dose administration of zileuton (600mg every 6 hours for 8 days) on the single dose pharmacokinetics of phenytoin 300mg. Serial blood samples were collected up to 72 hours after phenytoin administration and plasma concentrations were determined by high performance liquid chromatography. Pharmacokinetic data were analysed utilising noncompartmental and Michaelis-Menten-based population pharmacokinetic analysis. Zileuton did not significantly alter the peak plasma concentration, time to peak plasma concentration, and area under the plasma concentration-time curve of phenytoin. Moreover, population analysis revealed no significant effect of zileuton on the Michaelis-Menten parameters of phenytoin. Thus, coadministration of multiple doses of zileuton (2.4 g/day) did not significantly affect the single dose pharmacokinetics of phenytoin. PMID- 8620677 TI - Assessment of the pharmacokinetic interaction between zileuton and digoxin in humans. AB - The effects of coadministration of zileuton on the pharmacokinetic profile of digoxin were investigated in a double-blind placebo-controlled crossover study in 12 healthy male volunteers. During each study phase, the subjects received zileuton 600mg every 6 hours (regimen A) or placebo (regimen B) for 13 days. In addition, all subjects received concomitant digoxin 0.25 mg/day on study days 1 to 11 during both study phases. The study results provide no evidence of any significant overall effect of zileuton on digoxin plasma concentration-time profiles. Although the mean time to reach the maximum plasma concentration for digoxin was significantly shorter after concomitant administration of digoxin and zileuton than after concomitant administration of digoxin and placebo (0.95 vs 1.43 hours), there were no significant differences between the 2 regimens in the values for maximum plasma concentration, area under the plasma concentration-time curve from 0 to 24 hours, elimination half-life, oral clearance, and apparent volume of distribution associated with the terminal phase. Therefore, it is concluded that digoxin and zileuton may be coadministered without risk of clinically relevant effects on the pharmacokinetic profile of digoxin. PMID- 8620676 TI - The pharmacokinetics of single oral doses of zileuton 200 to 800mg, its enantiomers, and its metabolites, in normal healthy volunteers. AB - The pharmacokinetics of single oral doses of zileuton 200 to 800mg, its R(+) and S(-) enantiomers, and its N-dehydroxylated and glucuronide metabolites have been investigated in a randomised study in 16 normal male healthy volunteers. Zileuton was 93.4% bound to plasma proteins. The overall dispositional pharmacokinetics of zileuton racemate appeared to be linear. The mean dose-normalised area under the concentration-time curve from zero to infinity (AUC0-infinity) remained constant, while the mean dose-normalised peak plasma concentration (Cmax) decreased with the increase in dose, possibly because of dissolution rate-limited absorption at the higher doses. The R(+) and S(-) enantiomers of zileuton may have similar absorption profiles, although the apparent total plasma clearance of the S(-) enantiomer was 49 to 76% higher than the corresponding values for the R(+) enantiomer. The AUC0-infinity of each enantiomer increased proportionately with dose. The pharmacokinetics of the N-dehydroxylated metabolite of zileuton were highly variable, with a more than dose-proportional increase in the mean dose normalised Cmax and area under the concentration-time curve from zero to 24 hours. The elimination of the glucuronide metabolites of the R(+) and S(-) enantiomers of zileuton was formation rate-limited. The mean percentage of the administered zileuton dose recovered in urine as glucuronide metabolites ranged from 73.1 to 76.5% and showed no dose-related differences. The renal clearance of the glucuronide metabolites of zileuton exceeded the normal glomerular filtration rate, suggesting that these metabolites may be excreted through renal tubular secretion in addition to filtration. PMID- 8620679 TI - The role of routine patch tests in allergic contact dermatitis due to metal working fluids. PMID- 8620678 TI - The influence of multiple oral doses of zileuton on the steady-state pharmacokinetics of sulfasalazine and its metabolites, sulfapyridine and N acetylsulfapyridine. AB - The effects of zileuton (Abbott-64077) on the pharmacokinetics of sulfasalazine (SASP) and its metabolites, sulfapyridine (SP) and N-acetylsulfapyridine (ASP), were studied in a randomised double-blind placebo-controlled study enrolling 14 healthy male volunteers. All subjects received SASP 1 g every 12 hours for 8 days and zileuton 800mg or placebo administered twice daily from day 4 to day 8 inclusive. Coadministration of zileuton did not significantly affect the area under the plasma concentration-time curve, the maximum (Cmax) or minimum (Cmin) plasma concentration and the time to Cmax of SASP, SP or ASP. Likewise, zileuton did not modify the terminal elimination half-life of SASP. It is concluded that coadministration of zileuton 1.6 g/day has no significant effects on the pharmacokinetics of SASP 2 g/day or its metabolites, SP and ASP. PMID- 8620680 TI - Cutaneous and mucocutaneous leishmaniasis. PMID- 8620681 TI - Treating severe inflammatory acne: the last word. PMID- 8620682 TI - Cutaneous amyloidosis in patients with progressive systemic sclerosis. AB - It is rarely reported that amyloidosis occurs as a complication of progressive systematic sclerosis (PSS), in comparison with rheumatoid arthritis (RA). We observed pigmentation on the upper back of six of sixty-six patients with PSS (9 percent), and recognized amyloid deposits in the skin on histochemical and electron microscopic examinations. These amyloid deposits were localized cutaneous type, according to results of morphologic and immunologic studies. Patients' levels of serum amyloid A were normal or moderately elevated. Two of the six patients had Barnett type I disease and four had Barnett type II; none had severe visceral involvement with PSS. PMID- 8620683 TI - Rhinophymalike metastatic carcinoma. AB - Cutaneous metastases from internal carcinoma vary in their clinical appearance. An unusual presentation, initially resembling rhinophyma, is described in a 71 year-old man with lung cancer. The clinical variations of metastases to skin are reviewed. PMID- 8620684 TI - Condyloma latum of the toe webs: an unusual manifestation of secondary syphilis. A report of two cases. AB - The incidence of primary and secondary syphilis in the United States peaked in 1990, exceeding the number of reported cases since 1948. Uncommon manifestations of secondary syphilis seen more frequently in the preantibiotic era may be less well recognized today. One such manifestation is the condyloma latum, or split papule of the toe web. Two patients with condyloma of the toe web are presented and the previous literature is reviewed. Condyloma latum is a common manifestation of secondary syphilis; however, condyloma latum of the toe webs is unusual. Failure to recognize toe web condyloma latum may delay diagnosis and treatment. PMID- 8620685 TI - A unique case of long-term survival in a male patient with malignant melanoma of the distal urethra. AB - Primary malignant melanoma of the urethra in male patients is a rare entity, noted to have a dismal prognosis in recent reviews. No case of long-term disease free survival in patients with primary malignant melanoma has been previously reported. We report the first such case, and review briefly the factors that could have contributed to our patient's long-term survival. We also illustrate the precursor lesion present six years prior to diagnosis. PMID- 8620687 TI - The ethograms of Tetrahymena pyriformis GL and T. malaccensis. AB - The behaviour of Tetrahymena, one of the most intensively investigated laboratory organisms, was analysed, and ethograms of T. pyriformis and T. malaccensis were studied. The basic ethogram, which previously described the behaviour of ciliates was appropriate for T. pyriformis and T. malaccensis. Although quite similar to each other, the two new ethograms differed significantly in some aspects of ciliate behaviour. Four new behavioural patterns were recognized and described quantitatively and qualitatively, viz induced continuous trajectory change, induced continuous reorientation reaction, sliding and coiling. The complexity of these results is discussed per se and also from a comparative point of view. PMID- 8620686 TI - Use of a hydrocolloid dressing in combination with a topical steroid in plaque psoriasis. AB - The treatment of chronic stable plaque psoriasis continues to present a challenge to the practitioner. The use of topical steroids to treat lesions is often associated with resistance and rebound upon discontinuation of prolonged use. Use of hydrocolloid dressings enhances the efficacy of topical steroids and reduces the amount of medication required to control plaques. We report results obtained with a hydrocolloid dressing used for a seven-day period with triamcinolone acetonide 0.1 percent. In a group of six patients, an average of 44 percent improvement was achieved, while untreated sites remained stationary. Patient satisfaction with results and rating of convenience of the treatments was high. PMID- 8620688 TI - Comparative analysis of the rates of chromosome damage induced by bleomycin radiomimetic in human trisomic and diploid lymphocytes: in vitro cultures from a mosaic of a Down's syndrome individual. AB - Lymphocytes with different chromosome numbers (46,XX and 47,XX + 21) in cultures, were obtained from the blood of a mosaic of a Down's syndrome patient. Their distinctive susceptibility to chromosome damage induced by bleomycin radiomimetic was tested and compared with lymphocytes from healthy individuals. The test showed that the presence of an extra chromosome 21 occurred in parallel with the rise of an intrinsic basal rate of chromosome damage in trisomic cells. PMID- 8620690 TI - If you see no changes, you haven't lived long enough. PMID- 8620689 TI - Sulphonation of cytosine residues in fixed chromosomes. PMID- 8620691 TI - Asthma in athletes. Exercise-induced bronchoconstriction in figure skaters. PMID- 8620692 TI - Are advance directives becoming an endangered species? PMID- 8620693 TI - Pulmonary rehabilitation. Does the site matter? PMID- 8620694 TI - Sleep apnea and pulmonary hypertension. PMID- 8620695 TI - State-of-the-art tuberculosis prevention. PMID- 8620696 TI - Tidal volume, PEEP, and barotrauma. An open and shut case? PMID- 8620697 TI - Intraoperative multiplane vs biplane transesophageal echocardiography for the assessment of cardiac surgery. AB - This study was undertaken to test whether multiplane transesophageal echocardiography (TEE) offers advantages in comparison with biplane TEE in the intraoperative monitoring during cardiac surgery. A diagnostic multiplane TEE was performed in 400 patients in the immediate preoperative and postoperative periods. We systematically acquired cardiac images from the gastric fundus, lower esophagus, and upper esophagus; complete views of the descending aorta were also recorded. Usefulness of the different views in providing essential additional clinical information compared with exclusive transverse (0 to 20 degrees) and longitudinal (70 to 110 degrees) planes of the biplane TTE was assessed assuming that with manipulation of a biplane probe, a 20 degrees are could be added to the conventional horizontal and vertical planes. A high success rate of each view was demonstrated; anatomy and pathologic condition were best visualized in oblique planes. The method proved to be particularly useful in the preoperative and postoperative phases of aortic dissection (27 cases), aortic (65 cases) and mitral (35 cases) valve replacement, mitral valve repair (38 cases), left ventricular aneurysmectomy (25 cases), bleeding from proximal suture of an aortic heterograft (2 cases), and positioning of left ventricular hemopump (2 cases). Additional regional wall motion abnormalities of the right (four cases) and left ventricle (six cases) not appreciated in 0 to 20 degrees or 70 to 110 degrees planes were detected. Multiplane TEE is a useful clinical tool during intraoperative monitoring of cardiac surgery. Most structures of the heart and great vessels lie on oblique planes, while other views are optimized with the aid of slight angle corrections. This method improves the evaluation of anatomy and pathologic condition of the heart and great vessels, of native and prosthetic valves, and of left and right ventricular function. PMID- 8620698 TI - Exercise-induced asthma in figure skaters. AB - Many highly trained athletes experience exercise-induced bronchospasm (EIB): studies describing EIB in figure skaters, who may be at increased risk of EIB due to rink temperatures (7 to 10 degrees C), have not been published. We studied professionally coached figure skaters (n = 124) for EIB by spirometry at rinkside immediately before a simulated long program and at 0 to 1, 5, 10, and 15 min postexercise. Postexercise spirometry revealed the presence of EIB (a decrease from baseline in FEV1 of at least 10%) in 43 skaters, while the remainder (n = 81, control group) remained relatively stable. Pre-exercise FEV1, FVC, and FEV1/FVC ratio were not different between groups. The EIB group had significantly lower FEV1 vs baseline at each measurement following exercise: baseline, 3.08 +/- 0.13; 0 to 1 min postexercise, 2.81 +/- 0.13 (p < 0.05); 5 min postexercise, 2.77 +/- 0.14 (p < 0.05); 10 min postexercise, 2.78 +/- 0.13 (p < 0.05); 15 min postexercise, 2.78 +/- 0.13 (p < 0.05). The EIB group also had lower FVC: baseline, 3.48 +/- 0.16; 0 to 1 min postexercise, 3.16 +/- 0.15 (p < 0.05); 5 min postexercise, 3.19 +/- 0.15 (p < 0.05); 10 min postexercise, 3.27 +/- 0.16 (p < 0.05); 15 min postexercise, 3.26 +/- 0.16 (p < 0.05). Control subjects, however, experienced no decline in these variables. In conclusion, the incidence of EIB in the figure skaters measured during this investigation (43 of 124 = 35%) is greater than that of the population at large and other highly trained athletes, signifying that screening for EIB and therapeutic follow-up are reasonable considerations for participants in this sport. PMID- 8620699 TI - Prevalence of gastroesophageal reflux symptoms in asthma. AB - STUDY OBJECTIVE: To determine the prevalences of symptomatic gastroesophageal reflux (GER), reflux-associated respiratory symptoms (RARS), and reflux associated beta-agonist inhaler use in asthmatics. DESIGN: Questionnaire-based, cross-sectional analytic survey. SETTING: Outpatient asthma and clinical research clinics attached to the University of Calgary tertiary care centre and two family practices. PATIENTS: Asthma group consisted of 109 patients referred to an outpatient asthma clinic. First control group consisted of 68 patients visiting their family physicians. Second control group consisted of 67 patients with thyroid disease, hypercholesterolemia, or diabetes participating in drug trials. RESULTS: Among the asthmatics, 77%, 55%, and 24% experienced heartburn, regurgitation, and swallowing difficulties, respectively. Symptoms were less prevalent in the control groups. At least one antireflux medication was required by 37% of asthmatics (p < 0.001, vs controls). None of the asthma medications were associated with an increased likelihood of symptomatic GER. In the week prior to completing the questionnaire, 41% of the asthmatics noted RARS, including cough, dyspnea, and wheeze and 28% used their inhalers while experiencing GER symptoms. Inhaler use correlated with the severity of heartburn (r = 0.28, p < 0.05) and regurgitation (r = 0.40, p < 0.05) CONCLUSIONS: The questionnaire demonstrated a greater prevalence of GER symptoms, RARS, and reflux associated inhaler use in asthmatics. This excessive inhaler use may explain how GER indirectly causes asthma to worsen. PMID- 8620700 TI - High-intensity physical training in adults with asthma. A 10-week rehabilitation program. AB - Twenty-six adults (23 to 58 years) with mild to moderate asthma underwent a 10 week supervised rehabilitation program, with emphasis on physical training. In the first 2 weeks, they exercised daily in an indoor swimming pool (33 degrees C) and received education about asthma, medication, and principles of physical training. In the following 8 weeks, they exercised in the pool twice a week. Every training session lasted 45 min. The training sessions were made as suitable as possible for the individual subjects, in order to minimize "drop outs" from the program. The aim of the study was to evaluate the efficacy of the rehabilitation program and to determine if inactive asthmatic adults can exercise at high intensity. The rehabilitation program was preceded by a 6-min submaximal cycle ergometry test, a 12-min walking test, spirometry, and a methacholine provocation test. The subjects also responded to a five-item questionnaire related to anxiety about exercise, breathlessness, and asthma symptoms using a visual analogue scale. All subjects were able to perform physical training at a very high intensity, to 80 to 90% of their predicted maximal heart rate. No asthmatic attacks occurred in connection with the training sessions. Twenty-two of the 26 subjects completed the rehabilitation program, felt confident with physical training, and planned to continue regular physical training after the 10 week program. Improvements in cardiovascular conditioning, measured as a decreased heart rate at the same load on the cycle ergometer (average of 12 beats/min), and as a longer distance at the 12-min walking test (average of 111 m), were observed during the program. FEV1 increased significantly from 2.2 to 2.5 L. Forced expiratory flow at 25% of vital capacity also increased slightly but significantly. Methacholine provocation dose causing a fall in FEV1 by 20% was unchanged. Seventeen subjects had a peak expiratory flow reduction of more than 15% after the preprogram ergometry test and were classified as having exercise-induced asthma (EIA). Only three of these subjects had EIA after 10 weeks. The asthmatic subjects were less afraid of experiencing breathlessness during exercise and less anxious about exercising at a high intensity after 10 weeks (p < 0.05). The asthma symptoms abated significantly during the rehabilitation program and the subjects needed less acute asthma care after the rehabilitation program. The results indicate that asthmatic persons benefit from a rehabilitation period, including physical training. Rehabilitation programs are therefore of value as a supplement to conventional pharmacologic treatment of asthma. This rehabilitation program can be adapted for use in clinical practice. PMID- 8620701 TI - Exaggerated responses to chlorine inhalation among persons with nonspecific airway hyperreactivity. AB - Although chlorine gas is a common irritant exposure, little is known about airway responses to chlorine inhalation among persons with baseline airway hyperreactivity. We wished to determine whether such persons manifest an exaggerated response to chlorine compared with normal subjects. We studied 10 subjects, five with and five without airway hyperresponsiveness (HR) after exposure to 1.0 ppm chlorine and five persons, all with HR, to 0.4 ppm chlorine. After 1.0 ppm inhalation, there was a significant (p < 0.05) fall (mean +/- SE) in FEV1 immediately following exposure among normal (-180 +/- 37 mL) and HR subjects (-520 +/- 171 mL). The fall was greater among the HR compared with the normal subjects (p = 0.04). Specific airway resistance (Sraw) increased to a greater degree among the HR group compared with normal subjects (p = 0.04). Among all subjects (n = 10), the proportional change in FEV1 after 1.0 ppm chlorine correlated with baseline reactivity (Spearman rank correlation r = 0.64, p < 0.05). At 24-h follow-up, there were no significant chlorine-related pulmonary function deficits. After 0.4 ppm chlorine inhalation, there was no significant pulmonary function effect. These data indicated that persons with hyperreactive airways manifest an exaggerated airway response to chlorine at 1.0 ppm. This suggests that when large numbers of persons are exposed to chlorine, a susceptible subpopulation may acutely respond with a greater decrement in pulmonary function. PMID- 8620702 TI - BAY u3405, a thromboxane A2 antagonist, reduces bronchial hyperresponsiveness in asthmatics. AB - OBJECTIVE: Thromboxane A2 (TXA2) is reported to induce bronchial hyperresponsiveness along with the well-documented bronchoconstrictor action on smooth muscles. We examined the effect of the TXA2 antagonist, BAY u3405, on bronchial hyperresponsiveness to methacholine (MCh) in asthmatics. PATIENTS: Twelve adult asthmatics were studied in a randomized, double-blind, placebo controlled, crossover fashion. DESIGN: Following a 2-week run-in period, the subjects were administered 75 mg of BAY u3405 or placebo orally, twice a day for 2 weeks each in a crossover design, interposing a 2-week washout period. Bronchial hyperresponsiveness was measured by the astograph method. Briefly, the respiratory resistance (Rrs) was measured by the forced oscillation method during continuous inhalation of MCh in stepwise incremental concentrations, until Rrs reached twice the baseline value. Bronchial hyperresponsiveness was evaluated as the minimum cumulative dose (Dmin) of MCh that induced an increase in Rrs. Dmin was calculated so that 1 U of Dmin equals to 1 min of inhalation of aerosol solution at 1.0 mg/mL during quiet breathing. RESULTS: Three subjects were withdrawn from the evaluation because they had asthmatic attacks or wheezing during the study. The Dmin value of 0.533 U (GSEM 1.675) after the BAY u3405 treatment was significantly greater than that of 0.135 U (GSEM 1.969) after the placebo treatment (p = 0.0139). There were no safety concerns in either treatment group. CONCLUSION: We conclude that BAY u3405 may be a useful drug for attenuating bronchial hyperresponsiveness in bronchial asthma. PMID- 8620703 TI - Effects of inhaled fluticasone propionate and oral prednisolone on lymphocyte beta 2-adrenoceptor function in asthmatic patients. AB - The aim of the study was to evaluate the facilitatory effects of inhaled corticosteroid on in vitro parameters of lymphocyte beta 2-adrenoceptor function in asthmatic patients. Serum cortisol level was also evaluated as a measure of systemic bioactivity. Ten (four female) asthmatic subjects were evaluated, mean (SEM) age was 28.6(2.0) years, and FEV1 was 79.9%(8.7) predicted. Single doses of inhaled placebo (PL), fluticasone propionate, 1,000 micrograms (F1000), fluticasone propionate, 2,000 micrograms(F2000), or oral prednisolone, 50 mg(PRED), were given at 10 PM the previous night and measurements were made 10 h later. Values for beta 2-receptor density (logBmax: fmol/10(6)cells) were significantly (p < 0.05) greater than PL with PRED but not with inhaled fluticasone (as means and 95% confidence interval [CI] for difference vs PL): PL, 0.27; F1000, 0.30; F2000, 0.32; and PRED, 0.48 (95% CI vs PL, 0.075 to 0.341). Maximal cyclic adenosine monophosphate (cAMP) responses to isoproterenol hydrochloride (isoprenaline (Emax; pmol/10(6)cells) mirrored those for Bmax: PL, 4.00; F1000, 4.68; F2000, 4.26; and PRED, 7.46 (95% CI vs PL, -0.01 to 6.91). Receptor affinity (Kd) was not significantly altered by any treatment. There was significant (p < 0.05) suppression of serum cortisol (nmol/L) with F2000 and PRED compared with PL: PL, 307.9; F1000, 323.2; F2000, 130.1 (95% CI vs PL, 69.76 to 285.8) and PRED, 51.8 (95% CI vs PL, 144.11 to 368.01). Thus, high-dose inhaled fluticasone propionate did not have any facilitatory effects on lymphocyte beta 2 adrenoceptor parameters as compared with oral prednisolone which upregulated beta 2-receptor density and increased cAMP response. In contrast, high-dose inhaled fluticasone (2,000 micrograms) significantly suppressed serum cortisol. In conclusion, there would appear to be a dissociation in systemic sensitivity between effects of inhaled corticosteroid on adrenal suppression and lymphocyte beta 2-adrenoceptor regulation. PMID- 8620704 TI - Iodinated glycerol has no effect on pulmonary function, symptom score, or sputum properties in patients with stable chronic bronchitis. AB - STUDY OBJECTIVE: It has been reported that therapy with iodinated glycerol (IG) can improve the quality of life for patients with chronic bronchitis. The purpose of this study was to determine the effects of IG therapy on the quality of life, pulmonary function, and on the properties of sputum collected from adults with stable chronic bronchitis. DESIGN: Thirty-two week, double-blind, randomized, placebo-controlled cross-over study. SETTING: A university outpatient pulmonary clinic. PARTICIPANTS: Twenty-six adults with stable chronic bronchitis completed the study; 28 completed the first treatment arm. INTERVENTIONS: Sixteen weeks each of placebo or IG 60 mg qid. MEASUREMENTS: First, pulmonary function by spirometry and plethysmography. Second, symptom score measured using a questionnaire. Third, sputum bulk and surface rheology, spinnability, mucociliary transportability and cough transportability. RESULTS: There were no significant changes in pulmonary function, clinical scores, or sputum properties related to therapy with IG. There was a significant improvement in the Global Petty score after both IG (p = 0.01) and placebo (p < 0.01) when compared with baseline, but there was no difference between treatment periods. There was a positive correlation between changes in the Global score during therapy and changes in sputum spinnability (p < 0.01, r = 0.38). CONCLUSIONS: This study clearly demonstrates that in chronic bronchitis, 16 weeks of therapy with IG does not produce any appreciable effect on pulmonary function, well being, or on sputum viscoelasticity or clearability. PMID- 8620705 TI - Contribution of emphysema and small airways in COPD. AB - BACKGROUND: The contribution and role of emphysema and small airways disease in causing expiratory airflow limitation in COPD is controversial. METHODS: We obtained high-resolution thin-section 2-mm CT scans of the lung for emphysema grading and lung function in 116 consecutively seen COPD outpatients with fixed expiratory airflow limitation. In this group, inflated whole lung(s) were subsequently obtained in 24 patients (23 autopsy, 1 surgery) for morphologic studies and results compared with lung CT. Airway histologic condition was studied in 17 of the 24 patients. RESULTS: There was fair to weak negative correlation between CT emphysema score and either FEV1/FVC percent (r = -0.51, p = 0.001) or FEV1 percent predicted (r = -0.31, p = 0.001). In only 24 of the 81 patients (30%) with FEV1 less than 50% predicted, the CT emphysema score was 60 or more, indicating severe emphysema. In the 24 patients studied, there was a good correlation (r = 0.86, p = 0.001) between CT and pathologic grade of emphysema. While respiratory bronchioles (RBs) and membranous bronchioles (MBs) demonstrated marked morphologic abnormalities, there was a weak correlation with emphysema grade (for RB, r = 0.36, p = 0.16; for MB, r = 0.41, p = 0.10) or with FEV1 percent predicted (for RB, r = -0.21, p = 0.42; for MB, r = -0.28, p = 0.28). There was no correlation between emphysema and FEV1 percent predicted (r = -0.13, p = 0.54). CONCLUSIONS: High-resolution CT lung scans are an in vivo surrogate to quantitate moderate to severe morphologic emphysema. Emphysema does not appear to be primarily responsible for severe expiratory airflow limitation in most patients with severe COPD. There was no correlation between severity of small airway histologic condition and emphysema or FEV1 percent predicted. The causes of the lesions responsible for small airways obstruction need to be identified. PMID- 8620706 TI - Thin-section CT detection of emphysema associated with bronchiectasis and correlation with pulmonary function tests. AB - PURPOSE: To evaluate, on thin-section CT scans, the prevalence of emphysema in patients with bronchiectasis and to correlate the results of thin-section CT scans with the results of pulmonary function tests, in order to question whether there was a particular functional test profile in this group of patients. PATIENTS AND METHODS: This is a retrospective study including 90 patients having both thin-section CT scans and pulmonary function tests for bronchiectasis. A CT scoring system was established for assessing the airway disease by the severity and extent of bronchiectasis and by the extent of emphysema. CT scans were reviewed independently by two reviewers and final interpretation was obtained by consensus. Results of thin-section CT scans were correlated with results of pulmonary function tests, including FEV1 and FEV1/FVC to assess air-way obstruction, total lung capacity and residual volume to assess air trapping, and diffusing capacity for carbon monoxide/alveolar volume (DCO/VA). RESULTS: CT evidence of emphysema, which was noted in 45% of the patients (n = 41), was mainly localized in the same bronchopulmonary segments as bronchiectasis. The presence of emphysema was in relation to the extent and to the severity of bronchiectasis. Only eight patients with CT evidence of emphysema had functional evidence of emphysema (20%). When comparing the group of patients with CT evidence of emphysema with the group of patients with no CT evidence of emphysema, the group of patients with CT evidence of emphysema had significantly higher airflow obstruction and air trapping, had significantly lesser value of diffusing capacity, but with no decreased gas transfer (DCO/VA > 80%). CONCLUSION: Our series suggests that there is a high prevalence of emphysema in patients with bronchiectasis. Emphysema that was not suggested using pulmonary function tests in most of the cases could explain in part the higher airway obstruction observed in the group of patients with CT evidence of emphysema. This study could support the suggestive notion that emphysema, which was mainly localized in bronchiectatic lobes, could be due to the inflammatory airway process. PMID- 8620707 TI - A comparison between an outpatient hospital-based pulmonary rehabilitation program and a home-care pulmonary rehabilitation program in patients with COPD. A follow-up of 18 months. AB - AIM: In this study, the effects of a 12-week hospital-based outpatient pulmonary rehabilitation program (HRP) are compared with those of a 12-week home-care rehabilitation program (HCRP) in COPD patients. A control group received no rehabilitation therapy. METHODS: After randomization and stratification, effects on lung function, exercise performance (4-min walking test and cycle ergometer test), dyspnea, and leg effort during exercise, and well-being were assessed in 45 COPD patients with moderate to severe airflow limitation (mean [SD] FEV1 percent predicted, 42.8 [8.4]). RESULTS: After HRP and HCRP, at 3 to 6 months after the start of the study, equal improvements were detected in exercise capacity and in Borg dyspnea and leg effort scores at similar work levels during the cycle test. However, whereas after HRP at longer term values tended to return to baseline outcome, after HCRP a further ongoing significant improvement in exercise capacity was observed, while Borg dyspnea scores remained significantly improved over 18 months. Improvements in cycle workload and dyspnea score were significantly better maintained after HCRP as compared with HRP. Lung function, arterial oxygen saturation, and heart frequency during exercise did not change. A significant improvement in well-being was maintained over 18 months in both rehabilitation groups. CONCLUSION: Beneficial effects are achieved both after a HRP and a HCRP in COPD patients with moderate to severe airflow limitation. Yet we recommend to initiate HCRPs as improvements are maintained longer and are even further strengthened in this setting. PMID- 8620708 TI - Advance directive education during pulmonary rehabilitation. AB - We conducted a questionnaire survey of 346 pulmonary rehabilitation programs to determine the present utilization and potential value of these sites for promoting advance directive education for patients with chronic lung diseases. Responses were analyzed for all responding programs and for programs categorized by size. Eighty-two percent of the 218 responding programs discussed with patients prognostic information. Only 33% of programs asked patients if they had advance directives and 17% kept these documents on file. Thirty-three percent of programs provided some form of advance directive education, and 42% distributed directive educational material, usually through informal and unstructured methods. Seventy-seven percent of responders considered pulmonary rehabilitation an appropriate site for directive education, and 86% indicated willingness to incorporate directive education into their programs. Larger programs were more likely to present information about patient prognosis (p = 0.0003) and advance directives (p = 0.021). We conclude that most of the responding pulmonary rehabilitation programs do not educate patients about advance directives but are willing to do so if supplied with appropriate teaching materials. Rehabilitation programs may be valuable sites for educating patients with chronic disorders about advance directives and promoting an improved patient-physician dialogue about these issues. PMID- 8620709 TI - Pulmonary hemodynamics in the obstructive sleep apnea syndrome. Results in 220 consecutive patients. AB - We have investigated pulmonary hemodynamics in a large series of consecutive, unselected patients with obstructive sleep apnea syndrome (OSAS). The aims of this study were to evaluate the frequency of pulmonary artery hypertension (PH) in OSAS and to analyze, as far as possible, its mechanisms. Two hundred twenty patients were included on the basis of a polysomnographic diagnosis of OSAS (apnea+hypopnea index > 20). PH, defined by a resting mean pulmonary artery mean pressure (PAP) of at least 20 mm Hg, was observed in 37 of 220 patients (17%). Patients with PH differed from the others with regard to pulmonary volumes (vital capacity [VC], FEV1) and the FEV1/VC ratio that were significantly lower (p < 0.001); PaO2 (64.4 +/- 9.3 vs 74.7 +/- 10.1 mm Hg; p < 0.001); PaCO2 (43.8 +/- 5.4 vs 37.6 +/- 3.9 mm Hg; p < 0.001), apnea+hypopnea index (100 +/- 33 vs 74 +/- 32; p < 0.001), and mean nocturnal arterial oxygen saturation (SaO2) (88 +/- 6% vs 94 +/- 2%; p < 0.001). Patients with PH were also more overweight (p < 0.001). Multiple regression analysis showed that 50% of the variance of PAP could be predicted by an equation including PaCO2 (accounting for 32% of the variance), FEV1 (12%), airway resistance (4%), and mean nocturnal SaO2 (2%). In conclusion, PH is observed, in agreement with previous studies, in less than 20% of OSAS patients. PH is strongly linked to the presence of an obstructive (rather than restrictive) ventilatory pattern, hypoxemia, and hypercapnia, and is generally accounted for by an associated obstructive airways disease. In this regard, the severity of OSAS plays only a minor role. PMID- 8620710 TI - Ventilation and gas exchange during sleep and exercise in severe COPD. AB - Ventilation and gas exchange were studied during sleep and incremental treadmill exercise in 19 patients with severe stable COPD with the primary aim of comparing the pathophysiology of oxygen desaturation in the two conditions. A secondary aim was to determine whether exercise studies could aid in the prediction of sleep desaturation. Full polysomnography was used, and ventilation, arterial oxygen saturation (SaO2), and transcutaneous PCO2 (PtcCO2) were monitored continuously during sleep. No patient had significant sleep apnea. Mean (SD) FEV1 was 32 (9.1)% predicted, PaO2 was 71.2 (12.4) mm Hg, and PaCO2 was 44.5 (4.6) mm Hg. SaO2 fell twice as much during sleep as during maximum exercise: 13.1 (8.9) vs 6.0 (3.6)% (p < 0.001). The mean sleep and exercise SaO2, and minimum sleep and exercise SaO2 were well correlated on linear regression (r = 0.81 and 0.78, respectively, p < 0.001), but on multiple regression analysis, awake PaO2 was a better predictor of sleep desaturation than was exercise desaturation. The 12 major desaturators (minimum sleep SaO2 < 85%) had twice as great a fall in exercise SaO2 as the 7 minor desaturators (3.6 +/- 2.8 vs 7.4 +/- 3.3%, p < 0.05). The major desaturators also had a greater fall in estimated sleep PaO2: 19.8 (5.1) vs 6.4 (7.1) mm Hg (p < 0.01), which suggests that their greater sleep desaturation is not simply due to their position on the steep portion of the oxyhemoglobin dissociation curve. The rise in PtcCO2 during sleep was similar among major and minor desaturators: 7.5 (2.9) vs 5.8 (3.7) mm Hg (p = NS), suggesting that all patients had a similar degree of hypoventilation during sleep, and that the greater fall in SaO2 and estimated PaO2 among some patients was secondary to other factors such as increased ventilation-perfusion mismatching. PMID- 8620711 TI - Accuracy of oximetry for detection of respiratory disturbances in sleep apnea syndrome. AB - STUDY OBJECTIVE: The cost and inconvenience of polysomnography make simplified techniques of screening desirable in the strategy of diagnosis of sleep apnea syndrome (SAS). We have evaluated, in a prospective study of 301 consecutive patients referred for suspected sleep disorders, an index (delta index) that detects apneic events by quantifying arterial oxygen saturation (SaO2) variability. SETTING: Regional sleep laboratory taking referrals from general practitioners and specialists. DESIGN: Classic polysomnography was the gold standard, with 15 apneas plus hypopneas per hour (RDI) being used as a threshold for definition of obstructive sleep apnea (OSA). Oximetry was recorded over the same night. Signal variability was quantified as a function of time, using digital processing of oximetric data. Sensitivity, specificity, and positive and negative predictive values of oximetry testing were calculated. A receiver operating characteristic (ROC) curve was constructed representing the comparative courses of sensitivity and 1-specificity at different thresholds of delta index. RESULTS: Three hundred one patients were included (age, 56 +/- 12 years). Their RDI was 30 +/- 24. For a delta threshold at 0.6, the sensitivity of oximetry for the diagnosis of OSA was 98% and the specificity was 46%. The positive and negative predictive values for diagnosing SAS were 77% and 94%, respectively. The three false-negative cases had a relatively high awake SaO2 (97 vs 93.9 +/- 2.8%), a moderate RDI (23.3 +/- 1.6), and were less obese than the other patients (body mass index: 25 +/- 3 vs 33 +/- 8). The 58 false-positive cases had an RDI of 8 +/- 4, an awake SaO2 of 93.1 +/- 3.6 vs 94.1 +/- 2.6 for the rest of the population (p = 0.01). Finally, the false-positive cases had more airways obstruction (FEV1/VC = 72 +/- 13 vs 77 +/- 15%; p = 0.026). Using a delta value of 0.8 leads to a sensitivity of 90% with 19 false-negative cases but with a higher specificity of 75%. CONCLUSIONS: A nocturnal oximetry test with a delta index below 0.6 is helpful in ruling out the diagnosis of SAS in patients being screened for this condition, as this yielded only three negative test results in 301 screening procedures. PMID- 8620712 TI - Myasthenia gravis and upper airway obstruction. AB - Respiratory impairment in myasthenia gravis is usually attributed to weakness of the diaphragm and thoracic chest wall muscles, and is rarely attributed to upper airway obstruction. Myasthenia gravis is characterized by weakness of the striated muscles and usually affects those innervated by the bulbar cranial nerves. Weakness of these bulbar and upper airway muscles can lead to upper airway obstruction. To our knowledge, there are only five case reports in the literature associating upper airway obstruction with myasthenia gravis. Therefore, we attempted to further define its occurrence in myasthenia gravis patients by reviewing their flow volume loops. We present a case of upper airway obstruction causing respiratory symptoms in a myasthenia gravis patient. We then surveyed a total of 61 patients with myasthenia gravis who were tested in our pulmonary function laboratory between February 1990 and August 1993. Of these 61 patients, 12 had flow volume loops and 7 of these 12 disclosed a pattern of extrathoracic upper airway obstruction. The FVC was 80% or more in five of seven patients. Our data suggest that upper airway obstruction is much more common in patients with myasthenia gravis than previously recognized. In conclusion, we recommend the performance of flow volume loops in patients with myasthenia gravis to evaluate their respiratory impairment. PMID- 8620713 TI - Bronchodilator response at low lung volumes predicts bronchiolitis obliterans in lung transplant recipients. AB - BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long term lung allograft viability. The diagnosis often occurs after significant organ dysfunction is present, and BOS is often unresponsive to standard immunosuppressive agents. We have observed bronchodilator responses (BRs) at low lung volumes in many of our patients who have developed BOS. We therefore assessed whether BR could predict the development of BOS. METHODS: We conducted a retrospective review of the clinical and pulmonary function laboratory records of 146 patients who underwent transplantation between March 1983 and November 1993. BR was defined as 25% or more increase in forced expiratory flow at 50% of vital capacity or 30% or more increase in forced expiratory flow at 75% of vital capacity. BOS was defined according to recently published FEV1 criteria. Bronchiolitis obliterans was defined histologically according to criteria of the Lung Rejection Study Group. RESULTS: Of the total population, 52 were excluded because of death or insufficient information. BRs of the small airways were seen in 31 patients (33%), 25 of whom developed BOS (83%). Approximately half of those with BR who developed BOS had evidence of acute rejection in the month prior to the onset of BR. Two thirds (four of six) of patients with BR not developing BOS had acute rejection in the previous month. The sensitivity of BR in predicting BOS was 51% with a specificity of 87%. The positive predictive value was 81%. CONCLUSIONS: BR appears to be useful as an early marker of BOS. The development of BR in selected patients should lead to closer monitoring and possibly a trial of augmented immunosuppression to arrest the establishment of BOS. PMID- 8620714 TI - Three hundred patients referred for lung transplantation. Experiences of the Dutch Lung Transplantation Program. Groningen Lung Transplantation Group. AB - In November 1990, a lung transplantation program began at the University Hospital in Groningen, the Netherlands. As of April 1994, 300 patients were referred for lung transplantation and we investigated the decisions that have been made concerning these referrals up to January 1, 1995. The patients were evaluated according to a stepwise procedure. In stage 1, written information about the referred patients was discussed during the weekly, multidisciplinary lung transplantation meeting. In this stage, 14% of the patients were rejected and 2% were postponed. If no major objections for transplantation were identified, the patient was invited for a visit to the outpatient clinic, stage 2. The newly acquired information from that visit was discussed again at the transplantation meeting. In this stage, 11% of the patients were rejected and 18% postponed. The remaining patients underwent an (partial or complete) inpatient evaluation, stage 3. From all patients about whom a decision was made in this stage, only 5% were rejected, respectively 35% after partial evaluation and only 1.5% after complete evaluation. A total of 110 patients (37% of all referred patients) were listed for lung transplantation, stage 4. Of the listed patients, 20% died while awaiting an appropriate donor. The group of patients with COPD/emphysema had by far the lowest death rate on the waiting list. Patients with short stature (< or = 1.65 m) had a much higher risk to die on the waiting list compared with patients with longer stature, 42% vs 13%. As of January 1, 1995, 55 patients have undergone transplantation, which is 50% of all patients on the waiting list and 18% of all referred patients. The stepwise selection procedure identifies patients with potential contraindications at an early stage. In this way, unrealistic expectations and unnecessary examinations, expense, and/or hospital admissions may be prevented. Donor shortage, and thus waiting list problems, still remains a significant drawback in the further development of lung transplantation. PMID- 8620715 TI - Combined use of pleural adenosine deaminase with lymphocyte/neutrophil ratio. Increased specificity for the diagnosis of tuberculous pleuritis. AB - Increased pleural fluid adenosine deaminase (ADA) activity is classically associated with tuberculous pleuritis. However, increased activity can also occur in a number of other diseases and this may negatively affect the diagnostic utility of ADA measurements and decrease its specificity for the diagnosis of tuberculosis (TB). The presence of ADA in pleural fluids reflects the cellular immune response in the pleural cavity and in particularly, the activation of T lymphocytes. Different disease entities are typically associated with the presence of particular types of leukocytes. OBJECTIVE: To determine whether the combined use of ADA activity and differential cell counts would provide a more efficient means for diagnosing tuberculous pleurisy than the use of ADA levels alone. METHODS: Biochemistry, cytology, and microbiology studies were performed on 472 consecutive pleural fluids. ADA and differential cell counts were determined on all exudative effusions. RESULTS: ADA activity in tuberculous effusions was significantly higher than in any other diagnostic group (p < 0.005). At a level of 50 U/L, the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and efficiency for the identification of TB were calculated at 91%, 81%, 84%, 89%, and 86%, respectively. When the additional requirement of a lymphocyte neutrophil ratio of 0.75 or greater was included, the sensitivity, specificity, ppv, npv, and efficiency for the identification of TB were calculated at 88%, 95%, 95%, 88%, and 92%, respectively. CONCLUSION: ADA, especially when combined with differential cell counts and lymphocyte/neutrophil ratios, remains a useful test in the diagnosis tuberculous pleuritis. PMID- 8620716 TI - The validity of classic symptoms and chest radiographic configuration in predicting pulmonary tuberculosis. AB - OBJECTIVE: Patients admitted to the pulmonary isolation service to rule out tuberculosis (TB) were prospectively studied to identify predictors of smear positive TB. METHODS: History of symptoms--cough, sputum production, fever, weight loss, night sweats, hemoptysis, anorexia, and dyspnea; medical history- TB, tuberculin skin test (TST) status, TB contact; and social factors--crowding index, history of incarceration or living in a shelter, and employment status were obtained in face-to-face interviews. Chest x-rays (CXRs) were scored as typical, atypical, or negative. Serial morning sputa were collected. RESULTS: Complete data were collected on 101 patients; 44 had pulmonary TB; 33 patients were smear positive and considered infectious; 11 patients were smear negative but culture positive. There was no difference between TB and non-TB patients with respect to HIV status and social risk factors. Significant differences were found between patients who were smear positive for TB and smear negative with respect to cough, sputum, and typical CXR (79%, 76%, and 79% sensitivity, respectively). Eleven patients without TB had an atypical CXR and denied cough, sputum, and weight loss. Only one patient with TB presented this way. CONCLUSION: Even in high-risk populations, symptoms of cough, sputum, weight loss for less than 2 weeks, and the absence of a typical CXR are strong negative predictors for TB. PMID- 8620717 TI - Lisinopril attenuates acute hypoxic pulmonary vasoconstriction in humans. AB - OBJECTIVE: We have studied the effects of angiotensin-converting enzyme (ACE) inhibition with lisinopril on acute hypoxic pulmonary vasoconstriction (HPV). DESIGN: Randomized, double-blind, placebo-controlled study in ten healthy volunteers. Subjects received four daily doses of lisinopril or matched placebo before attending the laboratory 5 h after taking the final dose. After reaching a resting hemodynamic state, subjects were made hypoxemic (SaO2, 75 to 80%) for 30 min. MEASUREMENTS: Pulmonary and systemic hemodynamic parameters were measured noninvasively at baseline and after 30 min of hypoxemia. RESULTS: Mean pulmonary artery pressure (MPAP) and total pulmonary vascular resistance (TPR) were similar at baseline on both study days. The increase in MPAP induced by hypoxemia was significantly blunted by pretreatment with lisinopril (means and 95% confidence interval [CI] for difference) 13.4 mm Hg vs placebo 19.6 mm Hg (95% CI, 2.5, 9.9). Likewise, the TPR response to hypoxemia was significantly blunted by lisinopril: 124 dyne.s.cm-5 vs placebo 179 dyne.s.cm-5 (95% CI, 11, 99). Lisinopril had no confounding systemic effects on mean arterial pressure, cardiac output, or systemic vascular resistance at baseline or in response to hypoxemia. CONCLUSIONS: Lisinopril therefore significantly attenuated the pulmonary pressor response to hypoxemia without decreasing baseline MPAP or TPR. This suggests that angiotensin II might play a modulatory role during HPV in man and that ACE inhibition may be a useful adjunctive treatment in hypoxemic pulmonary hypertension. PMID- 8620718 TI - Wegener's granulomatosis in the elderly. AB - STUDY OBJECTIVE: To determine if elderly patients with Wegener's granulomatosis (WG) exhibit distinctive clinical features or outcomes compared with patients whose conditions were diagnosed at younger ages. DESIGN: Retrospective cohort study. SETTING: University medical center. PATIENTS: Thirty-three patients with WG diagnosed when 60 years old or older and 34 patients with WG diagnosed at age younger than 60 years, identified by record review of all WG patients seen over an 11-year period. RESULTS: The prevalence of specific clinical features, progression to end-stage renal disease, mortality rate, and infectious and noninfectious complications of therapy were examined. The prevalence of upper respiratory tract involvement (rhinitis, sinusitis, otitis, epistaxis) and hemoptysis were significantly less common as initial manifestations in the elderly patients, although pulmonary infiltrates were seen more commonly during the course of their disease. Renal insufficiency was more common at the time of diagnosis in the elderly patients (64% vs 35%; p < 0.05). Most notably, CNS involvement was 4.5-fold more common in elderly patients (27% vs 6%; p = 0.02). The overall incidence of infectious and noninfectious complications of therapy was similar between the groups, although the mortality rate was markedly higher in the elderly patients (54% vs 19%; p < 0.01). Almost all deaths were due to overwhelming infection. CONCLUSIONS: Elderly patients with WG present with distinctive clinical features, particularly a relatively low incidence of upper respiratory tract complaints and a high incidence of CNS involvement. The mortality risk from infectious complications of WG is substantially higher in elderly patients, although this cannot be attributed directly to adverse affects of therapy. PMID- 8620720 TI - Passive smoking in children. Racial differences in systemic exposure to cotinine by hair and urine analysis. AB - Passive smoking has been shown to adversely affect the health of infants and children. Black children and adults appear to be more susceptible to a variety of tobacco smoke health hazards for unknown reason. The objectives of this study were as follows: (1) to correlate the number of cigarettes reported to have been smoked by parents with urine and hair concentrations of cotinine in children; and (2) to identify race differences in systemic exposure to cotinine in children. This was an observational study in a consulting pediatric office on 169 nonsmoking children between 2 and 18 years of age, not actively smoking. The outcome measures of interest were urinary cotinine concentrations corrected for milligram of creatinine and hair concentration of cotinine (per milligram of hair). There were significant correlations between the number of cigarettes the child was exposed to and urinary cotinine (r = 0.68, p = 0.0001) or hair cotinine concentrations (r = 0.19, p = 0.02), and between urinary and hair cotinine (r = 0.3, p = 0.0005). In this cohort, parents of black children (n = 21) tended to smoke less (6.6 +/- 3/d, mean +/- SEM) than white parents (n = 97) (12 +/- 1.8, mean +/- SEM) (p = 0.2). Despite being exposed to less cigarettes, black children had higher hair concentrations of cotinine than white children (0.89 +/- 0.25 ng/mg vs 0.48 +/- 0.05 ng/mg; p = 0.05). The ratio hair/urine concentrations of cotinine was twofold higher in black children (0.035 +/- 0.01 vs 0.019 +/- 0.002; p = 0.004). White children with dark hair did not differ significantly from white children with fair hair in any of these indexes. The amount of urinary cotinine per milligram of creatinine caused by 1 cigarette per day was twofold higher in black children (14.7 +/- 5.2 ng/mg of creatinine) than in white children (6.3 +/- 1.2 ng/mg of creatinine) (p = 0.02). These data suggest that black children handle cigarette smoke differently from white children and that black children have higher systemic exposure to this constituent of cigarette smoke. PMID- 8620719 TI - Safety of nicotine polacrilex gum used by 3,094 participants in the Lung Health Study. Lung Health Study Research Group. AB - STUDY OBJECTIVE: To assess cardiovascular conditions and other side effects associated with the use of nicotine polacrilex (NP), 2 mg. DESIGN: A multicentered randomized control trial of early intervention for the prevention of COPD. SETTING: Ten university medical centers in the United States and Canada. PARTICIPANTS: Adult smoking volunteers with evidence of early COPD; 3,923 in intervention and 1,964 controls. INTERVENTION: Smoking cessation program, including NP. MEASUREMENTS: Data on hospitalizations were collected annually. Data on reported NP side effects were collected at 4-month intervals for intervention participants. RESULTS: The rates of hospitalization for cardiovascular conditions and cardiovascular deaths during the 5 years of the study were not related to use of NP, to dose of NP, or to concomitant use of NP and cigarettes. About 25% of NP users reported at least one side effect, but most were very minor and transient. Side effects associated with discontinuance of NP in 5% or more of users included headache, indigestion, mouth irritation, mouth ulcers, and nausea. There was no evidence that concomitant use of NP and cigarettes was associated with elevated rates of reported side effects. Participants in the smoking cessation intervention who received intensive levels of instruction and monitoring of NP use (initially at 12 meetings during 3 months) appeared to report significantly lower rates of side effects (dizziness, headache, and throat irritation) than control participants, presumed to have less instruction and monitoring. CONCLUSIONS: NP, as used in the Lung Health Study, appears to be safe and unrelated to any cardiovascular illnesses or other serous side effects. PMID- 8620721 TI - Cost-effectiveness of collecting routine cytologic specimens during fiberoptic bronchoscopy for endoscopically visible lung tumor. AB - STUDY OBJECTIVE: Fiberoptic bronchoscopy is the most common modality used to diagnose endobronchial carcinoma. Collection of brushing and washing specimens for cytology is common during bronchoscopy for endobronchial abnormality, but it is unknown if collection of these specimens is cost-effective. DESIGN: Retrospective review of a computerized database with cost-effectiveness analysis. SETTING: Tertiary care medical center. PATIENTS: Two hundred one patients undergoing bronchoscopy for endobronchial lung tumor. INTERVENTION: All patients in the study underwent fiberoptic bronchoscopy that included forceps biopsies, washings, and brushings. In addition to analyzing the sensitivity of forceps biopsy, washings, and brushings at diagnosing malignancy, we analyzed the cost effectiveness of three potential specimen collection strategies. These strategies were (1) collection of both washings and brushings in addition to forceps biopsy specimen, (2) collection of either washings or brushings in addition to forceps biopsy specimen, and (3) collection of forceps biopsy specimen only. MEASUREMENTS AND RESULTS: The sensitivity of bronchoscopy, including biopsy, washing, and brushing is 85.3% (95% confidence interval [CI], 80.1 to 90.5%). The sensitivity of forceps biopsy is 80.8% (95% CI, 75.0 to 86.6%). The addition of washings and brushings increases the sensitivity of bronchoscopy from 80.8 to 85.3% (McNemar's p = 0.01). Cost-effectiveness analysis reveals that forceps biopsy plus washing or brushing has a marginal cost-effectiveness ratio of $308 per reduced-quality day avoided compared with forceps alone. Adding an additional cytology specimen has a marginal cost-effectiveness ratio of $5,500 per reduced-quality day avoided. CONCLUSIONS: There is a modest but definite increase in the sensitivity of bronchoscopy in diagnosing endobronchial cancer with the addition of washings and brushings for cytology. Cost-effectiveness analysis reveals that collection of either washings or brushings is probably the best strategy. PMID- 8620722 TI - Identifying early predictors of mortality in pediatric patients with acute leukemia and pneumonia. AB - STUDY OBJECTIVE: To identify clinical variables of pneumonia in children with acute leukemia that predicted respiratory failure and mortality. DESIGN: A retrospective chart review of children with acute leukemia admitted to the hospital with the diagnosis of pneumonia or ARDS from March 1991 to April 1994. SETTING: Lucile Salter Packard Children's Hospital at Stanford, a 168-bed teaching hospital and regional tertiary referral center for children in northern California. PATIENTS: During this study period, 20% of the 174 admissions of children with acute leukemia had pneumonia at the time of admission or during the course of the hospitalization for a total of 36 admissions. The mean age of these children was 9.2 +/- 1.1 years. RESULTS: Eleven percent of the children with pulmonary infiltrates in one quadrant on the chest x-ray film at the onset of pneumonia and 53% of the children with pulmonary infiltrates in more than one quadrant at the onset of pneumonia died. Fifteen percent of the children without sepsis at the onset of pneumonia and 70% of the children with sepsis at onset died. Eighteen percent of the children without shock at the onset of pneumonia and 75% of the children with shock at the onset died. None of the children died who required < or = 3L/min of O2 to maintain SO2 > or = 95%, but 79% of the children who required > 3L/min O2 died. Using the criteria "> 3 L/min O2 by nasal cannula to maintain SO2 > or = 95%" to identify the nonsurvivors had a sensitivity of 100% and specificity of 88%. This specificity was not increased by combining the criteria "O2 requirements at any time" and "the extent of pulmonary infiltrates at the onset of pneumonia." All children who required mechanical ventilatory support for respiratory failure had previously received > 3 L/min O2 by nasal cannula to maintain SO2 > or = 95% for 37.8 +/- 12.9 h (range 3 to 96 h). Nine of the 10 children in our study who received mechanical ventilation died. CONCLUSION: In children with acute leukemia and pneumonia, the amount of O2 required to maintain SO2 > or = 95% may identify those who are likely to develop respiratory failure hours before mechanical ventilatory support is needed. The ability to identify children at risk for respiratory failure is not increased by combining the risk factors "oxygen requirements" and "extent of pulmonary infiltrates at the onset of pneumonia". Finally, only 10% of the children who required mechanical ventilatory support survived. PMID- 8620723 TI - Scintigraphic lung scans and clinical assessment in critically ill patients with suspected acute pulmonary embolism. AB - PURPOSE: The purpose of this investigation was to evaluate the diagnostic accuracy of radionuclide scintigraphic lung scans and clinical assessment in critically ill patients with suspected acute pulmonary embolism. MATERIALS AND METHODS: Critically ill patients were defined as follows: (1) patients who were hypoxemic on room air, and not given ventilatory support (n = 89); (2) patients given ventilatory support (n = 46); and (3) patients in ICUs, but not given ventilatory support (n = 85), and hypotensive patients who were not hypoxemic or given ventilatory support (n = 3). Comparisons were made with patients who had none of these characteristics of critically ill patients (n = 627). Data are from the Prospective Investigation of Pulmonary Embolism Diagnosis. RESULTS: The sensitivities, specificities, and positive predictive values of high probability lungs scans among each of the four categories of critically ill patients were not statistically significantly lower than values in noncritically ill patients. The positive predictive values of the clinical assessments did not differ to a statistically significant extent from noncritically ill patients. Clinical assessment, when concordant with the lung scan interpretation, usually increased the positive predictive value for pulmonary embolism. CONCLUSION: Scintigraphic lung scans and clinical assessment retain their diagnostic value even in critically ill patients. PMID- 8620724 TI - Clara cell protein (CC-16) and surfactant-associated protein A (SP-A) in asbestos exposed workers. AB - Asbestos-exposed workers (Asb) can sometimes develop lung impairments resembling idiopathic pulmonary fibrosis (IPF). Smoking is often a troubling confounder in the natural history of these lung diseases. Distal airspace epithelial cells, which are also altered in asbestosis, secrete Clara cell protein (CC-16, also designated CC-10) and surfactant-associated protein A (SP-A). By inhibiting phospholipase A2 (PLA2), CC-16 and SP-A are putative candidates for controlling lung inflammatory events. Both were measured with PLA2 activity in alveolar fluids (and sera for CC-16) of smoker and nonsmoker Asb and compared with smoking matched normal subjects (N). CC-16 (in mg/L) was slightly increased in Asb and affected by smoking: nonsmoker Asb: 3.1 +/- 0.5 vs nonsmoker N: 1.9 +/- 0.2 (p < 0.05), smoker Asb: 1.7 +/- 0.3 vs smoker N: 0.6 +/- 0.1 (p < 0.05). SP-A (in microgram/mL) was enhanced in Asb but not affected by smoking: 5.4 +/- 1.5 in Asb vs 1.6 +/- 0.4 in N (p < 0.05), whereas SP-A to phosphorus ratio was increased in Asb but affected by smoking. CC-16 to albumin and CC-16 in serum to alveolar fluid ratios were altered by cigarette consumption in Asb (p < 0.05 vs N). Secretory PLA2 activity was slightly enhanced in Asb (p < 0.05 vs N). All data were similar between stages of disease. In summary, alveolar CC-16, SP-A, and secretory PLA2 activity were increased in Asb. Smoking affected several parameters. By this habit, Asb might reinforce lung profibrotic factors and increase their risk in developing lung alterations resembling IPF. PMID- 8620725 TI - Effect of altitude on hand-held peak flowmeters. AB - OBJECTIVE: To quantify the effect of altitude on the operational characteristics of hand-held peak flowmeters. DESIGN: Altitude simulation within a hypobaric chamber combined with five constant simulated peak flows delivered from a computerized pump were used to test commercially available peak flowmeters. SETTING: F.G. Hall Hyperbaric/Hypobaric facilities located at Duke University School of Medicine. MEASUREMENTS: Two each of nine models of commercially available hand-held peak flowmeters and a volume spirometer were tested at six simulated altitudes (100, 500, 1,000, 1,500, 2,000, and 3,000 m) using five target peak flows. Each peak flow was injected into each meter twice. Forward stepwise regression was used to check for nonlinear relationships between altitude and peak expiratory flowmeter readings. Linear regression equations were fit to the data at each target flow across altitude. Effect of absolute peak flow was tested by analysis of covariance. RESULTS: For these altitudes, linear relationships were found between altitude and measured peak flow. For all meters tested, the average decrease in peak flow ranged from -8.7% at the lowest target flow (123 L/min) to -6.5% at the highest target flow (702 L/min) for each 100 mm Hg decrease in barometric pressure (PB). Individual meters ranged from -12.3% at the lowest target flow to -4.4% at the highest target flow for 100 mm Hg decrease in PB. The spirometer had no significant changes associated with changes in PB. In all cases, the magnitude of the altitude effect, measured by percent change, decreased with increasing peak flow. CONCLUSIONS: Peak expiratory flowmeters underread PEF as a function of both increasing altitude and increasing target peak flow. PMID- 8620726 TI - Positive end-expiratory pressure prevents the loss of respiratory compliance during low tidal volume ventilation in acute lung injury patients. AB - STUDY OBJECTIVE: To study the effect of positive end-expiratory pressure (PEEP) on the decay of respiratory system compliance (Cpl,rs) due to low tidal volume (VT) ventilation in acute lung injury (ALI) patients. SETTING: General ICU in a university hospital. PARTICIPANTS: Eight ALI patients with a lung injury score greater than 2.5. INTERVENTION: Pressure-controlled ventilation (PCV) and volume controlled ventilation (VCV), with an average VT of 8.5 +/- 0.4 mL/kg, were applied at three levels of PEEP (5, 10, and 15 cm H2O). Before each PCV and VCV period, lung volume history was standardized by manual hyperinflation maneuvers. MEASUREMENTS: We measured Cpl,rs at time 0 (start), 10, 20, and 30 (end) min from the beginning of each PCV and VCV period. Gas exchange and hemodynamic data were collected at end. RESULTS: At PEEP 5 and 10 cm H2O, we observed a progressive Cpl,rs decay with both PCV and VCV modes. At PEEP 5 cm H2O, we detected a higher Cpl,rs decrease during PCV, due to a higher Cpl,rs at start, compared with VCV. At PEEP 15 cm H2O, Cpl,rs did not decrease significantly. Cpl,rs values measured at end as well as oxygenation and hemodynamic data did not differ between PCV and VCV. At PEEP 15 cm H2O, PCV provided lower PaCO2 than VCV. CONCLUSIONS: A PEEP of at least 15 cm H2O was needed to prevent Cpl,rs decay. The progressive Cpl,rs loss we observed at lower PEEP probably reflects alveolar instability. PMID- 8620727 TI - Effectiveness of nitric oxide inhalation in septic ARDS. AB - STUDY OBJECTIVE: To evaluate the percentage of nitric oxide (NO) responders in septic shock patients with ARDS. Additionally, to investigate long-term NO effects on cardiac performance and oxygen kinetic patterns in NO responders vs nonresponders. DESIGN: Prospective cohort study. SETTING: ICU of a university hospital. PATIENTS: Twenty-five consecutive patients with a diagnosis of septic shock and established ARDS requiring inotropic and vasopressor support. INTERVENTIONS: After diagnosis of ARDS, NO was administered at 18 or 36 ppm. Patients demonstrating a NO-induced rise of arterial oxygen tension of 20% or more and/or a fall in mean pulmonary artery pressure of 15% or more were grouped as NO responders; others were grouped as nonresponders. MEASUREMENTS AND RESULTS: Ten patients (40%) were NO responders, while 15 patients (60%) were nonresponders. Mortality was 40% in NO responders and 67% in nonresponders (NS). NO responders developed a significantly lower mean pulmonary artery pressure (28 +/- 6 vs 33 +/- 6 mm Hg; p < 0.05), lower pulmonary vascular resistance (PVR: 258 +/- 73 vs 377 +/- 163 dyne.s.cm-5.m-2; p < 0.05), and higher PaO2/FIO2 ratio (192 +/- 85 vs 144 +/- 74 mm Hg; p < 0.05) within the study period. In responders, NO induced afterload reduction resulted in increased right ventricular ejection fraction (RVEF: 40 +/- 7 vs 35 +/- 9%; p < 0.05), significantly higher cardiac index (CI: 4.5 +/- 1.1 vs 4.0 +/- 1.2 L.min-1.m-2; p < 0.05) and oxygen delivery (DO2: 681 +/- 141 vs 599 +/- 160 mL.min-1.m-2; p < 0.05) compared with nonresponders. In NO nonresponders, RVEF was correlated with PVR, CI, DO2, mixed venous oxygen saturation (SvO2), and oxygen extraction ratio (O2ER) (r = +/- 0.60 to +/- 0.69; p < 0.05). No significant correlation between RVEF and any of these parameters was observed in responders. SvO2 (75 +/- 7 vs 69 +/- 8%; p < 0.05) and O2ER (0.24 +/- 0.06 vs 0.27 +/- 0.06; p < 0.05) were significantly different between responders and nonresponders, while no difference in oxygen consumption was observed (161 +/- 41 vs 153 +/- 43 mL.min.m-2). CONCLUSIONS: Inhaled NO is effective in only a subgroup of septic ARDS patients, with a higher, but insignificantly different percentage of survivors in the responder group. NO responders were characterized by increased RVEF accompanied by higher CI, DO2, and lower O2ER. In nonresponders, RVEF remained depressed, with a close correlation between RVEF and CO as well as DO2 and O2ER. Thus, nonresponders seem to suffer from impaired cardiac reserves and correspondingly lower oxygen transport variables. PMID- 8620728 TI - Volume expansion increases right ventricular infarct size in dogs by reducing collateral perfusion. AB - STUDY OBJECTIVE: Plasma volume expansion is frequently recommended to correct the low output state resulting from right ventricular (RV) infarction. However, any subsequent increase in pericardial and RV filling pressures from volume expansion could impair RV collateral blood flow. We examined whether volume expansion in dogs before right coronary ligation reduced collateral perfusion and worsened the extent of RV necrosis. DESIGN: Randomized experimental study. SETTING: Animal research laboratory in university medical center. PARTICIPANTS: Forty anesthetized, closed-chest dogs were randomly assigned to normovolemic, pericardium opened (n = 10) or intact (n = 10) groups, and hypervolemic, pericardium opened (n = 10) or intact (n = 10) groups. INTERVENTIONS: Hypervolemic animals received 24 mL/kg of 6% hetastarch. All animals underwent 90 min right coronary ligation, followed by 120 min reperfusion. Collateral coronary blood flow (radioactive microspheres) and area of necrosis (An) were determined in the area at risk (Ar). MEASUREMENTS AND RESULTS: Stroke volume decreased in all groups with ischemia but remained 25 to 40% greater in both hypervolemic groups than in normovolemic animals (p < 0.05). In hypervolemic animals with intact pericardium, RV end-diastolic pressure increased to 10.4 +/- 2.1 mm Hg (mean +/- SD), a value that significantly exceeded those of the other three groups. During RV ischemia, collateral perfusion in the Ar was similar in both normovolemic groups and in hypervolemic animals with opened pericardium (mean range, 12.9 +/- 8.8 to 13.8 +/- 7.6 mL/min/100 g; p = NS), and the An/Ar varied from 11.8 +/- 6.3 to 18.6 +/- 17.4% (p = NS). In contrast, in hypervolemic animals with intact pericardium, collateral perfusion decreased to 7.2 +/- 3.5 mL/min/100 g and the An/Ar was increased to 38.2 +/- 18.6% (p < 0.05 compared with other groups, respectively). Overall, An/Ar was inversely related to collateral blood flow in the Ar (r = -0.46; p < 0.05) and correlated positively with RV end-diastolic pressure (r = 0.61; p < 0.05). CONCLUSIONS: Volume expansion preserved stroke volume during RV ischemia, independent of pericardial integrity. However, volume expansion in animals with an intact pericardium increased RV infarct size by twofold to threefold secondary to reduced periischemic collateral perfusion. This detrimental effect of volume expansion on infarct size was prevented by opening the pericardium. PMID- 8620729 TI - Anion gap in turpentine-induced pleural effusions. Correlation with pH and protein level. AB - Since the pleural fluid proteins and lactate are unmeasured anions, the pleural fluid anion gap (Na+K-Cl-total CO2) should vary with the protein level and should be high in acidic effusions (which have high lactate levels). The anion gap is also convenient and inexpensive to measure, and less subject to artifact than the pH measurement. To test the hypothesis that the anion gap correlates with the pH, protein level, and other traditional pleural fluid measurements, we used a well described model of turpentine-induced effusions in nine New Zealand white rabbits. Nonacidic exudative effusions were induced by an intrapleural injection of turpentine; acidic exudative effusions were induced by a second injection. Pleural fluid and blood were obtained just before (0 h) and 9, 24, 48, and 72 h after the second injection. We found the anion gap correlated with pH, the glucose, protein, and lactate dehydrogenase levels, pleural-fluid/plasma protein and lactate dehydrogenase ratios, and WBC count (all p < 0.001). The pH and protein ratio together accounted for 95% of all anion gap variation within individual subjects. We also found the influence of the PCO2 level on pH was not significant after taking into account the influence of the anion gap. These results suggest the anion gap may be useful in the clinical evaluation of pleural effusions and could potentially replace the pH measurement. PMID- 8620730 TI - Optimizing coronary thrombolysis with i.v. administration of recombinant tissue plasminogen activator. Single bolus vs double bolus vs front-loading. AB - This study was designed to compare the efficacy of coronary thrombolysis obtained with i.v. administration of three dose regimens of recombinant tissue plasminogen activator (rtPA). Although many studies have confirmed the efficacy of thrombolytic therapy in treatment of acute myocardial infarction, few prospective studies have been designed to determine which dose regimen optimizes the rate of coronary thrombolysis. A canine model was used. Coronary thrombosis was induced by injection of radioactive, autologous blood clots through a catheter placed in the left anterior descending coronary artery. Subsequently, 15 dogs were randomized into 3 groups of 5 dogs each. In group 1 dogs, 1.25 mg/kg of rtPA was administered i.v. as a bolus; in the group 2 dogs, 1.25 mg/kg of rtPA was administered over 60 min. The administration was "front loaded" so that 15% was administered as a bolus, 60% over 30 min, and 25% over 30 min; in group 3, 1.25 mg/kg of rtPA was divided into two i.v. boluses and administered 15 min apart. Coronary thrombolysis was assessed with a gamma camera. Despite differences in rate of administration of rtPA, at 15, 30, and 90 min after onset of treatment, extent of clot lysis was similar between groups. These results indicate that despite differences in dose regimens, rates of thrombolysis are similar when i.v. rtPA is relatively rapidly administered. Further, the similar rates of clot lysis over time between groups suggest both an effective upper limit to the dose thrombolytic rate relationship and relatively high, sustained steady-state plasma concentrations of rtPA. PMID- 8620731 TI - The pathophysiology of hyperventilation disorders. PMID- 8620732 TI - Vitamin D, calcium, and sarcoidosis. AB - Hypercalcemia occurs in about 10% of the patients with sarcoidosis; hypercalciuria is about three times more frequent. These abnormalities of calcium metabolism are due to dysregulated production of 1,25-(OH)2-D3 (calcitriol) by activated macrophages trapped in pulmonary alveoli and granulomatous inflammation. Undetected hypercalcemia and hypercalciuria can cause nephrocalcinosis, renal stones, and renal failure. Corticosteroids cause prompt reversal of the metabolic defect. Chloroquine, hydroxychloroqune, and ketoconazole are the drugs that should be used if the patient fails to respond or develops dangerous side effects to corticosteroid therapy. PMID- 8620733 TI - Reduction pneumoplasty for giant bullous emphysema. Implications for surgical treatment of nonbullous emphysema. AB - A review of the literature on reduction pneumoplasty for giant bullous emphysema was undertaken to identify current criteria for this surgical treatment and in the hope of obtaining insights into evaluating reduction pneumoplasty for nonbullous emphysema. Twenty-two retrospective case series, published since 1950, were retrieved by a computer search of the literature and a search of the Index Medicus prior to 1966. Reduction pneumoplasty is most effective when bullae are larger than one third of a hemithorax with evidence of compression of adjacent lung tissue and an FEV1 of less than 50% predicted; the presence of emphysema in nonbullous lung and the amount of compression are best judged by CT. The rationale for reduction pneumoplasty for nonbullous emphysema is supported by the similar early functional changes after reduction pneumoplasty for bullous and nonbullous-improvement of blood gas values and lung mechanics. A single study showing that decline of lung function after surgery for bullous emphysema was less in those who stopped smoking than in those who continued to smoke supports the need for preoperative and maintained smoking cessation in patients receiving reduction pneumoplasty. After 4 decades, the duration of improvement in lung function, whether worsening of emphysema occurs in remaining lung, and late morbidity and mortality after reduction pneumoplasty for bullous emphysema are not well defined. A registry with an unoperated-on comparison group could more rapidly accumulate such data after reduction pneumoplasty for nonbullous emphysema. PMID- 8620734 TI - Virtual bronchoscopy. Relationships of virtual reality endobronchial simulations to actual bronchoscopic findings. AB - Advances in computer technology have permitted development of virtual reality images of the tracheobronchial tree using data sets derived from helical CT of the chest. To determine the relevance of these images to actual bronchoscopic findings, we compared "virtual bronchoscopy" images with videotaped bronchoscopy results in 20 patients who had undergone both helical chest CT and fiberoptic bronchoscopy during clinical evaluation of their thoracic problems. Suboptimal endobronchial simulations in ten patients identified important, readily-addressed technical requirements for this imaging procedure. In the ten patients with technically suitable renderings of the airway, virtual bronchoscopy simulations accurately demonstrated endobronchial obstructions by tumor in five, airway distortion and/or ectasia in four, and accessory bronchi in another. These preliminary observations suggest that virtual bronchoscopy simulations accurately represent major endobronchial anatomic findings. This technique may have a role in prebronchoscopy planning, endoscopy training, and/or endobronchial therapy, and merits further study. PMID- 8620735 TI - Thoracoscopic lobectomy for benign diseases. AB - The few reports in the literature on thoracoscopic anatomic lung resections were almost exclusively for early primary lung cancers. We report our combined experience on video-assisted thoracoscopic (VAT) lobectomy for benign diseases from two major hospitals in Hong Kong over a 20-month period. From August 1993 to March 1995, 66 VAT lobectomies were performed; of this number, 10 cases (15%) were for benign diseases (5 tuberculosis, 2 organized pneumonia, 1 bronchiectasis, 1 sclerosing hemangioma, 1 infected bronchogenic cyst). There was no mortality or incidence of intraoperative complications. Postoperative complications occurred in one patient with tuberculosis and consisted of persistent air leak and subsequent wound infection. There were no long-term complications after a mean follow-up of 11 months. The mean duration of chest tube drainage was 6.7 days and that of hospital stay was 9.8 days. These results were not statistically different from those achieved in VAT lobectomies performed for malignant tumors (5.0 days for drainage and 6.8 days for hospital stay) despite the fact that hilar dissection was sometimes more difficult in the former because of inflammatory changes. We conclude that VAT lobectomy for benign diseases is technically feasible even though its role in thoracic surgery remains to be defined. PMID- 8620736 TI - A 36-year-old comatose woman exhibiting decerebrate posturing. PMID- 8620737 TI - A 25-year-old man with back pain and an abnormal chest radiograph. PMID- 8620738 TI - Atypical electromechanical dissociation in a patient with recurrent pulmonary embolism. AB - A 47-year-old man experienced recurrent pulmonary embolism resistant to aggressive medical and surgical prophylaxis. Although paraneoplastic hypercoagulability was suspected, no endoscopic or radiologic signs of malignancy were detected. Death was the result of electromechanical dissociation, which was attributed to right ventricular outflow obstruction. At autopsy, anaplastic lung carcinoma was found in the left basal segment with superimposed pulmonary infarction. A huge pedunculated thrombus was attached to the left ventricular apex and extended into the ascending aorta, obstructing the left ventricular outflow. To our knowledge, this is the first case of electromechanical dissociation due to left ventricular thrombus in a patient with pulmonary embolism. Radiologic and echocardiographic evaluation of such patients should take into account possible masking of the underlying neoplasm by embolic or hemorrhagic phenomena, or both, and the presence of left-sided cardiac thombi, which may cause catastrophic events. PMID- 8620739 TI - Extensive idiopathic benign bilateral asynchronous pleural fibrosis. AB - We describe a young man who had been healthy until he developed extensive benign left pleural fibrosis. He subsequently developed disabling right pleural thickening a year later. No clear cause was discovered. PMID- 8620740 TI - Subacute rupture of the left ventricular free wall after acute myocardial infarction. Three cases of long-term survival without emergency surgical repair. AB - Rupture of the left ventricular free wall after acute myocardial infarction (AMI) has been regarded as uniformly fatal unless emergency surgical repair is performed. Among 2,862 patients admitted with AMI to our ICU during the last 8 years, 107 patients developed rupture of the left ventricular free wall. Twenty nine patients had a subacute course and three of them survived for prolonged periods without having to have emergency surgical repair. At the onset of rupture on day 1 through 7 after AMI, the three survivors developed sudden hypotension accompanied by a new pericardial effusion. They were initially managed with hemodynamic support. Two patients had elective open-heart surgery 2 to 3 months after AMI, whereas one patient did nt require surgery. All three survived 1 1/2 to 8 1/2 years after AMI. This report indicates that a small subset of patients with subacute ventricular free wall rupture has a benign course that may allow for prolonged survival without having to have emergency surgical repair. PMID- 8620741 TI - Treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis. AB - Postneumonectomy syndrome has only been described after a right pneumonectomy except in cases of congenital mediastinal anomalies or right-sided aortic arch. Placement of Silastic prostheses into the empty hemithorax is the preferred surgical treatment; however, other nonsurgical options exist. Herein, we report a case of left postpneumonectomy syndrome in an adult who was successfully treated with the placement of an endobronchial stent. PMID- 8620742 TI - Postcardiac injury syndrome. An immunologic pleural fluid analysis. AB - The postcardiac injury syndrome (PCIS) is characterized by inflammation of the pericardium, pleura, and pulmonary parenchyma following a variety of cardiac injuries. Although it has been clinically recognized for decades, confirmation of the syndrome has been problematic owing to lack of a sufficiently diagnostic test. Previously, we have reported pleural fluid characteristics which help to exclude other diagnoses that may mimic the syndrome. We describe the first immunologic assessment, including antimyocardial antibody testing, of pleural fluid from a patient with PCIS which supports a local immunologic mechanism in the pathogenesis of the syndrome. These results support the important role of pleural fluid analysis in the diagnosis of PCIS. PMID- 8620743 TI - Similar pleural fluid findings in pleuropulmonary tularemia and tuberculous pleurisy. AB - Biochemical and cellular characteristics of pleural fluid from two patients with pleuropulmonary tularemia and 39 patients with tuberculous pleurisy were compared. High pleural fluid concentrations of adenosine deaminase, lysozyme, and beta 2-microglobulin occurred in both diseases. As is the case with tuberculous pleural effusions, pleural fluid in tularemia showed an abundance of lymphocytes, predominantly CD4-positive T lymphocytes. The similar pleural fluid findings suggest analogous local pathogenetic mechanisms in tularemia and tuberculosis. In the diagnostic evaluation of a lymphocyte-rich exudative pleural effusion with a high adenosine deaminase concentration, a possible cause to consider is tularemia. PMID- 8620744 TI - Delayed right heart failure following lung transplantation. AB - Dynamic right ventricular outflow tract obstruction (RVOTO) has been reported following lung transplantation for pulmonary hypertension, usually in association with the use of inotropic agents. This report describes delayed severe right sided heart failure associated with right ventricular outflow tract obstruction following sequential bilateral lung transplantation and closure of a ventricular septal defect. The patient had no evidence of outflow tract obstruction in the early posttransplant period but developed progressive right heart failure more than 2 months later. Catheterization revealed dynamic RVOTO and an elevated right ventricular end-diastolic pressure. The patient was treated with metoprolol tartrate and diltiazem hydrochloride with resolution of the outflow tract obstruction and heart failure. This case demonstrates that RVOTO can occur in the late posttransplant period and must be included in the differential diagnosis for patients who develop right-sided heart failure. PMID- 8620745 TI - Prolonged survival of a patient with left ventricular pseudoaneurysm following myocardial infarction and mitral valve replacement. AB - We present the case of a patient with left ventricular pseudoaneurysm following acute myocardial infarction. Survival for 2 years following diagnosis, despite the large size of the aneurysm, and subsequent management with cardiac transplantation represent unusual and interesting aspects of this complication of myocardial infarction. PMID- 8620746 TI - Peritoneal-pleural communications in hepatic hydrothorax demonstrated by thoracoscopy. AB - To understand the mechanism of hepatic hydrothorax clearly, thoracoscopy was performed with a flexible bronchoscope. It revealed an intrathoracic influx of ascitic fluid via a bleb and two defects located in the tendinous portion of the right hemidiaphragm and confirmed the existence of transdiaphragmatic peritoneal pleural communications. PMID- 8620747 TI - Pulmonary tuberculosis and steroids. PMID- 8620748 TI - Hazards of ultraviolet lighting used for tuberculosis control. PMID- 8620749 TI - A matter of choice? PMID- 8620750 TI - Respiratory care education includes intubations. PMID- 8620751 TI - Nicotine is hazardous to your heart. PMID- 8620752 TI - Nutritional state and exercise tolerance in patients with COPD. PMID- 8620753 TI - The effect of prophylaxis on the outcome of HIV-associated Pneumocystis carinii pneumonia. PMID- 8620754 TI - Treating patients who have Klebsiella pneumoniae pneumonia. PMID- 8620755 TI - The contribution of respiratory viruses to severe exacerbations of asthma in adults. PMID- 8620756 TI - Dyspnea doesn't always signify bronchial asthma. PMID- 8620757 TI - Evaluating the bronchodilator response in elderly who have asthma. PMID- 8620758 TI - Effects of noninvasive ventilation on survival in patients with Duchenne's muscular dystrophy. PMID- 8620759 TI - Acute myocardial infarction. Then and now. PMID- 8620760 TI - Treatment of carbon monoxide poisoning. PMID- 8620761 TI - False-low carbon monoxide diffusing capacity measurement after general anesthesia. PMID- 8620762 TI - [Use of stomatological instruments for surgical treatment of recurrent dislocation of the shoulder]. PMID- 8620763 TI - [Advances in diagnosis and treatment of idiopathic scoliosis in children and adolescents]. PMID- 8620764 TI - [Degenerative spondylolisthesis]. AB - Results of surgical treatment for degenerative spondylolisthesis in 31 patients are presented. Disabling lumbar and radicular pain not responding to conservative treatment was the indication for surgery in all cases. Twenty-six patients underwent neural decompression and segmental fusion; in 5 patients with no radicular symptoms fusion alone has been performed. In 15 patients with partially preserved intervertebral disc the fusion embraced the pedicle and processus transversus, in other 15 with damaged disc posterior fusion was done and in one case intervertebral fusion was performed. In 7 cases of posterior fusion a wire loop was used for internal stabilization of vertebrae. There were 28 good and fair results (91%) and in 3 patients no improvement was attained. The authors conclude surgical treatment according to the indications presented in this paper is the most beneficial mode of treatment for degenerative spondylolisthesis. PMID- 8620765 TI - [The value of CT in diagnosis of low back pain syndromes]. AB - The authors discuss results of CT studies in 300 patients with low back pain syndromes. Apart from herniated discs (56.3% of cases) pain could be often explained by degenerative changes of facet joints or bulging disc leading to spinal canal stenosis. In the group of patients operated on due to disc herniation correlation between CT and surgical results has 85.4%. In the authors' opinion CT is an effective, non-invasive diagnostic method in patients with low back pain syndromes. PMID- 8620766 TI - [Use of multiplanar reconstruction of CT images (MPR) in diagnosis of low back pain syndromes]. AB - The authors describe program of multiplanar reconstruction of CT images (MPR) and discuss its usefulness in diagnosing low back pain syndromes. The program was especially useful in differentiation between herniated disc and osteophytes, in spondylolisthesis and in the cases of asymmetric spine. MPR enables also imaging of pathological spinal lesions in the operative view which is helpful for planning of the surgery. In the authors' opinion CT with MPR is not inferior and in some authors aspects even superior to MR imaging. PMID- 8620767 TI - [Transthoracic approach to the thoracic spine via the rib cage]. AB - Transthoracal approach to the thoracic spine is presented. It has been used in 19 patients with trauma or tumor within thoracic spine. This approach allowed for direct access to the vertebral body where the pathology was located. Transthoracal approach, however, should be performed by a surgeon with sufficient knowledge of thoracic surgery. PMID- 8620768 TI - [Anatomy and development of the hip joint during growth. I. anatomy and development of the acetabulum]. AB - Anatomy and development of the hip joint during growth is discussed on the basis of vast literature. The issue of acetabular growth zones and factors determining normal growth are presented. PMID- 8620769 TI - [Treatment of congenital dislocation or subluxation of the hip taken from personal material--factors determining the course and result of treatment]. AB - A series of 65 children (130 hips) treated for congenital dislocation or subluxation of the hip with Pavlik harness been retrospectively reviewed. All patients were managed by the same physician and according to previously established principles. Mean follow-up after the termination of treatment was 7.5 months. Age at the onset of treatment and initial state of the hip were recognized as two major factors influencing the course and result of treatment. It was found, that the harness treatment is best initiated in first 9 weeks after birth and even a few weeks delay may seriously affect its efficacy. A dynamic index on alfa angle changes on serial ultrasound images of the hip (Graf method) has been introduced. PMID- 8620770 TI - [Treatment outcome for dysplastic hip after overhead traction]. AB - Clinical and radiological analysis has been carried out in series of 71 dysplastic hips in children aged 7-21 months of life (mean 16 months) treated with an over head traction. At one year follow-up there were clinically 22% excellent results, radiologically 10% excellent results and 25% poor ones. At the age of 5 complete remodeling of the hip was found in 21% of cases, and poor radiological result in 29% of cases. Avascular necrosis of the femoral head was found in 25% of patients at 1 year follow-up. PMID- 8620771 TI - [Prospects of using ultrasonic examination in Perthes disease]. AB - The paper indicates benefits from ultrasound early diagnostics and management monitoring in Perthes disease. Fifty-two children with 56 hips (35 boys and 17 girls) aged 6-12 years treated because of aseptic of the femoral head to Orthopaedic Department Lodz were included in this study. Five MHz transducer was positioned anteriorly along the anterior margin of the femoral neck in external rotation of the limb, neutral and internal one as well as in perpendicular fashion to the long axis of the neck. These four images were compared with conventional a-p and Lauenstein radiographs. Ultrasound allowed for assessment of the synovial effusion, shape of the epiphysis, configuration of the physis and the shape of metaphysis. High correlation between ultrasound examination and radiography was found. Ultrasonography allowed for assigning a hip to one of Catterall's type. It has also served well for monitoring of the treatment and reduced the number of radiological exposures. PMID- 8620772 TI - [Use of muscular and cutaneous fascial flaps in treatment of open tibial fracture with soft tissue defects]. AB - Owen experience in treatment of 14 patients with tibial fracture complicated by wasted soft tissue defect is presented. External fixation has been combined with transposition of medial head of gastrocnemius, soleus muscle or transposition of cutaneous fascial flap has healed the infection ceased and the fracture united in all cases. Encouraging results prove efficacy and usefulness of this method. PMID- 8620773 TI - [Hemangioma of bone and soft tissue in the midfoot--case report]. AB - A case of in isolated arterovenous developmental disorder in 23 years old female is described. Bony changes were at the beginning obscured by skin and soft tissue changes. The diagnosis has been established on the basis of histopathological evaluation of the II metatarsal bone and soft tissue vascular changes removed at the operation. PMID- 8620774 TI - [Absorbable implants in traumatology of the musculoskeletal system]. AB - Absorbable implants made of synthetic biodegradable polymers were recently used clinically especially for the fixation of small intra-articular fractures and physical fractures. In the paper history research and current concepts concerning biological response to biodegradable polymers and main indications for using absorbable implants for fixation of fractures are presented. PMID- 8620775 TI - [Osteopoikilosis--case report]. AB - A case of osteopoikilosis in 15 years old female with painful hip is presented. Genetic background of this condition has been confirmed by finding mother, two brothers and sister of the patient who affected with osteopoikilosis. PMID- 8620776 TI - [Multifunctional external fixator compatible with the Zespol system and Schanz pins]. AB - The construction and use of new external fixator broadening the use and increasing efficacy of Zespol system is presented. The apparatus may be used primarily or may replace Zespol plate. It enables controlled interfragmental compression, limb elongation, correction of angular and translational dislocations. After bony union occurs the fixator may be replaced with Zespol plate and reapplied to another patient. It is also compatible with Schanz pins. PMID- 8620777 TI - Variations in colon and rectal surgical mortality. Comparison of specialties with a state-legislated database. AB - PURPOSE: This study was designed to examine variations in operative mortality among surgical specialists who perform colorectal surgery. METHODS: Mortality rates were compared between six board-certified colorectal surgeons and 33 other institutional surgeons using comparable colorectal procedure codes and a validated database indicating patient severity of illness. Thirty-five ICD-9-CM procedure codes were used to identify 2,805 patients who underwent colorectal surgery as their principal procedure between July 1986 and April 1994. Atlas, a state-legislated outcome database, was used by the hospital's Quality Assurance Department to rank the Admission Severity Group (ASG) of 1,753 patients from January 1989 to April 1994 (higher ASG, 0 to 4, indicates increasing medical instability). RESULTS: Colorectal surgeons had an eight-year mean in-hospital mortality rate of 1.4 percent compared with 7.3 percent by other institutional surgeons (P = 0.0001). There was a significantly lower mortality rate for colorectal surgeons compared with other institutional surgeons in ASG 2 (0.8 and 3.8 percent, respectively; P = 0.026) and ASG 3 (5.7 and 16.4 percent, respectively; P = 0.001). CONCLUSIONS: Board-certified colorectal surgeons had a lower in-hospital mortality rate than other institutional surgeons as patients' severity of illness increased. PMID- 8620778 TI - Perianal Crohn's disease--is it all bad news? AB - PURPOSE: The outcome of treatment of perianal Crohn's disease was assessed in 127 patients. METHODS: A retrospective review of the case notes of 415 patients who were seen in the North East of Scotland between 1985 and 1989 was undertaken. RESULTS: A total of 127 of 415 patients with Crohn's disease had perianal involvement. In 56 patients, perianal disease was the presenting complaint. Ninety-nine of the 127 patients had colonic involvement. Thirty-two were treated with metronidazole and 41 were treated with azathioprine, with at least temporary improvement in 91 and 68 percent, respectively. Seventy patients had treatment for fistula-in-ano, and in 50 percent of patients permanent healing was achieved. In general, treatment and outcome were largely related to the extent and severity of gut involvement. Proctectomy was performed in 32 patients (in 11 because of ongoing colonic disease). Only seven patients had proctectomy solely because of perianal disease. Proctectomy was necessary in 32 of 99 patients with colitis and perianal disease but in none of 28 patients without colonic involvement. Primary healing of the perineal wound was obtained in 17 patients, and only one patient has an unhealed perineal wound at the time of reporting. CONCLUSION: Perianal Crohn's disease does not inevitably lead to panproctocolectomy. Cautious surgery for fistula when rectal inflammation is quiescent is worthwhile. Loss of bowel continuity is more likely when colitis coexists with perianal disease. Panproctocolectomy is often indicated because of the combination of colitis and perianal disease rather than for perianal disease alone. PMID- 8620780 TI - Laparoscopic resections for colorectal carcinoma. A three-year experience. AB - Laparoscopic resection for carcinoma of the colon and rectum is currently under intense scrutiny. PURPOSE: The purpose of this study is to review our three-year experience of laparoscopic surgery for colon and rectal carcinoma. METHODS: From October 1991 to September 1994, 76 laparoscopic procedures were performed for colorectal neoplasia (32 males and 44 females; mean age, 69 years). Fifty-five procedures were done for carcinoma, 16 for large polyps, and five for diversion in patients with unresectable cancer. For resectable tumors, the average size was 4 cm; staging was as follows: Dukes A, 10 patients; Dukes B1, 11; Dukes B2, 18; Dukes C1, 1; Dukes C2, 9; and Dukes D, 8. Fourteen cases (25 percent) that were converted to open procedures were compared with the 41 cases that were completed laparoscopically for differences in tumor size, surgical margins, number of lymph nodes harvested, length of hospital stay, and evidence of recurrence. Procedures completed laparoscopically were then compared with a group of open controls completed during the same time period. RESULTS: During the first six months, the conversion rate was 32 percent but dropped to 8 percent in the last six months. There were a total of 19 complications (25 percent), of which 8 (14 percent) were directly related to the laparoscopic technique. The mean number of lymph nodes harvested in laparoscopic resection for carcinoma was 8.5, and the average closest tumor margin was 4.5 cm. When laparoscopic resections were compared with converted and standard open colectomies, there was no significant difference in tumor margins or numbers of nodes resected. Length of stay was significantly shorter for anterior resections completed laparoscopically than for converted or conventional colectomies. Although this was also the trend for right hemicolectomies, it did not reach statistical significance. Mean follow-up of the group completed laparoscopically was 16.7 months, during which there was one recurrence. There were no trocar site recurrences. CONCLUSIONS: This early experience seems to indicate that laparoscopic surgery for colorectal carcinoma does not per se compromise surgical oncologic principles and encourages us to continue our critical appraisal of this technique. PMID- 8620779 TI - Mutation of p53 tumor suppressor gene in flat neoplastic lesions of the colorectal mucosa. AB - PURPOSE: In a recent comparative histologic survey of flat colorectal neoplasias, we found more lesions with high-grade dysplasia (HGD) and carcinoma in Japanese than in Swedish patients. The purpose of this work was to assess the p53 protein overexpression in flat colorectal neoplasias in Swedish patients and to compare results with those reported in Japan. METHOD: A total of 57 neoplastic lesions of the colorectal mucosa were investigated: 29 had been regarded both at endoscopy and at histology as flat and the remaining 28 as exophytic. Deparaffinized, rehydrated sections were treated immunohistochemically to detect the p53 protein. Lesions having a moderate (++) or high ( ) staining were considered as overexpressing the p53 protein. RESULTS: Results indicated that 16.7 percent (1/6) of the exophytic adenomas with low-grade dysplasia (LGD) had distinct p53 overexpression as well as 57.1 percent (8/14) of those with HGD and 87.5 percent (7/8) with invasive growth. In flat neoplastic lesions, 7.7 percent (1/13) of the tubular adenomas with LGD, 25 percent (3/12) of tubular adenomas with HGD, and 75 percent (3/4) of adenocarcinomas arising in flat adenomas had p53 overexpression. CONCLUSIONS: In Swedish patients, the proportion of flat and exophytic colorectal neoplasias showing p53 immunoreactivity increased with increasing degree of dysplasia, the highest percent being recorded in lesions with invasive growth. Because a similar stepwise increase was reported for exophytic and flat colorectal neoplasias in Japan, it seems that the comparison of results in both countries is justifiable. One possible conclusion from this comparison is that the higher proportion of flat neoplastic colorectal lesions with HGD and carcinoma in the Japanese (compared with the Swedish) takes place for reasons extraneous to the overexpression of the p53 protein. PMID- 8620781 TI - Laparoscopic-assisted colorectal surgery. Lessons learned from 240 consecutive patients. AB - PURPOSE: To audit the development and outcomes of laparoscopic colorectal surgery at the Royal Brisbane Hospital. METHODS: Since July 1991, laparoscopic-assisted colectomy for benign and malignant colorectal disease has been performed on more than 300 patients at the Royal Brisbane Hospital. This paper summarizes the outcome for the first 240 patients who underwent a laparoscopic colorectal procedure. All laparoscopic data were collected prospectively, and for selected studies, data were compared with open surgical controls. RESULTS: Nineteen patients required open conversion (7.9 percent). There was a significant decrease in wound infection rates in patients having a laparoscopic-assisted colectomy (3.6 percent) compared with historical controls (7.9 percent) (P < 0.05; chi squared). There were five anastomotic leaks, five laparotomies for postoperative adhesive obstruction, and four perioperative deaths. A total of 103 patients had a procedure for colorectal cancer. Of the 79 potentially curative procedures, there have been 5 (6.3 percent) recurrences to date. CONCLUSION: The overall morbidity and mortality in this series seem to be acceptable compared with that of open procedures. PMID- 8620782 TI - Long-term results of total abdominal colectomy for chronic idiopathic constipation. Value of preoperative assessment. AB - PURPOSE: A small proportion of patients with chronic idiopathic constipation are incapacitated by the problem. We have assessed 1) the efficacy of total abdominal colectomy, and 2) the predictive value of preoperative testing. METHODS: Preoperative testing included complete history and physical examination, appropriate biochemical and hematologic assessment, psychiatric interview, colon transit studies using ingested radiopaque pellets, anorectal manometry, colonic intraluminal manometry, and measurement of colon diameters and length on barium enema examination. All patients were followed for 65 +/- 40 months. RESULTS: Seventy-one percent had excellent or very good results. Twenty-one percent were satisfied, had improved the quality of their life, and felt the operation was worthwhile despite frequent residual or new symptoms. Two (8 percent) patients did not improve. Patients with a psychiatric history or physiologic evidence of an afferent nerve defect had poorer results (P < 0.05). CONCLUSIONS: Total abdominal colectomy with ileorectal anastomosis is highly effective in alleviating symptoms in patients with chronic idiopathic constipation. PMID- 8620783 TI - Surgical outcome in acquired immunodeficiency syndrome patients with non Hodgkin's lymphoma of the gastrointestinal tract. AB - PURPOSE: Incidence of non-Hodgkin's lymphoma (NHL) has shown a dramatic increase, concurrent with the epidemic of acquired immunodeficiency syndrome (AIDS). In terms of surgical intervention, management of the patient with AIDS-NHL remains unclear. Purpose of this paper was to determine the role and outcome of surgical intervention in patients with AIDS-NHL of the gastrointestinal (GI) tract. METHODS: Data were obtained by retrospective chart review. RESULTS: From 1980 to 1993, charts of 22 patients with diagnosis of AIDS-NHL of the GI tract who underwent either biopsy or surgical procedure were reviewed. All patients were male, with a mean age of 35.7 years. Sixty-seven biopsies were performed in the 22 patients identified. No morbidity or mortality was associated with any of the biopsy procedures. Major intra-abdominal operations were performed in eight patients, including seven who underwent primary resections of lymphomas. Mean survival for the group as a whole was 18 months, although that for the seven patients undergoing resection was 20.4 months. CONCLUSIONS: Diagnosis of AIDS-NHL of the GI tract should not discourage performance of otherwise appropriate surgical procedures. PMID- 8620785 TI - Outcome of restorative perineal graciloplasty with simultaneous excision of the anus and rectum for cancer. A ten-year experience with 81 patients. AB - PURPOSE: To review the complications, survival, and long-term functional outcome of patients with anorectal cancer who had restorative perineal graciloplasty (RPG) simultaneously with abdominoperineal resection (APR). METHODS: Between 1985 and 1994, 81 patients underwent APR plus RPG. Gracilis muscles were then conditioned by electrostimulation, either intermittently or chronically. Thirtyseven surviving patients were followed for a mean of 78.6 months and were analyzed for long-term functional outcome of RPG. RESULTS: Postoperative complications occurred in 30 patients (37 percent). Crude five-year survival rate was 58 percent, and five-year estimated cumulative probability of survival was 65 percent. There was no statistically significant difference for probability of survival and for probability of disease-free interval between uncomplicated and complicated patients. Fecal continence was obtained in 90 percent of patients. CONCLUSION: RPG does not reduce the effectiveness of APR in the cure of cancer. Postoperative complications, though frequent, were not serious and resolved without sequelae. There was no statistically significant impact on the probability of survival and of disease-free interval by graciloplasty. Continence was achieved by most patients (90 percent) who underwent RPG simultaneously with APR. PMID- 8620784 TI - Escherichia coli heat-stable enterotoxin receptors. A novel marker for colorectal tumors. AB - PURPOSE: Receptors for Escherichia coli heat-stable toxin (ST) are selectively expressed in membranes of intestinal mucosa cells and colon carcinoma cells in vitro, suggesting their use as a marker for colorectal tumors in vivo. The present studies examined the expression and function of ST receptors in normal human tissues and primary and metastatic colorectal tumors obtained from patients at surgery. METHODS: Surgical specimens were obtained as follows: from normal colon; from primary adenocarcinomas from all anatomic divisions of the colon and rectum; from gallbladder, kidney, liver, lung, lymph node, ovary, peritoneum, stomach; and from colon carcinomas metastatic to liver, lung, lymph node, ovary, and peritoneum. Membranes prepared from these specimens were assessed for the presence and functional characteristics of ST receptors. RESULTS: ST bound specifically to membranes from each division of normal colon and rectum and all primary and metastatic colorectal tumors examined. The affinity and density of ST receptors were similar in tumors of different grades and from various metastatic sites. ST-receptor interaction was coupled to activation of guanylyl cyclase in all normal samples of colon and rectum and all primary and metastatic colorectal tumors examined. In contrast, neither ST binding nor ST activation of guanylyl cyclase was detected in any extraintestinal tissues examined. CONCLUSIONS: Functional ST receptors are expressed in normal colonic tissue and primary and metastatic colorectal tumors but not by extraintestinal tissues in humans. Expression of ST receptors does not vary as a function of the metastatic site or grade of these tumors. Receptors expressed by colorectal tumors retain their characteristic function, with binding of ST coupled to activation of guanylyl cyclase. These studies support the suggestion that ST receptors represent a specific marker for human colorectal tumors that may have use as a target for directing diagnostics and therapeutics to these tumors in vivo. PMID- 8620786 TI - Diverticular hemorrhage in the elderly--is it well tolerated? AB - PURPOSE: Elderly patients frequently develop lower gastro-intestinal bleeding secondary to diverticulosis. This select group of patients potentially tolerates blood loss poorly, often have coexisting cardiovascular morbidity, and may not tolerate surgical intervention. Thus, optimal management of elderly patients with diverticular hemorrhage remains difficult. METHODS: All patients who were admitted with the diagnosis of diverticulosis at the St. Louis University affiliated hospitals during the past 60 months were identified. Those with diverticular bleeding were extracted. Patients were reviewed as to age, sex, diagnosis of diverticular bleeding, number of bleeding episodes, lowest hemoglobin before transfusion, amount of blood received, treatment, operations, the presence of recurrent bleeding, morbidity, and mortality. RESULTS: One hundred fifteen consecutive patients, age 70 years admitted with lower gastrointestinal hemorrhage secondary to diverticulosis who required transfusion, were identified. Mean age was 79 years; 26 of 115 (23 percent) were more than 80 years of age; 78 of 115 (54 percent) were males; 39 of 115 (34 percent) had more than one previous admission for diverticular hemorrhage. The mean serum hemoglobin was 8.9 g/dl. All patients underwent colonoscopy; 34 of 115 (29 percent) underwent 99Tc scanning, of which 18 of 34 (54 percent) underwent arteriogram. Seven of 18 (39 percent) demonstrated extravasation secondary to bleeding diverticulosis. The mean transfusion requirement was 2.8 (range, 1-17) units; 21 of 115 (18 percent) required intestinal resection; 2 of 21 (9 percent) experienced a 30-day mortality. Among those, 94 of 115 were treated without surgery, and 3 of 94 (4 percent) died. Mortality was independent of initial hemoglobin (P = 0.21), previous diverticular hemorrhage (P = 0.44), amount of blood transfused (P = 0.36), and type of treatment (0.09). CONCLUSIONS: Most diverticular bleeding in the elderly is well tolerated using nonoperative management. Success and safety of treatment does not seem to depend on a history of previous diverticular bleeding, initial hemoglobin, or amount of blood transfused. The majority of patients are treated nonoperatively. Surgical intervention seems to be well tolerated. PMID- 8620787 TI - Formalin instillation for refractory radiation-induced hemorrhagic proctitis. Report of 16 patients. AB - PURPOSE: Our goal was to evaluate use of topical (4 percent) formalin in management of radiation-induced hemorrhagic proctitis, refractory to other methods of treatment. Specifically, we wished to determine its safety, ability to stop bleeding, and complications associated with therapy. METHODS: Sixteen patients with radiation-induced hemorrhagic proctitis were treated with topical (4 percent) formalin. All had been previously treated with conservative regimens such as cautery, topical steroids, or laser, but these had failed. Five-hundred milliliters (ml) of a 4 percent formalin solution was instilled into the rectum in 50-ml aliquots. Each aliquot was kept in contact with rectal mucosa for approximately 30 seconds. Treatments were performed under local anesthesia in nine patients, sedation only in four, spinal in two, and general in one patient. RESULTS: In 12 patients, bleeding stopped after a single formalin instillation; in 3, bleeding was considerably reduced but continued sporadically. One patient required three treatments before bleeding stopped. Four patients developed postoperative anal pain, of which one also had significant tenesmus and reduced capacity. Of these four patients, only two had significant anal pain and fissures that lasted longer than one month. CONCLUSIONS: Topical (4 percent) formalin is safe and effective in treatment of radiation-induced hemorrhagic proctitis. A single treatment will stop bleeding in 75 percent of patients. PMID- 8620788 TI - Wound recurrence following conventional treatment of colorectal cancer. A rare but perhaps underestimated problem. AB - Reports of trocar and extraction site tumor recurrences following laparoscopic colectomy raise concern that such recurrences may be occurring more frequently with laparoscopic compared with open colectomy. Contemporary data on the incidence of incisional recurrence following open colectomy, in the age of adjuvant therapies, are not available. PURPOSE: This study was undertaken to examine the incidence and clinical features of wound recurrence in current prospective trials including 1,711 patients with primary adenocarcinoma of the colon or rectum treated for cure. METHODS: Files of all patients with recurrence (n = 623) were reviewed. Each site of recurrence was recorded separately. All patients have been followed prospectively, and 3-year and 4-year data are mature on 100 and 70 percent, respectively. Stage at diagnosis was B2 in 344 patients and C in 1,367 patients (> 4 nodes positive in 346 patients). RESULTS: Recurrence was identified in 623 patients (36.4 percent) and occurred at a mean of 1.5 years following primary treatment. Eleven patients (0.6 percent) had documented incisional recurrences (9 abdominal wound, 1 perineal wound, and 1 stoma wound). Only four were diagnosed clinically, and the remaining seven were diagnosed incidentally at reoperation. Of 11 patients with incisional wound recurrences, 2 had primary Stage B2 and 9 had primary Stage C disease. Nine of 11 patients were found to have multiple sites of recurrence at time of recurrence. At a mean follow-up of 1.8 years after recurrence, 3 of 11 patients are alive with disease, although 8 have died because of disease. CONCLUSIONS: Incisional recurrence is uncommon, although likely underestimated, following conventional treatment of colorectal carcinoma. Its occurrence is usually a harbinger of diffuse intra abdominal disease. These data may provide useful information for investigations of laparoscopic approaches to colon cancer. PMID- 8620789 TI - Long-term seton drainage for high anal fistulas in Crohn's disease--a sphincter saving operation? AB - METHODS: Forty-one consecutive patients with Crohn's disease who underwent long term seton drainage for high transsphincteric, suprasphincteric, or extrasphincteric anal fistula from 1985 to 1993 were reviewed. The subsequent associated procedure was simple seton removal (18), secondary fistulotomy (7), rectal flap advancement (3), and proctectomy (2). Eleven patients still had the seton in place. RESULTS: Recurrence developed in seven patients (39 percent) undergoing simple seton removal and in one patient undergoing rectal flap advancement. None of the patients treated by secondary fistulotomy developed a recurrence. At the end of follow-up, five patients (12 percent) required proctectomy mainly for severe proctitis, and five patients (12 percent) developed anal incontinence, which was severe in two. CONCLUSION: Long-term seton drainage for high and fistula in Crohn's disease is efficacious in both treating sepsis and preserving anal sphincter function. PMID- 8620790 TI - Effect of nifedipine on rectoanal motility. AB - PURPOSE: Based on the rationale that the calcium channel blocker, nifedipine, decreases lower esophageal sphincter pressure in achalasia, a prospective controlled trial was performed to evaluate the effect of sublingual nifedipine on the anal sphincter of controls and patients with high anal resting pressures. METHODS: Ten age-matched and sex-matched controls without evidence of anal disorder and ten patients with hemorrhoids and/or fissure-in-ano were included in the study. Anorectal manometry, with an eight-channel, water-perfused catheter was performed on all patients before and 30 minutes after administration of 20 mg of sublingual nifedipine. RESULTS: Nifedipine significantly reduced anal resting pressure in both controls and patients by approximately 30 percent (P < 0.001 and P < 0.0001, respectively). A significant reduction was also noted in the length of high-pressure zone of the anal sphincter (P < 0.02 for both groups) and in the frequency (controls, P < 0.05; patients, P < 0.03) and amplitude (controls, P < 0.03; patients, P < 0.009) of slow waves in both groups, whereas the presence, frequency, and amplitude of ultraslow waves were significantly reduced only in the patient group (P < 0.05; P < 0.01; P < 0.0005, respectively). CONCLUSION: Nifedipine reduces the activity of the internal anal sphincter both in controls and patients with high anal resting pressure. The drug might be of some use in relieving symptoms in patients with hemorrhoids or anal fissure. PMID- 8620791 TI - Role of cytokines and platelet-activating factor in inflammatory bowel disease. Implications for therapy. AB - BACKGROUND: Platelet-activating factor (PAF) and cytokines, such as interleukins, tumor necrosis factor, and others, are thought to play a role in the inflammatory process involving gastrointestinal disorders such as Crohn's disease, ulcerative colitis, ischemic colitis, or antibiotic-associated colitis. PURPOSE: This study was undertaken to review the latest literature on the role of PAF and cytokines in the genesis of inflammatory bowel disease and implications for therapy and management. RESULTS: PAF is an endogenous phospholipid involved in hypersensitivity and inflammatory reactions such as platelet and neutrophil aggregation, vasodilation, increased vascular permeability, and leukocyte adhesion, which have been associated with inflammatory processes. Cytokines are peptides that regulate and coordinate inflammatory and immunologic responses. Increased production of cytokines has been reported during Crohn's disease and ulcerative colitis and is correlated with disease activity. CONCLUSIONS: Because PAF and cytokines may have an important role in the pathogenesis of inflammatory bowel disease, their inhibition by specific antagonists, mediators, or other agents such as steroids may have a potential therapeutic benefit in treatment and management of these inflammatory diseases in the near future. PMID- 8620792 TI - Island flap anoplasty for treatment of transsphincteric fistula-in-ano. AB - BACKGROUND: Treatment of fistula-in-ano often replaces one problem, risk of persistent anal sepsis, with another, either incontinence after fistulotomy or mucosal ectropion after rectal flap advancement. A new technique for treatment of transsphincteric fistulas is described that could eliminate risk of both complications. TECHNIQUE: Island flap anoplasty, previously used in management of anal strictures or ectropion, is modified to treat transsphincteric fistulas. RESULTS: The operation has been performed in 11 patients, 3 of whom had Crohn's disease. Follow-up varied from one to ten months. Early recurrences have occurred in three patients, two with Crohn's disease and one without. Remaining patients have done well. CONCLUSION: This procedure is technically easy to perform and appears to cure transsphincteric fistulas while preserving anal sphincter. In the event of persistence of fistula, other operative options are not eliminated by this procedure. We feel that further experience and longer follow-up is needed to define precise indications for this procedure and to determine if continence is improved more so than with standard fistulotomy. PMID- 8620793 TI - Reconstruction with bilateral gluteus maximus myocutaneous rotation flap after wide local excision for perianal extramammary Paget's disease. Report of two cases. AB - PURPOSE: Extramammary Paget's disease is a rare dermatosis. Wide local excision is recommended in patients with perianal extramammary Paget's disease. After wide local excision, it is necessary to do reconstruction, with preservation of bowel function. We present here two cases of perianal Paget's disease, in which the patients were treated by reconstruction with bilateral gluteus maximus myocutaneous rotation flap after wide local excision. PATIENTS AND METHODS: A 55 year-old woman and 58-year-old man were admitted with anal pain and bleeding. Histologic examination of the perianal lesion revealed the presence of typical Paget's cells, and no underlying carcinoma and no distant metastasis was detected in either patient. Wide local excision, including the rectal mucosa, was performed, with reference to intraoperative frozen sections. Surgical defect was overlapped with bilateral gluteus maximus myocutaneous rotation flap, and the anus was reconstructed. RESULTS: Postoperative bowel function and quality of life were well preserved, and flaps healed satisfactorily. Patients have had no recurrence and have been able to return to work. CONCLUSION: Bilateral gluteus maximus rotation flap may be useful after wide local excision of perianal extramammary Paget's disease without underlying invasive carcinoma. PMID- 8620795 TI - Relationship between colorectal and esophageal cancer. PMID- 8620794 TI - Appendiceal mucocele with concomitant colonic cancer. Report of two cases. AB - PURPOSE: Mucocele of the appendix is an uncommon disorder, usually found incidentally during ultrasonography or radiographic studies. We report two cases of combined appendiceal mucocele and colonic cancer. METHODS: The two cases were analyzed for the clinicopathologic characteristics such as history, presentation, laboratory data, radiologic and endoscopic studies, pathology, and p53 immunoreactivity. RESULTS: Two patients were diagnosed with an appendiceal mucocele by ultrasound of the abdomen, together with computed tomography. Colonoscopic examination subsequently revealed synchronous colonic adenocarcinoma in both patients. Ileocecal resection following endoscopic polypectomy and a right hemicolectomy was performed for each patient. An appendiceal mucocele was histologically diagnosed as a mucinous cystadenoma. Immunohistochemical detection of abnormally high level of p53 protein was observed in colonic adenocarcinomas of both patients, whereas both appendiceal cystadenomas were negative for p53. CONCLUSIONS: To be remembered is the high frequency of concomitant gastrointestinal tumors in patients with appendiceal mucocele, especially caused by mucinous neoplasms. A total colonoscopic surveillance will afford earlier diagnosis of synchronous colonic cancers in these patients. PMID- 8620796 TI - Does eversion of the anorectum during restorative proctocolectomy influence functional outcome? AB - PURPOSE: The aim of this study was to determine the effect of eversion of the anorectum during restorative proctocolectomy (RP) for ulcerative colitis on functional outcome. METHODS: One hundred seventeen patients underwent RP with stapled end-to-end ileal pouch-anal anastomosis (EEA), without resection of the anal mucosa. Sixty-four underwent EEA with eversion of the anorectum, and 53 underwent EEA without eversion. Each patient underwent paired studies of anorectal function before and a median of 12 months after RP. RESULTS: One year after RP, median (interquartile range) maximum resting pressure was 69 (range, 51 88) cmH2O in those patients who underwent eversion vs. 80 (range, 64-90) cmH2O in patients without eversion (P < 0.04). Threshold sensation in the upper, middle, and lower thirds of the anal canal were 9.1, 7.4, and 6.8 mA after eversion vs. 6.9, 4.9, and 3.8 mA without eversion (P = 0.003, P < 0.001, P < 0.001, respectively). Before operation, all patients had a rectoanal inhibitory reflex; however, after RP, 54 of 64 patients in the eversion group and 50 of 53 patients with a stapled EEA without eversion had an inhibitory reflex (P = not significant). Leakage of mucus was experienced by 11 patients who underwent eversion, compared with 9 patients without eversion. Fifty-six of 64 patients with eversion could defer defecation for more than 30 min compared with 43 of 53 patients without eversion. Twenty-two of 64 patients in the eversion group retained perfect discrimination between flatus and feces compared with 38 of 54 without eversion (P < 0.001). Level of the anastomosis was 1 (range, 0.5-3) cm above dentate line after eversion compared with 1.5 (range, 0-6) cm without eversion. CONCLUSION: Clinical outcome after RP with eversion was not as good as outcome after stapled EEA without eversion. Such a conclusion requires confirmation in a prospective control trial. PMID- 8620797 TI - Prognostic value of ploidy, cell proliferation kinetics, and conventional clinicopathologic criteria in patients with colorectal carcinoma: a prospective study. AB - PURPOSE: The aim of this study was to obtain additional biologic determinants that may be of use in segregating into subgroups with different prognosis patients with similarly staged colorectal cancers. METHODS: Between 1989 and 1991, a prospective study of prognostic factors has been performed in a group of 98 consecutive, unselected patients who underwent curative resections for primary untreated large bowel carcinoma. The fate of all patients is known at three years after operation. Clinical and pathologic data were recorded at the time of presentation and operation, and patients have been the subjects of regular follow up. Tumor DNA content was determined by flow cytometry, and cell proliferative activity was determined by autoradiography with tritiated thymidine labeling index (LI). RESULTS: Univariate analysis revealed that the most important predictors of survival (P < 0.001) were the presence of positive lymph nodes, the presence of preoperative complications, Dukes stage, and LI. The multivariate analysis showed that Dukes stage (P < 0.002) and LI (P < 0.0001) were the only factors significantly related to survival. Disease-free survival was influenced significantly by Dukes stage (P < 0.001), LI, according to the classification in the two groups of high and low proliferative activity, respectively, (P < 0.0001), LI, calculated as a continuous variable (P < 0.0002), and the presence of lymph node metastases (P < 0.003). Outcome (favorable/unfavorable) was influenced significantly by Dukes stage (P < 0.0001) and LI (P < 0.0001). Concordance for each patient between Dukes stage and outcome was 73.1 percent and between LI, calculated as a continuous variable, and outcome was 74.1 percent. If, on the other hand, Dukes stage and LI are used together, concordance with outcome reaches 89.2 percent. CONCLUSION: We can conclude that, from a practical point of view, LI is an essential factor that must be combined with pathologic variables for a better prediction of patient outcome. PMID- 8620798 TI - Effect of systemic steroids on ileal pouch-anal anastomosis in patients with ulcerative colitis. AB - BACKGROUND: Long-term steroid therapy predisposes to postsurgical complications, especially in patients with inflammatory bowel disease. PURPOSE: This study was undertaken to determine incidence of early septic complications after ileal pouch anal anastomosis (IPAA) in patients who are undergoing prolonged steroid therapy. METHODS: We reviewed charts of 692 patients undergoing restorative proctocolectomy and IPAA to treat ulcerative colitis. Incidence of early (within 30 days) septic complications and sepsis-related reoperations, in patients who were having high-dose (>20 mg of prednisone per day) and low-dose steroid therapy (<20 mg of prednisone per day) for more than one month before surgery, was compared with patients who were not receiving steroid therapy. Follow-up included an annual questionnaire and physical examination. RESULTS: Patients without steroid dose data recorded were excluded (n = 21). Of the 671 remaining patients, 310 received no steroids, 169 received low-dose steroids, and 192 received high dose steroids. These three groups were similar in gender composition, age at surgery, types of anastomosis (stapled or handsewn), and incidence of diabetes mellitus, peripheral vascular disease, and obesity. Early septic complications were found in 18 (6 percent), 14 (8 percent), and 12 (6 percent) patients without steroid therapy, those having low-dose steroid therapy, and those having high dose steroid therapy (P = 0.57), respectively. Sepsis- related reoperation rate (P = 0.73) and number of sepsis-related pouch excisions (P = 0.79) did not differ between groups. In patients undergoing IPAA without ileostomy, early septic complications were found in one (3.8 percent), two (20 percent), and five (50 percent) patients without steroid treatment, low-dose steroid therapy, and high dose steroid therapy (P = 0.004), respectively. CONCLUSION: In patients who are undergoing IPAA with diversion for ulcerative colitis, prolonged systemic steroid therapy before surgery is not associated with increased septic complications. PMID- 8620799 TI - Intra-abdominal patterns of disease dissemination in colorectal cancer identified using radioimmunoguided surgery. AB - PURPOSE: Patterns of metastatic spread are difficult to determine with routine postoperative follow-up. This study was undertaken to evaluate two selected populations of colorectal cancer patients injected and screened with anti-tumor antibody. METHODS: Eighty-six evaluable patients with colorectal cancer underwent exploratory laparotomy with both traditional surgical exploration and radioimmunoguided surgery (RIGS) following injection of 125I-labeled CC49 monoclonal antibody. RIGS-positive tissue detectable with a handheld gamma detecting probe was defined as tissue involved with the disease process. Comparisons were made between extent of disease using traditional exploration and extent using RIGS. RESULTS: In 41 patients with primary disease, traditional exploration detected 45 sites of disease (1.1 sites/patient) compared with 153 RIGS-positive sites (3.7 sites/patient). In 45 patients with recurrent disease, traditional exploration found 116 sites (2.6 sites/patient) vs. 184 RIGS-positive sites (4.1 sites/patient). Involvement by selected anatomic sites is shown below [Table: see text]. CONCLUSION: RIGS detected more tissue involved in disease process for all sites in both primary and recurrent disease except liver metastases. Areas with highest proportion of RIGS-positive tissue, the gastrohepatic ligament and celiac nodes, are rarely resected and are not pathologically examined. Positive RIGS localization of tumor antigen in these areas suggests more extensive dissemination of disease process. PMID- 8620801 TI - Clinical and physiologic effects of biofeedback in outlet obstruction constipation. AB - PURPOSE: We report the results of biofeedback (BF) on patients with outlet obstruction defecation (OOC), including those with and without measurable paradoxical puborectalis contractions (PP). Clinical and anorectal physiologic parameters (ARP) were assessed one week before and after a standardized course of BF. METHODS: Sixty-two consecutive patients (24 men, 38 women; mean age, 48 (standard error of the mean, 2.3) years) were recruited. All had persistent constipation despite six weeks of dietary fiber supplements. Colonic inertia was excluded by transit marker studies. Defecating proctography excluded anatomic abnormalities causing outlet obstruction. Patients underwent four outpatient sessions of biofeedback, each session lasting one hour. RESULTS: After BF, 56 patients (90.3 percent) were subjectively improved. Frequency of spontaneous bowel movements were significantly increased (P = 0.003). Frequency of laxative induced (P = 0.004) and enema-induced (P = 0.005) stools were reduced. Anal resting (P = 0.04) and squeeze (P = 0.002) pressures were increased. Number of patients with PP was reduced from 40 to 31 (P = 0.004). Presence of PP did not affect response to BF. There were no differences in ARP between the 56 patients who improved and the 6 who did not. There were no side effects or clinical regressions after a mean follow-up of 14.9 (standard error of the means, 0.9) months. CONCLUSIONS: BF effectively treated OOC in 90.3 percent, regardless of PP. Anal pressures were increased, and PP was decreased. PMID- 8620800 TI - 111In-CYT-103 scanning in recurrent colorectal cancer--does it affect standard management? AB - PURPOSE: In a blinded fashion, radiolabeled B72.3 was investigated in operative cases of recurrent colorectal cancer to determine if diagnostic accuracy would be improved to ultimately maximize curability and minimize interventional morbidities. METHODS: Study patients underwent conventional evaluation including history, physical examination, abdominal/pelvic computed tomographic scan (CT), colon examination, and carcinoembryonic antigen (CEA) determination, with select magnetic resonance imaging and ultrasonographic imaging as indicated. Murine monoclonal antibody B72.3 was labeled with indium-111 (111In-CYT-103 provided by Cytogen) and scans obtained at 48 hours and, selectively, at 72 and 96 hours. Unlike previous studies, the operating surgeon was blinded to 111In-CYT-103 abdominal scan results until surgical exploration was complete. RESULTS: Of 15 study patients (10 male; 5 female), average age was 57 years, and average CEA was 10 ng/ml (with eight elevated CEA levels). A single patient did not undergo surgery because of presence of pulmonary metastases identified on CT scan but not identified on a 111In-CYT-103 scan. Laparotomies included resection and intraoperative radiation (10), resection alone (1), and biopsy only (3). CT and 111In-CYT-103 scans were compared with operative findings. CT scans had an accuracy and positive predictive value of 47 and 100 percent, respectively, whereas those of 111In-CYT-103 scan were 60 and 82 percent, respectively. Contribution of the scan to diagnosis and management was graded by the surgeon as no effect (67 percent), beneficial effect (13 percent), or negative effect (20 percent). CONCLUSIONS: 111In-CYT-103 was more accurate compared with CT scan, but when value of the scan was examined with respect to its potential contribution to patient management, it was beneficial in only 13 percent of patients. Further refinements may enhance the value of antibody imaging techniques. PMID- 8620802 TI - Long-term causes of death following ileal pouch-anal anastomosis. AB - PURPOSE: The aim of this study was to identify the overall long-term causes of death in a large series of patients who were undergoing proctocolectomy with ileal pouch-anal anastomosis (IPAA). METHODS: Records of patients who underwent proctocolectomy with IPAA at the Mayo Clinic affiliated hospitals between January 1981 and October 1994 were reviewed to determine overall mortality, cause, and timing of death. RESULTS: A total of 1,603 patients underwent proctocolectomy with IPAA reconstruction (1,407 for chronic ulcerative colitis (CUC), 187 for familial polyposis (FAP), and 9 for other diagnoses). Thirty-two patients have died, with an overall mortality rate of 2 percent. Mean age at time of death was 40 (23-60) years. There was no significant difference in overall mortality between patients with CUC and patients with FAP. Three deaths occurred postoperatively (0.2 percent) because of pulmonary embolism, perforated gastric ulcer, and subarachnoid hemorrhage. Late deaths occurred in 29 patients (1.8 percent), 10 months to 10.4 years after the operation. The most common cause of late death was cancer, including colon and rectal carcinoma (10 patients), hematologic malignancies (4 patients), cholangiocarcinoma (3 patients), and germ cell carcinoma (1 patient). Four patients died from unrelated sepsis, two died following myocardial infarction, two patients died from complications of subsequent orthopedic surgery, and one patient died of cirrhosis. Two additional patients committed suicide. No late deaths were directly attributable to the IPAA procedure. CONCLUSIONS: Proctocolectomy with IPAA is a safe procedure. Operative mortality is low, and late deaths are related to carcinogenic and extracolonic manifestations of underlying or unrelated coexisting diseases and events. PMID- 8620803 TI - Perianal Crohn's disease. Results of local surgical treatment. AB - PURPOSE: This study relates our experience with local surgical management of perianal Crohn's disease. METHOD: Of 1,735 patients with Crohn's disease seen between 1980 and 1990, records of 66 patients (3.8 percent) with symptomatic perianal Crohn's disease treated by local operations were retrospectively reviewed to study outcome of local surgical intervention. RESULTS: All patients had intestinal disease that was limited to the colon in 32 patients (48 percent), ileocolonic region in 22 patients (33 percent), and ileum in 12 patients (18 percent). Types of perianal disease encountered included perianal suppuration (57), anal fistula (47), anal fissure (21), anal stenosis (5), gluteal abscess (3), scrotal abscess (2), and anovaginal fistula (2). A total of 321 episodes of anal complications necessitated 256 local surgical interventions. Local anorectal operations performed included simple incision and drainage of abscess (57), fistulotomy (35), incision and drainage of complex anorectal abscesses and fistulas and insertion of seton (24), internal sphincterotomy (6), fissurectomy (1), and anal dilation (3). Of 24 patients with horseshoe abscesses and fistulas managed with insertion of a seton and 35 patients who underwent fistulotomy as a primary procedure or in conjunction with drainage of an abscess, none experienced fecal incontinence as a direct result of the operation. Thirteen patients required proctectomy to control perianal disease, and a similar number underwent total proctocolectomy for extensive intestinal disease. Forty patients (61 percent) continue to retain a functional anus. CONCLUSION: Patients with symptomatic low anal fistula involving minimum sphincter musculature can be treated safely with fistulotomy. In treatment of patients with horseshoe abscesses and high fistulas, aggressive local surgical intervention using a seton permits preservation of the sphincter and good postoperative function. PMID- 8620804 TI - A twenty-year review of malignant colorectal neoplasms at University College Hospital, Ibadan, Nigeria. AB - PURPOSE: Colorectal malignancies are less common in developing than developed nations because of lower per capita income and higher dietary fiber consumption. This clinicopathologic study attempts to determine changes in the pattern of these neoplasms in Ibadan, Nigeria, during the last two decades. METHODS: The present study is based on the clinical Cancer Registry records and gross and morphologic surgical pathology findings of 526 patients with histologically verified malignant colorectal neoplasms received in the Department of Pathology, University College Hospital, Ibadan, between 1971 and 1990. RESULTS: Colonic malignancies increased by 81 percent, whereas rectal malignancies decreased 16.1 percent in frequency (P < 0.05). The modal ages were 55 to 60 years and 45 to 50 years for colonic and rectal neoplasms, respectively, in contrast to reported peak occurrence in the seventh decade among Caucasians. Colonic neoplasms were predominantly right-sided (34.3 percent cecal), abdominal mass and pain being major clinical manifestations. This differs from the pattern in American Negroes, among whom colonic carcinomas are predominantly left-sided, dyschezia being an important presentation. As in most other studies, adenocarcinomas were the predominant neoplasms. CONCLUSIONS: Further work is required to determine prognostically significant features of colorectal cancer in our environment. PMID- 8620805 TI - Brain metastases from colorectal cancer. AB - PURPOSE: We wished to determine the time interval before diagnosis of brain metastases from a colorectal origin, typical neurologic presentation, overall survival, and impact of type of treatment and metastatic growth patterns (solitary, location, etc.) on prognosis. METHODS: Between 1980 and 1994 we retrospectively identified 19 patients with metastatic colorectal cancer to the brain. Data were collected with regard to patient characteristics, location and stage of primary tumor, and extent and location of metastatic disease. Statistical analyses were performed with STATISTICA for Windows. RESULTS: Mean patient age was 66 (43-87) years. Primary tumor location was distributed throughout the colon; 58 percent had disseminated disease when first diagnosed. All patients were symptomatic. Lesions were solitary in 63 percent, unilateral in 89 percent, and cerebral in 53 percent. Overall median survival was 2.8 months; 1 year survival was 0 percent. Survival was longer following craniotomy (4.1 months) compared with radiation (2.8 months); however, survival was not affected by number or location of metastatic lesions or whether the brain was the sole site of metastatic disease. CONCLUSIONS: Brain metastases from colorectal cancer are infrequently the sole site of metastatic disease, and because survival is dismal regardless of form of therapy chosen, craniotomy is rarely indicated. Exception is the rare patient who has minimum neurologic impairment, a long disease-free interval, a solitary metastasis, and no extracranial disease. PMID- 8620806 TI - Immunohistochemical analysis of statin in colorectal adenocarcinoma, polyps, and normal mucosa. AB - PURPOSE: The search for an understanding of the kinetics of the malignant cell is an ongoing focus of research. The aim of the present study was to determine whether there were any differences in the expression of statin, a nonproliferation-specific nuclear protein, among different colorectal tissues and whether there is any relationship between statin presence and neoplastic aggressivity. METHODS: The study population consisted of specimens from 19 patients who underwent resection for carcinoma, one for villous adenoma, and seven for colonoscopic polypectomies. Tissue samples were taken from the center of the specimen and from mucosa 10 cm from the lesion. RESULTS: Statin immunoreactivity was evaluated by counting stained nuclei in ten randomly chosen fields, and percent of positive cells was calculated. Average percentage of statin-positive cells was 34.33 +/- 6.81 in the normal crypt, 44.42 +/- 7.28 for polyps, and 7.74 +/- 5.67 for carcinomas (significantly lower than normal mucosa and polyps; P < 0.001). CONCLUSION: Statin expression is dramatically diminished in invasive carcinoma tissue, but it did not help determine aggressivity with respect to Dukes stage. PMID- 8620807 TI - Stent endoprosthesis for obstructing colorectal cancers. AB - PURPOSE: Purpose of this study was to assess stent endoprosthesis for colorectal cancer (SECC) as an adjuvant to operative preparation in patients with obstructing colorectal cancers. METHODS: A self-expanding stainless steel stent was inserted in 15 patients with obstructing colorectal cancers under colonoscopic observation and fluoroscopic control. Following successful SECC, the colon was mechanically prepared using polyethylene glycol. Definitive surgical treatment then was undertaken. RESULTS: All 12 patients in whom the stent had been successfully placed recovered intestinal transit and tolerated mechanical preparation. A satisfactory preparation was confirmed during the operation. Two perforations and one dislocation were encountered. CONCLUSION: SECC is a new method for operative preparation of patients with obstructing colorectal cancers, which may reduce morbidity and mortality associated with this difficult problem. PMID- 8620809 TI - Overexpression of p53 protein and histologic grades of dysplasia in colorectal adenomas. AB - PURPOSE: To clarify the relation between tumor-suppressor gene p53 expression and histologic grades of dysplasia in colorectal adenomas, we performed immunohistochemical analysis in a series of 59 colorectal polyps and 40 advanced carcinomas. METHODS: Adenomatous polyps were stained by hematoxylin and eosin and classified into mild, moderate, and severe dysplasia (intramucosal carcinoma), according to the World Health Organization's classification. RESULTS: p53 was positive in 7.1 percent (2/28) of mild, 29.4 percent (5/17) of moderate, and 62.5 percent (5/8) of severe dysplasia. In submucosal and advanced carcinomas, positivity rates were 75 percent (3/4) and 47.5 percent (19/40), respectively. Different staining patterns were found, according to grades of dysplasia. In the adenomas with mild or moderate dysplasia, a few focal crypts showed localized p53 positive staining. Adenomas with severe dysplasia had two different staining types. One was a focal staining type as shown in mild or moderate dysplasia; the other was a diffuse staining type, in which glands with mild or moderate dysplasia, surrounding severe dysplasia area, were also stained. Submucosal and advanced carcinomas showed a strong positive staining in cancer cells only. CONCLUSIONS: Overexpression of p53 protein in adenomas with mild or moderate dysplasia and existence of two types of expression in adenomas with severe dysplasia were observed. These facts suggested the possible existence of different pathways in the adenoma to carcinoma progression. PMID- 8620808 TI - First experimental sutureless laser anastomosis of the large bowel: long-term study. AB - Large bowel anastomoses were successfully performed without sutures on New Zealand white rabbits using 1,064 nm, 0.4-W power pulsating Nd:YAG laser to create tissue welding. PURPOSE: The aim of this study was to assess long-term results of our experimental data and summarize our experimental work on laser colon anastomosis. METHODS: This experimental study investigated long-term integrity, degree of narrowing, animal body weight change, visual findings, microscopic appearance, and collagen concentration of laser colon anastomoses, compared with those of conventional sutured anastomoses at thirty and ninety postoperative days. RESULTS: Two animals in the laser group died without leakage. Postsurgical course in all remaining rabbits studied was uneventful. Bursting pressures in the two groups were equivalent, but the laser group exhibited a consistent narrowing tendency. However, laser anastomoses demonstrated fewer and milder adhesions, and animals showed a better recovery of body weight. Histologically, laser anastomoses showed better layer-to-layer reconstitution without foreign body response and with less fibrosis. Difference in collagen (hydroxyproline) concentration did not reach statistical significance. CONCLUSION: The nature of tissue welding via laser remains undefined, but there is definitely a future for laser bowel anastomosis. PMID- 8620810 TI - Vaginography--investigation of choice for clinically suspected vaginal fistulas. AB - PURPOSE: Vaginal fistulas are rare but can cause extremely distressing symptoms for patients and prove difficult to define anatomically. Barium studies have been reported as having a maximum sensitivity of only 34 percent for detection of vaginal fistulas. Vaginography is an alternative method for diagnosis and evaluation of suspected vaginal fistulas, which has been reported to have a sensitivity of 100 percent. We reviewed our total experience of vaginography to fully assess its capabilities. METHODS: Twenty-seven patients with clinically suspected vaginal fistulas were investigated with vaginography during a six-year period. Results of vaginograms were compared with final operative or clinical diagnosis and with results of other radiologic investigations. RESULTS: Vaginography successfully identified 19 of 24 fistulas, giving a sensitivity of 79 percent. In our series, barium enema was only able to identify 9 percent of fistulas arising from the colon. CONCLUSIONS: In this, the largest series of vaginograms, apparent reduction in sensitivity from the 100 percent quoted in earlier series to 79 percent probably represents a more accurate assessment of vaginography as a diagnostic investigation. Even allowing for this reduction, vaginography is still the most sensitive, economic, and informative investigation for identification and delineation of vaginal fistulas. We recommend that vaginography be the initial investigation of choice in patients with clinically suspected vaginal fistulas. PMID- 8620811 TI - Collagenous colitis: pathogenesis and management. AB - INTRODUCTION: Collagenous colitis is a rare disease of unknown etiology that primarily affects middle-aged women. It presents with chronic watery diarrhea and thickening of the subepithelial collagen layer of the colonic mucosa in the absence of endoscopic abnormalities. PURPOSE: This study was undertaken to review the current literature on clinical course, pathology, diagnosis, and current management of collagenous colitis. RESULTS: Collagenous colitis is an inflammatory disease of the colon, clinically characterized by a waxing and waning course of watery diarrhea, an inflammatory infiltration of the colonic mucosa, and a thickening of the subepithelial collagen layer. Its pathogenesis remains unclear, but there is evidence for an inflammatory process triggered possibly by an uncommon luminal agent. Diagnosis is established by colonic biopsies; in the setting of normal colonic mucosa, the disorder is primarily managed medically with virtually no role for surgery. CONCLUSIONS: Pathogenesis of collagenous colitis remains unclear. Current data favor an inflammatory etiology, possibly involving an initiating luminal insult. Guidelines for diagnosis are being established, and medical treatment options are variably effective in the majority of cases. Very unusual refractory cases may benefit from surgical management. PMID- 8620812 TI - Mesosigmoplasty as a definitive operation in treatment of acute sigmoid volvulus. AB - PURPOSE: The aim of this prospective study was to present our patients with managed mesosigmoplasty as a definitive method and to discuss the efficiency of this operation in the treatment of acute sigmoid volvulus. METHODS: Fifteen patients with acute sigmoid volvulus were treated by mesosigmoplasty between April 1992 and April 1995. RESULTS: Postoperatively, temporary abdominal distention and constipation were seen in two patients, and one patient died of myocardial infarction. Morbidity and mortality rates were 13.3 and 6.6 percent, respectively. The average follow-up was 28.09 +/- 9.60 months, and recurrences and complaints of undue constipation were not seen. CONCLUSIONS: Our results suggest that the mesosigmoplasty is a definitive procedure and that it is the first and most reliable choice that can be easily performed with minimum morbidity and mortality for patients with acute sigmoid colon volvulus who do not have sigmoid necrosis at laparotomy. PMID- 8620813 TI - Laparoscopic-assisted transvaginal resection of the rectum. AB - Laparoscopic resection of the low rectum is technically difficult. This article describes a technique for laparoscopic-assisted, transvaginal low anterior resection of the rectum, which is technically easier and leads to an excellent cosmetic result. PMID- 8620814 TI - Pouch polyposis after ileal pouch-anal anastomosis for familial adenomatous polyposis: report of a case. AB - PURPOSE: A case of a patient with familial adenomatous polyposis (FAP) is reported, in whom adenomas developed in an ileal pelvic pouch six years after it was made. This case is reported to serve as a warning that restorative proctocolectomy, a relatively recent addition to surgical options for FAP, does not remove the risk of metachronous intestinal neoplasia; it merely defers it. METHODS: Case of a patient with pouch polyposis was reviewed, and patient was prospectively studied after three months of sulindac therapy. RESULTS: Polyps not removed at first examination became much less prominent. Literature review reveals only one study of adenomas in pelvic pouches, with 7 cases of 38. CONCLUSION: Proctocolectomy and ileal pouch-anal anastomosis does not cure FAP, and multiple polyps can occur in the ileal pouch. PMID- 8620815 TI - Colitis cystica profunda: imaging diagnosis and conservative treatment: report of two cases. AB - Rectally localized colitis cystica profunda can simulate mucosecretory carcinoma. PURPOSE AND METHODS: Because endoscopic examination and barium enema do not clarify the diagnosis, other diagnostic imaging methods such as transrectal ultrasonography, computerized tomography, or magnetic resonance imaging are needed. RESULTS: Transrectal ultrasonography identifies multiple cysts in the rectal submucosa, with areas of echorefringent fibrosis between cysts, and confirms the absence of lymph node involvement or invasion of the muscular layer. Findings with computerized tomography and magnetic resonance imaging have not previously been described for colitis cystica profunda. With computerized tomography, the lesion appears as a noninfiltrating entity in the submucosa, with loss of perirectal layers of fatty tissue and thickening of the levator ani muscle. With nuclear magnetic imaging, nodulations produce intense signals that increase in T2, illustrating the mucoprotein content of the cysts. The presence in surgical biopsy material of large, whole cysts confirms the diagnosis. CONCLUSION: Reeducation of bowel habits aimed at avoiding straining and a high fiber diet together with bulk laxatives can lead to complete remission of lesions in 6 to 18 months. PMID- 8620816 TI - Retractor-induced femoral neuropathy. PMID- 8620817 TI - Can hydroxyapatite deposition in the eye cause a neutrophil-related inflammatory reaction? AB - Patients with chronic renal failure on intermittent dialysis sometimes develop an acute diffuse conjunctival and episcleral hyperaemia. In this study the hypothesis was tested whether the precipitation of hydroxyapatite crystals could result in an inflammatory reaction mediated by enzymes liberated from polymorphonuclear leucocytes (PMN). Ingestion of the crystals by PMN's can result in cell death and membranolysis and subsequent release of intracellular enzymes into the surrounding tissues. This 'suicide sac' hypothesis for the inflammatory reactions of the conjunctiva and episclera was rejected after histopathological examination of conjunctival biopsies failed to show complement activation or crystal ingestion by PMN's despite the presence of small subepithelial hydroxyapatite crystals. PMID- 8620818 TI - Recurrence rate of herpetic uveitis in patients on long-term oral acyclovir. AB - We examined the recurrence rate of herpetic uveitis (HU) in 13 patients (group A) treated prophylactically with long-term systemic acyclovir (600-800 mg/day) and compared it with that of 7 patients with no prophylactic therapy (group B). HU was diagnosed on the basis of a history of dendritic or disciform keratitis accompanied by iridocyclitis and iris atrophy. The study population consisted of 12 men and 8 women with a mean age at onset of uveitis of 52.9 years (range 19-78 years). All patients were followed for at least 8 months. The mean follow-up time of patients on long-term oral acyclovir was 26.0 months. In this group, only one patient experienced a single recurrent episode of uveitis while on 600-800 mg/day of acyclovir therapy; two additional patients had recurrence of HU within 16.2 months after the acyclovir dose was tapered below 600 mg/day. In striking contrast, 16 recurrences occurred in the 7 patients of group B (p < 0.05). Of these, the initial recurrence occurred within an average of 4.3 months following cessation of therapy. There was a significant difference (p < 0.05) in the mean recurrence-free interval between patients in group A (24.6 months) and those in group B (3.4 months). Herpetic uveitis is a serious ocular disease in which recurrence of inflammation results in severe ocular complications. The long-term use of oral acyclovir may be of benefit in the prevention of recurrences, and hence may reduce the blinding complications of this disease. Efforts at completing a randomized, placebo-controlled trial on this matter by the Herpes Epithelial Disease Study Group were unsuccessful due to insufficient patient recruitment. PMID- 8620820 TI - Colour contrast sensitivity in patients with soft drusen, an early stage of ARM. AB - The present institutional study was undertaken in order to determine whether testing of colour contrast sensitivity is valuable in detecting early functional changes in patients at initial stages of age-related maculopathy (ARM). The study included 27 patients with soft drusen as an early sign of ARM and 29 age-matched normals. The area occupied by drusen was determined using a computer program. Colour contrast sensitivity was measured with a computer graphics system. Visual acuity, central visual field (Humphrey Field Analyser) and colour vision (D-15 panel) were normal in both groups. Mean colour contrast sensitivity was significantly lower in patients with early ARM (for the protan axis: p = 0.00019, for the deutan axis: p = 0.000078 and for the tritan axis: p = 0.000096) than in the controls. Interindividual variations were large. There was a tendency towards a bimodal distribution for all three colour axes, most evident for the tritan axis. We found a correlation of the drusen area with the protan and deutan thresholds for the fundus colour photographs r = 0.5 (p < 0.01) and r = 0.4 (p < 0.05), respectively, and with the tritan threshold for the angiograms r = 0.5 (p < 0.05). The findings suggest that colour contrast sensitivity may offer an additional possibility of predicting exudative ARM. Whether the patients with the highest thresholds are those who will be the first to show progression with indications of exudative ARM is not yet known. The patient group will be followed up in order to elucidate this question. PMID- 8620821 TI - Vitrectomy techniques in late-stage Coats'-like exudative retinal detachment. AB - Retinal telangiectasia is the hallmark of Coats' disease. In the late stages, leakage from these abnormal vessels can result in a total, bullous exudative retinal detachment with cholesterol-laden subretinal fluid. Secondary angle closure glaucoma may result in a blind and painful eye which may require enucleation or evisceration. Surgical reattachment of the retina and destruction of the retinal telangiectasia may preserve these eyes. We have found that vitrectomy, internal drainage of subretinal fluid and cholesterol, direct treatment of the retinal telangiectasia with intraocular diathermy and intravitreal gas or silicone oil injection are effective surgical techniques for salvaging these severely damaged eyes. PMID- 8620819 TI - Retinal-image mediated ocular growth as a mechanism for juvenile onset myopia and for emmetropization. A literature review. AB - The very common ocular clinical ocular condition in children juvenile onset myopia results from axial elongation of the eye. In humans, some studies have found an association of myopia with greater levels of nearpoint activity and with differences in accommodation and convergence function. This paper reviews a variety of laboratory and clinical studies which are consistent with the hypothesis that retinal image defocus is biochemically transformed into an axial elongation expressed through increased posterior segment growth, and thus myopia. This paper also reviews theories of emmetropization, and classifies them as correlational, feedback, and combination. Evidence is presented to suggest that a combination theory, which combines both correlation of the ocular dioptric components and some feedback mechanism for growth of the eye, is the most correct. Current laboratory research suggests that quality and/or focus (defocus) of retinal imagery is involved in this feedback mechanism and that experimentally induced myopia might be enhanced, reduced or eliminated by pharmaceutical application. Direction of defocus may affect the rate of posterior segment growth, and thus the rate of ocular axial elongation. PMID- 8620822 TI - Bendazac decreases in vitro glycation of human lens crystallins. Decrease of in vitro protein glycation by bendazac. AB - Bendazac has been used as an anti-cataractogenic drug. It has been reported that this acts by preventing protein denaturation. In this study the ability of bendazac to inhibit in vitro glycation of human lens crystallins was evaluated. Possible effects of bendazac were detected by incubation of WS crystallins with the reducing sugars glucose and fructose. The efficiency of bendazac was evaluated by means of selected parameters including: browning, glycation (measured as tyrosine content) and specific NTP-fluorescence. The results showed clearly that bendazac (bendazac L-lysine and sodium) inhibits the early stages of protein glycation, as well as the formation of fluorescent advanced glycation products. Bendazac lysine (20 mM) proved to be more effective in inhibiting fluorescence development (67% inhibition) that the corresponding sodium salt (35% inhibition). No significant differences were found with respect to furosine levels; about 40% inhibition was produced with either bendazac lysine or sodium salt bendazac clearly inhibits glycation of human lens crystallins, as can be efficiently monitored by following specific changes in lens protein fluorescence. These results may constitute a new and relevant therapeutic approach to monitoring cataract development. PMID- 8620823 TI - [Comparison of the quality of life after subtotal and total gastrectomy for stomach carcinoma]. AB - OBJECTIVE: To compare quality of life after subtotal gastrectomy (STG) and total gastrectomy of various types, in view of the fact that, with T1 and T2 gastric carcinoma of intestinal type in the distal third of the stomach, subtotal gastrectomy is similar to total gastrectomy regarding the extent of lymphadenectomy and prognosis. PATIENTS AND METHODS: Quality of life was measured by standardised questionnaires given to 36 patients after subtotal gastrectomy (22 men, 14 women; mean age 63 [27-79] years): general physical complaints (GPC); contentment with life (CL); psychosocial stress (PSS). The results were compared with those previously obtained in 58 patients with total gastrectomy (46 men, 12 women; mean age 63.4 [36-74] years) and oesophagojejunostomy (OJS) (n = 29) or oesophagojejunoplication and pouch (OJPP) (n = 29). RESULTS: Weight loss of patients after OJPP was not significantly different from that of patients after STG, but it was significantly higher after OJS (13.5 +/- 8.6 kg; P < 0.0006). Patients with STG had significantly more general complaints (P < 0.05) and greater discontent with life (P < 0.05) than those with OJPP. Specific analysis of gastric complaints showed greatest dissatisfaction with gastrointestinal functions in patients after STG (P < 0.0004), less also after OJS compared with OJPP (P < 0.01). CONCLUSIONS: Subtotal gastrectomy for gastric carcinoma has no advantages over total gastrectomy with oesophagojejunoplication and pouch as regards weight loss, gastrointestinal complaints, psychosocial stress and general contentment. The poor quality of life seems to have its functional correlate in increased intestino-oesophageal reflux with incompetent cardia and after Billroth II reconstruction. PMID- 8620824 TI - [Aneurysm of the ascending aorta in tertiary syphilis]. AB - HISTORY AND CLINICAL FINDINGS: A 58-year-old man was admitted after he was found to have a huge aneurysm of the thoracic aorta. 38 months previously he had first experienced subacute pain in the right thorax. After this the aneurysm gradually increased in size. On admission a pulsating mass, 20 cm in diameter, was obvious, having broken through the right ventral thoracic wall. It was causing venous inflow congestion in the upper part of the body. The patient's general condition was poor: he had marked orthopnoea. INVESTIGATIONS: Serological tests for syphilis gave the following results: VDRL test, 1:32; FTA-ABS test reactive; TPHA test, 1:8000; treponema-specific IgM negative. Computed tomography with contrast medium injection exactly defined site and extent of the aneurysm. It started at the aortic root and extended to the aortic arch. Third-degree aortic valvar regurgitation was demonstrated on echocardiography. Pulmonary function tests indicated severe restrictive ventilatory abnormality with clearly reduced respiratory reserve. TREATMENT AND COURSE: The symptoms improved after 2 weeks on oral medication of captopril (25 mg/d), furosemide (80 mg/d), spironolactone (100 mg/d), codeine phosphate (90 mg/d) and thick paraffin (20 mg/d as needed). The patient declined further treatment. He died 8 weeks later at home. PMID- 8620825 TI - [Splenic infarction caused by paradoxical emboli in severe pulmonary hypertension]. AB - HISTORY AND CLINICAL FINDINGS: A 55-year-old woman developed increasing shortness of breath and breath-independent pain in the left lower chest. 20 years previously she had had an episode of pulmonary embolism and 10 years previously a central venous thrombosis in the left eye. No cause of the increased thrombogenesis had been found. On admission she had resting dyspnoea but a stable circulation. On auscultation the breath sounds were diminished over the left base and there was a diastolic murmur over the pulmonary area with an accentuated second sound. There was also marked tenderness below the left costal margin. Recurrent pulmonary embolism or left-sided pleuropneumonia was suspected. INVESTIGATION: Arterial blood gases (without additional oxygen) showed severe hypoxaemia (pO2 42.3 mm Hg, pCO2 27.8 mm Hg, pH 7.455, oxygen saturation 80.5%). Transthoracic and transoesophageal echocardiography showed normal left ventricular dimensions, right atrial and ventricular dilatation, and an atrial septal aneurysm with a right to left interatrial shunt. Right heart catheterisation demonstrated severe pulmonary hypertension. Sonography, computed tomography and scintigraphy revealed multiple splenic infarcts. TREATMENT AND COURSE: Heparinisation was instituted (partial thromboplastin time 70-90 s) and overlapping oral anticoagulation to a Quick value of 20%. Subsequently the calcium antagonist felodipine (15 mg daily) was given. The mean pulmonary artery pressure was 61 mm Hg before and 57 mm Hg after treatment. CONCLUSION: Splenic infarction resulting from paradoxical embolisation is rare, but should be routinely considered in the presence of thromboembolic phenomena. PMID- 8620826 TI - [Therapy of rheumatoid arthritis]. PMID- 8620827 TI - [Angiotensin receptor antagonists. A new class of antihypertensive agents]. PMID- 8620828 TI - [Von Gierke's glycogenosis. First description in a female patient in the Karlsruhe Franz Lust Pediatric Clinic in 1929]. PMID- 8620829 TI - [BCG vaccination]. PMID- 8620830 TI - [Drinking water filters]. PMID- 8620831 TI - [Successful treatment with methylene blue of ifosfamide-induced central nervous system effects]. PMID- 8620832 TI - A Cdc2 dependent checkpoint maintains diploidy in Drosophila. AB - DNA replication in G2 does not normally occur due to the checkpoint control. To elucidate its mechanism, the functions of the escargot and Dmcdc2 genes of Drosophila were studied. When escargot function was eliminated, diploid imaginal cells that were arrested in G2 lost Cyclin A, a regulatory subunit of G2/M cdk, and entered an endocycle. escargot genetically interacted with Dmcdc2 which encodes a catalytic subunit of G2/M cdk. The mutant phenotypes of Dmcdc2 itself was similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system entered an endocycle and sometimes formed polytene chromosomes. Since mitotically quiescent abdominal histoblasts still required Dmcdc2 to remain diploid, the inhibitory activity of G2/M cdk on DNA replication appeared to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk that actively inhibits the entry into S phase. PMID- 8620833 TI - Determination of wing cell fate by the escargot and snail genes in Drosophila. AB - Inset appendages such as the wing and the leg are formed in response to inductive signals in the embryonic field. In Drosophila, cells receiving such signals initiate developmental programs which allow them to become imaginal discs. Subsequently, these discs autonomously organize patterns specific for each appendage. We here report that two related transcription factors, Escargot and Snail that are expressed in the embryonic wing disc, function as intrinsic determinants of the wing cell fate. In escargot or snail mutant embryos, wing specific expression of Snail, Vestigial and beta-galactosidase regulated by escargot enhancer were found as well as in wild-type embryos. However, in escargot snail double mutant embryos, wing development proceeded until stage 13, but the marker expression was not maintained in later stages, and the invagination of the primordium was absent. From such analyses, it was concluded that Escargot and Snail expression in the wing disc are maintained by their auto- and crossactivation. Ubiquitous escargot or snail expression induced from the hsp70 promoter rescued the escargot snail double mutant phenotype with the effects confined to the prospective wing cells. Similar DNA binding specificities of Escargot and Snail suggest that they control the same set of genes required for wing development. We thus propose the following scenario for early wing disc development. Prospective wing cells respond to the induction by turning on escargot and snail transcription, and become competent for regulation by Escargot and Snail. Such cells initiate auto- and crossregulatory circuits of escargot and snail. The sustained Escargot and Snail expression then activates vestigial and other target genes that are essential for wing development. This maintains the commitment to the wing cell fate and induces wing-specific cell shape change. PMID- 8620834 TI - Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo. AB - The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiC1 treatment. Furthermore, both the negative spatial control functions and response to LiC1 require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study. PMID- 8620836 TI - Initiation patterns of flower and floral organ development in Arabidopsis thaliana. AB - Sector boundary analysis has been used to deduce the number and orientation of cells initiating flower and floral organ development in Arabidopsis thaliana. Sectors were produced in transgenic plants carrying the Ac transposon from maize inserted between the constitutive 35S promoter and the GUS reporter gene. Excision of the transposon results in a blue-staining sector. Plants were chosen in which an early arising sector passed from vegetative regions into the inflorescence and through a mature flower. The range of sector boundary positions seen in mature flowers indicated that flower primordia usually arise from a group of four cells on the inflorescence flank. The radial axes of the mature flower are apparently set by these cells, supporting the concept that they act as a structural template. Floral organs show two patterns of initiation, a leaf-like pattern with eight cells in a row (sepals and carpels), or a shoot-like pattern with four cells in a block (stamens). The petal initiation pattern involved too few cells to allow assignment. The numbers of initiating cells were close to those seen when organ growth commenced in each case, indicating that earlier specification of floral organ development does not occur. By examining sector boundaries in homeotic mutant flowers in which second whorl organs develop as sepal-like organs rather than petals, we have shown that their pattern of origin is position dependent rather than identity dependent. PMID- 8620835 TI - The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila. AB - Rows of cells that flank the parasegment boundary make up a signaling center within the epidermis of the Drosophila embryo. Signals emanating from these cells, encoded by hedgehog (hh) and wingless (wg), are shown to be required for all segment pattern dorsally. Wg activity is required for the differentiation of one cell type, constituting half the parasegment. The gene lines appears to act in parallel to the Wg pathway in the elaboration of this cell type. Hh activity is responsible for three other cell types in the parasegment. Some cell types are specified as Hh activity and interfere with the function of patched, analogous to patterning of imaginal discs. However, some pattern is independent of the antagonism of patched by Hh, and relies instead on novel interactions with lines. Lastly, we provide evidence that decapentaplegic does not mediate patterning by Hh in the dorsal epidermis. PMID- 8620837 TI - Embryonic taste buds develop in the absence of innervation. AB - It has been hypothesized that taste buds are induced by contact with developing cranial nerve fibers late in embryonic development, since descriptive studies indicate that during embryonic development taste cell differentiation occurs concomitantly with or slightly following the advent of innervation. However, experimental evidence delineating the role of innervation in taste bud development is sparse and equivocal. Using two complementary experimental approaches, we demonstrate that taste cells differentiate fully in the complete absence of innervation. When the presumptive oropharyngeal region was taken from a donor axolotl embryo, prior to its innervation and development of taste buds, and grafted ectopically on to the trunk of a host embryo, the graft developed well-differentiated taste buds. Although grafts were invaded by branches of local spinal nerves, these neurites were rarely found near ectopic taste cells. When the oropharyngeal region was raised in culture, numerous taste buds were generated in the complete absence of neural elements. Taste buds in grafts and in explants were identical to those found in situ both in terms of their morphology and their expression of calretinin and serotonin immunoreactivity. Our findings indicate that innervation is not necessary for complete differentiation of taste receptor cells. We propose that taste buds are either induced in response to signals from other tissues, such as the neural crest, or arise independently through intrinsic patterning of the local epithelium. PMID- 8620839 TI - hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila. AB - The hedgehog (hh) gene plays a role in regulating cell proliferation and specifying cell identity in diverse systems. We show that hh is expressed at the extreme apical end of Drosophila ovarioles in terminal filament cells and a newly identified group of associated somatic cells. Reducing or ectopically expressing hh affects somatic cells in region 2 of the germarium, 2-5 cells away from the cells in which Hh protein is detected. hh activity stimulates the proliferation of pre-follicle somatic cells, and promotes the specification of polar follicle cells. hh signaling during egg chamber assembly appears to be closely related to, or part of pathways involving the neurogenic genes. PMID- 8620838 TI - The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division. AB - The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle. PMID- 8620840 TI - The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf. AB - The sevenless gene encodes a receptor tyrosine kinase which is required for the development of the R7 photoreceptor cell in each ommatidium of the Drosophila eye. We have previously used a sensitized genetic screen to identify mutations, designated Enhancers of sevenless (E(sev)), which affect genes that encode components of the sevenless signaling pathway. Here, we report that one of these mutations, E(sev)1Ae0P is a dominantly inhibiting allele of corkscrew, which encodes an SH2 domain-containing protein tyrosine phosphatase (Perkins et al., 1992). We show that corkscrew function is essential for sevenless signaling and that expression of a membrane-targeted form of corkscrew can drive R7 photoreceptor development in the absence of sevenless function. Furthermore, we have used the dominantly inhibiting corkscrew allele to examine the role of corkscrew during signaling by activated forms of Ras1 and Raf. Our analysis indicates that corkscrew function is still required during signaling by activated forms Ras1 and Raf proteins. These results define a function for corkscrew that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development. PMID- 8620841 TI - Expression and regulation of Cek-8, a cell to cell signalling receptor in developing chick limb buds. AB - The Eph-related receptor tyrosine kinase gene, Cek-8, is expressed in mesenchyme at the tip of chick limb buds, with high levels of transcripts posteriorly and apically but fading out anteriorly. Expression of Cek-8 in distal mesenchyme is regulated by apical ridge- and FGF-polarising signals and retinoic acid, and is uniform across the anteroposterior axis in talpid3 mutants. These data indicate that Cek-8 expression responds to regulatory signals during limb patterning and suggest that this receptor tyrosine kinase may have a role in coordinating responses to signals in the progress zone of early buds. Later on in limb development, Cek-8 expression is associated with cell condensations that form tendons and their attachments to cartilage rudiments and then in developing feather buds. PMID- 8620842 TI - Peptide YY expression is an early event in colonic endocrine cell differentiation: evidence from normal and transgenic mice. AB - The hormone peptide YY is produced by endocrine cells in the pancreas, ileum and colon. We have previously shown that peptide YY is coexpressed in all four islet cell types in the murine pancreas when they first appear, suggesting a common peptide YY-producing progenitor. In the colon, peptide YY has been frequently identified in glucagon-expressing L-type endocrine cells. Characterization of colonic endocrine tumors in transgenic mice expressing simian virus 40 large T antigen under the control of the peptide YY gene 5' flanking region revealed tumor cells producing not only peptide YY and glucagon, but also neurotensin, cholecystokinin, substance P, serotonin, secretin, and gastrin. This suggested that multiple enteroendocrine lineages were related to peptide YY-producing cells. Subsequent examination of the ontogeny of colonic endocrine differentiation in nontransgenic mice revealed that peptide YY was the first hormone to appear during development, at embryonic day 15.5. Between embryonic days 16.5 and 18.5, cells expressing glucagon, cholecystokinin, substance P, serotonin, secretin, neurotensin, gastrin and somatostatin first appeared and peptide YY was coexpressed in each cell type at this time. Peptide YY coexpression continued in a significant fraction of most enteroendocrine cell types throughout fetal and postnatal development and into adulthood, with the exception of serotonin-producing cells. This latter population of cells expanded dramatically after birth with rare coexpression of peptide YY. These studies indicate that expression of peptide YY is an early event in colonic endocrine differentiation and support the existence of a common progenitor for all endocrine cells in the colon. PMID- 8620844 TI - A mutational analysis of the 5' HoxD genes: dissection of genetic interactions during limb development in the mouse. AB - Using gene targeting in mice, we have undertaken a systematic mutational analysis of the homeobox-containing 5' HoxD genes. In particular, we have characterized the limb defects observed in mice with independent targeted disruptions of hoxd 12 and hoxd-13. Animals defective for hoxd-12 are viable, fertile, and appear outwardly normal yet have minor autopodal defects in the forelimb which include a reduction in the bone length of metacarpals and phalanges, and a malformation of the distal carpal bone d4. The limb phenotypes observed in hoxd-13 mutant mice are more extensive, including strong reductions in length, complete absences, or improper segmentations of many metacarpal and phalangeal bones. Additionally, the d4 carpal bone is not properly formed and often produces an extra rudimentary digit. To examine the genetic interactions between the 5' HoxD genes, we bred these mutant strains with each other and with our previously characterized hoxd 11 mouse to produce a series of trans-heterozygotes. Skeletal analyses of these mice reveal that these genes interact in the formation of the vertebrate limb, since the trans-heterozygotes display phenotypes not present in the individual heterozygotes, including more severe carpal, metacarpal and phalangeal defects. Some of these phenotypes appear to be accounted for by a delay in the ossification events in the autopod, which lead to either the failure of fusion or the elimination of cartilaginous elements. Characteristically, these mutations lead to the overall truncation of digits II and V on the forelimb. Additionally, some trans-animals show the growth of an extra postaxial digit VI, which is composed of a bony element resembling a phalange. The results demonstrate that these genes interact in the formation of the limb. In addition to the previously characterized paralogous interactions, a multitude of interactions between Hox genes is used to finely sculpt the forelimb. The 5' Hox genes could therefore act as a major permissive genetic milieu that has been exploited by evolutionary adaptation to form the tetrapod limbs. PMID- 8620843 TI - Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. AB - A key event in the development of the mammalian cerebral cortex is the generation of neuronal populations during embryonic life. Previous studies have revealed many details of cortical neuron development including cell birthdates, migration patterns and lineage relationships. Programmed cell death is a potentially important mechanism that could alter the numbers and types of developing cortical cells during these early embryonic phases. While programmed cell death has been documented in other parts of the embryonic central nervous system, its operation has not been previously reported in the embryonic cortex because of the lack of cell death markers and the difficulty in following the entire population of cortical cells. Here, we have investigated the spatial and temporal distribution of dying cells in the embryonic cortex using an in situ endlabelling technique called 'ISEL+' that identifies fragmented nuclear DNA in dying cells with increased sensitivity. The period encompassing murine cerebral cortical neurogenesis was examined, from embryonic days 10 through 18. Dying cells were rare at embryonic day 10, but by embryonic day 14, 70% of cortical cells were found to be dying. This number declined to 50% by embryonic day 18, and few dying cells were observed in the adult cerebral cortex. Surprisingly, while dying cells were observed throughout the cerebral cortical wall, the majority were found within zones of cell proliferation rather than in regions of postmitotic neurons. These observations suggest that multiple mechanisms may regulate programmed cell death in the developing cortex. Moreover, embryonic cell death could be an important factor enabling the selection of appropriate cortical cells before they complete their differentiation in postnatal life. PMID- 8620845 TI - Coordination of cellular events that precede reproductive onset in Acetabularia acetabulum: evidence for a 'loop' in development. AB - Amputated apices from vegetative wildtype cells of the uninucleate green alga Acetabularia acetabulum can differentiate a reproductive structure of 'cap' in the absence of the nucleus (Hammerling, J. (1932) Biologisches Zentralblatt 52, 42-61). To define the limits of the ability of wildtype cells to control reproductive differentiation, we determined when during development apices from wildtype cells first acquired the ability to make a cap in the absence of the nucleus and, conversely, when cells with a nucleus lost the ability to recover from the loss of their apices. To see when the apex acquired the ability to make a cap without the nucleus, we removed apices from cells varying either the developmental age of the cells or the cellular volume left with the apex. Cells must have attained the adult phase of development before the enucleate apex could survive amputation and make a cap. Apices removed from cells early in adult growth required more cell volume to make a cap without the nucleus than did apices removed from cells late in adult growth. To define the limits of the cell to recapitulate development when reproduction falters, we analyzed development in cells whose caps either had been amputated or had spontaneously aborted. After loss of the first cap, cells repeated part of vegetative growth and then made a second cap. The ability to make a second cap after amputation of the first one was lost 15-20 days after cap initiation. Our data suggest that internal cues, cell age and size, are used to regulate reproductive onset in Acetabularia acetabulum and add to our understanding of how reproduction is coordinated in this giant cell. PMID- 8620846 TI - The Drosophila morphogenetic protein Bicoid binds DNA cooperatively. AB - The Drosophila morphogenetic protein Bicoid, encoded by the maternal gene bicoid, is required for the development of the anterior structures in the embryo. Bicoid, a transcriptional activator containing a homeodomain, is distributed in an anterior-to-posterior gradient in the embryo. In response to this gradient, the zygotic gene hunchback is expressed uniformly in the anterior half of the embryo in a nearly all-or-none manner. In this report we demonstrate that a recombinant Bicoid protein binds cooperatively to its sites within a hunchback enhancer element. A less than 4-fold increase in Bicoid concentration is sufficient to achieve an unbound/bound transition in DNA binding. Using various biochemical and genetic methods we further demonstrate that Bicoid molecules can interact with each other. Our results are consistent with previous studies performed in the embryo, and they suggest that one mechanism to achieve a sharp on/off switch of gene expression in response to a morphogenetic gradient is cooperative DNA binding facilitated by protein-protein interaction. PMID- 8620847 TI - Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns. AB - The injection of an antagonistic anti-murine c-kit monoclonal antibody ACK2 during mouse embryonic development produced three distinctive pigmentation patterns on the coat of the offspring. Pattern 1 consisted of pigmentation in craniofacial and caudal regions and was induced by an ACK2 injection between 9.5 and 11.5 days post coitum (dpc). In pattern 2, the entire coat was unpigmented and was induced by the injection at around 13.0 dpc. Pattern 3 consisted of pigmented patches spreading ventrolaterally from the dorsoanterior trunk regions towards the anterior and posterior directions and it was induced by ACK2 administered at 14.5-15.0 dpc. We investigated the embryological basis of these nonuniform pigmentation patterns to elucidate the process of melanoblast differentiation between lineage commitment and colonization into developing hair follicles. The results showed the following. (1) Melanocyte differentiation at the embryonic stage from 10.5 to 12.5 dpc progresses in a spatially nonuniform fashion, being faster in the craniofacial and caudal regions than in the trunk; pattern 1 reflects this. (2) Melanoblasts are activated to proliferate synchronously upon entering into the epidermis; pattern 2 correlates with this process. (3) c-kit functions as a survival signal for proliferating melanoblasts in the epidermis. (4) The melanoblasts that enter developing hair follicles can survive without a c-kit signal; pattern 3 essentially represents the hair follicles colonized by these cells. Analysis of the melanoblast distribution of ls/ls embryos that bear a loss-of-function mutation in the endothelin 3 gene suggested that endothelin 3 is required for early melanoblast differentiation before entering into the epidermis, whereas proliferation in the epidermis takes place without this molecule. Based on these data, we propose 4 distinct steps of embryonic melanocyte differentiation: (1) migration in the dermis, which requires both c-kit and endothelin 3; (2) a state before epidermal entry that is resistant to anti-c-kit mAb; (3) cell proliferation after entering the epidermal layer, which requires c-kit and endothelin receptor B but not endothelin 3 and (4) integration into developing hair follicles, which renders melanoblasts resistant to anti-c-kit mAb. Thus, melanoblast differentiation proceeds by alternately repeating c-kit -dependent and c-kit-independent stages and c-kit functions as a survival factor for the proliferating melanoblasts. PMID- 8620848 TI - Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis. AB - Tip morphogenesis during the Dictyostelium developmental life cycle is a process by which prestalk cells sort to form the anterior region of the multicellular organism. We show that the temporal regulation of this morphological process is dependent on the copy number of the Dictyostelium G alpha5 gene. Tip formation is delayed in aggregates of g alpha5 null mutant cells and accelerated in aggregates overexpressing the G alpha5 gene compared to tip formation in wild-type cells. The onset of cell-type-specific gene expression associated with mound formation and tip morphogenesis is also temporally altered in G alpha5 mutants. Tip morphogenesis in chimeric organisms of G alpha5 mutants and wild-type cells is dependent on the copy number of the G alpha5 gene, indicating that G alpha5 function plays an integral role in the intercellular signaling of this stage of development. The G alpha5 gene encodes a G alpha subunit that has 51% identity to the Dictyostelium G alpha4 subunit. Like the G alpha4 gene, the G alpha5 gene is expressed in a subset of cells distributed throughout the multicellular organism, with a distribution that is similar to the anterior-like cell population. Amino acid substitutions in the G alpha5 subunit analogous to substitutions altering guanine nucleotide binding and hydrolysis in other G alpha subunits had no apparent effect on the rate of tip formation when a single copy of the mutant gene was used to replace the wild-type gene. Overexpression of these mutant G alpha5 genes by increased gene dosage resulted in cell death, suggesting that high levels of the altered subunits have detrimental effects during vegetative growth. PMID- 8620849 TI - Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. AB - Hedgehog genes have been implicated in inductive signaling during development in a variety of organisms. A key element of the hedgehog signaling system is encoded by the gene patched. In Drosophila hedgehog regulates gene expression by antagonizing the action of patched. In addition, patched is itself a transcriptional target of hedgehog signaling. We have isolated a chicken patched homolog and find it to be strongly expressed adjacent to all tissues where members of the hedgehog family are expressed. As in Drosophila, ectopic expression of Sonic hedgehog leads to ectopic induction of chicken Patched. Based on this regulatory conservation, vertebrate Patched is likely to be directly downstream of Sonic hedgehog signaling. An important role of Sonic hedgehog is the regulation of anterior/posterior pattern in the developing limb bud. Since Patched is directly downstream of the hedgehog signal, the extent of high level Patched expression provides a measure of the distance that Sonic hedgehog diffuses and directly acts. On this basis, we find that Sonic hedgehog directly acts as a signal over only the posterior third of the limb bud. During limb patterning, secondary signals are secreted in both the mesoderm (e.g. Bone Morphogenetic Protein-2) and apical ectodermal ridge (e.g. Fibroblast Growth Factor-4) in response to Sonic hedgehog. Thus knowing which is the direct target tissue is essential for unraveling the molecular patterning of the limb. The expression of Patched provides a strong indication that the mesoderm and not the ectoderm is the direct target of Sonic hedgehog signaling in the limb bud. Finally we demonstrate that induction of Patched requires Sonic hedgehog but, unlike Bone Morphogenetic Protein-2 and Hox genes, does not require Fibroblast Growth Factor as a co-inducer. It is therefore a more direct target of Sonic hedgehog than previously reported patterning genes. PMID- 8620850 TI - Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells. AB - Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested. PMID- 8620851 TI - Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars. AB - The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the inner enamel epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar. PMID- 8620852 TI - The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana. AB - The role of the Arabidopsis homeobox gene, GLABRA 2 (GL2), in the development of the root epidermis has been investigated. The wild-type epidermis is composed of two cell types, root-hair cells and hairless cells, which are located at distinct positions within the root, implying that positional cues control cell-type differentiation. During the development of the root epidermis, the differentiating root-hair cells (trichoblasts) and the differentiating hairless cells (atrichoblasts) can be distinguished by their cytoplasmic density, vacuole formation, and extent of elongation. We have determined that mutations in the GL2 gene specifically alter the differentiation of the hairless epidermal cells, causing them to produce root hairs, which indicates that GL2 affects epidermal cell identity. Detailed analyses of these differentiating cells showed that, despite forming root hairs, they are similar to atrichoblasts of the wild type in their cytoplasmic characteristics, timing of vacuolation, and extent of cell elongation. The results of in situ nucleic acid hybridization and GUS reporter gene fusion studies show that the GL2 gene is preferentially expressed in the differentiating hairless cells of the wild type, during a period in which epidermal cell identity is believed to be established. These results indicate that the GL2 homeodomain protein normally regulates a subset of the processes that occur during the differentiation of hairless epidermal cells of the Arabidopsis root. Specifically, GL2 appears to act in a cell-position-dependent manner to suppress hair formation in differentiating hairless cells. PMID- 8620853 TI - Mutations in the PERIANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern. AB - An open question in developmental biology is how groups of dividing cells can generate specific numbers of segments or organs. We describe the phenotypic effects of mutations in PERIANTHIA, a gene specifically required for floral organ patterning in Arabidopsis thaliana. Most wild-type Arabidopsis flowers have 4 sepals, 4 petals, 6 stamens, and 2 carpels. Flowers of perianthia mutant plants most commonly show a pentamerous pattern of 5 sepals, 5 petals 5 stamens, and 2 carpels. This pattern is characteristic of flowers in a number of plant families, but not in the family Brassicaceae, which includes Arabidopsis. Unlike previously described mutations affecting floral organ number, perianthia does not appear to affect apical or floral meristem sizes, nor is any other aspect of vegetative or floral development severely affected. Floral organs in perianthia arise in a regular, stereotypical pattern similar to that in distantly related species with pentamerous flowers. Genetic analysis shows that PERIANTHIA acts downstream of the floral meristem identity genes and independently of the floral meristem size and floral organ identity genes in establishing floral organ initiation patterns. Thus PERIANTHIA acts in a previously unidentified process required for organ patterning in Arabidopsis flowers. PMID- 8620854 TI - Vegetal egg cytoplasm promotes gastrulation and is responsible for specification of vegetal blastomeres in embryos of the ascidian Halocynthia roretzi. AB - An animal-vegetal axis exists in the unfertilized eggs of the ascidian Halocynthia roretzi. The first phase of ooplasmic segregation brings the egg cortex to the vegetal pole very soon after fertilization. In the present study, when 5-8% of the egg cytoplasm in the vegetal pole region was removed between the first and second phase of segregation, most embryos exhibited failure of gastrulation, as reported previously in Styela by Bates and Jeffery (Dev. Biol, 124, 65-76, 1987). The embryos that were deficient in vegetal pole cytoplasm (VC deficient embryos) developed into permanent blastulae. They consisted for the most part of epidermal cells and most lacked the derivatives of vegetal blastomeres, such as endoderm, muscle and notochord. Removal of cytoplasm from other regions did not affect embryogenesis. The cleavage of the VC-deficient embryos not only exhibited radial symmetry along the animal-vegetal axis but the pattern of the cleavage was also identical in the animal and vegetal hemispheres. Examination of the developmental fates of early blastomeres of VC-deficient embryos revealed that the vegetal blastomeres had assumed the fate of animal cells. These results suggested that the VC-deficient embryos had been totally animalized. When vegetal pole cytoplasm was transplanted to the animal pole or equatorial position of VC-deficient eggs, gastrulation occurred, starting at the site of the transplantation and tissues derived from vegetal blastomeres formed. Therefore, it appears that vegetal pole cytoplasm specifies the site of gastrulation and the cytoplasm is responsible for the specification of vegetal blastomeres. It is suggested that during the second phase of ooplasmic segregation, cytoplasmic factors responsible for gastrulation spread throughout the entire vegetal hemisphere. PMID- 8620855 TI - Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation. AB - The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex relative to the inner cytoplasmic core. This cortical rotation depends on microtubules and is correlated with a parallel array of microtubules just inside the vegetal cortex. Since the parallel array moves with the inner cytoplasm and most of its microtubules are oriented with their plus ends facing the direction of cortical movement, it has been suggested that plus end-directed motor molecules attached to the cortex drive the rotation by moving along microtubules of the parallel array. Using an inverted confocal microscope to examine living eggs, however, we found that rotation movements precede the formation of a detectable parallel array at the vegetal pole, that the parallel array consists of multiple layers of microtubules at depths ranging from 4 to 8 microns inside the plasma membrane and that the velocity of rotation is immobilized eggs increases with depth in this region. These findings suggest that (1) early cytoplasmic movements are due to something other than the fully formed parallel array and (2) the motor molecules responsible for the bulk of the rotation movement are not restricted to a monolayer at the subcortical interface but may be distributed throughout the parallel array, perhaps causing microtubules to slide along other microtubules by a mechanism similar to that seen in cilia and eukaryotic flagella. PMID- 8620856 TI - A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation. AB - In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermatogenesis. Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells. These transcripts should encode a truncated version of the c-kit protein, lacking the extracellular, the transmembrane and part of the intracellular tyrosine-kinase domains. The 5' end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene. We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors. Deletion of both these sequences abolishes activity of the promoter in vitro. We have also established that this promoter is functional in vivo, in a tissue-and cell-specific fashion, since intronic sequences drive the expression of the E. coli lacZ reporter gene in transgenic mice specifically in the testis. Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis. In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa. An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems. PMID- 8620857 TI - Segregation of germ granules in living Caenorhabditis elegans embryos: cell-type specific mechanisms for cytoplasmic localisation. AB - Germ granules are ribonucleoprotein particles that are thought to function in germline specification in invertebrates and possibly in vertebrates. In Caenorhabditis elegans, these structures, termed P granules, are partitioned to the germline P cells during the early embryonic divisions. By injecting a fluorescently labelled anti-P-granule antibody into the C. elegans germline syncitium, we followed P-granule segregation in live embryos using laser-scanning confocal microscopy. We show that, in early P cells (P0 and P1), P-granule partitioning is achieved primarily by their migration through the cytoplasm towards the site of formation of the germline daughter cell. A different mechanism appears to operate in later P cells (P2 and P3): P granules associate with the nucleus and move with it toward the site of formation of the germline daughter cell, where they are then deposited. At each division, there is also disassembly or degradation of those P granules that remain in the cytoplasm destined for the somatic daughter cell. Microfilaments, microtubules and the product of the gene mes-1 are required for the normal pattern of P-granule segregation in P2. PMID- 8620858 TI - Characterizing the zebrafish organizer: microsurgical analysis at the early shield stage. AB - The appearance of the embryonic shield, a slight thickening at the leading edge of the blastoderm during the formation of the germ ring, is one of the first signs of dorsoventral polarity in the zebrafish embryo. It has been proposed that the shield plays a role in fish embryo patterning similar to that attributed to the amphibian dorsal lip. In a recent study, we fate mapped many of the cells in the region of the forming embryonic shield, and found that neural and mesodermal progenitors are intermingled (Shih, J. and Fraser, S.E. (1995) Development 121, 2755-2765), in contrast to the coherent region of mesodermal progenitors found at the amphibian dorsal lip. Here, we examine the fate and the inductive potential of the embryonic shield to determine if the intermingling reflects a different mode of embryonic patterning than that found in amphibians. Using the microsurgical techniques commonly used in amphibian and avian experimental embryology, we either grafted or deleted the region of the embryonic shield. Homotopic grafting experiments confirmed the fates of cells within the embryonic shield region, showing descendants in the hatching gland, head mesoderm, notochord, somitic mesoderm, endoderm and ventral aspect of the neuraxis. Heterotopic grafting experiments demonstrated that the embryonic shield can organize a second embryonic axis; however, contrary to our expectations based on amphibian research, the graft contributes extensively to the ectopic neuraxis. Microsurgical deletion of the embryonic shield region at the onset of germ ring formation has little effect on neural development: embryos with a well-formed and well-patterned neuraxis develop in the complete absence of notochord cells. While these results show that the embryonic shield is sufficient for ectopic axis formation, they also raise questions concerning the necessity of the shield region for neural induction and embryonic patterning after the formation of the germ ring. PMID- 8620860 TI - Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males. AB - Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and postmeiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, as spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man. PMID- 8620859 TI - IGF-I, insulin and FGFs induce outgrowth of the limb buds of amelic mutant chick embryos. AB - IGF-I, insulin, FGF-2 and FGF-4 have been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying mesoderm required for outgrowth and patterning of the developing limb. To study further the roles of these growth factors in limb outgrowth, we have examined their effects on the in vitro morphogenesis of limb buds of the amelic mutant chick embryos wingless (wl) and limbless (ll). Limb buds of wl and ll mutant embryos form at the proper time in development, but fail to undergo further outgrowth and subsequently degenerate. Wl and ll limb buds lack thickened AERs capable of promoting limb outgrowth, and their thin apical ectoderms fail to express the homeobox-containing gene Msx-2, which is highly expressed by normal AERs and has been implicated in regulating AER activity. Here we report that exogenous IGF-I and insulin, and, to a lesser extent, FGF-2 and FGF-4 induce the proliferation and directed outgrowth of explanted wl and ll mutant limb buds, which in vitro, like in vivo, normally fail to undergo outgrowth and degenerate. IGF-I and insulin, but not FGFs, also cause the thin apical ectoderms of wl and ll limb buds to thicken and form structures that grossly resemble normal AERs and, moreover, induce high level expression of Msx-2 in these thickened AER-like structures. Neither IGF-I, insulin nor FGFs induce expression of the homeobox containing gene Msx-1 in the subapical mesoderm of wl or ll limb buds, although FGFs, but not IGF-I or insulin, maintain Msx-1 expression in normal (non-mutant) limb bud explants lacking an AER. The implications of these results to the relationships among the wl and ll genes, IGF-I/insulin, FGFs, Msx-2 and Msx-1 in the regulation of limb outgrowth is discussed. PMID- 8620862 TI - Modifications occur at different structural levels during the heat denaturation of beta-lactoglobulin. AB - Heat-induced modifications in the tertiary and quaternary structure of beta lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near-ultraviolet-CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65-70 degrees C. Addition of iodoacetamide during the heating/cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein. Reaction of the native beta-lactoglobulin dimer with iodoacetamide or dithiobis(2 nitrobenzoic acid) was only observed upon heating at temperatures higher than 40 degrees C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate limiting step in the reaction of beta-lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (Ea = 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive species in heat-treated beta-lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like free monomer that has an exposed sulfhydryl group on its surface. PMID- 8620861 TI - cDNA cloning and primary structure of tryptase from bovine mast cells, and evidence for the expression of bovine pancreatic trypsin inhibitor mRNA in the same cells. AB - A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711-bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active-site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin-like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro. PMID- 8620863 TI - Glycosylation of recombinant ancrod from Agkistrodon rhodostoma after expression in mouse epithelial cells. AB - The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells. PMID- 8620864 TI - Comparison of the structural features of ubiquinone reduction sites between glucose dehydrogenase in Escherichia coli and bovine heart mitochondrial complex I. AB - To characterize the structural features of the ubiquinone reduction site of glucose dehydrogenase (GlcDH) in Escherichia coli, we performed structure/activity studies of a systematic set of synthetic ubiquinone analogues and specific inhibitors (synthetic capsaicins) of this site. Considering the proposed similarity of the quinone binding domain motif between GlcDH and one subunit of mitochondrial complex I [Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H. & Weiss, H. (1990) FEBS Lett. 265, 37-40], we compared the structure/activity profiles of the substrates and inhibitors for GlcDH with those for bovine heart mitochondrial complex i. With respect to GlcDH, replacement of one or both methoxy groups in the 2 and 3 positions of benzoquinone ring by ethoxy group(s) resulted in a drastic decrease in the electron accepting activity. The presence of a 5-methyl group and the conformational property of the 6-alkyl side chain did not significantly contribute to the activity. These results suggested that only half of the benzoquinone ring (the moiety corresponding to the 2 and 3 positions) is recognized by the quinone reduction site in a strict sense. In contrast, quinone analogues with structural modifications at all positions in the benzoquinone ring retained the activity with mitochondrial complex I. This finding indicated that the catalytic site of complex I is spacious enough to accommodate a variety of structurally different quinone derivatives. The correlation of the inhibitory potencies of a series of synthetic capsaicins between the two enzymes was very poor. These findings indicated that the binding environment of ubiquinone in GlcDH is very specific and differs from that in mitochondrial complex I. PMID- 8620865 TI - Analysis of the mechanism of activation of cAMP-dependent protein kinase through the study of mutants of the yeast regulatory subunit. AB - Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1 6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit. PMID- 8620866 TI - Translational control of poly(A)-binding protein expression. AB - Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5' untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system. Results of our studies show that removal of the putative PABP-binding sites from the 5' UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system. Furthermore, in vitro translation of the full length PABP mRNA was inhibited by addition of purified PABP to the cell-free system. In contrast, translation of truncated mRNA lacking the putative PABP binding sites at the 5' UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5' UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5' UTR of its full-length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5' UTR. The adenine-rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism. PMID- 8620867 TI - C23 interacts with B23, a putative nucleolar-localization-signal-binding protein. AB - The human protein C23 (nucleolin) is a major nucleolar protein. Its interactions with other proteins were studied with the two-hybrid system which identified nucleolar protein B23 (nucleophosmin) as being associated with C23. Both proteins were co-immunoprecipitated from HeLa cell nuclear extract by either monoclonal anti-C23 or monoclonal anti-B23. Binding studies utilizing deletion mutants indicated that the binding of C23 and B23 involves specific motifs. In addition to an approximately 46-amino-acid-binding domain in B23 (amino acids 194-239), amino acids 540-628 of C23 were required for binding; this region of C23 is required for the nucleolar localization. In addition, nucleolar protein p120 was also found to be co-immunoprecipitated with B23. A fragment of p120 containing a functional nucleolar localization signal bound to the truncated binding domain of B23, as did C23. These results suggest that the interaction of C23 and B23 may represent a nucleolar-targeting mechanism in which B23 acts as a nucleolar localization signal-binding protein. PMID- 8620868 TI - Complete cDNA sequence of the preproform of human pregnancy-associated plasma protein-A. Evidence for expression in the brain and induction by cAMP. AB - A cDNA that encodes the prepropeptide of pregnancy-associated plasma protein-A (preproPAPP-A), a putative metalloproteinase, has been cloned and sequenced. PAPP A is synthesized in the placenta as a 1627-residue precursor preproprotein with a putative 22-residue signal peptide and a highly basic propeptide of 58 residues. The prepro-PAPP-A-encoding transcript contains a region with an extremely high G+C content and has an unusually long 5' untranslated region with several upstream short ORF. No alternatively spliced products could be identified by means of Northern blotting experiments or with rapid amplification of 5' cDNA ends experiments. A stretch within the 5' untranslated region shows sequence identities to a partial cDNA isolated from brain and a to cAMP-inducible sequence from a choriocarcinoma cell line. PMID- 8620869 TI - The expression of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme-A synthase in neonatal rat intestine and liver is under transcriptional control. AB - Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HOMeGlt-CoA) synthase regulates ketogenesis in the liver of adult rat and in the intestine and liver of neonatal animals but whose mechanisms of regulation have not been fully defined. To investigate transcriptional control of this gene in intestine and liver of suckling rats a quantitative PCR amplification of the pre-mRNA (heteronuclear RNA), compose of part of the first exon and of the first intron, was carried out. Results show that the intestinal pre-mRNA for mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern that is nearly identical to that of mature mRNA, with maximum levels on the ninth postnatal day then decreasing smoothly so that at weaning there is no transcriptional activity. Mitochondrial HOMeGlt-CoA synthase protein follows a pattern that is identical to the pre-mRNA and mature mRNA, suggesting no translational regulation. The changes in transcriptional activity are not produced by the presence of an alternative promoter, since the transcription-initiation site is identical in several tissues assayed, including intestine and liver. Enterocytes are the only intestinal cells that express this ketogenic enzyme, as deduced from immunolocalization experiments. The mature intestinal protein is located in mitochondria and not in the cytosol, which coincides with what is found in liver. By using analogous techniques we conclude that hepatic pre-mRNA of mitochondrial HOMeGlt-CoA synthase from suckling rats follows a pattern of expression identical to that of mature hepatic mRNA, which also suggests a transcriptional modulation of this gene in the liver of neonatal rats. PMID- 8620870 TI - Dissociation kinetics of actinomycin D from individual GpC sites in DNA. AB - We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT)n than An.Tn. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC)n.(GT)n present good binding sites; in this context, dissociation from CGCG is faster than from TGCA. PMID- 8620871 TI - Purification and characterization of a 5' to 3' exoribonuclease from rabbit reticulocytes that degrades capped and uncapped RNAs. AB - The cytoplasm of mammalian cells of undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5' to 3' manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat-labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10-200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5' end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate-limiting step involves the positioning of the enzyme at the 5' end of the substrate and/or cleavage of the first internucleotide bond. PMID- 8620872 TI - Sequence requirements in the reactive-center loop of plasminogen-activator inhibitor-1 for recognition of plasminogen activators. AB - Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue-type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy-terminal side of the cleavage site of recombinant human PAI-1. Using site-directed mutagenesis, the P1'-P5' residues (P1' is the first residue on the carboxy-terminal side of the protease cleavage site) of the wild-type PAI-1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor-2, antithrombin, alpha 2-antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue-type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI-1. Analysis of substitutions at positions P4' and P5' both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue-type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI-1. Apparently, the determinants of the rapid inhibitory specificity of PAI-1 are localized to the P1'-P5' region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI-1 for plasminogen activators. PMID- 8620873 TI - Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2. AB - Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15 18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16 19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions. PMID- 8620874 TI - Thermal stability of individual domains in platelet glycoprotein IIbIIIa. AB - Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha IIb beta 3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching over 65-110 degrees C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15 degrees C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca(2+)-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca(2+)-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH2-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca(2+)-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa. PMID- 8620875 TI - Hydrogen exchange in the carbon monoxide complex of soybean leghemoglobin. AB - Hydrogen/deuterium exchange rates for individual amide protons have been measured for the carbon monoxide complex of soybean leghemoglobin. Fast two-dimensional NOESY experiments were performed, with 5.2-min data-collection time for each spectrum, which made possible the measurement of NOE cross-peaks of relatively rapidly exchanging amide protons at early time points. Exchange rates were measured for 61 backbone amides, the protection factors were calculated to provide information on the packing and local stability of the protein. The data are consistent with the presence of transient cooperative local unfolding of helical segments. The B-, E-, G- and H-helices have extensive regions of slow-, medium- and fast-exchanging amide protons. For each of these helices, there is a progressive decrease in protection on moving from the helix center to the termini. This is consistent with a stable helix center, with dynamic fraying at the ends. Amide exchange from the A-helix and C-helix is rapid except in small local regions. The F-helix, which is located on the proximal side of the heme pocket and is well formed in solution as demonstrated by characteristic medium range NOE connectivities [Morikis, D. Lepre, C.A. & Wright, P.E. (1994) Eur. J. Biochem. 219, 611-626], exhibits fast exchange for all amide protons. The implied flexibility and low stability of the F-helix may be functionally important in facilitating movement of the helix upon ligand binding. Fast exchange has also been observed for all amide protons in the CE-loop and in turns, as expected for flexible or solvent exposed regions. A strong tertiary contact has been established between the A-, G- and H-helices by the presence of a slowly exchanging indole N epsilon H of Trp129. PMID- 8620876 TI - Malonate decarboxylase of Klebsiella pneumoniae catalyses the turnover of acetyl and malonyl thioester residues on a coenzyme-A-like prosthetic group. AB - During aerobic growth of Klebsiella pneumoniae on malonate, a soluble malonate decarboxylase is induced. Malonate decarboxylation consumes a proton (not H2O) and forms acetate and CO2 (not HCO3-) as products. The enzyme was purified 56 fold to apparent homogeneity. It has a native molecular mass of 142 kDa and consists of four subunits alpha, beta, gamma and delta with molecular masses of 65, 34, 30, and 12 kDa, respectively. Two different forms of the enzyme were recognised: a catalytically inactive SH-enzyme and the catalytically active acetyl-S-enzyme which is formed by post-translational acetylation of the SH enzyme with ATP, acetate and a specific ligase. The acetyl-S-enzyme was converted into the SH-enzyme by incubation with hydroxylamine or dithioerythritol. Chemical reacylation of the SH-enzyme, which restores catalytic activity, was achieved with acetic anhydride or more efficiently with malonyl-CoA. This acylation of the SH group was prevented after incubation with various thiol-specific reagents. After incubation of the SH-enzyme with iodo[1-14C]acetate, the delta subunit became specifically labelled. This subunit was also labelled after incubation of the acetyl-S-enzyme with [2-14C]malonate. The radioactivity was completely liberated from the protein upon malonate addition. These results indicate that the delta subunit is the acyl-carrier protein of the complex and that malonate decarboxylation proceeds in two steps: the acetyl residue on the ACP is first replaced by a malonyl residue which subsequently undergoes decarboxylation thereby regenerating the acetyl-S-ACP. The binding site for the acyl residues on the acyl-carrier protein was shown to be 2'-(5"-phosphoribosyl)-3'-dephospho-CoA after alkaline cleavage of this prosthetic group from the enzyme and chromatographic as well as mass spectroscopic analyses. PMID- 8620877 TI - Structure of the O-specific polysaccharide of Acinetobacter baumannii O5 containing 2-acetamido-2-deoxy-D-galacturonic acid. AB - A polysaccharide containing 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2 deoxy-L-fucose (FucNAc), and 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid-free, isolated cell walls of the reference strain for Acinetobacter baumannii serogroup O5, by mild acid hydrolysis of the extract and chromatography of the water-soluble products on Sephadex G-50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide: [table: see text] PMID- 8620878 TI - Glucose uptake in Trypanosoma vivax and molecular characterization of its transporter gene. AB - A gene, TvHT1, encoding a glucose transporter protein, has been cloned from the haemoflagellate protozoon, Trypanosoma vivax, which has an active Kreb's cycle in the mammalian stage. The deduced polypeptide is similar in amino acid sequence to other kinetoplastid hexose transporters from Trypanosoma brucei (THT1 and THT2), Trypanosoma cruzi (TcrHT1) and Leishmania (Pro-1). The similarity is higher with THT2 (expressed in T. brucei insect forms) than with the other isoforms. The kinetic properties of glucose uptake in Chinese Hamster Ovary (CHO) cells expressing TvHT1 and in trypanosomes show s a saturable transport mechanism typical of a facilitated carrier system, with a similar affinity for D-glucose as that of the T. brucei bloodstream form carrier, THT1 (Km = 0.548 +/- 0.01 mM, Vmax = 4.26 +/- 0.12 nmol.min-1.mg protein-1 in CHO cells and Km = 0.585 +/- 0.068 mM, Vmax = 88.5 +/- 6.2 nmol.min-1.mg protein-1 in T. vivax). The specificity of the TvHT1 protein for various D-glucose analogues, as judged by inhibition of 2-deoxy-D-arabinose-hexose transport, shows properties that are intermediate between those of THT1 on the one hand and TcrHT1 and THT2 on the other. As with the hexose transporters in the other members of Kinetoplastida, the TvHT1-encoded system differs from erythrocyte-type glucose transport by its moderate sensitivity to cytochalasin B and its capacity to transport fructose. PMID- 8620879 TI - Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley. AB - The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da. A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5'untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley. PMID- 8620880 TI - Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues. AB - The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP. PMID- 8620881 TI - Structure of the 5'-flanking regulatory region of the mouse gene encoding the clearance receptor for atrial natriuretic peptide. AB - A full-length cDNA, encoding the mouse atrial natriuretic peptide clearance receptor (ANP-CR), was isolated from a mouse lung cDNA library. The deduced amino acid sequence of the mouse ANP-CR, showing a typical tripartite organization which lacks a guanylyl cyclase domain, was extremely well conserved compared with the ANP-CR homologs. To understand the molecular mechanisms underlying the regulation of mouse ANP-CR gene expression and to define the essential DNA sequences for the transcriptional activity, a genomic clone containing over 9 kb of the 5'-flanking region of the mouse ANP-CR gene has been isolated from a mouse genomic library. Sequence analysis revealed that the 2.3-kb region upstream from an ATG codon of the mouse ANP-CR gene contained a number of putative regulatory elements; TATA box, CAAT box, cAMP response element, AP-1 and two shear stress responsive elements. Additionally, an unusual feature was the presence of the tandem-repeated AP-2-like elements, which were closely overlapped with SP-1 element. Promoter analysis using deletion plasmids in mouse Balb/3T3 cells, highly producing ANP-CR mRNA, demonstrated that deletion of the sequence from 144 to +46 relative to the transcription start point caused a dramatic decrease of the transcriptional activity and that the TATA box at -269 was not essential for the basal transcriptional activity. Primer extension analysis indicated that transcription of the mouse ANP-CR gene starts from at least two major sites, suggesting that the sequence from -144 to +46, which was shown to involve a novel sequence composed of tandem-repeated TATA-box-like elements, contained promoter sequences. Furthermore, cis-acting negative elements were shown to be situated in three regions (from -1178 to -708, from -707 to -625 and from -248 to -145) of the mouse ANP-CR gene promoter. PMID- 8620882 TI - Two distinct quinoprotein amine oxidases are induced by n-butylamine in the mycelia of Aspergillus niger AKU 3302. Purification, characterization, cDNA cloning and sequencing. AB - Two distinct quinoprotein amine oxidases were found in Aspergillus niger mycelia grown on n-butylamine medium and purified using chromatographic techniques. The respective enzymes were termed AO-I, which had already been isolated, and AO-II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80 kDa, respectively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit dimeric AO-I and 80 kDa for monomeric AO-II. The absorption spectra of both enzymes were different from each other; the absorption maxima in the visible region were at 490 nm for AO-I and 420 nm for AO-II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity to inhibitors typical for copper/topa quinone-containing amine oxidases, but they had different copper contents and also differed in their N-terminal sequences. Their peptide maps showed almost identical patterns, with the exception of two additional bands for AO-II. Among the peptides obtained from digestion of AO-II, peptides with sequences corresponding to the N-terminal part of AO-I were detected. Polyclonal antibodies raised against AO-I and AO-II recognized both enzymes, but with different specificities. Using precipitation with AO-I, the antibody prepared against AO-II was purified and was shown to be specific only for AO-II. The cDNA of AO-I was cloned and sequenced. A highly conserved tetrapeptide sequence, Asn Tyr-Glu-Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinone was present in the 670-residue deduced amino acid sequence. Northern blot analysis indicated that AO-I was highly expressed in A. niger grown on n-butylamine as a single nitrogen source. Genomic Southern blot analysis confirmed that both enzymes are likely to be encoded by the same gene. PMID- 8620883 TI - Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O11. AB - A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy D-galactose was obtained after mild acid hydrolysis of the water-soluble material released by treatment of cell walls from Acinetobacter baumannii strain O11 with hot, aqueous phenol. By means of NMR studies, Smith degradation and N deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2-acetamido-2-deoxyglucose-and 2-acetamido-2-deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O-antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that O11 specificity was conferred by the major polymer. [formula: see text] PMID- 8620884 TI - Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin sensitive Klebsiella pneumoniae O3. AB - Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniae O3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4 deoxy-L-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-L-arabinopyranose. In the sensitive-type LEN 1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-1. PMID- 8620885 TI - Molecular sequencing and modeling of Neobellieria bullata trypsin. Evidence for translational control by Neobellieria trypsin-modulating oostatic factor. AB - Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut. PMID- 8620886 TI - Transient expression of a mitochondrial precursor protein. A new approach to study mitochondrial protein import in cells of higher eukaryotes. AB - In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit-9-H2folate reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that is was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature-sized protein was folded into a protease-resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit-9 H2folate-reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV-mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders or organelle protein import. PMID- 8620887 TI - Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. AB - Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP-2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP-2 protein of M(r) 26,000 was prepared with a purity greater 98%. The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15-20 nM). A peptide 1-17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N-terminai reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1-12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1-17. A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible. These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity. PMID- 8620888 TI - A class of highly potent antibacterial peptides derived from pardaxin, a pore forming peptide isolated from Moses sole fish Pardachirus marmoratus. AB - Pardaxin, a 33-amino-acid pore-forming polypeptide toxin isolated from the Red Sea Moses sole Pardachirus marmoratus, has a helix-hinge-helix structure. This is a common structural motif found both in antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Herein we show that pardaxin possesses a high antibacterial activity with a significantly reduced hemolytic activity towards human red blood cells (hRBC), compared with melittin. Its potency is comparable to that of other known native antibacterial peptides such as magainin, cecropins and dermaseptins. To determine the structural features responsible for the selective hemolytic and antibacterial activities, and the structural requirements for a high antibacterial activity, 8 truncated and modified pardaxin analogues were synthesized and structurally and functionally characterized. Each peptide was synthesized with a free carboxylate or amino group (i.e., aminated form) at its C-terminus. The aminated form of pardaxin has both high hemolytic and antibacterial activity. A truncated analogue, with 11 amino acids removed from the C-terminal domain, had dramatically reduced hemolytic activity. However, the aminated form of this analogue was significantly more potent that pardaxin against most bacteria tested, suggesting that the C-terminal tail of pardaxin is responsible for non selective activity against erythrocytes and bacteria. Furthermore, a positive charge added to its N-terminus significantly increased its antibacterial activity and abolished its low hemolytic activity. The 22-amino-acid C-terminal domain and the short 11-amino-acid N-terminal domain were, in their aminated forms, active only against gram-positive bacteria. Secondary-structure determination using circular dichroism spectroscopy revealed that all the aminated analogues had 25 80% more alpha-helical content in 40% CF3CH2OH/water than their non-aminated forms. Using model phospholipid membranes it was found that all the analogues that were less hemolytic but had retained antibacterial activity could permeate acidicly charged phospholipid vesicles better than zwitterionic phospholipid vesicles, a property characteristics of all the native antibacterial peptides tested so far (e.g., cecropins, magainins and dermaseptins). Pardaxin and its analogues therefore represent a new class of antibacterial peptides that can serve as a basis for the design of therapeutic agents. Furthermore, negative staining electron microscopy revealed that total inhibition of bacterial growth was due to total lysis of the bacterial wall. Therefore, it might be more difficult for bacteria to develop resistance to such a destructive mechanism, compared with the more specific mechanisms of the currently used antibiotics. PMID- 8620889 TI - A (G+C)-rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element. AB - The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA proteins interactions could be impaired by a DNA probe that contained an Sp1 binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors. PMID- 8620890 TI - Potassium ions and the molecular-chaperone activity of DnaK. AB - Potassium ions stabilize the DnaK.ADP complex that forms on incubation of nucleotide-free DnaK with ADP or ATP. Generation of the crystallographically defined Mg2+ cluster [Wilbanks, S.M. & McKay, D.B. (1995) J. Biol. Chem. 270, 2251-2257], in which two K+ and the nucleotide are bound together with Mg2+ in the ATPase site, appears to be essential for the ATP-induced acceleration of binding of peptide ligands, the ATP-induced release of peptide ligands and for the peptide-induced increase in ATPase activity. Thus, K+ is instrumental in signal transmission between the ATPase site and the peptide-binding site. PMID- 8620891 TI - Catalytic properties, molecular composition and sequence alignments of pyruvate: ferredoxin oxidoreductase from the methanogenic archaeon Methanosarcina barkeri (strain Fusaro). AB - Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate [Bock, A. K., Prieger-Kraft, A. & Schonheit, P. (1994) Arch. Microbiol. 161, 33-46]. The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl-CoA was purified about 90-fold to apparent electrophoretic homogeneity. The purified enzyme catalyzed the CoA dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate: ferredoxin oxidoreductase. The deazaflavin, coenzyme F420, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme. In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors. Pyruvate: ferredoxin oxidoreductase also catalyzed the oxidation of 2-oxobutyrate but not the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3-hydroxypyruvate and oxaloacetate. The apparent Km values of pyruvate:ferredoxin oxidoreductase were 70 microM for pyruvate, 6 microM for CoA and 30 microM for clostridial ferredoxin. The apparent Vmax with ferredoxin was about 30 U/mg (at 37 degrees C) with a pH optimum of approximately 7. The temperature optimum was approximately 60 degrees C and the Arrhenius activation energy was 40 kJ/mol (between 30 degrees C and 60 degrees C). The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0 degrees C. Sodium nitrite inhibited the enzyme with a Ki of about 10 mM. The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an alpha beta gamma delta structure. The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non-heme iron and acid-labile sulfur. FAD, FMN and lipoic acid were not found. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the alpha-subunit was similar to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate:ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the alpha subunit of the M. barkeri enzyme and the heterodimeric pyruvate:ferredoxin oxidoreductase of the archaeon Halobacterium halobium. PMID- 8620892 TI - Oxidation of dimethoxylated aromatic compounds by lignin peroxidase from Phanerochaete chrysosporium. AB - The stabilities of the cation radicals of veratryl alcohol, 3,4-dimethoxytoluene and 1,4-dimethoxybenzene were compared by monitoring the formation of dimeric products during the oxidation of these substrates by lignin peroxidase (LiP). LiP oxidized veratryl alcohol to generate veratraldehyde as the major product. Several other monomeric products were obtained in low yield. Dimeric products resulting from the coupling of two cation radicals, or a cation radical with a neutral molecule, were obtained only in trace amounts or not at all. This suggests that the cation radical of veratryl alcohol rapidly loses a benzylic proton to form a benzylic radical which undergoes further reactions to form veratraldehyde. In contrast, the LiP oxidation of 3,4-dimethoxytoluene generated the dimeric product 3-(2,3-dimethoxy-6-methylphenyl)-4-methyl-1,2-benzoquinone as the major product. Several other monomeric and dimeric products were produced in lower yields. The generation of these dimeric products indicates that the cation radical of 3,4-dimethoxytoluene is considerably more stable than that of veratryl alcohol. This suggests that the electronegative benzylic oxygen of veratryl alcohol increases the acidity of the benzylic protons, destabilizing the veratryl alcohol cation radical. LiP oxidized 1,4-dimethoxybenzene to generate 1,4 benzoquinone and 2-(2,5-dimethoxyphenyl)-1,4-benzoquinone as the major products. The formation of these products indicates that the cation radical of 1,4 dimethoxybenzene also is relatively stable, as previously demonstrated by ESR. All of these results indicate that the veratryl alcohol cation radical generated by LiP oxidation is unstable, suggesting that it would not act as a diffusable radical mediator in LiP-catalyzed reactions. PMID- 8620893 TI - Structural and functional characterization of two mutated R2 proteins of Escherichia coli ribonucleotide reductase. AB - The R2 protein of ribonucleotide reductase from Escherichia coli is a homodimeric tyrosyl-radical-containing enzyme with two identical dinuclear iron centers. Two randomly generated genomic mutants, nrdB-1 and nrdB-2, that produce R2 enzymes with low enzymatic activity, have been cloned and characterized to identify functionally important residues and areas of the enzyme. The mutations were identified as Pro348 to leucine in nrdB-1 and Leu304 to phenylalanine in nrdB-2. Both mutations are the results of single amino acid replacements of non-conserved residues. The three-dimensional structures of [L348]R2 and [F304]R2 have been determined to 0.26-nm and 0.28-nm resolution, respectively. Compared with wild type R2, [L348]R2 binds with higher affinity to R1, probably due to increased flexibility of its C-terminus. Since the three-dimensional structure, iron-center properties and radical properties of [L348]R2 are comparable to those of wild type R2, the low catalytic activity of the holoenzyme is probably caused by a perturbed interaction between R2 and R1. The [F304]R2 enzyme has increased radical sensitivity and low catalytic activity compared with wild-type R2. In [F304]R2 the only significant change in structure is that the evolutionary conserved Ser211 forms a different hydrogen bond to a distorted helix. The results obtained with [F304]R2 indicate that structural changes in E. coli R2 in the vicinity of this helix distortion can influence the catalytic activity of the holoenzyme. PMID- 8620894 TI - A beta A4 amyloid precursor protein gene and Alzheimer's disease. AB - Alzheimer's disease is a senile dementia caused by progressive neurodegeneration of the central nervous system. One of the most prominent pathological characteristics is beta A4 amyloid deposition in senile plaques in the brain parenchyma and in cerebral blood vessels. beta A4 amyloid is processed from a larger integral membrane protein, the beta A4 amyloid precursor protein. Different pathogenic mutations in this protein have been detected in a small number of Alzheimer's disease families. Here functional implications of these mutations on the processing of the precursor protein and the beta A4 amyloid deposition will be discussed with respect to the pathogenesis of Alzheimer's disease and related disorders. PMID- 8620895 TI - T-cell membrane-associated serine protease, tryptase TL2, binds human immunodeficiency virus type 1 gp120 and cleaves the third-variable-domain loop of gp120. Neutralizing antibodies of human immunodeficiency virus type 1 inhibit cleavage of gp120. AB - It has been suggested that the third variable domain (V3) loop of human immunodeficiency virus type 1 (HIV-1) gp120 has to interact with a cell-surface associated protease(s) that acts as a cofactor after binding of gp120 to the CD4 receptor during entry of HIV-1 into susceptible cells. We isolated the membrane associated serine protease, tryptase TL2, from human CD4-positive lymphocytes. This enzyme specifically binds gp120 through interaction with its V3 domain. To investigate the role of tryptase TL2 in HIV infection, we examined the affinity of the interaction and the proteolytic susceptibility of various recombinant gp120 expressed in mammalian cells to the enzyme, and we determined the cleavage sites. Tryptase TL2 bound gp120 with an apparent dissociation constant of 38 nM. The affinity was lower than that of gp120 for CD4 which suggests that gp120 initially binds to CD4, followed by interaction with tryptase TL2 which is localized close to CD4 on the cell surface. After binding, tryptase TL2 cleaved recombinant gp120 expressed in mammalian cells into two protein species of 70 kDa and 50 kDa but did not cleave gp120 expressed in insect cells, which indicates that the structure of the oligosaccharides linked to the polypeptide backbone of gp120 affects the proteolytic susceptibility. Cleavage was specifically inhibited by a neutralizing antibody against the V3 loop. Cleavage-site determination revealed that tryptase TL2 cleaved gp120 at various sites in the V3 in a strain dependent manner. The amino acid variability at the cleavage site(s) in almost all HIV-1 isolates was restricted to amino acids which are susceptible to the chymotryptic and/or tryptic activities of tryptase TL2. PMID- 8620896 TI - Isolation, characterisation and crystallisation of a water-soluble fragment of the Rieske iron-sulfur protein of bovine heart mitochondrial bc1 complex. AB - A water-soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was + 306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant alpha-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple wavelength anomalous-scattering technique is underway. PMID- 8620897 TI - Detailed oligosaccharide structures of human integrin alpha 5 beta 1 analyzed by a three-dimensional mapping technique. AB - Structures of N-linked oligosaccharides obtained from human integrin alpha 5 beta 1 are described. Integrin alpha 5 beta 1 (4.5 mg) was purified from human placenta and digested using trypsin and chymotrypsin. N-linked oligosaccharides were released from the glycopeptides by digestion with glycoamidase A (from almond). The reducing ends of the oligosaccharides were derivatized with 2 aminopyridine. The pyridylamino-oligosaccharides were separated and these structures were identified by a three-dimensional HPLC mapping technique on three kinds of HPLC columns [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem. 226, 139-146]. Finally, 35 different oligosaccharide structures were identified, 10 of which were neutral, 6 mono sialyl, 10 di-sialyl, 7 tri-sialyl and 2 tetra-sialyl. The molar ratio of neutral, mono-sialyl, di-sialyl, tri-sialyl and tetra-sialyl oligosaccharides was 20.8%, 24.8%, 27.7%, 18.1% and 8.6%, respectively. High-mannose-type oligosaccharides accounted for only 1.5% of the total. The remaining oligosaccharides were all complex type. The most predominant structure was the diantennary di-alpha-(2,3)-sialyl fucosyl. Major linking of sialic acid was alpha (2,3)-linkage, and over 50% of all oligosaccharides were fucosylated at the N acetylglucosamine residue of the reducing end. PMID- 8620898 TI - Biochemical and antibacterial analysis of human wound and blister fluid. AB - Fluid from a post-operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse-phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti-Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1-11,1-15 and 1-16, intact thymosin beta-4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL-39 and its precursor(s). The HPLC-separated blister fluid was extremely rich in anti-Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50-fold differences in anti-Bm activities and more than 10-fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further. PMID- 8620899 TI - Half-of-the-sites reactivity of bovine serum amine oxidase. Reactivity and chemical identity of the second site. AB - The organic cofactor of bovine serum amine oxidase was identified as 2,4,5 trihydroxyphenylalanine quinone by means of the phenylhydrazine adduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burligame, A.L. & Klinman, J.P. (1990) Science 248, 981-987]. A still debated question is, however, whether the dimeric protein binds two mol phenylhydrazine/mole or only one, that is whether it actually contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions with phenylhydrazine and other inhibitors such as semicarbazide and p-pyridine-2 yl-phenylacetohydrazide. The two latter reagents were found to bind in two steps, one mole/mole dimer in the first step with loss of catalytic activity but only about (0.10-0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or by reverse-phase HPLC of the labelled peptides produced on proteolysis. Irrespective of the inhibitor nature and reacted amount, all adducts formed on proteolysis a single labelled peptide, of same 25-amino acid composition, showing that the same cofactor is present in both subunits, in the same stretch of the polypeptide chain. The slow reaction of the second cofactor may be related to slow conformational equilibria, which are established after the first cofactor has reacted and are probably mediated by a change of the hydrogen bond pattern. The conformers spectroscopic properties suggest that they differ in whether the cofactor does or does not directly interact with copper. PMID- 8620900 TI - Usefulness of the DNA-fingerprinting pattern and the multilocus enzyme electrophoresis profile in the assessment of outbreaks of meningococcal disease. AB - The objective of the study was to assess whether genotypic characterization by means of DNA-fingerprinting pattern (DFP) and multilocus enzyme electrophoresis (MEE) profile as compared to phenotypic characterization would improve the differentiation of Neisseria meningitidis strains associated with outbreaks from strains associated with sporadic cases of meningococcal disease. In addition, the differentiation of serogroup C carrier strains from those associated with an outbreak of serogroup C meningococcal disease was investigated. A total of 118 N. meningitidis strains were available for the study: 59 from patients involved in outbreaks of meningococcal disease (2 serogroup B and 2 serogroup C), 37 patients considered to be sporadic cases and 22 serogroup C carrier strains. Among the 59 strains from patients involved in outbreaks the 4 strains isolated from the patient registered as the first in each outbreak were designated the index strains. Among the remaining 55 outbreak strains 52 were either DFP-identical or DFP-indistinguishable when compared with the one relevant out of the 4 index strains. This was only the case for 17 of the 37 strains isolated from sporadic cases caused by the same serogroup of meningococci during the outbreak periods, and 5 of the 22 meningococcal strains isolated from healthy carriers. Among the 56 (52 + 4) DFP-identical or DFP-indistinguishable outbreak strains 5 different electrophoretic types were identified by MEE. Among 59 assumed outbreak strains a total of 4 were identified as genotypically distinct. Among the 37 mainly DFP indistinguishable or DFP-different strains from sporadic cases 17 different ETs were identified, and among the 22 mainly DFP-different carrier strains 13 different ETs were identified. Two strains among those selected from sporadic cases were identical to the outbreak strain. None of the local serogroup C carrier strains isolated during the outbreak of serogroup C disease were identical to the outbreak strain. Both DNA-fingerprinting and MEE improved the differentiation of meningococci when compared with phenotypic characterization. The results indicate that tracing a virulent strain within a open group of contacts is irrelevant. PMID- 8620901 TI - Epidemics of serogroup A Neisseria meningitidis of subgroup III in Africa, 1989 94. AB - A total of 125 strains of Neisseria meningitidis recovered in the course of outbreaks from patients with systemic disease in 11 African countries between 1989 and 1994 were analysed by serogrouping, serotyping and multilocus enzyme electrophoresis. Of the 125 patient strains 115 (92%) belonged to the clone complex of serogroup A meningococci, designated subgroup III. Among the remaining strains, 4 were also serogroup A, but belonged to the clonal groups I and IV-1 (2 strains each), whilst 6 strains (4 serogroup C and 2 serogroup W135) represented clones of the ET-37 complex. Our results indicated that the second pandemic caused by clones of subgroup III is still spreading in Africa. Towards the West it has reached Niger, Mali, Guinea and The Gambia, and towards the South, the Central African Republic, Uganda, Rwanda, Burundi, Tanzania and Zambia. PMID- 8620902 TI - Epidemic cholera in Guatemala, 1993: transmission of a newly introduced epidemic strain by street vendors. AB - Epidemic cholera reached Guatemala in July 1991. By mid-1993, Guatemala ranked third in the hemisphere in reported cases of cholera. We conducted a case-control study with two age-, sex-, and neighbourhood-matched controls per patient in periurban Guatemala City. Twenty-six patients hospitalized for cholera and 52 controls were enrolled. Seven (47%) of 15 stool cultures obtained after admission yielded toxigenic Vibrio cholerae O1. All seven were resistant to furazolidone, sulfisoxazole, and streptomycin, and differed substantially by pulsed-field gel electrophoresis from the Latin American epidemic strain dominant in the hemisphere since 1991. In univariate analysis, illness was associated with consumption of left-over rice (odds ratio [OR] = 7.0, 95% confidence interval [CI] = 1.4-36), flavored ices (-helados') (OR = 3.6, CI = 1.1 - 12), and street vended non-carbonated beverages (OR = 3.8, CI = 1.2-12) and food items (OR = 11.0, CI = 2.3-54). Street-vended food items remained significantly associated with illness in multivariate analysis (OR = 6.5, CI = 1.4-31). Illness was not associated with drinking municipal tap water. Maintaining water safety is important, but slowing the epidemic in Guatemala City and elsewhere may also require improvement in street vendor food handling and hygiene. PMID- 8620905 TI - Presence of Salmonella spp. and Campylobacter spp. in shellfish. AB - Bivalve molluscs, (cockles, mussels, scallops and oysters) were examined according to EC shellfish bed classification regulations for faecal coliforms, Escherichia coli and salmonella, and for coliforms and campylobacter which are not specified by these regulations. Salmonella serotypes were detected in 8% of 433 molluscs. Seven salmonella isolations (2%) were made from category A beds, nominally suitable for immediate consumption according to E. coli counts. A higher percentage of salmonella isolates (6%) was detected in shellfish which require relaying or depuration prior to eating. In another survey, thermophilic Campylobacter spp. were found in 42% of 380 shellfish. These findings show bed classification on the basis of indicator organisms alone is not sufficient to assure the absence of bacterial, and no doubt viral, pathogens. Depuration and end product specifications which require the absence of salmonellae are an essential part of these regulations. Microbiologists may wish to consider whether tests for pathogens such as salmonella and campylobacter should be included when determining the suitability of shellfish for human consumption. PMID- 8620903 TI - Usefulness of ribotyping in a molecular epidemiology study of shigellosis. AB - Ribotyping performed with six restriction endonucleases was used to study the molecular epidemiology of shigellosis in Asturias, Spain. The series included Shigella sonnei from 34 sporadic cases, 3 outbreaks and 3 reference strains, and S. Flexneri from sporadic cases and 1 reference strain. The S. sonnei strains were grouped into 5 ribotypes with Sal I, 4 with Hind III and Pvu II, 3 with Bgl II and EcoR I and 2 with Hinc II (Discriminatory Index (DI) between 0.54 and 0.14); the S. flexneri into 5 ribotypes with Sal I, Hinc II and Hind III, and 4 with the other enzymes (DI = 0.71 - 0.63). The combination of results for 2 or more enzymes facilitated and additional discrimination, the highest values in S. sonnei were for the 6 enzymes (16 types, DI = 0.91) and in S. flexneri for some combinations of 3 or more enzymes (7 types, DI = 0.81). Ribotypes with the 6 enzymes defined 16 clonal lines in S. sonnei and 7 in S. flexneri, which showed a different degree of genetic heterogeneity, and all the lines of each species falling into a different cluster. No line appeared as clearly endemic in the bowels of Asturian people. PMID- 8620904 TI - Specific detection of Salmonella enterica serotype Enteritidis using the polymerase chain reaction. AB - An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis. PMID- 8620906 TI - A hospital outbreak of salmonella food poisoning due to inadequate deep-fat frying. AB - In an outbreak of plasmid-free Salmonella enteritidis phage type 4 (PT4) food poisoning at a hospital for mentally handicapped people in July 1990, 101 residents and 8 staff were affected and a cohort study implicated beef rissoles cooked by deep-fat frying as the vehicle of infection (relative risk 2.92, 95% confidence interval 1.73-4.93, P << 0.001). Replication of the cooking process demonstrated that the rissoles achieved core temperatures of only 48-60 degrees C despite external temperatures of 91-95 degrees C and an oil temperature of 142 154 degrees C. No residual food was available for microbiological testing but plasmid-containing S. enteritidis PT 4 was isolated in shell eggs from the hospital kitchen. PMID- 8620907 TI - Consecutive salmonella outbreaks traced to the same bakery. AB - Two consecutive community outbreaks of Salmonella enteritidis phage type 4 (PT4) traced to the same bakery occurred in Cardiff, Wales during August-September 1992. In the first outbreak, illness was associated with eating custard slices (odds ratio 23.8, 95% confidence interval 6.5-94.4, P < 0.0001), and in the second, with eating fresh cream cakes (odds ratio 15.8, 95% confidence interval 1.6-374, P = 0.004). Environmental investigations implicated cross-contamination during preparation of the cold-custard mix as the cause of the first outbreak, and inadequate cleaning and disinfection of nozzles used for piping cream in the second outbreak. S. enteritidis PT4 was isolated from fresh cream sponge cake retained by a case and from two fresh cream cakes and four environmental swabs obtained at the bakery. This incident illustrates the hazard of widespread environmental contamination with salmonella and the need for thorough environmental cleansing for any premises implicated in an outbreak of food poisoning. PMID- 8620908 TI - Resistotyping of campylobacters: fulfilling a need. AB - A 9-month trial of a simple typing scheme for ?thermophilic' enteric campylobacter isolates at a large Public Health Laboratory is described. Resistotyping was performed with six agents in a method modified by Bolton and colleagues from an earlier scheme, and biotyping was performed by a modified Lior scheme involving three tests. Reproducibility was excellent in both schemes, with test variation < 2%. Five household clusters and one larger presumptive milk borne outbreak were identified in this scheme, and confirmed in pyrolysis mass spectrometry. The 328 isolates from new patients, excluding duplication from these clusters, were divided into 35 resistotypes with the largest group comprising 22% of isolates. In combined bio- and resistotyping, 86 types were found, with the largest group comprising 9.5% of isolates. The results are contrasted with salmonella sero- and phage-typing, where, on the same basis, the 176 isolates in the same period were divided into 40 groups, with the largest comprising 45% of isolates. Resistotyping, with or without additional biotyping, proved to be a convenient, simple, rapid, highly discriminatory, reproducible and inexpensive method well suited to use in local laboratories. It is a strong candidate for first-line national and local surveillance of campylobacter infections, fulfilling a need for monitoring of this important cause of enteric disease. PMID- 8620909 TI - Carriage of multiple ribotypes of non-encapsulated Haemophilus influenzae in aboriginal infants with otitis media. AB - Ribotyping with the restriction enzyme XbaI was used to study the dynamics of Carriage of non-encapsulated Haemophilus influenzae (NCHi) in Aboriginal infants at risk of otitis media. Carriage rates of NCHi in the infants in the community were very high; the median age for detection was 50 days and colonization was virtually 100% by 120 days of age and persisted at a high level throughout the first year of life [1]. Eighteen different ribotypes of NCHi were identified from 34 positive swabs taken from 3 infants over a period of 9 months. The same ribotypes were recovered for up to 3 months from consecutive swabs of individual infants, and 12 of 27 swabs (44.4%) yielded two ribotypes from four colonies typed. Statistical analysis suggested that most swabs would have been positive for two ribotypes if enough colonies had been typed although the second most frequent ribotype was detected on average in only 13% of strains. Early colonization and carriage of multiple ribotypes of NCHi may help to explain the chronicity of carriage and thus the persistence of otitis media in Aboriginal infants. PMID- 8620910 TI - Legionellosis linked with a hotel car park--how many were infected? AB - An outbreak of legionellosis associated with a hotel in Sydney, Australia, and the subsequent epidemiological and environmental investigations are described. Four cases of Legionnaires' disease were notified to the Public Health Unit. A cross-sectional study of 184 people who attended a seminar at the hotel was carried out. Serological and questionnaire data were obtained for 152 (83%) of these. Twenty-eight (18%) respondents reported symptoms compatible with legionellosis. Thirty-three subjects (22%) had indirect fluorescent antibody (IFA) titres to Legionella pneumophila serogroup 1 (Lp-1) of 128 or higher. The only site which those with symptoms of legionellosis and IFA titre > or = 128 were more likely to have visited than controls was the hotel car park (adjusted odds ratio [OR] 14.7, 95% confidence interval [CI]: 1.8-123.1). Those with symptoms compatible with legionellosis, but whose IFA titres were < 128 were also more likely to have visited the hotel car park (adjusted OR 4.4, 95% CI: 1.5 12.9). Seroprevalence of Lp-1 antibodies was higher in those who attended the seminar than in a population sample of similar age. Findings suggested that the 4 cases represented a small fraction of all those infected, and highlighted difficulties in defining illness caused by Lp-1 and in interpreting serology. PMID- 8620911 TI - Acute poststreptococcal glomerulo-nephritis in general practice: the contribution of infection to its onset and course. AB - Twenty-one patients considered to have acute poststreptococcal glumerulo nephritis were encountered during 35 years of general practice. In ten of them good evidence of active streptococcal infection at the time of discovery of nephritis was recorded. The more complete the data the more convincing was the evidence of active infection. In over half of those whose urine were routinely cultured pathogens were isolated and over a third were treated for infection of the urinary tract. Such infections were associated with adverse effects and prolonged illness. As compared with children, adults in general had a longer history of ill-health, were less likely to present with acute infections and more likely to have urinary tract infections and prolonged illness. Vigorous antistreptococcal treatment was followed by rapid recovery in those patients so treated whose illnesses were not complicated by urinary tract infections. Concurrent streptococcal infection and secondary infection of the urinary tract may contribute more to the onset of acute poststreptococcal glomerulo-nephritis and to its course than is currently believed. PMID- 8620912 TI - An epidemiological investigation of Norwalk virus infection in South Africa. AB - A study was carried out to determine the incidence and seroprevalence of Norwalk virus (NV) in the Pretoria area, South Africa, using a recombinant NV (rNV) immunoassay for the detection of serum IgG antibodies. Maternal antibody was detectable in infants' sera up to approximately 6 months of age. Infection with NV was detected serologically in the second year of life and the seroprevalence of NV IgG rose from 37.1% at 7-11 months of age to 62.1% by the age of 40 years. No significant differences in seroprevalence of NV IgG antibody was evident between subjects of European or African ethnic origin, where overall seroprevalence rates were 56.4% and 53.9% respectively. PMID- 8620913 TI - Pigs experimentally infected with Serpulina hyodysenteriae can be protected from developing swine dysentery by feeding them a highly digestible diet. AB - Weaner pigs (n = 72) were fed 1 of 4 diets. These were based on either cooked rice and animal protein, cooked rice and lupin, wheat and lupin, or wheat and animal protein. Twenty-six of the pigs were slaughtered after 1 month. Those fed the highly digestible cooked rice and animal protein diet had drier colonic contents and faeces, lighter large intestines, and the contents of their large intestines had increased pH values and decreased total VFA concentrations. The other 46 were orally challenged with broth cultures of Serpulina hyodysenteriae, and were monitored for faecal excretion of the spirochaetes, and for the development of swine dysentery (SD). None of 18 pigs fed the cooked rice and animal protein diet developed colonic changes or disease, whereas most pigs on the other diets developed mucohaemorrhagic colitis and dysentery. The reduced fermentation that occurred in the large intestines of pigs fed cooked rice and animal protein was associated with a subsequent failure of colonization by S. hyodysenteriae, and resultant protection against SD. PMID- 8620914 TI - Parasite prevalence in free-ranging farm cats, Felis silvestris catus. AB - No animals tested were positive for feline leukemia virus antigen and Chlamydia psittaci antibodies, but all were positive for antibodies to feline calicivirus (FCV), feline herpesvirus 1 (FHV1) and rotavirus. They had antibodies to feline parvovirus (96%), feline coronavirus (84% and cowpox virus (2%). Antibody to feline immunodeficiency virus (FIV) was found in 53% of animals, which were less likely to be infected with Haemobartonella felis, and had higher FHV antibody titres than cats without FIV. FCV was isolated from 51% cats and FHV1 and feline reovirus each from 4%. H. felis was present in 42% of animals, and antibody to Toxoplasma gondii in 62%. Clinical abnormality had a significant association with FIV and feline calicivirus infections, but sex, age, social status and feeding group had no significant association with prevalence of any parasites. Toxocara cati and Toxascaris leonina eggs were found, respectively, in 91% and 82% of animals tested. PMID- 8620915 TI - The concurrent prevalence of chloramphenicol-sensitive and multi-drug resistant Salmonella typhi in Vellore, S. India. AB - A multidrug resistant (MDR) variety of Salmonella typhi emerged as the cause of epidemic typhoid fever in some Asian countries including India, during the late 1980s. We faced the epidemic from April 1990 to the first quarter of 1993. However, during this period we continued to isolate chloramphenicol sensitive (CS) S. typhi also. The relative prevalences showed that the frequency of CS variety was unaffected by the epidemic of MDR variety. This is an unusual epidemiological pattern, which indicates that there may have been factors which favoured the epidemic of the MDR variety but not the CS one. PMID- 8620916 TI - Microencapsulated ciliary neurotrophic factor: physical properties and biological activities. AB - Controlled drug release in the CNS and PNS is still an obstacle to the treatment of neurodegenerative disorders. We have prepared a variety of microspheres containing either ciliary neurotrophic factor (CNTF) or genetically engineered cells able to synthesize and release this cytokine. CNTF is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. However, when given in therapeutically effective doses by systemic injections, it produces numerous adverse side effects. In order to minimize these effects we have microencapsulated it in biopolymers (chitosans, alginates, and copolymers in various proportions to achieve different kinetic properties). Size distribution profiles were determined by an image analysis system and surface characteristics were assessed by electron microscopy. The total content of CNTF as well as the amounts released per day were determined by ELISA and in vitro bioassays. The results from the release kinetics demonstrate that long-term secretion (up to 24 days) of CNTF is achieved by combining chitosan with copolymerized lactic and glycolic acid, whereas microspheres made of alginate provided only relatively short-term release (2-12 days). Neuron survival and neurite outgrowth in cultures of ciliary ganglia were supported by microencapsulated CNTF, indicating biological stability of CNTF. Genetically engineered human kidney cells 293 continued synthesizing CNTF within spheres and the released amounts of CNTF in the culture medium were comparable to the amounts secreted from monolayers (1 ng/ml of supernatant from confluent cultures) or even higher. These studies provide a basis for future testing of CNTF in encapsulated preparations using animal models of neurodegenerative disorders. PMID- 8620917 TI - Enhancement of mouse sciatic nerve regeneration by the long chain fatty alcohol, N-Hexacosanol. AB - The purpose of the present study was to determine the effects of n-hexacosanol (hexa) on nerve regeneration. Hexa, a long chain fatty alcohol has been shown to possess neurotrophic properties on cultured neurons and to attenuate the degeneration of cholinergic neurons after injury. The effects of daily intraperitoneal injections of hexa (1 mg/kg) on regeneration of nerve fibers were studied in mice following a sciatic nerve crush. Measurement of axonal regeneration using the pinch test 7 days postlesion showed a 40% increase of the regeneration rate of sensory fibers in hexa-treated mice compared to controls (1.67 +/- 0.15 mm/day and 1.09 +/- 0.03 mm/day, respectively). The recovery of neuromuscular function was significantly improved, as shown by quantitative electromyography and and sensorimotor tests. Clinical signs of recovery evaluation with toe spreading reflex appeared earlier in hexa group than in control animals. Electrophysiological recordings were performed each 3 days during 34 days following nerve injury. Higher values of the compound muscle action potential (CMAP) were obtained in hexa-treated animals that correspond to an improved regeneration. Moreover, hexa induced a significantly faster regeneration rate (hexa: 2.87 +/- 0.15 mV/day; control: 2.00 +/- 0.06 mV/day), as measured by the slope of CMAP increase (44% enhancement). A morphometric analysis performed 7 days following crush showed an increased number of regenerating fibers, as well as increased diameter and thickness of the myelin in hexa-treated mice. Thus, hexa increased the regeneration of both sensory and motor axons in lesioned nerve, leading to an improved functional recovery. PMID- 8620918 TI - Perforant path transection induces complement C9 deposition in hippocampus. AB - The presence of complement system proteins in amyloid plaques and the up regulation of several complement mRNAs in neurons and glial cells in affected brain regions during Alzheimer disease (AD) provided a basis for further examination of complement protein expression in a rodent lesion model of AD. Perforant path transection in rats was used as a model for the degeneration of entorhinal cortex (EC) layer II neurons and the consequent deafferentation of the hippocampus that occurs during AD. Immunostaining for C9, a key terminal component of the complement cascade membrane attack complex (MAC), showed extracellular C9 deposition in parenchyma around the EC wound and in hippocampus as early as 1 day, and disappeared by 14 days postlesion. Apoptosis of EC layer II neurons was seen and was presumably due to severing of their axonal projections to the hippocampus by the transection lesion. However, apoptotic EC layer II neurons were not immunostained by anti-rat C9 antibody, suggesting complement was not involved in inducing apoptosis. In the deafferented hippocampus, extracellular C9 immunostaining was localized to the dentate gyrus middle molecular layer, a region of synaptic loss, dendritic degeneration, and early synaptogenesis. In addition, intracellular C9 immunostaining was seen only in select hippocampal interneurons. Dentate gyrus granule neurons and pyramidal neurons were not C9 immunostained. Clusterin (SGP-2), a soluble inhibitor of the MAC that is up-regulated in AD, was also detected in the wound area (extracellular), the dentate gyrus middle molecular layer (extracellular), and intracellularly in scattered hippocampal interneurons. The data support the hypothesis that the complement system generally participates in responses to brain injury, as well as in AD. PMID- 8620919 TI - Peripheral administration of Interleukin-1 Receptor antagonist inhibits brain damage after focal cerebral ischemia in the rat. AB - We assessed the efficacy of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) on brain injury and edema formation after permanent middle cerebral artery occlusion (MCAo) in the rat. Previous studies showed that low amounts of rhIL-1ra injected directly into the brain significantly decreased infarct size after MCAo or excitotoxic injury in rats. Peripheral administration of rhIL-1ra (100 mg/kg sc at 0, 4, 8, 12, and 18 h after MCAo) significantly inhibited infarct size, by 46% (P < 0.05), measured at 24h. This was greater than the effect of MK801 administered immediately after MCAo (4 mg/kg ip, 0 h) which did not significantly reduce infarct size. rhIL-1ra (100 mg/kg also significantly inhibited cerebral edema formation by 49% (p< 0.05 measured 24 h after MCAo, but did not reduce edema formation measured 2 h after MCAo, but did not reduce edema formation measured 2 h after MCAo. Inhibition of infarction by rhIL-1ra was dependent on dose and time of administration. Together the results demonstrate that peripherally administered rhIL-1ra at high doses is able to mimic the efficacy of low dose of rhIL-1ra administered directly into the brain in a rodent model of stroke and that protection observed with rhIL-1ra was better than that offered by MK801 in this model. PMID- 8620920 TI - Altered expression of microtubule-associated proteins in cat trochlear motoneurons after peripheral and central lesions of the trochlear nerve. AB - Neurons lesioned in the peripheral nervous system (PNS) generally regenerate and survive, while neurons lesioned in the central nervous system (CNS) do not regenerate and often die. Investigators have traditionally compared the neuronal responses to PNS and CNS lesions in two separate populations of neurons. In this study, we compared the effects of PNS and CNS lesions on the expression of cytoskeletal proteins in a single neuronal population, the trochlear motoneurons of the cat. The trochlear nerve was lesioned either unilaterally in the PNS or bilaterally in the CNS (within the anterior medullary velum), and animals were allowed to survive 1, 2, or 4 weeks. Brain sections were reacted immunocytochemically using antibodies against microtubule -associated protein-2 (MAP-2) and a phosphorylated isoform of MAP1B, termed MAP1B-P. MAP-2 immunoreactivity (IR) was significantly decreased in the CNS-lesioned trochlear nucleus, compared to the lesioned and the unlesioned trochlear nucleus of PNS lesioned animals. MAP1B-P IR was significantly increased in PNS- and CNS- lesioned trochlear axons, compared to axons in the unlesioned trochlear nerve of PNS-lesioned animals, and appeared in a small percentage of PNS- and CNS-lesioned cell bodies. These results support the growing body of evidence that MPA-2 can serve as a marker for cells that will eventually die following neuronal insult. The increased immunostaining of MAP1B-P in lesioned axons and its appearance in lesioned cell bodies are characteristic of the immature CNS and may reflect an initial recapitulation of early development, when the levels of this protein are high. PMID- 8620921 TI - Fetal neocortical tissue blocks implanted in brain infarcts of adult rats interconnect with the host brain. AB - The purpose of the present study was to study if the connectivity of fetal neocortical tissue blocks placed in ischemic brain infarcts of adult rats would be enhanced in rats housed in an enriched environment. We also investigated whether the enriched housing conditions could enhance the postischemic and postgrafting functional outcome, in terms of motor behavior. This part of the study has been published recently. The middle cerebral artery was ligated on the right side in 37 inbred, adult male spontaneously hypertensive rats. The rats were placed at random either in an enriched environment (groups A and B) or in standard laboratory cages (group C). Three weeks after the artery occlusion, blocks of fetal sensorimotor cortex (embryonic day 17) were transplanted into the infarct cavity of rats from groups B and C. After 9 weeks all transplanted rats received an injection, into the graft, of a mixture containing the two tracers Fluoro-Gold and biotinylated Dextran amine. The transplants revealed a structured morphology with whorls and bands of cells reminiscent of normal neocortex. Tracing of efferent transplant to host fibers with biotinylated Dextran amine showed pronounced intrinsic transplant projections, as well as fibers, although significantly fewer, to the host ipsilateral sensorimotor cortex, striatum, and thalamus. Host to transplant projections were revealed by Fluoro-Gold-labeled cells found in the ipsilateral host sensorimotor cortex, the basal nucleus of Meynert, the thalamic ventrobasal, ventrolateral and posterior nuclei, and in the dorsal raphe nuclei. We conclude that fetal frontal neocortical block grafts placed in brain infarcts of adult rats develop a morphology reminiscent of normal neocortex and that both afferent and efferent neural connections, although sparse, are established with the host brain, whether the rats are reared under enriched housing conditions or not. PMID- 8620922 TI - Manganese-induced hydroxyl radical formation in rat striatum is not attenuated by dopamine depletion or iron chelation in vivo. AB - The present studies were aimed at investigating the possible roles of dopamine (DA) and iron in production of hydroxyl radicals (OH) in rat striatum after Mn2+ intoxication. For this purpose, DA depletions were assessed concomitant with in vivo 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formation from the reaction of salicylate with OH, of which 2,3-DHBA is a nonenzymatic adduct. Following intrastriatal Mn2+ injection, marked 2,3-DHBA increases were observed in a time- and dose-dependent fashion reaching maximum levels at 6-18 h and a plateau beyond 0.4 micromol (fourfold increase). The delayed increase of 2,3-DHBA levels suggestS that Mn2+ induces OH formation in the living brain by an indirect process. The early DA depletion (2 h) and relatively late OH formation (6 h) indicate independent processes by Mn2+. In addition, depletion of DA (about 90%) by reserpine pretreatment not significantly alter Mn2+-induced 2,3-DHBA formation or the extent of DA depletion, suggesting that DA or DA autoxidation are not participating in Mn2+-induced OH formation in vivo. Furthermore, Mn2+ injection did not significantly alter the low molecular weight weight iron pool in striatum, and co-injections of the iron-chelator deferoxamine with Mn(2+) into striatum did not significantly attenuate Mn(2+)-induced 2,3-DHBA formation. These findings suggest no role of chelatable iron in generation of Mn(2+)-induced OH, but do not exclude a role for mitochondrial heme-iron or peroxynitrite (Fe indepeNdent) in Mn2+-induced OH formation. PMID- 8620923 TI - Progesterone rapidly decreases brain edema: treatment delayed up to 24 hours is still effective. AB - Cerebral edema is a serious side effect of traumatic brain injury. We have previously established that progesterone injections, initiated within 1 h after cortical contusion injury, reduced edema when assessed 3 days later. To determine how rapidly progesterone can reduce edema, male and female rats were given the hormone 1 h after damage to the medial frontal cortex, and edema levels were assessed between 2 h and 7 days postinjury. Progesterone decreased edema with 6 h of the injury and continued to be effective for the duration of treatment. In addition, we assessed whether progesterone injections are effective when delays are imposed between injury and initiation of treatment. Male and female rats received progesterone after postinjury delays 6, 24, or 48 h. Progesterone was effective in reducing edema when treatment was delayed until 24 h after injury. PMID- 8620924 TI - Differential binding of apolipoprotein E isoforms to tau and other cytoskeletal proteins. AB - The apolipoprotein E4 (apoE4) gene dose is a major risk factor for late-onset and sporadic Alzheimer's disease with 50% of homozygous patients developing the disease by age 70. Previous studies have shown localization of apoE to the cytoplasm of certain neurons within the brain. In addition, apoE3, but not apoE4, forms SDS-stable complexes with the microtubule-associated proteins tau and MAP 2. To extend these studies and quantitate the association of apoE with other proteins, the association of apoE3 and apoE4 with several cytoskeletal proteins was examined using both gel shift and overlay assays. In the gel shift assay, apoE3 formed SDS-stable complexes with the longest isoform of human recombinant tau (T4L), the shortest isoform of human recombinant tau (T3), and the 160-kDa neurofilament protein (NFM). ApoE4 did not bind T3, T4L, or NFM in this assay. The association of apoE3 and apoE4 with T4L, actin, or tubulin was further examined in an overlay assay with known amounts of the cytoskeletal proteins slot blotted onto nitrocellulose and incubated in 0.15 microM (5 microg/ml) apoE3 or apoE4. In this assay, apoE3 and apoE4 bound T4L and tubulin equally well. In contrast, apoE3 bound actin with a significantly greater affinity than did apoE4. These results indicate that apoE isoforms interact with cytoskeletal proteins with at least two different binding affinities. The more avid interaction results in the formation of complexes which are SDS stable and occurs almost exclusively with apoE3, while the other interactions between apoE and cytoskeletal proteins are specific for apoE3. PMID- 8620925 TI - Methylprednisolone administration improves axonal regeneration into Schwann cell grafts in transected adult rat thoracic spinal cord. AB - Schwann cell (SC) grafts support the regeneration of axons of numerous spinal cord neurons when placed into transected adult rat midthoracic spinal cord. Clinically, methylprednisolone (MP) has been shown to be neuroprotective if administered within 8 h after spinal cord injury. We investigated whether axonal regrowth into SC grafts is enhanced when MP is administered at the time of spinal cord transection and SC implantation. SCs from adult rat sciatic nerves were purified in culture, suspended in Matrigel, and drawn into semipermeable polymeric channels. MP (30 mg/kg) or vehicle (control) was administered intravenously at 5 min, 2 h, and 4 h to adult Fischer rats after transection at T8 and removal of the next three caudal segments. The rostral cord stump was inserted 1 mm into the channel; the distal end of the channel was capped. Thirty to forty-five days later, the SC/MP group showed large tissue cables in the channels and host cord tissue retained in the rostral end of the channels. Significantly more myelinated axons (1159 +/- 308) were present at the 5-mm level in SC/MP grafts (n = 6) than in SC/vehicle cables (355 +/- 108, n = 5). More unmyelinated than myelinated axons (approximately 4:1, n = 3) were resolved in the cables by electron microscopy. In the SC/MP group, unlike the SC/vehicle group, serotonergic and noradrenergic fibers were detected immunocytochemically 2.5 and 2.0 mm respectively, into the graft; astrocytes were also identified at similar distances from the interface. Fast Blue retrograde tracing (SC/MP, n = 4; SC/vehicle, n = 3) showed that more spinal cord neurons (1116 +/- 113 vs 284 +/- 88, respectively) and spinal cord neurons more distant from the graft (C8 vs C5) responded by extending axons into the graft in the presence of MP. Also, very significantly, supraspinal brain stem neurons extended axons into the graft only when MP was administered (mean 46 vs 0, n = 3). These results indicate that MP improves axonal regenerationn from both spinal cord and brain stem neurons into thoracic SC grafts, possibly by reducing secondary host tissue loss adjacent to the graft. PMID- 8620926 TI - Quantitative effects of peripheral monocytes and nerve growth factor on CNS neural morphometric outgrowth parameters in vitro. AB - Would healing of the central nervous system (CNS) is a complex process involving interactions between cells from both the vascular and the neural environments, extracellular matrix proteins, and a cocktail of agonistic and antagonistic bioactive molecules. Vascular cells, particularly peripheral monocytes and macrophages, are believed to play an important role in organizing and mediating CNS tissue reactions subsequent to penetrating injuries that compromise the blood brain barrier. Although many investigators have studied the effect of macrophages and microglia (resident brain macrophages) on neural outgrowth, little is known regarding monocyte effects. We have combined tissue culture, video microscopy, and digital image processing and analysis to quantify morphometric parameters of neurons exposed to monocyte secretory products in vitro. The experimental system developed is simple in design but provides a quantitative understanding of cellular function and molecular mechanisms and has the ability to both study processes of graded complexity and relate cellular function to overall systems behavior. We evaluate the efficacy of the experimental model developed by measuring morphometric parameters of human neural cells (hNT cell line) in the presence of nerve growth factor (NGF). Results suggest that monocyte-conditioned media (MCM) increases neuron outgrowth parameters, such as neuritic output, mean arbor output, neurite branching, and effective cell diameter. Moreover, we show that the bioactive factor present in MCM is not IL-1 and the activity of the factor with respect to neural outgrowth is between that of 10 and 100 ng/ml NGF. PMID- 8620927 TI - Intraseptal microinfusion of muscimol: effects on hippocampal formation theta field activity and phasic theta-ON cell discharges. AB - The effect of intraseptal microinfusions of the GABA-A agonist muscimol on spontaneously occurring or hypothalamically induced hippocampal formation (HPC) theta field activity and the simultaneously occurring discharge properties of CA1 pyramidal and dentate granule layer phasic theta-ON cells, was investigated in urethane-anesthetized rats. The microinfusion of 5.0-12.5 nmol of muscimol into the medical septum/vertical limb of the diagonal band of Broca (MS/vDBB) resulted in a progressive reduction (beginning 5 min postinfusion) in the power (amplitude) and finally the total loss of theta field activity. In contrast, theta field frequency remained unaffected during the entire postinfusion period that theta field activity was present. In the time immediately following the first 1-min intraseptal microinfusion of 5 nmol muscimol, (before changes in theta amplitude occurred) a brief period of increased phasic theta-ON cell excitability was noted. This was manifested as an increase in the number of discharges per rhythmic burst. Associated with the progressive reduction of the amplitude of theta field activity, phasic theta-ON cell discharge rates progressively decreased for a period beginning 5 min postinfusion of 5 nmol muscimol. Despite the progressive decrease in the number of discharges and a noticeable reduction in the degree of rhythmicity, phasic theta-ON cells maintained their preferred timing of discharges in relation to the phase of theta field activity, while the latter was present. Just prior to the complete abolishment of theta field activity, phasic theta-ON cells ceased discharging. During the period when theta field activity was replaced on low amplitude asynchronous activity, phasic theta-ON cells discharged in bursts correlated with every occurrence of sharp wave field activity. The results support the following conclusions: (1) the brief excitatory effect on HPC theta-ON cell discharges may be correlated pharmacologically with an initial brief increase in HPC ACh turnover. The reduction of phasic theta-ON cell discharges and theta field activity may be correlated with the longer lasting reduction of HPC ACh turnover, controlled by MS/vDBB GABA-A inputs to MS/vDBB cholinergic septohippocampal neurons, possibly along with a direct inhibition of the GABAergic septohippocampal projection; (2) the primary contribution of the MS/vDBB nuclei, as a nodal point in the ascending brainstem HPC synchronizing system, is the modulation of the amplitude of HPC formation theta field activity and secondarily to relay frequency-coded inputs from the posterior hypothalamic region (posterior and supramammillary nuclei); (3) HPC theta and sharp wave field activity represent functionally distinct neural inputs to the same population of phasic theta-ON cells located in both the CA1 pyramidal and dentate granule cell layers. PMID- 8620928 TI - Energy and glutamate dependency of 3-Nitropropionic acid neurotoxicity in culture. AB - 3-Nitropropionic acid (3-NP) irreversibly inhibits the activity of the mitochondrial enzyme succinate dehydrogenase, leading to selective striatal lesions when administered in vivo. We studied the effects of 3-NP on dissociated cultures of neurons and glia with the following findings: (a) 3-NP killed cultured striatal neurons with a median lethal dose of 2.5 mM after 20 h of incubation in 20.0 mM glucose medium. Despite its selective toxicity in vivo, cultured striatal, hippocampal, septal, and hypothalamic neurons were similarly sensitive to 3-NP incubation. (b) 3-NP's effects were remarkably energy substrate dependent, with the median lethal dose dropping over an order of magnitude when glucose concentrations were lowered to 3.0 mM, a condition that was itself nontoxic. Cultures exposed to 3-NP had a far greater sensitivity to energy availability than those exposed to glutamate. (c) Recent work suggests that 3-NP toxicity may be partially mediated by excitotoxins. Our experiments show that neither kynurenic acid, a nonspecific glutamate receptor antagonist, nor the NMDA receptor antagonist, DL-2-amino-7-phosphonoheptanoic acid, either in combination or alone, reduced 3-NP toxicity in striatal cultures. However, the noncompetitive NMDA antagonist MK-801 did attenuate 3-NP toxicity. PMID- 8620930 TI - Transplantation of mesencephalic cell suspension in dopamine-denervated striatum of the rat.I. Effects on spontaneous activity of striatal neurons. AB - These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway. Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine. Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side. Sham grafted rats received the suspension medium only. The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine. Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months. The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates. The signs of increased neuronal activity were also present in sham-grafted animals. The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate. The effects of the intrastriatal graft appeared to be present within a radius of 1.5-2 mm from the core of the grafted area. The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording. The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains. PMID- 8620929 TI - Identification in immunolocalization of a new class of proteoglycan (keratan sulfate) to the neuritic plaques of Alzheimer's disease. AB - Previous studies have demonstrated three distinct classes of proteoglycans (PGs)/glycosaminoglycans (GAGs) localized to the characteristic lesions (i.e., neuritic plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles) of Alzheimer's disease (AD). These include heparan sulfate (i.e., perlecan), dermatan sulfate (i.e., decorin), and chondroitin sulfate PGs/GAGs. In the present study, two different antibodies demonstrated the presence of a new class of PG (i.e., keratan sulfate) in the neuritic plaques of AD. Asynaptic vesicle keratan sulfate PG (known as SV2PG) was detected by the monoclonal antibodies, anti-SV2 and anti-SV4, which recognize the keratan sulfate core protein and GAG chains, of the SV2PG antigen, respectively. Both antibodies immunolocalized SV2PG primarily to synapses and to dystrophic neurites within neuritic plaques of AD and normal aged brain. The SV2PG was not immunolocalized to diffuse plaques, cerebrovascular amyloid deposits, or neurofibrillary tangles in AD or normal aged brain. SV2PG immunoreactivity in AD brain was similar in distribution to synaptophysin and showed apparent reduced immunoreactiviy+in AD cortex in comparison to age-matched controls. In conjunction with previous studies, these results now suggest that within the neuritic plaques of AD, there are at least four different classes of PGs present. Although heparan sulfate PGs are still the only class of PG immunolocalized to amyloid fibrils within the neuritic plaques of AD, the specific immunolocalization of keratan sulfate, dermatan sulfate, and chondroitin sulfate containing PGs to the periphery of plaques, suggests that these particular PGs/GAGs may also play distinct and important roles in neuritic plaque pathogenesis. PMID- 8620931 TI - Activity-dependent changes in "transplanted" cerebellar cultures. AB - Organotypic cerebellar cultures were used to assess the effects of increasing or blocking neuronal activity on circuit reconstruction in an in vitro transplantation model. Granule cells and oligodendrocytes were destroyed and astrocytes were functionally compromised by exposing newborn mouse-derived cerebellar explants to cytosine arabinoside for the first 5 days in vitro. Such cultures were "transplanted" at 9 days in vitro with granule cells and glia and maintained in standard nutrient medium; in medium with the GABA antagonist, picrotoxin, to increase neuronal activity; or with tetrodotoxin and elevated levels of magnesium to block neuronal activity. Transplanted cultures exposed to picrotoxin were not significantly different from control transplanted cultures. Transplanted cultures deprived of neuronal activity had reduced inhibitory synaptogenesis, greater persistence of heterotypical axospinous synapses, and hyperactive cortical spontaneous discharges after recovery from the blockade. Transplantation-induced changes that were not affected included myelination, reduction of sprouted Purkinje recurrent axon collaterals, astrocytic ensheathment of Purkinje cells, reduction of excess Purkinje cell axosomatic synapses, and formation of excitatory parallel fiber-Purkinje cell dendritic spine synapses. The results were consistent with previous studies indicating the necessity of neuronal activity for the full development of inhibitory circuitry, and suggested that neuronal activity is also necessary for the reconstruction of inhibitory circuitry after transplantation. PMID- 8620932 TI - A device for the implantation of multiple cellular deposits into a large volume of brain from a single cannula site. AB - Current grafting techniques for the treatment of Parkinson's disease incompletely restore the dopaminergic innervation of the caudate/putamen and while, in successful cases, bradykinesia and rigidity are reduced, tremor is largely unaffected. Increasing the number of cellular deposits would allow grafted neurons to be dispersed more widely within the host brain. This might be expected to lead to a more complete reinnervation of the caudate/putamen and therefore a better clinical result. In order to increase the volume of brain accessible to reinnervation without increasing the number of needle passages through the cortex, we have designed a device which allows a Teflon tube to be extruded sideways from the end of a stainless steel cannula. Through this tube, cells can be implanted at some distance in any radial direction from the axis of the cannula. Using such a device we have made up to 12 deposits of lac-z-labeled cells via a single cannula entry into the rat brain, at distances of up to 5 mm from the axis of the cannula. We propose that a similar device could be used to graft embryonic neurons into the human brain. PMID- 8620933 TI - Methodological developments in nucleic acid diagnosis II. Workshop report and abstracts. Berlin, December 3, 1994. PMID- 8620935 TI - The amphiphilic properties of novenamines determine their activity as inhibitors of HIV-1 RNase H. AB - Few inhibitors of the RNase H function associated with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U 34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles, and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules form stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted, the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system. PMID- 8620934 TI - Regulation of the cell cycle following DNA damage in normal and Ataxia telangiectasia cells. AB - A proportion of the population is exposed to acute doses of ionizing radiation through medical treatment or occupational accidents, with little knowledge of the immediate effects. At the cellular level, ionizing radiation leads to the activation of a genetic program which enables the cell to increase its chances of survival and to minimize detrimental manifestations of radiation damage. Cytotoxic stress due to ionizing radiation causes genetic instability, alterations in the cell cycle, apoptosis, or necrosis. Alterations in the G1, S and G2 phases of the cell cycle coincide with improved survival and genome stability. The main cellular factors which are activated by DNA damage and interfere with the cell cycle controls are: p53, delaying the transition through the G1-S boundary; p21WAF1/CIP1, preventing the entrance into S-phase; proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), blocking DNA replication; and the p53 variant protein p53 as together with the retinoblastoma protein (Rb), with less defined functions during the G2 phase of the cell cycle. By comparing a variety of radioresistant cell lines derived from radiosensitive ataxia telangiectasia cells with the parental cells, some essential mechanisms that allow cells to gain radioresistance have been identified. The results so far emphasise the importance of an adequate delay in the transition from G2 to M and the inhibition of DNA replication in the regulation of the cell cycle after exposure to ionizing radiation. PMID- 8620937 TI - Phosphotyrosine protein phosphatases and diabetic pregnancy: an association between low molecular weight acid phosphatase and degree of glycemic control. AB - Low molecular weight acid phosphatase encoded by the highly polymorphic locus ACP1 is a member of the protein-tyrosin phosphatase family (PTPases) which plays an essential role in the control of receptor signalling through phosphotyrosine pathways. Recent experiments have shown that purified rat liver ACP, corresponding to human ACP1, is able to hydrolyze a phosphotyrosine-containing synthetic peptide corresponding to the 1146-1158 sequence of the human insulin receptor, and shows a high affinity for it. This prompted us to analyze the degree of glycemic control in relation to ACP1 genetic variability in a sample of 214 diabetic pregnant women including IDDM, NIDDM and gestational diabetes. The ACP1 genotype was also determined in 482 non-diabetic pregnant women. In diabetic women glycemic levels in the last trimester of pregnancy appear to be significantly associated with the ACP1 genotype, and correlate positively with ACP1 enzymatic activity. The data suggest that quantitative variations of ACP1 may influence the clinical manifestations of diabetic disorders, and call for further studies on the role of this enzyme in the modulation of insulin-receptor phosphotyrosine pathways. PMID- 8620936 TI - Effect of various stressors on the level of lipid peroxide, antioxidants and Na+, K(+)-ATPase activity in rat brain. AB - The level of malondialdehyde (MDA), an index of lipid peroxidation, and the antioxidants superoxide dismutase (SOD) and glutathione (GSH), as well as the activity of Na+, K(+)-ATPase, were assessed in whole rat brain after immobilization, anemic hypoxia (NaNO2) and 72 h starvation. The effect of these stressors on plasma glucose and corticosterone levels was also observed. Hypoxia and starvation stimulated the lipid peroxide formation in brain as indicated by an increase in the level of MDA, being higher after starvation than hypoxia. Brain SOD activity was also increased in response to hypoxia and starvation while GSH content was only diminished in hypoxia. However, neither MDA nor antioxidants were affected by immobilization. On the other hand, the activity of brain Na+, K(+)-ATPase was significantly increased by immobilization and hypoxia but decreased in starvation. A similar pattern of change was also observed in plasma glucose and corticosterone levels in response to these stressors. These results elucidate differences in the biochemical response of animals towards various types of stress, with increased lipid peroxide formation in hypoxia and starvation. PMID- 8620938 TI - N-monomethyl-arginine and nicotinamide prevent streptozotocin-induced double strand DNA break formation in pancreatic rat islets. AB - The impact of short term in vitro exposure to the diabetogenic drug streptozotocin on pancreatic islet glucose metabolism, insulin secretion, DNA fragmentation and cell viability, was studied. Streptozotocin impaired cell viability as well as insulin secretion and the oxidation of glucose. These effects were partially counteracted by inhibition of the inducible form of nitric oxide synthase with N-monomethyl-arginine and by scavenging oxygen free radicals with nicotinamide. Isolated islets underwent double strand DNA fragmentation after 24 h in culture. The degree of DNA breakdown was strongly enhanced by exposure of the islets to 0.55 mM streptozotocin for 30 min before culture. Prevention of streptozotocin-induced cleavage of islet DNA was obtained with N monomethyl-arginine and nicotinamide. These data suggest that the generation of reactive oxygen and nitrogen species is involved in the deleterious action of streptozotocin on pancreatic islet tissue. A role for oxygen radicals generated during streptozotocin-induced islet cell damage as possible mediators of the expression of the inducible form of nitric oxide synthase and the scavenging action of nicotinamide on these radicals, is then proposed. PMID- 8620939 TI - Central nicotinic receptor blockade inhibits emotionally conditioned pressor responses in rats. AB - A conditioned stimulus previously paired with electric footshock produced an increase in blood pressure in conscious, freely moving rats. The conditioned pressor response was reproducible. Intracerebroventricular injection of the nicotinic receptor antagonists hexamethonium (1-10 micrograms) or pentolinium (10 micrograms) but not the muscarinic receptor antagonist methylatropine (3 micrograms) produced an inhibition of the conditioned pressor response, whereas intraarterial injection of hexamethonium (10 micrograms) did not affect the response. Intraventricular injection of the cholinesterase inhibitor physostigmine (3-10 micrograms) produced an enhancement of the conditioned pressor response. These results are consistent with the possibility that central nicotinic receptors play a role in the expression of the emotionally conditioned pressor response in rats. PMID- 8620941 TI - Mismatch repair as an important source of new mutations in non-dividing cells. AB - This paper describes a mechanism which permits somatic cells to generate random mutations in the complete absence of cell proliferation. Knowledge of the existence of this mechanism should provide us with the basis for a better understanding of a number of important biological phenomena, and in particular may help to explain the origins of many human cancers. PMID- 8620940 TI - The action of the cholecystokinin-A receptor antagonist, devazepide, on the digestive system of the chicken. AB - The influence of the cholecystokinin (CCK)-A receptor antagonist, devazepide (DVZ), on the chicken digestive tract was investigated. The passage of food from the crops of birds treated with DVZ was not significantly different from that of the control. DVZ treatment did not inhibit the biliary flow stimulated by the CCK analogue, caerulein. Dispersed chicken pancreatic acini stimulated with CCK were treated with various concentrations of DVZ. At 10-5 M, DVZ completely inhibited amylase release; this concentration was much higher than those reported to have similar effects in mammals. The results suggest that the action DVZ as a CCK antagonist in the chicken is very weak. PMID- 8620943 TI - The prey consumption and prey preference of the larvae of the mosquito Culex (Lutzia) raptor on the larvae of Culex quinquefasciatus. AB - The maximum consumption of the larvae of the pest and vector mosquito Culex quinquefasciatus by the predatory mosquito Culex (Lutzia) raptor was studied at various instars of both the predator and the prey. The prey preferences of the predator when given lavae of different instars were also investigated. The IVth instar of the predator consumed the maximum number of Ist instar and the maximum biomass of IVth instar larvae of the prey. Instars I and II of the predator preferred the Ist of the prey; instars III and IV of the predator preferred instars II and III of the prey respectively. The predator consumed an average of 157.1 larvae during its whole larval period, when each instar of the predator was given its preferred instar of the prey. PMID- 8620942 TI - Complement evasion by the Lyme disease spirochete Borrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance. AB - The effectiveness of complement-mediated killing of Borrelia burgdorferi, the causative agent of Lyme disease, in the presence of host-derived tissues was studied. Second and high passage forms of B. burgdorferi 297 isolate were grown in a LEW/N rat joint tissue co-culture system and in artificial BSK medium. Guinea pig complement and third week immune serum from hamsters with experimental Lyme disease were added to the cultures. Both high and low passage borrelia grown in BSK medium died and did not revive after 3 weeks incubation in BSK medium. However, 5-12% of tissue co-cultured borrelia survived the first complement mediated lysis. Repeated re-growth and lysis cycles in tissue co-culture resulted in isolation of an 85% complement-resistant population of B. burgdorferi. Joint tissue culture supernatant collected on the third day of tissue culture, and fibronectin (25 micrograms/ml), also protected spirochetes from complement mediated lysis in contrast to BSK or fresh co-culture medium. Complement-mediated lysis may not be an effective mechanism in eradication of borrelia, and the chronicity of Lyme disease may be due to resistance of B. burgdorferi variants to host immune defense mechanisms in the presence of host-derived tissues. PMID- 8620945 TI - Cavitary bronchiolitis obliterans organizing pneumonia. PMID- 8620944 TI - High altitude pulmonary oedema: who gets it and why? PMID- 8620946 TI - Ventilatory and pulmonary vascular response to hypoxia and susceptibility to high altitude pulmonary oedema. AB - Reduced tolerance to high altitude may be associated with a low ventilatory and an increased pulmonary vascular response to hypoxia. We therefore, examined whether individuals susceptible to acute mountain sickness (AMS) or high altitude pulmonary oedema (HAPE) could be identified by noninvasive measurements of these parameters at low altitude. Ventilatory response to hypoxia (HVR) and hypercapnia (HCVR) at rest and during exercise, as well as hypoxic pulmonary vascular response (HPVR) at rest, were examined in 30 mountaineers whose susceptibility was known from previous identical exposures to high altitude. Isocapnic HVR expressed as difference in minute ventilation related to difference in arterial oxygen saturation (delta V'E/ delta Sa,O2) (L.min-1/%) was significantly lower in subjects susceptible to HAPE (mean +/- SEM 0.8 +/- 0.1; n = 10) compared to nonsusceptible controls (1.5 +/- 0.2; n = 10), but was not significantly different from subjects susceptible to AMS (1.2 +/- 0.2; n = 10). Hypercapnic ventilatory response was not significantly different between the three groups. Discrimination between groups could not be improved by measurements of HVR during exercise (50% maximum oxygen consumption (V'O2,max)), or by assessing ventilation and oxygen saturation during a 15 min steady-state exercise (35% V'O2,max) at fractional inspiratory oxygen (FI,O2) of 0.14. Pulmonary artery pressure (Ppa) estimated by Doppler measurements of tricuspid valve pressure at an FI,O2 of 0.21 and 0.12 (10 min) did not lead to a further discrimination between subjects susceptible to HAPE and AMS with the exception of three subjects susceptible to HAPE who showed an exaggerated HPVR. It is concluded that a low ventilatory response to hypoxia is associated with an increased risk for high altitude pulmonary oedema, whilst susceptibility to acute mountain sickness may be associated with a high or low ventilatory response to hypoxia. A reliable discrimination between subjects susceptible to high altitude pulmonary oedema and acute mountain sickness with a low ventilatory response to hypoxia is not possible by Doppler echocardiographic estimations of hypoxic pulmonary vascular response. PMID- 8620947 TI - Thrombolytic therapy of right heart emboli-in-transit. AB - Currently, no consensus exists for the appropriate treatment of echocardiographically diagnosed mobile right heart masses giving rise to a high suspicion of migrant thromboembolism in patients with pulmonary embolism. This may lead to unnecessary delay in the implementation of the most appropriate treatment for these patients. Several earlier studies have supported the beneficial role of thrombolytic therapy. We report on an additional two patients with mobile right heart thromboemboli, refractory to systemic anticoagulation, who recovered quickly after initiation of thrombolytic therapy. PMID- 8620948 TI - Angiotensin II potentiates methacholine-induced bronchoconstriction in human airway both in vitro and in vivo. AB - Angiotensin II levels are elevated in patients with acute severe asthma. In addition, intravenous angiotensin II causes bronchoconstriction in mild asthmatic patients. In the present study, we examined the effects of this hormone on bronchi in vitro and its interaction with the cholinergic agonist methacholine both in vivo and in vitro. Contractions of rings of human bronchi were measured isometrically. Concentration-response curves were obtained to angiotensin II and to methacholine in the presence and absence of angiotensin II. In addition, seven asthmatic patients with mild bronchial hyperreactivity to methacholine received placebo, angiotensin II, 1 or 2 ng.kg-1.min, by infusion, followed by methacholine challenge. Forced expiratory volume in one second (FEV1) values were measured at baseline, at the end of the infusion and during methacholine challenge. Angiotensin II alone in vitro evoked small contractions of human bronchi ( < 0.25 g wt). Pre-incubation with low concentrations of angiotensin II significantly enhanced contractions to methacholine. In mild asthmatic patients, angiotensin II alone evoked no change in baseline FEV1 values at the levels studied. Compared to placebo, angiotensin II 2 ng.kg-1.min, but not 1 ng.kg 1.min, evoked a significant increase in bronchial reactivity to methacholine. Angiotensin II in subthreshold concentrations enhances methacholine-evoked bronchoconstrictions both in human in vitro and in mild asthmatic patients in vivo. Our findings suggest a novel role for angiotensin II as a putative mediator in asthma. PMID- 8620949 TI - High altitude exposure reduces bronchial responsiveness to hypo-osmolar aerosol in lowland asthmatics. AB - It is well-known that many patients with asthma undergo clinical improvement during a stay at high altitude. At high altitude, the atmospheric and climatic conditions (such as hypoxia, cold and dry air inhalation) could modify the bronchial responsiveness in asthmatics. Our study was designed to assess the difference in bronchial responsiveness to hypotonic aerosol between sea level and high altitudes in nonresident asthmatic subjects. The results were obtained during two mountaineering expeditions above 4,000 m i.e. at 4,559 m on Mt Rosa, Italy; and at 5,050 m near the Mt Everest base camp in Nepal. Eleven mild asthmatics performed standard bronchial challenges with ultrasonically nebulized distilled water (5 min inhalation, delivery 2 mL-min-1) at sea level and after staying at least 72 h at the above mentioned altitudes. The decrease in forced expiratory volume in one second (FEV1) from baseline was used as index of bronchial response. There was no significant difference in pre-challenge FEV1 between sea level and high altitude in either study. However, the bronchoconstriction response to ultrasonically nebulized distilled water was significantly reduced at high altitude in both studies. At sea level the mean FEV1 decrease was 22.2% (range 15-35%), whereas as the maximal altitude it was 6.7% (range 2-11%). Our results indicate that there is a reduction in bronchial responsiveness to hypoosmolar aerosol at high altitude. This suggests that atmospheric and climatic conditions, or physiological adaptations, via mediators such as atrial natriuretic peptide, are beneficial to patients with asthma at high altitude. PMID- 8620950 TI - Reduced dose salbutamol in comparison with standard dosage for symptom relief in asthma. AB - Regular treatment with beta 2-agonists has been reported to be associated with an increase in risk of asthma death or near death, and with a deterioration in asthma symptom control. Low-dose beta 2-agonists provide effective bronchodilatation and bronchoprotection, even though maximal bronchodilatation is not achieved, and they may offer a better safety profile. In a double-blind, randomized,, cross-over study, we evaluated the efficacy of low-dose salbutamol metered-dose inhaler (50 micrograms.puff-1), used over a period of 2 weeks, compared with a standard dose (100 micrograms.puff-1) in control of asthma symptoms in 20 moderately severe asthmatic subjects using inhaled glucocorticosteroid therapy. Asthma control was assessed by symptom scores, peak flow rates, spirometry, inhaler usage and, where possible, by bronchial responsiveness to methacholine. Despite a 46% reduction in mean weekly salbutamol dosage, mean forced expiratory volume in one second (FEV1), morning and evening peak expiratory flow (PEF), PEF variability, dose of methacholine provoking a 20% decrease in FEV1 (PC20) (n=9), and symptom scores showed no difference between low-dose and standard inhaler treatment periods. Low-dose inhaler administration resulted in a small but significant increase in number of inhaler actuations. Low dose salbutamol metered-dose inhaler may, thus, be useful for control of symptoms in moderately severe asthma. This strategy could be used to achieve a reduction in total beta 2-agonist usage, which may minimize any potential for adverse effects. PMID- 8620951 TI - Overnight protection by inhaled salmeterol on exercise-induced asthma in children. AB - The main aim of the present study was to evaluate whether inhaled salmeterol given in the evening protected against exercise-induced asthma the next morning. Twenty three children (12 males and 11 females) with a mean age of 11 yrs and with exercise-induced asthma participated in a double-blind, randomized, placebo controlled study. The children inhaled salmeterol 25 micrograms, salmeterol 50 micrograms and placebo by Diskhaler at 10 p.m. on 3 separate days. Next morning, half of the children ran on a motor-driven treadmill for 6 min at submaximal load at 8 a.m. and the remainder at 10 a.m. Lung function was measured by maximal expiratory flow-volume loops before running, immediately after, and 3, 6, 10 and 15 min after running. The mean maximum reduction in forced expiratory volume in one second (FEV1) after treadmill run was 34% before inclusion in the study. Mean maximum fall in FEV1 was significantly greater after placebo: 30% (23-36) 95% confidence interval) than after salmeterol 25 micrograms: 19% (12-23) or salmeterol 50 micrograms: 18% (12-25). In addition to the reduced postexercise bronchoconstriction, pre-exercise lung function (FEV1) was significantly higher both after salmeterol 25 micrograms: 2.4 L.s-1 (2.1-2.7) and salmeterol 50 micrograms: 2.5 L.s-1 (2.2-2.8) than after placebo: 2.2 L.s-1 (1.9-2.5). No significant differences in pre- and postexercise lung function were found between children tested at 8 or 10 a.m., or in relation to salmeterol dosage. Thus, inhaled salmeterol 25 and 50 micrograms offered similar overnight protection against exercise-induced asthma and improved baseline lung function in the morning as compared to placebo. PMID- 8620952 TI - Asthma medications and disease exacerbations: an epidemiological study as a method for asthma surveillance. AB - Recent experimental and epidemiological studies have suggested that outcomes of asthma are significantly influenced by treatment patterns. This study was conducted in order to investigate the links between treatment patterns in asthmatics and occurrence of disease exacerbations. We performed a nested case control study in a cohort of 680 asthmatics identified between 1986 and 1991 in a drug dispensing database. After validation in a pilot study, the intermittent use of oral corticosteroids was used as a proxy for asthma exacerbations. Cases with an exacerbation (n=133) were pair-wise matched with controls. The type of medications used for the usual treatment of asthma were examined in relation to the risk of asthma exacerbation. The use of oral xanthines and inhaled fenoterol but not of inhaled salbutamol, corticosteroids, cromoglycate and ipratropium bromide was associated with an increased probability of asthma exacerbation. Within the cohort, the proportion of subjects dispensed inhaled corticosteroids rose from 12 to 27% between 1986 and 1991. The proportion of subjects using inhaled bronchodilators without inhaled corticosteroids also decreased over this period of time. The identification of markers of asthma exacerbations made it possible to link the probability of adverse outcome risk for such exacerbations with treatment patterns. This method could be useful in further development of asthma surveillance using drug dispensing databases. PMID- 8620953 TI - IgE-induced passive sensitization of human isolated bronchi and lung mast cells. AB - Passive sensitization of human isolated lung with serum from atopic asthmatic patients provides an opportunity to study the link between airway hyper responsiveness and the allergic process. To directly demonstrate the role of immunoglobulin E (IgE) in the effect of the atopic serum, we have compared the effect of passively sensitizing both human bronchi and isolated lung mast cells with either serum from atopic asthmatic patients or human monoclonal IgE. Peripheral bronchi ( < 5 mm in internal diameter) were dissected out from human lung obtained at thoractomy and isometric contraction was studied in response to a variety of immunological stimuli according to the sensitization protocol. Mast cells were also isolated from human lung and histamine release was measured under similar experimental conditions. A contractile response was elicited by either the specific antigen or anti-IgE (0.6-600 ng.mL-1) but not anti-immunoglobulin G (IgG) 0.2-20 micrograms.mL-1) in airways sensitized with atopic serum (total IgE concentration of approximately 1,000 international units (IU).mL-1). The maximal contractile response to anti-IgE was 75 +/- 22% of the response to 1 mM acetylcholine. Similarly, anti-IgE released histamine from isolated lung mast cells sensitized with atopic serum up to 22.4 +/- 2% of total histamine measured within mast cells. When isolated airways or mast cells were sensitized with human monoclonal IgE (1,000 IU.mL-1), response to anti-IgE in terms of contractile response or histamine release, respectively, were not significantly different from those obtained following passive sensitization with atopic serum. Finally, the bronchial contractile response to anti-IgE depended not only on the concentration of anti-IgE but also on that of IgE (300-2,000 IU.mL-1) used to sensitize the airways. These results indicate that the effect of antigen or anti IgE in peripheral bronchi passively sensitized with atopic serum is mimicked when sensitization is carried out directly with human monoclonal IgE. PMID- 8620954 TI - Intrasubject variability in airway inflammation sampled by bronchoalveolar lavage in stable asthmatics. AB - Despite the increasing complexity of bronchoalveolar lavage (BAL) studies in asthmatics there are few published data concerning the variability of inflammatory parameters measured using this technique. We studied the intrasubject variability of cellular and solute parameters in 20 clinically stable, symptomatic, mild-to-moderate asthmatics, in repeat 180 mL BAL procedures performed 1 month apart. During the study, there was no change in disease activity or medication. Mean (SD) forced expiratory volume in one second (FEV1) was 3.2 (1.09) L at the first BAL and 3.05 (0.98) L at the second. The geometric mean dose of methacholine provoking a 20% decrease in FEV1 (PD20) was 23 micrograms (range 2-1,170 micrograms) at the first BAL and 28 micrograms (range 2 400 micrograms) at the second. There was considerable variability in the BAL cellular and solute parameters measured over the two procedures. Estimates of power calculated for subsequent studies involving this type of subject group were made from the observed variability. Sample sizes of less than 15 mean that differences have to be large in order to be detected in repeat BAL samples. However, there is little improvement in the power of BAL studies for sample sizes greater than 20, indicating that there is little gain in recruiting more than this number of subjects. Thus, our study indicates that although studies of the pathophysiology underlying asthma using BAL require considerable commitment, they are practicable. PMID- 8620955 TI - Effects of IL-2 treatment on different compartments of the irradiated rat lung analysed by bronchoalveolar lavage and lung tissue morphology. AB - In recent years, interleukin-2 (IL-2) has been used as an immunomodulatory agent in the treatment of various malignant tumours. However, this treatment has been limited by serious side-effects, including toxic reactions in the lung. The effects of IL-2 treatment on inflammatory cell populations in the normal and irradiated rat lung were investigated in this study. IL-2 was continuously administered as a subcutaneous infusion over a 6 week study period. Irradiation was given in a single dose (25 Gy) the day after starting IL-2 treatment. Evaluation with bronchoalveolar lavage fluid (BALF) analysis and lung tissue morphology was made 6 weeks after irradiation. In nonirradiated rats, IL-2 treatment induced significant increases in the total number of inflammatory cells in the perivascular, interstitial and peribronchial tissues as well as in the alveolar space. These increases were not reflected in BALF; on the contrary, a significant decrease of the total numbers of inflammatory cells was found in BALF. Irradiation alone caused a more pronounced inflammatory response was significant increases of all inflammatory cells in all lung compartments, which was also reflected in BALF. Concomitant treatment with IL-2 and irradiation induced an enhanced accumulation of inflammatory cells in the perivascular and peribronchial tissues compared with irradiation alone. Thus, both irradiation and IL-2 treatment induce inflammatory reactions in the lung, but there were signs of synergistic effects seen in this study. Furthermore, the results also emphasize the difficulties in making sophisticated conclusions from BALF analyses alone. PMID- 8620956 TI - Diagnosis of pulmonary lymphangioleiomyomatosis by HMB45 in surgically treated spontaneous pneumothorax. AB - Pulmonary lymphangioleiomyomatosis (PLAM) is a rare disease with poor prognosis, characterized by an abnormal proliferation of smooth muscle. The patients are females and recurrent pneumothorax is a frequent complication. HMB45 is a monoclonal antibody with specific immunoreactivity for malignant melanoma. Recently, it was reported that some of the smooth muscle cells in PLAM had reactivity for HMB45. The aim of this study was to assess the sensitivity and specificity of HMB45 for the diagnosis of PLAM in cystic pulmonary diseases that cause recurrent pneumothorax. We compared immunoreactivity of the specimens obtained by open lung biopsy at surgical resection of bullae in 72 patients. The specimens of five females with PLAM, one female with suspected PLAM, 49 patients with primary spontaneous pneumothorax (19 females and 30 males), four with pulmonary eosinophilic granuloma (2 females and 2 males), seven with pulmonary emphysema (7 males), and six with idiopathic pulmonary fibrosis with apical bullous change (2 females and 4 males) were stained with HMB45 and anti-smooth muscle actin. All PLAM cases had HMB45 positive cells, which also stained with anti-smooth muscle actin. The biopsy specimens of a PLAM suspected case also stained with HMB45. None of the specimens from other diseases reacted with HMB45. HMB45 appears to provide a highly specific and highly sensitive diagnosis for PLAM in females. It may also be useful in patients with subtle smooth muscle proliferation. where the diagnosis of PLAM is difficult to confirm by conventional histological examination. PMID- 8620957 TI - Elevated exhaled nitric oxide in patients with hepatopulmonary syndrome. AB - The hypoxaemia of hepatopulmonary syndrome, seen in severe chronic liver dysfunction, occurs as a result of precapillary pulmonary arterial dilatation and arteriovenous communications. These abnormalities contribute to the mismatch between ventilation and perfusion, and the right to left blood flow shunting. Nitric oxide (NO) is a powerful vasodilator concerned with the regulation of pulmonary vascular tone in man. Using a chemiluminescence analyser, we have measured endogenously produced NO in the exhaled air of three patients with the hepatopulmonary syndrome, six normoxaemic cirrhotic patients and six healthy volunteers. The subjects breathed NO-free air throughout the measurements. The molar rate of production of exhaled NO was raised almost threefold in the patients with hepatopulmonary syndrome compared with normal volunteers and with normoxaemic cirrhotic patients. Hypoxia per se, achieved in the normal volunteers by breathing a hypoxic gas mixture, reduced rather than increased the exhaled NO. One hepatopulmonary syndrome patient received an orthotopic liver transplant and achieved normoxaemia after 3 months. The exhaled NO also returned to normal. Increased pulmonary production of NO could contribute to the development of the hepatopulmonary syndrome. PMID- 8620958 TI - Repeatability of airway deposition and tracheobronchial clearance rate over three days in chronic bronchitis. AB - Previous investigations on tracheobronchial clearance in chronic bronchitis or chronic obstructive pulmonary disease (COPD) have usually referred to measurements during a short time-period, i.e. a few hours. The purpose of this study, therefore, was to study regional particle deposition and tracheobronchial clearance during 72 h. In 14 patients with chronic bronchitis clearance of 111In labelled 3.6 micrograms Teflon particles and lung function were measured on two occasions, with an interval of 2 weeks. Lung retention of test particles was measured at 0, 24, 48 and 72 h using a profile scanner. The weight of expectorated sputum samples was measured after the two clearance measurements. The particle retentions at all time-points were reproducible, as seen from the two measurements ( r > 0.90). The fast clearance phase was completed within 72 h. No correlation between sputum volume and clearance was seen. There was a significant negative correlation between airway resistance and the 72 h retention (r= -0.66), and an even better correlation between specific airway resistance and the 72 h retention (r = -0.82), indicating more central deposition in obstructed airways. There was no significant correlation between lung function tests reflecting smaller airways and the 72 h retentions. Deposition data agreed well with theoretical calculations and experimental data in healthy subjects. In spite of earlier findings that mucociliary transport is usually severely impaired in chronic bronchitis and COPD, the present results indicate that overall tracheobronchial mucus clearance in these patients is fairly effective, probably due to a productive cough. Alveolar deposition may be estimated by measurements of the 72 h retention in subjects with chronic obstructive pulmonary disease. The 72 h retention is dependent mainly on the calibre of larger airways. The present method of studying airway clearance during 3 days is highly reproducible. PMID- 8620959 TI - Efficacy of noninvasive CPAP in COPD with acute respiratory failure. AB - Dynamic hyperinflation and the development of intrinsic positive end-expiratory pressure (PEEPi) are commonly observed in patients with severe chronic obstructive pulmonary disease (COPD) in acute respiratory failure. Previous studies have shown that externally applied PEEP reduces PEEPi and its adverse effects in mechanically-ventilated COPD patients. The purpose of this study was to determine the effects of graded amounts of continuous positive airway pressure (CPAP) on the degree of inspiratory effort, pattern of breathing, gas exchange, and level of dyspnoea in a group of spontaneously breathing, nonintubated COPD patients in acute hypercapnic respiratory failure. Ten COPD patients admitted to the intensive care unit in acute hypercapnic respiratory failure were studied. Inspiratory effort was measured by the tidal excursions of oesophageal (Poes) and transdiaphragmatic (Pdi) pressure. Inspiratory effort and both the pressure-time product for the diaphragm (integral of Pdi-dt) and for the inspiratory muscles (integral of Poes.dt) were measured during the application of 5, 7.5, and 10 cmH2O of CPAP. Dyspnoea, gas exchange and pattern of breathing were also assessed. Inspiratory effort and the pressure-time product both for the diaphragm and the inspiratory muscles fell significantly with CPAP in a dose-dependent fashion. Both the pattern of breathing and level of dyspnoea improved with CPAP. End-expiratory lung volume remained stable at the lower levels of CPAP, with only modest increases at the higher levels. Arterial oxygen tension (Pa,O2) and arterial carbon dioxide tension (Pa,CO2) either improved or remained stable with CPAP. We conclude that the noninvasive application of CPAP to spontaneously breathing patients with severe COPD in acute respiratory failure decreases inspiratory effort and dyspnoea whilst improving breathing pattern. It is conceivable that the early institution of CPAP in this setting may obviate the need for intubation and conventional mechanical ventilation. PMID- 8620961 TI - Detection of flow limitation during tidal breathing by the interruptor technique. AB - In patients with airflow obstruction, flow limitation can be established in various ways. Using body plethysmography, flow limitation is assumed when expiratory flow decreases whilst alveolar pressure increases at the same time. During forced expiration, flow limitation can be established by means of the flow interruptor technique; flow limitation is assumed when, after release of an occlusion, a spike flow superimposed on the ongoing alveolar flow (delta peak flow) is detected. In this study, the flow interruptor technique was applied to detect flow limitation during tidal breathing. The results were compared to those obtained with the body plethysmograph. The expiratory flow pattern, post interruption, was analysed in 33 subjects; 11 patients with airflow obstruction and flow limitation established with the body plethysmograph (AO+); 11 patients with airflow obstruction without flow limitation (AO-); and 11 healthy volunteers. Mean spike areas were 27.6 +/- 18.3, 4.6 +/- 2.3 and 3.4 +/- 2.0 mL for the AO+, AO- and control group, respectively, showing a highly significant difference between the AO+ patients and the other groups. Also, significantly higher delta peak flows were found in the AO+ patients compared to the other groups. No differences in delta peak flows or spike areas could be established between patients without flow limitation and controls. We conclude that the interruptor technique may be a useful means of assessing flow limitation during tidal breathing. PMID- 8620960 TI - Comparative effects of pressure support ventilation and intermittent positive pressure breathing (IPPB) in non-intubated healthy subjects. AB - We compared the efficacy of three devices delivering assisted non-invasive ventilation with different working mechanisms, during room air breathing and during CO2-induced hyperventilation. In seven healthy volunteers, breathing pattern, respiratory muscle activity and comfort were assessed: during unassisted spontaneous breathing through a mouth-piece (SB); during assisted breathing with a device delivering inspiratory pressure support (IPS); and with two devices delivering intermittent positive pressure breathing (IPPB), the Monaghan 505 (IPPB1), and the CPU 1 ventilator (IPPB2). All three devices were set at 10 cmH2O of maximal pressure. During room air breathing, the work of breathing expressed as power, was significantly greater with the two IPPB devices than with the two other modes (IPPB1 and IPPB2 7.3 +/- 5.2 and 7.2 +/- 6.2 J.min-1, respectively, versus SB and IPS 2.4 +/- 0.7 and 2.3 +/- 3.3 J.min-1, respectively). The difference did not reach the statistical significance for the pressure-time product (PTP). Discomfort was also greater during the IPPB modes. During CO2 induced hyperventilation, considerable differences in power of breathing were found between the two IPPB devices and the other two modes. The PTP was also much higher with IPPB. Transdiaphragmatic pressure was significantly smaller during IPS than during the three other modes (IPS 18 +/- 2.6 cmH2O versus SB 22 +/- 2.6, IPPB1 32 +/- 5.2, and IPPB2: 28 +/- 5.2). Maximal discomfort was observed during the IPPB modes and was correlated with the magnitude of transdiaphragmatic pressure (r = 0.60). Despite similarities in their operational principles, IPS and IPPB had very different effects on respiratory muscle activity in healthy non intubated subjects. IPPB machines not only failed to reduce patient's effort but also induced a significant level of extra work by comparison to spontaneous ventilation at ambient pressure. Great caution is, therefore, needed in the use of patient-triggered devices for non-intubated patients with acute respiratory failure. PMID- 8620962 TI - Mechanism of theophylline-induced inotropic effects on foreshortened canine diaphragm. AB - The mechanisms of theophylline-induced inotropic effects at shorter diaphragm length have not yet been explored. We wondered whether the greater inotropic effects of the drug at shorter diaphragm length might result from an effect on intracellular calcium level. Forty pairs of diaphragm bundles were stimulated at 70% of optimal length in the presence of either verapamil (10(-5)M), calcium-free Krebs solution (buffered or not with 2 mM ethylene glycol tetra-acetic acid (EGTA)) or ryanodine (10(-6) M). Theophylline (1 mM) was subsequently added to one muscle bundle and, after 15 min, twitches were repeated. The twitch potentiation induced by theophylline (37 +/- 21%) was unaffected by verapamil (43 +/- 26%), or zero calcium (39 +/- 18%) and virtually unchanged when the latter was buffered with EGTA. By contrast, theophylline failed to increase twitch tension after pretreatment with ryanodine, a blocker of the calcium release by the sarcoplasmic reticulum. This decreased twitch tension in control (-5 +/- 11%) and experimental (-14 +/- 12%) bundles and prolonged half-relaxation time as a result of impaired sarcoplasmic reticulum calcium reuptake. We conclude that the inotropic effects of theophylline on twitch tension in foreshortened canine diaphragm bundles were not related to sarcoplasmic reticulum. This is consistent with an action of theophylline on the sarcoplasmic reticulum. PMID- 8620963 TI - Aetiology and prognostic factors of patients with AIDS presenting life threatening acute respiratory failure. AB - Respiratory failure is a significant contributor to morbidity and mortality in patients with the acquired immune deficiency syndrome (AIDS). We performed a study to investigate the aetiology, prognostic factors, and short- and long-term outcome of AIDS patients with life-threatening respiratory failure and pulmonary infiltrates. Forty-two AIDS patients (29 of whom required mechanical ventilation), admitted to a Respiratory Intensive Care Unit (ICU) from 1985 to 1992 because of severe respiratory failure (arterial oxygen tension/fractional inspiratory oxygen (Pa,O2/FI,O2) ratio at hospital admission 19 +/- 14 kPa (mean +/- SD)) and diffuse pulmonary infiltrates, were studied for evaluation of the aetiology and outcome. Necropsy studies were performed in 14 out of 23 (61%) patients who died. Pneumocystis carinii was the most common aetiology of pulmonary infiltrates (28 patients (67%)). Overall, 19 patients survived (45%) and 23 (55%) died. A multivariate analysis of prognostic factors influencing the outcome of the whole population showed that the presence of P. carinii pneumonia and the requirement for mechanical ventilation (MV) were the major determinants of outcome for this type of patient. The median survival time after ICU discharge for P. carinii pneumonia patients was lower (49 days) when compared to that of the remaining patients (154 days). Median survival time after ICU discharge for patients needing MV (112 days) did not differ from that observed in patients not requiring artificial ventilatory support (154 days). Although the ICU survival rate in this study was reasonable, 55% for the whole population, and 36% for P. carinii pneumonia patients, the poor outcome after ICU discharge, in particular for P. carinii pneumonia patients, deserves the reassessment of ICU admission criteria for this type of AIDS population. PMID- 8620964 TI - Nosocomial and community-acquired Legionella pneumonia: clinical comparative analysis. AB - Previous reports have suggested that nosocomial and community Legionella pneumonia cases are similar. However, community and hospital characteristics, such as aquatic environment, antibiotic pressure (usage) and populations, are quite different, leading to the suspicion that Legionella infection may differ in the two settings. Univariate and multivariate analyses were performed to compare demographic data, risk factors, clinical, radiological and outcome data between 125 nosocomial and 33 community-acquired cases of Legionella pneumophila infection. Patients in the nosocomially acquired Legionella pneumonia (NALP) group were older than those in the community-acquired Legionella pneumonia (CALP) group. Univariate analysis showed that smoking habit, cough, thoracic pain, and extrapulmonary manifestations were more prevalent in the CALP group, whilst chronic lung disease and cancer were more prevalent in the NALP group. Moreover, patients in the NALP group were more likely to have received oxygen and corticosteroid therapy and also to have altered creatinine values than patients in the CALP group, whilst more patients in the latter group had altered alanine amino-transferase values. However, multivariate analysis failed to confirm most of these differences. Smoking habit and blood creatinine levels were the only variables remaining significant. In conclusion, demographic, clinical, laboratory, radiological and outcome data in nosocomial and community-acquired Legionella pneumonia are quite similar. PMID- 8620965 TI - Parapneumonic effusions secondary to community-acquired bacterial pneumonia in human immunodeficiency virus-infected patients. AB - The purpose of this study was to determine whether the clinical and microbiological characteristics of parapneumonic effusions in patients with community-acquired pneumonia (CAP) infected with the human immunodeficiency virus (HIV) were different from those observed in patients without HIV infection. One hundred and thirty seven patients with parapneumonic effusions were included and divided into two groups depending on whether they had HIV infection or not. The parapneumonic effusion rate was significantly higher in HIV-positive than in noninfected patients (21 vs 13%). Their clinical course was more severe, presenting a higher rate of bacteraemias (58 vs 18%). Pleural fluid in patients infected with HIV had significantly lower glucose levels than that of patients without HIV infection. Chest tube drainage was more frequent in parapneumonic effusions of patients infected with HIV than in those without HIV infection (71 vs 44%). Staphylococcus aureus was the most common microorganism found in the bacteriological samples of patients with CAP infected with HIV (53 vs 12%). We conclude that patients with community-acquired pneumonia and HIV infection have a higher rate of parapneumonic effusions and a more severe clinical course than non HIV patients, and that Staphylococcus aureus predominates in their bacteriological samples. PMID- 8620966 TI - Peak inspiratory flow through Turbuhaler in acute asthma. AB - Efficient use of dry powder inhalers, such as Turbuhaler, is dependent on the generation of adequate inspiratory flow. It is not clear whether patients with acute asthma are able to generate adequate flow. Peak inspiratory flow (PIF) was measured through an empty Turbuhaler, and without this device, in 99 adults presenting to hospital with acute exacerbations of asthma. Where possible, patients were studied prior to nebulized bronchodilator therapy. Mean (SD) forced expiratory volume in one second (FEV1) was 1.2 (0.7) L, forced vital capacity (FVC) 2.1(1.0) L and peak expiratory flow (PEF) 199 (92) L.min-1. PIF without Turbuhaler was 152 (77) L.min-1 and correlated with PEF (r = 0.69). PIF through Turbuhaler was 60 (20) L.min-1 and weakly correlated with PEF (r = 0.35), and with PIF without Turbuhaler (r = 0.43). Two patients failed to generate the minimum inspiratory flow (30 L.min) required for efficient use of Turbuhaler; both recorded 26 L.min-1. Acute asthma is associated with considerable inspiratory, as well as expiratory airflow limitation. The relationship between inspiratory and expiratory airflow is not strong enough to predict whether patients with severe acute asthma will have difficulty using dry powder inhalers efficiently. Despite this, 98% of patients in this study generated inspiratory flow through Turbuhaler which would allow a therapeutically active amount of bronchodilator drug to be delivered to the airways. PMID- 8620967 TI - Wheezes. AB - Wheezes are continuous adventitious lung sounds. The American Thoracic Society Committee on pulmonary nomenclature define wheezes as high-pitched continuous sounds with a dominant frequency of 400 Hz or more. Rhonchi are characterized as low-pitched continuous sounds with a dominant frequency of about 200 Hz or less. The large variability in the predominant frequency of wheezes is one of the difficulties encountered with automated analysis and quantification of wheezes. The large variations observed in automated wheeze characterization emphasize the need for standardization of breath sound analysis. This standardization would help determine diagnostic criteria for wheeze identification. The mechanism of wheeze production was first compared to a toy trumpet whose sound is produced by a vibrating reed. The pitch of the wheeze is dependent on the mass and elasticity of the airway walls and on the flow velocity. More recently, a model of wheeze production based on the mathematical analysis of the stability of airflow through a collapsible tube has been proposed. According to this model, wheezes are produced by the fluttering of the airways walls and fluid together, induced by a critical airflow velocity. Many circumstances are suitable for the production of continuous adventitious lung sounds. Thus, wheezes can be heard in several diseases, not only asthma. Wheezes are usual clinical signs in patients with obstructive airway diseases and particularly during acute episodes of asthma. A relationship between the degree of bronchial obstruction and the presence and characteristics of wheezes has been demonstrated in several studies. The best result is observed when the degree of bronchial obstruction is compared to the proportion of the respiratory cycle occupied by wheeze (tw/ttot). However, the relationship is too scattered to predict forced expiratory volume in one second (FEV1) from wheeze duration. There is no relationship between the intensity or the pitch of wheezes and the pulmonary function. The presence or quantification of wheezes have also been evaluated for the assessment of bronchial hyperresponsiveness. Wheeze detection cannot fully replace spirometry during bronchial provocation testing but may add some interesting information. Continuous monitoring of wheezes might be a useful tool for evaluation of nocturnal asthma and its treatment. PMID- 8620968 TI - Objective assessment of cough. AB - Cough is a primitive reflex typically consisting of an initiating deep inspiration, glottal closure, and an explosive expiration accompanied by a sound. The flow characteristics of cough have been shown to differ between different pulmonary diseases. Cough sounds are generated at the larynx and in the lungs. Modern analysing techniques have also been applied in cough sound studies, and differences in cough sound duration and spectra have been found in pulmonary diseases with different bronchopulmonary pathophysiology. Since the objective assessment of cough is clinically important, automatic cough detectors and counters have been constructed, e.g. to assess the efficacy of antitussive drugs. Also, ambulatory methods for assessment of cough have been reported. This review includes a brief history of cough research and present methods available for objective assessment of cough. PMID- 8620969 TI - Occupational asthma caused by natural rubber latex. AB - IgE-mediated sensitization in protein allergens of natural rubber latex (NRL) can induce immediate hypersensitivity reactions ranging from mild urticaria in life threatening anaphylaxis after cutaneous, mucosal or visceral exposure. Elutable allergens from NRL gloves absorb to the cornstarch powder particles, become airborne, and have the potential to cause respiratory reactions. Recent studies indicate that asthma is a frequent manifestation of NRL allergy among workers manufacturing NRL materials and among health-care providers using NRL workers. NRL-induced asthma should receive increasing attention as it can lead to permanent respiratory sequelae and occupational disability. The need for early and accurate diagnosis is outlined and the different diagnostic approaches are reviewed. Specific issues pertaining to the management of affected subjects and to the prevention of exposure to airborne NRL are discussed. Ares of future research should include: 1) further characterization of relevant NRL allergens; 2) development and validation of methods for quantitative assessment of allergen content in NRL devices and workplace environment; 3) evaluation of the natural history and risk factors of NRL-induced asthma; and 5) analysis of effectiveness and cost of preventive strategies. PMID- 8620970 TI - Methacholine challenge does not affect bronchoalveolar fluid cell number and many indices of cell function in asthma. AB - Methacholine (MCh) challenge testing is often incorporated into clinical studies prior to performing bronchoscopy as a measure of bronchial hyperresponsiveness (BHR). However, the effect of methacholine on many aspects of bronchoalveolar lavage (BAL) fluid cell count and function have not been fully evaluated. Ten patients with asthma, maintained on inhaled beta 2-agonists, were studied. Each subject underwent two bronchoscopies in a random order, one preceded by methacholine challenge within 30 min of the BAL. The investigators were blinded to the regimen. Several markers of BAL fluid cell number and function were studied: cell count and differential histamine, eosinophil products, including eosinophil cationic protein and Charcot-Leyden crystal protein, macrophage production of thromboxane B2 and leukotriene B4, neutrophil lysozyme and lactoferrin, and lymphocyte typing and activation markers measured via flow cytometry. No significant differences were noted in any of these markers of cell number or function which could be ascribed to methacholine challenge. Thus, methacholine challenge does not appear to affect these markers of cell number and function. These findings indicate that a methacholine challenge can be used as a measure of bronchial hyperresponsiveness within 30 min prior to bronchoscopy without altering bronchoalveolar lavage fluid characteristics. PMID- 8620971 TI - BOOP presenting with haemoptysis and multiple cavitary nodules. AB - A 47 year old woman developed idiopathic bronchiolitis obliterans organizing pneumonia (BOOP) presenting with haemoptysis and diffuse multiple cavitary nodules. The disease was histologically confirmed by open lung biopsy after other entities had been excluded. The patient responded to a course of corticosteroids. This BOOP should be added to the list of diseases with multiple cavitary nodules. PMID- 8620972 TI - Idiopathic bronchiolitis obliterans organizing pneumonia with multiple cavitary lung nodules. AB - Bronchiolitis obliterans organizing pneumonia (BOOP) is a pulmonary disorder with a wide spectrum of radiological features. We report the case of a 58 year old woman, in whom the radiological appearance was multiple cavitary nodules in both lungs, that responded with a complete resolution after corticosteroid therapy. This finding justifies the inclusion of BOOP in the differential diagnosis of multiple cavitary nodules. PMID- 8620973 TI - Psychogenic vocal cord dysfunction simulating bronchial asthma. AB - Upper airway obstruction can mimic bronchial asthma. A particular type results from psychogenic dysfunction of the vocal cord, and presents as bronchial asthma or organic upper airways obstruction. If not recognized, ineffective and potentially harmful therapy is given instead of focusing on the underlying psychopathology. We report three Saudi females with this condition, in whom polygamy and a large family system created associated social stress. Spirometry with a flow volume loop showed characteristic features, and in all three this test was the most important clinical tool that led to the correct diagnosis. Clinicians should perform spirometry and flow-volume loop tests routinely in patients presenting with asthmatic symptoms, and look for clues suggestive of this condition, including a psychosocial assessment. PMID- 8620974 TI - Pulmonary veno-occlusive disease in a HIV-infected intravenous drug abuser. AB - Pulmonary veno-occlusive disease (PVOD) is a rare cause of pulmonary hypertension. Its origin remains unknown but in some cases a viral aetiology has been suggested. Recently primary pulmonary hypertension has been increasingly observed in patients with human immunodeficiency virus (HIV) infection, but only one case of pulmonary veno-occlusive disease has so far been described. We report the case of a 27 year old male intravenous drug abuser with HIV infection and pulmonary hypertension. Open lung biopsy led to the diagnosis of pulmonary veno occlusive disease. This second case of pulmonary veno-occlusive disease raises the question of a relationship between HIV infection and lesions involving the pulmonary veins. However, the pathogenesis of vascular changes remains to be elucidated. PMID- 8620975 TI - Comparison of nebulizer/compressor combinations for domestic use. PMID- 8620976 TI - Flow-volume indices in snorers with and without-OSAS. PMID- 8620977 TI - Pathogenesis of high-altitude pulmonary oedema. PMID- 8620978 TI - [Comparative evaluation of different instruments for root canal preparation tested on extracted human teeth III. Study of sonic and ultrasonic instruments]. AB - A total of 460 roots of extracted human teeth were divided into three groups i.e. straight, apically curved and entirely curved groups. Each of the three groups were divided into seven subgroups according to instrumentation by conventional hand, K-Flex file, 3-LD, 3-LDSY, Excalibur. MM 1400 and Endo Sonic. Radiographs were taken both clinical and proximal views before and after the enlargement of canals. An assessment of shaping characteristic of instrumentation was made on the basis of overimposed radiographs including perforation, ledge formation smoothness of wall contour incidence of elbow and zip measurements at the elbow level and asymmetry measurements. The majority of enlarged root canals were asymmetrical in shape. High differences were found between the incidence of elbow and zip formation. Under laboratory conditions the K-Flex file produced minor canal aberrations and significantly less asymmetry than the conventional instruments. But both the above mentioned instruments caused perforations on curved canals, such as the instrumentation by Excalibur. The loss of working distance was significant in all canal forms prepared with 3-LD and 3-LDSY instruments, which could have occurred through the packing of debris towards the apical constriction. The Excalibur appeared to be superior to the conventional hand instrument in straight canals, but its shaping characteristic was similar to conventional hand instrument in curved canals. The MM 1400 handpiece and ultrasonic instruments resulted in less aberration and significantly less asymmetry than the other tested instruments. PMID- 8620979 TI - [Dental enamel hypoplasia apropos of a case]. AB - The developmental anomalies of dental hard tissues are relatively common in children. These anomalies can involve separately the enamel and they are due to many factors acting during odontogenesis. The paper deals with the main ethological factors and describes a case of idiopathic hypercalcemia. It is normally accompanied by aortic stenosis, mental retardation and a characteristic elfin face. This is called Williams syndrome. In this case we only found enamel hypoplasia on the cusps of the first molars. PMID- 8620980 TI - [Bacteriological study of air in dental offices]. AB - Authors investigated the quality and quantity of the bacteria in the air of a dental surgery practical-room with 6 chairs. They measured the changes of bacterial count during the practical lessons. Equipment HYCON RCS Plus was used. RESULTS: Quantity of the bacteria in the air of this dental surgery was very high. The number of the staphylococci didn't meet the authors' previous expectations. Dispersity of the bacteria was not equal, the highest number was measured at the centre of the room. PMID- 8620981 TI - Prophylactic bilateral oophorectomy to prevent epithelial carcinoma. Number 2- December 1994. Committee on Quality Assessment. American College of Obstetricians and Gynecologists. PMID- 8620982 TI - Medical care of an alleged sexual assault victim. Number 3--January 1995. Committee on Quality Assessment. American College of Obstetricians and Gynecologists. PMID- 8620983 TI - Military pregnancies and adverse perinatal outcome. AB - OBJECTIVE: To identify significant risk factors for an adverse outcome in active duty military women. METHODS: A prospective study of 105 pregnancies and their outcome. RESULTS: The data revealed that: (1) single women more than married personnel had cesarean births when compared with forceps and vacuum (P < 0.03) or spontaneous vaginal delivery (P < 0.04); and (2) active-duty women who gained < 25 pounds during pregnancy developed preterm labor more often (P < 0.05). CONCLUSIONS: Risk factors for these adverse outcomes remain unknown. PMID- 8620984 TI - Trends in maternal mortality in Saudi Arabia. AB - OBJECTIVE: To identify trends and factors related to maternal death, and areas that need improvement. METHODS: A prospective national survey, a multistage sample of the hospitals of Saudi Arabia. The setting was a maternal mortality survey committee in Riyadh with field/area coordinators in different areas of the Kingdom. The subjects were women who died or who were dead on arrival in hospital during pregnancy or within 6 weeks of the end of pregnancy. All the data were coded and analyzed. EPINFO software was used to calculate the maternal mortality ratio (MMR) and the relative risk for the necessary variables. RESULTS: The MMR was 18 per 100,000 births (155/880 248). Mortality was higher in older multiparous women of low income and no education. Hemorrhage, both antepartum and postpartum, was the leading cause of maternal death, together with rupture of the uterus and abortive bleeding, constituting 43% of direct and 29% of total maternal deaths. Substandard care was identified in 73% of direct maternal deaths. CONCLUSIONS: The MMR in Saudi Arabia compares favorably with that of developed countries and the oil-producing Gulf states. Improving the number of booked patients, especially older grand multiparas, increasing the availability of banked blood and adopting a positive approach towards life-saving surgery are likely to reduce maternal deaths. PMID- 8620985 TI - Fetal fibronectin as a screening test for premature delivery in multiple pregnancies. AB - OBJECTIVE: To evaluate fetal fibronectin as a screening test for premature delivery in asymptomatic women with multiple pregnancies. METHODS: In the mid second trimester, the concentrations of fetal fibronectin in the cervical and vaginal secretions of 68 patients with multiple gestations were sampled weekly by monoclonal antibody immunoassay in order to predict preterm labor. RESULTS: The results for the prediction of preterm labor differ according to whether we consider a single positive result (fetal fibronectin > 50 ng/ml) as predictive of preterm labor or whether we only consider at least two consecutive positive results as predictive of perterm labor. The fetal fibronectin test had a sensitivity for preterm birth before 37 weeks of 90.9% and 86.6%, respectively, with a specificity of 68.5% vs. 78.9% and a positive and negative values of 73.1% vs. 76.4% and 88.8% vs. 88.2%, respectively. Similar results were obtained for perterm birth before 34 weeks. CONCLUSIONS: In a condition such as multiple pregnancy which is already at risk for premature delivery the possibility of raising the specificity of the test with virtually no decrease in sensitivity guarantees better recognition of patients likely to develop premature labor. This possibility can be achieved simply by considering two positive consecutive samples as predictive of preterm labor. PMID- 8620986 TI - Screening for anemia in pregnancy with copper sulfate densitometry. AB - OBJECTIVE: The copper sulfate method of screening for anemia was evaluated to determine its accuracy in antenatal patients. METHODS: In an antenatal clinic in a tertiary referral center, which also serves a local urban black community, 100 antenatal patients were prospectively tested for anemia by Coulter hemoglobin analysis in comparison with the copper sulfate test. The respective accuracy and costs of the tests were evaluated. RESULTS: Once initial technical difficulties had been overcome, the copper sulfate test proved accurate in detecting a hemoglobin level < 10 g% in pregnancy (sensitivity 94%, specificity 95%, positive predictive value 80%, negative predictive value 99%). The cost of the copper sulfate test is estimated to be less than 0.3% that of the Coulter test. CONCLUSION: The copper sulfate test is accurate and inexpensive, and can be recommended for screening for anemia in pregnancy. PMID- 8620987 TI - Pregnancy following rupture of the pregnant uterus. AB - OBJECTIVE: To review the cases of ruptured uterus at King Khalid University Hospital (KKUH) over the 11 years of the hospital's existence (1984-1994), to analyze the causative factors of uterine rupture with a view to its prevention, and to highlight the management approach in relation to maintaining the patients' future fertility. METHODS: Case notes were reviewed for all patients with ruptured uterus at KKUH over a period of 11 years from January 1984 to December 1994. Relevant data relating to the clinical features, characteristics of labor, operative procedures, and maternal and perinatal outcome were assessed. RESULTS: There were 11 cases of ruptured uterus, six of which occurred in patients with previous cesarean scars. Two patients were primigravidas, one of whom ruptured her uterus following a road traffic accident. In one patient with six previous preterm labors, rupture resulted from non-removal of cervical cerclage during labor. The rupture occurred in the fundus in one case, and in the lower segment in the remaining 10. Fetal heart abnormalities were observed in all cases in which the uterus ruptured during labor. Abdominal hysterectomy was performed in three cases, two of which were total and the third subtotal. The remaining eight patients had suture repair, all of whom became pregnant later and were delivered by cesarean section. CONCLUSION: Even though rupture of the uterus was a rare complication of pregnancy at KKUH, it occurrence should be suspected when there are sudden fetal heart abnormalities during labor, or unexplained postpartum shock. Suture repair should be considered whenever possible in order to preserve the patients' reproductive potential. PMID- 8620988 TI - Treatment of vaginitis. AB - OBJECTIVE: The purpose of this study was to evaluate the efficacy of metronidazole 500 mg and miconazole nitrate 100 mg (Neo-Penotran, Embil Pharmacy Company, Istanbul, Turkey) in the treatment of vaginitis. METHOD: One hundred patients (mean age 26.5 years, range 18-50) with a clinical diagnosis of vaginitis entered this open, noncomparative study. Each patient inserted a Neo Penotran pessary twice daily for 14 days. Assessments were carried out at the beginning of the study and 15 and 22 days after the start of therapy. Student's t test was used for the statistical analysis. RESULTS: Out of 100 patients who were initially recruited to the study, 20 were subsequently excluded, leaving a total of 80 patients (80%) who completed the full course of treatment. Vaginitis was resolved in 75% of cases, it improved in 18% and was unchanged in 7%. The success rate for the treatment of trichomoniasis was 80%, 93.4% for bacterial vaginosis and 84.4% for candidal vaginitis. CONCLUSION: Neo-Penotran pessaries represent a novel and effective formulation for the treatment of common types of vaginitis and this pessary may also be of particular value in the treatment of resistant or recurrent vaginitis. PMID- 8620989 TI - Transvaginal sonography and progesterone challenge for identifying endometrial pathology in postmenopausal women. AB - OBJECTIVE: To evaluate the usefulness of transvaginal sonographic (TVS) measurement of endometrial thickness combined with a progesterone challenge test (PCT) for identifying endometrial pathology in postmenopausal women. METHODS: Two hundred eighty-four postmenopausal women were examined by TVS: 130 asymptomatic women (group A) and 154 with uterine bleeding (group B). Endometrial thickness > 5 mm was considered pathological. All women with abnormal endometrium from group A underwent PCT. All women from group B underwent D&C. RESULTS: Thirty women with a positive PCT underwent D&C. There was no endometrial cancer. One hundred seven patients from group B had abnormal sonographic and histologic findings: benign (hyperplasia, polyp) or malign (endometrial cancer). There was no cancer in cases with endometrial thickness < or = 6 mm. The sensitivity, specificity and accuracy of TVS for detecting endometrial pathology were 99%, 89% and 96% if the cut-off limit of 5 mm was used. CONCLUSION: TVS combined with PCT is a simple, well tolerated, safe and reliable method for identifying endometrial pathology in postmenopausal women. PMID- 8620990 TI - Low-dose follicle-stimulating hormone treatment for polycystic ovarian disease. AB - OBJECTIVE: An attempt was made to induce ovulation with low-dose follicle stimulating hormone (FSH) in clomifene-resistant women with anovulatory polycystic ovarian disease (PCOD). METHODS: Twenty-two PCOD patients were treated with a low-dose protocol of purified urinary FSH, starting with 75 IU/day and increasing every 7 days by 37.5 IU/day, if necessary. Monitoring was based on ultrasound scanning and estradiol measurements. RESULTS: Twenty-eight of the thirty-one cycles induced were ovulatory, the majority being uniovulatory (58%). No multiple pregnancies occurred. There was a small number of cancelled cycles (12.9%). The prevalence of complications was low, with one case of ovarian hyperstimulation syndrome. Ovulation was induced by a small amount of FSH (15.1 +/- 4.9 ampules). CONCLUSION: The use of the low-dose protocol permitted induction of ovulation safely and successfully in a selected group of PCOD patients. This therapy was associated with a high incidence of single dominant follicles and a very low multiple pregnancy rate. PMID- 8620991 TI - Evaluation of the endometrial cavity during menopause. AB - OBJECTIVE: To compare transvaginal sonography (TVS), hysteroscopy and suction curettage in the evaluation of uterine bleeding during the menopause. METHODS: Forty-seven patients who presented with either postmenopausal bleeding (31 cases) or sonographic endometrial abnormalities at menopause (16 cases) were evaluated using TVS, hysteroscopy, and curettage with a Karman curette. RESULTS: When endometrial thickness measured by TVS was < 4 mm, there was no endometrial pathology. However TVS could not differentiate accurately between hyperplasia, polyps or endometrial carcinoma. In these cases, endometrial thickness was invariably greater than 5 mm. CONCLUSIONS: Hysteroscopy proved superior to curettage in the diagnosis of endometrial polyps. PMID- 8620992 TI - Maternal brain death during pregnancy. AB - A case of a 36-year-old woman diagnosed brain dead secondary to pneumococcal meningitis at 27 weeks of gestation is presented. In spite of aggressive therapy, supportive intensive care was possible for only 36 h. Signs of fetal distress appeared and a cesarean section was performed. The complexity of supportive care and its ethical implications are discussed. PMID- 8620993 TI - Congenital pelvic arteriovenous malformation with massive vaginal bleeding. PMID- 8620994 TI - Prevalence of hysterectomy of Chinese women in Taiwan. PMID- 8620995 TI - Urinary incontinence. Number 213--October 1995 (replaces No. 100, January 1987. Committee on Technical Bulletins of the American College of Obstetricians and Gynecologists. PMID- 8620996 TI - Ethical guidance for patient testing. Number 159--October 1995. Committee on Ethics. American College of Obstetricians and Gynecologists. PMID- 8620997 TI - The effect of chronic intestinal parasitic infection on maternal and perinatal outcome. AB - OBJECTIVE: The main objective of this prospective cohort study was to investigate the prevalence, risk factors and maternal-perinatal consequences of chronic asymptomatic intestinal parasitic infection during pregnancy. METHODS: Prenatal patients (n=91) attending a public clinic in Quito, Ecuador, were followed during the third trimester. Intestinal parasitic, nutritional status, sociodemographic/sanitation indicators and fetal outcome data were collected and analyzed using multivariate ANOVA and regression techniques. RESULTS: Most subjects (93%) were infected with at least one species of pathogenic intestinal parasite: 88% with Entamoeba histolytica. Greater parasite burdens were associated with poorer maternal iron status and reduced fetal growth. In particular, a high E. histolytica load was associated with decreased maternal serum hemoglobin (P=0.002) and hematocrit levels (P=0.01), iron deficiency anemia (P=0.026), and indicators of diminished intrauterine growth including a decreased ponderal index (P=0.04), mid-arm circumference (P=0.01), and mid-arm/head circumference ratio (P=0.003). CONCLUSION: Asymptomatic intestinal parasitic infection represents a hidden risk factor for maternal iron deficiency anemia and fetal growth retardation. PMID- 8620998 TI - Vaginal delivery after previous cesarean birth. Number 1--August 1995. Committee on Practice Patterns. American College of Obstetricians and Gynecologists. PMID- 8620999 TI - Endometrial ablation. Number 1--November 1994. Committee on Quality Assessment. American College of Obstetricians and Gynecologists. PMID- 8621000 TI - 1995 Presidential Address. Things will never be the same again. PMID- 8621001 TI - The search for IDDM susceptibility genes: the next generation. AB - Two human chromosomal regions, the HLA region on chromosome 6p2l and the insulin gene region on chromosome 11p15, have been investigated in detail for more than 10 years for the presence of IDDM susceptibility genes. Recent genome searches indicate the possible existence of many additional susceptibility genes in IDDM. The lengthy and protracted studies to prove the linkage and identity of the susceptibility genes in the HLA and insulin gene regions provide a perspective and background for understanding the complexities and time course for characterization of the putative additional IDDM susceptibility genes uncovered by genome searches. PMID- 8621002 TI - The effect of glucagon-like peptide I (GLP-I) on glucose elimination in healthy subjects depends on the pancreatic glucoregulatory hormones. AB - Glucagon-like peptide I (GLP-I) decreases plasma glucose in type II diabetic patients and in healthy subjects indirectly by stimulation of insulin and inhibition of glucagon secretion, whereby the hepatic glucose production decreases. However, recent studies indicate that GLP-I may also directly influence peripheral and hepatic glucose uptake. We infused somatostatin (SS) intravenously (500 or 1,000 microgram/h) in 13 healthy subjects to suppress insulin and glucagon secretion from the endocrine pancreas, together with infusion of either GLP-I (50 pmol / kg / h) or saline intravenously. After 30 min, a 25-g intravenous glucose tolerance test (IVGTT) was carried out, and plasma concentrations of glucose, insulin, glucagon, and GLP-I were measured during the following 2 h. IVGTT together with GLP-I infusion significantly elevated insulin during 500 microgram/h SS but not during 1,000 microgram/h SS. Plasma glucagon was strongly depressed in all experiments. During 500 microgram/h SS, the glucose disappearance constant, Kg, was 0.49 +/- 0.03% per minute with GLP-I and 0.39 +/- 0.04% per minute with saline (n = 8, P = 0.004). With 1,000 microgram/h SS, Kg was 0.42 +/- 0.03% per minute with GLP-I and 0.40 +/- 0.03% per minute without (NS). In conclusion, when endogenous insulin secretion is held at a constant low level, which may be accomplished only with very large doses of SS, GLP-I has no effect on glucose elimination. Thus, an insulin-independent effect of GLP-I on glucose disposal could not be demonstrated. PMID- 8621003 TI - Kilham rat triggers T-cell-dependent autoimmune diabetes in multiple strains of rat. AB - Kilham rat virus (KRV) infection of BB/Wor diabetes-resistant (DR) RT1(u) rats induces autoimmune diabetes without direct cytolytic infection of pancreatic beta cells and is a new model of virus-induced IDDM. To investigate genetic susceptibility to KRV-induced diabetes, major histocompatibility complex congenic and other inbred rats were infected with the virus and studied for the appearance of diabetes and insulitis. KRV infection alone induced insulitis, selective beta cell necrosis, and diabetes in BB/Wor DR and LEW1.WR1 (RT1 A(u) B/D(u) C(a)) but not other rats. Thus, KRV, an environmentally ubiquitous rat parvovirus, can precipitate autoimmune diabetes in rats that are not susceptible to spontaneous diabetes. If rats are injected with poly(I.C) immediately before KRV infection, diabetes frequency increases to >90% in BB/Wor DR and LEW1.WR1 rats, and PVG.RT1(u) rats are converted from KRV-resistant to KRV-susceptible status. Susceptibility to KRV-induced diabetes thus requires the presence of class I A(u) and class II B/D(u) gene products, which are shared by DR, LEW1.WR1, and PVG.RT1(u) rats. The RT1(u) haplotype is not sufficient for susceptibility, however, because while WF rats are RT1(u), they resist KRV-induced diabetes. If rats are depleted of RT6.1+ regulatory T-cells before KRV infection, the frequency of diabetes is dramatically increased in DR and LEW1.WR1, but not PVG.RT1(u) or other rats. These data confirm a regulatory role of RT6.1+ T-cells in diabetes induction, but indicate that they may not operate as such in all rat strains. KRV-induced diabetes is T-cell-mediated: DR and LEW1.WR1 rats are protected from diabetes by treatment with monoclonal antibodies directed against alpha beta T-cell receptor (TCR)+, CD5+, and CD8+ T-cells. Concanavalin A activated spleen cells from KRV-infected DR rats adoptively transfer diabetes and insulitis into class II(u) compatible rats, suggesting that KRV infection of susceptible rats leads to the activation of diabetogenic class II(u) restricted T cells. The ability of a common rat virus to initiate IDDM in multiple strains of rats strengthens the possibility that viruses may also initiate IDDM in human populations. PMID- 8621004 TI - Evidence for a major gene for type II diabetes and linkage analyses with selected candidate genes in Mexican-Americans. AB - We have carried out two independent family studies in low-income Mexican Americans from San Antonio, Texas. In the first study, probands were ascertained at random without regard to any medical condition (658 examined individuals from 50 families), and in the second study, probands were subjects with type II diabetes identified in a prior epidemiological survey (523 examined individuals from 29 families). Pedigrees ranging in size from 2 to 45 family members (median 11) in the first study and from 2 to 50 family members (median 12) in the second study were examined. Diabetes was diagnosed according to World Health Organization criteria. In both sets of families, segregation analyses revealed support for a major gene with an autosomal dominant mode of inheritance influencing early age of onset of diabetes. Non-Mendelian inheritance was rejected in both data sets. Individuals with the early age of onset allele had a mean age of diabetes onset of 51 years in the first data set and 60 years in the second data set. In the first data set, the major gene accounted for approximately 70% of the phenotypic variance in age of onset of diabetes, and there were no residual family effects once the major gene effect was taken into account. In the second data set, the major gene accounted for approximately 50% of the phenotypic variance, and residual family effects were statistically significant. Linkage analyses were performed with 11 candidate genes, and tight linkage with diabetes was rejected for Rh blood group, glucose transporter 2, fatty acid-binding protein, tumor necrosis factor beta, glucokinase, and lipoprotein lipase. A logarithm of odds (LOD) score of 0.92 at a recombination fraction of 0.05 was observed for insulin receptor substrate 1. This LOD score corresponds to a chi2 of 4.24 (P = 0.039). PMID- 8621005 TI - Potentiation of effects of weight loss by monounsaturated fatty acids in obese NIDDM patients. AB - Although moderate weight loss improves glycemic control in obese NIDDM patients, quite often it is not normalized. To determine whether the response to weight loss can be improved by altering the macronutrient composition of hypocaloric diets, 17 obese NIDDM patients were studied at I) baseline, 2) after dieting for 6 weeks on a formula diet enriched in either monounsaturated fatty acids (MUFAs, n = 9) or carbohydrates (CHOs, n = 8) at a 50% caloric deficit, and 3) after 4 weeks of postdiet refeeding on the respective formulas with caloric intake titrated to achieve weight maintenance. Fasting, 24-h, and oral glucose tolerance test (OGTT) blood glucose, plasma insulin, and C-peptide levels were measured. All prediet parameters were similar between groups. After dieting, although weight loss was similar between groups, the fasting glucose level decreased significantly more in the MUFA group (-4.6 +/- 0.7 mmol/l) than in the CHO group (-2.4 +/- 1.0 mmol/l; P < 0.05). Twenty-four-hour glycemia decreased in both groups after dieting, but the MUFA group had a greater decrease than the CHO group (P < 0.05, analysis of variance [ANOVA]). Although decreases in fasting glycemia were maintained in both groups after refeeding, postprandial glycemia deteriorated after refeeding with the CHO- but not the MUFA-enriched formula (P < 0.05). After dieting and refeeding, fasting C-peptide increased 204 +/- 47 pmol/l in the MUFA group, but the CHO group remained at prediet levels (P < 0.05). Twenty-four-hour C-peptide levels were similar between groups after dieting and refeeding, despite the lower glycemia and CHO content of the MUFA formula. However, when equal amounts of CHO were consumed during the OGTT, the MUFA group had significantly higher C-peptide levels after both dieting and refeeding (P < 0.05). Fasting, 24-h, and OGTT insulin levels were similar between groups throughout the study. These results indicate that macronutrient composition is an important determinant of the glycemic response to weight-loss therapy in obese NIDDM patients. Based on the C-peptide response during the OGTT, increased CHO induced insulin secretion is one possible mechanism by which this occurs. PMID- 8621006 TI - Hematocrit and risk of NIDDM. AB - There is limited evidence that raised hematocrit levels may be associated with insulin resistance, which links cardiovascular disease with NIDDM. The association between hematocrit level at screening and the subsequent development of physician-diagnosed NIDDM during 12.8 years of follow-up was examined in a prospective study of 7,735 middle-aged men drawn at random from general practice in 24 British towns. With the exclusion of men with missing hematocrit data and men with diabetes at screening, data were available for 7,193 men, in whom there were 187 new cases of NIDDM during follow-up. The risk of NIDDM increased significantly with increasing hematocrit levels. There was more than a fourfold increase in relative risk (RR) of diabetes among men with a hematocrit of > or = 48% relative to those with a hematocrit <42%, adjusted for age and BMI (RR 4.5; 95% CI 2.5-6.3). On further adjustment for predictors of NIDDM with which hematocrit is correlated, there remained a strong linear association with the risk of diabetes. There was a nearly fourfold increased risk of NIDDM in the highest relative to the lowest hematocrit group in the fully adjusted proportional hazard model (RR 3.6; 95% CI 1.7-7.6). The strong positive association between hematocrit level and risk of diabetes was seen even after exclusion of men with preexisting ischemic heart disease. The findings suggest that a raised hematocrit level, which is a major determinant of whole blood viscosity, should be added to the cluster of risk factors that link NIDDM with atheromatous, vascular disease. PMID- 8621007 TI - A fatty acid-induced decrease in pyruvate dehydrogenase activity is an important determinant of beta-cell dysfunction in the obese diabetic db/db mouse. AB - We studied the effects of fatty acid oxidation on insulin secretion of db/db mice and underlying molecular mechanisms of these effects. At 2-3 months of age, db/db mice were markedly obese, hyperglycemic, and hyperinsulinemic. Serum free fatty acid (FFA) levels were increased in 2-month-old (1.5 +/- 0.1 vs. 1.1 +/- 0.1 mmol/l, P < 0.05) and 3-month-old (1.9 +/- 0.1 vs. 1.2 +/- 0.1 mmol/l, P < 0.01) mice compared with the age and sex-matched db/+ mice serving as controls. Glucose induced insulin release from db/db islets was markedly decreased compared with that from db/+ islets and was specifically ameliorated (by 54% in 2-month-old and 38% in 3-month-old mice) by exposure to a carnitine palmitoyltransferase I inhibitor, etomoxir (1 micromol/l). Etomoxir failed to affect the insulin response to alpha-ketoisocaproate. The effect of etomoxir on glucose-induced insulin release was lost after culturing db/db islets in RPMI medium containing 22 mmol/l glucose but no fatty acid. Culture of db/+ islets with 0.125 mmol/l palmitate led to a decrease in glucose-induced insulin secretion, which was partially reversible by etomoxir. Both islet glucose oxidation and the ratio of glucose oxidation to utilization were decreased in db/db islets. Etomoxir significantly enhanced glucose oxidation by 60% and also the ratio of oxidation to glucose utilization (from 27 +/- 2.5 to 37 +/-3.0%, P < 0.05). Pyruvate dehydrogenase (PDH) activity was decreased in islets of db/db mice (75 +/-4.2 vs. 91 +/- 2.9 nU/ng DNA, P < 0.01), whereas PDH kinase activity was increased (rate of PDH inactivation -0.25 +/- 0.02 vs. - 0.11 +/- 0.02/min, P < 0.0 1). These abnormalities were partly but not wholly reversed by a 2-h preexposure to etomoxir. In conclusion, elevated FFA levels in the db/db mouse diminish glucose induced insulin secretion by a glucose-fatty acid cycle in which fatty acid oxidation inhibits glucose oxidation by decreasing PDH activity and increasing PDH kinase activities. PMID- 8621008 TI - Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen. AB - The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes. PMID- 8621009 TI - Islets of Langerhans generate wavelike electric activity modulated by glucose concentration. AB - The electric activity of whole islets of Langerhans was monitored for the first time in this study. Measurements were made from single islets isolated from mice, hamsters, gerbils, and rats by means of external electrodes. Well-structured synchronized potential spikes up to 0.5 mV in amplitude with a stable frequency of 0.5-2 Hz were measured. Spike generation had a glucose concentration threshold. In the physiological range of each animal species, firing rate was an approximate linear function of glucose concentration. At low glucose concentrations, firing became intermittent, i.e., in bursts, while in the physiological range and above, firing was typically continuous. Simultaneous measurements from two locations on an islet indicate that the measured activity reflects the propagation of an excitation wave throughout the islet. This, together with signal synchronization, suggests that the islets contain a functional pacemaker (FPM) from which excitation propagates by means of gap junctions to the rest of the islet cells (mostly beta-cells). Thus, the electric characteristics of the individual beta-cells are functionally masked so that the islet acts as a single functional unit. In view of the dependency of insulin secretion on the islet's electric activity, the islet glucose-insulin dose response characteristics must be determined by those of the FPM. PMID- 8621010 TI - The hemodynamic abnormalities in short-term insulin deficiency: the role of prostaglandin inhibition. AB - It has been suggested that the hemodynamic derangements present in diabetic ketoacidosis are the results not only of profound volume depletion but also of the effects of increased production of vasodilating prostaglandins (PGs), principally PGI2, released by adipose tissue. In animal and in vitro models, prostaglandin synthesis is increased during insulin deficiency. We assessed the effects of short-term ketosis on the metabolic and hemodynamic variables of 10 IDDM patients free from long-term complications and of 9 normal control subjects after a 7-day randomized double-blind indomethacin (INDO) (50 mg q.i.d.) or placebo treatment period. Calf blood flow (CBF), postocclusive reactive hyperemia (PORH), and recovery half-time (an index of overall perfusion) after PORH were measured by plethysmography. Left ventricular and myocardial functions were also studied in each different condition during placebo and INDO treatment in IDDM patients. During placebo treatment, the increase in CBF during ketosis was higher (1.75 +/- 0.29 ml / min / 100 ml muscle) than during INDO (0.85 +/- 0.17 ml / min) / 100 ml muscle; P = 0.007). PORH was similar in baseline conditions, during ketosis, and in recovery in both the placebo and INDO arms. Recovery half-time significantly increased during placebo (10 +/- 2; 200%; P < 0.01) but not during INDO (1 +/- 1; 106%; NS) treatment. In normal control subjects, insulin deficiency did not induce any significant effect on hemodynamic variables. In IDDM patients, during placebo treatment, ketosis increased both the cardiac index (from 3.4 +/- 0.7 to 4.1 +/- 0.81 / min / m; P < 0.01) and the stroke index (from 42 +/- 8 to 49 +/- 7 ml/m2; P < 0.01) without changes in left ventricular ejection fraction but with a significant increase in both left and right ventricular end-diastolic volumes. Metabolic recovery induced a normalization of these parameters. INDO treatment significantly blunted these alterations. In summary, we showed that during acute insulin deficiency, INDO-sensitive mechanisms mediate vascular disturbances. Moreover, INDO treatment was capable of completely preventing the cardiac venous return and the left ventricular alterations. INDO does not interfere with the overall ketogenetic process or with insulin-induced metabolic recovery. PMID- 8621011 TI - Association of HLA-DPB1*0301 with IDDM in Mexican-Americans. AB - Susceptibility to IDDM has been associated with specific alleles at the HLA class II loci in a variety of human populations. Previous studies among Mexican Americans, a group ancestrally derived from Native Americans and Hispanic whites, showed that the DR4 haplotypes (DRB1*0405-DQB1*0302 and DRB1*0402-DQB1*0302) and the DR3 haplotype (DRB1*0301-DQB1*0201) were increased among patients and suggested a role for both DR and DQ alleles in susceptibility and resistance. Based on the analysis of 42 Mexican-American IDDM families and ethnically matched control subjects by polymerase chain reaction/sequence-specific oligonucleotide probe typing, we report an association of IDDM with the DPB1 allele, *0301 (relative risk = 6.6; P = 0.0012) in this population. The analysis of linkage disequilibrium patterns in this population indicates that the observed increased frequency in DPB1*0301 among patients cannot be attributed simply to linkage disequilibrium with high-risk DR-DQ haplotypes. These data suggest that in addition to alleles at the DRB1 and DQB1 loci, polymorphism at the DPB1 locus may also influence IDDM risk. PMID- 8621012 TI - Mechanisms behind insulin resistance in rat skeletal muscle after oophorectomy and additional testosterone treatment. AB - The absence of female sex hormones, as well as testosterone treatment of oophorectomized (OVX) female rats has been demonstrated to result in decreased whole-body insulin-mediated glucose uptake. The cellular mechanism behind this insulin resistance and the role of low levels of female sex hormones as a risk factor for development of peripheral insulin resistance are not yet fully clarified. We assessed the protein expression of GLUT4 and glycogen synthase, as well as insulin-induced translocation of GLUT4 to the plasma membrane, in soleus skeletal muscle from control rats, OVX rats, and OVX rats treated for 8 weeks with testosterone (OVX + T). Whole-body insulin-mediated glucose uptake assessed by the hyperinsulinemic-euglycemic clamp procedure was 25% lower in OVX rats (P < 0.001) and addition of testosterone treatment further decreased insulin-mediated glucose uptake in OVX + T rats by 48% (P < 0.001) compared with controls. GLUT4 protein expression in soleus muscles was unaltered in the OVX and OVX + T rats compared with controls. Insulin induced a 3.7-fold increase (P < 0.05) in the plasma membrane content of GLUT4 in soleus muscle from control rats, whereas plasma membrane content of GLUT4 in soleus muscle from OVX or OVX + T rats was unaltered in response to insulin. Glycogen synthase protein expression in muscle homogenates was decreased by 25% in the OVX group (P < 0.05) and by 37% in the OVX + T group (P < 0.05) when compared with the control group. Insulin receptor and tyrosine kinase activities in the basal and insulin-stimulated states did not differ between the OVX and OVX + T rats. In conclusion, the absence of female sex hormones appears to decrease insulin-mediated whole-body glucose uptake via an impaired insulin-stimulated translocation of GLUT4 to the plasma membrane and by decreased protein expression of glycogen synthase. Testosterone treatment further impairs whole-body insulin-mediated glucose uptake, presumably by additional impairment of glycogen synthase expression. PMID- 8621013 TI - Small doses of subcutaneous insulin as a strategy for preventing slowly progressive beta-cell failure in islet cell antibody-positive patients with clinical features of NIDDM. AB - We report a pilot study to determine the preventive effect of small doses of insulin injected subcutaneously on slowly progressive beta-cell damage in islet cell antibody (ICA)-positive patients with apparent NIDDM. Ten NIDDM patients who were ICA' were divided into two groups of five. In the insulin group (age: 51 +/- 8 years [mean +/- SD], sex: 3 men and 2 women), intermediate-type insulin (3-16 U/day) was given once or twice daily as a subcutaneous injection. The sulfonylurea (SU) group (age: 48 +/- 11 years, sex: 3 men and 2 women) was initially treated with a SU agent. Changes in beta-cell function, as indicated by serum C-peptide responses and blood glucose values during a 100-g oral glucose tolerance test, as well as ICA and GAD antibody status, were evaluated for up to 30 months in both groups. ICA status became negative in four of five patients in the insulin group. ICA status did not become negative in any of the patients in the SU group (P = 0.047 vs. insulin group). ICA status was persistently positive in two patients whose beta-cell function eventually progressed to an insulin dependent state and fluctuated in the remaining three patients. In the insulin group, GAD antibody status became negative in one of four initially GAD antibody positive NIDDM patients. In the SU group, GAD antibody status was persistently positive in three NIDDM patients (NS vs. insulin group). The serum C-peptide response improved significantly within 6 and 12 months in the insulin group, whereas it decreased progressively in the SU group. The changes in C-peptide response were significantly different between the two groups at 6, 12, 24, and 30 months. Two-hour blood glucose and HbA1 values were unchanged in the insulin group, but they increased in the SU group. Subcutaneous small doses of insulin, resulting in a high rate of negative conversion of ICA and an improved serum C peptide response, may be effective in treating ICA+ NIDDM patients who are at high risk for slowly progressive beta-cell failure. PMID- 8621014 TI - Diabetes increases sciatic nerve susceptibility to endothelin-induced ischemia. AB - Endothelin-1 (ET) is a potent vasoconstrictor of vasa nervorum, the vascular supply of peripheral nerve trunks, that may, through elevated circulating levels, promote microangiopathy in human diabetes patients. In previous work, we observed that sciatic nerve trunks of rats exposed to epineurial ET developed transient dose-dependent endoneurial ischemia that might be associated with reversible motor conduction block. In the present study, we explored the possibility that ET ischemia might selectively damage axons of diabetic nerve trunks. We exposed the sciatic nerves of rats with streptozotocin-induced diabetes of 6-8 weeks duration and age-matched nondiabetic controls to supramaximal vasoconstrictive concentrations of epineurial ET and studied serial regional erythrocyte flux (laser Doppler flowmeter) and sciatic tibial motor conduction for up to 14 days. In diabetic rats, but not controls, ET ischemia damaged a large proportion of sciatic axons, predicted in each instance by focal motor conduction block across the site of ET application and eventual loss of distal motor nerve excitability. Histological studies confirmed the presence of severe axonal degeneration in diabetic sciatic nerves exposed to ET. Part of the susceptibility to ET in diabetes was accounted for by more intense and prolonged vasoconstriction. Diabetic nerves are selectively susceptible to ET-mediated ischemia. PMID- 8621015 TI - Abdominal fat and insulin resistance in normal and overweight women: Direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM. AB - Insulin resistance appears to be central to obesity, NIDDM, hyperlipidemia, and cardiovascular disease. While obese women with abdominal (android) fat distribution are more insulin resistant than those with peripheral (gynecoid) obesity, in nonobese women, the relationship between abdominal fat and insulin resistance is unknown. By measuring regional adiposity with dual-energy X-ray absorptiometry and insulin sensitivity by euglycemic-hyperinsulinemic clamp in 22 healthy women, with a mean +/- SE body BMI of 26.7 +/- 0.9 kg/m2 and differing risk factors for NIDDM, we found a strong negative relationship between central abdominal (intra-abdominal plus abdominal subcutaneous) fat and whole-body insulin sensitivity (r = -0.89, P < 0.0001) and nonoxidative glucose disposal (r = -0.77, P < 0.001), independent of total adiposity, family history of NIDDM, and past gestational diabetes. There was a large variation in insulin sensitivity, with a similar variation in central fat, even in those whose BMI was <25 kg/m2. Abdominal fat had a significantly stronger relationship with insulin sensitivity than peripheral nonabdominal fat (r2 = 0.79 vs. 0.44), and higher levels were associated with increased fasting nonesterified fatty acids, lipid oxidation, and hepatic glucose output. Because 79% of the variance in insulin sensitivity in this heterogeneous population was accounted for by central fat, abdominal adiposity appears to be a strong marker and may be a major determinant of insulin resistance in women. PMID- 8621016 TI - Mitochondrial FAD-glycerophosphate dehydrogenase and G-protein-coupled inwardly rectifying K+ channel: No evidence for linkage in maturity-onset diabetes of the young or NIDDM. AB - Two genes that have potentially important regulatory roles in insulin secretion are both located on chromosome 2q24.1. G-protein-coupled muscarinic potassium channel (GIRK1) is an inwardly rectifying K+ channel that helps to maintain the resting potential and excitability of cells. Mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH) catalyzes a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Reduced m-GDH activity has been demonstrated in islets isolated from diabetic subjects compared with islets from nondiabetic control subjects and from the diabetic GK rat. To study the relationship between these candidate genes and NIDDM, we have examined a simple tandem-repeat polymorphism (STRP) close to both the KCN J3 (GIRK1) locus and the m-GDH locus. In a linkage study of three maturity-onset diabetes of the young (MODY) pedigrees, not linked to MODY1, MODY2, or MODY3, a cumulative score of - 9.6 at a recombination fraction of theta = 0 excluded linkage. In a population association study, no linkage disequilibrium for the STRP was found between 190 unselected NIDDM patients and 60 geographically and age-matched white nondiabetic subjects (chi2 = 1.51 on 3 df, P = 0.68). Thus, mutations involving the genes for GIRK1 or FAD-glycerophosphate dehydrogenase are unlikely to cause MODY, and a common mutation in either gene is unlikely to contribute to NIDDM in whites. These data do not exclude mutations in some families or other ethnic groups. PMID- 8621017 TI - LY290181, an inhibitor of diabetes-induced vascular dysfunction, blocks protein kinase C-stimulated transcriptional activation through inhibition of transcription factor binding to a phorbol response element. AB - Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181. LY290181 prevented glucose-induced increases in blood flow and permeability in rat granulation tissue and corresponding vascular changes in the retina, sciatic nerve, and aorta of diabetic rats. Tested for its ability to inhibit PKC-regulated processes, LY290181 inhibited phorbol ester-stimulated plasminogen activator activity in a dose-dependent manner in bovine retinal endothelial cells and in human dermal fibroblasts. In addition, LY290181 inhibited phorbol ester-stimulated activation of the porcine urokinase plasminogen activator (uPA) promoter (-4600/+398) linked to the chloramphenicol acetyltransferase (CAT) reporter gene (p4660CAT). More detailed analysis of the uPA promoter revealed that LY290181 inhibited phorbol ester-stimulated activation of the uPA phorbol response element (-2458/-2349) located upstream of the thymidine kinase promoter (puPATKCAT). LY290181 appears to inhibit uPA promoter activation by blocking phorbol ester-stimulated binding of nuclear proteins to the uPA PEA3/12-0-tetradecanoylphorbol 13-acetate responsive element (TRE). These results suggest that LY290181 may inhibit diabetes-induced vascular dysfunction by inhibiting transcription factor binding to specific PKC-regulated genes involved in vascular function. PMID- 8621018 TI - Metabolic impairment precedes insulin resistance in skeletal muscle during high fat feeding in rats. AB - To examine whether impairment of intracellular glucose metabolism precedes insulin resistance, we determined the time courses of changes in insulin stimulated glucose uptake, glycolysis, and glycogen synthesis during high-fat feeding in rats. Animals were fed with a high-fat (66.5%) diet ad libitum for 0, 2, 4, 7, or 14 days (n = 10-11 in each group) after 5 days of a low-fat (12.5%) diet. Submaximal and maximal insulin-stimulated glucose fluxes were estimated in whole body and individual skeletal muscles using the glucose clamp technique combined with D-[3-3H]glucose infusion and 2-[1-14C]deoxyglucose injection. Both submaximal and maximal insulin-stimulated glucose uptake in whole body decreased gradually with high-fat feeding. However, the decreases were minimal and not statistically significant during the initial few days (i.e., 2 and 4 days) of high-fat feeding (P > 0.05). In contrast, insulin-stimulated whole-body glycolysis (both maximal and submaximal) significantly decreased by approximately 30% with 2 days of high-fat feeding and remained suppressed thereafter (P < 0.05). Similar patterns of changes in insulin-stimulated glucose uptake and glycolysis were also observed in skeletal muscle. Insulin-stimulated glycogen synthesis and glucose-6-phosphate (G-6-P) concentrations in skeletal muscle increased significantly during the initial few days of high-fat feeding and gradually returned to control levels by day 14, suggesting that increased G-6-P concentrations were responsible for increased glycogen synthesis. Thus, suppression of insulin-stimulated glycolysis and a compensatory increase in glycogen synthesis (presumably arising from the glucose-fatty acid cycle) preceded decreases in insulin-stimulated glucose uptake in skeletal muscle during high-fat feeding. These findings suggest that the insulin resistance may develop as a secondary response to impaired intracellular glucose metabolism. PMID- 8621020 TI - Increased urea synthesis in insulin-dependent diabetic dogs maintained normoglycemic: effect of portal insulin administration and food protein content. AB - In IDDM, the gluconeogenic turnover of amino acids is increased even if glycemia is well controlled and may be restored to normal by means of prehepatic insulin substitution. Therefore, the present study was designed 1) to investigate the influence of route of insulin administration (portal versus peripheral) on the urea production rate, which is considered to measure amino acid catabolism, and 2) to elucidate the impact of different food-protein intake. Paired studies were conducted in chronic insulin-dependent diabetic dogs maintained normoglycemic. Diabetic animals and nondiabetic controls were fed either a high-protein diet (46% of energy intake provided by proteins; study 1) or a low-protein carbohydrate-supplemented diet (20% of energy intake provided by protein; study 2) for 2 days, and flux rates of glucose and urea were measured using isotope dilution techniques. In both studies, the diabetic animals were maintained normoglycemic by glucose-controlled insulin infusion delivered either systemically or portally. In study 1 versus study 2, the animals showed lower alpha-amino nitrogen levels and concentrations of gluconeogenic amino acids, predominantly alanine. There were no significant differences in plasma glucose and glucose turnover between the experimental groups on either systemic or portal insulin infusion versus controls; however, peripheral insulin levels were higher for diabetic animals maintained with systemic versus portal insulin delivery (P < 0.05). No significant differences in glucagon, lactate, pyruvate, nonesterified fatty acids, or beta-hydroxybutyrate were observed. Urea production was significantly higher in study 1 compared with study 2: 7.48 +/- 0.83 vs. 5.97 +/- 0.59 micromol / kg / min (normal dogs); 12.97 +/- 1.86 vs. 5.54 +/- 0.60 micromol / kg / min (diabetic dogs on portal insulin); 16.11 +/- 2.59 vs. 6.82 +/- 0.70 micromol / kg / min (diabetic dogs on systemic insulin infusion); P < 0.05 for all. The diabetic dogs maintained normoglycemic with systemic insulin infusions had significantly higher rates of urea synthesis than those with portal insulin infusion (P < 0.05). It is concluded that in IDDM, even if normoglycemia is managed, there is significantly increased amino acid catabolism with posthepatic systemic insulin treatment. This increased catabolic rate is more pronounced during high-protein nourishment. PMID- 8621019 TI - Oral vanadyl sulfate improves insulin sensitivity in NIDDM but not in obese nondiabetic subjects. AB - We compared the effects of oral vanadyl sulfate (100 mg/day) in moderately obese NIDDM and nondiabetic subjects. Three-hour euglycemic-hyperinsulinemic (insulin infusion 30 mU / m / min) clamps were performed after 2 weeks of placebo and 3 weeks of vanadyl sulfate treatment in six nondiabetic control subjects (age 37 +/ 3 years; BMI 29.5 +/- 2.4 kg/m2 ) and seven NIDDM subjects (age 53 +/- 2 years; BMI 28.7 +/-1.8 kg/m2). Glucose turnover ([3-3 H]glucose), glycolysis from plasma glucose, glycogen synthesis, and whole-body carbohydrate and lipid oxidation were evaluated. Decreases in fasting plasma glucose (by approximately 1.7 mmol/l) and HbAlc (both P < 0.05) were observed in NIDDM subjects during treatment; plasma glucose was unchanged in control subjects. In the latter, the glucose infusion rate (GIR) required to maintain euglycemia (40.1 +/- 5.7 and 38.1 +/- 4.8 micromol / kg fat-free mass FFM / min) and glucose disposal (Rd) (41.7 +/- 5.7 and 38.9 +/-4.7 micromol / kg FFM / min were similar during placebo and vanadyl sulfate administration, respectively. Hepatic glucose output (HGO) was completely suppressed in both studies. In contrast, in NIDDM subjects, vanadyl sulfate increased GIR approximately 82% (17.3 +/- 4.7 to 30.9 +/- 2.7 micromol / kg FFM / min, P < 0.05); this improvement in insulin sensitivity was due to both augmented stimulation of Rd (26.0 +/-4.0 vs. 33.6 +/- 2.22 micromol / kg FFM / min, P < 0.05) and enhanced suppression of HGO (7.7 +/- 3.1 vs. 1.3 +/- 0.9 micromol / kg FFM / min, P < 0.05). Increased insulin-stimulated glycogen synthesis accounted for >80% of the increased Rd with vanadyl sulfate (P < 0.005), but plasma glucose flux via glycolysis was unchanged. In NIDDM subjects, vanadyl sulfate was also associated with greater suppression of plasma free fatty acids (FFAs) (P < 0.01) and lipid oxidation (P < 0.05) during clamps. The reduction in HGO and increase in Rd were both highly correlated with the decline in plasma FFA concentrations during the clamp period (P < 0.001). In conclusion, small oral doses of vanadyl sulfate do not alter insulin sensitivity in nondiabetic subjects, but it does improve both hepatic and skeletal muscle insulin sensitivity in NIDDM subjects in part by enhancing insulin's inhibitory effect on lipolysis. These data suggest that vanadyl sulfate may improve a defect in insulin signaling specific to NIDDM. PMID- 8621021 TI - Human obese gene: molecular screening in Japanese and Asian Indian NIDDM patients associated with obesity. AB - The mouse obese (ob) gene has recently been isolated through the positional cloning technique and has been proved to result in the obese and NIDDM phenotype in mice when mutated (Nature 372:425-432, 1994). More recently, it has been demonstrated, by experiments with recombinant ob protein, that ob gene product can cause mice, including ob/ob mice, diet-induced obesity mice, and normal mice, to lower their food intake and body weight (Science 269:540-549, 1995). To investigate the genetic and/or environmental influences underlying the development of NIDDM associated with obesity, we isolated and partially sequenced the human obese (OB) gene. The human OB gene isolated in this study encoded 167 amino acids and its open reading frame was revealed to be divided into two parts with an intermediate intron of approximately 2.4 kb. Using the single-strand conformation polymorphism (SSCP) technique, we screened Japanese and Asian Indian subjects for mutations in the protein coding regions of the OB gene. A total of 75 NIDDM patients with obesity (54 Japanese and 21 Asian Indians), 40 NIDDM patients without obesity (34 Japanese and 6 Asian Indians), and 34 Japanese patients with simple obesity showed no abnormal SSCP patterns in either component of the coding sequences. These results suggested that mutations in the coding regions of the OB gene are not likely to be commonly identifiable and that there would likely be a kind of obesity-associated NIDDM not caused by mutations of the OB gene. PMID- 8621022 TI - Absence of mutations in the human OB gene in obese/diabetic subjects. AB - The product of the obese (ob) gene, leptin, is a secreted protein that is important in the regulation of body weight. Mice with mutations in the ob gene are obese and diabetic and manifest reduced physical as well as metabolic activity. In this study, we tested the possibility that mutations in the OB gene may contribute to human obesity. We report the isolation and partial sequence of the human OB gene and the screening of 105 obese patients for mutations in the protein coding sequence using the technique of single-strand conformational polymorphism. No coding sequence polymorphism was found, suggesting that mutations in the coding sequence of the OB gene do not constitute a common cause of increased body weight in humans. We also identified a highly polymorphic simple dinucleotide repeat DNA polymorphism in this gene that will be useful for genetic studies. PMID- 8621023 TI - Association between insulin secretory pulse frequency and peripheral insulin action in NIDDM and normal subjects. AB - Abnormalities of both insulin secretion and insulin action occur in NIDDM. It is not clear, however, which is the primary defect. Recently, it has been suggested that the frequency of insulin pulses is an important factor regulating insulin action in normal humans. We examined the relationship between pulsatile insulin secretion and insulin action in eight NIDDM subjects and eight health matched control subjects. Insulin action was assessed prevailing fasting glucose levels before and after hype insulinemia (2-h insulin infusion at 2.0 mU / kg / min). Pulsatility of insulin was assessed by sampling every 2 min for 90 min after an overnight fast and identifying insulin pulses using the computer program Pulsar. Fasting plasma glucose and postabsorptive endogenous glucose production were both greater in diabetic subjects compared with control subjects (10.1 +/- 1.2 vs. 5.4 +/- 0.1 mmol/l, P < 0.01; 11.8 +/- 0.8 vs. 9.9 +/- 0.4 micromol / kg / min, P < 0.05). During the 2.0 mU insulin infusion, glucose clearance was lower in the diabetic subjects (3.6 +/- 0.7 vs. 6.9 +/- 0.5 ml / kg / min), P < 0.05), whereas endogenous glucose production was suppressed to a similar degree in both groups (4.5 +/- 0.8 vs. 3.6 +/- 0.7 micromol x kg(-1) x min(-1), NS). The frequency of insulin pulses and glucose clearance were negatively correlated in both diabetic subjects (r = -0.75, P < 0.05) and normal control subjects (r = -0.82, P < 0.01). This negative correlation was also present in both groups taken together (r = 0.72, P < 0.001). There was no correlation between insulin pulse frequency and endogenous glucose production either in the fasting state or during hyperinsulinemia. We concluded that the frequency of insulin pulses and peripheral insulin sensitivity are closely linked in NIDDM and normal subjects. PMID- 8621024 TI - Indication for linkage of the human OB gene region with extreme obesity. AB - Obesity is one of the most significant risk factors for hypertension, coronary heart disease, and NIDDM (Frayn KN, Coppack SW: Insulin resistance, adipose tissue and coronary heart disease. Clin Sci 82:1-8, 1992; Kaplan NM: The deadly quartet: upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension. Arch Intern Med 149:1514-1520, 1989). While family segregation, adoption, and twin studies have indicated that degree of adiposity has a significant genetic component (Stunkard AJ, Harris JR, Pedersen NL, McClearn GE: The body-mass index of twins who have been reared apart. N Engl J Med 322:1483 1487, 1990; Bouchard C, Despres J-P, Mauriege P: Genetic and nongenetic determinants of regional fat distribution. Endocr Rev 14:72-93, 1993), the genes and predisposing mutations remain poorly understood. This is in contrast to several well-defined genetic models for obesity in rodents, particularly the mouse obese (ob) gene, in which loss-of-function mutations cause severe obesity. Recent studies have demonstrated a substantial reduction in body fat when recombinant ob protein (leptin) is administered to mice. To test the relevance of these observations to human obesity, the location of the human homologue (OB) was established by radiation hybrid mapping and eight microsatellite markers spanning the OB gene region (7q3l.3) were genotyped in 101 obese French families. Affected sib-pair analyses for extreme obesity, defined by BMI >35 kg/m2, revealed suggestive evidence for linkage to three markers located within 2 cM of the OB gene (D7S514, D7S680, and D7S530). The OB gene is therefore a candidate for genetic predisposition to extreme obesity in a subset of these families. PMID- 8621025 TI - Extreme obesity may be linked to markers flanking the human OB gene. AB - Mice with mutations of the ob gene are extremely obese, and the human homologue (OB) has been cloned and physically mapped. The protein product of the ob gene (leptin) reduces body fat in mice when given exogenously, and leptin has been proposed to provide a lipostatic signal that regulates adiposity. Variation in the OB gene may be one genetically determined cause of obesity in human populations. To test this hypothesis, we genotyped siblings from 78 families at markers flanking the human OB gene. Pairs of siblings with extreme obesity (BMI > or = 40; n = 59) shared haplotypes identical-by-descent for the region containing the OB gene at greater than chance levels (corrected P = 0.04). Furthermore, one haplotype containing the OB gene was transmitted by heterozygous parents to extremely obese (BMI > or = 40) offspring more frequently than expected by chance, indicting significant allelic disequilibrium (corrected P = 0.027). One explanation for these linkage findings is that some individuals with extreme obesity have an allelic variant of the OB gene, although other nearby genes could contribute to obesity in these families. PMID- 8621027 TI - Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro. AB - This study was undertaken to investigate the changes in obesity (OB) gene expression and production of leptin in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions in humans. Three protocols were used: 1) euglycemic clamp with insulin infusion rates at 40, 120, 300, and 1,200 mU / m / min carried out for up to 5 h performed in 16 normal lean individuals, 30 obese individuals, and 31 patients with NIDDM; 2) 64-to 72-h hyperglycemic (glucose 12.6 mmol/l) clamp performed on 5 lean individuals; 3) long-term (96-h) primary culture of isolated abdominal adipocytes in the presence and absence of 100 nmol/l insulin. Short-term hyperinsulinemia in the range of 80 to > 10,000 microU/ml had no effect on circulating levels of leptin. During the prolonged hyperglycemic clamp, a rise in leptin was observed during the last 24 h of the study (P < 0.001). In the presence of insulin in vitro, OB gene expression increased at 72 h (P < 0.01), followed by an increase in leptin released to the medium (P < 0.001). In summary, insulin does not stimulate leptin production acutely; however, a long-term effect of insulin on leptin production could be demonstrated both in vivo and in vitro. These data suggest that insulin regulates OB gene expression and leptin production indirectly, probably through its trophic effect on adipocytes. PMID- 8621028 TI - Introduction to invited papers presented at the 1994 ADM Annual Meeting Southampton, Bermuda. PMID- 8621026 TI - Plasma leptin and insulin relationships in obese and nonobese humans. AB - Hyperinsulinemia. is associated with an overexpression of mRNA for the ob protein leptin in rodent models of genetic obesity, and insulin has been reported to directly stimulate leptin mRNA in rat adipocytes. Human obesity is also associated with increased leptin mRNA as well as plasma levels, but there have been no reports of the effect of insulin on leptin secretion. We, therefore, tested the hypothesis that insulin stimulates leptin secretion in humans. Using a newly developed leptin assay, immunoreactive leptin was measured in fasting and postprandial plasma samples from 27 healthy adults and in samples before and during euglycemic-hyperinsulinemic then stepped hypoglycemic (hourly steps at 85, 75, 65, 55, and 45 mg/dl) clamps from 10 healthy subjects and 11 patients with IDDM. Plasma leptin was correlated (r = 0.84, P = 0.0005) with BMI in obese but not nonobese subjects and with fasting (r = 0.75, P = 0.008) but not postprandial plasma insulin levels. (Leptin levels did not change postprandially.) Euglycemic hyperinsulinemia did not alter leptin levels, nor did hyperinsulinemic hypoglycemia. Thus, because circulating leptin levels are not increased during postprandial hyperinsulinemia or during euglycemic (or hypoglycemic) hyperinsulinemia, we conclude that, at least in the short term, insulin does not increase leptin secretion in humans and that hyperleptinemia in obese individuals is not likely the result of hyperinsulinemia. PMID- 8621029 TI - Perspectives on strength. AB - Strength values are often relied upon as indicators of structural performance for brittle dental materials. Strength, however, is more of a "conditional" than an inherent material property, and strength data alone cannot be directly extrapolated to predict structural performance. Strength data are meaningful when placed into context via knowledge of material microstructure, processing history, testing methodology, testing environment and failure mechanism(s). Structure failure is determined by additional failure probability variables (in concert with strength) that describe stress distributions, flaw size distributions, and that can account for either single or multiple failure modes. Lifetime predictions require additional information about the time dependence of slow crack growth. Basic fracture mechanics principles and Weibull failure modeling will be reviewed for the perspective they provide in understanding strength and the data obtained using various laboratory tests. Examples will be given to demonstrate how failed specimens can provide crucial information to either validate or question the failure mechanisms invoked during laboratory testing. The role of interfacial stresses is discussed as applied to dental structures of current interest. Overall, it is emphasized that an understanding of actual clinical failure modes is absolutely necessary before results of in vitro strength testing can be considered to have clinical validity. PMID- 8621030 TI - Fracture mechanics principles. AB - The principles of linear elastic fracture mechanics (LEFM) were developed in the 1950s by George Irwin (1957). This work was based on previous investigations of Griffith (1920) and Orowan (1944). Irwin (1957) demonstrated that a crack shape in a particular location with respect to the loading geometry had a stress intensity associated with it. He also demonstrated the equivalence between the stress intensity concept and the familiar Griffith criterion of failure. More importantly, he described the systematic and controlled evaluation of the toughness of a material. Toughness is defined as the resistance of a material to rapid crack propagation and can be characterized by one parameter, Kic. In contrast, the strength of a material is dependent on the size of the initiating crack present in that particular sample or component. The fracture toughness of a material is generally independent of the size of the initiating crack. The strength of any product is limited by the size of the cracks or defects during processing, production and handling. Thus, the application of fracture mechanics principles to dental biomaterials is invaluable in new material development, production control and failure analysis. This paper describes the most useful equations of fracture mechanics to be used in the failure analysis of dental biomaterials. PMID- 8621031 TI - Fractography: determining the sites of fracture initiation. AB - Fractography is the analysis of fracture surfaces. Here, it refers to quantitative fracture surface analysis (FSA) in the context of applying the principles of fracture mechanics to the topography observed on the fracture surface of brittle materials. The application of FSA is based on the principle that encoded on the fracture surface of brittle materials is the entire history of the fracture process. It is our task to develop the skills and knowledge to decode this information. There are several motivating factors for applying our knowledge of FSA. The first and foremost is that there is specific, quantitative information to be obtained from the fracture surface. This information includes the identification of the size and location of the fracture initiating crack or defect, the stress state at failure, the existence, or not, of local or global residual stress, the existence, or not, of stress corrosion and a knowledge of local processing anomalies which affect the fracture process. The second motivating factor is that the information is free. Once a material is tested to failure, the encoded information becomes available. If we decide to observe the features produced during fracture then we are rewarded with much information. If we decide to ignore the fracture surface, then we are left to guess and/or reason as to the cause of the failure without the benefit of all of the possible information available. This paper addresses the application of quantitative fracture surface analysis to basic research, material and product development, and "trouble-shooting" of in-service failures. First, the basic principles involved will be presented. Next, the methodology necessary to apply the principles will be presented. Finally, a summary of the presentation will be made showing the applicability to design and reliability. PMID- 8621032 TI - Adhesion testing of dentin bonding agents: a review. AB - Adhesion testing of dentin bonding agents was reviewed starting with the adhesion substrate, dentin, the variables involved in etching, priming and bonding, storage variables and testing variables. Several recent reports attempting to standardize many of these variables were discussed. Recent advances in the development of new bonding systems have resulted in bond strengths on the order of 20-30 MPa. At these high bond strengths, most of the bond failure modes have been cohesive in dentin. As this precludes measurement of interfacial bond strength, new testing methods must be developed. One such new method, a microtensile method, was described along with preliminary results that have been obtained. The last decade has produced major advances in dentin bonding. The next decade should prove to be even more exciting. PMID- 8621033 TI - Three-dimensional finite element analysis of the shear bond test. AB - OBJECTIVES: The purpose of this study was to use finite element analyses to model the planar shear bond test and to evaluate the effects of modulus values, bonding agent thickness, and loading conditions on the stress distribution in the dentin adjacent to the bonding agent-dentin interface. METHODS: All calculations were performed with the ANSYS finite element program. The planar shear bond test was modeled as a cylinder of resin-based composite bonded to a cylindrical dentin substrate. The effects of material, geometry and loading variables were determined primarily by use of a three-dimensional structural element. Several runs were also made using an axisymmetric element with harmonic loading and a plane strain element to determine whether two-dimensional analyses yield valid results. RESULTS: Stress calculations using three-dimensional finite element analyses confirmed the presence of large stress concentration effects for all stress components at the bonding agent-dentin interface near the application of the load. The maximum vertical shear stress generally occurs approximately 0.3 mm below the loading site and then decreases sharply in all directions. The stresses reach relatively uniform conditions within about 0.5 mm of the loading site and then increase again as the lower region of the interface is approached. Calculations using various loading conditions indicated that a wire-loop method of loading leads to smaller stress concentration effects, but a shear bond strength determined by dividing a failure load by the cross-sectional area grossly underestimates the true interfacial bond strength. SIGNIFICANCE: Most dental researchers are using tensile and shear bond tests to predict the effects of process and material variables on the clinical performance of bonding systems but no evidence has yet shown that bond strength is relevant to clinical performance. A critical factor in assessing the usefulness of bond tests is a thorough understanding of the stress states that cause failure in the bond test and then to assess whether these stress states also exist in the clinical situation. Finite element analyses can help to answer this question but much additional work is needed to identify the failure modes in service and to relate these failures to particular loading conditions. The present study represents only a first step in understanding the stress states in the planar shear bond test. PMID- 8621034 TI - Some effects of water on dentin bonding. AB - OBJECTIVES: The goals of this study were to determine how: 1) the bond strengths of hydroxyethylmethacrylate (HEMA)-based dentin primers are affected by different solvents (water or acetone), 2) the application time of these primers affect the bond strength, and 3) the conversion of water contaminated bonding resins. METHODS: HEMA (35%), mixed with water or acetone, was placed on moist dentin for 30 or 120 s, dried, covered with a bonding resin, and light-cured. Composite cylinders were bonded to these surfaces, and the shear bond strength was determined after 30 d of water storage at 37 degrees C. The conversion of bonding resins containing 0.05, 0.1, 0.2, 0.4 and 0.8 mL water per mL of bonding resin was determined with FTIR. RESULTS: The two acetone groups gave significantly (p<0.5) higher bond strength values (sigma 30s=22.2 +/- 2.1 MPa and sigma 120s=21.5 +/- 3.2 MPa) than the two water groups (sigma 30s=7.0 +/- 3.3 MPa and sigma 120s=16.2 +/- 4.8 MPa). In contrast to the acetone-based primer, the water based primer improved its bond strength with increased priming time without reaching that of the two acetone groups. The conversion of the bonding resin was 53.5%, which decreased to approximately 25% when 0.2 mL or more water was added per mL resin. SIGNIFICANCE: Compared to acetone, water is inferior as a solvent for HEMA-based dentin primers and gives both lower bond strength and requires longer priming time than acetone. A possible explanation of these results is the ability of water to interfere with the polymerization of the resin systems. PMID- 8621036 TI - Predicting lifetimes of materials and material structures. AB - The mechanical strength of brittle materials under stress is of prime importance in applications where allowable design stress, lifetime, and reliability are critical issues. A proper analysis should enable an engineer to select an allowable design stress that will permit a brittle component to function for the expected lifetime with an acceptable low probability of failure. It is the purpose of this paper to provide the background for assessing the mechanical reliability of brittle materials under tensile strength. PMID- 8621035 TI - In vitro fatigue behavior of restorative composites and glass ionomers. AB - OBJECTIVES: This in vitro study was conducted to investigate the fatigue behavior of several dental restoratives, including composites, glass ionomers and a resin reinforced glass ionomer. METHODS: Fatigue was imposed under a reverse stress controlled regimen, following a staircase approach. Samples were stored and tested under both dry and wet conditions. The following parameters were measured and analyzed: Young's modulus, restrained fracture strength, and flexural fatigue limit. RESULTS: As a general trend, all products showed a decrease in Young's modulus following water sorption. For all products except the resin-reinforced glass ionomer, the same trend was seen in the restrained fracture strength. This is, however, no longer valid for the flexural fatigue limit: the trend is steady state for the glass ionomers, status quo for the resin-reinforced glass ionomer, and all composites tested show a decrease. SIGNIFICANCE: The diversity in structure of both composites and glass ionomers does not allow findings for one product to be extrapolated to other similar products. PMID- 8621038 TI - Shear bond strength of four commercial bonding systems to cp Ti. AB - OBJECTIVES: The purpose of this study was to evaluate the bond strength of veneering composite to commercially pure titanium (cp Ti) using several different bonding systems and a post-cure heat treatment. METHODS: Four commercial bonding systems (Cesead, Kuraray; New Metacolor, Sun Medical; Silicaoater MD, Kulzer; Termoresin LC II, GC) were evaluated. Bonding was attempted with the opaque resin provided by each bonding system as well as with the New Metacolor opaque resin. New Metacolor resin composite was used for the veneering composite. Half of the specimens were subjected to a post-cure heat treatment at 100 degrees C for 30 min. The shear bond strengths were tested after aging the specimens in water at 37 degrees C for 1 d and also after thermocycling for 16.5 d (20,000 cycles). RESULTS: Strong bonds, exceeding 20 MPa, were achieved with all of the bonding systems with the exception of Thermoresin LC II, which is designed for noble metals. Bond strengths were only increased by the post-cure heat treatment for the New Metacolor system. Thermocycling caused a significant reduction in bond strength for the New Metacolor adn the Thermoresin LC II systems. The use of the New Metacolor opaque resin produced increased bonding for the Silicoater MD and the opaque resin produced increased bonding for the Silicoater MD and the Cesead systems, but the effect was eliminated after thermocycling. SIGNIFICANCE: Strong, durable bonds can be achieved between composite and sandblasted cp Ti, thus enhancing the usefulness of this metal for esthetic resin-veneered crowns and other fixed prosthetics. PMID- 8621037 TI - Critique of test methods for lifetime predictions. AB - Failure predictions for ceramics depend on the experimental parameters that measure the strength distribution (m and sigma 0) and the time-dependency of strength (n). These parameters can be determined by measuring strength as a function of stressing rate in a test environment that simulates the service environment. To minimize the uncertainty in these experimental parameters, at least 30 samples per stressing rate should be tested over a stressing rate range of at least 3 orders of magnitude. The uncertainty in these experimental parameters can be taken into account in design calculations by the use of appropriate safety factors. Thus, through well designed experiments coupled with a reliability analysis, rational design decisions can be made that ensure the successful use of ceramics in demanding structural applications. PMID- 8621040 TI - Porcelain bond to laser-welded titanium surfaces. AB - OBJECTIVES: The purpose of this investigation was to determine if laser-welding titanium had an effect on the bond strength of porcelain to metal. This is an important consideration since the metal copings for many multi-unit porcelain restorations are fabricated as single units and then either soldered or welded. METHODS: The bond strength of porcelain to laser-welded titanium was measured with a four-point bending test and the interface was examined using SEM/EDX analysis. RESULTS: The result showed no statistically significant differences (p<0.05) in bond strengths between laser-welded surfaces and mechanically machined surfaces and neither were there any alterations in composition identified after the welding. The use of a special bonding agent did not improve the bond strength significantly. SIGNIFICANCE: Fusing porcelain to laser-welded areas in titanium fixed restorations implies no deterioration of bond strength. PMID- 8621039 TI - Demonstration of a focused ion-beam cross-sectioning technique for ultrastructural examination of resin-dentin interfaces. AB - OBJECTIVES: focused ion-beam (FIB) etching, commonly used as a cross-sectioning technique for failure analysis of semiconductor devices, has recently been applied to biological tissues to expose their ultrastructure for examination. It was the aim of this investigation to determine the practical utility of FIB to cross-section resin-dentin interfaces in order to morphologically evaluate the completeness of resin penetration into the exposed collagen scaffold at the resin dentin bond interface. METHODS: Two representative commercially available dentin adhesive systems were bonded to mid-coronal dentin. After appropriate fixation and dehydration of the resin-bonded dentin samples, a scanned focused ion-beam of a few tens of nano-meters in diameter was used to cross=section the resin-dentin interface. Examination of the interfacial ultrastructure was accomplished using a field-emission SEM. RESULTS: Results indicate possible artifact production at the cross-sectioned interface, hiding its actual ultrastructure, probably due to a heat-effect with possible recrystallization. Further studies of FIB are needed to optimize its usefulness for resin-dentin interface examinations and other biological tissue applications. SIGNIFICANCE: Complete resin saturation of the demineralized dentin surface-layer has been claimed to be the key factor for a long-lasting resin-dentin bond. A "clean" artifact-free micro-cross-sectioning technique may provide indisputable ultra-structural information about the depth of resin penetration into the demineralized zone. Such a test would be useful in the development of dentin adhesive systems. PMID- 8621042 TI - Freeze-drying and scanning electron microscopy of setting dental gypsum. AB - OBJECTIVES: The initial and final forms of reactive gypsum products have been photomicrographed previously. However, the purpose of this project was to document the microscopic morphology of setting dental stone at various stages during the reaction. METHODS: Two dental products, a conventional (Type IV) die stone and a fast-setting (Type III) stone, were investigated. At selected times ranging from 1 min to 24 h after mechanically mixing the stone under vacuum, the conversion of calcium sulfate hemihydrate to a dihydrate was suspended by immersion into liquid nitrogen. Water was immediately removed by freeze-drying the specimen to prevent any further reaction so that the specimen could be returned to room temperature for examination in a scanning electron microscope (SEM). RESULTS: Crystal formation appeared to be nearly complete at the 20 min interval for the die stone and at the 10 min interval for the fast-setting dental stone. Transitions noted during these times include the nucleation and growth of small needle-like crystals on and near the larger prismatic-shaped hemihydrate crystals, the concurrent decrease in size and number of the hemihydrate crystals, and the progressive entanglement of the growing dihydrate crystals. SIGNIFICANCE: The two-step process of suspending the reaction, then freeze-drying the specimen made it possible to observe and document the intermediate stages during crystal growth of dental stone. These observations should be helpful in understanding the structural dynamics of crystal growth during the setting of gypsum dental products. This procedure should be applicable to the study of other water-based dental materials. PMID- 8621041 TI - Short-term fluoride release/uptake of glass ionomer restoratives. AB - OBJECTIVES: The short-term fluoride release/uptake of four glass ionomer restoratives was measured and compared to the fluoride release after exposure to three commercial fluoride gels. METHODS: Materials tested were: 1) Ketac-Fil (ESPE GmbH); 2) Ketac-Silver (ESPE GmbH); 3) Photac-Fil (ESPE GmbH); and 4) Fuji II LC (GC Corp.). Twenty discs of each material were fabricated and stored in deionized water at 37 degrees C. Initial fluoride release was measured at 24 h intervals for 7 d, and 24 h intervals at the end of each week for 5 wk. After 6 wk, samples were divided into groups and each group was treated for 6 min with one of the following commercial fluoride gels: acidulated phosphate fluoride (APF), neutral sodium fluoride (NaF), and stannous fluoride (SnF2). Control specimens were placed in deionized water. After exposure, fluoride measurements were carried out at 24 h intervals for 7 d, and 24 h intervals at the end of 2 wk. Fluoride exposure was repeated, and measurements were again recorded over 3 wk. RESULTS: Initial fluoride release by all materials was highest during the first 24 h and decreased sharply over the first week. After exposure to APF, fluoride release increased significantly for all materials. Exposure to NaF also resulted in increased fluoride release for all materials although it was not as high as the increase recorded after exposure to APF. Exposure to SnF2 did not result in significant fluoride release by any material. SIGNIFICANCE: The results of this study suggest that certain fluoride gels may replenish fluoride within some glass ionomers and thus prolong their cariostatic potential. PMID- 8621043 TI - Failure of ethylene oxide to sterilize extracted human teeth. AB - OBJECTIVES: The purpose of this study was to determine if ethylene oxide could sterilize extracted human teeth to be used in research. METHODS: An occlusal preparation was cut in freshly extracted molars and a small hole was drilled into the pulp chamber. A suspension of Bacillus subtilis (globigii) endospores, the standard biological monitor used for ethylene oxide sterilization, was injected into the pulp cavity, and the pulp cavity access was filled with composite material and sealed with a light-cured sealant. The teeth were exposed to either a 30 degrees C or 63 degrees C ethylene oxide sterilization process. Following exposure, the teeth were aseptically split and cultured to reveal viable spores. RESULTS: Sixty-four percent of the teeth exposed to "cold" ethylene oxide treatment and 80% of the teeth exposed to the "warm" treatment still contained viable spores. SIGNIFICANCE: Ethylene oxide cannot be relied on to sterilize extracted human teeth. Therefore, before they are used in research, other methods should be used to ensure killing of bloodborne pathogens that may be present within the teeth. PMID- 8621044 TI - Influence of filler addition to bonding agents on shear bond strength to bovine dentin. AB - OBJECTIVES: The purpose of this study was to investigate the influence of adding filler particles to a bonding agent on dentin bond strength and of the temperature change during curing in order to determine the optimum filler level for an experimental bonding agent. METHODS: Experimental light-cured bonding agents with microfiller (average size: 0.05 micrometers) content of 0, 10, 20, 30, 40, 50, 60, and 70 wt% were used with the Imperva Bond / Lite-Fil II A (Shofu) restorative material. Bovine incisors were mounted in self-cured resin, and the facial surfaces were prepared with 600-grit SiC paper. After dentin surface pretreatment with dentin primer, experimental bonding agents were applied to the dentin surface and bonded with resin composite. Ten samples per test group were stored in 37 degrees C water for 24 h, then shear tested at 1.0 mm/min. The temperature change of the bonding agent was monitored during the exthothermic polymerization reaction according to the method of ISO standard #4049. The peak temperature and the time required to reach peak temperature were recorded. RESULTS: Bond strength to dentin and the temperature change were greatly affected by the filler level. Maximum dentin bond strength (14.3 +/- 2.3 MPa) was obtained with a filler level of 10 wt% and decreased with filler level higher than 30 wt% (10.4 +/- 1.7 MPa - 5.3 +/- 2.6 MPa). Peak temperature decreased and the time required to reach peak temperature increased with the higher filler levels. There were strong correlations between the bond strength and temperature change of experimental bonding agents. SIGNIFICANCE: The initial setting behavior of bonding agents containing filler particles may be one of the important factors influencing dentin bond strength. When bonding agents with filler particles are used, it is important to determine if optimum filler levels exist in order to optimize the dentin bond strength. PMID- 8621045 TI - In vitro reaction of macrophages to metal ions from dental biomaterials. AB - OBJECTIVES: This study was conducted to 1) measure the sensitivity of human and mouse macrophages to metal ions which are released from dental biomaterials, 2) compare these sensitivities with those of other cell types in the oral cavity, and 3) determine if metal ions alter the metabolism and synthetic processes of these cells at lower concentrations than are required to lyse the cells. This information will help define the biological risks associated with the release of metal ions into the oral cavity. METHODS: Macrophages were exposed to a range of concentrations of Ag1+, Au3+, Cu2+, Hg2+, Ni2+, Pd2+, Pt4+, and Zn2+ for 24 h in cell culture. The concentrations which caused a 50% decrease in succinic dehydrogenase (SDH) activity, protein production, and lactate dehydrogenase (LDH) release were measured and compared with these values for fibroblasts and osteoblasts. RESULTS: Most metal ions caused alteration in SDH activity and protein production at lower concentrations than were required to induce LDH release. There were exceptions to this trend, and the differences were not always statistically significant. Furthermore, although the macrophages sometimes had statistically different sensitivities to metal ions than fibroblasts or osteoblasts, these differences were less than one order of magnitude. Macrophage response to the metal ions was highly dependent on the metal ion and the species of macrophage. SIGNIFICANCE: Macrophages react adversely to metal ions at similar concentrations as other cell types found in the oral cavity. Furthermore, the concentrations which affect cell metabolism and protein production are generally lower than those which lyse the cells. Thus, non-lethal concentrations of metal ions may alter the secretion of protein inflammatory mediators such as cytokines which direct the inflammatory response in tissues. PMID- 8621046 TI - In vitro evaluation of OCA wear resistance of posterior composites. AB - OBJECTIVES: Posterior composites undergo generalized (CFA) as well as localized (OCA) wear. The latter is caused by the contact of an antagonist cusp on the surface of the restoration. The purpose of this paper was to create OCA wear by means of a three-bodied wear device and also to develop a simple but reliable method for calculating loss of material in occlusal contact areas. METHODS: After repeated cycles of loading, the OCA wear loss was measured with a profilometer, and the worn surfaces were observed through scanning electron micrography. RESULTS: The results of this study have demonstrated significant differences in wear values among materials. These findings suggest that the difference in wear characteristics is derived from the mechanism by which the filler particle is bonded to the resin matrix. SIGNIFICANCE: If OCA wear is of sufficient magnitude, it can cause appreciable changes in functional occlusion. Thus, knowing the OCA wear resistance of various composites is a prerequisite for developing a new composite with improved longevity and better clinical performance. PMID- 8621047 TI - Mechanical longevity estimation model for post-and-core restorations. AB - OBJECTIVES: The aim of this study was to integrate existing knowledge of in vitro strength of post-and-cores and masticatory loading to arrive at longevity estimates for post-and-core restorations when subjected to clinically relevant loads. METHODS: A biomechanical model was developed to predict the in vivo longevity. This method was applied to direct post-and-core restorations with amalgam or composite cores. Both experimental laboratory strength values and theoretical clinical strength values were used in the model. The restorations made in the laboratory were assumed to be of a higher quality than clinically made restorations, due to factors such as ease of manipulation, absence of saliva, etc. Both a high and low level of average masticatory loading were considered. The model was used to estimate the probability of mechanical failure before 5 X 10(6) load cycles (5 to 15 years) for all combinations of load range and manufacturing quality. RESULTS: The calculated failure probability was effectively zero for most combinations except for a clinical quality core subjected to loads in the high range. There the probability of mechanical failure before 5 X 10(6) cycles was estimated to be 2 X 10(-5) for amalgam and 5 X 10(-5) for composite cores. These results agree with the overall observed clinical failure rate of about 1% per year for post-and-core restorations. SIGNIFICANCE: The mechanical properties of the post-and-core restorations were adequate for clinically relevant loading conditions. PMID- 8621048 TI - Effect of air-powder polishing on adhesion of bonding systems to tooth substrates. AB - OBJECTIVES: The air-powder polisher was introduced to provide a method to quickly remove stains and plaque. The purpose of this study was to evaluate what effect preparing teeth with an air-powder polisher (Air-flow 2, Shofu) has on bonding to enamel and dentin substrates. METHODS: The bonding systems used in this study were one-step etching/priming systems (KBT-5, Kanebo; and Clearfil Liner Bond II, Kuraray) and a two-step etching/priming system (Superbond D-Liner Plus, Sun Medical). Bovine enamel and dentin surfaces were prepared and finished with 600 grit silicon carbide paper. Then the surfaces were either not treated (control) or air-powder polished for 15 s. A 4-mm bonding area was demarcated, and the tooth substrates were bonded with the three bonding systems according to the manufacturers' instructions. Tensile bond strengths were measured 24 h after storage in water at 37 degrees C. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses were performed on air-powder polished specimens to determine changes in the surface morphology and detect residual material from the air-powder polisher. SEM and transmission electron microscopic (TEM) observations of the interface between the bonding resins and dentin were performed to detect variations in the hybrid layers of the control and treated specimens. RESULTS: The results indicated that air-powder polishing did not influence the tensile bond strength to enamel, but adversely affected the tensile bond strength to dentin for the one-step systems, but not for the two-step system. Changes to the enamel surface after air-powder polishing were minimal, but an increase in surface roughness and complete removal of the smear layer were observed on dentin. SEM and TEM observations of the resin/dentin interface suggested that the air-powder polisher might cause superficial maceration of the collagen fibers on the dentin surface. This change may have decreased the ability of the etching/priming agents and bonding resin to penetrate into the dentin, which would have resulted in a poor quality hybrid layer. SIGNIFICANCE: For those systems that contain only an acidic primer for dentin conditioning, the use of air-powder polishing for surface preparation prior to restoration of dentin margin cavities is contraindicated because of the loss of reliable bonding. PMID- 8621049 TI - Resin cure determination by polymerization shrinkage. AB - OBJECTIVES: This study was designed to develop a methodology that would measure the monomer conversion of dental restorative resins without the use of an infrared spectrometer. METHODS: Specimens were prepared from heat-cured TEGDMA/MMA copolymers consisting of 0, 20, 40, 50, 80, and 100 mol% TEGDMA. The pre- and post-cure volumes of bar-shaped specimens (45 X 7 X 1 mm) and thin films were measured. The cure of the thin specimens was determined using changes in resin volume as well as by an infrared spectroscopic method. The cure of thin specimens of a commercially available unfilled bonding resin was also determined using the shrinkage method and by conventional infrared spectroscopic methods. RESULTS: There was no difference (p=0.0617) between cure values of the commercial resin using either conventional IR methods or the shrinkage methods. For each copolymer combination of thin films, there was also no significant difference in the extent of cure between the IR method used and the shrinkage method presented (lowest p-value 9.29). The cure of TEGDMA/MMA copolymer bar specimens demonstrated a decreasing trend of cure with increasing proportion of TEGDMA, and had cure values which were similar to others reported in the literature. SIGNIFICANCE: The evidence from this study demonstrated that the specimen volume method can be used to determine the extent of resin cure of thin film or bulk resin specimens. This method represents an easily performed procedure that can be used by all investigators without expensive equipment. PMID- 8621050 TI - SEM observations on stress corrosion cracking of commercially pure titanium in a topical fluoride solution. AB - OBJECTIVES. The purpose of the present study was to determine whether commercially pure titanium is susceptible to stress corrosion cracking/hydrogen embrittlement in a topical fluoride solution used in preventive dentistry. METHODS: Thin electropolished titanium test specimens were previously cold-rolled or cold-rolled and annealed before testing. For the stress corrosion tests, the U shaped specimens of both treatment types were stressed into a radius of curvature of 30 mm. Then, the bent part was placed in the fluoride solution at 37 degrees C for 1, 5, 10, and 20 d. The effects of the fluoride solution on cold-rolled and annealed titanium were studied using a scanning electron microscope. In addition, mechanically fractured surfaces of cold-rolled titanium specimens exposed and not exposed to the fluoride solution were examined by SEM. A qualitative evaluation of the surfaces was conducted. RESULTS: Narrow cracks were observed in cold rolled specimens following exposure to the fluoride solution for 5 d. The cracks were associated with branching, a characteristic of stress corrosion cracking. The cold-rolled specimen exposed to the fluoride solution exhibited a brittle fracture. In contrast, the fracture mode of the unexposed specimen was ductile in nature. SIGNIFICANCE: Topical fluoride solutions can cause stress corrosion cracking of commercially pure titanium. PMID- 8621051 TI - Cyclic fatigue of a model feldspathic porcelain. AB - OBJECTIVES: This study was conducted to evaluate the fatigue parameters of a model porcelain based on the Weinstein patent using cyclic fatigue and to compare the parametric values obtained from cyclic fatigue tests with those from dynamic fatigue tests previously reported by Fairhurst et al. (1993). METHODS: Cyclical biaxial flexure of 1 mm thick and 12 mm diameter disks was performed at 37 degrees C in distilled water at a frequency of 4 Hz with constant stressing rates between a minimum and maximum stress. Three groups of samples (50, 40, 40) were tested with a maximum stress of 51, 47, and 43 MPa, respectively. The crack growth exponent, n, and the scaling constant, sigma fo, were derived from the regression constants obtained from a linear regression of the logarithm of the median time to failure with the logarithm of the maximum stress. RESULTS: No significant differences were found between the cyclic fatigue parameters, n and sigma fo, derived from the median time to failure and those obtained from dynamic fatigue data. SIGNIFICANCE: Within the limits of error in this determination, the median cyclic fatigue life can be estimated by the use of fatigue parameters obtained from dynamic fatigue testing. PMID- 8621052 TI - The physical properties of a gallium alloy restorative material. AB - OBJECTIVES: This study was conducted to compare the physical properties of a gallium alloy restorative material and a widely used mercury-containing dental amalgam. METHODS: Although the specimens were not prepared according to ISO specifications, specimens of both materials were subjected to ISO standard (ISO 1559; 1986) tests. The results were analyzed by ANOVA, Mann-Whitney U test and Student's t-test. RESULTS: The gallium alloy showed setting expansion which was greater than that for the other amalgam and greater than the upper limit set by the ISO standard. For other properties, gallium alloy performed as well as, and in the case of creep, was superior to that of the amalgam. SIGNIFICANCE: The gallium alloy proved difficult to manipulate even when using the PTFE coated instruments recommended by the manufacturer. It is suggested that while the material may prove to be a viable alternative to conventional dental amalgam, a considerable improvement in its handling characteristics would be required before it would gain widespread acceptance for clinical use. PMID- 8621053 TI - Review of dental materials citations: Part A, January to June 1995. AB - OBJECTIVE: Electronic databases are an excellent resource for identifying relevant references for research and education projects. However, these databases are not yet a substitute for direct inspection of the literature because: (a) there still is a lag of many months between journal issue publication and updates of the database, and (b) most databases selectively report a portion of the entire literature. The objective was to identify and categorize all the dental materials citations in biomedical journals that were published from January 1995 through June 1995. METHODS: Seventeen primary and 56 secondary dental journals were manually searched via their tables of contents to detect dental materials publications from January to June of 1995. Dental materials citations were categorized into 17 major sections and further divided into several subsections. The review excluded case reports and literature primarily related to dental implants or biomedical materials outside of dentistry. RESULTS: Three hundred sixty-nine citations were identified. The greatest number of citations were associated with dentin bonding, composites, and glass ionomers. There was no significant increase in dental materials publications vs. the number reported for 1994. SIGNIFICANCE: This list provides a comprehensive source for review by academicians and researchers to bridge the gap between initial publication and electronic citation. PMID- 8621054 TI - Absorption, metabolism, and transport of carotenoids. AB - Carotenoids are currently under intense scrutiny regarding their potential to modulate chronic disease risk and prevent vitamin A deficiency, and renewed emphasis has been placed on achieving a better understanding of the metabolic fate of these compounds in humans. The development of new animal models, and use of human metabolic studies and stable tracer methods have greatly improved our knowledge of how carotenoids are absorbed, metabolized, and transported to tissues; however, many important issues remain unresolved. For example, intestinal uptake of carotenoids occurs by passive diffusion, but the lumenal or intracellular factors limiting this process are obscure. The intestinal mucosa plays a key role in the metabolism of provitamin A carotenoids such as beta carotene, thus greatly influencing their bioavailability. Most recent evidence supports a central oxidation mechanism of cleavage of beta-carotene to retinal in the intestinal mucosa, but the extent and site(s) of postabsorptive metabolism in the human is unknown. While the human and other species clearly absorb non provitamin A carotenoids, little is known of the extent and pathways of their metabolism and elimination. The metabolic fate of cis isomers of beta-carotene is a subject of recent interest, since 9-cis retinoic acid can apparently be formed from 9-cis beta-carotene in vitro and in vivo. Substantial cis-trans isomerization of at least small oral doses of 9-cis beta-carotene occurs in the human, although the site of isomerization is not yet known. Carotenoids are transported in plasma exclusively by lipoproteins, with the distribution among lipoprotein classes determined in large part by the physical properties of the carotenoid. The consequences of differential distribution in terms of tissue uptake and retention are not clear at present. Improved knowledge of the metabolic fate of carotenoids will assist in the development and testing of hypotheses regarding their potential to influence biological processes in the human. PMID- 8621055 TI - Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal. AB - The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4). PMID- 8621056 TI - Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability. AB - The rentry of hepatocytes and nonparenchymal cells from the normal quiescent G0 phase into the cell cycle during liver regeneration after 70% partial hepatectomy results in the discrete modulation of mRNA transcripts for many different genes. The modulation of steady-state levels of transcripts for genes involved in hepatocyte growth and replication during liver regeneration indicates that gene expression is regulated not only transcriptionally but also posttranscriptionally. In fact, posttranscriptional control appears to be the primary mechanism of regulating gene expression after the first 3 h after partial hepatectomy. Alteration in transcript stability is a key posttranscriptional regulatory mechanism used by the regenerating liver to modulate the steady-state transcript levels of multiple genes. Even genes that are transcriptionally activated during liver regeneration exhibit posttranscriptional control at the level of transcript stability. Moreover, the abundance of mRNA binding proteins, as well as translational activity and rate of poly(A) tail removal, are modulated and appear to influence transcript stability during liver regeneration. However, alteration of transcript stability is not the sole posttranscriptional mechanism regulating steady-state levels. Posttranscriptional control also occurs at the level of alternative splicing, stabilization of heterogeneous nuclear (hn) RNA, and hnRNA processing. Moreover, the role of nucleocytoplasmic transport of mature mRNA during liver regeneration is still undefined. Thus, during liver regeneration gene expression is regulated at multiple levels after the initial synthesis of hnRNA. By understanding the role of posttranscriptional mechanisms in regulating steady-state transcript levels in an in vivo model of normal growth, we will begin to appreciate its role in the genesis of abnormal growth. PMID- 8621057 TI - Cell-surface engineering with GPI-anchored proteins. AB - Protein engineering of cell surfaces is a potentially powerful technology through which the surface protein composition of cells can be manipulated without gene transfer. This technology exploits the fact that proteins that are anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, incorporate into their surface membranes and are fully functional. By substituting 3'-mRNA end sequence of naturally GPI-anchored proteins (i.e., a sequence that contains the signals that direct GPI anchoring) for endogenous 3' mRNA end sequence, virtually any protein of interest can be expressed as a GPI anchored derivative. The GPI-anchored product then can be purified from transfectants and the purified protein used to "paint" any target cell. Such protein engineering or "painting" of the cell surface offers several advantages over conventional gene transfer. Among these advantages are that 1) GPI-anchored proteins can be painted onto cells that are difficult to transfect, 2) cells can be altered immediately without previous culturing, 3) the amount of protein added to the surface can be precisely controlled, and 4) multiple GPI-anchored proteins can be sequentially or concurrently inserted into the same cells. Emerging applications for the technology include its use for the analysis of complex cell surface interactions, the engineering of antigen presenting cells, the development of cancer vaccines, and possibly the protection against graft rejection. PMID- 8621058 TI - The role of proteases during apoptosis. AB - The importance of proteases during apoptosis is becoming increasingly apparent. Because apoptosis contributes to a diverse variety of disease processes, understanding the roles played by proteases and their inhibitors might provide insight into the pathogenesis of these conditions and suggest novel therapeutic strategies. In this review, we discuss the involvement and role of specific proteases, substrates, and protease inhibitors that appear to participate in the apoptotic process. PMID- 8621059 TI - Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. AB - This review focuses on the extracellular proteoglycans. Special emphasis is placed on the structural features of their protein cores, their gene organization, and their transcriptional control. A simplified nomenclature comprising two broad groups of extracellular proteoglycans is offered: the small leucine-rich proteoglycans or SLRPs, pronounced "slurps, " and the modular proteoglycans. The first group encompasses at least five distinct members of a gene family characterized by a central domain composed of leucine-rich repeats flanked by two cysteine-rich regions. The second group consists of those proteoglycans whose unifying feature is the assembly of various protein modules in a relatively elongated and often highly glycosylated structure. This group is quite heterogeneous and includes a distinct family of proteoglycans, the "hyalectans," that bind hyaluronan and contain a C-type lectin motif that is likely to bind carbohydrates, and a less distinct group that contains structural homologies but lacks hyaluronan-binding properties or lectin-like domains. PMID- 8621060 TI - The vascular protective effects of estrogen. AB - There is now strong epidemiological evidence that estrogen replacement therapy has a protective effect in postmenopausal women. The cardiovascular protective action of estrogen is reported to be mediated indirectly by an effect on lipoprotein metabolism and by a direct effect on the vessel wall itself. Estrogen is active both in vascular smooth muscle and endothelium. Functionally competent estrogen receptors have been identified in vascular smooth muscle cells, and specific binding sites have been demonstrated in endothelium. Estrogen administration promotes vasodilation both in human and experimental animals, in part by stimulating] prostacyclin and nitric oxide synthesis. Both the prostaglandin synthase and the constitutive nitric oxide synthase were recently reported to be induced by estrogen treatment. In vitro, estrogen exerts a direct inhibitory effect on the smooth muscle by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, estradiol 17 beta prevents neointimal thickening after balloon injury and in rabbit cardiac transplant allografts. These data are consistent with in vitro studies wherein estrogen inhibits [3H]thymidine uptake by arterial segments from porcine coronary artery as well as proliferation of rabbit aortic vascular smooth muscle cells induced by hyperlipedemic serum. Recent studies have also reported an effect of estrogen on directed vascular smooth muscle cell migration. Furthermore, like other steroids, the effect of estrogen on the vessel wall has a rapid nongenomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression. The nature of these estrogen response genes in the vessel wall and their relation to vasodilation and antiproliferation remain to be determined. PMID- 8621061 TI - The Ras superfamily of GTPases. AB - The Ras superfamily of small GTPases comprises a group of molecular switches that regulate an astonishing diversity of cellular functions. A deep understanding of mitogenesis, cytoskeletal organization, vesicle traffic, and nuclear transport now requires the inclusion of the small GTPases as essential components of the molecular machines that drive these processes. The rich complexity of the control mechanisms involved is evidenced by the recent discoveries of GTPase cascades, multiple downstream effectors, and interconnected networks of GTPase-regulated protein kinase cascades. The 1995 FASEB Summer Conference at Snowmass Village, Colorado, on the Ras GTPase superfamily provided testimony to the broad impact that the study of these proteins continues to exert on cell biology. PMID- 8621062 TI - Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. AB - We have recently shown that mechanical stress activates a phosphorylation cascade of protein kinases including Raf-1 and the extracellular signal-regulated kinases (ERKs) in cultured cardiac myocytes partially through the enhanced secretion of angiotensin II. Osmotic stress in budding yeast has been shown to activate similar signaling molecules including Hog-1, a distant relative of the ERK family. In the present study, we examined whether mechanical stretch of cardiac myocytes activates the stress-activated protein kinases (SAPKs)/c-Jun NH2 terminal kinase, the mammalian homologs of yeast Hog-1 that regulate gene expression through activation of the transcription factor, AP-1. When cardiac myocytes of neonatal rats cultured on a deformable silicone dish were stretched, activity of SAPKs was increased from 10 min, peaked at 30 min, and gradually decreased thereafter. The increase in activity of SAPKs was proportional to the stretch. Unlike ERKs, the activation of SAPKs by stretching cardiac myocytes was not dependent on the secreted angiotensin II. The chelation of extracellular Ca2+ or down-regulation of protein kinase C did not attenuate activation of SAPKs by stretch. Transfection experiments using an AP-1 binding site-containing reporter gene revealed that stretch increases AP-1 activity in cardiac myocytes. In conclusion, like osmotic stress in yeast, mechanical stretch activates SAPKs in cardiac myocytes without the participation of angiotensin II. These results suggest that the activation of SAPKs may regulate gene expression during mechanical stress-induced cardiac hypertrophy. PMID- 8621063 TI - Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase. AB - The Tec family is a recently emerging subfamily among nonreceptor type protein tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt. They have a long amino-terminal unique region containing a pleckstrin homology domain and a Tec-homology domain. We could previously show that, through the Tec homology domain, Tec is bound to Lyn kinase both in vitro and in vivo. Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec-Lyn association. Here we demonstrate that Lyn can phosphorylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts. However, coexpression of Tec has little effect on the phospho-tyrosine-contents of Lyn. By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo. From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways. This is a novel case where one PTK is phosphorylated and regulated by another. PMID- 8621065 TI - Functional glutamate receptors in a subpopulation of anterior pituitary cells. AB - We have studied the effects of glutamate receptor agonists on the cytosolic Ca2+ concentration ([Ca/+]i) of single rat anterior pituitary (AP) cells. Ionotropic (NMDA and kainate/AMPA) and, to a smaller extent, metabotropic glutamate receptors were both present in all the five AP cell types, defined by the hormone they store. Cells within all the types responded also to thyrotropin-releasing hormone (TRH). Alternative typing by the response to four well-established hypothalamic releasing hormones (HRHs), GHRH, GnRH, CRH, and TRH, was performed. One-third of the cells were not sensitive to any HRH, another third were sensitive to only one HRH, and the last third were sensitive to more than one HRH, frequently to all four. Only the cells responding to TRH showed functional glutamate receptors. Superimposed to the above association, the strongest responses to glutamate were found in the cells responsive to multiple HRHs. These results suggest that glutamate may act, by a nonsynaptic mechanism, as a new releasing factor for one or, like TRH, several AP hormones. Coexpression of glutamate and TRH receptors in the subpopulation of cells responsive to multiple HRHs might have a functional meaning, perhaps related to phenotypic plasticity and long-term regulation of hormone secretion by the anterior pituitary. PMID- 8621064 TI - Partial night sleep deprivation reduces natural killer and cellular immune responses in humans. AB - Prolonged and severe sleep deprivation is associated with alterations of natural and cellular immune function. To determine whether alterations of immune function also occur after even a modest loss of sleep, the effects of early-night partial sleep deprivation on circulating numbers of white blood cells, natural killer (NK) cell number and cytotoxicity, lymphokine-activated killer (LAK) cell number and activity, and stimulated interleukin-2 (IL-2) production were studied in 42 medically and psychiatrically healthy male volunteers. After a night of sleep deprivation between 10 P.M. and 3 A.M., a reduction of natural immune responses as measured by NK cell activity, NK activity per number of NK cells, LAK activity, and LAK activity per number of LAK precursors (CD16,56, CD25) was found. In addition, concanavalin A-stimulated IL-2 production was suppressed after sleep deprivation due to changes in both adherent and nonadherent cell populations. After a night of recovery sleep, NK activity returned to baseline levels and IL-2 production remained suppressed. These data implicate sleep in the modulation of immunity and demonstrate that even a modest disturbance of sleep produces a reduction of natural immune responses and T cell cytokine production. PMID- 8621066 TI - Atherosclerotic heart disease and cancer: looking for the "smoking guns". PMID- 8621067 TI - GCN4 eukaryotic transcription factor/FokI endonuclease-mediated 'Achilles' heel cleavage': quantitative study of protein-DNA interaction. AB - Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site. This is a novel version of the 'Achilles' heel cleavage' (AC) technique [Koob et al., Science 241 (1988) 1084-1086]. Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact. Thus, the same GCN4 site can be used in consecutive cleavage reactions. This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction. Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C. In addition, conditions for controlled, partial GCN4 IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes. PMID- 8621068 TI - Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS. AB - From Azospirillum lipoferum (Al) FS, a nitrogen-fixing bacterium isolated from the rhizosphere of rice, we cloned and sequenced draT, encoding dinitrogenase reductase ADP-ribosyltransferase, and draG, encoding dinitrogenase reductase activating glycohydrolase. The nucleotide sequences of draTG showed extensive similarity to the same genes from Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus, and they are assumed to be co-transcribed as a single operon. When this draTG operon was introduced into Klebsiella oxytoca, this organism acquired the ability to respond to extracellular NH(+4) ions with reversible inhibition of nitrogenase activity, similar to that seen in Al FS. We constructed a plasmid containing a draT::lacZ gene fusion and found that beta galactosidase activity was detected under microaerobic conditions, regardless of NH(+4) concentration, but not under aerobic conditions. This indicates that the transcription of draTG responds to the level of oxygen, but not to that of NH(+4) ions. PMID- 8621069 TI - A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae. AB - An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK). PMID- 8621070 TI - Schizosaccharomyces pombe rad23 is allelic with swi10, a mating-type switching/radioresistance gene that shares sequence homology with human and mouse ERCC1. AB - Schizosaccharomyces pombe (Sp) rad23-1 mutant cells are extremely sensitive to UV light and ionizing radiation. A genomic DNA fragment that contains wild-type (wt) rad23 has been cloned. The DNA sequence of this cloned gene has been determined and was found to be identical to the previously characterized mating-type switching/radioresistance gene, swi10. Complementation tests between rad23-1 and swi10-154 mutant cells exclusively produce UV-sensitive progeny and confirm that these two genes are allelic. The DNA sequences of rad23-1 and swi10-154 reveal that each contains a single, unique point mutation. In rad23-1, Glu231 changes to a stop codon, resulting in the production of a truncated protein. In swi10-154, a G to A transition mutation is within a splice consensus sequence for intron 1. Therefore, the corresponding mRNA is incapable of being processed appropriately. PMID- 8621071 TI - An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina. AB - To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa)that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYR1. Restriction-fragment length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes. PMID- 8621072 TI - Isolation and sequence analysis of Clpg1, a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum. AB - Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race beta and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed on ORF encoding a 363-amino-acid (aa) polypeptide beginning with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5' non coding region. This genomic clone was thereafter designated Clpg1. Southern analysis, performed with a Clpg1-specific probe, showed that this gene is present as a single copy in the Cl genome. PMID- 8621073 TI - Isolation and characterization of two novel genes expressed in germinating conidia of the obligate biotroph Erysiphe graminis f.sp. hordei. AB - A cDNA library was constructed from germinating conidia of the obligate biotrophic fungus, Erysiphe graminis DC ex Merat f.sp. hordei Em. Marchal (Egh). Subtractive hybridization and differential screening were carried out. Two cDNA clones, cEgh7 and cEgh16, which were highly expressed in germinating conidia, but not in ungerminated conidia, were selected for further characterization. The corresponding genomic sequences, gEgh7 and gEgh16, were isolated from a cosmid library and sequenced. The gEgh7 gene contains an open reading frame (ORF) that codes for a 249-amino-acid (aa), Pro-rich polypeptide with a repeated primary structure. Expression studies in planta indicated that gEgh7 may have a function in the development and maturation of conidia. The ORF of gEgh16 is interrupted by two introns of 91 and 119 bp. It encodes a 251-aa polypeptide of unknown function. This gene belongs to a multigene family and is expressed during all developmental stages of Egh in planta and may be associated with hyphal growth. PMID- 8621075 TI - Construction of mini-F plasmid vectors for plasmid shuffling in Escherichia coli. AB - We have constructed two Escherichia coli mini-F plasmid vectors, pJK286 and pJK289, which have unique EcoRI, BamHI and HindIII sites downstream from the lac promoter. The mini-F vectors are useful for plasmid shuffling, with which we can efficiently carry out localized mutagenesis. PMID- 8621074 TI - Cloning the gene encoding Schistosoma mansoni p50, an immunophilin. AB - A 2.2-kb fragment of genomic DNA encoding Schistosoma mansoni immunophilin p50 (Smp50) was identified on a 14-kb genomic clone. The sequence of Smp50 reveals seven exons interrupted by six small introns ranging from 28-35 bp in size. The transcription start point, defined by primer extension analysis of schistosome RNA, begins at 30 bp upstream from the start AUG codon. Smp50 lacks a TATA box and appears to be a single-copy gene. PMID- 8621076 TI - Construction of beta-lactamase-encoding ApR gene cassettes for rapid identification of cloned genes. AB - We have constructed two Escherichia coli plasmids, pYK18 and pYK19, from which the BamHI, SmaI or EcoRI DNA fragments containing the ApR gene, conferring resistance to ampicillin, can be excised. The ApR cassettes have an annealing site for the sequencing primer of pUC plasmids at each end. Therefore, when the cassette is inserted into a gene, we can determine the nucleotide sequence of the gene from the insertion site using the sequencing primers of the pUC plasmids. This method is useful for identifying a cloned gene. PMID- 8621077 TI - Introducing StuI sites improves vectors for the expression of fusion proteins with factor Xa cleavage sites. AB - The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins. Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained. PMID- 8621079 TI - Nucleotide sequence of the Rhodobacter capsulatus hemH gene. AB - The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence. PMID- 8621078 TI - Construction of an alkaline phosphatase fusion-generating transposon, mTn10phoA. AB - We constructed a derivative of the mini-transposon mTn10 that generates translational fusions to the phoA gene from Escherichia coli and carries the KmR determinant from Tn5. This new transposon, mTn10phoA, is carried on a mobilizable plasmid with both selectable and counterselectable markers. The plasmid carrying mTn10phoA was introduced into Legionella pneumophila. Southern hybridization analysis indicated that the mTn10phoA insertions were randomly distributed. PMID- 8621080 TI - Sequence of a Lactococcus lactis DNA fragment homologous to the recF gene of Bacillus subtilis. AB - The recF gene of Lactococcus lactis ATCC 7962 is located 3 kb downstream from the lacZ gene and is transcribed in the opposite orientation. The recF gene is immediately preceded by a 121-codon ORF, and both recF and orf121 may be transcribed from the same promoter. The deduced RecF amino-acid sequence shows high homology to that of the Bacillus subtilis and Streptococcus pyogenes RecF proteins. PMID- 8621081 TI - The Schizosaccharomyces pombe spqM gene is a new member of the Qm transcription factor family. AB - The Qm family of proteins, which are found in a wide variety of species such as budding yeast, plants and humans, are believed to play a role in gene expression. Here, we report the isolation ofaa gene, spqM, from the fission yeast Schizosaccharomyces pombe, whose deduced amino-acid sequence shared 71.6 to 61.36% identity with members of the Qm family. The high degree of conservation of the Qm members suggest that they were selectively conserved, because of an important biological role. PMID- 8621082 TI - Cloning and sequence of the Ascobolus immersus S-adenosyl-L-methionine synthetase encoding gene. AB - The structural gene encoding S-adenosyl-L-methionine synthetase (SAM-S) in the fungus Ascobolus immersus has been cloned and sequenced. It contains a 1179-bp ORF, interrupted by three introns, encoding a 393-amino-acid protein (42 978 Da) that is 90% homologous to the SAM-S of the filamentous fungus Neurospora crassa, indicating that these fungi are closely related species. PMID- 8621083 TI - Overproduction of mycobacterial ribosomal protein S13 induces catalase/peroxidase activity and hypersensitivity to isoniazid in Mycobacterium smegmatis. AB - A Bacillus Calmette Guerlin (BCG) DNA fragment was identified which conferred hypersensitivity to isoniazid (INH) upon Mycobacterium smegmatis (Ms) when present on a multicopy plasmid. The gene cluster present on this fragment contains the genes encoding ribosomal proteins L36 (rpmJ), S13 (rpsM), S11 (rpsK) and S4 (rpsD), as well as the gene encoding initiation factor-1 (infA), an open reading frame of unknown function (ORFX) and a putative promoter region. The rpsM gene, from either BCG or Ms is necessary and sufficient to produce the INH hypersensitive phenotype in Ms, but the gene cluster has no effect on INH sensitivity when introduced into BCG on a multicopy plasmid. The presence of rpsM on a multicopy plasmid also causes an increase in catalase/peroxidase (Kat/Prx) activity in Ms. The overproduction of S13 may induce a stress response, resulting in increased expression of katG (encoding Kat/Prx) in Ms, thereby causing hypersensitivity to INH. PMID- 8621084 TI - Expression of the mitochondrial RNase P RNA subunit-encoding gene from a variant promoter sequence in Saccharomyces cerevisiae. AB - Ribonuclease P (RNase P) is a common tRNA processing enzyme that removes the 5' leader sequence of precursor tRNAs. This activity is identified in yeast mitochondria as a separate enzyme from the nuclear RNase P. Like other RNase P enzymes, the mitochondrial (mt) RNase P is also a ribonucleoprotein composed of both RNA and protein subunits. The RNA subunit is encoded by a mt gene and the protein subunit is supplied by a nuclear gene. Earlier studies described one active promoter (FP1) located 5' to the mt tRNA(fMet)-RNase P RNA-tRNA(Pro) gene cluster, so that the mitochondrially encoded RNA subunit was thought to be co transcribed with two of its substrate tRNAs. However, the results of in vitro transcription and primer extension experiments presented here demonstrate that the mt RNase P RNA subunit-encoding gene (RPM1) is transcribed from a new promoter (SP)which is located between the tRNA(fMet) and RPM1 genes. The sequence [5'-TATAAGAA(+1)] of the new promoter varies from the conserved promoter sequence [5'-TATAAGTA(+1)], but is one of the sequences that is active in the in vitro transcription assay to determine the consensus promoter sequence [5'-T A T/a A A/g/c G T/a/c N(+1)]. This result demonstrates that a naturally occurring variant promoter is used by RPM1. Identification of the novel SP promoter suggests that the synthesis of the mt RNase P RNA subunit might be uncoupled from the expression of upstream tRNA(fMet) gene, and that RPM1 might be independently transcribed in Saccharomyces cerevisiae. PMID- 8621085 TI - A cluster of genes encoding major isozymes of lignin peroxidase and manganese peroxidase from the white-rot fungus Trametes versicolor. AB - A gene cluster from the white-rot basidiomycete Trametes (Coriolus) versicolor (Tv) PRL 572 containing three structural genes, LPGIII, LPGIV and MPGI, was characterized. The genes are arranged in the same transcriptional direction, within a 10-kb region, and found to encode quantitatively dominant isozymes of lignin peroxidase (LP) and manganese peroxidase (MP). The second gene in sequence, LPGIV, predicts a 346-amino-acid (aa) mature polypeptide (36.9 kDa, pI 4.31) which is identical with the partial aa sequence information available on the LP12 isozyme (43.1 kDa, pI 3.27). The first gene, LPGIII, encodes a 341-aa polypeptide (36.1 kDa, pI 3.93) which has not been identified at the protein level. However, the similarity of LPGIV would suggest that the predicted product is an LP-type enzyme. LPGIII and LPGIV are homologous to the tandemly arranged genes LPGII and LPGI, respectively, recently described by Jonsson and Nyman [Biochim. Biophys. Acta 1218 (1994) 408-412]. The homologous genes, LPGIII/LPGII and LPGIV/LPGI, are 99% and 96% identical in sequence, respectively, and are predicted to encode identical polypeptides, since base substitutions in the predicted exons are all synonymous. The third gene, MPGI, is different in intron exon organization and predicted to be disrupted by five rather than six introns, as are the LP genes. The deduced polypeptide, 339 aa in size (35.9 kDa, pI 4.07), is identical with the partial aa sequence information available for isozyme MP2 (44.5 kDa, pI 3.09). The MPGI- and LPGIV-encoded polypeptides are 70% identical in sequence which suggests that MP and LP from Tv may be regarded as members of the same family within the plant peroxidase superfamily. Most importantly, this study identifies a gene encoding the MP2 isozyme, and further shows that genes encoding MP and LP can be closely linked on the chromosome and may be coordinately transcribed. PMID- 8621086 TI - Insert selection by BamHI methyltransferase protection in P1 phage-based cloning. AB - A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 10(6) transformants per microgram vector, depending on vector concentration. Cloning of BglII fragments of gamma DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per microgram vector. Plasmids recovered from these clones were all found to contain a gamma BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHi digestion. PMID- 8621087 TI - In vivo intermolecular recombination in Escherichia coli: application to plasmid constructions. AB - Repair of a double-strand break (DSB) was investigated by intermolecular recombination in Escherichia coli (Ec) recBC sbcBC cells with restriction enzyme cleaved model plasmids. Circular plasmids were generated when a linearized plasmid (vector) containing an origin of replication was co-transformed with a DNA fragment (template) containing a homologous sequence. The influence of the position of the DSB in the vector was analyzed using templates which contain various genetic markers, non-homologous sequences and/or deletions relative to the vector. In all cases, when a DSB occurs within a marker, this marker is lost in the resulting plasmid, whereas markers flanked by homologous regions located in the vicinity of a DSB are transmitted. Insertions (deletions), substitutions and shuffling of genetic markers are possible by in vivo recombination using Ec and can be applied to plasmid constructions. It is shown that recombination can occur from both template ends or from one vector and one template end. A D-loop nuclease is suggested to participate in the resolution of the recombination intermediates. PMID- 8621088 TI - Increased efficiency of alkaline phosphatase production levels in Escherichia coli using a degenerate PelB signal sequence. AB - To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels. PMID- 8621090 TI - N-Acetylglucosaminidase (chitobiase) from Serratia marcescens: gene sequence, and protein production and purification in Escherichia coli. AB - The chitobiase (Chb) encoding gene (chb) from Serratia marcescens (Sm) has been cloned, sequenced and expressed in Escherichia coli (Ec). Sequencing has revealed an open reading frame encodinga protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can produce enzymatically active Sm Chb protein which is secreted into the periplasm. An efficient purification scheme using cation-exchange chromatographyis presented. This yields about 3 mg of > 95% pure Sm Chb per litre of Ec culture. The deduced aa sequence is 27-aa longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved central region which aligns with almost all members of this family. According to the crystal structure of Sm Chb, this region comprises the catalytic domain of Chb which has an alpha/beta barrel fold. PMID- 8621089 TI - Genetic organization of a DNA-processing region required for mobilization of a non-self-transmissible plasmid, pEC3, isolated from Erwinia carotovora subsp. carotovora. AB - A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp. carotovora, can be mobilized by an IncP-type plasmid. The hybrid plasmid vector, pETC3, constructed from pEC3 by fusion to markers conferring TcR and CmR, was transferred by conjugation from Escherichia coli (Ec) to various genera of Enterobacteriaceae and to other genera of Gram(-) bacteria which included Xanthomonas, Agrobacterium and Rhizobium. Deletion analysis and successive subcloning of pEC3 revealed that a cis-acting locus, oriT and a trans-acting locus, mob, were involved in mobilization of pEC3. Five open reading frames (ORFs) were found in the mob region, of which four were identified as mobA, B, C and D. The mobA gene overlapped with mobC, B, D and ORF1 that were transcribed polycistronically from upstream from mobC. The nature of the four products of mob genes, MobA, B, C and D, was verified by use of the T7 promoter system in Ec. PMID- 8621092 TI - Characterisation of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus. AB - Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec). The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp. The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies. PMID- 8621091 TI - Flagellar genes from Rhodobacter sphaeroides are homologous to genes of the fliF operon of Salmonella typhimurium and to the type-III secretion system. AB - A flagellar region of the genome of Rhodobacter sphaeroides was cloned and sequenced. Three ORFs were identified and arranged in the same order as fliH, fliI and fliJ of Salmonella typhimurium (St). ORF2 is highly similar to FliI from St (49% similarity) showing Walker's A and B motifs. Similar scores were found with proteins of the type-III secretion system of virulence factors. ORF3 shows 16.4 and 11.1% similarity to FliJ from St and Bacillus subtilis, respectively. This work also shows that ORF3 is similar to HrpJ5 from Pseudomonas syringae (19.2% similarity). It was found that ORF2 and ORF3 start immediately downstream from the adjacent coding region, suggesting a single transcriptional unit. PMID- 8621093 TI - Vectors using the phospho-alpha-(1,1)-glucosidase-encoding gene treA of Bacillus subtilis as a reporter. AB - The intracellular phospho-alpha-(1,1)-glucosidase, TreA, from Bacillus subtilis (Bs) hydrolyses trehalose 6-phosphate into glucose and glucose 6-phosphate. The enzyme is also able to cleave p-nitrophenyl alpha-D-glucopyranoside (PNPG). This enzymatic reaction can be easily monitored in a beta-galactosidase analogous enzyme assay. The vectors we have constructed can be used to study promoter activity in transcriptional treA fusions and may prove especially useful under high-salt conditions due to the halophilic character of TreA. The treA gene is useful as a reporter in either Bs or Escherichia coli (Ec). Such fusions can be integrated in the Bs amyE locus and selected on either kanamycin or chloramphenicol, or used as plasmids in Ec. As an example of the general utility, we demonstrate treA expression under xylA-operator-promoter control. PMID- 8621094 TI - Cloning and sequencing of the dnaK operon of Bacillus stearothermophilus. AB - Here, we report the cloning of a 5.8-kb PstI fragment of chromosomal DNA from Bacillus stearothermophilus (Bt) carrying the three genes, grpE, dnaK and dnaJ. This fragment contains, in addition, the 3'-end of an open reading frame which has been shown to be part of the dnaK operon in three bacterial species. The dnaJ gene could complement an Escherichia coli dnaJts mutant for growth at high temperature. Sequencing and hybridization data strongly suggest that the Bt chromosome contains an analog of dnaJ. PMID- 8621095 TI - A Bacillus sphaericus gene encoding a novel type of mosquitocidal toxin of 31.8 kDa. AB - A size-fractionated genomic library of Bacillus sphaericus strain SSII-1 was constructed and screened for toxicity against larvae of the mosquito Culex quinquefasciatus (Cq). One toxin-producing clone, pS35, was identified and a 2.7 kb subclone was completely sequenced. An open reading frame of 879 bp encoding a 31.8-kDa protein (designated Mtx2) was identified. Purified, recombinant Mtx2 was toxic to Cq larvae. Mtx2 shows no significant homology to known insecticidal toxins, but has homology to two toxins active against mammalian cells, namely the epsilon-toxin of Clostridium perfringens and the cytotoxin of Pseudomonas aeruginosa. Thus, Mtx2 represents a new type of mosquitocidal toxin. PMID- 8621096 TI - Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and El Tor strains of Vibrio cholerae O1. AB - A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and El Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans cytoplasmic membrane (CM) protein which may have a regulatory role. PMID- 8621097 TI - Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentum ATCC 13869. AB - A repetitive DNA element of the Gram+ Brevibacterium lactofermentum (Bl), cloned by a modification of the subtractive hybridization method, contained a 1.4-kb IS like element, IS13869, which included an open reading frame (ORF) inside a perfect 26-bp terminal inverted repeat (TIR). An 8-bp direct repeat (DR) was found outside each TIR. The ORF encoded a deduced protein of 436 amino acids (49 380 Da) with extensive similarity to other known transposases of insertion elements of Mycobacterium smegmatis (IS1096). Pseudomonas sp. (tpnA) and Corynebacterium glutamicum (IS31831). Distinct patterns were observed in different strains of Bl by hybridization with a probe internal to IS13869: four copies of IS13869 occurred in the wild type (wt) and R31 strains, but only three of them were observed in a recA derivative of the wt. Analysis by pulsed-field gel electrophoresis suggested that at least one copy of IS13869 had changed its position inside the chromosome during the lineage of a Bl derivative. PMID- 8621098 TI - Cloning, sequencing and characterization of a fatty acid synthase-encoding gene from Mycobacterium tuberculosis var. bovis BCG. AB - Mycobacterial cell walls contain unique lipids such as mycolic acids, very long chain fatty acids and multimethyl-branched fatty acids. A multifunctional fatty acid synthase (Fas) with the unique capability of catalyzing both de novo synthesis and chain elongation of fatty acids has been purified and characterized from Mycobacterium tuberculosis var. bovis BCG (Bacillus Calmette-Geurin) [Kikuchi et al., Arch. Biochem. Biophys. 295 (1992) 318-326]. To understand how the various domains that catalyze the reactions involved in both de novo synthesis and elongation are organized in the mycobacteria, a fas gene was cloned from a cosmid library of genomic DNA from M. bovis BCG. Sequencing of the cosmid clone revealed a contiguous sequence of 11 577 bp of mycobacterial genome containing a 8389-bp open reading frame that could code for a protein of 2797 amino acids (301 kDa). By comparing the Fas aa sequence with the sequences in the active site regions of known fas and polyketide synthase-encoding genes, the functional catalytic domains in Fas were identified. This analysis revealed that the domains are organized in the following order: acyltransferase, enoyl reductase, dehydratase, malonyl/palmitoyl transferase, acyl carrier protein, beta keto reductase, beta-ketoacyl synthase. This domain organization is like a head to tail fusion of the two yeast fas gene subunits. The results obtained constitute the first report of the cloning, sequencing and structural elucidation of a fas from the Mycobacteria. PMID- 8621099 TI - Geriatrics photo quiz. Raynaud's: classic "blue fingertips'. PMID- 8621100 TI - The ABCs of managing hyperthyroidism in the older patient. AB - Hyperthyroidism in the older patient often presents with nonspecific symptoms, such as apathy, weight loss, and atrial fibrillation. General screening is not cost-effective, with the exception of two populations: women over age 40 with one or more nonspecific complaints and patients being admitted to a specialized geriatric unit. The sensitive TSH test is an excellent screen; a low TSH should be confirmed by an actual or estimated high free thyroxine test (FT4). The RAIU can help narrow the differential diagnosis. Radioiodine is the preferred treatment for the older patient with Graves' disease, the most common form of thyrotoxicosis. Graves' must be differentiated from thyroiditis and toxic nodular goiters, as treatment regimens differ for each disorder. PMID- 8621101 TI - Palliative medicine: providing care when cure is not possible. A roundtable discussion: Part I. AB - Palliative medicine describes the care of patients with advanced disease. When cure is no longer possible, the goal becomes control of pain, other symptoms, and psychological distress. In the United States, palliation has been pioneered by the hospice movement for patients with disseminated cancer and AIDS. Palliative care is also appropriate for patients with many of the chronic diseases of aging. For medical, humanitarian, financial, and legal reasons, physicians are being called on to provide palliative care when they make the diagnosis of all illness that is unresponsive to curative treatment. PMID- 8621102 TI - Reversing sarcopenia: how weight training can build strength and vitality. AB - Sarcopenia, the loss of skeletal muscle mass with advancing age, results in lower basal metabolic rate, weakness, reduced activity levels, decreased bone density, and low calorie needs. The related increase in body fatness is linked to hypertension and abnormal glucose tolerance. Many consequences of sarcopenia are preventable or even reversible. Progressive resistance exercises can produce substantial increases in strength and muscle size, even in the oldest old. For many older patients, exercise represents the safest, least expensive means to lose body fat, decrease blood pressure, improving glucose tolerance, and maintain long-term independence. PMID- 8621104 TI - Suffering at the end of life. PMID- 8621103 TI - Swollen lesion on the abdomen. Could this violaceous blister be related to patient's treatment for UTIs? PMID- 8621105 TI - Factors affecting the metabolism of cinnamyl anthranilate in the rat and mouse. AB - The biological actions of cinnamyl anthranilate are dependent on both dose size and animal species. The present study aimed to examine metabolism as a possible source of explanation for these differences. [3-14C]Cinnamyl anthranilate was synthesized, injected ip into male Fischer 344 (F344) rats and CD-1 mice and urine and faeces collected for 3 days. The pattern of elimination of 14C was the same in both species, with the bulk of the administered material recovered in urine over the first 24 hr. Urinary metabolic profiles were compared by radioHPLC, which showed that the major radioactive excretion product in the rat was hippuric acid accompanied by smaller amounts of benzoic acid. In contrast, mouse urine contained relatively less hippuric acid, more benzoic acid and small amounts (approx. 3% of dose) of unchanged cinnamyl anthranilate. The effect of dose size on urinary metabolites produced by mice was examined using both 3-14C labelled and unlabelled cinnamyl anthranilate, detected by fluorescence HPLC. Over a dose range of 5 to 250 mg/kg body weight administered ip it was found that at 5 mg/kg body weight no intact ester was excreted in urine whereas at 20 mg/kg body weight or above, the proportion present as the intact ester remained constant. Dietary administration to male and female B6C3F1 mice for 21 days over a dose range of 0 to 30,000 ppm revealed the same qualitative picture with no intact cinnamyl anthranilate detected in urine at or below 1000 ppm (equivalent to 100 mg/kg body weight). A study in human volunteers using a single oral dose of 250 mg failed to reveal any intact cinnamyl anthranilate in 0-24-hr urine. These data support the hypothesis that the peroxisome proliferating action of cinnamyl anthranilate, which is mediated by the intact ester, is manifest only at high doses in species in which its metabolism by hydrolysis is saturated, as a consequence of which the intact ester 'overflows' into urine. PMID- 8621106 TI - Toxic oil syndrome: traceback of the toxic oil and evidence for a point source epidemic. AB - Rapeseed oil denatured with aniline was the vehicle of the causal agent of the toxic oil syndrome (TOS) epidemic that occurred in Spain in 1981. Although the precise aetiologic agent remains unknown, researchers established that increasing concentrations of oleyl anilide and other fatty acid anilides were associated with an increased risk for disease. To examine the hypothesis that 5-litre plastic containers of rapeseed oil associated with TOS, and which contained oleyl anilide had a characteristic shape, we measured fatty acid, sterol and fatty acid anilide levels in oil from containers of different shapes. We identified 1673 bottles of oil that had been collected during the Spanish Government's oil exchange programme and linked these bottles to people with TOS as reported in the official government census of patients with TOS. Although rapeseed oil (identified by the presence of brassicasterol) was found in 798 (47.7%) of the 1673 bottles examined, contamination with fatty acid anilide occurred in only 329 (19.6%) of the 1673 bottles and 319 (97%) of the 329 were oil containers of the shape sold by RAELCA, an oil company in Madrid. The first aniline-denatured oil that RAELCA had purchased to be refined specifically for distribution was refined at the ITH refinery of Seville, and this oil has been most directly associated with the epidemic. Previous work has shown that the only toxic oil linked to a specific refinery was that associated with rapeseed oil from the ITH refinery in Seville, and the epidemic began shortly after this oil was delivered to RAELCA for retail sale. On the basis of these findings, we conclude that oil refined by ITH and distributed by RAELCA was the principal, and probably the only, oil responsible for the TOS epidemic. Information about the history and treatment of this oil may yield important clues towards identifying the aetiologic agent of TOS. PMID- 8621107 TI - Change in glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase activities in the organs of mice treated with 2-chloroethyl ethyl sulfide or its oxidation products. AB - Various organs or skin from male ICR mice treated intraperitoneally with 2 chloroethyl ethyl sulfide (CEES) or its oxidation derivatives 2-chloroethyl ethyl sulfoxide (CESSO) and 2-chloroethyl ethyl sulfone were analysed for changes in two thiol-containing enzymes, namely glutathione S-transferase (GST) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CEES was more potent than its oxidation derivatives with respect to the decrease in organ weight and the loss in GAPDH activity, although the reverse was found in GST induction. Whereas the induction of GST was highest in the lung after multiple intraperitoneal intoxication with CEESO (8 and 32 mg/kg), the decrease in GAPDH activity after exposure to CEES (8 mg/kg body weight) was most remarkable in the spleen, the most susceptible organ to toxicity of CEES. GST and GAPDH activities in the skin of male hairless mice exposed subcutaneously to CEES (2 mg/kg body weight) were not altered significantly at 2-hr exposure, but decreased up to 60% of that of controls at 8 hr, when oedema formation was greatest. Taken together, it appears that GAPDH activity is a more sensitive biochemical parameter than GST activity in organs of mice treated with CEES or its oxidation products. PMID- 8621108 TI - Skin reservoir formation and bioavailability of dermally administered chemicals in hairless guinea pigs. AB - There is concern as to whether dermally applied chemicals that remain in the skin after exposure are bioavailable and should be included as part of the systemic dose; this study was conducted to investigate the temporal relationship between the skin depot and absorbed dose. Single doses of 14C-labelled phenanthrene, benzo[a]pyrene or di(2-ethylhexyl) phthalate were administered dermally to groups of four female, Hartley hairless guinea pigs which were housed individually in metabolism cages to collect urine and faeces for radioassay. The animals were sacrificed at 6 hr, 24 hr, 48 hr, 7 days or 14 days after dosing to harvest skin specimens for the determination of radioactivity by autoradiographic and liquid scintillation methods, and to determine the dose that remained in the body. It was found that for all three compounds the amount of chemical left in the skin decreased over time while the cumulative percent dose excreted in urine and faeces increased. The autoradiographic results were consistent with those obtained from the liquid scintillation method showing a gradual decrease in radioactivity grain accumulation over the time periods for the three compounds, with the highest grain density observed around hair follicles of the skin. The results of this study indicate that the chemicals left in the skin after surface washing eventually enter the systemic circulation and should be considered as part of the total dose absorbed, and that the hair follicle may play an important role in percutaneous penetration. PMID- 8621109 TI - Formulation effect on the dermal bioavailability of isothiazolone biocide. AB - A central tenet of the science of toxicology is that the toxic effect of any material monotonically increases with the amount of the material delivered to the target tissue. This is the so-called dose-response relationship. Previous work is discussed in which a dose-response relationship is established for the induction and elicitation of cutaneous contact allergy from the isothiazolone biocide which is a 3 to 1 mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4 isothiazolin-3-one. The commercial product name for this mixture is Kathon LX biocide. Given a tissue dose-response, this current work determines and demonstrates a difference in risk from aqueous formulations with and without polymer. A lower delivered dose of biocide goes into the skin from an aqueous based polymeric emulsion or paint than from an aqueous solution or oil-in-water emulsions without polymer. Thus, the estimate of risk, as a direct result of dose, and the determination of a safe use level of a biocide in any particular formulation matrix need to incorporate this reality. PMID- 8621110 TI - Carcinogenic and co-carcinogenic studies of thiram on mouse skin. AB - Thiram (tetramethyl thiuram disulfide), a carbamate fungicide, is used in the rubber processing industry as an accelerator and vulcanizing agent. Previous studies evaluated the tumorigenic potential of thiram in rodents, but failed to provide conclusive results. In the present study the tumorigenic potential of thiram was evaluated in Swiss albino mice by a two-stage initiation-promotion protocol and a long-term in vivo bioassay for carcinogenicity. Results revealed that following tumour initiation with thiram and promotion with 12-O tetradecanoyl phorbol 13-acetate, skin tumours developed, mostly at the site of treatment (dorsal skin) in single and multiple dose-initiated animals. Similarly, papillomatous growths were observed on the dorsal skin of the mice initiated with a single subcarcinogenic dose of dimethylbenzanthracene and promoted with thiram. Thiram failed to provoke tumorigenesis when tested as a complete carcinogen for up to 52 wk and thereafter the study was terminated due to increased mortality. It is concluded that thiram has both tumour initiating and tumour-promoting potential in both sexes of Swiss albino mice following topical exposure at the tested dose level. PMID- 8621112 TI - N-nitrosodimethylamine in Spanish beers. AB - A survey of 21 beers produced in Spain during 1994 was conducted to assess contamination with volatile N-nitrosamines. N-Nitrosodimethylamine (NDMA), the only volatile N-nitrosamine detected, was found in 52% of Spanish beers at or above the detection level (0.05 microgram/kg). The mean NDMA level for all samples was 0.11 microgram/kg, and the range was from not detected to 0.55 microgram/kg. No differences in the NDMA content were observed between the different beer types. Individual daily intake of NDMA may reach up to 0.021 microgram/person/day for Spanish people. PMID- 8621111 TI - The chicken enucleated eye test (CEET): a practical (pre)screen for the assessment of eye irritation/corrosion potential of test materials. AB - The enucleated eye test with chicken eyes (CEET) obtained from an abattoir proved to be a valuable and practical alternative for the 'traditional' enucleated eye test with eyes of laboratory rabbits. Since 1992, the CEET has been incorporated in standard contract toxicity testing at the Toxicology Division of the TNO Nutrition and Food Research Institute as a (pre)screen for the Draize eye test with rabbits. The results of the first 44 compounds tested showed excellent correlation with the in vivo results. The CEET identified non-irritating or severely irritating compounds, and predicted (slightly to moderately) irritating compounds. Statistical analysis of the CEET and the rabbit in vivo scores showed high linear correlations between the critical values of both tests and confirmed the relevance of this assay with respect to ocular effects. In general, tiered in vitro/in vivo testing was considered a meaningful approach for further validation of alternative methods and for reducing the use of suffering of laboratory animals to a minimum. Tiered testing of compounds in cases of eye irritation hazard assessment should be incorporated in the legislation of the European Community. PMID- 8621113 TI - A review of the role of tissue repair as an adaptive strategy: why low doses are often non-toxic and why high doses can be fatal. AB - The role of tissue repair as an adaptive strategy by species is important to consider in both evolutionary and toxicological perspectives. This paper assesses the distinct and integrative roles of early phase regeneration (EPR) (i.e. arrested G2 hepatocytes chemically activated to proceed through mitosis) and secondary phase regeneration (SPR) (i.e. hepatocytes mobilized principally from G0/G1 to proceed through mitosis) in the repair of carbon tetrachloride (CCl4) induced liver damage. The role of EPR as a triage system facilitating repair of minor toxic insults as well as providing an essential role in autoprotection as an initial step to augment and sustain SPR is proposed. The function of EPR is then compared with that of SPR in tissue recovery following more massive injury. The interrelationships of these two repair processes with EPR invoking and accelerated SPR following low-to-modest degrees of toxicant-induced hepatotoxicity as well as in auto- or hetero-protection supports the theory that the two responses are co-ordinated in time and functionality. The integration of these two repair processes as shown through experimental manipulation provides a new mechanistic framework to account for the previously reported profound (67 fold) potentiation of acute CCl4 hepatotoxicity by chlordecone (kepone) in adult male Sprague-Dawley rats as well as important interspecies variation in susceptibility to hepatotoxic agents in general and CCl4 in particular. On the basis of the distinct and integrative roles of EPR and SPR in liver responses to toxic injury, a generalized framework is presented that facilitates prediction of both toxic outcome, including shape of dose-response functions and interspecies variation to chemically induced liver damage. PMID- 8621114 TI - Capsaicin in hot chili pepper: carcinogen, co-carcinogen or anticarcinogen? AB - Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a major pungent ingredient of the Capsicum fruits such as hot green and red peppers. Besides its use as a food additive in various spicy cuisines, capsaicin is currently utilized for therapeutic purposes to treat various peripheral painful conditions such as rheumatoid arthritis and diabetic neuropathy. Considering consumption of capsaicin as a food additive and its current medicinal application in humans, correct evaluation and precise assessment of any harmful effects of this compound are essential from the public health standpoint. Numerous investigations have been conducted to determine the potential mutagenic and carcinogenic activity of capsaicin and chili pepper, but results are discordant. This review briefly examines findings in the literature of studies testing mutagenicity and tumorigenicity of capsaicin and presents a possible mechanistic basis for the dual effects exerted by the compound. PMID- 8621115 TI - Pesticide residues in food. PMID- 8621116 TI - Outdoor air pollution and asthma. PMID- 8621117 TI - PCR DNA typing of stamps: evaluation of the DNA extraction. AB - Today, the PCR analysis of DNA from the saliva deposited on a stamp of an anonymous letter can lead to an identification. However, this analysis still involves problems, and DNA extraction can be particularly difficult. A comparative study of two DNA extraction methods with two categories of postage stamps was carried out and a purification process was tested. This study shows that the extraction with phenol/chloroform gives much better results than Chelex extraction. A purification process such as the use of Centricon 100 microconcentrators is recommended when an inhibition of PCR is present. This operation, however, results in a large loss of material. PMID- 8621118 TI - Detection of toluene in an adipoceratous body. AB - A 24-year-old male was found dead in a car left in a river for about 3 months. The cadaver was almost adipoceratous and autopsy findings revealed that there were neither remarkable injuries nor lethal diseases. Toluene, ethanol, 1 propanol, 2-propanol, 1-butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate were detected in the specimens collected at the autopsy by head space gas chromatography/mass spectrometry (GC/MS). The toluene concentrations (micrograms/g) were 31.0 in brain, 10.6 in liver, 5.4 in kidney, 15.0 in skeletal muscle and 187.1 in adipose tissue. The presence of diatom in lung, liver and kidney suggested that death was caused by drowning. So far as we know, this is the first report of detection of toluene in an adipoceratous body. PMID- 8621119 TI - Sequence determination of an allele ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample. AB - The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4, was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8' to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8' was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.23). PMID- 8621120 TI - Analysis of the D1S80 (pMCT118) VNTR locus polymorphism in a native Kuwaiti population by the polymerase chain reaction. AB - We have determined the allele and genotype frequencies at the hypervariable locus D1S80 in a native Kuwaiti population using the polymerase chain reaction technique and subsequent high resolution gel electrophoresis. In a sample of 200 individuals, 21 alleles and 57 genotypes were detected. The alleles with 18 and 24 repeat units were most common with frequencies of 0.188 and 0.408 respectively. The distribution of the observed genotypes was in agreement with the Hardy-Weinberg equilibrium prediction. The observed heterozygosity for the population sample was 0.80 with the allelic diversity of 0.781 +/- 0.029 and the power of discrimination was 0.94. The data obtained in this study are potentially useful for individual identification in forensic casework. PMID- 8621121 TI - PCR typing of DNA fragments of the two short tandem repeat (STR) systems upstream of the human myelin basic protein (MBP) gene in Danes and Greenland Eskimos. AB - DNA from the double short tandem repeat (STR) system MBP (locus 18q23-pter) was amplified by the polymerase chain reaction (PCR) and the two polymorphic repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths of the DNA fragments of the two MBP STR systems MBP-A and MBP-B were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. DNA samples from 112 unrelated Danes, 140 unrelated Greenland Eskimos, and 88 Danish mother/child pairs were analyzed. The distributions of MBP phenotypes were in Hardy-Weinberg equilibrium in both the Eskimo and Danish populations. Significant differences were observed between the distribution of fragments ('alleles') in Greenland Eskimos and in Danes. The allele MBP-A7 was considerably more frequent in Eskimos (0.2214) than in Danes (0.0775) and also the allele MBP-B9 was considerably more frequent in Eskimos (0.225) than in Danes (0.06). Strong gametic associations were found between fragments from MBP-A and MBP-B series in both Danes and Eskimos. Some of the associations were different in Danes and Eskimos. In the 88 Danish mother/child pairs, the segregation of the MBP genotypes were in accordance with a genetic model of co-dominant inheritance and no mutation was found. Two MBP STR regions with irregular structures were sequenced. One fragment had a single base G to A transition at position 124 in the primer binding region between the MBP-A and MBP-B regions. In the other fragment, a deletion starting at position 117 and including the primer binding region between MBP-A and MBP-B regions was found. PMID- 8621122 TI - Pathoanatomic findings and blood alcohol analysis at autopsy (BAC) in forensic diagnoses of undetermined suicide. A cross-cultural study. AB - In Sweden, ca. 25% of unnatural deaths ascribed to self-inflicted injury are finally recorded as 'undetermined suicide' (abbreviated UMSA), i.e. the forensic pathologist has not been able to establish whether the fatality was an accident or a suicide. In the present study, a series of UMSA cases was investigated with the aims to study the impact of immigrant status, and alcohol abuse on the occurrence of this forensic diagnosis on the mode of death. The alcohol issue was addressed by focusing on blood alcohol concentrations at autopsy (BAC) and post mortem signs of alcohol-related organ pathology. The results can be summarised as follows: Positive BAC occurred at an equal rate in the UMSA group and in definite suicides, i.e. about 45%. Among non-Swedish UMSA victims positive BAC was more common (50%) than among the Swedish (41%), whereas no difference was found in the definite suicide group. The level of BAC at autopsy was significantly higher in Finnish immigrants than in other ethnic groups. Organic signs of chronic alcohol abuse were found in 13 of 40 cases testing positive for BAC; thus, presence of alcohol at autopsy may reflect incidental intake rather than habitual overconsumption. PMID- 8621123 TI - Report on the third EDNAP collaborative STR exercise. European DNA Profiling Group. AB - This report describes an inter-laboratory exercise completed on behalf of the European DNA Profiling (EDNAP) group. The exercise is one in a series designated to identify STR loci which could be used for harmonisation between participating European forensic science laboratories. Participants were asked to identify the alleles present in five bloodstains at the STR loci HUMTHO1 and HUMVWFA31/A. Two of the stains were prepared from mixtures of two different blood samples. There were no special instructions and each laboratory was requested to use the methodology normally employed for crime case investigations. All participating laboratories achieved the same results for both loci. In addition, the laboratories were also requested to report the results obtained from any other loci which would normally be used in crime case investigations. A comparison of these results showed some inter-laboratory variation. PMID- 8621124 TI - Rapid analysis of amphetamines in blood using head space-solid phase microextraction and selected ion monitoring. AB - A simple and rapid method for analysis of methamphetamine (MA) and amphetamine (AP) in blood was developed using head space-solid phase microextraction (HS SPME) and gas chromatography-mass spectrometry/electron impact ionization selected ion monitoring (GC-MS/EI-SIM). A vial containing a blood sample, sodium hydroxide, and pentadeuterated methamphetamine as an internal standard, was heated at 80 degrees C for 20 min. The extraction fiber of the SPME was exposed for 5 min in the head space of the vial. First, heptafluorobutyric anhydride solution was injected into the injection port of the GC-MS to make heptafluorobutyramide (HFB) derivatives of amphetamines, and compounds absorbed on the fiber were detached by exposing the fiber in the injection port. Straight calibration curves of MA and AP were obtained from 0.01 to 2 micrograms/g in blood, respectively. No interfering substances were found, and the time for analysis was 30 min for one sample. PMID- 8621125 TI - Relationship between hepatocyte proliferative activity and liver functional reserve in human cirrhosis. AB - Hepatocyte proliferative activity is elevated in cirrhotic patients who develop hepatocellular carcinoma (HCC) and decreased in alcohol-induced hepatitis patients with poor outcome. Hepatocyte proliferative activity has not been evaluated in an unselected population of cirrhotic patients regarding the severity of the disease. Forty-six cirrhotic patients (21 alcoholic, 20 viral, and 5 other) were prospectively analyzed by proliferating cell nuclear antigen (PCNA) immunostaining on methanol-fixed, paraffin-embedded liver biopsy specimens. In these conditions, the PCNA-labeling index (PCNA-LI) measures the number of cells in the S-phase and assesses tissue proliferation. The median value of the PCNA-LI for all samples was 4.3% (range, 0%-20.2%). It declined with worsening Child-Pugh score: 9.15% (range, 3.3%-20.2%), 5.3% (range, 1.2%-18%), and 2.4% (range, 0%-4.4%) in Child classes A, B, and C, respectively (P < .05). Using the best cutoff PCNA-LI value to divide cirrhosis into slowly and rapidly proliferating tissue subsets, the PCNA index was independently associated with serum albumin. The probability of survival in patients with a high PCNA-LI ( > 4.4%) was significantly higher than in those with a lower PCNA-LI (0.93 vs. 0.53, at a median follow-up of 153 days; P = .01). In all 6 patients undergoing placement of a transjugular intrahepatic portosystemic shunt (TIPS), the PCNA-LI decreased after the procedure. This early impairment of hepatocyte proliferative activity after TIPS placement might reflect the functional alterations induced by this treatment. In conclusion, hepatocyte proliferative activity assessed by PCNA LI is increased in cirrhotic patients and decreases with worsening of the disease. PMID- 8621126 TI - Cell cycle progression proteins (cyclins), oncogene expression, and signal transduction during the proliferative response of human hepatocytes to hepatocyte growth factor. AB - Human hepatocytes stimulated with human recombinant hepatocyte growth factor (h rHGF) (10 ng/mL) displayed a characteristic lag period before entering into the S phase. The duration of this delay was dependent on the timing of h-rHGF addition to cultures. The highest peak of DNA synthesis was observed at 120 hours of culture when hepatocytes were stimulated with h-rHGF at 72 hours of culture. This was accompanied by an early peak of c-jun and c-fos synthesis (3 hours after addition of h-rHGF) followed by c-myc (6 hours) and increased expression of cyclins A, B, D, and E (12 hours after h-rHGF). A significant dose-dependent increase in inositol 1,4,5-P3 was observed within 45 seconds after stimulation with the factor. This was followed by an immediate increase in the cytosolic-free calcium. Cyclic adenosine monophosphate (cAMP) levels did not change after stimulation with the factor. Tyrosine phosphorylation seems to be an early event in the course of the stimulatory effect of h-rHGF on DNA synthesis of hepatocytes; genistein, a tyrosine kinase inhibitor, impaired the stimulatory effect of h-rHGF on DNA synthesis dose dependently. On the other hand, the action of the factor was negatively regulated by protein kinase C activation, as shown by the increased stimulatory effect of h-rHGF on DNA synthesis upon inhibition of protein kinase C by H7. PMID- 8621127 TI - Specificity and sensitivity of gp210 autoantibodies detected using an enzyme linked immunosorbent assay and a synthetic polypeptide in the diagnosis of primary biliary cirrhosis. AB - Between 10% and 42% of patients with primary biliary cirrhosis (PBC) have been reported to have autoantibodies directed against a restricted epitope of gp210, a glycoprotein of the nuclear pore membrane. The prevalence and specificity of these antibodies was studied in a French series of 285 patients with PBC and 497 control individuals affected with other liver or autoimmune diseases. Sera were analyzed by an enzyme-linked immunosorbent assay (ELISA) that used a synthetic polypeptide containing the predominant autoepitope of gp210, in parallel to immunoblotting of gp210 protein and immunofluorescence microscopy. Autoantibodies to the gp210 epitope detected by ELISA were 25.5% sensitive and 99.5% specific for the diagnosis of PBC. These results were in agreement with a 99.4% specificity with immunoblotting analysis and a 96.6% specificity with immunofluorescence. In a subset of PBC patients without detectable antimitochondrial autoantibodies (AMA), gp210 autoantibodies were found in 7 of 15 patients (47%). Therefore, gp210 autoantibodies are highly specific for PBC and may be of particular utility in assessing patients without AMA or with other atypical presentations. PMID- 8621128 TI - Prediction of risk of liver disease by alcohol intake, sex, and age: a prospective population study. AB - The association between self-reported alcohol intake and the risk of future liver disease was studied in a large population-based prospective cohort with 12-year follow-up. Alcohol intake was assessed in 13,285 men and women aged 30 to 79 years by a self-administered questionnaire. Diagnoses indicating alcohol-induced liver disease (n = 261) or alcohol-induced cirrhosis (n = 124) were obtained from death certificates and the hospital discharge register, and data were analyzed by multiplicative Poisson regression models. The total cumulated observation time was 130,558 person-years. The overall incidence rates of alcohol-induced cirrhosis were 0.2% per year in men and 0.03% per year in women. The nadir of the estimated relative risk of developing liver disease was observed at an alcohol intake of 1 to 6 beverages per week, and above this level a steep increase in relative risk was observed. The risk function was independent of age and stable over time. The level of alcohol intake above which the relative risk was significantly greater than 1 was observed at 7 to 13 beverages per week for women and 14 to 27 beverages per week for men. Women had a significantly higher relative risk of developing alcohol-related liver disease than men for any given level of alcohol intake. We observed a dose-dependent increase in relative risk of developing alcohol-induced liver disease for both men and women, with the steepest increase among women. In the general population, self-reported current alcohol intake is a good predictor of the future risk of alcohol-induced liver disease. PMID- 8621129 TI - Liver function tests in normal pregnancy: a prospective study of 103 pregnant women and 103 matched controls. AB - Except for increased serum alkaline phosphatase (AP) levels, the changes in liver function test (LFT) values during normal pregnancy have not been clearly established, mainly because most studies do not include matched controls. We therefore measured the serum values of routine liver tests including 5' nucleotidase and total bile acids in 103 healthy pregnant women (first trimester, n = 34; second trimester, n = 36; third trimester, n = 33) and in 103 age-matched controls not receiving oral contraception. Fasting blood samples were taken. Because of hemodilution, serum albumin levels were significantly lower during all trimesters. As expected, AP activity was significantly higher in the third trimester. Serum aspartate transaminase (AST) activity and total bile acid (TBA) concentrations did not differ between pregnant and nonpregnant women. Serum alanine transaminase (ALT) activity was slightly higher in the second-trimester pregnant women than in controls (6.8 +/- 4.5 vs. 8.2 +/- 5.8, P = .04), although all values remained within normal limits. In pregnant women, total and free bilirubin concentrations were significantly lower during all three trimesters, as was conjugated bilirubin during the second and third trimesters. Serum gamma glutamyl transpeptidase (GGT) activity was significantly lower in the second and third trimesters. Serum 5'-nucleotidase activity was slightly but significantly higher in the second and third trimesters. The knowledge of these results is useful for the interpretation of LFT values and the management of liver diseases during pregnancy. PMID- 8621130 TI - Splenic Doppler impedance indices: influence of different portal hemodynamic conditions. AB - The spleen plays a pivotal role in the pathogenesis and maintenance of portal hypertension. Few data exist about splenic hemodynamics evaluated by duplex sonography in this condition. Twenty-six normal subjects, 207 patients with portal hypertension of various causes, and in different splenoportal hemodynamic conditions, and 31 patients with liver transplantation were evaluated. In each patient the splenic resistive index (RI = peak systolic--end diastolic velocity/peak systolic velocity) and pulsatility index (PI = peak systolic--end diastolic velocity/mean velocity) were measured. In 17 cirrhotic patients, splenic indices were compared with portal hemodynamics as invasively evaluated by hepatic vein catheterization. In the various groups, RI and PI were respectively: normal subjects, 0.51 +/- 0.05 and 0.72 +/- 0.11; cirrhotic patients with hepatopetal portal blood flow (n = 167), 0.64 +/- 0.08 and 1.03 +/- 0.24; cirrhotic patients with hepatofugal portal flow (n = 3), 0.74 +/- 0.08 and 1.27 +/- 0.08; cirrhotic patients with portal vein thrombosis (n = 9), 0.74 +/- 0.08 and 1.36 +/- 0.34; patients with noncirrhotic obstruction of the portal system (n = 7), 0.69 +/- 0.11 and 1.16 +/- 0.28; cirrhotic patients with surgical decompression of splenic vein system (n = 21), 0.54 +/- 0.07 and 0.76 +/- 0.15; patients with liver transplantation (n = 31), 0.50 +/- 0.08 and 0.70 +/- 0.15. Both RI and PI were significantly higher in cirrhotic patients with hepatopetal portal flow compared with controls (P < .0001), and even higher in cirrhotic patients with portal vein thrombosis (P < .004 and P < .001 in comparison with RI and PI values of cirrhotic patients). In patients with noncirrhotic portal vein thrombosis, splenic impedance indices were higher than those in controls (RI and PI P < .0001). Cirrhotic patients who underwent surgery for the therapy of portal hypertension showed splenic impedance indices significantly decreased compared with other cirrhotic patients (RI and PI P < .0001). In patients who underwent liver transplantation, splenic impedance indices were the same as those in controls. In 23 of the 52 patients surgically treated (surgical shunt or liver transplantation), impedance indices were evaluated both before and after surgical treatment. All these patients showed a decrease in splenic impedance indices (RI and PI, P < .0001) after surgical treatment. RI and PI values were higher in patients with large esophageal varices as compared with patients without or with small varices (P < .02 and P < .01). RI and PI values were not related to age, mean arterial pressure, sex, Child-Turcotte-Pugh score, presence of ascites, or cause. A significant correlation was found between splenic impedance indices and portal resistance as evaluated by hepatic vein catheterization (r = .80, P < .001 for RI values; r = .87, P < .001 for PI values). In conclusion, this study shows that splenic impedance indices are increased in cirrhotic patients, and seems to demonstrate that in patients with cirrhosis these indices reflect portal vein blood flow resistance. PMID- 8621131 TI - Does malnutrition affect survival in cirrhosis? PINC (Policentrica Italiana Nutrizione Cirrosi). AB - A total of 1,053 cirrhotic patients were included in a prospective study to determine whether malnutrition is a risk factor for mortality in cirrhotic patients. Child-Pugh classification as well as clinical and biochemical variables were used to assess the severity of cirrhosis. Nutritional status was evaluated both by anthropometric and clinical measurements. Patients were defined as malnourished when midarm muscle area (MAMA) and/or midarm fat area (MAFA) were below the 5th percentile of an age- and sex-matched population. During follow-up, 419 patients died. The estimated survival rate was 82.7% at 1 year, 65.1% at 3 years, and 50.7% at 5 years. The presence of muscle depletion and/or of a steep reduction in fat deposits was associated with a higher risk of mortality (midarm muscle area, < 5th percentile, relative risk = 1.79; midarm fat area, < 5th percentile, relative risk = 1.35). When patients were stratified according to the Child-Pugh classification, cumulative survival was lower in patients with a reduction in muscle mass in Child-Pugh classes A and B (log rank: P = .027; P = .022, respectively) but not in class C. Conversely, a significant reduction in adipose tissue deposits appeared to have no independent impact on survival in any Child-Pugh class. When examined using a multivariate Cox proportional hazard analysis, age, sex, bilirubin, cholinesterase, ascites, and esophageal varices were selected, whereas the parameters of nutritional status were not. This suggests that malnutrition, while strongly associated with the deterioration of liver function, cannot be considered an independent risk factor for mortality in a general population of cirrhotic patients. PMID- 8621132 TI - The effect of long-term treatment with spironolactone on variceal pressure in patients with portal hypertension without ascites. AB - The effect of spironolactone on esophageal variceal pressure (VP) in patients without ascites was investigated. VP was assessed using a noninvasive endoscopic gauge. Spironolactone was administered during a 6-week period at a dosage of 100 mg/d. This treatment decreased VP from 16.8 +/- 1.9 (SD) to 14.1 +/- 2.7 mm Hg (P < .001) in a group of 12 patients and from 18.6 +/- 2.1 to 13.7 +/- 4.1 mm Hg (P < .01) in another group of 8 patients who still had high VP despite chronic intake of propranolol. In both groups, placebo administration to 12 and 8 comparable patients did not significantly alter VP. Spironolactone induced a significant reduction of plasma volume (42.1 +/- 5.5 to 36.1 +/- 6.6 mL/kg body weight, P < .01) and of the concentration of alpha-atrial natriuretic peptide (alpha-ANP) (39.8 +/- 22 to 27.7 +/- 20 pg/mL, P < .01); in addition, a pronounced increase in plasma renin activity (PRA) (1.1 +/- 0.9 to 7.5 +/- 3.4 ng/mL/h, P < .001) was induced by the treatment. No significant changes in systemic hemodynamics were observed during the studies. Severe side effects were not observed except for a high incidence (55%) of painful gynecomasty in the male patients. In conclusion, chronic spironolactone administration effectively lowers VP, even in patients under chronic propranolol therapy. The combination of propranolol and spironolactone deserves further study as a prophylactic therapy of variceal hemorrhage, but development of gynecomasty might be a problem. Finally, we confirmed the reproducibility of VP measurements with the noninvasive gauge in chronic conditions. PMID- 8621133 TI - Molecular and functional characterization of bile acid transport in human hepatoblastoma HepG2 cells. AB - Bile acids are taken up into human liver by Na+-dependent and Na+-independent transport mechanisms. In hepatocarcinogenesis, numerous liver-specific functions are lost and the uptake of organic anions is markedly reduced. We have investigated the molecular and functional derangements of bile acid transport in the human hepatoblastoma cell line HepG2. Uptake of [3H]-taurocholic acid was saturable and entirely Na+ independent, with the kinetic characteristics of the human liver organic anion transporting polypeptide (OATP). OATP, but not the Na+ dependent bile acid transporter (Na+-taurocholate-cotransporting polypeptide [NTCP]), was detectable by reverse-transcription polymerase chain reaction (RT PCR) analysis of HepG2 RNA. The level of OATP expression in HepG2 cells was determined by Northern blot analysis and was found to be 40% in comparison with normal liver. Transfection of an OATP-derived phosphorothioate (PTO)-antisense oligonucleotide into HepG2 cells resulted in 77% inhibition of temperature dependent bile acid uptake. Injection of HepG2 messenger RNA (mRNA) into Xenopus laevis oocytes significantly stimulated Na+-independent taurocholate uptake, indicating the expression of a bile acid transport protein. We conclude that bile acid uptake into human hepatoblastoma HepG2 cells is mediated by the multi specific organic anion transporting polypeptide OATP. Therapeutic strategies employing bile acid-derived cytostatic agents for the treatment of hepatocellular carcinomas may therefore depend upon the expression of the Na+-independent bile acid transporter OATP in hepatic malignancies. PMID- 8621134 TI - Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. AB - The Dubin-Johnson syndrome is characterized by an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into the bile. We have recently identified the membrane protein mediating the adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronate conjugates as a multidrug-resistance protein (MRP) and localized it to the canalicular as well as to the lateral hepatocyte plasma membrane. In the present study we show the selective absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syndrome by double-label immunofluorescence and confocal laser scanning microscopy using antibodies directed against MRP and dipeptidyl-peptidase IV (DPPIV). Another isoform of MRP was detected, however, in the lateral hepatocyte membrane of the patient. Moreover, MRP was present on immunoblots of erythrocyte membranes from Dubin Johnson syndrome and normal humans. These findings are analogous to our recent observations on the localization of the rat homolog of MRP and its canalicular isoform, cMrp, in normal and transport-deficient GY/TR- Wistar rat liver. The elucidation of the selective absence of an isoform of MRP and from the canalicular membrane domain in conjunction with the defined substrate specificity of the MRP and cMRP gene-encoded conjugate export pumps contributes to the molecular definition of the transport defect in Dubin-Johnson syndrome. PMID- 8621135 TI - Evidence of increased guanylate cyclase activation by acetylcysteine in fulminant hepatic failure. AB - Patients with fulminant hepatic failure (FHF) have a severe microcirculatory disturbance causing tissue hypoxia. Infusion of acetylcysteine improves survival and reduces the incidence of multiorgan failure by enhancing tissue oxygenation. Because the observed circulatory effects of acetylcysteine in FHF are similar to and synergistic with those produced by the microcirculatory vasodilator prostacyclin, we postulated that acetylcysteine might potentiate an endogenous vasodilator. Nitric oxide, a vasodilator that activates soluble guanylate cyclase, is a possible candidate as plasma cyclic 3',5'-guanosine monophosphate (cGMP) is raised in FHF, and in vitro acetylcysteine has been found to enhance soluble guanylate cyclase activity. To investigate this possible mechanism further, plasma cGMP was measured before and after acetylcysteine infusion in 24 patients with FHF and again in 6 patients after recovery from acute illness. cGMP levels were high in FHF during acute illness (median, 7.0 nmol/L [interquartile range, 2.6-10.0]) in comparison with levels taken after recovery (1.5 nmol/L [1.0 1.9]; P < .05). Levels rose further after acetylcysteine infusion in the FHF cases (mean increase, 204% [95% CI; 49 to +360]; P < .01) but not in the cases after recovery (38% [-7 to +84]). There were no significant changes in levels of plasma atrial natriuretic peptide (ANP) or cyclic adenosine monophosphate (cAMP) (mean increases, 8% [-6 to +22] and 17% [-9 to +43], respectively). The findings further support the hypothesis that the beneficial hemodynamic effects of acetylcysteine in FHF are mediated by enhancing the activity of the nitric oxide/soluble guanylate cyclase enzyme system. PMID- 8621136 TI - Prognostic value of insulinlike growth factor I and its binding protein in patients with alcohol-induced liver disease. EMALD group. AB - Insulinlike growth factor I (IGF-I) is a single-polypeptide chain with important anabolic and endocrine activities. The liver is the major source of IGF-I and its binding protein, IGFBP-3. Circulating concentrations of IGF-I and IGFBP-3 are decreased in patients with chronic liver disease and correlate with the severity. The aim of this study was to assess the additional prognostic value of IGF-I and IGFBP-3 in patients entered in a large multicenter study (EMALD). Three hundred thirty-seven patients with alcohol-induced liver disease were studied in a randomized placebo-controlled trial of malotilate with a mean follow-up period of 569 days (range, 7-1,544). A multivariate Cox regression analysis of pertinent clinical and biochemical variables showed a significant independent prognostic value of years of alcohol intake, coagulation factors 2, 7, and 10, alkaline phosphatases, serum creatinine, and immunoglobulin (Ig) M. When IGF-I or IGFBP-3 were added into this model, a Cox regression analysis showed that either had a significant independent prognostic value. Because IGF-I and IGFBP-3 were closely correlated, they contained almost the same prognostic information. Inclusion of IGF-I gave these results: IGF-I (P < .03), alcohol intake (P < .02), coagulation factors 2, 7, and 10 (P < .01), creatinine (P < .001), and IgM (P < .01) contained independent prognostic information. Inclusion of IGFBP-3 gave these results: IGFBP-3 (P < .02), alcohol intake (P < .05), coagulation factors 2, 7, 10 (P < .01), creatinine (P < .001), and IgM (P < .02) were independent predictors of survival. In conclusion, IGF-I or IGFBP-3 provide important additional information on survival in patients with alcohol-induced liver disease. PMID- 8621137 TI - The role of ultrasonography and automatic-needle biopsy in outpatient percutaneous liver biopsy. AB - The risk of complications from percutaneous liver biopsy is low, but discomfort is common and complications require hospitalization in approximately 4% of patients. The optimal method of performing these biopsies is unknown. The goal of our study was to determine whether the use of ultrasonography in the biopsy room immediately prior to or during the procedure would lessen the risk of complications and to compare the safety and efficacy in obtaining tissue by use of a Trucut needle versus an automatic biopsy needle. Between 1992 and 1994, 836 patients were entered into a randomized study (489 in Rochester, MN; 347 in Barcelona, Spain). Patients were randomized immediately prior to liver biopsy into four groups: Trucut needle, or automatic biopsy needle, and with or without ultrasonography. Fisher's Exact Test and a logistic regression model were also used to assess the effect of needle and ultrasonography on the odds for complications. The four biopsy groups were well-matched at entry with respect to age, sex, underlying liver disease, hemoglobin, prothrombin time, and platelet count. The use of ultrasound was associated with a decreased rate of hospitalization for pain, hypotension, or bleeding (2 vs. 9, P < .05). No difference in safety was found between the two types of needles. The number of passes needed to obtain specimens was similar for all four groups. The average length of the specimen was slightly greater with ultrasonographic-guided biopsies (1.7 mm vs. 1.6 mm, P < .05) and with biopsies obtained using the automatic biopsy needle when compared with the Trucut needle (1.7 mm vs. 1.5 mm, P < .05), but this did not seem to be clinically important. The addition of ultrasonography reduces complications in patients undergoing percutaneous liver biopsy. The type of needle appears to offer little difference in safety or yield of diagnostic tissue. The use of ultrasonography for guidance of percutaneous liver biopsy will lead to a lower rate of complications. The value of this benefit must be weighed against the added cost of ultrasonographic guidance. PMID- 8621138 TI - Zinc supplementation and amino acid-nitrogen metabolism in patients with advanced cirrhosis. AB - Zinc deficiency is common in cirrhosis and has been involved in the altered nitrogen metabolism. In this study, we measured the effects of zinc supplementation on the dynamics of amino acid-derived urea synthesis in cirrhosis with mild or latent encephalopathy. The hepatic conversion of amino acids into urea was studied in eight patients with advanced cirrhosis under controlled conditions of substrate availability (continuous alanine infusion), before and after 3-month oral zinc sulfate supplementation (600 mg/d). Eight more patients, matched for hepatocellular failure and encephalopathy, served as controls. Plasma zinc levels were reduced in all patients and returned to normal after oral zinc. The alanine-stimulated urea nitrogen synthesis rate in relation to alpha-amino-N concentration--the functional hepatic nitrogen clearance--increased by 25% after zinc supplementation, i.e., more urea was produced at any alpha-amino-N concentration. Basal and alanine-induced glucagon decreased by 50%, and the ammonia response to alanine decreased by 30%. Psychometric tests improved, as did routine and dynamic liver function tests and the Child-Pugh score. Also, the plasma concentration of lipid peroxides was reduced by zinc. No significant changes were observed in the control group. Our data indicate that long-term oral zinc speeds up the kinetics of urea formation from amino acids and ammonia. Changes in the hormonal drive and/or the antioxidant activity of zinc might be involved in the general improvement in liver function, whereas the beneficial effects on encephalopathy might stem from decreased ammonia. PMID- 8621139 TI - Detection of a unique gamma-glutamyl transpeptidase messenger RNA species closely related to the development of hepatocellular carcinoma in humans: a new candidate for early diagnosis of hepatocellular carcinoma. AB - Many studies concerning gamma-glutamyl transpeptidase (GGTP) in hepatocellular carcinoma (HCC) have suggested that changes in hepatic GGTP expression may be closely related to the development of HCC. However, its mechanisms are not well known, and genomic analysis of the specific GGTP to HCC is also lacking. Recently, the human GGTP complementary DNA (cDNA) sequences from fetal liver, placenta, and HepG2 cells have been published. In the present study, we sought to clarify the distribution of the GGTP messenger RNA (mRNA) molecular species in human liver and determine whether alterations in GGTP mRNA expression occur upon the development of HCC. The specific primer sets for reverse-transcription polymerase chain reaction (PCR) corresponding to the 5'-noncoding human GGTP mRNA of fetal liver (type A), HepG2 cells (type B), and placenta (type C) were prepared. Oligonucleotide probes specific for each type of mRNA were also synthesized. Liver tissues were obtained from patients with or without HCC, and total RNA was extracted. Total RNA was also extracted from various organs obtained from one male patient upon autopsy. Types of GGTP mRNAs were analyzed using type-specific primer sets and oligonucleotide probes. The types of GGTP mRNA varied in different organs. In normal liver and diseased liver without HCC, the main type of GGTP mRNA was type A. The expression was monogenic in most cases but was polygenic in some cases. In the polygenic cases, type C was common, but type B was found occasionally. On the other hand, type B was predominant in cancerous tissues with HCC. In noncancerous tissues of livers with HCC, the main types were types A and B. The prevalence of type B was significantly higher in both cancerous and noncancerous tissues of livers with HCC than in livers without HCC. The prevalence of type A in cancerous tissue, but not in noncancerous tissue, was significantly lower than in livers without HCC. These results strongly suggested that the GGTP mRNA expression in human liver may shift from type A to type B during the development of HCC. The high prevalence of type B in noncancerous tissues suggested that the shift of the GGTP mRNA may occur from the preneoplastic stage of hepatocytes. PMID- 8621140 TI - Heat serum inactivation as a mandatory procedure for antiactin antibody detection in cell culture. AB - In autoimmune hepatitis (AIH), the smooth-muscle antibody is specific for polymerized actin. Detection of antiactin antibody (AAA) has been hampered by technical problems. We have investigated AAA in 30 sera from patients with liver diseases and smooth-muscle antibody. AAA was detected by indirect immunofluorescence in 1:40, 1:80, and 1:160 dilutions. Five techniques were performed using fibroblasts: with vinblastine (A); without drugs (B); with sodium citrate (C); without drugs but with heat serum inactivation (D); and with sodium citrate and heat serum inactivation (E). For comparative analysis, we considered: the total number of AAA-positive sera regardless of the dilution in which reactivity was observed, as well as in each dilution separately; and the comparison of AAA intensity between 1:40 x 1:80, 1:40 x 1:160, and 1:80 x 1:160 dilutions. AAA was more positive in techniques B, C, D, and E than in A (P < .001) in general, and in each dilution separately. AAA was more positive in technique D than in B in 1:40 (P = .0005) and 1:80 dilutions (P = .03), as well as in E than in C (P = .0001) in 1:40 dilution. Techniques B and D yielded results similar to C and E, respectively. AAA staining was significantly more intense in 1:80 and 1:160 than in 1:40 dilution in A, B, and C; it was both significantly less intense in 1:80 and 1:160 than in 1:40 dilution and in 1:80 than in 1:160 in techniques D and E. We concluded that heat inactivation increased AAA seropositivity/intensity in 1:40 and 1:80 dilutions, preventing false-negative results; actin polymerization with sodium citrate did not enhanced AAA seropositivity/intensity. The technique with vinblastine was the least effective. PMID- 8621141 TI - Biliary malignancies in primary sclerosing cholangitis: timing for liver transplantation. AB - Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease associated in 10% to 36% of those with hepatobiliary malignancies, which are, in the majority of cases, not known prior to transplantation. Diagnosis of carcinomas in a PSC setting at an early stage has not yet been achieved, because there are no differences in the age of patients or clinical course, particularly with regard to the time between diagnosis of PSC and detection of carcinomas. To assess optimal timing for transplantation in patients with PSC, we applied the Mayo survival model to 48 patients receiving transplants for that disease in our center between 1972 and 1994. Of these patients, 10 had a biliary malignancy, which was incidental in 9. According to the Mayo model, low-, moderate-, and high risk groups of patients could be formed. The actuarial patient survivals at 1 and 7 years were 100% and 100% (low risk), 68.6% and 68.6% (moderate risk), and 54.6% and 46.8% (high risk), respectively. Patients with a biliary malignancy had a 30% survival at 1 year; none survived 6 years. Local recurrence of the tumor was found in 3 patients, 2 of them with low tumor stages at the time of transplantation. Analysis of the Mayo Model risk scores demonstrated a marked increase in the incidence of biliary malignancies at a score above 4.4. All patients with tumors were found to have a score above 4. Moreover, the prevalence rate rose from 14.3% in the low-risk group to 33.3% in the moderate-risk group. There was no difference in the clinical courses at 6 to 12 months prior to transplantation; in particular, the bilirubin levels (PSC alone, 250 +/- 230 mumol/L; PSC with carcinoma, 288 +/- 182 mumol/L) did not differ significantly (P > .05) between both patient groups. Because the outcome after transplantation is poor even in patients with low-grade malignancies, early timing of transplantation in patients with PSC is suggested to prevent formation of biliary malignancies. Therefore, regular scoring of patients with the Mayo Model risk score is suggested, and transplantation should be taken into consideration at scores above 4. PMID- 8621142 TI - Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis. AB - The aim of this study was to identify high-risk patients for hepatocellular carcinoma (HCC). Among 151 patients with histologically proven cirrhosis hospitalized from 1987 to 1990 and prospectively followed-up until June 1994, 31 developed HCC. We assessed the predictive value of 22 variables recorded at enrollment for HCC occurrence by the log rank test and the Cox proportional hazards model. Six clinical and biological variables summarized predictive information of HCC: age > or = 50 years (P = .01), male (P = .01), large esophageal varices (EV) (P = .03), prothrombin activity < 70% (P = .04), serum alpha-fetoprotein (AFP) > or = 15 ng/L (P = .06), and anti-hepatitis C virus antibodies (P = .08). A clinicobiological predictive score identified two groups of patients at low (n = 67; 3-year cumulative incidence, 0%) and high risk for HCC (n = 84; 3-year cumulative incidence, 24%). The predictive value of this score was confirmed using an independent population of 49 patients with cirrhosis. Furthermore, liver large-cell dysplasia (LCD) had an additional predictive value in high-risk patients (P = 10(-4), which thus helped to define a subgroup at very high risk for HCC (n = 12; 3-year cumulative incidence, 72%). In Western patients with cirrhosis, a limited number of usual variables can identify a group of patients at high risk for HCC. Among these patients, liver biopsy allows for the determination a subgroup of patients at very high risk for HCC requiring intensive screening or preventive measures. PMID- 8621143 TI - Auxiliary liver transplantation: regeneration of the native liver and outcome in 30 patients with fulminant hepatic failure--a multicenter European study. AB - Auxiliary liver transplantation (LT) is a special procedure of LT which could be proposed to patients with fulminant hepatic failure (FHF) and has for aim that complete regeneration of the native liver (NL) left in place will allow the graft recipient to resume normal liver function after allograft withdrawal. We report 30 cases of auxiliary LT performed for FHF in 12 European centers. Twenty-five of 30 patients were younger than 50 years. The cause of FHF was hepatitis A virus (HAV) in 4 patients, hepatitis B virus (HBV) in 7, paracetamol overdose in 5, ecstasy in 2, hepatotoxic drugs in 4, autoimmune hepatitis in 2, liver lesions of preeclampsia in 1 and unknown in 5. A postoperative, both clinical and histological follow-up of more than 3 weeks was obtained in 22 patients, enabling us to look for indicators predictive of NL regeneration and outcome. Histological changes observed in the NL included complete regeneration in 68%, incomplete regeneration with obvious fibrous sequelae in 14% and severe liver fibrosis or cirrhosis in 18%, of the 22 patients studied. The percentage and distribution of necrosis observed in tissue samples of the NL at the time of transplantation was not related to the final outcome. Complete NL regeneration was observed in 15 patients, out of whom 14 were younger than 40 years. Patients with complete regeneration were mainly affected by FHF due to HAV, HBV, or paracetamol overdose. After a follow-up of 18/11 (mean/median) months (range, 3 to 67 months), 19 of the 30 patients (63%) survived and 13 of them (68%), i.e., 43% of the 30 patients, had resumed normal NL function, with interrupted immunosuppression, the ultimate goal of emergency auxiliary LT. We conclude that, in patients with FHF, auxiliary LT is a procedure feasible in a number of centers and is associated with a complete regeneration capability of the NL in a majority of survivors, especially in those younger than 40 years. Confirmation of these encouraging preliminary results by large-scale prospective studies is required. PMID- 8621144 TI - Effect of liver transplantation on QT interval prolongation and autonomic dysfunction in end-stage liver disease. AB - Both a prolonged QT interval and disturbance of autonomic nervous system function are markers of poor prognosis in patients with diabetes mellitus and alcoholic liver disease (ALD). We studied the prevalence of abnormal QT interval and autonomic nervous system dysfunction in 53 consecutive patients with end-stage liver disease before and after orthotopic liver transplantation (OLT). The maximum QT interval in any lead (QTmax) was assessed by two independent observers. The QTmax, corrected for heart rate (QTcmax) was prolonged in 44 patients (83%), although increased QT dispersion was not found. There was a significant correlation between the QTcmax and Child-Pugh score but not with etiology. Evidence of parasympathetic dysfunction was present in 41 patients (77%), and sympathetic dysfunction was present in 20 patients before OLT. Fifty two patients underwent transplantation. There was significant improvement in the QTcmax interval after OLT (P < .001); 32 of the 44 patients with prolonged QTcmax ( > 440 milliseconds) improved. Repeat testing was not performed in 7 patients, because they had died or had not undergone transplantation. Indices of parasympathetic function improved in 27 patients after OLT, but no improvement was observed in 8. Improvement in sympathetic dysfunction was observed in 13 of the 19 patients tested. There was no association between QTcmax, autonomic dysfunction, and survival. These results suggest that both prolonged QTcmax and some tests of autonomic function are temporary and arise as a consequence of liver dysfunction. PMID- 8621145 TI - Long-term administration of isosorbide-5-mononitrate does not impair renal function in cirrhotic patients. AB - Isosorbide-5-mononitrate (Is-5-Mn), alone or combined with beta-blockers, has been proposed for prophylaxis of variceal bleeding in cirrhosis. However, renal insufficiency, might be an important undesirable effect of this therapy, especially in patients with ascites. We assessed the changes in renal function induced in 26 cirrhotic patients by acute or chronic administration of Is-5-Mn. The acute administration of 20 mg of Is-5-Mn to 21 patients reduced mean blood pressure (83.4 +/- 2.4 vs. 92.8 +/- 3.4 mm Hg, P < .001), urine volume (5.5 +/- 0.8 vs. 8.7 +/- 1.1 mL/min, P < .05), urine sodium excretion (114 +/- 19 vs. 244 +/- 41 muEq/min, p < .001), urine potassium excretion (41 +/- 3.4 vs. 67 +/- 8.5 muEq/min, P < .001), and atrial natriuretic factor (74 +/- 10 vs. 98 +/- 12 pg/mL, P < .005). The glomerular filtration rate was decreased in the 11 patients with ascites (57 +/- 9 vs. 68 +/- 12 mL/min, P < .05), and plasma renin activity was increased in 4 ascitics. Twenty-one patients (16 from the acute study + 5 other patients) were given Is-5-Mn for 3 months at the dose of 80 mg/d. This did not affect blood pressure and renal function in patients without ascites, but reduced mean blood pressure (91.9 +/- 3.4 vs. 89.6 +/- 3 mm Hg, P < .05), urine volume (5.8 +/- 1.1 vs. 3.4 +/- 0.9 mL/min, P < .05), and urine sodium excretion (205 +/- 38 vs. 99 +/- 16 muEq/min, P < .01) in those with ascites. There were no changes in glomerular filtration rate and renal plasma flow, while plasma renin activity increased in only 3 patients with ascites and 1 without. Systemic hemodynamics and renal function of cirrhotic patients, especially those with ascites, are affected adversely by acute administration of Is-5-Mn. Long-term administration of the drug is well tolerated by compensated patients and does not affect renal plasma flow nor glomerular filtration rate, but can induce hypotension and sodium retention in patients with ascites. PMID- 8621146 TI - The effect of posture on central blood volume in patients with preascitic cirrhosis on a sodium-restricted diet. AB - The status of the central blood volume in cirrhosis is controversial. A combination of sodium restriction and upright posture, which redistributes intravascular volume to dependent parts of the body should further aggravate a contracted central blood volume reduction. The aim of this study was to determine the effect of upright posture and sodium restriction on central blood volume (CBV) in preascitic cirrhotic patients, compared with controls. Eight male, preascitic, alcoholic cirrhotic subjects and eight healthy male controls were studied while on a 20-mmol/d sodium diet. Measurements of CBV by radionuclide angiography, and neurohumoral factors were performed on day 7 in both supine and erect positions and cardiac output and systemic vascular resistance (SVR) was calculated. Sodium restriction resulted in less weight loss in the cirrhotic patients (P = .03), with significantly lower plasma renin activity (P = .001). Similar central blood volumes and systemic hemodynamics were observed in both groups in the supine posture. In contrast to the cirrhotic patients, in the control subjects, upright posture resulted in a significant reduction in cardiac output (P = .002) and increase in SVR (P = .005), associated with a decrease in all blood volumes which were significantly less than in the cirrhotic patients. Mean arterial pressure was maintained in both groups in both postures. In conclusion, with sodium restriction, preascitic cirrhotic patients have less intravascular volume contraction than control patients. Erect posture results in redistribution of this relatively expanded intravascular volume to the CBV. Therefore, a low-sodium diet can be safely administered in preascitic cirrhotic patients. PMID- 8621147 TI - Is the Mayo model for predicting survival useful after the introduction of ursodeoxycholic acid treatment for primary biliary cirrhosis? AB - Treatment of patients with primary biliary cirrhosis (PBC) using ursodeoxycholic acid (UDCA) leads to a reduction in serum bilirubin. The first objective of this study was to assess the performance of certain prognostic indicators for PBC after the introduction of treatment with UDCA. Serum bilirubin is an important prognostic indicator for PBC and an important component of the Mayo model for grading patients into risk categories. In an analysis of patients enrolled in the Canadian multicenter trial, the Mayo score was calculated before and after treatment with UDCA. After treatment, the Mayo score continued to divide patients with PBC into groups with varying risk. In addition, the serum bilirubin alone was shown to do the same even after the introduction of treatment with UDCA. A second objective was to establish whether UDCA had an effect on long-term (2- to 6-year) survival in patients with PBC. PMID- 8621148 TI - Relationship between serum and hepatic 7S fragments of type IV collagen in chronic liver disease. AB - We evaluated the mechanism of increased serum concentrations of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in chronic liver disease. We measured the concentrations of hepatic-free and deposited 7S collagens after extraction with Tris-HCl buffer and bacterial collagenase, then compared them with the serum levels in 8 normal controls and 48 patients with chronic liver disease. The hepatic 7S collagen levels extracted with Tris-HCl buffer and collagenase accounted for 7% and 93%, respectively, of the total 7S collagen levels in normal controls. Both hepatic 7S collagen levels as well as serum levels increased in accordance with the progress of liver disease. Serum levels of 7S collagen showed a closer correlation with the hepatic 7S collagen levels extracted with Tris-HCl buffer (r = .822), compared with those extracted with collagenase (r = .382). On the other hand, the histological degrees of liver fibrosis were highly correlated with the hepatic collagenase-extracted 7S collagen levels (r = .822), compared with serum and the hepatic Tris-HCl buffer extracted levels (r = .478 and r = .537, respectively). Although there was no difference in serum and hepatic 7S collagen levels between B and C viral patients, the serum and hepatic Tris-HCl buffer-extracted 7S collagen levels were higher in patients with alcoholic cirrhosis than patients with viral cirrhosis. However, the hepatic collagenase-extracted levels were similar in both groups. Gel filtration demonstrated that the serum and hepatic Tris-HCl buffer-extracted 7S collagens were mainly eluted in the macromolecular 7S collagen-reactive fraction in cirrhosis, whereas the hepatic collagenase-extracted 7S collagen was eluted in the authentic 7S collagen-reactive fraction. The results suggest that serum 7S collagen levels are not a particularly reliable measure of hepatic fibrosis but reflect the enhanced metabolism, especially synthesis of type IV collagen in the liver. PMID- 8621149 TI - Modulation of rat hepatocyte proliferation by bile salts: in vitro and in vivo studies. AB - In this study, the stimulatory effect of bile salts (BS) was evaluated both in vitro, using hepatocyte primary cultures, and in vivo, in normal and 40% partially hepatectomized rats previously fed on BS-enriched diets for 4 weeks. In vitro results show that conjugated cholate (CA) and chenodeoxycholate (CDCA) augmented proliferative activity in rat hepatocytes cultured in absence of mitogens, whereas conjugated deoxycholate (DCA), and ursodeoxycholate (UDCA) did not have any significant effect. None of these BSs increased significantly the replicative response induced by submaximal concentrations of epidermal growth factor (EGF). In vivo, at the end of dietary treatment all animals fed on CA or DCA but not those fed on either CDCA, or UDCA, or tauroursodeoxycholate (TUDCA) developed cholestatic hepatitis and a burst of damage-induced hepatocyte proliferation. After 40% partial hepatectomy (PH), CA- and DCA-treated groups underwent a deterioration of cholestatic hepatitis. On the other hand, in CDCA-, and UDCA-, and TUDCA-treated groups liver histology, serum glutamic pyruvic transaminase (SGPT) and cholestasis indices did not change significantly compared with controls. As far as the proliferative activity, a significant increase was observed not only in CA and DCA but also in UDCA- and TUDCA-fed groups compared with controls, whereas a slight decrease was observed in CDCA-treated animals. In conclusion, our data indicate that conjugated BSs had only a modest stimulatory effect on hepatocyte proliferation in vitro. However, in vivo, in PH rats, UDCA or TUDCA treatment determined a further increase of hepatocellular proliferation not attributable to hepatotoxic effects. Our result suggest that modifications of bile acid pool could modulate hepatocellular proliferation. PMID- 8621150 TI - Inhibitory actions of cyclic adenosine monophosphate and pertussis toxin define two distinct epidermal growth factor-regulated pathways leading to activation of mitogen-activated protein kinase in rat hepatocytes. AB - Increased intracellular cyclic adenosine monophosphate (cAMP) levels have been shown in some reports to inhibit and in other studies to stimulate growth factor mediated activation of the mitogen-activated protein kinase (MAP kinase) pathway, depending on the cell type examined. The relationship between cAMP and MAP kinase in hepatocytes has not been examined. In the current study, stimulation of primary cultures of rat hepatocytes with hepatocyte growth factor (HGF) or epidermal growth factor (EGF) increased Ras, Raf, and MAP kinase activity. Incubation of hepatocytes with cAMP-increasing agents blocked activation of Raf by both HGF and EGF, whereas activation of Ras was unaffected. MAP kinase activation by HGF was completely inhibited, whereas EGF-stimulated MAP kinase activity was only slightly reduced. Incubation of hepatocytes with pertussis toxin slightly blunted MAP kinase activation by EGF but not HGF. Increasing cAMP in hepatocytes preincubated with pertussis toxin completely inhibited the activation of MAP kinase by EGF. In conclusion, HGF activates MAP kinase in hepatocytes exclusively through an Raf-dependent pathway and this activation may be completely blocked by increasing cAMP. In contrast, EGF activates MAP kinase in hepatocytes through both Raf-dependent and Raf-independent pathways: the latter pathway probably involves a pertussis toxin-sensitive G protein. PMID- 8621151 TI - Vasoactive intestinal peptide in cirrhotic rats: hemodynamic effects and mesenteric arterial receptor characteristics. AB - Vasoactive intestinal peptide (VIP) blood levels in cirrhosis are elevated, but its hemodynamic and receptor characteristics remain unclarified. We aimed to quantify VIP receptor characteristics in mesenteric arteries, plasma VIP concentration by radioimmunoassay (RIA), and the hemodynamic effects of VIP infusion in bile duct-ligated (BDL) cirrhotic and sham-operated control rats. Mesenteric arterial membranes were prepared by ultracentrifugation, and receptor characteristics were studied using 125I-labeled VIP as a radioligand. For the hemodynamic study, there were four groups: cirrhotic and sham-operated rats were infused with either VIP (50 ng/kg/min for 15 minutes) or equivolumic isotonic saline. Regional blood flows were measured in conscious rats with radioactive microspheres. Receptor studies showed high- and low-affinity binding sites for VIP, which had similar equilibrium dissociation constants (binding affinities) and receptor densities for both the cirrhotic and control rats. Plasma VIP concentrations were significantly elevated in the cirrhotic rats. In both cirrhotic and sham-operated rats, VIP infusion produced plasma levels approximately twofold to threefold increased over the basal levels observed in cirrhotic rats. In cirrhotic rats, VIP infusion did not affect any hemodynamic parameter, whereas in the sham-operated rats VIP infusion significantly increased the mesenteric visceral blood flow. These results show that the hyporesponsiveness to VIP in cirrhotic rats is not attributable to receptor downregulation, implying postreceptor alterations. This suggests that VIP may not play a major role in the maintenance of splanchnic hyperemia in cirrhosis. PMID- 8621152 TI - Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma. AB - The expression of the HLA class I molecules on the cell surface was investigated in hepatocellular carcinoma (HCC) cell lines using complement-mediated cytotoxicity (CMC) and flow cytometric analysis. Although HLA-A antigens were detected by CMC in all cell lines tested, HLA-B and -C antigens were not detectable in six of seven HCC cell lines. These results were also confirmed by flow cytometric analysis focusing on HLA-Bw4 and Bw6 public antigens. Furthermore, complementary DNA (cDNA) from each cell line was tested for the expression of HLA-A, -B, -C and the transporter associated with antigen processing genes (TAP1 and TAP2). Two cell lines showed a reduced level of one or both of the TAP messenger RNAs (mRNAs), and one of these showed a reduction of HLA-B and -C gene expression as well, but the others had detectable mRNA levels. These results demonstrate that hepatocellular carcinoma cell lines tested in the current study lose or decrease the expression of HLA-B and -C alleles on the cell surface, even though mRNA encoding these alleles is present, suggesting that the loss of the HLA molecules might be caused by posttranscriptional events or failure to transport and load peptides necessary for HLA expression. The selective loss of HLA-B and -C, but not -A, molecules (which also excludes a beta 2-microglobulin defect) is intriguing, and may be attributable to the ability of some of the HLA-A molecules to load signal peptides not requiring TAP transport, or to natural selection of HLA-B or -C locus-specific immune surveillance. PMID- 8621153 TI - Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis. AB - Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation. PMID- 8621154 TI - Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1 induced apoptosis in primary cultures of hepatocytes. AB - Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death. PMID- 8621155 TI - Reversible impairment of neonatal hepatobiliary function by maternal cholestasis. AB - The effect of total blockage of maternal biliary excretion during the last third of the pregnancy on the maturation of hepatobiliary function was investigated in neonatal rats. Extrahepatic obstruction of the common bile duct on day 14 of pregnancy induced a marked enhancement in serum bilirubin--mainly conjugated bilirubin--and bile acid concentrations as compared with sham-operated pregnant rats. Excretion of bile acids by the kidney was significantly increased, whereas fecal elimination of these compounds was almost abolished. Most of the cholestatic mothers (CMs) (77%) were able to carry pregnancy to term and lactation until weaning (21 days after birth). The body and liver weights of their offspring were lower than for offspring of control healthy mothers in all postnatal periods considered. Serum bile acid concentrations were higher in the fetuses and neonates of CMs. This difference was evident up to 1 week after weaning and disappeared in young adult animals (8 weeks old). When the bile secretion rate was investigated in these animals at 4 or 8 weeks of age, no significant difference was found as far as nonstimulated bile flow and bile acid output was concerned. However, the biliary response to stepwise sodium taurocholate (TC) intravenous infusion showed that 4-week-old neonates of CMs had impaired bile acid secretion. Moreover, the maximal secretion rate (SRmax) for TC was significantly reduced (-30%), whereas the choleretic ability of taurocholate was not modified. This alteration was not selective for bile acids. The SRmax for bromosulfophthalein (BSP) was also significantly lowered (-40%). These dysfunctions were overcome during subsequent development. No impaired biliary response to either TC or BSP infusion was observed at 8 weeks of age. Morphological abnormalities in the canaliculi were found in animals with impaired biliary function. In summary, these results indicate that maternal cholestasis may profoundly but transiently impair the normal liver maturation. The importance of the implications derived from these findings both in the nutrition and management of human neonates demands further evaluation of the hepatobiliary function of babies born after alterations of fetal-maternal bile acid homeostasis, such as in maternal obstetric cholestasis. PMID- 8621156 TI - Long-term octreotide treatment prevents vascular hyporeactivity in portal hypertensive rats. AB - Chronically portal-hypertensive rats show in vitro vascular hyporeactivity in large part mediated by the endothelium-derived vasodilator nitric oxide. We tested whether long-term octreotide treatment (15 micrograms/kg subcutaneously in 5% D/W, 8-hourly) corrects the in vitro vascular hyporeactivity. Increases in perfusion pressures (delta mm Hg) to potassium chloride (30-300 mmol/L) of in vitro perfused superior mesenteric arterial vascular beds of partial portal vein ligated (PVL) rats were significantly (P < .05) higher in octreotide (n = 9) compared with placebo (n = 10, 5% D/W) treated animals. Octreotide significantly (P < .05) increased mean arterial pressure compared with placebo, the values being 129 +/- 3 and 117 +/- 4 mm Hg, respectively. Furthermore, a significant (P < .001) correlation was observed between in vitro vascular reactivity and mean arterial pressure. Incubation of separate vascular beds (n = 7 for both PVL and sham-operated rats) with octreotide (10(-6) mol/L) did not enhance pressure responses to 125 mmol/L potassium chloride, and failed to increase perfusion pressures in preconstricted vessel preparations (n = 6), excluding a direct inhibitory effect on NO. In summary, long-term octreotide treatment prevents in vitro vascular hyporeactivity in prehepatic portal-hypertensive rats, and octreotide does not exert its action through direct effects on endothelium derived NO. PMID- 8621157 TI - Role of Kupffer cells in arresting circulating tumor cells and controlling metastatic growth in the liver. AB - Metastasis to the liver is a common event in clinical oncology. Blood-borne tumor cells (TCs) arriving to the liver sinusoids run into a special vascular bed. The lining of liver sinusoids is shared by Kupffer cells (KCs) and endothelial cells. KCs, liver-fixed macrophages, are responsible for detection and removal of "non self" particles. To investigate their role in arresting blood-borne TCs and controlling tumor growth, we injected a syngeneic colon carcinoma cell line into a mesenteric vein of two groups of rats; one group was without Kupffer cells and the other normal controls. We removed the liver of these animals at different time intervals and performed immunohistochemical analysis with monoclonal antibodies (MoAbs) against our tumor cell line, three macrophage subpopulations, natural killer cells, and B and T lymphocytes. Additionally, we showed in vitro spontaneous cytotoxicity of KCs against our tumor cell line. Results suggest that KCs play a relevant role in arresting circulating TCs at the liver sinusoid, although it is limited to a small number of malignant cells. They also seem to play a major role in clearing neoplastic cells from the liver parenchyma, in controlling tumor growth in the very early stages of metastatic development, and in modulating the host immune response to cancer cells. PMID- 8621158 TI - Binding of apolipoprotein A-I and acetaldehyde-modified apolipoprotein A-I to liver extracellular matrix. AB - Apolipoprotein A-I (Apo A-I), a protein produced mainly by hepatocytes, is decreased in the sera of alcoholic patients with liver fibrosis and cirrhosis. To explain this decrease, we investigated possible interactions between liver extracellular matrix (ECM) and Apo A-I. Using a solid-phase binding assay, we evaluated the binding of Apo A-I to the different liver matrix components. Apo A I bound significantly to fibronectin (FN) (optical density [OD] = 1.11 +/- .26, P = .01) and collagen (C) I (OD = 0.91 +/- 0.22, P = .02) in comparison with bovine serum albumin (BSA) (OD = 0.26 +/- 0.16). Binding of Apo A-I to fibronectin was concentration dependent and saturable. Apo A-I bound also to ECM in vivo because Apo A-I was detected by immunofluorescence on fibrous septa in liver biopsy specimens of alcoholic patients. Because a negative correlation between Apo A-I and liver fibrosis is amplified in alcoholic patients, we investigated whether the in vitro formation of Apo A-I/acetaldehyde complex (adducts) increased the binding of Apo A-I to the ECM. We showed that the amount of Apo A-I that bound to FN was significantly higher with acetaldehyde-modified Apo A-I (OD = 2.18 +/- 0.19, P = .01) than with native Apo A-I. This increase was probably related to the formation and binding of Apo A-I dimers, because immunoblot of in vitro acetaldehyde-modified Apo A-I showed the formation of dimeric Apo A-I. In conclusion, FN binds both native and acetaldehyde-modified Apo A-I. Because FN is deposited early and in excess during liver fibrosis, a storage mechanism of Apo A I on newly deposited fibronectin would explain, in part, the decrease observed in alcoholic patients with liver fibrosis. PMID- 8621159 TI - Kupffer cell depletion by liposome-delivered drugs: comparative activity of intracellular clodronate, propamidine, and ethylenediaminetetraacetic acid. AB - Macrophages such as Kupffer cells in the liver are multifunctional cells. They are involved in host defense mechanisms and have a regulatory role in many biomedical processes. Their selective depletion, using liposome-encapsulated drugs, forms a widely accepted approach to studying their functional aspects in vivo. We have compared the Kupffer cell-depleting activities of liposome encapsulated clodronate, propamidine, and ethylenediaminetetraacetic acid (EDTA) for this purpose. These molecules represent the drug families of bisphosphonates, diamidines (or aromatic polyamidines), and polyaminopolycarboxylic acid-chelating agents, respectively. The Kupffer cell-depleting activity of the liposome encapsulated antimicrobial drug propamidine exceeded that of clodronate by about a factor of 10. EDTA appeared to be inefficacious for depletion of Kupffer cells in the rat. PMID- 8621160 TI - Mitogen-activated protein kinase activation in hepatocyte growth factor stimulated rat hepatocytes: involvement of protein tyrosine kinase and protein kinase C. AB - Hepatocyte growth factor (HGF) stimulated mitogen-activated protein (MAP) kinases and MAP kinase kinase in primary cultured rat hepatocytes. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced MAP kinase activity. A PKC activator, phorbol myristate acetate (PMA), induced MAP kinase activation in a concentration-dependent manner. Protein tyrosine kinase (PTK) inhibitors, genistein, and ST638 also inhibited HGF-induced MAP kinase activation. Furthermore, HGF increased formation of Ras guanosine triphosphate (GTP) complex, indicating Ras activation. Genistein inhibited HGF-induced Ras activation, but Ro31-8425 was without effect. On the other hand, Ro31-8425 decreased HGF-induced [3H]arachidonic acid (AA) release and [3H]thymidine incorporation. Genistein also prevented [3H]AA release and [3H]-thymidine incorporation. Moreover, a commonly used phospholipase A2 (PLA2) inhibitor, quinacrine, decreased HGF-induced [3H]AA release and [3H]thymidine incorporation. The inhibitory profile of [3H]AA release was well correlated with that of [3H]thymidine incorporation in Ro31-8425-, genistein-, and quinacrine-treated cells. A cyclooxygenase inhibitor, indomethacin, which suppressed HGF-induced DNA synthesis, had minimal effect on MAP kinase activation. In contrast, prostaglandin (PG) E1, E2, or F2 alpha, which stimulate [3H]thymidine incorporation to the same level as that caused by HGF in hepatocytes, caused very weak activation of MAP kinases. These results suggest that PTK, Ras, and PKC play roles in MAP kinase activation induced by HGF and that MAP kinase activation resulting in AA release is involved in DNA synthesis in rat hepatocytes. PMID- 8621161 TI - Expression of a rat liver phosphatidylcholine translocator in Xenopus laevis oocytes. AB - A phospholipid translocating protein from rat liver has been expressed in Xenopus laevis oocytes. Injection of oocytes with total rat liver messenger RNA (mRNA) resulted in the function expression of saturable uptake of the water soluble phophatidylcholine derivative L-alpha-dibutyroylglycero-3-phophatidylcholine (diC4PC), Kinetic studies revealed an apparent Km value of approximately 10 mmol/L, which is similar to the value previously obtained in isolated rat liver canalicular plasma membrane vesicles for an adenosine triphosphate (ATP) independent phosphatidylcholine translocator. Size fractionation of total rat liver mRNA yielded an active mRNA species between 1.8 and 2.6 kb, that stimulated the expressed phophatidylcholine uptake activity approximately fivefold as compared with differently sized mRNA subfractions. This active mRNA size class is too small to code for the mdr2 P-glycoprotein, which has been suggested to function as an ATP-dependent canalicular phosphatidylcholine translocator. Hence, the data indicate that there are at least two separate polypeptides involved in phospholipid translocation from hepatocytes into bile. PMID- 8621162 TI - Localization of alpha 2-macroglobulin protein and messenger RNA in rat liver fibrosis: evidence for the synthesis of alpha 2-macroglobulin within Schistosoma mansoni egg granulomas. AB - alpha 2-Macroglobulin (alpha 2M) in the rat is a strong-reacting acute-phase protein with potent protease-inhibiting and cytokine-binding properties. Production of alpha 2M is ascribed mainly to liver parenchymal cells. In the present study, we investigated, by means of immunohistochemistry and in situ hybridization, whether fibrosis in the rat liver induced by Schistosoma mansoni eggs leads to local production of alpha 2M. alpha 2M protein and messenger RNA (mRNA) in the unaffected liver tissue, as well as serum values of alpha 2M, were comparable in control rats and egg-injected rats, at 1, 3, and 8 weeks after injection of the eggs. alpha 2M was homogeneously distributed across the liver lobule. In contrast, at the sites of the granulomas, a strong increase in alpha 2M was observed. alpha 2M mRNA was expressed by granuloma cells, but not by the surrounding liver parenchymal cells. Within the granulomas, alpha 2M protein was present in numerous spindle-shaped cells and was diffusely distributed in the extra-cellular matrix. Using double-staining techniques, a subpopulation of the alpha 2M-positive cells in the granulomas appeared to be desmin-positive, suggesting a myofibroblast origin. In addition, parenchymal cells directly surrounding the granulomas contained alpha 2M protein in approximately 50% of the granulomas 1 week after injection of the eggs. In situ hybridization on consecutive sections revealed that these parenchymal cells showed only background activity of alpha 2M mRNA, suggesting uptake of alpha 2M-protein by these parenchymal cells and previous activation of alpha 2M by proteases within the granuloma. The significance of the present study is that alpha 2M is produced locally at sites of inflammation and liver fibrosis, without measurable increase of serum levels of alpha 2M. Unexpectedly, alpha 2M present at the sites of the granulomas is not produced by the liver parenchymal cells, but rather by granuloma cells. PMID- 8621164 TI - Endoscopic therapy for cholangitis in cirrhosis. PMID- 8621163 TI - Hepatic silicosis, cirrhosis, and liver tumors in mice and hamsters: studies of transforming growth factor beta expression. AB - Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma. PMID- 8621165 TI - Choledocholithiasis: in quest of the stone. PMID- 8621166 TI - The pruritus of cholestasis is relieved by an opiate antagonist: is this pruritus a centrally mediated phenomenon? PMID- 8621167 TI - Familial prevalence of gallstones in first-degree relatives of patients with gallstones. PMID- 8621168 TI - Quantity and location of stone compounds, not the mere presence in small amounts within the stone fabric, are important factors for the correct classification of gallstones and a better knowledge of their pathogenesis. PMID- 8621169 TI - Virological patterns of hepatitis C virus in chronic hepatitis C with biochemical sustained response to interferon therapy. PMID- 8621170 TI - Are hepatitis C virus genotypes 1a and 1b so different? PMID- 8621171 TI - Glutamine, myo-inositol, and brain edema in acute liver failure. PMID- 8621172 TI - Publishing trends in hepatology. PMID- 8621173 TI - Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection. AB - The significance of circulating antibody to hepatitis C virus (HCV) envelope glycoprotein 2 (E2)/nonstructural protein 1 (NS1) glycoprotein was studied in 83 patients with chronic HCV infection diagnosed by polymerase chain reaction (PCR). E2/NS1 antibody was quantitatively examined by a passive hemagglutination test using recombinant E2/NS1 glycoprotein encompassing amino acids 388 to 664 of the HCV-H strain. The results were correlated with clinical and virological features such as genotypes and viremic levels assessed by a competitive reverse transcription PCR assay. E2/NS1 antibody was found in 73 patients (88%), and its occurrence was related to viremic levels. E2/NS1 antibody titers were low in asymptomatic HCV carriers with low levels of viral replication; 9 of 17 such patients tested positive for E2/NS1 antibody (53%), compared with 64 of 66 chronic hepatitis C patients (97%) (P < .01). A significant direct relationship was observed between viremic levels and E2/NS1 antibody titers (r = .52, P < .01). Of the 13 patients with low viremic levels of < 10(6) copies/mL, only 5 tested positive for E2/NS1 antibody (38%), whereas 68 of the 70 patients with viremic levels of > or = 10(6) copies/mL had it (97%) (P < .01). As for the relation to HCV genotypes, no difference was seen in E2/NS1 antibody titers among genotypes examined (1b, 2a, and 2b). These findings suggest that the E2/NS1 antibody tested exhibits no neutralizing activity in chronic HCV infection but may serve as a serological indicator of active virus replication. PMID- 8621174 TI - A pilot study of corticosteroid priming for lymphoblastoid interferon alfa in patients with chronic hepatitis C. AB - Interferon treatment reduces the serum level of hepatitis C virus (HCV) and improves inflammatory activity, but relapse is frequently observed. In an attempt to develop a new therapeutic strategy that may reduce relapse and cure the disease, we evaluated the effect of corticosteroid priming on lymphoblastoid interferon alfa in an open randomized clinical trial. The level of HCV RNA increased significantly during corticosteroid priming (from 5.60 [median] to 21.0 x 10(5) Eq/mL; P = .0004) but decreased to the pretreatment level 4 weeks after cessation of corticosteroid (7.0 x 10(5) Eq/mL; P = .07). Sustained normalization of alanine transaminase (ALT) level and virus clearance, confirmed by negative results for HCV RNA using reverse-transcription nested polymerase chain reaction (PCR), were observed over a period of 6 months in 8 of 19 (42.1%) corticosteroid primed patients, compared with 6 of 19 patients (31.6%) treated with interferon only. A "rebound" of ALT after the withdrawal of corticosteroid was observed in only 2 of 19 patients primed with corticosteroid, but both showed sustained responses. Multivariate analysis for factors predictive of the sustained response indicated that HCV titers measured immediately before interferon therapy and HCV genotype were statistically significant (P = .006 and P = .025, respectively). Our results indicated that corticosteroid priming has a marginal benefit over treatment with interferon alone and that large-scale clinical trials are necessary to determine whether interferon with corticosteroid priming is more effective than interferon alone. PMID- 8621175 TI - Liver-targeted antiviral nucleosides: enhanced antiviral activity of phosphatidyl dideoxyguanosine versus dideoxyguanosine in woodchuck hepatitis virus infection in vivo. AB - It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3' Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP-ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP-ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis. PMID- 8621176 TI - Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus. AB - Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton mediated process occurred. The productive infection of SEC may contribute to the progression of the infection. PMID- 8621177 TI - Severe recurrent cholestatic hepatitis C following orthotopic liver transplantation. AB - Recurrent infection with hepatitis C virus (HCV) is almost universal following orthotopic liver transplantation although clinical severity varies. Data on 135 patients who underwent transplantation for hepatitis C cirrhosis were reviewed. We describe a progressive, severe cholestatic form of hepatitis occurring in a subgroup of patients with recurrent hepatitis C. Ten patients with severe recurrent hepatitis C were identified; 1 has died, 1 awaits retransplantation, and 8 have undergone retransplantation. All 10 developed severe progressive cholestatic hepatitis, with a mean rise in bilirubin to 24.7 mg/dL at the time of retransplantation. Histology at initial recurrence was of mild hepatitis without evidence of rejection. The failed grafts showed either cirrhosis or confluent hepatic necrosis. The onset of cholestasis preceded retransplantation by less than 5 months. Our study suggests that a minority of patients with recurrent hepatitis C after undergoing liver transplantation develop a severe progressive cholestatic hepatitis and liver failure. PMID- 8621178 TI - Hepatitis C virus RNA in peripheral blood mononuclear cells: comparing acute and chronic hepatitis C virus infection. AB - Hepatitis C virus (HCV) can infect peripheral blood mononuclear cells (PBMC) of patients with chronic HCV infection. No data are available on PBMC testing for HCV RNA in acute hepatitis C. This study investigated the presence of HCV RNA in PBMC of patients with acute posttransfusion hepatitis C, compared with those with chronic HCV infection. Nested polymerase chain reaction (PCR) was applied to detect HCV RNA in 111 and 48 paired samples of serum and PBMC of 11 patients with acute posttransfusion hepatitis C and 48 patients with chronic HCV infection, respectively. In patients with acute posttransfusion hepatitis C, HCV RNA was detected in 17 of 29 (59%) and 67 of 82 (82%) serum samples collected during the incubation period and acute phase, respectively. Meanwhile, of the 48 patients with chronic HCV infection, 41 had serum HCV RNA (85%). HCV RNA was not detected in PBMC samples from incubation period or from acute-phase hepatitis, although it was detected in 12 of the 48 PBMC samples of chronically infected patients (25%) P < .005). Of the 12 PBMC specimens positive for positive-stranded HCV RNA, 6 were also positive for negative-stranded HCV RNA. Among patients with chronic HCV infection, HCV infection of PBMC was not related to age, sex, blood transfusion, serum alanine transaminase (ALT) levels, or serum virus titers. In conclusion, HCV infection of PBMC rarely exists in patients with acute hepatitis C. As HCV infection persists, the incidence of HCV infection of PBMC becomes higher. PMID- 8621179 TI - Pulmonary hypertension after transjugular intrahepatic portosystemic shunt: effects on right ventricular function. AB - The short- and mid-term hemodynamic effects of transjugular intrahepatic portosystemic shunt (TIPS) were studied in 16 sedated cirrhotic patients. Indications included relapsing variceal bleeding (n = 10) and refractory ascites (n = 6). The decrease of porto-atrial pressure gradient (from 20.4 +/- 4.2 mm Hg to 10.1 +/- 2.4 mmHg; P < .05) was associated with an increase of mean pulmonary artery pressure (MPAP) (from 12.3 +/- 3.0 mm Hg to 20.3 +/- 5.3 mm Hg; P < .05) and of right atrial pressure (RAP) from 3.4 +/- 2.6 mm Hg to 8.3 +/- 3.7 mm Hg; P < .05), whereas right ventricular end-diastolic volume (RVEDVI) remained unchanged. The significant increase of cardiac index (CI) (from 4.5 +/- 1.2 L/min/m2 to 5.0 +/- 1.1 L/min/m2; P < .05) was essentially attributable to an increase of heart rate (HR) (from 81 +/- 11 to 88 +/- 10 beats/min; P < .05). Systemic vascular resistance (SVR) decreased (from 812 +/- 281 to 666 +/- 191 dynes/sec/cm5; P < .05), whereas pulmonary vascular resistance (PVR) increased (from 60.6 +/- 29.6 to 82.0 +/- 34.6 dynes/sec/cm5; P < .05). After transient shunt occlusion with a balloon catheter, all of the hemodynamic parameters returned to baseline values, except pulmonary artery pressure, which also decreased but remained significantly increased. One month after TIPS, pulmonary pressure remained elevated, and CI further increased. It is concluded that increased PVR is the major hemodynamic alteration occurring after TIPS placement. It correlates with the decrease of porto-atrial gradient and is probably mediated by both mechanical and neurohumoral factors. PMID- 8621180 TI - Elevation of gamma delta T lymphocytes in peripheral blood and livers of patients with primary sclerosing cholangitis and other autoimmune liver diseases. AB - Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease that is possibly an autoimmune disease. Although gamma delta T cells represent a small proportion of the total T-cell population in healthy individuals, there is evidence to suggest a role for these cells in autoimmunity. Accordingly, the aim of this study was to investigate the population of gamma delta T cells in patients with PSC, compared with other chronic liver diseases. An elevation in the percentage and absolute numbers of gamma delta T cells was found in the peripheral blood of patients with PSC (8.66% and 0.13 x 10(-6)/L [P < .01 and < .05, respectively]) and autoimmune hepatitis (AIH) (8.03% and 0.13 x 10(-6)/L [both P < 0.001]) compared with controls (4.10% and 0.06 x 10(-6)/L). We also found an elevation in the percentage and absolute numbers of gamma delta T cells in the portal areas of patients with PSC (10.55% and 4.33 [P < .001 and < .001, respectively]), AIH (7.16% and 4.55 [P = .001 and < .001, respectively]), and primary biliary cirrhosis (PBC) (5.57% and 3.49 [P = .008 and < .001, respectively]) when compared with controls (2.23% and 0.81). These findings suggest a role for gamma delta T cells in the mechanism of immune damage in autoimmune liver diseases. PMID- 8621181 TI - Prognosis of small hepatocellular carcinoma (less than 3 cm) after percutaneous acetic acid injection: study of 91 cases. AB - To assess the efficacy of ultrasound (US)-guided percutaneous acetic acid (in concentrations of 15%, 20%, 30%, 40%, and 50%) injection for small hepatocellular carcinomas (HCCs) for long-term prognosis, percutaneous acetic acid injection using 15% to 50% acetic acid was performed in 91 patients with one to four HCCs smaller than 3 cm during the past 6.5 years. During the series of treatment sessions for each patient, the same concentration of acetic acid was used. All tumors could be treated successfully with percutaneous acetic acid injection despite the differences in acetic acid concentration used. The number of treatment sessions to treat similar size of tumor was less when the higher concentration of acetic acid was used. No serious complications occurred as a direct sequela to percutaneous acetic acid injection. None of the tumor treated regrew. The 1-, 2-, 3-, 4-, and 5-year survival rates for 91 patients were 95%, 87%, 80%, 63%, and 49%, respectively. The 1-, 2-, 3-, 4-, and 5-year cancer-free survival rates of these patients were 83%, 54%, 50%, 37%, and 29%, respectively. Both liver function and size of tumor affected both survival rate and cancer-free survival rate significantly, but the number of tumors did not. The concentration of acetic acid did not affect the survival rate. Percutaneous acetic acid using 15% to 50% acetic acid will be effective therapy for small HCCs for long-term prognosis. PMID- 8621182 TI - Image cytometric analysis in pathology. PMID- 8621183 TI - Pitfalls in establishing a molecular diagnostic laboratory. AB - Diseases are increasingly being defined in terms of genetic alterations. A body of information termed "molecular pathogenesis" is evolving which provides a framework for integrating the rapidly accumulating genetic information with the related disease process. Molecular methods with their increased sensitivity and specificity are required to gather this information. A major challenge now lies in the transfer of this molecular technology from a research environment to the clinical testing arena. Technical issues, patented technology, special facilities, personnel, and regulatories issues imposed by CLIA'88, require those desiring to perform molecular tests to pay special attention to laboratory design, personnel training, and test menu development. Although establishing a successful molecular diagnostics laboratory is a complex and difficult task, the added value of these tests can have a tremendous impact in disease diagnosis and patient management. PMID- 8621184 TI - Molecular cytometry of cancer. AB - The application of molecular probes to diagnosis and prognosis of malignancies has redefined our perceptions of disease, allowing diagnosis by genotypic rather than phenotypic criteria. DNA analysis is especially useful when applied to pathological material in situ, because this allows the pathologist to combine information from both morphological and molecular observations. DNA in situ hybridization is a useful approach for the molecular pathologist, especially when combined with cytometric analysis. Potential clinical applications for in situ hybridization and the recently described technique of comparative genomic hybridization in tumor diagnosis and prognosis are described. PMID- 8621185 TI - Morphometric applications in anatomic pathology. AB - The components of the cell and tissue changes in many diseases are variable and can therefore be quantified. Characterization of these quantitative changes provides data that is useful not only for making a definitive, cell- and tissue based diagnosis of disease, but also for predicting the course of disease. The spectrum of changes found in malignant tumors, ie, cell grade, architecture, cellularity, extent of invasion, nature and extent of inflammatory reaction, exemplify this range of quantifiable features. The diagnosis and prognosis of nonneoplastic diseases, ie, myopathy and metabolic bone disease, can also be determined by quantitating tissue changes. Morphometry is the quantification of changes in the "objects" of tissues, ie, cells and organelles, and their organization, using quantitative evaluation tools. The principles of morphometry have been known for a century. With the increasing availability of affordable, powerful computer systems and increasingly flexible and user-friendly software has come easier ability to measure these changes. This article discusses the principles of morphometry with illustrations of types of analysis (ie, area fraction, object counting, shape and size analyses, and mutliparametric analyses) using examples of these applications with discussions of error sources and limitations of morphometry. PMID- 8621186 TI - Automated screening of cervical cytology specimens. AB - After more than four decades of research into automation of the process of screening Papanicolaou (Pap) smears, attempts to develop commercially viable automated screening machines have increased in recent years. These developments have been made possible in part because of the improving price-to-performance ratios in computers and other electronics. Although the Pap smear has been responsible for a very significant decrease in mortality of cervical cancer over the past 40 years, concern has arisen over false-negative cases, with their effects on patients, and the associated legal liability, particularly in the United States. In addition, shortages of cytotechnologists, which have been exacerbated by new regulations limiting the number of slides that may be examined per day, have caused concern about handling the workload, which will probably increase as more individuals gain access to preventive health care. Automated screening machines can potentially allow detection of abnormal cases that are missed with conventional screening, although they may substantially increase the cost of Pap smears. The use of automated screening machines represents a change in the way cervical cytology specimens are processed, and with some machines, a significant change in the operation of the cytology laboratory. Current methods for processing and evaluating Pap smears have not changed significantly for the past four decades. This review discusses some of the principles of operation and practical aspects of automated screening machines. PMID- 8621187 TI - Image cytometric analysis in pathology. AB - Image cytometry (ICM) is used in surgical pathology to quantify nuclear DNA content, nuclear and cytoplasmic immunostain. DNA aneuploidy is shown to be an independent negative prognostic factor in malignant melanoma, small cell carcinoma of the lung, esophageal, ovarian, endometrial, prostatic, urinary bladder, and papillary thyroid carcinoma. On bladder washings, DNA ploidy by ICM is used as an aid in diagnosis and management of recurrent transitional cell carcinoma of the bladder. Quantitation of nuclear immunostain for proliferation markers by ICM has clinical significance in prognosis and management of solid tumors of bladder, breast and ovary, astrocytoma, lymphoma, and malignant melanoma. Angiogenesis, measured by microvessel density is a predictor of prognosis in breast carcinoma and an independent predictor of metastasis for breast carcinoma, malignant melanoma, non-small cell lung carcinoma, and prostate carcinoma. Quantitated by ICM, angiogenesis is predictive of the presence or subsequent development of regional lymph node metastases in head and neck squamous carcinomas. Future prospects for ICM in pathology include the standardization of ICM techniques; extended clinical use of DNA ploidy for diagnosis, prognosis and as a help with therapeutic decisions; development of neural networks and quantification of fluorescence in situ hybridization to distinguish benign from malignant lesions of low malignant potential, and three dimensional reconstruction of morphology from two-dimensional sections measured for prognostic parameters. PMID- 8621188 TI - Multiparameter fluorescence imaging microscopy: reagents and instruments. AB - Fluorescence imaging microscopes and fluorescent reagents have both evolved greatly in the past 10 years. Sensitive imaging cameras developed over the last decade allow detection of fluorescence signals from even a single fluorescent tag on a protein. At the same time, numerous antibody markers for growth control proteins and DNA probes for chromosomal alterations have been discovered and these can be labeled with the new multicolor fluorescent dyes for detection by microscopy. Image analysis software automatically localizes and quantifies of as many as 5-6 of these different color fluorescent markers in the individual cells of a preparation. The results is that powerful methods for multiparameter analysis of molecular structures and dynamic processes are now accessible to the pathologist. PMID- 8621189 TI - Follicular lymphoma with involvement of the splenic marginal zone: a pitfall in the differential diagnosis of splenic marginal zone cell lymphoma. AB - Follicular lymphomas (FL) involving the spleen arise in and expand the germinal centers of the white pulp, whereas mantle cell lymphomas arise in the mantle zone and surround the benign germinal center. The recently described marginal zone cell lymphoma (MZCL) is a B-cell neoplasm characterized by concentric expansion of cells around the follicle, with or without infiltration of the mantle zone or germinal center. In addition, the immunoglobulin heavy chain gene is rearranged although none of the MZCL reported have been shown to be positive for the bcl-2 gene translocation by molecular studies. We report a patient with FL showing preferential involvement of the marginal zone of spleen morphologically mimicking a primary splenic MZCL. The patient reported here had a well-documented previous lymph node diagnosis of FL. The immunoglobulin heavy chain gene and bcl-2 gene rearrangement analysis confirmed the clonal origin of both the original FL, as well as the lymphoma, with a marginal zone pattern in the spleen. PMID- 8621191 TI - Demonstration of the oligoclonality of an enteropathy associated T-cell lymphoma by monoclonal antibodies and PCR analysis of the T-cell receptor V-beta repertoire on fixed tissue. AB - The apparent clonality of T cells present in enteropathy associated T cell lymphomas (EATCLs) has been previously reported by showing T cell receptor (TCR) gene rearrangement in fresh tumor tissue. The EATCL presented here exhibits the novel phenotype CD3+, HML-1+, CD4+, CD8+, and TCR Vbeta 8+. The oligoclonality of the tumor cells is shown using the polymerase chain reaction (PCR) on cDNA from RNA extracted from formalin-fixed paraffin-embedded tissue. The T cells present in the lymphoma were predominantly TCR Vbeta 8+. PMID- 8621190 TI - Ganglioneuroblastoma of the thymus: an adult case with the syndrome of inappropriate secretion of antidiuretic hormone. AB - A 61-year-old woman was admitted to the hospital because of general fatigue. Laboratory examinations showed hyponatremia, plasma hypo-osmolarity, and inappropriate increased concentration of the plasma antidiuretic hormone (ADH) in the presence of concentrated urine. Magnetic resonance imaging revealed a mass lesion in the anterior mediastinum. An extended thymectomy was performed under the diagnosis of thymoma with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Histologically the tumor was located in the thymic tissue and was diagnosed as ganglioneuroblastoma. Immunohistochemical studies showed the existence of ADH in the tumor cells. To the knowledge of the authors, this is the first case of ganglioneuroblastoma of the thymus with SIADH. PMID- 8621192 TI - Solitary fibrous tumor. PMID- 8621193 TI - Improvement of coronary flow reserve after long-term therapy with enalapril. AB - To date, no clinical study shows an improvement in coronary flow reserve due to long-term antihypertensive therapy. in view of the contribution of the renin angiotensin system to the process of hypertensive remodeling of the heart and coronary circulation, angiotensin-converting enzyme (ACE) inhibitors might act as cardioreparative drugs in arterial hypertension. Accordingly, our objective in this investigation was to examine under clinical conditions to what extent long term antihypertensive treatment with an angiotensin-converting enzyme inhibitor improved the diminished coronary flow reserve in hypertensive patients with microvascular angina pectoris. For the purpose of comparison, we also treated a normotensive control group of 6 patients with hypertrophic nonobstructive cardiomyopathy. Fifteen hypertensive individuals (10 men, 5 women; age, 58 +/- 6 years) were treated with enalapril (10 to 20 mg/d; mean, 16.7 +/- 4.9 mg/d) for 11 to 13 months. At the end of the treatment period, systolic pressure decreased from 178 +/- 14 to 137 +/- 12 mm Hg and diastolic pressure from 102 +/- 11 to 86 +/- 4 mm Hg under ambulatory conditions. Left ventricular muscle mass index decreased by 8%, from 149 +/- 32 to 137 +/- 28 g/m2 (P < .05). Maximal coronary blood flow after dipyridamole was increased by 43%, from 181 +/- 69 to 258 +/- 116 mL/min per 100 g (P < .001), and minimal coronary vascular resistance was diminished by 29%, from 0.66 +/- 0.23 to 0.47 +/- 0.24 mm Hg x min x 100g x mL-1 (P < .001) after enalapril treatment. Consequently, the calculated coronary reserve increased from 2.2 +/- 0.6 to 3.3 +/- 1.2 (P < .001). After enalapril therapy, the functional class of angina pectoris according to the Canadian classification system had changed from 2.5 +/- 0.6 to 1.5 +/- 0.6 (P < .01). The maximal working capacity had increased from 23.775 +/- 3.970 to 26.255 +/- 4.598 J (mean +/- SE, P < .05). The maximal ST-segment depression at maximal work-load was reduced from 0.18 +/- 0.02 to 0.06 +/- 0.02 (mean +/- SE, (P < .01). In summary, long-term therapy with the angiotensin-converting enzyme inhibitor enalapril must be considered a cardioreparative treatment with respect to the coronary microcirculation in hypertensive heart disease. PMID- 8621194 TI - Changes in left ventricular anatomy and function in hypertension and primary aldosteronism. AB - We investigated the effects on the heart of hypertension due to the excess of aldosterone and suppression of the renin-angiotensin system caused by primary aldosteronism with M-mode echocardiography and transmitral Doppler flow velocity measurements. We studied 34 consecutive patients with primary aldosteronism and 34 with essential hypertension individually matched for age, gender, race, body mass index, blood pressure values, and duration of hypertension. The groups were similar in age, body mass index, blood pressure, and duration of hypertension. However, lower serum potassium levels (3.5 +/- 0.6 versus 4.1 +/- 0.2 mmol/L, P < .0001) and plasma renin activity (0.53 +/- 0.45 versus 1.82 +/- 1.59 ng Ang I x mL-1 x h-1, P < .0001) and higher plasma aldosterone levels (1107 +/- 774 versus 206 +/- 99 pmol/L, P < .0001), left ventricular wall thickness, and left ventricular mass index (112 +/- 4.7 versus 98 +/- 3.7 g/m2, P = .029) were found in patients with primary aldosteronism compared with those with essential hypertension. Similarly, the PQ interval was longer (173 +/- 20 versus 141 +/- 14 milliseconds, P < .001) in primary aldosteronism than in essential hypertension patients. Significantly more primary aldosteronism than essential hypertension patients had left ventricular hypertrophy or left ventricular concentric remodeling (50% versus 15%, chi 2 = 11.97, P = .007). Both the E wave flow velocity integral (1063 +/- 65 versus 1323 +/- 78, P = .013) and the E/A integral ratio (0.91 +/- 0.05 versus 1.25 +/- 0.08, P < .001) were lower, and atrial contribution to left ventricular filling was higher (53.3 +/- 1.5% versus 45.5 +/ 1.3% P < .001) in patients with primary aldosteronism compared with essential hypertension patients. After 1 year of follow-up, highly significant decreases of left ventricular wall thickness and mass were observed in patients treated with surgical excision of an aldosterone-producing tumor, but not in those with medical therapy. Thus, in patients with primary aldosteronism, the excess aldosterone with suppression of the renin-angiotensin system is associated with both increased left ventricular mass and significant changes of left ventricular diastolic filling. The former changes appear to be reversible on removal of the cause of excessive aldosterone production. PMID- 8621195 TI - Cardiac and vascular structural changes. Prevalence and relation to ambulatory blood pressure in a middle-aged general population in northern Italy: the Vobarno Study. AB - The aims of this study were to determine the prevalence of structural changes in the carotid arteries and heart and the correlation between these changes and the commonly recognized cardiovascular risk factors in the general population. Structural changes in the carotid arteries were defined as the intima-media thickness of the artery measured by B-mode ultrasound. Changes in the heart were defined as left ventricular mass index (LVMI) measured by echocardiography. LVMI values greater than 134 g/m2 in men and greater than 110 g/m2 in women were considered abnormal, indicating the presence of left ventricular hypertrophy. Blood pressure (BP) was measured in the clinic setting with a mercury sphygmomanometer and by 24-hour noninvasive ambulatory monitoring. Hypertension was defined as a sustained systolic BP greater than or equal to 160 mm Hg and/or diastolic BP increase greater than or equal to 95 mm Hg. The study population consisted of 225 subjects (107 women and 118 men) 48 to 64 years old. Prevalence of intima-media thickening (intima-media thickness > 1 mm) was 11% in normotensive subjects and 44% in hypertensive subjects. The presence of plaque (wall thickening with either mineralization or focal protrusion in the lumen at least 50% greater than the surrounding wall, usually > 2 mm) was observed in 35% of normotensive subjects and 44% of hypertensive subjects. The prevalence of left ventricular hypertrophy was 13% in normotensive subjects and 19% in hypertensive subjects. Intima-media thickness in the common and bifurcation segments of carotid arteries correlated well with LVMI (r = .20 and r = .19, respectively; P < .01). Intima-media thickness and LVMI were both positively related to 24-hour monitored BP (P < .01). However, in the multivariate analysis, body mass index (P = .027), sex (P < .001), and 24-hour mean BP (P = .025) were the most significant determinants of LVMI, whereas carotid artery intima-media thickness was found to be associated best with age (P < .001), cigarette smoking (P = .009), serum cholesterol (P = .025), serum glucose (P = .038), and nighttime systolic BP (P = .006). Logistic regression analysis confirmed the association between the presence of plaque and age (P < .001), nighttime systolic BP (P < .05), and cigarette smoking (P < .05); a negative association between plaque and the decrease in mean systolic BP daytime to nighttime was also observed (P < .001). In conclusion, in a general population of unselected middle-aged subjects, carotid wall thickness and LVMI were associated with each other and related to 24 hour BP levels although the major determinants of carotid wall and cardiac structure were different. PMID- 8621196 TI - The fibrinolytic system is not impaired in older men with hypertension. AB - The fibrinolytic system is thought to be impaired in older hypertensive adults, thus contributing to the elevated risk of atherothrombosis, stroke, and acute myocardial infarction in this population. However, studies that have examined the fibrinolytic system in hypertensive individuals have failed to control for the confounding effects of other metabolic risk factors, making it difficult for one to determine the independent effect of hypertension on the fibrinolytic system. The purpose of the present study was to test the hypothesis that the fibrinolytic system is not impaired in older sedentary hypertensive men when the confounding effects of cardiovascular disease, diabetes, and dyslipidemia are controlled. Plasma concentrations of tissue-type plasminogen activator antigen and activity as well as plasminogen activator inhibitor-1 antigen and activity were measured under resting conditions in 12 hypertensive (69.4 +/- 1.4 years) and 11 normotensive 65.2 +/- 1.3 years) older men. The hypertensive and normotensive subjects had similar anthropometric and metabolic characteristics. There were no significant differences between the hypertensive and normotensive men in tissue type plasminogen antigen (7.3 +/- 0.5 versus 6.1 +/- 0.6 ng/mL) and activity (1.8 +/- 0.3 versus 1.7 +/- 0.2 IU/mL) or plasminogen activator inhibitor-1 antigen (14.1 +/- 2.3 versus 10.8 +/- 2.2 ng/mL) and activity (17.4 +/- 1.2 versus 17.5 +/- 1.8 arbitrary units [AU]/mL) levels. In addition, the molar concentration ratio of active tissue type plasminogen activator to active plasminogen activator inhibitor-1 did not differ between the hypertensive (1:9.7 +/- 2.3) mmol/L) and normotensive (1:10.5 +/- 2.2 mmol/L) subjects, indicative of no impairment in fibrinolytic potential in either group. These results support the hypothesis that hypertension does not directly result in impaired fibrinolytic function in older adults. Furthermore, our findings suggest that abnormalities in fibrinolytic function in older hypertensive men are likely due to the primary effects of other metabolic disorders that usually accompany hypertension, such as hyperinsulinemia and dyslipidemia. PMID- 8621197 TI - Anticipatory blood pressure response to exercise predicts future high blood pressure in middle-aged men. AB - Increases in blood pressure during the period of emotional arousal attendant to impending exertion are well documented, yet the etiologic significance of these elevations is unknown. Research suggests that exaggerated cardiovascular responses to psychological stress may be importantly related to hypertension. We examined blood pressure reactivity in anticipation of an exercise stress test in relation to future hypertension in the Kuopio Ischemic Heart Disease Risk Factor Study, a population-based study of middle-aged men from Eastern Finland. Subjects were 508 unmedicated men with resting blood pressure less than 165/95 mm Hg who completed a bicycle ergometer stress test at baseline and whose hypertensive status was assessed at 4 years of follow-up. Systolic and diastolic reactivity were calculated as the difference between blood pressure measured after seated rest on the bicycle ergometer before initiation of exercise and mean seated resting blood pressure measured 1 week earlier. Logistic regression models adjusted for age and resting blood pressure revealed a graded association between quartiles of reactivity and risk of subsequent hypertension ( > or = 165/95 mm Hg), with men showing systolic responses greater than or equal to 30 mm Hg or diastolic responses greater than 15 mm Hg at nearly four times the risk of becoming hypertensive (odds ratios, 3.80 [95% confidence interval, 1.90 to 7.63] and 3.65 [95% confidence interval, 1.86 to 7.17], respectively) relative to the least-reactive groups (systolic response, < 10 mm Hg; diastolic response, < 5 mm Hg). Adjustments for traditional risk factors for hypertension did not alter these associations. Results demonstrate the clinical significance of the pressor response in anticipation of exercise and support the hypothesis that cardiovascular reactivity to psychological challenge plays a role in the etiology of hypertension. PMID- 8621198 TI - Prospective study of nutritional factors, blood pressure, and hypertension among US women. AB - We examined prospectively the relation of nutritional factors with hypertension and blood pressure levels among 41,541 predominantly white US female nurses, aged 38 to 63 years, who completed a detailed semiquantitative food frequency questionnaire in 1984 and were without diagnosed hypertension, cancer, or cardiovascular disease. During 4 years of follow-up, from 1984 to 1988, 2,526 women reported a diagnosis of hypertension. Age, relative weight, and alcohol consumption were the strongest predictors for the development of hypertension. Dietary calcium, magnesium, potassium, and fiber were not significantly associated with risk of hypertension, after adjusting for age, body mass index, alcohol, and energy intake. Among women who did not report hypertension during the follow-up period, calcium, magnesium, potassium, and fiber were each significantly inversely associated with self-reported systolic and diastolic pressures, after adjusting for age, body mass index, alcohol consumption, and energy intake. When the four nutrients were added simultaneously to the regression model, only fiber and magnesium intakes retained significant inverse associations with systolic and diastolic pressures. In analyses of food groups, intakes of fruit and vegetables were inversely associated with systolic and diastolic pressures, and intakes of cereals and meat were directly associated with systolic pressure. These results support hypotheses that age, body weight, and alcohol consumption are strong determinants of risk of hypertension in middle aged women. They are compatible with the possibilities that magnesium and fiber as well as a diet richer in fruits and vegetables may reduce blood pressure levels. PMID- 8621199 TI - Pulse pressure correlates in humans with a proscillaridin A immunoreactive compound. AB - Endogenous digitalis-like factors in humans are presumably cardenolides and bufadienolides. To test whether bufadienolide-like substances may circulate in human blood, we used antibodies from rabbits against the bufadienolide proscillaridin A to measure the concentration of cross-reacting material in human plasma with an indirect enzyme-linked immunosorbent assay. IgG had an apparent affinity of 2 x 10(-9) mol/L for proscillaridin A. It was specific for bufadienolides and did not cross-react with cardenolides or several steroid hormones. Extraction of human plasma with ethanol and fractionation of this extract over a high-performance liquid chromatographic reverse-phase C18 column with a propanol/isopropanol gradient resulted in the separation of three peaks of increasing hydrophobicity (ED1, ED2, ED3) that inhibited the sodium pump of human red blood cells and cross-reacted with proscillaridin A antibodies. The concentration of the proscillaridin A immunoreactivity ED1 in normotensive subjects had a geometric mean of 0.1 nmol/L, with a dispersion factor of 8.77. ED1 correlated positively in a group of 60 normotensive subjects, 22 patients with hypertension, and 19 patients with chronic renal failure with mean arterial blood pressure (log ED1 [nmol/L] = 0.013 x mm Hg-2.17, r = .25, P < .05), systolic pressure (log ED1 [nmol/L] = 0.010 x mm Hg-2.23, r = .32, P < .01), and pulse pressure (log ED1 [nmol/L] = 0.019 x mm Hg-1.80, r = .38, P < .0001). There was no correlation with other parameters of the donors. We conclude that several substances cross-reacting with proscillaridin A antibodies and inhibiting the sodium pump of human red blood cells circulate in human blood. The level of one of these substances (ED1) correlates with mean arterial and pulse pressures. PMID- 8621200 TI - Wave propagation in coupled left ventricle-arterial system. Implications for aortic pressure. AB - The objective of this study was to examine the effects of wave propagation properties (global reflection coefficient gamma IG; pulse wave velocity, c(ph); and characteristic impedance zeta(o) on the mechanical performance of the coupled left ventricle-arterial system. Specifically, we sought to quantify effects on aortic pressure (P(ao)) and flow Q(ao) while keeping constant other determinants of P(ao) and Q(ao) (left ventricular end-diastolic volume, V(ed), and contractility, heart rate, and peripheral resistance, R(s)). Isolated rabbit hearts were subjected to real-time, computer-controlled physiological loading. The arterial circulation was modeled with a lossless tube terminating in a complex load. The loading system allowed for precise and independent control of all arterial properties as evidenced by accurate reproduction of desired input impedances and computed left ventricular volume changes. While propagation phenomena affected P(ao) and Q(ao) morphologies as expected, their effects on absolute P(ao) values were often contrary to the current understanding. Diastolic (Pd) and mean (Pm) P(ao) and stroke volume decrease monotonically with increases in gamma G, c(ph), or zeta(o) over wide ranges. In contrast, these increase had variable effects on peak systolic P(ao) (Ps): decreasing with gamma G, biphasic with c(ph), and increasing with zeta(o). There was an interaction between gamma G and c(ph) such that gamma G effects on P(m) and P(d) were augmented a higher C(ph) and vice versa. Despite large changes in system parameters, effects on Pm and Ps were modest ( < 10% and < 5%, respectively); effects on Pd were always two to four times greater. Similar results were obtained when the single-tube model of the arterial system was replaced by an asymmetrical T-tube configuration. Our data do not support the prevailing hypothesis that P(s) (and therefore ventricular load) can be selectively and significantly altered by manipulating gamma G, c(ph), and/or zeta o. PMID- 8621201 TI - Cytosolic calcium changes induced by angiotensin II in neonatal rat atrial and ventricular cardiomyocytes are mediated via angiotensin II subtype 1 receptors. AB - We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca2+]i) in the absence and presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan or the selective AT2 antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also Ang II receptor density, affinity, and mRNA expression. [Ca2+]i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of [Sar1,Ile8]Ang II, losartan, and PD 123319. AT1 receptor (types AT1A and AT1B) mRNA abundance was measured by reverse transcription-polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca2+]i values in atrial and ventricular cells were similar but Ang II (10-9 mol/L)-induced [Ca2+]i changes were significantly greater in atrial compared with ventricular cells Ang II responses were blocked by losartan (10-7 mol/L) but not PD 123319 (10-7 mol/L). Binding studies demonstrated a single class of high-affinity. Ang II binding sites on cardiomyocyte membranes (Kd = 0.71 +/- 0.11 mumol/L). 125I [Sar1,Ile8]Ang II was displaced by losartan but not by PD 123319. AT1 receptor mRNA was detected by reverse transcription-polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT1A and AT1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT1 receptor subtype that are linked to [Ca2+]i signaling pathways. The different Ang II-induced [Ca2+]i responses between atrial and ventricular cells may be related to differences in the distribution of AT1 receptor subtype subvariants. PMID- 8621202 TI - Tyrosine kinase signaling pathways modulate angiotensin II-induced calcium ([Ca2+]i) transients in vascular smooth muscle cells. AB - Tyrosine kinases have been implicated in vascular smooth muscle cell proliferation and contraction. Underlying mechanisms may involve C(a2+) dependent pathways. This study assesses relationships between angiotensin II (Ang II)-stimulated phospholipase C-mediated Ca2+ transients and tyrosine kinase dependent pathways in vascular smooth muscle cells. Intracellular free Ca2+ concentration ([Ca2+]i) was measured in primary cultured unpassaged vascular smooth muscle cells derived from mesenteric resistance vessels of Wistar-Kyoto rats with the use of fura 2 methodology. [Ca2+]i effects of Ang II (1 nmol/L) were determined in vascular smooth muscle cells in which tyrosine kinase pathways were stimulated by insulin (70 muU/mL; 0.5 nmol/L), insulin-like growth factor-I (1 ng/mL; 0.13 nmol/L), or platelet-derived growth factor-BB (1 ng/mL; 0.04 nmol/L) and in cells in which tyrosine kinase was inhibited by specific inhibitors (1 mumol/L tyrphostin A-23 and genistein). Ang II elicited a rapid and transient [Ca2+]i response (from 94 +/- 8 to 239 +/- 5.8 nmol/L). Activation of the receptor tyrosine kinase by insulin, platelet-derived growth factor, and insulin-like growth factor-I significantly reduced (P < .01) Ang II-induced [Ca2+]i to 161 +/- 7, 189 +/- 3.7, and 183 +/- 5 nmol/L, respectively. In the presence of tyrphostin A-23 and genistein, Ang II-stimulated [Ca2+]i remained persistently elevated and failed to return to basal levels. Tyrphostin A-1, the inactive tyrphostin analogue, had not significant effect on Ang II-induced [Ca2+]i. This study demonstrates that activation of tyrosine kinase pathways reduces Ang II-elicited [Ca2+]i responses, whereas tyrosine kinase inhibition prevents [Ca2+]i recovery after agonist stimulation. Interaction between tyrosine kinase- and phospholipase C-dependent signaling pathways modulates vascular smooth muscle cell [Ca2+]i responses to Ang II. PMID- 8621203 TI - Alpha 2-adrenergic agonists increase cellular lactate efflux. AB - We reported previously that genetic polymorphisms of the alpha 2-adrenergic receptor are associated with hyperinsulinemia, diabetes mellitus, and hypertension in blacks. The evolutionary driving force for maintaining such deleterious mutations in the black population is unknown. Recognizing that vascular alpha 2-adrenergic receptors mediate cold-induced vasoconstriction and that temperature maintenance is a primary thrust of cellular metabolism, we postulated that vascular alpha 2-adrenergic receptors contribute significantly to metabolic heat generation in homeotherms such as humans. Using aerobic lactate production as an indicator of thermogenesis, we measured metabolic heat production in HT29 cells that expressed the gene encoding human vascular alpha 2 adrenergic receptors. Epinephrine, an alpha 2-adrenergic receptor agonist, increased net lactate efflux from 226 +/- 20 to 280 +/- 20 nmol/min (mean +/- SE) (P = .06). Clonidine, a more specific alpha 2-adrenergic agonist, increased lactate efflux from 110 +/- 6 to 156 +/- 8 nmol/min (P < .01). Similarly, in the presence of physiological concentrations of glucose (5.5 mmol/L), insulin increased lactate production from 123 +/- 6 to 175 +/- 10 nmol/min (P < .01). Because differences in aerobic glycolysis may also explain the heat intolerance and abnormal fuel homeostasis found in genetically hypertensive rats, we also measured lactate production in cultured vascular smooth muscle cells isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control Wistar-Kyoto rats (WKY). Vascular smooth muscle cells from SHRSP had significantly greater lactate efflux compared with cells from normotensive WKY (296 +/- 4 versus 172 +/- 2 nmol/min, P < .001). These differences were not due to abnormalities in glucose uptake, as lactate efflux was greater in SHRSP cells compared with WKY cells when dextrose was replaced with equimolar concentrations of fructose (230 +/- 6 versus 138 +/- 2 nmol/min, P < .001). alpha 2-Adrenergic agonists increase lactate efflux in HT29 cells, and abnormalities in vascular smooth muscle lactate metabolism in genetically hypertensive rats is independent of altered glucose uptake. These data provide support for our hypothesis that balanced polymorphisms of the alpha 2-adrenergic receptor could offer protection against cold stress by increasing the thermogenic response associated with aerobic lactate production. PMID- 8621204 TI - Doxazosin prevents proteinuria and glomerular loss of heparan sulfate in diabetic rats. AB - We examined whether blood pressure reduction or good glycemic control equally lower albuminuria by preventing glomerular loss of heparan sulfate and progression of glomerulosclerosis in streptozotocin-induced diabetic rats. We used doxazosin, and alpha 1-adrenergic blocker, to lower systemic blood pressure, and good glycemic control was achieved by insulin treatment. Rats were killed after 20 weeks of treatment. Doxazosin significantly lowered systolic pressure in diabetic rats; however, it had no effect in normal rats. Good glycemic control also lowered systolic pressure. In diabetic rats with good glycemic control, doxazosin had an additive effect on blood pressure. Glomerular heparan sulfate synthesis was significantly lower and urinary albumin excretion higher in diabetic than in normal rats. Both doxazosin treatment and good glycemic control normalized these abnormalities in diabetic rats. Insulin normalized plasma glucose and glycosylated HbA1 concentrations in diabetic rats, as did doxazosin. Significant increases in mesangial area and glomeruloscelerosis were observed in diabetic rats. Only good glycemic control normalized these pathological changes in all diabetic rats. Two-way factorial ANOVA showed an interaction between the effects of doxazosin and insulin on systolic pressure and plasma glucose. The data show that after 20 weeks of doxazosin treatment, albuminuria was reduced by 80%; however, this treatment had no significant effect on mesangial expansion or progression to glomerulosclerosis. Conversely, good glycemic control prevented all three of the preceding sequelae. PMID- 8621205 TI - Preweanling administration of terazosin decreases blood pressure of hypertensive rats in adulthood. AB - To examine the contribution of the sympathetic nervous system to the development of hypertension, we injected spontaneously hypertensive rat (SHR) pups and normotensive Wistar-Kyoto rat (WKY) pups twice daily with saline (1.0 mL/kg SC) or terazosin (0.5 mg/kg SC), an alpha 1-adrenoceptor antagonist, from postnatal day 1 through 21. We determined the effectiveness and duration of action of this terazosin dose in pilot studies with adult SHR and WKY. Body weights of WKY pups were greater than body weights of SHR pups from postnatal day 1 through 21. In addition, body weights of terazosin-treated pups of both strains were comparable to body weights of saline-injected littermate controls. Indirectly measured systolic pressures of terazosin-treated SHR were reduced significantly at 60 and 90 days of age but not at 30 days of age compared with saline-injected littermate controls. Terazosin did not affect systolic pressures of WKY, measured at 30, 60, and 90 days of age. At 100 days of age, in chronically catheterized rats, mean arterial pressures of terazosin-treated SHR were reduced significantly compared with those of saline-injected littermate controls. In contrast, terazosin did not affect mean arterial pressures of WKY at 100 days of age. Finally, preweanling treatment with terazosin did not alter patterns of open field behavior of adult SHR or WKY. SHR were significantly more active and reared more frequently compared with WKY. These findings indicate that the time between birth and weaning at 21 days of age is critical for the full expression of the hypertensive phenotype in SHR. Chronic blockage of alpha 1-adrenoceptors during the preweanling period in SHR may reduce vascular hypertrophy, leading to long-term reductions in arterial pressure. PMID- 8621206 TI - Plasma and renal prorenin/renin, renin mRNA, and blood pressure in Dahl salt sensitive and salt-resistant rats. AB - We measured plasma prorenin and renin levels, renal renin mRNA, renal anti-renin and anti-prorenin-prosequence immunoreactivity, and blood pressure in maturing Brookhaven Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats during 14 days of low (0%), medium (0.4%), or high 4%) NaCl diets. Blood pressure was higher in Dahl S rats and did not increase with high NaCl. Seven-week-old Dahl R rats had twofold and sixfold higher levels of plasma prorenin and renal prosequence immunoreactivity, respectively, which by 9 weeks were the same as in Dahl S rats. The anti-renin antiserum, BR1-5, was found to detect prorenin better than renin; Dahl S rats had suppressed renal anti-renin immunoreactivity relative to Dahl-R rats. Dahl R rats were unresponsive to high NaCl, whereas in Dahl S rats, plasma renin and renal prosequence immunoreactivity fell by 90% (P < .01), renal anti-renin immunoreactivity and renal renin MRNA fell by 35% (P < .05 for both), and plasma prorenin fell by 30% (P = NS). NaCl depletion increased prorenin/renin parameters similarly in both strains. There were direct relationships among all of the prorenin/renin parameters. Between low and high salt diets in Dahl S rats, plasma renin increased 20-fold, plasma total renin (renin plus prorenin) and renal renin mRNA both increased threefold, and plasma prorenin increased twofold. The results indicate that under steady-state conditions, plasma and renal renin/prorenin parameters change concordantly and that plasma total renin (renin plus prorenin) reflects changes in renal renin mRNA. The lower blood pressure of Dahl R rats is associated with later maturation related declines in plasma and renal prorenin. Suppression of plasma renin may delay the salt-induced blood pressure rise in Dahl S rats. Finally, the renin system and blood pressure of Dahl R rats have remarkable disregard for a high salt diet. PMID- 8621207 TI - Angiotensinogen polymorphism M235T, hypertension, and nephropathy in insulin dependent diabetes. AB - The allele 235T (a threonine in place of a methionine at position 235) of angiotensinogen has been found to be associated with a predisposition to essential hypertension. We investigated whether this allele also confers increased susceptibility to nephropathy in patients with insulin-dependent diabetes mellitus (IDDM). A group of 380 patients who had had IDDM for 15 to 20 years were genotyped at the angiotensinogen 235 locus. Included were 75 patients with normoalbuminuria (albumin excretion rate < 30 micrograms/min), two series of patients with microalbuminuria (n = 30 and n = 136), and two series with overt proteinuria (n = 41 and n = 98). Allele 235T frequency was higher among cases with microalbuminuria (0.41 in the two series combined) or overt proteinuria (0.40) than in the normoalbuminuria group (0.36). However, this difference was not statistically significant with this sample size (chi 2 = 1.2, P = NS with 2 df). Under a recessive model, allele 235T homozygotes had a 1.6-fold risk of developing nephropathy relative to carriers of other genotypes, but this value was not significantly different from 1(95% CI = 0.8 to 3.5). The strength of the association did not improve after stratification by degree of glycemic control. With respect to the hypertension in these IDDM patients, no association with allele 235T was found. Allele 235T frequencies in normotensive and hypertensive individuals were 0.363 and 0.353, respectively, among normoalbuminuric IDDM individuals (chi 2 = 0.01, P = NS) and 0.411 and 0.414 among microalbuminuric IDDM subjects (chi 2 = 0.0, P = NS). We conclude that the angiotensinogen polymorphism M235T might influence susceptibility to nephropathy in insulin dependent diabetes, but its effect, if any, is rather small and independent of hypertension. PMID- 8621208 TI - Effects of cyclosporin A on the synthesis, excretion, and metabolism of endothelin in the rat. AB - Increasing evidence suggests that endothelin, a potent vasoconstrictor, is implicated in cyclosporin A (CsA)-induced nephrotoxicity. Increased levels of urinary and circulating endothelin have been described in CsA-treated humans and animals. The exact mechanisms by which CsA induces these increases are still unknown, and no data indicate whether these elevated levels reflect increased synthesis or decreased clearance of endothelin. In the present study, we investigated the effects of CsA administration (50 mg/kg per day i.p. for 6 days) to rats on plasma and urinary levels of endothelin; expression of endothelin-1 (ET-1), ET-3, and endothelin-converting enzyme in renal tissue; clearance of infused 125I-ET-1; and degradation of 125I-ET-1 by recombinant neutral endopeptidase. Rats given CsA for 6 days developed severe renal insufficiency, as shown by a 74% decrease in creatinine clearance rate (Ccr) (P < .006). Ccr was remarkably improved in CsA-treated rats that received bosentan, the combined antagonist of both endothelin A and endothelin B receptors. Urinary excretion of endothelin increased from an undetectable level to 31.7 +/- 6.0 pg/24 h (P < .001), and plasma levels of endothelin were unchanged (2.8 +/- 0.2 to 3.1 +/- 0.2 pg/mL). Reverse transcription followed by quantitative polymerase chain reaction revealed that ET-1 mRNA in the renal medulla increased by 59% (P < .006), whereas the expression of both ET-3 and endothelin-converting enzyme was unchanged. In other rats, neither acute nor chronic treatment with CsA affected either the clearance of 125I-ET-1 from the blood or the renal and pulmonary uptake of the peptide. Moreover, CsA did not affect the degradation of 125I-ET-1 by highly purified recombinant neutral endopeptidase, a well-known endothelinase. Taken together, these data suggest that the elevated urinary endothelin levels obtained after CsA treatment originate from the kidney and reflect increased renal synthesis of ET-1. Moreover, the production of endothelin appears to be regulated at the mRNA transcription level, and expressions of ET-1 and ET-3 are regulated independently. PMID- 8621209 TI - Platelet lipoxygenase in spontaneously hypertensive rats. AB - We have previously reported that the nonselective lipoxygenase inhibitor phenidone is a potent hypotensive agent in the spontaneously hypertensive rat (SHR). In the present study, we examined the relationship between production of platelet 12-hydroxyeicosatetraenoic acid (12-HETE) and intra-arterial blood pressure in SHR and Wistar-Kyoto rats (WKY) using both a cross-sectional analysis and an acute pharmacological intervention. Basal generation rate of 12-HETE by platelets collected from the SHR was approximately 3.7-fold higher than in the WKY (0.86 +/- 0.24 versus 0.23 +/- 0.05 nmol/mL per 10 minutes, respectively; P < .01). Systolic arterial pressure was positively related to platelet 12-HETE formation rate when the entire rat population was considered (r = .70, P < .001). The specific 12-lipoxygenase inhibitor cinnamyl-3,4-dihydroxycyanocinnamate induced lowering of both arterial blood pressure and platelet 12-lipoxygenase activity in SHR. At 15 mg/kg, cinnamyl-3,4-dihydroxycyanocinnamate elicited a marked hypotensive effect in SHR but not in WKY. This reduction in arterial pressure was accompanied by an approximate 70% inhibition in platelet 12-HETE production rate. The return of high blood pressure to basal levels was associated with a significant rise in the production of platelet 12-HETE toward control values (baseline, 0.97 +/- 0.33 nmol/mL per 10 minutes; nadir of blood pressure, 0.19 +/- 0.03; resumption of basal pressure, 0.42 +/- 0.14). In contrast, captopril (15 mg/kg) induced a quantitatively similar decrease in blood pressure but had no effect on platelet 12-HETE generation rate. Thus, hypertension in SHR is linked to increased production rate of platelet 12-HETE. Acute blood pressure reduction attained during lipoxygenase inhibition but not by angiotensin converting enzyme inhibition leads to a concomitant reduction in the production of platelet 12-HETE. We speculate that since rat arterial tissue produces 12 HETE, increased 12-lipoxygenase activity in SHR may contribute to the maintenance of elevated arterial pressure in this strain. PMID- 8621210 TI - Endothelin adrenocortical secretagogue effect is mediated by the B receptor in rats. AB - We investigated the gene expression and localization of endothelin-1 (ET-1) receptor subtypes ET(A) and ET(B) in the rat adrenal cortex as well as their involvement in the corticosteroid secretagogue effect of ET-1 in vitro. Reverse transcription-polymerase chain reaction with primers specific for ET(A) and ET(B) cDNAs demonstrated the expression of both receptor genes in homogenates of adrenocortical tissue. However, in isolated zona glomerulosa and zona fasciculata cells, only ET(B) mRNA was detected. Autoradiographic examination of the selective displacement of 125I-ET-1 binding by BQ-123 and BQ-788 (specific ligands for ET(A) and ET(B), respectively) indicated that zona glomerulosa possesses both ET(A) and ET(B), whereas zona fasciculata is exclusively provided with ET(B). ET-1 enhanced in a concentration-dependent manner aldosterone and corticosterone secretions of dispersed zona glomerulosa and zona fasciculata cells, respectively. The ET(B) antagonist BQ-788 markedly reduced the secretory response of zona glomerulosa cells and completely suppressed that of zona fasciculata cells, whereas the ET(A) antagonist BQ-123 was ineffective. These findings indicate that in the rat, the adrenocortical secretagogue action of ET-1 is mediated by the ET(B) receptor subtype and that the ET(A) receptor is not directly involved in such an effect. PMID- 8621211 TI - Renal function and blood pressure response to dietary salt restriction in normotensive men. AB - The interindividual variability of the blood pressure response to changes in dietary sodium intake might be traced in part to heterogeneity in renal adaptation. To further explore this possibility, we evaluated glomerular filtration rate and tubular sodium handling in 47 healthy male volunteers from the Olivetti factory in Naples who were studied on their habitual sodium-rich diet (urinary sodium, 184 +/- 9 mmol/24 h) and after 3 days of a salt-restricted diet (urinary sodium, 69 +/- 5 mmol/24 h). Individual salt sensitivity, defined as the mean blood pressure change recorded after the shift from habitual to low sodium diet, significantly and directly correlated with glomerular filtration rate and absolute proximal sodium reabsorption during the habitual diet. When the entire population was divided into tertiles of salt sensitivity, the group with the highest salt sensitivity showed higher blood pressure, glomerular filtration rate, and absolute proximal sodium reabsorption during the habitual diet compared with the least salt-sensitive group; however, during the low NaCl diet, no differences were detectable between the groups. Twenty-four-hour urinary sodium was similar across the groups. We conclude that relative hyperfiltration and altered tubular sodium handling may occur in salt-sensitive normotensive individuals on a high sodium diet and that NaCl restriction may offset these abnormalities. PMID- 8621212 TI - Effect of salt intake and inhibitor dose on arterial hypertension and renal injury induced by chronic nitric oxide blockade. AB - Long-term nitric oxide blockade by N omega -nitro-L-arginine methyl ester (L NAME) leads to severe and progressive hypertension. The role of salt intake in this model is unclear. To verify whether salt dependence in this model is related to the extent of nitric oxide inhibition, we gave adult male Munich-Wistar rats a low salt, standard salt, or high salt diet and oral L-NAME treatment at either 3 or 25 mg/kg per day. At 10 to 15 days of treatment, the slope of the pressure natriuresis line was decreased in rats receiving low-dose L-NAME compared with untreated controls. In rats treated with the higher dose, the line was shifted to the right but remained parallel to that obtained in untreated controls. Renal vascular resistance was moderately increased in rats receiving low-dose L-NAME, whereas high-dose L-NAME induced a marked vasoconstriction that was aggravated by salt overload. Low-dose L-NAME treatment induced hypertension only when associated with sodium overload. In rats receiving high-dose L-NAME, hypertension was aggravated by sodium excess but was not ameliorated by sodium restriction. Long-term (6 weeks) L-NAME treatment was associated with progressive hypertension, which was aggravated by salt overload, and with the development of albuminuria, focal glomerular collapse, glomerulosclerosis, and renal interstitial expansion. These abnormalities were worsened by salt overload and largely prevented by salt restriction. In the model of chronic nitric oxide blockade, salt dependence is a function of the inhibitor dose, and renal injury varies directly with the level of salt intake. PMID- 8621213 TI - Role of nitric oxide in short-term and prolonged effects of angiotensin II on renal hemodynamics. AB - Short-term infusions of angiotensin II (Ang II) increase renal vascular resistance and thereby endothelial shear stress and nitric oxide (NO) release. Prolonged stimulation of Ang II can decrease the expression of NO synthase isoforms in the macula densa, but prolonged increases in shear stress can increase transcription of endothelial NO synthase. Therefore, we designed these studies to test the hypothesis that Ang II exerts time-dependent effects on renal NO generation as assessed from renal excretion of nitrate and nitrite, percent increases in renal vascular resistance during inhibition of NO synthase with intravenous NG -nitro-L-arginine methyl ester (L-NAME), or decreases in renal vascular resistance during stimulation of endothelial NO synthase with intravenous acetylcholine. Rats were tested during graded short-term (30 to 90 minutes intravenous) or prolonged (5 to 6 days subcutaneous) Ang II infusions that led to dose-dependent increases in blood pressure and renal vascular resistance and reductions in renal blood flow. Captopril was administered for 3 to 4 days to suppress Ang II generation. The renal excretion of nitrate and nitrite was increased during short-term Ang II infusions (from 205 +/- 22 to 331 +/- 58 pmol.min-1, P < .05) but was unchanged during prolonged Ang II infusion (control group, 197 +/- 33 versus Ang II, 245 +/- 42 pmol.min-1, P=NS). The percent increase in renal vascular resistance with L-NAME was potentiated dose dependently by short-term but not long-term Ang II infusions. The increase in renal vascular resistance with L-NAME in control rats without Ang II infusions was +150 +/- 13%. At an Ang II infusion of 200 ng.kg-1.min-1, the L-NAME-induced percent increase in renal vascular resistance was significantly (P < .01) increased compared with controls in short-term Ang II-infused rats (+369 +/- 70%) but was not significantly different in prolonged infused rats (+190 +/- 33%). Intravenous acetylcholine caused dose-dependent renal vasodilation that was not significantly changed in rats receiving short-term intravenous Ang II but was significantly (P < .005) potentiated in those receiving prolonged Ang II infusions (change in renal vascular resistance with acetylcholine at 10 micrograms.kg-1.min-1 versus control, -21.5 +/- 5.0%; with short-term Ang II, 24.9 +/- 4.5%; with long-term Ang II, -52.1 +/- 7.2%). In conclusion, short- and long-term Ang II infusions caused equivalent changes in blood pressure and renal blood flow and hence presumably equivalent increases in endothelial shear stress. However, only short-term Ang II infusions increased NO generation and the dependence of the renal circulation on NO, whereas acetylcholine-induced NO release was enhanced selectively during long-term Ang II infusions. This suggests that during long-term Ang II, renal NO release may become uncoupled from shear stress yet remains highly responsive to receptor-mediated stimulation. PMID- 8621214 TI - Improvement of insulin sensitivity contributes to blood pressure reduction after weight loss in hypertensive subjects with obesity. AB - To access the role of insulin resistance in obesity hypertension, we examined the change of insulin sensitivity after weight loss in 24 obese hypertensive subjects by the euglycemic hyperinsulinemic glucose clamp method. The results of the 4 week calorie-restricted diet were a weight loss of 10.2% (from 74 +/- 12 to 67 +/ 11 kg, P < .01) and a decrease in mean blood pressure of 13.1% (from 124 +/- 7 to 107 +/- 9 mm Hg, P < .01). A decrease in plasma norepinephrine (from 208 +/- 74 to 142 +/- 52 pg/mL, P < .01) was associated with decreases in plasma renin activity (from 1.06 +/- 0.98 to 0.62 +/- 0.63 ng/mL per hour, P < .01) and serum aldosterone (from 70 +/- 28 to 57 +/- 24 pg/mL, P < .05). Glucose infusion rate increased significantly (42.9%), from 809 +/- to 1155 +/- 251 mumol/m2 per minute. The insulin sensitivity index, which is a measure of the glucose infusion rate divided by plasma insulin, increased significantly (42.6%), from 10.8 +/- 3.5 to 15.4 +/- 4.4 (mumol/m2 per minute)/(microU/mL). Stepwise multiple linear regression analysis showed that changes of plasma norepinephrine, insulin sensitivity index, plasma renin activity, and age were significant predictive factors for the change of mean blood pressure after weight loss. These results indicate a distinct relation between an improvement of insulin sensitivity and a decrease in blood pressure after weight loss in obese hypertensive subjects. The decrease in blood pressure after weight loss is probably related to the suppression of sympathetic nervous activity. PMID- 8621215 TI - Clonidine and heart failure. PMID- 8621216 TI - Assessment of arterial distensibility by automatic pulse wave velocity measurement. PMID- 8621217 TI - Abortion and breast cancer risk. AB - The relationship between spontaneous and induced abortions and breast cancer risk was analyzed using data from a case-control study conducted between June 1991 and February 1994 in 6 Italian centers on 2,569 histologically confirmed incident breast cancer cases and 2,588 controls admitted to hospital for a wide range of acute, non-neoplastic, non-hormone-related diseases. One or more abortions were reported by 31% of cases and 32% of controls, corresponding to a multivariate odds ratio (OR) of 1.0 (95% confidence interval [CI], 0.8-1.1). No trend in risk was observed with increasing number of total abortions or spontaneous and induced abortions separately. No significant relationship was found between the risk of breast cancer and history of spontaneous or induced or total abortions in separate strata of age at diagnosis, number of children, time of abortion in relation to first birth and family history of breast cancer. When abortion was the outcome of the first pregnancy, the OR was 1.2 for spontaneous and 1.3 for induced abortion, in relation to women with birth as outcome of the first pregnancy, and 1.0 and 1.1, respectively, when the reference category was nulligravidae. Thus, our results indicate a lack of association between induced and spontaneous abortions and breast cancer risk. PMID- 8621218 TI - Serological and immunochemical analysis of Lewis y (Ley) blood group antigen expression in epithelial ovarian cancer. AB - The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley-specific monoclonal antibody (MAb 3S193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley-positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC-1 mucins, lower m.w. glycoproteins and glycolipids. In cell lines, Ley was more commonly expressed on MUC-1 mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC-1. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP-1, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC-1), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies. PMID- 8621220 TI - The risk of brain tumours in hereditary non-polyposis colorectal cancer (HNPCC). AB - Hereditary non-polyposis colorectal cancer (HNPCC) is known to be associated with several extracolonic cancers, e.g., cancers of the endometrium, stomach, urinary tract, small bowel and ovary. An association between HNPCC and brain tumours has also been reported, although previous risk analysis did not reveal an excess of this type of tumour. To determine whether HNPCC predisposes patients to brain tumours, we used risk analysis to compare families with HNPCC to those in the general population. Of the 1,321 subjects from 50 HNPCC families (with 60,237 person-years of follow-up) in the Dutch HNPCC Registry which satisfy the Amsterdam Criteria, 312 had colorectal cancer. The registry revealed 14 brain tumours in the HNPCC-patients and their first-degree relatives: 5 astrocytomas, 3 oligodendrogliomas, 1 ependymoma and 5 tumours for which a pathological report was not available. The relative risk of brain tumour in patients with HNPCC and their first-degree relatives was 6 times greater than in the general population (95% confidence interval, 3.5 to 10.1). After exclusion of the cases based only on family history, the relative risk was 4.3 (95% confidence interval, 2.3 to 8.0). Although the relative risk of brain tumour was increased, the lifetime risk was low (3.35%). Because it is not certain whether an improvement of the overall prognosis can be achieved by early diagnosis and intervention, and in view of the low lifetime risk, we do not recommend screening for brain tumours in HNPCC families. PMID- 8621219 TI - Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5 year experience. AB - Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group. PMID- 8621221 TI - Genetic basis of drug sensitivity in human testis tumour cells. AB - Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98orC1). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning. PMID- 8621222 TI - Risk factors for adrenal cancer: an exploratory study. AB - Adrenal cancer is a heterogeneous group of neoplasms with unknown etiology. In search of risk factors, we conducted a case-control study based on data from the 1986 National Mortality Followback Survey, which included a questionnaire sent to the next of kin of almost 20,000 deceased adults (age > or = 25 year) in the United States. Information was obtained on a large number of items, including use of cigarettes, alcohol, oral contraceptives (OCs), height and weight and food consumption patterns. A total of 176 subjects who died of adrenal cancer (88 men and 88 women) and 352 controls (176 men and 176 women) who died of causes unrelated to smoking, drinking or OCs (for female controls) were included in the study. Although information on histologic type was not available, most cases were estimated from incidence surveys to be adrenocortical carcinoma, with a small percentage being malignant pheochromocytoma or neuroblastoma. An increased risk was associated with heavy smoking (> or = 25 cigarettes/day) among men (odds ratio [OR] = 2.0, 95% confidence interval [CI] 1.0-4.4) but not women. No clear association was seen for alcohol use, height and weight or food consumption patterns in either sex. Among women, increased risks were found for ever users of OCs (OR = 1.8, 95% CI 1.0-3.2) and especially those who used them before age 25 (OR = 2.5, 95% CI 1.2-5.5). When the analysis was restricted to subjects with spousal respondents, more pronounced risks were seen for ever users of OCs and for those who used OCs before age 25. Our findings suggest that cigarette smoking and use of OCs may increase the risk of adrenal cancer, but additional studies are needed with more detailed information on risk factors and histologic type of adrenal cancer. PMID- 8621223 TI - Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: evidence of persistent oxidative stress in cancer. AB - Data regarding oxidatively modified DNA bases suggest that cancer cells are more exposed to oxidative stress than adjacent non-tumorous tissue. This novel concept may contribute to the understanding of certain aspects of tumor biology such as activated transcription factors, genetic instability, chemotherapy-resistance and metastasis. We therefore tested this concept in human renal-cell carcinomas (RCCs) by evaluating the expression of hMTH1, an enzyme preventing the misincorporation into DNA of 8-oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate), an oxidized form of dGTP in the nucleotide pool. The expression of hMTH1 messenger RNA (mRNA) in RCC was significantly higher than that in adjacent non-tumorous kidney. Moreover, advanced-stage tumors showed significantly higher hMTH1 mRNA expression than early-stage tumors, and there was a modest linear correlation between hMTH1 expression and c-myc expression. The results provide logical support for the concept of "persistent oxidative stress in cancer" and suggest a role of hMTH1 mRNA level as a prognostic marker. PMID- 8621224 TI - Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study. AB - Cdk4-mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non-small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16 positive specimens did not stain for Rb. Only 1 of the p16-negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein. PMID- 8621225 TI - Male breast cancer in Iceland. AB - All malignant tumours of the male breast diagnosed in Iceland during the 40-year period 1955-1994 were studied with regard to histological classification, tumour grading and flow cytometric analysis. Of 31 malignant tumours diagnosed, 29 were primary breast carcinomas. Male breast carcinoma constitutes 1% of all breast malignancies in Iceland and 0.25% of all malignant tumours in males. About 80% of the male breast carcinomas were diagnosed during the latter half of the study period. The mean age of the patients was 66.3 years and the left-to-right ratio was 1.9:1.0. Right-sided tumours appeared to be more aggressive. The mean tumour size was 2.6 cm. The vast majority of the carcinomas (79%) were of the infiltrating ductal type. Of these 21.7% were grade I, 43.5% were grade II and 34.8% grade III. Papillary carcinomas made up 17% of the total. These occurred in slightly older patients than the infiltrating ductal carcinomas and were diploid tumours. In this study 57% of the tumours were found to be aneuploid, but nearly 70% of the invasive ductal carcinomas NOS were aneuploid. In general, the aneuploid tumours were larger, of higher average histological grade and had a higher mean S-phase value. The overall mean S-phase fraction was 7.2% which is similar to that found in female breast tumours in Iceland. It is concluded that male-to-female ratio of breast carcinoma in Iceland is similar to that found in other Western countries. The age-standardised incidence has increased considerably in the last 20 years, in contrast to the rates reported from most other countries. Papillary tumours are unusually common in Icelandic males. PMID- 8621226 TI - Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study. AB - In a pilot I/II study we have tested synthetic ras peptides used as a cancer vaccine in 5 patients with advanced pancreatic carcinoma. The treatment principle used was based on loading professional antigen-presenting cells (APCs) from peripheral blood with a synthetic ras peptide corresponding to the ras mutation found in tumour tissue from the patient. Peptide loading was performed ex vivo and the next day APCs were re-injected into the patients after washing to remove unbound peptide. Patients were vaccinated in the first and second week and thereafter every 4-6 weeks. In 2 of the 5 patients treated, an immune response against the immunising ras peptide could be induced. None of the patients showed evidence of a T-cell response against any of the ras peptides before vaccination. The treatment was well tolerated and could be repeated multiple times in the same patient. Side effects were not observed even if an immunological response against the ras peptide was evident. We conclude that ras peptide vaccination according to the present protocol is safe and may result in a potentially beneficial immune response even in patients with advanced malignant disease. PMID- 8621227 TI - Detection of beta-human chorionic gonadotropin mRNA as a marker for cutaneous malignant melanoma. AB - The beta chain of human chorionic gonadotropin (hCG) hormone is produced by fetal cells, gonadal cell tumors and several types of non-gonadal carcinoma. hCG is composed of an alpha and a beta chain, the latter of which can be used to distinguish the molecule from other related gonadotropin hormones. Detection of beta-hCG mRNA transcripts can be potentially useful as a marker to identify tumor cells. We devised a highly specific and sensitive assay to detect the atavistic expression of beta-hCG in cutaneous melanoma by RT-PCR. Twenty-four melanoma cell lines and 43 melanoma biopsies were evaluated for beta-hCG mRNA expression. An RT PCR assay was developed to specifically distinguish beta-hCG poly-A mRNA from other related gonadotropin beta chains. This was performed by endonuclease digestion of a unique Sty 1 site in the beta chain, followed by Southern blot analysis with a beta-hCG cDNA probe. Of the 24 melanoma cell lines analyzed, 18 expressed beta-hCG mRNA. Analysis of melanoma biopsy specimens revealed beta-hCG mRNA expression in 17/25 melanoma-positive TDLN, and in only 5/15 non-lymphoid melanoma metastases. Beta-hCG mRNA expression had a 53% correlation to tyrosinase mRNA, a predominant melanoma marker. Beta-hCG mRNA was not detected in normal donor PBL and normal lymph nodes. Detection of beta-hCG mRNA expression may be a useful molecular marker to define a subset of malignant melanoma. PMID- 8621228 TI - Expression of nma, a novel gene, inversely correlates with the metastatic potential of human melanoma cell lines and xenografts. AB - nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (1.5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and completely absent in highly metastatic human melanoma cell lines. Fluorescence in situ hybridization combined with the analysis of a panel of human-rodent somatic cell hybrids indicated that the nma gene is located on human chromosome 10, in the region p11.2-p12.3. Sequence analysis of nma showed no homologies with other known genes or proteins, except for several partially sequenced cDNAs. The predicted amino acid sequence suggests that the protein encoded by nma contains a transmembrane domain. Expression of nma is high in human kidney medulla, placenta and spleen, low in kidney cortex, liver, prostate and gut and absent in lung and muscle. Whereas nma is not expressed in normal skin tissue, expression is high in melanocytes and in 3 out of 11 melanoma metastases tested. PMID- 8621229 TI - Radiation-induced apoptosis in normal and pre-neoplastic mammary glands in vivo: significance of gland differentiation and p53 status. AB - The tumor suppressor gene p53 maintains the integrity of the genome by stimulating apoptosis in cells that have sustained DNA damage. The p53 gene is frequently altered in human cancers, including breast cancer, and such alterations are thought to result in genomic instability and aneuploidy, 2 hallmarks of anaplastic cells. We used radiation as a DNA-damaging agent to test the role of p53 in controlling apoptosis propensity in pre-neoplastic mammary lesions in mice. Four different pre-neoplastic mammary outgrowth lines, D1, TM2H, TM4 and TM12, were maintained by serial transplantation in the cleared mammary fat pads of syngeneic BALB/c mice. These lines have known alterations in p53 expression: TM12 has normal expression, D1 over-produces wild-type protein, TM2H contains a deletion resulting in a null phenotype and TM4 produces a mutant protein. Mice bearing the various outgrowths were irradiated with 5 Gy, and apoptosis was scored by examination of histological sections prepared from the outgrowths 6 hr after irradiation. The TM12 outgrowths, but not the TM2H outgrowths, exhibited radiation-induced apoptosis. The D1 and TM4 lines expressed radiation-induced apoptosis while the fat pads were being repopulated; however, the induced apoptosis declined to low levels as the mice aged. These results are consistent with the hypothesis that normal p53 function is important if mammary cells with DNA damage are to be deleted by apoptosis. PMID- 8621230 TI - Inhibition of protein kinase C-dependent protein phosphorylation correlates with increased polarity and locomotion in Walker 256 carcinosarcoma cells. AB - Signal transduction pathways controlling tumor cell locomotion are not yet well understood. We have studied the role of protein kinase C (PKC)-dependent protein phosphorylation associated with changes in cell shape and locomotor activity of Walker carcinosarcoma cells in culture. We show that the inhibitory effect of phorbol-12-myristate-13-acetate (PMA), an activator of PKC, on cell polarity and locomotion can be suppressed by the PKC-selective inhibitor Ro 31-8220. PMA induces increased phosphorylation of at least 2 proteins, of 65 and 80 kDa, in intact Walker carcinosarcoma cells. These bands are enriched in cytosolic fractions isolated from cells incubated with 32PO4. Pre-incubation with Ro 31 8220 inhibits the PMA-induced phosphorylation of both bands in a concentration dependent manner. This effect is very likely not due to inhibition of translocation of PKC to the membrane as Ro 31-8220 enhances, rather than inhibits, PMA-induced transfer of PKC beta(II) to the particulate fraction. We have carried out a quantitative analysis of phosphorylation of the 80-kDa band. Ro 31-8220 reverses both PMA-induced phosphorylation of this band and PMA-induced suppression of cell polarity in parallel. Increased phosphorylation of proteins via PKC may thus be a stop signal for locomoting Walker carcinosarcoma cells. PMID- 8621231 TI - Schedule-dependent reversion of acquired cisplatin resistance by 5-fluorouracil in a newly established cisplatin-resistant HST-1 human squamous carcinoma cell line. AB - In vitro continuous stepwise exposure of HST-1 human squamous carcinoma cell line to cisplatin (CDDP) for 12 months resulted in a 3.5-fold stably resistant subline designated HST-1/CP0.2. Compared with parental cells, this cell line showed a 1.8 fold increase in cellular glutathione (GSH) and a 50% reduction in initial numbers of DNA interstrand cross-links (ICLs), despite similar levels of intracellular platinum accumulation. Evaluation of the kinetics of DNA ICL removal at nearly equivalent levels of DNA ICL formation indicated that HST 1/CP0.2 cells appeared to remove DNA ICLs more rapidly than do HST-1 parental cells. Thus, both elevated cellular GSH and increased DNA repair capacity would be the major factors contributing to CDDP resistance. Pretreatment of HST-1/CP0.2 cells with 5-FU, with drug-free intervals of 24 to 48 hr before exposure to CDDP, completely reversed CDDP resistance, or even increased the sensitivity to a level greater than that of parental cells, whereas the opposite sequence had no effect on resistance. In parallel with augmentation of the cytotoxicity, the levels of cellular GSH were significantly reduced over 48 hr by 5-FU pretreatment. However, depletion of cellular GSH using buthionine sulfoximine resulted in partial reversal of CDDP resistance, indicating that reduction of cellular GSH alone is not sufficient for complete reversal of CDDP resistance. Our data, together with evidence that 5-FU modulates the repair of platinum-DNA cross-links, suggest that schedule-dependent, complete reversal of CDDP resistance by 5-FU might be attributed to its inhibitory effects on both GSH levels and the repair of platinum-DNA adducts. Thus, optimization for the drug administration schedule is important when aiming at therapeutic synergy and circumvention of acquired CDDP resistance. PMID- 8621232 TI - Toxicity of ribosome-inactivating proteins-containing immunotoxins to a human bladder carcinoma cell line. AB - Immunotoxins were prepared by linking the type 1 ribosome-inactivating proteins (RIP) momordin I, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6 to the 48-127 monoclonal antibody (MAb) recognising a glycoprotein (gp54) expressed on all human bladder tumours tested and on human bladder carcinoma cell lines, in particular on the T24 cell line. T24 cells required a 2 hr contact with immunotoxins to ensure binding and endocytosis. A time course of exposure, followed by further incubation without the immunotoxins, showed that maximum inhibition of protein synthesis by T24 cells was reached after 2 hr of contact followed by 3 days without the immunotoxins. Under optimal conditions, 48-127/RIP immunotoxins at nanomolar concentrations inhibited by 50% protein synthesis of target T24 cells. No toxicity was observed if (i) target cells were treated with non-conjugated RIP, (ii) target cells were treated with momordin I- or PAP-S containing immunotoxins made with an irrelevant antibody and (iii) a non-target cell line was treated with the same 2 RIP conjugated to 48-127 antibody. The in vitro selective toxicity of these immunotoxins encourages further studies in view of a possible use in clinical trials for the local therapy of human bladder carcinomas. PMID- 8621233 TI - Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression. AB - We previously reported that fenretinide (4HPR) is effective against a human ovarian carcinoma xenografted in nude mice. The effects of 4HPR on ovarian tumors have been further studied in in vitro ovarian carcinoma cell lines A2780, IGROV I, SW626 and OVCA432. A2780 was the most sensitive line: 50% growth inhibition was obtained after 3 days of exposure to 1 microM 4HPR, a pharmacologically achievable concentration, whereas approx. 10 microM 4HPR gave a similar inhibition in the other cell lines. All-trans retinoic acid (RA), at doses up to 10 microM, did not inhibit cell proliferation. Gel electrophoresis of DNA from either detached or attached A2780 cells treated with 4HPR revealed DNA ladders in detached cells. Apoptosis was also evidenced in detached 4HPR-treated cells by flow cytometry and microscopic observation. The difference in cell line sensitivity to the anti-proliferative effect of 4HPR was not related to drug uptake or efflux. Only A2780 cells, the most sensitive to 4HPR, expressed constitutive levels of RARbeta; moreover, the levels of RARalpha and RARgamma expression in these cells were higher than in the other cell lines. In A2780 cells, the association of an IC20 of 4HPR to cisplatin resulted in a strong potentiation of the anti-proliferative effect. These data show (i) that 4 HPR, in contrast to RA, has an anti-proliferative effect in human ovarian carcinoma cells which is related to induction of apoptosis and (ii) that among the tested lines, the most responsive to the drug expressed RARbeta and the highest levels of RARalpha and RARgamma. The results also suggest that 4HPR can potentiate the effects of cisplatin in ovarian carcinoma. PMID- 8621234 TI - Constituents of autocrine IL-6 loops in myeloma cell lines and their targeting for suppression of neoplastic growth by antibody strategies. AB - We examined the constitution and biological relevance of an autocrine IL-6/IL6 receptor (r) loop in 7 multiple myeloma and plasma-cell leukemia lines in order to determine its biological role and potential therapeutic impact on antibody strategies. The expression and constitution of the IL-6r [i.e. membrane-bound gp 80, soluble (s)gp-55 and the gp-130 IL-6 signal-transducing element (str)], the binding capacity of the membrane-associated receptor(s) for IL-6, the production and secretion of IL-6 by neoplastic plasma cells, and the effect of IL-6 on tumor cell proliferation were investigated. In the U-266 cell line, the growth inhibitory effects of antibodies (Abs) against IL-6 and IL-6-binding subunit of its receptor were compared with each other. From our results the following conclusions may be drawn: (i) Substantial differences in the quantificative assembly of the IL-6r constituents and in the response to recombinant (r) human (h) IL-6 became evident in the 7 myeloma cell lines. (ii) The components of an autocrine IL-6 loop may be regulated in an independent and, in the case of IL-6 and sgp-55, probably counteractive manner. (iii) The level of endogenous IL-6 and the reservoir of recruitable sgp-55 were important for the response to exogenous rhIL-6. (iv) Apart from IL-6, other growth factors are important for the propagation of myeloma cells but at least some of them exert their effect through an IL-6-dependent pathway. Their growth-promoting activity, as well as that of IL 6, may be successfully targeted by immunological means, with Abs against the IL 6r being more efficient than those against the ligand. PMID- 8621235 TI - Protective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs. AB - The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine in DNA and thus may protect cells against genotoxic effects of agents inducing this lesion. To analyze quantitatively the level of protection mediated by MGMT against antineoplastic drugs, we determined the cytotoxic and recombinogenic (sister-chromatid exchange inducing) effects of various chemotherapeutic agents in a pair of isogenic Chinese hamster cell lines deficient and proficient for MGMT, generated upon transfection with human MGMT cDNA. Furthermore, we compared the responses of the human cell lines HeLa MR (MGMT deficient) and HeLa S3 (MGMT proficient) to the various agents. It is shown that: (1) MGMT proficient cells are resistant in cell killing to the methylating drug streptozotocin and all the chloroethylating nitrosoureas tested. There was a marked agent specificity in protection. The level of resistance provoked by MGMT increased in the order BCNU < CCNU < ACNU < HeCNU < streptozotocin. (2) MGMT did not protect cells against killing induced by chlorambucil, cisplatin, melphalan, activated cyclophosphamide (malosfamide) and activated ifosfamide (4-hydroperoxy-ifosfamide). (3) MGMT caused protection against the recombinogenic effect of all nitrosoureas tested. The lowest level of protection was again observed for BCNU, followed by CCNU, ACNU < HeCNU < streptozotocin. (4) MGMT proficient cells did not exhibit resistance in SCE induction towards cyclophosphamide (activated by microsomes), 4-hydroperoxy ifosfamide, mafosfamide, chlorambucil and melphalan. Some protection was afforded, however, against cisplatin (and transplatin). This effect was abolished by pretreatment of cells with O6-benzylguanine, which depletes MGMT, indicating that some lesion(s) induced by cisplatin giving rise to SCEs can be repaired by MGMT. Taken together, these results indicate that streptozotocin, HeCNU and ACNU are more selective than CCNU and BCNU in killing MGMT deficient cells, and that in the cases of cyclophosphamide, ifosfamide, chlorambucil, cisplatin and melphalan MGMT is not involved in mediating cytotoxic drug resistance. PMID- 8621236 TI - Endothelial CD44H mediates adhesion of a melanoma cell line to quiescent human endothelial cells in vitro. AB - A critical step in the metastatic spread of tumour cells is the interaction of circulating tumour cells with the vascular endothelium. We have investigated the role of CD44 and its variants in the adhesion of a human melanoma cell line (RPMI 7951) and a breast adenocarcinoma cell line (MDA-MB-231) to quiescent human umbilical vein endothelial cells (HUVEC) in vitro. Both tumour cell lines express CD44H, CD44A and CD44v9, while HUVEC express only CD44H. Pre-treatment of endothelial cell monolayers with a blocking monoclonal antibody against CD44H (MAb 5A4) reduced the adhesion of RPMI-7951 cells but not that of MDA-MB-231. In contrast, pre-treatment of both tumour cell lines with the same antibody had no effect on adhesion. Digestion of the CD44 ligand hyaluronic acid (HA) on RPMI 7951 cells significantly reduced adhesion to endothelial monolayers, while digestion of HUVEC HA had no effect. We conclude that CD44H expressed on the surface of quiescent endothelial monolayers mediates in part the adhesion of the metastatic melanoma cell line RPMI-7951 but not that of a breast adenocarcinoma line. It does so by acting as a receptor for HA on the tumour cell surface. Tumour cell CD44H and variants CD44A and CD44v9 do not appear to be involved in adhesion to endothelial cells. PMID- 8621237 TI - Abrogation of lung metastasis of human fibrosarcoma cells by ribozyme-mediated suppression of integrin alpha6 subunit expression. AB - The interaction of tumor cells with the basement membrane plays a crucial role in tumor metastasis. VLA-6 (alpha6beta1) integrin is one of the major surface receptors for the basement membrane, specifically recognizing laminin. To study the role of VLA-6 integrin in tumor invasion and metastasis, we synthesized a ribozyme that selectively degrades the integrin alpha6 subunit mRNA. The catalytic activity of the ribozyme was verified by in vitro cleavage of alpha6 subunit mRNA. Introduction of the anti-alpha6 ribozyme gene into the human fibrosarcoma cell line HT1080 yielded stable transfectants, which expressed a significantly reduced level of integrin alpha6 mRNA. Flow cytometric analysis showed that the surface expression of VLA-6, but not other integrins, was reduced by approximately 70% in transfected cells. Ribozyme-transfected cells were less adherent to laminin-coated substrata and less invasive into reconstituted basement membrane than mock-transfected cells. When injected i.v. into nude mice, ribozyme-transfected cells produced no lung metastasis in all except 1 of 35 mice, though mock-transfected cells produced multiple lung metastases in 22 of 29 mice. Our results indicate that VLA-6 integrin plays a critical role in tumor invasion and metastasis and may serve as a potential target for eradication of tumor metastasis in the lung. PMID- 8621238 TI - Loss of responsiveness to transforming growth factor beta (TGFbeta) is tightly linked to tumorigenicity in a model of thyroid tumour progression. AB - It has been suggested that an important step in the progression of some epithelial tumours is the loss of responsiveness to the growth-inhibitory effects of transforming growth factor beta (TGFbeta). Here we describe the use of a model of thyroid tumorigenesis to investigate this question. Seven genetically closely related epithelial cell lines were derived following infection of primary cultures of rat thyroid epithelium with retroviral vectors encoding mutant ras. A strong negative correlation (p < 0.001) was found between the responsiveness of the lines to TGFbeta growth inhibition in vitro and their tumorigenicity in nude mice. Whereas TGFbeta-unresponsive and TGFbeta-stimulated lines formed rapidly growing, poorly differentiated tumours at all injection sites, cells that retained a partial inhibitory response formed much more slowly growing tumours, which showed a high degree of glandular differentiation. A line which retained full inhibition by TGFbeta formed slowly growing tumours at only 30% of injection sites, and cells explanted from these tumours subsequently showed a much reduced TGFbeta response in vitro. Our data using thyroid cells thus greatly strengthen the suggestion from previous studies that loss of growth inhibition by TGFbeta is associated with malignant progression of epithelial tumours. We also present an experimental model of papillary thyroid cancer which may prove useful in identifying the molecular changes involved in progression to the anaplastic form of the disease. PMID- 8621240 TI - A bivalent single-chain antibody-toxin specific for ErbB-2 and the EGF receptor. AB - ErbB-2 and EGF receptors are often co-expressed in human tumors and have been shown to synergize in the transformation of cells in experimental model systems. Transactivation of ErbB-2 can occur via ligand-induced heterodimerization with EGF receptor or other members of the ErbB family of receptor tyrosine kinases. We have previously described the potent anti-tumoral activity of the monospecific single-chain antibody-toxins scFv(FRP5)-ETA and scFv(225)-ETA binding to, respectively, ErbB-2 and the EGF receptor. Here we report the construction and functional characterization of a novel bivalent, bispecific single-chain antibody toxin, scFv2(FRP5/225)-ETA. The fusion protein consists of 2 scFv domains specific for ErbB-2 and the EGF receptor linked to a modified Pseudomonas exotoxin A. ScFv2(FRP5/225)-ETA displayed in vitro cell killing activity on tumor cells overexpressing either ErbB-2 or the EGF receptor similar to that of the monospecific toxins. It was more potent in vitro and in vivo in inhibiting the growth of tumor cells expressing both receptors. Treatment of A431 cells with scFv2(FRP5/225)-ETA led to an increase in EGF receptor and ErbB-2 phosphotyrosine content, most likely via the induction of receptor heterodimers. This may explain the enhanced toxicity of the bispecific antibody-toxin. PMID- 8621239 TI - Decreased expression of nucleoside diphosphate kinase alpha isoform, an nm23-H2 gene homolog, is associated with metastatic potential of rat mammary adenocarcinoma cells. AB - The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23 H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay. PMID- 8621242 TI - Evidence of a hereditary p53 syndrome in cancer-prone families. PMID- 8621241 TI - Monoclonal anti-idiotypic antibody functionally mimics the human gastrointestinal carcinoma epitope GA733. AB - Anti-idiotypic antibodies (Ab2) that bind to the antigen-combining region of anti tumor antibodies (Ab1) may functionally, and even structurally, mimic tumor antigen. We have previously demonstrated that polyclonal goat Ab2 directed against anti-human gastrointestinal carcinoma Ab1 GA733 induces anti-anti idiotypic antibodies (Ab3) in animals that are Ab1-like in their binding specificity and idiotope expression. To obtain more defined Ab2 vaccines with potentially increased specificity and efficacy, a monoclonal Ab2 (FG1) was produced against Ab1 GA733 in rats. The monoclonal Ab2 FG1, similar to the polyclonal Ab2 described previously, induced Ab3 in rabbits that were Ab1-like in their idiotope expression and binding specificity to tumor cells and antigen. Antigen-specific Ab3 induced by Ab2 FG1 were easily detected in unprocessed rabbit sera, whereas the demonstration of such Ab3 after polyclonal Ab2 immunization required purification of the Ab3 from the rabbit sera. In addition, Ab2 FG1 induced antigen-specific humoral and cellular immunity in mice. Murine Ab3 bound specifically to antigen-positive tumor cells. Ab2-immunized mice showed antigen-specific delayed-type hypersensitivity (DTH) reaction, and cultured splenocytes from the immune mice demonstrated specific proliferation and cytokine (interferon-gamma and interleukin-4) secretion upon stimulation with GA733 antigen. However, immune mice were not protected against a challenge with syngeneic GA733 antigen-expressing colon carcinoma cells. PMID- 8621243 TI - Mouse mammary-tumor virus: the basics of an oncogenic retrovirus. Steiner Young Investigator Award Lecture 1995. PMID- 8621244 TI - Risks of testicular cancer and cryptorchidism in relation to socio-economic status and related factors: case-control studies in Denmark. AB - To explore risk factors for testicular cancer and cryptorchidism, 2 parallel case control studies were conducted in Denmark. The testicular-cancer study was population-based and included 514 cases and 720 controls. The cryptorchidism study included 387 cases and 416 controls and was based on 2 hospital series of men treated for cryptorchidism and a control group sampled among residents in the Copenhagen area. The 2037 men were interviewed by telephone, and self administered questionnaires were sent to their mothers. A strong association was seen between low social class and cryptorchidism, with sons of unskilled workers having a 3-fold higher risk of cryptorchidism than sons of self-employed men. Testicular cancer was only moderately associated with high-social-class indicators, and only with such indicators pertaining to the mother. Both testicular cancer and cryptorchidism tended to occur more frequently in first born men and in sons of older women but these associations were not statistically significant. Late puberty was associated with reduced risk of testicular cancer. The effect of age at puberty may be due both to advanced age at diagnosis and to the existence of common determinants of age at puberty and testicular cancer. Men who had been treated for cryptorchidism entered puberty later than other men, possibly because of impaired hormonal function of the testes. There was no indication of increased risk of testicular cancer or cryptorchidism in sons of mothers who smoked around the time of conception or during the pregnancy. PMID- 8621245 TI - Time trends in lung-cancer mortality rates among men in Lithuania, 1965-1994. AB - This study examines time trends in lung-cancer mortality rates among men in Lithuania during the period 1965-1994. Age-standardized mortality rates increased from 32.5 per 100,000 in 1964-1969 to 62.9 per 100,000 in 1990-1994. Regression analysis indicates that net drift (sum of cohort and period slopes) was positive. Cohort effect was dominant in the trend observed. Mortality in cohorts born before 1945 showed an increase. In younger generations, born after 1945, the risk declined in each subsequent cohort. Higher prevalence of cigarettes with filters and lower tar content of cigarettes may be of importance. This analysis suggests that mortality will rise until cohorts born around 1950 reach middle age or old age. After that some stabilization can be expected if other conditions remain unchanged and previous trends continue. PMID- 8621246 TI - p53 gene mutations in oral carcinomas from India. AB - In this study, we analyzed 53 oral squamous-cell carcinomas among Indians for the presence of alterations in the tumor-suppressor gene p53 by PCR-SSCP and sequencing methods. Our results showed that 21% (11/53) of oral carcinomas analyzed carried mutations within the exons 5-8 of the p53 gene. We have identified 11 single-base pair substitutions consisting of 10 mis-sense mutations and one at the splice acceptor site, and one deletion mutation involving 4 consecutive bases. The majority of the base substitutions were transitions (5 TA to CG and 5 GC to AT), while only one transversion (TA to GC) was observed. Probable hot-spots for the mutation induction were identified at codons 149 and 274, which have not been observed before in head-and-neck cancers. The mutational spectrum might have originated from base alkylations at guanine and thymine residues, caused by some alkylating agents. The present results are thus consistent with the involvement of tobacco-related nitrosoamines in the etiology of oral squamous-cell carcinoma. PMID- 8621247 TI - Low frequency of p16/CDKN2 gene mutations in esophageal carcinomas. AB - Mutational analysis of the p16/CDKN2 gene was conducted by direct sequencing of the whole coding sequence (exons 1-3 and flanking splicing sites) in 21 esophageal squamous-cell carcinomas and 3 adenocarcinomas from a high-incidence area of Italy. Two inactivating mutations were found in exon 1 of the gene (both in squamous-cell carcinoma), whereas no mutations were detected in exon 2, where most of the sequence changes reported so far have been located, or in exon 3. Southern blot analysis of exon 2 in this set of samples and in a complementary set of 12 tumor samples from France did not show homozygous deletions or detectable gene rearrangements. Thus, p16/CDKN2 gene alterations do not appear to play a major role in the group of patients examined. PMID- 8621248 TI - Infrequent alterations of the p15, p16, CDK4 and cyclin D1 genes in non astrocytic human brain tumors. AB - While several genetic alterations associated with the evolution of the astrocytomas have been identified, the molecular basis of non-astrocytic brain tumors has remained largely unknown. In this study, p15, p16, CDK4 and cyclin D1 genes were analyzed in 69 nonastrocytic human brain tumors, including 17 oligodendrogliomas, 16 medulloblastomas/primitive neuroectodermal tumors (PNETs), 14 ependymomas and 22 meningiomas. Southern blot analysis of DNA from frozen samples showed no homozygous deletions in p15 or p16 genes in any of these tumors. No mobility shift was found by PCR-single-strand conformation polymorphism (PCR-SSCP) analysis in exons 1 and 2 of the p15 gene and exons 1 and 2 of the p16 genes, except for one oligodendroglioma. Direct sequencing of DNA from this tumor showed a G --> A transition at nucleotide 436 (codon 140) in exon 2 of the p16 gene, which is a common polymorphism. Southern blot analyses revealed no amplification of CDK4 and cyclin D1 genes in any of the neoplasms analyzed. In contrast to astrocytic brain tumors, which show frequent loss of the p16 gene and amplification of the CDK4 gene, alteration of these genes appears to be rare in other neoplasms of the human nervous system. PMID- 8621250 TI - Co-expression of heparin-binding EGF-like growth factor and related peptides in human gastric carcinoma. AB - Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor alpha(TGF-alpha), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9 fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-alpha, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB EGF, EGF and TGF-alpha enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder. PMID- 8621249 TI - Protective factor against progression from atrophic gastritis to gastric cancer- data from a cohort study in Japan. AB - To investigate the association between atrophic gastritis and gastric cancer and to identify the risk and protective factors for the progression of atrophic gastritis to cancer, we conducted a prospective study on 5,373 subjects with neither cancer nor resected stomach who underwent gastroscopic examination and completed a life-style questionnaire. After an average of 6 years of follow-up, 69 gastric-cancer cases were identified, 65 from the subjects without atrophic gastritis and 4 from the subjects without atrophic gastritis. The presence of atrophic gastritis increased the risk of gastric cancer 2.19-fold, the risk trend increasing with the degree and the extension of atrophy [relative risk (RR) 1.60 for mild atrophy and 2.85 for moderate and severe atrophy]. Among the subjects with atrophic gastritis, family history of gastric cancer (RR 2.27) and a preference for spicy food (RR 1.84) increased the risk and self-administered meal controls, such as portion reduction (RR 0.44) reduction of salty food (RR 0.56) and the change to the consumption of easily digested food (RR 0.57) decreased the risk of gastric cancer. The results of this study suggest that atrophic gastritis increases the risk of gastric cancer but that dietary modification prevents the progression from atrophic gastritis to gastric cancer, regardless of pre cancerous lesions. PMID- 8621251 TI - Frequent expression of Bcl-2 in renal-cell carcinomas carrying wild-type p53. AB - The p53 tumor-suppressor gene is the most commonly mutated gene in cancer. However, p53 gene alterations are infrequent in renal-cell cancer (RCC). Bcl-2 has been shown to inhibit apoptosis triggered by wild-type p53 and an inverse correlation between Bcl-2 expression and p53 mutation has been observed in breast cancer and glioma. To characterize the expression of bcl-2 in RCC and its relationship to the p53 status, we analyzed 25 RCCs by immunohistochemistry for Bcl-2 and p53, Southern hybridization for bcl-2, and PCR-SSCP and sequencing for p53. Positive Bcl-2 staining was detected in 17 of 25 RCCs, whereas positive p53 staining was seen in only 1. Amplification of bcl-2 or p53 mutation was not detected in any of the tumors. Bcl-2 protein was expressed in all 7 RCC cell lines examined. Only one of the 7 lines had p53 mutation. These results suggest that overexpression of bcl-2, rather than p53 mutation, may prevent apoptosis during RCC development. PMID- 8621253 TI - Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR. AB - Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkins lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene. PMID- 8621252 TI - Serum trypsinogen-2 and trypsin-2-alpha(1)-antitrypsin complex in malignant and benign digestive-tract diseases. Preferential elevation in patients with cholangiocarcinomas. AB - Serum concentrations of trypsinogen-2 and trypsin-2-alpha(1)-antitrypsin (trypsin 2-AAT) were determined in 145 patients with malignant and 61 with benign digestive-tract diseases. The validity of these tests for detection of cancer was compared with that of CA 19-9 and CEA. Elevated levels of trypsinogen-2 (>90 micrograms/l) and trypsin-2-AAT (>25 micrograms/l) were found in 46% and 42%, respectively, of patients with malignant disease and the levels of trypsinogen-2 were significantly higher than in those with benign disease (p<0.005). High trypsinogen-2 and trypsin-2-AAT concentrations were found most often in patients with biliary and pancreatic cancer, but also in benign obstructive biliary disease. Our results suggest that trypsinogen-2 and trypsin-2-AAT are new potential markers for cholangiocarcinomas. PMID- 8621254 TI - Purified recombinant EBV desoxyribonuclease in serological diagnosis of nasopharyngeal carcinoma. AB - To evaluate applications of highly purified recombinant EBV DNAase in the diagnosis and prognosis of NPC, we tested sera from patients with NPC, other EBV associated diseases and EBV-seropositive and -seronegative healthy subjects by immunoblotting and DNAase inhibitory assay. The results were compared with those obtained by the conventional immunofluorescence assays against the EBV-specified early antigens and capsid antigens. The antigenic specificity of the immunoblotting assay for IgG antibody against the viral enzyme, but not that for the IgA antibody, was correlated with DNAase-inhibitory activity of the sera and their titers of IgG antibodies against the viral early antigens. Purified IgA as well as IgG from NPC sera inhibited enzyme activity with similar efficiency. The use of highly purified viral DNase has increased the sensitivity of detection of the corresponding antibodies by immunoblotting, with the IgG antibody being detected in all but one, and IgA antibody in all but 2, of the 174 NPC sera tested. The IgG antibody was also commonly detected in the other groups of control sera, while the IgA antibody was detected in about 10% of African Burkitt's lymphoma and Algerian Hodgkin's lymphoma patients and less than 3% of the other control subjects. These results suggest that IgA antibody against recombinant EBV DNAase may be useful in the diagnosis of NPC, but the level of this antibody did not appear to be related to clinical stages of this cancer. PMID- 8621255 TI - Interaction of ionizing radiation with topotecan in two human tumor cell lines. AB - The effect of topotecan, a topoisomerase I inhibitor, on ionizing radiation induced cytotoxicity was studied in 2 human tumor cell lines characterized by a different expression of the target enzyme. The cytotoxicity of topotecan alone or in combination with radiation was assessed in exponentially growing non-small cell lung cancer (H460) and glioblastoma (GBM) cells using the colony-forming assay. An isobologram method was used to evaluate the treatment interaction. An apparent supra-additive effect in cell killing following drug-radiation-combined treatment was observed only in GBM cells exposed to topotecan for 24 hr. In the case of H460 cells, interaction varied from a strong infra-additive effect at low radiation doses to a slight supra-additive effect when cells were exposed to radiation doses greater than 3 Gy. Northern blot analysis indicated that topoisomerase I expression in H460 cells was 8-fold higher than that of GBM cells. Although the H460 cell line exhibited an increased sensitivity to topotecan, only in the GBM cell line (which expressed a lower level of topoisomerase I) did the drug potentiate the radiation cytotoxicity. The observation that the radiosensitization by topotecan was related to topoisomerase I level is consistent with a putative role of the enzyme in processes involved in the repair of radiation damage. It is conceivable that the modulation of enzyme function results in an effective reduction of cellular capability for repair of radiation damage only if the enzyme is not over-expressed. Although a precise role of topoisomerase I in the cellular response to ionizing radiations (in particular, in DNA repair) remains to be documented, such results suggest the potential interest of topoisomerase I inhibitors in combination with radiation therapy for tumors expressing low topoisomerase I levels. PMID- 8621256 TI - Hyperthermia induces apoptosis in malignant fibrous histiocytoma cells in vitro. AB - The effect of mild hyperthermia on a cultured rat malignant fibrous histiocytoma (MFH) cell line, MFH-2NR, was investigated. MFH cells in log-phase (growing phase) were heated at 41 degrees-44 degrees C for 1 hr. Hyperthermic treatment at 41 degrees C did not substantially affect cell proliferation and treatment at 44 degrees C caused necrosis. After hypothermic treatment at 42 degrees or 43 degrees C, proliferation of MFH cells was arrested and morphological changes characteristic of apoptosis, cell shrinkage accompanying apoptotic bodies and chromatin condensation, became apparent. Hyperthermia-induced apoptosis was further confirmed by terminal deoxynucleotidyl transferase staining and a ladder pattern on agarose gel electrophoresis. Flow cytometric analysis indicated that the population in the G1 phase of the cell cycle significantly decreased with a concomitant increase in apoptotic cells, indicating that apoptosis might occur mainly in the G1 phase population. PMID- 8621258 TI - Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines. AB - In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway. PMID- 8621257 TI - Increased expression of schwannoma-derived growth factor (SDGF) mRNA in rat tumor cells: involvement of SDGF in the growth promotion of rat gliomas. AB - Schwannoma-derived growth factor (SDGF) is a member of the epidermal growth factor (EGF) family, having mitogenic activity on rat astrocytes, fibroblasts and Schwann cells. The SDGF gene is significantly expressed in the newborn rat lung and in the adult rat sciatic nerve. However, except for one rat schwannoma cell line, from which SDGF and its cDNA were isolated, nothing is known about SDGF expression in established tumor cell lines. We examined the expression level of the SDGF gene in a variety of rat tumor cell lines by Northern blotting and found that it was increased in 11 of 25 established lines. The most abundant SDGF mRNA, which was about 50-fold higher than in the newborn rat lung, was expressed in rat liver adenoma dRLa74 cells. In rat glioma cell lines, such as C6, 9L and T9, and in the rat hepatoma dRLh84 and H411E cells, the SDGF expression level was about 10-fold higher than in the newborn rat lung. In 8 of 13 cell lines expressing SDGF mRNA, the EGF receptor (EGFR) gene, the product of which is regarded as a functional receptor of SDGF, was co-expressed. In addition, transfected gene dependent anti-sense SDGF RNA expression under the control of the human metallothionein promoter significantly suppressed the in vitro growth as well as in vivo tumorigenicity of 9L glioma cells. Our results suggest that SDGF acts as an autocrine growth factor in the development and growth of rat tumors such as gliomas. PMID- 8621259 TI - Direct killing of interleukin-2-transfected tumor cells by human neutrophils. AB - We have previously established that human polymorphonuclear cells (PMN) express IL-2R beta- and gamma-chains and that addition of IL-2 maintains the viability of PMN by preventing these cells from undergoing programmed cell death. The purpose of this study was to examine whether IL-2-releasing tumor cells are capable of stimulating PMN tumoricidal activity. We therefore investigated the ability of PMN to kill IL-2-transfected tumor cells using normal human PMN directed against the murine mammary adenocarcinoma TS/A engineered to release high amounts of murine IL-2 (3,600 U, B6) compared with TS/A parental cells and TS/A tumor cells transfected with the neomycin-resistance (NEO) gene only. The potency of PMN as IL-2-induced killer cells was indicated by the low number of cells required for killing (effector cell:target cell ratio 10:1) and the degree of tumor cell lysis (68+/-10%). Evidence for the role of IL-2 as a mediator of tumor cytotoxicity by PMN was substantiated by inhibition of tumor killing with anti-IL-2 and anti-IL 2R beta monoclonal antibodies (MAbs). Furthermore, in vivo depletion of mature granulocytes using MAb RB6-8C5 resulted in B6 adenocarcinoma growth, thereby confirming a direct role for IL-2-activated PMN in tumor cytolysis. Lastly, we suggest that one possible mechanism involved in IL-2-induced PMN cytotoxicity against the B6 clone occurs via the nitric oxide pathway, which could be inhibited upon addition of the arginine analog, N(G)-monomethyl-L-arginine. PMID- 8621261 TI - Establishment and characterization of primary and metastatic uveal melanoma cell lines. AB - We report on the establishment and characterization of 2 primary (EOM-3, EOM-29) and 3 metastatic uveal melanoma cell lines (OMM-1, OMM-2, OMM-3) and further cytogenetic characterization of a previously described primary uveal melanoma cell line (OCM-1). Only a few long-term growing primary uveal melanoma cell lines have as yet been established, while of metastatic uveal melanoma cell lines we have found no descriptions. The morphology of the in vitro cultured cells varied from spindle to epithelioid. The cell lines were characterized by immunocytochemistry, electron microscopy and cytogenetical analysis. The relative growth rate was determined by bromodeoxyuridine (BUdR) incorporation. The melanocytic origin of the cell lines was determined by positive staining with antibodies identifying melanoma-associated antigens. Melanosomes and pre melanosomes were indeed observed by electron microscopy in all cell lines. The stem-cell karyotype was found to be normal in 3 cell lines (EOM-29, OMM-2, OMM-3) and abnormal in 3 others (EOM-3, OCM-1, OMM-1) showing a net loss of chromosome 6. The OCM-1 and the OMM-1 cell lines even demonstrated a large amount of structural chromosomal aberrations, the former being near-tetraploid and the latter triploid. The EOM-29 cell line, cultured from a ciliary body melanoma, did not show the previously described chromosome 3 and 8 abnormalities. PMID- 8621260 TI - Potentiation of the anti-tumor effect of actinomycin D by tumor necrosis factor alpha in mice: correlation between in vitro and in vivo results. AB - The anti-tumor effects of actinomycin D (Act D) and recombinant human tumor necrosis factor (TNF)-alpha have been studied on 4 established murine tumor cell lines: MmB16 melanoma, Lewis lung (LL/2) carcinoma, L1 sarcoma and L1210 leukemia. During short-term incubation (24 hr) Act D produced dose-dependent cytostatic/cytotoxic effects against MmB16, LL/2 and L1 tumor cells but did not reduce the viability of these cells even at high concentration (10 micrograms/ml), below a threshold of 30-60%. However, L1210 leukemic cells were highly susceptible to Act D, and no viable cells were detected in cultures incubated with 1 microgram/ml of Act D. TNF-alpha alone, when used under the same culture conditions, had only a negligible effect on all cell lines tested. However, the combination of this cytokine with Act D produced synergistic cytotoxic effects against MmB16, LL/2 and L1 cells but not against L1210 leukemia cells. In an in vivo model of regional therapy in which tumor-bearing mice were treated with Act D and TNF-alpha, a correlation with in vitro results was observed. In mice bearing MmB16 melanoma, LL/2 carcinoma and L1 sarcoma, the most potent anti-tumor effects were observed in mice treated with Act D and TNF-alpha together. This treatment led to a delay of tumor growth and induced complete tumor regression in some cases. On the contrary, TNF-alpha did not enhance the effect of Act D in mice injected with L1210 leukemia cells. Our results show that TNF-alpha can potentiate the anti-tumor effects of Act D against tumors weakly susceptible to Act D and may be a useful adjuvant to chemotherapy in the local treatment of neoplasia. PMID- 8621262 TI - TNF promotes metastasis by impairing natural killer cell activity. AB - Inflammatory mediators such as tumor necrosis factor (TNF) and interleukin-1 enhance tumor colony formation in different models of experimental and spontaneous metastasis. The involvement of the natural killer (NK) cell system in this process was investigated. Tumor necrosis factor does not appear to act directly on tumor cells by reducing their susceptibility to the cytotoxic action of NK cells but rather impairs NK activity in tumor-bearing mice. Such impairment of the natural killer system might be one means by which TNF supports tumor colony formation. Even though the metastasis- enhancing effect of TNF remained detectable in mice which have a greatly reduced NK cell cytotoxic activity due to a defect in the bg locus, normal mice which were depleted of NK cells by antibody treatment did not show enhanced metastasis after TNF injection. Therefore, the TNF-enhanced metastasis can only be seen as long as some NK cell function is operating in the animals. PMID- 8621263 TI - Protective effects of interferon-gamma on squamous-cell carcinoma of head and neck targets in antibody-dependent cellular cytotoxicity mediated by human natural killer cells. AB - An in vitro model of antibody-dependent cellular cytotoxicity (ADCC) was established, using squamous-cell carcinoma of the head and neck (SCCHN) targets,human/mouse chimeric monoclonal antibodies (cMAbs) SF-25 and 323/A3 and human peripheral blood mononuclear cells (PBMC). We previously showed that natural killer (NK) cells are the main effector population mediating ADCC in the presence of the cMAbs. ADCC was significantly inhibited by the overnight pre treatment of SCCHN targets with exogenous interferon-gamma (IFN-gamma). This inhibition was dose-dependent, reproducible and consistently observed with various SCCHN cell lines. SCCHN cells pre-treated with IFN-gamma had a significantly higher expression of intercellular adhesion molecule-I (ICAM-I) and major histocompatibility complex (MHC) class I antigens compared with untreated target cells. No differences in expression of the SCCHN-associated antigens on these targets or in the formation of NK-SCCHN conjugates were found, using flow cytometry. IFN-gamma-pre-treated SCCHN cells were less effective in competing with untreated targets in cold target inhibition assays and in inducing cytokine production from NK cells in co-incubation experiments. Protective effects of IFN gamma on target cell sensitivity to lysis were blocked by pre-treatment of target cells with actinomycin-D or cycloheximide. The susceptibility of the target cells was restored by removal of MHC class I antigens from their surface by acid stripping before ADCC. Our results suggest that the decreased ADCC seen with SCCHN targets pre-treated with IFN-gamma is related to post-binding events, possibly altered signaling from targets to effector cells, and requires protein synthesis in the target cells. PMID- 8621264 TI - Optimal regimes for local IL-2 tumour therapy. AB - In this report we present studies on optimal regimes for regional IL-2 therapy, focused on dose, schedule and site of injection. Original data obtained in 2 murine tumour models show that all 3 factors are of importance. Anti-tumour responses were most effective when IL-2 was administered regionally 5 to 10 times, at doses ranging from 7,000 to 33,000 IU/day every day or every other day. This resulted in cure rates of more than 40% in mice bearing ascitic tumour that had also disseminated to liver and lungs. The importance of these data is discussed in the light of previous results of our group. These results illustrate that the doses and schedules used in this study are not effective exclusively in these 2 tumour models but may have a more general applicability. PMID- 8621265 TI - Effect of quercetin on the genotoxic potential of cisplatin. AB - The natural product flavonoid quercetin has been shown to sensitise cells to the cytotoxic potential of cisplatin. Both cisplatin and quercetin are genotoxicants. As quercetin is currently in clinical trial as a cytotoxicant-sensitising agent, we wanted to elucidate whether it affects the genotoxicity associated with cisplatin. The genotoxic potential of both agents alone and in combination was studied in Salmonella typhimurium strains TA 98, TA 100 and TA 102 and by assessment of unscheduled DNA synthesis (UDS) in rat hepatocytes. Furthermore, effects of quercetin on levels of cisplatin-DNA adducts were studied in hepatocytes by ELISA. Cisplatin was mutagenic in all 3 bacterial strains and quercetin in strain TA 98. The number of revertant Salmonella colonies observed with the combination did not differ significantly from that caused by the drugs on their own. In the UDS assay, cisplatin was genotoxic but quercetin was not. In combination, quercetin decreased the nuclear grain count caused by cisplatin, but quercetin did not alter the level of cisplatin-DNA adduct formation in hepatocytes. Our results suggest that the mutagenic potential of the combination cisplatin-quercetin, as judged by the bacterial short-term test, does not exceed that associated with the individual components. However, in hepatocytes, quercetin appears to inhibit repair of cisplatin-induced DNA damage. Therefore, in patients who are to be treated with a combination of cisplatin and quercetin, the risk of genotoxicity in normal tissues will have to be taken into consideration. PMID- 8621267 TI - High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma. AB - Plasma and ascitic fluid of rats bearing the Yoshida ascites hepatoma AH-130 were shown to contain high levels of proteolytic enzymes belonging to different classes active at neutral and acidic pH. Relative to those measured in control rat plasma, in tumor-bearing animals, the activity levels of lysosomal cathepsins B and L, in their latent, acidic-activatable form, were approximately 5-fold higher in plasma and 9-fold higher in ascitic fluid, and cathepsin D activity was about 5-fold higher in both plasma and ascitic fluid. Plasma and ascitic fluid of tumor-bearing rats also contained novel neutral and acidic gelatinolytic activities. The latter, as revealed by zymographic analysis conducted at pH 6.0, in the presence of dithiothreitol and in the absence of divalent metal ions, was sensitive to iodoacetamide inhibition but not to EDTA, showed a molecular mass of approximately 90 kD on SDS-PAGE, and was lost upon limited proteolysis with pepsin. Therefore, this enzyme is not identifiable as cathepsin B or L or their related latent forms and may represent a novel, so far undescribed, gelatinase. Its presence exclusively in the body fluids of AH-130-bearing rats suggests its possible use as a tumor marker. PMID- 8621266 TI - Expression of the alpha 6, beta 1 and beta 4 integrin subunits, basement membrane organization and proteolytic capacities in low and high metastatic human colon carcinoma xenografts. AB - Malignant transformation is associated with alterations in both cell-cell and cell-matrix interactions. The E2 and C5 clones, derived from the human colon adenocarcinoma LoVo cell line, show, respectively, low and high metastatic capacity as experimental xenografts in vivo. In this study, we have assessed the adhesion and spreading of E2 and C5 cells on basement membrane laminin, expression of the laminin receptor integrins alpha 6 beta 1 and alpha 6 beta 4 and expression of gelatinolytic and plasminogen-dependent activities. On days 5 and 7 after subcutaneous grafting to immunosuppressed newborn rats, well differentiated E2 tumors displayed a polarized expression of these integrin subunits, with the exception of the beta 1 subunit which remained pericellular. In contrast, C5 tumors were unorganized and the three integrin subunits remained nonpolarized and pericellular. Flow cytometry results showed that the expression of alpha 6 beta 1 and alpha 1 beta 4 integrins was weaker in the highly metastatic C5 clone than in the E2 clone whereas laminin expression was not significantly different. Under-expression and pericellular localization of these integrin receptors in C5 cells as compared to E2 cells may explain the difference in their binding and spreading capacity on laminin, organization of peritumoral basement membrane and maintenance of a differentiated phenotype. Whereas similar levels of gelatinolytic and plasminogen activator activities have been detected in the culture supernatant of the two clones, histozymograms showed that plasminogen-dependent caseinolysis appeared earlier in sections of C5 and parental tumors than in those of E2 xenografts. These results suggest that enhanced aggressiveness of C5 tumors in vivo may be linked to both an impairment of basement membrane setting due to integrin underexpression and distribution and of proteolytic activities modulated by tumor/host interactions. PMID- 8621268 TI - Immunophenotype analysis of dendritic cells and lymphocytes associated with cutaneous malignant melanomas. AB - The purpose of this study was to investigate the relationship between changes in density and distribution of dendritic cells, both in epidermis and in peritumoral infiltrate, and lymphocyte subset variations in malignant melanomas (MM) of patients belonging to different risk groups. The collective immunoreactive expression of six markers (S100 protein, CD1-a, HLA-DR, CD4, CD8 and CD25) was analyzed in 13 cutaneous malignant melanomas. Changes were observed in density and distribution of Langerhans cells (LC) (S100+, CD1-a+) in the epidermis overlying the tumor, as well as in peritumoral and intratumoral locations, independently of the tumor-invasion level. A decrease was recorded in LC (S100+, CD1-a+) in the epidermis overlying six tumors, whereas most of the MM studied showed an increase of LC (S100+, CD1-a+) in peritumoral infiltrate. The expression of HLA-DR in tumor cells was controversial; it was observed in three moderate-risk MM, but it was negative in high-risk tumors. The percentage of CD4+ cells was in most cases greater than that of CD8+ in the peritumoral infiltrate, irrespective of the degree of histopathological malignancy. The concomitant expression of the lymphocytic activation marker CD25 (receptor for interleukin 2) in lymphocytic infiltrate was variable. Peritumoral infiltrate in three high risk MM contained few CD25+ cells, and a concomitant decrease was recorded in LC. This preliminary report shows that alterations in the density and distribution of LC may be responsible for determining the degree or T lymphocyte activation, and this may be critical for the development of effective tumor-directed immunity. Further studies are required to demonstrate these hypothetical interrelations. PMID- 8621269 TI - Estrogen activates invasiveness of endometrial cancel cells to the interstitium. AB - Invasiveness of endometrial cancer cells such as Ishikawa, HEC-1-A and HHUA cells, to the interstitium was significantly enhanced by estradiol, while medroxyprogesterone acetate (MPA) significantly diminished the estradiol-enhanced invasive potential. All of these endometrial cancer cells possess estrogen receptors. It is suggested that invasiveness to the interstitium, and consequently infiltration to the uterine myometrium (which means local advance) are activated by estradiol via a mechanism related to the estrogen receptor, and that MPA as an antiestrogen agent partly inactivates the invasive ability. PMID- 8621270 TI - Stearate inhibits human tumor cell invasion. AB - Tumor growth and metastasis are affected by changes in membrane lipid composition, however, little is known regarding the role of specific fatty acids in these pathological events. We investigated the effects of the long-chain saturated fatty acids (LCSFA), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) on two key steps of metastasis: cell adhesion and invasion into extracellular matrix (ECM). Using a new 72-hour ECM (Amgel) invasion assay, we demonstrated that the exposure of highly invasive human fibrosarcoma HT-1080 cells to 0.3 mM stearate inhibited their ability to traverse Amgel by 59.4 +/- 8%. In contrast, treatment of tumor cells with 0.3 mM myristate or palmitate had no effect. Microscopic examination revealed a time-dependent inhibition of tumor cell adhesion to the Amgel in the stearate-treated group. Cell adhesion assays further showed a series of rapid morphological cellular changes, i.e. retraction of processes, cell rounding, and subsequent detachment in the presence of stearate. These morphological events were both dose- and time-dependent. Viability of LCSFA-treated cells exceeded 80%. This stearate inhibition of HT 1080 cell adhesion was also observed with two other invasive human tumor cell lines. Similar treatment of HT-1080 cell with the unsaturated long-chain fatty acid oleate (C18:1) did not alter tumor cell adhesiveness. In contrast, nontransformed human fibroblasts (Hs-68) were unaffected by stearate treatment. This inhibition of cell adhesion by stearate was determined to be dependent upon laminin-containing ECM. Pretreatment of HT-1080 cells with stearate dramatically abolished their capacity to attach to laminin but not to collagen type IV or fibronectin matrices. Immunofluorescent studies with anti-beta 1 integrin receptor and antivinculin antibodies demonstrated beta 1 subunit and vinculin colocalization to focal adhesions in untreated HT-1080 cells adherent to laminin, in contrast to stearate-treated tumor cells. Further, stearate-induced changes were shown to be functionally coupled to integrins as an anti-beta 1 antibody markedly diminishes the adhesive ability of tumor cells to laminin. These data demonstrate stearate inhibits tumor cell adhesion, and therefore invasion, via a mechanism involving a laminin integrin receptor. PMID- 8621271 TI - Synthetic cell-adhesive laminin peptide YIGSR conjugated with polyethylene glycol has improved antimetastatic activity due to a longer half-life in blood. AB - This study was conducted to determine the mechanisms for the enhanced inhibitory effect of cell-adhesive peptides conjugated to polyethylene glycol (PEG) on tumor metastasis. Tyr-Ile-Gly-Ser-Arg (YIGSR), a laminin-derived peptide, conjugated with amino-PEG (YIGSR-aPEG) inhibited lung metastasis of B16-BL6 melanoma cells more effectively than unconjugated YIGSR peptide. [125I]-YIGSR-aPEG and native [125I]-YIGSR showed similar biphasic elimination and profiles after intravenous injection into C57BL/6 mice. Both [125I]-YIGSR and [125I]-YIGSR-aPEG expressed similar plasma half-lives and organ distributions. The radioactivity of both compounds was transported rapidly from the blood to the kidneys, and immediately excreted into the urine. [125I]-YIGSR was almost completely degraded in the urine, but [125I]-YIGSR-aPEG was not. In an in vitro stability assay, [125I] YIGSR was degraded immediately upon incubation with mouse serum, whereas [125I] YIGSR-aPEG was not degraded after 180 min incubation in mouse serum. These findings indicate that the enhanced inhibitory effect of YIGSR-aPEG on lung metastasis might be due to its increased stability in the blood. PMID- 8621272 TI - Metastatic pleomorphic adenoma. AB - We report a case of pleomorphic adenoma of the parotid with metastasis to the liver, following resection for local recurrence, which has only been described once before. The diagnosis was made by fine needle aspiration and the lesion was resected without complications. The patient is free of disease 4 years later. Histological features of malignancy were not present in any specimens from the original resection, the local recurrence and the metastatic lesion. It is difficult to say which patients with pleomorphic adenoma should be observed for the development of treatable metastases, but metastases have been reported to occur mainly after repeated resections for local recurrences. PMID- 8621273 TI - Resistance mechanisms in human lung cancer. AB - Drug resistance is an important problem in the treatment of lung cancer. Patients become resistant not only to the drugs used initially, but also to those to which they have not yet been exposed. Multiple mechanisms contribute to drug resistance in this disease, all or any combination of which may occur simultaneously within each cell, producing an overall drug-resistant phenotype. The limited success of hitherto applied strategies in their ability to circumvent drug resistance in lung cancer suggests that new approaches are required. PMID- 8621274 TI - Increased expression of sialyl Lewis A and sialyl Lewis X in liver metastases of human colorectal carcinoma. AB - Sialyl Lewis A (SLA) and sialyl Lewis X (SLX) have been shown to be specific ligands for endothelial leukocyte adhesion molecule-1 (ELAM-1), and may be involved in the process of adhesion between cancer cells and endothelium. We used immunohistochemical methods to study the expression of SLA, SLX and CEA in both primary tumors and matched metastatic liver lesions of colorectal carcinomas. Specimens from primary tumors and matched liver metastases from 24 patients with colorectal carcinomas were studied immunohistochemically. The degree of expression of CEA in liver metastases was similar to that in primary tumors, but SLA and SLX were expressed on a larger proportion of tumor cells in liver metastases than in primary tumors. Our findings suggest that colorectal carcinoma cells expressing SLA and/or SLX form metastatic liver tumors. They also suggest that expression of SLA and SLX in primary of colorectal carcinoma can be used as a prognostic indicator of metastasis. PMID- 8621275 TI - The result of radical retropubic prostatectomy and adjuvant therapy for pathologic stage C prostate cancer. AB - PURPOSE: The results of therapy in 288 men with pathologic Stage C prostate cancer who underwent radical retropubic prostatectomy (RRP) were analyzed to determine the effects of adjuvant therapy. METHODS AND MATERIALS: Twenty-seven of the 288 patients received preoperative neoadjuvant hormonal therapy (leuprolide acetate). Postoperatively, 60 patients received adjuvant radiotherapy (RT) to the prostate bed. Follow-up ranged from 3 to 83 months (median = 32 months). Freedom from failure (FFF) was defined as maintaining a serum PSA level of < or = 0.3 ng/ml. RESULTS: The FFF was 61% at 3 years and 45% at 5 years for the entire group. The FFF following RRP plus RT was 75% at 3 years and 57% at 5 years as compared to 56% at 3 years and 40% at 5 years for RRP without RT (p=0.049). The FFF following RRP plus neoadjuvant hormonal therapy was 58% at 3 years and 40% at 5 years as compared to 60% at 3 years and 45% at 5 years following RRP without hormonal therapy (p=0.3). In patients without seminal vesicle (SV) invasion, the FFF was 81% at 3 years and 5 years for RRP plus RT as compared to 61% at 3 years and 50% at 5 years for RRP without RT (p=0.01). In patients with SV invasion, the FFF was 61% at 3 years and 36% at 5 years for RRP plus RT as compared to 44% at 3 years and 23% at 5 years for RRP without RT (p=0.23). The projected local control rate was 83% at 5 years for those with RRP alone as compared to 100% for RRP plus RT (p=0.02). Survival at 5 years was projected to be 92% and was not significantly altered by the administration of adjuvant therapies. CONCLUSIONS: Postoperative RT was associated with significantly improved local control and FFF rates, especially in patients with tumors which did not involve the seminal vesicles. PMID- 8621276 TI - Predicting the patient at low risk for lymph node metastasis with localized prostate cancer: an analysis of four statistical models. AB - PURPOSE: Statistical models using preoperative Prostate-Specific Antigen, Gleason primary grade or score of the biopsy specimen, and clinical stage have been developed to predict those patients with clinically localized prostate cancer at low risk for lymph node metastasis. It has been recommended that these patients do not require pelvic lymph node dissections. Four such models were evaluated to assess their accuracy in identifying this subgroup of patients. METHODS AND MATERIALS: We reviewed the records of 214 patients with clinically localized prostate cancer who underwent pelvic lymph node dissections. Data from these patients were entered into the four models. RESULTS: Lymph node metastasis was detected in 14% of patients. The results showed the following for each of the proposed models respectively: 78, 50, 76, and 42% of the patients were identified as low risk and, hence, would be spared pelvic lymph node dissections. The false negative rates are 13 (7.8%), 5 (4.6%), 14 (8.6%), and 1 (1.1%). Sensitivities are 56.7, 83.3, 53.3, and 96.7%. CONCLUSIONS: While the pelvic lymph node dissection is the most accurate method of detecting occult nodal metastasis, statistical models can identify a cohort of low risk patients that may be spared lymphadenectomy. PMID- 8621277 TI - Pretreatment prostate-specific antigen doubling times: clinical utility of this predictor of prostate cancer behavior. AB - PURPOSE: The distribution of pretreatment and posttreatment prostate specific antigen (PSA) doubling times (PSADT) varies widely. This report examines the pretreatment PSADT as an independent predictor of biochemical freedom from disease (bNED) and describes the clinical utility of PSADT. METHODS AND MATERIALS: Ninety-nine patients with T1-3 NX, M-0 prostate cancer treated between February 1989 and November 1993 have pretreatment PSADTs calculated from three or more PSA levels. Biochemical disease-free (bNED) survival (failure is PSA > or = 1.5 ngm/ml and rising) is evaluated by multivariate analysis of common prognostic indicators and PSADT. RESULTS: Prostate-specific antigen doubling time (PSADT) is a significant predictor of survival along with radiation dose. Patients with a pretreatment PSADT of < 12 months show 50% failure by 18 months, while those with a PSADT that is not increasing show only 3% failure at 3 years. CONCLUSIONS: Prostate-specific antigen doubling time (PSADT) is a predictor of bNED outcome in prostate cancer. Patients with PSADT < 12 months have aggressive disease, and should be considered for multimodal therapy. Slow PSADT (> or = 5 years) is observed in 57% of patients, and this end point may be considered in the decision to observe rather than to treat. After treatment failure, the PSADT may be used to determine which patients do not need immediate androgen deprivation. PMID- 8621278 TI - Conventional vs. conformal radiotherapy for prostate cancer: preliminary results of dosimetry and acute toxicity. AB - PURPOSE: To compare conformal radiotherapy using three dimensional treatment planning (3D-CRT) to conventional radiotherapy (Conven-RT) for patients with Stages T2-T4 adenocarcinoma of the prostate. METHODS AND MATERIALS: A Phase III randomized study was activated in May 1993, to compare treatment toxicity and patient outcome after 78 Gy in 39 fractions using 3D-CRT to that after 70 Gy in 35 fractions using Conven-RT. The first 46 Gy were administered using the same nonconformal field arrangement (four field) in both arms. The boost was given nonconformally using four fields in the Conven-RT arm and conformally using six fields in the 3D-CRT arm. The dose was specific to the isocenter. The first 60 patients, 29 in the 3D-CRT arm and 31 in the Conven-RT arm, are the subject of this preliminary analysis. RESULTS: The two treatment arms were first compared in terms of dosimetry by dose-volume histogram analysis. Using a subgroup of patients in the 3D-CRT arm (n=15), both Conven-RT and 3D-CRT plans were generated and the dose-volume histogram data compared. The mean volumes treated to doses above 60 Gy for the bladder and rectum were 28 and 36% for the 3D-CRT plans, and 43 and 38% for the Conven-RT plans, respectively (p < 0.05 for the bladder volumes). The mean clinical target volume (prostate and seminal vesicles) treated to 95% of the prescribed dose was 97.5% for the 3D-CRT arm, and 95.6% for the Conven-RT arm (p < 0.05). There were no significant differences in the acute reactions between the two arms, with the majority experiencing Grade 2 or less toxicity (92%). Moreover, no relationship was seen between acute toxicity and the volume of bladder and rectum receiving in excess of 60 Gy for those in the 3D-CRT arm. There was also no difference between the groups in terms of early biochemical response. Prostate-specific antigen levels at 3 and 6 months after completion of radiotherapy were similar in the two treatment arms. There was only one biochemical failure in the study population at the time of the analysis. CONCLUSIONS: Comparison of the Conven-RT and 3D-RT treatment plans revealed that significantly less bladder was in the high dose volume in the 3D-CRT plans, while the volume of rectum receiving doses over 60 Gy was equivalent. There were no differences between the two treatment arms in terms of acute toxicity or early biochemical response. Longer follow-up is needed to determine the impact of 3D CRT on long-term patient outcome and late reactions. PMID- 8621279 TI - The influence of older age on breast cancer treatment decisions and outcome. AB - PURPOSE: Information concerning the differences between older and younger women with breast cancer, treated with standard therapy, is lacking from many prospective series. The purpose of this study is to identify factors that influence treatment decisions and determine if women age 65 and older are treated differently than younger women. The outcomes of older women would then be compared to younger to determine if treatment differences influence outcome. METHODS AND MATERIALS: The records of 558 women with early invasive breast cancer who were treated with breast conserving surgery and radiation therapy were retrospectively reviewed. Four hundred thirty-two women under the age of 65 (range: 24-64) and 126 women age 65 and older (range: 65-85) were assessed for treatment differences including breast reexcision, extent of axillary dissection, extent of breast and nodal irradiation, and the use of chemotherapy or hormonal therapy. Differences in the treatment of the two groups were determined and the end points of local control, disease-free survival, and overall survival were compared. Median follow-up was 5.5 years. RESULTS: The two treatment groups had identical pathologic TNM staging with the exception that 21% of the older age group and 5% of the younger group did not undergo axillary dissection. Women age 65 and older were less likely to have a reexcision, extensive axillary dissection, chemotherapy, or nodal irradiation. They were more likely to receive hormonal therapy. Reexcision in older women was positively influenced by a family history of breast cancer and negatively influenced by a history of previous malignancy. None of the patients who were treated without and axillary dissection suffered a regional recurrence. Although local control was better in older patients, there were no differences in disease-free or overall survival for the two groups. DISCUSSION: The findings of this study reveal that older patients have significant treatment differences as compared to younger patients; however, despite these differences, similar local control and survival were achieved at 5 to 10 years. With the expected survival of older women increasing, the prospective evaluation of treatment options for older women should be considered. PMID- 8621280 TI - The use of mammography in breast preservation in locally advanced breast cancer. AB - PURPOSE: As the feasibility of breast preservation in locally advanced breast cancer is currently under evaluation, little information is available correlating mammographic changes to chemotherapy with local outcome. To evaluate the role of mammography in selecting candidates with locally advanced breast cancer for conservative local therapy, we analyzed mammographic changes in the breast to induction chemotherapy and correlated the radiologic appearance with pathologic outcome. METHODS AND MATERIALS: From 1985 through 1993, 91 patients with Stage III breast cancer were enrolled on a multimodality clinical trial using chemohormonal therapy followed by local treatment and maintenance therapy. Induction therapy consisted of cyclophosphamide, doxorubicin, methotrexate,and 5 fluorouracil with hormonal synchronization using tamoxifen and conjugated estrogens. After nine cycles, surgical biopsies of the breast were performed. Through 1988, clinical examination alone directed the site for postinduction biopsy; for patients treated after 1988, mammography, in addition to physical examination, determined the biopsy location. Local treatment was determined by biopsy result. Patients with a pathologic complete response received radiation only to the breast adn regional nodes, while those with pathologically proven residual disease underwent mastectomy and postoperative radiotherapy. Nine additional cycles of maintenance chemotherapy were administered. RESULTS: Fifty five of 91 patients (58%) obtained a clinical complete response (CR) to induction chemotherapy. Twenty-eight of the 53 women with a clinical CR had both pre- and postinduction mammograms. Of these 28 women, 9 obtained a pathologic CR and 19 obtained a pathologic partial response (PR). Fifty-five percent of the pathologic complete responders had resolution of mammographic abnormalities on the postinduction mammograms. Sixty-eight percent (13) of the pathologic partial responders had abnormal mammographic findings. The positive predictive value for residual cancer using physical examination was 92%, while the negative predictive value was only 36%. Among patients with a clinical complete response, the positive and negative predictive values for residual cancer using postinduction mammography were 79% and 56%, respectively. Limitations of mammography included uncertain significance of residual microcalcifications and residual masses on postinduction chemotherapy mammograms. CONCLUSIONS: Although mammography improved the accuracy of noninvasive evaluations in patients with a clinical complete response, pathologic assessment was still required to determine appropriate local therapy. More sensitive imaging modalities or modifications of film-screen mammography may improve noninvasive detection of residual disease following induction chemotherapy. PMID- 8621281 TI - Surgical clips in planning the electron boost in breast cancer: a qualitative and quantitative evaluation. AB - PURPOSE: To evaluate, qualitatively and quantitatively, the role of surgical clips in planning the tumor bed electron boost in patients undergoing breast conserving surgery and radiotherapy. METHODS AND MATERIALS: In 50 patients, the excision cavity boundaries were marked by clips at surgery. The electron boost field was first planned using clinical information, aiming to achieve a margin of 2 cm, and its accuracy evaluated by screening the surgical clips and, if necessary, adjusting the field to encompass all clips with 2 cm margins. Orthogonal radiographs were take with solder wire delineating the clinical and screened fields and the scar. Hypothetical clinical and radiological fields, with 1 and 3 cm margins, were reconstructed on the radiographs. RESULTS: The clinical field was inadequate in 34 patients (68%). The precision of each clinical setup was quantified by two indices. The Normal Tissue Index defined the percentage of the clinical field comprised of tissue, beyond the tumor bed, not at high risk of local recurrence, and gave an estimate of potential spring of normal tissue: median 14.6% (range 0-83.0), 17 out of 50 > 25%; median 13% (range 0-70.7), 12 out of 50 > 25%; median 9.7% (range 0-59.8), 10 out of 50 > 25%, for 1, 2, and 3 cm margins, respectively. The Geographical Miss Index defined the percentage of the radiologically defined field, at high risk of local recurrence, not predicted by the clinical field, and gave an estimate of the extent of geographical miss: median 32.9% (range 0-83.5), 28 out of 50 > 25%; median 26.1% (range 0-69.8%), 26 out of 50 > 25%; median 18.6% (range 0-60.3), 20 out of 50 > 25%, for 1, 2, and 3 cm margins, respectively. The median distance from the scar midpoint to the furthest clip was 3.8 (range 1.2-8.1) cm. The median maximal clip depth was 3.1 (range 1.4-5.2) cm. CONCLUSION: (a) Electron boost field planning by clinical landmarks alone was inaccurate in 68% of cases. (b) Quantitative measures, based on margins of 1, 2, and 3 cm, revealed that in 20-34% of patients more than one quarter of the clinical field covered tissue at low risk of local recurrence, and in 40-56% of patients less than three-quarters of the final radiological field was predicted clinically. (c) The relative positions of the scar and clips may be widely disparate. (d) Clip depth measurements reveal a significant risk of underdosing at depth. PMID- 8621282 TI - Analysis of outcome in patients reirradiated for brain metastases. AB - PURPOSE: Patients with newly diagnosed brain metastases generally benefit from whole brain radiation therapy (WBRT). However, the role of reirradiation for patients who develop progressive brain metastases has been controversial. This retrospective study examines our experience with reirradiation of patients for progressive brain metastases after an initial+ course of WBRT. METHODS AND MATERIALS: From 1975-1993, 2658 patients received WBRT for brain metastases at our institution. Eighty-six patients were subsequently reirradiated for progressive brain metastases. The median age of these patients was 58 (range: 31 81). The most common primary sites were breast and lung. Fifty patients had metastatic disease at other sites. Most patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 2 (40 patients) or 3 (38 patients). The median dose of the first course of irradiation was 30 Gy (range: 1.5-50.6 Gy). The median dose of the second course of irradiation was 20 Gy (range: 8.0 30.6 Gy). RESULTS: Twenty-three patients (27%) had resolution of neurologic symptoms, 37 patients (43%) had partial improvement of neurologic symptoms, and 25 patients (29%) had either no change or worsened after reirradiation. The median survival following reirradiation was 4 months (range: 0.25-72 months). The majority of patients had no significant toxicity secondary to reirradiation. Five patients had radiographic abnormalities of their brain consistent with radiation related changes. One patient had symptoms of dementia that was thought to be caused by radiotherapy. Various potential prognostic factors were evaluated for possible associations with survival, including age, sex, primary site, ECOG performance status, RTOG neurologic functional class, absence of extracranial metastases, and dose of irradiation. Absence of extracranial metastasis, solitary brain metastasis, and retreatment dose > 20 Gy were associated with improved survival in univariate analysis (p=0.025, 0.033, and 0.061, respectively). The absence of extracranial disease was the only significant factor in multivariate analysis (p=0.05). CONCLUSION: The majority of patients in our series had favorable symptomatic responses. Clinically significant complications were minimal. Reirradiation should be offered to patients who develop progressive brain metastases. PMID- 8621283 TI - The lazaroid U74389G protects normal brain from stereotactic radiosurgery-induced radiation injury. AB - PURPOSE: To test an established model of stereotactic radiosurgery-induced radiation injury with pretreatments of either methylprednisolone or the lazaroid U74389G. METHODS AND MATERIALS: Nine cats received stereotactic radiosurgery with a linear accelerator using and animal radiosurgery device. Each received a dose of 125.0 Gy prescribed to the 84% isodose shell to the anterior limb of the right internal capsule. One animal received no pretreatment, two received citrate vehicle, three received 30 mg/kg of methylprednisolone, and three received 5 mg/kg of U74389G. After irradiation, the animals had frequent neurologic examinations, and neurologic deficits developed in all of them. Six months after the radiation treatment, the animals were anesthetized, and had gadolinium enhanced magnetic resonance (MR) scans, followed by Evans blue dye perfusion, euthanasia, and brain fixation. RESULTS: Magnetic resonance scans revealed a decrease in the size of the lesions from a mean volume of 0.45 +/- 0.06 cm(3) in the control, vehicle-treated, and methylpredniosolone-treated animals to 0.22 +/- 0.14 cm(3) in the U74389G-treated group. The scans also suggested the absence of necrosis and ventricular dilatation in the lazaroid-treated group. Gross pathology revealed that lesions produced in the untreated, vehicle-treated, and methylprednisolone-treated cats were similar and were characterized by a peripheral zone of Evans blue dye staining with a central zone of a mature coagulative necrosis and focal hemorrhage. However, in the U74389G-treated animals, the lesions were found to have an area of Evans blue dye staining, but lacked discrete areas of necrosis and hemorrhage. CONCLUSION: These results suggest that the lazaroid U74389G protects the normal brain from radiation injury produced by stereotactic radiosurgery. PMID- 8621284 TI - Measurement of intracellular dna double-strand break induction and rejoining along the track of carbon and neon particle beams in water. AB - PURPOSE: The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. METHODS AND MATERIALS: An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated following irradiation with 250 kV x-rays and 109 MeV/u carbon- and 295 MeV/u neon-ions. RESULTS AND CONCLUSION: While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent. PMID- 8621285 TI - The use of 212Pb-labeled monoclonal antibody in the treatment of murine erythroleukemia. AB - PURPOSE: The goals of this study were to learn whether the DOTA chelator was useful for targeting lead radionuclides (203,212 Pb) to cells and tissues invaded by the Rauscher leukemia virus (RVB3) and to investigate the therapeutic efficacy of targeted 212Pb in treating the murine leukemia. METHODS AND MATERIALS: Five to 6-week-old BALB/c mice were inoculated i.v. with RVB3. This virus causes marked splenomegaly and death by day 13 and day 70 postinfection, respectively. Biodistribution, tumor targeting, and toxicity studies were performed using varying doses of 212Pb-DOTA-103A. A heavy metal chelator, DMPS, was administered orally and parenterally in two phases of the toxicity study. RESULTS: Biodistribution studies showed marked tumor targeting (58% ID/g spleen) in mice treated with 203Pb-103A as compared with mice treated with control antibody B3 (4.6% ID/g spleen). Histologic cure was achieved in all leukemic mice treated with 20 muCi212Pb-103A; however, all of the mice died with leukopenia and secondary++ bacterial infections due to severe bone marrow toxicity. Nonleukemic mice and mice treated with 20 muCi212Pb-B3 experienced less marrow toxicity and longer survival. Coadministration of the heavy metal chelator did not diminish the bone marrow toxicity. CONCLUSION: An effective, nonlethal dose could not be established to treat this tumor. The severe bone marrow toxicity associated with this radionuclide may limit its usefulness in systemic radioimmunotherapy. PMID- 8621286 TI - Effect of irradiation on bromodeoxyuridine incorporation in human colon cancer xenografts. AB - PURPOSE: Although we have characterized the incorporation of the thymidine analog bromodeoxyuridine (BrdUrd) into human colon cancer xenografts under a wide variety of conditions, little is known about the effect of radiation on subsequent incorporation. Because clinical protocols include, as one component, BrdUrd administration after radiation, it was important to confirm that irradiation did not prevent subsequent BrdUrd incorporation. Therefore, we studied the effect of irradiation on BrdUrd incorporation into HT29 human colon cancer xenografts. METHODS AND MATERIALS: Two types of experiments were performed. In the first, the effect of radiation on subsequent incorporation was measured. Tumors received doses of 0, 2, 8, and 12 Gy, animals were infused with BrdUrd for 4 days, and incorporation was assessed at the end of the infusion. In the second, the effect of radiation on the elimination of BrdUrd from tumors was determined. Animals were infused with BrdUrd, tumors were irradiated with either 0 or 12 Gy, and tumor incorporation of BrdUrd was measured 1 and 3 days later. RESULTS: Radiation affected neither the incorporation into nor the elimination of BrdUrd from human tumor xenografts. CONCLUSIONS: These findings support the feasibility of clinical trials interdigitating BrdUrd infusion and radiation. PMID- 8621287 TI - The effect of heat on Na+/H+ antiport function and survival in mammalian cells. AB - PURPOSE: Because intracellular pH (pHi) is a determinate of thermosensitivity, it is important to understand the relationship between heat cytotoxicity and the mechanisms responsible for pHi regulation, such as the Na+/H+ antiport. The objective of this study is to elucidate the relationship between heat damage and Na+/H+ antiport activity. METHODS AND MATERIALS: Various cell lines, EMT6, RIF-1, and its thermoresistant variant TR-4, and CCL39, and its variant that lacks the Na+/H+ antiport (PS120), were all heated using a water bath. Parallel assessments of antiport function and pHi were made using the fluorescent dye 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). RESULTS: Exposure of EMT6 cells to 43-46 degrees C for 30-60 min caused progressive decline in antiport activity, in parallel with cytotoxicity. When the same degree of cytotoxicity was induced by ionizing radiation, no alteration in Na+/H+ antiport function was observed. Despite a 10-fold lower survival in RIF-1 compared to TR-4 cells after heating, there was no difference in the thermosensitivity of their antiports. Antiport activity in the TR-4 cells, however, was higher than that of RIF-1 cells both before and during heating. Intracellular pH for TR-4 cells decreased minimally during heating, in contrast to a decline of 1 pH unit in RIF-1 cells despite similar relative levels of antiport activity, suggesting that in this pair of cell lines, antiport activity does not play a major pHi regulatory role. PS120 and CCL39 cells and similar survival levels when heated at pHe 7.2 in the presence of NaHCO3, which allows function of the other major regulator of pHi, the Na+ -dependent HCO3-/Cl- exchanger. This occurred despite a drop in pHi in the PS120 cells during heating. A reduced survival was observed, however, in PS120 cells after 43 degrees C for 30-60 min at either pHe 6.5 or pHe 7.2 in the absence of NaHCO3. Intracellular pH was consistently greater for PS120 than CCL39 cells. CONCLUSION: We demonstrated that damage to the Na+/H+ antiport likely reflects early heat-induced change in membrane function, but is not a primary target for heat cytotoxicity. Although there is an association between survival, antiport function, and pHi level under most treatment conditions, the precise role of the Na+/H+ antiport in mediating thermal cytotoxicity remains uncertain. PMID- 8621288 TI - Local hyperthermia of N2/N3 cervical lymph node metastases: correlationof technical/thermal parameters and response. AB - PURPOSE: Patients with advanced head and neck carcinomas, primarily nonresectable as well as recurrent cases, were treated in multimodality regimens with radiotherapy, chemotherapy, and local hyperthermia. Commercially available microwave and radiowave applicators were used in 50 patients with N2/N3 cervical lymph node metastases during more than 250 heat treatments. To assess technical suitability, the achieved power densities and thermal parameters were tested for correlation with anatomical and geometrical factors. To assess effectiveness, the response was compared with derived parameters of the achieved temperature distributions. METHODS AND MATERIALS: The temperature measurement points (in thermometry catheters) documented by computerized tomography are labeled according to tissue depth, shielding by osseous structures, and location in relation to the external applicators. Relative and absolute specific absorption rates (SAR) and cooling coefficients are extracted from the temperature-time curves. Time-averaged temperature-position curves are evaluated to obtain index temperatures (T90, T50, T20), minimum/maximum tumor temperatures, cumulative minutes T90 > or = 43 degrees C, and 43 degrees C-equivalent min T90. Radiation dose, treatment time, and chemotherapy regiment are also considered. A response parameter is defined using the pre- and posttherapeutic tumor volumes. A multivariate variance analysis is performed for the dependent variables power density, thermal parameters, and response. RESULTS: A significant correlation exists between power density and absorption, presence of a fat layer, and applicator illumination. The maximum depth is 5 cm, where SAR of >= 10 mW/g are registered. Achieved temperatures at individual measurement points are dependent on the SAR, and to a lesser extent, the perfusion-dependent cooling coefficients, but the index temperature T90 is only significantly related to intratumorally achieved SAR. The thermal gradient (T20-T50) and temperature peak (T20) are significantly influenced by the tumor volume. The response is directly related to the index temperature T90, equivalent minute T90 43 degrees C, and cumulative minutes T90 > or = 40.5 degrees C, and inversely related to the tumor volume. CONCLUSIONS: Local hyperthermia using microwave and radiowave applicators in the head and neck region is a tolerable and clinically practical supplementary therapy used as part of multimodal regimens, and has already been proven to be effective. However, the analyses also demonstrated the limits of currently available technology, and confirm the need for continued methodical research. PMID- 8621289 TI - Radiosurgery for the treatment of previously irradiated recurrent primary brain tumors and brain metastases: initial report of radiation therapy oncology group protocol (90-05). AB - PURPOSE: To determine the maximum acutely tolerable dose of single fraction radiosurgery in patients with recurrent previously irradiated primary brain tumors or brain metastases. METHODS AND MATERIALS: Between August 1990 and September 1993, 102 analyzable patients were entered on Radiation Therapy Oncology Group (RTOG) protocol 90-05, 38 of whom had recurrent primary brain tumors (median prior dose 60 Gy), and 64 of whom had recurrent brain metastases (median prior dose 30 Gy) < or = 40 mm in maximum diameter. Unacceptable toxicity was defined as irreversible Grade 3, any Grade 4, or Grade 5 central nervous system (CNS) toxicity according to the RTOG CNS criteria, occurring in > 20% of patients per treatment arm within 3 months of radiosurgery. RESULTS: Patients were initially entered onto one of three treatment arms according to the maximum diameter of the recurrent lesion. Twelve to 22 patients were entered on each arm. The dose levels were: arm 1, < or = 20 mm, 18 Gy; arm 2, 21-30 mm, 15 Gy; and arm 3, 31-40 mm, 12 Gy. Subsequently, doses were escalated as follows: arm 4, < or = 20mm, 21 Gy; arm 5, 21-30 mm 18 Gy; and arm 6, 31-40 mm, 15 Gy. Unacceptable acute toxicity secondary to cerebral edema occurred in 0, 7 and 5% of patients on Arms 1, 2 and 3, respectively, and in no patients on arms 4, 5, or 6. Multivariate analysis revealed that tumor volume > or = 8200 mm(3) and a ratio of maximum dose to prescription dose (MD/PD) > or = 2 were significantly associated unacceptable toxicity. Of 15 patients with both tumor volume > or = 8200 mm(3) and MD/PD > or = 2, unacceptable toxicity occurred in 2 of 4 treated with a single isocenter and 1 of 11 treated with multiple isocenters. Subsequently, operation for symptomatic radionecrosis was required in 6% of patients. CONCLUSION: We found that the incidence of acute toxicity was acceptable at 0-7% in patients with recurrent, previously irradiated primary brain tumors or brain metastases < or = 40 mm in maximum diameter treated according to the protocol described. PMID- 8621290 TI - Hyperfractionated conformal radiotherapy in locally advanced prostate cancer: results of a dose escalation study. AB - PURPOSE: This study was initiated to assess the incidence of chronic complications and histologic and biochemical control following hyperfractionated conformal radiotherapy in patients with locally advanced prostate cancer. METHODS AND MATERIALS: Between October 1991 and October 1994, 49 patients with locally advanced prostate cancer were entered on the first two dose levels of a prospective dose-escalation study using hyperfractionated three dimensional conformal radiotherapy. The first 25 patients received a minimum tumor dose of 78 Gy to the prostate and seminal vesicles in 6 weeks at 1.3 Gy, b.i.d. No increase in chronic toxicity compared with conventional radiotherapy was noted; therefore, an additional 24 patients were treated to a minimum tumor dose of 82.8 Gy to the prostate and seminal vesicles in 7 weeks at 1.15 Gy, b.i.d. Toxicity was scored according to the Radiation Therapy Oncology Group morbidity grading scale. Efficacy was assessed through scheduled postradiation prostate specific antigen values and ultrasound-guided biopsies. The median follow-up for the entire group was 20 months. RESULTS: The hyperfractionated external radiation was well tolerated with minimal acute morbidity. At 30 months, the actuarial probability of Grade 2 gastrointestinal toxicity was 17%. At 30 months, the actuarial probability of Grade 2 genitourinary toxicity was 16%. There was no statistically significant difference between the two dose levels. No Grade 3 or 4 gastrointestinal or genitourinary toxicity was noted. At 12 months, 84% of patients had a prostate specific antigen < or = 4; and 53% < or = 1 ng/ml. At 12 months, 71% of patients had post radiation biopsies that were either negative (55% or showed a marked therapeutic effect (16%). CONCLUSION: The use of hyperfractionated conformal radiotherapy facilitated dose escalation with no increase in chronic toxicity compared to standard doses. The initial tumor response based on prostate specific antigen measurements and postradiation biopsies is highly encouraging. Based on these results, an increase in dose to 87.4 Gy has been planned according to the schema of this ongoing dose escalation study. PMID- 8621291 TI - Radiosurgery for cerebral arteriovenous malformations: assessment of early phase magnetic resonance imaging and significance of gadolinium-DTPA enhancement. AB - PURPOSE: To evaluate the initial changes within the nidus of arteriovenous malformations (AVMs) and to assess the reaction to the brain tissue surrounding AVMs after radiosurgery by serial magnetic resonance (MR) imaging. METHODS AND MATERIALS: Twenty-one patients, treated using 60Co gamma knife unit with cerebral AVMs, were retrospectively evaluated. Forty-seven follow-up MR images of the 21 patients were performed including 10 patients with two or more serial gadolinium enhanced studies (Gd-MR). Two or more sequential MR angiographies (MRA) were obtained in 13 patients. Three-dimensional (3D) time-of-flight MRA and two dimensional (2D) phase contrast MRA were used in 13 patients for evaluating the flow changes of AVMs. The follow-up period after radiosurgery ranged from 3 to 30 months (average 10.8 months) and the interval time of MRI ranged from 34 days to 13 months (average 4.9 months). RESULTS: Reduction of nidus size was observed in 14 of 21 patients (67%) between 4 to 13 months on spin echo (SE) images. Complete obliteration was observed on SE images in 4 of these 14 patients; three were confirmed by conventional angiography. New hyperintense areas surrounding the nidus on T2s-weighted images (T2WI) developed in 9 of the 14 patients who showed nidus reduction between 5 to 17 months after radiosurgery; in three patients, size of the hyperintense area started to decrease between 6 to 7 months after its appearance. Probable radiation necrosis of pons developed in one patient 26 months after radiosurgery. The irradiated area within the AVM nidus was significantly enhanced in 8 of the 10 patients who underwent Gd-MR. The degrees of enhancement within the nidus increased with time in 7 of the 10 patients. Overall, total enhancement of irradiated areas was observed in four of the 10 patients; in three of the four, the enhancement decreased in size and degree, indicating nidus reduction. In three patients who had a partial volume irradiation within the nidus, the enhancing areas corresponded with the exact portions of irradiated volume. The nidus reduction was observed in 7 of the 13 patients on MRA during 5 to 13 months after radiosurgery. MRA was more useful compared to SE images in four of the seven patients in evaluating the size change of AVM nidus, feeding arteries, and draining veins. CONCLUSION: Magnetic resonance imaging and MRA were useful in assessing the progress of nidus reduction. T2-weighted imaging was sensitive to radiation-induced reaction in and around the AVM nidus. The enhancement within the AVM nidus on Gd-MR may represent the initial sign of nidus reduction and demonstrates the exact location of irradiation in the nidus. The changes of the enhancement pattern are presumed to represent the processes of nidus reduction and irradiated reaction within the AVM nidus. PMID- 8621292 TI - Endocavitary radiotherapy of rectal cancer. AB - PURPOSE: This analysis was performed to evaluate the results of endocavitary radiotherapy (RT) administered for early rectal cancer at our institution. METHODS AND MATERIALS: Patient charts were retrospectively reviewed to determine the results of endocavitary RT regarding survival, local control, and complications. Between 1987 and 1994, 25 patients were treated with endocavitary RT for early rectal cancer. Twenty had early, low grade tumors and met the criteria for treatment with curative intent. Five had more advanced, high grade, or multiple recurrent tumors and were treated with palliative intent. The tumors were treated to between 20 and 155 Gy in one to four fractions with 50 KV x-rays given through a specialized proctoscope. Patients were followed for 5 to 84 months (median = 55 months) after therapy. Local control and survival were determined using the Kaplan-Meier method. RESULTS: Local control was achieved in 18 of the 20 patients treated with curative intent and 4 of 5 treated with palliative intent. For those patients treated with curative intent, the 5-year local control rate was 89% and the 5-year survival rate was 76%. The most significant toxicity was ulceration that occurred in 5 of the 25 patients. The ulcers were asymptomatic in three cases and associated with bleeding in one case. The fifth patient had pain. One ulcer was biopsied, resulting in perforation that was treated with an abdominal perineal resection (APR). There was no tumor found upon pathologic evaluation. CONCLUSIONS: Endocavitary RT can be used to treat patients with early, low-grade rectal cancers and will yield a high level of disease control and a low risk of serious complications. Major advantages of this treatment technique are that it requires neither general anesthesia nor hospitalization. PMID- 8621293 TI - An analysis of intratreatment and intertreatment displacements in pelvic radiotherapy using electronic portal imaging. AB - PURPOSE: To evaluate the relative frequency and magnitude of intratreatment and intertreatment displacements in the patient positioning for pelvic radiotherapy using electronic portal imaging. METHODS AND MATERIALS: Five hundred ninety-four electronic portal images of seven patients treated with a four-field pelvic technique were evaluated. All patients were treated prone without an immobilization device. Two fields were treated per day, from which an average of two electronic portal images were obtained for each field. No treatment was interrupted or adjusted on the basis of these images. Each image was aligned to the corresponding simulation film to measure the displacements in the mediolateral, craniocaudal, and anteroposterior directions relative to the simulated center. The intertreatment displacement was the displacement measured from the initial image for each daily treated field. For each daily treated field the intratreatment displacement was calculated by subtracting the displacement measured on the initial image from the displacement measured on the final image. RESULTS: The frequency of the intertreatment displacements exceeding 10 mm was 3%, 16%, and 23% for the mediolateral, craniocaudal, and anteroposterior translations, respectively. There were no intratreatment displacements exceeding 10mm (p < 0.001). The frequency of intertreatment displacements exceeded 5 mm was 40, 52, and 51% for the mediolateral, craniocaudal, and anteroposterior translations, respectively; whereas, the frequency of intratreatment displacements exceeding 5 mm was 1, 5, and 7% for the same translations, respectively (p < 0.001). The standard deviation of the intertreatment displacements was at least three times as great as the standard deviation of the intratreatment displacements for all translations. These deviations were greater than the precision limit of the measurement technique, which is approximately 1mm. Each patient had one direction where systematic error predominated in intertreatment positioning. Random error predominated for intratreatment positioning and for the other two directions in intertreatment positioning. CONCLUSIONS: During a course of pelvic radiotherapy, the frequency of intertreatment displacements exceeding 5 and 10 mm is significantly greater than the frequency of intratreatment displacements of these magnitudes. Errors in intertreatment positioning are predominantly systematic in one direction for each patient, whereas intratreatment error is predominantly random. Because patients do not move considerably during the daily treatment of a pelvic field, a single electronic portal image per daily field may be considered representative of the treated position. PMID- 8621294 TI - Clinical results of computerized tomography-based simulation with laser patient marking. AB - PURPOSE: Accuracy of a patient treatment portal marking device and computerized tomography (CT) simulation have been clinically tested. METHODS AND MATERIALS: A CT-based simulator has been assembled based on a commercial CT scanner. This includes visualization software and a computer-controlled laser drawing device. This laser drawing device is used to transfer the setup, central axis, and/or radiation portals from the CT simulator to the patient for appropriate patient skin marking. A protocol for clinical testing is reported. Twenty-five prospectively, sequentially accessioned patients have been analyzed. RESULTS: The simulation process can be completed in an average time of 62 min. Under many cases, the treatment portals can be designed and the patient marked in one session. Mechanical accuracy of the system was found to be within +/- 1mm. The portal projection accuracy in clinical cases is observed to be better than +/- 1.2 mm. Operating costs are equivalent to the conventional simulation process it replaces. CONCLUSION: Computed tomography simulation is a clinical accurate substitute for conventional simulation when used with an appropriate patient marking system and digitally reconstructed radiographs. Personnel time spent in CT simulation is equivalent to time in conventional simulation. PMID- 8621296 TI - Variation in prostate position relative to adjacent bony anatomy. AB - PURPOSE: In prostatic cancer, the prostate cannot be discerned from a portal image. Setup information is, therefore, obtained from bony anatomy. To perform high-precision conformal therapy, knowledge about the variation in distance between the prostate and adjacent bony anatomy during an external radiotherapy treatment is mandatory. This report is concerned with that variation. METHODS AND MATERIALS: Nine patients previously treated interstitially for prostatic cancer with implantation of 125I seeds, agreed to cooperate in a study. They underwent a number of simulations of external radiotherapy treatment. After the first patient setup, this setup was repeated five times for each patient as if the patient was treated by external radiotherapy. Simulator radiographs were made from each setup in the anterior-posterior and left-right lateral directions. The seeds were clearly visible in the simulator film images and reflect the position of the prostate. No bladder instructions were given and the filling of the rectum was not quantified. RESULTS: Variation in distance between the prostate and the bony anatomy was measured and is presented in one standard deviation of the normal distribution function: 0.8 mm lateral, 1.5 mm ventrodorsal, and 1.7 mm craniocaudal. CONCLUSIONS: The bone structure of the pelvis reflects fairly well the position of the prostate. The prostate is least mobile in a lateral direction and most mobile in the craniocaudal and ventrodorsal directions. PMID- 8621295 TI - A quality control study of the accuracy of patient positioning in irradiation of pelvic fields. AB - PURPOSE: Determining and improving the accuracy of patient positioning in pelvic fields. METHODS AND MATERIALS: Small pelvic fields were studied in 16 patients treated for urological cancers using a three-field isocentric technique. Large pelvic fields were studied in 17 gynecological cancer patients treated with anterior and posterior (AP-PA) parallel opposed fields. Quantitative analysis of 645 megavolt images and comparison to 82 simulation images were carried out. RESULTS: Small pelvic fields: for the position of the patient in the field, standard deviations of the difference between simulation (SIM) and treatment (MV) images were 3.4 mm in the lateral direction, 5.3 mm in the cranio-caudal direction, and 4.8 mm in the ventro-dorsal direction. Alterations in the positioning technique were made and tested. Large pelvic fields: differences between simulation and treatment images for the position of the patient in the field were 4 mm [1 standard deviation (SD)] in the lateral direction and 6.5 mm in the cranio-caudal direction. A systematic shift of the treatment field in the cranial direction had occurred in the majority of patients. A positioning technique using laser lines and marking of the caudal field border was shown to be more accurate. CONCLUSIONS: Studies of positioning accuracy in routine irradiation techniques are needed to obtain data for definition of the margins for each treatment site at each institution. Random variations should be kept at a minimum by monitoring and improving positioning techniques. Treatment verification by megavolt imaging or film should be used to detect and correct systematic variations early in the treatment series. PMID- 8621297 TI - Limitations of the minimum peripheral dose as a parameter for dose specification in permanent 125I prostate implants. AB - PURPOSE: The objective of this work is to investigate whether the minimum peripheral dose is a practical parameter for dose specification in permanent 125I implants of the prostate. METHODS AND MATERIALS: The investigation was carried out by use of a computer model of ellipsoidal 125I implants in which the average dimension and elongation factor were varied to provide a wide range of geometries. Both ideal and nonideal implants were investigated. The 125I seeds were confined to the target volume except for a portion of the study in which the effect of placing seeds outside the target volume was investigated. RESULTS: The minimum peripheral dose was found to be very sensitive to the seed placement. The irregularities in the seed spacing that inevitably occur in actual implants tend to lower the minimum peripheral dose. As a result, the minimum peripheral dose is generally significantly less than planned by an amount that is unpredictable, and often exceeds 25%. However, the percentage of the target volume that receives a dose less that the prescribed minimum peripheral dose is generally less than 10%. Implanting seeds outside the target volume improves the dose uniformity, but does not appear to offer any advantage in dose coverage, and increases the volume of normal tissue irradiated. CONCLUSION: If a minimum peripheral dose is prescribed for a permanent 125I prostate implant, and the implant is planned using an idealized implant having precisely spaced seeds, the prescribed dose will rarely, if ever, be achieved. Reasonable agreement with the prescribed dose can be achieved only if the requirement for coverage is relaxed from 100 to 90%, or if the total source strength is increased by 20% to compensate for the anticipated imperfections in seed placement. PMID- 8621299 TI - Intra- and interfractional reproducibility of tangential breast fields: a prospective on-line portal imaging study. AB - PURPOSE: A perception exists that weekly verification films accurately reflect the setup of the tangential breast portals. This prospective study was undertaken to assess patient movement during treatment and setup reproducibility of tangential breast fields using electronic on-line portal imaging. METHODS AND MATERIALS: Thirteen patients with carcinoma of the breast were treated on a linear accelerator equipped with an on-line portal imaging system. Patients were immobilized daily with an alpha cradle. The medial and lateral tangential fields were imaged and 139 fractions, 225 portal fields, and 4450 images were obtained. Images were then analyzed off line and 22,250 measurements were made from these images. Anatomical features recorded include the lung area (LA), central lung distance (CLD), central breast distance (CBD), central flash distance (CFD), and inferior central margin (ICM). Intrafractional variations were calculated for every portal field and fraction for each patient. Interfractional variations were determined by finding the variance of intrafractional means for each patient. A population standard deviation for each of the five parameters for intra- and interfractional variations were determined. The simulation to treatment setup errors were calculated for all five variables. RESULTS: Lung area variation was 1.50 and 4.19 cm(2) [1 standard deviation (SD)] for intra- and interfractional movement. Intrafractional variation for the other four variables ranged from 0.85 mm for ICM to 2.1 mm (1 SD) for CBD, while interfractional variations ranged from 3.2 to 6.25 mm for CBD and ICM, respectively. The simulation-to-treatment setup variation was greater than the interfractional variation for three of the five variables and was similar for the other two. CONCLUSIONS: On-line verification of intrafractional variation shows a moderate deviation from the treatment setup position for all five parameters studied, while interfractional variation showed even greater deviations for these five parameters. To cover the breast target in 95% of cases, margins of 7.70, 7.70, and 10.30 mm corresponding to the CLD, CFD, and ICM distances, respectively, are required. PMID- 8621298 TI - Dose to contralateral breast: a comparison of four primary breast irradiation techniques. AB - PURPOSE: Contralateral breast dose from primary breast irradiation has been implicated in the risk of second breast malignancies. It has been previously shown that the use of half-beam blocking can increase the opposite breast dose by a factor of five. This study evaluates four different breast treatment techniques to compare the radiation dose to the contralateral breast. METHODS AND MATERIALS: Dose measurements were made using thermoluminescent dosimeters (TLD) capsules, which were placed in the Rando phantom in the following locations in the contralateral breast: seven along the central axis plane, on at 5 cm superior to, and one 5 cm inferior to the central axis plane. One TLD capsule was placed in the midcenter of the treated breast. The following radiation techniques were used: (a) half-beam with a custom block (HB+CB), (b) half-beam using asymmetric collimator jaw (HB/AJ), (c) half-beam using asymmetric collimator jaw with custom block (H/AJ+CB), and (d) isocentric technique with nondivergent posterior borders [Joint Center for Radiation Therapy (JCRT) techique]. For each technique, isodose distributions for the Rando phantom were optimized using (a) 15 degree medial and lateral compensating wedges, and (b) a single 30 degree lateral compensating wedge. The phantom was treated with 6 MV photons. Each technique was repeated six times, and the TLD readings were averaged. RESULTS: The custom cerrobend half beam block technique gives the highest contralateral breast dose, regardless of wedge. The remaining techniques give results in a similar range, with the asymmetric jaw with no medial wedge technique giving the lowest total dose (p = not significant). The use of a medial wedge increases the opposite breast dose for all techniques. The asymmetric half-beam technique gives significantly less dose than the cerrobend half-beam technique, due to decreased transmission through the asymmetric collimators. The asymmetric jaw vs JCRT technique results in similar contralateral breast dose. CONCLUSIONS: As expected, dose to the contralateral breast increases with the use of a medial wedge. Cerrobend half beam blocking gives the highest opposite breast dose. The lowest contralateral breast dose is with the asymmetric jaw with no medial wedge and no block. The asymmetric jaw technique with block yields equivalent contralateral breast doses to the JCRT technique. PMID- 8621300 TI - Past, present, and future of intraoperative irradiation for colorectal cancer. PMID- 8621301 TI - Adjuvant therapy for pathologic stage C prostate cancer: a casualty of the PSA revolution? PMID- 8621302 TI - You say either, I say either, but let's not call the whole thing off: models for predicting the risk of lymph node involvement in patients with prostate cancer. PMID- 8621303 TI - Ageism or acumen -- the treatment of older women with breast cancer. PMID- 8621304 TI - Neutron-photon treatment: modern day Pyrrhic victory? Regarding Haraf et al. IJROBP 33(1): 3-14; 1995. PMID- 8621305 TI - Small is beautiful--and often enough: in response to Drs. Mohan and Ling, IJROBP 33(1):235-237; 1995. PMID- 8621306 TI - Opinions on effects of reforming veterinary education. PMID- 8621307 TI - Opinions on effects of reforming veterinary education. PMID- 8621308 TI - More on fish anatomy. PMID- 8621309 TI - What is your diagnosis? Generalized loss of cortical bone density and a displaced compression fracture of 10th thoracic vertebra. PMID- 8621310 TI - ECG of the month. Third-degree atrioventricular block with a multifocal idioventricular escape rhythm in a dog. PMID- 8621311 TI - Animal behavior case of the month. Coprophagy, urination and defecation in the house, and general fearfulness in a dog. PMID- 8621312 TI - The dynamics of change. PMID- 8621313 TI - Establishing the standard of care. PMID- 8621314 TI - Impact and risk of foreign animal diseases. PMID- 8621315 TI - Case-control study of the association between intraoperative administration of nafcillin and acute postoperative development of azotemia. AB - OBJECTIVE: To describe 7 cases of acute postoperative azotemia in dogs and to examine by use of a case-control study the possible association between this complication and administration of nafcillin. DESIGN: Retrospective and case control study. ANIMALS: 7 case dogs and 28 matched control dogs. PROCEDURE: Cases of acute renal failure or acute renal insufficiency were identified by retrospective study of records of dogs treated between July 1, 1992, and Feb 28, 1995, and from information received from a practitioner. A random sample of records of dogs undergoing invasive procedures between Dec 1, 1992, and Nov 30, 1993, was examined to determine the prevalence of nafcillin use. Each case dog was matched with 4 control dogs, and data were subjected to logistic regression analysis, employing exact conditional inference on the parameter estimates. RESULTS: Case dogs were between 1 and 9 years old and weighed between 21 and 60 kg. Preoperatively, none of the dogs had a history of renal disease, and BUN concentrations, hematocrit, and plasma protein concentrations were within reference ranges. Postoperatively, each dog became azotemic and had clinical signs consistent with uremia. Hyponatremia was recorded in 6 case dogs. One dog did not respond to treatment and was euthanatized. Two dogs had persistent isosthenuria, and 4 dogs recovered. Nafcillin was used in approximately 502 of 2,184 (23%) dogs that underwent invasive procedures between Dec 1, 1992, and Nov 30, 1993. The use of nafcillin ceased on Nov 30, 1993, and no further cases were recorded in the following 15 months. In the case-control study, the only factor that was significantly associated with the occurrence of acute postoperative azotemia was administration of nafcillin. CLINICAL IMPLICATIONS: Intraoperative use of nafcillin maybe associated with development of acute postoperative azotemia in dogs. PMID- 8621316 TI - Quantity and distribution of Malassezia organisms on the skin of clinically normal dogs. AB - OBJECTIVE: To define the extent to which Malassezia organisms can be recovered from the skin of clinically normal dogs and to assess differences in organism recovery related to anatomic sampling site and to method of collection. DESIGN: Prospective, controlled study. ANIMALS: 19 clinically normal dogs. PROCEDURE: The number of Malassezia pachydermatis organisms were determined in fungal cultures of samples obtained from the skin of clinically normal dogs, using an adhesive tape method to obtain samples from 10 sites/dog. Additionally, 3 methods (direct impression, swabbing technique, and superficial skin scraping) that are commonly used for obtaining samples for cytologic examination were evaluated. RESULTS: Malassezia organisms were found in low numbers as part of the microflora of the skin of clinically normal dogs. Number of organisms differed significantly for various anatomic locations (chin, highest number; inguinal and axillary regions, lowest number). Malassezia organisms were identified more frequently by use of adhesive tape and fungal culturing than by the methods used for cytologic examination. However, comparing methods used for obtaining samples for cytologic examination with each other, marked differences were not detected in our ability to recover yeast organisms among the 3 techniques. CLINICAL IMPLICATIONS: Although Malassezia spp is part of the microflora of the skin of clinically normal dogs, it is extremely difficult to detect the organism by any of the 3 sampling methods used for sample collection for cytologic examination. Therefore, anatomic site and method of sample collection should be considered when attempting to make a diagnosis of Malassezia dermatitis. PMID- 8621317 TI - Methylphenidate toxicosis in a cat. AB - A 10-year-old cat with restlessness, vocalizing, and circling was examined 13 hours after it was inadvertently given a 5-mg tablet of the CNS stimulant methylphenidate hydrochloride. Physical examination findings (generalized tremors, agitation, mydriasis, tachycardia, tachypnea, and hypertension) were consistent with overstimulation of the CNS and excessive adrenergic activity resulting from methylphenidate toxicosis. Plasma methylphenidate concentration at admission (83 ng/ml) was 5 to 16 times greater than the concentration reported to provide therapeutic effect in human beings. The cat was placed in a dark, padded cage to minimize external stimuli, and supportive care consisting of fluids and diazepam were administered. Clinical signs resolved within 25 hours after ingestion of methylphenidate. PMID- 8621318 TI - Sensitivity and specificity analysis for somatic cell count (SCC) used to predict bacteriologically positive subclinical mastitis at calving in a dairy herd with low SCC. AB - OBJECTIVE: Validate, by sensitivity and specificity analyses, use of somatic cell count (SCC) to predict bacteriologically positive subclinical mastitis in a California dairy herd with low SCC. DESIGN: Study of monthly dairy herd improvement SCC obtained from the immediate preceding lactation and individual cow composite milk sample microbiologic isolates collected at calving. ANIMALS: 515 California dairy cows with SCC and culture data. PROCEDURE: Somatic cell count sensitivity and specificity analyses with combinations of SCC parameter and at various thresholds were done, using the bacterial isolates as the standard. RESULTS: Combination of SCC threshold and SCC parameters could not be developed that had sufficient sensitivity and specificity to be a useful predictor of cows that would calve with subclinical mastitis. CLINICAL IMPLICATIONS: Under the conditions at this particular dairy, SCC could not be used as a basis of prediction of cows that would calve with bacteriologically positive subclinical mastitis or require selective nonlactating-cow antibiotic treatment. PMID- 8621320 TI - Elemental sulfur toxicosis in a flock of sheep. AB - Two thousand Panama X Rambouillet ewes from a flock of 2,200 developed signs of acute toxicosis after being moved to a field that had been sprayed 16 hours earlier with elemental sulfur. Acute signs were lethargy, abdominal discomfort, and prostration. Two hundred six (10%) of the affected ewes died within 24 hours. Polioencephalomalacia that was unresponsive to thiamine treatment developed in another 40 (2%) of the ewes; 28 (70%) of the ewes with polioencephalomalacia recovered. Sulfur is converted to hydrogen sulfide in the rumen. Signs of sulfur toxicosis are a result of absorption of hydrogen sulfide and interaction with the cytochrome system and hemoglobin. Sulfide is detoxified in the RBC and by the liver. PMID- 8621321 TI - Proper drug use: an issue in milk screening tests. PMID- 8621319 TI - Epidemiologic study of decubital ulcers in sows. AB - OBJECTIVE: To determine prevalence and risk factors for decubital ulcers of the shoulder in sows. DESIGN: Descriptive cross-sectional study. SAMPLE POPULATION: All females of breeding age in a large confinement swine facility. PROCEDURE: 1,916 females were examined for lesions of the skin over the tuber of the spine of the scapula and for body condition scoring. Observational data were combined with sow data (parity, date of farrowing, litter size) contained in computerized records. RESULTS: Decubital ulcers were observed in 8.3% of females, predominantly lactating sows. Ulcer prevalence was strongly associated with time after farrowing. Lesions apparently healed rapidly after weaning. Ulcer prevalence was associated with low body condition scores, but was not associated with parity. IMPLICATIONS: Decubital ulcers are a multifactorial condition. Housing on concrete floors per se did not result in ulcers. Prolonged recumbency during parturition, reduced activity in early lactation, periparturient illness, thin body condition, moist skin, and floor type are potential risk factors. PMID- 8621322 TI - Salmonella in culled cows focus of study. PMID- 8621323 TI - An ultrastructural study of the phagocytic activity of astrocytes in adult rat brain. AB - The role of adult astrocytes in the removal of cell debris and foreign particles following injury to the brain is controversial. This study was undertaken to elucidate the response of adult astrocytes to needle injury of the rat cerebral cortex, using a suspension of colloidal carbon as a marker for phagocytosis. Either a single or 2 successive injections of colloidal carbon suspension were made into the cerebral cortex. The animals were allowed to survive for periods of from 1 to 30 d. Unequivocal involvement of astrocytes in the removal of carbon particles was evident only in those brains which had been subjected to 2 successive injections of carbon. The particles were located in membrane-bound vacuoles and were subsequently sequestered in lysosomes. Carbon-containing astrocytes were observed in the immediate vicinity of the lesion, in the adjacent parenchyma, around blood vessels and abutting carbon-containing macrophages. This study demonstrates that adult astrocytes are involved in phagocytosis, but only as a second line of defence. The possible significance of carbon-laden astrocytes further away from the site of the lesion is discussed. PMID- 8621324 TI - Does the nerve supply to both the superficial and deep surfaces of pectoralis major imply two separate developmental origins? AB - The nature of the nerve supply to the "pocket' of pectoralis major was examined on 7 randomly selected sides of 5 embalmed cadavers. The pocket was a U-shaped muscular fold, opening cranially. The anterior limb and inner surface of the fold were supplied by nerve branches that originated from the middle segment of the pectoral nerve loop and penetrated pectoralis minor. The outer surface of the posterior limb was supplied by one or two branches that extended from the caudal segment of the pectoral nerve loop. If the muscular U-shaped fold is unfolded, it becomes obvious that the posterior wall of the pocket forms the most caudal part of pectoralis major and is supplied from both the superficial (anterior) and deep (posterior) surfaces. This dual surface supply does not suggest any aspect of the developmental origin of the pocket but may simply be due to the relative positions of the pectoralis major and its nerve. PMID- 8621325 TI - The distribution of organised lymphoid tissue in the alimentary tracts of koalas (Phascolarctos cinereus) and possums (Trichosurus vulpecula and Pseudocheirus peregrinus). AB - The anatomical arrangement of organised lymphoid tissues of the alimentary tract for 3 Australian marsupials, the koala (Phascolarctos cinereus), the common brushtail possum (Trichosurus vulpecula and the common ringtail possum (Pseudocheirus peregrinus), was determined by gross dissection and acetic acid treatment. Oropharyngeal tonsils were consistently found in the dorsolateral wall of the caudal oropharynx in all 3 species and additionally in the ventral soft palate of the koala. Aggregated lymphoid nodules (Peyer's patches) were present in the small intestine of koalas, ringtail possums and brushtail possums and were of similar appearance for all 3 species. Bilateral large intestinal lymphoid patches were detected in the caecocolic lateral wall adjacent to the termination of the ileum for all 3 species. Caecocolic patches were more complex in koalas and had mucosal folds and a central recess. In addition, solitary and grouped large intestinal lymphoid nodules were variably present in the proximal colon and caecum of the koala. In contrast, possums had solitary and grouped large intestinal lymphoid nodules present in the proximal colon and rectum but not the caecum. Aggregated lymphoid tissue was not detected in the tongue, oesophagus or stomach for all 3 species. In contrast to a previous report, this study did not find a paucity of lymphoid tissue associated with the gut of the koala. The appearance and distribution of gut-associated lymphoid tissue in koalas and possums was found to be similar to that described in other marsupials and eutherian mammals, although some variations in appearance and anatomical location were observed. PMID- 8621326 TI - Histological and immunohistological investigation of alimentary tract lymphoid tissue in the koala (Phascolarctos cinereus), brushtail possum (Trichosurus vulpecula) and ringtail possum (Pseudocheirus peregrinus). AB - The histological appearance and distribution of T cells, B cells and plasma cells were investigated for oropharyngeal tonsils, small intestinal lymphoid aggregations (Peyer's patches), caecocolic lymphoid patches and mesenteric lymph nodes of koalas (Phascolarctos cinereus), common brushtail possums (Trichosurus vulpecula) and common ringtail possums (Pseudocheirus peregrinus). The histological organisation and distribution of lymphoid cell subpopulations of these tissues were similar to those described in eutherian mammals, although some differences were found in comparison with previous descriptions of American opossum tissues. The main variation among the 3 species was in the structural organisations of the oropharyngeal tonsil and the caecocolic lymphoid patch which were more complex in the koala than in possums. In the koala the extensive crypts of the oropharyngeal tonsils and folding of the mucosa of the caecocolic lymphoid patch increased their surface area and, in addition, both structures had areas of epithelium heavily infiltrated with T and B cells. These features could indicate that these structures are important in immunological surveillance of orally presented antigens in koalas. PMID- 8621327 TI - A quantitative morphological study of the recovery of cat lingual nerves after transection or crushing. AB - The morphological changes were examined proximal and distal to crush and transection injuries of the lingual/chorda tympani nerve. Under general anaesthesia the nerve was transected unilaterally in 6 adult cats and crushed with watchmakers forceps in 6 others. After 12 wk, again under general anaesthesia, the injured and contralateral (control) nerves were removed, fixed and embedded for histological examination. Sections were cut from sites proximal and distal to the injury and from a site equivalent to that of the injury on the control side. Using systematic randomised sampling techniques the number of nonmyelinated axons and the number and size of myelinated axons in each nerve at each location was estimated. In addition, the mean number of nonmyelinated axons in each Schwann cell unit was determined. The only significant difference between control and injured nerves proximal to either injury was a reduction in the number of myelinated axons in the chorda tympani after transection, and an increase in their mean size. This indicates a selective loss of smaller fibres and is consistent with the poor recovery of gustatory and thermosensitive fibres previously reported (Robinson, 1989). Distal to both types of injury there was an increase in the number of fascicles. The mean number of myelinated axons was reduced distal to a crush injury but unchanged distal to transection. The number of nonmyelinated axons distal to a transection injury was 5 times control counts and after a crush injury double. These findings suggest that sprouting persists 12 wk after both injuries but is much greater after transection. PMID- 8621328 TI - Muscle spindles in the jaw-closer muscles of the domestic cat. AB - The objectives of this study were to identify the exact location of spindles in jaw-closer muscles of the cat, to count the total number of spindles and to compare their distribution with the distribution of slow extrafusal fibres. The jaw-closer muscle group with all the skeletal attachments intact was fixed in a modified Carnoy solution, decalcified and processed through to wax. Complete series of sections were cut transverse, sagittal and perpendicular to the anterior temporalis muscle. At regular intervals, serial sections were stained by the Weigert-van Gieson method or immunostained for myosin isoforms. Spindle counts were made only from muscle areas where fibres were cut in transverse section, and the spindles were followed individually. The extrafusal fibres were identified by indirect immunoperoxidase staining with antibodies specific for the slow (type I) and fast (type IIM) isoforms of myosin found in jaw-closer muscles. The mean numbers and locations of spindles found were 13 in medial pterygoid (close to inferior border), 123 (one count only) in the deep anterior portion of the temporalis muscle (between the coronoid process of the mandible and the cranium), 50.5 in a small deep zone of superficial masseter anterior to the temporomandibular joint, and 50 in zygomaticomandibularis (the deepest portion of masseter). Most of the spindles were simple spindles. Spindle complexes (4 or more spindle units fused in parallel) were rare and were found only in zygomaticomandibularis and in masseter. Most parts of the jaw-closer muscles had no spindles and contained only fast fibres. Cosegregation of muscle spindles with slow fibres was found in most parts of this muscle group, but the distribution of spindles was more restricted than that of slow fibres. PMID- 8621329 TI - Cryoscanning electron microscopy of loaded articular cartilage with special reference to the surface amorphous layer. AB - The surface layer (i.e. the surface lamina) of articular cartilage, which is devoid of a collagen fibril network or cells, was investigated in the pig and human. It overlies the collagenous main part of the articular cartilage which contains chondrocytes and is thought to be important biomechanically. In order to examine morphological changes in this layer when under load, knee articular cartilage of the pig, along with the underlying subchondral bone, was compressed with a cylindrical indenter. The specimen was frozen by immersion in liquid nitrogen to maintain the loaded condition and was then freeze-fractured at the indented region. The fracture face was examined with a cryoscanning electron microscope. The surface layer was compressed beneath the indenter regardless of loading pressure or period and was expanded around the indenter to form a triangular bulge in cross section. The height of the bulge was related to the applied pressure and not to the loading period. Recovery of the cartilage from indentation was also examined. Immediately after removal of the indenter, the bulge of the surface layer moved back into the previously indented region. The region was covered by a thick surface layer after 2 s. The response of the surface layer to and recovery from indentation was largely instantaneous and elastic. Under heavy load conditions, the main part of the cartilage under the indenter was observed to have a striped pattern which was made up of bands of densely packed collagen fibrils with fibrillar networks remaining between them. These morphological findings agree well with previously reported biomechanical hypotheses and can be explained by the flow of interstitial fluid provoked by stress application. PMID- 8621330 TI - Morphological changes in the myenteric plexus of rat ileum after transection and end-to-end anastomosis. AB - Surgical interruption of the gastrointestinal tract is widely used for investigating the structure of the enteric nervous system and in the treatment of certain pathological conditions of the gastrointestinal tract. The effects of transection and end-to-end anastomosis and myotomy on nerve cells of the myenteric plexus were studied by light and electron microscopy, 1, 2 and 6 wk postsurgically. During the 1st wk, degeneration of some nerve cells was indicated by the dilation of the endoplasmic reticulum and dispersion of Nissl substance. The degenerative process (an early electron-dense lamellar degeneration and a late floccular degeneration) was accompanied by some regenerative changes during the recovery period. Quantitative light microscopic analysis demonstrated a significant decrease in the number of neurons located 1 and 5 mm from operation sites in the intestine. PMID- 8621331 TI - Immunocytochemical evidence that a specialised region of the rat oviduct secretes an oviductal glycoprotein. AB - The immunocytochemical localisation in the rat oviduct of an oviductal glycoprotein was investigated by light and electron microscopy. Using a monoclonal antibody (MAb) that cross-reacted with the rat oviductal glycoprotein ( > 330 kDa), we examined the epithelium of 4 regions (fimbriae, ampulla, isthmus and uterotubal junction) of the rat oviduct during the oestrous cycle. The MAb reacted specifically with the epithelial cells of the rat oviduct and not with the stromal cells. Intense labelling was observed in the isthmic epithelium throughout the oestrous cycle. Immunohistochemical staining was observed in the ampullary epithelium but the reaction was very weak. In the fimbriae and at the uterotubal junction, the immunoreaction was barely apparent in the epithelial cells. At the ultrastructural level, the MAb bound selectively to the putative secretory granules of nonciliated secretory cells in the isthmus, but no labelling was observed in most of the secretory granules in the ampullary segment. These results demonstrate that certain glycoproteins are secreted mainly by the isthmic secretory cells, suggesting regional specificity of the production of the glycoprotein in the rat oviduct. PMID- 8621332 TI - Quantitative histomorphology of the blind mole rat harderian gland. AB - Anatomical, histological and morphometric studies have been performed on the harderian gland and its surroundings in the blind mole rat (Spalax ehrenbergi). The gland is tubuloalveolar with no true duct system. All ducts within the gland are formed by a single epithelial cell type and drain into a wide secretory duct. This opens into the conjunctival sac which serves as a reservoir for harderian secretions. Drainage from the conjunctival sac follows 2 possible routes: one through the nasolacrimal duct to the external nasal cavity, the other through a unique excretory duct that emerges from the anteromedial part of the conjunctival sac and runs through the dermis to the skin, opening at the base of a hair follicle. The function of this newly described duct is discussed. Morphometric studies revealed that the lumen volume fraction in the female, slightly smaller than that of the male during the summer, becomes significantly greater during the winter breeding season. The dimorphism and seasonal variations found in the gland acini suggests that the gland may be implicated in pheromone production. PMID- 8621333 TI - Histological evaluation of the canine retinal vasculature following chronic systemic administration of basic fibroblast growth factor. AB - The purpose of the present study was to determine if prolonged systemic arterial administration of basic fibroblast growth factor (bFGF) at a dose sufficient to enhance collateral vessel formation in the ischaemic hearts of dogs would produce retinal neovascularisation in these same animals. Adult dogs (15-25 kg) were subjected to gradual occlusion of a coronary artery and randomised to receive 1 of 3 treatments via an indwelling left atrial catheter: (1) bFGF 1.74 mg/d, 5 d/wk for 63 d (n = 7); (2) bFGF 1.74 mg/d, 5 d/wk, for 35 d followed by physiological saline, 5 d/wk, for 28 d (n = 10); or (3) physiological saline, 5 d/wk, for 63 d (n = 10). After 63 d the retinal vasculatures from these dogs were isolated and examined for capillary varicosity, neovascularisation and other histopathological signs of angiopathy. All data were collected under masked conditions. The results suggest that chronic, systemic arterial administration of bFGF stimulates neovascularisation in the ischemic myocardium, but has no significant structural or vasoproliferative effect on the nonischaemic retina of the same animal. PMID- 8621334 TI - Matrix metalloproteinases in the formation of human synovial joint cavities. AB - Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling in growth and development. A histochemical study of human fetal limbs was undertaken to assess the presence, and consequently the possible role, of MMPs and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in synovial joint cavity formation. Cryostat sections of fetal limbs from 7 to 14 wk gestation were stained with specific antibodies to collagenase (MMP-1), gelantinases A (MMP-2) and B (MMP-9), stromelysin (MMP-3) and TIMP-1. Immunoreactive (IR) MMP-1, MMP-2 and MMP-3 were seen chiefly in chondrocytes, but in all cases in zones distant from the joint line before cavity formation. IR-MMP-1 and MMP-2 were also localised both in synovium and on the articular surfaces of joints after cavity formation. In addition IR-MMP-2 was seen in a "collar' of perichondrium alongside the hypertrophic zone of chondrocytes and weakly in bone marrow spaces. IR-MMP-9 was seen in neutrophil leucocytes and in bone marrow spaces. IR-TIMP-1 was generally distributed in connective tissue cells. No IR-MMP (1, 2,3 or 9) was seen along potential joint lines before or at the time of cavity formation, nor was there aspecific decrease in IR-TIMP-1 at this site. These findings confirm a role for metalloproteinases in developmental processes such as cartilage remodelling and bone marrow space formation. MMP-1 and MMP-2 may be involved in the remodelling of developing synovial tissue and the articular surfaces subsequent to cavity formation. However, we have failed to find evidence to indicate that the loss of tissue strength at the joint line which allows synovial joint cavity formation relates to high local levels of MMPS. PMID- 8621335 TI - Expression of the vascular endothelial growth factor receptor, KDR, in human placenta. AB - Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor known to act directly on vascular endothelial cells by promoting cell proliferation and permeability. To date, 3 structurally related cell surface receptors for VEGF, Flt-1, Flt-4 and KDR, have been identified and shown to be human type III receptor tyrosine kinases. The establishment of a vascular network is crucial to the development of the placenta and occurs through both angiogenesis and vasculogenesis. The signals controlling these processes are unclear. Immunohistochemical and in situ hybridisation techniques have localised VEGF in the trophoblast layers and VEGF binding to placental vascular endothelial cells and haemangioblasts has been shown, suggesting a role for VEGF and its receptors in development of the vascular network. In this study we have used specific antibodies to localise KDR and endothelial cells in 1st and 3rd trimester human placenta. The staining showed a colocalisation of KDR with endothelial cells and haemangioblasts. No staining of trophoblast cells was observed, but strong staining of the endothelial cells was seen in the villous stroma adjacent to areas of trophoblast proliferation. PMID- 8621336 TI - Quantitative comparison of soft tissue-bone interface at chondral ligament insertions in the rabbit knee joint. AB - At chondral ligament insertions the calcified fibrocartilage interdigitates deeply with the lamellar bone. The shape of this interface is formed under physiological loading conditions. For the purpose of morphological comparison between different ligament entheses in the rabbit knee, the number and frequency of interdigitations and thickness of calcified fibrocartilage were quantitated at the femoral insertion of the medial collateral ligament, both insertions of the cruciate ligaments, and the tibial insertion of the patellar ligament. Among the insertions, the femoral insertion of the medial collateral ligament showed the lowest frequency and depth of interdigitations at the soft tissue-bone interface, but had the thickest zone of calcified fibrocartilage. An inverse relationship was found at the insertion interface of the cruciate and patellar ligaments. The frequency and depth of interdigations at the bone-soft tissue interface at different chondral entheses seem to be related to the mechanical strength of the respective ligament; meanwhile it may be hypothesised that the thickness of the calcified fibrocartilage might be more related to the amount of motion which takes place at an insertion. PMID- 8621337 TI - Cell proliferation and renewal of mouse adrenal cortex. AB - Although many hypotheses concerning cell proliferation and renewal in the adrenal cortex of mammals have been proposed, this topic has so far not been elucidated. Adrenocortical cells of adult mammals have low proliferative activity and take a considerable length of time to be renewed. This makes it difficult to investigate the dynamic features of their proliferation. To clarify the cell kinetics, we undertook a long term study in mice using an autoradiographic technique. We radiolabelled almost all the cells throughout the body in newborn mice with the exception of the neurons in central nervous system by the frequent subcutaneous injections of [3H]thymidine every 6 h for 30 d (pulse labelling). After this sequence of pulse labelling, we observed autoradiographically a decrease in the number of 3H-labelled cells in the adrenal cortex as a result of replacement with proliferated unlabelled cells (renewed cells). Single injections of [3H]thymidine (flash labelling) was also performed to examine DNA synthesis in the adrenal cortex. The investigations indicated that the adrenocortical cells proliferate at the border between the zona glomerulosa and the zona fasciculata, and that renewed cells which proliferated in that region move with time bidirectionally towards the cortical surface and the inner (medullary) surface. Half of the cortical cells in the zona glomerulosa, zona fasciculata and zona reticularis were replaced by renewed cells in 30, 60 and 120 d respectively. It took 200 d for almost all cortical cells to be replaced by renewed cells. PMID- 8621338 TI - Morphometric study on the uterine horn and thyroid gland in hypothyroid, and thyroxine treated hypothyroid rats. AB - A wide range of reproductive disorders such as irregular menstruation and frank infertility is found in women with hypothyroidism. Most research done on these patients has focused on steroid and gonadotropin hormone profiles, whilst there has been little work on uterine morphology. Studies on hypothyroid animals have also demonstrated increases in fetal wastage, but there have been few studies of uterine structure in the hypothyroid rat. The present study has used hypothyroid Wistar rats as a model for investigating the effects of hypothyroidism on uterine structure. Three groups of Wistar rats were studied. One was made hypothyroid with methimazole (MMI), the 2nd was also made hypothyroid with methimazole but in addition the rats were simultaneously given daily thyroxine intraperitoneally (MMI + T4), and the 3rd was an untreated euthyroid group (control). Daily vaginal smears were obtained from rats in all 3 groups. All rats were aged 6 wk at the start of treatment and were killed after a further 6 wk. Uterine horns were removed and studied. Systematic random transverse sections were obtained from the proximal, middle, and distal regions of the horn and subjected to morphometric analysis. Difference between regions was assessed using 2-way analysis of variance. Absolute volume of endometrium in the uteri of hypothyroid rats was reduced by 45.1% (P < 0.05), whilst that of the muscle layer was decreased by 33.6% (P < 0.05). The cross-sectional area and absolute volume of the uterine horns were also reduced in hypothyroid animals (P < 0.05). In hypothyroid rats given thyroxine (MMI + T4) all variables increased significantly above those of hypothyroid rats. These changes suggest that hypothyroidism has an effect on uterine structure, which demonstrably improves under exogenous thyroxine administration. The observed structural changes might well play a significant role in the reproductive difficulties observed during hypothyroidism. PMID- 8621339 TI - Computer-assisted stereological analysis of gastric volume during the human embryonic period. AB - Morphometric data concerning human embryos and fetuses have become more clinically informative since ultrasound was employed to make prenatal measurements and software preprocessing techniques improved the previous fuzzy ultrasound signals (Mahoney, 1992). The aim of this study was to determine the volume of the human stomach during the embryonic period and to compare its rate of growth with that during the early fetal period. To calculate gastric volume, computer imaging techniques were applied on cross sections of a graded series of human embryos (from Carnegie stage 11) and fetuses. Gastric volume increased progressively, except for a decrease between stages 12 and 13 due principally to the reduction of the right gastric wall. The growth of the left wall of the stomach was predominant over that of the right. Until stage 20 the stomach volume increased due to the predominant growth of the walls, after this stage the gastric cavity volume increased rapidly, and the rate of growth of the gastric volume reached similar values to that of the early fetal period. We concluded that in the beginning the human stomach grows due to the predominant growth of its walls, chiefly of the left, and from stage 20 because of the predominant expansion of its cavity, which may be related to the capacity to swallow amniotic fluid at the end of the embryonic period. The diminution of the right gastric wall volume (stages 12-13) is consistent with an extension of the omental bursa into the mesodermal anlage of the stomach. PMID- 8621340 TI - A quantitative study of nerve distribution in the conduction system of the guinea pig heart. AB - Quantitative measurements of relative nerve density were achieved using computer assisted image analysis of immunohistochemically and histochemically defined nerves in the conduction system of the guinea pig heart. All regions of the conduction system possessed a similar density of nerve fibres and fascicles displaying immunoreactivity for the general neuronal marker protein gene product 9.5 (PGP 9.5), and this was 3 to 4-fold higher than in the adjacent myocardium. Acetylcholinesterase (AChE) positive and tyrosine hydroxylase (TH)-immunoreactive nerves were the main subtypes identified in the sinus and atrioventricular nodes, representing 40-45% of the stained area occupied by PGP 9.5-immunoreactive nerves. AChE-positive nerves were the dominant subtype identified in the left and right bundle branches, but were equal in proportion to TH-immunoreactive nerves in the penetrating bundle. Neuropeptide Y-immunoreactive nerves represented the main peptide-containing subpopulation in the nodal tissues, displaying a similar pattern of distribution and relative density to those nerves demonstrating TH immunoreactivity. Substance P and calcitonin gene-related polypeptide immunoreactive nerves were present throughout the conduction system and represented the main peptide-containing subpopulation in the ventricular conduction tissues. Nerve fibres showing immunoreactivity for either somatostatin or vasoactive intestinal polypeptide exhibited distinct patterns of distribution and comprised a relatively minor component of the innervation. The innervation of the guinea pig conduction tissues thus exhibits a uniform distribution and it comprises putative parasympathetic nerves and intrinsic neurons (AChE positive), sympathetic efferent nerves (NPY and TH-immunoreactive nerves) as well as other peptide-containing nerves, some of which (substance P and calcitonin gene-related polypeptide) are considered to represent afferent nerves. The distribution and density of nerve subpopulations in the guinea pig conduction system differ from those observed in the human conduction system, which suggests that the guinea pig may be an inappropriate model for comparative functional studies. PMID- 8621341 TI - The effects of isolation on the mechanics of the human heel pad. AB - In previous studies on the mechanical properties of the human heel pad (Bennett & Ker, 1990; Aerts et al. 1995) the fat pad and part of the calcaneus was removed from amputated test specimens. The present study tested whether this procedure influences the mechanical behaviour of the sample. Intact amputated feet were therefore mounted on steel rods driven through the calcaneus and placed in a mechanical test situation (pendulum or servohydraulic material tester). The mechanical properties of the pad were determined for a series of experiments in which the pad was gradually freed from the foot in the way done by Bennett & Ker (1990) and Aerts et al. (1995). The results showed no observable differences in the mechanics of the pad by isolating it from the rest of the foot. Thus, in relation to human locomotion, the load-deformation relation of heel pads as described by Aerts et al. (1995) is the most appropriate to date. PMID- 8621342 TI - MRI-based surface area estimates in the normal adult human brain: evidence for structural organisation. AB - There are a number of quantitative relationships between geometric parameters describing the structure of the normal human cerebral cortex examined in vivo using volumetric magnetic resonance imaging. A voxel-counting method is used to estimate grey-white interface surface area. The effects of bias associated with the method are considered. In 33 normal controls, the cerebral hemispheres were symmetric in terms of total volume, irrespective of handedness, but not in terms of surface areas for right-handers. The surface area of the grey matter-white matter interface was directly proportional to the cortical grey matter volume, suggesting that growth of the neocortex is primarily tangential, with repetition of a basic structural element rather than gross alterations in the thickness of the cortex. The majority of the surface area of the grey-white interface lies within gyral white matter cores. The mean thickness of the cortex of the right cerebral hemisphere in vivo was 3.0 mm and that of the left 3.3 mm. There was a relationship between the cross-sectional area of the corpus callosum and grey white interface surface area, suggesting that a fixed proportion and cortical neurons extend interhemispheric axons. These findings suggest that there are general architectural principles governing the organisation of the complex, but ordered, human cerebral cortex. PMID- 8621343 TI - Histology of the mucosa of the oesophagogastric junction and the stomach in adult Rana perezi. AB - The histological structure of the frog digestive mucosa changes at the oesophagogastric junction. The pseudostratified ciliated mucosal epithelium of oesophageal type changes to a simple mucus-secreting epithelium of gastric type. The glands straighten and the muscularis mucosae develops as a complete layer. The muscularis increases in thickness. Unlike the mammalian stomach, in the frog the surface of the plicae forms convoluted ridges that delimit furrow-shaped pits. Two types of gastric glands are distinguished, fundal and pyloric. The former consist of mucous, oxynticopeptic and endocrine cells. The pyloric glandular cells are mainly of mucus-secreting type with scattered endocrine cells. Scattered endocrine cells of P, D, G, A, EC, and EC-L-like types are found in the glands along the stomach. It is concluded that the mucosal structure of the anuran oesophagogastric junction and stomach is less complicated than that of mammals, including man. PMID- 8621344 TI - The vomeronasal organ of the cat. AB - The vomeronasal organ of the cat was studied macroscopically, by light microscopy and by immunohistochemical techniques. Special attention was paid to the general distribution of the various soft tissue components of this organ (duct, glands, connective tissue, blood vessels and nerves.) Examination of series of transverse sections showed that the wall of the vomeronasal duct bears 44 different types of epithelium: simple columnar in the caudal part of the duct, respiratory and receptor respectively on the lateral and medial walls of the middle part of the duct, and stratified squamous rostrally. The pattern of distribution of other soft tissue components was closely associated with that of epithelium types. In areas where the duct wall was lined with receptor epithelium, nerves and connective tissue were present between the epithelium and the medial sheet of the vomeronasal cartilage. Most glands and blood vessels were located lateral to those areas of the duct wall lined with respiratory epithelium. Numerous basal cells were present in the sensory epithelium. Understanding of the distribution of the soft tissue components of this organ may shed light on its function. PMID- 8621345 TI - Quantitative skeletal muscle histochemistry of four east African ruminants. AB - A quantitative histochemical study was made of superficial thigh muscle specimens (semimembranosus and some vastus lateralis) from topi, hartebeest, wildebeest and waterbuck (species listed in order of increasing size). Fibres were largest (up to 120 microns diameter) in waterbuck but smallest (maximum approximately 90 microns) in wildebeest. Type 2B fibres, most of them large, highly glycolytic and weakly aerobic, constituted approximately 75% of the cross-section of topi specimens and approximately 83% of the others, greater area fractions than in other large herbivores. Type 1 fibres, highly aerobic but weakly glycolytic, occupied only 2-3.5% of the area fractions, very low figures, even for these superficial sites. Type 2A fibres occupied > 20% areas in topi, approximately 15% in the other species. In waterbuck they were never more than moderately aerobic, but quite highly glycolytic; elsewhere their characteristic metabolic profiles were close to those of type 1 fibres. The 2B fractions indicate that glycolytic metabolism predominates over lipolytic in all 4 species. Mean enzymic capacities were compared semiquantitatively between species on the basis of wide-area photometric readings. Much the greatest difference was in aerobic (succinate dehydrogenase) capacities: the mean reading in topi was x 2.6 that in waterbuck, but wildebeest capacity came close to that of topi. These latter are the 2 most active species. Readings for the force-generating enzyme, actomyosin ATPase, were slightly weaker in the heavier species. This could be predicted on allometric grounds, but mass considerations appear to be overridden by behavioural differences in relation to metabolism. PMID- 8621346 TI - Emigration of neural crest cells from macaque optic vesicles is correlated with discontinuities in its basement membrane. AB - It is established that cranial neural crest cells play critical roles in normal development, but the production of neural crest cells from the prosencephalon has received little attention, especially in primates. We therefore investigated the emigration into adjacent mesenchyme of neuroepithelial cells from macaque optic vesicles. Paraffin sections prepared from 13 embryos (Macaca fascicularis) representing developmental stages 10-14 were examined following standard immunoperoxidase staining for laminin and type IV collagen. At stage 10 the optic vesicle basement membrane was closely applied to that of the surface ectoderm except at its posterior border, where it contacted mesenchyme. The basement membrane was continuous and showed no evidence of cell migration. By stage 11 the optic vesicle basement membrane exhibited numerous gaps along its posterior border. These defects were frequently occupied by cells emigrating from the optic vesicle epithelium, as judged by the deflection of basement membrane fragments. Gaps were also seen along the lateral border, where migrating neuroepithelial cells were positioned between the surface ectoderm and optic vesicle. This cell migration appeared to increase during stage 12, with deterioration of all areas of the optic vesicle basement membrane. Basement membranes of the surface ectoderm, adjacent mesencephalon, and telencephalon anlage remained intact. By stage 13 the basement membranes of the optic vesicles were repaired and nearly continuous, with migrating cells rarely seen. Development of the optic cups at stage 14 indicated a near absence of basement membrane defects and emigrating cells. PMID- 8621347 TI - The role of the vertebral laminae in the stability of the cervical spine. AB - The aim of this study was to determine the contribution of the vertebral laminae to the stability of the cervical spine since laminectomy may result in deformity of the neck. In 40 dry adult male cervical columns the weight-bearing areas of the inferior surfaces of the bodies and articular facets from C2 to C7 were measured and the means and S.D.S. calculated. In all columns the lamina index (height x thickness) of right and left halves of each lamina was calculated and summed at each cervical level. Means and S.D.S. were calculated for the series. The trabecular patterns in the laminae were studied in 6 of the columns. Results show that the laminae of C2 and C7 are heavily loaded, whilst the intervening ones are not. Thus laminectomy at C2 and C7 would tend to lead to instability, but between C3 and C6 this would be less likely. Significant segmental variation in weight transmission was not found for the facet joints. PMID- 8621348 TI - An immunohistochemical study of the intraventricular macrophages in induced hydrocephalus in prenatal rats following a maternal injection of 6 aminonicotinamide. AB - Hydrocephalus was induced experimentally in prenatal rats following an injection of 6-aminonicotinamide (6-AN) into pregnant rats. The most remarkable change of the dilated lateral ventricles was in a marked increase in the number of intraventricular macrophages, some of which were laden with ingested erythrocytes. The immunoreactivity of the intraventricular macrophages was noticeably enhanced with the monoclonal antibodies OX-42 and OX-18 which marked the complement type 3 receptors (CR3) and major histocompatibility complex (MHC) class I antigen, respectively. Many immunoreactive cells with similar external morphology were observed to penetrate the ependymal lining at the roof of the ventricles. This, coupled with the concomitant depletion of the conglomeration of amoeboid microglia in the supraventricular corpus callosum, suggests that the upsurge of immunoreactive intraventricular macrophages in hydrocephalus was partly due to the influx of amoeboid microglia probably in response to the damage of the ventricular walls and possible alteration in the contents of the cerebrospinal fluid. The significance of the upregulation of complement type 3 receptors and major histocompatibility complex class I antigens on epiplexus cells in hydrocephalic rats remains to be explored, although our results suggest that the surface antigens may be involved in increased phagocytosis and/or a possible immune response. PMID- 8621349 TI - Discovery of an angiotensin II binding inhibitor from a Cytospora sp. using semi automated screening procedures. AB - Cytosporin A, B and C, three antagonists of [125I]-angiotensin II binding to rat adrenal glands were discovered in fermentations of an endophytic Cytospora sp. during routine screening using semi-automated procedures. The most potent of these displayed an IC50 of 1.5-3 microM and was specific for angiotensin II AT2. PMID- 8621350 TI - Dibefurin, a novel fungal metabolite inhibiting calcineurin phosphatase activity. AB - The novel calcineurin inhibitor, dibefurin, has been isolated from the fungal culture AB 1650I-759. The isolation was bioactivity-directed fractionation using an assay which measures the phosphatase activity of calcineurin. The compound was purified by countercurrent, reverse phase and gel filtration chromatographies. Several studies, including crystallographic, NMR and MS, revealed that dibefurin is a novel dimeric compound of a unique structural type. PMID- 8621351 TI - Fusaricidin A, a new depsipeptide antibiotic produced by Bacillus polymyxa KT-8. Taxonomy, fermentation, isolation, structure elucidation and biological activity. AB - Fusaricidin A, a new depsipeptide antibiotic, was isolated from the culture broth of Bacillus polymyxa KT-8 obtained from the rhizosphere of garlic suffering from the basal rot caused by Fusarium oxysporum. The structure of fusaricidin A was determined by 1D and 2D NMR and MS experiments coupled with amino acid analysis to be a hexadepsipeptide containing 15-guanidino-3-hydroxypentadecanoic acid as a side chain. The absolute configuration of each amino acid residue was determined by chiral HPLC. Fusaricidin A is active against fungi and Gram-positive bacteria. PMID- 8621352 TI - Kalimantacins A, B and C, novel antibiotics from Alcaligenes sp. YL-02632S. I. Taxonomy, fermentation, isolation and biological properties. AB - Novel antibacterial antibiotics, kalimantacins A, B and C, have been isolated from the fermentation broth of Alcaligenes sp. YL-02632S. In this paper, the taxonomy of the producing strain, fermentation, isolation and biological activities of kalimantacins are reported. Kalimantacins inhibit the growth of Staphylococcus aureus and S. epidermidis including multiple-drug resistant strains. PMID- 8621353 TI - Kalimantacin A, B, and C, novel antibiotics produced by Alcaligenes sp. YL 02632S. II. Physico-chemical properties and structure elucidation. AB - Kalimantacin A, B and C are new antibiotics produced by Alcaligenes sp. YL 02632S. Their structures were elucidated to be novel long chain structure compounds containing O-carbamoyl, amide and carboxylic acid moieties based on various 2D NMR experiments and MS analysis. PMID- 8621354 TI - Antibiotics A21459 A and B, new inhibitors of bacterial protein synthesis. I. Taxonomy, isolation and characterization. AB - Novel cyclic peptide antibiotics A21459 A and B are produced by a member of the genus Actinoplanes sp. These antibiotics inhibit bacterial protein synthesis and have selective antimicrobial activity against clostridia, mycoplasma and some Gram-negative bacteria. PMID- 8621355 TI - Antibiotics A21459 A and B, new inhibitors of bacterial protein synthesis. II. Structure elucidation. AB - The structures of the antibiotics, active against a few Gram-negative bacteria and Clostridium difficile, were determined on the basis of physicochemical analyses on the intact molecules and on the acid hydrolysate of A21459 A. FAB-MS and 1H and 13C NMR investigations identified the amino acid units and determined their sequence. Antibiotics A21459 A and B are homodetic cyclic peptides constituted by eight amino acid units. They are glycine, methoxytryptophan, tryptophan, cysteine, alanine, sarcosine, dehydroalanine, and alpha-aminobutyric acid for A21459 A (alanine for A21459 B). Cysteine and alanine condensed to form a thiazole moiety, according to the biosynthesis of thiazole containing antibiotics. PMID- 8621356 TI - Inhibition of mouse tumor metastasis with nojirimycin-related compounds. AB - The antimetastatic activity of ten compounds structurally related to nojirimycin A was examined using a pulmonary metastatic model of mouse B16 melanoma. Nojirimycin B, deoxynojirimycin, D-gluco-delta-lactam, CP3068 and CP3069 are structural analogues of nojirimycin A, and showed potent or moderate antimetastatic activities. Nojirimycin A, nojirimycin B, deoxynojirimycin and D gluco-delta-lactam showed potent or moderate inhibitory activities against alpha glucosidase, beta-glucosidase and beta-mannosidase, but CP3068 and CP3069 in which the structures were related to D-gluco-delta-lactam showed no inhibitory activities. CP3041, CP3042, CP3043, CP3045 and CP3048 are analogues of sodium D glucaro-delta-lactam (ND2001), a carboxy derivative of nojirimycin A, and showed potent or moderate antimetastatic activities. But no analogue was superior to ND2001 concerning with antimetastatic and anti-beta-glucuronidase activities. CP3041 and CP3042 showed potent and moderate inhibitory activities against beta glucuronidase, respectively, but CP3043, CP3045 and CP3048 showed little or no activities. PMID- 8621358 TI - RES-1149-1 and -2, novel non-peptidic endothelin type B receptor antagonists produced by Aspergillus sp. III. Biochemical properties of RES-1149-1, -2 and structure-activity relationships. AB - RES-1149-1 and -2, produced by Aspergillus sp. RE-1149, were found to be non peptidic antagonists for endothelin type B receptor (ET(B) receptor). RES-1149-1 and -2 selectively inhibited the endothelin-1 (ET-1) binding to ET(B) receptor in a competitive manner with IC50 values of 1.5 microM and 20 microM, respectively. RES-1149-1 inhibited the increase in intracellular Ca2+ concentration elicited by 1 nM ET-1 in COS-7 cells expressing human ET(B) receptor, but not in the case of cells expressing ET(A) receptor. In addition, some structure-activity relationships are described. PMID- 8621357 TI - Structure of the spiroketal-macrolide ossamycin. AB - Ossamycin is a cytotoxic agent of undetermined structure that was originally isolated in 1965 from culture broths of Streptomyces hygroscopicus var. ossamyceticus. Its overall structure and relative stereochemistry have now been determined by single crystal X-ray diffraction studies. Absolute stereochemistry was established according to the previously determined configuration of its aminosaccharide constituent, ossamine. The aglycone of ossamycin possesses a 24 membered macrolide ring system onto which is incorporated both a 6,6-spiroketal and 5-membered hemiketal ring system. The overall three-dimensional structure possesses features in common with the related macrocyclic antibiotics dunaimycin, cytovaricin, and A82548A. PMID- 8621359 TI - Chemical modification of PA-48153C, a novel immunosuppressant isolated from Streptomyces prunicolor PA-48153. AB - 5beta-Methoxy (20), 14-methyl (24), 14,14-dibromo-15-nor (25), 8-O-acyl (26-45), 8-O-alkyl (46), 8-O-alkoxycarbonyl (47, 48), and 8-O-carbamoyl (49) derivatives of PA-48153C, a novel immunosuppressant isolated from fermentation products of Streptomyces prunicolor PA-48153, were prepared. These compounds were found to retain the inhibitory activity on the responses of both T and B cells to mitogens. Among them, the C-8 hexanoate 28 showed potent suppressive effects on mitogen responses with less cytotoxicity to EL4 cells and was selected for in vivo evaluation. PMID- 8621360 TI - Dimerization of A82846B, vancomycin and ristocetin: influence on antibiotic complexation with cell wall model peptides. AB - The thermodynamics of glycopeptide antibiotic dimerization have been studied by means of sedimentation equilibrium, using A82846B, vancomycin, ristocetin and complexes formed with several cell wall model peptides. These results indicate that vancomycin dimerization can be strongly promoted in two ways: i) stabilization of the antibiotic conformation in which the carbonyl group of residue three is on the back face of the molecule and ii) preferential interaction of the dimer with the lysine residue of N,N'-diacetyl-lysyl-D-alanyl D-alanine. This effect was not found in ristocetin. A82846B forms stable dimers at very low antibiotic concentration. Two conformational forms have been found for complexed A82846B by 1H NMR. However, calorimetric binding experiments have shown that all its binding sites are thermodynamically equivalent. The affinity of the A82846B dimer for the tripeptide has been estimated to be about 3kJ x mol 1 higher than that of the vancomycin monomer and about -2.6kJ x mol-1 lower than that of dimeric vancomycin. The possible role of dimerization in the biological activity of glycopeptide antibiotics is discussed further on the basis of present thermodynamic data. PMID- 8621361 TI - Carboxamides and hydrazide of glycopeptide antibiotic eremomycin. Synthesis and antibacterial activity. AB - Carboxamides and hydrazide of glycopeptide antibiotic eremomycin were obtained by a direct reaction of the carboxy group of eremomycin with an appropriate amine or hydrazine using diphenyl phosphorazidate as a condencing agent. Eremomycin hydrazide was also obtained by hydrazinolysis of the eremomycin methyl ester. Use of dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for amidation led to the corresponding eremomycin ureides. The ESI-MS data indicate that eremomycin and its amides exist as dimers. The carboxamide, methylamide and benzylamide of eremomycin were as active against Gram-positive bacteria as the parent antibiotic, and the methylamide, benzylamide and hydrazide were almost an order of magnitude more active than eremomycin against Staphylococcus epidermidis clinical isolates in vitro. Amide of eremomycin as well as ureides were devoid of histamine liberating properties, which demonstrates that protection of the carboxyl group leads to a decrease in the allergenic properties. PMID- 8621362 TI - A novel 1 beta-methylcarbapenem antibiotic, S-4661. Synthesis and structure activity relationships of 2-(5-substituted pyrrolidin-3-ylthio)-1 beta methylcarbapenems. AB - The synthesis and biological activity of (1R,5S,6S)-2-[(3S,5S)-5-substituted pyrrolidin-3-ylthio]-6-[(1R)-1-hydroxyethyl]-1- methylcarbapen-2-em-3-carboxylic acids are described. These compounds exhibit potent antibacterial activity against a wide range of both Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa. Of these new carbapenems, (1R,5S,6S)-2-[(3S,5S)-5 sulfamoylaminomethyl pyrrolidin-3-ylthio]-6-[(1R)-1-hydroxyethyl]-1-methylcarb apen- 2-em-3-carboxyli c acid (S-4661) showed the most potent and well balanced activity and was selected as a candidate for further evaluation. PMID- 8621363 TI - On an unstable antifungal metabolite from Trichoderma koningii. Isolation and structure elucidation of a new cyclopentenone derivative (3-Dimethylamino-5 hydroxy-5-vinyl-2-cyclopenten-1-one). PMID- 8621364 TI - In vitro antimicrobial activity and structure-activity relationship of C-2 triazolylthio and pyridinylmethylthio carbapenems. PMID- 8621365 TI - Acetophthalidin, a novel inhibitor of mammalian cell cycle, produced by a fungus isolated from a sea sediment. PMID- 8621366 TI - Inhaled corticosteroids in asthma--star wars. PMID- 8621368 TI - Risk factors for asthmatic patients requiring intubation. III. Observations in young adults. AB - During recent decades, asthma prevalence and mortality have increased rapidly worldwide among children, teenagers, and young adults. Little attention has been placed on the latter group. Therefore, we studied risk factors for intubation in young adults as potential severity markers predictive of death. We analyzed demographic data from a retrospective cohort of hospitalized asthmatic young adults, including all asthmatics aged 20-34 years admitted over a 10-year period (1984-1994) to the University of California, Davis, Medical Center, Sacramento, California. A total of 550 such asthma admissions were reviewed, involving 351 women and 199 men, mean age 27.9 +/- 4.2 years. Of this group, 180 young adults were white, 209 were black, 118 were Hispanic, 16 were Asian, and 27 were American Indian. By National Heart, Lung, and Blood Institute guidelines, there were 95 mild, 322 moderate, and 133 severe cases. Thirty-four young adults required intubation for their asthma. Significant risk factors identified for intubation were psychological factors and psychosocial problems odds ratio (OR) 25.0; 95% confidence interval (Cl) 12.4, 50.8, prior intubation (OR 23.6; 95% Cl 7.5, 42.8), language barrier (OR 17.3, 95% Cl 7.9, 38.0), prior asthma emergency room visit in past year (OR 10.2; 95% Cl 4.6, 16.0), crowding (OR 8.5; 95% Cl 4.6, 16.0), prior asthma hospitalization in past year (OR 8.3; 95% Cl 3.3, 20.8), family dysfunction (OR 7.2; 95% Cl 3.6, 14.3), active smoking/secondhand smoke exposure (OR 7.1; 95% Cl 5.1, 9.9), respiratory infection (OR 6.0; 95% Cl 3.2, 11.5), low formal education (OR 5.7; 95% Cl 2.9, 11.2), unemployment (OR 4.9; 95% Cl 2.5, 9.5), steroid dependence (OR 4.6; 95% Cl 3.2, 6.4), and atopy (OR 4.3; 95% Cl 2.1, 8.5). These variables are important determinants of baseline risk factors. PMID- 8621367 TI - Current prevalence of asthma-related symptoms in San Diego's predominantly Hispanic inner-city children. AB - Ethnic minorities of low socioeconomic status are disproportionately represented in the trends of increasing asthma prevalence, morbidity, and mortality. We surveyed a cohort of 998 fourth-grade students in an impoverished area of southeast San Diego with a high percentage of Hispanic Mexican-Americans. Of the 654 Hispanic 9-12-year-olds, 14.4% were categorized as probable current asthma (within the past year), based on symptom of wheezing or physician diagnosis of asthma [with respiratory symptom(s) or medication]. An additional 13.5% had respiratory symptoms indicating possible asthma. Differences by ethnic group in the percentage of probable asthma or related symptoms were highly significant (p < 0.0001). Among Hispanics with a category of probable asthma, only 57.4% had a physician diagnosis versus 80.6% of black and 85.7% of white students. The frequency of health insurance coverage differed significantly between ethnic groups (p < 0.0001), with Hispanics among the lowest (37.2%). PMID- 8621369 TI - An asthma self-management program for children, including instruction in peak flow monitoring by school nurses. AB - A randomized trial of an instructional method was conducted in which school nurses taught children asthma self-management principles and skills, including peak flow monitoring, in 20-min, individual sessions over an 8-week period. Thirty-six children participated. An intervention group of 18 children received the teaching sessions. A control group of 18 children received regular care by the nurses, but no teaching sessions. The sample included 64% boys, 69% African Americans, and 69% Medicaid recipients. The average age of subjects was 10.2 years. The two groups were demographically similar, but despite random assignment, the control group had a significantly earlier age of onset of asthma and tended to have had more asthma attacks in the preceding year. These factors were statistically controlled in outcome analyses. Results of group comparisons showed no significant differences in the number of postintervention emergency room visits and days absent from school. However, nurses reported that children who practiced breathing exercises had less anxiety during exacerbations, and the nurses' knowledge of the children's baseline peak expiratory flow rates facilitated care of the children. Nurses expressed the opinion that the individual sessions with students might be useful in motivating them to participate effectively in later group sessions. The intervention was well accepted by students, parents, and nurses. We believe that this intervention is promising as a practical, low-cost approach to enhancing children's asthma self management skills and warrants further testing in a larger sample, with the intervention conducted over a longer period. PMID- 8621371 TI - Inhaled corticosteroids: benefits and risks. PMID- 8621370 TI - Asthma and its association with cardiovascular disease in the elderly. The Cardiovascular Health Study Research Group. AB - Cardiovascular disease (CVD) is more prevalent in elderly than in middle-aged patients. Symptoms such as intermittent wheezing with dyspnea may then be due to either CVD or asthma. The objective of this study was to determine the prevalence and correlates of asthma in the elderly and their associations with CVD and CVD risk factors. A community sample of 5201 elderly persons from the Cardiovascular Health Study was asked if they had a physician diagnosis of asthma, and multiple cardiovascular risk and disease variables were measured. Six percent of the participants (309) recalled a history of asthma, and half of these were never smokers. Thirty percent of those with asthma were currently taking a bronchodilator, 14% inhaled steroids, and 10% oral prednisone. Men and women with asthma who were cigarette smokers were more likely to report a concurrent diagnosis of congestive heart failure than smokers without asthma (p = .04). However, when we determined the independent CVD correlates of asthma in this cohort, controlling for smoking status, age, gender, and diagnoses of chronic bronchitis and emphysema, only higher levels of high-density lipoprotein cholesterol (HDL-C) and higher plasma fibrinogen levels were significantly associated with asthma. It was concluded that asthma is as prevalent in the elderly as in middle-aged persons and is associated with higher HDL-C and higher fibrinogen levels, but not with prevalent cardiovascular disease. PMID- 8621372 TI - Maximal airway narrowing on the dose-response curve to methacholine is increased after exercise-induced bronchoconstriction. AB - The changes in airway responsiveness between before and after exercise in asthma are not well defined. We investigated the effect of exercise on PC20 (bronchial sensitivity) and maximal airway narrowing (MAN) on the dose-response curve to methacholine in 56 mildly asthmatic children. High-dose methacholine inhalation tests were performed before and 7 hr after exercise challenge. Methacholine PC20 was not changed by exercise, irrespective of exercise-induced bronchoconstriction (EIB). However, the subjects with (+)EIB displayed increased MAN after exercise, whereas those with (+/-)EIB or (-)EIB did not. The results showed that EIB may be followed by increased MAN but not by the change of bronchial sensitivity. PMID- 8621373 TI - The incidence of exercise-induced bronchospasm in competitive figure skaters. AB - Pediatric commitment to competitive sports is on the rise. Previous reports of the incidence of exercise-induced bronchospasm (EIB) have investigated high school, college, and Olympic athletes in traditional sports. The purpose of this study was to investigate the incidence of EIB in competitive figure skating, a high-intensity, cold-weather sport performed by young athletes. To investigate the incidence of EIB in skaters, 100 competitive skaters from five Mid-Atlantic rinks completed rinkside pulmonary function tests. Results showed an overall incidence of 30%, signaling the need for education and screening for EIB in youth participating in physically demanding, cold-weather sports. PMID- 8621374 TI - Exercised-induced asthma in the athlete. PMID- 8621375 TI - Protein prenyltransferases. PMID- 8621376 TI - Molecular determinants of high affinity dihydropyridine binding in L-type calcium channels. AB - The pore-forming alpha1 subunit of L-type voltage-gated Ca2+ channels is pharmacologically modulated by dihydropyridine (DHP) Ca2+ antagonists and agonists. Site-directed mutation of amino acids within transmembrane segments IIIS6 and IVS6 to those characteristic of DHP-insensitive channels revealed 2 mutations in IIIS6 (I1049F and I1052F) and 4 mutations in IVS6 (Y1365I, M1366F, I1372M, and I1373L) with increased KD values for (+)-[3H]PN200-110 binding. A tyrosine residue (Y1048) in IIIS6 that is conserved between DHP-sensitive and insensitive Ca2+ channels was also altered by mutagenesis. Y1048F had a KD for (+)-[3H]PN200-110 binding that was increased 12-fold, and Y1048A had a KD at least 1000-fold higher than that of wild-type. These results support the hypothesis that transmembrane segments IIIS6 and IVS6 both contribute critical amino acid residues to the DHP receptor site and that Tyr-1048 within transmembrane segment IIIS6 is required for high affinity DHP binding, even though it is conserved between DHP-sensitive and -insensitive Ca2+ channels. PMID- 8621377 TI - ADP-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells. AB - ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport. ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport. It also plays an important role in maintaining the function and morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH3 cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network. Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi. These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles. PMID- 8621378 TI - Regulation of expression of ob mRNA and protein by glucocorticoids and cAMP. AB - Regulation of obese gene (ob) expression in ob/ob and db/db mice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/ob mice. In the present report we show that leptin treatment down-regulates endogenous adipose ob mRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous ob mRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both ob mRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as beta adrenergic agonists or Bt2cAMP itself, decreased ob mRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated ob mRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis. PMID- 8621379 TI - Role of the nuclear localization sequence in fibroblast growth factor-1 stimulated mitogenic pathways. AB - Fibroblast growth factor-1 (FGF-1) is a potent mitogen for mesoderm- and neuroectoderm-derived cell types in vitro. However, a mutant FGF-1 with deletion in its nuclear localization sequence (NLS, residues 21-27) is not mitogenic in vitro. We demonstrated that synthetic peptides containing this NLS were able to stimulate DNA synthesis in a FGF receptor-independent manner after they were delivered into living NIH 3T3 cells by a cell-permeable peptide import technique. The stimulation of maximal DNA synthesis by these peptides required the presence of peptides during the entire G1 phase of the cell cycle. The mitogenic effect was specific for the NLS of FGF-1 because a peptide with double point mutations at lysine residues was inactive in stimulating DNA synthesis. Our results suggest that the NLS plays an important role in the mitogenic pathway initiated by exogenous FGF-1 by its direct involvement in the nuclear transport and signaling of internalized FGF-1. PMID- 8621380 TI - Oncogenic RET receptors display different autophosphorylation sites and substrate binding specificities. AB - The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities. PMID- 8621381 TI - The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein. AB - The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm. It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin beta (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin beta binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein). Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin beta was dependent on karyopherin beta concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin beta concentrations that were equimolar to Ran GTP. In gel filtration experiments, we found Ran-GTP and karyopherin beta to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin beta. We propose that stoichiometric complex formation between karyopherin beta and Ran GTP renders Ran-GTP inaccessible to RanGAP. PMID- 8621382 TI - A novel 55-kDa regulatory subunit for phosphatidylinositol 3-kinase structurally similar to p55PIK Is generated by alternative splicing of the p85alpha gene. AB - Phosphatidylinositol 3-kinase, which is composed of a 110-kDa catalytic subunit and a regulatory subunit, plays important roles in various cellular signaling mechanisms. We screened a rat brain cDNA expression library with 32P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85alpha gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55alpha) containing two SH2 domains and an inter-SH2 domain of p85alpha but neither a bcr domain nor a SH3 homology domain. Interestingly, p55 alpha contains a unique 34-amino acid sequence at its NH2 terminus, which is not included in the p85alpha amino acid sequence. This 34 amino acid portion was revealed to be comparable with p55PIK (p55gamma) in length, with a high homology between the two, suggesting that these NH2-terminal domains of p55alpha and p5 gamma may have a specific role that p85 does not. The expression of p55alpha mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85alpha mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85alpha SH3 domain, while the expression of p55alpha can be readily detected. These results suggest that p55 alpha may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle. PMID- 8621384 TI - Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C delta. AB - The expression of an oncogenic rasHa gene in epidermal keratinocytes stimulates the tyrosine phosphorylation of protein kinase C delta and inhibits its enzymatic activity (Denning, M. F., Dlugosz, A. A., Howett, M. K., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Keratinocytes expressing an activated rasHa gene secrete transforming growth factor alpha (TGFalpha) and have an altered response to differentiation signals involving protein kinase C (PKC). Because the neoplastic phenotype of v-rasHa expressing keratinocytes can be partially mimicked in vitro by chronic treatment with TGF alpha and the G protein activator aluminum fluoride (AlF4-), we determined if TGF alpha or AlF4- could induce tyrosine phosphorylation of PKCdelta. Treatment of primary keratinocyte cultures for 4 days with TGFalpha induced tyrosine phosphorylation of PKCdelta, whereas AlF4- only slightly stimulated PKCdelta tyrosine phosphorylation. The PKCdelta that was tyrosine-phosphorylated in response to TGFalpha had reduced activity compared with the nontyrosine-phosphorylated PKCdelta. Treatment of keratinocytes expressing a normal epidermal growth factor receptor (EGFR) with TGFalpha or epidermal growth factor for 5 min induced PKCdelta tyrosine phosphorylation. This acute epidermal growth factor treatment did not induce tyrosine phosphorylation of PKCdelta in keratinocytes isolated from waved-2 mice that have a defective epidermal growth factor receptor. In addition, the level of PKCdelta tyrosine phosphorylation in v-rasHa-transduced keratinocytes from EGFR null mice was substantially lower than in v-rasHa transduced wild type cells, suggesting that activation of the EGFR is important for PKC delta tyrosine phosphorylation in ras transformation. However, purified EGFR did not phosphorylate recombinant PKC delta in vitro, whereas members of the Src family (c-Src, c-Fyn) and membrane preparations from keratinocytes did. Furthermore, clearing c-Src or c-Fyn from keratinocyte membrane lysates decreased PKCdelta tyrosine phosphorylation, and c Src and c-Fyn isolated from keratinocytes treated with TGFalpha had increased kinase activity. Acute or chronic treatment with TGFalpha did not induce significant PKCdelta translocation in contrast to the phorbol ester 12-O tetradecanoylphorbol-13-acetate, which induced both translocation and tyrosine phosphorylation of PKCdelta. This suggests that TGFalpha-induced tyrosine phosphorylation of PKC delta results from the activation of a tyrosine kinase rather than physical association of PKCdelta with a membrane-anchored tyrosine kinase. Taken together, these results indicate that PKCdelta activity is inhibited by tyrosine phosphorylation in response to EGFR-mediated signaling and activation of a member of the Src kinase family may be the proximal tyrosine kinase acting on PKCdelta in keratinocytes. PMID- 8621385 TI - Temperature and pH dependence of fluorescein binding within the monoclonal antibody 9-40 active site as monitored by hydrostatic pressure. AB - In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40. PMID- 8621383 TI - Transcriptional glucose signaling through the glucose response element is mediated by the pentose phosphate pathway. AB - Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate. PMID- 8621387 TI - Enzyme inhibition in open systems. Superiority of uncompetitive agents. AB - Investigations of the open system behavior of reversible dead-end inhibitors were carried out by means of computer simulations and experimental studies. The results from both approaches indicate that substrate-competitive inhibition may often be an inappropriate basis for design of potential therapeutic agents. The use of uncompetitive (also called anticompetitive) inhibitors in this role is likely to be far more effective. Chemical analogs of pathogen-specific enzymic reaction products rather than analogs of substrates provide a promising basis for the systematic design of such uncompetitive inhibitors. PMID- 8621386 TI - Comparative properties of the single chain antibody and Fv derivatives of mAb 4-4 20. Relationship between interdomain interactions and the high affinity for fluorescein ligand. AB - Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2 3-fold), in Escherichia coli. Both variable heavy (VH) and variable light (VL) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20. Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein. In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations. The equilibrium dissociation constant (Kd) for the VL/VH association (1.43 x 10(-7) M), which was determined using the change in fluorescein spectral properties upon ligand binding, was relatively low considering the high affinity displayed by the Fv protein for fluorescein (Kd, 2.9 x 10(-10) M). Thus, domain-domain stability in the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced affinity (2-3-fold) for fluorescein as compared with the Fab or IgG form of 4-4-20. With their identical ligand binding and structural properties, the decreased SCA or Fv affinity for fluorescein must be an ultimate consequence of deletion of the CH1 and CL constant domains. Collectively, these results verify the importance of constant domain interactions in antibody variable domain structure-function analyses and future antibody engineering endeavors. PMID- 8621388 TI - Differential structural requirements for interaction of Ras protein with its distinct downstream effectors. AB - Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors. PMID- 8621390 TI - Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. AB - To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP 1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter. PMID- 8621389 TI - Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase. AB - Mitogen-activated protein/ERK kinase kinases (MEKKs) phosphorylate and activate protein kinases which in turn phosphorylate and activate the p42/44 mitogen activated protein kinase (MAPK), c-Jun/stress-activated protein kinases (JNKs), and p38/Hog1 kinase. We have isolated the cDNAs for two novel mammalian MEKKs (MEKK 2 and 3). MEKK 2 and 3 encode proteins of 69.7 and 71 kDa, respectively. The kinase domains encoded in the COOH-terminal moiety are 94% conserved; the NH2 terminal moieties are approximately 65% homologous, suggesting this region may encode sequences conferring differential regulation of the two kinases. Expression of MEKK 2 or 3 in HEK293 cells results in activation of p42/44MAPK and JNK but not of p38/Hog1 kinase. Immunoprecipitated MEKK 2 phosphorylated the MAP kinase kinases, MEK 1, and JNK kinase. Titration of MEKK 2 and 3 expression in transfection assays indicated that MEKK 2 preferentially activated JNK while MEKK 3 preferentially activated p42/44MAPK. These findings define a family of MEKK proteins capable of regulating sequential protein kinase pathways involving MAPK members. PMID- 8621391 TI - Sodium-calcium exchange and store-dependent calcium influx in transfected chinese hamster ovary cells expressing the bovine cardiac sodium-calcium exchanger. Acceleration of exchange activity in thapsigargin-treated cells. AB - The effects of extracellular Na+ on store-dependent Ca2+ influx were compared for transfected Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (CK1.4 cells) and vector-transfected control cells. Store-dependent Ca2+ influx was elicited by depletion of intracellular Ca2+ stores with ionomycin, thapsigargin, or extracellular ATP, a purinergic agonist. In each case, the rise in [Ca2+]i upon the addition of extracellular Ca2+ was reduced in CK1.4 cells compared with control cells at physiological [Na+]o. When Li+ or NMDG was substituted for Na+, the CK1.4 cells showed a greater rise in [Ca2+]i than control cells over the subsequent 3 min after the addition of Ca2+o. Under Na+ free conditions, SK&F 96365 (50 microM), a blocker of store-operated Ca2+ channels, nearly abolished the thapsigargin-induced rise in [Ca2+]i in the control cells but only partially inhibited this response in the CK1.4 cells. We conclude that in the CK1.4 cells, Ca2+ entry through store-operated channels was counteracted by Na+o-dependent Ca2+ efflux at physiological [Na+]o, whereas Ca2+ entry was enhanced through Na+i-dependent Ca2+ influx in the Na+-free medium. We examined the effects of thapsigargin on Ba2+ entry in the CK1.4 cells because Ba2+ is transported by the Na+-Ca2+ exchanger, but it enters these cells only poorly through store-operated channels, and it is not sequestered by intracellular organelles. Thapsigargin treatment stimulated Ba2+ influx in a Na+ free medium, consistent with an acceleration of Ba2+ entry through the Na+-Ca2+ exchanger. We conclude that organellar Ca2+ release induces a regulatory activation of Na+-Ca2+ exchange activity. PMID- 8621392 TI - Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. AB - Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr992 (EGFR988 998). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr992 in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the alpha2 region of PTP1, most likely Ile23, was labeled by the Tyr992-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis. PMID- 8621393 TI - HIV-1 protein expression from synthetic circles of DNA mimicking the extrachromosomal forms of viral DNA. AB - We have constructed circular forms of human immunodeficiency virus type 1 viral DNA in vitro that closely resemble the single and double long terminal repeat circular forms of unintegrated viral DNA formed in the nuclei of infected cells. We have analyzed viral protein expression after transient transfection of these circular DNAs into HeLa cells and compared it with expression from a transfected linearized plasmid containing an integrated provirus. Both circular forms are expressed, as judged by the appearance of extracellular p24, and expression is trans-activated by human immunodeficiency virus type 1 Tat. Viral p24 production, however, is approximately an order of magnitude lower than that obtained with transfected integrated viral DNA. Similar data were obtained when a luciferase reporter gene was substituted for the coding regions of the viral DNA. Positional effects of the transcriptional initiation and termination signals in the long terminal repeat appear to account for some of the low expression levels. These data suggest that unintegrated circular viral DNAs are transcriptionally active, although at low levels, and may contribute to overall viral replication in infected people under some conditions. PMID- 8621394 TI - Functional and genetic characterization of the (methyl)ammonium uptake carrier of Corynebacterium glutamicum. AB - Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 +/- 5 nmol.min-1.(mg dry weight)-1. The specific activity of this uptake was 10-fold lower when growing the cells under sufficient nitrogen supply, indicating a tight regulation on the expression level. The methylammonium uptake showed Michaelis-Menten kinetics with an Km of 44 +/- 7 microM and was completely inhibited by the addition of 10 microM ammonium. This finding and the fact that methylammonium was not metabolized by C. glutamicum strongly suggests that the uptake carrier actually represents an ammonium uptake system. Methylammonium uptake was strictly dependent on the membrane potential. From the pH optimum and the accumulation of methylammonium in equilibrium, it could be deduced that only one net charge is transported and, thus, that methylammonium is taken up in its protonated form via an uniport mechanism. The amt gene encoding the (methyl)ammonium uptake system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (Mr = 47,699) and shows 26-33% identity to ammonium transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile, it is an integral membrane protein containing 10 or 11 membrane-spanning segments. PMID- 8621396 TI - Intracellular assembly of inducible NO synthase is limited by nitric oxide mediated changes in heme insertion and availability. AB - Cytokines induce the mouse macrophage cell line RAW 264.7 to express cytokine inducible nitric oxide synthase (iNOS), which is active only in dimeric form. Because dimerization of purified iNOS subunits requires tetrahydrobiopterin, heme, and L-arginine, we investigated if availability of these factors also influences intracellular assembly of dimeric iNOS. Following exposure to cytokines, iNOS protein was found to accumulate in a near linear manner over 16 h of further culture. In contrast, dimeric iNOS accumulated at a slower rate that continuously decreased during culture, resulting in only 25% of the accumulated iNOS protein being in dimeric form by 16 h. Further experiments argued against dimer instability or L-arginine and tetrahydrobiopterin availability as factors limiting iNOS dimer accumulation. Blocking cellular NO synthesis with Nomega nitro-L-arginine methyl ester (L-NAME) greatly increased iNOS dimer assembly, indicating NO synthesis limited iNOS dimerization. NO synthesis was found to prevent an increase in soluble heme level that was associated with iNOS induction in Nomega-nitro-L-arginine methyl ester-treated cells and also diminished heme insertion into iNOS. These NO-related defects were not reversed by adding heme precursors or hemin to the activated cell cultures. Measurement of iron release from activated cells demonstrated that endogenous NO synthesis substantially increased the release of 59Fe to the medium. These observations suggest that iNOS dimerization is limited to a large extent by iNOS NO synthesis. NO appears to limit intracellular assembly of dimeric iNOS by preventing heme insertion and decreasing heme availability. PMID- 8621395 TI - A schiff base with mildly oxidized carbohydrate ligands stabilizes L-selectin and not P-selectin or E-selectin rolling adhesions in shear flow. AB - Selectins are a family of lectins, that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. Mild periodate oxidation of the L-selectin ligand CD34, or L-selectin ligands on leukocytes, enhanced resistance to detachment in shear and decreased rolling velocity equivalent to an 8-fold increase in ligand density, yet had little effect on the rate of tethering. Enhanced interactions were also seen with mildly oxidized sialyl Lewisa and sialyl Lewisx glycolipids. Enhancement was completely reversed by borohydride reduction, yielding a strength of interaction equivalent to that with the native ligands. No effect on the strength of P-selectin and E-selectin interactions was seen after mild oxidation of their ligands. Completeness of modification of sialic acid by mild periodate was verified with monoclonal antibody to sialyl Lewisx-related structures and resistance to neuraminidase. The addition of cyanoborohydride to leukocytes rolling through L-selectin on mildly oxidized but not native CD34 caused arrest of rolling cells and formation of EDTA-resistant bonds to the substrate, suggesting that a Schiff base was reduced. Cyanoborohydride reduction of mildly oxidized cells rolling on P-selectin and E selectin also caused arrest and formation of EDTA-resistant bonds but with slower kinetics. These data suggest that interactions with a sialic acid aldehyde group on mildly oxidized ligands that include interconversion to a Schiff base can occur with three selectins yet only stabilize binding through the selectin with the fastest koff, L-selectin. PMID- 8621397 TI - Adriamycin-induced DNA adducts inhibit the DNA interactions of transcription factors and RNA polymerase. AB - Adriamycin is known to specifically induce DNA interstrand cross-links at 5'-GC sequences. Because 5'-GC sequences are a predominant feature of 5'-untranslated regions (transcription factor-binding sites, promoter, and enhancer regions), it is likely that adriamycin adducts at GC sites would affect the binding of DNA interacting proteins. Two model systems were chosen for the analysis: the octamer binding proteins Oct-1, N-Oct-3 and N-Oct-5, which bind to ATGCAAAT and TAATGARAT recognition sites, and Escherichia coli RNA polymerase binding to the lac UV5 promoter. Electrophoretic mobility shift studies showed that adriamycin adducts at GC sites inhibited the binding of octamer proteins to their consensus motifs at drug levels as low as 1 micoM, but no effect was observed with a control sequence lacking a GC site. Adriamycin adducts at GC sites also inhibited the binding of RNA polymerase to the lac UV5 promoter. Adriamycin may therefore function by down-regulating the expression of specific genes by means of inactivation of short but critical motifs containing one or more GC sites. PMID- 8621398 TI - Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1. AB - The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+ dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first. PMID- 8621399 TI - Porcine 80-kDa protein reveals intrinsic 17 beta-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/dehydrogenase, and sterol transfer activities. AB - Four types of 17beta-hydroxysteroid dehydrogenases have been identified so far. The porcine peroxisomal 17beta-hydroxysteroid dehydrogenase type IV catalyzes the oxidation of estradiol with high preference over the reduction of estrone. A 2.9 kilobase mRNA codes for an 80-kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-hydroxysteroid dehydrogenases. The 80 kDa protein is N terminally cleaved to a 32-kDa fragment with 17beta hydroxysteroid dehydrogenase activity. Here we show for the first time that both the 80-kDa and the N-terminal 32 kDa (amino acids 1-323) peptides are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with 3-hydroxyacyl-CoA. The central part of the 80-kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80-kDa protein (amino acids 597-737) is similar to sterol carrier protein 2 and facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The unique multidomain structure of the 80-kDa protein allows for the catalysis of several reactions so far thought to be performed by complexes of different enzymes. PMID- 8621400 TI - Demonstration of cyclin-dependent kinase inhibitory serine/threonine kinase in bovine thymus. AB - A synthetic peptide corresponding in sequence to residues 6-20 of p34cdc2, cdc2(6 20), and a substitution analogue, cdc2(6-20)F15K19 , which contains Thr-14 as the only phosphorylation target were used as substrates to identify a novel protein kinase in bovine thymus cytosol. The kinase catalyzed the phosphorylation of Thr 14 in both peptides and was purified extensively on the basis of its peptide phosphorylation activity. Upon SDS-polyacrylamide gel electrophoresis analyses, the purified samples consistently displayed a prominent 43-kDa protein band which could undergo in gel autophosphorylation, thus suggesting that this band represented the kinase protein. The suggestion was supported further by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhibited by millimolar concentrations of cAMP. The kinase was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide analysis of the phosphorylated Nclk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this thymus kinase is a novel Cdk inhibitory protein kinase, distinct from the recently cloned dual functional and membrane-associated Cdc2 inhibitory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G. (1995) Science 270, 86-90). PMID- 8621401 TI - Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum. AB - A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45 enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD+ ratios associated with anaerobiosis. PMID- 8621402 TI - Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration. AB - The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I) (Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J. M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C., Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G. (1994) Biochemistry 33, 9405-9413), designed to better resist autolysis. Monitoring the intrinsic fluorescence of the two enzymes during urea-mediated denaturation has shown that at high NaCl concentration, both the conformational stability ( DeltaG0) and the transition midpoint (D1/2) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far- and near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a two-state equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity. PMID- 8621403 TI - The interleukin-6 (IL-6) partial antagonist (Q159E,T162P)IL-6 interacts with the IL-6 receptor and gp130 but fails to induce a stable hexameric receptor complex. AB - The extracellular "soluble" domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130) form a hexameric ternary receptor complex together with IL-6, consisting of two molecules of each component. In this report we have investigated the interactions of the partial IL-6 antagonist (Q159E,T162P)IL-6 ((QT)IL-6), with the sIL-6R and sgp130. The kinetic rate constants of the binding of sIL-6R to immobilized monomeric (QT)IL-6 or IL-6 were obtained using an optical biosensor with analysis of the primary data by linear and nonlinear regression. Both methods of analysis showed that, due to a higher off-rate, sIL-6R has lower apparent affinity for (QT)IL-6 than IL-6. The lower affinity of (QT)IL-6 was further confirmed by equilibrium binding measurements at the sensor surface and in solution. Using the biosensor it was also shown that the (QT)IL-6 complex interacts with sgp130, supporting the notion that the biological activity of (QT)IL-6 is mediated via gp130. However, the IL-6 mutant, when incubated with sIL 6R and sgp130, failed to induce a stable hexameric receptor complex, as shown by narrowbore size exclusion chromatography. PMID- 8621404 TI - Differential regulation of vitronectin in mice and humans in vitro. AB - To define the cis-acting elements involved in the regulation of the murine vitronectin (Vn) gene in inflammation, the 5'-flanking region was isolated, fused to the luciferase reporter gene, and the basal and interleukin 6 (IL-6) stimulated transcriptional activity was tested in transfection experiments using Hep3B cells. Treatment with IL-6 induced this construct by more than 20-fold, whereas the corresponding 5'-flanking region of the human Vn gene was not stimulated. Transfection studies using murine Vn constructs with serial 5' deletions revealed that two sequences were important in the IL-6 response, and specific mutations in both sequences abolished the response. A 2-base pair mutation converted the human sequence to that of a murine IL-6 responsive element and partially conveyed IL-6 inducibility. In contrast, transforming growth factor beta stimulated the human construct and the endogenous Vn gene in human Hep3B cells in a dose-dependent manner, whereas the murine construct was not responsive. The transforming growth factor beta responsive region was localized to a 30-base pair fragment with little homology to the murine sequence. These studies reveal that the structural basis for the differential regulation of the human and murine Vn genes resides in the differences in promoter sequence. PMID- 8621405 TI - Reduction in water activity greatly retards the phosphoryl transfer from ATP to enzyme protein in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase. AB - Cys-674 of the sarcoplasmic reticulum Ca2+-ATPase was labeled with N-acetyl-N'-(5 sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The ATP-induced drop in the fluorescence of the label, which was shown in our previous studies to reflect the conformational change upon formation of the calcium.enzyme.ATP complex, was followed by the stopped-flow method. The subsequent phosphoenzyme formation was followed by the rapid quenching method. Effects of a partial substitution of organic solvents for water in the medium on the conformational change and phosphoenzyme formation were investigated in the presence of 100 microM CaCl2 at pH 7.5, 0 degrees C. The rate of the conformational change increased with increasing ATP concentration (0.1 100 microM) and was unaffected by 30% (v/v) dimethyl sulfoxide. In contrast, the rate of phosphoenzyme formation decreased sharply with increasing concentration of dimethyl sulfoxide (20-40% (v/v)), even when phosphoenzyme formation was saturated with ATP. N,N-Dimethylformamide and glycerol had essentially the same effects as dimethyl sulfoxide. These results show that the reduction in water activity does not affect the rate of the conformational change upon formation of the calcium.enzyme.ATP complex, but greatly retards the subsequent phosphoryl transfer from ATP to the enzyme protein. This strongly suggests that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme. PMID- 8621406 TI - A di-leucine motif and an upstream serine in the interleukin-6 (IL-6) signal transducer gp130 mediate ligand-induced endocytosis and down-regulation of the IL 6 receptor. AB - The interleukin-6 (IL-6) receptor complex is composed of two different subunits, the IL-6 binding protein (IL-6R, gp80) and the signal transducing component gp130. Our previous studies revealed that the 10-amino acid sequence TQPLLDSEER within the intracellular domain of gp130 is crucial for the efficient internalization of IL-6. Since this sequence contains a putative di-leucine internalization motif, we further analyzed this region by constructing two additional deletions and a series of point mutants. Analyses of these mutants showed that the di-leucine pair (Leu-145 and Leu-146) is essential for ligand internalization, with leucine 145 being less resilient to exchanges. Furthermore, when a chimeric protein (Tac-STQPLL) composed of the Tac antigen fused to the hexapeptide STQPLL of gp130 was studied, we found that this sequence is sufficient to mediate endocytosis and lysosomal targeting of the chimera. Mutational analysis of three serine residues upstream of the di-leucine motif revealed that mutation of serine 139 to an alanine reduces the initial internalization rate by 50%. This finding suggests that a serine phosphorylation may be important for rapid endocytosis. PMID- 8621407 TI - Molecular basis of the soluble and membrane-bound forms of the murine leukemia inhibitory factor receptor alpha-chain. Expression in normal, gestating, and leukemia inhibitory factor nullizygous mice. AB - The murine leukemia inhibitory factor receptor alpha-chain (mLIFR) exists in a membrane-bound and a soluble form. The two major classes of mRNA transcript correspond to either the soluble or membrane-bound form of the mLIFR. In this study we have identified a complex and heterogeneous pattern of expression of mRNA transcripts for this receptor in normal mouse tissues and cell lines. In order to understand the molecular basis of these transcripts, genomic clones encompassing the region of divergence from the soluble to the membrane-bound form of the receptor were isolated. cDNAs encoding the membrane-bound form of the mLIFR were generated by an alternative splicing event where an exon that is specific to the soluble mLIFR was skipped. The membrane-bound form of the mLIFR was heterogeneously polyadenylated with at least five different sites of polyadenylation. The mRNA transcript encoding the soluble form of the mLIFR contained a region highly homologous to a murine B2 repetitive element, thus providing a possible explanation for the genesis of this transcript. The different forms of the mLIFR were analyzed in a wide range of mouse tissues in pseudopregnant mice and in mice at various stages of pregnancy. Only liver, placenta, and uterus showed an increase in the levels of mLIFR mRNA expression during pregnancy, indicating an important role for the LIFR in this process. However, somewhat surprisingly, there was no detectable difference in mLIFR mRNA levels or levels of soluble protein in leukemia inhibitory factor nullizygous mice when compared with normal mice. PMID- 8621408 TI - Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide. AB - The sensitivity of mu and delta receptor binding to dithiothreitol and N ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor. PMID- 8621409 TI - Functional analysis of Burkitt's lymphoma mutant c-Myc proteins. AB - The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt's lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived from Burkitt's lymphoma cell lines are resistant to inhibition by p107, thus providing a rationale for the increased oncogenic activity of these mutant c-Myc proteins. It has been suggested that these mutant c-Myc proteins resist down-modulation by p107 because they lack cyclin A-cdk2-dependent phosphorylation. Here, we have examined three different Burkitt's lymphoma mutant c-Myc proteins found in primary Burkitt's lymphomas and one mutant c-Myc protein detected in a Burkitt's lymphoma cell line. All four have an unaltered ability to activate transcription and are sensitive to inhibition of transactivation by p107. Furthermore, we provide evidence that down modulation of c-Myc transactivation by p107 does not require phosphorylation of the c-Myc transactivation domain by cyclin A-cdk2. Our data indicate that escape from p107-induced suppression is not a general consequence of all Burkitt's lymphoma-associated c-Myc mutations, suggesting that other mechanisms exist to deregulate c-Myc function. PMID- 8621410 TI - Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells. AB - VEGF121 and VEGF165 are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF165 contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF165 a heparin binding capability which VEGF121 lacks. 125I VEGF165 bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while 125I-VEGF121 bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of 125I-VEGF121 to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF165 and VEGF121 inhibited the binding of 125I VEGF121 to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF165 by 2-3-fold. These results contrast with previous observations which have indicated that the binding of 125I-VEGF165 to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF165 is susceptible to oxidative damage caused by oxidants such as H2O2 or chloramine-T. VEGF121 was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF165 but not the receptor binding ability of damaged VEGF121. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF165 may enable the restoration of damaged VEGF165 function in processes such as inflammation or wound healing. PMID- 8621411 TI - Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum. A dioxygenase with catalase activity. AB - Homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) cleaves the aromatic ring of its substrate with insertion of both atoms of oxygen from O2 to form alpha-hydroxy- delta-carboxymethyl cis-muconic semialdehyde. The enzyme has been purified from the Gram-positive bacterium Brevibacterium fuscum and characterized. The enzyme appears to have a range of quaternary structures with predominant components of alpha4 and alpha6 (alpha subunit Mr = 42500 +/- 1500) and binds approximately 1 Fe(II)/subunit. Although the substrate Km values are similar to those of other Fe(II) ring cleaving dioxygenases, the turnover number is lower by 90-97%, and the enzyme exhibits much higher stability to metal chelators and H2O2. The stability to H2O2 is shown to derive from an endogenous catalase activity of 2,3 HPCD (stoichiometry: 2 H2O2 --> 2 H2O + O2) that is novel for dioxygenases. H2O2 is a mixed-type inhibitor of the dioxygenase activity, suggesting that dioxygenase and catalase activities are both catalyzed by the enzyme, but at distinguishable sites. In contrast, catecholic substrates, including homoprotocatechuate and p-nitrocatechol, are nonessential activators of the catalase activity. The plot of 1/vi of catalase activity versus 1/[H2O2] is parabolic in the absence of catecholic substrates and linear in their presence, indicating that these reactions proceed by different mechanisms. A mechanism for catalase activity is proposed in which 2 H2O2 molecules bind simultaneously to the iron to account for the observed parabolic kinetic plot. Electron transfer between the peroxides mediated by the iron would yield 2 H2O and O2. Catecholic substrates are proposed to modify this reaction by excluding one H2O2 from the Fe(II), thereby causing the kinetic plots to appear linear. Electron donation by the catecholic substrates would facilitate O O bond cleavage of H2O2, but outer sphere electron transfer from a second H2O2 in another step would be necessary to complete the reaction. p-Nitrocatechol is shown to bind differently to 2,3-HPCD than to other Fe(II) ring cleavage dioxygenases. Possible explanations for this observation are considered in the context of the proposed catalase and normal dioxygenase mechanisms which may also have bearing on the unique catalase activity and low dioxygenase turnover number of the enzyme. PMID- 8621412 TI - Consequences of functional expression of the plasma membrane Ca2+ pump isoform 1a. AB - The plasma membrane Ca2+-ATPase pump (PMCA) is an integral component of the Ca2+ signaling system which participates in signal transduction during agonist stimulated cell activation. To better understand the physiological function of the pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter. The cell lines selected after transfection with PMCA1a construct, expressed 3-4-fold increased pump protein which was mostly targeted to the plasma membrane as indicated by immunoperoxidase staining. Ca2+ uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca2+ pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca2+ and calmodulin for stimulation of activity. Measurement of [Ca2+]i and [Ca2+]out showed that expression of PMCA1a had a profound effect on different aspects of the Ca2+ signal. The peak increase in [Ca2+]i evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells. Accordingly, titration with ionomycin of Ca2+ content of internal stores, measurement of Ca2+ uptake into the thapsigargin- and oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes, Ca2+ uptake into streptolysin O permeabilized cells, and analysis of SERCA mRNA and protein, showed that expression and activity of the SERCA pump was down-regulated in cells expressing PMCA1a pump. Expression of PMCA1a also down-regulated expression of the inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ channel and the rate of IP3-mediated Ca2+ release in permeable cells, without affecting the affinity of the channel for IP3. On the other hand the rate of store depletion-dependent Ca2+ and Mn2+ influx (Ca2+ entry) into PMCA1a expressing cells was increased by about 2.6-fold. These changes prevented estimating the rate of pump-mediated Ca2+ efflux from changes in [Ca2+]i. Measurement of [Ca2+]out showed that the rate of Ca2+ efflux in cells expressing PMCA1a was about 1.45-fold higher than Neo controls, despite the 4-fold increase in the amount of functional pump protein. The overall study points to the flexibility, interdependence, and adaptability of the different components of the Ca2+ signaling systems to regulate the expression and activity of each component and maintain a nearly constant Ca2+ signal. PMID- 8621413 TI - Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol 1,4,5-trisphosphate receptor and promote its phosphorylation. AB - Prostaglandin I2 (PGI2) and sodium nitroprusside (SNP) induce a rapid decay of the thrombin-promoted increase of [Ca2+]i in aspirin-treated platelets incubated in the absence of external Ca2+. The mechanism of their effect was studied with a new method which utilizes ionomycin to increase [Ca2+]i, followed by bovine serum albumin (BSA) to remove the Ca2+ ionophore. The rapid decay of [Ca2+]i after BSA is mostly due to the reuptake into the stores, since it is strongly inhibited by the endomembrane Ca2+-ATPase inhibitor thapsigargin. PGI2 and SNP are without effect on the BSA-promoted decay both with and without thapsigargin, showing that they do not affect the activity of the Ca2+-ATPases. The fast decay of [Ca2+]i after BSA is decreased by thrombin which produces the Ca2+ releaser inositol 1,4,5-trisphosphate (InsP3), thus counteracting the activity of the endomembrane Ca2+ pump. When added after thrombin, PGI2 and SNP accelerate the BSA-activated decay of [Ca2+]i. However, under the same conditions, they do not decrease the concentration of InsP3. In saponin-permeabilized platelets, cAMP and cGMP counteract the Ca2+ release induced by exogenous InsP3. Their inhibitory effect disappears at high InsP3 concentrations. This demonstrates that PGI2 and SNP potentiate Ca2+ reuptake by inhibiting the InsP3 receptor. Two bands of approximately 260 kDa are recognized by a monoclonal antibody recognizing the C terminal region of the InsP3 receptor. Both are phosphorylated rapidly, the heavier more intensely, in the presence of PGI2 and SNP. The phosphorylation of the InsP3 receptor is fast enough to be compatible with its involvement in the inhibition of the receptor by cyclic nucleotides. PMID- 8621415 TI - Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus. AB - Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe 188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors. PMID- 8621414 TI - Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles. AB - Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP 2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma 32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments. PMID- 8621416 TI - Probing the indirect readout of the restriction enzyme EcoRV. Mutational analysis of contacts to the DNA backbone. AB - According to the crystal structure of the specific EcoRV.DNA complex, not only the functional groups of the nucleobases but also the phosphate groups of the DNA backbone are contacted by the enzyme. To examine the contribution of backbone contacts to substrate recognition and catalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resulting EcoRV mutants and characterized them with respect to their DNA binding and cleavage activity. According to our steady state kinetic analysis, there are strong interactions between three basic amino acid residues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV.DNA complex. These contacts are important in both the ground state and the transition state. Other, uncharged residues (Thr-93 and Ser-112), which could be involved in hydrogen bonds to the phosphate groups, are needed primarily to stabilize the transition state. An especially important amino acid residue is Thr-37, which seems to couple recognition to catalysis by indirect readout. PMID- 8621417 TI - cGMP accumulation induced by hypertonic stress in Dictyostelium discoideum. AB - The change in extracellular osmolarity from 0.07 osm to 0.38 osm caused rapid cell shrinkage and loss of pseudopodes in Dictyostelium discoideum amoebae and induced elevation of total (cellular + extracellular) cGMP with a 2.5-min lag. cGMP accumulation reached a peak at 10-15 min after the change, and then the total cGMP gradually decreased. cGMP first accumulated intracellularly and was then secreted. A roughly identical osmotic concentration was required for the accumulation when the effect of KCl and glucose was tested. The non-osmolytes, formamide and ethanol, did not induce the accumulation. We concluded that hypertonic stress induces cGMP accumulation in D. discoideum amoebae. The hypertonic stress-induced accumulation of cGMP was observed in a streamer F mutant (NP368) that lacks cGMP-specific phosphodiesterase. While Dictyostelium cells also have nonspecific phosphodiesterases that degrade both cGMP and cAMP, hypertonic stress induced only a small increase in cAMP in wild type and streamer F cells. These results suggest that hypertonic stress-induced accumulation of cGMP is due to the activation of guanylate cyclase rather than the inhibition of phosphodiesterases. Binding of folic acid to the specific receptors on the cell surface induces a rapid transient accumulation of cGMP that reaches a peak at 10 s. When cells were stimulated by folic acid after the addition of 0.31 M glucose, rapid transient cGMP accumulation was observed immediately after the stimulation by folic acid and prolonged cGMP accumulation was induced 2-3 min after the addition of glucose irrespective of the timing of folic acid stimulation. These results suggest that the hypertonic stress-induced and the receptor-mediated accumulation proceed independently of one another. 2,3-Dimercapto-1-propanol, a thiol-reducing reagent, induces prolonged cGMP accumulation similar to hypertonic stress. However, the hypertonic stress-induced cGMP accumulation was enhanced by EDTA and was not suppressed by folic acid and cAMP. These characteristics are distinct from the reducing reagent-induced accumulation that is suppressed by EDTA, folic acid, and cAMP. These findings show that hypertonic stress has a unique effect on the activation of guanylate cyclase. PMID- 8621418 TI - Intermediates in the catalytic cycle of copper-quinoprotein amine oxidase from Escherichia coli. AB - Investigations on the reduction of copper quinoprotein amine oxidases (EC 1.4.3.6) by substrate indicate that the nature of the reduced enzyme species formed varies, as judged from the spectroscopic data reported in the literature for different enzymes and substrates. The availability of substantial amounts of overproduced, homogeneous Escherichia coli amine oxidase (ECAO) enabled us to investigate this aspect with a number of different approaches: quantitative titration of enzyme with substrate, stopped-flow kinetic spectrophotometry (anaerobic and semianaerobic), EPR spectroscopy of stable intermediates in the catalytic cycle, and conversions with H2O2 as the oxidant. Reduction of ECAO by a variety of substrates led to spectra (UV/Vis, EPR) identical to those that have been ascribed to the semiquinone form of the topaquinone cofactor. The extent of semiquinone formation was enhanced in the presence of KCN, but the properties of the artificially induced semiquinone were different from those of the spontaneously induced one, as shown by the spectroscopic data and the reactivity toward O2 and H2O2. On titrating ECAO at high concentrations with substrate, evidence was obtained that disproportionation takes place of the semiquinone formed, the reaction most probably proceeding via intermolecular electron transfer, leading to a topaquinone- and Cu1+-containing enzyme species that is able to perform substrate conversion. The latter, as well as OH*, is probably also formed when H2O2 replaces O2 as oxidant, explaining why substrate conversion with concomitant enzyme inactivation occurs under this condition. Formation of the semiquinone was always preceded by that of a hitherto unknown species with an absorbance maximum at 400 nm. The structure proposed for this species is a protonated form of the aminoquinol cofactor, the Zwitter ionic structure being stabilized by amino acid residues in the active site having opposite charges. Based on the properties observed and the moment of appearance during conversions, a proposal is made for the sequence in which the three reduced enzyme species convert into each other. PMID- 8621419 TI - Site-specific dephosphorylation of tau protein at Ser202/Thr205 in response to microtubule depolymerization in cultured human neurons involves protein phosphatase 2A. AB - Tau proteins isolated from paired helical filaments, the major building blocks of Alzheimer's disease neurofibrillary tangle, are abnormally phosphorylated and unable to bind microtubules. To examine the dynamics of tau phosphorylation and to identify specific tau phosphorylation sites involved in the stabilization of microtubules, we treated cultured postmitotic neuron-like cells (NT2N) derived from a human teratocarcinoma cell line (NTera2/D1) with drugs that depolymerize microtubules (i.e. colchicine or nocodazole). This led to the recovery of dephosphorylated tau from the NT2N cells as monitored by a relative increase in the electrophoretic mobility of tau and an increase in the turnover of [32P]PO4 labeled tau. However, not all phosphorylation sites on tau are affected by colchicine or nocodazole. Ser202/Thr205 appears to be completely and specifically dephosphorylated by protein phosphatase 2A since this dephosphorylation was blocked by inhibitors of protein phosphatase 2A but not by inhibitors of protein phosphatase 2B. These findings, together with the recent observation that protein phosphatase 2A is normally bound to microtubules in intact cells, suggest that the polymerization state of microtubules could modulate the phosphorylation state of tau at specific sites in the normal and Alzheimer's disease brain. PMID- 8621420 TI - CCAAT box enhancer binding protein alpha (C/EBP-alpha) stimulates kappaB element mediated transcription in transfected cells. AB - A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS 4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65 stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites. PMID- 8621421 TI - Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. AB - Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr 895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr 703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr 960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase mediated signaling pathways. PMID- 8621422 TI - Cellular retinol-binding protein-supported retinoic acid synthesis. Relative roles of microsomes and cytosol. AB - This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the quantitatively major share of retinal for retinoic acid (RA) biogenesis in rat tissues from the predominant substrate available physiologically, holo-cellular retinol-binding protein, type I (CRBP). With holo CRBP as substrate in the absence of apo-CRBP microsomal retinol dehydrogenases have the higher specific activity and capacity to generate retinal used for RA synthesis by cytosolic retinal dehydrogenases. In the presence of apo-CRBP, a potent inhibitor of cytosolic retinol dehydrogenases (IC50 = approximately 1 microM), liver microsomes provide 93% of the total retinal synthesized in a combination of microsomes and cytosol. Cytosolic retinol dehydrogenase(s) and the isozymes of alcohol dehydrogenase expressed in rat liver had distinct enzymatic properties; yet ethanol inhibited cytosolic retinol dehydrogenase(s) (IC50 = 20 microM) while stimulating RA synthesis in a combination of microsomes and cytosol. At least two discrete forms of cytosolic retinol dehydrogenase were observed: NAD- and NADP-dependent forms. Multiple retinal dehydrogenases also were observed and were inhibited partially by apo-CRBP. These results provide new insights into pathways of RA biogenesis and provide further evidence that they consist of multiple enzymes that recognize both liganded and nonliganded states of CRBP. PMID- 8621423 TI - Requirements for calcium and calmodulin in the calmodulin kinase activation cascade. AB - We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1 434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation. PMID- 8621424 TI - Binding of neurotrophin-3 to p75LNGFR, TrkA, and TrkB mediated by a single functional epitope distinct from that recognized by trkC. AB - Neurotrophins regulate differentiation and survival of vertebrate neurons through binding to members of the Trk family of receptor tyrosine kinases and to a common low affinity receptor, p75LNGFR. The specificity of neurotrophin action is determined by their selective interaction with the different members of the Trk family; TrkA, TrkB, and TrkC serve as cognate receptors for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3), respectively. Unlike nerve growth factor and brain-derived neurotrophic factor, NT-3 can to some extent also bind and activate non-cognate TrkA and B receptors, although the physiological relevance of these interactions is unclear. Previous studies established that neurotrophins use an extended surface for binding to cognate Trk receptors, while binding to p75LNGFR is mediated by a localized cluster of positively charged residues. Here we show that the binding site of NT-3 to its non-preferred receptors TrkA and TrkB is dominated by two positively charged residues, Arg-31 and His-33, previously shown to constitute a main determinant of binding to p75LNGFR. Simultaneous mutation of these two residues into Ala completely abolished NT-3 binding and signaling through TrkA and greatly diminished binding and activation of TrkB. However, NT-3 binding and signaling through its cognate receptor TrkC was unaffected by the mutation. These results show that binding of NT-3 to p75LNGFR, TrkA, and TrkB is mediated by a common determinant, which is distinct from that recognized by TrkC and also different and more localized than the one recognized by TrkA and TrkB in their cognate ligands. Thus, although homologous regions in all neurotrophins are used for binding to Trk receptors, a given Trk may actually contact different residues in different neurotrophins. The mutant NT-3 described here may be of greater advantage than native NT-3 when a trophic activity needs to be specifically targeted to TrkC-expressing neurons and provides a monospecific neurotrophin for future therapeutic development. PMID- 8621425 TI - Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells. AB - We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [35S]methionine/cysteine into the vitreous, 44% of [35S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [35S]Met/Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Muller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Muller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics. PMID- 8621426 TI - Defective fatty acid-mediated beta-cell compensation in Zucker diabetic fatty rats. Pathogenic implications for obesity-dependent diabetes. AB - Although obesity is associated with insulin resistance, most obese humans and rodents remain normoglycemic because of compensatory hyperinsulinemia. This has been attributed to beta-cell hyperplasia and increased low Km glucose metabolism of islets. Since free fatty acids (FFA) can induce these same beta-cell changes in normal islets of Wistar rats and since plasma FFA are increased in obesity, FFA could be the signal from adipocytes that elicits beta-cell compensation sufficient to prevent diabetes. To determine if FFA-induced compensation is impaired in islets of rats with a diabetogenic mutation, the Zucker diabetic fatty (ZDF) rat, we cultured islets from 6-week-old obese (fa/fa) rats that had compensated for obesity and apparently normal islets from lean ZDF rats (fa/+) in 0, 1, or 2 mM FFA. Low Km glucose usage rose 2.5-fold in FFA-cultured control islets from age-matched Wistar rats, but failed to rise in either the precompensated islets of ZDF rats or in islets of lean ZDF rats. Bromodeoxyuridine incorporation increased 3.2-fold in Wistar islets but not in islets from obese or lean ZDF rats. Insulin secretion doubled in normal islets cultured in 2 mM FFA (p < 0.01) but increased only slightly in islets from lean ZDF rats (not significant) and declined in islets from obese ZDF rats (p < 0.05). We conclude that, unlike the islets of age-matched Wistar rats, islets of 6-week old heterozygous and homozygous ZDF rats lack the capacity for FFA-induced enhancement of beta-cell function. PMID- 8621427 TI - Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site directed mutagenesis. AB - Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation. PMID- 8621428 TI - Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture. AB - To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM). In contrast, the order in reference to both the extent of Ca2+i reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by NG-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca2+i response in cocultured VSMCs to the isoculture level. In ECs, when the Ca2+ influx was blocked by Ni2+ or by chelating extracellular Ca2+, all four agonists markedly decreased NO production, the half decay time of the Ca2+i degenerating phase, and the area under the Ca2+i curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca2+i curve but not to the Ca2+i peak level. Thus, the sustained elevation of Ca2+i in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca2+i reduction in adjacent VSMCs. Furthermore, L-arginine but not D arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca2+i in ECs, and markedly decreased the Ca2+i in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T. (1992) J. Biol. Chem. 267, 20377-20382). PMID- 8621429 TI - DNA aggregation induced by polyamines and cobalthexamine. AB - We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 microM/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions. PMID- 8621430 TI - Differential regulation of fibroblast growth factor (FGF) receptor-1 mRNA and protein by two molecular forms of basic FGF. Modulation of FGFR-1 mRNA stability. AB - To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF 2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3 fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF 2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2. PMID- 8621431 TI - Mammalian vesicle trafficking proteins of the endoplasmic reticulum and Golgi apparatus. AB - Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations. As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins. Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood. In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport. rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions. Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus. Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes. Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment. The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus. PMID- 8621432 TI - A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase. AB - Phosphorylation and dephosphorylation of Tyr-530 in human c-Src (Tyr-527 in avian c-Src) is critical in regulating c-Src kinase activity. So far, it has not been possible to distinguish the active and inactive forms in vivo. We now report a new monoclonal antibody that selectively recognizes the active form of c-Src. This antibody, termed clone 28, recognized a region adjacent to Tyr-530 (Q529YQP532) in the C-terminal regulatory domain of c-Src, and its binding was hindered by phosphorylation of this tyrosine as determined by peptide competition assay. Combined immunoprecipitation/Western blotting revealed that clone 28 reacted with a 60-kDa protein that was precipitated by mAb 327, a well known monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage and two dimensional tryptic maps confirmed that clone 28 was specific for the active form (Tyr-530 not phosphorylated), whereas mAb 327 recognized the inactive form (Tyr 530 phosphorylated) as well as the active form. Clone 28 selectively immunoprecipitated the active form and augmented its kinase activity. Preabsorption experiments revealed that clone 28 could not completely immunoprecipitate the mAb 327 binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together, strongly suggest the existence of multiple forms of c-Src as proposed by Cooper and Howell (1993) (Cooper, J. A., and Howell, B. (1993) Cell 73, 1051-1054). Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibroblast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets. PMID- 8621433 TI - Jun kinases are rapidly activated by cholecystokinin in rat pancreas both in vitro and in vivo. AB - Stimulation of pancreatic acini from male Sprague-Dawley rats by both cholecystokinin (CCK)-8 and anisomycin caused an increase in p46jnk and p55jnk activities. Both forms of c-Jun amino-terminal kinase (JNK) were slightly activated at 5 min, reached a maximum at 30 min, and remained significantly increased at 60 min of CCK stimulation. By contrast, p42mapkwas activated fully by 5 min. In pancreatic acini stimulated with different concentrations of CCK for 30 min, the minimal and maximal JNK responses were observed at 30 pm and 100 nM CCK, respectively; p42mapk activation was, as previously reported, much more sensitive, with maximal activation by 1 nm CCK. Carbachol and bombesin also stimulated JNK activity, while vasoactive intestinal peptide did not. Neither activating protein kinase C nor increasing intracellular Ca2+ significantly activated JNK. In in vivo experiments, rats were infused intravenously for 5 and 15 min with a secretory (0.1 microg/kg/h) or supramaximal (10 microg/kg/h) dose of the CCK analog caerulein (CER). Secretory doses of CER induced a 4-fold increase of both forms of JNK in pancreatic tissue at 5 and 15 min, while at the same time points, supramaximal stimulation with CER caused 4- and 27-fold increases, respectively, of these kinase activities. The secretory dose of CER slightly increased the activities of both forms of mitogen-activated protein kinase, while the supramaximal dose induced a 10-fold increase of p42mapk at 5 min. In conclusion, JNKs and mitogen-activated protein kinases are rapidly activated in rat pancreatic acini stimulated with CCK as well as in pancreatic tissue during in vivo stimulation with CER. The large response to supramaximal CER stimulation may be of importance in the early pathogenesis of acute pancreatitis. PMID- 8621435 TI - Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide. AB - We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV 1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy. PMID- 8621434 TI - TrkC isoforms with inserts in the kinase domain show impaired signaling responses. AB - The genetic locus for the TrkC/neurotrophin 3 (NT-3) receptor tyrosine kinase encodes multiple isoforms including receptors with inserts in the catalytic domain. This study examines the signaling capabilities of TrkC and related kinase insert isoforms TrkC14 and TrkC25. We show that in PC12 cells expressing both TrkC and TrkA/nerve growth factor (NGF) receptors, different morphological changes occur upon addition of NGF or NT-3. NT-3-treated cells exhibit longer neurites and larger cell bodies as compared to NGF-treated cells. Both TrkC and TrkA mediate qualitatively similar increases in the tyrosine phosphorylation of phospholipase C (PLC)-gamma1, Shc, SNT, and MAPK and the transcription of the c fos, c-jun, NGFI-A, and NGFI-B immediate early genes. However, the TrkC kinase insert forms fail to stimulate these events. Furthermore, TrkC14 and TrkC25 have only a low intrinsic tyrosine kinase activity, and insertion of the TrkC14 kinase insert into TrkA at an equivalent position results in a dramatic reduction of the kinase activity and signaling capabilities of TrkA. The TrkC14 and -25 isoforms may fail to transmit signals due to their low intrinsic kinase activity and failure to activate and/or tyrosine phosphorylate targets shown to be involved in neurotrophin signal transduction pathways. PMID- 8621436 TI - A conditional lethal mutant in the fission yeast 26 S protease subunit mts3+ is defective in metaphase to anaphase transition. AB - We have isolated a conditional lethal mutant mts3 in the fission yeast Schizosaccharomyces pombe which at the permissive temperature is resistant to the mitotic poison MBC and at the restrictive temperature is defective in metaphase to anaphase transition. The predicted amino acid sequence of mts3+ is 36% identical with the budding yeast gene NIN1. NIN1 cloned into a fission yeast expression vector can rescue both mts3 temperature-sensitive and null alleles demonstrating that NIN1 is the budding yeast homologue of the fission yeast mts3+ gene. The phenotype of the mts3 null is identical with the mts3 ts mutant demonstrating that the phenotype of the mts3 ts mutant is due to loss of mts3+ function. The deduced amino acid sequences of both mts3+ and NIN1 show homology to peptide sequences obtained from subunit 14 of the 26 S protease purified from bovine or human cells. PMID- 8621437 TI - DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements. AB - Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3 thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate. PMID- 8621438 TI - Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange. AB - RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 +/- 20 bp min-1 at 37 degrees C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 degrees C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 +/- 1.1 kcal mole-1 for DNA strand exchange, and 14.4 +/- 1.4 kcal mole-1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min-1) is related to the kcat for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor. PMID- 8621439 TI - Interleukin-1 induces growth arrest by hypophosphorylation of the retinoblastoma susceptibility gene product RB. AB - Interleukin-1 (IL-1) causes G0/G1 phase growth arrest in human melanoma cells, A375-C6. Because hypophosphorylation of the retinoblastoma susceptibility gene product, RB, is one of the key events responsible for G0/G1 phase growth arrest, we investigated whether IL-1 altered the phosphorylation status of RB protein in these cells. Exposure to IL-1 caused a time-dependent increase in hypophosphorylated RB that correlated with an accumulation of cells arrested in the G0/G1 phase. The ability of IL-1 to cause hypophosphorylation of RB and growth arrest was abrogated by the SV40 large T antigen, which binds preferentially to hypophosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB. Furthermore, the cells were protected from IL-1-inducible growth inhibition by ectopic expression of dominant negative mutants of the Rb gene, or the transcription factor E2F-1, which is a downstream target of RB. These results suggest that hypophosphorylated RB mediates the growth arrest induced by IL-1. PMID- 8621440 TI - The cytoplasmic and transmembrane domains of AE2 both contribute to regulation of anion exchange by pH. AB - We have compared regulation by pH of AE1 (band 3)- and AE2-mediated 36Cl- uptake into Xenopus oocytes. 36Cl- influx was assayed at varying extracellular pH (pHo) values between 9.0 and 5.0 under conditions in which corresponding intracellular pH (pHi) values were at or near steady-state. Wild type (WT) AE1 displayed a broad convex pH versus activity curve, with peak activity at pHo 7.0 and 63% of maximal activity at pHo 5.0. In contrast, WT AE2 displayed a steep pH versus activity curve, with peak activity at pHo9.0 and full suppression at pHo 5.0. The structural basis of these differing pH sensitivities was examined by expression of cRNAs encoding chimeric and truncated proteins. Mutant polypeptides were expressed in oocytes and detected at the cell surface. The AE2cyto/AE1memb polypeptide displayed a broad pH versus activity curve similar to that of WT AE1. In contrast, the AE1cyto/AE2memb polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values from that of WT AE2 by 0.69 +/- 0.04 pHo units. Moreover, whereas the pH versus activity curves of AE2 Delta99 and WT AE2 were indistinguishable, AE2 Delta510 exhibited a pH versus activity curve acid-shifted from that of WT AE2 by 0.66 +/- 0.13 pHo units (indistinguishable from that of AE1cyto/AE2memb). The data suggest that a pH sensor resides within the transmembrane region of AE2. The affinity for protons of this pH sensor is influenced by a modifier site located between residues 99 and 510 of the N-terminal cytoplasmic domain of AE2. Acidification of oocytes with acetate suggested that pHi accounted for some but not all of the measured pH dependence of AE2. PMID- 8621442 TI - Requirement of cysteine residues in exons 1-6 of the extracellular domain of the luteinizing hormone receptor for gonadotropin binding. AB - The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain. PMID- 8621441 TI - The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2-terminal kinase groups of mitogen-activated protein kinases. AB - In airway smooth muscle cells ligand binding to the seven-transmembrane endothelin and thrombin receptors stimulates cell growth. Rapid activation of the extracellular regulated kinase 2 and c-Jun NH2-terminal kinase groups of mitogen activated protein kinases was also observed. The results demonstrate a novel mechanism of seven-transmembrane receptor signaling involving activation of the Jun kinase pathway. Receptor coupling to Jun kinase activation may involve heterotrimeric G proteins since the kinase was enzymatically activated in cells treated with aluminum fluoride. The activity of Raf-1, measured by immune complex kinase assay, revealed that platelet-derived growth factor and phorbol 12 myristate 13-acetate both stimulated Raf-1 activity, while thrombin and endothelin did not appreciably stimulate Raf-1. The data suggest that endothelin and thrombin stimulate Raf-1-independent mechanisms of mitogen-activated protein kinase activation. Endothelin- or thrombin-induced activation of mitogen activated protein kinases was significantly inhibited by activation of cyclic AMP dependent protein kinase by forskolin. Proliferation of airway smooth muscle cells, measured by incorporation of [3H]thymidine into DNA, was also greatly attenuated by forskolin. PMID- 8621444 TI - The novel anticonvulsant drug, gabapentin (Neurontin), binds to the alpha2delta subunit of a calcium channel. AB - Gabapentin (1-(aminomethyl)cyclohexane acetic acid; Neurontin) is a novel anticonvulsant drug, with a mechanism of action apparently dissimilar to that of other antiepileptic agents. We report here the isolation and characterization of a [3H]gabapentin-binding protein from pig cerebral cortex membranes. The detergent-solubilized binding protein was purified 1022-fold, in a six-step column-chromatographic procedure, with a yield of 3.9%. The purified protein had an apparent subunit Mr of 130,000, and was heavily glycosylated. The partial N terminal amino acid sequence of the Mr 130,000 polypeptide, EPFPSAVTIK, was identical to that reported for the alpha2delta subunit of the L-type Ca2+ channel from rabbit skeletal muscle (Hamilton, S. L., Hawkes, M. J., Brush, K., Cook, R., Chang, R. J., and Smilowitz, H. M. (1989) Biochemistry 28, 7820-7828). High levels of [3H]gabapentin binding sites were found in membranes prepared from rat brain, heart and skeletal muscle. Binding of [3H]gabapentin to COS-7 cells transfected with alpha2delta cDNA was elevated >10-fold over controls, consistent with the expression of alpha2 delta protein, as measured by Western blotting. Finally, purified L-type Ca2+ channel complexes were fractionated, under dissociating conditions, on an ion-exchange column; [3H]gabapentin binding activity closely followed the elution of the alpha2 delta subunit. [3H]Gabapentin is the first pharmacological agent described that interacts with an alpha2delta subunit of a voltage-dependent Ca2+ channel. PMID- 8621443 TI - Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF165 via its exon 7-encoded domain. AB - Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked 125I-VEGF165 to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF165 binding site, with a Kd of 2.8 x 10(-10) M and 0.95 1.1 x 10(5) binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a Kd of 7.5 x 10(-12) M, presumably FLK/KDR, and the other with a Kd of 2 x 10(-10) M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked 125I-VEGF165 but not 125I-VEGF121. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF165 that are absent in VEGF121, was fused to glutathione S-transferase (GST). The GST VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF165 and inhibited the binding of 125I-VEGF165 to 231 cells. Cross-linking of 125I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF165 receptors. It was concluded that certain tumor derived cell lines express novel surface-associated receptors that selectively bind VEGF165 via the exon 7-encoded domain, which is absent in VEGF121. PMID- 8621445 TI - T1/ST2 signaling establishes it as a member of an expanding interleukin-1 receptor family. AB - Through data base searches, we have discovered new proteins that share homology with the signaling domain of the type I interleukin-1 receptor (IL-1RI): human "randomly sequenced cDNA 786" (rsc786), murine MyD88, and two partial Drosophila open reading frames, MstProx and STSDm2245. Comparisons between these new proteins and known IL-1RI homologous proteins such as Toll, 18-Wheeler, and T1/ST2 revealed six clusters of amino acid similarity. We tested the hypothesis that sequence similarity between the signaling domain of IL-1RI and the three mammalian family members might indicate functional similarity. Chimeric IL-1RI receptors expressing the putative signaling domains of T1/ST2, MyD88, and rsc786 were assayed by three separate IL-1 responsive assays, NF-kappaB, phosphorylation of an epidermal growth factor receptor peptide, and an interleukin 8 promoter controlled reporter construct, for their ability to transduce an IL-1-stimulated signal. All three assays were positive in response to the T1/ST2 chimera, while the MyD88 and rsc786 chimeras failed to respond. These data indicate that the sequence homology between IL-1RI and T1/ST2 indicates a functional homology as well. PMID- 8621446 TI - Cloning of a putative ligand for the T1/ST2 receptor. AB - T1/ST2 is a receptor-like molecule homologous to the type I interleukin-1 receptor. Despite this sequence similarity, we have been unable to demonstrate binding of T1/ST2 to any of the three interleukin-1 species. In searching for a ligand for T1/ST2, we have cloned a cell surface protein to which it binds. This protein is unable to initiate signal transduction by the T1/ST2 receptor in several in vitro assays. PMID- 8621448 TI - Estrogen-related receptor, hERR1, modulates estrogen receptor-mediated response of human lactoferrin gene promoter. AB - We have shown previously that estrogen-stimulated transcription from the human lactoferrin gene in RL95-2 endometrium carcinoma cells is mediated through an imperfect estrogen response element (ERE) at the 5 -flanking region of the gene. Upstream from the ERE, a DNA sequence (-418 to -378, FP1) was selectively protected from DNase I digestion by nuclear extracts from endometrial and mammary gland cell lines. In this report, using the electrophoresis mobility shift assay, site-directed mutagenesis, and DNA methylation interference analyses, we show that three different nuclear proteins bind to the FP1 region (C1, C2, and C3 sites). The nuclear receptor, COUP-TF, binds to the C2 site. Mutations in the C1 binding region abolish C1 complex formation and reduce estrogen-dependent transcription from the lactoferrin ERE. When the imperfect ERE of the lactoferrin gene is converted to a perfect palindromic structure, the enhancing effect of the C1 binding element for estrogen responsiveness was abolished. We isolated a complementary DNA (cDNA) clone from an RL95-2 expression library that encodes the C1 site-binding protein. The encoded polypeptide maintains 99% amino acid identity with the previously described orphan nuclear receptor hERR1. A 2.2 kilobase mRNA was detected in RL95-2 cells by the newly isolated cDNA but not by the first 180 base pair of the published hERR1 sequence. By Western analysis, a major 42-kDa protein is detected in the RL95-2 nuclear extract with antibody generated against GST-hERR1 fusion protein. Finally, we show that the hERR1 interacts with the human estrogen receptor through protein-protein contacts. PMID- 8621447 TI - Formation of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene transcription by interferon-alpha. AB - An upstream inverted repeat (IR) element mediates transcriptional activation of the interferon response factor-1 gene (IRF-1) by interferon (IFN)-alpha and IFN gamma. IFN-alpha and IFN-gamma fail to induce IRF-1 in cells that lack signal transducer and activator of transcription 1 (STAT1), and STAT1 homodimers bind to IR elements in extracts of IFN-alpha-treated cells. We now report that STAT2 also plays an important role in the IFN-alpha-mediated transcriptional activation of the IRF-1 gene. A new factor, most likely a STAT1-STAT2 heterodimer, was detected with an IR probe in extracts of IFN-alpha-treated cells. STAT1 and STAT2 are already known to combine with p48, a DNA-binding protein, to form IFN-stimulated gene factor 3 (ISGF3), which binds to IFN-stimulated response elements (ISREs) distinct from the IR of the IRF-1 gene. In extracts of U2A cells, which lack p48, STAT1-STAT2 heterodimers were still formed, indicating that they do not contain p48. We manipulated the intracellular levels of STAT1-STAT2 heterodimers and STAT1 homodimers to examine their roles in the induction of IRF-1 by IFN-alpha. Although both dimers can induce IRF-1 transcription, the heterodimers are more potent and thus may be the major activators in vivo. Deletion analysis reveals that the C-terminal domain of STAT2 is important for transcriptional activation mediated by both STAT1-STAT2 heterodimers and ISGF3. PMID- 8621449 TI - Expression, purification, and mechanistic studies of bovine mitochondrial translational initiation factor 2. AB - A complete cDNA clone encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained. The regions of the cDNA corresponding to mature IF-2mt and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor (approximately 90 kDa) and mature (approximately 85 kDa) forms of IF-2mt are toxic to E. coli and can only be expressed at low levels. Shorter forms of this factor (approximately 80 and approximately 72 kDa) are also found during the expression of mature IF 2mt. The various forms of IF-2mt can be separated by high performance liquid chromatography. All of these forms are active in promoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovine mitochondrial ribosomes. IF-2mt can bind to mitochondrial ribosomes in the absence of GTP, initiator tRNA, or messenger RNA. The presence of GTP stimulates IF-2mt binding to ribosomes about 3-fold. IF-2mt interacts only weakly with GTP or with the initiator tRNA in the absence of ribosomes. Molecular dissection of IF-2mt shows that a long deletion (approximately 150 amino acid residues) from the NH2-terminal region does not affect its activity in vitro. The COOH domain of IF-2mt (amino acid residues 332-727) can bind to ribosomes even though it does not promote initiator-tRNA binding. PMID- 8621450 TI - Transphosphorylation of the neurotrophin Trk receptors. AB - The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. Cotransfection experiments in COS-7 cells and fibroblasts showed that despite the presence of different extracellular regions, intermolecular transphosphorylation of homologous cytoplasmic domains occurred between TrkA or TrkB and their cognate chimeras. Heterologous transphosphorylation between TrkB and TrkC kinase domains was also observed when one partner was a chimeric receptor; however, TrkA did not transphosphorylate the TrkB or TrkC kinase domains of chimeric receptors or act as a transphosphorylation substrate for these two receptors. The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions. PMID- 8621451 TI - A naturally occurring T14A11 tract blocks nucleosome formation over the human neurofibromatosis type 1 (NF1)-Alu element. AB - The nature of chromatin organization over Alu repetitive elements is of interest with respect to the maintenance of their transcriptional silencing as well as their potential to influence local chromatin structure. We previously demonstrated that the pattern of nucleosomal organization over Alu elements in native chromatin is specific and similar to the pattern observed with an in vitro reconstituted Alu template. This pattern, distinguished by a nucleosome centered over the 5 -end of the Alu element, is associated with repression of polymerase III-dependent transcription in vitro (Englander, E. W., Wolffe, A. P., and Howard, B. H. (1993) J. Biol. Chem. 268, 19565-19573; Englander, E. W., and Howard, B. H. (1995) J. Biol. Chem. 270, 10091-10096). In the current study, additional templates representing both evolutionarily old and young Alu subfamilies were found to direct a similar pattern of nucleosome assembly, consistent with the view that nucleosome positioning in vitro is shared by a majority of Alus. We discovered however, that the specific nucleosome positioning pattern was disrupted over one member of a young Alu subfamily, which recently transposed immediately downstream to a T14A11 sequence in the neurofibromatosis type 1 locus (Wallace, M. R., Andersen, L. B., Saulino, A. M., Gregory, P. E., Glover, T. W., and Collins, F. S. (1991) Nature 353, 864-866). Upon removal of this sequence motif, the expected pattern of assembly was restored to the neurofibromatosis type 1-Alu template. This finding indicates that, at least in vitro, certain sequences can override the propensity for positioning nucleosomes that is inherent to Alu elements. The finding also raises the possibility that a similar situation may occur in vivo, with potential implications for understanding mechanisms by which certain Alu elements may evade chromatin mediated transcriptional silencing. PMID- 8621453 TI - Identification of p90, a prominent tyrosine-phosphorylated protein in fibroblast growth factor-stimulated cells, as 80K-H. AB - Tyrosine phosphorylation of cellular proteins occurs rapidly upon treatment of fibroblasts with acidic or basic fibroblast growth factors (aFGF, bFGF), suggesting a role for protein phosphorylation in the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins, of which the most prominent has been designated as p90. The phosphorylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N terminal amino acid sequence which corresponded to a protein previously named as 80K-H. Polyclonal antibodies raised against the predicted C terminus of 80K-H recognized p90 on all Western blots. p90 was found to bind specifically to GRB-2 glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphotyrosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not from control fibroblasts. The biological function of 80K-H is yet unknown. However, from this study and a previous observation of the obligatory dependence of p90 phosphorylation on FGF receptor occupation, it appears that 80K-H is involved in FGF signaling. PMID- 8621452 TI - Type II human complement C2 deficiency. Allele-specific amino acid substitutions (Ser189 --> Phe; Gly444 --> Arg) cause impaired C2 secretion. AB - Type II complement protein C2 deficiency is characterized by a selective block in C2 secretion. The Type II C2 null allele (C2Q0) is linked to two major histocompatibility haplotypes (MHC) that differ from the MHC of the more common Type I C2 deficiency. To determine the molecular basis of Type II deficiency the two Type II C2Q0 genes were isolated and transfected separately into L-cells. Subsequent molecular biology, biosynthetic, and immunofluorescence studies demonstrated that C2 secretion is impaired in Type II C2 deficiency because of different missense mutations at highly conserved residues in each of the C2Q0 alleles. One is in exon 5 (nucleotide C566 --> T; Ser189 --> Phe) of the C2Q0 gene linked to the MHC haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G1930 --> A; Gly444 --> Arg) of the C2Q0 gene linked to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene product is retained early in the secretory pathway. These mutants provide models for elucidating the C2 secretory pathway. PMID- 8621454 TI - Alpha1-adrenergic receptor subtype mRNAs are differentially regulated by alpha1 adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo. Repression of alpha1B and alpha1D but induction of alpha1C. AB - The three cloned alpha1-adrenergic receptor (AR) subtypes, alpha1B, alpha1C, and alpha1D, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three alpha1-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the alpha1-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12 72 h). Alpha1B and alpha1D mRNA levels were repressed by NE, whereas alpha1C mRNA was induced. Changes in mRNA levels were mediated by an alpha1-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. alpha1-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2alpha, and phorbol 12-myristate 13 acetate, also induced alpha1C mRNA and repressed alpha1B mRNA. In myocytes from hearts with pressure overload hypertrophy, alpha1 mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among alpha1-AR subtypes expressed natively in the same cell. Transcriptional induction of the alpha1C-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes. PMID- 8621455 TI - A novel function for the second C2 domain of synaptotagmin. Ca2+-triggered dimerization. AB - Synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis from neurons. While the mechanism by which synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a multimeric complex and that its second C2 domain is essential for efficient excitation-secretion coupling. Here we describe biochemical data that may account for these phenomena. We report that Ca2+ causes synaptotagmin to oligomerize, primarily forming dimers, via its second C2 domain. This effect is specific for divalent cations that can stimulate exocytosis of synaptic vesicles (Ca2+ >> Ba2+, Sr2+ >> Mg2+) and occurs with an EC50 value of 3-10 microM Ca2+. In contrast, a separate Ca2+-dependent interaction between synaptotagmin and syntaxin, a component of the fusion apparatus, occurs with an EC50 value of approximately 100 microM Ca2+ and involves the synergistic action of both C2 domains of synaptotagmin. We propose that Ca2+ triggers two consecutive protein protein interactions: the formation of synaptotagmin dimers at low Ca2+ concentrations followed by the association of synaptotagmin dimers with syntaxin at higher Ca2+-concentrations. Our findings, in conjunction with physiological studies, indicate that the Ca2+-induced dimerization of synaptotagmin is important for the efficient regulation of exocytosis by Ca2+. PMID- 8621456 TI - The multiple endocrine neoplasia type 2B point mutation alters long-term regulation and enhances the transforming capacity of the epidermal growth factor receptor. AB - The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild type and M857T EGF receptors. However, significant differences in receptor down regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation. PMID- 8621457 TI - Cloning and characterization of the promoter for a potassium channel expressed in high frequency firing neurons. AB - The Kv3.1 potassium channel is expressed in neurons that generate trains of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterized its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltransferase reporter gene, this fragment was found to be active in the undifferentiated PC12 cell line, a neuron-like cell line, but not in a fibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silencing elements and enhancing element(s) are involved in the cell type-specific expression of this gene. Further regulatory elements, including one cyclic AMP/calcium response element (CRE) and one Ap-1 element were found in the upstream region of the promoter. Using a stable undifferentiated PC12 cell line transfected with the Kv3.1 5' flanking region, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobility shift assays confirmed that the Kv3.1 CRE sequence binds both a nuclear factor in undifferentiated PC12 cells and recombinant CRE binding protein. Our results suggest that the transcription of the Kv3.1 channel may be regulated by neurotransmitters that elevate cAMP levels in neurons. PMID- 8621458 TI - Inhibitory interactions between two inward rectifier K+ channel subunits mediated by the transmembrane domains. AB - Inwardly rectifying K+ channel subunits may form homomeric or heteromeric channels with distinct functional properties. Hyperpolarizing commands delivered to Xenopus oocytes expressing homomeric Kir 4.1 channels evoke inwardly rectifying K+ currents which activate rapidly and undergo a pronounced decay at more hyperpolarized potentials. In addition, Kir 4.1 subunits form heteromeric channels when coexpressed with several other inward rectifier subunits. However, coexpression of Kir 4.1 with Kir 3.4 causes an inhibition of the Kir 4.1 current. We have investigated this inhibitory effect and show that it is mediated by interactions between the predicted transmembrane domains of the two subunit classes. Other subunits within the Kir 3.0 family also exhibit this inhibitory effect which can be used to define subgroups of the inward rectifier family. Further, the mechanism of inhibition is likely due to the formation of an "inviable complex" which becomes degraded, rather than by formation of stable nonconductive heteromeric channels. These results provide insight into the assembly and regulation of inwardly rectifying K+ channels and the domains which define their interactions. PMID- 8621459 TI - Regulation of epidermal growth factor receptor signaling by phosphorylation of the ras exchange factor hSOS1. AB - In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 was phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resulted in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylated hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 was phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping showed that MAPK phosphorylated hSOS1 in vitro on sites which were also phosphorylated in vivo. Phosphorylation by MAPK did not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex showed that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 was unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway. PMID- 8621460 TI - Regulation of macrophage inflammatory protein-1alpha mRNA by oxidative stress. AB - Accumulation of inflammatory cells within the lung has been implicated in oxidative injury. Recruitment of these cells to a tissue site is a complex process that depends in part upon the local expression of appropriate proinflammatory chemokines. Macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the CC subfamily of chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1alpha mRNA expression in macrophages is induced by bacterial endotoxin. The objective of this study was to test the hypothesis that an oxidative stress alone may trigger expression of MIP-1alpha mRNA in macrophages and to determine the mechanism leading to increased expression. A rat alveolar macrophage cell line (NR8383) was exposed to H2O2 or menadione (2-methyl 1,4-naphthoquinone (MQ)), a quinone compound that undergoes redox cycling and generates reactive oxygen species continuously. Steady-state mRNA levels encoding MIP-1alpha were markedly increased (3-fold) in these cells after 1 h of exposure to 0.5 mM H2O2, remained higher than control levels after 4 h, and decreased after 6 h. Similarly, MQ (25 or 50 microM) caused a significant increase of MIP-1 alpha mRNA with a maximal induction after 4 h of exposure (5-fold). Both H2O2 and MQ-induced up-regulation of MIP-1 alpha mRNA was suppressed by co-treatment with N-acetylcysteine, a synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ induction of MIP-1alpha mRNA to a greater extent than the H2O2 induced increase. Transcription of the MIP-1alpha gene was increased by exposure to both H2O2 and MQ. H2O2 treatment also induced a marked increase of the MIP 1alpha mRNA half-life, indicating post-transcriptional stabilization. These observations indicate that an oxidative stress can regulate MIP-1alpha mRNA expression by two distinct mechanisms: transcriptional activation of the MIP 1alpha gene and post-transcriptional stabilization of MIP-1alpha mRNA. PMID- 8621461 TI - Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue. AB - The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl. PMID- 8621462 TI - Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow. AB - Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21 L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis. PMID- 8621463 TI - A novel subtype of the prostacyclin receptor expressed in the central nervous system. AB - By use of several prostacyclin analogs and an in vitro autoradiographic technique, we have found a novel subtype of the prostacyclin receptor, one having different binding properties compared with those of the known prostacyclin receptor in the rat brain. Isocarbacyclin, which is a potent agonist for the known prostacyclin receptor, had high affinity for the novel subtype (dissociation constant (Kd) of 7.8 nM). However, iloprost, which is usually used as a stable prostacyclin analog, showed low affinity binding (Kd = 159 nM) for the subtype. Other prostaglandins showed no or little affinity for the subtype. [3H]Isocarbacyclin binding was high in the thalamus, lateral septal nucleus, hippocampus, cerebral cortex, striatum, and dorsal cochlear nucleus. Although the nucleus of the solitary tract and the spinal trigeminal nucleus showed a high density of [3H]isocarbacyclin binding, [3H]iloprost also had high affinity in these regions, and the binding specificity was similar to that for the known prostacyclin receptor. Hemilesion studies of striatal neurons lesioned by kainate or of dopaminergic afferents lesioned by 6-hydroxydopamine revealed that the binding sites of the novel subtype exist on neuronal cells in the striatum, but not on the presynaptic terminal of afferents or on glial cells. Electrophysiological studies carried out in the CA1 region of the hippocampus revealed that prostacyclin analogs have a facilitatory effect on the excitatory transmission through the novel prostacyclin receptor. The widespread expression of the prostacyclin receptor in the central nervous system suggests that prostacyclin has important roles in neuronal activity. PMID- 8621464 TI - Expression and characterization of the flavoprotein subcomplex composed of 50-kDa (NQO1) and 25-kDa (NQO2) subunits of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. AB - This study reports the expression of the flavoprotein (FP) subcomplex of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from Paracoccus denitrificans, which is composed of the NQO1 (50 kDa) and the NQO2 (25 kDa) subunits. The two subunits are co-expressed in Escherichia coli using a double expression plasmid system. The expressed subunits form a water-soluble heterodimer complex with 1:1 stoichiometry. The expressed complex contained one [2Fe 2S] cluster but almost no FMN or [4Fe 4S] cluster. The two latter prosthetic groups could be partially reconstituted with FMN, Na2S, and (NH4)2Fe(SO4)2 in vitro under anaerobic conditions. The reconstituted FP subcomplex showed EPR signals from two distinct species of iron-sulfur cluster. One resonance transition originates from a [2Fe-2S] cluster with g values of gx,y,z = 1.92, 1.95, and 2.00 and slow spin relaxation, which was tentatively assigned to the cluster N1a. These EPR properties are very similar to those reported for the NQO2 subunit expressed alone (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). The other originates from a [4Fe 4S] cluster with g values of gx,y, z = 1.87, 1.94, and 2.04 and fast relaxing behavior, which are reminiscent of the cluster N3 in the membrane bound enzyme complex. After reconstitution with FMN, the FP subcomplex catalyzed electron transfer from NADH and from deamino-NADH to a variety of electron acceptors. The enzymatic properties of the FP subcomplex, reconstituted with FMN and iron-sulfur, correspond to those of the isolated P. denitrificans NADH-dehydrogenase complex. PMID- 8621465 TI - Human ryudocan from endothelium-like cells binds basic fibroblast growth factor, midkine, and tissue factor pathway inhibitor. AB - Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment. PMID- 8621466 TI - Multiple Sp1 binding sites in the cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase gene promoter are required for expression in Sol8 muscle cells. AB - The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene encodes a Ca2+ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (-1810 base pair (bp) to -1110 bp) and one proximal (-284 bp to -72 bp), within the SERCA2 promoter. The proximal promoter region from -284 bp to -80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1-like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells. PMID- 8621467 TI - Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation. AB - Intermittent plasma growth hormone (GH) pulses, which occur in male but not female rats, activate liver Stat 5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation of this latent cytoplasmic transcription factor (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262-13270). We demonstrate that physiological levels of GH can also activate Stat 1 and Stat 3 in liver tissue, but with a dependence on the dose of GH and its temporal plasma profile that is distinct from Stat 5 and with a striking desensitization following a single hormone pulse that is not observed with liver Stat 5. GH activation of the two groups of Stats leads to their selective binding to DNA response elements upstream of the c-fos gene (c sis-inducible enhancer element; Stat 1 and Stat 3 binding) and the beta-casein gene (mammary gland factor element; liver Stat 5 binding). In addition to tyrosine phosphorylation, GH is shown to stimulate phosphorylation of these Stats on serine or threonine in a manner that either enhances (Stat 1 and Stat 3) or substantially alters (liver Stat 5) the binding of each Stat to its cognate DNA response element. These findings establish the occurrence of multiple, Stat dependent GH signaling pathways in liver cells that can target distinct genes and thereby contribute to the diverse effects that GH and its sexually dimorphic plasma profile have on liver gene expression. PMID- 8621468 TI - A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. AB - Phospholamban is a phosphoprotein regulator of cardiac sarcoplasmic reticulum which is phosphorylated in response to beta-adrenergic stimulation. Previous results have shown that phospholamban forms Ca2+-selective channels in lipid bilayers. The channel-forming domain has been localized to amino acid residues 26 52, which form a stable pentameric, helical structure. The specific residues responsible for stabilizing the pentameric membrane domain of phospholamban have been identified by mutational analysis. Residues 26-52 were individually mutated to Ala or Phe, and the ability of the resulting mutant to form a pentamer or other oligomer was assessed by SDS-polyacrylamide gel electrophoresis analysis. Replacement of Leu37, Ile40, Leu44, Ile47, or Leu51 by Ala prevented pentamer formation, indicating their essential involvement in the oligomeric assembly. The heptad repeats, and 3-4-residue spacing of the essential amino acids suggest that residues 37-52 adopt a pentameric coiled-coil structure stabilized by a leucine zipper motif formed by the close packing of Leu37, Ile40, Leu44, Ile47, and Leu51. The resulting symmetric structure contains a central pore defined by the hydrophobic surface of the five stabilizing leucine zippers, which are oriented to the interior and form the backbone of the pentamer. PMID- 8621470 TI - Relating structure to function in phi29 DNA polymerase. PMID- 8621469 TI - Participation of JAK and STAT proteins in growth hormone-induced signaling. AB - The binding of growth hormone leads to dimerization of its receptor, accompanied by phosphorylation and activation of intracellular tyrosine kinases (JAKs) and the latent cytoplasmic transcriptions factors STAT1, STAT3, and STAT5. Both JAK1 and JAK2 are phosphorylated in response to growth hormone in mouse 3T3 F442A and human HT1080 cells. The roles of JAKs in growth hormone signal transduction were examined by using mutant HT1080 cells missing either JAK1 or JAK2. JAK2 is absolutely required for growth hormone-dependent phosphorylation of the receptor, STAT1 and STAT3, JAK1, and the SH2-containing adaptor molecule Shc. In contrast, JAK1 is not required for any of the above functions. These data indicate that JAK2 is both necessary and sufficient for the growth hormone-dependent phosphorylation events required to couple the receptor both to STAT-dependent signaling pathways and to pathways involving Shc. Furthermore, STAT5 is activated by growth hormone in 3T3 F442A cells, but not in HT1080 cells, revealing that the set of STATs activated by growth hormone can vary, possibly contributing to the specificity of the growth hormone response in different cell types. PMID- 8621471 TI - Activation and inactivation of Ca2+ release by NAADP+. AB - Nicotinic acid adenine dinucleotide phosphate (NAADP+) is a recently identified metabolite of NADP+ that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP+ in intact sea urchin eggs elicits long term Ca2+ oscillations. On the other hand, uncaging threshold amounts of NAADP+ produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP+ receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time dependent manner. Thus, the NAADP+-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization. PMID- 8621472 TI - Localization of subunits of transcription factors IIE and IIF immediately upstream of the transcriptional initiation site of the adenovirus major late promoter. AB - The assembly of a preinitiation complex containing RNA polymerase II on promoter DNA is a complex process that involves several general transcription factors. Using 5-[N-(p-azidobenzoyl)-3-aminoallyl] photocross-linking, we previously determined the locations of the two large subunits of transcription factor (TF) IIA (A35 and A21), TATA box-binding protein (TBP), RNA polymerase II-associated protein (RAP) 30, and TFIIB along the Ad2 ML promoter. We have now localized TFIIE34 and RAP74 just upstream of the transcription start site. The two subunits of TFIIF, RAP74 and RAP30, cross-linked to nucleotides that probed adjacent spaces on the same face of the DNA helix beginning just downstream of TBP at -19 and extending to -5. Specific photocross-linking of TFIIE34 required the presence TFIIE56. In addition, TFIIE and RAP74 strongly stimulated cross-linking of RAP30 and the large subunits of RNA polymerase II to position -19. Our topological data support the idea that RAP74 and TFIIE34 may be involved in melting of the promoter DNA upstream of the initiation site. PMID- 8621473 TI - A peptide sequence from Bax that converts Bcl-2 into an activator of apoptosis. AB - Bcl-2 and Bax are members of a family of cytoplasmic proteins that regulate apoptosis. The two proteins have highly similar amino acid sequences but are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect. The antagonism appears to depend upon dimerization between Bcl-2 and Bax, but its mechanism is otherwise unknown. Here we report that overexpressing Bax induces apoptosis in a mammalian fibroblast cell line, and we identify a novel, short "suicide domain" in Bax that is required for this effect. Inserting this domain in place of the corresponding, divergent sequence in Bcl-2 converts Bcl-2 from an inhibitor into an activator of cell death. These findings imply that a specific region in Bax confers an active propensity for apoptosis in mammalian cells and support the view that Bcl-2 may block death primarily by suppressing Bax activity. PMID- 8621474 TI - Identification, cloning, and sequencing of a cDNA coding for rat gamma-glutamyl hydrolase. AB - Purified gamma-glutamyl hydrolase secreted from rat H35 hepatoma cells has been characterized as a diffuse band of 55 kDa on SDS-polyacrylamide gel electrophoresis that is converted to bands of 35 and 33 kDa after enzymatic removal of N-linked carbohydrate. Polyclonal antibodies against 55-kDa gamma glutamyl hydrolase captured the enzyme activity and recognized the glycosylated and both deglycosylated forms of gamma-glutamyl hydrolase. A complete cDNA sequence of gamma-glutamyl hydrolase was obtained using degenerate oligonucleotides derived from peptide sequences, screening of a rat hepatoma cDNA library, and reverse transcription polymerase chain reaction. Based upon the deduced amino acid sequence the peptide component of gamma-glutamyl hydrolase had a molecular weight of 33,400. The results of amino acid analysis of the purified protein agreed with the deduced amino acid sequence in which there are seven potential asparagine-containing glycosylation sites. PMID- 8621475 TI - Interleukin-4 induces activation of mitogen-activated protein kinase and phosphorylation of shc in human keratinocytes. AB - Most cytokines stimulate the p21ras pathway, leading to MAP kinase activation. One exception is interleukin-4 (IL-4), which has been shown not to activate this pathway in hematopoietic cells. However, IL-4 acts on a broad range of cells, including keratinocytes, in which it induces IL-6 production. We report here that IL-4 stimulation of human keratinocytic cell lines or primary cultures activates MAP kinase. In these cells, IL-4 stimulation induces the tyrosine phosphorylation of p42/44 MAP kinase as well as its catalytic activity. We also observed an increased phosphorylation of p46shc, an SH2-containing protein involved in the Ras pathway, as a result of IL-4 stimulation in human keratinocytic cell lines but not in T lymphocytes. PMID- 8621476 TI - Sp3 represses transcription when tethered to promoter DNA or targeted to promoter proximal RNA. AB - Sp3 is a member of the Sp family of transcription factors, and it binds to the GC box with an affinity and specificity comparable with that of Sp1. Previous studies have shown that Sp3 repressed Sp1-mediated transcriptional activation, suggesting that Sp3 is an inhibitory member of the Sp family. The experiments described here demonstrate that Sp3 contains a portable repression domain that can function independently from the zinc finger DNA-binding domain. We found that the amino-terminal region of Sp3 tethered to a promoter DNA by connecting to a heterologous DNA-binding protein domain represses transcriptional activation by different positive regulators. Moreover, we determined that Sp3 targeted to a promoter-proximal RNA sequence acts as a transcriptional repressor. Taken together, our results suggest that Sp3 functions as a repressor by protein protein interaction with components of the general transcription complex. PMID- 8621477 TI - Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors. AB - In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3 kinase. PMID- 8621478 TI - Magnesium(II) is a crucial constituent of the blood coagulation cascade. Potentiation of coagulant activities of factor IX by Mg2+ ions. AB - We recently showed that not only Ca2+ ions but also Mg2+ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg2+ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg2+ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca2+ ions was accelerated by Mg2+ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg2+ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa*tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa*tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg2+ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations. PMID- 8621479 TI - Arrest of beta-amyloid fibril formation by a pentapeptide ligand. AB - Polymerization of amyloid beta-peptide (Abeta) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease. Here, we show that peptides incorporating a short Abeta fragment (KLVFF; Abeta16-20) can bind full-length Abeta and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys16, Leu17, and Phe20 are critical for binding to Abeta and inhibition of Abeta fibril formation. A mutant Abeta molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues Abeta16-20 serve as a binding sequence duringA beta polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and non-peptide agents aimed at inhibiting Abeta amyloidogenesis in vivo. PMID- 8621480 TI - A hydrophobic domain of Ca2+-modulating cyclophilin ligand modulates calcium influx signaling in T lymphocytes. AB - Ca2+-modulating cyclophilin ligand (CAML) was originally described as a cyclophilin B-binding protein whose overexpression in T cells causes a rise in intracellular calcium, thus activating transcription factors responsible for the early immune response. As reported here, structure-function analysis of the CAML gene in Jurkat T cells indicates that two of CAML's putative membrane-spanning domains are necessary and sufficient for the modulation of intracellular calcium. We propose that the hydrophobic C-terminal tail of CAML forms its effector domain, thus implicating the N-terminal hydrophilic domain in a regulatory role. These findings define a novel protein motif that functions in intracellular calcium signaling. PMID- 8621481 TI - Dissecting clot retraction and platelet aggregation. Clot retraction does not require an intact fibrinogen gamma chain C terminus. AB - Fibrinogen mediates the processes of platelet aggregation and clot retraction. Previous studies have demonstrated that fibrinogen binding to the platelet receptor alphaIIbbeta3 requires the C-terminal residues of the fibrinogen gamma chain. We made a recombinant human fibrinogen that lacks the gamma chain C terminal four residues (AGDV). As expected this fibrinogen did not support platelet aggregation. Unexpectedly, this variant did support clot retraction that was indistinguishable from retraction with normal recombinant or plasma fibrinogen. These results suggest that the site on fibrinogen that is required for platelet aggregation differs from the site on fibrin that is required for clot retraction. PMID- 8621482 TI - The polyelectrolyte nature of F-actin and the mechanism of actin bundle formation. AB - Polymerized (F-)actin is induced to form bundles by a number of polycations including divalent metal ions, Co(NH3)63+, and basic polypeptides. The general features of bundle formation are largely independent of the specific structure of the bundling agent used. A threshold concentration of polycation is required to form lateral aggregates of actin filaments. The threshold concentration varies strongly with the valence of the cation and increases with the ionic strength of the solution. Polyanions such as nucleoside phosphates or oligomers of acidic amino acids disaggregate actin bundles into single filaments. These features are similar to the phenomenon of DNA condensation and can be explained analogously by polyelectrolyte theories. Similar results were found when F-actin was bundled by the peptide corresponding to the actin binding site of myristoylated alanine-rich protein kinase C substrate protein (MARCKS) or by smooth muscle calponin, suggesting that a broad class of actin bundling factors may function in a common manner. Physiologic concentrations of both small ions and large proteins can induce actin interfilament association independent of a requirement for specific binding sites. PMID- 8621483 TI - Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors. AB - B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers. PMID- 8621485 TI - p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1. AB - Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain. PMID- 8621484 TI - Conserved Glu318 at the cytochrome P450 1A2 distal site is crucial in the nitric oxide complex stability. AB - Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). Both NOS and P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an NO-ferric heme complex of methemoglobin, that has an imidazole-coordinated heme active site, is easily reduced. The NO complex stability of the thiolate-coordinated hemoproteins, however, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu-318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetic data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS, is largely ascribed to an ionic bridge between NO and the distal carboxyl group. PMID- 8621486 TI - Intracellular ADP modulates the Ca2+ release-activated Ca2+ current in a temperature- and Ca2+-dependent Way. AB - The rat basophilic cell line RBL-1 is known to express high levels of the Ca2+ current activated by store depletion, known as Ca2+ release-activated Ca2+ current (ICRAC), the main Ca2+ influx pathway so far identified in nonexcitable cells. We show here that, as reported in other cell types, metabolic drugs strongly inhibit the Ca2+ influx operated by store depletion in RBL-1 cells also. We have tested the hypothesis that intracellular adenine and/or guanine nucleotide levels act as coupling factors between ICRAC and cell metabolism. Using the whole cell configuration of the patch-clamp technique, we demonstrate that addition of ADP to the intracellular solution significantly reduces ICRAC induced by inositol 1,4,5-trisphosphate. This phenomenon differs from other regulatory pathways of ICRAC, since it is highly temperature-dependent, is observable only in the presence of low intracellular Ca2+ buffering capacity, and requires a cytosolic factor(s) which is rapidly lost during cell dialysis. Moreover, the inhibition is specific for ADP and is partially mimicked by ADPbetaS and AMP, but not by GDP or GTP. PMID- 8621487 TI - Differential function of Wilms' tumor gene WT1 splice isoforms in transcriptional regulation. AB - The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor that can repress transcription of a number of genes. WT1 mRNA undergoes alternative splicing at two locations, yielding four different mRNA species and protein products. One alternative splice alters the zinc finger region of WT1, resulting in the addition of three amino acids, Lys-Thr-Ser (KTS), between zinc fingers 3 and 4, altering the binding of WT1 to DNA. Here, we show that the WT1 protein with and without the KTS tripeptide can repress transcription from the human full length WT1 promoter. Repression of transcription by WT1 has been shown to require two WT1 binding sites. We examined WT1 repression of the human minimal WT1 promoter, which contains two potential WT1 binding motifs. WT1 lacking the KTS tripeptide (WT1-KTS) was unable to repress transcription from a minimal WT1 promoter of 104 base pairs, whereas WT1 containing the KTS tripeptide (WT1+KTS) repressed transcription from the minimal promoter. The ability of WT1+KTS to repress transcription where WT1-KTS could not provided a functional assay to define differential WT1 binding motifs based on the presence or the absence of the KTS tripeptides. We present data defining the differential consensus DNA binding motifs for WT1-KTS and WT1+KTS. We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1-KTS and WT1+KTS. PMID- 8621488 TI - The interaction between Ku antigen and REF1 protein mediates negative gene regulation by extracellular calcium. AB - Through the specific binding of a negative calcium-responsive element to its binding protein in response to extracellular Ca (Ca2+e), negative calcium responsive element-bearing genes, such as the human parathyroid hormone gene, are negatively regulated by Ca2+e. The Ku antigen mediated negative gene regulation by Ca2+e by interacting with a redox factor protein, REF1. Although sequence nonspecific DNA binding activity of the Ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure. Here, we report that the specific binding of the Ku antigen to another protein, REF1, leads to DNA-protein complex formation with a novel sequence specificity and thereby regulates gene expression. PMID- 8621490 TI - Structure-function relations of smooth muscle calponin. The critical role of serine 175. AB - Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin dependent protein kinase II. To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II. The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively). Tissue purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective. Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases. These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate. PMID- 8621489 TI - The human Aquaporin-5 gene. Molecular characterization and chromosomal localization. AB - The cDNA for the fifth mammalian aquaporin (AQP5) was isolated from rat, and expression was demonstrated in rat salivary and lacrimal glands, cornea, and lung (Raina, S., Preston, G. M., Guggino, W. B., and Agre, P. (1995) J. Biol. Chem. 270, 1908-1912). Here we report the isolation and characterization of the human AQP5 cDNA and gene. The AQP5 cDNA from a human submaxillary gland library contains a 795-base pair open reading frame encoding a 265-amino acid protein. The deduced amino acid sequences of human and rat AQP5 are 91% identical with 6 substitutions in the 22-amino acid COOH-terminal domain. Expression of human AQP5 in Xenopus oocytes conferred mercurial-sensitive osmotic water permeability (Pf) equivalent to other aquaporins. The human AQP5 structural gene resides within a 7. 4-kilobase SalI-EcoRI fragment with four exons corresponding to amino acids 1 121, 122-176, 177-204, and 205-265 separated by introns of 1.2, 0.5, and 0.9 kilobases. A transcription initiation site was identified 518 base pairs upstream of the initiating methionine. Genomic Southern analysis indicated that AQP5 is a single copy gene which localized to human chromosome 12q13; this coincides with the chromosomal locations of the homologous human genes MIP and AQP2, thus confirming 12q13 as the site of an aquaporin gene cluster. The mouse gene localized to distal chromosome 15. This information may permit molecular characterization of AQP5 expression during normal development and in clinical disorders. PMID- 8621491 TI - A thermally induced reversible conformational transition of the tryptophan synthase beta2 subunit probed by the spectroscopic properties of pyridoxal phosphate and by enzymatic activity. AB - A reversible thermally induced conformational transition of the beta2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectroscopic probe. Increasing the temperature converts the major form of pyridoxal 5'-phosphate bound to the beta2 subunit from a ketoenamine species with lambdamax at 410 nm to a enolimine species with lambdamax at 336 nm (Tm = approximately 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm. The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5'-phosphate binding. The internal aldimine between pyridoxal 5'-phosphate and the epsilon-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60 degrees C results in covalent attachment of [4' 3H]pyridoxal 5'-phosphate. The thermal transition of the beta2 subunit below 60 degrees C produces reversible thermal inactivation (Ti = approximately 52 degrees C) and occurs at a much lower temperature than the major reversible unfolding at approximately 80 degrees C (Remeta, D. P., Miles, E. W., and Ginsburg, A. (1995) Pure Appl. Chem. 67, 1859-1866). Our new results indicate that the 410 nm absorbing species of pyridoxal 5'-phosphate is the catalytically active form of the cofactor in the beta2 subunit and that the low temperature reversible conformational transition disturbs the active site and causes loss of catalytic activity. PMID- 8621492 TI - Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta. AB - The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-beta molecule was engineered into a secreted form and co-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT- alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms. PMID- 8621493 TI - Interactions of cellular polypeptides with the cytoplasmic domain of the mouse Fas antigen. AB - The mouse Fas/APO-1 antigen represents a 45-kilodalton transmembrane receptor that initiates apoptosis by a poorly defined signaling mechanism. The cytoplasmic domain of Fas does not display any known enzymatic activities but is capable of interacting with a number of proteins that were identified recently using the yeast interactive cloning method. To investigate direct biochemical interactions from cellular lysates prepared from Fas-responsive cells, a series of recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the mouse Fas cytoplasmic domain was used. Polypeptides of 25, 50, and 70 kilodaltons were found to associate with the Fas intracellular domain, and this binding was stable in the presence of 1 M NaCl. These interactions were also detected using a mouse Fas fusion protein containing an Ile to Asn mutation, which is responsible for a lymphoproliferative disorder in certain strains of mice (lprcg). Furthermore, the binding of cellular proteins to Fas could be blocked upon incubation with a polyclonal antibody directed against the cytoplasmic domain of Fas. The strong association of cellular proteins with the cytoplasmic region implies that constitutive interactions may exist to regulate apoptotic signaling through the Fas antigen. PMID- 8621494 TI - Loop replacement and random mutagenesis of omega-loop D, residues 70-84, in iso-1 cytochrome c. AB - To study the role of omega loop D, residues 70-84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly 77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1 cytochrome c are discussed. PMID- 8621495 TI - A non-AUG translational initiation event generates novel WT1 isoforms. AB - The Wilms' tumor (WT) suppressor gene, WT1, is mutated in a small set of WTs and is essential for proper development of the urogenital system. The gene has three sites of transcriptional initiation and produces mRNA transcripts containing 5' untranslated regions of more than 350 nucleotides. The mRNA, through two alternative splicing events, is predicted to direct the synthesis of four protein isoforms with molecular masses of 47-49 kDa. In this report, we identify and characterize novel WT1 protein isoforms having predicted molecular masses of 54 56 kDa. Mutational analysis of the murine wt1 mRNA demonstrates that the novel isoforms are the result of translation initiation at a CUG codon 204 bases upstream of and in frame with the initiator AUG. We show that these isoforms are present in both normal murine tissue and in WTs. Like WT1, the larger isoforms localize to the cell nucleus and are capable of mediating transcriptional repression. Our results indicate that regulation of WT1 gene expression is more complex than previously suspected and have important implications for normal and abnormal urogenital system development. PMID- 8621496 TI - Partial C-terminal unfolding is required for channel formation by staphylococcal alpha-toxin. AB - The pore-forming alpha-toxin from Staphylococcus aureus is secreted as a soluble monomeric protein. In order to form a transmembrane channel, the protein has to undergo oligomerization and membrane insertion. Previous studies have shown that channel formation is favored by acidic pH. We have analyzed the effect of pH on the kinetics of channel formation as well as on the conformation of the toxin. Using a variety of spectroscopic probes for protein structure, we have shown that alpha-toxin unfolded upon acidification and that the unfolding process occurred in at least three steps. The various steps could be selectively affected by modifying the salt concentration or the temperature. This unfolding was, however, only partial as the secondary structure remained native-like as witnessed by far UV CD measurements. The first unfolding step, corresponding to a region of the C terminal half of the toxin, is of particular importance as it coincided with the exposure of hydrophobic patches on the surface of the protein as well as with the onset of channel formation. Our observations strongly suggest that transition of the C-terminal half of alpha-toxin to a molten globule-like state is required for channel formation. PMID- 8621497 TI - Phosphorylation of the DNA polymerase alpha-primase B subunit is dependent on its association with the p180 polypeptide. AB - The B subunit of the DNA polymerase (pol) alpha-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180.p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180.p86 subcomplex is required for B subunit phosphorylation. PMID- 8621498 TI - In vitro transcription of the rat insulin-like growth factor-I gene. AB - Although the liver is the major source of circulating insulin-like growth factor I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are initiated largely in exon 1, we established an in vitro transcription system to evaluate activation of transcription via the major exon 1 initiation site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter was used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared with liver. 54 base pairs (bp) of upstream sequence were sufficient to direct IGF I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and 60 bp; binding was confirmed by gel shift analysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to such sites was established by competition analysis, which revealed a specific decrease in IGF-I transcription in the presence of a competing fragment. Use of this in vitro transcription system should permit analysis of the function of individual transcription factors involved in regulation of IGF-I gene expression. PMID- 8621500 TI - The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness. AB - Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness. PMID- 8621499 TI - Non-catalytic beta- and gamma-subunit isoforms of the 5'-AMP-activated protein kinase. AB - The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta-, and gamma-subunits. The alpha-subunit is the catalytic subunit and is related to the yeast Snf1p kinase. In this study, we report the cloning of full-length cDNAs for the non-catalytic beta- and gamma subunits. The rat liver AMPK beta-subunit clone predicts a protein of 30,464 Da, which is related to the Sip1p, Sip2p, and Gal83p subfamily of yeast proteins that interact with Snf1p and are involved in glucose regulation of gene expression. The AMPK beta-subunit, when expressed in bacteria and in mammalian cells, migrates anomalously on SDS gels at an apparent molecular mass of 40 kDa. Rat and human liver AMPK gamma-subunit clones predict a protein of 37,577 Da (AMPK gamma1), which is related to the yeast Snf4p protein that copurifies with Snf1p and to a larger family of other human AMPK gamma-isoforms. The mRNAs for both AMPK- beta and AMPK-gamma1 are widely expressed in rat tissues, consistent with a broad role for AMPK in cellular regulation. These data reveal a mammalian multisubunit protein kinase strikingly similar to the multisubunit glucose sensing Snf1 kinase complex. The identification of isoform families for the AMPK subunits indicates the potential diversity of the roles of this highly conserved signaling system in nutrient regulation and utilization in mammalian cells. PMID- 8621501 TI - Bradyrhizobium japonicum porphobilinogen synthase uses two Mg(II) and monovalent cations. AB - Bradyrhizobium japonicum porphobilinogen synthase (B. japonicum PBGS) has been purified and characterized from an overexpression system in an Escherichia coli host (Chauhan, S., and O'Brian, M. R. (1995) J. Biol. Chem. 270, 19823-19827). B. japonicum PBGS defines a new class of PBGS protein, type IV (classified by metal ion content), which utilizes a catalytic MgA present at a stoichiometry of 4/octamer, an allosteric MgC present at a stoichiometry of 8/octamer, and a monovalent metal ion, K+. However, the divalent MgB or ZnB present in some other PBGS is not present in B. japonicum PBGS. Under optimal conditions, the Kd for MgA is <0.2 microM, and the Kd for MgC is about 40 microM. The response of B. japonicum PBGS activity to monovalent and divalent cations is mutually dependent and varies dramatically with pH. B. japonicum PBGS is also found to undergo a dynamic equilibrium between active multimeric species and inactive monomers under assay conditions, a kinetic characteristic not reported for other PBGSs. B. japonicum PBGS is the first PBGS that has been rigorously demonstrated to lack a catalytic ZnA. However, consistent with prior predictions, B. japonicum PBGS can bind Zn(II) (presumably as ZnA) at a stoichiometry of 4/octamer with a Kd of 200 microM; but this high concentration is outside a physiologically significant range. PMID- 8621503 TI - T cell antigen receptor ubiquitination is a consequence of receptor-mediated tyrosine kinase activation. AB - Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptor-mediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56lck. The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors. PMID- 8621502 TI - In vitro metabolism of the vitamin D analog, 22-oxacalcitriol, using cultured osteosarcoma, hepatoma, and keratinocyte cell lines. AB - Using four cultured cell models representing liver, keratinocyte, and osteoblast, we have demonstrated that the vitamin D analog, 22-oxacalcitriol is degraded into a variety of hydroxylated and side chain truncated metabolites. Four of these metabolic products have been rigorously identified by high pressure liquid chromatography, diode array spectrophotometry, and gas chromatography-mass spectrometry analysis as 24-hydroxylated and 26-hydroxylated derivatives as well as the cleaved molecules, hexanor-1alpha,20-dihydroxyvitamin D3 and hexanor-20 oxo-1alpha-hydroxyvitamin D3. Comparison with chemically synthesized standards has revealed the stereochemistry of the biological products. Although differences exist in the amounts of products formed with the different cell types, it is apparent that 22-oxacalcitriol is subject to metabolism by both vitamin D inducible and noninducible enzymes. Time course studies suggest that the truncated 20-alcohol is derived from a side chain hydroxylated molecule via a hemiacetal intermediate and the 20-oxo derivative is likely formed from the 20 alcohol. Biological activity measurements of the metabolites identified in our studies are consistent with the view that these are catabolites and that the biological activity of 22-oxacalcitriol is due to the parent compound. These results are also consistent with recent findings of others that the biliary excretory form of 22-oxacalcitriol is a glucuronide ester of the truncated 20 alcohol. PMID- 8621504 TI - Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme. AB - Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity. PMID- 8621505 TI - Genistein is a natural inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter, GLUT1. AB - Genistein is a dietary-derived plant product that inhibits the activity of protein-tyrosine kinases. We show here that it is a potent inhibitor of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, genistein inhibited the transport of dehydroascorbic acid, deoxyglucose, and methylglucose in a dose-dependent manner. Transport was not affected by daidzein, an inactive genistein analog that does not inhibit protein tyrosine kinase activity, or by the general protein kinase inhibitor staurosporine. Genistein inhibited the uptake of deoxyglucose and dehydroascorbic acid in Chinese hamster ovary (CHO) cells overexpressing GLUT1 in a similar dose dependent manner. Genistein also inhibited the uptake of deoxyglucose in human erythrocytes indicating that its effect on glucose transporter function is cell independent. The inhibitory action of genistein on transport was instantaneous, with no additional effect observed in cells preincubated with it for various periods of time. Genistein did not alter the uptake of leucine by HL-60 cells, indicating that its inhibitory effect was specific for the glucose transporters. The inhibitory effect of genistein was of the competitive type, with a Ki of approximately 12 microM for inhibition of the transport of both methylglucose and deoxyglucose. Binding studies showed that genistein inhibited glucose displaceable binding of cytochalasin B to GLUT1 in erythrocyte ghosts in a competitive manner, with a Ki of 7 microM. These data indicate that genistein inhibits the transport of dehydroascorbic acid and hexoses by directly interacting with the hexose transporter GLUT1 and interfering with its transport activity, rather than as a consequence of its known ability to inhibit protein tyrosine kinases. These observations indicate that some of the many effects of genistein on cellular physiology may be related to its ability to disrupt the normal cellular flux of substrates through GLUT1, a hexose transporter universally expressed in cells, and is responsible for the basal uptake of glucose. PMID- 8621506 TI - Characterization and analysis of conserved motifs in a peroxisomal ATP-binding cassette transporter. AB - The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. Both are implicated in genetic disorders of peroxisome biogenesis and function. Proteins homologous to ALDP and the 70-kDa peroxisomal membrane protein have been discovered in other eukaryotic organisms and form a growing group of peroxisomal half ABC transporters. Amino acid sequence alignment of these and other ABC transporters reveals several protein motifs that are highly conserved both in sequence and location. Here we characterize two of these, designated the EAA-like and the loop1 motifs. We study them by introducing missense mutations in Pxa1p, a Saccharomyces cerevisiae ortholog of ALDP, and show that both motifs are important for Pxa1p function. Interestingly, missense mutations in corresponding amino acids in ALDP cause adrenoleukodystrophy in humans. We conclude that these motifs are important for ABC transporter function and that the yeast protein Pxa1p is a useful system for understanding the molecular basis of adrenoleukodystrophy. PMID- 8621507 TI - Deglycosylated products of endogenous digoxin-like immunoreactive factor in mammalian tissue. AB - Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plant derived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr. (1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 10(3)-fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have lambdamax at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals. PMID- 8621508 TI - Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx. AB - Glucose stimulation raises the pHi of pancreatic beta-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high K+ and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pHi of pancreatic beta-cells isolated from normal mice, as assessed by BCECF fluorescence from single cells or islets in the presence of external bicarbonate. The typical acute effect of glucose (22-30 mM) on the pHi was a fast alkalinization of approximately 0.11 unit, followed by a slower acidification. The relative expression of the alkalinizing and acidifying components was variable, with some cells and islets displaying a predominant alkalinization, others a predominant acidification, and others yet a mixed combination of the two. The initial alkalinization preceded the [Ca2+]i rise associated with the activation of voltage-sensitive Ca2+ channels. There was a significant overlap between the glucose-evoked [Ca2+]i rise and the development of the secondary acidification. Depolarization with 30 mM K+ and tolbutamide evoked pronounced [Ca2+]i rises and concomitant cytosolic acidifications. Blocking glucose-induced Ca2+ influx (with 0 Ca2+, nifedipine, or the K-ATP channel agonist diazoxide) suppressed the secondary acidification while having variable effects (potentiation or slight attenuation) on the initial alkalinization. KIC exerted glucose-like effects on the pHi and [Ca2+]i, but the amplitude of the initial alkalinization was about twice as large for KIC relative to glucose. It is concluded that the acute effect of glucose on the pHi of pancreatic beta-cells is biphasic. While the initial cytosolic alkalinization is an immediate consequence of the activation of H+-consuming metabolic steps in the mitochondria, the secondary acidification appears to originate from enhanced Ca2+ turnover in the cytoplasm. The degree of coupling between glucose metabolism and Ca2+ influx as well as the relative efficacies of these processes determines whether the acute pHi response of a beta-cell (or of a tightly coupled multicellular system such as an islet of Langerhans) is predominantly an alkalinization, an acidification, or a mixed proportion of the two. PMID- 8621509 TI - Post-transcriptional regulation of chymase expression in mast cells. A cytokine dependent mechanism for controlling the expression of granule neutral proteases of hematopoietic cells. AB - Although all mouse mast cells are derived from a common progenitor, these effector cells exhibit tissue-specific differences in their expression of the chymase family of serine proteases whose genes reside on chromosome 14. Immature bone marrow-derived mast cells (mBMMC), developed in vitro with interleukin (IL) 3-enriched medium, were cultured in the presence or absence of IL-10 to determine at the molecular level how the expression of the individual chymases is differentially regulated. As assessed by RNA blot analysis, mBMMC contain high steady-state levels of the transcript that encodes mouse mast cell protease (mMCP) 5, but not the homologous chymase transcripts that encode mMCP-1, mMCP-2, or mMCP-4. Nevertheless, nuclear run-on analysis revealed that these cells transcribe all four mast cell chymase genes. IL-10 elicited high steady-state levels of the mMCP-2 transcript, and pulse-chase experiments revealed that the half-life of the mMCP-2 transcript in mBMMC maintained in the presence of IL-10 is approximately 4-fold longer than that in replicate cells subsequently cultured in medium without IL-10. Reverse transcription-polymerase chain reaction/nucleotide sequence analysis demonstrated that mBMMC cultured in the absence or presence of IL-10 correctly process mMCP-2 pre-mRNA. Experiments with cycloheximide and actinomycin D indicated that IL-10 induces expression of a trans-acting factor(s) that stabilizes the mMCP-2 transcript or facilitates its processing. The discovery that the expression of certain chymases in mBMMC is regulated primarily at the post-transcriptional level provides a basis for understanding the mechanism by which specific cytokines dictate expression of the chromosome 14 family of serine proteases in cells that participate in inflammatory processes. PMID- 8621510 TI - The role of the dodecamer subunit in the dissociation and reassembly of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin. AB - The dissociation of the approximately 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39]8- (SiW), [NaSb9W21O86]18- (SbW) and [BaAs4W40O140]27- (AsW) at neutral pH was followed by gel filtration, SDS polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (approximately 200 kDa), T+L (approximately 50 kDa), and M (approximately 25 kDa) (T = disulfide bonded trimer of chains a c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm*SCN > Gdm.Cl > urea > Gdm.OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed approximately 10-nm particles with masses of approximately 200 kDa, suggesting them to be dodecamers (a+b+c)3d3. OxyHb dissociations in urea and Gdm.Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (2-linked N acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N acetylglucosaminyltransferase or a hexosaminidase that could remove N acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids. PMID- 8621526 TI - Influence of the phosphate backbone on the recognition and hydrolysis of DNA by the EcoRV restriction endonuclease. A study using oligodeoxynucleotide phosphorothioates. AB - A set of phosphorothioate-containing oligonucleotides based on pGACGATATCGTC, a self-complementary dodecamer that contains the EcoRV recognition sequence (GATATC), has been prepared. The phosphorothioate group has been individually introduced at the central nine phosphate positions and the two diastereomers produced at each site separated and purified. The Km and Vmax values found for each of these modified DNA molecules with the EcoRV restriction endonuclease have been determined and compared with those seen for the unmodified all-phosphate containing dodecamer. This has enabled an evaluation of the roles that both of the non-esterified oxygen atoms in the individual phosphates play in DNA binding and hydrolysis by the endonuclease. The results have also been compared with crystal structures of the EcoRV endonuclease, complexed with an oligodeoxynucleotide, to allow further definition of phosphate group function during substrate binding and turnover. For further study, see the related article "Probing the Indirect Readout of the Restriction Enzyme EcoRV: Mutational Analysis of Contacts to the DNA Backbone" (Wenz, A., Jeltsch, A., and Pingoud, A. (1996) J. Biol. Chem. 271, 5565-5573). PMID- 8621527 TI - Structure of the heme d of Penicillium vitale and Escherichia coli catalases. AB - A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction. PMID- 8621528 TI - The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. I. Reexamination of the number and masses of its constituents. AB - The deep-sea tube worm Riftia pachyptila Jones possesses a well developed circulatory system and a large coelomic compartment, both containing extracellular hemoglobins. Fresh vascular blood is heterogeneous and contains two different hemoglobins (V1 and V2), whereas the coelomic fluid is homogeneous and comprises only one hemoglobin (C1). Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi angle laser light scattering (MALLS). Both methods yielded approximately the same molecular weights with masses significantly higher than the literature data for V1. V1, V2, and C1 had Mr of 3396 +/- 540 x 10(3), 393 +/- 71 x 10(3), and 410 +/ 51 x 10(3) by STEM, and 3503 +/- 13 x 10(3), 433 +/- 8 x 10(3), and 380 +/- 4 x 10(3) by MALLS, respectively. Transmission electron micrographs of V1 are typical of an hexagonal bilayer hemoglobin (HBL Hb). When submitted to dilution or osmotic shock, V1 dissociates into halves and one-twelfth subunits like annelid HBL Hbs. V1 is resistant to urea treatment, indicating that hydrophobic interactions play a small role in its quaternary structure. Conversely, V1 Hb is rather unstable in solution without denaturant, a property which seems to be characteristic of vestimentiferan HBL Hbs and could be explained by an important number of hydrogen bonds. PMID- 8621529 TI - The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. II. Complete polypeptide chain composition investigated by maximum entropy analysis of mass spectra. AB - The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (approximately 3500 kDa) and V2 (approximately 400 kDa), and one in the coelomic cavity, C1 (approximately 400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at approximately 16, 23 27, and 30 kDa, corresponding to (i) two monomeric globin chains, b (Mr 16,133.5) and c (Mr 16,805.9); (ii) four linker subunits, L1 L4 (Mr 23,505.2, 23,851.4, 26,342.4, and 27,425.8, respectively); and (iii) one disulfide-bonded dimer D1 (Mr 31,720.7) composed of globin chains d (Mr 15,578.5) and e (Mr 16, 148.3). V2 and C1 Hbs had no linkers and contained a glycosylated monomeric globin chain, a (Mr 15,933.4) and a second dimer D2 (Mr 32,511.7) composed of chains e and f (Mr 16,368.1). The dimer D1 was absent from C1 Hb, clearly differentiating V2 and C1 Hbs. These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. The following models are proposed ((cD1)(bD1)3) for the one-twelfth protomer of V1 Hb, ((cD)(bD)6(aD)) (D corresponding to either D1 or D2) for V2 and C1 Hbs. HBL V1 Hb would be composed of 180 polypeptide chains with 144 globin chains and 36 linker chains, each twelfth being in contact with three linker subunits, providing a total molecular mass = 3285 kDa. V2 and C1 would be composed of 24 globin chains providing a total molecular mass = 403 kDa and 406 kDa, respectively. These results are in excellent agreement with experimental Mr determined by STEM mass mapping and MALLS. PMID- 8621530 TI - Interaction between the Grb10 SH2 domain and the insulin receptor carboxyl terminus. AB - Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor beta-subunit using the yeast two hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding. PMID- 8621531 TI - The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity. AB - We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (Deltaper9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced Deltaper9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane. PMID- 8621532 TI - Variant AE2 anion exchanger transcripts accumulate in multiple cell types in the chicken gastric epithelium. AB - Molecular analyses have resulted in the isolation of two chicken stomach AE2 anion exchanger cDNAs, AE2-1 and AE2-2. The approximately 4.3-kilobase (kb) AE2-1 cDNA contains an open reading frame that encodes a predicted polypeptide of approximately 135 kDa that is homologous to AE2 anion exchangers from other species. The partial approximately 1.7-kb AE2-2 cDNA, which differs from the AE2 1 cDNA in two regions, would be predicted to encode an AE2 polypeptide with an alternative N-terminal cytoplasmic tail. Examination of the distribution of these variant transcripts has revealed that AE2 transcripts ranging in size from approximately 4.4 to approximately 7.3 kb accumulate in various adult tissues. However, in the stomach, the unique sequence at the 5'-end of AE2-1 is preferentially associated with transcripts that range in size from approximately 4.5 to approximately 4.9 kb, while the unique sequence at the 5'-end of AE2-2 is preferentially associated with the approximately 7.3-kb AE2 RNA species. In situ hybridization analyses have further revealed that AE2 transcripts accumulate to very high levels within the acid-secreting epithelial cells of the profound gland in the stomach and, to a lesser extent, within the mucus-secreting cells of the superficial gland that line the stomach lumen. This result suggests that AE2 anion exchangers are involved in the regulation of intracellular pH in each of these gastric epithelial cell types. PMID- 8621533 TI - Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome. AB - In yeast and humans, nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA requires a large number of highly conserved protein factors, which include the multisubunit RNA polymerase II transcription factor TFIIH. Here, we examine whether NER occurs by sequential assembly of different repair factors at the site of DNA damage or by the placement there of a "preformed" repairosome containing TFIIH and all the other essential NER factors. Contrary to the recent report (Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1995) Cell 80, 21-28), our results provide no evidence for a pre-assembled repairosome; instead, they support the sequential assembly model. By several independent criteria, including co purification, immunoprecipitation, and gel filtration of homogeneous proteins, we show that the damage recognition factor Rad14 exists in a ternary complex with the Rad1-Rad10 nuclease. We also find that Rad14 interacts directly with Rad1, but only slightly with Rad10, and that it interacts with the Rad1-Rad10 complex much more efficiently than with Rad1 alone. In the reconstituted NER system, a higher level of incision of UV-damaged DNA is achieved with the Rad1-Rad10-Rad14 complex, which we designate as nucleotide excision repair factor-1, NEF-1. PMID- 8621534 TI - Hormonally regulated double- and single-stranded DNA-binding complexes involved in mouse beta-casein gene transcription. AB - Transcription of the 252-base pair-long mouse beta-casein gene promoter is induced by the synergistic action of insulin, prolactin, and glucocorticoid in a primary mammary epithelial cell culture. The promoter contains a region termed block C having a highly conserved sequence and position among many casein genes. Mutation of block C reduced the response of the promoter to lactogenic hormones 84%. Nuclear extracts from lactating mouse mammary glands contained both a double stranded and a single-stranded DNA binding protein complex (DS1 and SS), which specifically bind to the sequences AAATTAGCATGT and CCACAA of block C, respectively. The DS1 and the SS protein complexes were approximately 400 and 280 kDa, respectively. Each complex contained a DNA-binding component(s) having a molecular mass of approximately 120 kDa for DS1 and 80 and 65 kDa for SS. Deoxycholate, which interferes with the protein-protein interactions, inhibited the binding activities of DS1 and SS. The maximal increase in the binding activity of DS1 and SS in the mammary gland occurred during pregnancy and during lactation, respectively. In organ culture, the DS1 activity is increased by epidermal growth factor or prolactin in combination with insulin, whereas the SS activity is enhanced by insulin, prolactin, and glucocorticoid. These results suggest that multiprotein complexes binding to the double- and single-stranded DNA of block C mediate hormonal induction of beta-casein gene transcription. PMID- 8621535 TI - Generation of 8-epiprostaglandin F2alpha by human monocytes. Discriminate production by reactive oxygen species and prostaglandin endoperoxide synthase-2. AB - F2-isoprostanes are free radical-catalyzed products of arachidonic acid. One of these compounds, 8-epiprostaglandin F2alpha (8-epi-PGF2alpha), is a mitogen and vasoconstrictor. We have shown that 8-epi-PGF2 alpha, unlike other F2 isoprostanes, is a minor product of the prostaglandin endoperoxide synthase-1 (PG G/H S-1) expressed in human platelets (Pratico, D., Lawson, J. A., and Fitzgerald, G. A. (1995) J. Biol. Chem. 270, 9800-9808). Human monocytes express PG G/H S-1 constitutively and exhibit regulated expression of PG G/H S-2. Induction of PG G/H S-2 by concanavalin A, the phorbol ester, phorbol 12 myristate 13-acetate, and bacterial lipopolysaccharide was confirmed with a specific antibody in monocytes pretreated with aspirin to inhibit PG G/H S-1. Induction of PG G/H S-2 by all three stimuli coincided with increased formation of prostaglandin E2 (PGE2), thromboxane B2 (TxB2), and 8-epi-PGF2 alpha, but not of other F2-isoprostanes. Confirmation of PG G/H S-2 as the source of 8-epi-PGF2 alpha formation was obtained by down-regulating the enzyme with dexamethasone; preventing protein synthesis with cycloheximide; and preventing synthesis of PGE2, TxB2, and 8-epi-PGF2 alpha with the specific PG G/H S-2 inhibitor, L 745,337. Monocytes also exhibit the facility to generate 8-epi-PGF2 alpha in a free radical-dependent manner. Thus, stimulation with opsonized zymosan or coincubation with low density lipoprotein was unassociated with product formation. However, coincubation of low density lipoprotein with zymosan stimulated human monocytes resulted in marked formation of 8-epi-PGF2alpha, but not of PGE2 or TxB2. Production of 8-epi-PGF2 alpha coincided with that of thiobarbituric acid-reactive substances and lipid hydroperoxides, but was unaccompanied by PG G/H S-2 induction. Pretreatment of monocytes with the antioxidant, butylated hydroxytoluene or with superoxide dismutase, but not with L 745,337, suppressed formation of 8-epi-PGF2alpha, thiobarbituric acid-reactive substances, and lipid hydroperoxides. In conclusion, human monocytes may form bioactive 8-epi-PGF2alpha either via free radical- or enzyme-catalyzed pathways. 8-Epi-PGF2alpha is a more abundant product of monocyte PG G/H S-2 than of platelet PG G/H S-1. Formation by inducible PG G/H S-2 must be considered as a source of this compound in vivo. PMID- 8621536 TI - Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina. cDNA and deduced amino acid sequences. AB - The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin 44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein. PMID- 8621537 TI - The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. AB - The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA PKcs. Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity. PMID- 8621538 TI - Interactions of a transcriptional activator in the env gene of the mouse mammary tumor virus with activation-dependent, T cell-specific transacting factors. AB - The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas. PMID- 8621539 TI - ERK3 is a constitutively nuclear protein kinase. AB - The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp171) expected to be involved in catalysis eliminated autophosphorylation. Ser189 of ERK3, which corresponds to Thr183, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions. PMID- 8621540 TI - Direct binding of C-terminal region of p130Cas to SH2 and SH3 domains of Src kinase. AB - p130Cas is a major tyrosine-phosphorylated protein that tightly binds v-Crk in v crk-transformed cells and v-Src in v-src-transformed cells. The "substrate domain" of p130Cas contains 15 possible Src homology (SH) 2-binding motifs, most of which conform to the binding motif for the Crk SH2 domain. Another region near its C terminus contains possible binding motifs for the Src SH2 domain and proline-rich sequences that are candidates for SH3-binding sites. Using GST fusion proteins, we revealed that both SH2 and SH3 domains of Src bind p130Cas, whereas v-Crk binds p130Cas through its SH2 domain. We located the binding site of p130Cas for the Src SH3 domain at the sequence RPLPSPP in the region near its C terminus. Mutations within this sequence or at Tyr762 of p130Cas caused a significant reduction in the association of p130Cas with Src, and no association was detected when both of them were deleted. The kinase activity in v-Crk transformed cells was also associated with p130Cas through this region. On the other hand, the deletion of the substrate domain abolished the binding with v Crk. The association through the C-terminal region of p130Cas with Src kinase may facilitate effective hyperphosphorylation of tyrosine residues in the substrate domain of p130Cas, resulting in the binding of SH2-containing molecules to p130Cas. PMID- 8621541 TI - Amyloids beta40 and beta42 are generated intracellularly in cultured human neurons and their secretion increases with maturation. AB - Previous studies have demonstrated the presence of amyloid beta (Abeta) in neurons (NT2N) derived from a human embryonal carcinoma cell line (NT2) by steady state metabolic radiolabeling and immunoprecipitation. We show here that Abeta is present intracellularly since trypsin digestion of intact NT2N cells at 4 degrees C did not eliminate the Abeta recovered in cell lysates. To determine whether both Abeta40 and Abeta42 are produced intracellularly, quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was performed using COOH-terminal end specific anti-Abeta monoclonal antibodies. Sandwich ELISA detected intracellular Abeta40 and A++beta42 in NT2N cell lysates at a ratio of 3:1, whereas secreted Abeta40 and Abeta42 were recovered in medium conditioned by NT2N cells at a ratio of approximately 20:1. Metabolic steady state and pulse-chase labeling studies demonstrated a 2-h delay in the detection of cell-associated Abeta40/Abeta42 in the medium, suggesting that Abeta is generated at a slow rate intracellularly prior to its secretion. Finally, as NT2N cells mature over time in culture, the secretion of Abeta40 and Abeta42 increases more than 5-fold over 7 weeks. This increase in the secretion of Abeta40/Abeta42 in NT2N cells as a function of time may recapitulate a similar phenomenon in the aging brain. PMID- 8621542 TI - Interaction between c-Rel and the mitogen-activated protein kinase kinase kinase 1 signaling cascade in mediating kappaB enhancer activation. AB - The Rel family of transcription factors are important mediators of various cytokine stimuli such as interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and CD28 costimulation in T cell effector responses. These stimuli induce Rel family DNA-binding activity to the kappaB enhancer and CD28 response elements of many cytokine gene promoters leading to cytokine production. Consistent with the importance of Rel family induction during immune responses, c-Rel knockout mice exhibit profound defects in T cell functions including IL-2 secretion and T cell proliferative responses to CD28 plus T cell receptor costimulation. The novel protein kinases, c-Jun NH2-terminal kinases (JNKs)/stress-activated protein kinases, are also activated by TNF-alpha, IL-1, and CD28 costimulation. Because of the common regulation of c-Rel and JNK1 by these agents in T cells, we investigated the role of JNK1 in c-Rel activation. We found that MAP kinase kinase kinase (MEKK) 1, a JNK1 activator, induced transcription from the human immunodeficiency virus-1 long terminal repeat and IL-2R alpha promoters in a kappaB-dependent manner. Coexpression of IkappaBalpha, a c-Rel inhibitor, inhibited the MEKK1-induced transcriptional activity. JNK1 synergized with MEKK1 in activating transcription from a kappaB-driven heterologous promoter. Furthermore, JNK1 associated with c-Rel in vivo in Jurkat T cells by coimmunoprecipitation assays and bound directly to c-Rel in a yeast two-hybrid assay. c-Rel also competed with c-Jun in in vitro kinase assays. However, JNK1 did not phosphorylate c-Rel, NF-kappaB, and IkappaB alpha in vitro, indicating that c-Rel may serve as a docking molecule to allow JNK1 phosphorylation of certain Rel-associated proteins. Transactivation of the IL-2Ralpha and HIV-kappaB driven promoters by c-Rel was augmented by coexpression of MEKK1. These results demonstrate the first significant role for the MEKK1 kinase cascade module in c Rel-mediated transcription. PMID- 8621543 TI - Alpha1 adrenergic receptors activate phosphatidylinositol 3-kinase in human vascular smooth muscle cells. Role in mitogenesis. AB - Activation of alpha1 adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of alpha1 receptors may induce mitogenesis in HVSMCs, we have found that alpha1 receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of alpha1 receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85 alpha subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration dependent activation of PI 3-kinase in the presence of a beta adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of alpha1 receptors and by pertussis toxin, suggesting that alpha1 receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca2+ L-channel blocker did not inhibit noradrenaline-stimulated PI 3 kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of alpha1 receptors activates PI 3-kinase in HVSMCs and that alpha1 receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to alpha1 receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs. PMID- 8621544 TI - A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate. AB - The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (approximately 75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported. The association of a small proportion of eIF4E with the ribosome fraction appears to be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate. PMID- 8621545 TI - The CD95 (APO-1/Fas) receptor activates NF-kappaB independently of its cytotoxic function. AB - Engagement of the CD95 (APO-1/Fas) receptor induces apoptosis in a variety of cell types. However, the nature of the cytotoxic signal and the intermediate messenger molecules remain to be elucidated. In an effort to understand CD95 mediated signaling, we assessed possible changes in the DNA binding activity of NF-kappaB as a result of CD95 engagement in various tumor cells. By performing electrophoresis mobility shift assays, we show that CD95 can stimulate the DNA binding activity of NF-kappaB in a variety of cells, irrespective of their sensitivity or resistance to CD95-mediated cytotoxicity. Moreover, deletion of 37 carboxyl-terminal residues from the cytoplasmic domain of CD95, which abrogates CD95-mediated apoptosis, only marginally affects NF-kappaB activation. Taken together, these observations indicate that CD95 has a function that involves activation of NF-kappaB and that appears to be unrelated to its role as an inducer of apoptotic cell death. PMID- 8621546 TI - Reconstitution of barley photosystem I with modified PSI-C allows identification of domains interacting with PSI-D and PSI-A/B. AB - The PSI-C subunit of photosystem I shows similarity to soluble 2[4Fe-4S] ferredoxins. Alignment analysis clearly shows that PSI-C contains an 8-residue internal loop and a 15-residue C-terminal extension that are absent in the ferredoxins. The remaining residues in PSI-C are likely to be folded in a way similar to the soluble 2[4Fe-4S] ferredoxins. Two modified PSI-C subunits lacking either the 8-residue loop or 10 residues of the C terminus were expressed in Escherichia coli and used to reconstitute a barley P700-FX core prepared to specifically lack PSI-C, PSI-D, and PSI-E. As shown by EPR spectroscopy, the modified proteins carry two [4Fe-4S] clusters with characteristics similar to those of native PSI-C. Western blot analysis of the reconstituted photosystem I complexes showed that the modified PSI-C proteins bind to the P700-FX core. Flash photolysis revealed that in photosystem I complexes reconstituted in the presence of PSI-D with the C-terminally deleted PSI-C, the FA/FB back-reaction was less efficiently restored than with wild-type PSI-C. The loop-deleted PSI-C was even less efficient. We attribute these differences to altered binding properties of the modified proteins. Comparison of reconstitutions performed in the presence and absence of PSI-D shows that the loop-deleted PSI-C is unable to bind without PSI-D, whereas the C-terminally deleted PSI-C binds only weakly with PSI-D. These results imply that the internal loop of PSI-C interacts with the PSI-A/B heterodimer and that the C terminus of PSI-C interacts with PSI-D. PMID- 8621548 TI - Human p300 protein is a coactivator for the transcription factor MyoD. AB - Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation. We show that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in MyoD-dependent transactivation. In addition, p300 is able to interact both in vivo and in vitro with MyoD through a portion at the carboxyl-terminal cysteine/histidine-rich domain and associates with the components of the basal transcriptional complex through its two separate transactivation domains at the amino and carboxyl termini. Consistent with its role as a coactivator, p300 potentiates MyoD-activated transcription. PMID- 8621547 TI - Morphogen-induced decline in Gialpha2 triggers F9 teratocarcinoma stem cell progression via phospholipase C and mitogen-activated protein kinase. AB - The linkage between Gialpha2 and morphogen-induced promotion of F9 embryonic teratocarcinoma stem (F9 stem) cells to primitive endoderm was explored using probes of the mitogen-activated protein (MAP) kinase network. The morphogen induced decline in Gialpha2 is shown to trigger activation of phospholipase C, thereby activating protein kinase C, MAP kinase, and cell progression to primitive endoderm. In the absence of retinoic acid, reduction-of-function mutants (Gialpha2-deficient) display the effects of morphogen, i.e. activation of phospholipase C, protein kinase C, MAP kinase, and progression to primitive endoderm. Gain-of-function mutants (expressing the Q205L activating-mutation of Gialpha2) displayed no activation of phospholipase C, protein kinase C, MAP kinase and no progression to primitive endoderm, even in the presence of retinoic acid. Selective inhibitors of protein kinase C, like the gain-of-function mutations, effectively block morphogen-induced progression to primitive endoderm. Morphogen triggers F9 stem cell progression by triggering Gialpha2 loss and thereby activation of downstream elements, including protein kinase C and MAP kinase. PMID- 8621549 TI - Alterations in the promoter-specific imprinting of the insulin-like growth factor II gene in Wilms' tumor. AB - The human IGF2 gene, which encodes a mitogenic peptide required for normal fetal development, is overexpressed in many types of tumors. IGF2 is transcribed from four promoters (P1-P4), and in most tissues, the gene is imprinted. In this study, we have analyzed IGF2 promoter usage and determined the allelic expression from each promoter in 19 ApaI- and 22 AluI-heterozygous Wilms' tumors. Loss of IGF2 imprinting (LOI) was observed in 8 ApaI-informative tumors. In these tumors, each parental allele was expressed in equal abundance, indicating that there was complete relaxation of IGF2 imprinting. In each LOI tumor, expression from promoter P1 as well as from the normally imprinted promoters P2-P4 was biallelic. In the 11 ApaI-informative tumors which maintained IGF2 imprinting (maintenance of imprinting), transcription from promoters P2-P4 was always monoallelic, while transcripts from P1 were derived from either one or both alleles. The lack of consistency of IGF2 imprinting of promoter P1 in maintenance of imprinting tumors was also observed in normal fetal tissues of 6 12 weeks gestation, suggesting a similarity in IGF2 regulation between Wilms' tumors and embryonic tissue development. These data suggest that the increased expression of IGF2 in Wilms' tumor may be caused either by biallelic gene expression in LOI tumors from promoters P2-P4 and/or by a reversion to an earlier stage of development which is characterized by increased synthesis of this fetal growth factor. PMID- 8621550 TI - Hepatocyte nuclear factor-4 is responsible for the liver-specific expression of the gene coding for hepatocyte growth factor-like protein. AB - In an attempt to understand the molecular mechanism regulating the expression of the gene coding for human hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL), our laboratory has isolated and characterized approximately 4200 bp of the 5'-flanking region of the HGFL gene. To determine the location of sites which may be critical for the function of the HGFL gene promoter, we constructed a series of hybrid genes containing serial deletions of this region attached to the coding sequences for chloramphenicol acetyltransferase. Expression of these chimeric plasmids was examined by transient transfection of HepG2 and 293 cells. Our results suggest that the transcriptional activity of the HGFL promoter is modulated in HepG2 cells by one positive element at position -135 to -105 (-135/-105). In contrast, only background levels of chloramphenicol acetyltransferase expression have been detected in 293 cells. The -135/-105 region appears to bind a liver-specific transcription factor essential for expression of this gene. Gel mobility shift experiments with antibodies against hepatocyte nuclear factor-4 (HNF-4) and transactivation of the HGFL promoter by a HNF-4 cDNA expression vector suggest that HNF-4 binds to the -135/-105 region and is responsible for the liver specific expression of HGFL. PMID- 8621551 TI - Cortisol inhibits the synthesis of insulin-like growth factor-binding protein-5 in bone cell cultures by transcriptional mechanisms. AB - Glucocorticoids inhibit the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5) in osteoblasts, but the mechanisms involved are unknown. IGFBP-5 stimulates bone cell growth, and its inhibition by glucocorticoids may be relevant to the action of this binding protein on bone formation. We tested the effects of cortisol on IGFBP-5 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). Cortisol decreased IGFBP-5 polypeptide levels in the extracellular matrix and caused a time- and dose-dependent decrease in IGFBP-5 mRNA. IGFBP-5 transcripts were markedly decreased by cycloheximide, and further suppressive effects of cortisol could not be determined. Cortisol did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells. Cortisol decreased IGFBP-5 hnRNA, the rate of IGFBP-5 transcription, and the activity of the murine IGFBP-5 promoter by 35% in transient transfection experiments. Deletion analysis showed that the region responsive to cortisol is from base pairs -70 to +22, and E-box-binding proteins or c-Myb-related nuclear factors may be involved in its regulation. In conclusion, cortisol inhibits IGFBP 5 transcription in Ob cells through the Myb-binding domain. This effect may be partly responsible for the effect of glucocorticoids on bone formation. PMID- 8621552 TI - The role of the carboxyl-terminal amino acid residues in Escherichia coli DNA topoisomerase III-mediated catalysis. AB - The role that the carboxyl-terminal amino acids of Escherichia coli DNA topoisomerase I (Topo I) and III (Topo III) play in catalysis was examined by comparing the properties of Topo III with those of a truncated enzyme lacking the generalized DNA binding domain of Topo III, Topo I, and a hybrid topoisomerase polypeptide containing the amino-terminal 605 amino acids of Topo III and the putative generalized DNA binding domain of Topo I. The deletion of the carboxyl terminal 49 amino acids of Topo III decreases the affinity of the enzyme for its substrate, single-stranded DNA, by approximately 2 orders of magnitude and reduces Topo III-catalyzed relaxation of supercoiled DNA and Topo III-catalyzed resolution of DNA replication intermediates to a similar extent. Fusion of the carboxyl-terminal 312 amino acid residues of Topo I onto the truncated molecule stimulates topoisomerase-catalyzed relaxation 15-20-fold, to a level comparable with that of full-length Topo III. However, topoisomerase-catalyzed resolution of DNA replication intermediates was only stimulated 2-3-fold. Therefore, the carboxyl-terminal amino acids of these topoisomerases constitute a distinct and separable domain, and this domain is intimately involved in determining the catalytic properties of these polypeptides. PMID- 8621553 TI - Conformational changes in the A1 domain of von Willebrand factor modulating the interaction with platelet glycoprotein Ibalpha. AB - The interaction between von Willebrand factor (vWF) A1 domain and platelet glycoprotein Ib alpha occurs in the presence of high shear stress or when vWF becomes immobilized onto a surface but not appreciably in the normal circulation. To investigate the structural properties regulating A1 domain function, we have used recombinant fragments prepared either in cyclic form with oxidized Cys509 Cys695 disulfide bond or reduced and alkylated. Interaction with glycoprotein Ibalpha was assessed by testing inhibition of monoclonal antibody LJ-Ib1 binding to platelets and inhibition of shear-induced platelet aggregation mediated by native vWF. Fragments exposed to pH between 2.5 and 3.5 adopted the molten globule conformation with loosened tertiary structure intermediate between native and completely unordered state. Maximal receptor binding activity was observed when fragments kept at acidic pH, particularly after reduction of the Cys509 Cys695 disulfide bond, were subjected to quick refolding by rapid pH increase. In contrast, slow refolding by incremental pH change over several hours resulted in at least 20-fold lower activity. A specific single point mutation (I546V) resulted in enhanced receptor binding, whereas another mutation (S561G) caused markedly reduced binding. These results provide experimental evidence that conformational transitions can modulate function of the vWF A1 domain in solution. PMID- 8621554 TI - Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication. AB - The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis. PMID- 8621555 TI - The Goodpasture autoantigen. Structural delineation of two immunologically privileged epitopes on alpha3(IV) chain of type IV collagen. AB - The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the alpha3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the alpha3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen. PMID- 8621556 TI - Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A. AB - Aminopeptidase A (EC 3.4.11.7, APA) is a homodimeric membrane-bound glycoprotein that contains the consensus sequence HEXXH(385-389) found in zinc metallopeptidases such as thermolysin. The x-ray structure of the latter enzyme revealed that the two histidines of this motif are two of the three zinc coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. Alignment of the sequence of mouse APA with those of the already characterized metallopeptidases showed the presence of several conserved amino acids such as a glutamate residue in position 408 which may constitute the putative third zinc ligand. The functional implication of this residue and the role of glutamate 386 in the HELVH(385-389) motif of APA have been investigated by replacing these residues with an aspartate (Asp-386, Asp-408) or an alanine (Ala-386, Ala-408) by site-directed mutagenesis. Expressed mutated proteins in position 386 showed no APA activity. Ala-408 was also inactive, and Asp-408 had 5% of the wild type enzyme activity and a similar Km. 65Zn incorporation measurements indicated that Ala-386 binds the zinc ion as well as the wild type enzyme, whereas the Ala-408 mutant did not. These results provide evidence that Glu-408 is the third zinc-coordinating residue of APA, confirm the presumed involvement of Glu-386 in the catalytic process of the enzyme, and identify APA as a zinc metallopeptidase functionally similar to thermolysin. PMID- 8621557 TI - Zipper protein, a B-G protein with the ability to regulate actin/myosin 1 interactions in the intestinal brush border. AB - We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin. PMID- 8621558 TI - Formation of sodium dodecyl sulfate-stable fibronectin multimers. Failure to detect products of thiol-disulfide exchange in cyanogen bromide or limited acid digests of stabilized matrix fibronectin. AB - Fibronectin exists in a soluble form in body fluids and as a fibrillar component of the extracellular matrix. Matrix fibronectin associates as large complexes in SDS unless a reducing reagent is also present. This observation suggests that complex formation is due to interprotomeric disulfides that form by thiol disulfide exchange. To localize the presumptive new disulfides, we labeled protomeric fibronectin by the chloramine-T method or with 125I-Bolton-Hunter reagent, incorporated 125I-fibronectin into the matrix of cultured fibroblasts, and subjected matrix fibronectin to acid or cyanogen bromide digestion. When cyanogen bromide digests of matrix 125I-fibronectin and protomeric 125I fibronectin labeled with Bolton-Hunter reagent were analyzed by two-dimensional polyacrylamide gel electrophoresis in SDS, with the first dimension being nonreducing and the second reducing, we were not able to identify any fragments of matrix fibronectin that migrated as high molecular weight complexes in the first dimension. Limited acid digestion of matrix 125I-fibronectin also dissociated the majority of the high molecular weight complexes. Since we could account for all of the parts of fibronectin that contain cysteine or cystine, we conclude that matrix fibronectin is not stabilized by interprotomeric disulfides. We propose, instead, that stabilization is mediated by noncovalent protein protein interactions that are sensitive to reduction, cyanogen bromide digestion, or limited acid digestion. PMID- 8621559 TI - Functional analysis of the human cyclin D2 and cyclin D3 promoters. AB - The D-type cyclins promote progression through the G1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5'-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions -306 and -114 relative to the ATG codon at +1. Additional positive elements were localized between -444 and -345, whereas sequences that reduced transcription were identified between nucleotides -1624 and -892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides -366 and -167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides -306 and -1624 of the cyclin D2 gene were necessary for serum inducibility. PMID- 8621560 TI - Enhanced release of amyloid beta-protein from codon 670/671 "Swedish" mutant beta amyloid precursor protein occurs in both secretory and endocytic pathways. AB - The mutation at codons 670/671 of beta-amyloid precursor protein (betaPP) dramatically elevates amyloid beta-protein (Abeta) production. Since increased Abeta may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimer's disease, evaluation of the cellular mechanism(s) responsible for the enhanced Abeta release may suggest potential therapies for Alzheimer's disease. In this study, we analyzed Chinese hamster ovary cells stably transfected with either wild type betaPP (betaPP-wt) or "Swedish" mutant betaPP (betaPP-sw) for potential differences in betaPP processing. We confirmed that increased amounts of Abeta and a beta-secretase cleaved COOH-terminally truncated soluble betaPP (betaPPs) were secreted from betaPP-sw cells. As shown previously for betaPP-wt cells, Abeta was released more slowly than the secretion of betaPPs from surface-labeled betaPP-sw cells, indicating that endocytosis of cell surface betaPP is one source of Abeta production. In contrast, by [35S]methionine metabolic labeling, the rates of Abeta and betaPPs release were virtually identical for both cell lines. In addition, the identification of intracellular betaPPs and Abeta shortly after pulse labeling suggests that Abeta is produced in the secretory pathway. Interestingly, more Abeta was present in medium from betaPP-sw cells than betaPP wt cells after either cell surface iodination or [35S]methionine labeling, indicating that betaPP-sw cells have enhanced Abeta release in both the endocytic and secretory pathways. Furthermore, a variety of drug treatments known to affect protein processing similarly reduced Abeta release from both betaPP-wt and betaPP sw cells. Taken together, the data suggest that the processing pathway for betaPP is similar for both betaPP-wt and betaPP-sw cells and that increased Abeta production by betaPP-sw cells arises from enhanced cleavage of mutant betaPP by beta-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH2 terminus of Abeta. PMID- 8621561 TI - The different activities of the two activation domains of the Brn-3a transcription factor are dependent on the context of the binding site. AB - The POU (Pit-Oct-Unc) family transcription factor Brn-3a contains two distinct activation domains, one at the N terminus of the molecule and one at the C terminus coincident with the DNA binding domain. These different activation domains have been shown previously to differ in their ability to activate an artificial test promoter containing a Brn-3a binding site and the naturally occurring alpha-internexin gene promoter. Here we identify the target site for Brn-3a in the alpha-internexin gene promoter and show that it can confer responsiveness to Brn-3a on a heterologous promoter. One of the single-stranded DNA sequences derived from either this novel Brn-3a binding site or from the previously characterized site in the test promoter are shown to bind Brn-3a preferentially compared with the complementary single strand or the corresponding double-stranded sequence. The pattern of responsiveness of these two sequences when cloned upstream of the same test promoter and co-transfected with constructs encoding various portions of Brn-3a indicates that the activity of the two Brn-3a activation domains is dependent upon differences in the context of the target sequence in each promoter rather than on differences in the target sequence itself. PMID- 8621562 TI - Assembly and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum. AB - The Bradyrhizobium japonicum cbb3-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme. PMID- 8621563 TI - Cloning and characterization of the yeast HEM14 gene coding for protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. AB - Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides. The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity). The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system. PMID- 8621564 TI - Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis. AB - The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease. PMID- 8621565 TI - Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. AB - Membrane-type matrix metalloproteinase-1 (MT-MMP-1) has been proposed to play a critical role in regulating the expression of tissue-invasive phenotypes in normal and neoplastic cells by directly or indirectly mediating the activation of progelatinase A. To begin characterizing MT-MMP-1 structure-function relationships, transmembrane-deletion mutants were constructed, and the processing of the zymogens as well as the enzymic activity of the mature proteinases was analyzed. We now demonstrate that pro-MT-MMP-1 mutants are efficiently processed to active proteinases following post-translational endoproteolysis immediately downstream of an Arg108-Arg-Lys-Arg basic motif by a proprotein convertase-dependent pathway. The secreted form of active MT-MMP-1 not only displays an N terminus identical with that described for the processed wild type enzyme at Tyr112 (Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grants, G. A., and Goldberg, G. I. (1995) J. Biol. Chem. 270, 5331-5338), but also directly mediated progelatinase A activation via a two-step proteolytic cascade indistinguishable from that observed with intact cells. Furthermore, although the only function previously ascribed to MT-MMP-1 is its ability to act as a progelatinase A activator, purified transmembrane deletion mutants also expressed proteolytic activities against a wide range of extracellular matrix molecules. Given recent reports that MT-MMP-1 ectodomains may undergo intercellular transfer in vivo (Okada, A., Bellocq, J.-P., Rouyer, N., Chenard, M.-P., Rio, M.-C., Chambon, P., and Basset, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2730-2734), our data suggest that soluble forms of the proteinase confer recipient cells with the ability to not only process progelatinase A, but also directly degrade extracellular matrix components. PMID- 8621566 TI - Preferential binding of MyoD-E12 versus myogenin-E12 to the murine sarcoma virus enhancer in vitro. AB - The MyoD family of transcription factors regulates muscle-specific gene expression in vertebrates. In the adult rat, MyoD mRNA accumulates predominately in fast-twitch muscle, in particular type IIb and/or IIx fibers, whereas Myogenin mRNA is restricted to slow-twitch type I muscle fibers. Transgenic mice expressing the avian v-ski oncogene from the murine sarcoma virus (MSV) promoter enhancer display preferential hypertrophy of type IIb fast-twitch muscle apparently because of the restricted expression of the transgene. We tested the hypothesis that preferential interactions of MyoD, as a heterodimer with E12, with the MSV enhancer, which has six E-box targets for MyoD family proteins, could contribute to v-ski gene expression in IIb muscle fibers. A series of quantitative binding studies was performed using an electrophoretic mobility shift assay to test MyoD-E12 versus Myogenin-E12 binding to the MSV enhancer. Our results indicate that MyoD-E12 binds the MSV enhancer with higher affinity and higher cooperativity than Myogenin-E12. Interestingly, MyoD-E12 bound all of the individual E-boxes tested with positive cooperativity indicating DNA-mediated dimerization of the protein subunits. PMID- 8621567 TI - Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules. AB - In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43 kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins. PMID- 8621568 TI - Sequence-dependent induction of base pair substitutions and frameshifts by propanodeoxyguanosine during in vitro DNA replication. AB - Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (C-PdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10(3) - 10(4)-fold compared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the Vmax/Kmvalues for incorporation of dGMP were similar in both sequence contexts, whereas the Vmax/Km for dAMP incorporation increased 4.7-fold when the base pair 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1- and 2-base deletions were major products of replication in both sequence contexts. Full-length products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation of dAMP opposite PdG. The observation of PdG --> T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter. PMID- 8621569 TI - Characterization of a nuclear protein conferring brefeldin A resistance in Schizosaccharomyces pombe. AB - The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum. Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe. Here we describe the characterization of one of these genes, hba1. This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex. The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo. The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S. pombe. PMID- 8621570 TI - Essential amino acids for substrate binding and catalysis of human flap endonuclease 1. AB - Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity. PMID- 8621571 TI - Enzymatic and electron transfer activities in crystalline protein complexes. AB - Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme. PMID- 8621572 TI - Synergistic induction of anchorage-independent growth of NIH3T3 mouse fibroblasts by cysteine proteinase inhibitors and a tumor promoter. AB - We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin alpha is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin alpha carrying a CAAX motif (cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, Ras delta 42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin alpha-CVLS or Ras delta 42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin alpha-CVLS- and Ras delta 42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis. PMID- 8621573 TI - STAT activation by epidermal growth factor (EGF) and amphiregulin. Requirement for the EGF receptor kinase but not for tyrosine phosphorylation sites or JAK1. AB - The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF- and AR induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1 independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation. PMID- 8621574 TI - The orphan nuclear hormone receptor LXR alpha interacts with the peroxisome proliferator-activated receptor and inhibits peroxisome proliferator signaling. AB - The yeast two-hybrid system was used to isolate novel cellular factors that interact with the mouse peroxisome proliferator-activated receptor alpha (PPARalpha). One of the interacting clones isolated encoded LXRalpha, a recently described human orphan nuclear hormone receptor. LXRalpha bound directly to PPARalpha, as well as to the common heterodimerization partner 9-cis-retinoic acid receptor (RXRalpha). LXRalpha did not form a DNA binding complex with PPARalpha on synthetic hormone response elements composed of direct repeats of the TGACCT consensus half-site or on naturally occurring peroxisome proliferator response elements (PPREs) or LXRalpha response elements. However, LXRalpha inhibited binding of PPARalpha/RXRalpha heterodimers to PPREs, and coexpression of LXRalpha in mammalian cells antagonized peroxisome proliferator signaling mediated by PPARalpha/RXRalpha in vivo. These findings identify a novel partner for PPARalpha and suggest that LXRalpha plays a role in modulating PPAR-signaling pathways in the cell. PMID- 8621575 TI - Activation of yeast protein kinase C by Rho1 GTPase. AB - We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1. PMID- 8621577 TI - A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. AB - The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa 4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa 4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir. PMID- 8621576 TI - Glucocorticoids block protein kinase A inhibition of calcium-activated potassium channels. AB - Adrenal corticosteroids have well known and profound effects on neurons and neuroendocrine cells, but the underlying cellular mechanisms are poorly understood. The present study analyzed membrane currents and ACTH release in AtT20 mouse pituitary corticotrope tumor cells. Patch-clamp analysis revealed a significant and selective inhibition of calcium-activated (BK-type) potassium channels upon activation of protein kinase A by corticotropin-releasing factor or 8-chlorophenylthio-cAMP. The synthetic glucocorticoid dexamethasone had no effect on potassium currents evoked by depolarization but prevented the inhibitory effect of protein kinase A activators. The action of dexamethasone had the hallmarks of protein induction, i.e. a lag time and sensitivity to inhibitors of DNA transcription and mRNA translation. In parallel, the specific BK channel blocker iberiotoxin abolished early glucocorticoid inhibition of corticotropin releasing factor-stimulated ACTH secretion. In summary, the present data show that glucocorticoid-induced proteins render BK-type channels resistant to inhibition by protein kinase A and that this action of the steroid is pivotal for its early inhibitory effect on the secretion of ACTH. PMID- 8621578 TI - A putative selectivity filter in the G-protein-coupled receptors for parathyroid hormone and secretion. AB - The seven transmembrane segments (TMs) of many G-protein-coupled receptors (GPCRs) are thought to form a cavity into which cognate ligands insert, leading to receptor activation. Residues lining the cavity are often essential for optimal ligand binding and/or signal transduction. The present studies evaluated whether residues lining the cavity also contribute to specificity, using GPCRs for the polypeptides parathyroid hormone (PTH) and secretin as models. These ligands display no sequence homology with one another, and neither ligand cross reacts with the other's receptor. However, mutation of a single amino acid in the second TM of the secretin receptor to the corresponding residue in the PTH receptor (N192I) resulted in a receptor that binds and signals in response to PTH. The reciprocal mutation in the PTH receptor (I234N) likewise unmasked responsiveness to secretin. Neither mutation significantly altered the response of the receptors to their own ligands. The results suggest a model of specificity wherein TM residues near the extracellular surface of the receptor function as a selectivity filter that restricts access of inappropriate ligands to an activation site in the transmembrane cavity. PMID- 8621579 TI - The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit. AB - The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence). PMID- 8621581 TI - beta-Thymosins are not simple actin monomer buffering proteins. Insights from overexpression studies. AB - beta-Thymosins are the currently favored candidates for maintaining the large actin monomer (G-actin) pool in living cells. To determine if beta-thymosin behaves like a simple G-actin buffering agent in the complex environment of a cell, we overexpressed thymosin beta10 (Tbeta 10) in NIH3T3 cells and determined the effect on the monomer/polymer equilibrium. Tbeta 10 is the predominant beta thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well characterized actin monomer binding proteins. Clonal cell lines that overexpressed three times more Tbeta 10 had 23-33% more polymerized actin than control cells, and the filaments appeared thicker after staining with fluorescent phalloidin. There was no change in total actin, profilin, and cofilin/actin depolymerizing factor content. The overexpressing cells were more motile; they spread faster and had higher chemotactic and wound healing activity. Assuming that there is no compensatory inactivation of the other classes of monomer binding proteins, our paradoxical observation can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239). beta-Thymosin at levels comparable with that found in the overexpressing cells binds actin filaments and decreases the critical concentration (C(c)) for actin polymerization. This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increase in thymosin does not lead to a corresponding increase in G-actin. Furthermore, the decrease in C(c) reduces the buffering capacity of the other actin monomer binding proteins. As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other actin monomer binding proteins. The outcome depends on the level of beta-thymosin expression relative to the composition of the other actin monomer binding protein. Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may have physiological significance. PMID- 8621580 TI - Cell cycle regulation of nuclear factor p32 DNA-binding activity by novel phase specific inhibitors. AB - The nuclear factor p92, originally discovered by its interaction with the human papillomavirus type 18 enhancer, is a cellular protein whose activity is restricted to S phase in human primary fibroblasts. The human papillomavirus type 18 p92 binding sequence confers enhancer activity on a heterologous promoter, suggesting that p92 acts as a transcription factor. We have identified a class of nuclear inhibitory proteins, I-92s, which noncovalently associate with p92 but not with other transcription factors such as AP1, E2F, or NF-kappaB. Different I 92s occur in G1, G2, and G0, while no I-92 is detectable in S phase. Phase specific inhibitors, therefore, are responsible for the cell cycle dependence of p92 activity and provide a novel mechanism linking transcription factor regulation with the cell cycle. PMID- 8621582 TI - Tbeta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentration. AB - Thymosin beta 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-actin to copolymerize with actin reduces the sequestering activity of G-actin binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins. PMID- 8621583 TI - Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence. AB - P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems. PMID- 8621584 TI - An alternative splicing product of the lamin A/C gene lacks exon 10. AB - Expression of the A-type lamins was studied in the lung adenocarcinoma cell line GLC-A1. A-type lamins, consisting of lamin A and C, are two products arising from the same gene by alternative splicing. Northern blotting showed in GLC-A1 a relatively low expression level of lamin C and an even lower expression level of lamin A as compared to other adenocarcinoma cell lines. Immunofluorescence studies revealed highly irregular nuclear inclusions of lamin A, suggesting protein or gene expression abnormalities. Reverse transcriptase-polymerase chain reaction-based cDNA analysis followed by sequencing indicated the presence of an as yet unidentified alternative splicing product of the lamin A/C gene. This product differs from lamin A by the absence of the 5' part of exon 10 (90 nucleotides). Therefore we propose to designate this product lamin Adelta10. Deletion of the 30 amino acids encoded by exon 10 was predicted to result in a shift in pI of the protein from 7.4 to approximately 8.6, which was confirmed by two-dimensional immunoblotting. mRNA analysis in a variety of cell lines, normal colon tissue as well as carcinomas demonstrated the presence of lamin Adelta 10 in all samples examined, suggesting its presence in a variety of cell types. PMID- 8621585 TI - EPR study of NO complex of bd-type ubiquinol oxidase from Escherichia coli. AB - The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex. PMID- 8621586 TI - Existence of novel branched side chains containing beta-1,2 and alpha-1,6 linkages corresponding to antigenic factor 9 in the mannan of Candida guilliermondii. AB - Isolation of beta-linkage-containing side chain oligosaccharides from the mannan of Candida guilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive beta-1,2-linked mannose units at the nonreducing terminal of alpha-linked oligosaccharides. The linkage sequence present in this mannan, Man beta 1-->2Man alpha 1-->3Man alpha-->, has also been found in the mannan of Saccharomyces kluyveri but not in the mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3 O-substituted mannose units as follows. (Carbohydrate sequence in text) Structure 1 and (Carbohydrate sequence in text) Structure 2 The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (delta delta = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9. PMID- 8621587 TI - Characterization of the sialic acid-binding site in sialoadhesin by site-directed mutagenesis. AB - The sialoadhesins are a distinct subgroup of the immunoglobulin superfamily, comprising sialoadhesin, CD22, the myelin-associated glycoprotein, and CD33. They can all mediate sialic acid-dependent binding to cells with distinct specificities. Sialoadhesin is a murine macrophage-restricted cell-surface molecule with 17 extracellular immunoglobulin-like domains that recognizes NeuAc alpha 2-3Gal in N- and O-glycans and interacts preferentially with cells of the granulocytic lineage. Its sialic acid-binding site is located within the NH2 terminal (membrane-distal) V-set domain. Here we have carried out site-directed mutagenesis in an attempt to identify the binding site of sialoadhesin. A subset of nonconservative mutations disrupted sialic acid-dependent binding without affecting binding of three monoclonal antibodies directed to two distinct epitopes of sialoadhesin. A CD8 alpha-based molecular model predicts that these residues form a contiguous binding site on the GFCC'C" beta-sheet of the V-set domain centered around an arginine in the F strand. A conservative mutation of this arginine to lysine also abolished binding. This amino acid is conserved among all members of the sialoadhesin family and is therefore likely to be a key residue in mediating sialic acid-dependent binding of sialoadhesins to cells. PMID- 8621588 TI - Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cell-surface molecule CD22. AB - B-lymphocyte antigen CD22 is a member of the recently described sialoadhesin family of immunoglobulin-like cell-surface glycoproteins that bind glycoconjugates terminating in sialic acid. One prominent ligand for CD22 is the highly glycosylated leukocyte surface protein CD45. Using surface plasmon resonance spectroscopy, we characterized the interaction of recombinant mouse CD22 with native CD45 purified from rat thymus (CD45-thy). By in situ desialylation and resialylation of immobilized CD45-thy, we show that mouse CD22 binds to the sialoglycoconjugate NeuGc alpha 2-6Gal beta 1-4GlcNAc carried on CD45-thy N-glycans. Previous studies have shown that the sialic acid-binding site lies within the two membrane-distal domains of CD22 (domains 1 and 2), which are V-set and C2-set immunoglobulin superfamily domains, respectively. To further localize the binding site, we have made 42 single amino acid substitutions throughout both domains. All 12 mutations that abrogated binding to CD45-thy without disrupting antibody binding were of residues within the GFCC'C" beta sheet of domain 1. These residues are predicted to form a contiguous binding site centered around an arginine residue in the F strand that is conserved in all members of the sialoadhesin family. Our results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC'C" beta-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition. PMID- 8621589 TI - Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C. AB - Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases. PMID- 8621591 TI - The role of arsenic-thiol interactions in metalloregulation of the ars operon. AB - The ars operon of the Escherichia coli plasmid R773 that confers arsenical and antimonial resistance is negatively regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates. However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coordinate. A site-directed mutagenic approach was employed in a search for a third arsenic ligand. ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-ars DNA complexes could not be dissociated by inducers. Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the operon. While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand. Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations. Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo. In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex. Thus, two ArsR-arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide affinity resin. ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin. Alteration of any two of the three resulted in loss of binding. Arsenic X-ray absorption spectroscopy of ArsR treated stoichiometrically with arsenite confirmed the average arsenic coordination as AsS3 These results suggest that all three cysteine thiolates are arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction. PMID- 8621590 TI - The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling. AB - The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1 related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS. PMID- 8621592 TI - Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression. AB - NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373). PMID- 8621593 TI - Genomic cloning and characterization of the human thrombin receptor gene. Structural similarity to the proteinase activated receptor-2 gene. AB - The seven-transmembrane segment thrombin receptor (TR) represents the prototype of a putative family of proteolytically cleaved receptors that may include the proteinase activated receptor-2. A panel of somatic cell hybrids retaining distinct portions of human chromosome 5 were used to establish that the human TR gene is present as a single-copy locus within the region 5q11.2 -->q13.3, confirming our previous localization using fluorescent in situ hybridization analysis. To further characterize the TR gene, overlapping clones from a human genomic library were isolated. Genomic analysis confirmed that the TR gene is of limited complexity, spanning approximately 27 kilobases and containing two exons separated by a large approximately 22-kilobase intron. The larger second exon contains the majority of the coding sequence and the thrombin cleavage site, remarkably similar to the organization of the proteinase activated receptor-2 gene in which the putative cleavage site is also contained within the large second exon. Primer extension analysis using two 30-mer oligonucleotide primers known to be contained within the first exon identified the predominant transcription initiation site 351 base pairs upstream from the initiator methionine in both human umbilical vein endothelial and human erythroleukemia cells. Sequence analysis of the 5'-flanking region revealed the TR promoter to be TATA-less, although nucleic acid motifspotentially involved in transcriptional gene regulation were evident and include a GATA motif, octamer enhancer sequences, AP-2-like sites, and Sp1 sites. These data provide evidence for remarkable similarity at the gene level between both proteolytically cleaved receptors described to date. PMID- 8621594 TI - Phosphorylation of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, by cyclic AMP-dependent protein kinase. AB - Activity of the recently cloned ATP-sensitive epithelial K+ channel, ROMK (Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Hebert, S. C. (1993) Nature 362, 31-38), is regulated by phosphorylation dephosphorylation processes with cAMP-dependent protein kinase (PKA)-dependent phosphorylation events being required for maintenance of channel activity in excised membrane patches (McNicholas, C. M., Wang, W., Ho, K., Hebert, S. C., and Giebisch, G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8077-8081; Kubokawa, M., McNicholas, C. M., Higgins, M. A., Wang, W., and Giebisch, G. (1995) Am. J. Physiol. 269, F355-F362). To determine whether this channel is a substrate for PKA, ROMK tagged with the hemagglutinin epitope was transiently transfected into HEK293 cells. In vitro labeling of immunoprecipitated proteins from transfected cells showed that ROMK could be phosphorylated by PKA. Metabolic labeling of ROMK resulted in a significantly increased phosphorylation upon pretreatment of the cells with forskolin, consistent with an action of cAMP-dependent protein kinase. Phosphoamino acid analyses of the ROMK phosphoproteins revealed that phosphate was attached exclusively to serine residues. Three putative PKA phosphorylation sites containing serine residues in the predicted ROMK proteins are shown directly to be substrates for PKA. Site-directed mutagenesis of each of these sites or double mutation of any two sites showed that ROMK proteins retained the ability to be phosphorylated by PKA both in vivo and in vitro to a variable extent, while triple mutation of all three PKA sites abolished the phosphorylation induced by cAMP agonists in transfected cells. Two-electrode voltage clamp experiments showed that PKA-dependent phosphorylation was required for ROMK channel activity and that at least two of the three sites were required for channel function when expressed in X. laevis oocytes. Taken together, these results provide strong evidence that direct phosphorylation of the channel polypeptide by PKA is involved in channel regulation and PKA-dependent phosphorylation is essential for ROMK channel activity. PMID- 8621595 TI - Enhancement of protein kinase C-dependent O2 production in Epstein-Barr virus transformed B lymphocytes by p120Ras-GAP antisense oligonucleotide. AB - The mammalian Ras GTPase-activating protein (p120Ras-GAP) interacts with activated members of the Ras superfamily of GTP-binding proteins to accelerate their deactivation by sharply increasing their rates of GTP hydrolysis. Among the Ras-family proteins interacting with p120Ras-GAP is Rap1A/Krev1, whose activity is not affected by p120Ras-GAP but which competes with Ras for p120Ras-GAP. A second protein that interacts with p120Ras-GAP is P190Rac-GAP, which activates the GTPase of guanine nucleotide-binding proteins of the Rho family (including Rac1 and Rac2). Both these p120Ras-GAP-binding proteins are of interest in connection with the regulation of the respiratory burst oxidase, Rap1A/Krev1 because it copurifies with cytochrome b558 and p190Ras-GAP because it inhibits the Rac2-dependent activation of the respiratory burst oxidase in a cell-free system. Using an 18-mer antisense oligonucleotide, we were able to decrease the expression of p120Ras-GAP in Epstein-Barr virus-transformed B lymphocytes. Under conditions where p120Ras-GAP expression was significantly depressed by antisense oligonucleotides, we observed a 40% increase in protein kinase C-dependent but not receptor-dependent O2 production. In contrast, sense and scrambled oligonucleotides had no effect on either p120Ras-GAP expression or O2 production. Our results suggest a role for p120Ras-GAP as a negative regulator in the protein kinase C-mediated activation of the respiratory burst oxidase. PMID- 8621596 TI - Topological mapping of the cysteine residues of N-carbamyl-D-amino-acid amidohydrolase and their role in enzymatic activity. AB - The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production Of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Only the mutant carrying the Cys172 substitution was catalytically inactive, and the other mutants maintained the same specific activity as the wild type enzyme. The crucial role of Cys172 in enzymatic activity was also confirmed by chemical derivatization of the protein with iodoacetate. Furthermore, chemical derivatizations using both acrylamide and Ellman's reagent revealed that (i) none of the five cysteines is engaged in disulfide bridges, (ii) Cys172 is easily accessible to the solvent, (iii) Cys193 and Cys250 appear to be buried in the protein core, and (iv) Cys243 and Cys279 seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions. These data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction. PMID- 8621597 TI - Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx. AB - The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2 fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx. PMID- 8621598 TI - Analysis of left-handed Z-DNA formation in short d(CG)n sequences in Escherichia coli and Halobacterium halobium plasmids. Stabilization by increasing repeat length and DNA supercoiling but not salinity. AB - To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG)4 6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M. PMID- 8621599 TI - A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. AB - The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34. PMID- 8621600 TI - New molecular and structural determinants involved in beta 2-adrenergic receptor desensitization and sequestration. Delineation using chimeric beta 3/beta 2 adrenergic receptors. AB - As the beta 3-adrenergic receptor (beta3AR) is resistant to short term agonist promoted desensitization and sequestration, chimeric beta3/beta2 receptors were generated to identify the molecular determinants responsible for these regulatory processes in the beta2AR. By exchanging single or multiple intracellular domains of the beta3AR for the corresponding regions of the beta2AR, we show that specific domains can be identified as additive determinants for desensitization, while sequestration is more dependent on global structural conformation. The carboxyl-terminal tail, the third and the second intracellular loops of the beta2AR provided additive contributions to the desensitization observed upon short term agonist stimulation. The second intracellular loop plays a role which is as important as that of third cytoplasmic loop and carboxyl-terminal tail which had previously been identified as the major determinants of agonist promoted desensitization. Additive contributions of the cytoplasmic domains of the beta2AR were also observed for agonist-promoted sequestration. The substitution of the first and second intracellular loops and the carboxyl tail were associated with a beta2-like sequestration phenotype. However, in contrast to what is observed for desensitization the co-substitution of the third cytoplasmic loop with any of the other domains completely suppressed sequestration. These results suggest that sequestration depends not only on appropriate interactions of multiple molecular determinants within the cytoplasmic region of the beta2AR but also on conformational determinants that may influence their orientation. PMID- 8621601 TI - B-myb promotes S phase and is a downstream target of the negative regulator p107 in human cells. AB - The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth arrested human osteosarcoma cells (SAOS2). p107 might exert its growth suppressive activity by regulating B-myb gene transcription. Indeed, p107 down modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity. PMID- 8621602 TI - Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression. AB - Recent studies indicate that sphingolipids mediate several cellular processes. We assessed roles of sphingolipids in the regulation of E-selectin expression in human umbilical vein endothelial cells. All exogenously-added sphingolipids (sphingosine, C2-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin expression by themselves. C2-ceramide at 5 micron enhanced interleukin-1 beta (IL-1 beta)-induced E-selectin expression 2.7-fold, whereas other sphingolipids tested had no effects on this process. Sphingomyelinase, but not phospholipases A2, C, or D, mimicked the enhancing effect of C2-ceramide. Northern blot analyses revealed that C2-ceramide and sphingomyelinase increased interleukin-1 beta-induced E-selectin gene transcription levels. C2-ceramide and sphingomyelinase induced NF-kappaB activation by themselves and enhanced activation by IL-1 beta, which is essential for E-selectin expression. Immunological analyses with anti-NF-kappaB antibodies showed that subunit composition of NF-kappaB activated by IL-1 beta differs from that activated by C2-ceramide, suggesting that signaling pathways utilized by these stimuli may be different. Treatment with C2-ceramide or sphingomyelinase did not alter NF-ELAM1 specific binding activity. IL-1 beta induced sphingomyelin hydrolysis to ceramide; intracellular ceramide level increased to 182% of control value at 30 min. Taken together, these findings suggest that (i) sphingomyelin hydrolysis to ceramide does not trigger, but rather enhances cytokine-induced E selectin expression, in part through NF-kappaB; (ii) sphingomyelin hydrolysis to ceramide does not mediate all the effects of IL-1 beta, although it may play important roles in IL-1 beta signal transduction in human umbilical vein endothelial cells. PMID- 8621603 TI - Prostaglandin A2 blocks the activation of G1 phase cyclin-dependent kinase without altering mitogen-activated protein kinase stimulation. AB - Prostaglandin A2 (PGA2) reversibly blocked the cell cycle progression of NIH 3T3 cells at G1 and G2/M phase. When it was applied to cells synchronized in G0 or S phase, cells were blocked at G1 and G2/M, respectively. The G2/M blockage was transient. Microinjected oncogenic leucine 61 Ras protein could not override the PGA2 induced G1 blockage, nor could previous transformation with the v-raf oncogene. The serum-induced activation of mitogen-activated protein kinase was not inhibited by PGA2 treatment. These data suggest that PGA2 blocks cell cycle progression without interfering with the cytosolic proliferative signaling pathway. Combined microinjection of E2F-1 and DP-1 proteins or microinjected adenovirus E1A protein, however, could induce S phase in cells arrested in G1 by PGA2, indicating that PGA2 does not directly inhibit the process of DNA synthesis. In quiescent cells, PGA2 blocked the normal hyperphosphorylation of the retinoblastoma susceptible gene product and the activation of cyclin dependent kinase (CDK) 2 and CDK4, in response to serum stimulation. PGA2 treatment elevated the p21Waf1/Cip1/Sdi1 protein expression level. These data indicate that PGA2 may arrest the cell cycle in G1 by interfering with the activation of G1 phase CDKs. PMID- 8621604 TI - Identification and characterization of an S-adenosyl-L-methionine: delta 24 sterol-C-methyltransferase cDNA from soybean. AB - In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e. as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids. The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine: delta 24-sterol-C-methyl-transferase (SMT), a rate-limiting enzyme for phytosterol biosynthesis. A cDNA clone (SMT1) encoding soybean SMT was isolated from an etiolated hypocotyl cDNA library by immunoscreening using an anti-(plasma membrane) serum. The deduced amino acid sequence of the SMT1 cDNA contained three conserved regions found in S-adenosyl-L methionine-dependent methyltransferases. The overall structure of the polypeptide encoded by the SMT1 cDNA is most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene. The polypeptide encoded by the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity. The growing soybean vegetative tissues had higher levels of SMT transcript than mature vegetative tissues. Young pods and immature seeds had very low levels of the SMT transcript. The SMT transcript was highly expressed in flowers. The expression of SMT transcript was suppressed in soybean cell suspension cultures treated with yeast elicitor. The transcriptional regulation of SMT in phytosterol biosynthesis is discussed. PMID- 8621605 TI - Metabolism of the "Swedish" amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the "beta-secretase" site occurs in the golgi apparatus. AB - The 4-kDa beta-amyloid peptide (Abeta), a principal component of parenchymal amyloid deposits in Alzheimer's disease, is derived from amyloid precursor proteins (APP). To identify potential intracellular compartments involved in Abeta production, we expressed human APP-695 (APPwt) and APP-695 harboring the Swedish double mutation (APPswe) associated with familial early-onset Alzheimer's disease, in mouse N2a cells. We demonstrate that cells expressing APPswe secrete high levels of Abeta peptides and beta-secretase-generated soluble APP derivatives (APP s beta) relative to cells expressing APPwt. In addition, we observed a concomitant diminution in the levels of alpha-secretase-generated soluble APP derivatives (APP s alpha). Our interpretation of these findings is that beta-secretase cleavage occurs in an intracellular compartment and disables those substrates which would normally be cleaved by alpha-secretase. As anticipated, the levels of APPswe are diminished relative to the steady-state levels of surface-bound APPwt; moreover, surface-bound APPswe and APPwt molecules are released from the plasma membrane after cleavage by alpha-secretase, but not by beta-secretase. Finally, by examining the rate of appearance of specific APP metabolites generated by beta-secretase, we now unequivocally demonstrate that beta-secretase cleavage of APPswe occurs within the Golgi apparatus, as early as the medial compartment. PMID- 8621606 TI - Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity. AB - Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity. PMID- 8621607 TI - Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association. AB - Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28 specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP). PMID- 8621608 TI - The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center. AB - During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process. PMID- 8621609 TI - Mannose enters mammalian cells using a specific transporter that is insensitive to glucose. AB - The concentration of D-mannose in serum is 20-50 micron, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K(uptake) of about 30-70 micron and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2 deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mM D-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis. PMID- 8621610 TI - Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltage-sensitive sodium channel. AB - Anthopleurin B is a potent anemone toxin that binds with nanomolar affinity to the cardiac and neuronal isoforms of the voltage-gated sodium channel. A cationic cluster that includes Arg-12, Arg-14 and Lys-49 has been shown previously to be important in this interaction. In this study, we have used site-directed mutagenesis to determine the contribution to activity of two aliphatic residues, Leu-18 and Ile-43, that have previously been experimentally inaccessible. Leu-18, a residue proximal to the cationic cluster, plays a critical role in defining the high affinity of the toxin. In ion flux studies, this is exemplified by the several hundredfold loss in affinity (231-672-fold) observed for both L18A and L18V toxins on either isoform of the sodium channel. When analyzed electrophysiologically, L18A, the most severely compromised mutant, also displays a substantial loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms. This difference in affinities may reflect an increased preference of the L18A mutant for the closed state of the neuronal channel. In contrast, Ile-43, a residue distal to the cationic cluster, plays at most a very modest role in affinity toward both isoforms of the sodium channel. Only conservative substitutions are tolerated at this position, implying that it may contribute to an important structural component. Our results indicate that Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date. These results have extended the binding site beyond the cationic cluster to include Leu-18 and broadened our emphasis from the basic residues to include the crucial role of hydrophobic residues in toxin-receptor interactions. PMID- 8621611 TI - Pro-OmpA derivatives with a His6 tag in their N-terminal "translocation initiation domains" are arrested by Ni2+ at an early post-targeting stage of translocation. AB - We examined in vitro translocation of pro-OmpA derivatives with a His6 tag at various positions in their mature proteins and with a c-Myc tag at their C termini across inverted membrane vesicles of Escherichia coli. Those with a His6 tag in the N-terminal region of the mature domain, which corresponds to the "translocation initiation domain" proposed previously (Andersson, H., and von Heijne, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9751-9754), could not be translocated in the presence of 100 micron Ni2+, while OmpA derivatives with a His6 tag in the middle of or at the C terminus did not show such Ni2+ sensitivity. The inhibitory action of Ni2+ on pro-3His-OmpA' (with a His6 tag after the third amino acid of the mature OmpA-c-Myc region) translocation was exerted only during early events, after which it became ineffective. The inhibition point of Ni2+ was suggested to lie between membrane targeting and exposure of the signal cleavage site to the periplasm since the unprocessed and membrane-bound form of pro-3His-OmpA' was accumulated by the addition of Ni2+. The Ni(2+)-"trapped" precursor was released from its translocation block by 30 mM histidine, which should compete with the His6 tag on the precursor protein for formation of a Ni2+ chelating complex. We propose that Ni2+ confers a reversible positive charge effect on the His6-tagged initiation domain of the pro-OmpA derivatives and inhibits an early event(s) of protein translocation, such as presentation of the precursor to the membranous part of the translocase. This system will be useful in dissecting early events of the protein translocation pathway. PMID- 8621612 TI - Pharmacologic manipulation of ob expression in a dietary model of obesity. AB - Mutation of the obese (ob) gene results in severe hereditary obesity and diabetes in the C57BL/6J and related strains of mice. In this study we examined the expression of the ob gene in a dietary model in which moderate obesity develops in response to fat (58% of calories from fat) without mutation of the ob gene, and in four genetic models of obesity in mice: ob/ob, db/db, tubby, and fat. Several white and brown adipose depots were examined (epididymal, subcutaneous, perirenal, and interscapular). Northern blot analysis shows that levels of ob mRNA are increased in all adipose depots examined in every model of obesity. The average fold increases were 12.0 +/ 2.1 (ob/ob), 4.8 +/- 1.5 (db/db), 2.8 +/- 0.1 (tubby), 2.4 +/- 0.3 (fat), and 2.1 +/- 0.2 (high fat diet-induced A/J). Moreover, we found that the expression of the ob gene could be manipulated by pharmacologically blocking the development of diet-induced obesity. Supplementation of a high fat diet with a beta 3-adrenergic receptor agonist (CL316,243) prevented obesity, but not hyperphagia associated with high fat feeding (body weights of high fat-fed A/J mice = 34.0 +/- 1.0 g; high fat plus CL316,243-fed mice = 26.8 +/- 0.5 g; n = 10). CL316,243-treated, high fat-fed animals contained levels of ob mRNA in all adipose depots that were equal to or less than levels in low fat-fed mice (average levels in high fat plus CL316,243 fed mice relative to low fat-fed mice: 0.93 +/- 0.09). Inasmuch as fat cell size, but not number, was increased in a previous study in diet-induced obese A/J mice, these results indicate that expression of the ob gene serves as a sensor of fat cell hypertrophy, independent of any effects on food intake. PMID- 8621613 TI - Protein-tyrosine kinases activate while protein-tyrosine phosphatases inhibit L type calcium channel activity in pituitary GH3 cells. AB - The aim of this study was to evaluate the effect of protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on Ca2+ channels in GH3 cells. The activity of Ca2+ channels was monitored either by single-cell microfluorometry or by the whole-cell configuration of the patch-clamp technique. Genistein (20-200 micron) and herbimycin A (1-15 micron) inhibited [Ca2+]i rise induced either by 55 mM K+ or 10 micron Bay K 8644. In addition, genistein and lavendustin A inhibited whole-cell Ba2+ currents. By contrast, daidzein, a genistein analogue devoid of PTK inhibitory properties, did not modify Ca2+ channel activity. The inhibitory action of genistein on the [Ca2+]i increase was completely counteracted by the PTP inhibitor vanadate (100 micron). Furthermore, vanadate alone potentiated -Ca2+-i response to both 55 mM K+ and 10 micron Bay K 8644. The possibility that genistein could decrease the [Ca2+]i elevation by enhancing Ca2+ removal from the cytosol seems unlikely since genistein also reduced the increase in fura-2 fluorescence ratio induced by Ba2+, a cation that enters into the cells through Ca2+ channels but cannot be pumped out by Ca2+ extrusion mechanisms. Finally, in unstimulated GH3 cells, genistein caused a decline of [Ca2+]i and the disappearance of [Ca2+]i oscillations, whereas vanadate induced an increase of [Ca2+]i and the appearance of [Ca2+]i oscillations in otherwise non-oscillating cells. The present results suggest that in GH3 cells PTK activation causes an increase of L-type Ca2+ channel function, whereas PTPs exert an inhibitory role. PMID- 8621614 TI - Magnesium-mediated conversion of an inactive form of a hammerhead ribozyme to an active complex with its substrate. An investigation by NMR spectroscopy. AB - The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. Elevated concentrations (1.0 M) of monovalent Na+ ions could not induce the formation of the R32-RdC11 complex. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme substrate complex. PMID- 8621615 TI - Down-regulation of the expression of the obese gene by an antidiabetic thiazolidinedione in Zucker diabetic fatty rats and db/db mice. AB - Obese (ob) is a recently identified gene involved in the regulation of energy balance in the mouse. We report here that AD-5075, a potent thiazolidinedione which lowered plasma glucose and triglyceride in Zucker diabetic fatty (ZDF) rats and db/db mice, decreased the expression of the ob gene in these animal models of obesity and non-insulin-dependent diabetes mellitus. The level of adipose ob mRNA in ZDF rats was 3-fold greater than that detected in the Zucker lean littermates. Chronic treatment with AD-5075 elicited a 67 and 70% reduction of ob mRNA in ZDF and control lean rats, respectively. Furthermore, the amount of adipose ob mRNA in db/db mice was 7 times higher than that detected in lean littermates. Treatment of db/db mice with AD-5075 resulted in a 78% reduction of the level of ob mRNA with parallel changes in circulating level of the ob gene product, leptin. The reduction of the ob mRNA in the Zucker lean rats was accompanied by significantly greater food intake and weight gain. However, in ZDF rats and db/db mice, there was profound increase in body weight without hyperphagia. The results demonstrate that the expression of the ob gene is up-regulated in these two rodent models of diabetes compared to their lean counterparts and that such overexpression is attenuated by treatment with an agent that improves insulin sensitivity and glucose homeostasis in vivo. PMID- 8621616 TI - Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes. AB - Compartmentalization of the type II cyclic AMP-dependent kinase (PKA) is achieved through association of the regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). Using an interaction cloning strategy with RIIalpha as a probe, we have isolated cDNAs encoding a novel 1129-amino acid protein that contains both a PKA binding region and a peroxisome targeting motif. Northern analysis detected mRNAs of 9.7 and 7.3 kb in several rat tissues with the highest levels present in the brain and testis. Western analysis and RII overlay experiments showed that the protein is approximately 220 kDa and was, therefore, named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis extracts resulted in co purification of the type II PKA holoenzyme. The specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the cell lysate to 3.3 nmol/min/mg in the immunoprecipitate. Immunohistochemical analysis of rat testicular TM4 cells showed that AKAP 220 and a proportion of RII were co-localized in microbodies that appear to be a subset of peroxisomes. Collectively, these results suggest that AKAP 220 may play a role in targeting type II PKA for cAMP-responsive peroxisomal events. PMID- 8621617 TI - Mitochondria are a major site for folate and thymidylate synthesis in plants. AB - The subcellular distributions of folate and folate-synthesizing enzymes were investigated in pea leaves. It was observed that the mitochondrial folate pool (approximately 400 micron) represented approximately 50% of the total pool. Furthermore, all the enzymes involved in tetrahydrofolate polyglutamate synthesis were present in the mitochondria. In marked contrast, we failed to detect any significant activity of these enzymes in chloroplasts, cytosol, and nuclei. The presence of the tetrahydrofolate synthesis pathway in mitochondria is apparently a general feature in plants since potato tuber mitochondria also contained a high folate concentration (approximately 200 micron) and all the enzymes required for tetrahydrofolate polyglutamate synthesis. The specific activities of tetrahydrofolate-synthesizing enzymes were rather low (1.5-15 nmol h-1 mg-1 matrix protein), except for dihydrofolate reductase (180-500 nmol h-1 mg-1 matrix protein). Dihydrofolate reductase was purified to homogeneity. The enzyme had a native molecular mass of approximately 140 kDa and was constituted of two identical 62-kDa subunits. Interestingly, this mitochondrial protein appeared to be a bifunctional enzyme, also supporting thymidylate synthesis. The cell distribution of thymidylate synthase was also investigated. No significant activity was observed in cell fractions other than mitochondria, indicating that plant cell mitochondria are also a major site for thymidylate synthesis. PMID- 8621618 TI - Equilibrium studies of kinesin-nucleotide intermediates. AB - We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule. PMID- 8621619 TI - Unexpectedly strong binding of a large metal ion (Bi3+) to human serum transferrin. AB - Large metal ions (>0.9 A ionic radius) have previously been found to bind only weakly to human serum transferrin (hTF, 80 kDa), presumably because the interdomain cleft cannot close around the metal and synergistic anion. Surprisingly, therefore, we report that Bi3+ (ionic radius 1.03 A), a metal ion widely used in anti-ulcer drugs, binds strongly to both the N- and C-lobes with log K1* = 19.42 and log K2* = 18.58 (10 mM Hepes, 5 mM bicarbonate, 310 K). The uptake of Bi3+ by apo-hTF from bismuth citrate complexes is very slow (hours), whereas that from bismuth nitrilotriacetate is rapid (minutes). Evidence from absorption and NMR spectroscopy is presented to show that Bi3+ binds to the specific Fe3+ binding sites along with carbonate as the synergistic anion. Under the conditions used, preferential binding of Bi3+ to the C-lobe of hTF is observed. Linear free energy relationships show that there is a strong correlation between the strength of binding of Bi3+ and Fe3+ to a wide variety of ligands which include transferrin. Therefore we conclude that the strength of metal ion binding to transferrin is determined more by the ligand donor set than by the size of the ion. PMID- 8621620 TI - Keratan sulfate modification of CD44 modulates adhesion to hyaluronate. AB - CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function. PMID- 8621621 TI - Receptor-mediated endocytosis of coagulation factor Xa requires cell surface bound tissue factor pathway inhibitor. AB - Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation. PMID- 8621622 TI - STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6. AB - Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N)5AA motifs. We report an IL 6 responsive element in the proximal region of the human CRP 5'-flanking region that bears a TT(N)4AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-gamma was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6. PMID- 8621624 TI - Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli. AB - PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli. The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. In the cleft, four acidic residues recognize the three positively charged nitrogen atoms of spermidine, while five aromatic side chains anchor the methylene backbone by van der Waals interactions. The overall fold of PotD is similar to that of other periplasmic binding proteins, and in particular to the maltodextrin-binding protein from E. coli, despite the fact that sequence identity is as low as 20%. The comparison of the PotD structure with the two maltodextrin-binding protein structures, determined in the presence and absence of the substrate, suggests that spermidine binding rearranges the relative orientation of the PotD domains to create a more compact structure. PMID- 8621623 TI - TEF-1 transrepression in BeWo cells is mediated through interactions with the TATA-binding protein, TBP. AB - Transcription enhancer factor-1 (TEF-1) has been implicated in transactivating a placental enhancer (CSEn) that regulates human chorionic somatomammotropin (hCS) gene activity. We demonstrated that TEF-1 represses hCS promoter activity in choriocarcinoma (BeWo) cells (Jiang, S.W., and Eberhardt, N.L. (1995) J. Biol. Chem. 270, 13609-13915), suggesting that TEF-1 interacts with basal transcription factors. Here we demonstrate that hTEF-1 overexpression inhibits minimal hCS promoters containing TATA and/or initiator elements, Rous sarcoma virus and thymidine kinase promoters in BeWo cells. Cotransfection of TEF-1 antisense oligonucleotides alleviated exogenous TEF-1-mediated repression and increased basal hCS promoter activity, indicating that endogenous TEF-1 exerts repressor activity. GST-TEF-1 fusion peptides fixed to glutathione-Sepharose beads retained in vitro-generated human TATA-binding protein, hTBP. The TEF-1 proline-rich domain was essential for TBP binding, but polypeptides also containing the zinc finger domain bound TBP with higher apparent affinity. TBP supershifted hTEF-GT IIC DNA complexes, but TEF-1 inhibited in vitro binding of TBP to the TATA motif. Coexpression of TBP and TEF-1 in BeWo cells alleviated TEF-1-mediated transrepression, indicating that the TBP-TEF-1 interaction is functional in vivo. The data indicate that TEF-1 transrepression is mediated by direct interactions with TBP, possibly by inhibiting preinitiation complex formation. PMID- 8621625 TI - Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class IV alcohol dehydrogenase gene expression. AB - Endogenous retinoic acid (RA) has been observed in vertebrate embryos as early as gastrulation, but the mechanism controlling spatiotemporal synthesis of this important regulatory molecule remains unknown. Some members of the alcohol dehydrogenase (ADH) family catalyze retinol oxidation, the rate-limiting step in RA synthesis. Here we have examined mouse embryos for the presence of endogenous RA and expression of ADH genes. RA was not detected in egg cylinder stage embryos but was detected in late primitive streak stage embryos. Detection of class IV ADH mRNA, but not class I or class III, coincided with the onset of RA synthesis, being absent in egg cylinder embryos but present in the posterior mesoderm of late primitive streak embryos. During neurulation, RA and class IV ADH mRNA were colocalized in the craniofacial region, trunk, and forelimb bud. Class IV ADH mRNA was detected in cranial neural crest cells and craniofacial mesenchyme as well as trunk and forelimb bud mesenchyme. The spatiotemporal expression pattern and enzymatic properties of class IV ADH are thus consistent with a crucial function in RA synthesis during embryogenesis. In addition, the finding of endogenous RA and class IV ADH mRNA in the craniofacial region has implications for the mechanism of fetal alcohol syndrome. PMID- 8621626 TI - Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. Assembly of sarcomeric units in series VIA gp130/leukemia inhibitory factor receptor-dependent pathways. AB - Cardiotrophin-1 (CT-1) was recently isolated by expression cloning based on its ability to induce an increase in cell size in neonatal rat ventricular cardiomyocytes. Sequence similarity data suggested that CT-1 is a novel member of a family of structurally related cytokines sharing the receptor component gp130. The present study documents that gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes. CT-1 induces a hypertrophic response in cardiomyocytes that is distinct from the phenotype seen after alpha adrenergic stimulation, both with regard to cell morphology and gene expression pattern. Stimulation with CT-1 results in an increase in cardiac cell size that is characterized by an increase in cell length but no significant change in cell width. Confocal laser microscopy of CT-1 stimulated cells reveals the assembly of sarcomeric units in series rather than in parallel, as seen after alpha adrenergic stimulation. CT-1 induces a distinct pattern of immediate early genes, and up-regulates the atrial natriuretic factor (ANF) gene, but does not affect skeletal alpha-actin or myosin light chain-2v expression. As evidenced by nuclear run-on transcription assays, both CT-1 and alpha-adrenergic stimulation lead to an increase in ANF gene transcription. Transient transfection analyses document that, in contrast to alpha-adrenergic stimulation, the CT-1 responsive cis regulatory elements are located outside of the proximal 3 kilobase pairs of the ANF 5'-flanking region. These studies indicate that CT-1 can activate a distinct form of myocardial cell hypertrophy, characterized by the promotion of sarcomere assembly in series, via gpl30/LIFRbeta-dependent signaling pathways. PMID- 8621627 TI - Effect of the R569W missense mutation on the biosynthesis of myeloperoxidase. AB - Human neutrophil microbicidal activity is largely mediated by reactive species generated by the oxygen-dependent myeloperoxidase (MPO) system. Peroxidase negative neutrophils from many patients with hereditary MPO deficiency possess a 90-kDa MPO-related protein. We recently identified a missense mutation, R569W, in the MPO gene of many subjects with MPO deficiency. In these studies we examined the consequences of R569W on MPO biosynthesis and processing, using stably transfected K562 cells expressing normal MPO or the R569W mutation. K562 cells expressing normal MPO mimicked faithfully many features of MPO biosynthesis in myeloid cells. 1) apopro-MPO was synthesized; 2) a functional heme group was inserted into apopro-MPO, and enzymatically active pro-MPO was thereby generated; 3) pro-MPO underwent proteolytic processing to mature MPO; and 4) hemin augmented the processing of pro-MPO. pREP-R569W cells synthesized apopro-MPO, but heme was not inserted. Neither enzymatically active pro-MPO nor mature MPO was synthesized by transfectants expressing mutated cDNA, confirming our hypothesis that the R569W mutation results in a form of apopro-MPO which does not undergo post translational processing to enzymatically active MPO species. In addition, these data support previous suggestions that heme insertion into apopro-MPO is necessary for its subsequent proteolytic processing into mature MPO subunits. PMID- 8621628 TI - The human glucocorticoid receptor beta isoform. Expression, biochemical properties, and putative function. AB - Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity. PMID- 8621629 TI - Enzymatic formation of dehydrodolichal and dolichal, new products related to yeast dolichol biosynthesis. AB - Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal. PMID- 8621630 TI - Molecular cloning and developmental expression of mouse p130, a member of the retinoblastoma gene family. AB - With sequence homology to the SV40 T antigen-binding domain of the retinoblastoma protein (Rb), p107 and p130 constitute two additional members of the Rb family. To explore the potential function of p130 in mouse development, we cloned the full-length mouse cDNA for p130 and characterized p130 mRNA expression in mice. The deduced mouse p130 protein sequence shares a higher degree of similarity with mouse p107 than with mouse Rb. In adult mice, p130 mRNA is found in all tissues examined. Levels of p130 mRNA vary among different adult tissues, with the highest level in testis. Within testis, p130 mRNA is found predominantly in Leydig cells. Additionally, p130 expression in testis correlates with sexual maturation, suggesting p130 is important for the development of testis and, in particular, Leydig cells. In situ hybridization shows that in post coitus day 12.5 and 14.5 mouse embryos, distribution of p130 mRNA is quite uniform with the exception of a few tissues. Little differences in mRNA levels of either p130 or p107 were found between normal and Rb-deficient embryos, suggesting that p130 and p107 are expressed independently of Rb. Our data are consistent with the hypothesis that p130 and p107 do not compensate for the loss of Rb and support the view that p130 is related to, yet distinct from, the RB gene. PMID- 8621631 TI - Two pathways for base excision repair in mammalian cells. AB - Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides. PMID- 8621632 TI - Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding. AB - Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits proinflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu4-Leu5-Arg6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe17, Phe2l, Ile22, and Leu43. These two receptor-binding regions are separated by over 20 A in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity. PMID- 8621633 TI - Transcriptional regulation of the gene coding for human protein C. AB - The promoter for the gene coding for human protein C has been characterized as to nucleotide sequences that regulate the synthesis of mRNA. The major transcription start site was found 65 nucleotides upstream from the first intron/exon boundary along with two minor sites. Functional characterization of 1528 base pairs at the 5'-end of the gene was then carried out by chloramphenicol acetyltransferase reporter assays, protection from DNase I digestion, and electrophoretic mobility shift assays employing HepG2 and HeLa cells. One of the upstream regions (nucleotides -25 to +9) contained binding sites for at least two different transcription factors, including a hepatic nuclear factor 1-binding site (-10 to +9) and two overlapping and oppositely oriented hepatic nuclear factor 3-binding sites (-25 to -11). A second major region (PCE1) (+12 to +30) appeared to be a unique, liver-specific regulatory sequence. An Sp1-binding site in exon I (+58 to +65) was also recognized by cotransfection experiments with an Sp1 expression plasmid. Specific mutations in these promoter elements reduced transcriptional activity and abolished the binding of hepatic nuclear proteins. Finally, a strong silencer element (PCS1) (between -162 and -82) and two possible liver-specific enhancer regions (PCE2 and PCE3), which interact coordinately with the promoter elements, were also found (between -1462 and -162). PMID- 8621634 TI - Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Engelbreth Holm-Swarm tumor matrix. AB - The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue specific differences in their distribution. PMID- 8621635 TI - Interactions between ifenprodil and the NR2B subunit of the N-methyl-D-aspartate receptor. AB - Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which demonstrates a 140-fold preference for NR2B over NR2A subunits, although the molecular basis for this subunit specificity is unknown. We have made chimeric receptors by fusing the murine forms of NR2A (epsilon 1) and NR2B (epsilon 2) to localize the high affinity determinants of ifenprodil inhibition on the 2B subunit. Binding experiments with 125I-MK-801 implicated the region between amino acids 198 and 356 of NR2B for high affinity ifenprodil interaction. Site-directed mutants at Arg-337 showed that this residue is absolutely required for high affinity ifenprodil inhibition. Polyamines also modulate the NMDA receptor with a preference for NR2B subunits, and the pharmacology of these agents overlaps with ifenprodil. Although the determinants of the polyamine enhancement of iodo-MK-801 binding also localize to the NH2 terminus of NR2B, the point mutants at Arg-337 form receptors that are polyamine stimulated at wild type levels. In addition, polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit. PMID- 8621636 TI - ATPase activity of UvrB protein form Thermus thermophilus HB8 and its interaction with DNA. AB - Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system. The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins. The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis. The T. thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity. The purified T. thermophilus UvrB protein was stable up to 80 degrees C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. coli UvrB protein alone has not been shown to exhibit detectable ATPase activity. The values of K(m) and k(cat) for the ATPase activity were 4.2 mM and 0.32 s-1 without DNA, and 4.0 mM and 0.46 s-1 with single stranded DNA, respectively. This suggests that T. thermophilus UvrB protein could interact with single-stranded DNA in the absence of UvrA protein. PMID- 8621637 TI - Kinetic and structural probing of the precleavage synaptic complex (type 0) formed during phage Mu transposition. Action of metal ions and reagents specific to single-stranded DNA. AB - In an earlier kinetic study (Wang, Z., and Harshey, R. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn2+ and Co2+ ions, like Mg2+ and Mn2+ ions, can efficiently cleave the type 0 complex. However, unlike Mg2+ and Mn2+ ions, Zn2+ and Co2+ ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition. PMID- 8621638 TI - Kinetic resolution of the incorporation of the D1 protein into photosystem II and localization of assembly intermediates in thylakoid membranes of spinach chloroplasts. AB - The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes. PMID- 8621639 TI - Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts. AB - To investigate the effect of the major UV-induced lesions on SV40 origin dependent DNA replication and mutagenesis in a mammalian cell extract, double stranded plasmids containing a single cis,syn-cyclobutane dimer or a pyrimidine pyrimidone (6-4) photoproduct at a unique TT sequence have been constructed. These plasmids have been used as templates in DNA replication-competent extracts from human HeLa cells. Plasmids containing a single pyrimidine cyclobutane dimer on the potential lagging strand for DNA replication are replicated with an efficiency approximately equal to that of an unmodified plasmid. A small decrease in replication efficiency of approximately 20% was observed when the lesion was located on the potential leading strand for DNA replication. In both orientations, DpnI-resistant, replicated closed circular plasmid DNA was sensitive to nicking by the pyrimidine dimer-specific enzyme, T4 endonuclease V, indicating that complete replication of the damaged plasmid occurs in vitro. In contrast, a (6-4) photoproduct, within the same site and sequence context on the lagging strand for DNA synthesis, inhibits replication in vitro by an average of approximately 50%, indicating that the mammalian replication complex responds differently to the two major UV-induced lesions during DNA replication in vitro. Analysis of the DpnI-resistant, replicated DNA for mutations targeted to the lesion site indicates that neither of these lesions resulted in significant mutagenesis. UV-induced lesions at TT sites may therefore be poorly mutagenic under these conditions for DNA replication in human cell extracts in vitro. PMID- 8621640 TI - Preparation of figure 8 and cruciform DNAs and their use in studies of the kinetics of branch migration. AB - We have re-examined the kinetics of the branch migration of double-stranded DNA that is mediated by the stepwise movement of the Holliday junction. This work revises and extends our previous treatment (Thompson, B. J., Camien, M. N., and Warner, R. C. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2299-2303). New methodology and new highly purified substrates have been used. The latter include figure 8s prepared from phage G4 DNA by annealing single-stranded components and two sizes of a novel cruciform. We treat the process as a one-dimensional diffusion based on the random walk, the mathematical basis of which is discussed in detail. The step rate is shown to be 3 orders of magnitude slower than we reported previously. The most important contribution to the erroneously high rate was a result of the presence of EDTA in the spreading solution used for electron microscopy at that time. A second contribution of about 4-fold resulted from catalysis by EcoRI and other proteins. The rates reported here are for the uncatalyzed reaction. PMID- 8621641 TI - Calnexin associates exclusively with individual CD3 delta and T cell antigen receptor (TCR) alpha proteins containing incompletely trimmed glycans that are not assembled into multisubunit TCR complexes. AB - Most T lymphocytes express on their surfaces an oligomeric protein complex consisting of clonotypic alpha beta polypeptides associated with invariant CD3 gamma delta epsilon and zeta chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oligomerization of TCR alpha and CD3 delta glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3 delta proteins and TCR alpha proteins occurred prior to their association with any other TCR components and that calnexin specifically interacted with unassembled TCR alpha and CD3 delta proteins containing incompletely trimmed oligosaccharides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient association of calnexin with TCR alpha glycoproteins but not with CD3 delta glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3 delta and TCR alpha proteins occurring coincident with or immediately after their translocation into the endoplasmic reticulum and preceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3 delta and TCR alpha glycoproteins involves both glycan-dependent and glycan-independent mechanisms. PMID- 8621642 TI - Cloning and kidney cell-specific activity of the promoter of the murine renal Na K-C1 cotransporter gene. AB - The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5' flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0 kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney. PMID- 8621643 TI - Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione dependent vincristine transport. AB - The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S conjugates. In the present study, we have examined the transport of leukotriene C4 (LTC4) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V(max) and K(m) values for LTC4 transport by membrane vesicles from T14 cells were 529 +/- 176 pmol mg(-1) min(-1) and 105 +/- 31 nM, respectively. At 50 nM LTC4, the K(m) (ATP) was 70 micron. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly hydrolyzable ATP analogs. LTC4 transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC4 and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC4 transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC4 was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC4 transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC4 transport (K(i(app)) 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC4 transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC4 transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC4 transport and prevents photolabeling of MRP by LTC4, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent. PMID- 8621644 TI - ATP-dependent 17 beta-estradiol 17-(beta-D-glucuronide) transport by multidrug resistance protein (MRP). Inhibition by cholestatic steroids. AB - In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site specific requirements for glucuronidation of the steroid nucleus. PMID- 8621645 TI - Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. AB - Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane. PMID- 8621646 TI - Csk is constitutively associated with a 60-kDa tyrosine-phosphorylated protein in human T cells. AB - The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP. PMID- 8621647 TI - A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain inhibits insulin action on glucose transport in rat adipocytes. AB - The present study was conducted to examine the involvement of Rab4, a low molecular weight GTP-binding protein, in the action of insulin on glucose transport. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl terminal domain, Rab4-(191-210), was successfully transferred into rat adipocytes by electroporation and inhibited insulin-stimulated glucose transport by about 50% without affecting the basal transport activity. In contrast, synthetic peptides corresponding to the Rab3C and Rab3D carboxyl-terminal hypervariable domain had little effect on insulin action on glucose transport. The Rab4-(191 210) peptide also reduced insulin-induced GLUT4 translocation from the intracellular pool to the plasma membrane. Furthermore, the Rab4-(191-210) peptide reduced both insulin-induced glucose transport and GLUT4 translocation in the presence of a major histocompatibility complex class I antigen-derived peptide, D(k)-(62-85), which is a potent inhibitor of GLUT4 internalization, suggesting that the peptide inhibited exocytotic recruitment of GLUT4-containing vesicles. The Rab4-(191-210) peptide also inhibited GTP gamma S-stimulated glucose transport. In addition, insulin-stimulated glucose transport was inhibited by the addition of anti-Rab4 antibody. These results suggest that Rab4 protein plays a crucial role in insulin action on GLUT4 translocation, especially in exocytotic recruitment by the hormone of the glucose transporter to the plasma membrane from the intracellular retention pool. PMID- 8621648 TI - The N-terminal domain of tomato 3-hydroxy-3-methylglutaryl-CoA reductases. Sequence, microsomal targeting, and glycosylation. AB - The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, considered the rate-limiting step in isoprenoid biosynthesis. In plants, isoprenoid compounds play important roles in mediating plant growth and development, electron transport, photosynthesis, and disease resistance. Sequence comparisons of plant HMGR proteins with those from yeast and mammalian systems reveal high levels of sequence identity within the catalytic domain but significant divergence in the membrane domain. Mammalian HMGRs are integral membrane proteins of the endoplasmic reticulum with eight membrane-spanning regions. In contrast, the membrane domain of plant HMGRs is predicted to contain only one to two transmembrane spans. We have isolated and sequenced a clone (pCD4) encoding exon 1 of tomato hmg1. The membrane domain structures of two differentially regulated tomato HMGR isoforms, HMG1 and HMG2, were analyzed using in vitro transcription and translation systems. Microsomal membrane insertion of the tomato HMGRs is co translational and does not involve cleavage of an N-terminal targeting peptide. HMGR membrane topography was established by protease protection studies of the HMG1 membrane domain and an analogous region of HMG2 engineered to contain a c myc epitope tag. The data indicate that both tomato HMGRs span the membrane two times with both the C and N termini located in the cytosol. Lumenal localization of the short peptide predicted to lie within the endoplasmic reticulum was further confirmed by in vitro glycosylation of an asparagine-linked glycosylation site present in HMG2. PMID- 8621649 TI - Structural analysis of the predicted coiled-coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1). Empirical localization of the N-terminal globular domain-rod boundary. AB - The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family. PMID- 8621650 TI - Mutations in the B subunit of Escherichia coli DNA gyrase that affect ATP dependent reactions. AB - We have previously reported specific labeling of Escherichia coli DNA gyrase by the ATP affinity analog pyridoxal 5'-diphospho-5'adenosine (PLP-AMP), which resulted in inhibition of ATP-dependent reactions. The analog was found to be covalently bound at Lys103 and Lys110 on the gyrase B subunit (Tamura, J. K., and Gellert, M. (1990) J. Biol. Chem. 265, 21342-21349). In this study, the importance of these two lysine residues is examined by site-directed mutagenesis. Substitutions of Lys 103 result in the loss of ATP-dependent functions. These mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a nonhydrolysable ATP analog, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP). The ATP-independent functions of gyrase, such as relaxation of negatively supercoiled DNA and oxolinic acid-induced cleavage of double-stranded DNA, are unaffected by these mutations, suggesting that the mutant B subunits are assembling correctly with the A subunits. Gyrase with substitutions of Lys110 retains all activities. However, the affinity of ATP is decreased. The DNA supercoiling activity of gyrase A2B2, tetramers reconstituted with varying ratios of inactive mutant and wild-type gyrase B subunits is consistent with a mechanism of DNA supercoiling that requires the interdependent activity of both B subunits in ATP binding and hydrolysis. PMID- 8621651 TI - Human T-cell leukemia virus type I tax masks c-Myc function through a cAMP dependent pathway. AB - Human T-cell leukemia virus type I Tax is a pleiotropic gene regulator that functions through CREB/ATF- and NF-kappaB-mediated pathways. In most contexts, Tax is a potent gene activator. Here, we describe an unexpected finding of Myc repression by Tax. In cells that overexpress human T-cell leukemia virus type I Tax, the detection of c-Myc protein in the nucleus by a monoclonal antibody was masked. Tax prevented immunological visualization of a Myc epitope contained within amino acids 45-104, resulting in interference with Myc function in transcription and in anchorage-independent cell growth. Tax did not affect steady state protein levels since detection of c-Myc with other antibodies was unperturbed. Four observations suggest that this Tax-Myc interaction is mediated through CREB/ATF signal transduction. 1) Tax point mutants, selectively defective for activation of CREB/ATF but not NF-kappaB, failed to mask c-Myc; 2) masking of Myc was abolished when Tax-expressing cells were treated with protein kinase inhibitor H-9; 3) Tax-specific shielding of Myc is absent in cells (B1R) that are genetically defective for cAMP signaling; and 4) forskolin treatment of cells mimicked Tax in masking the Myc epitope. Considered collectively, these findings suggest a regulation of Myc function at the level of localized protein conformation. PMID- 8621652 TI - Interactions between the repressor and the early operator region of bacteriophage Mu. AB - The repressor of bacteriophage Mu, c, binds to three operator sites, O1, O2, and O3, overlapping two divergent promoters, which regulate the lytic and lysogenic pathways. Its binding to this operator region generates several complexes, which were analyzed by DNase I protection experiments. We demonstrate that c first binds to two 11-base pair partially repeated sequences in O2 that could represent "core" binding sites for the repressor. This initial interaction serves as an organizer of a more complex nucleoprotein structure in which O2, O1, and O3 become successively occupied. The quaternary structure of the repressor was also investigated. Size exclusion chromatography and protein-protein crosslinking experiments with chemicals that possess linking arms of various lengths indicate that the repressor oligomerizes in solution. A model is proposed describing the successive interactions of c with the operator sites O2, O1, and O3 leading to the elaboration of a higher order structure in which the early lytic functions are repressed. PMID- 8621653 TI - Characterization of phenobarbital-inducible mouse Cyp2b10 gene transcription in primary hepatocytes. AB - The mouse phenobarbital (PB)-inducible Cyp2b10 gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405 base pair (bp) Cyp2bl0 promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (-123/-82 bp) into the middle of a consensus sequence to the so-called "Barbie box." In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10 promoter identified a basal transcription element at -64/-34 bp and a negative element at -971/-775 bp. Sequences contained within the -1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the -1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene 158, 263 268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10 gene are located in the distal promoter. PMID- 8621654 TI - Evidence for the existence of a sodium-dependent glutathione (GSH) transporter. Expression of bovine brain capillary mRNA and size fractions in Xenopus laevis oocytes and dissociation from gamma-glutamyltranspeptidase and facilitative GSH transporters. AB - Our laboratory previously has shown apparent carrier-mediated glutathione (GSH) uptake across the blood-brain barrier (BBB) in two animal models. In the present study, when Xenopus oocytes were injected with bovine brain capillary mRNA expression of intact GSH, uptake was observed after 3 days. When total mRNA was converted to cDNA and subfractionated with subsequent cRNA injection into oocytes, three distinct fractions (5, 7-8, and 11-12) expressed carrier-mediated intact GSH transport. Northern blot analysis established the presence of RcGshT, the previously cloned sodium-independent hepatic canalicular transporter, only in fraction 5. GSH transport activity in fraction 7 was significantly inhibited by replacement of NaCl with choline chloride and by sulfobromophthalein-GSH, neither of which affects RcGshT. The Na(+)-dependent GSH uptake kinetics exhibited high affinity (approximately 400 micron) and low affinity (approximately 10 mM) components. Fraction 11 expressed Na(+)-independent transport of intact GSH and also contained the GGT transcript. In conclusion, we have identified three distinct sized transcripts from bovine brain capillary mRNA which express GSH transport: one fraction expresses a novel Na(+)-dependent GSH uptake which can be dissociated unequivocally from both GGT and RcGshT for the first time and which may account for uptake of GSH against its electrochemical gradient at the BBB. PMID- 8621655 TI - Molecular cloning and tissue distribution of keratocan. Bovine corneal keratan sulfate proteoglycan 37A. AB - Previous studies showed that the keratan sulfate-containing proteoglycans of bovine corneal stroma contain three unique core proteins designated 37A, 37B, and 25 (Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W. (1991) J. Biol. Chem. 266, 14226-14231). Degenerate oligonucleotides designed from amino acid sequences of the 37A protein were used to screen a cDNA expression library from cultured bovine keratocytes. A cDNA clone coding for keratocan, a 37A protein, was isolated and sequenced. The deduced keratocan amino acid sequence is unique but related to two other keratan sulfate-containing proteins, lumican (the 37B core protein) and fibromodulin. These three proteins share approximately 35% amino acid identity and a number of conserved structural features. Northern hybridization and immunoblotting of tissue extracts found keratocan distribution to be more limited than that of lumican or fibromodulin. Keratocan is abundant in cornea and sclera and detected in much lesser amounts in skin, ligament, cartilage, artery, and striated muscles. Only in cornea was keratocan found to contain large, sulfated keratan sulfate chains. Keratocan, like lumican, is a core protein of a major corneal proteoglycan but is present in non-corneal tissues primarily as a nonsulfated glycoprotein. PMID- 8621656 TI - Evidence for the direct interaction of the nifW gene product with the MoFe protein. AB - The Azotobacter vinelandii nifW gene, under control of the nifH promoter, was subcloned into the broad host range multicopy plasmid pKT230 for overexpression in both wild-type and delta nifW strains of A. vinelandii. Unlike the parent delta nifW strain, which grows slowly relative to wild-type under N2-fixing conditions, both overproduction strains grow at the same rate, showing that the overexpressed nifW product is functional in vivo. The approximately 40-fold overexpressed protein was purified, and sequence analysis confirmed its identity. During purification it was observed that NifW in crude extracts ran above the predicted molecular weight on denaturing gels and that as the purification proceeded lower molecular weight forms appeared. Mass spectrometry and studies with protease inhibitors revealed that this abnormal behavior was due to proteolysis. Native molecular weight determinations demonstrate that NifW is a homomultimer, most likely a trimer. Native gel electrophoresis analysis shows that the behavior of wild-type and overexpressed NifW are identical and that when extracts are prepared anaerobically only the homomultimeric forms of NifW are observed. When extracts are exposed to oxygen, however, NifW becomes part of a very high molecular weight complex. Immunoprecipitation with NifW antibodies demonstrate that under those conditions NifW specifically associates with the MoFe protein. These data are consistent with a model whereby NifW is not involved in the initial assembly of an active MoFe protein but rather is part of a system design to protect the MoFe protein from O2 damage. PMID- 8621657 TI - Differential expression of the expression site-associated gene I family in African trypanosomes. AB - A minimum of 20 different mRNA species encoding related members of the expression site-associated gene I (ESAG-I) family occur in metacyclic variant antigen type 4 bloodstream trypanosomes. None of these ESAG-I mRNAs are derived from the metacyclic variant antigen type 4 variant surface glycoprotein (VSG) gene expression site, and some appear to come from pseudogenes. The ESAG-Is are transcribed in both procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a detectable steady state level only in bloodstream trypanosomes. At least five different groups of 3'-untranslated regions (3'-UTRs) are represented among these ESAG-I mRNAs, suggesting that the 3'-UTR does not contribute to their differential expression. Some ESAG-I mRNAs completely lack a 3'-UTR or have only a single nucleotide as a 3'-UTR. Transcription of the ESAG-Is is sensitive to alpha-amanitin, indicating that they are transcribed by a different RNA polymerase than the VSG genes. These results collectively demonstrate that ESAG-I's are a heterogeneous population that can be expressed independently of VSG genes, but like the VSG genes, their mRNAs are present in the bloodstream stage of the parasite and not in the procyclic stage. PMID- 8621658 TI - Distinct roles of the two tumor necrosis factor (TNF) receptors in modulating TNF and lymphotoxin alpha effects. AB - The role for the two tumor necrosis factor (TNF) receptors in discriminating TNF and lymphotoxin alpha (LTalpha) effects has been studied. TNF and LTalpha were equally mitogenic in Fs4 fibroblasts, which express a high amount of the p55 compared to the p75 TNF receptors (TNFRs). In contrast, TNF was more potent than LTalpha in mediating gene regulation and cytotoxicity in SW480-betaGal cells and KYM-1 cells, which have a high p75/p55 TNFR ratio. Both TNF and LTalpha showed comparable affinities for the two TNFRs. However, in contrast to LTalpha, TNF dissociated rapidly from the p75 TNFR, whereas both cytokines dissociated slowly from the p55 TNFR. Soluble p55 TNFR was much more potent than soluble p75 TNFR in inhibiting TNF cytotoxicity, whereas both soluble receptors moderately decreased LTalpha-mediated cytotoxicity with comparable efficacy. Antagonistic monoclonal antibodies against either TNFR types markedly inhibited TNF effects. However, only the p55 TNFR antagonistic antibody significantly decreased LTalpha-mediated cytotoxicity and cytomegalovirus promoter activation, whereas blocking of the p75 TNFR enhanced the LTalpha effects. These data suggest that whereas the p75 TNFR can both directly propagate TNF signals and "pass" TNF to the p55 TNFR, it attenuates LTalpha and may serve as a decoy receptor for this cytokine. PMID- 8621660 TI - The eta isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene. AB - Transglutaminase 1 (TGase 1) is expressed during the terminal differentiation of keratinized squamous epithelium to form cornified cell envelope in differentiated keratinocytes by the epsilon-(gamma-glutamyl) cross-linking reaction. The gene for human TGase 1 is responsible for autosomal recessive lamellar ichthyosis, a severe hereditary keratinizing disorder of the skin. We examined the transcriptional activity of the gene in FRSK, rat keratinocytic cells, transfected with the luciferase reporter gene under control of the 5' upstream region of human TGase 1 gene. Transfection of the reporter gene with an expression vector for the eta isoform of novel protein kinase C (nPKCeta), as well as exposure to 12-0-tetradecanoylphorbol-13-acetate, markedly increased the luciferase activity in FRSK, but not in HT-1080 fibrosarcoma cells, although exogenous nPKCeta was expressed in both. The induction was suppressed by deleting the TGase 1 upstream sequence from -95 to -67 and by deleting the kinase domain from exogenous nPKCeta. In comparison with other PKC isoforms, nPKCeta most effectively induced the luciferase activity. We suggest that nPKCeta, an epithelium-specific isoform of PKC, mediates the activation of the TGase 1 transcription. PMID- 8621659 TI - Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl. AB - Rse, Ax1, and c-Mer comprise a family of cell adhesion molecule-related tyrosine kinase receptors. Human Gas6 was recently shown to act as a ligand for both human Rse (Godowski et al., 1995) and human Ax1 (Varnum et al., 1995). Gas6 contains an NH2-terminal Gla domain followed by four epidermal growth factor-like repeats and tandem globular (G) domains. The G domains are related to those found in sex hormone-binding globulin and to those utilized by laminin and agrin for binding to the dystroglycan complex. A series of Gas6 variants were tested for their ability to bind to Rse and Ax1. The Gla domain and epidermal growth factor-like repeats were not required for receptor binding, as deletion variants of Gas6 which lacked these domains bound to the extracellular domains of both Rse and Axl. A deletion variant of Gas6 containing just the G domain region was shown to activate Rse phosphorylation. These results provide evidence that G domains can act as signaling molecules by activating transmembrane receptor tyrosine kinases. Furthermore, they provide a structural link between the activation of cell adhesion related receptors and the control of cell growth and differentiation by the G domain-containing superfamily of proteins. PMID- 8621661 TI - Cloning, characterization, and expression of cDNAs encoding human delta 1 pyrroline-5-carboxylate dehydrogenase. AB - Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDh; EC 1.5.1.12), a mitochondrial matrix NAD(+)-dependent dehydrogenase, catalyzes the second step of the proline degradation pathway. Deficiency of this enzyme is associated with type II hyperprolinemia (HPII), an autosomal recessive disorder characterized by accumulation of delta 1-pyrroline-5-carboxylate (P5C) and proline. As an initial step in understanding the biochemistry of human P5CDh and molecular basis of HPII, we utilized published peptide sequence data and degenerate primer polymerase chain reaction to clone two full-length human P5CDh cDNAs, differing in length by 1 kilobase pair (kb). Both cDNAs have the identical 1689-base pair open reading frame encoding a protein of 563 residues with a predicted molecular mass of 62 kDa. The long cDNA contains an additional 1-kb insert in the 3' untranslated region that appears to be an alternatively spliced intron. The conceptual translation of human P5CDh has 89% sequence identity with the published human P5CDh peptide sequences and 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively, as well as homology to several other aldehyde dehydrogenases. Both P5CDh cDNA clones detect a single 3.2-kb transcript on Northern blots of multiple human tissues, indicating the long cDNA containing the 3'-untranslated intron represents the predominant transcript. The P5CDh structural gene appears to be single copy with a size of about 20 kb localized to chromosome 1. To confirm the identity of the putative P5CDh cDNAs, we expressed them in a P5CDh-deficient strain of S. cerevisiae. Both conferred measurable P5CDh activity and the ability to grow on proline as a sole nitrogen source. PMID- 8621662 TI - Identification of a novel membrane transporter associated with intracellular membranes by phenotypic complementation in the yeast Saccharomyces cerevisiae. AB - A partial mouse cDNA was isolated by its ability to functionally complement a thymidine transport deficiency in plasma membranes of the yeast, Saccharomyces cerevisiae. The full-length cDNA encoded a previously unidentified 27-kDa protein (mouse transporter protein (MTP)) with four predicted transmembrane-spanning domains. MTP mRNA was detected in cells of several mammalian species, and its predicted protein sequence exhibited near identity (98%) with that of a human cDNA (HUMORF13). MTP and its homologs evidently reside in an intracellular membrane compartment because a protein (about 24 kDa) that was recognized by MTP specific antibodies was observed in a subcellular fraction of rat hepatocytes enriched for Golgi membranes. Deletion of the hydrophilic C terminus of MTP, which encompassed two putative signal motifs for intracellular localization (Tyr X-X-hydrophobic amino acid), allowed expression of recombinant protein (MTP deltaC) in plasma membranes of Xenopus laevis oocytes. MTP deltaC-expressing oocytes exhibited greater fragility than nonexpressing oocytes, and those that survived the experimental manipulations were capable of mediated uptake of thymidine, uridine, and adenosine. Thymidine uptake by MTP deltaC-expressing oocytes was inhibited by thymine and dTMP. MTP may function in the transport of nucleosides and/or nucleoside derivatives between the cytosol and the lumen of an intracellular membrane-bound compartment. PMID- 8621663 TI - A cell-specific glycosylated silk protein from Chironomus thummi salivary glands. Cloning, chromosomal localization, and characterization of cDNA. AB - Chironomid salivary glands contain 40 cells dedicated to the synthesis of a relatively small ensemble of silk proteins. Glands in some species contain a special lobe composed of 4 cells distinguishable from the others. We have cloned a special lobe-specific cDNA from Chironomus thummi salivary glands. Northern blots of salivary gland RNA demonstrated that the cDNA hybridizes to a 2.5 kilobase transcript present only in the special lobe. In situ hybridization mapped the gene encoding this cDNA to region A2b on polytene chromosome IV, the locus of the special lobe-specific Balbiani ring a. The deduced amino acid sequence encodes a protein with a calculated molecular mass of 77 kDa and numerous potential glycosylation sites; it appears unrelated to other known chironomid silk proteins. Polyclonal antibody, raised against a cDNA-encoded fusion protein, reacted exclusively with a special lobe-specific 160-kDa silk protein. Lectin binding studies indicate that the immunoreactive 160-kDa protein contains both N- and O-linked glycan moieties. We conclude that glycosylation most likely contributes to the difference between calculated and apparent molecular masses and that this cDNA encodes the special lobe-specific silk protein previously described as ssp160 (Kolesnikov, N. N., Karakin, E. I., Sebeleva, T. E., Meyer, L., and Serfling, E. (1981) Chromosoma 83, 661-677). PMID- 8621664 TI - PKN associates and phosphorylates the head-rod domain of neurofilament protein. AB - PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament. PMID- 8621665 TI - Biogenesis of the covalently flavinylated mitochondrial enzyme dimethylglycine dehydrogenase. AB - Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import. PMID- 8621666 TI - Molecular genetic analysis of the human Lewis histo-blood group system. II. Secretor gene inactivation by a novel single missense mutation A385T in Japanese nonsecretor individuals. AB - The Lewis histo-blood group system comprises two major antigens, Lewis a and Lewis b. The Lewis b antigen is a product of two fucosyltransferases, the alpha(1,3/1,4)fucosyltransferase (Lewis enzyme; Fuc-TIII) encoded by the Lewis gene and an alpha(1,2)fucosyltransferase which is not required for synthesis of Lewis a antigen. An enzyme responsible for secreting ABH antigens into body secretions (secretor enzyme) is also one of alpha(1,2)fucosyltransferases. A candidate gene encoding secretor enzyme Sec2 gene was recently cloned by Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D. ((1995) J. Biol. Chem. 270, 4632-4639) and Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. ((1995) J. Biol. Chem. 270, 4640-4649) who demonstrated a G428A nonsense mutation (Trp143 to terminal codon) in Sec2 of nonsecretors. However, the G428A nonsense mutation discovered in the Sec2 gene of nonsecretors in an ethnic group other than Japanese was not found in any of 45 Japanese nonsecretors, whereas one Filipino who had been erroneously registered as a Japanese possessed the G428A mutation heterozygously. In order to explore the Sec2 gene of a Japanese population, we performed a molecular genetic analysis of the Sec2 gene on 226 Japanese individuals, 21 in a family study and 205 in a random sampling study. We discovered two novel mutations in the Sec2 gene, an A385T missense mutation (Ile129 to Phe) that results in inactivation of Sec2 encoded alpha(1,2)fucosyltransferase and a C357T silent mutation which is irrelevant to amino acid substitution, in Japanese nonsecretors. The analysis of Japanese individuals using the polymerase chain reaction-restriction fragment length polymorphism method found three alleles in the Sec2 gene, the first having no mutation, the second having a C357T mutation, and the third having both C357T and A385T mutations, which we designated as Se1, Se2, and sej, respectively. Among 226 Japanese individuals, 40 having a Le(a+b-) phenotype and 5 having a Le(a-b-) nonsecretor phenotype were homozygous for sej/sej, whereas 149 having a Le(a-b+) phenotype and 32 having a Le(a-b-)-secretor phenotype possessed at least one Se1 or Se2. The frequencies of occurrence of Se1, Se2, and sej among 410 alleles examined in a random sample of 205 Japanese individuals were 15, 46, and 39%, respectively, indicating a rather wide distribution of the sej allele in the Japanese population. The results show that the Sec2 gene really encodes the secretor enzyme alpha(1,2)fucosyltransferase and indicate that a ethnic group specific nonsense or missense point mutation in the Sec2 gene determines nonsecretor status. The phylogenic aspect and biological significance of the Se and Le genes are discussed. PMID- 8621667 TI - The natural mutation Y248C of human angiotensinogen leads to abnormal glycosylation and altered immunological recognition of the protein. AB - Common molecular variants of the angiotensinogen gene have been associated with human hypertension. The rare Tyr to Cys change at residue 248 of mature angiotensinogen was identified in one pedigree. Heterozygous individuals (Y248C) had a 40% decrease in plasma angiotensinogen concentration and a 35% reduction of the angiotensin I production rate. Recombinant wild-type (Tyr-248) and mutant (Cys-248) proteins were stably expressed in Chinese hamster ovary cells. Angiotensinogen monoclonal antibodies revealed marked differences in the epitope recognition of the mutant protein and allowed the demonstration of its presence in plasma of Y248C individuals. Similar kinetic constants of angiotensin I production with human renin were observed for both proteins. Western blot analysis showed similar heterogeneities; however, a 3-kDa increase in molecular mass for the Cys-248 protein was observed after immunopurification. Metabolic labeling of the intracellular Cys-248 protein showed a 61-kDa band in addition to the 55.5- and 58-kDa bands observed for the Tyr-248 protein, with all bands being sensitive to endoglycosidase H. In addition, pulse-chase studies revealed a slower intracellular processing for the Cys-248 protein. In conclusion, the Cys 248 mutation alters the structure, glycosylation, and secretion of angiotensinogen in Chinese hamster ovary cells and is accompanied by a decrease in plasma angiotensinogen concentration in Y248C individuals. PMID- 8621668 TI - Porcine submaxillary mucin forms disulfide-bonded dimers between its carboxyl terminal domains. AB - COS-7 cells transfected with three different expression vectors encoding the 240 amino acid residue, disulfide-rich domain at the carboxyl terminus of porcine submaxillary mucin have been used to determine the possible function of the domain in forming higher oligomers of the mucin polypeptide chain. The domain is expressed as a disulfide-bonded dimer, as shown by SDS-gel electrophoretic analysis of the immunoprecipitated domain in the presence and absence of reducing agent and the cross-linking agent bis(sulfosuccinimidyl) suberate. Molecular weight determination by gel filtration on agarose columns in 6 M guanidine HCl confirmed dimer formation. However, the domain expressed is heterogeneous as the result of different extents of glycosylation. Pulse-chase studies with the 35S labeled domain show that dimer formation and secretion from cells occur very rapidly. Moreover, dimer formation is not dependent on the N-linked oligosaccharides on the domain. Evidence is presented that dimer formation most likely occurs in the endoplasmic reticulum before complex-type oligosaccharide synthesis is completed. Neither brefeldin A nor tunicamycin interferes with the rate of dimer formation. These studies suggest that the disulfide-rich domain acts to form dimers of the polypeptide chain of mucin. This role of the domain is consistent with its amino acid sequence similarity to the disulfide-rich domain of human prepro-von Willebrand factor, which also serves to form dimers of this blood coagulation factor. PMID- 8621669 TI - Induction of ferritin synthesis in cells infected with Mengo virus. AB - We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state. PMID- 8621670 TI - Systematic mutational analysis of the death domain of the tumor necrosis factor receptor 1-associated protein TRADD. AB - Tumor necrosis factor receptor 1 (TNF-R1) mediates most of the biological properties of TNF including activation of the transcription factor NF-kappaB and programmed cell death. An approximately 80-amino acid region within the intracellular domain of the receptor, termed the death domain, is required for signaling NF-kappaB activation and cytotoxicity. A TNF-R1-associated protein TRADD has been discovered that interacts with the death domain of the receptor. Elevated expression of TRADD in cells triggers both NF-kappaB activation and programmed cell death pathways. The biological activities of TRADD have been mapped to a 111-amino acid region within the carboxyl-terminal half of the protein. This region shows sequence similarity to the death domain of TNF-R1 and can self-associate and bind to the TNF-R1 death domain. We have performed an alanine scanning mutagenesis of TRADD's death domain to explore the relationship among its various functional properties. Mutations affecting the different activities of TRADD do not map to discrete regions but rather are spread over the entire death domain, suggesting that the death domain is a multifunctional unit. A mutant that separates cell killing from NF-kappaB activation by the TRADD death domain has been identified indicating that these two signaling pathways diverge with TRADD. Additionally, one of the TRADD mutants that fails to activate NF kappaB was found to act as dominant negative mutant capable of preventing induction of NF-kappaB by TNFalpha. Such observations provide evidence that TRADD performs an obligate role in TNF-induced NF-kappaB activation. PMID- 8621671 TI - Molecular determinants of the clearance function of type C receptors of natriuretic peptides. AB - Receptor-mediated endocytosis is the cellular mechanism by which type C receptors of natriuretic peptides exert their clearance function. In the present work, performed in recombinant Chinese hamster ovary cells stably transfected with wild type or mutated human kidney C receptors, we determined net endocytic rates (ER) of C receptor-ligand complexes, lysosomal hydrolysis of ligand (125I-labeled native atrial natriuretic factor, ANF1-28), and receptor recycling. Equilibrium ligand binding, immunocytochemistry, and immunoprecipitation were performed to characterize the transfected receptors. The net ER of recombinant wild type C receptors was approximately 6% of occupied receptors internalized per min, and C receptor-mediated lysosomal hydrolysis of ligand amounted to approximately 250% of specifically bound 125I-ANF1-28/h, with efficient recycling of internalized C receptors to the cell surface. Hypertonic sucrose reduced net ER and lysosomal hydrolysis of 125I-ANF1-28 more than 10-fold, indicating that endocytosis occurred via clathrin-coated pits. Total deletion of the cytoplasmic domain also reduced net ER and lysosomal hydrolysis of 125I-ANF1-28 by almost 10-fold, whereas deletion of the terminal 28 amino acids of the cytoplasmic tail led to a 4-fold reduction in these parameters. Replacement of cytoplasmic domain Tyr508 by Ala, or Tyr508 and Phe538 by Ala, reduced net endocytosis and lysosomal hydrolysis of 125I-ANF1-28 by 40-50%. Replacement of extracellular domain Cys473 by Ala impeded the constitutive formation of homodimers and reduced by approximately 50% the net ER and lysosomal hydrolysis of 125I-ANF1-28. These results demonstrate that the cytoplasmic domain of C receptors, Tyr508 within this domain, and constitutive receptor dimerization are the major molecular determinants of the clearance function of C receptors. PMID- 8621672 TI - Molecular basis of genetic instability of triplet repeats. PMID- 8621673 TI - Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets. AB - We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca2+ influx via receptor-operated cation channels and release of Ca2+ from intracellular stores. Fura-2 measurements of [Ca2+]i in stirred platelet suspensions showed that both ADP (40 microM) and the non-hydrolyzable ATP analogue alphabeta-meATP (alpha, beta-methyleneadenosine 5-triphosphate, 10 microM) activated a rapid Ca2+ influx whereas only ADP mobilized Ca2+ from internal stores. In "nystatin" whole cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, alpha, beta-meATP and ATPgammaS (adenosine 5 -O-(3-thiotriphosphate), all activated a cation channel permeable to both monovalent and divalent cations with a single-channel conductance of 11 picosiemens in NaCl saline. The current response to ATP (40 microM) was activated within 20 ms and desensitized with a time constant of 47-107 ms in the continued presence of agonist, which are characteristics of P2X1 receptors in other tissues. We conclude that human platelets possess a P2X1 purinoceptor, which mediates a rapid phase of ADP- or ATP-evoked Ca2+ entry via a cation channel, whereas one or more separate ADP selective P2 purinoceptors evoke release of calcium from intracellular stores. PMID- 8621674 TI - Amino acid side chains that define muscarinic receptor/G-protein coupling. Studies of the third intracellular loop. AB - Amino acids in the third intracellular loops of receptors play pivotal roles in G protein coupling. To define their structural requirements, we have subjected the N- and C-terminal regions of this loop (Ni3 and Ci3, respectively) of the m5 muscarinic receptor to random saturation mutagenesis. (see Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141 3146 and Hill-Eubanks, D., Burstein, E. S., Spalding, T. A., Brauner Osborne, H., and Brann, M. R. (1996) J. Biol. Chem. 271, 3058 3065). In the present study, we have extended our analysis of Ni3 by constructing libraries of receptors with all possible amino acid substitutions at the residues we previously identified as functionally important and characterizing their functional phenotypes. Numerous hydrophobic substitutions were well tolerated at Ile216 and Thr220 and caused constitutive activation in two cases, establishing that hydrophobicity is structurally favored at these positions and that many amino acid side chains are compatible with this structural role. Similarly, hydrophobic and polar, but not charged, substitutions were observed at Tyr217, but in contrast to results for Thr220, most substitutions at Tyr217 substantially decreased maximum response and increased the EC50 for carbachol, demonstrating that the specific side chain of residue 217 participates in G-protein coupling. Arg223 allowed the widest range of substitutions of the residues tested, but only basic residues were well tolerated. All other substitutions significantly increased (up to 100-fold) the EC50 for carbachol without significantly affecting maximal response. There were no significant changes in the ligand binding properties of these mutant receptors. We conclude that Ile216 and Thr220 fulfill a structural role, forming the foundation of the G-protein-coupling pocket, whereas Tyr217 and Arg223 contact G-proteins through specific side chain interactions. We propose that G-proteins are recruited to receptors by ionic interactions and that hydrophobic residues participate in activation. PMID- 8621675 TI - Characterization of the structure and function of a novel MAP kinase kinase (MKK6). AB - Mitogen-activated protein (MAP) kinases require dual phosphorylation on threonine and tyrosine residues in order to gain enzymatic activity. This activation is carried out by a family of enzymes known as MAP kinase kinases (MKKs or MEKs). It appears that there are at least four subgroups in this family; MEK1/MEK2 subgroup that activates ERK1/ERK2, MEK5 that activates ERK5/BMK1, MKK3 that activates p38, and MKK4 that activates p38 and Jun kinase. Here we describe the characteristics of a new MKK termed MKK6. The clones we isolated encode two splice isoforms of human MKK6 comprised of 278 and 334 amino acids, respectively, and one murine MKK6 with 237 amino acids. Sequence information derived from cDNA cloning indicated that MKK6 is most closely related to MKK3. The functional data revealed from co-transfection assays suggests that MKK6, like MKK3, selectively phosphorylates p38. Unlike the previously described MKKs (or MEKs), MKK6 exists in a variety of alternatively spliced isoforms with distinct patterns of tissue expression. This suggests novel mechanisms regulating activation and/or function of various forms of MKK6. PMID- 8621676 TI - Hydroxyalkenal formation induced by advanced glycosylation of low density lipoprotein. AB - Advanced glycosylation end products (AGEs) have been identified to be present on both the apolipoprotein and lipid components of low density lipoprotein (LDL) and to act to prevent its recognition and uptake by high affinity, tissue LDL receptors. Lipid-linked AGEs form readily in vitro by the covalent addition of glucose to the amine-containing head groups of phospholipids. This process is accompanied by oxidation of the unsaturated fatty acid side chains and occurs in the absence of exogenously added transition metals or free radical generating systems, suggesting that AGE formation may contribute significantly to lipid oxidation in vivo. To assess more precisely the chemical basis of AGE-induced oxidative modification, we performed gas chromatography-mass spectrometry analysis of the lipid products which form over time during LDL-advanced glycosylation in vitro. Negative ion chemical ionization mass spectroscopy of two major compounds that were identified were consistent with the structures of the fatty acid oxidation products 4-hydroxyhexenal and 4-hydroxynonenal. These data support the concept that AGE formation in close proximity to unsaturated fatty acyl groups leads to lipid oxidation and provide additional evidence that advanced glycosylation is an important pathogenic modification of the LDL particle in vivo. PMID- 8621677 TI - Thermodynamics of colchicinoid-tubulin interactions. Rrol of B-ring and C-7 substituent. AB - The quenching of tryptophan fluorescence has been used to determine the kinetic and thermodynamic parameters of binding of B-ring analogs of colchicine to tubulin. The on rate, activation energy, off-rate, and thermodynamics of binding reaction have been found to be controlled at different points of analog structure. The on-rate and off-rate of deacetamidocolchicine (DAAC) binding with tubulin is 17 times slower than that of 2-methoxy-5-(2',3',4' trimethoxyphenyl)tropone-tubulin (AC-tubulin) interaction, although both reactions have very similar activation energies. The presence of B-ring alone does not significantly affect the thermodynamics of the binding reactions either, since both AC-tubulin and DAAC-tubulin interactions are enthalpy driven. Introduction of a NH2 group at C-7 position of the B-ring, as in deacetylcolchicine (NH2-DAAC) lowers the on-rate further with a significant rise in the value of the activation energy. However, bulkier substitutions at the same position, as in demecolcine (NHMe-DAAC) and N-methyldemecolcine (NMe2-DAAC) have no significant additional effect either on the on-rate or on the value of activation energy. Introduction of NH2 group in the C-7 position of B-ring also increases the positive entropy of the binding reaction to a significant extent, and it is maximum when NMe2 is substituted instead of NH2 group. Thus, interaction of NH2-DAAC, NHMe-DAAC, and NMe2-DAAC with tubulin are entropy driven. Our results suggest that the B-ring side chain of aminocolchicinoids makes contact(s) with dimeric tubulin molecules. PMID- 8621678 TI - Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan. AB - Potato D-enzyme was purified from recombinant Escherichia coli, and its action on synthetic amylose (average Mr of 320,000) was analyzed. D-enzyme treatment resulted in a decrease in the ability of the amylose to form a blue complex with iodine. Analysis of the products indicated that the enzyme catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic alpha-1,4 glucan (cycloamylose). Confirmation of the cyclic structure was achieved by demonstrating the absence of reducing and nonreducing ends, resistance to hydrolysis by glucoamylase (an exoamylase), and by "time of flight" mass spectrometry. The degree of polymerization of cycloamylose products was determined by time of flight mass spectrometry analysis and by high-performance anion-exchange chromatography following partial acid hydrolysis of purified cycloamylose molecules and was found to range from 17 to several hundred. The yield of cycloamylose increased with time and reached >95%. D-enzyme did not act upon purified cycloamylose, but if glucose was added as an acceptor molecule, smaller cyclic and linear molecules were produced. The mechanism of the cyclization reaction, the possible role of the enzyme in starch metabolism, and the potential applications for cycloamylose are discussed. PMID- 8621679 TI - Induction, localization, and purification of a novel sialidase, deaminoneuraminidase (KDNase), from Sphingobacterium multivorum. AB - Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNalpha2-->3Galbeta1- >3GalNAc alpha1-->3[KDNalpha2--> (8KDN alpha2-->)n-->6]GalNAcol, as a sole carbon source induced KDNase SM activity 15 40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid. PMID- 8621680 TI - ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae. AB - Glutathione-dependent detoxification reactions are catalyzed by the enzyme glutathione S-transferase and are important in drug resistance in organisms ranging from bacteria to humans. The yeast Issatchenkia orientalis expresses a glutathione S-transferase (GST) protein that is induced when the GST substrate o dinitrobenzene (o-DNB) is added to the culture. In this study, we show that overproduction of the I. orientalis GST in Saccharomyces cerevisiae leads to an increase in o-dinitrobenzene resistance in S. cerevisiae cells. To recover genes that influence o-DNB resistance in S. cerevisiae, a high copy plasmid library was screened for loci that elevate o-DNB tolerance. One gene was recovered and designated ROD1 (resistance to o-dinitrobenzene). This locus was found to encode a novel protein with no significant sequence similarity with proteins of known function in the data base. An epitope-tagged version of Rod1p was produced in S. cerevisiae and shown to function properly. Subcellular fractionation experiments indicated that this factor was found in the particulate fraction by differential centrifugation. Overproduction of Rod1p leads to resistance to not only o-DNB but also zinc and calcium. Strains that lack the ROD1 gene are hypersensitive to these same compounds. Rod1p represents a new type of molecule influencing drug tolerance in eukaryotes. PMID- 8621681 TI - Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. AB - A monoclonal antibody has been produced which immunoprecipitates 58- and 53-kDa proteins which are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor related receptor. P58/53 did not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 did not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein which, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor. PMID- 8621682 TI - Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate stimulated lipid peroxidation. AB - Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances. PMID- 8621683 TI - Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. AB - Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2 terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP 3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12 myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells. PMID- 8621684 TI - An intact N terminus of the gamma subunit is required for the Gbetagamma stimulation of rhodopsin phosphorylation by human beta-adrenergic receptor kinase 1 but not for kinase binding. AB - Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved Ggamma fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, Ggamma contributes secondary and/or tertiary structural features to activate the kinase. PMID- 8621685 TI - Arachidonate and related unsaturated fatty acids selectively inactivate the guanine nucleotide-binding regulatory protein, Gz. AB - Gz is a member of the family of trimeric guanine nucleotide-binding regulatory proteins (G proteins), which plays a crucial role in signaling across cell membranes. The expression of Gz is predominately confined to neuronal cells and platelets, suggesting an involvement in a neuroendocrine process. Although the signaling pathway in which Gz participates is not yet known, it has been linked to inhibition of adenylyl cyclase. We have found that arachidonate and related unsaturated fatty acids suppress guanine nucleotide binding to the alpha subunit of Gz. This inhibition of nucleotide binding by cis-unsaturated fatty acids is specific for Gz alpha; other G protein alpha subunits are relatively insensitive to these lipids. The IC50 for inhibition by the lipids closely corresponds to their critical micellar concentrations, suggesting that the interaction of the lipid micelle with Gzalpha is the primary event leading to inhibition. The presence of the acidic group of the fatty acid is critical for inhibition, as no effect is observed with the corresponding fatty alcohol. While arachidonic acid produces near-complete inhibition of both GDP and guanosine 5-(3-O thio)triphosphate binding by Gzalpha, release of GDP from the protein was unaffected. Furthermore, the rate of inactivation of Gzalpha by arachidonate is essentially identical to the rate of GDP release from the protein, indicating that GDP release is required for inactivation. These observations indicate that the mechanism of inactivation of Gzalpha by unsaturated fatty acids is through an interaction of an acidic lipid micelle with the nucleotide-free form of the protein. Although the physiologic significance of this finding is unclear, similar effects of unsaturated fatty acids on other proteins involved in cell signaling indicate potential roles for these lipids in signal modulation. Additionally, the ability of arachidonate to inactivate this adenylyl cyclase inhibitory G protein provides a molecular mechanism for previous findings that treatment of platelets with arachidonate results in elevated cAMP levels. PMID- 8621686 TI - The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity. AB - Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+ permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin. PMID- 8621687 TI - Cyclophilin 40 (CyP-40), mapping of its hsp90 binding domain and evidence that FKBP52 competes with CyP-40 for hsp90 binding. AB - The structurally related immunophilins cyclophilin 40 (CyP-40) and FKBP52 have been identified as components of the unactivated estrogen receptor. Both immunophilins have a similar molecular architecture that includes a C-terminal segment with a tetratricopeptide repeat (TPR) domain predicted to mediate protein interaction. hsp90 is a common cellular target for CyP-40 and FKBP52. Deletion mutants of CyP-40 fused to glutathione S-transferase were immobilized on glutathione-agarose and then used in a rapid hsp90 retention assay to define regions of the CyP-40 C terminus that are important for hsp90 binding. Our evidence suggests that the TPR domain is not sufficient for stable association of CyP-40 with hsp90 and requires the participation of flanking acidic and basic residues clustered at the N- and C-terminal ends, respectively. Both microdomains are characterized by alpha-helical structures with segregated hydrophobic and charged residues. Corresponding regions were identified in FKBP52. By preincubating myometrial cytosol with lysates containing bacterially expressed FKBP52, we have shown that FKBP52 competes with CyP-40 for hsp90 binding. Our results raise the possibility of a mutually exclusive association of CyP-40 and FKBP52 with hsp90. This would lead to separate immunophilin-hsp90-receptor complexes and place the estrogen receptor under the control of distinct immunophilin signaling pathways. PMID- 8621688 TI - Rational design of granulocyte-macrophage colony-stimulating factor antagonist peptides. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the four helix bundle family of cytokines/growth factors which exhibit several activities. It is a hematopoietic growth factor, a cytokine involved in inflammatory and immune processes, an adjunct for cancer therapy, and an anti-tumor immunomodulator. Studies of interactions between GM-CSF and its receptor and identification of small peptides presenting binding capacity to the receptor are important goals for the development of GM-CSF analogs. Here we describe the study of two cyclic peptides, 1785 and 1786, developed based on structural analysis of the GM-CSF region mimicked by anti-anti-GM-CSF recombinant antibody 23.2. These peptides were designed to structurally mimic the positions of specific residues on the B and C helices of human GM-CSF implicated in receptor binding and bioactivity. Both 1785 and 1786 were specifically recognized by polyclonal anti GM-CSF antibody (stronger for 1786 than 1785). 1786 also competitively inhibited binding of GM-CSF to the GM-CSF receptor on HL-60 cells and demonstrated antagonist bioactivity, as shown by its reversal of GM-CSF's ability to inhibit apoptosis of the GM-CSF-dependent cell line MO7E. These studies support the role of residues on the GM-CSF B and C helices in receptor binding and bioactivity and suggest strategies for mimicking binding sites on four-helix bundle proteins with cyclic peptides. PMID- 8621690 TI - Chimeric vesicular monoamine transporters identify structural domains that influence substrate affinity and sensitivity to tetrabenazine. AB - The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. Two domains of VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 and the other from the end of TMD9 through TMD12, increase the affinity for serotonin and histamine as well as the sensitivity to tetrabenazine but only in the context of more C-terminal and more N-terminal VMAT2 sequences, respectively. In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein. PMID- 8621689 TI - Biosynthesis and processing of proteinase 3 in U937 cells. Processing pathways are distinct from those of cathepsin G. AB - Proteinase 3 is a human polymorphonuclear leukocyte serine proteinase that degrades elastin in vitro and causes emphysema when administered by intratracheal insufflation into hamsters. Proteinase 3, stored in the azurophilic granules, is expressed in progenitor cells of myeloid origin. In the present study, the biosynthesis, processing, and intracellular transport of the enzyme was investigated in the human myelomonocytic cell line U937. Proteinase 3 is initially identified as a 35-kDa precursor and converted into the 29-kDa mature form within 3 h. By using a combination of techniques including amino-terminal sequencing, we identified the 35-kDa form as a zymogen containing an activation dipeptide but lacking the amino-terminal 25 residues, presumably the result of cleavage by a signal peptidase. Tunicamycin treatment and alkalinization of acidic cell compartments with NH4Cl did not prevent the processing of the proteinase 3 zymogen into the mature form, suggesting that the enzyme is targeted to the cytoplasmic granules by a mechanism other than the mannose 6-phosphate receptor. Brefeldin A inhibited the zymogen processing, suggesting that the dipeptide cleavage occurred in a post-Golgi organelle. The enzyme responsible for the removal of the dipeptide is a cysteine proteinase since E-64d, a class specific inhibitor, prevented processing. However, treatment of cells with a dipeptidyl peptidase I inhibitor, Gly-Phe-diazomethyl ketone and with the lysosomotropic agents, NH4Cl and chloroquine, did not prevent dipeptide cleavage, indicating that the processing enzyme for proteinase 3 is not dipeptidyl peptidase I. In contrast, Gly-Phe-diazomethyl ketone inhibited cleavage of the dipeptide from cathepsin G. This indicates that processing of proteinase 3 is distinct from that of cathepsin G. Proteinase 3 is also processed at the COOH terminal extension. Cleavage takes place next to Arg-222, suggesting that a trypsin-like proteinase is involved in the COOH-terminal processing. PMID- 8621691 TI - Structural studies on folding intermediates of serine hydroxymethyltransferase using single tryptophan mutants. AB - Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4 degrees C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V.(1995) J. Biol. Chem. 270, 19294-19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state. PMID- 8621692 TI - The COQ7 gene encodes a protein in saccharomyces cerevisiae necessary for ubiquinone biosynthesis. AB - Ubiquinone (coenzyme Q) is a lipid that transports electrons in the respiratory chains of both prokaryotes and eukaryotes. Mutants of Saccharomyces cerevisiae deficient in ubiquinone biosynthesis fail to grow on nonfermentable carbon sources and have been classified into eight complementation groups (coq1 coq8; Tzagoloff, A., and Dieckmann, C. L.(1990) Microbiol. Rev. 54, 211-225). In this study we show that although yeast coq7 mutants lack detectable ubiquinone, the coq7 1 mutant does synthesize demethoxyubiquinone (2-hexaprenyl-3-methyl-6 methoxy-1,4-benzoquinone), a ubiquinone biosynthetic intermediate. The corresponding wild-type COQ7 gene was isolated, sequenced, and found to restore growth on nonfermentable carbon sources and the synthesis of ubiquinone. The sequence predicts a polypeptide of 272 amino acids which is 40% identical to a previously reported Caenorhabditis elegans open reading frame. Deletion of the chromosomal COQ7 gene generates respiration defective yeast mutants deficient in ubiquinone. Analysis of several coq7 deletion strains indicates that, unlike the coq7 1 mutant, demethoxyubiquinone is not produced. Both coq7 1 and coq7 deletion mutants, like other coq mutants, accumulate an early intermediate in the ubiquinone biosynthetic pathway, 3-hexaprenyl-4-hydroxybenzoate. The data suggest that the yeast COQ7 gene may encode a protein involved in one or more monoxygenase or hydroxylase steps of ubiquinone biosynthesis. PMID- 8621693 TI - Site-directed mutagenesis of residues at subunit interfaces of porcine fructose 1,6-bisphosphatase. AB - Mutation of Arg-15, Glu-19, Arg-22, and Thr-27 of porcine liver fructose-1,6 bisphosphatase was carried out by site-directed mutagenesis. These residues are conserved in all known primary sequences of mammalian fructose-1,6 bisphosphatase. On the basis of the crystal structure of the enzyme, Arg-15, Glu 19, and Arg-22 are located at the interface of the two dimers (C1-C2 and C3-C4), and Thr-27 is in the AMP binding site. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible differences were observed between the secondary structures of the wild-type and mutant forms of fructose-1, 6-bisphosphatase on the basis of CD data. Kinetic analyses revealed similar kcat values for mutants R15A, E19Q, R22K, and T27A of fructose-1,6 bisphosphatase; however, a 2-fold increase of kcat was observed with R22M compared with that of the wild-type enzyme. Small changes in Km values for fructose-1,6-bisphosphate were found in the five mutants. 4 6-fold decreases in Ki values for fructose 2,6-bisphosphate and 5-9-fold decreases in the binding affinity of Mg2+ relative to the wild-type enzyme were exhibited by R15A and E19Q. No alteration of Mg2+ cooperativity was found in the five mutants. Significant changes in Ki values for AMP were obtained in the case of R22K (30 fold) and T27A (1300-fold) with a Hill coefficient of 2.0. Replacement of Arg-22 with methionine, however, caused the total loss of AMP cooperativity without changing AMP affinity. Modeling of the mutant structures was undertaken in an attempt to define the functional role of Arg-22. These studies link specific interactions between subunits in fructose-1,6-bisphosphatase to observed properties of cooperativity. PMID- 8621694 TI - Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing. AB - Apolipoprotein B (apoB) RNA editing involves site-specific deamination of a cytidine to a uridine. A mooring sequence, a spacer region, and a regulator region are components of the apoB RNA editing motif of which only the mooring sequence is both necessary and sufficient for editosome assembly and editing. The catalytic component of the editosome is APOBEC-1. In rat hepatoma, stable cell lines, overexpression of APOBEC-1 resulted in 3 6-fold stimulation of the editing efficiency on either rat endogenous apoB RNA or transiently expressed human apoB RNA. In these cell lines, cytidines in addition to the one at the wild type site were edited. The occurrence and efficiency of this "promiscuous" editing increased with increasing expression of APOBEC-1. Promiscuous editing was restricted to cytidines 5' of the mooring sequence and only occurred on RNAs that had been edited at the wild type site. Moreover, RNAs with mutant editing motifs supported high efficiency but low fidelity editing in the presence of high levels of APOBEC-1. This study demonstrates that overexpression of APOBEC-1 can increase the efficiency of site-specific editing but can also result in promiscuous editing. PMID- 8621695 TI - Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase. AB - The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does not. This identifies sites around residue 70 and/or between 202 and 212 of the gamma subunit as involved in epsilon subunit binding. PMID- 8621696 TI - Identification of a thrombin response element in the human platelet-derived growth factor B-chain (c-sis) promoter. AB - Thrombin is a coagulation system protease that also serves as a potent stimulator of gene expression in several cell types, including endothelial cells (EC). We and others have previously demonstrated that the transcription of platelet derived growth factor (PDGF) B-chain (c-sis) by EC is stimulated severalfold by thrombin. Here we examine the molecular mechanism of this regulatory process using bovine aortic EC transiently transfected with a vector containing the chloramphenicol acetyltransferase (CAT) gene under the control of a 400-base pair fragment of the human PDGF B-chain promoter. Thrombin treatment of these cells caused a severalfold increase in CAT expression. Deletion analysis and site directed mutagenesis revealed that the region spanning nucleotides -61 to -53 from the transcription initiation site (referred to as the thrombin response, or ThR, region) was critical for the transcriptional response to thrombin. Electrophoretic mobility shift assays with an oligonucleotide corresponding to the region -64 to -44, which contained the ThR region, led to the identification of a thrombin-inducible nuclear factor (TINF) in extracts from thrombin-treated, but not control, EC. TINF was formed as early as 40 min post-thrombin treatment, persisted for at least 7 h, but was no longer present after 24 h. TINF appeared in the absence of de novo protein synthesis. The ThR region consists of a repeat of a CCACCC element in an ABBA configuration, which, based on mutation analysis and transfection assays, appears to be critical in mediating thrombin stimulation of the PDGF B-chain gene. The conservation of the ThR region in the promoter of the PDGF B-chain among three species (human, feline, and murine) further supports the importance of this region as a cis-acting regulatory element. PMID- 8621698 TI - Structural studies of a peptide activator of human lecithin-cholesterol acyltransferase. AB - The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase (LCAT) despite its lack of sequence homology to apolipoprotein A-I, the primary in vivo activator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP-20's ability to activate LCAT were studied by optical and two-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles. Analysis of the CD data shows that LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The helical nature of the peptides in the presence of both lipids was confirmed by upfield H alpha NMR secondary shifts relative to random coil values. Average structures for both peptides in aqueous solutions containing SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect based distance restraints. The backbone (N, Calpha, C=O) RMSD from the average structure of an ensemble of 17 out of 20 calculated structures was 0.41 +/- 0.15 Angstrom for LAP-20 in SDS and 0.41 +/- 0.12 A for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the presence of SDS, the distance geometry and simulated annealing calculations show that LAP-20 adopts a well defined class A amphipathic helix with distinct hydrophobic and hydrophilic faces. A similar structure was obtained for LAP-20 in the presence of DPC, suggesting that both detergents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are discussed relative to models for apolipoprotein A-I activation of LCAT. PMID- 8621697 TI - Activation of epidermal growth factor receptor gene transcription by phorbol 12 myristate 13-acetate is mediated by activator protein 2. AB - The response of the epidermal growth factor (EGF) receptor gene to phorbol 12 myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run off experiments showed that PMA increased EGF receptor gene transcription. Cell free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene. PMID- 8621699 TI - Control of beta1 integrin function. Localization of stimulatory epitopes. AB - The beta1 integrins can be expressed on the surface of cells in a latent form, which is activated by a variety of stimuli. As an approach to examining the transition to an active receptor, a panel of stimulatory antibodies to beta1 were produced and characterized. These antibodies induced adherence of the T-leukemic cell line Jurkat to collagen and fibronectin. Competitive antibody binding assays indicated the existence of at least three distinct epitope clusters A (B3B11, JB1B, 21C8), B (B44, 13B9), and C(N29) defined by the indicated antibodies. Two antibodies to the A site, JB1B and B3B11, were shown to localize to positions 671 703 and 657-670, respectively, of the beta1. This region is located in an area encompassing a predicted disulfide bond between linearly distant cysteines in beta1 (Cys415-Cys671). The homologous region of the beta3 integrin (490 690 and 602 690) has been shown to be one of the sites recognized by stimulatory antibodies to ligand-induced binding sites. The present results indicate the existence of multiple stimulatory regions and suggest considerable homology between the locations of beta1 and beta3 regulatory sites. PMID- 8621700 TI - Active site-directed inactivation of Escherichia coli glucosamine-6-phosphate synthase. Determination of the fructose 6-phosphate binding constant using a carbohydrate-based inactivator. AB - Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine 6 phosphate from fructose 6-phosphate using glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. N-Iodoacetylglucosamine 6-phosphate is an active site-directed irreversible inactivator of GlmS from Escherichia coli (kinact/KI = 17 (+/-3) m-1 s-1). Both fructose 6-phosphate and glutamine protect the enzyme from inactivation, indicating that this reagent is directed at both the sugar binding site and the glutamine binding site. Protection studies with fructose 6-phosphate demonstrate that the value of the dissociation constant for fructose 6-phosphate is 3.3 (+/-0.5) x 10(-7) m, approximately 3 orders of magnitude less than the Kia value for this substrate determined from initial velocity experiments (Badet, B., Vermoote, P., and Le Goffic, F. (1988) Biochemistry 27, 2282-2287). PMID- 8621701 TI - Structure of a G-protein-coupling domain of a muscarinic receptor predicted by random saturation mutagenesis. AB - The third intracellular loop (i3) plays a critical role in the coupling of many receptors to G-proteins. In muscarinic receptor subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are sufficient to direct appropriate G-protein coupling. The relative functional contributions of all amino acids within Ni3 was evaluated by constructing libraries of m5 muscarinic receptors containing random mutations in Ni3 and screening them using high throughput assays based on ligand dependent transformation of NIH 3T3 cells. In receptors that retained a wild type phenotype, the pattern of functionally tolerated substitutions is consistent with the presence of three turns of an alpha helix extending from the transmembrane domain. All of the amino acid positions that tolerate radical substitutions face away from a conserved hydrophobic face that ends with an arginine, and helix disrupting proline substitutions were not observed. All of the mutant receptors with significantly compromised phenotypes had amino acid substitutions in residues predicted to form the hydrophobic face. Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141-3146) are consistent with the presence of a single helical turn extending from the transmembrane domain, with an alanine that defines G protein affinity. Functionally critical residues of Ni3 and Ci3 are predicted to be in close proximity where they form the G-protein-coupling domain. PMID- 8621702 TI - Regulation of activating transcription factor-1 and the cAMP response element binding protein by Ca2+/calmodulin-dependent protein kinases type I, II, and IV. AB - The ability of activating transcription factor-1 (ATF1) or the cAMP response element-binding protein (CREB) to enhance transcription can be stimulated by increases in intracellular Ca2+ concentrations. To identify protein kinases which may mediate the ability of Ca2+ to activate these transcription factors, we compared the ability of constitutively active forms of several Ca2+/calmodulin dependent protein kinases (CaM kinases) to activate ATF1 or CREB. We find that constitutively active CaM kinase I and IV can activate both ATF1 and CREB. In addition, expression vectors for full-length CaM kinase I and IV were able to augment the ability of Ca2+ influx to activate ATF1 or CREB consistent with a role for these kinases in mediating transcriptional responses to Ca2+ signaling. In contrast, CaM kinase II was unable to activate either ATF1 or CREB. These findings provide a potential mechanism that may permit variation in the ability of ATF1 and CREB to respond to changes in intracellular Ca2+ concentrations depending on differences in the relative concentrations of specific CaM kinases. PMID- 8621703 TI - Parathyroid hormone-related protein antagonizes the action of parathyroid hormone on adult cardiomyocytes. AB - Ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH). A structurally related peptide hormone, parathyroid hormone related peptide (PTH-rP), is expressed in the heart. In the present study, it was investigated whether PTH-rP can mimic or modify effects of PTH on cardiomyocytes. The investigated effect was induction of creatine kinase (CK) activity, which is associated with cardiac hypertrophy. PTH and PTH-rP have a similar secondary structure within the active domain 28 34, with exception of amino acid 29. At this position the hydrophilic glutamine in the PTH molecule corresponds to hydrophobic alanine in the PTH-rP molecule. Synthetic PTH or PTH-rP peptides covering domain 28 34 and recombinant full-length PTH(1 84) were used. PTH(28 48) (100 nm) induced CK activity within 24 h (123 +/- 3%; means +/- S.D., n = 4). PTH rP(7-34) (1 nm to 1 microm) failed to induce CK activity in cardiomyocytes. Given simultaneously, PTH-rP (1 mum) reduced the stimulation of CK activity by PTH(1 84), PTH(1-34), and PTH(28-48) by 94 +/- 9, 79 +/- 8, and 69 +/- 14%, respectively (means +/- S.D., n = 4). In contrast, PTH-rP(7-34) was sufficient to stimulate proliferation of chicken chondrocytes. Thus, PTH-rP exerts different effects on cardiomyocytes and classical target cells for PTH. A synthetic hybrid peptide was synthesized, [Ala29]PTH(28-48), in which alanine replaced glutamine at position 29, as in the PTH-rP molecule. In contrast to PTH(28-48), this mutated peptide [Ala29]PTH(28-48) had no intrinsic activity but antagonized the effect of PTH(1-84) and PTH(28-48) on cardiomyocytes. The results demonstrate that on cardiomyocytes the effect of PTH can be antagonized by PTH-rP. This antagonism seems due to a hydrophobic replacement at position 29. PMID- 8621705 TI - Lateral organization of pyrene-labeled lipids in bilayers as determined from the deviation from equilibrium between pyrene monomers and excimers. AB - In lipid bilayers, pyrene and pyrene-labeled lipids form excimers in a concentration-dependent manner. The aromatic amine N, N-diethylaniline (DEA), which has a high membrane-to-medium partition coefficient, quenches the monomers only, and therefore it is expected that under conditions in which the monomers are in equilibrium with the excimers due to the mass law, the Stern-Volmer coefficient (Ksv) for monomers (M), defined as KM, should be identical to that of the excimer (E), defined as KE, and KE/KM = 1. 0. This is indeed the case for pyrene and pyrene valerate in egg phosphatidylcholine small unilamellar vesicles. However, for pyrene decanoate and pyrene dodecanoate in these vesicles, and for N [12-(1-pyrenyl)dodecanoyl]sphingosylphosphocholine in a matrix of either N stearoyl sphingosylphosphocholine or 1-palmitoyl-2-oleoyl phosphatidylcholine, KE < KM. This can be explained either by the existence of (a) two subpopulations of excimers, one in fast equilibrium with the monomers and the other, related to ground-state protoaggregates of pyrene lipids; (b) two monomer subpopulations where part of M cannot be quenched by DEA; or (c) two monomer subpopulations, both quenched by DEA, but only one of which produces excimers. The good agreement between the photophysical processes determined by steady state and time-resolved measurements supports the third explanation for the bilayers containing pyrene phospholipids. It also suggests that the main factors determining the immiscibility of pyrene lipids in phospholipid bilayers are the temperature, the difference in the gel-to-liquid-crystalline phase transition temperature (deltaTm) between the matrix and the pyrene lipid, and the structural differences between the matrix lipid and the pyrene-labeled lipid. These results indicate that the KE/KM ratio can serve as a very sensitive tool to quantify isothermal microscopic immiscibility in membranes. This novel approach has the following advantages: applicability to fluid phase immiscibility, requirement of a relatively low mol fraction of pyrene lipids, and conceivably, applicability to biological membranes. PMID- 8621704 TI - Exchange of aspartate and alanine. Mechanism for development of a proton-motive force in bacteria. AB - We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0. 4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (Kt = 0.4 mm) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37 degrees C prior to reconstitution was prevented by the presence of either aspartate (KD(aspartate) = 0.3 mm) or alanine (KD(alanine) > or = 10 mm), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3. PMID- 8621706 TI - Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function. AB - Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx (SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region. PMID- 8621707 TI - Characterization of the radical trapping activity of a novel series of cyclic nitrone spin traps. AB - alpha-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. Specifically, their ability to inhibit iron-induced lipid peroxidation in liposomes was assessed, as well as superoxide anion (O2(-.)) and hydroxyl radical ((.)OH) trapping activity as determined biochemically and using electron spin resonance (ESR) spectroscopy. All cyclic nitrones tested were much more potent as inhibitors of lipid peroxidation than was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the analogs of MDL 101,002 revealed a direct correlation of activity with lipophilicity. However, lipophilicity does not solely account for the difference between MDL 101,002 and PBN, inasmuch as the calculated octanol/water partition coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicated the cyclic nitrones are inherently more effective radical traps than PBN in a membrane system. The most active compound was a dichloro analog in the seven membered ring series (MDL 104,342), which had an IC50 of 26 mum, which was 550 fold better than that of PBN. The cyclic nitrones were shown to trap (.)OH with MDL 101,002 being 20 25 times more active than PBN as assessed using 2 deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of (.)OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the (.)OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O2(-.) radical to yield a six-line spectrum with coupling constants very distinct from that of the (.)OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of (.)OH and O2(-.) suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury. PMID- 8621708 TI - A heme-containing ascorbate oxidase from Pleurotus ostreatus. AB - A novel type of ascorbate oxidase was purified 420-fold from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield of 13%. The molecular mass of the native enzyme determined by high performance gel permeation chromatography was 94 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme consists of two subunits with a molecular mass of 46 kDa. The N-terminal amino acid sequence of the enzyme was Asp-Val-Lys-Thr-Leu-Gln-Glu-His-Leu-Gln-Leu-Ala-Leu-Met-Val-. The enzyme was optimally active at pH 5.2, monitored at 37 degrees C. The enzyme had affinity toward L-ascorbic acid, D-ascorbic acid, L-erythroascorbic acid, and D erythroascorbic acid. Under optimal conditions, the Km value of the enzyme toward L-ascorbic acid was 0.48 mm. The absorption spectra of the native enzyme exhibited a Soret maximum at 418 nm in its oxidized form and at 426 nm in its reduced form, and alpha and beta bands at 558 and 527 nm only in its reduced form, respectively. On the basis of spectral changes after treatment with cyanide and carbon monoxide, the enzyme is a hemoprotein, quite similar to b-type cytochrome, and contains 2 mol of heme per molecule. The reaction catalyzed by the enzyme was L-ascorbic acid + O2 --> dehydro-L-ascorbic acid + H2O2. PMID- 8621709 TI - Identification, purification, and characterization of a PA700-dependent activator of the proteasome. AB - The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000 Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity. PMID- 8621710 TI - Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by Galphai/o subunits. AB - A membrane-associated form of Raf-1 in v-Ras transformed NIH 3T3 cells can be inactivated by protein phosphatases regulated by GTP. Herein, a distinct protein tyrosine phosphatase (PTPase) in membrane preparations from v-Ras transformed NIH 3T3 cells was found to be activated by guanyl-5'-yl imidodiphosphate (GMPPNP) and was identified as an effector for pertussis toxin (PTx)-sensitive G-protein alpha subunits. PTPase activation was blocked by prior treatment of cells with PTx. PTPase activation by GTP, but not GMPPNP, was transient. A GMPPNP-stimulated PTPase (PTPase-G) co-purified with Galphai/o subunits during Superose 6 and Mono Q chromatography. PTPase-G activity in Superose 6 fractions from GDP-treated membranes was reconstituted by activated Galphai/o, but not G beta gamma, subunits. PTPase-G may contribute to GMPPNP-stimulated inactivation of Raf-1 in v Ras cell membranes because Raf-1 inactivation was PTx-sensitive and PTPase-G inactivated exogenous Raf-1. PMID- 8621711 TI - Heparinase I from Flavobacterium heparinum. Mapping and characterization of the heparin binding domain. AB - In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis. PMID- 8621712 TI - The OST4 gene of Saccharomyces cerevisiae encodes an unusually small protein required for normal levels of oligosaccharyltransferase activity. AB - Sodium vanadate is an effective drug for the enrichment of yeast mutants defective in glycosylation reactions that are carried out in the Golgi complex. We have isolated vanadate-resistant, hygromycin B-sensitive mutants that act at very early steps of N-linked glycosylation, occurring in the endoplasmic reticulum. Here we describe the phenotypic characterization of ost4, a vanadate resistant mutant that is defective in oligosaccharyltransferase (OTase) activity both in vivo and in vitro. The OST4 open reading frame is unusual in that it predicts a protein of only 36 amino acids. We demonstrate that the OST4 gene product is, in fact, an unusually small protein of approximately 3.6 kDa, predicted to lie almost entirely in the hydrophobic environment of the membrane. Strains carrying a disruption of the OST4 gene are viable but grow poorly at 25 degrees C. The null mutant is inviable at 37 degrees C, demonstrating that the OST4 gene product is essential for growth at high temperatures. Deletion of the OST4 gene greatly diminishes OTase activity but does not abolish it. These results suggest that the OST4 gene encodes a subunit or accessory component of OTase that is essential at high temperature. PMID- 8621713 TI - Ras involvement in signal transduction by the serotonin 5-HT2B receptor. AB - The family of serotonin 5-HT2 receptors stimulates the phospholipase C second messenger pathway via the alpha subunit of the Gq GTP-binding protein. Here, we show that agonist stimulation of the 5-HT2B receptor subtype stably expressed in the mouse fibroblast LMTK- cell line causes a rapid and transient activation of the proto-oncogene product p21ras as measured by an increase in GTP-bound Ras in response to serotonin. Furthermore, 5-HT2B receptor stimulation activates p42mapk/p44mapk (ERK2/ERK1) mitogen-activated protein kinases as assayed by phosphorylation of myelin basic protein. Antibodies against p21ras, Galphaq, beta, or -gamma2 subunits of the GTP-binding protein inhibit MAP kinase-dependent phosphorylation. The MAP kinase activation is correlated with a stimulation of cell division by serotonin. In addition to this mitogenic action, transforming activity of serotonin is mediated by the 5-HT2B receptor since its expression in LMTK- cells is absolutely required for foci formation and for these foci to form tumors in nude mice. Finally, we detected expression of the 5-HT2B receptor in spontaneous human and Mastomys natalensis carcinoid tumors and, similar to the 5 HT2B receptor transfected cells, the Mastomys tumor cells are also responsive to serotonin with similar coupling to p21ras activation. PMID- 8621714 TI - A molecular switch of chemokine receptor selectivity. Chemical modification of the interleukin-8 Leu25 --> Cys mutant. AB - Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu25 to cysteine. The protein folds correctly with two disulfide bonds and a free thiol group at Cys25. This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor binding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leucine. However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2 N-methyl, N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminoethyl)acetamido (NBD) group lowers potency in neutrophil chemotaxis and affinity in IL-8 receptor binding assays by 2 orders of magnitude. This Leu25 --> Cys-NBD mutant introduces monocyte chemoattractant activity and the ability to displace 125I-labeled macrophage inflammatory protein-1 alpha from the recombinant CC CKR-1 receptor. Additionally, we show a specific interaction between the fluorescent mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specificity between CXC and CC chemokines. Circular dichroism spectra of the IL-8 mutants having CC chemokine-like activity show a consistent drop in alpha-helical content compared with the spectra for wild-type IL-8. This suggests that distortion of the C terminal helix may play a role in chemokine receptor-ligand selectivity. PMID- 8621715 TI - Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk 1/KDR. AB - Here we show that the Escherichia coli expressed monomers of placenta growth factor (PLGF)129 and vascular endothelial growth factor (VEGF)165 can be re folded in vitro to form PLGF/VEGF heterodimers. The purified recombinant PLGF/VEGF heterodimers and VEGF homodimers have potent mitogenic and chemotactic effects on endothelial cells. However, PLGF/VEGF heterodimers display 20-50-fold less mitogenic activity than VEGF165 homodimers. In contrast, PLGF129 homodimers have little or no effect in these in vitro assays. We also demonstrate the presence of natural PLGF/VEGF heterodimers in the conditioned media of various human tumor cell lines. While PLGF/VEGF heterodimers bind with high affinity to a soluble Flk-1/KDR receptor, PLGF129 homodimers fail to bind to this receptor. Cross-linking of 125I-ligands to human umbilical vein endothelial cells reveals that PLGF/VEGF heterodimers and VEGF165 homodimers, but not PLGF129 homodimers, form complexes with membrane receptors. VEGF165 homodimers and PLGF/VEGF heterodimers stimulate tyrosine phosphorylation of a 220-kDa protein, the expected size for the KDR receptor in human umbilical vein endothelial cells, whereas PLGF129 homodimers are unable to induce tyrosine phosphorylation of this protein. These data indicate that PLGF may modulate VEGF-induced angiogenesis by the formation of PLGF/VEGF heterodimers in cells producing both factors. PMID- 8621716 TI - Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity. AB - The aim of the present study was to demonstrate that the modulation of P glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp. PMID- 8621717 TI - Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis. AB - We have recently described an ATP-driven, valinomycin-dependent 86Rb+uptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (1994) J. Biol. Chem. 269, 26058 26065). P-glycoprotein mediated the ATP-dependent uptake of 86Rb+-ionophore complex into the proteoliposomes, where the radioactive cation was accumulated, thus, circumventing the obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies. Taking advantage of this assay and of the high levels of P-glycoprotein expression in multidrug-resistant Chinese hamster ovary cells, we measured simultaneously both the ATPase and transport activities of P glycoprotein under identical conditions and observed 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed. The amount of 86Rb+ ions transported within 1 min via the ATP- and valinomycin-dependent P-glycoprotein was equivalent to an intravesicular cation concentration of 8 mM. Thus, this stoichiometry and transport capacity of P-glycoprotein resemble various ion-translocating ATPases, that handle millimolar substrate concentrations. This constitutes the first demonstration of comparable rates of P-glycoprotein-catalyzed substrate transport and ATP hydrolysis. PMID- 8621718 TI - Characterization of the Xenopus rhodopsin gene. AB - The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5 upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5 upstream region using a luciferase reporter. DNA sequences encompassing -5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes. PMID- 8621720 TI - Photoaffinity labeling of human recombinant sulfotransferases with 2 azidoadenosine 3',5'-[5'-32P]bisphosphate. AB - Photoaffinity labeling with 2-azidoadenosine 3', 5'-[5'-32P]bisphosphate was used to identify and characterize adenosine 3',5'-bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co incubation with unlabeled adenosine 3',5'-bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 microM. To verify that the 31 35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3',5'-[5'-32P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3',5'-[5' 32P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3',5'-bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites. PMID- 8621719 TI - p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. AB - We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3 kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors. PMID- 8621721 TI - Thrombin primes responsiveness of selective chemoattractant receptors at a site distal to G protein activation. AB - To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL 8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2. PMID- 8621722 TI - Beta subunit heterogeneity in N-type Ca2+ channels. AB - The beta subunit of the voltage-dependent Ca2+ channel is a cytoplasmic protein that interacts directly with an alpha1 subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the alpha1B subunit of the N-type Ca2+ channel associates with several different beta subunits. Polyclonal antibodies specific for three different beta subunits immunoprecipitated 125I-omega-conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca2+ channels with an alpha1B subunit specific monoclonal antibody showed the association of beta1b, beta3, and beta4 subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca2+ channel confirmed the presence of the beta3 and beta4 subunits. Each of the beta subunits bound to the alpha1B subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the beta subunit composition of the N-type Ca2+ channels, which may be responsible for some of the diverse kinetic properties recorded from neurons. PMID- 8621723 TI - Regulation of ribosomal DNA transcription during contraction-induced hypertrophy of neonatal cardiomyocytes. AB - Cardiac hypertrophy requires protein accumulation. This results largely from an increased capacity for protein synthesis, which in turn is the result of an elevated rate of ribosome biogenesis. The process of ribosome formation is regulated at the level of transcription of the ribosomal RNA genes. In this study, we examined the amounts and activities of various components of the ribosomal DNA transcription apparatus in contraction-arrested neonatal cardiomyocytes and in spontaneously contracting cardiomyocytes that hypertrophy. Nuclear run-on assays demonstrated that spontaneously contracting cardiomyocytes supported a 2-fold increased rate of ribosomal DNA transcription. However, enzymatic assay of total solubilized RNA polymerase I and Western blots demonstrated that contraction-induced increases in ribosomal RNA synthesis were not accompanied by increased activity or amounts of RNA polymerase I. In contrast, accelerated ribosome biogenesis was accompanied by an increased amount of the ribosomal DNA transcription factor, UBF. Immunoprecipitation of [32P]orthophosphate-labeled UBF from hypertrophying, neonatal cardiomyocytes indicated that the accumulated UBF protein was phosphorylated and, thus, in the active form. UBF mRNA levels began to increase within 3-6 h of the initiation of contraction and preceded the elevation in rDNA transcription. Nuclear run-on assays demonstrated increased rates of transcription of the UBF gene. Transfection of chimeric reporter constructs containing deletions of the 5' flanking region of the UBF gene revealed the presence of contraction response elements between -1189 and -665 relative to the putative start of transcription. These results are consistent with the hypothesis that UBF is an important factor in the regulation of rDNA transcription during contraction-mediated neonatal cardiomyocyte hypertrophy. PMID- 8621724 TI - Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. AB - We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET 1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10(-8) approximately 10(-7) M) maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist. BQ123 also suppressed stretch induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an approximately 3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy. PMID- 8621725 TI - Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. AB - Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response. PMID- 8621726 TI - Transcriptional regulation of alpha1,3-galactosyltransferase in embryonal carcinoma cells by retinoic acid. Masking of Lewis X antigens by alpha galactosylation. AB - Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media. The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1 3)GlcNAcbeta1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha galactosylation. Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens. PMID- 8621727 TI - Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways. AB - Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50 100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin. PMID- 8621728 TI - Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin. AB - P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1 3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1 4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E selectin. PMID- 8621729 TI - Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor stimulated Hela cells. AB - Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling. PMID- 8621730 TI - An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta. AB - Previous studies have shown that transforming growth factor-beta (TGF-be ta) and tumor necrosis factor-alpha (TNF-alpha) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human alpha2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/chloramphenicol acetyltransferase (CAT) reporter gene constructs. Transient cell transfection assays using human dermal fibroblasts and stable transfection experiments using NIH 3T3 fibroblasts identified the region located between residues -265 and -241, as critical for TGF-beta response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF-beta on COL1A2 promoter activity and allows antagonistic activity of TNF alpha on the TGF-beta effect. Gel mobility shift assays indicate that nuclear factor binding to this 25-base pair region of COL1A2 promoter is competed by AP 1, but not NF-1 or NF-kappaB, oligonucleotides. Transient cell transfection experiments with plasmid constructs in which the potential AP-1-binding site located within this short region of promoter was modified by site-directed mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF-beta response and does not require the presence of the three Sp-1-binding sites located further upstream, between nucleotides -273 and -304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF- beta response, further implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation of type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF-beta effects on gene transcription. PMID- 8621731 TI - Autocrine regulation of membrane transforming growth factor-alpha cleavage. AB - Transforming growth factor alpha (TGF-alpha) is biosynthesized as a membrane bound precursor protein, pro-TGF-alpha, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-alpha production in human tumor derived cell lines that endogenously express pro-TGF-alpha and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-alpha that accumulated on the cell surface. Membrane-bound pro-TGF alpha interacted with the EGF receptor, and complexes of receptor and pro-TGF alpha contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-alpha had a dual effect on TGF-alpha production: an increase in pro-TGF-alpha mRNA levels and an increase in pro-TGF-alpha cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF alpha could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-alpha and that TGF-alpha is able to induce its own endoproteolytic cleavage by activating the EGF receptor. PMID- 8621732 TI - Consequences for the organization of reaction center-light harvesting antenna 1 (LH1) core complexes of Rhodobacter sphaeroides arising from deletion of amino acid residues from the C terminus of the LH1 alpha polypeptide. AB - The light harvesting antenna 1 (LH1) complex of Rhodobacter sphaeroides is intimately associated with the reaction center (RC) as part of the reaction center RC-LH1 core complex. The pufA gene has been modified such that between 5 and 16 amino acid residues were progressively deleted from the C terminus of the LH1 alpha polypeptide. The two largest deletions produced strains which were deficient in LH1. The remaining four deletion mutants exhibited significant reductions in the average level of LH1 per reaction center. Analysis of detergent solubilized cores on sucrose gradients showed that the mutant strains had a sizeable population of antenna-deficient reaction centers in addition to core complexes with a reduced ratio of LH1:RC. The decrease in the ratio of LH1:RC in core complexes of the mutant strains was accompanied by a progressive blue shift of the absorbance maximum of LH1, which we attribute to the reduced aggregation state of LH1 in the smaller cores. The PufX polypeptide was not required for photosynthetic growth in mutants with reduced core sizes. We conclude that the level of LH1 in the bacterial membrane, and the aggregation state of LH1 in core complexes, are both influenced by the C terminus of the alpha polypeptide, and we discuss possible models for the organization of the core complex in Rb. sphaeroides. PMID- 8621733 TI - Characterization of the hypoxia-inducible protein binding site within the pyrimidine-rich tract in the 3'-untranslated region of the tyrosine hydroxylase mRNA. AB - Reduced tension of O2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552 1578 in the 3 -untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27 base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species. PMID- 8621734 TI - Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product. AB - Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance. PMID- 8621735 TI - The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies. AB - We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity. PMID- 8621737 TI - Neuron-specific gene expression of synapsin I. Major role of a negative regulatory mechanism. AB - The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif. PMID- 8621736 TI - Cloning of a retinoic acid-sensitive mRNA expressed in cartilage and during chondrogenesis. AB - Retinoic acid (RA) is known to play a role in various aspects of skeletal development in vivo, including morphogenesis, growth plate maturation, and apoptosis. In cell culture, RA treatment of chondrocytes suppresses the differentiated phenotype characterized by production of type II collagen and aggrecan. In an effort to discover molecules involved in regulation of the chondrocyte phenotype or related to developmental processes such as chondrogenesis, mRNAs from bovine chondrocytes cultured with and without RA were amplified by reverse transcription-polymerase chain reaction (PCR) and compared by differential display. PCR products whose expression was inhibited by RA treatment were cloned. One cDNA encodes a molecule we call cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its properties are described here. The full-length bovine CD-RAP mRNA was cloned after amplification by the rapid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specific primers. The bovine CD-RAP mRNA contains an open reading frame of 130 amino acids. CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and cartilage. The inhibition of CD-RAP mRNA expression by RA in vitro was time- and dose-dependent and was tested over concentrations from 10(-8) to 10(-6) M. Southern blot analysis of genomic DNA indicated that CD-RAP was encoded by a single copy gene and that no other genes were closely related. What appears to be the human homologue of CD-RAP was recently isolated and cloned from a melanoma cell line and shown to function as a growth inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner, R., and Bogdahn, U. (1994) Cancer Res. 54, 5695-5701). Neither CD-RAP nor this protein showed any homology to known proteins. We speculate that, in vivo, CD-RAP functions during cartilage development and maintenance. PMID- 8621738 TI - Identification of a 14-kDa subunit associated with the catalytic sector of clathrin-coated vesicle H+-ATPase. AB - The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti peptide antibody, directed against deduced protein sequence, was affinity purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue. PMID- 8621739 TI - Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes. AB - A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation. PMID- 8621740 TI - Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. AB - Cerebral infarction initiates a cascade of molecular events, leading to proteolytic cell death. Matrix-degrading metalloproteinases (MMPs) are neutral proteases involved in extracellular matrix damage. Type IV collagenase is an MMP that increases cerebral capillary permeability after intracerebral injection and may be important along with plasminogen activators (PA) in secondary brain edema in stroke. Therefore, we measured MMPs and PAs in spontaneously hypertensive (SHR) or Wistar-Kyoto (WKY) rats with permanent middle cerebral artery occlusion (MCAO). Brain tissue was assayed for MMPs and PAs at 1, 3, 12, and 24 h and 5 days after occlusion, using substrate gel polyacrylamide electrophoresis (zymography). SHR showed an increase in 92-kDa type IV collagenase (gelatinase B) in the infarcted hemisphere compared with the opposite side at 12 and 24 h (p < 0.05). Gelatinase A remained the same in both infarcted and normal tissue until 5 days after injury, when it increased significantly (p < 0.05). Urokinase-type PA was increased significantly at 12 and 24 h and 5 days, while tissue-type PA was decreased significantly at 1, 12, and 24 h in the ischemic compared with the nonischemic hemisphere. Gelatinase B was markedly increased in SHR at 12 and 24 h compared with WKY (p < 0.05). Secondary vasogenic edema is maximal 1-2 days after a stroke, which is the time that gelatinase B was elevated. The time of appearance of gelatinase B suggests a role in secondary tissue damage and vasogenic edema, while gelatinase A may be involved in tissue repair. PMID- 8621741 TI - The use of in vivo fluorescence image sequences to indicate the occurrence and propagation of transient focal depolarizations in cerebral ischemia. AB - A method for the detection and tracking of propagated fluorescence transients as indicators of depolarizations in focal cerebral ischemia is described, together with initial results indicating the potential of the method. The cortex of the right cerebral hemisphere was exposed for nonrecovery experiments in five cats anesthetized with chloralose and subjected to permanent middle cerebral artery (MCA) occlusion. Fluorescence with 370-nm excitation (attributed to the degree of reduction of the NAD/H couple) was imaged with an intensified charge-coupled device camera and digitized. Sequences of images representing changes in gray level from a baseline image were examined, together with the time courses of mean gray levels in specified regions of interest. Spontaneous increases in fluorescence occurred, starting most commonly at the edge of areas of core ischemia; they propagated usually throughout the periinfarct zone and resolved to varying degrees and at varying rates, depending on proximity of the locus to the MCA input. When a fluorescence transient reached the anterior cerebral artery territory, its initial polarity reversed from an increase to a decrease in fluorescence. An initial increase in fluorescence in response to the arrival of a transient may characterize cortex that will become infarcted, if pathophysiological changes in the periinfarct zone are allowed to evolve naturally. PMID- 8621742 TI - Inhibition of tumor necrosis factor alpha (TNFalpha) activity in rat brain is associated with cerebroprotection after closed head injury. AB - We recently demonstrated that closed head injury (CHI) in the rat triggers the production of tumor necrosis factor alpha (TNFalpha) in the contused hemisphere. Other investigations have shown that this cytokine plays a role in the inflammatory response following trauma. The present study was designed to determine whether inhibition of TNFalpha production or activity affects the development of cerebral edema as well as neurological dysfunction and hippocampal cell loss after CHI. To this end, we used two pharmacological agents, each acting via a different mechanism: pentoxifylline (PTX), which attenuates the production of TNFalpha, and tumor necrosis factor binding protein (TBP), a physiological inhibitor of TNFalpha activity. Both agents significantly lessened peak edema formation at 24 h and facilitated the recovery of motor function for < or = 4 days postinjury. In addition, TBP attenuated disruption of the blood-brain barrier and protected hippocampal cells. PTX significantly lowered the brain TNFalpha level (by approximately 80%), and TBP completely abolished the activity of recombinant human TNF when they were added at the same time in the in vitro bioassay. We suggest, therefore, that a decrease in TNFalpha level or the inhibition of its activity is accompanied by reduced brain damage. PMID- 8621743 TI - The metabolic topography of normal aging. AB - Normal aging is associated with the degeneration of specific neural systems. We used [18F] fluorodeoxyglucose (FDG)/positron emission tomography (PET) and a statistical model of regional covariation to explore the metabolic topography of this process. We calculated global and regional metabolic rates for glucose (GMR and rCMRglc) in two groups of normal subjects studied independently on different tomographs: Group 1--130 normal subjects (62 men and 68 women; range 21-90 years); Group 2--20 normal subjects (10 men and 10 women; range 24-78 years). In each of the two groups, the Scaled Subprofile Model (SSM) was applied to rCMRglc data to identify specific age-related profiles. The validity of these profiles as aging markers was assessed by correlating the associated subject scores with chronological age in both normal populations. SSM analysis disclosed two significant topographic profiles associated with aging. The first topographic profile, extracted in an analysis of group 1 normals, was characterized by relative frontal hypometabolism associated with covariate metabolic increases in the parietooccipital association areas, basal ganglia, mid-brain, and cerebellum. Subject scores for this profile correlated significantly with age in both normal groups (R2 = 0.48 and 0.33, p < 0.0001 for groups 1 and 2, respectively). Because of clinical similarities between normal motoric aging and parkinsonism, we explored the possibility of shared elements in the metabolic topography of both processes. We performed a combined group SSM analysis of the 20 group 2 normals and 22 age-matched Parkinson's disease patients, and identified another aging related topographic profile. This profile was characterized by relative basal ganglia hypermetabolism associated with covariate decreases in frontal premotor cortex. Subject scores for this profile also correlated significantly with age in both normal groups (group 1: R2 = 0.30, p < 0.00001; group 2: R2 = 0.59, p < 0.01). Healthy aging is associated with reproducible topographic covariation profiles associated with specific neural systems. FDG/PET may provide a useful metabolic marker of the normal aging process. PMID- 8621744 TI - Combined study of cerebral glucose metabolism and [11C]methionine accumulation in probable Alzheimer's disease using positron emission tomography. AB - There is a characteristic decrease in glucose metabolism in associative frontal and temporo-parietal cortices of patients suffering from Alzheimer's disease (AD). The decrease in metabolism might result from local neuronal loss or from a decrease of synaptic activity. We measured in vivo [11C]methionine accumulation into proteins with positron emission tomography (PET) to assess cortical tissue loss in AD. Both global regional activity and compartmental analysis were used to express [11C]methionine accumulation into brain tissue. Glucose metabolism was measures with [18F]fluorodeoxyglucose and autoradiographic method. Combined studies were performed in 10 patients with probable AD, compared to age-matched healthy volunteers. There was a significant 45% decrease of temporo-parietal glucose metabolism in patients with AD, and frontal metabolism was lowered in most patients. Temporo-parietal metabolism correlated to dementia severity. [11C]methionine incorporation into temporo-parietal and frontal cortices was not significantly decreased in AD. There was no correlation with clinical symptoms. Data suggest that regional tissue loss, assessed by the decrease of [11C]methionine accumulation, is not sufficient to explain cortical glucose hypometabolism, which reflects, rather, reduced synaptic connectivity. PMID- 8621745 TI - Stereotaxic summation analysis of human cerebral benzodiazepine binding maps. AB - Summation analysis strategies are recognized throughout diverse scientific fields as powerful means of differentially enhancing experimental signals over random fluctuations (noise). Such techniques, applied to emission tomographic cerebral blood flow scans, reveal subtle alterations in neuronal activity during specific behavioral states. In the present work, we extend the principles of intersubject image summation analysis to the evaluation of emission tomographic ligand-binding studies. A general methodology is presented that may be applied to a wide variety of binding site determinations. The procedure consists of anatomic standardization of individual brains to a common stereotaxic orientation, followed by statistical analyses of group versus group or individual versus group differences. We develop and evaluate performance of our technique with the use of positron emission tomographic [11C]flumazenil scans from normal volunteers, depicting the regional cerebral distribution of benzodiazepine binding sites. PMID- 8621746 TI - Positron emission tomographic imaging of serotonin activation effects on prefrontal cortex in healthy volunteers. AB - Serotonergic system abnormalities have been implicated in major depression, suicide, violence, alcoholism, and other psychopathologies. The prolactin response to fenfluramine has been widely used as a neuroendocrine probe to study brain serotonin responsivity. We have extended this methodology by using the positron emission tomography (PET) 18F-fluorodeoxyglucose (18FDG) method to examine the fenfluramine-induced changes in regional cerebral glucose metabolism (rCMRglu), an indicator of changes in regional neuronal activity. We report results on 16 healthy controls, each of whom underwent two PET studies. One group of six subjects had a placebo on day 1 and a single 60 mg oral dose of fenfluramine on day 2. The second group, of 10 subjects, was tested on two consecutive occasions without drug or placebo. Data were analyzed for significant rCMRglu changes on day 2 vs day 1 using the statistical parametric mapping method (p < 0.01). Subjects who did not receive drugs showed no statistically significant areas of rCMRglu increase or decrease on day 2 versus day 1. In contrast, the group that received fenfluramine showed significant fenfluramine induced responses. Areas of rCMRglu increases involved mainly the left prefrontal and left temperoparietal cortex. Within the prefrontal cortex, two major areas of rCMRglu increase included, first, an area centered on the anterior cingulate and, second, an area in the lateral prefrontal cortex involving principally the inferior, middle, and superior frontal gyri. Some decreases in rCMRglu were observed, principally in the right hemisphere. This PET-fenfluramine paradigm is a potentially useful method for studying abnormalities of serotonin function in the prefrontal cortex. PMID- 8621748 TI - Influence of glucose supply and demand on determination of brain glucose content with labeled methylglucose. AB - The equilibrium brain/plasma distribution ratio for 3-0-methyl-D-glucose (methylglucose) varies with plasma and tissue glucose contents and can be used to determine local glucose levels in brain. This ratio was previously found to rise as brain glucose concentration fell in response to lowered plasma glucose content. The ratios, however, differed with the same tissue glucose levels in conscious and pentobarbital-sedated rats, suggesting that changes in metabolic demand might alter the quantitative relationship between the methylglucose distribution ratio and brain glucose concentration. To examine this possibility, metabolic rate was varied by focal drug application, and hexose concentrations measured in treated and surrounding tissue. When tissue glucose levels were reduced by raised metabolic demand, methylglucose distribution ratios also fell. When brain glucose levels rose due to reduced consumption, the methylglucose distribution ratio also rose. Thus, in contrast to the inverse relationship between brain/plasma methylglucose ratio and brain glucose concentration when brain glucose content is altered secondarily to changes in plasma glucose level, changes in brain glucose content induced by altered glucose utilization cause the brain glucose level and methylglucose distribution ratio to rise and fall in a direct relationship. Determination of brain glucose content from methylglucose distribution ratios must take into account rates of glucose delivery and consumption. PMID- 8621747 TI - 1H NMR studies of glucose transport in the human brain. AB - The difference between 1H nuclear magnetic resonance (NMR) spectra obtained from the human brain during euglycemia and during hyperglycemia is depicted as well resolved glucose peaks. The time course of these brain glucose changes during a rapid increase in plasma glucose was measured in four healthy subjects, aged 18 22 years, in five studies. Results demonstrated a significant lag in the rise of glucose with respect to plasma glucose. The fit of the integrated symmetric Michaelis-Menten model to the time course of relative glucose signals yielded an estimated plasma glucose concentration for half maximal transport, Kt, of 4.8 +/- 2.4 mM (mean +/- SD), a maximal transport rate, Tmax, of 0.80 +/- 0.45 micromol g 1 min-1, and a cerebral metabolic glucose consumption rate (CMR)glc of 0.32 +/- 0.16 micromol g-1 min-1. Assuming cerebral glucose concentration to be 1.0 micromol/g at euglycemia as measured by 13CMR, the fit of the same model to the time course of brain glucose concentrations resulted in Kt = 3.9 +/- 0.82 mM, Tmax = 1.16 +/- 0.29 micromol g-1 min-1, and CMRglc = 0.35 +/- 0.10 micromol g-1 min-1. In both cases, the resulting time course equaled that predicted from the determination of the steady-state glucose concentration by 13C NMR spectroscopy within the experimental scatter. The agreement between the two methods of determining transport kinetics suggests that glucose is distributed throughout the entire aqueous phase of the human brain, implying substantial intracellular concentration. PMID- 8621750 TI - Hypertonic environment prevents depolarization and improves functional recovery from hypoxia in hippocampal slices. AB - Treatments that postpone hypoxic spreading depression (SD)-like depolarization (also called anoxic depolarization) facilitate recovery of function after transient cerebral hypoxia. Hypertonia reduces cerebral excitability, and we tested whether it also offers protection against SD-like depolarization and hypoxia. Oxygen was withdrawn from hippocampal slices bathed in normal artificial cerebrospinal fluid (ACSF) and, simultaneously, from slices cut from the same hippocampus but bathed in strongly hypertonic ACSF. Extracellular osmolarity (pi(o)) was increased by adding 100 mM mannitol or fructose to ACSF. Slices in normal pi(o) underwent SD-like negative extracellular voltage shift (delta Vo). The hypertonic slices usually showed no SD-like delta Vo but only a small, gradual negative voltage shift. Hypertonia also prevented the precipitate drop of interstitial calcium level ([Ca2+]o). When oxygenation and normal osmolarity were restored, synaptic transmission in the previously hypertonic slices recovered completely, but 3 h after reoxygenation orthodromically transmitted population spikes of the control slices recovered only 25.1% of the initial control amplitude. We conclude that hypertonic treatment during hypoxia improves subsequent recovery of synaptic function. The protection is probably due to the prevention of calcium uptake by blocking the SD-like depolarization, with the prevention of hypoxic cell swelling playing a lesser role. PMID- 8621749 TI - Non-invasive neurochemical analysis of focal excitotoxic lesions in models of neurodegenerative illness using spectroscopic imaging. AB - Water-suppressed chemical shift magnetic resonance imaging was used to detect neurochemical alterations in vivo in neurotoxin-induced rat models of Huntington's and Parkinson's disease. The toxins were: N-methyl-4 phenylpyridinium (MPP+), aminooxyacetic acid (AOAA), 3-nitropropionic acid (3 NP), malonate, and azide. Local or systemic injection of these compounds caused secondary excitotoxic lesions by selective inhibition of mitochondrial respiration that gave rise to elevated lactate concentrations in the striatum. In addition, decreased N-acetylaspartate (NAA) concentrations were noted at the lesion site over time. Measurements of lactate washout kinetics demonstrated that t1/2 followed the order: 3-NP approximately MPP+ >> AOAA approximately malonate, which parallels the expected lifetimes of the neurotoxins based on their mechanisms of action. Further increases in lactate were also caused by intravenous infusion of glucose. At least part of the excitotoxicity is mediated through indirect glutamate pathways because lactate production and lesion size were diminished using unilateral decortectomies (blockade of glutamatergic input) or glutamate antagonists (MK-801). Lesion size and lactate were also diminished by energy repletion with ubiquinone and nicotinamide. Lactate measurements determined by magnetic resonance agreed with biochemical measurements made using freeze clamp techniques. Lesion size as measured with MR, although larger by 30%, agreed well with lesion size determined histologically. These experiments provide evidence for impairment of intracellular energy metabolism leading to indirect excitotoxicity for all the compounds mentioned before and demonstrate the feasibility of small-volume metabolite imaging for in vivo neurochemical analysis. PMID- 8621751 TI - Flow threshold for reduction of cyclic AMP binding in the hippocampus CA1 and other brain regions during stroke development in gerbils. AB - The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions. PMID- 8621752 TI - Neuroprotection after several days of mild, drug-induced hypothermia. AB - Stroke trials are initiated after demonstrated pharmacological protection in animal models. NBQX protects CA1 neurons against global ischemia; however, this glutamate antagonist induces a period of subnormal temperature (e.g., a decrease of only 1.0-1.5 degrees C) lasting several days. In this study, NBQX (3 x 30 mg/kg, i.p.) was administered starting 60 min after reperfusion, and brain temperature had declined significantly below vehicle-treated animals by 2 h after reperfusion. When the postischemic brain temperature of NBQX-treated gerbils was regulated, no neuronal protection was found. Mimicking an NBQX-induced temperature profile for 28 h postischemia yielded histological protection 4 days later comparable to that of NBQX. However, both the NBQX and temperature simulation groups showed decreased protection after 10-day survival. Our data suggest that a protracted period of subnormal temperature during postischemic period can obscure the interpretation of preclinical drug studies. PMID- 8621753 TI - Widespread hemodynamic depression and focal platelet accumulation after fluid percussion brain injury: a double-label autoradiographic study in rats. AB - Cerebrovascular damage leading to subsequent reductions in local cerebral blood flow (lCBF) may represent an important secondary injury mechanism following traumatic brain injury (TBI). We determined whether patterns of 111-indium labeled platelet accumulation were spatially related to alterations in lCBF determined autoradiographically 30 min after TBI. Sprague-Dawley rats (n = 8), anesthetized with halothane and maintained on a 70:30 (vol/vol) mixture of nitrous oxide/oxygen and 0.5% halothane, underwent parasagittal fluid percussion brain injury (1.7-2.2 atm). 111-Indium-tropolone-labeled platelets were injected 30 min prior to TBI while [14C]-iodoantipyrine was infused 30 min after trauma. Sham-operated animals (n = 7) underwent similar surgical procedures but were not injured. In autoradiographic images of the indium-labeled platelets, focal sites of platelet accumulation within the traumatized hemisphere were restricted to the pial surface (five of eight rats), the external capsule underlying the lateral parietal cortex (five of eight rats), and within cerebrospinal fluid (CSF) compartments (six of eight rats). In contrast, mild-to-moderate reductions in lCBF, not restricted to sites of platelet accumulation, were seen throughout the traumatized hemisphere. Flow reductions were most severe in coronal sections underlying the impact site. For example, within the lateral parietal cortex and hippocampus, lCBF was significantly reduced [p <0.01; analysis of variance (ANOVA)] from 1.71 +/- 0.34 (mean +/- SD) and 0.78 +/- 0.12 ml/g/min, respectively, versus 0.72 +/- 0.17 and 0.41 +/- 0.06 ml/g/min within the traumatized hemisphere. Significant flow reductions were also seen in remote cortical and subcortical areas, including the right frontal cortex and striatum. These results indicate that focal platelet accumulation and widespread hemodynamic depression are both early consequences of TBI. Therapeutic strategies directed at these early microvascular consequences of TBI may be neuroprotective by attenuating secondary ischemic processes. PMID- 8621754 TI - Failure of an endothelin antagonist to modify hypoperfusion after transient global ischaemia in the rat. AB - The role of endogenous endothelins in mediating postischaemic hypoperfusion after transient global ischaemia was investigated in halothane-anaesthetised rats. Pretreatment with the broad-spectrum (ET (A) and ET (B)) endothelin antagonist. Bosentan (17 micromol/kg) had minimal effect on postischaemic hypoperfusion, measured by hydrogen clearance, in the caudate nucleus and the parietal cortex in the 3 h after bilateral common carotid artery occlusion with concomitant haemorrhagic hypotension (transient global ischaemia). In a separate series of rats with CBF measured by [14C]iodoantipyrine autoradiography at 90 min after carotid occlusion with concomitant haemorrhagic hypotension, Bosentan treatment failed to significantly alter CBF in any of the 35 brain regions examined. No significant alterations in CBF, measured by hydrogen clearance, were observed after transient bilateral common carotid artery occlusion. [14C]Iodoantipyrine autoradiography at 90 min after occlusion failed to demonstrate any significant increases in CBF after transient bilateral common carotid artery occlusion in any of the 35 brain regions examined in anaesthetised rats. The failure of the broad spectrum endothelin antagonist Bosentan, at concentrations known to inhibit the cerebrovascular effects of exogenous ET-1, provide no support for the view that endothelins have a major role in mediating acute postischaemic hypoperfusion. PMID- 8621755 TI - L-Arginine does not restore dilatation of the basilar artery during diabetes mellitus. AB - The goal of this study was to test the hypothesis that administration of L arginine, a substrate for the synthesis of nitric oxide, restores endothelium dependent dilatation of the basilar artery during diabetes mellitus. We measured the diameter, of the basilar artery in vivo in nondiabetic and diabetic (streptozotocin; 50-60 mg/kg i.p.) rats in response to endothelium-dependent agonists (acetylcholine and bradykinin) and an endothelium-independent agonist (nitroglycerin) before and during application of L-arginine. Topical application of acetylcholine (1.0 and 10 muM) and bradykinin (1.0 and 10 microM) produced dilatation in nondiabetic rats of the basilar artery which was impaired in diabetic rats. Topical application of nitroglycerin (0.1 and 1.0 microM) produced similar dilatation of the basilar artery in nondiabetic and diabetic rats. Topical application of L-arginine (0.1 and 3 mM) did not enhance dilatation of the basilar artery in response to acetylcholine and bradykinin in diabetic rats. Thus, impairment of dilatation of the basilar artery in diabetic rats in response to acetylcholine and bradykinin appears to be related to a mechanism unrelated to the availability of L-arginine for nitric oxide synthase. PMID- 8621756 TI - Autoregulation in the middle meningeal artery. AB - In cats anesthetized with alpha-chloralose, autoregulation of blood flow (F) in the middle meningeal and common carotid arteries was assessed by bleeding and subsequently reinfusing the animals to achieve a 25% step reduction in mean arterial blood pressure (P), while maintaining the systolic blood pressure >80 mmHg. The integrity of autoregulation was assessed by calculating the gain factor Gf = 1 - [(deltaF/F)/(deltaP/P)]. Cats were examined intact, after hexamethonium (10 mg/kg), and after papaverine (6 mg/kg). Reduction of blood pressure of 25 to 60 mmHg produced equivalent drops in carotid blood flow (Gf = 0.041 +/- 0.34; mean +/- standard deviation, n = 12). There were only small changes in flow in the middle meningeal artery during this procedure (Gf = 0.91 +/- 0.29). Hexamethonium did not block autoregulation in the middle meningeal artery (Gf = 0.92 +/- 0.13, n = 4). However, papaverine almost completely abolished the ability of the artery to autoregulate (Gf = 0.10 +/- 0.16, n = 7). The results suggest that the middle meningeal artery possesses an ability similar to that of the cortical circulation to autoregulate its blood flow through intrinsic, non neuronal mechanisms. This will have important implications for the study of disturbances of dural arterial control in migraine and other headaches. PMID- 8621757 TI - NADPH-diaphorase neurons contacting the cerebrospinal fluid in the ventricles of rat brain. AB - Two populations of scattered neurons containing nitric oxide synthase activity were detected in the wall of the third and lateral cerebral ventricles of rat brain, using histochemistry for NADPH-diaphorase activity. One type was multipolar and lay supraependymally, with dendrites oriented in the plane of the ependymal layer. The second type was bipolar and was situated subependymally, with dendrites extending in opposite directions, either into the surrounding brain tissue or to the ventricular surface. Moreover, multipolar neurons, situated in the corpus callosum and in the subcortical white matter, had long varicose dendrites extending toward the roof of the lateral ventricles. As a result, numerous NADPH-diaphorase neurites spread out on the free surface of the ependymal layer in contact with the CSF. These observations raise the possibility that periventricular nitrergic neurons play an essential role in registering the composition of the CSF and in modulating subcortical cerebral blood flow. A further possibility is that supraependymal nitrergic neuronal processes are effectors regulating activity of ependymal cells. PMID- 8621758 TI - Color duplex measurement of cerebral blood flow volume: intra- and interobserver reproducibility and habituation to serial measurements in normal subjects. AB - Color duplex flowmetry of internal carotid and vertebral arteries permits estimation of intravascular flow volumes and global cerebral blood flow volume (CBFV) by summing the flow volumes measured in each of the four extracranial vessels. Intravascular flow volumes were calculated as the product of angle corrected time-averaged flow velocity and the cross-sectional area of the vessel. The reliability of this new method was tested in a prospective, intra- and interdiane, intra- and interobserver reproducibility study of 32 healthy subjects aged 7-57 years. In each subject, CBFV was tested by each observer twice on day 1 and once on day 2 in consecutive recordings. In each artery, both examiners found closely similar mean intravascular flow volumes. Intradiane interobserver reproducibility of CBFV was high on both days (correlation coefficient, CC, 0.90 and 0.85, p < or = 0.0001; coefficient of variance, CV, 10.0 and 10.4%, respectively), as was the interdiane comparison (CC = 0.81, p < or = 0.0001; CV < or = 13.3%). Intraobserver reproducibility was even higher. On both days, there was a progressive decrease in CBFV from each subject's first to the last examination within a 1-h examination period (day 1: 717 +/- 150 ml/min to 690 +/- 120 ml/min; difference, p < or = 0.05; day 2: 700 +/- 120 ml/min to 665 +/- 126 ml/min; difference, p < or = 0.01). This habituation effect was more pronounced in subjects with high initial CBFV. Reproducibility of CBFV is comparable to that of mean CBF measurements with 133Xe inhalation and H2 15(O) positron emission tomography techniques reported by other groups. This method makes serial bedside monitoring of CBFV feasible without posing the risks of radiation exposure. PMID- 8621759 TI - A feedback-controlled pump produces stable hypotension in anaesthetised rabbits. AB - A method is described for the reliable production of controlled hypotension in experimental animals. Reduction in arterial blood pressure was obtained in rabbits by withdrawing arterial blood using a computer-driven pump operating within a feedback control system. Arterial blood pressure, blood flow velocity in the basilar artery (measured using transcranial Doppler), and anterior cortical microcirculation (measured using laser Doppler) were monitored. The aim of the experiments was to compare stability of hypotension produced using arterial blood pressure or basilar flow velocity as feedback control variables. Basilar artery flow velocity provided the most stable profound hypotension and during reinfusion when animals were not autoregulating. However, arterial blood pressure provided the most accurate stepwise control in autoregulating animals. PMID- 8621760 TI - The curtain rises on the renin-angiotensin system: AT2 receptors are in the spotlight. PMID- 8621761 TI - The zebrafish: heritable disorders in transparent embryos. PMID- 8621762 TI - Inhibition of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human cells. Evidence for a transcriptional block. AB - Cyclopentenone prostaglandins (PGs) inhibit virus replication in several DNA and RNA virus models, in vitro and in vivo. In the present report we demonstrate that the cyclopentenone prostaglandins PGA(1) and PGJ(2) at nontoxic concentrations can dramatically suppress HIV-1 replication during acute infection in CEM-SS cells. PGs did not affect HIV-1 adsorption, penetration, reverse transcriptase activity nor viral DNA accumulation in HIV-1 infected cells. A dramatic reduction in HIV-1 mRNA levels was detected up to 48-72 h after infection (p.i.) in PG treated cells, and HIV-1 protein synthesis was greatly reduced by a single PG treatment up to 96 h p.i. Repeated PGA(1)-treatments were effective in protecting CEM-SS cells by the cytopathic effect of the virus, and in dramatically reducing HIV-1 RNA levels up to 7 d after infection. The antiviral effect was not mediated by alterations in the expression of alpha-, beta-, or gamma-interferon,TNFalpha, TNFbeta, IL6, and IL10 in HIV-infected CEM-SS cells. The fact that prostaglandins are used clinically in the treatment of several diseases, suggests a potential use of cyclopentenone PGs in the treatment of HIV-infection. PMID- 8621763 TI - Defective anion transport and marked spherocytosis with membrane instability caused by hereditary total deficiency of red cell band 3 in cattle due to a nonsense mutation. AB - We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C --> T substitution resulting in a nonsense mutation (CGA --> TGA; Arg --> Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl-/HCO3- exchange, causing mild acidosis with decreases in the HCO3 concentration and total CO2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO2 transport, and acid base homeostasis, but is not always essential to the survival of this mammal. PMID- 8621764 TI - Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model. AB - Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death. PMID- 8621765 TI - Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. AB - Idiopathic pulmonary fibrosis (IPF) is a progressive disorder characterized by inflammation, fibroblast proliferation, and accumulation of extracellular matrix proteins. Leukotrienes (LTs) are pro-inflammatory and pro-fibrogenic mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism. They are thought to play a role in a number of disease processes, but have received relatively little attention in investigations into the pathogenesis of IPF. In this study, we measured the levels of immunoreactive LTs B(4) and C(4) in homogenates of lung tissue obtained from patients with newly diagnosed, untreated IPF, as compared to levels measured in homogenates of uninvolved nonfibrotic lung tissue from patients undergoing resectional surgery for bronchogenic carcinoma. Compared to homogenates on nonfibrotic control lung, homogenates from IPF patients contained 15-fold more LTB(4) and 5-fold more LTC(4). IPF homogenate levels of LTB(4) were significantly correlated with histologic indices of both inflammation (r=0.861) and fibrosis (r=0.926). Activation of 5-LO is known from in vitro studies to be associated with localization of the enzyme at the nuclear membrane. Immunohistochemical staining for 5-LO protein in alveolar macrophages (AMs) demonstrated that such an "activated" localization pattern was significantly more frequent in IPF lung (19.2+/-3.3% of cells) than in control lung (9.3+/-0.9%); this localization pattern was rarely seen (3.2%) in sections from a truly normal transplant donor lung. Consistent with these data, AMs obtained from IPF patients by bronchoalveolar lavage, purified by adherence, and cultured in the absence of a stimulus for 16 h elaborated significantly greater amounts of LTB(4) and LTC(4) than did control AMs obtained from normal volunteers. These data indicate that the 5-LO pathway is constitutively activated in the lungs of patients with IPF, and the AM represents at least one cellular source of LT overproduction in this disorder. We speculate that LTs participate in the pathogenesis of IPF, and their overproduction in this disorder may be amenable to specific pharmacotherapy. PMID- 8621767 TI - Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits. In vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner. AB - Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/ 18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration. PMID- 8621769 TI - Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+. AB - Pancreatic islets are known to respond to a raise of the glucose concentration with Ca2+ -induced 2-3-min pulses of insulin release. The reports of cyclic variations of circulating insulin in the fasting state made it important to explore whether insulin release is also pulsatile in the absence of stimulated entry of Ca2+. Individual pancreatic islets were isolated from a local colony of ob/ob mice and perifused under conditions allowing dual wavelength recordings of the cytoplasmic Ca2+ concentration ([Ca2+]i) with fura-2 and measurements of insulin with ELISA technique. At 3 mM of glucose, [Ca2+]i remained at a stable low level, but insulin was released in pulses with a frequency of 0.41+/-0.02 min 1, determined by Fourier transformation of original and autocorrelated data. Pulses of basal insulin release were also seen when glucose was omitted and 1 microM clonidine or 400 microM diazoxide was added to a glucose-free medium. The results indicate that pulsatile insulin release can be generated in the absence of stimulated entry of Ca2+. A tentative explanation for this phenomenon is inherent fluctuations in the ATP production of the beta cells. PMID- 8621766 TI - Nitric oxide inhibits viral replication in murine myocarditis. AB - Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo. PMID- 8621768 TI - Leukemia inhibitory factor (LIF) stimulates proopiomelanocortin (POMC) expression in a corticotroph cell line. Role of STAT pathway. AB - We recently described the expression of leukemia inhibitory factor (LIF) in human fetal and murine corticotrophs. LIF and the related cytokine oncostatin M induced basal, and corticotropin-releasing hormone (CRH) induced proopiomelanocortin (POMC) mRNA and ACTH secretion in AtT20 cells. LIF signaling and regulation of POMC gene transcription were therefore tested. Dexamethasone inhibited both basal and LIF-induced ACTH secretion (P<0.05) and LIF induction of ACTH was also attenuated by immuneutralization of either the LIF receptor (35%, P<0.05) or the gp130 affinity converter (41%, P<0.05). These antisera also attenuated basal ACTH secretion in the absence of added ligand (P<0.05). To examine intrapituitary LIF signaling, phosphorylation of post-receptor substrates was measured. 1 nM LIF rapidly induced tyrosyl phosphorylation of STAT 1 and STAT 3 proteins, as well as tyrosyl phosphorylation of a 115-kD protein, coimmunoprecipitated with STAT 1. The transfected rat POMC promoter -706/+64, fused to the luciferase reporter gene, was induced by LIF, which exerted strong (18-fold) synergy with CRH. Deletion of the major CRH responsive region in POMC (-323/-166) abolished CRH induction of transcription and severely limited LIF synergy. Although 8 bromo cAMP or forskolin modestly enhanced POMC transcription (2.8-fold), LIF markedly potentiated (7.4-fold) these cAMP activators. These results demonstrate that corticotroph LIF action is receptor mediated and involves activation of STAT signaling pathways. LIF potently synergizes with both CRH and cAMP induction of POMC transcription. This novel intrapituitary signaling mechanism may mediate a neuroimmune pituitary interface. PMID- 8621770 TI - Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome. AB - We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N-acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was approximately 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy. PMID- 8621771 TI - Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells. AB - The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues. In the absence of exogenous ligand, expression of sst2 in NIH 3T3 cells resulted in inhibition of cell growth. Polymerase chain reaction coupled to reverse transcription demonstrated that expression of sst2 in NIH 3T3 cells stimulated the expression of preprosomatostatin mRNA accompanied by a production of immunoreactive somatostatin-like peptide which corresponded predominantly to somatostatin 14. Moreover anti-somatostatin antibodies suppressed sst2-promoted inhibition of cell proliferation. Inhibition of cell proliferation associated with increased secretion of somatostatin-like immunoreactivity was also observed after expression of sst2 in human pancreatic tumor cells BxPC3 devoid of endogenous receptors. In addition, expression of sst2 in NIH 3T3 cells was associated with constitutive activation of tyrosine phosphatase PTP1C that resulted from enhanced expression of the protein. Blocking of PTP1C tyrosine phosphatase activity with orthovanadate or that of PTP1C protein with antisense PTP1C oligonucleotides decreased the sst2-induced inhibition of cell proliferation. These results, taken together, show that expression of sst2 in NIH 3T3 cells generated a negative autocrine loop by stimulating sst2 ligand production and amplifying PTP1C sst2-transducer. Sst2/ligand may function as a determinant factor involved in the negative growth control of cells. PMID- 8621772 TI - Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase. AB - Urea, in concentrations unique to the renal medulla, increases transcription and protein expression of several immediate-early genes (IEGs) including the zinc finger-containing transcription factor, Egr-1. In the present study, the proximal 1.2 kb of the murine Egr-1 5' -flanking sequence conferred urea-responsiveness to a heterologous luciferase reporter gene when transiently transfected into renal medullary mIMCD3 cells,and this effect was comparable with that of the extremely potent immediate-early gene inducer, O-tetradecanoylphorbol 13-acetate (TPA). Urea inducibility of Egr-1 expression was protein kinase C (PKC)-dependent because staurosporine and calphostin C abrogated the urea effect, and down regulation of PHC through chronic TPa treatment inhibited both urea-inducible Egr 1 protein expression and gene transcription. In addition, hyperosmotic urea increased inositol 1,4,5-trisphosphate (IP3) release from mIMCD3 cells and induced tyrosine phosphorylation of the receptor tyrosine kinase-specific phospholipase C (PLC) isoform, PLC-gamma. Importantly, urea-inducible Egr-1 expression was strongly genistein-sensitive, to a much greater extent than the comparable TPA-inducible Egr-1 expression. These data suggest that urea-inducible Egr-1 expression is a consequence of sequential PLC-gamma activation, IP3 release, and PKC activation. Urea-inducible PLC-gamma activation, in conjunction with the genistein-sensitivity of urea-inducible Egr-1 expression suggest the possibility of a cell surface or cytoplasmic urea-sensing receptor tyrosine kinase. PMID- 8621773 TI - Monocytic cell type-specific transcriptional induction of collagenase. AB - Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP 1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences. PMID- 8621774 TI - Primary preventive and secondary interventionary effects of acetyl-L-carnitine on diabetic neuropathy in the bio-breeding Worcester rat. AB - The abnormalities underlying diabetic neuropathy appear to be multiple and involve metabolic neuronal and vasomediated defects. The accumulation of long chain fatty acids and impaired beta-oxidation due to deficiencies in carnitine and/or its esterified derivatives, such as acetyl-L-carnitine, may have deleterious effects. In the present study, we examined, in the diabetic bio breeding Worcester rat, the short- and long-term effects of acetyl-L-carnitine administration on peripheral nerve polyols, myoinositol, Na+/K+ -ATPase, vasoactive prostaglandins, nerve conduction velocity, and pathologic changes. Short-term prevention (4 mo) with acetyl-L-carnitine had no effects on nerve polyols, but corrected the Na+/K+ -ATPase defect and was associated with 63% prevention of the nerve conduction defect and complete prevention of structural changes. Long-term prevention (8 mo) and intervention (from 4 to 8 mo) with acetyl-L-carnitine treatment normalized nerve PGE(1) whereas 6-keto PGF(1-alpha) and PGE(2) were unaffected. In the prevention study, the conduction defect was 73% prevented and structural abnormalities attenuated. Intervention with acetyl-L carnitine resulted in 76% recovery of the conduction defect and corrected neuropathologic changes characteristic of 4-mo diabetic rats. Acetyl-L-carnitine treatment promoted nerve fiber regeneration, which was increased two-fold compared to nontreated diabetic rats. These results demonstrate that acetyl-L carnitine has a preventive effect on the acute Na+/- K+_ATPase defect and a preventive and corrective effect on PGE1 in chronically diabetic nerve associated with improvements of nerve conduction velocity and pathologic changes. PMID- 8621775 TI - Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart. Regulation of NOS3 transcription in vitro and in vivo by cyclic adenosine monophosphate in rat cardiac myocytes. AB - Cardiac myocytes express the nitric oxide synthase isoform originally identified in constitutive nitric oxide synthase cells (NOS3), which mediates the attenuation by muscarinic cholinergic agonists of beta-adrenergic stimulation of L-type calcium current and contractility in these cells. However, calcium current and contractility in these cells. However, the reciprocal regulation of NOS3 activity in myocytes by agents that elevate cAMP has not been reported. In this study, we show that NOS3 and mRNA and protein levels in cardiac myocytes are reduced both in vitro after treatment with cAMP elevating drugs, and in vivo after 3 d of treatment with milrinone, a type III cAMP phosphodiesterase inhibitor. This effect on NOS3 activity by cAMP is cell type specific because treatment of cardiac microvascular endothelial cells in vitro or in vivo did not decrease NOS3 mRNA or protein in these cells. NOS3 downregulation in myocytes appeared to be at the level of transcription since there was no modification of NOS3 mRNA half-life by agents that increase intracellular cAMP. To determine the functional effects of NOS3 downregulation, we examined the contractile responsiveness of isolated electrically paced ventricular myocytes, isolated from animals that had been treated in vivo with milrinone, to the beta-adrenergic agonist isoproterenol and the muscarinic cholinergic agonist carbamylcholine. There was no difference in baseline contractile function in cells that had been pretreated with cAMP elevating agents compared to controls, but cells exposed to milrinone in vivo exhibited an accentuation in their contractile responsiveness to isoproterenol compared to controls and a loss of responsiveness to carbamylcholine. Downregulation of myocyte NOS3 by sustained elevation of cAMP may have important implications for the regulation of myocardial contractile state by the autonomic nervous system. PMID- 8621776 TI - Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. AB - We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation. PMID- 8621777 TI - Stromelysin-3 expression promotes tumor take in nude mice. AB - Stromelysin-3 (ST3) is a matrix metalloproteinase expressed in human carcinomas in ways suggesting that it may play a role in tumor progression. To test this possibility, we have performed gene transfer experiments using both anti-sense and sense ST3 expression vectors, and malignant cells either expressing (NIH 3T3 fibroblasts) or not (MCF7 epithelial cells) endogenous ST3. We have compared the ability of parental and transfected cells to cause subcutaneous tumor development in nude mice. 3T3 cells expressing anti-sense ST3 RNA showed reduced tumorigenicity, and MCF7 cells expressing mouse or human ST3 were associated with reduced tumor-free period leading to a significant increased tumor incidence(P<10(-4)). However, once established, the ST3 expressing tumors did not grow faster than those obtained with the parental MCF7 cell line. In addition, tumors obtained after sub-cutaneous injection of ST3-expressing or nonexpressing cells did not exhibit obvious histological differences, and careful examination did not reveal any local invasive tissue areas nor systemic metastases. These in vivo observations were in agreement with those obtained in vitro showing that ST3 expression did not modify proliferative nor invasive properties of transfected cells. Altogether, these results indicate that ST3 expression promotes tumor take in nude mice, presumably by favoring cancer cell survival in a tissue environment initially not permissive for tumor growth. These findings represent the first experimental evidence showing that ST3 can modulate cancer progression. PMID- 8621778 TI - T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. AB - IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL 8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID- 8621780 TI - The role of cytokines, adhesion molecules, and chemokines in interleukin-2 induced lymphocytic infiltration in C57BL/6 mice. AB - IL-2 mediates the regression of certain malignancies, but clinical use is limited because of associated toxicities, including parenchymal lymphocytic infiltration with multiple organ failure. Secondarily induced cytokines are important mediators of IL-2 toxicity and IL-2-induced lymphocyte-endothelial adherence and trafficking. The recently discovered C-C chemokines, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein 1alpha, have also been implicated in lymphocytic migration. We hypothesized that IL-2 alters cytokine, C-C chemokine, and adhesion molecule expression in association with parenchymal lymphocytic infiltration. C57BL/6 mice were injected with 3x10(5) IU of IL-2 or 0.1 ml of 5% dextrose intraperitoneally every 8 h for 6 d, then killed. IL-2 induced massive lymphocytic infiltration in the liver and lung and moderate infiltration in the kidney in association with organ edema and dysfunction. Immunostaining showed increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in association with this organ-specific lymphocytic infiltration. Flow cytometry showed increased expression of the corresponding ligands (lymphocyte function-associated antigen-1 and very late antigen-4) on splenocytes. IL-2 increased TNF-alpha mRNA and protein expression in the liver. Organs infiltrated by lymphocytes had increased TNF-alpha mRNA, whereas RANTES mRNA was increased in all organs, regardless of lymphocytic infiltration. IL-2 toxicity involves organ-specific TNF alpha and RANTES production with increased ICAM-1 and VCAM-1 expression as potential mechanisms facilitating lymphocytic infiltration and organ dysfunction. PMID- 8621779 TI - The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes. AB - Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion. PMID- 8621781 TI - Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. AB - Prolonged hypokalemia causes vasopressin-resistant polyuria. We have recently shown that another cause of severe polyuria, chronic lithium therapy, is associated with decreased aquaporin-2 (AQP2) water channel expression (Marples, D., S. Christensen, E.I. Christensen, P.D. Ottosen, and S. Nielsen, 1995. J. Clin. Invest., 95: 1838-1845). Consequently, we studied the effect in rats of 11 days' potassium deprivation on urine production and AQP2 expression and distribution. Membrane fractions were prepared from one kidney, while the contralateral kidney was perfusion-fixed for immunocytochemistry. Immunoblotting and densitometry revealed a decrease in AQP2 levels to 27+/-3.4% of control levels (n=11, P<0.001) in inner medulla, and 34+/-15% of controls (n=5, P<0.05) in cortex. Urine production increased in parallel, from 11+/-1.4 to 30+/-4.4 ml/day (n=11, P<0.01). After return to a potassium-containing diet both urine output and AQP2 labels normalized within 7 d. Immunocytochemistry confirmed decreased AQP2 labeling in principal cells of both inner medullary and cortical collecting ducts. AQP2 labeling was predominantly associated with the apical plasma membrane and intracellular vesicles. Lithium treatment for 24 d caused a more extensive reduction of AQP2 levels, to 4+/-1% of control levels in the inner medulla and 4+/-2% in cortex, in association with severe polyuria. The similar degree of downregulation in medulla and cortex suggests that interstitial tonicity is not the major factor in the regulation of AQP2 expression. Consistent with this furosemide treatment did not alter AQP2 levels. In summary,hypokalemia, like lithium treatment, results in a decrease in AQP2 expression in rat collecting ducts, in parallel with the development of polyuria, and the degree of downregulation is consistent with the level of polyuria induced, supporting the view that there is a causative link. PMID- 8621782 TI - Common T cell receptor clonotype in lacrimal glands and labial salivary glands from patients with Sjogren's syndrome. AB - Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS. PMID- 8621783 TI - The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3', 5' monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats. AB - The renal effects of angiotensin II(AII) are attributed to AT1 receptors. In contrast, the function of renal AT2 receptors in unknown. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) prostaglandin E2 (PGE2) and cyclic guanosine 3', 5'-monophosphate (cGMP) in response to dietary sodium (Na) depletion alone, or Na depletion or normal Na diet combined with the AT1 receptor blocker, Losartan, the AT2 receptor blocker, PD 123319 (PD), or angiotensin II, individually or combined in conscious rats. Na depletion significantly increased PGE2 and cGMP. During Na depletion, Losartan decreased PGE2 and did not change cGMP. In contrast, PD significantly increased PGE2 and decreased cGMP. Combined administration of Losartan and PD decreased PGE2 and cGMP. During normal Na diet, RIF PGE2 and cGMP increased in response to angiotensin II. Neither Losartan nor PD, individually or combined, changed RIF PGE2 or cGMP. Combined administration of angiotensin II and Losartan or PD produced a significant decrease in response of PGE2 and cGMP to angiotensin II, respectively. These data demonstrate that activation of the reninangiotensin system during Na depletion increases renal interstitial PGE2 and cGMP. The AT1 receptor mediates renal production of PGE2. The AT2 receptor mediates cGMP. AT2 blockade potentiates angiotensin-induced PGE2 production at the AT1 receptor. PMID- 8621786 TI - Complex trait genetics: new methods yield a result for essential hypertension. PMID- 8621784 TI - Tumor suppression and inhibition of aneuploid cell accumulation in human brain tumor cells by ectopic overexpression of the cyclin-dependent kinase inhibitor p27KIP1. AB - To investigate how overexpression of p27KIP1, a downstream effector of TGF-beta and a universal cyclin-dependent kinase (CDK) inhibitor could influence the malignant phenotype of malignant human brain tumor cells, an adenovirus vector system was used to transfer the human p27KIP1 gene (Adp27KIP1) into the human astrocytoma cell line, U-373MG. Inhibition of CDK activity in Adp27KIP1-infected cells was indicated by inhibition of [3H]thymidine incorporation, an increase in cell doubling time and by cell cycle arrest in G1. Notably, ectopic overexpression of p27KIP1 was associated with a marked decrease in the accumulation of aneuploid cells. Diminished malignant potential of Adp27KIP1 infected cells was manifested by the loss of anchorage-independent growth in soft agar and by the inability to induce tumorgenesis in a xenograft model. These studies suggest that p27KIP1 is a tumor suppressor gene and supports the use of Adp27KIP1 for gene therapy of human brain tumors. PMID- 8621787 TI - Recent advances in gene mutagenesis by site-directed recombination. PMID- 8621785 TI - Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults. AB - In hypercholesterolemic rabbits, oral L-arginine (the substrate for endothelium derived nitric oxide) attenuates endothelial dysfunction and atheroma formation, but the effect in hypercholesterolemic humans is unknown. Using high resolution external ultrasound, we studied arterial physiology in 27 hypercholesterolemic subjects aged 29+/-5 (19-40) years, with known endothelial dysfunction and LDL cholesterol levels of 238+/-43 mg/dl. Each subject was studied before and after 4 wk of L-arginine (7 grams x 3/day) or placebo powder, with 4 wk washout, in a randomized double-blind crossover study. Brachial artery diameter was measured at rest, during increased flow (causing endothelium-dependent dilation, EDD) and after sublingual glyceryl trinitrate (causing endothelium-independent dilation). After oral L-arginine, plasma L-arginine levels rose from 115+/-103 to 231+/-125 micromol/liter (P<0.001), and EDD improved from 1.7+/-1.3 to 5.6+/-3.0% (P<0.001). In contrast there was no significant change in response to glyceryl trinitrate. After placebo there were no changes in endothelium-dependent or independent vascular responses. Lipid levels were unchanged after L-arginine and placebo. Dietary supplementation with L-arginine significantly improves EDD in hypercholesterolemic young adults, and this may impact favorably on the atherogenic process. PMID- 8621788 TI - Cytoplasmic localization of FAC is essential for the correction of a prerepair defect in Fanconi anemia group C cells. AB - Mutations in the gene defective in Fanconi anemia complementation group C, FAC, are responsible for a subset of Fanconi anemia, a group of autosomal recessive disorders characterized by chromosomal instability, hypersensitivity to cross linking agents, and cancer susceptibility. Although abnormalities in DNA repair have been suspected, localization of the FAC gene product to the cytoplasm has cast doubt on such a mechanism. Monitoring of interstrand DNA cross-linking shows that the predominant defect in group C cells is in the initial induction of cross links, not in repair synthesis. Both the cross-linking defect and the enhanced cytotoxicity of cross-linkers on Fanconi anemia group C cells are corrected completely by cytoplasmic isoforms of the FAC protein, but not by an isoform targeted to the nucleus. The ability of FAC to correct these phenotypic abnormalities reaches a maximum threshold despite overexpression leading to higher levels of cytosolic protein. These results demonstrate that cytoplasmic localization is essential for the intracellular activity of the FAC protein. It is proposed that this activity is coupled to a cytoplasmic defense mechanism against a specific class of genotoxic agents. PMID- 8621789 TI - The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. AB - Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology. PMID- 8621790 TI - Early and late stimulation of ob mRNA expression in meal-fed and overfed rats. AB - ob protein is hypothesized to be a circulating feedback signal in the regulation of energy balance. Obese, overfed rats have high levels of ob mRNA expression and suppressed voluntary food intake, indicating the presence of a potent satiety factor. The objectives of this experiment were to determine whether feeding rats their normal daily intake in three meals, compared with ad libitum feeding, increased ob mRNA expression and to determine the degree of obesity required to stimulate expression of ob mRNA. Rats were fed ad libitum, were tube-fed their normal intake in three meals a day, or were tube-fed twice normal intake, ob mRNA was measured by Northern blot analysis after 0, 2, 7, 14, 21, and 32 d of tube feeding. After only 2 d ob mRNA was threefold higher in tube-fed animals than in ad libitum controls. By day 21 there was a further increase in ob mRNA expression in overfed rats which were at 130% control weight. These results suggest that a metabolic consequence of meal-feeding increases ob mRNA expression in the absence of increased food intake or weight gain. There is a further increase in ob mRNA expression once significant obesity is established. PMID- 8621791 TI - CD4+ CD7- CD28- T cells are expanded in rheumatoid arthritis and are characterized by autoreactivity. AB - Clonal expansion of CD4+ T cells is a characteristic finding in patients with RA and is only infrequently found in patients with psoriatic arthritis and healthy controls. Expanded CD4+ clonotypes are present in the blood, infiltrate into the joint, and persist over years. We have not addressed the question of whether the expanded clonotypes have unique functional and phenotypic properties which may explain the preferential in vivo expansion in RA. In contrast to most CD4+ T cells, expanded clonotypes lacked the expression of the CD28 and CD7 cell surface molecules. Accordingly, the subsets of CD4+ CD28- (9.7 vs 1.7, P = 0.00002) and CD4+ CD7- T cells (21.5 vs 12.26, P = 0.018) were increased in RA patients compared with age-matched normal individuals. Despite the lack of CD28 expression, clonally expanded CD4+ T cells were not anergic but proliferated in response to immobilized anti-CD3 and could be maintained in tissue culture. In vivo expanded CD4+ T cells were autoreactive to ubiquitously distributed autoantigens. They responded in an autologous mixed lymphocyte reaction, and T cell clones isolated from selected patients proliferated to autologous peripheral blood adherent cells. These data suggest that in RA patients selected CD4+ T cells which share the CD7- CD28- phenotype escape from peripheral tolerance. PMID- 8621792 TI - Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation. AB - Alpha-Melanocyte-stimulating hormone (MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. We showed previously that alpha-MSH inhibits nitric oxide (NO) production in cultured macro-phages. To determine how alpha-MSH acts in vivo, we induced acute hepatic inflammation by administering endotoxin (LPS) to mice pretreated with Corynebacterium parvum, alpha-MSH prevented liver inflammation even when given 30 min after LPS administration. To determine the mechanisms of action of alpha-MSH, we tested its influence on NO, infiltrating inflammatory cells, cytokines, and chemokines. Alpha-MSH inhibited systemic NO production, hepatic neutrophil infiltration, and increased hepatic mRNA abundance for TNF alpha, and the neutrophil and monocyte chemokines (KC/IL-8 and MCP-1). We conclude that alpha-MSH prevents LPS-induced hepatic inflammation by inhibiting production of chemoattractant chemokines which then modulate infiltration of inflammatory cells. Thus, alpha-MSH has an effect very early in the inflammatory cascade. PMID- 8621793 TI - Inferior petrosal sinus sampling in healthy subjects reveals a unilateral corticotropin-releasing hormone-induced arginine vasopressin release associated with ipsilateral adrenocorticotropin secretion. AB - Arginine vasopressin (AVP) acts synergistically with corticotropin-releasing hormone (CRH) to stimulate ACTH release from the anterior pituitary. In a previous study of bilateral simultaneous inferior petrosal sinus (IPS) sampling in healthy human subjects, we observed lateralized ACTH secretion, suggesting lateralized secretion of an ACTH-regulating hypothalamic factor. To investigate this possibility, we measured ACTH, CRH, AVP, and oxytocin (OT) levels in the IPS and the peripheral circulation in nine normal volunteers, before and after 1 microgram/kg i.v. bolus ovine CRH (oCRH). At baseline, ACTH, AVP, and OT exhibited a significant (P < 0.05) two to threefold intersinus gradient (ISG), indicating the existence of a dominant petrosal sinus. Endogenous CRH was undetectable in all samples. Despite similar exogenous oCRH levels in both petrosal sinuses, oCRH caused a significant increase (P < 0.001) in the ACTH ISG (15.8 +/- 5.6, mean +/- SEM), suggesting increased responsiveness of one dominant side of the anterior pituitary. This was associated with an ipsilateral CRH induced AVP release and a significant increase (P < 0.01) in the AVP ISG (8.6 +/- 2.3), suggesting lateralized AVP secretion by the hypothalamus. Furthermore, the increased AVP ISG after oCRH correlated strongly with the ACTH ISG (r = 0.92, P < 0.01). oCRH administration did not affect OT. These findings suggest that there is a dominant petrosal sinus in healthy volunteers that appears to reflect a dominant side of the adenohypophysis, characterized by increased functional activity and/or responsiveness of the pituitary corticotrophs. This may reflect lateralized hypothalamic and/or suprahypothalamic function resulting in CRH responsive lateralized secretion of AVP from parvocellular and/or magnocellular axons in the median eminence and the posterior pituitary. Although the functional and teleologic significance of these findings remains to be investigated, our data suggest a novel mechanism for CRH-mediated ACTH release, namely CRH-induced release of AVP which then enhances CRH action on the corticotrophs. Furthermore, our data represent the first direct evidence for the concept of brain lateralization with respect to neuroendocrine secretion. PMID- 8621794 TI - Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells. AB - Interleukin 1 and nitric oxide (NO) from infiltrating macrophages and activated mesangial cells may act in concert to sustain and promote glomerular damage. To evaluate if such synergy occurs, we evaluated the effect if IL-1 beta and NO on the formation of prostaglandin (PG)E2 and cyclooxygenase (COX) expression. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, alone did not increase basal PGE2 formation. However, these compounds amplified IL-1 beta induced PGE2 production. Similarly, sodium nitroprusside and S-nitroso-N acetylpenicillamine by themselves did not induce mRNA and protein for COX-2, the inducible isoform of COX; however, they both potentiated IL-1 beta-induced mRNA and protein expression of COX-2. The stimulatory effect of NO is likely to be mediated by cGMP since (a) an inhibitor of the soluble guanylate cyclase, methylene blue, reversed the stimulatory effect of NO donors on COX-2 mRNA expression; (b) the membrane-permeable cGMP analogue, 8-Br-cGMP, mimicked the stimulatory effect of NO donors on COX-2-mRNA expression; and (c) atrial natriuretic peptide, which increases cellular cGMP by activating the membrane bound guanylate cyclase, also amplified IL-1 beta-induced COX-2 mRNA expression. These data indicate a novel interaction between NO and COX pathways. PMID- 8621795 TI - Identification of cardiac myosin peptides capable of inducing autoimmune myocarditis in BALB/c mice. AB - Immunization with cardiac myosin induces T cell-mediated myocarditis in genetically predisposed mice and serves as a model for autoimmune heart disease. This study was undertaken to identify pathogenic epitopes on the myosin molecule. Our approach was based on the comparison of the pathogenicity between cardiac (alpha-)myosin and soleus muscle (beta-)myosin. We show that alpha-myosin is the immunodominant isoform and induces myocarditis at high severity and prevalence whereas beta-myosin induces little disease. Therefore the immunodominant epitopes of alpha-myosin must reside in regions of different amino acid sequence between alpha- and beta-myosin isoforms. Cardiac myosin peptides corresponding to these regions of difference were synthesized and tested for their ability to induce inflammatory heart disease. Three pathogenic peptides were identified. One peptide that is located in the head portion of the molecule induced severe myocarditis, whereas two others that reside in the rod portion possessed only minor pathogenicity. The identification of pathogenic epitopes on the cardiac myosin molecule will allow detailed studies on the recognition of this antigen by the immune system and might be used to downmodulate ongoing heart disease. PMID- 8621796 TI - Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. AB - We investigated the role of the costimulatory molecules, CD40 and its ligand CD40L, in the pathogenesis of human SLE. In comparison to normal subjects or patients in remission, PBMC from active lupus patients had a 21-fold increase in the frequency of CD40L-expressing, CD4+T cells. However, the expression of CD40L induced in either lupus or normal T cells by mitogenic stimulation could be down regulated equally well by CD40 molecules on autologous B cells. Active lupus patients also had a 22-fold increase in percentage of CD8+ T cells expressing CD40L, consistent with their unusual helper activity in SLE. Surprisingly, patients with active lupus had a 20.5-fold increase in B cells that spontaneously expressed high levels of CD40L, as strongly as their T cells. Although lupus patients in remission had low levels of CD40L+ cells in the range of normal subjects, mitogen-induced upregulation of CD40L expression in the T and B cells was markedly greater than normal, suggesting an intrinsic defect. A mAb to CD40L blocked significantly the ability of lymphocytes from lupus patients with active and established disease to produce the pathogenic variety of antinuclear autoantibodies in vitro, bolstering the possibility of anti-CD40L immunotherapy for lupus. Future studies on the hyperexpression of CD40L could elucidate a regulatory defect in the pathogenic T and B cells of lupus. PMID- 8621797 TI - Expression of the immunoglobulin VH gene 51p1 is proportional to its germline gene copy number. AB - 51p1 is an immunoglobulin VH gene that is frequently expressed in B cell chronic lymphocytic leukemia and early in B cell ontogeny. The 51p1 gene locus is highly polymorphic, consisting of 13 alleles that can be classified as being either 51p1 related or hy1263-related, based on distinctive sequence motifs in the second complementarity determining region. Two of the 51p1-related genes usually occur as a linked pair on the same haopltype, resulting from gene duplication. Consequently, a person can have a total of zero to four copies of 51p1-related genes. These genes are detectable in genomic DNA by sequence-specific RFLP analysis using oligonucleotide probes. Ig encoded by nonmutated 51p1-related genes can be detected by G6, a murine antiidiotypic mAb. We have now studied lymphocytes from 35 human tonsils to examine the relation between the number of 51p1-related germlime gene copies and the proportion of IgD-bearing tonsillar B cells that react with G6. All subjects who had zero copies of 51p1-related genes lacked any G6-reactive B cells, whereas those with four copies of 51p1-related genes had the highest proportions of G6-positive IgD B cells, up to 11.4%. Subjects with intermediate gene doses had intermediate proportions of G6-reactive B cells. Over the entire data set, the percentage of IgD-bearing B cells that reacted with G6 was proportional to the 51p1-related gene copy number (r = 0.92, p < 0.001), with each copy accounting for 2.4-4.0% of the IgD-bearing B cells. We conclude that 51p1-related genes are expressed by a relatively large percentage of IgD+ tonsillar B cells and this percentage is proportional to the germline copy number of 51p1-related genes. PMID- 8621798 TI - Human fatty acid synthesis is stimulated by a eucaloric low fat, high carbohydrate diet. AB - A new experimental approach was used to determine whether a eucaloric, low fat, high carbohydrate diet increases fatty acid synthesis. Normally volunteers consumed low fat liquid formula diets (10% of calories as fat and 75% as glucose polymers, n = 7) or high fat diets (40% of calories as fat and 45% as glucose polymers, n = 3) for 25 d. The fatty acid composition of each diet was matched to the composition of each subject's adipose tissue and compared with the composition of VLDL triglyceride. By day 10, VLDL triglyceride was markedly enriched in palmitate and deficient in linoleate in all subjects on the low fat diet. Newly synthesized fatty acids accounted for 44 +/- 10% of the VLDL triglyceride. Mass isotopomer distribution analysis of palmitate labeled with intravenously infused 13C-acetate confirmed that increased palmitate synthesis was the likely cause for the accumulation of triglyceride palmitate and "dilution" of linoleate. In contrast, there was minimal fatty acid synthesis on the high diet. Thus, the dietary substitution of carbohydrate for fat stimulated fatty acid synthesis and the plasma accumulation of palmitate-enriched, linoleate deficient triglyceride. Such changes could have adverse effects on the cardiovascular system. PMID- 8621800 TI - Cellular bioenergetics after erythropoietin therapy in chronic renal failure. AB - After erythropoietin (rHuEPO) therapy, patients with chronic renal failure (CRF) do not improve peak O2 uptake (VO2 peak) as much as expected from the rise in hemoglobin concentration ([Hb]). In a companion study, we explain this phenomenon by the concurrent effects of fall in muscle blood flow after rHuEPO and abnormal capillary O2 conductance observed in CRF patients. The latter is likely associated with a poor muscle microcirculatory network and capillary-myofiber dissociation due to uremic myopathy. Herein, cellular bioenergetics and its relationships with muscle O2 transport, before and after rHuEPO therapy, were examined in eight CRF patients (27 +/- 7.3 [SD] yr) studied pre- and post-rHuEPO ([Hb] = 7.8 +/- 0.7 vs. 11.7 +/- 0.7 g x dl-1) during an incremental cycling exercise protocol. Eight healthy sedentary subjects (26 +/- 3.1 yr) served as controls. We hypothesize that uremic myopathy provokes a cytosolic dysfunction but mitochondrial oxidative capacity is not abnormal. 31P-nuclear magnetic resonance spectra (31P-MRS) from the vastus medialis were obtained throughout the exercise protocol consisting of periods of 2 min exercise (at 1.67 Hz) at increasing work-loads interspersed by resting periods of 2.5 min. On a different day, after an identical exercise protocol, arterial and femoral venous blood gas data were obtained together with simultaneous measurements of femoral venous blood flow (Qleg) to calculate O2 delivery (QO2leg) and O2 uptake (VO2leg). Baseline resting [phosphocreatine] to [inorganic phosphate] ratio ([PCr]/[Pi]) did not change after rHuEPO (8.9 +/- 1.2 vs. 8.8 +/- 1.2, respectively), but it was significantly lower than in controls (10.9 +/- 1.5) (P = 0.01 each). At a given submaximal or peak VO2leg, no effects of rHuEPO were seen on cellular bioenergetics ([PCr]/[Pi] ratio, %[PCr] consumption halftime of [PCr] recovery after exercise), nor in intracellular pH (pHi). The post-rHuEPO bioenergetic status and pHi, at a given VO2leg, were below those observed in the control group. However, at a given pHi, no differences in 31P-MRS data were detected between post-rHuEPO and controls. After rHuEPO, at peak VO2, Qleg fell 20% (P < 0.04), limiting the change in QO2leg to 17%, a value that did not reach statistical significance. The corresponding O2 extraction ratio decreased from 73 +/- 4% to 68 +/- 8.2% (P < 0.03). These changes indicate that maximal O2 flow from microcirculation to mitochondria did not increase despite the 50% increase in [Hb] and explain how peak VO2leg and cellular bioenergetics (31P-MRS) did not change after rHuEPO. Differences in pHi, possibly due to lactate differences, between post-rHeEPO and controls appear to be a key factor in the abnormal muscle cell bioenergetics during exercise observed in CRF patients. PMID- 8621799 TI - Effects of erythropoietin on muscle O2 transport during exercise in patients with chronic renal failure. AB - Erythropoietin (rHuEPO) has proven to be effective in the treatment of anemia of chronic renal failure (CRF). Despite improving the quality of life, peak oxygen uptake after rHuEPO therapy is not improved as much as the increase in hemoglobin concentration ([Hb)] would predict. We hypothesized that this discrepancy is due to failure of O2 transport rates to rise in a manner proportional to [Hb]. To test this, eight patients with CRF undergoing regular hemodialysis were studied pre- and post-rHuEPO ([Hb] = 7.5 +/- 1.0 vs. 12.5 +/- 1.0 g x dl-1) using a standard incremental cycle exercise protocol. A group of 12 healthy sedentary subjects of similar age and anthropometric characteristics served as controls. Arterial and femoral venous blood gas data were obtained and coupled with simultaneous measurements of femoral venous blood flow (Qleg) by thermodilution to obtain O2 delivery and oxygen uptake (VO2). Despite a 68% increase in [Hb], peak VO2 increased by only 33%. This could be explained largely by reduced peak leg blood flow, limiting the gain in O2 delivery to 37%. At peak VO2, after rHuEPO, O2 supply limitation of maximal VO2 was found to occur, permitting the calculation of a value for muscle O2 conductance from capillary to mitochondria (DO2). While DO2 was slightly improved after rHuEPO, it was only 67% of that of sedentary control subjects. This kept maximal oxygen extraction at only 70%. Two important conclusions can be reached from this study. First, the increase in [Hb] produced by rHuEPO is accompanied by a significant reduction in peak blood flow to exercising muscle, which limits the gain in oxygen transport. Second, even after restoration of [Hb], O2 conductance from the muscle capillary to the mitochondria remains considerably below normal. PMID- 8621801 TI - Quantitative trait locus mapping of human blood pressure to a genetic region at or near the lipoprotein lipase gene locus on chromosome 8p22. AB - Resistance to insulin-mediated glucose disposal is a common finding in patients with non-insulin-dependent diabetes mellitus (NIDDM), as well as in nondiabetic individuals with hypertension. In an effort to identify the generic loci responsible for variations in blood pressure in individuals at increased risk of insulin resistance, we studied the distribution of blood pressure in 48 Taiwanese families with NIDDM and conducted quantitative sib-pair linkage analysis with candidate loci for insulin resistance, lipid metabolism, and blood pressure control. We found no evidence for linkage of the angiotensin converting enzyme locus on chromosome 17, nor the angiotensinogen and renin loci on chromosome 1, with either systolic or diastolic blood pressures. In contrast, we obtained significant evidence for linkage or systolic blood pressure, but not diastolic blood pressure, to a genetic region at or near the lipoprotein lipase (LPL) locus on the short arm of chromosome 8 (P = 0.002, n = 125 sib-pairs, for the haplotype generated from two simple sequence repeat markers within the LPL gene). Further strengthening this linkage observation, two flanking marker loci for LPL locus, D8S261 (9 cM telomeric to LPL locus) and D8S282 (3 cM centromeric to LPL locus), also showed evidence for linkage with systolic blood pressure (P = 0.02 and 0.0002 for D8S261 and D8S282, respectively). Two additional centromeric markers (D8S133, 5 cM from LPL locus, and NEFL, 11 cM from LPL locus) yielded significant P values of 0.01 and 0.001, respectively. Allelic variation around the LPL gene locus accounted for as much as 52-73% of the total interindividual variation in systolic blood pressure levels in this data set. Thus, we have identified a genetic locus at or near the LPL gene locus which contributes to the variation of systolic blood pressure levels in nondiabetic family members at high risk for insulin resistance and NIDDM. PMID- 8621802 TI - Large scale isolation, growth, and function of porcine neonatal islet cells. AB - Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice. PMID- 8621803 TI - Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. AB - T cell cytokines are known to play a major role in determining protection and pathology in infectious disease. It has recently become clear that IL-12 is a key inducer of the type 1 T cell cytokine pattern characterized by production of IFN gamma. Conversely, IL-10 down-regulates IL-12 production and type 1 cytokine responses. We have investigated whether IL-12 and IL-10 might be involved in a chronic inflammatory reaction, atherosclerosis. In atherosclerotic plaques, we found strong expression of IFN-gamma but not IL-4 mRNAs as compared to normal arteries. IL-12 p40 mRNA and IL-12 p70 protein were also found to be abundant in atherosclerotic plaques. IL-12 was induced in monocytes in vitro in response to highly oxidized LDL but not minimally modified LDL. The cross-regulatory role of IL-10 was indicated by the expression of IL-10 in some atherosclerotic lesions, and the demonstration that exogenous rIL-10 inhibited LDL-induced IL-12 release. These data suggest that the balance between IL-12 and IL-10 production contributes to the level of immune-mediated tissue injury in atherosclerotsis. PMID- 8621804 TI - Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. AB - Lymphocytes activate adhesion to intracellular adhesion mlecule 1 (ICAM-1) via leukocyte function associated antigen 1 (LFA-1), their major beta 2 integrin, in response to PMA (phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma membrane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV-transformed B cells before and after PMA activation. Diffusion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equivalent size. PMA caused a 10 fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton. PMID- 8621805 TI - Combined administration of recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor enhances multilineage hematopoietic reconstitution in nonhuman primates after radiation-induced marrow aplasia. AB - This study compared the therapeutic potential of recombinant, native versus pegylated megakaryocyte growth and development factor (rMGDF and PEG-rMGDF, respectively), as well as that of the combined administration of PEG-rMGDF and r methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF) on hematopoietic reconstitution after 700 cGy, 60Co gamma, total body irradiation in nonhuman primates. After total body irradiation, animals received either rMGDF, PEG-rMGDF, r-metHuG-CSF, PEG-rMGDF and r-metHuG-CSF or HSA. Cytokines in all MGDF protocols were administered for 21-23 d. Either rMGDF, PEG-rMGDF, or PEG-rMGDF and r-metHuG-CSF administration significantly diminished the thrombocytopenic duration (platelet count (PLT) < 20,000 per microliter)to o.25, 0, 0.5 d, respectively, and the severity of the PLT nadir (28,000, 43,000, and 30,000 per microliter, respectively) as compared with the controls (12.2 d duration, nadir 4,000 per microliter), and elicited an earlier PLT recovery. Neutrophil regeneration was augmented in all cytokine protocols and combined PEG-rMGDF and r metHuG-CSF further decreased the duration of neutropenia compared with r-metHuG CSF alone. These data demonstrated that the administration of PEG-rMGDF significantly induced bone marrow regeneration versus rMGDF, and when combined with r-metHuG-CSF significantly enhanced multilineage hematopoietic recovery with no evidence of lineage competition. PMID- 8621808 TI - Inter-observer variability of portal hemodynamics measured by Doppler ultrasound on three different locations of portal vein. AB - Doppler ultrasound is a noninvasive modality for portal hemodynamic study. However, inter-observer variability has been observed. This study has investigated ways to produce less inter-observer variability. Doppler ultrasound portal vein hemodynamic studies were carried out by three well-trained specialists on 20 healthy hospital staff members. The intra-hepatic, first branch, right portal vein, the hilar portal vein, and the extra-hepatic portal vein were chosen for study. With respect to the diameter of portal veins, a significant inter-observer variability was found for the first branch right portal vein and the extra-hepatic portal vein, but not for the hilar portal vein. For maximal portal vein velocity studies, inter-observer variability was not found at any location. A significant failure rate was noted for the measurement of extra-hepatic portal vein velocity. Only 8 volunteers had complete data from all of the three investigators. A significant variability was also noted for the average velocity of extra-hepatic portal vein. We conclude that Doppler ultrasound hemodynamic studies of the hilar portal vein has the most acceptable inter-observer variability and thus should be used for longitudinal portal hemodynamic studies. PMID- 8621807 TI - Prenatal prediction of neonatal growth status in twins using individualized growth assessment. AB - OBJECTIVE: To determine if the growth status at birth of twins can be predicted in the third trimester using the Prenatal Growth Assessment Score (PGAS). METHODS: The growth of 40 twin fetuses were studied with ultrasound from 14 weeks until delivery. Measurements of the head circumference (HC), abdominal circumference (AC), thigh circumference (ThC), femur diaphysis length (FDL), head cube (A), and abdominal cube (B) were made at 2 to 3 week intervals. Rossavik growth models for these parameters were determined from second trimester measurements. These models were used to define expected third trimester growth curves and birth characteristics. Comparisons of expected and actual third trimester measurements were used to calculate PGAS values after various time points (PGASAt) and after the last time point (PGASAT). Similar comparisons after birth were used to determine Growth Potential Realization Index (GPRI) values for HC, AC, ThC, weight (WT), and crown-heel length (CHL), with and without correction for decreased soft tissue deposition. These two sets of GPRI values were used to calculate two sets of Neonatal Growth Assessment Scores (NGASS, NGASTw). Using NGASS and NGASTw (as well as GPRI values in some cases), the twin neonates were classified as Normal (N), Decreased Soft Tissue Deposition, (DSTD), Intrauterine Growth Retardation (IUGR) and Macrosomia (M). RESULTS: At birth 22/40 (55%) were classified as N, 9/40 (22.5%) as DSTD, 6/40 (15.0%) as IUGR, and 3/40 (7.5%) as M. All -PGASAT values in the N group were greater than -0.40% with one exception (-PGASAT = -0.43%). All PGASAt values were above this same boundary except for one fetus. No differences were seen between the N and DSTD groups [mean -PGASAT (range): N, -0.12% (0% to -0.34%); DSTD, -0.10% (0% to -0.30%)]. The IUGR group had 4 fetuses with -PGASAT values between -0.65% and 2.79% and two with values of 0.0% and -0.12%. Growth retardation in the latter two was limited to a decrease in thigh soft tissue deposition. -PGASAt values in the first 4 fetuses were below -0.40% 1.6 to 9.5 weeks before delivery (mean: 6.1 weeks). Fetuses in group M had +PGASAT values of 0.0%, +1.8%, and +1.2%. PGASAt values were above +0.40% at 3.6 and 9.8 weeks before delivery in the latter two fetuses. CONCLUSIONS: These results support the concept that PGASAT and PGASAt values outside +/- 0.40% indicate either IUGR or macrosomia. Almost all fetuses with growth problems in the third trimester can be detected, on average, 6 weeks before delivery unless the growth abnormality is limited to decreased soft tissue deposition. PMID- 8621806 TI - Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. AB - The expression of leptin, the ob gene product, is increased in adipose tissue in response to feeding and energy repletion, while leptin decreases food intake. Because adipose tissue gene expression is regulated by cytokines induced during infection and because infection is associated with anorexia, we tested whether induction of leptin might occur during the host response to infection. Administration of endotoxin (LPS), a model for gram negative infections, induces profound anorexia and weight loss in hamsters. In fasted adipose tissue to levels similar to fed control animals. There is a strong inverse correlation between mRNA levels of leptin and subsequent food intake. TNF and IL-1, mediators of the host response to LPS, also induced anorexia and increased levels of leptin in mRNA in adipose tissue. As assessed by immuknoprecipitation and Western blotting, circulating leptin protein is regulated by LPS and cytokines in parallel to regulation of adipose tissue leptin mRNA. Induction of leptin during the host response to infection may contribute to the anorexia of infection. PMID- 8621809 TI - Use of sonography in the evaluation of the gastroesophageal junction. AB - To demonstrate the gastroesophageal junction with a real-time, transabdominal sonography through the window of left lobe of liver, the normal sonographic pattern and the thickness of the wall of abdominal esophagus were determined. The detection rate of the normal pattern in 30 control subjects was 93%, the normal thickness of the wall being 3.8 +/- 1.2 mm (range 2 mm to 5 mm). In 7 patients with severe acute esophageal inflammation, the thickness was 7.6 +/- 2.1 mm (range 5 mm to 10 mm). In 6 patients with an invading lesion in the gastroesophageal junction due to malignancy, the thickness of the wall was more than 10 mm in each case. This preliminary study indicates that the sonographic detection of gastroesophageal junction through the liver window can be included in routine abdominal sonography. An increased thickness of the wall needs further study to find the cause of the thickening. PMID- 8621810 TI - Antenatal sonographic features of 100 alpha-thalassemia hydrops fetalis fetuses. AB - OBJECTIVE: To characterize the sonographic findings of alpha-thalassemia hydrops fetalis. METHODS: Descriptive study of the ultrasound findings in 100 women with antenatal diagnosis of hydrops fetalis due to alpha-thalassemia. RESULTS: Mean (+/- SD) maternal age was 27.5 + 5.7 years (17-41 years). Mean menstrual age was 31.3 +/- 4.7 weeks (22-42 weeks). All fetuses were stillborn or died very shortly after birth. The indications for sonographic examination included early onset of pre-eclampsia, large for date, decreased fetal movement and nonreactive NST, or bradycardia and premature contractions. The sonographic features found in more than 90% of cases included hepatosplenomegaly, cardiac enlargement, edematous placenta, and ascites. Other common findings were oligohydramnios (82%), subcutaneous edema (75%), decreased fetal movement (74%), cord edema (63%), and enlarged umbilical vessel (62%). Pericardial or pleural effusion was seen in only 15% of cases. None of the fetuses showed any morphological abnormality. Early findings (22-28 weeks) included evidence of a thickened placenta and/or minimal ascites and/or slight cardiomegaly. CONCLUSIONS: The sonographic characteristics of alpha-thalassemia hydrops fetalis ranged from no obvious morphological abnormalities to evidence of fluid retention. These typical abnormal findings generally did not appear until the late second trimester or early third trimester. PMID- 8621811 TI - The sensitivity and specificity of vaginal sonography in detecting endometrial abnormalities in women with postmenopausal bleeding. AB - OBJECTIVE: To evaluate the sensitivity and specificity of vaginal sonography in the detection of endometrial abnormalities in patients with postmenopausal bleeding (PMB). METHODS: In a prospective study, 54 patients with PMB were examined with vaginal ultrasonography prior to dilatation and curettage. The mean endometrial thickness was compared with the histopathological results. A cutoff value of 5 mm was prospectively chosen to evaluate the sensitivity and specificity of this method. RESULTS: The calculated sensitivity for the measurement of endometrial thickness as a predictor of endometrial pathology was 89%, and the specificity was 83%. We found carcinoma associated with an endometrial thickness of 6 mm. CONCLUSIONS: Although its sensitivity was high, we feel that an endometrial thickness > 5 mm should constitute a cause for concern but not a definitive indication of pathology. Thus we believe that, at this point, the role of vaginal sonography as an aid in determining which women with PMB should undergo curettage has yet to be determined. PMID- 8621813 TI - Multi-septate gallbladder: an unusual sonographic pattern in acute hepatitis. PMID- 8621812 TI - Chest wall hamartoma diagnosed prenatally using ultrasonography and computed tomography. PMID- 8621814 TI - Female urethra diverticula: value of transrectal sonography. PMID- 8621815 TI - Duplex kidney with an ectopic ureter inserted into the urethra: report of a case. PMID- 8621816 TI - Prenatal sonographic diagnosis of anterior fossa porencephaly. PMID- 8621817 TI - Skin localization of alveolar echinococcosis of the liver. AB - Alveolar echinococcosis is a rare parasitic disease caused by the intrahepatic growth of Echinococcus multilocularis larvae. Secondary localizations can be observed; pulmonary metastases are the most frequent and are observed in 22% of patients. Other extrahepatic localizations are less frequent. We describe two patients with abdominal skin involvement. To our knowledge, this has never before been reported. In both patients, the liver lesion was located in the left lobe, and larvae probably spread to the skin via the falciform ligament. In one patient albendazole therapy was effective. PMID- 8621819 TI - Hypothenar hammer syndrome: an uncommon cause of digital ischemia. AB - Unilateral digital ischemia may occur as a result of repetitive use of the hypothenar eminence as a hammer, with resultant damage to the wall of the ulnar artery and the superficial palmar arch. Secondary thrombus formation from intimal damage and potential embolization may cause signs and symptoms of arterial insufficiency in the second through fifth digits. Three automobile mechanics with unilateral ischemia caused by hypothenar hammer syndrome are described. All three patients had improvement with conservative treatment and continued to work with only minimal disability. PMID- 8621818 TI - Association of scabies with a bullous pemphigoid-like eruption. AB - We describe a patient with scabies who had an associated bullous pemphigoid-like eruption. Previous reports of this association have appeared in the literature. However, most case reports have described negative or nonspecific findings with direct immunofluorescence in contrast to the intense linear homogenous band of IgG and C3 found in our patient. PMID- 8621820 TI - Coexistence of pemphigus vulgaris and ocular cicatricial pemphigoid. AB - We describe the case of a patient with both pemphigus vulgaris limited to the oral cavity and ocular cicatricial pemphigoid. The diagnoses were established by means of histopathologic examination and direct and indirect immunofluorescence studies and confirmed by immunoblot analysis of serum. Treatment with dapsone resulted in a prolonged remission of the ocular cicatricial pemphigoid. The pemphigus vulgaris has remained localized to the oral cavity and has responded to sublesional corticosteroid injections. PMID- 8621821 TI - Mycosis fungoides presenting as an acquired ichthyosis. AB - A 25-year-old patient with an acquired ichthyosis-like condition is described. Histologic and molecular biologic examination of the fine, whitish, scaling skin lesions suggested the diagnosis of mycosis fungoides. Typical lesions of mycosis fungoides, such as patches, plaques, or tumors, were not present. To the best of our knowledge, this is the first case of mycosis fungoides presenting solely with the clinical features of acquired ichthyosis. The sudden appearance of ichthyosiform skin changes in adults must be taken seriously and the underlying cause must be evaluated. A skin biopsy should be performed in all cases of acquired ichthyosis to rule out mycosis fungoides. PMID- 8621822 TI - Vancomycin-induced linear IgA bullous dermatosis (LABD). AB - We report the eleventh case of vancomycin-induced linear IgA disease. Our case is unusual because symptoms developed within minutes of administration of the drug. We discuss the pathogenesis and review the literature. PMID- 8621823 TI - Hyperandrogenism, insulin resistance, and acanthosis nigricans (HAIR-AN) syndrome: spontaneous remission in a 15-year-old girl. AB - Acanthosis nigricans is associated with many diseases, including internal malignancies, genetic disorders, and endocrine abnormalities. Insulin resistance frequently accompanies endocrine-associated acanthosis nigricans. We describe a patient with acanthosis nigricans associated with hyperandrogenism and extreme insulin resistance that spontaneously resolved after a marked decrease in insulin receptor antibodies. PMID- 8621824 TI - Cutaneous cryptococcosis and histoplasmosis coinfection in a patient with AIDS. AB - Patients with AIDS may have multiple infections at one time, and skin lesions resulting from simultaneous infections with more than one organism have been described. We report a case of disseminated cryptococcal and histoplasmosis infections with cutaneous lesions in a patient with AIDS. In addition, we demonstrate the first case of two coexisting fungal infections in a unique skin lesion. The cutaneous presentation of infectious disorders in HIV-infected patients is often nondescript and not diagnosed by clinical observation alone. Biopsies and cultures are essential for making accurate diagnoses in immunocompromised patients with unusual skin lesions. PMID- 8621825 TI - Pyoderma gangrenosum in a patient with cryoglobulinemia and hepatitis C successfully treated with interferon alfa. AB - We describe a patient with long-standing pyoderma gangrenosum unresponsive to therapy. The patient had concomitant cryoglobulinemia and hepatitis C. When the hepatitis C was treated with interferon alfa-2a his pyoderma gangrenosum resolved. Whether this was from the interferon alfa or spontaneous resolution is not known. PMID- 8621826 TI - Subcutaneous T-cell lymphoma with associated hemophagocytic syndrome and terminal leukemic transformation. AB - We report a case of subcutaneous T-cell lymphoma that presented as recurrent subcutaneous nodules, pancytopenia, and fever. Histopathologic examination revealed a dense infiltrate of markedly atypical lymphoid cells localized to the panniculus. These cells were identified as T cells by immunohistochemistry. There was associated karyorrhexis and fat necrosis. Hemophagocytosis was present both in the panniculus and in the bone marrow, with no tumor evident outside the subcutaneous tissue. Despite chemotherapy in conjunction with an autologous bone marrow transplant, the patient died after metastases, including explosive leukemic transformation, developed. Review of the literature shows subcutaneous T cell lymphoma to be a rare peripheral T-cell lymphoma, often mistaken initially as a benign panniculitis, that manifests an aggressive, fulminant presentation in approximately one half of the patients; the remainder transform into a high-grade malignancy after months to years. The hemophagocytic syndrome, though to be a reactive T-cell process mediated by cytokines, is a frequent complication of this lymphoma and is responsible for its poor prognosis. Our patient uniquely demonstrated fatal leukemic transformation. PMID- 8621827 TI - Infectious complications of erythrodermic psoriasis. AB - Severe morbidity and mortality may be associated with erythrodermic psoriasis, especially when complicated by septicemia. We describe five patients with erythrodermic psoriasis complicated by staphylococcal septicemia. In two, concurrent infection with HIV increased vulnerability to bacteremia. PMID- 8621828 TI - Atypical scabies in HIV-positive patients. AB - Five cases of Norwegian or keratotic scabies in HIV-positive patients are described. One of these patients was the source of an outbreak in a hospital, ultimately involving 72 persons. Three of our patients had a markedly pruritic eruption. This is unusual in crusted scabies in which pruritus is usually slight or absent. Two of the five patients had unusual CD4 counts of more than 200 cells per cubic millimeter. All our patients responded to lindane and keratolytic agents. When generalized papular, crusted, or eczematoid lesions are observed in HIV-positive patients, particularly if the CD4 count is less than 200/mm3, scabies should be included in the differential diagnosis. PMID- 8621829 TI - Trimethoprim-sulfamethoxazole-associated acute febrile neutrophilic dermatosis: case report and review of drug-induced Sweet's syndrome. AB - Sweet's syndrome, or acute febrile neutrophilic dermatosis, is characterized by fever, neutrophilia, and painful erythematous cutaneous plaques that contain a dense neutrophilic dermal infiltrate. Although the disorder is usually idiopathic, patients with drug-induced Sweet's syndrome have been described. We describe a 50-year-old woman with trimethoprim-sulfamethoxazole (TMP-SMX)- induced Sweet's syndrome and review the features of the 13 previously reported patients with drug-induced Sweet's syndrome. All patients had fever, painful skin lesions (most commonly on the upper extremities), and a biopsy-confirmed neutrophilic dermatosis. All patients also exhibited a temporal relationship between drug administration and clinical presentation and between drug withdrawal and healing. In patients with drug-induced Sweet's syndrome, neutrophilia is often absent. PMID- 8621830 TI - Congenital erythropoietic porphyria: clinical, biochemical, and enzymatic profile of a severely affected infant. AB - Blistering of light-exposed skin, pink-stained fluorescing diapers, and fluorescing peripheral erythrocytes led to diagnosis of congenital porphyria in an infant born to consanguineous parents. Although massive coproporphyrinuria and coproporphyrinemia initially suggested a coproporphyrinogen oxidase deficiency disorder, excess porphyrins were chiefly of the isomer I series, implicating a uroporphyrinogen III synthase defect. Congenital erythropoietic porphyria was confirmed by demonstration of a profound defect in the activity of the infant's uroporphyrinogen III synthase (4% of the mean value for nine normal controls) and in both parents at approximately 50% of the mean normal activity. Coinheritance of gene defects for either hereditary coproporphyria or erythropoietic protoporphyria in addition to those for congenital erythropoietic porphyria was excluded by demonstrating normal activities of both coproporphyrinogen oxidase and ferrochelatase in the infant. The complicated perinatal and postnatal clinical course and biochemical and enzyme assay data for the infant and his parents are described. PMID- 8621831 TI - Multiple periorbital cutaneous myxomas progressing to scleromyxedema. AB - Myxomas are rare cutaneous tumors that have been associated with a variety of other abnormalities including atrial myxomas, endocrine abnormalities, and bone malformations. We describe a 38-year-old white man with multiple periorbital myxomas in whom myalgias, fatigue, and more diffuse cutaneous involvement developed. These findings were consistent with scleromyxedema. He also had an associated left subclavian deep venous thrombosis. The patient responded well to therapy with cyclophosphamide. PMID- 8621832 TI - Induction of Darier-White disease with UVB radiation in a clinically photo insensitive patient. AB - Combination UVA/UVB radiation and UVB radiation alone have been shown to induce the lesions of Darier-White disease. However, 6% of patients with Darier-White disease claim that sunlight ameliorates their condition. We performed an unblinded, side-by-side controlled trial of UVB, UVA, and combination UVB/UVA phototherapy in a patient with historically photoameliorated Darier-White disease to determine whether phototherapy was beneficial, to determine whether phototherapy-related heat was detrimental, and to confirm, with appropriate controls, the action spectrum of the disease. Phototherapy with radiation in the UVB but not UVA spectrum evoked Darier-White disease in this patient, both clinically and histologically. UVB radiation was capable of inducing Darier-White disease in vivo in spite of a history of photoamelioration, whereas UVA radiation alone and the heat associated with phototherapy in our protocol had no effect on the disease. PMID- 8621833 TI - Spiny keratoderma in association with autosomal dominant polycystic kidney disease with liver cysts. AB - Spiny keratoderma of the palms and soles has been rarely reported. Debate exists regarding the proper nosologic classification of this disorder. We describe a patient and her mother with concurrent autosomal dominant polycystic kidney disease with liver cysts. PMID- 8621834 TI - Different treatment modalities for the management of a patient with the nevoid basal cell carcinoma syndrome. AB - Nevoid basal cell carcinoma (BCC) syndrome is a genetically linked disorder characterized by multiple BCCs associated with various skeletal abnormalities and sometimes with mental retardation. Because of the large number of lesions, treatment of BCCs in these patients may be extremely difficult. The value of different therapeutic options was assessed in a patient with multiple, disfiguring nevoid BCC syndrome. Surgical excision and split-skin grafting was used to remove three larger tumors. Photodynamic therapy led to healing of flat lesions; small papules within the treated areas, however, did not respond to this type of management. Three nodular BCCs treated with intralesional application of interferon alfa-2b were markedly reduced in size. Still, complete healing could not be achieved. Nodular lesions vaporized with the CO2 laser disappeared and showed no recurrence after 2 years of follow up. Our experience indicates that CO2 laser vaporization of BCCs allows the treatment of a large number of lesions in a single session, and is indicated when surgical treatment is not feasible for all lesions. Photodynamic therapy with 5-amino-levulinic acid may be a valid therapeutic option for flat lesions only. Intralesional application of interferon alfa-2b removes papular lesions of small size. PMID- 8621835 TI - Antiepiligrin cicatricial pemphigoid: the first case report from Japan. AB - We describe a Japanese man with antiepiligrin cicatricial pemphigoid and typical clinical features, including ocular involvement. Direct immunofluorescence showed IgG deposition at the basement membrane zone. Indirect immunofluorescence of 1M sodium chloride-split skin showed circulating antibasement membrane zone antibodies of IgG class reactive with the dermal side of the split. Immunoblotting of human epidermal and dermal extracts, as well as a bacterial fusion protein of BP180 NC16a domain, showed no specific reactivity. In contrast, with immunoprecipitation of either culture medium or cell lysate from normal keratinocytes, the patient's serum clearly reacted with the protein epiligrin, a laminin isoform present in the lamina lucida of the human epidermal basement membrane zone. This is the first confirmed case of a Japanese patient with this disease entity. PMID- 8621836 TI - Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle, Tenebrio molitor: purification, kinetic properties and localization of the enzyme. AB - Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6-5.8. The Km for trehalose was 4.4 mmol.1(-1). Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore. Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female. PMID- 8621837 TI - Identification of four ovarian receptor proteins that bind vitellogenin but not other homologous plasma lipoproteins in the rainbow trout, Oncorhynchus mykiss. AB - Membrane proteins from ovarian follicles, testis and somatic tissues of rainbow trout, Oncorhynchus mykiss, were extracted by ultracentrifugation, separated on sodium dodecyl sulphate gels and isolated on polyvinyl difluoride membranes. Vitellogenin receptor proteins were visualized using protein staining and hybridisation with 125I-vitellogenin. Four follicle-membrane proteins, with molecular masses of 220, 210, 110 and 100 kDa, showed a strong affinity for vitellogenin and were specific to the ovary. Other homologous lipoproteins (very low density lipoprotein, low density lipoprotein and high density lipoprotein) had a very limited ability to displace 125I-vitellogenin from its receptor, indicating that the ovarian receptor proteins were fairly specific for vitellogenin. Proteins with an affinity for very low density lipoprotein and low density lipoprotein were visualised in liver, spleen and muscle, eluting on sodium dodecyl sulphate gels with molecular masses of about 150 kDa. Peptides generated from trypsin digests of the receptor proteins with a high affinity for vitellogenin showed sequence homology with receptors in the lipoprotein family, including a sequence that is believed to act as the internalisation signal [Phe Asp-Phe-Tyr-] and a sequence identity with the recently characterised chicken vitellogenin/very low density lipoprotein receptor [Ser-Glu-Leu-Tyr-Glu-Pro-Ala ]. Together, the ligand blotting and peptide sequence data support the contention that the four ovarian membrane proteins isolated are receptor proteins specific for vitellogenin and they do not bind other plasma lipoproteins to any significant degree. PMID- 8621838 TI - Age and segmental differences in 5-hydroxytryptamine-induced hypersecretion in the pig small intestine. AB - 5-Hydroxytryptamine is a mediator in cholera toxin-induced hypersecretion in the small intestine. Our hypothesis is that the hypersecretion induced by 5 hydroxytryptamine in the small intestine decreases with increasing age and in an aboral direction in the small intestine. In vivo, measuring accumulated fluid in ligated loops, the apparent maximal efficacy of the 5-hydroxytryptamine-induced jejunal secretion in pig neonates was 4.8 +/- 1.1 mg x mg(-1) dry loop x 45 min( 1). The apparent maximal efficacy decreased by 23% and 63% in young and adult pigs, respectively, compared with neonates. In vitro, measuring changes in short circuit current in Ussing chambers, the apparent maximal efficacy was 66.7 +/- 4.8 micronA x cm(-2) in neonates and was reduced by 30% and 57% in young and adult pigs, respectively. Young pigs were used in the segmental study. The apparent maximal efficacy in vivo was 3.7 +/- 0.5 mg x mg(-1) dry loop and decreased by 22% and 56% in the mid and distal small intestine, respectively. By contrast, in vitro the apparent maximal efficacy was elevated by 56% to 72.0 +/- 5.0 micronA x cm(-2) in the distal compared with the proximal part. In conclusion, the secretory response to 5-hydroxytryptamine in pig small intestine decreases with increasing age and in the aboral direction according to in vivo results. We suggest that the decrease in sensitivity to 5-hydroxytryptamine can explain a part of the reduced secretory response to cholera toxin with age and in the aboral direction of the small intestine. PMID- 8621841 TI - Allergen nomenclature. PMID- 8621840 TI - Modified adipokinetic peptides containing two tryptophan residues and their activities in vitro and in vivo in Locusta. AB - Locusta migratoria has three adipokinetic hormones, adipokinetic hormone-I, II and III. Adipokinetic hormone-III ( or = 5.33 x 10(-10)mol x 1(-1)) at inhibiting acetate uptake into locust fat body in vitro, especially so when it is only moderately potent in mobilizing lipid in vivo. The Trp7 in adipokinetic hormones-III, alongside the Trp8 characteristic of adipokinetic hormones, is not seen in any other adipokinetic hormones. To test whether this is hormone-III in the assay in vitro, novel peptides were synthesised to include or remove this structural motif. Thus sweetened flaked corn cereal > sucrose (P < .001). Glucose at 3 hours was greater than sucrose (P < .001). There were no significant differences for free insulin, triglycerides, or free fatty acids. APPLICATIONS: Equivalent gram amounts of carbohydrate as presweetened breakfast cereals are not detrimental to persons with IDDM compared with unsweetened cereals. Therefore, presweetened cereals can be used in the correct portion sizes and based on the number of carbohydrate or starch servings in a person's diabetic meal plan. PMID- 8621872 TI - Salt taste perceptions and preferences are unrelated to sodium consumption in healthy older adults. AB - OBJECTIVE: Age-related deficits in salt taste perception are said to increase preferences for salty foods, thereby leading potentially to greater sodium consumption. This study examined the link between salt taste perceptions and preferences and sodium intakes as a function of age and gender. DESIGN: We studied 24 young adults (aged 20 to 30 years) and 24 healthy older adults (aged 60 to 75 years). The subjects tasted and rated five sodium chloride solutions and eight samples of salted chicken broth containing from 0.04 to 0.64 mol/L sodium. Food intakes were assessed using a 24-hour food recall and 14 consecutive days of diet records. RESULTS: Older and younger subjects did not differ in their sensory evaluations of chicken broth, including ratings of the intensity of saltiness. Older subjects preferred less salty soups than did young adults. Hedonic response profiles for salt in soup were not related to daily sodium intakes as assessed by diet records. APPLICATIONS: Salt taste perceptions and preferences were unrelated to sodium intakes in young adults and in older respondents. Factors other than taste may influence dietary sodium consumption. PMID- 8621873 TI - Effect of participation in congregate-site meal programs on nutritional status of the healthy elderly. AB - OBJECTIVE: This study was designed to evaluate whether participation in a congregate-site meal program influenced the nutritional status of a group of healthy elderly. DESIGN: Nutritional status, as defined by dietary intake and biochemical indexes, was assessed in free-living persons (aged 60 to 89 years) who either did (n = 70) or did not (n = 65) participate in the meal program. Three-day mean intakes of 17 nutrients and serum levels of 13 indexes of nutritional status were measured. STATISTICAL ANALYSES: Multifactorial analysis of variance was used to determine differences in nutrient intake data and biochemical indexes between the groups. By means of correlation analysis, relationships between income and main outcome measures were examined. chi 2 Analysis was used to determine differences in response to categorical variables of the questionnaire. RESULTS: In general, dietary intakes of participants did not differ significantly from those of nonparticipants, nor did the meal provided at the site significantly affect the overall dietary intake of participants. Mean biochemical indexes of nutritional status were within normal ranges for participants and nonparticipants, except for iron. However, 26% of the population consumed diets that may place them at risk for nutritional inadequacy. CONCLUSIONS: Mean dietary intake data and biochemical indexes of nutritional status suggest that the congregate-site meal program did not significantly affect the nutritional status of the population surveyed. Additional studies focusing on the nutritional intake and status of low-income, ethnic minority, and socially isolated participants in the congregate-site meal program are needed to assess which populations are at risk for nutritional deficiencies. PMID- 8621874 TI - Enhanced enteral and parenteral nutrition practice and outcomes in an intensive care unit with a hospital-wide performance improvement process. AB - To improve patient outcomes at a 455-bed community health care facility, a performance improvement process was implemented for the delivery of enteral and parenteral nutrition in a 28-bed intensive care unit (ICU). In 1992, the study group consisted of all patients who were started on either enteral or parenteral nutrition while in the ICU during a 2-month period. These patients were followed up until discharge from the hospital or death to determine practice patterns and outcomes. Three actions were identified as opportunities to change practice and improve outcomes: increase use of the enteral route of alimentation compared with the parenteral route; start alimentation sooner, especially via the enteral route; and meet protein and energy needs of patients. Educational programs were developed targeting physician and nursing staff. Through an interdisciplinary approach, a nutrition support decision tree and patient outcome statement were developed. In 1994, evaluation of a group meeting the same criteria as the original group indicated that the goals for nutrition support practice improvement were met in all three areas identified. Providing a systematic approach to an interdisciplinary performance improvement process, as part of an organization-wide plan, enhanced nutrition support practice in a community hospital and resulted in quality improvement and cost savings. PMID- 8621875 TI - Vitamin retention in cook/chill and cook/hot-hold hospital food-services. AB - The vitamins with the greatest losses during hot-holding of food (> 10% after 2 hours) are vitamin C, folate, and vitamin B-6; retinol, thiamin, riboflavin, and niacin appear to be relatively stable. The 66 studies reviewed in this article give inadequate information on the losses of many other vitamins. In cook/chill food-services, substantial losses of sensitive vitamins occur during each of the chilling, storage, and reheating stages. Different reheating methods have similar effects on the amount of vitamin retention. Losses of vitamin C and folate can be greater than 30% when food is reheated after storage for 24 hours at 3 degrees C. Current research indicates that under normal operating conditions, with hot holding limited to less than 90 minutes, vitamin retention is better in a conventional food-service than in a cook/chill system. PMID- 8621876 TI - Focus groups identify desirable features of nutrition programs for low-income mothers of preschool children. PMID- 8621877 TI - Role of the dietitian in hospital wellness centers in a southern region of the United States. PMID- 8621878 TI - Designing a client-administered food frequency questionnaire. PMID- 8621879 TI - Increased iron content of some Indian foods due to cookware. PMID- 8621880 TI - Fellow of the American Dietetic Association credentialing program: development and implementation of a portfolio-based assessment. AB - This report has described the measurement and operational procedures used for the design, development, and implementation of the newly inaugurated Fellow of The American Dietetic Association credentialing program. The program was established to identify and certify registered dietitians who possess the characteristics of advanced-level practice. The main features of the Fellow program are as follows. It is based on the results of an empirical study that identified the characteristics of advanced-level practice. It is guided by the Fellow Assessment Plan, which specifies the content and level of dietetics practice involved and the psychometric and operational requirements for sound measurement. It is a performance-based portfolio assessment of six professional characteristics (education, work experience, professional achievement, professional roles, professional contacts, and approach to practice). It uses objective automated computer scoring and independent peer-review grading of candidate materials in relation to an explicit scoring rubric. It requires statistical analysis of the database of candidate scores, which is conducted to ensure that the scoring of candidate materials is accurate and reliable and that the assessment, as a measurement instrument, is psychometrically sound. It involves a standard-setting workshop in which a panel of subject-matter experts establish the lowest level of acceptable candidate performance on Approach to Practice; the passing standard for the other five characteristics was preestablished by the empirical study. A careful review of all evidence on the development and implementation of the 1994 Fellow assessment suggests that these procedures are technically sound and produce psychometrically defensible results. Our evaluation and commentary from CDR and CTB staff, subject-matter expert participants, and the candidates themselves suggested a number of ways to improve the Fellow program in subsequent administrations of the assessment. Changes have been implemented and CDR will continue to consider all data in its ongoing improvement efforts. PMID- 8621881 TI - Investigations into the rheological characteristics of bovine amniotic fluid. AB - The rheological characteristics of bovine amniotic fluid have been studied at different shear rates. The viscosity of bovine amniotic fluid at 20 degrees C was found to increase with time at a constant low shear rate during the measurement. Additionally, the viscosity was observed to decrease with increasing shear rate, indicating that a shear thinning behaviour of the fluid was occurring. The log log plot of shear stress versus shear rate yielded a straight line consistent with non-Newtonian behaviour of the fluid and characteristic of pseudoplastic liquids. The data of shear stress versus shear rate could be represented by a power law model. The treatment of amniotic fluid with cetylpyridinium chloride (CPC) resulted in the precipitation of a mixture of components, including complex sulphated polysaccharides and extracellular proteoglycans, with the viscosity of the resulting liquid similar to that of water at 20 degrees C. In addition, the viscosity of the CPC-pretreated fluid did not increase with time at constant shear rate and remained constant with the increase in shear rate. The apparent increase in viscosity with time and the shear thinning behaviour of the amniotic fluid can thus be attributed to pseudoplastic liquid behaviour associated with the presence of structurally complex polysaccharides and extracellular proteoglycans. The implications of this fluid viscosity behaviour are discussed in terms of their impact on the operation of packed or expanded (fluidized) chromatographic bed systems when amniotic fluid biofeedstocks are used as a source of commercially important proteins. PMID- 8621882 TI - Electrical resistance measurements on cerebral capillary endothelial cells--a new technique to study small surface areas. AB - We present a new method which allows resistance measurements in selected cell monolayer areas with a size of less than one mm2. Up to now cell-covered macroscopic filters with areas up to 5 cm2 have been used giving reliable results only in the absence of inhomogeneities or contaminating cells. The new measuring device is posed on the microscope optic to allow an optical characterization of the measuring area and enables us to scan the cell-covered filter. This method has been applied to determine a reversible modulation of the tightness of intercellular contacts between epithelial or endothelial cells in culture. Tight junction resistance is modulated by Ca2+ and basic amino acids in cultured porcine cerebral microvascular endothelial cells that represent the blood-brain barrier. PMID- 8621883 TI - Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat. AB - To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5 dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5 tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level. PMID- 8621884 TI - An enzymatic method to determine receptor-mediated endocytosis. AB - Many studies have measured receptor-mediated endocytosis using radiolabeled ligands or antibodies. Upon ligation and cross-linking, the labeled ligand or antibody is endocytosed and the internalization of the radioisotope is assayed after stripping the uninternalized ligand from the cell membrane. This study reports on an enzymatic assay to measure receptor-mediated endocytosis and compares it with the radioactive method. The results show that receptor-mediated endocytosis measured using the peroxidase conjugated antibody is two fold higher than that measured with a radiolabeled antibody. Thus, approximately 38% endocytosis of CD3 is measured using an 125I-labeled antibody, whereas approximately 79% endocytosis is detected by peroxidase conjugated antibody method. Similar increases are also found with CD2 receptor-mediated endocytosis. Our study has demonstrated that the enzymatic method could be employed in determining receptor-mediated endocytosis. In addition to increased sensitivity, the enzymatic assay eliminates the use of radioactive materials. PMID- 8621885 TI - Model components of luminol chemiluminescence generated by PMNL. AB - The production of activated oxygen species (AOS) by neutrophils (PMNL) is thought to play a key role in the host defence against invading microorganisms. However, the oxygen metabolites are toxic not only to the invading bacteria but also to the surrounding tissue. The oxidative metabolites production can be evaluated by means of chemiluminescent methods. In this study, the possibility of a new analytical approach for quantitative assessment of chemiluminescent kinetics (AOS generation) of isolated PMNL was estimated. Based on the assumption that the kinetics of luminol-amplified chemiluminescence (LCL) of stimulated PMNL possesses a time-probabilistic nature, this kinetics was described with three components. These components, obtained from different investigated systems, were analyzed and a conclusion was made that the first and the second component represent the processes resulting in extra-and intracellular myeloperoxidase (MPO)-dependent light emission (AOS generation), respectively. The second component was found to be completely dependent on the stimulus ingestion. The third component was not completely MPO-dependent and complicated for interpretation. This component was weakly dependent on the stimulus ingestion, and presents at least some intracellular processes different from those presented by the second component. A conclusion is made that the examined approach for analysis of LCL kinetics allows an assessment of extra-and intracellularly generated quantities of AOS by stimulated PMNL. The assessment could be done for emitting systems in which no additional modifications are used. PMID- 8621886 TI - Determination of lipid ester ozonides and core aldehydes by high-performance liquid chromatography with on-line mass spectrometry. AB - Unsaturated triacylglycerols (TG) and choline (PC) and ethanolamine (PE) phosphatides of known structure were subjected to ozonization and reduction with triphenylphosphine to yield the corresponding lipid ester core aldehydes. Mono- and di-C9 aldehyde palmitoylglycerols were prepared from oleoyldipalmitoyl and oleoyllinoleoylpalmitoyl glycerols, respectively, while egg yolk PC and PE provided the mono-C5 and mono-C9 aldehydes of palmitoyl-and stearoyl glycerophospholipids. The aldehydes were isolated in the free form and as the dinitrophenylhydrazone (DNPH) derivatives by thin-layer chromatography (TLC). The intermediate ozonides, free aldehydes and hydrazones were identified by reversed phase high performance liquid chromatography (HPLC) with on-line negative ion thermospray and normal phase HPLC with on-line positive ion electrospray mass spectrometry (LC-MS). The synthetic aldehydes were used as carriers during isolation from natural sources and as reference compounds in quantitative analyses. PMID- 8621888 TI - A new luminometer for sensitive quantification by chemiluminescence of specific proteins in microtitre plates and on blot membranes. AB - Generally applicable technologies are described, which depend on the use of chemiluminescence and a new type of a versatile luminometer, that can measure also weak light in microtitre plates (microplates) and on blotting membranes that is especially useful for dot blot immunoassay. Applications are described using alkaline phosphatase conjugated antibodies against IgG and IgE and the reagent Lumi-Phos 530. This chemiluminescence offers advantages over the use of radioactive isotopes, densitometry and light reflection measurement on membranes and also ELISA, for sensitive quantification of e.g. specific proteins. Special procedures are described for the first time that with the mentioned reagent in agarose gel allows specific and very sensitive quantification of proteins on dot blot membranes. The luminometer, which has temperature control, is very sensitive, precise and allows efficient protocols for various assays. It thus fulfils many of the requirements for good quantification, is time-saving and in addition brings significant improvements due to very low detection limits and large linear concentration ranges that can be measured with excellent regression coefficients (r2 often about 0.999). PMID- 8621887 TI - Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label. AB - Electron paramagnetic resonance was used to characterize the first use of a thio specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl 2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions. PMID- 8621889 TI - Novel vaccine approaches. AB - Recent advances in immunology, molecular biology, and peptide biochemistry have allowed the construction of subunit vaccines based on viral or bacterial recombinants, peptides or plasmid vectors. Although none of these approaches is currently being used for mass vaccination (with the exception or vaccinia-rabies G protein recombinant virus for wildlife immunization); several of them are undergoing clinical trials. None of these different vaccine constructs is likely to be totally effective in either the prevention of infectious diseases or immunotherapy of cancer. Recombinant viral vaccines such as those based on vaccinia or adenovirus as a rule induce potent immune responses. Vaccinia viruses have the added advantage of being heat stable and immunogenic after oral application, making them good candidates for wildlife immunization. Recombinants based on replication-defective adenoviruses are safer compared with vaccinia virus recombinants and, as far as our data indicate, have superior efficacy. In addition, they induce excellent immunity upon application to mucosal membranes, suggesting their usefulness as vaccines for infectious agents that enter through the airways or the genital tract. Peptides are of limited benefit in infectious disease prevention but might provide custom-made vaccines for cancer therapy. Genetic vaccines that were first described less than 5 years ago have already progressed to phase I clinical trials in healthy human adults. Provided that their safety can be confirmed, they might be suited to induce immunity to numerous agents. PMID- 8621890 TI - "The good, the bad, and the ugly." The role of chemokines in models of human disease. PMID- 8621891 TI - Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts. AB - Long term in vitro culture of clonally expanded CD8+T cells, generally found within the CD57+ or CD28-subset, has generally been unsuccessful, suggesting that these cells may have a limited replicative potential. Telomeric shortening may reflect the action of a "mitotic clock" regulating the number of divisions a cell can undergo. In this study, we have compared the telomeric lengths of CD28-CD8+ and CD28+CD8+ T cells in 10 normal individuals to assess their replicative history. Overall, the telomeric lengths were found to be significantly shorter in the CD28-CD8+ T cell subset compared with the CD28+CD8+ subset. Furthermore, clonally expanded TCRBV11+CD8+ T cells from an individual exhibited telomeric lengths that were 2.9 kb shorter than those found in the polyclonal CD28+CD8+ T cell subset. These findings indicate that clonally expanded CD28-CD8+ T cells have undergone many more rounds of replication than CD28+CD8+ T cells, and consistent with the loss of CD28 expression, they may have reached a state of replicative senescence. PMID- 8621892 TI - Different patterns of TCR transgene expression in single-positive and double negative T cells. Evidence for separate pathways of T cell maturation. AB - Two lines of transgenic mice were established using the TCR alpha (V alpha 4.4-J alpha 24)- and beta (V beta 9-D beta 1.1-J beta 2.1)-chain genes from a cloned CD4-CD8-alpha beta + (double-negative; DN) T cell line from BALB/c mice. The TCR genes were expressed in CD4+CD8- and CD4-CD8+ (single-positive; SP) and double positive (DP) T cells in the thymus, and in SP T cells in the peripheral lymphoid tissues, and marrow in one transgenic mouse line, and predominantly in DN T cells in the other. Bone marrow precursor cells from only the DN mouse line generated T cells expressing the V beta 9 transgene during tissue culture. V beta 9+ T cells were found in DN but not SP transgenic mice backcrossed to BALB/c nu/nu mice. The results suggest two separate pathways of T cell maturation, one which generates SP T cells in the thymus, and another which generates DN T cells in both the thymus and bone marrow. PMID- 8621893 TI - T cell suppression in transplantation tolerance through linked recognition. AB - Allogeneic tissues transplanted to mice treated with CD4- and CD8-specific Abs are often accepted indefinitely due to the induction of immunologic tolerance. When transplantation tolerance was induced to grafts mismatched at multiple minor histocompatibility loci, Ag specificity was inferred because third party grafts, mismatched at the MHC, were rejected normally. However, some "third party" grafts were either accepted, or rejected more slowly. Tolerant mice possess CD4+ cells, which suppress rejection by T cells reacting to the same grafts. Therefore, we hypothesized that tolerated third party grafts might share Ags with the original tolerizing graft, and that these Ags are a target for such suppression. To test this idea, we tolerized mice to a set of minor Ags (B10 minors) and challenged them with third party grafts that carried those minors, as well as an additional strong transplantation Ag, the class I MHC molecule, H-2Kb. This class I molecule acts as a good target for rejection in both naive mice and in mice tolerized to B10 minors. However, when this third party class I molecule is provided "linked" to those B10 minors on an F1 graft, rejection was significantly impaired. The data suggest that suppression within tolerant animals operates locally (perhaps on the same APC) via linked recognition. In addition, our preliminary findings suggest that suppression via linked recognition can also lead to tolerance to the third party Ag. PMID- 8621894 TI - Staphylococcal enterotoxin D functions as a human B cell superantigen by rescuing VH4-expressing B cells from apoptosis. AB - Staphylococcal enterotoxins are potent superantigens, in that they activate T cells bearing specific V beta-chain gene segments. In this study, we analyzed the capacity of staphylococcal enterotoxin D (SED) to function as a B cell superantigen. SED induced T cell-dependent polyclonal proliferation and differentiation of B cells. In the absence of T cells, SED induced survival of B cells uniquely expressing VH4 containing IgM. The mechanism of survival of VH4 expressing B cells appeared to relate to the countering of apoptosis initiated by the engagement of HLA-DR by SED. Analysis of the VH4 gene products expressed by SED-stimulated B cells revealed the usage of six of the known functional VH4 genes with a variety of different CDR3 regions, employing different DH and JH gene segments. Moreover, the sequence analysis identified a possible site for SED binding of VH4 that includes the solvent-exposed surfaces of 3' CDR2/FR3 and/or FR1. Thus, SED appears to function as a unique B cell superantigen by inducing survival of VH4-expressing B cells. PMID- 8621895 TI - Cytokine production by T lymphocytes from young and aged mice. AB - We previously have shown that T cell proliferation in response to a primary signal through the CD3 epsilon chain and a costimulatory signal via the CD28 molecule is impaired in healthy, aged mice. To determine whether age-related alterations in cytokine production might explain the reduced proliferative responses of T cells from aged mice, we examined the secretion of the major T cell immunoregulatory cytokines, IFN-gamma, IL-4, and IL-2. Splenic T cells from young (2 to 4 mo) and aged (20 to 26 mo) mice were studied. T cells were stimulated with immobilized anti-CD3 epsilon chain mAb and soluble anti-CD28 mAb for 24 h. T cells from aged mice, when compared with young controls, showed increased IFN-gamma production, no difference in IL-4 production, and decreased IL-2 production. Most IFN-gamma was produced by CD8+ T cells, whereas most IL-2 and IL-4 was produced by CD4+ T cells. Both CD4+ and CD8+ T cells from aged mice produced significantly more IFN-gamma than corresponding cells from young mice. This increased production could be accounted for by increased numbers of CD4+CD44high and CD8+CD44high T cells in aged animals. CD4+CD44high and CD8+CD44high T cells from young mice produced comparable amounts of IFN-gamma as corresponding cells from aged mice. In contrast to unseparated splenic T cells, no age-related difference in IL-2 or IL-4 production by purified CD4+ T cells was observed. Similarly, when CD4+ T cells were further separated into CD44low and CD44high subpopulations, no age-related difference in IL-2 production was found. Therefore, we found no consistent evidence that diminished production of the major T cell growth factors, IL-2 and IL-4, is responsible for the age-related decrease in the proliferation of T cell subpopulations that were stimulated in vitro through the CD3 epsilon chain and costimulated via the CD28 molecule. The physiologic relevance of increased IFN-gamma production by T cells from aged mice is unknown. PMID- 8621896 TI - Aberrant regulation of cytokines in HIV-1 TAT72-transgenic mice. AB - The trans-activator of transcription or TAT gene from HIV-1 encodes a protein that increases the processivity of transcription from the HIV-1 genome. TAT protein can also affect cellular processes in the absence of its ribonucleic HIV target sequence trans activation response element and may be responsible for some aspects of HIV pathogenesis apart from infectious virus or other viral gene products. We have previously shown that TAT72 decreases CTL activity in TAT72 transgenic mice, and we now demonstrate aberrant regulation of mitogen-elicited IL-2 at both transcriptional and translational levels. In contrast, alloantigen stimulation resulted in increased IL-6 and IL-10 production in the TAT72 transgenic mice. Con A-stimulated cultures of splenic lymphocytes from TAT72 transgenic mice do not undergo clonal proliferation of CD4+ cells as compared with CD8+ cells monitored over 72 h. These results suggest that TAT is sufficient to induce some pathology associated with AIDS and is a potent immunologic manipulator apart from its function as trans-activator. PMID- 8621897 TI - Regulation of the Fas lytic pathway in cloned CTL. AB - Cloned murine CTL activated via the TCR or by PMA and ionomycin up-regulate surface Fas ligand and show an increased ability to kill non-Ag-specific Fas+ target cells. This up-regulation starts after 45 to 60 min and has a t1/2 for reversal of about 90 min. Up-regulation of lytic function is accompanied by up regulation of Fas ligand on the CTL surface, which can be blocked by protein synthesis inhibitors. When up-regulation of Fas lytic function was induced by PMA and ionomycin, EGTA blocked both activation of lytic function and expression of Fas ligand detected by FACS analysis. However, when up-regulation was induced by specific Ag, EGTA blocked activation of lytic function, but not up-regulation of Fas ligand. Moreover, EL-4 cells have very high levels of surface Fas ligand, although they are not cytotoxic. Thus, expression of surface Fas ligand may be required, but not sufficient, for Fas-mediated lysis. PMID- 8621898 TI - Specificity and degeneracy of minor histocompatibility antigen-specific MHC restricted CTL. AB - Random peptide libraries were employed to investigate the specificity of Ag recognition by H-3-specific, H-2K(b)-restricted CTL clones. The peptide libraries consist of octapeptides with one defined sequence position and mixtures of 19 amino acids (all proteinogenic amino acids except for cysteine) in the remaining seven sequence positions. The complete set of 152 peptide libraries includes all octapeptides possible with these amino acids. Responses of the CTL clones to these peptide libraries reveal patterns of preferred epitope amino acids. Depending on the CTL clone tested, varying numbers of different amino acids were identified for the different sequence positions indicating degeneracy of Ag recognition. Sequences for synthetic epitopes active at low pM concentrations could be deduced from these patterns. They confirm that TCRs of CTL clones do not exhibit specificity for unique ligand structures but rather can interact with sets of ligands. The sequences of peptides recognized by a single clone exhibit great sequence heterogeneity. PMID- 8621899 TI - Intercellular adhesion molecule-1 is necessary but not sufficient to activate CD4+ T cells. Discovery of a novel costimulator on kidney tubule cells. AB - Kidney tubule cells (KTC) are targets of T lymphocyte injury during allograft rejection and interstitial nephritis. KTC process and present self- and foreign Ags for immune recognition by CD4+ T cells in vivo and in vitro. However, it is not known whether KTC can provide the costimulatory signal required to fully activate CD4+ T cells. Using the MRL/MpJ fas model of lupus interstitial nephritis, we found that KTC did not express the costimulators B7-1 or B7-2. Nevertheless, KTC from both normal and systemically infected mice provided non-B7 costimulation to splenic CD4+ T cells. T cell proliferation was blocked by mAbs binding intercellular adhesion molecule-1 (ICAM-1) but not by mAb or fusion proteins binding B7-1, B7-2, heat-stable Ag, or vascular cell adhesion molecule 1. Importantly, ICAM-1 expression was necessary but not sufficient to provide costimulation. The transformed KTC line D3.B7- expressed high levels of ICAM-1 but did not provide costimulation. Interestingly, KTC provided costimulation to splenic T cells but not to a Th1 clone. These results show that freshly isolated KTC can provide non-B7 costimulation to splenic T cells via an unidentified costimulator and ICAM-1. Furthermore, these experiments demonstrate the complex nature of T cell activation and show that at least for splenic T cells, three or more signals may be required for full activation on live APC. PMID- 8621900 TI - Sustained T cell receptor-mediated Ca2+ responses rely on dynamic engagement of receptors. AB - We have investigated the functional advantage of surface-attached ligands for TCR mediated cell activation with flow cytometric measurements of cytoplasmic Ca2+ changes in T cells after aggregation of TCR by soluble and bead-attached mAb. Conjugation of HPB-ALL human leukemia cells with cell-sized beads coated with anti-TCR mAb causes a stronger, more sustained Ca2+ response than that produced by the soluble form of the same mAb. Addition of a large excess of the soluble mAb subsequent to stimulation with the beads causes a marked reduction in the response of the bead-conjugated cells, but only limited disruption of the conjugates. Free (nonconjugated) cells, sampled simultaneously in this mixture, respond to the soluble mAb with a transient Ca2+ increase that declines with the same kinetics as the bead-conjugated cells after addition of the soluble mAb. Fab fragments of the anti-TCR mAb cause a similar reduction in the response of the bead-conjugated cells, and they do not stimulate free cells. Following the Fab mediated decline in cytoplasmic Ca2+ of conjugated cells to near-baseline concentrations, the addition of a second, noncompetitive, anti-TCR mab causes a Ca2+ response that is substantially reduced in magnitude compared with that for the free cells. The results indicate that soluble and surface-attached ligands cause TCR-specific desensitization of the Ca2+ response. Surface-attached ligands are more effective than soluble ligands in sustaining signaling in T cells at least in part because they facilitate steady association and/or reassociation of TCR into the bound state in the surface contact area. PMID- 8621901 TI - Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes. AB - Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses. PMID- 8621902 TI - Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. AB - Perforin- and Fas-based cytolytic pathways are two major mechanisms of cell mediated cytotoxicity. Recently, we have shown that an inhibitor of vacuolar type H+-ATPase, concanamycin A (CMA), inhibits perforin-based cytotoxic activity, mostly due to accelerated degradation of perforin by an increase in the pH of lytic granules. Here we show that CMA failed to inhibit the cytolytic activity of CD4+ CTL clone and perforin-deficient CD8+ CTL clone, which exclusively mediate Fas-based cytotoxicity, although CMA inhibited acidification and induced drastic vacuolation of cytoplasmic granules in these clones. In a wide range of alloantigen-specific CTL, a significant amount of the lysis of Con A blasts from normal mice and of Fas-positive tumor cells remained unaffected even in excess concentrations of CMA. However, CMA almost completely inhibited the lysis of Con A blasts from lpr mice and of Fas low expressing or negative tumor cells. Cytolysis by alloantigen-specific CD8+ CTL derived from gld mice was completely prevented by CMA. Furthermore, CMA-insensitive cytolysis exerted by CD8+ CTL clone was completely inhibitable by soluble Fas molecules. Thus, these data clearly indicate not only that CMA-insensitive cytolysis mediated by alloantigen specific CTL is Fas dependent, but also that CMA is a selective inhibitor to block only the perforin-based killing pathway. In contrast, brefeldin A blocked the Fas-based cytotoxicity, but only marginally reduced the perforin-based cytotoxicity. Moreover, CMA and brefeldin A in combination completely abrogated all cytolytic activity of alloantigen-specific CTL. Taken together, these results reveal that CTL mainly exert perforin-based cytotoxicity and complementary Fas based cytotoxicity, and that CMA is a powerful tool to clarify the contributions of the two distinct cytolytic pathways. PMID- 8621903 TI - DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b mice. AB - The serum Ab response and the class I-restricted CTL response of C57BL/6 (H-2b) mice to hepatitis B (pre)core Ag (HBcAg, HBeAg) was studied. Injection of HBcAg particles without adjuvants into mice efficiently primed serum Ab responses but not CTL response. We constructed the expression plasmids pCMV-1/c and pCMV-1/e in which expression of HBcAg or HBeAg was driven by cytomegalovirus immediate early region promoter sequences. Stable murine RBL5/C transfectant lines expressing HBcAg were established. Intramuscular DNA immunization with plasmid pCMV-1/c (encoding intracellularly expressed core Ag) or pCMV-1/e (encoding secreted precore Ag) efficiently primed specific serum Ab responses and CTL responses. The CTL response elicited in this system was mediated by CD4-CD8+ effector cells primed in vivo. The CTL recognized the HBcAg93-100 8-mer peptide MGLKFRQL in the context of Kb. Hence, DNA immunization with HBcAg/HBeAg-expressing plasmids, but not immunization with exogenous HBcAg particles, elicits a class I-restricted CTL response of defined epitope/restriction specificity in H-2b mice. PMID- 8621904 TI - CD59 and CD48 expressed by rat retinal pigment epithelial cells are major ligands for the CD2-mediated alternative pathway of T cell activation. AB - The alternative CD2-mediated pathway of T cell activation, which is independent of MHC/peptide recognition by the TCR/CD3 complex, is dependent upon two signals being received by the CD2 molecule. The natural ligand for CD2 is CD58, but controversy exists over alternative or additional ligands that could deliver the second signal in vivo. We have used rat retinal pigment epithelial cells (RPE), which lack temperature-insensitive ligands for CD2 adhesion, to study Ag independent T cell activation. Rat RPE cells expressed high levels of CD59 and low levels of another potential CD2 ligand, CD48, both in vitro and in the in vivo model of experimental autoimmune uveoretinitis. When increasing numbers of syngeneic T cells were added to microwell cultures of rat RPE cells, the T cells, even in the absence of any exogenous stimulant in the cultures, underwent spontaneous proliferation. This effect required metabolically active RPE cells, and was IL-2 driven and enhanced in the presence of indomethacin. Proliferation was modulated by phosphatidylinositol-phospholipase C treatment of the RPE, and blocked by mAbs to CD59. Ab cross-linking of CD48 but not CD59 on the RPE was found to induce messenger RNA expression for IL-1 beta, which together with constitutively expressed IL-6 are required costimulatory factors for T cell activation through CD2. This is the first demonstration in a fully syngeneic system that bi-directional signaling involving CD59 and CD48 molecules expressed by physiologically normal, nonhematopoietic, cells can trigger T lymphocyte activation and proliferation through autocrine IL-2 production in the absence of Ag. PMID- 8621905 TI - Differential activity of dexamethasone on IL-2-, IL-4-, or IL-9-induced proliferation of murine factor-dependent T cell lines. AB - Mouse helper T cell lines were developed that proliferate permanently without Ag and APCs in response to either IL-2, IL-4, or IL-9, three cytokines whose receptors interact with the IL-2R gamma-chain for signal transduction. Depending on the growth factor, a marked difference was observed regarding the ability of dexamethasone (DEX) to inhibit cell proliferation. In three different cell lines, proliferation induced by IL-2 was completely arrested, while that supported by IL 9 was hardly affected. With IL-4, proliferation was also maintained but less markedly than with IL-9. Although DEX was able to induce apoptosis in these cells, the inhibition of IL-2-induced proliferation was not the result of apoptosis, as this process was equally antagonized by all three factors. Moreover, addition of IL-4 or IL-9 to cultures previously incubated with IL-2 and DEX for several days restored cell proliferation. Finally, autonomous cell variants derived from the factor-dependent cell lines were still protected by IL 4 and IL-9 against growth inhibition by DEX. Together, these results indicate that growth stimulation in the presence of glucocorticoids and inhibition of apoptosis involve distinct aspects of cytokine activities. PMID- 8621907 TI - Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway. AB - By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency. PMID- 8621906 TI - Expression of IFN regulatory factor family proteins in lymphocytes. Induction of Stat-1 and IFN consensus sequence binding protein expression by T cell activation. AB - Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the IFN regulatory factor (IRF) family. Evidence indicates that this family has a function in the immune system. Unlike other members of the family, ICSBP is expressed exclusively in the immune system. In this work, immunoblot analysis was performed to study expression of ICSBP and other members of the family in various murine lymphocytes. The results show that all IRF family members are expressed constitutively in B cells throughout development, and in resting and activated cells. In contrast, ICSBP expression was undetectable in thymocytes and resting T cells, while all other IRF proteins tested (IRF-1, IRF-2, and ISGF3-gamma) were detected in these cells. Induction of ICSBP (and weakly IRF-1, but not other members) was observed upon activation of T cells following anti-CD3 Ab binding or Con A stimulation. Once T cells were activated, ICSBP was expressed stably in both Th1 and Th2 cells. We show that Stat-1, which binds to the IFN-gamma responsive element of the ICSBP promoter, was induced following anti-CD3 Ab and Con A stimulation. Stat-1 induction was found in T cells of IFN-gamma+/+, but not of IFN-gamma-/- mice, indicating that T cell activation stimulates the Stat pathway of transcription that is mediated through IFN-gamma. IFN-gamma-activated Stat-1 partly accounted for ICSBP induction in activated T cells, as levels of induction were lower in IFN-gamma-/- than in IFN+/+ T cells. Taken together, these results show that activation of ICSBP is coupled with T cell activation that is partly due to IFN-gamma-induced Stat-1. PMID- 8621909 TI - CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes. AB - Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death. PMID- 8621908 TI - A fundamental subdivision of circulating lymphocytes defined by adhesion to mucosal addressin cell adhesion molecule-1. Comparison with vascular cell adhesion molecule-1 and correlation with beta 7 integrins and memory differentiation. AB - The leukocyte integrin alpha 4 beta 7 is a receptor for the vascular mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Most circulating B and T lymphocytes in man are alpha 4+, and on these cells the regulated display of the beta 7 integrin chain determines the expression of alpha 4 beta 7 and, in large part, binding to MAdCAM-1. Among CD4+ T cells, beta 7 high memory cells (including the L-selectin+ subset) bind MAdCAM-1 better than beta 7int naive cells; whereas beta 7- memory cells,including skin homing lymphocytes, interact poorly if at all. Circulating alpha E beta 7+ T cells are alpha 4 beta 7high and also bind MAdCAM-1 well. B cells are also subdivided by beta 7 expression, and beta 7+ B cells bind MAdCAM-1 better than the beta 7low/- subset. The related vascular ligand vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelium primarily in nonmucosal sites of inflammation, interacts with blood lymphocytes (including beta 7high T cells) almost exclusively via alpha 4 beta 1 and binds beta 7low/-(beta 1high) better than beta 7+ B cells and memory cells better than naive CD4+ cells. beta 7-(beta 1high) memory T cells are somewhat enriched over beta 7high memory cells at low (but not at high) VCAM-1 densities. Interestingly, CD56+ NK cells, which express both alpha 4 beta 7 and alpha 4 beta 1, bind well to VCAM-1 but poorly to MAdCAM-1. The findings indicate that the display and function of alpha 4 beta 7 determine integrin-dependent blood lymphocyte interactions with MAdCAM-1, thus delineating discrete mucosal vs nonmucosal lymphocyte populations in vivo; that alpha 4 beta 1 dominates blood lymphocyte interactions with VCAM-1; and that quantitative and qualitative regulation of MAdCAM-1 vs VCAM-1 can critically control the recruitment of specialized lymphocyte subsets during inflammation. PMID- 8621910 TI - TCR alpha beta gene usage for myelin basic protein recognition in healthy monozygous twins. AB - The pathogenic role of myelin basic protein (MBP)-specific T lymphocytes in multiple sclerosis (MS) has been suggested by the encephalitogenicity of MBP specific T cells in experimental allergic encephalomyelitis (EAE). In humans, extensive analysis of TCRs involved in MBP recognition has led to conflicting results, varying from an intra- and/or interindividual restriction to high diversity in TCRAV/TCRBV gene usage. We previously established MBP-specific T cell lines (TCLs) from healthy monozygous twins and characterized their fine epitope specificity. In this study, we report on the TCR alpha beta gene usage of 52 of these MBP TCLs that are specific for epitopes recognized by both co-twins within the same pair. High overall diversity in the TCR alpha and TCR beta genes used for recognition of this self-Ag, MBP, was observed. Variable genes belonging to 19 different TCRAV and 16 different TCRBV subfamilies are expressed by the 52 TCLs herein studied. In co-twins, TCLs utilized genes belonging to common TCRAV and/or TCRBV gene subfamilies in 7 of 13 instances of shared epitope recognition. Statistical analysis of intrapair concordance for TCR gene usage for the recognition of a given peptide did not show any significant deviation from values that would be anticipated in the absence of genetic background effect. PMID- 8621911 TI - Peptide-dependent expression of HLA-B7 on antigen processing-deficient T2 cells. AB - Class I MHC Ag presentation and cell surface expression largely depend on peptide transport into the ER/cis-Golgi by TAP, the transporter associated with Ag processing. Despite this dependency, class I MHC molecules are expressed at low levels on the surface of TAP-deficient T2 cells. We studied the peptide dependency of HLA-B7 expression in transfected T2 cells. HLA-B7 expression was affected by mutations at 19 out of 23 peptide-binding groove residues, but not by nine mutations outside of the peptide-binding groove. T2 cell surface HLA-A2, B7, and -B51 had similar stabilities, and approximately half of these class I molecules had a long t1/2 consistent with tight peptide binding. Using metabolically labeled T2 cells, HLA-A2-bound peptide eluted as five prominent peaks, but HLA-B7-bound peptide was not detected. In contrast, HLA-B7-eluted peptides were detected spectrophotometrically. These data suggest that HLA-A2 and HLA-B7 molecules utilize distinct TAP-independent peptide supply mechanisms to different degrees. Equivalent amounts of HLA-B7 from TAP- and TAP+ cells yielded similar amounts of peptide, which had the characteristic HLA-B7 peptide-binding motif. The dependency of HLA-B7 cell surface expression on peptide-binding groove residues, the stability of cell surface class I molecules, and the ability to detect HLA-B7-bound peptide indicate that the low level expression on T2 cells is largely peptide dependent. TAP-independent peptide Ag presentation may allow immune recognition of intracellular pathogens that interfere with TAP-dependent peptide transport. PMID- 8621912 TI - T cell tolerance is influenced by concomitant T cell recognition of cross reactive self-peptides. AB - Although the current dogma of T cell recognition stresses its exquisite specificity, T cell clones selected for a given peptide can recognize other sequentially or structurally related peptides. Here, we have examined the immunogenicity and tolerogenicity of various self-peptides derived from region 61 80 of different MHC class I proteins co-expressed in the same mouse. Following immunization of B10.A mice (K(k), A(k), E(k), L(d), D(d)) with self-L(d) 61-80 peptide, vigorous MHC class II-restricted T cell proliferation was elicited after restimulation with either the immunogen or with self-K(k) 61-80 but not with self D(d) 61-80. Furthermore, adult B10.A mice, tolerized with L(d) 61-80 prior to immunization with L(d) 61-80 did not respond to challenge with L(d) 61-80 and the cross-reactive K(k) 61-80. However, following K(k) 61-80 immunization, L(d) 61-80 tolerized mice responded to K(k) 61-80 but not to L(d) 61-80. Thus, tolerance induction to L(d) 61-80 resulted in the elimination/inactivation of L(d) 61-80 reactive T cells including the subpopulation that cross-reacted with K(k) 61-80. However, T cells that recognized K(k) 61-80 exclusively were preserved. Moreover, we showed that immunization with K(k) 61-80 resulted in tolerance breakdown to the cross-reactive, dominant self-peptide D(b) 61-80 in B10.A(4R) mice (K(k), A(k), L(d),D(b)). Together, these results show that the autoimmune T cell repertoire is influenced by the concomitant recognition of different cross reactive self-peptides within the same individual. PMID- 8621913 TI - Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family. AB - In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1 negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function. PMID- 8621914 TI - AP-1 regulates the basal and developmentally induced transcription of the CD11c leukocyte integrin gene. AB - The p150,95 integrin (CD11c/CD18) mediates leukocyte/endothelium interactions during inflammatory reactions and certain CTL-target interactions, and is also a receptor for fibrinogen, LPS, and the complement component iC3b. CD11c/CD18 is expressed primarily on cells of the myeloid lineage and activated B lymphocytes, and is an important diagnostic marker for hairy cell leukemia. To identify the transcription factors and cis-acting elements involved in the regulated expression of CD11c/CD18 during myeloid cell differentiation and B lymphocyte activation, we have performed structural and functional analysis on the CD11c gene promoter. Electrophoretic mobility shift assays identified an AP-1 binding site (AP1-60) within the proximal promoter region and evidenced differences in the pattern of the Fos family members bound to the AP1-60 element in undifferentiated and differentiated myeloid cells, as well as between B lineage derived cells. The involvement of the AP1-60 element in DNA-protein interactions was confirmed by means of in vivo footprinting experiments, and its functionality was demonstrated by trans activation of the CD11c promoter by c-Jun. Site directed mutagenesis of AP1-60 greatly reduced the basal CD11c promoter activity in myeloid and B cells. Furthermore, mutations at AP1-60 inhibited the induction of the CD11c promoter activity during the PMA-triggered U937 cell differentiation, pointing out a key role for the AP-1 transcription factor complex in both the basal and the developmentally regulated expression of the p150,95 leukocyte integrin. The involvement of AP-1 in the transcription of the CD11c gene raises the possibility of altering leukocyte integrin expression by pharmacologic means and will greatly contribute to the characterization of the intracellular signals controlling the expression of leukocyte adhesion molecules. PMID- 8621915 TI - Early B cell factor interacts with a subset of kappa promoters. AB - A conserved sequence element situated between the decamer and TATA box in V kappa II and V kappa V promoters has a high homology to the binding site for early B cell factor (EBF). The kappa promoter element was shown to bind EBF specifically using both in vitro-translated protein and nuclear extract. Concomitant binding of EBF and Oct proteins to a wild-type kappa promoter template was observed at low efficiency, and such dual occupancy was dependent on an intact amino terminus of the Oct protein. When the two binding sites were separated by a 10-bp spacer, this dependency disappeared. A single kappa promoter EBF site together with a TATA box and an Ig heavy chain enhancer showed marginal transcriptional stimulatory activity. In contrast, the EBF site acted synergistically with a decamer element in EBF-negative plasmacytoma cells, but not in B cells of an earlier differentiation stage. In these cells, a distinct protein was observed that interacted with the EBF binding motif, while overexpression of EBF down regulated the expression of a reporter construct containing Ig control elements. PMID- 8621916 TI - The generation of antibody diversity in the turtle. AB - The Ab response in reptiles has been studied at the protein level, and in turtles some aspects resemble those of cold-blooded vertebrates from other classes. The genetic bases for these features are not clear. The present study is the first on the IgH organization and complexity of a reptilian Ig gene system. The approach to cloning turtle (Pseudemys scripta) sequences is entirely PCR based, and its efficacy is demonstrated by obtaining extensive information on a heretofore unexplored Ig gene system. A number of genomic VH sequences, representing possibly four families, were isolated, as was a genomic C mu 4 clone. These sequences, used as probes, provided proof that in the turtle there is a single IgH locus with multiple VH genes and one C mu gene. In Northern hybridizations, the C mu 4 probe detected two transcripts; of the four VH groups, only one was expressed, and multiple bands indicated the presence of at least two non-mu transcripts. Using reverse transcription-PCR on spleen or liver RNA, an IgM heavy chain sequence was obtained, as were a number of VDJ rearrangements. Among 32 unique VDJ rearrangements from one animal, there were 22 sequence variants at framework 4, suggesting either a very large number of J segments or somatic modification in the variable region. The latter interpretation is supported by point mutations found in framework 3 and CDR3. The number of changes is considerably greater than the deduced Taq misincorporation rate (0.05%). PMID- 8621917 TI - A DNA binding protein in human thymocytes recognizes the T cell receptor-delta deleting element psi J alpha. AB - The TCR is a heterodimeric molecule composed of either gamma delta or alpha beta chains. The differentiation mechanisms that force thymocytes into the gamma delta alpha beta lineage are poorly understood, but rearrangement processes in the TCR delta alpha locus are likely to play an important role. The TCR-delta gene complex is flanked by the delta-deleting elements delta Rec and psi J alpha, which are assumed to delete the TCR-delta gene before V alpha-J alpha rearrangement. The nonproductive delta Rec-psi J alpha recombination occurs at high frequency in both fetal and postnatal immature thymocytes. To find DNA binding proteins involved in the delta Rec-psi J alpha preferential rearrangement, we performed electrophoretic mobility shift assays using the recombination signal sequence of psi J alpha with additional upstream and downstream sequences. We observed a 180-kDa DNA binding protein in nuclear extracts from human thymocytes that recognized a 46-bp binding site on the psi J alpha gene segment, containing the core motif GTTAATAGG. The psi J alpha binding protein, which we call PJA-BP, was also detected in immature CD3-T cell lines with TCR-delta genes deleted on both alleles, in a TCR-alpha beta+ cell line, and in two of four myeloid cell lines. This protein was absent in a TCR-gamma delta+ T cell line, in nonhemopoietic cell lines, and in all but one B cell lines tested. Although we could detect binding activity of the PJA-BP to some other TCR J alpha gene segments, we postulate that binding of PJA-BP to the psi J alpha gene segment is one of the factors involved in the preferential delta Rec-psi J alpha gene rearrangement process. PMID- 8621918 TI - T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31. AB - T cells recognize peptide fragments of Ags bound to MHC-encoded molecules. Pockets in the MHC peptide-binding groove accommodate a limited set of amino acid side chains present at anchor positions in peptide; however, the functional significance of accommodation of different side chains at an anchor position in peptide is not clear. A panel of T cell clones was evaluated to test the effect of conservative amino acid substitution at a primary peptide anchor position. Results of T cell stimulation studies were correlated with two well studied characteristics of the peptide/MHC complex, which are the affinity of peptide binding to MHC and the stability of the resulting complex upon PAGE in the presence of SDS. We found that formation of a functional complex required neither high affinity peptide binding nor SDS stability. Furthermore, T cell clones differed in their ability to recognize individual peptide variants, suggesting that some structural aspect of the peptide/MHC complex is influenced by interactions between peptide anchor residues and MHC pockets. PMID- 8621919 TI - Rejection of MHC class II-transfected tumor cells requires induction of tumor encoded B7-1 and/or B7-2 costimulatory molecules. AB - Many tumor cells that have been transfected with genes encoding B7 costimulatory molecules become effective cellular vaccines against wild-type tumor. The improved immunity is dependent on newly induced tumor-specific CD8+ and/or CD4+ T cells and presumably occurs because the B7 transfectants provide the requisite second signal for activation of T cells in conjunction with tumor cell-presented MHC class I/tumor peptide and/or MHC class II/tumor peptide complexes, respectively. Since B7 expression is such a potent enhancer of tumor immunity, and yet some tumors are immunogenic in the absence of B7 transfection, we have used class I+ class-II-transfected tumors to investigate whether costimulatory molecules are also involved in rejection of immunogenic, non-B7-transfected tumor. Blocking studies with B7 mAbs demonstrate that induction of tumor immunity in naive mice requires B7-1 and/or B7-2 expression, while experiments with tumor primed mice indicate that once antitumor immunity is established, expression of B7 is not necessary. Flow cytometry analyses demonstrate that costimulatory molecules are expressed by the tumor cells via an in vivo induction process. Experiments with class II genes with truncated cytoplasmic tails indicate that the cytoplasmic region of the tumor-expressed class II heterodimer is involved in induction of B7. We therefore conclude that for this class I+ class II transfected tumor, generation of tumor immunity requires induction of tumor cell encoded B7 molecules that are mediated by the cytoplasmic region of the transfected class II heterodimer. PMID- 8621920 TI - Chemotactic activity of mycobacterial lipoarabinomannans for human blood T lymphocytes in vitro. AB - A crucial early event in tuberculosis is the ingestion of Mycobacterium tuberculosis (Mtb) by alveolar macrophages. Chemotactic factors released by infected macrophages are likely to initiate a granulomatous response, a key feature of host resistance to tuberculosis. To date, the role of mycobacterial products in regulating the granulomatous response has not been clearly defined. Here we report that the mycobacterial cell wall glycophospholipid lipoarabinomannan (LAM) could specifically induce human peripheral blood T cell chemotaxis in vitro. Both terminally mannosylated LAM isolated from Mtb and LAM lacking the terminal mannosyl units isolated from an avirulent mycobacterium could induce T cell migration in the absence of serum. In contrast, terminally mannosylated LAM isolated from Mycobacterium bovis BCG failed to induce T cell chemotaxis. These observations represent the first report that LAM is capable of directly inducing biologic responses in human T cells. Flow cytometry analysis revealed that CD4+, CD8+, and CD45RO+ lymphocytes were present in the migrating cell populations at ratios similar to those found in nonmigrating cells. The chemotactic response was found to require new protein synthesis, and could be blocked by inhibitors of protein tyrosine kinases at concentrations that did not affect random migration. Acyl groups at the reducing terminus of LAM appear to be required for the chemotactic activity of this mycobacterial glycolipid. Lastly, culture supernatants from human alveolar macrophages infected in vitro with a virulent strain of Mtb could induce T cell migration. Much of the migratory activity present in these supernatants could be blocked using a mAb against LAM, suggesting that LAM is one of the chemotactic factors released by Mtb-infected alveolar macrophages. PMID- 8621921 TI - Oxidative killing of Cryptococcus neoformans by human neutrophils. Evidence that fungal mannitol protects by scavenging reactive oxygen intermediates. AB - Polymorphonuclear neutrophils (PMN) kill Cryptococcus neoformans (Cn) by oxidative mechanisms, but the roles of various reactive oxygen intermediates (ROIs) are not known. We used a mannitol low-producing Cn mutant (Cn MLP) and its wild-type parent (Cn H99) to examine the role of ROIs distal to H2O2 in PMN killing and to determine whether mannitol produced by Cn protects the fungus against ROIs. At PMN:Cn cell ratios of 1:1, 10:1, and 100:1, PMN killed significantly more Cn MLP than Cn H99 cells after 2 and 4 h (p less than 0.05). Superoxide dismutase and the hydroxyl radical (OH.) scavengers mannitol and DMSO inhibited killing of both strains (p less than 0.05), but catalase did not. Cn H99 and Cn MLP stimulated PMN to produce similar amounts of O2- and H2O2. In contrast, Cn MLP stimulated greater luminol-dependent chemiluminescence than did Cn H99 (p less than 0.05). Finally, H2O2 alone killed similar numbers of Cn H99 and Cn MLP cells, but oxidants generated by FeSO4 (1 microM), H2O2 (10 microM), and iodide (1 to 3 microM) killed significantly more Cn MLP than Cn H99 cells in 1 h (p less than 0.05). Mannitol, DMSO, and catalase completely inhibited killing of both Cn strains by this cellfree system, but superoxide dismutase did not. These results suggest that 1) distal ROIs such as OH. and HOCI are key effector molecules against Cn, and 2) mannitol produced by Cn may protect against oxidative killing by scavenging distal ROIs. PMID- 8621922 TI - Role of host antigen receptor-bearing and antigen receptor-negative cells in immune response to rat adenocarcinoma 13762. AB - Involvement of individual immune cell populations in the immune response to rat breast adenocarcinoma 13762 was studied by selective depletion treatments in vivo. Depletion of Ag nonspecific host defense cells from naive animals before tumor challenge resulted in statistically significant acceleration of tumor growth (p less than 0.01 or less), whereas depletion of either CD4+ T cells or CD8+ T cells had no effect on the incidence or kinetics of tumor development. In contrast to naive animals treated before tumor challenge, animals actively immunized by injection of irradiated tumor before depleting treatments were shown to require CD4+ T cells to reject tumorigenic challenge. Depletion of either macrophages or neutrophils from immune animals also increased tumor development, whereas NK cells were not involved. Depletion of CD8+ T cells from immune animals permitted transient growth of tumors that were subsequently rejected, implying a role in tumor rejection. Transfer of immune antiserum to naive animals at the time of tumor challenge was without effect on tumor development. Depletion of CD4+ T cells, neutrophils, or macrophages in the priming phase of antitumor immune response abrogated tumor immunity, but depletion of CD8+ T cells or NK cells was without effect on the ability to prime animals by immunization with irradiated cells. Collectively, these data suggest that host natural defense cells that do not express Ag receptor are primarily responsible for resistance of adenocarcinoma 13762 growth in naive animals. In contrast, tumor immunity induced by active immunization requires Ag receptor-bearing CD4+ T cells and involves participation of CD8+ T cells, neutrophils, or macrophages in elimination of tumor. PMID- 8621923 TI - Regulation of lymphocyte homing into the brain during viral encephalitis at various stages of infection. AB - The passage of circulating lymphocytes into the central nervous system (CNS) during acute viral encephalitis was studied in vivo using fluorescently labeled cells inoculated into Sindbis virus (SV)-infected mice. Donor lymphocytes were detected in the brains of recipient animals when mononuclear cells were isolated from the CNS and screened by flow cytometry. The magnitude of this accumulation related to the duration of encephalitis in recipient mice and to the activation state of the inoculated cells. While Ag specificity did not influence lymphocyte entry into the inflamed CNS at any stage of infection, SV-immune cells were retained selectively within the brains of infected animals compared with cells of an irrelevant specificity. Coincident with the onset of CNS inflammation, ICAM-1 and VCAM-1 were up-regulated on cerebrovascular endothelium. Lymphocyte entry into the brains of infected animals during maximal inflammation could be inhibited by pretreating inoculated cells with Abs that blocked LFA-1, but not with those that blocked VLA-4 or down-regulated CD44. None of these reagents prevented lymphocyte entry into the brain at the onset of inflammation, suggesting that the earliest recruited cells utilize presently uncharacterized receptor-ligand interactions. These data show that the degree of existing inflammation and the activation state of circulating cells, but not their Ag specificity, influence lymphocyte recruitment into the brain during SV encephalitis. While CNS homing can be blocked with Abs against known adhesion molecules during peak inflammation, lymphocyte entry into the brain during early infection remains poorly characterized. PMID- 8621924 TI - Immunization with granulocyte-macrophage colony-stimulating factor-transduced, but not B7-1-transduced, lymphoma cells primes idiotype-specific T cells and generates potent systemic antitumor immunity. AB - Recently, genetically modified tumor cell vaccines have been described for nonhematopoietic cancers in which the relevant Ags are unknown. Several of these cell-based vaccine strategies have been shown to induce T cell-mediated systemic antitumor immunity, either by enhancing the processing and presentation of tumor Ags by host APCs or by facilitating effective Ag presentation by the tumor vaccine itself. These strategies were compared in a model B cell lymphoma, a tumor derived from APCs, which have the inherent capacity to activate Ag-specific T cells. Eradication of pre-established systemic lymphoma was achieved following immunization with lymphoma cells engineered to produce granulocyte-macrophage (GM)-CSF, and to a lesser extent cells producing IL-4, whereas vaccination with lymphoma cells transfected with the genes encoding IL-2 or B7-1 had no effect. The systemic immunity generated by GM-CSF- or IL-4-transfected lymphoma required both CD4+ and CD8+ T cells. Previous immunotherapeutic strategies for the treatment of lymphoma have focused on the generation of Ab responses targeted to the unique Ig Id as a tumor-specific Ag. Anti-idiotypic Abs were undetectable in animals vaccinated with GM-CSF-transduced lymphoma cells. In contrast, such immunization did result in the induction of Id-specific T cell responses. This is the first demonstration that T cell responses specific for a native tumor Ag are generated by GM-CSF-transduced tumor cell-based vaccination, suggesting that B cell lymphoma may be a suitable disease for genetically modified tumor vaccine strategies. PMID- 8621925 TI - Mechanisms of Trypanosoma cruzi-induced down-regulation of lymphocyte function. Inhibition of transcription and expression of IL-2 receptor gamma (p64IL-2R) and beta (p70IL-2R) chain molecules in activated normal human lymphocytes. AB - Acute infection with Trypanosoma cruzi, the agent of Chagas' disease, is accompanied by multiple manifestations of immunosuppression, and this parasite has been shown to inhibit T and B lymphocyte functions in vitro through a mechanism involving impaired membrane expression of at least the p55IL-2R (IL-2R alpha) and p70IL-2R (IL-2R beta) components of the multimeric high affinity IL-2R complex. We document in this paper that addition of T. cruzi or a parasite conditioned medium (trypanosomal immunosuppressive factor (TIF) to cultures of PHA-stimulated normal human blood lymphocytes markedly decreases the cytoplasmic levels of p64IL-2R and p70IL-2R molecules, i.e., the two IL-2R chains that bind IL-2 and participate in signal transduction. These effects highlighted the ability of T. cruzi to curtail biosynthesis of these IL-2R chains and weakened the possibility that decreased membrane IL-2R expression results exclusively from altered chain transportation to the lymphocyte surface. Additional support for impaired lymphocyte production of IL-2R components was derived from northern blot analyses demonstrating TIF-induced decreases in p64IL-2R and p70IL-2R mRNA levels. These reductions are unlikely to be due to conditions affecting mRNA stability, since the rates of decay of both mRNA species were nearly identical in the presence and in the absence of TIF. In addition, nuclear run-on studies revealed that transcription of the p64IL-2R, p70IL-2R, and p55IL-2R genes, but not that of the CD69 gene, was below normal levels in PHA-stimulated lymphocytes incubated with TIF. These results suggest that altered gene transcription is involved in the mechanism by which T. cruzi down-regulates the expression of all the known components of the high affinity IL-2R. PMID- 8621926 TI - Adoptive immunotherapy with tumor-specific T lymphocytes generated from cytokine gene-modified tumor-primed lymph node cells. AB - Adoptive immunotherapy with immune T cells mediates regression of established tumors in animal models. We previously demonstrated that precursor lymphocytes of sensitized T cells can develop into mature effector cells after in vitro activation with anti-CD3 mAb and IL-2. We demonstrate here that tumor cells genetically modified to secrete IL-2 can enhance the precursor response in the tumor-bearing host and subsequently augment the antitumor efficacy of adoptive immunotherapy. MCA205 and MCA203, weakly immunogenic fibrosarcomas, were transfected in vitro with cDNA encoding for IL-2, IL-4, or IL-6. Lymph nodes (LN) draining these cytokine-producing tumors for 7 days were harvested, activated in vitro with anti-CD3/IL-2, and adoptively transferred into mice bearing established parental MCA205 pulmonary metastases. The effector cells generated from LN draining the IL-2 producing tumor exhibited enhanced antitumor activity compared with cells from LN draining parental, IL-4-producing, or IL-6-producing tumor. Phenotype analysis of cells from LN draining the IL-2-producing tumor revealed selective expansion of V beta 8+ cells. Depletion of V beta 8+ effector cells abrogated the antitumor efficacy indicating that V beta 8+ cells constituted the majority of antitumor reactivity and that secretion of IL-2 from tumor cells promoted the priming of V beta 8+ precursor cells, which can develop into mature effector cells. These results have important clinical implications that the method presented here could be applicable to the treatment of human cancer as more effective immunotherapy. PMID- 8621927 TI - Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy. AB - Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes. PMID- 8621928 TI - Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein. AB - The protective roles of influenza viral nucleoprotein (NP), together with the cellular mechanism of the protection in the nasal site, were examined in BALB/c mice immunized intranasally with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin)-combined A or B virus recombinant NP. The NP-immune mice, when challenged intranasally with a sublethal dose of the virus 3 wk after immunization, had accelerated virus clearance from the nasal site in both an influenza type-specific and a nonspecific manner, as shown by the protection from high morbidity from the second day after challenge. Both type-specific and nonspecific acceleration of recovery was confirmed by the increased survival rate after challenge with a lethal dose of virus in mice immunized and boosted with adjuvant-combined NP. The acceleration of nasal virus clearance was accompanied with acceleration of type-specific systemic delayed-type hypersensitivity (DTH) and with IFN-gamma production by nasal lymphocytes. The nasal lymphocytes from the immunized and challenged mice generated a significantly high level of DTH when transferred locally, but no class I MHC-restricted CTL response. Moreover, nasal CD4+ T cells, induced by NP immunization and increased in number by the subsequent challenge, were involved in the accelerated IFN-gamma production. These results suggest that nasal Th1 cells, capable of producing IFN-gamma and mediating DTH, are involved in the type-specific acceleration of recovery from influenza after challenge in mice immunized intranasally with adjuvant-combined NP, although the nonspecific mechanism of accelerated recovery remains to be solved. PMID- 8621929 TI - Human herpes simplex virus (HSV)-specific CD8+ CTL clones recognize HSV-2 infected fibroblasts after treatment with IFN-gamma or when virion host shutoff functions are disabled. AB - Herpes simplex virus (HSV)-specific CD8+ CTL cloned from individuals infected with HSV-2 efficiently lyse HSV-infected EBV-transformed B lymphoblastoid cells; however, these same CTL fail to lyse infected dermal fibroblasts. By 3 h after infection (early), class I MHC expression is reduced to less than 20% of that in uninfected fibroblasts, and expression is further reduced to less than 1% of the level in uninfected cells between 6 and 18 h after infection (late). We used an HSV-2 mutant that lacked the virion host shutoff (vhs) function to demonstrate that vhs plays a role in the loss of class I expression. While fibroblasts infected with this mutant are lysed by CTL that recognize virion proteins presented early as a consequence of introduction into the cytoplasm by the infecting virus, they are resistant to lysis by CTL that recognize viral proteins that must be synthesized de novo to be presented as class I Ags. Fibroblasts infected with a mutant that lacks the transporter-associated protein inhibitor ICP47 and is partially vhs defective are sensitive to CTL lysis. Pretreatment of fibroblasts with IFN-gamma prior to HSV infection sustained the level of class I expression for longer periods after infection, and these fibroblasts, infected with wild-type HSV-2, were partially sensitive to lysis by HSV-specific CTL. Taken together, these results suggest that the combined effects of the HSV-2 vhs and ICP47 gene products are to block Ag presentation by class I MHC. However, this effect can be transiently counteracted by IFN-gamma providing an early role for CD8+ CTL in the cellular immune response to HSV-2. PMID- 8621930 TI - Enhanced tumor outgrowth after peptide vaccination. Functional deletion of tumor specific CTL induced by peptide vaccination can lead to the inability to reject tumors. AB - CTL can play an important role in the defense against tumors. Protective CTL mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1 expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases. PMID- 8621931 TI - Protective immunity in BALB/c mice against the simian virus 40-induced mKSA tumor resulting from injection of recombinant large T antigen. Requirement of CD8+ T lymphocytes. AB - BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes. PMID- 8621932 TI - Down-regulation by IL-4 and up-regulation by IFN-gamma of mast cell induction from mouse spleen cells. AB - While investigating an involvement of other factors aside from endogenous IL-3 and prostaglandin E (PGE) in mast cell induction from mouse splenocytes, we found that the mast cell induction was inversely proportional to IL-4 levels and tended to directly proportionate IFN-gamma levels in the supernatants recovered on days 2 and 4. Thereafter, we examined the effects of rIFN-gamma, rIL-4, and rIL-10 on mast cell induction. IFN-gamma and IL-10 dose-dependently induced mast cells. Time course study showed an importance of adding rIFN-gamma into the cultures at the early phase (on days 0 and 2 of a 12-day culture). When endogenous IFN-gamma at the early phase was neutralized by anti-IFN-gamma Ab, all stimulants, including rIFN-gamma, rIL-10, and PGE1, failed to induce mast cells. On the contrary, rIL-4 dose-dependently suppressed the mast cell induction by rIFN gamma, rIL-10, LPS, PGE, and dibutyryl cAMP. The inhibitory effect of IL-4 was observed when IL-4 was added into the cultures at the early phase, but not after day 4. The suppressive action of IL-4 was diminished completely by the addition of neutralizing anti-IL-4 Ab. IL-12, a key regulator of IFN-gamma and IL-4 production, also induced mast cells. These results revealed, for the first time, that IFN-gamma is crucial for the survival and/or differentiation of splenic mast cell precursors and that IL-4 is a key inhibitor for the precursors, although IFN gamma is not a mast cell growth factor and IL-4 is a growth factor for immature and mature mast cells. PMID- 8621933 TI - Induction of IL-12 p40 messenger RNA expression and IL-12 production of macrophages via CD40-CD40 ligand interaction. AB - The mechanism of IL-12 production has been studied by stimulating macrophages or B cell lines with LPS, Staphylococcus aureus, or phorbol diester. However, since IL-12 plays an important role in the activation of T cells interacting with APC, it is important to study the mechanism of IL-12 production induced by T helper cell-APC interaction. We and others have demonstrated that IL-12 is produced in cultures where Th1 cells are stimulated with Ag or APC. In the present experiments, we studied a role of CD40-CD40 ligand (CD40L) interaction in IL-12 production and obtained the following results: 1) incubation of normal Th1 clone with APC in the presence of Ag induced IL-12 p40 and p35 mRNA accumulation and IL 12 production, and the addition of anti-CD40L blocked the p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation; 2) when Th1 clone from a CD40L-deficient mouse was used in the incubation, p35 mRNA accumulation was induced, but neither p40 mRNA accumulation nor IL-12 production was induced; 3) CD40L+ Th1 clone, or insect cell membrane expressing mouse CD40L, induced p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation. These results indicate that the CD40-CD40L interaction plays a critical role in IL-12 p40 mRNA accumulation and bioactive IL-12 production and that p35 mRNA accumulation was regulated via a different mechanism than CD40-CD40L interaction. Most of the cells producing IL-12 were Mac-1+ macrophages. PMID- 8621934 TI - Galectin-3 promotes adhesion of human neutrophils to laminin. AB - Galectin-3 is a member of a growing family of animal lectins composed of three domains, with the amino-terminal half consisting of a short segment followed by tandem repeats, and the carboxyl-terminal half representing the carbohydrate recognition domain. Previously, we have shown that galectin-3 binds to the surface of human neutrophils and is capable of activating these cells. We have now studied the effect of exogenous galectin-3 on adhesion of human neutrophils to laminin-coated microtiter plates and found that this lectin promotes the adhesion in a dose-dependent manner. The effect was dependent on the lectin's carbohydrate-binding function, as well as its amino-terminal region. The galectin 3-induced adhesion was reduced significantly in the presence of EDTA, even though Ca2+ and Mg2+ are not required for the lectin binding, and the adhesion was significantly less at 4 degrees C, as compared with 37 degrees C. Galectin-3 also induced neutrophil adhesion to fibronectin, which is not recognized by the lectin, but much higher concentrations of the lectin were required, and the effect is completely dependent on Ca2+ and Mg2+. We conclude that galectin-3 induces neutrophil adhesion to laminin through a combination of two distinct mechanisms: 1) the lectin bridges neutrophils to laminin, in a carbohydrate dependent and Ca2+-, Mg2+-independent manner, and 2) the lectin induces activation of neutrophils, in the presence of the divalent cations, resulting in the positive regulation of other cell adhesion molecules and enhanced adhesion to laminin. The results suggest that galectin-3 may play a role in the traversing of neutrophils through the basement membrane at inflammation sites. PMID- 8621935 TI - Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation. AB - The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation. PMID- 8621936 TI - Activation of human monocytes through CD40 induces matrix metalloproteinases. AB - The activation of monocytes/macrophages to secrete pro-inflammatory cytokines and matrix metalloproteinases (MMPs) is critically important in the development of chronic inflammatory diseases. However, the consequence of interactions between activated T cells and monocytes in these inflammatory processes is not well understood. In this study we have investigated the induction of MMPs in human monocytes by activated T cells. We show that fixed cells and the cell membranes from a T cell line, BMS-2, that expresses high levels of the CD40 ligand gp39 (also called TRAP, TBAM, or CD40L) stimulate both the expression of mRNA and the production of MMPs by human monocytic cells. Activation of monocytes by the human T cells could be significantly inhibited by a F(ab')2 fragment of a neutralizing Ab specific for human gp39, but not by an Ab that recognizes murine gp39. Furthermore, recombinant soluble gp39 (sgp39) alone induced marked increases in the levels of a 92-kDa metalloproteinase (gelatinase) in both the human monocytic cell line, THP-1, and peripheral human monocytes, and induction was blocked by the anti-human gp39 Ab. Pretreatment with IFN-gamma significantly enhanced gp39 induction of MMPs in THP-1 cells but not in peripheral monocytes. Up-regulation of mRNA for the 92-kDa MMP by gp39 could be detected within 6 h of stimulation and was maximal 24 h after treatment. MMP enzymatic activity was detectable in the culture medium 12 to 18 h following stimulation of the cells and remained high through 48 h. These results suggest the interaction of T cells with monocytes/macrophages via the gp39-CD40 counter receptors may be significant in development or maintenance of chronic inflammatory lesions. PMID- 8621937 TI - Salicylates inhibit I kappa B-alpha phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration. AB - The expression of leukocyte adhesion molecules on endothelial cells is induced by TNF-alpha and other inflammatory cytokines. This induction of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 requires the transcription factor nuclear factor-kappa B (NF-kappa B). Recent work has suggested that some nonsteroidal anti-inflammatory agents, including sodium salicylate and aspirin, can inhibit NF kappa B-dependent gene activation. We studied the effects of salicylates on expression of adhesion molecules in HUVECs. We found that sodium salicylate inhibited activation of NF-kappa B (p50/p65 and p65/p65) by preventing phosphorylation and subsequent degradation of the inhibitor 1 kappa B-alpha. Salicylate treatment had no effect on TNF-alpha-induced phosphorylation of the transcription factor ATF-2. Salicylate blocked the TNF-alpha-induced increase in mRNA levels of adhesion molecules and gave a dose-dependent inhibition of TNF alpha-induced surface expression of vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with higher doses required to inhibit endothelial-leukocyte adhesion molecule-1 expression. Indomethacin, a nonsalicylate cyclooxygenase inhibitor, had no effect on surface expression of adhesion molecules, suggesting that the effects were not due to inhibition of cyclooxygenase. Treatment of endothelial cell monolayers with sodium salicylate inhibited transendothelial migration of neutrophils but had no significant effect on neutrophil adhesion under flow conditions. The clinical importance of high dose salicylates in inflammation may be due, in part, to the ability to prevent expression of inducible adhesion molecules and recruitment of leukocytes. PMID- 8621938 TI - Extracellular ATP activates mast cells via a mechanism that is different from the activation induced by the cross-linking of Fc receptors. AB - In this study, the extracellular ATP (ATPo)-induced biochemical events were elucidated by comparing them with either the Fc epsilon RI- or Fc gamma R-induced events in the mouse mast cell line MC9. The omission of extracellular Ca2+ almost completely abolished the elevation of intracellular Ca2+ ([Ca2+]i) in the ATPo stimulated cells, but only suppressed the second phase of the increase of [Ca2+]i in FcR-stimulated cells, thus suggesting that the ATPo-induced elevation of [Ca2+]i is totally dependent on the entry of extracellular Ca2+. Pretreatment with genistein, which inhibits protein kinases, especially protein tyrosine kinase, inhibited the FcR-triggered increase of [Ca2+]i, but not the ATPo triggered one; however, such pretreatment did suppress both ATPo- and FcR mediated beta-hexosaminidase release. An immunoblot analysis revealed that both ATPo and the cross-linking of FcRs led to tyrosine phosphorylation of 44- and 110 kDa proteins, which thus suggested that these tyrosine-phosphorylated proteins are involved in a modulation of the degranulation process following an elevation of [Ca2+]i. Pretreatment with PMA inhibited the FcR-induced [Ca2+]i increase, while not inhibiting the ATPo-induced one, thus suggesting that ATPo can mobilize [Ca2+]i even when protein kinase C (PKC) has already been activated. Pretreatment of calphostin C, a specific PKC inhibitor, had little effect on the ATPo-mediated beta-hexosaminidase secretion, thus indicating that the ATPo-induced degranulation is not mediated by PKC. Taken together, these results demonstrate that ATPo activates MC9 mast cells by a mechanism that is different from the activation induced by the cross-linking of FcRs. PMID- 8621939 TI - Eosinophil degranulation in the presence of bronchial epithelial cells. Effect of cytokines and role of adhesion. AB - It has been suggested that eosinophils (Eos) are responsible for damage to bronchial epithelial cells by releasing toxic eosinophil granule proteins in bronchial asthma. We examined eosinophil cationic protein (ECP) release from human Eos cultured in the presence of human bronchial epithelial cell line BEAS 2B (Ep). ECP release was potentiated only when both Eos and Ep were activated by IL-5 and TNF, respectively, while it was not potentiated when either Eos or Ep were activated. ECP release from Eos activated by IL-5 was also enhanced when Ep was stimulated by IFN-gamma. Paraformaldehyde fixation of Ep had no effect on ECP enhancement, excluding the possibility that soluble factors from Ep contribute to ECP potentiation. Coculture of Eos and Ep with cytokine treatment resulted in the enhancement of eosinophil adhesion and ECP release, and eosinophil adhesion preceded ECP release in the kinetic study. The enhancement of ECP release was partially inhibited by anti-CD18 mAb, which caused partial and comparable inhibition on the potentiation of eosinophil adhesion. These results suggest that the activation of Ep may profoundly affect the ability of cocultured Eos to release ECP and that CD18-dependent adhesion of Eos to Ep may be considered as one of the mechanisms of ECP enhancement. PMID- 8621940 TI - Neutrophils undergo apoptosis following ingestion of Escherichia coli. AB - Apoptosis is a distinct mechanism by which eukaryotic cells die. Neutrophils (PMN) play a fundamental role in the systemic inflammatory response syndrome. Clearance of PMN during resolution of the acute inflammatory process occurs by apoptosis, but factors inducing this process are unknown. The aims of this study were to determine whether PMN ingestion of Escherichia coli would result in PMN apoptosis and whether the mechanism was related to the respiratory burst. PMN from 10 healthy volunteers were cultured with different ratios of PMN:E. coli (1:0 to 1:25) for 12 h. Apoptosis was then assessed by propidium iodide DNA staining, morphology, gel electrophoresis, and Fc gamma RIII expression. There was a significant induction of PMN apoptosis on incubation with E. coli at a ratio of 1:10 and 1:25 PMN:E. coli as well as decreases in Fc gamma RIII. This correlated with increased ingestion of FITC-labeled E. coli and intracellular reactive oxygen intermediates after a 2-h coculture. To clarify the role of reactive oxygen intermediates in E. coli-induced PMN apoptosis, we assessed the effects of the antioxidants catalase, DMSO, glutathione, and N-acetylcysteine. There was a significant decrease in E. coli-induced PMN apoptosis on incubation with DMSO (1.0%), glutathione (25 mM), and N-acetylcysteine (25 mM) compared with control PMN:E. coli. This study demonstrates for the first time that E. coli induces PMN apoptosis through an oxygen-dependent mechanism. The removal of effete PMN by the process of apoptosis rather than necrosis may be teleologically beneficial during Gram-negative septicemia. PMID- 8621941 TI - Binding of HIV-1 virions or gp120-anti-gp120 immune complexes to HIV-1-infected quiescent peripheral blood mononuclear cells reveals latent infection. AB - HIV-1-infected quiescent CD4+ cells harbor the virus in an inactive state until subsequent activation. The possibility that HIV-1 itself and the virus envelope glycoprotein 120 (gp120) might be important agents of this activation was investigated. The present data indicate that binding of heat-inactivated HIV-1 (iHIV-1) to infected resting PBMCs was sufficient to activate NF-kappa B and AP 1, to induce transition from the G0/G1 stage of the cell cycle to the S/G2/M stage, to induce cell surface expression of CD25, to stimulate provirus integration, and to commit cells to produce virus. The cumulative amount of HIV-1 produced by iHIV-1-stimulated cells strictly depended on the concentration of p24gag in the virion preparations used for stimulation. Moreover, virus production was not evidenced in infected resting cells exposed to iHIV-1 previously incubated with soluble CD4 (sCD4), indicating that activation requires a contact between HIV-1 envelope glycoproteins and cell surface CD4. Although soluble gp120 did not stimulate virus production, we found that transition to the S/G2/M stage of the cell cycle, cell surface expression of activation Ags, and virus production were stimulated by cross-linking of CD4 by gp120-anti-gp120 immune complexes. Finally, incubation of gp120-anti-gp120 immune complexes with sCD4 inhibited these effects. These findings suggest that virions and gp120 anti gp120 immune complexes found in infected patients at all times of infection can stimulate virus production in CD4+ cells harboring HIV-1 in an inducible state. PMID- 8621942 TI - Phage display cloning and characterization of an immunogenetic marker (perinuclear anti-neutrophil cytoplasmic antibody) in ulcerative colitis. AB - Ulcerative colitis (UC) is genetically associated with a marker serum Ab (pANCA), identified by its reactivity with a neutrophil Ag. This study utilized phage display technology to clone and characterize pANCA, which has resisted conventional isolation strategies. Since spontaneous pANCA-secreting B cells are detectable in UC lamina propria lymphocytes, this cell source was used to construct a complete IgG1-kappa Ig library. Selection of phage by panning with fixed neutrophils yielded a 195-fold enrichment after five cycles of panning. BstN1 fingerprinting of the enriched library revealed two predominant clones, and DNA sequencing demonstrated highly homologous heavy and light chain variable region segments. Clones were reengineered to express soluble Fab, and neutrophil binding was verified by ELISA. Detailed studies with the two recombinant Fabs, NANUC-1 and -2, validated their identity with serum pANCAs by the criteria of immunofluorescence, confocal microscopy, and DNase I sensitivity. NANUC-1 and -2, like serum UC-pANCA, lack reactivity with previously characterized ANCA-reactive neutrophil proteins and thus detect a novel Ag(s). This study demonstrates the feasibility of selecting phage display Ab libraries on uncharacterized biologic substrates to isolate marker Abs of pathogenic importance. PMID- 8621943 TI - Intact antigen receptor-mediated calcium signals in patients with early stage HIV 1 infection. AB - A variety of deficiencies in T cell activation have been described in HIV-1 infection. To determine whether one component of Ag receptor signal transduction might be impaired and contribute to the immunopathology of HIV infection, we tested CD4 cells from patients with early to mid-stage HIV infection for TCR induced calcium mobilization. There was no detectable difference between patients and controls in the mean CD4 cell calcium response or in the fraction of responding CD4 cells after cross-linking the TCR with OKT3 Ab. In addition, in HIV-infected patients, there was no correlation between calcium mobilization and the CD4 cell count. These results indicate that there are no intrinsic impairments of Ag receptor calcium signaling in circulating CD4 cells from HIV infected patients with more than 400 CD4 cells/mm3, although abnormalities in patients with later stage infections cannot be excluded. PMID- 8621944 TI - Human lupus anti-spliceosome A protein autoantibodies bind contiguous surface structures and segregate into two sequential epitope binding patterns. AB - High levels of anti-spliceosomal autoantibodies are commonly found in systemic lupus erythematosus (lupus) sera. We have evaluated the binding of lupus autoantibodies to octapeptides of nuclear ribonucleoprotein (nRNP) A, identified patterns of binding that vary between sera, quantified the proportion of anti nRNP that binds individual peptides, and related these data to the partial tertiary structure of nRNP A. Anti-nRNP A-positive lupus sera share binding to eight antigenic groups of octapeptides from nRNP A. The four shared antigenic sites in the known nRNP A tertiary structure are contiguous on its surface and include surface loops between beta sheets and/or alpha helices. Anti-peptide Abs account for a significant portion of the human autoimmune response to nRNP A. Lupus sera are also obviously divisible into two subsets by their binding to nRNP A-derived peptides. Eight of the thirteen anti-nRNP A sera tested bind most of the eight common antigenic regions of nRNP A. All five sera in the other subset bind to two and only two groups of nRNP A octapeptides. Of this subset, absorption experiments show that as much as 75% of the anti-nRNP found in a patient serum is bound to these two octapeptides from nRNP A. The 70K spliceosome protein contains similar peptides that are also bound by these sera (OR=13.5, p<0.001). Lupus autoantibodies bind to peptides of nRNP A in discernibly consistent patterns that identify serologic subsets and in a manner that provides useful insights into the autoimmunity and antigenic structure of the nRNP A spliceosomal protein. PMID- 8621945 TI - Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. AB - Several lines of evidence indicate that immunologic effectors, particularly suppressor T cells and NK cells, may play a role in the pathogenesis of idiopathic repetitive abortions. To investigate the involvement of these immune cell populations, we determined the immunophenotypic characteristics of endometrial leukocytes from nonpregnant recurrent aborters. Habitual aborters with a negative investigation underwent an endometrial biopsy during their secretory phase and were followed prospectively to assess clinical outcome. Endometrial leukocytes were evaluated by two-color flow cytometric analysis. The percentage of endometrial CD8+ T lymphocytes was significantly decreased in recurrent aborters, and their CD4:CD8 ratio was increased. In contrast, the proportion of B lymphocytes (CD20+) was strikingly increased in these patients' endometria. The proportion of NK cells was identical in recurrent aborters and normal controls, but the CD16-CD56 bright NK cell subset, which is predominant in normal decidua and endometrium, was significantly decreased in favor of an important contingent of CD16+CD56 dim NK cells in all habitual aborters. Repetitive aborters who had normal CD8+ and CD20+ cell numbers and a normal CD4:CD8 ratio subsequently underwent successful pregnancies, while patients with continuing abortions presented lymphoid populations observed in the habitual aborters group. In conclusion, endometrial lymphocytes of recurrent spontaneous aborters harbor a distinct immunophenotypic profile that antedates implantation and suggests that endometrial immunologic conditions are intrinsically altered in recurrent aborters. Also, the prognostic impact of CD8 and CD20 expression supports their predominant role in the development of fetal tolerance. Finally, a role for NK cells in the abortion process is suggested by their altered subsets in all repetitive aborters. PMID- 8621946 TI - Selective reduction of V alpha 14+ NK T cells associated with disease development in autoimmune-prone mice. AB - A novel peripheral T cell subset characterized by the expression of a NK marker and invariant TCR encoded by V alpha 14 J alpha 281 gene segments with a 1-base N region was investigated in relation to autoimmune disease development. First, we observed that invariant V alpha 14+ NK T cells are specifically reduced with aging in C57BL/6 lpr/lpr or MRL lpr/lpr mice, whereas no change was observed in age-matched control C57BL/6 or MRL +/+ mice as determined by FACS analysis and RNase protection assay. This reduction precedes the disease development and could also be detected in other autoimmune disease-prone mice, such as C3H gld/gld and (NZB x NZW)F1 mice. These results suggest that the specific decrease in invariant V alpha 14+ NK T cells correlates strongly with the development of autoimmunity. Second, injection of MRL lpr/lpr mice with anti-V alpha 14 mAb resulted in the early onset and exacerbation of lymphosplenomegaly due to the accumulation of abnormal CD3+ B220+ CD4-CD8- T cells as well as an increase in the titers of anti dsDNA autoantibodies. These results indicate that V alpha 14+ NK T cells regulate autoimmune responses and play a crucial role in controlling the development of autoimmune diseases. PMID- 8621947 TI - Murine lupus in the absence of alpha beta T cells. AB - To investigate the possibility that non-alpha beta T cell-dependent mechanisms can induce systemic autoimmune disease, and to address the roles of alpha beta T cells in murine lupus, we analyzed lupus-prone MRL mice congenitally deficient in alpha beta T cells. Surprisingly, TCR-alpha-/- MRL mice developed several characteristics of human systemic lupus erythematosus, including hypergammaglobulinemia, autoantibodies against DNA and small nuclear ribonucleoproteins, and immune deposits in kidneys. These results, which contrast with past studies concluding that MRL autoimmunity requires CD4+ alpha beta T cells, demonstrate that non-alpha beta T cell-dependent mechanisms are capable of inducing lupus phenomena, and further suggest that MRL disease may consist of both alpha beta T cell-independent and alpha beta T cell-dependent mechanisms. PMID- 8621948 TI - Flow cytometric and functional analyses of central nervous system-infiltrating cells in SJL/J mice with Theiler's virus-induced demyelinating disease. Evidence for a CD4+ T cell-mediated pathology. AB - Theiler's murine encephalomyelitis viruses (TMEVs) are endemic enteric pathogens of mice that cause immune-mediated, chronic, progressive, central nervous system (CNS) demyelinating disease in susceptible strains. Analysis of T cell phenotype and functional state from TMEV-infected SJL/J mice by flow cytometry reveals that 13.5 to 25% of the CD4+ T cells in the CNS express high affinity IL-2R, a marker of recent T cell activation, whereas splenic levels of CD4+IL-2R+ T cells generally range between 2 and 8.5%. In contrast, very few CD8+ T cells (<1-2%) from either site express IL-2R. From days 20 to 119 postinfection, the percentage of CD4+IL-2R+ T cells increases gradually in the CNS, but varies little in the spleen. CD4+ T cells isolated from the spinal cord of infected mice proliferate in vitro in response to viral Ag. Similar T cell phenotypes were found in experimental autoimmune encephalomyelitis, an established model of CD4+ T cell mediated demyelination. In addition, most CD4+ and CD8+ T cells in CNS isolates from TMEV-infected mice are CD44+, indicating that prior activation may be required to traffic through and/or be retained in the CNS. Finally, TCR V beta region usage as well as IL-2R expression by individual V beta region subsets are heterogeneous in both the CNS and spleen. These results are consistent with a model in which a polyclonal population of TMEV-specific, CD4+ Th1 cells plays a major effector role in the demyelinating process. PMID- 8621949 TI - Immunization with short peptides from the sequence of the systemic lupus erythematosus-associated 60-kDa Ro autoantigen results in anti-Ro ribonucleoprotein autoimmunity. AB - Systemic lupus erythematosus is characterized by the production of Abs to ribonucleoproteins, including Ro (or SS-A) and La (or SS-B). The mechanism by which these autoantibodies arise is unknown. We have immunized rabbits with peptides from the sequence of the 60-kDa Ro. Animals so immunized not only produce Abs to the immunogen peptide, but also develop an autoimmune response to the entire 60-kDa Ro Ag. In about 20% of these animals, autoimmunity also spreads to the La Ag. Anti-native DNA was present in one of nine rabbits. All animals had anti-nuclear Ab, but even among those immunized with the same peptides, different patterns of immunofluorescence were found. Thus, among these outbred animals immunized with peptides from 60-kDa Ro, distinct serologic manifestations were present, showing heterogeneity in the development of autoimmunity. PMID- 8621950 TI - Application of experimental design techniques to optimize a competitive ELISA. AB - This paper describes the application of experimental design techniques to optimize a sensitive ELISA for a hapten molecule with a calibration range of 0 1000 pg/ml. Ten factors that were expected to affect the assay performance were initially screened, followed by factorial experiments to delineate the effects of the critical factors identified at the screening stage. Assay performance was evaluated using a unique rating system based on standard curve reproducibility, assay detection limits and the use of desirability functions. This rating system allowed multiple responses to be evaluated simultaneously. It was found that the substrate incubation time and enzyme label lot played an important role, while dilutions of the enzyme label and the anti-hapten antibody showed significant interaction. These observations were in good agreement with optimal assay conditions based on historical data collected over a period of two to three years. Application of experimental design techniques enabled us to confirm the significance of the factors affecting the assay within a three month period, with a minimum number of experiments. In addition, information on interaction between factors were determined. PMID- 8621951 TI - Production of a monoclonal antibody against the 128 kDa (CagA) protein of Helicobacter pylori. AB - Helicobacter pylori is recognised as an important factor in gastroduodenal pathology. The 128 kDa CagA protein has been established as a useful marker of H. pylori strains associated with more severe forms of disease. A mouse monoclonal antibody raised against the CagA protein has been produced and characterised as belonging to the IgG1 subtype. It identified the protein in all clinical isolates (10/10) from this laboratory and in two NCTC reference strains (NCTC 11637 and NCTC 11961). No cross-reacting proteins were detected in H. pylori L2, a well characterised strain known not to contain the cagA gene, or in four Helicobacter sp. from non-human sources (H. canis, H. mustelidae, H. muridarum and H. acinonyx). The monoclonal antibody was used to develop an antigen capture ELISA system for detecting the presence of antibodies to the CagA protein in human serum samples. PMID- 8621952 TI - Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay. AB - A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies. PMID- 8621953 TI - Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS). AB - Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria. The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS). Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specificity of antigen antibody interactions. Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties. In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB03 used was screened for lipid A/LPS reactivity in ELISA experiments. The binding was confirmed in the described blot system and depends on the membranes and blocking agents used. The use of nitrocellulose versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining. PMID- 8621954 TI - Luminometry: a novel bioluminescent immunoassay enhances the quantitation of mucosal and systemic antibody responses. AB - We have directly compared enzyme-linked immunoassays (ELISAs) with bioluminescent immunoassays employing derivatives of the bioluminescent molecule aequorin, and have shown that detection of mucosal and serum antibodies is considerably more sensitive when detected by luminometry. Luminometry is based upon counting photons of light via phototubes and is generally similar to scintillation spectrometry. Current commercial luminometric technology employs a phototube which is most efficient for light emission in the 400-420 nm wavelength range. For this reason, we have chosen the bioluminescent molecule, aequorin, which upon the addition of Ca2+ undergoes a conformational change resulting in the emission of blue light at 469 nm. The high quantum yield is reflected by the fact that addition of Ca2+ to 1 ng of recombinant streptaequorin, a covalent conjugate of streptavidin and aequorin, resulted in the production of 7 x 10(8) relative light units. In this study, we show the superior sensitivity of biotin-streptaequorin when directly compared with biotin-streptavidin linked horseradish peroxidase commonly used for ELISA. For example, mice orally immunized once with cholera toxin (CT) did not exhibit detectable fecal IgA antibodies as determined by ELISA, whereas use of streptaequorin and the bioluminescent immunoassay revealed fecal IgA anti-CT-B subunit antibody titers of 1:24 500. In addition, no detectable anti-CT-B antibodies were noted in saliva samples by ELISA 7 days following oral immunization with CT, while IgA endpoint titers could be extrapolated to 1:393 000. The 21 day fecal IgA anti-CT-B titers were 1:512 by ELISA, whereas titers determined by luminometry reached 1:10(7) when Neutralite avidin and biotinylated aequorin were employed. In general, the bioluminescent immunoassay was > 10(4)-fold more sensitive when compared with ELISA for detection of mucosal and serum antigen- and isotype-specific antibody responses. Thus, the bioluminescent immunoassay is a more sensitive assay for detection of antibodies in dilute external secretions. PMID- 8621955 TI - Amplification of genes, single transcripts and cDNA libraries from one cell and direct sequence analysis of amplified products derived from one molecule. AB - We report a procedure to generate and amplify cDNA libraries and to amplify and sequence genes and single RNA transcript molecules from the same cell without cloning. An absence of cloning steps minimizes potential sources of contamination, which can be especially problematic when working at the single cell level. Potential contamination is further reduced by an absence of any purification step prior to PCR amplification. Amplifications are designed to minimize the production of aberrant molecules in favor of full-length products, which is especially advantageous when generating cDNA libraries. Genes are amplified from isolated single nuclei, which are segregated from cytoplasmic lysates by microcentrifugation. Specific cDNA, total cDNA or both are synthesized from aliquots of the cytoplasmic lysate, and single cDNA molecules are isolated from others of the same species by limiting dilution prior to PCR amplification. In this way, the frequency of amplified products provides for a direct calculation of cDNA copy number by a Poisson analysis. Incorporation errors by Taq DNA polymerase occur at a low frequency and can be eliminated by sequencing independently amplified cDNA molecules from the same cell. Single molecule amplifications provide sufficient material for numerous (approximately 150) direct DNA sequencing reactions. The limiting dilution approach also permits sequence information to be obtained from a single cDNA, when highly related transcripts derived from distinct genes are present in the same cell and simultaneously amplified with the same primers. In sum, this method provides for a maximum amount of nucleic acid information to be extracted from one cell. It has a wide range of applications to studies of the immune system where, to a first approximation, each lymphocyte has a unique receptor identity, where specific states of differentiation may be difficult to assess in a mixed cell population, and where cell immortalization procedures are not always possible nor practical. PMID- 8621956 TI - Comparison of antibodies raised against the peptide 10-24 of chicken riboflavin carrier protein (cRCP) by classical and multiple antigen peptide (MAP) approaches. AB - Riboflavin carrier protein is an essential protein required for the growth and development of the embryo and hence for the maintenance of pregnancy. Our efforts to delineate the antigenic determinants of chicken riboflavin carrier protein (cRCP) resulted in the identification of a bioneutralization epitope in the region 10-24 of cRCP. The present work compares the properties of the antibodies raised against the peptide epitope by classical and multiple antigen peptide (MAP) system approaches. The extent of cross-reaction of the antibodies to the MAP construct with the parent protein was found to be significantly less as compared to the antibodies raised against the peptide-diphtheria toxoid conjugate. Furthermore, the bioneutralizing ability of the antisera to the MAP construct was also found to be very poor. The results suggest that there are serious limitations in the ability of antibodies raised against MAP constructs to cross-react with the native proteins. PMID- 8621957 TI - A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents. Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B. AB - We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-kappa (in the standard) and each anti-IgG subclass specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing kappa chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5-6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation. PMID- 8621958 TI - Bioimmunoassays for proinflammatory cytokines involving cytokine-induced cellular adhesion molecule expression in human glioblastoma cell lines. AB - Twelve human glioblastoma/astrocytoma cell lines were tested for cellular adhesion molecule expression following cytokine induction in order to identify a cell line that would be suitable for functional cytokine bioimmunoassays. Many of the glioblastoma/astrocytoma cell lines were shown to inducibly express intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1) following stimulation with interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), tumour necrosis factor-beta (TNF-beta), and interferon-gamma (IFN-gamma), but not with any of the several other cytokines tested. The cell line U-138MG, a human glioblastoma-derived line, was the most sensitive one to IL-1 alpha/beta, TNF alpha/beta and IFN-gamma for ICAM-1 expression, comparing well with proinflammatory cytokine-induced ICAM-1 expression in the endothelial cell hybrid EA-hy926 line, and was shown to be useful for the functional assay of the biological potencies of these individual cytokines. Such bioimmunoassays, which are developed by routine ELISA techniques, should provide valuable alternatives to existing bioassays for these cytokines. PMID- 8621959 TI - A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils. AB - Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in unseparated clinical samples. After fixation with parabenzoquinone and permeabilization with n-octyl-beta-D-glucopyranoside, the detection of intracellularly stored proteins was achieved using of monoclonal antibodies against ECP (EG1, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal microscopy was used to demonstrate the intracellular origin of the fluorescent signal. Fixation with parabenzoquinone was superior to a previously described protocol using paraformaldehyde, since it reduces non-specific binding of FITC to the basic proteins in eosinophils. Fixation and permeabilization do not alter the light scatter characteristics of eosinophils in contrast to other leukocytes and thus permit gating on eosinophils without prior purification. Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and simultaneous measurements of surface antigens and intracellular proteins is possible. We have used different clinical samples (peripheral blood, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of intracellular eosinophil proteins by flow cytometry is a rapid, easy and semiquantitative procedure which may be used to study their expression in diseases where eosinophils are involved. PMID- 8621960 TI - A new method for double immunolabelling with primary antibodies from identical species. AB - There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (1) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; "-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron. PMID- 8621961 TI - Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA. AB - Conventional solid-phase immunoassays measuring interactions between anti phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131-135). PMID- 8621962 TI - [Characteristics of enterotoxigenic Escherichia coli and E. coli harboring enteroaggregative E. coli heat-stable enterotoxin-1 (EAST-1) gene isolated from a water-borne outbreak]. AB - A water-borne outbreak occurred in A Town in Akita prefecture on March 1995. Enterotoxigenic Escherichia coli (ETEC) strains were isolated from 6 of 13 feces of patients with food poisoning disease and from 1 of 4 drinking water samples. In addition, E. coli strains harboring Enteroaggregative E. coli (EAggEC) heat stable enterotoxin-1 (EAST-1) gene were isolated from 5 of 13 patient's feces and 1 feces sample obtained from the septic tank. Both of the E. coli strains were isolated from the 3 patient's feces, suggesting that this outbreak was a mixed infectious case. All of the ETEC strains possessed both heat-stable enterotoxin (ST) and EAST-1 genes and their serotype was O148:H28. The EAST-1 gene was detected on a ca. 80 kb plasmid by a southern blot analysis using EAST-1 DNA probe in the 5 of 7 ETEC strains. The southern blot analysis suggested that the location of the EAST-1 gene was genome in the rest of the 2 ETEC strains. A southern blot analysis using ST DNA probe also suggested that the location of the ST gene was genome in all of the ETEC strains. On the other hand, all of the 6 E. coli strains harboring EAST-1 gene could not be serotyped with commercially available OH sera. The location of the EAST-1 gene in all of the isolates was suggested to be genome by the southern blot analysis. All of the isolates lacked aggA gene which has been demonstrated to be involved in expression of aggregative adherence phenotype in EAggEC, suggesting that the EAST-1 gene-harboring strains isolated in this case were distinct from EAggEC. These results indicated that the EAST-1 gene was also harbored by E. coli strains distinct from EAggEC. In addition, a possibility was also suggested that the EAST-1 gene might be a transposon, as well as ST gene. Further study should be conducted in order to elucidate the significance of EAST-1 as a vilurence factor of diarrheagenic E. coli. PMID- 8621963 TI - Evaluation of a direct fluorescent antibody assay for detection of Chlamydia pneumoniae. AB - We examined the utility of the direct immunofluorescent antibody test kit, IMAGEN (DAKO Diagnostic Co., Ltd.). The stainability of inclusions and purified elementary bodies (EBs) of all the Chlamydia pneumoniae strains used in this study was highly specific. Immunoelectron microscopy and light microscopy of stained EBs revealed the target antigen of IMAGEN to be located on the surface of the EB outer membrane. In a clinical study, we tested oropharyngeal swab specimens obtained from 41 patients (45 specimens). The results were compared with those obtained by isolation in cell culture, the indirect immunofluorescent antibody (IFA) assay and serum antibody titration. C. pneumoniae was isolated from six specimens (13.3%), and the organisms were detected in 11 specimens (24.4%) with IMAGEN and nine specimens (20%) with IFA. A diagnosis of C. pneumoniae infection was made in six patients (13.3%) by the serological examination. Six specimens positive in isolation were positive in the IMAGEN but there were three cases which seemed to be false-positive by the IMAGEN. We conclude that the use of IMAGEN is an easy, rapid and sensitive method for detecting C. pneumoniae when there is a large amount of chlamydial antigen in the clinical specimen and when identification of the chlamydial species forming inclusions in cell cultures is required. PMID- 8621964 TI - [Detection of chlamydial antibodies by immunoblotting technique--in comparison of three commercially available test kits and micro-IF]. AB - We reexamined the assay conditions of immunoblotting (W-B) technique to detect antibodies (IgG and IgA) to C. trachomatis serovar L2. Partially purified Chlamydia (35% urografin) was used as the antigen. The marker bands of W-B for positive and/or negative are major outer membrane protein (MOMP) band and either one or more bands staining in 40-62 KDa area. We then compared the sensitivity and specificity of three commercially available test kits and micro-IF by using W B as a standard. The kits compared were sero-IPALISA-IgG-IgA, IP-Azyme-IgG-IgA and HITAZYME-IgG-IgA, and micro-IF. Serum samples were collected from the outpatient departments of gynecology, urology, and internal medicine and pediatrics. The results of agreement between W-B and test kits in IgG detection were as follows: in sero-IPALISA, total agreement was 85.7%, positive agreement 83.3%, negative agreement 90.9% and in IPAzyme: 85.7%, 83.3%, 90.9%, respectively and in HITAZYME: 82.9%, 75%, 100%, respectively. These results are almost the same as micro-IF: 85.7%, 79.2%, 100%, respectively. These kits may have relatively high sensitivity and specificity in IgG antibody detection. In IgA detection, the total agreement between W-B and sero-IPALISA was 82.9%, positive agreement 100%, negative agreement 66.7%, in IPAzyme: 77.1%, 58.8%, 94.4%, respectively and in HITAZYME: 65.7%, 64.7%, 66.7%, respectively and in micro-IF: 82.9%, 70.6%, 94.9%, respectively. Although these agreements are not so high in IgA detection, the W-B technique gives fairly consistent results as well as those kits. This indicates that W-B technique with MOMP and other protein bands (40-62 KDa) as marker for positive reaction is a useful method for detection of chlamydial antibodies. PMID- 8621965 TI - [Epidemiology of rotavirus infection in five different locations in the Asian area]. AB - Between 1991 and 1994, serotypes and electropherotypes were determined for clinical specimens of group A human rotavirus (HRV) rotavirus collected from five different locations in the Asia, Saitama, Shizuoka, Taichung, Hong Kong, and Beijing. HRVs were detected through the year in Taichung and Hong Kong. The peaks of each rotavirus season were in January in Hong Kong, in March in Taichung, and in February and March in Saitama and Shizouka. We set one study year as that from July to next June. Serotype G1 was the most prevalent strain in the five different settings. Most of serotype G3 strains were identified in the study year 1991. Serotype G2 was the most predominant in Shizuoka and Taichung in the year 1993. Electropherotypes indicated the possible G types except in six cases of an unusual variant type whereas the serotype was G2 and the electropherotype was the "long" type. Five of the 502 cases and three of the 622 cases were identified as group C HRV by PAGE in Saitama and Shizouka respectively. In 216 samples in Taichung, no group C HRV strain was detected. PMID- 8621966 TI - [A case of amoebiasis as a latent infection]. AB - A 69-year-old male was admitted because of constipation and abdominal distension. A colon fiberscopic examination revealed the formation of ulceration. The trophozite of Entamoeba histolytica was detected from from the specimens of colon mucosal biopsy of the ulcer. The stool was positive for the cysts of E. histolytica too. He had been in China and Southeast Asia during World War II. He returned home and experienced amoebic dysentry, and was treated with Emethin hydrochlolide. He suffered from amoebic disentry in 1970 and was treated with Emethine hydrochrolide again. After the War he had not gone abroad, and was not a homosexual. So this case was considered a latent infection of E. histolytica from the War. This time he was treated with metronidazol and minocycline. After the therapy, cysts were negative in the stool and the ulcer was cured on the colon fiberscopic examination. PMID- 8621967 TI - [A fatal case of acute enteritis caused by Salmonella Weltevreden after travel to Indonesia]. AB - A 67-year-old male was admitted to our hospital because of watery diarrhea and pre-shock status at 10:30 am on March 20, 1995. He had travelled to Bali Island in Indonesia from March 13 to March 18, 1995. On admission, his systolic blood pressure was 60 mmHg and body temperature was 35.2 degrees C. His skin was very dry. Laboratory tests showed that s-Cr was 6.3 mg/dl and CPK was 5620 IU/l. A massive fluid transfusion was given immediately and then his blood pressure rose to 158/92 about two hours after admission. However, he developed a high grade fever and systemic cyanosis in the evening of the first hospital day and died at 0:20 am on March 21st. Salmonella Weltevreden was detected in the fecal and blood cultures obtained on admission. We considered that his acute renal failure was attributable to rhabdomyolysis due to dehydration and that the cause of death was probably septic shock. The patient had a previous history of cholecystectomy ten years ago and also suffered from hypertension, but his general condition was not so bad before this episode. Therefore, we were surprised that his illness became so severe. This case emphasizes that Salmonella enteritis may occasionally be a serious and lethal disease. PMID- 8621968 TI - [A case of schistosomiasis suspected by circumoval precipitin test and diagnosed by rectal biopsy]. AB - A forty-year-old female from Brazil was admitted to Teikyo Hospital because of easy fatigability, fullness of the abdomen and left hyochondralgia. She was anxious about Schistosoma mansoni infection, because three of her relatives died of the infection. Physical examinations revealed a tenderness at the left hypochondrium. Laboratory data showed no abnormal finding. No egg of S. mansoni was found in the stool. A circumoval precipitin test (COPT) with the serum showed a deposite around the egg. Enzyme-linked immunosorbent assay (ELISA) revealed the presence of antibody against S. mansoni in the serum. A colonoscopy showed no abnormal finding macroscopically. The rectal biopsy showed the existence of mild procitis. The diagnosis was made by finding the characteristic lateral-spined eggs in the biopsy specimens from the rectum. Treatment of 3 g of prazicantel per day for three days was started. She complained of mild nausea at the first dosing. A month later, another three-day-treatment was given. In the case where there are no eggs found in the stool, COPT and ELISA are usefull in detecting the disease, and colonoscopy is recommended in diagnosing the disease. PMID- 8621969 TI - [A case of bacterial empyema caused by Gemella morbillorum]. AB - A 44-year-old female with diabetic ketoacidosis was admitted due to right back pain and dyspnea. A chest roentgenogram showed accumulation of fluid in the right pleural space. A drain was inserted into the right thoracic cavity and pleural fluid yielded a putrid odor. Gemella morbillorum was diagnosed based on culture of the fluid. She was treated with clinidamycin and panipenem/betamipron intravenously and insertion of a drainage tube. The in vitro activity of CLDM and PAPM/BP against G. morbillorum were less than 0.025 micrograms/ml and 0.05 micrograms/ml, respectively. She was cured and discharged after 28 days of hospitalization, the portal of entry was thought to be connected with dental caries. PMID- 8621970 TI - [A case of Lyme borreliosis which was suspected to be caused by Borrelia japonica infection in Shizuoka, Japan]. AB - We report a case of Lyme borreliosis (Lyme disease) found in Shizuoka City, Japan which was suspected to be caused by Borrelia japonica infection. A 8-year-old female was bitten on her head by a tick at a camping ground, near Tamagawa, Shizuoka. The tick was removed by the patient and was discarded before species identification. After one week, lymph node swelling with tenderness developed on her left neck. She consulted a local pediatrician and was suspected to have upper respiratory infection. As oral antibiotic, cefaclor was not effective, the patient was referred to us. The patient's serum showed positive reaction with Lyme Borreliosis ELISA kit (Dakopatts, Denmark) using Borrelia burgdorferi flagellum as antigen. The serum also gave positive results with home-made ELISA to B. japonica strain IKA2, which was isolated from I. ovatus, but not with other borrelial strain isolated in the United States, Europe, and from I. persulcatus and wild rodent in Japan. In western blotting, the serum reacted with flagellin and outer surface protein A (OspA) of B. japonica. We diagnosed her as Lyme disease and got a successful result with oral penicillin, sultamicillin. From a result of our field tick survey, we have not collected I. persulcatus around the area where the patient had a tick bite. These findings indicated that Lyme disease was caused by B. japonica infection with I. ovatus bite. PMID- 8621971 TI - [A case of Legionella micdadei pneumonia]. AB - A 60-year-old male with type C chronic hepatitis was admitted to Kibikogen Rehabilitation Center with high fever, cough and general fatigue. Chest X-ray film on admission showed consolidation in the left middle and lower lung lung field. Initial treatment with intravenous ceftazidime, imipenem/cilastatin and clindamycin were ineffective due to continuous high fever and cough and spread of the pneumonia shadow. Administration of minocycline was started for suspected non bacterial pneumonia whereupon his symptoms improved and the pneumonia shadow began to decrease in size. However, his symptoms and pneumonia shadow worsened after taking him off of minocycline due to progressive pancytopenia and liver dysfunction. He was transferred to our hospital and intravenous erythromycin treatment was initiated for suspected Legionell pneumonia because of the elevation of Legionella micdadei serum antibody titer. Immediately after starting treatment, his symptoms improved and the pneumonia shadow decreased in size. Erythromycin was stopped after the 14th day of administration. In this case, diagnosis of L. micdadei pneumonia was made because of the positive results of the polymerase chain reaction test and elevation of the L. micdadei serum antibody titer (from 0 to 1,024). This is the second report of a L. micdadei pneumonia case here in Japan. PMID- 8621972 TI - [Necrotizing fasciitis due to invasive group A streptococcal infection: a case report]. AB - Necrotizing fasciitis is a relatively rare but potentially fatal soft tissue infection. We report a case of invasive group A streptococcal necrotizing fasciitis. Patient was a 55-year-old healthy male who presented an expanding suppurative lesion over the left lower extremity within one day. Streptococcus pyogenes was a sole microorganism isolated from the infection site. The key to successful treatment in this case was suggested to be early diagnosis combined with aggressive debridement followed by open drainage and high dose administrations of piperacillin before complications such as liver dysfunction and renal failure became serious. PMID- 8621973 TI - [Properties of a hemolysin produced by group B streptococci]. AB - The hemolysin produced by group B streptococci (GBH) has an isoelectric point (pI) of 5.8 and it shows a hemolytic activity in the absence of 2-mercaptoethanol (2-ME). The hemolytic activities of GBH were compared to that of streptolysin O (SLO) and streptolysin S (SLS). These hemolysins differed with respect to the binding and release of hemoglobin (Hb). GBH was bound to phospholipids on the membranes of target erythrocytes, followed by the gentle release of K+ and slow Hb release without lag time. Incontrast SLO released Hb as rapidly as K+. GBH induced hemolysis was inhibited by the addition of 30 mM raffinose. These results indicate that the effective diameter of the pores formed by GBH was about 1.1 nm. GBH showed a lower hemolytic efficiency than SLO, reflecting the fact that these hemolysins destroy erythrocytes by a different mechanism. Intracellular K+ and Hb were released at a different rate in GBH treated cells, indicating that a colloid osmotic process is involved in the lytic mechanism. PMID- 8621974 TI - [Typing of group A streptococci by pulsed field gel electrophoresis]. AB - Restriction endonuclease (Sma I) digestion patterns of chromosomal DNAs of T types 1 and 3 (T1 and T3) group A streptococci (GAS), isolated at two hospitals in Toyama Prefecture from 1983 to 1994, were analysed by pulsed-filed gel electrophoresis (PFGE). One hundred twenty seven isolated of T1 GAS were further divided into 5 PFGE types, which were provisionally designated as T1-1 approximately T1-5. Two epidemics of T1 GAS were observed during the 1983 approximately 1994 period, the first of which (1887 approximately 1988) seemed to be caused by T1-2, and the second (1991 approximately 1993) by T1-5. Some differences in biological characteristics between the two PFGE type strains were also observed. On the other hand, eighty one T3 GAS strains were divided into only two PFGE types, provisionally designated as T3-1 and T3-2, most of which belonged to T3-2. Naturally, both epidemics by T3 GAS, first from 1985 to 1986 and second from 1993 to 1994, seemed to be caused by the same PFGE type, T3-2. No differences in biological characteristics were observed between T3-1 and T3-2 strains. PMID- 8621975 TI - Body composition and in vivo neutron activation. PMID- 8621976 TI - Hemoglobin-based blood substitutes: a dream-like trade of blood and guile? PMID- 8621977 TI - Heparin-induced thrombocytopenia: understanding improves but questions remain. PMID- 8621978 TI - Reliability of in vivo neutron activation analysis for measuring body composition: comparisons with tracer dilution and dual-energy x-ray absorptiometry. AB - In vivo neutron activation (IVNA) analysis has the capacity to measure several total body elements in human subjects. Although it has been considered a criterion method for the past 3 decades, the reliability of IVNA analysis has been tested only in phantom calibrations. In 5 male weight-stable patients with AIDS, total body N, Ca, Cl, Na, P, and C were measured three times in 16 weeks at Brookhaven National Laboratory. With tracer dilution methods for total body water (TBW) by 3H2O and for extracellular water (ECW) by 35SO4 and NaBr, and dual energy x-ray absorptiometry (DXA), total body calcium (TBCa) and fat percentage were measured within 2 weeks of IVNA measurements. For comparison, tracer dilution for TBW by D2O and ECW by NaBr, plus DXA measurements, were performed three times in 5 weight-stable healthy volunteers. The reliability of the IVNA technique was very high in patients with AIDS; it ranged from 0.99 for total body chloride (TBCI) to 0.84 for total body phosphorus (TBP), and it agreed with phantom calibration results in the literature. The reliability for measuring fat percentage and TBCa by DXA was similar in patients with AIDS and in healthy volunteers. Tracer dilution for measuring TBW by 3H2O in patients with AIDS and by D2O in healthy volunteers had a reliability score similar to those found with IVNA and DXA. The reliability scores for measuring ECW in patients with AIDS by 35SO4 and NaBr, 0.66 and 0.68, respectively, were the lowest among all measurements, whereas the reliability score for NaBr in healthy volunteers was 0.96, as with the other measurements. PMID- 8621979 TI - Safety evaluation of a polymerized hemoglobin solution in a murine infection model. AB - Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals. This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial. Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication. Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection. At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXl) has been developed that has improved oxygen affinity and prolonged vascular retention. In the present study the potential risk of this solution on the promotion of infections has been evaluated. PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E. coli, or Candida albicans organisms. PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice. Also, the in vitro proliferation of L. monocytogenes, S. typhimurium, and E. coli was not increased by PolyHbXl. In conclusion, PolyHbXl does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections. PMID- 8621980 TI - Platelet binding of IgG from patients with heparin-induced thrombocytopenia. AB - Although heparin induces immune-mediated thrombocytopenia, it has been difficult to demonstrate heparin specificity of the putative immunoglobin. Recently, however, a body of data has indicated that platelet factor 4 (PF4) is required for heparin-induced thrombocytopenia (HIT) antibody to bind to heparin. Using viable platelets in a physiologic buffer, we have now documented specific and reversible platelet binding of iodine 125-labeled IgG from 5 patients with HIT and binding of 125I-labeled F(ab')2 from 2 of them. The binding requires the presence of both heparin and PF4 in a molar ratio of approximately 1:1. We have also shown that platelet activation increases HIT antibody binding. Our data suggest that the F(ab')2 domain of HIT antibody binds to heparin-PF4 complexes that accumulate on the platelet surface when equimolar concentrations of heparin and PF4 are present. PMID- 8621982 TI - Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity. AB - Fibronectin is a major product of fibroblasts and can mediate diverse functions including wound healing. Chronic bacterial infections are generally associated with a marked decreased in the ability to repair. We therefore hypothesized that bacterial endotoxin, lipopolysaccharide (LPS), might alter fibroblast fibronectin production. LPS augmented fibronectin production by fibroblasts and also stimulated the release of fibronectin from cell layers. An increase in new protein synthesis appeared to account for part of the increased fibronectin, because the inhibitor of protein synthesis, cycloheximide, inhibited the increase in total production of fibronectin. Cycloheximide did not attenuate the increased release of fibronectin into the culture medium. This increased release appeared to be caused, at least in part, by fragmentation of fibronectin by proteases contained in LPS preparations. In this regard all preparations of LPS tested were found to cleave fibronectin. Finally, zymograms indicated that LPS could also cleave gelatin with at least two bands of proteolytic activity but that it did not cleave bovine serum albumin or ovalbumin. These results indicate that the ability of bacterial products to alter fibronectin production and to degrade this macromolecule may account for altered wound repair that occurs with chronic bacterial infection. PMID- 8621981 TI - Risk factors for immunologically mediated disease in workers with respiratory symptoms when exposed to hexahydrophthalic anhydride. AB - Occupational immunologic lung diseases caused by exposures to substances such as hexahydrophthalic anhydride are significant health problems. It would be useful to identify risk factors, other than exposure, for occupational immunologic lung diseases such as occupational asthma. Our objective was to identify risk factors for development of immunologically mediated disease in workers with respiratory symptoms associated with exposure to hexahydrophthalic anhydride. A medical and immunologic survey study was conducted of 33 employees with respiratory symptoms associated with hexahydrophthalic anhydride exposure. Of the 33 employees with respiratory symptoms, 20 had no immunologically mediated disease. Seven had both immunoglobulin E-mediated and immunoglobulin G-mediated disease, 5 had immunoglobulin E-mediated disease only, and 1 had immunoglobulin G-mediated disease only. Although larger samples would have rendered atopy a statistically significant risk factor (assuming effect replication), the associated effect strength represents less than a 16% improvement versus chance, which is indicative of marginal clinical significance. However, as expected, elevated levels of specific antibodies were statistically and clinically significant risk factors. Development of one type of immunologically mediated disease was highly predictive of development of the other type. In hexahydrophthalic anhydride exposed employees with respiratory symptoms, development of immunologically mediated respiratory disease is most closely associated with presence of specific immunoglobulin E or immunoglobulin G antibodies. Neither race, age, smoking status, atopy, nor exposure levels emerged as significant risk factors in this symptomatic study population. PMID- 8621984 TI - Bennhold's analbuminemia: a follow-up study of the first two cases (1953-1992). AB - A pair of siblings with analbuminemia were followed for 38 years. The female patient received replacement therapy with human serum albumin. Extreme lipodystrophy developed in this patient by the fourth decade of life. She had juvenile osteoporosis, which normalized under albumin replacement. She died from a granulosa cell cancer at age 69. Her brother never received albumin, even though his serum contained only 60 micrograms/ml of an albumin-like protein. He suffered from severe osteoporosis with gibbus formation, and he died from a colon carcinoma at age 59. Despite high cholesterol values and high levels of several blood clotting factors, neither of the patients had severe atherosclerosis or thrombotic events. Laboratory findings before and after infusion of large amounts of albumin into the sister point to a mechanism whereby albumin-bound substances can be passively transported from the bloodstream into the extravascular space and vice versa. PMID- 8621983 TI - Infusion of ovine C5a into sheep mimics the inflammatory response of hemodialysis. AB - Previous studies in our group have explored the inflammatory response in sheep to dialysis with a variety of different hemodialysis membranes. In the present study we investigated the potential role of C5a in mediating inflammatory responses that have been attributed to complement activation in the extracorporeal setting. Sheep C5a was infused into sheep in a manner that simulated exposure to this anaphylatoxin during dialysis. C5a infusion into sheep was shown to produce a dose-dependent neutropenia that was quantitatively and temporally identical to the response of sheep undergoing dialysis with complement-activating membranes. The two lowest doses used (0.25 and 0.50 micrograms/kg), which resulted in concentrations below the detectable limits of current assays (10 ng/ml), produced significant neutropenia (21.8% and 78.1%, respectively). The ability of the neutrophils (PMNs) to bind fluorescein isothiocyanate-C5a or initiate a respiratory burst in response to phorbol myristate acetate were also affected in a dose-dependent manner. In contrast, C5a alone was not able to produce significant release of lactoferrin, a specific granule constituent, suggesting that degranulation of PMN-specific and primary granules requires secondary stimuli. The production of thromboxane A2 and thromboxane's consequent cardiopulmonary effect of increasing mean pulmonary artery pressure were both observed in a dose-dependent fashion. However, larger amounts of C5a were required to elicit these latter responses as compared with the PMN activities. These results suggest that C5a may be a primary mediator of complement-dependent events that occur during extracorporeal therapies such as hemodialysis, and they also suggest that very little complement activation is necessary to activate leukocytes, whereas higher thresholds are required to produce cardiopulmonary responses. PMID- 8621985 TI - The pathophysiology of alveolar osteonecrosis of the jaw: anticardiolipin antibodies, thrombophilia, and hypofibrinolysis. AB - We studied 55 patients (50 women, 5 men) with severe facial pain and biopsy proven neuralgia-inducing cavitational osteonecrosis (NICO) of the alveolar bone of the jaws. Our aim was to assess the pathophysiologic contributions to NICO of anticardiolipin antibodies (aCLA), thrombophilia (increased tendency to intravascular thrombi), and hypofibrinolysis (reduced ability to lyse thrombi). Of the 55 patients, 43 (78%) had one or more tests positive for thrombophilia or hypofibrinolysis (or both), and only 12 (22%) were normal. Eighteen of 55 (33%) patients had high aCLA (> 2 SD above mean value for control subjects); immunoglobulin G (IgG) (p = 0.01) and immunoglobulin A (IgA)(p = 0.001) levels were higher in patients than in controls. The distribution of elevated aCLA immunoglobulin classes among patients was as follows: IgG alone, 5 (9%); IgA alone, 7 (13%); and IgM alone, 3 (5%). Three patients (5%) had high levels of both IgG and IgA aCLA. Other defects of the thrombotic or fibrinolytic systems in the 55 patients included high lipoprotein(a) in 36% (vs 20% in control subjects (p = 0.03)), low stimulated tissue plasminogen activator activity (tPA-Fx) in 22% (vs 7% in control subjects (p = 0.08)), high plasminogen activator inhibitor activity (PAI-Fx) in 18% (vs 8% in control subjects (p = 0.03)), resistance to activated protein C in 16% (vs 0% in control subjects (p = 0.007)), low antigenic protein C in 4+ (vs 0% in control subjects (p > 0.2)), and low antigenic protein S in 4% (vs 0% in control subjects (p > 0.2)). Anticardiolipin antibodies and other defects of the thrombotic and fibrinolytic systems appear to be common, potentially reversible pathogenetic risk factors associated with osteonecrosis of the jaw. PMID- 8621986 TI - Separation of mixed red cell populations by using microbead columns. AB - The methods available for separating mixed populations of red blood cells (RBCs) are not completely satisfactory. The purpose of this study was to evaluate the utility of microbead columns for the separation of mixtures of RBCs of different blood groups. Suspensions of RBCs positive for nine different blood group antigens were mixed with RBCs lacking the antigen so that 1%, 5%, 10%, 15%, or 25% at the antigen-positive RBCs were represented. After agglutination by the appropriate antiserum, the antigen-positive RBCs were separated from the antigen negative RBCs by using microbead columns. The average recovery of the antigen negative RBCs in the effluent of the columns of the 135 mixtures of RBCs tested was 83% +/- 5% (mean +/- SD). The absence of contaminating antigen-positive RBCs was established serologically and by flow cytometry. The procedure was effective in removing as little as 1% of antigen-positive cells from a mixture. Microbead columns offer a simple and efficient method for separating mixtures of RBCs for biochemical, clinical, and serologic studies. PMID- 8621987 TI - Mononuclear cell conditioned medium enhances bronchial epithelial cell migration but inhibits attachment to fibronectin. AB - The attachment and migration of bronchial epithelial cells are important features in re-epithelialization after tissue injury. We hypothesized that inflammatory cytokines might alter bronchial epithelial cell attachment and migration. To test this hypothesis, we evaluated the effects of mononuclear cell conditioned medium (MNCCM) on attachment and migration of bronchial epithelial cells to fibronectin in vitro. MNCCM was prepared from bovine blood mononuclear cells that were stimulated with concanavalin A. MNCCM stimulated bronchial epithelial cell migration and spreading. Sephadex G-75 column chromatography of MNCCM found two peaks of migration-stimulatory activity. Activity in the higher molecular weight peak was partially inhibited by anti-tumor necrosis factor-alpha antibodies. Activity in the low-molecular-weight peak was lipid-extractable, suggesting the possibility that the activity was an arachidonate metabolite. We evaluated the effects of protein kinase C (PKC) inhibitors on enhancement of bronchial epithelial cell migration by MNCCM under the hypothesis that stimulated bronchial epithelial cell migration by MNCCM was elicited through PKC-dependent signaling pathways. PKC inhibitors, calphostin and H-7, inhibited the effect of MNCCM on bronchial epithelial cell migration. In addition, MNCCM stimulated PKC translocation and activity in these cells. Thus mononuclear cells produce inflammatory cytokines with important effects on bronchial epithelial cell migration and spreading. The stimulatory effect may be mediated in part through PKC signaling pathways. PMID- 8621988 TI - Down-regulation of glucose transport by elevated extracellular glucose concentrations in cultured rat aortic smooth muscle cells does not normalize intracellular glucose concentrations. AB - Vascular disease is a prominent complication of diabetes mellitus, and hyperglycemia has been implicated as a risk factor for the development of these vascular complications. It has previously been suggested that down-regulation of glucose transport in response to hyperglycemia might serve a protective role by decreasing intracellular glucose concentrations. In the present study, regulation of glucose transport by extracellular glucose concentrations was investigated in cultured rat vascular smooth muscle cells (VSMCs). Confluent quiescent VSMCs were exposed to medium containing either normal (5 mmol/L) or elevated (20 mmol/L) extracellular glucose concentrations for 24 hours. VSMCs exposed to elevated extracellular glucose concentrations (with or without serum) for 24 hours exhibited significant decreases in 2-deoxyglucose (2-DG) and D-glucose uptake rates. This decreased glucose transport was associated with a decrease in the Vmax of D-glucose transport without a change in KM. In the absence of serum, a decrease in the quantity of GLUT-1 transport protein at the plasma membrane was noted in cells exposed to elevated extracellular glucose concentrations for 24 hours. Intracellular glucose concentrations were estimated by using two methods, and the results revealed significantly higher intracellular glucose concentrations in the cells exposed to elevated extracellular glucose concentrations for 24 hours. These results suggest that down-regulation of glucose transport in cultured VSMCs exposed to elevated extracellular glucose concentrations for 24 hours does not occur to an extent that normalizes intracellular glucose concentrations. This prolonged increase in intracellular glucose concentrations and the potential associated toxicity may explain the increased incidence of vascular complications in patients with diabetes mellitus. PMID- 8621989 TI - Can treatment that is helpful on average be harmful to some patients? A study of the conflicting information needs of clinical inquiry and drug regulation. AB - Randomized controlled trials are conducted with heterogeneous groups of patients, and the trial results represent an estimate of the average difference in the responses of the treatment groups. Clinicians, however, engage in a process of clinical inquiry, assembling data that will allow an assessment of the appropriate choice of treatment according to more narrowly defined clinical features. We describe a method of clinical inquiry within RCTs that can enhance the applicability of results to clinical decision making. Our methods included the use of data from the Beta-Blocker Heart Attack Trial, which enrolled 3837 subjects in 31 clinical centers. The 31 centers were divided into 21 dominant centers (mortality rates higher for placebo than propranolol) and 10 divergent centers (higher mortality rates for patients randomized to propranolol). Overall, compared to placebo, propranolol reduced the risk of dying for the "average" patient from 9.8 to 7.2%. Results for patients in dominant centers (RR = 0.50) were significantly different from those in divergent centers (RR = 1.33). We identified two cotherapies--aspirin use and coronary artery surgery--that subsequently affected the benefits of propranolol in divergent centers. For patients in divergent centers, propranolol reduced the risk of dying for patients treated with aspirin and/or coronary surgery (RR = 0.39), but not for patients not receiving these therapies (RR = 1.42). We conclude that differences in results across centers of a multicenter RCT may reflect important distinctions in the clinical conditions of enrolled subjects. These distinctions help to identify subgroups of patients in which treatment that has an average overall benefit may be harmful for some patients. PMID- 8621990 TI - Reproducibility and validity of a food frequency questionnaire in a case-control study on breast cancer. AB - A 110-item food frequency questionnaire was tested among 152 community controls of the Kuopio Breast Cancer Study. They completed the questionnaire twice and kept two 7-day diet records at 3-month intervals. The intraclass correlations for nutrients varied from 0.49 (thiamine with supplements) to 0.81 (lactose), and for foods from 0.52 (poultry) to 0.84 (alcoholic drinks). The Pearson correlations between the first food frequency questionnaire and the 14-day diet records, after adjustment for energy, varied for nutrients from 0.18 (thiamine without supplements) to 0.80 (alcohol), and for foods from 0.30 (inner organs) to 0.90 (coffee). Comparison of quintile classification between the two methods is reasonably accurate when observed restrictions concerning some nutrients and foods are taken into account. A low association (r = 0.12) was observed between toenail selenium and dietary selenium intake, indicating the difficulty of estimating selenium intake in the Finnish diet. PMID- 8621991 TI - Tracking of glycated hemoglobin in the original cohort of the Framingham Heart Study. AB - Glycated hemoglobin measures average blood glucose over the preceding 2 to 3 months. The authors examined the tracking of the major glycated hemoglobin A1c (HbA1c), over a period of 4 to 6 years. Two HbA1c measurements were obtained between 1986 and 1993 from 639 elderly, presumptively nondiabetic members of the original cohort of the Framingham Heart Study, Framingham, Massachusetts. Mean +/ standard deviation (SD) baseline and follow-up HbA1c were 5.43% +/- 0.7 and 5.71% +/- 0.9, respectively. Intraclass correlation of 0.59 between baseline and follow-up measurements indicated good reliability of a single HbA1c measurement. Ninety-one percent of follow-up measurements were within +/- 20% of baseline value; HbA1c values tended to move 15% closer to the baseline mean over time. There was a modest tendency for HbA1c values to increase with time; the mean difference between measurements was 0.28% +/- 0.7 SD (p < 0.0001). Change in HbA1c was positively associated with age and body mass index at baseline examination, and negatively associated with cigarette smoking, even after controlling for age and body mass index. These effects were very small, however. We conclude that glycated hemoglobin reliably categorizes the glucose control of nondiabetic subjects over a period of 4 to 6 years, confirming its value as an epidemiological measure. PMID- 8621992 TI - Standard error and sample size determination for estimation of probabilities based on a test variable. AB - A method of sample size determination for estimation of probabilities based on a test variable is presented. Applications to estimation of sensitivity and specificity of medical tests are the focus of this research, although the methods can be applied to other areas of study such as engineering reliability. Examples are given for determining sample sizes required for the classification of patients with cutaneous lupus erythematosus based on the incidence of several markers. In this example, the test variable is the number of markers present. The methodology employs a weighted average of model-based and non-model-based estimates of the probability with the weights determined by the closeness to or the confidence in the given model. Formulas and charts required for determining sample size are provided for test variables that can be modeled by the binomial, Poisson, or normal distributions, i.e., for the most commonly encountered distributions for counting events (binomial and Poisson) and for measurements (normal). However, the methods given can be applied to any distribution, including multivariate. Especially when relatively small probabilities (the rare events) are being estimated, the techniques provided assistance in safeguarding against undersampling brought on by unwarranted confidence in a test variable distribution and against oversampling required for high accuracy in non-model based probability estimators. PMID- 8621993 TI - Behavior and interpretation of the kappa statistic: resolution of the two paradoxes. AB - Two apparent paradoxes have been identified for the kappa (kappa) statistic: (1) high levels of observer agreement with low kappa values; (2) lack of predictability of changes in kappa with changing marginals. The first paradox is a function of prevalence of the trait in the sample, while the second is related to symmetry of observations in the disagreement categories. While examining the behavior of kappa as a function of the distribution of responses in a contingency table, it was discovered that for any measured level of observer agreement (Po) there are three characteristic values of kappa: kappa max, kappa min, and kappa nor, each of which is a function only of Po. The characteristic values allow an observed kappa (kappa o) to be placed into perspective. By observing symmetry in agreement and disagreement categories, the behavior of kappa is readily understood and predictable. We define symmetry expressions for agreement (SA) and disagreement (SD) in order to represent and quantify these effects. Kappa alone has little interpretive value and we recommend that studies reporting kappa also report Po, SD, and P++ (agreement on the presence of the trait). PMID- 8621994 TI - Statistical considerations in the design and analysis of community intervention trials. AB - Community intervention trials are often characterized by the allocation of intact social units to different intervention groups. The assessment of adequate sample size for such trials must take into account the statistical dependencies among responses observed within an allocated unit. However, the small numbers of units typically involved in such trials imply that many methods of analysis that have been proposed for analyzing correlated data, particularly in the case of a dichotomous outcome variable, are not applicable to such designs. In this article we investigate this issue and determine the minimum number of units required per group, for the case of both a dichotomous and a continuous outcome variable, needed to provide adequate statistical power for detecting various levels of treatment effect. The use of significance testing as a method of detecting intracluster correlation is also investigated, and, in general, discouraged. PMID- 8621995 TI - Determinants of preterm delivery in low-risk pregnancies. The RADIUS Study Group. AB - From 14,948 low-risk singleton pregnancies, we calculated incidence, risk ratios, and attributable risks for characteristics associated with spontaneous and medically induced preterm delivery. There were 754 women who gave birth prior to 37 weeks of gestation (50.4/1000 deliveries). The greatest fraction of the incidence of prematurity among low-risk pregnancies was due to unknown factors associated with carrying a first live birth, regardless of preterm delivery mechanism (i.e., spontaneous labor, PROM, medical intervention), with population attributable risk percents (PAR%) ranging from 16.0 to 30.5%. Other than nulliparity, male sex of the fetus accounted for the greatest fraction of spontaneous labor-induced prematurity incidence (PAR% = 13.6%), and maternal age greater than 30 years or a positive urine culture accounted for the greatest fraction of PROM-induced prematurity incidence (PAR% = 7.9 and 6.7, respectively). All other risk factors for either preterm labor or PROM accounted for less than 5% of the incidence. Three characteristics explained a large fraction of medically induced prematurity: women over 150 pounds at the onset of pregnancy (PAR% = 23.8), a > or = 2+ prenatal urine protein (PAR% = 18.7%), and cigarette smoking during the first trimester (PAR% = 8.6). Our results suggest known risk factors may explain only a small fraction of spontaneous preterm delivery incidence in low-risk pregnancies. PMID- 8621996 TI - Hematological variables in cord blood of neonates of smoking and nonsmoking mothers. AB - Smoking during pregnancy is associated with maternal and fetal complications. In the present study the effect of maternal smoking on neonatal cellular blood components was investigated. The values of whole blood cell count, leukocyte differential count, and thrombocyte and reticulocyte counts were determined and compared in cord blood of neonates of nonsmoking (n = 89) and smoking (n = 53) mothers. The variables of the erythrocyte and thrombocyte count were not different in cord blood of neonates who were exposed to smoke and in those who were not. In the reticulocyte range the reticulocyte count was significantly lower in the smoking group, while the reticulocyte subsets remained stable. The neutrophils were significantly lower in cord blood of neonates of smoking mothers (p < 0.05). The latter finding might be an explanation for the enhanced incidence of postnatal infection seen in children of smoking mothers. PMID- 8621997 TI - A comparison of prospective and retrospective assessments of sleep. AB - The main purpose of this study was to evaluate the correlations between retrospective and prospective assessments of sleep. Subjective reports of sleep characteristics and sleep quality were obtained from 146 young (20-40 years, average age 29.15) healthy men (n = 43), nonpregnant (n = 70) and pregnant women (n = 33). Three types of subjective sleep reports were obtained: (1) general estimates of sleep habits (retrospective measures); (2) sleep logs completed upon awakening on three successive workdays (prospective measures); and (3) general sleep complaints. Men showed the greatest stability in the sleep log reports, followed by nonpregnant women, with pregnant women showing the least stability. Significant positive correlations between prospective (log reported) and retrospective (general estimates) sleep measures were found only for men. However, for measures of sleep quality, such as feelings of fatigue upon awakening, women showed greater agreement between their general estimates and three nightly reports. The limitations of using retrospective estimates in sleep surveys are discussed. PMID- 8621998 TI - Psychosocial characteristics of asthma. AB - The objective of this study was to compare psychosocial characteristics of children with asthma and children with bronchial hyperreactivity with those of normal children. A population-based study of 2634 children (mean age, 10 years) was carried out. Pulmonary function tests of children were performed in children before and after cold air hyperventilation challenge to determine bronchial hyperreactivity. Parental assessment of children's behavior was evaluated with 15 questions about school/learning habits, level of activity, communication/affection, and sleeping patterns. A factor analysis was performed and the factor loading adjusted for confounders compared in the different groups. Asthmatic children sleep less well than normal and hyperreactive children (p < 0.001). Unexpectedly, however, all other single items did not differ significantly. As a result of the factorial analysis we obtained two factors. On the first factor, measuring school behavior and learning, there was a small difference between asthmatic and normal children, which could not be found on the second factor indicating activity and communication. We conclude that psychosocial differences of asthmatic children are less remarkable than expected. As a result of the examination of the hyperreactive children it is likely that asthmatic children are influenced more by secondary psychosocial factors than by any primary effect of asthmatic disease. PMID- 8621999 TI - Psychosocial and neurological predictors of mental health in multiple sclerosis patients. PMID- 8622000 TI - Comparing period prevalences with application to drug utilization. AB - Period prevalence is frequently measured in studies based on administrative data such as that from health maintenance organizations. For example, treated prevalence and drug utilization prevalence are important measures that are typically defined in relationship to a specified time period. Often one wishes to compare administrative data with period prevalences based on national surveys. It may also be of interest to compare period prevalences from two (or more) different data sources. This comparison is not straightforward owing to the problem of "person-time at risk." This article reviews the values and drawbacks of period prevalence as compared with cumulative incidence. L ife table methodology is described for comparing period prevalence data from administrative databases with survey results. This technique can be extended to the comparison of period prevalence observations from two or more administrative data bases. Examples are given pertaining to hypnotic drug use and the treatment of schizophrenia. PMID- 8622001 TI - Adverse effects of medications on urinary symptoms and flow rate: a community based study. AB - The relationship between urinary symptoms and medication use was investigated in a community-based cross-sectional study involving a random sample of 2115 men 40 79 years of age in Olmsted County, Minnesota. The American Urological Association Symptom Index (AUASI) was generated from a validated self-administered questionnaire. Medication use was assessed by in-person interviews. While 1087 men reported daily medication use, only 136 reported daily use of medications known to affect urinary function adversely, including antidepressants (42), antihistamines (23), and bronchodilators (43). Age-adjusted AUASI scores were higher in men reporting daily use of antidepressants, and the association persisted after additionally adjusting for the Depression and Anxiety subscales of the General Psychological Well-Being Scale (adjusted mean difference, 2.1; 95% confidence interval (CI), 0.5-3.6; p = 0.008). The adjusted AUASI was also higher among men who took antihistamines daily (adjusted mean difference, 2.3; 95% CI, 0.3-4.3; p = 0.03). Lower age-adjusted urinary flow rates occurred with antidepressants, but not with antihistamines or bronchodilators. Clinicians evaluating men for causes of voiding dysfunction in accordance with the Agency for Health Care Policy and Research practice guideline for the diagnosis and management of benign prostatic hyperplasia should be aware that daily use of antidepressants or antihistamines may be associated with AUASI scores that are two to three points higher than in men not taking these medications. PMID- 8622002 TI - Results of an epidemiological study on drug-treated intraocular hypertension in Belgium. AB - Some epidemiological aspects of drug-treated intraocular hypertension in Belgium were established in 1992 from questionnaires filled in by 1513 patients who attended a sample of 209 pharmacies open to the public. The mean age of the patients was 67 years (range: 3-95). After standardization for age, the prevalence of diabetes mellitus among patients was found to be 1.7 times higher than in the general population. Family history of glaucoma and/or blindness was reported by 28% of the patients. Beta-blocker eye drops were used by 96% of the sample. An ophthalmologist was consulted more than four times a year by 24% of the patients who had been drug-treated for less than one year; the proportion, however, decreases to 11% for those treated for at least five years. The annual consumed dose of eye drops containing parasympathomimetics, beta-blockers, and epinephrine or dipivefrine was 12.4, 13.6, and 12.3 bottles, respectively, and that of ointments containing parasympathomimetics was 9.9 tubes. From these results and using sales figures, the prevalence of drug-treated intraocular hypertension was found to be 77 per 10,000 inhabitants (95% CI: 31-123). PMID- 8622003 TI - Selection bias, indexes of "burden," and risk. PMID- 8622004 TI - Italy publishes anesthesia monitoring protocols. PMID- 8622005 TI - Chemotherapy in malignant pleural mesothelioma. A review. AB - PURPOSE AND DESIGN: We reviewed the published literature of clinical studies in malignant pleural mesothelioma, including phase II trials of the newer antifolates and plant derivatives, as well as older single-agent and combination chemotherapy trials. We excluded trials with less than 15 patients, although we have mentioned smaller trials in the text to make a specific point, as well as ones that show promise. We have also included confidence intervals when cited in the original reports, or calculated them when absent. RESULTS: No drugs have consistently induced a response greater than 20%. Higher response rates have been reported with detorubicin, high-dose methotrexate, and edatrexate at 26%, 37%, and 25%, respectively, but these have yet to be confirmed. Agents that produce response rates in 10% to 20% of patients include doxorubicin, epirubicin, mitomycin, cyclophosphamide, ifosfamide, cisplatin, and carboplatin. Combination chemotherapy trials do not demonstrate a consistently greater response rate than single-agent trials. However, the combination of doxorubicin, cisplatin, bleomycin, and mitomycin demonstrated a response rate of 44% (95% confidence interval, 27% to 63%), but this remains unconfirmed. Intrapleural therapy using interferon gamma, particularly for small-volume disease, shows promise. CONCLUSION: The successful treatment of unresectable pleural mesothelioma awaits the discovery of active drugs. Recent trials of high-dose methotrexate and other antifolates are encouraging. Newer agents, including suramin, should be evaluated in phase II trials. Off-protocol combination therapy cannot be recommended over single-agent therapy, but studies that use combinations of the newer agents should be conducted. PMID- 8622006 TI - Tamoxifen-associated eye disease. A review. AB - PURPOSE: The oral antiestrogen tamoxifen has demonstrated efficacy in the treatment of metastatic breast cancer and as adjuvant therapy in early-stage disease. Clinical trials of tamoxifen in chemoprevention of breast cancer among high-risk women have focused attention on potential adverse effects of long-term tamoxifen use, including the possibility of ocular toxicity. This review evaluates the published case reports, clinical series, and clinical trial data on ocular toxicities attributed to tamoxifen. Clinical issues of surveillance, differential diagnosis, and management of tamoxifen-related eye disease are discussed. DESIGN: National Library of Medicine online bibliographic services were used to identify case reports and clinical studies of ocular adverse effects that occurred in patients receiving tamoxifen published through the fall of 1994. The medical literature relevant to issues raised by the reports and studies was similarly identified and reviewed. RESULTS: Case reports and case series identify crystalline retinal deposits, macular edema, and corneal changes as potential tamoxifen ocular toxicities. Extensive retinal lesions and macular edema with visual impairment have been reported in a few patients receiving high-dose tamoxifen. Less extensive retinal changes may occur in patients receiving low doses for long periods, and isolated retinal crystals may be observed in patients without visual symptoms. CONCLUSION: Ocular toxicity is uncommon in the current clinical setting of long-term, low-dose tamoxifen use. Physicians should be aware of the potential for ocular toxicity among patients receiving the drug and should assure appropriate surveillance and prompt evaluation of visual complaints. PMID- 8622008 TI - Medical oncology for the general internal medicine trainee. American Society of Clinical Oncology. PMID- 8622007 TI - Commentary on endometrial cancer deaths in tamoxifen-treated breast cancer patients. PMID- 8622009 TI - Paclitaxel hypersensitivity revisited. PMID- 8622010 TI - Strange bedfellows. PMID- 8622011 TI - Major toxicity of cisplatin, fluorouracil, and leucovorin following chemoradiotherapy in patients with nasopharyngeal carcinoma. PMID- 8622012 TI - Positron emission tomography in the clinical evaluation of metastatic cancer. PMID- 8622013 TI - Should we treat HER, too? PMID- 8622015 TI - Irinotecan is an active agent in untreated patients with metastatic colorectal cancer. AB - PURPOSE: To determine the response rate, survival, and toxicity of the new anticancer agent, irinotecan (CPT-11), in the treatment of metastatic colorectal cancer. PATIENTS AND METHODS: Forty-one chemotherapy-naive patients with measurable metastatic colorectal cancer were treated with a 90-minute infusion of irinotecan 125 mg/m2 administered weekly for 4 weeks every 6 weeks. Pretreatment tumor biopsies to assess topoisomerase-I (Topo-I) activity were obtained from 11 patients. The pharmacokinetics for irinotecan and its active metabolite, SN-38, were determined in 18 patients. RESULTS: Thirteen of 41 patients (32%) had a partial response (PR; 95% confidence interval, 18% to 46%). The median response duration was 8.1 months (range, 4.0 to 16.0) and the median survival time was 12.1 months (range, 2.1 to 21.7) for all 41 patients. Grade 3 or 4 toxicities were diarrhea (29% of patients) and neutropenia (22% of patients). Grade 3 or 4 diarrhea was substantially more prevalent in the initial 18 patients on study, with an incidence rate of 56%; a significant reduction in the incidence of severe diarrhea to 9% was noted with strict adherence to an antidiarrheal regimen of loperamide and diphenyldramine. No correlations were seen between pharmacokinetics of irinotecan/SN-38 and the clinical parameters of response, survival, or incidence of diarrhea. CONCLUSIONS: Irinotecan has activity in the treatment of patients with metastatic colorectal cancer. Strict adherence to an antidiarrheal regimen of diphenhydramine/loperamide significantly reduced the incidence of diarrhea; the agent was thereafter well tolerated in the majority of patients. PMID- 8622014 TI - Noninvasive monitoring of tumor metabolism using fluorodeoxyglucose and positron emission tomography in colorectal cancer liver metastases: correlation with tumor response to fluorouracil. AB - PURPOSE: To investigate and measure the metabolism of colorectal cancer liver metastases using 18F-fluorodeoxyglucose positron emission tomography (FDG PET), before and during the first month of chemotherapy. The findings were compared with tumor outcome conventionally assessed using changes in tumor size. PATIENTS AND METHODS: Patients with colorectal cancer liver metastases were treated with fluorouracil (5FU) as a protracted venous infusion (300 mg/m2/d), with or without interferon-alpha 2b for two 10-week blocks separated by a 2-week break. Before and at 1 to 2 and 4 to 5 weeks on treatment, FDG PET scans were performed. Patients fasted, were injected intravenously with FDG (50 to 100 MBq), and scanned using a large-area positron camera; the image data was processed such that regions of interest could be identified. The results were expressed as a ratio of FDG uptake in the tumor and normal liver (T:L) or as a semiquantitative standardized uptake value (SUV). These measures were compared with the tumor dimensions measured on a computed tomographic (CT) scan performed at 12 weeks from commencement of chemotherapy. RESULTS: Twenty patients were studied; however, two did not have assessable liver metastases. Objective partial responses were observed in 11 of 18 patients. A total of 27 metastatic lesions were assessable. Pretreatment T:L ratios and SUVs did not correlate with tumor response, although response was associated with lower 1- to 2-week (1.84 v 2.17; t=2.667; P < .02) and 4- to 5-week (1.36 v 2.28; t=5.02; P < .001) T:L ratios, and 4- to 5-week (3.57 v 4.95; t=2.492; P < .05) SUVs. Expressed as a percent of the baseline values of the T:L ratio, responding lesions had a greater reduction in metabolism (67% v 99%; t=7.53; P < .001). The 4- to 5-week T:L ratio was able to discriminate response from nonresponse both in a lesion-by-lesion and overall patient response assessment (sensitivity 100%; specificity 90% and 75%, respectively). CONCLUSION: Positron emission tomography used to evaluate the uptake of FDG in tumors yields data that correlate with the antitumor effect of chemotherapy in patients with liver metastases from colorectal cancer. PMID- 8622016 TI - ZD1694: A novel thymidylate synthase inhibitor with substantial activity in the treatment of patients with advanced colorectal cancer. Tomudex Colorectal Study Group. AB - PURPOSE: Tomudex (ZD1694; Zeneca Ltd, Macclesfield, United Kingdom) appears to have a favorable toxicity profile (defined in phase I studies) and antitumor activity in a broad range of epithelial tumors. We report here the results of a large phase II study of Tomudex in advanced colorectal cancer (CRC). PATIENTS AND METHODS: One hundred seventy-seven patients were entered onto the study between October 1992 and September 1993. Patients were required to have advanced CRC without prior chemotherapy (adjuvant chemotherapy was permissible) and at least one measurable lesion. Tomudex (ZD1694) was administered at a dose of 3 mg/m2 intravenously once every 3 weeks in the absence of toxicity or disease progression. Patients were assessed for objective response, progression, and survival. RESULTS: Of 177 patients entered onto the study, 5% had received prior adjuvant chemotherapy and 83% had liver metastases. Objective responses were seen in 26% of patients (95% confidence interval, 19% to 33%; four complete responses [CRs] and 41 partial responses [PRs]) while median time to progression was 4.2 months and median survival 9.6 months. All sites were audited, and responses were reviewed by an independent panel. Common toxicities included mild reversible transaminitis, nausea and vomiting, and asthenia or flu-like symptoms, and World Health Organization (WHO) grade 3 and 4 leukopenia and diarrhea were seen in 6% and 9.8% of patients, respectively. Stomatitis and alopecia were common. CONCLUSION: In this large multicenter phase II study of patients with advanced CRC, interesting activity was seen (objective response rate, 26%). In addition, Tomudex has an acceptable toxicity profile and a convenient dosing schedule (single intravenous injection every 3 weeks) and thus appears to offer real potential as a novel agent for the treatment of patients with advanced CRC. PMID- 8622017 TI - Combined liver radiation and chemotherapy for palliation of hepatic metastases from colorectal cancer. AB - PURPOSE: To report the effects of boost dose radiation on palliation, survival, and toxicity in patients undergoing palliative treatment for hepatic metastases from colorectal cancers and to assess the potential benefits of higher doses of radiation to partial liver volumes. MATERIALS AND METHODS: Forty-five patients with hepatic metastases from colorectal cancers were treated with a course of palliative irradiation. Eligible patients included those with radiographically or histologically proven liver metastases. All patients but one received chemotherapy, either pretreatment (one patient) and/or concurrently with radiation (43 patients) via intravenous or hepatic intraarterial infusion. Patients were divided into two groups based on whether or not boost radiation was given. Thirty-three of the 45 patients (group 1) received whole-liver irradiation at doses that ranged from 8 to 31 Gy at 2.0 to 3.0 Gy per fraction (median dose, 21 Gy). The remaining 12 patients (group 2) received liver irradiation to 20 to 30 Gy followed by a boost dose to the area of dominant disease for a total dose of 33 to 60 Gy. The extent of liver involvement was similar between the two groups. Palliation, overall survival, and toxicity were analyzed with respect to radiation dose. RESULTS: There was no increase in acute effects observed in treating partial liver volumes to higher doses in conjunction with systemic chemotherapy. No cases of radiation-induced hepatitis or nephritis were documented. Hematologic toxicity (> or = grade 3) was observed in four patients with thrombocytopenia, three with leukopenia, and two with anemia. Pain was relieved in 71% and hepatomegaly in 59% of group 1 patients, as compared with 100% and 89%, respectively, of group 2 patients. Other symptoms such as nausea, fever, fatigue, and jaundice were palliated in 35% of group 1 and 90% of group 2 patients. The median survival time for group 1 patients was 4 months (range, 1 week to 26 months), which is consistent with that reported in the literature. The median survival time for group 2 patients was 14 months (range, 2 to 32 months) (P=.01). CONCLUSION: Standard hepatic irradiation followed by boost radiation to partial liver volumes in combination with chemotherapy is well tolerated without significant acute/late morbidity. Higher radiation doses to partial liver volumes offers improved palliative benefit and may prolong survival without an increase in morbidity. PMID- 8622018 TI - Intravenous azidothymidine with fluorouracil and leucovorin: a phase I-II study in previously untreated metastatic colorectal cancer patients. AB - PURPOSE: To determine the plasma pharmacokinetics and the maximum-tolerated dose (MTD) of intravenous (IV) azidothymidine (AZT) administered 90 to 120 minutes after fluorouracil (5-FU) and leucovorin and to preliminarily evaluate the antitumor activity of this combination in metastatic colorectal cancer. PATIENTS AND METHODS: 5-FU 500 mg/m2 IV bolus was administered once a week in the middle of a 2-hour infusion of leucovorin; AZT was given as a 90 to 120-minute IV infusion 60 minutes after 5-FU. Initial AZT dose was 0.5 g/m2, and it was escalated in successive cohorts of three patients by 0.5 to 2 g/m2. RESULTS: Thirty-five chemotherapy-naive metastatic colorectal cancer patients were entered onto the study, and AZT doses ranged from 0.5 to 10 g/m2. The peak AZT plasma concentration increased from 21.9 to 995.6 micromol/L. The area under the concentration/time curve (AUC) also showed a progressive, but not linear increase from 40.34 to 3,108 h x micromol/L. The most relevant toxicity was diarrhea, which was severe in six patients (17%). Toxicities were not AZT-dose-related, except fpr hypotension, which occurred in patients treated at AZT doses > or = 7 g/m2 and became dose-limiting for AZT 10 g/m2. Among 34 assessable patients, 15 objective responses were observed (44%; 95% confidence interval 27 to 62), lasting a median of 44 weeks; five (15%) were complete. CONCLUSION: AZT doses > or = 6 g/m2 administered IV over 90 to 120 minutes produce maximum plasma concentration and AUC similar to those previously reached in murine tumor models. Dose-limiting toxicity is hypotension, which occurs at AZT 10 g/m2. The recommended AZT dose for further studies is 8 g/m2. The combination of 5-FU plus leucovorin plus AZT is feasible with acceptable toxicities, and has promising activity in metastatic colorectal cancer. PMID- 8622019 TI - Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. AB - PURPOSE: Breast cancer frequently overexpresses the product of the HER2 proto oncogene, a 185-kd growth factor receptor (p185HER2). The recombinant humanized monoclonal antibody (rhuMAb) HER2 has high affinity for p185HER2 and inhibits the growth of breast cancer cells that overexpress HER2. We evaluated the efficacy and toxicity of weekly intravenous administration of rhuMAb HER2 in patients with HER2-overexpressing metastatic breast cancer. PATIENTS AND METHODS: We treated 46 patients with metastatic breast carcinomas that overexpressed HER2. Patients received a loading dose of 250 mg of intravenous rhuMAb HER2, then 10 weekly doses of 100 mg each. Patients with no disease progression at the completion of this treatment period were offered a maintenance phase of 100 mg/wk. RESULTS: Study patients had extensive metastatic disease, and most had received extensive prior anticancer therapy. Adequate pharmacokinetic levels of rhuMAb HER2 were obtained in 90% of the patients. Toxicity was minimal and no antibodies against rhuMAb HER2 were detected in any patients. Objective responses were seen in five of 43 assessable patients, and included one complete remission and four partial remissions (overall response rate, 11.6%; 95% confidence interval, 4.36 to 25.9). Responses were observed in liver, mediastinum, lymph nodes, and chest wall lesions. Minor responses, seen in two patients, and stable disease, which occurred in 14 patients, lasted for a median of 5.1 months. CONCLUSION: rhuMAb HER2 is well tolerated and clinically active in patients with HER2-overexpressing metastatic breast cancers that had received extensive prior therapy. This is evidence that targeting growth factor receptors can cause regression of human cancer and justifies further evaluation of this agent. PMID- 8622020 TI - Improved local control and disease-free survival after perioperative chemotherapy for early-stage breast cancer. A European Organization for Research and Treatment of Cancer Breast Cancer Cooperative Group Study. AB - PURPOSE: To investigate whether a short intensive course of perioperative polychemotherapy can change the course of early breast cancer. PATIENTS AND METHODS: A total of 2,795 women with early breast cancer, stage I to IIIA, were randomized onto a trial (European Organization for Research and Treatment of Cancer [EORTC] 10854) to compare surgery followed by one course of perioperative chemotherapy versus surgery alone. Patients assigned to the chemotherapy arm received one course of fluorouracil 600 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide 600 mg/m2 (FAC) intravenously, within 24 hours after surgery. In both randomized treatment arms, a recommendation was made for premenopausal women with positive axillary nodes to receive prolonged courses of cyclophosphamide, methotrexate, and fluorouracil (CMF), according to the standard treatment for this subgroup. RESULTS: At a median follow-up time of 41 months, local control was significantly better in the perioperative treatment arm as compared with the observation arm (hazards ratio, 0.60; 95% confidence interval, 0.44 to 0.83; P < .01). Disease-free survival was significantly prolonged in the chemotherapy arm (hazards ratio, 0.84; 95% confidence interval, 0.70 to 0.99; P = .04). Premenopausal node-negative patients especially showed an advantage for the perioperative chemotherapy arm. No advantage for perioperative chemotherapy was observed in premenopausal node-positive women who also had received prolonged chemotherapy. CONCLUSION: We conclude that one course of perioperative FAC is able to improve local control and can prolong disease-free survival in women with early breast cancer. However, our results also suggest that a perioperative timing cannot improve the results of standard prolonged chemotherapy in premenopausal women with positive axillary nodes. PMID- 8622021 TI - Fifteen-year results of breast-conserving surgery and definitive breast irradiation for the treatment of ductal carcinoma in situ of the breast. AB - PURPOSE: To determine the 15-year outcome for women with ductal carcinoma in situ (DCIS, intraductal carcinoma) of the breast treated with breast-conserving surgery followed by definitive breast irradiation. PATIENTS AND METHODS: An analysis was performed of 270 intraductal breast carcinomas in 268 women from 10 institutions in Europe and the United States. In all patients, breast-conserving surgery included complete gross excision of the primary tumor followed by definitive breast irradiation. When performed, pathologic axillary lymph node staging was node-negative (n=86). The median follow-up time was 10.3 years (range, 0.9 to 26.8). RESULTS: The 15-year actuarial overall survival rate was 87%, and the 15-year actuarial cause-specific survival rate was 96%. The 15-year actuarial rate of freedom from distant metastases was 96%. There were 45 local recurrences in the treated breast, and the 15-year actuarial rate of local failure was 19%. The median time to local failure was 5.2 years (range, 1.4 to 16.8). A number of clinical and pathologic parameters were evaluated for correlation with local failure, and none were predictive for local failure (all P > or = .15). CONCLUSION: The results from the present study demonstrate high rates of overall survival, cause-specific survival, and freedom from distant metastases following the treatment of DCIS of the breast using breast-conserving surgery and definitive breast irradiation. These results support the use of breast-conserving surgery and definitive breast irradiation for the treatment of DCIS of the breast. PMID- 8622022 TI - Effect of adjuvant chemotherapy with or without anthracyclines on the activity and efficacy of first-line cyclophosphamide, epidoxorubicin, and fluorouracil in patients with metastatic breast cancer. AB - PURPOSE: To evaluate the effect of previous adjuvant chemotherapy with or without anthracyclines on overall survival (OS), progression-free survival (PFS), and objective response (OR) rates of metastatic breast cancer patients treated with cyclophosphamide, epidoxorubicin, and fluorouracil (CEF) as first-line chemotherapy. PATIENTS AND METHODS: Three-hundred twenty-six assessable metastatic breast cancer patients entered onto four consecutive randomized trials performed in our Institution and North-West Oncology Group (GONO) cooperative centers from 1983 to 1994. Patients received CEF-based chemotherapy as first-line therapy and were then evaluated. One hundred forty-four patients (44%) did not receive previous adjuvant chemotherapy, and 143 (44%) and 39 (12%) patients received cyclophosphamide, methotrexate, and fluorouracil (CMF)-based and anthracycline-based adjuvant chemotherapy, respectively. RESULTS: ORs to CEF chemotherapy were observed in 161 patients (49.4%). On univariate analysis, patients who had received prior adjuvant chemotherapy had a significantly lower probability of response than patients who did not: 43% versus 58% (P=.02). No difference between CMF-based (OR rate, 43%) and anthracycline-based (OR rate, 44%) adjuvant chemotherapy was observed. Stepwise logistic regression analysis indicated that adjuvant chemotherapy (P=.005), bone as dominant metastatic site (P=.02), and previous hormonotherapy for metastatic disease (P=.005) were the most important factors in predicting a poor OR rate. The median PFS and OS times of the whole group were 9.8 and 17.9 months, respectively. Patients who did not receive adjuvant chemotherapy had a longer survival time (21.1 months) compared with patients previously treated with CMF-based (15.3 months) or anthracycline based (15.8 months) adjuvant chemotherapy. Multivariate analysis confirmed adjuvant chemotherapy to be among the strongest prognostic factors associated with both a poor PFS and OS. CONCLUSION: Previous adjuvant chemotherapy adversely affects OR, PFS, and OS in metastatic breast cancer patients treated with the CEF regimen as first-line chemotherapy. No difference was observed between patients previously treated with CMF-based or anthracycline-based adjuvant chemotherapy. PMID- 8622023 TI - Phase I/II study of 72-hour infusional paclitaxel and doxorubicin with granulocyte colony-stimulating factor in patients with metastatic breast cancer. AB - PURPOSE: We conducted a phase I/II trial of concurrently administered 72-hour infusional paclitaxel and doxorubicin in combination with granulocyte colony stimulating factor (G-CSF) in patients with previously untreated metastatic breast cancer and bidimensionally measurable disease. PATIENTS AND METHODS: We defined the maximum-tolerated dose (MTD) of concurrent paclitaxel and doxorubicin administration and then studied potential pharmacokinetic interactions between the two drugs. Forty-two patients who had not received prior chemotherapy for metastatic breast cancer received 296 total cycles of paclitaxel and doxorubicin with G-CSF. RESULTS: The MTD was determined to be paclitaxel 180 mg/m2 and doxorubicin 60 mg/m2 each by 72-hour infusion with G-CSF. Diarrhea was the dose limiting toxicity (DLT) of this combination, with three of three patients developing abdominal computed tomographic (CT) scan evidence of typhlitis (cecal thickening) at the dose level above the MTD. All patients developed grade 4 neutropenia (absolute neutrophil count [ANC] < 500 microL), generally less than 5 days in duration. This combination was generally safely administered at dose levels at or below the MTD. The overall response rate was 72% (28 of 39 patients; 95% confidence interval [CI], 55% to 85%), with 8% complete responses (CRs) (three of 39; 95% CI, 2% to 21%) and a median response duration of 9 months. The median overall survival time for all patients is 23 months, with a median follow up duration of 28 months. Pharmacokinetic studies showed that administration of paclitaxel and doxorubicin together by 72-hour infusion did not affect the steady state concentrations of either drug. CONCLUSION: Concurrent 72-hour infusional paclitaxel and doxorubicin can be administered safely, but is associated with significant toxicity. The overall response rate of this combination in untreated metastatic breast cancer patients is similar to that achieved with other doxorubicin-based combination regimens. The modest complete response rate achieved suggests that this schedule of paclitaxel and doxorubicin administration does not produce significant additive or synergistic cytotoxicity against breast cancer. PMID- 8622025 TI - Paclitaxel has moderate activity in squamous cervix cancer. A Gynecologic Oncology Group study. AB - PURPOSE: Taxol (paclitaxel; Bristol-Myers Squibb, Princeton, NJ) is a new antineoplastic drug with broad-spectrum activity in solid tumors, including epithelial ovarian cancer, head and neck cancer, esophageal cancer, breast cancer, bladder cancer, and lung cancer. Its unique mechanism of action, polymerization of tubulin monomers, has stimulated both clinical and preclinical research on this agent. As limited drug supplies became more plentiful, a phase II trial of Taxol was initiated in patients with advanced squamous cervix cancer who had received no prior chemotherapy. PATIENTS AND METHODS: In this trial, 30 assessable patients were initially entered onto the study and four partial responses were seen. Further accrual of 22 assessable patients was then accomplished to define better the response rate with smaller confidence intervals. The starting dose of Taxol was 170 mg/m2 (135 mg/m2 for patients with prior pelvic radiation) given as a 24-hour continuous intravenous infusion with courses repeated every 3 weeks. Dose escalations to 200 mg/m2 and deescalations to 110 mg/m2 were allowed based on adverse effects. RESULTS: The final response rate was 17% (two complete responses and seven partial responses). The primary and dose-limiting toxicity was neutropenia. CONCLUSION: The response rate makes Taxol a drug with sufficient activity to explore it in combination with other agents with similar activity. PMID- 8622024 TI - Phase I and pharmacologic study of sequences of paclitaxel and cyclophosphamide supported by granulocyte colony-stimulating factor in women with previously treated metastatic breast cancer. AB - PURPOSE: Pacltaxel is active in metastatic breast cancer, but limited information is available on combinations of this agent with other cytotoxic agents. Study aims were to determine the maximum-tolerated doses (MTDs) of paclitaxel (24-hour infusion) and cyclophosphamide (1-hour infusion) administered every 21 days with granulocyte colony-stimulating factor (G-CSF, filgrastim) to determine the effect of drug sequence on toxicity and pharmacology and to evaluate the activity of this combination in women with anthracycline-resistant disease. PATIENTS AND METHODS: Thirty-seven women with metastatic breast cancer were treated. Starting doses were paclitaxel 135 mg/m2 and cyclophosphamide 750 mg/m2, with filgrastim 5 microG/kg/d subcutaneously beginning 24 hours after chemotherapy. Four patients were treated at each dose level. The sequence of drug administration was alternated between sequential patients, and with subsequent courses of therapy in each patient, to enable evaluation of effects of drug sequence on toxicity and pharmacology. Patients were treated every 21 days and disease status was reevaluated every two courses. RESULTS: Paclitaxel 200 mg/m2 and cyclophosphamide 1,250 mg/m2 is the MTD for this combination on this schedule. The hematopoietic toxicity of therapy was sequence-dependent. Paired analysis of toxicity data indicated more severe toxicity in courses in which paclitaxel was administered first. Sequence-dependent pharmacologic effects did not account for this phenomenon. Responses were noted in 29% of patients with anthracycline-resistant disease. CONCLUSION: Paclitaxel 200 mg/m2 and cyclophosphamide 1,250 mg/m2 with filgrastim administered every 21 days are the doses recommended for further study. PMID- 8622026 TI - Follow-up of Memorial Sloan-Kettering Cancer Center patients treated on National Cancer Institute Treatment Referral Center protocol 9103: paclitaxel in refractory ovarian cancer. AB - PURPOSE: To examine the short-term and long-term results of paclitaxel therapy in patients with advanced heavily pretreated, cisplatin-refractory ovarian cancer. PATIENTS AND METHODS: The results of treatment for patients entered onto National Cancer Institute (NCI) Treatment Referral Center protocol 9103 at the Memorial Sloan-Kettering Cancer Center (MSKCC) were reviewed to evaluate toxicity, efficacy, and survival. RESULTS: Of 46 individuals with measurable disease treated on the protocol at MSKCC, the objective response rate was only 4%. However, the 2- and 3-year survival rates for all 103 patients (including both measurable and nonmeasurable populations) entered onto this study at MSKCC were 18% and 11%, respectively. Twenty-one percent of patients received > or = six courses of paclitaxel, which suggests treatment-related stabilization of disease may have had a greater impact on the natural history of the malignancy than indicated by the objective response rate. CONCLUSION: This experience supports the hypothesis that a more prolonged delivery of paclitaxel (ie, > six courses), a cell-cycle-specific cytotoxic agent with limited or no cumulative toxicity, may result in an improved therapeutic outcome in ovarian cancer. This concept will need to be tested in a randomized phase 3 clinical trial. PMID- 8622027 TI - Phase I study of pharmacologically based dosing of carboplatin with filgrastim support in women with epithelial ovarian cancer. AB - PURPOSE: The aim of this study was to increase the dose intensity of carboplatin in women with International Federation of Gynecology and Obstetrics (FIGO) Stage Ic-IV epithelial ovarian cancer with the use of granulocyte colony-stimulating factor (G-CSF; filgrastim; Amgen, Thousand Oaks, CA). PATIENTS AND METHODS: A phase I study of escalating target area under the curves (AUCs) of carboplatin with G-CSF (filgrastim) ws undertaken. The target AUCs were 5 mg/mL.min every 21 days for four cycles, 5 mg/mL.min every 14 days for four cycles, 7 mg/mL.min every 14 days for four cycles, 9 mg/mL.min every 14 days for four cycles, and 11 mg/mL.min every 14 days for four cycles. G-CSF was given at a dose of 5 microg/kg/d starting 24 hours after carboplatin administration and lasting until 24 hours before the next cycle and until day 14 after the last cycle. RESULTS: We were able to escalate to an AUC level of 9 mg/mL.min every 14 days for four cycles. At this dose, severe thrombocytopenia, that necessitated dosage delays, and failure to give subsequent cycles of carboplatin were observed. We then reduced the AUC level to 8 mg/mL.min every 14 days for four cycles. However, severe thrombocytopenia was also observed at this level. CONCLUSION: An AUC of 7 mg/mL.min every 14 days for four cycles is the maximum tolerated AUC level that can be achieved with G-CSF. Further escalations may be possible using either combinations of cytokines or peripheral stem-cell collections. PMID- 8622029 TI - Cisplatin and etoposide combination chemotherapy for locally advanced or metastatic thymoma. A phase II study of the European Organization for Research and Treatment of Cancer Lung Cancer Cooperative Group. AB - BACKGROUND: Thymomas are rare neoplasms of the mediastinum. The role of chemotherapy in advanced thymomas is not fully established. PATIENTS AND METHODS: In the European Organization for Research and Treatment of Cancer (EORTC) Lung Cancer Cooperative Group, 16 patients with recurrent or metastatic malignant thymoma were entered over 6 years onto a study of combination chemotherapy that consisted of cisplatin 60 mg/m2 on day 1 and etoposide 120 mg/m2 on days 1, 2, and 3, every 3 weeks. RESULTS: A median of six courses per patient was administered. Main side effects of treatment were leukopenia, nausea and vomiting, and alopecia. Five complete responses and four partial responses were obtained, with a median response duration of 3.4 years. The median progression free survival and survival times were 2.2 years and 4.3 years, respectively, with a median follow-up duration of 7 years. CONCLUSION: The combination of cisplatin and etoposide is highly effective and well tolerated in advanced thymoma. The investigation of this combination in a neoadjuvant setting in unresectable invasive thymoma is warranted. PMID- 8622028 TI - Patients with limited-stage small-cell lung cancer treated with concurrent twice daily chest radiotherapy and etoposide/cisplatin followed by cyclophosphamide, doxorubicin, and vincristine. AB - PURPOSE: A phase II trial in patients with limited-stage small-cell lung cancer treated with induction etoposide/cisplatin plus twice-daily chest radiotherapy was conducted in an attempt to increase response rates and prolong survival. PATIENTS AND METHODS: Fifty-four previously untreated patients with limited-stage small-cell cancer were treated with etoposide/cisplatin and concurrent radiotherapy at 1.5 Gy twice daily for 3 weeks to a total dose of 45 Gy. Patients then received three more cycles of etoposide/cisplatin followed by four cycles of vincristine, doxorubicin, and cyclophosphamide or an individualized chemotherapy regimen. RESULTS: Nine patients are alive and free of cancer a median of 4 years (range, 2 to 7) from the start of treatment. Thirty-eight have had progression of their cancer at a median of 1.2 years (range, 0.5 to 5.4) and all have died of small-cell cancer. Thirteen of these 38 patients' (34%) only site of initial relapse was in the CNS and all died of CNS metastases. Five patients died during therapy or from its complications and two patients died of causes other than relapsed small-cell lung cancer and toxicity. The median survival time is 21.3 months, with an actual survival rate of 83% at 1 year, and actuarial survival rates of 43% at 2 years and 19% at 5 years. CONCLUSION: This combined modality regimen for patients with limited-stage small-cell lung cancer results in a 2 year survival rate of 43%, but the principal cause of death in these patients is still relapse of the original cancer. Isolated CNS metastases caused more than 30% of the cancer deaths. PMID- 8622030 TI - Influence of age on the treatment of limited-stage small-cell lung cancer. AB - PURPOSE: To evaluate the prognostic importance of age on response rate and survival in patients with limited-stage small-cell lung cancer (SCLC), and to determine the effect of age on chemotherapy dose delivery and toxicity. PATIENTS AND METHODS: We undertook a retrospective analysis of data from two multicenter, randomized trials conducted by the National Cancer Institute of Canada (NCIC) in which 608 SCLC patients who presented with limited disease (LD) all received the same chemotherapy. Treatment consisted of cyclophosphamide, doxorubicin, and vincristine (CAV), and etoposide plus cisplatin (EP) administered in an immediate or delayed alternating fashion, plus cranial and thoracic irradiation. RESULTS: There were 520 patients aged less than 70 years, and 88 > or = 70. No significant differences existed between the two age groups in baseline characteristics, including treatment protocol, performance status, and serum lactate dehydrogenase (LDH) level. There were more men in the older group (P = .05). Overall response rates were comparable (78% v 82%, P = .50), and 5-year survival rates were also similar (P = .14), with 11% alive in the younger group and 8% in the older group. Age was a significant predictor of overall survival when analyzed as a continuous variable in a univariate model (P = .01), but it was no longer an independent prognostic factor in our multivariate regression analysis. An analysis of chemotherapy delivery between the two age groups showed that patients aged > or = 70 years received lower total doses of each drug compared with the intended full protocol dose, primarily as a result of dose omissions, rather than dose reductions. The frequency of dose delays was not different between groups. No significant differences were seen in the incidence of either hematologic or most nonhematologic toxicities. CONCLUSION: Age is not a significant adverse prognostic variable in SCLC patients with LD. Moderately aggressive chemotherapy may be delivered safely to elderly patients with a good performance status, although modest attenuation of therapy through either dose reduction or omission may occur more frequently in this population. PMID- 8622032 TI - Adjuvant and adjunctive chemotherapy in the management of squamous cell carcinoma of the head and neck region. A meta-analysis of prospective and randomized trials. AB - PURPOSE: Using the technique of meta-analysis, we aim to illustrate the potential benefit, or lack of it, in adding chemotherapy to locoregional definitive treatment in a prospective randomized setting. PATIENTS AND METHODS: Mantel Haenszel summary analyses were used to test 42 prospective and properly randomized trials for statistically significant differences in the proportion with side effects and in the proportion with response to treatment between the experimental treatment arm (including chemotherapy) and control arm (local definitive treatment only) of the study. Summarized estimates of relative risks of side effects and relative proportions of positive responses were obtained using the summarizing options in PROC FREQ in the SAS computer package. In 25 of 42 studies, sufficient survival information was available to estimate the effect of chemotherapy on the rate of dying per person per unit of time. RESULTS: Chemotherapy, when added to local definitive treatment, was found to increase toxicity. This increase is statistically significant. The relative proportion of side effects was 2.17, with a 95% confidence interval of 1.84 to 2.56 and P less than .001. Addition of chemotherapy to local treatment has reduced the mortality rate for treated patients by 11% in the total group (all 25 studies), with a 95% confidence interval of 1% to 19%. This reduction means that at the time 50% of patients in the control arm were still alive, 54% of patients who received chemotherapy would be expected to be alive. Concurrent treatment (11 studies) has reduced the mortality rate by 22%, with a 95% confidence interval of 8% to 33%, which means that at the time 50% of patients in the control arm were still alive, 58% of patients who received chemotherapy would be expected to be alive. CONCLUSION: Addition of chemotherapy to local definitive treatment has significantly increased the morbidity of treatment as well as the chance of initial tumor response and local control. A statistically significant improvement in survival was found for the simultaneous use of chemotherapy and local definitive treatment. PMID- 8622031 TI - Combined preoperative chemotherapy and radiotherapy in patients with locally advanced esophageal cancer. Interim analysis of a phase II trial. AB - PURPOSE: The prognosis of patients with locally advanced esophageal cancer (LAEC) remains poor when treated with local modalities. An intensive preoperative program with chemoradiotherapy was used to evaluate the curative resection rate, pathologic response, and survival of patients with LAEC. PATIENTS AND METHODS: Ninety patients with LAEC were treated preoperatively with chemotherapy (three courses of fluorouracil, leucovorin, etoposide, and cisplatin [FLEP]) followed by concurrent chemoradiotherapy (one course of cisplatin plus etoposide in combination with 40 Gy of radiation). Transthoracic esophagectomy was performed 4 weeks after the end of radiation. RESULTS: Seventy-two patients were included in this evaluation. Forty-four (61%) underwent a complete tumor resection, and 16 (22%) had no tumor in the resected specimen (pathologic complete response [PCR]). The operative mortality rate was 15%. At a median follow-up time of 22 months (range, 12 to 41), the median survival duration of all 72 patients was 17 months (range, 1 to 41+). The calculated survival rates at 3 years were 33%, 42%, and 68% for all patients, patients after complete resection, and patients with PCR, respectively. CONCLUSION: This combined treatment modality is active in LAEC, with a PCR in 33% of the patients undergoing surgery. The results appear improved compared with those reported with surgery alone, by approximately doubling the 3 year survival rate. The high efficacy of preoperative chemoradiation warrants evaluation of the role of surgery in LAEC. PMID- 8622033 TI - Tumor size and prognosis in aggressively treated osteosarcoma. AB - PURPOSE: The aim of this retrospective analysis was to investigate the prognostic significance and optimal measures of tumor size in osteosarcoma treated with intensive neoadjuvant chemotherapy. PATIENTS AND METHODS: Initial anterior posterior (AP) and lateral x-ray films of 128 patients treated within the trials Cooperative Osteosarcoma Study (COSS)-80, -82, and -86, were evaluated for the following three tumor diameters: length, width, and depth. Metastasis-free survival (MFS) analyses were performed in univariate and multivariate models with one, two, and three dimensions of the tumor as absolute or relative measures (tumor length, referred to bone length, plane and volume to body-surface area). RESULTS: Univariate analyses of MFS showed a high prognostic significance of all absolute measures. Relative measures, at best, showed a comparable predictive value. Cox regression analysis indicated the high prognostic significance of absolute tumor volume (ATV; P < .0001) and histologic response (P < .0001). None of 19 patients with an ATV < or = 70 cm3 and only four of 53 with an ATV < or = 150 cm3 relapsed, while in patients with an ATV more than 150 cm3, the relapse rate remained 40% to 60%, irrespective of further increase in volume. CONCLUSION: Initial tumor size is an important and easily obtainable prognostic factor in osteosarcoma and may serve as a basis for risk-adapted therapy. It is best represented by the absolute three-dimensional measure ATV. There is a cut-off point regarding the incidence of metastases at a tumor volume of approximately 150 cm3 as calculated from two-plane x-ray films. PMID- 8622034 TI - Long-term results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma. AB - PURPOSE: This trial was performed to evaluate the impact of adjuvant brachytherapy on local and systemic recurrence rates in patients with soft tissue sarcoma. PATIENTS AND METHODS: In a single-institution prospective randomized trial, 164 patients were randomized intraoperatively to receive either adjuvant brachytherapy (BRT) or no further therapy (no BRT) after complete resection of soft tissue sarcomas of the extremity or superficial trunk. The adjuvant radiation was administered by iridium-192 implant, which delivered 42 to 45 Gy over 4 to 6 days. The two study groups had comparable distributions of patient and tumor factors, including age, sex, tumor site, tumor size, and histologic type and grade. RESULTS: With a median follow-up time of 76 months, the 5-year actuarial local control rates were 82% and 69% in the BRT and no BRT groups (P = .04), respectively. Patients with high-grade lesions had local control rates of 89% (BRT) and 66% (no BRT) (P = .0025). BRT had no impact on local control in patients with low-grade lesions (P = .49). The 5-year freedom-from-distant recurrence rates were 83% and 76% in the BRT and no BRT groups (P = .60), respectively. Analysis by histologic grade did not demonstrate an impact of BRT on the development of distant metastasis, despite the improvement in local control noted in patients with high-grade lesions. The 5-year disease-specific survival rates for the BRT and no BRT groups were 84% and 81% (P = .65), respectively, with no impact of BRT regardless of tumor grade. CONCLUSION: Adjuvant brachytherapy improves local control after complete resection of soft tissue sarcomas. This improvement in local control is limited to patients with high-grade histopathology. The reduction in local recurrence in patients with high-grade lesions is not associated with a significant reduction in distant metastasis or improvement in disease-specific survival. PMID- 8622035 TI - Prognostic factors in adult patients with locally controlled soft tissue sarcoma. A study of 546 patients from the French Federation of Cancer Centers Sarcoma Group. AB - PURPOSE: To define the prognostic factors in adult patients with locally controlled soft tissue sarcoma (STS) and to determine which patients should be considered for adjuvant treatment. PATIENTS AND METHODS: Five hundred forty-six patients with a nonmetastatic and locally controlled STS, collected in a cooperative data base by the French Federation of Cancer Centers (FNCLCC) Sarcoma Group from 1980 and 1989, were studied. Histologic slides of all patients were collegially reviewed. Initial treatment consisted of complete tumor resection with amputation in only 4% of the patients. Adjuvant radiotherapy was administered to 57.9% and adjuvant chemotherapy to 31%. Relationships between tumor characteristics were analyzed, and univariate and multivariate analyses were performed using Cox models for the hazards rate of tumor mortality, development of distant metastasis, and strictly local recurrence. RESULTS: Unfavorable characteristics with an independent prognostic value for tumor mortality were: grade 3 (P = 3 x 10(-10)), male sex (P = 1.5 x 10(-5)), no adjuvant chemotherapy (P = 5.4 x 10(-5)), tumor size > or = 5 cm (P = 3.8 x 10( 3)), and deep location (P = 4.6 x 10(-3)). Unfavorable characteristics for the development of distant metastasis were: grade 3 (P = 4 x 10(-12)), no adjuvant chemotherapy (P = 6.4 x 10(-4)), tumor size > or = 10 cm (P = 9.8 x 10(-4)), and deep location (P = 1.3 x 10(-3)). For the development of local recurrence, the unfavorable characteristics were: no adjuvant radiotherapy (P = 3.6 x 10(-6)), poor surgery (local excision) (P = 2 x 10(-4)), grade 3 (P = 7.6 x 10(-4)), and deep location (P = 10(-2)). Grade, depth, and tumor size were used to define groups of patients according to the metastatic risk. Adjuvant chemotherapy was beneficial in terms of overall survival and metastasis-free survival in grade 3 tumor patients only. Despite worse characteristics concerning tumor depth, tumor node-metastasis (TNM) and American Joint Committee (AJC)/International Union Against Cancer (UICC) classifications and grade in patients with adjuvant radiotherapy, the latter experienced significantly fewer local recurrences than patients with no radiotherapy. CONCLUSION: Grade, tumor depth, and tumor size could be used to select patients with a high metastatic risk, for which adjuvant chemotherapy could be beneficial. PMID- 8622036 TI - Combined systemic chemoimmunotherapy in advanced diffuse malignant mesothelioma. Report of a phase I-II study of weekly cisplatin/interferon alfa-2a. AB - PURPOSE: To assess the tolerance, toxicity, and antitumoral activity of the weekly combination of cisplatin (CDDP) and interferon alfa-2a (IFNalpha2a) in advanced diffuse malignant mesothelioma (DMM). PATIENTS AND METHODS: Twenty-six patients with DMM (23 pleural and three peritoneal), previously untreated, were enrolled onto this study between August 1991 and December 1992. All patients had measurable disease defined by computed tomographic (CT) scan and diagnostic confirmation by histopathology review panel. IFNalpha2a (3 x 10(6) IU subcutaneously on days 1 to 4) and CDDP (60 mg/m2/wk on day 2) were given weekly. Initially planned as a 5-weeks-on/3-weeks-off treatment cycle, poor patient tolerance observed in the first 12 patients treated (group A) led to schedule adaptation with a shorter treatment sequence and prolongation of the rest period (4 weeks on/4 weeks off) in the following 14 patients (group B). At least two cycles were administered to each patient in the absence of tumor progression. RESULTS: Twenty-six patients were assessable for toxicity and 25 for efficacy (World Health Organization [WHO] criteria). Sixty-eight cycles of IFN/CDDP were given, with a median of three cycles per patient (range, one to five). Toxicity was mainly clinical, with progressive anorexia, asthenia, and prolonged nausea/emesis; these side effects have limited treatment acceptance in many patients. Thrombocytopenia and leukopenia were rarely noted as treatment-limiting toxicities. Objective responses (all partial) were obtained in 10 patients (95% confidence interval [CI], 20% to 60%). The median response duration was 11 months (range, 6 to 18). The median time to progression (TTP) for the whole cohort was 6 months and the median survival time was 12 months (range, 5 to 32). Objective responders had a significantly longer median TTP (21 months) and survival time (25 months) than nonresponders (3 and 8 months, respectively). CONCLUSION: The results of this pilot phase I-II study show encouraging antitumor activity in this traditionally resistant tumor, even if the specific contribution of IFN remains speculative and needs further clinical research. Our ongoing program is exploring the dose-intensity impact of IFN dose within the same combination. PMID- 8622037 TI - P-glycoprotein expression at diagnosis may not be a primary mechanism of therapeutic failure in childhood rhabdomyosarcoma. AB - PURPOSE: To evaluate the prognostic significance of tumor cell P-glycoprotein (Pgp) expression at diagnosis in children with rhabdomyosarcoma. PATIENTS AND METHODS: A panel of three anti-Pgp monoclonal antibodies (mAb) (C219, C494, and JSB-1) that recognize different Pgp epitopes was used to measure Pgp expression in rhabdomyosarcoma specimens obtained at diagnosis from 76 patients treated at St Jude Children's Research Hospital from 1969 to 1991. Two separate experiments using different immunohistochemical methods (immune alkaline phosphatase and immunoperoxidase) were performed to evaluate Pgp expression. The immunostaining was graded using a semiquantitative scale corresponding to the percentage of tumor cells with detectable staining. The influence of Pgp expression on outcome was assessed by the Kaplan-Meier method and Cox regression analysis with stepwise selection. The relationship between Pgp expression and clinical features was assessed using the Mantel-Haenszel method. RESULTS: Pgp expression at diagnosis did not predict worse overall survival or progression-free survival when tested in either experiment with C219, C494, or JSB-1 separately. No association was shown between Pgp expression and clinical features (clinical group, primary site, or histology) or response. However, in the immune alkaline phosphatase experiment, patients whose tumors had more than 10% tumor cell staining with all three mAbs had a significantly higher rate of estimated 5-year survival (78% +/- 10%) than did all other patients (38% +/- 8%; P = .025). In this instance, Pgp expression had independent prognostic value after adjusting for clinical group. CONCLUSION: We found no strong association between Pgp expression at diagnosis and clinical features or extent of disease in pediatric rhabdomyosarcoma. Depending on the criteria used to define it, high Pgp expression at diagnosis does not predict poor outcome. Although a large prospective study is needed to provide definitive conclusions, our findings suggest that Pgp-mediated multidrug resistance may not be a primary mechanism of therapeutic failure in rhabdomyosarcoma. PMID- 8622039 TI - High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group. AB - PURPOSE: Here we report the results of a nationwide cooperative study in the Netherlands on acute lymphoblastic leukemia (ALL) in children. The aim of the study was to improve the cure rate and to minimize side effects in a group of non high-risk ALL patients, especially with regard to the CNS. A second aim was to study potential prognostic factors. METHODS: Children (age 0 to 15 years) with non-high-risk ALL (WBC count < 50 x 10(9)/L, no mediastinal mass, no B-cell phenotype, and no CNS involvement) were treated with a uniform protocol, ALL VI. The treatment protocol used 6-week induction regimen with three drugs (vincristine, dexamethasone, and asparaginase), three weekly doses of intravenous (IV) medium high-dose methotrexate (2 g/m2), and 2-year maintenance therapy that consisted of alternating 5-week periods of methotrexate and mercaptopurine and 2 week periods of vincristine and dexamethasone. In the first year of maintenance, triple intrathecal therapy was administered every 7 weeks. RESULTS: From December 1, 1984 until July 1, 1988, 291 children with ALL were diagnosed; 206 were categorized as non-high-risk (71%), and 190 were treated according to protocol ALL VI. At 8 years, the event-free survival (EFS) rate was 81% (SE = 3%) and survival rate 85% (SE = 2.9%); the median follow-up time was 7.3 years (range, 36 to 117 months). The CNS relapse rate was 1.1% (two of 184 patients who achieved a complete remission [CR]). The only factor found to be of negative prognostic importance in terms of EFS (P = .05) was a positive acid phosphatase reaction. CONCLUSION: For children with non-high-risk ALL, the combination of IV medium high-dose methotrexate (2 g/m2 times three), triple intrathecal therapy in the first year of maintenance treatment, and the use of dexamethasone for induction and pulses during maintenance treatment has proved to be highly effective, especially in the prevention of CNS relapse. A high cure rate was achieved without the use of anthracyclines, alkylating agents, and cranial irradiation. PMID- 8622038 TI - Randomized trial of recombinant human granulocyte-macrophage colony-stimulating factor in pediatric patients receiving intensive myelosuppressive chemotherapy. AB - PURPOSE: To evaluate whether recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) reduces the hematologic toxicities and supportive care requirements of an intensive combination chemoradiotherapy regimen in pediatric and young adult sarcoma patients. PATIENTS AND METHODS: Thirty-seven newly diagnosed patients age 1 to 25 years were randomized to receive 18 cycles of chemotherapy alone or with GM-CSF beginning in cycle 3. GM-CSF (5 to 15 micrograms/kg/d subcutaneously) was begun 24 hours after the completion of chemotherapy and continued through day 19 of each cycle or until the absolute granulocyte count (AGC) was > or = 500/microliter on 2 consecutive days. RESULTS: GM-CSF reduced the median duration of grade 4 granulocytopenia from 9.0 days (range, 2 to 24) to 7.0 days (range, 1 to 21) (P < .0001), but did not significantly affect the grade of granulocyte nadir. No differences were seen in the incidence or types of infectious complications, incidence or duration of hospitalization and antimicrobial therapy, response to chemotherapy, or event free or overall survival. GM-CSF was associated with more severe and protracted thrombocytopenia (median platelet nadir, 29,500/microliter [range, 3,000 to 288,000] v 59,000/microliter [range, 3,000 to 309,000], P < .0001; median time to recovery > 75,000/microliter, 16.0 days [range, 0 to 61] v 14.0 days [range, 0 to 38], P < .0001). CONCLUSION: GM-CSF does not produce clinically meaningful reductions in the degree or duration of severe granulocytopenia following intensive multiagent chemotherapy, but is associated with worsened thrombocytopenia. GM-CSF also does not reduce the need for hospitalization or the incidence of febrile neutropenia and infectious complications. We conclude that the costs and increased toxicities associated with the use of this agent are not justified by its minimal clinical benefit for regimens of this level of intensity. PMID- 8622040 TI - Predicting the risk of bacteremia in childen with fever and neutropenia. AB - PURPOSE: We sought to identify factors assessable at the time of admission for fever and neutropenia that predict bacteremia in children with cancer. PATIENTS AND METHODS: One hundred fifteen consecutive episodes of fever and absolute neutrophil count (ANC) less than 500/microliter in 72 children with cancer were studied prospectively to determine the risk of bacteremia using data assessable at the time of presentation. After exploratory analysis identified admission temperature and absolute monocyte count (AMoC) as the strongest predictive factors, recursive partitioning was used to determine cutpoints for these variables that resulted in discrimination between episodes associated with a lower or higher risk of bacteremia. RESULTS: There were 24 episodes of bacteremia (21% of episodes). Episodes were grouped using the cutpoints for AMoC and temperature: 17% were classified as low risk for bacteremia (AMoC > or = 100/microliter), 65% as intermediate risk (AMoC < 100/microliter and temperature < 39.0 degrees C), and 18% as high risk (AMoC < 100/microliter and temperature > or = 39.0 degrees C). No episodes classified as low risk were associated with bacteremia; 19% of intermediate-risk and 48% of high-risk episodes were associated with bacteremia. The odds ratio of bacteremia for the high-risk versus the intermediate-risk group is 4.4 (95% confidence interval, 1.6 to 12.9). The risk classification was validated using data from 57 different episodes of fever and neutropenia treated in the same hospital. CONCLUSION: Three levels of risk for bacteremia are defined using the AMoC and temperature at the time of admission for fever and neutropenia. Trials now should be conducted to test whether these factors may be used to assign some children to less intensive or outpatient antibiotic therapy at the time of presentation with fever and neutropenia. PMID- 8622041 TI - Adults and children with small non-cleaved-cell lymphoma have a similar excellent outcome when treated with the same chemotherapy regimen. AB - PURPOSE: We have used identical treatment protocols for adults and children with small non-cleaved-cell lymphoma (SNCL) for many years and report here the results of two successive treatment regimens in these age groups. PATIENTS AND METHODS: Seventy-two patients (39 adults and 33 children) were treated with protocol 77-04 between 1977 and 1985. All patients, except those with resected abdominal disease, received 15 cycles of a combination of cyclophosphamide (CTX), doxorubicin (ADR), prednisone (PRED), vincristine (VCR), high-dose methotrexate (MTX), and intrathecal (IT) therapy. Forty-one patients (20 adults and 21 children) were treated with protocol 89-C-41, which has been used since 1989. High-risk patients received four alternating cycles (with a total duration of 12 to 15 weeks) of an intensified version of protocol 77-04 without PRED (CODOX-M), and a new drug combination consisting of ifosfamide, etoposide, high-dose cytarabine (ara-C), and IT MTX (IVAC). Low-risk patients received three cycles of the CODOX-M regimen. High-risk patients were randomized to either receive or not receive granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS: Event free survival (EFS) in protocol 77-04 was 56% at 2 years and beyond. EFS in protocol 89-C-41 was 92% at 2 years and beyond. GM-CSF was associated with increased thrombocytopenia. CONCLUSION: Adults and children with SNCL have a similar prognosis when treated with the same chemotherapy. EFS in high-risk patients has been markedly improved by including IVAC in protocol 89-C-41, and excellent results can be achieved with only four cycles of therapy. In protocol 89-C-41, GM-CSF was not beneficial. PMID- 8622042 TI - Severe atypical neuropathy associated with administration of hematopoietic colony stimulating factors and vincristine. AB - PURPOSE: We have observed a severe atypical neuropathy (SAN) in patients with small non-cleaved-cell (SNCL) and large-cell lymphoma (LCL) treated with intensive chemotherapy and hematopoietic colony-stimulating factors (CSFs). The present analysis was undertaken in an attempt to identify factors associated with the development of this syndrome. PATIENTS AND METHODS: Fifty-four adult and pediatric patients consecutively treated according to the same chemotherapy protocol were included in the analysis. Low-risk patients received three cycles of cyclophosphamide, vincristine, doxorubicin, and high-dose methotrexate (CODOX M) while in high-risk patients this drug combination was alternated with high dose cytarabine (ara-C), etoposide, and ifosfamide (IVAC) for a total of four cycles. Twenty-eight patients received a CSF (granulocyte [G]- or granulocyte macrophage [GM]-CSF), and 26 patients received no CSF. A statistical analysis, which included a logistic regression model, was undertaken to examine the importance of potential contributing factors to the development of SAN. RESULTS: SAN, which consisted of excruciating foot pain, usually associated with marked motor weakness, was observed in 12 patients. There was a highly significant association between the occurrence of this syndrome and the administration of CSFs, and an independent association with the cumulative dose of vincristine given in the first cycle of chemotherapy. Furthermore, the analysis suggested a synergistic effect between administration of the CSFs and vincristine in the genesis of this neuropathy. CONCLUSION: Our results indicate that CSFs can precipitate SAN when given in conjunction with vincristine. The development of SAN was associated most strongly with the cumulative dose of vincristine -- the size of individual doses and the number of doses given in cycle 1 were important to the extent that they influenced the cumulative dose. PMID- 8622043 TI - CNS involvement in mantle-cell lymphoma. AB - PURPOSE: In non-Hodgkin's lymphomas, CNS involvement is highly dependent on the histology of the lymphoma. Mantle-cell lymphoma (MCL) is a lymphoma type with distinctive histologic, biologic, and clinical features in which CNS involvement has only been rarely described. The purpose of this report is to describe the incidence, clinical characteristics, and outcome of CNS infiltration in patients with MCL seen at a single institution. PATIENTS AND METHODS: Twenty-two patients with MCL, who account for 6% of all patients with nodal lymphomas diagnosed and monitored at a university hospital from 1987 to 1994, were studied. Analysis of the incidence of CNS involvement by the disease was performed. RESULTS: Five of 22 patients (22%; exact 95% confidence interval [CI], 7.8% to 45.4%) with MCL developed CNS involvement at a median of 18 months (range, 6 to 59) from diagnosis. All of these patients presented with poor MCL histologic subtypes and advanced disease. When the CNS infiltration became apparent, all of the patients displayed neurologic signs and had lymphoid cells consistent with the diagnosis of MCL in the CSF. In most of the cases, CNS infiltration was part of resistant disease or generalized relapse and had an ominous significance. CONCLUSION: The incidence of CNS involvement in MCL might be higher than previously recognized. The frequency of CNS infiltration in MCL deserves to be investigated in other series and, if a high incidence is confirmed, the risk factors, mechanisms, and clinical implications of such a complication should be further studied. PMID- 8622044 TI - Primary non-Hodgkin's lymphoma of the CNS treated with BVAM or CHOD/BVAM chemotherapy before radiotherapy. AB - PURPOSE: To assess whether chemotherapy that includes drugs that cross the blood brain barrier improves survival in primary CNS non-Hodgkin's lymphoma (PCNSL) when combined with radiotherapy. PATIENTS AND METHODS: Thirty-four patients, with no evidence of human immunodeficiency virus type 1 (HIV-1) infection, were treated with the related chemotherapy regimens of carmustine (BCNU), vincristine, cytarabine, and methotrexate (BVAM; 12 patients), cyclophosphamide, doxorubicin, vincristine, and dexamethasone (CHOD)/BVAM (17 patients) and intensified CHOD/BVAM (five patients) between 1986 and 1994. The median age was 60 years (range, 16 to 73) and 47% had a performance status of 3 or 4 (Eastern Cooperative Oncology Group [ECOG]/World Health Organization [WHO]). Ten patients were treated with BVAM chemotherapy between 1986 and 1989, and subsequently 17 patients were treated with CHOD/BVAM (cytarabine 3 g/m2). Twenty of these 27 patients received whole-brain radiotherapy (craniospinal in four). RESULTS: The complete response (CR) rate at the completion of chemotherapy was 63% for BVAM and 67% for CHOD/BVAM; more neutropenia occurred with CHOD/BVAM. The 5-year actuarial probability of survival of all 34 patients was 33% (95% confidence interval [CI], 14% to 52%), with so far only one recurrence after 2 years. Using multivariate analysis, age (P = .0005) and number of tumors at diagnosis (P = .0358) were prognostic factors. All five patients aged > or = 70 years died during or shortly after chemotherapy. Performance status was not an independent variable. CONCLUSION: The BVAM or CHOD/BVAM regimens can be delivered despite neutropenia without significant treatment delay or dose reduction in patients less than 70 years of age. Further intensification of this type of chemotherapy is probably not possible with patients of this age, many of whom have a poor performance status. PMID- 8622046 TI - Detection of the t(14;18) chromosomal translocation by interphase cytogenetics with yeast-artificial-chromosome probes in follicular lymphoma and nonneoplastic lymphoproliferation. AB - PURPOSE: The aim of this study was to establish a fluorescence in situ hybridization (FISH) technique for the detection of t(14;18)(q32;q21), characteristic for follicular lymphoma (Kiel classification: centroblastic centrocytic [cb-cc] lymphoma). MATERIALS AND METHODS: After the FISH system had been established, parallel studies of lymph node biopsy specimens from 30 patients with cb-cc lymphoma and from 32 patients with nonneoplastic lymphoproliferation were performed by means of chromosome analysis, polymerase chain reaction (PCR), and FISH analysis. Two differently labeled yeast-artificial chromosome (YAC) probes that contained the entire bcl-2 gene and the C-region of the immunoglobulin H (IgH) gene, respectively, were used to detect t(14;18) by FISH. RESULTS: The presence of the translocation is indicated by a red (Cy3)/green (fluorescien isothiocyanate [FITC]) double signal, which corresponds to the IgH/bcl-2 fusion gene, whereas in normal cells the signals are separate. Control studies showed that the double signal is visible in less than 1% of normal cells. FISH analysis was able to identify the t(14;18) in all cases of cb cc lymphoma we studied. All bcl-2 breakpoints can be detected. Combined immunophenotyping and interphase cytogenetics demonstrated that t(14;18) was restricted to CD22+ B lymphocytes and never occurred in CD3+ T lymphocytes. In four of 32 cases of nonneoplastic lymphoproliferation, t(14;18) was also detected. CONCLUSION: FISH turned out to be the most sensitive method to detect t(14;18). Our FISH results confirm PCR data from other groups that found evidence for the presence of t(14;18) in nonneoplastic lymphoproliferation. It needs to be determined whether, in morphologically nonneoplastic processes, t(14;18) is associated with an increased risk for the development of non-Hodgkin's lymphoma. PMID- 8622045 TI - Anaplastic large-cell lymphoma: clinical and prognostic evaluation of 90 adult patients. AB - PURPOSE: During the last few years, the application of CD30 monoclonal antibodies has led to the identification of a new lymphoma entity, termed anaplastic large cell lymphoma (ALCL). This tumor includes four distinct histologic subtypes, among which the Hodgkin's-like/Hodgkin's-related one (ALCL-HL) shares morphologic and phenotypic features with Hodgkin's disease (HD). PATIENTS AND METHODS: From September 1988 to October 1993, 90 ALCL patients were treated with third generation chemotherapy regimens (either vincristine, cyclophosphamide, fluorouracil, cytarabine, doxorubicin, methotrexate with leucovorin, and prednisone [F-MACHOP] or methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin [MACOP-B]) during the course of an Italian multicentric randomized trial on high-grade non-Hodgkin's lymphomas (HG-NHL). In particular, 47 patients had ALCL of the common type (ALCL CT) and 43 ALCL-HL. Null phenotype was the most common (39.8%), while T-cell, B cell, and hybrid forms accounted for 35.5%, 22.2%, and 2.5%, respectively. RESULTS: Complete remission (CR) was achieved in 66 of 90 (73.5%) patients (33 of 47 [70%] with ALCL-CT and 33 of 43 [77%] with ALCL-HL). The majority of the patients in CR (56.5%) were alive and well at a median follow-up time of 38 months; no significant differences were observed between the two histologic groups, with the rate of complete responders being 49% and 65% in ALCL-CT and ALCL-HL, respectively. The probability of relapse-free survival (RFS), projected at 63 months, was 67% for ALCL-CT and 82% for ALCL-HL. The risk of lower CR and RFS rates was associated with the presence of bulky disease, advanced stage, and B symptoms. CONCLUSION: The data of the present study confirm that ALCL responds to third-generation chemotherapy regimens similarly to other aggressive malignant lymphomas in terms of both CR and RFS rates. PMID- 8622047 TI - When to harvest peripheral-blood stem cells after mobilization therapy: prediction of CD34-positive cell yield by preceding day CD34-positive concentration in peripheral blood. AB - PURPOSE: To evaluate whether the CD34+ yield from a single peripheral-blood stem cell (PBSC) harvest could be predicted by measurement of the patient's circulating WBC and CD34+ cell concentrations on the day before harvest. PATIENTS AND METHODS: Thirty-nine patients with hematologic or nonhematologic malignancy underwent 41 stem-cell mobilization episodes with cytotoxic chemotherapy and/or granulocyte colony-stimulating factor (G-CSF), and a total of 63 leukapheresis procedures were performed. Peripheral-blood samples were analyzed for WBC and CD34+ cell concentration both on the day before and the day of leukapheresis. RESULTS: The median WBC and CD34+ concentrations on the day preceding leukapheresis were 10.0 x 10(9)/L (range, 0.4 to 44.4) and 24.9 x 10(6)/L (range, 0.1 to 349.4), respectively. On the day of harvest, the corresponding figures were 15.1 x 10(9)/L (range, 1.5 to 52.6) and 29.3 x 10(6)/L (range, 0.1 to 543.1), respectively. The median CD34+ cell number collected in a single leukapheresis was 2.6 x 10(6)/kg body weight (range, 0.1 to 26.1). Both the preceding day (r = .84, P < .001) and harvest day (r = .95, P < .001) CD34+ circulating concentrations correlated significantly with the number of CD34+ cells per kilogram collected at leukapheresis. The correlation between CD34+ cells per kilogram collected and harvest day WBC count was also significant (r = .43, P <.001), but with the preceding day WBC count was nonsignificant. CONCLUSION: The number of CD34+ cells harvested in a single leukapheresis can be predicted by measurement of the preceding day peripheral-blood circulating CD34+ concentration, and on the basis of these data a table of probable CD34+ cell yield has been constructed. This correlation may facilitate the efficient organization of leukapheresis procedures. PMID- 8622048 TI - Evaluation of race as a prognostic factor in multiple myeloma. An ancillary of Southwest Oncology Group Study 8229. AB - PURPOSE: The objective of this investigation was to assess the impact of race (black v white) on the survival of patients with multiple myeloma treated within the context of a large clinical trial. PATIENTS AND METHODS: A cohort of patients randomized to receive one of two treatment regimens and monitored for at least 10 years was studied to assess the impact of race as a prognostic factor, after adjusting for other known factors such as stage of disease. Patients were recruited from the referral network of the Southwest Oncology Group (SWOG), a national multiinstitutional consortium that includes both academic and community treatment centers. Patients had a diagnosis of multiple myeloma and had not previously been treated for this disease. They were carefully characterized as to demographic and clinical features, and were randomized to receive one of two treatment regimens, which proved to have virtually identical outcomes. The outcome measure was survival, measured from the date of randomization to the date of last contact. Patients still alive at last contact date were treated as censored observation. RESULTS: Survival for black myeloma patients was similar to that for white patients, both overall and adjusted for prognostic factors such as stage. CONCLUSION: Observed differences in mortality between blacks and whites cannot be attributed to differences in survival after diagnosis, given comparable treatment. PMID- 8622049 TI - Pharmacokinetic study of oral and bolus intravenous 2-chlorodeoxyadenosine in patients with malignancy. AB - PURPOSE: This study was designed to evaluate the absolute bioavailability (F value) of 2-chlorodeoxyadenosine (cladribine; 2-CdA) after multiple oral administrations, and the intersubject variability after oral and 2-hour intravenous (IV) administration schedules in patients with malignancy. PATIENTS AND METHODS: Patients with advanced malignancies were eligible. There were two treatment cycles; during cycle 1, patients received 2-CdA solution at 0.28 mg/kg/d orally under fasting conditions for 5 consecutive days concomitantly with omeprazole, and 4 weeks later during cycle 2 patients received 2-CdA as a 2-hour IV infusion of 0.14 mg/kg/d for 5 consecutive days. Serial blood samples for 2 CdA plasma levels were obtained after drug administrations on days 1 and 5 during each treatment cycle. RESULTS: Ten patients completed cycles 1 and 2. The F value of oral 2-CdA measured on days 1 and 5 was 37.2% and 36.7%, respectively. For both oral and IV multiple administrations, there was no significant accumulation in maximum concentration (Cmax), and the intersubject variabilities (coefficient of variation [CV], approximately 40%) in Cmax and area under the concentration time curve from 0 to 24 hours [AUC(0-24)] values were comparable for both routes on days 1 and 5. A three-compartment open model was applied to the plasma concentration data after oral and IV administrations and resulted in good agreement between observed and simulated concentration-time profiles. Neutropenia was the principal adverse event observed when 2-CdA was administered orally and IV. CONCLUSION: The F value of 2-CdA after oral administration was approximately 37% and there were no cumulative differences in bioavailability observed on multiple dosing of the drug. The absorption and disposition characteristics of oral 2-CdA were linear and predictable with this dosing regimen. PMID- 8622050 TI - Telephone-based nursing intervention improves the effectiveness of the informed consent process in cancer clinical trials. AB - PURPOSE: Here we report the results of a randomized study undertaken to test the efficacy of a supplementary, telephone-based nursing intervention in increasing patients' awareness and understanding of the clinical trials in which they are asked to participate. METHODS: During a 12-month period, 180 cancer patients who were approached to participate in a phase II or III clinical trial were randomized to undergo either of the following: (1) standard informed consent procedures based on verbal explanations from the treating physician plus written information (controls); or (2) standard informed consent procedures plus a supplementary, telephone-based contact with an oncology nurse (intervention). For purposes of evaluation, face-to-face interviews were conducted with all patients approximately 1 week after the informed consent process had been completed. RESULTS: The two groups were comparable with regard to sociodemographic and clinical variables. Both groups had a high level of awareness of the diagnosis and of the nature and objectives of the proposed treatments. The intervention group was significantly (P < .01) better informed about the following: (1) the risks and side effects of treatment; (2) the clinical trial context of the treatment; (3) the objectives of the clinical trial; (4) where relevant, the use of randomization in allocating treatment; (5) the availability of alternative treatments; (6) the voluntary nature of participation; and (7) the right to withdraw from the clinical trial. The intervention did not have any significant effect on patients' anxiety levels or on rates of clinical trial participation. Patients reported high levels of satisfaction with the intervention. CONCLUSION: The use of a supplementary, telephone-based nursing intervention is a feasible and effective means to increase cancer patients' awareness and understanding of the salient issues that surround the clinical trials in which they are asked to participate. PMID- 8622051 TI - Hormone replacement therapy in women with breast cancer. Do the risks outweigh the benefits? AB - PURPOSE: To review critically the literature regarding effects of estrogen replacement therapy (ERT)/combined estrogen and progesterone replacement therapy (HRT) on the risk of breast cancer and on other health risks and benefits in postmenopausal women, with a focus on risks and benefits in women with a previous diagnosis of breast cancer. METHOD: A literature search was conducted using Medline, Cancerline, and the bibliographies of reports published as of March 1995. All five published meta-analyses that examined the risk of breast cancer in relation to ERT/HRT in otherwise healthy women were critically reviewed. All known reports of women with a history of breast cancer given ERT/HRT subsequent to diagnosis and additional reports regarding the benefits of ERT/HRT were also reviewed. RESULTS: None of the five meta-analyses demonstrated a significantly increased risk of developing breast cancer in ever users compared with never users of ERT/HRT. Current use may be associated with a small increased risk. This increased risk should be balanced by the expected benefits of ERT/HRT on quality of life, bone metabolism, and cardiovascular function. Preliminary information does not suggest a major detrimental effect of ERT/HRT in women with a previous diagnosis of breast cancer, but these reports include few women with limited follow-up data. There are no randomized trials in women with a previous diagnosis of breast cancer. CONCLUSION: In healthy postmenopausal women, the benefits associated with ERT/HRT outweigh the risks. In women with a previous diagnosis of breast cancer, the balance of risks and benefits should be explored in randomized controlled trials. PMID- 8622052 TI - Economics of bone marrow transplantation. PMID- 8622053 TI - CA-125: an evolving role in the management of ovarian cancer. PMID- 8622054 TI - Economic evaluation of allogeneic bone marrow transplantation: a rudimentary model to generate estimates for the timely formulation of clinical policy. AB - PURPOSE: To provide an evidence-based approach to the formulation of clinical policy with respect to allogeneic bone marrow transplantation (BMT) that involves perceived trade offs between two major factors: costs and consequences. The report also highlights key informational deficiencies. PATIENTS AND METHODS: Adults with acute myeloid leukemia (AML) in second complete remission (2CR) and those with acute lymphoblastic leukemia (ALL) in first complete remission (1CR) were assigned to BMT or control groups solely on the availability of a suitable donor. All hospital-borne costs were estimated, based on services used according to manual chart review, in four categories: diagnostic and therapeutic costs, professional fees, drug costs, and ward costs. Incremental costs and incremental life-years were calculated, and the quotient determined a cost per life-year gained by BMT for AML (2CR) and ALL (1CR). RESULTS: The incremental cost (in 1992 Canadian dollars) per life-year gained by BMT (cost-effectiveness) for AML (2CR) was $29,200; and for ALL (1CR) it was minus $29,200. CONCLUSION: For AML (2CR), allogeneic BMT creates better outcomes than standard treatment, but is more costly. For ALL (1CR), both the costs and outcomes are similar for BMT and standard therapy. Quality adjustments made to life-years gained did not change these conclusions. PMID- 8622055 TI - Alternating versus hybrid MOPP and ABVD combinations in advanced Hodgkin's disease: ten-year results. AB - PURPOSE: To compare, in a prospective randomized trial, the efficacy of two different sequences of mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) and doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) chemotherapy in untreated advanced Hodgkin's disease. PATIENTS AND METHODS: From June 1982 to September 1990, 427 consecutive previously untreated patients with pathologic stage IB, IIA bulky, IIB, III (A and B), and IV (A and B) disease were prospectively randomized to receive two different sequences of MOPP and ABVD for a minimum of six cycles followed by radiotherapy (median dose, 30 Gy) to the nodal site(s) of pretreatment bulky disease. Of 415 assessable patients, 211 received one cycle of MOPP monthly, alternated with one cycle of ABVD (alternating regimen), and 204 patients received one-half cycle of MOPP alternated with one-half cycle of ABVD within a 1-month period (hybrid regimen). RESULTS: The complete remission (CR) rate was 91% with the alternating regimen and 89% with the hybrid regimen. At 10 years, the freedom-from-progression (FFP) rate was 67% versus 69% and the overall survival (OS) rate was 74% versus 72%, respectively. After attainment of CR, 85 patients relapsed in nodal (n = 60) versus extranodal with or without nodal (n = 25) sites. In patients given consolidative radiation because of bulky lymphoma, the true recurrence rate was 13%. A total of 23 second malignancies (6%) were documented, including 11 cases of acute nonlymphocytic leukemia. No cases of congestive heart failure attributable to doxorubicin or pulmonary toxicity related to bleomycin were documented. CONCLUSION: By delivering MOPP and ABVD, it is possible to cure approximately 70% of patients with advanced Hodgkin's disease. The two different drug sequences yielded superimposable results. PMID- 8622056 TI - Recovery of overall and local lung function loss 18 months after irradiation for malignant lymphoma. AB - PURPOSE: To determine the local and overall pulmonary injury 3 to 18 months after irradiation and to investigate whether the changes in overall lung function can be predicted using the three-dimensional (3-D) dose distribution in combination with dose-effect relations for local injury; and to study the influence of chemotherapy on the injury. PATIENTS AND METHODS: Local perfusion (Q), ventilation (V), and tissue density were measured in 25 patients treated for malignant lymphoma, before, 3 to 4 months after, and 18 months after irradiation. Dose-effect relations for local injury, calculated using correlated single-photon emission computed tomographic (SPECT) and computed tomographic (CT) data, were combined with the 3-D dose distribution, to calculate the estimated mean local changes over the complete lung for each patient. The result was correlated with the actual changes in pulmonary function. RESULTS: A dose-dependent increase with injury was observed at 3 to 4 months after irradiation, which at 18 months had recovered by approximately 50% to 60%. The estimated mean relative reduction of local Q predicted the change in overall lung function within 10% of the actually observed values in 63% to 73% of patients. Chemotherapy given before radiotherapy enhanced radiation-induced reduction of local Q significantly, with dose modifying factors of 1.22 and 1.37 at 3 to 4 months and 18 months, respectively. CONCLUSION: Partial recovery of radiation-induced reduction of local and overall lung function was observed at 18 months after irradiation. The overall functional outcome of most patients could be well predicted, based on the estimated mean local injury over the complete lung. Chemotherapy given before radiotherapy enhanced the radiation-induced reduction of local Q. PMID- 8622057 TI - Risk of subsequent malignant neoplasms among 1,641 Hodgkin's disease patients diagnosed in childhood and adolescence: a population-based cohort study in the five Nordic countries. Association of the Nordic Cancer Registries and the Nordic Society of Pediatric Hematology and Oncology. AB - PURPOSE: To assess the risk of subsequent malignant neoplasms among Hodgkin's disease patients diagnosed before 20 years of age in the five Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden). PATIENTS AND METHODS: There were 1,641 Hodgkin's disease patients identified through the national cancer registries since the 1940s or 1950s. The patients were monitored for 17,000 person-years until the end of 1991. Expected figures were derived from the age specific incidence rates in each country and standardized incidence ratios (SIR) were calculated. RESULTS: A total of 62 subsequent neoplasms were diagnosed (SIR, 7.7; 95% confidence interval [CI], 5.9 to 9.9). The overall cumulative risk of subsequent neoplasms was 1.9% at the 10-year follow-up point, 6.9% at 20 years, and 18% at 30 years. There were 26 subsequent neoplasms among males (SIR, 6.5; 95% CI, 4.3 to 9.6) and 36 among females (SIR, 8.9; 95% CI, 6.2 to 12), of which 16 were breast cancers (SIR, 17; 95% CI, 9.9 to 28). High risks were seen for thyroid cancer (SIR, 33; 95% CI, 15 to 62), for secondary leukemia (SIR, 17; 95% CI, 6.9 to 35), and for non-Hodgkin's lymphoma (SIR, 15; 95% CI, 4.9 to 35). The relative risk increased from 3.3 (95% CI, 1.2 to 7.1) for Hodgkin's disease patients diagnosed in the 1940s and 1950s to 15 (95% CI, 7.4 to 27) in the 1980s. The highest risk of secondary leukemia (SIR, 68; 95% CI, 18 to 174) was seen among those diagnosed with Hodgkin's disease in the 1980s. CONCLUSION: Patients who survive Hodgkin's disease at a young age are at very high relative risk of subsequent malignant neoplasms throughout their lives. In particular, the high relative risk of breast cancer following Hodgkin's disease in the teenage years calls for enhanced activity for early diagnosis. PMID- 8622058 TI - High-dose therapy followed by autologous hematopoietic stem-cell infusion for patients with multiple myeloma. AB - PURPOSE: To evaluate the outcome of patients with multiple myeloma (MM) who received high-dose therapy followed by autologous bone marrow (BM) or peripheral blood stem-cell (PBSC) infusion. PATIENTS AND METHODS: Sixty-three consecutive patients with MM received autologous BM (n = 13) or PBSC with or without BM (n = 50) following regimens that contained busulfan (Bu) and cyclophosphamide (Cy) (n = 18), modified total-body irradiation (TBI) followed by Bu and Cy (n = 36), or Bu, melphalan, and thiotepa (n = 9). Two thirds of the patients had resistant disease and 69% had received more than 6 months of previous chemotherapy. RESULTS AND CONCLUSION: Recovery of peripheral-blood cell counts was more rapid in patients who received PBSC with or without BM than in patients who received BM alone. Sixteen of 63 patients (25%) died of complications of treatment within 100 days. Nineteen (40%) of 48 assessable patients achieved a complete response (CR), 23 (48%) had a partial response (PR), and six (12%) had no response. The probabilities of survival and survival without relapse or progression for all 63 patients at 3.0 years were .43 and .21, respectively. The probability of relapse or progression at 3 years was .69, and 17 patients (27%) have died of progressive MM. The probabilities of survival and relapse-free survival at 3 years for the 19 patients who achieved a CR were .42 and .17, respectively. In the multivariate analysis, beta2-microglobulin levels more than 2.5 micrograms/mL, more than two regimens of prior therapy and eight cycles of treatment, time to transplant longer than 3 years from diagnosis, and prior radiation were associated with adverse outcomes. Additional strategies, such as intervention earlier in the disease course, improved treatment regimens, sequential high-dose treatments, and posttransplant therapies may improve outcome of selected patients with MM. PMID- 8622059 TI - Psychologic and neuropsychologic impact of autologous bone marrow transplantation. AB - PURPOSE: The major purpose of the current study was to evaluate the psychologic and neuropsychologic functioning of patients undergoing treatment with autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Fifty-four patients with hematologic disorders or breast cancer completed a battery of psychologic and neuropsychologic tests before ABMT, at mid-treatment (1 to 3 days following bone marrow reinfusion), and predischarge (within 1 to 2 days before discharge from the hospital). RESULTS: Analysis of pretransplant data showed significantly higher scores on the State-Trait Anxiety Inventory (STAI) and Profile of Mood States (POMS) for patients with hematologic disorders as compared with patients with breast cancer. However, no baseline differences on neuropsychologic measures were found when patients were divided into groups based on prior exposure to cranial radiation and/or intrathecal chemotherapy. Serial evaluations at pretransplant, following return of bone marrow, and at predischarge were available for 34 patients. For the psychologic data, patients with hematologic disorders tended to be more distressed than breast cancer patients at baseline, but became less distressed over time. By contrast, breast cancer patients were relatively less distressed at baseline, demonstrated a significant increase in distress midtreatment, and returned to baseline levels at predischarge assessment. Scores on neuropsychologic measures that assessed higher order cognitive functioning generally worsened over time. CONCLUSION: The results suggest a differential response on psychologic measures when comparing patients with hematologic disorders with those with breast cancer. However, both groups demonstrated a general decline in performance on neuropsychologic measures over the course of treatment. PMID- 8622060 TI - High-dose paclitaxel, cyclophosphamide, and cisplatin with autologous hematopoietic progenitor-cell support: a phase I trial. AB - PURPOSE: To determine the maximal-tolerated dose (MTD) of paclitaxel in combination with high-dose cyclophosphamide (CPA) and cisplatin (cDDP) followed by autologous hematopoietic progenitor-cell support (AHPCS). PATIENTS AND METHODS: Forty-nine patients with poor-prognosis breast cancer, non-Hodgkin's lymphoma (NHL), or ovarian cancer were treated with escalating doses of paclitaxel infused over 24 hours, followed by CPA (5,625 mg/m2 intravenously over 1 hour in three divided doses) and cDDP (165 mg/m2 intravenously as a continuous infusion over 72 hours) and AHPCS. Pharmacokinetic measurements for each drug were performed. RESULTS: Dose-limiting toxicities were encountered in two patients at 825 mg/m2 of paclitaxel; one patient died of multiorgan failure that involved the lung, CNS, and kidneys, and the other developed grade 3 respiratory, CNS, and renal toxicity, which resolved. The MTD of this combination was determined to be paclitaxel 775 mg/m2, CPA 5,625 mg/m2, and cDDP 165 mg/m2 followed by AHPCS. Sensory polyneuropathy and mucositis were prominent toxicities, but both were reversible and tolerable. The pharmacokinetics of paclitaxel correlated significantly with the severity of mucositis (P < .001) and peripheral neuropathy (P < .00004). Eighteen of 33 patients (54%) with measurable, heavily pretreated metastatic breast cancer achieved a partial response (PR). Responses were also observed in patients with NHL (four of five patients) and ovarian cancer (two of two). CONCLUSION: It is possible to escalate the dose of paclitaxel to 775 mg/m2 in combination with 5,625 mg/m2 of CPA, 165 mg/m2 of cDDP, and AHPCS. An encouraging response rate in poor-prognosis patients with breast cancer, NHL, and ovarian cancer warrants further study. PMID- 8622061 TI - Feasibility of multiple courses of high-dose cyclophosphamide, thiotepa, and carboplatin for breast cancer or germ cell cancer. AB - PURPOSE: To determine the feasibility and safety of multiple, closely timed courses of high-dose cyclophosphamide, thiotepa, and carboplatin (CTC) with peripheral-blood progenitor-cell transplantation (PBPCT). PATIENTS AND METHODS: Forty-eight patients with advanced cancer were scheduled to undergo either two or three courses of CTC with PBPCT. All PBPCs were harvested before high-dose therapy began. Full-dose CTC courses incorporated cyclophosphamide (6,000 mg/m2), thiotepa (480 mg/m2), and carboplatin (1,600 mg/m2) divided over days -6, -5, -4, and -3. Tiny CTC courses (tCTC) contained 67% of the doses of each of these agents. Second or third courses of CTC or tCTC began on day 28. RESULTS: A sufficient number of PBPC could be harvested from all but two patients. Thirty five first full-dose courses of CTC were given, 28 second courses, and 10 third courses. Second courses could be given on time and at full dose in 80% of the patients, but there was one toxic death from venoocclusive disease (VOD). Only four of 12 patients scheduled to receive three courses of full-dose CTC could be treated at the time and dose planned. There were three toxic deaths: one of VOD, one of sepsis, and one of hemolytic uremic syndrome (HUS). Eight patients were scheduled to receive three courses of tCTC. Eight first, seven second, and six third courses were given. One of the third courses had to be delayed and one had to be reduced in dose. CONCLUSION: A sufficient number of PBPCs for two or three transplantations can be harvested from most patients without much difficulty before high-dose therapy. Two full-dose CTC courses or three tCTC courses can be given safely and with acceptable toxicity at 5-week intervals. Organ toxicity rather than bone marrow toxicity has become dose-limiting for alkylating agents. PMID- 8622062 TI - Conditioning regimen-dependent disposition of cyclophosphamide and hydroxycyclophosphamide in human marrow transplantation patients. AB - PURPOSE: The pharmacokinetics of cyclophosphamide (CY) and 4 hydroxycyclophosphamide (HCY) were studied in 14 patients being prepared for bone marrow transplantation with either busulfan (BU)/CY (n = 7) or CY/total-body irradiation (TBI) (n = 7) to determine whether exposure to CY and its proximate toxic metabolite HCY is modulated by other agents used in the preparative regimen. PATIENTS AND METHODS: HCY was assayed by a new method that stabilized the metabolite at bedside. In BU/CY patients (who also received phenytoin), CY clearance was 112% greater (P = .0014), half-life 54% less (P = .0027), peak HCY concentration in plasma/CY dose 113% greater (P = .0006), and the ratio of area under the plasma concentration-time curves (AUCs) of HCY to CY 166% greater (P = .0116) than in CY/TBI patients. The ratio of the AUC of HCY/CY dose was 48% greater in BU/CY patients than in CY/TBI patients when one CY/TBI patient with an apparent impaired ability to eliminate HCY was excluded from analysis. In CY/TBI patients, there was an inverse correlation between the AUC of HCY and that of CY (R2 = .740, P = .028). Also, the ratio of the AUC of HCY/CY dose was correlated with the average concentration of BU at steady-state (Css, Bu) (R2 = .646, P = 0.29). Variability in CY and HCY pharmacokinetics among the 14 patients overall was pronounced, with the highest variability (15-fold) observed in the ratio of the AUC of HCY to that of CY. CONCLUSION: Prior administration of BU and/or phenytoin significantly alters exposure to CY and HCY. Interpatient variability in HCY exposure at a given CY dose is substantial. PMID- 8622063 TI - Phase I trial of ZD1694, a new folate-based thymidylate synthase inhibitor, in patients with solid tumors. AB - PURPOSE: To perform a phase I clinical and pharmacologic study of ZD1694 (Tomudex, Alderley Park, United Kingdom), a new folate-based thymidylate synthase (TS) inhibitor, in patients with advanced malignancy. PATIENTS AND METHODS: From February 1991 to January 1993, 61 patients with a range of solid tumor received 161 courses of ZD1694 given as a single 15-minute intravenous infusion every 3 weeks, at escalating doses from 0.1 to 3.5 mg/m2. Pharmacokinetic (PK) analysis was performed with the first two courses of treatment. There were 33 men and 28 women with a median age of 53 years (range, 21 to 73). Fifty-five patients (90%) had previously received chemotherapy. RESULTS: Reversible liver toxicity and dose related gastrointestinal (GI) and bone marrow toxicity occurred at > or = 1.6 mg/m2. Liver function usually returned to normal with repeated treatment, but GI and bone marrow toxicities generally became more severe. No renal toxicity was observed. The maximum-tolerated dose (MTD) was 3.5 mg/m2, at which, in addition to antiproliferative toxicities, four of six patients (67%) developed severe malaise that consisted of anorexia, nausea, and asthenia, with rapidly decreasing performance status that limited re-treatment. Abnormal liver function was also seen in four patients (67%). At 3.0 mg/m2, grades III and IV diarrhea were seen in six of 23 patients (26%) and grade IV myelosuppression in two others. Liver toxicity was self-limiting and not associated with severe malaise. Two patients had a partial response to treatment. PK analysis showed that plasma elimination was triexponential, with pronounced variability in the mean terminal half-life (t1/2gamma) for a given dose ranging from 8.2 to 105 hours. There was a linear relationship between dose and both the area under the concentration-time curve (AUC) and maximum concentration (Cmax), but no clear association between these parameters and response or toxicity. CONCLUSION: The dose of ZD1694 recommended for phase II trials is 3.0 mg/m2. PMID- 8622064 TI - Escalating systemic exposure of continuous infusion topotecan in children with recurrent acute leukemia. AB - PURPOSE: To determine the maximum-tolerated systemic exposure (MTSE) and exposure limiting toxicity of continuous infusion topotecan in children with recurrent acute leukemia. PATIENTS AND METHODS: Patients received escalating levels of topotecan systemic exposure as measured by steady-state topotecan lactone concentration (Css). Samples obtained within the first 24 hours were measured by high-pressure liquid chromatography (HPLC) for topotecan. A two-compartment model was fit to the data using a Bayesian algorithm. Css was calculated for each patient; if it differed by more than 20% of target, a new dosage was begun within 6 hours. Follow-up concentrations were obtained as well as serial plasma samples postinfusion. Toxicity and evidence of activity were assessed after each course. RESULTS: Thirteen boys and five girls received 23 courses of topotecan. Target Css ranged from 1.0 to 5.3 ng/mL (topotecan doses, 0.5 to 3.3 mg/m2/d). Nineteen of 23 courses were within +/- 20% of target after adjustment (range, 77% to 139%). The MTSE was 4.0 ng/mL, and mucositis was exposure-limiting at 5.3 ng/mL. A significant relation between topotecan lactone Css and the severity of mucositis was observed. Myelosuppression was experienced but was not considered exposure-limiting. One complete response and one partial response were noted. CONCLUSION: The MTSE for continuous infusion topotecan was 4.0 ng/mL. Responses were noted at Css comparable to those producing responses in a severe combined immunodeficiency (SCID) mouse model. Further studies of topotecan are warranted. PMID- 8622065 TI - Prognostic importance of p15INK4B and p16INK4 gene inactivation in childhood acute lymphocytic leukemia. AB - PURPOSE: The present study explores the prognostic importance of p16INK4/p15INK4B gene inactivation in childhood acute lymphocytic leukemia (ALL). MATERIALS AND METHODS: Cells from 79 pediatric ALL patients were investigated for inactivation of the p15INK4B and p16INK4 genes or loss of heterozygosity (LOH) for chromosome 9p markers by use of Southern hybridization, restriction fragment length polymorphism (RFLP) analysis, microsatellite analysis as well as single-strand conformation polymorphism (SSCP) analysis, and nucleotide sequencing of the p15INK4B and p16INK4 genes. Genetic data were correlated to clinical outcome and established prognostic factors. RESULTS: Inactivation of the p15INK4B and/or p16INK4 genes by homozygous deletion or loss of one allele and mutation of the other was detected in 24 cases (30%). Another 12 patients (15%) showed loss of one allele. A statistically significant correlation was found between inactivation of the p15INK4B/p16INK4 genes and poor prognosis (P < .01). Furthermore, inactivation proved to be an independent factor that predicted relapse, ranking second to WBC count. The trend toward overrepresentation of treatment failure was strongest in the high-risk (HR) group patients with p16INK4/p15INK4B gene inactivation. Patients with deletion of genetic material on 9p21 and normal coding sequence of the remaining p16INK4 and p15INK4B genes had a similar prognosis to that of nondeleted cases. CONCLUSION: The data suggest that analysis of p15INK4B/p16INK4 genes may contribute prognostic information in pediatric ALL. PMID- 8622066 TI - Therapy of refractory or recurrent childhood acute myeloid leukemia using amsacrine and etoposide with or without azacitidine: a Pediatric Oncology Group randomized phase II study. AB - PURPOSE: A randomized study compared the combination of amsacrine (100 mg/m2/d on days 1 to 5) and etoposide (200 mg/m2/d on days 1 to 3) with the same two agents plus azacitidine (250 mg/m2/d on days 4 to 50) for the therapy of induction resistant or relapse childhood acute myeloid leukemia (AML). PATIENTS AND METHODS: One hundred sixty-seven assessable children with AML who either had failed to respond to primary induction therapy (group 1, n = 41) or had relapsed (group 2, n = 126) were randomized. RESULTS: Overall, there were 56 complete responses (34%; SE 4%). Among primary refractory patients (group 1), the complete response rate was higher with the three-drug regimen (18% vs 53%, P = .03). In the relapsed patients (group 2), there was no difference in complete response rates related to treatment (31% vs 35%, P = .3). There were 17 early deaths. The major toxicities for both regimens were myelosuppression and infection. CONCLUSION: The overall complete response rate of 34% in this patient population is indicative of effective antileukemic activity. For patients with relapsed leukemia, the addition of azacitidine to etoposide and amsacrine did not improve response. The suggested advantage of the three-drug regimen for induction failures warrants further investigation. PMID- 8622067 TI - Desmoplastic small round-cell tumor: prolonged progression-free survival with aggressive multimodality therapy. AB - PURPOSE: To test intensive alkylator-based therapy in desmoplastic small round cell tumor (DSRCT). PATIENTS AND METHODS: Patients received the P6 protocol, which has seven courses of chemotherapy. Courses 1, 2, 3, and 6 included cyclophosphamide 4,200 mg/m2, doxorubicin 75 mg/m2, and vincristine (HD-CAV). Courses 4, 5, and 7 consisted of ifosfamide 9 g/m2 and etoposide 500 mg/m2 for previously untreated patients, or ifosfamide 12 g/m2 and etoposide 1,000 mg/m2 for previously treated patients. Courses started after neutrophil counts reached 500/microL and platelet counts reached 100,000/microL. Tumor resection was attempted. Post-P6 treatment options included radiotherapy and a myeloablative regimen of thiotepa (900 mg/m2) plus carboplatin (1,500 mg/m2), with stem-cell rescue. RESULTS: Ten previously untreated and two previously treated patients have completed therapy. The male-to-female ratio was 11:1. Ages were 7 to 22 years (median, 14). The largest masses were infradiaphragmatic (n = 11) or intrathoracic (n = 1). Other findings included serosal implants (n = 11), regional lymph node invasion (n = 8), ascites or pleural effusion (n = 7), and metastases to liver (n = 5), lungs (n = 4), distant lymph nodes (n = 3), spleen (n = 2), and skeleton (n = 2). Tumors uniformly responded to HD-CAV, but there were no complete pathologic responses. One patient died at 1 month from tumor related Budd-Chiari syndrome. Of seven patients who achieved a complete remission (CR), five remain in CR 9, 12, 13, 33, and 38 months from the start of P6, one patient died of infection at 12 months (autopsy-confirmed CR), and one patient relapsed 4 months off therapy. Of four patients who achieved a partial remission (PR), one remains progression-free at 34 months and three developed progressive disease. Five patients received local radiotherapy: three were not assessable for response, but in two patients, antitumor effect was evident. Four patients received thiotepa/carboplatin: two were in CR and remain so, and two patients had measurable disease that did not respond. CONCLUSION: For control of DSRCT, our experience supports intensive use of HD-CAV, aggressive surgery to resect visible disease, radiotherapy to high-risk sites, and myeloablative chemotherapy with stem-cell rescue in selected cases. PMID- 8622068 TI - Results of a stage-based protocol for the treatment of retinoblastoma. AB - PURPOSE: To describe the treatment of retinoblastoma at a single institution using a prospective protocol based on histopathologic staging. PATIENTS AND METHODS: We included 116 consecutive patients (101 eligible, 46 bilateral) from August 1987 to December 1993. Treatment was enucleation or conservative therapy for intraocular disease (stage I patients). Stage II patients (orbital or postlaminar invasion) received vincristine, cyclophosphamide, and doxorubicin for 57 weeks. Patients with orbital mass and extension beyond the cut end of the optic nerve also received orbital radiotherapy (45 Gy). The latter received intrathecal therapy. In those with CNS (stage III) or hematogenous metastasis (stage IV), cisplatin and etoposide were added along with cranial (in patients with a CNS mass and prophylactically in stage IV) or craniospinal (in patients with positive CSF) radiotherapy. RESULTS: The median follow-up time was 39 months (range, 12 to 84). The overall survival rate was 0.84. Survival rates according to stage were as follows: stage I probability of overall survival [pOS] = 0.97) (alive/total), 59 of 60; stage II (pOS = 0.85) including patients with scattered episcleral cells, three of three; orbital mass, one of one; postlaminar invasion up to and beyond the cut end of optic nerve, 10 of 11 and 11 of 14, respectively; of stage III (pOS = 0), zero of six; and stage IV (pOS = 0.50), three of six. Only those patients with preauricular adenopathy as the only metastatic site survived in the latter group. Acute toxicity was mild. CONCLUSION: Chemotherapy is not warranted to prevent systemic metastasis for intraocular disease. Patients with extraocular orbital disease and had a good outcome with this therapy. Patients with metastatic disease fared poorly, except for those with isolated malignant preauricular adenopathy. PMID- 8622069 TI - Resection of primary tumor at diagnosis in stage IV-S neuroblastoma: does it affect the clinical course? AB - PURPOSE: To determine whether resection of primary tumor has a favorable influence on outcome of infants (age 0 to 11 months) with stage IV-S neuroblastoma. PATIENTS AND METHODS: Between March 1976 and December 1993, 97 infants with previously untreated neuroblastoma diagnosed in 21 Italian institutions were classified as having stage IV-S disease. Seventy percent were younger than 4 months. Adrenal was the primary tumor site in 64 of 85 patients with a recognizable primary tumor. Liver was the organ most often infiltrated by the tumor (82 patients), followed by bone marrow and skin. RESULTS: The overall survival (OS) rate at 5 years in 80% and event-free survival (EFS) rate 68%. In 24 infants, the effect of resection of primary tumor could not be evaluated because of rapidly fatal disease progression (n = 8), absence of a primary tumor (n = 12), or partial resection (n = 4). Of 73 assessable patients, 26 underwent primary tumor resection at diagnosis: one died of surgical complications, one relapsed locally and died, and two others relapsed (one of these two locally) and survived, for a 5-year OS rate of 92% and EFS rate of 84%. Of the remaining 47 patients who did not undergo primary tumor resection at diagnosis 11 suffered unfavorable events, of whom five died, for an OS rate of 89% and EFS rate of 75% (no significant difference from previous group). Disease recurred at the primary tumor site in only one five who died, and in only one of six survivors of progression or relapse; in these patients, the primary tumor, located in the mediastinum, was successfully resected. CONCLUSION: Infants who underwent resection of the primary tumor at diagnosis had no better outcome than those in whom the decision was made not to operate. PMID- 8622070 TI - Defining response of ovarian carcinoma to initial chemotherapy according to serum CA 125. AB - PURPOSE: To produce definitions based on serial CA 125 levels to measure response of ovarian carcinoma in patients receiving first-line chemotherapy. PATIENTS AND METHODS: Definitions were derived from analysis of 277 patients in North Thames Ovary Trial 3. Patient data were then incorporated into a computer program and tested against 254 patients in North Thames Ovary Trial 4 and 458 patients in Gynecologic Oncology Group (GOG) protocol 97. For optimum detection of response, three response definitions have been combined into a computer program. The precise definitions use mathematic logic and take account of factors such as intervening samples. Response to a specific treatment has occurred if after two samples there has been a 50% decrease, confirmed by a fourth sample (50% response), or a serial decrease over three samples of greater than 75% (75% response). The final sample has to be at least 28 days after the previous sample. RESULTS: Six hundred twenty of 989 patients were considered assessable for response according to CA 125 level. Only two patients (0.3%) had a CA 125 response at the time of clinical progression. The CA 125 response rate was 62% and 54% in the North Thames trials. In the GOG trial, it was 66% in all 317 patients assessable for CA 125 and 67% in 221 patients whose CA 125 level was not measurable according to GOG criteria, compared with a GOG-defined response rate of 62%. The sensitivity for detecting GOG-defined response was at least 68%. CONCLUSION: Definitions based on a 50% or 75% decrease of CA 125 levels have been shown reliably to define partial response of ovarian cancer in patients receiving first-line chemotherapy. These definitions should be used in addition to or instead of standard response criteria. PMID- 8622072 TI - Conservative treatment versus mastectomy in early breast cancer: patterns of failure with 15 years of follow-up data. Institut Gustave-Roussy Breast Cancer Group. AB - PURPOSES: A randomized trial was conducted to compare tumorectomy and breast irradiation with modified radical mastectomy. We have analyzed the patterns of failure in each arm of the trial and the prognostic factors that have an independent effect on treatment failures and overall survival. PATIENTS AND METHODS: The trial included 179 patients with breast cancer of up to 20 mm in diameter at macroscopic examination. Eighty-eight patients had conservative management and 91 a mastectomy. All patients had axillary dissection with frozen section examination. For patients with positive axillary nodes (N+), a second randomization was performed: lymph node irradiation versus no further regional treatment. Patterns of failure were determined by a competing-risk approach and multivariate analysis. A prognostic-score was determined by multivariate analysis. RESULTS: Overall survival, distant metastasis, contralateral breast cancer, new primary malignancy, and locoregional recurrence rates were not significantly different between the two surgical groups, or between lymph node irradiation groups. Most recurrences appeared during the first 10 years. Three distinct prognostic groups were determined taking into account age, tumor size, histologic grading, and number of positive axillary nodes. CONCLUSION: Long-term results support conservative treatment with limited surgery and systematic breast irradiation as a safe procedure for the management of small breast cancers. Four easily obtainable clinical and histologic factors may be combined in a prognostic score that is highly predictive of overall and event-free survival. PMID- 8622071 TI - Phase II study of intravenous topotecan as a 5-day infusion for refractory epithelial ovarian carcinoma. AB - PURPOSE: To determine the efficacy and toxicity of topotecan administered as a 5 day intravenous infusion in patients with advanced ovarian cancer refractory to cisplatin-based chemotherapy. PATIENTS AND METHODS: Thirty patients with advanced epithelial ovarian cancer refractory to cisplatin-based chemotherapy received intravenous infusions of topotecan 1.5 mg/m2 delivered over 30 minutes each day for 5 days. A course was repeated every 21 days. The patient eligibility requirements included age > or = 18 years, Zubrod score < or = 2, measurable disease, adequate hepatic and renal function, neutrophil count > or = 1,500/microL, platelet count > or = 100,000/microL, and anticipated survival > or = 3 months. RESULTS: Twenty eight patients were assessable for response and toxicity. All patients were assessable for survival. The major toxicity from administration of topotecan at this dose schedule was myelosuppression; 21 patients required dose reductions. Four patients had neutropenic fever that required hospitalization, and seven patients required platelet transfusions. Maculopapular pruritic exanthema occurred in 20% of patients; gastrointestinal side effects were mild. No deaths were reported on the study. At dose levels of 1.5, 1.25, and 1.0 mg/m2, 61%, 31%, and 25% of patients, respectively, required dose reductions. Of 28 assessable patients, four (14%; 95% confidence interval [CI], 4% to 34%) achieved a partial response (PR) at a median of 1.4 months and lasting 8.9 months, and 17 had stable disease (SD). The overall median survival time was 10.0 months (95% CI, 8.1 to 13.5). CONCLUSION: Topotecan shows modest clinical activity against cisplatin-refractory ovarian cancer, although the dose intensity is compromised by the depth of the granulocyte nadir and the duration of granulocytopenia. Further studies of topotecan may necessitate a reevaluation of optimal dose schedule, with the possible incorporation of multilineage cytokines, and its activity in taxane-resistant tumors. PMID- 8622073 TI - Intensive outpatient adjuvant therapy for breast cancer: results of dose escalation and quality of life. AB - PURPOSE: A dose-escalation study was conducted to determine the maximum-tolerated dose (MTD) and dose-limiting toxicities (DLTs) of cyclophosphamide (CY) in combination with granulocyte colony-stimulating factor (G-CSF0 and doxorubicin (DOX) given every 2 weeks for eight cycles as outpatient adjuvant therapy for node-positive breast cancer. A pilot study to assess quality of life (QOL) was performed. PATIENTS AND METHODS: From March 1991 to April 1993, 19 patients were entered. Patients received escalating doses of CY intravenously (i.v.) (1,000 mg/m2, 1,500 mg/m2, 2,000 mg/m2, or 2,500 mg/m2) with DOX 40 mg/m2, G-CSF 10 micrograms/kg/d on days 2 to 12, and mesna, every 2 weeks for eight cycles. QOL was measured by the Profile of Mood States (POMS), the Psychosocial Adjustment to Illness Scale-Self Report (PAIS-SR), and a 27-item QOL scale. RESULTS: The CY dose of 2,500 mg/m2 every 2 weeks elicited toxicities that required dose reductions secondary to a combination of thrombocytopenia, hematuria, and anemia that required transfusion. The dose of 2,000 mg/m2 resulted in an acceptable toxicity profile. Ninety-two percent of cycles at the 2,000-mg/m2 dose were delivered on schedule and 77% without hospitalization. QOL assessments indicated high levels of distress measured by POMS in 47%, poor overall quality of life in 40%, and significant problems with physical symptoms in less than 27% of all patients for any given cycle. CONCLUSION: A dose of CY at 2,000 mg/m2 can be administered every 2 weeks with DOX and G-CSF for eight cycles in the outpatient setting with manageable toxicity. The majority of women described levels of physical symptoms and emotional distress as tolerable during treatment. PMID- 8622074 TI - Five-day course of granulocyte colony-stimulating factor in patients with prolonged neutropenia after adjuvant chemotherapy for breast cancer is a safe and cost-effective schedule to maintain dose-intensity. AB - PURPOSE: To analyze the safety and efficacy of a short course of granulocyte colony-stimulating factor (G-CSF) to maintain dose-intensity of subsequent cycles of chemotherapy after a prior episode of prolonged neutropenia, without febrile complications, in patients receiving adjuvant treatment for breast cancer. PATIENTS AND METHODS: Thirty-two patients undergoing adjuvant cyclophosphamide, methotrexate, and fluorouracil (CMF) or doxorubicin-CMF for stages I to II breast cancer were included after having chemotherapy delays due to neutropenia (absolute neutrophil count [ANC] < 1.5 x 10(9)/L) on day 22. G-CSF was administered subcutaneously on days 15 to 19 of each subsequent cycle. RESULTS: None of the patients included in this study had to be admitted to the hospital for fever and neutropenia. The median percentage of the projected dose-intensity for CMF or doxorubicin-CMF on an intent-to-treat basis was 0.994, which was significantly higher than the delivered dose-intensity before the start of G-CSF treatment (P < .0001). Patients who received concomitant G-CSF and radiotherapy achieved a similar dose-intensity as patients who did not undergo radiotherapy. Seven patients discontinued G-CSF treatment due to musculoskeletal pain. These patients had more subsequent cycle delays because of day 22 neutropenia than the 25 patients who followed the G-CSF schedule (P = .0028). CONCLUSION: A 5-day course of G-CSF in patients with prior chemotherapy delays due to prolonged neutropenia seems to be a safe and cost-effective schedule to maintain CMF or doxorubicin-CMF dose-intensity in the adjuvant treatment of breast cancer. PMID- 8622075 TI - Lack of relationship between systemic exposure for the component drug of the fluorouracil, epirubicin, and 4-hydroxycyclophosphamide regimen in breast cancer patients. AB - PURPOSE: The aim of this study was to investigate the covariance between the pharmacokinetics of the three components of the FEC regimen, epirubicin (EPI), fluorouracil (5-FU), and the cyclophosphamide (CP) metabolite 4 hydroxycyclophosphamide (4-OHCP), in breast cancer patients. PATIENTS AND METHODS: Data from 21 women were collected over a total of 35 cycles. 5-FU (300 to 600 mg/m2) and CP (300 to 600 mg/m2) were administered as bolus injections, whereas EPI (15 to 60 mg/m2) was administered either as a bolus injection or as an infusion. The pharmacokinetics of the component drugs were monitored using a limited sampling scheme. Population pharmacokinetic models for each of the three drugs were developed using the program NONMEM. RESULTS: The data for 5-FU were best described by a one-compartment model with nonlinear elimination, where the maximal rate of elimination (Vmax) and the concentration at which the elimination was half-maximal (Km) were 105 mg/L.h and 27 mg/L, respectively. EPI concentration-time profiles showed a triexponential decline, with a mean terminal half-life of 24 hours and a clearance (CL) of 59 L/h. The elimination of 4-OHCP was monoexponential, with a mean half-life of 7 hours. The interindividual coefficients of variation (CVs) in CL were 30%, 22%, and 41% for 5-FU, EPI, and 4 OHCP, respectively. The corresponding values for intrapatient course-to-course variability in CL were 11%, 8%, and 27%. No significant correlation in any of the pharmacokinetic parameters between the drugs was found. CONCLUSION: Individualization of dosing of the FEC regimen using therapeutic drug monitoring and attempts to find concentration-response relationships may be successful, but requires that the exposure of all three drugs is considered simultaneously. PMID- 8622076 TI - Postsurgical adjuvant chemotherapy of stage II breast carcinoma with or without crossover to a non-cross-resistant regimen: a Cancer and Leukemia Group B study. AB - PURPOSE: To compare two cyclophosphamide, methotrexate, fluorouracil, vincristine, and prednisone (CMFVP) regimens with a doxorubicin-based regimen- vinblastine, doxorubicin, thiotepa, and Halotestin (Upjohn, Kalamazoo, MI) (VATH) -in patients with stage II node-positive breast carcinoma. METHODS: Nine hundred forty-five women were treated with a 6-week induction course of CMFVP. They were then randomized to receive one of two consolidation CMFVP regimens: 6-week courses or 2-week courses. Following completion of CMFVP consolidation, patients were again randomized to either continue the CMFVP regimen or to receive six escalating doses of VATH. RESULTS: Among all patients, with a median follow-up time of 11.5 years, there is no statistically significant difference in disease free survival (DFS) between the two consolidation CMFVP regimens. VATH intensification treatment is statistically significantly superior to CMFVP in terms of DFS (P = .0040). For patients with one to three involved nodes, there is currently no significant difference between VATH and CMFVP; however, among those with four or more positive lymph nodes, there is a significant difference in favor of VATH (P = .0037). There is also improved overall survival with VATH (P = .043; median, > 14 years v 10 years). This difference is also statistically significant in patients with four or more involved lymph nodes, among postmenopausal patients, and among postmenopausal estrogen receptor-positive patients. CONCLUSION: Chemotherapy with crossover to escalating doses of VATH following CMFVP was well tolerated and effective. Inauguration of VATH as a treatment intensification at the eighth month produced a major increase in relapse-free and overall survival. The observation that sensitivity to VATH is retained so long after mastectomy raises questions about the proper duration of adjuvant chemotherapy and lends support to further investigation of cross-over designs in future trials to postoperative adjuvant chemotherapy regimens. PMID- 8622077 TI - Radioimmunoguided surgery after primary treatment of locally advanced breast cancer. AB - PURPOSE: To assess the role of radioimmunoguided surgery (RIGS) using a handheld intraoperative gamma-detecting probe (GDP) to identify neoplastic disease after primary chemotherapy in locally advanced breast cancer (LABC) patients injected with iodine 125-labeled monoclonal antibodies (MAbs). PATIENTS AND METHODS: Twenty-one patients with histologically documented LABC were treated with a combined modality approach. After three courses of primary chemotherapy and before modified radical mastectomy, the 125I-radiolabeled MAbs B72.3 (anti-TAG72) and FO23C5 (anti-carcinoembryonic antigen [CEA]) were administered to 11 patients (group A) and 10 patients (group B), respectively. At surgery, a GDP was used to locate the primary tumor and to assess possible tumor multicentricity and the presence of ipsilateral axillary metastases. Routine pathologic examination was performed in neoplastic and normal tissue specimens of all 21 patients. In addition, immunohistochemical assay for TAG72 and CEA expression was performed. RESULTS: In group A patients, RIGS identified primary tumor in seven of 11 patients (63.3%) and unpalpable multicentric tumor lesions were located in two of four (50%). Positive axillary lymph nodes were histologically documented in eight of 11 patients (72.7%) and RIGS identified three of eight (37.5%). In group B, RIGS located the primary tumor lesion in four of 10 patients (40%); in two cases, the tumor was not clinically evident. Multicentricity was observed in one of two patients and lymph node involvement in three of nine (33.3%). No false-positive results were observed in either group A or B. CONCLUSION: RIGS appears to be a safe and reliable technique. However, the MAbs used in this study are not sufficiently specific. RIGS represents a technique for which the full potential for intraoperative assessment of breast cancer lesions can be reached when more specific antibodies become readily available. PMID- 8622078 TI - p53 and bcl-2 expression correlates with clinical outcome in a series of node positive breast cancer patients. AB - BACKGROUND AND PURPOSE: The tumor-suppressor gene TP53 and the proto-oncogene bcl 2 encode, respectively, for a nuclear phosphoprotein and for a mitochondrial protein involved in multiple cellular functions. The proteins provide prognostic information in node-negative breast cancer and are supposed to influence treatment responsiveness. We analyzed the predictive role of p53 and bcl-2 expression, alone and in association with other variables, in postmenopausal women with node-positive, estrogen receptor-positive (ER+) breast cancers treated with radical or conservative surgery plus radiotherapy and adjuvant tamoxifen for at least 1 year. PATIENTS AND METHODS: On 240 resectable cancers, we determined the expression of p53 and bcl-2, using immunohistochemistry, cell proliferation (3H-thymidine labeling index [3H-dT LI]), and ER and progesterone receptors (PgR). RESULTS: p53 expression and 3H-dT LI were weakly related to one another and both were unrelated to bcl-2. Relapse-free and distant metastasis-free survival at 5 years were significantly lower for patients with tumors that highly expressed p53 (P = .0001) and for those that weakly expressed or did not express bcl-2 (P = .02). However, p53, but not bcl-2, provided prognostic information independent of tumor size, axillary node involvement, steroid receptors, and 3H dT LI. Moreover, the simultaneous p53 overexpression and lack of PgR identified patients at maximum risk of relapse, whereas bcl-2 overexpression, associated with a low 3H-dT LI or the presence of PgR, improved the prognostic resolution for low-risk patients. CONCLUSION: p53 expression appears to be indicative of clinical outcome in postmenopausal patients treated with tamoxifen. Whether p53 overexpression and weak bcl-2 expression are indicators of biologic aggressiveness, regardless of treatment, or of hormone resistance remains to be defined. PMID- 8622079 TI - Paclitaxel with mitoxantrone, fluorouracil, and high-dose leucovorin in the treatment of metastatic breast cancer: a phase II trial. AB - PURPOSE: Paclitaxel is a highly active single agent in the treatment of breast cancer. However, its optimal incorporation into combination regimens awaits definition. In this phase II study, we added paclitaxel, administered by 1-hour infusion, to a previously described combination regimen that included mitoxantrone, fluorouracil (5-FU), and high-dose leucovorin (NFL). PATIENTS AND METHODS: Forty-six patients with metastatic breast cancer received the following regimen as first- or second-line treatment: paclitaxel 135 mg/m2 by 1-hour intravenous (i.v.) infusion on day 1, mitoxantrone 10 mg/m2 by i.v. bolus on day 1, 5-FU 350 mg2/m by i.v. bolus on days 1, 2, and 3, and leucovorin 300 mg i.v. over 30 to 60 minutes immediately preceding 5-FU on days 1, 2, and 3. Courses were administered at 3-week intervals for a total of eight courses in responding patients. RESULTS: Twenty-three of 45 assessable patients (51%) had major responses. Previous chemotherapy, and in particular previous treatment with doxorubicin, did not affect response rate. The median response duration was 7.5 months. Myelosuppression was moderately severe, with 76% of courses resulting in grade 3 or 4 leukopenia. Hospitalization for treatment of fever during neutropenia was required in 13% of courses, and two patients died as a result of sepsis. Two patients developed severe congestive heart failure after a large cumulative anthracycline dose. CONCLUSION: This combination regimen was active as first- or second-line therapy for metastatic breast cancer, although its activity compared with other combination regimens or with paclitaxel alone is unclear. Myelosuppression was more severe than anticipated based on previous results with the NFL regimen or with paclitaxel administered at this dose and schedule as a single agent. The infrequent development of cardiotoxicity in these patients suggests that the paclitaxel/mitoxantrone combination may not share the problems previously reported with the paclitaxel/doxorubicin combination. PMID- 8622080 TI - Doxorubicin and dose-escalated cyclophosphamide with granulocyte colony stimulating factor for the treatment of hormone-resistant prostate cancer. AB - PURPOSE: The goals of this study were to define the efficacy and toxicity of doxorubicin and dose-escalated cyclophosphamide (Cy) along with granulocyte colony-stimulating factor (G-CSF) in the treatment of hormone-refractory prostate cancer (HRPC), to determine the maximal-tolerated dose (MTD) of Cy in this regimen, and to evaluate the impact of prior pelvic irradiation (XRT) on MTD and toxicity. PATIENTS AND METHODS: Thirty-five patients were treated every 21 days with fixed-dose doxorubicin (40 mg/m2) and Cy 800 to 2,000 mg/m2 (in a cohort dose-escalation schema) along with G-CSF. RESULTS: Five of 15 patients (33%) with measurable disease obtained an objective response. Sixteen of 35 patients (46%) had a greater than 50% decrease in prostate-specific antigen (PSA) level (95% confidence interval [CI], 28.8% to 63.4%). Ten of 35 patients (28.6%) had a greater than 75% decrease in PSA level. The median survival time was 11 months. The median survival duration of patients with a greater than 50% decrease in PSA level was 23 months, versus a median survival time of 7 months in patients without a PSA response (P = .02). Although 33% of cycles were associated with grade 4 neutropenia, febrile neutropenia occurred in only 7.8% of all cycles. Thrombocytopenia and anemia were rare. Nonhematologic toxicity was minimal. Patients who had received prior pelvic XRT had a lower Cy MTD, but their hematologic toxicity was not appreciably different. CONCLUSION: This is a well tolerated, active regimen for the treatment of HRPC. Toxicity was not different in patients with prior pelvic XRT, although these patients had a lower MTD. PMID- 8622081 TI - Suramin in hormone-refractory metastatic prostate cancer: a drug with limited efficacy. AB - PURPOSE: To confirm the previously reported high response rates and prolonged survival in hormone-refractory prostate cancer treated with suramin. PATIENTS AND METHODS: Thirty-six eligible patients with hormone-refractory prostate cancer with either measurable disease or bone disease only and a prostate-specific antigen (PSA) level greater than 50 ng/mL were enrolled. Treatment consisted of two 8-week courses of outpatient-based therapy with an interposed rest period. A bayesian adaptive control strategy and a three-compartment pharmacokinetic model that accommodates clearance changes was used to guide individual dosing. A rapid infusion of 1,000 mg/m2 suramin was followed by five daily infusions that targeted 285 micrograms/mL peak plasma levels during the first week. All patients received concomitant hydrocortisone. For the next 7 weeks, patients received one to two doses per week that targeted levels in the 150 to 285 micrograms/mL range and integrated weekly averages of 200 ug/mL. RESULTS: Nine patients (28%) had a partial response to suramin based on a > or = 50% decrease in PSA levels coupled with either relief of bone pain or by a 50% decrease in measurable disease. The median overall survival time for all patients is 31 weeks (95% confidence interval [CI], 23 to 51). Treatment was generally well tolerated, with fatigue being the most common significant toxicity, but fatal idiosyncratic myelosuppression (grade V) was observed in one patient. CONCLUSION: Using this dosing schedule, suramin has limited activity against hormone-refractory metastatic prostate cancer. Recent data suggest that hydrocortisone administered with suramin may be partly responsible for the benefit attributed to the drug. Although a small cohort of patients appeared to benefit, we were unable to confirm the previously reported high rate of activity and durability of remission using this agent. PMID- 8622082 TI - Positron emission tomography evaluation of residual radiographic abnormalities in postchemotherapy germ cell tumor patients. AB - PURPOSE: This study was performed to assess the ability of positron emission tomography (PET) to differentiate residual radiographic abnormalities in postchemotherapy nonseminomatous germ cell tumor (GCT) patients. MATERIALS AND METHODS: Thirty patients with nonseminomatous GCT were evaluated with PET scans before surgical resection of a residual mass or masses. Standardized uptake values (SUV) were calculated for the region of maximal 2-fluoro-2-deoxyglucose (FDG) uptake and compared with histologic findings. RESULTS: Eleven patients had necrosis/fibrosis in the resected specimen, 15 had teratoma, and four viable GCT. The median SUV for the necrosis/fibrosis group was 2.86, teratoma 3.07, and viable GCT 8.81. A significant association between SUV and histology was found when comparing viable GCT versus necrosis/fibrosis plus teratoma (P = .004). Patients with an SUV greater than 5 were 75 times more likely to have viable cancer than teratoma or necrosis/fibrosis (odds ratio; 95% confidence interval, 3.66 to 1,536). PET did not differentiate necrosis/fibrosis from teratoma. However, PET was able to differentiate viable GCT from residual necrosis/fibrosis or teratoma. CONCLUSION: PET-FDG imaging can be useful for detection of residual viable carcinoma following chemotherapy in nonseminomatous GCT patients with residual masses. It may be a valuable adjunct in the determination of which patients should undergo postchemotherapy resection. PMID- 8622083 TI - Paclitaxel in combination chemotherapy with radiotherapy in patients with unresectable stage III non-small-cell lung cancer. AB - PURPOSE: The addition of combination chemotherapy to standard radiation therapy has improved treatment for locally unresectable non-small-cell lung cancer. In this phase II study, we evaluated the toxicity and efficacy of a novel chemotherapy regimen that included paclitaxel, cisplatin, and etoposide plus concurrent radiation therapy in this group of patients. PATIENTS AND METHODS: Thirty-three patients with previously untreated, unresectable stage III non-small cell lung cancer (stage IIIA, 11 patients; stage IIIB, 22 patients) initially received two courses of chemotherapy, which included paclitaxel 135 mg/m2 by 1 hour infusion on day 1, cisplatin 60 mg/m/ intravenously (i.v.) on day 2, and etoposide 100 mg/m2 i.v. on days 1, 2 and 3. On week 6, radiation therapy (60 Gy in 30 fractions) was initiated in conjunction with two additional courses of chemotherapy: paclitaxel 135 mg/m2 i.v. by 1-hour infusion on day 1, cisplatin 5 mg/m2 i.v. on days 2- to 10, and etoposide 25 mg/m2 on days 1 to 10. RESULTS: This combined modality program was feasible and well tolerated by most patients. During the two courses of induction chemotherapy, grade 3 or 4 myelosuppression occurred in only six patients (18%). Esophagitis was common during combined modality therapy (grade 3, 10 patients; grade 4 five patients). Forty-two percent of patients had partial response after two courses of induction therapy, and 82% of patients had an objective response at completion of therapy. Twelve patients (36%) had a complete response. Nineteen patients remain progression-free at a median of 8 months; the median survival time has not been reached. CONCLUSION: This paclitaxel-containing combined modality therapy is feasible and highly active in patients with inoperable stage III lung cancer. Esophagitis is the most common severe toxicity with this program. Further studies with paclitaxel containing combination regimens in patients with stage III non-small-cell lung cancer are indicated. PMID- 8622084 TI - Phase II trial of docetaxel in previously untreated advanced non-small-cell lung cancer: a Japanese cooperative study. AB - PURPOSE: This phase II study was conducted to evaluate the efficacy and toxicity of moderate-dose (60 mg/m2) docetaxel in Japanese patients with previously untreated advanced (stage IIIB or IV) non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Docetaxel 60 mg/m2 was administered intravenously over 1 to 2 hours to patients with previously untreated stage IIIB or IV NSCLC. Treatment was repeated every 3 weeks. No routine premedication was given. The patients' median age was 67 years (range, 40 to 80). Forty-four patients (59%) had adenocarcinoma and 55 (73%) had stage IV disease. The median Eastern Cooperative Oncology Group (ECOG) performance status (PS) was 1. RESULTS: Seventy-five patients were eligible and treated with docetaxel. Fourteen patients (19%) achieved a partial response (PR); response was not significantly affected by histology or clinical stage. The median survival time for all patients was 297 days. The predominant toxicity was neutropenia, with 87% of patients experiencing grade 3 or 4. Febrile neutropenia was seen in eight patients. Hypersensitivity and edema each occurred in only 4% of patients and were easily manageable. There was no possible treatment-related death of acute exacerbation of pneumonitis. CONCLUSION: Docetaxel 60 mg/m2 showed significant activity in advanced NSCLC, with a low incidence of hypersensitivity or peripheral edema. Further investigation of this agent in NSCLC is warranted, especially in combination with other active drugs. PMID- 8622085 TI - Phase I dose-escalation trial of gemcitabine and cisplatin for advanced non-small cell lung cancer: usefulness of mathematic modeling to determine maximum tolerable dose. AB - PURPOSE: This study was undertaken to determine the maximum-tolerated doses of gemcitabine and cisplatin, each given weekly for 3 weeks with a 1-week rest. PATIENTS AND METHODS: Patients less than 75 years of age were eligible if they had stage III/IV non-small-cell lung cancer (NSCLC), life expectancy > or = 12 weeks, hemoglobin level > or = 10 g/dL, granulocyte count > or = 2 x 10(9)/L, platelet count > or = 100 x 10(9)/L, hepatic enzymes < or = three times the upper limit of normal, and creatinine concentration < or = 130 mumoles/L. The starting doses for gemcitabine and cisplatin were 1,000 mg/m2 and 25 mg/m2 per week for 3 weeks. At dose level 2, cisplatin was increased to 30 mg/m2/wk for 3 weeks, and thereafter only gemcitabine was increased by 250 mg/m2/wk at each dose level to a maximum of 2,250 mg/m2/wk. RESULTS: There were 33 men and 17 women, with a median age of 62 years. Pathology included adenocarcinoma in 35 patients, squamous in eight, large cell in six, and mixed histology in one. Sixteen patients had stage III and 34 had stage IV tumors. The median nadir granulocyte and platelet counts decreased with each dose level, but cycle 1 dose-limiting toxicity (DLT) in > or = two patients was not encountered in cycle 1, even at the highest dose level. Cumulative marrow toxicity was seen at all levels, which resulted in frequent dose reductions or omissions. A mathematic model of all toxicities over time suggested that dose level 4 (cisplatin 30 mg/m2/wk and gemcitabine 1,500 mg/m2/wk) would be the maximum dose at which grade 4 toxicity would be expected in < or = 33% of patients over four cycles. Of 47 assessable patients, 14 achieved a partial response (30%; confidence interval, 17% to 43%). The median duration was 16 weeks and the median survival time was 24 weeks (range, 3.5-64+). CONCLUSION: Weekly gemcitabine and cisplatin are active against NSCLC, and the recommended phase II doses are 30 and 1,500 mg/m2/wk for 3 weeks, respectively. PMID- 8622086 TI - Pharmacodynamics of fluorouracil-based induction chemotherapy in advanced head and neck cancer. AB - PURPOSE AND METHODS: To optimize the biochemical modulation of fluorouracil (5 FU), we administered the pure I-stereoisomer of leucovorin (LV) as a 132-hour continuous intravenous infusion (CIV) with cisplatin 100 mg/m2, 5-FU 640 mg/m2/d as a 120-hour CIV, and interferon alfa-2b (IFN) at 2 MU/m2/d for 6 days for three cycles (I-PFL-IFN). Pharmacologic parameters included morning (AM) and afternoon (PM) plasma concentrations of 5-FU, LV and its active metabolite 5-methyl tetrahydrofolate (MTHF), and dihydropyrimidine dehydrogenase (DPD) activity in peripheral mononuclear cells. RESULTS: Eighty-nine patients were treated (86 stage IV). Neutropenia and mucositis were the most common toxicities. Sixty-six percent achieved a complete remission (CR). There was a trend for higher PM versus AM 5-FU concentrations (median, 1.64 v 1.51 mumoles/L; P = .08), but not for LV plus MTHF (P = .66). The mean +/- SD DPD activity was 0.21 +/- 0.14 nmol/min/mg and did not correlate with plasma concentrations of 5-FU or LV plus MTHF or clinical toxicities. Higher PM 5-FU concentrations correlated with worse leukopenia (P = .04) and severity of mucositis (P = .04). PM 5-FU concentration was higher in women than in men (P = .07), with no apparent difference in severity of toxicities. The maximum 5-FU concentration was higher in CR than non CR patients (median, 2.01 v 1.54 mumoles/L; P = .02) and higher in women than men who achieved a CR (median, 2.77 v 1.91 mumoles/L; P = .03). No correlation of CR with dose-intensity was found. CONCLUSION: L-PFL-IFN is active in stage IV head and neck cancer. 5-FU concentration is a significant predictor of toxicity. In women, optimization of response outcome requires a higher 5-FU concentration. Individualized 5-FU dosing to obtain higher 5-FU plasma concentrations may be indicated. PMID- 8622087 TI - Docetaxel: an active drug for squamous cell carcinoma of the head and neck. AB - PURPOSE: We conducted a phase II study designed to evaluate the activity, safety, and tolerability of docetaxel (Taxotere: Rhone-Poulenc Rorer Pharmaceuticals Inc, Collegeville, PA) in patients with advanced, incurable, or recurrent squamous cell carcinoma of the head and neck (SCCHN) who had not received prior palliative chemotherapy. PATIENTS AND METHODS: Thirty-one patients with measurable, locoregional, or metastatic SCCHN were treated with docetaxel, administered at a dose of 100 mg/m2 as a 1-hour intravenous (i.v.) infusion once every 21 days on an outpatient basis. All patients were premedicated with dexamethasone, diphenhydramine, and cimetidine. Prophylactic administration of growth factors or antiemetics was not permitted. RESULTS: Thirty-one patients were treated. Twenty nine patients were assessable for response and 30 for toxicity. Four of 31 patients (13%) achieved complete response (CR), nine (29%) achieved partial response had stable disease (SD) and seven (23%) experienced progression of disease (PD). The major response rate was 42% (95% confidence interval [CI], 24% to 60%). The median duration of responses was 5 months (range, 2 to 14). The principal toxicity was leukopenia, which occurred with rapid onset and brief duration. Sixteen patients (53%) experienced nadir fever, and 13 required dose reduction. Hypersensitivity reactions occurred in four patients. Grade 3 peripheral neuropathy occurred in two patients; grade 2 or 3 fatigue occurred in six (20%) and 10 (33%), respectively. Minimal edema (grade 1) occurred in five patients (17%). Clinically significant mucositis, diarrhea, or dermatitis were not observed. CONCLUSION: Docetaxel has major activity against SCCHN. It appears to be well tolerated in this group of patients and can be safely administered on an outpatient basis. Premedication with dexamethasone, cimetidine, and diphenhydramine is associated with a reduced incidence of significant edema, hypersensitivity reactions, and dermatologic toxicities. PMID- 8622088 TI - Analysis of prognostic factors in 1,041 patients with localized soft tissue sarcomas of the extremities. AB - PURPOSE: To identify specific independent adverse clinicopathologic factors for event-free survival in a cohort of consecutively treated patients with extremity soft tissue sarcomas. PATIENTS AND METHODS: Prospectively collected data from a population of 1,041 adult patients with localized (American Joint Committee on Cancer [AJCC] stage IA to IIIB) extremity soft tissue sarcomas were analyzed. Patients were treated at a single institution between 1982 and 1994. Patient, tumor, and pathologic factors were analyzed by univariate and multivariate techniques to identify independent prognostic factors for the end points of local recurrence, distant recurrence, disease-specific survival, and post-metastasis survival. RESULTS: The 5-year survival rate for this cohort of patients was 76%, with a median follow-up time of 3.95 years. Significant independent adverse prognostic factors for local recurrence were age greater than 50 years, recurrent disease at presentation, microscopically positive surgical margins, and the histologic subtypes fibrosarcoma and malignant peripheral-nerve tumor. For distant recurrence, intermediate tumor size, high histologic grade, deep location, recurrent disease at presentation, leiomyosarcoma, and nonliposarcoma histology were independent adverse prognostic factors. For disease-specific survival, large tumor size, high grade, deep location, recurrent disease at presentation, the histologic subtypes leiomyosarcoma and malignant peripheral nerve tumor, microscopically positive surgical margins, and lower extremity site were adverse factors. For post-metastasis survival, only large tumor size ( > 10 cm) was an adverse prognostic factor. CONCLUSION: The independent adverse prognostic factors for distant recurrence and disease specific survival differ from those identified for subsequent local recurrence. Patients with microscopically positive surgical margins or patients who present with locally recurrent disease are at increased risk for subsequent local recurrence and tumor related mortality. Specific histopathologic subtypes are associated with increased risks for local failure and tumor-related mortality. PMID- 8622089 TI - The CD4+/CD8+ ratio as a prognostic factor in patients with metastatic melanoma receiving chemoimmunotherapy. AB - PURPOSE: As reported earlier, a chemotherapy regimen that consisted of dacarbazine, vincristine, lomustine, and bleomycin (DOBC) combined with natural leukocyte interferon (IFN) has been administered with favorable results to patients with metastatic melanoma. In this study, lymphocyte subsets (CD4+ and CD8+) were analyzed before and during treatment to elucidate if alterations in the CD4+/CD8+ ratio had any prognostic value. MATERIALS AND METHODS: Blood samples were systematically obtained from 54 patients with metastatic melanoma who received this chemoimmunotherapy. The frequencies of peripheral-blood lymphocyte subsets were monitored by flow cytometry using the monoclonal antibodies OKT4 (CD4+, T-helper cells) and OKT8 (CD8+, T-suppressor cells). RESULTS: Twenty-seven patients had a constantly increasing ratio, while the remaining 27 patients had a fluctuating or constantly decreasing ratio. The former group had a median survival time of 11.8 months, as compared with 6.5 months for the latter (P = .008, log-rank test). This difference was generated among patients who had an objective response. Responding patients with a constantly increasing ratio had a median survival time of 21.7 months, as compared with 10.2 months for patients with no constant increase in the ratio (P = .038, log-rank test). In nonresponders, no difference in survival was observed between the two groups. CONCLUSION: The monitoring of early changes in the CD4+/CD8+ ratio can provide valuable information that predicts the prognosis of metastatic melanoma patients receiving chemoimmunotherapy. PMID- 8622090 TI - Predictors of clinical response to interleukin-2--based immunotherapy in melanoma patients: a French multiinstitutional study. AB - PURPOSE: Various parameters have been reported to be correlated with response to interleukin-2 (IL-2) therapy. A multiinstitutional study was performed to assess by multivariate analysis the predictive value of known clinical and biologic melanoma prognostic markers recorded before the onset of IL-2 therapy on the likelihood of objective clinical response. PATIENTS AND METHODS: Serum C-reactive protein (CRP), IL-6, and lactate dehydrogenase (LDH) levels were measured in 81 metastatic melanoma patients included in different IL-2-based regimens before the starting of IL-2-therapy. Clinically defined prognostic groups, i.e., patients with superficial or visceral metastases, were also analyzed for response correlates. Patients were evaluated for response to treatment 4 to 6 weeks after completion of one course of therapy. RESULTS: On univariate analysis, the pretreatment values of CRP (P = .001), IL-6 (P = .007), and LDH (P = .02) and site of metastases (P = .0004) were correlated with clinical response. However, only CRP (P < .007) and clinically defined group (P < .004) were independent predictors on multifactorial analysis. Indeed, when adjusted to CRP, IL-6 tended to improve patient selection, but did not reach statistical significance (P = .07). Furthermore, using multivariate survival analysis based on the Cox proportional hazards model, only CRP was found to be an independent prognostic factor for survival (P < .0001). CONCLUSION: In this study, patients with high serum levels of CRP and/or visceral organ involvement before therapy were unlikely to respond to IL-2 therapy. Therefore, clinical classification based on the site of metastases and serum CRP determination before the start of IL-2 therapy may help to improve selection of melanoma patients who may benefit from IL-2 and could prevent unnecessary morbidity. PMID- 8622091 TI - Needle aspiration techniques in preoperative selection of patients with thyroid nodules: a long-term study. AB - PURPOSE: Long-term evaluation of the combination of two needle aspiration techniques (NAT) (fine-needle aspiration [FNA] and aspiration needle biopsy [ANB]) in performing an efficient preoperative selection of palpable thyroid nodules. PATIENTS AND METHODS: Eight years of extensive use of surgery for the detection of thyroid cancer was compared with 12 years of preoperative selection of by NAT. RESULTS: A total of 1,140 operations were performed from 1972 to 1979, and 35 malignant nodules were discovered (3.1%). Five thousand four hundred three patients were examined by NAT from 1980 to 1992; 483 (9%) underwent surgery and 158 malignant nodules were excised. The number of malignant nodules identified by NAT was 166 (eight were not excised) (3.1% of the total population examined). The principal clinical and pathologic features were similar in both groups. ANB yielded a definite benign diagnosis in 88 patients with inadequate FNA findings, it correctly identified four malignant nodules diagnosed as benign by FNA, it showed a macrofollicular component in 115 nodules diagnosed by FNA as microfollicular nodules, and it significantly changed the predictive value of 79 suspicions FNA diagnoses. CONCLUSION: Introduction of NAT reduced the number of operations for palpable thyroid nodules from 143 to 40 per year and increased from four to 13 the number of malignant nodules excised without any change in the overall incidence of malignant nodules. The combination of ANB to FNA significantly contributed to the high and efficient preoperative patient selection, principally by reducing the number of indeterminate or suspicious, as well as false-negative, preoperative FNA diagnoses. PMID- 8622092 TI - A randomized, double-blind, double-dummy, crossover trial comparing the safety and efficacy of oral sustained-release hydromorphone with immediate-release hydromorphone in patients with cancer pain. Canadian Palliative Care Clinical Trials Group. AB - PURPOSE: To evaluate the safety and efficacy of a new slow-release preparation of hydromorphone (SRH) in the treatment of cancer pain. PATIENTS AND METHODS: Ninety five adult patients from three Canadian Palliative Care Centers with no evidence of mental impairment received treatment for cancer pain with an oral opioid analgesic. After informed consent was obtained, patients underwent titration to a stable dose of immediate-release hydromorphone (IRH) for 48 hours, and were then randomized to receive IRH or SRH for 5 days in a double-blind basis. During day 6, a crossover took place, and patients received the alternate drug for 5 days. Pain intensity was assessed using a visual analog scale (VAS) and ordinal scale (OS). Side effects were assessed using VAS. Patients and investigators made a blinded global rating of efficacy a blinded final choice between SRH and IRH. RESULTS: In 75 assessable patients, pain intensity of the VAS and OS were (mean +/- SD) 27 +/- 21 and 1.3 +/- 0.6 on IRH, versus 29 +/- 21 (P = .13) and 1.3 +/- 0.6 (P = .19) on SRH, respectively. The total number of extra doses of opioids, global rating, and final blinded choice by both patients and investigators were not significantly different between IRH and SRH. Differences in side effects were not significant. CONCLUSION: Our findings suggest that SRH is as safe and effective as IRH in the treatment of cancer pain. PMID- 8622093 TI - Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer. AB - PURPOSE: Adjuvant chemotherapy for breast cancer causes significant changes in ovarian function. More young women survive breast cancer than ever before and they are at risk of the sequelae of early menopause. We attempted to (1) define menopausal status in the setting of adjuvant chemotherapy; (2) define chemotherapy-related amenorrhea (CRA); (3) document rates of permanent amenorrhea, temporary amenorrhea, and oligomenorrhea among different regimens; and (4) analyze variables that influence ovarian function. DESIGN: We reviewed reports of the effects of adjuvant chemotherapy for breast cancer on ovarian function in premenopausal women. We searched Medline and Cancerlit from 1966 to 1995 on the following terms: breast neoplasms; chemotherapy, adjuvant; menstruation disorders; premature menopause, and amenorrhea. Further references were obtained from reports retrieved in the initial search. RESULTS: A uniform definition of menopause and CRA is lacking. The wide range of CRA rates reported in adjuvant chemotherapy trials is a result, at least in part, of this problem. The average CRA rate reported in regimens based on cyclophosphamide, methotrexate, and fluorouracil (CMF) is 68% (95% confidence interval [CI], 66% to 70%), with a range of 20% to 100%. CRA incidence varies with age, cytotoxic agent, and cumulative dose. CONCLUSION: Ovarian damage is the most significant long-term sequela of adjuvant chemotherapy in premenopausal breast cancer survivors. We suggest a common definition of the following important terms: menopausal status, CRA (early and late), temporary CRA, and oligomenorrhea in the setting of adjuvant treatment. With uniform definitions in place, regimens can be more precisely compared with respect to this important complication. PMID- 8622095 TI - Carboplatin and cisplatin in medulloblastoma at diagnosis: "comparable efficacy?". PMID- 8622096 TI - Role of granulocyte-macrophage colony-stimulating factor in chemotherapy-induced oral mucositis. PMID- 8622094 TI - Statement of the American Society of Clinical Oncology: genetic testing for cancer susceptibility, Adopted on February 20, 1996. AB - As the leading organization of physicians who treat people with cancer, the American Society of Clinical Oncology (ASCO) recognizes that cancer specialists must be fully informed of the range of issues involved in genetic testing for cancer risk. The newly discovered and still developing ability to identify individuals at highest risk for cancer holds the promise of improved prevention and early detection of cancers. It also poses potential medical, psychological, and other personal risks that must be addressed in the context of informed consent for genetic testing. ASCO firmly believes that any physician who offers genetic testing should be aware of, and able to communicate, the benefits and limits of current testing procedures, and the range of prevention and treatment options available to patients and their families. For these reasons, ASCO endorses the following principles: ASCO affirms the role of clinical oncologists in documenting a family history of cancer in their patients, providing counseling regarding familial cancer risk and options for prevention and early detection, and recognizing those families for which genetic testing may serve as an aid in counseling. To the greatest extent possible, genetic testing for cancer susceptibility should be performed in the setting of long-term outcome studies. ASCO endorses the formulation and implementation of a national cooperative study/registry with appropriate confidentiality to define the clinical significance of mutations in known cancer susceptibility genes. ASCO is committed to providing educational opportunities for physicians concerning methods of quantitative cancer risk assessment, genetic testing, and pre- and post-test genetic counseling so that oncologists may more responsibly integrate genetic counseling and testing into the practice of clinical and preventive oncology. Oncologists must assure that informed consent has been given by the patient as an integral part of the process of genetic predisposition testing, whether such testing is offered on a clinical or research basis. ASCO recommends that cancer predisposition testing be offered only when: 1) the person has a strong family history of cancer or very early age of onset of disease; 2) the test can be adequately interpreted; and 3) the results will influence the medical management of the patient or family member. As clinical testing becomes more widely available, the Society encourages oncologists to utilize laboratories committed to the validation of testing methodologies, and to facilitate families' participation in long-term outcome studies. ASCO recommends that oncologists include in pre- and post-test counseling discussion of possible risks and benefits of cancer early detection and prevention modalities, which have presumed but unproven efficacy for individuals at the highest hereditary risk for cancer. ASCO endorses efforts to strengthen regulatory authority over laboratories that provide cancer predisposition tests that will be utilized to inform clinical decisions. These regulatory requirements should include appropriate oversight of the products used in genetic testing, interlaboratory comparisons of reference samples, as well as quality control mechanisms. ASCO endorses all efforts including legislation to prohibit discrimination by insurance companies or employers based on an individual's inherited susceptibility to cancer. All individuals at hereditary risk for cancer should have access to appropriate genetic testing and associated medical care, which should be covered by public and private third-party payers. ASCO endorses continued support of patient oriented research to analyze the psychological impact of genetic testing of at risk populations. PMID- 8622097 TI - Maculopapular rashes secondary to gemcitabine injection for non-small-cell lung cancer. PMID- 8622098 TI - Isepamicin Once a Day--Enhancing Traditional Aminoglycoside Therapy. Proceedings of a meeting. Rotherwick, England, September 24-25, 1994. PMID- 8622099 TI - Isepamicin once daily plus ceftriaxone versus amikacin plus ceftriaxone in febrile neutropenic patients. AB - Isepamicin is a new aminoglycoside with in-vitro activity superior to amikacin. It is a poor substrate for the 6'-aminoacetyltransferase-I enzyme which inactivates amikacin and therefore organisms possessing this enzyme are not resistant to isepamicin. The aim of this study was to compare the efficacy and safety of co-administration of isepamicin once daily plus ceftriaxone to amikacin twice daily plus ceftriaxone to amikacin twice daily plus ceftriaxone in febrile neutropenic cancer patients. Febrile episodes in 235 patients (156 in isepamicin group and 79 in amikacin group) were treated in this study. They occurred in 218 different patients. Fifteen patients were enrolled twice and one three times. Response rates to the two treatment regimens for microbiologically documented episodes, clinically documented episodes and further unexplained fever were similar. Tolerance of the treatment regimens, as measured by serum creatinine levels, hypoaccousia and cutaneous allergy was also similar in both treatment groups. In conclusion, isepamicin given once daily when combined with ceftriaxone in the treatment of febrile episodes in neutropenic cancer patients was as effective and no more toxic than amikacin. PMID- 8622100 TI - The role of aminoglycosides in modern therapy. AB - Aminoglycosides remain a mainstay of antimicrobial therapy, especially for treatment of serious Gram-negative infections. Aminoglycosides represent cornerstone of antibiotic combination therapy and, despite their well-documented toxicity, continue to be used because of their excellent bactericidal efficacy and their limited tendency towards the development of resistance during therapy. Various factors, particularly their concentration-dependent bactericidal activity and prolonged post-antibiotic effect, indicate that aminoglycosides can be given effectively in a once-daily dosing regimen. Once-daily dosing has also been shown to reduce toxicity in animal studies. Although once-daily administration of amino glycosides also has a number of practical advantages, questions remain concerning the indications that should be treated, the optimal peak and trough serum concentrations that should be attained, and the requirement for an initial loading dose. The present article examines the role of aminoglycosides in the treatment of serious infections and reviews the evidence presently available to answer questions on their appropriate clinical use. PMID- 8622101 TI - The efficacy and safety of isepamicin and ceftazidime compared with amikacin and ceftazidime in acute lower respiratory tract infection. AB - Isepamicin is a new aminoglycoside antibiotic which has a superior stability to aminoglycoside-inactivating enzymes compared with other available aminoglycosides. In this multicentre, randomised, open study, the safety and efficacy of isepamicin plus ceftazidime was compared with that of amikacin plus ceftazidime in adults with acute lower respiratory tract infection. Patients with severe infections received intravenous administration of isepamicin 15 mg/kg once daily + ceftazidime 2g twice daily (n = 121) or amikacin 7.5 mg/kg twice daily + ceftazidime 2g twice daily (n = 61). Those with less severe infection received intramuscular or intravenous administration of isepamicin 8 mg/kg once daily + ceftazidime 1g twice daily (n = 56) or amikacin 7.5 mg/kg twice daily + ceftazidime 1g twice daily (n = 28). In the efficacy populations, the proportion of patients clinically cured in the isepamicin group (87/100; 87%) was similar to that in the amikacin group (36/47; 77%). Significantly more patients in the isepamicin group were cured or improved compared with the amikacin group (97% vs 89%; p = 0.042). The difference between treatment groups was also significant in patients with pneumonia (p = 0.05). The most commonly isolated target organisms were Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The proportion of patients in the efficacy population whose pretreatment valid target organisms were eliminated was similar in each treatment group (90% isepamicin vs 89% amikacin). A retrospective analysis showed there were slightly fewer clinical successes and a higher death rate in patients with nosocomial rather than community acquired pneumonia. Both treatments were well tolerated . Fourteen per cent of isepamicin and 11% of amikacin patients experienced adverse events. The incidence of ototoxicity and nephrotoxicity was low. PMID- 8622102 TI - Comparison of the efficacy and safety of isepamicin and amikacin in the treatment of acute lower respiratory tract infections caused by gram-negative organisms. AB - In a prospective multicentre open trial, hospitalised adult patients with acute lower respiratory tract infections, mainly pneumonia or bronchitis, were randomised to receive either isepamicin 8 or 15 mg/kg once daily depending on the severity of the infection or amikacin 7.5 mg/kg twice daily. Patients with infections known to be caused by Pseudomonas aeruginosa were to be given concomitant treatment with ceftazidime. In the intent-to-treat population, i.e. patients who received at least one dose randomised treatment, a clinical cure or improvement at the end of treatment was seen in 112/125 (90%) isepamicin patients and 55/60 (92%) amikacin patients. The corresponding rates for patients with a primary diagnosis of pneumonia were 45/52 (87%) and 25/28 (89%). Cure/improvement rates for patients with P. aeruginosa as the causative pathogen (34 of whom also received ceftazidime) were 28/30 (93%) and 16/18 (89%), respectively. In the efficacy population (patients who had a valid pretreatment culture and who met other evaluability criteria), total elimination (documented or presumed if infection had resolved) of target pathogens occurred in 54/63 (86%) of isepamicin patients and 25/30 (83%) of amikacin patients. P. aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus were commonly isolated pathogens. Treatment-related adverse were mainly mild or moderate in severity and occurred in 10% of isepamicin patients and 13% of amikacin patients. Four patients (3 isepamicin and 1 amikacin) discontinued treatment because of severe adverse events and a further isepamicin patient withdrew because of a mild adverse event. Nephrotoxicity and ototoxicity occurred infrequently. PMID- 8622103 TI - The efficacy and safety of isepamicin compared with amikacin in the treatment of intra-abdominal infections. AB - This multicentre, randomised, open-label, parallel group study compared the efficacy and safety of isepamicin (15 mg/kg once daily) and amikacin (7.5 mg/kg twice daily) when given intravenously in combination with metronidazole to 267 hospitalised adults with intra-abdominal infections. Clinical cure or improvement was achieved in 96.3% (130/135) evaluable patients (efficacy population) in the isepamicin group and 94.3% (66/70) patients in the amikacin group. Bacteriological elimination occurred in 93.3% (126/135) evaluable isepamicin patients and 95.7% (67/170) amikacin patients. there was not statistically significant differences between the groups. Adverse events were reported by 9% of patients in the isepamicin group (16/178) and 10% of patients in the amikacin group (9/89). Events considered to be related to treatment occurred in 6% of patients in both groups. The most frequent adverse events were diarrhoea, nausea and vomiting. Renal problems caused three patients (2 isepamicin, 1 amikacin) to withdraw from the study. Ototoxicity (detected by audiometric testing) occurred in one patient (treated with isepamicin). In conclusion, isepamicin at a dose of 15 mg/kg once daily was shown to be as effective as amikacin (7.5 mg/kg twice daily) in the treatment of intra-abdominal infections in hospitalised adults also treated with metronidazole. Both treatments were well tolerated. PMID- 8622104 TI - Isepamicin versus amikacin in the treatment of urinary tract infection. AB - In a series of three prospective, randomised, multicentre trials, isepamicin (15 mg/kg or 8 mg/kg once daily depending on severity of infection) was compared with amikacin (7.5 mg/kg twice daily) in a total 252 adult hospitalised patients (mean age 51-54 years) with urinary tract infection. Pretreatment pathogens included Escherichia coli, which was isolated from approximately 50% of patients, and Pseudomonas aeruginosa, which was isolated from approximately 10% of patients with severe infections. The most commonly occurring primary diagnoses were complicated pyelonephritis, uncomplicated pyelonephritis and complicated lower urinary tract infection. For the patients included in the efficacy population, elimination of the pathogens occurred for all infections combined, in 92/101 (91%) patients in the isepamicin group and 51/55 (93%) patients in the amikacin group. Adverse events occurred in 15% of isepamicin patients and 6% of amikacin patients. Ototoxicity at the > or = dB threshold was noted in one isepamicin and two amikacin patients, but none of these patients had associated clinical signs of auditory or vestibular toxicity. Four isepamicin and four amikacin patients had potentially significant increases in serum creatinine indicative of possible nephrotoxicity. PMID- 8622105 TI - Comparison of the efficacy and safety of isepamicin and amikacin in the treatment of skin and skin structure infections. AB - Two hundred and three patients with skin and skin structure infections were treated with isepamicin once daily or amikacin twice daily in an open, randomised, comparative multicentre trial. Patients were randomised to treatment with isepamicin or amikacin in a 2:1 ratio. Severe infections (63 patients) were treated with isepamicin 15 mg/kg once daily (n = 15) or amikacin 7.5 mg/kg twice daily (n - 18), less severe infections (140 patients) with isepamicin 8 mg/kg once daily (n = 93) or amikacin 7.5 mg/kg twice daily (n = 47). The overall clinical response rate at the end of treatment was excellent in all treatment groups (94-96% cured or improved) with no significant differences between isepamicin and amikacin in patients with either server or less severe infections. The most commonly isolated target pathogens were Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Staphylococcus aureus. Overall, in patients who had a valid target pathogen isolated prior to treatment and who met other evaluability criteria, bacteriological eradication was achieved in over 90% of patients; amikacin patients with severe infections had a somewhat lower eradication rate (82%). Over all infections, 4/110 (4%) patients in the isepamicin group and 5/54 (9%) patients in the amikacin had organisms which persisted. Adverse events were reported in 12% of patients in the isepamicin group and 6% in the amikacin group. The most frequently reported adverse event in the isepamicin group as headache. Two patients (one in each treatment group), both of whom experienced skin rashes, were withdrawn. Potentially clinically significant changes in serum creatinine occurred in two patients, who received isepamicin and one who received amikacin (who was withdrawn from the study). Ototoxicity was rare, occurring in one patient treated with isepamicin. PMID- 8622106 TI - Evaluation of the efficacy and safety of isepamicin in the treatment of various bacterial infections. AB - A multicentre, non-comparative study evaluated the efficacy and safety of the new aminoglycoside isepamicin in hospitalised patients with various infections. Isepamicin was administered once daily with the daily dosage stratified according to the severity of infection: 15 mg/kg isepamicin for severe potentially systemic infections (53 patients) or 8 mg/kg isepamicin for less severe and localised infections (56 patients). The largest groups of patients had urinary tract infection (n = 54) or lower respiratory tract infection (n = 31); smaller numbers of patients were enrolled with skin and soft tissue infections (n = 9), intra abdominal infections (n = 8) or obstetric and gynaecological infections (n = 7). In the patients receiving 15 mg/kg isepamicin, clinical cure or improvement occurred in 19/21 patients with lower respiratory tract infections, 8/13 patients with urinary tract infections, 6/6 patients with skin infections, 5/6 patients with intra-abdominal infections and 6/7 patients with obstetric gynaecological infections. In the patients receiving 8 mg/kg isepamicin, 40 out of 41 patients with urinary tract infections were considered cured or improved as were 8/10 patients with lower respiratory tract infections, 1/3 patients with skin infections and 1/2 patients with intra-abdominal infections. Nine per cent of patients reported at least on adverse event during the study. Two patients (one from each dosage group) discontinued treatment because of adverse events, respiratory disorder and erythematous rash, but neither event was considered to be severe of life threatening No patients had evidence of ototoxicity by pure tone audiometry and no patients had potentially significant increases in serum creatinine which were considered to be treatment related. The results of this study indicate that treatment with isepamicin once daily is effective and well tolerated in hospitalised adults with various infections. PMID- 8622107 TI - Evaluation of the efficacy and safety of isepamicin compared with amikacin in the treatment of nosocomial pneumonia and septicaemia. AB - Isepamicin is a new aminoglycoside antibiotic which possesses greater stability to aminoglycoside-inactivating enzymes compared with other available aminoglycosides. In this prospective, randomised, open trial, the safety and efficacy of intravenous administration of isepamicin was compared with that of intravenous amikacin in seriously ill adults with nosocomial pneumonia or septicaemia. Each study aminoglycoside was administered concurrently with ceftazidime or imipenem. Patients were randomised to receive isepamicin 15 mg/kg once daily, isepamicin 7.5 mg/kg twice daily or amikacin 7.5 mg/kg twice daily. For patients with nosocomial pneumonia, the proportions of patients in the intent to-treat population (n = 130) who were clinically cured at the end of treatment were similar in each treatment group: 18/44 (41%) isepamicin once daily; 19/45 (42%) isepamicin twice daily; and 17/41 (42%) amikacin. Corresponding results for the efficacy population (n = 58) were: 12/20 (60%) isepamicin once daily; 14/21 (67%) isepamicin twice daily; 9/17 (53%) amikacin. In patients with septicaemia, clinical cure was achieved in 8/10 (80%) patients treated with isepamicin once daily, compared with 8/13 (62%) patients who received isepamicin twice daily, and 7/12 (58%) patients treated with amikacin. For both diagnoses, there were no statistically significant differences between the treatment groups in clinical cure rate. The most commonly isolated target pathogen was Pseudomonas aeruginosa. For both nosocomial pneumonia and septicaemia, the proportion of patients in the intent-to-treat population whose pretreatment valid target pathogens were eliminated was similar in each treatment group. In total, 51 patients (30%) died during study, mostly due to disease progression or complications, or concurrent illness. All three treatment regimens were well tolerated. The proportion of patients experiencing at least one adverse event was 11%, 25% and 9% for isepamicin once daily, isepamicin twice daily and amikacin, respectively. The incidence of ototoxicity and nephrotoxicity was relatively low in both treatment groups. PMID- 8622108 TI - The most frequently occurring aminoglycoside resistance mechanisms--combined results of surveys in eight regions of the world. The Aminoglycoside Resistance Study Groups. AB - Aminoglycoside-resistant isolates from different patients were collected in 149 hospitals in eight regions of the world. Aminoglycoside resistance mechanisms were determined in 11,079 of these isolates by the correlation of resistance phenotypes to 12 aminoglycosides and DNA hybridisation with up to 19 resistance genes. A very large diversity of different resistance mechanisms was found. For example, in Escherichia-Morganella-Proteus-Salmonella-Shigella, at total of 53 different mechanisms was found among the 2080 isolates studied. Therefore, the most common resistance mechanisms in the seven different pathogen groups were summarised. The seven pathogen groups were chosen so that each one had a unique mixture of resistance mechanisms. Among Enterobacteriaceae, the Citrobacter Enterobacter-Klebsiella, Providencia and Serratia groups had a particularly high incidence of combinations of resistance mechanisms especially when compared with data from eight early surveys. These mechanisms often tended to be combinations of previously common gentamicin-modifying enzymes with AAC(6')-I, an enzyme which can acetylate tobramycin, netilmicin and amikacin but which does not usually cause resistance to isepamicin. Resistance rates among the four Enterobacteriaceae groups reflected the incidence of these combinations; that is, amikacin resistance was 43.1%, 21.7%, 4.2% and 37.6% while that to isepamicin was 9.9%, 9.7% 2.7% and 7.6%, respectively. A very large diversity of mechanisms was found in Pseudomonas. However, three mechanism - permeability, ACC(6')-II and ANT(2")-I - were very common, both alone as single mechanisms and in combination with each other or other mechanisms. Because of the high incidence of permeability resistance in Pseudomonas, resistance to all aminoglycosides was quite high and the difference between amikacin (41.7%) and isepamicin (36.8%) was smaller. The greatest diversity of resistance mechanisms (67 in 1189 isolates) was found in Acinetobacter. The most common mechanisms - APH(3')-VI and AAC(3)-? were generally very rare in other genera but found frequently in Acinetobacter in combinations with a wide variety of other mechanisms. The most frequent resistance mechanisms in Acinetobacter varied by geographic region much more than did mechanisms in other genera. The resistance rates to all of the clinically useful aminoglycosides were generally higher in Acinetobacter than in any other pathogen group. In contrast to the other pathogen groups, only seven resistance mechanisms were found in the 898 resistant Staphylococcus. These were composed of the three known single mechanisms and four combinations of these three mechanisms. Because the principal resistance mechanism, (APH(2") + ACC(6'), confers high-level resistance to gentamicin and tobramycin and a lower level of resistance to amikacin, isepamicin and netilmicin, the observed resistance rate to netilmicin (15.0%) was the lowest for any of the aminoglycosides. PMID- 8622109 TI - The changing nature of aminoglycoside resistance mechanisms and the role of isepamicin--a new broad-spectrum aminoglycoside. The Aminoglycoside Resistance Study Groups. AB - Aminoglycoside resistance mechanisms from recent studies were compared with those found in earlier studies in the USA and Europe for three pathogen groups. Among Citrobacter-Enterobacter-Klebsiella, four single mechanisms (AAc(3)-II, AAC(3)-I, ANT(2")-I and AAC(6')-I were found in all studies, but the most recent studies showed a significant increase in combinations of AAC(6')-I with the other common mechanisms. Since AAC(6')-I confers resistance to tobramycin, netilmicin and amikacin, combinations of it with the other gentamicin modifying enzymes conferred broad-spectrum resistance to all clinically available aminoglycosides except isepamicin. Similar changes occurred in Escherichia-Morganella-Proteus Salmonella-Shigella except that the frequency of combinations was much lower and two additional single mechanisms - AAC(3)-IV and permeability - were also found frequently. Among aminoglycoside-resistant Pseudomonas, three mechanisms, AAC(6') II, ANT(2")-I and permeability, were always common and remained common. However, combinations of the three mechanisms with each other and with other mechanisms were more common in the recent surveys. Different genes which produce different proteins with the same aminoglycoside-modifying activity are now known. The results of hybridisation studies with two aac(3)-I, 2 aac(6')-II and 4 aac(6')-I gene probes are presented. The most commonly occurring genes were: aac(3)-Ia, aac(3)-IIa, aac(6')-IIa, aac(6')-Ib and, in Serratia, aac(6')-Ic. The activity of isepamicin against amikacin resistant strain which produce AAC(6')-I can be related to differences in the structure of these two similar aminoglycosides at Position 3". Amikacin may form a stable complex with AAC(6')-I enzymes via binding interaction at Position 3 and 3". Isepamicin, which has a secondary amino group at Position 3", may only be able to interact at Position 3 and enzyme isepamicin complexes are likely to be less stable. PMID- 8622110 TI - Once-daily versus multiple-daily dosing of aminoglycosides. AB - The pharmacodynamic characteristics of isepamicin and other aminoglycosides, both in terms of efficacy and toxicity, explain why once-daily administration of these agents should be the optimal dosing regimen. Isepamicin, as with other aminoglycosides, exhibits concentration-dependent bactericidal activity and produces prolonged post-antibiotic effects against susceptible organisms. High concentrations of these drugs would be expected to produce more rapid and extensive bacterial killing than lower levels. Furthermore, the post-antibiotic effect would protect against bacterial regrowth when serum and tissue concentrations fall below inhibitory levels. In animal models, the magnitude of the peak serum concentration or the area under the concentration-time curve, are the important determinants of efficacy for isepamicin and the other aminoglycosides. Isepamicin also exhibits the "first-exposure effect", i.e. initial exposure of bacteria to isepamicin down-regulates subsequent uptake of the drug. During this period of down-regulation, bacteria exhibit decreased killing and shorter post-antibiotic effects. Since the first-exposure effect lasts for several hours, once-daily administration of the aminoglycosides allows for this effect to dissipate completely between doses. High peak concentrations, greater than 8-10 times the minimum inhibitory concentration (MIC), will also decrease the emergence of resistant strains. With regard to toxicity, one of the first steps in the uptake of aminoglycosides into sites of toxicity is their binding to the brush borders of renal cells and to the cochlea and vestibular membranes. Binding to these membranes demonstrates saturable kinetics. As a result, uptake of the aminoglycosides is more efficient with low sustained concentrations than with high intermittent levels. Once-daily dosing of aminoglycosides has consistently been less toxic than more frequent dosing in animals. In clinical studies, once-daily dosing of aminoglycosides compared to two-or three-times daily administration has generally exhibited similar efficacy and toxicity. However, a few studies has shown greater efficacy or lower toxicity with once-daily dosing of aminoglycosides. Once-daily dosing of the aminoglycosides has the potential to enhance efficacy, reduce toxicity, and lower administration costs for this drug class. PMID- 8622111 TI - Pharmacokinetics of isepamicin. AB - Isepamicin is a new aminoglycoside that has activity against many bacteria resistant to other aminoglycosides. The pharmacokinetics of isepamicin have been characterized in neonatal, pediatric, adult, elderly and renally impaired human populations as well as in clinical trials using the techniques of population pharmacokinetics. The pharmacokinetics of isepamicin are uncomplicated and generally similar to those of other aminoglycosides, although there is some evidence that it may have less tissue accumulation. The drug is completely absorbed following intramuscular administration. The drug is not metabolized and unchanged isepamicin accounts for all of the drug substance in plasma and urine. It is completely eliminated via the renal route; consequently dosing in patients with renal insufficiency has to be adjusted according to the degree of renal impairment. The pharmacokinetics of isepamicin are generally linear. Thus peak plasma concentrations and area under the plasma concentration curve (AUC) values are proportional to the administered dose while clearance (1.1-1.3 mL/min/kg), volume of distribution at steady state (0.23-0.29 L/kg) and half-life (2-2.5 h) are independent of dose. There is no significant accumulation of drug in the plasma with once- or twice-daily dosing. The isepamicin plasma concentration curve following a 1 g intravenous dose to healthy volunteers can be best characterized by a tri-exponential curve corresponding to a t1/2 alpha of 0.17 h, a t1/2 beta of 2.1 h, and a gamma-phase of 34 h. The t1/2 beta represents the elimination phase and changes with age and renal functions, while the gamma-phase represents the return of drug to plasma from a deep compartment including binding in renal tissue. The gamma-phase represents less than 3% of the total AUC and does not change with age. Isepamicin readily distributes to extracellular fluid and pulmonary tissue. In conclusion, isepamicin demonstrates predictable linear kinetics and is similar pharmacokinetically to other aminoglycosides. Preliminary indications of decreased tissue accumulation implied from pharmacokinetic and pharmacodynamic characteristics of isepamicin favour once-daily dosing. PMID- 8622112 TI - Pharmacokinetics of isepamicin in paediatric patients. AB - The pharmacokinetics of isepamicin were evaluated in 50 paediatric patients ranging from newborn to 13 years old. Children with subdivided according to age: Group I (6-13 years); Group II (4 months to 6 years); Group III (16 days to 4 months); and Group IV (newborn to 16 days). All patients received isepamicin 7.5 mg/kg every 12 hours except those in Group IV who received 7.5 mg/kg once daily. Isepamicin was administered initially as an intravenous 30-minute infusion and then either intravenously or intramuscularly for between 4 and 12 days. Plasma samples were obtained after the first or second dose on day 1 at 0, 0.5, 1, 4, 6, 8 and 12 hours after the initiation of dosing and at 0.5 and 12 hours on other dosing days. Additional samples were collected in the Group IV patients at 18, 20 and 24 hours. Isepamicin showed a similar plasma concentration-time profile in Groups I, II and III (children from 16 days to 13 years), and in these groups the profile was generally similar to that observed in adults. Neonates up to the age of 16 days (Group IV) showed a distinctly different pharmacokinetic profile: a significantly larger AUC, longer half-life, lower Cmax and lower total body clearance. Isepamicin 7.5 mg/kg administered once daily to children less than 16 days old and twice daily to children aged 16 days to 13 years appears to be pharmacokinetically appropriate. The drug was very well tolerated by children of all age groups. PMID- 8622113 TI - Isepamicin (SCH 21420, 1-N-HAPA gentamicin B): microbiological characteristics including antimicrobial potency of spectrum of activity. AB - Isepamicin (formerly SCH 21420 or 1-N-HAPA-gentamicin B) is a novel broad spectrum aminoglycoside which possesses a high level of stability to aminoglycoside inactivating enzymes and low levels of toxicity to the kidney and inner ear. The only modifying enzymes capable of inactivating isepamicin are ANT(4')-I (staphylococci), ANT(4')-II and APH(3')-VI, in addition to resistance mediated by permeability mutations. The spectrum of isepamicin is most similar to that of amikacin, another aminoglycoside with high enzyme stability. Reviews of isepamicin activity demonstrate MIC90s ranging from 1.1 to 8.5 mg/L for members of the Enterobacteriaceae, slightly more potent than amikacin. Pseudomonas aeruginosa, Acinetobacter spp. and other pseudomonads had isepamicin consensus MIC90s of 7.8, 7.2 and 6.8 mg/ml, respectively. Staphylococci were generally very susceptible to isepamicin (MIC90s 0.5-6.9 mg/L), but enterococci and Streptococcus spp. were resistant (MIC90s > or = 64 mg/L), as were anaerobes, Xanthomonas (Stenotrophomonas) maltophilia, pathogenic Neisseria spp., Flavobacterium spp., Pseudomonas (Burkholderia) cepacia, Alcaligenes spp. and Vibrio spp. Additional studies of isepamicin microbiology revealed: 1) MICs were adversely influenced by elevated divalent cation content of the medium; 2) minimum inoculum effects were observed by using elevated concentrations; 3) bactericidal action and concentration dependent killing was the rule; 4) excellent stability in the presence of high beta-lactam co-drug concentrations was documented in several studies; 5) predictable synergistic or additive interactions with broad spectrum antimicrobial agents such as cephalosporins, penicillins, carbapenems and fluoroquinolones was observed by numerous investigators; and 6) in vitro susceptibility testing criteria (National Committee for Clinical Laboratory Standards) and quality control guidelines are established for routine clinical use. Isepamicin's antimicrobial qualities position it as a potential alternative aminoglycoside in hospitals or in geographical areas where resistance to existing aminoglycosides has emerged. The wider stability of isepamicin to contemporary aminoglycoside inactivating enzymes, its predictable pharmacokinetics, lower toxicity risks and enhanced activity (synergy) with other broad spectrum antimicrobial agents, will make isepamicin a valuable addition to the antimicrobial armamentarium in areas where ACC(6') enzymes are prevalent (Europe, Latin America, Western Pacific) and amikacin has become less efficacious. PMID- 8622114 TI - Auditory impairment in guinea pigs treated with isepamicin. AB - Groups of adult guinea pigs were treated intramuscularly with isepamicin at doses of 100, 150 and 225 mg/kg/day for 21 days. For comparison, two other groups were administered either saline or amikacin 225 mg/kg/day. Auditory function, as measured by the Preyer pinna reflex and the brain stem evoked response, was impaired in both the isepamicin and amikacin groups treated with 225 mg/kg/day, and to a lesser extent in the group receiving isepamicin 150 mg/kg/day. Morphological evaluation of the organ in Corti, performed either by scanning electron microscopy or by light microscopy, showed the typical pattern of damage associated with the aminoglycosides. The greatest damage was observed at 225 mg/kg/day, with no difference between isepamicin and amikacin. Isepamicin 100 mg/kg/day produced no impairment in auditory function and very little change in the morphology of the organ of Corti. PMID- 8622115 TI - Overview of the efficacy of isepamicin in the adult core clinical trial programme. AB - The efficacy of once-daily administration of isepamicin in hospitalized adult patients has been assessed in a multinational clinical trails programme. Following a small phase II programme, the phase III programmed assessed four main indications: lower respiratory tract infections (including nosocomial pneumonia), urinary tract, intra-abdominal and skin and soft tissue infections. The phase III trials were open, prospective, multicentre studies in which 1443 patients were randomised to receive either isepamicin (n = 1005) or amikacin (n = 438). The daily dose of isepamicin was dependent on the severity of infection (8 or 15 mg/kg once daily) while all patients received amikacin 7.5 mg/kg twice daily. A study of patients with nosocomial pneumonia had an additional treatment arm of isepamicin 7.5 mg/kg twice daily. The aminoglycosides were combined with other antimicrobial agents in accordance with current clinical practice depending on the site and severity of the infection and the type of organism isolated. Overall, clinical cure or improvement response rates of the isepamicin and amikacin regimens were comparable, ranging from 76-95% in the intent-to-treat population. Lower clinical response rates (62-63%) was observed in severely ill patients with nosocomial pneumonia in both the isepamicin and amikacin treatment groups. In the efficacy population, organism elimination rates of 90% were achieved with isepamicin and amikacin. Therefore, in adult patients with a wide range of infections requiring aminoglycoside therapy, once-daily dosing with isepamicin is as effective as twice- daily dosing with amikacin. PMID- 8622116 TI - An overview of the safety of isepamicin in adults. AB - The safety profile of isepamicin has been assessed from a series of phase II and III clinical trials. A total of 1243 patients were randomised to treatment with isepamicin, mainly administered once daily at a dose of 15 mg/kg or 8 mg/kg depending on the severity of infection. A small proportion of patients were randomised to isepamicin at a dosage of either 7.5 mg/kg twice daily or 4 mg/kg twice daily. In the majority of studies, isepamicin was compared with a standard twice-daily dosing regimen of 7.5 mg/kg amikacin = (n - 552). The clinical studies included patients with a variety of bacterial infections, including lower respiratory tract, urinary tract, intra-abdominal and skin and skin structure infections. The study aminoglycosides were co-administered with other antimicrobial agents in line with normal clinical practice depending on the site, nature and severity of infection. Most patients received isepamicin or amikacin as a 30-minute intravenous infusion and a small proportion of patients with less severe infections received intramuscular injections. The mean duration of treatment was nine days for both the isepamicin and amikacin treatment groups, and was similar for patients with both severe and less severe infections. Overall, the proportion of patients reporting any adverse event was comparable between the isepamicin (13%) and amikacin (11%) groups. No individual adverse event was reported in more than 2% of patients, the most commonly reported events being phlebitis, rash, headache and renal compromise. The frequency of adverse events was not influenced by treatment duration, age or gender. Treatment-related adverse events which were considered severe or life-threatening were reported in 1.8% of patients receiving isepamicin and 2.0% of patients receiving amikacin. Two per cent of patients in each treatment group discontinued the study because of adverse events and 2% of patients in each treatment group died during treating. Four per cent of patients in each treatment group died within 30 days after the end of treatment. Changes in laboratory tests were similar in both treatment groups; few changes were considered by the investigators to be treatment-related. Increases in serum creatinine indicative of possible renal compromise occurred in 4.6% of isepamicin and 5.1% of amikacin patients. The occurrence of ototoxicity as measured by standard frequency pure tone audiometry was low. In summary, isepamicin is a sell-tolerated aminoglycoside with a safety similar to that of amikacin. PMID- 8622117 TI - A randomised comparison of isepamicin and amikacin in the treatment of bacterial infections in paediatric patients. AB - The efficacy and safety of isepamicin 7.5 mg/kg of body weight twice daily or amikacin the same dosage regimen for the treatment of various infections in neutropenic and non-neutropenic paediatric patients were compared in a prospective randomised trial. In total, 306 patients were enrolled and received at least one dose of randomised treatment (204 isepamicin, 102 amikacin: intent to-treat population); 181 patients satisfied all criteria for evaluability (120 isepamicin, 61 amikacin: efficacy population). Clinical cure or improvement rates in the isepamicin and amikacin groups were: intent-to-treat population, 188/204 (92%) and 94/102 (92%), respectively; efficacy population, 117/120 (98%) and 58/61 (95%), respectively. The bacteriological elimination rate (efficacy population) in the isepamicin and amikacin treatment groups was 75/76 (99%) vs 35/38 (92%). Nephrotoxicity, defined as an increase in serum creatinine of 0.5 mg/dL or > or = 44.2 mumol/L from baseline, occurred in 4/187 (2%) and 1/191 (1%) children treated with isepamicin and amikacin, respectively. Definite ototoxicity at the > or = 20 dB threshold occurred in 3 (1 isepamicin and 2 amikacin) out of 56 children evaluated with at least two audiograms. Thus isepamicin was as effective and as well tolerated as amikacin in the treatment of various infections in paediatric patients. PMID- 8622118 TI - Structure and pharmacological properties of a molluscan glutamate-gated cation channel and its likely role in feeding behavior. AB - We describe the isolation of a molluscan (Lymnaea stagnalis) full-length complementary DNA that encodes a mature polypeptide (which we have named Lym eGluR2) with a predicted molecular weight of 105 kDa that exhibits 44-48% identity to the mammalian kainate-selective glutamate receptor GluR5, GluR6, and GluR7 subunits. Injection of in vitro-transcribed RNA from this clone into Xenopus laevis oocytes results in the robust expression of homo-oligomeric cation channels that can be gated by L-glutamate (EC50 = 1.2 +/- 0.3 micron) and several other glutamate receptor agonists; rank order of potency: glutamate >> kainate > ibotenate > AMPA. These currents can be blocked by the mammalian non-NMDA receptor antagonists 6,7-dinitroquinoxaline-2,3-dione, 6-cyano-7-nitroquinoxaline 2,3-dione, and 1-(4-chlorobenzoyl)piperazine-2,3-dicarboxylic acid. Ionic replacement experiments have shown that the agonist-induced current is carried entirely by sodium and potassium ions. In situ hybridization has revealed that the Lym-eGluR2 transcript is present in all 11 ganglia of the Lymnaea CNS, including the 4-cluster motorneurons within the paired buccal ganglia. The pharmacological properties and deduced location of Lym-eGluR2 are entirely consistent with it being (a component of) the receptor, which has been identified previously on buccal motorneurons, that mediates the excitatory effects of glutamate released from neurons within the feeding central pattern generator. PMID- 8622119 TI - Inhibition of cGMP breakdown promotes the induction of cerebellar long-term depression. AB - The effects of the nonspecific cyclic nucleotide inhibitors 1-methyl-3 isobutylxanthine (IBMX) and dipyridamole, and the cGMP-specific phosphodiesterase inhibitor Zaprinast were studied on parallel fiber-Purkinje cell synaptic responses in rat cerebellar slices. Bath application of all three compounds, at concentrations shown to inhibit cGMP breakdown, led to stable and robust long term depression of PF responses. Injections of dipyridamole directly into the Purkinje cell dendrites were similarly effective as bath applications, confirming a postsynaptic site of action. Inhibitors of both protein kinase G and C and also the metabotropic glutamate receptor antagonist MCPG completely prevented the induction of LTD by dipyridamole and Zaprinast. The extent of phosphodiesterase induced synaptic depression was dependent on the frequency of parallel fiber stimulation, and this form of LTD both occluded and was occluded by LTD induced by pairing parallel and climbing fiber inputs. The degree of LTD induced by IBMX was dose-dependent, and also required PKC and PKG activity, but was preceded by a large, transient potentiation of parallel fiber responses occurring by a postsynaptic mechanism independent of cGMP. These data not only confirm that cGMP is capable of inducing cerebellar LTD when paired with parallel fiber stimulation but indicate that cGMP is an endogenous intermediate in this form of synaptic plasticity. PMID- 8622120 TI - Inositol 1,4,5-trisphosphate-gated calcium transport through plasma membranes in nerve terminals. AB - We developed new biochemical approaches to demonstrate the presence of inositol 1,4,5-triphosphate (InsP3)-gated calcium channels in presynaptic plasma membranes (SPM) and their involvement in the presynaptic receptor-mediated Ca2+ influx into nerve terminals. In perfusion experiments using SPM vesicles preloaded with 45Ca2+, InsP3 elicited the release of 45CA2+ into perfusates in a saturable manner. The InsP3- evoked 45Ca2+ release from resealed SPM vesicles was more potent than that from resealed vesicles using any other subcellular fractions. Here we also report the involvement of InsP3-gated mechanisms in the presynaptic receptor-mediated Ca2+ influx into synaptosomes (nerve terminals) by use of such resealed vesicles reconstituted with purified Gi1. PMID- 8622121 TI - Differential expression of the p75 nerve growth factor receptor in glia and neurons of the rat dorsal root ganglia after peripheral nerve transection. AB - Sympathetic nerve terminals on blood vessels within the dorsal root ganglia sprout after sciatic nerve lesions in the rat. The mechanism underlying this phenomenon is not clear, but might be predicted to involve nerve growth factor or its homologs because these factors are known to trigger collateral sprouting of undamaged sympathetic noradrenergic terminals. We have found that sciatic nerve lesions lead to a decreased expression of neuronal p75, the low-affinity receptor for the neurotrophins, but an increased expression of glial p75 in ipsilateral dorsal root ganglia. Intriguingly, the increased expression of p75 was found primarily in association with glia surrounding large-diameter neurons, which are those associated with the noradrenergic sprouts. A smaller but significant glial response was also found in contralateral ganglia. The glial response in ipsilateral ganglia could be mimicked by ventral, but not dorsal, root transection. The dorsal root lesion-induced glial responses in contralateral ganglia were greater than those induced by ventral root or sciatic nerve lesions. Combined lesions of dorsal root and either ventral root or sciatic nerve did not prevent the glial responses of ipsilateral ganglia, suggesting that a peripheral signal is involved. Colocalization studies indicate that tyrosine hydroxylase immunoreactive nerve sprouts were associated with p75-immunoreactive glial cells. Thus, increased glial synthesis of p75 might provide an explanation for the abnormal growth of sympathetic fibers in dorsal root ganglia after peripheral nerve injury. PMID- 8622122 TI - Upregulation of GABAA current by astrocytes in cultured embryonic rat hippocampal neurons. AB - Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or a monolayer of postnatal cortical astrocytes to reveal putative changes in neuronal physiology that involve astrocyte-derived signals during the first 4 d of culture, GABA-induced Cl- current (IGABA) was quantified using outside-out and whole-cell patch-clamp recordings beginning at 30 min, when cells had become adherent. The amplitude and density (current normalized to membrane capacitance) of IGABA in neurons grown on astrocytes became statistically greater than that recorded in neurons grown on PDL after 2 hr in culture (HIC). Although the current density remained unchanged in neurons on astrocytes, that in neurons on PDL decreased and became statistically lower beginning after 2 HIC. The differences in amplitude and density of IGABA in the two groups of neurons were maintained during the 4 d experiment. The upregulation effect of astrocytes on neuronal IGABA required intimate contact between the neuronal cell body and underlying astrocytes. Suppression of spontaneous Cac2+ elevations in astrocytes by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid that was loaded intracellularly decreased their modulatory effects on IGABA. IGABA in all cells was blocked completely by bicuculline and exhibited virtually identical affinity constants, Hill coefficients, and potentiation by diazepam in the two groups. Outside-out patch recordings revealed identical unitary properties of IGABA in the two groups. More channels per unit of membrane area could explain the astrocyte enhancement of IGABA. The results reveal that cortical astrocytes potentiate IGABA in hippocampal neurons in a contact-dependent manner via a mechanism involving astrocyte Cac2+ elevation. PMID- 8622123 TI - INDO-1 measurements of absolute resting and light-induced Ca2+ concentration in Drosophila photoreceptors. AB - Absolute Ca2+ levels in dissociated Drosophila photoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca2+ influx that saturated the dye (>10 microM Ca2+), but lagged the electrical response by 2.8 msec. Resting Ca2+ levels in the dark, measured during the latent period before the response, averaged 160 nM in normal Ringer's (1.5 mM Ca2+). Ca2+ increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca2+ during the latent period; and (2) by using ninaE mutants with greatly reduced rhodopsin levels. Ca2+ rose linearly as a function of the time integral of the light sensitive current with a slope of 2.7 nM/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nM/pC, indicating a 2.5-fold reduction in the fractional Ca2+ current. From these data, it can also be estimated that >99% of the Ca2+ influx is effectively buffered by the cell. In Ca2+-free Ringer's, resting cytosolic Ca2+ was reduced (to 30-70 nM), but contrary to previous reports, significant light-induced increases (approximately 250 nM) could be elicited. This rise was reduced to <20 nM when extracellular Na+ was replaced with N-methyl-D-glucamine, suggesting that it could be attributed to Na+ influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nM. PMID- 8622124 TI - Glutamate responses of bipolar cells in a slice preparation of the rat retina. AB - Whole-cell currents from >70 voltage-clamped bipolar cells were recorded in a slice preparation of the rat retina. The recorded cells were identified and classified by intracellular staining with Lucifer yellow. Glutamate, the specific agonists (+/-)-2-amino-4-phosphonobutyric acid (AP-4) and kainate (KA), and the antagonist 6-cyanoquinoxaline-2,3-dione (CNQX) were applied. The cells could be isolated from presynaptic influences by the co-application of bicuculline, strychnine, and cobalt ions. Responses to AP-4 were elicited only from bipolar cells with axons stratifying in the inner part of the inner plexiform layer (IPL). AP-4 caused an outward current in these cells attributable to the closure of nonspecific cation channels. Responses to kainate representing a direct action of the drug on the recorded cells were observed only in bipolar cells with axons stratifying in the outer part of the IPL. KA caused a CNQX-sensitive inward current in these cells, associated with openings of nonspecific cation channels. The results predict that cone bipolar (CB) cells with axons terminating in the outer IPL are OFF-bipolars, whereas those with axons terminating in the inner IPL are ON-bipolars. Most of the cells expressed GABA-gated Cl- conductances. In rod bipolar and in some CB cells, only part of the GABA-induced currents could be blocked by the application of bicuculline, suggesting the presence of GABAc receptors in addition to GABAA receptors. PMID- 8622125 TI - Attenuation of astroglial reactivity by interleukin-10. AB - Prominent responses that follow brain trauma include the activation of microglia, recruitment of blood-derived macrophages, and astroglial reactivity. Based on evidence that cytokines produced by macrophages/microglia may cause astrocytes to become reactive, the aim of this study was to determine whether astroglial reactivity could be attenuated by interleukin (IL)-10, a potent inhibitor of cytokine synthesis by macrophages/microglia. Four days after the local application of IL-10 to the site of corticectomy in adult mice, the number of reactive astrocytes and their state of hypertrophy was reduced (by 60%) when compared with vehicle controls. In the majority of IL-10-treated mice, but not in any vehicle controls, the tissue in the immediate vicinity of IL-10 application contained viable but non reactive astrocytes. The mechanism by which IL-10 attenuates astroglial reactivity is likely via the reduction of cytokine production by macrophages/microglia because, based on Mac-1 immunohistochemistry, the macrophages/microglia of IL-10 brains had a decreased activation state compared with vehicle-controls. Another macrophage/microglia deactivating agent, macrophage inhibitory factor, also reduced astroglial activity in vivo. Furthermore, IL-10 had no direct effect on purified astrocytes in culture, indicating that its in vivo action on astroglial reactivity is likely via indirect mechanisms. Finally, injury resulted in the substantial rise of tumor necrosis factor-alpha mRNA levels, and this elevation was significantly inhibited by IL-10. The ability to manipulate the extent of astrogliosis should provide a means of addressing the neurotrophic or inhibitory role of reactive astrocytes in neurological recovery. PMID- 8622126 TI - Regulation of alpha7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents. AB - Distributions of alpha7 nicotinic acetylcholine receptor (nAChR) mRNA and [125]alpha-bungarotoxin (alpha-BTX) binding sites in the developing rat somatosensory cortex were characterized in relation to acetylcholinesterase (AChE) histochemical staining of thalamocortical terminals to investigate the role of this receptor in cortical development. Using quantitative in situ hybridization and receptor autoradiography, elevated levels of mRNA and binding site expression were first detected at post-natal day 1 (P1) in deep and superficial layers, just beneath the AChE-stained thalamocortical terminals. Onset of expression occurred approximately 1 d after ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and binding-site expression exhibited a disjunctive, barrel-like pattern in layer IV and, more clearly, in layer VI. The mRNA and binding-site expressions peaked at approximately 1 week postnatal and then declined to adult levels. Unilateral electrolytic or cytochemical lesions placed in the thalamic ventrobasal complex at P0 (just as thalamocortical afferents are innervating the cortex) and at P6 (when the somatotopic map is well established) resulted in a marked reduction of alpha7 nAChR mRNA and [125]alpha BTX binding-site levels in layers IV and VI, indicating their regulation by thalamocortical afferents. With P6 lesions, this reduction was observed as early as 6 hr postlesion. These results suggest that alpha7 nAChRs are localized primarily on cortical cells in rat somatosensory cortex and provide further evidence for thalamocortical influence on cortical ontogeny. These data also suggest a role for cholinergic systems during a critical period of cortical synaptogenesis. PMID- 8622127 TI - The divergent homeobox gene PBX1 is expressed in the postnatal subventricular zone and interneurons of the olfactory bulb. AB - In the mammalian brain, an important phase of neurogenesis occurs postnatally in the subventricular zone (SVZ). This region consists of a heterogeneous population of cells, some mitotically active, others postmitotic. A subset of mitotically active SVZ precursor cells gives rise to a population of neurons that migrates over a long distance to their final destination, the olfactory bulb. Other SVZ precursor cells continue to proliferate or undergo cell death. The combination of genes that regulates proliferation and cell fate determination of SVZ precursor cells remains to be identified. We have used the rat homolog of the human homeobox gene PBX1 in Northern analysis and in situ hybridization studies to determine the temporal and regional localization of PBX1 expression during embryonic and postnatal rat brain development. PBX1 is expressed embryonically in the telencephalon. In addition, it is expressed at high levels postnatally in the SVZ, in the migratory pathway to the olfactory bulb, and in the layers of the olfactory bulb that are the targets of these migratory neurons. Combining in situ hybridization for PBX1 with immunostaining for markers of cell proliferation (PCNA), postmitotic neurons (class III beta-tubulin), and glia (GFAP), we show that SVZ proliferating cells and their neuronal progeny express rat PBX1 mRNA, whereas glial cells do not express detectable levels of PBX1. The expression of PBX1 in SVZ precursor cells and postmitotic neurons suggests a role for PBX1 in the generation of olfactory bulb interneurons and in mammalian neurogenesis. PMID- 8622128 TI - The dynamics of dendritic structure in developing hippocampal slices. AB - Time-lapse fluorescence confocal microscopy was used to directly visualize the formation and dynamics of postsynaptic target structures (i.e., dendritic branches and spines) on pyramidal neurons within developing tissue slices. Within a 2 week period of time, pyramidal neurons in cultured slices derived from early postnatal rat (postnatal days 2-7) developed complex dendritic arbors bearing numerous postsynaptic spines. At early stages (1-2 d in vitro), many fine filopodial protrusions on dendrite shafts rapidly extended (maximum rate approximately 2.5 microM/minute) and retracted (median filopodial lifetime, 10 min), but some filopodia transformed into growth cones and nascent dendrite branches. As dendritic arbors matured, the population of fleeting lateral filopodia was replaced by spine-like structures having a low rate of turnover. This developmental progression involved a transitional stage in which dendrites were dominated by persistent (up to 22 hr) but dynamic spiny protrusions (i.e., protospines) that showed substantial changes in length and shape on a timescale of minutes. These observations reveal a highly dynamic state of postsynaptic target structures that may actively contribute to the formation and plasticity of synaptic connections during CNS development. PMID- 8622130 TI - Whole-cell patch-clamp recording reveals subthreshold sound-evoked postsynaptic currents in the inferior colliculus of awake bats. AB - The inferior colliculus receives excitatory and inhibitory input from parallel auditory pathways that differ in discharge patterns, latencies, and binaural properties. Processing in the inferior colliculus may depend on the temporal sequence in which excitatory and inhibitory synaptic inputs are activated and on the resulting balance between excitation and inhibition. To explore this issue at the cellular level, we used the novel approach of whole-cell patch-clamp recording in the midbrain of awake bats (Eptesicus fuscus) to record EPSCs or IPSCs. Sound-evoked EPSCs were recorded in most neurons. These EPSCs were frequently preceded by an IPSC, followed by an IPSC, or both. These findings help explain the large latency range and transient responses that characterize inferior colliculus neurons. The EPSC was sometimes followed by long-lasting oscillatory currents, suggesting that a single brief sound sets up a pattern of altered excitability that persists far beyond the duration of the initial sound. In three binaural neurons, ipsilateral sound evoked a large IPSC that partially or totally canceled the EPSC evoked by contralateral sound. In one binaural neuron with ipsilaterally evoked IPSCs, contralaterally evoked IPSCs occurred in response to frequencies above and below the neuron's best frequency. Thus, both monaural and binaural interactions can occur at single inferior colliculus neurons. These results show that whole-cell patch-clamp recording offers a powerful means of understanding how subthreshold processes determine the responses of auditory neurons. PMID- 8622129 TI - Retrograde transport of neurotrophins from the eye to the brain in chick embryos: roles of the p75NTR and trkB receptors. AB - The receptors involved in retrograde transport of neurotrophins from the retina to the isthmo-optic nucleus (ION) of chick embryos were characterized using antibodies to the p75 neurotrophin receptor and trkB receptors. Survival of neurons in the ION has been shown previously to be regulated by target-derived trophic factors with survival promoted or inhibited by ocular injection of brain derived neurotrophic factor (BDNF) or nerve growth factor (NGF), respectively. In the present paper, we show that during the period of target dependence, these neurons express trkB and p75 neurotrophin receptor but not trkA or trkC mRNAs. We also show that BDNF and NT-3 were transported efficiently at low doses, whereas NGF was transported significantly only at higher doses. The transport of BDNF and NT-3 was reduced by high concentrations of NGF or by antibodies to either trkB or the p75 neurotrophin receptor. Thus both receptors help mediate retrograde transport of these neurotrophins. Ocular injection of the comparatively specific trk inhibitor K252a did not reduce transport of exogenous BDNF, but did induce significant neuronal death in the ION, which could not be prevented by co injection of BDNF. Thus, transport of BDNF alone does not generate a trophic signal at the cell body when axonal trkB is inactivated. In summary, our results indicate that both p75 neurotrophin and trkB receptors can mediate internalization and retrograde transport of BDNF, but activation of trkB seems to be essential for the survival-promoting actions of this neurotrophin. PMID- 8622131 TI - Chronic 3-nitropropionic acid treatment in baboons replicates the cognitive and motor deficits of Huntington's disease. AB - We showed recently that chronic administration of the mitochondrial inhibitor 3 nitropropionic acid (3NP) in primates produces various dyskinetic movements and dystonic postures associated with selective striatal lesions displaying many similarities with the pathological features of Huntington's disease (HD). In the present study, we examined whether such a toxic treatment could also induce frontal-type deficits similar to those observed in HD patients. Cognitive performances of 3NP-treated and control baboons were compared using the object retrieval detour task (ORDT), a test designed to assess the functional integrity of the frontostriatal pathway in human and nonhuman primates. During the same time, the motor function of each animal was assessed under spontaneous "no drug" conditions, and time-sampled neurological observations were used after apomorphine administration. A significant impairment in the ORDT was observed in the 3NP animals after 3-6 weeks of treatment, occurring in the absence of spontaneous abnormal movements by in the presence of apomorphine-inducible dyskinesias. Prolonged 3NP treatment resulted in the progressive appearance of spontaneous abnormal movements. Histological evaluation of these animals showed selective bilateral caudate-putamen lesions with sparing of the cerebral cortex, notably the prefrontal cortex. The present study demonstrates that chronic 3NP treatment replicates in primates the basic pathophysiological triad of HD, including spontaneous abnormal movements, progressive striatal degeneration, and a frontostriatal syndrome of cognitive impairment. PMID- 8622132 TI - Possible role of protein kinase C in the sensitization of primate spinothalamic tract neurons. AB - The responsiveness of spinal cord nociceptive neurons to innocuous mechanical stimuli can be increased by the release of excitatory amino acids (EAAs) and peptides attributable to an injury-induced barrage of impulses. This sensitization of spinal dorsal horn neurons can also result from administration of phorbol ester by microdialysis, presumably by direct activation of protein kinase C (PKC). This study was designed to examine the effects of central sensitization of spinothalamic tract (STT) neurons produced by intradermal injection of capsaicin on the descending inhibition driven from the periaqueductal gray (PAG) and the possible role of PKC in this process in anesthetized monkeys. Sensitization of responses of STT cells to mechanical stimuli was induced by intradermal injection of capsaicin. PAG inhibition was significantly attenuated when sensitization of responses to mechanical stimuli occurred. However, perfusion of the spinal cord with NPC15437 (a selective PKC inhibitor) by microdialysis could prevent the sensitization of the responses to mechanical stimuli and the reduction in PAG inhibition of these responses induced by capsaicin injection. Results similar to those produced by capsaicin injection were observed when a PKC activator, phorbol ester (12-O-tetradecanoylphorbol-13 acetate), was infused within the dorsal horn by microdialysis. An inactive phorbol ester (4 alpha-phorbol 12,13-didecanoate) had no effect. These results provide evidence that the activation of PKC contributes to the development of central sensitization in dorsal horn neurons produced by chemical stimulation with capsaicin. Attenuation of the effectiveness of PAG inhibition takes place when the sensitization of dorsal horn cells develops, and PKC may play a significant role in this process. PMID- 8622133 TI - Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus. AB - The anteroventral periventricular nucleus (AVPV) is a nodal point in neural circuits regulating secretion of gonadotropin and contains sexually dimorphic populations of hormonally regulated dopamine-, dynorphin-, and enkephalin containing neurons. Because the tyrosine hydroxylase (TH), prodynorphin (PDYN), and proenkephalin (PENK) genes contain cAMP response elements that control their expression in their promoters, we used histochemical methods to determine whether ovarian steroids alter expression of the cAMP response element-binding protein (CREB) in the AVPV. Because the ability of CREB to activate transcription depends on phosphorylation at Ser133, we also evaluated the effects of acute steroid treatment on levels of phosphorylated CREB (pCREB) in AVPV neurons by using an antibody that differentiates between CREB and pCREB. Treatment of ovariectomized rats with estradiol treatments caused a significant induction in the number of pCREB-immunoreactive nuclei within 30 min that was maintained for at least 4 hr, but did not alter CREB immunostaining in the AVPV. Pretreatment with the estrogen antagonist Nafoxidine blocked this induction. In contrast, acute administration of progesterone to estrogen-primed animals suppressed and then increased pCREB staining in the ASVPV at 30 and 60 min, respectively; no significant differences between experimental and control animals were apparent by 2 hr after progesterone treatment. Double-labeling experiments showed that pCREB was colocalized with PDYN, PENK, or TH mRNA in the AVPV, suggesting that pCREB may mediate the effect of steroid hormones on gene expression in these neurons. PMID- 8622134 TI - Immunological instability of persistent adenovirus vectors in the brain: peripheral exposure to vector leads to renewed inflammation, reduced gene expression, and demyelination. AB - Nonreplicating adenovirus vectors are being developed as vehicles for the delivery of therapeutic genes in vivo. Whereas in many organs an antiviral T cell response eliminates the vector and damages local tissue, when adenovirus vectors are injected into the brain the subsequent immune attack can be ineffective, allowing the vector to persist. In the present study, E1-deleted human adenovirus vectors were injected into the caudate nucleus of rats. Two months later, expression of protein from the vector was still evident and little inflammation was seen. A subcutaneous injection of adenovirus vector at this time, however, led within 2 weeks to severe mononuclear inflammation and microglial activation in the caudate. This caused local demyelination and a decrease in detectable protein expression from the vector. Interestingly, intense microglial activation and numerous lymphocytes and monocytes were also seen in brain areas containing neurons capable of retrogradely transporting the adenovirus vector from the caudate. Control experiments established that this inflammation in distant brain areas was not a nonspecific consequence of degeneration. These experiments demonstrate that although adenovirus vectors can persist in the brain without causing chronic inflammation, they remain the potential target of a damaging cell mediated immune response brought about by a subsequent peripheral exposure to vector. The finding of lymphocytes in brain areas that project to the caudate further shows that viral antigens that are retrogradely transported by neurons can also be the target of a T cell attack. PMID- 8622135 TI - High-frequency oscillations in the output networks of the hippocampal-entorhinal axis of the freely behaving rat. AB - Population bursts of the CA3 network, which occur during eating, drinking, awake immobility, and slow-wave sleep, produce a large field excitatory postsynaptic potential throughout stratum radiatum of the CA1 field (sharp wave). The CA3 burst sets into motion a short-lived, dynamic interaction between CA1 pyramidal cells and interneurons, the product of which is a 200 Hz oscillatory field potential (ripple) and phase-related discharge of the CA1 network. Although many CA1 pyramidal neurons discharge during the time (50-100 msec) of each sharp wave, each wave of a ripple (approximately 5 msec) reflects the synchronization of more discrete subsets of CA1 neurons. When we used multi-site recordings in freely behaving rats, we observed ripples throughout the longitudinal extent (approximately 4-5 mm) of the dorsal CA1 region that were coherent for multiple cycles of each ripple. High-frequency ripples were also observed throughout the hippocampal-entorhinal output pathway that were concurrent but less coherent on a cycle-by-cycle basis. Single and multiunit neuronal activity was phase-related to local ripples throughout the hippocampal-entorhinal output pathway. Entorhinal ripples occurred 5-30 msec after the CA1 ripples and were related to the occurrence of an entorhinal sharp wave. Thus, during each hippocampal sharp wave, there is powerful synchronization among the neuronal networks that connect the hippocampus to the neocortex. We suggest that this population interaction (1) biologically constrains theoretical models of hippocampal function and dysfunction and (2) has the capacity to support an "off-line" memory consolidation process. PMID- 8622136 TI - Evolution of directional preferences in the supplementary eye field during acquisition of conditional oculomotor associations. AB - We assessed the preferred directions (PDs) of supplementary eye field (SEF) neurons during conditional visuomotor learning. Monkeys learned to select one of four saccadic eye movements in response to a foveal instruction stimulus (IS). ISs were either familiar or novel. Each familiar IS reliably evoked one saccade: 7 degrees left, right, up, or down form the central fixation point. Novel ISs initially triggered virtually random responses among those four possibilities, but the monkeys ultimately learned to select the instructed saccade. As reported previously, activity rates on novel IS trials significantly changed during learning. Some of these cells (learning-dependent) also have significant modulation on familiar IS trials, but others (learning-selective) lack such activity. Of the former, the familiar IS activity can be either directionally selective or omnidirectional. For most neurons, PDs were apparent during all phases of learning, but they were rarely constant. Only infrequently did a neuron's PD for novel ISs closely match that for familiar ISs throughout the learning process. In directional learning-dependent cells, the PD usually reoriented near the end of learning to resemble that for familiar IS trials. In omnidirectional cells, initially evident PDs dissipated with learning, even as the cell became more strongly modulated. Learning-selective cells typically began with significant PDs, but became unmodulated as learning progressed. Our findings show a pervasive lability in SEF PDs that may reflect a flexible and rapid remapping between inputs and responses within the premotor cortical network. PMID- 8622137 TI - Covert orienting of attention in the rat and the role of striatal dopamine. AB - Attention can be directed to a location in the absence of overt signs of orienting, a phenomenon termed "covert orienting." The ability to orient attention covertly has been well documented in humans, but recent progress has been made with the operational definition of the processes involved in covert orienting. Reaction times to visual targets are quickened when attention is drawn to the location of the subsequent target, and processes such as disengagement, maintenance, and movement of attention can be dissociated by using this method. The possible involvement of striatal dopamine in covert orienting is disputed, with conflicting reports of deficits in covert orienting in patients with Parkinson's disease. To examine the significance of dopamine in the striatum in attentional processes, a test of covert orienting, analogous to that used in humans, was devised for the rat. Unilateral dopamine-depleting lesions of the striatum resulted in increases in mean reaction times contralateral to the side of the lesion, but reaction times did not change differentially as a function of the requirements to maintain, disengage, or shift attention. These findings add additional support to the hypothesis that the deficit that appears as hemineglect observed after striatal damage reflects a motor impairment rather than damage in neural systems underlying mechanisms for directing attention. PMID- 8622138 TI - Conditioned and unconditioned stimuli increase frontal cortical and hippocampal acetylcholine release: effects of novelty, habituation, and fear. AB - Recent evidence showing that basal forebrain cholinergic neurons with projections to the frontal cortex and hippocampus are activated by behaviorally salient stimuli suggests that these neurons are involved in arousal and/or attentional processes. We sought in the present experiments to test this hypothesis by examining whether unconditioned stimuli (a tone and flashing light) that normally increase cortical nad hippocampal acetylcholine (ACh) release would fail to do so after habituation (i.e., repeated presentation with no programmed consequences). In addition, the extent to which presentation of these stimuli would continue to increase ACh release when they had previously been paired with an aversive stimulus was investigated. Three experimental groups were used: habituation, novel stimuli, and conditioned fear. Subjects in each of these groups were placed in a training apparatus for twelve 200 min sessions. While the habituation group received extensive exposure to the tone and light during the training sessions, subjects in the novel stimuli group were placed in the apparatus but were never exposed to the tone or light during these sessions. The conditioned fear group was treated identically to the habituation group, with the addition that the tone and light were paired with footshock. On completion of these training schedules, all animals were implanted with microdialysis probes in the frontal cortex and hippocampus. Two days later, they were placed in the apparatus and the tone and light were presented to all subjects during microdialysis. In the novel stimuli group, the tone and light (unconditioned stimuli) produced significant increases in frontal cortical and hippocampal ACh release. Similarly, in the conditioned fear group, presentation of the tone and light (conditioned stimuli) also significantly increased ACh release in frontal cortex and hippocampus. In contrast, in the habituation group the tone and light failed to significantly enhance ACh release in either structure. During the test session, the tone and light elicited a variety of arousal- and fear-related behaviors in the novel stimuli and conditioned fear groups. In contrast, subjects in the habituation group generally failed to respond to these stimuli. These data indicate that cortically and hippocampally projecting basal forebrain cholinergic neurons are activated by conditioned and unconditioned stimuli that produce arousal in rats (novelty or conditioned fear). In contrast, presentation of these stimuli to habituated animals fails to enhance ACh release. These findings are consistent with a growing body of information indicating that ACh release in the cortex and hippocampus is reliably activated by behaviorally relevant stimuli. They also provide strong support for the hypothesis that cholinergic neurons in the basal forebrain are involved in arousal and/or attentional processes. PMID- 8622139 TI - Somatostatin in the pontine reticular formation modulates fear potentiation of the acoustic startle response: an anatomical, electrophysiological, and behavioral study. AB - The amplitude of the acoustic startle response (ASP) in rats is increased in the presence of a cue that has previously been paired with an aversive stimulus such as a footshock. This phenomenon is called fear-potentiated startle and is a model to study the neuronal and neurochemical mechanisms of the acquisition and expression of fear. The present study investigated the role in fear-potentiated startle of somatostatin in the caudal pontine reticular nucleus (PnC) by a combination of anatomical, electrophysical, and behavioral methods. The PnC is an essential part of the primary startle circuit and is also the recipient of modulatory influences. First, we showed that the central gray (CG), which is involved in fear conditioning, is the main source of somatostatinergic input to the PnC. In the second experiment, we iontophoretically applied the somatostatin receptor agonist sandostatin on PnC neurons and extracellularly recorded the activity of PnC neurons. Sandostatin had no effect on tone-evoked or spontaneous activity, but markedly attenuated the increase of neuronal activity seen after the administration of glutamate. In our third experiment, we injected different doses of sandostatin into the PnC of awake rats. Sandostatin blocked fear potentiation of the ASR but had no effect on the baseline ASR amplitude. The present study indicates that the somatostatinergic projection from the CG to the PnC is important for the modulation of fear-potentiated startle. We present a possible neural circuitry for the expression of fear-potentiated startle based on these data and previous findings. PMID- 8622140 TI - Mouse model of hyperkinesis implicates SNAP-25 in behavioral regulation. AB - Although hyperkinesis is expressed in several neurological disorders, the biological basis of this phenotype is unknown. The mouse mutant coloboma (Cml+) exhibits profound spontaneous locomotor hyperactivity resulting from a deletion mutation. This deletion encompasses several genes including Snap, which encodes SNAP-25, a nerve terminal protein involved in neurotransmitter release. Administration of amphetamine, a drug that acts presynaptically, markedly reduced the locomotor activity in coloboma mice but increased the activity of control mice implicating presynaptic function in the behavioral abnormality. In contrast, the psychostimulant methylphenidate increased locomotor activity in both coloboma and control mice. When a transgene encoding SNAP-25 was bred into the coloboma strain to complement the Snap deletion, the hyperactivity expressed by these mice was rescued, returning these corrected mice to normal levels of locomotor activity. These results demonstrate that the hyperactivity exhibited by these mice is the result of abnormalities in presynaptic function specifically attributable to deficits in SNAP-25 expression. PMID- 8622141 TI - Rewarding actions of phencyclidine and related drugs in nucleus accumbens shell and frontal cortex. AB - Rats learned to lever-press when such behavior was reinforced by microinjections of phencyclidine (PCP) directly into the ventromedial (shell) region of nucleus accumbens, indicating that the drug has direct rewarding actions in that region. Separate groups of rats learned to lever-press when reinforced with microinjections of dizoclipine (MK-801) or 3-((+/-)2-carboxypiperazin-4yl)propyl 1-phosphate (CPP), drugs known to block NMDA receptor function but not dopamine uptake, into the same region. Each drug was ineffective or markedly less effective when injected at a slightly more dorsal and lateral site in the core of nucleus accumbens. Self-administration of PCP, MK-801, or CPP directly into nucleus accumbens was not altered by co-infusion of a dose of the dopamine antagonist sulpiride that effectively blocked intracranial self-administration of the dopamine uptake inhibitor nomifensine, suggesting that the rewarding actions of the NMDA receptor antagonists are not dopamine-dependent. Rats also developed lever-pressing habits when PCP, MK-801, and CPP were each microinjected directly into frontal cortex, a region previously associated with the rewarding actions of cocaine but not nomifensine. Thus nucleus accumbens and frontal cortex are each potential substrates for the rewarding properties of PCP and related drugs, and the ability of these drugs to disrupt NMDA receptor function seems sufficient to account for their rewarding actions. When considered with independent evidence, the present results suggest a model of drug reward within which the critical event is inhibition of medium spiny neurons in nucleus accumbens. PMID- 8622142 TI - Postoperative surveillance imaging in children with cerebellar astrocytomas. AB - The standard follow-up care for children with cerebellar astrocytomas includes regular surveillance imaging of the brain with computerized tomography or magnetic resonance. The purpose of surveillance imaging is to detect asymptomatic tumor recurrence at an early stage and permit safer reoperation. The authors evaluated the effectiveness of an intensive surveillance program for cerebellar astrocytoma and tested different models of surveillance frequency and duration to arrive at a specific recommended program. Review of the records of 93 children with typical cerebellar astrocytomas who received follow-up care between 1975 and 1993 was performed. Immediate postoperative and surveillance images were classified as showing definite equivocal, or no tumor based on the radiology report at the time the image was obtained. Various surveillance models were then tested for their predictive value for detecting tumor recurrence. Seventeen (18%) of the 93 children had tumor recurrence or progression. Eleven of these tumors were asymptomatic and detected only by surveillance image. Tumor recurred in only one patient with a total resection, whereas tumor progression occurred in five of 21 patients with equivocal postoperative images and in 11 of 14 patients with residual tumor. A model in which patients with possible or definite residual tumor after surgery undergo surveillance at 12, 18, 30, 42, and 66 months, and later have one additional image, yielded optimum predictive value for recurrence and/or progression with the fewest images. Patients with tumor recurrence were satisfactorily treated, and only one patient died. Children with totally resected cerebellar astrocytomas do not appear to benefit from routine surveillance, because the likelihood of recurrence is small. Surveillance is of benefit in those who may have subtotal resection based on the immediate postoperative imaging. PMID- 8622143 TI - Value of postoperative surveillance imaging in the management of children with some common brain tumors. AB - The rationale for obtaining surveillance computerized tomography (CT) scans or magnetic resonance (MR) images in pediatric patients with brain tumors is that early detection of recurrence may result in timely treatment and better outcome. The purpose of this study was to investigate the value of surveillance cranial images in a variety of common pediatric brain tumors managed at a tertiary care pediatric hospital. A retrospective chart review was performed of children with astrocytoma of the cerebral hemisphere, cerebellum, optic chiasm/hypothalamus, or thalamus; cerebellar or supratentorial high-grade glioma; supratentorial ganglioglioma; posterior fossa or supratentorial primitive neuroectodermal tumor (PNET); and posterior fossa ependymoma. Data were analyzed to determine the frequency with which recurrences were identified on a surveillance image and how the type of image at which recurrence was identified related to outcome. In 159 children, 17 of 44 recurrences were diagnosed by surveillance imaging. The percentage of recurrences identified by surveillance imaging was 64% for ependymoma, 50% for supratentorial PNET, 43% for optic/hypothalamic astrocytoma, and less than 30% for other tumors. The rate of diagnosis of recurrence per surveillance image varied from 0% to 11.8% for different tumor types. Only for ependymomas did there appear to be an improved outcome when recurrence was identified prior to symptoms. Our results indicate that, using the protocols outlined in this study, surveillance imaging was not valuable in identifying recurrence of cerebellar astrocytoma or supratentorial ganglioglioma during the study period, but was probably worthwhile in identifying recurrence of posterior fossa ependymoma and optic/hypothalamic astrocytoma and, possibly, medulloblastoma. Surveillance protocols could be made more effective by individualizing them for each type of tumor, based on current data on the patterns of recurrence. PMID- 8622144 TI - Adjuvant combined modality therapy for malignant meningiomas. AB - Malignant meningiomas constitute 10% to 15% of all meningiomas and limited information exists regarding adjuvant treatment of these aggressive primary brain tumors. Fourteen patients (eight men, six women), ranging in age from 28 to 61 years (median 51 years), were prospectively treated for primary malignant meningiomas according to an institutional protocol. All patients underwent surgery (gross-total in four and subtotal resection in 10 patients) followed in 2 to 4 weeks by involved-field radiotherapy (range 59-60 Gy, median dose 60 Gy). Two to 4 weeks after radiotherapy all patients were treated with adjuvant chemotherapy that included cyclophosphamide, adriamycin, and vincristine (CAV). Patients who underwent gross-total resection received three cycles, whereas those with subtotal resection received six cycles of CAV. Four patients required CAV dose reduction due to myelosuppression, and in three patients, myelosuppression prevented administration of the planned course of CAV. Four patients required transfusions (four received red blood cells, three received platelets), and two developed neutropenic fever without bacteriological documentation. Neuroradiographic response included three partial responses and 11 with stable disease. The median time to tumor progression was 4.6 years (range 2.2-7.1 years) and median survival was 5.3 years (range 2.6-7.6 years). The author concludes that combined modality therapy for the treatment of malignant meningiomas is associated with acceptable toxicity and a modest improvement in survival when compared to patients treated with surgery alone. PMID- 8622145 TI - Use of intraoperative ultrasound for localizing tumors and determining the extent of resection: a comparative study with magnetic resonance imaging. AB - A prospective study of 70 patients with intraparenchymal brain lesions (36 gliomas and 34 metastases) was performed to evaluate the efficacy of intraoperative ultrasound (IOUS) in localizing and defining the borders of tumors and in assessing the extent of their resection. Eighteen of the 36 glioma patients had no previous therapy. All of these 18 tumors were well localized by IOUS; margins were well defined in 15 and moderately defined in three. The extent of resection was well defined on IOUS in all 18 patients, as confirmed by measurements taken on postoperative magnetic resonance (MR) images (p = 0.90). The remaining 18 patients with gliomas had undergone previous surgery and/or radiation therapy; five had recurrent tumors and 13 had radiation-induced changes. The extent of resection of the recurrent tumors was well defined in all but one patient, as confirmed by postoperative MR imaging. The extent of resection was poorly defined in all 13 patients whose pathology showed radiation effects. All 34 metastatic lesions were well localized and had well-defined margins. In addition, IOUS accurately determined the extent of resection in all cases, the results were confirmed with postoperative MR imaging. In conclusion, IOUS is not only helpful in localizing and defining the margins of gliomas and metastatic brain lesions, it also accurately determines the extent of resection, as confirmed by postoperative MR imaging. This assessment does not apply, however when the lesion is due primarily to radiation effect. PMID- 8622146 TI - Central neurocytoma: proliferative potential and biological behavior. AB - The authors analyzed 13 central neurocytomas diagnosed at Seoul National University Hospital between January 1982 and December 1993 to clarify the proliferative potential and biological behavior of these tumors. The tumor was confined to the lateral and third ventricles in 12 cases and in one case extended from the posterior thalamus to the body and trigone area of the lateral ventricle. In all 13 cases, typical clinical and radiological findings were observed, and histological diagnosis was performed via craniotomy. The diagnosis was made using light microscopic examination, immunohistochemical staining for neuronal markers, and electron microscopic findings of neuronal differentiation. One patient died due to tumor progression with recurrence 26 months after subtotal removal plus radiation therapy. Another patient had a recurrence 18 months after total tumor removal. The remaining 11 patients are free of recurrent tumor after a follow-up period that ranged from 14 to 109 months (median 50 months). To predict the proliferative potential, immunoreactivity to proliferating cell nuclear antigen (PCNA), silver colloid staining for nucleolar organizing regions (AgNORs), and DNA flow cytometry were performed in 10 of the 13 cases. The proportion of PCNA-positive cells was less than 1% in all cases and the AgNORs score ranged from 1.11 to 2.0 (mean 1.67). The DNA flow cytometry revealed diploidy in all cases and the calculated proliferation index ranged from 5.1% to 9.6% (mean 7.8%). The one case of tumor recurrence, in which the authors performed the study of proliferative potential, and another case that demonstrated mild nuclear pleomorphism also showed low percentages of PCNA positive cells, low AgNORs scores, and diploidy in DNA flow cytometry. It is suggested that most central neurocytomas follow a benign clinical course with low proliferative potential assessed by PCNA, AgNORs, and DNA flow cytometry; however, recurrence is possible within a relatively short time period. PMID- 8622147 TI - Surgery versus radiosurgery in the treatment of brain metastasis. AB - Surgery and radiosurgery are effective treatment modalities for brain metastasis. To compare the results of these treatment modalities, the authors followed 13 patients treated by radiosurgery and 62 patients treated by surgery who were retrospectively matched. Patients were matched according to the following criteria: histological characteristics of the primary tumor, extent of systemic disease, preoperative Karnofsky Performance Scale score, time to brain metastasis, number of brain metastases, and patient age and sex. For patients treated by radiosurgery, the median size of the treated lesion was 1.96 cm3 (range 0.41-8.25 cm3) and the median dose was 20 Gy (range 12-22 Gy). The median survival was 7.5 months for patients treated by radiosurgery and 16.4 months for those treated by surgery; this difference was found to be statistically significant using both univariate (p = 0.0018) and multivariate (p = 0.0009) analyses. The difference in survival was due to a higher rate of mortality from brain metastasis in the radiosurgery group than in the surgery group (p < 0.0001) and not due to a difference in the rate of death from systemic disease (p = 0.28). Log-rank analysis showed that the higher mortality rate found in the radiosurgery group was due to a greater progression rate of the radiosurgically treated lesions (p = 0.0001) and not due to the development of new brain metastasis (p = 0.75). On the basis of their data, the authors conclude that surgery is superior to radiosurgery in the treatment of brain metastasis. Patients who undergo surgical treatment survive longer and have a better local control. The data lead the authors to suggest that the indications for radiosurgery should be limited to surgically inaccessible metastatic tumors or patients in poor medical condition. Surgery should remain the treatment of choice whenever possible. PMID- 8622148 TI - Medical management in the endovascular treatment of carotid-cavernous aneurysms. AB - Carotid-cavernous aneurysms account for between 1.9% and 9.0% of intracranial aneurysms. Entirely intercavernous aneurysms are believed to have a relatively benign course, with cranial nerve findings or headache being the usual initial symptomatology; however, subarachnoid hemorrhage or carotid-cavernous fistula formation can result from rupture. Over the past 15 years endovascular parent artery occlusion has essentially replaced surgical carotid occlusion as the treatment of choice. The authors describe a series of 39 consecutive patients at the University of Virginia Health Sciences Center who underwent endovascular treatment of a carotid-cavernous aneurysm. Aggressive invasive hemodynamic monitoring and maintenance of a state of normo- to mild hypervolemia in the asymptomatic patient was used throughout the periprocedural period. Rapid institution of hypervolemic-hypertensive therapy can reverse early neurological deficits related to hypoperfusion in these patients. Only one individual managed with this protocol developed neurological deficits not reversible with hypertensive-hypervolemic therapy. Heparin therapy was administered for 48 hours after occlusion, with patients receiving subsequent aspirin therapy for 6 months to combat distal embolism secondary to thrombosis. Long-term complications were not seen in patients receiving aneurysm trapping; however, two individuals with proximal carotid occlusion developed late optic neuropathy and one had recurrent transient ischemic attacks that ceased with supraclinoidal carotid clipping. PMID- 8622149 TI - Traumatic subarachnoid hemorrhage as a predictable indicator of delayed ischemic symptoms. AB - This report provides findings of an investigation of the influence of traumatic subarachnoid hemorrhage on the development of delayed cerebral ischemia caused by vasospasm. The authors prospectively studied 130 patients with closed-head trauma, who exhibited subarachnoid blood on admission computerized tomography (CT) scans. Ten (7.7%) of these patients developed delayed ischemic symptoms between Days 4 and 16 after the head injury. They consisted of three (3.0%) of 101 patients with small amounts of subarachnoid blood and seven (24.1%) of 29 patients with massive quantities of subarachnoid blood on admission CT scans. In each of the 10 patients, severe vasospasm was demonstrated by angiography performed soon after development of ischemic symptoms. There was a close correlation between the main site of the subarachnoid blood and the location of severe vasospasm. In seven of the patients, follow-up CT scans showed development of focal ischemic areas in the cerebral territories corresponding to the vasospastic arteries. These results demonstrate that traumatic subarachnoid hemorrhage, especially if massive, is a predictable indicator of delayed ischemic symptoms. PMID- 8622150 TI - Traumatic aneurysms and arteriovenous fistulas of intracranial vessels associated with penetrating head injuries occurring during war: principles and pitfalls in diagnosis and management. A survey of 31 cases and review of the literature. AB - In the early days of the war between Iran and Iraq, reports of the sudden deaths of soldiers who previously had survived a penetrating head injury suggested the possibility that a late complication, traumatic aneurysm (TA), could be the cause of this catastrophe. In response, the authors planned a prospective study to perform cerebral angiography in victims with penetrating head traumas, especially in those who had artillery shells or bone fragments passing through areas of dense vasculature. Thirty-one TAs and arteriovenous fistulas were documented. Not all of the lesions, however, were deemed appropriate for surgical intervention. Six aneurysms (19.4%) healed spontaneously and shrank or disappeared on repeated serial angiograms. The authors present their cases and discuss the incidence of TAs, their natural course and behavior, and the special problems encountered in managing these interesting and potentially fatal complications of penetrating head injuries. PMID- 8622151 TI - Alpha-1-antitrypsin phenotypes among patients with intracranial aneurysms. AB - A deficiency of alpha 1-antitrypsin has been implicated in the development of arterial aneurysms, including intracranial aneurysms. The authors determined the prevalence of alpha 1-antitrypsin deficiency of different phenotypes in 100 consecutive patients with intracranial aneurysms and compared the distribution of alpha 1-antitrypsin phenotypes to that in the general population (904 people). The study population consisted of 44 men and 56 women with a mean age of 52 years (range 15-81 years). The heterozygous alpha 1-antitrypsin deficiency states (PiMS and PiMZ) were more common in patients (16%) than in the general population (7%), providing an odds ratio of 2.56 (95% confidence interval (CI) 1.32-4.75; p = 0.005). In addition, one patient (1%) was homozygous for the deficient allele (PiZZ) compared to an expected number of 0.015, providing an odds ratio of 67.0 (95% CI 2.0-363.3; p = 0.015). These findings lead the authors to suggest that the heterozygous and homozygous alpha 1-antitrypsin deficiency states are genetic risk factors for the development of intracranial aneurysms. PMID- 8622152 TI - Temporary vessel occlusion for aneurysm surgery: risk factors for stroke in patients protected by induced hypothermia and hypertension and intravenous mannitol administration. AB - Temporary vessel occlusion is an effective technique used by microvascular surgeons to facilitate dissection and permanent clipping of cerebral aneurysms; however, several questions remain regarding the overall safety of this technique. To identify technical and patient-specific risk factors for perioperative stroke, the authors examined a series of patients in whom induced hypertension and mild hypothermia and intravenous mannitol administration were used as protection during temporary vessel occlusion for aneurysm clipping. The study comprises a nonconcurrent prospective analysis of 132 consecutive aneurysm clippings performed with the aid of temporary vascular occlusion and a specific antiischemic anesthetic protocol at the Massachusetts General Hospital from 1991 to 1993. Factors studied included duration of the temporary clip application, number of occlusive episodes, patient age and neurological status, presence of preoperative subarachnoid hemorrhage (SAH), and intraoperative aneurysm rupture ("forced" temporary clipping), as well as whether proximal vessel occlusion or complete aneurysm trapping was used. In a univariate analysis, patient age, intraoperative aneurysm rupture, temporary clipping lasting more than 20 minutes, clipping between the 4th and 10th day after SAH, and multiple clipping episodes were all significantly associated with stroke outcome. Multivariate logistic regression revealed that intraoperative aneurysm rupture (relative risk 5.6, p = 0.02) and a duration of temporary clip application that lasted more than 20 minutes (relative risk 9.4, p = 0.04) were independently associated with stroke outcome. Overall, 5.2% of the patients had postoperative clinical strokes. Based on their findings the authors conclude that temporary clipping is a safe adjunct to aneurysm surgery, particularly when the duration of clipping is short. PMID- 8622153 TI - Effects of a hydroxyl radical scavenger on delayed ischemic neurological deficits following aneurysmal subarachnoid hemorrhage: results of a multicenter, placebo controlled double-blind trial. AB - A water-soluble, novel synthetic compound, AVS ((+/-)-N, N' propylenedinicotinamide; nicaraven) has no demonstrable vasoactive properties but scavenges hydroxyl radicals in aqueous environmental conditions at neutral pH. Based on the results of preceding experimental and clinical studies showing marked ameliorative effects of AVS on cerebral vasospasm and ischemic brain damage, a multicenter, placebo-controlled double-blind clinical trial was undertaken to verify its beneficial effects on delayed ischemic neurological deficits (DINDs) due to vasospasm and on the overall outcome of patients with subarachnoid hemorrhage (SAH). A total of 162 patients with SAH who had Glasgow Coma Scale scores between 7 and 15 on admission were enrolled in the trial. Drug administration (4 g AVS or 4 g glucose as placebo; infused intravenously for 6-8 hours once a day) was begun within 5 days post-SAH and continued for 10 to 14 days. Intent-to-treat analysis of these patients revealed that the overall incidence of DINDs, which was defined as an exacerbation of impaired consciousness and/or focal neurological deficits, was significantly reduced, by 34.5% (placebo 54.2%, AVS 35.5%; p < 0.05, Mann-Whitney U-test). The Glasgow Outcome Scale (GOS) score at 1 month was significantly improved by AVS (p < 0.05, U-test). At 3 months, the difference in the GOS scores between the groups became marginal on U-tests (p < 0.10), but the percentage of good outcome tended to increase, with a relative increase of 20.3% (AVS 76.3%, placebo 63.4%; p < 0.10, chi-square test), and the cumulative incidence of death was significantly reduced (p < 0.05, log-rank test). No significant adverse reaction attributable to treatment was observed. the usefulness of AVS in therapy for SAH is strongly indicated by the fact that the agent significantly ameliorated DINDs, leading to a marked improvement in the GOS scores at 1 month, as well as a reduction in the cumulative incidence of death by 3 months. PMID- 8622155 TI - Surgical interruption of leptomeningeal drainage as treatment for intracranial dural arteriovenous fistulas without dural sinus drainage. AB - Intracranial dural arteriovenous fistulas (AVFs) have been recognized as acquired lesions that can behave aggressively depending on the pattern of venous drainage. Based on the type of venous drainage, they can be classified as fistulas drained only by venous sinuses, those drained by venous sinuses with retrograde flow in arterialized leptomeningeal veins, and fistulas drained solely by arterialized leptomeningeal veins. Serious symptoms, including hemorrhage and focal deficit, are related to the presence of arterialized leptomeningeal veins. In this paper, the authors report a consecutive series treated between 1988 and 1993 of 20 cases of intracranial dural AVFs with "pure leptomeningeal drainage." All patients underwent surgical interruption of the leptomeningeal draining veins. Based on the arterial supply, nine patients were managed by direct surgery, whereas 11 patients were prepared for surgery by means of preoperative arterial embolization. Radioanatomical cure of the fistula and good neurological recovery were achieved in 18 cases. Complete obliteration of the fistula was documented angiographically in two cases, but fatal hemorrhage occurred, probably due to partial thrombosis of the venous drainage. Based on this experience, the authors believe that surgical interruption of the draining veins is the best treatment option for intracranial dural AVFs. However, surgical results may be affected by the extension of postoperative thrombosis, which in turn may be related to the degree of preoperative venous engorgement. PMID- 8622154 TI - The role of radiosurgery and particulate embolization in the treatment of dural arteriovenous fistulas. AB - Over the past 5 years 29 patients with dural arteriovenous fistulas (AVFs) were treated by the authors using the Leksell radiosurgical gamma knife unit. Within 2 days after radiosurgery, 17 patients with AVFs that exhibited retrograde pial or cortical venous drainage (12 patients) and/or produced intractable bruit (eight patients) underwent particulate embolization of external carotid feeding vessels. The rationale for this treatment strategy was that radiosurgery was expected to cause obliteration of most fistulas after 12 to 36 months. In patients with bruit, ocular symptoms, or in those at risk for hemorrhage, treatment with embolization after radiosurgery kept the fistulas angiographically visible for radiosurgical targeting yet offered palliation of symptoms and temporary, partial protection from hemorrhage during the latency period. In 12 patients, preobliteration embolization immediately reduced (10 patients) or eliminated (two patients) retrograde pial venous drainage. To date, no lesion has hemorrhaged after treatment. Angiography 1 to 3 years posttreatment in 18 patients showed total obliteration of 13 fistulas (72%) and partial obliteration of five (28%). Radiosurgery, followed by embolization when retrograde pial venous drainage, intractable bruit, and/or major external carotid artery supply is present, appears to be a promising treatment for selected patients with symptomatic dural AVFs. PMID- 8622156 TI - Long-term outcome after operation for trigeminal neuralgia in patients with posterior fossa tumors. AB - During a 20-year period, 26 patients with typical symptoms of trigeminal neuralgia were found to have posterior fossa tumors at operation. These cases included 14 meningiomas, eight acoustic neurinomas, two epidermoid tumors, one angiolipoma, and one ependymoma. The median patient age was 60 years and 69% of the patients were women. Sixty-five percent of the symptoms were left sided. The median preoperative duration of symptoms was 5 years. The distribution of pain among the three divisions of the trigeminal nerve was similar to that found in patients with trigeminal neuralgia who did not have tumors; however, more divisions tended to be involved in the tumor patients. The mean postoperative follow-up period was 9 years. At operation, the root entry zone of the trigeminal nerve was examined for vascular cross-compression in 21 patients. Vessels compressing the nerve at the root entry zone were observed in all patients examined. Postoperative pain relief was frequent and long lasting. Using Kaplan Meier methods the authors estimated excellent relief in 81% of the patients 10 years postoperatively, with partial relief in an additional 4%. PMID- 8622157 TI - Prevention of cerebrovasospasm following subarachnoid hemorrhage in rabbits by the platelet-activating factor antagonist, E5880. AB - Recently, an important role of platelet-activating factor (PAF), an inflammation mediator, has been demonstrated in the genesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). In the current study, the authors examined whether intravenous administration of the novel PAF antagonist, E5880, can prevent vasospasm following SAH in rabbits. A vasospasm model was produced in three groups of rabbits using two subarachnoid injections of autologous arterial blood, followed by intravenous administration of distilled water (control), a low dose of E5880 (0.1 mg/kg in distilled water), or a high dose of E5880 (0.5 mg/kg in distilled water). Neurological deterioration was largely prevented in the rabbits that received E5880. Basilar artery constriction was also reduced by both doses of E5880. Histological examination at autopsy predominantly showed ischemic changes in the brain. Animals in each E5880-treated group exhibited ischemic changes less frequently than those in the control group. Plasma thromboxane B2 concentrations were reduced in rabbits treated with E5880. Platelet-activating factor was immunolocalized in the intima and media of the basilar artery in the control group. The PAF immunoreactivity demonstrated in the basilar artery was decreased in the E5880 groups in a dose-dependent manner. Thus, this study provides evidence that PAF may play a role in the pathogenesis of vasospasm after SAH and that intravenous administration of E5880 is a promising approach in preventing vasospasm. PMID- 8622158 TI - Modulation effects of hexamethylene bisacetamide on growth and differentiation of cultured human malignant glioma cells. AB - The modulation effects of hexamethylene bisacetamide (HMBA), a differentiation inducing agent, on growth and differentiation of cells from human malignant glioma cell line SHG-44 were studied. At cytostatic doses (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 15 days), HMBA exerted a marked inhibitory effect on cell proliferation. Exposure to HMBA (5 mM and 10 mM for 12 days) also resulted in an accumulation of cells in G0/G1 phase and a decrease of cells in S phase as analyzed by flow cytometry. The reversible effects of 7.5 mM HMBA and 10 mM HMBA on cell proliferation and 10 mM HMBA on disruption of cell cycle distribution were observed when HMBA was removed from culture media on Day 6 and replaced with HMBA-free media. Colony-forming efficiency (CFE) in soft agar was remarkably decreased by HMBA (2.5 mM, 5 mM, 7.5 mM, and 10 mM for 14 days), and in 7.5 mM HMBA- and 10 nM HMBA treated cells, the CFEs were reduced to 25% and 12.5%, respectively, of that in untreated cells. Cells treated with HMBA (5 mM and 10 mM for 15 days) remained tumorigenic in athymic nude mice, but the growth rates of the xenografts were much slower than those in the control group. The effects of HMBA on cell proliferation, cell cycle distribution, CFE, and growth of xenografts were dose dependent. A more mature phenotype was confirmed by the morphological changes from spindle shape to large polygonal stellate shape and remarkably elevated expression of glial fibrillary acidic protein in cells exposed to HMBA (5 mM, 10 mM for 15 days). Our results showed that a more differentiated phenotype with marked growth arrest was induced in SHG-44 cells by HMBA. PMID- 8622160 TI - Molecular genetic investigation of the neurofibromatosis type 2 tumor suppressor gene in sporadic meningioma. AB - The authors investigated the role of somatic mutations of the neurofibromatosis type 2 (NF2) gene in sporadic meningioma. Neurofibromatosis 2 is a dominantly inherited familial tumor syndrome predisposing affected patients to a variety of central nervous system tumors including vestibular schwannoma and meningioma. Neurofibromatosis type 2 is caused by germline mutations in the NF2 tumor suppressor gene. In addition, the authors and others have reported that somatic NF2 gene mutations occur frequently in nonfamilial vestibular schwannoma. In this study, molecular genetic analysis was performed on 23 nonfamilial meningiomas. Paired DNA samples extracted from the blood and tumors of the patients were analyzed for loss of heterozygosity (LOH) in the region of the NF2 gene on chromosome 22 using closely linked DNA markers. The NF2 gene mutations were sought by single-stranded conformation polymorphism analysis and DNA sequencing. Fourteen (61%) of 23 meningiomas showed LOH in the region of the NF2 gene on chromosome 22. Somatic NF2 gene mutations were detected in eight meningiomas (35%) after screening all 17 exons. All tumors with NF2 gene mutations showed simultaneous chromosome 22 LOH. Review of the histopathological findings of the cases studied did not demonstrate any predominance of genetic abnormalities in a particular histological type of meningioma. These results are compatible with the hypothesis that the NF2 gene acts as a tumor suppressor and that its inactivation is important in the pathogenesis of sporadic meningioma. PMID- 8622159 TI - Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy. AB - The expression of connexin43, the primary gap-junction constituent of glial cells, was evaluated at the messenger RNA and protein levels in different grades of astrocytoma to investigate the relevance of gap junctions in herpes simplex virus-thymidine kinase (HSV-tk)-mediated gene therapy of brain tumors. Transduction of the retroviral-mediated HSV-tk gene into tumor cells with subsequent administration of ganciclovir has recently been used as an experimental therapeutic strategy for treatment of brain tumors. One aspect of this approach is the bystander effect, which augments the efficacy of this therapeutic approach. Glioblastoma cells with minimum levels of connexin43 protein were transfected with a connexin43 complementary DNA. These cells manifested a marked increase in the in vitro bystander effect, supporting the contention that the in vitro bystander effect is a consequence of metabolic cooperation between cells mediated by gap junctions. To assess relative levels of gap-junction protein expression in the relevant tumor type, we examined primary astrocytomas, primary astrocytoma cell cultures, and glioblastoma cell lines. Although most astrocytoma tumor samples expressed connexin43, they differed in the level of expression, with the greatest variation exhibited in high-grade astrocytomas. Primary glioblastoma cell cultures and established glioblastoma cell lines also displayed some variability in connexin43 levels. In aggregate, our results anticipate that glioblastomas will have a varied bystander effect during HSV-tk gene therapy depending on the level of connexin43 expression. PMID- 8622162 TI - Dose escalation safety and tolerance study of the N-methyl-D-aspartate antagonist dextromethorphan in neurosurgery patients. AB - Experimental studies have shown that dextromethorphan, a noncompetitive N-methyl D-aspartate antagonist is neuroprotective in experimental models of ischemic cerebral injury. The authors studied the safety and tolerability of oral dextromethorphan (DM) in humans, and correlated serum levels of this drug with cerebrospinal fluid (CSF) and brain levels. Neurosurgical patients undergoing intracranial surgery or endovascular procedures were given ascending doses of oral DM prior to and 24 hours after surgery. Serum, CSF, and brain levels of DM and its active metabolite, dextrorphan, were measured. One hundred eighty-one patients received a total of 212 courses of DM treatment in dose ranges of 0.8 to 9.64 mg/kg. Serum DM levels correlated highly with CSF and brain DM levels. Brain levels were 68-fold higher than serum levels, whereas CSF levels were fourfold lower than serum levels. The maximum DM levels attained were 1514 ng/ml (serum) 118 ng/ml (CSF), and 92,700 ng/g (brain). The maximum dextrorphan levels were 501 ng/ml (serum), 167 ng/ml (CSF), and 6840 ng/g (brain). In 11 patients, brain and plasma levels of DM were comparable to levels that have been shown to be neuroprotective in animal studies. Frequent side effects occurring at neuroprotective levels of DM included nystagmus (64%), nausea and vomiting (27%) distorted vision (27%), feeling "drunk" (27%), ataxia (27%), and dizziness (27%). All symptoms were reversible and no patient suffered severe adverse reactions. This study demonstrates that potentially neuroprotective doses of DM can be administered safely to neurosurgical patients. Brain and CSF levels of DM can be estimated from serum levels of the drug. Side effects, even at the highest levels, proved to be tolerable and reversible. Administration of DM to patients at risk for cerebral injury should be further explored. PMID- 8622161 TI - Autocrine growth stimulation of human meningioma cells by platelet-derived growth factor. AB - The authors have previously shown that meningioma-derived conditioned medium profoundly stimulates the in vitro proliferation of meningioma cells. In this paper, self mitogenic agents found in the conditioned medium-autocrine growth stimulatory factors actually secreted by human meningioma cells-are characterized as proteins related to the B chain of platelet-derived growth factor (PDGF) and possibly to the A chain of PDGF as well. The addition to conditioned medium of a neutralizing antibody against PDGF-BB caused a significant inhibition of the conditioned medium-stimulated DNA synthesis in all three meningioma cultures studied. A similar neutralizing effect was observed with an anti-PDGF-AA antibody in one meningioma culture studied. Gel filtration chromatography of concentrated conditioned medium from two different meningiomas using a Sephadex G-100 column revealed similar profiles from both conditioned media with a major peak of mitogenic activity against meningioma cells at a molecular weight (M(r)) of approximately 32 to 36 kD, accompanied by a minor peak at approximately 22 kD. The major peak mitogenic activity was significantly reduced by addition of an anti-PDGF-BB antibody. Western blot analysis of protein extracts from five meningioma specimens was performed using a monoclonal antibody against the B chain of PGDF, and a major band of PDGF-B immunoreactivity was detected at an M(r) of approximately 19 kD in all five meningiomas under both reducing and nonreducing conditions. Exogenous human and porcine PDGFs both exhibited a significant dose-dependent stimulation of DNA synthesis in two of three and three of five meningioma cultures examined, respectively. Although not all meningiomas investigated proved to share the biological activity associated with PDGF and these results may be preliminary, it seems that the autocrine growth-stimulatory loop established by PDGF-B-related molecules plays an important functional role in meningioma cell proliferation. PMID- 8622163 TI - Acute presentation of a neurogenic sarcoma in a patient with neurofibromatosis type 1: a pathological and molecular explanation. Case report. AB - Neurofibromatosis type 1 (NF1) is the most common familial cancer-predisposing syndrome in humans, for which the gene (NF1) and its gene product (neurofibromin) have been identified. The majority of tumors occurring in patients with NF1 are benign neurofibromas, sarcomatous transformation is uncommon and most often occurs within the larger plexiform neurofibromas. Such malignant transformation in a known neurofibroma is often heralded by either radiological evidence of growth or a progression in clinical symptoms (pain and neurological deficit). This progression in symptoms is usually gradual in onset, typically occurring over a period of months. In this report the authors document a neurogenic sarcoma presenting with rapid clinical and radiological growth. The pathological basis of this acute presentation was increased cellular proliferation, with invasion of blood vessels resulting in tumor infarction. The molecular basis of neurofibroma development in NF1 is loss of expression of the NF1 gene and its gene product, neurofibromin, resulting in elevated levels of Ras-guanosine triphosphate. Subsequent molecular events result in sarcomatous transformation. PMID- 8622164 TI - Chronic encapsulated expanding hematoma in association with gamma knife stereotactic radiosurgery for a cerebral arteriovenous malformation. Case report. AB - The authors report a rare case of a patient with a chronic encapsulated expanding hematoma and progressive neurological deterioration who presented 2 years after gamma knife radiosurgery for a cerebral arteriovenous malformation (AVM). A tough capsule containing multiple layers of organized hematoma resulting from previous bleeding was confirmed surgically. Histological examination revealed that the capsule consisted of a dense collagenous outer layer and a granulomatous newly vascularized inner layer with marked fibrosis. Hemosiderin deposits were frequently observed in the inner layer, which suggested recurrent minor bleeding from fragile vessels in this layer. An AVM was found in the hematoma, which had degenerated as the result of radiosurgery. A cross-section of the abnormal vessels showed various stages of obliteration due to intimal hypertrophy. The clinical course, radiological features, and histological findings in this case were compatible with those of previously reported chronic encapsulated hematomas. A possible mechanism of hematoma formation and its expansion are discussed. PMID- 8622165 TI - Recurrence of a cerebral arteriovenous malformation after surgical excision. Case report. AB - Complete excision of a cerebral arteriovenous malformation (AVM) should eliminate the future risk of an associated intracranial hemorrhage. Because total removal of an AVM may be difficult to assess at the time of surgery, postoperative angiography has become the accepted standard for documenting that the removal has been accomplished. However, even angiography confirmed excision of an AVM does not completely ensure against rebleeding. Regrowth of an AVM with subsequent hemorrhage can occur. This has been documented in children and is attributed to forces acting on the immature vasculature of these younger patients. The authors report the case of an older patient whose AVM recurred when he was 28 years of age, despite an angiography proven complete excision, and emphasize that, even in adults, angiography documentation of total removal does not always eliminate the risk of reformation of an AVM. PMID- 8622166 TI - Vertebral artery stenting following percutaneous transluminal angioplasty. Technical note. AB - The authors report initial results and follow up using stent placement to treat atherosclerotic stenosis in vertebral arteries. Three patients with severe atherosclerotic vascular disease underwent vertebral artery stent placement using a balloon expandable stent. Medical therapy (aspirin and warfarin) and conventional percutaneous angioplasty failed to resolve the disease and the patients developed symptomatic restenosis within 3 months of angioplasty. Two patients had symptoms of anterior circulation ischemia with carotid artery occlusions and reduced supply to the anterior circulation from the stenosed vertebral arteries. One patient had recurrent posterior circulation symptoms. Stents were successfully placed in all three, resulting in immediate reversal of stenosis and resolution of symptoms. Clinical follow-up study (mean 9 months) has shown no recurrent symptoms in the patient with posterior circulation symptoms, but the two patients with anterior circulation ischemia did develop recurrent symptoms. Angiographic follow up in these two patients at 3 months and 1 year, however, demonstrated continued patency of vertebral artery lumina. They underwent extracranial-intracranial bypass surgery to relieve their symptoms. This experience suggests stents can be placed without complication in the proximal vertebral arteries and may have an adjunctive role in the treatment of atherosclerotic cerebrovascular disease following unsuccessful angioplasty. PMID- 8622167 TI - Exposure versus Instability. PMID- 8622168 TI - Dermatomes: pain and temperature. PMID- 8622169 TI - Dermatomes: pain and temperature. PMID- 8622170 TI - Stereotactic or stereotaxic: time to resolve the age-old controversy. PMID- 8622171 TI - Cavernous malformations. PMID- 8622172 TI - Cavernous malformations. PMID- 8622173 TI - Lessons from a case of kleeblattschadel: addendum. PMID- 8622174 TI - Radiosurgery for arteriovenous malformations. PMID- 8622175 TI - Glioblastoma of the optic chiasm. PMID- 8622176 TI - Coherent imaging of the cone mosaic in the living human eye. AB - A new system for the recording of high-resolution images of the cone mosaic in the living room fovea has been developed. The experimental method is inspired by stellar speckle interferometry, used in astronomy to resolve binary stars. Series of short-exposure images of small areas of the fovea are registered under coherent illumination. These images show speckle patterns that have some correlation with the topography of the cone mosaic and retain high-resolution information. Such correlation is better revealed in the power spectrum (square modulus of the Fourier transform). The signal-to-noise ratio is increased, without loss of high frequencies, by averaging the power spectra of a number of such speckle patterns. The average power spectra show, in most of the cases, an elliptical ring (or hexagon), whose mean radius corresponds to the characteristic spatial frequency of the cone mosaic (or the inverse of the mean row-to-row cone spacing) at a given retinal location. Good results are obtained in the five normal observers tested, at various retinal eccentricities, up to 1 visual degree, including the center of the fovea for two eyes. We find a decrease in the spatial frequency of the mosaic with the eccentricity and an important intersubject variability, in agreement with anatomical studies. PMID- 8622177 TI - Efficient Monte Carlo simulation of confocal microscopy in biological tissue. AB - A variance-reduction technique is described that greatly improves the efficacy of Monte Carlo simulations of reflection-mode confocal microscopy in anisotropically scattering media. The efficiency gain is large enough that the performance of confocal microscopes probing as deep as 5 scattering lengths can be simulated with a desktop computer. We use the technique to simulate the response of a true confocal microscope probing biological tissue, a problem that has been impractical to undertake by using conventional Monte Carlo methods. Our most important finding is that operation of a confocal microscope in the true confocal mode enables much more effective rejection of undesired scattered light than operation in the partially coherent mode, but the maximum probing depths of microscopes operated in either mode are similar (2-3) scattering lengths) in practice because of sensitivity limitations. PMID- 8622178 TI - Meso-substituted cationic porphyrins as efficient photosensitizers of gram positive and gram-negative bacteria. AB - Previous studies on the photosensitization of bacterial cells with different neutral or negatively charged porphyrins and phthalocyanines have demonstrated that, although Gram-positive bacteria are efficiently photoinactivated, Gram negative bacteria become photosensitive only after modification of the permeability of their outer membrane. The results described in this paper show that two meso-substituted cationic porphyrins, namely tetra(4N-methyl pyridyl)porphine tetraiodide and tetra(4N,N,N-trimethyl-anilinium)porphine, efficiently photosensitize the inactivation of Gram-negative bacteria, such as Vibrio anguillarum and Escherichia coli. A negatively charged meso-substituted porphyrin, tetra(4-sulphonatophenyl)porphine, has no appreciable photosensitizing activity towards Gram-negative bacteria, although all three porphyrins exhibit a similar subcellular distribution pattern, being mainly localized in the protoplasts or spheroplasts. Moreover, the three porphyrins show similar efficiency in the photoinactivation of the Gram-positive bacterium Entorecoccus seriolicida. PMID- 8622179 TI - Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both gram-negative and gram-positive bacteria. AB - The photosensitization of microorganisms is potentially useful for sterilization and for the treatment of certain bacterial diseases. Until now, any broad spectrum approach has been inhibited because, although Gram-positive bacteria can be photoinactivated by a range of photosensitizers, Gram-negative bacteria have not usually been susceptible to photosensitized destruction. In the present work, it has been shown that the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Enterococcus seriolicida, can be photoinactivated when illuminated in the presence of a cationic water-soluble zinc pyridinium phthalocyanine (PPC). The degree of photoinactivation is dependent on both the concentration of PPC and the illumination time. In contrast, the three bacteria are not photoinactivated by illumination in the presence of a neutral tetra-diethanolamine phthalocyanine (TDEPC) or negatively charged tetra-sulphonated phthalocyanine (TSPC). Uptake studies have revealed that the lack of activity of TSPC is due to the fact that it has very little affinity for any of the organisms. However, the issue appears to be more complex than simply the gross levels of cellular uptake, since TDEPC and PPC are both taken up by the organisms but only PPC shows activity. This indicates that the localization and subcellular distribution of the phthalocyanines may be a crucial factor in determining their cell killing potential. Further analysis of the uptake data has revealed a cell-bound photosensitizer fraction, which remains tightly associated after several washings, and another weakly bound fraction, which is removed by successive washings. Analysis of the cell killing curves, carried out after successive washings of E. coli exposed to PPC, has revealed that it is the tightly associated fraction that is involved in the photosensitization. Taken together with other data, these results suggest that cationic photosensitizers may have a broader application in the photoinactivation of bacterial cells than the anionic or neutral photosensitizers commonly used in photodynamic therapy. PMID- 8622180 TI - The UVR wavelength dependence for lomefloxacin photosensitization of human skin. AB - Lomefloxacin is a new fluoroquinolone with effective broad-spectrum antimicrobial activity. However, in common with other structurally related drugs, skin photosensitization reactions have been reported. The wavelength dependence for such photosensitization has been investigated on the previously unexposed buttock skin of 12 normal healthy human volunteers of skin types I and II. Using geometric square root of 2 dose increments, baseline 24 h minimal erythema doses were assessed at 300, 320, 330, 340, 350 and 360 nm, and with broad-band UVA. In addition, dose-response curves were constructed for erythema as measured by a reflectance device. Subjects received single daily oral doses of 400 mg lomefloxacin at specified times for 4 days. At 2 h after the final dose, new areas of buttock skin were irradiated to assess changes in minimal erythema dose and erythema dose-response. Convolution of the erythema action spectra obtained pre- and on-drug with a terrestrial solar spectrum showed that, although the UVA sensitivity on-drug was enhanced, most of the erythemally effective solar energy was still in the UVB region. An action spectrum derived for lomefloxacin skin photosensitization showed peak activity at 320 nm, the same spectral region as that for maximal absorption of the drug. There was no evidence of skin photosensitization at 300 nm. PMID- 8622181 TI - Tris(2,2'-bipyridyl)ruthenium(II)-mediated photoinduced electron transfer of engineered cytochrome P450 1A2. AB - In this study, it is shown that an electron from photoreduced tris(2,2' bipyridyl)Ru2+ ion reaches the haem iron of engineered wild-type cytochrome P450 1A2 (P450 1A2) with an electron transfer rate of 6.04 x 10(-3) min(-1). The electron transfer rate, 4.05 x 10(-2) min(-1), of a His163Glu mutant, which has a redox potential 40 mV lower than that of the wild type, is more than sixfold faster than that of the wild type. The photoinduced electron transfer rates of the present system are strongly influenced by detergents, cholic acid and Emulgen 913. We discuss the intermolecular and intramolecular electron transfer mechanism of the P450 1A2 system based on the kinetic data. PMID- 8622183 TI - Functional morphology and homology in the odontocete nasal complex: implications for sound generation. AB - The site and physiologic mechanism(s) responsible for the generation of odontocete biosonar signals have eluded investigators for decades. To address these issues we subjected postmortem toothed whale heads to interrogation using medical imaging techniques. Most of the 40 specimens (from 19 species) were examined using x-ray computed tomography (CT) and/or magnetic resonance imaging (MR). Interpretation of scan images was aided by subsequent dissection of the specimens or, in one case, by cryosectioning. In all specimens we described a similar tissue complex and identified it as the hypothetical biosonar signal generator. This complex includes a small pair of fatty bursae embedded in a pair of connective tissue lips, a cartilaginous blade, a stout ligament, and an array of soft tissue air sacs. Comparing and contrasting the morphologic patterns of nasal structures across species representing every extant odontocete superfamily reveals probable homologous relationships, which suggests that all toothed whales may be making their biosonar signals by a similar mechanism. PMID- 8622182 TI - Biological dosimetry of solar radiation for different simulated ozone column thicknesses. AB - During the Spacelab mission D-2, in the experiment RD-UVRAD, precalibrated biofilms consisting of dry monolayers of immobilised spores of Bacillus subtilis (strain Marburg) were exposed, for defined intervals, to extraterrestrial solar radiation filtered through an optical filtering system, to simulate different ozone column thicknesses. After the mission, the biofilms were processed and optical densities indicative of any biological activity were determined for each exposure condition by image analysis. For the different simulated ozone column thicknesses, biologically effective irradiances were experimentally determined from the biofilm data and compared with calculated data using a radiative transfer model and the known biofilm action spectrum. The data show a strong increase in biologically effective solar UV irradiance with decreasing (simulated) ozone concentrations. The full spectrum of extraterrestrial solar radiation leads to an increment of the biologically effective irradiance by nearly three orders of magnitude compared with the solar spectrum at the surface of the Earth for average total ozone columns. PMID- 8622184 TI - Functional morphology of the glomerular filtration barrier of Gallus gallus. AB - The anionic charge barrier and the endothelial and epithelial pore sizes on the glomerular filtration barrier (GFB) were examined in white leghorn chickens (Gallus gallus). Ruthenium red was used to stain anionic charge sites on the GFB. The tissue was treated by normal dehydration and freeze substitution dehydration for transmission electron microscopy (TEM). In addition, the basal lamina was isolated for study. The results of our study indicate that G. gallus possess a thick, negatively charged glycocalyx surrounding the podocytes and slit diaphragm and on the endothelium. However, in all cases, little anionic charge is present in the basal lamina. The pores on the endothelium are elliptical and have mean dimensions of 148 x 110 nm. This is in contrast to mammals, which have smaller, round pores. The epithelial pores in G. gallus measure approximately 35 nm in length, approximately 4 times larger than those found in mammals. These results indicate that the avian glomerulus may allow the filtration of larger molecules from the plasma than occurs in mammals and that the charge on the molecule may not be as restrictive a filtration characteristic as in mammals. PMID- 8622186 TI - Transvaginal sonographic orientation detection system using ceramic gyroscopes. AB - Use of three-dimensional ultrasonograms in daily clinical medicine requires the development of data accumulation techniques and image reconstruction techniques . We describe an image position and orientation detection technique using small piezoelectric vibratory gyroscopes. Such gyroscopes, of the same type that is used in an automobile navigation system, produce angular velocity by Coriolis force. After attaching three gyroscopes to an ordinary transvaginal probe, we attempted to detect image orientation to assist transvaginal scanning and to reconstruct three-dimensional projection images. Three-dimensional images were reconstructed by simple white and black inversion thresholding technique. Using free hand transvaginal scanning, the direction of the transvaginal ultrasound beam was displayed with B-mode ultrasonography. Using a personal computer, three dimensional images of follicles and fetuses were successfully reconstructed within a few minutes. Our system provides easy understanding of transvaginal sonographic images and potentially might allow ordinary ultrasound machines to handle three-dimensional images in a day-to-day clinical practice. PMID- 8622185 TI - Extracellular matrix and transmembrane linkages at the termination of intrafusal fibers and the outer capsule in chicken muscle spindles. AB - Attachments of intrafusal fibers and of the outer spindle capsule at the far polar region were examined by immunohistochemistry in serially sectioned chicken leg muscles. Patterns of distribution of connective tissues and intracellular filaments suggest that, in this segment of the muscle spindle, intrafusal fibers bind laterally with the capsule. Contrary to extrafusal fibers at myotendinous junctions, folded plasmalemmas at the ends of intrafusal fibers were rare. Thus, there was little end-to-end interlocking between intrafusal fibers and the extracellular matrix. The tapered contours of terminating intrafusal fibers resembled those of extrafusal fibers which end in fascicles without tendinous connections. At points where the distal portions of intrafusal fibers closely adjoined and overlapped extrafusal fibers, alpha-actinin, vinculin, filamin, talin, beta 1 integrin, spectrin, and dystrophin occurred with moderate to great frequency. It is generally accepted that these compounds are links in molecular chains that extend from the intracellular space across cell membranes to the extracellular matrix. Their location along substantial lengths of extrafusal fibers, distal capsule, and terminating intrafusal fibers suggests the presence of numerous transverse connections between elements of the terminal portion of the spindle and nonspindle tissues. Hence, it is likely that forces monitored by chicken spindles in muscles undergoing length changes are transferred from extrafusal fibers and extracellular matrix to the receptors in large part via lateral shear instead of by longitudinal tension. PMID- 8622187 TI - Assessment of postnatal gestational age using sonographic measurements of femur length. AB - Assessment of gestational age in the premature newborn infant is both essential and difficult. The purpose of this study is to evaluate a new method for the assessment of gestational age in premature neonates using femur length measurements obtained by ultrasonography and compare these with an established clinical method. Forty-seven newborn infants with true gestational age calculated by reliable last menstrual period, further confirmed by obstetric ultrasonographic estimation performed at <18 weeks, were enrolled within 3 days of birth. Birth weight, length, and head circumference were appropriate for the true gestational age. The modified Ballard maturational scoring system was used to determine gestational age. A trained sonographer imaged the femur and measured the shaft of the femur using electronic calipers. Gestational age was estimated from an average of six femur length measurements by a radiologist blinded to the study using tables of fetal femur length measurements. Results indicate that gestational age assessment by ultrasonography has an excellent correlation with true gestational age (r = 0.93), as does assessment by the Ballard score (r = 0.87), with no statistical difference between both the correlations' coefficients. We conclude that gestational age assessment in newborn infants weighing <1500 g, without intrauterine growth retardation, by sonographic measurement of femur length is an excellent means to estimate true gestational age. Furthermore, the sonographic method is ideal for sick, paralyzed neonates and may be useful in the design of future clinical trials. PMID- 8622188 TI - Duplex Doppler sonography of changes in portal vein flow in healthy term newborn infants after feeding. AB - To establish reference values for changes in portal venous diameter, angle- corrected maximal flow velocity, and flow in healthy term newborn infants after formula feeding, we studied 20 subjects using duplex Doppler sonography. After feeding, portal venous diameter increased from 3.6 +/- 0.1 (mean +/- standard error of the mean) to 3.9 +/- 0.1 mm at 15 min and decreased to 3.8 +/- 0.1 at 60 min. Maximal flow velocity increased from 24.1 +/- 1.3 cm/s to a maximum of 35.9 +/- 2.4 cm/s at 15 min and decreased to 28.8 +/- 1.5 cm/s at 60 min. Flow increased from 85.0 +/- 7.5 ml/min to a maximum of 153.6 +/- 14.9 ml/min at 15 min with decrease similar to the maximal flow velocity curve. We conclude that formula feeding produces peak portal blood flows of nearly twice the fasting values at 15 min after feeding and returns almost to fasting value by 60 min. PMID- 8622189 TI - Color amplitude imaging: preliminary results using vascular sonographic contrast agents. AB - Conventional (mean Doppler frequency shift) color Doppler imaging and a new method of displaying blood flow in color that uses the amplitude of the Doppler signal were utilized to evaluate three vascular sonographic contrast agents. To compare the two color flow detection modalities, a total of 20 pairs of contrast agent injections were performed while imaging a variety of abdominal organs and tumors in experimental animal models. The 20 paired injections were scored independently to indicate whether color amplitude imaging better demonstrated the effects of contrast enhancement (n = 14), both techniques were equivalent in demonstrating the effects of contrast enhancement (n = 4), or color Doppler imaging better demonstrated the effects of contrast enhancement (n = 2). These results indicate that compared to color Doppler imaging, color amplitude imaging improved visualization of both normal and abnormal blood flow in 70% of these cases (P < 0.0001). Specifically, with contrast enhancement of the Doppler signals, organ vascularity and regional differences in parenchymal blood flow were better demonstrated with color amplitude imaging than with color Doppler imaging. In addition, since color amplitude imaging is nondirectional and less angle dependent than color Doppler imaging, it was possible to visualize vessel continuity more completely and to demonstrate vessel branching more clearly with this modality. However, owing to the lack of directivity and poor temporal resolution, information obtained with color amplitude imaging appears to be complementary to that of color Doppler imaging. In conclusion, color amplitude imaging is a reliable method of determining the effectiveness of vascular sonographic contrast agents and should be considered one of the primary flow imaging modalities used for the assessment of these agents. PMID- 8622190 TI - Color Doppler sonographic assessment of placental circulation in the first trimester of normal pregnancy. AB - Our aim was to study placental circulation during the first trimester of normal pregnancy. For this purpose, 108 single pregnancies from 4 to 15 gestational weeks were evaluated through conventional Doppler ultrasonography. The flow velocity waveforms from the retrochorionic arteries (spiral-radial arteries) and the umbilical artery were assessed using the peak systolic velocity, resistive index, and pulsatility index). Intervillous flow velocity waveform was evaluated from the maximum velocity. The earliest color signal from the retrochorionic circulation was registered at 4.5 weeks along with gestational sac visualization. The venous Doppler signal from the intervillous space and the Doppler signal from the umbilical artery were recorded with an embryo visible from the end of week 5 onward. The retrochorionic, intervillous, and umbilical peak systolic velocities increase, whereas the resistive and pulsatility indices decrease progressively during early pregnancy with a significant correlation with gestational age. Similarly, intervillous maximum velocity gradually increases throughout the first trimester of pregnancy. Despite some methodologic problems related to Doppler technology and the vessels studied color Doppler sonography appears to be an adequate tool to assess the physiologic changes in the placental circulation during early pregnancy. PMID- 8622191 TI - Preoperative imaging of lower extremity varicose veins: color coded duplex sonography or venography. AB - We prospectively examined 137 limbs in 112 consecutive patients with clinical evidence of severe varicosis by color coded duplex sonography and ascending venography (including varicography in 48 limbs) to evaluate the diagnostic capabilities of color coded duplex sonography in the assessment of venous anatomy, variant varicosis, postthrombotic changes, and incompetence of the superficial and perforating venous system. Additionally, descending venography was performed in the first 52 limbs and compared to color coded duplex sonography in the diagnosis of deep and superficial venous reflux. Variant venous anatomy (21 cases) was missed in two limbs and misinterpreted in one limb by ascending venography compared to surgery. Color coded duplex sonography was inconclusive in two cases. Variant varicosis (59 cases) was missed in seven surgically proved cases by venography and in one case by color coded duplex sonography. Color coded duplex sonography was inconclusive in five cases. Ascending venography was slightly superior to color coded duplex sonography in the detection of postphlebitic changes. Good agreement was found between color coded duplex sonography and descending venography in the grading of superficial (k = 0.75) and deep venous reflux (k = 0.79). Excellent agreement was found between ascending venography in the grading of long (k = 0.96) and short (k = 0.94) saphenous vein reflux. More incompetent perforating veins were detected by ascending venography, (and varicography) than by color coded duplex sonography, but the latter technique allows direct preoperative marking of the skin, which is beneficial for the surgeon. We conclude that color coded duplex sonography is a valuable imaging tool before venous stripping and is capable of replacing invasive ascending and descending venography. Only patients with inconclusive color coded duplex sonographic results (e.g., complex variant venous anatomy) should proceed to venography. PMID- 8622192 TI - Percutaneous intravascular ethanol injection of the supplying tumor vessel in the treatment of hepatocellular carcinoma larger than 3 cm. AB - Percutaneous intravascular ethanol injection of the supplying vessel of the tumor using color Doppler imaging was performed in three patients (three lesions) with hepatocellular carcinoma measuring more than 3 cm. Intravascular injection was achieved in six out ot seven attempts. The total amount of ethanol injected intravascularly was small (10, 15,0 and 26 ml for each patient). One attempt failed and the ethanol was injected perivascularly. No serious complication was noted. After treatment, two patients showed remarkable decrease in tumor size and one showed massive tumor necrosis. In conclusion, ethanol injection using the intravascular approach may be of value in the palliative management of large hepatocellular carcinomas. PMID- 8622193 TI - Intratesticular vasculitis simulating a testicular neoplasm. PMID- 8622194 TI - Power Doppler sonography. PMID- 8622195 TI - Sonography of a solidified benign breast cyst. PMID- 8622196 TI - Anomalous origin of left coronary artery from right coronary artery (single coronary artery): diagnosis by transesophageal echocardiography. PMID- 8622197 TI - Intrascrotal paratesticular accessory spleen. PMID- 8622198 TI - Prenatal diagnosis of a grade IV sacrococcygeal teratoma. PMID- 8622199 TI - Paradoxical dilatation of the gallbladder after fat ingestion in patients with acute hepatitis. PMID- 8622200 TI - Power Doppler sonographic evaluation of acute pyelonephritis in children. AB - Twelve children admitted to the hospital with a clinical diagnosis of acute pyelonephritis were studied with power Doppler sonography and either renal cortical scintigraphy or contrast-enhanced CT. Imaging studies were normal in four patients. A single focus of pyelonephritis was seen by cortical scintigraphy in five children, with a matching defect observed on power Doppler sonography in four patients and a small focus of infection not detected on power Doppler sonography in one child. Multifocal pyelonephritis was identified correctly by power Doppler sonography in two children, although this imaging modality underestimated the extent of involvement in one of the children. One child with diffuse pyelonephritis had a normal power Doppler sonogram. In this study, power Doppler sonography had an overall sensitivity of 75% and specificity of 100% in the evaluation of children with acute pyelonephritis. PMID- 8622202 TI - Will altruism endanger prevention? PMID- 8622201 TI - Interobserver and inter-equipment variability of echo-Doppler sonographic evaluation of the superior mesenteric artery. AB - The aim of this study was to assess the interobserver and inter-equipment variabilities of echo-Doppler sonographic measurement of superior mesenteric artery flow parameters. In the first part of the study the echo-Doppler sonographic parameters (maximum systolic velocity, minimum diastolic velocity, mean velocity, restrictive index, and pulsatility index) were measured independently in nine patients by four skilled operators using four different equipment models. In the second part of the study measurements were taken by the four operators in seven different patients, after one day of cooperative training; the purpose of this part was to define a strict and uniform protocol of Doppler sonographic examination. Significantly different values of maximum systolic velocity, minimum diastolic velocity, mean velocity, and pulsatility index were obtained by the different operators, whereas the inter-equipment variability was significant only for maximum systolic velocity, mean velocity, and pulsatility index. The analysis of the components of variance showed that a large part of this variance was nonsystematic. After training and definition of the protocol no significant differences were found among the operators for any of the parameters, and the 95% confidence limits and coefficients of variation showed a decrease as for maximum systolic velocity, mean velocity, and pulsatility index. This study demonstrates that a significant systematic variability exists among mesenteric Doppler measurements obtained by different operators using different commercially available equipment. Cooperative training can reduce the interobserver variability significantly and bring to an acceptable level the reproducibility of Doppler measurements of superior mesenteric artery flow parameters. PMID- 8622203 TI - Findings on host resistance genes for infectious diseases are pointing the way to drugs, vaccines. PMID- 8622204 TI - Spring brings tick threat to peace enforcers. PMID- 8622205 TI - Update: influenza activity--United States and worldwide, 1995-96 season, and composition of the 1996-97 influenza vaccine. From the Centers for Disease Control and Prevention. PMID- 8622206 TI - Carbon monoxide poisoning at an indoor ice arena and bingo hall--Seattle, 1996. From the Centers for Disease Control and Prevention. PMID- 8622207 TI - Tick paralysis--Washington, 1995. From the Centers for Disease Control and Prevention. PMID- 8622208 TI - Measles--United States, 1995. From the Centers for Disease Control and Prevention. PMID- 8622209 TI - Hepatitis C virus infection among health care workers. PMID- 8622210 TI - Angioplasty for renal artery stenosis. PMID- 8622211 TI - A 47-year-old woman with ductal carcinoma in situ of the breast. PMID- 8622212 TI - A 47-year-old woman with ductal carcinoma in situ of the breast. PMID- 8622213 TI - A 47-year-old woman with ductal carcinoma in situ of the breast. PMID- 8622214 TI - A 47-year-old woman with ductal carcinoma in situ of the breast. PMID- 8622215 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622217 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622216 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622218 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622220 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622219 TI - Carcinogenicity of lipid-lowering drugs. PMID- 8622221 TI - Malaria: submerged awareness. PMID- 8622222 TI - Perinatal HIV infection and the effect of zidovudine therapy on transmission in rural and urban counties. AB - OBJECTIVES: To assess health care providers' identification of human immunodeficiency virus (HIV)-exposed infants, to ascertain the prevalence of transplacental or oral zidovudine treatment among infants exposed to HIV, and to estimate the impact of zidovudine use on perinatal transmission in rural and urban North Carolina. DESIGN: Survey of North Carolina newborns tested for HIV infection in 1993 and 1994 compared with the number of anonymous HIV-positive childbearing women. SETTING: North Carolina hospitals, public health clinics, and private physicians' offices. MAIN OUTCOME MEASURES: Rates of identification of HIV-exposed infants and of perinatal HIV-1 transmission, determined by HIV culture and polymerase chain reaction testing in the infants. RESULTS: The proportion of HIV-exposed children in North Carolina who were identified and tested increased from 60% in 1993 to 82% for all of 1994, and to more than 90% for the last quarter of 1994. The HIV-exposed infants born in rural counties were more likely to be recognized than those born in urban counties (P<.001). In 1994, most infants were evaluated relatively early in life: 39% by 1 week of age, 63% by 6 weeks, and 76% by 3 months. Among infants with recognized HIV exposure, transmission decreased significantly between 1993 and 1994, from 21% to 8.5%, respectively (P=.009). After the announcement of the results of the AIDS Clinical Trials Group Protocol 076, zidovudine was given to 75% of HIV-positive women who delivered infants in North Carolina. Only 5.7% of infants who received any zidovudine became infected, compared with 18.9% of infants who received no zidovudine (P=.007). CONCLUSIONS: Health care providers in North Carolina are identifying most of the state's HIV-seropositive pregnant women, treating them with zidovudine, and testing their infants soon after birth for HIV infection. The use of zidovudine in pregnant women and their infants has reduced perinatal HIV transmission in the state. PMID- 8622223 TI - The effect of acute renal failure on mortality. A cohort analysis. AB - OBJECTIVE: To determine if the high mortality in acute renal failure is explained by underlying illnesses (comorbidity). DESIGN: Cohort analytic study. SETTING: An 826-bed general hospital providing primary, secondary, and tertiary care. PATIENTS: From 16,248 inpatients undergoing radiocontrast procedures between 1987 and 1989, we identified 183 index subjects who developed contrast media associated renal failure (defined as an increase in serum creatinine level of at least 25%, to at least 177 micromol/L [2 mg/dL], within 2 days of receiving contrast material) and 174 paired subjects, matched for age and baseline serum creatinine level, who underwent similar contrast procedures without developing renal failure. MAIN OUTCOME MEASURE: Death during hospitalization. RESULTS: The mortality rate in subjects without renal failure was 7%, compared with 34% in the corresponding index subjects with renal failure (odds ratio, 6.5; P<.001). After adjusting for differences in comorbidity, renal failure was associated with an odds ratio of dying of 5.5. Subjects who died after developing renal failure had complicated clinical courses characterized by sepsis, bleeding, delirium, and respiratory failure; most of these complications developed after the onset of renal failure. Deaths from renal causes were rare. CONCLUSIONS: The high mortality rate in acute renal failure is not explained by the underlying conditions alone. Renal failure appears to increase the risk of developing severe nonrenal complications that lead to death and should not be regarded as a treatable complication of serious illness. PMID- 8622224 TI - Normal serum free thyroid hormone concentrations in patients treated with phenytoin or carbamazepine. A paradox resolved. AB - OBJECTIVE: To address the paradox that phenytoin- and carbamazepine-treated patients have decreased serum free thyroxine (T4) and triiodothyronine (T3) concentrations but appear clinically euthyroid and have normal serum thyroid stimulating hormone (TSH) concentrations. DESIGN: In vitro studies comparing measurements of total and free T4 and T3 by ultrafiltration assay (undiluted serum) and a commercial free T4 estimate kit in control serum samples or serum samples containing added therapeutic levels of phenytoin or carbamazepine. These measurements were made in serum samples diluted 1:5 with either identical serum or phospate buffer, pH 7.4, and in serum samples from patients with seizure disorders who were treated with phenytoin or carbamazepine. SETTING: A 650-bed teaching hospital. PATIENTS: Selected patients (n=19) who were in good health except for seizure disorder, with stable anticonvulsant drug levels in the upper half of the therapeutic range, and were not taking any other drugs that could affect thyroid parameters. MAIN OUTCOME MEASURE: Serum concentrations of free T4 and free T3 in patients taking phenytoin or carbamazepine vs normal controls. RESULTS: Addition of phenytoin or carbamazepine to normal human serum in vitro resulted in a significant increase in free T4 fraction and free T4 (P<.001). In patients taking phenytoin or carbamazepine, serum total T4 decreased significantly to 60% and 74%, respectively, of the control serum concentration (P<.001 for both phenytoin and carbamazepine); free T4 fraction (by ultrafiltration assay) increased 65% and 44%, respectively (P<.001 for phenytoin, P<.01 for carbamazepine); and free T4 remained unchanged. Free T4 concentration measured by a commercial kit (1:5 serum dilution) was significantly lower than the control concentration in both phenytoin- and carbamazepine-treated patients. Serum free T3 and serum TSH were also normal in phenytoin- and carbamazepine treated patients. CONCLUSIONS: Therapeutic levels of phenytoin and carbamazepine displace T4 and T3 from serum binding proteins. When added to serum, the drugs effect an increase in free hormone fractions and free T4 and T3. In drug-treated patients, increased free T4 and T3 fractions offset the significant decrease in serum T4 and T3, resulting in normal free T4 and free T3 concentrations. Since currently available clinical tests will continue to show decreased free T4 concentrations in patients taking phenytoin or carbamazepine, clinicians should rely on serum TSH measurements to confirm the euthyroid status of these patients. PMID- 8622225 TI - Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial. AB - OBJECTIVE: To assess the safety and immunogenicity of a bivalent serogroups A/C meningococcal oligosaccharide-protein conjugate vaccine compared with the licensed meningococcal polysaccharide vaccine. DESIGN: Randomized controlled trial. STUDY POPULATION: Ninety healthy 18- to 24-month-old children who were seen at a southern California Kaiser Permanente clinic. INTERVENTIONS: Vaccination with either the meningococcal conjugate vaccine (at 1 of 2 dosages) or the polysaccharide vaccine, with 2 doses given 2 months apart. MAIN OUTCOME MEASUREMENTS: Immune response to each vaccine dose as determined by measurement of serogroup-specific total antibodies by enzyme-linked immunosorbent assay (ELISA) and by assessment of serum bactericidal activity. RESULTS: Both vaccines appeared to be safe, and nearly all children responded with greater than 4-fold increases in antibody levels. The 2 dosages of the conjugate vaccine induced similar antibody responses; therefore, the data for the 2 conjugate vaccine groups were combined. Following 2 doses, ELISA antibody levels against group C meningococcus were significantly higher in conjugate vaccine recipients than in polysaccharide vaccine recipients (16.66 microg/mL vs. 8.31 microgm/mL; P<.001), but antibody levels against group A were not significantly different 22.75 microg/mL vs 21.24 microg/mL; P=.70). The serum bactericidal assays showed striking differences between the conjugate and polysaccharide vaccine groups. Geometric mean serum bactericidal titers were significantly higher in conjugate vaccine recipients (755.6 vs 37.6 for group A, P<.001; 3197.9 vs 11.4 for group C, P<.001). CONCLUSIONS: The immune response induced by this meningococcal oligosaccharide-protein conjugate vaccine was qualitatively different from that induced by the polysaccharide vaccine, and the antibodies it elicited provided greater functional activity. PMID- 8622226 TI - Zidovudine use to reduce perinatal HIV type 1 transmission in an urban medical center. AB - OBJECTIVE: To evaluate whether zidovudine treatment was accepted and used by pregnant women subsequent to the release of the results of a multicenter, randomized, placebo-controlled trial (AIDS Clinical Trial Group [ACTG] Protocol 076) that showed that zidovudine significantly reduced maternal-infant human immunodeficiency virus (HIV) type 1 transmission. DESIGN: Prospective study. SETTING: A community hospital with an integrated, multidisciplinary HIV-dedicated program located in an impoverished, HIV-endemic urban setting. PARTICIPANTS: All HIV-infected pregnant women identified after the release of the ACTG 076 results who were offered zidovudine therapy to reduce maternal-infant transmission. RESULTS: Only 49 of the 125 HIV-infected pregnant women delivering at our site during this study period were identified prenatally. Perinatal zidovudine therapy was chosen by 37 (75%) of 49 women. Women refusing zidovudine were more likely to report injection drug use as their HIV risk factor and to continue to use drugs during their pregnancy. Of women choosing zidovudine and delivering, 24 of 36 received all components of their elected therapy. The intrapartum dose was missed by 12 women, 4 of whom also missed their prescribed prenatal oral therapy. Lack of adherence to chosen therapy was associated with continued cocaine use during pregnancy. CONCLUSIONS: Zidovudine therapy to interrupt vertical transmission of HIV was not widely used by these HIV-infected pregnant women. Further studies evaluating factors affecting the acceptance and use of recently published public health recommendations are needed. PMID- 8622228 TI - Preventing perinatal HIV infection. How far have we come? PMID- 8622227 TI - Reversal of left ventricular hypertrophy in essential hypertension. A meta analysis of randomized double-blind studies. AB - OBJECTIVE: To determine the ability of various antihypertensive agents to reduce left ventricular hypertrophy, a strong, blood pressure-independent cardiovascular risk factor, in persons with essential hypertension. DATA SOURCES: MEDLINE, DIMDI, RINGDOC, ADES, EMBASE, and review articles through July 1995 (English language and full articles only). STUDY SELECTION: Meta-analysis of all published articles including only double-blind, randomized, controlled clinical studies with parallel-group design. DATA EXTRACTION: Intensive literature search and data extraction according to a prefixed scheme performed independently by 2 investigators. Reduction of left ventricular mass index after antihypertensive therapy with placebos, diuretics, beta-blockers, calcium channel blockers, or angiotensin-converting enzyme (ACE) inhibitors was the principal parameter. DATA SYNTHESIS: Of 471 identified references describing the effects of antihypertensive drugs on left ventricular hypertrophy, only 39 clinical trials fulfilled the inclusion criteria of our study. We found that the decrease in left ventricular mass index was more marked the greater was the decline in blood pressure (systolic r=0.46, P<.001; diastolic r=0.21, P=.08) and the longer was the duration of therapy (r=0.38, P<.01). After adjustment for different durations of treatment (mean duration of treatment, 25 weeks), left ventricular mass decreased 13% with ACE inhibitors, 9% with calcium channel blockers, 6% with beta blockers, and 7% with diuretics. There was a significant difference between drug classes (P<.01): ACE inhibitors reduced left ventricular mass more than beta blockers (significant, P<.05) and diuretics (tendency, P=.08). Similar differences between drug classes were found with regard to effect on left ventricular wall thickness (P<.05). CONCLUSIONS: The database of articles published through July 1995 is small and incomplete, and most of the articles are of poor scientific quality. In this first meta-analysis including only double blind, randomized, controlled clinical studies, decline in blood pressure, duration of drug treatment, and drug class determined the reductions in left ventricular mass index. The ACE inhibitors seemed to be more potent than beta blockers and diuretics in the reduction of left ventricular mass index; calcium channel blockers were somewhat in the intermediate range. The ACE inhibitors and, to a lesser extent, calcium channel blockers emerged as first-line candidates to reduce the risk associated with left ventricular hypertrophy. PMID- 8622230 TI - Regression of left ventricular hypertrophy. How and why? PMID- 8622229 TI - Acute renal failure--a dangerous condition. PMID- 8622231 TI - Visionary medical and other leaders meet to plan healthier communities. PMID- 8622233 TI - Gene therapy for HIV. PMID- 8622232 TI - A role for mitochondria in age-related disorders? PMID- 8622235 TI - Asthma mortality and hospitalization among children and young adults--United States, 1980-1993. From the Centers for Disease Control and Prevention. PMID- 8622234 TI - From the Food and Drug Administration. PMID- 8622236 TI - Survey of knowledge of and awareness about melanoma--United States, 1995. From the Center for Disease Control and Prevention. PMID- 8622237 TI - Risk factors for HIV-1 seroconversion may not be what they seem. PMID- 8622238 TI - The American Society of Anesthesiologists physical status score and risk of perioperative infection. PMID- 8622239 TI - Counting generalist physicians. PMID- 8622240 TI - Kaposi sarcoma of the conjunctiva. PMID- 8622241 TI - Weight loss vs exercise to reduce coronary artery disease risk factors. PMID- 8622242 TI - Weight loss vs exercise to reduce coronary artery disease risk factors. PMID- 8622243 TI - Cognitive effects of marijuana. PMID- 8622244 TI - Minorities in medicine: the Flexner Report. PMID- 8622245 TI - Long-term effects on plasma lipids of diet and drugs to treat hypertension. Treatment of Mild Hypertension Study (TOMHS) Research Group. AB - OBJECTIVE: - To compare long-term plasma lipid changes among 6 antihypertensive treatment interventions for stage I (mild) hypertension. DESIGN: - Multicenter, randomized, double-blind, parallel-group clinical trial. SETTING: - Four academic clinical research units in the United States. PARTICIPANTS: - A total of 902 men and women, aged 45 to 69 years, with stage I diastolic hypertension (diastolic blood pressure <100 mm Hg), recruited from 11914 persons screened in their communities. INTERVENTIONS: - Participants were randomized to 1 of 6 treatment groups: (1) placebo, (2) beta-blocker (acebutolol), (3) calcium antagonist (amlodipine), (4) diuretic (chlorthalidone), (5) alpha1-antagonist (doxazosin), and (6) angiotensin-converting enzyme inhibitor (enalapril). All groups received intensive lifestyle counseling to achieve weight loss, dietary sodium and alcohol reduction, and increased physical activity. MAIN OUTCOME MEASURES: - Changes in plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides from baseline to annual visits through 4 years. RESULTS: - Mean changes in all plasma lipids were favorable in all groups. The degree of weight loss with fat-modified diet and exercise was significantly related to favorable lipid changes. Significant differences (P<.01) among groups for average changes during follow-up in each lipid were observed. Decreases in plasma total cholesterol and LDL cholesterol were greater with doxazosin and acebutolol (for plasma total cholesterol, 0.36 and 0.30 mmol/L [13.8 and 11.7 mg/dL], respectively), less with chlorthalidone and placebo (0.12 and 0.13 mmol/L [4.5 and 5.1 mg/dL], respectively). Decreases in triglycerides were greater with doxazosin and enalapril, least with acebutolol. Increases in HDL cholesterol were greater with enalapril and doxazosin, least with acebutolol. Significant relative increases in plasma total cholesterol with chlorthalidone compared with placebo at 12 months were no longer present at 24 months and beyond, when mean plasma total cholesterol for the chlorthalidone group fell below baseline. Analyses of participants continuing to receive chlorthalidone throughout the 4 years of follow-up indicated this was not due solely to an increasing percentage of participants changing or discontinuing use of medication during follow-up. CONCLUSIONS: - Weight loss with a fat-modified diet plus increased exercise produces favorable long-term effects on blood pressure and all plasma lipid fractions of adults with stage I hypertension; blood pressure reduction is enhanced to a similar degree by addition of a drug from any one of 5 classes of antihypertensive medication. These drugs differ quantitatively in influencing the degree of long-term favorable effects on blood lipids obtained with nutritional-hygienic treatment. PMID- 8622246 TI - The progression from hypertension to congestive heart failure. AB - OBJECTIVES: - To study the relative and population-attributable risks of hypertension for the development of congestive heart failure (CHF), to assess the time course of progression from hypertension to CHF, and to identify risk factors that contribute to the development of overt heart failure in hypertensive subjects. DESIGN: - Inception cohort study. SETTING: - General community. PARTICIPANTS: - Original Framingham Heart Study and Framingham Offspring Study participants aged 40 to 89 years and free of CHF. To reflect more contemporary experience, the starting point of this study was January 1, 1970. EXPOSURE MEASURES: - Hypertension (blood pressure of at least 140 mm Hg systolic or 90 mm Hg diastolic or current use of medications for treatment of high blood pressure) and other potential CHF risk factors were assessed at periodic clinic examinations. OUTCOME MEASURE: - The development of CHF. RESULTS: - A total of 5143 eligible subjects contributed 72422 person-years of observation. During up to 20.1 years of follow-up (mean, 14.1 years), there were 392 new cases of heart failure; in 91% (357/392), hypertension antedated the development of heart failure. Adjusting for age and heart failure risk factors in proportional hazards regression models, the hazard for developing heart failure in hypertensive compared with normotensive subjects was about 2-fold in men and 3-fold in women. Multivariable analyses revealed that hypertension had a high population attributable risk for CHF, accounting for 39% of cases in men and 59% in women. Among hypertensive subjects, myocardial infarction, diabetes, left ventricular hypertrophy, and valvular heart disease were predictive of increased risk for CHF in both sexes. Survival following the onset of hypertensive CHF was bleak; only 24% of men and 31% of women survived 5 years. CONCLUSIONS: - Hypertension was the most common risk factor for CHF, and it contributed a large proportion of heart failure cases in this population-based sample. Preventive strategies directed toward earlier and more aggressive blood pressure control are likely to offer the greatest promise for reducing the incidence of CHF and its associated mortality. PMID- 8622247 TI - Lead exposure and conventional and ambulatory blood pressure: a prospective population study. PheeCad Investigators. AB - OBJECTIVE: - To evaluate in a prospective fashion the association between low level lead exposure and blood pressure. DESIGN: - Prospective cohort study. SETTING: - General population. PARTICIPANTS: - A random population sample (N=728; 49% men; age range, 20-82 years) was studied in Belgium for 1985 through 1989 and reexamined for 1991 through 1995. MEAN OUTCOME MEASURES: - At baseline and follow up, blood pressure was measured by conventional sphygmomanometry (15 total readings) and at follow-up also by 24-hour ambulatory monitoring. Lead exposure was estimated from blood lead and zinc protoporphyrin concentrations. Multivariate analyses controlled for sex, age, body mass index, smoking and drinking habits, physical activity, exposure at work, social class, menopausal status, use of medications (antihypertensive medication, oral contraceptives, hormonal replacement therapy), hematocrit or hemoglobin, serum total calcium concentration, 24-hour urinary sodium and potassium excretion, and gamma glutamyltransferase activity. RESULTS: - At baseline, mean (SD) systolic/diastolic conventional blood pressure was 130 (17)/77 (9) mm Hg. The mean blood lead concentration was 0.42 micromol/L (8.7 microgram/dL), and the mean zinc protoporphyrin concentration was 1.0 microgram per gram of hemoglobin. Over the 5.2-year median follow-up, the mean blood lead concentration dropped by 32% (0.14 micromol/L [2.9 microgram/dL]) (P<.001). Small but significant (P<.01) changes occurred in systolic (-1.5 mm Hg) and diastolic (+1.7 mm Hg) conventional blood pressure and in zinc protoporphyrin concentration (+0.5 microgram per gram of hemoglobin). Over the follow-up period, no consistent associations emerged between the changes in conventional blood pressure and in blood lead or zinc protoporphyrin concentrations. In addition, after adjustment for sex, age, and body mass index, blood lead and zinc protoporphyrin concentrations at baseline did not predict the development of hypertension in 47 patients (risk ratio for doubling of the initial lead concentration, 1.2; 95% confidence interval, 0.7 2.0). In a time-integrated analysis in which each person was characterized by all available measurements, conventional blood pressure did not correlate with blood lead or zinc protoporphyrin concentrations in a consistent manner. Similarly, the mean (SD) 24-hour blood pressure at follow-up (119 [11]/71 [8] mm Hg; N=684) did not show a consistent relationship with blood lead or zinc protoporphyrin concentrations at baseline or at follow-up. CONCLUSIONS: - Lead exposure at the intensity studied (60 days) mechanical ventilation. A total of 31 patients were studied, 23 with pulmonary disease, 4 with neurologic disease and 4 with other conditions. Many of these patients were elderly. The survival rates one year and two years after the start of mechanical ventilation were 40% and 33%, respectively. Respiratory tract infection was the most common and most serious complication, and ventilator-associated pneumonia was the main cause of death. It is difficult but quite important to prevent these complications, especially ventilator-associated pneumonia. PMID- 8622287 TI - [Endobronchial malignant lymphoma of mucosa-associated lymphoid tissue]. AB - A 56-year-old man presented with pulmonary infiltrates. A chest roentgenogram revealed infiltrates in the left upper lobe and a tomogram showed obstruction of the left upper lobe bronchus. Computed tomography of the chest confirmed the presence these infiltrates, and no lymphadenopathy or mediastinal involvement was detected. At bronchoscopic examination, endobronchial masses were found. These masses almost completely obstructed the distal portion of the left upper lobe bronchus and partly obstructed the right upper lobe bronchus. A left upper sleeve lobectomy was done. Macroscopically, the tumor mass looked like a polypoid growth in the bronchial lumen. Microscopic examination showed that the tumor was composed mainly of small cleaved cells. These cells were observed to have infiltrated the epithelium to form lymphoepithelial lesions. Their B-cell origin was shown by the positive CD19 stain. Monoclonality of tumor cells was shown immunohistochemically and by hybridization techniques. Evidence of monoclonal B cell proliferation was found in the rearrangement of Ig heavy- and light-chain genes, with Bam HI and Hind III digests. The final diagnosis was distinctive B cell lymphoma (diffuse, small cell type) of mucosa-associated lymphoid tissue. PMID- 8622288 TI - [Levels of melatonin in serum of patients with retinitis pigmentosa]. AB - PURPOSE: To elucidate melatonin secretion through the pineal gland in patients with night blindness. MATERIAL AND METHODS: Blood samples for melatonin RIA assay were taken from 14 patients at 8 o'clock a.m. and p.m., before and after 3 weeks therapy with Trental, Cocarboxylase and vitamin B12. RESULTS: Usual rhythm of melatonin secretion was demonstrated. The applied treatment influenced melatonin secretion in the youngest group of patients. CONCLUSION: Damage of the photoreceptors in the retina affects melatonin secretion and its circadian rhythm. PMID- 8622289 TI - [New technique for implanting Bomirski melanoma into the anterior chamber of Syrian hamster eyes]. AB - PURPOSE: To describe a newly elaborated ocular tumor model, obtained by implanting Bomirski melanoma into the anterior chamber of the eye in Syrian hamsters (Mesocricetus auratus). MATERIAL AND METHODS: The experiments were carried out using pigmented and non-pigmented lines of the tumor. Attempted injections of suspension of malignant melanoma cells in physiological salt solution to the anterior chamber with an infusion pump did not give the expected results. The growth of the tumor occurred only in 8% of eyes. The research material contained 132 adult pigmented hamsters. Tumor fragments of the size of 0,4-1,0 mm were implanted to the anterior chamber of the eyes. Melanotic cells were implanted in 71 hamsters, amelanotic ones in 61 hamsters. Hamsters were anesthetized with intravisceral vetbutal. The examination was carried out in 24 hour intervals. RESULTS: During the first 2 to 3 days the disappearance of the cells were observed, and then iris tumors appeared, in the cases of amelanotic cells after 4-6 days, and in the cases of melanotic cells after 8-10 days. Enucleated eye-balls were examined histopathologically. In all cases confirmation of the growth of melanoma in the iris was obtained. PMID- 8622291 TI - [Value of vitrectomy in treatment of lens dislocation into the vitreous]. AB - PURPOSE: To present own experiences in vitrectomy in cases of subluxation or luxation of the lens to the vitreous cavity. MATERIAL AND METHODS: 41 eyes with subluxation (16) and luxation (25) of the lens, operated on between May 1992 and April 1994 were analysed. Before the surgery, secondary glaucoma was observed in 18 eyes, hypotony in 8. The aim of pars plana vitrectomy was to release the lens from the surrounding tissues, then the lens was put into anterior chamber and removed by limbal incision. RESULTS: Visual acuity improvement was achieved in 73% of cases. Intraocular pressure was normalized in 14 eyes. Retinal detachment developed in 2 eyes after the surgery, and in one retina was reattached using silicone oil tamponade. CONCLUSION: Our results indicate that it is possible to remove the dislocated lens using vitrectomy without perfluorocarbon. PMID- 8622290 TI - [Image and diagnostic value of ultrasonography and magnetic resonance in selected cases of orbital diseases]. AB - PURPOSE: To compare diagnostic efficiency of USG and MR in orbital diseases. MATERIAL AND METHODS: The clinical material comprised 25 cases of various orbital changes, mostly tumor-like, examined between January 1994 and March 1995 using USG and MR. The value of both methods was compared as well as the results of histopathological studies performed in 15 eyes. Four selected cases are presented in details. RESULTS: In most cases proper diagnosis was state basing both on USG and MR examinations. In 2 cases of USG examination and in 2 of MR misdiagnosis was state as compared with histopathological results. In small tumors and those localised in the anterior part or apex of the orbit MR gives better images. CONCLUSION: USG and MR are complementary diagnostic methods of orbital soft tissue changes, very efficient if made by an experienced examiner. PMID- 8622292 TI - [Use of cryopexy in treatment of retinoblastoma]. AB - The aim of this work is to present our own experience in the treatment of retinoblastoma with cryopexy. MATERIAL AND METHODS: 24 children were observed, among whom 23 had bilateral and 1 unilateral retinoblastoma. We applied transconjunctival cryopexy to small neoplasmatic foci in retina located anterior to the equator. In 21 eyes this treatment was supplementary to brachytherapy and photocoagulation. We used probes of temperature -65C. Each freezing lasted about 20 seconds. The treatment was repeated every 6-8 weeks. In some cases different degree of post-surgical hemorrhages occurred. RESULTS: In 1/4 of cases already after a single freezing the tumour was destroyed and cicatrized. Only in 2 out of 24 treated eyes the desired effect was not reached. CONCLUSION: Cryopexy is an effective method of treatment of small foci of retinoblastoma located in the periphery of the eye fundus. This method is usually supplementary to brachytherapy. PMID- 8622293 TI - [Treatment of bullous keratopathy with excimer laser]. AB - PURPOSE: To evaluate the usefulness of excimer laser photokeratectomy in bullous keratopathy. MATERIAL AND METHODS: Treatment with excimer laser photokeratectomy was performed in 22 eyes with bullous keratopathy which developed after cataract extraction with IOL implantation. RESULTS: During 1 month follow-up a slight improvement of visual acuity, local state improvement and subjective symptoms regression were noted. This state was stable in 14 eyes for 6-12 months, in 8 eyes the recurrence of the disease occurred, but without pain symptoms. CONCLUSION: Excimer laser photokeratectomy does not restore corneal transparency but its benefit in bullous keratopathy is to decrease pain while awaiting for keratoplasty. PMID- 8622295 TI - [A case of unilateral eyelid edema in ascariasis]. AB - A case of ascariasis with unilateral eyelids edema in a boy, aged 3 years, is presented. The diagnostic difficulties connected with the unusual clinical picture and course are discussed. PMID- 8622294 TI - [Eye-strain symptoms after work with a computer screen]. AB - BACKGROUND: In a laboratory arranged according to ergonomic rules, the influence of the work with screen monitor computer on the visual function was evaluated. MATERIAL AND METHODS: Visual efficiency before and after one-hour reading of a text from monitor was compared in 60 persons. There was 50 women and 10 men, aged 22-47. The research scheme comprised 10 trials. RESULTS: After work with the monitor, the most important changes were the following: diminished power of accommodation, removal of the near point of convergence and deviation of phoria for near vision. Statistical analysis, using Wilcoxon's test revealed significant differences between the results achieved before and after work concerning accommodation and convergence (p < 0.05). CONCLUSION: The results suggest that weakness of these important visual functions could be the cause of eye-strain in computer operators. PMID- 8622296 TI - [Biochemical aspects of the development of lens opacity]. PMID- 8622297 TI - [Influence of pattern stimulus modality on visual evoked potentials]. PMID- 8622298 TI - Coronary artery bypass grafting in patients with an ejection fraction of twenty percent or less. AB - Over a 7-year period, 5.8% (n = 210) of patients who underwent coronary artery bypass grafting at our institution had severely impaired global left ventricular function with an ejection fraction of 20% or less. Mean age at operation was 66 years (+/- 0.7; standard error), and 76% of patients were male. Primary indications for operation were unstable angina (73 patients, 35%), return of symptoms with previous bypass grafting (41 patients, 20%), congestive heart failure with reversible ischemia (55 patients, 26%), and recurrent ventricular arrhythmias (41 patients, 20%). Overall, actuarial survival (n = 210) was 82%, 79%, and 73% at 1, 2, and 5 years. Risk of death was highest early after the operation, and then declined rapidly to a constant level. Patients who did not receive retrograde coronary sinus cardioplegia (p = 0.05), older patients (p = 0.004), and those with preoperative ventricular arrhythmias (p = 0.003) or renal failure (p < 0.0001) had an increased risk of death early after operation. Patients with congestive symptoms and those requiring extensive or redo bypass grafting (p = 0.02) were found to be at an increased risk of death throughout the follow-up period. When the number of distal anastomoses performed increased, survival was found to decrease (p < 0.003), and to a greater extent in women than in men (p = 0.02). Of the four primary indications for operation, unstable angina yielded the highest risk-adjusted survival. Successful results after surgical revascularization in patients with severe impairment of ventricular function can be achieved by careful patient selection and management. PMID- 8622299 TI - Long-term survival benefits of coronary artery bypass grafting and percutaneous transluminal angioplasty in patients with coronary artery disease. AB - The purpose of this study was to evaluate long-term survival benefits of bypass surgery and angioplasty versus medical therapy in 9263 patients at Duke University Medical Center between 1984 and 1990 with coronary artery disease confirmed by cardiac catheterization to involve one, two, or three vessels. Clinical data were prospectively entered into an established cardiovascular database, and annual follow-up was 97% complete for a mean interval of 5.3 years and a maximal interval of 10 years. Outcomes were analyzed with the Coronary Artery Surgery Study "method A" to define patient groups treated by medicine (n = 2449), angioplasty (n = 2924), or bypass surgery (n = 3890). Differences among treatment groups in baseline characteristics were adjusted by Cox proportional hazard models. The anatomic severity of coronary artery stenosis best defined survival benefit from bypass surgery and angioplasty versus medical treatment. One or both interventional treatments provided better long-term survival than did medical treatment for all levels of disease severity. All patients with single vessel disease, except those with at least 95% proximal left anterior descending stenosis, benefited from angioplasty versus bypass. All patients with three vessel disease and those two-vessel patients with > or = 95% proximal left anterior descending stenosis benefited from bypass surgery versus angioplasty. All other patients with two-vessel disease and those with > or = 95% proximal left anterior descending stenosis only had similar survival with either interventional treatment. The absolute survival benefit was greatest for patients with severe three-vessel disease treated with bypass surgery. PMID- 8622300 TI - Aortic valve replacement in patients aged eighty years and older: early and long term results. AB - We have studied 322 patients, 80 years of age or older, who underwent aortic valve replacement between June 1971 and December 1992. Two hundred six patients (64%) have had surgery since the end of 1985. Their mean age was 82.7 years (range 80 to 92 years). One hundred seventy-one (53%) were male and most (86%) were in New York Heart Association class III-IV. Fifty-seven patients (18%) required admission to the coronary care unit before the operation. One hundred seventy-nine patients (56%) underwent an urgent or emergency operation. Known cerebrovascular disease was present in 77 (24% of patients), aortic stenosis in 79%, aortic incompetence in 9%, and combined stenosis and incompetence in 12%. Associated procedures included bypass grafting in 139 (43%), mitral valve replacement/repair in 20 (6%), tricuspid valve repair in 6 (2%), and aortic annular enlargement in 38 (12%). Thirty patients (9.3%) were undergoing reoperation. Hospital mortality was 44 of 322 (13.7%). The median hospital stay was 11 days. On univariate analysis, significant predictors of hospital mortality were female sex, preoperative rest pain, New York Heart Association class III-IV, admission to the coronary care unit, heart failure, mitral valve disease, emergency/urgent operation, chronic obstructive pulmonary disease, bypass grafting, valve size, peripheral vascular disease, and ejection fraction less than 0.35. On multivariate analysis the most important independent predictors of operative mortality were female gender (p = 0.0001), renal impairment (p = 0.001), bypass grafting (p = 0.005), ejection fraction less than 0.35 (p = 0.01), and chronic obstructive pulmonary disease (p = 0.028). Age and year of operation did not influence mortality. Five-year survivals for all patients and for operative survivors were 60.2% +/- 3.2% and 70.3% +/- 3.4%, respectively. On univariate analysis, factors that adversely affected long-term survival were coronary bypass grafting (p = 0.007), more than two comorbidities (p = 0.02), male gender (p = 0.04), and ejection fraction less than 0.35 (p = 0.04). On multivariate analysis, no factor was consistently significant for long-term survival. At most recent clinical follow-up 85% were angina free and 82% were in class I-II. At least 92% of patients, both at 1 year and at most recent clinical follow-up, believed they had significantly benefited from the operation: CONCLUSION: Risk factors for aortic valve replacement in octogenarians include female gender, unstable symptoms, poor ejection fraction, renal impairment, and bypass grafting. However, despite a hospital mortality higher than that reported for younger patients, the outlook for operative survivors is excellent, with good relief of symptoms and an expected survival normal for this particular age group. If possible, aortic valve replacement should be done before development of unstable symptoms. PMID- 8622301 TI - Reexploration for bleeding is a risk factor for adverse outcomes after cardiac operations. AB - OBJECTIVE: Although previous studies have included early reexploration for bleeding as a risk factor in analyzing adverse outcomes after cardiac operations, reexploration for bleeding has not been systematically examined as a multivariate risk factor for increased morbidity and mortality after cardiac surgery. Furthermore, multivariate predictors of the need for reexploration have not been identified. Accordingly, we performed a retrospective analysis of 6100 patients requiring cardiopulmonary bypass from January 1, 1986, to December 31, 1993. METHODS: Eighty-five patients who had ventricular assist devices were excluded from further analysis because of the prevalence of bleeding and the significant morbidity and mortality associated with placement of a ventricular assist device, unrelated to reexploration. In the remaining 6015 patients, potential adverse outcomes analyzed included operative mortality, mediastinitis, stroke, renal failure, adult respiratory distress syndrome, prolonged mechanical ventilation, sepsis, atrial arrhythmias, and ventricular arrhythmias. To control for the confounding effects of other risk factors, we performed a multivariate logistic regression analysis. Potential covariates considered in the logistic model included age, sex, race, history of reoperation, urgency of the operation, congestive heart failure, prior myocardial infarction, renal failure, diabetes, hypertension, chronic obstructive pulmonary disease or stroke, and the bypass and crossclamp time. RESULTS: The overall incidence of reexploration was 4.2% (253/6015). Four independent risk factors--increased patient age (p < 0.001), preoperative renal insufficiency (p = 0.02), operation other than coronary bypass (p < 0.001), and prolonged bypass time (p = 0.0.3)--were identified as predictors of the need for reexploration. The preoperative use of aspirin, heparin, or thrombolytic agents and the bleeding time were not identified as predictors. Reexploration for bleeding was identified as a strong independent risk factor for operative mortality (p = 0.005), renal failure (p < 0.0001), prolonged mechanical ventilation (p < 0.0001), adult respiratory distress syndrome (p = 0.03), sepsis (p < 0.0001), and atrial arrhythmias (p = 0.006). CONCLUSION: These data indicate that meticulous attention to surgical hemostasis and possibly application of recently developed modalities designed to facilitate perioperative correction of coagulopathy could improve outcomes after cardiac operations. PMID- 8622303 TI - Endovascular stent-graft repair of thoracic aortic aneurysms. AB - Conventional repair of aneurysms of the descending thoracic aorta entails thoracotomy and graft interposition. For elderly patients and those with previous operations, obesity, respiratory insufficiency, or other comorbidities, such a procedure entails significant mortality and morbidity. Transluminal stent-graft placement offers an alternative approach with potentially less morbidity and quicker recovery; however, the effectiveness and durability of stent-grafts remain uncertain. METHODS: Since July 1992, thoracic aortic stent-grafts have been placed in 44 patients with a variety of pathologic conditions. Each graft was individually constructed from self- expanding, stainless-steel Z stents covered with a woven Dacron polyester fabric graft. Craft dimensions were determined from spiral computed tomographic scans. All implants were performed in the operating theater under fluoroscopic and transesophageal echocardiographic guidance. Follow-up was by computed tomography and contrast angiography in all cases. PATIENT DATA: There were 36 men and 8 women. Mean age was 66 years (range 35 to 88 years). Mean aneurysmal diameter was 6.3 cm (range 4.0 to 9.4 cm). Etiologies included 23 degenerative aneurysms, four posttraumatic aneurysms, four pseudoaneurysms, and one chronic aortic dissection. RESULTS: There were three early deaths (<30 days) and two late deaths. One early death resulted from graft failure. There were two instances of paraparesis or paraplegia, with one associated early death. A single stent was deployed in 27 patients, two stents were required in 14 patients, and three stents were required in three patients. In 23 patients, vascular access was attained through the femoral artery; abdominal aortic access, either native or graft, was necessary in the remaining 21 patients. Twelve grafts were placed in conjunction with open abdominal aortic surgical procedures. Mean follow-up (98% complete) was 12.6 months (range 1 to 34 months). One late death occurred from aneurysmal expansion and rupture in a patient with a persistent midgraft leak. The second late death may have resulted from aneurysmal rupture. Immediate thrombosis was achieved in 36 patients, and late thrombosis was achieved in three others. Failure to achieve complete aneurysmal thrombosis occurred in five patients, however, and one individual (previously noted) died of aneurysmal expansion and rupture; the remaining four are being carefully monitored. Only one patient has required conversion of the stent to an open procedure; a contained rupture of the false lumen of a chronic dissection eventually necessitated total descending thoracic aortic exclusion. CONCLUSIONS: These early results support the hypothesis that endovascular stent graft placement may be a safe and durable treatment for selected patients with aneurysmal disease of the descending thoracic aorta. Large introducer size (26F outer diameter) and relatively limited angulation capability, as well as imprecise deployment techniques, currently limit its applicability. Distal embolization and stent migration have not been observed. Failure to achieve complete aneurysmal thrombosis may allow continued aneurysmal expansion and rupture. Further follow-up is clearly necessary to evaluate the true long-term effectiveness of this procedure. PMID- 8622302 TI - Transmyocardial laser revascularization: operative techniques and clinical results at two years. AB - OBJECTIVES: A new technique, transmyocardial laser revascularization, provides direct perfusion of ischemic myocardium via laser-created transmural channels. From 1993 to 1995, we have treated 20 patients (mean age 61 years, four women and 16 men) with transmyocardial laser revascularization. Preoperatively, the average angina class was 3.7. The patients were screened before the operation by a technetium sestamibi perfusion scan to identify the location and extent of their reversible ischemia. METHODS: Operative exposure is gained via a left anterior thoracotomy. With the use of a 850-watt carbon dioxide laser, an average of 21 +/ 4 channels were created in 22 minutes with a total operative time of less than 2 hours. RESULTS: The in-hospital mortality was two of 20 patients. Three additional patients died after discharge. After an accumulated 172 patient-months (mean follow-up 11 +/- 8 months, range 1 to 26 months), the mean angina class is I (p = 0.01). Postoperative sestamibi scans were obtained at 3, 6, and 12 months. Using the septum as a control and comparing the postoperative results with the preoperative baseline, we noted a significant improvement in perfusion particularly in the areas of reversible ischemia. CONCLUSION: These early results indicate that transmyocardial laser revascularization is a simple operative technique that may improve myocardial perfusion and provide angina relief for patients in whom standard methods of revascularization is contraindicated. PMID- 8622304 TI - Influence of suture technique and suture material selection on the mechanics of end-to-end and end-to-side anastomoses. AB - Experiments were performed in dogs to evaluate the mechanics of 26 end-to-end and 42 end-to-side artery-vein graft anastomoses constructed with continuous polypropylene sutures (Surgilene; Davis & Geck, Division of American Cyanamid Co., Danbury, Conn.), continuous polybutester sutures (Novafil; Davis & Geck), and interrupted stitches with either suture material. After construction, the grafts and adjoining arteries were excised, mounted in vitro at in situ length, filled with a dilute barium sulfate suspension, and pressurized in 25 mm Hg steps up to 200 mm Hg. Radiographs were obtained at each pressure. The computed cross sectional areas of the anastomoses were compared with those of the native arteries at corresponding pressures. Results showed that for the end-to-end anastomoses at 100 mm Hg the cross-sectional areas of the continuous Surgilene anastomoses were 70% of the native artery cross-sectional areas, the cross sectional areas of the continuous Novafil anastomoses were 90% of the native artery cross-sectional areas, and the cross-sectional areas of the interrupted anastomoses were 107% of the native artery cross-sectional areas (p < 0.05). At physiologic pressures, there were no differences in compliance among the three types of anastomosis. These data suggest that when constructing an end-to-end anastomosis in a small vessel, one should use an interrupted suture line or possibly continuous polybutester suture. Forty-two end-to-side anastomoses demonstrated no differences in cross-sectional areas or compliance for the three suture techniques. This suggests that, unlike with end-to-end anastomoses, when constructing an end-to-side anastomosis in patients any of the three suture techniques may be acceptable. PMID- 8622306 TI - Esmolol and percutaneous cardiopulmonary bypass enhance myocardial salvage during ischemia in a dog model. AB - Despite recent advances in techniques of reperfusion for acute myocardial ischemia, myocardial salvage remains suboptimal. Beta-blockers have been shown to limit infarct size during acute ischemia, but their negative inotropic properties have limited their use. Cardiopulmonary bypass is an attractive technique for cardiac resuscitation because it can stabilize a hemodynamically compromised patient and potentially reduce myocardial oxygen consumption. In an attempt to maximize myocardial salvage in the setting of acute ischemia, the combination of esmolol, an ultrashort-acting beta-blocker, with percutaneous cardiopulmonary bypass was evaluated. Four groups of instrumented dogs underwent 2 hours of myocardial ischemia induced by occlusion of the proximal left anterior descending coronary artery, followed by 1 hour of reperfusion. Throughout the period of ischemia and reperfusion, esmolol plus percutaneous cardiopulmonary bypass was compared with esmolol alone, percutaneous cardiopulmonary bypass alone, and control conditions. After the reperfusion period, the extent of infarction of the left ventricle at risk was determined. Four animals had intractable arrhythmias: one in the esmolol plus bypass group, one in the esmolol group, and two in the control group. The extent of infarction of the left ventricle at risk was significantly reduced in the esmolol plus bypass group (30%) compared with bypass alone (52%), with esmolol alone (54%), and with the control groups (59%; p < 0.05). We conclude that in this experimental model the combination of esmolol with bypass improves myocardial salvage after ischemia and reperfusion. PMID- 8622307 TI - Analysis of right ventricular function during bypass of the left side of the heart by afterload alterations in both normal and failing hearts. AB - This study investigated the mechanism of right ventricular failure during bypass of the left side of the heart by precisely assessing right ventricular function with use of a conductance catheter. Bypass of the left side of the heart was established with a centrifugal pump in 10 mongrel dogs weighing 11 to 19 kg. Right ventricular function during left heart bypass was evaluated by two parameters that were both derived from measurement of relative change in right ventricular volume by the conductance catheter technique. One parameter was the right ventricular end-systolic pressure-volume relationship as a load-independent index, and the other was the peak right ventricular pressure-right ventricular stroke volume relationship as a "force-velocity relationship." These parameters were measured in both normal and failing hearts while afterload was increased by bilateral intrapulmonary balloon inflation. Moreover, changes in these relationships were observed by varying assist ratios of left heart bypass from 0% to 100%. Failing heart models were induced by normothermic aortic clamping for 20 minutes. The right ventricular end-systolic pressure-volume relationship in normal hearts did not change, irrespective of the assist ratio of left heart bypass, whereas that in failing hearts decreased from 4.25 +/- 1.41 mm Hg/ml without bypass of the left side of the heart to 3.53 +/- 1.30 mm Hg/ml after 100% assist of left heart bypass (p < 0.05). In the peak right ventricular pressure right ventricular stroke volume relationship, right ventricular stroke volume was almost constant in normal hearts when afterload was increased regardless of the assist ratio of left heart bypass. Moreover, right ventricular stroke volume was maintained at a higher level during bypass of the left side of the heart compared with that without left heart bypass. However, that slope of the relationship in failing hearts was inversely linear and became significantly steeper after 100% assist of bypass of the left side of the heart compared with that without left heart bypass (-0.131 +/- 0.042 versus -0.051 +/- 0.038, p < 0.005). Therefore ++these two slopes of the relationship intersected at a point that was considered the critical point of afterload during bypass of the left side of the heart. In other words, right ventricular stroke volume was decreased by 100% left heart bypass above the critical point of afterload. In conclusion, this study demonstrates not only that bypass of the left side of the heart results in an increase in right ventricular stroke volume in both normal and failing hearts at the physiologic range of afterload, but also that right ventricular function against higher afterload is impaired by 100% assist of bypass of the left side of the heart in failing hearts. PMID- 8622305 TI - The effects of Carmeda Bioactive Surface on human blood components during simulated extracorporeal circulation. AB - Postoperative morbidity after cardiopulmonary bypass most commonly manifests as bleeding diatheses or pulmonary dysfunction. The pathophysiology has been attributed to the activation of cellular and humoral components of blood after contact with an artificial surface. Development of a surface that would be nonthrombogenic and also would constitute a less potent inflammatory stimulus would therefore be beneficial. In the following experiments, we evaluated the heparin-bonded Carmeda Bioactive Surface (Medtronics Cardiopulmonary, Anaheim, Calif.) in an in vitro model of extracorporeal circulation at standard-dose heparin (5 U/ml), to examine the effects of the surface treatment on activation of blood elements, and at reduced-dose heparin (1 U/ml), to determine whether surface-bound heparin would serve as an effective anticoagulant. During the initial recirculation period, platelet counts in the Carmeda (n = 12) circuits were preserved at both doses of heparin and compared with control values (n = 12): At 5 U/ml, control 36% +/- 4% (mean +/- standard error of the mean) versus Carmeda 81% +/- 5%; at 1 U/ml, 43% +/- 3% versus 61% +/- 10%, expressed as a percent of baseline at 30 minutes, p < 0.05. Furthermore, plasma levels of platelet factor 4 and beta-thromboglobulin were significantly reduced in the Carmeda circuits throughout the experiment: At heparin 5 U/ml, 2500 +/- 340 ng/ml versus 604 +/- 191 ng/ml; at 1 U/ml, 2933 +/- 275 ng/ml versus 577 +/- 164 ng/ml of platelet factor 4 at 2 hours (p < 0.05). The pattern of beta-thromboglobulin release was similar, with effects more pronounced at the lower dose of heparin. Surface modification also reduced leukocyte depletion (p < 0.05) and release of elastase at both concentrations of heparin (5 U/ml, 0.72 +/- 0.29 ng/ml versus 0.33 +/- 0.23 ng/ml; 1 U/ml, 0.85 +/- 0.08 ng/ml versus 0.20 +/- 0.05 ng/ml, at 2 hours, p < 0.05). Moreover, as heparin concentration was reduced, Carmeda surface treatment significantly decreased generation of C3a des Arg (1 U/ml, 14,410 +/- 3558 ng/ml versus 3053 +/- 1039 ng/ml at 2 hours, p < 0.05). Although heparin bonding was originally intended to obviate the need for systemic heparinization, Carmeda treatment did not reduce fibrinopeptide A generation at the lower dose of heparin. In summary, Carmeda treatment failed to exhibit anticoagulant efficacy in this model; however, the data suggest that surface modification may have a role in ameliorating the typical inflammatory response initiated by blood contact with an artificial surface. PMID- 8622308 TI - Splenothoracic fistula complicating primary splenic hydatidosis. PMID- 8622309 TI - Hemoptysis caused by an endobronchial lipoma. PMID- 8622310 TI - Venoarterial connections in visceral heterotaxy. PMID- 8622311 TI - Systemic and venous connection in asplenia syndrome. PMID- 8622312 TI - Does video-assisted thoracic surgery disseminate tumor? PMID- 8622313 TI - Inhaled nitric oxide reduces human lung allograft dysfunction. AB - OBJECTIVE: Early severe graft dysfunction, as manifested by hypoxia and pulmonary hypertension, occurs in 10% to 20% of lung transplant recipients. We retrospectively investigated whether inhaled nitric oxide would reduce human lung allograft dysfunction by comparing postoperative hemodynamic data, gas exchange, and outcome in lung transplant recipients with early graft dysfunction treated with or without nitric oxide. METHOD: Among 243 adult lung transplant procedures, there were 32 patients (13.2%) in whom immediate severe allograft dysfunction developed (arterial oxygen tension/inspired oxygen concentration ratio <150). Group 1 (n = 17) included patients who underwent transplantation before nitric oxide became available in our center and were treated conventionally. Group 2 (n = 15) included those treated with nitric oxide as soon as severe allograft dysfunction was diagnosed. Duration of nitric oxide therapy (20 to 60 ppm) was 15 to 217 hours (average 84 hours). RESULTS: In group 2, nitric oxide lowered mean pulmonary artery pressure from 30 +/- 2 to 26 +/- 2 mm Hg (p < 0.05), improved the ratio of arterial oxygen tension to inspired oxygen fraction from 88 +/- 10 to 153 +/- 30 (p < 0.05) within 1 hour, and caused a sustained improvement in these parameters during extended therapy. Mean arterial pressure and cardiac index were unchanged during nitric oxide therapy. Transient methemoglobinemia (>6%) developed in two patients. However, no complications were associated with nitric oxide use. Duration of mechanical ventilation was 17 +/- 5 days in group 1 and 12 +/- 3 days in group 2. Four patients had airway complications in group 1, whereas no airway complication was encountered in group 2. Mortality was 24% (4/17) in group 1 and 7% (1/15) in group 2. CONCLUSION: Nitric oxide improves oxygenation and decreases pulmonary artery pressure without systemic circulatory effects in patients with severe allograft dysfunction. Furthermore, in these patients, nitric oxide may shorten postoperative mechanical ventilation time and reduce airway complications and mortality. PMID- 8622314 TI - In vitro evaluation of neonatal human immunity against the pig. AB - The critical shortage of organ donors has greatly limited the number of clinical allotransplantations. This is particularly true for neonatal patients, for whom xenotransplantation could provide an alternative therapeutic option to allotransplantation. The role of neonatal infant immunity in xenotransplantation is not, however, clearly understood. We examined both the proliferative responses of human neonatal lymphocytes to pig aortic endothelial cells and serum levels of neonatal natural antipig xenoantibody. Neonatal human lymphocytes and serum were isolated from umbilical cord blood. Adult human lymphocytes and serum were used as controls. A one-way xenogeneic mixed lymphocyte-endothelial cell reaction was performed, and lymphocyte proliferation was measured by tritiated thymidine uptake. Neonatal human lymphocytes recognized and proliferated in response to pig aortic endothelial cells (mean 63,926 +/- 26,054 counts per minute). The level of xenogeneic mixed lymphocyte-endothelial cell reaction of neonatal lymphocytes was significantly lower (p < 0.004) than that of adult human lymphocytes (mean 122,444 +/- 33,132 counts per minute). An enzyme-linked immunosorbent assay was performed to determine the binding of natural immunoglobulin M and G antibodies to pig endothelial cells. Whole-cell enzyme-linked immunosorbent assay demonstrated neonatal human serum to contain very low binding levels of natural antipig immunoglobulin M xenoantibody compared with adult serum. Like adult serum, neonatal human serum contained natural antipig immunoglobulin G xenoantibody. Neonatal serum was not cytotoxic to pig endothelial cells, suggesting that immunoglobulin G was not the predominant xenoreactive antibody. To assess whether neonatal pig endothelial cells also expressed xenoantigens, adult and neonatal cultured pig endothelial cells were examined by enzyme-linked immunosorbent assay with adult human serum. Adult human natural immunoglobulin M xenoantibody recognized not only adult pig endothelial cell xenoantigens but also neonatal pig endothelial cell xenoantigens. The binding levels of adult natural antipig immunoglobulin M xenoantibodies to adult and neonatal pig endothelial cells were similar, suggesting that neonatal pig aortic endothelial cells express xenoantigens. The findings of low binding levels of cytotoxic antipig immunoglobulin M xenoantibody and low levels of lymphocyte xenoreactivity to pig endothelial cells in human neonates suggests that pig organs may eventually be a suitable source of xenografts for human neonates. PMID- 8622315 TI - Long-term cryopreservation can prevent rejection of canine tracheal allografts with preservation of graft viability. AB - We previously reported that cryopreservation of tracheas for 1 month was possible in a canine tracheal autotransplantation model with use of a preservative solution containing trehalose. Realizing that the allogenicity of many organs is decreased by freezing, we examined the possibility of immunosuppressant-free canine tracheal allotransplantation after long-term cryopreservation. Six to 10 rings of the trachea were removed from donor dogs (n = 12), immersed in the preservative solution, and cryopreserved in a deep freezer at -85 degrees C for 285 +/- 28 days (cryopreservation group). Five rings of the mediastinal trachea of recipient dogs were removed. The cryopreserved tracheas were thawed and transplanted to replace the excised mediastinal tracheas. In a control group (n = 6), the graft was preserved in Euro-Collins solution at 10 degrees C for 16 to 17 hours. Allotransplantation of tracheas was done as in the cryopreservation group. The anastomotic site and graft were covered with an omental pedicle in both groups. In the cryopreservation group, every animal, except one that was killed for pathologic examination, survived more than 2 months. All the grafts of this group were viable, and no stenosis or tracheomalacia was observed. In the control group, most of the animals died within 1 month of tracheal stenosis caused by rejection. These findings reveal that immunosuppressant-free canine tracheal allotransplantation was consistently possible after long-term cryopreservation of the graft in a preservative solution containing trehalose. This simple method could solve both donor shortage and immunosuppression problems. PMID- 8622316 TI - Stage II esophageal carcinoma: the significance of T and N. AB - OBJECTIVE: Stage II esophageal carcinomas are a heterogeneous group of uncommon malignant tumors that include both node-negative (IIA; T2 N0 M0 and T3 N0 M0) and node-positive (IIB; T1 N1 M0 and T2 N1 M0) carcinomas. The purpose of this study was to evaluate this heterogeneity and to identify predictors of improved survival. RESULTS: Ninety-four of 345 patients undergoing esophageal resection at the Cleveland Clinic Foundation between 1985 and 1994 had stage 11 carcinomas; 70 stage IIA (24 T2 N0 M0 and 46 T3 N0 M0) and 24 stage IIB (9 T1 N1 M0 and 15 T2 N1 M0). Pathologic stage and T and N status were the only identifiable predictors of survival. Stage IIA survival was significantly better than stage IIB (p = 0.01). T2 N0 M0 survival was not different from T1 N0 M0 survival (p = 0.83). T3 N0 M0 survival was significantly worse than T1 N0 M0 (p = 0.03) and intermediate between T2 N0 M0 survival (p = 0.06) and T1 N1 M0 and T2 N1 M0 survivals (p = 0.07). T1 N1 M0 and T2 N1 M0 survival was not significantly different from T3 N1 M0 survival (p = 0.63). CONCLUSIONS: (1) N1 disease is the principal predictor of reduced survival and N1 is independent of T. Therefore the distinction between T1 N1 M0, T2 N1 M0, and T3 N1 M0 carcinomas is not warranted. (2) N0 disease is the principal predictor of improved survival but N0 is not independent of T. T1 N0 M0 and T2 N0 M0 survivals are similar and therefore distinction between these subgroups is not warranted. T3 N0 M0 survival is intermediate between T1 N0 M0 and T2 N0 M0 carcinomas and between T1 N1 M0, T2 N1 M0, and T3 N1 M0 carcinomas. Therefore stratification by T for N0 carcinomas is warranted. PMID- 8622317 TI - Enhanced isolated lung function after ischemia with anti-intercellular adhesion molecule antibody. AB - The binding of leukocytes to intercellular adhesion molecules expressed on endothelial surfaces during ischemia and subsequent reperfusion initiates leukocyte-mediated reperfusion injury. Interruption of this leukocyte-endothelium interaction may therefore prevent reperfusion injury. In an isolated, ventilated, blood-perfused rabbit lung preparation, we studied the effect of a monoclonal anti-intercellular adhesion molecule antibody on lung function during reperfusion. Lungs were harvested with 50 ml/kg cold Euro-Collins flush and 30 micrograms prostaglandin E1 before storage for 18 hours at 4 degrees C. Experimental groups received low-dose (100 micrograms) or high-dose (200 micrograms) anti-intercellular adhesion molecule antibody added to the pulmonary flush at harvest and to the initial reperfusate. Eighteen-hour control preparations were preserved for 18 hours and received saline solution vehicle. Immediate control preparations were harvested and immediately reperfused. The oxygen tension in the recirculated pulmonary venous effluent was measured after 30 minutes of reperfusion. Histologic specimens were graded by blinded observers for degree of leukocyte infiltration (0, normal, to 4, severe infiltration). The mean oxygen tensions (+/-standard error of the mean) were 138.29 +/- 6.23, 58.86 +/- 9.14, 86.87 +/- 11.32, and 139.33 +/- 16.15 mm Hg in immediate control preparations, 18-hour control preparations, low-dose antibody group, and high dose antibody group, respectively (p = 0.0001). The leukocyte grades (mean +/- standard error of the mean) were 1.5 +/- 0.723, 3.0 +/- 0.955, 1.9 +/- 0.899, and 1.2 +/- 0.834, respectively (p = 0.0002). We conclude that anti-intercellular adhesion molecule antibody added to the pulmonary flush and initial reperfusate results in a dose-dependent enhancement of the reperfused lung's ability to oxygenate blood, possibly as a result of decreased leukocyte sequestration. PMID- 8622318 TI - Survival and function after sleeve lobectomy for lung cancer. AB - Between 1962 and 1991, 72 patients (mean age 63.4 years) underwent sleeve lobectomy for primary lung cancer. Thirty-seven patients had adequate lung function and 35 were deemed unsuitable for pneumonectomy on the basis of inadequate pulmonary reserve (n = 31) or cardiac risk factors (n = 4). Squamous cell carcinomas (68%) and adenocarcinomas (26%) predominated. Upper lobectomy was performed in 48 patients, lower and middle lobectomy in 13, and right upper and middle bilobectomy in 11. Hospital mortality was 4% (3/72) and compares with a hospital mortality of 9% in 56 consecutive pneumonectomies between 1986 and 1990. Major complications occurred in 11% (bronchopleural fistula 1, persistent atelectasis 4, pneumonia 4). Adjusted actuarial survival after sleeve lobectomy at 1 and 5 years was 84% and 42%, compared with 76% and 44% after pneumonectomy. Five-year survival after lower and middle lobectomy in 13 patients (52%) was similar to that after upper lobectomy (46%), suggesting that in carefully selected patients the concept of sleeve lobectomy can be applied to all pulmonary lobes. N1 disease and compromised lung function were associated with lower survival (N1 38% vs N0 57%; compromised 20% vs adequate 55%). Comparison of preoperative and postoperative lung function and quantitative ventilation perfusion isotope studies substantiated the preservation of pulmonary function in this group of patients. Sleeve lobectomy is the procedure of choice for anatomically suitable carcinomas or when reduced pulmonary reserve precludes extensive resection. PMID- 8622320 TI - Subglottic stenosis complicating Wegener's granulomatosis: surgical repair as a viable treatment option. AB - Wegener's granulomatosis frequently involves the subglottis and trachea, often leading to compromise of the upper airway. Moreover, the stenotic segments may persist or progress despite control of the disease elsewhere in the body. In this report, we describe the cases of five patients with Wegener's granulomatosis who, in addition to nasal, sinus, pulmonary and renal involvement, had symptomatic subglottic or tracheal stenosis. Biopsy specimens from involved sites in the subglottis and trachea were often not diagnostic, and the diagnosis was later confirmed by a positive antineutrophil cytoplasm antibody titer. All patients had clinical remission on standard therapeutic regimens with prednisone and cyclophosphamide but continued to have symptoms of extrathoracic airway obstruction. Three of the five patients underwent primary thyrotracheal anastomosis while their disease was in clinical remission, without postoperative compromise of anastomotic integrity or wound healing despite concurrent use of prednisone and cyclophosphamide. There has been no evidence of local disease recurrence during follow-up periods ranging from 3 months to 14 years. We conclude that surgical intervention is a viable treatment option for patients who have symptomatic stenotic segments of the subglottis and trachea as a result of Wegener's granulomatosis in clinical remission. PMID- 8622319 TI - Dissemination of malignant tumors after video-assisted thoracic surgery: a report of twenty-one cases. The Video-Assisted Thoracic Surgery Study Group. AB - Video-assisted thoracic surgical techniques are widely used for biopsy and resection of thoracic tumors, but studies of long-term outcomes have not been reported. Dissemination of tumor by these techniques is a potential hazard. Therefore we surveyed the surgical members of the Video-Assisted Thoracic Surgery Study Group to determine whether tumor implants thought to be directly related to video-assisted techniques had occurred. Surgeons reported 21 cases. The sites of recurrence were the incision (n = 14), pulmonary staple line (n = 2), pleura (n = 2), both staple line and incision (n = 1), both pleura and incision (n = 1), and both pleura and staple line (n = 1). Review of these cases illustrates the pitfalls of present video-assisted techniques for malignant tumors of the thorax. PMID- 8622321 TI - Functional effects of decortication after penetrating war injuries to the chest. AB - Decortication was performed in 32 persons with penetrating war injuries to the chest. The indications were acute and chronic post-traumatic empyema, incompletely evacuated hemothorax, chylothorax, and chronic pneumothorax. Decortications were done through a thoracotomy in 29 cases and by thoracoscopy in three cases. Results of overall lung function tests and blood gas analyses were studied in all patients before operation, after immediate postoperative recovery, and 6 months after operation. Significant improvement in lung function was observed after decortication in all patients, particularly after thoracoscopic decortication. Restrictive pattern decreased moderately (p < 0.01). Blood gas analyses did not show significant changes after operation. There were no intraoperative or postoperative deaths. PMID- 8622322 TI - Histamine liberation related to cardiopulmonary bypass in children: possible relation to transient postoperative arrhythmias. AB - Tumor necrosis factor-alpha production and products of mast cell, basophil, and eosinophil degranulation (prostaglandin D2, histamine, and eosinophil cationic protein) were prospectively studied in 26 children undergoing cardiac operations. The relationship between inflammatory response to cardiopulmonary bypass and transient postoperative arrhythmias was analyzed. Cardiopulmonary bypass was conducted with circulatory arrest and deep hypothermia in 10 patients and with continuous low-flow and moderate hypothermia in 16 patients. Transient postoperative arrhythmias diagnosed on standard or atrial electrocardiograms (or both) were seen in eight of the 26 examined children: accelerated junctional rhythm (n = 3), junctional ectopic tachycardia (n = 3), second-degree atrioventricular block (n = 1), and third-degree atrioventricular block (n = 1). Children with transient postoperative arrhythmias were younger than those without (p < 0.05). Compared with baseline values, there was in all patients a significant release of histamine and eosinophil cationic protein (p < 0.05) related to cardiopulmonary bypass, reaching peak values 4 hours after the operation. In contrast, tumor necrosis factor-alpha production and prostaglandin D2 release were not significant. This suggests that activated basophils but not mast cells are the major sources of histamine liberated during and after cardiopulmonary bypass. Histamine release but not eosinophil cationic protein release correlated with circulatory arrest and deep hypothermia (p < 0.05), suggesting the participation of physicochemical alterations of circulating basophils leading to histamine liberation. Four hours after the operation, patients with transient postoperative arrhythmias had significantly higher blood concentrations of histamine (p < 0.02) and eosinophil cationic protein (p < 0.05) than did those without transient postoperative arrhythmias. On the first postoperative day, four of the eight patients with transient postoperative arrhythmias had persisting elevated histamine levels, whereas in patients without transient postoperative arrhythmias histamine reached baseline values. The multivariate analysis retained histamine release and eosinophil cationic protein variations related to cardiopulmonary bypass for the emerging model to predict transient postoperative arrhythmias. The results of this study show significant histamine release related to cardiopulmonary bypass. Furthermore, they document a possible relationship between circulating histamine and transient postoperative arrhythmias. The latter may therefore be suspected among the consequences of the inflammatory response to cardiopulmonary bypass. PMID- 8622323 TI - The effect of preoperative tranexamic acid on blood loss after cardiac operations in children. AB - Children undergoing cardiac operations in which cardiopulmonary bypass is used are at risk of significant postoperative blood loss. The acquired coagulopathy is complex but is thought to be due, in part, to excessive fibrinolysis. We examined the possibility of reducing postoperative blood loss in children by using the antifibrinolytic drug tranexamic acid. Using a prospective, randomized, double blind study design, we administered a single dose of tranexamic acid (50 mg/kg intravenously) or saline placebo, before skin incision, in 88 children undergoing cardiac operations. Post-operative blood loss and fluid replacement were recorded for the next 24 hours. In addition, hemoglobin, platelet counts, and coagulation measures were recorded every 6 hours. When all patients were examined, there was no significant difference in postoperative blood loss between the treated and placebo groups (21.2 +/- 12 ml/kg per 24 hours, tranexamic acid, vs 27.2 +/- 20.3 mls/kg per 24 hours, placebo). However, when the children with cyanosis were analyzed separately, there was a highly significant difference in blood loss between the groups during the first 6 hours (11.2 +/- 3.7 ml/kg per 6 hours, tranexamic acid, vs 27.2 +/- 11.4 mls/kg per 6 hours, placebo; p < 0.002), as well as the overall 24 hour study period (23.7 +/- 7.5 mls/kg per 24 hours, tranexamic acid, vs 48.9 +/- 27.6 mls/kg per 24 hours, placebo; p < 0.02). Also significantly less blood and blood products were administered to the treated cyanosed group. Tranexamic acid produced a significant reduction in postoperative blood loss and blood product requirements in children with cyanosis undergoing heart operations. The drug had no effect in children without cyanosis or those requiring a second thoracotomy. PMID- 8622324 TI - The coronary arteries in hearts with discordant atrioventricular connections. AB - Gross morphologic study of 14 hearts with congenitally corrected transposition or discordant atrioventricular connections and double-outlet right ventricle or pulmonary atresia disclosed consistently the origins of the coronary arteries from two aortic sinuses. With usual atrial arrangement, the artery arising in sinus 1 (right-hand facing), as seen from the noncoronary aortic sinus, supplied the morphologically right ventricle. Coronary blood supply to the morphologically left ventricle usually came from sinus 2 (left-hand facing), although in two hearts the circumflex branch was a continuation of the artery from sinus 1. The sinus nodal artery arose from the circumflex coronary artery, and histologic studies of two hearts demonstrated that blood supply to the anterior atrioventricular node also came from this vessel. Early branching and entrapment in fat or right ventricular muscle, as well as malalignment of aortic and pulmonary sinuses, occurred frequently. These findings may have implications for coronary arterial transfer in the double switch operation in hearts with discordant atrioventricular connections. PMID- 8622325 TI - Coronary artery bypass grafting in patients with an ejection fraction of twenty percent or less. PMID- 8622326 TI - A fat little earner. PMID- 8622327 TI - Coronary heart disease and vitamin E. PMID- 8622328 TI - Unnatural death, AIDS, and coroners. PMID- 8622329 TI - Antiepileptic drugs, clinical trials, and the marketplace. PMID- 8622330 TI - Choledochal cyst, a disease for all ages. PMID- 8622331 TI - Signing up for authorship. PMID- 8622332 TI - Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS) AB - BACKGROUND: Vitamin E (alpha-tocopherol) is thought to have a role in prevention of atherosclerosis, through inhibition of oxidation of low-density lipoprotein. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of alpha-tocopherol and lower rates of ischaemic heart disease. We tested the hypothesis that treatment with a high dose of alpha-tocopherol would reduce subsequent risk of myocardial infarction (MI) and cardiovascular death in patients with established ischaemic heart disease. METHODS: In this double-blind, placebo-controlled study with stratified randomisation, 2002 patients with angiographically proven coronary atherosclerosis were enrolled and followed up for a median of 510 days (range 3 981). 1035 patients were assigned alpha-tocopherol (capsules containing 800 IU daily for first 546 patients; 400 IU daily for remainder); 967 received identical placebo capsules. The primary endpoints were a combination of cardiovascular death and non-fatal MI as well as non-fatal MI alone. FINDINGS: Plasma alpha tocopherol concentrations (measured in subsets of patients) rose in the actively treated group (from baseline mean 34.2 micromol/L to 51.1 micromol/L with 400 IU daily and 64.5 micromol/L with 800 IU daily) but did not change in the placebo group. Alpha-tocopherol treatment significantly reduced the risk of the primary trial endpoint of cardiovascular death and non-fatal MI (41 vs 64 events; relative risk 0.53 [95% Cl 0.34-0.83; p=0.005). The beneficial effects on this composite endpoint were due to a significant reduction in the risk of non-fatal MI (14 vs 41; 0.23 [0.11-0.47]; p=0.005); however, there was a non-significant excess of cardiovascular deaths in the alpha-tocopherol group (27 vs 23; 1.18 [0.62-2.27]; p=0.61). All-cause mortality was 36 of 1035 alpha-tocopherol-treated patients and 27 of 967 placebo recipients. INTERPRETATION: We conclude that in patients with angiographically proven symptomatic coronary atherosclerosis, alpha tocopherol treatment substantially reduces the rate of non-fatal MI, with beneficial effects apparent after 1 year of treatment. The effect of alpha tocopherol treatment on cardiovascular deaths requires further study. PMID- 8622333 TI - Cyclical variation in paroxysmal supraventricular tachycardia in women. AB - BACKGROUND: Paroxysmal supraventricular tachycardia (SVT) in premenopausal women is often judged to be related to anxiety, and may be associated with the menstrual cycle. The aim of this study was to determine whether a cyclical variation of episodes of SVT exists and to correlate such variation with cyclical variation in plasma ovarian hormones. METHODS: 26 women (mean age 36 [SD 8] years; with paroxysmal SVT were screened; those with regular menses who experienced at least three episodes of paroxysmal SVT in two consecutive 48-hour ambulatory ECG recordings were included. 13 patients (aged 32 [6] years) met these criteria. Patients underwent 48-hour ambulatory ECG monitoring and determination of plasma concentrations of oestradiol-17 beta and progesterone on day 7, 14, 21, and 28 of their menstrual cycle. FINDINGS: An increase in the number and duration of episodes of paroxysmal SVT was observed on day 28 as compared to day 7 of the menstrual cycle. A significant positive correlation was found between plasma progesterone and number of episodes and duration of SVT (5.6 [2.2] ng/mL; r=0.83, p=0.0004; and r=0.82, p=0.0005), while a significant inverse correlation was found between plasma oestradiol-17 beta and number of episodes and duration of SVT (155 [22] pg/mL; r=0.89, p<0.0001; and r=0.81, p=0.0007). INTERPRETATION: Women with paroxysmal SVT and normal menses exhibit a cyclical variation in the occurrence of the arrhythmia with their menstrual cycle. There is a close correlation between the episodes of paroxysmal SVT and the plasma concentrations of ovarian hormones. These data suggest that changes in plasma levels of ovarian hormones (and their interaction) may be of importance in determining episodes of arrhythmia in such patients. The mechanisms of these effects are unknown. PMID- 8622334 TI - Role of gamma delta T cells in pathogenesis and diagnosis of Behcet's disease. AB - BACKGROUND: Behcet's disease (BD) is a multisystem disorder of unknown pathogenesis. The diagnosis is based on a set of international clinical criteria. Previous investigations have suggested that immunological cross-reactivity between peptides within streptococcal heat-shock proteins and human peptides might be involved in the pathogenesis of BD. We tested four peptides from mycobacterial heat-shock proteins to see if they specifically stimulated gamma delta T cells from BD patients. We then investigated this response to see whether it could be used as a laboratory test to diagnose BD. METHODS: We used a T-cell proliferative test to assay responses to four mycobacterial 65 kDa heat-shock protein peptides and to four homologous peptides derived from the sequence of the human 60 kDa heat-shock protein. FINDINGS: We elicited significant gamma delta T cell responses to the mycobacterial peptides in 25 (76%) of 33 patients with BD, compared with 2 (3.6%) of 55 controls with recurrent oral ulcers, systemic disease, or no disorders. The proportion of BD patients who had false-negative results decreased if the test was done during clinical manifestation of disease activity. There was a correlation between disease activity and T-cell responses. Four homologous peptides from human 60 kDa heat-shock protein also specifically stimulated T cells from patients with BD but with lower stimulation indices. INTERPRETATION: Activation of peripheral-blood mononuclear cells with the four heat-shock-protein peptides elicited significant T-cell proliferative responses by the gamma delta subset of T cells, which may regulate alpha beta T cells. Because these peptides have a high specificity for BD, this assay can be used as a laboratory diagnostic test for BD. PMID- 8622335 TI - Visual hallucinations in psychologically normal people: Charles Bonnet's syndrome. AB - BACKGROUND: Charles Bonnet's Syndrome (CBS), characterised by the presence of complex visual hallucinations in psychologically normal people, was considered for a long time to be rare. Systematic research on CBS has been limited. However, it has been realised that CBS occurs frequently in elderly, visually handicapped patients, and we have been able to study the syndrome in a large number of patients. METHODS: After screening 505 visually handicapped patients, 60 were found to meet proposed diagnostic criteria for CBS (generally, the existence of hallucinations without delusions or loss of insightful cognition.) Psychopathological characteristics, personal meaning, and the emotional impact of hallucinations, as well as factors influencing the hallucinations, were analysed. FINDINGS: Although diagnostic criteria demand merely "partial insight", all patients had full insight into the unreal nature of their hallucinations. Other characteristics varied. In 46 (77%) patients, hallucinations lacked a personal meaning. Sensory deprivation and a low level of arousal seemed to favour the occurrence of hallucinations. CBS caused considerable distress in only 17 (28%) patients. However, all patients were glad to be told that their hallucinations were not due to mental disease. The proper diagnosis had been made in only one of the 16 patients who had consulted a doctor. INTERPRETATION: Although largely unrecognised in clinical practice, CBS should be considered as a diagnosis in patients who complain of hallucinations and who meet defined diagnostic criteria. There is no proven treatment, but many patients will benefit from reassurance that their hallucinations do not imply mental illness. PMID- 8622336 TI - Preliminary evidence for genetic anticipation in Crohn's disease. AB - BACKGROUND: The term genetic anticipation is used to describe earlier onset of disease, increased severity, or both, in succeeding generations of families affected by a particular disease. This process has been linked with expanded genomic trinucleotide repeat regions in some neurological disorders. Crohn's disease, an inflammatory bowel disorder, has genetic influences, which remain undefined. We studied pairs of two-generation first-degree relatives with Crohn's disease to seek evidence for genetic anticipation in this disorder. METHODS: Through retrospective review of the records of 552 patients treated for Crohn's disease at Johns Hopkins Hospital, we identified 27 pairs of two-generation first degree relatives. We also studied 32 such pairs identified through a multicentre survey. After exploratory analyses by t tests, a generalised estimating equations approach was used to fit a marginal regression model. FINDINGS: The age at diagnosis was earlier in the younger member of the pair both in the Johns Hopkins Hospital dataset (18.9 [SE 6.9] vs 31.4 [12.0] years) and in the multicentre survey dataset (16.9 [7.4] vs 33.1 [11.9] years). The regression model confirmed the findings: the best-fitting model for the Johns Hopkins Hospital data showed an average 10.8 (SE 4.2) year difference in age at diagnosis between parent and child; that for the multicentre data showed an average difference of 15.1 (1.5) years. There was evidence of a further difference between the second and third generations. Disease was more extensive in the offspring than in the parent for 15 of the 27 pairs at Johns Hopkins Hospital; in 13 of these pairs the affected parent was the father. INTERPRETATION: The evidence of a lower age at diagnosis and a greater extent of disease in the younger member of two-generation pairs affected by Crohn's disease, as well as the association with paternal transmission, suggest that genetic anticipation does occur in Crohn's disease. A search for triplet repeat regions is warranted. PMID- 8622337 TI - Graft-versus-myeloma effect in two cases. AB - BACKGROUND: Allogeneic bone-marrow transplantation (BMT) is associated with the graft-versus-leukaemia effect because of the antileukaemic action of donor lymphocytes. We describe a graft-versus-myeloma effect after BMT in multiple myeloma. METHODS: Two patients with recurrent multiple myeloma after allogeneic BMT (T cells partly depleted, 10(5) T cells infused per kg) received leucocyte infusions obtained by leukapheresis from their original marrow donors. The patients were a 48-year-old woman and a 49-year-old man. FINDINGS: Both patients developed graft-versus-host disease and achieved complete remission of myeloma. Chimerism was complete in both patients in that all peripheral blood cells were of donor origin. INTERPRETATION: We see our results as evidence for a graft versus-myeloma effect. Using this form of adoptive immunotherapy, we could administer 1000-3000 times more T cells than with the earlier BMT. PMID- 8622338 TI - A woman with fever and a jejunal stricture. PMID- 8622339 TI - Melanoma. PMID- 8622340 TI - Tuberculosis programme changes and treatment outcomes in patients with smear positive pulmonary tuberculosis in Blantyre, Malawi. PMID- 8622341 TI - Euthanasia by stages. PMID- 8622342 TI - Betrayal by the surgeons. PMID- 8622343 TI - Prospects for antisense therapy are looking brighter. PMID- 8622344 TI - Are big casualty departments better? PMID- 8622345 TI - French patients will get protease inhibitors. PMID- 8622346 TI - Report calls on US to revamp AIDS research effort. PMID- 8622347 TI - US tobacco company settles law suit and more. PMID- 8622348 TI - Reduced quality of in-vitro clot formation with gelatin-based plasma substitutes. PMID- 8622349 TI - Seizures in healthy people with repeated "safe" trains of transcranial magnetic stimuli. PMID- 8622351 TI - Mortality of cadaveric kidney transplantation versus combined kidney-pancreas transplantation in diabetic patients. PMID- 8622350 TI - Mortality of cadaveric kidney transplantation versus combined kidney-pancreas transplantation in diabetic patients. PMID- 8622352 TI - Blood sterilisation with high temperature terminal dry heat. PMID- 8622353 TI - Fulminant meningitis due to Bacillus anthracis in 11-year-old girl during Ramadan. PMID- 8622354 TI - Use of electronic callipers for radiological and clinical measurements. PMID- 8622355 TI - A woman with bone pain, fractures, and malabsorption. PMID- 8622356 TI - A woman with bone pain, fractures, and malabsorption. PMID- 8622357 TI - A woman with bone pain, fractures, and malabsorption. PMID- 8622358 TI - Angioplasty for intermittent claudication. PMID- 8622359 TI - Hormonal contraception for men. The Task Force on Methods for the Regulation of Male Fertility. PMID- 8622360 TI - High syphilis and low but rising HIV seroprevalence rates in Madagascar. PMID- 8622361 TI - Altruism and the scientific congress. PMID- 8622362 TI - Criticism of Servier re perindopril. PMID- 8622363 TI - Jet flight leg. PMID- 8622364 TI - Ecstasy and serotonin depletion. PMID- 8622366 TI - Ethics committees. Wandsworth District Research Ethics Committee. PMID- 8622365 TI - Media wars. PMID- 8622367 TI - Hippocratic Oath. PMID- 8622368 TI - Improvised drug package for tuberculosis patients. PMID- 8622369 TI - Sputum smears for diagnosis of smear-positive pulmonary tuberculosis. PMID- 8622370 TI - Lessons to be learned from tuberculosis control in China. PMID- 8622372 TI - Epilepsy in a focus of onchocerciasis in Burkina Faso. PMID- 8622371 TI - Lessons to be learned from tuberculosis control in China. PMID- 8622373 TI - Safety of tamoxifen. PMID- 8622374 TI - Homocystinuria and transsexualism. PMID- 8622375 TI - Antimicrobial treatment in expectant management of preterm premature rupture of membranes. PMID- 8622376 TI - Rupture of the scarred uterus: prediction and diagnosis. PMID- 8622377 TI - Caesarean sections in Brazil. PMID- 8622378 TI - Growth charts for ethnic populations in UK. PMID- 8622379 TI - Thrombolytic therapy for acute myocardial infarction in older patients. PMID- 8622380 TI - Group A streptococcal skin infections after indoor association football tournament. PMID- 8622381 TI - The fourth sound of Korotkoff in pregnancy: a myth? PMID- 8622382 TI - Sick-building syndrome in a tropical city. PMID- 8622383 TI - Dealing with deception. PMID- 8622384 TI - Pathogenesis of polycythaemia vera. PMID- 8622386 TI - Perioperative steroid cover. PMID- 8622385 TI - Excess zinc and progressive cholestasis: a new disease? PMID- 8622387 TI - High-dose chemotherapy with autologous bone marrow/stem-cell rescue in lung cancer. PMID- 8622388 TI - No to DNR orders in acute stroke. PMID- 8622389 TI - Angiographic assessment of effects of bezafibrate on progression of coronary artery disease in young male postinfarction patients. AB - BACKGROUND: Bezafibrate has effects on lipid metabolism and haemostatic function. We undertook a double-blind, placebo-controlled intervention trial, the Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT), to establish whether bezafibrate (200 mg three times daily) could retard or prevent the progression of atherosclerotic lesions in dyslipidaemic male survivors of myocardial infarction who were younger than 45 years at the time of the event. METHODS: 92 patients completed an initial 3-month period of dietary intervention and were randomly assigned to treatment with bezafibrate or placebo. Dietary intervention continued throughout the trial. Coronary angiography was done at baseline and after 2 and 5 years. 81 patients (42 bezafibrate treated and 39 placebo treated) who underwent baseline angiography and at least one post treatment angiogram were included in the efficacy analysis. The primary endpoint was change in mean minimum lumen diameter. FINDINGS: The mean minimum lumen diameter decreased from baseline to the last angiographic assessment (2 or 5 years) by 0.06 mm (95% CI 0.15 reduction to 0.01 increase) in the bezafibrate group and by 0.17 mm (0.33 reduction to 0.09 increase) in the placebo group. The treatment effect was therefore 0.13 mm (95% CI 0.10 to 0.15; p=0.049). Parallel treatment effects, although not statistically significant, were observed for the secondary angiographic endpoints (mean segment diameter 0.02 mm [0.01-0.04] and percentage stenosis -3.41% [-4.00 to -2.98]). The cumulative coronary event rate was significantly lower among bezafibrate-treated than among placebo-treated patients (three vs 11 patients; p=0.02). There were significant treatment effects of bezafibrate for serum concentrations of cholesterol (-9%; p<0.001), very-low density-lipoprotein (VLDL) cholesterol (-35%; p<0.001), serum triglycerides ( 31%; p<0.001), VLDL triglycerides (-37%; p<0.001), and plasma fibrinogen (-12%; p=0.001), whereas low-density (LDL) cholesterol concentrations did not change. High density lipoprotein (HDL) cholesterol increased significantly with bezafibrate (9%; p=0.02). INTERPRETATION: The results show that bezfibrate improves dyslipidaemia, lowers plasma fibrinogen, slows the progression of focal coronary atherosclerosis, and reduces coronary events in young survivors of myocardial infarction. PMID- 8622390 TI - Ileal pouch/anal anastomosis for Crohn's disease. AB - BACKGROUND: Patients with Crohn's disease (CD) are not commonly considered as candidates for ileal pouch/anal anastomosis (IPAA). This approach has been avoided because of the poor results observed, retrospectively, in patients with an initial diagnosis of ulcerative colitis who were found to have CD on examination of the resected specimen. However, in 1985, we decided to investigate an alternative to coloproctectomy with definitive end-ileostomy by a prospective study of IPAA for selected patients with CD. METHODS: Between 1985 and 1992, 31 patients with CD, but with no evidence of anoperineal or small-bowel disease, were recruited to our study. They comprised 15 men and 16 women whose mean age was 36 years (SD 14; range 16-72). All CD patients underwent IPAA. The short-term and long-term functional results of this procedure were compared with those of 71 ulcerative colitis patients who also underwent IPAA during the same period in our unit. Mean follow-up was 59 (SD 25) months. FINDINGS: No significant differences were observed between patients with CD and ulcerative colitis in the postoperative complication rate. Of the 31 CD patients, six (19%) experienced specific complications 9 months to 6 years after surgery: three had pouch perineal fistulas, which required pouch excision in two cases; one had a pouch vaginal fistula that was treated by gracilis muscle interposition; and one had an extrasphincteric abscess, which was treated surgically. Two patients (6%), one of whom was treated for an extrasphincteric abscess, experienced CD recurrence on the reservoir, and were treated successfully with azathioprine. At 5-year follow up, there were no significant differences between patients with CD and ulcerative colitis in stool frequency (5.0 [2.0] vs 4.7 [1.4] per day; p=0.68), continence, gas/stool discrimination, leak or need for protective pads, and sexual activity. INTERPRETATION: Our results show that in selected cases of CD without anoperineal or small-bowel manifestations, IPPA can be recommended as an alternative to coloprotectomy with definitive end-ileostomy, when rectal resection is essential. PMID- 8622391 TI - Randomised, double-blind, placebo-controlled study of fluoxetine in chronic fatigue syndrome. AB - BACKGROUND: No somatic treatment has been found to be effective for chronic fatigue syndrome (CFS). Antidepressant therapy is commonly used. Fluoxetine is recommended in preference to tricyclic agents because it has fewer sedative and autonomic nervous system effects. However, there have been no randomised, placebo controlled, double-blind studies showing the effectiveness of antidepressant therapy in CFS. We have carried out such a study to assess the effect of fluoxetine in depressed and non-depressed CFS patients. METHODS: In this randomised, double-blind study, we recruited 44 patients to the depressed CFS group, and 52 to the non-depressed CFS group. In each group participants were randomly assigned to receive either fluoxetine (20 mg once daily) or placebo for 8 weeks. The effect of fluoxetine was assessed by questionnaires, self observation lists, standard neuropsychological tests, and a motion-sensing device (Actometer), which were applied on the day treatment started and on the last day. FINDINGS: The two groups were well matched in terms of age, sex distribution, employment and marital status, and duration of CFS. There were no significant differences between the placebo and fluoxetine-treated groups in the change during the 8-week treatment period for any dimension of CFS. There was no change in subjective assessments of fatigue, severity of depression, functional impairment, sleep disturbances, neuropsychological function, cognitions, or physical activity in the depressed or the non-depressed subgroup. INTERPRETATION: Fluoxetine in a 20 mg daily dose does not have a beneficial effect on any characteristic of CFS. The lack of effect of fluoxetine on depressive symptoms in CFS suggests that processes underlying the presentation of depressive symptoms in CFS may differ from those in patients with major depressive disorder. PMID- 8622392 TI - Apolipoprotein E allele distribution in parents of Down's syndrome children. AB - BACKGROUND: An increased risk of Alzheimer's disease (AD) has been reported in young mothers of Down's syndrome (DS) probands. Allele epsilon4 of the apolipoprotein E (apoE) gene is a genetic susceptibility factor for AD. We examined the distribution of apoE alleles in people with DS and their parents. METHODS: We studied 188 Danish people with non-mosaic free trisomy 21 of known parental origin (determined by DNA polymorphism analysis), and their parents, chosen from a population-based study of DS, and compared the frequency of apoE alleles with a previously published Danish control sample. FINDINGS: In people with DS, there was no significant difference in apoE allele distribution compared with controls. The frequency of allele epsilon4 in the fathers (11.8%) was significantly lower than in controls (17.4%, p=0.02). The frequency of allele epsilon4 in the mothers (19.4%) was not significantly different from that of controls. Nevertheless, in young mothers with a meiosis II error, epsilon4 frequency was 30.0%, significantly higher than in older mothers with a meiosis II error (13.0%, p=0.03). INTERPRETATION: We suggest that apoE allele epsilon4 is a risk factor for meiosis II non-disjunction in young mothers, but the biological role of apoE in oocytes remains to be investigated. PMID- 8622394 TI - Human parvovirus B19 infection associated with acute hepatitis. AB - BACKGROUND: Human parvovirus (HPV) B19 infection produces a range of clinical manifestations including erythema infectiosum in children. Here we describe seven children who had acute hepatitis with HPV B19 infection. METHODS: Hepatic dysfunction was noted in three children referred to our hospital during the course of erythema infectiosum caused by HPV B19 infection diagnosed by ELISA and PCR. The role of HPV B19 in the pathogenesis of hepatic involvement was investigated retrospectively by PCR assay of stored serum samples from 773 patients admitted to our hospital. FINDINGS: 15 patients admitted to our hospital from January, 1991, to June, 1992, were HPV B19 DNA positive, of whom four had acute hepatitis of unknown origin. These four patients were aged between 7 months and 5 years. Of the seven patients, infection with hepatitis A, B, or C viruses or Epstein-Barr virus was ruled out in six by virological examinations. INTERPRETATION: Epidemiological evidence suggests that HPV B19 can be the cause of acute hepatitis. PMID- 8622393 TI - Excess zinc associated with severe progressive cholestasis in Cree and Ojibwa Cree children. AB - BACKGROUND: High hepatic copper concentrations have been reported in several liver disorders. We report six Native Canadian children with severe chronic cholestatic liver disease, who had excess hepatic copper and zinc. METHODS: The children, aged 22 months to 8 years, came from northern Ontario, Canada. All were referred for possible liver transplantation because of end-stage liver disease. We examined explanted liver samples (or liver biopsy material in one case) by scanning transmission electronmicroscopic (STEM) X-ray elemental microanalysis and atomic absorption spectrophotometry. Samples from four controls (two with no liver pathology, one with biliary atresia, and one with Wilson's disease) were also analysed by atomic absorption spectrophotometry. FINDINGS: The explanted livers showed similar distinctive signs of advanced biliary cirrhosis, and on electronmicroscopy there were dense deposits in enlarged lysosomes and in cytoplasm. Hepatic copper concentrations were many times higher in the five patients with measurements (47.6-56.9 microgram/g dry weight) than in two samples of normal control liver tissue (2.3 and 2.9 microgram/g). Similarly, hepatic zinc concentrations were many times higher in the patients than in controls (104-128 vs 1.9-3.2 microgram/g dry weight). INTERPRETATION: The excess copper may be due to chronic cholestasis but the excess zinc is unexplained. Since three of the patients are related (shared grandparents), a genetic disorder of metal metabolism is possible, but we cannot exclude environmental factors. PMID- 8622395 TI - A woman with two young children and left chest pain. PMID- 8622396 TI - Cutaneous T-cell lymphoma (mycosis fungoides) PMID- 8622397 TI - Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder. AB - BACKGROUND: 138 infants and young children died from an endemic infantile liver cirrhosis in a circumscribed rural area of western Austria between 1900 and 1974. Frequency of the disease peaked between 1930 and 1960. It has disappeared from this area since 1974. METHODS: Clinical and genetic data on the patients was gathered; pedigrees analysed and ethnographic studies and interviews were undertaken. FINDINGS: The disease, which was clinically and pathologically indistinguishable from Indian childhood cirrhosis and hepatic copper toxicosis, was transmitted by autosomal recessive inheritance. Cow's milk, contaminated with copper from untinned copper or brass vessels, may have contributed to the development of copper toxicosis. Replacement of untinned copper cooking utensils by modern industrial vessels has eradicated the disease. INTERPRETATION: Our findings strongly suggest that the endemic Tyrolean childhood cirrhosis-and by analogy non-Wilsonian hepatic copper toxicosis occurring elsewhere-is an ecogenetic disorder requiring the involvement of both genetic and environmental factors for the disease to become manifest. PMID- 8622398 TI - Screening mammography in young women: a different perspective. PMID- 8622399 TI - Trust me, I'm a scientist: will urologists set a lead for geneticists to follow? PMID- 8622400 TI - Advice raises concern over safety of British beef. PMID- 8622401 TI - Uncertainty about mefloquine will take time to resolve. PMID- 8622402 TI - Camptothecins: new enthusiasm for an old drug. PMID- 8622403 TI - Good omens for NIH in a bad budget year. PMID- 8622404 TI - Dutch parliament supports drugs policy. PMID- 8622405 TI - An American in Keppel Street. Interview by David Sharp. PMID- 8622406 TI - The Internet: a global coffee room. PMID- 8622407 TI - Erythema annulare centrifugum and Escherichia coli urinary infection. PMID- 8622408 TI - Making sense. PMID- 8622409 TI - Making sense. PMID- 8622410 TI - Making sense. PMID- 8622411 TI - HIV quantification: useful for prediction of vertical transmission? PMID- 8622412 TI - High maternal vitamin A intake and risk of anomalies of structures with a cranial neural crest cell contribution. PMID- 8622414 TI - Testicular-cancer incidence and mortality in Slovakia, 1968-90. PMID- 8622413 TI - Hydroxyurea reacts with heme proteins to generate nitric oxide. PMID- 8622415 TI - Hair abnormalities in Alzheimer's disease. PMID- 8622416 TI - Mycoplasma-like organisms in Hodgkin's disease. PMID- 8622417 TI - Strong preference "to donate" among HIV-positive blood donors in Zimbabwe. PMID- 8622418 TI - HIV-1 group O and group M dual infection in Benin. PMID- 8622419 TI - Management of acute bacterial meningitis. PMID- 8622420 TI - Gluten sensitivity and neurological dysfunction. PMID- 8622421 TI - Anaesthesia or sedation for drug misusers. PMID- 8622422 TI - Gluten sensitivity and neurological dysfunction. PMID- 8622423 TI - Non-steroidal anti-inflammatory drugs, salicylates, and colitis. PMID- 8622424 TI - Classic Kaposi's sarcoma and volcanic soil in southern Italy. PMID- 8622425 TI - Priorities in Mostar and Manica. PMID- 8622426 TI - Weapons and health: bureaucrats beguiled by "virginity" of the atom. PMID- 8622427 TI - Down's syndrome screening in the UK. PMID- 8622428 TI - Down's syndrome screening in the UK. PMID- 8622429 TI - Poetry on rounds. PMID- 8622430 TI - Completeness of reporting of trials published in languages other than English. PMID- 8622431 TI - Adult coeliac disease. PMID- 8622432 TI - Does GBV-C cause fulminant hepatitis in Japan? PMID- 8622433 TI - Molecular evidence for transmission of hepatitis G virus by blood transfusion. PMID- 8622434 TI - Destruction of hepatitis C virus particles by haemodialysis. PMID- 8622435 TI - Familial herpes encephalitis. PMID- 8622436 TI - Myoclonic encephalopathy after exposure to aluminum. PMID- 8622437 TI - CD4 lymphopenia in elderly patients. PMID- 8622438 TI - CD4 lymphopenia in elderly patients. PMID- 8622439 TI - A young child with Kawasaki syndrome and AIDS. PMID- 8622440 TI - Circulating micrometastases following oncological surgery. PMID- 8622441 TI - Role of parasites in the pathogenesis of Chagas' cardiomyopathy. PMID- 8622442 TI - Is health a moral responsibility? PMID- 8622443 TI - Inflammatory bowel disease: germs or genes? PMID- 8622444 TI - Aminoacid infusions to prevent postoperative hypothermia. PMID- 8622445 TI - Carvedilol for heart failure, with care. PMID- 8622446 TI - Mosquitoes, models, and dengue. PMID- 8622447 TI - Reproducibility of patch-test results. PMID- 8622448 TI - Translation of clinical trials into practice: a European population-based study of the use of thrombolysis for acute myocardial infarction. European Secondary Prevention Study Group. AB - BACKGROUND: There is conclusive evidence from clinical trials that thrombolytic therapy reduces mortality in acute myocardial infarction (AMI). But still only a minority of patients admitted with AMI receive a thrombolytic drug. We have looked at a sample of AMI patients from several centres to study which factors limit the widespread use of thrombolytic therapy. METHODS: From eleven European countries, we drew a sample of 4035 patients who were discharged or died in hospital with a diagnosis of AMI between January, 1993, and June, 1994. From the medical records, we obtained the observed rate of thrombolytic use, and we defined the shortfall as the proportion of patients with no contraindication but who did not receive a thrombolytic. FINDINGS: Thrombolytic treatment had been used in 13-52% (median 36%) of the patients. Among untreated patients, we identified three groups of similar size: those whose symptom onset was more than 12 h (or unknown) before presentation; those causing diagnostic difficulty at presentation and/or lacking ECG criteria for treatment; and those with no apparent reason for withholding thrombolytic treatment (ie, the shortfall, which was 20%). Logistic regression analysis in all patients without contraindications showed that older patients and women were less likely to receive thrombolytic treatment. The adjusted odds ratio for female sex was 0.69 (95% CI 0.53-0.89), and that for age 65-74, for instance (versus 0-44), was 0.55 (0.34-0.89). These factors of age and sex were independent, and we noted that older patients and women were under-represented in the clinical trials of thrombolytic therapy for AMI. INTERPRETATION: We confirmed that only about one-third of patients admitted to European hospitals with AMI receive a thrombolytic drug. Allowing for delays to presentation and difficulty of early diagnosis, the maximum rate of thrombolysis is about 55%. The lower use of thrombolysis in the elderly may be due to their under-representation in the clinical trials; the sex difference is unexplained. PMID- 8622449 TI - Natural evolution of late whiplash syndrome outside the medicolegal context. AB - BACKGROUND: In Lithuania, few car drivers and passengers are covered by insurance and there is little awareness among the general public about the potentially disabling consequences of a whiplash injury. We took this opportunity to study the natural course of head and neck symptoms after rear-end car collisions. METHODS: In a retrospective questionnaire-based cohort study, 202 individuals (157 men; 45 women) were identified from the records of the traffic police department in Kaunas, Lithuania. These individuals were interviewed 1-3 years after experiencing a rear-end car collision. Neck pain, headache, subjective cognitive dysfunction, psychological disorders, and low back pain in this group were compared with the same complaints in a sex-matched and age-matched control group of uninjured individuals selected randomly from the population register of the same geographic area. FINDINGS: Neck pain was reported by 71 (35% [95% CI 29 42]) accident victims and 67 (33% [27-40]) controls. Headache was reported by 107 (53% [46-60]) accident victims and 100 (50% [42-57]) controls. Chronic neck pain and chronic headache (more than 7 days per month) were also reported in similar proportions (17 [8.4%; 5-13] vs 14 [6.9%; 4-12] and 19 [9.4%; 6-15] vs 12 [5.9%; 3-10]) by the two groups. Of those who reported chronic neck pain or daily headache after the accident, substantial proportions had had similar symptoms before the accident (7/17 for chronic neck pain; 10/12 for daily headache). There was no significant difference found. No one in the study group had disabling or persistent symptoms as a result of the car accident. There was no relation between the impact severity and degree of pain. A family history of neck pain was the most important risk factor for current neck symptoms in logistic regression analyses. INTERPRETATION: Our results suggest that chronic symptoms were not usually caused by the car accident. Expectation of disability, a family history, and attribution of pre-existing symptoms to the trauma may be more important determinants for the evolution of the late whiplash syndrome. PMID- 8622450 TI - Contribution of genes of the major histocompatibility complex to susceptibility and disease phenotype in inflammatory bowel disease. AB - BACKGROUND: Despite strong evidence implicating immune dysfunction and genetic predisposition in the pathogenesis of the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis, the importance of the genes of the major histocompatibility complex remains uncertain. We have investigated the contribution of HLA DRB1 and DQB genes by the strategies of non-parametric linkage analysis (affected sibling pair method) as well as association study. The relation between genotype and phenotype was examined in detail. METHODS: For linkage analysis 74 families in whom two or more siblings had inflammatory bowel disease were studied. A total of 83 affected sibling pairs were involved: in 42 pairs both siblings had Crohn's disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohn's disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohn's disease, and 472 controls. Details of sex, age of onset, disease extent, and family history were analysed. 24 patients with ulcerative colitis and 92 with Crohn's disease required surgery for refractory disease. HLA DRB1 and DQB1 gene typing was performed by polymerase chain reaction with sequence-specific primers. FINDINGS: In ulcerative colitis, the sharing of alleles among affected sibling pairs provided evidence for linkage with DRB1 locus (p = 0.017, chi2 = 5.32). Of 29 affected sibling pairs studied, only one pair shared no DRB1 DQB haplotypes. 15 shared two DRB DQB haplotypes. In contrast, no linkage was noted for Crohn's disease (42 sibling pairs; p = 0.30, chi2 = 0.16) or for inflammatory bowel disease overall (83 sibling pairs, p = 0.16, chi2 = 2.28). In the association study the rare DRB1*103 (8.3% vs 3.2% in controls) and DRB1*12 (8.6% vs 2.1% in controls) alleles were associated with ulcerative colitis (p = 0.0074, chi2 = 7.22, odds ratio OR = 2.9 [95% CI 1.3-6.4] and p = 0.0056, chi2 = 12.63, OR = 4.33 [1.8-11.0] respectively). No association with alleles representing DR2 (p = 0.55, chi2 = 0.34) was noted. No overall association was seen in Crohn's disease. In ulcerative colitis, the frequency of DRB1*0301 DQB*0201 (DR3 DQ2) was reduced in females (9.8% vs 26.3% in controls, p = 0.037, chi2 = 8.39 OR = 0.34 [0.15 0.71]), particularly in those with distal disease (2.3%, p = 0.001 vs controls, chi2 = 11.35, OR = 0.07 [0.00-0.39]). In both males and females, the DR3 DQ2 haplotype was predictive of extensive ulcerative colitis (32.9% vs 10.7% in distal disease, p < 0.01, chi2 = 10.94, OR 4.09 [1.70-10.6]) but not of need for surgery (p = 0.93, chi2 = 0.01). INTERPRETATION: These data provide strong evidence for genetic heterogeneity in inflammatory bowel disease. Genes of the major histocompatibility complex are implicated as important inherited determinants of susceptibility to ulcerative colitis and may also influence the pattern of disease. In Crohn's disease, important susceptibility genes are likely to exist outside the HLA region. PMID- 8622451 TI - Manganese toxicity in children receiving long-term parenteral nutrition. AB - BACKGROUND: In patients receiving long-term parenteral nutrition (PN), cholestatic disease and nervous system disorders have been associated with high blood concentrations of manganese. In such patients, the normal homoeostatic mechanisms of the liver and gut are bypassed and the requirement for this trace element is not known; nor has it been certain whether hypermanganesaemia causes the cholestasis or vice versa. We explored the direction of effect by serial tests of liver function after withdrawal of manganese supplements from children receiving long-term PN. We also examined the relation between blood manganese concentrations and brain lesions, as indicated by clinical examination and magnetic resonance imaging (MRI). METHODS: From a combined group of 57 children receiving PN we identified 11 with the combination of hypermanganesaemia and cholestasis; one also had a movement disorder. Manganese supplements were reduced in the first three and withdrawn in the remainder. MRI was done in two of these children. We also looked at manganese concentrations and MRI scans in six children who had received PN for more than 2 years without developing liver disease. FINDINGS: In the hypermanganesaemia/cholestasis group, four of the 11 patients died. In the seven survivors baseline whole-blood manganese was 615-1840 nmol/L, and after 4 months it had declined by a median of 643 nmol/L (p < 0.01). Over the same interval total bilirubin declined by a median of 70 mumol/L (p < 0.05). Two of these children had movement disorders, one of whom survived to have an MRI scan; this showed, with T1 weighted images, bilateral symmetrically increased signal intensity in the globus pallidus and subthalamic nuclei. Such changes were also seen in five other children--one from the hypermanganesaemia/cholestasis group and four of six in the long-term PN group without liver disease (in all of whom blood manganese was above normal). INTERPRETATION: The cholestasis complicating PN is multifactorial, but these results add to the evidence that manganese contributes. In view of the additional hazard of basal ganglia damage from high manganese levels in children receiving long-term PN, we recommend a low dose regimen of not more than 0.018 mumol/kg per 24 h together with regular examination of the nervous system. PMID- 8622452 TI - Prognostic implications of monosomy 3 in uveal melanoma. AB - BACKGROUND: A high proportion of patients with uveal melanoma die of metastatic disease. In a subgroup of uveal melanomas there is the loss of one chromosome 3. To assess the prognostic implications of this genetic anomaly, we studied 54 patients for a median of 3.4 years. METHODS: 180 patients underwent primary enucleation for malignant uveal melanoma at the Ophthalmology Department of the Universitatsklinikum Essen between 1987 and 1993. Tumour material was available for chromosome analysis and DNA preparation from 69 of these patients (for logistic reasons unlikely, we believe, to introduce bias). 15 patients were excluded from our study: nine because the methods for assessment of monosomy 3 were unsuccessful; five because of insufficient information about their relapse status; one because histopathological data were incomplete. Of the 54 remaining patients, the tumours of 16 were assessed for copy number of chromosome 3 by karyotype analysis, of 30 by comparative genomic hybridisation, and of eight by both techniques. Clinical status was assessed by contact with family doctor or a clinical check up. Statistical analysis was by the log-rank test and Cox proportional-hazard regression. FINDINGS: The tumours of 30 patients had monosomy 3. 17 (57%) of these patients relapsed with metastatic disease, and the 3-year relapse-free survival rate was 50%. By contrast, of the 24 patients whose tumours had retained both chromosomes 3, none developed metastatic disease. In univariate analysis monosomy 3 was the most significant (p < 0.0001) predictor of poor prognosis in uveal melanoma, followed by tumour location (p < 0.0007) and tumour diameter (p < 0.0021). Histopathological subtype, age, sex, extrascleral growth, and tumour thickness had no additional predictive value. INTERPRETATION: In uveal melanoma, monosomy 3 is a significant predictor of both relapse-free and overall survival. PMID- 8622454 TI - Dutch health care--a study in purple. PMID- 8622453 TI - Skin involvement in amyotrophic lateral sclerosis. AB - BACKGROUND: Patients with sporadic amyotrophic lateral sclerosis (ALS) show disorganised collagen and elastin of the dermis. We looked for inflammatory alterations to cutaneous blood vessels. PATIENTS AND FINDINGS: Seven patients with sporadic ALS were investigated; five were confined to bed, but none had bedsores. Light and electron microscopy of skin showed an oedematous dermis with collagen fibrils of irregular diameter. Small blood vessels were characterised by duplicated basement membranes and deposition of beta-amyloid protein, the main component of the neuronal and non-neuronal amyloid deposits in Alzheimer's disease. These skin changes were seen in all degrees of disability, but none was found in age-matched and sex-matched controls. INTERPRETATION: The skin in ALS is characterised by a distinctive pattern of alterations of connective tissue and blood vessels. Examination of skin in an additional and easily accessible investigation which may help elucidate the pathogenesis of ALS. PMID- 8622455 TI - The Netherlands. Discipline-planning: blessing or curse? PMID- 8622456 TI - The Netherlands. The dichotomy of medicine. PMID- 8622457 TI - The Netherlands. Randomised trials. PMID- 8622458 TI - The Netherlands. The Wageningen approach to human nutrition. PMID- 8622459 TI - The Netherlands. Research in general practice. PMID- 8622460 TI - The Netherlands. At the interface. Interview by Robin Fox. PMID- 8622461 TI - Television entertainment and the US health-care debate. AB - Some experts on the media say that entertainment can be more successful than news at providing insights into certain institutions, medicine being a good example. US television series that feature physicians as the central characters have been immensely popular. In the early series, dating back to the 1952 debut of City Hospital, the physician was an all-powerful hero working in a sparkling centre of healing, with medicine portrayed as a resource freely available to all. The programmes began to change in the 1970s. Plots centred more around the physicians' personal problems than on the patients, but economic and health policy issues were still rarely discussed adequately. In the end, what viewers come away with may lead them towards false expectations, and they may increasingly blame doctors for decisions that others make and enforce. PMID- 8622462 TI - Challenges to improving maternal health in rural Nepal. PMID- 8622463 TI - Getting ready for the next influenza pandemic. PMID- 8622464 TI - Misoprostol for third stage of labour. PMID- 8622466 TI - Case holding for tuberculosis in Africa: the patients' perspective. PMID- 8622465 TI - Foreign aid and tuberculosis control policy in the Federated States of Micronesia. PMID- 8622467 TI - Terminology for carcinoma-in-situ of the breast. PMID- 8622468 TI - Terminology for carcinoma-in-situ of the breast. PMID- 8622469 TI - Terminology for carcinoma-in-situ of the breast. PMID- 8622470 TI - Terminology for carcinoma-in-situ of the breast. PMID- 8622471 TI - Terminology for carcinoma-in-situ of the breast. PMID- 8622472 TI - Pregnancy terminations after oral contraception scare. PMID- 8622473 TI - Computed tomography in staging of primary gastric lymphoma. PMID- 8622474 TI - Hypothyroid nails and evolution. PMID- 8622475 TI - Rubella in mothers. PMID- 8622476 TI - Morphine for relief of cancer pain. PMID- 8622477 TI - Low-molecular-weight heparin during instability in coronary artery disease. PMID- 8622478 TI - New Austrian mutation in BRCA1 gene detected in three unrelated HBOC families. PMID- 8622479 TI - HMO formularies and care costs. PMID- 8622480 TI - A tranquil city with a high tranquilliser intake. PMID- 8622481 TI - Alleged role of medical personnel in genocide in Rwanda. PMID- 8622482 TI - USA aborts international family planning. PMID- 8622483 TI - Trial by ordeal in clinical research. PMID- 8622485 TI - Treatment of Crohn's disease. PMID- 8622484 TI - Correlation of Pneumocystis carinii PCR with clinical diagnosis in immunocompromised patients. PMID- 8622486 TI - Irritable bowel syndrome: psychology, biology, and warfare between false dichotomies. PMID- 8622487 TI - Are the effects of cholesterol lowering drugs always equal? PMID- 8622488 TI - Antidepressant-associated fatal intrahepatic cholestasis. PMID- 8622489 TI - Selective embryonic survival of conceptuses with sickle-cell and beta thalassaemia traits? PMID- 8622490 TI - The role of television in public health efforts in Lebanon. PMID- 8622492 TI - Different origins of expanded repeats for Haw River syndrome and dentatorubral pallidoluysian atrophy. PMID- 8622491 TI - Treatment of benign prostatic hyperplasia. The Scandinavian BPH Study Group. PMID- 8622493 TI - Value of preoperative pregnancy test in risk management. PMID- 8622494 TI - Group A streptococcus, pyoderma, and rheumatic fever. PMID- 8622495 TI - K-RAS mutation in the tumour of Ferrante I of Aragon, King of Naples. PMID- 8622496 TI - Dementia after Edinburgh. PMID- 8622497 TI - Schizophrenia and the 5-HT2A receptor gene. PMID- 8622498 TI - Pregnancy-associated osteoporosis. PMID- 8622499 TI - Primary angioplasty for acute myocardial infarction: is the balloon half full or half empty? PMID- 8622500 TI - Genetic abnormalities, male infertility, and ICSI. PMID- 8622501 TI - Barriers to controlling pain in patients with cancer. PMID- 8622502 TI - Impact of adjuvant therapy on quality of life in women with node-positive operable breast cancer. International Breast Cancer Study Group. AB - BACKGROUND: Adjuvant therapy for early breast cancer is effective but may be toxic. Our aim was to investigate the impact of the presence, timing, and duration of adjuvant chemotherapy on patients' perceptions of their quality of life (QL). METHODS: International Breast Cancer Study Group trial VI assessed adjuvant chemotherapy in 1475 premenopausal and perimenopausal patients, and trial VII assessed adjuvant tamoxifen or chemoendocrine therapy in 1212 postmenopausal patients with node-positive breast cancer. Patients were asked to complete a QL questionnaire-single-item linear analogue self-assessment scales measured physical wellbeing, mood, appetite, and perceived adjustment/coping. QL was assessed in this way at the beginning of treatment, 2 months after the start of treatment, every 3 months, and at 1 and 6 months after recurrence. FINDINGS: Baseline QL scores decreased as the number of involved axillary nodes increased (for example, mean mood score: 66.1 for women with one positive node, 66.4 for two to four positive nodes, 61.3 for five to nine positive nodes, and 59.1 for ten or more positive nodes; p = 0.008 for trends), and were lower in patients with oestrogen-receptor-negative than in patients with oestrogen-receptor positive tumours (61.4 vs 66.3, p = 0.0009). All treatment groups showed substantial improvement in QL scores during adjuvant therapy. Patterns of QL scores reflected presence, duration, and timing of cytotoxic treatment. Longer initial cytotoxic therapy delayed improvement in QL scores. Later cytotoxic therapy had transient adverse effects. Anticipation of future therapy also affected QL scores. INTERPRETATION: Overall, chemotherapy had a measurable adverse effect on QL, but this effect was transient and minor compared with patients' adaptation/coping after diagnosis and surgery. This finding should encourage patients and doctors to choose appropriate adjuvant therapy with less concern for initial toxicity. PMID- 8622503 TI - Mortality in adults with self-reported asthma. Copenhagen City Heart Study Group. AB - BACKGROUND: On the question of whether asthma shortens survival the published work gives no clear answer. We have prospectively analysed overall and cause specific mortality in persons with self-reported asthma. METHODS A sample of 13 540 individuals (6104 men) 20 years of age or older, randomly selected from the general population of the city of Copenhagen, was followed for 17 years. FINDINGS: Survival in participants with self-reported asthma was significantly poorer than in non-asthmatics, the excess mortality being limited to pulmonary mortality. After statistical adjustment for age, length of school education, and smoking, women with asthma had a 1.7 higher risk of dying than women without asthma (95% confidence interval 1.3--2.2). Although the relative risk (RR) of dying with asthma was slightly lower in men (RR = 1.5, 95% Cl 1.2-1.9) the difference between sexes was not significant. The results were similar within smoking groups and the highest risk of death associated with asthma was seen among never-smokers (RR = 2.1, 95% Cl 1.6-2.3). Inclusion of one-second forced expiratory volume, in % predicted, in the mortality analyses showed that the increased risk of death associated with asthma was mediated mainly through reduced lung function. INTERPRETATION: We conclude that, in the general population, self-reported asthma is associated with a slight excess of mortality, mainly from respiratory diseases. PMID- 8622504 TI - Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. AB - BACKGROUND: About 13% of cases of non-obstructive azoospermia are caused by deletion of the azoospermia factor (AZF), a gene or gene complex normally located on the long arm of the Y chromosome. Oligozoospermia is far more common than azoospermia, but little is known about genetic causes. We investigated whether severe oligozoospermia is caused by AZF deletions and, if so, whether those deletions are present in mature spermatozoa. METHODS: By PCR, we tested leucocyte DNA, from 35 men who presented at infertility clinics and who had severe oligozoospermia, for the presence of 118 DNA landmarks scattered across the Y chromosome. In the two men in whom Y-chromosome deletions in leucocyte DNA were detected, we also tested leucocyte DNA from the individuals' fathers, and in one man we tested sperm DNA. FINDINGS: In two men with ejaculate sperm counts of 40 000-100 000 per mL, we detected Y-chromosome deletions in leucocyte DNA similar in location to those previously reported in azoospermic individuals. No Y chromosome deletions were detected in the fathers of the two men. For one of the two men, sperm DNA was tested, and it showed the same Y-chromosome deletion seen in leucocytes. INTERPRETATION: The Y-chromosome deletions in these two men are de novo mutations, and are therefore the cause of their severe oligozoospermia. Not only is the absence of AZF compatible with spermatogenesis, albeit at reduced rate, but also the resultant sperm bear the mutant Y chromosome. Because intracytoplasmic sperm injection is increasingly used as a means of circumventing oligozoospermia, AZF deletions could be transmitted by this practice, and would probably result in infertile sons. In cases of severe oligozoospermia, it may be appropriate to offer Y-DNA testing and genetic counselling before starting assisted reproductive procedures. PMID- 8622505 TI - Association between schizophrenia and T102C polymorphism of the 5 hydroxytryptamine type 2a-receptor gene. European Multicentre Association Study of Schizophrenia (EMASS) Group. AB - BACKGROUND: An association between schizophrenia and the T102C polymorphism of the gene for 5-hydroxytryptamine type 2a (5-HT2a) receptor has been reported; the proportion of allele 2 of this polymorphism is higher than expected among schizophrenic patients. We looked for an association between schizophrenia and this variant of the 5-HT2a-receptor gene in a large multicentre study. METHODS: Seven countries recruited 1210 participants: 571 white schizophrenic patients and 639 ethnically matched controls. All patients had a diagnosis of schizophrenia or schizoaffective disorder. High-molecular-weight DNA was isolated from lymphocytes. PCR amplification and restriction enzyme digestion was used to examine sequence variation of the 5-HT2a-receptor gene. Genotypes 1/1, 1/2, and 2/2 were assigned. Woolf's method was used to look for an association between schizophrenia and allele 2 and the 2/2 genotype. FINDINGS: We found a significant overall association between schizophrenia and allele 2 with an odds ratio of 1.3 (95% Cl 1.1-1.53, p = 0.003). No evidence for heterogeneity was observed between samples. We found a highly significant excess of the 1-2/2-2 genotypes in schizophrenia (p = 0.008) with a relative risk of 1.7 (1.22-2.36) and an attributable fraction of 0.35. INTERPRETATION: Our findings suggest that the gene for 5-HT2a-receptor, or a locus in linkage disequilibrium with it, confers susceptibility to schizophrenia. Allele 2 is common in the population and it is, therefore, likely that this variant, or a nearby polymorphism, may affect a significant proportion of schizophrenic patients. PMID- 8622506 TI - Nuclear magnetic resonance imaging with hyperpolarised helium-3. AB - BACKGROUND: Magnetic resonance imaging (MRI) relies on magnetisation of hydrogen nuclei (protons) of water molecules in tissue as source of the signal. This technique has been valuable for studying tissues that contain significant amounts of water, but biological settings with low proton content, notably the lungs, are difficult to image. We report use of spin-polarised helium-3 for lung MRI. METHODS: A volunteer inhaled hyperpolarised 3He to fill the lungs, which were imaged with a conventional MRI detector assembly. The nuclear spin polarisation of helium, and other noble gases, can be greatly enhanced by laser optical pumping and is about 10(5) times larger than the polarisation of water protons. This enormous gain in polarisation easily overcomes the loss in signal due to the lower density of the gas. FINDINGS: The in-vivo experiment was done in a whole body MRI scanner. The 3He image showed clear demarcation of the lung against diaphragm, heart, chest wall, and blood vessels (which gave no signal). The signal intensity within the air spaces was greatest in lung regions that are preferentially ventilated in the supine position; less well ventilated areas, such as the apices, showed a weaker signal. INTERPRETATION: MRI with hyperpolarised 3He gas could be an alternative to established nuclear medicine methods. The ability to image air spaces offers the possibility of investigating physiological and pathophysiological processes in pulmonary ventilation and differences in its regional distribution. PMID- 8622507 TI - Congenital enterocyte heparan sulphate deficiency with massive albumin loss, secretory diarrhoea, and malnutrition. AB - BACKGROUND: The molecular basis of protein-losing enteropathy is unknown. However it has been shown that sulphated glycosaminoglycans may be important in regulating vascular and renal albumin loss. METHODS: We describe three baby boys who presented within the first weeks of life with massive enteric protein loss, secretory diarrhoea, and intolerance of enteral feeds. All required total parenteral nutrition and repeated albumin infusions. No cause could be found in any case despite extensive investigations, including small intestinal biopsy sampling, which were repeatedly normal. FINDINGS: By specific histochemistry, we detected gross abnormality in the distribution of small intestinal glycosaminoglycans in all three infants, with complete absence of enterocyte heparan sulphate. The distribution of vascular and lamina propria glycosaminoglycans was, however, normal. INTERPRETATION: The presentation of these infants suggests that enterocyte heparan sulphate is important in normal small intestinal function. PMID- 8622508 TI - A new treatment for severe malabsorption due to radiation enteritis. PMID- 8622509 TI - The challenge of the dementias. Writing Committee, Lancet Conference 1996. AB - Cognitive decline is a common feature of ageing, sometimes gentle at other times less so. It manifests as a wide spectrum from cognitive impairment that is not dementia to Alzheimer's disease and to the much rarer but potentially enlightening familial forms. The Lancet's 1996 international conference was held in Edinburgh, UK, on April 25 and 26. The meeting brought together epidemiologists, geneticists, neuropsychologists, neuropathologists, clinicians, and imaging specialists, and those concentrating on the social and ethical aspects of the dementias. Half the conference was devoted to discussion that crossed specialty boundaries. This report highlights the major areas of agreement and controversy and points to opportunities for future multidisciplinary research. PMID- 8622510 TI - Sources in science: who can we trust? AB - Journalists' sources of medical and scientific news seem limitless--journals, press releases, press conferences, newsletters, scientific meetings, and "tipoffs". But whether any one piece of information satisfies two important criteria-journalistic interest and scientific credibility-is another matter. Peer reviewed journals, in particular, are perceived to be trustworthy sources. Yet, there are increasing concerns in scientific publishing about commercial pressures from pharmaceutical companies, honorary authorship, scientific error, and outright fraud, which journalists cannot be expected to detect. That is down to the scientific community, which must recognise the importance of maintaining impartial sources of public information. PMID- 8622511 TI - Non-linear dynamics for clinicians: chaos theory, fractals, and complexity at the bedside. PMID- 8622512 TI - Artificial blood--bad news for vampires? PMID- 8622513 TI - HIV tests for babies to be mandatory in USA. PMID- 8622514 TI - Clinical algorithm for malaria in Africa. PMID- 8622515 TI - Clinical algorithm for malaria in Africa. PMID- 8622516 TI - Clinical algorithm for malaria in Africa. PMID- 8622517 TI - Clinical algorithm for malaria in Africa. PMID- 8622518 TI - Prostate-specific antigen in black men. PMID- 8622519 TI - Severe adverse reaction to moclobemide. PMID- 8622520 TI - Hypertension with moclobemide. PMID- 8622521 TI - Haemolytic anaemia after mitral valve repair. PMID- 8622522 TI - Do specific hyperimmunoglobulins aggravate clinical course of tick-borne encephalitis? PMID- 8622523 TI - Excess zinc associated with cholestasis. PMID- 8622524 TI - Creutzfeldt-Jakob disease. PMID- 8622525 TI - Creutzfeldt-Jakob disease. PMID- 8622526 TI - Creutzfeldt-Jakob disease. PMID- 8622527 TI - Creutzfeldt-Jakob disease. PMID- 8622528 TI - Euthanasia. PMID- 8622529 TI - Euthanasia. PMID- 8622530 TI - Illicit fentanyl in Europe. PMID- 8622531 TI - Long-case clinical examinations. PMID- 8622532 TI - Uninsured in the USA. PMID- 8622533 TI - Clot formation and gelatin-based plasma substitutes. PMID- 8622534 TI - Effect of increasing dietary folate on red-cell folate. PMID- 8622535 TI - Non-invasive detection of endothelial dysfunction with 30 MHz transducer. PMID- 8622536 TI - Metronome in Parkinson's disease. PMID- 8622537 TI - HHV8 DNA in normal gastrointestinal mucosa from HIV seropositive people. PMID- 8622538 TI - HHV8 cell-associated viraemia and clinical presentation of Mediterranean Kaposi's sarcoma. PMID- 8622540 TI - HCV and non-Hodgkin lymphoma. PMID- 8622539 TI - Direct vasoactivity of frusemide. PMID- 8622541 TI - Convulsive effects of tacrine. PMID- 8622542 TI - Serotonin transporter gene polymorphism and affective disorder. PMID- 8622543 TI - Serotonin transporter gene polymorphism and affective disorder. PMID- 8622544 TI - Clinical staging system for AIDS in Edinburgh. PMID- 8622545 TI - Renal transplantation despite systemic viral infections. PMID- 8622546 TI - Mouth-to-mouth resuscitation and Helicobacter pylori infection. PMID- 8622547 TI - Risk of iatrogenic transmission of Helicobacter pylori by gastroscopes. PMID- 8622548 TI - Amnesia from sarin poisoning. PMID- 8622549 TI - Ethics and validity of hypoglycaemia unawareness studies. PMID- 8622550 TI - Ginseng as a cause for Stevens-Johnson syndrome? PMID- 8622551 TI - Management of impending sterility. PMID- 8622552 TI - Inducible nitric oxide synthase in uterine smooth muscle. AB - The expression of inducible nitric oxide synthase (iNOS) mRNA in rat uterus upon in vivo stimulation with lipopolysaccharide (LPS) was studied by reverse transcription and polymerase chain reaction. The injection of LPS induced an increase in mRNA levels of a macrophage-type iNOS. In unstimulated rats, low levels of iNOS mRNA was detected in the uterus and lungs, but absent or negligible in the kidneys and liver. NO was produced in the LPS-treated uterus by addition of 1 to 1000 microM L-arginine. The production of NO in uterine tissue that faces the outside of the body may provide a bacteriocidal protective function against microorganisms in physiological condition. However, NO produced in a large amounts by cytokine and LPS may play some pathological reaction during septic shock or infection. PMID- 8622553 TI - Cocaine and butyrylcholinesterase (BChE): determination of enzymatic parameters. AB - In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogeneous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of Km and Vmax with cocaine as the substrate. The KM (11.9 microM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The Vmax (1.17 microM/min) is 50 fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity. PMID- 8622555 TI - The effect of aminophylline on the contraction threshold of rat diaphragm fibers and its modification by 9-aminoacridine. AB - We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (200 X) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT toward more negative values of the resting membrane potential (Vm). 2) 9 aminoacridine, a drug that diminishes Ca2+ release from the sarcoplasmic reticulum (SR), shifts the CT toward more positive values: 3) this effect is overcome by aminophylline. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca2+ release from the SR. PMID- 8622554 TI - Glutamate-independent long term depression in rat hippocampus by activation GABAA receptors. AB - Long-term depression (LTD) of synaptic transmission is a candidate for a neuronal model of forgetting and is considered to be important in learning and memory. The present study employed extracellular recording in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum. Muscimol induced a time and concentration-dependent LTD at a frequency of stimulation of 0.01 Hz or in the absence of stimulation. The LTD was reversed by stimulation at 1 Hz. The ability of muscimol to act via GABAA receptors was confirmed by the ability of bicuculline (5 microM) to reverse the LTD. The NMDA non-selective glutamate antagonists kynurenate, failed to modify the LTD. receptor antagonist 2-AP5, the selective metabotropic antagonist L(+)AP3 and the non-selective glutamate antagonists kynurenate, failed to modify the LTD. (1S,3R)-ACPD, a selective agonist at metabotropic receptors, did not induce LTD. The lack of involvement of glutamate receptors in muscimol induced LTD in our protocol may indicate a novel type of long-lasting synaptic depression PMID- 8622556 TI - Phosphorylation of enkephalins enhances their proteolytic stability. AB - Pharmacological action of enkephalins as opioid peptides is limited because of their rapid degradation by endoproteases. A novel approach is used in this study to prolong the life of those peptides. Phosphorylation of N-terminal tyrosine residue is found to have a profound influence in improving the stability of [Met]enkephalin and [Leu]enkephalin against the action of aminopeptidase M. Whereas, breakdown of [Met]enkephalin and [Leu]enkephalin is essentially complete in less than one min when incubated at 37 degree C with purified aminopeptidase M (EC3.4.11.2; substrate:enzyme = 1:0.1) in Tris buffer (pH 7.02), the corresponding phospho analogs are still detected 60 min after start of incubation. The rate of disappearance of phospho-[Met]enkephalin and phospho [Leu]enkephalin follows first-order kinetics with half-lives of 7.3 and 8.3 min, respectively. PMID- 8622557 TI - 2-[125I]Iodomelatonin binding sites in the quail heart: characteristics, distribution and modulation by guanine nucleotides and cations. AB - To investigate whether melatonin has a direct action on the cardiovascular system, putative melatonin receptors were studied in quail heart membrane preparations using the specific melatonin agonist 2-[125I]iodomelatonin (125I]Mel, as the radioligand. The [125I]mel binding demonstrated in the mature quail heart was saturable, highly 5.2 pM; Bmax = 1.32 +/- 0.25 fmol/mg protein; n = 8). The linear Scatchard plots and the close to unity Hill coefficient indicated a single class of binding sites. The pharmacological profile was in the affinity order of 2-iodomelatonin = 2-phenylmelatonin > melatonin > 6 chloromelatonin >> 6-hydroxymelatonin > 6-sulphatoxymelatonin >> N acetylserotonin>>>5-hydroxytryptamine. Guanosine 5'-triphosphate and guanosine 5' O-(3-thiotriphosphate) (GTP gammaS) dose dependently inhibited the binding. Ten microM GTPgammaS lowered the binding affinity by 50% in saturation studies. The order of potency of inhibition by cations was: Ca2+ > Mg2+ > Li+ > Na+ > K+ > choline chloride. Contrary to most other melatonin binding sites, millimolar concentrations of Ca2+ and Mg2+ did not promote binding in the quail heart membranes. In vitro autoradiography indicated homogenous labeling in the heart. Our results demonstrated [125I]Mel binding sites in the quail heart. That guanine nucleotides and Na+ inhibited the binding indicated that these putative melatonin receptors are coupled to guanine nucleotide-binding proteins (G-proteins). PMID- 8622558 TI - Baclofen, a gamma-aminobutyric acid B agonist, modifies hormonal secretion in pituitary cells from infantile female rats. AB - Recent work from our laboratory has demonstrated that the activation of GABA B adenohypophyseal receptors by baclofen inhibits pituitary hormone secretion under basal (PRL) or stimulated conditions (PRL and LH) in adult female rats, suggesting a hypophyseal site of action in addition to the central site previously described. Since different patterns of hormone secretion are observed in infantile and adult rats, the purpose of the present study was to determine whether GABA B pituitary receptors were involved in endocrine responses at early stages of development. Pituitary cells of 12 day-old female rats were cultured in vitro and the effect of baclofen was determined in the presence or absence of stimulatory factors. Baclofen (1.10(-9), 1.10(-7) and 1.10(-5) M) did not alter basal LH or FSH secretion but significantly inhibited the LHRH induced gonadotropins release after 30 or 60 minutes of incubation (after 60 minutes of incubation LH (%): control: 100 +/- 5.6; BACL(1.10(-7)): 134.5 +/- 25.8; LHRH(1.10(-7)): 596.7 +/- 85.9; LHRH(1.10(-7))-BACL(1.10(-7)): 374.7 +/- 48.0; p<0.01. FSH (%): control: 100 +/- 6.5; BACL(1.10(-7): 103.7 +/- 6.5; LHRH(1.10( 7)): 283.9 +/- 29.3; LHRH(1.10(-7))-BACL(1.10(-7): 183.0 +/- 20.0; p<0.01). Baclofen did not significantly modify either basal or TRH-stimulated PRL or TSH secretion. These results show that baclofen has direct effects on the of adenohypophyseal cells of immature rats and such effects are different from those observed in adult rats, and depend on the stage of development of the neuroendocrine controls of each cellular type. PMID- 8622559 TI - Plasmin stimulates expression of endothelin-1 mRNA and endothelin-1 release in vascular endothelial cells. AB - Incubation of cultured porcine aortic endothelial cells (ECs) with plasmin resulted in a significant and concentration-dependent increase in endothelin-1 (ET-1) release from the cells. This increasing effect was completely inhibited by aprotinin but not by tranexamic acid, thereby suggesting that the plasmin-induced stimulation of ET-1 release requires the catalytic site but not the lysine binding site, in plasmin molecule. Plasmin stimulated the expression of prepro ET 1 mRNA in ECs. Actinomycin D chase experiments suggested that enhanced stability of ET-T mRNA could not account for the above plasmin-induced stimulation. It is likely that plasmin potentiates the endothelial ET-1 production, probably by the stimulation of ET-1 gene transcription. It remains to be seen whether the endothelial ET-1 production is enhanced after the thrombolytic therapy. PMID- 8622561 TI - Chronic lateral ventricle infusion of a pineal gland-derived decapeptide alters pulsatile secretion of LH in rats. AB - The object of this investigation was to determine whether chronic lateral ventricle infusion of a pineal gland-derived antigonadotropic decapeptide (AGD) would affect pulsatile luteinizing hormone (LH) release in conscious, unrestrained male rats. Adult male Harlan SD rats were bilaterally orchiectomized and maintained under conditions of controlled photoperiods and temperature. After three (Experiment one) or four (Experiment two) weeks each was fitted stereotaxically with a stainless steel cannula for infusion into the right lateral ventricle. Each cannula was attached to a subcutaneous osmotic minipump filled either with artificial cerebrospinal fluid (CSF) or AGD in CSF (0.5 microgram/microliter). CSF (1.0 microliter/hr) or the AGD (0.5 microgram/microliter hr) was infused over a period of four days. Blood samples for determined of LH by radioimmunoassay were obtained at five minute intervals from a Tygon microbore cannula inserted via a femoral artery into the abdominal aorta. LH pulses were defined and identified with a computerized deconvolution algorithm designed to determine spontaneous LH secretory events. Although mean LH levels were not significantly reduced, LH secretory pulse frequency and nadirs were significantly decreased by ADG infusion (p<0.01). Additionally, LH secretory pulse amplitude and LH secretory response to LHRL administration were significantly increased (p<0.01) by AGD treatment. These results confirm initial reports of depressive effects of the AGD on LH secretion and support its hypothesized central site of action. PMID- 8622560 TI - Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide. AB - Neem seed preparations contain not only azadirachtin as the active insect antifeedant or growth regulator but also a variety of their limonoids, some of which are cytotoxic to N1E-115 neuroblastoma (mouse), 143B.TK- osteosarcoma (human) and Sf9 (insect) cultured cell lines. The most potent of these limonoids is nimbolide with an IC50 ranging from 4 to 10 microM, and averaging 6 microM for the three cell lines. Other limonoids of decreasing potency and their average IC50 values (microM) are epoxyazadiradione 27 microM, salannin 112 microM, and nimbin, deacetylnimbin and azadirachtin each >200 microM (practically nontoxic). Nimbolide at 10 microM acts rapidly in the neuroblastoma cells to induce blebbing associated with disruption of plasma membranes almost instantaneously and 50% loss of cell viability with 30 min. At 5 microM nimbolide, the cells become elongated and assume a neuronal shape accompanied by spikes and lamellipodia within 1-2 hr followed shortly thereafter by extensive cytological changes and and vacuolization associated with irreversible processess leading to cell death. Calcium is apparently not involved the cytotoxic effect since a calcium-free medium, leading to profound morpholigical changes, does not alter the sensitivity to nimbolide. In contrast, epoxyazadiradione requires higher concentrations and a few hr for 50 % viability loss without major morphological changes, indicating a difference in mode of action for nimbolide and epoxyazadiradione. and epoxyazadiradione. PMID- 8622562 TI - Messenger RNA expression of somatostatin receptor subtypes in human and rat gastric mucosae. AB - In several tissues including gastric mucosa, somatostatin displays various biological effects. Five seven-transmembrane-domain somatostatin receptor subtypes (SSTR1-5) have been recently cloned and only SSTR1 has been shown to be present in the human stomach. We used the polymerase chain reaction on reverse transcripts (RT-PCR) to characterize further the SSTR's mRNAs in human and rat gastric mucosae and in the human gastric tumoral cell-line HGTL. The SSTR1-5's mRNAs were found in both human fundic and antral mucosae as well as in the HGT1 cell and rat antrum. The four SSTR2-5's mRNA's but not SSTR1's were detected in the rat fundic mucosa. Furthermore, the use of rat isolated and purified fundic mucosal cells allowed us to localize SSTR2-5 in the parietal cell-enriched fraction, whereas SSTR2 and SSTR5 were the only subtypes found in the endocrine cell-enriched fraction. These results are the first to demonstrate the presence of five SSTR's mRNA subtypes in the stomach. PMID- 8622563 TI - Immunosuppressants and TGF-beta 1 accelerated and prolonged the nitric oxide/oxyradicals-dependent suppression by dexamethasone in paw edema of mice. AB - Dexamethasone (Dex, 0.3 mg/kg, s.c.) did not suppress histamine and ischemic paw edema of mice up to 1 hr. However, given TGF-beta 1 (0.3 microgram/kg, i.p.), Dex suppression appeared early as 30 min (36% and 42%). When Dex (0.1 mg/kg, s.c.) was injected 6 hr before the assay, Dex alone, TGF-beta 1 +/- Dex, FK506 (10 mg/kg, oral) +/- Dex, cyclosporin (CsA, 30 mg/kg, oral) +/- Dex, rapamycin (Rapa, 10 mg/kg, i.p.) +/- Dex, deoxyspergualin (DSP, 10 mg/kg, i.p.) +/- Dex, did not suppress the edemata (less than 11%). Nevertheless, if Dex and TGF-beta 1 were dosed together with one of these immunosuppressants, suppressions of histamine and ischemic edema were 53%, 45% (FK506), 45%, 49% (CsA), 44%, 48% (Rapa) and 39%, 51% (DSP), respectively. Glucocorticoid (GC) receptor (GR) complex contains heat shock proteins such as hsp56 (or CsA-binding protein: CyP-40), hsp70 and hsp90. FK506, Rapa and TGF-beta 1 receptor I (TR-I) bind FK-binding protein-12 (FKBP-12). FK506 and Rapa bind also hsp56. CsA binds CyP-40. DSP binds hsp70 and/or hsp90. These bindings might change or stabilize the conformation of GR complex resulting in edema suppressions. Nitric oxide synthase (NOS) inhibitors, superoxide dismutase (SOD), catalase, mannitol and cycloheximide, reversed the edema suppressions by TGF-beta1 +/- immunosuppressant at 30 min and 6 hr after Dex. Endogenous NO, O2- and/or .OH seemed to be essential for edema suppressions. Our demonstration in vivo may offer a theoretical support for clinicians to adopt combination therapy of immunosuppressant(s) and GC. PMID- 8622564 TI - Age-related effects of aluminum ingestion on brain aluminum accumulation and behavior in rats. AB - Both aluminum and aging have been associated with neurobehavioral changes in mammals. This study assessed in young (21 day old), adult (8 months), and old rats (16 months) the effects of prolonged aluminum ingestion on open-field activity and passive-avoidance conditioning. Aluminum was administered in drinking water as aluminum nitrate at doses of 0, 50, and 100 mg Al/kg/day over a 6.5 month period. There were no aluminum effects on the horizontal and vertical activity in an open-field, or in passive-avoidance learning in any group. On the other hand, measurement of aluminum concentrations in a number of brain regions indicated that the olfactory bulb and the rhachidical bulb were the regions with the highest aluminum levels, while the cortex and the thalamus were the cerebral regions showing the lowest aluminum content. For most brain regions analyzed the highest aluminum concentrations were found in young rats, which would indicate that early stages of the life cycle must be considered for enhanced brain aluminum accumulation. PMID- 8622565 TI - Inhibition by noncompetitive NMDA receptor antagonists of apomorphine-induced climbing behavior in mice. AB - The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is an important mediator of several forms of neural and behavioral plasticity. In the present study, we examined the potential role of NMDA receptors in the glutamatergic modulation of dopaminergic function at the postsynaptic dopamine receptor by determining the effects of NMDA antagonists on apomorphine-induced climbing behavior in mice. The noncompetitive NMDA receptor antagonists, MK-801, ketamine, dextrorphan, and dextromethorphan attenuated the apomorphine-induced climbing behavior at does well below those that produce untoward side effects. These results suggest that the NMDA receptors play important roles in the glutamatergic modulation of dopaminergic function at the postsynaptic dopamine receptors that mediate the apomorphine-induced climbing behavior in mice. PMID- 8622566 TI - A Ca2+/CAM protein kinase associated with Ca2+ transport in sarco(endo)plasmic vesicles from tracheal smooth muscle. AB - In this work, we show evidence to support the existence of a Ca2+ calmodulin (CAM) dependent protein kinase and a substrate, a 17 kilodaltons (KDA) polypeptide being both associated to sarco(endo)plasmic reticulum vesicles from tracheal smooth muscle. Anti-CAM drugs such as compound 48/80 inhibited this protein kinase activity and this inhibition was reversed in the presence of Ca2+CAM. Moreover, as a result of this phosphorylation, there is a significant increase in the ATP dependent Ca2+ transport in these sarco(endo)plasmic vesicles. PMID- 8622567 TI - Effect of motilin, somatostatin and bombesin on gastroduodenal myoelectric activity in sheep. AB - The effects of motilin, erythromycin, somatostatin and bombesin on antroduodenal myoelectric activity were investigated in conscious sheep. Myoelectric recordings were obtained from electrodes chronically implanted on the antrum and duodenal bulb. Peptides or erythromycin were infused intravenously (i.v.) during 5 min. Antagonists were injected i.v. as a bolus. Neither motilin (2.5-80 ng/kg/min) nor erythromycin (2-16 micrograms/kg/min) modified the antroduodenal myoelectric activity, although a single bolus of these compounds (250 ng/kg and 50 micrograms/kg respectively) increased the antral activity. Somatostatin at 5 ng/kg/min induced a decrease in the myoelectric activity of antrum and duodenum. However, doses of 10 to 40 ng/kg/min evoked a duodenal phase III-like activity with a subsequent quiescence period and a concomitant inhibition of the antral activity. These effects were reproduced by bombesin (2.5 to 40 ng/kg/min). Furthermore, an initial increase in the myoelectric activity and in the frequency of slow waves were recorded in the antrum when the highest doses were used. On the other hand, atropine (0.2 mg/kg) or hexamethonium (2 mg/kg) caused a long lasting inhibition of antroduodenal myoelectric activity. These cholinergic antagonists abolished the effects induced by somatostatin (20 ng/kg/min) but not those evoked by bombesin but not motilin are putative modulators of the migrating myoelectric complex (MMC) in sheep. Moreover, a cholinergic neural pathway is involved in the somatostatin but not in the bombesin-induced effects. PMID- 8622568 TI - Elevated norharman plasma levels in alcoholic patients and controls resulting from tobacco smoking. AB - Plasma norharman and harman levels were measured by solvent extraction and HPLC with fluorescence detection in alcohol-dependent patients undergoing in-patient abstinence treatment and in control subjects. In both groups, randomly collected samples from smokers contained higher mean norharman levels than those from non smokers. In three volunteers norharman concentrations rose sharply after smoking of one or two cigarettes and declined to near-basal levels within one hour after one cigarette. When 12 patients kept a smoking-free interval of at least 6 h, they had similarly low plasma norharman concentrations (20 +/- 8 pg/ml) as 18 non smoking control subjects (17 +/- 8 pg/ml) or as 13 smoking controls who had abstained from smoking (20 +/- 6 pg/ml). Ten of the patients smoked one cigarette and within 5-10 min attained norharman levels of 177 +/- 147 pg/ml plasma. The high prevalence of smokers among chronic alcoholics probably explains the previous finding of elevated norharman plasma levels in these patients. PMID- 8622570 TI - Evidence of sodium-dependent glucose transport in human erythroleukemia cells. AB - Sodium-dependent transport of D-glucose has been reported only in epithelial cells of small intestine and kidney, and well-differentiated tumors thereof. We observed a two-fold decrease (p < 0.05) in the intracellular distribution volume (Vic, defined as steady-state intracellular uptake divided by extracellular concentration) of the non-metabolized D-glucose analog 3-O-methylglucose (3-O-MG) when logarithmically growing K562 cells (an anaplastic human erythroleukemia) were incubated 3 h in choline-substituted, phosphate buffered saline (PBS) rather than Na+ PBS, each containing a glucose concentration ([Glu]) of 5.6 mM. Electromechanically measured cellular volume Vc differed < 10% between the different media. In Na+ PBS, Vic (3-O-MG) was approximately twice Vc and declined progressively when [Glu] was reduced to 2.8 and 0.1 mM. We conclude that, in a balanced salt medium containing glucose as the only energy source, K562 cells express a concentrative mechanism having characteristics consistent with Na(+) dependent transport of glucose. PMID- 8622569 TI - Diet-induced changes in serum cholesterol concentrations do not alter tryptophan hydroxylation rate or serotonin concentrations in gerbil brain. AB - The relationship between serum cholesterol concentrations and serotonin synthesis rate in brain was examined in Mongolian gerbils chronically fed diets containing 20% fat (safflower oil, beef tallow or butterfat) with or without added cholesterol (0.5%, dry weight). After 22 days on these diets, circulating cholesterol concentrations ranged between approximately 1.5 and approximately 20 mumol/ml. Despite this enormous range, in vivo tryptophan hydroxylation rate, and serotonin and 5-hydroxyindoleacetic acid concentrations in cerebral cortex, hypothalamus and brainstem did not differ significantly among the diet groups. Tryptophan concentrations in serum and brain were also unaffected. These results do not support the hypothesis that the link between depression, suicide and violent deaths and below-normal or reduced serum cholesterol concentrations in humans involves an alteration in serotonin synthesis and/or release by brain neurons. PMID- 8622571 TI - The catecholaminergic stimulation of gonadotropin-releasing hormone release by GT1-1 cells does not involve phosphoinositide hydrolysis. AB - Gonadotropin-releasing hormone (GnRH) secretion is modulated by a large number of neuromediators, among which catecholamines play a central role. Previous results have shown that both dopamine (DA) and norepinephrine (NE) stimulate GnRH secretion in GT1 neuronal cell lines. These stimulatory effects appear to involve D1-dopaminergic and beta 1-adrenergic receptors positively coupled to adenylate cyclase. However, in spite of a similar efficacy of these catecholamines to stimulate GnRH secretion, DA is two-fold more efficacious than NE to stimulate the formation of cyclic AMP. This rises the possibility that other signaling pathways and other receptor subtypes could be involved in the catecholaminergic stimulation of GnRH release. Since the signaling pathway triggered by phosphoinositide hydrolysis is a potent stimulator of GnRH secretion and appears to mediate the releasing actions of neuromediators such as histamine and endothelin, we investigated if this signaling pathway was also involved in the catecholaminergic stimulation of GnRH release in GT1 cells. Both DA and NE stimulated inositol phosphates production in GT1-1 cells with a very low potency and long latency with respect to GnRH secretion. Inositol phosphates production was stimulated by DA and NE only at a concentration of 100 microM, i.e. two to three orders of magnitude higher than the effective concentrations to maximally stimulate GnRH secretion. The effects of both catecholamines do not appear to be secondary to the stimulation of cyclic AMP production, since treatment of GT1-1 cells with forskolin did not affect inositol phosphates production. The effects of DA and NE on inositol phosphates production were blocked by specific antagonists such as SCH-23390, spiroperidol and phentolamine. However, specific dopaminergic agonists such as SKF-38393 and bromocriptine, or adrenergic agonists such as clonidine, methoxamine and isoproterenol were not capable of stimulating inositol phosphates production. Thus, due to the low potency and apparent non specificity of these effects, we conclude that inositol phosphates production is not involved in the catecholaminergic stimulation of GnRH release. PMID- 8622572 TI - The endogenous estrogen metabolite 2-methoxyestradiol induces apoptotic neuronal cell death in vitro. AB - We examined the effects of 2-methoxyestradiol, a metabolite of estradiol, on cell death in retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cell cultures. Cell death was induced by 2-methoxyestradiol in a concentration-dependent manner. Estradiol and 2-methoxyestradiol failed to induce cell death. The cell death response to 2-methoxyestradiol was sensitive to the protein synthesis inhibitor cycloheximide and the apopain inhibitor Ac-Asp-Glu-Val-Asp-H(aldehyde). 2 Methoxyestradiol also induced internucleosomal for and endogenous neuroactive steroid metabolite in the etiology of some neurodegenerative diseases. PMID- 8622573 TI - Effects of passive immunization against alpha-2u-globulin and supplementation with alpha-2u-globulin on serum gonadotrophins and testicular activity. AB - The purpose of the present study was to evaluate the effects of anti-alpha-2U globulin on pituitary-gonadal functions in male rats. Adult male rats were given injections of anti-alpha-2u-globulin (1 mg/day) for 14 days, when they were killed 7 days after the last injection, serum levels of gonadotrophins and testosterone measured by radioimmunoassays, were less, testicular delta 3 beta- and 17 beta-hydroxy-steroid dehydrogenase (3 beta- and 17 beta-HSD) activities were suppressed, spermatogenesis was inhibited and serum level of alpha-2u globulin was decreased in anti-alpha-2u-globulin treated rats. Administration of alpha-2u-globulin (1.5 mg/day) for 7 days to anti-alpha-2u-globulin treated rats reversed the 3beta-HSD and 17beta-HSD activities and serum levels of gonadotrophins, testosterone and alpha-2u-globulin, while spermatogenesis was restored to normal. The results indicate that changes in testicular steroidogenesis and spermatogenesis in rats after passive immunization against alpha-2u-globulin may be due to decrease in availability of endogenous alpha-2u globulin. PMID- 8622574 TI - Stress- and treatment-induced elevations of cortisol levels associated with impaired declarative memory in healthy adults. AB - Two studies investigated the association between cortisol levels and memory performance in healthy adults. In a first study, 13 subjects were exposed to a brief psychosocial laboratory stress ("Trier Social Stress Test") with a subsequent test of declarative memory performance. Results indicated a significant negative relationship between stress-induced cortisol levels and performance in the memory task, i.e. subjects with high cortisol response to the stressor showed poorer memory performance. In a second experiment it was investigated if cortisol, alone, i.e. independent of psychological stress, would also impair memory function. In this study, 40 healthy subjects received either 10 mg cortisol or placebo orally. One hour later they were tested for procedural and declarative memory and spatial thinking. Subjects who received cortisol showed impaired performance in the declarative memory and spatial thinking tasks but not in the procedural memory task. From these results we conclude that in healthy adults elevated free cortisol levels are associated with impaired memory function. PMID- 8622575 TI - Dynamic uncoupling and recoupling of perfusion and oxidative metabolism during focal brain activation in man. AB - Changes in glucose consumption, lactate production, and blood oxygenation were measured during prolonged neuronal activation (4-6 min) in human primary visual cortex using dynamic magnetic resonance spectroscopy and imaging. A decrease of steady-state glucose by 40% because of enhanced use by 21% was accompanied by a transient accumulation of lactate with a peak value of 170% 2.5 min after stimulation onset. Rapid blood hyperoxygenation indicating "uncoupling" of blood flow and oxidative metabolism was followed by a return to basal levels over 3 min. Thus, initial nonoxidative glucose consumption during functional activation is gradually complemented by a slower adjustment of oxidative phosphorylation that "recouples" perfusion and oxygen consumption at a new equilibrium. PMID- 8622576 TI - Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates. AB - For the first time, 13C NMR signals are shown from 13C-enriched, low-level tricarboxylic acid (TCA) cycle intermediates from extracts of normal cardiac tissue. As the low tissue content of the key intermediates alpha-ketoglutarate (alpha-KG) and succinate (SUC) in normal, well perfused tissues has until now precluded direct NMR detection from intact tissues and tissue extracts, 13C NMR signal from glutamate has generally been used to infer the isotopomer patterns of intermediates that are in chemical exchange with glutamate. However, the required assumptions regarding intracellular compartmentation for such indirect analysis have not been previously tested, as glutamate is largely cytosolic while the TCA cycle enzymes are located in the mitochondria. Chromatographic isolation of alpha KG and SUC from heart tissue extracts allowed isotopomer analysis to be performed for comparison with that of glutamate. At steady state, a direct relationship between glutamate and alpha-ketoglutarate isotopomers was found, but succinate isotopomers matched those of glutamate only in hearts that displayed negligible contributions from the oxidation of unlabeled endogenous carbon sources. PMID- 8622577 TI - Diffusion weighted fMRI at 1.5 T. AB - Functional magnetic resonance imaging (fMRI) is capable of detecting task-induced blood oxygenation changes using susceptibility sensitive pulse sequences such as gradient-recalled echo-planar imaging (EPI). The local signal increases seen in the time course are believed to be due to an increase in oxygen delivery that is incommensurate with oxygen demands. To help isolate the sources of functional signal changes, the authors have incorporated various forms of diffusion weighting into EPI pulse sequences to characterize the apparent mobility of the functionally modulated protons. Results suggest that the majority of the functional signal at 1.5 T arises from protons that have apparent diffusion coefficients that are approximately four or five times higher than that of brain tissue. This implies that significant functional signal sources are either protons within the vascular space or protons from the perivascular space that is occupied by cerebrospinal fluid. PMID- 8622578 TI - Noninvasive measurement of protein concentration. AB - At selected magnetic field strengths, protein and water proton spin-lattice relaxation rates are sensitive to the concentration of rotationally immobilized peptide nitrogen because of field dependent heteronuclear cross relaxation coupling between protein proton and nitrogen-14 spins that is carried to the water by proton homonuclear cross-relaxation. Measurement of the water proton spin-lattice relaxation time, or a signal amplitude proportional to it, may provide a noninvasive measure of peptide bond concentration, which provides a direct measure of immobilized protein content in most tissues. The approach using protein gels in two magnetic field strengths is demonstrated. At 66.7 mT; the proton Zeeman energy matches one of the peptide nitrogen transitions dominated by the unaveraged nuclear electric quadrupole interaction; cross-relaxation between the protons and nitrogen-14 is efficient. At 77.5 mT, the proton Zeeman energy is not matched with the nitrogen energy and proton-nitrogen cross-relaxation is not efficient. It is shown that the difference in the water proton spin-lattice relaxation rates on and off the energy level match condition is a linear function of the rotationally immobilized protein concentration. PMID- 8622579 TI - Effects of osmotically driven cell volume changes on diffusion-weighted imaging of the rat optic nerve. AB - The apparent diffusion coefficient (ADC) of the rat optic nerve was measured in vitro, using magnetic resonance imaging, to determine the effects of changes in cellular volume fraction on the diffusion of tissue water. Nerve ADC was determined under conditions of cell membrane depolarization and (i) increased intracellular volume, (ii) decreased intracellular volume, and (iii) negligible volume change. Depolarization alone had little affect on ADC, whereas volume changes produced strong, reversible effects. Increased cell volume decreased ADC and vice versa. These results are consistent with the view that changes in the extracellular space are the major source of ADC changes in brain tissue. PMID- 8622580 TI - Continuous assessment of relative cerebral blood volume in transient ischemia using steady state susceptibility-contrast MRI. AB - The utility of a noninvasive steady state susceptibility-contrast MRI technique for continuous measurement of relative cerebral blood volume (rCBV) during global transient ischemia and subsequent hyperemia in a feline ischemia model is demonstrated. The measurements were obtained during a 10-min period of occlusion and 1-h period of reperfusion. Maximal hyperemic responses in gray matter, basal ganglia, and white matter (observed at 7,7, and 5 min, respectively) were 1.9 +/- 0.5, 1.8 +/- 0.3, and 1.7 +/- 0.6 times greater than baseline CBV (mean +/- SEM). Thirty to forty minutes after onset of reperfusion, CBV returned to normal. Thereafter, it decreased below baseline, nearing the control level by 1 h after onset of reperfusion. Steady state susceptibility-contrast MRI permits continuous, in vivo mapping of alterations in CBV. PMID- 8622581 TI - A critical assessment of noise-induced errors in 31P MRS: application to the measurement of free intracellular magnesium in vivo. AB - Phosphorus magnetic resonance spectroscopy (31P MRS) is a noninvasive technique that has been used to estimate free intracellular magnesium concentration (free [Mg2+]). Free [Mg2+] is computed from the chemical shift separation between the alpha- and beta-phosphate resonances of ATP. The current study was undertaken to critically assess the influence of noise effects in estimating free [Mg2+] in rat brain subjected to moderate parasagittal fluid percussion-induced injury. We show that contrary to published data, free [Mg2+] does not significantly change for up to 4 h after moderate trauma in different rat strains and using different surface coils. Before injury, free [Mg2+] = 0.56 +/- 0.11 (mean +/- SD, n = 36) and 4 h post-trauma, free [Mg2+] = 0.56 +/- 0.28. Our results suggest that explanations for this discrepancy comprise errors of chemical shift assignments accompanying low signal-to-noise ratios and the method of analysis employed. Indeed, the authors propose that spectra of beta-ATP signal-to-noise ratio less than 5:1 will produce significant noise-induced errors. We conclude that without knowledge of the inherent errors in 31P MRS spectroscopy and appropriate statistical analysis, caution should be exercised in calculating free [Mg2+] and using these changes as a basis for proposing pharmacotherapeutic interventions. PMID- 8622582 TI - In vivo phosphorus spectroscopy of human skin. AB - Skin 31P MRS measurements might detect metabolic damage from irradiation, chemotherapy, or ischemia. Although rat and cadaver data have demonstrated this potential (C.D. Cuono, et al., Plast. Reconstr. Surg. 81, 1-11 (1988), H.W. Klein, et al., Ann. Plast. Surg. 20, 547-551 (1988)), few studies of in vivo phosphorus human skin spectra have been published (A. Zemtsov, et al., J. Dermatol. Surg. Oncol. 15, 1207-1211 (1989), A Zemtsov, et al., J. Am. Acad. Dermatol. 30, 959-965 (1994)), and those likely reflect underlying muscle as much as skin. To separate 31P skin and muscle spectra, we have developed a unique two layer "flotation" phantom for mapping coil sensitivity and an associated semiempirical two-power RF depth-resolved technique. Phantom and method have been applied in a study of 17 normal volunteers to obtain human in vivo 31P skin spectra uncompromised by muscle contamination and to quantitate ratios of major phosphometabolites. Skin results consistently showed low ratios of phosphocreatine (PCr) to adenosine triphosphate (ATP), high levels of phosphomonoester (PME), P(i), and phosphodiester (PDE) relative to PCr, and demonstrated a shift in pH toward greater alkalinity, compared to that with simultaneous muscle results. PMID- 8622583 TI - Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy. AB - A method for simultaneous extraction of lipids and water-soluble metabolites from a single cell sample was developed and optimized for NMR spectroscopy. Intermediary metabolites in cultured M2R mouse melanoma cells and changes therein in response to challenge with melanotropin were studied by 31P and 13C NMR. Cells were extracted with methanol, chloroform, and water (1:1:1, v/v/v). The contents of the chloroform and methanol-water phases were separated and quantitatively recovered. The contents of the upper and lower phases compared well with the homologous fractions obtained by perchloric acid and Folch's lipid extraction methods. The pH of the extracts remained within the physiologic range, eliminating potential deleterious effect on cellular metabolites. The water phase contained minimal amounts of salts, making these extracts amenable to subsequent analytical procedures. Obtaining lipid- and water-soluble metabolites from the same sample enables characterization of metabolic pathways that bridge the two cellular components in a quantitative manner. PMID- 8622584 TI - Lanthanide-based susceptibility contrast agents: assessment of the magnetic properties. AB - The T2* contrast efficacy of paramagnetic contrast agents is dependent on their magnetic properties. Vibrating sample magnetometry (VSM) and the Live Chan NMR method have been used to evaluate the influence of ligand structure on the bulk magnetic susceptibility (BMS) of low-molecular weight (LMW) lanthanide chelates. VSM was also used for the BMS assessment of LMW lanthanide chelates covalently attached to cross-linked starch particles. The ligand structure had no influence on the BMS of the gadolinium (Gd) and dysprosium (Dy) chelates. The mean BMS value of the Dy-chelates was 1.8 fold higher than that of the Gd-chelates. The holmium (Ho) DTPA-BMA chelate had a similar BMS to that of Dy-DTPA-BMA while the lowest BMS was found for europium (Eu(III)) DTPA-BMA. The covalent attachment of Gd-DTPA and Dy-DTPA to a cross-linked starch particle had no impact on their intrinsic magnetic properties. The BMS data were in good accordance with those obtained for non-particulate bound LMW Dy- and Gd-chelates. The magnetic susceptibility of the Gd-DTPA labeled particles was described by the Curie law, indicative of no magnetic interactions between Gd-DTPA molecules. The magnetic susceptibility of the Dy-DTPA labeled particles followed the Curie-Weiss law with a Curie-Weiss temperature of about-2 K, indicating magnetic interactions. The magnetic susceptibility of Dy-DTPA will, however, not be affected by such magnetic interactions at physiological temperatures. PMID- 8622585 TI - Multiexponential T2 relaxation in degenerating peripheral nerve. AB - The multiexponential T2 relaxation spectrum of peripheral nerve undergoing Wallerian degeneration has been measured both in vivo and in vitro. Degeneration of the sciatic nerve of the amphibian Xenopus laevis was induced by crush injury, and T2 relaxation spectra of the nerve were measured at several times up to 35 days following injury. Histologic evidence verified that the nerve underwent Wallerian degeneration. Relaxation spectra were observed to undergo measurable changes as degeneration progressed, the most evident being a reduction from three well-resolved T2 components to one and a decline in the fraction of the spectra associated with the shortest T2 component. The former appears to reflect the collapse and loss of myelinated fibers, while the latter a combination of interstitial edema and myelin loss. PMID- 8622586 TI - Effect of repetitive ischemia on myocardial oxygen tension in isolated perfused and hypoperfused rat hearts. AB - The objective of this study was to determine the effects of repetitive ischemia on myocardial oxygen tension (pO2), consumption, and delivery in crystalloid normoperfused (perfusion pressure>70 mmHg) and hypoperfused (perfusion pressure approximately 50 mmHg) constant flow isolated rat hearts. EPR oximetry with lithium phthalocyanine was used to measure myocardial pO2. Baseline myocardial pO2 (means +/- SE) was 185 +/- 13 mmHg (normoperfused) and 162 +/- 14 mmHg (hypoperfused). Myocardial pO2 fell to < 1 mmHg during no-flow ischemia. After recovery from repetitive ischemia, myocardial pO2 and coronary resistance increased significantly in all hearts; oxygen consumption and left ventricle work decreased in normoperfused hearts, although not significantly compared with controls, and did not change significantly in hypoperfused hearts. Increased myocardial pO2 in the normoperfused group may be due to decreased oxygen consumption and/or increased local delivery, while increased myocardial pO2 in the hypoperfused hearts is due to increased local oxygen delivery. PMID- 8622587 TI - Comparison of ultrafast dipyridamole magnetic resonance imaging with dipyridamole SestaMIBI SPECT for detection of perfusion abnormalities in patients with one vessel coronary artery disease: assessment by quantitative model fitting. AB - The value of ultrafast MRI for detection of myocardial perfusion abnormalities in patients with coronary artery disease (CAD) was assessed in 10 patients with stable angina pectoris and angiographically proven one-vessel CAD using double level short-axis ultrafast MRI with bolus injection of gadolinium-DTPA and tomographic technetium-99m SestaMIBI imaging (SPECT) during dipyridamole-induced coronary hyperemia. Abnormally perfused regions were assessed with SPECT and MRI in all (100%) patients. Agreement in localization between arteriography and SPECT was 80%; between arteriography and MR, 70%; and between SPECT and MR, 90%. The signal intensity increase after the bolus injection of gadolinium-DTPA using a linear fit, and the slope of gadolinium-DTPA wash-in using double exponential model fitting were significantly different between abnormally and normally perfused regions. These preliminary results demonstrate the potential of dipyridamole ultrafast MR to monitor stress-induced flow maldistribution in patients with single vessel CAD. PMID- 8622588 TI - Calibration of the radio frequency field for magnetic resonance imaging. AB - We have developed and validated the performance of a novel slice selective pulse sequence that allows direct calibration of the RF field using a simple rectangular pulse. The new sequence offers a number of substantial advantages. It operates at steady state and has an accurate calibration response at short repetition times. The slice selection train is insensitive to RF field strength changes caused by patient loading. The issue of patient motion has been addressed in our data collection and analysis routines. The applicability of the method to human scanning has been demonstrated in the automated RF power calibration routine of a commercial imaging system. PMID- 8622589 TI - Spoiling of transverse magnetization in gradient-echo (GRE) imaging during the approach to steady state. AB - The signal evolution behaviors and corresponding image appearances for different methods of spoiling or refocusing the transverse magnetization in short TR gradient-echo imaging during the approach to steady state were investigated experimentally and using computer simulations based on the Bloch equations. Specifically, ideally spoiled, gradient-spoiled, gradient-refocused, and RF spoiled pulse sequence configurations were studied. This study showed that, for the gradient-spoiled configuration, the signal evolution is position and phase encoding order-dependent and, under typical imaging conditions, can deviate substantially from the ideally spoiled signal evolution at some spatial positions, resulting in intensity banding image artifacts. For the gradient refocused configuration, the signal evolution oscillates toward the steady state and, generally, does not closely approximate that of ideal spoiling, resulting in different image contrast or image blurring. Using RF spoiling, the signal evolution closely approximates the ideally spoiled case for flip angles less than approximately 20 degrees and T2 values of less than approximately 200 ms and results in relatively artifact-free images. Also, this study showed that, for RF spoiling, an RF-pulse phase-difference increment other than 117 degrees, such as 84 degrees may be optimal for gradient-echo imaging during the approach to steady state. PMID- 8622590 TI - Imaging of the active B1 field in vivo. AB - The authors describe a method for accurate in vivo multislice imaging of the active component of the B1 field which is based on a previously proposed method, which uses the signal intensity ratio of two images measured with different excitation angles, and a repetition time TR 5 > or = 5 T1. The new method essentially reduces repetition and scan time by means of an additional compensating pulse. The suppression of T1 effects by this pulse are verified with simulations and measurements. Further investigations concerned the influence of slice selective excitation and magnetization transfer in multislice imaging to the B1 field determination. The stability and accuracy of the presented method is shown by several phantom and in vivo measurements. With the described method the active B1 field can be determined in vivo in 23 cross-sections in less than 6 min. PMID- 8622591 TI - An algorithm for eddy currents symmetrization and compensation. off. AB - Eddy currents, which are induced in the magnet cryostat by pulsed magnetic field gradients in MRI, generate undesired eddy fields within the imaging volume. In this work, an automated and computerized algorithm to compensate these eddy currents is presented. The compensation is done in two steps: (i) Eddy fields are symmetrized electronically with an R-C filter. (ii) The symmetric eddy fields are compensated by another R-C filter. The compensation algorithm is iterative; therefore, errors that remain from one iteration are eliminated in the next iteration. Hence, the compensation process is very robust and accurate. It is shown that all the even harmonics of the eddy fields are eliminated by the symmetrization process, but the odd field harmonics remain. The amplitude of these odd harmonics can be significantly reduced if the gradient coils are designed so that the field they generate is spatially similar to the eddy fields. PMID- 8622592 TI - Statistical methods of estimation and inference for functional MR image analysis. AB - Two questions arising in the analysis of functional magnetic resonance imaging (fMRI) data acquired during periodic sensory stimulation are: i) how to measure the experimentally determined effect in fMRI time series; and ii) how to decide whether an apparent effect is significant. Our approach is first to fit a time series regression model, including sine and cosine terms at the (fundamental) frequency of experimental stimulation, by pseudogeneralized least squares (PGLS) at each pixel of an image. Sinusoidal modeling takes account of locally variable hemodynamic delay and dispersion, and PGLS fitting corrects for residual or endogenous autocorrelation in fMRI time series, to yield best unbiased estimates of the amplitudes of the sine and cosine terms at fundamental frequency; from these parameters the authors derive estimates of experimentally determined power and its standard error. Randomization testing is then used to create inferential brain activation maps (BAMs) of pixels significantly activated by the experimental stimulus. The methods are illustrated by application to data acquired from normal human subjects during periodic visual and auditory stimulation. PMID- 8622593 TI - Inhomogeneity correction using an estimated linear field map. AB - A fast and robust method for correcting magnetic resonance image distortion due to field inhomogeneity is proposed and applied to spiral k-space scanning. The method consists of acquiring a local field map, finding the best fit to a linear map, and using it to deblur the image distortions due to local frequency variations. The linear field map is determined using a maximum likelihood estimator with weights proportional to the pixel intensity. The method requires little additional computation and is robust in low signal regions and near abrupt field changes. Additionally, it can be used in combination with other deblurring methods. The application of this method is illustrated in conjunction with a multislice, T2-weighted, breath-held spiral scan of the liver. PMID- 8622594 TI - Effects of trandolapril and verapamil on glucose transport in insulin-resistant rat skeletal muscle. AB - We have used an animal model of insulin resistance-the obese Zucker (fa/fa) rat to test whether oral administration of the non-sulfhydryl-containing angiotensin converting enzyme (ACE) inhibitor, trandolapril, alone or in combination with the Ca2+-channel blocker, verapamil, can induce a beneficial effect on insulin stimulated glucose transport and metabolism in skeletal muscle. Insulin stimulated 2-deoxyglucose (2-DG) uptake in the isolated epitrochlearis muscle was less than 50% as great in obese animals compared with lean (Fa/-) controls (P < .05), but was significantly improved in the obese group by both short-term (6 hours, +33%) and long-term (14 days,+70%) oral treatment with trandolapril. Verapamil treatment alone did not alter insulin-stimulated 2-DG uptake in muscle, but simultaneous administration of verapamil and trandolapril resulted in the most pronounced effect on insulin-stimulated 2-DG uptake (+106%). Long-term treatment with trandolapril alone and in combination with verapamil significantly increased muscle glycogen (+26% to 27%), glucose transporter GLUT-4 protein (+27% to 31%), and hexokinase activity (+21% to 49%), and decreased plasma insulin levels (-23% to -29%). Muscle citrate synthase activity was enhanced only when trandolapril and verapamil were administered in combination (+24%). We conclude that the long-acting, non-sulfhydryl-containing ACE inhibitor, trandolapril, alone and in combination with the Ca2+-channel blocker, verapamil, can significantly improve insulin-stimulated glucose transport activity in skeletal muscle of the insulin-resistant obese Zucker rat, and that this improvement is associated with favorable adaptive responses in GLUT-4 protein levels, glycogen storage, and activities of relevant intracellular enzymes of glucose catabolism. PMID- 8622595 TI - Hyperglycemia results in an increase in myocardial interstitial glucose and glucose uptake during ischemia. AB - The purpose of this investigation was to assess the effects of hyperglycemia, in the absence of changes in plasma insulin and arterial free fatty acid (FFA) levels, on interstitial glucose levels and glucose uptake across the left ventricular wall during ischemia in domestic swine. Insulin secretion was suppressed with a continuous infusion of somatostatin. Arterial FFA levels remained stable due to the suppression of insulin. Microdialysis probes were used to estimate changes in interstitial glucose and lactate, and were placed in the subepicardium and the subendocardium of the left anterior descending ([LAD] ischemic) coronary artery perfusion bed and in the midmyocardium of the circumflex ([CFX] nonischemic) perfusion bed. The LAD coronary artery was cannulated and perfused with blood from the femoral artery through an extracorporal perfusion circuit. Ischemia was induced in the LAD perfusion bed by reducing the flow of the LAD perfusion pump by 60% for 50 minutes, and was followed by 30 minutes of reperfusion. Twenty minutes into the ischemic period, seven animals were given a bolus injection of 50% glucose (200 mg/kg) followed by a glucose infusion (10 mg/kg/min), resulting in an increase in arterial glucose levels from 5 to 13 mmol/L in the hyperglycemic group. Hyperglycemia resulted in a marked increase in dialysate glucose during ischemia and a greater than twofold increase in glucose extraction and uptake. Dialysate glucose correlated with plasma glucose in all three perfusion beds. In conclusion, hyperglycemia, in the absence of an increase in insulin and a decrease in arterial FFA, resulted in a doubling of glucose extraction, delivery, and uptake, which corresponded to the twofold elevation in interstitial glucose during ischemia. PMID- 8622596 TI - Relative effects of high saturated fatty acid levels in meat, dairy products, and tropical oils on serum lipoprotein and low-density lipoprotein degradation by mononuclear cells in healthy males. AB - To determine the effects of three saturated fatty acid combinations on lipoprotein metabolism, we fed 18 21- to 32-year-old men three diets in a crossover design for 28-day periods separated by washout periods of 4 to 6 weeks. The men self-selected a prescribed diet at home emphasizing saturated fat as the visible fat for 1 week. Then, they ate experimental diets providing 40%, 15%, 17%, and 7% of food energy, respectively, as total, saturated, monounsaturated, and polyunsaturated fatty acids, levels representing amounts available in the US diet. Different test fatty acid combinations, given at 4 to 6 energy% (en%) each, were incorporated into food products: 12:0 + 14:0, 14:0 + 16:0, and 16:0 + 18:0. Test fatty acids were equalized by giving free myristic acid (14:0) with palm kernel oil or butter and sheanut butter (high in 18:0) with lard. The diet highest in 12:0 + 14:0 also provided 4.2 en% 16:0, the most common saturated fatty acid in the US diet. Mean apparent absorption of all fatty acids was at least 90%. The three diets produced similar concentrations of serum total and low density lipoprotein (LDL) cholesterol and apolipoprotein (apo) B-100 regardless of the apo E phenotype of the subjects. Compared with baseline, the experimental diets affected serum high-density lipoprotein (HDL) concentrations (P < .06), with the highest values occurring on diet 12:0 + 14:0. When the change from baseline in receptor-mediated degradation of 125I-LDL in freshly isolated mononuclear cells (MNC) was stratified by apo E phenotype, diet 16:0 + 18:0 produced a 30% increase, compared with a 9% decrease on diet 12:0 + 14:0 and a 6% increase on diet 14:0 + 16:0 in subjects with the apo E3/3 phenotype. These results suggested that different saturated fatty acid combinations, consumed at levels typical of availability in the United States and with diets providing ample unsaturated fat, had similar cholesterolemic properties in healthy males despite some subtly different effects on lipoprotein metabolism. PMID- 8622597 TI - Lysophosphatidylcholine stimulates the expression and production of MCP-1 by human vascular endothelial cells. AB - Lysophosphatidylcholine (LPC increased monocyte chemoattractant protein-1 (MCP-1) messenger RNA concentrations in human umbilical vein endothelial cells (HUVECs). A time-course study showed that the increase in MCP-1 mRNA levels peaked at 6 hours after treatment with LPC. The effect of LPC on the accumulation of MCP-I mRNA levels in HUVECs depended on LPC concentration, and the maximal effect was obtained at 50 micromol / L LPC, which induced a sixfold increase in MCP-1 mRNA levels. The amount of MCP-1 released from HUVECs measured using an enzyme-linked immunosorbent assay (ELISA) showed a 38% increase in the presence of 50 micromol/L LPC, but not in the presence of phosphatidylcholine or lysophosphatidylethanolamine. Coincubation with staurosporine, a potent inhibitor of protein kinase C (PKC) activity, attenuated the LPC-induced increase in MCP-1 mRNA levels by 53%. These results indicate that LPC can induce an increase in MCP 1 mRNA concentrations and stimulate the release of MCP-1 protein from HUVECs, and that the effect of LPC on the MCP-1 gene may be mediated through activation of the PKC pathway. PMID- 8622598 TI - Evidence for the paracrine action of islet-derived corticotropin-like peptides on the regulation of insulin release. AB - In view of recent evidence for the endogenous synthesis of proopiomelanocortin (POMC) by pancreatic islets, we have assessed (1) the release of POMC-derived corticotropin (ACTH)-like peptides (ACTH-LP) from isolated perifused rat islets, and (2) the potential paracrine modulatory effect on insulin output of these putative secretagogues. Islets perifused at a glucose concentration of 3.3 mmol/L secreted ACTH-LP at 0.15 +/- 0.005 ng/islet/10 min, which was increased by 17 fold at 16.7 mmol/L glucose. Islets statically incubated with different concentrations of medium glucose plus synthetic 1-39ACTH at 55 pmol/L showed a significant increase of insulin release at 8 (by 79%) and 16 (by 119%) mmol/L glucose, but not at 4 mmol/L. To determine the possible cis-directed effects of these endogenously released islet ACTH-LP on insulin secretion, we either blocked their biological action by immunoneutralization with an ACTH-specific antiserum or prevented their receptor interaction by addition of the ACTH-inhibiting polypeptide (CIP) to the incubation medium. In the presence of 16.7 mmol/L glucose, the rate of insulin output decreased by approximately 25% upon exposure to the antiserum and by approximately 50% in the presence of CIP. The foregoing observations would therefore suggest that both (1) the elaboration of ACTH-LP by isolated perifused islets and (2) the stimulation of islet insulin release by exogenous 1-39ACTH in static incubation occur as a function of glucose concentration in the incubation medium, and that (3) the newly-secreted endogenous ACTH-LP operate in a cis mode to enhance islet insulin output in a manner analogous to that of exogenously added ACTH species. These results strongly support the view that islet-elaborated ACTH-LP are important physiological paracrine modulators of insulin secretion. PMID- 8622599 TI - Role of cortisol in the metabolic response to stress hormone infusion in the conscious dog. AB - The role of cortisol in directing the metabolic response to a combined infusion of glucagon, epinephrine, norepinephrine, and cortisol (stress hormones) was investigated. Chronically catheterized, conscious fasted dogs were studied before hormone infusion and after a 70-hour stress hormone infusion containing glucagon, epinephrine, norepinephrine, and cortisol (n = 11) or containing all these hormones except cortisol (n = 5). Combined stress hormone infusion increased arterial plasma glucagon, cortisol, epinephrine, and norepinephrine approximately sixfold. Whole-body glucose production (Ra), glycogenolysis, and gluconeogenesis were assessed using tracer and arteriovenous-difference techniques. The absence of an increase in cortisol during stress hormone infusion attenuated the increase in arterial plasma glucose concentration and Ra (delta 81 +/- 16 v 24 +/- 3 mg/dL and 1.7 +/- 0.3 v 0.8 +/- 0.4 mg/ kg/min, respectively). However, it did not alter the increase in net hepatic glucose output (delta 0.7 +/- 0.3 v 0.8 +/- 0.4 mg/kg/min). When the increase in cortisol was absent, the increase in net hepatic gluconeogenic precursor uptake was attenuated (delta 0.7 +/- 0.3 v 0.1 +/- 0.3 mg glucose/kg/min) due to a decrease in gluconeogenic precursor levels. The efficiency of gluconeogenesis increased to a greater extent (delta 0.19 +/- 0.07 v 0.31 +/- 0.11) when cortisol was not infused. The absence of an increase in cortisol also led to marked glycogen depletion in the liver (10 +/- 4 v 55 +/- 10 mg/g liver). Cortisol thus plays a pivotal role in the metabolic response to stress hormone infusion by sustaining gluconeogenesis through a stimulatory effect on hepatic gluconeogenic precursor supply and by maintaining hepatic glycogen availability. PMID- 8622600 TI - Glyburide increases the secretion, tissue uptake, and action of insulin in conscious normal dogs. AB - The action of glyburide on glucose homeostasis involves pancreatic and extrapancreatic mechanisms. The relative importance of each of these processes in the hypoglycemic response to sustained administration of glyburide is unknown. In addition, the effect of this drug on the hepatic extraction of insulin is controversial. This investigation uses direct techniques in conscious normal dogs to examine the impact of glyburide therapy (2.5 mg twice daily for 4 weeks) on glucose homeostasis. Preparatory surgery included placement of Doppler flow probes on hepatic vessels and insertion of catheters in carotid artery, portal vein, hepatic vein, and renal vein. After recovery from surgery, animals underwent an intravenous glucose tolerance test ([IGTT] 0.3 g - kg (-1) intravenous glucose bolus) and an insulin infusion clamp test ([IICT] 2 mU - kg ( 1) - min (-1) intravenous insulin during 150 minutes) followed by glyburide therapy. After 4 weeks, the IGTT and IICT were repeated. Glyburide increased the insulin secretory response during the late phase of the IGTT and augmented glucose clearance during the IICT. Hepatic extraction of insulin was also stimulated by glyburide. We conclude that the hypoglycemic action of long-term glyburide administration involves stimulation of both insulin secretion by the pancreas and glucose disposal by peripheral tissues. In addition, glyburide augments the extraction of insulin by the liver, and such an effect might prevent the development of sustained high levels of insulin in blood perusing peripheral tissues. PMID- 8622601 TI - A moderate decline in specific activity does not lead to an underestimation of hepatic glucose production during a glucose clamp. AB - We have previously shown that modeling errors lead to underestimation of hepatic glucose production (HGP) during glucose clamps when specific activity (SA) declines markedly. We wished to assess whether the failure to keep SA constant substantially affects calculation of HGP during insulin infusion when glucose requirements to maintain the glucose clamp are moderate. Therefore, 150-minute hyperinsulinemic (5.4 pmol - kg (-1) - min (-1) clamps were performed in depancreatized dogs that were maintained hyperglycemic (approximately 10 mmol/L with either (l) unlabeled glucose infusate (COLD Ginf, n = 5) or (2) labeled glucose infusate (HOT Ginf, n = 6) containing high-performance liquid chromatography (HPLC purified [6-3H]glucose. Insulinemia and glucagonemia were similar between the two groups. Additionally, glucose infusion rates were equivalent with COLD and HOT Ginf, indicating comparable insulin effects on overall glucose metabolism. The SA decreased a maximum of 32% with COLD Ginf, but remained constant with HOT Ginf. HGP was suppressed equally with COLD or HOT Ginf treatments at each time point during the clamp (mean suppression during last hour of clamp, 69% +/- 4% and 69% +/- 5%, P = NS, COLD and HOT Ginf, respectively). We conclude that when glucose requirements are moderate and SA changes slowly, as in the diabetic dog, it is not necessary to keep SA perfectly constant to avoid significant modeling errors when calculating HPG during hyperinsulinemic clamps. PMID- 8622602 TI - Long-term administration of acipimox potentiates growth hormone response to growth hormone-releasing hormone by decreasing serum free fatty acid in obesity. AB - Obesity is associated with an impairment of normal growth hormone (GH) secretion and blunted responses to all stimuli. A high plasma free fatty acid (FFA) level is frequently observed in obesity. FFA participates in the regulation of pituitary GH secretion. To determine whether the derangement of GH secretion in obesity is associated with high plasma FFA levels, tests with GH-releasing hormone (GHRH) and acipimox (ACX), an antilipolytic agent able to decrease FFA, were undertaken in six obese subjects and seven normal control subjects. In addition, the effect of prolonged suppression of FFA level on GH response to GHRH after administration of ACX for 1 month was also examined in each of the obese subjects. The GH response in obese subjects (median, 9.1 microg/L) to GHRH (1-29) (1 microg/kg intravenously [IV]) was significantly blunted as compared with normal control subjects (23.5 microg / L, P < .05). Basal FFA levels were higher in obese subjects (855.2 microEq / L than in normal control subjects (514.6 microEq / L, P < .05). One-dose ACX (500 mg) decreased FFA levels in both obese and normal subjects: the lowest FFA levels in obese subjects (158.3 microEq/L 2 to 2.5 hours after ACX were similar to those of normal control subjects (108.7 microEq/L). One-dose ACX potentiated GHRH-stimulated GH response in both obese and normal subjects. GH responses potentiated by ACX in obese subjects (27.1 microg/L) were similar to GH responses to GHRH in normal control subjects, but lower than in normal subjects treated with ACX plus GHRH (58.5 microg / L, P < .05). Thereafter, all of the obese subjects were treated with ACX for 1 month, after which the ACX plus GHRH tests were repeated. After 1 month of acipimox administration in the obese subjects, GH responses (38.8 microg/L) were significantly higher than those of obese subjects treated with GHRH and one-dose ACX plus GHRH (P < .05). They were similar to GH responses of normal control subjects receiving the one-dose ACX plus GHRH test. In conclusion, in obesity the prolonged suppression of FFA levels induced by long-term administration of ACX potentiated somatotrope responsiveness, likely acting at the pituitary level, suggesting that the duration of FFA suppression had an important relation to the magnitude of GH response. PMID- 8622603 TI - Glucose processing during the intravenous glucose tolerance test. AB - The impact of the dynamic changes in plasma glucose and insulin levels observed during a frequently sampled intravenous (IV) glucose tolerance test (FSIGT) on whole-body glucose processing and muscle glycogen metabolism is not known. Paired randomized FSIGTs were performed in eight healthy subjects (age, 31 years; range, 28 to 35; BMI, 25.4 kg/m2; range, 22.3 to 32.1), one with muscle biopsy samples and one without. The mean time average (0- to 40- and 0- to 120-minute) insulin levels during the test were 26.6 and 11.4 mU/1, respectively. Glucose oxidation increased following the IV glucose bolus (basal 1.34 +/- 0.21 v mean value at 0 to 120 minutes 2.09 +/- 0.22 mg/kg fat-free mass [FFM]/min, P < .02). In contrast, fractional glucose-6-phosphate [G-6-P]) (0.1/10 mmol/L) skeletal muscle glycogen synthase activity in muscle biopsies obtained before and following the IV glucose bolus (-30, 30,60, and 120 minutes, respectively) were unchanged (38.1% +/- 2.3%, 38.3% +/- 2.9%, 38.1% +/- 2.3%, 35.4% +/- 2.3%, NS). Skeletal muscle glycogen concentration decreased slightly (449 +/- 54, 439 +/- 55, and 383 +/- 29, and 438 +/- 48 mmol/kg dry weight, P =.05), indicating no net storage of glucose into glycogen during the FSIGT. G-6-P decreased (0.77 +/- 0.08, 0.64 +/- 0.07, 0.66 +/- 0.07, and 0.54 +/- 0.04 mmol/kg dry weight, P < .05). Levels of the insulin-regulatable glucose transporter, GLUT-4, were unchanged. Insulin sensitivity (Si), glucose effectiveness, and insulin secretion parameters (01 and 02) were not affected by the muscle biopsy procedure. In conclusion, the FSIGT is associated predominantly with increased whole-body glucose oxidation with no apparent activation of muscle glucose storage as glycogen. Thus, the Si measured by the FSIGT, although similar in magnitude to the clamp-derived parameter, represents primarily glucose oxidation, in contrast to the euglycemic clamp, which involves glucose oxidation and storage. PMID- 8622604 TI - Increased nonoxidative glucose metabolism in idiopathic reactive hypoglycemia. AB - Idiopathic reactive hypoglycemia (IRH) is responsible for postprandial hypoglycemia. Normal insulin secretion and reduced response of glucagon to acute hypoglycemia, but mostly increased insulin sensitivity, represent the metabolic features of this syndrome- The present study has two aims: first, to investigate the fate of glucose utilization inside the cells to assess whether increased glucose disposal in IRH is due to the oxidative and/or nonoxidative pathway; and second, to evaluate glucagon response to prolonged insulin-induced hypoglycemia. In eight patients with IRH and eight normal (N) subjects, we performed two studies on different days: (1) 120-minute euglycemic-hyperinsulinemic (1.0 mU . kg-1 . min-1 regular human insulin) clamp associated with indirect calorimetry; and (2) 180-minute hypoglycemic (2.22 to 2.49 mmo/L achieved through 0.85 mU . kg 1 . min-1 intravenous [IV] regular human insulin) clamp. The results showed an increased insulin-mediated glucose uptake in IRH (9.10 +/- 0.19 v 6.78 +/- 0.18 mg kg-1 . min-1, P < .005). Glucose oxidation was similar in IRH subjects and controls both in basal conditions (1.39 +/- 0.16 v 1.42 +/- 0.15 mg . kg-1 . min 1 and during the clamp studies (2.57 +/- 0.21 v 2.78 +/- 0.26 mg . kg-1 . min-1. In contrast, nonoxidative glucose disposal was significantly higher in IRH than in N subjects (6.53 +/- 0.30 v 4.00 +/- 0.21 mg . kg-1 . min-1, P < .001). During insulinization, fat oxidation was reduced slightly more in IRH than in control subjects. During the hypoglycemic clamp, a significant (P < .01) increase in plasma glucagon concentrations was observed in normal subjects as compared with baseline, whereas no change occurred in IRH patients. In conclusion, in IRH: (1) increased insulin-mediated glucose disposal is due to the increase of nonoxidative glucose metabolism; and (2) glucagon secretion has been confirmed to be inadequate. The increase of insulin sensitivity associated with a deficiency in glucagon secretion can widely explain the occurrence of hypoglycemia in the late postprandial phase. PMID- 8622605 TI - Relationship between glutathione and sorbitol concentrations in erythrocytes from diabetic patients. AB - Red blood cell (RBC) concentrations of sorbitol and reduced glutathione (GSH) were evaluated in 29 type 11 diabetic subjects and eight normal controls. In erythrocytes from diabetic subjects, sorbitol levels were higher (18.7 +/- 1.33 v 11.2 +/- 0.7 nmol/g hemoglobin [Hb], P < .001) and GSH levels were lower (5.48 +/ 0.19 v8.33 +/- 0.24 micromol/g Hb, P < .01) than in nondiabetics. RBC sorbitol levels were positively correlated with fasting blood glucose (r =.57, P < .001) but not with HbAlc (r =.16, P < .05). RBC GSH levels showed a negative correlation with fasting blood glucose (r = -.35, P <.05) and with HbA1c (r = .34, P < .05) and a significant negative correlation with RBC sorbitol levels (r = -.62, P < .001). Stepwise regression analysis highlighted the fact that the hyperglycemia-dependent increase in RBC sorbitol was significantly influenced by GSH concentrations (partial F = 14.6, P < .001). These data suggest the hypothesis that the hyperglycemia-induced enhanced activity of the polyol pathway leads to GSH depletion and, in turn, GSH depletion, reducing the glycolytic flux to pyruvate, enhances the rate of glucose metabolism through the polyol pathway. The overall effect is a progressive worsening of metabolic pseudohypoxia and depletion of GSH, resulting in lower defense against oxidative stress. PMID- 8622606 TI - Insulin resistance syndrome and autonomically mediated physiological responses to experimentally induced mental stress in adolescent boys. AB - We investigated the relationship between hemodynamic and other autonomically mediated responses to experimentally induced mental stress and the parameters of the insulin resistance syndrome (IRS) in 48 healthy adolescent boys. Mental stress was induced with mental arithmetic and the Stroop Color-Word Test. Heart rate (HR), finger blood volume (FBV), and skin conductance level (SCL) were recorded continuously during task performance. IRS parameters measured were serum insulin, high-density lipoprotein (HDL) cholesterol, serum triglyceride (TG), systolic blood pressure (SBP), subscapular skinfold (SSF), and subscapular to triceps skinfold ratio (STR). The results indicated that a high level and an increasing linear trend of HR and FBV during task performance were related, independently of each other and of body mass index (BMI), to a high insulin concentration. An increasing linear trend of HR during mental stress was also related to high SSFs independently of MI. In addition, a high SCL during task performance was associated with high TG levels, SSFs, and STRs. It is discussed whether stress-induced sympathetic overactivity might contribute to the development of the IRS. PMID- 8622607 TI - Impaired regulation of hepatic fructose-1,6-biphosphatase in the New Zealand Obese mouse: an acquired defect. AB - Increased hepatic glucose production, a feature of (non-insulin-dependent diabetes mellitus [NIDDM]), is present at an early age in the New Zealand Obese (NZO) mouse and is associated with impaired suppression of the gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). The aim of this study was to further characterize the abnormality in the regulation of hepatic FBPase in NZO mice versus New Zealand Chocolate (NZC) control mice. At 20 weeks of age, NZO mice have elevated FBPase activity (65.3 +/- 7.9 v 46.7 +/- 5.0 micromol/min/mg protein, P =.07) and protein levels (31.7 +/- 3.1 v 22.5 +/- 2.8 arbitrary units, P < .05), but not mRNA levels (0.18 +/- 0.03 v 0.16 +/- 0.03 arbitrary units). Elevated FBPase activity and protein levels in NZO mice were also shown at 4 to 6 weeks of age, but not in 1-day-old mice, suggesting that the increase occurs between birth and weaning. The Km of the enzyme was the same in NZO and NZC mice (3.7 +/- 0.5 v 5.0 +/- 0.9 micromol/L, NZO v NZC). The regulation of FBPase by the competitive inhibitor, fructose-2,6-bisphosphate ([Fru(2,6)Pz] 5 micromol/L) measured over a range of substrate concentrations (2.5 to 80 micromol/L) was similar between NZO and control mice (Km in the presence of Fru(2,6)Pz, 10.8 +/- v 1.9 v 13.2 +/- 3.3 micromol/L, NZO v NZC). It is concluded that increased FBPase activity in the NZO mouse is due to elevated protein levels, and that this appears to be due to a failure of the normal decrease that occurs following birth in control animals. PMID- 8622608 TI - Relationship of insulin resistance to weight gain in nondiabetic Asian Indian, Creole, and Chinese Mauritians. Mauritius Non-communicable Disease Study Group. AB - There is evidence from animal models that postprandial insulin hypersecretion may precede the development of obesity and insulin resistance, but it is not clear if this is the case in humans. Recently, two longitudinal studies have suggested that insulin resistance acts to limit further weight gain rather than to promote it. The relationship of markers of insulin sensitivity and secretion to changes in weight and the waist to hip ratio (WHR) was therefore examined in nondiabetic Asian Indian (n = 2,169), Creole (n = 798), and Chinese (n = 189) Mauritians over a 5-year follow-up period. Younger age and lower initial body mass index (BMI) were consistent independent predictors of increase in weight in all sex-ethnic subgroups, and older age, higher BMI, and lower WHR were associated with change in WHR. Insulin sensitivity was assessed by homeostatic model assessment (HOMAS), as well as by fasting insulin and the ratio of fasting insulin to glucose. Insulin resistance predicted weight gain in Chinese men independently of baseline age and BMI. In Asian Indian and Creole men and women, these correlations were in the opposite direction (ie, insulin sensitivity predicted weight gain) but became nonsignificant when age and BMI were controlled. There was little relationship of insulin resistance/sensitivity to the change in WHR once baseline BMI was controlled. These data provide suggestive but not convincing evidence that insulin resistance may limit weight gain, and contradictory evidence in one ethnic group that insulin resistance promotes weight gain. PMID- 8622609 TI - Computed tomography-determined body composition in relation to cardiovascular risk factors in Indian and matched Swedish males. AB - Relationships between cardiovascular risk factors, body composition, and tissue distributions were examined in 10 Indian and 10 Swedish males matched by age, height, and weight. The body was divided into 29 compartments by means of a multiscan computed tomography (CT) technique. Fasting glucose, insulin, and triglycerides (TG) were higher in Indians than in Swedes. During the oral glucose tolerance test (OGTT), the glucose area was similar in both groups, whereas the insulin area was 80% larger in Indians. Adipose tissue (AT) and skin volumes were larger and remaining lean tissues were smaller in Indians. Indians had proportionally less muscle and more skeleton in the legs, but no ethnic difference could be demonstrated with respect to AT distribution. The visceral AT to total AT volume ratio was positively related to insulin and TG, and with higher risk factors for Indians at any given ratio. TG and glucose were negatively related to the leg muscle to total muscle volume ratio, and this ratio was smaller in Indians. It is concluded that the metabolic disturbances of Indians are not necessarily dependent on a preponderance of visceral AT, and also that an upper-body muscle distribution-recognized as a new phenotypic companion to the metabolic syndrome-is statistically related to cardiovascular risk factors. PMID- 8622610 TI - Mechanisms of cachexia induced by T-cell leukemia in the rat. AB - Body wasting (cachexia) is a common feature of cancer and a major cause of morbidity and mortality. The mechanisms underlying cachexia are largely unknown, and studies in experimental animals have focused mainly on solid tumors. Therefore, the objective of the present study was to quantify and investigate cachexia in experimentally induced T-cell leukemia in the rat. Induction of leukemia by serial passage (injection of cervical lymph node suspension) resulted in a rapid increase in white blood cell (WBC count, hypertrophy of the spleen (by day 11), and severe morbidity within 17 to 18 days. Body weight gain and food intake declined steadily in leukemic animals from day 12, although weight loss was significantly greater in pair-fed, nonleukemic animals. However, leukemic rats had a lower body fat content and higher water content than pair-fed animals on day 18, so the measurement of body weight significantly underestimated the severity of cachexia. Resting oxygen consumption (VO2), measured during the light phase, declined in pair-fed animals from day 13, but was elevated in leukemic rats on days 12 to 18 by 25% (P < .05, one-way ANOVA) compared with pair-fed rats and by 7% (P < .05, one-way ANOVA) relative to free-feeding controls. Hypermetabolism was associated with an increase in brown adipose tissue (BAT) activity (74% and 89%, respectively, P < .05, one-way ANOVA) in leukemic rats compared with control and pair-fed groups. Effects of leukemia on VO2 and BAT were prevented by administration of the adrenergic antagonist, propranolol. These results indicate that T-cell leukemia in the rat results in rapid and severe cachexia, which is largely due to marked hypophagia, but is also accompanied by inappropriately high rates of energy expenditure that are mediated by sympathetic activation of BAT thermogenesis. PMID- 8622611 TI - Glucose intolerance in thalassemia major is related to insulin resistance and hepatic dysfunction. AB - Glucose intolerance is a common consequence of transfusion therapy in patients with thalassemia major (TM), but the relative contribution of pancreatic damage and insulin resistance to glucose intolerance is unclear. We have investigated oral (OGTT) and intravenous (IVGTT) glucose tolerance, insulin sensitivity, and fasting concentrations of insulin, proinsulin, and des 31,32 proinsulin in 12 patients with TM (seven hepatitis C virus [HCV] antibody-negative and five positive), eight patients with hepatic cirrhosis, and nine healthy controls. Two hour plasma glucose concentrations were marginally higher in anti-HCV-negative (median, 7.4 mmol/ L; range, 4.0 to 8.2) and significantly so in anti-HCV positive thalassemics (median, 8.5 mmol/L; range, 6.4 to to 23.0) and cirrhotics (median, 8.0 mmol/L; range, 4.7 to 17.6) than in controls (median, 5.5 mmol/L; range, 3.0 to 6.3). Insulin sensitivity was also reduced in the three patient groups (P < .05). Insulin resistance was the main determinant of oral glucose intolerance in all patient groups (partial r2 = .49, P < .0001, n = 28). In turn, the main determinants of insulin insensitivity in TM patients were liver damage (albumin, r = .67, P = .02) and serum ferritin concentration (r = -.62, P = .03). There was no relationship of either 2-hour or incremental insulin concentrations with ferritin levels or with HCV status in TM subjects. Moreover, these patients showed no elevation of concentrations of proinsulin and des 31,32 proinsulin, markers of pancreatic beta-cell damage, in excess of those observed in cirrhotic patients. In conclusion, the glucose intolerance of TM, like that of cirrhosis, is associated with insulin resistance, not insulin deficiency, and may be a direct or indirect consequence of hepatic damage. PMID- 8622612 TI - Effect of endothelin-1 in man--impact on basal and stimulated concentrations of luteinizing hormone, follicle-stimulating hormone, thyrotropin, growth hormone, corticotropin, and prolactin with and without pretreatment with nifedipine. AB - In healthy men, intravenous (IV) endothelin-1 suppresses the growth hormone (GH) releasing hormone (GHRH)-stimulated increase in GH and prolactin (PRL) and augments corticotropin (ACTH)-releasing factor (CRF)-stimulated secretion of ACTH. Since some actions of endothelin-1 on pituitary function in vitro are antagonized by calcium channel antagonists, we have studied the effect of pretreatment with oral nifedipine (10 mg, given before infusion of endothelin-1 or vehicle) on basal and stimulated concentrations of pituitary hormones in a group of healthy men (N = 6). The augmentative effect of endothelin-1 on CRF induced ACTH secretion (P < .05) was counteracted by pretreatment with nifedipine. Pretreatment with nifedipine further inhibited (P < .01) the GHRH induced increase in plasma concentrations of GH (P < .05), which, in keeping with previous data, had already been reduced by IV endothelin-1 alone (P < .05). Thus, both endothelin-1 and nifedipine influence pituitary hormone secretion in healthy man. However, nifedipine does not ubiquitously counteract the effects of endothelin-1 since it enhances some of its actions on the pituitary and diminishes others. Endothelin-1 may therefore influence pituitary function by mechanisms other than activation of calcium channels alone. PMID- 8622614 TI - Altered lipid composition and enzyme activities of erythrocyte membranes in hepatic cirrhosis. PMID- 8622613 TI - Contribution of leg and splanchnic free fatty acid (FFA) kinetics to postabsorptive FFA flux in men and women. AB - We have previously shown that upper-body adipose tissue is more lipolytically active than lower-body adipose tissue in lean and obese women. The present studies were conducted to determine whether these regional differences are also present in men. Twenty-five lean, healthy men and 24 lean, healthy women underwent measures of body composition, postabsorptive systemic free fatty acid (FFA) flux, and leg and splanchnic FFA uptake and release. Upper-body adipose tissue was more lipolytically active than lower-body adipose tissue in both men (53.4 +/- 32.2 v 26.6 +/- 12.9 micromol x kg fat(-1) x min(-1), P < .001, respectively) and women (41.2 +/- 22.3 v 18.4 +/- 8.2 micromol x kg fat(-1) x min(-1), P < .001, respectively). The correlations between leg FFA release and systemic FFA flux were modest in women and men (r = .38, P = .07 and r = .56, P = .003, respectively) as were the correlations between splanchnic FFA release and systemic FFA flux (r = .41, P = .06 and r = .40, P = .07, respectively). No effect of gender on the relationship between leg or splanchnic FFA release and systemic FFA flux was detected. In summary, upper-body FFA release is greater than lower-body FFA release in both men and women, and the relationship between leg or splanchnic FFA release and systemic FFA release is weak and similar in men and women. These findings suggest that regional differences in postabsorptive FFA kinetics are unlikely to be responsible for differences in regional fat distribution. PMID- 8622615 TI - Scopolamine poisoning among heroin users--New York City, Newark, Philadelphia, and Baltimore, 1995 and 1996. AB - Heroin is mixed ("cut") frequently with other substances primarily to increase its weight for retail sale (e.g., mannitol and starch) and to add pharmacologic effects (e.g., dextromethorphan and lidocaine). During 1995 and 1996, health departments and poison-control centers in New York City (NYC); Newark, New Jersey; Philadelphia; and Baltimore reported at least 325 cases of drug overdoses requiring medical treatment in persons who had used "street drugs" sold as heroin that probably also contained scopolamine, an anticholinergic drug. This report summarizes the clinical and epidemiologic features of these cases, which represent a new type of drug overdose. PMID- 8622616 TI - Trends in rates of homicide--United States, 1985-1994. AB - During 1993, a total of 26,009 homicides were reported in the United States; 71% were firearm-related, and one third of all homicides occurred among persons aged 15-24 years . Since 1985, national homicide rates have increased sharply, especially firearm-related homicides and homicides among persons aged 15-24 years. However, based on data from the Supplementary Homicide Report compiled by the Federal Bureau of Investigation and reports from some cities, homicide rates have been stable or declining since 1993. To examine this trend and to assess the relative contributions of firearm- and nonfirearm-related homicide to these recent changes, CDC analyzed national vital statistics data for 1985-1994. This report summarizes this analysis, which indicates that overall rates of homicide increased from 1985 to 1991 and decreased from 1992 to 1994, and that during these two periods, rates for total firearm-related homicides and homicide among persons aged 15-24 years increased then stabilized but remained at record-high levels. PMID- 8622617 TI - Work-related injuries and illnesses associated with child labor--United States, 1993. AB - During 1993, an estimated 2.1 million persons aged 16-17 years in the United States were employed. Although many children aged <16 years work, employment data are neither routinely collected nor reported for this age group, and there are no reliable estimates of the number of children in this age group who work. During summer months, when most children are not in school, employment and hours worked by children aged <18 years increase substantially. To characterize workplace related health and safety hazards for children, CDC's National Institute for Occupational Safety and Health (NIOSH) analyzed 1993 data for workers aged <18 years from the Survey of Occupational Injuries and Illnesses (SOII), a survey administered by the Bureau of Labor Statistics (BLS), U.S. Department of Labor. This report summarizes the results of this analysis and indicates that substantial numbers of persons aged <18 years sustain work-related injuries and illnesses each year. PMID- 8622618 TI - Update: provisional Public Health Service recommendations for chemoprophylaxis after occupational exposure to HIV. AB - Although preventing blood exposures is the primary means of preventing occupationally acquired human immunodeficiency virus (HIV) infection, appropriate postexposure management is an important element of workplace safety. Information suggesting that zidovudine (ZDV) postexposure prophylaxis (PEP) may reduce the risk for HIV transmission after occupational exposure to HIV-infected blood prompted a Public Health Service (PHS) interagency working group, with expert consultation, to update a previous PHS statement on management of occupational exposure to HIV with the following findings and recommendations on PEP. PMID- 8622619 TI - Update: mortality attributable to HIV infection among persons aged 25-44 years- United States, 1994. AB - During the 1980s, human immunodeficiency virus (HIV) infection, the cause of acquired immunodeficiency syndrome (AIDS), emerged as a leading cause of death in the United States. In 1993, HIV infection became the most common cause of death among persons aged 25-44 years. This report updates national trends in deaths caused by HIV infection in 1994, which continue to increase. PMID- 8622620 TI - Accessibility of tobacco products to youths aged 12-17 years--United States, 1989 and 1993. AB - Although the sale of tobacco products to minors is illegal in all states and the District of Columbia, the prevalence of cigarette smoking among adolescents has continued to increase, and most minors are able to purchase tobacco products. Reducing sales to minors is believed to be an effective measure for reducing the prevalence of tobacco use. To determine recent patterns of minors' access to tobacco products from retail outlets and vending machines, data were analyzed from the 1989 and 1993 Teenage Attitudes and Practices surveys (TAPS I and TAPS II). This report summarizes the results of that analysis, which indicate that most minors who use tobacco purchase their own tobacco and that small stores are the sources of most purchases. PMID- 8622621 TI - Postnatal causes of developmental disabilities in children aged 3-10 years- Atlanta, Georgia, 1991. AB - Primary prevention of developmental disabilities requires knowledge of the specific causes of these conditions. Postnatal causes account for 3%-15% of all developmental disabilities and often are preventable. To assess the prevalence and determine the specific etiology of postnatally acquired developmental disabilities, CDC analyzed data from its ongoing Metropolitan Atlanta Developmental Disabilities Surveillance Program (MADDSP) for 1991 (the most recent year for which complete data were available). This report summarizes the findings of the analysis, which indicate that bacterial meningitis and child battering were the leading postnatal causes of developmental disabilities and that children with postnatally acquired developmental disabilities had a higher average number of disabilities than all other children with developmental disabilities. PMID- 8622622 TI - Update: influenza activity--United States, 1995-96 season. PMID- 8622624 TI - DNA repair activity in protein extracts of fresh human malignant lymphoid cells. AB - Nucleotide excision repair (NER) activity was investigated in lymphocytes from patients with chronic lymphocytic leukemia (CLL). The NER process consists of two broad stages: incision/excision of the damaged oligonucleotide and resynthesis of the repair patch. NER in CLL lymphocytes was monitored with the use of in vitro biochemical assays, allowing the determination of either the extent of repair synthesis or the incision activity on damaged plasmid DNA during incubation with whole-cell protein extracts. Fresh CLL tumor cells were purified from the blood of 7 untreated patients and 11 patients who had been treated with chemotherapy. No repair activity was found in 14 extracts (7 treated and 7 untreated) or in normal blood peripheral lymphocytes. The defect was at the level of both repair synthesis and incision/excision activity of DNA damage. In contrast, 4 of the extracts exhibited 25-60% of the repair activity measured in an extract from a control repair-proficient cell line. A linear relationship was found between the values of DNA-repair synthesis and incision activities, which indicates that the extent of significant incision was the limiting factor in these protein extracts. All of the extracts that exhibited DNA-repair activity were purified from lymphocytes of treated patients. These data suggest that chemotherapy might exert an effect on the status of repair activity in the lymphoid tumor cells of patients. PMID- 8622623 TI - 5-Carboxamido-tryptamine, CP-122,288 and dihydroergotamine but not sumatriptan, CP-93,129, and serotonin-5-O-carboxymethyl-glycyl -tyrosinamide block dural plasma protein extravasation in knockout mice that lack 5-hydroxytryptamine1B receptors. AB - We studied the dural plasma protein extravasation response after unilateral electrical stimulation of the trigeminal ganglion in mice lacking serotonin 5 HT1B (5-HT1D beta) receptors by modifying a technique previously described in rats or guinea pigs. We investigated the inhibitory effects of six 5-HT1 receptor agonists in this model: 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), sumatriptan, serotonin-5-O-carboxymethyl-glycyl -tyrosinamide (GTI), 5-methylaminosulfonylmethyl-3-(N-methylpyrrolidin-2R -ylmethyl)-1H-indole (CP 122,288), 5-carboxamido-tryptamine (5-CT), and dihydroergotamine. The plasma extravasation response did not differ between wild-type and mutant after vehicle injection. The potency of sumatriptan, CP-122,288, CP-93,129, and 5-CT in wild type mice was similar to that previously reported for rats. CP-122,288 (1 nmol kg), 5-CT (1 nmol/kg), and dihydroergotamine (72 nmol/kg) inhibited plasma protein extravasation within dura mater after electrical trigeminal ganglion stimulation in both wild-type and knockout mice, which suggests that these agonists act predominantly via receptors other than 5-HT1B. Unlike the wild-type mice, CP-93,129 (1.4 mumol/kg), a specific 5-HT1B receptor agonist, had no effect in knockout mice. The same held true for sumatriptan (0.7 mumol/kg) and GTI (0.6 mumol/kg). These results suggest that CP-93,129, sumatriptan, and GTI exert their effects via 5-HT1B (5-HT1D beta) receptors in mice. PMID- 8622625 TI - Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors. AB - Imidazoacridinones are new antitumor compounds that exhibit strong antitumor effect against solid tumors such as human colon and breast carcinomas. The mechanism of action of imidazoacridinones is unknown, although a similarity in the chemical structure between active imidazoacridinones and mitoxantrone suggests common cellular targets. We show that imidazoacridinones inhibit the catalytic activity of purified topoisomerase II as determined by both relaxation and decatenation assays. All biologically active compounds stimulated the formation of cleavable complexes in vitro, whereas inactive compounds did not. The pattern of DNA cleavage in SV40 DNA was similar to that obtained for 4'-(9 acridinylamino)methane-sulfon-m-aniside, particularly within the matrix associated region. Significant levels of DNA complexes were observed when DC-3F fibrosarcoma cells were treated with active compounds, whereas negligible amounts of these complexes were induced by inactive analogues. DC-3F/9-OHE cells, which are resistant to other topoisomerase II inhibitors, are 30-125-fold cross resistant to active imidazoacridinones. The resistance is associated with a reduction in the formation of DNA/protein complexes and is highest for compounds that are potent topoisomerase II inhibitors in vitro. Interestingly, the two most active derivatives, C-1310 and C-1311, were equally cytotoxic toward fast-growing monolayer cultures and cells growing in three dimensions as multicellular spheroids, which have a slower growth fraction. In contrast, 4'-(9 acridinylamino)methanesulfon-m-aniside, mitoxantrone, and doxorubicin were more cytotoxic toward monolayer cultures. Taken together, the results suggest that DNA topoisomerase II is a major cellular target of biologically active imidazoacridinones and that these drugs show both similarities and dissimilarities compared with classic topoisomerase II inhibitors. PMID- 8622626 TI - Transforming growth factor-beta 1 induces transcriptional down-regulation of m2 muscarinic receptor gene expression. AB - In human embryonic lung fibroblasts, transforming growth factor-beta 1 (TGF-beta 1) induced a time-dependent down-regulation of M2 muscarinic receptor binding sites as measured with the nonselective hydrophilic ligand [3H]N methylscopolamine (NMS). This down-regulation was slow, with 58% loss of all receptors after 24 hr of treatment. The affinity of [3H]NMS for the remaining sites was unaltered by TGF-beta 1. The loss in [3H]NMS binding was accompanied by reduced adenylyl cyclase activity and functional desensitization of M2 muscarinic receptors. Northern blot analyses showed a 72% decrease in the steady state levels of m2 muscarinic receptor mRNA after 24-hr TGF-beta 1 treatment. Recovery of m2 muscarinic receptor mRNA after TGF-beta 1 treatment was slow, with a half life of approximately 8 hr. There was no effect of TGF-beta 1 on the m2 muscarinic receptor mRNA half-life measured in the presence of actinomycin D, but the rate of m2 muscarinic receptor gene transcription measured with nuclear run on assay was reduced by 50%, indicating reduced gene transcription. Cycloheximide (10 micrograms/ml) pretreatment abolished the TGF-beta 1 effect, indicating that de novo protein synthesis was required for receptor downregulation. In summary, we have shown that TGF-beta 1 induced desensitization and down-regulation of M2 muscarinic receptor protein and gene that was mediated through reduction in the rate of m2 receptor gene transcription. PMID- 8622627 TI - Role of c-jun in human myeloid leukemia cell apoptosis induced by pharmacological inhibitors of protein kinase C. AB - Recent study results suggest that protein kinase C [PKC (EC 3.1.4.3)] -dependent up-regulation of c-jun may be involved in leukemic cell programmed cell death, or apoptosis, occurring in response to various chemotherapeutic agents. The current study was undertaken to further evaluate the contribution of c-jun in apoptosis with the use of two highly specific pharmacological inhibitors of PKC (calphostin C and chelerythrine). To address this issue, two human leukemic cell lines, HL-60 and U937, and a U937 subline stably expressing a dominant negative c-jun mutant (TAM67) were exposed to calphostin C and chelerythrine, and c-jun expression was monitored at both the mRNA and protein levels. Both PKC inhibitors induced the classic morphological features of apoptosis as well as internucleosomal DNA degradation in a concentration- and schedule-dependent manner. Concomitant with these changes, unequivocal increases were observed in c-jun mRNA (U937 and HL-60) and protein (U937). In contrast, up-regulation of c-jun mRNA and protein in TAM67 expressing cells exposed to both PKC inhibitors was markedly attenuated relative to effects observed in parental U937 cells. Importantly, despite impaired up regulation of c-jun at both the message and protein levels, TAM67-expressing cells were equally susceptible to PKC inhibitor-induced apoptosis as parental and empty vector U937 cells. Collectively, these findings raise the possibility that c-jun up-regulation in human myeloid leukemia cells undergoing PKC inhibitor associated apoptosis represents a response to, rather than a cause of, apoptotic events. They further suggest that this phenomenon involves pathways that do not require PKC activation. PMID- 8622628 TI - Role of nitric oxide in the cytokine-mediated regulation of cytochrome P-450. AB - We explored the effects of cytokines on cytochrome P-450 (CYP) in rat hepatocyte primary cultures. CYP content and several CYP protein levels were assessed in hepatocytes treated with a cytokine combination consisting of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN gamma). The combination was found to depress CYP content by 69 +/- 6%. Protein levels of CYP forms 1A2, 2C11, 2B1/2, and 3A2 were assessed with immunoblotting. Treatment with the cytokine combination resulted in a decrease in each CYP enzyme, with CYP2B1/2 exhibiting the greatest loss, to 33 +/- 9% of untreated cells. The addition of inhibitors of nitric oxide synthase (NOS) significantly prevented the cytokine-mediated decrease in each CYP protein, indicating a role for nitric oxide (NO) in the down-regulation. Treatment of hepatocytes with the NO donor 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene (300 microM) caused a decrease in each CYP apoprotein, with CYP2B1/2 exhibiting the greatest decrease, to 33 +/- 8% of untreated cells. Decreases in CYP protein levels were observed in response to treatment with TNF alpha, IL-1 beta, or IL-6 alone. With IL-1 beta treatment, increased levels of NO production were accompanied by decreased levels of each CYP protein. With TNF alpha treatment, increased levels of NO production were accompanied by decreased levels of CYP2B1/2 and CYP3A2. The effects of IL-1 beta and TNF alpha were blocked by the inclusion of the NOS inhibitors. Conversely, IL-6 caused a decrease in each of the CYP enzymes but did not affect NO production. The results indicate a dissociation in vitro between NOS induction and CYP down-regulation for IL-6 treatment, whereas the down-regulation of CYP by TNF alpha and IL-1 beta in vitro is directly associated with NO production. PMID- 8622629 TI - Expression of cytochrome P4502E1 in rat fetal hepatocyte culture. AB - Cytochrome P450 (CYP) 2E1 is present at very low levels or cannot be detected in rat fetal liver. Experiments were carried out to develop an ex vivo model of CYP2E1 expression in fetal liver. Fetal hepatocytes were prepared from pregnant rats on gestation days ranging from 12 to 19 and placed into culture for 2 days. Expression of CYP2E1 was observed at all gestational periods as evident from immunoblots and oxidation of paranitrophenol and N,N-dimethylnitrosamine by fetal liver microsomes. Northern blot analysis indicated production of CYP2E1 mRNA by the fetal hepatocytes cultured for 2 days but not by freshly isolated fetal rat hepatocytes. The addition of ethanol to the hepatocyte cultures did not have a significant effect on CYP2E1 catalytic oxidation of substrates or CYP2E1 mRNA levels. The content of CYP2E1, CYP2E1 mRNA levels, and CYP2E1 catalytic activity was greater in the fetal cultures grown in the presence of 2.5% fetal calf serum than in that grown with 15% fetal calf serum, suggesting that factors present in the serum limit expression or stability of CYP2E1. CYP2E1 was not detectable in two human fetal livers; however, expression did occur when human fetal hepatocytes were placed into culture for 4 days. These results suggest that cultures of rat and human fetal hepatocytes may be a valuable model with which to study factors that regulate expression of CYP2E1 and the influence of ethanol and other inducers on expression and stabilization of CYP2E1. PMID- 8622630 TI - Photoaffinity labeling of the human substance P (neurokinin-1) receptor with [3H2]azido-CP-96,345, a photoreactive derivative of a nonpeptide antagonist. AB - An azido derivative of [3H2](2S, 3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl) methyl)-1-azabicyclo[2.2.2]octon-3-amine (CP-96,345), a potent nonpeptide antagonist of the substance P (SP) (neurokinin-1) receptor, was synthesized and shown to have an affinity for the human SP receptor similar to that of the parent compound, CP-96,345. When Chinese hamster ovary cells expressing the human SP receptor were photolabeled with this compound and analyzed with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, several radioactive bands were observed, including a major band centered at molecular mass 80 kDa, the expected value for the SP receptor expressed in Chinese hamster ovary cells. Only the labeling of the 80-kDa protein was specific: nonradiolabeled CP-96,345 but not its optical enantiomer, CP-96,344 was a potent inhibitor of photoincorporation. SP prevented photolabeling only at concentrations higher than expected from its binding affinity but similar to those shown in a competition binding assay to displace radioiodinated analogue of CP-96,345. Antiserum generated against a synthetic peptide corresponding to the carboxyl terminus of the human SP receptor immunoprecipitated only the 80-kDa photoaffinity labeled protein, confirming that it is the human SP receptor. Interestingly, a second antiserum that was generated against the third extracellular loop of the G protein-coupled receptor no longer immunoprecipitated the receptor when covalently labeled with [3H2]azido-CP-96,345. This result indicates either that attachment of the antagonist modified the antigenic region directly, suggesting involvement of this domain in the binding of CP-96,345, or that the loss of recognition by the antiserum is secondary to a change in conformation induced by the covalent attachment of the antagonist at a different site. PMID- 8622631 TI - 1-Hydroxyethyl radical formation during NADPH- and NADH-dependent oxidation of ethanol by human liver microsomes. AB - Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. Experiments were carried out to evaluate the ability of human liver microsomes to catalyze this reaction, compare the effectiveness of NADH with that of NADPH, and assess the possible role of cytochrome b5 in HER formation. HER was detected as the alpha-(4-pyridly-1 -oxide)-N-t butylnitrone/HER adduct. Human liver microsomes catalyzed HER formation with either NADPH or NADH as cofactor; rates with NADH were approximately 50% those found with NADPH. Chelex-100 treatment of the reaction mixture produced marked inhibition of HER formation, suggesting that a transition metal, such as iron, was required to catalyze the reaction. The addition of ferric chloride restore HER formation. Catalase (2600 units/ml) and superoxide dismutases (500 units/ml) nearly completely inhibited the reaction with either NADPH or NADH. The NADH dependent rates of superoxide production, detected as 5,5-dimethyl-1-pyrroline-N oxide-O2H, were approximately 50% the NADPH-dependent rates, which is consistent with the rates of HER formation. Anti-cytochrome b5 IgG decreased NADPH- and NADH dependent HER formation, and this was associated with inhibition of superoxide formation with both reductants. These results indicate that human liver microsomes can catalyze the oxidation of ethanol of HER with either NADPH or NADH as reductant. The effectiveness of NADH may be significant in view of the increased NADH/NAD+ redox ratio in the liver as a consequence of ethanol oxidation by alcohol dehydrogenase. HER formation by human liver microsomes seems to be catalyzed by an oxidant derived from the interaction of iron with superoxide or H2O2, and a close association exists between HER formation and superoxide production. Cytochrome b5 seems to play a role in HER formation, most likely due to its effect on superoxide production. PMID- 8622632 TI - Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam. AB - We evaluated whether tolerance to the antagonism of bicuculine-induced seizures by diazepam is associated with changes (i) in the content of mRNAs encoding for gamma-aminobutyric acidA (GABAA) receptor subunits, (ii) in the expression density of these subunits, and (iii) in the 1,4-benzodiazepine binding site characteristics in discrete neocortical structures. We found that in diazepam tolerant rats, the content of the mRNA encoding for the alpha 1 subunit of the GABAA receptor decreased in the frontoparietal motor (FrPaM) cortex and in the hippocampus (42% and 20%, respectively) but not in the frontoparietal somatosensory (FrPaSS) cortex, striatum, olfactory bulb, and cerebellum. In the FrPaM cortex, gamma 2S and gamma 2L subunit mRNA contents were also decreased (48% and 30%, respectively), whereas that of alpha 5 was increased (30%). In the FrPaM and FrPaSS cortices as well as in cerebellum of diazepam-tolerant rats, the content of alpha 2, alpha 3, alpha 6, beta 2, and delta subunit mRNA was unchanged, as was the content of alpha 2, alpha 5, gamma 1, and gamma 2S subunit mRNA in the hippocampus. Furthermore, the reduction in alpha 1 subunit mRNA content in the FrPaM cortex and the anticonvulsant tolerance to diazepam returned to control values 72 hr after termination of the protracted diazepam treatment. Rats receiving a treatment with imidazenil in doses equipotent and with a schedule identical to that of diazepam failed to exhibit tolerance to the anticonvulsant action of this drug or cross-tolerance to diazepam. In these rats, the content of mRNA encoding for alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, gamma 1, gamma 2S, gamma 2L, and delta GABAA receptor subunits failed to change in the FrPaM and FrPaSS cortices, in the hippocampus, and in the other brain areas that were studied in diazepam-tolerant rats. Although the density and affinity of [3H]flumazenil and [3H]imidazenil binding failed to change in the FrPaM and FrPaSS cortices of diazepam-tolerant rats, the expression density of alpha 1 subunit immunogold labeling decreased by 37%, whereas that of alpha 5, gamma 2L/S, and beta 2/3 increased by 158%, 50%, and 47%, respectively, in the FrPaM cortex, and the density of the alpha 5 subunit selectively increased (209%) in the FrPaSS cortex. In contrast, the immunogold labeling density of the alpha 1, alpha 5, gamma 2L/S, and beta 2/3 subunits failed to change in either the FrPaM or FrPaSS cortex of rats receiving protracted imidazenil treatment. PMID- 8622633 TI - Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells. AB - We studied the effect of doxorubicin (Dox) on cell cycle progression and its correlation with DNA damage and cytotoxicity in p53-mutant P388 cells. P388 cells synchronized in S and G2/M phases were > 3-fold more sensitive to Dox than were cells in G1 phase (Dox ID50 = 0.50 +/- 0.16 microM in cells synchronized in S phase versus 1.64 +/- 0.12 microM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 microM Dox (2 x ID50) for 1 hr induced a marked cell arrest at G2/M phase at 6-12 hr after drug incubation. We then studied the effect of Dox on the p34cdc2/cyclin B1 complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34cdc2 kinase localizes in the nucleus and cytoplasms, particularly in the centrosomes, and p34cdc2 kinase activity is dependent on cell cycle progression, with the enzyme activity increasing steadily from G1/S to G2/M and markedly declining thereafter. Treatment of synchronized P388 cells in early S phase with 1 microM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for > or = 10 hr. In a cell-free system, Dox did not inhibit p34cdc2 kinase activity, indicating that is has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34cdc2 kinase dephosphorylation without altering its synthesis, and this effect was due to neither down-regulation of cdc25C nor inhibition of protein-tyrosine phosphatase activity. In contrast, Dox treatment was found to induced cyclin B1 accumulation as a result of stimulating its synthesis and inhibiting its degradation. A good correlation was found between extent of DNA double-strand breaks and p34cdc2 kinase activity inhibition. Our results suggest that anthracycline-induced cytotoxicity is cell cycle dependent and is mediated, at least in part, by disturbance of the regulation of p34cdc2/cyclin B1 complex, thus leading to G2/M phase arrest. PMID- 8622634 TI - Selective inhibition of topoisomerase II by ICRF-193 does not support a role for topoisomerase II activity in the fragmentation of chromatin during apoptosis of human leukemia cells. AB - Specific inhibitors of topoisomerase II (e.g., ICRF-193, an inhibitor of the catalytic activity of topoisomerase II and etoposide that stabilizes enzyme/DNA cleavable complexes) have been used to probe the role of topoisomerase II in the fragmentation of DNA during drug-induced apoptosis of human HL-60 leukemia cells. Topoisomerase II plays a role in the attachment of 50-kilobase domains of DNA to the nuclear matrix; fragments of this size are cleaved during apoptosis. Apoptosis was induced by 50 microM etoposide or 300 mM N-methylformamide (NMF), a nongenotoxic agent. Treatment with etoposide or NMF induced the morphology of apoptosis within 4 hr. Analysis of DNA integrity by electrophoresis showed coincident fragmentation from 50 kb and to integers of 200 bp. Transient protein associated DNA strand breaks, characteristic of etoposide-induced damage, were visualized as DNA fragments of > 600 kb. Preincubation with ICRF-193 (100 microM) reduced the number of etoposide-induced DNA strand breaks by 50% and delayed the appearance of DNA fragmentation by approximately 18 hr. However, ICRF-193 had no effect on either NMF- or camptothecin-induced DNA fragmentation. The induction of apoptosis by both etoposide and NMF was associated with a reduction in the cellular levels of topoisomerases II alpha and II beta. ICRF-193 inhibited proteolytic cleavage of topoisomerase II induced by etoposide but not by NMF. The data suggest that the activity of topoisomerase II is not required for the cleavage of DNA to 50-kb fragments but that proteolysis of topoisomerase II represents a conserved event of apoptosis. PMID- 8622635 TI - Multiplicity of glutamate receptor subunits in single striatal neurons: an RNA amplification study. AB - The RNA amplification technique was used to examine the pattern of coexpression of mRNAs encoding 16 subtypes/subunits of the glutamate receptor (GluR) in acutely dissociated neurons from adult rat striata. THe signal intensity for each mRNA varied within single neurons, but the general pattern of low versus high expression signals was similar among neurons, except for the GluR4 subunit of the (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. The mRNAs for GluR1-3 subunits of the AMPA receptor were present in all cells, with the signal intensity of GluR1 mRNAs usually the lowest. The kainate receptor subunit mRNAs (GluR5-7) were present in most neurons, and the signal intensity for GluR6 mRNA was the highest. The signals for N-methyl-D-aspartate (NMDA)R1 and NMDAR2B mRNAs were high in most neurons; however, NMDAR2A and NMDAR2C mRNAs gave low or undetectable signals. For mRNAs encoding metabotropic GluRs (mGluRs), signals for mGluR1, mGluR2, and mGluR3 mRNAs were low or undetectable, whereas mGluR4 and mGluR5 mRNA signals were high in most neurons. In most cases (12 of 16 mRNAs), the results agreed with data from in situ hybridization experiments in which individual mRNAs were examined. All neurons expressed subtypes/subunits mRNAs for all four types of GluRs; however, there were differences in the relative intensity of the mRNA signals detected in individual cells, suggesting that these receptors could exist in various combinations within individual neurons and thus confer synapse-specific function for information processing in the striatum. PMID- 8622636 TI - Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B. AB - (2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B. PMID- 8622637 TI - Ahr locus phenotype in congenic mice influences hepatic and pulmonary DNA adduct levels of 2-amino-3-methylimidazo[4,5-f]quinoline in the absence of cytochrome P450 induction. AB - The potent food mutagen/carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) undergoes metabolic N-hydroxylation by cytochromes P450, including cytochrome P450 1A2, followed by generation of an unstable ester catalyzed by acetyltransferases; promutagenic DNA adducts result. Genetic polymorphisms in these enzymes have been implicated in human cancer risk related to arylamine exposure. We investigated the effects of Ahr locus and acetylator polymorphisms on 32P-postlabeled IQ/DNA adducts in lungs and livers of female C57BL/6 mice congenic for slow acetylation and/or Ahr-nonresponsiveness; some groups were pretreated with beta-naphthoflavone (beta NF), a cytochrome P450 1A inducer. Total adducts in lung were doubled by beta NF pretreatment in Ahr-responsive mice only and consisted of < or = 30% adduct 2 and < or = 60% adduct 3. In contrast, in Ahr-nonresponsive mice, adducts 2 and 3 were each < or= 7% of the total. Livers of noninduced Ahr-responsive mice formed 6-18-fold more adducts than those of nonresponsive mice. This striking difference was not due to altered levels of cyp1a-2, as indicated by specific enzyme assays and immunoblotting, and was not accompanied by a comparable increase in the ability of liver preparations to activate IQ to a mutagen in the Ames test. Pretreatment of responsive mice with beta NF to induce cyp1a-1 and cyp1a-2 led to a reduction in liver adduct levels. Acetylation phenotype also had a significant effect in Ahr-responsive mice, with 3-fold more adducts in slow than in rapid acetylators. These results indicate that in uninduced mice, the normal Ah receptor facilitates formation of IQ/DNA adducts in liver and alters the profile of adducts in lung, via an unknown mechanism, whereas the Ah receptor-dependent enzyme induction reduces adducts in liver, probably due to increased detoxification, but increases them in lung. PMID- 8622638 TI - Marked inhibitory activity of non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 when combined with (-)2',3'-dideoxy 3'-thiacytidine. AB - Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated (as single agents or in combination) with (minus)-2', 3'-dideoxy-3'-thiacytidine (3TC) and the following HIV-1-specific non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs): 2', 5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino 1',2'-oxathi ole)-2',2'-dioxide derivative of 3-methylthymidine (TSAO-m3T), the thiocarboxanilides UC10 and UC42, bis(heteroaryl)piperazine (BHAP) derivative U90152, and the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivative 5-isopropyl-1-ethoxymethyl-6-benzyluracil (MKC-442). When used individually, the compounds led to the emergence of HIV-1 strains containing the following mutations in the RT: Glu138 to lysine for TSAO-m3T, Met184 to valine for 3TC, Lys103 to threonine/asparagine for the thiocarboxanilides, and Tyr181 to cysteine for BHAP and MKC-442. When 3TC was combined with TSAO-m3T, UC10, UC42, BHAP, or MKC-442, breakthrough of virus was markedly delayed or even suppressed. For these drug combinations, the concentrations of the individual drugs could be lowered by > or = 25-50-fold to suppress virus breakthrough compared with the individual use of the compounds. The concomitant presence of the Lys138 and Ile/Val184 mutations was found in the RT of the mutant viruses that emerged with combination therapy of the lowest concentrations of 3TC with either the lowest concentrations of TSAO m3T or UC10 (approximately 0.5-3-fold the EC50 value). These virus strains retained high sensitivity to other NNRTIs such as BHAP or HEPT. The virus mutants that arose in the presence of combinations of the lowest concentrations of 3TC with either BHAP or HEPT predominantly contained the Cys181 mutation in the RT. In one case, the Ile181 mutation was found. The latter mutations, particularly the Ile181 mutation, resulted in markedly decreased sensitivity to the NNRTIs but not to 3'-azido-2', 3'-dideoxythymidine or 3TC. PMID- 8622639 TI - A lysine residue of the cannabinoid receptor is critical for receptor recognition by several agonists but not WIN55212-2. AB - Lys192 in the third transmembrane domain of the human CB1 cannabinoid receptor was converted to an alanine to study its role in receptor recognition and activation by agonists. HU-210, CP-55940, WIN55212-2, and anandamide, four cannabinoid agonists with distinct chemical structures, were used to characterize the wild-type and the mutant receptors. In human embryonal kidney 293 cells stably expressing the wild-type receptor, specific binding to [3H]WIN55212-2 and inhibition of cAMP accumulation by cannabinoid agonists were demonstrated, with different ligands exhibiting the expected rank orders of potency and stereoselectivity in competition binding and functional assays. In cells expressing the mutant receptor, the binding affinity of the receptor for [3H]WIN55212-2 was only slightly affected (the Kd for the mutant receptor was twice that of the wild-type), and the ability of WIN55212-2 to inhibit cAMP accumulation was unchanged. However, HU-210, CP-55940, and anandamide were unable to compete for [3H]WIN55212-2 binding to the mutant receptor. In addition, the potencies of HU-210, CP-55940, and anandamide in inhibiting cAMP accumulation were reduced by > 100-fold. These results demonstrate that Lys192 is critical for receptor binding by HU-210, CP-55940, and anandamide. Because Lys192 is not important for receptor binding and activation by WIN55212-2, WIN55212-2 must interact with the cannabinoid receptor through at least one point of interaction that is distinct from those of the three other agonists. PMID- 8622640 TI - 3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor. AB - Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17 fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors. PMID- 8622641 TI - Isoform-specific sensitization of adenylyl cyclase activity by prior activation of inhibitory receptors: role of beta gamma subunits in transducing enhanced activity of the type VI isoform. AB - Many different types of cells develop increased adenylyl cyclase activity (sensitization) on prior treatment with drugs such as opiates that acutely inhibit the enzyme. We found that human embryonic kidney (HEK) 293 m2 cells, which express the inhibitory m2 muscarinic cholinergic receptor, exhibit a large increase in forskolin-stimulated cAMP synthesis when the cells are preincubated with the muscarinic agonist carbachol for > or = 5 min and forskolin stimulation is performed in the presence of the muscarinic antagonist atropine. To determine whether a specific isoform of adenylyl cyclase is susceptible to the adaptation induced by prior activation of inhibitory receptors, cells were transfected with expression vectors encoding adenylyl cyclase types I, II, and VI, representing three major groups of the adenylyl cyclase family. Preincubation of the cells with carbachol for 30 min resulted in a significant increase in prostaglandin E1 stimulated cAMP accumulation in cells expressing type VI, but not type I or type II, adenylyl cyclase. A similar selective increase in activity from type VI adenylyl cyclase was observed for prior treatment with the D2 dopamine agonist quinpirole and stimulation of cAMP synthesis with human chorionic gonadotropin in cells transfected with expression vectors coding for the cognate receptors. We next investigated whether beta gamma subunits play a role in the sensitization of type VI adenylyl cyclase activity; using expression of alpha tau to inhibit beta gamma-mediated effects, we found that the quinpirole-induced sensitization of type VI adenylyl cyclase was abolished. However, beta gamma subunits do not seem to directly activate type VI adenylyl cyclase, in contrast with their ability to directly activate the type II enzyme. Therefore, beta gamma subunits liberated after activation of inhibitory receptors seem to indirectly cause an increase in activity of type VI adenylyl cyclase. Indirect activation of the type VI enzyme by beta gamma subunits is a novel mechanism contributing to the sensitization of adenylyl cyclase. PMID- 8622642 TI - Interaction of full and partial agonists of the A1 adenosine receptor with receptor/G protein complexes in rat brain membranes. AB - Full and partial agonists of the A1 adenosine receptor were characterized with respect to their influence on G protein activation and their thermodynamic parameters of receptor binding in rat brain membranes. G protein activation was determined through measurement of [35S]guanosine-5'-(gamma-thio)triphosphate ([35S]GTP[S]) binding, and receptor binding was studied under identical conditions through the displacement of [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) in equilibrium binding studies. The intrinsic activity in stimulating [35S]GTP[S] binding did not correlate with the affinity of the ligands. 5'-Deoxy 5'-methylthioadenosine, 2-phenylaminoadenosine, and 2-chloro-2'-deoxyadenosine were identified as partial A1 agonists in the G protein activation assay. Depending on the temperature, these ligands showed agonistic and antagonistic properties to varying extents. EC50 values for G protein stimulation and KH and KL values of the partial agonists decreased when the incubations were performed at lower temperatures, indicating a mainly enthalpy-driven process of interaction with the receptor. Thermodynamic parameters of receptor binding of the partial agonists resembled the characteristics of the antagonist DPCPX more closely than those of the agonist 2-chloro-N6-cyclopentyladenosine. In addition, partial agonists detected fewer A1 adenosine receptors in the high affinity state than did full agonists. The lower efficacy in stimulation of the binding of [35S]GTP[S] is probably the consequence of an impaired ability of the partial agonists to release GDP from the G protein, as was shown by an impaired release of prebound [35S]GDP[S] from the membranes. PMID- 8622643 TI - Comparison of duration of agonist action at beta 1- and beta 2- adrenoceptors in C6 glioma cells: evidence that the long duration of action of salmeterol is specific to the beta 2-adrenoceptor. AB - The C6 glioma cell line, which expresses beta 1- and beta 2-adrenoceptors at a ratio of 80:20, was used to investigate the durations of action of formoterol at beta 1-adrenoceptors and of salmeterol at both beta 1- and beta 2-adrenoceptors in an attempt to determine whether the sustained duration of action of salmeterol was unique to beta 2-adrenoceptors or, as with formoterol, resulted from its lipophilic nature and partitioning into the bulk lipid of the plasma membrane. In this cell line, formoterol, like the nonselective beta-adrenoceptor agonist isoprenaline, behaved as a potent, full agonist at beta 1-adrenoceptors and did not seem to exhibit a high degree of selectivity for beta 2-adrenoceptors. Salmeterol seemed to stimulate cAMP accumulation in C6 cells predominantly via activation of the subpopulation of beta 2-adrenoceptors. However, at high (micromolar) agonist concentrations, salmeterol also activated beta 1 adrenoceptors, albeit with low potency and efficacy. At high concentrations (30 microM), salmeterol attenuated cAMP responses mediated by activation of beta 1 adrenoceptors by isoprenaline (Kp = 1.6 microM), indicating that salmeterol exhibited a low affinity for beta 1-adrenoceptors in C6 cells. In multiple washout experiments, cAMP responses to isoprenaline and formoterol waned with increasing numbers of washing processes. Therefore, it seemed that formoterol relied on its moderately lipophilic nature to partition into bulk lipid of the plasma membrane to produce sustained activity, particularly at high agonist concentrations. Salmeterol was found to persist at beta 2-adrenoceptors in C6 cells despite washing cell monolayers up to four times. To determine the duration of action of salmeterol at beta 1-adrenoceptors expressed on the same cells, use was made of full/partial agonist interactions. In cells exposed to a single washout of agonist-containing medium, salmeterol (30 microM) lost its ability to attenuate responses to the more efficacious agonist, isoprenaline. This observation provided convincing evidence to support the hypothesis that salmeterol exhibits sustained agonist activity at beta 2-adrenoceptors, but not beta 1-adrenoceptors, expressed on the same cells. Therefore, the sustained activity of salmeterol at beta 2-adrenoceptors seems to be unique and does not result solely from its partitioning into bulk lipid of the plasma membrane. PMID- 8622644 TI - Ability of nondepolarizing neuromuscular blocking drugs to act as partial agonists at fetal and adult mouse muscle nicotinic receptors. AB - We studied the ability of four nondepolarizing neuromuscular blocking agents (atracurium, gallamine, metocurine, and pancuronium) to act as competitive antagonists at mouse adult- and fetal-type muscle nictinic receptors. Receptor subunits for the fetal type (alpha, beta, gamma, and delta) and adult-type (alpha, beta, epsilon, and delta) receptors were stably expressed in quail fibroblasts. Binding for each drug was determined by the ability of the agents to reduce the initial rate of labeled alpha-bungarotoxin binding, and functional consequences were determined with the use of voltage-clamp studies of their ability to elicit currents or to block currents elicited by acetylcholine. Each agent has a different affinity for the two acetylcholine-binding sites on a single receptor; the rank order of affinities is the same for both fetal- and adult-type receptors. All agents inhibited activation of adult-type receptors by ACh, consistent with the idea that occupation of either the high or low affinity site completely blocks activation when acetylcholine binds to the other site on the receptor. The concentration dependence of the inhibition of acetylcholine elicited current was predictable from the affinities estimated from independent measurements of the inhibition of alpha-bungarotoxin binding. Gallamine and pancuronium also acted as competitive inhibitors of fetal-type receptors, and, again, the concentration dependence of the inhibition was predictable from binding data. However, metocurine and atracurium could potentiate the responses of fetal-type receptors to low concentrations of acetylcholine. The interaction of metocurine and atracurium with acetylcholine at fetal-type receptors could be accounted for by a weak partial agonist activity. It has been suggested that some pairs of nondepolarizing neuromuscular blocking agents might be more efficacious because the high affinity site for one agent might be the low affinity site for another. This hypothesis was tested for the pair of agents metocurine and gallamine by determining the ability of a mixture of agents to inhibit the binding of alpha-bungarotoxin. The results are consistent with the idea that both metocurine and gallamine have a high affinity for the same site on the receptor. The ability of gallamine to block the partial agonist action of metocurine at fetal-type receptors was tested as well and also indicated that both agents share the same high affinity site. PMID- 8622645 TI - Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor mediated gene activation in vivo. AB - Steroid/nuclear hormone receptors are ligand-regulated transcription f factors that play key roles in cell regulation, differentiation, and oncogenesis. Many nuclear receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA either as homodimers or as heterodimers with the 9-cis retinoic acid (RA) receptor (retinoid X-receptor [RXR]). We have previously reported that the ligands for VDR and RXR can differentially modulate the affinity of the receptors' interaction with DNA in vitro, primarily by modulating the dimerization status of these receptors. These experiments suggested a complex interaction between VDR and RXR and their respective ligands on inducible target genes in vivo. To examine these effects in cells, we used a transient transfection strategy whereby we simultaneously introduced two different reporter plasmids that are selectively inducible by each ligand. Although VDR can bind as a homodimer to the osteopontin gene vitamin D response element, we find that a RXR-VDR heterodimer must be the transactivating species from the element in vivo, since RXR enhances and 9-cis RA and other RXR-specific ligands attenuate this induction. Conversely, when VDR is overexpressed, vitamin D3 attenuates 9-cis RA induction from an RXR-responsive element. These effects, however, appear to be very sensitive to both the relative ratios of the two receptors and their respective target elements. Functional RXR-VDR complexes are strictly dependent on the DNA-binding polarity. Chimeric versions of VDR and RXR were also constructed to examine the putative activities of homodimeric receptors; a VDR chimera can transactivate in the absence of RXR, demonstrating that VDR has intrinsic transactivation properties. Taken together, these results establish a complex, sensitive cross talk in vivo between two ligands and their receptors that signal through two distinct endocrine pathways. PMID- 8622647 TI - Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. AB - Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady state activated Ras is insufficient to activate B-Raf in vivo. PMID- 8622646 TI - A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission. AB - The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation. PMID- 8622648 TI - Physical and functional sensitivity of zinc finger transcription factors to redox change. AB - Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites. PMID- 8622650 TI - Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation. AB - Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by an ankyrin-rich inhibitory protein termed I kappa B alpha. I kappa B alpha specifically binds to and masks the nuclear localization signal of the RelA subunit of NF-kappa B, thereby effectively sequestering this transcription factor complex in the cytoplasm. Specific cellular activation signals lead to the rapid proteolytic degradation of I kappa B alpha and the concomitant nuclear translocation of NF-kappa B. However, the precise biochemical mechanisms underlying the inhibitory effects of I kappa B alpha on RelA and its inducible pattern of degradation remain unclear. By using HeLa cells transfected with various cDNAs end-coding epitope-tagged mutants of I kappa B alpha, our studies demonstrate the following: (i) sequences within the 72-amino-acid N-terminal region of I kappa B alpha are required for tumor necrosis factor alpha (TNF alpha)-induced degradation but are fully dispensable for I kappa B alpha binding to and inhibition of RelA; (ii) serine residues located at positions 32 and 36 within the N-terminal region of I kappa B alpha represent major sites of induced phosphorylation (substitution of these serine residues with alanine abrogates TNF alpha-induced degradation of I kappa B alpha); (iii) the C-terminal 40 residues of I kappa B alpha (amino acids 277 to 317), which include a PEST-like domain, are entirely dispensable for TNF-alpha-induced degradation and inhibition of RelA; (iv) a glutamine- and leucine-rich (QL) region of I kappa B alpha located between residues 263 and 277 and overlapping with the sixth ankyrin repeat is required for both inducible degradation and inhibition of RelA function; (v) regulation of I kappa B alpha degradation by this QL-rich region appears to occur independently of phosphorylation at serines 32 and 36. These findings thus indicate that I kappa B alpha is generally organized within distinct modular domains displaying different functional and regulatory properties. These studies have also led to the identification of a novel class of dominant-negative I kappa B alpha molecules that retain full inhibitory function on NF-kappa B yet fail to undergo stimulus-induced degradation. These molecules, which lack N-terminal sequences, potently inhibit TNF-alpha-induced activation of the human immune deficiency virus type 1 kappa B enhancer, thus indicating their possible use as general inhibitors of NF-kappa B. PMID- 8622649 TI - Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. AB - The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated ED2Fs (E2F-1, E2F-2, and E2F 3) all induce S phase in quiescent rate fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody to cyclin D1. The p16INK4 induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S-phase entry, and overcomes a p16-mediated G1 block, and since E12F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p126-cyclin D-pRB pathway. Furthermore, our date suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer. PMID- 8622651 TI - Activation of signalling by the activin receptor complex. AB - Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals. PMID- 8622653 TI - Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae. AB - Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100 fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes. PMID- 8622652 TI - Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells. AB - The ability of thymocytes to express cytokine genes changes during the different stages of thymic development. Although CD4- CD8- thymocytes are able to produce a wide spectrum of cytokines in response to a T-cell receptor (TcR)-independent stimulus, as they approach the double-positive (DP) CD4+ CD8+ stage, they lose the ability to produce cytokine. After the DP stage, thymocytes become single positive CD4+ or CD8+ thymocytes which reacquire the ability to secrete cytokines. In an attempt to understand the molecular basis of this specific regulatin, we use AP-1-luciferase and newly generated NFAT-luciferase transgenic mice to analyze the transcriptional and DNA-binding activities of these two transcription factors that are involved in the regulation of cytokine gene expression. Here, we show that both AP-1 and NFAT transcriptional activities are not inducible in the majority of DP cells but that during the differentiation of DP cells to the mature single-positive stage, thymocytes regain this inducibility. Subpopulation analysis demonstrates that this inducibility is reacquired at the DP stage before the down-modulation of one of the coreceptors. Indeed AP-1 inducibility, just like the ability to express the interleukin-2 gene, is reacquired during the differentiation of DP TcRlow CD69low heat-stable antigen (HSA)high thymocytes to DP TcRhigh CD69high HSAhigh cells, which is considered to be the consequence of the first signal that initiates positive selection. We therefore propose that the inability of DP thymocytes to induce AP 1 and NFAT activities is one of the causes for the lack of cytokine gene expression at this stage and that this inducibility is reacquired at the latest stage of DP differentiation as a consequence of positive selection. This could be a mechanism to prevent the activation of DP thymocytes before selection has taken place. PMID- 8622654 TI - Phosphorylation-dependent formation of a quaternary complex at the c-fos SRE. AB - The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum responses element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex including the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. In this report, we describe a quaternary complex over the SRE which, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S 383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effect on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo. PMID- 8622655 TI - Regulated nuclear import of the Drosophila rel protein dorsal: structure-function analysis. AB - The formation of a gradient of nuclear Dorsal protein in the early Drosophila embryo is the last step in a maternally encoded dorsal-ventral signal transduction pathway. This gradient is formed in response to a ventral signal, which leads to the dissociation of cytoplasmic Dorsal from the I kappa B homolog Cactus. Free Dorsal is then targeted to the nucleus. Dorsal is a Rel-family transcription factor. Signal-dependent nuclear localization characterizes the regulation of Rel proteins. In order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus, we have performed an in vivo structure-function analysis. Our results show that all these functions are carried out by regions within the conserved Rel homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N terminal 40 amino acids abolished the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region. PMID- 8622656 TI - Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-met signalling in human cells concomitant with induction of the urokinase proteolysis network. AB - Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. Although HGF/SF is synthesized by mesenchymal cells and acts predominantly on epithelial cells, we have recently demonstrated that human sarcoma cell lines often inappropriately express high levels of Met and respond mitogenically to HGF/SF. In the present report we show that HGF/SF-Met signalling in the human leiomyosarcoma cell line SK-LMS-1 enhances its in vivo tumorigenicity, an effect for which the mitogenicity of this signalling pathway is likely to play a role. In addition, we found that HGF/SF Met signalling dramatically induces the in vitro invasiveness and in vivo metastatic potential of these cells. We have studied the molecular basis by which HGFSF-Met signalling mediates the invasive phenotype. A strong correlation has previously been demonstrated between the activation of the urokinase plasminogen activator (uPA) proteolysis network and the acquisition of the invasive metastatic phenotype, and we show here that HGF/SF-Met signalling significantly increases the protein levels of both uPA and its cellular receptor in SK-LMS-1 cells. This results in elevated levels of cell-associated uPA and enhanced plasmin-generating ability by these cells. These studies couple HGF/SF-Met signalling to the activation of proteases that mediate dissolution of the extracellular matrix-basement membrane, and important property for cellular invasion-metastasis. PMID- 8622657 TI - Cytoplasmic sequestration of wild-type p53 protein impairs the G1 checkpoint after DNA damage. AB - Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations. PMID- 8622658 TI - Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells. AB - Retinoids are known to inhibit the growth of hormone-dependent but not that of hormone-independent breast cancer cells. We investigated the involvement of retinoic acid (RA) receptors (RARs) in the differential growth-inhibitory effects of retinoids and the underlying mechanism. Our data demonstrate that induction of RAR beta by RA correlates with the growth-inhibitory effect of retinoids. The hormone-independent cells acquired RA sensitivity when the RAR beta expression vector was introduced and expressed in the cells. In addition, RA sensitivity of hormone-dependent cells was inhibited by a RAR beta-selective antagonist and the expression of RAR beta antisense RNA. Introduction of RAR alpha also restored RA sensitivity in hormone-independent cells, but this restoration was accomplished by the induction of endogenous RAR beta expression. Furthermore, we show that induction of apoptosis contributes to the growth-inhibitory effect of RAR beta. Thus, RAR beta can mediate retinoid action in breast cancer cells by promoting apoptosis. Loss of RAR beta, therefore, may contribute to the tumorigenicity of human mammary epithelial cells. PMID- 8622659 TI - A highly amplified mouse gene is homologous to the human interferon-responsive Sp100 gene encoding an autoantigen associated with nuclear dots. AB - In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment. PMID- 8622660 TI - Analysis of ATF3, a transcription factor induced by physiological stresses and modulated by gadd153/Chop10. AB - We demonstrate that ATF3, a member of the ATF/CREB family of transcription factors, is induced in a variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-deprived heart, and postseizure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop10, forms a nonfunctional heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does not bind to the ATF/cyclic AMP response element consensus site and does not repress transcription. Interestingly, ATF3 and gadd153/Chop10 are expressed in inverse but overlapping manners during the liver's response to carbon tetrachloride (CCl4): the level of gadd153/Chop10 mRNA is high in the normal liver and greatly decreases upon CCl4 treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver and greatly increases upon CCl4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibits the limited amount of ATF3 by forming an inactive heterodimer with it, whereas in CCl4-injured liver, the synthesis of gadd153/Chop10 is repressed, allowing the induced ATF3 to function. PMID- 8622661 TI - Interaction of the v-Rel oncoprotein with NF-kappaB and IkappaB proteins: heterodimers of a transformation-defective v-Rel mutant and NF-2 are functional in vitro and in vivo. AB - The v-Rel oncoprotein of the avian Rev-T retrovirus is a member of the Rel/NF kappa B family of transcription factors. The mechanism by which v-Rel malignantly transforms chicken spleen cells is not precisely known. To gain a better understanding of functions needed for transformation by v-Rel, we have now characterized the activities of mutant v-Rel proteins that are defective for specific protein-protein interactions. Mutant v-delta NLS, which has a deletion of the primary v-Rel nuclear localizing sequence, does not interact efficiently with I kappa B-alpha but still transforms chicken spleen cells approximately as well as wild-type v-Rel, indicating that interaction with I kappa B-alpha is not essential for the v-Rel transforming function. A second v-Rel mutant, v-SPW, has been shown to be defective for the formation of homodimers, DNA binding, and transformation. However, we now find that v-SPW can form functional DNA-binding heterodimers in vitro and in vivo with the cellular protein NF-kappa B p-52. Most strikingly, coexpression of v-SPW and p52 from a retroviral vector can induce the malignant transformation of chicken spleen cells, whereas expression of either protein alone cannot. Our results are most consistent with a model wherein Rel homodimers or heterodimers must bind DNA and alter gene expression in order to transform lymphoid cells. PMID- 8622662 TI - The scabrous gene encodes a secreted glycoprotein dimer and regulates proneural development in Drosophila eyes. AB - R8 photoreceptor cells play a primary role in the differentiation of Drosophila eyes. In scabrous (sca) mutants, the pattern of R8 photoreceptor differentiation is altered. The sca gene is predicted to encode a secreted protein related in part to fibrinogen and tenascins. Using expression in Drosophila Schneider cells, we showed that sca encoded a dimeric glycoprotein which was secreted and found in soluble form in the tissue culture medium. The sca protein contained both N- and O-linked carbohydrates and interacted with heparin. This Schneider cell protein was similar to protein detected in embryos. We showed that sca mutations, along with conditional alleles of Notch (N) and Delta (Dl), each affected the pattern of cells expressing atonal (ato), the proneural gene required for R8 differentiation. In normal development, about 1 cell in 20 differentiates into an R8 cell; in the others, ato is repressed. N and Dl were required to repress ato in the vicinity of R8 cells, whereas sca had effects over several cell diameters. Certain antibodies detected uptake of sca protein several cells away from its source. The overall growth factor-like structure of sca protein, its solubility, and its range of effects in vivo are consistent with a diffusible role that complements mechanisms involving direct cell contact. We propose that as the morphogenic furrow advances, cell secreting sca protein control the pattern of the next ommatidial column. PMID- 8622664 TI - The recessive phenotype displayed by a dominant negative microphthalmia associated transcription factor mutant is a result of impaired nucleation potential. AB - In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF. PMID- 8622665 TI - Evidence for two catalytically active kinase domains in pp90rsk. AB - Mitogen-activated protein kinase and one of its targets, pp90rsk (ribosomal S6 kinase [RSK]), represent two serine/threonine kinases in the Ras-activated signalling cascade that are capable of directly regulating gene expression. pp90rsk has been shown to have two highly conserved and distinct catalytic domains. However, whether both domains are active and which domain is responsible for its various identified phosphotransferase activities have not been determined. Here we demonstrate that the N-terminal domain is responsible for its phosphotransferase activity towards a variety of substrates which contain an RXXS motif at the site of in vitro phosphorylation, including serum response factor, c Fos, Nur77, and the 40S ribosomal protein S6. We also provide evidence that the C terminal domain is catalytically active and can be further activated by mitogen activated protein kinase phosphorylation. PMID- 8622663 TI - Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. AB - Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable. PMID- 8622666 TI - An RNA polymerase II promoter in the hsp70 locus of Trypanosoma brucei. AB - To study of structure of RNA polymerase (pol) II transcription units a nd the influence of temperature on the regulation of gene expression in Trypanosoma brucei, and hsp70 intergenic region promoter was characterized. In T. brucei, the hsp70 locus contains, from 5' to 3', a cognate hsp70-related gene (gene 1) which is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2 to 6). Transcription proceeds on the entire 23-kb locus, and polycistronic transcription occurs in hsp70 genes 2 to 6. Transcription of hsp70 genes 2 to 6 is only moderately sensitive to UV irradiation, indicating that it cannot be driven by a single far-upstream promoter, which suggests that promoters could be located in the region close to the hsp70 coding region. Transient transformations demonstrated that sequences located upstream of hsp70 gene 2 and in the intergenic region between hsp70 genes 2 and 3 are able to direct transcription of the reporter gene, the chloramphenicol acetyltransferase (CAT) gene. The plasmid DNA driven by the hsp70 intergenic region promoter gave CAT activity approximately 85-fold above to background level. This is equivalent to approximately 1% of that derived from a CAT plasmid driven by the procyclic acidic repetitive protein gene promoter, which is controlled by RNA pol I. The hsp70 intergenic region promoter can drive alpha-amanitin-sensitive transcription at an internal position of the chromosome as well as an episome, suggesting that it is controlled by RNA pol II. However, this hsp70 intergenic region promoter, along with the 3' splice site and the 5' untranslated region of the hsp70 genes that controls the transcription of the reporter gene, cannot up-regulate the expression of the reporter gene during heat shock. This result is consistent with the previous observation that expression of the hsp70 genes in T. brucei is mainly controlled at the posttranscriptional level. PMID- 8622667 TI - CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. AB - Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members. PMID- 8622668 TI - A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein. AB - The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center. PMID- 8622669 TI - MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. AB - The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway. PMID- 8622671 TI - New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila. AB - Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation. In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila. Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites. They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs. This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs. In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G. When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs. This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion. These results suggest a strong mechanistic link between chromosome breakage and telomere formation. PMID- 8622670 TI - Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage. AB - We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced. PMID- 8622673 TI - Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells. PMID- 8622672 TI - Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs. AB - The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis. PMID- 8622674 TI - p21 Disrupts the interaction between cdk2 and the E2F-p130 complex. AB - In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F p130 complex plays a role in the growth suppression function of p53. PMID- 8622675 TI - Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties. AB - Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer. PMID- 8622676 TI - Functional domains in the Mig1 repressor. AB - Mig1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. It is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 proteins and binds to a GC-rich motif that resembles the GC boxes recognized by these proteins. We have performed deletion mapping in order to identify functional domains in Mig1. We found that a small C-terminal domain comprising the last 24 amino acids mediates Mig1-dependent repression of a reporter gene. This effector domain contains several leucine-proline dipeptide repeats. We further found that inhibition of Mig1 activity in the absence of glucose is mediated by two internal elements in the Mig1 protein. A Mig1-VP16 hybrid activator was used to further investigate how Mig1 is regulated. Mig1-VP16 can activate transcription from promoters containing Mig1-binding sites and suppresses the inability of Snf1-deficient cells to grow on certain carbon sources. We found that a deletion of the SNF1 gene increases the activity of Mig1 VP16 fivefold under derepressing conditions but not in the presence of glucose. This shows that the hybrid activator is under negative control by the Snf1 protein kinase. Deletion mapping within Mig1-VP16 revealed that regulation of its activity by Snf1 is conferred by the same internal elements in the Mig1 sequence that mediate inhibition of Mig1 activity in the absence of glucose. PMID- 8622677 TI - Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2. AB - Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function. PMID- 8622678 TI - Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia. AB - A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I. PMID- 8622680 TI - Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins. AB - We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others. PMID- 8622679 TI - Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1. AB - vHNF1 (also termed HNF1 beta) is a member of the hepatocyte nuclear fa ctor 1 (HNF1; also termed HNF1 alpha) family of homeodomain-containing transcription factors that interact with a sequence motif found in the regulatory regions of a large number of genes expressed mainly in the liver. It has been suggested that vHNF1 plays a role in early differentiation of specialized epithelia of several endoderm- and mesoderm-derived organs, with HNF1 playing a role in later stages. In support of this idea, expression of vHNF1 but not HNF1 is induced upon treatment of the embryonal carcinoma cell line F9 with retinoic acid. We have cloned and analyzed the vHNF1 promoter to gain a better understanding of the regulation of vHNF1 expression and how it relates to the expression of HNF1. We have identified five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site, which involve at least three different families of transcription factors. Two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity. The DR-1 motif interacts with several members of the steroid hormone receptor superfamily including HNF4, COUP-TFI/Ear3, COUP-TFII/Arp1, and RAR alpha/RXR alpha heterodimers. The vHNF1 promoter is transactivated by COUP-TFI/Ear3 and COUP TFII/Arp1 and, unlike the HNF1 promoter, is virtually unaffected by HNF4. Interestingly, the proximal octamer site and not the DR-1 site is required for COUP-TFI/Ear3 and COUP-TFII/Arp1 transactivation of the vHNF1 promoter. COUP TFI/Ear3 does not bind directly to this proximal octamer site. We present evidence of an interaction between COUP-TFI/Ear3 and the octamer-binding proteins in vitro and in the cell, suggesting that COUP-TFI and COUP-TFII activate the vHNF1 promoter via an indirect mechanism. PMID- 8622682 TI - Postreplicative chromatin assembly by Drosophila and human chromatin assembly factor 1. AB - To study the relationship between DNA replication and chromatin assembly, we have purified a factor termed Drosophila chromatin assembly factor 1 (dCAF-1) to approximately 50% homogeneity from a nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA replication-coupled chromatin assembly revealed that both dCAF-1 and human CAF-1 mediate chromatin assembly preferentially with previously yet newly replicated DNA relative to unreplicated DNA. Moreover, the preferential assembly of the postreplicative DNA was observed at 30 min after inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it was no longer apparent at 120 min after inhibition of replication. These findings suggest that the coupling between DNA replication and chromatin assembly may not necessarily involve a direct interaction between the replication and assembly factors at a replication fork. PMID- 8622681 TI - Inhibition of v-Mos kinase activity by protein kinase A. AB - We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase. PMID- 8622683 TI - Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits. AB - U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA. PMID- 8622684 TI - A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene. AB - The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus. PMID- 8622685 TI - The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress. AB - Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1. PMID- 8622686 TI - Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae. AB - Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1. PMID- 8622687 TI - Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. AB - p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G1. By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings. PMID- 8622688 TI - 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region. AB - NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine threonine kinase with a novel substrate specificity. PMID- 8622689 TI - Functional dissection of the human Bcl2 protein: sequence requirements for inhibition of apoptosis. AB - Overexpression of the cytoplasmic oncoprotein Bcl2 blocks programmed cell death (apoptosis) in many cellular systems. To map the sequences in Bcl2 that are necessary for its activity, we created a library of deletion-scanning mutants of this 239-amino-acid protein and tested their abilities to block staurosporine induced fibroblast apoptosis, using a novel transient-transfection assay. Phenotypes of informative mutants were then confirmed by assaying for inhibition of steroid-induced apoptosis in stably transfected T-lymphoid cells. In accordance with earlier results, we found that Bcl2 activity was only partially reduced after deletion of the hydrophobic tail that normally anchors it in cytoplasmic membranes. Essential sequences were found in the remainder of the protein and appeared to be organized in at least two discrete functional domains. The larger, more C-terminal region (within residues 90 to 203) encompassed, but extended beyond, two oligopeptide motifs called BH1 and BH2, which are known to mediate dimerization of Bcl2 and related proteins. The second, more N-terminal regions (within residues 6 to 31) was not required for protein dimerization in vivo, but its deletion imparted a dominant negative phenotype, yielding mutants that promoted rather than inhibited apoptotic death. Residues 30 to 91 were not absolutely required for function; by deleting most of this region along with the hydrophobic tail, we derived a 155-residue mini-Bcl2 that retains significant ability to inhibit apoptosis. PMID- 8622690 TI - Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas. AB - Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic. PMID- 8622691 TI - Casein kinase II is required for efficient transcription by RNA polymerase III. AB - Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III. PMID- 8622692 TI - Casein kinase II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and is required for its degradation. AB - The phosphoprotein I kappa B alpha exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-kappa B (p50-p65). In response to specific cellular stimulation, I kappa B alpha is further phosphorylated and subsequently degraded, allowing NF-kappa B to translocate to the nucleus and transactivate target genes. To identify the kinase(s) involved in I kappa B alpha phosphorylation, we first performed an I kappa B alpha in-gel kinase assay. Two kinase activities of 35 and 42 kDa were identified in cellular extracts from Jurkat T and U937 promonocytic cell lines. Specific inhibitors and immunodepletion studies identified the I kappa B alpha kinase activities as those of the alpha and alpha' subunits of casein kinase II (CKII). Immunoprecipitation studies demonstrated that CKII and I kappa B alpha physically associate in vivo. Moreover, phosphopeptide maps of I kappa B alpha phosphorylated in vitro by cellular extracts and in vivo in resting Jurkat T cells contained the same pattern of phosphopeptides as observed in maps of I kappa B alpha phosphorylated in vitro by purified CKII. Sequence analysis revealed that purified CKII and the kinase activity within cell extracts phosphorylated I kappa B alpha at its C terminus at S-283, S-288, S-293, and T-291. The functional role of CKII was tested in an in vitro I kappa B alpha degradation assay with extracts from uninfected and human immunodeficiency virus (HIV)-infected U937 cells. Immunodepletion of CKII from these extracts abrogated both the basal and enhanced HIV-induced degradation of I kappa B alpha. These studies provide new evidence that the protein kinase CKII physically associates with I kappa B alpha in vivo, induces multisite (serine/threonine) phosphorylation, and is required for the basal and HIV-induced degradation of I kappa B alpha in vitro. PMID- 8622693 TI - Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene. AB - As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner. PMID- 8622694 TI - Characterization of a transcription terminator of the procyclin PARP A unit of Trypanosoma brucei. AB - The polycistronic procylcin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5- to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II. PMID- 8622695 TI - Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes. AB - Hepatocyte nuclear factor 4 (HNF4) was first identified as a DNA binding activity in rat liver nuclear extracts. Protein purification had then led to the cDNA cloning of rat HNF4, which was found to be an orphan member of the nuclear receptor superfamily. Binding sites for this factor were identified in many tissue-specifically expressed genes, and the protein was found to be essential for early embryonic development in the mouse. We have now isolated cDNAs encoding the human homolog of the rat and mouse HNF4 splice variant HNF4 alpha 2, as well as a previously unknown splice variant of this protein, which we called HNF alpha 4. More importantly, we also cloned a novel HNF4 subtype (HNF4 gamma) derived from a different gene and showed that the genes encoding HNF 4 alpha and HNF4 gamma are located on human chromosomes 20 and 8, respectively. Northern (RNA) blot analysis revealed that HNF4 GAMMA is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4 alpha RNA was found in all of these tissues. By cotransfection experiments in C2 and HeLa cells, we showed that HNF4 gamma is significantly less active than HNF4 alpha 2 and that the novel HNF4 alpha splice variant HNF4 alpha 4 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF4 proteins may play a key role in the differential transcriptional regulation of HNF4-dependent genes. PMID- 8622696 TI - Drosophila homologs of the proto-oncogene product PEBP2/CBF beta regulate the DNA binding properties of Runt. AB - The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt. PMID- 8622697 TI - Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases. AB - The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation. PMID- 8622699 TI - Identification of Prp40, a novel essential yeast splicing factor associated with the U1 small nuclear ribonucleoprotein particle. AB - We have used suppressor genetics to identify factors that interact with Saccharomyces cerevisiae U1 small nuclear RNA (snRNA). In this way, we isolated PRP40-1, a suppressor that restores growth at 18 degrees C to a strain bearing a cold-sensitive mutation in U1 RNA. A gene disruption experiment shows that PRP40 is an essential gene. To study the role of PRP40 in splicing, we created a pool of temperature-sensitive prp40 strains. Primer extension analysis of intron containing transcripts in prp40 temperature-sensitive strains reveals a splicing defect, indicating that Prp40 plays a direct role in pre-mRNA splicing. In addition, U1 RNA coimmunoprecipitates with Pro40, indicating that Prp40 is bound to the U1 small nuclear ribonucleoprotein particle in vivo. Therefore, we conclude that PRP40 encodes a novel, essential splicing component that associates with the yeast U1 small nuclear ribonucleoprotein particle. PMID- 8622700 TI - Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum. AB - We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum. Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins. Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site specific, bidirectional origin of replication is localized within the promoter region of the gene. We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon. Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins. Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions. Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P. polycephalum. PMID- 8622698 TI - Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. AB - The Notch/Lin-12/Glp-1 receptor family participates in cell-cell signaling events that influence cell fate decisions. Although several Notch homologs and receptor ligands have been identified, the nuclear events involved in this pathway remain incompletely understood. A truncated form of Notch, consisting only of the intracellular domain (NotchIC), localizes to the nucleus and functions as an activated receptor. Using both an in vitro binding assay and a cotransfection assay based on the two-hybrid principle, we show that mammalian NotchIC interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. Cotransfection assays using segments of mouse NotchIC and CBF1 demonstrated that the N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and that the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. Furthermore, transfection assays in which mouse NotchIC was targeted through Gal4-CBF1 or through endogenous cellular CBF1 indicated that NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization. PMID- 8622703 TI - Inhibition of Bcr serine kinase by tyrosine phosphorylation. AB - The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID- 8622702 TI - Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues. AB - Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage. PMID- 8622704 TI - A new method for estimating high mutation rates in cultured cells. AB - Fluctuation analysis allows for the determination of mutation rates in cell cultures in vitro. As originally described by Luria and Delbruck and extended by Lea and Coulson and by Capizzi and Jameson, this analysis has been useful in estimating mutation rates in cultured cells where the frequency of mutational events is low. However. in cultures where high mutation rates and multiple independent mutation events occur, leading to the accumulation of many mutant cells, these standard methods may not apply. Here, we present a new method for the estimation of mutation rates based on the assumption that multiple events may contribute to the accumulation of mutant cells. We compared mutation rates determined by Lea and Coulson's and by Capizzi and Jameson's methods with those determined by our method using experimental and stimulated data from our studies of immunoglobulin gene mutation and isotype switching in B lymphocyte cultures. The three methods resulted in very different calculated rates when many mutants were present in the culture, such as when mutation rates were high, while only small differences in calculated rates were found when mutants were rare. Unlike previous fluctuation analysis calculations, our method is applicable for the estimation of both low and high rates. PMID- 8622701 TI - Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. AB - Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2 associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation. PMID- 8622705 TI - Rec-A protein-mediated irreversible fixation of an oligodeoxyribonucleotide to specific site in DNA. AB - RecA protein can polymerize on an oligodeoxyribonucleotide to form a filament that finds its homologous sequence in double-stranded DNA. When such an oligonucleotide is linked to psoralen, a photoactivatable DNA intercalator, it irreversibly binds to the homologous site in double stranded DNA as a result of psoralen photoadduct formation at thymidines. The relative efficiency of specific vs. nonspecific binding of an oligonucleotide depended upon the ratio of psoralenated oligonucleotide to total DNA. Na+ ions at concentrations greater than 50 mM eliminated specific binding. Under optimal conditions. the probability of binding of an 80-mer oligonucleotide to a specific site was > 10(5) times greater than that of binding to any single nonspecific site. Under the conditions described, RecA-mediated photoadduction was equally efficient with superhelical and linear double-stranded DNA. PMID- 8622706 TI - A C3H strain-specific allele (alpha va126) of the murine alpha-globin gene newly detected by UT-PAGE and RT-PCR-SSCP analysis. AB - An extra band. distinct from the well-characterized globin chains (alpha, beta maj, beta-min, beta-s), was detected in an adult erythrocyte sample of the C3H strain by urea triton polyacrylamide gel electrophoresis (UT-PAGE) analysis. The extra band was recognized by an antibody against the alpha-globin chain by Western blot analysis. Reverse transcription, polymerase chain reaction and single strand conformation polymorphism (RT-PCR-SSCP) analysis and direct sequencing analysis of cDNA of the alpha-globin gene revealed a nucleotide substitution (GGA to GTA) corresponding to an amino acid substitution (Gly to Val) at codon 26 in the alpha-globin gene only in the erythrocyte sample of the C3H strain. Polypeptides generated by in vitro translation from the alpha-globin gene with the nucleotide substitution at codon 26 (alpha Val26) had the same mobility as that of the extra band of the C3H strain in UT-PAGE. These results suggest that the substitution GGA (Gly) to GTA (Val) at codon 26 of the murine alpha-globin gene may directly affect the mobility of alpha-globin in UT-PAGE and the base substitution may be a C3H strain-specific polymorphism. PMID- 8622707 TI - Analysis of mitotic recombination induced by several mono- and bifunctional alkylating agents in the Drosophila wing-spot test. AB - Mitotic recombination induced by six alkylating agents has been studied in the wing-spot test of Drosophila melanogaster. The model mutagens chosen have different models of action at the DNA level. These are: the direct-acting small alkylating agent methylmethanesulfonate (MMS), the small promutagens N dimethylnitrosamine (DMN) and N-diethylnitrosamine (DEN), the bifunctional cross linking alkylating agents mitomycin C (MMC), chlorambucil (CLA) and monocrotaline (MCT). Flies of the standard cross (flr3 / TM3, Bds females and mwh males) were used to produce the larvae to be treated. Three-day old Drosophila larvae were exposed by chronic feeding for 48 h to three different concentrations of all six alkylating agents. Acute feeding for only 2 h was used in addition with DEN and MMC. Wings of the marker-heterozygous (mwh+ / + flr3) as well as of the balancer heterozygous (mwh+ / TM3, Bds) progeny were analysed. The ranking of the compounds with respect to their genotoxic potency, based on mwh clone formation frequency in marker-heterozygous wings was: MMS > MNC > DMN > CLA approximately MCT > DEN. The ranking with respect to the induction of twin spots, which are produced by mitotic recombination exclusively, was: MMS > DMN > MMC > MCT > CLA > DEN. The quantitative determination of recombinagenic activity, based on mwh clone formation frequencies obtained in both types of wings, gave the following values: MMS, 93%; MCT, 87%; CLA, 80%; MMC, 73%; DMN, 67%; DEN, 22%. A clear relationship exists between the extent of N-alkylation of DNA and the efficiency of the monofunctional agents MMS and DMN as well of the bifunctional agents MCT, CLA and MMC to induce mitotic recombination. This contrasts with the ethylation of base oxygen atoms and the resulting lower efficiency of DEN to produce mitotic recombination. PMID- 8622708 TI - Comparison of spontaneous hprt mutation spectra at the nucleotide sequence level in the endogenous hprt gene and five other genomic positions. AB - Mutation spectra at the nucleotide sequence level of five hprt cDNA genes integrated in different genomic positions of a HPRT(-) derivative of the human lymphoblastoid TK6 cell line were compared with each other and with the spectrum of mutations confined to the 657 bp coding region of the endogenous hprt gene in the parental TK6 cells. The mutation rates in these genomic positions vary significantly and also the mutation spectra are different. In each genomic position the majority of mutations are basepair substitutions and deletions. the ratios of which vary among the genomic positions. Although it is likely that the different rates of deletion are to a large extent the net result of different rates of misalignment and repair of these errors in the various genomic positions, for the basepair substitutions it is not possible to deduce which mechanisms have caused these mutations and what causes the differences among the genomic positions. Taken together, the differences in mutation rates and spectra cannot be explained by a single mutagenic process. PMID- 8622709 TI - Radiation-induced chromosomal aberrations among TENORM workers: amang- and ilmenite-processing workers of Malaysia. AB - The usefulness of peripheral human lymphocytes as a bioindicator for ionizing radiation effect was tested in a survey of Malaysian workers in two industries producing technologically enhanced naturally occurring radioactive material (TENORM). Workers in amang processing plants who have been with the plant for an average of 12.9 years and who were exposed to radioactive dust showed significantly higher frequencies of chromosomal aberration compared to control and even ilmenite-processing workers. Such frequency was not significantly different between workers in ilmenite-processing plant and control. The differences in duration of employment, occupational hygiene, together with the difference in the percentage of 'old' and 'new' aberrations among the groups sampled were used to explain the high chromosomal aberration frequency among amang workers. The presence of significantly high chromosome damage (dicentrics and fragments) in workers who were chronically exposed to doses below 50 mSv per year or 20 mSv per year averaged over 5 years (ICRP, 1991) provided additional experimental data on the dose-effect relationship at these low-dose ranges. The results confirm the usefulness of using human lymphocytes as a bioindicator for chronic exposure to ionizing radiation and in cases where physical radiation detectors are not available. PMID- 8622710 TI - Inhibition of the phosphorylation of a myristoylated alanine-rich C kinase substrate by methyl methanesulfonate in cultured NIH 3T3 cells. AB - The effect of methyl methanesulfonate (MMS) on the phosphorylation of an acidic 80-kDa myristoylated alanine-rich C kinase substrate (MARCKS) protein was investigated in NIH 3T3 fibroblasts. An alkylating agent, MMS inhibited protein kinase C activity and the phosphorylation of MARCKS. MMS treatment also lowered the cellular amounts of second messengers of inositol-1,4,5-trisphosphate and diacylglycerol. Data suggest that MMS decreased the phosphorylation of phospholipase C, a protein whose activity is influenced by its phosphorylation state. We present here the first report that MMS intervenes in a signal cascade by inhibiting the phosphorylation of phospholipase C, which in turn leads to the inactivation of protein kinase C and the subsequent inhibition of MARCKS phosphorylation. PMID- 8622711 TI - 32P-Postlabeling analysis of a DNA adduct, an N2-acetyl derivative of guanine, formed in vitro by methylglyoxal and hydrogen peroxide in combination. AB - Methylglyoxal is a direct-acting mutagen in Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide. In addition, a mixture of methylglyoxal and hydrogen peroxide reacts with 2' deoxyguanosine to form N2-acetyl-2'-deoxyguanosine. We examined whether the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide using the 32P-postlabeling method. First, N2-acetyl-2' deoxyguanosine 3'-monophosphate and N2-acetyl-2'-deoxyguanosine 3,5'-diphosphate were chemically synthesized as standard compounds for the analysis. Then calf thymus DNA (3.24 micromol) was treated with methylglyoxal (64.8 micromol) at pH 7.4 for 3 h at 37 degrees C, and subsequently with hydrogen peroxide (64.8 micromol) at 37 degrees C for 2 h. The adduct formation was analyzed using HPLC in combination with the 32P-postlabeling method under the standard conditions. N2 Acetyl-2'-deoxyguanosine was detected at levels of 2/10(6) nucleotides in double stranded DNA and 1/10(5) nucleotides in single-stranded DNA. The estimated limit of detection by our method was 3 per 10(8) nucleotides. PMID- 8622712 TI - Post-UV survival of Escherichia coli strains carrying more than one UV sensitising plasmid. AB - The post-UV phenotypes conferred by wild-type plasmids R391 and pYD1, which increase UV-induced mutagenesis but sensitise Escherichia coli AB1157 umuC+ uvrB+ to UV, were compared, alone and in combination with that of plasmid pGW16, which sensitises AB1157 to low, but protects against high UV doses. All three plasmids increased UV resistance when present in Shigella sonnei. No plasmid significantly affected the UV sensitivity of E. coli TK501 umuC uvrB, in which pKM101, the parent of pGW16 increases UV resistance up to 1000-fold. Both pYD1 and R391 reduced the UV protective effect of pKM101, and increased UV-sensitisation conferred by pGW16. UV-sensitisation conferred by pYD1 and R391 was additive when the plasmids were together in strain AB1157, and both pKM101 and pGW16 reduced this additive sensitisation. PMID- 8622713 TI - Effect of melatonin on mitotic and proliferation indices, and sister chromatid exchange in human blood lymphocytes. AB - Cells from human peripheral blood were cultured in vitro in the presence of 0.05 to 1.00 mM melatonin, 10(-7) M mitomycin C (positive control) and 0.5% ethanol (solvent control) for 72 h at 37 +/- 1 degree C. Lymphocytes were examined for mitotic and proliferation indices, and for the incidence of sister chromatid exchange. The results indicate that the lymphocytes which were cultured in the presence of > or = 0.20 mM concentrations of melatonin exhibited a significant and concentration-dependent decrease in mitotic index and alteration in proliferation kinetics. This was demonstrated by an increase in the frequency of lymphocytes in their first division, with a concomitant decrease in the second and third or later division cells. The incidence of sister chromatid exchange was similar in the lymphocytes exposed to 0.05 to 1.00 mM melatonin and untreated controls. Exposure of the cells to ethanol, the solvent used, did not alter either the mitotic or proliferation indices, or the frequency of sister chromatid exchange. The lymphocytes treated with mitomycin C showed the expected decrease in mitotic and proliferation indices, and an increased incidence of sister chromatid exchange. These observations indicate that melatonin, when continuously present in the cultures for 72 h at the concentrations tested, while not genotoxic as indicated by the sister chromatid exchange assay, inhibits the proliferation of mitogen stimulated (and proliferating) human blood lymphocytes at supraphysiological concentrations. PMID- 8622714 TI - Effects of low-dose (2 cGy) X-ray on cell-cycle kinetics and on induced mitotic delay in human lymphocyte. AB - Experiments were carried out with human lymphocytes to test the effect of low dosage X-ray irradiation (2 cGy) on cell-cycle kinetics and on the mitotic delay induced by the conditioning pretreatment alone or by a subsequent high dose of X ray. All the tests were performed using lymphocytes from two donors who had previously displayed considerable differences in the interaction between a low and a high dose of ionizing radiation. A dose of 2 cGy led to significant variations in mitotic indices (MI) which differed for the two donors in relation to variations in the times of irradiation and fixation after stimulation with PHA. Moreover in one of the two donors 4-6 h after challenge the pretreated cultures have a higher MI that the controls; on the other hand, conditioning treatment alone induces in the other donor an extension of both G2 and of the time taken by cells in S at the time of challenge to reach mitosis. These findings could in the future provide some insight into the problem of the variability of the adaptive response in human lymphocytes. PMID- 8622716 TI - The first EEMS Frits Sobels Award to prof. H.J. Evans. PMID- 8622715 TI - Smoking and low antioxidant levels increase oxidative damage to sperm DNA. AB - Our previous studies have shown that men with low ascorbate intake have markedly increased oxo8dG in the DNA of their sperm. Because cigarette smoke is high in oxidants and depletes plasma and tissue antioxidants, oxidative DNA damage in sperm and tocopherol and ascorbate levels in seminal plasma were determined in smokers and non-smokers. The level in sperm DNA of oxo8dG, an oxidative lesion of guanine, was 50% higher in smokers compared to nonsmokers (p = 0.005). The concentration of alpha-tocopherol in seminal plasma was decreased in smokers by 32% (p = 0.03). Smoking and low antioxidant levels increase oxidative damage to sperm DNA. We discuss the possibility that paternal smoking causes mutations in sperm that lead to cancer, birth defects, and genetic diseases in offspring. PMID- 8622717 TI - Mutation and mutagenesis in inherited and acquired human disease. The first EEMS Frits Sobels Prize Lecture, Noordwijkerhout, The Netherlands, June 1995. PMID- 8622718 TI - The critical importance of stimulus intensity in intraoperative monitoring for partial dorsal rhizotomy. AB - During partial lumbosacral dorsal rhizotomy (PDR), intraoperative dorsal rootlet stimulation (drs) evokes motor responses, presumed to be reflexes, which are used to select rootlets for section. However, dr stimuli may also costimulate ventral root (vr) and evoke an M rather than a reflex response, the two being distinguishable only by comparison of response latencies after drs at two separate sites. In 15 consecutive spastic cerebral palsy patients undergoing PDR, we asked whether reflex and M responses were distinguishable on the basis of stimulus intensity (SI). For soleus H reflexes evoked by percutaneous tibial nerve stimulation, the SI for reflex afferents was usually subthreshold for exciting motor fibers. Similarly, for nerve roots, reflexes were evoked by drs at SIs generally less than that for M responses evoked by vr stimulation (vrs). In contrast, M responses evoked by drs required SIs that were on average 20 times greater. Finally, costimulation of contralateral vr after ipsilateral vrs occurred at SIs shown to evoke M responses after drs. We conclude that: (1) reflex and M responses evoked by drs are distinguishable on the basis of the required SI; and (2) drs employing SIs greater than required for vrs evokes M rather than reflex responses due to costimulation of ipsilateral and contralateral vr. PMID- 8622719 TI - Fibrosis and intercellular collagen connections from four weeks of muscle strains. AB - The effect of repeated cycles of muscle strain was studied in the soleus muscle of female rats. Muscle strains were repeated 3X/week for 1 month using two different strain protocols. Striking changes, including marked variability in fiber size, evidence of degradation and regeneration, and an expanded extracellular matrix were pronounced in the fast-stretched muscles but not in the slow-stretched muscles. However, the slow-stretched muscles did contain struts of connective tissue joining adjacent myofibers. Therefore, repeated muscle strains at high strain rates produced morphological changes similar to many myopathies, including fibrosis, whereas adaptation occurred in response to the same number of strains at slow strain rates. Such diverse tissue responses have relevance to the understanding of the mechanisms of skeletal muscle dysfunction in cumulative trauma disorders and in the design of preventive actions and treatments. PMID- 8622720 TI - Ulnar neuropathy and dystonic flexion of the fourth and fifth digits: clinical correlation in musicians. AB - Peripheral nerve lesions are sometimes associated with focal dystonia. We diagnosed ulnar neuropathy in 28 of 73 (40%) cases of occupational cramp in musicians. Focal slowing of ulnar conduction across the elbow was identified in 15 of 19 (79%) patients using the near nerve technique and in 5 of 17 (29%) patients using surface recording. Ulnar neuropathy was present in 24 of 31 (77%) cases with flexion dystonia of the fourth and fifth digits and only 4 of the remaining 42 (10%) cases with other patterns of focal dystonia. Focal dystonia improved in 13 of 14 patients whose ulnar neuropathy improved and appeared or worsened in 2 patients following ulnar nerve injury. These data, together with our recent observation of a dystonic pattern of antagonist bursting in patients with isolated ulnar neuropathy (Muscle Nerve 1995, 18:606-611), suggest that ulnar neuropathy may initiate or sustain a specific dystonia, flexion of the fourth and fifth digits, by inducing a central disorder of motor control. PMID- 8622721 TI - "Sunbath polyneuritis": subacute axonal neuropathy in perazine-treated patients after intense sun exposure. AB - This article aims at drawing attention to the peculiar association of intense exposure to sunlight and subacute development of sensory neuropathy which was seen in 7 psychiatric patients treated with the phenothiazine derivative, perazine. Three patients additionally developed bilateral VII nerve palsy. Symptoms followed a monophasic course with almost complete remission. Routine neurophysiology suggested axonal neuropathy confirmed by sural nerve biopsy in 1 patient. A toxic origin of neuropathy is supposed, possibly induced by phenothiazine photoproducts, which may cause cell damage via lipid peroxidation. PMID- 8622722 TI - Dermatomal/segmental somatosensory evoked potential evaluation of L5/S1 unilateral/unilevel radiculopathies. AB - Dermatomal and segmental somatosensory evoked potentials (SEPs) have been reported to be of diagnostic utility in unilateral/unilevel L5 and S1 radiculopathies. This investigation employs history, physical examination, imaging studies, and electrodiagnostic medicine evaluations to clearly define unilateral/unilevel L5 or S1 nerve root compromise. Inclusion criteria require all of the preceding diagnostic methods to corroborate a specific nerve root lesion. Regression equation analysis for cortical P1 latencies evaluating age and height based on comparable patient and control reference populations reveals segmental and dermatomal sensitivities for L5 radiculopathies to be 70% and 50%, respectively, at 90% confidence intervals. Similar sensitivities are obtained for 2 standard deviation mean cortical P1 latencies. Side-to-side cortical P1 latency difference data reveal segmental and dermatomal sensitivities for S1 radiculopathies to be 50% and 10%, respectively, at two standard deviations. The clinical utility of both segmental and dermatomal SEPs are questionable in patients with known unilateral/unilevel L5 and S1 nerve root compromise. PMID- 8622723 TI - Slow calcium current is not reduced in malignant hyperthermic porcine myotubes. AB - Malignant hyperthermia-susceptible (MHS) pigs express a sarcoplasmic reticulum (SR) Ca(2)+-release channel mutation that results in lower than normal contractile thresholds in skeletal muscles. In adult MHS pig muscles the L-type calcium current (ls) is also reduced. We tested the hypothesis that there is a causal relationship between ls and the lower contractile threshold by recording ls from MHS and normal porcine myotubes using the whole cell patch-clamp technique. Current voltage relationships for both MHS and normal myotubes were similar, with peak ls between +20 and +30 mV. Maximum ls amplitudes were not different from (normal: 4976 +/- 566 pA; MHS:6516 +/- 1088 pA) nor was ls specific density (normal: 9.0 +/- 0.8; MHS: 8.8 +/- 1.1 pA/pF). In both MHS and normal myotubes, both the dihydropyridine antagonist PN200-110 (200 nmol/L) and holding the membrane potential at -10mV for 5 min decreased ls significantly (by more than 50%). There was no apparent direct relationship between the mutation in the SR Ca(2)+ -release channel mutation on muscle development. PMID- 8622724 TI - Muscle fatigue, lactate, and pyruvate in mitochondrial myopathy with progressive external ophthalmoplegia. AB - We studied muscle fatigue and serum lactate and pyruvate levels in 20 patients with mitochondrial myopathy with progressive external ophthalmoplegia (PEO). Fatigue was assessed in the adductor pollicis muscle (AP) using a low-intensity exercise protocol (20 min). Forces (TFs) and relaxation times of ulnar nerve evoked twitches, compound muscle action potentials (CMAPs), and maximal voluntary contractions (MVCs) were monitored. Serum lactate and pyruvate levels were independently measured at rest and after exercise on a bicycle (15 min, 30 W). Most patients showed abnormal fatigue of the AP with a reduction of TFs and MVCs and normal CMAPs. The reduced TFs were significantly correlated with lactate levels at rest (r= - 0.60, P<0.05) and less so with those after exercise (r=- 0.47,P<0.05). Pyruvate levels revealed a similar correlation although they were widely scattered. We conclude that abnormal fatigue in PEO is metabolic, is localized beyond the muscle fiber membrane, and involves the electrome-chanical coupling and the contractile apparatus. Serum lactate levels at rest are good predictors of fatigue in PEO. PMID- 8622725 TI - Acute painful diabetic neuropathy following severe weight loss. AB - A 34-year-old man, recently diagnosed as diabetic, presented an acute painful neuropathy. He reported a profound weight loss during the months preceding onset. There were no motor symptoms, and only mild neurological signs were observed on examination. Improvement was related to a good glycemic control and weight gain. Acute painful diabetic neuropathy is a condition that may affect diabetic patients shortly after development of the disease. The pathogenetic roles played by different factors are reviewed. PMID- 8622726 TI - Motor cortex excitability during ballistic forearm and finger movements. AB - In a ballistic forearm flexion movement, a centrally programmed triphasic pattern of electromyogram (EMG) is seen with two bursts in biceps and a single burst in triceps. Rapid abduction of the index finger, in contrast, is achieved with a single agonist burst. Transcranial magnetic and electrical stimuli, triggered at the onset of the EMG burst, have been used to probe cortical and spinal cord excitability during and after self-paced ballistic finger and forearm movements. The first phase is coincident with the initial agonist burst. The second phase in biceps is associated with the second agonist burst, but in the finger movement, the raised motor cortical excitability is not associated with any EMG. It is argued that the motor program for the two movements may be similar, despite there being large differences in the EMG pattern generated. PMID- 8622727 TI - Inflammatory infiltrates in sural nerve biopsies in Guillain-Barre syndrome and chronic inflammatory demyelinating neuropathy. AB - Prompted by observations in experimental autoimmune neuritis we reanalyzed immunohistochemically the inflammatory infiltrates in sural nerve biopsies of 22 cases with Guillain-Barre syndrome (GBS) and 13 cases with chronic inflammatory demyelinating polyneuropathy (CIDP). Endoneurial infiltration of CD3+ T cells was found in 20 cases of GBS (median 5.5 cells/mm(2)) and in 10 cases of CIDP (5 cells). Epineurial T cells were present in all GBS cases (19.5 cells) and in 11 CIDP cases (21 cells). CD68+ macrophages were abundant in these neuropathies and often occurred in endoneurial perivascular clusters. In GBS subgroups the number of endoneurial T cells was significantly higher in patients with hypoesthesia and abnormal electrophysiological findings in the sural nerve. In CIDP hypoesthesia was associated with significantly higher numbers of macrophages. Our study also indicates that other factors including the time point of biopsy or previous corticosteroid treatment may influence the inflammatory cell profile. Quantifying cell infiltration may aid in establishing the diagnosis of an immunoneuropathy in patients with mild and noncharacteristic pathology. PMID- 8622728 TI - Extrafusal and intrafusal muscle effects in experimental botulinum toxin-A injection. AB - The effects of botulinum toxin-A was compared on both extrafusal and intrafusal muscle fibers in the biceps femoris of Wistar rats. Four days after injection no action potentials were elicited with stimulation single-fiber electromyography on the injected side. Fourteen days after injection, jitter became measurable and these values were increased on the injected side. Extrafusal muscle fibers began to atrophy on the 4th day and this continued to the 14th day postinjection. Atrophy was also evident and progressive in intrafusal muscle fibers. Increased terminal innervation ratios, end-plate spread of cholinesterase, and increased density of very small myelinated fibers in large intramuscular nerves were observed 14 days postinjection. Both extrafusal and intrafusal fibers are cholinergically innervated, and both were progressively affected by botulinum toxin, perhaps varying in degree only. In addition to partial denervation, Botulinum toxin effects in dystonia may also be related to modified spindle afferent discharge. PMID- 8622729 TI - Time course of muscle atrophy and recovery following a phenol-induced nerve block. AB - Clinically, phenol is used often as a neurolytic agent to treat pain and spasticity. The purpose of this study was to examine the time course of denervation and recovery in several hindlimb muscles following application of a 5% aqueous solution of phenol to the sciatic nerve. Phenol was applied to the sciatic nerve of adult female rats either by intraneural or perineural injection. Axonal degeneration was evident within the sciatic nerve 2 days following phenol application, although variable amounts of damage were observed. By 2 weeks, the soleus and tibialis anterior had atrophied to 63% and 51% of control. Reinnervation of hindlimb muscles occurred between 2 and 4 weeks following the nerve block. Following denervation, the soleus became slower in that all of the fibers expressed the slow myosin heavy chain (MHC). At 5 months, maximum tension of the soleus was 74% of control and the muscle consisted of more fast fibers on average, some of which expressed IIx MHC. These data suggest that 5% phenol causes an injury to the nerve that is more severe than a crush injury, and that reinnervation of denervated muscles may be by motoneurons other than those that originally innervated the muscles. PMID- 8622730 TI - Neuromyotonia, peripheral neuropathy and myasthenia gravis. AB - A patient with neuromyotonia, peripheral neuropathy and myasthenia gravis (MG) is described. Neurophysiological studies, at rest, showed continuous muscle discharges of motor unit action potentials (MUAPs) in duplets and triplets. Motor (MNCV) and sensory (SNCV) nerve conduction studies revealed mild axonal and demyelinating peripheral neuropathy. Plasma exchange was followed by disappearance of clinical and electrophysiological signs of neuromyotonia and MG, as well as peripheral neuropathy. PMID- 8622731 TI - A clinical trial of verapamil in amyotrophic lateral sclerosis. AB - Seventy-two patients with amyotrophic lateral sclerosis (ALS) were enrolled in a clinical trial of the efficacy of verapamil in the treatment of ALS. In period 1 (pretreatment, months 1-3) and period 3 (posttreatment, months 10-12), patients received no drug. In period 2 (months 4-9), patients received verapamil. The slopes of declining pulmonary function and limb megascores were not significantly different during drug treatment compared to natural history and washout periods. Thus, verapamil was ineffective in slowing the clinical progression in ALS patients. Controlled trials using a natural history period may represent a faster and less expensive method of screening drugs for ALS compared to placebo controlled trials. PMID- 8622732 TI - Critical care myopathy: an electrophysiological and histological study. PMID- 8622733 TI - Tissue distribution of mutant mitochondrial DNA in a patient with MERRF syndrome. PMID- 8622734 TI - Meralgia Paraesthetica: differential diagnosis and follow-up. PMID- 8622735 TI - Compressive ulnar neuropathy in the proximal forearm caused by a gouty tophus. PMID- 8622737 TI - Treatment of choice for carpal tunnel syndrome. PMID- 8622738 TI - Indications for operative versus conservative approach in CTS. PMID- 8622736 TI - IgM M-protein with antibody activity against gangliosides with disialosyl residue in sensory neuropathy binds to sensory neurons. PMID- 8622739 TI - Another case of chronic relapsing axonal neuropathy. PMID- 8622740 TI - On the heterogeneity of neurogenic facioscapulohumeral muscular atrophy. PMID- 8622741 TI - Myogenic hyperuricemia: what can we learn from metabolic myopathies? PMID- 8622742 TI - Optimal interelectrode recording distances. PMID- 8622743 TI - Aseptic technique in needle EMG: common sense and common practice. PMID- 8622745 TI - Republicans seek freeze on health research. PMID- 8622744 TI - One way out of a patent quagmire. PMID- 8622746 TI - Death of molecular biologist stuns San Diego science. PMID- 8622748 TI - Advisers deny pressure over Japan's 'first' AIDS case. PMID- 8622747 TI - NIH institute limits scope of 'AIDS research'. PMID- 8622749 TI - Once upon a smoggy day in London town... PMID- 8622750 TI - 'Indian ginseng' brings royalties for tribe. PMID- 8622751 TI - French cancer charity introduces new rules to restore probity. PMID- 8622752 TI - Impact factors can mislead. PMID- 8622753 TI - Sexual conflict as fuel for evolution. PMID- 8622754 TI - Protein targeting. The ribosome talks back. PMID- 8622755 TI - Human genetics. Blindness and the X. PMID- 8622756 TI - Red/blue chaotic power spectra. PMID- 8622757 TI - Red/blue chaotic power spectra. PMID- 8622758 TI - Red/blue chaotic power spectra. PMID- 8622760 TI - Whose teeth? PMID- 8622759 TI - Radiation doses from Ural region. PMID- 8622761 TI - Cosmological baryon density derived from the deuterium abundance at redshift z = 3.57. AB - The primordial ratio of deuterium to hydrogen nuclei (D/H), created as a result of the Big Bang, provides the most sensitive measure of the cosmological density of baryons. Measurements of the D/H ratio in the interstellar medium of our Galaxy place a strict lower limit on the primordial ratio, because processing of gas by stars reduces the abundance of deuterium relative to hydrogen. Absorption of radiation from distant quasars by intervening clouds of gas offers a means of probing D/H ratios at large redshifts, where the effects of stellar processing should be negligible. Measurements on one absorption system have indicated an extremely high primordial abundance ratio of 24 x 10(-5). Here we report a measurement of the D/H ratio in another high-redshift absorption system, and obtain a value that is an order of magnitude lower than that reported previously. The measured ratio of 2.3 x 10(-5) is consistent with that in the interstellar medium (after allowing for Galactic chemical evolution), and indicates that the absorption spectra on which the earlier estimates are based may have been subject to strong contamination. We calculate a baryon density that is 5% of the critical density required to close the Universe. PMID- 8622762 TI - A late Neanderthal associated with Upper Palaeolithic artefacts. AB - The French site of Arcy-sur-Cure is a key locality in documenting the Middle Upper Palaeolithic transition in Europe. Reliable attribution of the fragmentary hominid fossils associated with its early Upper Palaeolithic Chatelperronian industry has not been possible. Here we report the first conclusive identification of one of these fossils as Neanderthal on the basis of newly discovered derived features of the bony labyrinth. Dated at about thirty-four thousand years (34 kyr) ago, the fossil is representative of the youngest known Neanderthal populations, and its archaeological context indicates that these hominids used a rich bone industry as well as personal ornaments. The evidence supports the hypothesis of a long term coexistence with technocultural interactions between the first modern humans and the last Neanderthals in Europe. However, the complete absence of the derived Neanderthal traits in labyrinths of modern Upper Palaeolithic specimens from western Europe argues against phylogenetic continuity between the two populations in this region. PMID- 8622763 TI - Continental breakup and the ordinal diversification of birds and mammals. AB - The classical hypothesis for the diversification of birds and mammals proposes that most of the orders diverged rapidly in adaptive radiations after the Cretaceous/Tertiary (K/T) extinction event 65 million years ago. Evidence is provided by the near-absence of fossils representing modern orders before the K/T boundary. However, fossil-based estimates of divergence time are known to be conservative because of sampling biases, and some molecular/time estimates point to earlier divergences among orders. In an attempt to resolve this controversy, we have estimated times of divergence among avian and mammalian orders with a comprehensive set of genes that exhibit a constant rate of substitution. Here we report molecular estimates of divergence times that average about 50-90% earlier than those predicted by the classical hypothesis, and show that the timing of these divergences coincides with the Mesozoic fragmentation of emergent land areas. This suggests that continental breakup may have been an important mechanism in the ordinal diversification of birds and mammals. PMID- 8622764 TI - Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. AB - Each sex is part of the environment of the other sex. This may lead to perpetual coevolution between the sexes, when adaptation by one sex reduces fitness of the other. Indirect evidence comes from experiments with Drosophila melanogaster indicating that seminal fluid reduces the competitive ability of sperm from other males, thereby increasing male fitness. It also reduces a female's propensity to remate and increase her egg-laying rate. In contrast to these benefits to males, seminal fluid has substantial toxic side effects in females, with increasing quantity leading to decreasing female survival. Here I show that when female D. melanogaster are experimentally prevented from coevolving with males, males rapidly adapt to the static female phenotype. This male adaptation leads to a reduction in female survivorship, which is mediated by an increased rate of remating and increased toxicity of seminal fluid. PMID- 8622765 TI - Transcription factor AP-2 essential for cranial closure and craniofacial development. AB - During closure of the neural tube in the mouse, transcription factor AP-2 is expressed in ectoderm and in neural-crest cells migrating from the cranial neural folds. Cranial neural crest cells provide patterning information for craniofacial morphogenesis, generate most of the skull bones, and together with placodal ectoderm, form the cranial ganglia. To study the role of AP-2 during embryogenesis, we undertook a targeted mutagenesis of the AP-2 gene in the mouse. Here we report that AP-2(-/-) mice died perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs and cranial ganglia. Failure of cranial closure between 9 and 9.5 days postcoitum coincided with increased apoptosis in the midbrain, anterior hindbrain and proximal mesenchyme of the first branchial arch, but did not involve loss of expression of twist or Pax-3, two other regulatory genes known to be required for cranial closure. PMID- 8622766 TI - Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. AB - The retinoic acid-inducible transcription factor AP-2 is expressed in epithelial and neural crest cell lineages during murine development. AP-2 can regulate neural and epithelial gene transcription, and is associated with overexpression of c-erbB-2 in human breast-cancer cell lines. To ascertain the importance of AP 2 for normal development, we have derived mice containing a homozygous disruption of the AP-2 gene. These AP-2-null mice have multiple congenital defects and die at birth. In particular, the AP-2 knockout mice exhibit anencephaly, craniofacial defects and thoraco-abdominoschisis. Skeletal defects occur in the head and trunk region, where many bones are deformed or absent. Analysis of these mice earlier in embryogenesis indicates a failure of cranial neural-tube closure and defects in cranial ganglia development. We have shown that AP-2 is a fundamental regulator of mammalian craniofacial development. PMID- 8622767 TI - A cortical neuropeptide with neuronal depressant and sleep-modulating properties. AB - Acetylcholine (ACh) plays a key role in the transitions between the different phases of sleep: Slow-wave sleep requires low ACh concentrations in the brain, whereas rapid-eye-movement (REM) sleep is associated with high levels of ACh. Also, these phases of sleep are differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which has been shown to increase REM sleep without significantly affecting other phases. Here we report the cloning and initial characterization of cortistatin, a neuropeptide that exhibits strong structural similarity to somatostatin, although it is the product of a different gene. Administration of cortistatin depresses neuronal electrical activity but, unlike somatostatin, induces low-frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability. This suggests a mechanism for cortical synchronization related to sleep. PMID- 8622768 TI - Localization of dopamine D4 receptors in GABAergic neurons of the primate brain. AB - Dopamine receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five mammalian dopamine-receptor subtypes, the D4 subtype is of particular interest because of its high affinity for the atypical neuroleptic clozapine. Interest in clozapine stems from its effectiveness in reducing positive and negative symptoms in acutely psychotic and treatment resistant schizophrenic patients without eliciting extrapyramidal side effects. We have produced a subtype-specific antibody against the D4 receptor and localized it within specific cellular elements and synaptic circuits of the central nervous system. The D4-receptor antibody labelled GABAergic neurons in the cerebral cortex, hippocampus, thalamic reticular nucleus, globus pallidus and the substantia nigra (pars reticulata). Labelling was also observed in a subset of cortical pyramidal cells. Our findings suggest that clozapine's beneficial effects in schizophrenia may be achieved, in part, through D4-mediated GABA modulation, possibly implicating disinhibition of excitatory transmission in intrinsic cortical, thalamocortical and extrapyramidal pathways. PMID- 8622769 TI - Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting. AB - The signal-recognition particle (SRP) is important for the targeting of many secretory and membrane proteins to the endoplasmic reticulum (ER). Targeting is regulated by three GTPases, the 54K subunit of SRP (SRP54), and the alpha- and beta-subunits of the SRP receptor. When a signal sequence emerges from the ribosome, SRP interacts with it and targets the resulting complex to the ER membrane by binding to the SRP receptor. Subsequently, SRP releases the signal sequence into the translocation channel. Here we use a complex of a ribosome with a nascent peptide chain, the SRP and its receptor, to investigate GTP binding to SRP54, and GTP hydrolysis. Our findings indicate that a ribosomal component promotes GTP binding to the SRP54 subunit of SRP. GTP-bound SRP54 is essential for high-affinity interaction between SRP and its receptor in the ER membrane. This interaction induces the release of the signal sequence from SRP, the insertion of the nascent polypeptide chain into the translocation channel, and GTP hydrolysis. The contribution of the ribosome had previously escaped detection because only synthetic signal peptides were used in the analysis. PMID- 8622770 TI - Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. AB - Transcriptional silencing of the HM mating-type loci in the yeast Saccharomyces cerevisiae is caused by the localized formation of an altered chromatin structure, analogous to heterochromatin in higher eukaryotes. Silencing depends on cis-acting sequences, termed silencers, as well as several trans-acting factors, including histones H4 and H3, proteins RAP1 and ABF1, and the four SIR proteins (SIR1-4). Each of the four HM silencers contains an autonomously replicating sequence (ARS) to which the origin replication complex (ORC) binds. This six-protein complex is required for initiation of DNA replication, as well as for silencing. Efficient establishment of the silenced state requires both passage through the S phase of the cell cycle and SIR1 protein. Previous experiments suggested that SIR1 might be localized to the silencers by binding to ORC and/or RAP1. Here we report that SIR1 can bind directly to ORC1, the largest of the ORC subunits, and that targeting of SIR1 to ORC1 at a silencer is sufficient to establish a silenced state. PMID- 8622771 TI - [Evolution of cells]. AB - Life has existed on earth for some 4 x 10(9) years. During most of this time, evolution took place at the level of cell evolution. The cells of presently existing organisms belong to two fundamentally different cell types, protocytes (of bacteria and archaea) and eucytes (of eukarya). Thanks to molecular phylogenetics, the path of evolution can now be traced back to its very beginnings, although the picture may be blurred by repeated horizontal gene transfer. A symbiogenetic origin of plastids and mitochondria is now very well documented, and it is being discussed also for some other constituents of eucytes, including even the cells nucleus. It could be demonstrated that not only did bacterial cells become incorporated into protoeucytes and transformed into organelles of their respective hosts, but also that endocytic eucytes have apparently been transformed to complex organelles by coevolution with host cells. PMID- 8622772 TI - Parasites and carotenoid-based signal intensity: how general should the relationship be? AB - Evidence that selection by parasites maintains heritable variation in sexually selected signals (Hamilton-Zuk model) has proved equivocal. Bright individuals do not always have fewer parasites in intraspecific comparisons. Because the lymphocyte-based defence system and the production of some colors used in sexual signaling require carotenoids, we consider a trade-off between defence against parasites and sexual signals. The nature and the sign of the covariance between defence and signal brightness can vary. Depending on carotenoid availability and allocation, and the type of sexual signal, various relationships between parasite load and signal intensity are expected. PMID- 8622773 TI - AD progression as a function of disease stage. PMID- 8622774 TI - Methodological aspects of evaluating effects of AD progression. PMID- 8622775 TI - Influence of the patient's environment on the progression of Alzheimer's disease. PMID- 8622776 TI - Possible pharmacological approaches to slowing down Alzheimer's disease progression. PMID- 8622777 TI - Clinically meaningful interference in AD progression. PMID- 8622778 TI - SCG10, a neuron-specific growth-associated protein in Alzheimer's disease. AB - Neuronal growth-associated proteins (nGAPs) are markers of neuronal process outgrowth and are associated with both degenerative and sprouting responses in Alzheimer's disease (AD) brain. To study possible involvement of SCG10, an nGAP, in AD, we cloned human SCG10 cDNA and analyzed SCG-10 at mRNA and protein levels in control and AD brains. The deduced amino acid sequence of human SCG10 was 69% identical to stathmin, another nGAP. By in situ hybridization, both SCG10 and stathmin mRNAs were detected in selected neuronal populations in aged human brains. Quantitative analysis by RNase protection revealed that levels of neither SCG10 nor stathmin mRNAs were significantly altered in AD. Using an SCG10 specific antibody, Western blot analysis did not reveal any quantitative changes of SCG10 in AD. However, when the concentration of SCG10 protein was plotted against the number of tangles, a positive correlation was found. SCG10 levels did not correlate with plaque numbers. Furthermore, immunohistochemical study revealed that neuronal SCG10 protein accumulated in the cell bodies in AD affected regions. Thus, SCG10 compartmentalization and metabolism may be altered in AD possibly due to mechanisms related to tangle formation in this disease. PMID- 8622779 TI - Changes in platelet phospholipase C protein level and activity in Alzheimer's disease. AB - We have previously demonstrated that PLC-delta was abnormally accumulated in autopsied brains with Alzheimer's disease (AD). As nonneuronal tissue involvement in AD is also suggested and PLC activity is reduced in AD platelets, we examined the changes of the protein level of PLC-delta and its enzyme activity in platelets taken from patients with AD and age-matched controls. PLC-delta in human platelets was detected as a 72 kDa protein using a specific antibody against PLC-delta. Western blots revealed that the protein level of PLC-delta was significantly higher in the cytosolic fraction prepared from AD platelets compared to controls. We investigated the activity of PLC-delta which hydrolyzes phosphatidylinositol and found that the PLC-delta activity in the cytosolic fraction from AD platelets was significantly reduced compared to the control. This finding that the enzyme activity per PLC-delta molecule is reduced in AD platelets is consistent with the study using Alzheimer brains. These results suggest that aberrant phosphoinositide metabolism is present in nonneuronal tissues as well as the brains of patients with AD. PMID- 8622780 TI - Increased proteolytic activity in lymphocytes from patients with early onset Alzheimer's disease. AB - The levels of calpains (m-calpain and mu-calpain) in peripheral blood lymphocytes from patients with Alzheimer's disease were determined via Western blotting. The Ca-dependent proteolytic activity and the calpastatin activity were estimated using incubation with exogenous substrate. Evidence was obtained for an increased Ca-dependent proteolytic activity in lymphocytes from patients with early onset Alzheimer's disease. There was also an increased level of membrane-bound mu calpain in this group of patients. The observed changes may be caused by a general dysregulation of Ca homeostasis in peripheral cells of early onset Alzheimer victims. PMID- 8622781 TI - Delay in manifestations of aging by grafting NGF cultured chromaffin cells in adulthood. AB - Dopamine agonists or grafts compensate impaired motor functions in aged rats. However, there is no evidence showing whether grafting in adulthood retard aging manifestations. Motor performance of 13-month-old rats was tested on 2 meter-long wooden beams which had a 15 degree inclination and whose thickness varied from 3, 6, 12, 18, to 24 mm. Rats at 14 months were randomly assigned to 3 groups: sham graft (Group 1); intrastriatal graft of chromaffin cells cultured with NGF (Group 2); intrastriatal graft of chromaffin cells (Group 3). Motor performance was tested at monthly intervals up until rats were 26 months old. Two more groups were included: 26-month-old naive rats (Group 4); and 3- to 5-month-old naive rats (Group 5) both evaluated only once. At 26 months, the basal activity of ventral mesencephalic dopaminergic neurons was recorded. Results showed in Group 2 delay of motor detriments seen in aged rats, maintenance of basal firing rates of nigral cells compared to those of younger rats, and greater survival of substantia nigra cells. It is suggested that NGF cultured chromaffin cells produce a delay of motor detriments in aged rats, as a result of inducing survival and firing rates of nigral cells comparable to those seen in young rats. PMID- 8622782 TI - GM1 and NGF synergism on choline acetyltransferase and choline uptake in aged brain. AB - In the brain of aged rats high affinity choline uptake (HAChU) of the striatum, hippocampus, and frontal cortex is lower than in young rats, while choline acetyltransferase (ChAT) activity is lower in striatum and frontal cortex. Infusion into the lateral cerebral ventricle with nerve growth factor (NGF) enhances the low values of these cholinergic markers in a dose- and region dependent manner. GM1 ganglioside infused into the lateral ventricle, at a dose that is ineffective alone, together with NGF synergistically enhances the effect of NGF on ChAT and HAChU activities in the brain of aged animals. The pharmacology of this GM1/NGF synergism suggests potentiation of response. PMID- 8622783 TI - GDNF mRNA expression in normal postnatal development, aging, and in Weaver mutant mice. AB - Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to enhance the survival and process outgrowth of mesencephalic dopamine neurons. A nuclease protection assay was utilized to determine whether GDNF mRNA is expressed in the ventral mesencephalon and/or striatum during normal mouse postnatal development. While no GDNF mRNA was detected in the ventral mesencephalon, expression was detected in the striatum throughout postnatal development and maturity with the peak of expression being in the second postnatal week. In the process of normal aging, no change in the levels of GDNF mRNA was observed in the striatum, while a 10-fold increase in glial fibrillary acid protein (GFAP) mRNA was detected in 24-month-old relative to either 4.5- or 11-month-old mice. Further analysis addressed whether there are changes in GDNF gene expression associated with the neurodegeneration of dopamine neurons that occurs in the weaver mutant mouse. A transient 65% increase in the expression of GDNF mRNA was observed in weaver mutant striatum on postnatal day 22. The results of this study suggest that GDNF could provide target derived of dopaminergic neurotrophic support and stimulate fiber outgrowth during development and that decreased levels of GDNF expression are not responsible for either aging associated decreases in dopaminergic neuronal plasticity or neurodegeneration in the weaver mutant mouse. PMID- 8622784 TI - Increased susceptibility to MPTP toxicity in middle-aged rhesus monkeys. AB - In the present study, age-associated effects of the neurotoxin 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) administered via the right carotid artery were evaluated pre- and post-MPTP treatment in 26 female rhesus monkeys ranging in age from young adulthood to middle age (5 to 23 years old). A significant inverse correlation was seen between age and MPTP dose needed to produce stable, moderate parkinsonian features. Rhesus in the 5- to 9-year-old group required approximately three times the amount of MPTP as 20- to 23-year-old animals. Even though they received less MPTP, the older animals consistently displayed more severe bradykinesia, upper limb rigidity, and balance and gait abnormalities. Prior to MPTP treatment, home cage activity levels were strongly age dependent, with animals in the 10- to 19-year and 20- to 23-year groups displaying significantly less daytime activity than 5- to 9-year-old rhesus. Home cage activity levels tended to decrease in all age groups following MPTP treatment, but significant decreases were only measured in daytime activity in the 10- to 19 and 20- to 23-year age groups. PMID- 8622785 TI - Age-related changes in potassium-evoked overflow of dopamine in the striatum of the rhesus monkey. AB - Rapid (5 Hz) chronoamperometric recordings using Nafion-coated carbon fiber electrodes (30-90 microns o.d.) combined with pressure-ejection of potassium from micropipettes were used to investigate potassium-evoked overflow of dopamine (DA) in the striatum of young (5 to 10 years old) and middle-aged (19 to 23 years old) anesthetized rhesus monkeys. The potassium-evoked DA-like signals from the 19- to 23-year-old animals were significantly lower in amplitude than those recorded in the young animals. In addition, the temporal dynamics of DA signals in the caudate nucleus of middle-aged animals were faster, while the time courses of the signals recorded in the putamen of middle-aged monkeys were significantly longer as compared to the signals recorded from young animals. Moreover, home cage activity levels of the middle-aged animals were significantly lower. Taken together, these data support age-related changes in the output of DA from DA fibers in the striatum of middle-aged monkeys. PMID- 8622786 TI - Executive system dysfunction in the aged monkey: spatial and object reversal learning. AB - As part of the effort to characterize age-related cognitive changes in executive system function in a nonhuman primate model of human aging, the performance of seven rhesus monkeys, 20 to 28 years of age, was compared to that of five young adult monkeys, 6 to 11 years of age, on spatial and object reversal tasks. No differences in performance were found between the two groups in the initial learning of either task. On spatial reversals, aged monkeys were impaired relative to young adults, but there was no difference in overall performance between the groups on object reversals. Central to this article, a perseverative tendency was noted in the aged group on both spatial and object reversal tasks. Changes in executive system dysfunction may represent an important aspect of age related cognitive decline. PMID- 8622788 TI - Influence of aging on the seasonal rhythm of the vasopressin-expressing neurons in the human suprachiasmatic nucleus. AB - The mammalian suprachiasmatic nucleus (SCN) is considered to be a major component of the biological clock implicated in the temporal organization of a variety of physiological, endocrine, and behavioral processes. There is now a great deal of evidence indicating that many of these rhythms are progressively disturbed during senescence. The present study was aimed at investigating the influence of aging on the seasonal rhythm of the vasopressin (VP)-expressing neurons in the human SCN. To that end, brains obtained at autopsy of 48 human subjects, ranging in age from 6 to 91 years, were studied. Subjects were divided into two age groups, viz. "young subjects" (up to 50 years) and "elderly subjects" (over 50 years). It is shown that the number of VP-immunoreactive neurons in the human SCN exhibits a marked annual oscillation in young but not in elderly people. Whereas in young subjects low VP-immunoreactive neuron numbers were found during the summer (May July) and peak values in autumn (September-November), the SCN of elderly people showed a disrupted annual cycle with a reduced amplitude. These data suggest that the biosynthesis of vasopressin in the human SCN exhibits a seasonal rhythm that becomes disturbed later in life. PMID- 8622787 TI - Hippocampal muscarinic receptor function in spatial learning-impaired aged rats. AB - Efficiency of coupling of hippocampal muscarinic receptors to phosphoinositide (PI) turnover was investigated in behaviorally characterized young and aged Long Evans rats using hippocampal minces and the method of partial receptor alkylation of Furchgott. Densities of the m1, m2, and m3 receptor proteins were determined using specific antibodies and immunoprecipitation. Spatial learning ability was quantified using a water maze. There were no differences in the levels of muscarinic receptor proteins between young and aged (27 months) rats or in rats with impaired spatial learning. The dissociation constant (KD) for the agonist oxotremorine-M and the KD/EC50 ratio, an indicator of receptor-effector coupling efficiency were similar in young and aged rats. However, the maximal PI turnover response to oxotremorine-M was decreased in impaired aged rats and this parameter was highly correlated with the spatial learning index (R = -0.825; p < 0.001). A reduction in effector stimulation in the absence of changes in receptor protein or coupling efficiency suggests that dysfunction in the hippocampal muscarinic receptor systems occurs at the level of phospholipase C or beyond. PMID- 8622789 TI - Changes in brain membrane phospholipid and high-energy phosphate metabolism precede dementia. AB - A 52-year-old Caucasian male was followed with Mattis and 31P MRS examinations every 6 months for 33 months. At entry into the study, the subject had a normal clinical examination and normal Mattis scores but had alterations in MRS measures of membrane phospholipid and high-energy phosphate metabolism indistinguishable from those previously reported in mildly demented AD patients. After 33 months of follow-up, the subject had clinical and Mattis findings suggestive of possible incipient dementia and after 46 months of follow-up there was sufficient cognitive decline to make the diagnosis of dementia with a frontal lobe preponderance. The findings in this subject support the contention that alterations in brain membrane phospholipid and high-energy metabolism can be noninvasively detected by 31P MRS years before any clinical manifestations of the disease. PMID- 8622790 TI - Estrogen induction of glial heat shock proteins: implications for hypothalamic aging. AB - In the aging mammalian hypothalamus, a unique subpopulation of glial cells accumulates peroxidase-positive cytoplasmic inclusions distinct from lipofuscin. In adult rodents, this senescence-dependent glial granulation is accelerated by administration of estradiol valerate. In the present study, brain sections derived from male rats given 3 monthly intramuscular injections of estradiol valerate (0.2 mg or 2.0 mg) were immunostained for heat shock proteins and glial fibrillary acidic protein to determine whether a glial stress response is implicated in estrogen-induced granulation. Our findings indicate that estrogen elicits a heat shock response and subsequent granulation in astrocytes residing in estradiol receptor-rich brain regions including the arcuate nucleus and the wall surrounding the third ventricle but not in estradiol receptor-deficient regions such as the striatum and corpus callosum. The heat shock proteins induced by estrogen, namely, the 27, 72, and 90 kDa stress proteins, are upregulated in astrocytes in response to oxidative challenge supporting our hypothesis that estrogen mediates senescent changes in the rodent hypothalamus through oxidative mechanisms. PMID- 8622791 TI - Changes in brain amino acid content induced by hyposmolar stress and energy deprivation. AB - The changes in endogenous amino acids in brain extracellular and intracellular compartments evoked by hyposmotic stress and energy deprivation were compared. Tissue content and release of ten amino acids were measured simultaneously in rat hippocampal slices by means of high performance liquid chromatography. Hyposmotic stress induced a large release of taurine (25568 pmol mg-1 protein), and a smaller release of glutamate, accompanied by an inverse change in tissue content. Adding mannitol to correct osmolarity, blocked these changes. Energy deprivation caused an increase in the release of all amino acids except glutamine. The release was particularly large for glutamate and GABA (31141 and 13282 pmol mg-1, respectively). The intracellular concentrations were generally reduced, but the total amount of the released amino acids increased In contrast to the effect seen during hyposmolar stress, mannitol enhanced the changes due to energy deprivation. The results show that hyposmolar stress and energy deprivation cause different content and release profiles, suggesting that the mechanisms involved in the two situations are either different or modulated in different ways. The intracellular amino acid depletion seen during energy deprivation shows that increased outward transport is probably a primary event, and increased amino acid formation likely secondary to this release. PMID- 8622792 TI - Lesion of dentatothalamic pathways in severe post-traumatic tremor. AB - The present study investigates evidence of dentatothalamic pathway lesions in nineteen patients with severe kinetic post-traumatic tremor respectively by magnetic resonance imaging (MRI). Kinetic tremor is thought to be characteristic of lesions of the cerebellar outflow. While this hypothesis is supported by experimental data, neuropathological and neuroradiological findings have been limited. The appendicular tremors were unilateral in 13 patients and bilateral in 6, accounting for 25 instances of tremor. The tremor developed after severe head trauma in 18 patients. These patients had evidence of diffuse axonal injury on MRI. Postural and kinetic tremor was present in all patients, and was accompanied by tremor also present at rest in 14 instances. Multiplanar MRI studies were performed on a high-field MRI system operating at 2.0 T in 13 patients and on intermediate-field strength MRI systems in 6 patients according to a standardized protocol. To detect small deposits of hemosiderin after post-traumatic lesions, the protocol included a heavily T2-weighted spin-echo pulse sequence. Lesions of the dentatothalamic pathways were found in 22 instances. The lesions were classified into different types of according to their distribution. A lesion of the dentate nucleus ipsilateral to the tremor (type 1) was found in one instance (4%), lesions involving the ipsilateral predecussational dentatothalamic pathway (type II and III) were found in 14 instances (56%), and lesions involving the contralateral post-decussational course (type IV) in 7 instances (28%). One patient with a mild head trauma had a lesion of the contralateral thalamus. The lesions appeared as hypointense, hyperintense or mixed. Two of three patients with a parkinsonian-like rest tremor had type IV lesions involving the substantia nigra. The nosological concepts of tremors are discussed. 'Midbrain' tremor may have distinct pathoanatomical lesion sites. PMID- 8622793 TI - Brain tissue acid-base response to hypercapnia in neurosurgical patients. AB - These studies were conducted in neurosurgical patients to determine brain tissue nonbicarbonate buffering of pH changes during hypercapnia. Following a craniotomy, a sensor which continuously measures oxygen pressure, carbon dioxide pressure, pH and temperature was inserted into cortex tissue of nine subjects. Bicarbonate concentration was calculated from the Henderson-Hasselbach equation. Following baseline measures, PaCO2 was increased 10mmHg for 10 min. Tissue pCO2 increased 9 mmHg (p < 0.05) without a change in tissue pO2. In six patients, tissue bicarbonate concentration increased from 18 to 20 meq L-1 (p < 0.05), indicating a 40-50% attenuation of the increase in hydrogen ion (H+) by nonbicarbonate buffering mechanisms. Three patients showed no increase in tissue bicarbonate during hypercapnia; 2 had baseline tissue pH less than 6.5 and one displayed signs of tissue hypoxia during the CO2 challenge. In all patients, increases in tissue H+ during hypercapnia were related to baseline tissue bicarbonate concentration. Marked increases in H+ were seen when baseline bicarbonate decreased below 10 meq L-1. These results suggest that when tissue bicarbonate is depleted, the risk of H+ induced injury during hypercapnia is increased. PMID- 8622794 TI - Measurement of treatment response to sertraline in depressed multiple sclerosis patients using the Carroll scale. AB - We studied 11 patients with stable multiple sclerosis (MS) with major depression in terms of response to Sertraline at 100 mg q.d. in an open label trial. Patients were evaluated with self assessment measurements (Carroll scale) prior to and during treatment. Only one patient discontinued the drug during the three month treatment trial, and this was due to perceived lack of efficacy by the patient. The remainder of the patients completed at least three months of treatment and had significant improvement in depressive symptoms by self assessment measurements. No patients experienced side effects. Sertraline appears to be well tolerated and effective in treatment of major depression in MS. The Carroll scale is an easily administered means of assessing treatment response, and correlated highly with our clinical impressions. PMID- 8622795 TI - Influence of a 'brain protector' drug 21-amino steroid on the effects of experimental embolic stroke treated by thrombolysis. AB - This study was designed to evaluate the effects of tissue-type plasminogen activator (tPA) and 21- amino steroid (U74006F) in experimental embolic stroke in rabbits. The size of infarction from embolism was compared to controls with tPA alone, 21-amino steroid alone, and in combination. The middle cerebral artery of the rabbit was embolized by injecting an arterial ('white') thrombus in the right internal carotid artery. The rabbit treatment was 2 mg kg-1 of tissue-type plasminogen activator and/or 3 mg kg-1 of 21 amino steroid started at 2 h post embolization. The animals were terminated 4 h post-treatment and brains were examined for evidence of ischemia and/or hemorrhage. Administration of tissue type plasminogen activator and/or 21-amino steroid in the raw data show that there is a tendency for all treatments to reduce the ischemic volume when compared to the control group, also it is evident the standard deviation of these estimates is rather large when compared to the differences between treatments. The results of the analysis of variance shows that the differences expressed are not statistically significant. (No statistical differences were found between the treatment groups and the control group.) The results show that administration of tissue-type plasminogen activator and/or 21 amino steroid at 2 h post embolization alone or in simultaneous administration does not significantly reduce the volume of infarction. Further studies need to be addressed in regards to the region of viable brain in the peri-infarct area, in reducing the time to treatment. PMID- 8622796 TI - Hemodynamic assessment of the development and rupture of intracranial aneurysms using computational simulations. AB - Intracranial aneurysms manifest themselves as sacculations within a weakened region of the vessel wall and pose substantial neurological risks upon rupture. A primary factor in the development and rupture stages of an aneurysm is hemodynamics and its degrading effects on the aneurysm wall. Wall dynamics and hemodynamics within a fully developed aneurysm were investigated using computational simulation techniques. To study wall dynamics, the aneurysm was modeled as a thing spherical shell with linearly elastic and plastic (viscoelastic) wall behavior. The sensitivity of this model to the biophysical parameters which describe it will assist in the quantitative assessment of factors predisposing to aneurysm rupture and subarachnoid hemorrhage. Flow dynamics simulations were performed for spherical aneurysms with rigid walls. We observed the development and motion of an annular vortex within the lateral sacculation. We also simulated a ruptured aneurysm by placing a tear near the neck of the aneurysm. Flow patterns showed blood flowing out during the initial stages of the flow, but displayed an inflow of blood soon thereafter, as the internal pressure dropped. These results are substantiated by the clinical observations that turbulent flow is observed within the aneurysm as evidenced by reduced bruits. PMID- 8622797 TI - A strategy for Compton scatter correction in brain SPECT by orthogonal image expansions assisted by neural networks. AB - The simultaneous dual energy acquisition is often required to accurate co registration of brain images of both energy windows. However, the reconstructed images in the lower energy window are usually distorted by a significant Compton scatter component originating from the higher energy photons. We are proposing two methods for calculating the Compton scatter correction. Both of them utilize sets of TC99m and associated Compton scatter images in TI(201) energy window that represent a patient population and calculate the sought Compton image estimate 'by comparison' of the given new Tc99m image with the Tc99m set. The first method is based on the principal component image expansion and the second one utilizes in addition learning neural networks. Means to measure the accuracy of results are proposed as well. The proposed methods can be modified for application to other anatomical domains. PMID- 8622798 TI - Slit ventricle syndrome successfully treated by a lumboperitoneal shunt. AB - The authors present the case of a 17-year old man with slit ventricle syndrome, presenting as progressive neurological deterioration after head trauma. Serial computed tomography scans revealed slight ventricular enlargement, suggesting shunt malfunction. Communication between the lumbar subarachnoid space and the lateral ventricles was confirmed by computed tomography cisternography. He underwent a lumboperitoneal shunt, resulting in complete resolution of the symptoms. A lumboperitoneal shunt is considered to be a promising option for the treatment of slit ventricle syndrome. PMID- 8622799 TI - Brain temperature before and after brain death. AB - Although there has been renewed interest in human brain temperature, very little information is available on the association between brain temperature and cerebral perfusion pressure. In this study, we measured brain, tympanic, and rectal temperatures, arterial blood pressure and intracranial pressure in a case of massive hemorrhage deteriorating to brain death, and showed for the first time that when cerebral perfusion pressure began to decrease markedly brain temperature fell rapidly. Rectal and tympanic temperatures were higher than brain temperatures during the period of very low cerebral perfusion pressure. Circadian change in temperature (high at day, low at night) was preserved during the period of brain death. PMID- 8622800 TI - Neuroprotective effect of basic fibroblast growth factor on wobbler mouse motor neuron disease. AB - Basic fibroblast growth factor (bFGF) possesses neuroprotective effects on a variety of neurons. Here we report that it delays progression of motor neuron disease (MND) in the wobbler mouse. After initial diagnosis of MND at post-natal age 3-4 weeks, wobbler mice receive either recombinant human bFGF (1 mg kg-1, n = 10) or vehicle (n = 10), daily for weeks by subcutaneous injection in a blind fashion. We performed symptomatic and neuropathological assessments in both groups. The treatment was fulfilled at 7-8 weeks of age. In comparison with vehicle, bFGF treatment potentiated grip strength (p < 0.008), attenuated forelimb contracture (p < 0.003), and increased weight of the biceps muscle (p < 0.008). bFGF-treated mice retarded denervation muscle atrophy (p < 0.001) and degeneration of spinal motoneurons (p < 0.001). Our study shows that bFGF treatment is beneficial in a murine MND model. We provide a rationale that bFGF may have therapeutic potential in peripheral motor neuropathy or MND. PMID- 8622801 TI - Diffusion-weighted magnetic resonance imaging during brief focal cerebral ischemia and early reperfusion: evolution of delayed infarction in rats. AB - The purpose of this study was to ascertain if the signal intensity ratio and the lesion area determined by diffusion-weighted magnetic resonance imaging during brief focal ischemia and early reperfusion predict outcome determined by diffusion-weighted magnetic resonance imaging and T2-magnetic resonance imaging at 24 h. Seventeen rats were imaged before and during 30 min of endovascular middle cerebral artery occlusion and at 15 min, and 23.5 h after the onset of reperfusion. Both hemisphere and basal ganglia signal intensity ratio increased significantly from baseline during ischemia, decreased significantly from ischemic levels during early reperfusion, and increased again at 24 h. However, signal intensity ratio during ischemia or after 45 min of reperfusion did not correlate statistically with diffusion-weighted-signal intensity ratio at 24 h. Both hemisphere signal intensity ratio and basal ganglia signal intensity ratio at 15 min of reperfusion correlated, but only moderately, with diffusion-weighted signal intensity ratio at 24 h (r = 0.52, p < or = 0.05). Although lesion areas during ischemia were comparable to those observed at 24 h, lesion areas at both 15 and 45 min of reperfusion were significantly smaller than those observed during ischemia and at 24 hr. Thus, sequential imagining demonstrated partial resolution and delayed recurrence of magnetic resonance-defined ischemic lesions during reperfusion after brief focal ischemia. PMID- 8622802 TI - Selective vulnerability of hippocampal CA3 neurons to hypoxia after mild concussion in the rat. AB - Immunohistochemical staining for microtubule-associated protein 2 (MAP2) and synaptophysin was used to investigate the effect of hypoxia on hippocampal neurons after mild concussion in the rat. Male Sprague-Dawley rats were divided into four groups: Group 1 (n = 3) was subjected to a mild impact-acceleration closed head injury, group 2 (n = 3) was subjected to 30 min of moderate hypoxia, group 3 (n = 5) was subjected to head trauma followed by 30 min of moderate hypoxia, and group 4 (n = 3) comprised sham-operated controls. All rats were fixed by transcardial perfusion 24 h after insult. No damage was observed in CA1 or CA2 neurons in any of the rats. However, rats in group 3 showed selective damage of hippocampal CA3 neurons manifested by a pycnosis and a marked decrease in MAP2 immunoreactivity. Presynaptic terminals visualized by synaptophysin immunostaining showed no differences among groups. The loss of immunoreactivity for the post-synaptic somal and dendritic protein marker MAP2 from the CA3 subfield 24 h after combined insults indicates an increased vulnerability of pyramidal cells in this brain area. PMID- 8622803 TI - Hypothermic prevention of nuclear DNA fragmentation in gerbil hippocampus following transient forebrain ischemia. AB - The protective effect of hypothermia on DNA fragmentation following transient forebrain ischemia in mongolian gerbils was investigated. The DNA fragmentation demonstrated in situ in gerbil hippocampal CA1 was compared between intra- and post-ischemic hypothermia. Intra-ischemic hypothermia prevented the DNA fragmentation in hippocampal CA1 completely, while severe DNA damage was observed in post-ischemic hypothermia group. the degree of DNA fragmentation of hippocampal CA1 in the post-ischemic hypothermia group was equal to that in the ischemic control group. The results suggest that hypothermia during a transient forebrain ischemia exerts a protective effect on the post-ischemic hippocampal damage by preventing the DNA fragmentation in CA1 neurons. PMID- 8622804 TI - Early hemodynamic changes at the microcirculatory level and effects of mannitol following focal cryogenic injury. AB - Changes in cerebral blood flow due to infusion of hyperosmolar solutions are of considerable importance in states of raised intracranial pressure. The present study was aimed to evaluate the effects of mannitol on the cerebral microcirculation, in a model of vasogenic brain edema. A right fronto-parietal craniotomy was performed in 30 adult Sprague-Dawley rats. Vasogenic edema was produced by placing dry-ice over the dura for 1 min. The cortical blood flow was monitored for 120 min using a laser-Doppler flowmeter (Perimed, Stockholm, Sweden), and graphics were recorded using a personal computer. Animals were randomly divided into three groups: group 1 (control group) received no mannitol; group 2 was treated with a bolus injection of 20% mannitol (1 mg kg-1); group 3 received the same dose over a 30 min infusion. Mean blood pressure, temperature, and respiratory rate were continuously monitored. At the end of the procedure, an intravenous injection of Evan's blue 2% was given. Results were compared by using repeated measures of analysis of variance and a two-sample t-test at each time. After the production of a cryogenic injury, we found a marked decrease in the cerebral blood flow, whereas mannitol partially reversed that effect. There was not significant difference between groups 2 and 3; however, there was a significant difference between mannitol and control groups after 15 min. During the early phase of vasogenic edema, early use of mannitol did not increase the blood flow, but stabilized it, preventing further decrease. Laser-Doppler flowmetry is a valuable method for continuous estimation of hemodynamic changes in the cerebral microcirculation. PMID- 8622805 TI - Origin of muscle action potentials evoked by transcranial magnetic stimulation in cats. AB - We studied the effects of transcranial magnetic stimulation on ipsilateral and contralateral forelimb extensor muscles in anesthetized cats. A magnetic stimulator, operating at 100% intensity, was used through a circular coil, which was placed tangentially over the midline scalp. Bilateral activation of extensor muscles was readily obtained in all animals. The onset latencies were 7.3 +/- 1.1 and 7.07 +/- 0.8 msec for the contralateral and ipsilateral muscles, respectively. The amplitude of muscle response was unstable in magnitude, nevertheless, it did not show any significant difference between the two sides. The latency of response for ipsilateral and contralateral muscles was similar, which suggests simultaneous activation of motor pathways servicing forelimb muscles. Lesioning or ablation of the motor cortex and decerebration at mid colliculi level did not abolish the evoked responses elicited at high intensity magnetic stimulation. Stereotactic electrical stimulation of the vestibular nuclei complex was performed, and satisfactory ipsilateral motor responses were obtained. Subsequently, a stereotactic radiofrequency lesion was made at the vestibular nuclei complex, with morphological confirmation. After this lesion, the motor evoked potentials (MEPs) were significantly diminished in amplitude. This finding strongly suggests that the generator of the MEPs resides in the brainstem, mainly at the vestibular nuclei complex. PMID- 8622806 TI - [Diagnostic value of gamma glutamyl transpeptidase and the mean corpuscular volume in chronic hepatitis of alcoholic etiology]. AB - In order to assess the diagnostic value of serum gamma-glutamyl-transpeptidase (GGT) and mean corpuscular volume (MCV) as markers of alcoholism in chronic liver diseases, 107 patients with non-alcoholic chronic liver disease and 192 patients with alcoholic liver diseases have been compared. GGT and MCV values were checked two times: the day of admission to hospital and 10 days after complete withdrawal from alcohol. The patients with alcoholic liver diseases present significantly higher values of GGT and MCV in respect with patients with non-alcoholic liver diseases. A significant (p < 0.05) decrease of about 50% in serum GGT levels and of about 3% in MCV was observed after alcohol withdrawal only in the group of alcoholic liver diseases whereas no changes were found in the other group of patients: For the diagnosis of alcoholism in chronic liver diseases, while the sensitivity and the specificity of the several markers vary from 50% to 86%, the positive predictive values of GGT and MCV at admission were 92.2% and 73.4%, and the negative predictive values were 40.2% and 75.7%, respectively. Moreover, the positive predictive values of GGT and MCV after 10 days of alcohol withdrawal were 95.3% and 85.9% and the negative predictive values were 31.8% and 46.7%, respectively. The contemporary decrease in GGT and MCV values does not seem to offer better informations than GGT decrease. These data suggest that, even if GGT and MCV do appear per se as weak indicators of alcoholism during chronic liver diseases, the early decrease in their values, especially in serum GGT, are good and specific markers of alcohol abuse and, consequently, of the alcoholic etiology of liver disease. PMID- 8622807 TI - [Cortical function in 107 patients with supratentorial expanding mass. Pre- and postoperative study]. AB - A clinical study on functions of the cerebral cortex is reported. In this regard, 107 patients with a supratentorial expanding mass have been investigated before and after surgical removal of the lesion. The results obtained are discussed in the light of what is available in the literature. PMID- 8622808 TI - [Continuous spinal analgesia in home care of oncologic pain]. AB - The authors in this study, after a short survey of the most important therapeutic techniques for cancer pain, report their results in the treatment of 18 patients suffering from incurable disease. It was impossible to dismiss them from hospital care on account of a painful symptomatology not controllable by oral morphine or owing to excessive collateral morphinic consequences. The analgetic technique employed was continuous intrathecal infusion of morphine, clonidine, droperidol and, in 10 cases, bupivacaine. Drug delivery systems, totally internalized, except infusion pump, were always utilized. Adequate pain relief was obtained, within - 5 days, in all the patients. Family membres, in the same period, learnt the infusion circuit action. At this point the patients were dismissed and treated with home care. The average time of assistance was 140 days, and very moderate variations in posology were necessary. Hospital reentrance, really little numerous, happened only when no member of palliative care service was present. Reasons were no bodily pain, but the total suffering of cancer disease. No complication nor collateral consequences were never found. PMID- 8622809 TI - [Zinc and diabetes mellitus]. AB - In patients with type 1 and 2 diabetes was frequently found: low blood zinc levels, high zincuria, severe and ubiquitous cellular depletion of zinc, increased basal and after loading blood mineral clearance, and hyperglycaemia due to the reduction of pancreatic insulin secretion and to the reduced biological action of the hormone on liver, as a consequence of chronic zinc deficit. Strong endocellular zinc depletion in diabetics; low insulin secretion; insulin biological action decrease for zinc deficit; IG-I concentration decrease, that happens in this condition; insulin and IGF-I resistance; insulin and IGF-I receptors depletion in diabetics: are strong arguments for zinc pharmacological supplementation, in gastric protective formulation, to avoid gastroenteric problems. PMID- 8622810 TI - [Evaluation of the biocompatibility of medical devices based on European standards]. AB - Several issues concerning the biocompatibility) testing of medical devices as stated in European directive 90/385 and 93/42 are discussed. The authors describe the fundamental characteristics that biomaterials should have to be employed in the human body, the assays useful for testing different aspects of biocompatibility and the harmonized regulations already available or under development. The testing protocol applied by the a. in their Lab covering many issues of biocompatibility is presented: a body of experience was gained over the years and it has been revised under the new regulatory items. In conclusion, the biocompatibility of devices must be assessed following the European harmonized standards, the available. Where proper standards are still lacking, feasible testing methods, even if not officially adopted yet, have to be used. PMID- 8622812 TI - [Interhemispheric epidermoid cyst. A clinical case]. AB - The authors report a rare case of epidermoid cyst with interhemispheric growth closely connected to the anterior portion of the corpus callosum. Although extremely sensitive in determining the site, dimensions and relations of the lesion, NMR does not allow the nature of this pathology to be diagnosed owing to the lack of signal specificity. CT, using the measurement of Hounsfield units, allows a differential diagnosis of epidermoid cysts and arachnoid cysts and lipomas to be made in almost all cases. Treatment is surgical and the complete excision of the capsule avoids recidivation. PMID- 8622811 TI - [Malignant peritoneal mesothelioma: its relation to asbestos]. AB - Chronic exposure to asbestos can induce malignant peritoneal mesothelioma (PMM) without pulmonary or pleural involvement (PIMM). The localization to the peritoneum depends on the different susceptibility of the two mesotheliums and, perhaps, on the length of asbestos fibers which can facilitate their direct translocation. PMID- 8622813 TI - Marine natural products. PMID- 8622814 TI - Food reward: brain substrates of wanting and liking. AB - What are the neural substrates of food reward? Are reward and pleasure identical? Can taste pleasure be assessed in animals? Is reward necessarily conscious? These questions have re-emerged in recent years, and there is now sufficient evidence to prompt re-examination of many preconceptions concerning reward and its relation to brain systems. This paper reviews evidence from many sources regarding both the psychological structure of food reward and the neural systems that mediate it. Special attention is paid to recent evidence from "tasty reactivity" studies of affective reactions to food. I argue that this evidence suggests the following surprising possibilities regarding the functional components and brain substrates of food reward. (1) Reward contains distinguishable psychological or functional components--"liking" (pleasure/palatability) and "wanting" (appetite/incentive motivation). These can be manipulated and measured separately. (2) Liking and wanting have separable neural substrates. Mediation of liking related to food reward involves neurotransmitter systems such as opioid and GABA/benzodiazepine systems, and anatomical structures such as ventral pallidum and brainstem primary gustatory relays. Mediation of wanting related to food reward involves mesotelencephalic dopamine systems, and divisions of nucleus accumbens and amygdala. Both liking and wanting arise from vastly distributed neural systems, but the two systems are separable. (3) Neural processing of food reward is not confined to the limbic forebrain. Aspects of food reward begin to be processed in the brainstem. A neural manipulation can enhance reward or produce aversion but no single lesion or transection is likely abolish all properties of food reward. (4) Both wanting and liking can exist without subjective awareness. Conscious experience can distort or blur the underlying reward process that gave rise to it. Subjective reports may contain false assessments of underlying processes, or even fail at all to register important reward processes. The core processes of liking and wanting that constitute reward are distinct from the subjective report or conscious awareness of those processes. PMID- 8622815 TI - Role of peripheral angiotensin in salt appetite of the sodium-deplete rat. AB - Lines of evidence indicate that the importance of peripherally derived angiotensin II as a stimulus for salt appetite in rats has been underestimated. First, a series of observations is consistent with the idea that peripherally derived angiotensin acts at circumventricular organs of the brain to stimulate salt appetite following sodium depletion. Second, recent experiments show that depletion-induced salt appetite is abolished by the i.v. infusion of converting enzyme inhibitor (captopril) at a dose that totally prevents the formation of angiotensin II within the peripheral circulation. This same dose of converting enzyme inhibitor does not penetrate the blood-brain barrier to affect the actions of centrally derived angiotensin. These findings suggest that angiotensin II of peripheral origin is critical for the expression of salt appetite following extracellular fluid depletions. Together, these two lines of evidence suggest that the role of circulating angiotensin II in the stimulation of salt appetite in the rat should be re-examined. PMID- 8622816 TI - Human eating: diagnosis and prognosis. AB - Despite substantial recent progress, we remain without a comprehensive theory of human eating. The constraining influence of the single-factor, hunger-satiety model of feeding in animals is addressed. Three aspects of human eating--counter regulation in dieters, the effects of social models, and the influence of distress on eating--are reviewed briefly, in an attempt to demonstrate that a simple hunger-satiety model cannot handle the data. It is imperative that we consider social, cognitive, and other influences on eating as important casual agents in their own right; these influences are not necessarily mediated by their effect on hunger-satiety. A comprehensive theory of human eating is not likely to appear soon, but there are grounds for optimism in the process (rather than the final result) of research. PMID- 8622817 TI - Sensory-specific satiety and its importance in meal termination. AB - Pleasantness is important in influencing food choice, and may play a role in determining the amount of food consumed. Judgements of pleasantness decrease as the food is eaten. It has been proposed that his reflects the development of satiety to a specific food. However, consumers may not rate these changes as important in meal termination. Fifty-seven subjects were given ad lib access to a test meal of cheese on crackers and at the end of this meal recorded the main reason for stopping from a possible seven statements. They then rank ordered the importance of each reason. One hour later, subjects were offered a choice of the same food, a different food, or no second course. Again reasons for stopping were recorded by those who selected a second course. The most common reason given for a meal termination in the first course was "I got tired of eating that food" (40%) and for the second course "I felt full" (48%). Subjects were divided into those who rated fatigue and changes in pleasantness as important and those who rated fullness as more important. Significant differences in intake between these groups indicated that those who rated fatigue/hedonics as important consumed significantly fewer calories (275 +/- 23 kcal) than those who rated fullness as more important (424 +/- 65 kcal). It is argued that fatigue experienced by subjects may reflect sensory fatigue and that this is an important part of the development of sensory-specific satiety. Since subjects who rated gastric fullness as the most important reason for terminating the meal consumed more calories, it is suggested that this index of satiety may be relatively more crude than sensory or hedonic variables. PMID- 8622818 TI - How can eating behavior be regulated in the complex environments of free-living humans? AB - The eating behavior of humans in their natural environments is complexly affected by a myriad of nutritional, physiological, psychological, sociological and cultural factors. We have employed the diet diary technique to document the influence of: the subjective states of hunger, the amount of food or fluid remaining in the stomach at the onset of ingestion, and its composition, the time of day, day of the week, phase of the moon, month of the year, the location, the number of people present, the relationship of eating companions to the subject, dietary restraint, and eating disorders. Recently we have demonstrated, with twins, significant heritabilities for the amounts of foods and fluids ingested, independent of body size. By viewing behavior in free-living conditions the relative importance of these factors in the determination of the amounts and timing of intake can be discerned and measured. The analysis indicates that immediate environmental, psychological, social, and cultural stimuli exert powerful but short-lived effects on intake. Physiological stimuli, on the other hand, appear to influence intake subtly but persistently. Their influences are difficult to document on the short-term, but clear over long periods of time. Short-term intake for the most part would appear to be unregulated and allow to vary spontaneously within a relatively wide range. Physiological variables appear to feed back after a delay of at least a day and usually longer to alter the overall level of intake of bias. The persistent bias continues to shift intake, producing a cumulative net alteration of intake, while the effects of random short-term influences average over tome, and result in no effect on intake. PMID- 8622819 TI - Human eating: evidence for a physiological basis using a modified paradigm. AB - The aim of these studies was to determine if meal requests and changes in hunger ratings in humans were related to spontaneous changes in blood glucose concentration. In our first study, 18 healthy subjects were acutely isolated from food ant time cues. Blood glucose was continuously monitored online and visual analog ratings of hunger were obtained following an overnight fast. Spoken meal requests, if they occurred, were also recorded. In 83% of the subjects, both the perception and behavioral expression of hunger, as assessed by changes in hunger ratings and meal requests, were preceded by, and correlated with, brief, transient declines in blood glucose (nadir: -10% at 27 min). The pattern, magnitude and time course of these declines was similar to those observed in rats. This significant association, between increased expression of hunger and declines in blood glucose, is being tested in a second, ongoing study using acute insulin infusions to mimic spontaneous transient declines in blood glucose. Each subject was studied twice: either insulin or saline was infused while hunger ratings were obtained. Preliminary results in five subjects indicate that hunger ratings increased after insulin-induced transient declines in blood glucose. No change in hunger ratings occurred when blood glucose concentration was stable. These results suggest that this temporal pattern of blood glucose reflects an antecedent physiological event or provides a signal related to the expression of hunger in humans. Further understanding of human eating may result from investigation of the complex interaction of physiological and other factors in an experimental setting that allows the expression the behavior under study. PMID- 8622820 TI - The evaluation of insulin as a metabolic signal influencing behavior via the brain. AB - The intent of this paper is to evaluate decreases of food intake and body weight that occur when a peptide is administered to an animal. Using the pancreatic hormone insulin as an example, the case is made that endogenous insulin is normally secreted in response to circulating nutrients as well as in proportion to the degree of adiposity. Hence, its levels in the blood are a reliable indicator of adiposity. A further case is then made demonstrating that insulin is transported through the blood-brain barrier into the brain, where it gains access to neurons containing specific insulin receptors that are important in the control of feeding and metabolism. Finally, experimentally-induced changes of insulin in the brain cause predictable changes of food intake and body weight. Given these observations, the question is then asked: since endogenous insulin, acting within the brain, appears to decrease food intake, can a decrease of food intake caused by exogenous insulin administered into the same area of the brain be ascribed to the same, naturally-occurring response system, or should it be attributed to malaise or a non-specific depression of behavior? Arguments are presented supporting the former position that exogenous insulin, when administered in small quantities directly into the brain, taps into the natural caloric/metabolic system and hence influences food intake and body weight. PMID- 8622821 TI - Role of the liver in the metabolic control of eating: what we know--and what we do not know. AB - Profound metal-related changes in the supply of metabolites to t he liver and in the hepatic metabolism occur, and there is ample evidence that neural signals from hepatic metabolic sensors can affect eating. Hepatic afferent nerves presumably represent glucosensors which contribute to the control of eating by monitoring their own glucose utilization. Yet, the nature of the putative sensors that respond to the oxidation of other metabolites than glucose had not been identified. ATP and sodium pump activity may link hepatic oxidative metabolism and membrane potential, because hepatic phosphate-trapping by 2,5-anhydro mannitol, and inhibition of sodium pump activity by ouabain is associated with a stimulation of eating. Hepatocyte membrane potential is also subject to changes in transmembranal potassium flow through volumetrically controlled membranal potassium channels. Yet it is unknown if and how hepatocytes are linked to afferent nerves. It is also unclear how the effects of glucagon and insulin fit into the hepatic metabolic control of eating. Glucagon appears to induce satiety through its actions in the liver, but the involved mechanism is still unclear. Recent studies suggest that insulin, which has mainly been explored as a centrally acting long-term satiety signal, has an immediate effect on meal size, but is presently unknown whether an hepatic action of insulin is involved. PMID- 8622822 TI - Probing the causes of high-fat diet hyperphagia: a mechanistic and behavioral dissection. AB - High-fat diets promote hyperphagia in both rats and humans; however, understanding of the process by which dietary fat increases intake is incomplete. Since altering the fat content of a diet simultaneously changes both its sensory properties and postingestive effects, it is unclear whether high-fat diet hyperphagia is driven by oral influences, postingestive factors, or both. Previous findings from both animal and human studies indicate that relatively "less palatable" high-fat diets are overeaten relative to high-carbohydrate diets, indicating that the postingestive effects of high-fat foods are sufficient to promote hyperphagia. A program of research on rats is described, which isolates and assesses the independent effects of sensory and postingestive influences on intake of liquid high-fat and high-carbohydrate diets. An integrated series of experiments investigates both short-term (meal size, postprandial satiety) and long-term (ad lib intake over weeks) effects of diet composition on intake in order to "dissect" the causes of high-fat diet hyperphagia. Preliminary findings from this approach indicate that the postingestive effects of a high-fat diet promote longer meal size, less postprandial satiety per calorie, and greater daily calorie intake than a high carbohydrate diet. PMID- 8622823 TI - Cytokines and food intake: the relevance of the immune system to the student of ingestive behavior. AB - The aims of this paper are to provide a selective review of the literature relating immune system mediators, especially cytokines, to the control of eating and to indicate why this literature is particularly relevant to the student of ingestive behavior. Four reasons are given. Firstly, many immune system mediators influence eating, providing excellent examples of neuroimmunological controls of behaviour. Secondly, the immune system appears to be involved in the profound eating pathology associated with several clinical conditions. Thirdly, cytokines affect both energy intake and energy expenditure. Fourthly, the anorexia typically associated with activation of the immune system provides an informative model for the analysis of gut-brain communication in the control of eating. PMID- 8622824 TI - Mechanisms of sickness-induced decreases in food-motivated behavior. AB - Interleukin-1 beta (IL-1 beta) is a cytokine released by activated macrophages and monocytes, which mediates many of the local and systemic responses to inflammation. Interleukin-1 beta induces anorexia in rats when administered peripherally or centrally. An endogenous antagonist for the IL-1 type I receptor has been characterized and cloned (IL-1ra). We have used this protein to ascertain the site of action for the anorexic effects of IL-1 beta. Male rats were food restricted and trained on an operant schedule for food reinforcement. Administration of recombinant human IL-1 beta (4 micrograms i.p. or 40 ng i.c.v.) induced profound decreases in operant responding, with maximal effects 1-4 h post injection. Interleukin-1ra pretreatment (2.4 mg i.p. or 24 micrograms i.c.v.) completely blocked these effects when administered by the same route. In contrast, i.c.v. Il-1ra only partially blocked the effects of i.p. IL-1 beta, and i.p. IL-1ra was unable to block the effects of i.c.v. IL-1 beta. Interleukin-1ra did not affect responding by itself. These results suggest that IL-1 beta acts as both peripheral and central IL-1 receptors to reduce food motivated behavior. To determine the central site of action of IL-1 beta, small quantities of IL-1 beta (5 and 30 ng) were infused into the ventromedial hypothalamus of male rats. Both doses produced profound decreases in responding; the magnitude and time course of these effects were nearly identical to those observed after i.c.v. administration. These results suggest that the VMH may serve as a central site of action for the depressive effects of IL-1 beta on food intake. There is much controversy over the pathways of communication from the immune system to the brain. To test the hypothesis that the peripheral immune stimulus is transmitted to the brain via a neutral communication pathway, mice were injected with lipopolysaccharide at a behaviorally active dose (10 micrograms i.p.). This treatment increased the concentrations of substance P, neurokinin A, and calcitonin gene-related peptide in mouse spinal cord in a prostaglandin-dependent manner. Maximal increases in neuropeptide content were observed 1 h post injection. Finally, subdiaphragmatic vagotomy was found to attenuate the reduction in food-motivated behavior induced by both IL-1 beta and lipopolysaccharide in mice. PMID- 8622825 TI - Neutral mediation of food aversions and anorexia induced by tumor necrosis factor and tumors. AB - Tumors often cause declines in food intake and body weight, a condition referred to as tumor anorexia. A macrophage-derived peptide known as tumor necrosis factor (TNF) has been proposed as an important mediator of cancer anorexia and cachexia. Our work with an animal model of this condition indicates that strong learned aversions to the available diet arise in rats bearing implanted tumors and that these aversions contribute significantly to depressions in food intake and body weight. Lesions of the area postrema and nearby caudal medial nucleus of the solitary tract (APcmNTS) markedly attenuate both the learned aversions and the anorexia induced by tumor growth. A strikingly similar pattern of effects on tumor-induced aversions and anorexia was seen after subdiaphragmatic vagotomy and after capsaicin treatments. The similarity in the effects of all three treatments suggest that they involve interruption of the same system, presumably afferent signals conveyed by the vagus nerve to the nucleus of the solitary tract. The extent to which peripheral TNF administration generates symptoms similar to those produced by tumor growth was also examined. TNF administration was associated with the development of strong aversions to a novel but not a familiar diet. Area postrema lesions were found to significantly attenuate the effects of TNF on novel diet intake and preference. These observations provide parallels between the effects of TNF and the effects of tumor growth on diet aversions and food intake. PMID- 8622826 TI - Effects of oral experience on rewarding properties of oral stimulation. AB - The oropharyngeal sensory and motor experiences of ingestion have long been considered to be rewarding or motivating. Recent work has suggested that the reinforcing properties of oral stimulation are modulated by experience with an ingestive bout. Two processes that result from the repeated oral experience that occurs during an ingestive bout, sensitization and oral habituation, may influence the reinforcing or rewarding properties of oral stimulation. The first process, sensitization, describes an initial increase in responsiveness following the first presentation of a stimulus: such an increase in responding has been noted in the ingestive behavior of both rats and humans. The second process, oral habituation, occurs after sensitization of responding, and results from continued repeated exposure to oral stimulation. During oral habituation, ingestive responsiveness declines; decreased ingestive responding to oral stimulation is demonstrated even in the absence of substantial post-ingestive signals. Both oral habituation and sensitization reflect process through which experience may modulate the rewarding properties of oral stimulation. PMID- 8622827 TI - On the origin of consciousness, a postulate and its corollary. AB - A hypothesis is presented according to which structure of consciousness is quadri dimensional. The four dimensions are duration, quality, intensity, and affectivity. One may reach this conclusion by introspection alone, or by deduction along the following steps: (1) consciousness should be examined from the point of view of evolutionary psychology; (2) this leads to the following postulate--consciousness evolved from sensation; (3) the postulate entails a corollary-- consciousness has kept the quadri-dimensional structure of sensation. The above postulate could explain the phylogenetic origin of pleasure as the common currency, and in turn the tradeoffs for access to the behavioral final common path. It would also entail that joy is the transient sign of a useful conscious event, whereas happiness is the indifferent experience of a satisfied mind. PMID- 8622828 TI - The direct and indirect controls of meal size. AB - Meal size is a major determinant of energy intake and an important phenotype in animal models of obesity and in human eating disorders. Successful analysis of the controls of meal size is a fundamental goal of the science of ingestion. This paper proposes a new classification of the controls of meal size based on an unambiguous physical criterion. The criterion is food stimuli contacting preabsorptive receptors along the surface of the gut from the tip of the tongue to the end of the small intestine. Direct controls depend upon such contact. Indirect controls, e.g., rhythmic metabolic, cognitive, etc., do not have such contact. Instead, indirect controls change meal size by modulating the potency of direct controls. A method of measuring the potency of direct and indirect controls is described. The classification is unambiguous, comprehensive, and explicates the functional relationship between indirect and direct controls. Because the method of measurement is quantitative, this classification is heuristic foe mechanistic research and provides a common theoretical framework for diverse investigators interested in different aspects of the controls of eating and meal size. PMID- 8622829 TI - Sub-diaphragmatic vagal afferent integration of meal-related gastrointestinal signals. AB - We have established a method to investigate the range of mechanical, nutrient chemical and peptidergic meal-related stimuli t hat may generate vagal afferent neurophysiological signals critical to the negative feedback control of food intake in the rat. We have identified populations of fibers that respond with increased neurophysiological discharge rates to gastric loads, duodenal loads, and close celiac arterial administration of a brain-gut peptide, cholecystokinin. Load-sensitive fibers with gastric and duodenal mechanoreceptive fields are able to integrate information arising from mechanical and peptidergic stimulation, where cholecystokinin octapeptide (CCK) administration potentiates subsequent responses to distending loads, and synergizes with distending loads to produce greater excitation than either load stimulus alone or peptide stimulation alone. In addition, we have identified situations where the duodenal presence of nutrients modifies the vagal afferent activity of gastric load-sensitive fibers. Thus, our approach can mimic the temporal and spatial distribution of meal related stimuli in the gut, and reveals the potential for nutrients in one gastrointestinal compartment to affect neutral signals arising from another gut compartment. PMID- 8622830 TI - Vagal control of digestion: modulation by central neural and peripheral endocrine factors. AB - Vago-vagal reflex control circuits in the dorsal vagal complex of the brainstem provide overall coordination over digestive functions of the stomach, small intestine and pancreas. The neural components forming these reflex circuits are under significant descending neural control. By adjusting the excitability of the different components of the reflex, alterations in digestion control can be produced by the central nervous system. Additionally, the dorsal vagal complex is situated within a circumventricular region without an effective "blood-brain barrier". As a result, vago-vagal reflex circuitry is also exposed to humoral influences which profoundly alter digestive functions by acting directly on brainstem neurons. Behavioral and endocrine physiological observations suggest that this "humoral afferent pathway" may significantly alter the regulation of food intake. PMID- 8622831 TI - Food selection: problems in understanding how we choose foods to eat. AB - Understanding food selection will require considerably more than reductionist analyses of the internal workings of individual animals. To understand food choice we will have to examine not only the physiology and behavior of individuals, but also the biological and social environments within which individuals select items to ingest. The biological environment determines patterns of food availability and, over evolutionary time, provides selective pressures which shape sensory-affective responses to flavors, making them adaptive with respect to local conditions. Direct experience of the consequences of ingesting potential foods and interaction with conspecifics that have eaten various foods both affect food choices. These multiple influences, acting at different levels of organization, can bias food selection by individuals in either adaptive or maladaptive directions, depending on the characteristics of the environment in which feeding occurs. The need to understand the relationship between internal organization, individual and social experience and ecological demands may make food choice the most difficult of the core aspects of feeding behavior to analyze satisfactorily. PMID- 8622832 TI - The politics of nutrition in North America. AB - In the 100-odd years since the rise of modern nutritional science, North American' ideas about food and health have been influenced by much more than the scientists' findings. They have invariably been affected by political considerations. War, fear of revolution, leftist ideology, and the enormous financial stakes of giant food producers have all played important roles in shaping ideas about nutrition. PMID- 8622833 TI - Supertasting, earaches and head injury: genetics and pathology alter our taste worlds. AB - Family studies using thresholds showed that PROP (6-n-propylthiouracil) tasting is produced by a dominant allele, T. Nontasters have two recessive alleles and tasters have one or two dominant alleles. The bitterness of suprathreshold PROP and anatomical criteria subdivide tasters into medium and supertasters. Supertasters may be TT tasters, but this has yet to be demonstrated. Supertasters preceive the greatest bitterness and sweetness from many stimuli as well as the greatest oral burn from alcohol and capsaicin. Women are more likely than men to be supertasters. Otitis media and head trauma can alter taste and thus PROP classifications, complicating studies on PROP genetics. Some subjects with a history of otitis media show taste reductions, but others show enhanced tastes and appear to have more taste buds per fungiform papilla. Subjects with head trauma show reduced tastes on some oral loci, but there is evidence that severe reductions on the front of the tongue ameliorate reductions at the circumvallate papillae on the back of the tongue by a release of inhibition mechanism. PMID- 8622834 TI - The importance of calcium in the control of salt intake. AB - There is no doubt that the renin-angiotensin-aldosterone system (RAAS) can have a major influence on salt intake but the conditions used to demonstrate this are rare outside the laboratory. Changes in RAAS activity do not explain the voluntary intake of NaCl solution shown by non-deprived rats or the preference for salty foods shown by humans. As a first attempt to investigate an alternative mechanism, my colleagues and I are studying the effects of dietary calcium on salt appetite. Even though calcium-deprived rats have normal RAAS activity, they show a striking increase in NaCl intake, which can surpass the effects of the most severe manipulations of the RAAS. The physiological mechanism underlying this behavior is unknown but it probably involves an adrenal factor, perhaps corticosterone. Whatever the mechanism, the calcium-deprived rat and other low renin models of salt intake provide a promising approach to help understand the physiological basis of excess salt consumption by humans. PMID- 8622835 TI - Tuberculosis: current implications and management in obstetrics. AB - After years of decline, tuberculosis has again emerged as a serious public health issue. Following the introduction of effective chemotherapy at mid-century, cases of tuberculosis decreased until 1986. Since that time, the number of cases of tuberculosis have dramatically increased, particularly among young persons. The reemergence of tuberculosis is localized to urban areas and is linked to the increase in the incidence of human immunodeficiency virus infection and drug resistance. Since pregnant women are at risk for tubercular infection, an effective method exists to identify women who are asymptomatically infected, provide them with treatment to prevent progression of disease, and investigate their contacts. This review will discuss the issues of diagnosis and treatment of asymptomatic infection and active tuberculosis in pregnant women. Additionally, the need for universal screening of all prenatal patients and the benefit and potential perinatal toxicity of chemotherapy in both immunocompetent patients and those infected with human immunodeficiency virus will be addressed. PMID- 8622836 TI - Cancer in pregnancy: a review of the literature. Part I. AB - Cancer is an important cause of death in the United States in women of childbearing age. Approximately 1 per 1000 pregnant women will develop cancer. This review (Part II follows in this issue) examines the diagnosis, prognosis, and management of cancer during pregnancy; both in terms of the cancer's effect on the pregnancy, and the pregnancy's effect on the cancer. Some diagnostic modalities and some therapies are problematic to the fetus and placenta. However, in most cases and the pregnancy can be managed concurrently with a good outcome for the baby and without compromising the mother's prognosis. PMID- 8622837 TI - Cancer in pregnancy: a review of the literature. Part II. PMID- 8622838 TI - [Surgical use of laser on the locomotor apparatus]. PMID- 8622839 TI - [Laser-assisted induction of arthrosis]. AB - In a controlled experimental trial the induction of gonarthrosis following laser assisted meniscectomy was assessed. A XeCl excimer laser (lambda = 308 nm, pulsewidth 20 ns, repetition rate 10-70 Hz, radiant exposure 10-40 mJ/mm2) and a continuous wave Nd:YAG laser (lambda = 1064 nm, energy density 21-93 W/mm2) were employed in connection with 600-micrometer and 800-micrometer quartz fibers, respectively. In an animal model, effects on the remaining meniscus and the corresponding cartilage surfaces were studied in vivo within an observation period of 6 months. For evaluation of the pathogenesis in laser induced arthrosis, morphological and biomechanical studies were performed on native human menisci. Compared to conventional meniscectomy, laser energy effectively prevents further fibrillation of the meniscal structure. A remodelling of the meniscus did not occur in any experimental group. The degree of reactive synovitis, assessed by macroscopic and histologic scoring, was significantly greater following Nd:YAG laser surgery than after excimer laser resection and conventional surgery (P < 0.05). On X-ray, more progression of degenerative changes was observed following laser procedures than after conventional meniscectomy. The proteoglycan content in the femoral cartilage corresponding to the zone of meniscus resection was significantly lower in the laser group than after conventional meniscectomy (P < 0.01), which substantiates macroscopic and radiographic findings indicating degenerative derangement of the hyaline cartilage. On light microscopy and scanning electron microscopy of meniscus specimens, excimer laser irradiation led to well-delineated resection borders with a mean transitional zone of 20 micrometers, whereas menisci exposed to Nd:YAG laser irradiation presented extensive vaporization zones with the surface covered by an amorphous structure. Menisci irradiated by excimer or Nd:YAG laser energy showed significantly increased rigidity (P < 0.05 and P < 0.01); the maximum value of radial strain at flexion was lower following Nd:YAG laser irradiation (mean 1.5 +/- 0.3%) and following excimer laser irradiation (mean 2.4 +/- 0.3%) than in non-irradiated menisci (mean 3.0 +/- 0.5%). These observations lead to the conclusion that laser assisted meniscectomy promotes the early manifestation of gonarthrosis by virtue of a reduced femorotibial contact area and alterations in the biomechanical properties of the meniscus. PMID- 8622840 TI - [Magnetic resonance imaging and temperature measurement in cartilage following laser therapy]. AB - The aim of this study was the dynamic measurement, with magnetic resonance imaging, of temperature in cartilage irradiated by laser. Fresh specimens, each with chondromalacia grade II and III, were irradiated with a Holmium:YAG laser under MRI control. Temperature was measured by means of the proton-shift method, which allows high temporal and spatial resolution. With this method a maximum increase in temperature of 35 degrees was found after irradiation with 1.0 J and 10 Hz for 5 s at a distance of 1 mm. In no case at all was an increase in temperature of more than 10 degrees observed in the cartilage adjacent to the subchondral bone. These results exclude the possibility of significant thermal damage from laser treatment as described. PMID- 8622841 TI - [Biophysical bases of the effects of holmium laser on articular cartilage and their impact on clinical application technics]. AB - The in vitro study presented helps to clarify the biophysical mechanisms and tissue interactions of the holmium laser at the point of impact on the surface of cartilage-bone specimens investigated in different experimental settings. A striking event is the creation of a vapor bubble that opens up access for the laser beam through the fluid medium. This bubble shows a reproducible dynamic behavior function of the laser irradiance and the angle of incidence of the delivery fiber. These determine directly the amplitude of the pressure waves induced when the bubble collapses. Apart from this acoustic effect, which is correlated with epicentric histological features that can hardly be considered biologically relevant, a thermal effect is recognized that is finally responsible for the ablation and tissue damage. It induces typical histological alterations that can be observed along the laser beam axis, with a penetration function mainly of the irradiance but also of the angle of incidence. Nevertheless, at a pulse energy of 1 J and an irradiation angle of 30 degrees, the recorded overall depth of the immediate histological changes was down to 500-600 micrometers. Thus, in realistic working conditions, the damage observed after cartilage sealing with the holmium laser remains within an absolutely acceptable range. This is in agreement with the better results compared to mechanical cartilage debridement that have been reported in previous prospective clinical studies. PMID- 8622842 TI - [Temperature and ablation measurements in laser therapy of intervertebral disk tissue]. AB - We experimentally tested the effect of Laser light at wavelengths of 1064 nm, 1320 nm, 1440 nm and 2100 nm on intervertebral disc tissue. The tested parameters were tissue ablation, determined by weight reduction, and temperature change, measured with thermoelements. We used bovine nucleus pulposus tissue for ablation measurement and human spine segments for temperature measurement. We found that variation of single parameters such as power, frequency and energy per pulse had only a slight effect on ablation. The decisive factor for the mass of tissue ablated seems to be the total applied energy. One indispensable requirement is a suction unit; we procured a manifold increase in ablation rate using suction. In the investigations of tissue temperature, we found no risk to upper or lower plate when the fiber was centered correctly in the disc. Within a distance of 10 mm from the fiber tip, the temperature development was found to be radial, so no more tissue damage is to be expected in front of the tip than to the side of it. Although the various wavelengths tested are absorbed differently in water there were only small differences in temperature. Using a neodymium: YAG laser with a fiber of 400 micron diameter, we sometimes measured high cannula temperatures, showing that case one has to be exercised, especially with high energies. In conclusion, it can be said that both the Holmium:YAG laser and the neodymium:YAG laser are suitable for disc decompression with the parameters used currently in surgery. PMID- 8622843 TI - [Experimental bases for photodynamic laser therapy in chronic polyarthritis]. AB - The principle of photodynamic laser therapy (PDT) for chronic polyarthritis consists in specifically concentrating a drug (photosensitizer) in the synovium. Subsequent activation of the photosensitizer by means of laser leads to a cytotoxic effect. The practicability of PDT was first shown in cell cultures of human synovial fibroblasts. For further tests an animal model consisting of IgG induced arthritis in rabbits was used. In this model, concentration of the photosensitizer in the synovial lining cells, in the media of arteries and in the lymphoid infiltrate was seen. After laser irradiation there was total selective demarcation of the synovium. In contrast, bradytrophic tissues such as cartilage, meniscus and ligament structures were changed neither macroscopically nor microscopically. In the animal model PDT combines high selectivity with minimal invasiveness and can be used in small joints. PDT thus offers ideal preconditions for future minimal invasive treatment of chronic inflammatory joint diseases. PMID- 8622844 TI - [Changes in articular capsular tissue using holmium:YAG laser at non-ablative energy densities. Potential application in non-ablative stabilization procedures]. AB - Multidirectional and unidirectional glenohumeral instability constitute an important clinical problem, particularly in the athlete. Numerous methods have been described for treatment of glenohumeral instability, including closed management, open and arthroscopic procedures, but none have achieved universal success. The purpose of this study was to evaluate the effect of laser energy at non-ablative energy densities on the mechanical properties of joint capsular tissues in a rabbit model. Femoropatellar joint capsular tissues from 12 mature New Zealand white rabbits were harvested and divided using a randomized block design into three treatment groups with laser at 5 W, 10 W and 15 W, and one control group. Mechanical testing was performed before and after application of laser energy. The application of laser energy resulted in 9%, 26% and 38% reduction in capsular tissue length for the 5-W, 10-W and 15-W group, respectively. Laser energy caused a significant decrease in tensile stiffness in the 10-W and 15-W groups only (P < 0.05). Laser energy did not change the relaxation properties of capsular tissue at any energy density. The loads required to return specimens to their original length were significantly lower for the 5-W group than for the 10-W and 15-W groups. This study demonstrates that significant capsular shrinkage can be achieved with the application of non ablative Ho:YAG laser energy without detriment to the relaxation properties of the tissue, although at higher energy densities, laser energy did lessen capsular stiffness properties. PMID- 8622845 TI - [Percutaneous laser disk decompression. Experience since 1989]. AB - Since 1987/1988 percutaneous laser disc decompression (PLDD) has been used clinically for treatment of intervertebral disc prolapses. Credible prospective investigations that have been conducted since 1989 with large patient collectives are now available for analysis of their medium-term results and comparison with other minimally invasive procedures. Our follow-up examination of the first 180 patients treated with PLDD from 1989 to 1993 shows a success rate of 72.8%, similar to that with other percutaneous techniques (automated percutaneous lumbar discectomy, percutaneous lumbar discectomy, chemonucleolysis). To guarantee success, the spinal surgeon must have command of the correct technique and also use the appropriate instruments. Good results with the PLDD procedure can be procured when contraindications and indications for patient selection are strictly observed. Overall, our 5 year results seem encouraging. PMID- 8622847 TI - [Open laser surgery on the locomotor apparatus]. AB - The first applications of laser in surgery of the locomotor apparatus in the early 1980s used the haemostatic properties of laser to diminish the amount of substitution of coagulation factors in haemophiliac patients. Only since the early 1990s has a device been available in corporating the pulsed holmium:YAG laser which works in a fluid medium without relevant side effects. Apart from haemostasis, the cutting function and tissue ablation, together with the thermal shrinking effect, are exploited in arthroscopy and percutaneous disc decompression. Now that the biophysical mechanisms of action have been elucidated, nothing stands in the way of the use of infrared lasers in open surgery of the locomotor apparatus in some indications. In a prospective clinical study we included 30 consecutive patients who underwent open laser surgery from November 1992 to August 1994, for the following indications: the sparing haemostatic tissue ablation was used for synovectomy or for bony resection in osteophytes and osteochondromas of different locations, an osteoid osteoma and a painful sacral hyperplasia in the presence of incomplete sacral meningomyelocele. With bleeding eliminated, the shaping was much easier. The non-ablative shrinking produced less tissue loss and a stabilizing strengthening of tissue at the margins of soft tissue resections, e.g. in jumper's knee, tennis elbow and Achilles tendon cysts. All laser functions that are useful in open surgery have also been used in sequestered disc herniations that are inaccessible a percutaneous procedure and, in spinal decompression, for remodelling of the posterior spine contour. An analgesic effect of laser limited the postoperative administration of analgesic drugs to an average of 3 days. No complications related to the laser treatment were observed. At follow-up 12-21 months after operation, 25 of the 30 patients in this heterogeneous population showed complete or near-total healing of the operated pathological finding, and a further 3 patients showed significant improvement. To what extent these very encouraging results will persist will be shown by long-term observation. PMID- 8622846 TI - [Comparison of percutaneous manual and endoscopic laser diskectomy with chemonucleolysis and automated nucleotomy]. AB - The combination of percutaneous manual and endoscopic Ho:YAG laser discectomy (PELD) is a new minimal intervention technique in treating patients with herniated lumbar discs that do not penetrate the posterior longitudinal ligament. The results in 100 patients treated with PELD were compared randomly with those in 100 patients treated by chemonucleolysis with chymopapain (CN) and 100 patients treated by automated percutaneous lumbar discectomy (APLD) at the same hospital. We followed the 300 patients postoperatively for 1 year, with physical examination, postoperative plain lumbosacral radiography, CT, MRI and a self assessment questionnaire. Some 68% of the patients in the PELD group considered the outcome as excellent or good and 23% as fair; the corresponding figures were 55% and 27% in the CN group, and 48% and 32% in the APLD group. Nine percent of the patients in the PELD group underwent open microdiscectomy or were suffering from back pain with sciatica, compared with 18% in the CN group and 20% in the APLD group. PELD showed better extraction of the hernia mass than APLD and a lower rate of low back pain and less decrease in disc height than CN. PMID- 8622848 TI - [Arthroscopic holmium-YAG laser compared to conventional procedures on the knee joint. 2-year results of a randomized prospective study]. AB - The results of 320 patients after holmium:YAG laser treatment in comparison to conventional mechanical methods during a 2-year period were analyzed in a prospective, randomized study. The following knee injuries were included in the study: meniscal lesion, chondromalacia, combined meniscal/cartilage lesion, rheumatoid synovitis and femoropatellar pain syndrome. Because strict inclusion criteria were used, the patient series is homogeneous, not only in terms of sex, age, injured side and intrasurgical diagnosis, but also because the initial values of the Lysholm score modified after Klein are similar. After 2 years, the results of the laser series were significantly improved, whereas the results for the conventional series, especially for chondromalacia and synovitis, did not show the same improvement. Analysis of the effect of various instrumentation and the laser system itself show differing results for the different knee disorders. The hemostatic effect of the holmium-YAG laser was excellent during surgery for all knee disorders, including surface treatment. The operation time for laser surgery was not prolonged, as is often stated. This study shows that chondromalacia, combined meniscal-cartilage lesions and chronic rheumatoid synovitis are treated more effectively and with better results with the holmium YAG laser than with conventional arthroscopic methods. Furthermore, laser treatment of meniscectomy and lateral retinacular release is also better than mechanical techniques. This study demonstrates the feasibility of the holmium-YAG laser for arthroscopic surgery. A combination of different instruments seems to be most effective. PMID- 8622849 TI - [Use of laser in arthroscopy of the ankle. Indications, method, first results]. AB - As in the knee joint, lasers can be used during ankle arthroscopy for their resective and ablative properties. Reports on arthroscopic treatment of degenerative and post-traumatic disorder of the ankle by conventional techniques are encouraging. We have used laser in ankle arthroscopy in three main situations: (1) When exuberant scar tissue from previous capsulo-ligamentous damage (e.g. after ankle sprain) symptomatically restricts range of motion and causes pain (ankle impingement). Laser is used in this case at middle energy, as a resector. (2) In the presence of cartilaginous lesions, as in osteoarthritis, flake fractures or osteoarthritis, flake fractures or osteochondrosis dissecans. Here laser is used at lower energy to reshape the cartilaginous surface without resective effect ("welding"). (3) When impaired range of motion is due to osteophytic rims, mainly at the anterior tibia, resulting from previous capsular lesions. Laser is then used at high energy to cut excessive bone. A series of 16 patients underwent ankle arthroscopy at our clinic, mainly for post-traumatic disorders, including impingement, osteochondrosis dissecans and osteoarthritis. The most frequently encountered intraarticular findings were impingement by post traumatic synovitis and scar tissue of the anterio-lateral and postero-lateral compartments, with or without an osteophytic rim of the distal anterior tibial border. Scar tissue, synovitis and osteophytes were debrided in half of the patients purely mechanically (with arthroscopic scissors or shaver) and in the other half with the holmium laser. The results are encouraging; 50% of patients had no symptoms at all at follow-up (9.5 months on average), and another 38% were satisfied with a significant improvement. In two cases, no improvement at all occurred: one patient complained of persistent pain with lack of objective findings and is believed to have developed "insurance neurosis." The other had severe postinfectious osteoarthritis that was operated too late in the course to influence pain positively. Concerning the clinical, functional and subjective follow-up results there was no relevant difference between the two groups. We recommend ankle arthroscopy to treat post-traumatic impingement syndromes of the antero-lateral, antero-medial and postero-lateral compartments of the ankle joint; the use of lasers seems to have a slightly better analgetic effect, allows an easier approach than is the case with shavers or other bulky arthroscopic resectors, and allows shaping of convex resection surfaces, which cannot be performed with a shaver. PMID- 8622850 TI - [Shoulder arthroscopy using the holmium-YAG laser method. State of the art 1994]. AB - The Ho:YAG laser has been found to be a safe and efficacious adjunct to many arthroscopic shoulder procedures. The curved, thin handpieces are easily manipulated around the surfaces of the joint. Postoperative inflammation is minimized, and a versatile instrument that can cut, coagulate, ablate, and cauterize has obvious benefit where optimal visualization is required. The potential for chondral contouring and bone resection is currently being studied, and the use of laser-induced thermal contraction of soft tissue collagen may add some exciting possibilities to the treatment of joint instabilities. PMID- 8622851 TI - [Holmium-YAG laser in outlet impingement of the shoulder. Mid-term results]. AB - Arthroscopic widening of the subacromial space in the case of outlet impingement syndrome has many advantages over the open procedures. Arthroscopic subacromial decompression in case of impingement syndrome if refractory to conservative therapy, is an established procedure. The purpose of this study was to evaluate the results of arthroscopic subacromial decompression using the holmium:YAG laser and compare them with the result of the conventional arthroscopic procedure. In a prospective study, 47 patients with stage II and III impingement syndrome were divided into two groups. The first group (20 patients) underwent conventional arthroscopic subacromial decompression. The second group (27 patients) underwent arthroscopic subacromial decompression performed with the holmium:YAG laser. The results were assessed using the constant score preoperatively and postoperatively with a follow-up of at least 1 year for each patient. Improvement in the laser group was seen in the criteria of motion and pain. Abduction power, found to be improved after 1 week and 6 weeks in earlier investigation, showed no significant improvement at the follow-up time of 1 year. The average score of both groups increased after 1 year: in the laser group from 57.4 preoperatively to 75.3, in the conventional group from 49.7 to 65.7. There were no specific complications due to the laser application. PMID- 8622852 TI - [Arthroscopic management of recurrent anterior shoulder dislocation by combining a labrum suture with antero-inferior holmium:YAG laser capsular shrinkage]. AB - Current arthroscopic treatments do not address satisfactorily the capsular redundancy frequently associated with the Bankart lesion in recurrent anterior dislocation. Although the Bankart lesion heals, many of the recurrences after arthroscopic procedures are due to capsular redundancy and the laxity of glenohumeral ligaments. We propose that laser-assisted capsular shrinkage (LACS) be combined with arthroscopic labrum reattachment. As shown by Market et al., significant capsular shrinkage can be achieved by the application of non-ablative Ho:YAG laser energy without detrimental effects to the relaxation properties of the tissue. For 1 year we have used LACS together with labrum suture in 18 shoulders in 18 patients (mean age 24.6 years). All patients suffered from chronic anterioinferior recurrent dislocation. The labrum suture was realized by an anterior reattachment (REVO screws, Linvatec, USA) or by transglenoid suture. Two or three sutures were passed through the torn labrum with 2/0 non-absorbable suture material. The LACS procedure was performed with a holmium:YAG laser (VersaPulse, Coherent, USA) at an energy of 10 W (1 J, 10 Hz) with a 30 degrees curved handpiece. All patients were immobilized in a sling for 4 weeks postoperatively. Physical therapy was begun at 1 month with passive and active exercise. To date, none of the patients have had a recurrence. Seven of 18 patients returned to their previous sports activity, and at the same level. None of the patients had an iatrogenic lesion due to the laser application or labrum suture. Compared to the other shoulder, the loss of external rotation with the arm 90 degrees abducted was 30 degrees at 4 weeks and 10 degrees at 4 months. We think that the LACS procedure is a good treatment for the capsular redundancy that is frequently associated with Bankart's lesion in recurrent anterior dislocation and is probably responsible for the high failure rate in current arthroscopic procedures. Our results are short-term results, but we expect the capsular shrinkage associated with the labrum reattachment will provide a long term success rate that is comparable to open procedures. PMID- 8622853 TI - New insights into p53 function from structural studies. AB - Recent structural analysis of p53 has greatly enhanced our understanding of the biochemical activities of this protein by presenting us with a detailed picture of the chemical groups in the protein that are involved in protein stability, conformation and functional interactions. The current structures form the basis for the design of potential therapeutics which could, for example, revert a DNA binding mutant back to a DNA-binding competent conformation. The structure of the tet domain forms the basis for designing an active therapeutic p53 with an oligomerization domain which would not cross react with a DNA-binding mutant p53. However, as useful as these structures have been in providing insight into the structure/function relationship for p53, a complete understanding of this protein awaits more detailed information on the full-length protein. In this respect, one of the most useful roles for future structural studies will be to help identify the nature of the conformational transition between latent and active p53, and how it can be modulated. PMID- 8622854 TI - UVB radiation induces p21Cip1/WAF1 and mediates G1 and S phase checkpoints. AB - In a search for effectors and targets of UVB signaling in mammalian cells, we screened a keratinocyte cDNA library with differentially subtracted UVB-enriched cDNA probes. One of the UVB induced cDNA clones proved to be the rat p21Cip1/WAF1 homologue. UVB irradiation caused a rise in p53 protein levels, in association with induction of p21Cip1/WAF1 and cyclin G expression. The effects of UVB irradiation induced p21Cip1/WAF1 on the cell cycle were examined. In contrast to gamma irradiation, which caused G2 arrest, UVB treatment of asynchronous neonatal rat keratinocytes (NK) led to a marked inhibition of replicative DNA synthesis and prolonged G1 and S phase arrests, persisting to 18-24 h, with recovery of cycling by 36 h post-UVB. G1 arrest was accompanied by inhibition of cyclin D-, E and A-associated kinases. Kinase inhibition was not due to reduction in cyclin or cdk proteins. While the association of cyclin E with Cdk2 was moderately reduced, cyclin D1/Cdk4 and cyclin A/Cdk2 complexes were not disrupted. The activating threonine 160 phosphorylation of Cdk2 in cyclin complexes was not inhibited. An incremental binding of p21 with Cdk4 paralleled the inhibition of cyclin D1/Cdk4 kinase and a similar rise in Cdk2 binding to p21 was associated with inhibition of cyclin E and cyclin A dependent kinases. Furthermore, a rise in measurable p21Cip1/WAF1-Cdk2 inhibitory activity paralleled the loss of G1 cyclin-dependent kinase activity, supporting a role for p21Cip1/WAF1 in the UVB induced checkpoints. PMID- 8622855 TI - Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody. AB - Activation of the cyclin dependent kinases (CDK4/CDK6 and CDK2) is required for G1 phase progression and entry into S-phase. The activation of these kinases is regulated by checkpoints that monitor environmental and intracellular conditions. Progression into S-phase is controlled, in part, by the availability of growth factors, and we have investigated the relationship between growth factor availability and the activation of the CDK kinases. Blocking activation of epidermal growth factor (EGF) receptor tyrosine kinase with anti-EGF receptor monoclonal antibody (mAb) 225 induces G1 phase cell cycle arrest in DiFi human colon adenocarcinoma cells. When DiFi cells are treated with mAb 225 for 24 h, we observe marked decreases in the activities of CDK2 kinase and cyclin E-associated CDK kinase which are not accompanied by reduced levels of cyclin E and CDK2 proteins. However, the amount of cyclin/CDK kinase inhibitor p27KIP1 increases in the mAb-treated cells and p27KIP1 is bound to CDK2 in increasing amounts. Immunodepletion of p27KIP1 removes an inhibitory activity from lysates of mAb treated cells: the immunodepleted and heated lysates lose the capacity to inhibit cyclin E/CDK2 activity in an in vitro assay. The results suggest that G1 arrest in the cell cycle induced by EGF receptor blockade involves p27KIP1. PMID- 8622856 TI - Activation of the erythropoietin gene in the majority of F-MuLV-induced erythroleukemias results in growth factor independence and enhanced tumorigenicity. AB - Retroviral insertional activation of Fli-1 is the first detectable genetic alteration associated with F-MuLV-induced primary erythroleukemias, while mutations within p53 are only observed in Epo-dependent (ED) cell lines derived from syngeneic mice serially transplanted with F-MuLV-induced primary erythroleukemias. In this study we have determined the mechanism of growth factor independence in several Epo-independent (EI) cell lines established from adult mice previously injected with ED-erythroleukemia cell lines or serially transplanted primary tumor cells. Here we have shown constitutive expression of the Epo gene in 12 of 15 (80%) EI-erythroleukemia cell lines. Among these 12 cell lines, eight were shown to possess clonal rearrangement of the Epo gene which could be detected in the tumors used to establish the majority of these EI-cell lines. Analysis of the pattern of proviral integration revealed that the activation of the Epo gene in these cell lines is independent of retroviral insertional mutagenesis, but apparently the result of genomic rearrangements. Furthermore, the acquisition of growth factor independence by these leukemic cells confers a selective growth advantage in vivo and is associated with enhanced tumorigenicity. Together these observations suggest that the activation of the Epo gene in the large majority of these F-MuLV-induced erythroleukemic cell lines establishes an autocrine loop resulting in the constitutive activation of the Epo receptor signal transduction pathway, thereby conferring a growth and survival advantage in vito and in vitro. PMID- 8622857 TI - Microsatellite instability analysis of primary human brain tumors. AB - Microsatellite instability, as shown by the presence of additional alleles or shifts of electrophoretic mobility at simple sequence tandem repeat loci, has been demonstrated in hereditary and sporadic colorectal tumors and many other tumor types. To study microsatellite instability in human brain tumors, we examined a total of 144 sporadic neoplasms. These included 33 astrocytic tumors, 23 oligodendrogliomas, six gangliogliomas, 41 meningiomas, 10 vestibular schwannomas and 31 pituitary adenomas. Di-, tri- and tetranucleotide repeat microsatellite markers localized on chromosome 4 and 9, X, 13 and 22, respectively, were used to assess whether instability was a significant aspect of their abnormal chromosomal pattern. Instability of microsatellite markers was detected in four oligodendrogliomas (17.4%), one pituitary adenoma (3.2%), one meningioma (2.4%), one astrocytic tumor (3.0%) and not at all in gangliogliomas and schwannomas. Therefore, our results suggest that the microsatellite instability which occurs in colorectal cancers with defective mismatch repair is infrequent in many types of human brain tumors and that the lower level of instability observed in brain tumors may be reflective of other mechanisms of genetic instability. PMID- 8622858 TI - Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability. AB - Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis. Recently, we have shown RER colon cancers also demonstrate a marked 100-fold increase in mutation rates measured within an expressed gene, hprt, suggesting the mutator phenotype in these tumors extends beyond microsatellite sequences. To determine whether the RER phenotype indeed destabilizes non-repetitive DNA sequences we have sequenced hprt gene mutations recovered from the RER colon cancer cell line RKO. Greater than 10% of hprt mutants proved to be a single 3 bp deletion located in a nonrepetitive ATTAT sequence motif. Additionally, 1-4 bp deletions or insertions were found to be randomly located throughout the hprt gene. Lastly, one third of hprt mutations proved to be transitions or transversions. The microsatellite instability demonstrated in RKO is thus a global mutator phenotype which destabilizes DNA sequences both inside and outside of repetitive sequence elements and which augments base substitutions as well as frameshifts. These findings extend the characteristics of mutations associated with RER tumors and suggest additional mechanisms by which mutator phenotypes may alter oncogenes and tumor suppressor genes. PMID- 8622859 TI - Cloning and expression of the Rb-related mouse p130 mRNA. AB - The mouse p130 cDNA was cloned from a thymus-derived library using the human p130 cDNA as a probe. The 4515-bp mouse cDNA encodes a putative 1092 aa protein with predicted molecular mass of 123 kD. Comparison of mouse and human sequences reveals the mouse protein lacks 43 aa in a conserved domain, relative to the human sequence, located amino-terminal to the pocket region. Northern analysis of P19 embryonal carcinoma (EC) and RA-induced P19 cultures indicates p130 mRNA is not expressed at detectable levels in undifferentiated stem cells and is strongly upregulated after post-mitotic neurons begin to accumulate. Northern analysis of adult mouse tissues indicated that the 4.8 kb p130 mRNA is expressed ubiquitously, however, a putative 5'-truncated 1.7 kb isoform is detected solely in testis. Forced expression of mouse p130 induced growth suppression of P19 EC cells and Western analysis of transfected P19 cells suggested the cloned cDNA encodes the full-length p130 protein. PMID- 8622860 TI - Cis/trans-activation of the interleukin-9 receptor gene in an HTLV-I-transformed human lymphocytic cell. AB - The MT-2 cell-line, which had been established through in vitro cell to cell transmission of human T-cell leukemia virus type I (HTLV-I) among human primary lymphocytes, was shown to possess multiple copies of integrated proviruses, including defective proviral genomes. By analysing a genomic clone, we identified the integration site of a single HTLV-I long terminal repeat (LTR) in the interleukin-9 (IL-9) receptor (IL-9R) gene. The integrated HTLV-I-LTR was shown to be functional as a promoter and the integration site was located in an intron upstream of the first coding exon of the IL-9R gene. Upon analysis of total cellular RNA, specific expression of HTLV-I-LTR Il-9R chimeric mRNAs in MT-2 cells was demonstrated. Cloning and characterization of these cDNAs have identified HTLV-I-IL-9R chimeric splicing, using either intact or alternative splice sites within the IL-9R gene. The potential roles of multiple interactions between IL-9, IL-9R and HTLV-I in the monoclonal expansion and transformation of MT-2 cells are explored. PMID- 8622861 TI - Back-mutation of the V-Ets to the C-Ets carboxy-terminal amino acids in the P135gag-myb-ets results in chicken neuroretina cells transformation and loss of basic fibroblast growth factor responsiveness. AB - The v-Myb, v-Ets containing E26 retrovirus (called in this work E26ABC) induces the proliferation of chicken neuroretina (CNR) cells in minimal medium, strongly stimulated by basic Fibroblast Growth Factor (bFGF) which confers on them the ability to form colonies in soft agar. V-Ets differs from its cellular counterpart c-Ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids by 16 unrelated residues as a consequence of DNA segment inversion in the viral sequence. It has been documented that this different C terminal sequence influences DNA binding activity and specificity. Replacement in E26ABC virus of the sequence encoding the 16 v-Ets last C-terminal amino acids by the sequence encoding the 13 c-Ets-1 derived C-terminus (virus E26ABO), results in the production of a P135gag-myb-ets with modified biological properties on CNR cells. E26ABO infected CNR cells proliferate in minimal medium more efficiently than E26ABC, are unresponsive to bFGF and able to grow in soft agar. In contrast, CNR cells infected by viruses encoding Myb and Ets proteins either in the E26ABO or in the E26ABC configuration are bFGF responsive. Since Myb alone is sufficient to induce bFGF responsiveness on CNR cells, these results suggest that the c-Ets 1 C-terminus interferes with the Myb activity of the E26ABO P135gag-myb-ets protein in CNR cells. PMID- 8622862 TI - Cells in vivo and in vitro from osteopetrotic mice homozygous for c-src disruption show suppression of synthesis of osteopontin, a multifunctional extracellular matrix protein. AB - Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src. PMID- 8622863 TI - Identification of two isoforms of the Cak receptor kinase that are coexpressed in breast tumor cell lines. AB - The Cak receptor kinase is a member of a novel family of receptors that are characterized by the unique structure of the ectodomains. We have identified a new isoform of Cak that differs from the original isolate by the deletion of 37 amino acids in the cytoplasmic juxtamembrane sequence. Analysis of the genomic sequence suggests that the two isoforms arise by exon skipping. The isoform specific insert contains the motif NPXY, which was previously shown to be involved in diverse signaling function in a number of receptors. By RNase protection analyses, we found that the long isoform, Cak I is expressed at three- to sevenfold the abundance of the short isoform (Cak II). By Western blotting, Cak I receptor was found to be expressed in mouse embryos and in adult brain. Cak II protein was not detected in mouse embryos or adult tissues, but is abundantly expressed in some breast tumor cell lines. The expression profile of Cak suggests that its primary function is likely to be in developmental regulation. The coexpression of the Cak isoforms in some epithelial cell lines suggests that heterodimer formation may be a key feature in the function of the receptor. PMID- 8622864 TI - Regulation of nov by WT1: a potential role for nov in nephrogenesis. AB - The nov gene encodes a putative Insulin-like-Growth Factor-Binding-Protein (IGFBP) of a novel type which is structurally related to a family of growth factors likely to play a role in the control of cell proliferation. In the kidney, nov is expressed essentially at the embryonic stage and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms' tumors. The levels of human nov (novH) and WT1 mRNA in individual Wilms' tumors have been shown to be inversely correlated, suggesting that the expression of novH could be under the negative control of WT1. We have now established the nucleotide sequence of the 5' flanking region and identified two transcription start sites by RNase protection assays and primer extension. We report that in transient cotransfection experiments the transcription activity of novH promoter constructs was repressed by two isoforms of WT1 proteins (WT1 and WT1+KTS). Repression of the novH promoter required both intact zinc finger regions and the NH2 transcription repression domain of WT1. Inasmuch as the minimal region of novH promoter required to mediate WT1 repression in vivo failed to bine recombinant WT1 protein in in vitro footprinting assays this repression may be mediated by either (i) low affinity sites cooperative interactions or (ii) indirectly via protein-protein interactions with another factor(s). Furthermore, constitutive expression of wild type WT1 into 293 cells resulted in a decrease of endogenous NOVH protein levels, suggesting that novH may be a physiological target for WT1. The downregulation of novH expression by WT1 might represent a key element in normal and tumoral nephrogenesis. PMID- 8622865 TI - Phosphorylation of c-Fos at the C-terminus enhances its transforming activity. AB - c-Fos is phosphorylated by MAP kinase and the 90 kDa-ribosomal S6 kinase (RSK) in vitro at serines 362 and 374 (rat) which we demonstrate are major in vivo phosphorylation sites in early G1. We have constructed c-Fos mutants with these serines changed to aspartic acid residues (FosD) to mimic phosphorylation or to alanine residues (FosA) to prevent phosphorylation. Cells expressing FosD exhibited a more extensive transformed phenotype than those expressing either FosA or wild type c-Fos (FosWT). We also observed that FosA has a reduced half life in comparison with FosD in G1. Furthermore, we observed enhanced AP-1 transactivation activity in cells expressing FosD. These results indicate that phosphorylation of c-Fos at its extreme carboxyterminus, possibly by MAP kinase and RSK, supports the proliferative response by increasing c-Fos stability and/or by increasing its transactivation activity. Under conditions in which the MAP kinase pathway is constitutively activated, c-Fos phosphorylation probably contributes to cellular transformation. The highly conserved nature of these phosphorylation sites in other c-fos family members suggests that these may also be targets of MAP kinase and RSK. PMID- 8622866 TI - Secretion and mitogenic activity of zebrafish FGF3 reveal intermediate properties relative to mouse and Xenopus homologues. AB - Zebrafish (Brachyodanio rerio) Fgf-3 cDNAs expressed in COS-1 cells give rise to the heterogeneous set of secreted proteins with relative molecular masses in the range of 29-30.5 kDa. These proteins associate strongly with the extracellular matrix but are quantitatively released into the culture medium in the presence of heparin (5 micrograms/ml). Extracellular zebrafish FGF3 (ZFGF3) also contains a smaller sized component that appears to result from an amino-terminal proteolytic cleavage. These properties are similar to those described for Xenopus FGF3 (XFGF3). Receptor binding experiments indicate that ZFGF3 has a higher affinity for the IIIb rather than the IIIc isoform of FGFR2; properties that are more reminiscent of the mouse than the Xenopus homologue. Consistent with the FGF receptor binding properties, ZFGF3 shows a restricted mitogenic potential and a reduced transforming activity on NIH3T3 cells compared to XFGF3. Hybrid proteins made between Xenopus and zebrafish FGF3 implicate the C-terminal region in determining the differences in receptor potential affinities, mitogenic potency and transforming activity. Thus, ZFGF3 shows the structural and secretory properties of XFGF3, but has biological properties more akin to those of the mouse homologue. PMID- 8622867 TI - Analysis of human c-Abl tyrosine kinase activity and regulation in S. pombe. AB - c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells. Several mutations, including deletions of the SH3 domain, can activate abl and convert it into an oncogene. To study c-Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion. c-Abl, but not a kinase inactive form of the molecule, causes growth arrest followed by death of the cells. Concomitant to Abl expression we observed extensive phosphorylation of endogenous proteins on tyrosine. Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme. An SH3 domain deletion mutant of Abl is equally active as wild type c-Abl in yeast, even under conditions allowing detection of subtle differences. These results demonstrate that there is no intrinsic regulation of c-Abl kinase activity via the SH3 domain and suggest that the inhibitory effect of the SH3 domain observed in mammalian cells is medicated by a factor that is absent in fission yeast. Expression of Ab1 S.pombe provides a novel quantitative assay for ab1 activity and regulation. PMID- 8622868 TI - Expression of Epstein-Barr nuclear antigen 2 in kidney tubule cells induce tumors in transgenic mice. AB - The effect of EBNA2 in normal cells in vivo has not as yet been explored. The experiments described here were initiated to follow the consequences of the expression of EBNA2 in different tissues in transgenic mice. EBNA2 transgenic strains were generated using a vector containing EBNA2 encoding sequences under the control of the simian virus 40 (SV 40) early enhancer/promoter fused to the endogenous EBNA2 Wp promoter. Control mice carrying a transgene with the same sequence but lacking the EBV DNA part remained healthy during observation periods of up to 15 months. The SV-EBNA2 transgenic animals, however, over time developed abdominal masses that on necropsy showed to be due to kidney tumors. Histological examination revealed the presence of tumors with the morphology of kidney adenocarcinoma with a solid growth pattern. At the age of 20 weeks the kidneys of all animals investigated showed disseminated islands of tubular hyperplasia but no true malignant neoplasms. At about 50 weeks of age multiple foci of microscopic tubular adenocarcinomas were found in both kidneys. Eventually, tumors could be diagnosed in about 90% of the SV-EBNA2 transgenic mice. EBNA2 encoding RNA was expressed in both non-malignant kidney tissue and in tumors as shown by cDNA/PCR analysis. Immunoprecipitation and immunoblot analysis showed that the tumor cells contained a polypeptide of the same size as EBNA2 in B95-8 cells that reacted with a monoclonal anti-EBNA2 antibody. Immunohistochemistry demonstrated nuclear expression of EBNA2 in hyperplastic tubules and in tumor tissue. PMID- 8622869 TI - The adenovirus E1A-associated 300 kDa adaptor protein counteracts the inhibition of the collagenase promoter by E1A and represses transformation. AB - Adenovirus E1A proteins modulate the expression of a large variety of genes in transformed cells by either stimulating or repressing their promoters. For example, the E1A proteins inhibit the collagenase promoter, whereas they activate the c-jun promoter. Both effects are mediated through AP-1/ATF-binding sites. Repression of transcription of the collagenase gene requires the amino-terminus and conserved region 1 (CR1) of Ad5 E1A, two regions that are also crucial for interaction of E1A with the recently isolated transcriptional adaptor protein p300. We show here that overexpressed p300 can counteract the repressive effect of E1A on the collagenase promoter. Using the CREB-binding protein (CBP), which is highly homologous to p300, the same results were obtained. The domains in E1A required for binding to p300 are also essential for E1A-mediated cell transformation. We therefore tested the effect of p300 and CBP on the transforming potential of Ad5 E1 in baby rat kidney (BRK) cells. It was found that E1A-induced focus formation was strongly inhibited by overexpression of p300 or CBP. Moreover the BRK cell colonies, obtained after cotransfection with Ad5E1 and p300, could not be established. These results indicate that one of the mechanisms by which E1A modulates transcription and transforms cells is via transcriptional adaptors like p300 and CBP. PMID- 8622870 TI - Use of the two hybrid system to detect the association of the protein-tyrosine phosphatase, SHPTP2, with another SH2-containing protein, Grb7. AB - SHPTP2 is a ubiquitously-expressed SH2-containing tyrosine phosphatase that is tyrosine phosphorylated in response to activation of various receptor and nonreceptor tyrosine kinases. SHPTP2 associates with the platelet-derived growth factor (PDGF) receptor after ligand stimulation, and binding of SHPTP2 to this receptor promotes tyrosine phosphorylation of SHPTP2. The yeast two-hybrid system was modified to identify partners of tyrosine-phosphorylated proteins. Using SHPTP2 as bait and supplying an exogenous tyrosine kinase gene to the yeast cells, we have found that SHPTP2 interacts with another signaling protein, Grb7. We have localized the region of interaction to tyrosine 580 in the carboxyl end of SHPTP2 and to the SH2 domain in the carboxy-terminus of Grb7. We demonstrate that Grb7 binds to SHPTP2 in vitro under conditions where the latter is tyrosine phosphorylated. These experiments show that this modified two hybrid technique may be useful for the identification of proteins involved in tyrosine kinase signal transduction cascades. PMID- 8622871 TI - Linking protein kinase C to the cell cycle: ectopic expression of PKC eta in NIH3T3 cells alters the expression of cyclins and Cdk inhibitors and induces adipogenesis. AB - Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that ectopic expression of PKC eta in NIH3T3 fibroblasts blocks the normal phosphorylation of the Rb protein in quiescent cultures restimulated to enter the cell cycle; PKC eta activates a cellular program that includes increased expression of cyclins E (but not cyclin D), as well as the induced expression of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1. The increased expression of the latter inhibitors and their association with the cyclin E-Cdk2 complex results in decreased cyclin E associated kinase activity. Furthermore, in contrast to the control NIH3T3 cells, the cell that express PKC eta can be induced to undergo adipocyte differentiation in response to adipogenic hormones. Thus, PKC eta induces altered expression of several cell cycle related functions, which may contribute to its ability to promote cellular differentiation. PMID- 8622872 TI - Regulation of p21WAF1/CIP1 expression by p53-independent pathways. AB - The CDK-inhibitor p21WAF1/CIP1 has been implicated as a growth arrest mediator in p53-tumour suppression, cellular senescence and terminal differentiation. Cell type specific differences in p53-independent p21 expression and cell cycle arrest were found following treatment of human tumour cell lines with serum, 12-O tetradecanoyl-phorbol-13-acetate (TPA), or okadaic acid (OA). TPA induced p21 in ML1, K562 and HL60 leukemia cells, whereas OA induced p21 in SW480 and GM4723 carcinoma cells as well as in leukemic cells. In addition, TPA- and serum- but not OA-induced cell cycle arrest was reversed upon return of p21 to basal levels. To further investigate the mechanisms underlying p53-independent regulation of p21, the transcription inhibitor, Actinomycin D (AMD), was used to block p21 expression. The results showed a complete inhibition of p21 mRNA and protein induction by TPA or adriamycin but little effect on p21 mRNA induced by OA in the presence of AMD. These results suggested that TPA-induced p21 expression requires transcription initiation, while a post-transcriptional mechanism may be involved in OA-induction as well. Transient transfection assays with p21 promoter luciferase reporters and TPA or OA treatment further confirmed that TPA, and to a lesser extent, OA, initiated transcription of p21. Finally, the protein kinase C inhibitor, staurosporine, was found to interfere with p21 induction and prevent cell cycle arrest following treatment with TPA but not OA, suggesting a requirement for PKC in TPA activation of p21 expression. PMID- 8622873 TI - p300 gene alterations in colorectal and gastric carcinomas. AB - Colorectal tumors frequently have loss of heterozygosity on chromosome 22q, suggesting that inactivation of tumor suppressor gene(s) on 22q participates in the tumor development. Neurofibromatosis 2 (NF2) gene and E1A binding protein p300 gene, recently identified on 22q, are thought to be candidates for tumor suppressor genes. In this study, mutation of the NF2 gene in 59 colorectal carcinomas, and mutation of the p300 gene in 27 colorectal and two gastric carcinomas, were analysed using PCR-SSCP, RT-PCR-SSCP and direct sequencing methods. Missense mutations of p300 gene were detected in a colorectal carcinoma, and in a gastric carcinoma, though no mutation of NF2 gene was detected. Both p300 mutations were somatic and coupled to deletion of the second allele of the gene, which suggests inactivation of the p300 gene, in these carcinomas. The mutations are located within the Cys/His-rich regions, which are assumed to play important roles in the function of p300. These are the first cases in which p300 gene has been found to be altered in both alleles, suggesting that inactivation of the p300 gene may be involved in the development of carcinomas, and that this gene may be the target of loss of 22q in carcinomas of the digestive tract. PMID- 8622874 TI - Further characterization of the c-mos transcript and its cell cycle specific expression in NIH3T3 cells. AB - The mouse c-mos proto-oncogene is primarily expressed in germ cells. Our previous studies demonstrated c-mos RNA expression in mouse somatic cells, with the highest level present in the G2 phase of the cell cycle (Tsui et al., 1993). We have identified the transcription start site of this G2 specific c-mos transcript to be located about 1580 bp upstream from the open reading frame based on RT-PCR and RNase protection experiments. Upstream sequences containing this transcription start site directed highest expression of the luciferase reporter gene in M phase of the cell cycle. These results suggest that c-mos transcripts are produced in G2 phase and that c-Mos protein albeit at extremely low levels would accumulate in M phase. PMID- 8622875 TI - SH3 domain-dependent interaction of the proto-oncogene product Vav with the focal contact protein zyxin. AB - Scr homology 3 (SH3) domain-mediated protein-protein interactions have been implicated in the localization of proteins to specific sites within the cell. We present evidence that the product of the vav proto-oncogene, p95vav, interacts specifically with the focal adhesion protein zyxin both in vitro and in yeast two hybrid system. Solution binding and two-hybrid system experiments demonstrate that association of Vav with the LIM domain protein zyxin is mediated by the C terminal SH3 domain of the Vav and involves the proline-rich N-terminus of zyxin. The interaction appears to be selective, since no binding of the proline-rich N terminus of zyxin with other SH3 domain-containing proteins such as GRB-2, phospholipase C gamma, GTPase-activating protein, or p85 was detected. PMID- 8622876 TI - Investigation of co-amplification of the candidate genes ornithine decarboxylase, ribonucleotide reductase, syndecan-1 and a DEAD box gene, DDX1, with N-myc in neuroblastoma. United Kingdom Children's Cancer Study Group. AB - Although N-myc amplification is strongly associated with a poor prognosis, not all patients with neuroblastomas having N-myc amplification fare badly. To investigate whether genes other than N-myc are responsible for contributing to the prognosis, we examined seven cell lines and 87 primary tumours for co amplification of candidate genes known to be present near the normal N-myc locus: ornithine decarboxylase (ODC), ribonucleotide reductase (RRM2), syndecan-1 and a DEAD box protein gene, DDX1. Sequence analysis of the pG21 cDNA clone previously reported to represent an expressed gene frequently co-amplified with N-myc, showed this to be from the DDX1 gene. No co-amplification with the first three genes was found in any of the cell lines or tumour samples. DDX1, however was found to be amplified along with N-myc in 4/6 (67%) cell lines and 6/16 (38%) of the N-myc amplified tumours. Co-amplification of DDX1 and N-myc was found more frequently in stage 4 or 4S tumours than lower stage (1-3) tumours. With the exclusion of a single 4S case, there was a highly significant reduction in the mean disease-free interval from 24.4 +/- 4.7 (SE, n = 10) months for cases with co-amplification of N-myc and DDX1 compared with 9.2 +/- 1.8 (SE, n = 5) months for those cases showing amplification of N-myc alone (P = 0.0056, Welch's unpaired t-test). No amplification of DDX1, ODC, RRM2, or syndecan-1 was found in the absence of N-myc amplification. These observation indicate that the N-myc amplicon is of varied size and/or position relative to the N-myc gene, with DDX1 representing at least one other gene frequently co-amplified with N-myc. Further studies are required to confirm the biological and prognostic significance of DDX1 co-amplification and to elucidate the role that DDX1 plays in tumour genesis and progression. PMID- 8622877 TI - Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease. AB - It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor type specific events in the development of human cancers. PMID- 8622878 TI - Expression of the protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated leukemias. AB - The transcription factor IRF-1 (interferon regulatory factor 1) is an activator of type I interferon and interferon-inducible genes. IRF-1 manifests tumor suppressor activity. Its overexpression results in inhibition of cell growth, and deletions of the IRF-1 gene were demonstrated in a number of human leukemias and myelodysplasias. Although the mechanism by which IRF-1 affects cell growth is unknown, it is believed that IRF-1 activates a set of genes that negatively regulate cell growth. The double-stranded RNA-dependent protein kinase (PKR), which is an interferon-inducible gene, contains a promoter element for the binding of IRF-1 and exhibits antiproliferative properties. Consequently, we investigated the role of IRF-1 in PKR expression. Here, we show that in IRF-1 deficient embryonic fibroblasts, PKR expression is reduced relative to wild-type cells. This result predicts diminished expression of PKR as a potential consequence of deletion of the IRF-1 gene in human leukemias. We show that cells of the human leukemic U937 cell line contain a deletion of one IRF-1 gene and express low levels of PKR. We demonstrate that upregulation of IRF-1 expression in U937 cells by transfection is sufficient to induce PKR expression. We also found a marked reduction in the expression of PKR in blood samples from two patients with myelodysplasias, carrying a deletion of chromosome 5q, a locus to which IRF-1 was mapped. These results show that IRF-1 activates PKR expression and suggest that loss of one allele of the IRF-1 gene is sufficient to affect PKR expression. Therefore, PKR is a strong candidate for a mediator of the tumor suppressor activity of IRF-1. PMID- 8622879 TI - Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro. AB - The hepatoma-derived hepatitis B virus (HBV) DNA insert HU-a has recently been shown to contain two viral transactivator genes, X and preS2 /S. We report here that HU-a induces malignant transformation after stable transfection of the fetal mouse hepatocyte line FMH202, as indicated by soft agar growth and nude mouse tumorigenicity. Transfections with HU-a subclones, containing the X gene of the preS2 /S gene alone or sequences without transactivator gene, respectively, suggested that the X gene is essential for transformation. Sequential stages of transformation and tumor progression were analysed by injection of the stably transfected FMH202 lines into nude mice, explanation of the resulting tumors and re-establishment of cell lines from the tumors. Comparison of two HU-a transformed cell lines by HBV mRNA hybridization, Southern analysis and chromosomal in situ hybridization revealed that integrated HBV DNAs were involved in major chromosomal rearrangements in both cases. Interestingly, recombination of the HBV Dna insert during the nude mouse passage had completely abolished HBV specific transcription in one case, indicating that expression of integrated HBV genes, while presumably involved in early transformation, is dispensable at later stages of tumor progression. The sequential transformation observed in this experimental system suggests that expression of the X gene by integrated viral DNA and subsequent hepatocyte genome mutations might both contribute to HBV associated liver carcinogenesis. PMID- 8622881 TI - Adenoviral-mediated p53 tumor suppressor gene therapy of human ovarian carcinoma. AB - Mutations of p53 gene are reported in 50-60% of human cancers and reintroduction of wild-type p53 can suppress cell proliferation. In this study, replication deficient recombinant adenovirus encoding wild-type p53 (ACN53) under the control of the human cytomegalovirus (CMV) promoter was constructed. A specific incorporation of the p53 gene with ACN53 reduced 3 (deleted p53 gene) cells was observed. ACN53 reduced the colony-forming ability of SK-OV-3 cells 72-216 h after single infection. A highly aggressive ovarian xenograft model was established in which animals die between 25-45 days. A localization study with the adenovirus-containing beta galactosidase reporter gene showed effective gene transfer in the tumor tissues. Ex vivo treatment of SK-OV-3 cells with ACN53 followed by injection into nude mice, increased the survival of the p53 treated mice by more than 50% compared with control animals. Gene therapy with ACN53 in intraperitoneal model of SK-OV-3 cells in two independent experiments revealed that there were some long-term survivors in the group of mice [2/5 (66 and 120 days) and [2/8 (166 and 423 days)] treated with ACN53. These findings demonstrate the potential of the p53 tumor suppressor gene therapy in human ovarian carcinoma. PMID- 8622880 TI - A Herpes saimiri oncogene causing peripheral T-cell lymphoma in transgenic mice. AB - Herpesvirus saimiri is an oncogenic virus causing rapid T-cell lymphomas in New World primates and rabbits. Deletion analysis of one strain of H saimiri has indicated an open reading frame, StpA, necessary for oncongenicity in monkeys. We have investigated the function of StpA in tumor induction by the generation of transgenic mice. Expression of two different constructs caused the development of peripheral lymphomas. The infiltrating cells were of T-cell origin, expressing mainly the CD4 phenotype and restricted sets of V beta chains. Thus, StpA is not only necessary for the oncogenicity of Herpesvirus saimiri, but is also sufficient for the induction of peripheral pleomorphic T-cell lymphomas. PMID- 8622882 TI - Regulation of epidermal growth factor receptor by activated H-ras and V-myc oncogenes in mouse Balb/3T3 cells: possible roles of AP-1. AB - We previously reported that introduction of H-ras oncogene decreases the epidermal growth factor (EGF) binding activity to cell surface EGF receptor in mouse Balb/3T3. In this study, we have further isolated four H-ras transfectants, four v-myc transfectants and three both H-ras and v-myc (H-ras/v-myc) transfectants of mouse Balb/3T3 cells. In comparison with introduction of v-myc alone or both H-ras and v-myc oncogene, introduction of H-ras alone resulted in a loss of [125I]EGF binding activity to the cell surface EGF receptor. RT-PCR analysis also showed much lower levels of EGF receptor gene expression in H-ras transfectants compared to that of parental untransformed cells (Balb-Neo1), v-myc and H-ras/v-myc transfectants. Our results demonstrated the activated binding of a transcription factor, Stat1 p84/p91, which directly interacts with EGF receptor, to c-sis-inducible element (SIE) in both v-myc and H-rs/v-myc transfectants, but not in H-ras transfectants. Among transcription factors which we have analysed, activator protein 1 (AP-1) but not SP-1 was modulated by H-ras. Gel shift assays demonstrated the mobility pattern of TPA-responsive element (TRE) binding complex with AP-1 derived from H-ras transfectants migrated faster than those from Balb-Neo1, v-myc and H-ras/v-myc. Expression of c-Jun and Fra-1 was increased more than threefold in H-ras transfectants compared with Balb-Neo1, v-myc and H-ras/v-myc transfectants, but that of c-Fos, Jun B and SP-1 was unchanged. Both transient and permanent expression of H-ras enhanced AP-1 activity in mouse cells, but further co-introduction of dominant negative c-jun mutant encoding a transcriptionally inactive product inhibited the H-ras dependent AP-1 induction. Transfection of the dominant negative c-jun mutant also restored down-regulation of EGF binding by activated H-ras oncogene. Down regulation of EGf receptor by activated H-ras and the possible involvement of a transcription factor, AP-1 will be discussed. PMID- 8622883 TI - The role of p16 in the E2F-dependent thymidine kinase regulation. AB - The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein. PMID- 8622884 TI - Expression of cell-cycle regulatory genes in HTLV-I infected T-cell lines: possible involvement of Tax1 in the altered expression of cyclin D2, p18Ink4 and p21Waf1/Cip1/Sdi1. AB - To understand how the growth of T-cells transformed by Human T-cell leukemia virus type I (HTLV-I) is deregulated, we analysed the expression of cell-cycle regulatory genes in HTLV-I infected and non-infected T-cell lines. We investigated the gene for 6 cyclins, 4 cyclin-dependent kinases, and 5 cyclin dependent kinase inhibitors, and found the following: (1) HTLV-I infected T-cell lines preferentially expressed cyclin D2, whereas cyclin D3 was the major D-type cyclin in HTLV-I negative T-cell lines; (2) HTLV-I infected T-cell lines expressed strikingly low levels of p18Ink4 compared with those that were HTLV-I negative; (3) HTLV-I infected T-cell lines expressed high levels of p21Waf1/Cip1/Sdi1, whereas p21Waf1/Cip1/Sdi1 was undetectable in HTLV-I negative T-cell lines. These features were also found in T-cells immortalized by Tax1, which we established. Therefore, it is strongly suggested that Tax1 alters the expression of these cell-cycle regulatory genes. PMID- 8622885 TI - Widespread microsatellite instability occurs infrequently in adenocarcinoma of the gastric cardia. AB - Heredity non-polyposis colorectal cancer (HNPCC) is associated with an increased predisposition to colorectal cancer and extra-colonic cancers of the gastro intestinal, urological and female reproductive tracts. These tumours are characterised by an underlying defect in DNA mismatch repair and exhibit numerous replication errors throughout the genome (RER+ phenotype). HNPCC-associated gastric tumours, and a subset of sporadic, distally-located gastric tumours exhibit this RER+ phenotype. It is recognised that proximal and distal gastric tumours exhibit distinct epidemiological features. In this study we investigated the occurrence of microsatellite instability in a series of 38 primary gastric adenocarcinomas, arising in the proximal stomach. A total of 138 microsatellite markers, comprising mainly dinucleotide and tetranucleotide repeat units and covering all autosomal arms, excluding acrocentric arms, were analysed. One tumour demonstrated somatic microsatellite alterations at 62% (26 of 42) of loci tested. A further 32 tumours demonstrated levels of microsatellite instability ranging from 0.8% (1 of 28)-11.4% (15 of 132) of loci tested. Five tumours demonstrated no microsatellite alterations at any of the loci tested. These findings suggest that a high percentage of proximal gastric carcinomas exhibit a low level of microsatellite alterations at dinucleotide and tetranucleotide repeat loci. However, ubiquitous somatic alterations at these loci, characteristic of HNPCC-associated tumours, occur in a relatively small proportion of tumours. PMID- 8622886 TI - Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity. AB - The p53 gene has been indicated to be a tumour suppressor gene that is found in mutated form in common human cancers. Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs). The E6 protein of HPV is known to bind with p53 protein and inactive the tumor suppressor activity by promoting p53 degradation. Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs. Exons 5 through 8 of the p53 Mutations were observed in nine cases (24%). HPV-DNA detection and typing were performed using PCR with ?high risk group' HPV specified primers. HPV DNA sequences were detected in eight cases (21%). The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases. Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/ HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/ HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P < 0.01). We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% integral of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppressor genes may be crucial in the development of oral SCC if the prognosis is poor. PMID- 8622887 TI - Regulation of A-raf expression. AB - The Raf family proto-oncogenes encode cytoplasmic protein serine/threonine kinases which play a critical role in cell growth and development. A-raf shares several functional properties with Raf-1 including transforming activity, stimulation of the Raf/MAPK pathway and the ability of dominant negative versions to functionally block Ras signalling. A-raf transcripts are predominantly expressed in the mouse urogenital tissues. Interestingly, the human A-raf promoter region contains three potential glucocorticoid response elements GRE-1, GRE-2 and GRE-3, at positions -17, -34 and -168 respectively from the transcriptional start site. DNA sequence analysis of the mouse A-raf promoter region demonstrated that GRE-1 and -2 were conserved evolutionarily. To determine whether the human A-raf GREs represent functional motifs, an expression vector for the glucocorticoid receptor was cotransfected with A-raf promoter/reporter constructs into HeLa cells. A fivefold dexamethasone-dependent induction of A-raf promoter activity was observed using constructs containing all three GRE motifs whereas point mutations in the GREs either diminished or abolished dexamethasone induction. Electrophoretic mobility shift assays (EMSAs) using purified glucocorticoid receptor DNA binding domain (DBD) demonstrated that both GRE-2 and -3 motifs interact with DBD and oligonucleotide competition experiments established that these have different affinities for DBD. Using nuclear extracts from human and rodent cell lines in EMSAs, a specific protein-DNA complex was observed with GRE-1 which displayed binding properties unlike that of glucocorticoid receptor. These results demonstrate that the A-raf promoter is regulated in part by members of the glucocorticoid family of steroid hormone receptors and suggest a model for the regulation of A-raf expression in urogenital tissues. PMID- 8622888 TI - Signal transduction pathways induced by heregulin in MDA-MB-453 breast cancer cells. AB - Heregulins (HRGs) induce tyrosine phosphorylation of several members of the erb-B family of receptors. Although originally isolated as the ligands for p185c-erb-2, recent evidence suggests that other receptors of the erbB family, including p180erbB-3 and p180erbB-4, are their true cognate receptors. Stimulation of MDA MB-453 cells with HRG beta 2 resulted in the tyrosine phosphorylation of p185c erbB-2 and p180erbB-4 in a time- and dose-dependent fashion. This event was accompanied by the formation of multimeric complexes between the activated receptors and SH2-containing proteins. Ligand caused p120-rasGTPase activating protein (GAP), SHC and the p85 subunit of phosphatidylinositol-3'-kinase (PI3K) to be associated with both p185c-erbB-2 and p180erbB-4. In addition, tyrosine phosphorylation of p85-PI3K and SHC, but not of GAP or of its associated p62 and p190 proteins, was also detected. HRG also induced the association of GRB2 with tyrosine phosphorylated p185c-erbB-2, p180erbB-4 and SHC. Activation of mitogen activated protein kinase (MAPK) ( > 30-fold over untreated controls) was observed upon receptor(s) activation, as it was the induction of the immediate early gene c-fos ( > 200-fold). These observations suggest that p21ras activation plays a role in the HRG pathway. Furthermore, comparative analysis of the binding of p85 PI3K to 185c-erbB-2 and p180erbB-4, revealed a preferential association with activated p180erbB-4. These findings might suggest a model of HRG action in which the relative expression of the various erb-B family members and the partitioning of signal transduction molecules between each type of receptor might determine the nature of the signal elicited by the ligand and the biological response attained. PMID- 8622889 TI - Delay in serum stimulation of Erk activity caused by oncogenic transformation. AB - Mitogenic growth factor stimulation activates several signal transduction pathways, including the well-characterized Ras-Erk pathway, resulting in transient activation of Erk1 and Erk2. Oncogenic transformation, however, causes constitutive activation of growth signalling pathways, resulting in an accelerated rate of cell division. We investigated the effects of transformation on serum and growth factor stimulation of Erk1 and Erk2, and show that stimulation of these MAP kinases, as well as the Erk activator Mek, is delayed in oncogene transformed cells. Possible mechanisms of this delay are explored. In addition, our data indicate that prolonged mitogenic stimulation does not necessarily result in constitutive activation of Erk1 and Erk2. PMID- 8622890 TI - Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas. AB - The MET oncogene encodes the receptor for HGF/Scatter Factor, known to control cell motility and invasion in epithelial cells. We report that the Met/HGF receptor, absent in differentiated adult skeletal muscles, is aberrantly expressed in clinical samples and in established cell lines of human rhadbomyosarcomas. In both the embryonal and alveolar histotypes the oncogene is overexpressed and, in some cases, amplified. The Met receptor is exposed at the cell surface and is functionally active in response to HGF/Scatter Factor. Accordingly, rhabdomyosarcoma cells exhibit an invasive phenotype in vitro in response to exogenous HGF/Scatter factor. As the factor is known to be produced by connective tissues, a paracrine stimulation of rhabdomyosarcoma invasiveness in vivo is hypothesized. Two alveolar rhabdomyosarcomas were found in co-express the ?two-kringle' alternatively-spliced HGF/Scatter Factor variant, which has been previously shown to stimulate cell motility and matrix invasion in vitro. These cells displayed the invasive phenotype in the absence of exogenous HGF/Scatter Factor, suggesting an autocrine mechanism in vivo. These data indicate that aberrant expression of the MET proto-oncogene provides rhabdomyosarcoma cells with the same property as embryonal myoblasts to migrate into the surrounding connective tissues. PMID- 8622891 TI - Unmasking of a proliferation-restraining activity of the anti-apoptosis protein EBV BHRF1. AB - The BHRF1 protein of Epstein-Barr virus (EBV) is a structural and functional homolog of the Bcl-2 protein. Both BHRF1 and Bcl-2 proteins promote the survival of cells exposed to various apoptotic stimuli. This promotion of cell survival is associated with a block in proliferation. It is believed that the Bcl-2 family of anti-apoptosis proteins contribute to oncogenesis merely by promoting cell survival. We have discovered that mutations within a regulatory domain of the BHRF1 protein not only suppress apoptosis induced by the tumor suppressor protein p53, but also permit efficient proliferation of cells that would otherwise undergo total apoptosis. These gain-of-function mutants of BHRF1 cooperate more efficiently with the E1a oncogene in transformation of primary rat kidney cells where E1A expression results in apoptosis. Our results suggest that such mutational inactivation of a proliferation-restraining activity in the BHRF1 gene may play a direct role in oncogenesis. PMID- 8622892 TI - Repression of c-Jun-induced mouse major histocompatibility class I promoter (H 2Kb) activity by the Adenovirus type 12-unique 52R E1A protein. AB - Down-regulation of major histocompatibility (MHC) class I gene expression by protein products of the early region 1A (E1A), which might allow transformed cells to escape the host immune system, is discussed as one cause for the oncogenicity of Adenovirus (Ad) subtype 12-transformed cells. The MHC class I promoter is activated through several cellular-transcription factors among them AP-1, whose target sequences are located in the enhancers A and B, and NF kappa B. In this report we present evidence that the Ad12-unique 52R E1A protein inhibits c-Jun-induced activation of MHC class I gene expression. Repression occurs through both AP-1 recognition sequences with the AP-1 binding site of Enhancer A, which can be bound by c-Jun dimers in vitro, being the main target for c-Jun activation as well as 52R-mediated down-regulation. Furthermore our data revealed that both promoter elements of Enhancer A, the AP-1 and NF kappa B binding sites, are necessary for full promoter activity. As NF kappa B is down regulated by the 266R protein of Ad12 E1A our results suggest a model in which two Ad12 E1A proteins co-operate in the repression of MHC class I gene expression. PMID- 8622893 TI - A juxtamembrane autophosphorylation site in the Eph family receptor tyrosine kinase, Sek, mediates high affinity interaction with p59fyn. AB - The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signalling pathways utilised by this family are poorly characterised. We have now identified two in vitro autophosphorylation sites within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies defined Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, whilst a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylated exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis defined juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, whilst Y596 was phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis revealed a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signalling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signalling through fyn, or fyn-related, tyrosine kinases may be utilised by many members of this large subclass of transmembrane receptors. PMID- 8622894 TI - Novel AP-1 binding site created by DNA-methylation. AB - DNA-methylation is known to repress transcription either by inactivation of positive regulatory cis-elements containing CpG dinucleotides or via the sequence nonspecific and methylation-specific binding of inhibiting methyl-CpG dinucleotides or via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG binding protein 1 (MeCP1). In the present work we describe the novel way DNA-methylation can influence gene expression: a binding site for transcription factors AP-1 might be created by DNA-methylation. Such a DNA methylation-dependent AP-1 binding site was found in the first intron of the metastasis-associated mts1 gene. The expression level of this gene correlates with the hypomethylation of the mts1 first intron sequence in mouse adenocarcinoma cells. The DNA - methylation-dependent AP-1 binding site was found to be functionally active in the nucleotide context of the mts1 gene. When methylated, this site reproducibly repressed transcription of CAT-containing DNA that had been transiently transfected into mouse adenocarcinoma CSML100 cells. PMID- 8622895 TI - Expression of cyclin D1 and EMS1 in bladder tumours; relationship with chromosome 11q13 amplification. AB - 11q13 amplifications have been found in several cancers, including bladder tumours. However, the biological significance of this genetic alteration is not yet fully understood. To get more insight into the role of 11q13 amplification in bladder tumour development, we have studied the level of amplification and expression of 4 (protoonco)genes lying within the amplicon; cyclin D1, FGF3, FGF4 and EMS1 DNA amplification was found in 5/46 tumours. There was no correlation between amplification and clinico-pathological data. No expression of FGF3 and FGF4 was detected whereas both cyclin D1 and EMS1 were expressed at higher level in tumours with amplifications. Thus cyclin D1 and EMS1, but not FGF3 and FGF4, are likely to play a pathogenic role in the 11q13 amplification in bladder cancer. However, amplification is not the unique way of activation of these genes. Indeed, in situ hybridisation and Northern blot analysis have shown that most bladder tumours have a fair to high expression of cyclin D1 and EMS1 in contrast to normal urothelium with a moderate expression. Interestingly, a trend towards higher expression occurs in superficial versus invasive tumours (8.8 +/- 2.0 versus 1.9 +/- 0.4; P approximately equal to 13% for cyclin D1 and 4.5 +/- 1.4 versus 2.0 +/- 0.4; P approximately equal to 8% for EMS1). Moreover, the 9 tumours with low expression are all highly malignant, leading to the hypothesis that the tumours developing through a cyclin D1/EMS1 independent pathway are more aggressive. PMID- 8622896 TI - Abrogation of wild-type p53 mediated growth-inhibition by nuclear exclusion. AB - We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53val135 and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition. When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53 specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins. Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53val135, there occurring without specific selection. Also in these cells nuclear exclusion of the tsp53 correlated with loss of p53 mediated growth inhibition. Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53. PMID- 8622897 TI - Evidence for genomic instability in human colonic aberrant crypt foci. AB - Aberrant crypt foci (ACF) are morphologically abnormal structures that can be identified in whole amounts of colonic tissue from rodents treated with colon carcinogens and from patients at risk for development of colon tumors. ACF are heterogeneous and exhibit properties, such as altered patterns of cell proliferation, the presence of dysplasia, and mutations in protooncogenes and tumor formation. In this study, we have investigated the presence of genomic instability in DNA isolated from human ACF from patients with colon cancer. Altered allele length detected by electrophoretic separation of PCR amplified oligo A or microsatellite loci was used to identify candidate samples which were then more rigorously investigated by sequence analysis for instability. Of 20 patients examined, two exhibited alterations at two loci, and this instability could be confirmed by sequence analysis. An additional seven of the 20 patients had evidence for instability at a single locus. Quantitative sequence analysis of the DNA from an ACF of one of these seven patients was consistent with alteration of allele length in this patient, but the alteration was not sufficiently different from normal to reach statistical significance. Thus, genomic instability, manifest as altered length of microsatellite and oligo A sequences, in present in some ACF, and therefore can be a very early event in the development of some human colon cancers. PMID- 8622898 TI - Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. AB - Human mammary epithelial cells (HMEC) isolated from reduction mammoplasty tissue are proliferative for several passages but then enter a period termed ?selection' or M0 during which the majority of the cells become larger, flattened, and less proliferative. Early passage HMEC (prior to M0) were transduced with human papillomavirus type-16 E6, E7, or E6/E7 by using recombinant retroviral vectors. E7 alone or E6/E7, but not E6 alone, alleviated the M0 proliferation block, suggesting a possible role for Rb or Rb-related proteins, but not p53, in M0. In addition, cells in M0 did not have increased levels of p53 or p21 proteins, further indicating that induction of these proteins is not required for the M0 proliferation block. Early passage cells contained Rb which was primarily hyperphosphorylated while cells in M0 contained Rb protein which was predominantly underphosphorylated. Cells in M0 accumulated in G1 or B0 and expressed reduced levels of cyclin A and CDK2 proteins. PMID- 8622899 TI - NDF/heregulin induces persistence of terminal end buds and adenocarcinomas in the mammary glands of transgenic mice. AB - Neu differentiation factor (NDF), a member of the neuregulin family of ligands of erbB receptors, induces both differentiative and mitogenic effects on cultured human mammary epithelial cells. Since members of the epidermal growth factor receptor family, including Neu/erbB2, have been implicated in mammary carcinoma, we wished to know whether a potential ligand of this family, NDF, could induce such effects in the mammary gland in vivo. We therefore targeted expression of NDF to the mammary gland of transgenic mice using the mouse mammary tumor virus (MMTV) promoter in a fusion construct. There was a clear, but subtle effect on development of the adult virgin gland of female transgenic animals. Terminal end bud structures (TEBs), which normally disappear from the mammary gland at the age of approximately 8 weeks in wild type mice, persist in glands of virgin MMTV-NDF transgenic females, suggesting that NDF inhibits signals that normally lead to the terminal differentiation of these structures. Further, female mice, bred continuously to maximize expression of the transgene in the mammary gland, develop mammary adenocarcinomas at a median age of 12 months. Since these tumors arise in a solitary fashion, we infer that NDF is necessary, but not sufficient for their formation. In order to explore the signal transduction pathways potentially activated by NDF, we examined expression of the receptors erbB2, erbB3 and erbB4 in mammary epithelial cells established from an NDF-induced tumor. All three receptors were expressed, though only the erbB3 receptor was phosphorylated, suggesting that overexpression of NDF might operate through this receptor. Additionally, about 50% of MMTV-NDF transgenic mice developed Harderian (lachrymal) gland hyperplasia, a benign tumor that does not progress to frank malignancy. PMID- 8622900 TI - Zinc finger protein GFI-1 cooperates with myc and pim-1 in T-cell lymphomagenesis by reducing the requirements for IL-2. AB - The clonality of lymphomas that originate in myc/pim-1 bitransgenic mice due to synergistic action of both oncogenes indicates the requirement of additional events for progression to full malignancy. To isolate genes that cooperate with both myc and pim-1, we have used provirus tagging with E mu L-myc/pim-1 double transgenic mice. We find accelerated tumour formation in infected animals and show that the gfi-1 gene and neighbouring loci on mouse chromosome 5 are occupied by proviruses in about 53% of the tumours leading in all cases to high level gfi 1 expression. In agreement with data from Gilks et al. (1993) we find that forced expression of the gfi-1 encoded zinc finger protein in IL-2 dependent T-cells provokes increased survival upon IL-2 depletion and we present evidence that this occurs at least in part through stimulation of proliferation. Our data suggest that gfi-1 is a proto-oncogene cooperation with both myc and pim-1 genes in T cell lymphomagenesis. PMID- 8622901 TI - Detailed physical and deletion mapping of 8p with isolation of YAC clones from tumour suppressor loci involved in colorectal cancer. AB - Loss of heterozygosity (LOH) of markers at chromosome 8p is frequently noted in many different tumour types, including colorectal cancer. Numerous investigations indicate the presence of more than tumour suppressor gene (TSG) located on 8p. In this study, we describe a detailed LOH map in colorectal cancer and relate this to physical mapping data from reduced radiation 8p hybrids, yeast artificial chromosome (YAC) co-localisation of markers and fluorescence in situ hybridisation data. These data indicate the presence of two regions harbouring putative TSG's between the polymorphic markers for the LPL gene-D8S298 (approximately 4 Mb) and the markers D8S136-D8S137 (approximately 8 Mb). Yeast Artificial Chromosomes (YAC) have been isolated from these regions of interest to aid the localisation of the putative TSG's. PMID- 8622902 TI - Retinoblastoma protein inhibits IFN-gamma induced apoptosis. AB - Regulation of apoptosis (programmed cell death) is critical for maintaining tissue homeostasis. Recent studies indicate a tight coupling between cellular proliferation and apoptosis as cell cycle regulators such as Cyclin D, E1A and E7 appear to influence both events. Each of these modulators is able to bind to and inhibit the function of the retinoblastoma tumor suppressor protein (RB). RB functions, in part, by binding to and inactivating E2F transcription factors, preventing expression of E2F-activated genes associated with G1/S cell-cycle progression. Loss of functional RB deregulates E2F activity and, depending on cell type and environmental factors, promotes tumorigenesis or apoptotic death. To determine the effect of RB on IFN-gamma induced apoptosis, we treated RB defective carcinoma cell lines and their respective RB-constituted sister clones with IFN-gamma and examined the cells for alterations characteristic of apoptosis. We observed that RB-defective cells, but not the RB-reconstituted clones, decreased in size following IFN-gamma treatment. IFN-gamma treatment caused increased cell detachment in the RB-defective lines but did not affect adherence of the RB-reconstituted clones. Assays for DNA fragmentation revealed lower molecular weight DNA and the apoptosis-associated oligo-nucleosomal ladder following IFN-gamma treatment of the RB-defective cells while higher molecular weight DNA was present in the IFN-gamma treated, RB-reconstituted lines. IFN gamma-induced apoptosis in RB-defective cells was enhanced by serum stimulation, which is also characteristic of p53-dependent E2F-1-mediated apoptosis. However, IFN-gamma induced apoptosis in RB-defective lines does not require wild-type p53 suggesting that, upon IFN-gamma induction, deregulated E2F-mediated apoptosis can also proceed via p53-independent pathways. PMID- 8622903 TI - Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice. AB - Gene rearrangements activating the RET proto-oncogene are frequently associated with human thyroid carcinomas belonging to the papillary subtype. These arrangements cause the fusion of the tyrosine-kinase domain of RET to the 5' terminal region of different genes creating the RET/PTC chimeric oncogenes. Here we report the generation of transgenic mice lines expressing the RET/PTC1 oncogene under the control of the thyroid-specific rat thyroglobulin promoter. RET/PTC1-transgenic mice developed thyroid tumors displaying the histological aspect of papillary carcinomas. These tumors were slowly progressive and did not cause premature death of the animals. Two additional mice developed areas of thyroid hyperplasia. Immunohistochemical and reverse-transcriptase polymerase chain reaction analyses confirmed the thyroid-specific expression of the transgene. Given the frequency of activating rearrangements of RET in human papillary thyroid carcinomas we conclude that this animal system could be a good model for studying the neoplastic progression of thyroid carcinomas. PMID- 8622904 TI - Expression of the D-MEF2 transcription in the Drosophila brain suggests a role in neuronal cell differentiation. AB - D-MEF2 is a MADS domain transcription factor expressed in the cardiac, somatic, and visceral muscle cell lineages in the Drosophila embryo. Genetic studies have demonstrated that D-mef2 gene function is required for the proper differentiation of all three of these muscle types. We show that D-MEF2 is also expressed in a limited number of other cells types during development, including Kenyon cells present in the mushroom bodies of the Drosophila brain. This finding suggests a role for D-mef2 in neuron differentiation. To investigate D-mef2 expression in muscle and Kenyon cells, we assayed 26 kb of D-mef2 5'-flanking and intragenic DNA for regulatory sequences controlling the expression of the gene. Our results show that separable enhancer sequences direct D-mef2 gene expression in the myogenic and neuronal cell lineages. The identification of these regulatory DNAs provides a starting point for the analysis of transcriptional regulators controlling the cell-specific expression of D-mef2 and a means to address the function of D-mef2 in Kenyon cell differentiation. PMID- 8622905 TI - Over-expression of fibroblast growth factor-8 in human prostate cancer. PMID- 8622907 TI - Plant genetic engineering may help with environmental cleanup. PMID- 8622906 TI - Facilitating oligonucleotide delivery: helping antisense deliver on its promise. PMID- 8622908 TI - Smaller fleas ... ad infinitum: therapeutic bacteriophage redux. PMID- 8622909 TI - A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. AB - Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo. PMID- 8622910 TI - Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. AB - With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution. PMID- 8622911 TI - Long-circulating bacteriophage as antibacterial agents. AB - The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents. PMID- 8622912 TI - Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production. AB - In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB. PMID- 8622913 TI - Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate. AB - A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2 e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents. PMID- 8622914 TI - Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues. AB - Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21. PMID- 8622915 TI - Adenylyl cyclase inhibition and altered G protein subunit expression and ADP ribosylation patterns in tissues and cells from Gi2 alpha-/-mice. AB - The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1. PMID- 8622916 TI - A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation. AB - Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions. PMID- 8622917 TI - Protein-RNA interactions in the active center of transcription elongation complex. AB - By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment. PMID- 8622918 TI - X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis. AB - The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored. PMID- 8622919 TI - Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane associated motor protein expressed in developing sensory epithelia. AB - The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia. PMID- 8622920 TI - SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis. AB - Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F. PMID- 8622921 TI - The C-terminal region of human angiogenin has a dual role in enzymatic activity. AB - The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln 117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro 123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function. PMID- 8622922 TI - Fos and Jun do not bend the AP-1 recognition site. AB - We have used a solution-based DNA cyclization assay and a gel-phasing method to show that contrary to previous reports [Kerppola, T. K. & Curran, T. (1991) Cell 66, 317-326], basic region leucine zipper proteins Fos and Jun do not significantly bend their AP-1 recognition site. We have constructed two sets of DNA constructs that contain the 7-bp 5'-TGACTCA-3' AP-1 binding site, from either the yeast or the human collagenase gene, which is well separated from and phased by 3-4 helical turns against an A tract-directed bend. The cyclization probabilities of DNAs with altered phasings are not significantly affected by Fos Jun binding. Similarly, Fos-Jun and Jun-Jun bound to differently phased DNA constructs show insignificant variations in gel mobilities. Both these methods independently indicate that Fos and Jun bend their AP-1 target site by <5 degrees, an observation that has important implications in understanding their mechanism of transcriptional regulation. PMID- 8622923 TI - The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication. AB - The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains. PMID- 8622925 TI - Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. AB - In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis. PMID- 8622924 TI - The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons. AB - Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells. PMID- 8622926 TI - Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex. AB - The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system. PMID- 8622928 TI - Chemical egg defense in a green lacewing (Ceraeochrysa smithi) AB - The green lacewing Ceraeochrysa smithi (Neuroptera, Chrysopidae), like other members of its family, lays its eggs on stalks, but it is unusual in that it coats these stalks with droplets of an oily fluid. The liquid consists of a mixture of fatty acids, an ester, and a series of straight-chain aldehydes. Relative to the eggs of a congeneric chrysopid that lacks stalk fluid, the eggs of C. smithi proved well protected against ants. Components of the fluid, in an assay with a cockroach, proved potently irritant. Following emergence from the egg, C. smithi larvae imbibe the stalk fluid, thereby possibly deriving nutritive benefit, defensive advantage, or both. PMID- 8622927 TI - DUB-1, a deubiquitinating enzyme with growth-suppressing activity. AB - Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s). PMID- 8622929 TI - Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees. AB - Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host species shifts for PIV1s. PMID- 8622930 TI - A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin. AB - All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution. PMID- 8622931 TI - 4-Hydroxylation of estrogens as marker of human mammary tumors. AB - Estrogen is a known risk factor in human breast cancer. In rodent models, estradiol has been shown to induce tumors in those tissues in which this hormone is predominantly converted to the catechol metabolite 4-hydroxyestradiol by a specific 4-hydroxylase enzyme, whereas tumors fail to develop in organs in which 2-hydroxylation predominates. We have now found that microsomes prepared from human mammary adenocarcinoma and fibroadenoma predominantly catalyze the metabolic 4-hydroxylation of estradiol (ratios of 4-hydroxyestradiol/2 hydroxyestradiol formation in adenocarcinoma and fibroadenoma, 3.8 and 3.7, respectively). In contrast, microsomes from normal tissue obtained either from breast cancer patients or from reduction mammoplasty operations expressed comparable estradiol 2- and 4-hydroxylase activities (corresponding ratios, 1.3 and 0.7, respectively). An elevated ratio of 4-/2-hydroxyestradiol formation in neoplastic mammary tissue may therefore provide a useful marker of benign or malignant breast tumors and may indicate a mechanistic role of 4-hydroxyestradiol in tumor development. PMID- 8622932 TI - Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals. AB - The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease. PMID- 8622933 TI - Self-organized phase transitions in neural networks as a neural mechanism of information processing. AB - Transitions between dynamically stable activity patterns imposed on an associative neural network are shown to be induced by self-organized infinitesimal changes in synaptic connection strength and to be a kind of phase transition. A key event for the neural process of information processing in a population coding scheme is transition between the activity patterns encoding usual entities. We propose that the infinitesimal and short-term synaptic changes based on the Hebbian learning rule are the driving force for the transition. The phase transition between the following two dynamical stable states is studied in detail, the state where the firing pattern is changed temporally so as to itinerate among several patterns and the state where the firing pattern is fixed to one of several patterns. The phase transition from the pattern itinerant state to a pattern fixed state may be induced by the Hebbian learning process under a weak input relevant to the fixed pattern. The reverse transition may be induced by the Hebbian unlearning process without input. The former transition is considered as recognition of the input stimulus, while the latter is considered as clearing of the used input data to get ready for new input. To ensure that information processing based on the phase transition can be made by the infinitesimal and short-term synaptic changes, it is absolutely necessary that the network always stays near the critical state corresponding to the phase transition point. PMID- 8622934 TI - Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B. AB - The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu (O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB. PMID- 8622935 TI - Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium. AB - We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level. PMID- 8622936 TI - Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies. AB - Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG. PMID- 8622937 TI - Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state. AB - The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg 75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other. PMID- 8622938 TI - Why are some proteins structures so common? AB - Many biological proteins are observed to fold into one of a limited number of structural motifs. By considering the requirements imposed on proteins by their need to fold rapidly, and the ease with which such requirements can be fulfilled as a function of the native structure, we can explain why certain structures are repeatedly observed among proteins with negligible sequence similarity. This work has implications for the understanding of protein sequence structure relationships as well as protein evolution. PMID- 8622939 TI - Ecdysone-inducible gene expression in mammalian cells and transgenic mice. AB - During metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy. PMID- 8622940 TI - Improved adenovirus packaging cell lines to support the growth of replication defective gene-delivery vectors. AB - Adenovirus (Ad) vectors have been extensively used to deliver recombinant genes to a great variety of cell types in vitro and in vivo. Ad-based vectors are available that replace the Ad early region 1 (E1) with recombinant foreign genes. The resultant E1-deleted vectors can then be propagated on 293 cells, a human embryonal kidney cell line that constitutively expresses the E1 genes. Unfortunately, infection of cells and tissues in vivo results in low-level expression of Ad early and late proteins (despite the absence of E1 activity) resulting in immune recognition of virally infected cells. The infected cells are subsequently eliminated, resulting in only a transient expression of foreign genes in vivo. We hypothesize that a second-generation Ad vector with a deletion of viral genes necessary for Ad genome replication should block viral DNA replication and decrease viral protein production, resulting in a diminished immune response and extended duration of foreign gene expression in vivo. As a first step toward the generation of such a modified vector, we report the construction of cell lines that not only express the E1 genes but also constitutively express the Ad serotype 2 140-kDa DNA polymerase protein, one of three virally encoded proteins essential for Ad genome replication. The Ad polymerase-expressing cell lines support the replication and growth of H5ts36, an Ad with a temperature-sensitive mutation of the Ad polymerase protein. These packaging cell lines can be used to prepare Ad vectors deleted for the E1 and polymerase functions, which should facilitate development of viral vectors for gene therapy of human diseases. PMID- 8622941 TI - Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. AB - Complement receptor 1 (CR1, CD35) and complement receptor 2 (CR2, CD21) have been implicated as regulators of B-cell activation. We explored the role of these receptors in the development of humoral immunity by generating CR1- and CR2 deficient mice using gene-targeting techniques. These mice have normal basal levels of IgM and of IgG isotypes. B- and T-cell development are overtly normal. Nevertheless, B-cell responses to low and high doses of a T-cell-dependent antigen are impaired with decreased titers of antigen-specific IgM and IgG isotypes. This defect is not complete because there is still partial activation of B lymphocytes during the primary immune response, with generation of splenic germinal centers and a detectable, although reduced, secondary antibody response. These data suggest that certain T-dependent antigens manifest an absolute dependence on complement receptors for the initiation of a normally robust immune response. PMID- 8622942 TI - N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons. AB - Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression. PMID- 8622943 TI - Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide. AB - To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6 20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles. PMID- 8622944 TI - Transcription termination factor La is also an initiation factor for RNA polymerase III. AB - La RNA-binding protein is a transcription termination factor that facilitates recycling of template and RNA polymerase (pol) 111. Transcription complexes preassembled on immobilized templates were depleted of pol III after a single round of RNA synthesis in the presence of heparin and sarkosyl. The isolated complexes could then be complemented with highly purified pol III and/or recombinant La to test if La is required for transcription reinitiation. VA1, 7SL, and B1 transcription complexes cannot be transcribed by supplemental pol III in single or multiple-round transcription assays unless La is also provided. La mediates concentration-dependent activation of pol III initiation and thereby controls the use of preassembled stable transcription complexes. The initiation factor activity of La augments its termination factor activity to produce a novel mechanism of activated reinitiation. A model in which La serves pol III upon transcription initiation and again at termination is discussed. PMID- 8622945 TI - cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases. AB - By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel. PMID- 8622946 TI - Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment. AB - To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation. PMID- 8622947 TI - Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly. AB - A number of alternatively spliced epsilon transcripts have been detected in IgE producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains. PMID- 8622948 TI - Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor. AB - The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF. PMID- 8622949 TI - Homing events in the gyrA gene of some mycobacteria. AB - The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity. PMID- 8622950 TI - Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase. AB - Two-component signal transduction systems are most often found in prokaryotic organisms where they are responsible for mediating the cellular responses to many environmental stimuli. These systems are composed of an autophosphorylating histidine kinase and a response regulator. We have found evidence for the existence of two-component histidine kinases in the eukaryotic filamentous fungus Neurospora crassa based on screening with degenerate primers to conserved regions of these signaling proteins. Subsequent cloning and sequencing of one member of this newly discovered group, nik-1+, shows that the predicted protein sequence shares homology with both the kinase and response regulator modules of two component signaling proteins. In addition, the N-terminal region of the protein has a novel repeating 90-amino acid motif. Deletion of the nik-1+ gene in N. crassa results in an organism that displays aberrant hyphal structure, which is enhanced under conditions of high osmostress. Increased osmotic pressure during growth on solid medium leads to restricted colonial growth, loss of aerial hyphae formation, and no subsequent conidiophore development. This finding may have implications for mechanisms of fungal colonization and pathogenicity. PMID- 8622951 TI - A complexity measure for selective matching of signals by the brain. AB - We have previously derived a theoretical measure of neural complexity (CN) in an attempt to characterize functional connectivity in the brain. CN measures the amount and heterogeneity of statistical correlations within a neural system in terms of the mutual information between subsets of its units. CN was initially used to characterize the functional connectivity of a neural system isolated from the environment. In the present paper, we introduce a related statistical measure, matching complexity (CM), which reflects the change in CN that occurs after a neural system receives signals from the environment. CM measures how well the ensemble of intrinsic correlations within a neural system fits the statistical structure of the sensory input. We show that CM is low when the intrinsic connectivity of a simulated cortical area is randomly organized. Conversely, CM is high when the intrinsic connectivity is modified so as to differentially amplify those intrinsic correlations that happen to be enhanced by sensory input. When the input is represented by an individual stimulus, a positive value of CM indicates that the limited mutual information between sensory sheets sampling the stimulus and the rest of the brain triggers a large increase in the mutual information between many functionally specialized subsets within the brain. In this way, a complex brain can deal with context and go "beyond the information given." PMID- 8622953 TI - Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity. AB - Mutations in the obese (ob) gene lead to obesity. This gene has been recently cloned, but the factors regulating its expression have not been elucidated. To address the regulation of the ob gene with regard to body weight and nutritional factors, Northern blot analysis was used to assess ob mRNA in adipose tissue from mice [lean, obese due to diet, or genetically (yellow agouti) obese] under different nutritional conditions. ob mRNA was elevated in both forms of obesity, compared to lean controls, correlated with elevations in plasma insulin and body weight, but not plasma glucose. In lean C57BL/6J mice, but not in mice with diet induced obesity, ob mRNA decreased after a 48-hr fast. Similarly, in lean C57BL/6J controls, but not in obese yellow mice, i.p. glucose injection significantly increased ob mRNA. For up to 30 min after glucose injection, ob mRNA in lean mice significantly correlated with plasma glucose, but not with plasma insulin. In a separate study with only lean mice, ob mRNA was inhibited >90% by fasting, and elevated approximately 2-fold 30 min after i.p. injection of either glucose or insulin. These results suggest that in lean animals glucose and insulin enhance ob gene expression. In contrast to our results in lean mice, in obese animals ob mRNA is elevated and relatively insensitive to nutritional state, possibly due to chronic exposure to elevated plasma insulin and/or glucose. PMID- 8622952 TI - Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat. AB - Marked increases in intracellular calcium may play a role in mediating cellular dysfunction and death following central nervous system trauma, in part through the activation of the calcium-dependent neutral protease calpain. In this study, we evaluated the effect of the calpain inhibitor AK295 [Z-Leu-aminobutyric acid CONH(CH2)3-morpholine] on cognitive and motor deficits following lateral fluid percussion brain injury in rats. Before injury, male Sprague-Dawley rats (350-425 g) were trained to perform a beam-walking task and to learn a cognitive test using a Morris water maze paradigm. Animals were subjected to fluid percussion injury (2.2-2.4 atm; 1 atm = 101.3 kPa) and, beginning at 15 min postinjury, received a continuous intraarterial infusion of AK295 (120-140 mg/kg, n = 15) or vehicle (n= 16) for 48 hr. Sham (uninjured) animals received either drug (n = 5) or vehicle (n = 10). Animals were evaluated for neurobehavioral motor function at 48 hr and 7 days postinjury and were tested in the Morris water maze to evaluate memory retention at 7 days postinjury. At 48 hr, both vehicle- and AK295-treated injured animals showed significant neuromotor deficits (P< 0.005). At 7 days, injured animals that received vehicle continued to exhibit significant motor dysfunction (P< 0.01). However, brain-injured, AK295-treated animals showed markedly improved motor scores (P<0.02), which were not significantly different from sham (uninjured) animals. Vehicle-treated, injured animals demonstrated a profound cognitive deficit (P< 0.001), which was significantly attenuated by AK295 treatment (P< 0.05). To our knowledge, this study is the first to use a calpain inhibitor following brain trauma and suggests that calpain plays a role in the posttraumatic events underlying memory and neuromotor dysfunction. PMID- 8622954 TI - Vestibular navigation directed by the slope of terrain. AB - Slope of terrain is an important orienting gradient affecting the goal-directed locomotion of animals. Its significance was assessed in experiment 1 by training rats to find in darkness a feeder on the top of a low cone (80-cm base, 0- to 4 cm high). A computerized infrared tracking system monitoring the rat's position in darkness showed that the path length on the cone surface was inversely proportional to cone height. A device allowing continuous generation of slope guided locomotion was used in experiment 2. This device consists of a 1-m arena, the floor of which can be supported at a point corresponding to the position of one of three equidistant feeders located 17 cm from its center. The arena is inclined by the locomotion of the rat to a plane passing through the elevated (2- or 4-cm) feeder, the rat's center of gravity, and a point at the edge of the arena resting on the floor. The multitude of such planes generated by the rat's locomotion forms the surface of a virtual cone, the top of which is formed by the feeder. Additional path (difference between distance traveled and shortest distance of the animal from the goal at the onset of inclination) is inversely related to the incline of the arena and is a sensitive measure of performance in this type of vestibular navigation. PMID- 8622955 TI - Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. AB - The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells. PMID- 8622956 TI - Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies. AB - The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution. PMID- 8622957 TI - Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay. AB - An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals. PMID- 8622958 TI - Fumonisins and Alternaria alternata lycopersici toxins: sphinganine analog mycotoxins induce apoptosis in monkey kidney cells. AB - Fusarium moniliforme toxins (fumonisins) and Alternaria alternata lycopersici (AAL) toxins are members of a new class of sphinganine analog mycotoxins that occur widely in the food chain. These mycotoxins represent a serious threat to human and animal health, inducing both cell death and neoplastic events in mammals. The mechanisms by which this family of chemical congeners induce changes in cell homeostasis were investigated in African green monkey kidney cells (CV-1) by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. Structurally, these mycotoxins resemble the sphingoid bases, sphingosine and sphinganine, that are reported to play critical roles in cell communication and signal transduction. The addition of fumonisin B1 or AAL toxin, TA, to CV-1 cells induced the stereotypical hallmarks of apoptosis, including the formation of DNA ladders, compaction of nuclear DNA, and the subsequent appearance of apoptotic bodies. Neither mycotoxin induced cell death, DNA ladders, or apoptotic bodies in CV-1 cells expressing simian virus 40 large T antigen (COS-7) at toxin concentrations that readily killed CV-1 cells. Fumonisin B1 induced cell cycle arrest in the G1 phase in CV-1 cells but not in COS-7 cells. AAL toxin TA did not arrest cell cycle progression in either cell line. The induction of apoptosis combined with the widespread presence of these compounds in food crops and animal feed identifies a previously unrecognized health risk to humans and livestock. These molecules also represent a new class of natural toxicants that can be used as model compounds to further characterize the molecular and biochemical pathways leading to apoptosis. PMID- 8622959 TI - Definition of the HLA-A29 peptide ligand motif allows prediction of potential T cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy. AB - The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy. PMID- 8622960 TI - Logarithm of odds (lods) for linkage in complex inheritance. AB - Lod scores provide a method to unify linkage tests based on identity by descent and identity in marker state while permitting selection of the most informative individuals through their disease-related phenotypes and markers in relatives. After parametric lods are reviewed, a nonparametric approach that depends on a single logistic parameter beta is introduced. Lods for parents tested or unknown are derived, multiple pairwise mapping is presented, and power is shown to be good even for moderately small values of beta. Comparison of parametric and nonparametric approaches (yet to be made) will provide for polygenes the efficiency and reliability that lod scores gave to mapping of major loci 40 years ago. PMID- 8622961 TI - Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. AB - A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes. PMID- 8622962 TI - Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors. AB - A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans. PMID- 8622963 TI - Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta glucanase during germination. AB - The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3 1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant. PMID- 8622964 TI - Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide. AB - Pancreatic islet amyloid deposits are a characteristic pathologic feature of non insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia. PMID- 8622965 TI - Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. AB - Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM 1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins. PMID- 8622966 TI - Variant antigens and endothelial receptor adhesion in Plasmodium falciparum. AB - Parasite-derived proteins expressed on the surface of erythrocytes infected with Plasmodium falciparum are important virulence factors, since they mediate binding of infected cells to diverse receptors on vascular endothelium and are targets of a protective immune response. They are difficult to study because they undergo rapid clonal antigenic variation in vitro, which precludes the derivation of phenotypically homogeneous cultures. Here we have utilized sequence-specific proteases to dissect the role of defined antigenic variants in binding to particular receptors. By selection of protease-resistant subpopulations of parasites on defined receptors we (i) confirm the high rate of antigenic variation in vitro; (ii) demonstrate that a single infected erythrocyte can bind to intercellular adhesion molecule 1, CD36, and thrombospondin; (iii) show that binding to intercellular adhesion molecule 1 and CD36 are functions of the variant antigen; and (iv) suggest that binding to thrombospondin may be mediated by other components of the infected erythrocyte surface. PMID- 8622967 TI - Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. AB - The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1 converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype. PMID- 8622968 TI - Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus. AB - High-level globin expression in erythroid precursor cells depends on the integrity of NF-E2 recognition sites, transcription factor AP-1-like protein binding motifs, located in the upstream regulatory regions of the alpha- and beta globin loci. The NF-E2 transcription factor, which recognizes these sites, is a heterodimer consisting of (i) p45 NF-E2 (the larger subunit), a hematopoietic restricted basic leucine zipper protein, and (ii) a widely expressed basic leucine zipper factor, p18 NF-E2, the smaller subunit. p18 NF-E2 protein shares extensive homology with the maf protooncogene family. To determine an in vivo role for p18 NF-E2 protein we disrupted the p18 NF-E2-encoding gene by homologous recombination in murine embryonic stem cells and generated p18 NF-E2-/- mice. These mice are indistinguishable from littermates throughout all phases of development and remain healthy in adulthood. Despite the absence of expressed p18 NF-E2, DNA-binding activity with the properties of the NF-E2 heterodimer is present in fetal liver erythroid cells of p18 NF-E2-/- mice. We speculate that another member of the maf basic leucine zipper family substitutes for the p18 subunit in a complex with p45 NF-E2. Thus, p18 NF-E2 per se appears to be dispensable in vivo. PMID- 8622969 TI - Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation. AB - The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser 208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription. PMID- 8622970 TI - Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. AB - Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV 1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir. PMID- 8622971 TI - cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes. AB - Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I. PMID- 8622972 TI - Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins. AB - The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action. PMID- 8622973 TI - Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. AB - Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells. PMID- 8622974 TI - A functional Rev-erb alpha responsive element located in the human Rev-erb alpha promoter mediates a repressing activity. AB - Rev-erb alpha belongs to the nuclear receptor superfamily, which contains receptors for steroids, thyroid hormones, retinoic acid, and vitamin D, as well as "orphan" receptors. No ligand has been found for Rev-erb alpha to date, making it one of these orphan receptors. Similar to some other orphan receptors, Rev-erb alpha has been shown to bind DNA as a monomer on a specific sequence called a Rev erb alpah responsive element (RevRE), but its transcriptional activity remains unclear. In this paper, we characterize a functional RevRE located in the human Rev-erb alpha promoter itself. We also present evidence that (i) Rev-erb alpha mediates transcriptional repression of its own promoter in vitro, (ii) this repressing effect strictly depends on the binding of Rev-erb alpha to its responsive element and is transferable to a heterologous promoter; and (iii) Rev erb alpha binds to this responsive sequence as a homodimer. PMID- 8622976 TI - Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha. AB - A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV. Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure. Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml. When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml. Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity. Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G - >V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml. These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation. PMID- 8622975 TI - Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins. AB - The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control. PMID- 8622977 TI - Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene. AB - Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer. PMID- 8622979 TI - Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. AB - We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis. PMID- 8622978 TI - Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease. AB - The same heterozygous T -> C transition at nt 8567 of the von Willebrand factor (vWF) transcript was found in two unrelated patients with type III) von Willebrand disease, with no other apparent abnormality. In one family, both alleles were normal in the parents and one sister; thus, the mutation originated de novo in the proposita. The second patient also had asymptomatic parents who, however, were not available for study. The structural consequences of the identified mutation, resulting in the CyS2010 -> Arg substitution, were evaluated by expression of the vWF carboxyl-terminal domain containing residues 1366-2050. Insect cells infected with recombinant baculovirus expressing normal vWF sequence secreted a disulfide linked dimeric molecule with an apparent molecular mass of 150 kDa before reduction, yielding a single band of 80 kDa after disulfide bond reduction. In contrast, cells expressing the mutant fragment secreted a monomeric molecule of apparent molecular mass of 80 kDa, which remained unchanged after reduction. We conclude that CyS2010 is essential for normal dimerization of vWF subunits through disulfide bonding of carboxyl-terminal domains and that a heterozygous mutation in the corresponding codon is responsible for defective multimer formation in type III) von Willebrand disease. PMID- 8622980 TI - Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts. AB - The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5 iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects. PMID- 8622981 TI - Protein-free cell culture on an artificial substrate with covalently immobilized insulin. AB - Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation. In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin. Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin. Finally the immobilized-insulin film was usable repeatedly without marked loss of activity. PMID- 8622982 TI - The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. AB - In late 1994 and early 1995, Ebola (EBO) virus dramatically reemerged in Africa, causing human disease in the Ivory Coast and Zaire. Analysis of the entire glycoprotein genes of these viruses and those of other EBO virus subtypes has shown that the virion glycoprotein (130 kDa) is encoded in two reading frames, which are linked by transcriptional editing. This editing results in the addition of an extra nontemplated adenosine within a run of seven adenosines near the middle of the coding region. The primary gene product is a smaller (50-70 kDa), nonstructural, secreted glycoprotein, which is produced in large amounts and has an unknown function. Phylogenetic analysis indicates that EBO virus subtypes are genetically diverse and that the recent Ivory Coast isolate represents a new (fourth) subtype of EBO virus. In contrast, the EBO virus isolate from the 1995 outbreak in Kikwit, Zaire, is virtually identical to the virus that caused a similar epidemic in Yambuku, Zaire, almost 20 years earlier. This genetic stability may indicate that EBO viruses have coevolved with their natural reservoirs and do not change appreciably in the wild. PMID- 8622983 TI - Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases. AB - In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model. PMID- 8622985 TI - The resource consumption principle: attention and memory in volumes of neural tissue. AB - In the cerebral cortex, the small volume of the extracellular space in relation to the volume enclosed by synapses suggests an important functional role for this relationship. It is well known that there are atoms and molecules in the extracellular space that are absolutely necessary for synapses to function (e.g., calcium). I propose here the hypothesis that the rapid shift of these atoms and molecules from extracellular to intrasynaptic compartments represents the consumption of a shared, limited resource available to local volumes of neural tissue. Such consumption results in a dramatic competition among synapses for resources necessary for their function. In this paper, I explore a theory in which this resource consumption plays a critical role in the way local volumes of neural tissue operate. On short time scales, this principle of resource consumption permits a tissue volume to choose those synapses that function in a particular context and thereby helps to integrate the many neural signals that impinge on a tissue volume at any given moment. On longer time scales, the same principle aids in the stable storage and recall of information. The theory provides one framework for understanding how cerebral cortical tissue volumes integrate, attend to, store, and recall information. In this account, the capacity of neural tissue to attend to stimuli is intimately tied to the way tissue volumes are organized at fine spatial scales. PMID- 8622984 TI - Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes. AB - In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL 7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division. PMID- 8622986 TI - Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein. AB - Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid. PMID- 8622987 TI - Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity. AB - The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments. PMID- 8622988 TI - Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. AB - We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. PMID- 8622989 TI - Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni. AB - Adult Schistosoma mansoni blood flukes reside in the mesenteric veins of their vertebrate hosts, where they absorb immense quantities of glucose through their tegument by facilitated diffusion. Previously, we obtained S. mansoni cDNAs encoding facilitated-diffusion schistosome glucose transporter proteins 1 and 4 (SGTP1 and SGTP4) and localized SGTP1 to the basal membranes of the tegument and the underlying muscle. In this study, we characterize the expression and localization of SGTP4 during the schistosome life cycle. Antibodies specific to SGTP4 appear to stain only the double-bilayer, apical membranes of the adult parasite tegument, revealing an asymmetric distribution relative to the basal transporter SGTP1. On living worms, SGTP4 is available to surface biotinylation, suggesting that it is exposed at the hose-parasite interface. SGTP4 is detected shortly after the transformation of free-living, infectious cercariae into schistosomula and coincides with the appearance of the double membrane. Within 15 min after transformation, anti-SGTP4 staining produces a bright, patchy distribution at the surface of schistosomula, which becomes contiguous over the entire surface of the schistosomula by 24 hr after transformation. SGTP4 is not detected in earlier developmental stages (eggs, sporocysts, and cercariae) that do not possess the specialized double membrane. Thus, SGTP4 appears to be expressed only in the mammalian stages of the parasite's life cycle and specifically localized within the host-interactive, apical membranes of the tegument. PMID- 8622990 TI - Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family. AB - The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond. PMID- 8622991 TI - Human TOP3: a single-copy gene encoding DNA topoisomerase III. AB - A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. PMID- 8622993 TI - Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation. AB - The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene. PMID- 8622992 TI - Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus. AB - Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency. PMID- 8622995 TI - The invariant system of coordinates of antibody molecules: prediction of the "standard" C alpha framework of VL and VH domains. AB - A new approach of comparing protein structures that does not involve the procedure of superposition is suggested. An invariant system of coordinates for immunoglobulin molecules that is based on the geometrical symmetry inherent to the variable domain light-chain (VL)-heavy-chain (VH) complex is described. The coordinates of the Calpha atoms in 22 immunoglobulin structures are calculated in the invariant system of coordinates. We found that 76 identical positions in this Calpha framework are symmetrical about the twofold axis. Comparison of the identical positions in these molecules allows us to select 96 positions in the light chains and 87 positions in the heavy chains whose Calpha atom coordinates are approximately the same. To check whether the average coordinates of Calpha atoms in these positions complies with the stereochemical requirements, we calculated Calpha-Calpha distances. Seventy-three positions of the light chains and 72 positions of the heavy chains satisfy the Calpha-Calpha distance criterion. The Calpha atoms in these positions are used for constructing the "standard" Calpha framework of VL and VH complexes. The average coordinates of Calpha atoms are presented. PMID- 8622994 TI - Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor infiltrating antigen-presenting cells. AB - We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H 2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy. PMID- 8622996 TI - Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions. AB - The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation. PMID- 8622997 TI - P2X4: an ATP-activated ionotropic receptor cloned from rat brain. AB - Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues. PMID- 8622998 TI - Temporal events in cyclopean vision. AB - The majority of neurons in the primary visual cortex of primates can be activated by stimulation of either eye; moreover, the monocular receptive fields of such neurons are located in about the same region of visual space. These well-known facts imply that binocular convergence in visual cortex can explain our cyclopean view of the world. To test the adequacy of this assumption, we examined how human subjects integrate binocular events in time. Light flashes presented synchronously to both eyes were compared to flashes presented alternately (asynchronously) to one eye and then the other. Subjects perceived very-low frequency (2 Hz) asynchronous trains as equivalent to synchronous trains flashed at twice the frequency (the prediction based on binocular convergence). However, at higher frequencies of presentation (4-32 Hz), subjects perceived asynchronous and synchronous trains to be increasingly similar. Indeed, at the flicker-fusion frequency (approximately 50 Hz), the apparent difference between the two conditions was only 2%. We suggest that the explanation of these anomalous findings is that we parse visual input into sequential episodes. PMID- 8622999 TI - The wagon wheel illusion in movies and reality. AB - Wheels turning in the movies or in other forms of stroboscopic presentation often appear to be rotating backward. Remarkably, a similar illusion is also seen in continuous light. The occurrence of this perception in the absence of intermittent illumination suggests that we normally see motion, as in movies, by processing a series of visual episodes. PMID- 8623000 TI - Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. AB - Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value. PMID- 8623002 TI - Refeeding in fasted rats: dietary self-selection according to metabolic status. AB - We investigated the influence of the metabolic status rats have reached during a fast on their selection of protein, fat and carbohydrate when allowed to refeed after fasting for different lengths of time. They were refed either while essentially relying on lipid fuels (group 1), or while in the further stage when there is a rise in protein breakdown (group 2). In contrast to previous studies in which rats could not select macronutrients, there was no transitory anorexia during refeeding. Macronutrient selection as well as food and energy intake on the first day of refeeding were the same for both groups, total food intake being significantly larger than prior to the fast. However, there was thereafter a distinct pattern of diet selection: while fat intake progressively decreased in group 1, it increased until the fifth day in rats of groups 2. These rats, moreover, exhibited a further rise in protein intake. The data are discussed in relation to the optimization of the restoration of body fuel reserves according to the metabolic status reached at the end of a long fast. PMID- 8623001 TI - The use of spreading depression waves for acute and long-term monitoring of the penumbra zone of focal ischemic damage in rats. AB - Slow potential recording was used for long-term monitoring of the penumbra zone surrounding an ischemic region produced by middle cerebral artery (MCA) occlusion in adult hooded rats (n = 32). Four capillary electrodes (El-E4) were chronically implanted at 2-mm intervals from AP -3, L 2 (El) to AP 0, L 5 (E4). Spontaneous or evoked slow potential waves of spreading depression (SD) were recorded during and 4 h after a 1-h MCA occlusion and at 2- to 3-day intervals afterward for 3 weeks. Duration of the initial focal ischemic depolarization was maximal at E4 and decreased with distance from the focus. SD waves in the penumbra zone were high at El and E2, low and prolonged at E3, and almost absent at E4. Amplitude of elicited SD waves was further reduced 3 days later and slowly increased in the following week. Cortical areas displaying marked reduction of SD waves in the first days after MCA occlusion either remained low or showed substantial (60%) recovery, the probability of which decreased with the duration of the initial focal ischemic depolarization and increased with the distance from the focus. It is concluded that the outcome of ischemia monitored by long-term SD recovery in the perifocal region can be partly predicted from the acute signs of MCA occlusion. PMID- 8623003 TI - Influence of local environmental olfactory cues on place learning in rats. AB - The aim of the present study was to assess the influence of local environmental olfactory cues on place learning in rats. We developed a new experimental design allowing the comparison of the use of local olfactory and visual cues in spatial and discrimination learning. We compared the effect of both types of cues on the discrimination of a single food source in an open-field arena. The goal was either in a fixed or in a variable location, and could be indicated by local olfactory and/or visual cues. The local cues enhanced the discrimination of the goal dish, whether it was in a fixed or in a variable location. However, we did not observe any overshadowing of the spatial information by the local olfactory or visual cue. Rats relied primarily on distant visuospatial information to locate the goal, neglecting local information when it was in conflict with the spatial information. PMID- 8623004 TI - Food intake and the menstrual cycle: a retrospective analysis, with implications for appetite research. AB - The biological regulation of appetite is currently an important topic in nutrition, since hyperphagia has been implicated as the prime cause of obesity. Cyclical fluctuations in food intake occur in women across the menstrual cycle, with a periovulatory nadir and a peak in the luteal phase. These alterations in food intake, in response to ovarian steroid hormone changes may be more than 2.5 MJ/day, with the mean reported changes shown in 19 separate studies of 1.0 MJ/day. Hormonal induced fluctuations in food intake could, therefore, contribute to energy imbalance and consequent weight gain. Further, in nutrition studies involving women subjects where the menstrual cycle phase is not controlled, hormonally induced changes in food selection and intake may mask the often considerably smaller changes in response to experimental variables in appetite research. PMID- 8623005 TI - Cardiac and behavioral responses of long-term obese and lean Zucker rats to emotional stress. AB - Obesity is known as a risk factor in stress-related cardiovascular pathology in man. The length of obesity can be an important interacting variable. Therefore, cardiac and behavioral responses to emotional stress were studied in 1-year-old, genetically obese (fa/fa) and lean(Fa/-) male Zucker rats, a frequently used animal of genetic obesity. An early bradycardic response to emotional stress evoked by stimuli associated with brief previous inescapable foot shock, as observed in lean rats, was absent in the fatty Zuckers. This difference was not due to a learning deficit: obese and lean Zuckers showed the same degree of conditioned behavioral responses to the emotional stress. Furthermore, the magnitude of the novelty induced behavioral arousal was also comparable. As far as the regulation of body temperature is concerned, the fa/fa rats displayed a diminished increment in rectal temperature in response to the emotional stress. In conclusion, the results showed an impairment of phasic change in the parasympathetic drive of heart to emotional stress in the long-term obese animals. The diminished activation of heat production points to a blunted response of certain branches of the sympathetic nervous system to emotional stress. The findings favor the hypothesis that dysfunction of cardiac vagal drive in relation to stress is of pathologic importance in long-term obesity. PMID- 8623006 TI - Effects of housing on male and female rats: crowding stresses male but calm females. AB - Housing conditions affect behavioral and biological responses of animals. Effects of same-sex grouped, crowded, or individually housed conditions on plasma corticosterone levels of male and female Wistar rats were examined in two experiments. Experiment 1 examined the effects of individual vs. crowded housing conditions on corticosterone, a biochemical index of stress, in seven male and seven female rats. Experiment 2 extended the findings of Experiment 1 by separately manipulating spatial and population aspects of housing with 50 male and 50 female rats. Male rats had higher corticosterone levels under crowded conditions. In contrast, female rats had higher levels when individually housed. Spatial crowding was the key variable for males, whereas the number of other animals was more important for females. These results indicate that investigators must consider housing conditions as an intervening variable that is likely to differentially affect behaviors of male and female rats. PMID- 8623007 TI - Histamine H3 receptors contribute to drinking elicited by eating in rats. AB - A role for endogenous histamine and its H3 receptor subtype for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats. The i.p. injection of the H3 agonist R-alpha-methylhistamine (Ramh, 2.5 mg/kg) shortened the latency to initiate drinking and increased 1-h water intake in nondeprived rats freely eating pellets and drinking water. The ICV injection (through a surgically implanted chronic cannula) of 10 micrograms Ramh increased water intake; this Ramh-induced drinking was abolished by previous ICV injection of the H3 antagonist thioperamide (Th, 60 micrograms). For rats drinking and eating after 24-h food deprivation, s.c. Th inhibited drinking behavior: for example, 10 mg/kg Th s.c. delayed the latency to initiate drinking and inhibited 1-h water intake without inhibition of food intake. In contrast, 60 micrograms Th ICV failed to inhibit food-related drinking in rats eating after food deprivation. For nondeprived rats eating a small cracker, 10 mg/kg Th s.c. delayed the latency to initiate drinking and abolished water intake without effect of eating, and 60 micrograms Th ICV had similar effects upon drinking elicited by ingestion of cracker. The IG infusion (through a surgically implanted gastric catheter) of 2 ml 600 or 900 mOsm/kg NaCl, a treatment that is subthreshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was abolished by 10 mg/kg Th s.c. and attenuated by 60 micrograms Th ICV. The IG infusion of 2 ml 1800 mOsm/kg NaCl, a treatment that is above threshold for increase in systemic plasma osmolality, elicited drinking that was attenuated by 10 mg/kg s.c. or 60 micrograms Th ICV. These results demonstrate that peripheral and central H3 receptors for histamine have a role in drinking elicited by eating and the postprandial gastrointestinal osmotic consequences of eating. These findings extend the evidence demonstrating a histaminergic contribution to food-related drinking in rats. PMID- 8623009 TI - Effect of hepatic vagotomy on plasma insulin levels during fasting in rats. AB - The present investigation was designed to evaluate the effect of a selective hepatic vagotomy (HV) on the insulin response in rats fasted for 24 h when blood glucose levels were or were not maintained by a constant glucose infusion. Rats were divided into three dietary groups: one group of normally fed rats, one group of 24-h fasted rats, and one group of 24-h fasted rats infused with glucose throughout the fasting period. Each of these groups was subdivided into HV and sham-operated (SHM) rats. Fasting without glucose infusion resulted in a significant (p < 0.05) decrease in plasma glucose, liver glycogen, and insulin concentrations and in a significant (p < 0.01) increase in beta-hydroxybutyrate and FFA concentration. Despite the maintenance of plasma glucose concentrations in the glucose-infused groups, the concentrations of liver glycogen and insulin were still decreased (p < 0.01) and the concentrations of beta-hydroxybutyrate were still increased (p<0.05) at the end of the fasting period. However, no significant differences in insulin or in beta-hydroxybutyrate concentration were found between HV and SHM rats. It is concluded that the decline in plasma glucose concentration during fasting does not totally explain the insulinopenic response to fasting, and that the liver, through the mediation of the hepatic vagus nerve, does not seem to contribute to insulinopenia in 24-h fasted rats. PMID- 8623008 TI - Angiotensin AT1 and AT2 receptors contribute to drinking elicited by eating in rats. AB - A role for endogenous angiotensin II and its AT1 and AT2 receptor subtypes for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats. The ability of pharmacological antagonism of AT1 and/or AT2 receptors to abolish drinking elicited by exogenous angiotensin II was established first. The s.c. injection of the AT1 antagonist losartan (DuP 753) was sufficient to abolish drinking elicited by s.c. angiotensin II. The ICV injection (through a surgically implanted chronic cannula) of losartan inhibited drinking elicited by ICV angiotensin II; the combined ICV injection of losartan plus the AT2 antagonist PD123319 was sufficient to abolish drinking elicited by ICV angiotensin II. For rats drinking and eating after 24-h food deprivation, s.c. losartan plus PD123319 inhibited water to food ratio, but ICV losartan and/or PD123319 failed to inhibit food-related drinking. For nondeprived rats eating a small cracker, s.c. losartan and/or PD123319 attenuated water intake, but only ICV losartan produced statistically significant inhibition of drinking elicited by ingestion of cracker. The IG infusion (through a surgically implanted gastric catheter) of 2 ml 600 or 900 mOsm/kg NaCl, a treatment that is subthreshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was attenuated by s.c. losartan and/or PD123319 and attenuated by ICV losartan only. The IG infusion of 2 ml 1800 mOsm/kg NaCl, a treatment that is above threshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was not inhibited by S or ICV losartan and/or PD123319. These results demonstrate that peripheral AT1 and AT2 and central AT1 receptors for angiotensin II contribute to drinking elicited by eating and the gastrointestinal osmotic consequences of eating. These findings extend the evidence demonstrating a renal renin-angiotensin contribution to food-related drinking in rats. PMID- 8623010 TI - Effects of oral chemical irritation on tastes and flavors in frequent and infrequent users of chili. AB - The studies reported here addressed the question of whether the pungent principle in chilies, capsaicin, suppresses taste and flavor intensity. Over a period of several minutes, groups of frequent and infrequent eaters of chili repeatedly rated the taste and flavor intensities of sweet and sour solutions that also contained either orange or vanilla flavor, and capsaicin at 0, 2, 4, and 16 ppm. As well as the intensity of the qualities while in the mouth, measures of the number of rating periods for the intensity to dissipate to zero, and the summed total intensity were also derived. Infrequent chili users rated the capsaicin burn as more intense than did the frequent users. With few exceptions, and for groups, sweetness was suppressed by the presence of capsaicin. By contrast, sourness was unaffected by capsaicin. Flavor intensities also showed suppression by capsaicin. High correlations between ratings of sweetness and flavor were found, suggesting that perceptual confusion between the two qualities may have been responsible for the flavor suppression. A second experiment examined the effects of capsaicin on ratings of strawberry flavor alone. This study produced little evidence of flavor suppression by capsaicin. These results are discussed in terms of an attentional model of capsaicin's effects. PMID- 8623011 TI - Renal nerve transection inhibits drinking elicited by eating and by intragastric osmotic loads in rats. AB - Adult Sprague-Dawley male rats surgically equipped with a chronic gastric catheter were tested for drinking after 2 ml intragastric infusions of 290, 600, 900, 1200, or 1800 mOsm/kg Nacl. Rats with bilateral transection of renal nerves (RD) drank less than neurologically intact rats (S) after 600 and 900 mOsm/kg NaCl infusions. The RD rats were capable of a rapid and robust drinking response to relatively mild experimental challenges, because they drank appropriately in response to s.c. angiotensin II and to dose of histamine that is a threshold dose for increasing water intake. The RD rats ate less than S rats, but RD rats drank appropriate amounts of water when eating and drinking after 24-h food deprivation. When nondeprived and ingesting one, two, or four small (0.57 g) salted crackers, the RD rats drank significantly less water than S rats. At the time drinking was initiated after ingestion of one salted cracker, plasma osmolality, sodium, protein, and packed cell volume were not changed from baseline conditions (i.e., no eating) in neurologically intact rats; plasma renin activity was significantly elevated at the time drinking was initiated following the ingestion of one or two small crackers. These findings (a) demonstrate a role for renal nerves in drinking behavior in rats, and (b) suggest the working hypothesis that the ingestion of a small meal, and the subsequent delivery of a relatively small osmotic load to the gastrointestinal tract and/or hepatic-portal vein, activates mechanisms for drinking that include a change in activity of renal nerves to increase plasma renin activity without cellular dehydration or hypovolemia. Such mechanisms may be activated to mobilize drinking behavior in advance of postprandial fluid deficit. PMID- 8623012 TI - Endogenous histamine contributes to drinking initiated without postprandial challenges to fluid homeostasis in rats. AB - A role for endogenous histamine and its receptor subtypes for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats in two experimental paradigms in which drinking was initiated without accompanying change in systemic fluid balance: (a) nondeprived rats eating a small (0.57 g) salted cracker; (b) nondeprived rats receiving IG infusion (through a chronic gastric catheter) of 2 ml 900 mOsm/kg NaCl. The ability of i.p., s.c., or ICV (through a surgically implanted chronic cannula in a lateral ventricle) injections of antagonists to H1, H2, or H3 receptors for histamine to inhibit drinking was examined. For rats ingesting a small cracker: systemic injection of H1 antagonists dexbrompheniramine (DXB) or pyrilamine (PYR) delayed the latency to initiate drinking. Systemic injection of H1 antagonists DXB, PYR, or terfenadine (TER; which fails to penetrate the blood-brain barrier) and ICV injection of DXB or PYR inhibited water intake. The H2 antagonist cimetidine (C) given i.p. or ICV failed to affect drinking. The H3 antagonist thioperamide (Th) given s.c. or ICV delayed the latency to drink and inhibited water intake. For rats receiving IG NaCl: systemic injection of H1 antagonists DXB or PYR or the H3 antagonist Th inhibited water intake, but the H2 antagonist C failed to inhibit drinking. Our findings extend the evidence for the involvement of endogenous histamine in drinking elicited by eating in rats by suggesting roles for peripheral and brain H1 and H3 receptors for drinking initiated by postprandial gastrointestinal osmotic consequences that are subthreshold for changes in systemic fluid balance. PMID- 8623013 TI - Rectal adaptation to distention: implications for the determination of perception thresholds. AB - Chronic changes in rectal compliance and perception are often associated with constipation, but the mechanisms responsible for these changes are not known. These studies evaluated the dynamic response of the rectal wall to distention and, in a separate investigation, the influence of adaptive relaxation on perception thresholds. In Study 1, seven healthy volunteers were evaluated using a computer-controlled barostat to maintain continuous isobaric distention of the rectum at urge threshold over a 25-min period. Changes in intrabag volume were evaluated at minutes 1, 5, and 25. Study 2 investigated changes in perception thresholds with different interstimulus intervals (30 s vs. 60 s) in 16 healthy subjects. Pressure was incremented in steps of 2 mmHg up to discomfort threshold. The mean intrabag volume, pressure, and the compliance index for the first and second 5-s intervals were compared to the last 5 s interval. Statistical analyses were performed using the Wilcoxon Sign-Rank test with Bonferonni corrections. Study 1 showed a significant relaxation of the rectal wall in response to balloon distention with volumes consistently increasing from minute 1 to minute 25. Study 2 showed a significant change in the compliance index at the threshold for moderate urge and intense urge during the 60-s distention that resulted from progressive relaxation of the rectal wall. Study 1 showed an adaptive response of the rectum to distention. Study 2 confirmed these findings and implied a role for this adaptive response in the determination of rectal sensory thresholds. PMID- 8623014 TI - Testosterone-induced changes of call structure, midbrain and syrinx anatomy in partridges. AB - Testosterone (T) treated Grey partridges (Perdix perdix) of both sexes uttered significantly longer and lower-pitched calls than controls; both these acoustic features play a critical role in mate choice. A morphometrical analysis of the midbrain nucleus intercollicularis showed a cell size increase in T-treated birds regardless of their sex. Histological study of the syrinx did not reveal any sexually dimorphic structure in experimental and control birds; the major T induced change was a thickening of the external membranes, reported to be the main sound source in Galliforms. In conclusion, T appears able to modify not only some acoustic parameters, but also certain anatomical structures at the peripheral and central levels of the vocal system in a nonoscine species. PMID- 8623015 TI - Fat increases vection-induced nausea independent of changes in gastric emptying. AB - Vection and fat delay gastric emptying and can induce nausea. Therefore, we studied the interaction between vection and fat on the production of nausea and the relationship between the severity of symptoms and changes in gastric emptying. Twelve healthy males consumed two liquid test meals, a zero-fat and a high-fat meal. Nausea was induced by seating the subjects inside a rotating vection drum. In protocol 1, drum rotation started immediately after ingestion of either meal; in protocol 2, when approximately 50% of each meal had emptied into the intestine. Gastric emptying was determined using gamma scintigraphy. Severity of symptoms was assessed throughout. Symptoms were similar for the high-fat and the zero-fat meal when vection was induced immediately after meal ingestion. Scores were only elevated by the high-fat meal when about half the meal had entered the intestine before vection was induced. No correlation was found between gastric emptying and the severity of symptoms. In conclusion, the interaction of vection and intraduodenal fat exacerbates nausea, but this effect is independent of a delay in gastric emptying. PMID- 8623016 TI - Unfreezing the behaviour of two orb spiders. AB - Spider's webs reflect the builders behaviour pattern; yet there are aspects of the construction behaviour that cannot be "read" from the geometry of the finished web alone. Using computerised image analysis we developed an automatic surveillance method to track a spider's path during web-building. Thus we collected data on two orb-weaving spiders--the cribellate Uloborus walckenaerius and the ecribellate Araneus diadematus--for web geometry, movement pattern and time allocation. Representatives of these two species built webs of similar geometry but they used different movement patterns both spatially (which we describe qualitatively) and temporally (which we analyse quantitatively). Most importantly, temporal analysis showed that the two spiders differed significantly in some but not all web-building stages; and from this we deduce that Uloborus- unlike Araneus--was constrained by speed of silk production during the construction of its capture but not its auxiliary spiral. PMID- 8623017 TI - Prefeeding potentiates anorectic actions of neuromedin B and gastrin releasing peptide. AB - We report that the anorectic potency of neuromedin B (NMB) and gastrin-releasing peptide (GRP) is potentiated using a prefeed paradigm. During the light phase, adult male Sprague-Dawley rats (groups of n = 6) were given 30-min (pre-feed) access to a liquid diet. Thirty min later rats were injected i.p. with GRP or NMB (0, 8, 16, 32 or 64 micrograms kg-1) and given further access to the liquid diet. In prefed (PF) rats, high doses of NMB (32 and 64 micrograms kg-1) delayed the onset of eating in the test by > 10 min, significantly suppressing 30-min intake. The same doses of NMB in non prefed (NPF) rats were ineffective. Weak effects of GRP in NPF rats to reduce meal size were also enhanced under PF conditions. Exogenous GRP and NMB, in particular, appear to interact with processes stimulated by the postingestive actions of food, supporting a role for peripheral, endogenous GRP and NMB in the development of satiation and the maintenance of postprandial satiety, respectively. PMID- 8623018 TI - Telemetered recording of blood pressure and heart rate in different strains of rats during chronic social stress. AB - The role of stress in the etiology of high blood pressure and the biological mechanisms involved are still not clear. We have recently developed a paradigm of chronic social stress based on social instability and cohabitation with females, in which the different neuroendocrine responses to stress can be independently triggered. In this work, we used a telemetry technique to record blood pressure and heart rate chronically in freely moving undisturbed rats to study the influence of chronic social stress on blood pressure and heart rate in normotensive rats (Wistar and Long-Evans) and in Borderline Hypertensive Rats (BHR). No increase of blood pressure could be seen for one month of social stress in either strain. Wistar and Long-Evans rats were fully sensitive to social pressure, as shown by the changes in body weight, but may lack a specific vulnerability of the cardiovascular system. Conversely, Borderline Hypertensive rats have the genetic predisposition to develop hypertension but do not appear to be sensitive to social stimulations in the present experimental conditions. The experimental protocol used here should allow further investigation of the various possible sources of failure to induce chronic cardiovascular changes by social stress, such as blood pressure measurement techniques, social stress protocols, and genetic aspects of psychobiological and cardiovascular vulnerability to stress. PMID- 8623019 TI - Radial maze learning using exclusively distant visual cues reveals learners and nonlearners among inbred mouse strains. AB - Spatial working memory on the radial maze was studied in 8 groups of isogenic mice. The device and procedure were specially designed to prevent the mice from using a response strategy or taking advantage of olfactory trails or other proximal cues. The results showed that the strains of mice were clearly split between those which succeeded (C57BL/6, DBA/2, CB6F1, B6D2F1) and those which failed (NZB, CBA, C3H/HE, BALB/c) to learn the task. A second experiment established that when more extended training was given, the four strains which had performed poorly in experiment 1 still did not improve their performance. In the conclusion, we discuss the possible reasons for the deficits of nonlearners and emphasize the importance of using proper tools to ensure the unambiguous assessment of the cognitive processes underlying behavioral adaptation. PMID- 8623020 TI - Amylin decreases meal size in rats. AB - Adult male rats were intraperitoneally (i.p.) injected with 1.0 microgram/kg amylin at the beginning of the dark phase in 24 h food deprived or undeprived rats, and a computerized system measured feeding behavior. In food deprived rats, amylin reduced the size of the first postdeprivation meal without affecting intrameal feeding rate or the size or timing of subsequent meals. The same pattern was observed in undeprived rats, but amylin also increased the latency to the first postinjection meal. In a conditioned taste aversion test, i.p. amylin (1 microgram/kg) injection just prior to rats' first access to a saccharine flavored version of their maintenance diet, failed to affect their subsequent selection of that diet relative to the maintenance diet 2 d later. Finally, 2-min meal-contingent hepatic portal infusions of amylin (1-3.2 microgram/rat) during nocturnal spontaneous meals in undisturbed, ad lib fed rats reduced the meal size and meal duration, and increased the postprandial satiety ratio. Again, feeding rate and the size and duration of subsequent meals were not affected. These results suggest that amylin inhibits feeding by facilitating meal-ending satiety processes. PMID- 8623021 TI - Hormonal imprinting: neonatal treatment of rats with the peroxysome proliferator clofibrate irreversibly affects sexual behaviour. AB - In the rat, the peroxysome proliferator activated receptor (PPAR) inducer clofibrate can moderately influence the hormone (testosterone) level and, after single perinatal treatment, irreversibly affects sexual behavior through the mechanism of hormonal imprinting. The thymic glucocorticoid and estrogen receptors weren't significantly influenced. The experiments call the attention to the universality of false imprinting by molecules able to bind to the steroid/thyroid receptor superfamily, and point to the different sensitivity to different ligands. PMID- 8623022 TI - Behavior of the Roman/Verh high- and low-avoidance rat lines in anxiety tests: relationship with defecation and self-grooming. AB - The Swiss sublines of Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats have been selected and bred for rapid (RHA/Verh) vs. extremely poor (RLA/Verh) acquisition of two-way active avoidance. Behavioral and physiological measures of emotionality, or reactivity to stress, appear to be among the most prominent characteristics differentiating both rat lines. The present study shows that RLA/Verh rats are more sensitive, as compared to their RHA/Verh counterparts, to the conflict involved in the shock-induced suppression of drinking paradigm, as well as in a hyponeophagia test. RLA/Verh rats also showed higher defecation values which were significantly correlated with the main hyponeophagia test variables. Likewise, self-grooming was more frequent in RLA/Verh rats than in their RHA/Verh counterparts and showed significant correlations with conflict related behaviors (i.e., latency to start eating and time spent eating) from the hyponeophagia test. These results give additional support to the contention that RLA/Verh rats present higher anxiety (emotionality) than their RHA/Verh counterparts. PMID- 8623023 TI - Mercaptoacetate-induced feeding is impaired by central nucleus of the amygdala lesions. AB - Systemic administration of mercaptoacetate (MA) blocks beta oxidation of fatty acids and stimulates food intake. The present experiment examined MA-induced feeding in rats with bilateral lesions of the central nucleus of the amygdala (CNA) and sham-operated controls. Food intake was measured for 6 h immediately following i.p. injection of 400, 600, or 800 mumol/kg of MA or saline. Feeding was also measured in these rats in response to 2-deoxy-D-glucose (2DG) (100, 200, and 300 mg/kg, SC), a glucose analogue that competitively inhibits glucose utilization. We found that CNA lesions blocked feeding in response to all three doses of MA. Feeding in response to 2DG was significantly reduced, but not abolished, by the lesion. These findings suggest that the CNA is a crucial component of the neural pathway for feeding in response to MA and may also participate in 2DG-induced feeding. PMID- 8623024 TI - Effects of estrogen and male head coloration on chemosensory investigation of female cloacal pheromones by male broad-headed skinks (Eumeces laticeps). AB - Indirect experimental evidence suggests that pheromone production and responsiveness to pheromones in a lizard, the broad-headed skink (Eumeces laticeps), are regulated by sex steroid hormones. For study of estrogenic effects on female pheromone levels, tongue flicking by males was recorded in response to chemical samples from the female cloaca, the secretion site of the female sex pheromone. Cloacal chemicals from estrogen-treated females elicited higher tongue flick rates by all males than samples from sham-injected females. Male head coloration is bright orange in the breeding season, but fades to tan outside the breeding season. The availability of males having the full range of head coloration due to asynchronous onset of breeding condition in a laboratory population made it possible to examine the relationship between head coloration and responsiveness to female pheromones. Males with brightly colored heads, presumably reflecting higher underlying androgen levels, performed more tongue flicks in response to cloacal chemicals from estrogen-treated females than did males with tan heads. Male head coloration did not affect responsiveness to cloacal chemicals from sham-injected females. PMID- 8623025 TI - Two possible determinants of the timing of daily episodes of behavior in rats. AB - The present experiment examined endogenous ultradian rhythms and regulatory metabolic processes as two potential determinants of daily out-of-nest episodes (ONEs) and feeding episodes (FEs) in rats living in a 24-h environment. Two types of photoperiod were used: a Standard 12:12 h L/D schedule and a Skeleton 12:12 h schedule. During each type of photoperiod rats were exposed to 4 feeding conditions of 7 to 12 days each: (a) a baseline of ad lib food and water; (b) restricted to the diurnal (inactive) portion of the cycle; (c) both food and water restricted to the inactive period; and (d) return to baseline. Time series analyses of nocturnal ONEs in baseline revealed a strong circadian rhythm and weaker ultradian rhythms with periods between 2 and 6 h. Analyses of FEs, though, revealed a general absence of circadian rhythms but strong ultradian rhythms with periods similar to those observed in ONEs. When food and water were restricted to the inactive part of the cycle, ONEs showed no change in frequency, but a decrease in average duration and changes in ultradian periodicities. The results indicated control of daily episodes of behavior in rats by ultradian oscillators that are weaker and more variable in affect than those found in voles, but similar in period. PMID- 8623026 TI - Discriminative cues produced by NPY do not generalize to the interoceptive cues produced by food deprivation. AB - Exogenous administration of neuropeptide Y (NPY) into the third ventricle causes a rapid and robust feeding response in sated rats. The current experiment was designed to assess whether the interoceptive cues produced by exogenous NPY administration generalize to those produced by another orexigenic treatment: 24-h food deprivation. Rats were placed in a chamber after receiving either NPY or saline infused into the third ventricle for a total of six sessions (three after NPY and three after saline). Half of the rats were given a brief foot shock after NPY and not after saline, while the other half received the opposite contingency. The pattern of behavioral immobility for the two groups differed with the rats "freezing" significantly more to the treatment (NPY or saline) paired with shock. Then rats were tested for generalization by placing them in the chamber either after 0- or 24-h food deprivation with no NPY or saline administration or any foot shock. Behavioral immobility during the generalization test of rats that had been trained with shock following saline was greater than that for rats that had been trained with shock following NPY, independent of whether testing occurred when the rats were 0- or 24-h food deprived. This outcome indicates that interoceptive stimuli produced by NPY are no more similar to internal cues produced by 24-h food deprivation than to the stimulus consequences of ad lib feeding. These and other recent findings suggest that food deprivation activates processes or mechanisms different from those that underlie the orexigenic effects of NPY. PMID- 8623027 TI - Food restriction, gonadotropins, and behavior in the lactating rat. AB - This study sought to quantify effects of undernutrition on behaviors and to relate these to gonadotropin and prolactin concentrations in the lactating dam. Dams were studied in a 2 x 3 factorial design with litter size and food intake as the two factors. Behavioral data were collected from each dam and her litter on day 9, day 14, and day 19 of lactation, and maternal blood samples collected. Plasma was analyzed for luteinizing hormone, follicle stimulating hormone and prolactin. On day 15 of lactation, percent time nursing, number of pups actively nursing, total number of pups nursing and dam location acted as mediating factors of the effect of diet group on plasma luteinizing hormone concentration. No such relationships were seen for plasma follicle stimulating hormone, and only nest condition score appeared to be a mediator for plasma prolactin concentration. In conclusion, this analysis suggests that food restriction indirectly influences plasma concentration of luteinizing hormone, but not follicle stimulating hormone, by changing maternal and pup behaviors. The relationship among diet, behavior and circulating prolactin was less clear. PMID- 8623028 TI - Performance on a smell screening test (the MODSIT): a study of 510 predominantly illiterate Chinese subjects. AB - The 12-item Modular Smell Identification Test (MODSIT) was administered to 239 male and 271 female Chinese subjects whose age ranged from 50 to 92 years (68.3 +/- 10.9) and whose education ranged from 0 to 20 years (2.5 +/- 4.3). Every participant was examined by a physician and was found to be free of dementia, stroke, and Parkinson's disease. Different from the standard procedures, only one third of each odor pad was used for each subject, the four odor choices were presented orally for the majority of subjects, and they were not forced to make a selection when they could not detect or identify the odor. The average level of performance was 46% correct. The score was negatively associated with age, positively associated with education and with performance on a dementia screening test, and corroborated with subjects' report of smell deterioration in recent years. Nonsmokers and women performed better than smokers and men. The 12-item MODSIT had an internal consistency reliability of 0.73 and a 7-month retest stability of 0.57 with different examiners. The MODSIT is satisfactory for group studies, even when administered with suboptimal procedures such as those used in the present study. PMID- 8623029 TI - Neonatal testosterone and handedness in yearling rhesus monkeys (Macaca mulatta). AB - This study investigated the relationship between neonatal testosterone (T) and hand bias in young rhesus monkeys (Macaca mulatta). Subjects (n = 8 per group) included: neonatally androgen-suppressed males, using a Nal-Lys gonadotropin releasing hormone (GnRH) antagonist (Antide); androgen-suppressed males receiving T replacement by a long-acting T preparation (CDB); control males; and control females. Antide suppressed T to the female range, whereas CDB replacement produced supranormal levels. Visually guided reaching, in a social context, showed a population-level left-hand bias. Males with elevated T did not show a stronger left-hand bias than males with normal T, but did show a stronger bias for the preferred hand whether left or right. Males with Antide-suppressed T showed an intermediate degree of hand bias. Results suggest that high neonatal T levels affect laterality and raise the possibility that GnRH analogues influence brain development. These data suggest a broad influence of the CNS-pituitary testicular axis on brain asymmetries and provide support for an early neonatal period of T-influenced brain differentiation. PMID- 8623030 TI - Effects of medial septal lesions on an operant go/no-go delayed response alternation task in rats. AB - Previous work in our laboratory has found that whereas medial septal lesions impaired an operant left-right delayed alternation task in rats, the lesion also facilitated the performance of rats on a cued go/no-go discrimination task with a delay between the cue and the required response. These findings suggested to us that the medial septal lesions impaired "response" working memory, which in turn led to a compensatory enhancement of attention to stimulus cues. If this hypothesis is true, then the lesions should impair a go/no-go task based on "response" working memory. The current experiment tested this hypothesis. Rats (12 with medial septal lesions and 12 with sham operations) were tested on a discrete trial operant go/no-go response alternation task. The rats were first tested for 20 days without a delay contingency, followed by 35 days of testing with a 15-s delay between "go" and "no-go" trials. Both groups became proficient at the task under nondelay conditions and their terminal performance (averaging about 85% correct) did not differ. However, under delay conditions the performance of the lesioned rats was significantly impaired compared to the controls. As the go/no-go task does not require a spatial discrimination, the best explanation for our findings is that the lesions impaired response working memory. PMID- 8623031 TI - Heart rate variability during sleep in Down's syndrome. AB - We observed heart rate (HR) variability in the sleep-wake cycle of young adults with Down's syndrome (DS) and their age-matched normal controls. Spontaneous HR fluctuations for 10 min during each sleep stage were analyzed by spectral analysis. Only during rapid eye movement (REM) sleep, a middle-frequency (0.05 0.15 cycles/beat) component of the HR fluctuations jointly mediated by the sympathetic and parasympathetic nerves, was significantly lower in the DS than in the control subjects. As the high-frequency (0.15-0.40 cycles/beat) component mediated by the parasympathetic nerve was not different in the two groups, it is suggested that the sympathetic activity during REM sleep is lower in DS than in controls. PMID- 8623032 TI - Effect of sucrose and fructose macronutrient diets on feeding behavior of rats. AB - Different carbohydrate sources in animal diets can affect feeding behavior. The absence of a diet standard, thus, has the potential to introduce a confounding factor into experiments. The main objective of this study, therefore, was to determine if the choice of either sucrose or fructose as the pure carbohydrate in a carbohydrate diet ration would affect feeding behavior in rats. It was found that during the light and dark phases: 1) fructose-fed rats selected significantly less energy from carbohydrate than sucrose fed rats, 2) fructose fed rats selected more protein and lipid energy than sucrose fed rats, and 3) the total caloric intakes of the two groups were not significantly different. Differing postingestive effects of sucrose and fructose with subsequent compensatory intake may explain these results. Two different carbohydrate sources resulted in different macronutrient selection patterns, thus demonstrating the importance of the nature of dietary carbohydrate in the regulation of feeding behavior in rats. PMID- 8623033 TI - Effects of a cold-water stressor on psychomotor and cognitive functioning in humans. AB - The effects of an acute stressful and painful stimulus, cold water, on psychomotor and cognitive functioning, was assessed in 14 healthy volunteers. Subjects immersed their forearm in ice-cold water (2-3 degrees C) and luke-warm water (37 degrees C) for 3 min, and during this time period a psychomotor or cognitive test was performed. These immersions were done over the course of two experimental sessions, spaced at least 2 days apart, with six trials in each session. Within each session, cold and warm water immersions alternated. Results indicated that flicker-from-fusion threshold from the critical flicker frequency test was higher in the cold-water condition than in the luke-warm-water condition, indicative of increased alertness from the cold stimulus. Short-term memory was attenuated, however, in the cold-water condition. Performance on other tests including those that required speed and/or concentration were not affected by the manipulation. Subjects rated the cold-water stimulus as painful and bothersome, and their blood pressure was significantly elevated by the stimulus. We conclude that a painful stimulus may affect psychomotor and/or cognitive functioning, but the relationship is somewhat complex and depends on the particular tests used. PMID- 8623034 TI - Phase-shifting human circadian rhythms with exercise during the night shift. AB - Appropriately timed exercise can phase shift the circadian rhythms of rodents. The purpose of this study was to determine whether exercise during the night shift could phase delay the temperature rhythm of humans to align with a daytime sleep schedule. Exercise subjects (N = 8) rode a stationary cycle ergometer for 15 min every h during the first 3 of 8 consecutive night shifts, whereas control subjects (N = 8) remained sedentary. All subjects wore dark welder's goggles when outside after the night shift until bedtime, and then slept in dark bedrooms. Sleep was delayed 9 h from baseline. Rectal temperature was continuously measured. There were fewer evening-types and more morning-types in the exercise group than in the control group, which should have made phase delay shifts more difficult for the exercise group. Nevertheless, a majority of the exercise subjects (63%) had large temperature rhythm phase delay shifts ( > 6 h in the last 4 days relative to baseline), whereas only 38% of the control subjects had large shifts. An ANCOVA showed that, when morningness-eveningness was accounted for (as the covariate), the exercise group had a significantly larger temperature rhythm phase shift than the control group. As expected, there was a correlation between the temperature rhythm phase shift and morningness-eveningness in the control group, with greater eveningness resulting in larger phase shifts. However, there was no such relationship in the exercise group; exercise facilitated temperature rhythm phase shifts regardless of circadian type. These results suggest that exercise might be used to promote circadian adaptation to night shift work. PMID- 8623035 TI - The extreme regressive reaction of a psychopath. AB - Recent work on the phenomena associated with the concept of psychopathy has made more confident the clinician's identification of the syndrome, and enables a better examination of certain behaviors related to the diagnosis. One of these is an extreme form of regressive reaction often associated with adaptation to a new restrictive environment. In dynamic terms this can be seen as a primitive attempt to seize control of the maternal universe, perhaps re-enacting a successful strategy of the infant past. The case of a convicted offender with an extensive psychiatric history is described, and consideration is given to the management of such a case in the institutional environment. PMID- 8623036 TI - Psychiatrists as treatment team leaders: pitfalls and rewards. AB - The multidisciplinary treatment team has become a conventional component of inpatient psychiatric care delivery. Treatment team dynamics and their implications for the patient, the team members, the organization, and the team leader in particular have been generally understated in the training of psychiatrists, however, as has its value as a model for learning about administration. This article highlights the history and evolution of the multidisciplinary treatment team in psychiatry, the mix of mental health disciplines, philosophies, and roles involved, the characteristics of mental health care professionals, the conflicting manifest and latent work group tasks, and the dynamics and functions of team leadership. While reviewing information on which to base a systematic approach to team leadership, the author advocates for application of universal standards for education in administrative psychiatry including supervised leadership of multidisciplinary treatment teams and discussions of complimentary readings, examples of which are provided. PMID- 8623037 TI - Recurrent self-injurious behavior in forensic patients. AB - A high prevalence of self-injurious behavior has been reported in the forensic psychiatric population and the correctional psychiatric population. Severely and recurrently self-destructive patients pose great therapeutic challenges. The present study examined forensic patients who engaged in multiple acts of self injury while hospitalized and compared them to forensic patients who engaged in a single act of self-injury. The groups did not differ on demographic or diagnostic measures, but the recurringly self-injurious patients were more frequently and more severely aggressive against others (verbally as well as physically), and required longer hospitalization. The results are interpreted to suggest that the high cost of recurring self-injury in human and financial terms may be reduced by a strategy of early and vigorous intervention. PMID- 8623038 TI - Violence and the lax milieu?: preliminary data. AB - Recently, Rosenbaum noted a rise in the number of patient assaults on staff. He attributed this increase to a lax milieu of impaired structures, boundaries, and leadership. This case study reports on a sharp decline in violence on three lax units in one state mental hospital, when new management fielded initiatives to strengthen the structures and boundaries on these three units. The implications of the findings are discussed. PMID- 8623039 TI - Axis II diagnoses and treatment refractoriness in schizophrenia. AB - In a previous medical record review study we identified a subset of chronic schizophrenic patients with comorbid Axis II personality disorder diagnoses, who had significantly longer lengths of inpatient stay as compared to schizophrenic patients without Axis II diagnoses. This study describes a detailed review of a subset of these records. Eighteen records of schizophrenic patients with comorbid Axis II diagnoses were matched with records of patients without comorbid diagnoses. Demographic and premorbid data revealed no differences between the groups, indicating that the Axis II diagnoses were markers for current as opposed to past psychopathology. Negative symptoms were lower in the cohort with comorbid Axis II personality disorder diagnoses on admission only, while affective symptomatology was increased in this group throughout treatment. Ratings of personality pathology showed a trend towards significance, with the comorbid group demonstrating greater amounts of maladaptive personality traits. These data support the notion that personality traits may be independent of Axis I symptomatology in chronic schizophrenia, and can influence the course of treatment. PMID- 8623040 TI - Dose related response to clozapine in a state psychiatric hospital population: a naturalistic study. AB - After noting a striking difference in the dosing practices of two treating psychiatrists, each responsible for the operation of a clozapine unit in a state psychiatric hospital, the authors conducted a retrospective chart review to assess the clinical efficacy of low dose x = 294 mg. per day) versus high dose (x = 525 mg. per day) clozapine treatment for a cohort of 31 inpatients. Levels of psychopathology, behavior, and social functioning were assessed six months pre and during clozapine treatment for 16 patients who received low dose clozapine treatment and 15 patients who received high dose clozapine treatment. Patients on both units demonstrated significant reductions in their levels of psychopathology, improved social functioning and improvement in their behavior following six months clozapine treatment. This naturalistic study suggests that the use of low dose clozapine provides effective treatment for chronic, severely treatment resistant inpatients with schizophrenia or schizo-affective illness, at the same time reducing the potential for significant side effects. PMID- 8623041 TI - Pathological grief: two Victorian case studies. AB - Despite 75 years of investigation, the concept of pathological grief remains tenuous and controversial. The author turns to the stories of two nineteenth century women, one real and the other fictitious to examine the syndrome of grief gone away. He concludes that pathological grief may be best viewed on a continuum of psychopathology, the expression of which depends upon the interaction between the personality of the patient, the nature of the lost relationship, and the circumstances of its loss. PMID- 8623042 TI - [Relationship between cortisol and testosterone during resting conditions, after acute stress and hormone stimulation in steers]. AB - In this study the relationship between peripheral concentrations of cortisol and testosterone was investigated in 7 adult bulls under resting conditions as well as after sexual and hormonal stimulation. The animals were kept under natural photoperiods of 16L:8D and for hormone determinations blood was withdrawn from an indwelling jugular vein catheter every 30 minutes for 24 and 48 h, respectively. Evaluation of the hormonal profiles revealed that both steroids are secreted episodically characterized by irregular, short term fluctuations with high individual variability. A clear circadian rhythm with high levels in the morning and during the day and low concentrations in the evening and during night was observed for cortisol only. Concentrations of both steroidal hormones were not significantly correlated with each other. Intense sexual stimulation (mounting several times with ejaculation) during 30 minutes caused a rapid increase of cortisol without affecting testosterone secretion. After intravenous application of 1500 IU hCG and 0.5 mg GnRH a sharp rise of testosterone concentrations occurred with high values remaining for several hours but leaving the circadian cortisol pattern unchanged. The intravenous injection of 5 mg PGF2 alpha stimulated both the cortisol and testosterone secretion. Results from this investigation show that cortisol and testosterone are secreted episodically without any interrelationship under resting conditions. Sexual stress induced an increase of cortisol which did not inhibit testosterone secretion and high testosterone levels did neither influence the cortisol pattern. The concomitant increase of cortisol and testosterone after PGF2 alpha suggests a central (hypothalamus/hypophysis) action for prostaglandin. PMID- 8623043 TI - [Measurement of stress parameters in farm animals using active telemetry]. AB - We investigated the effect of an acute stressor on body temperature and heart rate in cows. Both parameters were recorded by active telemetry. For the experiments, five cyclic Brown Swiss cows were used, each of them exposed to an acute stressor during estrus and the luteal phase of the cycle. The stressor consisted in restraining the cows in a crush for hoof treatments. During the 2 hour stress period and for additional 6 hours, body temperature and heart rate were measured every 10 minutes. Control animals remained in their accustomed environment during the whole experiment of 8 hours. The course of body temperature and heart rate was clearly influenced by the stressor. While heart rate was maximal already at the beginning of the stress period, the body temperature showed highest values only one hour after stress and then continuously decreased. Changes in body temperature and heart rate under stress differed significantly from the values of control animals. Both parameters are reliable indicators of stress in the bovine species. The transmitters used in this investigation allowed us to register exact data without manipulation of the animal, which could possibly falsify the results. This advantage as well as the functional longevity of the transmitters (ca. 6 months) make active telemetry a useful tool for stress research in farm animals. PMID- 8623044 TI - [Gynecomastia in a goat buck]. AB - A 3-year old horned buck of the Toggenburger breed was referred to our clinic with signs of gynecomastia. Cytogenetic abnormalities could not be detected in the karyogram and compared to control animals the buck had normal plasma concentrations of testosterone and estradiol but not prolactin. Hyperprolactinemia has been considered the cause of gynecomastia leaving the semen quality undisturbed. PMID- 8623045 TI - [Reproductive medicine in transition: new developments in embryo transfer]. AB - Successful application of embryo transfer (ET) has become common practice in cattle, horses, sheep, goats and a variety of other species held in captivity. Yet in cattle only has the technique been established commercially. In 1994 more than 100,000 bovine embryos have been transferred in European countries. Important progress in transvaginal ovum pick up (OPU), in vitro production (IVP) and cryopreservation have further improved the applicability of ET. Direct transfer simplifies the procedure considerably allowing individual transfers and eliminating the need of synchronizing recipients. In Switzerland the organization 'Veterinary Society for Embryo Transfer' (TIGET) has been founded in 1995 to support practitioners performing embryo transfer. PMID- 8623046 TI - [Zoo, domestic and wild animal medicine--new veterinary problems]. PMID- 8623047 TI - [The animal health card for dairy cows--an instrument for veterinary herd health care and quality assurance in milk production]. AB - A chart (62 x 82 cm) for veterinary herd health management based on a hand written card system is presented. The chart serves basic needs especially in small Swiss dairyherds. It is easily understood and immediately installed for the end user, the farmer and veterinarian. Furthermore an EXCEL-based tool is programmed to assist in standard data analyses. This chart meets the needs for documentation in quality assurance programs within dairy production. PMID- 8623049 TI - Hepatobiliary scintigraphy in children. AB - Hepatobiliary scintigraphy using iminodiacetic (IDA) radiopharmaceuticals provides clinically useful information on the function of the biliary tract in a variety of pathological processes in children, including neonatal jaundice, gallbladder dysfunction, trauma, and liver transplantation. Phenobarbital premedication (5 mg/kg per day for a minimum of 5 days in divided doses) is used in infants who are being examined for neonatal jaundice to increase the accuracy of 99mTc-IDA scintigraphy in differentiating extrahepatic biliary atresia from neonatal hepatitis. Biliary atresia can be ruled out in an infant if a patent biliary tree is shown with passage of activity into the bowel. If no radiopharmaceutical is noted in the bowel on imaging up to 24 hours, distinction between severe hepatocellular disease and biliary atresia cannot be made. The literature reports 91% accuracy, 97% sensitivity, and 82% specificity for hepatobiliary imaging in the diagnosis of biliary atresia. The impairment of both intrahepatic and extrahepatic biliary drainage is an important cause of liver disease in cystic fibrosis. Hepatobiliary scintigraphy in cystic fibrosis has shown characteristic patterns of dilatation of mainly the left hepatic duct, narrowing of the distal common bile duct, gallbladder dysfunction, and delayed bowel transit. Cholecystitis in children may be acalculous. Sensitivity and specificity for the scintigraphic diagnosis of acute acalculous cholecystitis is reported to range from 68% to 93% and 38% to 93%, respectively. Cholescintigraphy in a suspected bile leak provides information generally not available with other techniques, except for direct cholangiography. If the amount of intraperitoneal accumulation of the tracer is greater than that entering the gastrointestinal tract, surgery is usually indicated. Hepatobiliary imaging in children who have undergone liver transplantation will assess graft vascularity, parenchymal function, biliary drainage, presence of a leak, and obstruction. PMID- 8623048 TI - Cholecystokinin and morphine pharmacological intervention during 99mTc-HIDA cholescintigraphy: a rational approach. AB - Pharmacological intervention with either cholecystokinin-8 (CCK-8) or morphine during 99mTc- hepatoiminodiacetic acid (HIDA) cholescintigraphy is required primarily for the assessment of the diseases affecting the gallbladder, the common bile duct, or the sphincter of Oddi. For imaging, the patient should be prepared by an overnight fast, or with 4 hours of minimum fast. Pre-emptying with CCK-8 is probably undesirable and should either be avoided or one should wait for at least 4 hours after CCK-8 to begin the 99mTc-HIDA study to achieve higher specificity of the test for acute cholecystitis. When he gallbladder is not observed by 60 mins in a clinical setting of acute cholecystitis, a dose of 0.04 mg/kg of morphine is administered intravenously and imaging continued for an additional 30 mins. Nonvisualization of the gallbladder by 90 mins with morphine in an appropriate clinical setting is diagnostic for acute cholecystitis. When the gallbladder is not observed by 60 min but is seen with morphine administered after 60 mins, a positive diagnosis of abnormal gallbladder function can be made. When the gallbladder is observed in a clinical setting of biliary pain or chronic calculous or acalculous cholecystitis, CCK-8 at a dose rate of 3.3 ng/kg/min is infused intravenously for 3 mins (10 ng/kg/3 min) for the measurement of the ejection fraction. An ejection fraction value of less than 35% is indicative of calculous or acalculous chronic cholecystitis. The gallbladder emptying is directly related to the total number of cholecystokinin receptors in the smooth muscle. The ejection fraction can be controlled to any desired level simply by controlling the dose rate or the duration of infusion of CCK-8. Morphine and other opiate metabolites circulate for many hours in blood and act on the sphincter of Oddi and decrease the gallbladder ejection fraction. Careful drug history, especially that of opiates, is very critical in all subjects with a low ejection fraction before assigning an abnormality to the gallbladder motor function. PMID- 8623050 TI - Scintigraphic evaluation of hepatic mass lesions: emphasis on hemangioma detection. AB - Nuclear medicine imaging techniques continue to play a unique role in the evaluation of hepatic masses. Although many useful radiodiagnostic agents are available, the predominant nuclear medicine study used to evaluate hepatic masses in this decade is technetium-labeled red blood cell "blood-pool" scintigraphy. Hepatic blood-pool scintigraphy is extremely useful for the confirmation or exclusion of benign hepatic hemangiomas. This technique was first described in the 1970s and has vastly improved since that time. This improvement has been caused predominantly by advancements in instrumentation, especially the development of single photon emission computed tomography (SPECT) imaging. However, a perfusion/blood-pool mismatch remains unchanged as the hallmark finding for hepatic hemangiomas. The sensitivity and specificity of blood-pool scanning for the detection of hemangiomas has continued to increase over the years, and has not been equaled or surpassed by other radiographic modalities, with the possible exception of magnetic resonance imaging. Furthermore, blood pool imaging is relatively inexpensive, simple to perform, and highly accurate. When a suspected hepatic hemangioma is confirmed by a positive radionuclide blood pool study, the clinical evaluation of patients with hepatic masses can generally be terminated. PMID- 8623051 TI - Gastrointestinal bleeding and cine-scintigraphy. AB - Two recent advances have significantly improved the ability of labeled red cell imaging to localize and to detect sites of active gastrointestinal bleeding. The first is the use of continuous dynamic imaging. The second is the commercial availability of an in vitro red cell labeling method that significantly improves image quality. The computer equipment required for dynamic imaging is available in most nuclear medicine laboratories. Because cinematic display of the dynamic images increases the accuracy and ease of interpretation it should be considered the method of choice for performing gastrointestinal bleeding studies. High-risk patients can be identified if very active bleeding is noted early. However, a study with negative results, after monitoring the patient over 1 to 2 hours, suggests that conservative medical management may be adequate. Therefore, with proper use scintigraphy can provide both diagnostic and prognostic information to aid in the management of patients with gastrointestinal bleeding. PMID- 8623052 TI - Abdominal abscess detection: gallium, 111In-, and 99mTc-labeled leukocytes, and polyclonal and monoclonal antibodies. AB - Computed tomography (CT) and magnetic resonance imaging (MRI) are excellent modalities for imaging patients with localizing signs of an intra-abdominal abscess. However, radionuclide techniques have an advantage over CT and MRI because they can evaluate the entire body for infection. In addition, radionuclides can noninvasively differentiate infection and inflammation from benign fluid collections. Radiopharmaceuticals available for imaging abdominal infection include gallium-67 citrate, indium-111, and technetium-99m-labeled leukocytes, and radiolabeled whole antibodies. The use of these radiopharmaceuticals for abdominal imaging is dependent on their biodistribution and mechanism of abscess localization. PMID- 8623053 TI - Role of positron emission tomography scanning in evaluating gastrointestinal neoplasms. AB - Positron Emission Tomography (PET) is rapidly evolving into a useful imaging modality for early and accurate detection of malignant tumor sites. Several recent studies have documented improved efficacy of detecting recurrent colorectal and hepatic (primary and metastatic) tumor sites with a sensitivity ranging from 92% to 100% and an accuracy of 90% to 96%. PET-FDG imaging using 2 [18F]-fluoro-2-deoxy-D-glucose has been found to be superior to computed tomography (CT) in detecting recurrent colorectal, hepatic, and abdominopelvic recurrent tumor sites from different primary cancers. PET-FDG imaging can be a cost-effective tool in the screening of patients with an elevated carcinoembryonic antigen and/or equivocal CT findings and suspected colorectal cancer. The role of PET scanning using FDG or C-11-5-HTP or C-11-L-DOPA appears promising in pancreatic carcinoma and functional endocrine tumors. Further studies are being carried out to assess the role of PET scanning in other gastrointestinal cancers. PMID- 8623055 TI - Pancreatic cancer. PMID- 8623054 TI - Extensive stage small cell lung cancer: determining suitability for outpatient management of febrile neutropenia. PMID- 8623056 TI - Surgical management of pancreatic carcinoma. AB - Since 1935, when Whipple originally described the removal of periampullary tumors, both the surgical method and the outcome associated with it have changed extensively. The mortality rates associated with the procedure as performed today have decreased from 20% to less than 5%; the 5-year survival rate after resection of pancreatic cancer has increased from 3.5% to more than 20% in some studies. These improvements are the result of improved perioperative care and the numerous modifications and technical refinements to the procedure itself. The most important determinants of long-term survival, however, relate to the biology of the tumor. Unfortunately, most pancreatic cancers are diagnosed at an advanced stage and are often unresectable. In this setting, any surgical intervention will be purely palliative in nature. Such procedures can reduce the symptoms associated with the disease and can slightly extend survival times. Several nonoperative approaches can also offer significant palliation to patients with inoperable pancreatic cancer. PMID- 8623057 TI - Radiotherapy for carcinoma of the pancreas. AB - Many carcinomas of the pancreas are locally unresectable but have no gross evidence of metastases at diagnosis. Initial experience showed the disease to be relatively unresponsive to radiotherapy. However, results from Duke University and the Mayo Clinic led to the study of the disease by the Gastrointestinal Tumor Study Group (GITSG). The GITSG studies showed that radiotherapy and 5 fluorouracil chemotherapy in a relatively nonaggressive regimen prolonged the survival following apparent curative resection of carcinoma of the pancreas. In addition, for locally unresectable disease, GITSG showed that both chemotherapy and radiotherapy were necessary for the best results. However, even with optimal therapy the median survival in this group of patients is no more than 1 year. Many studies of specialized radiation techniques have also been evaluated. These included preoperative radiotherapy and chemotherapy, use of radiosensitizers, particle irradiation, interstitial irradiation, intraoperative irradiation, and hyperthermia. Results of these studies have not shown major improvement over the GITSG experience. In general, patients with pancreatic cancer who are irradiated appear to do better than those who are not. No other single modality has been more effective. It is therefore hoped that successful chemotherapeutic agents or other modalities can be developed and combined with the limited efficacy of radiotherapy. PMID- 8623058 TI - Chemotherapy of adenocarcinoma of the pancreas. AB - Adenocarcinoma of the pancreas is the fifth leading cause of cancer death in the United States. Although surgery and radiation therapy may improve prognosis in patients with localized and resectable tumors, chemotherapy has produced minimal benefit. Patients with unresectable and/or metastatic pancreatic cancer are most frequently treated with 5-fluorouracil-based regimens which have produced little palliation with no improvement in overall survival. Newer treatment modalities including octreotide, biologic response modifiers, and monoclonal antibodies have been explored and have resulted in some minor responses. Other chemotherapeutic agents such as the taxanes (ie, paclitaxel and docetaxel) have not been fully evaluated. Initial evaluations of paclitaxel and docetaxel have shown less than 20% response rates. Attempts at dose escalation with growth factor support are also being pursued. The most exciting new agent being tested currently in pancreatic cancers is gemcitabine which has produced overall clinical benefits in as many as 25% of cases. PMID- 8623059 TI - Palliative and supportive care of patients with pancreatic cancer. AB - Pancreatic cancer tends to be diagnosed at a relatively late stage of disease and often secondary to significant complaints of pain. In addition there is evidence of higher rates of depressive symptoms at diagnosis in pancreatic cancer than in other forms of cancer. These factors, along with the specific tumor anatomy and pathophysiology of pancreatic cancer make palliative considerations central to the care of patients with the disease. The palliative and supportive approach must first include an aggressive evaluation of pain, mood, and emotional symptoms. Attention should be paid to the specific nature of pain complaints and attempts made to make accurate clinicopathological correlates for the pain. Assessment should be complete and ongoing. Pain treatments include pharmacotherapy, invasive anesthetic and surgical procedures, and supportive attention to side effects and other symptoms of disease and treatment. Depression often appears at higher rates than documented in other cancer patients and can be independent of pain complaints and other symptoms present in the preterminal phases of illness. Depression should be treated with pharmacotherapy and supportive psychotherapy as indicated. Hospice should be considered early on in the treatment relationship and can provide pain and symptom management services as well as play an important role in providing emotional support to the patient and family. Attention to pain, mood, psychological distress, and other quality of life issues can often allow for successful treatment of symptoms and improvement in functioning even in the setting of late stage pancreatic cancer. PMID- 8623060 TI - Epidemiology and risk factors in pancreatic cancer. AB - Pancreatic cancer is one of the most lethal neoplasms. Incidence in the United States has remained fairly stable over the past 25 years, with about 25,000 cases annually. Almost 100% of cases are fatal. Incidence in the developed world parallels that in the United States. Incidence in undeveloped nations is lower but may be underreported. Worldwide incidence is about 185,000 cases per year. There are no striking environmental risk factors, and geographic variation is less than with other gastrointestinal cancers. The most significant risk appears to be cigarette smoking, with a risk ratio of about 2. Alcohol and coffee consumption have been reported as possible risks in some (but not in most) studies. Diet is probably a significant factor, but is difficult to evaluate quantitatively. Other putative associations, including diabetes, probably are unimportant. PMID- 8623061 TI - Familial pancreatic cancer: a review. AB - The cause of pancreatic cancer remains elusive. The most consistently identified epidemiological risk factor is cigarette smoking. Genetic factors are known to play a significant role in perhaps 5% of the total pancreatic cancer burden. Recent discoveries in molecular biology, particularly germline mutations in inherited conditions which feature pancreatic cancer as an integral part of the tumor spectrum such as in adenomatosis polyposis and hereditary nonpolyposis colorectal cancer, provide powerful incentive to search for other "cancer genes" in this heterogeneous disease. Early detection of this dreadful disease is crucial because its mortality rate approximates its incidence; the ability to identify high-risk patients on the basis of genetic analysis would significantly enhance the potential for early diagnosis. This review addresses the genetic epidemiology of pancreatic cancer and updates our views on screening, surgery, chemotherapy, and genetic counseling, all of which must be used to gain value from genetic predictability of risk status. PMID- 8623062 TI - Anatomic relationships of the cervicothoracic junction. AB - STUDY DESIGN: This study analyzed the anatomic relationships between bony structures and soft tissues of the cervicothoracic junction. OBJECTIVES: To provide composite reference data for intrasegmental and intersegmental gradients of anatomic variation within the cervical-thoracic junction. SUMMARY OF BACKGROUND DATA: Because the risk of soft tissue damage during posterior spinal stabilization, an understanding of bony and soft tissue changes in the cervicothoracic junction is necessary. METHODS: Three-hundred-twenty-four cross sectional spinal segments from nine spines were analyzed to characterize cervicothoracic junctional anatomy. RESULTS: There were predictable cranial-to caudal alterations in both bone and soft tissue anatomy of the cervicothoracic junction. Neural and vascular structures directly anterior to the lateral mass or transverse process and lateral to the pedicle tend to decrease in frequency, whereas measured parameters of the vertebrae increase in size from C5-T3, except for pedicle dimensions that tend to increase at the C7-T1 junction. CONCLUSION: The anatomic changes that occur within the cervicothoracic junction are consistent and predictable, and their recognition should lead to a better appreciation of their clinical implications. PMID- 8623063 TI - A biomechanical assessment and model of axial twisting in the thoracolumbar spine. AB - STUDY DESIGN: Measured trunk kinematics, applied moments, and trunk muscle activities were employed in a biomechanical model to determine load experiences by the spine during dynamic torsional exertions. OBJECTIVES: The purpose of this investigation was to examine the influence of dynamic twisting parameters on spinal load. SUMMARY OF BACKGROUND DATA: Axial twisting of the torso has been identified as a significant risk factor for occupationally related low back disorders. However, previous studies have had difficulty describing how twisting is accomplished biomechanically, or how the spine is loaded during twisting motions. METHODS: Electromyograph activity of 10 trunk muscles was monitored while 12 subjects performed twisting exertions under various conditions of force, velocity, position, and direction. An electromyograph-assisted biomechanical model was developed to interpret the effects of those twisting parameters on spine loading. RESULTS: Significant flexion-extension and lateral moments were generated during the twisting exertions. Muscle co-activity associated with twisting exertions was significantly greater than that associated with lifting exertions. Employing electromyograph data to represent muscle co-activity, the model accurately predicted trunk moments and hence was assumed to reasonably reflect spine loading. CONCLUSIONS: Under the conditions tested, the results indicated that relative spinal compression during dynamic twisting exertions was twice that of static exertions. Spine loading also varied as a function of whether the trunk was twisted to the left or right and according to the direction of applied torsion--i.e., clockwise or counterclockwise. The results may help explain, biomechanically, why epidemiologic findings have repeatedly identified twisting as a risk factor for low back disorder. PMID- 8623064 TI - Instability of the lumbar burst fracture and limitations of transpedicular instrumentation. AB - STUDY DESIGN: This study analyzed the changes in the load-displacement behavior of lumbar spine segments caused by burst fractures that were experimentally produced in fresh human cadaveric spines. The effect of three transpedicular surgical constructs on stability was investigated in each specimen. OBJECTIVES: To quantify the loss of mechanical stiffness caused by the injury, and to evaluate the stiffness of three transpedicular surgical constructs. SUMMARY OF BACKGROUND DATA: Although various investigators have studied the biomechanical characteristics of the burst fracture and surgical stabilization techniques, few have reported quantitative data on the three-dimensional biomechanical instability of these fractures. METHODS: Load-displacement data were acquired in flexion, lateral bending, and axial rotation for intact specimens, after the L1 burst fracture was created and after the T12-L2 segments were stabilized using Luque plates, VSP plates, and Isola rods with one transverse connector. RESULTS: Spines with burst fractures showed a bilinear load-displacement behavior with significant instability (loss of stiffness relative to intact) at low loads (up to 3 N.m) in flexion, lateral bending, and axial rotation. The loss of stiffness was greatest in axial rotation over the entire load range (up to 10 N.m). If posterior element injury also was present, a significantly larger loss of stiffness was observed in flexion and axial rotation. The three transpedicular constructs improved the stability of the injured spine beyond that of the intact spine in flexion and lateral bending at low loads. At high loads, they restored the stiffness to intact levels. However, in axial rotation they did not restore the stiffness to pre-injury level, particularly when the posterior column was disrupted. CONCLUSIONS: Reduction of the burst fracture returns the spine to its position of greatest inherent instability, essentially requiring the transpedicular instrumentation to be load bearing. To enhance mechanical stability, it may be necessary to augment the transpedicular construct, particularly when the posterior column is disrupted. PMID- 8623065 TI - Form and function of the musculoskeletal system as revealed by mathematical analysis of the lumbar spine. An essay. AB - Wolff's law describes the mechanical ultrastructure of tissue and indicates a design for minimal stress or minimal energy expenditure. Formation of this theory for the mechanism of the back reveals the critical design of members of this musculoskeletal system, which is comparable with other anthropoids. It also reveals that the pre-adaptations that enable the individual to lift heavy weights are the same adaptations that allow him or her to walk and run. PMID- 8623066 TI - Evaluation of the effectiveness of the Minerva cervicothoracic orthosis. AB - STUDY DESIGN: This study evaluated a lightweight Minerva cervicothoracic orthosis with an occipital flare and forehead strap. OBJECTIVE: The orthosis was evaluated for its ability to immobilize the cervical spine in normal healthy volunteers. SUMMARY OF BACKGROUND DATA: Previous studies have been performed to evaluate cervical orthoses. Exception for the halo brace, none have controlled the upper cervical spine very well. The brace tested in the present report incorporates an occipital flare and forehead strap to better control the upper cervical spine. METHODS: Sixteen healthy male volunteers were evaluated in and out of the orthosis in three planes of motion. Maximal active cervical flexion, extension, and lateral bending were recorded and measured radiographically. Rotation was measured from overhead photographs. RESULTS: In a comparison of the present results with those of similar previous studies, improvement in control of flexion/extension of the upper cervical spine and in control of rotation was found. The occiput to C1 level, however, remained poorly controlled. CONCLUSION: This orthosis provides good control of the cervical spine below C1. PMID- 8623067 TI - Efficacy of multimodality spinal cord monitoring during surgery for neuromuscular scoliosis. AB - STUDY DESIGN: This study determined the relative efficacy of somatosensory-evoked potentials and motor-evoked potentials in monitoring spinal cord function during surgery for patients with idiopathic versus neuromuscular scoliosis. OBJECTIVES: To determine whether patients with idiopathic versus neuromuscular scoliosis demonstrate significantly different somatosensory-evoked potentials and motor evoked potentials recorded during surgery. SUMMARY OF BACKGROUND DATA: Ashkenaze et al (1993) and others have reported that cortical somatosensory-evoked potentials are unreliable when used to monitor spinal cord function in patients with neuromuscular scoliosis. It was recommended that other neurophysiologic tests be used. METHODS: Somatosensory-evoked potentials and motor-evoked potentials were recorded from two groups of patients: those with idiopathic scoliosis and those with neuromuscular scoliosis. Somatosensory-evoked potentials were obtained before and during surgery. Motor-evoked potentials were obtained during surgery. Normal variability, as indicated from idiopathic scoliotic results, was compared with data obtained from patients with neuromuscular scoliosis. Motor-evoked potentials and somatosensory-evoked potentials were obtained sequentially during the duration of surgery. RESULTS: Single-channel cortical somatosensory-evoked potentials demonstrated a 27% positive rate, which was consistent with results (28%) from Ashkenaze et al. The use of multiple recording sites for the somatosensory-evoked potentials and the addition of motor evoked potential procedures indicated that a reliable response could be obtained in more than 96% of the patients. It also was found that cortical somatosensory evoked potentials were more affected by anesthetic agents when recorded from patients with neuromuscular scoliosis compared with patients with idiopathic scoliosis. CONCLUSIONS: Single-channel cortical somatosensory-evoked potentials demonstrated a high level of unreliability, which reduced their clinical effectiveness. However, by using multiple recording sites with the somatosensory evoked potentials and by administering motor-evoked potential procedures, it was possible to monitor spinal cord function in neuromuscular patients and avoid postoperative neurologic deficits. PMID- 8623068 TI - Variance in the measurement of sagittal lumbar spine range of motion among examiners, subjects, and instruments. AB - STUDY DESIGN: Repeated measurements were made of lumbar sagittal range of motion by 14 examiners using three different measurement instruments. OBJECTIVES: To determine the reliability of lumbar range of motion measurements among examiners, subjects, and instruments, and to determine whether variance is due to subject inconsistency, examiner inconsistency, differences between examiners, or differences between instruments. SUMMARY OF BACKGROUND DATA: Measurements of lumbar spine range of motion are widely used in research and clinical applications as well as in disability rating systems for patients with low back pain. METHODS: Fourteen examiners measured the sagittal range of motion. Using three instruments, 18 healthy subjects were measured twice in a randomized sequence with blinded readings when performing full flexion, and partial flexion to a defined midpoint. None of the examiners routinely used the particular instruments in their practices. RESULTS: The mean test-retest reliability was 4.9 degrees. The intraexaminer reliability did not differ significantly among the examiners. Furthermore, there was no systematic difference resulting from instruments or posture condition. However, there was a statistically significant variance among examiners--i.e., a poor interexaminer reliability. CONCLUSION: The most likely explanation for these findings is the variability among examiners in locating bony landmarks. The results indicate that range of motion measurements must be interpreted with caution in clinical, research, and disability applications. Test administrator training may improve results, but this could not be determined from this study. PMID- 8623069 TI - Accuracy of pedicle screw placement in lumbar fusions by plain radiographs and computed tomography. AB - STUDY DESIGN: Postoperative radiographs and computed tomography scans were used to evaluate 74 pedicle screws in 16 consecutive patients who underwent lumbar spine fusion with pedicle screw fixation. OBJECTIVE: To evaluate pedicle screw placement using plain radiographs versus computed tomographic scans. SUMMARY OF BACKGROUND DATA: Plain radiographs are the primary means of assessing pedicle screw placement. Comparison of plain radiographs and computed tomography has not been done. METHODS: Screws were graded as IN, OUT, or QUESTIONABLE; the direction of misplacement was noted. All evaluations were performed independently by three observers. RESULTS: Fewer screws were clearly within the pedicle on computed tomography when compared with plain radiographs. Computed tomography showed 10 times as many screws violating the medial cortex as did radiographs. Interobserver differences were not statistically significant. Intraobserver differences approached statistical significance when the two tests were compared. No recognized neurologic complications resulted from pedicle screw placement. CONCLUSIONS: Plain radiographs alone may not accurately reveal pedicle screw placement. Plain radiographs and thin section computed tomographic scans should be used to evaluate postoperative neurologic deficits in patients undergoing instrumented lumbar spine fusion with pedicle screws. PMID- 8623070 TI - Far lateral disc herniations treated by microscopic fragment excision. Techniques and results. AB - STUDY DESIGN: This was a retrospective review of a consecutive series of patients who underwent excision of far lateral disc herniations by a paraspinal, muscle splitting incision that spares the facet. OBJECTIVE: The authors describe the surgical technique in detail and review the results of surgical treatment. SUMMARY OF BACKGROUND DATA: As the lumbar nerve roots exit the intervertebral foramen, they once again lie in juxtaposition to an intervertebral disc and are susceptible to compression with subsequent radiculopathy. Radiculopathy caused by disc herniations in this area is less common, and the anatomy is less familiar to spinal surgeons than that of paramedian disc herniations. The involved root can be approached by a transfacetal approach or by a muscle-splitting approach that spares the facet. METHODS: Thirty-one consecutive patients had undergone the muscle-splitting approach to far-lateral disc herniations. Twenty-five (80.6%) of these patients were available for evaluation at a minimum of 2 years after surgery. Evaluation consisted of history, pain questionnaires, visual analogue scales, physical examination, and plain radiographs. RESULTS: Twelve patients (48%) had excellent results, eight (32%) had good, and five (20%) had either fair or poor results. Low back pain and dysesthesias were causes of unsatisfactory results. No radiographic sign of instability developed postoperatively. CONCLUSION: The anatomy of the nerve root lateral to the pedicle and our technique for microscopic excision of herniated disc fragments lateral to the facet are described. Our overall results were encouraging. The described microscopic technique will hopefully minimize dysesthetic pain that appears to be the result of the manipulation of the dorsal root ganglion. PMID- 8623071 TI - Ligamentum flavum hematoma presenting as progressive root compression in the lumbar spine. AB - STUDY DESIGN: This is a case report. OBJECTIVES: To provide a better understanding of a rare entity, ligamentum flavum hematoma, based on a successfully treated patient. SUMMARY OF BACKGROUND DATA: Lumbar root compression is frequently caused by disc herniation. Canal stenosis, epidural hematoma, or tumors are less common. Few cases of hematoma in the ligamentum flavum causing lumbar root compression have been described. METHODS: A patient was treated for ligamentum flavum hematoma causing progressive lumbar root symptoms. Clinical and radiologic features, including magnetic resonance images, were recorded. RESULTS: Symptoms consisted of progressive lumbar root deficit after mild physical exertion. On magnetic resonance images, a mass continuous with the ligamentum flavum, compressing the dural sac and roots, was found. Areas of high signal in T1 were present in it. In this patient, removal of the ligamentum flavum (degenerated tissue containing hematoma) resolved all of the symptoms. The diagnosis could have been suspected based on clinical history and magnetic resonance imaging. CONCLUSIONS: Magnetic resonance imaging can provide a basis for diagnosing ligamentum flavum hematoma. Removal of ligamentum flavum is the treatment of choice. PMID- 8623072 TI - Maffucci's syndrome involving hemangioma in the cervical spine. AB - STUDY DESIGN: This is a case report of a patient with Maffucci's syndrome, with hemangioma located in the cervical spine causing paraplegia. OBJECTIVE: To present a rare case of Maffucci's syndrome with hemangioma located in the cervical spine, and to discuss the incidence and treatment of hemangioma located in the central nerve system in Maffucci's syndrome. SUMMARY OF BACKGROUND DATA: Maffucci's syndrome is characterized by enchondromatosis and soft tissue hemangioma. Hemangioma usually is located in the subcutaneous tissue and rarely in the mucosa and visceral organs. There have been no reports in the literature on the incidence of hemangioma in the cervical spine occurring with Maffucci's syndrome. METHODS: The patient was an 11-year-old girl, who complained of muscle weakness of the upper extremity and gait disturbance. After clinical and radiologic evaluation, the patient was diagnosed as having dumbbell tumor of the cervical spine associated with Maffucci's syndrome. RESULTS: The tumor was successfully excised after selective angiography and embolization of the feeding artery from the ascending cervical artery and costocervical trunk, the result being excellent neurologic recovery for the patient. Histologic study showed the tumor was hemangioma similar to the red-blue tissue tumor in the hand. CONCLUSION: The possibility of hemangioma being localized in the spinal column, as well as in the subcutaneous tissue, mucosa, and visceral organ, should be considered in cases of Maffucci's syndrome. PMID- 8623073 TI - Subarachnoid-pleural fistula as a complication of the lateral-extracavitary approach to thoracic intraspinal neurinoma. AB - STUDY DESIGN: This report describes an infrequent but major complication resulting from a lateral extracavitary approach to the spinal cord. The diagnosis was made via myelography-computed tomography. OBJECTIVES: The authors emphasize the importance of a proper approach in diagnosing a subarachnoid-pleural fistula and treating this clinical condition correctly. SUMMARY OF BACKGROUND DATA: Myelography-computed tomography was used to diagnose the subarachnoid-pleural fistula. It was necessary to re-open the thoracotomy to seal the dura mater because the pleuroperitoneal shunting was not effective. METHODS: The patient presented with an intradural and extramedullary thoracic neurinoma located on the anterior part of the spinal canal that was causing anterior spinal cord compression. A lateral extracavitary approach was taken with a thoracotomy, with the tumor being completely removed. During the postoperative period, the patient had a persistent pleural effusion. The diagnosis of a cerebrospinal fluid fistula was made via myelography-computed tomography. Implantation of a pleuroperitoneal shunt was unsuccessful, and it was necessary to re-open the thoracotomy to seal the dura mater. RESULTS: Myelography-computed tomography successfully helped diagnose the subarachnoid-pleural fistula and identify the precise anatomic location of the leakage. Pleuroperitoneal shunting was not effective in dealing with the pleural effusion. CONCLUSIONS: This complication should be taken into account when this kind of surgical approach is performed. Myelography-computed tomography is the most reliable test for diagnosing this clinical condition and pinpointing the exact location of the leakage. PMID- 8623074 TI - Modified anterior approach to the cervicothoracic junction. AB - STUDY DESIGN: This study reports the experience with four patients regarding a modified anterior approach to the cervicothoracic junction. OBJECTIVES: This technique was evaluated with respect to extent of exposure, ease of technique, and postoperative morbidity. SUMMARY OF BACKGROUND DATA: Previously reported anterior approaches to the cervicothoracic junction have described either full sternotomy resection of the left sternoclavicular junction or osteotomy of the clavicle. A simplified approach was chosen using a partial sternotomy, which has not been described previously for approaches to the spine. METHODS: Four patients with metastatic disease, in the region of the cervicothoracic junction, required decompression and stabilization for palliation of symptoms. An anterior approach was required for decompression. A standard cervical approach was combined with a partial median sternotomy and transverse osteotomy through the synostosis between the manubrium and body of the sternum. In three patients, the left innominate vein was divided. Decompression and anterior stabilization were followed by posterior stabilization at an interval of 4 to 7 days. RESULTS: This procedure was simple to perform, requiring little additional operative time for opening or closure. It provided excellent exposure from C3-T4. There was no associated morbidity related to the division of the manubrium or innominate vein. CONCLUSION: Partial sternotomy combined with a standard cervical incision provides excellent exposure to the cervicothoracic junction from C3-T4. It is technically simple to perform and avoids the risk of injury to subclavian vessels inherent in resection of the clavicle or sternoclavicular junction. There is no additional morbidity associated with this approach. PMID- 8623075 TI - Low back pain and illness behavior (inappropriate, maladaptive, or abnormal). AB - Patients with low back pain without a sufficient physical cause attract labels such as "hysteria" and "hypochondriasis," often on the basis of exclusion. The concept of "abnormal illness behavior" was introduced to clarify the classification and diagnosis of these conditions, in which the behavior of the doctor plays a particularly important part. Principles of management are discussed. PMID- 8623076 TI - Description of variations of the sciatica stretch phenomenon. PMID- 8623077 TI - Supervisor personal liability for ADA discrimination. Emerging issues for hospital administrators and physicians. AB - This article discusses emerging and conflicting trends in recent Americans with Disabilities Act discrimination legal decisions in which plaintiffs seek compensatory or punitive damages directly from supervisors and their employers, creating potential issues of personal liability for physicians or hospital administrators, for example. Also addressed in the article are potential problems employers face if supervisors are held personally liable for Americans with Disabilities Act discrimination. PMID- 8623078 TI - Use of Oswestry Disability Index (ODI) PMID- 8623079 TI - [The effect of local control on overall survival after the breast-conserving therapy of breast carcinoma]. AB - BACKGROUND: The impact of local control on survival in breast carcinoma has been controversially discussed depending upon the specific view of disease as being systemic from the initial onset or locally confined with spread sequentially from the primary tumor to the regional lymph nodes and then to distant sites. Results of recent studies provide a good example of why controversy persists over the benefit of local control on survival. PATIENTS AND METHODS. There are 4 recently updated randomised trials that compare conservation surgery alone or with radiation: Milan III (follow-up 3.5 years), Canada (follow-up 5 years), Uppsala/Orebro (follow-up 5 years), and NSABP-B06 (follow-up 10 to 12 years). In addition, the reanalysis of NSABP-B06-protocol reported by the EMMES Corporation has become available. Of further importance is the meta-analysis of Levitt et al. [7], which has used special statistical tools, the BAYESIAN-technique. RESULTS: All 4 trials report statistically significant increased local control and improved survival for the irradiated patients. Based on p-values and confidence intervals, survival was statistically improved in long-term follow-up of the NSABP-B06 trial, but not in the other trials, probably because of small sample sizes and short follow-up. At 10 years, the overall survival rates for the NSABP B06 were 65 +/- 2.1% and 71 +/- 2.0% for lumpectomy alone or with radiation respectively (p = 0.04), the distant-disease-free survival 55 +/- 2.3% vs. 62 +/- 2.2% (p = 0.03), and the disease-free survival 48 +/- 2.2% vs. 56 +/- 2.2% (p < 0.01). Within the Bayesian framework, the expected advantage in 10 year survival was 6% (the mean of NSABP-B06 10 year survival) with an 83% probability that the 10-year-survival difference ranges between 2% and 10%. CONCLUSIONS: The careful analysis of these randomized trials comparing lumpectomy with or without radiation clearly indicate an improvement in overall survival for the irradiated patients which is associated with increased local control. Combination of this survival trend with the reduced psychological and economic costs associated with local recurrence provides excellent arguments for radiation therapy as the standard treatment concept in breast conservation therapy. PMID- 8623080 TI - Results of radiotherapy for 230 patients with stage I-II seminomas. AB - PURPOSE: The outcome of treatment of testicular seminoma in 230 patients with stage I and II disease was retrospectively evaluated in regard of survival, pattern of failure, radiation dose, treatment volume, acute and chronic side effects. PATIENTS AND METHODS: From 1978 to 1992, 230 male patients with the diagnosis of pure seminoma of the testis were treated by postoperative radiotherapy at the University of Essen. According to the Royal Marsden Staging System, 188 patients were presenting with stage I disease, 24 with stage IIA, 13 with stage IIB and 5 with stage IIC disease. All patients received irradiation to the paraaortic lymph nodes (median dose: 36 Gy). In 154 patients the ipsilateral iliac lymph nodes were additionally irradiated with or without inguinal lymph nodes and in 66 patients the contralateral pelvic nodes were included. Since 1987, the total dose was reduced to 26 Gy for microscopic disease. A mediastinal irradiation (median dose: 30 Gy) was performed in 22 patients. Eight patients with stage IIB and IIC disease were additionally treated with chemotherapy. RESULTS: Overall actuarial survival (Kaplan-Meier method) for all patients was 97.8% at 5 years and 96.5% at 10 years. Ten-year survival corrected for intercurrent mortality (n = 8) was 100%. In 5 patients recurrent disease (n = 5) was observed, in 6 patients seminoma occurred in the contralateral testis. For stage I seminoma the disease-free survival was 96.8%. For the whole group of stage II seminoma the DFS was 88.1%, for stage IIA 91.7% and for stage IIB 76.9%. In stage IIC no recurrences occurred. In general, the radiation therapy was well tolerated with minor side effects only. CONCLUSIONS: Postoperative radiotherapy for seminoma stage I, IIA and IIB alone offers excellent control and survival rates with tolerable side effects. PMID- 8623081 TI - [Hormonally inactive hypophyseal adenomas: the results and late sequelae after surgery and radiotherapy]. AB - PURPOSE: External radiation therapy for non functioning pituitary adenomas is clearly indicated in inoperable patients, after incomplete transsphenoidal microsurgery and in case of tumor progression. This retrospective analysis gives results and toxicity following surgery and postoperative radiotherapy of non functioning pituitary adenomas. PATIENTS AND METHODS: Between 1983 and 1990, 50 patients with non-functioning pituitary adenomas received combined treatment at our institutions. Surgical approaches were transsphenoidal (61%), transcranial (24%) or a combination of both (15%) in short intervals. All 50 patients received a full-dose radiotherapy with single fractions between 1.9 and 2.1 Gy (median 1.9 Gy) and total doses between 46 and 63 Gy (median 48 Gy). Field sizes ranged between 20 and 72 cm2 (median 30 cm2). Endpoints of this study were: progression free survival, late effects of brain, optic nerve and intracranial vessels, visual impairment and hypothalamic-pituitary dysfunction. Median follow-up was 54 months. RESULTS: Forty-seven of the 50 patients remained progression-free. Patients over age 60 years progressed more frequently (3/17) as compared to younger patients (0/33, p = 0.034). Following radiotherapy vision improved in 8 patients (16%), 14 patients (28%) experienced a visual impairment between 10% and 60% (according to the grading system of the German Ophthalmologic Society). In multivariate analysis no influence of age (p = 0.097) or biologically effective dose (BED) (p = 0.11) was seen on visual impairment. Three patients (6%) experienced late effects with optic neuropathy in 2 cases and temporal lobe necrosis in 1 case. Only the BED had a significant impact on late effects (p = 0.0094). Adrenal, thyroid and gonadal function abnormalities were present in 58%, 66% and 96% of the patients at last follow-up, respectively. CONCLUSIONS: Radiotherapy after incomplete transsphenoidal or transcranial surgery of non functioning pituitary adenoma is an effective treatment. Future perspectives must include the better defining of high-risk patients and the reduction of late morbidity. PMID- 8623082 TI - [Primary cerebral non-Hodgkin's lymphomas. The results of radiotherapy]. AB - BACKGROUND: In the last years many therapeutic attempts were made to improve the poor prognosis of primary cerebral non Hodgkin's lymphoma. The aim of this study was to report on own experiences concerning this rare disease. PATIENTS AND METHODS: In 1986 to 1994 26 patients were treated for primary cerebral non Hodgkin's lymphoma. 15% were HIV-positive. High-grade non-Hodgkin's lymphomas were diagnosed in 62%. 46% of all cases showed multiple lesions. 23% of our patients received a dose less than 25.5 Gy, 27% received 37.5 Gy (whole brain) and 39% were treated with 51 Gy (37.5 Gy whole brain and 13.5 Gy boost dose). Radiation technique included regular and irregular fields (single dose: 1.5 Gy). RESULTS: 62% of patients achieved a complete or partial remission directly after irradiation, in 4% no change or progression was seen. 31% could not be examined due to their bad status or death. The mean follow-up time for all patients was 19.6 months, the median survival was 3.6 months. The 1-year-survival rate was 41%, 3-year survival rate was 28%. Patients who received a tumor dose of 51 Gy had better survival times in comparison to patients who received less (p = 0.01). Prognostic parameters (Cox regression analysis) were: tumor dose, grading and local result after irradiation. The Karnofsky performance status was not an independent parameter (p = 0.12). Side effects were low. Long-term survivors had relatively good quality of life. CONCLUSION: Taking into consideration the small numbers of patients in all studies all results or conclusions have to be made with great reservations. Despite the high proportion of HIV-positive patients our results are in compliance with other authors using similar therapy protocols. Reports dealing with combined radio-chemotherapy have published good results concerning survival time. But long-term morbidity due to these protocols needs further investigation and has to be awaited. PMID- 8623083 TI - [Radiotherapy in tubal carcinomas]. AB - PURPOSE: Cancer of the fallopian tube is one of the rarest gynecological malignancies. The treatment of choice for this tumor is not well defined because of its very low incidence. The preferred primary treatment is surgical resection consisting of abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy. The value and extent of adjuvant radiotherapy is unclear so far. This retrospective analysis shows the experience at our clinic, which will be discussed in comparison with the existing literature. PATIENTS AND METHODS: From 1967 to 1994, 9 patients were treated at the Clinic and Polyclinic for Radiotherapy--Radio-Oncology of the University of Munster with adjuvant radiotherapy following surgery for carcinoma of the fallopian tube. The staging according to FIGO yielded 2/9 stage I, 4/9 stage II and 3/9 stage III patients. In 5/9 patients the tumor could be resected completely. In 3/9 cases the pelvis alone, in 5/9 cases the pelvis and para-aortal region were irradiated with doses ranging from 45 Gy to 50 Gy. In 1/9 cases the target volume comprised the whole abdomen. The treatment results were acquired retrospectively by analysis of the patients' records and inquiries among their general practitioners. RESULTS: Median survival of our patients was 25 months, 6/9 developed recurrence after a median time of 10.5 months. All of these were localized in the abdominal cavity; 1 patient also developed liver metastases. All patients with recurrent tumor died from their disease within a median period of 9 months. Of the other 3/9 patients, 2/3 died after 6 and 36 months, and 1 patient is still alive after 42 months; all of them without any sign of recurrence. CONCLUSION: Adjuvant radiotherapy of carcinoma of the fallopian tube seems to be indicated with the exception of little invasive tumors of FIGO stage I. From our findings, the target volume should comprise the whole abdomen. A smaller treatment volume (pelvis alone or plus the para-aortal region) can only be of any use in a palliative situation or as a salvage therapy. PMID- 8623084 TI - On the measurement of the effective linear attenuation coefficient mueff in 60Co wedge fields. AB - PURPOSE: It is difficult to obtain a correct measurement of mueff. Moreover, wedge calculations using a single mueff value are not correct. We present a program for the analysis of wedge measurements. MATERIALS AND METHODS: The program employs parameters of an open field to correct the dosimetry of a wedge field. For this the ASCII export files of an MP3 (PTW) system are automatically imported into our program. The principle is illustrated for ionization measurements from a Chisobalt 2B75 cobalt unit. The wedge field dose profiles are corrected by the off-centre ratio of the open field and mueff is analysed as well as its dependents on geometry. RESULTS: We found that the dose attenuation by a wedge can be described by the product of wedge thickness and the effective linear attenuation coefficient mueff. However, mueff values for a given field size decrease with depth. This is interpreted as an effect of beam hardening. For the usual fields in radiotherapy this effect is independent of field size and can be described as an exponential function of wedge thickness. CONCLUSION: The effective linear attenuation coefficient mu(eff) is depth-dependent. A correction could be added to planning programmes, i.e.: exp. (delta mu.tw.[d-dmax]). PMID- 8623085 TI - [Spiral computed tomography for treatment planning and quality assurance in radiotherapy]. AB - BACKGROUND: The possibilities of spiral CT for radiotherapeutic treatment planning and quality assurance have been systematically investigated. PATIENTS AND METHODS: The influence of parameters such as slice thickness, table speed and increment on geometric accuracy was studied. Ring-, spheric-, PMMA- and humanoid Alderson phantoms were used. Furthermore, patients with infradiaphragmatic irradiation of Hodgkin's disease or with mediastinal irradiation were studied using CT-angiography. Patients with carcinomas of the head and neck before HDR- and PDR-brachytherapy were examined as well. RESULTS: Spiral CT offers 3D volume data information with excellent reduction of breath and motion artefacts for virtual simulation. 2D multiplanar reconstructions with excellent local resolution may be obtained. 3D MIP (Maximum Intensity Projection), based on CT angiographic imaging, is a good tool for infradiaphragmatic treatment planning of Hodgkin's disease, if small numbers for slice thickness, table speed and increment are used. SSD (Surface Shaded Display) offers good 3D visualization and good geometric control of intracavitary and interstitial brachytherapy applicators. High qualitative multiplanar reconstructions are useful for CT-based brachytherapy planning. CONCLUSIONS: Spiral CT is a precious tool for 3D treatment planning and virtual simulation in radiotherapy and superior to conventional CT data acquisition. Quality assurance is improved for dose-volume histograms and for brachytherapy. PMID- 8623086 TI - [Radiochemotherapy and simultaneous hormonal therapy of locally advanced prostatic carcinoma]. AB - PURPOSE: The results of radiotherapy alone in the prostate cancer stage T3-T4 are not satisfactory. We investigated, whether the combination of radio-chemotherapy with hormone therapy, possible most effective therapy in stage III, will be tolerated without serious side effects. PATIENTS AND METHODS: Eight patients with prostate cancer T3/T4 were treated with concurrent radio-chemotherapy and hormone therapy. The mean doses in reference point was 61.7 Gy, the single dose was 1.8 Gy. The patients received epirubicin 20 mg/m2 and 5-fluorouracil 800 mg/m2 on day 1 to 5 and 29 to 33 of radiotherapy. The hormone therapy began before the radio chemotherapy started. RESULTS: Only the hematological side effects were relevant. Three out of 8 patients had leucopenia grade III and 1 of 8 patients leucopenia grade IV according WHO. Other serious complications could not be observed. CONCLUSION: The concurrent radio-chemotherapy with epirubicin and 5-fluorouracil in the combination with hormone therapy was tolerated well. The effectiveness of this new treatment should be examined prospectively in prostate cancer stage T3 T4. PMID- 8623087 TI - [Irradiation in cervical carcinoma after radical surgery: what is the target volume? Is prophylactic irradiation of the para-aortal area effective?]. PMID- 8623088 TI - [The 3rd Intergroup Rhabdomyosarcoma Study (IRS III)]. PMID- 8623089 TI - [The prognostic significance of intramammary recurrence in breast-conserving therapy]. PMID- 8623090 TI - Carotid artery thrombus associated with severe iron-deficiency anemia and thrombocytosis. AB - BACKGROUND: Thrombus within the carotid artery usually occurs in vessels with severe atherosclerotic disease and may embolize to cause transient ischemic attacks and cerebral infarctions. The risk factors for carotid artery thrombus formation in the absence of atherosclerosis are not well characterized. A case series is presented that suggests an association of carotid artery thrombus with severe iron-deficiency anemia and thrombocytosis. CASE DESCRIPTIONS: We describe three women with severe iron-deficiency anemia and thrombocytosis secondary to menorrhagia who developed carotid artery thrombi. Thrombi were detected radiographically. The patients were treated with anticoagulation and antiplatelet therapy. In two patients, follow-up neuroimaging 10 to 14 days later demonstrated resolution of the thrombus and no identifiable vascular disease. CONCLUSIONS: Severe iron-deficiency anemia with thrombocytosis may be a risk factor for carotid artery thrombus formation. Medical management with anticoagulation and antiplatelet therapy is a reasonable approach for these patients while the thrombus resolves. PMID- 8623091 TI - Color-coded transcranial duplex sonography in a patient with transient stenosis of the middle cerebral artery due to bacterial meningitis. PMID- 8623092 TI - Natural beta 2-glycoprotein I-independent anticardiolipin autoantibodies are not age-related. PMID- 8623093 TI - Neuropathological findings of a Sneddon's syndrome presenting with dementia not preceded by clinical cerebrovascular events. PMID- 8623094 TI - Carotid pseudovalvular fold: a case diagnosed by angiography and helical cervical tomography. PMID- 8623095 TI - Mean platelet volume and acute ischemic stroke. PMID- 8623096 TI - US National Survey of Physician Practices for the Secondary and Tertiary Prevention of Ischemic Stroke. Carotid endarterectomy. AB - BACKGROUND AND PURPOSE: Data from several randomized clinical trials concerning the efficacy of carotid endarterectomy (CE) in patients with symptomatic and asymptomatic stenoses of the extracranial carotid artery are now available. Yet, there are few data concerning the patterns of use of CE by physicians for their patients at risk for stroke. These data are critical for the rational allocation of resources and targeting of educational efforts. METHODS: Between August 1993 and February 1994, we surveyed the stroke prevention practices of a stratified random sample of 2000 US physicians. The survey queried the perceived availability and use of diagnostic studies and surgery for specific types of patients who might be considered candidates for CE. RESULTS: Of eligible physicians, 67% (n = 1006) completed the survey. Seventy percent reported that they always or often obtain carotid ultrasonography for evaluation of patients with asymptomatic bruits; 89% do so in patients with recent transient ischemic attack or minor stroke (P < .001). For asymptomatic patients, 13% always or often obtain a cerebral angiogram if carotid ultrasonography indicates 50% to 70% stenosis versus 33% if carotid ultrasonography indicates > 70% stenosis (P < .001). For asymptomatic patients with > 70% stenosis, a cerebral angiogram was reported as seldom or never used by 42% of physicians who viewed the test as readily available versus 67% if cerebral angiography was perceived as not readily available (P = .005). Multinomial multiple logistic regression analysis showed that symptom status, the degree of stenosis, perceived availability of CE, and physician specialty independently contributed to the explained variance in the reported use of CE (P < .001). The odds of performing CE were approximately four times greater in patients recent symptoms compared with asymptomatic patients (P < .001) and four times greater in patients with > 70% stenosis compared with patients with 50% to 70% stenosis (P < .001). Physicians who perceived CE as not being readily available were one third as likely to report using the procedure compared with physicians who reported having ready access (P = .004). CE was reported as being always or often used by more than 80% of neurologists and surgeons but by only about half of internists and noninternist primary care physicians for patients with newly symptomatic high-grade stenosis (P < .001). Almost one in four noninternist primary care physicians responded that they would seldom or never use CE for these patients. CONCLUSIONS: These data show that (1) symptom status and degree of carotid artery stenosis strongly influence the reported frequency with which CE is used by practicing physicians; (2) the perceived availability of cerebral angiography and CE significantly affects their reported frequency of use; and (3) physician specialty significantly influences the reported frequency of use of CE. PMID- 8623097 TI - Will increased awareness among physicians of the significance of sudden agonizing headache affect the outcome of subarachnoid hemorrhage? Coventry and Warwickshire Study: audit of subarachnoid hemorrhage (establishing historical controls), hypothesis, campaign layout, and cost estimation. AB - BACKGROUND AND PURPOSE: The most common symptom associated with aneurysmal minor bleed ("warning leak") is a sudden agonizing headache. Early screening of these patients may improve the outcome of subarachnoid hemorrhage and may be highly cost-effective. METHODS: We conducted an extensive retrospective audit of subarachnoid hemorrhage over the last 10 years in the region of Coventry and Warwickshire, England, and initiated a continuous campaign among all physicians in the region for early neurosurgical referral of patients with sudden agonizing headache. RESULTS: Over the last 10 years the incidence of subarachnoid hemorrhage in the region was 8.7/100 000 per year. Surgical activity was 34% and early mortality 45.2%. Functional outcome, both overall and by grade on admission, was within internationally accepted levels. Warning leak symptoms before admission were experienced by 20% of patients. These patients sought medical advice but were not referred immediately to the neurosurgical unit. CONCLUSIONS: We have established our population as valid historical controls and outlined our campaign strategy. Lowering the clinical threshold at which patients with sudden agonizing headache are screened for aneurysms or arteriovenous malformations will undoubtedly increase diagnostic costs. However, for reasons given in the text, we estimated the cost per quality-adjusted life year gain to be 1000 pounds ($1500). PMID- 8623098 TI - Effect of dietary calcium and milk consumption on risk of thromboembolic stroke in older middle-aged men. The Honolulu Heart Program. AB - BACKGROUND AND PURPOSE: Evidence suggests that dietary calcium is protective against hypertension. This report examines whether the effect has an influence on thromboembolic stroke. METHODS: Since 1965, the Honolulu Heart Program has followed a cohort of men in a study of cardiovascular disease. This report examines the effect of baseline dietary calcium and milk intake on stroke risk in 22 years of follow-up in 3150 older middle-aged men (55 to 68 years). RESULTS: Men who were nondrinkers of milk experienced stroke at twice the rate (P < .05) of men who consumed 16 oz/d or more (7.9 versus 3.7 per 100, respectively). While the rate of stroke decreased with increasing milk intake (P < .05), the decline in stroke risk with increased consumption was modest for those who consumed under 16 oz/d. Intake of dietary calcium was also associated with a reduced risk of stroke (P < .01), although its association was confounded with milk consumption. Calcium intake from nondairy sources was not related to stroke, suggesting that other constituents or covariates related to milk consumption may be important. CONCLUSIONS: We conclude that an association between milk consumption and a reduced risk of stroke in older middle-aged men cannot be explained by intake of dietary calcium. Since milk is often part of a diverse pattern of dietary intake, it is difficult to determine whether milk consumption has a direct role in reducing the risk of stroke. Data suggest that consumption of milk in older middle age is not harmful, and when combined with a balanced diet, weight control, and physical activity, reductions in the risk of stroke may occur. PMID- 8623099 TI - Age-related changes in stroke risk in men with hypertension and normal blood pressure. AB - BACKGROUND AND PURPOSE: Stroke is a major contributor to total morbidity and mortality in older individuals, and hypertension is an important risk factor for stroke. Relatively few data exist on whether this relationship changes with age. METHODS: To examine age-related changes in the relationships between risk of stroke and hypertension, we examined the 6-year incidence of stroke among men aged 45 to 81 years using updated blood pressure data from three examinations of Japanese-American men from the Honolulu Heart Program. RESULTS: Both the prevalence of hypertension (systolic blood pressure > or = 160 mm Hg or diastolic blood pressure > or = 95 mm Hg or the use of antihypertensive medication) and the 6-year incidence of stroke increased significantly with increasing age (P < .01). The increase in thromboembolic stroke incidence with age was more marked in those who were normotensive at baseline (2.7/1000 in those aged 45 to 54 years to 23.9/1000 in those > or = 65 years; P < .001) than in hypertensive men (20.6/1000 in those aged 45 to 54 years to 33.5/1000 in those > or = 65 years; P < .01). The age-related increase in risk of thromboembolic stroke among normotensive men resulted in a decrease in the percentage of strokes attributable to hypertension (50% in those aged 45 to 54 years to 18% in those > or = 65 years; P < .05). Similar trends were seen for hemorrhagic stroke. There were no age-related changes in the relationships of other major atherosclerotic risk factors with stroke. The hypertension/stroke relationships were present after multivariate adjustment for age, smoking, cholesterol, and other factors. CONCLUSIONS: In view of the greater prevalence of hypertension and the proven efficacy of treatment in the elderly, these findings do not negate the value of aggressive screening and treatment of hypertension in this age group. However, it appears that other unidentified factors have an increasing role in the causation of stroke with advancing age. PMID- 8623100 TI - Ten-year trends in stroke incidence and mortality in the FINMONICA Stroke Study. AB - BACKGROUND AND PURPOSE: The trends in stroke incidence reported so far have not been entirely consistent, although declining trends in mortality from stroke have been reported from a number of studies around the world. This study aims to evaluate the 10-year trends (from 1983 through 1992) in incidence, attack rate, and mortality of stroke in the Finnish population. METHODS: A population-based stroke register was set up in the early 1980s to collect data on all suspected events of acute stroke that occurred in the population aged 25 to 74 years permanently residing in three geographic areas of Finland: the provinces of Kuopio and North Karelia in eastern Finland and the Turku-Loimaa area in southwestern Finland. Trends in age-standardized attack rates, incidence, and mortality were calculated for the period studied. RESULTS: During the 10-year study period, 11 392 acute stroke events occurred in the monitored populations. A statistically significant decline was observed in the pooled FINMONICA data, both in the incidence of stroke (-1.7% with 95% confidence interval [CI], -3.0% to 0.5% per year in men; -2.2% with 95% CI, -3.6% to -0.7% per year in women) and in mortality from stroke (-5.2% with 95% CI, -8.2% to -2.2% per year; -4.7% with 95% CI, -8.2% to -1.2% per year). The attack rate of stroke also declined significantly in both sexes. When the areas were considered separately, the declining trends were observed within each area. The decline in incidence of stroke was, however, statistically significant only among men and women in Kuopio and among women in Turku/Loimaa. Mortality declined significantly in all three areas among men but among women only in Kuopio. The incidence to mortality rate ratio increased during the study period, indicating a steeper fall in mortality than in incidence. CONCLUSIONS: A substantial decline in both stroke incidence and mortality was observed in the adult and elderly population in the FINMONICA study areas. Part but not all of the decline in stroke mortality, observed also in the official mortality statistics, can be attributed to the decline in stroke incidence during this 10-year period. PMID- 8623101 TI - Genetic basis of variation in carotid artery wall thickness. AB - BACKGROUND AND PURPOSE: Other than the documented associations of risk factors and carotid artery wall thickness, the genetic basis of variation in carotid artery intimal-medial thickness (IMT) is unknown. The purpose of this study was to examine the extent to which variation in common carotid artery (CCA) IMT and internal carotid artery (ICA) IMT are under genetic control. METHODS: The sibship data used for this analysis were part of an epidemiological survey in Mexico City. The CCA and ICA analyses were based on 46 and 44 sibships of various sizes, respectively. The CCA and ICA IMTs were measured with carotid ultrasonography. Using a robust variance decomposition method, we performed genetic analyses of CCA IMT and ICA IMT measurements with models incorporating several cardiovascular risk factors (eg, lipids, diabetes, blood pressure, and smoking) as covariates. RESULTS: After accounting for the effects of covariates, we detected high heritabilities for CCA IMT (h2 = 0.92 +/- 0.05, P = .001) and ICA IMT (h2 = 0.86 +/- 0.13, P = .029). Genes accounted for 66.0% of the total variation in CCA IMT, whereas 27.7% of variation was attributable to covariates. For ICA IMT, genes explained a high proportion (74.9%) of total phenotypic variation. The covariates accounted for 11.5% of variation in ICA IMT. CONCLUSIONS: Our results suggest that substantial proportions of phenotypic variance in CCA IMT and ICA IMT are attributable to shared genetic factors. PMID- 8623102 TI - Stroke in the young in Israel. Incidence and outcomes. AB - BACKGROUND AND PURPOSE: Data on stroke in the young in Israel are fragmentary. To obtain an overall perspective and to assess the nature and magnitude of the problem, a study was conducted on stroke occurrence in the young population during 1 year. Incidence and outcomes are reported in this communication. METHODS: We conducted a prospective ascertainment of first stroke in all permanent residents of Israel aged 17 to 49 years who were referred to all acute care hospitals in the country or died before reaching them. RESULTS: We identified 253 first stroke victims in the studied population; 62.8% were male. The age- and sex-adjusted incidence rate for all types of stroke was 10.36/100 000 per year (males, 13.00; females, 7.71). The majority of strokes (80.6%) were cerebral infarctions, with 9.9% intracerebral hemorrhages, 7.9% subarachnoid hemorrhages, and 1.6% strokes of unspecified type. The case-fatality rate for all types of stroke was 9.9% (mortality within the first 4 weeks after the event, on average 6 days). The survival rate was 95% for cerebral infarctions, 64% for intracerebral hemorrhages, and 80% for subarachnoid hemorrhages; 86.7% of all survivors remained with an impairment resulting in a disability. CONCLUSIONS: Incidence rates were similar to those reported from developed Western countries. The case-fatality rate of 9.9% and the considerable percentage of survivors with a disability in a population at the beginning of their family, professional, and social lives indicate the magnitude of the problem. PMID- 8623103 TI - Functional outcome in patients with lacunar infarction. AB - BACKGROUND AND PURPOSE: Little is known about the prognosis and the predictive factors for functional outcome after lacunar infarction. Our aim was to analyze this issue in more detail and with a longer follow-up than in previous reports. METHODS: Functional outcome was assessed in 81 consecutive patients with a first ever stroke and clinical and MRI findings compatible with lacunar infarction. We measured impairment (motor, sensory, and cognitive function), disability (Katz's Index of Activities of Daily Living [ADL] and four instrumental activities), and handicap (Oxford Handicap Scale). The patients were followed up for 3 years. RESULTS: During follow-up, 6% of the patients died and 21% had recurrent strokes, mostly new lacunar infarcts. A fast initial recovery was found in most patients. At 1 year, 12% were dependent in personal ADL, which after 3 years had increased to 24%, mostly as a result of the effects of recurrent strokes. In a logistic multivariate regression model, moderate or severe hemiparesis 1 month after stroke onset was the strongest predictor of physical dependence or death at 3 years (P < .001), followed by white matter hyperintensities on MRI (P < .01). Age, vascular risk factors, and recurrent stroke were not statistically significant independent predictors of functional outcome. CONCLUSIONS: Functional outcome regarding physical independence was favorable in most patients. Motor impairment and white matter disease were the strongest predictors of a poor functional outcome. Recurrent stroke increased disability and handicap but was not a statistically significant independent risk factor. Measurements of personal ADL alone were insensitive in detecting the consequences of stroke in many patients with preserved self-care ability, who still experienced disability and handicap. PMID- 8623104 TI - Risk of stroke in a cohort of 815 patients with calcification of the aortic valve with or without stenosis. AB - BACKGROUND AND PURPOSE: We sought to establish the possible role of calcification of the aortic valve with or without stenosis as a risk factor for stroke. METHODS: Occurrences of stroke, stroke subtypes, and concomitant cardiovascular risk factors were prospectively analyzed in 300 patients with echocardiographic evidence of aortic valve calcification, 515 patients with calcified aortic valve stenosis, and 562 control subjects. RESULTS: Twenty-four patients with aortic valve calcification, 24 patients with calcified aortic valve stenosis, and 27 control subjects had a stroke during follow-up. Using Cox proportional hazards models, we found that strokes were not significantly associated with aortic valve calcification with or without stenosis, but hypertension and any carotid stenosis were associated. On multiple logistic regression analysis, we did not find any association between one of the two valve lesions and indirect possible indications of cardiogenic embolism such as territorial as opposed to small deep brain infarcts or the presence of silent brain infarcts. CONCLUSIONS: Aortic valve calcification with or without stenosis is not a risk factor for stroke. PMID- 8623105 TI - Increased expression of TGF-beta 1 in brain tissue after ischemic stroke in humans. AB - BACKGROUND AND PURPOSE: Occlusion in cerebral vessels results in ischemic stroke and is followed by proliferation of microvessels, ie, angiogenesis. The process is particularly marked in the border zone of the infarct, known as the ischemic penumbra. This increase in vascularization is likely to be caused by the action of angiogenic factors, such as TGF-beta 1, which is a powerful regulator of angiogenesis. METHODS: In this study we examined 10 brain samples from patients who suffered from ischemic stroke for the expression of mRNA encoding TGF-beta 1. RESULTS: The ischemic penumbra contained the highest levels of TGF-beta 1 mRNA, whereas the normal contralateral hemispheres had the least (P < .001, Mann Whitney U test). Unlike those from normal brain, protein extracts from infarcted tissue contained active TGF-beta 1 as a 25-kD band in Western blot analysis. Extracts from the penumbra also contained a 12.5-kD isoform of TGF-beta 1. Both penumbra and infarct contained TGF-beta 1 immunoreactive products as assessed with immunohistochemistry, whereas very weak staining was observed in the contralateral hemisphere. CONCLUSIONS: These results suggest that TGF-beta 1 is important in the pathogenesis of the angiogenic response in ischemic brain tissue and its modulation may be used for therapeutic purposes. PMID- 8623106 TI - Evaluating neuroprotective agents for clinical anti-ischemic benefit using neurological and neuropsychological changes after cardiac surgery under cardiopulmonary bypass. Methodological strategies and results of a double-blind, placebo-controlled trial of GM1 ganglioside. AB - BACKGROUND AND PURPOSE: Many neuroprotective agents (NPAs) are effective in acute experimental cerebral ischemia in animals. None have proven effective in human stroke trials. Even short treatment delays cause substantial efficacy loss. Cardiac surgery under cardiopulmonary bypass (CS-CPB) causes cerebral ischemia with cognitive impairment at a predeterminable time point and should permit efficient screening of NPAs for stroke benefit. We sought to develop sensitive methods to assess dysfunction from CS-CPB in a double-blind trial of the NPA GM1 ganglioside. METHODS: Eighteen GM1 and 11 Control patients received GM1 300 mg or placebo, two doses intravenously, before nonemergency CS-CPB. Independent examiners administered structured neurological examinations and neuropsychological test batteries at Baseline and 1 day (Acute Postop; neurological only), 1 week (Early F/U), and > or = 6 months (Long-term F/U) postoperatively; using defined procedures they employed ordinal Clinical Change Scores (CCSs) to quantify neurological cerebral, neurological noncerebral, and neuropsychological performance changes. Several methods to analyze CCSs and neuropsychological test score changes were evaluated. RESULTS: The most sensitive indicators were the mean Acute Postop Neurologist's CCS-Cerebral (P < 10(-5)) and the mean Early F/U Neuropsychologist's CCS (P < .01), with statistically nonsignificant differences favoring GM1. No significant mean changes in Neurologist's CCS-Noncerebral or any Long-term F/U CCSs occurred. CCS distributions and neuropsychological test score mean changes showed similar temporal patterns, with less sensitivity to change. When, as usual in prior CS CPB studies, impairment was defined by neuropsychological test score declines (increases ignored), results were spurious. CONCLUSIONS: The strokelike cerebral dysfunction (maximal acutely, with eventual recovery) that occurs after CS-CPB is useful to screen NPAs for clinical efficacy. CCSs based on detailed neurological examination and neuropsychological testing are sensitive measures; refinement of this approach should enhance the efficiency of the CS-CPB model. Further testing of GM1 is warranted. PMID- 8623107 TI - Thrombolytic therapy of acute basilar artery occlusion. Variables affecting recanalization and outcome. AB - BACKGROUND AND PURPOSE: Thrombolysis may reduce mortality after acute basilar artery (BA) occlusion. We intended to find variables affecting recanalization and clinical outcome in patients with BA occlusion undergoing thrombolytic therapy. METHODS: We analyzed in retrospect the clinical and angiographic data of a consecutive series of 51 patients treated with intra-arterial urokinase (n = 44; 0.3 to 1.5 mIU) or intravenous or intra-arterial recombinant tissue plasminogen activator (n = 7; 22 to 100 mg). We identified effective variables by multiple logistic regression analyses and univariate tests. RESULTS: Sites of occlusion were the caudal (n = 23), middle (n = 18), and distal (n = 10) segments of the BA. The pathogenesis was embolism in 35 and local atherothrombosis in 16 patients. Collateral circulation was good in 32 patients and poor or absent in 19 patients. Recanalization was achieved in 26 of 51 (51%) patients and was associated with occlusions of embolic etiology (P = .0025). Mortality was 46% (12/26) in the recanalization group and 92% (23/25) in the nonrecanalization group (P = .0004). Other independent variables affecting mortality were length of BA obstruction (P = .0011), age (P = .0008), and collateral state (P = .0454). After follow-up (median, 32 months), 10 of the 16 survivors were only minimally impaired, with a Barthel Index score of 95 or greater; 5 patients were moderately and 1 severely disabled. CONCLUSIONS: Recanalization of acute BA occlusion reduces mortality significantly. Length of BA obstruction and state of the collaterals are additional independent variables affecting survival. Young patients with monosegmental embolic occlusion of the BA seem to have the best chance to considerably profit from thrombolysis. PMID- 8623108 TI - Open trial of intravenous tissue plasminogen activator in acute carotid territory stroke. Correlations of outcome with clinical and radiological data. AB - BACKGROUND AND PURPOSE: Pilot studies using early thrombolytic therapy in stroke have suggested that recombinant tissue plasminogen activator (rTPA) might be effective. While large, double-blind, randomized studies are needed, open trials could generate hypotheses concerning (1) the clinical correlations of outcome, (2) the significance of CT scan data during the first week, and (3) the use of adjunctive therapies. METHODS: We performed an open trial of intravenous rTPA on patients referred to our emergency service with all types of ischemic stroke in the carotid territory. All patients between 20 and 81 years hospitalized during 1994 with completed stroke in the internal carotid artery territory and a baseline Scandinavian Stroke Scale score lower than 48, even with severe disturbances of consciousness, were included. The inclusion time was within 7 hours after stroke onset. A 0.8-mg/kg dose of rTPA was infused for 90 minutes. Intravenous heparin was given either immediately at efficient dosage or after 24 hours. Mannitol was used in patients with severe presentation. The Scandinavian Stroke Scale evaluation was done at baseline, 3 hours, and 1, 7, 30, and 90 days. The CT scan was performed before the treatment and at days 1 (24 +/- 6 hours) and 7. RESULTS: Forty-three consecutive patients met the criteria of the protocol. The mean age at inclusion was 65 +/- 10.4 years, and the mean interval to treatment was 232 +/- 79 minutes. At day 90, 25 patients (58.1%) exhibited a complete regression of symptoms, and 3 had moderate neurological sequelae. Thirteen patients had severe neurological sequelae, 11 with infarcts and 2 with secondary parenchymal hematomas. Two patients died (4.6%), 1 with hematoma. The overall hematoma rate was 6.9%. Excellent outcome at day 90 was significantly correlated with major neurological improvement at day 1. Intravenous immediate heparin versus delayed heparin after 24 hours improved the ischemic outcome but not the overall outcome. Reinfarction syndromes after major neurological improvement, likely to be rethrombosis syndromes, were observed in 3 patients (6.9%). For the day 1 CT scan, poor outcome was associated with the presence of structured and homogeneous hypodensities likely to represent classic infarcts, as confirmed by day 7 CT scan. Conversely, total recovery was significantly associated with the absence of any image or with unstructured hypodensities, a particular type of image characterized by its heterogeneous darkness and often polylobar shape. This type of image disappeared at day 7 in 17.6% of the cases and is likely to represent reperfusion images and/or incomplete ischemic damage. CONCLUSIONS: The results obtained in this open, small study suggest safety and effectiveness of rTPA thrombolysis at the dose of 0.8 mg/kg within 7 hours in acute strokes of the carotid territory, including highly serious baseline neurological presentations, until age 81 years and under special therapeutic conditions. Complete recovery is significantly associated with major neurological improvement during the first 24 hours and the presence of a particular type of image at day 1 CT scan characterized by an unstructured hypodensity, often polylobar and heterogeneous, which is likely to correspond to reperfusion images. PMID- 8623109 TI - Clinical relevance of intracranial microembolic signals in patients with left ventricular assist devices. A prospective study. AB - BACKGROUND AND PURPOSE: The use of left ventricular assist devices has become an established method in bridging patients with end-stage cardiac failure to heart transplantation. Since thromboembolism is one of the major complications, we undertook this study to evaluate the clinical significance of Doppler microembolic signals (MES) in patients with left ventricular assist devices. METHODS: Six patients with left ventricular assist devices were monitored for MES with transcranial Doppler ultrasonography during the first 30 postoperative days. Additionally, repeated (10 per day and patient) and prolonged (3 hours per patient) monitorings were performed to assess the adequacy of the 30-minute recordings. Three observers evaluated 30 randomly assigned monitorings in a blinded fashion to assess the interobserver variability. The relation between MES counts and clinical, radiological, hemostaseological, and pump flow parameters and the predictive value of MES counts regarding the occurrence of embolic events was evaluated. RESULTS: Ten ischemic cerebrovascular accidents and 2 peripheral thromboembolic events occurred during the observation period of 177 days (total incidence, 6.8%). MES were found in 143 of 170 monitorings (84.1%). Their counts were significantly higher on days with clinically manifest embolic events as compared with event-free days (18.5 [3-74] versus 4 [0-52], respectively, median and 95% CI; P < .001, Mann-Whitney). The predictive value of MES counts above 7 per 30 minutes was high (75%). Significant differences in the incidence and counts of MES as well as in the incidence of clinically manifest embolic events were noted among the six patients (all P < .01) without equal differences in anticoagulant treatment or pump flow. Interobserver agreement was high (p = .78 to .89, unpaired Student's t test). Considerable short- and long-term intrapatient variations of MES counts, without consistent pattern, were noted. CONCLUSIONS: Serial monitoring for MES is prognostically superior to single monitorings in patients with left ventricular assist devices. In the future, this new application mode may individually guide anticoagulation strategies and even influence the decision regarding early cardiac transplantation versus long-term use of the assist devices. PMID- 8623110 TI - Role of the nondominant hemisphere and undamaged area during word repetition in poststroke aphasics. A PET activation study. AB - BACKGROUND AND PURPOSE: Although the resting regional cerebral blood flow (rCBF) in aphasic patients has been thoroughly investigated with positron emission tomography (PET) and single-photon emission CT, and PET studies in normal subjects have elucidated the functional localization of language processing, little is known about the activation pattern of language processing in aphasic patients. METHODS: We measured the changes in rCBF during a repetition task (hearing a single word and repeating it aloud) and the resting state using the H2(15)O PET activation technique in 6 normal subjects (mean +/- SD age, 58.3 +/- 8.1 years) and 16 aphasic patients: 10 fluent aphasics (age, 60.3 +/- 12.5 years) and 6 nonfluent aphasics (age, 50.5 +/- 8.3 years). RESULTS: In normal subjects, the posteroinferofrontal area (PIF) including Broca's area, the posterosuperotemporal area (PST) including Wernicke's area, the rolandic areas, and a few other areas were activated with left side dominance by the repetition task. In the resting state, the rCBF in the left PIF and the left posterotemporal area was reduced in both fluent and nonfluent aphasics. In aphasic patients, the magnitude of activation in the right PIF and PST by the repetition task was greater than in normal subjects. The increase in rCBF during the repetition task in the left PIF correlated with the Western Aphasia Battery score of spontaneous speech in the nonfluent aphasics with a left inferofrontal lesion. CONCLUSIONS: This study shows the importance in aphasic patients of the mirror regions of the left PIF and PST in the nondominant (right) hemisphere for performing the word repetition task. The results also show the importance for nonfluent aphasic patients of the recruitment of the undamaged PIF for spontaneous speech. PMID- 8623111 TI - Do silent brain infarctions predict the development of dementia after first ischemic stroke? AB - BACKGROUND AND PURPOSE: Silent brain infarctions (SBI) are common findings in advanced age, but their relationship to dementia is still uncertain. The present study was designed to evaluate whether SBI predict the development of dementia after first clinical ischemic stroke. METHODS: We blindly studied admission CT scans of 175 consecutive nondemented patients presenting with ischemic stroke that clinically was their first stroke episode. SBI were defined as CT evidence of infarcts not compatible with the acute event. The patients were subsequently followed for their mental state for 5 years. Survival analysis, wherein onset of dementia was the end point, was performed on the total sample population and conducted separately on those with and without SBI at admission. RESULTS: Dementia developed in 56 patients (32%), including 22 of the 63 (35%) with SBI and 34 of the 112 (30%) without SBI. Thus, dementia was not related to SBI. CONCLUSIONS: Our data indicate that SBI do not predict the development of dementia after stroke. PMID- 8623112 TI - Optimal timing of hemodilution for brain protection in a canine model of focal cerebral ischemia. AB - BACKGROUND AND PURPOSE: Hemodilution is known to ameliorate the effects of focal ischemia when used shortly after cerebral arterial occlusion; however, it remains to be proved whether hemodilution will be effective when used at more clinically relevant times, ie, with some delay between the onset of ischemia and initiation of therapy. METHODS: Thirty-two dogs were selected for inclusion in this study. Cerebral infarction was induced by permanent occlusion of the middle cerebral and the azygos anterior cerebral arteries. The animals were allocated to 1 of 4 groups of eight animals each: arterial occlusion without hemodilution (group 1); hemodilution immediately after occlusion (group 2); hemodilution 3 hours after occlusion (group 3); and hemodilution 6 hours after occlusion (group 4). Isovolemic hemodilution to a hematocrit of 30% was performed. The animals were killed 6 days after induction of ischemia, and the infarct size was determined. RESULTS: Groups 2 and 3 showed significant reduction of infarct size (P < .0001) when compared with group 1. The neurological grade of group 3 on postoperative days 4, 5, and 6 was significantly better than those of groups 1 and 4 (P < .01). Group 4 showed a significant increase in the incidence of hemorrhagic infarction when compared with groups 1 and 2 (P < .01). CONCLUSIONS: The current study indicates that hemodilution administered as much as 3 hours after ischemia is effective in reducing infarct size and improving neurological status. When administered 6 hours after ischemia, hemodilution is not helpful and may be harmful. PMID- 8623113 TI - Effects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization. AB - BACKGROUND AND PURPOSE: Hypothermia slows the increase in extracellular excitatory amino acid (EAA) concentrations during temporary cerebral ischemia. However, it is unclear whether hypothermia slows the rate of EAA release or just delays the time until the first sharp increase (which occurs coincident with terminal depolarization). METHODS: Pericranial temperatures were adjusted to 38 degrees C, 34 degrees C, 31 degrees C, or 25 degrees C in halothane-anesthetized rats. The cortical DC voltage was recorded from a glass microelectrode while the cortical concentrations of glutamate, aspartate, glycine, and gamma-aminobutyric acid (GABA) were measured by microdialysis. A cardiac arrest was induced with intravenous KCl, and the times until electroencephalograph isoelectricity and terminal depolarization were recorded. Dialysate concentrations of the four compounds were measured at 10, 20, and 30 minutes after depolarization. RESULTS: The times to isoelectricity and depolarization varied inversely with temperature; depolarization time increased from 70 +/- 9 seconds at 38 degrees C (mean +/- SD) to 294 +/- 34 seconds at 25 degrees C. The dialysate concentrations of all four compounds increased during ischemia, and the rate of increase was inhibited by cooling. After 30 minutes of ischemia, glutamate concentration in 38 degrees C animals was 58.4 +/- 31.8 mumol/L; this decreased to 15.9 +/- 8.4 mumol/L at 25 degrees C. The magnitude of the effects of temperature on amino acid release differed with the compound measured. For glutamate, the calculated Q10 was 3.63. Corresponding values for aspartate and glycine were 3.68 and 1.95, respectively. By contrast, Q10 for GABA release was 6.31, indicating greater sensitivity to cooling. CONCLUSIONS: These results suggest that effects of hypothermia on EAA concentrations during cerebral ischemia may be the result of both a delay until initial EAA release as well as a direct effect of temperature on the rate of amino acid release. The observed temperature effects are more consistent with carrier-mediated processes controlling EAA release. PMID- 8623114 TI - Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic-ischemic brain damage in the immature rat. AB - BACKGROUND AND PURPOSE: Intraischemic hypothermia (34 degrees C and 31 degrees C) has a profound neuroprotective effect on the brain of the immature rat. Hypothermia immediately after hypoxia-ischemia is not beneficial. To determine the mechanisms by which mild to moderate hypothermia affects cerebral energy metabolism of the brain of the newborn rat pup, we examined alterations in cerebral glycolytic intermediates and high-energy phosphate compounds during intraischemic and postischemic hypothermia and correlated these findings with known neuropathologic injury. METHODS: Seven-day-old rat pups underwent unilateral common carotid artery ligation and exposure to hypoxia in 8% oxygen at either 37 degrees C, 34 degrees C, or 31 degrees C for 3.0 hours. Separate groups were exposed to hypoxia-ischemia at 37 degrees C for 3 hours but recovered at either 37 degrees C, 34 degrees C, or 31 degrees C. At 60, 120, and 180 minutes of intraischemic hypothermia and at 10, 30, 60, and 240 minutes of postischemic hypothermia, individual rat pups were quick-frozen in liquid nitrogen for later determination of cerebral concentrations of glucose, lactate, ATP, and phosphocreatine. RESULTS: Cerebral glucose was significantly higher and lactate significantly lower in the 31 degrees C animals during hypoxia-ischemia than either the 34 degrees C or 37 degrees C groups. Brain ATP concentrations were completely preserved during hypoxia-ischemia at 31 degrees C, whereas 34 degrees C of hypothermia had no effect on preserving high-energy phosphate compounds compared with those animals in the 37 degrees C group. Postischemic hypothermia of either 34 degrees C or 31 degrees C had no effect on the rate or extent of recovery of glycolytic intermediates or high-energy phosphate compounds compared with the normothermic 37 degrees C rat pups. CONCLUSIONS: Moderate hypothermia of 31 degrees C completely inhibits the depletion of ATP during hypoxia-ischemia, a mechanism that likely accounts for its neuroprotective effect. No preservation of ATP was seen, however, during intraischemic mild hypothermia of 34 degrees C despite the relatively profound neuroprotective effect of this degree of temperature reduction. Thus, the mechanisms by which mild hypothermia is neuroprotective are temperature dependent and may act at more than one point along the cascade of events eventually leading to hypoxic-ischemic brain damage in the immature rat. PMID- 8623115 TI - Effect of short-term regression of atherosclerosis on reactivity of carotid and retinal arteries. AB - BACKGROUND AND PURPOSE: This study tested the hypothesis that functional abnormalities of carotid and ocular arteries may improve after short-term regression of atherosclerosis, before regression of structural abnormalities. METHODS: We examined effects of short-term dietary treatment of atherosclerosis on carotid and ocular vascular responses to serotonin and to platelet activation by collagen in vivo. Three groups of monkeys were studied: normal cynomolgus monkeys, monkeys fed an atherogenic diet for 34 months, and atherosclerotic monkeys that were fed a regression diet for 8.6 +/- 1.1 months (mean +/- SE). We measured changes in carotid blood flow (using a Doppler probe), retinal blood flow (using microspheres), and diameter of the internal carotid artery (using quantitative angiography). Endothelium-dependent relaxation to acetylcholine was studied in rings of internal carotid artery in vitro. RESULTS: Carotid blood flow increased in response to both serotonin and collagen in normal monkeys, decreased in response to both agents in atherosclerotic monkeys, and was restored toward normal after regression. Serotonin had little effect on retinal blood flow in normal monkeys and produced a marked decrease in retinal blood flow in atherosclerotic monkeys; the vasoconstrictor response to serotonin was reduced after regression. Activation of platelets by collagen increased blood flow in normal monkeys, decreased blood flow in atherosclerotic monkeys, and had little effect after regression. Alterations in responses of the internal carotid artery were consistent with changes in carotid and ocular blood flow. Endothelium dependent relaxation in vitro was impaired by atherosclerosis and was restored toward normal by regression. There was no reduction in intimal area of the atherosclerotic lesion in common carotid and ophthalmic arteries from regression monkeys, despite a marked reduction in cholesteryl ester. CONCLUSIONS: Within a few months of regression of atherosclerosis, endothelial function and hyperresponsiveness of carotid and ocular arteries to serotonin and platelet activation return toward normal. Functional improvement is associated with resorption of lipid from atherosclerotic lesions, but with little reduction in size of intimal lesions. PMID- 8623116 TI - Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Effect of polyethylene glycol-conjugated superoxide dismutase. AB - BACKGROUND AND PURPOSE: There is abundant evidence that after in vivo traumatic brain injury, oxygen radicals contribute to changes in cerebrovascular structure and function; however, the cellular source of these oxygen radicals is not clear. The purpose of these experiments was to use a newly developed in vitro tissue culture model to elucidate the effect of strain, or stretch, on neuronal, glial, and endothelial cells and to determine the effect of the free radical scavenger polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; pegorgotein, Dismutec) on the response of each cell type to trauma. METHODS: Rat brain astrocytes, neuronal plus glial cells, and aortic endothelial cells were grown in cell culture wells with 2-mm-thick silastic membrane bottoms. A controllable, 50 millisecond pressure pulse was used to transiently deform the silastic membrane and thus stretch the cells. Injury was assessed by quantifying the number of cells that took up the normally cell-impermeable dye propidium iodide. Some cultures were pretreated with 100 to 300 U/mL PEG-SOD. RESULTS: Increasing degrees of deformation produced increased cell injury in astrocytes, neuronal plus glial cultures, and aortic endothelial cells. By 24 hours after injury, all cultures showed evidence of repair as demonstrated by cells regaining their capacity to exclude propidium iodide. Compared with astrocytes or neuronal plus glial cultures, endothelial cells were much more resistant to stretch-induced injury and more quickly regained their capacity to exclude propidium iodide. PEG SOD had no effect on the neuronal or glial response to injury but reduced immediate posttraumatic endothelial cell dye uptake by 51%. CONCLUSIONS: These studies further document the utility of the model for studying cell injury and repair and further support the vascular endothelial cell as a site of free radical generation and radical-mediated injury. On the assumption that, like aortic endothelial cells, stretch-injured cerebral endothelial cells also produce oxygen radicals, our results further suggest the endothelial cell as a site of therapeutic action of free radical scavengers after traumatic brain injury. PMID- 8623117 TI - Relationships between ATP depletion, membrane potential, and the release of neurotransmitters in rat nerve terminals. An in vitro study under conditions that mimic anoxia, hypoglycemia, and ischemia. AB - BACKGROUND AND PURPOSE: It is known that the extracellular accumulation of glutamate during anoxia/ischemia is responsible for initiating neuronal injury. However, little information is available on the release of monoamines and whether the mechanism of its release resembles that of glutamate, which may itself influence the release of monoamines by activating presynaptic receptors. This study was designed to characterize the release of both amino acids and monoamines under chemical conditions that mimic anoxia, hypoglycemia, and ischemia. METHODS: The contents of synaptosomes in adenine nucleotides (ATP, ADP, and AMP), amino acids (aspartate, glutamate, taurine, and gamma-aminobutyric acid), and monoamines (dopamine, noradrenaline, and 5-hydroxytryptamine) were measured by high-performance liquid chromatography, after the synaptosomes were subjected to anoxia (KCN + oligomycin), hypoglycemia (2 mmol/L 2-deoxyglucose in glucose-free medium), and ischemia (anoxia plus hypoglycemia). RESULTS: The anoxia- and ischemia-induced release or noradrenaline, dopamine, 5-hydroxytryptamine, and glutamate correlated well with ATP depletion. The correlation observed between glutamate levels and the release of dopamine and 5-hydroxytryptamine in ischemic conditions suggests a functional linkage between the two transmitter systems. However, the antagonists of presynaptic glutamate receptors failed to alter the amount of monoamines released. The inhibition of Na+,K+-ATPase by ouabain had an effect similar to that produced by ischemia. CONCLUSIONS: The decrease in Na+ and K+ gradients resulting from the energy depletion of the synaptosomes under ischemic conditions or resulting from the inhibition of Na+, K+-ATPase by ouabain promotes the reversal of the neurotransmitter transporters. The decrease in uptake of neurotransmitters may also contribute to the rise in the extracellular concentration of different transmitters observed during brain ischemia. PMID- 8623118 TI - Treatment of focal cerebral ischemia with synthetic oligopeptide corresponding to lectin domain of selectin. AB - BACKGROUND AND PURPOSE: Synthetic oligopeptides with amino acid sequences of the lectin domain of selectin block selectin-mediated cell adhesion in vitro, which may be applied to a therapeutic intervention to attenuate acute inflammatory reactions. To evaluate the efficacy of such treatment against ischemic brain injury, the effects of administering a selectin oligopeptide that selectively blocks selectin-mediated cell adhesion on histological outcome and on cerebral blood flow (CBF) were studied in models of rodent focal cerebral ischemia. METHODS: Spontaneously hypertensive rats were anesthetized with halothane. Permanent focal cerebral ischemia was induced by tandem left middle cerebral artery (MCA) and common carotid artery (CCA) occlusion. Focal cerebral ischemia with partial reperfusion was introduced by reperfusing the CCA after 2 hours of tandem MCA/CCA occlusion. A synthetic oligopeptide (amino acid residues 23-30 from N terminal) of E-selectin was dissolved in physiological saline and was injected intravenously at a dosage of 2 mg/kg or 10 mg/kg before artery occlusion. Control animals received equivalent volumes of physiological saline or 10 mg/kg of synthetic oligopeptide with a scrambled amino acid sequence. Twenty four hours after the occlusion, seven coronal brain slices were stained with 2,3,5-triphenyltetrazolium chloride, and the volume of ischemic injury was calculated. In a separate set of animals, regional CBF was monitored with laser Doppler flowmetry at the dorsolateral cerebral cortex during 2-hour ischemia and 30 minutes after partial reperfusion. RESULTS: The volume of ischemic injury did not differ among groups in permanent ischemia. In ischemia with partial reperfusion, 10 mg/kg selectin oligopeptide, but not the same dosage of scrambled oligopeptide, significantly reduced the volume of ischemic injury (95 +/- 13, 73 +/- 11, 55 +/- 6, and 111 +/- 14 mm3 for saline [n = 11]; 2 mg/kg [n = 10] and 10 mg/kg [n = 16] selectin oligopeptide and 10 mg/kg scrambled oligopeptide [n = 6], respectively; P < .01 by one-way ANOVA followed by the Tukey test). Laser-Doppler flowmetry demonstrated a larger increase in CBF after reperfusion of the CCA in the 10-mg/kg selectin oligopeptide group. CONCLUSIONS: Our data demonstrate that administration of a synthetic oligopeptide corresponding to the lectin domain of selectin decreases the size of ischemic injury after transient, but not after permanent, focal cerebral ischemia as evaluated at 24 hours after onset of ischemia. These effects were associated with an improved CBF at the dorsolateral cerebral cortex after partial reperfusion. PMID- 8623119 TI - 23Na nuclear magnetic resonance spectral changes during and after forebrain ischemia in hypoglycemic, normoglycemic, and hyperglycemic rats. AB - BACKGROUND AND PURPOSE: The severity of brain injury in animal models of forebrain ischemia increases with blood glucose level. During ischemia, energy failure is slower and maintenance of ion gradients is prolonged as the level of glycemia increases. It is not clear how the level of glycemia influences recovery of ion homeostasis on reperfusion. It has been shown that changes in the intensity of the multiple-quantum 23Na nuclear magnetic resonance (NMR) signals reflect changes in intracellular Na+ levels. We have used 23Na NMR spectroscopy to evaluate the influence of the level of glycemia on changes in Na+ concentration during and after forebrain ischemia in rats. METHODS: Single quantum (SQ) and double-quantum (DQ) 23Na NMR spectra were measured before and during 10-minute forebrain ischemia and during reperfusion in hypoglycemic, normoglycemic, and hyperglycemic rats. RESULTS: The DQ 23Na NMR signal increased to 210% of preischemia intensity in all rats, but a delay in this increase was observed in normoglycemic and hyperglycemic animals. The rate of the DQ 23Na NMR signal increase was fastest in hypoglycemic (apparent first-order rate constant 0.673 +/- 0.046 min-1, P < .002 compared with normoglycemic animals) and slowest in hyperglycemic (0.285 +/- 0.024 min-1, P < .03) rats. During reperfusion, the signal intensity recovered rapidly in hypoglycemic (0.385 +/- 0.050 min-1) and normoglycemic (0.464 +/- 0.047 min-1) rats, whereas in hyperglycemic animals recovery was slow (0.108 +/- 0.044 min-1, P < .0001 compared with normoglycemic animals). The SQ 23Na NMR signal intensity increased to 117% of preischemia level in hypoglycemic (P < .05 compared with normoglycemic animals) and to 107% in normoglycemic and hyperglycemic animals during reperfusion. CONCLUSIONS: The slower increase in the 23Na DQ NMR signal intensity during forebrain ischemia in rats with higher blood glucose levels suggests that Na+ homeostasis is maintained longer in these animals. On reperfusion, the slower recovery of the DQ 23Na NMR signal intensity in hyperglycemic animals likely indicates a slower recovery of Na+ homeostasis, perhaps contributing to the increased neuronal injury after cerebral ischemia in hyperglycemic animals. PMID- 8623120 TI - Glutamate-induced disruption of the blood-brain barrier in rats. Role of nitric oxide. AB - BACKGROUND AND PURPOSE: The first goal of this study was to determine the effect of glutamate on permeability and reactivity of the cerebral microcirculation. The second goal of this study was to determine a possible role for nitric oxide in the effects of glutamate on the cerebral microcirculation. METHODS: We examined the pial microcirculation in rats with intravital microscopy. Permeability of the blood-brain barrier was quantified by the clearance of fluorescent-labeled dextran (molecular weight, 10 000 D; FITC-dextran-10K) before and during application of glutamate (0.1 and 1.0 mmol/L). In addition, we examined the permeability of the blood-brain barrier during application of a nitric oxide donor, S-nitroso-acetyl-penicillamine (SNAP; 10 mumol/L). Diameter of pial arterioles was measured before and during application of glutamate or SNAP. To determine a potential role for nitric oxide in glutamate-induced effects on the cerebral microcirculation, we examined the effects of NG-monomethyl-L-arginine (10 mumol/L). RESULTS: In control rats, clearance of FITC-dextran-10K from pial vessels was minimal, and the diameter of pial arterioles remained constant during the experimental period. Topical application of glutamate (0.1 and 1.0 mmol/L) and SNAP (10 mumol/L) produced an increase in clearance of FITC-dextran-10K and in diameter of pial arterioles. In addition, NG-monomethyl-L-arginine (10 mumol) attenuated glutamate-induced increases in permeability of the blood brain barrier and glutamate-induced dilatation of cerebral arterioles. CONCLUSIONS: The findings of the present study suggest that glutamate, a major neurotransmitter in the brain, increases permeability of the blood-brain barrier to low-molecular weight molecules and dilates cerebral arterioles via a nitric oxide-dependent mechanism. PMID- 8623121 TI - Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. AB - BACKGROUND AND PURPOSE: Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P450 (P450) epoxygenase to epoxyeicosatrienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ current in vascular smooth muscle cells from large cerebral arteries. Because of the close association between astrocytes and the cerebral microcirculation, we hypothesized that brain epoxygenase activity originates from astrocytes. This study was designed to identify and localize an AA epoxygenase in rat brain astrocytes. We also tested the effect of EETs on whole-cell K+ current in rat cerebral microvascular smooth muscle cells. METHODS: A functional assay was used to demonstrate endogenous epoxygenase activity of intact astrocytes in culture. Oligonucleotide primers derived from the sequence of a known hepatic epoxygenase, P450 2C11, were used in reverse transcription/polymerase chain reaction of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned into a plasmid vector and sequenced. A polyclonal peptide antibody was raised against P450 2C11 and used in Western blotting and immunocytochemical staining of cultured astrocytes. A voltage-clamp technique was used to test the effect of EETs on whole-cell K+ current recorded from rat cerebral microvascular muscle cells. RESULTS: Based on elution time of known standards and inhibition by miconazole, an inhibitor of P450 AA epoxygenase, cultured astrocytes produce 11,12- and 14,15-EETs when incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunoreactivity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staining of cultured astrocytes. EETs enhanced outward K+ current in muscle cells from rat brain microvessels. CONCLUSIONS: Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels. PMID- 8623122 TI - Recovery of apparent diffusion coefficient after ischemia-induced spreading depression relates to cerebral perfusion gradient. AB - BACKGROUND AND PURPOSE: Transient decreases of the apparent diffusion coefficient (ADC) of water as measured by fast diffusion-weighted imaging (DWI) in the ischemic border zone are thought to reflect cellular swelling associated with spreading depression. DWI and dynamic contrast-enhanced MRI were applied to study the characteristics of spreading depression and the correlation between ADC recovery time and tissue perfusion in focal ischemia. METHODS: Serial DWI was performed during remote middle cerebral artery occlusion in rats (n = 5) with an echo-planar imaging technique. ADC maps were calculated and ADC values displayed as a function of time in user-defined regions of interest with a time resolution of 12 to 16 seconds. Dynamic contrast-enhanced MRI was performed for qualitative correlation of ADC changes with tissue perfusion. RESULTS: Recovery time of transient ADC decreases correlated with the degree of the perfusion deficit (r = .81, P < .001). Slowly recovering ADC declines were found close to the ischemic core and correlated with severe perfusion deficit, while short-lasting ADC declines were typically found in moderately malperfused or normal tissue. Transient ADC decreases originated in the subcortical and cortical ischemic border zones and propagated along the cortex with a velocity of 2.9 +/- 0.9 mm/min. CONCLUSIONS: The variation in the recovery time of transient ADC decreases in the ischemic periphery reflects the gradient of the tissue perfusion. Severely delayed recovery time after spreading depression is thought to represent the ischemic penumbra. PMID- 8623123 TI - Restricted nonacral sensory syndrome. AB - BACKGROUND: Restricted sensory symptoms due to stroke most often occur at distal parts of the body, manifesting as cheiro-oral or cheiro-oral-pedal syndrome. Sensory symptoms restricted to proximal body parts have rarely been recognized. CASE DESCRIPTIONS: I describe four patients presenting with restricted sensory disturbances at the proximal parts of the body. The sensory symptoms were restricted to discrete areas of the contralateral proximal arm, shoulder, trunk, and upper thigh. The face and the distal parts of the extremities were largely spared. On MRI, three patients had a small thalamic infarction and one had a putaminal infarction. CONCLUSIONS: Strategically located minor strokes can produce restricted sensory syndromes at discrete areas of nonacral parts of the body. These observations highlight the diverse patterns of restricted sensory syndromes after unilateral stroke and support the theory of somatotopic-anatomic proximity of certain parts of the body in the human sensory pathway. PMID- 8623124 TI - Late contralateral hyperhidrosis in lateral medullary infarcts. AB - BACKGROUND AND PURPOSE: This study describes unilateral increases of sweating reactions observed in the months after contralateral medullary infarct; evaluation of sympathetic cutaneous response may help to explain sweating disorders. SUMMARY OF REPORT: After the discovery of the clinical phenomenon in one case, patients admitted between 1990 and 1993 were systematically evaluated clinically and electrophysiologically. In a group of five patients presenting with lateral or dorsal medullary lesions, two exhibited an increase of contralateral sweating reactions that appeared 6 to 8 months after stroke, were elicited by effort and exposure to heat and stress, and were more severe over the forehead, face, and upper trunk. In one case, this was clinically associated with an absence of sweating on the side of the lesion. During the late phase after stroke, in three patients presenting with lateral medullary lesions, electrophysiological evaluation revealed significant asymmetry of the sympathetic skin response, which was higher on the side contralateral to the lesion than on the ipsilateral side. In one patient, no response could be elicited by stimulations applied on the side of the lesion. CONCLUSIONS: Contralateral hyperhidrosis can be observed in the late phase after lateral medullary infarct and is likely due to lesion of the sympathetic pathway passing through the lateral medulla, which inhibits sudomotor neurons. Evaluation of sympathetic skin response may help to explain such clinical disorders. PMID- 8623125 TI - Neurological manifestations of acute posterior multifocal placoid pigment epitheliopathy. AB - BACKGROUND: Acute posterior multifocal placoid pigment epitheliopathy (APMPPE) is a chorioretinal disease that causes acute visual symptoms with characteristic fundus findings. Although this entity has been associated with a variety of neurological complications, it has received little attention in the neurological literature. We wanted to emphasize the spectrum of neurological involvement, in particular the occurrence and management of strokes in patients with APMPPE. CASE DESCRIPTIONS: We report three patients with APMPPE and neurological disease. All three presented with marked visual disturbances and headaches. One patient developed recurrent strokes involving different vascular territories of the brain and required immunosuppressive treatment for presumed cerebral vasculitis. The other two patients had cerebrospinal fluid pleocytosis and persistent headaches but recovered spontaneously. The review of the literature demonstrates a particular pattern of neurological complications in a subgroup of patients with APMPPE. CONCLUSIONS: APMPPE should be considered among the causes of stroke and aseptic meningitis in young adults. The diagnosis is critically dependent on a thorough ophthalmologic examination. Severe neurological complications are difficult to predict at the onset of the ophthalmologic disease. The patients should be monitored closely. If investigations suggest cerebral vasculitis, immunosuppressive treatment may be helpful to prevent recurrences. PMID- 8623126 TI - Blood group antigens: so many jobs to do. PMID- 8623128 TI - Effects of the temperature, the duration of frozen storage, and the freezing container on in vitro measurements in human peripheral blood mononuclear cells. AB - BACKGROUND: Bone marrow, peripheral blood, and umbilical cord blood have been used to prepare autologous and allogeneic pluripotential mononuclear cells for use in the repopulation of bone marrow. STUDY DESIGN AND METHODS: The purpose of this study was to evaluate how the temperature and duration of frozen storage of human peripheral blood mononuclear cells (PBMCs), as well as the freezing container, affected the in vitro recovery and viability of the mononuclear cells and their growth in colony-forming unit-granulocytic-erythroid-monocytic megakaryocytic (CFU-GEMM) tissue culture assay. PBMCs were isolated from ficoll hypaque-treated cellular residue obtained during the plateletpheresis of blood from 15 healthy donors. The PBMCs were treated with dimethyl sulfoxide (DMSO) to achieve a final DMSO concentration of 10 percent. Each unit was then separated into six aliquots: one stored in a polyvinylchloride (PVC) plastic bag, one in a polyolefin plastic bag, and four in polyethylene cryostorage vials. Each aliquot was frozen in a -80 degrees C mechanical freezer at a freezing rate of 2 to 4 degrees C per minute. The frozen PBMCs in PVC bags were stored in a -80 degrees C mechanical freezer and those in polyolefin bags in a -135 degrees C mechanical freezer. Each of the four frozen samples in a vial was stored at a different temperature: one in the -80 degrees C freezer, one in the -135 degrees C freezer, one in the vapor phase of liquid nitrogen at -150 degrees C, and one in liquid nitrogen at -197 degrees C. Some of the frozen PBMCs were stored for periods of 1 to 1.5 years and others for 2 to 2.4 years, after which they were thawed, washed, and tested. RESULTS: The samples stored in PVC bags and those stored in polyolefin bags exhibited in vitro recoveries that were 90 percent of the recovery of fresh PBMCs and viabilities of 90 percent after 2.4 years of frozen storage. The PBMCs stored in PVC bags exhibited no loss of CFU-GEMM activity after 1 to 1.5 years, but a 40-percent loss of activity was observed after 2 to 2.4 years. PBMCs stored in polyolefin bags, however, exhibited no loss of CFU GEMM activity, even after 2 to 2.4 years of storage. In vitro recovery was significantly lower in PBMCs stored in vials at -80 degrees C or -135 degrees C than in cells stored in PVC or polyolefin bags at these temperatures, both in the 1- to 1.5-year and the 2- to 2.4-year time frames. In vitro recovery and viability were similar in PBMCs stored in vials at -80 degrees C, -135 degrees C, -150 degrees C, and -197 degrees C. The growth patterns in the CFU-GEMM assay in PBMCs stored in vials were significantly lower after storage at -80 degrees C than after storage at -135 degrees C, -150 degrees C, or -197 degrees C. CONCLUSION: PBMCs isolated by leukapheresis and ficoll-hypaque treatment can be frozen with 10-percent DMSO in a -80 degrees C mechanical freezer. When a PVC bag is used for freezing and storage of PBMCs at -80 degrees C, the duration of frozen storage should not exceed 1.5 years, whereas PBMCs frozen in a polyolefin bag can be stored in a -135 degrees C freezer for as long as 2.4 years. When these guidelines were followed, in vitro recovery was 90 percent that of fresh PBMCs, viability was 90 percent, and growth in the CFU-GEMM tissue culture assay was similar to that of fresh PBMCs. The PBMCs frozen and stored in PVC or polyolefin bags exhibited satisfactory results, whereas those stored in cryostorage vials did not. PMID- 8623127 TI - A prospective, randomized study of the use of platelet concentrates irradiated with ultraviolet-B light in patients with hematologic malignancy. AB - BACKGROUND: Irradiation of platelet concentrates (PCs) with ultraviolet-B (UVB) light inactivates the contaminating white cells and might be an alternative to filtration for the prevention of alloimmunization to HLA antigens and subsequent refractoriness to further platelet transfusions in multiply transfused patients with bone marrow failure. STUDY DESIGN AND METHODS: Patients with hematologic malignancy, mainly acute myeloid leukemia, were prospectively assigned in a random manner to receive either UVB-irradiated or control, nonirradiated PCs. All patients were given red cells that were white cell reduced by filtration. Transfusion efficacy and alloimmunization were assessed by means of corrected count increments, requirement for red cells and PCs, and measurement of lymphocyte-reactive antibodies. RESULTS: UVB-irradiated PCs had a clinical efficacy similar to controls as judged by corrected count increments at 1 to 6 and 12 to 24 hours and by the median requirement for red cell and platelet transfusions. Alloimmunization determined by measurements of lymphocyte-reactive antibodies using both conventional and antiglobulin-augmented lymphocytotoxicity techniques was not abolished in recipients of UVB-irradiated PCs (4/30, 13%) but was less than that in controls (5/20, 25%; p = NS). The mean number of platelet transfusion episodes prior to the occurrence of alloimmunization was greater in the control group (27 vs. 10; p = 0.017). CONCLUSION: In this trial, UVB irradiation did not diminish the clinical efficacy of platelet transfusions. There was a small but nonsignificant reduction alloimmunization, but no difference in refractoriness of the two groups was observed. Larger prospective randomized studies are required to confirm these findings and to compare UVB irradiation with white cell reduction. PMID- 8623129 TI - Polymorphisms at the ABO locus in subgroup A individuals. AB - BACKGROUND: The common ABO allele sequences are known, but little or no genetic information is available on the rare but important A subgroups. STUDY DESIGN AND METHODS: Blood group ABO polymorphism was analyzed in genomic DNA from 45 rare subgroup A individuals by sequence-specific primer polymerase chain reaction and amplified fragment length polymorphism investigating exons VI and VII in the ABO genes. These methods are used to detect specific mutations only, and not all changes that might be present can be detected. ABO genotypes discriminating six alleles (A1, A2, B, O1, O1var, and O2) were determined. RESULTS: The C-->T substitution at nucleotide position 467 (C467T) is not restricted to A2 and cis AB individuals, but was found also in some A subgroups. Detection of the functionally more relevant C1060-single-point deletion in A2 was accomplished by a novel sequence-specific primer polymerase chain reaction approach. A 100 percent correlation between the C467T and the C1060-mutations was found. Fifteen of 17 samples showing the T646A mutation (described earlier in one case of Ax) showed a positive correlation with the C771T mutation in a frequently occurring O1var allele. The two exceptions were defined serologically as Ax. CONCLUSION: Indications have been found of an evolutionary relationship between A1 alleles and Ael and A3 subgroups as well as between A2 alleles and Aend and Aweak subgroups. Genetic heterogeneity within the Ax and Aint subgroups was also seen. PMID- 8623130 TI - Circulating red cells usually remain of host origin after bone marrow transplantation for severe combined immunodeficiency. AB - BACKGROUND: Patients with severe combined immunodeficiency (SCID) treated with allogeneic bone marrow transplantation often receive a milder conditioning regimen than patients who undergo transplantation for hematologic malignancy, and they regularly retain circulating white cells of host origin. The origin of circulating red cells following successful bone marrow transplantation to treat SCID is not known. STUDY DESIGN AND METHODS: Review of the medical records identified all patients with SCID who underwent ABO-mismatched bone marrow transplantation at the University of California, San Francisco, between 1982 and 1994. The ABO and Rh phenotype at >6 months after transplantation was determined for all successful transplants by review of the medical record or the taking of a fresh blood sample for analysis. Patient-conditioning and donor bone marrow preparative regimens were reviewed to assess their possible influence on the red cell phenotype after successful bone marrow transplantation. RESULTS: Nine of 35 SCID patients who underwent successful transplantation received marrow from ABO mismatched donors. Eight of the nine patients had only host red cells circulating at 6 to 84 months after transplantation, while one patient had only donor red cells circulating at 48 months after transplantation. None of the patients had circulating red cells of both host and donor origin. Conditioning regimens included cyclophosphamide and antithymocyte globulin for all nine patients; only three patients also received total body irradiation. Seven of the nine patients received related-donor, HLA-mismatched bone marrow, and two patients received HLA identical bone marrow; eight patients received T-cell-depleted bone marrow. The one patient whose red cell phenotype converted to that of the donor received T cell-depleted, haploidentical marrow, and the preparative regimen included chemotherapy and total body irradiation. CONCLUSION: SCID patients successfully treated with allogeneic bone marrow transplantation typically fail to show circulating red cells of donor phenotype; this finding is in contrast to the universal presence of circulating donor red cells following successful bone marrow transplantation to treat hematologic malignancies and other diseases. The milder conditioning regimens typically given to patients with SCID, along with T cell depletion and HLA mismatching, may play a role in this different outcome. It is not known whether the inability to find circulating red cells of donor origin is due to a failure to engraft donor pluripotent stem cells or a failure of engrafted donor stem cells to differentiate along the erythroid lineage. PMID- 8623131 TI - In vitro and in vivo persistence of reticulocytes from donor red cells. AB - BACKGROUND: Reticulocytes are important in the phenotyping of transfused patients. Reticulocytes can persist in blood units for the shelf life of the unit. STUDY DESIGN AND METHODS: Temperature dependence of reticulocyte persistence was examined in vitro at 4, 24, and 37 degrees C by using thiazole orange staining and flow cytometric analysis. Two-color flow cytometric analysis was used to evaluate the persistence of donor reticulocytes in transfused patients. RESULTS: Flow cytometric analysis using thiazole orange demonstrated that persistence of reticulocytes in units of stored CPDA-1 blood was temperature dependent. Reticulocytes disappeared over 13 and 6 days at 24 degrees C and 37 degrees C, respectively, but at 4 degrees C the reticulocyte count changed little over 35 days. Two-color flow cytometric analysis of reticulocyte antigens was used to follow donor reticulocytes in 14 transfusion events in nine different patients. Donor reticulocytes persisted through 24 hours in 75 percent of the patients and were detectable at 48 hours in three patients. CONCLUSION: This study demonstrates that reticulocytes persist during refrigerated storage; they are detectable in the circulation of most recipients for the first 24 hours after transfusion and in the circulation of a few recipients after 48 hours. These findings may have relevance for separation techniques based on reticulocyte density in samples drawn shortly after transfusion and for evaluation of reticulocyte counts in patients with hematologic abnormalities. PMID- 8623132 TI - Optimal conditions for white cell reduction in red cells by filtration at the patient's bedside. AB - BACKGROUND: A quality control program of white cell (WBC) reduction in red cells at the bedside was implemented, based on postfiltration counting in a Nageotte chamber of the residual WBCs from samples taken from a segment of the transfusion set, after 1-in-10 sample dilution with Turks's solution. During a 1-year quality control program, 5.1 percent of counted units had apparent filtration failures, that is, WBC counts exceeding 5 x 10(6) per unit. The cause(s) for these apparent failures were investigated. STUDY DESIGN AND METHODS: In Study 1, residual WBCs from 150 buffy coat-free red cells filtered through one type of filter at 4 degrees C, 20 to 24 degrees C, or 27 degrees C in 5 to 10 minutes, 50 to 100 minutes, or 100 to 200 minutes were counted as described above. In Study 2, residual WBCs in samples collected from segments of the transfusion set and from the postfiltration bags were counted in parallel by a new, more sensitive counting method. In this method, 5 mL of filtered red cells was diluted with 20 mL of 3-percent paraformaldehyde and centrifuged, the pellet was resuspended to 500 microL with a lysis solution, and the WBCs were counted in a Nageotte chamber. In Study 3, residual WBCs were counted by the 3-percent paraformaldehyde method in samples from postfiltration bags of 1- to 2-day-old buffy coat-rich red cell units filtered through a second type of filter. Filtration was started within 30 minutes of the removal of the unit from the refrigerator, ambient temperature was 20 to 24 degrees C, and the median filtration time was 90 minutes per unit. RESULTS: Study 1: Median WBC counts per unit increased progressively from 51,000 at 4 degrees C to 934,000 at 27 degrees C, with intermediate values at 20 to 24 degrees C. In no unit did the WBC count exceed 5 x 10(6) if filtration at 20 to 24 degrees C was completed within 100 minutes, while counts in excess of 50 x 10(6) were found at 20 to 24 degrees C and at 27 degrees C with filtration times of 100 to 200 minutes, and 50 to 100 minutes, respectively. Study 2: The relation between segment and postfiltration bag WBC counts obtained by the 3-percent paraformaldehyde method was poor, with the latter being almost always lower than the former. Study 3: None of the 120 units filtered through the second type of filter at 20 to 24 degrees C in 50 to 100 minutes contained more than 3.2 x 10(6) WBCs; the median value was 147,000 WBCs per unit. CONCLUSION: On the basis of the results with the 3-percent paraformaldehyde method, which showed the unreliability of segment counts, a new policy was adopted for quality control of bedside WBC reduction, based on controlling the time of and temperature at transfusion. Bedside WBC reduction in 1- to 2-day-old red cells performed with the second type of filter at 20 to 24 degrees C in less than 100 minutes per unit allowed the preparation of units that meet the standard of fewer than 5 x 10(6) WBCs in all tested cases. Bedside WBC reduction with the second type of filter and under the controlled conditions reported seems effective. PMID- 8623133 TI - Treatment of chronic autoimmune thrombocytopenic purpura with monoclonal anti-D. AB - BACKGROUND: The platelet count increases transiently after treatment with polyclonal anti-D in about 50 percent of D+ patients with autoimmune thrombocytopenic purpura (AITP). The effect is usually attributed to macrophage Fc-receptor blockade by antibody-coated red cells. As polyclonal anti-D is in limited supply, prospective testing was performed on a monoclonal anti-D (MoAb D) in such patients. STUDY DESIGN AND METHODS: Seven D+ patients with chronic AITP received MoAb D intravenously at doses of 47 to 95 microg per kg of body weight. Response was assessed by studying platelet count increment. Hemolysis and red cell-bound MoAb D were measured before and after MoAb D administration. RESULTS: MoAb D red cell binding was demonstrated in all patients at a ratio higher than that observed in AITP patients successfully treated with polyclonal anti-D. However, little or no platelet count increment was observed in six patients, while a transient response was observed in only one (platelet count 97 x 10(9)/L before MoAb D infusion and 163 x 10(9)/L 4 days later). Furthermore, because five patients showed signs of hemolysis and two became anemic, higher doses of MoAb D should be used only with caution in patients with AITP. CONCLUSION: The MoAb D used in this study cannot be proposed as an alternative treatment for patients with AITP. PMID- 8623134 TI - Incidence of the Nak(a)-negative platelet phenotype in African Americans is similar to that of Asians. AB - BACKGROUND: About 5 to 10 percent of Asians have platelets that lack the major membrane glycoprotein (GP) IV (CD36, GPIIIb) that carries the isoantigen Naka. The GPIV-negative platelet phenotype is extremely rare among whites, but its frequency in persons of African ancestry has not yet been determined. Isoimmunization against GPIV can occur in GPIV-negative persons and can lead to platelet transfusion refractoriness. Therefore, the expression of GPIV on platelets from unrelated African Americans was studied. STUDY DESIGN AND METHODS: Platelets were obtained from 250 African American and 280 white blood donors. Flow cytometry was used to determine the ability of these platelets to bind a monoclonal antibody that reacted with GPIV. Platelets that failed to react with this probe were tested with other GPIV-specific monoclonal antibodies and with anti-Naka, an isoantibody that recognizes an epitope on GPIV. RESULTS: Platelets from 6 of the 250 African American donors (2.4%) lacked GPIV and failed to bind anti-Naka, whereas platelets from all of the white donors were GPIV positive (p>0.05). No platelet-reactive antibodies were identified in the serum of the GPIV-negative donors. CONCLUSION: The frequency of the GPIV-negative platelet phenotype in African Americans is comparable to that in Asians and much greater than that in whites. Studies are needed to determine the frequency with which African Americans become isoimmunized to GPIV by transfusions and the possible contribution of this isoimmunization to platelet transfusion refractoriness in this population. PMID- 8623135 TI - Development and evaluation of a shipping system for platelet components. AB - BACKGROUND: Platelet concentrates and apheresis platelets must be maintained at a temperature as close as possible to 20 to 24 degrees C during transport. To improve temperature control, ensure component quality, and meet handling and freight carrier needs, a new insulated shipping container system was developed and evaluated. STUDY DESIGN AND METHODS: Molded polyurethane-insulated shipping containers were loaded with different payloads of simulated platelet components, with or without gel-based temperature stabilizing packs (TSPs). The containers were subjected to constant ambient temperature of 37, 4 or -10 degrees C. Payload temperatures were continuously monitored, in situ, for 24 hours. RESULTS: Temperature data are reported as the mean number of hours needed for components to warm or cool by 1 degree C. The temperature of payloads exposed to a constant 37 degrees C ambient temperature increased by 1 degree C in 2.5 to 3.8 hours when no TSPs were included in the shipment and in 6.1 to 6.9 hours when TSPs were used. Exposure to a constant 4 degrees C ambient temperature resulted in a 1 degree C temperature decrease in 1.8 to 3.4 hours without TSPs and in 4.6 to 5.6 hours with TSPs. At a -10 degrees C ambient temperature, there was a 1 degree C drop within 1.0 to 1.6 hours without TSPs and within 2.7 to 2.9 hours with TSPs. CONCLUSION: The container and packing methods described moderate the rate of change in the temperature of platelet components during their exposure to challenging ambient conditions. The use of TSPs substantially improves the performance of the system. In addition, the system meets freight carrier requirements and is easy to use, environmentally friendly, and durable. PMID- 8623136 TI - Unexplained hepatitis C virus antibody seroconversion in established blood donors. AB - BACKGROUND: Understanding of the epidemiology and natural history of hepatitis C virus (HCV) infection is incomplete without reference to the early phase of infection. The prevalence of HCV infection is well documented in numerous reports. The seroconversion pattern in previously antibody-negative blood donors provides a model for the study of the incidence and transmission of HCV infection. STUDY DESIGN AND METHODS: Records of HCV antibody tests at the West Midlands Blood Transfusion Centre were reviewed to determine the seroconversion rate in 1994 among previously anti-HCV-negative blood donors. Seroconverting donors were counseled to investigate the possible routes of infection. RESULTS: In 1994, blood donations (n = 256,935) were collected from 149,370 donors; 24 donors (0.016%; 1/6224) were positive in the screening enzyme-linked immunosorbent assay (ELISA) and the third-generation recombinant immunoblot assay (RIBA-3). Two donors previously negative for HCV antibody in ELISA were positive in both tests in 1994. Four donors positive in ELISA and indeterminate in RIBA-3 in 1993 reacted positively in both tests in 1994. One donor negative for HCV antibody on previous screening reacted positively in ELISA and was indeterminate in RIBA-3 in 1994 and has become positive in both tests in 1995. A further 43 donors negative for HCV antibody on previous screening reacted positively in ELISA and were indeterminate in RIBA-3 in 1994. CONCLUSION: Documented seroconversion can take place in the absence of exposure to recognizable risk factors for the infection. The index donation or the donation immediately preceding seroconversion may be positive for HCV RNA in the polymerase chain reaction. PMID- 8623137 TI - Sensitivity and specificity of four assays to detect human T-lymphotropic virus type I or type I/II antibodies. AB - BACKGROUND: Assays that detect human T-lymphotropic virus type I and type II antibody (HTLV-I/II) are widely used in the routine screening of blood donors. STUDY DESIGN AND METHODS: Four commercially available anti-HTLV-I (Fujirebio and Organon Teknika) or -HTLV-I/II assays (Murex and Ortho) were evaluated in various serum panels: A) HTLV-I-positive specimens (n = 41), confirmed by Western blot and polymerase chain reaction; B) a commercially available anti-HTLV-I/II panel; C) serial dilutions of sera from HTLV-I-positive individuals (n = 30), confirmed by immunofluorescence assay and Western blot: D) serial dilutions of HTLV-II positive blood donors (n = 20), confirmed by Western blot and polymerase chain reaction, and E) sera from first-time blood donors (n = 1055). RESULTS: All four assays elicited reactions in all 82 HTLV-I-positive samples in Panels A, B, and C. Of 32 HTLV-II-positive specimens in Panels B and D, 31 (96.9%) reacted in the Organon Teknika assay and all 32 reacted in the remaining tests. Probit analysis of test results in Panels C and D indicated that the Fujirebio test was the most sensitive assay, followed by Organon Teknika, Ortho, and Murex. The specificities of Fujirebio, Murex, Organon Teknika, and Ortho tests in 1055 first-time blood donors were 99.9, 100, 99.6, and 99.9 percent, respectively. CONCLUSION: All four studied assays for detecting HTLV-I or HTLV-I/II antibodies are appropriate as screening tests. PMID- 8623139 TI - Fatal hemolytic transfusion reaction resulting from ABO mistyping of a patient with acquired B antigen detectable only by some monoclonal anti-B reagents. AB - BACKGROUND: Some monoclonal anti-B reagents are prepared exclusively from an anti B clone, ES4, that is known to detect acquired B antigens that are not detectable by other anti-B clones or polyclonal anti-B reagents. CASE REPORT: A 92-year-old group A, Rh-negative man with diverticulitis was mistyped as group AB with the use of a monoclonal anti-B. The hospital did not detect anti-B in the patient's serum. After a negative antibody screen, blood was issued through an abbreviated crossmatch (i.e., immediate-spin crossmatch). The patient was given 3 units of group AB blood and 1 unit of group A blood, and no problems were reported. After the transfusion of a ?fourth unit of AB blood the patient had a severe hemolytic transfusion reaction which resulted in kidney failure and death 10 days later. After the transfusion reaction, the patient's pretransfusion red cells were found to be group A with an acquired B antigen. The monoclonal anti-B used the hospital was formulated from the ES4 clone. A sample of the patient's serum taken before the transfusion was later found to contain a weak anti-B, detectable most obviously by the antiglobulin test, which was not performed at the crossmatch stage. The manufacturers of monoclonal anti-B reagents prepared from ES4 have since modified their reagents (i.e., lowered the pH) so that they now detect only the strongest examples of acquired B antigen. CONCLUSION: A fatal hemolytic transfusion reaction resulted because a monoclonal anti-B that detected acquired B antigen was used to type red cells from an elderly man whose serum had weak anti-B that was not detected by abbreviated compatibility testing. PMID- 8623138 TI - A novel system for providing compatible blood to patients during surgery: "self service" electronic blood banking by nursing staff. AB - BACKGROUND: A good blood bank must be able to provide compatible blood units promptly to operating room patients with minimal wastage. A "self-service" by nursing staff blood banking system that is safe, efficient, and well-accepted has been developed. STUDY DESIGN AND METHODS: Specific blood units are no longer assigned to surgical patients who have a negative pretransfusion antibody screen, irrespective of the type of surgery. A computer-generated list of the serial numbers of all group-identical blood units currently in the blood bank inventory is provided for each patient. The units themselves are not labeled with a patient's name. The group O list will be provided for group O patients, the group A list for group A patients, and so forth. Should the patient require transfusion during surgery, the operating room nurses go to the refrigerator, remove any group-identical unit, and check the serial number of the unit against the serial numbers on the patient's list. If the serial number is on that list, the blood bank will accept responsibility for compatibility. The system was implemented in 1995. RESULTS: Since implementation, a total of 2154 patients have undergone operations at this hospital. Thirty-two patients received more than 10 units of red cells each. There were no transfusion errors. The crossmatch-to-transfusion ratio was reduced from 1.67 to 1.12. Turnaround time for supplying additional or urgent units to patients in operating room was shortened from 33 to 2.5 minutes. There was no incidence of a blood unit's serial number not being on the list. Work by nurses and technical staff was reduced by nearly 50 percent. CONCLUSION: The "self-service" (by nursing staff) blood banking system described is safe and efficient. It saves staff time and can be easily set up. PMID- 8623140 TI - Anaphylaxis after treatment with recombinant factor VIII. AB - BACKGROUND: Treatment of hemophilia patients with recombinant factor VIII concentrates has not previously been associated with anaphylaxis. STUDY DESIGN AND METHODS: A 5-week-old boy with severe hemophilia A developed dyspnea, cyanosis, hypotension, and a diffuse urticarial rash following treatment with a recombinant factor VIII (Recombinate). To identify the cause of anaphylaxis in this patient, the vial lot was examined for the presence of endotoxin, and a checkerboard immunoblotting technique was used to test serum and/or plasma samples from the patient and mother for the presence of antibodies (IgA, IgG, IgE, and IgM) to Recombinate-related antigens (recombinant factor VIII, von Willebrand factor, human serum albumin, Chinese hamster ovary proteins, bovine serum albumin, mouse monoclonal anti-human factor VIII, polyethylene glycol 3350), and to ethylene oxide, the agent used to sterilize the infusion equipment. RESULTS: No immune response directed against the Recombinate-related antigens or ethylene oxide that could be associated with the anaphylactic reaction was identified. Endotoxin was not present upon rabbit pyrogen testing of the therapeutic product. CONCLUSION: These studies failed to show any association between Recombinate and the onset of the allergic reaction. This seems to be the first reported case of anaphylaxis following the infusion of a recombinant form of factor VIII concentrate. PMID- 8623141 TI - Human blood groups: incidental receptors for viruses and bacteria. PMID- 8623142 TI - Data on apheresis, blood collection, and transfusion-related activities: statistical analyses of the American Association of Blood Banks institutional membership questionnaires. AB - BACKGROUND: The American Association of Blood Banks annually surveys institutional members on activities pertinent to blood collections, apheresis, and transfusions. STUDY DESIGN AND METHODS: Retrospective descriptive statistics and comparative statistical analyses including trend tests were performed on selected topics from the 1989, 1990, and 1991 Institutional Questionnaires. The data were compiled by institution type, namely, regional and community blood donor collection centers and hospital-based facilities. Evaluated topics included the apheresis and therapeutic procedures performed, transfusion-associated AIDS and hepatitis, and the blood components (red cells, platelets, fresh-frozen plasma, and cryoprecipitate) that were collected, transfused, or outdated or discarded. RESULTS: Significant findings (p<0.05) included upward trends over time in the numbers of donor plateletpheresis units collected and transfused and in the numbers of random-donor platelet concentrates collected by hospitals. There was an upward trend over time in the outdating or discarding of all blood component types that was reported by hospitals. Data from blood centers showed the outdating or discarding of significant numbers of apheresis platelets, fresh frozen plasma, and cryoprecipitate. No significant trends were identified in the reported cases of transfusion-associated hepatitis or AIDS. CONCLUSION: Ongoing data analysis of the institutional questionnaires provides information on trends in blood collection and transfusion-related activities. PMID- 8623143 TI - Cost-effectiveness of p24 antigen testing in preventing transfusion transmission of human immunodeficiency virus infection. PMID- 8623145 TI - Autoadsorptions for the detection of alloantibodies--should polyethylene glycol be used? PMID- 8623144 TI - Testing for human immunodeficiency virus p24 antigen. PMID- 8623146 TI - Quality system for test laboratories. PMID- 8623147 TI - Hepatitis B surface antigenemia in blood donors after vaccination. PMID- 8623148 TI - Identification and characterization of a galactosyl peptide mimetic. Implications for use in removing xenoreactive anti-A Gal antibodies. AB - Gal alpha 1,3 Gal is thought to be the major antigenic epitope present on pig tissues to which XNAs bind. Removal of antibodies directed against that structure may be critical to the success of pig to human xeno-transplantation. As a first step toward the development of ligands capable of removing XNAs, we have used a phage-displayed peptide library to identify a six-amino-acid peptide that binds to the lectin GS-1-B4 (which binds the carbohydrate Gal alpha 1,3 Gal). This peptide blocks the binding of GS-1-B4 to pig aortic endothelial cells. The carbohydrate Gal alpha 1,3 Gal competes with the binding of GS-1-B4 to the peptide, suggesting that they may bind the same site. Using a RBC agglutination assay, we show that this peptide inhibits the agglutination of pig RBCs by heat inactivated human serum at concentrations similar to that of Gal alpha 1,3 Gal. PMID- 8623149 TI - Concurrent MCMV infection augments donor antihost-specific activity and alters clinical outcome following experimental allogenic bone marrow transplantation. AB - The present studies were undertaken to examine whether concurrent MCMV infection during allogeneic bone marrow transplantation (BMT) could alter the developing donor-host immune interactions and affect the overall outcome of the transplant. In order to determine the effect of MCMV on antihost activity arising following an allogeneic BMT, specific donor antihost cytotoxicity was examined. The results demonstrated that concurrent virus infection in mice receiving a BMT from donors either H2-matched and non-MHC-mismatched or mismatched at both MHC and non-MHC transplantation loci, augmented antihost cytotoxic activity mediated by CD8+ T cells assayed directly from the recipient's spleen 10-14 days posttransplant. Notably, allogenic BMT recipients receiving either lethal or nonlethal numbers of donor T cells and inoculated with MCMV exhibited more rapid and profound weight loss compared with uninfected allogeneic and syngeneic BMT recipients. Concurrent virus presence also resulted in a markedly increased incidence of mortality in allogeneic BMT recipients of nonlethal numbers of T cells. We conclude from these findings that when virus is present early after allogeneic BMT, the resulting interactions can potentiate T cell-mediated donor-antirecipient--i.e., graft vs. host-reactivity. In total, the results support the notion that pathogens could complicate allogeneic BMT by contributing to the development of graft vs. host disease. PMID- 8623150 TI - Thrombin inhibition in an ex vivo model of porcine heart xenograft hyperacute rejection. AB - Prominent components of vascularized xenograft rejection such as platelet activation and microvascular thrombosis may be dependent upon thrombin generation in vivo. To study potential therapeutic benefits of a synthetic low-molecular weight thrombin inhibitor, SDZ MTH 958, in hyperacute porcine heart rejection by human blood ex vivo, a working model of hyperacute rejection of porcine by fresh, heparinized (6 microM/ml) human blood with or without 1 microM SDZ MTH 958 was used. Thrombin-antithrombin complexes (TAT) and prothrombin fragment F1.2 levels as markers of thrombin activation were determined, and biopsies from rejected hearts were analyzed by immunohistopathology. Control porcine hearts (n=8) underwent a rapid and consistent decline in cardiac output, ceasing function by 60 min. Experimental cardiac output values of 14 ml/g (SEM 1.2) were significantly higher than seen in controls (5 ml/g SEM 0.6) after 5 min of cardiac work, and prolonged survival times up to 120 min were noted (P<0.05). Activity of SDZ MTH 958 was confirmed by functional assays throughout perfusion. Levels of TAT and F1.2 increased consistently in control samples when compared with plasma samples containing SDZ MTH 958. Immunohistopathological examination confirmed diminished fibrin deposition, reduced leukocyte adherence to endothelium, impaired diapedesis and less tissue necrosis in the hearts perfused with SDZ MTH 958. SDZ MTH 958, in this xenoperfusion model, prolonged survival, enhanced function of the explanted organ, and improved histological features at the time of rejection. Effective and specific antagonism of thrombin may be useful as an adjunct therapy to complement inhibition for xenograft rejection PMID- 8623152 TI - The pharmacokinetics of a microemulsion formulation of cyclosporine in primary renal allograft recipients. The Neoral Study Group. AB - This study was a randomized, double-blind, 12-week comparison of the pharmacokinetics, safety, and tolerability of two cyclosporine (CsA) formulations, cyclosporine emulsion capsules and oral solution for microemulsion and cyclosporine, in the postoperative management of renal transplant patients. Of the 101 patients, aged 18 to 65, who entered the study, 89 were evaluable for pharmacokinetics. Initial dosage was 10 mg/kg per day, administered twice daily in two equal doses. Dosages were adjusted to achieve target CsA concentrations. The pharmacokinetic (PK) parameters (dose-normalized) of greatest interest were maximum blood concentration (C(max)/dose), time to reach maximum concentration (t(max), area under the blood concentration-vs.-time curve (AUC/dose), and trough blood concentrations (Co h/dose). The relative CsA bioavailabilty was found to be significantly enhanced with cyclosporine emulsion compared with cyclosporine with a 16% to 31% increase in AUC and a 32% to 42% increase in C(max). Intrapatient variability of PK parameters was significantly lower with cyclosporine emulsion than with cyclosporine for AUC, C(oh), t(max), and C(max) in many instances. This indicates a more consistent, rapid, and more complete total absorption of CsA. Despite higher CsA C(max) levels and AUCs with cyclosporine emulsion, safety and tolerability (detailed in a parallel report) were comparable to those of cyclosporine. The PK advantages of cyclosporine emulsion over cyclosporine are either independent of food conditions or possibly reflective of more consistent absorption of CsA with cyclosporine emulsion. The findings suggest that de novo use of cyclosporine emulsion may simplify and improve management of organ transplant recipients and that the PK advantages of cyclosporine emulsion may translate into clinical benefits. PMID- 8623151 TI - Changes in glucose transporter 2 and carbohydrate-metabolizing enzymes in the liver during cold preservation and warm ischemia. AB - In order to examine glucose metabolism in liver grafts during cold preservation (24 and 48 hr), warm ischemia (60 and 120 min), a combination of the two and reperfusion, the amount of protein and mRNA of glucose transporter 2 and the activities of enzymes in glycolysis (glucokinase, phosphofructokinase, pyruvatekinase), gluconeogenesis (glucose 6-phosphatase, fructose 1,6 bisphosphatase), and the pentose phosphate pathway (glucose 6-phosphate dehydrogenase) were measured. It appeared that glucose transport, the pentose phosphate pathway, and gluconeogenesis were maintained during cold preservation and warm ischemia. The activity of glucokinase significantly decreased from the control value of 1.33 +/- 0.23 IU/g protein to 0.70 +/- 0.17 (24 hr, P<0.05) and 0.57 +/- 0.12 (48 hr, P<0.01) only during cold preservation. However, the activity of phosphofructokinase significantly decreased from the control value of 4.37 +/- 0.06 IU/g protein to 2.67 +/- 0.15 (60 min, P<0.0001) and 1.53 +/- 0.06 (120 min, P<0.0001) only during warm ischemia. This indicates that glycolysis deteriorates during both cold preservation and warm ischemia and demonstrates further that the balance between glycolysis and gluconeogenesis shifts to gluconeogenesis. Even when cold preservation was combined with warm ischemia, the activity of glucokinase decreased only during cold preservation and the activity of phosphofructokinase decreased only during warm ischemia. Furthermore, these changes were time-dependent. It is suggested that they can be used as a clock to measure the durations of cold preservation and warm ischemia separately and that the magnitude of an ischemic injury to a liver and a liver graft's viability can be indirectly estimated before transplantation. PMID- 8623153 TI - HLA-identity--long-term renal graft survival, acute vascular, chronic vascular, and acute interstitial rejection. AB - The object is analysis of the impact of acute and chronic rejection on long-term function in HLA-identical renal transplants performed from 1967 to 1995 by the Saskatchewan Renal Transplant Unit. Forty-eight grafts in 46 patients were studied, of which 39 were first and nine second grafts. Forty-two were for primary and six for secondary renal disease. Thirty-five received azathioprine/prednisone prophylaxis, and 13 received cyclosporine/prednisone with/without azathioprine. Ten-year all graft actuarial survival was 84%, 10-year actuarial graft survival in patients with primary renal disease 90%, and with subsequent graft after first HLA graft failed 97.5%, for age-matched population 98.5% (P=NS). Overall death rate was 8.7% (4/46); in secondary renal disease patients 50% (3/6); in primary renal disease patients 2.5% (1/40, P=0.004). All (9/9) HLA-identical second grafts functioned. Acute rejection with azathioprine/prednisone prophylaxis occurred in 55% (9/17) of grafts treated with <6 pre-graft blood transfusions, with the same prophylaxis but >5 units in 12% (2/16, P=0.015), and with cyclosporine prophylaxis in 13% (2/15, P=0.021). Pulse steroids alone reversed all acute rejection. Grafts failed in 6.2% (3/48), all in primary renal disease patients and one from technical one noncompliance, and one chronic rejection. Graft cost/patient/year amortized over 9 years is $3,855 and comparable dialysis cost would be $35,650; cost for all patients on dialysis for 9 years would be $11,293,320 while comparable graft cost was 1,221,418, a savings of 89.2%. Our conclusions are that HLA-identity associates with the following: (1) a 10-year actuarial survival in primary renal disease that equals that of the age-matched population, (2) uniform success in repeat grafts, (3) virtual absence of chronic rejection despite a high incidence of acute rejection in azathioprine/prednisone grafts that (4) always reversed on pulse steroids, and (5) a cost reduction for grafting of 93.2% compared with dialysis therapy. PMID- 8623154 TI - A prospective study of hepatitis C virus infection in renal allograft recipients. AB - Hepatitis C virus (HCV) is the predominant cause of posttransplant non-A, non-B hepatitis among renal allograft recipients. Prior studies evaluating the impact of HCV in kidney transplantation have been retrospective in design and based largely on changes in serum transaminases. We studied a group of HCV-infected end stage renal disease patients prospectively with pretransplant liver biopsies and close virologic and biochemical follow-up posttransplant. Fourteen patients have been followed a mean of 11.6 +/- 5.6 months posttransplant (range, 5-21 months). Six had changes of chronic hepatitis on pretransplant liver biopsy while 8 showed only mild histologic abnormalities. Circulating viral titers increased several fold over baseline levels during posttransplant follow-up. Viral replication was particularly enhanced immediately following a course of antilymphocyte therapy. Although all patients showed a 2-3 fold increase in alanine aminotransferase (ALT) following transplantation, there were no association noted between pretransplant liver histology, the use of FK506 and/or cyclosporine-based immunosuppression, and the magnitude of ALT change posttransplant. The only clinical outcome found to differ significantly was a higher incidence of cytomegalovirus infection among patients with chronic hepatitis. All patients are alive with functioning grafts. There have been no episodes of fulmiinant or subfulminant liver failure. We conclude that HCV-infected patients can be safely transplanted with excellent short-term follow-up. Continued monitoring with sequential liver biopsies will be needed to define the long-term course of HCV infection in an immuno-suppressed population. PMID- 8623155 TI - The effect of (steroid) immunosuppression on skeletal muscle glycogen metabolism in patients after kidney transplantation. AB - To examine the mechanisms by which immunosuppression by steroids impairs glycogen synthesis in human skeletal muscle, we measured glycogen synthase protein content and activity in muscle samples from 14 patients receiving corticosteroid therapy after kidney transplantation and in 20 healthy control subjects. A percutaneous muscle sample was taken before and at the end of a euglycemic hyperinsulinemic insulin clamp. Insulin-stimulated glucose disposal was reduced by 33% in kidney transplant patients compared with healthy controls (33.8 +/- 4.2 vs. 50.5 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.01), primarily due to a decrease in nonoxidative glucose metabolism (14.2 +/- 3.3 vs. 32.3 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.001). Glycogen synthase activity measured at both 0.1 mmol/L (17.6 +/- 2.6 vs. 24.0 +/- 2.2 nmol min-1 mg protein-1; P<0.05), and at 10 mmol/L glucose 6 phosphate (24.1 +/- 3.5 vs. 33.7 +- 2.4 nmol min-1 mg protein-1; P<0.05) and glycogen synthase protein concentrations (8.8 +/- 1.8 vs. 18.9 +/- 1.9 relative units per ng DNA; P<0.01) were lower in kidney transplant patients compared with controls. Glycogen synthase protein correlated with nonoxidative glucose metabolism (r=0.42; P=0.04). Alpha-actinin (used as a control of general protein degradation) was lower in kidney transplant patients compared with controls (4.4 +/- 0.8 vs. 9.6 +/- 1.1 cpm/ng DNA; P<0.01). In conclusion, corticosteroids cause insulin resistance, which correlates with impaired activation of glycogen synthase and decreased enzyme protein content. The decrease in glycogen synthase protein may reflect increased degradation rather than a defect in translation. PMID- 8623156 TI - The effect of kidney size on cadaveric renal allograft outcome. AB - Chronic rejection is the commonest cause of long-term renal allograft loss. Though immunologic factors are thought dominant in its pathogenesis, nonimmunologic factors, in particular, hyperfiltration damage related to reduced renal mass, have also been proposed as factors in the causation of chronic allograft rejection. We assessed the influence of renal size on graft survival and function in all cyclosporine-treated cadaver donor adult renal allograft recipients engrafted at a single center between June 1989 and July 1994, whose grafts functioned for > or = to 3 months (n=169). Patients were divided into 4 groups based on the ratio of kidney volume to recipient body surface area (volume/BSA) (ml/m2), and outcome in groups compared by methods including Cox's proportional hazards and Kaplan-Meier analysis. No significant differences between groups existed for serum creatinine levels, presence of significant proteinuria, or 1- and 5-year graft survival. There was no correlation between volume/BSA and either serum creatinine or degree of proteinuria at 3, 6, 12, 36, and 60 months posttransplant. Volume/BSA was similar in patients with good or poor renal function (58 +/-21 vs. 56 +/- 28 ml/m2), with or without significant proteinuria (57 +/- 24 vs. 60 +/- 25 ml/m2) or in patients who lost their grafts to chronic rejection compared with those with stable allograft function (64 +/- 34 vs. 59 +/- 24 ml/m2). Volume/BSA was not a predictor of graft survival on multivariate regression. We conclude that donor kidney size has no apparent effect on cadaveric renal allograft outcome in the short and intermediate-term, suggesting that close matching of donor kidney size to recipient size is not presently indicated. PMID- 8623157 TI - Recombinant human growth hormone treatment. Its effect on renal allograft function and histology. AB - Our objective was to investigate the effects of recombinant human growth hormone (rhGH) treatment on long-term renal allograft function and histopathology. RhGH is a potent therapy for poor growth after renal transplantation. However, rhGH has proinflammatory properties and may induce acute rejection or accelerate chronic rejection. Nine prepubertal rhGH-treated renal transplanted children and nine pair-matched controls were studied 18 (before the start of rhGH) and 36 months after transplantation (mean duration of rhGH-treatment 14 months). 51Cr EDTA- and PAH-clearances were performed. A protocol renal biopsy was done at 36 months. Growth showed significant improvement during rhGH (P<0.01). One graft loss occurred in both groups. One acute rejection was seen in the control group. There was no difference in the rate pf change in 51Cr-EDTA-or PAH-clearance between the two groups. Histopathological findings were mostly mild. One new onset chronic rejection developed in both groups. Proximal tubular atrophy was more extensive in the rhGH-treated patients (P<0.05), but there was no uniform trend toward more severe findings. RhGH improved growth, and no significant differences were seen in allograft function or histopathology; however, larger trials controlled for pretreatment renal function and immunosuppression are needed. PMID- 8623158 TI - Hepatocellular integrity in liver donors and recipients indicated by glutathione transferase alpha. AB - Glutathione transferase Alpha (GSTA) is a sensitive indicator of hepatocellular integrity. Its reference range is low (0.7-14 microgram/L) and its half-life is short (1 hr) in serum. We evaluated the changes in GSTA concentration in 18 recipients during and after liver transplantation. The respective liver donors were also included in 13 cases. The baseline GSTA concentrations were normal or slightly elevated in all donors, 1.2-79 micrograms/L (median 5.1 micrograms/L) and recipients, 1.1-34 micrograms/L (median 6.4 micrograms/L). Surgical dissection of donor liver caused a moderate or even large increase in GSTA concentration, peak 80-6500 microgram/L (median 800 micrograms/L). In the recipients the peak of GSTA concentrations varied from 1400 to 47000 micrograms/L (median 5000 micrograms/L), and it was always observed within 45 min after reperfusion of the graft. The highest GSTA values were observed after long cold ischemia and in patients transplanted for acute liver failure. However, they were not associated with early graft dysfunction. There was a correlation between the AUC of GSTA and cold ischemia time in the recipients with chronic nonalcoholic liver failure (r=0.94). There was no correlation between GSTA values in the donors and recipients (r=0.14). The apparent half-life of GSTA in serum was 56 min (median). Perioperative GSTA concentrations in the donors had no obvious predictive value. In the recipients an exceptionally long apparent half-life of GSTA immediately after transplantation or a large second increase in GSTA were predictors of postoperative complications. PMID- 8623159 TI - The paradox of cytokine monitoring-predictor of immunologic activity as well as immunologic silence following cardiac transplantation. AB - The utility of cytokine monitoring to predict the onset of significant rejection was evaluated in 34 patients following heart transplantation. Serial blood levels of 5 cytokines involved in inflammation and immune activation(IL-1, IL-2, IL-6, IL-8, and TNF) were correlated with clinical outcome and endomyocardial biopsy scores. The majority of patients (68%) experienced a significant rejection during the study period. IL-6 and IL-8 levels were effective markers of significant rejection 2-4 days before diagnosis with EMBX. IL-6 and IL-8 levels of 15 and 1000 pg/ml predicted the onset of rejection with sensitivities of 75% and 66% and specificities of 86% and 76%, respectively. In contrast, IL-6 and IL-8 levels less than 15 and 400 pg/ml predicted a rejection-free course with sensitivities of 91% and 91% and specificities of 81% and 68%, respectively. The remaining cytokines differentiated patients experiencing a clinically unremarkable course from those experiencing mild-to-moderate rejection but did not discriminate rejection severity. IL-6 levels identified steroid and OKT3 resistance within 48 hr of antirejection therapy. IL-6 levels elevated to 197 +/- 20 pg/ml among steroid-resistant patients and normalized to 20 +/- 5 pg/ml among responders. IL 8 levels delineated OKT3 resistance. IL-8 levels rose to 3496 +/- 500 pg/ml among nonresponders, whereas levels fell to 152 +/- 50 pg/ml among responders. This study demonstrates that IL-6 and IL-8 are useful markers of rejection and therapeutic efficacy following heart transplation. PMID- 8623160 TI - Aspergillus infection in single and double lung transplant recipients. AB - To investigate the clinical manifestations of Aspergillus infections in lung transplant recipients, we reviewed the mycology and autopsy reports of all double (DLT=93) and single (SLT=48) lung transplant recipients from November 1983 to May 1993. Positive Aspergillus cultures were identified in 22% of the recipients (DLT=21, SLT=10). Colonization alone was present in 19 recipients (DLT=16, SLT=3). Complicated Aspergillus infection included Aspergillus bronchitis (DLT=1, SLT=1), aspergilloma (SLT=2), pulmonary invasive aspergillosis (DLT=1, SLT=2), disseminated aspergillosis (DLT=1, SLT=2), empyema (DLT=1), and a retroperitoneal abscess (DLT=1). Symptoms were seen only in patients with complicated lung infections and CXR abnormalities began in the native lung of four SLT recipients. Twenty patients survived (DLT=17, SLT=3) and 11 died (DLT=4, SLT=7) of disseminated aspergillosis (SLT=2), pulmonary invasive disease (DLT=1), bronchiolitis obliterans (DLT=2, SLT=2, CMV pneumonitis (SLT=1), diffuse alveolar damage (SLT=2), and hyperacute rejection (DLT=1). Complicated infection and mortality were more common in SLTs than DLTs (P<0.05). We conclude that infection with Aspergillus is not infrequent in the lung transplantation population. Single lung recipients develop more complicated infection than double lung recipients after Aspergillus infection with native lung being a potential source of infection. PMID- 8623161 TI - The effect of oral immunization on corneal allograft survival. AB - The present study examined the potential of orally induced tolerance for preventing immunological rejection of corneal allografts. Orthotopic corneal allografts were transplanted from either C3H (MHC + multiple minor H-mismatched) or NZB (multiple minor H-mismatched only) donors to CB6F1 recipients on day 0. Tissue cultured corneal epithelial and endothelial cells from relevant donor strains were administered orally from day -14 to day -4 on a daily basis, The incidence of graft rejection, graft mean survival time (MST), and alloimmune responses, and the antigen specificity of induced tolerance were studied. Oral immunization induced a remarkable tolerance such that only 55% of the orally immunized hosts rejected their fully allogeneic corneal grafts (MST = 43 days) compared with 100% rejection (MST = 18 days) in normal controls. Likewise, rejection of MHC-matched, multiple minor H-mismatched corneal grafts fell from 80% in untreated controls to 36% in orally immunized hosts. Oral immunization was effective in desensitizing previously immunized hosts. Rejection of MHC-matched, multiple H minor-mismatched corneal allografts fell from 93% in preimmune, unfed hosts to 36% in preimmune, orally tolerized mice. Thus, oral immunization is a safe and effective method for desensitizing high-risk, preimmune hosts and promoting corneal allograft survival. PMID- 8623162 TI - Depletion of the mature CD4+8- thymocyte subset by FK506 analogs correlates with their immunosuppressive and calcineurin inhibitory activities. AB - FK506 blocks T cell activation by preventing the transcription of lymphokine genes through binding to the intracellular protein FKBP12 and formation of complex that inhibits the phosphatase activity of calcineurin. Beside exerting potent suppressive activity on cellular and humoral immune responses, in vivo treatment with FK506 in rodent models induces thymic alterations characterized by a selective reduction of mature CD4+8- cells. The potential relationship between such thymic alterations and the immunosuppressive and calcineurin inhibitory activities of FK506 has not been defined. Here, we took advantage of the availability of FK506 analogs with different immunosuppressive potencies to address this question. Intravenous daily administration of FK506 in Sprague Dawley rats for 4 days was found to be sufficient to cause a depletion of CD4+8- thymocytes with an ED50=0.06 mg/kg/day. Under the same conditions, L-683,590 which is 2-3-fold less potent than FK506 in inhibiting T cell activation and calcineurin function gave an ED50=0.17 mg/kg/day. In contrast, the nonimmunosuppressive, calcineurin noninhibitory antagonist L-685,818, failed to deplete the CD4+8- thymocyte subset but could reverse the reducing effect of FK506 on this subset. Another analog, L-688,617, which does not completely inhibit T cell activation in vitro, also behaved as a partial agonist of CD4+8- cell depletion. Therefore, the ability of FK506 analogs to deplete the CD4+8- thymocytes subset correlates with their immunosuppressive and calcineurin inhibitory potencies. This suggests that calcineurin is involved in the intra thymic maturation processes of CD4+8- T cells. Moreover, the short-term treatment protocol described here provides a rapid and quantitative assay to determine the immunosuppressive potency of FK506-like compounds in vivo PMID- 8623163 TI - Identification of a calcium-inducible, cyclosporine sensitive element in the IFN gamma promoter that is a potential NFAT binding site. AB - Cyclosporine (CsA) inhibits cytokine transcription by preventing the activation of key promoter sites, in particular the binding of nuclear factor of activated T cells (NFAT) to the IL-2 NFAT site and the "P" site in IL-4. To identify potential NFAT-like sites in the IFN-gamma promoter, we sought areas of homology with the known sites in other promoters. In the promoter region of the mouse and human IFN-gamma gene, we identified two repeats of a consensus sequence ATTTCCnnT, designated P1 and P2 because of their homology to the calcium inducible and CsA-sensitive "P" sequences in the IL-4 promoter. In electrophoretic mobility shift assay (EMSA), a probe containing the second P sequence "P2" in the human IFN-gamma gene bound nuclear proteins from stimulated, but not unstimulated, humans T cells. The cytosol of unstimulated cells contained similar binding activity that decreased after stimulation, indicating that this binding activity translocated to the nucleus after stimulation. CsA inhibited nuclear translocation. Competition studies demonstrated that oligomers containing the sequences P1 and P2 in IFN-gamma gene, the NFAT site in the IL-2 gene, and the IL-4 P site competed with the P2 probe for protein binding, whereas an oligomer containing mutations in the P2 site did not. Addition of anti-NFAT antiserum altered protein binding to P2, indicating that the proteins were either identical or related to NFAT. Stimulation of T cells transfected with constructs containing three copies of the P2 sequence enhanced CAT activity in response to ionomycin, and this effect was blocked by CsA. These results suggest that the P2 sequence, and probably the P1 sequence, in the IFN-gamma promoter are NFAT binding sites and contribute to the calcium inducibility and CsA sensitivity of IFN-gamma production. PMID- 8623164 TI - Cardiac allograft vasculopathy. Preferential regulation of endothelial cell derived mesenchymal growth factors in response to a donor-specific cell-mediated allogeneic response. AB - We have previously reported that cell-mediated immunity to vascular endothelium is associated with the development of cardiac allograft vasculopathy (CAV). The mechanism by which a cell-mediated immune response to the coronary vascular is translated into the development of CAV is, however unknown. Peripheral blood mononuclear cells (PBMCs) obtained serially following cardiac transplantation were cocultured with donor-specific human aortic endothelial cells (HAECs) in 47 allograft recipients, 9 of whom had CAV (CAV+) at 1 year by angiography. At 20 hr following coculture, HAEC poly (A+) RNA was isolated, reverse-transcribed, and the cDNA-amplified (PCR) for a panel of growth factors (GFs) known to alter smooth muscle cell proliferation or migration. Relative quantitation of PCR product was performed using high-pressure liquid chromatography (HPLC). Three patterns of GF regulation were observed depending on the GF, the time posttransplant, and whether the patient had CAV: (1) no regulation (TGF-beta, PDGF-A early post-tx); (2) upregulation irrespective of CAV (bFGF, PDGF-B, TGF alpha early post-tx); and (3) preferential or exclusive upregulation by CAV+ patients (PDGF-A and TGF-alpha late post-tx, HB-EGF early and late post-tx). For example, using PBMCs as stimulators, obtained 6 months posttransplant from CAV+ patients, increases in HAEC-derived PDGF-A chain (31 +/- 7 to 69 +/- 11), TGF alpha (97 +/- 27 to 201 +/- 23), and HB-EGF (78 +/- 16 to 173 +/- 27) mRNA were demonstrated (all P<0.05 or greater using HPLC peak area as units). These data demonstrate that cell-mediated activation of vascular endothelial cells in patients with CAV results in preferential upregulation of certain endothelial derived mesenchymal growth factors capable of stimulating smooth muscle cell proliferation and migration. PMID- 8623165 TI - alpha/beta-T cell receptor-directed therapy in rat allograft recipients. Long term survival of cardiac allografts after pretreatment with R73 mAb is associated with upregulation of Th2-type cytokines. AB - Rejection of vascularized allografts still poses the major problem in organ transplantation. Therefore, transplant rejection of cardiac allografts was investigated in a rat model (BN-to-LEW) under alpha/beta-TCR (R73) mAb-targeted therapy. Two protocols were studied: posttransplant ("immunosuppressive") and pretransplant conditioning therapy. In posttransplant therapy over a wide dose range, R73 mAb only marginally improved cardiac allograft survival (7.8 +/- 0.8 days vs. 12.5 +/- 0.8 days at 0.1 mg/kg for 7 days). In contrast, conditioning treatment with low-dose (0.1 mg/kg) anti-alpha/beta-TCR mAb given 3 to 7 days prior to organ transplantation was highly effective and resulted in 50% permanent acceptance of cardiac allografts. R73 mAb-treated rats were monitored with respect to peripheral lymphocyte populations and intragraft cytokine levels. A temporary, incomplete reduction (CD5+ cells) and partial modulation (alpha/beta TCR/CD5 double+ cells) in the peripheral blood was observed. In contrast to untreated controls, intragraft production of IL-2 and IFN-gamma at the mRNA and protein level was abolished in both post- and pretreated recipients. Interestingly, pretransplant mAb application was associated with augmented in situ elaboration of the Th2-type cytokines, IL-4 and IL-10, together with up regulated TGF-beta and PGE. Increased expression of IL-4 and IL-10 continued to be observed in long-term surviving allografts. In conclusion, the mechanism of conditioning therapy with alpha/beta-TCR mAb prior to alloantigen exposure appears to be a switch from Th1 to Th2 response allowing long-term acceptance of allogeneic grafts. PMID- 8623166 TI - Anti-carbohydrate antibodies associated with hyperacute rejection in a vascularized mouse heart-to-rat xenotransplantation model. AB - Specificity of immune reactions has always been sought, because it facilitates intervention with unwanted mechanisms. Specific carbohydrate antigens have been proposed to be targets of antibodies in early immune responses in pig-to-man xenografts. This work was undertaken to determine carbohydrate structure for antibody response in the experimental xenograft model mouse-to-rat. Glycolipids were prepared from nine different mouse organs and separated for carbohydrate size on thin layer plates. Sera taken from normal untreated rats showed only weak or absent IgM antibody-binding to the separated mouse glycolipids. This is in accordance with the observation that mouse heart grafts are not hyperacutely rejected by the rat. However, sera taken from mouse heart xenografted rats show clear IgG and IgM antibody binding to neutral glycolipids migrating in the five sugar region of the thin-layer plate. These rats have previously been reported to hyperacutely reject a second xenograft. Glycolipids with this particular mobility and immunostaining properties are the dominant ones in the mouse caval vein preparation, which probably represents a rather pure vascular structure. The target antigen structure was identified, by mass spectrometry and proton nuclear magnetic resonance spectroscopy, to be the Forssman pentaglycosylceramide. A commercial monoclonal antibody directed toward the Forssman antigen bound the same biochemical structure as the antibodies derived from the mouse heart xenografted rats. Most of the IgM activity, but very little of the IgG activity was adsorbed using the Forssman terminal disaccharide solid phase. PMID- 8623167 TI - Increased expression of the HDJ-2 heat shock protein in biopsies of human rejected kidney. AB - The presence of donor-specific alloreactive helper and cytotoxic T cells has been described in allograft biopsies obtained from individuals undergoing acute allograft rejection of various solid organs. However, not all of these lymphocytes demonstrated specificity to mismatched donor HLA antigens. The identity of the antigens to which these T cells are directed to is still unknown at present. The possibility that heat shock proteins (Hsp) could serve as antigenic determinants to which these T cells respond has been raised. We have recently cloned and characterized a novel Hsp of 45Kd molecular weight. In the present study we show that the synthesis of this Hsp (HDJ-2) as well as Hsp60 is significantly elevated in kidney biopsies from individuals undergoing acute and chronic rejection. No message was detected either for HDJ-2 or Hsp60 in biopsies obtained from normal pretransplant kidneys or posttransplant kidneys with no rejection. However, there was some increase in Hsp in miscellaneous causes of allograft dysfunction such as infection and drug allergy. But, this was not as consistent as that noted for allograft rejection. This marked increase in Hsp expression during allograft rejection suggests Hsps as potential candidates for antigenic determinants contributing to kidney rejection. PMID- 8623168 TI - The safety and tolerability of cyclosporine emulsion versus cyclosporine in a randomized, double-blind comparison in primary renal allograft recipients. The Neoral Study Group. AB - A 12-week, randomized, double-blind, multicenter pharmacokinetics study was conducted to compare the clinical safety and tolerability of cyclosporine capsules and oral solution for microemulsion and cyclosporine in 101 primary renal transplant recipients Cyclosporine emulsion has more complete absorption and improved bioavailability compared with cyclosporine, and dosing of both cyclosporine formulations was adjusted to achieve comparable whole-blood trough levels. Mean serum creatinine values were higher in the cyclosporine emulsion group at baseline, 8, and 12 weeks (P<0.05). The incidence of acute rejection was similar in both treatment groups although fewer patients required monoclonal antibody therapy in the cyclosporine group (31% vs. 82%, respectively). Despite the increased bioavailability of cyclosporine emulsion, no significant differences in the incidence of adverse events were observed; the safety, tolerability, and efficacy of cyclosporine emulsion and cyclosporine were comparable. PMID- 8623169 TI - A time table for infections after renal transplantation in the tropics. PMID- 8623170 TI - Gastric aspiration for diagnosis of pulmonary tubercolosis in adult renal allograft recipients. AB - Of 213 renal allgraft recipients suspected to have had pulmonary tuberculosis, 132 had sputum examinations and 14 showed acid-fast bacilli. Of the remaining 118 patients, 25 had gastric aspirations, 18 had bronchoalveolar lavage, and 75 did not require further investigation because of spontaneous improvement or confirmation of an alternative diagnosis. While 9 of the 25 patients' gastric aspirate examination was positive, all the 18 who had bronchoalveolar lavage were negative for acid-fast bacilli. Eighty-one patients without expectoration had gastric aspiration directly and 14 showed acid-fast bacilli. Of the remaining 67 patients only 17 had bronchoalveolar lavage, of which three were positive for AFB and the rest did not require further testing for tuberculosis. A total of 106 patients had gastric aspiration. Acid-fast bacillus positivity was significantly more (P<.01) in patients with abnormal chest radiographs as compared with patients with normal chest radiographs as compared with patients with normal chest radiograph results. We suggest gastric aspiration for AFB in all renal transplant recipients who have fever, scanty expectoration, and abnormal chest radiograph with clinical suspicion of pulmonary tuberculosis. PMID- 8623171 TI - Isolated pancreas rejection in combined kidney pancreas tranplantation. AB - The clinical success of pancreas transplantation is limited by the difficulty in diagnosing rejection. In simultaneous pancreas kidney (SPK) transplantation, the diagnosis of pancreatic rejection is particularly difficult in the absence of clinical evidence of kidney rejection. Moreover, patients receiving only pancreas grafts will not have a concomitantly grafted kidney to serve as a "sentinel" for rejection. Percutaneous pancreas graft biopsy has been reported in a few small series but has not been adopted for broad clinical use. We describe the evaluation of 69 consecutive episodes of suspected isolated pancreas allograft rejection by percutaneous pancreas allograft biopsy. These rejection episodes occurred in 41 patients with bladder-drained pancreas transplants (25 SPK, 14 pancrease after kidney transplants [PAK], amd two pancreas transplant alone [PTA]). The indications for percutaneous pancreas biopsy were a twofold or greater increase in serum amylase or lipase, or a sustained 40% to 50% drop in urine amylase in the setting of no evidence of renal allograft dysfunction in SPK transplants. Biopsies were performed with color-flow Doppler ultrasound localization using an 18-gauge automated biopsy needle. Pancreatic tissue adequate for histologic evaluation was obtained in 61 of 69 cases (88%). There were two cases of intraabdominal bleeding, one of which required surgical intervention; the other resolved spontaneously. Histologic assessment of the biopsies demonstrated varying degrees of acute cellular rejection in 48 of 61 specimens (79%). Twelve specimens (20%) were free of histologic evidence of rejection, and one specimen (2%) showed acute pancreatitis. At the time of suspected rejection mean serum amylase and lipase values were increased 3.6 and 8.3-fold, respectively, and urine amylase was decreased by a mean of 45%. We conclude that the commonly used markers for pancreas allograft rejection are only about 80% specific for acute rejection. Percutaneous pancreas allograft biopsy is safe and allows the avoidance of unnecessary antirejection therapy with its attendant side effects and cost. PMID- 8623172 TI - Case report--sirolimus rescue therapy for refractory renal allograft rejection. AB - Sirolimus not only displays prophylactic activity, it also reverses acute rejection in rats with ongoing renal allograft rejection. The present case of a human renal allograft recipient documents the utility of dual-drug cyclosporine (CsA)/sirolimus therapy for a patient experiencing ongoing acute rejection and renal dysfunction that had not abated after treatment with corticosteroids equine antilymphocyte globulin (ATGAM), or the mouse anti-human CD3 monoclonal antibody OKT3. The patient described herein underwent induction therapy with 7 days of OKT3, and anti-rejection treatment with 14 days of ATGAM followed by 6 days of OKT3 therapy for biopsy-proved rejection episodes that rendered the patient dependent on dialysis treatments, oliguric with a urine output less than 650 ml per day, and uremic with a serum creatinine (sCr) value above 7 mg/dl. When OKT3 therapy was ceased because of the presence of human anti-mouse antibodies, sirolimus was added to the CsA/steroid regimen to treat the ongoing rejection and to reverse the markedly decreased renal perfusion, as revealed by a 99mTc-DTPA scan. Within 6 weeks the patient's sCr value decreased to 1.4 mg/dl, and urine output returned to greater than 2000 ml per day. To date the patient has exhibited no significant side effects after over 4 months of sirolimus therapy and the withdrawal of corticosteroids. PMID- 8623173 TI - Rhodococcus equi infection after liver transplantation. PMID- 8623174 TI - The "white clot syndrome" in hepatic transplantation. AB - Heparin-associated thrombocytopenia and thrombosis (Type II HAT), the "white clot syndrome," has not been previously reported as a cause for fulminant hepatic failure after liver transplantation. Thrombocytopenia and the use of heparin are common events in the newly transplanted patient. A man who was transplanted for sclerosing cholangitis, and re-exposed to heparin, is described with thrombocytopenia, thrombosis of all hepatic vessels, and heparin antibodies. Type II HAT is an immune phenomenon that can apparently occur despite T-cell-directed immunosuppression. Suspicion is a key element in establishing diagnosis. We no longer use heparin routinely in liver transplant cases. PMID- 8623175 TI - Association of serum concentration of soluble class I HLA with HLA allotypes. AB - We correlated serum concentrations of soluble class I HLA antigens (S-HLA-I) with HLA allotypes in 82 unrelated Caucasian and 58 unrelated African-American putatively normal subjects, as well as in 31 individuals with stable, normally functioning liver transplants. Caucasian and African-American subjects with HLA A23 or HLA-A24 were high secretors of S-HLA-I. We also observed that some HLA-A allotypes associated with high serum concentrations of S-HLA-I were ethnicity specific. HLA-A33 was associated with high S-HLA-I secretion in African-Americans but not in Caucasians. HLA-A29 was associated with high S-HLA-I secretion in Caucasians but not in African-Americans. All liver transplant recipients studied who were high secretors of S-HLA-I postoperatively carried HLA-A24 or HLA-A29. (There were no HLA-A33 or HLA-A23 allotypes in this group.) The "secretor genes," however, may be autogenous or allogenic (i.e., either donor or recipient HLA-A24 or HLA-A29 resulted in the observed high secretor status in liver transplant recipients after transplantation). It is noteworthy that serum S-HLA-I concentrations were low in all subjects with HLA-A2 regardless of whether the HLA A2 was of recipient or donor origin. This finding suggests that HLA-A2 could have a suppressive effect on S-HLA-I secretion. PMID- 8623176 TI - Pancreas autotransplantation in pig with systemic or portal venous drainage. Effect on the endocrine pancreatic function after transplantation. AB - The effect of the type of venous drainage of the graft on its endocrine function was studied in two groups of pigs after segmental pancrease autotransplantation. Group 1 comprised 10 pigs with portal venous drainage (PVD) and group 2 comprised 10 pigs with systemic venous drainage (SVD). The graft consisted of body and tail of the pancreas. The pigs were totally pancreatectomized. The pancreatic duct was occluded by neoprene injected into the duct. One week before and 1 and 3 months after transplantation, intravenous glucose tolerance (IVGTT) and meal stimulation tests (MST) were performed. Plasma glucose (PG), insulin (INS), C-peptide, glucagon (GLU),and pancreatic polypeptide (PP) were measured during the tests. All pigs had normal fasting PG, 1 and 3 months after PanTx, although MST disclosed significantly higher PG (P<0.05) during the test after transplantation. In the PVD group, a decrease in INS level during both test was recorded after PanTx (P<0.05), while in the SVD group a nonsignificant rise in INS level was recorded compared with before transplant. A significant difference (P<0.05) in INS levels were present both 1 and 3 mon. after PanTx between the two groups. Pigs with PVD showed a higher (P<0.05) C-peptide level than pigs with SVD during IVGTT. The initial significant rise in PP during MST and the initial fall in PP during IVGTT recorded in all pigs before transplantation were totally lost after transplantation in both groups. During the tests, PP remained steady and significantly lower than the pretransplantation levels in both groups. A significantly higher GLU level during both IVGTT and MST was observed in SVD compared with PVD 1 month after PanTx (P<0.05), being more pronounced during MST. This accentuated GLU concentration decreased by 3 months after transplantation, although it was still significantly greater than pretransplantation levels. We concluded that the unnatural mode of delivery of pancreas endocrine secretion to systemic rather than to portal circulation leads to derangements in pancreatic endocrine function in order to maintain glucose homeostasis. This may cause earlier exhaustion of islet cells. Segmental rather than whole organ and duct occlusion rather than exocrine drainage may further contribute to this, shortening the effective life of the graft. PMID- 8623177 TI - Cadaver lungs for transplantation. Effect of ventilation with alveolar gas. AB - In an effort to increase the donor pool for lung transplantation (LTX), we have demonstrated the feasibility of LTX from circulation-arrested cadavers in a canine LTX model. We hypothesized that ventilation of the cadaver lung with alveolar gas (20% O2, 5% CO2, balance N2) (AG) would be superior to ventilation with 100% oxygen (O2) after circulatory arrest of the donor. Twelve mongrel dogs were intubated, heparinized and euthanized by pentothal injection and ventilated with AG (n=6) or O2 (n=6). Four hours later, donor animals underwent sternotomy, and the lungs were flushed with cold modified Euro-Collins solution, harvested, and stored inflated in ice slush. Left lung allotransplantation was performed, and recipients were made dependent o n the transplanted lung by occlusion of the contralateral bronchus and pulmonary artery. Recipient animals were ventilated with an FiO2 of 0.4 and followed for 8 hr. Total ischemic time was 7.9 hr for both groups. Pulmonary edema developed in all recipients of AG lungs; one recipient survived the 8-hr observation period with poor oxygenation. In contrast, three of six recipients of O2-ventilated lungs survived for 8-hr with excellent gas exchange. Specimens of donor lungs before and after transplant were evaluated histologically utilizing trypan blue exclusion as an indicator of cell viability. At the time of organ retrieval 4 hr after death, 6% of cells were nonviable in the O2-ventilated cadaver lungs. Circulation-arrested cadaver lungs ventilated with 100% O2 prior to organ retrieval have superior pulmonary function after transplant compared with lungs ventilated with AG. Ventilation of cadaver lungs with AG induces pulmonary injury in this model. retrieval of donor lungs from circulation-arrested cadavers has potential for increasing the pulmonary donor pool. PMID- 8623178 TI - Effects of University of Wisconsin and Euro-Collins solutions on interstitial pulmonary edema in isolated rat lungs. AB - Macromolecules are present in lung preservation solutions to limit liquid filtration out of the pulmonary circulation and minimize pulmonary edema. We tested the effectiveness of these molecules by measuring interstitial edema in rat lungs perfused with macromolecular solutions (University of Wisconsin [UW] solution and Euro-Collins solution supplemented with modified pentastarch [pentafraction, PEN]) or with solutions that lacked macromolecules (UW solution with PEN and Euro-Collins solution.) The lungs were inflated with air and perfused with one of the test solutions, then rapidly frozen and prepared for histological analysis. From tissue sections, we measured cross-sectional areas of pulmonary arteries and veins, and also measured cross-sectional areas of the interstitial spaces surrounding arteries and veins. We then calculated the interstitium-to-vessel cross-sectional area ratio. In lungs perfused with macromolecular solutions these ratios were 0.09+/-0.15 and 0.53+/-0.56 (mean +/- SD) for UW solution and Euro-Collins solutions solution with PEN, respectively (P100,000 islet equivalents at a purity >50%) were analyzed retrospectively using univariate and multivariate analysis. Critical factors in the multiorgan cadaveric donors that were identified using univariate analysis included donor age (P<0.01), body mass index (BMI)(P<0.01), cause of death (P<0.01), and prolonged hypotensive episodes (systolic blood pressure <90 mmHg or mean arterial pressure <60 mmHg for > 15 min) requiring high vasopressors (>15 microgram/kg/min dopamine or >5 microgram/kg/min Levophed) (P>0.01). Independent analysis of 19 donor variables using multivariate logistic stepwise regression showed six factors were statistically significant. Odds ratio (OR) showed that donor age (OR 1.1, P<0.01), local procurement team (OR 10.9, P<0.01), and high BMI (OR 1.4, P<0.01) had a positive correlation with islet recovery. In contrast, hyperglycemia (all blood glucose >10 mmol/L) (OR 0.63, P<0.01), frequency and duration of cardiac arrest (OR 0.7, P<0.01), and increased duration of cold storage before islet isolation (OR 0.83, P<0.01) had negative correlation. Using these combinations of factors, the prediction of success was 85% accurate. By donor age, success was 13% for 2.5- to 18-year-old donors (n=23), 37% for 19- to 28-year-old donors (n=30), 65% for 29- to 50-year-old donors (n=70), and 83% for 51- to 65-year-old (n=29) donors. However, when vitro function was assessed by perifusion, the insulin secretory capabilities of islets isolated from the >50-year-old donor group was significantly reduced as compared with the 2.5- to 18-year-old group (P<0.02). Multiple regression analysis using postdigestion and postpurification islet recovery as outcome variables identified BMI, procurement team, pancreas weight, and collagenase digestion time factors tht can affect the recovery of human islets. Locally procured pancreases and donors with elevated minimum blood glucose levels were identified as factors that affect the insulin secretory capabilities of the isolated islets. This review of parameters suggests an improved approach to the prediction of successful islet isolation from human pancreases. Selection of suitable pancreases for processing may improve consistency in human islet isolation and thereby decrease costs. PMID- 8623182 TI - Randomized, prospective trial of cyclosporine monotherapy versus azathioprine prednisone from three months after renal transplantation. AB - Cyclosporine (CsA) and prednisone (Pred) are the mostly used drugs for immunosuppression after renal transplantation, but both drugs have marked side effects. Either replacement of CsA by azathioprine (Aza) or withdrawal of prednisone (Pred) resulting in CsA monotherapy can be employed to circumvent the adverse effects in the long run. Both treatment regimens were compared to this prospective randomized trial in patients who were treated with CsA and Pred during the first 3 months after renal transplantation (CsA: n=64, Aza-Pred: n=63, median duration of follow-up: 3.9 years). Estimated graft survival rates at 5 years after transplantation (in patients with a functioning graft at 3 months) were 78% in the CsA group and 87% in the Aza-Pred group. The incidence of a rejection within 3 months after start of steroid withdraw or conversion from CsA to Aza was 30% and 25% respectively (NS). At 2 years after transplantation, serum creatinine levels were lower in the Aza-Pred group (126+/-35 micromol/L) than in the CsA group (180+/-78 micromol/L; P>0.001). There were no differences in blood pressure or incidence of infections between the treatment groups. Treatment related costs were measured during the first year after transplantation and were lower in the Aza-Pred group (DFL 40,882+/-18,895 vs. DFL 53,484+/-44,828; 1 DFL [Dutch guilder] is about US $0.60; P<0.005). In conclusion, CsA monotherapy and Aza-Pred treatment from 3 months after renal transplantation are comparably effective immunosuppressive treatment regimens, although Aza-Pred therapy results in better graft function. Withdrawal of steroids and replacement of CsA by Aza both carry a substantial risk of rejection. The previously demonstrated cost effectiveness of CsA-containing therapies seems to be limited to the first phase after transplantation. Conversion to Aza-Pred at 3 months after transplantation reduces costs. PMID- 8623185 TI - One year of experience with extended application and modified techniques of split liver transplantation. AB - As organ donation rates decreased in Europe, the authors started a systematic approach of liver splitting in their center in 1994. During this 1-year experience, 73 cadaveric liver transplantations were performed in 66 patients. Sixteen of these transplantations were the result of split-liver transplantation (21.9% of grafts, 24.2% of patients). Patient and graft survival rates at 3 months were 81.2% and 75%, compared with 89.1% and 76.9 % for whole organs. Two modified techniques were developed, based on the technique of living related liver procurement, and applied in 10 cases. With these new techniques, patient and graft survival rates were 90% and 90%. This systematic approach allowed the total number of transplantations in our program to be maintained, despite the decrease in organ availability. PMID- 8623184 TI - Clinical correlates of chronic rejection in pediatric renal transplantation. A report of the North American Pediatric Renal Transplant Cooperative Study. AB - A total of 1699 patients with living donor transplants and 1795 patients with cadaver donor transplants entered between January 1987 and November 1994 in the North American Pediatric Renal Transplant Cooperative Study registry form the cohort for this report. Failure from chronic rejection occurred in 76 (4.5%) of living donor transplants and 149 (8.3%) of cadaver transplants. Chronic rejection is the leading cause of graft failure, with 27% of living donor and 26.7% of cadaver donor graft failures attributed to chronic rejection. Univariate and multivariate analyses of various risk factors revealed acute rejection (relative risk [RR]=3.1, P<0.001) and greater than two acute rejections (RR=4.3, P<0.001)to be the most common correlates of chronic rejection. Additionally, late initial acute rejection (>365 days; RR=2.3, P<0.001) was also correlated. Cadaver donor source (RR=1.6, P=0.001) and African-American race (RR=1.6, P<0.003) were weaker but significant risk factors. An analysis of cyclosporine dosing revealed tht a dose of <5mg/kg/day at day 30 was also a risk factor (RR=1.5, P=0.027). Our study concludes that acute rejection is the single most critical element in the genesis of chronic rejection. Thus, measures to prevent the first episode of acute rejection could ameliorate the incidence of chronic rejection. PMID- 8623186 TI - Graft function and outcome of older (> or = 60 years) donor livers. AB - Livers from donors > or = 60 years of age are often considered inadequate for transplantation by many centers. With waiting times exceeding 1 year in our region, we have aggressively used livers from this donor age group. Between 1990 and 1994, 209 patients received 223 liver grafts at our institution. Of these, 29 (13%) were from donors > or = 60 years of age (group A) and 194 (87%) were from donors < 60 years of age (group B). The two groups were matched for recipient diagnosis and severity of disease. Group A and B donors had similar liver, renal, and hematologic studies prior to donation. Weight, sex, race and vasopressor requirement were also similar. Postoperative alanine aminotransferase, aspartate aminotransferase,and prothrombin time were not significantly different over the first 10 postoperative days. Group A grafts were significantly more cholestatic than group B grafts on postoperative days 6-10. The retransplantation rate for primary graft nonfunction was not significantly different from group A (6.7%) and group B (3.4%; P=0.04). Patient and graft survival rates at 1 year were 58.6 % and 44.8% for group A and 79.2% and 74.5% for group B (P<0.001 for both). Four of 12 deaths in the first year in group A were completely unrelated to graft function. If these are excluded, patient and graft survival rates were 68% and 52%, which are better but still significantly less than in group B. Initial graft function of older donor livers are similar to that of the matched younger group. However, patient and graft survival rates were significantly worse for the older donors, even when corrected for unrelated deaths. Livers should not be discarded based on age alone without inspection and/or biopsy to rule out significant steatosis. Prompt retransplantation for primary graft nonfunction of older donors are generally more cholestatic than those from the younger donor age group; however, many of them function quite well. At the present time, given the inability to identify donor variables associated with decreased recipient survival, we recommend cautious use of older liver grafts in healthier recipients. PMID- 8623188 TI - Detection of human cytomegalovirus myocardial involvement by polymerase chain reaction during systemic infection and correlation with pp65 antigenemia and DNAemia in infected heart recipients. AB - The presence of human cytomegalovirus DNA was investigated in 103 unfixed endomyocardial biopsies, performed during the first 4 months in 17 heart transplant recipients by polymerase chain reaction. Results were correlated with human cytomegalovirus systemic infection, as detected by the test for the viral lower matrix phosphoprotein pp65 (antigenemia) and by polymerase chain reaction for viral DNA in blood leukocytes (DNAemia). Three patients out of 17 did not develop cytomegalovirus infection and 14 did: 5 had symptomatic disease treated with ganciclovir and 9 developed asymptomatic infection and were not treated. Viral DNA was detected in 24 out of 103 biopsies (23%) from 13 patients: 5 with symptomatic infection during the acute phase of disease (mean levels of pp65: 125+/-232 pp65 positive leukocytes/200,000 examined cells) and 8 patients with asymptomatic infection when the mean antigenemia was 5+/-15/200,000 (4 patients) or when DNAnemia was present in the blood (4 patients). No histological evidence of myocarditis was shown in viral DNA-positive biopsies. No difference in acute rejection was found in viral DNA-positive and DNA-negative biopsy specimens in symptomatic and asymptomatic infected patients. Our experience suggests that during systemic symptomatic and asymptomatic cytomegalovirus infection, polymerase chain reaction can detect a relatively frequent myocardial involvement, but this involvement is not associated with myocarditis or with a higher incidence of acute rejection. THe presence of viral DNA in myocardial biopsies can be a result of high viremia, but it also can be due to low level of viral DNA in circulating infected leukocytes. Polymerase chain reaction is the most sensitive method for cytomegalovirus DNA detection in biopsies, but its results need to be evaluated together with morphology-preserving methods and systemic markers of infection in order to make a correct diagnosis. PMID- 8623187 TI - Low molecular weight heparin for the prevention of veno-occlusive disease of the liver in bone marrow transplantation patients. AB - Hepatic veno-occlusive disease (VOD), a common complication of bone marrow transplantation (BMT), is a result of intensive conditioning by chemo radiotherapy. Endometrial injury causes fibrin deposition in the affected hepatic venules, leading to abnormal laboratory parameters followed by lethal full-blown disease. Previous studies have shown that unfractionated heparin can prevent VOD in BMT patients. Since low molecular weight heparin (LMWH) preserves the antithrombotic, but not the anticoagulant, activity of unfractionated heparin, we initiated a pilot study to determine the safety of LMWH for the prevention of VOD. Sixty-one patients undergoing BMT (allogeneic, n=24; autologous, n=37) were randomized to receive subcutaneous injection of enoxaparin (40 mg/day x 1) or a placebo prior to BMT conditioning and until day 40 after transplantation or discharge from the hospital. LMWH administration did not influence marrow engraftment, nor was it associated with bleeding tendency. Hemorrhagic events occurred significantly less frequently (P=0.025) were shorter duration (P=0.006) in the LMWH group than in the placebo group. Time to platelet recovery was significantly shorter (16.5 vs 29.6 days, (P=0.0075), and platelet transfusion requirements were lower (p=0.05) in the LMWH patients. VOD parameters occurred less frequently in the experimental group, including duration of elevated bilirubin levels (P=0.01) and incidence of hepatomegaly (P=0.04). LMWH, which seems to enhance platelet recovery, may be safely administrated to BMT patients in an attempt to prevent VOD of the liver. PMID- 8623189 TI - Prevention of Th1 response is critical for tolerance. AB - We investigated the role of Th1 ad Th2 cytokines in rejection and tolerance using the neonatal tolerance model. We reported previously that lymph nodes that drained immunogen-bearing tolerant grafts produced a 10- to 100-fold higher ratio of interleukin (IL)-4 to interferon (IFN)-gamma compared with lymph node cells from rejected grafts. Moreover, because neonatal antigen exposure triggers allospecific Th2 CD4 memory cells, whereas antigen exposure during adulthood triggers Th1 CD4 memory cells, we speculated that immunoredirection toward Th2 and away from Th1 functions as another mechanism of tolerance. To test the immunoredirection hypothesis, we examined whether recovery of Th1 cytokine responses abrogates tolerance. We now show that treatment with exogenous IFN gamma at the time of neonatal priming recovered mixed lymphocyte reaction hypoproliferation and restored the ability of mice to reject skin grafts. Mice that received IFN-gamma at the time of neonatal priming produced more IFN-gamma and contained more A/J-reactive IFN-gamma producing CD4 cells compared with untreated neonatal primed mice, but failed to recover A/J-specific INF-gamma producing CD8 cells or CTL responses, which suggests that graft rejection occurred via Th1 CD4 cells. Interestingly, draining lymph node cells from rejected grafts in IFN-gamma-treated neonatal primed mice also produced more IL 4, compared with cells from healthy grafts on untreated neonatal primed mice. Nonetheless, lower IL-4 to IFN-gamma ratio predicted graft rejection and higher ratios predicted acceptance. We conclude that neonatal tolerance depends on the ability to block generation of allospecific Th1 responses that lead to rejection. Thus, immunoredirection involves both the inhibition of Th1 and expansion of Th2 immune responses. PMID- 8623190 TI - T cells receptor beta-chain repertoires are nonrandomly selected in responses to HLA-DR1. AB - T cell receptor (TCR) beta-chain usage by HLA-DR1 alloreactive T cell lines was examined to determine whether common TCR gene segments were used preferentially. We have demonstrated previously that a DR1-specific, human renal allograft derived T cell line and replicate, anti-DR1 mixed lymphocyte reactions (MLR), established from an unrelated responder/stimulator pair, were selected for T cells expressing V beta 8. In this report V beta 8+ beta-chains from these T cell lines were sequenced to assess clonality and determine the contribution made by the beta-chain junctional regions. All 11 V beta 8+ cDNA clones sequenced from the allograft-derived T cell lines used J beta 2.7 and C beta 2 and had identical junctions, indicating the presence of a predominant V beta 8+ clone. All seven V beta 8+ sequences from the first anti-DR1 MLR and eight of the nine fron the second also used J beta 2.7 and C beta 2 were identical to one another, indicating that a common V beta 8+ clone was selected in these replicate cultures. The sequences of the predominant V beta 8+ beta-chains from the allograft-derived T-cell line and the MLR differed by only 10 nucleotides and four amino acids at the VDJ beta junction. To determine the reproducibility of TCR V beta selection in responses to DR1, additional MLR were established by pairing three different DR1+ stimulators with the same responder. The TCR repertoires of the resulting DR1-specific cell lines were examined. A preference was seen for utilization for certain homologous TCR V beta segments. The data suggest that particular TCR V beta or V beta/J beta combinations may be selected in alloresponses as evidenced either utilization of highly similar beta-chains or homologous V beta segments. PMID- 8623191 TI - A three-cell cluster hypothesis for noncognate T-B collaboration via direct T cell recognition of allogeneic dendritic cells. AB - In this article, we propose that T cell help for B cells can occur via an unusual three-cell cluster, with recipient CD4+ T helper cells interacting via direct allorecognition with donor dendritic cell class II MHC antigens, recipient B cells interacting with MHC class I (or any other) antigen on the donor dendritic cell surface, and noncognate (i.e., antigen nonspecific) T-B collaboration. In this noncognate pathway, antigen processing by B cells is not required and T cell help is potent because of the high precursor T cell frequency for direct recognition of allogeneic class II MHC molecules. The data supporting this hypothesis are: 1. LEW rat strain recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts were shown to have no detectable antibody to donor class I MHC antigens at day 7 after grafting. By contrast, LEW recipients of normal (DAxLEW)F1 kidneys had strong antibody responses. 2. Consistent wih important role for donor dendritic cells in the early antibody response to donor class I MHC antigens was the finding that it was dependent on donor class II MHC antigens. PVG recipients, previously immunized with pure DA RT1.B class II MHC antigens, had virtually no antibody response to the class I MHC antigens of DA kidney allografts. 3. We confirmed the low and high responder status of PVG and LEW rats, respectively, to DA class I antigens by studying antibody responses to pure DA class I antigens. However, PVG and LEW recipients of DA kidney allografts did not differ in their antibody response to the donor DA class I MHC antigens. This is consistent with this response not requiring the processing and presentation of DA class I antigen by PVG recipients. 4. LEW recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts did eventually develop a strong antibody response to DA class I antigens, but this was delayed by several weeks. That this delayed antibody response was probably mediated by conventional T-B collaboration and that T help was rate limiting in this situation, was demonstrated by immunizing LEW recipients with a DA class I peptide. This markedly accelerated the kinetics of the antibody response to the dendritic cell-free (DAxLEW)F1 kidneys. PMID- 8623192 TI - Prolonged survival of discordant porcine islet xenografts. AB - Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets. PMID- 8623193 TI - Neutrophil elastase inhibitor (ONO-5046) decreases cytokine-induced neutrophil chemoattractant after reperfusion of pancreaticoduodenal transplantation in rats. AB - The protective effects of a neutrophil elastase inhibitor (ONO-5046) on reperfusion injury following pancreaticoduodenal transplantation in rats were studied by measuring serum concentrations of cytokine-induced neutrophil chemoattractant (CINC). Male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. ONO-5046 was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. No significant differences were observed in the peak serum concentrations of amylase between the groups treated with and treated without ONO-5046. The serum lipase concentrations in the untreated animals increased and peaked 3 hr after reperfusion. ONO-5046 significantly decreased the peak serum lipase concentration. The serum CINC concentrations, which were determined by enzyme linked immunosorbent assay, increased and peaked 3 hr after reperfusion, decreasing gradually thereafter. However, pretreatment with ONO-5046 significantly inhibited the rise in serum CINC concentrations after reperfusion. Expression of CICN transcripts in the pancrease grafts was evaluated by Northern blot analysis and peaked 3 hr after reperfusion in untreated animals. Pretreatment with ONO-5046 also significantly inhibited the expression of CINC mRNA transcripts in the graft. ONO-5046 significantly decreased the number of neutrophils accumulated in the pancreas graft 24 hr after transplantation. In vitro CINC production by peritoneal macrophages was increased by neutrophil elastase in dose-dependent fashion. However, ONO-5046 decreased CINC production by peritoneal macrophages in response to neutrophil elastase. These results suggest that ONO-5046 prevents early neutrophil accumulation in the pancreas following ischemia/reperfusion of pancreaticoduodenal transplantation. PMID- 8623194 TI - Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study. AB - The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use. PMID- 8623195 TI - Transforming growth factor-beta fails to inhibit allograft rejection or virus induced autoimmune diabetes in transgenic mice. AB - Transgenic mice whose pancreata express transforming growth factor-beta (TGF beta) directed by an insulin promoter (Ins-TGF-beta mice) were used to assess the effect of local TGF-beta1 on allograft rejection and on autoimmune diabetes occurring as a cross-reaction to viral antigens. Pancreatic TGF-beta1 did not delay allograft rejection, nor did it inhibit autoimmune diabetes after lymphocytic choriomeningitis infection of double transgenic mice (LCMV/TGF-beta1 mice). These results suggest that local TGF-beta1 does not serve as an immunosuppressive agent for allograft rejection or virus-mediated autoimmune diabetes. PMID- 8623197 TI - Sarcoidosis recurrence following lung transplantation. AB - Sarcoidosis is a rare indication for lung transplantation. In this article, our experiences with recurring sarcoidosis following lung transplantation are described. Literature concerning recurrence of the disease in kidney, liver, heart and lung transmission of sarcoidosis via transplanted organs are discussed. PMID- 8623196 TI - Association of interleukin-10 with rejection-sparing effect in septic kidney transplant recipients. AB - Certain cytokines, particularly gamma-interferon (IFN) and interleukin (IL)-2 associated with TH1 cell function, have been shown to play a role in allograft rejection. One paradigm for long-term allograft acceptance involves TH2 cytokine predominance (IL-4 and IL-10). We describe two renal allograft recipients for whom immunosuppression was discontinued due to serious sepsis and who maintained stable renal function over 2-6 months without immunosuppression. During this time, there were higher levels of both IFN-gamma and IL-10 in the peripheral blood than in stable control kidney transplant recipients on immunosuppression. In one of the patients, levels of IL-10 fell, while those of IFN-gamma remained persistently elevated. This was associated with biopsy-proven rejection. Although peripheral blood cytokine levels may not reflect intragraft events, these data are consistent with an allograft protective role for IL-10 offsetting that of IFN gamma in both patients off immunosuppression. PMID- 8623198 TI - Continuous improvement in organ donation. The Spanish experience. PMID- 8623199 TI - Granulocyte colony-stimulating factor immunomodulation in the rat cardiac transplantation model. AB - Granulocyte colony-stimulating factor (G-CSF) administration decreases tumor necrosis factor(TNF) release, an important mechanism in allograft rejection. to study G-CSF's possible antirejection effects, 30 Lewis rats underwent heart transplantation using Brown-Norway donors and were assigned varying dosages of recombinant human G-CSF (0, 20, 100, 250 and 500 microgram/kg/day) for 14 days following the operation. Recipients receiving 250 microgram/kg/day experienced an improvement in graft survival (12.3+/-4 days vs. 7.0+/-0.6 days, P>0.05, Breslow). In a separate cohort, G-CSF-treated recipients (250 microgram/kg/day x 14) killed at 2,4,and 6 days after transplantation revealed improved serial allograft biopsy grading scores versus untreated controls (P<0.001 stratified Wilcoxon). Significant reduction in serum TNF levels was noted in the G-CSF treated animals (P<0.025, analysis of variance). These data describe a moderate antirejection effect of G-CSF administration. Inhibition of circulating TNF in the G-CSF-treated recipients may describe a marker or possible mechanism of this antirejection effect. PMID- 8623200 TI - Heterogenous distribution of chimerism produced by rat organ and bone marrow allotransplantation. PMID- 8623201 TI - Hepatitis C virus and transplantation. PMID- 8623202 TI - In support of the findings of Christopher F. Bryan et al. PMID- 8623203 TI - Effect of donor age liver allograft function. PMID- 8623204 TI - Intracardiac thrombus formation during orthotopic liver transplantation: a new entity or an old enemy? PMID- 8623205 TI - Characterization of baboon class I major histocompatibility molecules. Implications for baboon-to-human xenotransplantation. AB - Increasingly strong medical and political pressures are stimulating consideration of the transplantation of baboon organs and cells into humans. Critical to the success of these xenotransplants is management of the immune system such that graft rejection and, in the case of bone marrow transplantation, graft-versus host disease do not result in transplant failure. The polymorphic products of the major histocompatibility complex (MHC) are the primary barrier to successful allotransplantation, and here we describe class I MHC molecules from baboon (Papio anubis) to gain an understanding of how similarities and differences between baboon and human MHC molecules might affect xenograft survival and function. Comparative analyses of our five novel baboon class I molecules with defined HLA class I molecules demonstrate that the baboon class I molecule are up to 90% identical. Disparity between baboon class I proteins and their human homologues lies predominately at positions in the antigen-binding groove, while C terminal portions of the class I heavy chain are more conserved between the two species. Such concentration of cross-species differences within the alpha1 and alpha2 domains involves a majority of substitutions at positions demonstrating polymorphism in human alleles; the location of substitutions distinguishing baboon and human molecules thus resembles the positioning of human class I allopolymorphisms. Because this preliminary characterization indicates that both baboon and human T cells with be restricted by xenogeneic class I molecules, immune responses triggered during baboon-to-human transplantation should mimic those arising during MHC mismatched human allotransplantation. PMID- 8623206 TI - CTLA4-Ig plus bone marrow induces long-term allograft survival and donor specific unresponsiveness in the murine model. Evidence for hematopoietic chimerism. AB - Allograft rejection is dependent on T cell activation, which requires both the engagement of the T cell receptor by antigen in the context of the MHC molecules and costimulatory signals delivered by cell surface molecules such as B7 CD28/CTLA4 pathway. CTLA4-Ig is a fusion protein that blocks this pathway and has previously been shown to prolong both allograft and xenograft survival. The current study demonstrates markedly prolonged murine cardiac allograft survival and specific prolongation of secondary skin grafts using a combination of CTLA4 Ig plus donor bone marrow. A role for hematopoietic chimerism in the establishment of CTLA4-Ig-induced transplantation tolerance was investigated using reverse transcriptase polymerase chain reaction analysis of recipient tissues. Expression of donor-specific MHC class II transcripts in both peripheral and lymphoid tissues was demonstrated at greater than 200 days after transplant. To investigate the functional significance of this observation, heart donors, and donor bone marrow were irradiated before transplantation in CTLA4-Ig-treated recipients. A reduction in allograft survival was associated with irradiation of both the donor heart and the bone marrow. These results suggest that there may be a donor-derived radiosensitive element that enhances allograft survival in this model. Reverse transcriptase polymerase chain reaction analysis of allografts of tolerant and control animals at days 5, 8, and 12 after transplantation failed to demonstrate a dramatic difference in the expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma message. Cytotoxicity effector transcripts were largely intact in CTLA4-Ig + bone marrow-treated recipients as they showed no decrease in intragraft granzyme, perforin, Fas, or Fas ligand transcripts during thr first 8 days after transplant. These results imply that complex mechanisms may be important for the induction and maintenance of transplantation tolerance in the CTLA4-Ig plus bone marrow murine cardiac allograft model. PMID- 8623207 TI - Current advantage of FK 506 in cadaveric kidney transplantation. PMID- 8623208 TI - Tacrolimus (FK 506) in clinical cardiac transplantation: a five-year experience. PMID- 8623209 TI - Pharmacokinetics of FK 506 in living-related liver transplantation. PMID- 8623210 TI - Glucose and lipid metabolism in liver transplant patients under long-term tacrolimus (FK 506) monotherapy. PMID- 8623211 TI - Tacrolimus (FK 506) therapy is associated with a significant reduction in immunosuppressive treatment failures following primary liver transplantation. PMID- 8623213 TI - Treatment of steroid-resistant rejection with tacrolimus prior to OKT3 in liver transplant recipients. PMID- 8623212 TI - Tacrolimus (FK 506) for rescue of chronic rejection following orthotopic liver transplantation. PMID- 8623214 TI - Tacrolimus rescue therapy for renal allograft rejection refractory to cyclosporine-based immunosuppression. PMID- 8623216 TI - Neoral in liver transplantation. AB - In conclusion, Neoral gives more consistent drug absorption, achieving better pharmacokinetic predictability. Among other advantages, this results in a close correlation between trough blood levels and drug exposure (AUC) so that trough blood levels can be used as a more meaningful monitoring parameter when using the new formulation. Studies have also now confirmed that absorption of Neoral is bile independent, making it more useful in the early postoperative period and in the setting of cholestasis and rejection. Furthermore, studies have now demonstrated that in patients who have problems absorbing Sandimmune such as patients with cystic fibrosis, pancreatitis, or Crohn's disease, conversion to Neoral results in correction of malabsorption of CyA. Issues that need to be addressed in the future will include long-term toxicity associated with maintaining high Cmax and AUC; whether the introduction of Neoral can result in steroid sparing; and whether the introduction of Neoral will result in a reduced incidence of acute and chronic rejection. PMID- 8623215 TI - Interaction between FK 506 and diltiazem in an animal model. PMID- 8623217 TI - In vitro and in vivo studies to prevent hyperacute rejection. PMID- 8623218 TI - Immunosuppressant switching between cyclosporine and tacrolimus after liver transplantation. PMID- 8623220 TI - Specific acceptance of cardiac allografts after treatment with antibodies to CD80 and CD86 in mice. PMID- 8623219 TI - Donor-derived microchimerism in heart transplant recipients: a parameter for immunological risk? PMID- 8623221 TI - Long-term and multilineage bone marrow reconstitution in normal untreated recipients. PMID- 8623222 TI - Effects of exogenous IL-2 on tolerance induction by anti-ICAM-1 and anti-LFA-1 monoclonal antibodies. PMID- 8623224 TI - The inhibition of T-cell-receptor-induced Fas ligand upregulation by cyclosporine and FK 506. PMID- 8623223 TI - Immunosuppressive effects of bactobolamine in vitro and in vivo. PMID- 8623225 TI - FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin allograft survival and synergistic effect in combination with cyclosporine in rats. PMID- 8623226 TI - FTY720, a novel immunosuppressant possessing unique mechanisms. II. Long-term graft survival induction in rat heterotopic cardiac allografts and synergistic effect in combination with cyclosporine A. PMID- 8623227 TI - FTY720, a novel immunosuppressant possessing unique mechanisms. III. Synergistic prolongation of canine renal allograft survival in combination with cyclosporine A. PMID- 8623228 TI - FTY720, a novel immunosuppressant, possessing unique mechanisms. IV. Prevention of graft versus host reactions in rats. PMID- 8623229 TI - Different immunosuppressive effects of liposomal FK506 in liver and kidney transplantation. AB - The efficacy of liposomal FK506 was compared between a canine liver transplantation model and a canine kidney transplantation model. The present study revealed that liposomal FK506 increased immunosuppressive efficacy of FK506 in liver transplantation but decreased in kidney transplantation. Because liposomal FK506 increased FK506 levels in the liver and spleen, and decreased FK506 levels in the kidney, it was suggested that enhanced immunosuppressive efficacy in liver transplantation should be attributed to the local immunosuppressive effects in the hepatic allograft rather than effective suppression of splenocyte activity. PMID- 8623230 TI - Auxiliary partial orthotopic liver transplantation from a living related donor: a report of two cases. PMID- 8623231 TI - Role of the graft as a source of donor-type microchimerism in liver transplant patients. PMID- 8623232 TI - A comparative study of morphological characteristics of renal injuries of tacrolimus (FK506) and cyclosporin (CyA) in renal allografts: are the morphologic characteristics of FK506 and CyA nephrotoxicity similar? PMID- 8623233 TI - Long-term graft function of right single-lung transplantation after contralateral pneumonectomy in the rat. PMID- 8623234 TI - Loss of antigenicity at one month after liver transplantation and change of serum antibody titer to donor antigens. PMID- 8623235 TI - Protective effect of a prostaglandin I2 analogue on grafted livers subjected to in situ warm ischemia in porcine orthotopic liver transplantation: immunohistochemical analysis of thrombomodulin. PMID- 8623236 TI - HGF prevents tacrolimus (FK 506)-induced nephrotoxicity in SHR rats. PMID- 8623237 TI - Immunosuppressive mechanism and immune tolerance of FK 506 in rat small bowel transplantation. PMID- 8623238 TI - Efficacy of microencapsulation of a pancreatic B-cell line (MIN6) in an agarose/PSSa microbead as a bioartificial pancreas. PMID- 8623239 TI - Prolongation of survival of a xenografted bioartificial pancreas with amesh reinforced polyvinyl alcohol hydrogel bag employing a B-cell line (MIN6). PMID- 8623240 TI - Beneficial effects of combined use of phosphoenolpyruvate and gadolinium chloride on preservation-reperfusion liver injury in rats. PMID- 8623241 TI - Expression of ICAM-1 is involved in the mechanism of liver injury during liver transplantation: therapeutic usefulness of the F(ab')2 fragment of an anti-ICAM-1 monoclonal antibody. PMID- 8623242 TI - Involvement of ICE-like proteases in hypoxic cell death. PMID- 8623243 TI - Protective effect of FK 506 on hepatic energy metabolism in warm ischemic canine livers induced by total hepatic vascular exclusion. PMID- 8623244 TI - Genistein suppresses cellular injury following hepatic ischemia/reperfusion. PMID- 8623245 TI - Reliable long-term cryopreservation of trachea for tissue banks. PMID- 8623246 TI - Establishment of an islet bank and its future perspectives. PMID- 8623247 TI - Initial trial to organize a cryopreserved allograft valve bank in Japan. PMID- 8623248 TI - Intervention study on the introduction of free delivery donor cards in Japan. PMID- 8623249 TI - Cellular xenografting: from diabetes to AIDS. PMID- 8623250 TI - Experimental discordant xenotransplantation. PMID- 8623251 TI - Inhibition of complement-mediated porcine endothelial cell cytotoxicity by human IgM natural antibody. PMID- 8623252 TI - Potential mechanism of abnormal thromboregulation in xenograft rejection: loss of ecto-ATPases upon endothelial cell activation. PMID- 8623253 TI - Strategies to overcome the anti-Gal alpha (1-3)Gal reaction in xenotransplantation. PMID- 8623254 TI - Xenoislet rejection following pig-to-rat, pig-to-primate, and pig-to-man transplantation. PMID- 8623255 TI - Two genetic approaches to the galactose alpha 1,3 galactose xenoantigen. PMID- 8623256 TI - Germline VH gene usage in xenoreactive monoclonal antibody. PMID- 8623257 TI - Human xenoreactive natural antibodies against Gal alpha (1-3) pig terminal residues are not produced by CD5+ B-lymphocytes. PMID- 8623258 TI - The humoral immune response in humans following ex vivo perfusion of porcine livers. PMID- 8623259 TI - Characterization of the human alpha 1,3Gal-reactive natural antibody population. PMID- 8623260 TI - Characterization of xenoreactive natural antibodies: interactions with carbohydrate determinants. PMID- 8623261 TI - Polymorphism within the human anti-pig repertoire. PMID- 8623262 TI - The effect of plasmapheresis and deoxyspergualin or cyclophosphamide treatment on anti-porcine Gal-alpha (1,3)-Gal antibody titres in humans. PMID- 8623263 TI - Overlapping recognition of xenogeneic carbohydrate ligands by human natural killer lymphocytes and natural antibodies. PMID- 8623264 TI - Converting alpha-Gal epitope of pig into H antigen. PMID- 8623265 TI - Identification and characterization of a peptide mimeotope of galactose alpha 1,3 galactose. PMID- 8623266 TI - Variation in expression of biologically reactive xenoantigen does not correlate with expression of Gal alpha (1-3)Gal. PMID- 8623267 TI - alpha 1,3-Galactosyltransferase-deficient mice produce naturally occurring cytotoxic anti-Gal antibodies. PMID- 8623268 TI - Potent inhibition of human and baboon anti-alpha Gal antibodies by a subfraction of oligosaccharides derived from porcine stomach mucin. PMID- 8623269 TI - Murine monoclonal anti-idiotypic antibodies directed against human anti-alpha Gal antibodies prevent rejection of pig cells in culture: implications for pig-to human organ xenotransplantation. PMID- 8623270 TI - Delayed xenograft rejection in C3-depleted discordant (pig-to-baboon) cardiac xenografts treated with cobra venom factor. PMID- 8623271 TI - Ratites (ostrich, emu) as potential heart donors for humans: immunologic considerations. PMID- 8623272 TI - Efficiency of different synthetic oligosaccharides for blocking the natural anti g Gal antibodies found in human and baboon sera. PMID- 8623274 TI - Increased anti-Gal activity in diabetic patients transplanted with porcine islet cells. PMID- 8623273 TI - Human anti-B alloantibodies recognize xenoantigens on pig red blood cells and endothelial cells. PMID- 8623275 TI - New World monkeys as a primate model for xenografts in the absence of anti-Gal. PMID- 8623276 TI - The rejection of hamster xenografts by rats is mediated by a restricted repertoire of Ig heavy chain variable genes. PMID- 8623277 TI - Targeting of activated T-cells with natural cytotoxic antibodies via an IL2 hapten conjugate prolongs graft survival. PMID- 8623278 TI - Xenoreactive natural antibodies and isohemagglutinins: a distinct class of antibodies. PMID- 8623279 TI - Processing of IgM/Anti-IgM immune complexes by the liver of rats treated with anti-IgM monoclonal antibodies. PMID- 8623281 TI - Different action of xenoreactive natural and immune antibodies on functioning cardiomyocytes. AB - PNAB and IAB have totally different action on BCM. PNAB inhibit the contractile function of BCM by producing a reversible standstill and by disturbing the synchronization of the BCM-monolayer. IAB are cytotoxic and depend on the presence of complement. PMID- 8623280 TI - Identification of Forssman as a major guinea pig xenoantigen. PMID- 8623283 TI - Up-regulation of human decay accelerating factor on cultured endothelium from transgenic pigs in response to inflammatory stimuli. PMID- 8623282 TI - Soluble complement receptor type 1 and cobra venom factor in discordant xenotransplantation. PMID- 8623284 TI - Decay-accelerating factor transgenic mouse hearts are protected from human complement-mediated attack. PMID- 8623285 TI - Difference of expression levels between gene technological product delta CYT MCP(CD46) and intact MCP(CD46) in transgenic mice. PMID- 8623286 TI - Role of human decay accelerating factor expression on porcine kidneys during xenogeneic ex vivo hemoperfusion. PMID- 8623287 TI - Human MCP and DHF double transgenic mice are protected from human complement attack in an in vivo model. PMID- 8623289 TI - Protection of guinea pig cells transfected with the 512 gene for complement dependent cytolysis in rats. PMID- 8623288 TI - The effect of h-DAF expression on endothelial cell activation of porcine xenografts. PMID- 8623291 TI - Donor graft perfusion pre-transplant prolongs xenograft survival. PMID- 8623290 TI - A synthetic dextran derivative inhibits complement activation and complement mediated cytotoxicity in an in vitro model of hyperacute xenograft rejection. PMID- 8623292 TI - The role of C3a and C5a in hyperacute rejection of guinea pig-to-rat cardiac xenografts. PMID- 8623293 TI - Functional feature of C5b-8-step lysis of swine endothelial cell- transfected CD59. PMID- 8623294 TI - How can human DAF and HRF20 prevent HAR in transgenic mice? PMID- 8623295 TI - Studies on transfer of primate membrane-associated complement inhibitors from recipient blood to porcine donor organs. PMID- 8623296 TI - Inactivation of C3 and C5 prolongs cardiac xenograft survival and decreases leukocyte infiltration in a model of delayed xenograft rejection. PMID- 8623297 TI - In vitro and in vivo investigation of anticomplement agents FUT-175 and K76COOH, in the prevention of hyperacute rejection following discordant xenotransplantation in a nonhuman primate model. PMID- 8623298 TI - Porcine endothelial cell membrane antigens recognized by human and baboon xenoreactive antibodies during organ perfusion. PMID- 8623299 TI - Direct cell contact is required to induce expression of E-selectin and IL-8 secretion in pig endothelial cells by human natural killer cells. PMID- 8623300 TI - Human natural killer cells induce expression of E-selectin and interleukin-8 mRNA in porcine endothelial cells. PMID- 8623301 TI - Activation of human prothrombin by porcine endothelium. PMID- 8623302 TI - Human serum-induced porcine endothelial cell apoptosis--another pathway to xenograft rejection. PMID- 8623303 TI - Mutation at ser32 of I kappa B alpha blocks NF-kappa B activation. PMID- 8623304 TI - Role of adhesion molecules in islet allo- and xenograft rejection. PMID- 8623305 TI - Expression of a truncated form of the human p55 TNF-receptor in bovine aortic endothelial cells renders them resistant to human TNF. PMID- 8623306 TI - Analysis of alpha 1,3-galactosyltransferase knockout mice. PMID- 8623307 TI - Variability of alpha 1,3-galactosyltransferase splicing isoforms in pig tissues. PMID- 8623308 TI - Modulation of endothelial metabolism by xenogeneic serum: implications for vasoconstriction and permeability. PMID- 8623310 TI - The role of macrophages in endothelial cell injury--a possible mechanism for delayed xenograft rejection. PMID- 8623309 TI - Thromboxane mediates pulmonary vasoconstriction and contributes to cytotoxicity in pig lungs perfused with fresh human blood. PMID- 8623311 TI - Expression of newborn pig endothelial cell xenoantigens recognized by baboon natural xenoantibody. PMID- 8623313 TI - Fluorescence in situ hybridisation to analyse DAF transgenic jigsaw pigs. PMID- 8623312 TI - Effect of antisense ribozyme to pig alpha (1,3) galactosyl transferase gene on the expression of Gal alpha (1,3)Gal epitope. PMID- 8623314 TI - The anticoagulant effect of antithrombin III on hyperacute xenograft rejection. PMID- 8623315 TI - Assessment of hyaluronate clearance and endothelin production during extracorporeal xenogeneic pig liver perfusion. PMID- 8623316 TI - Nitric oxide and endothelins are effector agents of hyperacute vascular rejection. PMID- 8623317 TI - Reduced human neutrophil adhesion to porcine endothelia expressing human decay accelerating factor. PMID- 8623318 TI - Effect of Gal alpha 1,3 Gal-beta 1,4 GlcNAc and complement depletion on haemostatic activation in an in vitro model of the pig-to-human xenograft reaction. PMID- 8623319 TI - Deletion analysis of the porcine E-selectin promoter. PMID- 8623320 TI - The fertility and breeding potential of boars expressing a functional regulator of human complement activation. PMID- 8623321 TI - Comparative study of porcine and human blood coagulation systems: possible relevance in xenotransplantation. PMID- 8623322 TI - Discordant xenogeneic cellular interactions when hyperacute rejection is prevented: analysis using an ex vivo model of pig kidney perfused with human lymphocytes. PMID- 8623323 TI - Induction of human chimerism and functional suppressor cells in fetal pigs: feasibility of surrogate tolerogenesis for xenotransplantation. PMID- 8623324 TI - Human T-cell activation is mediate predominantly by direct recognition of porcine SLA and involves accessory molecule interaction of ICAM1/LFA 1 and CD2/LFA3. PMID- 8623325 TI - Newborn discordant cardiac xenotransplantation in primates: a model of natural antibody depletion. PMID- 8623326 TI - Direct human T-cell anti-pig xenoresponses are vigorous but significantly weaker than direct alloresponses. PMID- 8623327 TI - Significant primary indirect human T-cell anti-pig xenoresponses observed using immature porcine dendritic cells and SLA-class II-negative endothelial cells. PMID- 8623328 TI - Enhanced porcine hematopoiesis in mice receiving donor-specific interleukin-3. PMID- 8623329 TI - In vitro comparison of human xenogeneic and allogeneic proliferative cellular immune responses using serum-free media. PMID- 8623330 TI - Concordant xenoreactive and alloreactive cell-mediated immune responses are relatively comparable. PMID- 8623332 TI - Rejection of human xenoskins by non-CD4 T-cell populations in mice. PMID- 8623331 TI - Combined CTLA4Ig and anti-CD4/DC8 monoclonal antibody treatment prolongs survival of rat-to-mouse heterotopic cardiac xenografts. PMID- 8623333 TI - Cross-species chimerism and graft-versus-host disease (GVHD) induced by transplantation of mouse fetal hematopoietic cells into newborn rats. PMID- 8623334 TI - Induction of primate TH2 lymphokines to suppress TH1 cells. PMID- 8623335 TI - The antigenicity of serum proteins and their role in xenograft rejection. PMID- 8623336 TI - In vivo evaluation of human membrane cofactor protein in transgenic mice. PMID- 8623337 TI - Experimental cardiac xenografting with irradiation and intrathymic and intravenous xenogeneic cells. PMID- 8623338 TI - The different influences of allosensitization and xenosensitization on cellular rejection. PMID- 8623339 TI - Intrathymic inoculations of donor leukocytes evoke differential cellular immune responses during cardiac allogenic and xenogenic transplant. PMID- 8623340 TI - Delayed xenograft rejection in complement-depleted T-cell-deficient rat recipients of guinea pig cardiac grafts. PMID- 8623341 TI - Tacrolimus (FK 506)-dependent tolerance after liver and heart xenotransplantation: inhibition of humoral response and acceptance of donor organs. PMID- 8623342 TI - Xenogeneic bone marrow transplantation in the hamster-to-rat model prevents rejection of subsequently grafted hamster hearts. PMID- 8623343 TI - Mechanisms involved in long-term hamster-to-rat cardiac xenograft survival. PMID- 8623344 TI - Complete control of humoral and cell-mediated xenoreactions with the combination of leflunomide and cyclosporine. PMID- 8623345 TI - Prolongation of xenograft survival by soluble complement receptor type 1 and antithrombin-III combination therapy. PMID- 8623346 TI - Sequential morphological, functional and xenoreactive antibody analysis of hamster kidneys transplanted into unmodified rats. PMID- 8623347 TI - Cardiac xenotransplantation into complement 5-deficient mice. PMID- 8623348 TI - Control and reversal of chronic xenograft rejection in hamster-to-rat cardiac transplantation. PMID- 8623349 TI - High-level tissue specific expression of human CD59, MCP, and DAF proteins from genomic clones in transgenic mice. PMID- 8623350 TI - Functional analysis of hamster kidney xenografts in the rat: possible functional incompatibility and adaptation of hamster kidney grafts in a xenogenic rat environment. PMID- 8623351 TI - Pretreatment of recipients (nude rat) with donor antigens leads to prolonged survival of hamster heart xenografts. PMID- 8623352 TI - Effect of leflunomide, cyclosporine, and splenectomy in two different organ systems of concordant xenotransplantation in rats. PMID- 8623353 TI - Prolonged survival of discordant xenografts in a subset of complement-depleted nude rats. PMID- 8623354 TI - Xenogeneic fetal liver fragment transplantation with macrocapsule is beneficial therapy for acute liver failure in rats. PMID- 8623355 TI - Inhibition of platelet GPIIbIIIa prolongs survival of discordant cardiac xenografts. PMID- 8623356 TI - Efficacy of leflunomide to reduce xenoantibody titers in vivo: an evaluation of the prolongation of discordant xenograft survival. PMID- 8623357 TI - Hematopoietic competition limits xenogenic myeloid reconstitution in SCID mice. PMID- 8623358 TI - Tetrahydropyranyladriamycin with FK 506 combination therapy for hamster-to-rat concordant xenotransplantation. PMID- 8623359 TI - Specific xenogeneic (human) tolerance in immunosuppressed mice by combined donor derived human transferrin and antigens. PMID- 8623360 TI - Videomicroscopic analysis of the renal microcirculation during xenograft hyperacute rejection in the rat. PMID- 8623361 TI - CTLA4Ig Inhibits humoral and cellular immune responses to concordant xenografts. PMID- 8623362 TI - Limitations to the feasibility of discordant liver transplantation. PMID- 8623363 TI - Tolerance to experimental cardiac allografts but not xenografts is induced after simultaneous neonatal intrathymic inoculation with allo and xenogeneic cells. PMID- 8623364 TI - Induced tolerance and flow cytometry analysis of lymphocyte phenotypes in concordant xenograft transplantation. PMID- 8623365 TI - Intravital microscopy for evaluation of early xenograft viability of isolated perfused rat livers. PMID- 8623366 TI - Prevention of hyperacute xenograft rejection by polyclonal antibodies against donor blood cells. PMID- 8623367 TI - Use of a novel CD11b/CD18 inhibitory agent in a C6-deficient rat to evaluate delayed xenograft rejection. PMID- 8623368 TI - Obliterative airway disease after heterotopic tracheal xenotransplantation in a concordant rodent model: pathogenesis and treatment. PMID- 8623369 TI - Success in guinea pig to rat orthotopic corneal xenotransplantation with cyclosporin A. PMID- 8623370 TI - Use of MHC class I or II "knock out" mice to delineate the role of these molecules in acceptance/rejection of xenografts. PMID- 8623371 TI - Prolonged survival of hearts obtained from chimeric donors in a mouse to rat xenotransplant model. PMID- 8623372 TI - Xenotransplantation of hamster liver into Gunn rats reserves congenital hyperbilirubinemia. PMID- 8623373 TI - Effect of tacrolimus and splenectomy on engraftment and GVHD after bone marrow xenotransplantation in the reciprocal hamster to rat animal models. PMID- 8623375 TI - Prolongation of discordant cardiac xenotransplantation survival by Lisofylline, a phosphatidic acid inhibitor. PMID- 8623374 TI - Perioperative treatment with phosphatidic acid inhibitor (Lisofylline leads to prolonged survival of hearts in the guinea pig to rat xenotransplant model. PMID- 8623376 TI - Difference in tolerization of T-independent and T-dependent xenoantibody formation after cardiac xenotransplantation in rats. PMID- 8623377 TI - Immunologic studies in primates undergoing concordant xenotransplantation as a bridge allotransplantation. PMID- 8623378 TI - Retrovirus-mediated transfer and expression of swine MHC class II genes in CD34+ monkey stem cells. PMID- 8623379 TI - Extracorporeal auxiliary xenoperfusion: animal model of support in fulminant liver failure. PMID- 8623380 TI - Successful long-term concordant xenografts in primates: alteration of the immune response with methotrexate. PMID- 8623381 TI - Human decay accelerating factor expressed on endothelial cells of transgenic pigs affects complement activation in an ex vivo liver perfusion model. PMID- 8623382 TI - Continuous complement (C) inhibition using soluble C receptor type 1 (sCR1): effect on hyperacute rejection (HAR) of pig-to-primate cardiac xenografts. PMID- 8623383 TI - The mechanism of xenogeneic cell-mediated lympholysis between human and pig cells. PMID- 8623384 TI - Human complement regulatory proteins expressed in transgenic swine protect swine xenografts from humoral injury. PMID- 8623385 TI - Development and analysis of transgenic pigs expressing the human complement regulatory protein CD59 and DAF. PMID- 8623386 TI - Intrathymic stem-cell transplantation produces hematopoietic microchimerism in human to baboon xenografts. PMID- 8623387 TI - A mixed chimerism approach to renal transplantation between concordant nonhuman primate species. PMID- 8623388 TI - Long-term survival and functional tolerance of baboon to monkey kidney and liver transplantation: a preliminary report. PMID- 8623389 TI - Targeted in vivo gene transfection modulated hyperacute rejection of pig lungs perfused with human blood. PMID- 8623390 TI - Intravenous immunoglobulin delays xenogeneic hyperacute rejection in a model of pig liver perfused with human blood. PMID- 8623391 TI - Effect of LFA-1 inhibition on immediate organ function in concordant ex-vivo hemoperfusion of primate kidneys. PMID- 8623392 TI - Delay of xenogeneic hyperacute rejection by BN 52021 in a model of rabbit to pig liver transplantation. PMID- 8623393 TI - Human decay accelerating factor successfully protects pig hearts from hyperacute rejection by human blood. PMID- 8623395 TI - Transgenic porcine livers reduce liberation of humoral mediators during xenoperfusion with human blood. PMID- 8623394 TI - Transgenic human DAF-expressing porcine livers: their function during hemoperfusion with human blood. PMID- 8623397 TI - Phenotype of infiltrating cells in an ex vivo model of human antiporcine hyperacute cardiac xenograft rejection. PMID- 8623396 TI - Importance of cell-mediated immune responses in rejection of concordant heart xenografts in primates. PMID- 8623398 TI - In vitro and in vivo effects of polyclonal human intravenous immunoglobulins in discordant xenotransplantation. PMID- 8623399 TI - Xenogeneic orthotopic liver transplantation from sheep to pig: delay of rejection by FUT 175 Infusion. PMID- 8623400 TI - Histological findings in heart grafts after orthotopic pig to baboon cardiac transplantation. PMID- 8623401 TI - Derivation of immortalized swine stromal cell lines. PMID- 8623402 TI - Methotrexate in rescue therapy for xenotransplanted lungs in primates. PMID- 8623403 TI - Major oligosaccharide epitopes found in tissues of 23 animal species, potential donors for organ xenotransplantation. PMID- 8623404 TI - Thomsen-Friedenreich and PK antigens in pig-to-human xenotransplantation. PMID- 8623405 TI - Enhanced survival of porcine endothelial cells and lung xenografts expressing human CD59. PMID- 8623406 TI - Extracorporeal immunoadsorption of xenoreactive natural antibodies in the pig/rhesus model: comparison of three methods. PMID- 8623408 TI - Long-term culture or complement inhibition improves early islet function in dog to rat islet xenotransplantation. PMID- 8623407 TI - Perfusion of pig hearts with anti-CD45 monoclonal antibody to label interstitial dendritic cells before xenotransplantation. PMID- 8623409 TI - Long-term function of porcine islets in xenogeneic hosts. PMID- 8623410 TI - Xenoreactivity in the pig islet to human combination: feasibility of adenovirus mediated gene transfer into pig islets. PMID- 8623411 TI - Expression of cytokines during rejection of fetal pig islet xenografts in nonobese diabetic mice. PMID- 8623412 TI - Coherent microcapsules for pancreatic islet transplantation: a new approach for bioartificial pancreas. PMID- 8623414 TI - Prolongation of concordant islet xenografts with leflunomide. PMID- 8623413 TI - Tolerance to islet xenografts induced by dual manipulation of antigen presentation and co-stimulation. PMID- 8623415 TI - Survival of transplanted porcine neural cells treated with F(ab')2 antibody fragments directed against donor MHC class-I in a rodent model. PMID- 8623416 TI - Immunosuppression with cyclosporin A in combination with leflunomide and mycophenolate mofetil prevents rejection of pig-islets transplanted into rats. PMID- 8623417 TI - Xenotransplantation of bovine islets into dogs using biodegradable, injectable microreactors. PMID- 8623418 TI - CTLA4-Ig prolongs survival of microencapsulated rabbit islet xenografts in spontaneously diabetic Nod mice. PMID- 8623419 TI - The effect of gnotobiotic rearing on porcine islet isolation and function. PMID- 8623420 TI - Immunohistological study of islet xenograft rejection in immunosuppressed recipients. PMID- 8623421 TI - Hepatocyte xenotransplantation for Nagase's analbuminemic rats: the effect of FK506. PMID- 8623422 TI - Long-term culture of non-purified rat islets embedded in hydrogel matrix. PMID- 8623423 TI - Survival of hamster heart xenografts in rat recipients on a relatively mild immunosuppressive protocol. PMID- 8623424 TI - A simple method for xenotransplanting cells and tissues into rats using uncoated alginate microreactors. PMID- 8623425 TI - Influence of complement in human serum on isolated porcine islets. PMID- 8623426 TI - Porcine islet cell antigens are recognized by xenoreactive natural human antibodies of both IgG and IgM subtypes. PMID- 8623427 TI - Survival of human islet xenografts irradiated with ultraviolet B in diabetic rats. PMID- 8623428 TI - Role of humoral immunity in pancreatic islet allo- and xenograft rejection. PMID- 8623429 TI - Impact of antipassenger lymphocyte globulin on functional graft survival of discordant xenoislet grafts from a large animal donor. PMID- 8623430 TI - Interprimate stem cell transplantation. PMID- 8623431 TI - Increasing donor-recipient imbalance justifies xenograft research. PMID- 8623432 TI - Xenogeneic study of the isolated liver perfusion. PMID- 8623433 TI - Normal human polyclonal immunoglobulins delay xenograft rejection through distinct F(ab')2 and Fc mediated mechanisms. PMID- 8623434 TI - Screening of baboons as potential liver donors for humans. PMID- 8623436 TI - Cross Species Interaction of xenogeneic interleukins. PMID- 8623435 TI - Xenozoonoses: assessing activation of latent/unknown viruses in immunosuppressed baboons. PMID- 8623437 TI - Longitudinal analysis of the expression of human decay accelerating factor on peripheral blood mononuclear cells and in the plasma of transgenic pigs. PMID- 8623438 TI - Production of pigs transgenic for human regulators of complement activation using YAC technology. PMID- 8623439 TI - Neoral cyclosporine. PMID- 8623440 TI - 15-Deoxyspergualin in experimental transplant models: a review. PMID- 8623441 TI - Deoxyspergualin: clinical experience. PMID- 8623442 TI - Deoxyspergualin: mechanism of action and pharmacokinetics. PMID- 8623443 TI - Cancers in cyclosporine-treated vs azathioprine-treated patients. PMID- 8623444 TI - Do early cyclosporine levels affect the incidence of acute rejection in renal transplant recipients? PMID- 8623445 TI - Effect of Neoral or cyclosporine on the development of chronic rejection in an aortic allograft rat model. PMID- 8623446 TI - Comparison of the absorption pharmacokinetics of Sandimmune and Neoral following porcine small bowel transplantation. PMID- 8623447 TI - Addition of the colon to small bowel grafts causes lethal graft-versus-host disease in FK 506-treated pigs. PMID- 8623448 TI - Immunosuppression with the vitamin D analogue MC 1288 in experimental transplantation. PMID- 8623449 TI - Comparative pharmacokinetics of cyclosporine A and cyclosporine G in renal allograft recipients. PMID- 8623450 TI - Cyclosporine A enhances the vascular reactivity to angiotensin II in the renal microcirculation in rodents. PMID- 8623451 TI - Reduction in hospital stay after liver transplantation. PMID- 8623452 TI - Comparison of cyclosporine- vs tacrolimus-based immunosuppression in pediatric liver transplantation. PMID- 8623453 TI - Tacrolimus-based vs cyclosporine-based immunotherapy in combined kidney-pancreas transplantation. PMID- 8623454 TI - CyA-NOF for early oral CyA-based immunosuppression in patients after liver transplantation. PMID- 8623455 TI - Economic evaluation of immunosuppressive drugs: an empirical example using a secondary database of hospital charges. PMID- 8623456 TI - Cost-containment strategies in transplantation: the utility of cyclosporine ketoconazole combination therapy. PMID- 8623458 TI - The effect of 15-deoxyspergualin on islet allograft survival in the canine model. PMID- 8623457 TI - Results with histological diagnosis in kidney transplant rejections treated with 15-deoxyspergualin. PMID- 8623459 TI - Prolonged immunosuppressive effect and minimal immunogenicity from chimeric (CD25) monoclonal antibody SDZ CHI 621 in renal transplantation. PMID- 8623460 TI - In vivo (phase I) trial and in vitro efficacy of humanized anti-Tac for the prevention of rejection in renal transplant recipients. PMID- 8623461 TI - A prospective randomized trial of OKT3 vs ATGAM induction therapy in pancreas transplant recipients. PMID- 8623462 TI - Effect of MC1288, a vitamin D analogue, on lymphocyte proliferation in vitro and rat aortic allograft arteriosclerosis in vivo. PMID- 8623463 TI - Two new DNA synthesis inhibitors with potent immunosuppressive activity prolong allograft and xenograft survival in small and large animals. PMID- 8623464 TI - Overview of a 10-year experience on methods and compositions for inducing site specific immunosuppression with topical immunosuppressants. AB - Methods and formulations have been successfully developed to bring about site specific immune suppression of local T-cell-mediated immune responses involved in contact hypersensitivity, skin allograft rejection, and, putatively, autoimmune inflammatory conditions such as psoriasis. The induction of site-specific immune suppression results in reduced systemic pharmacology and toxicity. Certain formatulations have been devised that can effect dramatic transdermal drug delivery and systemic immunopharmacology. Local site-specific or systemic efficacy by transdermal delivery can be dependent upon carrier composition with respect to the hydrophilic/lipophilic nature of the solvent system, active principal solubilization, and concentration. Multiple classes of active immunosuppressive agents can be successfully combined to produce novel and extremely potent topical drugs. Specifically, either cyclosporine or rapamycin inhibit local inflammatory/immune responses by topical application to skin tissue in vivo. Rapamycin is particularly efficacious during the late local inflammatory/immune phase. Cyclosporine is particularly efficacious during the early local inflammatory-immune phase. Also, skin allograft survival may be prolonged via topical use of CyA, alone and in combination with other anti inflammatory agents. This includes combined immunosuppressant and steroidal anti inflammatory agents that can produce synergistic results. Systemic immunity in these instances is normal. Expression of MHC class I and MHC class II molecules is dramatically decreased in these CyA/steroid SITE-treated grafts. In summary, the induction of local immune suppression at the tissue site and focal responding immunocytes can result in surprising efficacy when used in conjunction with limited systemic administration, which could have significant immunologic and clinical ramifications. PMID- 8623465 TI - Islet protective effect of pravastatin from nonspecific inflammation in mouse pancreatic islet isografts. PMID- 8623466 TI - Pharmacokinetics of mycophenolate mofetil (RS61443): a short review. AB - Overall, the PK of MMF is reasonably straightforward and relatively unaffected by the complex pathophysiological changes involved in the management of renal transplant recipients. The ability to relate plasma MPA concentrations to efficacy through the PK/PD correlation implies that, unlike for many other drugs, plasma PK has direct relevance to the clinical outcome. These features should not only greatly help the exploration of immunosuppressive regiments using MMF but also the development of clinical use of MMF in situations other than renal transplantation. PMID- 8623467 TI - Immunosuppressive effect of RS-61443 on rat cardiac allograft survival in combination with leflunomide or FK 506. PMID- 8623468 TI - Prolongation of pancreas allograft survival by mycophenolate mofetil in the rat. PMID- 8623469 TI - Pharmacodynamic assessment of mycophenolic acid in a canine model. PMID- 8623470 TI - The effect of a nonsteroid immunosuppressive regimen with RS-61443 and cyclosporine on kidney allograft survival in dogs. PMID- 8623471 TI - Improved high-performance liquid chromatographic assay for the measurement of mycophenolic acid in human plasma. PMID- 8623472 TI - Bone marrow augmentation of kidney allografts can cause graft-versus-host disease in immunosuppressed recipients. PMID- 8623473 TI - Human donor bone marrow cells can enhance hyporeactivity in renal transplantation using maintenance FK 506 and OKT3 induction therapy. PMID- 8623474 TI - Combined immunosuppression with leflunomide and cyclosporine prevents MLR mismatched renal allograft rejection in a mongrel canine model. PMID- 8623475 TI - Spectrum of susceptibility to rejection of heart and multivisceral small bowel allografts using leflunomide immunosuppression. PMID- 8623476 TI - Immunosuppressive effect of brequinar on rat cardiac allograft survival in combination with leflunomide or FK 506. PMID- 8623477 TI - Effect of leflunomide on T-independent xenoantibody formation in rats receiving hamster heart xenografts. PMID- 8623478 TI - Prevention of chronic rejection and puromycin-induced glomerulosclerosis in rats by gamma-lactone, an inhibitor of macrophage-effector activation. PMID- 8623479 TI - Brequinar sodium significantly reduces the incidence of steroid-resistant rejection and resource utilization in primary renal transplant patients compared with azathioprine. PMID- 8623480 TI - Ling-Zhi-8: a fungal protein with immunomodulatory effects. PMID- 8623481 TI - Brequinar sodium. AB - The future use of BQR as a component of a treatment regimen for preventing graft rejection is uncertain. The drug exhibits a number of characteristics that are considered desirable for inclusion in a multidrug antirejection protocol. BQR is an effective immunosuppressive agent and can act as a single agent to prevent the rejection of allografts and xenografts. Its immunosuppressive activity is especially useful in those situations for which inhibition of antibody production is an important objective of the treatment protocol. The activity of BQR is substantially improved by including the drug with other immunosuppressive agents, such as CsA, that differ in the mechanisms by which they prevent immune responses. This type of combination therapy provides for effective and safe immunosuppression in rodent models. Additional desirable characteristics include the high level of bioavailability, patient acceptance, and ease of monitoring plasma drug levels. The primary difficulties associated with the application of BQR therapy to humans is the narrow ratio of the level of drug therapy necessary to provide for effective prevention of graft rejection and the appearance of side effects that limit the use of the drug. The principal feature of BQR's pharmacologic activity that appears to be responsible for this narrow therapeutic window may be the extended plasma half-life of the drug. The most effective treatment schedule for preventing graft rejection in rodents is administration of the drug on alternate days. Treating with smaller doses more frequently does not improve graft survival, suggesting that reducing the plasma half-life may allow for more frequent peaks of drug activity without increasing the severity of the side effects. The phase I trials of BQR have been completed and the extension of these studies for a more careful examination of the efficacy of the drug may depend upon biochemical modification of the parent drug. PMID- 8623482 TI - Mechanism of action of rapamycin (Sirolimus, Rapamune). PMID- 8623483 TI - Pharmacokinetics of rapamycin. AB - Based on the present findings, a number of preliminary conclusions can be made regarding the distribution, pharmacokinetics, and therapeutic range investigations with RAPA: (a) the majority of the drug is sequestered in erythrocytes, resulting in whole blood concentrations; (b) the drug has a relatively long half-life in both humans and animals with 24-hour trough concentrations being within the analytical range of HPLC when immunosuppressive doses are administered; (c) the drug exhibits a proportionality between trough concentrations and dose; (d) trough concentrations of the drug appear to be related to immunosuppressive efficacy and drug-related side effects. The studies described here should provide a basis for the establishment of therapeutic monitoring protocols for RAPA. PMID- 8623484 TI - Clinical use of FK 506 in liver transplantation. AB - In controlled clinical trials, primary immunosuppression with FK 506 has been shown to be comparable to a cyclosporine-based regimen for patient and graft survival following liver transplantation. FK 506 was superior to cyclosporine for treating acute, steroid-resistant, and refractory rejection. FK 506 also has been shown to be effective rescue therapy in patients with refractory rejection on conventional immunosuppression following liver transplantation. Generally, FK 506 allows the use of lower doses of corticosteroids and earlier discontinuation of azathioprine than does cyclosporine. In these clinical trials, FK 506 was associated with an increased incidence of side effects, in particular nephro- and neurotoxicity and abnormal glucose metabolism. However, preliminary results from a 2-year follow-up indicate that FK 506 and cyclosporine are comparable with respect to safety and tolerability. As increased experience is gained with the use of FK 506 for liver transplantation, the efficacy and safety profile of FK 506 should improve. PMID- 8623485 TI - Immunosuppressants suppressing signal 2 of T-cell activation enable CsA to induce operational transplantation tolerance in a strongly immunogenic heart allograft model in rats. PMID- 8623486 TI - Molecular basis of immunosuppressive drug action: regulation of steroid receptor mediated transcription by FK 506. PMID- 8623487 TI - Perioperative immunosuppression as a critical determinant of early outcome after discordant xenoislet transplantation: a comparative study. PMID- 8623488 TI - Combined rapamycin and cyclosporine immunosuppression in a porcine renal transplant model. PMID- 8623489 TI - A randomized, double-blind, placebo-controlled study to determine safety, tolerance, and preliminary pharmacokinetics of ascending single doses of orally administered sirolimus (rapamycin) in stable renal transplant recipients. PMID- 8623490 TI - A randomized, double-blind, placebo-controlled study of the safety, tolerance, and preliminary pharmacokinetics of ascending single doses of orally administered sirolimus (rapamycin) in stable renal transplant recipients. PMID- 8623491 TI - Tacrolimus (FK 506): a whole blood assay as measured on the Abbott IMx analyzer. PMID- 8623492 TI - FK 506 induction and rescue therapy in pancreas transplant recipients. PMID- 8623494 TI - Conversion from cyclosporine to tacrolimus in kidney, kidney/pancreas, and pancreas alone transplant recipients: the Memphis experience. PMID- 8623493 TI - Preliminary experience with FK 506 in pediatric renal transplant recipients: a single-center report. PMID- 8623495 TI - Role of protocol biopsies in the treatment of refractory renal allograft rejection with FK 506. PMID- 8623496 TI - Hepatocarcinogenesis: a polygenic model of inherited predisposition to cancer. AB - The murine inbred strain C3H provides an experimental model of inherited predisposition to hepatocellular cancer. Hepatocellular neoplastic lesions induced by chemical carcinogens reach a volume 10-100-fold greater in C3H mice than in genetically resistant strains. However, the huge strain differences in tumor size are explained by relatively small differences (10%-30%) in tumor cell kinetics. Genetic linkage experiments in different crosses demonstrated that six unlinked hepatocarcinogen sensitivity (Hcs) and two hepatocarcinogen resistance (Hcr) loci determined quantitative variations in susceptibility to hepatocarcinogenesis. Such results provide the genetic basis for the strain variations in susceptibility to hepatocarcinogenesis and demonstrate a new model of polygenic inheritance of predisposition to cancer. PMID- 8623497 TI - Applications of 99mTc-sestamibi in oncology. AB - Hexakis (2-methoxyisobutylisonitrile) technetium-99m (99mTc-SestaMIBI) is a radiopharmaceutical used in nuclear medicine for myocardial perfusion imaging. In the literature different non-cardiac applications of 99mTc-SestaMIBI have been reported. Clinical studies have been performed also in non-oncologic disease (such as thyroid adenoma, diabetic foot, osteomyelitis, pulmonary actinomycosis, aneurysmal bone cyst. Sudeck's atrophy). Several models for the uptake mechanism of this radiopharmaceutical have been proposed such as binding to an 8-10 kDa cytosolic protein, simple lipid partitioning, or a membrane translocation mechanism involving diffusion and passive transmembrane distribution. Most evidence points in the direction of the third hypothesis. Many studies have indicated that uptake of hexakis (alkylisonitrile) technetium complexes is dependent on mitochondrial and plasma membrane potentials like other lipophilic cations. This explains the initial biodistribution of 99mTc-SestaMIBI to tissues with negative plasma membrane potentials and with relatively high mitochondrial content (like heart, liver, kidney and skeletal muscle tissue). Malignant tumours also possess these properties in order to maintain their increased metabolism. This behaviour encouraged the study of 99mTc-SestaMIBI as an interesting tracer imaging various tumour types: osteosarcoma, brain, lung, breast, nasopharyngeal, parathyroid and thyroid cancer. Recent research on cell cellular physiology has further revealed an active transport of 99mTc-SestaMIBI out of the tumour cells, against the potential gradient. The same mechanism is also responsible for resistance to a structurally and functionally different group of cytotoxic agents such as vinca alkaloids, epipodophyllotoxins, anthracyclins and actinomycin D. This peculiar type of resistance is due to amplification of the mammalian MDR1 gene, located on chromosome 7. For this reason the 99mTc-SestaMIBI uptake in vivo could permit the prediction of the response to the chemotherapy, when the decreased accumulation of 99mTc-SestaMIBI implies the presence of P-gp enriched tissues. In the next future a particular attention should be dedicated to this matter since one of the most important goals of the clinical trials is the demonstration of the usefulness of 99mTc-SestaMIBI for in vivo assessment of multidrug resistance. PMID- 8623498 TI - Decreased expression and function of Vbeta6+ and Vbeta14+ T cells is associated with decreased Th1 cytokine production in mice with plasma cell tumors. AB - AIMS AND BACKGROUND: We have found that polyclonally stimulated T cells from mice bearing ascitic plasma cell tumors demonstrate specific decreases in Th1 cytokine production. In this study we investigated whether loss of Th1 responses in the plasma cell tumor system was associated with alterations in the Vbeta T cell receptor repertoire. METHODS: We examined the cell surface expression of specific Vbeta expressing splenic CD4+ or CD8+ T cells from normal and tumor bearing mice using direct three-color flowcytometry. In order to determine the Th phenotype of Vbeta expressing T cells, we enriched for Vbeta6, Vbeta14 or Vbeta8.1,8.2 cells, polyclonally stimulated them and measured the levels of the sytokines interleukin 4 (IL-4), IL-2 and interferon-gamma (IFN-gamma). RESULTS: We find there is a statistically significant decrease in the frequency of Vbeta6+ and Vbeta14+ CD8+ T cells in mice bearing a plasma cell tumor (B53) as compared to normal (p<0.05). Stimulated Vbeta6+ and Vbeta14+ T cells exhibit an exclusively Th1 phenotype. Stimulated Vbeta6+ and Vbeta14+ T cells from B53 mice are deficient in production of the Th1 cytokines. In contrast, stimulated Vbeta8.1,8.2+ T cells, which are not altered in B53 mice, reveal a Th2 phenotype. CONCLUSIONS: The significance of this study is our demonstration that decreased expression and function of Vbeta6+ and Vbeta14+ T cells may be, at least in part, responsible for the decrease in the production of IL-2 and/or IFN-gamma observed in hosts with tumors. PMID- 8623500 TI - Breast cancer screening: characteristics and results of the Italian programmes in the Italian group for planning and evaluating breast cancer screening programmes (GISMa). AB - In 1990, GISMa (Italian Group for planning and evaluating Mammographic Screening Gruppo Italiano per la pianificazione e la valutazione dei programmi di Screening Mammografico), a working group of operators (radiographers, radiologists, epidemiologists, clinicians, surgeons) involved in screening programmes ongoing in Italy, was created within the Italian School of Senology. The aim of this study is to illustrate data, presented at the GISMa meeting held in April 1994, concerning the characteristics of each programme and some early indicators of effectiveness. To assess these parameters (concerning compliance level, recall rate, benign/malignant biopsy ratio, detection rate, stage distribution, nodal involvement and number of cancers with a diameter under 1 cm, rate of cancer, etc.), 'acceptable' and 'desirable' standards obtained from Italian and North-European cancer screening experiences have been adopted. Most programmes have shown an acceptable standard for most of the indicators, and many of them have attained desirable levels. In most screening programmes the occurrence of interval cancers has not yet been measured, but all centres have (or are working to set up) a systematic active procedure to collect the data. The results indicate that common guidelines can be adopted, even when working in very heterogeneous contexts, and that it is possible to achieve a very high effectiveness and efficacy level. As regards quality control and cost/benefit issues, the goal of extending centralised, population-based screening programmes to other Italian regions becomes a priority. PMID- 8623499 TI - The potent angioinhibin AGM-1470 stimulates normal but not human tumoral lymphocytes. AB - BACKGROUND: AGM-1470 is a newly synthesized molecule developed as an analog of the potent anti-angiogenic fumagillin. Its efficacy in restraining tumor growth in vivo and the absence of major side effects have already led to phase I clinical trials in patients with solid cancers. However, neither the exact mechanisms of action of AGM-1470 nor its effects on the host of normal cells have been extensively studied. Recently, we showed that AGM-1470 enhanced the proliferation of B lymphocytes in the presence of T cells. Since AGM-1470 could potentially be used in patients with lymphoma, it was urgent to test the effect of the molecule on the proliferation of tumor lymphocytes. METHODS: The possible effect of AGM-1470 on the proliferation of normal or tumor lymphocytes was evaluated by thymidine-incorporated assays. Normal T and B lymphocytes were purified from human tonsils. The tumor lymphocytes used in the study were Molt 3, Molt 4 and Jurkatt cell lines for the T lineage and Daudi and Radji cell lines for the B lineage. RESULTS: As shown previously, AGM-1470 stimulates the proliferation of normal B lymphocytes through an action on normal T cells. THe angioinhibin was ineffective ont eh proliferation of both T and B transformed cells. Moreover, in the presence of the drug, tumor T cells co-cultured with normal B lymphocytes did not induce any increase in B cell proliferation, as previously observed with normal T lymphocytes. Inversely, tumor B cells co cultured with normal T lymphocytes were insensitive to the drug. CONCLUSIONS: Our results demonstrate that AGM-1470 is ineffective on lymphoid tumor cell proliferation and could potentially be safely administered to lymphoma patients. PMID- 8623501 TI - Endometrial ultrasonography - An alternative to invasive assessment in women with postmenopausal vaginal bleeding. AB - AIMS AND BACKGROUND: To test the reliability of endometrial sonography in selecting women with abnormal postmenopausal vaginal bleeding for further diagnostic assessment. METHODS: Endometrial thickness was measured in 368 consecutive women by abdominal or vaginal sonography prior to invasive assessment (hysteroscopy, curettage). The association of abnormal and endometrial thickness (4 mm or greater) with endometrial cancer was determined. RESULTS: Abnormal endometrial thickness was observed in 116 of 368 women. Subsequent assessment diagnosed endometrial carcinoma in 16 subjects, 15 of whom had abnormal endometrial thickness. One case with normal endometrial thickness was suspected at sonography because of the irregular appearance of the endometrium. CONCLUSIONS: Had it been used to select subjects for further assessment, sonography would have missed no cancer, and unnecessary invasive assessment (under general anesthesia in 20% of cases) would have been spared in 68% (251/368) of the subjects. Endometrial sonography should be routinely used to select women with postmenopausal vaginal bleeding for further investigations. PMID- 8623502 TI - Helicobacter pylori infection in patients with gastric adenocarcinoma. AB - We examined the biologic tumor behavior in Helicobacter pylori-seropositive patients with gastric adenocarcinoma. A total of 214 consecutive patients with pathologically confirmed adenocarcinoma of the stomach who underwent gastric resection were studied. The stored serum samples were tested for serum antibody to H. pylori by using a highly sensitive and specific IgG enzyme-linked immunosorbent assay. The difference in H. pylori-seropositive and seronegative patients with gastric adenocarcinoma was evaluated in terms of various clinicopathologic parameters. A multivariate logistic regression analysis was used to adjust for potential confounding variables. Antibodies to H.pylori were detected in 65.9% of patients with gastric adenocarcinoma. H.pylori-seropositive patients were younger than seronegative patients and had infiltrative tumor according to Ming's criteria. When adjusted for age, infiltrative tumor come out stronger. These findings suggest that H.pylori infection may be related to infiltrative type gastric adenocarcinoma; further study is necessary. PMID- 8623503 TI - Steroid hormone levels in patients with advanced breast cancer during therapy with droloxifene: a pilot study. AB - Antiestrogens, particularly tamoxifen, are effective in the treatment of pre- and postmenopausal women suffering from all stages of breast cancer. Unfortunately, many patients become resistant to tamoxifen during therapy, which allows the tumor to progress. Thus, a preclinical recognition of tumor progression, i.e. by monitoring serum hormone levels, could be worthwhile. The serum levels of dehydroepiandrosterone sulfate and estradiol of postmenopausal women with advanced breast cancer treated by the new antiestrogen droloxifene were therefore checked. However, only non-significant changes in the hormone levels during droloxifene therapy were observed, and no relation was found between hormone levels and the course of the disease, success or exhaustion of droloxifene application, or development of tumor progression. Our data do not confirm earlier findings reported in the literature that measurement of hormones seems to be suitable for an early indication of tumor progression during an antiestrogen therapy before its clinical manifestation. PMID- 8623504 TI - Hodgkin's disease stage I and II with exclusive subdiaphragmatic presentation. The experience of the Departments of Radiation Oncology and Hematology, University "La Sapienza" of Rome. AB - During the period 1978 to 1994, 1054 patients with Hodgkins's disease were evaluated and treated at the Departments of Radiation Oncology and Hematology, University "La Sapienza", Rome. A total of 549 patients presented with clinical or pathological stage I and II; 37 of these had Hodgkin's disease below the diaphragm (BDHD), and 512 above the diaphragm (ADHD). A comparison of patients with BDHD versus those with ADHD showed that the first group had a higher male to female ratio. A comparison of cases with stage II BDHD versus those with stage II ADHD showed that patients with BDHD were older (48 years vs 28 years), had different histologic features and a higher incidence of systematic symptoms (67% vs 33%). Stage II BDHD patients had a worse prognosis; in fact, there were significant differences in the overall survival and relapse-free-survival rates for cases with stage II BDHD versus those with stage II ADHD (overall survival, 46% vs 80%, P<0.001; relapse-free survival, 44% vs 69%, P<0.005). Stage was found to be the most important prognostic factor for BDHD cases without systematic symptoms treated with radiation therapy alone. The type of infradiaphragmatic presentation (intra-abdominal vs peripheral disease) did not influence outcome, probably due to the more aggressive therapy received by the intra-abdominal group. Treatment recommendations for BDHD cases should be tailored to the stage and the presence or absence of intra-abdominal localization. For patients with stage IA extended fields, irradiation (inverted Y) is sufficient. However, combined modality therapy should be the treatment of choice for stage II cases, particularly in the presence of intra-abdominal disease. Patients with systematic symptoms also require combined modalities. PMID- 8623505 TI - Surgery followed by intracavitary plus systemic chemotherapy in malignant pleural mesothelioma. AB - AIMS AND BACKGROUND: Malignant mesothelioma is associated with a median survival of 4 to 12 months. Data from the literature indicate that single modality treatment (surgery or intrapleural and/or systemic chemotherapy) does not significantly affect survival. METHODS: We therefore evaluated a combined approach consisting of surgery (pleurectomy + diaphragmatic or pericardial resection), intrapleural chemotherapy with cisplatin (100 mg/m2) and cytarabine (1,000 mg/m2) for 4 h immediately after pleurectomy, and systemic chemotherapy consisting of epirubicin (60 mg/m2) and mitomycin-C (10 mg/m2) day 1 every 4 weeks for 4 cycles. RESULTS: Twenty patients were enrolled in the study and were evaluable. Thirteen cases had residual gross disease after pleurectomy and 7 patients only minimal disease. Median time to disease progression was 7.4 months, and median survival was 11.5 months (range, 2-25+). No treatment-related death have been observed. Side effects after intracavitary chemotherapy included renal toxicity, anaemia and pain. Myelosuppression and alopecia were recorded during systematic chemotherapy. CONCLUSIONS: The results of the study indicate that the schedule is feasible, with encouraging results in terms of survival for patients with minimal residual disease after surgery. PMID- 8623506 TI - Cisplatin-vinorelbine combination chemotherapy in locally advanced non-small cell lung cancer. AB - AIM: The North Milan Group presents the results of a phase II study on a cisplatin-vinorelbine combination schedule in the treatment of locally advanced non-small cell lung cancer to evaluate its activity and tolerability. METHODS: Seventy-six consecutive patients entered the study. Patients' characteristics were the following: males/females 69/7; median age, 61.4 years (range, 40-73); ECOG performance status, 0-1; 17 stage IIIa and 59 stage IIIb. There were 49 squamous cell carcinomas, 20 adenocarcinomas, and 7 large cell carcinomas. All patients had not been previously treated and showed measureable disease. Treatment consisted of vinorelbine, 25 mg/m2 on days 1 and 8, plus cisplatin, 80 mg/m2 on day 1, administered intravenously every 21 days for three standard courses. RESULTS: Seventy-four patients were evaluable for response. Objective responses were documented in 42/74 patients with an overall response rate (CR+PR) of 56.7%; 18/74 patients (24.3%) showed stable disease and the remaining 14/74 (18.9%) went into progression. Twelve patients (16.2%) were suitable for a subsequent surgery. The median duration of response was 13.3 months. Survival time ranged from 4 to 36 months; it was 14.6 months for PR patients, 8.6 months for NC and 5 months for PD. Mean survival time is presently 12.85 months (SE, 1.2 months). Toxicity evaluated on 222 cycles administered was acceptable, and it was necessary to use G-CSF or delay the treatment because of severe leukopenia in only a few cases. CONCLUSIONS: The regimen is active and safe: the slight survival increase is likely due to the small amenability to surgery achieved (16.2%). However, our results are fully comparable to others obtained with vinorelbine in two/three drug combination chemotherapy regimens. PMID- 8623507 TI - Antioxidant agents and colorectal carcinogenesis: role of beta-carotene, vitamin E and vitamin C. AB - In consideration of findings reported in the literature and of our study, we examined the correlation between antioxidants (beta-carotene, vitamin C, vitamin E) and colorectal carcinogenesis. Although diagnostic progress has been made in the last decades, no significant improvements in death rates have been achieved in the western world. Exogenous factors might be responsible for a complex alteration process of might be responsible for a complex alteration process of normal colonic mucosa into adenoma and carcinoma. Free radicals and reactive oxygen metabolites, due to increased production or to reduced inactivation, following a decrease in the antioxidant burden in the mucosa, might cause damage to DNA, thereby resulting in genetic alterations. This might represent the cause of the transformation process: normal mucosa --> adenoma --> carcinoma. In a prospective study, we observed a reduction of beta-carotene levels in normal colonic mucosa in patients with polyps and colorectal cancer. We also showed that beta-carotene supplementation raises levels of this micronutrient in the colonic mucosa of these patients. Findings from the literature and our trials show a significant decrease in the antioxidant capacity of colorectal mucosa in patients affected by colorectal cancer, although there is a significant interindividual variability. Such results suggest a possible chemopreventive role of antioxidant agents in colorectal cancer. PMID- 8623508 TI - Phase II trial of oral tegafur and folinic acid with mitoxantrone as first-line regimen in patients with metastatic breast cancer. AB - BACKGROUND: Tegafur acts as a deport form of 5-fluorouracil when administered orally for long periods of time since it is an active drug in metastatic breast cancer, with response rates of 29-44%. Biochemical modulation with folinic acid and the addition of mitoxantrone could increase the efficacy of tegafur in patients with metastatic breast cancer. METHODS: A prospective phase II trial in patients with previously untreated metastatic breast cancer was carried out. The scheme consisted of mitoxantrone, 12 mg/m2 intravenous day 1, oral tegafur, 750 mg/m2/day divided in three equal doses, and leucovorin 15 mg/8 h orally for days 1-21, given in a 4-week schedule. None patient had received chemotherapy for metastatic breast cancer, although 16 patients had received previous adjuvant chemotherapy. RESULTS: Thirty-four patients were included. Objective responses were achieved in 20 of 32 patients assessable for response, with 1 complete response and 19 partial responses. The objective response rate was 62.5% (95% confidence intervals, 48-76%). The median duration of response was 10 months. Grade III-IV toxicity according to WHO criteria was digestive (nausea/vomiting) in 12.5%, diarrhea in 25% and stomatitis in 25% of patients. Other toxicities were low. Eight patients required dose-reduction. CONCLUSIONS: We achieved a significant response rate with the scheme, which was administered on an outpatient basis. It seems to be safe and effective as first-line treatment in metastatic breast cancer, with a short median response duration. The size of the trial does not permit definitive conclusions, and the role of biochemical modulation of tegafur in combination with mitoxantrone remains to be defined. PMID- 8623509 TI - Effects of one-year adjuvant treatment with tamoxifen on bone mineral density in postmenopausal breast cancer women. AB - In this study, the authors have analyzed the possible effects of one-year adjuvant treatment with tamoxifen on bone mineral density in postmenopausal breast cancer women. Bone mineral content was studied by photon absorptiometry (I 125), whereas bone balance was analyzed indirectly by serum PTH, osteocalcin, calcitonin, calcium and alkaline phosphatase levels. Bone mineral content and serum bone-related substances were measured before starting treatment and after one year. Results were analyzed using Student's t test for paired data. No difference was found between the two measurements for bone mineral content, PTH, calcitonin, calcium and alkaline phosphatase levels. Measurements at entry and after one year of treatment showed a statistically significant difference (P<0.001) only for osteocalcin. In accordance with other authors, we can conclude that treatment with tamoxifen does not cause an increase in menopausal bone resorption. The finding that osteocalcin levels decreased after one year of therapy with tamoxifen is interesting, but further studies are necessary to clarify the role of such levels in predicting a turnover of bone balance towards osteoblastic activity. PMID- 8623510 TI - A phase II study of mitomycin C, vindesine and cisplatin combined with alpha interferon in advanced non-small cell lung cancer. AB - AIMS AND BACKGROUND: MVP chemotherapy (mitomycin C, vindesine or vinblastine, cisplatin) is one of the most commonly used regimens for advanced non-small cell lung cancer (NSLCLC). Experimental data suggest a synergistic cytotoxic activity of alpha-interferon (alpha-IFN) when combined with cisplatin, mitomycin C and vinca alkaloids. In an effort to improve MVP chemotherapy activity, we have combined this regimen with alpha-IFN. PATIENTS AND METHODS: Thirty-five patients with advanced NSCLC (19 stage IV) were treated with the MVP regimen (mitomycin C, 8 mg/m2; vindesine, 3 mg/m2, cisplatin, 75 mg/m2, all on day 1) plus alpha-2a IFN, 3x10(6) U from day 1 to 7. The cycles were repeated every 28 days. RESULTS: There were no complete responses and 18 partial responses, for an overall response rate of 51%. Median time to treatment failure was 6 months (range, 1 18), and the median survival was 9.5 months (range, 1-32). WHO grade 3 toxicity was recorded in up to 8% of patients, flu-like syndrome was a common complaint; one toxic death occurred. CONCLUSIONS: The combination yielded a level of response comparable to that of other cisplatin-based regimens. Larger randomized trials are needed to assess the role of alpha-IFN combined with chemotherapy in advanced NSCLC. PMID- 8623511 TI - Biochemical characteristics and recent biological knowledge on prostate-specific antigen. AB - Since its identification in seminal fluid in 1971, much new information has been obtained about the biology and expression of prostate-specific antigen (PSA). PSA is a glycoprotein composed of 93% amino acids and 7% carbohydrates, with a molecular weight of about 30,000 Da. Functionally and structurally PSA is a kallikrein-like serine protease, and its physiologic role is degradation of the major proteins of seminal coagulum (semenogelin I and II, fibronectin), which leads to semen liquefaction. The PSA gene is located on the 13q region of chromosome 19, and it has a high degree of homology (more than 80%) with genes of the human glandular kallikrein (hKGK1). PSA production and expression are preferentially but not exclusively associated to the normal, benign hyperplastic and cancerous tissues of the prostate. In fact, it has been demonstrated that PSA is also present in accessory male sex glands and breast cancer. It was recently reported that PSA was also present in milk of lactating women. Many factors may influence PSA synthesis and production, and among them the most important are androgen, retinoic acid and growth factor stimulation. Significant advances have been recently made as regards the molecular isoforms of PSA. In the seminal fluid PSA seems partially bound to a serpine (protein C inhibitor), whereas in serum it is predominantly associated to alpha-1-antichymotrypsin and in a small quantity to alpha-2-macroglobulin. These new findings will have implications for the clinical application of PSA as a tumor marker for prostate cancer. PMID- 8623512 TI - Characterization of cancer-related disseminated intravascular coagulation in relation to tumor necrosis factor-alpha blood concentrations: possible therapeutic role of pentoxifylline. AB - AIMS AND BACKGROUND: Preliminary experimental data suggest the involvement of tumor necrosis factor (TNF) in determining endothelial damage related to disseminated intravascular coagulation (DIC). The present study was performed to investigate TNF secretion in DIC occurring in metastatic solid tumor patients and to evaluate the possible therapeutic role of pentoxifylline, which has been proven to have a TNF-lowering activity. METHODS: The study included 20 metastatic solid tumor patients who showed clinical and laboratory signs of DIC. Pentoxifylline was given orally at a dose of 1200 mg/day for 28 days. RESULTS: Abnormally high levels of TNF were found in 13/20 patients, and mean TNF serum levels observed in patients were significantly higher than those seen in a control group of 50 healthy subjects. Fibrinogen plasma concentrations were low in 11 cases. Patients with low fibrinogen values showed significantly higher mean TNF levels than those with normal or elevated concentrations. Pentoxifylline therapy induced a significant decrease in mean TNF concentrations and a significant increase in mean platelet number, which returned to within the normal range in 11/20 patients. An increase in platelets in response to pentoxifylline was more evident in patients with elevated pretreatment TNF values. CONCLUSIONS: Our results suggest the existence of abnormally high blood levels of TNF in cancer-related DIC, mainly in the presence of low fibrinogen values. Moreover, they indicate that pentoxifylline therapy may determine a decrease in TNF levels in DIC patients, an event associated with an increase in platelet number. PMID- 8623513 TI - Second surgical treatment of retroperitoneal persistent disease in epithelial ovarian cancer. AB - AIMS AND BACKGROUND: This report retrospectively analyzes 9 cases of epithelial ovarian cancer with persistent retroperitoneal metastasis after intraperitoneal surgery (without systematic lymphadenectomy) and chemotherapy. METHODS: All 9 patients were diagnosed as FIGO stage I to IV at the time of primary surgery. They received combined postoperative chemotherapy (8 cases with a cisplatin-based regimen and 1 with adriamycin and endoxan). They were submitted to pelvic and paraaortic lymphadenectomy at the National Cancer Institute of Milan during the period 1990-1994. RESULTS: All patients presented no evidence of disease in the abdominal cavity but retroperitoneal metastasis, which was the unique metastatic site. Chemotherapy was administered as adjuvant therapy after lymphadenectomy. Six patients were free of disease for 14 to 61 months. One patient with vaginal recurrence at the 18th month was treated with radiotherapy and chemotherapy, but died of widespread disease 25 months after lymphadenectomy. Two patients with massive positive lymph nodes dies of brain and lung metastasis 20 and 6 months later, respectively. CONCLUSIONS: We conclude that retroperitoneal metastasis may be the only site of persistent disease and that systematic lymphadenectomy technically feasible in this situation to increase the opportunity for local disease control and to obtain a good result. PMID- 8623514 TI - Oncostress: evaluation of burnout in Lombardy. AB - AIMS: This study was carried out to evaluate how working with oncologic patients (from various points of view) can cause personal discomfort for the operator. METHODS: Questionnaires (n=406) consisting of many requisites concerning personal and professional factors, organizational and work aspects were evaluated. They were given to oncologic doctors, radiotherapists, various specialists, nurses and radiotherapy technicians in Lombardy. RESULTS: The study revealed a state pf anxiety in 52.7% of subjects and depression in 16%, mainly in the professional nursing categories and in particular in women and the young. The most singular aspects observed were not related to work organization. CONCLUSIONS: Working in an oncologic environment is, in different ways the cause of "oncostress" and distinctly induces burnout. However, it appears to be less significant in Italy than indicated in the literature. PMID- 8623515 TI - Malignant rhabdoid tumor of the vulva: a case report and review of the literature. AB - Malignant rhabdoid tumor (MRT) is an uncommon aggressive neoplasm which usually occurs in the kidney of children, but it has also been found in extrarenal sites. MRT arising in the vulva is extremely rare. Only four cases of MRT of the vulva have been reported in the English literature. We herein present another case. The diagnosis and management of vulva MRT are reviewed. PMID- 8623517 TI - Follow-up of colorectal cancer. PMID- 8623516 TI - Pulmonary lymphangioleiomyomatosis: a case report in postmenopausal woman treated with pleurodesis and progesterone (medroxyprogesterone acetate). AB - The main problem in the treatment of pulmonary lymphangioleiomyomatosis, which frequently occurs in the reproductive age, is the control of chylothorax and disease progression. We herein report a case of a 62-year-old woman who underwent surgery for recurrent chylothorax. Histologic examination of lung and lymph node biopsies demonstrated lymphangioleiomyomatosis. Thirty-six months after tetracycline pleurodesis and high-dose medroxyprogesterone acetate therapy, the disease was stable and cyclothorax effectively controlled. It would therefore appear that hormonal treatment with medroxyprogesterone acetate may be beneficial in postmenopausal women. PMID- 8623518 TI - Chemical cleavage of 5'-linked protein from tobacco ringspot virus genomic RNAs and characterization of the protein-RNA linkage. AB - Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5' linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5' nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting to in vivo labeling with 32P, in vitro radioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5' region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3'-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5'-oligo) by its sensitivity to proteinase. In a presumed beta-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5'-oligo. Phosphatase-treated 5'-oligo accepted 5'-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5' terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5' uridylate of each genomic RNA. PMID- 8623519 TI - Defining the SOS operon of coliphage 186. AB - We have sequenced the LexA-controlled operon of coliphage 186 that carries the tum gene, whose product is necessary for UV induction of the 186 prophage. The operon consists of orf95 and orf97, and we have identified orf95 as the tum gene. The major translation products from orf95 result from internal initiations and modulate Tum activity. Tum is the product of the full-length Orf95 protein. The second gene of the operon, orf97, is of unknown function but, while it has little effect on prophage induction, its presence in the cell totally blocks infection of that cell by 186. PMID- 8623520 TI - The Escherichia coli retrons Ec67 and Ec86 replace DNA between the cos site and a transcription terminator of a 186-related prophage. AB - Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there are a number of indications that retrons are mobile elements, their transposition has not been observed. The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition. We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186. Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced approximately 180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene. These features--DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites--are shared with other retrons and suggest ways in which retron transposition might have occurred. PMID- 8623521 TI - Assembly of the Sindbis virus spike protein complex. AB - The Sindbis virus glycoproteins E1 and E2 are organized into 80 trimers of heterodimers within the virus envelope. Using pulse-chase protocols and chemical crosslinkers, we have found that E1 and E2 precursor, PE2, rapidly assemble into heterodimers and then into trimers of heterodimers after translocation into the endoplasmic reticulum. E1 folds into its mature conformation within the endoplasmic reticulum via at least three intermediates differing in the configurations of their disulfide bonds. PE2 can pair with the second of these E1 folding intermediates. The remaining E1 folding steps, therefore, occur after E1 PE2 multimers begin to form. Quaternary interactions between E1 and PE2 may help guide the folding of E1. While no PE2 folding intermediates have yet been detected, we have found that PE2 transiently enters into large, noncovalent complexes or aggregates with other PE2 molecules and/or with unknown host factors prior to pairing with E1. PMID- 8623522 TI - Analysis of the contribution of CTL epitopes in the immunobiology of morbillivirus infection. AB - In Balb/c (H-2d) mice, the nucleoprotein (NP) of measles virus (MV) induces a MHC class I restricted-CTL response to a single 9-amino-acid epitope (aa 281--289). This L(d)-restricted epitope is also present in the NP of the closely related canine distemper virus (CDV). To investigate whether this epitope is immunologically effective when it is present within the primary sequence of a nonviral protein, we have incorporated the 281--289 motif into the human CD36 protein. When cells are infected with vaccinia virus (VV) recombinants expressing this protein, CD36NP, the MV epitope is correctly processed and the cells are lysed by MVNP-specific CTLs. In vivo, VV-CD36NP induced CTLs which protected mice from a lethal dose of CDV, but did not block virus replication. The MVNP contains four other potential L(d)-restricted motifs. To investigate if these could be utilized in the absence of the dominant epitope, a mutant NP was produced in which one of the anchor residues in the aa 281--289 motif was mutated. Cells infected with a VV recombinant expressing this protein (VV-NP F289S) were only poorly lysed by MVNP-specific CTLs. Similarly, immunization of Balb/c mice with VV-NP F289S induced a lower level of CTL activity compared to the VV-NP, but the activity was now directed to three other epitopes. When mice were vaccinated with VV-NP F289S they were only partially protected from a lethal CDV challenge. The significance of these results for MV vaccine development is discussed. PMID- 8623524 TI - Analysis of cis-acting elements required for replication of barley stripe mosaic virus RNAs. AB - The replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNA alpha and -gamma transcripts. The 5' and 3' noncoding regions were required for replication, and lack of a 5' cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNA alpha abrogated replication in trans. A 2-base change that produced a truncated alpha a protein lacking the first 16 amino acids also compromised the ability of RNA alpha to be replicated. In contrast, RNA beta transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs alpha and gamma, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between beta a and beta b was not absolutely required for replication, but small deletions within this region reduced the abundance of RNA beta by at least 10-fold. Deletions within the first 507 nt of the gamma a ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the gamma a ORF, the gamma a--gamma b intergenic region, and the gamma b ORF could be amplified in trans. Two mutants containing extensive deletions encompassing the central region of the gamma a ORF and most of gamma b behaved like defective interfering RNAs because they multiplied to high levels in trans and caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs alpha and gamma. PMID- 8623523 TI - Poliovirus replicons that express the gag or the envelope surface protein of simian immunodeficiency virus SIV(smm) PBj14. AB - Poliovirus genomes encoding the complete gag or env surface gene of the simian immunodeficiency virus SIV(smm) PBj14 (SIV-PBj14) were constructed. The in vitro transcribed RNA from these genomes, referred to as replicons, have the capacity for self-replication when transfected into tissue culture cells. Serial passage of the replicons containing the SIV-PBj14 gag or SIV-PBj14 env (SU) genes with a recombinant vaccinia virus, VV-P1, which provides P1 in trans, resulted in the encapsidation of these replicons. Infection of cells with the encapsidated replicons that encode gag, referred to as vIC-SIV-PBj14 Gag, resulted in the production of a 55-kDa protein that was released from the infected cells. Using a sucrose density-gradient analysis, the protein was found to sediment at a density consistent with that of a virus-like particle. Infection of cells with a replicon that encodes the env SU gene, referred to as vIC-SIV-PBj14 SU, resulted in the production of two SIV-PBj14 envelope-related intracellular proteins. One of these proteins had a molecular mass consistent with that of the unglycosylated SIV PBj14 SU protein (63 kDa); the second protein had a higher molecular mass (>160 kDa). Characterization of this larger protein revealed that it was glycosylated and possibly represented a dimer of the SU protein. A pulse-chase analysis of cells infected with vIC-SIV-PBj14 SU demonstrated that a 110- to 130-kDa protein was released, which is consistent with the molecular mass of the SIV-PBj14 SU protein. The results of these studies demonstrate that poliovirus replicons can be used to express foreign proteins, including glycoproteins, which retain many of the physical features of the native protein. PMID- 8623525 TI - A novel ribozyme target site located in the HIV-1 nef open reading frame. AB - We have tested the sequence UUC CAG UCA GAC CU, at position 9016--9029 within the HIV-1(SF2) nef open reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate modified 14-nt DNA oligo (complementary to the nef9016--9029 site). A dose dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for the nef9016 -9029 site (hhRz.nef9016--9029) was constructed and transfected into the human T cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016--9029 were infected with HIV-1(SF2). A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (10(3)-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviral pol and gag sequences in hhRz.nef9016--9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support the nef9016--9029 site as a strong new candidate for ribozymal gene therapy against HIV-1 infection. PMID- 8623526 TI - Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes. AB - In order to examine genetic relatedness among viruses that infect microalgae, DNA polymerase gene (DNA pol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (Chlorella strains NC64A and Pbi, Micromonas pusilla and Chrysochromulina spp.). Phylogenetic trees based on DNA pol sequences and hybridization of total genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequence data showed good concordance (r=0.90), indicating that DNA pol sequences can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses. The phylogenetic tree inferred from the deduced amino acid sequences of DNA pol from 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses, and African swine fever virus corresponded well with groupings based on the International Committee on Taxonomy of Viruses. Microalgal viruses are more closely related to each other than to the other dsDNA viruses and form a distinct phyletic group, suggesting that they share a common ancestor and belong to the Phycodnaviridae. Moreover, the Phycodnaviridae are more closely related to the Herpesviridae than to other virus families for which DNA pol sequences are available. PMID- 8623527 TI - The low pH-dependent entry of avian reovirus is accompanied by two specific cleavages of the major outer capsid protein mu 2C. AB - Avian reoviruses are capable of inducing rapid and extensive syncytium formation, a process that occurs preferentially under conditions of neutral or alkaline pH. In order to ascertain whether the membrane fusion-inducing capability of avian reovirus confers a pH-independent entry mechanism on the virus, virus entry was investigated using internalization assays and several lysomotropic agents that inhibit endosomal acidification. The ability of avian reovirus to infect cells was severely restricted under all conditions that prevented endosomal acidification. The decreased infection efficiency in the presence of the lysomotropic agents correlated with an inhibition in the proteolytic processing of the major outer capsid protein mu 2C. The importance, with respect to virus infection, of the low pH-dependent cleavage of the avian reovirus mu 2C protein was confirmed by demonstrating that infectious subviral particles, generated by proteolytic processing in vitro, were capable of efficiently infecting cells in the presence of the lysomotropic agents. These results indicated that avian reovirus entry-specific membrane interactions are largely dependent on an endosome-mediated proteolytic processing of the virus particle, suggesting that the syncytium-inducing properly of the sigma 3 protein is not sufficient to promote virus uptake. Furthermore, avian reovirus internalization was associated with two distinct cleavages of the major outer capsid protein mu 2C, unlike the entry-specific processing of the analagous mammalian reovirus major outer capsid protein mu 1C. The mu 2C cleavages occured sequentially and appeared to involve distinct cleavage specificities. Moreover, the second cleavage event was observed to be both virus strain- and cell type-independent, suggesting that the cleavage is both specific and biologically significant. PMID- 8623528 TI - Identification of the receptor-binding regions of pb5 proteins of bacteriophages T5 and BF23. AB - The receptor-binding protein pb5(T5) of bacteriophage T5, when expressed from the oad gene cloned in pVK88 under the control of the phage T7 promoter/polymerase system, has been shown to bind to its FhuA receptor on the surface of E. coli, where it blocks FhuA for subsequent adsorption of T5 (Mondigler et al., FEMS Microbiol. Lett., 130, 293-300, 1995). In the present study the blocking assay has been applied to analyze the effects of several mutations within oad on the FhuA-binding properties of corresponding pb5 derivatives. Three classes of mutations were tested: (i) oad deletion derivatives, (ii) the oad mutation known to interfere with FhuA-binding of T5 (Heller and Bryniok, J. Virol., 49, 20-25, 1984), and (iii) linker-insertion mutations at a site very close to the oad mutation. Of the corresponding pb5 derivatives only one, a deletion derivative lacking the 153 C-terminal amino acids, was as active in the blocking assay as wild-type pb5(T5). All other derivatives were inactive or almost inactive. Isolation and molecular characterization of phenotypic revertants of T5oad showed that all revertants were true genotypic revertants of the oad mutation. The oad mutation has been identified as a G to T exchange resulting in a substitution of Gly for Trp at position 166 of pb5(T5). DNA sequencing of the hrs gene of bacteriophage BF23 and comparing the deduced amino acid sequence of pb5(BF23) with that of pb5(T5) revealed distinct regions of similarity and nonsimilarity. We propose that the receptor-binding region of pb5(T5) (pb5(BF23)) is formed by the region of nonsimilarity extending from amino acid position 89 (88) to position 305 (283). PMID- 8623529 TI - Preliminary crystallographic studies of bacteriophage T4 fibritin confirm a trimeric coiled-coil structure. AB - Fibritin, a 52-kDa product of gene wac of bacteriophage T4, forms fibrous "whiskers" that connect to the phage tail and facilitate the later stages of phage assembly. Preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel alpha-helical coiled-coil structure. To investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted M and E, that consist of its last 75 and 120 amino acids, respectively. Both proteins contain part of the coiled coil region and the 29 amino acid carboxy-terminal domain essential for the trimerization of fibritin. The proteins are expressed as a soluble product in an Escherichia coli system. We have obtained crystals of fibritins M and E. Complete native X-ray diffraction data sets have been collected to 1.85 and 2.7 A resolution, respectively. The crystals have space group P3 with a=44.3 A, c=91.3 A (fibritin M) and R32 with a=41.2 A, b=358.7 A (fibritin E) in the hexagonal setting. Symmetry and packing considerations show that fibritin is a triple coiled coil. PMID- 8623530 TI - Protection from pathogenic SIVmac challenge following short-term infection with a nef-deficient attenuated virus. AB - Infection of rhesus macaques with attenuated SIVmac is, at present, the only strategy which confers significant protection from challenge with wild-type SIVmac grown in monkey PBMC. However, initial results suggest that the protective mechanism does not develop until late after "vaccination" (approx 10 months). As part of a European study using the C8 variant of SIVmac251-32H (containing an in frame 12-bp deletion in the nef gene), we wished to determine (a) if protection could be achieved against challenge with a "swarm" of SIVmac251-32H produced in monkey cells and (b) if protection could be demonstrated after a short period of infection with the attenuated virus. Eight Indian rhesus macaques were infected with C8 and four were challenged after 10 weeks with 50 MID50 of an uncloned stock of SIVmac251-32H grown in rhesus cells, and the other four were challenged after 20 weeks. Four animals served as naive controls. Three of the four monkeys challenged at 10 weeks and three of those challenged at 20 weeks were protected from productive superinfection. From one monkey in each group it was, however, possible to demonstrate the presence of the wild-type provirus in monkey PBMC by diagnostic PCR and anamnestic immune response. There was no apparent correlation between the levels of binding or neutralizing antibodies on the day of challenge and subsequent protection. Approximately 1 year after infection with the attenuated virus all monkeys were rechallenged with the heterologous SIVsm strain, first with 10-20 MID50 and then with 1000 MID50. Although not all of the SIVsm-inoculated naive controls became productively infected, PCR analysis failed to reveal any evidence for infection of the "immunized" monkeys. PMID- 8623531 TI - Perturbation of the host cell cycle and DNA replication by the bovine papillomavirus replication protein E1. AB - A stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence. PMID- 8623532 TI - A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier. AB - In this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant. PMID- 8623533 TI - Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase. AB - The Nef protein of primate immunodeficiency viruses is essential for establishing a highly productive pathogenic infection in vivo. In tissue culture, Nef is not required for infection but enhances viral infectivity. This effect is most pronounced in unstimulated primary lymphocytes and occurs in the early phase of infection prior to viral gene expression. Since Nef expression does not lead to obvious changes in virus composition, it was of interest to analyze whether Nef is incorporated into virus particles. Here, we show that Nef is specifically immunoprecipitated from radioactively labeled human immunodeficiency virus type 1 (HIV-1)-infected cells and virus particle preparations. Quantitative analysis revealed Nef to be incorporated on the order of 10% of reverse transcriptase incorporation, which corresponds to 5 to 10 molecules of Nef per virion. In infected cells, Nef was detected as a full-length 27-kDa protein. In contrast, approximately 50% of particle-associated Nef corresponded to an 18-kDa species which comigrated with the larger product after in vitro cleavage of purified HIV 1 Nef by the viral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Nef is cleaved concomitantly with viral structural proteins on maturation of virus particles. This cleavage is likely to be functionally significant because it dissociates the conserved core domain from the N-terminal membrane attachment region. Our results suggest that the profound influence of Nef on establishing infection of unstimulated cells in tissue culture and in vivo is mediated by virion-associated Nef which functions in early infection before viral gene expression. PMID- 8623535 TI - Domains of the E1 protein of human papillomavirus type 33 involved in binding to the E2 protein. AB - Papillomavirus E1 and E2 proteins are essential for the initiation of viral DNA replication. We have now analyzed the interaction of E1 and E2 of human papillomavirus type 33, which is associated with cervical carcinoma. When synthesized in insect cells using the baculovirus expression system, the E1 and E2 proteins interacted efficiently at 4 degree. A monoclonal antibody recognizing E1 amino acids 584--600 inhibited the binding of E2 and vice versa, indicating that these amino acids are involved in E2 binding. To confirm this result, a mutational analysis of E1 was performed. The E2 binding activity of E1 deletion and point mutant proteins was assayed using glutathione S-transferase E1 fusion proteins and in vitro translated proteins. At 4 degree, the C-terminal portion of E1 including amino acids 312--644 was sufficient for E2 binding. Introduction of C-terminal deletions or a point mutation at position 586 (Pro --> Glu) resulted in the loss of the E2 binding activity. A second more N-terminally located binding domain (E1 amino acids 312--450) became active when the assays were performed at 22 degrees. The monoclonal antibody still inhibited E2 binding at this temperature, indicating that both E2 binding domains are engaged in the context of the full-length protein. PMID- 8623534 TI - p53 inhibits JC virus DNA replication in vivo and interacts with JC virus large T antigen. AB - The onset of DNA replication is an important step within the life cycle of the human neurotropic polyomavirus JC. In this report, evidence that both the human and the murine tumor suppressor protein p53 strongly inhibit JCV DNA replication in vivo is presented. This inhibition is dose-dependent and not a secondary effect of a decreased expression of JCV large T-antigen in response to p53. Using deletion mutants of murine p53 and tumor-derived point mutations of human p53, the basis of the suppression of JCV DNA replication by p53 was dissected. Deletion of either the amino- or the carboxy-terminal domain of murine p53 did not interfere with the repression of JCV DNA replication. However, deletion of the highly conserved central region of p53 abolished the inhibitory effect on replication. The tumor-derived human mutant p53(His273) inhibited JCV DNA replication significantly, whereas another tumorigenic mutant, p53(His175), had no inhibitory effect Concomitantly, a direct protein-protein interaction between p53 and JCV large T-antigen was lost in mutants which did not affect JCV DNA replication. These results strongly suggest that p53 inhibits JCV DNA replication by interacting with JCV large T-antigen. PMID- 8623536 TI - Genetic control of infection of primary macrophages with T-cell-tropic strains of HIV-1. AB - Human immunodeficiency virus type 1 (HIV-1) NDK, a Zairian subtype D virus highly cytopathic for CD4-positive lymphocytes, and the prototype subtype B virus HIV-1 LAV are about 10(4) and 10(5) times more infectious, respectively, for T lymphocytes than for blood-derived macrophages (BDM). Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control and the step of the virus/cell cycle responsible for infection of BDM with T-cell-tropic viruses. We found that recombinants bearing the envelope glycoprotein of HIV-1 NDK are able to enter more efficiently into BDM than recombinants with HIV-1 LAV envelope glycoprotein. We also found that a genetic region outside of the env gene is responsible for production of HIV-1 NDK infectious progeny from BDM. This region consists of the vif gene and the C- and N-terminal portions of pol and vpr genes, respectively. Our results suggest that productive infection of primary macrophages with T-cell-tropic strains of HIV-1 is determined by two different genetic mechanisms: one effective at the virus/cell entry, controlled by the env gene, and the second after entry, controlled by genes vif and vpr. In comparison with HIV-1 LAV, HIV-1 NDK has been able to more easily overcome both restriction mechanisms. PMID- 8623538 TI - The complete nucleotide sequence of barley mild mosaic virus RNA1 and its relationship with other members of the Potyviridae. AB - The complete nucleotide sequence of RNA1 of a French barley mild mosaic bymovirus isolate has been determined. The molecule is composed of 7261 nucleotides and contains a single large open reading frame encoding a protein of 2258 amino acids with a calculated Mr of 256,375. Amino acid sequence comparison with poty- and rymoviruses reveals eight domains corresponding to the potyviral P3, 6K1, Cl, 6K2, Nla-VPg, Nla-Pro, Nlb and capsid proteins, respectively. Seven putative cleavage sites, similar to those mediated by potyvirus Nla proteinases, were identified. The presence of two, so far undescribed putative cleavage sites (6K2/Nla-VPg and Nla-VPg/Nla-Pro) in the polyproteins encoded by RNA1 of barley yellow mosaic virus and wheat spindle streak mosaic virus, was predicted. These data indicate that the bymovirus RNA1 fits the consensus potyviral genetic map downstream of the helper component gene. PMID- 8623537 TI - Heat-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing HIV-1 envelope glycoproteins. AB - It has been shown that human CD4 expressed in nonhuman cells does not support HIV 1 entry into those cells and that components from human cells in addition to CD4 are required to overcome the block. We have used human red blood cells (huRBC) as a source for the accessory components since their membrane composition is less complex than that of nucleated cells and they are well characterized. Components were transferred by fusion of huRBC to nonhuman CD4(+) cells mediated by influenza hemagglutinin or polyethylene glycol. The RBC-modified nonhuman CD4(+) cells were labeled with fluorescent markers and incubated with gp 120-gp41 expressing cells labeled with a different fluorescent probe. Fusion between RBC- modified nonhuman CD4(+) cells and gp 120--gp41-expressing cells was quantified by fluorescence video microscopy. Human erythrocyte components transferred to nonhuman CD4+ cells conferred HIV-1 envelope glycoprotein-mediated fusion susceptibility to those cells. The fusion was enhanced by pretreatment of the erythrocytes for 10 min at 56 degrees. No gp 120--gp41-mediated fusion was observed when components from nonhuman RBC were transferred to nonhuman CD4+ cells. Human cell lines with pre-RBC characteristics (K562-CD4) also supported HIV-1 envelope glycoprotein-mediated fusion. PMID- 8623539 TI - Expression of the 69K movement protein of turnip yellow mosaic virus in insect cells. AB - The nonstructural 69-kilodalton (K) protein of turnip yellow mosaic virus is necessary for systemic spread of the virus within the plant. To examine the behavior of the 69K protein in vivo, antibodies were raised against the carboxy terminal region of this protein. The full-length 69K protein was also expressed in insect cells using a recombinant baculovirus. Studies on the posttranslational modifications of the 69K protein in insect cells revealed that the protein is phosphorylated but not glycosylated. Further experiments of subcellular fractionation and indirect immunolocalization in insect cells showed that the 69K protein is localized in the cytoplasm and/or in the plasma membrane. PMID- 8623540 TI - Deletion of glycoprotein gE reduces the propagation of pseudorabies virus in the nervous system of mice after intranasal inoculation. AB - A pseudorabies virus (PrV) mutant, deficient in the nonessential glycoprotein E (gE) and expressing the LacZ gene (gE- beta gal+ PrV), and its rescued virus were inoculated intranasally in mice. The median lethal dose of gE- beta gal+ PrV was similar to that of the parental Kaplan strain, but mice survived longer and did not develop symptoms of pseudorabies. In the nasal mucosa, gE- beta gal+ PrV replicated less efficiently than rescued virus. gE- beta gal+ PrV could infect first-order trigeminal and sympathetic neurons innervating the nasal mucosa. However, transneuronal transfer to second-order cells groups did not occur in trigeminal pathways and was severely reduced in sympathetic pathways. The mutant was also unable to propagate in the parasympathetic system. In contrast, gE rescued virus was transferred transneuronally in trigeminal, sympathetic, and parasympathetic pathways, like wild-type PrV. These findings provide further evidence that deletion of gE specifically affects transneuronal transfer of PrV more than penetration and multiplication of the virus in first-order neurons. PMID- 8623542 TI - Extensive regions of pol are required for efficient human immunodeficiency virus polyprotein processing and particle maturation. AB - Human immunodeficiency type 1 particle maturation is dependent upon proteolytic cleavage of the gag and gag-pol precursors by the pol-encoded viral protease. We have investigated the importance of domains of pol other than the protease for particle maturation and gag proteolytic processing. Truncations of the gag-pol polyprotein precursor of HIV-1 were created by deleting segments of the reverse transcriptase coding region or by introducing stop codons in the integrase region of an HIV-1 infectious molecular clone. In these mutants, the protease coding sequence was left intact. Particles produced by all of the mutants displayed abnormal morphologies and impaired proteolytic processing of gag. The severity of particle morphology abnormalities and of gag polyprotein processing impairment appeared to be affected both by the size and by the position of the deletions in pol, suggesting that the integrity of several pol domains within the gag-pol precursor is required for optimal protease activation and particle maturation. Additionally, cotransfection of a deletion mutant with wild-type provirus led to a marked reduction in the titer of infectious virus, suggesting that truncated gag-pol precursors can interfere with wild-type virus assembly and maturation. PMID- 8623541 TI - The phylogeny of New World (Tacaribe complex) arenaviruses. AB - Several New World (Tacaribe complex) arenaviruses (Arenaviridae) are known to cause severe hemorrhagic disease in humans. Phylogenetic reconstruction of the Tacaribe complex arenaviruses previously has been limited by the relative scarcity of sequence data for arenavirus genomes. In the present study, oligonucleotide primers were designed based on conserved regions of the nucleocapsid (N) protein gene and then used to amplify, by reverse transcription- polymerase chain reaction, a 613-to 649-nucleotide region of the N gene of all known Tacaribe complex arenaviruses. This has allowed completion of the first detailed genetic characterization and phylogenetic analysis of all known members of the Tacaribe complex. These viruses formed three lineages. Lineage A contained Flexal, Parana, Pichinde, and Tamiami viruses; lineage B contained Amapari, Guanarito (GUA), Junin (JUN), Machupo (MAC), Sabia (SAB), and Tacaribe viruses. Latino and Oliveros viruses occupied lineage C. The highly pathogenic Tacaribe complex arenaviruses (GUA, JUN, MAC, SAB) were all members of lineage B, suggesting the possibility that the highly pathogenic phenotype is the result of evolutionary radiation from a common ancestor. The approach described here provides a rapid method for characterization of novel Tacaribe complex arenaviruses and may provide clues as to their potential public health importance. PMID- 8623543 TI - Reduction of human immunodeficiency virus production and cytopathic effects by inhibitors of the Na+/K+/2Cl- cotransporter. AB - Infection of CD4+ T-lymphoblastoid cells by cytopathic strains of HIV-1 results in an increase in cell volume that leads to lysis and cell death. The increase in volume is attributable in part to an HIV-induced increase in intracellular monovalent ion concentrations mediated by the plasma membrane-associated Na+/K+/2 Cl- cotransporter. Loop diuretics, which inhibit cotransporter activity, blocked HIV-induced HIV production and cytopathic effects at physiologically employed concentrations. PMID- 8623545 TI - A brown algal virus genome contains a "RING" zinc finger motif. AB - The brown filamentous alga Feldmannia sp. contains a large icosahedral dsDNA virus, FsV, of which there are multiple variants. A 4.5-kb SstI-HindIII fragment (SH4.5) that is conserved among all genome variants was sequenced. Three open reading frames (ORF-1, -2, and -3, containing 555, 2022, and 411 bp, respectively) were shown to be transcriptionally active by ribonuclease protection assay. A "RING" zinc finger motif and a nucleotide binding site motif were identified in ORF-2. PMID- 8623544 TI - HSV-1-mediated modulation of cytokine gene expression in a permissive cell line: selective upregulation of IL-6 gene expression. AB - Pathological effects of herpes simplex virus (HSV) can result due to a combination of direct viropathic effects and immunological reactions to viral antigens. The immunological reactions are orchestrated by a variety of cytokines and chemokines released by the host cells. Therefore, the cytokine gene expression in response to HSV-1 infection in a permissive murine cell line was investigated. The data demonstrate that HSV-1 induced a selective activation of IL-6 gene expression at the mRNA and protein levels, in the permissive cell line. The cell line used was capable of expressing IL-1, IL-7, and IL-10 in addition to IL-6, upon lipopolysaccharide stimulation. UV or heat-inactivated viruses were unable to upregulate IL-6 expression. However, mutant HSV-1 strains lacking fully functional ICP0, ICP4, ICP8, or ICP27 genes, thereby rendering them replication incompetent or impaired in in vitro cell growth (ICP0), enhanced IL-6 expression selectively. Considering the role of IL-6 in inflammation, immune response, and its known association with increased levels of MyD116 and GADD 34 mRNAs (genes involved in the prevention of apoptotic death of cells), the present data may have relevance to HSV-1-mediated diseases as well as to the prevalence of HSV-1 in the host. PMID- 8623546 TI - Nitric oxide and viral infection: NO antiviral activity against a flavivirus in vitro, and evidence for contribution to pathogenesis in experimental infection in vivo. AB - Upon stimulation murine macrophages produce high levels of nitric oxide (NO), a potent microbicidal and tumoricidal agent recently also implicated as a mediator of antiviral defense. As dysregulated production of NO may lead to extensive tissue damage, the production of this powerful mediator is tightly regulated. Viral infection, however, may alter the regulation of certain macrophage functions, and recent work from our group demonstrated that viral infection--via induction of interferon-alpha beta synthesis -- may either prime for or down modulate NO production. In light of antiviral activities of NO, down-modulation of NO production in viral infection would seem contradictory to antiviral defense. As others, however, have provided evidence that NO production may contribute to pathogenesis of infection with several neurotropic viruses, the role of NO production was investigated in vitro and in vivo in murine macrophages and in BALB/c mice infected with tick-borne encephalitis virus (TBE-V), a flavivirus. Macrophages from TBE-V-infected mice, but not from control mice, spontaneously produced NO upon culture in vitro. In contrast to the inhibitory effect of NO on replication of several poxviruses and herpes simplex virus, high levels of NO production did not display an inhibitory influence on TBE-V replication in vitro. And finally, in vivo administration of a competitive inhibitor of NO production, aminoguanidine, to TBE-V-infected mice significantly increased their mean survival time. Our results thus demonstrate that the antiviral activity of NO in vitro may be confined to certain viruses, whereas others remain unaffected. Furthermore, we provide evidence that NO production may even contribute to pathogenesis of viral infection in vivo. PMID- 8623548 TI - Complete sequence of Venezuelan equine encephalitis virus subtype IE reveals conserved and hypervariable domains within the C terminus of nsP3. AB - The complete nuleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97--100% identity) and more distantly related to VEE strains of other antigenic varieties (83--93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94--95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1--329), which is highly conserved among all alphaviruses, was 93--94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330--563) was only 59--61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts. PMID- 8623547 TI - Proteolytic activity of human immunodeficiency virus Vpr- and Vpx-protease fusion proteins. AB - In addition to Gag, Pol, and Env, primate lentiviruses encode other virion associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressed in trans with HIV proviruses (Wu et al., J. Virol. 69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIV PR coding region was fused in-frame with the HIV-2 vpx and HIV-1 vpr genes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gag demonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles. Trans-expression of a PR Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gag precursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr arguments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function. PMID- 8623549 TI - Simian virus 40 large-T bypasses the translational block imposed by the phosphorylation of elF-2 alpha. AB - One of the cellular defense mechanisms against virus infection is mediated by activating the interferon-induced, double-stranded-RNA-activated protein kinase, PKR. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor, elF-2, leading to cessation of protein synthesis. Viruses have evolved diverse strategies to counteract this cellular antiviral response. A majority of these strategies target PKR to prevent its activation. Recently, we showed that simian virus 40 (SV40) large-T antigen reverses PKR mediated translational inhibition at a step downstream of PKR activation (Rajan et al., J. Virol. 69, 785--795, 1995). In this paper, we present evidence showing that SV40 can restore efficient translation in cells despite the elevated levels of phosphorylated elF-2 alpha resulting from PKR activation. Thus, SV40 large-T mediated translational rescue occurs at a step downstream of elF-2 alpha phosphorylation. PMID- 8623550 TI - The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins. AB - Tumor-inducing viruses like simian virus 40 or the human adenovirus produce oncoproteins which interfere with the cellular retinoblastoma (Rb) tumor suppressor protein to create an appropriate molecular environment in the nucleus for viral transcription and replication. Such a strategy has been considered to be restricted to animal viruses. Here we demonstrate that plant viruses may use similar mechanisms for recruiting host factors. Wheat dwarf virus (WDV) encodes two potential nonstructural proteins, C1 and C1:C2, both containing the consensus Rb-binding motif LeuXCysXGlu that allows the oncoproteins from animal viruses to inactivate Rb. C1:C2 is a key determinant of viral replication and V(virion) sense expression. Using a yeast two-hybrid protein assay, we demonstrate for the first time that the C1:C2 protein from WDV interacts with a retinoblastoma protein, providing an explanation for the previously observed dependence of viral replication on an intact Rb-binding motif. We also show that C1, for which no function had been demonstrated, is required for V-sense gene expression. This suggests that V-sense expression might be dependent on the interaction of C1 with Rb. Our findings provide further evidence for the presence of transforming-like proteins in a plant virus and will help to explain the production of symptoms in a plant viral infection through a mechanism mediated by a key regulator of cell cycle and differentiation. PMID- 8623551 TI - Resistance to pepper mild mottle tobamovirus conferred by the 54-kDa gene sequence in transgenic plants does not require expression of the wild-type 54-kDa protein. AB - We previously reported that Nicotiana benthamiana plants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenllado et al., 1995, Virology 211, 170--183). Here we show that some of the N. benthamiana plants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype. PMID- 8623553 TI - Role of the variable domain in modulating potato spindle tuber viroid replication. AB - Potato spindle tuber viroid (PSTVd) is believed to undergo a series of specific structural transitions during replication. The variable domain of PSTVd is known to contain sequences that are important for replication/accumulation as well as one of three "premelting regions" which control breakdown of the native structure in vitro. We have examined the structural and biological effects of five single and two double nucleotide substitutions within premelting region 3 in an effort to isolate temperature-sensitive mutations affecting PSTVd replication or pathogenesis. None of these mutants replicated as rapidly as the wild type, and a variety of spontaneous sequence changes were detected in their progeny. Higher temperatures were able to partially overcome the inhibition of replication associated with a more stable secondary structure, but no well-defined temperature-sensitive PSTVd mutants were identified. Selective pressures arising from the interaction of assay temperature and structural stability in vivo appear capable of moving PSTVd populations between peaks of relatively high fitness. Depending on the exact nature and location of the mutation, selection may occur at the level of either the plus or the minus strand. PMID- 8623552 TI - Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization. AB - Virus-like particles were produced in insect cells containing either the L1 and L2 capsid proteins of bovine papillomavirus type 4 (BPV-4) or only the L1 protein. Both preparations of VLPs proved to be extremely effective prophylactic vaccines. Thirteen of 15 calves immunised with either L1-L2 VLPs or L1-VLPs were refractory to experimental challenge with high doses of BPV-4 and did not develop papillomas, while 9 of 10 control animals developed multiple oral papillomas. VLPs were not efficient as therapeutic vaccine in calves with established papillomas, although VLP-vaccinated animals appeared to undergo tumour regression more rapidly than nonvaccinated control animals. Antibody responses in VLP vaccinated calves were associated with prevention of disease but not with regression of papillomas. Thus prophylactic VLP vaccination is effective in preventing disease in this model of mucosal papillomavirus infection. VLPs and native virus share at least some conformational epitopes, as shown by the cross reactivity of their antibodies. PMID- 8623554 TI - Aphid transmission and systemic plant infection determinants of barley yellow dwarf luteovirus-PAV are contained in the coat protein readthrough domain and 17 kDa protein, respectively. AB - Proteins encoded by open reading frames (ORF) 3, 4, and 5 of the barley yellow dwarf luteovirus genome are translated from a single subgenomic RNA. The structural proteins are encoded by ORF 3 (coat protein) and ORF 5 (readthrough domain) and contain undefined domains that regulate the movement of virus through aphid vectors. The biological function of the nonstructural 17-kDa protein encoded by ORF 4 is unknown. A complementation method was employed to test the ability of barley yellow dwarf virions carrying mutations within the readthrough domain and the 17-kDa protein to be transmitted by aphids and to cause systemic infections in plants. We show that the readthrough domain is required for aphid transmission; however, it is not required for virus to be taken up by aphid hindgut cells and released into the hemocoel. The circulative pathway of luteoviruses in aphid vectors requires that virus be actively transported from the hemolymph into the salivary system. Thus, it appears that the readthrough domain is required for transport of virus through membranes of the aphid salivary glands. Furthermore, the readthrough domain was not required for systemic infection of plants, but did influence the accumulation of virus in infected plants. The 17-kDa protein is required for the systemic infection of plants. PMID- 8623555 TI - Identification and characterization of a bovine herpesvirus-1 (BHV-1) glycoprotein gL which is required for proper antigenicity, processing, and transport of BHV-1 glycoprotein gH. AB - DNA sequence analysis of the bovine herpesvirus-1 (BHV-1) genome revealed the presence of an open reading frame named UL1 which exhibited limited homology to glycoprotein gL of herpes simplex virus-1 (S. K. Khattar, S. van Drunen Littel van den Hurk, L. A. Babiuk, and S. K. Tikoo, Virology 213, 28-37). To identify the BHV-1 UL1 protein, rabbit antisera were prepared against two synthetic peptides that were predicted by computer analysis to encompass antigenic epitopes. Sera against both peptides immunoprecipitated a 16- to 17-kDa protein from in vitro translated in vitro transcribed mRNA, BHV-1-infected MDBK cells, and purified virions. Enzymatic deglycosylation and lectin binding assays confirmed that the BHV-1 UL1 protein contains only O-linked oligosaccharides and was named glycoprotein gL. Sera against UL22 protein immunoprecipitated a protein of 108 kDa from BHV-1-infected MDBK cells and purified virions, which was modified only by N-linked oligosaccharides and was named glycoprotein gH. Glycoprotein gL expressed by recombinant vaccinia virus was properly processed and secreted into the medium. In contrast glycoprotein gH expressed by recombinant vaccinia virus was found to be retained in the rough endoplasmic reticulum. However, gH coexpressed with gL by recombinant vaccinia viruses was properly processed and transported to the cell surface, suggesting that complex formation between gH and gL is necessary for the proper processing and transport of gH but not gL. In addition gH--gL complex formation is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane. PMID- 8623556 TI - Catalytic activities of the human T-cell leukemia virus type II integrase. AB - Despite the widespread nature of HTLV-II in New World populations and intravenous drug users, the enzymatic activities of the pol genes have not been reported. To ascertain the activity of the HTLV-II(G12) integrase (IN), the coding region was isolated and the encoded protein was purified, using nickel-affinity chromatography, to greater than 90% homogeneity. HTLV-II(G12) IN proved active on HTLV-II(G12) and HIV-1 integration and disintegration substrates. Distinct differences in requirements for enzyme concentration for 3'-processing, strand transfer, and disintegration reactions were observed. Catalysis of integration reactions occurred in the presence of either Mn2+ or Mg2+, although strand transfer activity preferred Mn2+. In comparison, HTLV-II(G12) IN catalyzed disintegration reactions with almost 10-fold less protein, was not selective for Mn2+ or Mg2+, and tolerated higher NaCl concentrations than integration. HTLV II(G12) IN was unable to catalyze the "splicing" reaction, which suggests that this may not be an activity ubiquitous to all retroviral integrases. PMID- 8623557 TI - Broader tropism and higher cytopathicity for CD4+ T cells of a syncytium-inducing compared to a non-syncytium-inducing HIV-1 isolate as a mechanism for accelerated CD4+ T cell decline in vivo. AB - The emergence of syncytium-inducing (SI) HIV-1 isolates in infected individuals precedes an accelerated CD4+ T cell decline and is associated with high virus load and rapid disease progression. The exact mechanism by which SI HIV-1 variants may cause this enhanced clinical progression is unknown. Here we demonstrate that an SI HIV-1 isolate had a broader tropism for CD4+ T cell clones (TCC) compared to a macrophage-tropic non-syncytium-inducing (NSI) HIV-1 isolate. Whereas the NSI isolate replicated poorly in 6 of 12 TCC and completely failed to replicate in 3 of 12 TCC, the SI isolate replicated efficiently in all 12 TCC tested. Restriction for replication occurred early in the viral replication cycle, before provirus formation. Infection of TCC with the SI but not with the NSI HIV-1 isolate resulted in massive death of T cells, independent of the extent of virus replication and proportion of infected cells. The high cytopathicity and broader tropism of the SI isolate for primary CD4+ T cells may be directly related to the increased rate of CD4 cell decline and rapid disease progression in carriers of SI variants. PMID- 8623558 TI - Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus. AB - Nucleotide sequences of the genomic RNA beta components of hordeiviruses poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV) were determined. PSLV and LRSV closely resemble barley stripe mosaic virus (BSMV), type hordeivirus, in the gene arrangement of their RNAs beta, comprising 5'-proximal beta a (coat protein) gene and downstream triple gene block (TGB) coding for the beta b, beta c, and beta d putative transport proteins. The beta a, beta b, beta c, and beta d proteins of the three hordeiviruses showed significant sequence similarity, with the respective proteins of PSLV and BSMV being closer to each other than to their counterparts of LSRV. Comparisons of the TGB-encoded proteins of hordeiviruses, potexviruses, carlaviruses, and furoviruses indicate that the first and second TGB genes belong to the monophyletic groups, whereas the third gene may have multiple ancestry. LRSV, PSLV, and BSMV showed remarkable variation in the 3' untranslated regions of their genomic RNAs. Among the three hordeiviruses, LRSV has the shortest 3'-noncoding region that lacks tentative pseudoknot-forming elements conserved upstream of the 3'-tRNA-like structure in the BSMV and PSLV genomes. On the other hand, LRSV RNA beta, like that of BSMV, contained the internal poly(A) sequence that is absent from PSLV RNA. PMID- 8623559 TI - Site-specific spontaneous deletions in three genome regions of a temperate Streptococcus thermophilus phage. AB - Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperate Streptococcus thermophilus phage phi SFi21. Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3). Combinations of types 1 and 3 and 2 and 3 were observed. The mutants grew lytically although with reduced burst sizes. Type 2 mutants lost the capacity to lysogenize host cells. Upon serial passage, the deletion mutants overgrew the wild-type phage. No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites. Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site-specific recombination system. Sequencing of an Xbal restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KiIA, Ant). Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF. PMID- 8623560 TI - [II. European symposium on Quantification of Rehabilitation Training Programs. Vienna, Austria, 2-5 June 1994. Abstracts]. PMID- 8623561 TI - Failed innovations--no concern of ours? PMID- 8623562 TI - The economic impact of failures in total hip replacement surgery: 28,997 cases from the Norwegian Arthroplasty Register, 1987-1993. AB - The Norwegian Arthroplasty Register was established in 1987. Until January 1994, approximately 200 different implant combinations had been used in total hip replacements (THR) in Norway. About 5,500 THR were performed each year in this period with a total cost of 70 million USD per year. We analyzed the economic consequences related to the use of some inferior primary hip arthroplasties in this period. As the reference arthroplasty, we chose the most commonly used prosthesis in Norway, i.e., the Charnley prosthesis fixed with high viscosity cement containing antibiotic and with systemic antibiotic prophylaxis (n 4,970). We compared this reference group to all other primary THR registered in the same time period (n 24,027), and to the following sub-groups of primary THR: 1) uncemented Ti-Fit/ Bio-Fit (acetabulum/femur) combination (n 173), 2) uncemented Coxa/Femora combination (n 153), 3) THR with low-viscosity cement (n 1,807) and 4) THR with Boneloc cement (n 1,250). We estimated the number of additional revisions compared to the reference arthroplasty after a follow-up of 3-5 years in the different groups, with adjustment for age, sex and diagnosis. The direct extra revision costs were calculated. Compared to the reference arthroplasty, the group of all other primary THR gave an extra revision cost estimated at about 1.7 million USD per year. About 1,000 uncemented Bio-Fit femoral prostheses have been applied in Norway, including those implanted before the registration started (1985-1987). The extra revision costs the first postoperative years for these 1,000 prostheses amount to about 0.7 million USD per year. Corresponding figures in the Coxa/ Femora group were 0.08 million USD, in the group with low-viscosity cement, 0.3 million USD and in the Boneloc group, 0.4 million USD per year. PMID- 8623563 TI - The Harris-Galante cementless femoral component: poor results in 57 hips followed for 3 years. AB - Between 1986 and 1991, 68 Harris-Galante femoral prostheses were implanted in 63 patients. 53 patients (57 hips) attended for follow-up after a mean of 3.4 (2-6) years. The average Harris Hip Score (HHS) was 39 preoperatively and 81 postoperatively. Only 34 hips were excellent (27) or good (7). Mid-thigh pain occurred in 30 cases. 10 femoral components were radiographically loose because of osteolysis. Although 7 cups seemed to be radiographically loose, they probably played only a minor role in the poor overall early clinical results. PMID- 8623564 TI - Polyethylene wear in Scanhip arthroplasty with a 22 or 32 mm head: 62 matched patients followed for 7-9 years. AB - We measured radiographic polyethylene wear in patients with Scanhip arthroplasty and no clinical or radiographic signs of loosening. The patients were divided into 2 groups according to head sizes. 32 patients (33 hips) had an implant with a 22 mm and 30 patients (34 hips) with a 32 mm head. They were followed for 7-9 years. The groups were matched for diagnosis, sex, weight, age, and time of follow-up. The mean linear wear with a 22 mm head was 1.1 mm and with a 32 mm head 1.5 mm (p 0.004), which corresponds to a yearly wear rate of 0.15 mm and 0.18 mm, respectively. The mean difference in volumetric wear was greater, 420 mm3, as compared to 1239 mm3. PMID- 8623565 TI - Hip-simulator ranking of polyethylene wear: comparisons between ceramic heads of different sizes. AB - We carried out simulator studies on ceramic-polyethylene total-hip combinations to determine the volumetric wear-rates of 22 mm, 26 mm and 28 mm femoral-head sizes. Bovine-serum lubrication and 2 kN peak sinusoidal load-profile were used with polyethylene (UHMWPE) cups. Wear was assessed by gravimetric technique. Precision (9%) was ensured by the use of multiple specimens, multiple wear events, and the linear-regression method of estimating the average wear trend, thereby reducing the inherent, unpredictable nature of each wear-event. Volumetric wear-rates for polyethylene averaged 23 mm3 per 10(6) cycles for the 22 mm ceramic head and up to 32 mm3 per 10(6) cycles for the 28 mm head. The difference between 22 mm and the larger head-sizes was significant. This may well be the first laboratory confirmation of Charnley's original clinical Low-Friction Arthroplasty concept with regard to wear rate. The wear penalty increased linearly at the rate of 6% to 9% per mm of diameter increase. PMID- 8623566 TI - High failure rate of cementless threaded acetabular cups: a radiographic and histologic study in the goat. AB - We studied the fixation of the Mecron cementless titanium screw cup radiographically and histologically in 20 dwarf-goats after periods of 0, 6, 26 and 52 weeks. In only 3 goats did histology show good bone-implant contact, whereas in the other 17 goats a fibrous membrane interface was seen. This high failure rate is caused by the poor primary fixation and should be a warning against the use of this implant. PMID- 8623567 TI - Bone scintigraphy as an indicator for dome osteotomy of the pelvis: comparison between scintigraphy, radiography and outcome in 57 hips. AB - We performed dome pelvic osteotomy in 57 hips (54 patients) because of acetabular dysplasia. All patients had preoperative scintigraphy and were followed for more than 2 years. Excellent or good results were obtained in 49 hips, but 8 hips deteriorated. All hips which deteriorated had preoperatively a spotty isotope uptake image localized to the weight bearing area and the medial area of the joint. No hip without this image deteriorated, even when preoperative radiography showed advanced arthrosis. Our findings indicate that bone scintigraphy may be a more reliable predictor of failure for this operation than radiography. PMID- 8623568 TI - The plasminogen activation system is upregulated in loosening of total hip prostheses. AB - Interface tissues and pseudocapsules from loose total hip replacements were removed during revision of 11 cases and were investigated for the plasminogen activation system and IL-1beta. Control samples of synovium were taken during knee arthroscopy (n 8), and from the hip joint during primary total hip replacement (n 5). The concentrations of urokinase plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI 1) and interleukin 1beta were all found to be significantly different in interfaces and in pseudocapsules, compared to controls. Immunohistochemistry disclosed localization in periprosthetic tissues of uPA, uPA-receptor and tPA in macrophages with phagocytosed metal, polyethylene, cement particles or accompanying pieces of necrotic bone. PAI-1 staining was present in the neighboring areas that stained for uPA or tPA, but PAI-1 staining was also found overlapping and outside these areas. These findings suggest a role for the uPA/uPA- receptor and PAI-1 in activation and focalization of extracellular matrix degradation in periprosthetic tissues. The expression of the plasminogen activation system by macrophages containing phagocytosed material suggests undegradable microdebris as a possible initiating and perpetuating stimulus for a proteolytic activation cascade, which may contribute to loosening of the prosthesis. PMID- 8623569 TI - Bilateral synovitis in symptomatic unilateral transient synovitis of the hip: an ultrasonographic study in 56 children. AB - 56 children with a clinical diagnosis of unilateral transient synovitis of the hip underwent bilateral sonographic assessment. On the anterior scan, the distance between the femoral neck and the fibrous joint capsule was measured. This distance, which we call the synovial capsular complex distance, was compared with age-dependent normal values. An increased distance was found in all 56 symptomatic hips (mean 10 mm, SD 1.8). This distance was also increased in 14 hips on the contralateral side (mean 8 mm, SD 1.6). An effusion was demonstrated in 53 symptomatic hips and in 8 hips on the contralateral side. These findings indicate that in one quarter of children with symptoms of unilateral transient synovitis the contralateral hip may have an increased synovial capsular complex distance due to synovial swelling or joint effusion, suggesting an asymptomatic synovitis. We therefore recommend a comparison of the synovial capsular complex distance on the symptomatic side with age-related normal values, in addition to a comparison with the asymptomatic hip. PMID- 8623570 TI - Cement augmentation of intertrochanteric fracture fixation: a cadaver comparison of 2 techniques. AB - We evaluated 2 techniques of cement augmentation to enhance fixation of intertrochanteric hip fractures. 4 fixation groups with 6 cadaver femurs in each group were compared: stainless steel lag screw and side plate with and without cement augmentation and a titanium alloy expandable dome plunger and side plate with and without cement augmentation. Gauges were used to establish the mechanical behavior of intact and then fractured femurs to simple uniaxial loads. Subsequent loading to failure allowed determination of maximum fixation strengths and modes of failure. Cement augmentation of each device increased its load to failure. There was no significant difference between the cemented lag screw and the uncemented dome plunger groups with average loads to failure of 4.0 x 10(3) N. The greatest average load to failure was in the cemented dome plunger group (5.6 x 10(3) N) with the lowest in the uncemented sliding hip screw group (3.6 x 10(3) N). Device cut-out as a cause of failure occurred mostly in the uncemented lag screw group. Sliding was enhanced by those methods that increased the fixation surface area within the femoral head, unless cement encroached in the region of the barrel-screw junction. Strain analysis showed that the dome plunger unloaded the bone at the calcar, regardless of cement augmentation, while the sliding hip screw allowed for compressive stresses in this area. Proper cement augmentation increases load to failure and minimizes nail cut-out for both devices studied. However, the dome plunger, a device with a large fixation area in the femoral head, was equally effective and eliminated potential cement encroachment. Failure of intertrochanteric fracture fixation in osteoporotic bone may be minimized by an appropriate choice of device or cement augmentation. PMID- 8623571 TI - Callus formation in femur and tibia during leg lengthening: 7 patients examined with DXA. AB - We examined the callus formation during leg lengthening in 7 achondroplastic patients who underwent 3 bilateral femoral and 4 bilateral tibial lengthenings. Bone mineral content and bone mineral density (BMD) in the lengthened callus space were evaluated every 1 or 2 weeks for 10 weeks after the start of distraction using dual energy X-ray absorptiometry. The mean rate of callus mineralization in femurs (0.64 g/wk) was higher than in tibias (0.22 g/wk). The mean BMD at 10 weeks after the start was 0.35 g/cm2 in the femur and 0.14 g/cm2 in the tibia. Different rates of callus formation in different kinds of long tubular bones have not been reported previously. PMID- 8623572 TI - 11 femoral fractures with vascular injury: good outcome with early vascular repair and internal fixation. AB - We reviewed 11 consecutive cases with combined femoral fracture and vascular injury presenting with acute ischemia. 6 cases had ischemia exceeding 8 hours and 4 of them developed massive muscle necrosis in the lower leg. 5 cases with ischemia less than 8 hours had no muscle necrosis. Vascular repair preceded fracture stabilization in 5 cases; there were no vascular complications during the subsequent fracture stabilization. 6 fractures treated with internal fixation had uneventful fracture-healing, whereas the 4 which were treated with external fixation needed later reoperations to obtain fracture-healing. We conclude that the limb must be reperfused within 6-8 hours. Vascular repair should be the first procedure, and fracture fixation by internal fixation is then preferred. PMID- 8623573 TI - The long-term prognosis for severe damage to weight-bearing cartilage in the knee: a 14-year clinical and radiographic follow-up in 28 young athletes. AB - We examined 28 young athletes with isolated severe chondral damage in the weight bearing area of the knee joint clinically and radiographically 14 years after arthroscopic diagnosis. Except for Pridie drilling in 3 cases and occasional cartilage shaving or removal of free bodies, no special treatment was given initially. 21 patients were able to return to preinjury team sport activity levels. During the follow-up period, only 3 patients needed repeat surgery with removal of free bodies, and another 2 underwent diagnostic arthroscopy because of persistent pain. At the latest follow-up evaluation, 22 patients had excellent or good knee function. At this time, the patients were mainly involved in individual sports on a physical fitness level. 12 cases had radiographic joint space reduction (< 50%) which was limited to the compartment concerned. PMID- 8623574 TI - Scoliosis elasticity assessed by manual traction: 49 juvenile and adolescent idiopathic cases. AB - We assessed preoperative curve elasticity in 49 consecutive patients with juvenile or adolescent idiopathic scoliosis who were operated on with Harrington distraction rods. Preoperatively, the curve was determined from posteroanterior radiographs taken in the standing position and in the supine position, with traction. In the latter, the radiographs were taken at the moment of maximal traction when one technician applied traction to the ankles and another to the wrists. The scoliotic curve in the 10 patients with juvenile scoliosis averaged 59 degrees and 32 degrees in the standing and supine positions with traction, respectively. Immediately postoperatively, the curve averaged 19 degrees. 39 patients with adolescent scoliosis had a scoliotic curve which averaged 58 degrees in the standing position and 32 degrees in the supine position with traction. The mean postoperative measurement was 21 degrees. These findings suggest that manual traction is a simple and reliable means of predicting the minimal correction of the scoliotic curve to be expected, using Harrington distraction rods. PMID- 8623575 TI - Fracture of the humeral condyles in children: 49 cases evaluated after 18-45 years. AB - We evaluated 49 children with fracture of the humeral condyles 18-45 years after the injury. 20 fractures with a displacement of 2-1 0 mm with no tilting of the osteochondral fragment had been treated without reduction and with good results. 16 fractures with marked displacement and tilting of the osteochondral fragment had been treated surgically, with good results. 13 patients had been treated for old, displaced fractures both operatively and nonoperatively, with poor results. Nonunion developed in 4 cases and aseptic necrosis in 6. Arthrosis of the elbow was found in cases complicated by aseptic necrosis and nonunion or in old fractures when resection of the humeral condyle was done, but it was never observed in uncomplicated fractures. PMID- 8623576 TI - Malunited fractures of the distal radius with volar angulation: corrective osteotomy in 6 cases using the volar approach. AB - We treated 6 patients with volarly angulated malunions after distal radius fractures by corrective radius osteotomy via the volar approach. No complications related to this approach were seen. Postoperatively, ulnar head pain was alleviated and forearm rotation was within 10 degrees of normal. PMID- 8623577 TI - External tissue stretching for closing skin defects in 22 patients. AB - In this prospective study, we treated 12 women and 10 men with a newly developed skin-stretching system. The stretching device was used for the closure of 9 fasciotomies and for preoperative skin extension before excision of 6 tattooes, 7 splitskin transplants, 4 giant naevi, and 3 scars. The stretching device was placed under the skin, using local anesthesia. The patient stretched the skin. The mean stretching time was 4 (2-11) days. 20 patients completed the expansion successfully, with no pain or only slight discomfort. In 2 cases, we observed minor complications during the expansion. The external skin-stretching system can be used to obtain primary closure of defects where splitskin transplants otherwise would be necessary. The method can be applied in out-patients, using local anesthesia. PMID- 8623578 TI - Concentrations of proteoglycan fragments in relation to maturation, sex and time of day: physiologic variations in knee joint fluid of rabbits. AB - We analyzed the concentrations of proteoglycan fragments in knee joint fluid in 142 rabbits to investigate the effect of physiologic variations--i.e., maturation, sex and time of day. The concentrations of proteoglycan fragments differed significantly between young, adolescent and adult animals and showed an inverse correlation to the stage of maturation of the rabbit. Adolescent male rabbits had higher concentrations than age-matched females. Morning and evening samples had similar concentrations. No relation was found between the proteoglycan fragment concentrations in joint fluid and the cartilage mass. The proteoglycan fragment concentrations in knee joint fluid apparently reflect the metabolic status of growing articular cartilage. There are considerable physiologic variations associated with maturation and sex, and these need to be taken into account when using the proteoglycan fragment concentration as a marker for joint diseases. PMID- 8623579 TI - Neuropeptide-converting enzymes in cerebrospinal fluid: activities increased in pain from herniated lumbar dis, but not from coxarthrosis. AB - We measured activities of dynorphin-converting enzyme (DCE), substance P endopeptidase (SPE) and angiotensin-converting enzyme (ACE) in cerebrospinal fluid (CSF) in 13 patients with rhizopathic pain from an herniated lumbar disc, in 9 patients with pain from coxarthrosis and in 11 control patients without pain. In the patients with disc hernia and coxarthrosis, another sample of CSF was analyzed 3-12 months after treatment, when pain had subsided. The DCE activity in the patients was higher than that in both the control patients and the patients with pain from coxarthrosis (nociceptive pain). Similarly, the activity of SPE was lower in the patients with herniated lumbar disc than in controls and in the patients with coxarthrosis. After treatment, the difference in activity compared to controls was lower, but still significant in patients with herniated discs. The ACE activity did not differ from controls in patients with ischialgia, while it was increased in patients with coxarthrosis. This increase also remained after arthroplasty with pain relief. In conclusion, measurements of neuropeptides may be useful for evaluating neuropathic pain. PMID- 8623580 TI - A severely overcorrected high tibial osteotomy: revision by osteotomy and a long stem component. PMID- 8623581 TI - 3 deep-seated glomus tumors in the lower arm. PMID- 8623582 TI - Reactive arthritis, diagnosis and treatment: a review. AB - The diagnosis of reactive arthritis (ReA) is easy in typical cases with a history of an infection within 3 weeks in combination with an asymmetric mono or oligoarthritis with or without extra-articular manifestations. Subclinical microbial infections, a possible co-existing inflammatory bowel disease and the fact that in 25% of the cases the microbial agent remains unidentified, make the diagnosis more troublesome. The course of disease is usually self-remittent within 6 months but a less good long-term prognosis is pre-determined by two factors-namely, the presence of HLA-1327 and the recurrence of triggering infections. The finding of microbial fragments in the joint cavity have led to new treatment strategies especially in Chlamydia-triggered ReA. It must, however, be remembered that the antibiotics mostly used (namely, tetracyclines) also possess immunoregulatory and anticollagenolytic potential. In chronic destructive cases, antirheumatic treatment, similar to that used in rheumatoid arthritis, is recommended. PMID- 8623583 TI - Increased risk of dislocation with collar reinforced modular heads of the Lubinus SP-2 hip prosthesis. PMID- 8623584 TI - Polyethylene particles or formaldehyde reaction. PMID- 8623585 TI - Stem fracture with the Exeter prosthesis. PMID- 8623586 TI - A large collar in hip replacement. PMID- 8623587 TI - Surgical treatment of tuberculous spondylitis. PMID- 8623588 TI - Orthopaedica Belgica Congress. Rotator cuff ruptures. Mons, June 1-3, 1994. Proceedings. PMID- 8623589 TI - [Intermediate results in Neer's unrestricted total shoulder prosthesis inserted in glenohumeral arthropathies of arthrotic or arthritic origin]. PMID- 8623590 TI - [Results of Grammont's inverted prosthesis in omarthritis associated with major cuff destruction. Apropos of 16 cases]. PMID- 8623592 TI - [The role of glenohumeral arthrodesis compared to prosthetic arthroplasty of the shoulder]. PMID- 8623591 TI - [Hemi-arthroplasty of the shoulder: radiological, functional and dynamic studies]. AB - From September 1973 to October 1992, forty-three shoulder arthroplasties were performed in forty-one patients with complicated fractures or degenerative disease. Radiological and functional assessment with a mean follow-up time of forty-five months permitted us to review twenty patients with fifteen Neer II prosthesis, three isoelastic prosthesis and two total shoulder replacements. Of these, eleven presented with traumatic injuries and four with degenerative disease. The mean age at operative time was 61 years. Radiological evaluation showed that all cases had a retroversion of about 30 degrees. Normal humeral length and lateral shifting were not achieved in most patients; in fact only one had a restored length and lateral shifting. The mean scapulothoracic range of motion was found to be half that of a normal gliding shoulder (angle 30 degrees instead of 60 degrees). The subacromial space was diminished by one third in all cases. Relief of shoulder pain was the most significant finding. Mobility was restored to a level of about 75% of normal according to Constant's Scale. Dynamometric measures showed an important loss of force (47% of normal) despite a normal external rotation force. Recent improvements in implant conception (modular prosthesis) and a better understanding of physiological shoulder mechanisms should improve the functional results of this arthroplasty in the future. PMID- 8623593 TI - [Intramedullary osteosynthesis: elastic or rigid?]. PMID- 8623594 TI - Bundle nailing of diaphyseal fractures of the humerus. AB - The principles of bundle nailing--jamming of the nails in the cortical window, jamming in the waist of the medullary cavity, spreading the bunch of nails in the metaphysis and filling up the conus of the medullary cavity with short nails should be observed. Then this method of intramedullary osteosynthesis is of diaphyseal fractures with slight damage to the soft tissues is indicated ideally in the second to fifth sixth of the humerus. The complication rate is low. If there is a primary traumatic lesion of the radial nerve or if there are open fractures and/or multiple fractures in case of polytraumatized patients, operative stabilization is mandatory using plate osteosynthesis or external fixation. PMID- 8623595 TI - [Hackethal's method of bundle nailing for proximal or diaphyseal fractures of the humerus]. PMID- 8623596 TI - Indications for immediate percutaneous intramedullary nailing of complete diaphyseal forearm shaft fractures in children. PMID- 8623598 TI - ['Endolock' femoral and tibial nail. Concept and laboratory studies]. PMID- 8623597 TI - [Surgical repair of total rupture of the rotator cuff. Results and limitations]. PMID- 8623599 TI - ["Endolock" locking femoral nail without radiologic imaging (66 cases)]. PMID- 8623601 TI - The new unreamed AO nails for the tibia and the femur. AB - The advantages of the unreamed nailing technique compared to the reamed procedure are less damage to bone vascularity, lower intramedullary pressure during the operation and a fast operation time. These new solid nails with high mechanical stiffness and no central dead space have extended the nail indications for tibial as well as for femoral fractures, particularly in tibial shaft fractures with severe soft tissue damage and in polytraumatized patients with a femoral shaft fracture. Besides these advantages the new AO unreamed femur nail has a modular proximal interlocking system, which increases significantly the range of indications for nailing. Not only very high subtrochanteric femur fractures can be stabilized with greater stability, but also combined ipsilateral fractures of the shaft and the proximal femur can be treated with this new nail. The first clinical results of the AO unreamed nails for the tibia as well as for the femur are very encouraging. The unreamed nails prove to be excellent implants in the treatment of long bone fractures of the lower extremity. PMID- 8623600 TI - [Initial results of the treatment of unstable femoral and tibial fractures using centro-medullary flexible nail osteosynthesis]. PMID- 8623602 TI - [Risk of infection in centro-medullary locking nailing of open fractures of the femur and tibia]. AB - Intramedullary reamed locking nail of open fractures remains controversial because of the risk of infection. 1,474 closed reamed locked nailings were performed between 1974 and 1989 for femoral (744 cases) or tibial (730 cases) fractures. 349 fractures were open: 100 femoral fractures (51 Gustilo and Anderson Grade I and 49 Grade II) and 249 tibial fractures (140 Grade I, 99 Grade II et 10 Grade III). 24 femoral (3.2%) and 46 tibial (6.3%) nails were followed by infection. This difference is significant (p < 0.01). Reoperations for infection occur more frequently for femoral than tibial fractures (p < 0.05). There is no difference between the results of infection treatment between femoral or tibial fractures. Traumatic opening of the femoral fracture site does not affect the occurrence of an infection, its severity or the results of its treatment. Traumatic opening of the tibial fracture site significantly increases the infection rate (p < 0.001), and the incidence of infection increases with the severity of the soft tissue lesions; but the severity of the infection and the results of its treatment are not modified. Acute closed reamed intramedullary locking nail is the best treatment for open femoral or tibial fractures with respect to the bone healing and infection rate for Grade I and II fractures. For Grade III fractures, nailing must be followed by a coverage flap. PMID- 8623603 TI - [Multicenter study of 210 rotator cuff ruptures treated with arthroscopic acromioplasty]. PMID- 8623604 TI - [Clinical and anatomic results of a series of 20 rotator cuff ruptures treated with endoscopic stapler]. PMID- 8623605 TI - [History and development of the shoulder prosthesis]. PMID- 8623606 TI - [Contribution to the biomechanics of the shoulder]. PMID- 8623607 TI - [Adaptability and modulation in shoulder prosthesis]. PMID- 8623608 TI - [Classification of articular fractures of the upper extremity of the humerus]. PMID- 8623609 TI - [Preoperative planning in shoulder prosthesis]. PMID- 8623610 TI - [The value of MRI in the evaluation of lesions of the supraspinous muscle. Multicentric retrospective study of 66 records]. PMID- 8623611 TI - [Involvement of the shoulder in rheumatoid arthritis]. AB - The course of rheumatoid arthritis (R.A.) in the shoulder is evaluated in 100 patients in a retrospective study and in 73 patients in a prospective study. The involvement of the shoulder may occur at an early stage of the disease. However, usually it causes a significant handicap at a later stage. The retrospective study shows that 62% of the patients hospitalized between 1976 and 1992 for R.A. complained of pain, reduction in range of motion and functional disability of one or both shoulders. However, these historic cases cannot be used for a radiological study because the shoulders have been radiographed in 17% of cases only, probably because of the poor possibilities of surgical therapies available at this time. The survey of 72 patients received at the out- patient clinic between september 1992 and december 1993 who had a radiograph of the shoulder is the basis for the description of the progress of the disease. The erosions appear at the border of the cartilage where the synovium is attached and refers mainly to the cephalic articular cartilage. Narrowing of the subacromial space is often present with early appearance of subluxation in the cranial direction. The destruction often prevails in the area of humeral head but cases with predominant global joint space narrowing or with destruction of glenoid labrum and fossa exist as well. Secondary osteoarthritis was often observed. The involvement of the shoulders is not always symmetric. Unilateral involvement has been observed. The different evolutive processes will be illustrated and their frequency evaluated. The forms with predominant cephalic destruction constitute the best indications for total shoulder arthroplasty. PMID- 8623612 TI - [Neer's total shoulder prosthesis. Indication, technique and preliminary results]. PMID- 8623613 TI - In vivo stationary flux analysis by 13C labeling experiments. AB - Stationary flux analysis is an invaluable tool for metabolic engineering. In the last years the metabolite balancing technique has become well established in the bioengineering community. On the other hand metabolic tracer experiments using 13C isotopes have long been used for intracellular flux determination. Only recently have both techniques been fully combined to form a considerably more powerful flux analysis method. This paper concentrates on modeling and data analysis for the evaluation of such stationary 13C labeling experiments. After reviewing recent experimental developments, the basic equations for modeling carbon labeling in metabolic systems, i.e. metabolite, carbon label and isotopomer balances, are introduced and discussed in some detail. Then the basics of flux estimation from measured extracellular fluxes combined with carbon labeling data are presented and, finally, this method is illustrated by using an example from C. glutamicum. The main emphasis is on the investigation of the extra information that can be obtained with tracer experiments compared with the metabolite balancing technique alone. As a principal result it is shown that the combined flux analysis method can dispense with some rather doubtful assumptions on energy balancing and that the forward and backward flux rates of bidirectional reaction steps can be simultaneously determined in certain situations. Finally, it is demonstrated that the variant of fractional isotopomer measurement is even more powerful than fractional labeling measurement but requires much higher numerical effort to solve the balance equations. PMID- 8623614 TI - Analysis and modeling of substrate uptake and product release by prokaryotic and eukaryotic cells. AB - Translocation of molecules and ions across cell membranes is an important step for a complete description of the metabolic network in terms of kinetics, energetics and control. With a few exceptions, most molecules cross the permeability barrier of the membrane with the aid of membrane-embedded carrier proteins. Uptake of nutrients (carbon, energy and nitrogen sources as well as supplements) and excretion of the majority of products are thus carrier-mediated transport processes. Consequently, they are characterized by particular kinetic properties of the respective carrier systems, they depend on energy sources (driving forces) which must be provided by the cell, and they are subject to regulation both on the level of activity and expression. They are thus fully integrated into the functional and regulatory networks of the cell. Structural (primary structure, conformation and topology) and functional properties (kinetics, energetics and regulation) of the different classes of carrier systems from both prokaryotic and eukaryotic membranes are summarized. The methodical requirements for a quantitative measurement of their function and possible pitfalls in transport studies are described, both for determination using isolated cells and for analysis in a bioreactor. The significance of transport reactions for biotechnological processes in general and for metabolic design in particular is discussed, with respect to nutrient uptake, product excretion and the occurrence of energy wasting combinations of transport reactions (futile cycles). Some examples are given where transport reactions have been incorporated into modeling approaches with respect to metabolic control, to flux analysis, to kinetic properties and to energetic demands. PMID- 8623615 TI - Reaction engineering methods to study intracellular metabolite concentrations. AB - The analysis of intracellular metabolite concentrations is of basic importance for metabolic engineering of microorganisms. In vivo NMR-spectroscopy as a non invasive technique to measure intracellular metabolite concentrations and rapid sampling devices as invasive techniques are reviewed. The methods are discussed from a reaction engineering point of view. The objective is to obtain intracellular concentration data under well defined physiological conditions in balanced steady state and defined transitional states as well. Application examples are given for a membrane-cyclone-reactor configuration designed to achieve high signal sensitivity with in vivo 31P-NMR and 13C-NMR spectroscopy as well as for a sampling tube device designed for high sampling rates (2s-1). This sampling device enables the measurement of dynamic metabolite profiles at a time scale of a few seconds. PMID- 8623616 TI - Medical-legal issues in teleradiology. AB - With the advent of high-speed, high-resolution digital imaging and network transmission, teleradiology promises to dramatically change the mechanism for interpreting radiologic images and the traditional relationships between radiologists and patients and referring physicians. We discuss the current status of medical-legal issues pertaining to privacy, licensing, credentialing, liability, and fraud for teleradiology. Special emphasis is placed on the technical factors that may result in differences in image properties, consequently affect radiologic diagnosis, and bring the potential for liability. PMID- 8623617 TI - Medical-legal issues in teleradiology: a commentary. PMID- 8623618 TI - Communication of the urgent finding. PMID- 8623619 TI - Simple steps for improving multiple-reader studies in radiology. AB - Multiple-reader study designs have become popular in the radiology literature. We reviewed the major papers published in the American Journal of Roentgenology in the first 4 months of each of the years 1990 and 1995. The review was restricted to prospective studies of image interpretation. In the 1990 literature, we noted eight multiple-reader and 18 single-reader studies; in contrast, in the 1995 literature, we found 29 multiple-reader and eight single-reader studies. This trend reflects an increased awareness of the importance of multiple-reader studies. We examined the Results sections of the 29 multiple-reader studies from 1995 to assess the authors' motives for incorporating such a design. In 16 studies (55%), readers independently interpreted all images. However, the authors usually reported only the average interpretation of the readers; in only seven of the 29 studies (24%) did the authors describe differences among readers' interpretations. In 13 studies, interpretations were performed exclusively through "consensus reading." The method(s) used to achieve a consensus often were not explained. Only two of the 29 studies had more than three readers. In contrast, all of these studies included multiple patients. The average patient sample size was 45. Furthermore, differences observed among patients were routinely reported and/or depicted. PMID- 8623620 TI - Nonspecificity of short inversion time inversion recovery (STIR) as a technique of fat suppression: pitfalls in image interpretation. AB - Short inversion time inversion recovery (STIR) and the rapid acquisition with relaxation enhancement (RARE) version of STIR are commonly used pulse sequences that are sensitive enough to detect a broad range of pathologic conditions. In addition to suppressing the signal from fat, the STIR sequence achieves additive T1-weighted, T2-weighted, and proton density-weighted contrast to facilitate lesion conspicuity [1, 2]. Fat suppression with STIR sequences is based on short T1 relaxation rates and therefore is not tissue specific. The signal from any tissue with a short T1, similar to that of fat, may be nulled as well. The signal from tissues that accumulate paramagnetic contrast agents also may be suppressed with STIR sequences when an appropriate degree of T1 shortening results. PMID- 8623621 TI - Morphing radiologic images: applications on a desktop computer. AB - OBJECTIVE: Morphing is an image processing technology that transforms one image into another by generating a series of intermediate synthetic images. The ability to perform morphing, once restricted to high-end graphics workstations, is now widely available for desktop computers. We investigated the potential use of morphing for displaying radiographic images. MATERIALS AND METHODS: Morphing was performed with commercially available software (Morph 2.5; Gryphon Software, San Diego, CA) on a Macintosh (Apple Computer, Cupertino, CA) computer. Images from 26 patients with serial radiologic studies were selected, digitized, and morphed. Key points and key surfaces were identified on the images to improve the quality of the morph movie, a process that took approximately 15 min. The images were then morphed into a continuous Quick Time (Apple) movie lasting 5-7 sec. When a sequence contained more than two images, a single movie incorporating all the images was created. The intervals between segments of the movie were made proportional to the actual time elapsed between the images. Images obtained with different techniques (positron emission tomography and MR imaging) were also morphed. Three observers judged the quality of the morph movies as satisfactory, good, or excellent. RESULTS: Twenty of the 26 movies were judged to be of good or excellent quality. The movie format allowed the rapid display of multiple images in a concise 5- 7-sec time frame. Moreover, the movie allowed the recognition of several simultaneous processes more easily than was possible with static images. Morphing two images of the same anatomic site using different techniques (MR imaging and positron emission tomography) proved to be a simple method of superimposing images. CONCLUSION: Morphing is a readily available and easily learned technique for displaying serial studies. The movie format is a more interesting, intuitive, and concise summary of events than can be provided by serial static images. Morphing may be useful for displaying large numbers of serial images in a short time for occasions such as clinical conferences or teaching purposes. PMID- 8623622 TI - Differentiation of adrenal adenomas from nonadenomas using CT attenuation values. AB - OBJECTIVE: The purpose of our study was to determine whether unenhanced CT attenuation value, enhanced CT attenuation value, or lesion size can be used to differentiate adrenal adenomas from nonadenomatous adrenal masses. MATERIALS AND METHODS: We retrospectively assessed the CT scans of 135 adrenal masses in 124 patients with a variety of adrenal masses. There were 93 cortical adenomas (85 nonhyperfunctioning adenomas, four Cushing's adenomas, and four primary aldosteronism adenomas). The nonadenomas consisted of 34 metastases, four cortical carcinomas, and four pheochromocytomas. The scattergrams and mean values of the size and attenuation values on enhanced and unenhanced scans were correlated with the final diagnoses. Results were also subjected to receiver operating characteristic analysis. RESULTS: Forty-one adenomas and 20 nonadenomas had unenhanced CT. The mean attenuation value of the 41 adenomas was significantly lower (p < .001) than that of the nonadenomas (2.5 H +/- 14 compared with 32 H +/- 6.4). The lowest unenhanced CT attenuation value of the nonadenomas was 18 H; therefore, the sensitivity:specificity ratio for the diagnosis of adenomas was 85%:100% at a threshold value of 18 H. At this threshold, the positive predictive value was 100% and the negative predictive value was 77%. For the 85 masses with enhanced CT, the mean attenuation of the 60 adenomas was also significantly lower (p < .01) than for the 25 nonadenomas (47 H +/- 24 compared with 62 H +/- 21). The lowest enhanced CT attenuation value of the nonadenomas was also 18 H, but the sensitivity:specificity ratio was only 10%:100% at this threshold value of 18 H. Although the mean diameter of the adenomas was significantly lower (p < .001) than for the nonadenomas (2.4 cm +/- 0.9 compared with 4.5 cm +/- 2.5), there was sufficient overlap between the two groups at the smallest sizes that a threshold value for a highly specific diagnosis of adenoma was not present. The area under the receiver operating characteristic curve for unenhanced CT attenuation values (0.98 +/- 0.02) was significantly greater than the area for enhanced CT values (0.68 +/- 0.06, p < .001) and the area for size (0.79 +/- 0.04, p < .001). CONCLUSIONS: Unenhanced CT attenuation values can characterize an adrenal mass as a benign adenoma with high specificity and acceptable sensitivity. Adrenal masses cannot be characterized using enhanced CT attenuation values or lesion size. PMID- 8623623 TI - Gantry angulation in CT-guided percutaneous adrenal biopsy. AB - OBJECTIVE: This study was performed to test a new method for CT-guided adrenal biopsy using angulation of the CT gantry. MATERIALS AND METHODS: In 23 patients, 26 biopsies were performed on patients in the prone position using CT gantry angulation and the introduction of the biopsy needles along the same angle, guided by the gantry locating light. The use of angulation ensured a safe needle path that avoided vital structures, such as the pleura, while monitoring of the needle course and its tip on one or two CT slices. RESULTS: Twenty-two of 23 adrenal masses (96%) were successfully biopsied without complications. Fifteen (65%) of the target masses were 1.5 cm or smaller. CONCLUSION: Using an angled gantry during percutaneous biopsy is a safe and preferred method over other techniques for biopsy of adrenal masses and can be used easily for small adrenal masses. PMID- 8623624 TI - Peritoneal calcification associated with continuous ambulatory peritoneal dialysis. PMID- 8623625 TI - Breath-hold MR urography using the HASTE technique. PMID- 8623626 TI - Local staging of endometrial carcinoma: comparison of transvaginal and intraoperative sonography and gross visual inspection. AB - OBJECTIVE: The purposes of this study were to compare transvaginal sonography (TVS), intraoperative sonography (IOS), and gross visual inspection of the uterus with the histopathologic findings in patients with endometrioid adenocarcinoma, and to compare the accuracies of TVS, IOS, and gross visual inspection in staging of the tumor. SUBJECTS AND METHODS: Sixteen patients with endometriod carcinoma were prospectively evaluated with TVS and IOS. Intraoperative gross visual inspection was also performed. Gray-scale, duplex, and color Doppler findings were used to stage patients. The location and depth of myometrial invasion and the presence of cervical involvement were recorded. At gross visual inspection, only the absence or presence and the depth of myometrial invasion (< or = 50% or >50%) were recorded. The data were analyzed three ways. First, in uterine specimens with myometrial invasion, a site-by-site comparison was made among the TVS and IOS findings and the final histologic results regarding location and depth of tumor invasion. Next, to determine tumor stage, myometrial invasion was defined in two ways: (1) absent, 50% or less, or greater than 50%; and (2) 50% or less or greater than 50%. Then imaging findings, gross visual inspection, and the final histologic results were compared. RESULTS: Of the 16 uterine specimens, eight had myometrial invasion, with 13 separate sites of tumor invasion. IOS correctly identified the location and depth (+/- 10% of the histologic depth) of tumor invasion at four (31%) sites, and TVS at one (8%) site. TVS and IOS overestimated myometrial invasion due to adenomyosis, bulky intraluminal tumor, and lymphovascular invasion. When myometrial invasion was defined as absent, 50% or less, or greater than 50%, TVS was correct in 60% of cases, IOS in 56%, and gross visual inspection in 53%. When myometrial invasion was defined as 50% or less or greater than 50%, TVS was correct in 93% of cases, IOS in 81%, and gross visual inspection in 80%. CONCLUSION: TVS and IOS are inaccurate in predicting the precise location and depth of myometrial tumor invasion. However, when a less rigorous definition of invasion is used, the accuracies of TVS and IOS are comparable to gross visual inspection in staging of the tumor. PMID- 8623627 TI - High-resolution MR imaging of stage I cervical neoplasia with a dedicated transvaginal coil: MR features and correlation of imaging and pathologic findings. AB - OBJECTIVE: The purposes of this study were to assess the appearance of stage 1 neoplasia of the cervix by high-resolution MR imaging with an enveloping transvaginal receiver coil and to correlate the imaging findings with the pathologic findings. SUBJECTS AND METHODS: Fifteen patients (25-73 years old; mean, 40 years old) with clinical stage I disease were examined with a 37-mm diameter ring-design solenoid receiver coil placed around the cervix. Axial 2.5 mm contiguous slices were obtained with a field of view of 10-15 cm on a 1.0-T HPQ Vista scanner with T1-weighted (660/20 msec [TR/TE]) and T2- weighted (2500/80 msec) spin-echo sequences and dynamic gradient-echo sequences during injection of gadopentetate dimeglumine (0.1 mmol/kg). Ten patients subsequently underwent Wertheim's hysterectomy, two underwent radiotherapy, two underwent extended cone biopsy for microinvasive disease, and one underwent a punch biopsy. For seven of 10 patients who had a hysterectomy, the widths of the tumor and the residual stroma were measured at eight radial points on the transverse images and at corresponding points on the histologic specimens at 5, 10, 15, 20, and 25 mm from the ectocervix. We then compared the widths of the tumor and the stroma on images and histologic specimens at each of these 40 points. Tumor volumes were calculated from the MR imaging and pathologic data and compared. For the other three patients, detailed MR imaging-pathology correlation was not possible because of multifocal tumor distribution (two patients) and insufficient detailed pathologic data (one patient). RESULTS: Three carcinoma types were recognized. Squamous carcinoma (nine cases) was seen as a centrally expanding intermediate signal-intensity mass, whereas oat (small)-cell carcinoma (one case) and clear cell carcinoma (one case) showed a multifocal distribution. For patients who had a radical hysterectomy, we noted good agreement between the widths of the tumor and the stroma determined by MR imaging and histology. Tumor volumes were determined to be 0-28.2 cm3 by MR imaging and 0-18.4 cm3 by pathology. We observed tumor extension into the immediate parametrium in four patients by MR imaging; one of these cases was not confirmed at surgery. Parametrial extension was not underestimated by MR imaging in any case. CONCLUSION: High-resolution imaging of the cervix with a transvaginal coil provides accurate assessment of the intra- and extracervical extents of tumors in clinical stage 1 cervical neoplasia. PMID- 8623628 TI - Voiding cystourethrography in female stress incontinence. AB - OBJECTIVES: The aims of this study were to determine the sensitivity of voiding cystourethrography (VCUG) in detecting incontinence in women with a history of leakage, the positive and negative predictive values of VCUG for stress incontinence, and how often a history of leakage is accompanied by urethrocele on VCUG and to correlate urethral anatomic measurements with the presence of urethrocele and stress incontinence on VCUG. SUBJECTS AND METHODS: A total of 159 women with incontinence or voiding dysfunction were evaluated by VCUG and urodynamic study (UDS). RESULTS: VCUG detected stress incontinence in 76% of women with a specific history of stress incontinence. Of 61 women with genuine stress incontinence proven on UDS, only 37 (61%) were identified on VCUG, yielding a positive predictive value of 56%. Twenty-one of 29 women without genuine stress incontinence on UDS but with apparent stress incontinence on VCUG had detrusor instability on UDS, so that urge incontinence was falsely diagnosed as stress incontinence. VCUG had a negative predictive value of 74%. In 40 women with urethroceles, 26 had stress incontinence on VCUG. The anatomic changes in posterior urethrovesical angle, urethral descent, and urethral inclination denote urethroceles but do not correlate with the presence of stress incontinence. CONCLUSION: In the evaluation of stress incontinence, VCUG is limited because of detrusor instability producing urge incontinence and therefore resulting in false positive stress incontinence. The anatomic measurements of posterior urethrovesical angle change, urethral descent, and urethral inclination as well as the presence of urethrocele have limited ability in predicting stress incontinence. PMID- 8623629 TI - Meckel's diverticulum: imaging diagnosis. AB - Despite the availability and wide use of modern imaging techniques, the diagnosis of Meckel's diverticulum is difficult. The signs and symptoms vary from none to those of an acute abdomen or gastrointestinal bleeding. Findings on physical examination may be inconsistent because of the variable location of the diverticulum, and bleeding may occur with no appreciable physical findings. Finally, small diverticula are often concealed by overlying small-bowel loops on routine small-bowel barium studies. The purposes of this article are to review the use of available techniques for the imaging diagnosis of Meckel's diverticulum, to discuss the relative advantages and indications for the various procedures, and to emphasize the role each plays in specific clinical circumstances. The embryology, anatomy, and clinical presentation of Meckel's diverticulum are also briefly discussed. PMID- 8623630 TI - Celiac disease: how common is jejunoileal fold pattern reversal found at small bowel follow-through? AB - OBJECTIVE: Celiac disease, or nontropical sprue, is a cause of mucosal malabsorption. A decreased number of jejunal folds and an increased number of ileal folds (jejunoileal fold pattern reversal) found at small-bowel follow through have been reported for patients with celiac disease. We asked three questions regarding jejunoileal fold pattern reversal found at small-bowel follow through in patients with celiac disease. (1) How often is it present, either partially or completely? (2) How often is it associated with other findings of malabsorption? (3) How reliably can it be distinguished from the normal pattern? MATERIALS AND METHODS: Twenty-eight small-bowel follow-through examinations performed on 25 adult patients with celiac disease (confirmed by characteristic small-bowel biopsy and clinical response to a gluten-free diet) were reviewed retrospectively by two authors, who agreed by consensus on partial or complete jejunoileal fold pattern reversal and on other findings of malabsorption. Two methods were used to control for retrospective bias. (1) The prospective and retrospective readings of fold pattern reversal were compared for agreement. (2) The author who had not participated in the retrospective review was asked to distinguish, on the basis of the presence or absence of fold pattern reversal, 24 cases of celiac disease (all of which showed partial or complete fold pattern reversal on retrospective review) from 25 normal control cases (patients with diarrhea) (conformed by normal small-bowel biopsy). RESULTS: Partial or complete jejunoileal fold pattern reversal was identified retrospectively in 24 of the 28 small-bowel examinations (86%) performed on patients with celiac disease. One half lacked other findings of malabsorption. The prospective and retrospective readings of fold pattern reversal agreed in 21 of the 28 examinations (75%). Forty-four of 49 examinations (90%) were correctly identified by the third author on the basis of fold pattern reversal. CONCLUSION: In patients with celiac disease, partial or complete jejunoileal fold pattern reversal discovered at small-bowel follow-through is common, is often not associated with other findings of malabsorption, and can be reliably distinguished from the normal pattern. Identification of jejunoileal fold pattern reversal found at small-bowel follow through should prompt an appropriate clinical evaluation for celiac disease. PMID- 8623631 TI - Heterotopic ossification of midline abdominal incisions: CT and MR imaging findings. AB - OBJECTIVE: Heterotopic ossification of a midline surgical incision in a form of myositis ossificans traumatica in which osseous, cartilaginous, and, occasionally, myelogenous elements develop within an abdominal wound. When large amounts of internal ossification are present, the scar may demonstrate a complex radiologic appearance and potentially may be misinterpreted as a retained foreign body or incisional neoplastic recurrence. This report describes the CT and MR imaging findings of this entity. SUBJECTS AND METHODS: The authors retrospectively reviewed the cross-sectional imaging findings of 11 patients with ossified midline abdominal wounds. All but one of the patients were men, and the median age at diagnosis was 40 years old (range, 20-76 years old). Initial imaging was performed 7 days to 36 months after surgery (mean, 6.7 months). CT and MR imaging scans were reviewed, and lesion size, location, distance from the xiphoid, shape, and stability were assessed. Pathologic proof was obtained in one patient. RESULTS: CT and MR imaging examination in all patients showed ossified surgical scars, with the attenuation or signal intensity of the ossified components equivalent to that of the spine. Intralesional, fat-density components suggestive of marrow were present in two patients. All scars were located in the upper abdomen between the anterior abdominal fascia and the peritoneal surface, at the level of or inferior to the xiphoid process. Scars ranged in length from 0.7 to 13.4 cm (mean, 6.9 cm). Distances from the inferior tip of the xiphoid to the superior aspect of the ossified scar ranged from 0 to 4.9 cm (mean, 2.2 cm). Time from surgery to the initial postoperative demonstration of scar ossification ranged from 11 days to 36 months (mean, 6.8 months). None of the five patients who underwent preoperative CT examinations had abnormalities in the location of subsequent scar ossification. Of the nine patients with multiple postoperative examinations, scar size and appearance remained stable in six. In the remaining three patients, scar size was stable but showed progressive internal ossification. CONCLUSION: Heterotopic ossification within midline abdominal scars can be diagnosed by both CT and MR imaging examination. Recognition of the imaging appearances of such ossification should help prevent diagnostic confusion when attending postoperative patients. PMID- 8623632 TI - Value of three-dimensional spiral CT hepatic angiography. AB - OBJECTIVE: The aim of this study was to compare with conventional angiography images the diagnostic utility of three-dimensional CT hepatic angiography (CTHA) images reconstructed from spiral CT images obtained during direct injection of contrast material into hepatic arteries. MATERIALS AND METHODS: Three-dimensional CTHA images were reconstructed from spiral CTHA images obtained during direct hepatic arterial contrast material injection in 22 patients. We used maximum intensity-projection (MIP) and shaded-surface-display (SSD) techniques. We compared matched pairs of MIP CTHA images and SSD CTHA images with conventional angiograms for depiction of peripheral (segmental to subsegmental order branches) and proximal (common to proper) hepatic arteries as well as for conspicuity of the entire hepatic arterial tree in 22 patients and for conspicuity of tumors, recognition of tumor sites, and depiction of tumor-feeding arteries in 21 patients with malignant liver tumors. Our evaluations were performed in a blinded fashion by two experienced radiologists, who reached a consensus. (These radiologists also counted the number of tumors depicted by each method in the 21 patients with malignant liver tumors.) RESULTS: Depiction of peripheral and proximal hepatic arteries and conspicuity of the entire hepatic arterial tree were significantly better on conventional angiograms than on MIP CTHA images and SSD CTHA images. Conspicuity of tumors and recognition of tumor sites were significantly better on MIP CTHA images and SSD CTHA images than on conventional angiograms. Depiction of tumor-feeding arteries was significantly better on MIP CTHA images than on SSD CTHA images and conventional angiograms. For 47 malignant liver tumors, MIP CTHA images showed 40 (85%), SSD CTHA images showed 34 (72%), and conventional angiograms showed 20 (43%). CONCLUSION: MIP CTHA images showed liver tumors, tumor sites, and tumor-feeding arteries better than conventional angiograms. MIP was the preferred method for three-dimensional CTHA analysis. PMID- 8623633 TI - T2-weighted spin-echo MR imaging of the liver: breath-hold fast spin-echo versus non-breath-hold fast spin-echo images with and without fat suppression. AB - OBJECTIVE: The goal of our study was to compare a T2-weighted breath-hold fast spin-echo (BHSE) technique with T2-weighted non-breath-hold fast spin-echo techniques for imaging the liver. SUBJECTS AND METHODS: Thirty-three patients with hepatic lesions had T2-weighted BHSE images obtained in 22 sec and conventional T2-weighted non-breath-hold fast spin-echo images obtained in 3 min 12 sec with and without fat suppression. Images were analyzed quantitatively by measuring the lesion-liver contrast, spleen-liver contrast, and signal-to-noise ratios of lesions and qualitatively by evaluating the sharpness of hepatic contours, visibility of intrahepatic vessels and other segmental landmarks, and presence of artifacts. RESULTS: Quantitatively, lesion-liver contrast, spleen liver contrast, and signal-to-noise ratios obtained with the BHSE technique were inferior to those obtained with fast spin-echo techniques with and without fat suppression (11.2 +/- 7.1 versus 15.4 +/- 10.6 and 14.5 +/- 9.8, p < .001; 5.3 +/ 3.7 versus 8.7 +/- 3.5 and 7.0 +/- 3.8, p < .001; 16.2 +/- 8.2 versus 20.1 +/- 10.9 and 19.7 +/- 9.5, p < .01, respectively; Student's t test). Qualitatively, image artifacts and intrahepatic vessel depiction on BHSE images were similar to those obtained with the fast spin-echo techniques. The BHSE technique was superior to fat-suppressed fast spin-echo technique for showing hepatic contours (p < .01; Wilcoxon signed-rank test). CONCLUSION: The BHSE technique is quantitatively inferior to non-breath-hold fast spin-echo techniques. However, further studies with a surgical standard of reference are needed to compare the three techniques in terms of sensitivity. PMID- 8623634 TI - CT of focal hepatic injury due to surgical retractor. AB - OBJECTIVE: This study reports the CT appearance of changes in the liver due to retractor injury during surgery for upper gastrointestinal malignancy. SUBJECTS AND METHODS: Ten patients underwent CT examination of the abdomen before (n = 6) and after (n = 10) IV contrast enhancement 2-6 months after surgical resection of malignancy involving the gastric cardia. At each operation, a modified Weinberg's retractor was placed under the lateral segment of the left lobe of the liver to retract it and expose the esophagogastric junction. RESULTS: A sharply marginated, focal, hypodense lesion with no mass effect was shown in the lateral segment of the left lobe of the liver on unenhanced scans in six patients. Irregular enhancement was present after contrast injection in all 10 patients. All patients had a normal preoperative CT of the liver. No patient revealed clinical or laboratory evidence of liver metastasis or abscess. CONCLUSION: Focal hepatic injury due to intraoperative retraction has a typical location and CT appearance that can aid in recognition of the injury. PMID- 8623635 TI - Laparoscopic cholecystectomy with bile duct injury: percutaneous management of biliary stricture and associated complications. PMID- 8623636 TI - Avulsion fractures of the volar aspect of triquetral bone of the wrist: a subtle sign of carpal ligament injury. AB - OBJECTIVE: We report five examples of a triquetral fracture pattern not previously described to our knowledge. These fractures from the volar aspect of the triquetral bone are important because they are easily missed with conventional wrist radiographs and yet are associated with significant ligament injuries and carpal instability. MATERIALS AND METHODS: Five volar triquetral avulsion fractures occurred in young men (20-28 years old) during falls while playing sports. The initial evaluation consisted of a three-view radiographic series. Subsequently, an instability series was obtained in each case. Four patients were also evaluated by stress videofluoroscopy and arthrography, one by CT, and four by MR imaging. RESULTS: None of the five fractures was recognized on radiographs obtained in the emergency room. Each fracture was identified on an instability series and involved the radial aspect of the volar surface of the triquetral bone. All four wrists evaluated by stress videofluoroscopy showed lunatotriquetral joint instability. One also showed instability of the scapholunate joint. All four wrists evaluated by arthrography showed tears of the lunatotriquetral ligament and three had an associated tear of the scapholunate ligament. MR examination of four wrists showed a volar capsular ligament attached to the avulsion fracture fragment in each wrist. Two of the patients were treated by arthroscopic debridement and three were treated by prolonged splinting. All five patients had persistent pain and carpal instability of variable severity at least 1 year after injury. CONCLUSION: This avulsion fracture of the radial aspect of the volar triquetral bone is a subtle, easily missed sign of a significant injury of the perilunate ligaments. When this fracture is identified, we recommend further evaluation for associated ligament injury and carpal instability. PMID- 8623637 TI - Flexor tendon tears in the hand: use of MR imaging to diagnose degree of injury in a cadaver model. AB - OBJECTIVE: Treatment of flexor tendon lacerations of the finger partly depends on the degree of injury, which is difficult to determine clinically. We used a cadaver model to investigate the potential of MR imaging in evaluating these injuries. MATERIALS AND METHODS: A scalpel was drawn transversely across the volar surface of four cadaver hands, producing various flexor tendon injuries. MR imaging of each hand was performed using axial two-dimensional spin-echo and three-dimensional gradient-recalled-echo sequences. The three-dimensional data sets were interactively reformatted along the long axis of each tendon. The hands were then dissected; injury to each digit was categorized, measured, and compared with the prospective MR interpretations. RESULTS: Twelve high-grade flexor tendon tears (10 complete tears, with 1- to 14-mm separation of the torn ends, and two partial tears involving 50% or more of the total tendon cross-sectional area) and two partial tears of less than 50% of tendon area were produced; four tendons were not injured. Using MR imaging, we diagnosed 11 of the 12 high-grade lesions (those involving at least 50% of the total tendon cross-sectional area); the MR images did not show one complete tear whose separation measured 2 mm long at dissection. All intact tendons were correctly identified. We underestimated the extent of five lesions but overestimated none. Using the reformatted images, we reduced the number of errors that we would have made interpreting the transverse images alone. CONCLUSION: In this cadaver model, using MR imaging we accurately distinguished different degrees of flexor tendon tears. The potential of this technique for noninvasively diagnosing flexor tendon injury in patients awaits clinical studies. PMID- 8623638 TI - Distribution of serouslike bone marrow changes in the lower limbs of patients with anorexia nervosa: predominant involvement of the distal extremities. AB - OBJECTIVE: To assess the distribution of marrow changes in the bones of the lower limbs of patients with anorexia nervosa (AN) and to correlate the importance of the extent of marrow changes with clinical and hematologic data. MATERIALS AND METHODS: The frequency of serouslike marrow (low signal intensity on T1-weighted images and very high signal intensity on T2-weighted images) was determined with MR imaging in the bones of the feet, distal tibias, and proximal femurs in 19 patients with AN. RESULTS: Serouslike marrow was found with an increasing frequency from proximal to distal in the lower-limb bones (femur, 31%; distal phalanx of the first toe, 79%). The patients with abnormal femoral marrow (n = 6) were more severely emaciated and had lower leukocyte and neutrophil counts than the patients with normal femoral marrow (n = 13). Among these patients, the six patients with at least two involved bones in the feet had lower blood cell counts than the seven patients with fewer than two involved bones. CONCLUSION: In the lower limbs of patients with AN, marrow changes predominate in the distal aspects. This distribution pattern is the reverse of that seen in most bone marrow disorders but is similar to the normal conversion of hematopoietic to fatty marrow. The importance of the blood cell count changes is correlated with the extent of marrow changes in the limbs. PMID- 8623639 TI - Device for accurate localization of vertebrae before spinal surgery. PMID- 8623640 TI - Appendicitis in children and young adults: Doppler sonographic-pathologic correlation. AB - OBJECTIVE: The purpose of this study was to determine the following: whether the arterial supply of a normal appendix is visible with Doppler sonography; whether the physiologic vasodilatation that accompanies childhood appendicitis is visible; what Doppler patterns appear once necrosis of the appendix has occurred; what Doppler shifts are visible with chronic, recurrent appendicitis; and whether other conditions in the right lower quadrant can mimic the Doppler sonographic patterns of appendicitis. MATERIALS AND METHODS: Twenty-five fasting patients without abdominal pain or intestinal disease and 45 patients (1-25 years old; mean, 8 years old) with right lower quadrant pain and suspected appendicitis were examined sonographically, using 5- and 7-MHz linear, color, and pulsed Doppler transducers. Arterial signals were sought within the appendix and neighboring tissues, counted, and classified as sparse (1-2), moderate (3-4), or abundant (>4). The resistive index (RI) was measured. Thirty patients who underwent surgery were retained for surgical and pathologic correlation, and only they formed the appendicitis study. RESULTS: A normal appendix was found in 10 patients. Doppler shifts were sparse, and diastolic flow was low or absent (RI, 0.85-1). Acute uncomplicated appendicitis was found in 13 patients, who had abundant color Doppler signals throughout most of the appendix, with high diastolic flow (RI, 0.40-0.77; mean, 0.54). Acute necrotic appendicitis with perforation was found in 11 children, eight of whom showed no signals in the necrotic area at the tip. Few or no signals were seen in the remainder of the appendix (RI, 0.33-0.90; mean, 0.54). In two patients, signals were abundant in the tissues surrounding the appendix. Recurrent or chronic appendicitis was found in three patients, previously diagnosed as having Crohn's disease, psychosomatic illness, or nonspecific abdominal pain. The appendix had Doppler signals confined to the tip (RI, 0.63-0.83; mean, 0.75). Other diagnoses were found in three patients, in whom the appendix was not seen. There were abundant color signals in the intestinal wall and adjacent tissues in the right lower quadrant. Two patients had Crohn's disease, and the third had an unsuspected early pregnancy. The appendix was normal in all. CONCLUSION: Acute appendicitis is accompanied by inflammatory hypervascularity reflected as an increased number of color signals and higher diastolic Doppler shifts as compared with those found in normal persons. No Doppler shifts are identified in areas of appendiceal ischemia. Other acute inflammation in the right lower quadrant also produces numerous Doppler shifts with high diastolic flow, as does ovulation. Care must be taken to identify the source of these Doppler signals. The changing vascularity of healing, recurrent, and chronic appendicitis promises to further our understanding of the pathogenesis and evolution of this disease. PMID- 8623641 TI - Normal changes in the MR appearance of the spleen during early childhood. AB - OBJECTIVE: During the first year of life, the spleen undergoes histologic changes as the lymphoproliferative system matures. The purposes of this study were to describe the normal MR appearance of the spleen during infancy and early childhood and to correlate imaging findings with age-related histologic changes. MATERIALS AND METHODS: The spleen was evaluated on 28 abdominal MR studies obtained during the first 23 months of life. Splenic signal intensity was compared to that of the liver. Selected autopsy specimens were compared with the imaging results. RESULTS: The T2-weighted signal intensity of the spleen gradually increased from isointense to hypointense relative to the liver during the first week of life to moderately hyperintense to the liver by 8 months of age. T1-weighted splenic signal intensity gradually decreased. These findings corresponded to histological increases in white-pulp-red-pulp ratios. CONCLUSION: Before the lymphoid system completely matures, the T2-weighted signal intensity of the normal neonatal spleen appears decreased compared with the moderately hyperintense splenic signal seen in older children and adults. This finding should not be mistaken for disease. PMID- 8623642 TI - Mitochondrial myopathy-encephalopathy-lactic acidosis-and strokelike episodes (MELAS) syndrome: CT and MR findings in seven children. AB - OBJECTIVE: The purpose of this study was to describe the imaging characteristics of mitochondrial myopathy-encephalopathy-lactic acidosis-and strokelike episodes (MELAS) syndrome. MATERIALS AND METHODS: Twelve CT scans and 15 MR images were retrospectively reviewed in seven patients with proven MELAS syndrome. Follow-up studies were performed in all patients, and the total follow-up period ranged from 3 months to 3 years. Images were analyzed in terms of lesion distribution, enhancement pattern, presence of mass effect or atrophy, calcification, and changes on follow-up studies. RESULTS: CT and MR imaging showed multiple cortical and subcortical infarctlike lesions, which crossed vascular boundaries, and various degrees of generalized cerebral and cerebellar atrophy. Lesions were distributed in the posterior, parietal, and occipital areas in six patients, the putamen in five, the caudate nucleus in two, the thalamus in two, the frontal lobe in two, the globus pallidus in one, and the brainstem in one. Contrast enhancement was noted in two of seven cases on MR images and in three of five cases on CT scans. In the early stage, infarctlike lesions showed swelling and mass effect. Lesions resolved with or without local tissue loss in five patients, and new lesions appeared in another part of the brain in five patients. Generalized atrophy progressed in six cases and was most severe in the posterior part of the cerebral hemispheres. CONCLUSION: Our results indicate that imaging findings of multiple migrating infarctlike lesions that are not limited to a specific vascular territory, especially in the basal ganglia and posterior part of the cerebral hemisphere in children, are diagnostic of MELAS syndrome. PMID- 8623643 TI - The CT appearance of aortic transection. AB - Aortic transection is a frequently fatal traumatic injury. Patients who survive the initial event require rapid diagnosis and treatment. To this end, enhanced CT of the chest has been used to detect both mediastinal hematoma and direct vessel injury [1-6]. This essay shows the CT features associated with aortic transection, particularly the direct signs. PMID- 8623644 TI - Artifacts in chest radiographs with a third-generation computed radiography system. AB - Photostimulable phosphor computed radiography (CR) is a developing and increasingly widespread technology. The purpose of this pictorial essay is to familiarize readers with the appearance and cause of image artifacts that can occur in a third-generation computed radiographic system. Artifacts are described that relate to imaging plates, image readers, image processing, and film processing. PMID- 8623645 TI - Screening for deep venous thrombosis in asymptomatic postoperative orthopedic patients using color Doppler sonography: analysis of prevalence and risk factors. AB - OBJECTIVE: The purpose of our study was to assess the prevalence and risk factors for sonographically detectable lower extremity deep venous thrombosis (DVT) in asymptomatic patients following major orthopedic surgery. SUBJECTS AND METHODS: We performed color Doppler sonography of the lower extremities in 474 asymptomatic patients following major hip or knee surgery. We determined the prevalence of lower extremity DVT and used stepwise logistic regression to identify factors predictive of DVT. All patients received routine prophylactic measures. RESULTS: The prevalence of DVT was 7%. Laterality of surgery, age, and gender were all independent predictors of DVT (p < or = .01): the odds of having DVT were 20 times higher in the leg that was operated upon than in the leg that was not; the odds of DVT rose by a factor of 1.5 per decade of life; and the odds of DVT were 3.4 times greater in men than in women. DVT was more common in patients who had received general rather than epidural anesthesia, with borderline significance (p = .06). The length of anesthesia and the joint involved (hip or knee) were not predictive of DVT (p > .10). CONCLUSION: Despite prophylaxis, DVT is a relatively common postoperative complication in patients who undergo major orthopedic procedures. Routine screening for DVT is warranted in asymptomatic patients who have undergone hip or knee surgery, and color Doppler sonography, despite its limitations, offers a reasonably accurate noninvasive method for screening these patients. Subsets of patients who are at particular risk include the elderly, male patients, and patients who have undergone general anesthesia. The low prevalence of DVT in limbs not operated upon suggests that routine screening may be limited to evaluating the affected limbs only, thus helping to minimize the cost of screening. PMID- 8623646 TI - MR venography of the calf: value of flow-enhanced time-of-flight echoplanar imaging. AB - OBJECTIVE: Release of a tourniquet on the thigh and termination of Valsalva's maneuver result in a transient increase in venous blood flow in the leg. The purpose of this study was to determine the value of time-of-flight echoplanar imaging performed with these flow-enhancing methods to image the veins of the calf. SUBJECTS AND METHODS: Flow volumes in the femoral veins in eight volunteers were determined with a two-dimensional phase-contrast MR imaging technique before and immediately, 20, 40, and 60 sec after termination of Valsalva's maneuver and thigh vein occlusion. Subsequently, the calf veins of 11 healthy volunteers were imaged with a two-dimensional four-shot echoplanar MR imaging technique. Forty one 5-mm-thick sections were obtained over 10 sec immediately after termination of venous occlusion. Visibility of the three venous bundles was analyzed in the proximal, middle, and distal portions of the calf on the basis of a four-point scale. Finally, the calf of a single patient with documented DVT was imaged by the same MR imaging technique. RESULTS: Both Valsalva's maneuver and venous occlusion caused significant increases in venous flow (p < .05) only during the first 20 sec after termination of the maneuver and the occlusion. Venous occlusion had a significantly greater effect than did Valsalva's maneuver (p < .05). Flow augmentation by venous occlusion of the thigh improved calf vein visualization with time-of-flight echoplanar MR imaging (p < .0001). Of the 99 calf vein segments examined, 93 were clearly seen. Thrombi were seen in the patient study. CONCLUSION: Multishot time-of-flight echoplanar imaging can exploit the short-lived effect of mechanical flow-enhancing measures, resulting in good visualization of calf veins. The true diagnostic impact of this technique needs to be evaluated in a patient study. PMID- 8623647 TI - Automated bone editing algorithm for CT angiography: preliminary results. PMID- 8623648 TI - Percutaneous closure of an arteriovenous iliac fistula with a Cragg endoluminal graft. PMID- 8623649 TI - Unusual manifestations of head trauma. AB - Head injury is the leading cause of death in young adults. More than 2 million head injuries occur each year in the United States. Accurate and rapid diagnosis is imperative for the successful management of the trauma patient. The radiologic findings of common manifestations of head trauma have been well described; however, the patient who presents in the post-traumatic period with atypical radiographic findings is at risk for misdiagnosis and delay in treatment. In this essay, we illustrate some unusual findings in head injury that may complicate diagnosis. PMID- 8623650 TI - Peroneal nerve ganglion cyst. PMID- 8623651 TI - Neuroimaging findings in patients on immunosuppressive therapy: experience with tacrolimus toxicity. AB - OBJECTIVE: The purpose of this study is to describe the neuroimaging (CT and MR imaging) findings in liver transplant patients who develop severe neurologic side effects during immunosuppressive therapy with tacrolimus and to correlate these findings with clinical signs and tacrolimus levels in blood. SUBJECTS AND METHODS: Brain CT and/or MR imaging was performed on six patients who developed neurologic symptoms while receiving tacrolimus in the post-transplant period. All patients were evaluated by the neurology staff, and imaging studies were independently interpreted by three neuroradiologists. Trough tacrolimus levels in blood were measured with the IMX immunoassay and were correlated with neurologic symptoms and imaging findings. RESULTS: Imaging abnormalities were observed in five of six patients during the course of their neurologic illnesses. For each patient, neurologic symptoms began when the tacrolimus level in blood was at a peak, exceeding the therapeutic limit in all but one case. In five patients, neurologic symptoms eventually resolved after the tacrolimus dose was reduced or after the drug was stopped. Multifocal low attenuation of white matter was the predominant finding seen on CT images, and matching hyperintense white matter foci were observed on long-TR MR images. In three patients, clinical recovery was accompanied by reversal of the white matter abnormalities seen on CT and MR images. CONCLUSION: Immunosuppressive therapy with tacrolimus may produce neurologic side effects that are associated with brain CT and MR imaging abnormalities. Resolution of symptoms and reversal of imaging findings occur when the tacrolimus dose is reduced. PMID- 8623652 TI - Improved tissue characterization in adrenoleukodystrophy using magnetization transfer imaging. AB - OBJECTIVE: We performed this study to evaluate the usefulness of magnetization transfer (MT) imaging techniques and magnetization transfer ratio (MTR) measurements in assessing patients with adrenoleukodystrophy (ALD). SUBJECTS AND METHODS: Twelve patients who encompassed the range of ALD phenotypes were evaluated prospectively with MR and MT imaging (including digital subtraction images). The MT images and spin-echo MR images were compared by two staff neuroradiologists and a staff neurologist for extent of disease, different zones of involvement, and the presence of enhancement. MTR measurements were obtained from affected and unaffected regions of the brain. RESULTS: Eight of the 12 patients exhibited gadolinium enhancement. In seven of these patients, the MT subtraction images showed two zones of abnormal signal within the affected white matter. Their MTR measurements were classified as zone 1 (mean MTR, 35%; SD, 6%) and zone 2 (mean MTR, 20%; SD, 6%). Three of the other patients showed a single zone of involvement corresponding to the affected white matter on the standard spin-echo MR images. Their MTR measurements were similar to that of zone 1. The remaining two patients had no demonstrable signal abnormalities on any of the images. We noted no difference in any of the 12 subjects in the extent of white matter involvement on any of the imaging techniques. The MTR measurements in the unaffected white matter (mean MTR, 46%; SD, 5%) approximated established MTRs in white matter of normal volunteers. CONCLUSION: MT imaging and MTR measurements are helpful clinical techniques that may improve the evaluation of patients with ALD, particularly in the proposed ability of these techniques to divide the affected white matter into different zones that correspond to specific pathologic areas. These techniques can help the understanding of pathogenetic mechanisms and the evaluation of therapies. PMID- 8623653 TI - Primary craniosynostosis: imaging features. AB - In 1851, Virchow introduced the term craniosynostosis to describe a variety of abnormalities in calvarial growth. These skull deformities are usually apparent in infancy. When an abnormal calvarial configuration is detected, a radiologic evaluation is necessary to characterize the deformity and to guide the corrective surgical procedure. Affected children are believed to have an improved outcome when diagnosis and surgical intervention occur at an early age. CT with three dimensional reconstruction optimally evaluates the presence and degree of sutural involvement and assesses associated facial and intracranial abnormalities. This pictorial essay illustrates the imaging findings, nomenclature, and associated abnormalities of the various types of primary craniosynostosis. PMID- 8623654 TI - Endovascular treatment of a kitchen knife pseudoaneurysm of the cervical internal carotid artery. PMID- 8623655 TI - Preoperative MR imaging in hyperparathyroidism: results and factors affecting parathyroid detection. AB - OBJECTIVE: To determine the sensitivity of MR imaging for the detection of abnormal parathyroid glands in patients with biochemical evidence of hyperparathyroidism and to identify the factors affecting detection. SUBJECTS AND METHODS: Between 1985, 82 patients with biochemical proof of hyperparathyroidism were referred for MR imaging of the parathyroid glands prior to surgery. Axial T1 (600/20 [TR/TE]) and T2-weighted (2500/40, 80) spin-echo images were obtained using an anterior neck surface coil. The interpretation of the MR image was compared with the findings at surgery and also correlated with gland histology, volume, and weight. Cases in which a false-positive or false-negative diagnosis was made were reviewed to determine the factors affecting detection. RESULTS: MR imaging detected 71 of 92 (77%) surgically proven abnormal glands (sensitivity, 77%; 95% confidence interval (CI), 68-86%) and misdiagnosed five (1.6%) of 314 normal glands as abnormal. There was no difference in the detection of enlarged glands in patients presenting for the first time (n = 71) (sensitivity, 77%; 95% CI, 66-86%) compared with patients with recurrent hyperparathyroidism (n = 12) (sensitivity, 77%; 95% CI, 46-95%). There was no significant difference in the detection of adenomas (sensitivity, 77%; 95% CI, 65-86%) compared with hyperplasia (sensitivity, 71%; 95% CI, 42-92%). Of five patients with ectopic parathyroid glands (1.6%), four had had previous surgery. All five glands were successfully located (three mediastinal, two in the neck). Factors contributing to a false-negative MR imaging diagnosis included small gland size and thyroid disease. Four of five false-positive diagnoses were due to enlarged lymph nodes being mistaken for parathyroid glands. CONCLUSIONS: MR imaging is an accurate technique for investigation of hyperparathyroidism. Pitfalls include low sensitivity for the identification of small glands, misinterpretation of enlarged lymph nodes as parathyroid adenomas, and misinterpretation because of thyroid disease. MR imaging is particularly useful in the investigation of patients who remain hypercalcemic following initial surgery. PMID- 8623657 TI - With helical CT, is nonionic contrast a better choice than ionic contrast for rapid and large IV bolus injections? PMID- 8623656 TI - Tension pneumocephalus: a complication of invasive ossifying fibroma of the paranasal sinuses. PMID- 8623658 TI - When should we use dual helical CT of the liver? PMID- 8623659 TI - What radiographic views constitute acute abdominal series? PMID- 8623660 TI - At what age should screening mammography no longer be performed? PMID- 8623661 TI - Thyroid sonography in the workup of a questionably palpable thyroid lesion. PMID- 8623662 TI - Bowel preparation for excretory urography. PMID- 8623663 TI - Bowel preparation for excretory urography. PMID- 8623664 TI - Relationship between retroperitoneal compartments and pleural sinus. PMID- 8623665 TI - Defending malpractice actions involving barium enema examinations. PMID- 8623666 TI - Voiding cystourethrography in boys: the presence of the catheter does not obscure the diagnosis of posterior urethral valves but prevents estimation of the adequacy of transurethral fulguration. PMID- 8623667 TI - Osteomyelitis and septic arthritis in children: guidelines for the use of imaging. PMID- 8623668 TI - Sonography of pedunculated exogastric leiomyoblastoma. PMID- 8623669 TI - Blastomycosis of the breast. PMID- 8623671 TI - "Eraseroma" in a nasal cavity. PMID- 8623670 TI - Hypoechoic periportal encasement in Chediak-Higashi syndrome. PMID- 8623672 TI - Diagnosis of non-Hodgkin's lymphoma using flow cytometry. PMID- 8623673 TI - Spontaneous regression of postangioplasty restenosis in common iliac artery. PMID- 8623674 TI - Life-threatening anaphylactoid reaction after iv injection of gadoterate meglumine. PMID- 8623675 TI - Regurgitated giant fibrovascular polyp of the esophagus. PMID- 8623676 TI - Clear cell tumor of the lung. PMID- 8623677 TI - Paying heed to the treatment of tuberculosis. PMID- 8623678 TI - Aminoglycosides: once-daily dosing regimen. PMID- 8623679 TI - Family medicine faculty recruitment crisis of the '90s. PMID- 8623680 TI - Physicians' fears about treating anxiety disorders. PMID- 8623681 TI - The 'moving target syndrome' in publication endeavors. PMID- 8623682 TI - Domestic violence in men. PMID- 8623683 TI - Paper mill workers and chronic extrinsic allergic alveolitis. PMID- 8623684 TI - Relevance of race or ethnic group in case reports. PMID- 8623685 TI - Tobacco use and dental caries. PMID- 8623686 TI - Low-dose antidepressant therapy for mild depression. PMID- 8623687 TI - Outpatient management of tuberculosis. AB - All patients with a positive reaction to the tuberculin skin test should be evaluated for weight loss, night sweats, fever, chronic cough and other signs of active tuberculosis. Chest radiographs should also be obtained in these patients. All converters receiving isoniazid should be examined monthly for signs of hepatitis and neurotoxicity. Liver function tests should be obtained at initiation of isoniazid therapy and again during the first two to four months in patients age 35 or older or with a history of alcoholism, liver disease or intravenous drug use. All cases of active tuberculosis should be reported to the local public health department. Most patients with active tuberculosis should receive isoniazid, rifampin, ethambutol and pyrazinamide daily for two weeks, followed by directly observed administration twice weekly for six additional weeks. If sputum cultures show no resistance after two months of therapy, pyrazinamide and ethambutol may be discontinued, with isoniazid and rifampin taken for an additional 16 weeks. Patients with human immunodeficiency virus infection or acquired immunodeficiency syndrome should receive the initial treatment for at least nine to 12 months, and isoniazid and rifampin for at least six months after culture conversion has occurred. PMID- 8623688 TI - Preeclampsia and hypertensive disorders of pregnancy. AB - Hypertensive conditions encountered during pregnancy are classified as preeclampsia, transient hypertension and chronic hypertension. The pathophysiology, consequences and management of these disorders differ, but their clinical presentations overlap substantially. Preeclampsia is a syndrome of the second half of pregnancy, characterized by hypertension, edema and proteinuria, but all three findings are not required to make the diagnosis. Preeclampsia can progress unpredictably to a variety of crises, including eclamptic seizures, and contributes significantly to maternal and perinatal mortality. Management consists of prompt delivery for a mature fetus. Management of preeclampsia at earlier stages of gestation requires balancing the risks of immediate delivery of an immature fetus against the risks to both mother and child of a complication of preeclampsia. Transient hypertension is a clinically benign condition characterized by isolated high blood pressure in late pregnancy; its significance lies in the difficulty of distinguishing it from early preeclampsia. Chronic hypertension is a risk factor for intrauterine growth restriction and intrauterine fetal demise, as well as for preeclampsia. The management strategy consists of control of maternal blood pressure, ongoing antepartum assessment of fetal well-being and surveillance for superimposed preeclampsia. PMID- 8623689 TI - The athlete with type I diabetes: managing insulin, diet and exercise. AB - The numerous benefits of exercise for patients with insulin-dependent (type I) diabetes mellitus include an increase in insulin sensitivity and a reduction of blood glucose levels. However, competitive athletes with type I diabetes must be careful when planning to exercise. The most common potential complications in these athletes include exercise-induced hypoglycemia or hyperglycemia and postexercise hypoglycemia due to increased insulin sensitivity. With proper modifications of insulin dose and diet, plus careful blood glucose monitoring, athletes with type I diabetes can exercise safely and regularly. To prevent hypoglycemia, the insulin dose may need to be reduced by 30 or 50 percent before exercise. Avoiding regular insulin at bedtime and reducing the evening insulin dose may help prevent nocturnal hypoglycemia after exercise. A tailored diet should reduce the chance of hypoglycemia. PMID- 8623690 TI - Cutaneous manifestations of selected rheumatologic diseases. AB - Systemic lupus erythematosus, chronic cutaneous lupus erythematosus and subacute and acute cutaneous lupus erythematosus are associated with distinct cutaneous manifestations, and each disease has a particular clinical course and prognosis. Dermatomyositis, an immune-mediated disorder of unknown etiology that is often associated with a malignancy, consists of an inflammatory myopathy combined with characteristic cutaneous findings. Some patients with dermatomyositis also have clinical features that overlap with those of systemic lupus erythematosus or scleroderma. Manifestations of scleroderma range from cutaneous involvement alone to multisystem internal disease. Morphea, progressive systemic sclerosis, eosinophilic fascitis, eosinophilia myalgia syndrome and mixed connective tissue disease are all within the spectrum of scleroderma. PMID- 8623691 TI - Shoulder pain: the role of diagnostic injections. AB - Many different shoulder disorders cause similar symptoms and pain patterns. An accurate diagnosis can generally be made by obtaining a detailed history, performing a comprehensive, directed physical examination and obtaining selected radiographs. Occasionally, shoulder injections can be of great assistance in establishing a clear diagnosis and providing relief of symptoms. Subacromial space injection, acromioclavicular joint injection, intra-articular injection and injection of the biceps tendon are helpful in identifying such disorders as subacromial bursitis, acromioclavicular arthritis, injury to the glenohumeral joint and bicipital tendinitis. PMID- 8623692 TI - Breaking the barriers to childhood immunization. AB - Although about 98 percent of children have received their basic series of immunizations at the time of school entry, only 67 percent of two-year-old children are appropriately immunized. Parental, provider, economic and system barriers to immunization must be overcome if the United States is to achieve the objective that 90 percent of two-year-old children receive the basic vaccination series against the major preventable childhood diseases by 1996. Parents must be educated about the importance of vaccines and the hazards of the diseases they prevent, and they must be given proper information about vaccine side effects and contraindications. Physicians should take advantage of all appropriate patient contacts, including acute office visits for minor illnesses, to keep children's immunizations current. Audits of office records, as well as tracking or reminder systems, improve vaccination rates. The Vaccines for Children Program is intended to remove cost as a barrier to the immunization of some children. The single immunization schedule endorsed by the American Academy of Family Physicians, the American Academy of Pediatrics and the Advisory Committee on Immunization Practices may alleviate confusion about the appropriate time to administer different vaccines. PMID- 8623693 TI - Chemically dependent patients in recovery: roles for the family physician. AB - Successful recovery from chemical dependency usually requires at least two years of abstinence and has complex medical, psychologic, and social aspects. In addition to the multiple medical problems associated with substance abuse, patients are at risk for conditions resulting from impaired judgment during addiction, including sexually transmitted diseases. In addition, therapeutic doses of ongoing medications (e.g., antihypertensive agents) may need to be reestablished because of the physiologic changes that occur during recovery. Patient attitudes toward physicians and medications may range from overuse to avoidance, depending on personal experiences and vulnerabilities. These factors may also influence the efforts of physicians, family members and support groups who are trying to help recovering patients. Twelve-step programs, supportive medical follow-up and the judicious use of medications in specific circumstances can greatly increase the probability of successful and sustained recovery for previously addicted patients. PMID- 8623694 TI - Adolescent chest pain. AB - Chest pain in adolescents rarely indicates serious organic pathology. Musculoskeletal disorders are the most common identifiable causes; psychogenic, respiratory and gastrointestinal disorders are also part of the differential diagnosis. Although cardiac disease is part of the differential diagnosis, it is very rare in this age group. Routine tests usually do not help establish a diagnosis and should be ordered only if indicated on the basis of the history and findings of the physical examination. Although most cases of chest pain are not caused by serious disease, the complaint should not be casually dismissed. An important role for primary care physicians is to provide support and adequate follow-up, because adolescent chest pain is often chronic. PMID- 8623695 TI - Necrotizing fasciitis. AB - Necrotizing fasciitis is a life-threatening, invasive soft-tissue infection that is characterized by widespread, rapidly developing necrosis of the subcutaneous tissue and fascia. It is more likely to occur in patients with a compromised immune system. In type I necrotizing fasciitis, anaerobes and gram-negative bacteria are predominant; in the type II form, the bacterial etiology is group A beta-hemolytic streptococci. The diagnosis must be made on the basis of clinical grounds and is characterized by rapidly developing, painful erythema that progresses to bullous formation and gangrenous necrosis. PMID- 8623696 TI - Fibromyalgia syndrome: a review. AB - Fibromyalgia syndrome includes symptoms of widespread, chronic musculoskeletal aching and stiffness and soft tissue tender points. It is frequently accompanied by fatigue and sleep disturbance. Fibromyalgia is more common in women than in men, and it occurs at a mean age of 49 years. Differential diagnosis includes myofascial pain syndrome and chronic fatigue syndrome. Fibromyalgia is a multifactorial problem and no universal treatment guidelines apply to all cases. Pharmacologic therapy may incude tricyclic antidepressants. In addition to commonly used pharmacologic therapies, patient education, reassurance and an exercise program can each play an important role in relieving the symptoms associated with this common musculoskeletal syndrome. PMID- 8623697 TI - Sunscreens: the ounce of prevention. AB - Sun exposure is linked to visible signs of skin aging, skin cancer, photodermatoses, exacerbation of systemic disease and photoallergic, as well as phototoxic, drug eruptions. Sunscreens vary considerably in their ability to protect patients from exposure to ultraviolet light and its effects. Inappropriate choice and use of sunscreen products can lead to worse problems than using no sunscreen at all. Controversies about sunscreen include adequate level of sun protection factor, appropriate age of users, and whether use of sunscreen products can prevent skin cancer. Instructing patients in how to select and use sunscreen can help prevent or mitigate a variety of cutaneous and systemic diseases. PMID- 8623698 TI - Diagnostic imaging of patients with acute scrotal pain. AB - Common causes of acute scrotal pain include testicular torsion, epididymo orchitis and trauma. Epididymitis in adult men is typically associated with a history of urinary tract infection or prostatitis. Testicular torsion typically presents in young adults with a sudden onset of severe scrotal pain and, frequently, a history of recurrent episodes that have spontaneously resolved. With scrotal trauma, ultrasound may demonstrate testicular fracture, hematoceles and areas of hemorrhage or testicular infarction. Since both epididymitis and testicular torsion present with scrotal pain and swelling, and may be accompanied by fever and pyuria, Doppler ultrasound or radionuclide imaging may be necessary to make the diagnosis. In acute testicular torsion, color Doppler ultrasound shows absent flow to the epididymis and testis, while nuclear imaging shows central photon-deficient areas in the ischemic hemiscrotum. In epididymo orchitis, color Doppler ultrasound shows increased flow to the epididymis and testis, while nuclear imaging shows increased perfusion of the affected testis and hemiscrotum. PMID- 8623699 TI - Preserving confidentiality in occupational medical practice. AB - The United States does not have a uniform law regarding medical confidentiality. However, codes of conduct from the American Medical Association, the American College of Occupational and Environmental Medicine and the International Commission of Occupational Health, as well as relevant parts of the Americans with Disabilities Act and the Occupational Safety and Health Act of 1970, can help physicians make informed decisions about requests for medical information. Such activities include preplacement and fitness-for-work evaluations medical surveillance examinations (including drug testing) and evaluation of symptoms and treatment of injuries that may be work-related. The patient should be the one to decide whether and when to release medical records to employers, unless overruled by public health risks or laws. PMID- 8623700 TI - Recreational scuba diving injuries. AB - Because of the increasing popularity of recreational scuba diving, primary care physicians should be familiar with common diving injuries. One form of barotrauma, middle ear squeeze, is the most common diving injury. Other important diving injuries include inner ear barotrauma and pulmonary barotrauma. Arterial gas embolism, a potentially life-threatening form of pulmonary barotrauma, requires hyperbaric treatment. Decompression sickness is the result of bubble formation in body tissue. Symptoms of decompression sickness range from joint pain to neurologic or pulmonary problems. Recompression is the mainstay of treatment. PMID- 8623701 TI - Gamekeeper's thumb: ulnar collateral ligament injury. AB - Injury to the ulnar collateral ligament of the thumb, once a common chronic occupational injury occurring in British gamekeepers, is now most frequently an acute sports-related injury in skiers, football players and other athletes. Differentiation of complete versus partial tears of the ligament is crucial because the treatment for complete tears is surgical. Radiographic evaluation, while important, has a limited role. Partial ligamentous tears, or those associated with uncomplicated avulsion fractures of the proximal phalanx, can be adequately treated by the family physician using simple shortarm thumb spica casting. PMID- 8623702 TI - The evolution of aminoglycoside therapy: a single daily dose. AB - After five decades of clinical use, aminoglycosides continue to be important antibiotics in the management of gram-negative infections. However, antimicrobial therapy with gentamicin, tobramycin, amikacin and netilmicin has been complicated by the risk of nephrotoxicity and ototoxicity. Considerable research has been conducted to determine the optimal aminoglycoside dosing method that will produce maximum efficacy with minimal toxicity. Experimental data and clinical experience suggest that aminoglycoside regimens modified from standard dosing (every eight to 12 hours) to once-daily dosing may provide improved clinical outcomes, with reduced toxicity and cost of therapy. Gentamicin, tobramycin and netilmicin may be administered in single daily dosages ranging from 4 to 7 mg per kg, and amikacin may be given in a dosage of 15 to 20 mg per kg once daily. PMID- 8623703 TI - Significant FDA approvals in 1995. PMID- 8623704 TI - NIH releases statement on behavioral and relaxation approaches for chronic pain and insomnia. PMID- 8623705 TI - American Psychiatric Association issues guidelines on treatment of substance use disorders. PMID- 8623706 TI - ACOG Technical Bulletin summarizes current approaches for induction of labor. PMID- 8623707 TI - Genetic testing: are family physicians ready? PMID- 8623708 TI - New guidelines on treating hypertension in diabetics. PMID- 8623709 TI - Cautious history-taking with potentially violent patients. PMID- 8623710 TI - Cryosurgery and podophyllum in combination for condylomata. PMID- 8623711 TI - Plantar fasciitis, posterior night splints and activity during recovery. PMID- 8623713 TI - Etiology of cat-scratch disease. PMID- 8623712 TI - Plantar fasciitis, posterior night splints and activity during recovery. PMID- 8623714 TI - Plantar fasciitis, posterior night splints and activity during recovery. PMID- 8623715 TI - Treating patients from other cultures. PMID- 8623716 TI - Common elbow fractures in children. AB - Approximately 75 percent of all fractures sustained by children occur in the upper extremities and frequently occur during a fall onto an outstretched hand. The majority of these injuries involve the wrist and forearm, but the elbow alone accounts for approximately 10 percent of all fractures in children. Elbow fractures in children are challenging because of the abundance of unossified cartilage and the high potential for limb-threatening damage to neurovascular structures. Common types of elbow fractures include supracondylar, lateral condylar, medial epicondylar, radial neck and transphyseal fractures. PMID- 8623717 TI - Endocarditis in injection drug users. AB - Injection drug use is an important risk factor for endocarditis. The clinical manifestations of endocarditis associated with injection drug use differ from those in person who do not use drugs. Endocarditis in drug users more often affects the right side of the heart and presents with fever and pulmonary emboli rather than left-sided emboli. Blood cultures and echocardiography are the mainstay of diagnosis; these tests are particularly helpful in identification of endocarditis associated with injection drug use because of the high frequency of right-sided valvular involvement and the low incidence of culture-negative endocarditis in this population. Since staphylococcal species are the dominant causative organism, penicillin and an aminoglycoside are the treatments of choice. Injection drug users with left-sided endocarditis or with symptomatic human immunodeficiency virus infection have poor prognoses. PMID- 8623718 TI - Diagnosis and management of gestational diabetes. AB - Gestational diabetes affects 3 to 6 percent of all pregnancies. Proper screening, diagnosis and management of this condition can reduce both maternal and neonatal morbidity. Family physicians can readily manage most patients with gestational diabetes by prescribing an appropriate diet, encouraging regular exercise and providing careful follow-up with regular glucose monitoring for the remainder of the pregnancy. Up to 90 percent of these patients do not need more intensive therapy. Since patients with gestational diabetes are at high risk for developing type II (non-insulin-dependent) diabetes mellitus later in life, they should be counseled and tested during the postpartum period and encouraged to lose weight (if necessary) and follow a diet to minimize the development of glucose intolerance. PMID- 8623719 TI - Transvaginal ultrasonography in nonpregnant and postmenopausal women. AB - Compared with transabdominal pelvic ultrasonography, transvaginal ultrasonography provides improved resolution for visualization of female pelvic organs, with less artifact. Transvaginal ultrasonography has superseded the transabdominal approach in many situations. Identification of uterine and adnexal masses can be facilitated with use of transvaginal ultrasonography. Transvaginal ultrasonography is also useful in the evaluation of pelvic pain and suspected pelvic inflammatory disease. Postmenopausal patients with vaginal bleeding or a palpable ovary can be triaged on the basis of transvaginal ultrasonographic findings, avoiding invasive diagnostic procedures in selected patients. Screening average-risk patients for ovarian cancer is not an accepted indication for transvaginal ultrasonography at this time. PMID- 8623720 TI - Clinical applications of genetic testing: implications for the family physician. AB - Genetic testing may be applied in a variety of clinical situations, including preconception counseling, prenatal diagnosis and postnatal determination of genetic predisposition to disease. The family physician needs to become familiar with the full range of genetic testing possibilities in all phases of the human life cycle. Cystic fibrosis, Huntington's disease and cancer are three diseases for which genetic testing has become a reality, and they serve to illustrate the clinical and ethical dilemmas that arise with this type of testing. PMID- 8623721 TI - Tuberculosis infection and disease in children. AB - Children with primary tuberculosis are more likely than adults to be asymptomatic and to have extrapulmonary disease. If tuberculosis infection or disease is suspected in a child, it is imperative to obtain a detailed medical, family and social history, which must include an investigation of the child's contact with an infectious adult. Because it is difficult to isolate a Mycobacterium tuberculosis strain from children, the treatment regimen must often be based on the susceptibility pattern of the M. tuberculosis strain in the infecting adult. Patients with fully susceptible pulmonary tuberculosis are treated with isoniazid, rifampin and pyrazinamide for two months and then with isoniazid and rifampin for four more months. If infection with a drug-resistant organism is suspected, other medications should be used for treatment. When treating a child with possible tuberculosis disease, the primary care physician generally should consult with a pediatric tuberculosis specialist. Identifying the adult source of a child's tuberculous infection and marshalling the resources needed for treatment are best done in conjunction with the local health department. PMID- 8623722 TI - Evaluation and treatment of erectile dysfunction. AB - Erectile dysfunction has an organic etiology in most cases, with vascular disease the single most common cause. In addition to a thorough history and physical examination, clinical evaluation may include hormonal assessment, diabetes screening, nocturnal tumescence testing and color Doppler flow studies of the penile vasculature. Therapy for erectile dysfunction has progressed rapidly during the past decade, with alternatives including hormone supplements, vacuum constriction devices, penile self-injection therapy and penile prostheses. The majority of patients and their partners report satisfactory results with these treatments. PMID- 8623723 TI - Evaluating asymptomatic patients with abnormal liver function test results. AB - Asymptomatic patients with abnormal results on liver function test pose a diagnostic challenge. In general, determinations of routinely ordered tests of liver function are neither sensitive nor specific for liver disease. Fatty liver, alcohol-related liver damage and chronic viral hepatitis are the most common causes of abnormal liver function test results in asymptomatic patients. Causes of asymptomatic liver disease include hemochromatosis, Wilson's disease, drug toxicity, chronic autoimmune hepatitis, biliary cirrhosis, sclerosing cholangitis, alpha1-antitrypsin deficiency and sarcoidosis. The most efficient screening tests for liver damage are alanine transaminase, alkaline phosphatase and bilirubin. Repeat testing when results are abnormal, and use of ancillary tests, such as creatine phosphokinase or gamma-glutamyl-transferase, may confirm liver damage. Imaging studies help exclude biliary obstruction or neoplasm. Treatable illnesses should be ruled out. Three to six months of observation for progressive symptoms and liver dysfunction may follow. After the period of observation, further laboratory tests, a diagnostic liver biopsy and/or referral to gastroenterologist may be needed. PMID- 8623724 TI - Management of depression in patients with coexisting medical illness. AB - Depressive illness is common in the general population, with a prevalence of 5 percent. However, 10 to 15 percent of any general medical population has clinically significant depression; in patients with selected chronic illnesses, prevalence rates between 25 to 50 percent are noted. In patients with coexisting medical illness, the diagnosis of depression requires differentiating symptoms of the medical illness from symptoms of the comorbid depression. The Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) can be helpful in this endeavor. An understanding of the effect of particular medications on neurotransmitters is required and can guide the clinician in selecting therapeutic agents that have a low incidence of side effects and toxicity. PMID- 8623725 TI - American Psychiatric Association releases guideline on the psychiatric evaluation of adults. PMID- 8623726 TI - ACC/AHA collaborate on guidelines for the evaluation and management of heart failure. PMID- 8623727 TI - Indinavir approved for HIV infection. PMID- 8623728 TI - Conditions of the eye. Recommended core educational guidelines for family practice residents. American Academy of Family Physicians. PMID- 8623729 TI - A randomized factorial trial of reperfusion strategies and aspirin dosing in acute myocardial infarction. The DUCCS-II Investigators. AB - The focus of new research efforts to improve the morbidity and mortality associated with acute myocardial infarction (AMI) has turned to adjuvant agents that show promise of improving outcomes following coronary thrombolysis. We enrolled 162 patients with AMI in a randomized trial comparing front-loaded tissue-plasminogen activator (t-PA) plus weight-adjusted heparin with anisoylated plasminogen streptokinase activator complex (APSAC) without heparin as well as standard-dose (325 mg) and low-dose (81 mg) aspirin. The primary end point was an in-hospital morbidity profile; secondary end points were clinical and angiographic potency and hemorrhagic events. Selected sites performed an electrocardiographic substudy to determine the time to 50% ST-segment recovery and the time to steady state. Although the trial was terminated when the Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries-I trial showed that t-PA had a significant mortality advantage over streptokinase, important trends were evident. Patients given t-PA and heparin were better anticoagulated (p = 0.001), yet AP-SAC-treated patients had more bleeding complications. The primary end point favored t-PA (25.4% vs 31.3%), and the secondary end points were similar in both groups. In the electrocardiographic substudy, the t-PA group achieved both 50% ST-segment recovery and steady-state recovery sooner than the APSAC group. Patients taking low-dose aspirin had lower in-hospital mortality and less recurrent ischemia but more strokes than the standard-dose aspirin group. Thus, this trial demonstrated trends favoring front loaded t-PA with weight-adjusted heparin over APSAC without heparin in the treatment of AMI. The use of low-dose aspirin did not appear to impose a loss of protection from adverse events, nor did standard-dose aspirin increase serious bleeding. PMID- 8623730 TI - Detection and significance of myocardial ischemia in women versus men within six months of acute myocardial infarction or unstable angina. The Multicenter Myocardial Ischemia Research Group. AB - Ischemia detection after an acute coronary event predicts subsequent cardiac events. However, gender-related aspects in the prevalence and prognostic significance of ischemia detection after an acute coronary event have not been reported. Noninvasive tests, which included resting 12-lead electrocardiogram (ECG), 24-hour ambulatory ECG, exercise ECG, and thallium-201 stress scintigraphy were performed in 936 stable patients (224 women and 712 men) 1 to 6 months (average 2.7) after an acute coronary event (i.e., myocardial infarction or unstable angina). Primary end points during an average follow-up of 23 months included cardiac death, nonfatal myocardial infarction, and unstable angina, while restricted end points included the first 2. Ischemia detection was significantly less frequent among women than among men on 24-hour ambulatory ECG, exercise ECG, and thallium-201 stress scintigraphy. Primary end points occurred in 19.2% of women and in 19% of men, and restricted end points occurred in 5.8% of women versus 8%. of men (p = NS). Cox analyses revealed that gender and its interaction with each of the ischemia tests did not contribute to the prediction of the primary or restricted end points. We conclude that in stable patients 1 to 6 months after an acute coronary event, ischemia detection by noninvasive tests was significantly less prevalent in women than in men. However, subsequent cardiac event rates in women were similar to those observed in men, and there was no gender-ischemic detection interaction regarding subsequent events. PMID- 8623731 TI - Angiographic correlates of lesion relevance and suitability for percutaneous transluminal coronary angioplasty and coronary artery bypass grafting in the Bypass Angioplasty Revascularization Investigation study (BARI). AB - The Bypass Angioplasty Revascularization Investigation (BARI) randomized 1,829 patients to percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG). Clinical site angiographers categorized lesions of > or = 50% diameter stenosis (n = 4,977) as clinically significant (86.4%) or nonsignificant (13.6%), and as favorable or nonfavorable for PTCA or CABG. More lesions were considered favorable for revascularization by CABG than by PTCA (91.5% vs 78.4%; p <0.001), particularly in the subgroup of 99% to 100% lesions (77.6% for CABG vs 21.9% for PTCA, p <0.001). Lesion features, characterized by the BARI core laboratory, were correlated with clinical site angiographers' assessment of clinical importance and suitability for PTCA or CABG. By multivariate analysis, positive predictors of clinical importance for 50% to 95% stenoses were greater stenosis severity, more jeopardized myocardium, larger reference diameter, and proximal vessel location. For 99% to 100% occlusions, predictors were shorter duration of occlusion and more jeopardized myocardium. PTCA suitability for 50% to 95% stenoses was inversely related to lesion length, ostial location, location on a bend, difficult access, and age, and was directly associated with greater Thrombolysis in Myocardial Infarction (TIMI) trial flow rate and more jeopardized myocardium. Predictors of PTCA suitability for 99% to 100% lesions were a lower American College of Cardiology/American Heart Association class and higher TIMI grade. Predictors for 50% to 95% stenoses were more jeopardized myocardium, larger reference diameter, and more proximal vessel location, and for 99% to 100% occlusions, more jeopardized myocardium and shorter duration of occlusion. Suitability for PTCA depended on lesion potency (<99%) and multiple morphologic characteristics that contrasted with the few angiographic features that adversely affect CABG suitability. PMID- 8623732 TI - Performance standards and edge detection with computerized quantitative coronary arteriography. The Lovastatin Restenosis Trial Group. AB - Quantitative coronary angiography (QCA) has become an important tool for evaluating coronary angiograms. Many methodologic factors, such as the choice of frame to analyze, the selection of the "normal," segment and the method of edge detection used may affect the results of QCA. The sequential steps in performing QCA, including a comparison of visual and automated edge-detection methodologies, were evaluated using 12 precision-drilled phantoms and 20 patient films. Normal diameter, minimal lumen diameter, and diameter stenosis were measured. In the phantom studies, the measurements from both visual and automated systems correlated well with the true measurements of the phantoms and between systems (all r values >0.92). To study the difference between methodologies on QCA results as influenced by the choice of frame and normal segment analyzed, the patient films were analyzed independently in 3 separate rounds of interpretation. In round 1, each system's operator individually chose frames and normal segments for analysis. In round 2, both systems analyzed the same preselected frames, but independently chose normal segments. In round 3, both systems analyzed the same preselected normal segments and frames. The intersystem correlations between visual and automatic systems for rounds 1, 2, and 3 were: normal diameter, r = 0.25, r = 0.37, and r = 0.75, respectively; minimal lumen diameter, r = 0.79, r = 0.86, and r = 0.85, respectively; and diameter stenosis, r = 0.65, r = 0.73, and r = 0.87, respectively. The manual edge-detection and automated edge-detection systems used in this study are reasonably accurate and consistent on phantom studies. In patient studies, the nonautomated processes (choice of frame and normal segment for analysis) produced significant differences in the QCA results, thus illustrating that operator-dependent factors other than edge detection are very important in QCA. PMID- 8623733 TI - Newly developed ST-T abnormalities on the electrocardiogram and chronologic changes in cardiovascular risk factors. AB - An ST-T abnormality on an electrocardiogram (ECG) is known to independently predict subsequent morbidity and mortality from cardiovascular diseases. But how ST-T abnormality develops in relation to chronologic changes in cardiovascular risk factors has not been fully discussed. Sixty-eight men whose ECG had been initially normal but who exhibited ST-T abnormality later (ST-T subjects) were identified among 21,579 apparently healthy adults who had undergone comprehensive health examinations for > 10 years. Echocardiography proved that 26 of 29 examinees among ST-T subjects had left ventricular hypertrophy. Antihypertensive drugs were given to 26 of the ST-T subjects. Their cardiovascular risk factors were chronologically reviewed from 10 years before the onset of definite ST-T abnormality, and were compared with those of 68 men whose ECG had remained consistently normal for 10 years (controls). Mean values of systolic and diastolic blood pressure gradually increased over 10 years (from 127/78 to 144/84 mm Hg) among ST-T subjects, but showed little change (from 122/76 to 124/77 mm Hg) during the same period in controls. The time course of blood pressure over 10 years was similar in ST-T subjects, irrespective of final blood pressure level. Mean serum cholesterol and glucose increased over 10 years in both ST-T and control subjects. Uric acid decreased over 10 years (from 6.1 to 5.6 mg/dl) only in ST-T subjects. Multivariate analysis revealed that blood pressure and uric acid before onset of ST-T abnormality were chronologically changed independent of other risk factors. The time course of risk factors may be of great importance in the development of cardiovascular disorders. PMID- 8623735 TI - Patterns of angiotensin-converting enzyme inhibitor use in congestive heart failure in two community hospitals. AB - Because they provide relief of symptoms and reduce mortality, angiotensin converting enzyme (ACE) inhibitors have become a highly recommended part of the pharmacologic treatment of patients with congestive heart failure (CHF). Although clinical trials suggest that 80% to 90% of patients with CHF tolerate ACE inhibitors, recent surveys reveal that for fewer than this number of patients are actually receiving these drugs. The reasons for this discrepancy are not known. To better understand physician-prescribing behavior, the current study examined the demographic, clinical, laboratory, and medical care characteristics of patients treated and not treated with ACE inhibitors during hospitalization for decompensated CHF. The charts of a consecutive series of patients admitted to 2 acute care hospitals during 1992 (n = 424) were reviewed and comparisons made between those receiving and not receiving ACE inhibitors at the time of hospital admission and hospital discharge. In addition, measures of in-hospital and postdischarge outcome were compared between the groups. The results revealed significant differences in certain demographic variables (e.g., patient age), clinical measures (e.g., left ventricular ejection fraction and serum creatinine), management issues (e.g., documentation of left ventricular function and documentation of etiology of CHF), and treatment strategies (e.g., ancillary drug use). Few differences were noted in measures of severity of CHF (e.g., New York Heart Association functional class and serum sodium level). Death rates were significantly higher for those not receiving ACE inhibitors. Patterns that emerged that could explain under-prescription ACE inhibitors included older age, worse renal function, left ventricular diastolic dysfunction, use of alternate vasodilators, and overall less intense medical management. Programs to educate care providers regarding the proper use of ACE inhibitors in CHF are recommended. PMID- 8623734 TI - Comparison of atrial natriuretic peptide B-type natriuretic peptide, and N terminal proatrial natriuretic peptide as indicators of left ventricular systolic dysfunction. AB - We have directly compared atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and N-terminal pro-ANP (N-ANP) as markers of patients with left ventricular ejection fraction (LVEF) < or = 35%, as measured by radionuclide ventriculography. Venous blood samples were obtained from an unselected group of 87 patients who had been referred for assessment of ventricular function. ANP, BNP, and N-ANP were measured by radioimmunoassay using commercial kits. Receiver operating characteristic analysis was used for the objective assessment of the diagnostic performance of each assay. There was a weak negative correlation between LVEF and plasma levels of ANP-li (r = -0.50,), BNP-li (r = -0.57), and N ANP-li (r = -0.49) (p <0.01 for each peptide). Areas under the receiver-operating characteristic curves for BNP (0.880) and N-ANP (0.832) were not significantly different from each other, but were both significantly greater than the value for ANP (0.761): BNP versus ANP, p <0.01; and N-ANP versus ANP, p <0.05. The optimal sensitivity and specificity of each assay for the detection of patients with LVEF < or = 35% were: BNP > 4 pmol/L-sensitivity 1.0, specificity 0.58; N-ANP >200 pmol/L-sensitivity 0.95, specificity 0.35; and ANP >10 pmol/L-sensitivity 0.90, specificity 0.30. Plasma concentrations of BNP and N-ANP provide sensitive indicators of moderate to severe LV dysfunction; both peptides, are objectively superior to ANP for identifying patients with LVEF < or = 35%. These simple tests could be used to screen patients with suspected ventricular dysfunction to reduce the demand for further cardiac investigations. PMID- 8623736 TI - Malalignment-type ventricular septal defect in double-chambered right ventricle. AB - Double-chambered right ventricle (DCRV) is commonly associated with ventricular septal defect (VSD). In this study, an assessment was made of the relevance of a malalignment-type VSD to hemodynamic and morphologic features in DCRV. During an 8.5-year period, 53 patients with DCRV were enrolled after study with echocardiography, catheterization, and angiography. Patients were divided into 2 groups: group I included 40 patients, aged 3.7 +/- 3.2 years, with a malalignment type VSD; group II consisted of 13 patients, aged 8.6 +/- 2.7 years, without a malalignment-type VSD. History of congestive heart failure in infancy was present in 21 group I and 2 group II patients (53% vs 15%, respectively, p <0.05). The mean pulmonary-to-systemic flow ratio was significantly higher in group I than in group II (1.89 +/- 0.74 vs 1.14 +/- 0.21, respectively, p <0.05). The mean pressure gradient across the right ventricular outflow tract was lower in group I than in group II (41 +/- 16 vs, 73 +/- 33 mm Hg, respectively, p <0.05). Among 42 patients who had a series of echocardiograms recorded, progression of pressure gradient was evident in 35: 28 in group I and 7 in group II. A subaortic ridge was present exclusively in 29 group I patients (73%). Prolapse of the aortic valve was present in 26 (49%): 20 group I (50%) and 6 group II (46%) patients. Aneurysm formation of the septal defect was found in 17 (43%) and 7 (54%) group I and II patients, respectively. It can be concluded that a history of congestive heart failure was more common in DCRV patients with a malalignment-type VSD. Malalignment-type VSD is significantly associated with a larger pulmonary-to systemic flow ratio and subaortic ridge. PMID- 8623737 TI - Comparison and reproducibility of visual echocardiographic and quantitative radionuclide left ventricular ejection fractions. AB - Left ventricular (LV) ejection fraction (EF) is commonly assessed by equilibrium radionuclide angiography and echocardiography. These methods are presumed to be interchangeable for this purpose. This study (1) compares quantification of LVEF by equilibrium radionuclide angiography with visual estimation of LVEF by echocardiography, (2) determines the reproducibility of both methods, and (3) evaluates whether differences in determinations of LVEF are of clinical relevance. Seventy-three clinically stable patients had both equilibrium radionuclide angiography and echocardiography performed within a 4-day period. LVEF by both techniques was compared after blinded analysis by 3 echocardiographers and 3 nuclear technologists. Reproducibility was assessed by blinded repeat analysis after a 1-week interval. The frequency of differences in repeat assessments of EF that the authors considered to be of potential clinical relevance (i.e., difference > or = 10% EF units) was assessed for both techniques. Correlation of LVEF determined by both methods was good (r = 0.81, SEE = 3.5) but with substantial differences in individual patients (limits of agreement, 23.6%). Intra- and inter-observer reproducibility was good for both methods, but better for radionuclide LVEF than for echocardiographic LVEF. Limits of agreement were substantially better for radionuclide LVEF than for echocardiographic LVEF (1.8% to 3.6% versus 13.4% to 17.4%, respectively). Clinically relevant differences did not occur on repeat processing of equilibrium radionuclide angiography. In contrast, potentially clinically relevant differences occurred in 8% to 26% of studies on repeat analysis of echocardiography. Thus, LVEF determined by equilibrium radionuclide angiography and echocardiography show good agreement. Both methods provide clinically valuable measurements for LV function. However, when a precisely reproducible measurement is required for patient management decisions, equilibrium radionuclide angiography is the method of choice. PMID- 8623738 TI - The effect of simvastatin treatment on natural antioxidants in low-density lipoproteins and high-energy phosphates and ubiquinone in skeletal muscle. AB - It has been hypothesized that treating hypercholesterolemic patients with statins will lead not only to a reduction in cholesterol, but also to inhibited synthesis of other compounds which derive from the synthetic pathway of cholesterol. In theory, this could further lead to ubiquinone deficiency in muscle cell mitochondria, disturbing normal cellular respiration and causing adverse effects such as rhabdomyolysis. Furthermore, ubiquinone is one of the lipophilic antioxidants in low-density lipoprotein (LDL), and therefore it has also been hypothesized that statin treatment will reduce the antioxidant capacity of LDL. We investigated the effect of 6 months of simvastatin treatment (20 mg/day) on skeletal muscle concentrations of high-energy phosphates and ubiquinone by performing biopsies in 19 hypercholesterolemic patients. Parallel assays were performed in untreated control subjects. The muscle high-energy phosphate and ubiquinone concentrations assayed after simvastatin treatment were similar to those observed at baseline and did not differ from the values obtained in control subjects at the beginning and end of follow-up. These results do not support the hypothesis of diminished isoprenoid synthesis or energy generation in muscle cells during simvastatin treatment. Furthermore, the results of analysis of antioxidant concentrations in LDL before and after simvastatin treatment indicate that the antioxidant capacity of LDL is maintained in simvastatin-treated patients. PMID- 8623739 TI - Snow shoveling: a trigger for acute myocardial infarction and sudden coronary death. PMID- 8623740 TI - Prospective trials of implantable cardioverter defibrillators versus drugs: are they addressing the right question? PMID- 8623741 TI - Risk factors for new coronary events in a large cohort of very elderly patients with and without coronary artery disease. AB - Independent risk factors for new coronary events in older men include age, prior coronary artery disease, cigarette smoking, systemic hypertension, diabetes mellitus, serum total cholesterol, and serum high-density lipoprotein cholesterol (inverse association). Independent risk factors for new coronary events in older women include age, prior coronary artery disease, cigarette smoking, systemic hypertension, diabetes mellitus, serum total cholesterol, serum high-density lipoprotein cholesterol (inverse association), and serum triglycerides (weak association). PMID- 8623742 TI - Usefulness of the transcendental meditation program in the treatment of patients with coronary artery disease. AB - Twenty-one patients with documented coronary artery disease were tested at baseline by exercise tolerance testing, and assigned to either stress reduction using the Transcendental Meditation (TM) program or to a wait-list control, After 8 months, the TM group had a 14.7% increase in exercise tolerance, an 11.7% increase in maximal workload, an 18% delay in onset of ST-segment depression, and significant reductions in rate-pressure product at 3 and 6 minutes, and at maximal exercise compared with the control group. PMID- 8623743 TI - Utility of 10 MHz ultrasound catheters in the intraaortic assessment of coronary artery ostial stenoses. AB - Our preliminary observations show that intraaortic intravascular ultrasound using 10 MHz transducer catheters is safe and feasible in the assessment of coronary ostial lesions. This imaging modality is potentially useful in decision making regarding the choice of coronary intervention. PMID- 8623744 TI - Effects of coronary artery aneurysms on intracoronary flow velocity dynamics in Kawasaki disease. AB - In coronary artery aneurysms associated with Kawasaki disease, a significant decrease in coronary flow velocity together with an abnormal flow profile and a reduction in coronary flow reserve were observed, and were significantly correlated with the size of the aneurysm. These results suggest that coronary arterial aneurysms impair coronary circulation. PMID- 8623745 TI - Angiotensin-converting enzyme insertion/deletion polymorphism and restenosis after coronary angioplasty in unstable angina pectoris. AB - We studied the relation between angiotensin-converting enzyme insertion/deletion gene polymorphism and restenosis in Caucasian patients who underwent coronary angioplasty for management of unstable angina pectoris. Our results indicate that, in contrast to previous reports in Japanese patients, no association exists between angiotensin-converting enzyme gene polymorphism and the development of restenosis in Caucasian patients with acute coronary syndromes.) PMID- 8623746 TI - Angiotensin-converting enzyme insertion/deletion polymorphism in angina pectoris with normal coronary arteriograms. AB - We investigated the relation between angiotensin-converting enzyme gene insertion/deletion polymorphism and syndrome X (angina with normal coronary arteriogram). The results of our study suggest that this polymorphism does not play a major role in the pathogenesis of microvascular angina. PMID- 8623747 TI - Acetylcholine-induced prolongation of the QT interval in idiopathic long QT syndrome. AB - Intracoronary acetylcholine prolonged the QT interval in 5 patients with congenital long QT syndrome but not in subjects with normal QT intervals. Prolongation was not due to bradycardia or adrenergic drive, and atropine was suggested to attenuate the response. PMID- 8623748 TI - Value of multiplane transesophageal echocardiography in determining aortic valve area in aortic stenosis. AB - Aortic valve area determination by transesophageal planimetry and by the continuity equation was compared with hemodynamic measurements in 45 patients with symptomatic aortic stenosis. The correlations between transesophageal echocardiography and hemodynamics were dependent on the amount of valvular calcium, whereas the continuity equation correlated well with hemodynamics in all patients. PMID- 8623749 TI - Long-term outcome in patients with pacemakers following the Fontan operation. AB - Patients with pacemakers after Fontan surgery compared favorably with nonpaced patients with respect to survival. In patients with atrioventricular block, dual chamber pacing was superior to VVI pacing. PMID- 8623750 TI - Clinical impact of transcatheter closure of secundum atrial septal defects with the double umbrella device. AB - The clinical impact of transcatheter closure of the isolated secundum atrial septal defect was reviewed. Closure by echocardiographic evaluation was 23 +/- 14% at 6 months, 49 +/- 16% at 2 years, and 64 +/- 16% at 4 years, and right ventricular end-diastolic dimensions in patients without residual shunts did not differ significantly from those with residual shunts. PMID- 8623751 TI - Cardiac output response to dynamic exercise after atrial switch repair for transposition of the great arteries. AB - Results from this study showed that patients who underwent successful operation for transposition of the great arteries had no appropriate increase in stroke volume in response to exercise of a nature similar to common recreational activities. The impairment, most likely due to disturbances in both venous return and ventricular systolic function, is compensated for by an increase in peripheral oxygen extraction; however, this increase may not be adequate with maturation or during prolonged exercise when cardiovascular constraints are more important. PMID- 8623753 TI - Experimental fetal transesophageal and intracardiac echocardiography utilizing intravascular ultrasound technology. AB - Fetal transesophageal and intracardiac echocardiography by utilizing ultrasound technology permits accurate definition of cardiac anatomy in fetal sheep. Because fetal transesophageal echocardiography is less invasive than intracardiac echocardiography, it has the potential to serve as a monitoring tool for currently developed open and fetoscopic fetal cardiac interventions. PMID- 8623754 TI - In vitro evaluation of coronary artery endothelial reactivity 11 years after transplantation. AB - We studied in vitro the vascular reactivity of a transplanted heart which was explanted 11 years later for chronic rejection. Radiologic, histologic, and vascular aspects are presented and correlated. PMID- 8623755 TI - Atypical electrocardiographic changes in severe hyperkalemia. AB - The electrocardiogram is often used to gauge the severity of hyperkalemia. We present a case of severe hyperkalemia associated only with pseudonormalized T waves and sinoatrial exit block. PMID- 8623752 TI - The varying evolution of Friedreich's ataxia cardiomyopathy. AB - During a mean follow-up period of 8 years, 17% of 66 patients with Friedreich's ataxia developed hypokinetic-dilated cardiomyopathy; most patients originally had a hypertrophic left ventricle. The presence of pathologic Q waves identifies a subgroup of patients with wall motion abnormalities; these patients are more likely to develop a hypokinetic left ventricle, and the prognosis is ostensibly poorer. PMID- 8623756 TI - Limited toxicology screening: end of a controversy. PMID- 8623757 TI - Video microscopy and gynecologic cytopathology. PMID- 8623758 TI - Detection of the bcl-1 rearrangement at the major translocation cluster in frozen and paraffin-embedded tissues of mantle cell lymphomas by polymerase chain reaction. AB - The t(11;14)(q13;q32) translocation and its molecular counterpart bcl-1 rearrangement are highly characteristic of mantle cell lymphomas (MCLs). Most of these translocations occur at the major translocation cluster (MTC) in a tight area that makes this rearrangement identifiable by the polymerase chain reaction (PCR). In this study, the specificity and sensitivity of the PCR technique in the identification of bcl-1 rearrangement and its suitability to amplify the t(11;14) MTC in fixed, paraffin-embedded tissues were analyzed. Genomic DNA was obtained from 21 MCLs and 1 chronic lymphocytic leukemia (CLL) with the t(11;14) translocation. The bcl-1 rearrangement was studied by Southern blot with the MTC, p94PS, and PRAD-1 probes. Polymerase chain reaction was performed using a JH consensus primer and specific primers for chromosome II in the MTC region. bcl-1 rearrangement was identified by Southern blot in the MTC in nine (43%) MCLs and in the p94PS region in the CLL. Polymerase chain reaction analysis of genomic DNA showed that the nine MCLs with MTC rearrangement also had an amplifiable band of the expected size (100%). No amplifiable products were detected in the negative MCLs or in the CLL. The specificity of the PCR products was confirmed by hybridization with an internal MTC oligonucleotide probe. Amplifiable DNA was obtained from the paraffin blocks of 7 cases with MTC rearrangement and 11 negative tumors. bcl-1 rearrangement was detected in this DNA of 6 positive MCLs (86%) by PCR and in none of the negative cases. In conclusion, this study demonstrates that the PCR technique is highly sensitive and specific for the detection of the bcl-1 rearrangement at the MTC. It can be used with both high molecular weight DNA and DNA obtained from formalin-fixed, paraffin-embedded tissues. PMID- 8623759 TI - Immunohistochemical detection of p53, bcl-2, and retinoblastoma proteins in follicular lymphoma. AB - Mutations of p53 have been suggested to be involved in the histologic transformation of follicular lymphoma, but the role of the retinoblastoma (RB) gene, another tumor suppressor gene, in lymphomagenesis has not been established. To determine the roles of these tumor suppressor genes and their relationship with the anti-apoptotic bcl-2 gene in follicle center lymphoma, the immunohistochemical expression of p53, bcl-2, and RB proteins was correlated with cytologic grade in 50 cases of follicular lymphoma, and the results were compared to 23 cases of diffuse large B-cell lymphoma. The results showed that only 2 of 25 grade 1 follicular lymphoma were p53-positive compared to 16 of 25 grade 3 cases (P <.0001). A significantly lower number (13 of 25) of grade 3 follicular lymphomas expressed bcl-2 compared to grade 1 cases (23 of 25) (P <.004). Eight of 14 bcl-2-negative follicular lymphomas expressed p53, compared with 10 of 36 bcl-2-positive cases (P = .1). Twenty-four of 25 grade 3 follicular lymphomas showed 2+ to 3+ staining for RB protein compared to 9 of 21 grade 1 cases (P <.0002). Expression of p53 protein correlates significantly with higher cytologic grade in follicular lymphoma. Similar to earlier studies of breast cancer and lymphoma, there appears to be an inverse relationship between p53 and bcl-2 expression in follicular lymphoma. Inactivation of the retinoblastoma gene does not seem to be involved in the histogenesis of follicle center lymphoma or diffuse large B-cell lymphoma. PMID- 8623760 TI - Blast-like cells in the cerebrospinal fluid of young infants: further characterization of clinical setting, morphology and origin. AB - Blast-like cells in the cerebrospinal fluid (CSF) of neonates have been identified and previously suggested to be of germinal matrix origin. Twelve additional CSF specimens with blast-like cells collected at the University of Texas Medical Branch, Galveston, between 1985-1992 were analyzed. The cytological features of the blast-like cells as well as their associated clinical setting were further characterized by the authors. All patients in the study were young infants with hydrocephalus and nearly all underwent placement of a ventriculoperitoneal (VP) shunt at the time the CSF specimen was collected. In addition, a cytologic preparation of germinal matrix cells obtained from an autopsy specimen was analyzed, which closely resembled the blast-like cells. These data provide additional evidence that blast-like cells originate from the germinal matrix. PMID- 8623761 TI - A simple splenic reticuloendothelial function test: counting erythrocytes with argyrophilic inclusions. AB - The presently accepted methods for evaluation of splenic reticuloendothelial (RE) function include 99mTc sulfur colloid spleen scan, antibody-coated autologous erythrocyte clearance, and pocked erythrocyte count. All methods involve special equipment and/or risk and inconvenience to patients. A simple method of assessing splenic RE function was developed by counting erythrocytes with argyrophilic inclusions using a simple silver stain and an ordinary microscope. To test the validity of this method, blood samples were collected from patients suspected of having hyposplenia or asplenia, including patients with history of splenectomy, sickle cell disease or trait, and newborns. Blood samples were also collected from normal adults and from patients without hyposplenia or asplenia as controls. The samples were tested by this method and compared to the pocked erythrocyte count that served as a gold standard. The results obtained by the two methods were found to be very comparable with little overlap between those from controls and patients with definite hyposplenia or asplenia. With the pocked erythrocyte count as the gold standard, this method has a sensitivity of 88.9% and a specificity of 97.1%. However, this method requires no special equipment. Staining can be applied to fresh blood smears as well as to Wright-stained smears, and the silver-stained smears are permanent. PMID- 8623762 TI - Evaluation of the Abbott Cell-DYN 3500 hematology analyzer in university hospital. AB - The Abbott (R) Cell-Dyn 3500 (Abbott CD 3500, Abbott Diagnostics Division, Mountain View, CA) is a fully automated hematology analyzer capable of providing a complete blood count (CBC) profile, including a five-part differential leukocyte count (DLC) and flagging system in this study. The CBC profile and DLC flagging system of the Abbott CD 3500 were evaluated according to the HA-20 protocol of the National Committee for Clinical and Laboratory Standards (NCCLS) and compared to the Technicon H*2 blood analyzer currently used in the authors' laboratory. Linearity, carryover, precision, and stability were all within the limits established by the manufacturer. No significant break-downs were found during the evaluation period. Evaluation of DLC indicated an excellent correlation with the manual reference method for neutrophils, lymphocytes, and eosinophils (r = 0.916, 0.936, 0.967, respectively), a good correlation for monocytes (r = 0.811) and a poor correlation for basophils (r = 0.224). Overall flagging for morphologic abnormalities displayed higher sensitivity (85%) than specificity (67%), with a false-positive ratio of 33%. In general, these results are in accordance with those obtained by other authors in the same period of time. PMID- 8623763 TI - Gastric lymphoepithelioma-like carcinoma and jejunal B-cell MALT lymphoma with large cell transformation. Demonstration of EBV with identical LMP gene deletions in the carcinoma and large cell lymphoma. AB - The authors describe a patient with gastric lymphoepithelioma-like carcinoma (LELC) and jejunal low grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) with histologic transformation to large cell lymphoma. In situ hybridization studies for Epstein-Barr virus (EBV) demonstrated abundant EBV RNA (EBER) within the neoplastic cells of the gastric LELC and the jejunal large cell lymphoma. The low grade MALT lymphoma was EBER negative. Polymerase chain reaction (PCR) studies confirmed the in situ hybridization results, and demonstrated a 30 base pair deletion in the 3' end of the latent membrane protein gene in both the gastric LELC and the jejunal large cell lymphoma. These results suggest that the same viral strain infected both the stomach and jejunal tumors. In addition, the finding of EBV in the large cell lymphoma, but not the low grade component suggests that EBV may have played a role in large cell transformation. PMID- 8623764 TI - Ovarian dermoid cyst-associated autoimmune hemolytic anemia: a case report with emphasis on pathogenic mechanisms. AB - Autoimmune hemolytic anemia (AIHA) as a manifestation of ovarian dermoid cyst (ODC) is a rare paraneoplastic syndrome of unknown pathogenesis. Among mechanisms postulated to explain this association, cross-reactivity between cyst and red blood cell (RBC) antigens, and local production of RBC autoantibodies by intracyst B lymphocytes are the most likely. Studies to test these hypothesis were done in a patient diagnosed of AIHA associated with a nonpalpable ODC, in whom the AIHA subsided after tumor excision. The RBC-bound autoantibody was an IgG directed against the Rh complex. The cyst's fluid content lacked detectable RBC autoantibodies or immunoglobulins, the latter being measured by a high sensitivity assay. It also failed to inhibit the ability of the purified autoantibody to agglutinate RBCs. Ovarian dermoid cyst histology disclosed that (1) the biotin-labelled RBC autoantibody did not bind to ODC structures; (2) scanty amounts of small mature lymphocytes (50% CD45RO+; 50% CD20+) were present only in a few tissue sections; (3) plasma cells producing IgM or IgG were extremely scarce; and (4) deposits of immunoglobulins were not detected into the ODC. These data fail to support any of the aforementioned hypotheses on the pathogenesis of this paraneoplastic syndrome. Other possible mechanisms are discussed, and a wider use of imaging diagnosis to search for ODC in women with AIHA is emphasized. PMID- 8623765 TI - Results of limited versus comprehensive toxicology screening in a university medical center. AB - Both comprehensive toxicology screening (COM) and limited toxicology screening (LIM) were performed on the same urine in 1,734 consecutive cases over a 6-month period at a university medical center. About half of the screens originated from inpatient services and half from outpatient services (mostly emergency department). In 71 % of the cases, there was agreement between the results of LIM and COM, with 47% of cases being negative. For 500 screens (accounting for 655 individual discrepant findings), at least one discrepancy was noted between LIM and COM. Of these, 399 cases demonstrated at least one finding with COM, but not with LIM ("false-negative" LIM). Topical anesthetics (including cocaine and cocaethylene), antiepileptics, and sympathomimetic amines accounted for 65% of findings. In contrast, 147 cases showed a finding with LIM, but not with COM ("false-positive" LIM), mostly amphetamines. In certain specified clinical circumstances, LIM may be a more cost effective and efficient approach than COM. PMID- 8623767 TI - Evaluation of the Technicon Immuno 1 free thyroxine assay. AB - The Technicon Immuno 1 free thyroxine (fT4) assay, a modified two-step procedure that is fully automated on a random access analyzer, was evaluated-at two clinical sites. The method had excellent precision and correlated well overall with three other estimates of free thyroxine: free thyroxine index (FTI) measured on the Immuno 1; the Abbott IMx fT4 assay (Abbott Park, IL); and the Clinical Assays two-step manual fT4 assay. Using a combination of thyrotropin and FTI assay results as a "gold standard" for defining thyroid status, the Immuno 1 fT4 method had a sensitivity of 100% and specificity of 98.3% for hyperthyroidism, versus 93.8% and 99.3%, respectively, for hypothyroidism. In conclusion, the Immuno 1 fT4 assay is useful in screening for thyroid disease. PMID- 8623766 TI - Fourier transform infrared microscopy identification of crystal deposits in tissues: clinical importance in various pathologies. AB - The presence of crystal deposits in tissues is associated with various pathologies. Sometimes their identification is useful for understanding the etiology or the mechanism of the disorder. The authors applied Fourier transform infrared microscopy (FTIRM) to the molecular characterization of crystal deposits in tissue and compared the results with those provided by histologic studies using polarized light microscope and histochemical reactions. Twenty-five biopsies were investigated. In 10 cases, the results were in good agreement. In 15 cases only FTIRM could precisely identify the crystals. In three cases, this technique allowed to characterize dihydroxyadenine crystals revealing an adenine phosphoribosyltransferase deficiency previously undiagnosed in patients presenting severe chronic renal failure. In three cases, crystal deposition was related to drug therapy. In other cases, crystal identification was useful to understand the mechanism of the pathology responsible for tissue damage and crystal deposition. PMID- 8623768 TI - Cytokine function: a study in biologic diversity. AB - Cytokines are a group of hormone-like polypeptide mediators that play a variety of regulatory roles in both host defense and normal and abnormal homeostatic mechanisms. They may he produced by diverse cell types and exert their function on a variety of cells. Their effects (which may be suppressive or enhancing) are on cellular proliferation, differentiation, activation, and motility. In addition, cytokines can exert cytodestructive effects on infectious agents or tumor cells, either directly or by activating cells with cytodestructive potential. Any given cytokine may have many different biologic effects. However, two different cytokines may have similar or identical activities. Cytokines may be classified on the basis of their cell of origin, their spectrum of activity, the category of activity they influence, the cells that are their targets, or on specific features of their ligand-receptor interaction. The mode of action of many of the cytokines involves typical signal transduction events such as protein phosphorylation, and to date there is only limited understanding of the mechanisms that lead to one activity over another when a specific cytokine is involved in a specific biologic reaction. Nevertheless, elucidation of their role in other pathologic processes has provided insight into autoimmune and allergic diseases, as well as a variety of systemic disorders. Because of their broad spectrum of activity, cytokines have been used in a variety of therapeutic settings involving both infectious diseases and neoplasia. As the number of known cytokines continues to grow, it will be increasingly difficult for the non "cytokinologist" to follow the exponentially expanding literature. Hopefully, this brief review will provide an overview that can serve as a framework for the understanding of this important area of biology and pathology. PMID- 8623769 TI - Telecytology: diagnostic accuracy in cervical-vaginal smears. AB - Although cervical-vaginal telecytology is a promising tool, diagnostic accuracy has not been extensively evaluated. The authors examined the accuracy of five cytotechnologists who retrospectively reviewed 50 cervical-vaginal smears using the video monitor, and 2 months later, using the light microscope. Accuracy was expressed in terms of crude agreement with the original diagnosis and number of false positives (FPs) and false negatives (FNs). With a greater than one step difference as discrepant, the group crude agreement using the video monitor and the light microscope was 85.6% and 95.6%, respectively. The group number of FNs and FPs for the light microscope was 8 and 7, respectively, and for the video monitor was 34 and 7, respectively. There was a wide range of individual performance. We conclude that accuracy of telecytology is high, but less than that of light microscopy. The major reason for lower telecytologic accuracy was undercalling dysplasia. PMID- 8623770 TI - p53 expression in colorectal cancer: relation to tumor type, DNA ploidy pattern and short-term survival. AB - p53 protein expression was evaluated in a series of 204 primary colorectal adenocarcinomas by immunohistochemistry using frozen tissue sections and monoclonal antibody DO-7. Nuclear staining of more than 5% of neoplastic cells was observed in 124 (60.8%) adenocarcinomas, which were classified as p53 positive. p53 immunoreactivity was found to he unrelated to several clinical and pathologic variables, including age and sex of patient, tumor site, tumor stage, grade of differentiation, pattern of growth, degree of peritumoral lymphocytic infiltration, and venous invasion. A strong association was demonstrated between p53 immunostaining and tumor type. Only 4 of 21 mucinous carcinomas examined (19%) were p53 positive. Conversely, 120 of 183 (65.6%) nonmucinous adenocarcinomas showed positive p53 immunostaining (P <.0001). p53 expression also was related to the flow cytometric DNA ploidy pattern, aneuploid carcinomas with DI >1.20 showing higher frequency of p53 overexpression than DNA diploid, and aneuploid tumors with DI < or = 1.20 (P = .0003). No relationship was found between p53 expression and the Ki-67 proliferation index. With respect to the total study population (mean follow-up 33.4 months; range 19-47 months) the duration of overall survival was independent of p53 expression. In the group of 141 patients with stage I, stage II, and stage III disease who had undergone curative resection, positive p53 immunostaining was associated with poorer overall survival (P = .029). Subgroup analysis showed that the reduced survival conferred by p53 overexpression was confined to patients with stage III tumors (P = .027). However, in multivariate analysis, p53 expression failed to demonstrate independent prognostic significance. Our results indicate that immunohistochemical analysis of p53 expression provides valuable information for the understanding of colorectal cancer biology and clinical behavior. PMID- 8623771 TI - Expression of mucin carbohydrate antigens (T, Tn and sialyl Tn) and MUC-1 gene product in intraductal papillary-mucinous neoplasm of the pancreas. AB - Aberrant or incomplete glycosylation of mucins results in expression of T, Tn, and sialyl-Tn (STn) antigens in various malignant neoplasms. MUC-1 gene product (a mucin core protein of mammary type) is known to alter or overexpress in several malignant tumors. However, expression of these mucin-related antigens has rarely been examined in intraductal papillary-mucinous neoplasm (IPN) of the pancreas. The authors examined immunohistochemically the expression of these antigens and MUC-1 gene product in nine IPN. In normal pancreas (n = 5), pancreatic ducts did not express T, Tn, or STn antigens, but expressed MUC-1 gene product. Among the nine IPNs, two (22%) expressed T antigen, nine (100%) expressed Tn antigen, and seven (78%) expressed STn antigen. MUC-1 gene product was expressed in nine (100%) IPNs. In invasive ductal adenocarcinoma of the pancreas (n = 6), all cases showed strong expression of Tn and STn antigens and the MUC-1 gene product, but expressed no T antigen. The expression of these antigens and MUC-1 gene product was focal in IPN, whereas it was diffuse in invasive ductal adenocarcinoma of the pancreas. These data suggest that aberrant or incomplete glycosylation occurs in epithelial mucins of IPN, that IPN is a borderline or low grade malignant neoplasm in terms of mucin-related antigen expression, and that mucin core protein (MUC-1 gene product) does not alter during pancreatic ductal carcinogenesis. PMID- 8623772 TI - Metastatic carcinoma resulting in hepar lobatum. AB - Hepar lobatum is an acquired liver deformity mostly known as the end-stage of tertiary syphilis. The authors report two cases of hepar lobatum resulting from metastatic mammary ductal carcinoma in the liver and reassess the clinicopathologic features of seven previously reported cases (two in the German language). A liver of near-normal weight with an irregularly lobulated contour, capsular indentations/crevices from which intersecting (carcinoma-bearing) fibrous septa extended deep into the parenchyma, a predominant centrifugal distribution of lesional areas, and many septa abutting on the degenerated center of tumor nodules were the salient gross features. No significant tumor/fibrous occlusion of intrahepatic branches of portal or hepatic veins, nor cirrhotic type nodular hepatocellular regeneration was observed. Both of these patients experienced a drastic decrease in CEA serum levels during multiagent palliative chemotherapy. In one patient, abundant macrophages in conjunction with minimal residual tumor were present within intrahepatic septa. The pathogenesis of this condition appears largely related to an active phase of chemo-induced tumor regression with subsequent tissue collapse, followed by an organizing phase of healing and scar contraction. PMID- 8623773 TI - Assessment of the role of GM-CSF in the cellular transformation and the development of erosive lesions around orthopaedic implants. AB - Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor with a regulatory effect on the transformation of immature macrophages into multinucleated giant cells (MNGC) that exhibit phenotypic and functional characteristics of osteoclasts (OC). The authors analyzed the bone implant interface membranes harvested from 15 patients with failed total joint replacements for the production and tissue distribution of GM-CSF and interleukin 1 (IL-1). Immunohistology and liquid culture were employed to assess the contribution of these factors in the recruitment of macrophages and the development of bone resorbing MNGC at these sites. This process has been implicated in osteoclastic bone resorption, bone, and bone marrow necrosis adjacent to orthopaedic implants. Histologic assessment of the interface indicated the presence of granuloma and a variable number of MNGC in 11 cases. Four cases showed sites of intramembranous formation of osteoid and mineralized bone that was accompanied by normal bone marrow in two cases. Granulocyte macrophage colony stimulating factor was expressed by a distinct subset of phagocytic macrophages in the lining layer on the implant side. interleukin-1 positive cells outnumbered those stained for GM-CSF. Stimulation of cultured cells with prosthetic metal particulate material showed marked similarity in the expression of these cytokines to cultures treated with lipopolysaccharide (LPS) or phytohemagglutinin (PHA). The induction of GM-CSF production in the lining layer where small MNGC develop indicates that these cells differentiate locally following the phagocytosis of particulate wear debris. In conclusion, GM-CSF promotes the proliferation and early stages of fusion and development of MNGC responsible for osteolysis at these sites. These results also highlight the capacity of the interface to display both osteogenic and inflammatory characteristics. Collectively, the findings suggest that the local bone marrow could participate in the development of the interface as a source of myeloid cells in addition to the capacity of marrow stroma to generate various osteogenic cells essential for the ingrowth of bone into prosthetic implants. PMID- 8623774 TI - Apoptosis of gamma/delta T cells in human ehrlichiosis. AB - Expansion of activated T cells expressing the T-cell receptor (TCR) gamma/delta, CD45RO, and HLA-DR antigens is a prominent feature of acute infection with Ehrlichia chaffeensis in humans. The fate of these activated cells and the resolution of the gamma/delta T-cell response with return to the usual alpha/beta T-cell populations in this disease are not clearly understood. At a morphologic level, apoptotic cells are present in the peripheral blood during the acute and resolution phases of the infection. Simple culture of density gradient-separated lymphocytes from the blood of patients with acute ehrlichiosis produced cell death rapidly in the media compared to alpha/beta T cells. This loss of viability after incubation was apparently mediated by apoptosis, based on flow cytometric and morphologic analyses. The results suggest that most primed (CD45RO+) and activated (HLA-DR+) gamma/delta T cells in acute ehrlichiosis might be subject to removal from the body by programmed or apoptotic cell death. PMID- 8623775 TI - Polymerase chain reaction detection of Lyme disease: correlation with clinical manifestations and serologic responses. AB - The authors have developed a simple, nested polymerase chain reaction (PCR) assay for amplification of an outer surface protein A (OspA) gene fragment of Borrelia burgdorferi using rapid temperature cycling and ethidium bromide detection on agarose gels, and applied it to the diagnosis of Lyme disease in humans. With denaturing and annealing temperature spikes instead of holds, cycle times were less than 20 minutes for a 30-cycle amplification. Using this rapid cycle PCR technique, as few as 5 spirochetes per mL of phosphate buffered saline were detected. In addition, B burgdorferi DNA was detected from spirochetes that had been spiked into one of several types of human body fluids including serum, synovial fluid, and cerebrospinal fluid (CSF). A number of clinical samples, which had been tested for Lyme immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody were also examined. In 29 serologic positive samples (14 IgG and IgM positive, 9 IgM alone and 6 IgG alone), B burgdorferi DNA was not detected. In contrast, nine serum samples and one synovial fluid from patients with definite clinical features of Lyme disease were found to be negative by EIA and Western blot analysis for IgG and IgM antibody, but contained B burgdorferi DNA, as detected by PCR. Polymerase chain reaction analysis of serum and synovial fluid may be of significant diagnostic value in Lyme disease, especially in the absence of a serologic response in early, partially treated and seronegative chronic disease. This is the first study to report an association between PCR positivity and the absence of a serologic response to Lyme borreliosis. PMID- 8623776 TI - Vernix caseosa peritonitis. PMID- 8623777 TI - p53 protein expression and p53 mutation in thymic epithelial tumors. PMID- 8623778 TI - Preterm delivery from 34 to 37 weeks of gestation: is respiratory distress syndrome a problem? AB - Our purpose was to evaluate the necessity of using tocolytic agents for preterm labor and the benefit of conservative management for preterm premature rupture of membranes from 34 to 37 weeks' gestation. All patients who had accurate obstetric dates and were delivered between 34 and 37 weeks' gestation with no other medical or obstetric problems were retrospectively evaluated for inclusion in the study. The rates of respiratory distress syndrome and other neonatal outcomes were evaluated. A total of 416 patients met criteria for admission into the study. During the thirty-fourth week of pregnancy the incidence of respiratory distress syndrome was 14.9% (p<0.05). Other neonatal complications were also more frequent in deliveries occurring during the thirty-fourth week of pregnancy than in those occurring in the thirty-fifth or thirty-sixth week. Fetal lung maturity studies should be considered and delivery possibly delayed through the thirty-fourth week of gestation to decrease neonatal morbidity in our population. PMID- 8623779 TI - Management of endometrial cancer. AB - Preoperative assessment requires only endometrial sampling for diagnosis. Curettage is needed when endometrial sampling is unsatisfactory. Transvaginal ultrasonography may be useful in screening high-risk patients, as well as in assessing myoinvasion or cervical extension. Postsurgical pathologic prognostic factor analysis is most accurate in assigning risk for recurrence. Once the extent of disease is confirmed by the surgical staging procedure of hysterectomy, bilateral removal of the ovaries, and selective pelvic and periaortic node dissection, adjunctive therapy can be considered. Patients with low-risk stage IA and IB grade 1 disease require hysterectomy and removal of the adnexa. The poorer prognosis of patients with grade 2 or 3 histologic features in stages IB to IIB dictates considerations for adjunctive therapy. Soon randomized controlled trials will elucidate objectively what may be optimal adjunctive therapy. Ongoing prospective trials will clarify the role of operative laparoscopy. Current management guidelines are based on independent prognostic factors derived from analysis of surgicopathologic studies. PMID- 8623780 TI - Oxygenation in fetal lambs supported by extrauterine right atrium to artery extracorporeal membrane oxygenation. AB - OBJECTIVE: Our purpose was to determine the adequacy of oxygenation, particularly cranial and cardiac oxygenation, in exteriorized fetal lambs on right atrium to artery extracorporeal membrane oxygenation. STUDY DESIGN: Thirteen fetal lambs were placed on right atrium to artery extracorporeal membrane oxygenation between the gestational ages of 113 and 133 days. Various PO2 and oxygen saturation (SO2) values were obtained by varying the oxygen concentrations at the oxygenator membrane. Blood gases, pH, and SO2 were observed on samples taken before and after membrane oxygenation from the left ventricle and through the cranial carotid arterial catheter. These were compared with control values obtained before the cessation of umbilical circulation. Fetal coronary oxygenation was represented by left ventricle PO2 and SO2 and cranial oxygen by carotid artery PO2 and SO2. RESULTS: We classified oxygen saturation as low, medium, and high on the basis of the level of postmembrane SO2. Carotid artery cranial oxygenation in the low SO2 group was equivalent to control values, but that in the medium and high SO2 groups was significantly higher than in the control group. Left ventricle oxygenation was consistently lower than cranial oxygenation in any SO2 group. In the low group left ventricle oxygenation was significantly lower than the control values. CONCLUSIONS: Right atrium to artery extracorporeal membrane oxygenation appears sufficient to oxygenate the fetal cranial circulation but may be inadequate for the efficient distribution of oxygenated blood into the left ventricle and thus the coronary circulation. PMID- 8623781 TI - The fetus with gastroschisis: impact of route of delivery and prenatal ultrasonography. AB - OBJECTIVES: Our purpose was (1) to assess the influence of delivery route on neonatal outcome in fetuses with gastroschisis and (2) to correlate ultrasonographic appearance of fetal bowel with immediate postnatal outcome. STUDY DESIGN: Forty-seven cases (1986 to 1994) were reviewed; three abortions and two stillbirths were excluded. Ultrasonographic appearance of fetal bowel (small bowel dilatation > 10 mm) was evaluated in 27 cases. RESULTS: Twenty-six infants (61.9%) were delivered vaginally and 16 (38.1%) by cesarean section (11 elective, 5 in labor). Delivery route was not significantly associated with indicators of neonatal outcome (rate of primary closure, postoperative complications, days of parenteral nutrition, days to oral feeding, hospital days, or mortality). When ultrasonographic appearance of fetal bowel was correlated with outcome, fetuses with prenatally dilated bowel had significantly more bowel edema at birth (p=0.038), longer operative time (p=0.013), and higher overall rate of postoperative complications (p=0.037). CONCLUSIONS: (1) Elective cesarean delivery does not improve neonatal outcome in infants with gastroschisis. (2) Abnormal ultrasonographic appearance of fetal bowel is associated with a more difficult repair and a higher overall incidence of postoperative complications. PMID- 8623782 TI - Decreased fetal erythropoiesis and hemolysis in Kell hemolytic anemia. AB - OBJECTIVE: Lower changes in optical density (450 nm) measurements have been reported in fetuses with anti-Kell anemia compared with those anti-D anemia. The purpose of this investigation was to determine if hemolysis and erythropoiesis differ between anti-Kell and anti-D hemolytic disease. STUDY DESIGN: Ninety-three pregnancies complicated by either anti-D or anti-Kell alloimmunization were evaluated. Fetal blood samples obtained at the first cordocentesis were tested for the red blood cell antigen type, hemoglobin, hematocrit, reticulocyte count, nucleated red blood cells, total serum bilirubin concentration, umbilical venous respiratory blood gases, serum erythropoietin level, and strength of the direct Coombs test. To determine the evolution of hemolytic anemia in the two antigen groups, these laboratory parameters were repeated on the fetal blood samples triggering the decision to perform a fetal intravascular transfusion (hematocrit <30%). RESULTS: A total of 65 of 93 fetuses were antigen positive (11 for Kell and 54 for RhD). The mean gestational age and laboratory measurements of antigen- positive, nonanemic fetuses at first blood sampling did not differ significantly between groups. There was a strong inverse relationship observed between the hemoglobin concentration and reticulocyte count independent of gestational age in the anti-D group but not in the anti-Kell group. Eight (73%) fetuses with anti Kell antibodies and 37 (69%) with anti-D antibodies underwent intravascular transfusion. At the cordocentesis when the decision for transfusion was made, anti-Kell anemic fetuses had lower reticulocyte counts and total bilirubin concentrations. The strong inverse relationship between the hemoglobin and reticulocyte count was again seen only in the anti-D group. In both groups, fetal erythropoietin increased significantly between the first and last blood samplings and in each group were negatively correlated with hemoglobin independent of gestational age. CONCLUSION: Anti-Kell anemic fetuses have lower reticulocyte counts and total serum bilirubin levels than do comparable anti-D anemic fetuses. This finding argues in favor of fetal blood sampling rather than amniotic fluid analyses for the management of fetal hemolytic disease resulting from Kell antibodies. Unlike RhD alloimmunized fetuses, these fetuses do not manifest an inverse relationship between hemoglobin concentration and reticulocyte count. We speculate that compared to anti-D fetal anemia, anti-Kell anemia is associated with increased hemolysis of nonhemoglobinized or incompletely hemoglobinized erythroid precursors. PMID- 8623783 TI - Acceleration of fetal head induced by vibration of maternal abdominal wall in sheep. AB - OBJECTIVE: The human body is often exposed to significant vibration stress in the workplace, at home, and during recreational activities. The current study was designed to evaluate whether low- to midfrequency vibrations present at the extraabdominal wall would be attenuated across this wall and what the levels of exposure would be once these vibrations reached the fetal head. STUDY DESIGN: Four pregnant sheep were instrumented with acceleration transducers to obtain acceleration levels at the extraabdominal and intraabdominal walls and at the fetal head. Sine-wave vibration stimulation was applied over a frequency range of 3 to 150 Hz at a constant acceleration level of 2.5 m/sec2 (root-mean-square). RESULTS: Vibration of the extraabdominal wall resulted in a frequency-dependent rise in vibration levels at the intraabdominal wall, from 4% to 140% of the input level. At the fetal head a broad peak in response was noted between 6 and 12 Hz, but the overall levels never exceeded 4% of the input level. CONCLUSION: Fetal exposure to localized vibratory stimulation of the maternal abdomen is maximal in the range of 6 to 12 Hz. PMID- 8623784 TI - Increased incidence of placental chorioangioma in high-altitude pregnancies: hypobaric hypoxia as a possible etiologic factor. AB - OBJECTIVES: Our purpose was to determine the effects of pregnancy at high altitude on the human placental structure. STUDY DESIGN: Histologic material from 22 term placentas delivered at altitudes above 3600 m was examined and compared with control material from 760 m. RESULTS: Intraplacental chorioangiomas were identified in 5 of the 22 high-altitude placentas but in none of the 59 controls. The lesions were not visible on the external surface of the placentas and were not encapsulated. The state of differentiation varied; some contained numerous capillaries that showed sinusoidal dilations, whereas in others at the opposite extreme mesenchymal cells predominated. The presence of chorioangiomas was frequently associated with threatened premature delivery, a circumvallate placenta, and multiple infarction. The lesions represented only a small percentage of the overall placental volume (<10%). CONCLUSIONS: The increased incidence of placental chorioangioma observed at high altitude (above 3600 m) may be due to the prevailing hypobaric hypoxia. Overexpression of angiogenic cytokines such as vascular endothelial growth factor, which is known to be up regulated by this factor in vitro, may mediate this effect. PMID- 8623785 TI - Dynamics of the fetal adrenal, cholesterol, and apolipoprotein B responses to antenatal betamethasone therapy. AB - OBJECTIVE: Prior studies suggest that fetal plasma cholesterol is regulated in part by the rate of uptake and utilization of low-density lipoprotein cholesterol by the fetal adrenals for use in steroid biosynthesis. Direct evidence for this phenomenon and the kinetics of this process is, however, virtually impossible to obtain in a controlled experiment in the developing human. In the current study we sought to take advantage of the anticipated transient inhibition of the hypothalamic-pituitary-adrenal axis that occurs after antenatal therapy with glucocorticosteroids, to evaluate the temporal relationship between fetal adrenal steroids and plasma lipoprotein cholesterol levels in umbilical cord blood at delivery. STUDY DESIGN: Umbilical cord serum was obtained at delivery from 136 infants (30.5 +/- 2.7 weeks' gestation) who previously had been treated in utero with betamethasone, 12 mg per 12 or 24 hours for one or two doses and from 308 preterm infants (30.5 +/- 2.1 weeks) who had not been exposed to such therapy. We quantified the concentrations of dehydroepiandrosterone sulfate and cortisol as representative fetal adrenal steroids and also measured the total cholesterol and apolipoprotein B; the relationship between the steroids and lipids as a function of the interval between initial treatment and delivery was analyzed. RESULTS: Umbilical cord levels of dehydroepiandrosterone sulfate and cortisol were significantly reduced within the first 24 hours after initial treatment and remained significantly lower than in control infants through 4 days after initial treatment. In contrast, serum levels of cholesterol were significantly increased 3 to 4 days after treatment but fell on day 5. Serum levels of apolipoprotein B generally followed the same pattern as cholesterol. Cholesterol levels also were higher than normal in infants delivered >1 week after initial betamethasone treatment. CONCLUSIONS: The results of this study are consistent with the view that the plasma cholesterol pool in the fetus is regulated, at least in part, by the rate of uptake of low-density lipoprotein cholesterol and utilization by the fetal adrenals as substrate for steroidogenesis. Betamethasone also may influence cholesterol and lipoprotein synthesis in the fetus. PMID- 8623786 TI - Prenatal maternal dried blood screening with alpha-fetoprotein and free beta human chorionic gonadotropin for open neural tube defect and Down syndrome. AB - OBJECTIVE: Our purpose was to evaluate second-trimester prenatal screening for open neural tube defects and Down syndrome by use of dried blood specimen collection and transport. STUDY DESIGN: A prospective study of 7497 dried blood specimens from patients <35 years old was performed. Specimens were assayed for maternal blood alpha-fetoprotein and free beta-human chorionic gonadotropin. Patient-specific risks for both disorders were calculated and used to determine whether further evaluation was indicated. The study included an evaluation of the median and SD of analyte multiple of the median levels. RESULTS: The initial positive rate for open neural tube defect was 4.4% adjusted to 2.7% after ultrasonographic revision and collection of a second sample. The initial positive rate for Down syndrome was 3.6% adjusted to 2.8% after ultrasonographic revision. All seven cases of open neural tube defect were detected within the increased risk group. Six of 8 (75%) cases of Down syndrome were detected. The median alpha fetoprotein multiple of the median was 3.5 in open neural tube defect cases and 0.6 in Down syndrome cases. The median free beta-human chorionic gonadotropin multiple of the median was 2.4 in Down syndrome cases. The SD (log e) of alpha- fetoprotein and free beta-human chorionic gonadotropin in 5868 unaffected white patients was 0.4022 and 0.5635, respectively. CONCLUSION: Second-trimester dried blood screening for open neural tube defects and Down syndrome can achieve screening efficiency comparable to serum-based protocols with distinct advantages over the conventional method of blood collection. PMID- 8623788 TI - Diagnosis of fetal rubella infection with reverse transcription and nested polymerase chain reaction: a study of 34 cases diagnosed in fetuses. AB - OBJECTIVE: Our purpose was to develop a reliable method for prenatal diagnosis of fetal rubella infection through the detection of viral ribonucleic acid extracted from the chorionic villi, amniotic fluid, or fetal blood in pregnant women. STUDY DESIGN: Double amplification of rubella viral ribonucleic acid by nested polymerase chain reaction after reverse transcription was applied to samples from 34 women suspected of having rubella. The results were compared with those of serum antibody and levels of rubella virus-specific immunoglobulin M antibodies in fetal blood. RESULTS: Viral ribonucleic acid was revealed in 8 of 34 cases (23.5%). In the remaining 26 cases, healthy babies were born in 24, 1 was electively aborted, and 1 died in the thirty-sixth week of pregnancy of unknown causes. CONCLUSIONS: This method allowed very early detection of fetal rubella infection by sampling of chorionic villi and amniotic fluid compared with evaluation of the maternal symptoms and serum antibody levels. Fetal blood was also more useful for making a diagnosis up to the twentieth week of pregnancy than was measuring rubella virus-specific immunoglobulin M antibodies. PMID- 8623787 TI - Elevation of nitrate levels in pregnant ewes and their fetuses. AB - OBJECTIVE: Nitric oxide is a potent vasodilator released by endothelial cells that may play an important role in modulating maternal and fetal vascular tone in normal pregnancy. The current study was designed to evaluate whether plasma or urine nitrite and nitrate (the metabolites of nitric oxide) concentrations are elevated in pregnant compared with those of nonpregnant sheep and whether the nitrate concentrations in the fetal circulation were increased in comparison with the maternal circulation. STUDY DESIGN: Eleven pregnant sheep and seven nonpregnant oophorectomized sheep were instrumented with catheters in the maternal and fetal femoral arteries and veins, uterine and umbilical veins, and amniotic cavity. Blood, urine, and amniotic fluid samples were collected for nitrate determination at least 5 days after surgery. After extraction nitrate was reduced to nitrite and quantitated with the Greiss reagent. RESULTS: Arterial plasma nitrate concentrations in the pregnant sheep were significantly elevated compared with those of nonpregnant sheet (5.0 +/- 0.9 vs 2.5 +/- 0.6 micromol/L, p < 0.05). The urinary nitrate concentrations were also significantly increased in the pregnant sheep compared with those of nonpregnant sheet (89.9 +/- 16.3 vs 23.1 +/- 4.5 nmol/mg creatinine, p < 0.01). Fetal plasma nitrate concentrations were ninefold higher than the maternal nitrate concentrations (43.9 +/- 7 vs 5.0 +/- 0.9 micromol/L, p < 0.01), whereas amniotic fluid concentrations were extremely high (133.8 +/- 13.8 micromol/L, n = 3). No venous-arterial differences were measurable across either the maternal or fetal sides of the placenta. CONCLUSION: Nitrate concentrations in pregnant sheet and their fetuses are increased. The increased nitrate concentrations in the maternal and fetal circulations may reflect the increased nitric oxide synthesis, which may in part mediate the cardiovascular adaptations to normal pregnancy and the low systemic and umbilical vascular resistance in the fetus. PMID- 8623789 TI - Nitric oxide is involved in flow-induced dilation of isolated human small fetoplacental arteries. AB - OBJECTIVES: The aim of this study was to determine the dilation that occurs in response to increments of intraluminal flow in isolated human small fetoplacental arteries and to investigate the role played by nitric oxide. STUDY DESIGN: Small fetoplacental arteries (mean luminal diameter 482 +/- 31 micrometers, n = 17, at zero flow and pressure) were dissected from samples of placental tissue obtained from normal term vaginal deliveries and elective term cesarean sections for breech presentation. The arteries were mounted on a pressure myograph, and the response to increasing intraluminal flow was investigated in the presence and absence of a nitric oxide synthase inhibitor (N-omega-nitro-L-arginine methyl ester, 10(-4) mol/L). Basal tone was assessed in a separate group of arteries (n=7) by the removal of extracellular calcium. RESULTS: The presence of significant basal tone was demonstrated in these arteries. The arteries dilated in response to increasing luminal flow, and the dilation was significantly reduced by inhibition of nitric oxide synthase (control, 5.5% +/- 1.0% increase in artery diameter, n=10, vs 0.95 +/- 0.94, n=10, in the presence of N-omega nitro-L-arginine methyl ester, 10(-4) mol/L, p<0.01). CONCLUSIONS: The data substantiate previous indirect studies suggesting that nitric oxide plays a role in the fetoplacental circulation. Flow-induced nitric oxide release in the stem villous arteries may make an important contribution to maintenance of this low resistance circulation. PMID- 8623790 TI - Antibiotic treatment in preterm premature rupture of membranes and neonatal morbidity: a metaanalysis. AB - OBJECTIVE: We performed a metaanalysis of seven published randomized clinical trials to estimate more precisely the effect of prophylactic antibiotics on neonatal mortality, clinical sepsis of the neonate, respiratory distress syndrome, intraventricular hemorrhage, and necrotizing enterocolitis. STUDY DESIGN: To evaluate the effect of antibiotic treatment unaffected by other forms of treatment such as tocolytics or corticosteroids, investigations in which these additional measures were used were not included. We analyzed study patients and methods and abstracted quantitative outcome data. For each outcome both odds ratios and 95% confidence intervals were calculated. RESULTS: Among the 657 patients from seven trials published between 1989 and 1994, antibiotic therapy significantly reduced the risk of neonatal sepsis by 68% (odds ratio 0.32, 95% confidence interval 0.16 to 0.65, p=0.001) and that of intraventricular hemorrhage by 50% (odds ratio 0.50, 95% confidence interval 0.28 to 0.89, p=0.019). In contrast, no significant effect of antibiotics on overall neonatal mortality (odds ratio 0.92, 95% confidence interval 0.46 to 1.81), respiratory distress syndrome (odds ratio 0.84, 95% confidence interval 0.58 to 1.22), or necrotizing enterocolitis (odds ratio 1.27, 95% confidence interval 0.61 to 2.62) was found. CONCLUSION: This metaanalysis supports an improvement of neonatal morbidity in mothers with preterm premature rupture of membranes treated prenatally with different antibiotic regimens. PMID- 8623791 TI - Hydrostatic and osmotic pressure gradients produce manifestations of fetofetal transfusion syndrome in a computerized model of monochorial twin pregnancy. AB - OBJECTIVE: In spite of recent advances in the assessment and treatment of fetofetal transfusion syndrome, its underlying mechanism remains controversial. We aimed to determine whether the clinical features of fetofetal transfusion syndrome could be explained by unidirectional or bidirectional intertwin transfusion along placental vascular anastomoses. STUDY DESIGN: We constructed a dynamic computerized model of monochorial twin fetoplacental units on the basis of numerous interrelated hemodynamic, osmotic, and metabolic physiologic variables. The circulations were then linked by various combinations of direction and number of arteriovenous anastomoses. RESULTS: With unidirectional anastomoses disease severity, characterized by disparity in blood solids, depended on donor arterial pressure but not on the number of anastomoses. In the chronic state water movement resulting from raised osmotic pressure in the recipient and reduction in the donor produced hydroosmotic pressure equilibrium, reducing anastomotic flow to near zero. Atrial natriuretic peptide-driven urine production was markedly increased in the recipient because of the raised vascular hydrostatic pressure component. With bidirectional anastomoses recirculation between twins reduced discordancy in colloids and hematocrit, and the clinical picture was determined by the degree of asymmetry in the number of connections. CONCLUSIONS: Severe manifestations of fetofetal transfusion syndrome can be explained by unidirectional intertwin transfusion and lesser degrees by asymmetric bidirectional transfusion. PMID- 8623792 TI - Nicked free beta-subunit of human chorionic gonadotropin: a potential new marker for Down syndrome screening. AB - OBJECTIVE: Our purpose was to investigate maternal serum levels of nicked free beta-subunit in normal and Down syndrome pregnancies. STUDY DESIGN: Serum specimens were obtained from 64 karyotypically normal and 6 Down syndrome pregnancies before amniocentesis. Two immunoenzymometric assays for the beta- subunit of human chorionic gonadotropin were used to determine the level of free beta-subunits with peptide linkage breaks ("nicks") between residues 43 to 48. RESULTS: The mean level of nicked free beta-subunit in Down syndrome was 4.76 multiples of the median, which was significantly elevated compared with normal controls (1.07 multiples of the median, p<0.05). In 5 of 6 cases levels were > or = 2 multiples of the median, with a range of 1.96 to 15.43 multiples of the median, which was 2- to 3-fold higher than the regular free beta-subunit assay (mean level 1.53 multiples of the median, range 0.70 to 3.10 multiples of the median). In one case no nicked free-beta subunits were detectable. CONCLUSION: Our preliminary data indicate that nicked free beta-subunit of human chorionic gonadotropin might be a sensitive marker for Down syndrome screening. PMID- 8623793 TI - First-trimester Down syndrome screening: free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A. AB - OBJECTIVE: Our purpose was to determine the feasibility of a first-trimester Down syndrome screening protocol including free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A. STUDY DESIGN: First-trimester maternal blood samples from 22 Down syndrome and 483 control cases were assayed for free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A by enzyme-linked immunosorbent assay procedures. False-positive and detection rates were determined on the basis of Down syndrome risks calculated from the levels of biochemical markers and maternal age. Because 11 of the 22 Down syndrome cases were from older pregnancies (> or = 35 years old), rates were recalculated with the United States age distribution of live births to get a more representative estimate of false positives and detection efficiency. RESULTS: The median free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A levels in cases of Down syndrome was 2.09 (95% confidence interval 1.69 to 2.62) and 0.405 multiples of the median (95% confidence interval 0.28 to 0.67), respectively. At a 5.0% false-positive rate, 15 (68.2%) Down syndrome cases were detected. By the use of the age distribution of live births, 63% of cases could be expected to be detected at a 5.0% false-positive rate. CONCLUSION: First trimester free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A screening for Down syndrome can achieve detection rates as high as those associated with alpha-fetoprotein and human chorionic gonadotropin or alpha fetoprotein, human chorionic gonadotropin, and unconjugated estriol screening in the second trimester. Prospective studies are needed to further assess first trimester screening. PMID- 8623794 TI - Female urinary stress incontinence--does it have familial prevalence? AB - OBJECTIVE: Many factors have been suggested to contribute to the development of urinary stress incontinence in women. One of the suggested factors has been attributed to a genetic defect in the connective tissue in such patients. The purpose of this study was to evaluate the prevalence of urinary stress incontinence among relatives of patients with urinary stress incontinence. STUDY DESIGN: The prevalence of urinary stress incontinence among first-degree relatives was investigated in 259 females with urinary stress incontinence. A control group comprising 165 women without micturition disorders was randomly selected from our gynecologic outpatient clinic. The two groups were matched according to age, parity, maximal birth weight, and percentage of first-degree relatives on whom information was obtained. RESULTS: Data from 780 first-degree relatives in the study group and 474 in the control group were evaluated. We found that there was an overall prevalence of 20.3% (178/780) first-degree relatives in the study group compared with 7.8% (37/474) in the relatives of the control group (p<0.05). Among the mothers we found the prevalence of urinary stress incontinence to be 34.9% (71/203) in the study group compared with 12.7% (19/149) of mothers in the control group (p<0.005). Among the sisters in the study group the prevalence of urinary stress incontinence was 19.9% (73/367) compared with 6.8% (15/220) of sisters in the control group (p<0.005). Among the daughters we found the prevalence in the study group (6.7%) to be twice as high as in the control group (2.9%), but the difference did not reach statistical significance. CONCLUSIONS: The results of this study indicated a three-fold prevalence of urinary stress incontinence among first-degree relatives of female patients with urinary stress incontinence. This finding may support the theory of a genetic factor in the cause of urinary stress incontinence. PMID- 8623795 TI - Are there any clinical signs and symptoms that are related to endometriosis in infertile women? AB - OBJECTIVE: Our purpose was to assess the physical signs and clinical symptoms associated with endometriosis in infertile women. STUDY DESIGN: This case-control study was carried out in an academic tertiary hospital. There were 174 infertile women with endometriosis and 174 infertile women without endometriosis, all of them studied by laparoscopy. Before laparoscopy a standard interview and a standard physical examination were performed. RESULTS: Cul-de-sac nodularity was more frequent in infertile women with endometriosis than in infertile women without endometriosis (6.3% vs 0%). Although uterosacral tenderness was also more frequent in infertile women with endometriosis (7.5% vs 1.7%), uterosacral tenderness without nodularity was similar in both populations. Uterine retroversion and cul-de-sac obstruction frequencies were somewhat higher in the endometriosis group (p<0.10). The remaining signs and symptoms analyzed, including pelvic pain and dysmenorrhea, were similarly frequent in both populations. Symptoms were similarly frequent in all American Fertility Society stages, although adnexal mass was higher in stage IV. CONCLUSION: Uterosacral nodularity was pathognomonic of endometriosis in infertile women. Uterosacral nodularity and uterosacral tenderness (associated with uterosacral nodularity) were the only symptoms or signs of value to indicate endometriosis in infertile patients. The remaining clinical signs, as well as clinical symptoms, were of no value in diagnosing endometriosis in infertile women. PMID- 8623796 TI - Age and the ovarian follicle pool assessed with transvaginal ultrasonography. AB - OBJECTIVE: We tested whether transvaginal ultrasonography could detect the age related decrease in follicle counts that has been observed in autopsy studies. STUDY DESIGN: Thirty-one healthy volunteers in three age groups (22 to 25, 30 to 33, and 39 to 42 years) underwent ultrasonography in the follicular and luteal phases of the menstrual cycle. At the conclusion of the study the 124 ovarian scans were randomly ordered and antral follicles > or = 2 mm were counted by an evaluator unaware of age. Ordinary least-squares linear regression was used to estimate the associations of age with the total antral follicle count and with ln (1 + follicle count). RESULTS: The numbers of antral follicles > or = 2 mm decreased by about 60% between 22 and 42 years. Age-related decreases were similar for both phases of the cycle and held for both smaller (2 to 3.5 mm) and larger (>3.5 mm) follicles. CONCLUSION: We hypothesize that ultrasonographically derived counts of follicles provide a measure of reproductive age that may help to predict age-related phenomena. PMID- 8623797 TI - Smoking and cycle control among oral contraceptive users. AB - OBJECTIVE: Because cigarette smoking has a variety of antiestrogenic actions, we investigated the possibility that smoking may adversely affect spotting and bleeding among women using oral contraceptives. STUDY DESIGN: Three open-label, randomized clinical trials involving 16,506 cycles among 2956 oral contraceptive users were performed. RESULTS: Smokers reported a consistently higher frequency of spotting or bleeding than did nonsmokers. After recency and consistency of oral contraceptive use and progestin component were controlled for, smokers were, on average, 47% more likely to have spotting or bleeding than nonsmokers were over six cycles of oral contraceptive use, with higher levels of smoking associated with a greater frequency of spotting or bleeding. By the sixth cycle women who smoked > or = 16 cigarettes per day were almost three times more likely to have spotting or bleeding than were nonsmokers. CONCLUSION: Cigarette smoking adversely affects cycle control among oral contraceptive users, possibly by increasing estrogen catabolism. Although these findings also raise the possibility that oral contraceptive efficacy may also be impaired in smokers, an immediate concern is that oral contraceptive users who have spotting and bleeding are more likely to discontinue their use, placing them at risk of unintended pregnancy. PMID- 8623799 TI - Ovarian remnant syndrome: experience at Jackson Memorial Hospital, University of Miami, 1985 through 1993. AB - OBJECTIVE: Ovarian remnant syndrome has become increasingly recognized as a cause of pelvic pain after extirpative surgery. The purpose of this review was to compare our patient population with those reported in the literature, as well as to address the usefulness of careful retroperitoneal discussion in the surgical management of these patients. STUDY DESIGN: This article discusses the presentation and surgical management of eight cases of ovarian remnant. RESULTS: Data were obtained through a retrospective chart review of pathologically confirmed cases of ovarian remnant. Discussion focuses on the surgical management of these cases. CONCLUSION: In summary, extensive and careful retroperitoneal dissection is typically required to facilitate identification and removal of the ovarian remnant tissue. PMID- 8623798 TI - Development and characterization of an interleukin-2-transduced human ovarian carcinoma tumor vaccine not expressing major histocompatibility complex molecules. AB - OBJECTIVE: We initiated studies to develop cytokine-secreting human ovarian carcinoma cells for the purpose of using these cells as vaccines for the treatment of advanced epithelial ovarian cancer. STUDY DESIGN: A human ovarian carcinoma cell line (UCI-107) was genetically engineered to secrete the cytokine interleukin-2 by retroviral-mediated gene transduction. RESULTS: One clone, termed UCI-107A IL-2 AS, constitutively secreted high levels of interleukin-2 (i.e., 2000 to 2300 pg/ml/10(5) cells per 48 hours) for > 55 passages and 8 months of study. Unlike parental- and vector-transduced cells, UCI-107A IL-2 AS cells were aneuploid and failed to express major histocompatibility complex class I and HER2/neu surface antigens. UCI-107A IL-2 AS cells were highly resistant to killing by gamma irradiation and continued to produce high levels of interleukin 2 even after irradiation with 10,000 cGy. Balb/C nude mice injected intraperitoneally with UCI 107-A IL-2 AS cells survived significantly longer than control animals, with 25% of the animals totally rejecting their tumors. UCI-107A IL-2 AS was totally resistant to killing by fresh allogeneic peripheral blood lymphocytes in four hour chromium 51 release assays but induced high levels of killing in 72-hour long-term cytotoxic assays. CONCLUSION: The potential use of these interleukin-2-secreting ovarian carcinoma cells as vaccines for women with advance ovarian cancer will be discussed. PMID- 8623800 TI - The prevalence of urinary incontinence or prolapse among white and Hispanic women. AB - OBJECTIVE: Our purpose was to study the distribution of symptoms and disorders of urinary incontinence or prolapse among white and Hispanic women. STUDY DESIGN: Data were collected for all new patients referred to the urogynecology clinic over a 2-year period. One hundred twenty-one Hispanic and 50 white women consecutively referred to the urogynecology clinic as new patients over 2 years were included in the study. All patients underwent a detailed history and physical examination and multichannel urodynamic studies. Differences between the two groups were analyzed for significant differences by use of demographic data, presenting symptoms, urodynamic profiles, and final diagnosis or disorder. RESULTS: The symptoms of stress, urge, or mixed incontinence and prolapse were noted in 26%, 18%, 30%, and 14% of white women, respectively, compared with 41%, 9%, 21%, and 26% of Hispanic women (p=0.019). The diagnosis of genuine stress incontinence, motor urge incontinence, mixed incontinence, and pelvic organ prolapse without incontinence was made, respectively, in 16%, 44%, 6%, and 18% of white women versus 30%, 27%, 5%, and 18% of Hispanic women (p=0.33). The nondiagnostic rate after a complete evaluation for both groups was 10%. Hispanic women were of significantly higher gravidity (5.6 vs 3.8, p=0.001) and parity (4.7 vs 3.0, p=0.0006) than white women but were of comparable age. Medical problems and medications were too infrequent to allow meaningful comparison. White women were much more likely to have undergone a hysterectomy (36% vs 11.5%, p=0.0004, 95% confidence interval 1.8 to 10.3). CONCLUSION: Although the distribution of presenting symptoms of incontinence differs between Hispanic and white women, the final diagnosis after a complete urogynecologic evaluation was similar. Therefore presenting complaints in patients of different ethnic groups appears to be a poor predictor of the type of incontinence. Hispanic women were of higher gravidity and parity than white women were, but white women were more likely to have undergone a hysterectomy. Because the power of this study was limited by the 50 white women, larger prospective and longitudinal studies are needed to determine the significance of possible difference in etiologic factors. PMID- 8623801 TI - Delivery of dehydroepiandrosterone to premenopausal women: effects of micronization and nonoral administration. AB - OBJECTIVES: This single-dose study compares three dehydroepiandrosterone delivery methods (oral crystalline steroid, micronized steroid, and vaginal administration) to ascertain whether physiologic levels of circulating dehydroepiandrosterone and dehydroepiandrosterone sulfate can be obtained while increases in testosterone are minimized. STUDY DESIGN: Two randomized, double blind, placebo-controlled single-dose comparisons were made. For oral micronized versus crystalline dehydroepiandrosterone 300 mg doses of micronized or crystalline dehydroepiandrosterone were administered, followed by 6 hours of blood sampling (n=7). Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone levels were measured; areas under the curve and mean peak values were analyzed by Student-Newman-Keuls tests. For oral versus vaginal micronized dehydroepiandrosterone 150 mg oral or vaginal doses of micronized dehydroepiandrosterone were administered, followed by blood sampling over 12 hours (n=5). Data analysis was as described. RESULTS: Oral micronized and unmicronized dehydroepiandrosterone resulted in increases in serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone. Micronization increased the area-under-the-curve ratios for dehydroepiandrosterone sulfate/dehydroepiandrosterone and dehydroepiandrosterone sulfate/testosterone. Vaginal administration provided equivalent serum dehydroepiandrosterone; however, it failed to increase dehydroepiandrosterone sulfate or testosterone over placebo. CONCLUSION: Micronization of oral dehydroepiandrosterone diminishes bioconversion to testosterone. Vaginal dehydroepiandrosterone delivers equivalent dehydroepiandrosterone but substantially diminishes dehydroepiandrosterone bioconversion. PMID- 8623802 TI - Laparoscopic versus abdominal myomectomy: a prospective, randomized trial to evaluate benefits in early outcome. AB - OBJECTIVE: Our purpose was to investigate the advantages of laparoscopic myomectomy versus laparotomy. STUDY DESIGN: A prospective, randomized trial was performed on 40 women, 22 to 44 years old, undergoing myomectomy. Patients were randomized to have laparoscopy (n=20) or laparotomy (n=20). The intensity of pain was assessed by a visual analog scale at 0, 1, 2, and 3 days postoperatively. The proportions of patients who were analgesic free on day 2, discharged from the hospital by day 3, and feeling fully recuperated on day 15 were also compared. RESULTS: The intensity of postoperative pain was lower (p<0.05) after laparoscopy than after laparotomy. A higher (p<0.05) proportion of patients was analgesic free on day 2, discharged from hospital by day 3, and feeling fully recuperated on day 15 after laparoscopy compared with laparotomy. CONCLUSION: Laparoscopic myomectomy may offer the benefits of lower postoperative pain and shorter recovery time in comparison with laparotomy. PMID- 8623803 TI - Transferrin increases adherence of iron-deprived Neisseria gonorrhoeae to human endometrial cells. AB - OBJECTIVE: Our purpose was to study the effects of iron deprivation with and without human transferrin supplementation on the adherence and invasion of Neisseria gonorrhoeae to human endometrial cells. STUDY DESIGN: N. gonorrhoeae grown with our without iron was placed in media alone or media containing 2.5 mg/ml saturated human transferrin or unsaturated transferrin. N. gonorrhoeae was inoculated onto polarized human endometrial carcinoma cell (HEC 1-B) monolayers, and at various intervals monolayers were washed and incubated with media containing gentamicin or media alone. Colony-forming units per milliliter of N. gonorrhoeae associated with HEC 1-B cells were then determined. N. gonorrhoeae strains tested included both a transferrin receptor-positive (wild-type) and a transferrin receptor-negative mutant. Differences in percent of original inoculum remaining at varying time points were analyzed by the Mann-Whitney U test. Transmission electron microscopy using a primary endometrial cell line was used to verify findings. RESULTS: Iron-negative N. gonorrhoeae exhibited less adherence than did iron-positive N. gonorrhoeae. No difference in HEC 1-B adherence was seen when either saturated transferrin or unsaturated transferrin was added to the iron-positive N. gonorrhoeae. With iron-negative N. gonorrhoeae addition of either saturated transferrin or unsaturated transferrin significantly increased N. gonorrhoeae adherence although unsaturated transferrin did not permit growth of iron-negative N. gonorrhoeae in tissue culture media alone. Transmission electron microscopy confirmed increased adherence of iron-negative N. gonorrhoeae supplemented with unsaturated transferrin. An iron-negative N. gonorrhoeae mutant lacking the transferrin receptor exhibited no adherence regardless of addition of saturated transferrin or unsaturated transferrin. Invasion could not be quantitated reliably because of persistence of gentamicin effect. CONCLUSION: Iron and transferrin increased attachment of N. gonorrhoeae to human endometrial cells. PMID- 8623804 TI - Peripheral joint laxity increases in pregnancy but does not correlate with serum relaxin levels. AB - OBJECTIVE: Our purpose was to evaluate peripheral joint laxity during pregnancy and to determine whether serum relaxin levels are associated with increased joint laxity. STUDY DESIGN: A prospective observational study was performed. RESULTS: A significant increase in joint laxity was found in five of seven peripheral joints over the course of the pregnancy and post partum. There was no correlation with serum relaxin levels. There were no significant differences in joint laxity on the basis of parity, age, or prepregnancy exercise levels. CONCLUSIONS: Peripheral joint laxity is noted to increase as pregnancy progresses. The cause of this change is undetermined. PMID- 8623805 TI - Accuracy of maternal perception of preterm uterine activity. AB - OBJECTIVE: Our purpose was to compare the accuracy of maternal perception of preterm uterine activity by self-palpation versus home uterine activity monitoring. STUDY DESIGN: A total of 72,962 uterine activity records of 778 women receiving home uterine activity monitoring services were analyzed. Simultaneously with home uterine activity monitoring, the women indicated by an electronic marker when they felt a "contraction" through self-palpation. The perceptions of the women were compared to the tocodynamometrically measured uterine activity. RESULTS: Using self-palpation, women correctly identified 17.2% of contractions recorded by tocodynamometry. Overall mean percent correct correlation per patient was only 14.1%. Patients missed an average of 85.7% of their contractions. Patients incorrectly perceived contractions that were not present an average of 40.3% of the time. Singleton gestations had significantly better correct correlations than twin gestations. Multiparous women had improved correlations compared with primiparous women. No significant correlation was found between maternal perceptive ability and gestational age. CONCLUSION: Women were unable to perceive accurately the presence or absence of preterm uterine activity through self-palpation compared with simultaneous measurement by home uterine tocodynamometry. PMID- 8623806 TI - Epidermal growth factor increases phosphoinositide turnover and intracellular free calcium in an immortalized human myometrial cell line independent of the arachidonic acid metabolic pathway. AB - OBJECTIVES: The objectives of this study were to determine whether epidermal growth factor increases intracellular calcium and phosphoinositide turnover in human myometrial cells by a tyrosine kinase-mediated mechanism, to evaluate an obligatory role for arachidonic acid metabolites in these actions, and to compare the actions of epidermal growth factor and oxytocin. STUDY DESIGN: Intracellular calcium and phosphoinositide turnover were measured in a myometrial cell line after stimulation with epidermal growth factor (0.1 to 100 nmol/L) or oxytocin (20 nmol/L). The effects of nifedipine, thapsigargin, genestein and tyrphostin, the guanosine triphosphate binding protein antagonist GPA-7, indomethacin, and nordihydroguaiaretic acid were determined. Data were analyzed by analysis of variance and Duncan's multiple-range test. RESULTS: Epidermal growth factor stimulated phosphoinositide turnover and increased intracellular calcium in a dose-dependent manner (median effective concentration 2.6 nmol/L). In contrast to oxytocin, the effects of epidermal growth factor were inhibited by tyrosine kinase inhibitors but not by GPA-7. Indomethacin and nordihydroguaiaretic acid did not inhibit the epidermal growth factor-stimulated increase in intracellular calcium. CONCLUSIONS: The acute epidermal growth factor-stimulated increase in intracellular calcium in this myometrial cell line is primarily derived from release of calcium from intracellular stores, and it involves the activation of a tyrosine kinase, presumably the epidermal growth factor receptor. Arachidonic acid metabolites are not obligatory intermediates. Oxytocin increases phosphoinositide turnover and intracellular calcium by a distinctly different pathway. PMID- 8623807 TI - Intrauterine infection and the effects of inflammatory mediators on prostaglandin production by myometrial cells from pregnant women. AB - OBJECTIVE: Our purpose was to evaluate the effects of known stimulants of prostaglandin production on cultured myometrial cells from women in labor with and without intrauterine infection. STUDY DESIGN: Myometrial segments were obtained from 16 patients between 33 and 40 weeks' gestation who had been in labor for > or = 8 hours at cesarean delivery; 8 patients had clinical chorioamnionitis and 8 did not. Myometrial cells were isolated and grown in culture. Incubations were conducted with interleukin-1 beta, tumor necrosis factor-alpha, or epidermal growth factor. Prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) were measured by radioimmunoassay, and cellular protein was determined. RESULTS: Cultured human myometrial cells from patients with and without prior intrauterine infection produced prostaglandins in response to interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor at a significantly increased rate (p<0.05 vs controls at and above 10 ng/ml of interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor). The major prostaglandin produced in response to each stimulant was 6-keto-prostaglandin F1 alpha; however, this response was attenuated in cells from patients with intrauterine infection. CONCLUSIONS: Cultured human myometrial cells from patients with and without prior intrauterine infection respond to known stimulants of prostaglandin production. Prior intrauterine infection has no effect on baseline prostaglandin production, but the amount of prostacyclin produced as a response to cellular stimulants is decreased with prior intrauterine infection. This effect may have a role in regulating myometrial function in intrauterine infection. PMID- 8623808 TI - Effects of selected vasoconstrictor agonists on isolated omental artery from premenopausal nonpregnant women and from normal and preeclamptic pregnant women. AB - OBJECTIVE: Our purpose was to compare the responsiveness of omental resistance arteries from nonpregnant women and from normotensive and preeclamptic pregnant women to selected contractile agonists. STUDY DESIGN: Omental artery rings with intact endothelium from normotensive premenopausal nonpregnant women and from normal and preeclamptic pregnant women were mounted in Krebs-bicarbonate solution in organ baths for isometric tension recording. After the presence of endothelium was confirmed, cumulative concentrations of norepinephrine, serotonin, U46619, and endothelin-1 were added. Concentration-response curves were constructed and expressed as percentage of a reference 60 mmol/L potassium chloride contraction. Data analysis was by repeated-measures analysis of variance. Newman-Keuls test, and paired or unpaired Student t test, as appropriate. Statistical significance was by two-tailed p<0.05. RESULTS: Endothelin-1 and U46619 increased tension similarly in all three groups. Norepinephrine increased tension in nonpregnant vessels to a greater extent than in either preeclamptic or pregnant vessels (nonpregnant 114.3 +/- 5.42% vs pregnant 65.2 +/- 10.5%, p<0.05). Nonpregnant omental artery developed significantly greater tension than did pregnant tissue at three concentrations of norepinephrine (10(-5) mol/L, 3 x 10(-5) mol/L, 10(-4) mol/L), and preeclamptic vessels developed more tension than that from normal pregnant vessels at 3 x 10(-6) mol/L (p=0.06) and 10(-5) mol/L (p<0.05). There was a negligible change in tension with increasing concentrations of serotonin in the vessels from nonpregnant women; serotonin-induced contraction in the omental arteries from normotensive pregnant women and preeclamptic patients was <6% of the potassium chloride reference contraction, but this was significantly (p<0.05) different from that of the nonpregnant women. CONCLUSIONS: Omental artery segments from nonpregnant, normotensive pregnant and preeclamptic women contract similarly to endothelin-1 and U46619 but exhibit variable responses to norepinephrine and serotonin. PMID- 8623809 TI - Vertical transmission of human papillomavirus from infected mothers to their newborn babies and persistence of the virus in childhood. AB - OBJECTIVE: The purpose of this study was to determine the potential for human papillomavirus to be transmitted vertically. STUDY DESIGN: We started a systematic study of children 0.3 to 11.6 years old born to mothers included in the cohort of 530 women prospectively followed up for genital human papillomavirus infections in Kuopio since 1981. So far 98 children have been examined. The examinations included medical history, clinical examination of the oral cavity and hand warts, and cytologic samples from the oral mucosa for detection of human papillomavirus deoxyribonucleic acid with polymerase chain reaction with subsequent Southern blot hybridization. RESULTS: Human papillomavirus deoxyribonucleic acid was found in 31 of the 98 (31.6%) oral scrapings. with MY09 and MY11 human papillomavirus primers, 12 of the 98 were positive for human papillomavirus deoxyribonucleic acid in the electrophoresis gel and in subsequent hybridization. Nineteen of the positive samples were not visible in the gel but become positive when hybridized. At delivery, 5 mothers had genital human papillomavirus infection with the same virus type found in her child. In the additional 11 mothers genital human papillomavirus infection with the same virus type as in the child was diagnosed a few months before or after delivery. Mothers of the 25 children shown to be negative for oral human papillomavirus were also human papillomavirus deoxyribonucleic acid negative at delivery. Minor hyperplastic growths of the oral mucosa were found in 21 of the 98 children (21%). One child had a papilloma where human papillomavirus 16 deoxyribonucleic acid was detected, as was also found in her mother's genital area at delivery. CONCLUSIONS: Our results support the concept that an infected mother can transmit human papillomavirus to her child. PMID- 8623810 TI - A model for the antiphospholipid antibody syndrome: monoclonal antiphosphatidylserine antibody induces intrauterine growth restriction in mice. AB - OBJECTIVE: Antiphospholipid antibodies are associated with clinical intrauterine growth restriction. In this study we investigated whether immunoglobulin M monoclonal antibodies against phosphatidylserine or cardiolipin or cross-reactive with both phospholipids would induce intrauterine growth restriction in an experimental model of the antiphospholipid antibody syndrome. STUDY DESIGN: Balb/c or CD-1 mice were injected intraperitoneally on day 8 of pregnancy with three immunoglobulin M monoclonal antibodies that differentiated between cardiolipin- and phosphatidylserine-dependent antigens or with control immunoglobulin M monoclonal antibodies against irrelevant antigens. The animals were killed on day 15 of pregnancy and placental and fetal weights were measured. RESULTS: Monoclonal antibody 3SB9b, which reacted in enzyme-linked immunosorbent assays with phosphatidylserine but not cardiolipin, induced a significant reduction in both fetal and placental weights. Monoclonal antibodies BA3B5C4, which was cross-reactive with cardiolipin and phosphatidylserine, and D11A4, which reacted with cardiolipin, did not alter fetoplacental weights. CONCLUSION: An antiphospholipid antibody that reacts with phosphatidylserine induces significant fetal and placental intrauterine growth restriction in a mouse model for the antiphospholipid antibody syndrome, but those that react with cardiolipin do not. PMID- 8623811 TI - Efficacy of P6 acupressure in the treatment of nausea and vomiting during pregnancy. AB - OBJECTIVE: Our purpose was to investigate the efficacy of P6 acupressure in reducing or relieving symptoms of nausea with or without vomiting and retching during pregnancy. STUDY DESIGN: Symptomatic pregnant volunteers (n=161) participated in a 7-day community-based clinical trial. All participants were assigned to one of three groups (i.e., P6 acupressure, placebo [acupressure bands inappropriately placed], or control) on the basis of a process of blocked randomization. Data were analyzed by error bar charts and analysis of variance of difference scores. RESULTS: Of 161 women, 149 (92.5%) completed the protocol. Irrespective of group assignment, participants reported significant decreases in nausea (p<0.0009) and vomiting or retching (p<0.0009). However, there was no differential treatment effect as a result of acupressure. CONCLUSION: There was no apparent medical benefit from the use of P6 acupressure. Our findings differ from other recently published studies that did not include a control group. PMID- 8623812 TI - The cost-effectiveness of human immunodeficiency virus screening in pregnancy. AB - OBJECTIVE: My purpose was to evaluate the cost-effectiveness of screening for human immunodeficiency virus during pregnancy as part of a protocol in which zidovudine was used to reduce the risk of vertical transmission. STUDY DESIGN: This mathematic model used decision analysis to calculate the marginal cost effectiveness of screening for human immunodeficiency virus in pregnancy and treating human immunodeficiency virus-positive women with zidovudine. Cost and probability assumptions were drawn from a literature review. Sensitivity analyses were performed for important costs and probabilities. RESULTS: When baseline cost and probability assumptions were used, the marginal cost-effectiveness of human immunodeficiency virus screening was $436,927 when the prevalence of human immunodeficiency virus in the population was low (0.00075) and $198,510 when the prevalence was average (0.0015). Above a prevalence of human immunodeficiency virus of 0.009, testing is both cheaper and more effective than not testing. Of the cost variables examined, the charge for a negative testing sequence had the greatest impact on cost-effectiveness. CONCLUSION: Human immunodeficiency virus testing in pregnancy is cost-effective in populations in which the prevalence of human immunodeficiency virus exceeds 9 per 1000 population. Depending on how individual lives saved are valued, screening may also be warranted in populations with lower prevalences of infection. PMID- 8623813 TI - Impaired insulin-stimulated nonoxidative glucose metabolism in glucose-tolerant women with previous gestational diabetes. AB - OBJECTIVE: Our purpose was to investigate insulin sensitivity and insulin secretion in women with previous gestational diabetes. STUDY DESIGN: Twelve women with previous gestational diabetes and 11 controls were examined by oral and intravenous glucose tolerance tests and a hyperinsulinemic euglycemic clamp including indirect calorimetry. All women were lean and had normal oral glucose tolerance test results. Activities of glycogen synthase, phosphofructokinase, and hexokinase were measured in vastus lateralis muscle biopsy specimens obtained in the basal state and after insulin stimulation. RESULTS: Women with previous gestational diabetes had a decreased glucose disposal rate (p<0.01) because of a reduced insulin-stimulated nonoxidative glucose metabolism (6.63 +/- 0.47 vs 9.04 +/- 0.57 mg/kg fat-free mass per minute, p<0.01). The muscle activities of glycogen synthase, phosphofructokinase, and hexokinase were similar in the two groups. The first-phase insulin response to the intravenous glucose tolerance test was, in absolute terms, comparable in the two groups. However, when the decreased insulin sensitivity was taken into account, women with previous gestational diabetes had a relative insulin secretion deficiency. CONCLUSION: Women with previous gestational diabetes have a decreased insulin sensitivity and a relative impairment of insulin secretion. PMID- 8623814 TI - Mechanical stress eliminates the effects of plasma from patients with preeclampsia on endothelial cells. AB - OBJECTIVE: Our purpose was to determine whether mechanical deformation alters in vitro effects of plasma from patients with preeclampsia on endothelial cell function to produce a paradigm similar to the in vivo disease state. STUDY DESIGN: The effects of 2% plasma from 12 patients with preeclampsia and 12 normal pregnant women on prostacyclin, nitric oxide, and endothelin production by cultured endothelial cells were measured in the presence or absence of cyclic stretch and laminar shear stress. RESULTS: In the absence of mechanical stress plasma from patients with preeclampsia resulted in greater prostacyclin and nitric oxide production (but no change in endothelin production) compared with plasma from normal pregnant women. Cyclic stretch did not affect prostacyclin or endothelin production but produced similar increases in nitric oxide production in cells exposed to plasma from the two groups. Shear stress markedly increased prostacyclin and nitric oxide production (but did not alter endothelin production). In the presence of shear stress there were no differences in production rates of nitric oxide or prostacyclin between cells exposed to plasma from the two groups. CONCLUSION: Shear stress alters the effects of plasma on endothelial cells. This is an important caveat in the interpretation of previous static in vitro culture studies and may partially explain the dichotomy between in vitro studies and in vivo findings in preeclampsia. PMID- 8623815 TI - Preeclampsia: the effects of serum on endothelial cell prostacyclin, endothelin, and cell membrane integrity. AB - OBJECTIVE: Our purpose was to determine whether serum from women with preeclampsia or gestational hypertension (1) decreased endothelial cell prostacyclin, (2) increased endothelial cell endothelin, and (3) caused endothelial cell damage. STUDY DESIGN: Production of 6-keto-prostaglandin F1 alpha and endothelin by cultured endothelial cells was measured after 48 hours' incubation with sera from 23 nonpregnant women, 23 normal pregnant women, 12 women with preeclampsia, and 11 women with gestational hypertension. Structure damage of endothelial cells was assessed by a chromium release assay. RESULTS: Serum from normal pregnant women induced more endothelial prostacyclin but less endothelin than did serum form nonpregnant women (p<0.05). No difference was found between normal pregnant and hypertensive pregnant women for prostacyclin production, but serum of preeclamptic women induced less endothelin production than did that of normal pregnant women (p<0.05). Chromium 51 release by endothelial cells was similar between normal pregnant and hypertensive pregnant groups. CONCLUSIONS: Serum from preeclamptic women stimulates less endothelin production than does serum from normal pregnant women but does not alter prostacyclin production and is not cytotoxic to endothelial cells after short term incubation. PMID- 8623816 TI - Uterine contractility as assessed by abdominal surface recording of electromyographic activity in rats during pregnancy. AB - OBJECTIVES: The primary aim of this study was to develop a noninvasive method for recording uterine contractile activity during pregnancy by systematically evaluating whether abdominal surface electromyography is representative of uterine electrical and contractile events. A secondary purpose was to use these techniques to determine when the uterus is in a state of preparedness for labor. STUDY DESIGN: Electrical activity was acquired by use of unipolar electrodes attached simultaneously to the uterine wall and to the abdominal surface of pregnant rats. Intrauterine pressure was recorded with a pressure transducer inserted in the uterine cavity. Computer-acquired records of electrical events (electromyography) and pressure were compared on different days of gestation, during spontaneous labor at term, or during preterm birth induced with an antiprogestin. Similarly, electrical activity was assessed after administration of agents that either stimulate (oxytocin) or inhibit (isoproterenol) contractility. Correlation analyses were performed between uterine electromyographic activity, surface electromyographic activity, and concomitant intrauterine pressure. The effects of vaginal wall stimulation were also evaluated at different times of pregnancy. RESULTS: The electrical activity recorded early in pregnancy (day 18) from the uterus consisted of irregular electrical bursts with little correspondence to the signals recorded from the surface (R2=0.006). Later in gestation (days 19 through 21) the electrical activity of the uterus became more regular, consisting of frequent bursts with accordance between the signals recovered from the uterus and those collected from the surface (R2=0.95). During labor (preterm or term) bursts recorded from either the uterus or the abdominal surface were of large amplitude and corresponded to ample changes in intrauterine pressure. Correlation coefficients between uterus and surface and uterus and pressure were R2=1 and 0.96, respectively. Similarly, during preterm labor the coefficients were R2=1 for uterine electromyographic activity versus surface electromyographic activity and R2=0.99 for uterine electromyographic activity versus intrauterine pressure. Vaginal stimulation in early gestation was not followed by subsequent signal conduction to the uterus, whereas during delivery activity induced in the vagina propagated to the uterus and consequently to the abdominal surface. CONCLUSIONS: Abdominal surface recording of uterine electrical events are representative of the activity generated by the muscle cells of the uterus. During term and preterm labor uterine electrical activity and intrauterine pressure achieve maximum activity. Electromyographic monitoring with vaginal stimulation of the uterus may allow prediction of when the uterus is in a state required for labor. PMID- 8623817 TI - Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assessment of the therapeutic ability of drugs against preterm delivery. AB - OBJECTIVE: Our purpose was to establish a new animal model for human preterm delivery for assessment of the protective effect of drugs against preterm delivery. STUDY DESIGN: C3H/HeN, C3H/HeN, and BALB/c female mice impregnated by C3H/HeN, B6D2F1, and B6D2F1 male mice, respectively, were treated intraperitoneally with Escherichia coli lipopolysaccharide (0 to 100 microgram/kg, single dose or repeated doses at 1- to 6-hour intervals) on days 12 through 17 of pregnancy. On day 15 of pregnancy, the C3H/HeN females that had been impregnated by B6D2F1 males and administered lipopolysaccharide were treated intraperitoneally with indomethacin (1000 microgram/kg), ritodrine hydrochloride (1000 microgram/kg), urinary trypsin inhibitor (25 x 10(4) units/kg), or gabexate mesylate (100 mg/kg); preterm or term delivery was recorded for these mice. RESULTS: C3H/HeN females impregnated by B6D2F1 males revealed the highest (100%) incidence of preterm delivery when the females were treated with 50 microgram/kg lipopolysaccharide twice at a 3-hour interval on day 15 or 17 of pregnancy. Indomethacin and urinary trypsin inhibitor used separately significantly decreased the incidence of preterm delivery, but only urinary trypsin inhibitor, and not any of the other drugs, significantly increased the incidence of term delivery in the mice. CONCLUSION: A new animal model for investigation of preterm delivery was established, and its usefulness for assessment of the protective effect of drugs against preterm delivery was demonstrated. PMID- 8623818 TI - Pregnancy outcomes and health care use: effects of abuse. AB - OBJECTIVE: Our purpose was to determine whether pregnancy and neonatal outcomes differed between abused and nonabused women. STUDY DESIGN: Women (N=1014) who completed an abuse questionnaire during pregnancy were followed up after delivery. The 242 women reporting past abuse and the 59 women reporting abuse in pregnancy were grouped in terms of recency and severity of domestic abuse, and their pregnancy and birth outcomes were compared with those of nonabused women with the chi-square test, analysis of variance, and multivariate logistic regression techniques. RESULTS: Abused women smoked more cigarettes (p<0.0001) and took more prescription drugs (p<0.03) and antidepressants (p>0.05) than nonabused women; they were more likely to have epilepsy (p=0.0002) and asthma (p=0.0018), and also they used social work services more often (p>0.0001). Obstetric histories revealed a higher incidence of miscarriage (p=0.0014), two or more pregnancy terminations (p>0.0001), and neonatal death (p=0.0503) among the abused group. Although abused women delivered infants whose mean birth weight was 132 gm lower than that of nonabused women, the difference was not significant after adjustments were made. Mildly and moderately abused women were admitted to the hospital more frequently during pregnancy (p=0.0067). CONCLUSION: Domestic abuse adds significantly to the cost of health care during pregnancy and is associated with poor maternal and fetal outcomes. PMID- 8623819 TI - Uteroplacental and luteal circulation in normal first-trimester pregnancies: Doppler ultrasonographic and morphologic study. AB - OBJECTIVE: Our purpose was to establish reference data representative of normal findings at transvaginal color and spectral Doppler ultrasonographic examination of the uteroplacental and luteal circulation in the first trimester and to relate the Doppler findings to morphologic data. STUDY DESIGN: A cross-sectional study was performed of 64 uncomplicated pregnancies of 5 to 11 completed gestational weeks terminated by legal abortion for psychosocial reasons. Doppler examinations of the main uterine arteries, subchorionic arteries, intrachorionic area, and corpus luteum were performed. Abortion material was examined morphologically. RESULTS: In the uterine and subchorionic arteries blood flow velocity increased and pulsatility index values decreased with advancing gestation. Arterial or venous Doppler shift spectra were recorded from the intrachorionic area in 91% (58/64) of the pregnancies. Intrachorionic arterial blood flow velocities and pulsatility index values were low and did not change with advancing gestation; the mean time-averaged maximum velocity, peak systolic velocity, and pulsatility index values were 4.6 cm/sec, 6.0 cm/sec, and 0.47, respectively. Morphologic examination showed trophoblast plugs in the spiral arteries to consistently manifest multiple spaces containing red blood cells at the choriodecidual junction. CONCLUSION: Blood flow velocity in the uteroplacental arteries increases and pulsatility index values decrease with advancing gestation, indicating a steady increase in uterine perfusion during the first trimester. Our findings suggest intervillous flow to be present as early as the first trimester. PMID- 8623820 TI - Change in serum beta-human chorionic gonadotropin after abortion with methotrexate and misoprostol. AB - OBJECTIVE: The purpose of this study was to determine the normal beta-human chorionic gonadotropin change within 24 hours after a medical abortion. Because a medical abortion creates a "miscarriage," these data can represent the serum beta human chorionic gonadotropin changes that would occur with a complete spontaneous abortion. Knowledge of normal beta-human chorionic gonadotropin changes after a spontaneous abortion may help to differentiate within a 24-hour period a complete from an incomplete spontaneous abortion or an ectopic pregnancy. STUDY DESIGN: Data from recent trials that used methotrexate and misoprostol for abortion at < or = 56 day's gestation were reviewed. Patients from each of four trials were included in this analysis if (1) they received both methotrexate intramuscularly and misoprostol vaginally and (2) they had serum beta-human chorionic gonadotropin levels drawn on both the day of misoprostol administration and the next day. RESULTS: The change in serum beta-human chorionic gonadotropin was evaluated in 86 patients. Subjects who had a complete abortion after receiving methotrexate and a single dose of misoprostol had a decline in serum beta- human chorionic gonadotropin of 66% +/- 8%. All other subjects had a decline of 25% +/- 19% (p=0.0001). CONCLUSIONS: An aborting pregnancy, if the abortion has occurred, should have a beta-human chorionic gonadotropin decrease of at least 48% within approximately 24 hours. This decline, however, does not guarantee that the abortion is complete. A patient with a serum beta-human chorionic gonadotropin level that has not declined by a minimum of approximately 50% over 24 hours is unlikely to have a complete abortion. PMID- 8623821 TI - Myometrial arginase activity increases with advancing pregnancy in the guinea pig. AB - OBJECTIVES: Arginase has been suggested to play an important role in cellular growth and development, particularly important to the fetus, by supplying L ornithine for the synthesis of polyamines. The purpose of this investigation was to determine whether pregnancy alters myometrial arginase activity and whether estradiol was responsible for the change. STUDY DESIGN: Myometrium and kidney were obtained from nonpregnant and pregnant guinea pigs of known gestational age. Arginase activity was measured under physiologic conditions by the conversion carbon 14-labeled guanidino-L-arginine to carbon 14-labeled urea. The concentrations of the enzyme's substrate, L-arginine, and its principal metabolite, L-ornithine, were measured in myometrium from near-term pregnant animals by use of an amino acid analyzer. Finally, a group of random cycle guinea pigs received 500 microgram/kg estradiol for 5 days before the myometrium was removed. RESULTS: Myometrial arginase activity in pregnant animals was more than double that of myometrium from nonpregnant animals by the time the first measurement was made at 0.14 gestation. It continued to rise, peaking at values >25-fold higher than the nonpregnant activity by 0.90 gestation. Arginase activity in the myometrium underlying the placental implantation site was >25 fold higher (p<0.05) than myometrium from nonpregnant animals when first studied at 0.63 gestation and 10-fold higher than the contralateral fundal myometrium at the same time of gestation. Myometrial arginase activity in the sterile horn of six pregnant animals was half that of the horn containing one or more pups, but still five times higher than that of nonpregnant animals. Renal arginase activity also rose with advancing pregnancy, but the magnitude of the increase (up to 2 fold) was much smaller than that observed in either the fundal or placental implantation site myometrium. Estradiol had no significant effect on myometrial arginase activity. CONCLUSIONS: These studies demonstrate that pregnancy increases myometrial arginase activity and that the presence of placenta or fetus is necessary for the maximal effect. PMID- 8623822 TI - Amnioinfusion in women with previous cesarean births: a preliminary report. AB - OBJECTIVE: Our purpose was to evaluate the use of intrapartum amnioinfusion in women undergoing a trial of labor after a previous cesarean delivery. STUDY DESIGN: Labor and delivery records of women undergoing intrapartum amnioinfusion over a 1-year period were reviewed retrospectively. Neonatal data were obtained by chart review. RESULTS: During the study period 936 women underwent intrapartum amnioinfusion for the following indications: oligohydramnios (76.6%), meconium stained amniotic fluid (12.8%),and variable decelerations (7.5%). Among these, 122 (13%) had previous cesarean births. Seventy-one (58.2%) women were delivered vaginally; the remaining 51 (41.8%) were delivered by repeat cesarean. Among women undergoing intrapartum amnioinfusion there were no statistically significant differences (p<0.05) between those with previous cesarean births and those with unscarred uteri with respect to the following: cesarean section for fetal distress (1.6% vs 1.4%), meconium aspiration (0% vs 0.9%), or low 5-minute Apgar scores (2.5% vs 0.9%). There were no perinatal or maternal deaths in either group. One uterine rupture occurred in a woman with one previous cesarean birth. CONCLUSIONS: Amnioinfusion appears to be an acceptable procedure in women with previous cesarean births. The incidence of uterine rupture (0.8%) noted was similar to the 10-year institutional rate at Los Angeles County-University of Southern California Women's Hospital (0.7%). PMID- 8623824 TI - De mulierum organis generationi inservientibus tractatus novus... PMID- 8623823 TI - Endothelial cell proliferation is suppressed by plasma but not serum from women with preeclampsia. AB - OBJECTIVES: Evidence has been sought for a circulating factor derived from the placenta that suppresses endothelial cell proliferation and hence contributes to the maternal endothelial cell disturbances of preeclampsia. STUDY DESIGN: The effects of sera and plasmas from women with proteinuric preeclampsia and from matched normal pregnant control women on endothelial cell proliferation were compared. The recovery of endothelial cell inhibitory activity from syncytiotrophoblast microvesicles added to male blood and prepared as plasma or serum was determined to investigate the possible placental origin of the inhibitory factor. RESULTS: Sera from women with preeclampsia did not inhibit endothelial cell proliferation. In contrast, plasma from preeclamptic women significantly suppressed endothelial cell growth at 20% dilution compared with controls, and suppression was more pronounced in severe preeclampsia. The inhibitory activity of syncytiotrophoblast microvesicles added to blood could not be recovered from serum, only from plasma, which may explain why there was no suppression with sera from preeclamptic women. CONCLUSIONS: These results confirm that there is a blood-borne endothelial cell suppressive factor in preeclampsia that may be derived from the placenta. PMID- 8623825 TI - Impact of laparoscopically assisted vaginal hysterectomy: the missing link. PMID- 8623826 TI - Misoprostol for induction of labor. PMID- 8623827 TI - Fetal fibronectin and the safety of outpatient management of preterm labor symptoms. PMID- 8623828 TI - Of policies and palsies. PMID- 8623829 TI - Placental vascular anatomy and twin transfusion syndrome. PMID- 8623830 TI - The need for seizure prophylaxis in preeclampsia is still unresolved. PMID- 8623831 TI - Activated protein C resistance and pregnancy complications. PMID- 8623833 TI - But isn't that your job, son? PMID- 8623832 TI - The T/QRS ratio of the fetal electrocardiogram--how to (in)validate experimental data. PMID- 8623834 TI - The wealth of nations: knowledge as a national resource. PMID- 8623835 TI - Transgene expression in the rhesus cervix mediated by an adenovirus expressing beta-galactosidase. AB - OBJECTIVES: More than 90% of cervical cancers are positive for human papillomavirus, which functionally represses p53 and pRb. The remainder have been found to contain p53 mutations. Gene therapy involves insertion of a functioning gene into a patient to correct a genetic abnormality. STUDY DESIGN: The ability of a beta-galactosidase adenovirus to mediate transgene expression in the rhesus cervix was evaluated. Three different doses and two different entry techniques of virus were investigated. RESULTS: The ideal dose determined by X-galactosidase staining was 2 x 10(10) plaque-forming units, and the injection method yielded better staining than did abrasion with topical application. Increased adenoviral specific immunoglobulin G antibody response in the injected monkeys confirmed the results. CONCLUSION: High transduction efficiency by use of adenoviral vectors can be achieved in the cervix. Reversing the effects of human papillomavirus and p53 mutations with gene therapy may become a novel therapy for invasive and preinvasive cervical cancer. PMID- 8623836 TI - Evidence for a unifocal origin in familial ovarian cancer. AB - OBJECTIVE: The purpose of this investigation was to determine the pattern of loss of heterozygosity in multiple tumor sites from familial ovarian cancer cases. If ovarian cancer arises focally in one ovary and then metastasizes to other sites, a similar pattern should be seen in all tumor sites. However, if ovarian cancer arises multifocally throughout the peritoneal cavity, a different pattern of loss would be expected among the different sites. STUDY DESIGN: The presence or absence of loss of specific alleles for 9 loci on chromosomes 1, 6, 11, 13, 16, and 17 was determined in multiple tumor sites from 12 familial ovarian cancer cases. RESULTS: The frequency of loss of heterozygosity was as follows: chromosome 17 (100%), chromosome 13 (82%), chromosome 6 (80%), chromosome 16 (73%), chromosome 1 (57%), and chromosome 11 (22%). In every case an identical pattern was present for at least one locus. In four cases loss of the same allele was present in tumor from the ovary and all metastatic sites for all informative loci. In the remaining eight cases loss of the same allele for one to five (mean three) loci was detected. CONCLUSIONS: The pattern of loss of heterozygosity in the 12 familial ovarian cancers included in this investigation favors a unifocal origin of disease. A dual primary origin could not be absolutely excluded in 3 cases. High frequencies on chromosomes 17q and 13 suggest that loss of whole or part of these chromosomes is important in ovarian carcinogenesis. PMID- 8623837 TI - The effect of transforming growth factor-beta on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model. AB - OBJECTIVE: Our purpose was to determine the effects of transforming growth factor beta on steroidogenesis and regulation of steroidogenic enzyme expression by use of a human ovarian thecal-like tumor cell culture system. STUDY DESIGN: Human ovarian thecal-like tumor cells were treated in serum-free medium in the presence or absence of forskolin and transforming growth factor-beta 1. The accumulation of progesterone and androstenedione in the culture medium was evaluated by radioimmunoassay. The effects of forskolin with or without transforming growth factor-beta 1 on the enzymatic activity of P450c17 and 3 beta HSD, the expression of immunodetectable P450c17 protein, and the expression of messenger ribonucleic acid for P450scc, P450c17, and 3 beta HSD were determined. RESULTS: Basal steroid secretion, steroidogenic enzyme activity, enzyme protein, and messenger ribonucleic acid expression were not affected by transforming growth factor-beta 1 alone. Forskolin treatment significantly stimulated steroid production and the enzymatic activity of P450c17 and 3 beta HSD up to 10-fold above basal levels. However, transforming growth factor-beta 1 inhibited forskolin-stimulated androstenedione production to near basal levels and increased progesterone 1.4- to 2-fold while suppressing P450c17 enzyme activity to near basal levels, but it did not affect 3 beta HSD activity. Forskolin-stimulated immunodetectable P450c17 alpha protein was markedly inhibited by transforming growth factor-beta 1. In addition, transforming growth factor-beta 1 markedly inhibited the forskolin stimulation of P450c17 messenger ribonucleic acid, while not significantly altering P450scc or 3 beta HSD messenger ribonucleic acid expression. CONCLUSION: Forskolin stimulated human ovarian thecal-like tumor cell steroidogenesis, P450c17 and 3 beta HSD activity, immunodetectable P450c17, and messenger ribonucleic acid content for P450scc, P450c17, and 3 beta HSD. Transforming growth factor-beta 1 inhibited forskolin stimulation of androstenedione production through the inhibition of P450c17 expression. PMID- 8623838 TI - 1-Deamino-[8-D-arginine] vasopressin-induced maternal plasma hypoosmolality increases ovine amniotic fluid volume. AB - OBJECTIVE: Maternal 1-deamino-[8-D-arginine] vasopressin (a selective antidiuretic agonist) and oral water loading decrease maternal and fetal plasma osmolality and markedly increase fetal urine flow in sheep over 24 hours. We hypothesized that a sustained reduction in plasma osmolality would increase the amniotic fluid volume. STUDY DESIGN: Pregnant sheep (130 +/- 1 days, n = 6) were given oral water loading (2000 ml) and intravenous 1-deamino-[8-D-arginine] vasopressin (20 microgram injection followed by 4 micrograms/hr) to induce maternal urinary antidiuresis and to maintain a maternal plasma osmolality decrease of approximately 20 mOsm/kg for 48 hours. Fetal plasma osmolality, electrolytes, atrial natriuretic factor, and arginine vasopressin levels, fetal swallowing activity, and fetal urine flow and composition were measured each study day, and amniotic fluid volume was measured by tracer dilution before and at 48 hours of plasma hypoosmolality. RESULTS: In response to 1-deamino-[8-D arginine] vasopressin and water loading, maternal plasma osmolality decreased (298 +/- 1 to 275 +/- 3 mOsm/kg), mirrored by fetal plasma hypoosmolality (293 +/ 1 to 271 +/- 3 mOsm/kg). Fetal urine osmolality (155 +/- 16 to 117 +/- 10 mOsm/kg) and amniotic fluid osmolality significantly decreased (281 +/- 3 to 269 +/- 5 mOsm/kg). Fetal urine flow rate significantly increased (0.33 +/- 0.06 to 0.51 +/- 0.04 ml/min), whereas fetal swallowing activity decreased (56 +/- 7 to 33 +/-5 swallows per hour). In response to alterations in fetal fluid dynamics, amniotic fluid volume significantly increased from 493 +/- 80 to 839 +/- 160 ml. CONCLUSION: Chronic maternal 1-deamino-[8-D-arginine vasopressin-induced plasma hypoosmolality induces a sustained reduction in fetal swallowing activity and an increase in fetal urine production, which results in a marked increase in amniotic fluid volume. Use of maternal 1-deamino-[8-D-arginine] vasopressin and oral fluid loading has the potential for manipulation of amniotic fluid volume homeostasis and may prove useful in the treatment of oligohydramnios. PMID- 8623839 TI - Sites of failure and times to failure in carcinoma of the vulva treated conservatively: a Gynecologic Oncology Group study. AB - OBJECTIVE: The objective of this review was to examine the patterns of failure after conservative therapy for vulvar carcinoma to establish a basis for the design of prospective cooperative trials in this disease. STUDY DESIGN: The records and characteristics of all patients in whom cancer recurred after conservative therapy in two prospective clinical trials were analyzed. RESULTS: Of 143 patients who received one or more modifications of therapy, 37 patients had recurrence of cancer and 20 died of the disease. The median intervals to recurrence were 35.9 months for vulvar cancer and 7.0 months for groin cancer (p = 0.0002). There were 12 groin recurrences; 11 (91.7%) of these patients have died of the disease. The median survival time after recurrence was 52.4 months for patients with vulvar cancer and 9.4 months for those with groin cancer (p = 0.0025). CONCLUSIONS: Groin cancer recurs earlier than vulvar cancer among patients treated conservatively. Future trials that evaluate modified therapy to the groin need to include early interim analyses. Follow-up for > 36 months after study entry may not be necessary to evaluate the impact of new treatments. PMID- 8623840 TI - Outcomes of newborns with gastroschisis: the effects of mode of delivery, site of delivery, and interval from birth to surgery. AB - OBJECTIVE: Our purpose was to determine the impact of delivery site, delivery mode, and delivery-to-surgery interval on outcomes for neonates diagnosed with gastroschisis. STUDY DESIGN: Data were obtained retrospectively by chart review on 56 newborns diagnosed with gastroschisis. Outcome measures examined included primary closure, days to enteral feeding, days in intensive care, total length of stay, and hospital charges. RESULTS: Inborn infants experienced fewer days to enteral feeding (p < 0.01)., shorter total lengths of hospital stay (p < 0.01), and lower hospital charges (p < 0.01). Newborns delivered by cesarean section tended to have longer lengths of stay (p = 0.07), greater hospital charges (p = 0.06), and significantly longer lengths of stay in intensive care (p = 0.05). Shorter intervals from delivery to surgery were observed for inborn neonates (p < 0.01) and for those delivered by cesarean section (p < 0.05). No relationships between hours from delivery to surgery and neonatal outcomes were observed. CONCLUSIONS: Delivery at a regional center is associated with improved outcomes, whereas cesarean deliveries were associated with worse outcomes. We observed no salutary effect related to the interval between delivery and initial surgical repair. PMID- 8623841 TI - Expanded pelvic radiotherapy fields for treatment of local-regionally advanced carcinoma of the cervix: outcome and complications. AB - OBJECTIVE: Recent anatomic and radiographic studies have indicated that standard external beam radiation portals may not adequately treat the volume at risk in patients with local-regionally advanced cervical cancer. A feasibility study was undertaken to evaluate toxicity, outcome, and patterns of failure in patients with advanced cervical cancer treated by expanded pelvic radiation fields. STUDY DESIGN: Thirty-eight women with stages IIB and III cancers of the cervix confined to the pelvis were irradiated with curative intent with expanded pelvic radiation portals. Anteriorly and posteriorly, the median field length and width were 20 and 17.5 cm, respectively. Lateral fields had a median width of 16.5 cm, and the posterior border encompassed the entire sacral silhouette. The median external beam whole-pelvis dose was 4140 cGy, with overall point A dose boosted by brachytherapy to 8315 cGy. RESULTS: Stage IIB patients (n = 22) had a 4-year actuarial local control rate of 70%, freedom from distant metastases rate of 62%, and disease-specific survival rate of 76%. In stage III disease (n = 16), the 4 year actuarial local control, freedom from distant metastases, and disease specific survival rates were 80%, 48%, and 53%, respectively. Radiographically determined nodal status was an important predictor of disease-specific survival and distant metastases but not local control. The 4-year disease-specific survival rate was 40% in 11 patients with nodal disease compared with 71% in 27 node-negative patients (p < 0.01). The rate of freedom from distant metastases was 36% in node-positive patients versus 67% in node-negative cases (p < 0.01). The actuarial overall 4-year severe late complication rate was 14.8%. CONCLUSION: This study has demonstrated that expanded fields for pelvic radiotherapy is feasible, well tolerated, and therapeutic. The pelvic field design concepts presented should be integrated into radiation oncology practice. PMID- 8623842 TI - Phase I/II study of PIXY321 in combination with cyclophosphamide and carboplatin in the treatment of ovarian cancer. AB - OBJECTIVE: Our purpose was to evaluate the safety and biologic effects of PIXY321 after chemotherapy in patients with ovarian carcinoma. STUDY DESIGN: A multicenter, nonrandomized, phase I/II study of subcutaneously administered PIXY321 after the second cycle of chemotherapy in cohorts of three or more patients at 50, 125, 250, 500, 750, or 100 micrograms/m2 per day. RESULTS: Cyclophosphamide (600 mg/m2) and carboplatin (400 mg/m2) were administered every 28 days to 34 patients. At doses > or = 500 mg/m2 per day, the median nadir platelet and median nadir absolute neutrophil counts in cycle 2 (with PIXY321) compared with cycle 1 (control) were both higher in 13 of 26 (50%) patients. Twenty-one patients were withdrawn from the study. A total of 17 of 21 (81%) were removed for myelosuppression (n = 15) or PIXY321 toxicity (n = 2). A total of 28 of 34 (82%) patients had injection site reactions. Thirty-seven nonhematologic grade 3 events occurred. CONCLUSIONS: At these doses and schedules PIXY321 can be safely administered. Aggressive dosing of cyclophosphamide and carboplatin could not be maintained for six cycles in the majority (62%) of patients. PMID- 8623843 TI - The risk of pregnancy after tubal sterilization: findings from the U.S. Collaborative Review of Sterilization. AB - OBJECTIVE: Our purpose was to determine the risk of pregnancy after tubal sterilization for common methods of tubal occlusion. STUDY DESIGN: A multicenter, prospective cohort study was conducted in U.S. medical centers. A total of 10,685 women who underwent tubal sterilization was followed up for 8 to 14 years. The risk of pregnancy was assessed by cumulative life-table probabilities and proportional hazards models. RESULTS: A total of 143 sterilization failures was identified. Cumulative 10-year probabilities of pregnancy were highest after clip sterilization (36.5/1000 procedures) and lowest after unipolar coagulation (7.5/1000) and postpartum partial salpingectomy (7.5/1000). The cumulative risk of pregnancy was highest among women sterilized at a young age with bipolar coagulation (54.3/1000) and clip application (52.1/1000). CONCLUSIONS: Although tubal sterilization is highly effective, the risk of sterilization failure is higher than generally reported. The risk persists for years after the procedure and varies by method of tubal occlusion and age. PMID- 8623844 TI - Obesity and prognosis in endometrial cancer. AB - OBJECTIVE: We tested the null hypothesis that morbid obesity as measured by the Quetelet index has no influence on survival in endometrial cancer. STUDY DESIGN: A retrospective study of 492 women with endometrial carcinoma was performed. Age, height, weight, Quetelet index, stage, cell type, grade, node status, peritoneal cytologic findings, and depth of myometrial invasion were analyzed for influence on survival. RESULTS: Mean Quetelet index was 34 (range 16 to 89). Quetelet index was < 30 in 45% of patients, 30 to 40 in 33%, and > 40 in 22%. Five percent of those with a Quetelet index > 40 had positive nodes, but 64% of patients with a Quetelet index > 40 did not have lymph node sampling done. Lack of sampling of lymph nodes in the entire group had no adverse effect on survival. In a proportional hazards regression model for time from diagnosis to death from disease, grade, node status, myometrial invasion, and stage had highly significant effects. When Quetelet index was analyzed as a continuous variable, as Quetelet index increased, time to recurrence was significantly increased (p = 0.0136), and significance was approached for survival (p = 0.0645). Quetelet index was strongly related to grade (p = 0.013), depth of myometrial invasion (p = 0.031), negative cytologic findings (p = 0.004), and stage (p = 0.011) with obese patients having better differentiated, less invasive tumors of lower stage with negative washings. CONCLUSIONS: Morbid obesity positively affects survival in endometrial carcinoma. This effect is accounted for by the association of obesity with less aggressive disease. Morbid obesity is not associated with increased death from other causes. Lack of sampling of negative lymph nodes does not adversely affect survival. PMID- 8623845 TI - Does pregnancy increase the risk for development and progression of diabetic nephropathy? AB - OBJECTIVE: This study was designed to determine whether pregnancy and increasing parity in women with insulin-dependent diabetes mellitus (1) increases the risk for diabetic nephropathy and (2) accelerates the progression of diabetic nephropathy. STUDY DESIGN: The study included women with insulin-dependent diabetes mellitus who enrolled in our diabetes-in-pregnancy trial with a pregnancy that continued beyond 20 weeks' gestation and who were delivered between 1978 and December 31, 1991, to allow for a minimum of 3 years' follow-up. Pregnancy and follow-up information was obtained from the medical records and from our computerized database. For patients followed up elsewhere, information was obtained from their current physicians. Life-table analysis was used to determine (1) the risk for nephropathy developing de novo as a function of duration of disease and the association of this risk with parity and (2) the risk of renal failure developing in women with preexisting nephropathy and its association with parity. RESULTS: The study population included 182 pregnant women with insulin-dependent diabetes mellitus: 46 with overt nephropathy (group F) and 136 without nephropathy (group NF). Pregnancy and increasing parity did not increase the overall risk for nephropathy (44% after 27 years of diabetes). In group NF 10% had nephropathy within 10.1 +/- 4.2 years of the pregnancy. Proteinuria appearing during pregnancy and glycemic control during pregnancy were significantly associated with the subsequent development of nephropathy. In group F 26% had end-stage renal disease after a median period of 6 years from the pregnancy. Pregnancy or increasing parity did not increase the risk for renal failure in women with nephropathy. CONCLUSIONS: Our data support the premise that pregnancy in women with insulin-dependent diabetes mellitus does not increase the risk of subsequent nephropathy and does not accelerate progression of renal disease in women with preexisting nephropathy. PMID- 8623846 TI - The effect of changes in atmospheric pressure on the occurrence of the spontaneous onset of labor in term pregnancies. AB - OBJECTIVE: Our purpose was to determine whether there is a relationship between changes in atmospheric pressure and spontaneous onset of labor in term pregnancy. STUDY DESIGN: All women admitted to Medical Center of Central Massachusetts Memorial Hospital with spontaneous onset of labor at term and who were delivered on the service during a 12-month period represent the cohort for this study. Each maternal chart was abstracted to ensure that each member of the cohort met the inclusion criteria. Hourly recordings of atmospheric pressure made at the Worcester Station of the National Weather Service, Department of Commerce, were used as the meteorologic data points of interest. Least-squares regression was used to determine an equation that expresses the probability of the onset of labor in this cohort as a function of gestational age, which was used to calculate expected numbers for the statistical analyses. Two relationships were studied: (1) the ratio of the observed to the expected number of onsets of labor and (2) the initiation of labor and atmospheric pressure changes in the preceding 3 hours. RESULTS: Three-hour periods of falling atmospheric pressure were less often followed by initiation of labor than were the periods with other types of pressure sequences. No association was observed between the onset of labor and days of low mean pressure. CONCLUSION: Although there was an observed statistically significant association between falling barometric pressure and onset of labor, the magnitude of the difference is not of clinical significance. PMID- 8623847 TI - The economics of infertility: developing an infertility managed-care plan. AB - Infertility is a natural disorder for developing a managed-care plan. Infertility has a clearly defined end point. The treatment regimens and protocols used for treating infertile couples have predictable outcomes; it is possible to develop rational treatment plans over a limited period of time. It is not unreasonable for infertile couples to share the cost of treatment and for the physician providers to share the economic risk by providing a bundle of optimal services at a fixed price. We present the theoretic basis for an infertility managed-care plan and the requirements for developing a plan that can be sold in the marketplace. Finally, we discuss some of the consequences of an infertility managed-care plan on physician relationships, physician manpower, research, and education. PMID- 8623848 TI - In vitro fertilization, gamete intrafallopian transfer, and superovulation with intrauterine insemination: efficacy and potential health hazards on babies delivered. AB - OBJECTIVE: The purpose of this article was to review the efficacy and potential hazards of assisted conception. STUDY DESIGN: A review of pertinent scientific articles published in English was done. RESULTS: There are no adequate prospective, randomized, controlled, or comparative studies of sufficient power on the efficacy of in vitro fertilization, gamete intrafallopian transfer, and superovulation with intrauterine insemination in well-defined infertile couples. In vitro fertilization can overcome tubal sterility. The pregnancy per cycle is 19.8% and delivery per cycle is 16.0% for in vitro fertilization (all indications) and 29.5% and 19.8%, respectively, for gamete intrafallopian transfer. In limited prospective studies, in vitro fertilization, gamete intrafallopian transfer, and superovulation with intrauterine insemination have similar fecundity. Multiple births from in vitro fertilization and gamete intrafallopian transfer are increased, whereas preterm labor and low-birth-weight babies are significantly more common, even in singletons. CONCLUSION: The efficacy or relative superiority of IVF, gamete intrafallopian transfer, and superovulation with intrauterine insemination in nontubal subfertility remains to be shown by properly designed, prospective, randomized, controlled, or comparative studies. Therefore less invasive and less expensive methods such as expectant management or superovulation with intrauterine insemination should be used before embarking on in vitro fertilization and gamete intrafallopian transfer. Further studies on the outcome of babies delivered after assisted conception are required. PMID- 8623849 TI - Is there ever a role for tubal surgery? PMID- 8623850 TI - Carotid atherosclerosis in women with polycystic ovary syndrome: initial results from a case-control study. AB - OBJECTIVE: Our purpose was to determine whether women with polycystic ovary syndrome have greater subclinical atherosclerosis as measured by carotid artery ultrasonography. STUDY DESIGN: Sixteen premenopausal women > or = 40 years old with a history of clinical polycystic ovary syndrome and a current total testosterone concentration > or = 2.0 nmol/L and 16 age-matched (+/- 5 years) cycling women underwent carotid scanning. Intima-media thickness and plaque were compared between cases and controls, as were risk factors for atherosclerosis including body mass index and fasting insulin and lipid levels. Statistical analysis included t tests, Fisher's exact test, and multiple linear regression. RESULTS: Mean +/- SE intima-media thickness was found to be significantly greater for cases with polycystic ovary syndrome (0.680 +/- 0.019 mm) than for controls (0.630 +/- 0.012 mm) (t = 2.31, p = 0.035). Five cases (31.3%) and two controls (12.5%) had ultrasonographic evidence of plaque (not significant). Univariate regressions of intima-media thickness yielded significant coefficients for insulin, total cholesterol, low-density lipoprotein cholesterol and body mass index. When either total cholesterol or low-density lipoprotein were included in the model simultaneously with polycystic ovary syndrome, each retained significance. This was not true for insulin and body mass index, however, suggesting that these factors covaried with polycystic ovary syndrome in a dimension affecting intima-media thickness. CONCLUSIONS: In spite of a major limitation of small sample size, these data suggest that women with polycystic ovary syndrome have an increased risk of subclinical atherosclerosis in their 40s. PMID- 8623851 TI - Multifetal pregnancy reduction: evaluation of fetal growth in the remaining twins. AB - OBJECTIVE: Our purpose was to study fetal growth after reduction of high-order multiple gestations to twins. STUDY DESIGN: Birth weight and gestational age data were collected for 236 triplet and greater multiple pregnancies reduced to twins (113 triplets, 89 quadruplets, and 34 quintuplets or above) and was compared with those of a control group of unreduced twins. RESULTS: Rates of intrauterine growth restriction per pregnancy were significantly different between the nonreduced and all categories of reduced multifetal pregnancies. The incidence of intrauterine growth restriction was 19.4% in the nonreduced twins, 36.3% in pregnancies reduced from triplets, 41.6% in pregnancies reduced from quadruplets, and 50% from higher-order multiple gestations. There was a statistically significant trend toward increasing frequency of intrauterine growth restriction with increasing starting fetal number (p = 0.04). The increase in intrauterine growth restriction was primarily accounted for by twin pairs with only one growth restricted newborn. CONCLUSION: Multifetal pregnancy reduction does not reduce the incidence of intrauterine growth restriction in the remaining fetuses to that of nonreduced twins. PMID- 8623852 TI - CA 125 kinetics: a cost-effective clinical tool to evaluate clinical trial outcomes in the 1990s. AB - OBJECTIVE: Our purpose was to determine the importance of the rate of decline of CA 125 relative to conventional prognosticators of ovarian cancer survival to develop a cost effective management algorithm that supports clinical trial research. STUDY DESIGN: By use of a retrospective chart review the slope of the CA 125 exponential regression curve was calculated for 126 women undergoing combination chemotherapy for epithelial ovarian cancer. Univariate and multivariate survival analyses evaluated conventional parameters including age, grade, stage, histologic features, time to initial chemotherapy, dose and treatment intensity, number of cycles to normal CA 125 levels, the intercept from the regression equation, and the slope of the exponential curve. RESULTS: The ideal CA 125 regression rate was calculated at 7.6 days (95% confidence interval 5.9 to 10.7). Univariate analysis determined slope of the CA 125 exponential regression curve (p = 0.0003), number of cycles to normal CA 125 levels (p = 0.0001), residual disease (p = 0.0006), and platinum treatment intensity (p = 0.0001) as the most important predictors of survival. Cox proportional-hazard regression analysis identified slope of the CA 125 exponential regression curve and number of cycles to normal CA 125 levels as the most significant factors for actuarial survival, replacing such conventional parameters as patient age, stage, grade, chemotherapy intensity, and residual disease. None of the factors investigated predicted treatment outcome for patients without residual disease. Multiple linear regression analysis of the slope of the CA 125 exponential regression curve identified intercept of the regression equation, stage, age, and time to initial chemotherapy as important determinants of the slope. CONCLUSION: The slope of the CA 125 exponential regression curve is the single most important prognosticator of actuarial survival for the patient with a CA 125-positive ovarian carcinoma. Treatment algorithms based on this slope may be helpful in developing novel cost-effective clinical trials. PMID- 8623853 TI - Fetal surgery. AB - Although most fetal defects are best managed after birth, a few with predictable, life-threatening developmental consequences have been successfully corrected in utero. Many technical problems have been solved, but preterm labor remains a significant risk to mother and fetus. Less invasive interventional techniques and fetal stem cell transplantation promise to extend the indications for intervention. PMID- 8623854 TI - The current status of multifetal pregnancy reduction. AB - The number of women conceiving three or more fetuses has increased dramatically as a result of successful infertility therapy with ovulation-inducing agents and assisted reproductive technology. Higher-order multiple gestations have an increased risk of premature delivery and its attendant sequelae of increased neonatal mortality or irreversible morbidity. Multifetal pregnancy reduction is a procedure designed to decrease the increased propensity to deliver very prematurely in these patients by reducing the number of live fetuses they are carrying. The procedure has proved to be both safe and effective, and pregnancies reduced to twins proceed as if that were the number of fetuses originally conceived. Nevertheless, this invasive procedure does have the potential to result in loss of the entire pregnancy and causes considerable emotional distress for some couples who view it as their "least bad" alternative. The medical benefits of performing multifetal pregnancy reduction in women with four or more fetuses seem fairly well established, but this is less true for triplets. Serious attention should be paid to reducing the number of higher-order multiple pregnancies resulting from infertility therapy. In the meantime, when three or more fetuses have been conceived, multifetal pregnancy reduction offers a reasonable option to patients whose only choices in the past were either to accept the risk of delivering extremely prematurely or to terminate the entire pregnancy. PMID- 8623855 TI - Common dermatoses. Part II. AB - Skin diseases are an essential part of primary care medicine. Most dermatologic care is delivered in an outpatient setting. The initial evaluation is usually performed by the primary care physician. Women account for nearly 60% of all visits for dermatologic complaints. PMID- 8623856 TI - The impact of the Women, Infants and Children Food Supplement Program on birth outcome. AB - OBJECTIVE: Our purpose was to compare the birth outcomes of pregnant women in the Women, Infants and Children Food Supplement Program with women not in the program. STUDY DESIGN: The vital records of 4713 women, 2895 enrolled in the Women, Infants and Children Food Supplement Program and 1812 not enrolled in the program, whose infants were delivered at Wishard Memorial Hospital over 18 months were reviewed with respect to age, education, race, substance habits, trimester of entry into prenatal care, maternal weight gain, and status in the program. The primary outcome variables evaluated were low birth weight, as defined by the delivery of an infant < 2500 gm, and infant mortality. The primary predictor was program participation versus nonparticipation. Analysis was by t test, chi2, and logistic regression models. RESULTS: Black women, women with no prenatal care, and women who smoke were more likely to deliver a low-birth-weight infant. The incidence of low birth weight was 13.1% for nonparticipants versus 10.2% for program participants (p < 0.05). Univariate analysis confirmed program participants to be at significantly less risk for a low-birth-weight delivery (odds ratio = 0.75, p < 0.05). This relation, however, was not significant in the multivariate model (odds ratio = 0.88), indicating that the effect of participating in the program is being confounded by race, entry into prenatal care, and smoking. The overall infant mortality rate was 12.4 per 1000 for participants and 16 per 1000 for nonparticipants (p = not significant). CONCLUSION: Nutritional and nonnutritional benefits to participation in the Women, Infants and Children Food Supplement Program were confirmed. Women enrolled in the program were less likely to deliver a low-birth-weight infant. Multiple variables likely contribute to the poorer outcome for nonparticipants. PMID- 8623857 TI - Synchronization of the factors critical for diabetic teratogenesis: an in vitro model. AB - OBJECTIVE: Our goal was to determine the relationship between critical factors and conditions such as gestational age and exposure time to elevated glucose levels in diabetic embryopathy. STUDY DESIGN: A postimplantation rat embryo culture was used as a model for investigation. The effect of various factors on embryonic development was studied. Experiments were conducted with increasing glucose concentrations (150 to 905 mg/dl, n = 186), at various gestational ages (10 to 12 days, n = 169), and for varying durations of exposure (30 to 180 minutes, n = 169). Gross morphologic characteristics of the yolk sac and embryo were assessed. RESULTS: Embryopathy was induced by hyperglycemia in a dose related fashion: a 20% rate at two times control glucose concentration, almost a 50% rate at four times control, and approximately a 100% abnormality rate at more than six times control. A critical window in gestational age, days 10 to 11, and a minimum exposure time to hyperglycemia of 2 hours were necessary to induce teratogenesis. CONCLUSIONS: Diabetic teratogenesis occurs in a dose-related fashion and requires a minimum exposure time and critical gestational age. Only synchronization of these critical conditions induces embryonic maldevelopment. Furthermore, nonsynchronized aberrant conditions may result in apparently normal embryonic development. PMID- 8623858 TI - Right ventricular function in chronically anemic fetal lambs. AB - OBJECTIVE: Our purpose was to determine whether the increase in extravascular fluid in chronic fetal anemia occurs either because of heart failure or despite successful cardiac adaptation. STUDY DESIGN: Right ventricular function curves were obtained in five ovine fetuses at the start, midpoint, and end of 5 to 8 days of anemia induced by isovolemic daily hemorrhage. Least-squares fit of the ascending and plateau lines of stroke volume versus right atrial pressure were used to establish breakpoints (intersection of the ascending and plateau lines), which were compared by analysis of variance for repeated measures. Myocardial blood flow was measured by microspheres. RESULTS: Carotid arterial oxygen content was reduced from 7.0 +/- 0.3 to 2.1 +/- 0.1 ml/dl and the hematocrit from 29% +/- 1.8% to 13% +/- 0.6%. Breakpoint analysis of function curves showed that although right atrial pressure remained unchanged (3.4 +/- 0.7 and 3.6 +/- 0.6 mm Hg) stroke volume increased from 1.03 +/- 0.14 to 1.62 +/- 0.25 ml/kg. Both right and left ventricular coronary blood flow were increased, 1351 +/- 313 and 1166 +/- 264 ml/min per 100 gm. Excess fluid was present in abdomen and chest of most animals at autopsy. CONCLUSION: Tissue edema during severe anemia occurs despite normal right atrial pressure, increased stroke volume, and markedly increased coronary blood flow, markers of successful cardiac adaptation. PMID- 8623859 TI - Improved intrapartum surveillance with PR interval analysis of the fetal electrocardiogram: a randomized trial showing a reduction in fetal blood sampling. AB - OBJECTIVE: Our goal was to test the hypothesis that the addition of fetal electrocardiogram time-interval analysis to conventional electronic fetal monitoring would significantly reduce the number of cases requiring fetal scalp blood sampling without an increase in adverse outcome. STUDY DESIGN: A randomized prospective trial was performed in 214 women with high-risk labor. RESULTS: There was a significant reduction in the number of cases that had fetal blood sampling performed in the fetal electrocardiogram plus electronic fetal monitoring group (risk ratio for electronic fetal monitoring alone 3.53; p < 0.01, 95% confidence interval 1.39 to 8.95). The fetal blood samplings performed in the electronic fetal monitoring alone group were less likely to be abnormal (pH < 7.25, base excess < -8.0) than those performed in the fetal electrocardiogram plus electronic fetal monitoring group (risk ratio for electronic fetal monitoring alone 0.62, p = 0.05, 95% confidence interval 0.35 to 1.10). There was a trend of more infants with an arterial umbilical pH < 7.15 and a base excess less than 8.0 mmol/L at birth being unsuspected and more instrumental deliveries for presumed fetal distress being performed in the electronic fetal monitoring alone than in the fetal electrocardiogram plus electronic fetal monitoring group. CONCLUSION: The addition of fetal electrocardiogram analysis to conventional electronic fetal monitoring during labor can reduce significantly the number of parturients undergoing fetal scalp blood sampling and can simultaneously increase its efficiency without an increase in adverse outcome. PMID- 8623860 TI - RhD genotype determination by single sperm cell analysis. AB - OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of her Rh-positive partner. Currently, a reliable technique for an absolute determination of RhD genotype is not available. This study was initiated to develop an accurate method for RhD genotyping in men. STUDY DESIGN: RhD genotype was determined by deoxyribonucleic acid amplification of a D-specific sequence in single sperm cell samples. Micromanipulation techniques were used for sampling of single sperm cells, which were further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in all of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately determined in 10 of 10 donors. A total of 132 single sperm cells were analyzed (8 to 17 samples per donor), of which 96 were successfully amplified as assessed by an internal control. As expected, the specific region of the RhD gene was amplified in all, some, and none of the signal-positive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh negative subjects, respectively, allowing accurate determination of the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the management of Rh isoimmunization. PMID- 8623861 TI - Transplacental passage of recombinant human granulocyte colony-stimulating factor in women with an imminent preterm delivery. AB - OBJECTIVE: We attempted to determine, in a pilot study, whether recombinant human granulocyte colony-stimulating factor, administered to women with an imminent delivery at < or = 30 weeks' estimated gestational age, crosses to the fetal circulation and stimulates fetal neutrophil production. STUDY DESIGN: We measured granulocyte colony-stimulating factor and neutrophil concentrations in the blood of the mothers and in the umbilical venous cord blood after a single intravenous dose (25 micrograms/kg) of recombinant granulocyte colony-stimulating factor given to 11 women in whom preterm delivery was imminent and compared these with values from 34 control women and 35 cord sera. RESULTS: Ten infants were delivered within 30 hours (10.8 +/- 8.9, mean +/- SD) of the granulocyte colony stimulating factor administration ("early delivery"), and two were delivered after 54 and 108 hours, respectively. In the early delivery group maternal granulocyte colony-stimulating factor concentrations and blood neutrophil levels were higher than in controls. However, no difference was seen in the cord blood neutrophil concentrations. In the late delivery group, although maternal serum and cord blood granulocyte colony-stimulating factor concentrations did not differ from controls, cord blood neutrophil levels were higher (25,900 and 17,700 cells/microliters) than controls (3500 +/- 2000 cells/microliters, p < 0.05) and remained elevated for 1 week. Specifically, the blood neutrophil levels on days 5 and 8 were 12,000 and 17,000, respectively, for these patients, whereas the reference range upper limit for controls was 6000 cells/microliters. CONCLUSION: Administration of recombinant granulocyte colony-stimulating factor to pregnant women with an imminent preterm delivery may result in the transplacental passage of a measurable quantity of granulocyte colony-stimulating factor, an amount that can have a biologic effect on the fetus. These events were most noticeable in those patients who received granulocyte colony-stimulating factor at least 30 hours before being delivered of their infants. PMID- 8623862 TI - Methodology citations and the quality of randomized controlled trials in obstetrics and gynecology. AB - OBJECTIVES: Randomized controlled trials offer the best chance for valid treatment comparisons, yet most trials are of poor quality. This may reflect a lack of awareness of the requirements for conducting and reporting this type of research. If so, then citation of methodology references might indicate knowledge of how to conduct these studies and vice versa. Our study tests the hypothesis that the methodologic quality of published trials is related to citation of methodology references. STUDY DESIGN: We performed a hand search of the American Journal of Obstetrics and Gynecology, the British Journal of Obstetrics and Gynaecology, the Journal of Obstetrics and Gynaecology, and Obstetrics and Gynecology to identify all randomized controlled trials published in 1990 and 1991 (N = 206). We reviewed the reference lists of all reports of randomized controlled trials and evaluated the adequacy of randomization methods by accepted criteria. RESULTS: Most reports (81.6%) cited no methodology text or article. Although lack of any methodology reference was not significantly related to failure to report an adequate random method of sequence generation, this was highly related (p < 0.001) to failure to report adequate allocation concealment. Scanning the reference list of reports took a mean of 16 seconds and identified most poorly done trials. CONCLUSIONS: Investigators who conduct randomized controlled trials should be thoroughly familiar with this type of research or should get expert help. Poorly done trials are wasteful and often misleading. PMID- 8623863 TI - Preoperative evaluation of macrophage colony-stimulating factor levels in patients with endometrial cancer. AB - OBJECTIVE: Our purpose was to examine the relationship between preoperative serum levels of macrophage colony-stimulating factor, alone and in combination with CA 125, and the presence of prognostic clinicopathologic factors and subclinical metastases in women with endometrial cancer. STUDY DESIGN: Ninety-two women who underwent primary exploration for endometrial adenocarcinoma had preoperative serum samples evaluated for macrophage colony-stimulating factor and CA 125 levels. Multivariate analysis was used to determine the associations of surgicopathologic findings with macrophage colony-stimulating factor and CA 125 levels. Logistic regression analysis was used to identify factors associated with the risk of extrauterine disease. The association of macrophage colony stimulating factor and CA 125 levels with stage, grade, and depth of myometrial invasion and histologic characteristics were analyzed with Fisher's two-tailed exact test. RESULTS: Elevated levels of macrophage colony-stimulating factor were not associated with depth of myometrial invasion, histologic grade, or histologic cell type; however, advanced stage (p = 0.02) and the presence of lymph node metastases (p = 0.04) were associated with elevated levels. Sensitivity and specificity of macrophage colony-stimulating factor for predicting extrauterine disease were 42% and 89%, respectively. If either an elevated macrophage colony stimulating factor or an elevated CA 125 level was used to predict extrauterine disease, the sensitivity was increased to 67% but the specificity was decreased to 78%. Macrophage colony-stimulating factor elevations predicted lymph node metastases with a sensitivity of 50% and a specificity of 86%. A multivariate regression model showed CA 125 to be the most significant predictor of extrauterine disease; macrophage colony-stimulating factor also contributed prognostic information (p = 0.02). The sensitivity and specificity of the multivariate model for predicting extrauterine disease were 75% and 73%, respectively. CONCLUSION: Macrophage colony-stimulating factor and CA 125 are neither sensitive nor specific enough to be used as predictors of the presence or absence of extrauterine disease in patients with endometrial cancer. PMID- 8623864 TI - Vaginal hysterectomy versus abdominal hysterectomy for the treatment of stage I endometrial adenocarcinoma. AB - OBJECTIVE: The aims of the current study were to (1) determine the effectiveness of vaginal hysterectomy for the treatment of stage I endometrial cancer and (2) analyze which clinical pathologic parameters were independent predictors of clinical outcome. STUDY DESIGN: In a retrospective analysis, 5- and 10-year results of vaginal hysterectomy were compared with those of abdominal hysterectomy in 327 cases of stage I adenocarcinoma. No preoperative irradiation was given. Overall, 180 patients underwent vaginal hysterectomy, whereas 147 patients had abdominal hysterectomy (106 cases with lymphadenectomy). The log rank test was used for evaluation of survival differences. RESULTS: The 5- and 10 year survival rates (Kaplan-Meier method) were, respectively, 90% and 87% in the vaginal hysterectomy group and 91% and 90% in the abdominal hysterectomy group (difference not significant). The grade of differentiation, depth of myometrial invasion, and age were significantly correlated with survival, whereas histologic type, mode of surgery, lymphadenectomy, and adjuvant radiotherapy were not. In a multivariate analysis (Cox proportional hazards), grade of differentiation and age were independent predictors of clinical outcome, whereas depth of myometrial invasion lost significance. CONCLUSIONS: Vaginal hysterectomy showed a high rate of cure in stage I endometrial cancer. Therefore it can be used as an alternative to the abdominal operation in obese and poor surgical risk patients and, possibly, in selected low-risk cases. PMID- 8623865 TI - Comparison of saline infusion sonography with office hysteroscopy for the evaluation of the endometrium. AB - OBJECTIVE: Intrauterine infusion of saline solution during transvaginal ultrasonography enhances visualization of the endometrium. We compared the accuracy and pain rating of saline infusion sonography with those of flexible office hysteroscopy. STUDY DESIGN: The uterine cavities of 130 patients with abnormal bleeding were evaluated by two physicians in an office setting. Findings of endometrial polyps, submucous myomas, synechiae, endometrial hyperplasia, or cancer were recorded independently and subsequently compared. Patients rated their pain after each procedure. RESULTS: Both procedures were performed in 113 of 130 patients. With saline infusion sonography pathologic findings were identified in 61 patients (54%). For all findings combined, sensitivity was 0.96 and specificity was 0.88, compared with hysteroscopy. The results of saline infusion sonography and hysteroscopy did not differ significantly (p = 0.18). The former was less painful for patients than hysteroscopy (p < 0.0001). CONCLUSION: Saline infusion sonography is an accurate and well-tolerated method to evaluate abnormal uterine bleeding, compared with hysteroscopy. PMID- 8623866 TI - Supplementation with omega-3 polyunsaturated fatty acids in the management of dysmenorrhea in adolescents. AB - OBJECTIVES: The purpose of the study was to examine whether dietary supplementation with omega-3 fatty acids can relieve symptoms of dysmenorrhea in adolescents. STUDY DESIGN: Forty-two adolescents with dysmenorrhea were randomly allocated to two groups. In the first group 21 girls received fish oil (1080 mg icosapentaenoic acid, 720 mg docosahexaenoic acid, and 1.5 mg vitamin E) daily for 2 months followed by a placebo for an additional 2 months. In the second group 21 girls received placebo for the first 2 months, followed by fish oil for 2 more months. The Cox Menstrual Symptom Scale was used to assess response to treatment. RESULTS: There were no significant differences in the Cox Menstrual Symptom Scale between the two groups at baseline after 2 months of placebo administration. After 2 months of treatment with fish oil there was a marked reduction in the Cox Menstrual Symptom Scale from a baseline mean value of 69.9 to 44.0 (p < 0.0004). CONCLUSIONS: This study suggests that dietary supplementation with omega-3 fatty acids has a beneficial effect on symptoms of dysmenorrhea in adolescents. PMID- 8623867 TI - Evaluation of a deoxyribonucleic acid probe for the detection of Trichomonas vaginalis in vaginal secretions. AB - OBJECTIVE: Our purpose was to compare Affirm VP, a new deoxyribonucleic acid probe test, with standard "wet preparation" microscopic examinations and culture for the identification of Trichomonas vaginalis organisms in vaginal secretions. STUDY DESIGN: We examined vaginal samples from 615 women with symptoms or signs of vaginitis for T. vaginalis using the deoxyribonucleic acid probe test, microscopic examination of wet preparations of vaginal secretions, and culture in modified Diamond's medium. RESULTS: T. vaginalis was identified in specimens from 95 (15.4%) of the 615 patients. Cultures in Diamond's medium identified 93 (98%) of the 95 infected patients. Vaginal wet preparation identified 76 (80%) of the infected women. The deoxyribonucleic acid probe test detected 86 (90.5%) of the 95 infected patients. There was one false-positive deoxyribonucleic acid probe test (specificity 519/520: 99.8%). CONCLUSION: The Affirm VP deoxyribonucleic acid probe test had a sensitivity of 90% and a specificity of 99.8% for the identification of T. vaginalis organisms in women with symptoms with a high prevalence of trichomoniasis. Such a nonculture test may be of considerable benefit in diagnosing T. vaginalis infections, especially in settings where microscopy, culture, or both are unavailable, inconvenient, or unreliably performed. PMID- 8623868 TI - Pregnancy outcomes in the Diabetes Control and Complications Trial. AB - OBJECTIVE: Our purpose was to examine the maternal and fetal outcomes of the 180 women with insulin-dependent diabetes mellitus who became pregnant during the Diabetes Control and Complications Trial. STUDY DESIGN: The Diabetes Control and Complications Trial was a multicenter controlled clinical trial that compared intensive with conventional therapy in insulin-dependent diabetes mellitus. Intensive therapy, with the aim of achieving normal glycemic control, was initiated in women originally assigned to conventional therapy who actively sought to become pregnant or who became pregnant. RESULTS: A total of 180 women completed 270 pregnancies between 1983 and 1993, with 191 total live births. The mean glycosylated hemoglobin level was significantly different between the treatment groups at conception (7.4% +/- 1.3% in intensive vs. 8.1% +/- 1.7% in conventional therapy, p = 0.0001) but was similar during gestation (6.6% +/- 0.8% and 6.6% +/- 1.3%). Nine congenital malformations were identified (4.7%), eight of which occurred in women originally assigned to conventional therapy (p = 0.06). There were 32 spontaneous abortions, 13.3% in the intensive and 10.4% in the women originally assigned to conventional treatment (not significant). There were no significant differences in outcome between the women with conventional treatment who initiated intensive therapy preconception and those who began it after conception. CONCLUSIONS: Timely institution of intensive therapy is associated with rates of spontaneous abortion and congenital malformation similar to those in the nondiabetic population. PMID- 8623869 TI - Colonization with group B streptococci in pregnancy and adverse outcome. VIP Study Group. AB - OBJECTIVE: Our purpose was to study the association of cervicovaginal colonization with group B streptococci with pregnancy and neonatal outcome. STUDY DESIGN: A prospective study was conducted at seven medical centers between 1984 and 1989. Genital tract cultures were obtained at 23 to 26 weeks' gestation and at delivery. Prematurity and neonatal sepsis rates were compared between group B streptococci positive and negative women. RESULTS: Group B streptococci was recovered from 2877 (21%) of 13,646 women at enrollment. Heavy colonization was associated with a significant risk of delivering a preterm infant who had a low birth weight (odds ratio = 1.5, 95% confidence interval 1.1 to 1.9). Heavily colonized women given antibiotics effective against group B streptococci had little increased risk of a preterm, low-birth-weight birth. Women with light colonization were at the same risk of adverse outcome as the uncolonized women. Neonatal group B streptococci sepsis occurred in 2.6 of 1000 live births in women with and 1.6 of 1000 live births in women without group B streptococci at 23 to 26 weeks' gestation (p = 0.11). However, sepsis occurred in 16 of 1000 live births to women with and 0.4 of 1000 live births to women without group B streptococci at delivery (p < 0.001). CONCLUSIONS: Heavy group B streptococci colonization of 23 to 26 weeks' gestation was associated with an increased risk of delivering a preterm, low-birth-weight infant. Cervicovaginal colonization with group B streptococci at 23 to 26 weeks' gestation was not a reliable predictor of neonatal group B streptococci sepsis. Colonization at delivery was associated with sepsis. PMID- 8623870 TI - Differences in practice patterns between obstetricians and family physicians: use of serum screening. AB - OBJECTIVE: Our purpose was to compare the use of the Iowa Expanded Serum Screening program (maternal serum alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol) by obstetricians and family physicians. STUDY DESIGN: A registry was used to identify all obstetricians (160) and family physicians (404) who practice obstetrics in Iowa. A questionnaire exploring attitudes and practice patterns related to serum screening was mailed to these physicians. RESULTS: The response rate was 80.3% overall. There were significant differences in the offering of serum screening (p < 0.001) for following three responses: obstetricians were more likely to offer screening through the Iowa Expanded Serum Screening Program than were family physicians (89.3% vs 57.2%) and less likely to offer screening outside the state program (9.9% vs 39.0%), whereas family physicians were more likely to not offer screening (0.8% vs 3.8%). When the responses of the physicians who offer screening were analyzed, several differences emerged: (1) obstetricians were more likely than family physicians to offer screening to all obstetric patients (99.2% vs 92.1%, p < 0.05), whereas family physicians were more likely to offer testing to patients with perceived risks for neural tube defects or chromosomal abnormalities and to those patients who requested it, (2) obstetricians were more likely to recommend screening than family physicians (39.0% vs 25.7%, p < 0.05), who more often discouraged testing, although the latter did not reach statistical significance, (3) family physicians more than obstetricians felt that screening was not necessary if the patient would not terminate the pregnancy for neural tube defects (41.6% vs 19.1%, p < 0.001) or chromosomal abnormalities (39.7% vs 21.4%, p < 0.01), and (4) obstetricians used nurses to counsel patients to a much greater extent than family physicians did (44.6% vs 14.0%, p < 0.001). CONCLUSION: There is a significant difference in practice patterns between obstetricians and family physicians in their reported use and presentation of maternal serum screening. Guidelines based on outcome studies should be developed and followed by obstetricians and family physicians. PMID- 8623871 TI - Closure or nonclosure of the visceral peritoneum at cesarean delivery. AB - OBJECTIVE: Our purpose was to determine whether nonclosure of the visceral peritoneum at low transverse cesarean delivery has advantages over suture peritonization with regard to postoperative morbidity. STUDY DESIGN: A prospective randomized trial of 549 women undergoing cesarean section was carried out; 262 were randomized to nonclosure and 287 to closure of the visceral peritoneum. Perioperative, intraoperative, and postoperative management decisions were made without reference to treatment groups. Statistical analysis compared intraoperative and postoperative outcome between the two groups. RESULTS: Operating and anesthesia times were significantly shorter in patients receiving nonclosure. The incidence of febrile morbidity and cystitis and the need for antibiotics and narcotics were all significantly greater when the peritoneum was closed. Hospital stay was significantly shorter after nonclosure. CONCLUSION: Nonclosure of the visceral peritoneum is associated with lower febrile and infectious morbidity. Routine closure of the visceral peritoneum should be abandoned at cesarean delivery. PMID- 8623872 TI - Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes. AB - OBJECTIVES: It has been suggested that increased matrix metalloproteinases activity promotes the weakening of the amniochorion during normal and premature rupture of membranes. This study was designed to determine whether levels of matrix metalloproteinases and the tissue inhibitor of metalloproteinases-1 in amniotic fluid change in a pattern consistent with this hypothesis. STUDY DESIGN: Gelatinolytic activity, measured by a soluble substrate assay and zymography, and the concentrations of tissue inhibitor of metalloproteinases-1 were estimated in amniotic fluid obtained from (1) normal early gestations, (2) normal term pregnancies with labor, (3) normal term pregnancies without labor, and (4) pregnancies complicated by premature rupture of membranes. The 92 kd type IV collagenase (matrix metalloproteinase-9) was also detected in amniotic fluid by Western blotting. RESULTS: Matrix metalloproteinase activities were higher in amniotic fluid from normal term pregnancies with labor and pregnancies complicated by premature rupture of membranes than from early pregnancies and term gestations without labor. The amniotic fluid from term pregnancies with labor or pregnancies with premature rupture of membranes contained several gelatinases, as revealed by zymography. The major amniotic fluid gelatinolytic activity in premature rupture of membranes and term pregnancies with labor corresponded to matrix metalloproteinase-9. Tissue inhibitor of metalloproteinases-1 concentrations were highest in early-pregnancy amniotic fluid, followed by term gestation with labor, term gestation without labor, and premature rupture of membranes. CONCLUSIONS: Normal labor and premature rupture of membranes are associated with increased levels of matrix metalloproteinases, particularly matrix metalloproteinase-9 in amniotic fluid. Premature rupture of membranes is associated with reduced levels of tissue inhibitor of metalloproteinases-1. The imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in amniotic fluid may reflect a disorder that promotes premature rupture of membranes. PMID- 8623873 TI - Congenital diaphragmatic hernia: can prenatal ultrasonography predict outcome? AB - OBJECTIVE: We wanted to establish whether prenatal ultrasonography predicts postnatal outcome in congenital diaphragmatic hernia. STUDY DESIGN: We designed a retrospective multicenter cohort study of 135 patients with congenital diaphragmatic hernia (122 left, 10 right, 2 bilateral, 1 anterior). In isolated left congenital diaphragmatic hernia five potential prenatal prognostic factors were studied: diagnosis at < or = 25 weeks' gestation, polyhydramnios, intrathoracic stomach, small abdomen, and major mediastinal shift. RESULTS: None of the 44 fetuses or infants who had multiple malformations survived. Of the 91 cases of isolated congenital diaphragmatic hernia, there were 82 live births; 76 of these infants had a left congenital diaphragmatic hernia. Of these, 51 (67%) died postnatally. A statistically significant relation was found between mortality and polyhydramnios, intrathoracic stomach, and major mediastinal shift. Mortality increased as a function of the number of these prognostic factors from 20% when none was present to 94% when all three were present. CONCLUSION: In the majority of cases of isolated left congenital diaphragmatic hernia the prognostic value of fetal ultrasonography is too low to alter perinatal management. PMID- 8623874 TI - On intrapartum fetal monitoring. AB - The currently advised conduct for intrapartum surveillance of the fetus is either intermittent auscultation of continuous electronic monitoring, depending on the physician's preference. This applies to all, normal or high-risk, conditions. The bases for this recommendation, a number of controlled studies comparing the two methods, showed no better neonatal outcomes and increased cesarean section rates with electronic fetal monitoring. A review of the works pertaining to fetal development of cardiovascular and central nervous systems and their response to various pathophysiologic conditions (in animals and humans) was carried out in an effort to find an explanation for this apparently uncongruous position. It was found that fetal responses to seemingly comparable conditions are radically different depending on age of gestation. Many authors have pointed this out for the human fetus. However, for interpretation of electronic fetal monitoring in labor, various standard, nondescriptive, confusing words are used to imply the need for rapid intervention. The complete lack of uniform interpretation has been shown in studies comparing interobserver and intraobserver variations. This may be the consequence of poor or superficial teaching of a tool that requires much study and hard work for useful application. The inescapable conclusion is unpleasant but inevitable: to use electronic fetal monitoring properly it is necessary to start a new learning of the physiology of the fetus, its changing evolution as pregnancy advances, its different responses under stress or distress, and the various ways these are represented in electronic fetal monitoring tracings. These efforts take dedication and time spent in labor suites collating tracings with neonatal condition. Only by doing this will it be possible to assist the laboring patients with a useful tool that, so far, has not been adequately applied because of insufficient understanding. PMID- 8623875 TI - A preliminary report on the intravenous use of magnesium sulphate in puerperal eclampsia. 1925. PMID- 8623876 TI - Evaluation of preterm birth misleading. PMID- 8623877 TI - Feasibility of a randomized controlled trial of planned cesarean section versus planned vaginal delivery for breech presentation at term. PMID- 8623878 TI - The Kustner technique: a neglected transverse lower abdominal incision. PMID- 8623879 TI - Intrapartum fetal heart rate assessment: which method is superior. PMID- 8623880 TI - The validation of automated blood pressure monitors by intraarterial comparison. PMID- 8623881 TI - Confocal microscopy findings of Acanthamoeba keratitis. AB - PURPOSE: Tandem scanning confocal microscopy was performed on two patients with Acanthamoeba keratitis to provide images detailing characteristic findings of the disease. Although tandem scanning confocal microscopy of Acanthamoeba has been described in previous reports, Acanthamoeba keratitis has not been fully characterized with this instrument. In vivo confocal micrographs showed the double-walled structure of the Acanthamoeba cyst and associated radial keratoneuritis (perineuritis). METHODS: We reviewed the records of two patients with a clinical diagnosis of Acanthamoeba keratitis, one with culture-proven Acanthamoeba and the other with a suspected Acanthamoeba infection. Slit-lamp biomicroscopy and in vivo tandem scanning confocal microscopy were performed. The images obtained were compared with images from patients without corneal disease. RESULTS: High-contrast round bodies suggestive of Acanthamoeba cysts, as previously described, and irregular forms suggestive of Acanthamoeba trophozoites were found by tandem scanning confocal microscopy. Additionally, we showed conclusively that under certain circumstances (that is, corneal scarring) tandem scanning confocal microscopy can resolve the double-walled structure of the Acanthamoeba ectocyst surrounding the endocyst. Furthermore, radial keratoneuritis was demonstrated, consisting of an irregularly swollen nerve fiber with probable amoebic infiltration. CONCLUSIONS: Confocal microscopy can be a useful, noninvasive imaging technique helpful in the study, diagnosis, and treatment of Acanthamoeba keratitis. PMID- 8623882 TI - Outbreak of keratitis presumed to be caused by Acanthamoeba. AB - PURPOSE: A sharp increase of Acanthamoeba keratitis from two cases per year to 30 cases per year at our institution prompted this study to determine whether there was a change in the clinical characteristics, basic epidemiology, and outcome of this disease. METHODS: We reviewed all cases of Acanthamoeba keratitis diagnosed at the University of Iowa Hospitals and Clinics from mid-1993 through 1994. RESULTS: We screened 217 patients with keratitis by tandem scanning confocal microscopy and suspected Acanthamoeba in 51 patients. Diagnosis was confirmed by cytology in 43 patients (48 eyes). There were no positive cultures. Patients examined within four weeks of onset of symptoms were younger (mean age, 32.6 +/- 15.4 years) and wore contact lenses (11 of 18 patients), and infrequently herpes simplex keratitis (four of 18 patients) was diagnosed. Patients examined after four weeks were older (mean age, 54.0 +/- 19.5 years), infrequently wore contact lenses (six of 25 patients), and often had herpes simplex keratitis (12 of 25 patients). CONCLUSIONS: Corneal examination with tandem scanning confocal microscopy was associated with a marked increase in the detection of Acanthamoeba, strongly suggesting that the disease is more prevalent than suspected. Acanthamoeba may account for many cases of clinically presumed herpes simplex keratitis, the leading cause of corneal blindness in the United States. Acanthamoeba should be considered in the differential diagnosis of any unexplained keratitis, even those of short duration. PMID- 8623883 TI - Excimer laser in situ keratomileusis under a corneal flap for myopia of 2 to 20 diopters. AB - PURPOSE: We studied the efficacy and safety of a recent technique of keratomileusis for myopia, excimer laser in situ keratomileusis. METHODS: We studied retrospectively 88 eyes of 63 patients who received excimer laser in situ keratomileusis with the Chiron Automated Corneal Shaper and the Summit OmniMed laser under a hinged corneal flap without sutures. RESULTS: Mean follow-up was 5.2 months. Mean spherical equivalent of the manifest refraction before surgery was -8.24 diopters (range, -2.00 to -20.00 diopters). Mean spherical equivalent refraction after surgery was +0.22 +/- 1.42 diopters. Of 40 eyes with a baseline refraction from -2.00 to -6.00 diopters, 25 eyes (63%) had refraction within +/- 0.50 diopter of emmetropia, and 37 eyes (93%) had refraction within +/- 1.00 diopter. In eyes with baseline refraction of -6.12 to -12.00 diopters, postoperative refraction was within +/- 1.00 diopter in 19 (65%) of 29 eyes. In eyes with baseline refraction of -12.10 to -20.00 diopters, postoperative refraction was +/- 1.00 diopter in eight (43%) of 19 eyes. Overall, 64 (72.8%) of 88 eyes had a refraction within +/- 1.00 diopter after surgery. Between three weeks and five months after surgery the change in the mean spherical equivalent refraction was -0.61 diopter in the myopic direction. Uncorrected visual acuity after surgery was 20/20 or better in 31 eyes (36%) and 20/40 or better in 61 eyes (71%). Three eyes (3.6%) lost two lines or more of spectacle-corrected visual acuity, two from progressive myopic maculopathy and one from irregular astigmatism. No eyes had vision-threatening complications. CONCLUSION: Excimer laser in situ keratomileusis under a corneal flap can be an effective method of reducing myopia between -2.00 and -20.00 diopters, with minimal complications. Current surgical algorithms need modification to improve predictability of outcome. Stability of refraction after surgery requires further study. PMID- 8623885 TI - Autosomal dominant zonular cataract with sutural opacities in a four-generation family. AB - PURPOSE: We identified and examined four generations of a family with coexisting autosomal dominant zonular cataracts and sutural opacities and sought to determine their genetic basis. METHODS: Twenty-four of the 48 members in the family were examined. Systemic and ocular histories were obtained, and a detailed ophthalmic examination was performed. From each individual, 20 ml of blood was drawn for linkage studies with microsatellite markers in regions to which zonular cataracts had previously been localized (chromosomes 1, 2, and 16). RESULTS: Individuals of the first generation were reportedly asymptomatic. Several members of the second generation had morphologically identical zonular cataracts. Affected members of the third generation showed morphologic heterogeneity, with the zonular opacity varying from a uniform lamella to a segregation of dots. A high degree of consanguinity in the second generation suggested recessive inheritance with a pseudodominant inheritance pattern. However, examination of one member of the asymptomatic first generation disclosed senile cataractous changes superimposed on a faint zonular cataract enclosing sutural opacities and a pulverulent fetal nucleus. The latter findings were reconfirmed to be present in affected members of all generations, suggesting an autosomal dominant mode of inheritance. Initial efforts at linkage analysis excluded the gene locus causing this cataract from the Duffy, haptoglobin, and gamma-crystallin regions. CONCLUSIONS: The cataract in this family is both phenotypically and genetically distinct from previously described and mapped cataracts. PMID- 8623884 TI - An immunohistochemical study of topical cyclosporine in vernal keratoconjunctivitis. AB - PURPOSE: To determine the immunomodulating effects of topical cyclosporine on the immune cells in the conjunctival biopsy specimens obtained from patients with active vernal keratoconjunctivitis. METHODS: We studied six patients who had severe active vernal keratoconjunctivitis. Each patient was given topical cyclosporine 2% eyedrops four times daily. A 2 x 2-mm limbal conjunctival biopsy specimen was obtained from each patient before and three weeks after treatment. Using a panel of monoclonal and polyclonal antibodies and immunohistochemical techniques, we analyzed the conjunctival immune cells before and after cyclosporine treatment. RESULTS: Three weeks after topical cyclosporine treatment, there was marked clinical improvement and a statistically significant reduction in the number of epithelial and stromal class II MHC+ cells, UCHL1+ T cells, and stromal IgA+ and IgG+ plasma cells. The mean number of cells before and after therapy, respectively, were: class II MHC+ (epithelium), 31.5 +/- 13.1 and 8.3 +/- 5.6 (P = .031); class II MHC+ (stroma), 77.0 +/- 28.7 and 24.7 +/- 17.5 (P = .031); UCHL1+ T cells (epithelium), 24.5 +/- 14.1 and 4.2 +/- 2.9 (P = .031); UCHL1+ T cells (stroma), 78.7 +/- 31.1 and 44.5 +/- 27.5 (P = .031); IgA+ plasma cells, 66.7 +/- 32.1 and 22.2 +/- 7.8 (P = .031); and IgG+ plasma cells, 37.3 +/- 30.0 and 9.0 +/- 6.4 (P = .031). There was a statistically insignificant decrease in the epithelial class II MHC+ dendritic Langerhans cells, epithelial and stromal KP1+ macrophages, stromal OPD4+ helper/inducer T cells, and stromal L26+ B cells. The numbers of IgE+ plasma cells and mast cells were unaltered. CONCLUSION: The clinical improvement in vernal keratoconjunctivitis after topical cyclosporine therapy may result from its immunomodulating effect on the components of cell-mediated and humoral immune responses. In contrast, the drug has no immunomodulatory effect on mast cells and IgE-mediated allergic response. PMID- 8623887 TI - Imaging of epiretinal membranes in macular holes by scanning laser ophthalmoscopy. AB - PURPOSE: Because recognition and removal of an epiretinal membrane are important in macular hole surgery, we used the scanning laser ophthalmoscope preoperatively to study epiretinal membranes in patients with idiopathic macular holes. METHODS: We studied 67 eyes (60 consecutive patients) with idiopathic macular holes. We evaluated the thickness and the extent of the epiretinal membrane by using a scanning laser ophthalmoscope. We then compared the fundus images obtained with the scanning laser ophthalmoscope with red-free monochromatic fundus photographs. RESULTS: Confocal imaging of the fundus with either argon blue (488 nm) or argon green (514 nm) laser illumination clearly showed the epiretinal membranes. In all eyes, we observed lesions ranging from a patchy glinting light reflex to a dense epiretinal membrane. A well-demarcated dense epiretinal membrane around the macular hole was observed in seven (44%) of 16 eyes with stage 2 macular holes and in 12 (40%) of 30 eyes with stage 3 holes, but in only two (10%) of 21 eyes with stage 4 holes. The prevalence of the dense epiretinal membrane in stage 2 or 3 holes was significantly higher than in stage 4 holes (P = .025 and .024, respectively). CONCLUSIONS: Fundus imaging using the scanning laser ophthalmoscope with argon laser illumination is useful preoperatively to evaluate epiretinal membranes in eyes with idiopathic macular holes. PMID- 8623886 TI - A randomized, placebo-controlled, double-blind crossover study of the effect of pentoxifylline on ocular fundus pulsations. AB - PURPOSE: To estimate the short-term effects of pentoxifylline on ocular blood flow in healthy volunteers. METHODS: In ten healthy subjects, either 200 or 400 mg of pentoxifylline or placebo was administered intravenously over 90 minutes in a randomized, double-blind, placebo-controlled, three-way crossover study design. Noninvasive measurements of blood pressure, pulse rate, flow variables in the radial artery, ocular fundus pulsations, and whole blood viscosity and filterability were performed at baseline and at 30-minute intervals until four hours after the start of drug infusion. Ocular fundus pulsation amplitude, which has been shown to estimate the pulsatile component of the ocular blood flow, was recorded with a laser interferometer in the macula, the optic disk, and a peripheral region. RESULTS: Fundus pulsation amplitude significantly increased after infusion of pentoxifylline, with maximum effect 150 to 180 minutes after the start of the infusion. This effect was dose-dependent and more pronounced in the macula (+17%, P < .001 vs baseline, after 200 mg; and +27%, P < .001 vs baseline, after 400 mg of pentoxifylline) and the peripheral region (+14%, P < .001; and +26%, P < .001) than in the optic disk (+11%, P < .002; and +13%, P < .001). Viscosity but not filterability of whole blood dose-dependently decreased. Peak systolic flow velocity in the radial artery decreased after infusion of 400 mg of pentoxifylline. Blood pressure and pulse rate were unchanged during the observation period. CONCLUSIONS: These findings demonstrate that pentoxifylline increases pulsatile ocular blood flow in healthy volunteers. Our results support the possibility that pentoxifylline could be used therapeutically in several eye diseases, such as diabetic retinopathy. PMID- 8623888 TI - Retinal pigment epithelial cell count, distribution, and correlations in normal human eyes. AB - PURPOSE: To determine the number of the retinal pigment epithelial cells, their regional distribution, and correlations with general variables in normal human eyes. METHODS: We examined 53 normal human donor eyes from individuals with a mean age of 58.6 +/- 18.8 years and an axial length of less than 27 mm. After opening the globes by a 16-mm corneoscleral trephination, up to 25 biopsy specimens of the retina and retinal pigment epithelium were obtained in four meridians. The retinal pigment epithelial cells and the retinal photoreceptors were counted on photographs taken from the apical retinal pigment epithelial surface and the inner photoreceptor segments. RESULTS: Total number of the retinal pigment epithelial cells was 3,556,290 +/- 490,700 (mean +/- S.D.; range, 2,130,500 to 4,653,200), and it was positively correlated with the number of rods and cones, the total area of the retina, the optic disk size, and the retinal pigment epithelial cell density but was independent of gender. The retinal pigment epithelial cell density decreased significantly (P < .001) from the fovea (4,220 +/- 727 cells/mm2) to the midperiphery (3,002 +/- 460 cells/mm2) and to the outer peripheral fundus regions (1,600 +/- 411 cells/mm2). In the retinal periphery, the retinal pigment epithelial cell density was the highest (P < .01) in the nasal fundus region compared with any other fundus quadrant. The retinal pigment epithelial cell density decreased by about 0.3% per year with increasing age. CONCLUSIONS: These data on the quantitative anatomy of the retinal pigment epithelium may be important for physiology and pathophysiology of the visual process, especially in evaluating the effects of aging. PMID- 8623889 TI - Cobalt-60 treatment of choroidal hemangiomas. AB - PURPOSE: We investigated the therapeutic possibilities of gamma brachytherapy to improve the final functional results of eyes with choroidal hemangiomas, which are benign vascular tumors that can induce progressive impairment of visual acuity. METHODS: We treated 41 patients with choroidal hemangioma with cobalt-60 applicators. The lesions consisted of 39 circumscribed hemangiomas and two diffuse hemangiomas in patients with Sturge-Weber syndrome. Before treatment, visual acuity in the affected eye was 20/200 in ten patients, 20/200 to 20/50 in 17 patients, 20/40 to 20/25 in 11 patients, and 20/20 in three patients. All patients were symptomatic. The macula was infiltrated by the tumor in 12 eyes (29.3%). There was retinal detachment in 40 eyes (97.6%), cystoid edema in ten eyes (24.4%), subretinal fibrosis in eight eyes (19.5%), and areolar atrophy in two eyes (4.9%). RESULTS: After treatment, the retina was reattached in all eyes, and the tumor progressively transformed into a flat scar. The postirradiation macular lesions that we identified were pigment migrations in the macular region, subretinal fibrosis, and an areolar atrophic scar. We correlated the functional results at two, five, and ten years after treatment with the initial visual acuity, and with pre-existing and posttreatment macular lesions. CONCLUSIONS: Our results suggest that radiotherapy is a valuable therapeutic modality for choroidal hemangiomas, particularly in hemangiomas that involve the macula, and for tumors associated with bullous retinal detachment. PMID- 8623890 TI - The treatment of Graves' ophthalmopathy in an incidence cohort. AB - PURPOSE: To determine the frequencies of medical and surgical treatments in an incidence cohort of 120 patients with Graves' ophthalmopathy. METHODS: We reviewed the community medical records and administered a follow-up questionnaire. RESULTS: Of the 120 patients, 89 (74.2%) required either no therapy or only supportive measures. Six patients (5.0%) were treated with systemic corticosteroids. One patient had orbital radiotherapy. Twenty-four patients (20.0%) underwent one or more surgical procedures. The cumulative probabilities of undergoing ophthalmic surgery of any type were 5.0% by one year after the diagnosis of ophthalmopathy, 9.3% after two years, 15.9% after five years, and 21.8% after ten years. The need for surgery was significantly related to age (P < .01; Cox proportional hazards model) but was not significantly dependent on gender (P = .5) or the interaction of age and gender (P = .15). The overall risk of the need for surgery was 2.6 times greater in patients older than 50 years (95% confidence interval, 1.2 to 5.8) than in younger patients. There were no significant differences between tobacco smokers and nonsmokers in the cumulative probabilities of undergoing surgery. CONCLUSION: In 24 (20%) patients, one or more surgical procedures were used to treat Graves' ophthalmopathy. The probability of surgical intervention was significantly related to patient age (older than 50 years), but it was not related to gender or smoking. PMID- 8623891 TI - In vivo confocal microscopy and Acanthamoeba keratitis. PMID- 8623892 TI - Ocular signs and symptoms caused by exposure to sarin gas. AB - PURPOSE: Ocular signs and symptoms that occurred in people exposed to sarin gas in a subway sarin gas attack were studied. METHODS: Among victims of sarin gas exposure, 96 were treated by us. RESULTS: Ocular signs and symptoms caused by sarin gas exposure included miosis, conjunctival injection, and ocular pain. CONCLUSIONS: Ocular signs and symptoms spontaneously resolved between three and 21 days after exposure in most cases. Treatment with 0.5% tropicamide ophthalmic solution was effective in decreasing ocular pain. PMID- 8623893 TI - Accommodation in primate eyes after implantation of refilled endocapsular balloon. AB - PURPOSE: To restore accommodation after crystalline lens removal. METHODS: The crystalline lens of one eye of each of four primates was removed by phacoemulsification. Each operated-on eye underwent endocapsular silicone balloon implantation followed by filling with an organosilicone mixture. Seven months postoperatively, the anterior chamber depth and refractive power changes were measured before and after topical application of 4% pilocarpine. RESULTS: In the refilled eyes, the average anterior chamber depth reduction was 0.50 mm and the average maximal myopic change was 6.74 diopters. CONCLUSION: After lens removal, measurable accommodation was restored in the eyes with the endocapsular balloon filled with an organosilicone mixture. PMID- 8623894 TI - Reading-evoked visual dimming. AB - PURPOSE: To carry out a neuroradiologic investigation in a monocular 49-year-old patient who during the past five years described symptoms of dimming of central vision in his left eye, which was provoked only by reading. METHODS: Computed tomography and magnetic resonance imaging were performed. RESULTS: An orbital apex intraconal tumor situated laterally to and above the optic nerve was found. CONCLUSIONS: Reading-evoked visual dimming can be a variant of gaze-evoked amaurosis. The optic nerve displaced laterally and superiorly, and stretched by the act of reading, may be compressed between the tumor above and the contracted inferior rectus muscle inferiorly. PMID- 8623895 TI - Ultrasound biomicroscopy in pars planitis. AB - PURPOSE: To report the diagnosis of pars planitis on the basis of ultrasound biomicroscopy images. METHODS: The Humphrey ultrasound biomicroscope was used for examination of the right eye of a 17-year-old boy, referred to our clinic because of blurred vision. The lens showed a posterior subcapsular cataract precluding fundus visualization. RESULTS: Ultrasound biomicroscopy showed a homogeneous mass of medium reflectivity over the pars plana. Lensectomy and vitrectomy confirmed the diagnosis. CONCLUSION: This brief case report points out the utility of ultrasound biomicroscopy in diagnosing pars planitis in cases of media opacities. PMID- 8623896 TI - Choroidal neovascular membrane associated with optic nerve head drusen in a child. AB - PURPOSE: To illustrate the diagnosis, evaluation, and complications of pseudopapilledema in children. METHODS: We examined a 9-year-old boy who had suspected papilledema and a retinal mass. He had undergone neuroradiologic imaging at an outside facility. RESULTS: Clinical examination of the patient provided the diagnosis of optic nerve head drusen, pseudopapilledema, and a cicatrized choroidal neovascular membrane. Examination of the boy's parents disclosed optic nerve head drusen in the father. CONCLUSIONS: Choroidal neovascular membranes caused by optic nerve head drusen are uncommon in children. Clinical examination of the patient and family members, along with B-scan ultrasonography, can establish this cause. Neuroradiologic testing is unnecessary, and carries risk related to the need for sedation. PMID- 8623897 TI - Presumed ocular and central nervous system tuberculosis in a patient with the acquired immunodeficiency syndrome. AB - PURPOSE: To elucidate a case of tuberculous choroiditis in a patient with the acquired immunodeficiency syndrome (AIDS). METHODS: We treated a 35-year-old woman who had AIDS with neurologic involvement caused by Mycobacterium tuberculosis. She developed a yellow-white chorioretinal infiltrate with indistinct borders and mild vitreitis in the right eye, probably caused by this pathogen. RESULTS: The patient's visual acuity improved in the right eye with healing of the ocular lesion and her neurologic condition improved after specific therapy with isoniazid, rifampin, and ethambutol. CONCLUSION: Tuberculosis must be considered in the differential diagnosis of posterior uveitis and choroiditis in AIDS patients. PMID- 8623898 TI - Squamous cell carcinoma of the eyelid and the acquired immunodeficiency syndrome. AB - PURPOSE: We studied a case of squamous cell carcinoma of the eyelid in a 42-year old homosexual man with the acquired immunodeficiency syndrome (AIDS) to explore the link between squamous cell carcinoma and human Papillomavirus infection. METHOD: A rapidly growing, 3-mm lesion was removed from the right lower eyelid by excisional biopsy. RESULTS: Examination of the lesion confirmed squamous cell carcinoma. Polymerase chain reaction for human Papillomavirus 16 was positive. CONCLUSION: This case suggests a role for human Papillomavirus infection in the cause of squamous cell carcinoma in AIDS patients. PMID- 8623899 TI - Postmortem diagnosis of Fusarium panophthalmitis by the polymerase chain reaction. AB - PURPOSE: We undertook this study to determine if a polymerase chain reaction based test that we developed for the filamentary fungus, Fusarium, could be used to detect the organism in postmortem ocular tissues. METHODS: We applied the polymerase chain reaction to amplify a target fragment of Fusarium DNA from formalin-fixed ocular tissues from a patient with endogenous Fusarium panophthalmitis. RESULTS: By using the polymerase chain reaction-based test, we were able to amplify the target fragment of DNA from the infected eyes, but not from uninfected control eyes. CONCLUSIONS: The technique appears to hold promise to be a sensitive, specific, and rapid method of diagnosing Fusarium infections. PMID- 8623900 TI - Cataract surgery and intraocular lens implantation in children, and intraocular lens implantation in children. PMID- 8623901 TI - Cataract surgery and intraocular lens implantation in children. PMID- 8623902 TI - Charting a new course through the chaos of KS (Kaposi's sarcoma) PMID- 8623903 TI - Molecular analysis of the pathological autoimmune antigens of Heymann nephritis. PMID- 8623904 TI - Molecular anatomy and the pathological expression of antibody light chains. PMID- 8623905 TI - Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix. PMID- 8623906 TI - Evidence that intestinal intraepithelial lymphocytes are activated cytotoxic T cells in celiac disease but not in giardiasis. AB - To further define intraepithelial lymphocytes (IELs) in celiac disease (CD) and giardiasis, IELs were probed for the presence of cytolytic granules containing granzyme B (GrB) and T-cell-restricted intracellular antigen (TIA)-1. The expression of TIA-1, GrB, CD3 (T-cell-receptor-associated complex), and Leu-7 (subset of natural killer cells) was studied by a sensitive three-step immunoperoxidase technique. Stained IELs were determined quantitatively, and results were expressed as number of stained IELs per 100 epithelial cells (ECs). The relative content in labeled lamina propria lymphocytes was determined and expressed as the percentage of all lamina propria cells counted. When compared with controls, CD3+ and GrB+ IELs were significantly increased (P < 0.0004) in CD paralleled by an increase in TIA-1+ IELs (P < 0.0004). In CD, the highest numbers of IELs containing GrB were found in subjects with a flat mucosa (median, 38 IELs/100 ECs, P < 0.0004), followed by cases with shortened and blunted villi (median, 8 IELs/100 ECs, P < 0.0004) and, finally, CD patients with an intact villous architecture (median, 0.5 IELs/100 ECs, P < 0.02). Except for cases with giardiasis, Leu-7+ IELs were virtually absent in all groups as were GrB+ IELs in the controls and in subjects with giardiasis. In the lamina propria of CD subjects, GrB+ lymphocytes were also significantly increased (P < 0.001), whereas controls and cases with giardiasis were essentially free of GrB+ cytotoxic T lymphocytes. The percentage of CD3+ lamina propria lymphocytes was nearly equal in all groups. In humans and mice, extensive studies revealed a GrB expression to be absolutely restricted to activated cytotoxic T lymphocytes and natural killer cells. TIA-1, on the other hand, is considered a marker of resting T lymphocytes possessing cytolytic potential. We therefore conclude that IELs are cytotoxic T cells that are in a resting state in the normal small bowel and in giardiasis. In CD, they become activated as suggested by the GrB positivity of their granules. PMID- 8623907 TI - Thymoma-associated myasthenia gravis. Transplantation of thymoma and extrathymomal thymic tissue into SCID mice. AB - To study the possible role of thymomas and of extrathymomal thymic tissue in the development and maintenance of myasthenia gravis, we transplanted fragments of either tissue into SCID mice and monitored the production of anti-acetylcholine receptor antibodies in the recipients. Furthermore, the transplants were characterized by immunohistochemistry. Unlike after transplantation of thymus with lymphofollicular hyperplasia that induced high titers of anti-acetylcholine receptor antibodies, thymoma transplants never produced autoantibodies. Mice transplanted with extrathymomal thymic tissue also failed to produce anti acetylcholine receptor antibodies except one group that received transplants containing hyperplastic extrathymomal tissue. These findings may explain the refractoriness of thymomatous myasthenia to thymectomy. PMID- 8623908 TI - Chemokines and T lymphocyte recruitment to lymph nodes in HIV infection. AB - Recruitment of T lymphocytes to lymph nodes in patients with HIV infection is critical to the pathogenesis of disease. Chemokines are a family of cytokines, which are potent regulators of leukocyte migration. We studied the leukocyte populations and expression of chemokines known to be active upon T cells in lymph nodes of four HIV infected patients and seven control subjects using in situ hybridization, immunohistochemistry, and FACS analysis. The HIV lymph nodes showed CD8+ T lymphocyte accumulation and strongly enhanced chemokine expression, notably for the CD8+ T cell chemoattractant, macrophage inflammatory protein (MIP)-1 alpha. Resident macrophages appeared to be a major cellular source of chemokines in the HIV nodes. RANTES expression was present in both HIV and control lymph nodes, suggesting a physiological role for this chemokine in T lymphocyte recirculation. Chemokines may be important determinants of T lymphocyte accumulation in lymphoid tissue of patients with HIV/AIDS. PMID- 8623909 TI - Expression of E-cadherin in primary and metastatic prostate cancer. AB - Immunohistochemical studies have suggested that E-cadherin may be a useful prognostic marker in prostate cancer. Previous studies have depended on cryostat sections of tissues selected grossly. Many prostate cancers, even when extensive, are not visible grossly; many others cannot be demarcated sharply grossly. The wide applicability of prognostic markers after total prostatectomy will depend upon methods that can be applied to tissue selected based upon the histopathological examination of the entire prostate. Our purpose was to investigate the possibility that E-cadherin could be demonstrated in paraffin embedded whole prostates and metastatic prostate cancer. Microwaving in citrate buffer was the best of five methods tested for the demonstration of E-cadherin in paraffin-embedded prostate and was used to investigate 53 primary prostate cancers from 44 patients and lymph node metastases from 14 patients. Metastases of prostate cancer to lymph nodes expressed less (P = 0.008) E-cadherin than primary prostate cancers. The expression of E-cadherin correlated with the histopathological differentiation (Gleason grade) of primary prostate cancers (P = 0.03, Ptrend = 0.003). The use of monoclonal anti-human E-cadherin (HECD-1) with microwaving in citrate buffer followed by immunoperoxidase staining with heavy metal enhancement for the demonstration of E-cadherin in paraffin-embedded tissue will, for the first time, allow the use of archival tissue for prognostic studies of E-cadherin in prostate cancer and other tissue. Our results are consistent with the hypothesis that aggressive prostate cancers exhibit decreased expression of E-cadherin and demonstrate the feasibility of long-term prognostic studies of this molecule in the usually multiple prostate cancers found in whole, formalin-fixed, paraffin-embedded resected prostates. PMID- 8623910 TI - The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplasmic localization. AB - Mutations of the p53 tumor suppressor gene are rarely found in neuroblastoma. Though typically a nuclear protein, a number of tumor cell types have recently been reported to exhibit cytoplasmic p53 immunostaining, and it has been suggested that altered cellular localization is another mechanism of inhibiting p53 function. We examined p53 protein expression, localization, and function in neuroblastoma cell lines with wild-type p53 genes. Basal p53 levels were largely confined to the cytoplasmic compartment in these cells. However, after irradiation, p53 protein levels increased predominately in the nucleus. Transcriptional activity of p53 was intact in these cells because "downstream" proteins, p21WAF1 and MDM2, were induced by irradiation. In contrast to a neuroblastoma cell line harboring a mutant p53 gene, the neuroblastoma cells with wild-type protein were associated with an intact G1 arrest after DNA damage. The induced nuclear protein in these neuroblastoma cells also appeared functional as measured by its capacity to bind to a DNA oligomer containing a p53-consensus sequence. We have concluded that although p53 expression in neuroblastoma cells is primarily localized to the cytosol, ionizing radiation induces a functional p53 protein in the nucleus and that this cytoplasmic sequestration of p53 in human neuroblastoma is not a mechanism of inactivating p53 function. PMID- 8623911 TI - CD40 antigen is expressed by endothelial cells and tumor cells in Kaposi's sarcoma. AB - The CD40 antigen is a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily and is involved in cell proliferation, differentiation, and survival. Using different monoclonal antibodies, we found CD40 expression by immunohistochemistry on CD31- and CD34-positive Kaposi's sarcoma spindle cells in all tumors of 18 HIV-1 seropositive and 4 HIV-1 seronegative patients. Western blot analysis of tumor lysates detected a 48- to 50-kd glycoprotein corresponding to the CD40 antigen expressed by B lymphocytes. CD40 expression was also detectable in one of four cultures of spindle cells derived from Kaposi sarcoma tissue. Treatment of the CD40-positive spindle cells but not of the CD40-negative ones with interferon-gamma up-regulated CD40 surface expression. Besides on Kaposi sarcoma tumor cells, CD40 was distinctly present on vascular endothelial cells in areas within and adjacent to the tumors and in benign inflammatory lesions such as granulation tissue of HIV-1-negative patients. In contrast, CD34-negative endothelia of thin walled vessels, most likely lymphatics, were predominantly CD40 negative. Only faint or no CD40 expression was found on endothelial cells in normal skin. We conclude from our data that expression of the CD40 antigen by endothelial cells is up-regulated during tissue inflammation. As signaling through CD40 is able to increase cell survival, expression of CD40 by Kaposi sarcoma tumor cells might play an important role in the pathogenesis of this neoplasm. PMID- 8623912 TI - Light chain cardiomyopathy. Structural analysis of the light chain tissue deposits. AB - Cardiomyopathy due to monoclonal light chain deposits is a complication of plasma cell disorders. The deposits may be either fibrillar as in light chain amyloid or nonfibrillar as in light chain deposition disease. The reasons for these structural differences are still unknown. We characterized the myocardial deposits by immunohistochemical examination of sections and extraction and biochemical analysis of the tissue deposits in a patient (MCM) who died of myeloma and systemic light chain deposition disease. Amino acid sequence analysis of the extracted nonfibrillar MCM kappa-light chain reveals that it belongs to the L12a germline subset of the kappa(I) protein and contains five distinctive amino acid substitutions (three in the framework region III and two in the complementarity-determining region III) that have not been reported previously in the same positions in other kappa(I) light chains. The theoretically determined isoelectric point (pI 8.21) of the MCM light chain is high compared with the low isoelectric point of other Bence Jones proteins from subjects without light chain deposition disease. The diffuse binding to basement membranes and the high isoelectric point of the MCM kappa-light chain suggest electrostatic interaction as a possible mechanism of tissue deposition. The spatial locations of the five distinctive residues and a sixth rare substitution of the MCM protein modeled on the backbone structure of REI, a kappa(I)-soluble Bence Jones light chain of known three-dimensional structure, may be responsible for protein destabilization, partial unfolding, and aggregation leading to tissue deposition. PMID- 8623914 TI - Stage-specific expression of integrin alphaVbeta3 in neuroblastic tumors. AB - The ligand specificity of the integrin cell adhesion receptors probably determines the ability of specific integrins to promote tumor cell proliferation and metastasis. Therefore, we compared the expression of integrin alphaVbeta3, a promiscuous receptor that binds with high affinity to numerous cell matrix proteins, with the expression of integrin alphaVbeta5 and the integrin beta 1 subunit (which pairs with multiple alpha subunits) in neuroblastic tumors at various stages of differentiation. Undifferentiated neuroblastoma tumors rapidly invade and metastasize, whereas ganglioneuroblastomas rarely metastasize. Differentiating neuroblastomas are associated with an intermediate prognosis. Paraffin sections of neuroblastic tumors at various stages of differentiation obtained at biopsy from 17 patients were hybridized with antisense integrin subunit-specific alphaV, beta3, beta1, and beta5 riboprobes. All neuroblastic tumors and seven adrenal glands obtained at autopsy were analyzed immunohistochemically with antibodies directed toward the alphaV, beta3, beta1, and beta5 subunits. The alphaV subunit was expressed in neuroblastic tumors independent of the stage of differentiation, although mRNA and protein expression were generally weak in ganglioneuroblastomas, and was also detected in adrenal gland medullae. The beta1 subunit was detected in most neuroblastic tumors independent of the stage of differentiation as well as in adrenal gland medullae. In contrast, the beta3 subunit, which was not expressed in adrenal gland medullae, was expressed at the protein and mRNA levels in undifferentiated neuroblastomas (six of seven and seven of seven, respectively) but was not expressed in neuroblasts or ganglion cells in ganglioneuroblastomas (one case weakly positive out of five). The beta 5 subunit was expressed at the protein (five of five) and mRNA (four of five) levels in the ganglion cells of ganglioneuroblastomas and, although mRNA for this subunit was detectable in undifferentiated tumors, the protein was not detectable. The expression of integrin alphaVbeta3 in undifferentiated neuroblastomas may contribute to the rapid growth of these tumors and their tendency to metastasize. PMID- 8623913 TI - Transient functional expression of alphaVbeta 3 on vascular cells during wound repair. AB - During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound. PMID- 8623915 TI - Normal human ovary and ovarian tumors express glycodelin, a glycoprotein with immunosuppressive and contraceptive properties. AB - Glycodelin is a glycoprotein with potent immunosuppressive and contraceptive activities. It reacts with antibodies against placental protein 14, or progesterone-associated endometrial protein, and has a unique carbohydrate structure. Previous nomenclature is misleading, because glycodelin is neither synthesized in the placenta nor is it endometrium specific. No ovarian synthesis of glycodelin has been demonstrated. We present evidence for glycodelin synthesis in the human ovary and ovarian tumors. In follicular phase, immunoperoxidase staining of microwave-treated tissue sections employing affinity-purified polyclonal antibodies localized glycodelin to areas of stromal cell condensation in ovarian cortex, theca interna, and the granulosa. In luteal phase, cortical stroma was negative or only weakly positive, whereas glycodelin was present in theca interna of the corpus luteum and luteinized granulosa cells and also in corpus albicans and Leydig cells of the ovarian hilus. In situ hybridization gave negative results for glycodelin mRNA in normal ovary, whereas in ovarian tumors strong expression of both the glycodelin mRNA and the protein were found in benign and malignant serous cystadenomas, mucinous ovarian tumors being negative. We conclude that glycodelin is synthesized in human ovarian tumors, and its occurrence in normal human ovary may represent either synthesis or a site of glycodelin action. PMID- 8623916 TI - Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin. AB - The surface of a normal ovary is covered by a monolayer of epithelial cells that rest on a basement membrane. The glycoprotein laminin is the major noncollagenous protein present in the basement membrane. The integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4 serve as cell surface receptors for laminin. During the progression of serous ovarian carcinoma, tumor cells are frequently exfoliated from the surface of the ovary, thereby losing contact with the basement membrane. This study was designed to determine whether alterations in integrin expression may be associated with the malignant phenotype of the primary ovarian tumor and exfoliated ovarian carcinoma cells in the ascites fluid. By immunohistochemical staining, the entire surface of epithelial cells of normal ovaries stained positively for beta 1, alpha 2, and alpha 3 integrins, whereas only the basal surface of the epithelial cells, where they are in contact with laminin, stained positively for alpha 6 and beta 4. The entire surface of epithelial cells of solid tumors from patients with serous ovarian carcinoma stained positively for beta 1, alpha 2, and alpha 3 integrins. In most cases, no intact basement membrane surrounded the tumor nests, and staining for alpha 6 and beta 4 was irregular. When present, the basement membrane stained positively for laminin, and the basal surface of the epithelial cells stained positively for alpha 6 and beta 4. Ovarian carcinoma ascites cells exhibited a distinct phenotype, with a significant decrease in expression of the alpha 6 and beta 4 integrin subunits. As alpha 6 and beta 4 integrin subunits are present at the basal surface of many epithelial cells and serve as receptors for laminin, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of alpha 6 and beta 4 integrin subunits. PMID- 8623917 TI - Expression of extracellular matrix proteoglycans perlecan and decorin in carbon tetrachloride-injured rat liver and in isolated liver cells. AB - Proteoglycans are important components of the extracellular matrix. They are involved in liver regeneration as well as in liver fibrosis. The distribution and cellular source of proteoglycans under normal as well as pathological conditions is still under debate. Localization of decorin and perlecan was studied in normal, acutely damaged, and cirrhotic liver by histochemistry. Furthermore, their synthesis was analyzed in different liver cell populations isolated from normal rat liver. In normal liver, decorin positivity was observed in the perisinusoidal space and in the portal area. Perlecan was clearly detectable in the portal area (blood vessels and bile ducts); a moderate reaction was also seen along the sinusoids. Strong positivity for both proteoglycans was detectable in the necrotic areas of acutely damaged liver. Chronic liver damage was characterized by the deposition of decorin and perlecan in the fibrotic septa. Immunocytochemical reactions were positive for perlecan and decorin in cultured Ito and endothelial cells but not in hepatocytes and Kupffer cells. Northern hybridization confirmed the capacity of Ito cells and endothelial cells to express the two genes. Interestingly, although rat skin fibroblasts expressed strong messages for both proteoglycans, rat aortic smooth muscle cells did not synthesize decorin. PMID- 8623918 TI - Blast-like cell compartment in carcinogen-induced proliferating bile ductules. AB - Small non-epithelial cells with morphological features of blast-like cells are found within a proliferating intrahepatic biliary system after institution in rats of a diethylnitrosamine, 2-acetylaminofluorene, partial hepatectomy carcinogenesis protocol. Two to three days after the partial hepatectomy step of the carcinogen protocol, the small blast-like cells are evident beneath a layer of bile ductule epithelial cells that line the walls of the bile ductules. The basally located small cells are not exposed to the bile ductule lumen or to the surrounding basal lamina. They ranged in size from 3.0 to 5.0 microns, exhibit an undifferentiated phenotype, including a high nucleus-to-cytoplasm ratio and no to minimal differentiated cytoplasmic and surface structures. Mitosis of blast-like cells are evident, and their nuclei express proliferating nuclear cell antigen. The ductal blast-like cells do not express cytokeratin 19, oval cell antigen 270.38, or actin immunoreactivity, in contrast to bile ductule epithelial cells. The basal cells, as well as bile ductule epithelial cells, are negative for a panel of T and B lymphocyte surface markers in contrast to lymphocytes present in the connective tissue stroma surrounding the bile ductules and throughout the hepatic parenchyma. Within some segments of the biliary system, some of the ductal blast-like cells increased in size to approximately 10 microns and showed increased amounts of cytoplasmic organelles and plasma membrane filapodia but did not develop the polarized phenotype of bile ductule epithelial cells (ie, apical microvilli, desmosomes, connections to bile ductule cells, and exposure to duct lumen); however, their nuclear morphology was essentially similar to the smaller basal cells. We also found bile ductules to contain two types of polarized epithelial cells, one with the characteristic oval nucleus of the oval/bile ductule epithelial cells and the other, transitional epithelial cells with a rounder nucleus and prominent nucleoli. The transitional cells exhibit a similar apical-basal polarity and antigenic phenotype as the oval/bile ductule epithelial cells. However, transitional cells are larger and have an overall less dense cytoplasm than the bile ductule epithelial/oval cells, and some show apical microvilli changes and small catalase-positive peroxisomes. These observations indicate that a greater diversity of cell types exist within intrahepatic bile ductules of rats treated with carcinogens. Furthermore, the nonpolarized ductal blast-like cells undergo proliferation and are significantly different in phenotype from other hepatic cells previously reported as candidates for liver progenitor cells. PMID- 8623919 TI - Increased expression of interleukin-4 receptors on psoriatic epidermal cells. AB - Abnormal keratinocyte proliferation and differentiation, the main characteristics of psoriasis, may be induced and maintained by cytokines produced by activated resident and recruited inflammatory cells in lesional skin. As the epidermal cytokine profile is clearly altered in psoriasis and because increased expression of interleukin-4 receptor (IL-4R) has been reported in some epithelial proliferative diseases, we investigated the expression of IL-4R on psoriatic epidermal cells (ECs). The expression of IL-4R on freshly isolated ECs from healthy skin and untreated psoriatic lesions was studied by immunostaining using an IL-4R-specific antibody and by examining their capacity to bind biotinylated recombinant human IL-4 using flow cytometry. The number of IL-4R+ ECs and the number of binding sites per cell were significantly increased on psoriatic ECs as compared with healthy control ECs. Immunostaining confirmed these results, whereby staining was mainly observed in the lower epidermal layers. In addition, an increased IL-4R mRNA expression was also observed in psoriatic epidermis using a digoxigenin-labeled IL-4R RNA probe. In short-term in vitro cultures, lipopolysaccharide/phorbol-myristate-acetate-stimulated and unstimulated psoriatic ECs did not produce any immunoreactive IL-4. The results of this study together with the reported increased expression of IL-4R in epithelial neoplasias suggest an association between overexpression of IL-4R and abnormal keratinocyte activation and proliferation. PMID- 8623920 TI - Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells. AB - Chronic inflammation developed spontaneously in the large intestine of C.B-17 scid mice restored with the CD45RBhigh subset of CD4+ T cells obtained from normal BALB/c mice. The inflammation, which extended diffusely from the cecum to the rectum, was localized to the lamina propria of mildly affected mice but became transmural in severely affected mice. Immunohistochemical and flow cytometric analyses showed that the inflammatory infiltrate contained numerous macrophages accompanied by moderate numbers of activated CD4+ lymphocytes. Some mice also had scattered multinucleated giant cells. Mucin depletion and epithelial hyperplasia resulting in glandular elongation and mucosal thickening were also consistently seen. Less frequent findings included ulceration with fibrosis, crypt abscesses, crypt loss, and granulomatous inflammation. Immunofluorescent analysis of inflamed large intestinal sections demonstrated increased epithelial expression of major histocompatibility class II antigens. The changes in the large intestine of these mice are similar to those seen in patients with idiopathic inflammatory bowel disease (Crohn's disease and ulcerative colitis). This murine model may be useful for studying mucosal immunoregulation as it relates to the pathogenesis and treatment of chronic inflammatory bowel diseases in the large intestine of human patients. PMID- 8623921 TI - Purification and characterization of Lewy bodies from the brains of patients with diffuse Lewy body disease. AB - Lewy bodies (LBs) are the pathological hallmarks of degenerating neurons in the brains of patients with Parkinson's disease and diffuse Lewy body disease. We developed a novel purification procedure for LBs using sucrose density separation followed by fluorescence-activated particle sorting, and we raised > 15 monoclonal antibodies to LBs purified from diffuse Lewy body disease brains. The monoclonal antibody that stained the largest number of LBs most intensely did not recognize ubiquitin in free or monoubiquitinated forms nor the ubiquitin conjugating enzymes, but it did react with polyubiquitin chains as well as with high molecular weight polyubiquitinated LB-derived proteins. Thus, these results suggest that LBs contain polyubiquitin chains. Although polyubiquitination of LB proteins may trigger ubiquitin-proteasome proteolytic pathways, the incomplete activation of these pathways could play a mechanistic role in the formation of LBs in neurodegenerative diseases. PMID- 8623922 TI - Analysis of p53, K-ras-2, and C-raf-1 in pulmonary neuroendocrine tumors. Correlation with histological subtype and clinical outcome. AB - Neuroendocrine tumors of lung, including typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC) constitute a spectrum of malignancies in which the pathologist at times has difficulty in discerning tumor subtype and aggressiveness in a reproducible fashion. Therefore, 59 primary neuroendocrine lung tumors including 10 TCs, 26 ACs, 15 LCNECs, and 8 SCLCs were selected from cases collected from 1976 to 1988 and immunostained for p53 protein. All of these tumors were also genotyped for specific point mutational damage affecting p53 (exons 5, 7, and 8; with ACs additionally sequenced for p53 exon 6); 13 tumors for K-ras-2 (exon 1); and 31 tumors for c-raf-1 (exon 15) growth-regulatory genes. Genotyping was performed on topographically selected, minute tumor samples removed from unstained formalin-fixed, paraffin-embedded tissue sections (topographic genotyping) using polymerase chain reaction and direct sequencing. The distribution of p53 immunohistochemical staining had four patterns: negative in TCs, one-half of ACs, 3 of 15 LCNECs, and 1 of 8 SCLCs; less than 10% but more than five tumor cells per 10 high power fields (focal) in a subset (7 of 26) of aggressive ACs; 10 to 49% of tumor cells (patchy) in a subset (6 of 26) of ACs with a higher grade of aggressiveness; and 50 to 100% of tumor cells (diffuse), exclusively seen in LCNECs (12 of 15) and SCLCs (7 of 8). Three patterns of immunohistochemical staining intensity of p53 protein were seen: negative, weak or mild, and moderate to marked. SCLCs and LCNECs accounted for cases of moderate to marked staining and were the only ones to have mutations in p53 exons 5, 7, or 8. No mutations were found in AC and TC, showing absent to weak staining and no staining, respectively. The difference in distribution and staining intensities between LCNEC and SCLC compared with AC and TC was statistically significant (P < 0.001). Patients having AC with patchy p53 immunostaining usually had survival limited to 3 years, whereas those having AC with focal p53 immunostaining subsequently developed metastatic or recurrence of AC disease (P < 0.05). The absence of point mutations in cases with patchy or focal immunostaining suggests increased expression of wild-type p53 tumor suppressor protein likely in response to growth deregulation in a more aggressive subtype of AC. A novel hypothesis is presented in regard to these findings. K-ras-2 and c-raf-1 gene sequence analysis showed no evidence of point mutational change in any of the tumors studied. The TC and AC categories are therefore genetically distinct from the higher grade neuroendocrine SCLC and LCNEC. Immunohistochemistry for p53 on AC lung tumors may be helpful to delineate cases at higher risk for aggressive behavior. Additionally, although LCNEC is categorized as a non-small-cell carcinoma, it is more akin genetically and immunohistochemically to SCLC. PMID- 8623923 TI - Monoclonal antibodies PG-B6a and PG-B6p recognize, respectively, a highly conserved and a formol-resistant epitope on the human BCL-6 protein amino terminal region. AB - The human BCL-6 gene, which is rearranged in approximately 30% of diffuse large B cell lymphomas, encodes a 706-amino-acid nuclear protein of the Kruppel-type zinc finger transcription factors mainly expressed in normal germinal center B cells and related lymphomas. Four monoclonal antibodies (PG-B6, PG-B6a, PG-B6p, and PG B6m), specifically directed against the human BCL-6 protein, were generated by immunizing BALB/c mice with a recombinant protein corresponding to the BCL-6 amino-terminal region (amino acids 3 to 484). The PG-B6 monoclonal antibody reacted with a BCL-6 epitope sensitive to fixatives and preserved in all mammalian species. PG-B6a (a is for avian) recognized the most evolutionarily conserved BCL-6 epitope (expressed in all animal species including avian). PG-B6p (p is for paraffin) recognized a fixative-resistant epitope of BCL-6 that was detectable on paraffin sections after microwave heating in 1 mmol/L EDTA buffer. PG-B6m (m is for mantle) was the least specific monoclonal antibody as, in addition to BCL-6, it reacted with a yet undefined antigen selectively located in the cytoplasm of mantle and marginal zone B cells. All monoclonal antibodies detected strong nuclear expression of BCL-6 in follicular lymphomas, diffuse large B cell lymphomas, Burkitt's lymphomas, and nodular, lymphocyte-predominance Hodgkin's disease. In diffuse large B cell lymphomas, BCL-6 expression was independent of BCL-6 gene rearrangements and did not correlate with expression of other markers or the proliferation index. BCL-6 was not expressed in B-CLL, hairy cell leukemia, mantle-cell- and marginal-zone-derived lymphomas. Labeling of paraffin sections with PG-B6p proved useful for differentiating proliferation centers in B-CLL (BCl-2+/BCL-6-) from trapped germinal centers in mantle cell lymphomas (BCL-2-/BCL-6+) and for identifying neoplastic cells in cases of nodular, lymphocyte-predominance Hodgkin's disease. Because of their high specificity, wide reactivity in humans and animal species including avians (PG B6a), and suitability for labeling routine paraffin sections (PG-B6p), the reagents described in this paper should prove valuable in both research and diagnostics. PMID- 8623924 TI - Prognostic significance of Bcl-2 in clinically localized prostate cancer. AB - The clinical course of prostate cancer is highly variable and cannot satisfactorily be predicted by histological criteria alone. To study the prognostic significance of Bcl-2 and p53 overexpression in prostate cancer, 137 consecutive radical prostatectomy specimens were examined by immunohistochemistry. Both Bcl-2 and p53 were associated with malignant phenotype. Bcl-2 expression was more frequent in pT3 tumors (31% positive) than in pT2 tumors (5% positive, P = 0.001). p53 overexpression (found in 8%) was associated with high Gleason score (P = 0.03) and increased tumor growth fraction (Ki67 labeling index (LI); P = 0.017). Survival analysis showed that Bcl-2 expression (P = 0.03), high Ki67 LI (P = 0.018), high grade (P = 0.0037), advanced local stage (P = 0.0005), and positive lymph nodes (P = 0.026) were predictors of progression. The combined analysis of Ki67 LI and Bcl-2 allowed the distinction of three groups with different clinical outcome. Prognosis was best in Bcl-2-negative tumors with low Ki67 LI, worst in Bcl-2-positive tumors with high Ki67 LI, and intermediate in the remaining tumors (P = 0.03). These data suggest that altered expression of both Bcl-2 and p53 play a role in prostate cancer progression. Combined analysis of factors regulating both apoptosis and cell proliferation may be relevant in prostate cancer. PMID- 8623926 TI - Immunohistochemical detection of p53 in Wilms' tumors correlates with unfavorable outcome. AB - The role of p53 in the pathogenesis and progression of Wilms' tumors is only partly understood. Although p53 mutations were initially reported only in anaplastic Wilms' tumors, we had reported that, of two of twenty-one cases that had a p53 mutation, one tumor showed no evidence of anaplasia. To determine the significance of p53 expression in all clinical stages of Wilms' tumor, twenty eight cases were analyzed for p53 immunoreactivity. Paraffin sections were immunolabeled with two different monoclonal antibodies, recognizing both mutant and wild-type p53. Fifteen of sixteen tumors in the recurrent/metastatic group and three of twelve tumors in the nonmetastatic/nonrecurrent group showed p53 immunopositivity. Only one of three positive tumors in the latter group showed moderate to strong positivity, whereas twelve of sixteen metastatic/recurrent tumors revealed a similar degree of p53 positivity. The positivity was stronger in the metastasis/recurrences as compared with the corresponding primary tumor. Western blot analysis revealed p53 expression in all of the Wilms' tumors tested, suggesting its involvement in the development of Wilms' tumors. Single-strand conformation polymorphism analysis performed on twenty-three of these tumors revealed p53 mutations in four of fourteen recurrent/metastatic tumors and none in the nonmetastatic/nonrecurrent group. Our results show that, whereas 60% of cases were immunopositive for p53 protein, mutations were detected in only 16% of tumors, indicating that wild-type p53 protein is retained in the other tumors. We conclude that p53 immunopositivity strongly correlates with recurrence/metastasis in Wilms' tumors. Furthermore, the accumulation of p53 in these tumors is not only due to mutations but may also involve stabilization of normal p53 with other proteins. PMID- 8623925 TI - Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers. AB - Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed in all prostate cancers evaluated, with high percentages of immunopositive cells and strong immunointensity typically occurring regardless of tumor grade. The findings suggest that expression of several anti-apoptotic members of the bcl-2 gene family, including bcl-2, bcl-X, and mcl-1 increases during progression of prostate cancers, a finding that may be relevant to the hormone-insensitive, metastatic phenotype of most advanced adenocarcinomas of the prostate. PMID- 8623927 TI - Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity. AB - Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. PMID- 8623928 TI - Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl 2-deficient mice. AB - Renal development in bcl-2-deficient mice was monitored to examine the temporal and spatial function of this gene during nephrogenesis in vivo. Extensive apoptosis occurred during abnormal nephrogenesis in bcl-2-deficient mice. In embryos and newborn mice, the sequence of morphological events was monitored by morphology in conjunction with morphometry, and bcl-2 -/-, bcl-2 +/-, and bcl-2 +/+ mice were compared. In bcl-2 -/- mice, initial induction of nephrons was detected by embryonic day 13 (E-13) as normal. Then, apoptotic cells became five times more frequent at E-13 to E-16 with a significant reduction (1/5) in nephron number at E-17 to E-19 in bcl-2 -/- mice compared with bcl-2 +/+ mice. No morphological difference was evident between bcl-2 +/- mice and bcl-2 +/+ mice by morphometry. Apoptotic cells were found mainly among the mesenchyme and less frequently in tubuli. Little apoptosis among ureteric buds was noted. In bcl-2 -/ mice at E-17 to E-19, inactive branching and insufficient convolution of ureteric buds were accompanied by fulminant apoptosis in the mesenchyme. Neonatal bcl-2 -/- mice lacked the nephrogenic zone, exhibiting renal hypoplasia. Thus, bcl-2 seems to inhibit apoptosis in renal stem cells during the induction of nephrons in vivo. PMID- 8623929 TI - Induction of Heymann nephritis with a gp330/megalin fusion protein. AB - There is considerable evidence that glomerular deposits in Heymann nephritis, a rat model of membranous nephritis, result from shedding of immune complexes formed on podocytes and that the principal antigen is part of the extracellular domain of a cell surface glycoprotein receptor called gp330 or megalin. It has also been reported that the immunogen that induces Heymann nephritis is a complex formed between gp330 and the receptor-associated protein RAP. The recent elucidation of the primary structure of gp330 makes it possible to investigate the ability of defined portions of gp330, devoid of RAP, to induce Heymann nephritis. In the present study we show that a gp330-glutathione-S-transferase fusion protein, containing 137 amino acid residues (1114 to 1250) of the ectodomain, induces active Heymann nephritis and that heterologous antibodies against this fusion protein produce passive Heymann nephritis. By immunofluorescence, typical glomerular immunoglobulin deposits were found, but complement components were lacking and the rats did not develop proteinuria. In the active model, we obtained evidence indicating that the deposits contained portions of the ectodomain of gp330, including regions other than those of the fusion protein. Thus, the deposits were stained by polyclonal antibodies to gp330 and to the gp330 fusion protein, as well as by two monoclonal antibodies reactive with portions of the ectodomain of gp330, only one of which reacted with the fusion protein in vitro. Antibodies against the cytoplasmic domain of gp330 did not stain. Furthermore, we found that RAP was able to bind to gp330 in the glomerular deposits but not to the gp330 fusion protein in vitro. The results show that the region of gp330 spanning amino acid residues 1114 to 1250 contains peptides capable of inducing pathogenic antibodies of Heymann nephritis without a contributory role of RAP. PMID- 8623930 TI - Induction of apoptosis in endothelial cells treated with cholesterol oxides. AB - Cholesterol oxides have a wide range of cytotoxic effects on vascular cells. Therefore, 7-ketocholesterol, 7 beta-hydroxycholesterol, 19-hydroxycholesterol, cholesterol 5 alpha, 6 alpha-epoxide, and 25-hydroxycholesterol, identified in various foodstuffs and human tissues, were chosen to compare and characterize the mode of cell death they induce, apoptosis or necrosis, on bovine aortic endothelial cells. The toxic potency differed from one compound to another, and 7 beta-hydroxycholesterol and 7-ketocholesterol exhibited the most potent effects. Cytotoxicity was accompanied by a decreased number of adherent cells, an increased number of non-adherent cells, and an enhanced permeability to propidium iodide. By electron and fluorescence microscopy performed after staining with Hoechst 33342, apoptotic cells with fragmented and condensed nuclei were identified mainly among non-adherent cells. By flow cytometry, cells with a lower DNA content than cells in the G0/G1 phase were apparent, giving a characteristic sub-G1 peak. Quantification of apoptosis evaluated either by the proportion of apoptotic cells identified by fluorescence microscopy after staining with Hoechst 33342 or by the percentage of cells present in the sub-G1 peak indicated that the ability of cholesterol oxides in inducing apoptosis was in the following order: 7 beta-hydroxycholesterol > 7-ketocholesterol > 19-hydroxycholesterol > cholesterol 5 alpha, 6 alpha-epoxide > 25-hydroxycholesterol. By using electrophoresis on agarose gel, typical internucleosomal DNA fragmentations were detected; they were no longer observed when bovine aortic endothelial cells were simultaneously incubated with 0.5 mmol/L zinc chloride, known to inhibit Ca2+/Mg2+-dependent endonucleases. None of the cholesterol-oxide-induced apoptotic features described above were noted with cholesterol. It is concluded that cholesterol oxides constitute a new class of cholesterol derivatives that can induce cell death by apoptosis in cultured endothelial cells. PMID- 8623931 TI - Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy. Role of metalloproteinases and pyridinoline cross-links. AB - A major contribution to the mechanical strength of the heart is provided by a continuous fibrillar collagen network embracing individual myocytes and forming an interstitial and perivascular framework. This study explores the possibility that idiopathic dilated cardiomyopathy may involve extensive changes in this collagenous framework. Idiopathic dilated cardiomyopathy hearts were obtained at transplant and compared with control hearts from autopsy. Idiopathic dilated cardiomyopathy showed a doubling of collagen concentration and a quadrupling of the total collagen per heart, whereas the stable mature cross-link, pyridinoline, diminished from 2.07 mol/mol of collagen to 1.0. Neutrophil-type collagenase activity is elevated approximately 30-fold as is the activity of gelatinase. Tissue inhibitor of metalloproteinase activity falls to negligible levels in idiopathic dilated cardiomyopathy, whereas alpha 2-macroglobulin is high. It is postulated that collagen critical to mechanical stability of the heart is degraded by metalloproteinase activity and is replaced by fibrous intercellular deposits of poorly cross-linked collagen. These changes contribute to weakening and dilatation of the ventricular wall. PMID- 8623932 TI - Tumor autocrine motility factor responses are mediated through cell contact and focal adhesion rearrangement in the absence of new tyrosine phosphorylation in metastatic cells. AB - Autocrine motility factor (AMF) is a tumor-secreted cytokine that acts as a motogen as well as a mitogen via a receptor-mediated signaling pathway(s). Expression of the AMF receptor (AMF-R) in normal cells is regulated by cell contact whereas in transformed cells AMF-R is constitutively expressed irrespective of cell density. Here we have analyzed the regulation of AMF-R expression in a BALB/c angiosarcoma tumor system that allows investigation of cellular characteristics associated with tumor progression. The metastatic cell variant (A31-M) displayed a higher rate of unstimulated motility and responded to tumor-derived AMF locomotory stimulus as compared with the nonmetastatic cell variants (A31-TR and A31-TU) and, although a similar level of receptor expression was detected in cellular extracts from subconfluent cultures of these sublines, surface localization differed and cell contact down-regulated AMF-R expression in the normal but not the transformed cell counterparts. AMF promoted marked rearrangement of focal adhesion plaque proteins in the AMF migration-responsive cells exclusively. Reorganization of vinculin after AMF stimulation was paralleled by morphological redistribution of tyrosine-phosphorylated proteins and the tyrosine kinase pp125FAK in the migration-responsive cells; however, we did not observe a concomitant change in the pp125FAK phosphorylation state or the general level of cellular tyrosine phosphorylation in response to treatment, suggesting that the induction of cellular migration by AMF is independent of tyrosine phosphorylation events at the focal contacts and may therefore represent a novel pathway of cytokine-induced migration regulation. PMID- 8623933 TI - Human T lymphotropic virus-I infection of human T lymphocytes induces expression of the beta-galactoside-binding lectin, galectin-3. AB - Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin previously designated as epsilon BP (IgE-binding protein), CBP35, Mac-2, L 29, and L-34, and its expression has been associated with various physiological and pathological processes, including cell growth, tumor transformation, and metastasis. Galectin-3 is widely distributed in various tissues and cell types and is expressed in many leukocytes, with the notable exception of B and T lymphocytes. We now report that galectin-3 is abundantly expressed in a number of human T lymphotropic virus (HTLV)-I-infected human T cell lines, including F6T, HUT 102, K3T, MT-2, and SLB-I, but is not expressed in non-HTLV-I-infected T cell lines such as Jurkat, CEM, and MOLT-4. In addition, the galectin-3 level was markedly increased in human thymocytes after infection with HTLV-I as compared with uninfected thymocytes. The up-regulation of galectin-3 expression appeared to correlate well with HTLV-I gene expression, as undetectable or very low levels of galectin-3 were found in the S1T and ATL-1K cell lines, which are nonproductively infected with HTLV-I. In co-transfection experiments, the galectin-3 promoter was significantly up-regulated by expression vectors encoding the 40-kd Tax protein, a potent transactivator in HTLV-I. Analysis of various Tax mutants suggested that galectin-3 promoter induction is dependent on activation of the cyclic-AMP-responsive element binding protein/activation transcription factor family of transcription factors and, to a lesser extent, nuclear factor kappa B/Rel induction. Transfection of human promonocytic U-937 cells with an HTLV-I Tax expression vector induced galectin-3 expression in this cell line. Functionally, galectin-3 was shown to activate interleukin-2 production in Jurkat T cells. Together, these findings raise the possibility that HTLV-I Tax production induces the transcription and subsequent synthesis and secretion of galectin-3, which in turn may further activate these T cells and contribute to the altered properties of cell growth found in adult T cell leukemia induced by HTLV-I. PMID- 8623934 TI - Clinical and pathological significance of microsatellite instability in sporadic endometrial carcinoma. AB - Defective DNA mismatch repair in neoplasia is manifested by extra, aberrant bands within multiple microsatellite markers. The replication error (RER) phenotype is present in most colorectal and endometrial carcinomas in patients with the hereditary nonpolyposis colorectal carcinoma syndrome. In addition, a minority of sporadic colorectal and endometrial carcinomas are RER positive. RER in sporadic colorectal carcinomas has been associated with improved prognosis, but its clinical significance in sporadic endometrial cancer has not been characterized. We analyzed DNA extracted from 109 formalin-fixed sporadic endometrial carcinomas for microsatellite instability. The RER-positive phenotype was demonstrated by microsatellite instability in more than one of the eight dinucleotide markers tested. RER was correlated with pathological and clinical parameters as well as with immunohistochemical staining for the p53 gene product and alterations in codon 12 of Ki-ras. Nine percent of the endometrial carcinomas were RER positive, and RER was significantly associated with high grade and adverse outcome. We found no significant correlation of RER with histological subtype, stage, depth of invasion, mutations in the 12th codon of Ki-ras, or p53 immunoreactivity. We conclude that the RER phenotype is present in a minority of sporadic endometrial carcinomas and is associated with high grade and poor prognosis. PMID- 8623935 TI - Transforming growth factor-beta synthesis by human peritoneal mesothelial cells. Induction by interleukin-1. AB - Peritoneal mesothelial cells are uniquely located to regulate cellular events in the peritoneal cavity and are a potentially important source for various cytokines. The present study was designed to elucidate the capacity of human peritoneal mesothelial cells (HPMCs) to synthesize and secrete the transforming growth factor (TGF)-beta isoforms 1, 2, and 3 and to characterize their regulation by inflammatory cytokines. HPMCs constitutively released appreciable amounts of TGF-beta 1 and low amounts of TGF-beta 2 as detected by specific immunoassays. TGF-beta 1 levels secreted within 48 hours (45 +/- 8.9 pg/10(5) cells) were 60-fold higher than TGF-beta 2 levels (0.9 +/- 0.1 pg/10(5) cells), respectively. Treatment of HPMCs with interleukin (IL)-1 beta (10 ng/ml) resulted in a significant increase of both TGF-beta 1 (mean, 5-fold; P < 0.001) and TGF beta 2 (mean, 6-fold; P < 0.01) generation. After 48 hours of IL-1 beta treatment the levels were 185 +/- 17.1 pg/10(5) cells for TGF-beta 1 and 5.3 +/- 1.5 pg/10(5) cells for TGF-beta 2, respectively. Neither tumor necrosis factor (TNF) alpha nor interferon (IFN)-gamma (both 10 ng/ml) affected TGF-beta 1 or TGF-beta 2 synthesis by HPMCs. TGF-beta 3 could not be detected in any of the supernatant media. Stimulation of HPMCs with IL-1 beta increased steady-state levels of TGF beta 1- and TGF-beta 2-specific mRNA. Western blot analysis of supernatants revealed the presence of an immunoreactive band at 25 kd. Indirect competition assays confirmed receptor-binding activity of HPMC-derived TGF-beta. Appreciable amounts of TGF-beta were present in a bioactive form. Our results demonstrate that HPMCs synthesize the TGF-beta isoforms 1 and 2 and that the levels of mRNA and protein release can be up-regulated by the proinflammatory cytokine IL-1 beta. PMID- 8623936 TI - Testicular dysgenesis does not affect expression of anti-mullerian hormone by Sertoli cells in premeiotic seminiferous tubules. AB - Anti-Mullerian hormone (AMH) immunoreactivity was studied on paraffin sections obtained from archival testicular biopsies of 29 children with intersex disorders and of 22 controls. Strong AMH immunoreactivity was observed in Sertoli cell cytoplasm from 8 fetal weeks until puberty. During pubertal maturation, in both normal and intersex patients, AMH expression was present in premeiotic seminiferous tubules, but was no longer detected in neighboring tubules with meiotic development. AMH immunostaining was abolished in the testis of one patient with persistent Mullerian ducts due to a mutation of the AMH gene, but was conserved in the testes of two patients with mutations of the AMH receptor gene. Testicular dysgenesis usually results in sexual ambiguity, with low testosterone and AMH serum levels and persistence of Mullerian derivatives. AMH immunoreactivity was conserved in premeiotic seminiferous tubules of dysgenetic testes, and also in sex-cord cells of a gonadoblastoma. In patients with asymmetric gonadal differentiation, the streak gonad was AMH-negative. In conclusion, secretion of AMH is a constitutive feature of the immature Sertoli cell and its expression is altered only by mutations of the AMH gene, but not by gonadal dysgenesis. The degree of regression of Mullerian ducts and serum AMH levels reflect the number, not the functional value, of Sertoli cells present in the immature testis. PMID- 8623937 TI - Canine cutaneous histiocytoma is an epidermotropic Langerhans cell histiocytosis that expresses CD1 and specific beta 2-integrin molecules. AB - Canine cutaneous histiocytoma (CCH) is a common, benign neoplasm of the dog. Histiocytomas most commonly occur as solitary lesions that undergo spontaneous regression. The age-specific incidence rate for histiocytomas drops precipitously after 3 years, although histiocytomas occur in dogs of all ages. Langerhans cells (LCs) in humans and dogs express abundant major histocompatibility complex class II molecules and a variety of leukocyte antigens characteristic of dendritic cell differentiation including CD1a, CD1b, CD1c, and CD11c. The immunophenotype of CCH resembled that of cutaneous LCs by virtue of the expression of CD1 molecules (CD1a, -b, and -c), CD11c, and major histocompatibility complex class II. Furthermore, histiocytoma cells had a tropism for epidermis, which was also consistent with an epidermal LC lineage. The expression of adhesion molecules such as CD11b (variable), CD44, CD54 (ICAM-1), and CD49d (VLA-4) in CCH indicated that the infiltrating cells had some of the characteristics of activated LCs, as these molecules are not expressed by normal, resting canine epidermal LCs. CCH did not express Thy-1 or CD4. Thy-1 expression is a characteristic of human and canine dermal dendrocytes, which are perivascular dendritic antigen-presenting cells closely related to epidermal LCs. CD4 expression is prevalent in human LC histiocytosis, and in this respect CCH differed from human LC histiocytosis. Here we demonstrate that CCH is a localized form of self-limiting LC histiocytosis, which predominantly expresses an epidermal LC phenotype. CCH occurs as solitary or, less commonly, as multiple cutaneous nodules or plaques, which rarely may extend beyond the skin to local lymph nodes. Regression of CCH occurs spontaneously in the vast majority of cases in primary and secondary sites, and is mediated by CD8+ alpha beta T cells. The high frequency of CCH within the general canine population offers the potential that the dog may provide an interesting model system to further the understanding of LC proliferative disorders, particularly the self-limiting, cutaneous form of human LC histiocytosis. PMID- 8623939 TI - How much do they really want to know? Preoperative patient interviews and the anesthesiologist. PMID- 8623938 TI - Expression of S-100 protein and glial fibrillary acidic protein in cultured submandibular gland epithelial cells and salivary gland tissues. Histogenetic implication for salivary gland tumors. AB - S-100 protein and glial fibrillary acidic protein (GFAP) were studied in human salivary gland tissues and human cultured submandibular gland epithelial cells. Immunohistochemically, ductal cells in normal salivary gland tissues were positive for S-100 protein and GFAP, but myoepithelial cells were uniformly negative. Immunocytochemically, cultured submandibular gland ductal cells were positive for S-100 protein and GFAP. By immunoblotting analysis of the cultured cell lysates, a 6.5-kd S-100 protein was detected. This band corresponded to S 100 protein purified from bovine brain. The cultured submandibular gland cells expressed 49- and 54-kd GFAP polypeptides. These results have important implications for the histogenesis of salivary gland tumors. PMID- 8623940 TI - The Amsterdam Preoperative Anxiety and Information Scale (APAIS) AB - The purpose of the present study was to assess patients' anxiety level and information requirement in the preoperative phase. During routine preoperative screening, 320 patients were asked to assess their anxiety and information requirement on a six-item questionnaire, the Amsterdam Preoperative Anxiety and Information Scale (APAIS). Two hundred patients also completed Spielberger's State-Trait Anxiety Inventory (STAI-State). Patients were able to complete the questionnaire in less than 2 min. On factor analysis, two factors emerged clearly: anxiety and the need for information. The anxiety scale correlated highly (0.74) with the STAI-State. It emerged that 32% of the patients could be considered as "anxiety cases" and over 80% of patients have a positive attitude toward receiving information. Moreover, results demonstrated that 1) women were more anxious that men; 2) patients with a high information requirement also had a high level of anxiety; 3) patients who had never undergone an operation had a higher information requirement than those who had. The APAIS can provide anesthesiologists with a valid, reliable, and easily applicable instrument for assessing the level of patients' preoperative anxiety and the need for information. PMID- 8623941 TI - Processing familiar and unfamiliar auditory stimuli during general anesthesia. AB - We tested memory priming for auditory stimuli presented during general propofol sufentanil anesthesia in 58 patients undergoing day-case arthroscopic surgery. Stimuli were presented via headphones and consisted of common facts (Group A, 29 patients), or familiar or unfamiliar full names of fictitious people (GRoup B, 29 patients). Group A was expected to give more correct answers to questions about the common facts than Group B, when tested postoperatively, and Group B to attribute more fame to presented names than Group A (famous names test). Because the process for learning new or unfamiliar stimuli (elaboration) in particular may be impaired under general anesthesia, more memory priming was expected for familiar than for unfamiliar material. No significant differences were demonstrated between the two groups in performance on common facts or in fame attributed to the names. The amount of memory priming, however, was positively related to one of two measures of preoperative anxiety. PMID- 8623942 TI - Force, torque, and stress relaxation with direct laryngoscopy. AB - The anesthetist exerts axial force on the laryngoscope handle to expose the glottis. The anesthetist must also apply a perpendicular force to balance the torque on the laryngoscope. Several studies have measured axial force during direct laryngoscopy, but none has measured torque. This study used a newly designed laryngoscope handle to measure force and torque simultaneously during direct laryngoscopy of ASA grade I and II patients requiring general anesthesia and endotracheal intubation for elective surgery. In 58 patients, peak force averaged 38 +/- 2 newtons. Peak torque averaged 4 +/- 0.2 newton-meters, and the perpendicular force was estimated as 40 +/- 2 newtons. The peak torque that can be balanced by the wrist is approximately 6 newton-meters, suggesting that torque may be a limiting factor for laryngoscopy in some situations. Peak force and torque demonstrated stress relaxation, a viscous property of biologic tissues. Force and torque decreased monoexponentially to approximately 70% of peak values with a half-time of 4 +/- 0.3 s. The phenomenon occurred in spite of administration of muscle relaxants, and was probably due to stress relaxation of pharyngeal tissues that are passively stretched during laryngoscopy. PMID- 8623943 TI - Force and torque vary between laryngoscopists and laryngoscope blades. AB - Several studies have examined the effects of patient characteristics on force of laryngoscopy, but little attention has been paid to the importance of technique and equipment. This study investigated whether force, torque, head extension, and view varied significantly between laryngoscopists and compared force and torque using Macintosh 3 and Miller 2 blades. The study population consisted of ASA grade I and II patients requiring general anesthesia and endotracheal intubation for elective surgery. Force, torque, head extension, and laryngeal view were highly reproducible when laryngoscopy was repeated by the same individual, Force and torque showed great variation between laryngoscopies performed by different anesthetists, For example, peak force varied over a range of 56 newtons among patients, but could also vary as much as 30 newtons between different anesthetists repeating laryngoscopy in the same patient. Force and head extension were 30% less with Miller laryngoscope compared to the Macintosh. Thus, laryngoscopic force and torque depend on technique and equipment. Further studies of force and torque may lead to improved techniques. The force-measuring laryngoscope could be a useful tool in teaching laryngoscopy. PMID- 8623945 TI - Reducing pain during propofol injection: the role of the solvent. AB - We hypothesized that the concentration of propofol in the aqueous phase may be the most important variable responsible for the pain experienced during injection of the drug. The concentration of propofol in the aqueous phase (18.57 micrograms/mL) can be decreased by increasing the fat content of the solvent. To test this hypothesis, 36 patients were randomly allocated to one of three groups, each receiving a different formulation of propofol. Group A received 20 mL of propofol alone in a commercial preparation (Diprivan(R) with 10 mL of saline); Group B, 20 mL of propofol to which 5 mL of long-chain triglyceride (LCT) fat emulsion and 5 mL of saline and been added; and Group C, 20 mL of propofol and 10 mL of LCT fat emulsion. The propofol emulsion was injected over 30-60 s into a dorsal vein of the hand. Patients reported pain during injection as none, mild, moderate, or severe (almost intolerable). In Group A, 8 of 12 patients reported moderate or severe pain upon injection whereas in Group C only mild pain was reported by 6 of 12 patients. Our results suggest that a smaller concentration of propofol in the aqueous phase of the emulsion reduces pain on injection. With the addition of more lipid (10 mL), a higher percentage of propofol is absorbed by fat particles. If solvents that permit a smaller concentration of the drug in the aqueous phase of oil-in-water emulsions were used for propofol and other drugs that cause pain on injection, pain would be reduced and patient satisfaction may be increased. PMID- 8623944 TI - Prevention of pain on injection of propofol: a comparison of lidocaine with alfentanil. AB - We undertook a randomized, placebo-controlled, double-blind study to compare the use of alfentanil 1 mg and lidocaine 40 mg for the reduction of pain during injection of propofol. Eighty-nine patients were randomly allocated to one of three groups: Group L, lidocaine 40 mg added to 180 mg propofol; Group A, alfentanil 1 mg 30 s prior to propofol; or Group P, placebo (normal saline). The incidence of pain in the placebo group was 67%. Both treatment groups had a significantly lower incidence of pain than the placebo group (P < 0.002). There was no significant difference in the incidence of pain between the groups receiving lidocaine or alfentanil (13% and 24%, respectively). There was no significant difference in the induction dose of propofol between the groups. Fifty-two percent of patients who experienced pain at induction had recall of that pain in the recovery room. Alfentanil 1 mg and lidocaine 40 mg are both effective in reducing pain during injection of propofol. PMID- 8623946 TI - Growth of microorganisms in propofol, thiopental, and a 1:1 mixture of propofol and thiopental. AB - To assess and compare the growth of four microorganisms in solutions of intravenous anesthetics, known quanta of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans were inoculated into propofol, thiopental, a 1:1 mixture of propofol and thiopental, and normal saline. All microorganisms were taken from standard stock cultures and incubated for 24 h (48 h for C. albicans). Growth of microorganism in each drug was compared by counting the number of colony forming units (CFUs) grown from a subculture of each inoculated anesthetic at 0, 3, 6, 12, and 24 h. The study shows that propofol strongly supports the growth of E. coli and C. albicans but is bacteriostatic toward S. aureus and weakly bactericidal toward P. aeruginosa. In contrast, both thiopental and the 1:1 mixture of propofol and thiopental behaved differently, exhibiting markedly bactericidal properties toward E. coli, S. aureus, and P. aeruginosa and a bacteriostatic effect on C. albicans. This finding supports recommendations that a strict aseptic technique should be used when handling propofol and that the contents of an ampoule should be used within 6 h of aspirating. The measured high pH of both thiopental and the 1:1 mixture of propofol and thiopental compared to propofol alone suggests pH to be a major factor in determining whether a given drug will support microbial growth. PMID- 8623947 TI - The comparative effects of sevoflurane versus propofol in the induction and maintenance of anesthesia in adult patients. AB - A randomized, prospective study was performed at four institutions to compare anesthetic induction, maintenance, and recovery characteristics between sevoflurane- and propofol-based anesthesia in 186 ASA physical status I and 11 patients undergoing elective surgical procedures of 1-3 h. Group 1 (n = 93) patients received sevoflurane-nitrous oxide for both induction and maintenance of anesthesia while Group 2 (n = 93) received propofol-nitrous oxide anesthesia. Induction of anesthesia and tracheal intubation times were significantly shorter with propofol (2.21 +/- 0.2 min, 5.11 +/- 0.3 min, respectively) than with sevoflurane (3.11 +/- 0.2 min, 7.21 +/- 0.3 min, respectively). Emergence times after sevoflurane (8.81 +/- 1.2 min) were significantly shorter than with propofol (13.21 +/- 1.2 min). Overall frequency of complication-free induction, maintenance, and emergence did not differ between the two anesthetic groups. However, side effects involving airway excitement were more prevalent during mask induction with sevoflurane as compared to propofol. Patients in the sevoflurane group were oriented and required postoperative analgesia much earlier than those who received propofol. Both groups were hemodynamically stable throughout the study period. The incidence of postoperative nausea, vomiting, and pain discomfort scores were similar between the two groups. Urinary specific gravity decreased in the sevoflurane-treated group while serum creatinine and urinary pH were unchanged from preoperative values in both groups. Sevoflurane compared favorably with propofol when used for anesthesia for elective procedures of 1-3 h duration. PMID- 8623948 TI - The effects of sevoflurane on contractile and electrophysiologic properties in isolated guinea pig papillary muscles. AB - We examined, in guinea pig papillary muscles, whether the negative inotropic effect of sevoflurane is due to the depression of the influx of extracellular Ca2+ or to inhibition of the availability of intracellularly stored Ca2+. Sevoflurane decreased action potential duration and contractile force in a concentration-dependent fashion in normally polarized guinea pig papillary muscles. Sevoflurane produced a depression of contractile force with different rates or patterns of stimulation in the rested state and at low stimulation frequencies. In a potentiated state, sevoflurane did not depress contractile forces. Although sevoflurane decreased action potential duration and contractile force in a concentration-dependent fashion in normal Tyrode's solution, in high K+ Tyrode's solution, it caused a depression of contractile force without a shortening of action potential duration. Sevoflurane also depressed contractile force in normal and high K+ Tyrode's solution with ryanodine 1 microM. Our results suggest that in myocardial contractile force the negative inotropic effect of sevoflurane might be caused by depression of transsarcolemmal Ca2+ influx, accompanied by shortening of the action potential duration. PMID- 8623949 TI - Effects of anesthesia based on large versus small doses of fentanyl on natural killer cell cytotoxicity in the perioperative period. AB - Surgical stress and general anesthesia suppress immune functions, including natural killer cell cytotoxicity (NKCC). This suppression could be attributable, at least in part, to opiates. We have previously shown that large-dose fentanyl administration suppressed NKCC in rats. The present study sought to compare the effects of two anesthetic protocols, based on large- (LDFA) versus small (SDFA) dose fentanyl anesthesia on NKCC in the perioperative period. Forty patients were included in this study; half were assigned to each protocol of anesthesia. In each anesthetic group, half the patients were undergoing surgery for malignant diseases, and half for benign conditions. Blood samples were collected during the perioperative period. NKCC was assessed using the chromium release assay. Initially, both types of anesthesia similarly suppressed NKCC, with a peak effect 24 h after surgery. The two types of anesthesia, however, differed in the rate of recovery of NKCC suppression. By the second postoperative day, NKCC returned to control values in the SDFA patients, whereas NKCC was still significantly suppressed after LDFA. These results indicate that LDFA causes prolonged suppression of NK cell function. Whether this suppression might have a long-term impact on the overall outcome, especially in cancer patients, remains to be determined. PMID- 8623950 TI - Ondansetron blocks nifedipine-induced analgesia in rats. AB - The serotonergic system is involved in pain transmission and the 5 hydroxytryptamine (5-HT3) receptor subtype mediates some of these effects at the spinal level. Therefore, we explored the effects of the serotonergic system on nifedipine-induced analgesia by using the 5-HT3 receptor antagonist ondansetron. Male Sprague-Dawley rats were pretreated with ondansetron (1 mg/ kg intraperitoneally) or normal saline. After 15 min, rats received injections of nifedipine (15 mg/kg intraperitoneally) or dimethylsulfoxide (DMSO), solvent for nifedipine, as a control. Nociception was assessed by tail-flick method. Rats treated with nifedipine alone had an increase in tail-flick latency of 122%, as measured by the area under the curve, compared to rats treated with DMSO alone. Pretreatment with ondansetron, however completely blocked the analgesic effect of nifedipine, with tail-flick latency remaining at baseline throughout the measurement period. These results indicate that the 5-HT3 receptor plays an important role in the analgesic response to nifedipine and that medications that block this receptor may decrease the analgesic effectiveness of this type of therapy. PMID- 8623951 TI - Changes in lung and chest wall properties with abdominal insufflation of carbon dioxide are immediately reversible. AB - Previously we have reported that large increases in lung and chest wall elastances as well as lung resistance occur with abdominal insufflation of carbon dioxide during laparoscopic surgery. To examine whether these effects were reversible with abdominal deflation, we calculated lung and chest wall elastances and resistances from measurement of airway flow and pressure and esophageal pressure in 17 anesthetized/paralyzed patients undergoing laparoscopic surgery. Measurements were made immediately prior to abdominal insufflation and after deflation. Lung and chest wall elastances and resistances were not changed from baseline (P > 0.05), although total respiratory elastance remained slightly increased compared to baseline (P < 0.05). The change in total respiratory elastance did not correlate with abdominal insufflation time, surgical site, smoking history, or physical characteristics of the patients. There were no differences in frequency and tidal volume dependences of the elastances and resistances before and after abdominal insufflation (P > 0.5). We conclude that residual changes in respiratory mechanics caused by carbon dioxide insufflation during laparoscopic surgery are minor, and that the reported compromise of respiratory function indicated by pulmonary function tests after laparoscopy does not appear to be due to changes in passive mechanical properties of the lungs or chest wall. PMID- 8623952 TI - Cost-benefit analysis of nasal cannulae in non-tracheally intubated subjects. AB - We evaluated four nasal cannulae used to deliver oxygen and measure PETC02 in a non-tracheally intubated, healthy population. The effect of various oxygen flow rates on PETC02 and respiratory rate (RR), as well as the cost and relative patient comfort of the cannulae, was compared. In this controlled study, 20 healthy volunteers tested the cannulae using oxygen flow rates of 0 (breathing room air), 2, 4, and 6 L/min. The volunteers rated the comfort of each cannula on a scale from 1 (uncomfortable) to 10 (comfortable). Hospital costs of the cannulae were also compared. All of the cannulae, except the Hospitak nasal cannula (HOS), demonstrated an oxygen flow dependency with respect to PETC02 and RR. The HOS cannula was judged by the study population to be the most comfortable and was the second least expensive cannula tested. From a qualitative standpoint, the carbon dioxide waveform recorded with the HOS cannula was the one least affected by oxygen flow. Of the nasal cannula systems evaluated in this study, the HOS system demonstrated the best cost-benefit ratio, performing well clinically while being comfortable to wear and relatively inexpensive. These conclusions are specific to a healthy population and not to patients with lung disease,those who smoke, or those having a higher ASA classification status. Our evaluation suggests that comfort and clinical performance of nasal cannulae may well depend on device design. PMID- 8623953 TI - The effects of the systemic antimycotics, itraconazole and fluconazole, on the pharmacokinetics and pharmacodynamics of intravenous and oral midazolam. AB - We studied the interaction of azole antimycotics with intravenous (IV) and oral midazolam using a cross-over design in 12 volunteers, who ingested placebo, itraconazole, or fluconazole for 6 days. A 7.5-mg dose of midazolam was ingested on the first day, 0.05 mg/kg was administered IV on the fourth day, and 7.5 mg orally on the sixth day. Itraconazole reduced the clearance of IV midazolam by 69% and fluconazole reduced the clearance of IV midazolam by 51% (P < 0.001). A single dose of itraconazole and fluconazole increased the area under the oral midazolam concentration-time curve [AUC(0-infinity)] 3.5-fold (P < 0.001) and the peak concentration two-fold (P < 0.05) compared to placebo. On the sixth day the AUC(0-infinity) of oral midazolam was almost seven times greater with itraconazole (P < 0.001) and 3.6 times greater with fluconazole (P < 0.001) than without the antimycotics. The psychomotor effects of midazolam were also profoundly increased (P < 0.001). The psychomotor tests demonstrated only a weak interaction between the antimycotics and IV midazolam. When bolus doses of midazolam are given for short- time sedation, the effect of midazolam is not increased to a clinically significant degree by itraconazole and fluconazole, and it can be used in normal doses. However, the use of large doses of IV midazolam increases the risk of clinically significant interactions also after IV midazolam. Use of oral midazolam with itraconazole and fluconazole should be avoided. PMID- 8623954 TI - Simulated clinical evaluation of conventional and newer fluid-warming devices. AB - The purpose of the study was to evaluate the ability of five commercially available devices utilizing a variety of heat exchange technologies to deliver normothermic (37 degrees C) fluids. Conditions of slow (6.5 mL/ min), moderate (13-25 mL/min), and rapid (gravity and pressure driven flows, roller clamp wide open) infusion were simulated. Fluid temperatures were measured using rapid response thermistors after the fluid exited the heat exchanger (T outlet) and before delivery to the patient intravenously (IV) (T distal). Devices tested were the FW537, H1000, Hotline, BairHugger, and Flotem IIe. Fluids tested were crystalloid at room temperature and red cells diluted with saline (11-19 degrees C). At slow and moderate flows, T distal of crystalloid was between 35.3 and 37.9 degrees C for Hotline at 42 degrees C, 33.8 and 37.7 degrees C for H1000 at 42 degrees C, 29.4 and 34.2 degrees C for BairHugger, 26.1 and 31.5 for Flotem IIe, and 23.8 and 32.1 for FW537 at 42 degrees C. With gravity and pressure driven flows, T distal of crystalloid were 39.0 and 38.9 for H1000 at 42 degrees C, 38.7 and 38.4 degrees C for FW537 at 42 degrees C, 34.7 and 28.9 degrees C for Hotline at 42 degrees C, 29.2 and 24.2 degrees C for BairHugger, and 29.7 and 24.2 degrees C for Flotem. In conclusion, only the H1000 at 42 degrees C was effective at delivering normothermic fluids at all clinically relevant flow rates. The Hotline at 42 degrees C was effective at slow and moderate flow, whereas the FW537 was effective only at rapid flow. PMID- 8623955 TI - The effects of neuromuscular block on peak airway pressure and abdominal elastance during pneumoperitoneum. AB - Administration of muscle relaxants is considered as necessary to prevent high intraabdominal and peak inspiratory pressures induced by pneumoperitoneum during laparoscopy. In the present study, we hypothesized that neuromuscular block does not alter pulmonary or abdominal elastic properties in pigs receiving general anesthesia. To test this hypothesis, changes in peak airway pressure and abdominal elastance during intraabdominal CO(2), insufflation from 0 to 15 mm Hg were recorded in anesthetized pigs, with or without muscle relaxants. A 100% increase in peak inspiratory airway pressure was obtained. This was unaffected by neuromuscular block induced by atracurium (13.2 +/- 5.0 mm Hg vs 25.0 +/- 4.8 mm Hg for the control group and 12.6 +/- 5.0 mm Hg vs 23.5 +/- 6.2 mm Hg for the paralyzed group). Abdominal pressure/volume relationships were independent of muscle relaxant administration (calculated elastance was 3.98 +/- 1.56 mm Hg/L without muscle relaxant vs 3.86 +/- 1.37 mmHg/L in the atracurium group). We conclude that high peak inspiratory airway pressures and intraabdominal pressures during laparoscopy are not affected by neuromuscular block. These findings also question the necessity of muscle relaxants in clinical anesthetic practice during laparoscopic surgery. PMID- 8623956 TI - Sevoflurane versus isoflurane: induction and recovery characteristics with single breath inhaled inductions of anesthesia. AB - Because of its nonpungent odor and low blood-gas solubility coefficient, sevoflurane might be an ideal drug for single-breath inhaled induction of anesthesia. Fifty ASA grade I-III ambulatory surgical patients (18-76 yr old) received a single-breath induction with either 5.0% sevoflurane or 5.0% isoflurane (randomized) in a 1:1 N2O/O2 mixture. Anesthesia was maintained with the same anesthetic in 70% N2O until the end of surgery, when anesthetics were abruptly discontinued. Induction times (loss of eyelash reflex) were similar for sevoflurane (75 +/- 3 s, mean +/- se) and isoflurane (67 +/- 4 s, P = not significant). Sevoflurane patients were less likely to have complications during induction (P < 0.005); coughing occurred more frequently with isoflurane (P < 0.001). During induction, heart rate increased with both sevoflurane (from 73 +/- 3 to 90 +/- 4 bpm, P < 0.05) and isoflurane (from 70 +/- 2 to 92 +/- 2 bpm, P < 0.05); the increase with isoflurane was greater than that with sevoflurane. Times to eye opening for sevoflurane (8.1 1 +/- 1.0 min) did not differ significantly from those for isoflurane (10.6 +/- 1.3 min). Patients opened their eyes at lower end-tidal minimum alveolar anesthetic concentration (MAC)-fractions of sevoflurane (0.12 +/- 0.01 MAC) than isoflurane (0.15 +/- 0.01 MAC, P < 0.01). During recovery, patients who received sevoflurane felt less clumsy (P < 0.001) and less confused (P < 0.005) but had higher pain scores (P < 0.005) than those who received isoflurane. Sevoflurane is more suitable than isoflurane for single breath induction, because it produces a smoother induction with a lower incidence of complications and better patient acceptance. PMID- 8623957 TI - Recovery profile after desflurane with or without ondansetron compared with propofol in patients undergoing outpatient gynecological laparoscopy. AB - We studied the effect of combining prophylactic ondansetron (4 mg intravenously [IV]) to desflurane-based anesthesia in 90 ASA grade I or 11 women undergoing outpatient gynecological laparoscopy. Recovery after anesthesia, with special focus on postoperative nausea and vomiting (PONV), was assessed. Control groups received a similar desflurane anesthetic (placebo) or a propofol-infusion-based (active control) anesthetic. The study design was randomized, controlled, and double-blind (regarding ondansetron) and single-blind (regarding the anesthetic technique). Early recovery (eye opening, orientation, following commands, sitting) was similar in the three groups. However, overall home readiness (toleration of oral fluids, walking, pain tolerable by oral analgesics, no or only mild nausea) was achieved faster in the desflurane group receiving ondansetron (109 [21-937] min, P < 0.01) and in the propofol group (110 [33-642] min, P < 0.001) when compared to the desflurane only group (372 [45-723] min) (median [range]). The total incidence of PONV in the desflurane-only group was 80% (P < 0.01), compared to 40% and 20% in the desflurane group receiving ondansetron and the propofol group, respectively. The postoperative antiemetic requirements were consistently and significantly (P < 0.01) higher in the desflurane-only group compared to the other two groups. Postoperative sedation, analgesic requirements, and psychomotor recovery (assessed by the Maddox Wing and the Digit Symbol Substitution Tests) were similar in the three groups. Our results suggest that in order to achieve a propofol-like recovery profile in patients with a high likelihood of PONV, desflurane should be combined with a potent antiemetic (e.g., ondansetron). PMID- 8623958 TI - High reproducibility in the interpretation of intraoperative transesophageal echocardiographic evaluation of aortic atheromatous disease. AB - Intraoperative decisions are often based on interpretation of results from transesophageal echocardiography (TEE). One such area is the intraoperative evaluation of atheromatous disease of the thoracic aorta and subsequent classification or grading. These grading schemes are predictive of stroke after cardiac surgery. Since intraoperative strategies may be modified based on this TEE aortic atheroma grading, assessment of the interobserver variability of TEE findings between observers is essential. Forty TEE videotape segments imaging three portions of the thoracic aorta (ascending, arch, descending) were selected from 189 reports of a larger cohort. Three independent, blinded observers, experienced in TEE, evaluated these examinations for atheroma severity. If a TEE segment had insufficient data, "uninterpretable" was recorded. Weighted kappa coefficients of agreement were calculated on the three data sets. Mean weighted kappa coefficients for the three observers A, B, and C were 0.69, 0.74, and 0.72, for the ascending, arch, and descending aorta segments, respectively, representing excellent agreement. We have demonstrated uniformly high agreement for interpretation of TEE, which indicates the excellent reproducibility of TEE grading and stratification of aortic atheroma. Reproducibility within and across specialties and institutions is essential for widespread application of TEE for evaluation of the thoracic aorta. PMID- 8623959 TI - The effect of dexmedetomidine on the balance of myocardial energy requirement and oxygen supply and demand. AB - The effect of the alpha(2)-adrenergic agonist dexmedetomidine on the balance between myocardial energy requirement and oxygen supply and demand was investigated in 16 open-chest dogs anesthetized with either chloralose/urethane (CU) or fentanyl/halothane (FH). Myocardial energy requirement (estimated from the pressure work index), blood flow and its transmural distribution (radioactive microspheres), as well as myocardial oxygen and lactate extraction, were measured before and after administration of dexmedetomidine in doses ranging from 0.1 to 10 micrograms/kg intravenously. Under CU anesthesia, dexmedetomidine decreased heart rate, arterial blood pressure, and cardiac output. During FH anesthesia, dexmedetomidine reduced heart rate and cardiac output whereas arterial blood pressure increased. Dexmedetomidine decreased myocardial energy requirement only during CU anesthesia; myocardial oxygen supply and demand decreased in parallel. At the (large) dose of 10 micrograms/kg, myocardial oxygen extraction increased during both types of anesthesia. Dexmedetomidine >/= 1 microgram/kg increased endocardial/epicardial blood flow ratio during FH anesthesia. These data indicate that dexmedetomidine >/= 1 microgram/kg reduces myocardial energy requirements, especially when baseline heart rate and blood pressure are increased. Dexmedetomidine preserves endocardial perfusion and reduces oxygen demand in parallel with oxygen supply and energy requirements. PMID- 8623961 TI - Preoperative oral ondansetron for pediatric tonsillectomy. AB - This prospective, randomized, double-blind, placebo-controlled study evaluated the antiemetic efficacy of preoperative oral ondansetron, 0.075 mg/kg or 0.15 mg/kg, in 136 preadolescent children premedicated with midazolam 0.5 mg/kg per os and dexamethasone 0.1 mg/kg intravenously prior to undergoing tonsillectomy with isoflurane anesthesia. The incidence of vomiting during the 24 h after tonsillectomy was significantly reduced (P < 0.04) by ondansetron 0.15 mg/kg compared with placebo and ondansetron 0.075 mg/kg (15%, 38%, and 36%, respectively). There was a significant reduction (P < 0.03) in the mean number of vomiting episodes per patient during the 24 h immediately after tonsillectomy in the ondansetron 0.15 mg/kg group compared with the placebo and ondansetron 0.075 mg/kg study groups (0.2 +/- 0.6, 0.8 +/- 1.3, and 0.8 +/- 1.3, respectively). The need for antiemetic rescue therapy (ondansetron 0.15 mg/kg intravenously after three episodes of emesis prior to discharge) was significantly greater in children who received placebo compared with the ondansetron 0.15 mg/kg study group (13% vs 0%, P < 0.05). We conclude that ondansetron 0.15 mg/kg, administered orally prior to tonsillectomy, is associated with reduced postoperative vomiting in preadolescent children. In addition, the preoperative oral administration of ondansetron 0.075 mg/kg is no more effective than placebo in preventing posttonsillectomy vomiting in preadolescent children. PMID- 8623960 TI - The effects of dobutamine and phenylephrine on atrioventricular conduction during combined use of halothane and thoracic epidural lidocaine. AB - The purpose of this study was to measure cardiac sympathetic nerve activity (CSNA) and atrioventricular (AV) conduction and to test the effects of dobutamine (DOB) and phenylephrine (PHE) on AV conduction during combined use of halothane and thoracic epidural lidocaine. Cats were anesthetized with 1 % halothane and an epidural catheter was inserted through T-9 laminectomy. His bundle and atrial electrocardiograms were obtained and atrial electric stimulation was performed using quadripolar catheter electrodes. Cats underwent left thoracotomy, and CSNA was recorded directly from the left ventrolateral or ventromedial nerve. In addition to sinus cycle length (SCL) measurement during spontaneous beating, the functional refractory period (FRP) of the atrioventricular node (AV node), effective refractory period (ERP) of the atrium, atrium-His (A-H) intervals were determined just before and 10, 20, and 30 min after epidural administration of 1% lidocaine (0.2 mL/kg) in Group C. DOB 5 micrograms/kg/min (Group DOB) and PHE 0.5 1.0 micrograms/kg/min (Group PHE) were intravenously administered from 12 to 22 min after epidural lidocaine. CSNA and mean arterial pressure (MAP) were markedly decreased and SCL, FRP of AV node, ERP of atrium and A-H interval were significantly prolonged after epidural lidocaine. MAP increased to baseline level during DOB or PHE infusion. Worsening of cardiac electrophysiological variables was improved with DOB infusion, but did not change with PHE infusion. We conclude that thoracic epidural lidocaine during halothane anesthesia almost eliminates CSNA, and thereby attenuates sinus node automaticity and AV node function. DOB restored normal cardiac electrophysiological variables, and therefore is preferable to phenylephrine as a pressor drug. PMID- 8623962 TI - Video-assisted thoracoscopic surgery for the treatment of congenital cardiac defects in the pediatric population. AB - Recent technologic advances have contributed to a renewed interest in thoracoscopic surgery. In our institution, thoracoscopy through video-assisted technology has been successfully applied to congenital heart surgery. We reviewed the charts of 45 consecutive patients (ASA physical status 11-IV) who underwent video-assisted thoracoscopic surgery (VATS) for various congenital heart defects. The mean age of the patients was 2.65 yr and the mean weight was 11.78 kg. The surgical procedures included patent ductus arteriosus interruption (n = 28) and vascular ring division (n = 8), and 9 patients had miscellaneous procedures. The most commonly used anesthetic regimen consisted of isoflurane, pancuronium, fentanyl, air, and oxygen. Seven patients were managed with one-lung ventilation, the remainder by two-lung ventilation with surgical lung retraction. Intraoperative desaturation occurred in 12 patients (26.7%) but resolved quickly with brief reexpansion of the lungs. Postoperative complications included: pleural effusions (n = 3), chylothorax (n = 2), right upper lobe atelectasis (n = 1), small pneumothorax (n = 1), and vocal cord paralysis (n = 1). Seven patients (15.5%) required conversion to a thoracotomy for insufficient exposure (n = 4) or due to concern over bleeding (n = 3). This experience with VATS in pediatric patients with congenital heart disease may provide a database for comparison with others who work with the VATS technique. PMID- 8623963 TI - Hemodynamic effects of intravenous ephedrine in infants and children anesthetized with halothane and nitrous oxide. AB - The aim of this study was to evaluate the effect of age on the hemodynamic responses to intravenous (IV) ephedrine in pediatric patients anesthetized with halothane, nitrous oxide, and oxygen. One hundred ten pediatric patients, ranging in age from 0.1 to 15 yr, were assigned to receive 0.1 mg/kg (n = 55) or 0.2 mg/kg (n = 55) IV ephedrine. General anesthesia was maintained with 1.0 minimum alveolar anesthetic concentration (MAC) of halothane and 67% nitrous oxide in oxygen after tracheal intubation. Measurements of arterial blood pressure and heart rate were made at 1-min intervals for 10 min after ephedrine 0.1 or 0.2 mg/kg was injected IV as a bolus. Significant correlations were noted between age and changes in mean blood pressure (r = 0.37, P < 0.01 for the subjects receiving ephedrine 0.1 mg/kg; r = 0.63, P < 0.001 for the subjects receiving ephedrine 0.2 mg/kg), but not between age and changes in heart rate. The present results indicate that age correlates with the pressor but not the chronotropic effects of ephedrine in pediatric patients anesthetized with 1 MAC halothane and nitrous oxide. PMID- 8623964 TI - The response to varying concentrations of inhaled nitric oxide in patients with acute respiratory distress syndrome. AB - We investigated the response to varying concentrations of inhaled nitric oxide (NO) in 18 patients with acute respiratory distress syndrome (ARDS). The study was divided into two parts. In Part 1, 5-40 ppm of inhaled NO was evaluated in 10 patients with ARDS. In Part 2, 0.1-10 ppm of inhaled NO was evaluated in eight patients with ARDS. Inhaled NO significantly (P < 0.05) decreased the mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance index (PVRI), and increased the arterial oxygenation (PaO2) at concentrations of 0.1 to 40 ppm. No dose response was detectable for the pulmonary artery pressure (PAP) or PVRI over this dose range. The increase in PaO2 at 10 ppm of NO was significantly greater than that at 0.1 ppm but not 1 ppm. The decrease in PVRI and the increase in PaO2 were both significantly correlated with the baseline PVRI. While the maximum hemodynamic and oxygenation responses to inhaled NO are achieved at approximately 1 ppm, it appears that the maximum hemodynamic response is observed at lower concentrations (0.1 ppm) of inhaled NO than the improvement in oxygenation (1-10 ppm). Higher concentrations of NO do not produce any further change in these variables. It appears that the baseline PVRI may be the best marker predicting a beneficial response to NO. PMID- 8623965 TI - Brain tissue oxygen, carbon dioxide, and pH in neurosurgical patients at risk for ischemia. AB - A sensor that measures oxygen pressure (PO2), carbon dioxide pressure (PCO2), and pH was evaluated in brain tissue of patients at risk for ischemia. The sensor is 0.5 mm in diameter and was inserted into cortex tissue in 14 patients undergoing craniotomy for cerebrovascular surgery. A compromised cerebral circulation was identified in 8 of 14 patients by single photon emission computed tomography (SPECT) scan, cerebral angiography, and transient ischemic episodes before surgery. Under baseline conditions with isoflurane anesthesia and normal blood gases, tissue P02 was lower in the eight compromised compared to six noncompromised patients (noncompromised 37 +/- 12 mm Hg, compromised 10 +/- 5 mm Hg; P < 0.05), PCO2 was increased (noncompromised 49 +/- 5 mm Hg, compromised 72 +/- 23 mm Hg; P < 0.05), and pH was decreased (noncompromised 7.16 +/- 0.08, compromised 6.82 +/- 0.21; P < 0.05). Critical tissue values for the identification of ischemia were a P02 of 20 mm Hg, PCO2 of 60 mm Hg, and a pH of 7.0. These results suggest that brain tissue measures of P02, PCO2, and pH provide information on the adequacy of cerebral perfusion in neurosurgical patients. PMID- 8623966 TI - Cerebral microregional oxygen balance during chronic versus acute hypertension in middle cerebral artery occluded rats. AB - This study was performed to compare microregional 0(2) supply and consumption balance in spontaneously hypertensive rats (SHR), normotensive Wistar Kyoto rats (WKY), and in phenylephrine-induced acutely hypertensive WKY (WKY + ph) rats. Under isoflurane anesthesia, a middle cerebral artery (MCA) of SHR (n = 7) and WKY (n = 14) rats was occluded. Seven of the WKY rats were infused with phenylephrine (WKY + ph) to keep the mean arterial pressure (MAP) at the same level as that of the SHR. In all animals, 1 h after MCA occlusion, regional cerebral blood flow (rCBF) was determined using an autoradiographic technique, and microregional arterial and venous 02 saturations were determined using microspectrophotometry. MAP was 76 +/- 4 (SD), 136 +/- 15, and 132 +/- 12 mm Hg for the WKY, WKY + ph, and SHR groups, respectively. All variables describing regional O2 balance and rCBF were similar between the SHR and the WKY groups in the ischemic cortex as well as in the contralateral cortex. With phenylephrine infusion, rCBF of both the ischemic cortex and the contralateral cortex were increased in the WKY group. The average 02 supply-to-consumption ratio in the ischemic cortex was higher in the WKY + ph than in the WKY or SHR group. In the ischemic cortex, heterogeneity of venous 02 saturation (SvO2), expressed as a coefficient of variation (CV = 100 X SD/mean), was significantly lower in the WKY + ph (18.3 +/- 2.4) group than in the SHR (30.5 +/- 11.8) or in the WKY (31.3 +/- 9.0) group. The number of veins with low 02 saturation (SvO2 < 40%) in the ischemic cortex was significantly lower in the WKY + ph than in the SHR or in the WKY group. Our data suggest that in chronically hypertensive animals, cerebrovascular adaptations enable the microregional 02 balance in focal ischemia to be maintained at a level similar to that of normotensive animals. However, in normotensive animals with focal cerebral ischemia, an acute increase of MAP improves microregional O2 balance. PMID- 8623967 TI - Intraoperative myogenic motor evoked potentials induced by direct electrical stimulation of the exposed motor cortex under isoflurane and sevoflurane. AB - We monitored myogenic motor evoked potentials (MEPS) during intracranial surgery in 21 patients anesthetized with nitrous oxide in oxygen, fentanyl, and 0.75-1.5 minimum alveolar anesthetic concentration (MAC) isoflurane (n = 11) or sevoflurane (n = 10). The exposed motor cortex was stimulated with a single or train-of-five rectangular pulses at a high frequency (500 Hz), while the compound muscle action potentials (CMAPS) were recorded from the abductor pollicis brevis muscle. Neuromuscular block was monitored by recording the CMAPs from the abductor pollicis brevis muscle in response to electrical stimulation of the median nerve at the wrist (M-response). Stimulation of the motor cortex with a single pulse elicited MEPs in none of the patients, while stimulation with a train-of-five rectangular pulses at high frequency elicited MEPs in all patients. The relationship between MEP amplitude and the level of neuromuscular block induced by vecuronium infusion was evaluated in seven patients. For comparison of the individual measurements, the MEP amplitude at a M-response amplitude of 100% was calculated by means of the individual regression curve as 100% of MEP amplitude. There was a linear correlation between percent MEP amplitude and percent M-response amplitude (r = 0.81; P < 0.01). Intraoperative monitoring of MEP could be performed at a M-response amplitude above 90 % of the baseline value in 10 patients and at a M-response amplitude of 20%-50% of the baseline value in 11 patients. During monitoring of the 21 patients, MEPs did not change in 18 patients and disappeared in two patients. In the remaining patient, MEP amplitudes were attenuated to approximately 10% of the baseline value and recovered after cessation of surgical manipulation. In the two patients in whom MEPs disappeared, motor paresis developed postoperatively. We conclude that 1) intraoperative myogenic MEP monitoring is feasible during isoflurane or sevoflurane anesthesia if stimulation is performed with a short train of rectangular pulses, and 2) that electromyographic monitoring of neuromuscular block is useful to assess intraoperative MEP changes under partial neuromuscular block. PMID- 8623968 TI - The effects of bolus administration of opioids on cerebrospinal fluid pressure in patients with supratentorial lesions. AB - In many studies reporting an increase in cerebrospinal fluid pressure (CSFP) after opioid administration, concomitant decreases in mean arterial pressure (MAP) have been observed. Autoregulatory cerebral vasodilation may therefore have been a factor in the CSFP increases. We tested the hypothesis that increases in CSFP after bolus injection of opioids could be minimized by modifying concomitant decreases in MAP with phenylephrine. Thirty-three patients with supratentorial mass lesions were studied in a randomized, prospective, double-blind, saline controlled comparative trial. The principal outcome measures were lumbar CSFP, MAP, and heart rate (HR). Study drugs, sufentanil 0.8 micrograms/kg (n = 12), fentanyl 4.5 micrograms/kg (n = 11), or normal saline (n = 10), were injected intravenously (IV) during stable general anesthesia with 0.3-0.7 minimum alveolar anesthetic concentration (MAC) of isoflurane in oxygen and controlled ventilation (end-tidal carbon dioxide 32-35 mm Hg). Phenylephrine 50-100 micrograms was injected IV when MAP decreased by more than 15% of initial values, and atropine 0.5 mg IV when HR decreased to less than 45 bpm. Opioid administration was associated with significant decreases in MAP, 21 +/- 9 mm Hg (mean +/- SD) in the sufentanil group and 16 +/- 7 mm Hg in the fentanyl group; P < 0.001. These decreases in MAP were of short duration (i.e., corrected with 1-2 min). Patients in the sufentanil group needed more phenylephrine than patients in the fentanyl group (170 +/- 89 micrograms vs 100 +/- 47 micrograms; P < 0.05). No significant change in the CSFP was seen in either the sufentanil (1 +/- 6 mm Hg) or fentanyl treated patients (O +/- 2 mm Hg). No significant changes in MAP or CSFP were observed in the saline-treated patients. HR decreased after injection of either study drug (P < 0.01) but remained unchanged in the saline group. In summary, during stable anesthesia with isoflurane in oxygen, bolus injections of fentanyl or sufentanil, despite producing rapidly corrected mean decreases in MAP of 18% and 25%, respectively, were not associated with any change in CSFP. PMID- 8623969 TI - Metoclopramide exaggerates stress-induced tachycardia in pregnant sheep. AB - Metoclopramide is often administered to hasten gastric emptying prior to cesarean section and during labor, but has also been demonstrated to increase catecholamine release during stress and to increase heart rate and blood pressure in nonpregnant humans. The purpose of this study was to examine the maternal and fetal effects of metoclopramide during maternal stress in pregnant ewes. After baseline measures, eight ewes were randomly allocated to receive either intravenous metoclopramide, 10 mg, or saline, followed in 10 min by nonpainful stress to increase mean arterial pressure 40%-45% for 30 s. At least 2 days later, the ewes received the alternate treatment. Metoclopramide, but not saline, increased resting maternal heart rate. In the 10 min after maternal stress, maternal heart rate was increased more after metoclopramide than after saline treatment, whereas maternal blood pressure, uterine blood flow, and fetal hemodynamic variables and arterial blood gas tensions did not differ between the two treatments. Whereas these results are not consistent with a generalized increase in sympathetic nervous system tone after a single dose of metoclopramide, they do suggest that metoclopramide may exaggerate tachycardia after stress, encountered frequently both during and after cesarean section. PMID- 8623970 TI - Adding fentanyl 0.0002% to epidural bupivacaine 0.125% does not delay gastric emptying in laboring parturients. AB - Previous studies have shown that bolus doses of fentanyl (50 and 100 micrograms) with epidural bupivacaine delay gastric emptying by up to 45 min. We studied the effect of the addition of small-dose fentanyl to epidural bupivacaine infusions on gastric emptying during labor. The acetaminophen absorption technique was used to infer gastric emptying. Twenty-eight patients in established labor consented to participate in the study. They were randomized to receive either 1) 10 mL bupivacaine 0.125% followed by an infusion of 0.125% bupivacaine at 10 mL/h or 2) 10 mL bupivacaine 0.125% with 50 micrograms fentanyl followed by an infusion of 0.125% bupivacaine and 0.0002% fentanyl at 10 mL/h. Two hours after initiation of epidural analgesia, each patient ingested 20 mg/kg acetaminophen in a suspension of 150 mL water. Venous blood samples were drawn for a baseline and then every 15 min for 2 1/2 h. There were no significant demographic differences between the groups. There were no differences detected between groups in the peak plasma concentrations of acetaminophen, the time to achieve the peak plasma concentrations, or the area under the curve at 45 and 90 min. Our results indicate that epidural infusions for labor analgesia using 0.125% bupivacaine and 0.0002% fentanyl do not delay gastric emptying compared to infusions of bupivacaine 0.125% alone. PMID- 8623971 TI - The effects of intrathecal neostigmine on somatic and visceral pain: improvement by association with a peripheral anticholinergic. AB - This study was designed to qualitatively evaluate the analgesic actions of intrathecal neostigmine alone and with intravenous (IV) N-butyl-scopolamine on somatic and visceral pain. Twenty-seven patients scheduled for both tubal ligation and vaginoplasty were divided into three groups. Patients received a standard anesthetic with thiopental, atracurium, and N2O/O2/enflurane. N-butyl scopolamine, 20 mg, or saline was administered as a 2-mL IV bolus 20 min before the end of the surgical procedure. The control group (CG) received spinal and IV saline; the neostigmine group (NG), spinal neostigmine and IV saline; and the neostigmine-N-butyl-scopolamine group (NSG), spinal neostigmine and IV N-butyl scopolamine. Postoperatively, patients assessed their pain on a 10-cm visual analog scale (VAS). The CG had both visceral and somatic pain at the first 30-min assessment, and all patients requested morphine. Patients from the NG had only visceral pain from the first assessment; however, they had lower VAS scores (P = 0.026) and requested less morphine (P = 0.037). Patients from the NSG were pain free during all assessment times (P < 0.0001). Neostigmine was more effective for somatic pain than visceral pain. N-butyl-scopolamine administration acted peripherally as an effective complement for treatment of visceral pain, reflecting an association between central cholinergic effects and peripheral anticholinergic effects in the treatment of visceral postoperative pain. PMID- 8623972 TI - Cerebrospinal fluid norepinephrine and acetylcholine concentrations during acute pain. AB - Painful stimulation increases spinal cord norepinephrine (NE) in animals, and spinally released NE induces acetylcholine (ACh) release to cause analgesia. The purpose of this study was to determine the relationship between NE and ACh in cerebrospinal fluid (CSF) in sheep and humans during painful stimulation. CSF was sampled in anesthetized sheep before and during electrical nerve stimulation at an intensity sufficient to increase mean arterial pressure 15%-20%. To determine whether spinally released NE caused ACh release by stimulation of alpha(2) adrenoceptors, seven sheep received intrathecal (IT) idazoxan whereas seven sheep received IT saline before stimulation. To examine the effect of pain on CSF NE and ACh in humans, CSF was sampled in 33 women after at least 4 h of painful labor and in 22 pregnant women without pain. Painful stimulation in sheep increased CSF NE and ACh. IT idazoxan blocked the increase in both NE and ACh. Although mean concentrations of CSF NE and ACh did not differ between parturients with and without pain, there was a significant correlation between NE and ACh concentrations only in those with pain. These data provide evidence in animals for activation of spinal cord noradrenergic-cholinergic systems in response to pain. There is only weak evidence for such activation, however, in women with painful labor. PMID- 8623974 TI - Spinal cord blood flow after intrathecal injection of ropivacaine: a screening for neurotoxic effects. AB - The study of spinal cord blood flow (SCBF) after spinal drug application is an important aspect of preclinical neurotoxicological screening. This investigation was designed to study how a new local anesthetic, ropivacaine, affects SCBF after intrathecal (IT) administration in the rat. SCBF was measured continuously in spontaneously breathing, enflurane/N2O-anesthetized rats, using the laser-Doppler flowmetry technique. The spinal cord was exposed by laminectomy, and a laser Doppler probe was placed over the dorsal horn, allowing on-line registration of spinal blood flow in a tissue hemisphere of 1-2 mm. Relative changes in spinal blood flow over time were then measured after IT administration of either 0.9% saline, 5 mg/mL ropivacaine, i.e., a concentration within the pharmacological range, or a high, provocative concentration of 20 mg/mL ropivacaine. A minor and transient decrease in SCBF was seen after administration of 5 mg/mL ropivacaine (50 micrograms given IT), but this decrease was not significantly different from that in the saline group. SCBF decreased significantly to approximately 45% of the predrug value after the high concentration of 20 mg/mL ropivacaine (200 micrograms given IT), and this reduction was reversible within a period of 20 - 40 min after the injection. Whereas a high concentration of ropivacaine caused a definite reduction in spinal cord blood flow when administered IT to anesthetized rats, clinically relevant concentrations induced only minor changes. These results suggest that ropivacaine may be used to induce spinal anesthesia without causing clinically relevant effects on SCBF. PMID- 8623973 TI - Intrathecal clonidine and tizanidine in conscious dogs: comparison of analgesic and hemodynamic effects. AB - Intrathecal delivery of alpha(2)-adrenergic agonists produces an analgesic effect. However, hemodynamic side effects limit their clinical usage. To more fully characterize the effects on heart rate and arterial blood pressure of alpha(2)-adrenergic agonists, clonidine and tizanidine were injected intrathecally in conscious dogs. Both compounds produced a potent inhibition of thermal foot-withdrawal latencies at 1000 micrograms, which was blocked by the alpha(2)-adrenergic antagonist yohimbine. Tizanidine (250-500 micrograms) did not change heart rate. Clonidine (500 -2000 micrograms) and tizanidine (1000-2000 micrograms) decreased heart rate. The tizanidine effect was inhibited by yohimbine and the alpha(2)/imidazoline antagonist idazoxan, as well as the parasympathetic blocker glycopyrrolate. No drug completely inhibited the clonidine-induced bradycardia. Clonidine had a biphasic effect on arterial blood pressure, a decrease at 500 micrograms and an increase at 2000 micrograms. Tizanidine decreased arterial blood pressure at all doses. The results indicate that, while the analgesic effects of both drugs are similar, the hemodynamic responses differ. While the decrease in heart rate with tizanidine is consistent with alpha(2)-adrenergic binding and vagal action, the bradycardia induced by clonidine is more complex. In addition, the increased arterial blood pressure with high doses of clonidine, which is suggestive of a peripheral vasoconstrictive effect, does not occur with tizanidine. PMID- 8623975 TI - Naloxone versus nalbuphine infusion for prophylaxis of epidural morphine-induced pruritus. AB - This randomized, double-blind study compared the efficacy of two mu-receptor antagonists, naloxone and nalbuphine, in the prophylactic management of pruritus in postcesarean section patients receiving epidural morphine. Dosages of study drugs were individualized by the use of a patient self-administration (PSA) device. All 51 patients were healthy women who received a uniform epidural anesthetic and epidural morphine (5 mg). Coded solutions were infused for 24 h, with 5-min PSA lockout times: Group A (n = 17), nalbuphine 2.5 mg/h, PSA nalbuphine 1 mg; Group B (n = 16), naloxone 50 micrograms/hr, PSA saline; Group C (n = 18), naloxone 50 micrograms/h, PSA naloxone 40 micrograms. Patients were assessed for pruritus and pain every 8 h for 24 h. Both naloxone and nalbuphine provided good relief for pruritus; median pain and pruritus scores were in the none-to-mild range (0-3) for all groups at all assessment intervals. The pruritus scores of the PSA saline group were higher during the 16- to 24-h period (P < 0.05) than the scores of either group receiving A-receptor antagonist by PSA. There was evidence of shortening of the duration of analgesia in patients receiving naloxone who required treatment for pruritus after 16 h. Patients who self-administered large doses of nalbuphine over the first 8 h also reported pain scores consistent with reversal of analgesia. The potency ratio for naloxone:nalbuphine for antagonism of the pruritic effects of epidural morphine was approximately 40:1. Intervention to treat either unrelieved pruritus or pain, respectively, was necessary in the following numbers of patients: Group A, 0/1; Group B, 1/1; Group C, 2/2. Prophylactic infusions offer the potential for labor cost savings by minimizing the need for episodic therapeutic interventions to treat pruritus. PMID- 8623976 TI - Comparative electrophysiologic and hemodynamic effects of several amide local anesthetic drugs in anesthetized dogs. AB - Large and equipotent doses of several local anesthetics were administered in a cardiac electrophysiologic model on closed-chest dogs. Five groups of pentobarbital-anesthetized dogs were each given intravenously 16 mg/kg lidocaine, 12 mg/kg mepivacaine, 4 mg/kg or 8 mg/kg etidocaine, and 4 mg/kg bupivacaine. Lidocaine induced bradycardia, slowing of atrioventricular node conduction (AH), and marked hemodynamic depression, represented by a decrease in mean aortic pressure (MAoP), in the peak of first derivative of left ventricular pressure (LVdP/dt(max)) and by an increase in left ventricular end-diastolic pressure (LVEDP). Atrial pacing at pacing cycle length (PCL) of 298 ms did not enhance the alteration of variables of ventricular conduction (His ventricle [HV] interval and QRS duration). Mepivacaine induced slight alteration of electrophysiologic variables. Atrial pacing at PCL of 312 ms did not enhance the alteration of HV and QRS duration. Mepivacaine induced transient hemodynamic depression. Etidocaine (4 mg/kg) induced electrophysiologic and hemodynamic alterations similar to mepivacaine but artrial pacing at PCL of 330 ms enhanced HV lengthening and QRS widening (P < 0.05). Etidocaine (8 mg/kg) induced marked impairment of PR, HV, QRS, and QT, and dramatic hemodynamic depression represented by a decrease in MAoP from 123.5 +/- 16.2 at baseline to 36.5 +/- 8.3 mm Hg at 1 min (P < 0.001) and of LVdP/dtmax) from 1446 +/- 379 to 333 +/- 93 mm Hg/s (P < 0.001). Bupivacaine induced dramatic impairment of electrophysiologic variables. Bupivacaine also decreased LVDP/dtmax (from 1333 +/- 347 to 617 +/- 299,P < 0.001) and increased LVEDP. We conclude that mepivacaine induced moderate cardiotoxicity. In contrast, lidocaine induced dramatic hemodynamic depression while etidocaine and bupivacaine markedly impaired both electrophysiologic and hemodynamic variables. This double impairment could explain the great difficulty in resuscitating patients who have had cardiotoxic accidents induced by etidocaine or bupivacaine. PMID- 8623977 TI - The effect of muscle relaxants on cricothyroid muscle: a report of three cases. PMID- 8623978 TI - Total intravenous anesthesia and endotracheal oxygen insufflation for repair of tracheoesophageal fistula in an adult. PMID- 8623979 TI - Intraoperative cardiac tamponade after central venous cannulation. PMID- 8623980 TI - Epidural blood patch and late postpartum eclampsia. PMID- 8623981 TI - Sodium nitroprusside administration. PMID- 8623982 TI - Regional anesthesia and local anesthetic-induced seizures. PMID- 8623984 TI - Arterial pressure waveform analysis during hypovolemia. PMID- 8623983 TI - Anticholinergic treatment for choreoathetosis in a child after induction with propofol. PMID- 8623985 TI - Deliberate oxygen deprivation in children: ethical considerations. PMID- 8623986 TI - Isoflurane, fentanyl, thiopental, and brain edema. PMID- 8623987 TI - Extracorporeal shunt: a theoretical approach to the prevention of arterial hyperoxia and the reduction of gaseous emboli during cardiopulmonary bypass. PMID- 8623988 TI - Preoperative pregnancy testing in adolescents. PMID- 8623989 TI - Mechanisms of local anesthetic action. PMID- 8623990 TI - Continuous ventilation during transnasal fiberoptic bronchoscope-aided tracheal intubation. PMID- 8623991 TI - A puzzling accidental intraoperative finding: an arteriovenous fistula? PMID- 8623992 TI - Massive subcutaneous emphysema during emergence from anesthesia followed by seizures. PMID- 8623993 TI - Preemptive analgesia. Why its effect is not always obvious. PMID- 8623994 TI - Effect of preemptive nerve block on inflammation and hyperalgesia after human thermal injury. AB - BACKGROUND: Postoperative pain relief may be improved by reducing sensitization of nociceptive pathways caused by surgical trauma. Such a reduction may depend on the timing and efficacy of analgesia and the duration of the nociceptive block versus the duration of the nociceptive input. We examined whether a prolonged nerve block administered before a superficial burn injury could reduce local inflammation and late hyperalgesia after recovery from the block. METHODS: The effects of a preemptive saphenous nerve block on primary and secondary hyperalgesia, skin erythema, and blister formation, were compared to the opposite unblocked leg for 12 h after bilateral thermal injuries (15 x 25 mm, 49 degrees C for 5 min) in 20 healthy volunteers. Recovery from the block was identified by return of sensation to cold. RESULTS: Six subjects were excluded because of insufficient initial block (2 subjects) or because the block lasted beyond the study period (4 subjects). The remaining 14 subjects experienced significantly reduced primary (P = 0.005) and secondary hyperplasia (P = 0.01) in the blocked leg after return of cold sensation compared to the unblocked leg. Erythema intensity and blister formation were not significantly affected by the blockade (P = 0.94 and P = 0.07, respectively). CONCLUSIONS: These data suggest that a prolonged, preemptive nerve block reduced late hyperalgesia after thermal injury, whereas the erythema and blister formation were not significantly affected. PMID- 8623995 TI - Prophylactic use of epidural mepivacaine/morphine, systemic diclofenac, and metamizole reduces postoperative morphine consumption after major abdominal surgery. AB - BACKGROUND: Surgical trauma induces nociceptive sensitization leading to amplification and prolongation of postoperative pain. While preemptive analgesic treatment with numerous agents has been successful in experimental animals, results of human studies remain conflicting. The authors used a multimodal approach for preemptive analgesia before abdominal surgery: diclofenac and metamizole inhibit prostaglandin synthesis, thus influencing peripheral sensitization; epidural local anesthetics induce conduction block, epidural opioids inhibit nociceptive synaptic transmission, and metamizole induces descending inhibition. The interaction of these drugs might suppress spinal nociceptive sensitization and postoperative analgesic demand. METHODS: One hundred forty-two patients scheduled for major abdominal surgery were randomly assigned to one of three groups and studied prospectively. Epidural catheters in groups 1 and 2 were placed at interspaces T8-T10, the position of the catheter was confirmed by epidurography, and sensory testing after administration of 5 ml mepivacaine 1%. Group 1 received 75 mg intramuscular diclofenac, 1000 mg intravenous metamizole, 5.3 +/- 1 mg epidural morphine, and 15-20 ml mepivacaine 1% 85 +/- 41 min before skin incision. Epidural analgesia was maintained by injections of 0.1 ml.kg-1.h-1 mepivacaine 1%. Group 2 patients received the balanced analgesia regimen before wound closure (221 +/- 86 min after skin incision). Group 3 patients did not receive any study substances. General anesthesia was induced with 5 mg/kg thiopental and 2 micrograms/kg fentanyl and maintained with enflurane and nitrous oxide. Postoperative analgesia consisted of patient-controlled intravenous morphine over 5 days. RESULTS: Median visual analog scale pain intensities were < 3 cm and did not differ among the groups. Morphine consumption per hour on postoperative day 2 was 0.8 +/- 0.1 mg/h (group 1) < 1.2 +/- 0.1 mg/h (group 2) = 1.1 +/- 0.1 mg/h (group 3) and cumulative morphine consumption (in mg) on the morning of day 5 was 95 +/- 9 (group 1) < 111 +/- 11 (group 2) < 137 +/- 10 (group 3). CONCLUSIONS: A significant reduction of patient controlled analgesia requirements could be achieved by our preincisional balanced analgesia regimen compared to application before wound closure. The more distinct difference between patients receiving balanced analgesia and those in the control group is based on the analgesic action of the study substances, which lasted about 14 h. PMID- 8623996 TI - Desflurane-mediated neurocirculatory activation in humans. Effects of concentration and rate of change on responses. AB - BACKGROUND: Rapid increases in the inspired concentration of desflurane have been associated with sympathetic activation, tachycardia, hypertension, and in select cases, myocardial ischemia. The current study examined the effects of the rate of change of the desflurane concentration on the sympathetic and hemodynamic responses to desflurane and sought to determine whether a finite concentration (end-tidal) of desflurane consistently initiated these responses. METHODS: After Institutional Review Board approval, 23 healthy male volunteers were instrumented for electrocardiogram (heart rate (HR)), intraarterial blood pressure, and peroneal nerve microneurography (sympathetic nerve activity (SNA)). Subjects were given propofol (2.5 mg/kg) and vecuronium (0.15 mg/kg), and their lungs were mechanically ventilated for 30 min at a minimum alveolar concentration of 0.5 MAC with either desflurane or isoflurane (random assignment). The end-tidal concentration was increased at either 1% per min (n = 7) or 0.5% per min (n = 7) for desflurane or 0.16% per min (n = 9) for isoflurane (MAC-multiple comparable to 1% per min desflurane group) until 1.5 MAC was reached. HR, blood pressure, and SNA were averaged over 1-min segments from 0.5 to 1.5 MAC levels. RESULTS: Awake neurocirculatory variables did not differ among the three groups. At 0.5 MAC, blood pressure had decreased (12-15%) and HR increased (12-20%) similarly in both groups. SNA decreased 77% in the isoflurane group but was not significantly changed in the desflurane groups. In the desflurane groups, the threshold (end tidal concentration associated with a 10% increase in the measured variable) ranged between 4% and 10% for HR and between 4% and 7.7% for SNA. In the isoflurane group, the threshold occurred between 1.0% and 1.6% for HR and between 0.7% and 1.3% for SNA. The rate of change did not affect the threshold concentration or the peak HR increase in the desflurane groups. In contrast, SNA responses to desflurane were directly proportional to the rate of change. CONCLUSION: There was no consistent threshold for the neurocirculatory activation associated with desflurane, and the HR and SNA thresholds generally were less than 1 MAC. The HR increase associated with desflurane was not rate- or concentration-dependent. In contrast, SNA responses were proportional to the rate of change and the concentration of desflurane. PMID- 8623997 TI - Derivation and cross-validation of pharmacokinetic parameters for computer controlled infusion of lidocaine in pain therapy. AB - BACKGROUND: Lidocaine administered intravenously is efficacious in treating neuropathic pain at doses that do not cause sedation or other side effects. Using a computer-controlled infusion pump (CCIP), it is possible to maintain the plasma lidocaine concentration to allow drug equilibration between the plasma and the site of the drug effect. Pharmacokinetic parameters were derived for CCIP administration of lidocaine in patients with chronic pain. METHODS: Thirteen patients (mean age 45 yr, mean weight 66 kg) were studied. Eight subjects received a computer-controlled infusion, targeting four increasing lidocaine concentrations (1-7 micrograms.ml-1) for 30 min each, based on published kinetic parameters in which venous samples were obtained infrequently after bolus administration. From the observations in these eight patients, new lidocaine pharmacokinetic parameters were estimated. These were prospectively tested in five additional patients. From the complete data set (13 patients), final structural parameters were estimated using a pooled analysis approach. The interindividual variability was determined with a mixed-effects model, with the structural model parameters fixed at the values obtained from the pooled analysis. Internal cross-validation was used to estimate the residual error in the final pharmacokinetic model. RESULTS: The lidocaine administration based on the published parameters consistently produced higher concentrations than desired, resulting in acute lidocaine toxicity in most of the first eight patients. The highest measured plasma concentration was 15.3 micrograms.ml-1. The pharmacokinetic parameters estimated from these eight patients differed from the initial estimates and included a central volume one-sixth of the initial estimate. In the subsequent prospective test in five subjects, the new parameters resulted in concentrations evenly distributed around the target concentration. None of the second group of subjects had evidence of acute lidocaine toxicity. The final parameters ( +/- population variability expressed as %CV) were estimated as follows: V1 0.101 +/- 53% 1.kg-1, V2 0.452 +/- 33% 1.kg-1, Cl1 0.0215 +/- 25% 1.kg-1.min-1, and Cl2 0.0589 +/- 35% 1.kg-1.min-1. The median error measured by internal cross-validation was +1.9%, and the median absolute error was 14%. CONCLUSIONS: Pharmacokinetic parameters for lidocaine were derived and administration was prospectively tested via computer-controlled infusion pumps for patients with chronic neuropathic pain. The estimated parameters performed well when tested prospectively. A second estimation step further refined the parameters and improved performance, as measured using internal cross validation. PMID- 8623998 TI - Enzymatic versus pharmacologic antagonism of profound mivacurium- induced neuromuscular blockade. AB - BACKGROUND: Mivacurium, a nondepolarizing muscle relaxant, is hydrolyzed by butyrylcholinesterase. The use of butyrylcholinesterase for antagonism of profound mivacurium-induced blockade has not been studied in humans. In part 1 of this two-part study, the authors examined the relationship between the posttetanic count (PTC) and recovery from profound mivacurium-induced blockade. In part 2, an attempt was made to antagonize a quantified level of profound mivacurium-induced blockade using either butyrylcholinesterase, edrophonium, or neostigmine. METHODS: Eighty-seven ASA physical status 1 or 2 adult patients were given 0.15 mg.kg-1 mivacurium during fentanyl-thiopental-nitrous oxide-isoflurane anesthesia. They were randomly assigned to eight groups. Neuromuscular function was monitored by recording the mechanomyographic response of the adductor pollicis to PTC and train-of-four (TOF) stimulation in all patients except those in group 1 where the TOF was the only pattern used. In part 1, neuromuscular function was allowed to recover spontaneously in ten patients (group 1; control TOF) until TOF ratio (the amplitude of the fourth evoked response as a fraction of the first evoked response T4/T1) had reached 0.75. The temporal relationship between PTC and the first reaction to TOF stimulation was determined in another 31 patients, and neuromuscular function in 10 of these patients was allowed to recover spontaneously until TOF ratio had reached 0.75. The temporal relationship between PTC and the first reaction to TOF stimulation was determined in another 21 patients, and neuromuscular function in 10 of these patients was allowed to recover spontaneously, until TOF ratio had reached 0.75 (group 2; control-PTC). In part 2, the antagonism of mivacurium-induced profound (PTC > or = 1; groups 3 6) and 90% block (groups 7-8) of twitch height were investigated in another 56 patients. Groups 3 and 7 received neostigmine 0.06 mg.kg-1 whereas groups 4 and 8 received edrophonium 1 mg.kg-1, respectively. Groups 5 and 6 received exogenous human butyrylcholinesterase equivalent to activity present in 25 or 70 ml.kg-1 of human plasma, respectively. RESULTS: Neither butyrylcholinesterase nor edrophonium shortened the times from first PTC response to TOF = 0.75 compared to group 2. Neostigmine resulted in prolongation of recovery time. There was a linear relationship (r = -0.80; P = 0.00001) between PTC and time of onset of TOF response. CONCLUSIONS: There appears to be no clinical advantage in attempting to antagonize profound mivacurium-induced neuromuscular blockade. PMID- 8623999 TI - Parental presence during induction of anesthesia. A randomized controlled trial. AB - BACKGROUND: To determine whether parental presence during induction of anesthesia is an effective preoperative behavioral intervention, a randomized controlled trial with children undergoing outpatient surgery was conducted. METHODS: Eighty four children were randomly assigned to a parent-present or parent-absent group. Using multiple behavioral and physiologic measures of anxiety, the effect of the intervention on the children and their parents was assessed. Predictors for the response to the intervention were examined using multivariate linear regression analysis. RESULTS: When the intervention group (parent-present) was compared to the control group (parent-absent), overall there were no significant differences in any of the behavioral or physiologic measures of anxiety tested during induction of anesthesia. Using the child's serum cortisol concentration as the outcome, parental presence, the child's age and baseline temperament, and trait anxiety of the parent, were identified as predictors of the child's anxiety during induction. Analysis of variance demonstrated that three groups showed diminished cortisol concentrations with parental presence: children older than 4 yr (P = 0.001), children whose parent had a low trait anxiety (P = 0.02), and children who had a low baseline level of activity as assessed by temperament (P = 0.05). CONCLUSIONS: Children who were older than 4 yr or those with a parent with a low trait anxiety or who had a low baseline level of activity/temperament benefited from parental presence during induction. PMID- 8624000 TI - Alterations in spectral characteristics of heart rate variability as a correlate of cardiac autonomic dysfunction after esophagectomy or pulmonary resection. AB - BACKGROUND: Both esophagectomy and pulmonary resection are associated with postoperative cardiac complications, partly because of autonomic perturbations involving the heart. This study was undertaken to determine whether heart rate variability (HRV), employed as an index of cardiac autonomic function, changes in patients undergoing esophagectomy or pulmonary resection. METHODS: Electrocardiographic RR intervals were measured in 20 esophagectomized patients, 10 undergoing right and 10 undergoing left pulmonary resection on the preoperative day as baseline data and on postoperative days 1, 3, 5, 7, 14, and 30. Instantaneous heart rate was calculated every 250 ms from 416-s data of RR intervals. Power spectra of HRV for 128 s were computed using a fast Fourier transform and normalized by squared mean heart rate. The average ten sets of normalized HRV power were obtained by integrating the following power spectral bands: the low-, (0.06-0.10 Hz), high- (0.15-0.40 Hz), and total-frequency regions (0.01-0.40 Hz). RESULTS: In the esophagectomy group, mean low-, high-, and total-frequency HRV power decreased after surgery to 17%, 6%, and 15% of their preoperative values, respectively, and these indexes remained suppressed for up to 30 days. After right pulmonary resection, low- and total-frequency HRV power decreased through 30 and 7 postoperative days, respectively. In the left pulmonary resection group, HRV remained unchanged. In the esophagectomy group, mean (+/- SEM) heart rate increased from 78 (+/- 3) bpm to more than 90 bpm throughout the study, and body temperature from 36.5 (+/- 0.1) degrees C to more than 37.0 degrees C through 14 postoperative days. Heart rate and body temperature remained increased for 3 days after pulmonary surgery. Mean arterial pressure remained unchanged in the three surgical groups. CONCLUSIONS: Reductions indicate HRV after esophagectomy or right pulmonary resection indicate a substantial and prolonged surgical injury to the autonomic nervous control of pulse rate. PMID- 8624001 TI - In vitro 31P-magnetic resonance spectroscopy of muscle extracts in malignant hyperthermia-susceptible patients. AB - BACKGROUND: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition. METHODS: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed. RESULTS: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found. CONCLUSIONS: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients. PMID- 8624002 TI - Pharmacokinetics and pharmacodynamics of cisatracurium in young and elderly adult patients. AB - BACKGROUND: The effects of a muscle relaxant may differ in elderly compared with young adult patients for a variety of reasons. The authors compared the effects of a new muscle relaxant (cisatracurium) in young and elderly adults and used pharmacokinetic/pharmacodynamic modeling to identify factors explaining differences in time course of effect. METHODS: Thirty-one young (18-50 yr) and 33 elderly ( > 65 yr) patients anesthetized with nitrous oxide, isoflurane, and fetanyl were studied. Cisatracurium (0.1 mg/kg) was given after induction of anesthesia and later additional boluses of 0.025 mg/kg or an infusion of cisatracurium was given. Neuromuscular transmission was measured using the first twitch of the train-of-four response at the adductor pollicis after supramaximal stimulation of the ulnar nerve at 2 Hz every 15 s. Five venous blood samples were obtained for plasma drug concentration at intervals ranging from 2 to 120 min from every patient. Three additional samples were obtained from those who received an infusion. A population pharmacokinetic/pharmacodynamic model was fitted to the plasma concentration and effect data. The parameters of the model were permitted to vary with age to identify where differences existed between young and elderly adults. RESULTS: Onset of block was delayed in the elderly; values being mean 3.0 (95% confidence interval 1.75-11.4) min and 4.0 (2.4-6.5) min in the young and elderly, respectively (P < 0.01). Duration of action was similar in the two groups. Plasma clearance was 319 (293-345) ml/min in the study population and did not differ between young and elderly patients. Apparent volume of distribution was 13.28 (9.9-16.7) 1 and 9.6 (7.6-11.7) 1 in the elderly and young adults, respectively (P < 0.05). There also were differences in pharmacodynamic parameters between the young and elderly; the predominant change being a slower rate of biophase equilibration (ke0) in the elderly (0.060 [0.052 0.068])/min compared with the young (0.071 [0.065-0.077]/min; P < 0.05). CONCLUSIONS: The pharmacokinetics of cisatracurium differ only marginally between young and elderly adults. Onset is delayed in the elderly because of slower biophase equilibration. PMID- 8624003 TI - Repeated doses of rocuronium bromide administered to cirrhotic and control patients receiving isoflurane. A clinical and pharmacokinetic study. AB - BACKGROUND: Steroid muscle relaxants often display pharmacodynamic changes in patients with cirrhosis because of alterations in elimination processes. Rocuronium is a new steroid muscle relaxant possibly eliminated through the liver. This study was designed to compare rocuronium pharmacodynamics and pharmacokinetics in cirrhotic and healthy patients. METHODS: Rocuronium was administered to 26 cirrhotic patients and 24 control subjects anesthetized with isoflurane for an elective procedure. Patients were randomly allocated to received an initial dose of rocuronium: 120, 180, 250, or 300 micrograms.kg-1. Dose-response curves were established, and ED50 was calculated. Preselected maintenance doses (75, 150, or 225 micrograms.kg-1) were administered at 25% recovery of twitch height to compare clinical duration of action. At the end of the procedure, relaxation was reversed in half of the patients, and the time course of recovery was compared in the two groups. Blood samples drawn during the procedure and after the last maintenance dose allowed pharmacokinetic analysis in six cirrhotic patients and six control subjects. RESULTS: ED50 of the initial dose was 144 micrograms.kg-1 in cirrhotic patients and 60 micrograms.kg-1 in control subjects, related to a higher initial volume of distribution (cirrhotic 78.5 +/- 31.7 ml.kg-1, control 29.8 +/- 17.3 ml.kg-1). Time from complementary dose to 25% recovery was longer in cirrhotic patients (41.0 +/- 20.7 min vs 30.2 +/- 9.7 min), but time course of action during maintenance was not statistically different in the two groups. In cirrhotic patients receiving five maintenance doses or more, prolongation of the duration of action with successive maintenance doses could be statistically demonstrated. Spontaneous recovery was delayed in cirrhotic patients, because of impaired elimination processes: greater volume of distribution at steady-state (264 +/- 92 vs. 151 +/- 59 ml.kg-1); trend toward a lower clearance (189 +/- 60 vs. 296 +/- 169 ml.min-1). CONCLUSIONS: Rocuronium pharmacodynamics are moderately altered by cirrhosis, possible because of pharmacokinetic alterations. Individual variability in response to rocuronium is great, and dosage should be carefully titrated to that required. PMID- 8624004 TI - Fiberoptic intubation using anesthetized, paralyzed, apneic patients. Results of a resident training program. AB - BACKGROUND: There is no consensus about the best way to teach fiberoptic intubation. This study assesses the effectiveness of a training program in which novice anesthetic residents routinely were taught fiberoptic tracheal intubation of anesthetized, paralyzed, apneic patients. METHODS: Eight inexperienced anesthetic residents learned fiberoptic and conventional tracheal intubation simultaneously during their first 4 months of training. All intubations were performed using general anesthesia and muscle paralysis. Of these intubations, 223 (23%) were fiberoptic and 743 (77%) were laryngoscopic. Subsequently, their intubation skills with the two techniques were studied in a prospective, single blind randomized trial involving 131 elective patients. Intubation times, SpO2, ETCO2, hemodynamic changes on intubation, and complications were recorded for 71 fiberoptic and 57 laryngoscopic intubations. RESULTS: There were two failures of the rigid and one failure of the fiberoptic technique due to inability to intubate within 180 s. In cases of failure, the tracheas were intubated successfully after mask ventilation by the alterative technique. No hypoxemia or hypercarbia occurred in any patient. There were no differences in hemodynamic indexes nor incidence of sore throat or hoarseness between the two groups. Mean intubation times were 56 +/- 24 s (mean +/- SD) for fiberoptic and 34 +/- 10 s (mean +/- SD) for laryngoscopic (P < 0.001). CONCLUSIONS: Novices taught fiberoptic intubation and rigid laryngoscopic intubation under similar conditions, with similar volumes of experience, learn both techniques well. The safety and effectiveness of this training regimen commend it for inclusion in any residency program. PMID- 8624005 TI - Rate of coronary flow adaptation in response to changes in heart rate before and during anesthesia for coronary artery surgery. AB - BACKGROUND: The rate of adaptation of coronary blood flow in response to stepwise changes in heart rate (HR) has been extensively studied in dogs and goats to improve our understanding of the dynamics of coronary regulation processes and their pathophysiology and to obtain time constants for mathematical modeling of the coronary regulation. However, little is known about the dynamic characteristics of coronary flow adaptation in humans. In patients undergoing coronary artery surgery, we investigated the rate of coronary adaptation in response to stepwise changes in HR, in the awake and anesthetized states. METHODS: In 11 patients with stable coronary artery disease, arterial blood pressure, right atrial pressure, and coronary sinus blood flow, measured by continuous thermodilution, were calculated per beat. The ratio of beat-averaged arterial blood pressure minus right atrial pressure and coronary sinus blood flow was calculated to obtain an index of coronary resistance. The rate of change of coronary resistance index was quantified by t50, defined as the time required to establish 50% of the total change in coronary resistance index. Responses of coronary resistance index after HR changes, before and after induction of anesthesia, were compared. The anesthesia technique consisted of 100 micrograms.kg-1 fentanyl and 0.1 mg.kg-1 pancuronium bromide in combination with oxygen in air ventilation (FIO2 = 0.5). RESULTS: In the awake situation, t50 values of the dilating and constricting responses, induced by an increase and a decrease in HR were 5.0 +/- 2.1 (SD) s (range 2.6-9.0 s) and 5.7 +/- 1.2 s (range 4.1-7.8 s), respectively. During fentanyl/pancuronium anesthesia, the rate of coronary flow adaptation was significantly slower, with t50 values of 10.2 +/- 2.1 s (range 7.7-13.1 s) after an HR step-up and 9.8 +/- 2.1 s (range 6.6-13.2 s) after an HR step-down. Compared to the awake situation, arterial blood pressure was significantly reduced during anesthesia, but coronary vascular resistance remained unchanged. This implies that the steady-state static regulation of coronary blood flow had not changed. CONCLUSIONS: These preliminary data suggest that, in patients with coronary artery disease, the rate of change in coronary vascular resistance in response to pacing-induced changes in HR is mitigated by fentanyl/pancuronium anesthesia during positive pressure ventilation. A further qualification of our findings in a larger number of patients is warranted. PMID- 8624006 TI - Effects of preemptive or postinjury intrathecal local anesthesia on persistent nociceptive responses in rats. Confounding influences of peripheral inflammation and the general anesthetic regimen. AB - BACKGROUND: Although experimental evidence indicates that preemptive intrathecal treatment with local anesthetics reduces postinjury neuronal hyperexcitability, clinical evidence indicates that preemptive treatments do not consistently reduce postoperative pain. The current study used experimental models of postinjury nociception, in which rats received subcutaneous or intraarticular injections of the irritant formalin, to evaluate the effects of peripheral inflammation, or the use of agents supplemental to anesthesia, as possible confounding influences on the effectiveness of preinjury and postinjury intrathecal local anesthetic treatments. METHODS: In experiment 1, lumbar intrathecal lidocaine (30 microliters, 2%), given either 5 min before or 5 min after hind paw injection of 50 microliters of varying concentrations of formalin, was compared with intrathecal cerebrospinal fluid, for their effects on nociceptive responses in the late phase of the formalin test. Furthermore, the effect of hind paw injection of 50 microliters of 2.5, 3.75, or 5.0% formalin on peripheral inflammation was assessed by measuring plasma extravasation in the hind paws of rats given Evans Blue dye (50 mg/kg, intravenous). In experiment 2, rats received a deep tissue injury (100 microliters of 5.0% formalin into the knee joint) while under halothane anesthesia. In addition to halothane (3-4%), rats received either saline, pentobarbital (13 mg/kg, intraperitoneal), or pentobarbital + morphine (0.5 mg/kg, intravenous), with or without preinjury or postinjury spinal anesthesia using intrathecal bupivacaine (30 microliters, 0.75%), to assess the effects of supplemental treatments on the preemptive effects of intrathecal bupivacaine. RESULTS: Lumbar intrathecal lidocaine pretreatment, but not posttreatment, significantly reduced late phase nociceptive responses to hind paw injections of 2.5% formalin. The preemptive effects of lidocaine were overridden in rats that received hind paw injections of 3.75 and 5.0% formalin. Hind paw injection of 50 microliters of 3.75 or 5.0%, but not 2.5% formalin produced an increase in plasma extravasation. Either pentobarbital or pentobarbital + morphine treatment, or a pentobarbital + morphine treatment and postinjury treatment with intrathecal bupivacaine failed to produce a significant reduction in the nociceptive response to the deep tissue injury. However, rats that received pentobarbital + morphine treatments and intrathecal bupivacaine before the injury had significantly reduced nociceptive responses to deep tissue injury when compared to the saline control group, but not to the group that received pentobarbital + morphine treatment and postinjury treatment with bupivacaine. CONCLUSIONS: The current results attest to the important effects of ongoing inputs from inflamed tissue, and the use of supplemental treatments, as important confounding factors that may influence the effectiveness of preemptive spinal anesthesia for postoperative pain. PMID- 8624007 TI - Influence of timing of administration on the analgesic effect of bupivacaine infiltration in carrageenin-injected rats. AB - BACKGROUND: Recent evidence has suggested that the timing of administration of analgesic drugs could influence their efficacy by reducing the sensitization of the nervous system induced by the nociceptive inputs, but this concept of preemptive analgesia is still debated in both clinical and basic research. METHODS: The model of acute inflammatory pain induced by carrageenin was used to study the influence of timing of administration of bupivacaine (0.2 ml of a 0.5% solution with 0.005 mg/ml epinephrine) on the development of hyperalgesia, edema, and increase in temperature. The animals received bupivacaine 5 min before (BUPI PRE group, n = 20) or 60 min after (BUPI POST group, n = 20) carrageenin (1 ml/kg of 1% solution) was injected into the left hind paw. Two control groups (n = 15 in each) received saline 5 min before or 60 min after administration of carrageenin. Hyperalgesia of the injected paw was evaluated by the vocalization threshold to paw pressure, edema by measuring paw circumference with a thread, and plantar temperature with a thermocouple thermometer. All measurements were done before carrageenin injection then every 30 min thereafter for 240 min. Another series (n = 24), with the same four groups was also evaluated at 24 h. RESULTS: Local injection of bupivacaine 60 min after carrageenin partially reduced the edema and hyperalgesia. The injection of bupivacaine 5 min before carrageenin was more efficient than the delayed injection and reduced hyperalgesia, edema and the increase in temperature temporarily, but did not totally prevent their development. All groups were similar at 240 min and 24 h. CONCLUSIONS: These results show that a slight advantage of infiltration with bupivacaine before injury exists in this carrageenin model of acute inflammatory pain. However, this benefit is limited in time and bupivacaine did not have any preemptive analgesic effect. PMID- 8624008 TI - Isoform-dependent effects of halothane in human skinned striated fibers. AB - BACKGROUND: Reports of the effects of halothane on isoform contractile proteins of striated muscles are conflicting. To determine whether halothane affects cardiac and skeletal contractile proteins differently, the authors examined the effects of two doses of halothane (0.44 and 1.26 mM, equivalent to 0.75 and 2.25 vol%, respectively) on the Ca++ sensitivity and maximal force in human skinned cardiac, type I (slow twitch), and type II (fast twitch) skeletal muscle fibers. METHODS: Left ventricular muscle strips and skeletal muscle biopsy specimens were obtained from eight and ten patients undergoing cardiac and orthopedic surgery, respectively. Sarcolemma and sarcoplasmic reticulum were destroyed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus Brij 58. Ca++ sensitivity was studied by observing the isometric tension developed by skinned fibers challenged with increasing concentrations of Ca++. Muscle fiber type was determined in each skeletal fiber by the difference in strontium-induced tension measurements. RESULTS: Halothane shifted the Ca++ tension curves toward higher Ca++ concentrations and increased the Ca++ concentrations for half-maximal activation in both cardiac and type I skeletal muscle fibers (from 1.96 microM and 1.06 microM under control conditions to 2.92 microM and 1.71 microM in presence of 0.75 vol% halothane, respectively) without changing the slope of this relationship (Hill coefficient). In contrast, no significant effect was observed in type II fibers. Halothane also decreased the maximal activated tension in the three groups of fibers with a lesser effect in type II fibers. CONCLUSIONS: Halothane decreases Ca++ sensitivity and maximal force in human skinned cardiac and type I fibers at 20 degrees C. It is concluded that the negative inotropic effects of halothane depend on contractile proteins isoforms. PMID- 8624009 TI - Pentobarbital enhances cyclic adenosine monophosphate production in the brain by effects on neurons but not glia. AB - BACKGROUND: Cyclic adenosine monophosphate (cAMP) is an important regulator of neuronal excitability. The effects of barbiturates on cAMP production in intact neurons are not known. This study used cultures of cortical neurons, cultures of glia, and slices of cerebral cortex from the rat to study the effects of barbiturates on cAMP regulation in the brain. METHODS: Primary cultures of cortical neurons or glia were prepared from 17-day gestational Sprague-Dawley rat fetuses and were used after 12-16 days in culture. Cross-cut slices (300 microns) were prepared from cerebral cortex of adult rats. Cyclic AMP accumulation was determined by measuring the conversion of [3H]adenosine triphosphate (ATP) to [3H]cAMP in cells preloaded with [3H]adenine. RESULTS: Pentobarbital enhanced isoproterenol- and forskolin-stimulated, but not basal, cAMP accumulation in cultures of cerebral neurons. Cyclic AMP production was enhanced by pentobarbital in a dose-dependent fashion up to a concentration of 250 microM; This concentration of pentobarbital increased cAMP production by 40-50% relative to that in controls without pentobarbital. At 500 microM pentobarbital, the magnitude of the enhancement was less. Pentobarbital had no effect on isoproterenol-stimulated cAMP production in cultures containing only glia. Pentobarbital also enhanced isoproterenol-stimulated, but not basal, cAMP production in slices of cerebral cortex by approximately 30% at concentrations of 62.5-250 microM and by almost 100% at 500 microM. CONCLUSIONS: Pentobarbital enhances stimulated cAMP accumulation in cultured preparations from brain and fresh cortical slices. Neurons are required for this effect. Because cAMP modulates neuronal excitability, this effect of pentobarbital may be an important mechanism by which this anesthetic influences brain function. PMID- 8624010 TI - Halothane and isoflurane inhibit vasodilation due to constitutive but not inducible nitric oxide synthase. Implications for the site of anesthetic inhibition of the nitric oxide/guanylyl cyclase signaling pathway. AB - BACKGROUND: Inhalational anesthetics inhibit the nitric oxide-guanylyl cyclase signaling pathway, but the site of this inhibition is not yet clear. This study was designed to test the hypothesis that receptor activation or downstream signaling events leading to nitric oxide synthase activation are important sites for this inhibition by comparing the effect of anesthetics on vasodilation caused by the calcium-dependent constitutive endothelial nitric oxide synthase versus the calcium-independent inducible nitric oxide synthase. METHODS: Endothelium intact or -denuded rat thoracic aorta rings preincubated with or without lipopolysaccharide were mounted for isometric tension measurement, constricted with phenylephrine, then relaxed with methacholine in the presence or absence of halothane (1-3%) or isoflurane (1-3%). The cyclic guanosine 3,5-monophosphate content in the endothelium-denuded rings preincubated with or without lipopolysaccharide in the presence or absence of 3% halothane or 3% isoflurane was quantified by radioimmunoassay. The activity of partially purified inducible nitric oxide synthase from activated mouse macrophage was assayed in the presence or absence of halothane (1-4%) or isoflurane (1-5%) by the conversion of 3H-L arginine to 3H-L-citrulline. RESULTS: Halothane and isoflurane inhibited methacholine-stimulated, nitric oxide-mediated vasorelaxation in endothelium intact aortic rings. Neither halothane nor isoflurane affected the vasorelaxation caused by basal endothelial nitric oxide synthase or inducible nitric oxide synthase activity. Neither anesthetic altered the cyclic guanosine 3,5 monophosphate increase caused by inducible nitric oxide synthase in the lipopolysaccharide-treated rings. CONCLUSIONS: The results demonstrated that halothane and isoflurane inhibit only receptor/calcium-activated nitric oxide synthase action and that direct inhibition of nitric oxide synthase, soluble guanylyl cyclase, or an interaction with nitric oxide are not responsible for anesthetic inhibition of endothelium-dependent vasorelaxation. PMID- 8624011 TI - Myocardial depressant effects of sevoflurane. Mechanical and electrophysiologic actions in vitro. AB - BACKGROUND: The effects of anesthetic concentrations of sevoflurane were studied in isolated myocardial tissue to delineate the mechanisms by which cardiac function is altered. METHODS: Isometric force of isolated guinea pig ventricular papillary muscle was studied at 37 degrees C in normal and 26 mM K+ Tyrode's solution at various stimulation rates. Normal and slow action potentials were evaluated using conventional microelectrodes. Effects of sevoflurane on sarcoplasmic reticulum function in situ were also evaluated by its effect on rapid cooling contractures, which are known to activate Ca2+ release from the sarcoplasmic reticulum, and on concentrations of rat papillary muscle. Finally, Ca2+ and K+ currents of isolated guinea pig ventricular myocytes were examined using the whole-cell patch clamp technique. RESULTS: Sevoflurane equivalent to 1.4% and 2.8% depressed guinea pig myocardial contractions to approximately 85 and approximately 65% of control, respectively, although the maximum rate of force development at 2 or 3 Hz and force in rat myocardium after rest showed less depression. In the partially depolarized, beta-adrenergically stimulated myocardium, sevoflurane selectively depressed late peak force without changing early peak force, whereas it virtually abolished rapid cooling contractures. Sevoflurane did not alter the peak amplitude or maximum depolarization rate of normal and slow action potentials, but action potential duration was significantly prolonged. In isolated guinea pig myocytes at room temperature, 0.7 mM sevoflurane (equivalent to 3.4%) depressed peak Ca2+ current by approximately 25% and increased the apparent rate of inactivation. The delayed outward K+ current was markedly depressed, but the inwardly rectifying K+ current was only slightly affected by 0.35 mM sevoflurane. CONCLUSIONS: These results suggest that the direct myocardial depressant effects of sevoflurane are similar to those previously described for isoflurane. The rapid initial release of Ca2+ from the sarcoplasmic reticulum is not markedly decreased, although certain release pathway, specifically those induced by rapid cooling, appear to be depressed. Contractile depression may be partly related to the depression of Ca2+ influx through the cardiac membrane. The major electrophysiologic effect of sevoflurane seems to be a depression of the delayed outward K+ current, which appears to underlie the increased action potential duration. PMID- 8624012 TI - Concurrent spinal infusion of MK801 blocks spinal tolerance and dependence induced by chronic intrathecal morphine in the rat. AB - BACKGROUND: MK801, an N-methyl-D-aspartate receptor antagonist, has recently been reported to attenuate tolerance to, and withdrawal from morphine. This study analyzes tolerance and withdrawal in a chronic intrathecal coinfusion model of morphine and MK801. METHODS: Intrathecal catheters, attached to 7-day miniosmotic infusion pumps, were implanted in rats and infused with saline, 20 nM/h morphine, MK801 (10 and 3 nM/h) + morphine; and 10 nM/h MK801. Analgesia was measured on the hot plate daily. On the day 7, groups received 3 mg/kg intraperitoneal naloxone and six signs of withdrawal were assessed: vocalization to air motion or light touch, abnormal posture, spontaneous vocalization, escape attempts, "wet dog shakes," and ejaculation. Similar groups were tested only on days 1 and 7. Intrathecal morphine dose-response curves were obtained on day 8. A separate morphine-tolerant group received 10 nM MK801 on day 7. Rats from each group received 10 nM intrathecal morphine 1 week later. RESULTS: Coinfusion of MK801 with morphine resulted in a dose-dependent preservation of effect, and attenuated three of six signs of withdrawal. Coinfusion of MK801 (10 and 3 nM/h) prevented the reduction of potency observed with morphine alone. ED50 values (maximum percent effect, nM morphine) were: saline (16), morphine (496), MK801 (10 nM/h) + morphine (4), and 10 nM/h MK801 (0.3). Acute administration of MK801 was ineffective in restoring sensitivity to morphine. One week after cessation of infusion, there was no significant difference between groups. CONCLUSIONS: Chronic spinal MK801 attenuates tolerance to, and withdrawal from spinal morphine in a dose-dependent fashion, supporting the hypothesis that N-methyl-D-aspartate receptor activity plays a role in the reorganization of spinal function produced by chronic opioid receptor activation. Chronic intrathecal MK801 appears to sensitize the spinal cord to intrathecal morphine. PMID- 8624013 TI - Volatile anesthetics selectively inhibit the Ca(2+)-transporting ATPase in neuronal and erythrocyte plasma membranes. AB - BACKGROUND: The activity of the plasma membrane Ca(2+)-transporting adenosine triphosphatase (PMCA) is inhibited by volatile anesthetics at clinical concentrations. The goal of the current study was to determine whether the inhibition is selective as compared to other adenosine triphosphatases (ATPases) and another group of general anesthetics, barbiturates. In addition, the authors determined whether the response to anesthetics of the enzymes in neuronal membranes is similar to that in erythrocyte membranes. METHODS: The effects of halothane, isoflurane, and sodium pentobarbital on four different ATPase activities were studied at 37 degrees C in two distinct plasma membrane preparations, human red blood cells and synaptosomal membranes from rat cerebellum. RESULTS: Inhibition patterns of the PMCA by halothane and isoflurane at anesthetic concentrations were vary similar in red blood cells and synaptosomal membranes. The half-maximal inhibition (I50) occurred at 0.25-0.30 mM halothane and 0.30-0.32 mM isoflurane. The PMCA in both membranes was significantly more sensitive to the inhibitory action of volatile anesthetics (I50 = 0.75-1.15 minimum alveolar concentration) than were other ATPases, such as the Na+,K+-ATPase (I50 approximately 3 minimum alveolar concentration) or Mg(2+) ATPase (I50 > or = 5 minimum alveolar concentration). In contrast, sodium pentobarbital inhibited the PMCA in both membranes only at approximately 100-200 fold above its anesthetic concentrations. The other ATPases were inhibited at similar pentobarbital concentrations (I50 = 11-22 mM). CONCLUSIONS: The findings demonstrate analogous response of the PMCA of neuronal and erythrocyte cells to two groups of general anesthetics. The PMCA activity is selectively inhibited by volatile anesthetics at their clinical concentrations. The enzyme in vivo may then be a pharmacologic target for volatile anesthetics but not for barbiturates. PMID- 8624014 TI - Dose-dependent increases in the renal sympathetic nerve activity during rapid increase in isoflurane concentration in intact, lower airway-deafferented, and baroreceptor-deafferented rabbits. AB - BACKGROUND: The inhalation of high concentrations of isoflurane has been reported to increase the heart rate and the concentration of serum catecholamines. Although the precise mechanisms for the sympathetic activation of isoflurane have yet to be clearly elucidated, they are considered to possibly originate from the stimulation of airway sensory afferents, the baroreceptor reflex, or the direct stimulation of the central nervous system. To determine how these three mechanisms contribute to sympathetic augmentation, the effects of lower airway deafferentation and baroreceptor deafferentation on the isoflurane-induced changes in the renal sympathetic nerve activity (RSNA) in tracheally intubated rabbits were examined. METHODS: Twenty rabbits were given basal anesthesia. After tracheotomy and during mechanical ventilation, the changes in the heart rate, mean arterial pressure, and RSNA in response to random exposures to 1%, 2%, 3%, and 4% isoflurane were examined. The animals were assigned to one of three groups; 1, the intact group (n = 6); 2, the baroreceptor-deafferented group (n = 9), in which the sinoaortic plus vagal nerves were cut; and 3, the lower airway deafferented group (n = 5), which underwent a bilateral vagotomy. The exposure to isoflurane was for 10 min in group 1 and 5 min in groups 2 and 3. At least 1 h was allowed for the recovery interval between exposures to isoflurane. RESULTS: The inhalation of isoflurane caused dose-dependent increases in RSNA in all three groups. RSNA during high concentrations of isoflurane began to increase at 1 min, reaching the maximum at 4 or 5 min in group 1 (2.8- and 3.8-fold at 3% and 4% isoflurane, respectively) and group 3 (2.8- and 4.5-fold at 3% and 4% isoflurane, respectively), but it reached the peak at 2 or 3 min in group 2 (1.7- and 2.4 fold at 3% and 4% isoflurane, respectively) after the initiation of inhalation, in association with early slight increases followed by decreases of mean arterial pressure in groups 1 and 2 but only gradual decreases of mean arterial pressure in group 3. The increases in RSNA in group 3 were similar to group 1, however, those in group 2 were significantly attenuated compared with group 1. CONCLUSIONS: The inhalation of isoflurane caused an increase of RSNA in intact, baroreceptor-deafferented, and lower airway-deafferented rabbits. The extent of the increases in RSNA was greater in intact and lower airway-deafferented rabbits than in baroreceptor-deafferented rabbits. Therefore, it is suggested that isoflurane may increase the efferent sympathetic nerve activity via the direct stimulation of the central nervous system and via the arterial baroreceptor reflex reflecting the reduction in arterial blood pressure. The stimulation of the vagally innervated airway may not contribute to the increase in the sympathetic nerve activity by isoflurane. PMID- 8624015 TI - Antagonism of the antinocifensive action of halothane by intrathecal administration of GABAA receptor antagonists. AB - BACKGROUND: The hind brain and the spinal cord, regions that contain high concentrations of gamma-aminobutyric acid (GABA) and GABA receptors, have been implicated as sites of action of inhalational anesthetics. Previous studies have established that general anesthetics potentiate the effects of gamma-aminobutyric acid at the GABAA receptor. It was therefore hypothesized that the suppression of nocifensive movements during anesthesia is due to an enhancement of GABAA receptor-mediated transmission within the spinal cord. METHODS: Rats in which an intrathecal catheter had been implanted 1 week earlier were anesthetized with halothane. Core temperature was maintained at a steady level. After MAC determination, the concentration of halothane was adjusted to that at which the rats last moved in response to tail clamping. Saline, a GABAA, a GABAB, or glycine receptor antagonist was then injected intrathecally. The latency to move in response to application of the tail clamp was redetermined 5 min later, after which the halothane concentration was increased by 0.2%. Response latencies to application of the noxious stimulus were measured at 7-min intervals during the subsequent 35 min. To determine whether these antagonists altered baseline response latencies by themselves, another experiment was conducted in which the concentration of halothane was not increased after intrathecal administration of GABAA receptor antagonists. RESULTS: Intrathecal administration of the GABAA receptor antagonists bicuculline (0.3 micrograms) or picrotoxin (0.3, 1.0 micrograms) antagonized the suppression of nocifensive movement produced by the small increase in halothane concentration. In contrast, the antinocifensive effect of the increase in halothane concentration was not attenuated by the GABAB receptor antagonist CGP 35348 or the glycine receptor antagonist strychnine. By themselves, the GABAA receptor antagonists did not alter response latency in rats anesthetized with sub-MAC concentrations of halothane. CONCLUSIONS: Intrathecal administration of bicuculline or picrotoxin, at doses that do not change the latency to pinch-evoked movement when administered alone, antagonized the suppression of noxious-evoked movement produced by halothane concentrations equal to or greater than MAC. These results suggest that enhancement of GABAA receptor mediated transmission within the spinal cord contributes to halothane's ability to suppress nocifensive movements. PMID- 8624016 TI - Involvement of glutamate receptors in strychnine- and bicuculline-induced allodynia in conscious mice. AB - BACKGROUND: Glycine and gamma-aminobutyric acid (GABA) are inhibitory neurotransmitters that appear to be important in sensory processing in the spinal dorsal horn. Intrathecal administration of strychnine (strychnine-sensitive glycine receptor antagonist) or bicuculline (GABAA antagonist) was reported to induce allodynia. Although the strychnine-induced allodynia was shown to be mediated through the N-methyl-D-aspartate (NMDA)-type glutamate receptor, it is not clear whether the bicuculline-evoked-allodynia is mediated through the glutamate receptor system or how different the allodynia induced by strychnine and bicuculline are. METHODS: Male ddY mice weighing 20 +/- 2 g were used in this study. A 27-G stainless-steel needle attached to a microsyringe was inserted between the L5 and L6 vertebrae by a slight modification of the method of Hylden and Wilcox. Drugs in vehicle were injected slowly into the subarachnoid space to conscious mice at 22 +/- 2 degrees C. The volume of the intrathecal injection was 5 microliters. Studies on allodynia were carried out essentially according to the method of Yaksh and Harty. RESULTS: The intrathecal administration of strychnine or bicuculline in conscious mice resulted in allodynia elicited by nonnoxious brushing of the flanks. The maximum allodynia induced by strychnine was observed 5 min after intrathecal injection, but that induced by bicuculline was observed 10 min after intrathecal injection. Both responses gradually decreased over the experimental period of 50 min. The allodynia induced by strychnine was dose dependently relieved by NMDA receptor antagonists (D-AP5, ketamine, and 7-C1 KYNA) and non-NMDA receptor antagonists (GAMS and CNQX) but not by metabotropic receptor antagonists (L-AP3 and L-AP4). On the other hand, allodynia induced by bicuculline was dose-dependently relieved by GAMS, L-AP3, and L-AP4, but not by D AP5, ketamine, 7-C1-KYNA, and CNQX. Whereas the strychnine-evoked allodynia was dose-dependently relieved by the nitric oxide synthase inhibitor N(omega)-nitro-L arginine methyl ester (L-NAME) and the soluble guanylate cyclase inhibitor methylene blue, the bicuculline-induced one was dose-dependently relieved by methylene blue but not by L-NAME. CONCLUSIONS: These results demonstrate that both strychnine- and bicuculline-evoked allodynia were mediated through pathways that include the glutamate receptor and nitric oxide systems but in a different manner. the current study suggests that GABA and glycine may modulate responses to an innocuous tactile stimulus as inhibitory neurotransmitters at presynaptic and postsynaptic sites in the spinal cord, respectively. PMID- 8624017 TI - Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. AB - BACKGROUND: Although voltage-dependent sodium channels have been proposed as possible molecular sites of anesthetic action, they generally are considered too insensitive to be likely molecular targets. However, most previous molecular studies have used peripheral sodium channels as models. To examine the interactions of volatile anesthetics with mammalian central nervous system voltage-gated sodium channels, rat brain IIA sodium channels were expressed in a stably transfected Chinese hamster ovary cell line, and their modification by volatile anesthetics was examined. METHODS: Sodium currents were measured using whole cell patch clamp recordings. Test solutions were equilibrated with the test anesthetics and perfused externally on the cells. Anesthetic concentrations in the perfusion solution were determined by gas chromatography. RESULTS: All anesthetics significantly suppressed sodium currents at clinical concentrations. This suppression occurred through at least two mechanisms: (1) a potential independent suppression of resting or open sodium channels, and (2) a hyperpolarizing shift in the voltage-dependence of channel inactivation resulting in a potential-dependent suppression of sodium currents. The voltage-dependent interaction results in IC50 values for anesthetic suppression of sodium channels that are close to clinical concentrations at potentials near the resting membrane potential. PMID- 8624018 TI - Endobronchial inflammatory polyp after thoracoabdominal aneurysm surgery: a late complication of use of a double-lumen endobronchial tube. PMID- 8624019 TI - Heparinase and thromboelastography in liver transplantation for a patient with von Willebrand's disease. PMID- 8624020 TI - Cervical dural puncture in a neonate: a rare complication of internal jugular venipuncture. PMID- 8624021 TI - Practice guidelines for cancer pain management. A report by the American Society of Anesthesiologists Task Force on Pain Management, Cancer Pain Section. PMID- 8624022 TI - Forget the costs: use what is best. PMID- 8624023 TI - Preoperative pregnancy testing in ambulatory surgery. PMID- 8624024 TI - Preoperative pregnancy testing in ambulatory surgery. PMID- 8624025 TI - Preoperative pregnancy testing in ambulatory surgery. PMID- 8624026 TI - Discrepancy between thromboelastography and prothrombin time. PMID- 8624027 TI - Double-lumen laryngeal mask airway. PMID- 8624028 TI - You found it where?! PMID- 8624029 TI - Difficulty using a laryngeal mask airway in a patient with lingual tonsil hyperplasia. PMID- 8624030 TI - Postoperative management strategies may obviate the need for most preoperative cardiac testing. PMID- 8624031 TI - Genetic markers linked to quantitative traits in poultry. AB - This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, backcross (BC1) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F2 progeny were produced in phase II from the phase I BC1 population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P < or = 0.05) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL. PMID- 8624032 TI - Efficient resolution of parentage in dogs by amplification of microsatellites. AB - Parentage control has been performed for 15 litters from 12 different dog breeds by amplification of microsatellites. As it was possible to include all putative parents in all cases, they were solved by exclusion. Discrimination between parents/non-parents was made after genotyping of 6-9 microsatellite loci. In 12 of the cases all but one of the alleged fathers were excluded while in three cases it was unambiguously shown that superfecundation had taken place. Furthermore, one inclusion case concerning disputed maternity has been investigated. Maternity indices were calculated for 12 loci and probability of maternity was estimated to be 99.99%. These results testify that microsatellites can be applied very efficiently for resolution of parentage in dogs. PMID- 8624033 TI - Variation in the control region sequence of the sheep mitochondrial genome. AB - The DNA sequences of the control region of the mitochondrial genome of fifty unrelated sheep were determined in order to ascertain the extent and distribution of its variability. A consensus sequence was derived, and 1081 differences from it were observed amongst the fifty animals. Some constant groups of differences were observed that were held in common by a number of animals, which thus fell into two main groups, although neither group was typical of any of the breeds sampled. The consensus sequence also allowed comparison between the control region sequences of sheep and other mammals. The sequence contains four tandem repeats of a 75 base-pair motif that accounts for the difference in its size from the cattle control region, to which it is otherwise very similar. Comparison with the cattle sequence allowed the determination of the homologues of various functionally important sites. The homologues of the transcription promoters, the origin of replication and the central conserved sequence block were all identified by this method. PMID- 8624034 TI - Conservation of the RD-BF-C2 organization in the pig MHC class-III region: mapping and cloning of the pig RD gene. AB - The RD gene, named after the arginine (R) and aspartic acid (D) repeat in the central part of its protein, was initially mapped in the mouse H-2S subregion between C4 and BF. It was later mapped in the same position in the human MHC and here we show it is also conserved in the pig MHC class III region, close to the complement BF gene. A pig RD genomic clone was isolated from a lambda-phage library. Hybridizations on genomic DNA separated with pulsed field gel electrophoresis identified common 220 kb NruI, 130 kb EagI and 200 kb MluI bands for RD, BF and C2. The RD gene has also a 17 kb Kp nI and 11 kb SacI fragment in common with BF but not with C2. The close linkage of the RD and BF genes was further established by hybridization of BF to a genomic lambda-phage clone also containing the RD gene. This genomic RD clone overlaps with a lambda-phage clone previously isolated and containing the complete BF gene and the 3' part of C2. The distance between RD and BF is about 6 kb. The junction between the two complement genes BF and C2 was sequenced and the BF 5' promoter region, overlapping the 3' noncoding region of C2, was compared with that of the human BF promoter. The overall homology was about 80% and all but one identified promoter elements were found in the same position in both genes. The results obtained demonstrate the RD-BF-C2 organization is strongly conserved between human, mouse and pig. No polymorphisms were detected in either the RD gene or in the BF promoter region using polymerase chain reaction and restriction fragment polymorphism analysis. PMID- 8624035 TI - Isolation and characterization of 45 polymorphic microsatellites from the bovine genome. AB - A small-insert bovine genomic library was constructed in pBluescript II SK(+) and enriched for microsatellites by selective rescue of single-stranded pBluescript DNA carrying (CA)n/(TG)n tandem repeats. Approximately 50% of the clones in the enriched library contained (CA)n repeats or CA-rich sequences. Sequencing of clones selected for (CA)n repeats resulted in the identification and characterization of 45 (CA)n polymorphic microsatellites. Genotyping in 9 large paternal half-sib families indicated that 40 of these microsatellite markers exhibit autosomal Mendelian inheritance. The numbers of alleles range from 2 to 18, with an average of 6.3 per locus. The polymorphic microsatellite markers we have identified and characterized will contribute to the construction of a high resolution linkage map of bovine genome. PMID- 8624036 TI - PCR amplification of a polymorphic minisatellite VNTR locus in whiting (Merlangius merlangus L.). AB - An approach has been developed for the screening of allelic variation of minisatellite DNA loci that substantially reduces the time and hazards involved. Primers were designed for a minisatellite region isolated from a gadoid fish species (Merlangius merlangus L.), enabling amplification by polymerase chain reaction, so that differences in the number of minisatellite repeat units (allelic variability) were detectable by ethidium bromide fluorescence (over UV light) following separation by agarose gel electrophoresis. This amplifiable minisatellite variable number tandem repeat region, the first non-primate marker of its kind can be used successfully with DNA extracted by a rapid Chelex protocol. From a sample of 97 individuals, 24 alleles were resolved (750-2200 kb) and heterozygosity was estimated at 0.94. PMID- 8624037 TI - Gene frequencies of DRB3.2 locus of Argentine Creole cattle. AB - Gene and genotype frequencies of BoLA-DRB3 were studied in seven herds of Argentine Creole cattle. Twenty-one out of thirty previously identified alleles were detected in this breed. The F statistics showed high degree of variability among the studied subpopulations suggesting that subdivision and genetic drift, rather than inbreeding, have been the major factors acting on the observed interpopulation variability. The observed high degree of genetic variation in Argentine Creole cattle could be crucial for the long-term survival of this population. Maintenance of such polymorphism, as a genetic resource, could be an important issue that will demand attention in future breeding programmes in species under high selective pressures. PMID- 8624038 TI - Ovine dinucleotide repeat polymorphism at the McM218, McM150 and McM138 loci. PMID- 8624039 TI - Polymorphic Atlantic salmon, Salmo salar L., microsatellites at the SSOSL438, SSOSL439 and SSOSL444 loci. PMID- 8624040 TI - A highly polymorphic bovine dinucleotide repeat D19S4 (IOBT 34) at chromosome 19q21. PMID- 8624041 TI - A highly polymorphic bovine dinucleotide repeat D3S40 (IOBT 250) derived from a chimeric cosmid clone. PMID- 8624042 TI - A polymorphic (GAAA)n microsatellite in a canine Wilms tumor 1 (WT1) gene intron. PMID- 8624043 TI - Two polymorphic microsatellite markers close to the ovine NRAMP gene. PMID- 8624044 TI - FBN3 (D6S24): a bovine microsatellite derived from cosmid cIOBT475 at chromosome 6q12-14. PMID- 8624046 TI - MspI RFLP at the CETP locus in baboons. PMID- 8624045 TI - Seven bovine dinucleotide repeat polymorphisms: RM042, RM128, RM134, RM154, RM162, RM181, RM509. PMID- 8624047 TI - TaqI RFLP at the LIPC locus in baboons. PMID- 8624048 TI - The equine MSH-R TaqI RFLP is not informative for hair colour in Arabian horses. PMID- 8624049 TI - Single-parent segregant pools for allocation of markers to a specified chromosomal region in outcrossing species. AB - Bulked co-segregant analysis is a method of rapidly allocating unmapped genetic markers to a specific chromosomal region. Although originally developed for utilization in populations derived from crosses between fully inbred lines, it has been proposed that co-segregant pools could also serve the same purpose in outbreeding populations, if individuals from only a single large family are pooled. Large, fully mapped, single-sire backcross and half-sib families are presently available as part of the international chicken and bovine reference family panels respectively. In this study, power and tests of significance for single-parent co-segregant analysis are derived for full-sib, single-parent back cross and single-parent half-sib families, as a function of proportion of recombination between index marker and linked marker, pro-portion of single parent alleles among the mates, number of individuals in each segregant pool and technical error variance. Power was found to be greater than 0.80 for many reasonable parameter combinations. The method is illustrated using microsatellite markers and a large single-sire bovine family, part of the international bovine reference family panel. PMID- 8624050 TI - Does the brain know when the heart is ischemic? PMID- 8624051 TI - Changing focus on unexplained esophageal chest pain. PMID- 8624052 TI - Outpatient clinic. PMID- 8624053 TI - Identification of K-ras mutations in pancreatic juice. PMID- 8624054 TI - Misoprostol and nonsteroidal anti-inflammatory drugs. PMID- 8624055 TI - Misoprostol and nonsteroidal anti-inflammatory drugs. PMID- 8624056 TI - Misoprostol and nonsteroidal anti-inflammatory drugs. PMID- 8624057 TI - Corticosteroid-induced pancreatitis. PMID- 8624058 TI - Fact or fiction? PMID- 8624059 TI - Advance medical planning. PMID- 8624060 TI - Advance medical planning. PMID- 8624061 TI - Silent ischemia as a central problem: regional brain activation compared in silent and painful myocardial ischemia. AB - OBJECTIVE: To test whether the silence of painless myocardial ischemia is caused by abnormal handling by the central nervous system of afferent messages from the heart. DESIGN: Nonrandomized study. SETTING: A tertiary referral center (postgraduate medical school). PATIENTS: 2 matched groups of nondiabetic patients with coronary artery disease. Group A consisted of nine patients with reproducible stress-induced angina; group B consisted of nine patients with reproducible stress-induced myocardial ischemia but no angina. INTERVENTIONS: Intravenous placebo infusion and low-dose (5 and 10 micrograms/ kg per minute) and high-dose (20 to 35 micrograms/kg per minute) dobutamine infusions. MEASUREMENTS: Positron emission tomography was used to measure regional cerebral blood flow changes as an index of neuronal activation during painful and silent myocardial ischemia induced by intravenous dobutamine. RESULTS: Regional cerebral blood flow changes during myocardial ischemia were compared with those during baseline conditions and during placebo infusion. During myocardial ischemia, regional cerebral blood flow increased bilaterally in the thalami and prefrontal, basal frontal, and ventral cingulate corticles in patients in group A. Both thalami were activated in group B, but cortical activation was limited to the right frontal region. A formal comparison of groups A and B showed significant differences (P < 0.01) in activation of the basal frontal cortex, ventral cingulate cortex, and left temporal pole. In both groups, thalamic regional cerebral blood flow remained increased after the symptoms and signs of ischemia had ceased. CONCLUSIONS: Bilateral activation of the thalamus can be shown in both angina and silent ischemia; thus, peripheral nerve dysfunction cannot completely explain silent ischemia. Frontal cortical activation appears to be necessary for the sensation of pain. Abnormal central processing of afferent pain messages from the heart may play a determining role in silent myocardial ischemia. PMID- 8624062 TI - Unexplained chest pain: the hypersensitive, hyperreactive, and poorly compliant esophagus. AB - OBJECTIVE: To determine whether neuromuscular dysfunction of the esophagus causes chest pain in patients in whom no disease is found on cardiac work-up, upper gastrointestinal endoscopy, esophageal manometry, and 24-hour pH studies. DESIGN: Prospective study. SETTING: Tertiary referral center. PATIENTS: 24 consecutive patients and 12 healthy controls. MEASUREMENTS: A new technique, impedance planimetry, was used to measure the sensory, motor, and biomechanical properties of the human esophagus. The impendance planimeter, which consists of a probe with four ring electrodes, three pressure sensors, and a balloon, simultaneously measures intraluminal pressure and cross-sectional areas. This allows calculation of the biomechanical variables of the esophageal wall. RESULTS: Stepwise balloon distentions from 5 to 50 cm H2O induced a first sensation at a mean pressure (+/- SD) of 15 +/- 9 cm H2O in patients and 30 +/- 11 cm H2O in controls (P < 0.001). Moderate discomfort and pain were reported by 20 of 24 patients (83%) at 26 +/- 9 cm H2O and at 36 +/- 9 cm H2O, respectively, but by none of the controls (P < 0.001). Typical chest pain was reproduced in 20 of 24 patients (83%). In patients, the reactivity of the esophagus to balloon distention was greater (P = 0.01), the pressure elastic modulus was higher (P = 0.02), and the tension-strain association showed that the esophageal wall was less distensible (P = 0.02). Distention excited tertiary contractions and secondary peristalsis at a lower threshold of pressure (P = 0.05) and with a higher motility index in patients than in controls (P = 0.04). CONCLUSION: In patients with chest pain and normal cardiac and esophageal evaluations, impedance planimetry of the esophagus reproduces pain and is associated with a 50% lower sensory threshold for pain, a 50% lower threshold for reactive contractions, and reduced esophageal compliance. PMID- 8624063 TI - Diagnostic value of esophageal studies in patients with angina-like chest pain and normal coronary angiograms. AB - OBJECTIVE: To study 1) variations in esophageal motility and pH values and 2) electrocardiographic ST-segment changes in patients with angina-like chest pain but normal coronary angiograms. DESIGN: Cross-sectional study. SETTING: Tertiary cardiologic referral center. PATIENTS: 63 consecutive patients referred to the study center over a 3-year period and 22 healthy controls. Patients were grouped according to the results of exercise electrocardiography: normal response to exercise (n = 28) and ischemic response to exercise (n = 35). MEASUREMENTS: 1) 24 hour three-channel esophageal manometry and two-channel pH monitoring, 2) provocation testing with intravenous edrophonium chloride and esophageal acid perfusion, and 3) Holter electrocardiography conducted during the first two tests. In conventional time-weighted analyses, values during periods of pain and the 2 minutes before pain developed were compared with baseline values. RESULTS: Regardless of the outcome of exercise testing, no differences were seen in 24 hour esophageal variables between patients and controls. Forty-six patients had a total of 248 spontaneous episodes of chest pain. Only minor differences were seen between baseline and the prepain and pain periods. Edrophonium chloride provocation and acid perfusion caused chest pain in 9 patients (14%) and 10 patients (16%), respectively. Esophageal monitoring variables did not differ between patients with a positive response to one or both provocation tests (n = 16 [25%]) and controls and did not change between baseline and the prepain or pain perioids. Forty-eight ST-segment depressions were recorded on Holter monitoring in 16 patients (25%), but these depressions were associated with one prepain period and four pain periods. No ST-segment changes were seen during esophageal provocation testing. CONCLUSION: The rationale for routine esophageal investigations in this patient group is questionable. PMID- 8624064 TI - The risk for and severity of bleeding complications in elderly patients treated with warfarin. The National Consortium of Anticoagulation Clinics. AB - OBJECTIVE: To determine whether increasing age is associated with an increased risk for bleeding during warfarin treatment. DESIGN: Combined retrospective and prospective cohort studies. SETTING: 6 anticoagulation clinics. PATIENTS: 2376 patients receiving warfarin for various indications. MEASUREMENTS: Bleeding events categorized as minor (resulting in no costs or consequences), serious (requiring testing or treatment), life-threatening, or fatal. RESULTS: 812 first bleeding events (4 fatal, 33 life-threatening, 222 serious, and 553 minor) occurred during 3702 patient-years. Age was inversely related to the mean warfarin dose and dose-adjusted prothrombin time ratio. The unadjusted incidence of minor bleeding complications did not vary according to age group: 18.0 per 100 patient-years for patients younger than 50 years of age, 21.5 for patients 50 to 59 years of age, 24.0 for patients 60 to 69 years of age; 23.5 for patients 70 to 79 years of age, and 16.3 for patient 80 years of age and older. The unadjusted incidence of serious bleeding complications also did not vary according to age group: 9.3 per 100 patient-years for patients younger than 50 years of age, 7.1 for patients 50 to 59 years of age, 6.6 for patients 60 to 69 years of age, 5.1 for patients 70 to 79 years of age, and 4.4 for patients 80 years of age and older. The unadjusted incidence of life-threatening or fatal complications combined was significantly higher among the oldest patients: 0.75 per 100 patient years for patients younger than 50 years of age, 0.97 for patients 50 to 59 years of age, 1.10 for patients 60 to 69 years of age, 0.68 for patients 70 to 79 years of age, and 3.38 for patients 80 years of age and older. Patients 80 years of age and older had a relative risk of 4.5 (95% CI, 1.3 to 15.6) compared with patients younger than 50 years of age. After adjustment for the intensity of anticoagulation therapy and the deviation in the prothrombin time ratio using Cox and Poisson regression, age was not generally associated with the occurrence of bleeding; relative risk estimates ranged from 0.99 to 1.03 per year of age (lower bound 95% CI, 0.97 to 1.01; upper-bound 95% CI, 1.00 to 1.09). The single exception was life-threatening and fatal complications in patients 80 years of age or older (relative risk, 4.6 [CI, 1.2 to 18.1]). CONCLUSIONS: Age did not appear to be an important determinant of risk for bleeding in patients receiving warfarin, with the possible exception of age 80 years or older. The intensity of anticoagulation therapy and the deviation in the prothrombin time ratio were much stronger predictors of risk for bleeding. PMID- 8624065 TI - Heart transplantation is associated with an increased risk for pancreaticobiliary disease. AB - OBJECTIVE: To determine the risk factors for and the incidence, morbidity, mortality, and outcome of pancreaticobiliary disease in patients who have had orthotopic heart transplantation. DESIGN: Retrospective case-control analysis. SETTING: University hospital-based heart transplantation center. PATIENTS: 20 case-patients with pancreaticobiliary disease and 40 controls; all patients received heart transplants between 1985 and 1994. Controls were matched to case patients by time of transplantation and length of survival. MEASUREMENTS: Charts were reviewed and data were extracted using a structured data abstraction protocol. Risk factors assessed before transplantation were cause of heart disease, history of diabetes, reproductive history, and sex. Risk factors assessed at presentation of pancreaticobiliary disease were weight change after transplantation, alcohol use, use of medications, recent total parenteral nutrition, age at symptom onset, recent rejection episode, cyclosporine level, complete blood count, time between transplantation and onset of symptoms, body mass index, calcium level, liver function test results before and at symptom onset, and lipid profile. RESULTS: Pancreaticobiliary disease occurred in 20 of 255 transplant recipients (7.8%). The incidence of disease in these patients within 1 year after transplantation was 3.9% compared with an expected rate of 0.2% per year (P < 0.01). A decreased serum calcium level was the only risk factor found to differ significantly between the two groups (mean +/- SD, 2.19 +/ 0.17 mmol/L in case-patients and 2.31 +/- 0.14 mmol/L in controls; P = 0.005). The duration of hospitalization for the treatment of pancreaticobiliary disease was longer for patients who received transplants than for patients who did not receive transplants and were treated at Temple University Hospital during a similar period (17.1 days compared with 7.2 days; P < 0.001). However, the outcome was excellent in most patients. CONCLUSIONS: Pancreaticobiliary disease occurs 17.4 times (95% CI, 9.2 to 32.7 times) more frequently in patients receiving transplants than in the general population. It is a seldom recognized but apparently common complication of orthotopic heart transplantation that results in substantial morbidity and health care resource use. Further study is needed to ascertain why this condition occurs and how the risk for developing it can be reduced. PMID- 8624066 TI - Viral dynamics of HIV: implications for drug development and therapeutic strategies. AB - The ability to quantitate human immunodeficiency virus (HIV) in blood and tissues from patients at all stages of disease has provided new insights into the pathogenesis of HIV disease. There is a dynamic equilibrium between HIV production and clearance even during the period of clinical latency, which may permit resistant virus to emerge with the imposition of drug pressure. Disruption of the equilibrium with effective drugs reduces circulating levels of HIV within 1 week, thus allowing the rapid assessment of new candidate drugs. To maximize the magnitude and durability of HIV RNA suppression, therapeutic strategies must be implemented that are effective against high levels of rapidly replicating virus that consist of many genetic variants. PMID- 8624067 TI - Match and mismatch: identifying the neuronal determinants of pain. AB - Despite the increased intensity and sophistication of research on pain mechanisms in the past three decades, serious uncertainties remain about the neuronal origin of pain, especially in painful clinical conditions. Although a positive correlation between nociceptive afferent activity and the subjective perception of pain has been seen under controlled experimental conditions, important mismatches point to the critical importance of central nervous system processes as determinants of pain. Multiple peripheral, segmental, and supraspinal neuronal activities control nociceptive processing at all levels of the neuraxis. Three studies in this issue highlight the problem of identifying the neuronal determinants of pain by addressing contrasting mismatches: angina-like chest pain without an obvious cause and a potential source of angina (myocardial ischemia) without pain. The results of these studies suggest that selective visceral hyperalgesia and hypoalgesia of peripheral or central origin may be present without other clinical evidence for neurologic abnormality. Complex mechanisms interacting at several levels of the nervous system appear to be involved. PMID- 8624068 TI - Survivor treatment selection bias in observational studies: examples from the AIDS literature. AB - Unlike patients in a randomized, clinical trial, patients in an observational study choose if and when to begin treatment. Patients who live longer have more opportunities to select treatment; those who die earlier may be untreated by default. These facts are the essence of an often overlooked bias, termed "survivor treatment selection bias," which can erroneously lead to the conclusion that an ineffective treatment prolongs survival. Unfortunately, misanalysis of survivor treatment selection bias has been prevalent in the recent literature on the acquired immunodeficiency syndrome. Approaches to mitigating this bias involve complex statistical models. At a minimum, initiation of therapy should be treated as a time-dependent covariate in a proportional hazards model. Investigators and readers should be on the alert for survivor treatment selection bias and should be cautious when interpreting the results of observational treatment studies. PMID- 8624069 TI - [Laboratory biosafety manual. World Health Organization]. PMID- 8624070 TI - In vivo structural studies of the hippocampus in normal aging and in incipient Alzheimer's disease. AB - Population trends indicate that in the near future the size of the elderly population will increase. This will result in a large increment in the numbers of persons suffering mild to severe levels of cognitive impairment. While considerable efforts continue to be made to explain brain changes associated with Alzheimer disease (AD), little is known of the brain changes in aging without dementia or so-called normal aging. Pathologic studies suggest that the medial temporal lobe is informative in the examination of the early brain changes related to AD. However, pathologic studies only offer a single observation and considerable uncertainty exists regarding the likelihood of progression of disease and the development of dementia. Several structural neuroimaging studies have recently investigated this anatomy and recent reports are encouraging for a medial temporal lobe based diagnosis for age-related cognitive impairments. We will present our findings on the MRI anatomy of the hippocampal formation as well as data bearing on the use of hippocampal formation imaging in the diagnosis of AD and as a predictive marker for future dementia. Our findings suggest an anatomically specific relationship between hippocampal volume and secondary memory performance. Because these observations apply to nondemented and normal elderly subjects, we are encouraged that the anatomy of age-related cognitive impairments can be reliably recognized and possibly put to use in therapeutic studies. PMID- 8624071 TI - The Neurobiology of Alzheimer's Disease. Proceedings of a meeting. Zurich, Switzerland, February 17-19, 1995. PMID- 8624072 TI - Regulation of tau phosphorylation in Alzheimer's disease. AB - Alzheimer's disease (AD) is a heterogeneous dementing disorder of the elderly that is characterized by progressive cognitive impairments and the accumulation of abundant amyloid or senile plaques (SPs) and neurofibrillary tangles (NFTs) as well as the massive loss of neurons in the AD brain. Indeed, a secure diagnosis of AD in patients with a chronic progressive dementia requires evidence of numerous SPs and NFTs in the postmortem brain. Although the deposition of fibrillar amyloid or A beta-peptides in extracellular plaques and the accumulation of tau-rich intraneuronal NFTs are not restricted exclusively to AD, there is a close correlation between the burden of tau-rich neurofibrillary lesions in selected telencephalic regions of the brain and the dementia in AD. Since the formation of neurofibrillary lesions from hyperphosphorylated tau proteins may compromise the function and viability of neurons in the AD brain, this review summarizes recent insights into mechanisms that regulate the phosphorylation state of tau in AD. PMID- 8624073 TI - Cellular signaling pathways and cytoskeletal organization. AB - We describe a possible mechanism by which the amyloid precursor protein (APP) may be linked to the intraneuronal pathology of Alzheimer's disease (AD). Extracellular proteolytic products of APP may directly or indirectly transduce signals to cells under normal as well as pathological conditions. This activity appears to reside in the cysteine-rich amino terminus of APP. How APP or its metabolic products may affect the phosphorylation of tau is considered. PMID- 8624074 TI - Tau protein and the neurofibrillary pathology of Alzheimer's disease. AB - Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major component. PHFs are composed of the microtubule-associated protein tau in a hyperphosphorylated state. PHF-tau is hyperphosphorylated on all six adult brain isoforms. As a consequence, tau is unable to bind to microtubules and is believed to self-assemble into the PHF. Several candidate protein kinases and protein phosphatases have been identified through in vitro experiments. PMID- 8624075 TI - Molecular mechanism of Alzheimer's neurofibrillary degeneration and therapeutic intervention. AB - Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer's disease (AD) and the abnormal tau is the major protein subunit of paired helical filaments (PHF). The abnormal phosphorylation of tau probably precedes its polymerization into PHF. The abnormal tau does not bind to tubulin, but competes with tubulin in binding to normal tau and thereby inhibits the assembly of microtubules in the affected neurons. The abnormal tau can be dephosphorylated enzymatically and by this way its microtubule assembly promoting activity can be restored. The activities of protein phosphatases might be decreased in the affected neurons in AD brain, allowing the abnormal hyperphosphorylation of tau. Neurofibrillary degeneration can probably be inhibited by increasing the activities of protein phosphatases in the brain of patients with Alzheimer's disease. PMID- 8624076 TI - Apolipoprotein E. Structure, function, and possible roles in Alzheimer's disease. AB - Apolipoprotein (apo) E is associated with the two characteristic neuropathologic lesions of Alzheimer's disease--extracellular neuritic plaques representing deposits of amyloid beta (A beta) peptide and intracellular neurofibrillary tangles representing filaments of a microtubule-associated protein called tau. Incubation of the apoE4 isoform with the A beta peptide in vitro results in the formation of a dense, stable network of very long monofibrils, while incubation of apoE3 with the A beta peptide results in the formation of a less dense, less stable network. The more complex nature of the plaques formed with the A beta peptide in the presence of apoE4 in vivo may impair the normal clearance process and enhance plaque formation. Alternatively or additionally, apoE may alter the cytoskeletal structure and function and, under certain conditions, may promote the formation of the neurofibrillary tangles. Our studies have demonstrated that apoE3 and apoE4 exert differential effects on neuronal growth (i.e., neurite extension and branching) in vitro. When combined with a source of lipid, apoE3 stimulated neurite extension in peripheral nervous system neurons (dorsal root ganglia), whereas apoE4 inhibited it. Similar results were obtained with central nervous system neurons (murine neuroblastoma Neuro-2a cells). Addition of free apoE3 or apoE4 without beta-VLDL had no effect on neurite outgrowth. There was also differential accumulation of apoE3 and apoE4 by the neuroblastoma cells: apoE3 accumulated within cell bodies and neurites to a greater extent than apoE4. Thus, apoE3 may facilitate cytoskeletal activity, whereas apoE4 may inhibit it, which would be detrimental during synaptic remodeling. PMID- 8624077 TI - Age-related changes in cortical blood flow activation during perception and memory. AB - Although many cognitive functions are affected by age, some are relatively maintained. There also are numerous age-related changes in brain structure, but not much is known about how these changes impact upon the alterations seen in cognition. In order to understand the basis for the reductions and sparings of cognitive function in the aged, experiments were carried out to compare regional cerebral blood flow (rCBF) and performance in young and old subjects on visual perceptual and memory tasks. In the first experiment, which examined the perception of faces and spatial locations, old subjects were as accurate as young subjects, and both groups had rCBF activation in fusiform gyrus during face matching and in superior parietal cortex during location matching. However, old subjects had less activation of prestriate cortex and more activation of frontal and lateral temporal cortex than did young subjects. The second experiment tested recognition memory for faces, a task on which old subjects were impaired. Young subjects had rCBF activation in left prefrontal and inferior temporal cortex and in right hippocampus during encoding of the faces, and in right prefrontal and parietal cortex during recognition. Old subjects showed no significant activation of the areas involved in encoding, but did have increased rCBF in right prefrontal cortex during recognition. These results suggest that the aging brain can demonstrate alterations of the functional systems involved in some cognitive processes, such as perception, that may serve as a compensatory mechanism to maintain performance. Failure to show such compensation, coupled with dysfunction of the areas primarily involved in processing may lead to more marked deficits in performance. PMID- 8624078 TI - Morphological, biochemical, and genetic support for an apolipoprotein E effect on microtubular metabolism. AB - There are two distinct viewpoints on the association of the inheritance of apolipoprotein E (APOE) alleles and the age of onset distribution of Alzheimer's disease (AD): genetic and phenotypic expression. There have been multiple corroborations of the APOE-epsilon 4 association with Alzheimer's disease in populations around the world in clinic based studies as well as emerging epidemiological studies. The genetic data do not imply mechanism of pathogenesis. The phenotypic expression of AD has been based in theories based on amyloid plaques or neurofibrillary tangles. ApoE protein interacts with both beta-amyloid and tau in an isoform-specific manner. The interaction with tau had been thought to be an in vitro artifact, since apoE had not been previously localized to the neuronal cytoplasm. Immuno-EM studies have localized apoE in neuronal cytoplasm. ApoE3 interacts with both tau and MAP2c at the microtubule binding repeat domain under conditions in which apoE4 is less tightly bound. These data further support a hypothesis that apoE3 (and apoE2) protect the microtubule binding domain of tau from binding to itself to form paired helical filaments and neurofibrillary tangles, while protecting the site for microtubule stabilizing interactions with beta-tubulin. These data are supported by recent data from APOE knock-out mice demonstrating dendritic alterations leading to synaptic simplification similar to that observed in AD. PMID- 8624079 TI - Clinical and neuropathological correlates of apolipoprotein E genotype in Alzheimer's disease. Window on molecular epidemiology. AB - Inheritance of the apolipoprotein E (apo E) epsilon 4 allele has recently been found to be associated with Alzheimer's disease. We have studied the clinical and neuropathological correlates of apolipoprotein E genotype in a large group of Alzheimer's patients. The primary influence on clinical presentation is a shift towards earlier age of onset in individuals who have the apo E epsilon 4 gene: no change in clinical course was observed. In neuropathological studies, we find that the major influence of apo E epsilon 4 is on increased A beta deposition. These results led to a model of the biological interaction between the apo E protein and Alzheimer's disease. PMID- 8624080 TI - Apolipoprotein E, plaques, tangles and cholinergic dysfunction in Alzheimer's disease. AB - Apolipoprotein E is a plasma cholesterol and phospholipid transporter which plays a central role in lipoprotein metabolism in the brain. Apolipoprotein E is a polymorphic protein with three common alleles in the general population, designated epsilon 2, epsilon 3 and epsilon 4 coding for proteins ApoE2, ApoE3 and ApoE4, respectively. Recent findings have demonstrated a significant relationship between the epsilon 4 allele and late onset familial and sporadic Alzheimer's disease. We examined several classical neuropathological hallmarks of Alzheimer's disease to determine whether they might be related to apolipoprotein E genotype: the presence of intracellular neurofibrillary tangles, extracellular senile plaques, and the attenuation of choline acetyltransferase activity. Significant correlations were found between epsilon 4 allele copy number and senile plaque density in the frontal, parietal and fusiform cortical areas. Similarly, significant correlations were also found with increased neurofibrillary tangle number in the frontal and fusiform cortex. Interestingly, there was an inverse correlation between the epsilon 4 allele with temporal cortical choline acetyltransferase activity. To further define the specific function of ApoE4, cultured rat hippocampal neurons were used to investigate interactions involving beta-amyloid protein. In this model, ApoE4 (but not ApoE2) was able to reverse the neuroprotective effects of beta-amyloid. ApoE3 was demonstrated to increase the internalization of beta-amyloid peptide into these neurons. Taken together, these results support the involvement of ApoE4 in the pathogenesis of Alzheimer's disease and also provide some explanations for the possible function of this protein. PMID- 8624081 TI - Regulation of APP processing. Potential for the therapeutical reduction of brain amyloid burden. AB - The role of brain amyloid in the pathogenesis of Alzheimer's disease (AD) is discussed controversially, but combined genetic and biochemical evidence points to a central role of the gene encoding the amyloid precursor APP in at least some forms of AD. This article proposes that preventing brain amyloid formation is a rational concept for drug treatment of AD. We suggest that pharmacologically active ligands for specific cell surface receptor subtypes--normally stimulated by neurotransmitters, growth factors, and cytokines--constitute a class of chemicals that might be useful to accelerate processing of APP into non amyloidogenic, and biologically active, derivatives. This class of agents includes muscarinic m1 and m3 agonists, serotoninergic 5-HT2a and 5-HT2c agonists, glutamatergic mGluR1 agonists, as well as agonists for bradykinin and vasopressin receptors. PMID- 8624082 TI - A novel brain cysteine protease forms an SDS stable complex with the beta-amyloid precursor protein. AB - Alzheimer's disease (AD) brain accumulates beta-protein (A beta) a peptide proteolytically derived from the beta-amyloid precursor protein (APP). The abnormal production and aggregation of A beta have been implicated in the pathogenesis of the disease. The mechanism of production of A beta in vivo is not yet clear; but endoproteases capable of degrading APP are likely to be involved in the process. We have isolated a protease from AD brain by following its activity in digesting a synthetic peptide of 10 amino acids derived from the APP sequence flanking the N-terminus of A beta. The protease was purified by a fractionation scheme including ammonium sulfate precipitation and column chromatography using hydrophobic interaction, anion exchange, affinity, hydroxyapatite and size exclusion gels. The purity of the final product was assessed on a silver stained SDS gel by the presence of a single band. Microsequencing was performed following trypsin digestion of the sample. Internal peptide sequences were found to have sequence homology to cysteine proteases in the database. The enzyme requires DTT for activity and can be inhibited by specific inhibitors of cysteine but not serine proteases. The purified enzyme has a pI of 5.0 and a native tetrameric structure with subunits of 48 kD each. The enzyme is capable of digesting APP and generating a short peptide recognizable by antibodies specific to the C-terminus of APP. Interestingly, the purified protease also forms heat- and SDS-stable complexes with APP. PMID- 8624084 TI - High affinity choline transporter status in Alzheimer's disease tissue from rapid autopsy. AB - The degeneration of nucleus basalis cholinergic neurons in Alzheimers disease (AD) has led to therapies that attempt to increase the synaptic availability of acetylcholine in the remaining cholinergic nerve terminals and to thereby reverse or slow the progressive dementia accompanying the disease process. The inadequacy of current choline-replacement therapies suggests that utilization of choline may be disordered and the rate-limiting step in acetylcholine synthesis, the high affinity choline transporter, may be involved. An adequate test of this hypothesis requires the use of fresh, unfrozen tissue, as the transporter activity declines rapidly after death. Using tissue acquired within two hours of death, the activity of the high affinity choline transporter was shown to be increased in cortical brain regions from AD patients compared to non-AD controls. Further studies using frozen tissues with similar short postmortem acquisition times, revealed the expression of the high affinity uptake transporter to be increased in AD cortex as well. When the ratio of regional uptake activity or expression to the regional level of choline acetyltransferase was calculated, the increase in choline transporter activity and expression was clearly statistically significant. Further statistical significance in the choline transporter activity of the AD group was achieved when the putamen, a region without marked pathology in AD, was used as an internal standard to control for agonal state differences in the individual patients contributing tissue to this study. These increases in choline transporter expression and activity in AD indicate disordered regulation of this rate-limiting component of acetylcholine synthesis above and beyond that required to compensate for the reduced cholinergic synaptic availability in AD. PMID- 8624083 TI - M1 agonists for the treatment of Alzheimer's disease. Novel properties and clinical update. AB - The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151 (S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single-blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales. PMID- 8624085 TI - Occurrence of interleukin-6 in cortical plaques of Alzheimer's disease patients may precede transformation of diffuse into neuritic plaques. AB - Interleukin-6 (IL-6) immunoreactivity has previously been shown in plaques in Alzheimer's disease (AD), and elevated IL-6 concentrations have been measured biochemically in brains of AD patients. In this report, we present data on the appearance of IL-6 immunoreactivity in AD plaques according to the stage of plaque formation. Diffuse plaques are found in the early stages of plaque formation, whereas primitive and classic plaques are thought to represent later stages of plaque pathology. We classified plaques using the Bielschowsky silver stain method in serial sections of paraffin-embedded cortices of clinically diagnosed and histopathologically confirmed AD patients and patients with no clinical history of dementia. In the brains of nondemented and demented persons, we found plaques using the silver staining method or immunohistochemistry with antibodies against the amyloid precursor protein. In the nondemented group, diffuse plaques were the predominant plaque type, whereas primitive plaques formed the larger proportion of lesions in the group of AD brains. IL-6 was only detectable in plaques of demented patients. In AD cases, IL-6 was found in a significantly higher ratio in diffuse plaques as would have been expected from a random distribution of IL-6 in all plaque types. We conclude that the presence of IL-6 immunoreactivity correlates with clinically detectable dementia. In addition to the ubiquitous presence of amyloid in nondemented and demented brains, an IL-6 related immunological mechanism may be involved both in the transformation from diffuse to primitive plaques in AD and in the development of dementia. PMID- 8624086 TI - Anti-inflammatory drugs in the fight against Alzheimer's disease. AB - Lesions in Alzheimer disease are characterized by the assembly of a variety of cells and proteins associated with the immune system. Activated microglia express high levels of MHC glycoproteins and receptors for complement. Small numbers of T lymphocytes infiltrate tissue. Proteins of the classical complement pathway are closely connected with beta-amyloid deposits. Several materials associated with senile plaques, including beta-amyloid protein itself, bind C1q in vitro and activate the pathway. The membrane attack complex of complement, as well as proteins which defend against that complex, colocalize with dystrophic neurites. These data imply that an autodestructive process is occurring in Alzheimer's disease, and that anti-inflammatory drugs might be an effective form of therapy. Some epidemiological evidence and results of a pilot clinical trial support this hypothesis. PMID- 8624088 TI - Disorders of visual and spatial perception in the early stage of Alzheimer's disease. AB - A battery of visual and spatial perception tests (VOSP) has been administered to a sample of 25 mild Alzheimer's disease (AD) patients and 25 age- and education matched controls in order to assess visuospatial skills in the early phase of the disease. Among visual object perception tests, AD patients were impaired only in the task where they had to name the stimulus (Silhouettes task). The score in this task significantly correlated with the Boston Naming Test score (r = .73; p < 0.0001). The performance on the Object decision task (which does not require naming), figure-ground analysis, and identification of degraded stimuli was unimpaired. Similarly there were no significant differences for any of the spatial perception tests. The results showing that the performances on most measures of visual and spatial perception is unimpaired, indicate that the "early" perceptual processes, as well as the access to structural knowledge, are not defective in early AD. On the other hand, AD patients are impaired at the level of semantic access and name retrieval. The finding of an highly significant correlation among tests requiring visual access to structural and semantic knowledge suggests that a rigid separation between perceptual and semantic mechanisms may be unwarranted. PMID- 8624087 TI - Near infrared spectroscopy in the diagnosis of Alzheimer's disease. AB - Near infrared spectroscopy (NIRS) is a new technique that permits noninvasive monitoring of cerebral blood and tissue oxygenation. Recently, we and others have shown that NIRS measurements are sensitive enough to follow changes in cerebral hemoglobin oxygenation due to activation of brain function. Based on these findings we have assessed the influence of aging as well as the influence of neurodegeneration on cerebral hemoglobin oxygenation during mental work. The typical NIRS pattern in young healthy subjects while performing calculation tasks measured in the frontal cortex were increases in oxygenated hemoglobin [HbO2] and total hemoglobin [HbT] while reduced hemoglobin [HbR] decreased. Elderly healthy subjects showed a significant lower mean increase in [HbO2] and [HbT] levels. Regression analysis revealed an age-dependent decline in activation-induced local increase of [HbO2] as well as [HbT]. Furthermore, we monitored changes in cerebral hemoglobin oxygenation in the frontal cortex while patients with probable Alzheimer's disease (AD) performed cognitive tasks. Whereas elderly healthy subjects (as well as patients with major depression, age-associated memory impairments or vascular dementia) again showed clear increases in the local concentrations of [HbO2] and [HbT] during brain activation, AD patients showed significant decreases compared to the baseline levels in both variables that were most pronounced in the parietal cortex. To clarify whether the different patterns in cerebral hemoglobin oxygenation during cognitive activation were due to an altered functional brain organization in AD or to alterations in the cerebrovascular response to neuronal activation, we are currently performing simultaneous NIRS and (015-H20-)PET measurements during performance of a cognitive task (Stroop test). Our finding of a regional reduced oxygen supply during activation of brain function may be of relevance to the development and the time course of neurodegeneration. PMID- 8624089 TI - Atrophy of the hippocampal formation in early familial Alzheimer's disease. A longitudinal MRI study of at-risk members of a family with an amyloid precursor protein 717Val-Gly mutation. AB - The hippocampal formation (HF) is known from pathological and magnetic resonance imaging (MRI) studies to become severely atrophied in established Alzheimer's disease (AD). This study examined whether changes in the HF could also be detected in very early AD by scanning subjects at risk of developing familial AD (FAD). Five at risk members of a pedigree with the amyloid precursor protein (APP) 717 valine to glycine mutation underwent serial MRI scanning with volumetric measurement of the HF as well as neurological and neuropsychological assessments. Over a period of two years two subjects became clinically affected, a loss of up to 20% of the volume of the HF occurred in the two years over which symptoms first appeared. Asymmetrical HF atrophy was shown to have been present before the development of overt symptoms. This may have important implications for early diagnosis in AD more generally. PMID- 8624090 TI - Visual versus auditory memory stimulation in patients with probable Alzheimer's disease. A PET study with 18 FDG. AB - Patients with probable Alzheimer's disease were either stimulated with a continuous visual recognition task (n = 18) or with a continuous auditory recognition task during PET measurement with 18 FDG. The PET scanner was a Siemens CTI, ECAT EXACT with 5 mm transaxial and 6 mm axial resolution. The global stimulation effects were nearly identical in both groups and increased of 4.17 +/- 8.14% in the auditory stimulated group and of 4.03 +/- 11.78% in the visual stimulated group. Beyond regional stimulation effects in both groups a common cerebellar stimulation effect was measured. It is concluded that the cerebellar stimulation effect reflects a modality independent cognitive process and that the only small enhancement of global glucose metabolism indicates disturbed stimuli processes and finally explained the patient's failure in memory tasks. PMID- 8624092 TI - Varieties of progressive non-fluent aphasia. AB - We report four patients with progressive aphasia of the non-fluent type as the presenting clinical manifestation. The patients were included in a longitudinal study of focal progressive neuropsychological syndromes, and were periodically submitted to neuropsychological evaluations and neuroimaging studies (TC, MRI, SPET or PET). The pattern of neuropsychological impairment was in good agreement with the results of functional imaging studies, which indicated involvement of the anterior regions of the left hemisphere. The evolution of the clinical picture was extremely heterogeneous in the four patients, ranging from a relatively stable picture of transcortical motor aphasia to a severe progressive frontal lobe syndrome. Progressive non-fluent aphasia appears to be a reliable clinical marker of the localization of the pathological process; whether this is related to specific neuropathological conditions, such as Pick's disease, remains for the moment a matter of speculation. PMID- 8624091 TI - Alterations in functional neuroanatomical connectivity in Alzheimer's disease. Positron emission tomography of auditory verbal short-term memory. AB - Conscious recall of past events which have specific temporal and spatial contexts, termed episodic memory, is mediated by a system of interrelated brain regions. In Alzheimer's disease (AD) this system breaks down, resulting in an inability to recall events from the immediate past. Studies of normal human auditory-verbal short-term memory suggest that the brain system underlying these processes has distinct components, and the present study utilized the methods of functional brain mapping to determine the nature and extent of the breakdown that occurs in AD. Using subtraction techniques of PET-acquired images of regional cerebral blood flow we demonstrate that AD patients show a compensatory hyperactivation of various regions of cerebral cortex normally involved in these tasks, as well as activation of cortical areas not activated by normal elderly subjects. These results provide clear evidence of functional plasticity in the AD patient's brain even if those changes do not result in normal memory function. PMID- 8624093 TI - Computer-based cognitive training in Alzheimer's disease patients. AB - Memory training programs for cognitively impaired patients have often been criticized for their lack of relevance to everyday activities. We therefore report our experience with four patients suffering from probable Alzheimer's disease, who were trained with a new computer-based program recently developed by our research group. An everyday task of personal relevance to the patient is simulated and trained on a PC-touch-screen using personal photographs of the patient's surroundings and biography. According to the degree of cognitive impairment, training has three major aspects 1) social competence in patients with beginning deficiencies, 2) orientation in patients with moderate disease, and 3) emotional aspects in patients with advanced deficiencies. The patient's training performance improved substantially. While psychopathometric tests showed no significant effects with regard to general cognitive performance, levels of motivation were high and there was a positive acceptance of the training and signs of emotional activation. PMID- 8624094 TI - ApoE2 allele, Down's syndrome, and dementia. AB - All individuals with Down's syndrome (trisomy 21-DS) develop the pathogenic hallmarks of Alzheimer's disease in old age (+40 years). The extent of pathology is variable, but it has been shown that the amount of beta-amyloid pathology is variable and related to age and the degree of dementia. Thus, in DS, growing old is associated with a progressive pathological process which results in cognitive decline. However, neuropsychological studies of older DS subjects have identified a clinical dementia in only a proportion of cases. These contradictory observations could be reconciled if some factor existed which modulated the rate and amount of beta-amyloid pathology. Recent studies demonstrate an association between the apolipoprotein E4 (ApoE4) allele and the earlier age of onset in both sporadic and familial AD. Increased amounts of beta-amyloid pathology can also be related to the E4 allele. However, at present there are no data documenting the effects of ApoE genotype on the expression or degree of clinical symptoms of the disease. We have examined the ApoE genotype in a cohort of clinically evaluated elderly patients with DS in order to examine the effects of ApoE genotype on the clinical symptoms of dementia. We report here that, despite the presence of an active disease process, the ApoE E2 allele is associated with longevity and preservation of cognitive functioning. PMID- 8624095 TI - Alzheimer's disease and apolipoprotein E in Italy. AB - Recent studies have provided evidence of association of apolipoprotein E (ApoE) epsilon 4 allele and late onset familial and sporadic Alzheimer's disease (AD). Epidemiological studies have established allelic variation at the ApoE locus. We have analyzed the ApoE gene polymorphism in a sample of 416 Italian subjects. Our data confirm a significant association between epsilon 4 allele and sporadic AD. The frequency of epsilon 4 allele in early onset familial AD patients was comparable to control values suggesting that epsilon 4 allele does not represent a risk factor for early onset familial AD (EOFAD). Moreover, we found a not previously reported association between ApoE epsilon 2 allele and sporadic AD and EOFAD. We included in this study two EOFAD families with the APP717 Val-->Ile mutation in the Amyloid Precursor Protein (APP) gene on chromosome 21. In any of the EOFAD families there was a significant effect of the ApoE genotype on the age of onset with the exception of one of the two mutated EOFAD families in which the 2 allele delays the age of onset. PMID- 8624096 TI - Apolipoprotein E alleles and brain vascular pathology in Alzheimer's disease. AB - The presence of apolipoprotein E (ApoE)-E4 allele has been implicated as a risk factor for Alzheimer's disease (AD). We examined the occurrence of ApoE 4 alleles in AD associated with cerebral amyloid angiopathy and other vascular lesions. We found significantly high frequency of the ApoE 4 allele in AD with moderate to severe CAA. The frequency of the allele was also high in AD cases with other vascular lesions such as multiple infarcts and lacunes. As previously reported, we confirm a greater frequency of the ApoE 4 allele in the diffuse Lewy body variant of AD. Our results suggest ApoE 4 allele to be a significant factor in the development of CAA in AD. While this may be related to increased brain amyloid load as a consequence of ApoE genotype, the possibility exists that ApoE may be a specific factor in vascular abnormalities associated with AD. PMID- 8624097 TI - Amyloid beta-protein, APOE genotype and head injury. AB - Deposition of amyloid beta-protein (A beta) in the brain plays a key role in the pathogenesis of Alzheimer's disease. Head injury is an epidemiological risk factor for Alzheimer's disease, and deposition of A beta occurs in approximately one third of individuals dying shortly after a severe head injury. Of the three common apolipoprotein E alleles (APOE-epsilon 2, epsilon 3, and epsilon 4) APOE epsilon 4 allele is a strong risk factor for both sporadic and some familial cases of Alzheimer's disease and there is in vitro evidence that apolipoprotein E is directly involved in A beta deposition. We have examined the frequency of APOE epsilon 4 in those individuals with A beta deposition following head injury and found that the APOE-epsilon 4 frequency (0.52) is higher than in most studies of sporadic Alzheimer's disease. In those head-injured individuals without amyloid deposition the APOE-epsilon 4 frequency (0.16) is similar to that in non Alzheimer's disease controls (p < 0.00001). Our data indicate an interaction between known environmental and genetic risk factors for Alzheimer's disease and underlines the importance of convergence of data around the common mechanism of A beta deposition. Furthermore, it indicates a genetic susceptibility to the effects of a head injury which may be of significance both to those who have recently sustained such an injury and to those whose activities put them at risk of trauma. PMID- 8624098 TI - Apolipoprotein E polymorphism is associated with both senile plaque load and Alzheimer-type neurofibrillary tangle formation. AB - Recent work provided evidence that the apolipoprotein (apo) E polymorphism is associated with late-onset sporadic Alzheimer's disease. The major histological hallmarks of Alzheimer's disease are the extraneuronal deposition of A4/beta amyloid and the intraneuronal formation of neurofibrillary tangles, the latter correlating strongly with the psychometric status. We examined the relationship between the apo E polymorphism and Alzheimer's disease-related histological changes using a staging system which accounts for the progression of the disease over time and correlates well with the cognitive decline ante mortem. We observed a significant positive correlation between both neurofibrillary changes and A4/beta-amyloid deposits and the epsilon 4 gene dose. We estimated that the presence of one apo E4 allele leads to an earlier onset of the histopathological process of about one decade. The association of both types of Alzheimer's disease related changes with the prevalence of the epsilon 4-allele suggests that the apo E polymorphism causally contributes to the development of Alzheimer's disease. PMID- 8624099 TI - APP gene family. Alternative splicing generates functionally related isoforms. AB - The Alzheimer's beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. APP exists in 8 isoforms generated by alternative splicing of exons 7, 8, and 15, of which the L-APP mRNAs lacking exon 15 are ubiquitously expressed in rat tissues but not in neurons. Rat APLP2, the nearest relative of APP, is similarly expressed in 4 different isoforms due to alternative splicing of inserts encoding a Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a divergent region of 12 amino acids on the NH2-terminal side of the transmembrane domain (12 aa exon). KPI-APLP2 transcripts are highly expressed in neurons, in contrast to KPI-APPs, while L-APLP2 mRNA isoforms lacking the 12 aa exon are predominantly expressed in non-neuronal rat tissues, similar to L-APPs. Further examination of the divergent domains in APP and APLP2 harboring the similarly alternatively spliced APP exon 15 and the 12 aa exon of APLP2 revealed some structural similarities of the amino acid sequences and the predicted secondary structures. In both L-APLP2 and L-APP, a putative xylosyl-transferase recognition site for chondroitin sulfate glycosaminoglycan attachment is present that is interrupted in APP and APLP2 isoforms expressing APP exon 15 or the 12 aa exon of APLP2. Thus, a related function of the divergent domains and the corresponding alternatively spliced APP and APLP2 isoforms in regulation of the binding properties of the ectodomain is suggested. Additionally, beta-secretase cleavage of APP might be sterically hindered selectively in proteoglycan L-APP but not in APP lacking the proteoglycan attachment site. Neurons which have a uniquely low portion of L-APP and high content of APP might therefore be especially susceptible to beta A4-protein liberation. This could explain the selective vulnerability of neurons that is observed in Alzheimer's disease. PMID- 8624100 TI - Altered expression of amyloid beta precursor mRNAs in cerebral vessels, meninges, and choroid plexus in Alzheimer's disease. AB - Altered tissue-specific processing or production of the amyloid precursor protein (APP) is thought to be central to amyloid deposition in cerebrovascular and the neocortical tissues in Alzheimer's disease (AD). We demonstrate that A beta peptide(s) is readily detectable and increased in cerebral vessels, meninges and choroid plexus obtained at autopsy from AD subjects compared to age-matched controls. Using the reverse transcription (RT)-polymerase chain reaction (PCR), we further found that A beta transcripts encoding the A beta sequence in all forms of APP containing exons 16 and 17 (of APP770) were significantly increased in vessel samples in AD subjects. This was also evident in the neocortical samples and not related to pre-mortem factors or postmortem interval. It is possible that the increased A beta mRNAs reflect enhanced expression of the L-APP isoform (APP770 without exon 15) expressed in leukocytes and glia alike. We also found evidence for changed proportions of APP 770, 756 and 695 mRNAs in cerebral vessel samples from AD subjects compared to controls. Whereas APP770 and APP751, the predominant forms, were significantly decreased, APP695 transcript was increased in vessel samples from AD subjects. Such changes were not evident in neocortical samples from the same subjects. These observations suggest tissue specific changes in expression of APP isoforms implicating one of the mechanisms for increased accumulation of A beta in cerebrovascular tissues in AD. PMID- 8624101 TI - beta-APP cognitive function versus beta-amyloid--induced cell death. AB - The beta-amyloid precursor protein (beta-APP) has been hypothesized to play an important role in the establishment of synaptic connections. Icv injections of anti-beta APP antibodies into rat brains produced no appreciable effect on subsequent learning of a passive avoidance task whereas memory assessed 1 day later in a retention test was impaired in anti-beta-APP--but not control-IgG injected animals. This suggests a possible involvement of beta-APPs in cognitive functions. In order to evaluate the properties of the proteolytic A beta-fragment accumulating in Alzheimer's disease brains, four different neuronal cell types were exposed to A beta 1-42 for 24 hours. All cells degenerated in response to A beta, yet chromosomal condensation and internucleosomal DNA fragmentation, typical for apoptosis, occurred in only three of the cell types tested. These findings suggest that beta-APPs may play an important role in cognitive processes and additionally, that their alternative proteolytic product A beta may be differentially toxic to neuronal cell types, inducing cell death either by necrosis or by apoptosis. PMID- 8624103 TI - Functional imaging patterns in Alzheimer's disease. Relationships to neurobiology. AB - Brain imaging with functional techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have been widely applied to the study of dementia. While the pattern of temporal and parietal hypometabolism and hypoperfusion have often been suggested to be of diagnostic utility in ascertaining that a dementia is due to Alzheimer's disease (AD), the exact sensitivity and specificity of this pattern in clinically important situations is unclear. These imaging findings have been of considerable interest, however, in describing the regional patterns of predilection in the disease. Evidence supports the contention that the earliest sites of functional impairment in AD are in the temporal lobes. Surprisingly, however, mesial temporal lobe hypometabolism was difficult to detect in a group of mildly demented AD patients in comparison to a group of healthy older subjects. These results suggest that simple use of mesial temporal lobe metabolic rates as a diagnostic for AD may not be fruitful, and that evaluation of the earliest stages of AD can be most productively studied by investigating healthy older individuals. PMID- 8624104 TI - Participation of gene expression in the protection against amyloid beta-peptide toxicity by the beta-amyloid precursor protein. AB - The amyloid beta-peptide (A beta) is a toxic derivative of the beta-amyloid precursor protein. Alternative processing of this precursor also yields large soluble forms (APPSs) which are secreted from many cell types. These APPSs have neuritogenic and neuroprotective activities; indeed, APPSs can protect primary neurons from the toxicity of A beta itself. To begin to explore the regulation of gene expression by APPS, we have focused on the NF-kappa B transcription factor family. NF-kappa B is induced by conditions of stress, including cellular oxidation. We report that NF-kappa B can also be induced by APPS. Furthermore, we effected direct activation of NF-kappa B through disinhibition using antisense oligonucleotide technology. This means of activating NF-kappa B resulted in protection of neuroblastoma cells from the toxicity of a calcium ionophore and protection of primary hippocampal neurons from the toxicity of A beta. Together, these data suggest that NF-kappa B may exist as a common agent inducing a neuroprotective pattern of gene expression in response to either trophic cytokines or stress itself. PMID- 8624102 TI - Amyloid beta-protein induces the cerebrovascular cellular pathology of Alzheimer's disease and related disorders. AB - One of the hallmark pathologic characteristics of Alzheimer's disease (AD) and related disorders is deposition of the 39-42 amino acid amyloid beta-protein (A beta) in the walls of cerebral blood vessels. The cerebrovascular A beta deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall which have been implicated in the expression of the amyloid beta protein precursor (A beta PP) and formation of A beta. We have established primary cultures of human cerebrovascular smooth muscle cells as a model for investigating the cellular pathologic processes involved in the cerebral amyloid angiopathy of AD and related disorders. Recently, we have shown that A beta 1-42, the predominant pathologic cerebrovascular form of A beta, causes extensive cellular degeneration that is accompanied by a striking increase in the levels of cellular A beta PP, potentially amyloidogenic carboxyl terminal A beta PP fragments, and soluble A beta peptide in the cultured human cerebrovascular smooth muscle cells. Together, these studies provide evidence that A beta contributes to the onset and progression of the cerebrovascular pathology associated with AD and related disorders and suggests the mechanism involves a molecular cascade with a novel product-precursor relationship that results in the adverse production and accumulation of A beta. PMID- 8624105 TI - The role of arachidonic acid in the secretion of the amyloid precursor protein (APP). AB - We have studied the activation of human ml-muscarinic receptors in a genetically engineered Chinese hamster ovary cell line (CHO-ml) to determine which second messenger systems affect the secretion of APP via the non-amyloidogenic route. Carbachol activation of the signaling pathways in CHO-ml cells promotes APP secretion by activation of both protein kinase C (PKC)-dependent or Ca(++) dependent second messenger pathways. Both pathways converge to increase the enzyme activity of phospholipase A2 (PLA2), the enzyme that releases arachidonic acid from cellular stores. Directly activating PLA2 with melittin, a peptide from bee venom, or by adding arachidonic acid directly to cultured cells increases the secretion of APP. Thus, our results indicate that arachidonic acid is yet another cellular second messenger involved in regulating the metabolism of APP in addition to PKC and cytoplasmic Ca++. Moreover, activation of PLA2 appears to be an obligatory event in increasing the secretion of APP from CHO-ml cells by the various methods of activation that we have tried thus far. PMID- 8624106 TI - The role of heparan sulfate proteoglycans in the pathogenesis of Alzheimer's disease. AB - The hallmark of Alzheimer's disease (AD) is the deposition of amyloid plaques and neurofibrillary tangles in the brain. The relationship between amyloid deposition and the cognitive deficit is still unclear. The amyloid beta A4 protein is produced by proteolytic cleavage of the amyloid protein precursor (APP). Very little is known about the normal function of APP and the role the protein may play in pathogenesis. Several studies have shown that APP is important for the regulation of neurite outgrowth. Our studies support these findings and indicate that the neurite outgrowth-promoting effects of APP are stimulated by an interaction between APP and specific proteoglycans. Using site-directed mutagenesis, a heparan sulfate binding site which mediates this effect has been mapped to the N-terminus of APP (residues 96-110, HBD-1). A peptide homologous to HBD-1 blocks the trophic effects of APP in cell culture. To purify specific proteoglycans which stimulate the action of APP, an affinity column was constructed using a biotinylated peptide homologous to HBD-1 coupled to streptavidin-agarose. Two proteoglycans were isolated from a crude brain cell conditioned medium by affinity chromatography. The purified proteoglycans bound APP saturably with high affinity and stimulated the action of APP on neurite outgrowth from chick sympathetic neurons. Digestion of the proteoglycan fraction with heparitinase I or chondroitinase ABC demonstrated the presence of two major proteins, a heparan sulfate proteoglycan with a core protein of 63-67 kD molecular mass and a chondroitin sulfate proteoglycan with a core protein of 100 110 kD molecular mass. The results demonstrate that APP binds to at least two proteoglycans and that this interaction may regulate the trophic effects of the protein. The interaction of specific APP-binding proteoglycans with amyloid plaques may disturb the normal function of APP and contribute to the neuritic degeneration that is commonly seen around the amyloid plaque cores. PMID- 8624107 TI - Differential regulation of APP secretion by apolipoprotein E3 and E4. AB - The apolipoprotein E isozyme, apolipoprotein E4, has been implicated as a risk factor for Alzheimer's disease. One reason for the increased risk may be that apolipoprotein E binds to the A beta peptide, but there may be other factors as well. We show that apolipoprotein E is a potent regulator of the secretion of amyloid precursor protein. In cultures of PC12 cells, nanomolar levels of apolipoprotein E3 induce a rapid decrease in the secretion of APP, being observable in 30 min. and stable over 24 hours. Apolipoprotein E4, in contrast, increases secretion of APP over a similar time course. Reciprocal changes occur in cellular amyloid precursor protein. Differential characteristics are also seen in apo E binding to the cells, where apo E4 binds over a slower time course than apo E3. These results suggest a novel mechanism by which apolipoprotein E may be influencing the metabolism of amyloid precursor protein. PMID- 8624108 TI - Regulation of APP processing by intra- and intercellular signals. AB - APP processing appears to be under complex regulation. This regulation is apparently important under both normal and pathological conditions. Of direct clinical interest is the observation that A beta formation can be regulated by various means. This raises the possibility that altered APP processing may cause an increase in A beta formation in AD, and suggests that it may be possible to regulate the production of A beta as a therapeutic approach in AD. As an example of the utility of the latter approach, consider a patient carrying the Swedish APP mutation. If it is true that the cause of AD in such a patient is due to increased A beta production, then decreasing A beta production should delay the onset of the disease. Even in individuals where increased A beta formation is not the cause of AD but there is some other causes, such as the presence of an allele of apolipoprotein E which causes A beta accumulation and hence synaptic loss, decreasing A beta formation may be beneficial. It is of course a very long way from in vitro experiments to therapy. The current emphasis on studying APP processing in vivo represents the next step towards this goal. PMID- 8624109 TI - Defective protein kinase C alpha leads to impaired secretion of soluble beta amyloid precursor protein from Alzheimer's disease fibroblasts. AB - The present study shows that cultured fibroblasts from sporadic AD patients present: a) reduced (-30%) cytosolic protein kinase C (PKC) activity; b) increased KD of phorbol ester binding (+94%) in cytosolic fractions; c) reduced ( 30%) soluble protein kinase C alpha immunoreactivity; d) lower (-27.5%) basal soluble APP secretion and e) reduced soluble APP secretion in response to low phorbol ester concentrations (over threefold difference using 9 nM phorbol-12,13 dibutyrate-PdBu). Since the PKC-stimulated secretion of APP leads to the cleavage of the precursor within the amyloidogenic beta-A4 fragment, the reduced PKC activity in AD patients may lead to accumulation of potentially amyloidogenic or toxic APP fragments. A defect in the secretion of soluble amyloid beta-protein precursor is indeed suggested by literature data on familial AD fibroblasts as well as by the reported results. PMID- 8624110 TI - Metabotropic glutamate receptors regulate APP processing in hippocampal neurons and cortical astrocytes derived from fetal rats. AB - It has previously been shown that stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol (DAG) and activating protein kinase C (PKC), accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, non-amyloidogenic peptides (APPs). This relationship has been demonstrated in human glioma and neuroblastoma cells as well as in transfected human embryonic kidney (HEK) cells and PC12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to DAG and PKC, similarly accelerates processing of APP into non-amyloidogenic APPs in hippocampal neurons and cortical astrocytes derived from normal fetal rats. The mGluR antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), and GF 109203X, an inhibitor of PKC, both blocked the release of APPs from hippocampal neurons and astrocytes evoked by glutamate receptor stimulation. Inasmuch as glutamatergic neurons in cortex and hippocampus are known to be damaged in Alzheimer's disease, our findings suggest that amyloid formation may be enhanced by the resulting glutamate deficiency and that selective mGluR agonists may be useful in facilitating synaptic efficacy and treating the disease. PMID- 8624111 TI - Development and characterization of a monoclonal antibody 369.2B specific for the carboxyl-terminus of the beta A4 peptide. AB - The beta-amyloid deposits in Alzheimer's diseased (AD) brain are made up mainly of beta A4 peptides which show both N- and C-terminal heterogeneity. The predominant C-terminal species, identified by peptide sequencing of purified beta amyloid, end either at position 40 or 42 of the beta A4 peptide. The distribution of these two beta A4 species in postmortem tissue as well as their generation in vitro could not be addressed previously due to the lack of specific antibodies that could differentiate them. This report describes the generation of a highly specific monoclonal antibody, MAb 369.2B which was raised against a synthetic peptide consisting of the C-terminus of the 1-42 beta A4 species. MAb 369.2B does not recognize the shorter beta A4 species 1-40 in solution or in solid phase. Furthermore, both beta A4 1-40 and 1-43 were unable to absorb out the antibody when used immunocytochemically. The regional distribution of MAb 369.2B immunoreactivity in AD and non-AD brain samples were compared to MAb 286.8A, an antibody raised against the N-terminal region of beta A4. Overall, the staining patterns were very similar. In AD cases with extreme vascular involvement, the N terminal (MAb 286.8A) antibody was more likely to label the vascular basement membrane of affected vessels, and to label them more uniformly. In addition, preliminary quantitative analyses revealed that the C-terminal antibody labeled fewer, larger plaques than the N-terminal antibody. Qualitative analyses revealed that these smaller plaques were typically subregions of the larger plaques. The data corroborate the biochemical findings of N-terminal raggedness in beta A4 plaques. Further studies are required to explain this differential pattern of deposition. PMID- 8624113 TI - Free radicals in the neurotoxic actions of beta-amyloid. AB - The fragment of the beta-amyloid protein that contains the peptide sequence 25 to 35 of the parent compound, increases cytosolic calcium and is directly cytotoxic to neurons in tissue culture. The cytotoxic action of beta-amyloid has been considered to be the primary determinant of the neurodegeneration observed in Alzheimer's disease. The cytotoxic effects of beta-amyloid 25-35 or beta-amyloid 1-40 added to primary cultures of rat hippocampal or cortical neurons can be reduced or prevented by anti-oxidant lazaroid drugs such as U-74500A, U-78517F, or U-83836E. beta-amyloid 25-35 evokes an immediate increase in cytosolic calcium when added to suspensions of PC 12 cells, and is cytotoxic to PC 12 cells in tissue culture. Both the calcium-elevating and the cytotoxic actions of beta amyloid 25-35 on PC 12 cells are reduced or prevented by lazaroid anti-oxidant drugs. Since the lazaroids have been shown to scavenge free radical species, the present results indicate that the actions of beta-amyloid 25-35 on calcium regulation and on cell survival are mediated by free radicals. PMID- 8624112 TI - Nicotine-induced protection against glutamate cytotoxicity. Nicotinic cholinergic receptor-mediated inhibition of nitric oxide formation. AB - Cortical neurodegeneration in Alzheimer's disease (AD) is suggested to be attributable not only to beta-protein but also to glutamate. Although degeneration of cholinergic projection to the cerebral cortex is recognized to be one of the most prominent pathological changes in AD, there is only limited information concerning the cholinergic interaction with the cortical neurodegeneration. This study was performed to examine the protective effect of nicotine against glutamate-induced cytotoxicity using rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate or N-methyl-D-aspartate (NMDA). The simultaneous addition of nicotine did not reduce glutamate cytotoxicity. In contrast, the simultaneous application of NMDA receptor antagonists such as MK-801 reduced glutamate cytotoxicity. Incubating the cultures with nicotine (10 microM) for 0.5-24 h prior to glutamate exposure reduced its cytotoxicity. Neuroprotection by nicotine was dependent on both the concentration and incubation period. In contrast to nicotine, muscarine (10 microM) weakly potentiated glutamate cytotoxicity. The neuroprotective effect of nicotine against glutamate cytotoxicity was antagonized by hexamethonium but not by artopine. Nicotine prevented NMDA cytotoxicity but did not affect cytotoxicity induced by either kainate or alpha-amino-3-hydroxy-5 methyl-4-isoxazole propionic acid (AMPA). Cell viability was significantly reduced by a brief exposure of cultures to ionomycin, a calcium ionophore. Ionomycin-induced cytotoxicity was abolished by removing Ca2+ from incubating medium. S-nitrosocysteine (SNOC), which spontaneously releases nitric oxide (NO), also induced delayed cell death. Nicotine prevented ionomycin-induced cytotoxicity without affecting SNOC-induced cytotoxicity. These results suggest that nicotinic cholinergic receptor stimulation induces neuroprotection against glutamate cytotoxicity by its inhibitory action on NO-formation. Therefore, we propose that acetylcholine, acting through nicotinic cholinergic receptors, can function as a putative neuroprotective factor against neurodegeneration caused by the excessive release of glutamate and/or NMDA receptor activation. PMID- 8624114 TI - Molecular membrane interactions of a phospholipid metabolite. Implications for Alzheimer's disease pathophysiology. AB - Alzheimer's disease is characterized by changes in phospholipid metabolism leading to a perturbation in the levels of phosphomonoesters, including L Phosphoserine (L-PS). These early changes in lipid metabolism may result in a defect in membrane bilayer structure, leading to increased rates of beta-amyloid formation. To investigate the effect of L-PS on membrane lipid bilayers, small angle x-ray diffraction and high resolution differential scanning calorimetry (DSC) approaches were used with liposomes composed of lecithin and cholesterol. A one-dimensional electron density profile of a control dimyristoyl phosphatidylcholine (DMPC)/cholesterol lipid bilayer with a unit cell dimension of 52 A at 37 degrees C was generated from the x-ray diffraction data. Following incubation with 2.0 mM L-PS, a broad decrease in electron density +/- 4.12A from the lipid bilayer center was observed concomitant with an increase in the width of the phospholipid headgroup electron density and a 3A reduction in lipid bilayer width. The interactions of L-PS with DMPC lipid bilayers were concentration-dependent, highly affected by cholesterol content and reproduced in egg phosphatidylcholine/cholesterol liposomes. DSC analysis showed that millimolar (1.0-5.0 mM) L-PS levels decreased the phase transition cooperative unit size of DMPC liposomes in a highly concentration-dependent manner which was significantly greater in preparations containing 10 mol% cholesterol. These data provide direct evidence that phosphomonoester levels modulate the biophysical properties of the membrane lipid bilayer which may, in turn, lead to altered structure/function relationships in AD. PMID- 8624115 TI - Molecular and functional magnetic resonance neuroimaging for the study of dementia. AB - The living brain's structure, function, and underlying chemistry are increasingly being revealed in sharpened detail by the extraordinary evolution of magnetic resonance (MR) technology. Recent years have ushered in a wealth of new information about neurophysiology and pathological states owing to such technologies as functional MR imaging (fMRI), MR spectroscopy (MRS), and MR spectroscopic imaging (MRSI). These advances are of substantial benefit in the study of the dementias, especially Alzheimer's disease (AD). One primary objective of our laboratory at the Massachusetts General Hospital NMR Center is to utilize these extant and emerging MR technologies to further understanding of the human brain as it undergoes assault by AD. Our approach is guided by the belief that the pathological states observed in the AD brain must be accompanied by structural, chemical and/or physiological changes that can be made visible in an in vivo MR study. Quantitative measurements by MRI medical temporal lobe structures have been shown to be abnormal by several groups including our own. This use of MRI will be reviewed by others. In this paper, I will review recent advances in the application of MR for the study of chemical and functional brain abnormalities in dementia, and in particular to the investigation of AD. PMID- 8624116 TI - Brain glucose metabolism is controlled by amplification and desensitization of the neuronal insulin receptor. AB - Glucose metabolism is essential for brain function and structure. Glucose contributes to the formation of neurotransmitters and is normally the only source for energy formation. There is increasing evidence that brain glucose metabolism is under control of the neuronal insulin/insulin receptor signal transduction. The present data clearly show that intracerebroventricularly administered insulin exerts anabolic effects on cerebral glucose/energy metabolism (amplification of the neuronal insulin receptor complex) whereas cortisol (corticosterone) acts antagonistically (desensitization of the neuronal insulin receptor complex). It is also shown that short-term cortisol (corticosterone) enhanced energy turnover in temporoparietal cortex and hippocampus. In contrast, long-term cortisol (corticosterone) reduced energy turnover in both brain structures studied. This metabolic pattern is reminiscent of that found in very old age. Therefore, it is assumed that long-term cortisol accelerates the aging process in the brain and thus the risk for age-related disorders such as dementia. PMID- 8624117 TI - Cellular mechanisms of brain energy metabolism. Relevance to functional brain imaging and to neurodegenerative disorders. AB - Astrocyte end-feet surround intraparenchymal microvessels and represent therefore the first cellular barrier for glucose entering the brain. As such, they are a likely site of prevalent glucose uptake. Astrocytic processes are also wrapped around synaptic contacts, implying that they are ideally positioned to sense and be functionally coupled to increased synaptic activity. We have recently demonstrated that glutamate, the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 2-DG uptake and phosphorylation by astrocytes in primary culture. The effect is not receptor-mediated but rather proceeds via one of the recently cloned glutamate transporter. The mechanism involves an activation of the Na+/K+ ATPase. Concomitant to the stimulation of glucose uptake, glutamate causes a concentration-dependent increase in lactate efflux. These observations suggest that glutamate uptake is coupled to aerobic glycolysis in astrocytes. In addition, since glutamate release occurs following the modality specific activation of a brain region, the glutamate-evoked uptake of glucose into astrocytes provides a simple mechanism to couple neuronal activity to energy metabolism. These data also suggest that modality-specific activation visualized using 2DG-based autoradiography or PET may primarily reflect glutamate-mediated uptake of 2DG into astrocytes. PMID- 8624118 TI - Beta-amyloidosis in normal aging and transmitter signaling in human temporal lobe. AB - Interactions between abnormal amyloid precursor protein metabolism and cholinergic dysfunction are increasingly apparent. Both of these major features of Alzheimer's disease occur in restricted loci in normal aging--a potential model for early Alzheimer type pathology. Entorhinal cortex is particularly vulnerable to beta-amyloidosis and compared with other cortical areas is remarkable for the relatively high density of nicotinic (3H-nicotine) but not other cholinergic or glutamate receptor binding. With increasing age, post maturity, there is a persistent decline in nicotinic receptor binding in entorhinal cortex whereas muscarinic M1 and non-M1, glutamate NMDA and non-NMDA receptors are spared. Normal elderly individuals, distinguished by the absence of beta A4 immunoreactive plaques in this area, are differentiated from those with plaques by higher nicotine binding. Amongst individuals with an established history of smoking tobacco, nicotinic receptor binding and hippocampal choline acetyltransferase were elevated compared with non-smokers and preliminary evidence indicates a reduced density of cortical plaques. These findings are consistent with the hypothesis that down regulation of the nicotinic cholinergic receptor--a ligand gated calcium channel known to control the expression of neurotrophins--plays a role in the evolution of Alzheimer-type pathology. PMID- 8624119 TI - The effect of cholinesterase inhibitors on the secretion of APPS from rat brain cortex. AB - In this study we examined the question whether cholinesterase inhibitors (ChEI) could alter the release of amyloid precursor protein (APP) from superfused brain cortical slices of the rat following electrical as well as pharmacological stimulation with bethanechol (BETHA). Three ChEI, both reversible and irreversible were tested for their ability to enhance the release of non amyloidogenic soluble derivatives (APPs). These included physostigmine (PHY), heptyl-physostigmine (HEP) and 2,2-dichlorovinyldimethyl phosphate (DDVP), at the concentrations producing cholinesterase (ChE) inhibition ranging from 5% to 95%. All three ChEI elevated APPs release significantly above control levels. Electrical field stimulation significantly increased the release of APPs within 50 min. Similar increase was observed after muscarinic receptor stimulation with BETHA. Tetrodotoxin (TTX) completely blocked the effect of electrical stimulation. These findings suggest that long-term administration of ChEI to Alzheimer's disease (AD) patients may have a neuroprotective effect by activating normal APP processing and decreasing the formation of amyloidogenic APP products. PMID- 8624120 TI - Therapeutic effects of CDP-choline in Alzheimer's disease. Cognition, brain mapping, cerebrovascular hemodynamics, and immune factors. AB - CDP-choline was given to patients with Alzheimer's disease (AD) at a daily dose of 1000 mg/day p.o. for one month. This compound slightly improved mental performance, tended to reduce theta activity in fronto-temporal regions, increasing alpha power in occipital areas, and enhanced cerebrovascular perfusion by increasing blood flow velocity and reducing pulsatility and resistance indexes. In addition, CDP-choline diminished histamine and interleukin-1 levels in blood and serum, respectively, and increased plasma TNF. PMID- 8624121 TI - HWA 285 (propentofylline)--a new compound for the treatment of both vascular dementia and dementia of the Alzheimer type. AB - The pharmacological profile of HWA 285 favors its use in patients with both Alzheimer's disease (PDD) and/or vascular dementia (MID). Clinical trials showed clinically relevant, statistically significant efficacy in the domains of cognitive function, global function and activities of daily living (ADL) in both PDD and MID. HWA 285 had a prolonged symptomatic effect for at least 12 months, although therapeutic effects were seen already after the first 3 months of treatment. HWA 285 was very well tolerated for at least 1 year. PMID- 8624122 TI - Preliminary evaluation of besipirdine for the treatment of Alzheimer's disease. Besipirdine Study Group. AB - Besipirdine hydrochloride (HP 749) is an indole-substituted analog of 4 aminopyridine. Besipirdine enhances both cholinergic and adrenergic neurotransmission in the central nervous system, and may have greater efficacy than purely cholinergic agents in treating dementia due to Alzheimer's disease. The present study examined the efficacy and tolerability of two doses of besipirdine (5 and 20 mg BID) in 275 patients with Alzheimer's disease during 3 months of treatment and during 3 months after withdrawal of treatment. Besipiridine was generally well tolerated. The level of performance on a cognitive test was sustained during 3 months of treatment with besipirdine, whereas the performance of patients treated with placebo deteriorated over the same time period. The results suggest a dose-response relationship, with greater efficacy after 3 months of treatment and longer persistence after treatment withdrawal for besipiridine 20 mg BID than for 5 mg BID. A clinical global rating did not detect a besipirdine treatment effect. The full efficacy after 3 months of treatment did not persist after withdrawal of treatment, suggesting that the benefit is primarily symptomatic. Treatment with higher doses and for longer periods may enhance efficacy on both cognitive and clinical global assessments. PMID- 8624123 TI - Immortalized neural cells from trisomy 16 mice as models for Alzheimer's disease. AB - The trisomy 16 mouse (Ts16) is a general accepted animal model for both Downs syndrome (DS) and Alzheimer's Disease (AD). However, the efficacy of this model is severely hampered by the fact that Ts16 is lethal after about 18-20 days of gestation. Chimeras, long-term tissue culture and neural transplantation of Ts16 material have previously been used to overcome this limitation presented by death in utero of the Ts16. In this paper we describe a new strategy to overcome this limitation, i.e. immortalization of primary cells from Ts16 mice with retrovirus mediated gene transfer of a temperature sensitive immortalizing oncogene. By this method we have obtained a total of 21 stable cell lines from Ts16 hippocampus, Ts16 cortex, normal hippocampus, and normal cortex. So far, two of the cell lines have been karyotyped and as expected, the cell line immortalized from Ts16 embryos has retained three copies of chromosome 16. We are currently characterizing these cell lines with respect to expression of APP, T-antigen, Nestin, GFAP, NF and Map-2. Moreover, the processing and secretion of APP fragments are being investigated by immunoblotting. In summary, we have immortalized CNS cells from Ts16 mice and we expect that these cell lines will be useful as in vitro and in vivo models for studying various aspects of the pathology of Alzheimer's disease. PMID- 8624124 TI - Mice deficient for the amyloid precursor protein gene. AB - To understand the in vivo function of the amyloid precursor protein (APP) we generated an APP null mutation in mice by homologous recombination in embryonic stem (ES) cells. We show here that homozygous APP deficient mice were produced at expected frequencies. Neither APP mRNA nor protein could be detected in these animals. Yet the homozygous APP mutant mice are fertile and do not show overt abnormalities at up to 12 weeks of age. Neuroanatomical studies of the brain did not reveal significant differences in the knockout mice as compared to the wild type controls. These results argue against an essential function of APP in mouse embryonic and early neuronal development. PMID- 8624125 TI - The D1 agonist dihydrexidine releases acetylcholine and improves cognition in rats. AB - Neurochemical and behavioral studies have elucidated extensive interactions between dopaminergic and cholinergic systems in brain areas associated with movement and cognition. The initial goal of these studies was to evaluate the effect of the anti-Parkinson drug dihydrexidine (DHX), a dopamine D1 full efficacy agonist, on brain acetylcholine (ACh) release using in vivo microdialysis techniques. Moderate doses of DHX (3 and 10 mg/kg) produced approximately a 50% increase in striatal ACh release that was blocked by the D1 agonist SCH23390 (0.3 mg/kg). A higher dose of DHX (17.5 mg/kg) was less effective in raising striatal ACh, possibly due to D2 receptor activation. In frontal cortex, DHX (10 mg/kg) evoked a more robust increase in ACh release (+200%) that was blocked by SCH23390 (0.3 mg/kg). Since elevations in brain ACh are associated with cognitive improvement, the effectiveness of DHX in a passive avoidance model of learning and memory was also evaluated. These studies revealed a significant improvement in performance by 0.3 mg/kg DHX in scopolamine-induced amnestic rats. These results provided support for the hypothesis that DHX improves cognitive performance as a consequence of ACh release in relevant brain regions. Further, D1 agonists may have novel therapeutic potential in the treatment of dementia. PMID- 8624126 TI - Clinical and neuroimaging features of familial Alzheimer's disease. AB - Subtle phenotypic differences between familial Alzheimer's disease (FAD) pedigrees can be identified which may reflect the genetic and allelic heterogeneity of the disease. Positron emission tomography (PET) of APP mutation and chromosome 14-linked FAD pedigree members reveals biparietal bitemporal hypometabolism. Scanning of asymptomatic at-risk individuals reveals a similar, but quantitatively less severe, pattern of hypometabolism. PMID- 8624127 TI - The role of APP processing and trafficking pathways in the formation of amyloid beta-protein. AB - The amyloid beta-protein (A beta) is a proteolytic fragment of the beta-amyloid precursor protein (beta APP). We previously reported the constitutive secretion of A beta peptides from a variety of cells expressing beta APP under normal culture conditions. These endogenously produced A beta peptides have heterogeneous N- and C-termini that vary as a function of beta APP missense mutations. Treatment of A beta-secreting cells with agents that alter intravesicular pH showed that an acidic compartment is required for proper A beta generation. One such compartment appears to be the endosome. Immunolabeling of cell-surface beta APP in living neurons and non-neuronal cells directly demonstrated the endocytosis of the protein and its rapid recycling (within 5-10 minutes) to the cell surface, as well as the trafficking of some beta APP to lysosomes. Expression of beta APP with various deletions of the cytoplasmic domain, including the NPTY motif, leads to decreased internalization and an associated decrease in the production of A beta peptides that begin at the usual asp1 start site. These and other data suggest that A beta production begins with cleavage of beta APP by a still unknown protease(s) (beta-secretase[s]) at the met-asp bond proceeding the A beta N-terminus and that this occurs in part in early endosomes. To characterize the substrate requirements of beta-secretase, beta APP was mutagenized by placing stop codons within or at the end of the transmembrane domain or substituting other amino acids for the wild-type met and asp at the P1 and P1' positions. These experiments showed that proper beta secretase cleavage requires the precursor to be membrane-anchored and is highly sequence specific; most substitutions at met or asp substantially decrease A beta production. Analogous mutagenesis experiments around the A beta C-terminus revealed that the unknown protease(s) cleaving here ("gamma-secretase[s]") does not show such specificity. Cells secreting A beta may also be useful for examining the critical issue of the aggregation of A beta into its neurotoxic polymeric form under physiological conditions. In this regard, we have found that beta APP-expressing CHO cells show aggregation of > or = 10-20% of their secreted A beta peptides into SDS-stable dimers, trimers and sometimes tetramers under normal culture conditions. The identity of these small multimers was confirmed by extensive immunochemical characterization and radiosequencing. They are present at approximately 100-500 pM levels in conditioned medium of CHO transfectants. Using this endogenous A beta aggregating system, we have begun to examine variables that influence aggregation and compounds which may retard it. In conclusion, studies of the regulation of A beta production and aggregation in cell culture can provide information under physiological conditions that can complement analyses of these processes in vivo. PMID- 8624128 TI - Mice homozygous for a modified beta-amyloid precursor protein (beta APP) gene show impaired behavior and high incidence of agenesis of the corpus callosum. AB - The amyloid precursor protein (beta APP) gene of the mouse was disrupted by homologous recombination; however, contrary to expectation, brain and other tissues still contained beta APP-specific RNA, albeit at a level 5-10 fold lower than wild-type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum. PMID- 8624129 TI - Regulation of APP expression, biogenesis and metabolism by extracellular matrix and cytokines. AB - We have identified and characterized the ligand binding properties of the Alzheimer's disease (AD) beta A4 amyloid protein precursor (APP), mapped the APP ligand binding sites and analyzed the regulation of APP expression, biogenesis and metabolism by components of the extracellular matrix (ECM) and cytokines. PMID- 8624130 TI - Studies on the metabolism and biological function of APLP2. AB - Amyloid precursor proteins (APP) are a member of a larger family of proteins that include the amyloid precursor-like proteins (APLP) APLPI and APLP2. We have examined the expression and metabolism of APLP2 and document that APLP2 is expressed at high levels in the nervous system and in peripheral tissues. Furthermore, several APLP2 isoforms encoded by alternatively spliced transcripts are posttranslationally modified by a chondroitin sulfate glycosaminoglycan (CSGAG) chain. Furthermore, CSGAG modification is regulated by the insertion of sequences encoded by an alternatively spliced exon. Notably, expression of the CSGAG form of APLP2 appears restricted to embryonic neurons and mature neuronal populations that undergo regeneration, such as olfactory sensory neurons. Thus, differences in posttranslational modifications between the APLP2 isoforms and APP are likely to underlie differences in the regulation and function of these homologues. Our present efforts are directed towards using gene targeting strategies to disrupt the expression of the mouse APP/APLP2 genes to define the normative roles of the encoded molecules in development, plasticity, regeneration, and repair. PMID- 8624131 TI - Neurotrophic and neuroprotective effects of hAPP in transgenic mice. AB - To better understand the role the human amyloid precursor protein (hAPP) plays in Alzheimer's disease (AD), it is essential to define its primary function(s). Here we expressed different hAPPs in neurons of transgenic (tg) mice to characterize their effects on the intact central nervous system (CNS). Immunolabeled brain sections of tg and non-tg mice were compared quantitatively by microdensitometry and computer-aided analysis of laser scanning confocal digitized images. Compared with non-tg mice, tg mice overexpressing hAPPs showed an increase in the number of synaptophysin immunoreactive presynaptic terminals as well as in the expression of the growth-associated marker GAP-43. While non-tg controls and tg mice expressing hAPP751 at moderate levels displayed a normal pattern of reinnervation of the dentate gyrus following perforant pathway transection, tg mice expressing hAPP695 at severalfold higher levels showed an accentuation of the synaptic loss and no sprouting reaction. In addition, expression of hAPP751 at moderate levels effectively protected neurons against excitotoxic injury induced either acutely by systemic injection of kainic acid or chronically by transgene-driven glial production of the soluble HIV-1 protein gp120. Neuronal expression of hAPP695 at higher levels provided less excitoprotection. Our findings are consistent with the postulate that APP plays a role in the formation/maintenance of synapses and that processes which affect this function could contribute to the synaptic pathology seen in AD. Our study also revealed that hAPPs can exert important excitoprotective functions in vivo and that the efficiency of this protection may depend on the hAPP isoform expressed as well as on the level of neuronal hAPP expression. Neuronal overexpression of hAPP beyond a certain level may have detrimental effects on the CNS, particularly in the context of secondary neural injuries. PMID- 8624133 TI - Structure, microtubule interactions, and phosphorylation of tau protein. AB - This paper summarizes recent structural and functional studies on tau protein, its interactions with microtubules, its self-assembly into paired helical filaments (PHF)-like fibers, and its modification by phosphorylation. The structure of tau in solution resembles that of a random coil. Both tau and Alzheimer PHFs have very little secondary structure, making it improbable that the assembly of tau into PHFs is based on interacting beta sheets. Tau's binding to microtubules can be described by a "jaws" effect. The domain containing the repeats binds very weakly, while the flanking regions (jaws) bind strongly, even without the repeats. However, only the combination of flanking regions and repeats makes binding productive in terms of microtubule nucleation and assembly. Although the majority of Alzheimer-like phosphorylation sites are outside the repeats they have only a weak influence on binding, whereas the phosphorylation at Ser262 inside the repeats inhibits binding and makes microtubules dynamically unstable. This site can be phosphorylated by kinases present in brain tissue, and it is uniquely phosphorylated in Alzheimer brain. PMID- 8624132 TI - Amyloid fibril toxicity in Alzheimer's disease and diabetes. AB - Several lines of evidence suggest that amyloid deposition in the brain contributes to neuronal degeneration in Alzheimer's disease (AD). In the AD brain, diffuse plaques composed mostly of amorphous beta amyloid (Am-beta A) are inert, whereas compact plaques composed of beta amyloid fibrils (Fib-beta A) are associated with neurodegenerative changes. The effects of these two types of amyloid were tested on primary rat hippocampal neurons. Fib-beta A induced the formation of dystrophic neurites and caused neuronal cell death, whereas Am-beta A was not toxic. In addition, Fib-beta A caused synapse loss in the remaining viable neurons, whereas Am-beta A did not significantly affect synapse number. We also examined the effects of amylin, the primary constituent of the amyloid fibrils that form in the pancreas in adult-onset diabetes. Amylin was toxic to rat and human insulin-producing islet cells in the concentration range of fibril formation. The relative toxic potencies of amylin peptides of different species correlated with their fibril-forming capacity. Soluble amylin was not toxic. The amyloid fibril-binding dye Congo red inhibited the toxicity of both beta A and amylin. Congo red afforded protection against toxicity by a dual mechanism. When present during the phase of fibril polymerization, Congo red could inhibit fibril formation from some peptides. When added to preformed fibrils, Congo red bound to fibrils rendering them nontoxic. These results suggest that fibril formation is necessary for both beta A and amylin toxicity. Congo red appears to be a general inhibitor of amyloid fibril toxicity and may therefore be a useful prototype for drugs targeted to the amyloid pathology of AD and adult-onset diabetes. PMID- 8624134 TI - Aspects of the search for neural mechanisms of memory. AB - The search for neural mechanisms of memory has been under way for more than a century. The pace quickened in the 1960s when investigators found that training or differential experience leads to significant changes in brain neurochemistry, anatomy, and electrophysiology. Many steps have now been identified in the neurochemical cascade that starts with neural stimulation and ends with encoding information in long-term memory. Applications of research in this field are being made to child development, successful aging, recovery from brain damage, and animal welfare. Extensions of current research and exciting new techniques promise novel insights into mechanisms of memory in the decades ahead. PMID- 8624135 TI - Health psychology: psychological factors and physical disease from the perspective of human psychoneuroimmunology. AB - This review addresses the importance of studies of human psychoneuroimmunology in understanding the role of psychological factors in physical illness. First, it provides psychologically and biologically plausible explanations for how psychological factors might influence immunity and immune system-mediated disease. Second, it covers substantial evidence that factors such as stress, negative affect, clinical depression, social support, and repression/denial can influence both cellular and humoral indicators of immune status and function. Third, at least in the case of the less serious infectious diseases (colds, influenza, herpes), it considers consistent and convincing evidence of links between stress and negative affect and disease onset and progression. Although still early in its development, research also suggests a role of psychological factors in autoimmune diseases. Evidence for effects of stress, depression, and repression/denial on onset and progression of AIDs and cancer is less consistent and inconclusive, possibly owing to methodological limitations inherent in studying these complex illnesses, or because psychological influences on immunity are not of the magnitude or type necessary to alter the body's response in these cases. What is missing in this literature, however, is strong evidence that the associations between psychological factors and disease that do exist are attributable to immune changes. PMID- 8624136 TI - Long-term potentiation and learning. AB - Long-term potentiation (LTP), a relatively long-lived increase in synaptic strength, remains the mot popular model for the cellular process that may underlie information storage within neural systems. The strongest arguments for a role of LTP in memory are theoretical and involve Hebb's Postulate, Marr's theory of hippocampal function, and neural network theory. Considering LTP research as a whole, few studies have addressed the essential question: Is LTP a process involved in learning and memory? The present manuscript reviews research that attempts to link LTP with learning and memory, focusing on studies utilizing electrophysiological, pharmacological, and molecular biological methodologies. Most evidence firmly supports a role for LTP in learning memory. However, an unequivocal experimental demonstration of a contribution of LTP to memory is hampered by our lack of knowledge of the biological basis of memory and of the ways in which memories are represented in ensembles of neurons, the existence of a variety of cellular forms of LTP, and the likely resistance of distributed memory stores to degradation by treatments that incompletely disrupt LTP. PMID- 8624137 TI - Theoretical foundations of cognitive-behavior therapy for anxiety and depression. AB - Cognitive-behavior therapy (CBT) involves a highly diverse set of terms and procedures. In this review, the origins of CBT are briefly considered, and an integrative theoretical framework is proposed that (a) distinguishes therapy interventions targeted at circumscribed disorders from those targeted at generalized disorders and (b) distinguishes interventions aimed at modifying conscious beliefs and representations from those aimed at modifying unconscious representations in memory. Interventions aimed at altering consciously accessible beliefs are related to their theoretical bases in appraisal theories of emotion and cognitive theories of emotion and motivation. Interventions aimed at modifying unconscious representations are related to their theoretical bases in learning theory and findings from experimental cognitive psychology. In the review, different formulations of CBT for anxiety disorders and depression are analyzed in terms of this framework, and theoretical issues relating to self representations in memory and to emotional processing are considered. PMID- 8624139 TI - Origins and early development of perception, action, and representation. AB - Research relevant to the origins and early development of two functionally dissociable perceptual systems is summarized. One system is concerned with the perceptual control and guidance of actions, the other with the perception and recognition of objects and events. perceptually controlled actions function in real time and are modularly organized. Infants perceive where they are and what they are doing. By contrast, research on object recognition suggests that even young infants represent some of the defining features and physical constraints that specify the identity and continuity of objects. Different factors contribute to developmental changes within the two systems; it is difficult to generalize from one response system to another; and neither perception, action, nor representation qualifies as ontogenetically privileged. All three processes develop from birth as a function of intrinsic processing constraints and experience. PMID- 8624138 TI - Methodological issues in psychopathology research. AB - We present an overview of methodological issues involved in conducting psychopathology research, including conceptual, analytic, and interpretive considerations. Research issues germane to structured diagnostic interviewed, comorbidity of mental disorders, and ascertainment and sampling are reviewed. Further, the problem of specificity (with respect to disorder, to differential deficit, and to time) is discussed. Specific issues concerning risk vs protective factors, conducting research with special populations, and the continuity of abnormal and normal functioning are highlighted. Finally, various analogue strategies (human subclinical syndromes, experimental study of "pathological" processes in normals, animal models, and computer simulations) are critiqued. Our review documents many of the impressive methodological developments that have emerged in this field, and we hope our review stimulates additional research that exploits recent methodological advances. PMID- 8624140 TI - Auditory psychophysics and perception. AB - In this review of auditory psychophysics and perception, we cite some important books, research monographs, and research summaries from the past decade. Within auditory psychophysics, we have singled out some topics of current importance: Cross-Spectral Processing, Timbre and Pitch, and Methodological Developments. Complex sounds and complex listening tasks have been the subject of new studies in auditory perception. We review especially work that concerns auditory pattern perception, with emphasis on temporal aspects of the patterns and on patterns that do not depend on the cognitive structures often involved in the perception of speech and music. Finally, we comment on some aspects of individual difference that are sufficiently important to question the goal of characterizing auditory properties of the typical, average, adult listener. Among the important factors that give rise to these individual differences are those involved in selective processing and attention. PMID- 8624141 TI - Environmental psychology 1989-1994. AB - A review of research and theory on transactions between people and physical environments emphasizes new contributions to theory and empirical research published in major journals of environmental psychology, 1989-1994. Theories focused on arousal, load, stress, privacy-regulation, behavior settings, and transactional analysis; new theory increasingly incorporated situational and contextual variables. Empirical research emphasized field settings over the laboratory and employed increasingly diverse methods, populations, and cultures. Environmental design studies integrated scientific and applied goals through post occupancy evaluation. New findings concerned features of residences, work places, hospitals, schools, prisons, and larger community environments. New studies also addressed environmental stressors (e.g., temperature, noise); effects of attitudes and behaviors on conservation, crime, pollution, and hazards; and issues for neighborhoods, public places, and natural environments. Directions for the future include integrated theory to guide research, more design experiments, and development of conventions for case studies. PMID- 8624142 TI - Attachment and separation in young children. AB - Attachment theory is criticized for being based on momentary stressful situations, for being limited to behaviors and occur with the primary attachment figure, for including only overt behaviors in its paradigm, and for failing to consider multiple attachments at different stages of life. A model of psychological attonement is then presented and supported by several studies documenting behavioral, physiological, and biochemical responses to separations from parents and peers. PMID- 8624143 TI - The design and analysis of social-interaction research. AB - Static models of interacting persons measured at the interval level are reviewed. A discussion of the fundamental sources of variance and key design decisions in social-interaction research is presented. Outlined are the basic designs for social-interaction research and their proper analysis. Multilevel modeling is likely to become the most common data analysis method. Critical issues unique to social-interaction research are examined, particularly the effect of the partner on the interaction actor. Finally, illustrations of analyses from four extended examples are presented. PMID- 8624144 TI - Personality: individual differences and clinical assessment. AB - Research in clinical personality assessment continues to be produced at a high rate. The MMPI/MMPI-2 remains the most popular instrument for both clinical application and psychopathology research. Two other clinical personality instruments, the Rorschach and TAT, continue to find a place in research and clinical assessment. Some new instruments have surfaced recently to deal with areas, such as personality disorders, that have been considered inadequately addressed. There is a growing recognition that the Five-Factor Model is too superficial for clinical assessment that requires more refined and broadened patient information. Clinical personality assessment has successfully survived a number of past challenges. The newest challenge stems from the health-care revolution, in which managed-care providers are reluctant to pay for assessment because of shrinking funds. Psychologists need to develop models for incorporating assessment information into the treatment process. The future is likely to see more extensive research and theoretical development in this endeavor. PMID- 8624145 TI - Treatment of recalcitrant leg ulcers in cryoglobulinemia types I and II with plasmapheresis. PMID- 8624146 TI - Less than human, more than human. The use of mouse models in dermatologic research. PMID- 8624147 TI - Allergic contact dermatitis from doxepin cream. One-year patch test clinic experience. AB - BACKGROUND AND DESIGN: Several topical antihistamines are known to cause contact allergy. Premarket predictive patch testing with doxepin cream showed it to have "low risk of irritation and sensitization" on normal human skin. In our patch test clinic, we routinely test topical preparations, and over a recent 1-year period, we patch-tested doxepin cream, the standard screening tray, and other topical preparations on 97 patients with various pruritic dermatoses. When possible, patients with positive reactions to doxepin cream were patch-tested with its ingredients, and repeated open-application use tests were also performed with the product. RESULTS: Seventeen patients had relevant positive patch test reactions to doxepin cream and 80 had negative reactions. In 13 of the 17 patients with positive reactions, the diagnosis of allergic contact dermatitis to doxepin cream was confirmed by positive patch test reactions to both the active ingredient and the whole formulation of doxepin cream, by an observed positive use test reaction to doxepin cream, or by both. Of 14 patients who completed testing with doxepin cream ingredients, all had positive reactions to the whole formulation, and 12 had positive reactions to doxepin hydrochloride. Repeated open-application use tests with doxepin cream on normal skin resulted in positive eczematous responses in eight of 10 patients. Eight of the 17 patients had concurrent, relevant positive reactions to other patch tests, especially to fabric-finish resins and to ingredients of other topical preparations. Many had long-standing dermatitis, and each had used doxepin cream for several days to 1 year. Two patients appeared to have had systemic contact dermatitis. CONCLUSIONS: Diagnostic patch test clinic experience illustrates that doxepin cream is a contact sensitizer on inflamed skin. Oral doxepin should be avoided in patients with doxepin contact allergy. PMID- 8624149 TI - Similar Ro/SS-A autoantibody epitope and titer responses in annular erythema of Sjogren's syndrome and subacute cutaneous lupus erythematosus. AB - BACKGROUND AND DESIGN: Studies were conducted to determine whether certain types of Ro autoantibodies are unique to subacute cutaneous lupus erythematosus and annular erythema of Sjogren's syndrome. Ten American subacute cutaneous lupus erythematosus sera, 11 Japanese annular erythema of Sjogren's syndrome sera, and 39 control sera were tested by enzyme-linked immunosorbent assay with recombinant 52- and 60-kd Ro fusion proteins, native 60-kd Ro protein, and native La/SS-B protein. RESULTS: Japanese annular erythema of Sjogren's syndrome sera and American subacute cutaneous lupus erythematosus sera share several types of anti 52-kd and anti-60-kd Ro autoantibodies. However, these antibodies were found significantly more often and in significantly higher titers in sera from patients with Sjogren's syndrome who did not manifest these skin diseases. Patients with high Ro autoantibody titers were likely to have overt exocrine gland dysfunction. CONCLUSIONS: The failure to identify disease-specific Ro autoantibodies in subacute cutaneous lupus erythematosus and annular erythema of Sjogren's syndrome sera suggests that additional factors influence the development of these skin diseases. However, similarities in the Ro autoantibody responses and in the clinical features of subacute cutaneous lupus erythematosus and annular erythema of Sjogren's syndrome suggest that these two skin diseases might arise from a similar pathogenic process. PMID- 8624148 TI - Topical tretinoin (retinoic acid) improves early stretch marks. AB - BACKGROUND AND DESIGN: Stretch marks are disfiguring lesions usually caused by excessive stretching of skin. We investigated the response of early, clinically active stretch marks to topical 0.1% tretinoin (retinoic acid) cream. In a double blind, randomized, vehicle-controlled study, 22 patients applied either 0.1% tretinoin (n = 10) or vehicle (n = 12) daily for 6 months to the affected areas. Patients were evaluated by physical examination monthly and by analysis of biopsy specimens of stretch marks obtained before and at the end of therapy in comparison with untreated normal skin. RESULTS: After 2 months, patients treated with tretinoin had significant improvements in severity scores of stretch marks compared with patients who received vehicle (P < .05). After 6 months, eight (80%) of the 10 tretinoin-treated patients had definite or marked improvement compared with one (8%) of the 12 vehicle-treated patients (P = .002). Targeted stretch marks in patients treated with tretinoin had a decrease in mean length and width of 14% and 8%, respectively, compared with an increase of 10% (P < .001) and 24% (P = .008), respectively, in patients who received vehicle. There were no significant differences in various measures of quality and quantity of dermal collagen and elastic fibers in stretch marks when tretinoin and vehicle treatments were compared. CONCLUSIONS: Topical application of tretinoin significantly improves the clinical appearance of early, active stretch marks. The processes that are responsible for the clinical improvement remain unknown. PMID- 8624150 TI - Specific mucosal erosions in hypereosinophilic syndrome. Evidence for eosinophil protein deposition. AB - BACKGROUND: Mucosal erosions can be a presenting feature of the hypereosinophilic syndrome. The aim of this study was to analyze in situ the presence of eosinophil proteins and the state of eosinophil activation. Biopsy specimens of mucosal lesions and normal skin were taken from two men with oral and genital erosions typical of hypereosinophilic syndrome. Tissue sections were immunohistochemically labeled with anti-major basic protein, anti-eosinophil-derived neurotoxin, and anti-eosinophil peroxidase antibodies. The same specimens were also subjected to electron microscope examination. OBSERVATIONS: Eroded specimens displayed areas of eosinophil spongiosis within which extracellular deposits of eosinophil peroxidase, major basic protein, and eosinophil-derived neurotoxin were present. In normal skin, only a few eosinophils were present within the capillary lumen, and no extracellular deposits of these proteins were seen. Under the electron microscope, the cytoplasmic membranes of eosinophils located around the erosion were disrupted. Remnants of necrotic keratinocytes were found near these lysed eosinophils. CONCLUSION: As with other involved organs in hypereosinophilic syndrome, mucosal erosions seem to be the consequence of eosinophil protein release. PMID- 8624151 TI - Cutaneous Cryptococcus infection and AIDS. Report of 12 cases and review of the literature. AB - BACKGROUND: Cryptococcal infections occur in 6% to 13% of patients with acquired immunodeficiency syndrome (AIDS), most commonly infecting the central nervous system. Cutaneous lesions have been described morphologically as umbilicated papules, nodules, and violaceous plaques and can mimic molluscum contagiosum and Kaposi's sarcoma. Cutaneous lesions can present months prior to other signs of systemic infection. OBSERVATIONS: Cases of infection with cutaneous Cryptococcus and AIDS were reviewed and compared with cases reported in the literature. Among patients with Cryptococcus infection and AIDS seen at our institutions, 5.9% had skin lesions. All patients with cutaneous lesions had systemic involvement. Women were less commonly infected than men. There was no apparent predisposition associated with age, race, or human immunodeficiency virus infection risk factors. The median CD4 helper T-cell count was 0.024 X 10(9)/L (24/microL), and 44% (16/36) of the patients had previous opportunistic infections. Lesions were most commonly seen on the head and neck (78% [36/46]) and often mimicked molluscum contagiosum (54% [25/46]). The median serum and cerebrospinal fluid cryptococcal antigen titers were 1:32,768 and 1:512, respectively. Patients in our group did well with therapy (one death at 6 weeks, compared with 38% [13/34] mortality in the literature). There was no correlation between onset of lesions, number of lesions, CD4 helper T-cell count, or histopathologic characteristics. CONCLUSIONS: Disseminated Cryptococcus infection in AIDS presents with cutaneous lesions in up to 6% of cases. Clinicians need to be aware of the varied morphologic characteristics, since cutaneous lesions may present well in advance of other signs of systemic infection. PMID- 8624152 TI - A primary cutaneous non-T, non-B CD4+, CD56+ lymphoma. AB - BACKGROUND: Cutaneous lymphomas are heterogeneous clonal lymphoproliferative disorders originating from B or T lymphocytes. OBSERVATION: We describe a patient with a unique primary cutaneous lymphoma characterized by a bruise-like aspect of the skin lesions, a CD4+, CD43+, CD56+, CD2-, CD3-, CD8-, T-cell receptor negative phenotype of the medium-sized to large lymphoid tumor cells and an undetermined genotype (T-cell receptor beta and immunoglobulin heavy chain in germline configuration, no clonal T-cell receptor gamma population as detected after analysis with polymerase chain reaction combined with denaturing gradient gel electrophoresis) and fast relapse after radiotherapy. CONCLUSIONS: This non B, non-T cutaneous lymphoma cannot be classified by any current lymphoma classification. It seems to represent a new disease entity with peculiar clinical, histologic, and molecular features. PMID- 8624153 TI - Immunoregulatory events in the skin of patients with cutaneous T-cell lymphoma. AB - BACKGROUND: Involved skin of patients with cutaneous T-cell lymphoma, mycosis fungoides type, contains an increased number of bone marrow-derived epidermal cells that express class II major histocompatibility complex molecules and an infiltrate of both activated non-malignant and malignant T cells. However, the mechanism by which the T cells achieve and maintain their activated state is uncertain. The aim of this article is, therefore, to review recent studies from the literature dealing with immunoregulatory events in patients with mycosis fungoides and Sezary syndrome. OBSERVATIONS: The nonmalignant T cells seem to be activated through the T-cell receptor by lesional epidermal CD1a+CD36+ macrophagelike cells that, on a cell per cell basis, are more potent antigen presenting cells than normal CD1a+ Langerhans' cells present in uninvolved epidermis. In contrast, the malignant T cells have different activation requirements, because they can only be stimulated through antigen independent pathways, such as CDw60, CD28, and CD2. The malignant T cells produce T-helper (Th)-2 cytokines, and because interferon gamma (IFN-gamma)-producing Th1 cells are present in the early lesions of mycosis fungoides, nonmalignant tumor infiltrating T cells may represent Th1 cells. Because Th1 cytokines counteract Th2 cytokines, tumor-infiltrating T cells may potentially have the capacity to downregulate the growth of the malignant cells. CONCLUSION: The balance between progression vs remission in mycosis fungoides is related to complex interactions between the malignant T cells, nonmalignant T cells, and hyperstimulative antigen presenting cells present within the skin. PMID- 8624154 TI - If small plaque (digitate) parapsoriasis is a cutaneous T-cell lymphoma, even an 'abortive' one, it must be mycosis fungoides! PMID- 8624155 TI - Cutaneous lymphomas consist of a spectrum of nosologically different entities including mycosis fungoides and small plaque parapsoriasis. PMID- 8624156 TI - A child with a localized hair abnormality. Woolly hair nevus. PMID- 8624157 TI - Hyperkeratotic nodule. Keratoacanthomalike pilomatricoma. PMID- 8624158 TI - Hair loss in a 6-month-old child. Monilethrix. PMID- 8624160 TI - The idiopathic hypereosinophilic syndrome. PMID- 8624159 TI - Hair abnormality present since childhood. Pili annulati. PMID- 8624161 TI - Health care system reform and the American Academy of Dermatology. PMID- 8624162 TI - Hypertrichosis cubiti. PMID- 8624163 TI - Sertraline in the treatment of neurotic excoriations and related disorders. PMID- 8624164 TI - High-dose methotrexate-induced bullous variant of acral erythema. PMID- 8624165 TI - Risk of rupture of large abdominal aortic aneurysms. Disagreement among vascular surgeons. AB - BACKGROUND: Many patients with an abdominal aortic aneurysm (AAA) who are cared for by internists are not good candidates for surgery. Elective repair is usually deferred in these patients until the AAA reaches a diameter at which the estimated risk of rupture is believed to outweigh the operative risk. The risk of rupture is usually estimated by a consulting vascular surgeon, but whether these estimates are well-supported or consistent has not previously been assessed. OBJECTIVE: To determine the agreement among vascular surgeons about the risk of rupture of large AAAs. METHODS: All individual members of the Society for Vascular Surgery (Manchester, Mass) residing in the United States were mailed a survey asking for their estimates of the likelihood of rupture of large AAAs. RESULTS: The response rate was 66% and the 257 respondents who reported that they were practicing vascular surgeons constitute the study group. The median estimates of the 1-year risk of rupture were 20% for 6.5-cm AAAs and 30% for 7.5 cm AAAs, with one third of respondents estimating 50% or greater risk of rupture for 7.5-cm AAAs and nearly one third estimating 50% or greater risk of rupture for 6.5-cm AAAs. The responses spanned a wide range and were generally much higher than would be expected based on published data and estimates. CONCLUSIONS: This survey demonstrates profound disagreement among vascular surgeons about the risk of rupture of large AAAs, reflecting a lack of pertinent published data. Better data are necessary and are being collected. PMID- 8624166 TI - The cost of a food-borne outbreak of hepatitis A in Denver, Colo. AB - BACKGROUND: In 1992, a food-borne outbreak of hepatitis A associated with a catering facility in Denver, Colo, resulted in 43 secondary cases of hepatitis A and the potential exposure of approximately 5000 patrons. OBJECTIVES: To assess (1) disease control costs, including state and local health department personnel costs, provision and administration of immune globulin, and cost of extra hepatitis A serologic tests performed; (2) business losses; and (3) cost of the cases' illnesses. METHODS: Cost data were collected from hospitals, health maintenance organizations, health departments, laboratories, the caterer's insurance company, and the catering facility involved in the outbreak. RESULTS: The total costs assessed in the outbreak from a societal perspective were $809,706. Disease control costs were $689,314, which included $450,397 for 16,293 immune globulin injections and $105,699 for 2777 hours of health department personnel time. The cases' medical costs were $46,064, or 7% of the disease control costs. CONCLUSIONS: The cases' medical costs and productivity losses were only a minor component of the total cost of this outbreak. The high cost of food borne outbreaks should be taken into account in economic analyses of the vaccination of food handlers with inactivated hepatitis A vaccine. PMID- 8624167 TI - The use of living wills at the end of life. A national study. AB - BACKGROUND: Knowing more about who uses living wills may help explain their limited acceptance. METHODS: We analyzed the 1986 National Mortality Followback Survey, a random sample of all US deaths linked to a survey about decedents' use of living wills, their social and health status, and their use of medical services. Decedents with and without living wills were compared for differences in social and health characteristics and use of medical services. RESULTS: There were 16,678 decedents; 9.8% had a living will. Rates of use were higher for decedents who were white (10.7%), were female (11.0%), had private insurance (13.8%), had incomes of $22,000 or more (14.5%), or had college educations (18.7%). The use of living wills was lower among blacks (2.7%), Medicaid recipients (6.3%), those with incomes of less than $5,000 (7.5%), or those with less than 8 years of education (4.0%). Health was also related to use of living wills. Functionally independent persons were unlikely to have a living will (5.5%); use increased with dependency. Cognitive impairment made it less likely that a decedent had a living will (6.7%). Persons who died of cancer (16.4%) or pulmonary disease (11.4%) were more likely to have one. All demographic and health characteristics remained significant in multivariate analyses. Controlling for health status, decedents with living wills used more physician visits (five to nine vs two to four, P < .001) and hospital days (37 vs 30, P < .001). Although more likely to use hospices (19.5% vs 8.4%, P < .001) and half as likely to receive cardiopulmonary resuscitation or ventilatory support, they were still 20% more likely to die in the hospital. CONCLUSIONS: Patients who are black, poorly educated, underinsured, or cognitively impaired are least likely to prepare a living will. Decedents with living wills forgo specific treatments, but remain intensive users of routine medical services. PMID- 8624168 TI - Prognostic stratification in acute renal failure. PMID- 8624169 TI - Aspartame as a cause of allergic reactions, including anaphylaxis. PMID- 8624170 TI - Alcohol intake, cigarette smoking, and sleep disturbance. PMID- 8624171 TI - Perforation of great vessels during central venous line placement. PMID- 8624172 TI - Acute pancreatitis associated with sodium stibogluconate treatment in a patient with human immunodeficiency virus. PMID- 8624173 TI - Safety of antioxidant vitamins. AB - As a result of the many scientific and popular press reports of the benefits of antioxidant vitamins (vitamin A, beta-carotene, vitamin E, and ascorbic acid), it is estimated that 40% of the US population is consuming vitamin supplements. The efficacy of these supplements is not yet proved, and some have questioned their safety. Approximately 10 to 15 cases of vitamin A toxic reactions are reported per year in the United States, usually at doses greater than 100,000 IU/d. No adverse effects have been reported for beta-carotene. The frequency of vitamin E toxic reactions is not well delineated, but case reports are few at dosages less than 3200 mg/d. Ascorbic acid toxic reactions are rare at dosages less than 4 g/d. Despite a lack of clinical trial data, it seems that antioxidant vitamins are safe, although prudence might dictate their avoidance by women of childbearing potential, persons with liver disease or renal dysfunction, and those taking certain medications or undergoing specific laboratory tests. PMID- 8624174 TI - D-dimer testing and acute venous thromboembolism. A shortcut to accurate diagnosis? AB - D-dimer fragments can be measured easily in plasma and whole blood, and the presence or absence of D-dimer could be useful in the diagnostic evaluation of venous thromboembolism. We systematically reviewed the English literature for articles that compared D-dimer results with those of other tests for deep venous thrombosis or pulmonary embolism. Twenty-nine studies were selected for detailed review, and we noted wide variability in assay performance, heterogeneity among subjects, and failure to define absence or presence of venous thromboembolism by a comprehensive criterion standard for diagnosis. These methodologic problems limit the generalizability of the published estimates of D-dimer accuracy for deep venous thrombosis or pulmonary embolism, and the clinical utility of this potentially important test remains unproved. PMID- 8624175 TI - The internist's role in addressing violence. A review of current recommendations and a model for intervention. AB - Approximately 25,000 people die in the United States each year as the result of intentional homicide, and many millions more suffer the physical and emotional consequences of nonfatal interpersonal violence. Violence affects everybody's quality of life. The cost to the health care system is in the billions of dollars annually. Over the past 2 decades epidemiologic studies have defined risk factors for violence, and a number of physicians and medical groups have made recommendations for preventive action. As the largest physician group in the United States, internists have a clear role to play in carrying out the medical responsibility to help prevent violence. Internists should be aware of the signs and symptoms of violence in patients and be prepared to assist victims. To facilitate action against violence, a model for intervention is proposed that involves screening, counseling, and treatment for patients at greatest risk. PMID- 8624177 TI - The natural history of eosinophilia-myalgia syndrome in a tryptophan-exposed cohort in South Carolina. AB - BACKGROUND: In a previous study, we did follow-up on 418 patients who were exposed to tryptophan in 1989, of whom 47 (11%) had definite and 63 (9%) possible eosinophilia-myalgia syndrome (EMS). METHODS: We assessed mortality and clinical spectrum of illness since 1989 for 242 (58%) of the 418 tryptophan-exposed patients from the original study. To assess outcomes, we used hospital and death records, interviewer-administered questionnaires, physical examinations, and laboratory tests. RESULTS: During the follow-up interval, mortality from all causes was 19% in those with definite EMS, 7% in possible EMS, and 3% in those who were not ill. The age- and sex-adjusted mortality in those with definite EMS was more than 3 times that of the general population or of tryptophan users in the practice who were not ill. Six deaths (66%) among the definite EMS case patients occurred during the 18 months immediately after symptom onset. Compared with the tryptophan users who were not ill, survivors with definite EMS continued to report excess morbidity for 6 major EMS symptoms (myalgia, arthralgia, weakness, rash, alopecia, and sclerodermiform skin changes), but they also reported that the symptom number and severity diminished with time. None of the tryptophan users who were not ill in 1989 developed a symptom complex suggesting new EMS during the follow-up interval. CONCLUSIONS: This study assessing a tryptophan-exposed population found those persons who developed EMS during the 1989 epidemic were at increased risk for death, particularly early after disease onset. Survivors reported improvement or resolution of major symptoms, suggesting that the severity of EMS diminishes with time. We found no evidence of delayed onset of EMS in tryptophan users who were not ill in 1989, regardless of the brand used. PMID- 8624176 TI - Overweight, underweight, and mortality. A prospective study of 48,287 men and women. AB - BACKGROUND: The relative contributions of a low and high body mass index (BMI [weight in kilograms divided by height in meters squared]) to all-cause and cause specific mortality are still controversial. OBJECTIVE: To examine mortality rates in relation to BMI in a prospective cohort study of 48,287 Dutch men and women aged 30 to 54 years at baseline from 1974 to 1980. METHODS: During an average 12 year follow-up, 1319 deaths occurred. Relative risks (RRs) were calculated from the Cox proportional hazard model by using a BMI between 18.5 and 24.9 kg/m2 as the reference category. RESULTS: All-cause mortality was significantly increased in obese men (BMI, > or = 30 kg/m2; RR, 1.5; 95% confidence interval [CI], 1.1 2.0) and in underweight men (BMI, < 18.5 kg/m2; RR, 2.6; 95% CI, 1.8-3.9) but not in women. The increased risk in underweight men could be attributed to deaths within the first 5 years of follow-up and to lung cancer mortality among smokers. Coronary heart disease (CHD) mortality was about 3-fold higher among obese men and women. About 21% and 28% of CHD mortality in men and women, respectively, could be attributed to being overweight (BMI, > or = 25 kg/m2). The RR (but not the absolute risk) for CHD among obese men was still significant after adjustment for the presence of smoking, hypertension, hypercholesterolemia, and diabetes mellitus at baseline, and it was more pronounced for CHD among nonsmokers than among smokers (RR, 7.1; 95% CI, 2.3-21.7; and RR, 2.7; 95% CI, 1.5-4.7, respectively). CONCLUSIONS: Total mortality was increased in obese and underweight men but not in women. The increased mortality in overweight men was mainly attributable to CHD and, in underweight men, to early mortality and especially lung cancer mortality among smokers. PMID- 8624178 TI - Characteristics predicting incorrect metered-dose inhaler technique in older subjects. AB - OBJECTIVE: To determine whether cognitive status, hand strength, and demographic variables are predictive of correct use of metered-dose inhalers by older subjects. METHODS: Clinic patients (n = 29) and healthy volunteers (n = 42) older than 50 years with no previous or limited metered-dose inhaler use were enrolled. After cognitive (Mini-Mental State Examination) and hand strength assessments, subjects received extensive instruction in proper metered-dose inhaler technique. Technique was independently assessed by two evaluators immediately after instruction and 1 week later. Correct technique was defined as (1) activating the canister in the first half of inhalation, (2) continuing to inhale slowly and deeply, and (3) holding breath at full inspiration (5 seconds). Data for the two subject groups were pooled for analyses. RESULTS: The mean age of the subjects was 69.7 years. Forty subjects (56%) demonstrated correct metered-dose inhaler technique at 1 week. Logistic regression showed that hand strength measurement (odds ratio, 0.68; 95% confidence interval, 0.55 to 0.84), Mini-Mental State Examination score less than 24 (odds ratio, 3.66; 95% confidence interval, 1.07 to 12.4), and male gender (odds ratio, 5.01; 95% confidence interval, 1.07 to 23.5) were significant predictors of incorrect inhaler use. Correct use of the metered-dose inhaler was unrelated to age, education, or subject status. CONCLUSIONS: Clinicians should consider cognitive status and hand strength when metered-dose inhaler therapy is initiated for an older adult. Patients with cognitive impairment and hand strength deficits may require more extensive training, frequent follow-up, or alternative dosage forms. PMID- 8624179 TI - Weight change between age 50 years and old age is associated with risk of hip fracture in white women aged 67 years and older. AB - BACKGROUND: Although changes in body weight with aging are common, little is known about the effects of weight change on health in old age. OBJECTIVES: To study the effects of weight loss and weight gain from age 50 years to old age on the risk of hip fracture among postmenopausal white women aged 67 years and older and to determine if the level of weight at age 50 years modifies this risk. METHODS: The association between weight change and the risk of hip fracture was studied in 3683 community-dwelling white women aged 67 years and older from three sites of the Established Populations for Epidemiologic Studies of the Elderly. RESULTS: Extreme weight loss (10% or more) beginning at age 50 years was associated in a proportional hazards model with increased risk of hip fracture (relative risk [RR], 2.9; 95% confidence interval [CI], 2.0-4.1). This risk was greatest among women in the lowest (RR, 2.3; CI, 1.1-4.8) and middle (RR, 2.8; CI, 1.5-5.3) tertiles of body mass index at age 50 years. Among the thinnest women, even more modest weight loss (5% to < 10%) was associated with increased risk of hip fracture (RR, 2.3; CI, 1.0-5.2). Weight gain of 10% or more beginning at age 50 years provided borderline protection against the risk of hip fracture (RR, 0.7; CI, 0.4-1.0). The RRs for weight gain of 10% or more were protective only among women in the middle and high tertiles of body mass index at age 50 years and were not significant (middle tertile RR, 0.8; CI, 0.3-1.8; high tertile RR, 0.6; CI, 0.2-1.9). CONCLUSIONS: Weight history is an important determinant of the risk of hip fracture. Weight loss beginning at age 50 years increases the risk of hip fracture in older white women, especially among those who are thin at age 50 years; weight gain of 10% or more decreases the risk of hip fracture. Physicians should include weight history in their assessment of postmenopausal older women for risk of hip fracture. PMID- 8624180 TI - Predictive values of the character, timing, and complications of chronic cough in diagnosing its cause. AB - BACKGROUND: It is not clear whether careful history taking with detailed questioning of the characteristics of cough is diagnostically useful. OBJECTIVE: To determine if the character, timing, or complications of chronic cough were helpful in determining its cause. METHODS: A prospective, descriptive study of consecutive, unselected, immunocompetent patients referred to our university outpatient clinic because of chronic cough. All patients were evaluated by a previously published and validated systematic diagnostic protocol, a self administered questionnaire, and by observing the character of involuntary and voluntary coughs. The final diagnosis of the cause of cough required fulfillment of pretreatment criteria plus having cough disappear or substantially improve as a complaint with specific therapy. RESULTS: Eighty-eight patients met inclusion criteria and were fully evaluated. The mean +/- SD age was 53.1 +/- 16 years (range, 15-83 years) and 24 were males and 64 were females with a mean +/- SD history of cough for 6.6 +/- 9.8 years (range, 1 month-44 years). The cause of chronic cough was established in 86 (98%) of 88 patients. Eighty-one (92%) of 88 had cough disappear as a complaint. Cough was as a result of a single cause in 39% and multiple causes in 59%. Gastroesophageal reflux disease, postnasal drip syndrome, and asthma were the 3 most common causes of chronic cough and accounted for 90% of diagnoses. Gastroesophageal reflux disease, postnasal drip syndrome, and asthma were again found to be the 3 most common causes of chronic cough irrespective of patient estimated quantity of daily sputum production. These 3 conditions caused chronic cough in 99.4% of patients with the following characteristics: (1) nonsmoker; (2) not receiving an angiotensin-converting enzyme inhibitor drug; and (3) normal or nearly normal and stable chest radiograph. With respect to the spectrum and frequency of diagnoses and their interrelationships with the character, timing, and complications of cough, multiple stepwise linear regression analysis showed that none of the variability of the character, timing, or complications of cough could be explained by any specific diagnosis. CONCLUSIONS: A carefully taken history with detailed questioning of the character, timing, and complications of chronic cough is not likely to be useful in diagnosing the cause of cough. The cause can be determined and successfully treated with specific therapy in the greatest majority of cases. Chronic cough is often caused by multiple, simultaneously contributing causes. Postnasal drip syndrome, asthma, and gastroesophageal reflux disease remain the 3 most common causes of chronic cough and there is a clinical profile that nearly always predicts their presence in immunocompetent patients. PMID- 8624181 TI - A symptom provocation study of posttraumatic stress disorder using positron emission tomography and script-driven imagery. AB - BACKGROUND: Previous studies have used symptom provocation and positron emission tomography to delineate the brain systems that mediate various anxiety states. Using an analogous approach, the goal of this study was to measure regional cerebral blood flow changes associated with posttraumatic stress disorder (PTSD) symptoms. METHODS: Eight patients with PTSD, screened as physiologically responsive to a script-driven imagery symptom provocation paradigm, were exposed sequentially to audiotaped traumatic and neutral scripts in conjunction with positron emission tomography. Heart rate and subjective measures of emotional state were obtained for each condition. Statistical mapping techniques were used to determine locations of significant brain activation. RESULTS: Increases in normalized blood flow were found for the traumatic as compared with control conditions in right-sided limbic, paralimbic, and visual areas; decreases were found in left inferior frontal and middle temporal cortex. CONCLUSIONS: The results suggest that emotions associated with the PTSD symptomatic state are mediated by the limbic and paralimbic systems within the right hemisphere. Activation of visual cortex may correspond to the visual component of PTSD reexperiencing phenomena. PMID- 8624182 TI - The identification and validation of distinct depressive syndromes in a population-based sample of female twins. AB - BACKGROUND: Depression, a clinically heterogeneous syndrome, may also be etiologically heterogeneous. Using a prospective, epidemiologic, and genetically informative sample of adult female twins, we identify and validate a typology of depressive syndromes. METHODS: Latent class analysis was applied to 14 disaggregated DSM-III-R symptoms for major depression reported over the last year by members of 1029 female-female twin pairs. RESULTS: Seven classes were identified, of which 3 represented clinically significant depressive syndromes: (1) mild typical depression, (2) atypical depression, and (3) severe typical depression. Severe typical depression was characterized by comorbid anxiety and panic, long episodes, impairment, and help seeking. Atypical depression was similar in severity to mild typical depression, but was characterized by increased eating, hypersomnia, frequent, relatively short episodes, and a proclivity to obesity. Individuals with recurrent episodes tended to have the same syndrome on each occasion. The members of twin pairs concordant for depression had the same depressive syndrome more often than expected by chance and this resemblance was greater in monozygotic than in dizygotic pairs. CONCLUSION: In an epidemiologic sample of female twins, depression is not etiologically homogeneous, but is instead made up of several syndromes that are at least partially distinct from a clinical, longitudinal, and familial/genetic perspective. PMID- 8624183 TI - A controlled trial comparing buprenorphine and methadone maintenance in opioid dependence. AB - BACKGROUND: Buprenorphine is a partial agonist at the mu-opioid receptor that has been proposed as an alternative to traditional full agonist maintenance therapy for the treatment of opioid addiction. We report on a clinical trial in which the relative safety and efficacy of long-term fixed-dose buprenorphine maintenance was examined in comparison to low- and high-dose methadone maintenance. METHODS: Two hundred twenty-five treatment-seeking opioid addicts (46 women, 179 men) were randomly assigned to receive, in a double-blind manner, either 8 mg/d of buprenorphine, 30 mg/d of methadone, or 80 mg/d of methadone maintenance over a 1 year period. Objective and subjective measures of efficacy (urine toxicology, retention, craving, and withdrawal symptoms) were examined at the study midpoint and at termination, and safety data were tabulated over the entire 52-week study period. RESULTS: Patients assigned to high-dose methadone maintenance performed significantly better on measures of retention, opioid use, and opioid craving than either the low-dose methadone or the buprenorphine group at both 26-week and 52-week time points. Performance on these measures was virtually identical between the latter two groups. No serious adverse health effects attributable to buprenorphine were noted. CONCLUSIONS: Buprenorphine maintenance at 8 mg/d appears to be less than optimally efficacious under the conditions of the present study. Continued research is needed to reconcile these findings with the more positive results reported by other investigative groups. There are no apparent health risks associated with long-term buprenorphine maintenance at this dosage. PMID- 8624184 TI - Sustained cocaine abstinence in methadone maintenance patients through voucher based reinforcement therapy. AB - BACKGROUND: Chronic cocaine abuse remains a serious and costly public health problem. This study assessed the effectiveness of a voucher-based reinforcement contingency in producing sustained cocaine abstinence. METHODS: A randomized controlled trial compared voucher-based reinforcement of cocaine abstinence to noncontingent voucher presentation. Patients were selected from 52 consecutively admitted injecting heroin abusers in a methadone maintenance treatment program. Patients with heavy cocaine use during baseline period (N = 37) participated. Except where otherwise indicated, the term cocaine abuse is used in this article in a generic sense and not according to the DSM-III-R definition. Patients exposed to abstinence reinforcement received a voucher for each cocaine-free urine sample (ie, negative for benzoylecgonine) provided three times per week throughout a 12-week period; the vouchers had monetary values that increased as the number of consecutive cocaine-free urine samples increased. Control patients received noncontingent vouchers that were matched in pattern and amount to the vouchers received by patients in the abstinence reinforcement group. RESULTS: Patients receiving vouchers for cocaine-free urine samples achieved significantly more weeks of cocaine abstinence (P = .007) and significantly longer durations of sustained cocaine abstinence (P = .001) than controls. Nine patients (47%) receiving vouchers for cocaine-free urine samples achieved between 7 and 12 weeks of sustained cocaine abstinence; only one control patient (6%) achieved more than 2 weeks of sustained abstinence. Among patients receiving vouchers for cocaine free urine samples, those who achieved sustained abstinence ( > or = 5 weeks) had significantly lower concentrations of benzoylecgonine in baseline urine samples than those who did not achieve sustained abstinence (P < or = .01). Patients receiving voucher reinforcement rated the overall treatment quality significantly higher than controls (P = .002). CONCLUSION: Voucher-based reinforcement contingencies can produce sustained cocaine abstinence in injecting polydrug abusers. PMID- 8624185 TI - Israeli preschoolers under Scud missile attacks. A developmental perspective on risk-modifying factors. AB - BACKGROUND: The devastating effects of traumatic events on children are modulated by risk and protective factors. This study examines the differential effects of traumatic displacement of preschool children and their families following Scud missile attacks on Israel during the Persian Gulf War. METHODS: Three groups participated in the study: families displaced after their houses were damaged, undisplaced families from the same neighborhood (without home damage), and families from a distant city that was threatened but not directly attacked. Data concerning the traumatic event, the child (personality, internalizing, externalizing, and stress symptoms), the mother (Symptom Checklist-90-Revised), and the family (Family Adaptability and Cohesion Evaluation Scales) were gathered 6 months after the end of the war. RESULTS: Displaced children and mothers showed higher externalizing and stress symptom levels compared with undisplaced and threatened subjects. Destruction of the house and displacement, but not mere distance from the missile impact, explained symptomatic behavior. Inadequate family cohesion predicted symptomatic reaction for 3- and 4-year-old children but not for older ones. CONCLUSION: Both human and nonhuman factors contribute to the preschool child's adaptive mechanisms that regulate environmental stressful stimuli. These risk-modifying factors become more autonomous of caretakers with increasing age. PMID- 8624186 TI - Maldistribution of interstitial neurons in prefrontal white matter of the brains of schizophrenic patients. AB - BACKGROUND: The cortical subplate is a transitory structure involved in the formation of connections in developing cerebral cortex. Interstitial neurons, normally present in subcortical white matter (WM) of the adult brain, have escaped the programmed cell death that eliminates most subplate neurons. Previous investigations indicated a maldistribution of one population of interstitial neurons in the WM of brains of schizophrenic patients, suggesting a defect of the subplate during brain development. METHODS: Three histochemically or immunocytochemically defined neuronal populations were studied in WM beneath the middle frontal gyrus of 20 schizophrenic patients and 20 matched control subjects. RESULTS: Brains of schizophrenic patients showed significant changes in the distribution of the three neuronal populations: microtubule-associated protein 2 and nonphosphorylated neurofilament-immunoreactive neurons showed a decreased density in superficial WM and an increased density in deeper WM. Nicotinamide adenine dinucleotide phosphate-diaphorase neurons were reduced in superficial WM and showed variable densities in deeper WM. Thirty-five percent of the brains of schizophrenic patients but no brains of the control subjects showed a maldistribution of neurons toward deeper WM with at least two of the three markers. Changes in neuronal distribution were not linked to age, gender, autolysis time, or subtype of schizophrenia. CONCLUSIONS: Selective displacement of interstitial WM neurons in the frontal lobe of brains of schizophrenic patients may indicate alteration in the migration of subplate neurons or in the pattern of programmed cell death. Both could lead to defective cortical circuitry in the brains of schizophrenic patients. PMID- 8624187 TI - A prospective 4-year follow-up study of attention-deficit hyperactivity and related disorders. AB - BACKGROUND: Previous cross-sectional data showed that children and adolescents with attention-deficit hyperactivity disorder (ADHD) are at increased risk of comorbid conduct, mood, and anxiety disorders as well as impairments in cognitive, social, family, and school functioning. However, longitudinal data were needed to confirm these initial impressions. METHODS: Using DSM-III-R structured diagnostic interviews and raters blinded as to diagnosis, we reexamined psychiatric diagnoses at 1- and 4-year follow-ups in children with ADHD and controls. In addition, subjects were evaluated for cognitive, achievement, social, school and family functioning. RESULTS: Analyses of follow up findings revealed significant differences between children with ADHD and controls in rates of behavioral, mood, and anxiety disorders, with these disorders increasing markedly from baseline to follow-up assessments. In addition, children with ADHD had significantly more impaired cognitive, family, school, and psychosocial functioning than did controls. Baseline diagnosis of conduct disorder predicted major depression and bipolar disorder at follow-up, and anxiety disorders at baseline predicted anxiety disorders at follow-up. CONCLUSION: These results confirm and extend previous retrospective results indicating that children with ADHD are at high risk of developing a wide range of impairments affecting multiple domains of psychopathology such as cognition, interpersonal, school, and family functioning. These findings provide further support for the value of considering psychiatric comorbidity in both clinical assessment and research protocols involving children with ADHD. PMID- 8624188 TI - The contribution of alpha 2-noradrenergic mechanisms of prefrontal cortical cognitive function. Potential significance for attention-deficit hyperactivity disorder. AB - This article aims to review research in nonhuman primates demonstrating that norepinephrine can enhance the cognitive functioning of the prefrontal cortex through actions at alpha 2 A-adrenergic receptors postjunctional to noradrenergic terminals. As prefrontal cortex cognitive deficits are prominent in several psychiatric disorders, including attention-deficit hyperactivity disorder, these basic findings may have relevance for the development of novel pharmacotherapies. PMID- 8624189 TI - Catching up on schizophrenia. The Fifth International Congress on Schizophrenia Research, Warm Springs, Va, April 8-12, 1995. AB - It is axiomatic that the diversity and ingenuity of strategies in the study of schizophrenia bear testimony to the complexity of this disorder. Accordingly, the opportunity for investigators of diverse interests to share their latest findings and be informed of developments in key areas of science is critical to advance our understanding of schizophrenia. Nearly 700 investigators from around the world gathered at Warm Springs, Va, to present their data at the Fifth International Congress on Schizophrenia Research (April 8-12, 1995). This 5-day conference, held every other year alternating with the European Winter Workshop on Schizophrenia, is co-organized by S. Charles Schulz, MD, Case Western Reserve University, Cleveland, Ohio, and Carol Tamminga, MD, Maryland Psychiatric Research Center, University of Maryland at Baltimore. Drs Schulz and Tamminga first held the Congress in 1987 in Clearwater, Fla, with 170 investigators presenting. The Congress has flourished since then and is now the largest research meeting devoted solely to schizophrenia. The Congress organizers, the program coordinator (Jeffrey Lieberman, MD, Hillside Hospital, Glen Oaks, NY), and the Congress coordinator (Susan Nusbaum, Maryland Psychiatric Research Center) are congratulated on the success of this event and its contribution to fostering initiatives and collaborations within schizophrenia research. PMID- 8624190 TI - The evolution of endocrine surgery as a subspecialty of general surgery. Fragmentation or enhancement? PMID- 8624191 TI - Prospective surveillance for perioperative venous thrombosis. Experience in 2643 patients. AB - BACKGROUND: Patients who undergo neurosurgical procedures are at high risk for perioperative deep vein thrombosis (DVT) and pulmonary embolism (PE), which have been reported in 6% to 43% of these patients. OBJECTIVES: To (1) determine the utility of prospective DVT surveillance in patients who undergo neurosurgical procedures by using venous duplex ultrasound scanning (VDUS), (2) assess the efficacy of DVT prophylaxis (elastic stockings and intermittent pneumatic compression), (3) identify subgroups of patients who are at higher risk, and (4) determine whether DVT surveillance would reduce the incidence of fatal PE. DESIGN: All patients had undergone preoperative VDUS of both lower extremities, and postoperative VDUS was performed on days 3 and 7, and weekly thereafter until patients were ambulatory or discharged. PATIENTS: During a 5-year period, 2643 patients who underwent neurosurgical procedures were enrolled in prospective DVT surveillance. SETTING: University-affiliated community hospital. RESULTS: Acute DVT was diagnosed in 147 (5.6%) of the 2643 patients. Eighty-one percent of the patients with acute DVT were asymptomatic at the time of diagnosis. Deep vein thrombosis developed de novo in the proximal veins in 98% of the patients. Patients in whom a craniotomy was done had a significantly higher risk for DVT (7.7%, P = .006), and patients who underwent cervical or lumbar spinal surgical procedures had a significantly lower risk (1.5%, P < .001). Among those patients in whom a craniotomy was performed for treatment of a tumor and who had DVT, 87% had malignant neoplasms. Significant lower-extremity neuromotor dysfunction was present in 69% of all patients with DVT, and this finding predominated among patients with DVT in the subgroups with a lower risk. A PE was diagnosed in 5 patients (0.19%) while they were hospitalized, and a PE was fatal in 2 (0.07% of all patients). CONCLUSIONS: Most perioperative DVTs were clinically silent and formed spontaneously in proximal venous segments where there would be a risk for a PE. The overall incidence of DVT (5.6%) was low, suggesting effective DVT prophylaxis. Patients who underwent spinal surgical procedures were at a significantly lower risk for DVT, and future surveillance is not indicated in this patient group unless other conditions exist (paralysis, malignancy). Patients in whom a craniotomy was performed had a significantly higher risk of DVT, particularly when other risk factors existed. The low incidence of a fatal PE (0.07%) reflected that early detection and treatment of proximal DVT were facilitated by prospective VDUS surveillance in these patients. PMID- 8624192 TI - Surgical aspects of patients with adenocarcinoma of the stomach operated on for cure. AB - BACKGROUND: A retrospective study was performed to evaluate our recent results of curative gastric resections for adenocarcinoma. METHODS: Between 1979 and 1988, 187 patients fulfilled study entry criteria. This group of patients composes 64% of all patients with tumors arising distal to the gastroesophageal junction. Tumors arising in the region of the gastroesophageal junction were excluded. Patients were classified according to the American Society of Anesthesiologists physical status classification ( > or = 3, 56%) and Eastern Cooperative Oncology Group performance status ( > or = 2, 44%). Histologic characteristics were re reviewed. INTERVENTIONS: Subtotal and total gastrectomies were performed in 78% and 22% of the patients, respectively. Extended lymph node dissections were performed selectively (5%). Adjuvant chemotherapy and radiotherapy were employed in 3% and 2% of patients, respectively. RESULTS: Postoperative morbidity and mortality were 27% and 4%, respectively. Synchronous splenectomy (P = .06) and type of gastric resection (P = .06) showed a borderline association with postoperative complications, but did not affect postoperative mortality. With a median follow-up time of 47 months in all patients, and a median of 9 years in patients still alive, the 5- and 10-year overall survival rates (Kaplan-Meier method) were 48% and 32%, respectively. In univariate survival analysis, age, American Society of Anesthesiologists classification, stage, tumor diameter, serosal extension of tumor lymph node metastases, and type of resection showed prognostic significance. In the Cox multivariate analysis, however, only serosal extension of tumor (P < .001) and lymph node metastases (P = .02) were independent prognostic factors. CONCLUSIONS: Despite the older age and comorbid conditions of patients with gastric cancer, 5-year survival was achieved in half the patients by standard radical operations. Until appropriate controlled prospective studies are performed, total gastrectomy, splenectomy, and extended lymph node dissection should not be routinely adopted, given their unproven efficacy and potentially increased morbidity and mortality. PMID- 8624193 TI - Outcome of respiratory symptoms after antireflux surgery on patients with gastroesophageal reflux disease. AB - OBJECTIVE: To investigate factors predictive of relief of respiratory symptoms associated with gastroesophageal reflux disease (GERD). DESIGN: A case series of patients with GERD and respiratory symptoms undergoing fundoplication from 1987 to 1994 at a tertiary care university hospital. PATIENTS: Of 118 patients undergoing fundoplication for cardinal symptoms of GERD, 63 had respiratory symptoms. Postoperative follow-up information was available in 50 patients at a median of 3 years. INTERVENTIONS: The presence of GERD was documented on the basis of barium swallow, esophagoscopy, esophageal manometry, and 24-hour pH studies. A standardized questionnaire was used to score symptoms. A Nissen fundoplication was performed in 39 patients, a Collis-Belsey fundoplication in 3 patients. MAIN OUTCOME MEASURES: A repeat standardized questionnaire was used to evaluate the response to surgery for each symptom experienced. Univariate analysis was performed to evaluate factors influencing outcome. RESULTS: Respiratory symptoms were present in 53% (63/118) of patients with GERD. Fundoplication relieved the respiratory symptoms in 76% (38/50) of the patients. Reflux symptoms were relieved in 86% (43/50) of the patients. Abnormalities of esophageal motility were present in 34% (17/50) of the patients, and these were significantly more common in patients who did not experience relief of their respiratory symptoms (9/12 vs 8/38, chi 2 = 9.54, P = .002). CONCLUSIONS: Respiratory symptoms are common in patients with GERD. Unlike classic reflux symptoms, the beneficial effects of antireflux surgery on respiratory symptoms are less predictable. The probability of relief of these respiratory symptoms with antireflux surgery is directly dependent on esophageal motor function. PMID- 8624194 TI - Long-term results with the Kasai operation for biliary atresia. AB - OBJECTIVE: To evaluate long-term outcome in a series of children with biliary results atresia treated by portoenterostomy. DESIGN: Case series of consecutive infants with biliary atresia with 10-year follow-up. Data were obtained by retrospective chart review or phone interview. SETTING: A tertiary academic medical center and regional children's hospital. PATIENTS: A consecutive series of 104 infants diagnosed with biliary atresia more than 10 years ago were evaluated. Eighty-nine had totally obliterated extrahepatic ducts, 4 had proximal hilar cysts (correctable type), and 11 had patency of the gallbladder and distal common duct. INTERVENTIONS: Ninety-eight patients underwent biliary reconstruction and 6 had exploration only. Seventy-four infants underwent reconstruction using a Rouxen-Y with exteriorization. The 11 infants with distal patency underwent a portocholecystostomy ("gallbladder Kasai"). The remainder had various modifications of the Kasai operation. MAIN OUTCOME MEASURES: Survival, liver function, complications, growth, and development. RESULTS: The 6 patients who did not have a portoenterostomy died. Of the 98 who had a reconstruction, 63 died (mean age at death, 27 months; median, 13.4 months), 10 following liver transplantation. Twelve of the 35 survivors ultimately required liver transplants. Twenty-three children are alive more than 10 years after portoenterostomy without the need for transplantation. Two thirds have experienced some manifestation of portal hypertension (ie, variceal bleeding, hypersplenism, ascites). Nineteen patients (79%) are anicteric with normal liver synthetic function and are in an age-appropriate school grade or working and living independently. CONCLUSIONS: We found that surgical correction of biliary atresia offers long-term survival for about one quarter of patients, provides palliation until liver transplantation becomes necessary, and if surgical correction is not feasible, biliary atresia is uniformly fatal. The outlook is good for those children who survived more than 10 years and justifies continued attempts to establish bile flow in infants with biliary atresia. PMID- 8624195 TI - Prospective study of the incidence, timing and treatment of pouchitis in 104 consecutive patients after restorative proctocolectomy. AB - OBJECTIVE: To determine the incidence timing and effectiveness of treatment of symptomatic pouchitis following restorative proctocolectomy with ileal J-pouch anal anastomosis. DESIGN: A cohort analytical study. SETTING: University hospitals, a tertiary referral center; all subjects entered into the study followed up for a minimum of 12 months (mean follow-up, 40 months). PATIENTS: One hundred four consecutive patients undergoing restorative proctocolectomy with ileal J-pouch anal anastomosis for either ulcerative colitis (n = 97) or familial adenomatous polyposis (n = 7) between June 1986 and December 1994. INTERVENTIONS: Patients with symptomatic pouchitis were treated with either oral metronidazole or ciprofloxacin. OUTCOMES: Diagnosis of pouchitis was determined by clinical symptoms and confirmed with endoscopy. Response to oral antibodies was determined by resolution of symptoms. RESULTS: Fifty-two patients (50%) experience at lest 1 episode of pouchitis. The first episode of pouchitis occurred within the first 12 months after restoration of intestinal continuity in 56% of the cases. In 2 patients it occurred after 30 months. Response to antibiotic treatment was 96%. Two thirds of patients had multiple episodes. Chronic pouchitis occurred in 6 patients, necessitating pouch removal in 2. CONCLUSIONS: The incidence of pouchitis after ileal J-pouch anal anastomosis is approximately 50% with two thirds of these patients having multiple episodes. Chronic pouchitis occurs in a minority of patients. In chronic pouchitis, the risk of pouch loss is substantial. PMID- 8624196 TI - A prospective study of carbon dioxide-digital subtraction vs standard contrast arteriography in the evaluation of the renal arteries. AB - OBJECTIVE: To compare carbon dioxide-digital subtraction arteriographic (CO2-DSA) images of renal artery anatomy with standard iodinated contrast arteriographic (ICA) images. DESIGN: One hundred patients with vascular disease who required abdominal aortography were evaluated by both CO2-DSA and ICA modalities. Two blinded readers interpreted arteriograms for the degree of renal artery stenosis, and a third reader was employed to resolve differences in reader interpretations. SETTING: University medical center. MAIN OUTCOME MEASURES: The sensitivity, specificity, negative predictive value, positive predictive value, and accuracy were calculated for the ability of CO2-DSA to demonstrate a 60% or greater stenosis of the main renal artery; kappa values for CO2-DSA and ICA were calculated to assess intraobserver variability. RESULTS: Of the 200 main renal arteries imaged, 17 (9 by means of CO2-DSA), 8 means of ICA) were eliminated because of inadequate visualization of the renal artery. In identifying a renal artery stenosis of 60% or greater, CO2-DSA had a sensitivity of 0.83, specificity of 0.99, positive predictive value of 0.94, and negative predictive value of 0.98. The overall accuracy was 0.97. The kappa was 0.75 for CO2-DSA and 0.70 for ICA, hence, the variation in the interpretations of CO2-DSA and ICA were comparable. CONCLUSION: Images by means of CO2-DSA accurately reflect pathologic changes in renal arteries and are thus useful in the diagnosis of clinically occult occlusive renal artery disease in patients at risk of contrast medium related nephrotoxicity. PMID- 8624197 TI - Laparoscopic appendectomy for complicated appendicitis. AB - BACKGROUND: Acute gangrenous and perforating appendicitis are associated with an increased risk for postoperative complications and have been considered a relative contraindication of laparoscopic appendectomy. OBJECTIVE: To determine the complication rate following laparoscopic appendectomy for gangrenous of perforating appendicitis. DESIGN: A retrospective analysis of patients who underwent laparoscopic appendectomy for gangrenous or perforating appendicitis. SETTING: A multispecialty clinic. RESULTS: Fifteen patients underwent laparoscopic appendectomy for gangrenous appendicitis and 19 patients for perforating appendicitis. In the gangrenous appendicitis group, average operating time was 85 minutes; average length of hospitalization, 2 days; and morbidity rate, 7% (one patient with abdominal abscess). The perforating appendicitis group had an average operating time of 84 minutes, hospitalization of 7 days, and a morbidity rate of 42%. This morbidity included five patients (26%) who developed intra-abdominal abscesses, two patients (10%) in whom wound infections developed, and one patient (5%) who died of Candida sepsis and multisystem organ failure. PMID- 8624198 TI - Preoperative "chemoradiation" for stages II and III rectal carcinoma. AB - OBJECTIVE: To determine whether preoperative administration of combination chemotherapy and external beam irradiation ("chemoradiation") for patients with stage II or stage III rectal carcinoma had an impact on perioperative morbidity on oncologic outcome, as compared with patients not receiving preoperative chemoradiation. DESIGN: A group of patients with stage II or stage III rectal carcinoma receiving preoperative chemoradiation were followed up prospectively and compared in a nonrandomized fashion with an inception cohort group of similar patients. SETTING: Northwestern Memorial Hospital, Chicago, Ill, a tertiary care academic medical center. PATIENTS: Thirty patients with rectal carcinoma undergoing preoperative chemoradiation were compared with 56 patients not undergoing preoperative chemoradiation, and also with a subset group of 24 patients who received standard postoperative adjuvant chemoradiation. INTERVENTION: External beam radiation, 45 to 50 Gy, was delivered concurrently with fluorouracil and mitomycin 4 to 8 weeks prior to surgical resection. MAIN OUTCOME MEASURES: Patients were followed up at regular intervals for either tumor recurrence or death. In addition, the group receiving preoperative chemoradiation was evaluated for major preoperative morbidity. RESULTS: All patients agreeing to preoperative chemoradiation completed therapy. Perioperative major morbidity in this group (13%) was comparable to previously published results. Of the 56 patients with stage II or stage III rectal carcinoma not receiving preoperative chemoradiation, only 24 (43%) completed standard postoperative adjuvant chemoradiation. Patients receiving preoperative chemoradiation (n = 30), patients not receiving preoperative chemoradiation (n = 56), and the subset of the group not receiving preoperative chemoradiation who completed standard postoperative chemoradiation (n = 24) were followed up for a mean of 39 months, 31 months, and 32 months, respectively. Five-year actuarial local control rates were 96%, 83%, and 88%, respectively. Disease-free-survival rates were 80%, 57%, and 47%, respectively. Overall survival rates were 85%, 48%, and 78%, respectively. CONCLUSIONS: Preoperative chemoradiation in the treatment of stage II or stage III rectal carcinoma is well tolerated and not associated with an increase in subsequent perioperative major morbidity. In addition, local control, disease free survival, and overall survival compare favorably with a nonrandomized inception cohort group of patients receiving standard postoperative adjuvant chemoradiation. PMID- 8624199 TI - Critical analysis of the operative treatment of Hirschsprung's disease. AB - OBJECTIVE: To critically analyze complications and long-term results of the operative treatment of Hirschsprung's disease. DESIGN: Medical records of patients with Hirschsprung's disease were reviewed retrospectively. Follow-up was obtained using a standardized telephone questionnaire. SETTING: Major pediatric referral center. PATIENTS: Eighty-two infants and children (68 boys, 14 girls) were treated for Hirschsprung's disease during a 20-year period (1975 to 1994). The age at diagnosis was younger than 30 days in 47 neonates (57%), 30 days to 1 year in 22 infants (27%), and older than 1 year in 13 children (16%). Aganglionosis was limited to the rectosigmoid region in 66 patients (81%). Fifty five Soave (endorectal) and 27 Duhamel (retrorectal) primary pull-through operations were performed. MAIN OUTCOME MEASURES: Postoperative complications, reoperations, hospitalization, and current bowel habits. RESULTS: Eighteen children (67%) undergoing the Duhamel operation recovered uneventfully compared with 33 children (60%) undergoing the Soave operation. The complications following the Duhamel operation included enterocolitis in five cases (19%), rectal achalasia in four cases (15%), and persistent rectal septum in two cases (7%). Additional operations, which included myomectomy, rectal septum division, diverting enterostomy, and sphincterotomy, were required in seven patients (26%). Only one patient required more than one reoperation. In contrast, complications following the Soave operation included enterocolitis in 15 cases (27%), rectal stenosis in 12 (22%), anastomotic leak in four (7%), late perirectal fistula in three (5%), rectal prolapse in one (2%), and recurrent severe constipation in one (2%). Sixteen patients (29%) required additional operations, including diverting enterostomy, myomectomy, redo pull-through, sphincterotomy, fistulectomy, and revision of rectal prolapse. In this group nearly two reoperative procedures per patient were required. Telephone follow-up (mean, 89.3 months) after pull-through operations in 61 patients (74%) showed a mean of 2.8 stools per day, with 13 patients (21%) requiring daily medications. CONCLUSIONS: The most common operations (Soave and Duhamel) for Hirschsprung's disease result in an uneventful recovery in only 60% to 67% of patients. Although both Soave and Duhamel pull through operations have nearly identical reoperation rates (26% vs 29%), complications after Soave pull-through operations often require multiple, more extensive procedures. Short-term total continence rates for both procedures are less than 50%, however, 100% became continent by 15 years after the pull-through procedure. Further refinement in operative technique and close follow-up are warranted. PMID- 8624200 TI - Mechanisms of neurologic deficits and mortality with carotid endarterectomy. AB - OBJECTIVE: To evaluate the incidence and etiology of perioperative complications of carotid endarterectomy. DESIGN: Retrospective review of carotid endarterectomies performed over 13 years. Risk factors, indications, results of electroencephalographic (EEG) monitoring, and outcomes were evaluated. SETTING: University medical center. PATIENTS: Three hundred sixty-seven consecutive primary carotid endarterectomies were performed on 336 patients. Indications for operation included transient ischemic attack (48.5%), asymptomatic stenosis (24%), stroke (17%), nonlateralizing ischemia (9.5%), and stroke-in-evolution (1%). MAIN OUTCOME MEASURES: Postoperative neurologic deficits (permanent and transient) and deaths were correlated with preoperative symptoms, probable mechanism of the neurologic event, intraoperative EEG changes, and the use of intraoperative shunts. RESULTS: Four new permanent neurologic deficits (1.1%) and one transient postoperative deficit were noted. Of the five deficits, three were related to undiagnosed intraoperative cerebral ischemia and two were related to perioperative emboli. Three perioperative deaths (0.8%) occurred: two of myocardial infarction and one of an intracerebral hemorrhage from a ruptured arteriovenous malformation. Intraoperative EEG tracings for the most recent consecutive 175 procedures were analyzed. Shunts were used in 45 patients (26%), 38 of whom demonstrated significant EEG changes with carotid clamping. CONCLUSIONS: Carotid endarterectomy can be performed with a low risk of stroke (1.1%) and death (0.8%). Stroke was due to cerebral ischemia or embolization. With meticulous surgical technique, death is due to myocardial ischemia and not neurologic events. PMID- 8624201 TI - Transient systolic hypotension. A serious problem in the management of head injury. AB - OBJECTIVE: To determine the frequency and clinical impact of transient systolic hypotension (systolic blood pressure < 100 mm Hg) in patients with severe anatomic head injury. DESIGN: Retrospective case-control study. SETTING: Urban level 1 trauma center. PATIENTS: Consecutive trauma patients admitted to the intensive care unit (ICU) with severe anatomic head injury, defined as Head and Neck Abbreviated Injury Scale Score of 4 or higher. One thousand thirteen trauma patients were admitted to the ICU during the study period, 157 of whom met inclusion criteria. MAIN OUTCOME MEASURES: Acute mortality, defined as death during initial ICU admission, and functional status of ICU survivors, assessed as level of function sufficient for discharge to home. RESULTS: One hundred fifty seven patients with severe head injury had a total of 831 episodes of systolic hypotension. Fifty-five percent of the patients suffered at least one event. Patients were grouped by total number of low systolic blood pressure events and by average number of events per ICU day. The total number of hypotensive events was associated with increased mortality rates and decreased rate of discharge to home. Average daily frequency of events was associated with increased mortality rates. After stratification by admission Glasgow Coma Scale score, the effects were most dramatic in patients with an initial Glasgow Coma Scale score higher than 8. CONCLUSIONS: Transient hypotension is common in the ICU and is associated with increased acute mortality and decreased functional status in patients with head injury. The impact of this secondary insult is greatest in patients with less severe primary injury. Strict avoidance of hypotension through enhanced monitoring and active treatment appears to be important, especially in patients with higher presenting Glasgow Coma Scale scores. PMID- 8624202 TI - Laparoscopic cholecystectomy in acute cholecystitis. What is the optimal timing for operation? AB - OBJECTIVE: To review the results of laparoscopic cholecystectomy (LC) in patients with acute cholecystitis with attention to cost and clinical outcome. DESIGN: Retrospective study. SETTING: Large private metropolitan teaching hospital. PATIENTS: Four hundred forty-six patients had LCs at our institution between January 1993 and February 1995. Acute cholecystitis, confirmed by clinical, laboratory, operative, and histopathological findings, was present in 60 patients. MAIN OUTCOME MEASURES: The medical history, laboratory findings, gallbladder ultrasounds, timing of operation from the onset of symptoms, conversion rates to open procedures, operative times, intraoperative findings, complications, postoperative length of stay, cost of operative procedures and hospitalizations, and convalescence times were collected. RESULTS: Laparoscopic cholecystectomy was attempted in 16 patients within 72 hours of the onset of symptoms of acute cholecystitis (group 1), in 19 patients with symptoms between 4 and 7 days (group 2), and in 25 patients with symptoms lasting more than 7 days (group 3). The only factor (eg, preoperative laboratory and ultrasound findings) that affected the outcome of the operation was duration of symptoms prior to operation. Patients who had LC done within 72 hours of the onset of symptoms had lower rates of conversion to open procedures, less difficult operations, shorter operative times, less costly procedures, and a shorter convalescence than those with symptoms for longer than 72 hours prior to operation. The conversion rates in patients operated within and after 72 hours were 12% and 30%, respectively. There were no bile duct injuries and no mortalities. CONCLUSIONS: Laparoscopic cholecystectomy can be performed safely in most patients with acute cholelithiasis. However, we found that the duration of symptoms prior to LC affected the outcome; the conversion rates, hospital costs, and convalescence times increased in operated-on patients with symptoms for more than 72 hours. In our opinion, interval cholecystectomy may be a superior option in this latter group of patients. PMID- 8624203 TI - Laparoscopy during pregnancy. AB - OBJECTIVE: To compare the safety and efficacy of laparoscopic surgery with that of open laparotomy in pregnant patients. DESIGN: Six-year case-control study. SETTING: Tertiary care, university and community hospitals. PATIENTS: Population based sample. From 1990 through 1995, 16 pregnant patients underwent laparoscopic surgery (study group) and 18 underwent open laparotomy (control group) during the first or second trimester. Follow-up ranged from 1 month to 6 years. INTERVENTION: In the study group, 4 patients underwent appendectomies and 12 underwent cholecystectomies. The control group included 7 appendectomies and 11 cholecystectomies. MAIN OUTCOME MEASURES: The 2 groups were compared for age, trimester, surgical time, oxygen saturation, end-tidal carbon dioxide, return of gastrointestinal tract function, duration of intravenous or intramuscular narcotics, postoperative stay, gestational age of delivery, 1- and 5-minute Apgar scores, birth weights, and complications. RESULTS: Age, trimester, oxygenation, end-tidal CO2, gestational age at delivery, Apgar scores, and birth weights were not different between the 2 groups. The patients who underwent laparoscopy had significantly longer operative times 82 vs 49 minutes), shorter stay (1.5 vs 2.8 days), earlier resumption of regular diet (1.0 vs 2.4 days), and shorter duration of intravenous or intramuscular narcotics (1.2 vs 2.6 days) (all P < .01). Four complications were found in the laparotomy group vs 6 in the laparoscopy group. CONCLUSIONS: Laparoscopic surgery in pregnant women significantly decrease hospitalization, decreases narcotic use, and quickens return to a regular diet when compared with open laparotomy in pregnant women. No significant differences between the 2 groups in perioperative morbidity or mortality were present. These data suggest that therapeutic laparoscopy during pregnancy in the first or second trimester is safe. PMID- 8624204 TI - Bedside tracheostomy in the intensive care unit. AB - OBJECTIVE: To prove that tracheostomy performed at the bedside in the intensive care unit is a safe, cost-effective procedure. DESIGN: Retrospective review of all adult patients undergoing elective bedside tracheostomy in the intensive care unit between January 1983 and December 1988. Two hundred four patients were identified. SETTING: A private 1200-bed tertiary care center with a 120-bed critical care facility. MAIN OUTCOME MEASURES: Major and minor perioperative complications, cost savings, and comparison of risk between bedside tracheostomy and that performed in the operating room. RESULTS: There were six major complications (2.9%): one death due to tube obstruction, two bleeding episodes requiring reoperation, one tube entrapment requiring operative removal, one nonfatal respiratory arrest, and one bilateral pneumothorax; and seven minor complications (3.4%): five episodes of minor bleeding, one tube dislodgement in a tracheostomy with a well-developed tract, and one episode of mucus plugging. One late complication (tracheal stenosis) was identified. CONCLUSIONS: Bedside tracheostomy in the intensive care unit can be performed with morbidity and mortality rates comparable to operative tracheostomy. In addition, it provides a significant cost savings for the patient. PMID- 8624205 TI - The role of prophylactic cholecystectomy in the short-bowel syndrome. AB - OBJECTIVES: To determine the factors associated with cholelithiasis and define the role of prophylactic cholecystectomy in the short-bowel syndrome. DESIGN: Retrospective clinical review of cohort of consecutive patients. SETTING: Tertiary care, academic medical center. PATIENTS: Fifty adult (age, > 16 years) patients with intestinal remnants less than 180 cm were evaluated over a 15-year period. MAIN OUTCOME MEASURES: Incidence and natural history of cholelithiasis, postoperative morbidity, and mortality rates. RESULTS: Prophylactic cholecystectomy was performed in 5 patients (10%). Ten patients (20%) died within 20 days without evidence of gallstones. Eleven (31%) of the other 35 patients at risk developed biliary disease; 6 of these patients had inflammatory complications or common bile duct stones. Biliary disease was more likely (P < .05) in patients with intestinal remnant length less than 120 cm (47% vs 13%), an absent ileocecal junction (41% vs 0%), long-term total parenteral nutrition (45% vs 13%), and Crohn's disease (67% vs 24%). Patients with mesenteric vascular disease had high initial mortality (50%) and a 38% incidence of biliary disease. Patients with cancer and/or irradiation had a lower initial mortality (7%) and no biliary disease. Patients with benign conditions had a significant incidence of cholelithiasis (57%). CONCLUSIONS: Patients with the short-bowel syndrome have a significant risk for cholelithiasis if the intestinal remnant length is less than 120 cm, total parenteral nutrition is required, and the terminal ileum is resected. Prophylactic cholecystectomy is indicated in patients with benign conditions and anticipated long-term survival. It should also be considered in patients with mesenteric vascular disease who, despite significantly shortened survival, may have a high incidence of early biliary problems. PMID- 8624206 TI - Recurrent acute pancreatitis caused by afferent loop stricture after gastrectomy. AB - Afferent loop obstruction after gastrectomy and Billroth II gastrojejunostomy is only rarely diagnosed as the cause of recurrent acute pancreatitis. Three patients are described in whom afferent loop stricture after gastrectomy and Billroth II reconstruction manifested as recurrent pancreatitis 13 to 24 years after the initial procedure. Late onset, nonspecific symptoms, and other simultaneous gastrointestinal pathologic features promoted a chronic clinical course in all patients. Symptoms included acute abdominal pain, vomiting, jaundice, hyperamylasemia, weight loss, and anemia. A thorough history, barium examination, cholescintigraphy, and endoscopy were central in establishing the diagnosis. The pathogenesis of stricture formation is thought to be ischemic mucosal damage from intestinal crossclamping. Surgical decompression provided lasting relief of the symptoms. Afferent loop stricture should be considered in the different diagnosis in patients with recurrent acute pancreatitis and previous gastrectomy with Billroth II reconstruction. PMID- 8624207 TI - Gossypiboma of the abdomen. AB - Intra-abdominal cysts may rise from a variety of organs. However, foreign-body reaction and cyst formation should be considered in the differential diagnosis. In this report, we describe the finding of a preoperatively undetected gossypiboma. A gossypiboma is a mass within the body that is composed of a cotton matrix; in this case, an unmarked laparotomy sponge. The evaluation, findings, and prevention of gossypiboma are discussed. PMID- 8624208 TI - Blind drive. PMID- 8624209 TI - Blind drive. PMID- 8624210 TI - Cerebral and extracerebral abnormalities in Parkinson's disease. PMID- 8624211 TI - Limited resources, competition and health care reform: witnessing the evolution of academic neurology. PMID- 8624212 TI - Conditions that mimic stroke. PMID- 8624213 TI - Antihistaminics in idiopathic dystonia. PMID- 8624214 TI - Airline policy relating to passengers with epilepsy. PMID- 8624215 TI - Crashes and violations among drivers with Alzheimer disease. AB - BACKGROUND: Several small studies have found a high automobile crash rate for drivers with Alzheimer disease (AD) compared with unaffected elderly drivers, prompting the suggestion that the diagnosis of AD mandate cessation of driving. OBJECTIVES: To compare automobile crash and violation rates of a large number of patients with AD with appropriately matched elderly subjects. To determine if neuropsychological test scores predict these adverse driving events. To determine if intervention by physicians or family members influences driving cessation. DESIGN: Review of crashes and violations from 1986 to 1993 in police-filed Michigan State driving records of 143 licensed patients with AD and 715 licensed comparison subjects matched 5 to 1 in age (+/- 6 years), sex, and county of residence. We correlated crashes and violations with neuropsychological test scores. A questionnaire-based inquiry on the influence of physician, family, and state interventions on driving cessation was administered. RESULTS: The crash and violation rates of patients with AD were not significantly different from those of comparison subjects. However, patients with AD probably drove fewer kilometers than did comparison subjects. Neuropsychological test scores did not predict future crashes or violations. CONCLUSIONS: This study, the largest to our knowledge involving state driving records of patients with AD, does not confirm the previously reported excessive crash rate among drivers with AD relative to an appropriate comparison population. Reduced driving exposure of patients with AD probably kept their crash adverse equal to that of comparison subjects. Intervention by physicians and family members was major factor in reducing driving exposure. These findings affirm that the mere diagnosis of AD does not justify license revocation. PMID- 8624216 TI - Apolipoprotein E genotype determines survival in the oldest old (85 years or older) who have good cognition. AB - OBJECTIVE: To quantify the influence of apolipoprotein E (APOE) polymorphism on cognition and survival in a population sample aged 75 years or older. DESIGN: The Kungsholmen Project established a cohort of 1810 residents in a district in Stockholm, Sweden, aged 75 years or older in 1987. Information on cognition at cohort inception is available for all subjects. Subjects were followed up for mortality to January 1, 1995. SUBJECTS: Included in this study are 1077 subjects (of 1124 genotyped for APOE) with the common epsilon 2/3, epsilon 3/3, and epsilon 3/4 APOE genotypes. RESULTS: The odds of cognitive impairment for the epsilon 3/4 vs epsilon 3/3 genotype declined with age: 4.8 for age 75 through 79 years; 1.7 for age 80 through 84 years; and 1.0 (i.e., no association) for age 85 years or older. Despite this association, APOE polymorphism did not significantly predict survival in subjects younger than 85 years, nor did it predict survival in subjects 85 years or older who were cognitively impaired. Instead, survival varied fourfold with respect to APOE polymorphism in those 85 years or older who had good cognition: Mortality in subjects with the epsilon 2/3 genotype was half that in those who carried the epsilon 3/3 genotype (hazard ratio, 0.5; 95% confidence interval, 0.2 to 0.9), and mortality in subjects with the epsilon 3/4 genotype was twice that in those who carried the epsilon 3/3 genotype (hazard ratio, 2.0; 95% confidence interval, 1.1 to 3.5). This fourfold variation resulted in 2-year differences in survival. CONCLUSIONS: The minor sequence variation in the apolipoprotein E isoforms resulted in a fourfold difference in the risk of death among the oldest old (age > or = 85 years) with good cognition. The observed variation in mortality was unlikely to have been caused by cognitive impairment, as APOE polymorphism was not a risk factor for cognitive impairment in this age group. PMID- 8624217 TI - The prediction of Alzheimer disease. The role of patient and informant perceptions of cognitive deficits. AB - OBJECTIVE: To determine whether the perceptions of patients' cognitive deficits by either the patient or an informant could predict who would develop Alzheimer disease (AD) in a group of 120 memory-impaired patients without dementia. METHODS: At entry into the study, patients were assessed by several measures that included neuropsychological tests and the Cambridge Mental Disorders of the Elderly Examination Interview Schedule. The latter schedule asks patients and their informants about their perceptions of cognitive deficits in the patients. After 2 years, patients underwent a diagnostic workup for AD: 29 had developed probable AD, whereas the other 91 did not develop dementia. We used logistic regression analyses to examine the predictive accuracy of patients' and informants' perceptions of deficits at entry into the study. RESULTS: Informants' perceptions, not patients' perceptions, contributed significantly to the prediction of AD. The best prediction of AD was obtained by the regression model that included both informants' perceptions and 2 neuropsychological tests. CONCLUSIONS: These results demonstrate the clinical use of including informant perceptions about patients' cognitive deficits in the diagnostic assessment of AD. They also indicate that patients' perceptions of their own deficits are not predictive of AD, but are related to depressive affect. PMID- 8624218 TI - Physical aggression is associated with preservation of substantia nigra pars compacta in Alzheimer disease. AB - OBJECTIVE: To investigate specific neuropathologic correlates of agitation and physical aggression in Alzheimer disease (AD). DESIGN: Neuronal counts in the nucleus basalis, locus ceruleus, and substantia nigra were compared in the brains of patients with pathologically definite AD with or without histories of agitation or interpersonal violence. SETTING: Alzheimer disease center of a university department of neurology. MAIN OUTCOME MEASURES: Neuron densities in the nucleus basalis and absolute neuron counts in the locus ceruleus and substantia nigra pars compacta. RESULTS: The patients with AD who had histories of unequivocal interpersonal violence had significantly greater neuron counts in the substantia nigra pars compacta than did the nonviolent patients with AD. This finding remained significant after multiple clinical and neuropathologic variables were adjusted for. Neuropathologic findings in the nucleus basalis and locus ceruleus were not different between violent and nonviolent patients. CONCLUSION: Preservation of pigmented substantia nigra neurons may be a risk factor for physical aggression in AD. PMID- 8624219 TI - Gender comparisons of cognitive performances among vascular dementia, Alzheimer disease, and older adults without dementia. AB - BACKGROUND: We hypothesized that women with Alzheimer disease (AD) would perform worse on a test of semantic memory but not on tests of other cognitive domains. We did not expect that women without dementia would perform more poorly than men without dementia on the same task. OBJECTIVE: To explore the specificity of a semantic memory deficit among women with AD by exploring gender differences among a group of subjects with vascular dementia (VD). DESIGN: A case-control study in which differences between men and women were explored using regression models to control for the potentially confounding effects of age, education, duration of dementia, and severity of dementia. SETTING: Alzheimer's Disease Research Center Consortium of Los Angeles and Orange Counties, California. SUBJECTS: Volunteers, recruited from the community or clinic referrals, who met clinical criteria for AD (n = 159) or VD (n = 117) or met criteria for control status without dementia (n = 134). MAIN OUTCOME MEASURES: Five neuropsychological measures, commonly used in the diagnosis and assessment of dementia. RESULTS: Women with VD scored lower than men with VD on 3 tests. However, when controlling for potential confounds, the gender difference was maintained only for the semantic memory task. Women with AD showed a strong trend to perform worse than men with AD on the test of semantic memory only. No gender differences were found among subjects without dementia. CONCLUSIONS: Findings support the existence of a semantic memory deficit for women with AD and suggest that a similar deficit may exist among women with VD. PMID- 8624221 TI - Delayed-onset cerebellar syndrome. AB - BACKGROUND: Delayed-onset involuntary movements, including dystonia and myoclonus, have been reported after stroke or head trauma. Moreover, there have been reports of delayed-onset isolated intention tremor and, in several of these cases, gait ataxia. OBJECTIVE: To further define the clinical features of a delayed-onset cerebellar syndrome. DESIGN: Subjects with cerebellar tremor and either head trauma or stroke were identified using a computerized database, providing detailed demographic and clinical information of 4002 patients with involuntary movements other than Parkinson's disease seen at our center between 1983 and 1995. Medical records and videotaped neurological examinations were retrospectively reviewed. SETTING: The Center for Parkinson's Disease and Other Movement Disorders at Columbia-Presbyterian Medical Center, New York, NY. PATIENTS: Five patients with delayed-onset cerebellar syndromes. RESULTS: Five patients with stroke or head trauma developed a cerebellar syndrome 3 weeks to 2 years after the initial insult. The syndrome, characterized by intention tremor, ataxic dysarthria, nystagmus, dysmetria, dysdiadochokinesis, and gait ataxia, was progressive in at least one patient. In four patients, lesions were present on neuroimaging in the thalamus or brain stem (especially in the midbrain). CONCLUSIONS: A delayed-onset cerebellar syndrome may follow head trauma or stroke. The syndrome is sometimes progressive and often disabling. The delayed onset implies that the syndrome is not caused by the initial lesion itself but may be caused by development of post-synaptic supersensitivity or secondary reorganization of involved pathways. PMID- 8624220 TI - Citicoline improves verbal memory in aging. AB - OBJECTIVE: To test the verbal memory of older volunteers given citicoline. DESIGN: A randomized, double-blind, placebo-controlled, parallel group design was employed in the initial study. After data analysis, a subgroup was identified whose members had relatively inefficient memories. These subjects were recruited for a second study that used a crossover design. The subjects took either placebo or citicoline, 1000 mg/d, for 3 months in the initial study. In the crossover study, subjects took both placebo and citicoline, 2000 mg/d, each for 2 months. SUBJECTS: The subjects were 47 female and 48 male volunteers 50 to 85 years old. They were screened for dementia, memory disorders, and other neurological problems. Of the subjects with relatively inefficient memories, 32 participated in the crossover study. MAIN OUTCOME MEASURE: Verbal memory was tested at each study visit using a logical memory passage. Plasma choline concentrations were measured at baseline; at days 30, 60, and 90 in the initial study; and at day 60 of each treatment condition in the crossover study. Plasma choline concentrations and memory scores were analyzed using repeated-measures analysis of variance and covariance, followed by planned comparisons when appropriate. RESULTS: In the initial study, citicoline therapy improved delayed recall on logical memory only for the subjects with relatively inefficient memories. In the crossover study, the higher dosage of citicoline was clearly associated with improved immediate and delayed logical memory. CONCLUSIONS: Citicoline therapy improved verbal memory functioning in older individuals with relatively inefficient memories. Citicoline may prove effective in treating age-related cognitive decline that may be the precursor of dementia. PMID- 8624222 TI - Diagnosing narcolepsy through the simultaneous clinical and electrophysiologic analysis of cataplexy. AB - OBJECTIVE: To demonstrate the utility of accurate clinical and electroencephalographic characterization of provoked cataplexy spells in the diagnosis of narcolepsy. METHODS: Four individuals, three with suspected and one with known narcolepsy, were clinically assessed during split-screen, video polysomnographic monitoring sessions after cataplectic events were induced by emotional provocation. RESULTS: The subjects experienced a total of nine cataplectic-like events, one occurring spontaneously (sleep paralysis) in association with a hypnagogic hallucination. During all events, the patients appeared to be sleeping with polysomnographic rapid eye movement sleep patterns, but when questioned they were able to give appropriate verbal responses. The diagnosis of narcolepsy was substantiated in all cases using standard overnight polysomnograms and multiple sleep latency tests. CONCLUSION: The diagnosis of narcolepsy can be greatly enhanced by documenting cataplexy with thorough clinical assessment and demonstration of typical rapid eye movement sleep patterns during provoked spells in the course of polysomnographic monitoring sessions. PMID- 8624223 TI - Effect of an antibacterial varnish and amine-fluoride/stannous fluoride (AmF/SnF2) toothpaste on Streptococcus mutans counts in saliva and dental plaque of children. AB - The purpose of this study was to evaluate the effect of the simultaneous application of a chlorhexidine and thymol-containing varnish (Cervitec) and an amine fluoride/stannous fluoride containing toothpaste (Meridol) on Streptococcus mutans counts in saliva and dental plaque of school children 12-14 years of age, during a six-week period. The children were separated into group 1 (Cervitec varnish + fluoride-containing toothpaste), group 2 (Cervitec varnish + Meridol toothpaste), and group 3 (Meridol toothpaste alone). Over the six weeks the greatest improvement in salivary Streptococcus mutans count occurred in group 2. Overall, a statistically significant decrease in total microbiological count, and Streptococcus mutans was found in all three groups. PMID- 8624224 TI - Intra-oral comparison and evaluation of the ability of fluoride dentifrices to promote the remineralization of caries-like lesions in dentin and enamel. AB - The effect of three types of fluoride dentifrices and one non-fluoride dentifrice were evaluated and compared intra-orally for their ability to promote the remineralization of caries-like lesions in thin sections of human dentin and enamel. The three fluoride dentifrices were silica based. One dentifrice contained 0.76% sodium monofluorophosphate (MFP), another contained 0.247% sodium fluoride (NaF), and the third contained 0.247% NaF plus 1.3% pyrophosphate and 1.5% polyvinylmethyl ether/maleic acid (PVM/MA) copolymer (denoted NaF/PPi). The fourth dentifrice (placebo) was also silica based, but contained no fluoride, and served as a negative control. Two week treatment resulted in the following percent mineral changes for the enamel specimens: +21 +/- 16 for MFP, +10 +/- 30 for NaF, +16 +/- 15 for NaF/PPi, and -30 +/- 30 for the placebo. The respective percent mineral change values for the dentin specimens were +28 +/- 20 for MFP, +22 +/- 19 for NaF, +28 +/- 18 for NaF/PPi, and -34 +O/- 61 for the placebo. A statistical analysis revealed that the three fluoride dentifrices were significantly better (p<0.05) than the placebo at promoting remineralization for both types of specimens. No significant differences were found among the three fluoride dentifrices. The following can be concluded from this study: 1) silica based dentifrices containing MFP and NaF are effective at remineralizing caries like lesions in both enamel and dentin; 2) there was no statistical difference between NaF and MFP in their ability to promote remineralization; and 3) pyrophosphate does not interfere with the remineralizing effects of NaF. PMID- 8624225 TI - Effect of a pre-brush mounthrinse containing triclosan and a copolymer on calculus formation: a three-month clinical study in Thailand. AB - A three-month, double-blind, parallel clinical study was conducted on a population of Thai adults to evaluate the effect of the twice daily use of a commercially available pre-brush mouthrinse on supragingival calculus formation. The mouthrinse test product contained 0.03% triclosan and 0.13% PVM/MA copolymer with the absence of fluoride. The subjects were initially examined for calculus using the Volpe-Manhold procedure. All subjects received an oral prophylaxis and were assigned to the use of either 1) a triclosan-copolymer mouthrinse, or 2) a matching flavored/colored water placebo mouthrinse. Subjects were instructed to rinse twice daily with 10 cc of the assigned mouthrinse for 1 minute, followed by brushing with the provided toothpaste containing fluoride for 45 seconds. After three months of using the assigned mouthrinse, the subjects were reexamined for calculus formation. The results indicated that the subjects using triclosan/copolymer mouthrinse had 23.17% less supragingival calculus than the placebo mouthrinse subjects. This reduction was statistically significant at the 99% or greater (F = 24.35, p<0.001) level of confidence. PMID- 8624226 TI - Effect of a triclosan/copolymer pre-brush mouthrinse on established plaque formation and gingivitis: a six-month clinical study in Thailand. AB - A six-month, double-blind parallel clinical study was conducted to evaluate the efficacy on existing plaque and gingivitis of a pre-brush mouthrinse (Colgate Plax, Thailand) containing 0.03% triclosan and 0.13% of a polyvinylmethyl ether/maleic acid (PVM/MA) copolymer in the absence of fluoride, as compared to a matching placebo rinse. A total of 121 subjects were stratified into two balanced groups according to baseline Quigley-Hein Plaque Index scores and Loe-Silness Gingivitis Index scores. Each group was randomly assigned to the use of either the triclosan/copolymer pre-brush rinse or the placebo pre-brush rinse. No subjects received an oral prophylaxis. They were instructed to rinse twice daily for 60 seconds using the provided fluoride dentifrice and soft-bristled toothbrush. After six months' use of their assigned mouthrinse, 118 subjects who completed the study were evaluated for plaque index score and gingivitis index score using the same scoring procedure. After six months, the triclosan/copolymer mouthrinse group provided a 35.48% reduction of plaque (p<0.001) and an 18.82% reduction of gingivitis (p<0.001), as compared to the placebo mouthrinse group. The reduction of the triclosan/copolymer group was most pronounced in the severity manifestation of plaque and gingivitis (p<0.001). The results indicate that twice daily use of a pre-brush mouthrinse containing triclosan/copolymer in the absence of fluoride significantly reduces pre-existing plaque and gingivitis as compared to the placebo rinse. PMID- 8624227 TI - Intra-oral remineralization of enamel with a MFP/DCPD and MFP/silica dentifrice using surface microhardness. AB - The present study was undertaken to ascertain the effect of dicalcium phosphate dihydrate (DCPD) abrasive in a dentifrice on the remineralizaton of enamel using a surface microhardness technique. The method of assessing enamel remineralization via surface microhardness (SMH) was validated in a randomized, crossover, double-blind, intra-oral remineralization study conducted with 12 healthy adults. Enamel demineralization was achieved in vitro by covering bovine enamel blocks with exogenous oral bacteria, S. Mutans 1600 Ingbritt, containing glucan which was then exposed to sucrose. In the intra-oral treatment phase, subjects were fitted with oral maxillary palatal retainers, each holding four demineralized enamel blocks. Subjects brushed their teeth for 30 seconds with a test dentifrice, swished for an additional 60 seconds, rinsed with water and then retained the blocks intra-orally for 4 hours. Percent mineral recovery for each enamel block was calculated as the ratio of the changes in enamel microhardness due to treatment (remin) and sucrose challenge (demin). Treatments included DCPD based dentifrices containing 0, 250 and 1000 ppm fluoride (F) from sodium monofluorophosphate (MFP). Using SMH, respective mean percent mineral recoveries of 5.7, 18.7 and 41.4% were obtained. All ADA criteria for model validation were fulfilled. This same model was then used to compare the remineralization effects of a silica placebo, DCPD placebo, 1000 ppm F MFP/silica and 1000 ppm F MFP/DCPD dentifrice. Mean percent mineral recoveries of -0.9, 24.1, 30.2 and 55.7% were obtained, respectively. The MFP/DCPD dentifrice was superior to MFP/silica (<0.01) with use of the MFP/DCPD dentifrice when compared to MFP/silica or the silica placebo. These results indicate that more active calcium and a higher degree of saturation (DS(EN)) with respect to enamel exists for an extended period of time after use of a MFP/DCPD dentifrice. Since an elevation in DS(EN) is considered a major parameter controlling the extent of enamel remineralization, this finding may partly explain the superior remineralization of enamel observed with the MFP/DCPD dentifrice. The significant increases in calcium activity and intra-oral enamel remineralization by the DCPD-based dentifrice are consistent with earlier findings that a DCPD abrasive provides added benefit for enamel remineralization. PMID- 8624228 TI - The effect of two toothpastes on plaque and gingival inflamation. AB - In this study on 60 adult subjects, the effective of Parodontax, a dentifrice containing herbal ingredients and sodium bicarbonate abrasive, was compared to a non-marketed new toothpaste containing herbal ingredients and calcium hydrogen phosphate as the abrasive. Plaque, gingivitis and gingival bleeding parameters were scored. The periodontal probe bleeding index of Ainamo and Bay was modified to score slight and moderate bleeding. In this first four-week period all subjects used the new toothpaste. After this period the new toothpaste produced a significant decrease (p<0.01) in gingivitis and bleeding on probing, but no effect on plaque was observed. During the second period of eight weeks the subjects were randomly divided into two groups, one using Parodontax and the other group continuing with the new toothpaste. The study design was a double blind procedure. At the end of the 12-week study period the plaque index showed no changes in both groups. The gingivitis and bleeding indices decreased significantly (p<0.001) by 40% in both groups compared to the baseline examination. PMID- 8624230 TI - A comparative study of stain removal with two electric toothbrushes and a manual brush. AB - Recent studies have suggested that a sonic electric toothbrush is more effective than a manual brush at removing extrinsic dental stain. There have been few studies of the comparative stain removal properties of different electric brushes. The study reported here was conducted to compare the efficacy of the sonic toothbrush (Sonicare) with an oscillating/rotating brush (Braun Oral-B Plaque Remover) and a conventional manual brush (Crest Complete). The study was a single-blind, randomized, cross-over design, balanced for residual effects and employing 24 subjects. Stain was enhanced over a 21-day period by twice-daily rinses with chlorhexidine and frequent intakes of tea and/or coffee. At the end of each period, tooth stain intensity and area, tongue stain intensity and area, lower lingual calculus and subjective tooth sensitivity were recorded together with preference for the brushes determined at the study's completion. Similar levels of tongue staining were recorded for the three periods, with no significant differences between the three groups. Tooth stain intensity, for most sites, was not significantly different between the three groups. For mean total stain area and for lingual and lingual interproximal sites, a significant reduction in stain was seen following use of the oscillating/rotating brush compared to the manual brush. The reductions in stain with the sonic brush were not significantly different from the manual brush. With the exception of maximum stain intensity, there were no significant differences between the oscillating/rotating and sonic brushes. Significantly less tooth sensitivity was found following use of the oscillating/rotating brush compared to both the manual and sonic brushes. All three brushes were found to be safe, but volunteer preference significantly and predominantly favored the oscillating/rotating brush. The results suggest that the oscillating/rotating brush is superior to a manual brush for stain removal. PMID- 8624229 TI - Reduction of calculus and Peridex stain with Tartar-Control Crest. AB - Regular use of an effective oral rinse (0.12% chlorhexidine [CHX]) may be accompanied by tooth staining and slightly increased calculus formation. Since dentifrices containing soluble pyrophosphates are known to significantly reduce calculus formation, this study was designed to investigate whether CHX-induced staining and increased calculus may be reduced by the use of such dentifrices. The study compared stain and calculus formation in 163 subjects using a CHX oral rinse (Peridex, Procter & Gamble) according to label directions and either a pyrophosphate-containing anticalculus toothpaste (Tartar-Control Crest, 3.3% pyrophosphate) or an otherwise similar toothpaste without pyrophosphate (Regular Crest). Subjects were instructed to brush and floss ad lib and were examined after three and six months. Whole-mouth calculus occurrence was significantly reduced in the anticalculus toothpaste group at three and six months. Also, staining on the cosmetically important facial-anterior surfaces was significantly reduced at the 3-month examination. After six months the difference in facial anterior staining was still directionally favorable to the anticalculus toothpaste group but no longer statistically significant. These results indicate that routine brushing with an anticalculus toothpaste such as Tartar-Control Crest significantly reduces both facial-anterior staining and calculus occurrence in subjects using a CHX oral rinse. Whether more frequent or more thorough brushing would lead to still greater reductions remains to be investigated. PMID- 8624231 TI - The effect of an oscillating/rotating electric toothbrush and a sonic toothbrush on removal of stain from enamel surfaces. AB - This laboratory investigation compared the Sonicare toothbrush, the Braun Oral-B Plaque Remover and a manual brush, in terms of removal of stained pellicle from the surface of enamel specimens. Enamel specimens were stained over a period of 4 days to produce a stained pellicle film. Staining was assessed photometrically before and after brushing 16 specimens per group for 4.5 minutes with the three test toothbrushes and dentifrice slurry prepared from Crest toothpaste. The positive control consisted of the ADA reference abrasive and a manual toothbrush. Results were expressed as a cleaning ratio (CR), with higher values representing more efficient stain removal. Analysis of results revealed that removal of stain from the enamel surfaces was similar in the Braun Oral-B Plaque Remover group (CR 94) and the reference standard group (CR 100). There was no statistically significant difference between these 2 groups. In contrast, stain removal in the Sonicare toothbrush group and the manual toothbrush group was significantly less effective (p<0.01), with a mean CR of 66 and 76, respectively. There was no statistical difference between the cleaning ratio scores for these 2 groups. It is concluded that extrinsic stain removal with the Sonicare toothbrush is similar to that achieved with a manual brush, but that stain removal with the Braun Oral B Plaque Remover is significantly more effective. PMID- 8624232 TI - Effect of a clamp on toothbrush bristle deterioration. AB - The purpose of this study was to evaluate, in vitro, the effectiveness of a device to prevent toothbrush deterioration. Ten toothbrushes from each of four brands, Oral-B 40, Py-co-pay Softex 12.0 row, Butler #330, and Oral-B 20, were divided into control and experimental groups. Toothbrushes were fixed into a machine that simulated toothbrushing by producing a reciprocating motion 41.5 mm in length at a rate of 380 strokes/min. Bristles slid over a ceramic disc that had a hardness similar to dental enamel. Pressure (450 g) was applied at a right angle to the toothbrush while it moved back and forth in a slurry of toothpaste. The pressure was calibrated with load cells. Toothbrushes were run for 15 minutes followed by a drying cycle for a total of four hours, or approximately equivalent to brushing twice daily for six months. During the drying cycle the bristles of the toothbrushes in the experimental group were compressed 3/4 their length from the brush head with a clamp (Gregory Patent #4884311). Bristle deterioration was evaluated based on Wear Index (WI), a quantitative means of ranking brushes in various stages of deterioration. Data on WI and WR showed that toothbrushes in the experimental group had significantly (< or = 0.005) less deterioration. Improvement in wear resistance was dependent on size and configuration of the bristles. Compressing the bristles between brushings retains the non-matted shape of the bristles and reduces their deterioration. PMID- 8624233 TI - A comparative clinical evaluation of the Wisdom Straight, Plaque Control and Angled toothbrushes compared to the Oral-B 35. AB - Three Wisdom toothbrushes, the Straight, Plaque Control and Angled, were compared to the Oral-B 35 toothbrush for clinical safety and efficacy. The 30-day testing protocol conformed to the American Dental Association's program guidelines for manual toothbrushes. No irritation was observed nor reported at anytime for any of the toothbrushes evaluated. All toothbrushes tested were effective in reducing plaque area, gingivitis and bleeding on probing over the 30 days of the study. PMID- 8624235 TI - 8-Hydroxy-7-nitroquinoline-5-sulfonic acid monohydrate. AB - The title compound, C9H6N2O6S.H2O, is zwitterionic (i.e. 8-hydroxy-7 nitroquinolinium-5-sulfonate monohydrate) with a deprotonated sulfonic group and a protonated quinoline N atom which interacts with the hydroxy O atom. The H atom of the hydroxy group is distal to the N atom and is involved in an interaction with an O atom of the nitro group, which is oriented at 32.2(1) degrees with respect to the quinoline ring system. PMID- 8624234 TI - Laboratory and clinical stain removal evaluations of two tartar control dentifrices. AB - The purpose of this research was to evaluate Colgate original Tartar Control dentifrice (TC) and new Colgate Micro Cleansing Tartar Control (MCTC) dentifrice in a laboratory and clinical study for their ability to remove induced stain. In the laboratory study, polished and etched bovine enamel specimens were stained for 4 days with a coffee, tea, and mucin, Sarcina lutea tartox culture in trypticase soy broth. Sixteen specimens were then brushed for 200 strokes with a sodium carboxymethyl cellulose solution to remove loose stain, and then brushed for 300 strokes with a 1:1 toothpaste slurry. Tooth color was measured by reflectance with the CLE L*a*b* scale. The calculated stain removal, 37% for MCTC and 24% for TC, was significantly different (p<0.01) favoring the MCTC dentifrice. In the clinical study, eight-six subjects were given Peridex (0.12% chlorhexidine gluconate) mouthrinse, a low abrasive dentifrice and a soft toothbrush to use twice a day for 8 weeks. Stain was monitored on the buccal and lingual surfaces of the anterior teeth using the Lobene index. The seventy-seven subjects with sufficient total stain were stratified into two balanced groups, given a new soft toothbrush and were randomly assigned to use one of the dentifrices tested, twice a day for 8 weeks. At 4, 6 and 8 weeks in the study, the MCTC dentifrice product had consistent, significantly (p<0.05-0.001) lower total stain scores than the TC dentifrice. In the laboratory and clinical studies conducted, even though stain induction and evaluation procedures differed, the new Colgate Micro Cleansing Tartar Control dentifrice was significantly more effective at removing stain than the Colgate original Tartar Control dentifrice formula. PMID- 8624236 TI - A non-peptide angiotensin II receptor antagonist: 2-butyl-6-dimethoxymethyl-5 phenyl-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4- yl]methyl]-1H-imidazo[5,4-b]pyridine. AB - The title compound, C33H33N7O2, is one of a series of imidazo[4,5-b]pyridine based angiotensin II receptor antagonists showing high antihypertensive activity. The biphenyltetrazole moiety assumes the same conformation as in related compounds, but its relative orientation with respect to the central fused ring is different to that in these compounds, indicating that there is considerable conformational flexibility about the methylene bridge joining the two ring systems. PMID- 8624237 TI - Ipriflavone, an antiostheophorotic agent. AB - The antiostheophorotic agent ipriflavone [7-(1-methyl-ethoxy)-3-phenyl-4H-1 benzopyran-4-one, C18H16O3] is an important member of the isoflavone family. The structure of the molecule is dominated by the dihedral angle (50 degrees) between the planes of the phenyl and benzo-pyran moieties. A structural comparison with other related published structures is represented. PMID- 8624238 TI - X-ray structure investigations of potential beta-blockers. II.6,7-Dimethoxy-1 phenyl-1,2,3,4-tetrahydroisoquinoline. AB - The saturated part of the isoquinoline moiety in the title compound, C17H19NO2, has a twist conformation and the phenyl ring substituent is in an equatorial position. The absolute configuration of the molecule is R and molecules form infinite chains by means of weak hydrogen bonds. PMID- 8624239 TI - mu-Oxo-bis[(5,10,15,20-tetraphenylporphyrinato)oxomolybdenum(V)]. AB - The crystal structure of a new solvated crystal form of the title complex, [?Mo(O)(TPP)?2O].1.5H2O.0.5CH2Cl2 (TPP = C44H28N4), has been determined. The dimeric molecule is in a general position, and the overall structural features are extremely similar to those of the previously reported non-solvated form. PMID- 8624240 TI - Fluorene-1-carboxylic acid. AB - In fluorene-1-carboxylic acid, C14H10O2, the sole hydrogen bond is of the cyclic dimer type about a center of symmetry. The carboxyl H atom is ordered. Distances in the fluorene core are very similar to those in fluorene itself; the fluorene core dihedral angle is, however, larger than in fluorene. PMID- 8624241 TI - Three protected tetrapeptides. AB - The structures of three protected tetrapeptides, containing the Boc-Gly-Gly-Phe-X OMe chain, tert-butoxycarbonyl-glycy-glycl-phenylalanine-leucine methyl ester dihydrate, Boc-Gly-Gly-L-Phe-D-Leu-OMe, C25H38N4O7.2H2O, tert-butoxycarbonyl glycy-glycl-phenylalanine-methionine methyl ester dihydrate, Boc-Gly-Gly-L-Phe-D Met-OMe, C24H36N4O7S.2H2O and tert-butoxycarbonyl-glycy-glycl-phenylalanine norleucine methyl ester dihydrate, Boc-Gly-Gly-D-Phe-L-Nle-OMe, C25H38N4O7.2H2O, are described. The three molecules have the same conformation on the tetrapeptide chain and display the same packing, consisting of couples of molecules linked head-to-tail by two hydrogen (N--H...O) bonds; other hydrogen bonds, also involving two water molecules of crystallization, link these couples together and give rise to a planar structure. PMID- 8624242 TI - 7-Benzyl-3-thia-7-azabicyclo[3.3.1]nonane 3-oxide. AB - The molecular geometry of a major metabolite, C14H19NOS, of a potent anti arrhythmic drug, 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane (BRB-I-28), has been determined by X-ray diffraction. The 3,7-dihetero bicyclic system of the sulfoxide molecule adopts a chair--chair conformation like that of the HCLO4 salt of the BRB-I-28 molecule. The S...N contact distance of 2.863(2)A in the present molecule is significantly shorter than that found in the crystal structure of its precursor [3.038(4)A]. The overall molecule possesses pseudo-mirror symmetry. PMID- 8624243 TI - Effects of H/D substitution on thermal vibrations in piperazinium hexanoate-h11, d11. AB - The crystal structures of piperazinium hexanoate-h11, 1/2C4H12N2(2+).C6H11O2-, and piperazinium hexanoate-d11, 1/2C4H12N2(2+).C6D11O2-, have been determined from neutron diffraction data collected at 15 K. Nuclear anisotropic displacement parameters have been analyzed to obtain the internal molecular displacements of the H and D nuclei, given by (u(obs)2)-(u(ext)2) where (u(ext)2) is the contribution assuming all H/D to be carried rigidly on the vibrating molecular framework consisting of the heavier nuclei. In both crystal structures the cation ring is well fitted by the rigid-body model and the anion chain by a model with two rigid segments. In the piperazinium cations the corresponding protons in the two structures have about the same internal vibrational directions and magnitudes except for the two N--H protons, perhaps owing to differences in N--H...O hydrogen bonding. The internal vibrations of corresponding H/D in the h11 and d11 anions have approximately the same vibrational directions. The internal mean square displacements of the H nuclei are systematically greater than the values of the corresponding D nuclei by an average factor 1.7(3). For both anions, normal-mode analyses have been carried out using the force fields derived from ab initio quantum-mechanical calculations with HF/3-21G and HF/6-31G** basis sets. The values of the resultant H/D internal displacements for C--H(D) bond stretching and methylene out-of-plane vibrations are in good agreement with experiment. However, with either basis set, theory predicts methylene in-plane mean-square displacements significantly greater than the experimental values. PMID- 8624244 TI - Roles of bombesin-like peptides in lung development and lung injury. PMID- 8624245 TI - Expression of gastrin-releasing peptide receptor gene in developing lung. AB - Bombesin (BN) or its mammalian counterpart, Gastrin-releasing peptide (GRP) is produced by pulmonary neuroendocrine cells (PNEC). The function of GRP as a growth factor involves lung morphogenesis, but the precise mechanism and the target cells have not been defined. We used a nonradioactive in situ hybridization (NISH) and Northern blot analysis for both GRP receptor (GRP-R) and its ligand GRP on samples of fetal and newborn human and rabbit lung. For immunolocalization of BN/GRP and the proliferating lung cell population we used anti-BN/GRP or MIB-1 antibodies, respectively. NISH and Northern blot showed peak expression of GRP-R mRNA on day 24 gestation in the fetal rabbit lung. At the cellular level, GRP-R mRNA was localized mainly in the distal airway epithelial tubes and surrounding mesenchyme, whereas proximal airways showed decreased signal. Epithelium expressing GRP-R showed positive labeling for proliferation antigen in contrast to PNEC, which were negative. In human post-natal lung, strong signal for GRP-R mRNA was localized in the airway epithelial cells and submucosal glands. PNEC in both rabbit and human lung expressed mRNA for GRP, but only in human lung were they immunoreactive for the peptide. However BN/GRP immunoreactive cells were detected in rabbit fetal lung culture. The expression of GRP-R in mammalian lung is developmentally regulated, peaking both spatially and temporally during the phase of rapid airway growth and differentiation. Both epithelial and mesenchymal components express GRP-R consistent with paracrine mechanism for GRP activity during lung morphogenesis. PMID- 8624246 TI - Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5. AB - Exposure of animals to adenoviral gene transfer vectors has been associated with respiratory tract inflammation. The pathogenesis of this inflammation is unclear. One hypothesis is that viral vectors directly induce production of inflammatory cytokines by host cells in the airways. We exposed cultured human lung cells to an adenovirus-5--based vector containing the cytomegalovirus promoter and lacZ reporter gene (Ad.CMV.lacZ) and to wild-type adenovirus 5 (wtAd5) and measured subsequent release of cytokines into cell culture supernatants. Inoculation of human bronchial epithelial (HBE) cells with Ad.CMV. lacZ at 10(1) to 10(4) plaque forming units (pfu)/cell resulted in dose-related expression of lacZ by both X gal staining and immunohistochemistry but did not increase release of interleukin (IL)-8 or IL-6 at 24, 48, or 96 h after inoculation. In the same cultures, tumor necrosis factor-alpha induced marked increases in release of both IL-8 and IL-6 at 24 and 48 h after stimulation. Similar data were observed in the BEAS-2B HBE cell line. HBE cells incubated with wtAd5 at doses of 10(1) to 10(3) pfu/cell did not release increased amounts of IL-6 or IL-8 up to 48 h after inoculation, though wild-type respiratory syncytial virus (3 pfu/HBE cell) infection resulted in increases in both cytokines. Human alveolar macrophages obtained by bronchoalveolar lavage also showed no increases in cytokine release after incubation with Ad.CMV.lacZ, though relatively little gene transfer occurred in macrophages. These data do not support a role for direct induction of airway epithelial or alveolar macrophage inflammatory cytokines in the pathogenesis of inflammation associated with exposure of airways to adenovirus or to adenoviral gene transfer vectors. PMID- 8624247 TI - Lung inflammation and epithelial changes in a murine model of atopic asthma. AB - A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer. PMID- 8624248 TI - Immunolocalization of interleukin-4 in eosinophils in the bronchial mucosa of atopic asthmatics. AB - Several studies have shown that human eosinophils can synthesize and release a number of cytokines. The aim of this study was to investigate whether eosinophils contain interleukin (IL)-4 protein. We examined the ultrastructural localization of IL-4 in eosinophils in bronchial mucosal biopsies of 29 nonsmoking atopic asthmatics and 7 controls. No eosinophils were detected in the bronchial mucosa of controls. In the eosinophils (n = 42) of the asthmatics, IL-4 was localized to the electron-dense crystalloid core compartment of 85% of the secondary or specific eosinophil granules (n = 468). Other structures in the eosinophils were unlabeled. Control sections, incubated with an irrelevant primary antibody, were negative. This study demonstrates that pre-formed IL-4 is stored in the secondary eosinophil granules. These results were extended by light microscopic immunodouble-staining for IL-4 protein and eosinophil cationic protein, which showed that a subpopulation of activated eosinophils express IL-4 immunoreactivity in bronchial mucosal biopsies of asthmatics as well as controls. These data indicate that eosinophils may be an important source of IL-4 in allergic inflammation. PMID- 8624249 TI - Diminished expression of CYP1A1 in urethane-induced lung tumors in strain A/J mice: analysis by in situ hybridization and immunohistochemical methods. AB - We have investigated the regulation and expression of CYP1A1 in solid and papillary lung tumors induced by the carcinogen urethane. Female strain A/J mice were administered urethane (1 mg/g body wt) intraperitoneally, and when lung tumors were established at 16 weeks, mice were treated with 3-methylcholanthrene to induce CYP1A1. CYP1A1 mRNA was detected by in situ hybridization with a 3H labeled RNA probe and quantitated by image analysis, whereas CYP1A1 protein was detected by immunohistochemical staining with an avidin-biotin complex procedure. Our results showed that in untreated control and tumor-bearing mice, the CYP1A1 mRNA was present at low levels, but the CYP1A1 protein was not detectable. Treatment with 3-methylcholanthrene induced increased levels of both CYP1A1 mRNA and protein in lung parenchyma and tumor foci. This induction was markedly higher in the parenchyma of control and tumor-bearing lungs than in either solid or papillary tumors. Differences in CYP1A1 mRNA and protein expression were not evident in solid and papillary tumors. In the parenchyma, induced CYP1A1 mRNA and protein were localized in the endothelial and alveolar septal cells. Endothelial cells in tumors also contained substantial CYP1A1 mRNA and protein, whereas low levels of both were found in lung tumor cells. Our finding of significant reduction in inducibility of CYP1A1 protein in conjunction with reduced CYP1A1 mRNA in tumor foci suggests reduced transcriptional activation as a regulatory mechanism. The lack of association between diminished CYP1A1 expression and either the tumor type or the mechanism mediating metabolic activation supports the hypothesis that the persistence of lung tumors may be due, in part, to a restricted capability for the formation of reactive intermediates. PMID- 8624250 TI - Inhibition of Sephadex-induced lung injury in the rat by Ro 45-2081, a tumor necrosis factor receptor fusion protein. AB - Tumor necrosis factor (TNF) is an inflammatory cytokine produced by many cell types which may contribute to the pathophysiology of a variety of lung diseases. In this study we have used Ro 45-2081 (a soluble receptor composed of the human p55 TNF receptor and human heavy-chain immunoglobulin G) to explore the role of TNF in the acute inflammatory response in the rat lung to intravenous injection of Sephadex beads. The effects of Ro 45-2081 have also been compared with those of dexamethasone. At 24 and 72 h after Sephadex, there was a significant increase in the total number of leukocytes in bronchoalveolar lavage fluid (BALF). At 24 h, the number of neutrophils comprised around 50% of the total leukocyte number, decreasing to around 10% of total by 72 h. The eosinophil count was maintained at around 10% of the total leukocyte number. Pretreatment with either Ro 45-2081 [1 and 3 mg kg-1, intraperitoneally (i.p.)] or dexamethasone (0.1 and 0.3 mg kg-1, i.p.) inhibited the neutrophilia at 24 h after Sephadex, although Ro 45-2081 had no significant effect on total cell number. At 72 h after Sephadex, Ro 45-2081 (1 and 3 mg kg-1, i.p., daily) significantly reduced the neutrophil influx into BALF but had no inhibitory effect on eosinophil number. In contrast, dexamethasone (0.1 and 0.3 mg kg-1, i.p., daily) virtually abolished the infiltration of neutrophils and eosinophils into BALF. The lack of effect of Ro 45-2081 on eosinophil infiltration into the rat lung and the inhibition caused by dexamethasone suggest that factors other than TNF are involved in this part of the inflammatory response induced by Sephadex. However, the inhibitory effects of Ro 45-2081 show that TNF may play an important role in the recruitment of neutrophils into the lungs of Sephadex-treated rats. PMID- 8624251 TI - PAF-induced airways hyperreactivity is modulated by mast cells in mice. AB - We tested the hypothesis that mast cells contribute to platelet-activating factor (PAF)-induced airways hyperreactivity and hyperpermeability in mice. Airways reactivity to acetylcholine (ACh) and lung permeability to Evans blue (EB) dye were measured before and after PAF challenge in genetically mast cell-deficient (WBB6F1 W/Wv) and normal congenic (WBB6F1 +/+) mice, as well as mast cell reconstituted (BMT W/Wv) mice. In addition, prostaglandin D2 (PGD2), a mast cell specific mediator, was measured in the bronchoalveolar lavage (BAL) from +/+ and W/Wv mice to determine if lung mast cell activation was a consequence of PAF challenge. Genetically PAF-sensitive AKR/J mice were also treated with the mast cell stabilizer nedocromil prior to assessment of PAF effects on ACh reactivity. Intravenous PAF (10 micrograms/kg) induced a significant (P < 0.05) increase in airways reactivity to ACh (25 micrograms/kg) in both +/+ (371 +/- 52%) and W/Wv (122 +/- 24%) mice. There was a significantly greater increase in +/+ compared with W/Wv mice. PAF-induced hyperreactivity to ACh in BMT W/Wv mice (191 +/- 44%) was significantly (P < 0.05) greater than age-matched W/Wv mice (80 +/- 16%), but not significantly different from age-matched +/+ mice (153 +/- 44%). PAF (10 micrograms/kg) also significantly (P < 0.5) increased lung permeability in +/+ and W/Wv mice, but there was no significant difference between groups. BAL PGD2 increased significantly in +/+ mice following PAF challenge (559 +/- 24 ng/ml) compared with vehicle controls (152 +/- 8 pg/ml). There was no significant increase in BAL PGD2 from W/Wv mice. Nedocromil pretreatment significantly (P < 0.05) decreased PAF-induced hyperreactivity in AKR/J mice but not in W/Wv mice (P > 0.05). We conclude that mast cells contribute significantly to PAF-induced hyperreactivity but not hyperpermeability in mice. PMID- 8624252 TI - Oligoclonal V gene usage by T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients. AB - The T-cell antigen receptor (TCR) repertoire was examined in lymphocytes isolated from the lungs and blood of 12 sarcoidosis patients and nine control patients. This analysis, by polymerase chain reaction (PCR), examined the variable (V) domain genes of both the alpha and beta chains of the TCR. This is the first study to examine the usage of all known V alpha gene segments in sarcoidosis. A similar degree of diversity was observed in the TCR repertoire in the lungs and blood of the sarcoidosis patients. However, 11 of the 12 sarcoidosis patients showed an increased use of particular TCR V alpha and V beta genes in lung T cells as compared with blood. The pattern of TCR V gene bias in the lung T cells was specific for each patient. The clonality of selected V genes was examined by determining the third complementarity-determining region (CDR3) length polymorphism of particular PCR products. The majority of lung T cells with biased TCR V gene segments were oligoclonal. Altogether, these results suggest oligoclonal expansion of lung T cells in response to a local antigenic stimulus, with additional nonspecific T-cell accumulation. The variability in the V gene segments used by the expanded T-cell subsets in different sarcoidosis patients may reflect different epitopes or antigens being recognized in the lung, as well as variations in major histocompatibility complex (MHC) haplotype between the patients. PMID- 8624253 TI - A small proline-rich protein, SPRR1, is upregulated early during tobacco smoke induced squamous metaplasia in rat nasal epithelia. AB - Small proline-rich proteins, believed to be precursor proteins for the crosslinked envelope formation in cells undergoing squamous differentiation, are encoded by the SPRR genes. To further investigate the role of these proteins, the time course of increased synthesis of SPRR1 mRNA in nasal epithelia of rats exposed to cigarette smoke was determined, and the deduced amino acid sequence of the rat SPRR1 was compared with those of other species. Using the pig homologue (20K) antisense cRNA probe, high levels of SPRR1 transcript were detected by in situ hybridization in squamous epithelia that line the nasal vestibule and hard palate of the rat. Basal cells of both the vestibule and palate contained low levels of the transcript, and increasing amounts were detected in the squamous layers. In rats exposed to 250 mg/m3 (total particulate matter) cigarette smoke 6 h/day for 5 days, the number of small mucous cells increased in the respiratory epithelium of the nasal septum in the early stages of squamous differentiation, but were gradually replaced by squamous metaplastic cells. During this transition, hybridization of the 20K antisense cRNA probe increased in the epithelial and mesenchymal cells, indicating that SPRR1 protein could have roles in cellular differentiation other than as a building block of the crosslinked envelope. Similarly, high levels of SPRR1 transcript were detected in the nasal transitional epithelium lining internal walls and maxilloturbinates that had undergone squamous metaplasia after cigarette smoke exposure. At 5 days after the withdrawal of cigarette smoke exposure, the morphology of the midseptal epithelium returned to that of a pseudostratified mucociliary epithelium and the epithelia lining the maxilloturbinates to that of a transitional epithelium. Accompanying this change in morphology of the tissues, the levels of SPRR1 transcripts significantly decreased in the epithelia. However, in the mesenchyme no significant decrease was observed during this recovery. RNA prepared from the external nose surrounding the nasal vestibule contained a transcript of about 0.9 kb that hybridized to the 20K cDNA probe on Northern blot analysis. DNA sequence analysis of the transcript confirmed the identity as that of the SPRR mRNA with its characteristic repeat encoding the oligopeptide with the general consensus EPC*PKVP-. However, the rat homologue rSPRR1 contained more repeats of the oligopeptide compared with those of higher mammals such as the rabbit, pig, and human, suggesting a possible inverse relation between number of repeats and evolution development. This finding suggests that the number of repeats in the protein may be redundant; however, the conserved sequence of the peptide indicates that this region is essential for the function of this protein. PMID- 8624254 TI - Airway hyperresponsiveness to acetylcholine: segregation analysis and evidence for linkage to murine chromosome 6. AB - A genetic predisposition to nonspecific airway hyperresponsiveness (AHR) can be demonstrated in humans and in many animal models. The goal of the current study was to gain insight into the molecular mechanisms that determine AHR by mapping the genes that control this phenotype. We describe genetic studies in a mouse model of differential sensitivity to acetylcholine (ACh)-induced AHR. This model was used to ascertain the number, magnitude of effect, and chromosomal location of quantitative trait loci (QTL) providing susceptibility to ACh-induced AHR. Segregation analyses indicated that a major locus acting additively with a polygenic effect segregates with the airway pressure-time index (APTI) in the progeny of hyperresponsive A/J and hyporesponsive C3H/HeJ mice. Additionally, four loci segregate with respiratory system resistance (Rrs). Examination of the genome for markers linked to these phenotypes indicated that a QTL on chromosome 6 was common to both traits. QTL analysis in the [(C3H/HeJ x A/J)F1 x A/J] backcross generation revealed significant linkage for ACh-induced AHR within the interval spanning the chromosome 6 deoxyribonucleic acid (DNA) markers D6Mit16 and D6Mit13. A/J alleles in this interval were associated with significantly greater airway responsiveness than were C3H/HeJ alleles. Several important candidate genes map to this region, including the locus for the interleukin-5 (IL 5) receptor. This mapping information in the mouse may relate to human studies in which bronchial hyperresponsiveness links to the chromosomal region containing the gene for IL-5 (1). PMID- 8624256 TI - WHO Expert Committee on Biological Standardization. PMID- 8624255 TI - Inhibition of the human neutrophil respiratory burst by native and synthetic surfactant. AB - Production of oxygen radicals by phagocytic cells and loss of surfactant function have each been implicated in the pathogenesis of acute lung injury. Therapeutic administration of exogenous surfactant to injured lungs in which neutrophils are the dominant cell type has been proposed. To understand the role of surfactant in modulating pulmonary inflammation and the impact of surfactant supplementation on diseased lungs, we studied the effect of native porcine and synthetic surfactant preparations on human neutrophil respiratory burst oxidase activity in vitro. We found that surfactant inhibited neutrophil superoxide production induced by either receptor-mediated [formylmethionylleucylphenylalanine (fMLP)] or non receptor-mediated [phorbol myristate acetate (PMA)] agonists with an IC50 of approximately 0.015 mg phospholipid/ml for porcine surfactant or approximately 0.050 mg phospholipid/ml for synthetic surfactant. Surfactant had no effect on detection of superoxide generation in a noncellular system using xanthine and xanthine oxidase and only minimally inhibited superoxide generation by neutrophils that had been fully stimulated by prior exposure to PMA. There was no effect of surfactant on neutrophil calcium mobilization in response to fMLP, on lactoferrin release in response to PMA, or on membrane protein kinase C activity in response to PMA. Suspensions of dipalmitylphosphatidylcholine alone had no effect on neutrophil superoxide production. Taken together, these findings indicate that certain components of lung surfactant may effect relatively late steps in the activation of the respiratory burst or may alter subsequent steps involved in the assembly of the respiratory burst oxidase. PMID- 8624257 TI - Bovine spongiform encephalopathy (BSE). WHO Consultation on public health issues related to BSE and the emergence of a new variant of Creutzfeldt-Jakob disease. PMID- 8624258 TI - Hepatocellular carcinomas with a high proliferation index and a low degree of apoptosis and necrosis are associated with a shortened survival. AB - In this study we investigated tumour growth in relation to the immunohistochemical expression of p53 and bcl-2 and to patient survival data in 33 operated hepatocellular carcinomas (HCCs). In order to estimate the growth, a growth index, based on the degree of cell proliferation, apoptosis and necrosis, was calculated for each tumour. Cell proliferation was determined immunohistochemically by the number of proliferating cell nuclear antigen (PCNA) positive cells in tumours, the extent of apoptosis was determined by counting the number of cells labelled by the in situ 3'-end labelling technique and tumour necrosis was estimated as the percentage of necrotic areas in haematoxylin--eosin stained tissue sections. In our analysis we found that the survival of patients with HCCs showing a high growth index (i.e. tumours showing a high proliferation and simultaneously a low degree of apoptosis and necrosis) was significantly shorter than with other patients (P = 0.004, log-rank test). When analysed separately, cell proliferation, apoptosis or necrosis did not show any significant association with survival. p53 positivity was found in 8/33 (24%) of tumours. There were significantly more p53-positive cases in tumours with a high growth index (P = 0.01, Fisher's exact test) suggesting that dysfunction of the p53 gene may affect tumour growth. p53-positive cases did not, however, have a significantly shorter survival time than p53-negative cases (P = 0.3, log-rank test). bcl-2 positivity was found in only 1/33 (3%) of the HCCs. Thus bcl-2 overexpression does not seem to play an important role in hepatocellular carcinogenesis. In summary, our results suggest that in HCCs a compound score based on the evaluation of the degree of cell proliferation, apoptosis and necrosis is a biologically more relevant prognostic indicator than any of its composite parameters alone. PMID- 8624259 TI - Apoptosis of human seminoma cells upon disruption of their microenvironment. AB - One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis. Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels. In contrast, three SEs with over 12% of apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions, obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders. Flow cytometric analysis of end labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells. As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions. Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro. PMID- 8624260 TI - Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines. AB - In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co expression of beta-glycan, RI and RII. PMID- 8624261 TI - pMel17 is recognised by monoclonal antibodies NKI-beteb, HMB-45 and HMB-50 and by anti-melanoma CTL. AB - Recently, we cloned the cDNA encoding the melanocyte lineage-specific antigen gp100 and demonstrated that gp100 is recognised by three different monoclonal antibodies (MAbs) used to diagnose malignant melanoma. In addition, we showed that tumour-infiltrating lymphocytes (TIL 1200) from a melanoma patient reacted specifically with cells transfected with the gp100 cDNA. Molecular characterisation of the gp100 cDNA revealed that the gp100 antigen is highly homologous, but not identical, to another melanocyte-specific protein, pMel17. Here, we report that cells transfected with pMel17 cDNA also react with all three MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-45 and HMB-50. Moreover, pMel17 transfectants are specifically lysed by TIL1200. These data demonstrate that antigenic processing of both gp100 and pMel17 give rise to peptides seen by anti-melanoma cytotoxic T lymphocytes (CTL) and are therefore potential targets for immunotherapy of malignant melanoma. PMID- 8624262 TI - O6-benzylguanine enhances the sensitivity of a glioma xenograft with low O6 alkylguanine-DNA alkyltransferase activity to temozolomide and BCNU. AB - The effect of the O6-alkylguanine-DNA alkyltransferase (AGT) inhibitor, O6 benzylguanine (O6-BG), on the anti-tumour activity of 8-carbamoyl-3-methylimidazo [5,1-d]-1,2,3,5-tetrazine-4(3H)-one (temozolomide) or 1,3-bis(2-chloroethyl) nitrosourea (BCNU) was evaluated in athymic mice bearing subcutaneous (s.c.) human glioma (U87MG) xenografts. The activity of AGT in U87MG xenografts was 4.3 +/- 1.5 fmol mg-1 protein (mean +/- s.d). These xenografts were inherently sensitive to treatment with alkylating compounds alone, with non-toxic doses of temozolomide (35 mg kg-1) or BCNU (10 mg kg-1) producing tumour growth delays of 23.3 and 11.8 days respectively. O6-BG (40 mg kg-1) did not inhibit tumour growth when administered alone, but was found to enhance significantly the anti-tumour activity of temozolomide or BCNU when administered 1 h before therapy (P < 0.002, Mann-Whitney test). AGT activity measured 24 h after the administration of 40 mg kg-1 O6-BG, was only 0.9 +/- 0.2 fmol mg-1 protein. These results are in contrast to previous studies in vitro with tumour cell lines of low AGT activity (< 15 fmol mg-1 protein), in which the cytotoxicity of temozolomide or BCNU was unaffected by AGT depletion. PMID- 8624263 TI - Effects of AGM-1470 and pentosan polysulphate on tumorigenicity and metastasis of FGF-transfected MCF-7 cells. AB - Previously, we described FGF-1- or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM 1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM 1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGF- or FGF-4-transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 10(5) times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen. PMID- 8624264 TI - Comparison of staurosporine and four analogues: their effects on growth, rhodamine 123 retention and binding to P-glycoprotein in multidrug-resistant MCF 7/Adr cells. AB - The potent kinase inhibitor staurosporine and its protein kinase C (PKC) selective analogue CGP 41251 are known to sensitise cells with the multidrug resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) to cytotoxic agents. Here four PKC-selective staurosporine cogeners, CGP 41251, UCN-01, RO 31 8220 and GF 109203X, were compared with staurosporine in terms of their MDR-reversing properties and their susceptibility towards P-gp-mediated drug efflux from MCF 7/Adr cells. Staurosporine was the most potent and the bisindolylmaleimides RO 31 8220 and GF 109203X the least potent cytostatic agents. When compared with MCF-7 wild-type cells, MCF-7/Adr cells were resistant towards the growth-arresting properties of RO 31 8220 and UCN-01, with resistance ratios of 12.6 and 7.0 respectively. This resistance could be substantially reduced by inclusion of the P-gp inhibitor reserpine. The ratios for GF 109203X, staurosporine and CGP 41251 were 1.2, 2.0 and 2.9 respectively, and they were hardly affected by reserpine. These results suggest that RO 31 8220 and UCN-01 are avidly transported by P-gp but that the other compounds are not. Staurosporine and CGP 41251 at 10 and 20 nM, respectively, decreased efflux of the P-gp probe rhodamine 123 (R123) from MCF-7/Adr cells, whereas RO 31 8220 and GF 109203X at 640 nM were inactive. CGP 41251 was the most effective and GF 109203X the least effective inhibitor of equilibrium binding of [3H]vinblastine to its specific binding sites, probably P gp, in MCF-7/Adr cells. Overall, the results imply that for this class of compound the structural properties that determine susceptibility towards P-gp mediated substrate transport are complex. Comparison with ability to inhibit PKC suggests that the kinase inhibitors affect P-gp directly and not via inhibition of PKC. Among these compounds CGP 41251 was a very potent MDR-reversing agent with high affinity for P-gp and least affected by P-gp-mediated resistance, rendering it an attractive drug candidate for clinical development. PMID- 8624265 TI - Increased insulin-like growth factor binding protein-2 (IGFBP-2) gene expression and protein production lead to high IGFBP-2 content in malignant ovarian cyst fluid. AB - Expression of insulin-like growth factor-I (IGF-I), its receptor and IGF-binding proteins (IGFBPs) by ovarian cancer cells and its mitogenic effect on these cells in vitro, suggest that IGF-I may have a role in regulation of human ovarian cancer. We have recently shown IGFBP-2 to be markedly elevated in malignant ovarian cyst fluid in vivo. To identify the origin of increased IGFBP-2 in these cyst fluids, the gene expression and protein content of IGFBP-2 were investigated in 14 malignant and four benign epithelial ovarian neoplasms. IGFBP-2 mRNA was detected in all ovarian specimens and was 2- to 30-fold higher in malignant than in benign neoplasms. Within the malignant tissues IGFBP-2 mRNA levels correlated with the aggressiveness of the tumour and were higher in invasive tumours than in those with borderline pathology. Southern blot analysis revealed no amplification of IGFBP-2 gene in the DNA samples from ovarian tumours regardless of their nature. IGFBP-2 was the major binding protein in tissue extracts, as measured by both Western ligand blotting and immunoblotting, and was significantly higher in malignant than in benign neoplasms. These findings were further supported by immunohistochemical detection of IGFBP-2 in tumour sections. Our data suggest that increased local production by the tumour in vivo is responsible for the increased IGFBP-2 levels in the cyst fluid bathing the ovarian malignancy. This may represent an autocrine regulatory mechanism for IGF-I proliferative effect of ovarian cancer. PMID- 8624266 TI - Markers of type I collagen degradation and synthesis in the monitoring of treatment response in bone metastases from breast carcinoma. AB - Thirty-six patients with bone metastases included in a trial of supportive calcitonin on the treatment response to systemic therapy were monitored by conventional radiography, conventional indicators of bone metabolism [alkaline phosphatase (AP), osteocalcin (gla), urinary hydroxyproline excretion (OHP), urinary calcium (uCa), serum calcium (sCa)] and collagen metabolites (ICTP, the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen; PICP, the carboxy-terminal propeptide of type I procollagen; and PIIINP the amino terminal propeptide of type III procollagen). All patients had been on the same systemic treatment for at least 3 months at the start of the trial. There was a positive correlation between the concentrations of ICTP and PICP at baseline (Spearman's rank-order correlation coefficient rs = 0.62). Both ICTP and PICP showed statistically significant correlations to the other markers of bone metabolism (except sCa and uCa) as well as to the number of bone metastases on bone scans. Reduction in ICTP correlated significantly with the treatment response at three months (rs = - 0.57). while PICP showed a borderline negative correlation to therapy response (rs = - 0.37). Of all the biochemical parameters studied the changes in ICTP showed the best correlation with the treatment response. PICP and ICTP changes in patients with progressive disease differed significantly from those in patients with responding and stable metastases, whereas no difference was found between responders and stable patients. PMID- 8624267 TI - Efficacy of up-front 5-fluorouracil-epidoxorubicin-cyclophosphamide (FEC) chemotherapy with an increased dose of epidoxorubicin in high-risk breast cancer patients. AB - The prognosis of patients with stage IIIB breast carcinoma with tumour spread to the apical axillary lymph nodes has hardly improved despite adequate locoregional control and the introduction of systemic adjuvant therapy. A combined modality regimen that includes anthracyclin-based chemotherapy, high-dose chemotherapy with peripheral stem cell support and radiation and hormonal therapy is currently under investigation in this subset of patients. The present study aims to document the efficacy and feasibility of dose-intensive epidoxorubicin in combination with a standard dose of 5-fluorouracil and cyclophosphamide as up front chemotherapy in this setting. A preoperative chemotherapy regimen consisting of three courses of 5-fluorouracil 500 mg m-2, epidoxorubicin 120 mg m 2 and cyclophosphamide 500 mg m-2 (FE120C) was administered at 21 day intervals without haematopoietic growth factors to 70 patients with apex node-positive disease. All patients were below 60 years of age and had not had prior chemotherapy or radiotherapy. Sixty-six patients were evaluable for clinical response and histopathological examination could be performed in 62 of these. Thirteen patients achieved a clinical complete response (20%). Of these patients, microscopic examination of the mastectomy specimen revealed absence of malignant cells in two and exclusively ductal carcinoma in situ (DCIS) in another two patients. In addition, of the 46 patients (70%) with a clinical partial response, at pathological examination one patient had sclerosis only and four had DCIS. This results in a pathological complete response in three (5%) of all patients and absence of invasive carcinoma in 10%. None of the patients progressed during chemotherapy. The major toxicity was moderate bone marrow suppression with a median white blood count (WBC) nadir of 1800 microliters-1 (range 500-4900). Other toxicities were mild. The full planned dose could be given without delays in 66 of 70 patients FE120C is well tolerated and is highly effective as up-front chemotherapy in relatively young patients with high-risk breast cancer, with a 90% (CI 74-98%) clinical objective response rate. PMID- 8624268 TI - Hormone replacement therapy as treatment of breast cancer--a phase II study of Org OD 14 (tibilone). AB - Org OD 14 (tibilone) is a synthetic steroid, designed to combine the favourable effects of oestrogens, progestagens and androgens into a single substance for use as hormone replacement therapy (HRT). Given its antiovulatory properties, the ability to control menopausal symptoms and blocking action on progesterone receptors, Org OD 14 was considered as an agent with potential anti-cancer activity while at the same time helping existing menopausal symptoms. In this phase II study, 14 post-menopausal women with advanced or metastatic breast cancer, who had failed on tamoxifen, were treated with Org OD 14. The median duration of treatment was 12 weeks and all patients stopped because of progressive disease with or without toxicity. Vaginal bleeding occurred in four patients, three of whom had recently stopped tamoxifen. One response was seen: an 82-year-old patient had a partial response in an axillary soft tissue mass, improvement in liver function tests and an improvement in her performance status that lasted over 6 months. One patient with progressive disease on Org OD 14 improved on stopping the drug. In view of the vaginal bleeding, Org OD 14 should not be given to patients who have recently stopped tamoxifen. PMID- 8624269 TI - Metabolic effects of pamidronate in patients with metastatic bone disease. AB - We have evaluated the value of specific bone resorption markers in monitoring metastatic bone disease to define the duration of action of a single high-dose pamidronate infusion. Twenty patients received a single infusion of pamidronate 120 mg for painful bone metastases. Ten out of these 20 patients also received a second infusion. They were evaluated at baseline, 2, 4 and 8 weeks after each infusion. A composite pain questionnaire, serum and urine tests were carried out at these time points. Bone resorption markers measured included urinary calcium, hydroxyproline and two new markers: pyridinoline and deoxypyridinoline. Reference values were defined by 20 healthy controls matched by age and sex. Pamidronate induced a profound fall in bone resorption with a maximal effect within the first month after therapy. Changes in urinary calcium levels were confounded by a rise of 100% in the parathyroid hormone levels. Before treatment, pyridinoline and deoxypyridinoline were increased in 70% of patients, while urinary calcium was increased in only 40% of them. Thirteen patients had a > or = 50% fall in deoxypyridinoline levels and were considered as biochemical responders. These patients had a mean reduction in pain score of about 30% of baseline levels, which was significantly higher than the seven non-biochemical responders. In conclusion, urinary calcium is not a precise marker of bone resorption. Deoxypyridinoline seems to be the most specific bone resorption marker in cancer patients. Biochemical responders have the most benefit from pamidronate in terms of pain relief. This suggests that patients may benefit from more potent or repeated infusions of bisphosphonates. PMID- 8624270 TI - A phase II study of cisplatin, vindesine and continuously infused 5-fluorouracil in the treatment of advanced non-small-cell lung cancer. Osaka Lung Cancer Chemotherapy Study Group. AB - Fifty-two previously untreated patients with advanced non-small-cell lung cancer (NSCLC) were treated on a 14 day cycle with cisplatin (60 mg m-2 i.v.) and vindesine (3 mg m-2 i.v.) on day 1, followed by a 3 day continuous infusion of 5 fluorouracil (800 mg m-2 day-1) starting on day 8. An overall response rate of 40.4% was observed in 47 evaluable patients, which included one complete response and 18 partial responses. Responses were achieved in 61.1% of stage 3 patients and 27.6% of stage 4 patients. The median progression-free interval was 19.3 weeks, and median survival time was 41.6 weeks (47.1 weeks for patients with stage 3 disease and 38.7 weeks for those with stage 4 disease). Toxicity was well tolerated. Gastrointestinal and renal toxicities did not exceed WHO grade 2. Grade 3 or 4 leucopenia and anaemia occurred in nine (19%) and four (9%) patients respectively, but only grade 2 thrombocytopenia was observed. Phlebitis at the infusion site was observed in 24 patients (53%). This treatment programme achieved a response rate similar to other active combination regimens for the treatment of advanced NSCLC, and was less toxic. PMID- 8624272 TI - Endocrinological late effects after chemotherapy for testicular cancer. AB - Type and extent of endocrinological alterations were studied in long-term disease free survivors after cisplatin-based chemotherapy for testicular cancer. A total of 63 patients with a median age of 30 (19-53) years, and median follow-up of 42 (16-128) months were included. Elevated serum follicle-stimulating hormone (FSH) levels were found in 63% of patients, 24% showed pathologically elevated luteinising hormone (LH) levels with normal and 10% with subnormal testosterone levels. The degree of gonadotropin elevation was highly significantly correlated with the cumulative platinum (P) dose. Patients treated with platinum-vinblastine bleomycin regimens showed higher gonadotropin levels than those treated with platinum-etoposide-bleomycin. The adrenal androgen dehydroepiandrosterone (DHEA), pathologically elevated in 68% of patients, was significantly correlated with the cumulative doses of chemotherapy (ctx) used and to the gonadotropin levels. Treatment variables, such as type and dose of cytotoxic agents used, as well as degree of gonadotropin elevation were further correlated with changes in oestron, testosterone and 17 alpha-OH-progesterone levels. Cholesterol levels were elevated in 32% of patients and significant interactions between the steroid hormone levels and cardiovascular risk factors could be shown. PMID- 8624271 TI - A prognostic index for multiple myeloma. AB - The current prognostic systems have failed to identify multiple myeloma (MM) patients who require aggressive therapy. These staging systems do not reliably distinguish patients with different prognoses. This paper explores the possibility of improving the prognostic forecast in MM by considering some clinical characteristics at diagnosis together with response to first-line chemotherapy. A total of 231 patients were prospectively randomised in a multicentre trial to no therapy vs melphalan + prednisone (MP) for stage I, MP in stage II, and MP vs peptichemio, vincristine and prednisone for stage III. The clinical features of these groups were evaluated for prognostic variables predictive of overall survival by means of univariate and multivariate analysis. The independently significant variables were incorporated into a model that identified three groups of patients with different risks of death and different overall survival. Three variables retained statistical significance: the staging system proposed by the British Medical Research Council, a composite parameter integrating the percentage of bone marrow plasma cells with cytological features of the infiltrating elements (plasma cell vs plasmablast), and response to 6 months of first-line chemotherapy. These three variables led the proposal of a scoring system able to identify three different risk classes (with median overall survival of 52, 28 and 13 months respectively) and to estimate individual patient prognosis more flexibly. The proposed risk classes, drawn from both diagnostic and therapeutic parameters, are thought to be a clinical and investigational instrument for separating MM patients into comparable groups, for selecting the best available therapy and for evaluating response with respect to the disease of each new patient. PMID- 8624273 TI - Will the Scottish Cancer Target for the year 2000 be met? The use of cancer registration and death records to predict future cancer incidence and mortality in Scotland. AB - Cancer mortality data reflect disease incidence and the effectiveness of treatment. Incidence data, however, reflect the burden of disease in the population and indicate the need for prevention measures, diagnostic services and cancer treatment facilities. Monitoring of targets mandates that both be considered. The Scottish Cancer Target, established in 1991, proposed that a reduction of 15% in mortality from cancer in the under-65s should be achieved between 1986 and 2000. Each year in Scotland approximately 8300 persons under 65 are diagnosed with cancer and 4500 die from the disease. The most common malignancies, in terms of both incident cases and deaths, in the under-65s, are lung and large bowel cancer in males, and breast, large bowel and lung cancer in females. A decrease of 6% in the number of cancer cases diagnosed in males under 65 is predicted between 1986 and 2000, whereas the number of cases in females in the year 2000 is expected to remain at the 1986 level. In contrast, substantial reductions in mortality are expected for both sexes: 17% and 25% in males and females respectively. Demographic changes will influence the numbers of cancer cases and deaths in the Scottish population in the year 2000. However, long-term trends in the major risk factors, such as smoking, are likely to be the most important determinants of the future cancer burden. PMID- 8624274 TI - Dietary intake and the risk of malignant mesothelioma. AB - A high consumption of fruit and vegetables reduces the risk of several types of cancer. There is little information on the association between dietary intake and mesothelioma. A hospital-based case-control study of 94 men and women with malignant mesothelioma and 64 control patients without cancer was conducted to determine the odds associated with consumption of carotenoid-containing fruits and vegetables. After statistical adjustment for occupational asbestos exposure, the odds ratio was 0.2 [95% confidence interval (CI) 0.1-0.8] for carrot consumption and 0.5 (95% CI 0.2-1.4) for tomato consumption. However, the frequency of consuming other foods that have a high vitamin A or carotenoid content was not associated with a decreased risk of cancer. These results provide some justification for the hypothesis that provitamin A or beta-carotene may decrease the risk of mesothelioma. The body mass index was unrelated to the risk of mesothelioma. PMID- 8624276 TI - Classical Kaposi's sarcoma in north-east Sardinia: an overview from 1977 to 1991. AB - The incidence of classical Kaposi's sarcoma in 1977-91 was studied in north-east Sardinia. In this period, 160 new cases were observed in a defined area, of which 124 were in males. This represented a standardised incidence of the disease of 1.58/100,000 inhabitants per year (2.43 for males and 0.77 for females). This is the highest incidence of classical Kaposi's sarcoma so far recorded. The incidence increased with age, particularly after the age of 70 in males. PMID- 8624275 TI - Risk of malignant melanoma in relation to drug intake, alcohol, smoking and hormonal factors. AB - In a population-based, matched case-control study from southern Sweden of 400 patients with a first diagnosis of malignant melanoma and 640 healthy control subjects aged 15-75 years, the association between commonly prescribed drugs, alcohol, smoking and malignant melanoma was evaluated. In addition, the relation between reproductive and hormonal factors and melanoma in women was studied. It was found that certain specific types of prescribed drugs, i.e. beta-blockers, hydralazines and benzodiazepines, may increase the risk of melanoma development. However, these associations were diminished, at least for benzodiazepines, after controlling for host factors. As these findings are unconfirmed, and may be due to chance or confounding, further studies are warranted. The risk of malignant melanoma was not influenced by alcohol consumption or smoking habits. Our results do not suggest an association between oral contraceptives and melanoma. Furthermore, reproductive factors were not independent risk factors for melanoma. However, increasing number of live births seemed to be protective (P for trend = 0.01). There is a need for further research to be able to draw firm conclusions on the relation between number of live births and melanoma. The results based on histopathological re-examinations and those based on tumour registry data were essentially the same. PMID- 8624277 TI - Physical activity and risk of colorectal cancer in men and women. AB - We examined the association between self-reported occupational and recreational physical activity and the subsequent risk of colorectal cancer in a population based cohort in Norway. During a mean follow-up time of 16.3 years for males and 15.5 years for females, 236 and 99 colon cancers and 170 and 58 rectal cancers were observed in males and females, respectively, among 53,242 males and 28,274 females who attended the screening between 1972 and 1978. Physical activity at a level equivalent to walking or bicycling for at least four hours a week during leisure-time was associated with decreased risk of colon cancer among females when compared with the sedentary group (RR = 0.62, 95% CI 0.40-0.97). Reduced risk of colon cancer was particularly marked in the proximal colon (RR = 0.51, 95% CI 0.28-0.93). This effect was not observed for occupational physical activity alone, probably due to a narrow range of self-reported physical activity at work among females. However, by combining occupational and recreational physical activity we observed an inverse dose-response effect as increasing total activity significantly reduced colon cancer risk (P for trend = 0.04). Among males 45 years or older at entry to the study, an inverse dose-response effect was observed between total physical activity and colon cancer risk (P for trend = 0.04). We also found in males a stronger preventive effect for physical activity in the proximal as compared to distal colon. In addition, we found a borderline significant decrease in colon cancer risk for occupational physical activity in males 45 years or older when compared to the sedentary group (RR = 0.74, 95% CI 0.53-1.04). All results were adjusted for age, body mass index, serum cholesterol and geographic region. No association between physical activity and rectal cancer was observed in males or females. The protective effect of physical activity on colon cancer risk is discussed in regard to energy balance, dietary factors, age, social class, body mass index and gastrointestinal transit time. PMID- 8624278 TI - Childhood leukaemias in New Zealand: time trends and ethnic differences. AB - Registrations from the New Zealand Cancer Registry were used to examine time trends in the incidence of leukaemias among children aged 0-14. There was a statistically significant increase in the incidence of leukaemia among children aged 0-4 during 1953-57 to 1988-90. In this age group, the recorded incidence rate increased from 4.89 per 100,000 person-years in 1953-57 to 7.92 in 1988-90. During 1973-77 to 1988-90 (and probably in earlier years), the increase was due to an increase in acute lymphoblastic leukaemia (ALL). The trends were unlikely to be due to changes in diagnosis or case ascertainment. The childhood leukaemia trends might be related to trends in family size, maternal age, socioeconomic level or exposure to infections. However, there are uncertainties about the importance of these factors or about their trends. The incidence of acute non lymphoblastic leukaemia (ANLL) decreased between 1968-72 and 1988-90. The time trends highlight the likely importance of environmental factors in the aetiology of childhood leukaemias in New Zealand. The risk of ALL was lower in the Maori than in the non-Maori population (relative risk Maori/non-Maori 0.74). The risk of ANLL was higher among Maori (relative risk 1.84). PMID- 8624279 TI - Blood transfusion as a risk factor for non-Hodgkin lymphoma. AB - In a case-control study of 280 out of 426 consecutive patients with a recent diagnosis of non-Hodgkin lymphoma (NHL) and 1827 control subjects, 53 (19%) and 230 (13%) respectively had received blood transfusions 1 year or more before the interview. Using an age- and sex-stratified analysis the odds ratio (OR) for transfusion was 1.74 (95% CI 1.24-2.44). ORs were also determined for transfusions received in the intervals 1-5, 6-15, 16-25 and > or = 26 years before diagnosis. In the interval 6-15 years, the OR for transfusion was 2.83 (95% CI 1.60-4.99) whereas ORs for transfusions received in other intervals were lower and not significantly elevated. Histological diagnoses (Kiel classification) and results of staging procedures were known for 185 patients. For low-grade NHL of nodal B-cell chronic lymphocytic leukaemia (B-CLL) or immunocytoma type, the OR for transfusions was 4.15 (95% CI 1.92-9.01). For low grade nodal lymphomas of follicle centre cell type and high-grade nodal lymphomas, no relation to transfusions could be demonstrated. For high-grade extranodal lymphoma as sole manifestation, OR for transfusions was 3.27 (95% CI 1.30-8.24). It is concluded that blood transfusion may be a risk factor for NHLs especially those of B-CLL or immunocytoma type and for high-grade extranodal lymphoma. PMID- 8624280 TI - Mortality pattern among biological research laboratory workers. AB - A cohort study was conducted to investigate the mortality of individuals employed by biological research institutes in the UK. The inclusion criteria were met by 12,703 individuals, of whom 95% were traced (11,502 alive, 395 deaths, 246 embarkations). All-cause mortality was significantly reduced in men (standardised) mortality ratio (SMR) 55 and women (SMR 52). Mortality was also significantly reduced for circulatory and respiratory diseases, and overall there was low mortality from malignant neoplasms. SMRs exceeded 100, but were not statistically significant, for infective and parasitic diseases. There were no statistically significant raised SMRs for any cancer site. Workers were categorised as ever worked in a laboratory (laboratory workers) and never worked in a laboratory (non-laboratory workers). The all-cause SMR was significantly reduced in both groups, as was mortality from circulatory and respiratory diseases. The SMR for malignant neoplams was also significantly reduced in laboratory workers. On the basis of follow-up to 31 December 1994, there is no evidence of any overall increased risk of mortality in biological research laboratory workers. However, the power of the analysis is limited by the young age of many cohort members and short duration of follow-up. Follow-up is continuing and the data will be reanalysed once more deaths have accumulated. PMID- 8624282 TI - Drugs: Holland bows to international pressure. PMID- 8624281 TI - Occurrence of cancer in women with Turner syndrome. AB - A study of cancer incidence in a cohort of 597 women with Turner syndrome (TS) and a virtually complete follow-up is presented. The cohort was established from the Danish Cytogenetic Register. Information on cancer incidence was obtained from the Danish Cancer Registry and compared with the expected number calculated from the age-, period- and site-specific cancer rates for Danish women. A total of 21 neoplasms was observed, of which 13 occurred more than 1 year after diagnosis of TS, corresponding to a relative risk of cancer of 1.1. Wilms' tumour was the only identified childhood cancer. No case of gonadoblastoma or dysgerminoma was identified in the 29 women with a Y chromosome or in the women in whom no Y chromosome material was detected by standard cytogenetic methods, suggesting that the risk of ovarian germ cell tumours may be lower than previously estimated. Colon cancer was observed in five patients (relative risk 6.9, 95% confidence interval 2.2-16.2). Further studies are needed to assess whether colon cancer in TS is related to Turner-associated genes on the sex chromosome(s). PMID- 8624284 TI - Turbulent times for cancer structures in the U.S. PMID- 8624283 TI - Breast cancer: to screen or not to screen? PMID- 8624285 TI - FDA: quality of life matters! PMID- 8624286 TI - Theoretical and practical aspects of paclitaxel scheduling. PMID- 8624287 TI - Early ovarian cancer: treat now, later or never? PMID- 8624288 TI - The adequacy of consent forms for informing patients entering oncological clinical trials. AB - The impact on 100 patients of information and consent forms signed prior to medical oncology clinical trials was evaluated by a survey at a subsequent visit. Only 40 patients believed that the purpose of the form was to explain the treatment. The form was listed as the major source of information by 12 patients while 52 listed a doctor and 26 a nurse. Although 21 patients believed that the form mad them less anxious, 19 patients believed that it made them more anxious. Despite 80 patients reading all of the form, 60 claiming to understand all of it and 68 claiming that in contained adequate information, in tests of recall only 52 patients could name all of their drugs and only 4 all of the side effects. The number of drugs named correlated with how much of the consent form had been read (p = 0.003) and the highest education level achieved by the patient (p = 0.0003). Patients under 55 years had significantly better recall. Patients with a better ECOG performance status were more likely to find the form very helpful. Such forms may not ensure that the requirements for informed consent are satisfied. PMID- 8624289 TI - Folate-based thymidylate synthase inhibitors as anticancer drugs. AB - The enzyme, thymidylate synthase (TS) is considered an important target for the development of new anticancer agents. Moreover, the folate-binding site in TS is believed to offer better opportunities for the design of highly specific inhibitors than the pyrimidine (dUMP) binding site. This belief led to the design of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline-based drug which had antitumour activity in clinical studies. Occasional, but serious nephrotoxicity led to the withdrawal of CB3717 from further clinical study. More water-soluble and non-nephrotoxic analogues were developed with an interesting diversity in biochemical profile, particularly with respect to interactions with the reduced-folate cell membrane carrier (RFC) and folylpolyglutamate synthetase (FPGS). An example of a compound that uses both of these processes well is the quinazoline, ZD1694 (Tomudex), a drug which is about to complete phase III evaluation for colorectal cancer. High chain length polyglutamates are formed that are up to 70-fold more potent TS inhibitors than the parent drug (Ki tetraglutamate = 1 nM). Furthermore they are retained in cells/tissues for a prolonged period. A number of other novel folate-based TS inhibitors are currently in pre-clinical or clinical study. For example, LY231514 is a pyrrolopyrimidine analogue in phase I study and, although less potent as a TS inhibitor, has biochemical properties similar to ZD1694. Another compound in phase I study is the benzoquinazoline, BW1843U89 which has somewhat different properties. It is a very potent TS inhibitor (Ki = 0.09 nM) and an excellent substrate for the RFC (human) and FPGS, but polyglutamation proceeds to diglutamate only and is not accompanied by increased TS inhibition. Another highly water-soluble compound in pre-clinical development is ZD9331 which was specifically designed to use the RFC but not be a substrate for FPGS. Potent TS inhibition (Ki = 0.4 nM) was achieved through a rational programme of computerised molecular modelling of the active site of TS and a large database of structure-activity relationships. Two lipophilic compounds were designed to be devoid of interactions with either the RFC or FPGS. High resolutions crystal complexes of E. coli TS were central to obtaining potent TS inhibitors and both AG337 (Ki human recombinant TS = 16 nM) and AG331 (Ki = 12 nM) are in clinical studies. This portfolio of novel compounds therefore comprehensively addresses the potential of TS as a target for cancer chemotherapy. PMID- 8624290 TI - Multi-day fractionated administration schedule for paclitaxel. AB - PURPOSE: To establish the feasibility of fractionating paclitaxel administration by utilizing daily one-hour infusions for three, four or five days with dose escalating to determine the patterns of hematologic and non hematologic toxicities. PATTERNS AND METHODS: Forty patients received 87 courses of daily fractionated paclitaxel for three, four or five days; cycles were repeated every 21 days. Six patients received concomitant daily cisplatin. The median number of cycles delivered per patient was two with a range of one to six. RESULTS: Cumulative doses per cycle ranged from 120 mg/m2 to 250 mg/m2 with 25% of the cycles delivering 200 mg/m2 or more. Ten cycles (11.5%) were associated with dose limiting neutropenia (grade 3 [7 cycles]; grade 4 [3 cycles]). No hypersensitivity reactions were observed and no patient required cytokine support. No patient required hospitalization. CONCLUSIONS: Administering paclitaxel on a daily fractioned schedule in an ambulatory setting is logistically feasible; does not require premedication; is associated with a toxicity pattern similar to single day schedules (e.g. 24-hour or three-hour infusion); is capable of delivering a higher dose per cycle than published 96- or 120-hour infusion schedules; and could possibly be escalated to doses higher than 250 mg/m2 in carefully selected patients. The optimal dose rate for five-day multifractionated administration of paclitaxel is 40 to 50 mg/m2/d or a cumulative cycle dose of 200 to 250 mg/m2 and does not require cytokine usage. Adding cisplatin on a fractionated daily schedule may accentuate the neurotoxicity associated with both agents. A prospective comparison of four-day fractionated vs. four-day continuous infusional paclitaxel has been proposed as a randomized study to determine clinical differences in response, dose intensity and toxicity. PMID- 8624291 TI - Adjuvant treatment for early epithelial ovarian cancer: results of two randomised clinical trials comparing cisplatin to no further treatment or chromic phosphate (32P). G.I.C.O.G.: Gruppo Interregionale Collaborativo in Ginecologia Oncologica. AB - BACKGROUND: From 1983 to 1990, 271 consecutive patients with stage I ovarian cancer entered two randomised trials, aimed at assessing the role of adjuvant chemotherapy after radical surgery in early stages of ovarian cancer. Trial I compared cisplatin (50 mg/m2 with repeated courses every 28 days for 6 cycles) to no further therapy in F.I.G.O. stage Ia & b Grade II-III patients; trial II compared cisplatin (same dose and schedule) to 32P in Iaii & bii and Ic patients. METHODS: Both studies were multicentric and centrally randomized. Treatment was allocated by phone and stratified by center. All patients satisfying major eligibility criteria (histological and grade, no previous neoplasms) were analysed according to treatment allocated by randomisation. RESULTS: With a median observation time of 76 months, cisplatin significantly reduced the relapse rate by 65% (HR = 0.35; 95% CI = 0.14-0.89, p = 0.028; Cox Model) in trial I and 61% (HR = 0.39; 95% CI = 0.19-0.77, p = 0.007; Cox Model) in trial II. Survival was not significantly different (trial I - Kaplan-Meier overall 5-year survival: cisplatin = 88%, control = 82%, HR = 1.15; 95% CI = 0.44-2.98; p = 0.773; Cox Model); trial II - overall 5-year survival: cisplatin = 81%, 32P = 79%, HR = 0.72; 95% CI = 0.37-1.43; p = 0.354; Cox model). In both studies the risk of dying after relapse increased for patients originally randomized to the cisplatin arms: in trial I, 6 of 7 patients in the cisplatin relapsed arm and died of tumor compared with 8 of 14 patients in the control arm. In trial II 11 of 12 patients on cisplatin, and 18 of 26 on 32P succumbed to tumor recurrence. CONCLUSION: Adjuvant cisplatin treatment in early ovarian cancer significantly prevents relapse in comparison to 32P in stage IC patients or to no immediate treatment in earlier stage women. The impact of cisplatin adjuvant treatment on survival remains, however, unclear. PMID- 8624292 TI - Fixed versus response-adapted MOPP/ABVD chemotherapy in Hodgkin's disease. A prospective randomized trial. AB - BACKGROUND: The optimal number of chemotherapy courses in responding patients with advanced-stage Hodgkin's disease (HD) is unknown. PATIENTS AND METHODS: With minimizing chemotherapy and thereby reducing late complications as the objective, patients with advanced HD were randomized to receive either 4 full MOPP/ABVD courses or treatment up to complete remission (CR). Forty-seven patients were given the fixed (FT) and 41 patients the individual treatment (IT). The two groups were balanced according to age, histopathology and sex, although stage IVB dominated in the IT group (20 vs. 8). RESULTS: Sixty-six of 88 patients (75%) achieved CR. No difference between the two treatment groups in the proportion of stage IVB patients was seen when those achieving CR, i.e., the efficacy population were compared. The mean number of single chemotherapy courses given was 3.7 of MOPP and 3.5 of ABVD in the FT group, compared to 2.6 of MOPP and 2.5 of ABVD in the IT group (p < 0.001). The predicted progression-free survival at 10 years was 81% in the FT and 68% on the IT arm, respectively (p < 0.05). No statistically significant difference in cause-specific 10 year survival was observed (82% and 83%, respectively; p = 0.18). Long-standing CRs were achieved following minimal chemotherapy. CONCLUSIONS: Since there are no available methods to identify long-term disease-free survivors among CR patients following a limited induction treatment, we suggest that the policy of giving 3-4 full MOPP/ABVD courses should continue. The price for such an approach is the overtreatment of a subset of already cured patients. PMID- 8624293 TI - Further chemotherapy versus low-dose involved-field radiotherapy as consolidation of complete remission after six cycles of alternating chemotherapy in patients with advance Hodgkin's disease. German Hodgkins' Study Group (GHSG). AB - OBJECTIVE: It was the aim of this prospective randomized multicenter study to compare chemotherapy and radiotherapy as consolidation treatments in patients achieving complete remission (CR) after 6 cycles of doxorubicin-containing chemotherapy in advanced-stage Hodgkin's disease (HD). METHODS: A total of 288 previously untreated patients aged 18-60 years with stage IIIB or IV HD received induction chemotherapy with 3 X (COPP + ABVD). Patients achieving CR were eligible for randomisation to either 20 Gy radiotherapy to initially involved fields (RT-arm) or to an additional 1 X (Copp + ABVD) (CT-arm ). Patients with nodal PR were allocated to more intense radiotherapy (IRT-arm: 20 Gy IF, 40 Gy to persisting tumor). Four patients with persisting organ involvement after induction received salvage chemotherapy. RESULTS: Of 288 patients, 171 (59%) achieved CR after induction chemotherapy. Of these, 100 patients were successfully randomized to RT or CT. In the CT arm relapses were observed on 10 of 49 patients compared with 13 of 51 patients in the RT arm (p = n.s.). Fifty patients refused randomisation and for them a treatment was chosen, and 21 patients refused any further treatment. Of these 21 patients with no consolidation therapy, 9 relapsed, indicating an approximately 3-fold increased relapse risk compared with those receiving either of the consolidation therapies. No relapse was observed in initially involved lung or liver sites. Adverse prognostic factors for freedom from treatment failure and survival were low hemoglobin and large mediastinal mass at initial presentation. CONCLUSIONS: No statistically significant differences in treatment efficacy were detected between 20 Gy IF radiotherapy and 1X (COPP + ABVD) chemotherapy following CR after six cycles of alternating chemotherapy in patients with advanced-stage HD. However, limited observations in a non-randomized cohort indicate that patients without consolidation treatment of CR after 6 cycles of chemotherapy may have an elevated risk of relapse. PMID- 8624294 TI - A phase I trial of intermittent high-dose alpha-interferon and dexamethasone in metastatic renal cell carcinoma. AB - BACKGROUND: Evidence exists that the toxic of alpha-interferon can be ameliorated by co-administration of dexamethasone without compromise of therapeutic efficacy. We therefore conducted a phase I trial to determine the maximum tolerated dose of intermittent interferon when combined with oral dexamethasone. PATIENTS AND METHODS: Thirty patients with metastatic renal cell carcinoma were enrolled. The starting dose of interferon was 20 million IU/m2/day given as a subcutaneous injection days 1 to 4 of each 14 day cycle. Dose levels were escalated at increments of 5 million IU/m2. Dexamethasone 4 mg was administered orally every 6 hours during administration of high-dose interferon. Low-dose maintenance interferon, 3 million IU/m2/day, was administered without dose escalation on days 5 to 14 of each cycle. RESULTS: The maximum tolerated dose of intermittent high dose interferon was 40 million IU/m2/day. The dose limiting toxicity was fatigue. EEG abnormalities developed in five patients and neuropsychiatric parameters deteriorated significantly in seventeen. CONCLUSIONS: We conclude that co administration of dexamethasone improves the tolerance of intermittent high-dose interferon. The results of this trial may be useful in designing high-dose interferon regimens for renal cell carcinoma and other interferon-sensitive diseases. PMID- 8624295 TI - Ki-1 (CD30) anaplastic large-cell lymphoma in children. AB - BACKGROUND: Ki-1 (CD30) ALCL is a rare and distinct type of high-grade NHL. A relevant feature is young age at presentation. Most reported pediatric series are retrospective analyses of heterogeneously-treated cases. PATIENTS AND METHODS: Between 1976 and 1993, Ki-1 ALCL was diagnosed in 32 children (20 males 12 females; median age 9 years; 10.4% of childhood NHL). Before 1987, original diagnoses had been malignant histiocytosis in 14 cases, and immunoblastic NHL in 2. The treatment program for childhood T-lymphoblastic NHL, except for CNS prophylaxis, was applied in 28/32 cases. Radiotherapy to involved sites not in complete remission within 4 weeks after treatment start was used only prior to 1987. RESULTS: In 78% of cases, Ki-1 ALCL presented in skin, bone, spleen and lung. Lymph nodes were frequently involved, often in association with periadenitis. Bone marrow and spinal fluid were negative in all cases. Eleven patients were classified as stage I/II, 21 as stage III. Fewer was present in 65% of patients. Cell phenotype was T in 21/32, and null in the remaining 11. Treatment results were evaluable in 27/32 children, all of whom achieved complete remission. Seven relapsed and 4 died of their disease. Five-year survival and progression-free survival were 84% and 72% respectively. Involvement of peripheral lymph nodes, absence of spleen, liver, lung and mediastinum involvement, and male sex were favourable indicators. CONCLUSIONS: Ki-1 ALCL is a high-grade NHL with a relatively good prognosis when treated with multiagent intensive chemotherapy. Because of its clinical peculiarities a unique treatment approach in probably warranted. PMID- 8624296 TI - Selective suppression of cytokine secretion in patients with small-cell lung cancer. AB - BACKGROUND: Whether or not cytokine secretion is impaired in patients with small cell lung cancer (SCLC), is unknown. We therefore investigated whether cytokine secretion by immunocompetent cells may be suppressed in patients with SCLC. PATIENTS AND METHODS: We determined cytokine secretion by lymphocytes and monocytes in whole blood cell cultures from 58 patients with SCLC, 95 patients with non-small-cell lung cancer (NSCLC), 10 patients with nonmalignant lung disease and from 44 normal healthy individuals by using an enzyme-linked immunosorbent assay (ELISA) specific for the different cytokines measured. RESULTS: Compares to normal controls, immunocompetent cells from patients with SCLC secreted significantly lower amounts of IL-2, IFN alpha, and IFN gamma upon mitogen stimulation. TNF alpha-secretion was significantly reduced in SCLC extensive disease but not in SCLC limited disease. In contrast, secretion of IL-1 alpha and IL-1 beta was not reduced. In patients with NSCLC, secretion of IL-2 and IFN alpha was significantly reduced. Reduction of IFN gamma secretion was significant in metastasized NSCLC and marginally significant in localized NSCLC. Secretion of TNF alpha, IL-1 alpha and IL-1 beta was not impaired. In addition, cytokine secretion in SCLC patients substantially improved upon successful reduction of tumor load by chemotherapy but not upon ineffective chemotherapy. Furthermore, TGF beta 1 suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha but not of IL-1 alpha and IL-1 beta in whole blood cell cultures from healthy individuals. CONCLUSIONS: Suppression of cytokine secretion in patients with SCLC was selective, dependent on tumor load, different from immunosuppression in NSCLC and seemed to be reconstituted upon reduction of tumor load. These results may suggest interactions between tumor cells and the immune system. TGF beta 1 secreted by SCLC cell lines induced the same selective cytokine suppression as that found in SCLC patients. However, whether or not tumor-derived TGF beta 1 is a factor inducing selective immunosuppression in SCLC patients is presently unclear. PMID- 8624297 TI - Effects of the hematopoietic growth factors GM-CSF, IL-3, and IL-6 on human tumor colony-forming units taken directly from patients. AB - BACKGROUND: One concern regarding the use of hematopoietic growth factors (e.g., GM-CSF, IL-3, and IL-6) to accelerate hematologic recovery after treatment of solid tumors with high doses of chemotherapy is that these factors may stimulate tumor growth. MATERIALS AND METHODS: We tested the effects of GM-CSF, IL-3 or IL 6 (continuous exposure to 1, 10, and 100 ng/ml of each cytokine) on tumor cells taken directly from patients with solid tumors using the human tumor cloning assay. The range of concentrations of the cytokines used in our study included the concentrations that appear to be clinically relevant. RESULTS: Of the evaluable samples, stimulation of tumor growth was noted in 0/16 exposed to GM CSF, in 3/72 (4%) exposed to IL-3, and in 1/65 (2%) exposed to IL-6. Inhibition of tumor proliferation was noted in no sample exposed to GM-CSF, in 7 (10%) exposed to IL-3 and in 7 (10%) exposed to IL-6. CONCLUSIONS: The use of GM-CSF, IL-3 or IL-6 to reduce myelosuppression after high dose chemotherapy appears unlikely to result in stimulation of the growth of the most common solid tumors. It is also unlikely that either IL-3 and IL-6 alone will be useful as antitumor agents against solid tumors. PMID- 8624298 TI - Recurrences and second primary tumours in the head and neck region: differentiation by p53 mutation analysis. AB - BACKGROUND: Patients with head and neck cancer are at high risk of developing additional primary tumours in the aerodigestive tract as a result of field cancerization phenomena. In this context, the appearance of a new neoplasm often poses a problem of differential diagnosis between recurrence and new primary tumour. The differentiation between the two entities in essentially clinical and conventionally based on the histological and spatio-temporal relations between the two lesions; however, the validity of these criteria has still to be assessed. DESIGN: To evaluate whether field cancerization phenomena may affect the clinical diagnosis of relapse/metastasis in the head and neck region, p53 mutation pattern was analysed in a series of primary tumours and corresponding recurrences/metastases. The rationale was that, since p53 mutations are a very early and polymorphic phenomenon, a recurrence/metastasis must retain the same mutation as the the primary tumour, whereas independent tumours are likely to display a different p53 gene status. RESULTS: Molecular analysis provided conclusive results in 9 of 12 cases analysed. The clinical diagnosis of recurrence was confirmed by molecular analysis in 4 of these cases. In contrast, a differential p53 mutation pattern supported an independent origin for 3 presumed local recurrences and 2 lung lesions. CONCLUSIONS: The use of p53 mutation analysis as a clonality marker allowed us to ascertain the inadequacy of the current diagnostic criteria for the differentiation between a new independent tumour and recurrence/metastasis. These findings substantiate the relevance of field cancerization phenomena in the head and neck region and prompt the use of p53 mutation analysis as a fundamental tool to improve the diagnosis and management of head and neck cancers. PMID- 8624299 TI - Brain metastases in small-cell lung cancer. PMID- 8624300 TI - Construction, expression and characterisation of a single chain anti-tumour antibody (scFv)-IL-2 fusion protein. AB - We describe the production and preliminary characterisation of a fusion protein between interleukin-2 and a single-chain Fv version of the H17E2 anti-placental alkaline phosphatase (PLAP) antibody. This molecule could be used to target interleukin-2 to PLAP-expressing tumours. PMID- 8624302 TI - Bronchoscopic treatment modalities in lung cancer, indications and limitations. PMID- 8624301 TI - Two phase III trials of tauromustine (TCNU) in advanced colorectal cancer. PMID- 8624303 TI - Hypoxemia due to right-to-left shunt in the carcinoid syndrome; beneficial response to octreotide. PMID- 8624304 TI - Ambulatory urodynamic monitoring. PMID- 8624305 TI - Revised FIGO staging for early invasive carcinoma of the vulva and cervix. PMID- 8624307 TI - Treating all babies with vitamin K: an 'unnatural' policy? PMID- 8624306 TI - Biochemical prediction of pre-eclampsia. PMID- 8624308 TI - Ambulatory urodynamic monitoring. PMID- 8624309 TI - Treating all babies with vitamin K: an "unnatural" policy? PMID- 8624310 TI - A method of proportional audit of perinatal care. PMID- 8624311 TI - Cervical and vulva cancer: changes in FIGO definitions of staging. PMID- 8624312 TI - Serum screening for Down's syndrome between 8 and 14 weeks of pregnancy. International Prenatal Screening Research Group. AB - OBJECTIVE: To determine the value of serum screening for Down's syndrome at 8-14 weeks of pregnancy using seven potential serum markers (alpha-fetoprotein, unconjugated oestriol, total human chorionic gonadotrophin (hCG), free alpha-hCG, free beta-hCG, pregnancy associated plasma protein A (PAPP-A), and dimeric inhibin A). DESIGN: Stored blood samples collected from women at about 10 weeks of pregnancy, prior to having chorionic villus sampling procedure on account of advanced maternal age, were retrieved from pregnancies associated with Down's syndrome and from matched unaffected pregnancies. SETTING: Twenty-one obstetric centres in nine countries. SUBJECTS: Seventy-seven pregnancies associated with Down's syndrome each matched with five controls (except in two cases that were matched with four controls) for maternal age (same five year age groups), duration of storage of the serum sample (same calendar year), and gestational age (usually same week of pregnancy). RESULTS: The levels of two potential markers differed between affected and unaffected pregnancies sufficiently to be of value in screening--free beta-hCG and PAPP-A. The median free beta-hCG level in affected pregnancies was 1.79 times the median level for unaffected pregnancies, and the median PAPP-A level was 0.43 times the normal median. These two markers were combined with maternal age to estimate a woman's risk of having a fetus with Down's syndrome. A screening programme that used a risk cutoff level of 1:300 would detect 63% of affected pregnancies and also classify 5.5% of unaffected pregnancies as screen positive. None of the other five markers added more than 2% detection for the same false-positive rate. CONCLUSION: The performance of screening using maternal age and serum-free beta-hCG and PAPP-A at 10 weeks of pregnancy was better than the double test (alpha-fetoprotein and hCG with maternal age) and similar to the triple test (alpha-fetoprotein, unconjugated oestriol and hCG with maternal age) at 15-22 weeks. PMID- 8624313 TI - Effects of hypoglycaemia on fetal heart activity and umbilical artery Doppler velocity waveforms in pregnant women with insulin-dependent diabetes mellitus. AB - OBJECTIVE: To study the effect of induced hypoglycaemia on fetal wellbeing as indicated by fetal heart rate and umbilical artery flow velocity waveforms. DESIGN: A prospective experimental investigation. SETTING: High risk pregnancy unit and diabetes research unit at Karolinska Institutet, Danderyd Hospital, a university affiliated hospital. PARTICIPANTS: Ten women with insulin-dependent diabetes mellitus in the third trimester of pregnancy. INTERVENTIONS: The fetal heart rate, the blood flow velocity waveforms in the umbilical artery and the maternal catecholamine levels were investigated during a 150-minute hyperinsulinaemic hypoglycaemic clamp with induction and maintenance of an arterial blood glucose level of about 2.2 mmol/l. MAIN OUTCOME MEASURES: 1. Fetal: changes of fetal heart rate pattern and pulsatility index of the umbilical artery flow velocity waveforms. 2. Maternal: levels of plasma adrenaline and plasma noradrenaline. RESULTS: Maternal hypoglycaemia was associated with an increase in frequency and amplitude of fetal heart rate accelerations, a slight decrease in the pulsatility index of the umbilical artery and a rise in the maternal catecholamine levels. CONCLUSIONS: We speculate that the increased number of fetal heart rate accelerations reflects an increased sympathico-adrenal activity during the hypoglycaemia clamp. No potentially harmful effects on the fetus were observed in the fetal heart rate or in the umbilical artery Doppler waveform analysis during hypoglycaemia. PMID- 8624314 TI - Early prediction of pre-eclampsia by measurement of kallikrein and creatinine on a random urine sample. AB - OBJECTIVE: To assess the measurement of inactive urinary kallikrein (IUK) to creatinine (Cr) ratio (IUK:Cr) on an untimed urine sample, collected between 16 and 20 weeks of pregnancy, as a predictive test for the development of both proteinuric and nonproteinuric pre-eclampsia. DESIGN: A prospective longitudinal study. SETTING: A clinic for antenatal care and a university research department. PARTICIPANTS: Three hundred and seven normotensive women randomly selected (124 nulliparous and 183 parous) attending the antenatal clinic for their booking visit. MAIN OUTCOME MEASURES: 1. Nonproteinuric pre-eclampsia: a rise in diastolic blood pressure of 25 mmHg or more and a crossing of the threshold of 90 mmHg; 2. Proteinuric pre-eclampsia: same as 1. plus the development of significant proteinuria ( > 1 + on urine dipstick). RESULTS: Thirty-seven women developed pre-eclampsia, 12 of whom had proteinuria. Median IUK:Cr ratio in this group was 78.27, compared with 358.19 in the remainder. Analysis of receiver operator characteristics gave an area under the curve of 0.803. An IUK:Cr ratio of 170 or less in this study predicted nonproteinuric or proteinuric pre eclampsia with a sensitivity of 70% and a specificity of 86%. Ten of the twelve women who had proteinuria had an IUK:Cr below 170. Median IUK:Cr for those with proteinuric pre-eclampsia was 72.91. CONCLUSIONS: Measurement of IUK:Cr on a urine sample, collected between 16 and 20 weeks of gestation, represents a simple and practical test for the risk of subsequent pre-eclampsia, with a sensitivity and specificity comparable to those reported by other investigators using the widely recognised, but less practical, angiotensin II sensitivity test. PMID- 8624315 TI - Fetal size at birth in relation to quality of blood glucose control in pregnancies complicated by pregestational diabetes mellitus. AB - OBJECTIVE: To determine the relation between maternal levels of blood glucose and glycated haemoglobin (HbA1c) and infant size at birth in pregestational diabetes. DESIGN: Longitudinal study from 6 to 14 weeks gestation. Women were treated intensively with insulin, aiming at normoglycaemia but avoiding hypoglycaemia. Blood glucose was determined six times daily, HbA1c every four weeks. Individual mean fasting and postprandial glucose levels were calculated for three-week periods of gestation. Birthweight > 2 SD or within +/- 2 SD for gestational age and gender was classified as large (LGA) or appropriate (AGA), respectively. Birthweight ratio was calculated as the ratio of birthweight to normal mean birthweight after correction for gestational age and gender. PARTICIPANTS: One hundred and thirteen consecutive pregnant women with pregestational diabetes and their newborn infants. RESULTS: Perinatal mortality was nil, the rates of spontaneous preterm delivery (8.9%) and severe maternal hypoglycaemia (4.4%) were low. Mothers with LGA infants (26%) had a significantly higher fasting glucose between weeks 27 and 32 than mothers of AGA infants (P < 0.01). Relative birthweight was significantly and independently associated with pre-pregnancy bodyweight (r = 0.24, P < 0.05) and fasting glucose at weeks 27 to 29 (r = 0.27, P < 0.01) but together could only explain 12.3% of the variation in birthweight (mult. r = 0.35, P < 0.01). HbA1c correlated with glucose levels but was unrelated to birthweight ratio. The fasting glucose level between weeks 30 and 32 was significantly interrelated with the fasting glucose level from each of the six preceding three-week periods. CONCLUSION: Near normoglycaemia cannot be obtained in all patients, presumably due to intrinsic differences in glucoregulatory ability between individuals. The incidence of LGA infants was unexpectedly high. The modest abnormality in glycaemic control in mothers with LGA infants could only partly explain fetal oversize, suggesting that other factors must be implicated to explain fetal growth acceleration. PMID- 8624316 TI - Ambulatory monitoring and conventional cystometry in asymptomatic female volunteers. AB - OBJECTIVES: Ambulatory monitoring continues to gain acceptance and increasing clinical application. There remains, however, a sparsity of published data asymptomatic volunteers, particulary women. Our aim was, by study of such a group, to establish normal ranges for cystometric variables on ambulatory monitoring. PARTICIPANTS: We recruited 22 women from staff and gynaecological inpatients. A standard questionnaire of urinary complaints was administered and any woman with significant symptoms was excluded. Conventional cystometry and ambulatory monitoring were undertaken in all volunteers in random order. Volunteers were asked to go about everyday activities and to keep a detailed event diary whilst undergoing ambulatory monitoring. RESULTS: Using standard International Continence Society terminology, detrusor instability was found in 18% of volunteers on conventional cystometry and in 68% on ambulatory monitoring. Significant differences were found between ambulatory monitoring and conventional cystometry with respect to the detrusor pressure rise on filling (P < 0.001) and voided volumes (P < 0.001). A significant difference was also present with respect to voiding pressures (P < 0.001), although the absence of simultaneous flow rate measurement makes absolute separation of voiding pressure from post voiding after contractions difficult. CONCLUSIONS: Ambulatory monitoring detects a greater number of abnormalities based on conventional cystometric criteria of normality. Before we can usefully apply the method to symptomatic patients we must first define normal ranges for filling and voiding cystometry for the technique. This study moves towards this goal. (The addition of flow rate data will permit accurate identification and measurement of voiding pressures.) PMID- 8624317 TI - Serum levels of endometrial proteins during transcervical resection of the endometrium. AB - OBJECTIVE: To study the potential for dissemination of endometrial tissue substances during transcervical resection of the endometrium (TCRE). DESIGN: Prospective study. SETTING: One university and two county hospitals. PARTICIPANTS: Forty-eight women with dysfunctional bleeding. INTERVENTIONS: The serum levels of two endometrial proteins, insulin-like growth factor binding protein-1 (IGFBP-1) and placental protein 14 (PP14), were measured before and every 10 min during the operations. Blood loss was also measured by a photometer together with absorption of the irrigating fluid containing glycine 1.5% and ethanol 1% by expired-breath tests, and serum sodium and volumetric fluid balances. MAIN OUTCOME MEASURES: Linear correlations between changes in IGFBP-1 and PP14 during TCRE and operating parameters such as operating time, blood loss and fluid absorption. RESULTS: The baseline levels of IGFBP-1 were normal but PP14 could only be detected in one third of the patients, which was due, in part, to pre-operative treatment with danazol. The highest levels of IGFBP-1 and PP14 during surgery correlated positively with the baseline concentrations. Fluid absorption (median 405 ml, range 0-2177) was the only surgical factor associated with increasing serum levels of endometrial proteins. CONCLUSION: Absorption of the solution used to irrigate the uterus is associated with dissemination of endometrial products in the bloodstream during TCRE. PMID- 8624318 TI - Vulvar intraepithelial neoplasia: long term follow up of treated and untreated women. AB - OBJECTIVE: To investigate the long term outcome of patients with vulvar intraepithelial neoplasia. DESIGN: A retrospective study using information obtained from patient casenotes. SAMPLE: One hundred and thirty-three women with a primary diagnosis of vulvar intraepithelial neoplasia (VIN), identified during a 15-year period. RESULTS: The diagnosis of vulvar intraepithelial neoplasia increased throughout the study period. Human papilloma virus changes were noted in 104 patients (78%); these women were significantly younger than those without (P < or = 0.001). Nineteen (14%) were managed by observation or medical treatment and the remainder by surgical methods. Histological or symptomatic recurrence after surgical treatment occurred in 55 (48%). When disease recurred, it usually did so within four years of treatment. Recurrence was more common following laser vapourisation than after local excision (75% vs 40%; P < or = 0.01). Progression to invasive disease occurred in nine patients (7%), none of whom were in the group being observed. Four deaths occurred in this group, three from gynaecological malignancies of the lower genital tract. CONCLUSION: Patients with vulvar intraepithelial neoplasia require long term follow up, and the risk of invasion may be higher than previously thought. Surgical treatment when required should be by excisional rather than ablative methods in most instances. In selected cases it is also possible to safely manage patients by more conservative methods. PMID- 8624319 TI - Vulvar intraepithelial neoplasia with superficially invasive carcinoma of the vulva. AB - OBJECTIVE: To investigate the long-term outcome of patients presenting with vulvar intraepithelial neoplasia (VIN) with superficially invasive carcinoma of the vulva (SICa). DESIGN: A retrospective study using information obtained from patient case notes. SAMPLE: Twenty-six women found at presentation to have VIN in association with superficially invasive carcinoma were identified during a 15 year period. RESULTS: Pruritus vulvae was the most frequent presenting symptom in 18 patients (69%). Sixteen women (61.5%) had multiple symptoms. Features noted at vulvar examination were variable and none were pathognomonic of either VIN or of superficial invasion. All patients had VIN 3 in association with a superficially invasive carcinoma. Histological changes associated with human papillomavirus were found in 19 (73%) women. Half had a co-existent or previous abnormality of the lower genital tract. Local excision was the most frequent initial treatment (n = 9 [35%]). Mean follow up time was 65 months (range 12-174). Disease persisted after primary treatment in five women (19%). Both histological recurrence (of either VIN or SICa) or symptomatic recurrence occurred in 10 patients (38%). All patients who experienced recurrence did so within 36 months of treatment. Overall, 12 patients (46%) relapsed (histological or symptomatic recurrence); the mean time was 18 months. Fourteen patients (54%) were managed satisfactorily by their initial treatment. One patient died of recurrent cervical cancer. Three progressed to frankly invasive disease: two (aged 31 and 39 years) with carcinoma of the vulva and one aged 34 years with carcinoma of the perianal margin. All are alive and well after treatment. One patient had recurrence of superficially invasive carcinoma treated by local excision with no further problems. No episode of metastasis via lymphatic or vascular channels has been seen. CONCLUSIONS: Patients with superficially invasive carcinoma of the vulva may be safely treated by local excisional methods without recourse to lymphadenectomy. Relapse after primary treatment is common, and there appears to be a significant risk of progression to frankly invasive carcinoma. PMID- 8624320 TI - The effect of directed biopsy on the atypical cervical transformation zone: assessed by digital imaging colposcopy. AB - OBJECTIVE: To determine the effect of directed punch biopsy trauma on the natural history of atypical cervical transformation zones. DESIGN: A prospective randomised clinical trial. SETTING: Academic Unit colposcopy clinics in Birmingham. PARTICIPANTS: One hundred and eighty women attending the colposcopy clinics were recruited over an 18 month period, of which 161 were eligible for analysis. METHODS: Three-way randomisation of patients into 1. no biopsy, 2. central biopsy and 3. peripheral biopsy groups. Quantitative assessment of the change in surface area and severity of cervical intra-epithelial neoplasia (CIN) lesions in each group measured six weeks apart using digital imaging colposcopy. RESULTS: No significant difference in change in lesion size (P = 0.40) was noted in the three treatment groups. Results suggest that the severity of the lesion was underestimated by the peripheral biopsy. CONCLUSIONS: The results of this study suggest that directed punch biopsy trauma does not have a significant effect on the immediate natural history of CIN. No statistically significant differences were found in lesion size whether biopsy was employed or not. In addition, the site of biopsy had no influence on the outcome. It appears, therefore, that tissue trauma from punch biopsy and the subsequent inflammatory and wound healing processes do not modify the course of CIN. The regressive changes observed after punch biopsy in previous natural history studies are probably not a result of the initial inflammatory response to biopsy trauma and subsequent re-epithelialisation with normal cells, but may result from processes that continue long after tissue repair is completed. PMID- 8624322 TI - Elective late fetal karyotyping. PMID- 8624321 TI - Characterisation of a sperm coating auto-antigen reacting with antisperm antibodies of infertile males using monoclonal antibodies. AB - OBJECTIVE: In a previous study a number of sperm-specific antigens were identified which reacted with antisperm antibodies from both infertile and vasovasostomised males. To investigate the localisation and distribution of these antigens and their role in male fertility, monoclonal antibodies were raised against them; immunoblotting techniques were used to select only those antibodies which competed with human antisperm antibodies for these human auto-antigens. DESIGN: One antibody, NW21, reacted with an 18 kDa auto-antigen present on epididymal sperm but absent from testicular sperm. Immunohistochemical studies showed that the antigen is produced in small basal cells between the columnar epithelium of the corpus epididymis, passes up into the tubule and then coats sperm passing along the epididymis. Sperm stored in the cauda epididymis and ductus deferens stain strongly for this sperm coating glycoprotein. CONCLUSIONS: The localisation of this antigen supports the suggestion that auto-immune infertility may represent a response to epididymal rather than testicular sperm. Monoclonal antibodies raised to unique and immunologically accessible sperm coating proteins, produced in the epididymis rather than in the testis, would seem to present an excellent theoretical solution to male contraception. PMID- 8624323 TI - Unrecognised HIV infection among gynaecology patients. PMID- 8624324 TI - The application of standard and morphometric dating criteria for luteal phase endometrial specimens collected by outpatient sampling. PMID- 8624325 TI - A new approach to reconstruction following vulval excision. PMID- 8624326 TI - An N-of-1 trial for treatment of hyperemesis gravidarum. PMID- 8624327 TI - The management of recurrent genital herpes infection in pregnancy: a postal survey of obstetric practice. PMID- 8624328 TI - Ovarian cancers related to minimal access surgery. PMID- 8624329 TI - Biochemical screening for Down's syndrome. PMID- 8624330 TI - Laser Doppler-recorded reactive hyperaemia in the forearm skin during the menstrual cycle. PMID- 8624331 TI - Elaboration of stem villous vessels in growth restricted pregnancies with abnormal umbilical artery Doppler waveforms. PMID- 8624332 TI - A multicentre comparative study of 17 experts and an intelligent computer system for managing labour using the cardiotocogram. PMID- 8624333 TI - Diagnosis, prevention and management of ovarian hyperstimulation syndrome. PMID- 8624334 TI - Postmenopausal women: HRT today and into the future. Proceedings of the 6th Novo Nordisk International Symposium. Copenhagen, Denmark, 20-30 September 1995. PMID- 8624335 TI - What do women want? PMID- 8624336 TI - The oestrogen receptor in bone-evolution of our knowledge. PMID- 8624337 TI - Screening and treatment in the elderly to reduce osteoporotic fracture risk. PMID- 8624338 TI - Is osteoporosis a genetically determined disease? PMID- 8624339 TI - Relationship of common allelic variation at the vitamin D receptor locus to bone mineral density and bone turnover. PMID- 8624340 TI - Osteoporosis into the year 2010. PMID- 8624341 TI - Are changes in lipoproteins during HRT important? PMID- 8624342 TI - Combined hormone replacement therapy with oestradiol and norethisterone acetate: effects in hyperlipidaemia. PMID- 8624343 TI - Hormone replacement therapy and cardiovascular disease: the rabbit model. PMID- 8624344 TI - Hormone replacement therapy and coronary artery atherosclerosis: the monkey model. PMID- 8624345 TI - Hormone replacement therapy and cardiovascular disease: the human model. PMID- 8624346 TI - Hormone replacement therapy and syndrome X. PMID- 8624348 TI - Oestrogen replacement therapy and Alzheimer's disease. PMID- 8624347 TI - Benefits of exogenous oestrogen in inhibiting stress-related coronary artery atherosclerosis. PMID- 8624349 TI - Hormone replacement therapy and breast cancer: an overview. PMID- 8624350 TI - The role of bone turnover in the pathophysiology of osteoporosis. PMID- 8624351 TI - Decision factors influencing hormone replacement therapy. PMID- 8624352 TI - Accumulated knowledge of Kliogest safety aspects. PMID- 8624353 TI - Polarity-dependent conformational switching of a peptide mimicking the S4-S5 linker of the voltage-sensitive sodium channel. AB - The S4-S5 linker (or S45) in voltage-sensitive sodium channels was previously shown to be involved in the permeation pathway. The secondary structure, investigated by circular dichroism, of a S4-S45 peptide from domain IV and its fragments (including S45) is reported here and compared with that of the homologous peptide from domain II as a function of the solvent dielectric constant. The reduction in helicity seen for S4-S45 (II) in polar media is cancelled in membrane-like environment. The most striking result-- a sharp alpha helix --> beta-sheet transition upon exposure of the S45 moiety to aqueous solvents-- is discussed as regards channel activation and selectivity. PMID- 8624354 TI - Two pathways for electrogenic bicarbonate ion movement across the rabbit corneal endothelium. AB - Amiloride (0.5 mM) inhibited the rate of entry of Na+ into corneal endothelial cells by more than half ((0.76 +/- 0.10) to (0.21 +/- 0.10) microEq cm(-2)h(-1)). The same concentration of amiloride caused only minimal disturbance to corneal hydration control by the endothelium (range 0-12%). Amiloride (0.5 mM) and acetazolamide (1 mM) reversibly inhibited trans-endothelial short circuit current by about a half. Their combined effect was not additive. Acetazolamide (1 mM) reduced net HCO3- flux across the short-circuited endothelium by about the same amount ((0.50 +/- 0.11) microEq cm(-2)h(-1)) that amiloride (0.5 mM) reduced Na+ entry into the cells ((0.55 +/- 0.14) microEq cm(-2)h(-1)). Low concentrations of amiloride (10 microM) had little effect on the transport characteristics of the endothelium, indicating that Na+ entry into the endothelial cells under physiological conditions is not primarily through Na+ channels. The data are consistent with an Na+/H+ exchanger acting in tandem with carbonic anhydrase through a pathway which could have a regulatory role on endothelial transport via its effect on Na+ re-entry. Residual trans-endothelial HCO3- transport, apparently unaffected by amiloride or acetazolamide inhibition, is calculated to be of sufficient magnitude to maintain corneal hydration. PMID- 8624355 TI - Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both D-glucose and peptide transport. AB - We have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of D-[14C]glucose and [14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [14C]gly-sar transport was first optimised by imposing a pH gradient (pHo = 5.7, pHi = 8.4) and an interior negative p.d. (using outwardly directed K+ gradient plus valinomycin). Under identical conditions (pHo = 5.7, pHi = 8.4), an acceleration of initial D [14C]glucose (at 100 microM) transport by 2.0 +/- 0.7-fold was observed compared to no proton gradient (pHo = 8.4, pHi = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pHo = pHi = 5.7) also resulted in acceleration of D-glucose influx (2-fold). The increase in D-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased D-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K+ gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation of D-[14C]glucose observed in H+ gradient conditions. H(+)-stimulated D-[14C]glucose transport showed a linear dependence on D-glucose concentration up to 20 mM D-glucose, unlike electrogenic Na(+)-dependent D-glucose transport, whose Km was 1.77 +/- 0.35 mM. In contrast, the initial rate of [14C]gly-sar (100 microM) transport by the renal H+/di-tripeptide transporter was accelerated 15.7 +/- 3.3-fold and stimulated a marked overshoot of 5.1 +/- 0.4-fold over equilibrium values. Conversely, the electrogenic, Na+/glucose transporter could be readily demonstrated, whilst [14C]gly-sar transport could not be energised by an inward Na+ gradient. The absence of electrogenic D-glucose transport in H+ gradient conditions is clear evidence against H+/glucose cotransport in Na(+)-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membrane D glucose uptake in Na(+)-rich media, it is unlikely that in vivo renal D-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient. PMID- 8624356 TI - Anomalous phase behavior of long chain saturated lecithin bilayers. AB - X-ray scattering has been performed on fully hydrated unoriented multilamellar vesicles of lecithins with even chain lengths n from 16 to 24 as a function of temperature in chain ordered phases. The longer chain lengths, n > or = 20, show anomalous behavior compared to the shorter chain lengths, n < 20. This report concentrates on n = 24. Although the history and time dependence shows that equilibrium was not always achieved, it appears that there is a second gel-like phase G2 below 40 degrees C. The G2 phase has a small tilt angle and opposite hexagonal symmetry breaking from the usual G1 gel phase. Also, as T is raised above 45 degrees C, the wide-angle data suggest the appearance of a phase with hexagonal chain packing and small chain tilt angle. PMID- 8624357 TI - Triggerable plasmalogen liposomes: improvement of system efficiency. AB - A photoactivated liposome release system that is generally applicable for triggered release of encapsulated hydrophilic materials is described. This approach to phototriggered release, derived from the known effects of plasmalogen photooxidation on membrane permeability in whole cells and model membrane systems, relies on producing a lamellar phase change or increase in permeability upon cleaving its constitutive lipids to single-chain surfactants using 630-820 nm light to sensitize the photooxidation of the plasmalogen vinyl ether linkage. Semi-synthetic plasmenylcholine liposomes containing encapsulated calcein and a membrane-bound sensitizer, such as zinc phthalocyanine, tin octabutoxyphthalocyanine, or bacteriochlorophyll a, were prepared by extrusion. Irradiation of air-saturated liposome solutions enhanced membrane permeability toward calcein and Mn2+, and promoted membrane fusion processes compared to non irradiated or anaerobic controls. Bacteriochlorophyll a sensitization produced the fastest observed photoinitiated release rate from these liposomes (100% calcein release in less than 20 min; 800 nm irradiation at 300 mW); the observed release rate was two orders of magnitude slower for egg lecithin liposomes prepared and irradiated under identical experimental conditions. Liposome aggregation, interlipidic particle formation, and membrane fusion between adjoining liposomes was observed by 31P-NMR, freeze-fracture/freeze-etch TEM, and cryo-TEM as a function of irradiation time. The use of near-infrared sensitizers and the capacity of photolyzed plasmenylcholine liposomes to undergo membrane fusion processes make photodynamic therapy with these liposome-borne sensitizers an attractive adjunct to biochemical targeting methods. PMID- 8624358 TI - The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione by rat liver plasma membrane vesicles. AB - The effect of thiol/disulfide exchange on ATP-dependent S-(2,4 dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent Km of 9.6 microM for GS-DNP and 124 microM for ATP. 5,5'-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half maximal inactivation occurred in 10 min at 40 microM for DTNB and 550 microM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the gamma-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo. PMID- 8624359 TI - Activation of Na+/K(+)-ATPase by fatty acids, acylglycerols, and related amphiphiles: structure-activity relationship. AB - A number of fatty acids and derivatives have been shown to activate Na+/K(+) ATPase when ATP is suboptimal. To explore the relation of the structures of these amphiphiles to enzyme activation, the effects of varying amphiphile concentrations on the activity of the highly purified kidney Na+/K(+)-ATPase at 50 microM ATP were determined. Among fatty acids, efficacy (maximal level of activation) and potency were found to be dependent, in different ways, on chain length and unsaturation. Compared to fatty acids, the corresponding alcohols had lower efficacies. Methyl esters of fatty acids inhibited, but CoA esters and monoacyl esters of glycerol activated the enzyme. Relation between chain length and potency among CoA esters and monoacylglycerols was the same as that observed with acids. Diacylglycerols did not activate, but they antagonized the effects of the activator amphiphiles. The substantial specificities of the amphiphile effects support the hypothesis that these ligands bind to a distinct amphipathic peptide segment of the intracellular central loop of the alpha-subunit to regulate ATP binding to the enzyme. The findings also suggest that direct effects of the changing intracellular levels of fatty acids and derivatives on Na+/K(+) ATPase should be considered as a possible mechanism for the regulation of its function in the intact cell. PMID- 8624360 TI - Fluctuations in IR spectral parameters detected in mixed acyl chain membranes of Acholeplasma laidlawii B. AB - Acholeplasma laidlawii B cells were grown at 37 degrees C on three binary C16:0 d(31)/C18:1 fatty acid mixtures at initial mol ratios of 3:2, 1:1, and 2:3. These mol ratios produced final C16:0-d(31)/C18:1 lipid acyl chain mol ratios of 1.66 +/- 0.23 (n=6), 1.3 +/- 0.20 (n=6) and 0.58 +/- 0.09 (n=10), respectively, in the membrane of the microorganism. Membrane conformational order for the deuterated and proteated acyl chains in intact cells was monitored by FT-IR spectroscopy through the thermotropic response of the acyl chain CD2 and CH2 stretching frequencies. Intact cells and isolated membranes revealed broad phase transitions centered well below the growth temperature. This result differs from previous studies (Moore, D.J. and Mendelsohn, R. (1994) Biochemistry 33, 4080-4085) of cells grown on a single saturated fatty acid source, where Tm was close to the growth temperature. Fluctuations in IR spectral parameters from the liquid crystalline phases were detected in ten separate samples of cells grown on a 2:3 mixture (final mol ratio 0.58:1) of C16:0-d(31)/C18:1, and in no other cell preparation. These were manifest by reduced precision in the measurement of CH2 and CD2 stretching frequencies and are attributed to fluctuations in the membrane conformational order. In addition to conformational order fluctuations in intact cells, similar behavior was noted for the simple binary phosphatidylcholine (PC) mixture, DOPC/1-C16:0-d(31),2-C18:1 PC (2:1 molar ratio). In this instance, the fluctuations were also detected through the temporal and thermotropic response of the relative intensity of the 1341 cm(-1) band assigned to end-gauche conformers about the penultimate C-C bond in the oleoyl chains. The relationship of these observations to the Raman spectroscopic detection of packing fluctuations in highly unsaturated PC's (Litman, B.J., Lewis, N., and Levin, I.W. (1991) Biochemistry 30, 313-319) is considered. PMID- 8624361 TI - The effect of high external pressure on DPPC-cholesterol multilamellar vesicles: a pressure-tuning Fourier transform infrared spectroscopy study. AB - We have investigated the effect of incorporation of cholesterol on the barotropic phase behavior of aqueous dispersions of 1,2-dipalmitoylphosphatidylcholine (DPPC) using Fourier-transform infrared spectroscopy (FTIR) in combination with the diamond anvil technique. Infrared spectral parameters, such as the frequencies, intensities, bandshapes and band splittings have been used to detect structural and dynamical changes upon incorporation of cholesterol into the DPPC bilayer. Analysis of these spectral parameters yields information on conformer population, reorientational fluctuations, interchain interaction, hydrogen bonding, interdigitation packing, and phase transformations of the DPPC/cholesterol mixtures. We present FTIR data of aqueous DPPC dispersions at 0, 10, 20, 30, 40, and 50 mol% cholesterol in the pressure range from 0.001 to 20 kbar at two temperatures, 25 degrees C and 55 degrees C. In addition, comprehensive temperature dependent measurements in the range from 20 degrees C to 80 degrees C were performed at ambient pressure. Analysis of the CH2 symmetric and antisymmetric stretching modes yields information of the effect of cholesterol concentration on the phase transition phenomena occurring in the lipid bilayer. Observation of the correlation field splittings of the CH2 bending and rocking modes monitors structural changes and dynamical properties of the lipid mixtures. Cholesterol induces more orientational disorder of the lipid molecules in terms of an increase of the reorientational fluctuations of the molecules and twisting/torsion motions of the acyl chains in the gel phase even at elevated pressures. It therefore appears that one important role of cholesterol is to make the membrane insensitive to changes in external environment, such as high hydrostatic pressure. Increase of pressure leads to a decrease in half width of the C = O band contour of pure DPPC and of DPPC/cholesterol mixtures, especially for cholesterol concentrations equal and higher than 30 mol%, which might be due to a marked increase in free carbonyl groups. At high pressure part of the bound water from the interfacial zone of the membrane is withdrawn. Increase of the cholesterol concentration and increase in pressure have opposite effects on the population of free and hydrated carbonyl ester groups of DPPC in the gel phases. PMID- 8624362 TI - Interactions of annexin V with phospholipid monolayers. AB - To understand the mechanism of annexin V-membrane interactions, we measured the interaction of human recombinant annexin V with phospholipid monolayers with differing head group and acyl group structures. Annexin V interacted with anionic phospholipid monolayers via non-specific electrostatic interactions, which was highly dependent on the surface pressure of monolayer with a sharp maximum. The unique surface pressure dependence of the annexin V-monolayer binding is strikingly similar to that observed for the binding of Ca2+ to anionic phospholipid monolayers, which indicates that the annexin V-bound Ca2+ binds two phospholipids at the membrane surface and that factors governing the Ca(2+) phospholipid complex formation regulate the overall annexin V-Ca(2+)-membrane interactions. PMID- 8624363 TI - The water adsorption characteristics of charged phospholipids. AB - The hydration isotherms of the negatively charged phospholipids, egg phosphatidic acid, bovine heart cardiolipin and two phosphatidylserines as well as one positively charged phospholipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, have been obtained gravimetrically. The presence of an electrical charge on these phospholipids does not, in itself, determine whether the water binding to the phospholipids is 'strong' or 'weak'. Interestingly, hysteresis effects were present for certain charged phospholipids suggesting some rearrangement of the lipid molecular organization upon hydration and dehydration, perhaps due to the presence of the ionizable moiety. The hydration isotherms of the charged phospholipids have been analyzed by BET theory, although, of the charged lipids studied, all may not be amenable to the application of BET theory. The hydration isotherms and the resulting parameters obtained from the BET analysis are compared to those found previously for zwitterionic phospholipids, especially egg phosphatidylcholine. The water adsorption characteristics of phospholipids are found to depend mainly on the total head-group structure including the presence of hydrophobic groups as well as electrical charge on the head group. PMID- 8624364 TI - Antitumor activity of vincristine encapsulated in glucuronide-modified long circulating liposomes in mice bearing Meth A sarcoma. AB - Liposomes modified with the uronic acid derivative palmityl-D-glucuronide (PGlcUA) have a long circulation time and tend to accumulate in the tumors of tumor-bearing mice. Taking advantage of this character, we investigated the therapeutic effect of vincristine (VCR) encapsulated in liposomes containing PGlcUA (dipalmitoylphosphatidylcholine/cholesterol/PGlcUA = 4:4:1 as a molar ratio) on tumor-bearing mice. VCR was loaded into liposomes by a remote loading method, and then free or liposomal VCR was injected intravenously into BALB/c mice bearing Meth A sarcoma implanted subcutaneously 5 days before hand. Single dose administration of VCR (3.0 mg/kg) in PGlcUA-liposomes significantly suppressed tumor growth, and prolonged the survival time (T/C = 1.37). Furthermore, two-dose administration of the liposomes cured one third of the animals. The therapeutic effect of PGlcUA-liposomes was greater than that of control liposomes containing dipalmitoylphosphatidylglycerol instead of PGlcUA. PGlcUA-liposomes might thus be a useful tool for delivering antitumor agents to tumor tissues. PMID- 8624365 TI - Poly(ethylene glycol)-coated anti-cardiac myosin immunoliposomes: factors influencing targeted accumulation in the infarcted myocardium. AB - Biodistribution and infarct accumulation of different liposome preparations in rabbits with experimental myocardial infarction have been investigated. The influence of such parameters as liposome size, and presence or absence of poly(ethylene glycol) (PEG) and infarct-specific antimyosin antibody (AM) on liposome behavior in vivo was studied. All three variables were shown to affect liposome biodistribution, liposome size being the least significant variable. Statistical analysis of the data obtained demonstrated that of all variables, PEG coating expresses the strongest influence on the liposome blood clearance, significantly (P=0.0001) increasing the mean level of blood radioactivity under all circumstances. Infarct accumulation depended upon the presence of both PEG (P=0.0013) and AM (P=0.005). The infarct-to-normal ratio was affected by the presence of AM (P=0.0002), but the extent of the effect depended also on the presence of PEG (P=0.01). Two differing mechanisms can be seen in infarct accumulation of PEG-liposomes (slow accumulation via the impaired filtration) and AM-liposomes (specific binding of immunoliposomes with the exposed antigen). Both mechanisms are supplementary in case of liposomes carrying PEG and AM at the same time. An optimization strategy is suggested for using liposomes as carriers for diagnostic (a high target-to-nontarget ratio is required) and therapeutic (a high absolute accumulation in the target is required) agents. PMID- 8624366 TI - Expression of insulin-like growth factor (IGF)-I receptors, IGF-II/cation independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells. AB - We have analyzed the surface distribution and functional expression of the insulin-like growth factor I (IGF-I) receptor and the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CI-MPR) in the polarized human colon adenocarcinoma cell line, Caco 2. Domain-selective biotinylation of the apical and basolateral surfaces of Caco-2 cells grown on filter supports revealed a 3-4-fold enrichment of these receptors on basolateral membranes. In addition, the biotinylation studies revealed the presence of the cation-dependent MPR on both membrane surfaces, with a 3.4-fold enrichment on basolateral membranes. Binding of 125I IGF-I at 4 degrees C confirmed similar higher levels of expression of the IGF-I receptor at the basolateral surface than at the apical surface. Cell surface specific binding of the iodinated lysosomal enzyme beta-glucuronidase was detected at 4 degrees C on both plasma membrane domains. However, significant uptake of beta-glucuronidase at 37 degrees C was observed only from the basolateral surface. These results indicate that the MPRs and the IGF-I receptor are expressed in a polarized fashion in Caco-2 cells and that the IGF-II/CI-MPR present on apical membranes, unlike the IGF-II/CI-MPR expressed on the basolateral surface, is not functional in endocytosing lysosomal enzymes. PMID- 8624367 TI - Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform. AB - Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes. PMID- 8624368 TI - Pathophysiology and treatment of severe chronic neutropenia. AB - Severe chronic neutropenia (SCN) include a heterogeneous group of diseases characterized by blood neutrophil counts chronically less than 0.5 x 10(9)/ L. In phase I-III studies in SCN patients, treatment with recombinant human granulocyte colony stimulating factor (r-metHuG-CSF; Filgrastim) resulted in a rise in the absolute neutrophil counts (ANC) to above 1.0 x 10(9)/L associated with a reduction in bacterial infections. Long-term treatment with filgrastim up to 8 years demonstrate a sustained ANC response, a significant reduction of the need for intravenous antibiotics and a dramatic improvement in the quality of life. In 1994 an international registry for severe chronic neutropenia (SCNIR) was established to improve care for chronic neutropenia and for further understanding the pathophysiology of this rare disease. Three-hundred and ten patients have been enrolled to this registry so far. Worldwide phase I-III studies with filgrastim and SCNIR provide information on 424 patients with severe chronic neutropenia. Adverse events include the development of acute myeloid leukemia in approximately 7% of the patients within the cohort of patients with congenital neutropenia (Kostmann's syndrome) suggesting that congenital neutropenia is a preleukemic syndrome. None of the patients with cyclic of idiopathic neutropenia developed leukemia suggesting that filgrastim is not involved in the development of leukemia. PMID- 8624369 TI - Resistance to activated protein C, the FV:Q506 allele, and venous thrombosis. AB - Vitamin K-dependent protein C is an important regulator of blood coagulation. After its activation on the endothelial cell surface by thrombin bound to thrombomodulin, it cleaves and inactivates procoagulant cofactors Va and VIIIa, protein S and intact factor V working as cofactors. Until recently, genetic defects of protein C or protein S were, together with antithrombin III deficiency, the established major causes of familial venous thromboembolism, but they were found in fewer than 5-10% of patients with thrombosis. In 1993, inherited resistance to activated protein C (APC) was described as a major risk factor for venous thrombosis. It is found in up to 60% of patients with venous thrombosis. In more than 90% of cases, the molecular background for the APC resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine (R) at position 506 by a glutamine (Q). Mutated factor V (FV:Q506) is activated by thrombin or factor Xa in normal way, but impaired inactivation of mutated factor Va by APC results in life-long hypercoagulability. The prevalence of the FV:Q506 allele in the general population of Western countries varies between 2 and 15%, whereas it is not found in several other populations with different ethnic backgrounds. Owing to the high prevalence of FV:Q506 in Western populations, it occasionally occurs in patients with deficiency of protein S, protein C, or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting severe thrombophilia to be a multigenetic disease. PMID- 8624370 TI - Clinical applications of new antithrombotic agents. AB - The clinical targets for which new generations of antithrombotics have been or are currently under clinical development are those associated with a high risk for thromboembolism, (a) patients undergoing general, orthopedic, major abdominal, and cancer surgery, to prevent venous thromboembolism; (b) patients with deep-vein thrombosis and/or pulmonary embolism who are at high risk for extension or recurrence of thromboembolism; (c) patients with unstable angina or myocardial infarction treated with percutaneous transfemoral coronary angioplasty, in whom antithrombotic treatment aims to prevent subsequent arterial thrombus formation and reocclusion. In most of these conditions standard heparin at prophylactic or therapeutic doses has proven to be only moderately effective. Furthermore, standard heparin has a rather poor bioavailability, a large and unpredictable variability in anticoagulant response, and a non-specific binding to many plasma proteins. Moreover, heparin treatment is associated with side effects such as bleeding, heparin-induced thrombocytopenia, and osteopenia. New antithrombotics have been designed which more specifically inhibit pivotal activated coagulation factors, i.e., Xa and IIa, and have potentially fewer undesired interactions and side effects. In this review we first provide an overview of recent insights on the mechanism of blood coagulation and the levels at which antithrombotics interfere with this system. Subsequently, we discuss specific new antithrombotic agents, and in particular the results of recent clinical investigations. PMID- 8624371 TI - The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders. AB - Chemoresistance genes have been identified as an impediment to anticancer drug treatment. In particular, P-glycoprotein, the product of the multidrug-resistance (MDR1) gene, plays a major role in clinical treatment failure. Conversely, expression of an MDR1 cDNA in bone marrow of transgenic animals renders hematopoietic cells chemoresistant. Efficient transfer of drug-resistance genes to normal hematopoietic progenitor cells has been achieved with the use of retroviral vectors. In this article we review approaches which use the multidrug resistance gene to protect bone marrow from myelosuppression following chemotherapy and as a selectable markerin vivo to increase the expression of nonselectable genes which correct hereditary diseases of the hematopoietic system. PMID- 8624372 TI - High-dose methotrexate in the treatment of adult acute lymphoblastic leukemia. AB - The application of high-dose treatment elements has an increasing importance in the therapy of acute lymphoblastic leukemia (ALL). High-dose methotrexate (HDMTX) has been introduced in clinical trials more than 20 years ago, since it has several theoretical advantages compared to conventional dose methotrexate. These trials revealed that the efficacy and toxicity of HDMTX depends on features such as dose level, infusion time, combination regimen and schedule of leucovorin rescue. Particularly the application of controlled leucovorin rescue and improved supportive care enabled the application of increasing doses of HDMTX in the treatment of childhood and adult ALL within a variety of schedules and at dose levels mostly ranging between 0.5 g/m2 and 5.0 g/m2. In childhood ALL, first devised to replace CNS irradiation in the prophylaxis of CNS relapse HDMTX has contributed to the reduction of bone marrow relapses particularly in low risk B lineage ALL. In addition it proved to be an effective therapy element for prophylaxis and treatment of CNS disease. At least in low risk ALL CNS irradiation could be safely replaced by repeated cycles of HDMTX with additional intrathecal therapy. In adult ALL only few of the successful treatment approaches with HDMTX have been investigated up to now. The results indicate that HDMTX has a beneficial effect with regard to overall outcome in adult B-lineage ALL. It provides effective CNS prophylaxis in combination with intrathecal therapy. In mature B-ALL HDMTX proved to be one of the most effective treatment elements and contributed to an impressive improvement of outcome in this subgroup. For T-ALL however sufficient data do not exist either in childhood or in adult ALL. Since HDMTX is an effective treatment element with manageable acute and long-term toxicity, its role in the management of adult ALL should be explored more intensively. PMID- 8624373 TI - Role of 67Ga scintigraphy in localization of lymphoma. AB - This review deals with the applications of 67Gallium (Ga) scintigraphy for the initial staging and follow-up during and after treatment of patients with Hodgkin's or non-Hodgkin's lymphoma (NHL). During the last decade, the technique of visualization has been largely improved by using higher doses and additional tomography. Here, the indications for 67Ga scintigraphy in comparison with CT and MRI are discussed. The conditions resulting in false positive and false negative results have been outlined. 67Ga may detect unusual involved sites during staging procedures. However, the major contribution to the management of Hodgkin's disease and NHL is the evaluation of residual masses, because 67Ga uptake reflects the metabolic activity of the tumor. The review ends with an additional short overview of other radionuclide imaging methods useful for malignant lymphoma. PMID- 8624374 TI - Minimally differentiated acute myeloid leukemia (AML-M0): a distinct clinico biologic entity with poor prognosis. AB - FAB proposals for the diagnosis of AML-M0 represent the formal recognition of a distinct entity which has been described over the past few years by several authors and called minimally differentiated acute myeloid leukemia. By definition, AML-M0 includes acute leukemias which do not fit morphological and cytochemical criteria for the diagnosis of AML, and for which myeloid lineage assignment can be made by immunological assay showing positivity for MPO, CD13, and CD33 and negativity for lymphoid markers. Involvement of an early myeloid progenitor in the leukemic process is a possible theory hypothesized to explain the existence of such a form. Validity of this assumption has been based on the observation that AML-M0 frequently bears "stem cell" markers such as CD34, HLA DR, Tdt, CD7, and promiscuous IgH/TCR gene rearrangements, which are thought to occur in uncommitted cells. Finally, AML-M0 very frequently carries cytogenetic abnormalities common to MDS or secondary AML, such as -5/5q- or -7/7q- deletions and or complex karyotype. In our experience, AML-M0 is also very often associated with the MDR phenotype, which in turn has been found strictly linked to "stem cell" features, especially in MDS. These biological aspects, altogether, translate into a very unfavorable prognosis, confirming even from a clinical point of view that AML-M0 is a distinct entity. In conclusion, "stem cell" markers, MDR phenotype, complex chromosome lesions, frequent occurrence in elderly patients, and intrinsic chemoresistance characterize AML-M0 and indicate the need for tailored treatments, possibly involving the use of MDR modulators and/or differentiating agents. PMID- 8624375 TI - Duration of second complete remission in patients with acute myeloid leukemia treated with chemotherapy: a retrospective single-center study. AB - A total of 168 patients with de novo AML were retreated with chemotherapy at relapse following first CR; 66 patients (39%) achieved a second complete remission (CR). The probability of achieving a second CR was highly dependent on the duration of the first remission. Patients who received no or conventional postremission chemotherapy after second CR had a median remission duration of 7.5 months, and the probability of remaining in remission at 3 years was 24%. Patients with a first CR of more than 12 months had a median second remission duration of 18 months. The probability of a second CCR was 35% at 3 years and 24% at 5 years, whereas none of the patients with a first CR of less than 12 months was in remission at 3 years. Only a poor correlation (p = 0.31) was found when the durations of the first and second CR were compared in patients with a second relapse. Patients with long-lasting remissions and long-term survivors after second CR are characterized by a first CR duration of > 12 months and favorable or normal cytogenetics. The type of salvage treatment seems to be less important for achievement of long-term remission, but it is probably important to administer consolidation chemotherapy after second CR. Other so-far ill-defined factors may be responsible for the suppression of the leukemic clone in patients with long-lasting remissions following chemotherapy for relapse after second CR. PMID- 8624376 TI - Allogenic bone marrow transplantation or chemotherapy for patients with acute myeloid leukemia in first complete remission: a decision analysis approach. AB - The methodology of decision analysis was originally developed to improve clinical decisions of physicians for individual patients. However, it is also well suited to support consensus procedures. We have used this methodology to analyse the question whether allogeneic bone marrow transplantation (BMT) or consolidation chemotherapy (CCT) should be used as first line postremission treatment in patients with acute myeloid leukemia. Main risk factors relevant for the outcome after BMT and CCT are therapy-related mortality and leukemic relapse, respectively. If the possibility of salvage BMT for patients relapsing after CCT is included, the outcomes of the two strategies come rather close. However, they are clearly different in subtypes of leukemia with high or low risk of relapse, and in patients at high risk for BMT-related mortality. Sensitivity analysis considering the variation of more than one risk factor provides valuable information for decision making for both individual patients and particular subgroups of patients with acute myeloid leukemia. PMID- 8624377 TI - Detection of partial cDNA sequences differentially expressed in patients with myelodysplasia. AB - The most common chromosomal aberrations in myelodysplastic syndromes (MDS) are complete or partial loss of chromosomes 5 and 7, and trisomy 8. To identify genes important in the pathogenesis of this disease that could be associated with these gross chromosomal defects, we have employed the differential display PCR (DDPCR) procedure developed by Liang and Pardee. This method allows simultaneous comparison of several cDNA sources for the presence of differentially expressed genes. Polymorphonuclear cells (PMNs) from two MDS patients, containing a 5q deletion or a trisomy 8, and three healthy controls were used. Initial screening resulted in the identification of five and three partial cDNA sequences, respectively that were either differentially expressed in both patient samples or in individual patients, as compared with the controls. The authenticity of aberrant expression was verified by reanalyzing the same primer combinations on newly prepared cDNA. Differential expression of the three remaining fragments was subsequently checked on a larger panel of MDS patients, using amplicon-specific primer sets. These were obtained by cloning and sequencing of the fragments. For one partial cDNA (DC3), the original expression pattern, i.e., decreased expression in individual MDS patients, was confirmed. These results demonstrate the utility of the DDPCR procedure to isolate differentially expressed sequences in primary patient samples where the availability of cells is a limiting factor. PMID- 8624378 TI - Improved multilineage response of hematopoiesis in patients with myelodysplastic syndromes to a combination therapy with all-trans-retinoic acid, granulocyte colony-stimulating factor, erythropoietin and alpha-tocopherol. AB - Differentiation induction therapy is being tested in myelodysplastic syndromes to ameliorate maturation defects and to restore normal hematopoietic function. To this end, 17 patients (eight with refractory anemia, two with refractory anemia and ring sideroblasts, and seven with refractory anemia and excess of blast cells) were treated with a combination of all-trans-retinoic acid (ATRA), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and alpha tocopherol for durations of 8-16 weeks. Absolute neutrophil counts increased in all patients; platelet counts increased in five patients with discontinuation of transfusion needs in two of four transfusion-dependent patients. Stimulation of erythropoiesis was seen in eight patients with an increase in hemoglobin concentration in three, a discontinuation of transfusion requirements in another three, and a significant increase in reticulocyte counts as the only parameter in two patients. Clinically important multilineage responses with increases of hemoglobin levels or discontinuation of transfusion needs were thus seen in six patients (35.3%) with three patients having a trilineage response. Serum erythropoietin concentrations did not differ significantly between responders and nonresponders, but the erythroid response was accompanied by a rise in the serum transferrin receptor levels. In the bone marrow, the myeloid-to-erythroid ratio and the maturation index of myeloid cells increased during therapy, while the percentage of blast cells did not change. Cytogenetic analysis demonstrated the persistence of the abnormal clones. Prior to therapy, nonresponders had a significantly higher serum TNF level than responders. Serum concentrations of TNF alpha and soluble TNF-alpha receptor significantly increased during therapy, but mainly in the patients without an erythroid and platelet response. Soluble IL-2 receptor and soluble ICAM-1 concentrations both increased. This pilot study demonstrates that treatment with ATRA/G-CSF/EPO/tocopherol is well tolerated, leading to normalization of neutrophil counts in most, and to improvement of platelets and red blood cells in a significant subgroup of patients. PMID- 8624379 TI - Interleukin-10, interleukin-12, and tumor necrosis factor-alpha differentially influence the proliferation of human CD8+ and CD4+ T-cell clones. AB - Activation and proliferation of human T lymphocytes in vitro can be obtained by various stimuli including specific antigens, mitogens, and cytokines. Here we describe the effect of interleukin-10, interleukin-12 and tumor necrosis factor alpha on the interleukin-2 dependent proliferation and function of established human CD4+ and CD8+ alloreactive T-cell clones in the absence of antigen presenting cells. IL-12 and TNF-alpha both demonstrated an inhibitory effect on the proliferation of CD8+ cytotoxic T lymphocyte clones, whereas IL-10 enhanced the proliferation. IL-12-induced inhibition of CD8+ CTL clones was not mediated by the endogenous production of TNF-alpha by these clones. The strong inhibitory effect of IL-12 and TNF-alpha did not result in apoptosis. These cytokines did not alter the cytotoxicity of CD8+ CTL clones. When CD4+ T-cell clones were tested in the absence of APC, no significant change in IL-2-dependent proliferation due to IL-10, IL-12, and TNF-alpha could be measured. Since these effects on established CTL clones are in contrast to the effects of IL-10, IL-12, and TNF-alpha during the induction phase of immune responses, a dichotomy of immunomodulatory cytokines such as IL-10, IL-12, and TNF-alpha early and late in the immune response is suggested. PMID- 8624380 TI - Neutralization of stem cell factor in vitro and in vivo: dose-dependent inhibition of stromal cells and induction of granulocyte/monocyte differentiation. AB - Stem cell factor (SCF), also termed mast cell growth factor or c-kit ligand, plays a central role in the regulation of hematopoiesis and maintenance of viability of hematopoietic cells. We used a new murine monoclonal antibody (MAb) specific for canine SCF to further dissect the role of SCF in vitro and in vivo. This neutralizing MAb, RG7.6 (IgG1), recognizes the soluble form as well as the membrane-bound form of SCF on marrow-derived stromal cells. Treatment of long term bone marrow cultures (LTMC) with RG7.6 suppressed or stimulated the production of CFU-GM, depending on the MAb concentration and the time of addition to cultures. At concentrations of 0.1-10 micrograms/ml given on the day of recharge of the LTMC, RG7.6 resulted in sustained suppression of CFU-GM grown from nonadherent cells. In contrast, higher doses of RG7.6 (20-100 micrograms/ml) led to a two- to threefold increase in CFU-GM formation from nonadherent cells after 3 days of RG7.6 exposure; after longer RG7.6 exposure there was a rapid decline in the number of CFU-GM. The early increase of CFU-GM was even more distinct when RG7.6 addition to LTMCs was delayed until 1 day before cells were plated for the CFU-GM assay. The early increase of CFU-GMs in the presence of high-dose RG7.6 was mimicked by the addition of granulocyte colony-stimulating factor (G-CSF) to cultures containing suboptimal concentrations of RG7.6, suggesting the possibility that the "positive" response to high-dose RG7.6 was due to an overriding effect of other growth factors, e.g., G-CSF. In stromal cells expressing the membrane-bound form of SCF, the presence of MAb RG7.6, even at low concentrations, interfered with thymidine uptake and proliferation. RG7.6 was also tested in vivo. RG7.6 was given intravenously immediately (days 0-4) after total body irradiation and autologous bone marrow transplantation, and granulocyte counts were followed. The post-irradiation nadir of peripheral blood granulocytes was indistinguishable from controls at low doses of RG7.6 but became more shallow as higher doses of RG7.6 were infused, again suggesting a positive effect on granulocyte differentiation. Thus, the SCF-specific MAb appears to interfere with both stromal and hematopoietic cell function. While only inhibition was observed at lower concentrations, a transient increase in granulocyte production was seen at higher MAb concentrations. PMID- 8624381 TI - Umbilical cord blood contains normal frequencies of cytotoxic T-lymphocyte precursors (ctlp) and helper T-lymphocyte precursors against noninherited maternal antigens and noninherited paternal antigens. AB - Umbilical cord blood (UCB) has been successfully used as an alternative source of hematopoietic stem cells for allogeneic transplantation. A relatively low incidence and severity of graft-versus-host disease (GVHD) following UCB transplants has been reported, and it has been suggested that this may be caused by a low frequency of alloreactive lymphocytes in UCB. Low frequencies of alloreactive T lymphocytes in UCB may allow transplantation across major MHC barriers with an acceptable risk of GVHD. We investigated cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies in UCB. Normal frequencies of CTLp and HTLp were measured against 1-2 HLA class I and 0-1 HLA-DR mismatched stimulator cells. Since it has been postulated that due to maternal fetal transfusion during pregnancy, fetal blood lymphocytes may become tolerant for noninherited maternal antigens (NIMA), allowing transplantation over certain HLA barriers, reactivity of 24 umbilical cord blood samples was analyzed against both parents. The median frequencies of CTLp against NIMA with 1 class-I mismatch was 79 per 10(6) nucleated cells (range 16-428) and with 2 class I mismatches 121 (range 33-748). CTLp frequencies against noninherited paternal antigens (NIPA) were not statistically different from those against NIMA, with a median of 115 (range 8-336) and 176 (range 50-725) for 1 or 2 HLA class I mismatches, respectively. HTLp frequencies in UCB against parents with 0 or 1 HLA DR antigen mismatches were similar with respect to NIMA (median 74, range 43-233 and 88, range 16-777, respectively) and NIPA (median 125, range 18-174, and 110, range 28-350, respectively). In four cases, UCB from two HLA-identical siblings was tested against both parents. A correlation between the frequencies of CTLp and HTLp from HLA-identical individuals was found, illustrating that these frequencies are genetically determined. These results illustrate that UCB contains normal frequencies of CTLp and HTLp against MHC alloantigens. PMID- 8624382 TI - Study of hematopoietic chimerism following allogeneic peripheral blood stem cell transplantation using PCR amplification of short tandem repeats. AB - Allogeneic peripheral blood progenitor cell transplantation (PBPCT) is increasingly being used to treat hematologic malignancies. However, the capacity of PBPC to maintain long-term hematopoiesis remains controversial. To add further information to this issue we studied the chimeric status in 12 patients receiving G-CSF-mobilized PBPC from HLA-identical sibling donors. All patients were conditioned with cyclophosphamide and total body irradiation. In six cases the apheresis product was partially T-cell depleted by counterflow centrifugation (n = 2) or the immunoadsorption biotin-avidin method (n = 4). The follow-up was longer than 6 months in five patients, with a maximum of 420 days. Molecular analysis of the engraftment was done using PCR amplification of short tandem repeats. Apparent complete donor chimerism was detected in all patients between 28 and 420 days after engraftment. This study indicates that full short-term engraftment is achieved in patients receiving allogeneic G-CSF-mobilized PBPC from healthy donors and suggests that this might also be true for long-term engraftment. PMID- 8624383 TI - Effects of G-CSF administration and peripheral blood progenitor cell collection in 20 healthy donors. AB - The effects of both daily G-CSF administration and subsequent peripheral blood progenitor cell collection (PBPCC) by apheresis on 20 healthy adult donors were studied. All received daily G-CSF (filgrastim) 10 micrograms/kg for 5-7 days by subcutaneous injection. G-CSF administration was well tolerated, except for moderate bone pain and headache. Peak values of CD34+ cells were observed on days 5 (n = 12) or 6 (n = 8). In all donors a significant increase in CD3+, CD4+, CD8+, CD19+, and NK cells was observed on day 5 in relation to the baseline values. CD4/CD8 lymphocyte ratio was unmodified by G-CSF. None of the donors required a central venous line for PBPCC. Immediately after PBPCC, a platelet count below 100 x 10(9)/1 was observed in nine of 18 cases, although in all donors platelet counts were over 100 x 10(9)/1 7 days later. A lymphocytopenia on day 7 following PBPCC was observed, although there was a tendency to achieve baseline values 30-90 days after the procedure. Mean numbers ( +/- SD) of collected cells x 10(6)/kg after a median of two (1-4) apheresis sessions and a median of 20 1 (10-40) processed were: CD34+ 5.5 ( +/- 2.3), CD3+ 326 ( +/- 105), CD4+ 207 ( +/- 64), CD8+ 164 ( +/- 60), CD19+ 88 ( +/- 32), and NK cells 32 ( +/- 14). We conclude that G-CSF administration to healthy donors is a well-tolerated procedure which is associated with (a) obtaining a high number of hematopoietic progenitor cells, and (b) a significant increase in T, B, and NK cells in donors' blood. In addition, PBPCC by apheresis results in a moderate, rapidly reversible, and clinically irrelevant thrombocytopenia and a moderate lymphocytopenia, which tends to resolve within 3 months following the procedure. PMID- 8624384 TI - Antibiotic strategy after the empiric phase in patients treated for a hematological malignancy. AB - Empiric broad-spectrum antibiotic therapy has become a generally accepted strategy in the treatment of febrile neutropenic patients. Particularly in patients with prolonged neutropenia, subsequent adaptation of such a regimen will be the rule rather than exception. Since there are no uniformly accepted guidelines for the modification of antibiotic therapy during the post-empiric phase, we assessed the impact of a set of rules that evolved during the first randomized trials. Evaluation of the clinician's compliance with these rules in 1951 febrile neutropenic episodes was the subject of the present analysis. Treatment was modified in 761 (39%) cases, and these changes were made according to the rules in 76%. For 75% of the alterations in treatment during the evening and night shifts, no reasonable explanation was established, while 93% of the modifications during the normal working hours were made for objective reasons. The empiric regimen was more frequently changed in patients with a clinical focus of infection at the onset of fever than in patients who showed fever as the only symptom of a possible infection. The perceived need for modification amounted to 69% in pulmonary infections, to 51% in skin and soft-tissue infections, to 44% in patients with abdominal complaints, and to 37% in upper respiratory tract infections. Glycopeptides constituted 22% of modifications, particularly in patients with a central venous catheter, and systemically active antifungals were administered in 16% of cases. Especially inexperienced clinicians tend to adjust antibiotic therapy, in spite of the fact that persistence of fever alone seldom reflects inadequate treatment when the clinical condition of the patient is stable or improving. On the other hand, the development of subsequent infectious events emphasizes that a genuine need for modification does frequently exist. PMID- 8624385 TI - Platelet autoantibodies (IgG, IgM, IgA) against glycoproteins IIb/IIIa and Ib/IX in patients with thrombocytopenia. AB - Autoimmune thrombocytopenic purpura (AITP) is most frequently induced by platelet specific autoantibodies against epitopes on platelet GP Ib/IX or GP IIb/IIIa. These antibodies are reliably detected on the patients' autologous platelets. So far, studies on the characterization of platelet autoantibodies have been restricted to IgG antibodies. We used the monoclonal antibody immobilization of platelet antigens assay (MAIPA) in a modified version to detect GP-specific IgG, IgM, and IgA antibodies. Platelets of 46.2% of patients carried elevated amounts of IgG antibodies. IgM and IgA antibodies were observed less frequently and showed only weak OD signals in the MAIPA assay. Circulating IgG antibodies in serum were found in 11.5% of patients. Circulating IgM autoantibodies were observed in 8.9% and IgA antibodies in no patient with AITP. Results of direct MAIPA assay were compared with the reactivity of eluates in the platelet adhesion immunofluorescence assay and were found to be highly concordant. Patients with AITP in remission carried high percentages of anti-GP IIb/IIIa. Findings made in this study suggest that autoantibodies of the IgM and IgA classes play only a minor role in the pathogenesis of AITP. PMID- 8624386 TI - Fever and progressive pancytopenia in a 20-year-old woman with Crohn's disease. PMID- 8624387 TI - Mechanisms of polymer degradation and erosion. AB - The most important features of the degradation and erosion of degradable polymers in vitro are discussed. Parameters of chemical degradation, which is the scission of the polymer backbone, are described such as the type of polymer bond, pH and copolymer composition. Examples are given how these parameters can be used to control degradation rates. Degradation leads finally to polymer erosion, the loss of material from the polymer bulk. The resulting changes in morphology, pH, oligomer and monomer properties as well as crystallinity are illustrated with selected examples. Finally, a brief survey on approaches to polymer degradation and erosion is given. PMID- 8624388 TI - Stabilized polyglycolic acid fibre-based tubes for tissue engineering. AB - Polyglycolic acid (PGA) fibre meshes are attractive candidates to transplant cells, but they are incapable of resisting significant compressional forces. To stabilize PGA meshes, atomized solutions of poly(L-lactic acid) (PLLA) and a 50/50 copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) dissolved in chloroform were sprayed over meshes formed into hollow tubes. The PLLA and PLGA coated the PGA fibres and physically bonded adjacent fibres. The pattern and extent of bonding was controlled by the concentration of polymer in the atomized solution and the total mass of polymer sprayed on the device. The compression resistance of devices increased with the extent of bonding, and PLLA bonded tubes resisted larger compressive forces than PLGA bonded tubes. Tubes bonded with PLLA degraded more slowly than devices bonded with PLGA. Implantation of PLLA bonded tubes into rats revealed that the devices maintained their structure during fibrovascular tissue ingrowth, resulting in the formation of a tubular structure with a central lumen. The potential of these devices to engineer specific tissues was exhibited by the finding that smooth muscle cells and endothelial cells seeded onto devices in vitro formed a tubular tissue with appropriate cell distribution. PMID- 8624389 TI - Computer-aided design of bioerodible devices with optimal release characteristics: a cellular automata approach. AB - The development of a computational tool to design bioerodible devices with optimal release characteristics is presented. This computational tool uses cellular automata and parallel iterations to model and simulate the release of bioactive agents (drugs) from bioerodible matrices. The simulations can accurately model surface erosion processes in multicomponent systems of arbitrary geometry and with different dissolution rates for each component. Simulation results are analysed to show how the overall release rates are affected by the intrinsic dissolution rates, drug loading, porosity and the dispersion of the drug in the bioerodible matrix. A strongly non-linear dependence of release rates on drug loading and the intrinsic dissolution rates of the solid components is obtained and the effects of phase dispersion on the variability of release rates are elucidated. Finally, guidelines are presented for screening of alternatives to minimize the development effort and experimentation required for designing devices with desired release characteristics. PMID- 8624390 TI - Role of material surfaces in regulating bone and cartilage cell response. AB - Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Studies comparing chondrocyte response to TiO2 of differing crystallinities show that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. Optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biologicals is another. PMID- 8624391 TI - Healing of chondral and osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage. AB - In this study a canine model was developed to investigate the nature of early healing responses to both chondral and osteochondral defects and to evaluate the tissue regenerative capacity of cultured autologous chondrocytes in chondral defects. The healing response to surgically created chondral defects was minor, with little cellular infiltration. In contrast, osteochondral defects exhibited a rapid cellular response, resulting ultimately in the formation of fibrous tissue. The lack of significant cellular activity in chondral defects suggests that an evaluation of the capacity of cultured autologous chondrocytes to regenerate articular cartilage is best studied in chondral defects using the canine model. When dedifferentiated cultured articular chondrocytes were implanted into chondral defects, islands of type II collagen staining were demonstrated in the regenerative tissue within 6 weeks. The relatively early expression of cartilage specific markers by the implanted chondrocytes, coupled with the inability of untreated chondral defects to repair or regenerate, demonstrates the utility of the canine model in evaluating novel materials for cartilage repair and regeneration. PMID- 8624392 TI - Effect of basic fibroblast growth factor on cartilage regeneration in chondrocyte seeded collagen sponge scaffold. AB - A chondrocyte-collagen composite was prepared in an attempt to regenerate cartilage by its subcutaneous implantation in nude mouse. When the composite was impregnated with basic fibroblast growth factor (bFGF) prior to implantation, regeneration of the cartilage tissue was remarkably accelerated. Histological staining of the implanted composites with Safranin O-fast green revealed that the cells incorporated in the composites exhibited their phenotype and formed a new matured cartilage. A thin layer of fibrous capsule was observed surrounding the implanted composite and the inflammatory response of the host to the implant was mild. Specific proteoglycans were accumulated in the composite even 1 week after implantation. At 2 weeks after implantation, the chondrocytes regenerated the cartilage tissue, although still immature, but at 4 weeks almost all of the chondrocytes transferred to the mature stage. Conversely, such mature cartilage tissue was not noticed up to 4 weeks after implantation if the collagen scaffold was not impregnated with bFGF. Moreover, the mature area was limited to only a small fraction of the implanted composite, unless bFGF was incorporated in it. PMID- 8624393 TI - Use of porous polyurethanes for meniscal reconstruction and meniscal prostheses. AB - In the past, porous materials made of an aromatic polyurethane (PU) were successfully used to meniscal reconstruction in dogs. Since aromatic PUs yield very toxic fragments upon degradation, a linear PU was synthesized by curing a poly(epsilon-caprolactone) and 1,4-trans-cyclohexane diisocyanate based prepolymer with cyclohexanedimethanol. Porous materials of this polymer were also implanted for meniscal reconstruction. The results were comparable with the most successful implant series so far. Additionally, a porous meniscal prosthesis was developed to replace a total meniscus. Due to the very high shear stresses to which the prosthesis would be exposed, the stress hysteresis phenomenon linear PUs are known to exhibit could be of great consequence. Therefore an aliphatic PU network, synthesized by cross-linking poly(epsilon-caprolactone) and 1,4-trans cyclohexane diisocyanate with glycerol, was used. Dislocation caused by tearing out of the sutures was found to be a problem because the tear resistance of the material was relatively low. In this study the tearing problem has been partly circumvented by using a complex suturing technique. Meniscal prostheses turned out to induce fibrocartilage upon implantation, and degeneration of articular cartilage was less severe than after meniscectomy. PMID- 8624394 TI - Evolution of bone transplantation: molecular, cellular and tissue strategies to engineer human bone. AB - Bone defects occur in a wide variety of clinical situations, and their reconstruction to provide mechanical integrity to the skeleton is a necessary step in the patient's rehabilitation. The current gold standard for bone reconstruction, the autogenous bone graft, works well in many circumstances. However, autograft reconstruction, along with the available alternatives of allogenous bone graft or poly(methylmethacrylate) bone cement, do not solve all instances of bone deficiency. Novel materials, cellular transplantation and bioactive molecule delivery are being explored alone and in various combinations to address the problem of bone deficiency. The goal of these strategies is to exploit the body's natural ability to repair injured bone with new bone tissue, and to then remodel that new bone in response to the local stresses it experiences. In general, the strategies discussed in this paper attempt to provide the reconstructed region with appropriate initial mechanical properties, encourage new bone to form in the region, and then gradually degrade to allow the new bone to remodel and assume the mechanical support function. Several of the concepts presented below are already finding clinical applications in early patient trials. PMID- 8624395 TI - Poly(alpha-hydroxy acids): carriers for bone morphogenetic proteins. AB - A broad spectrum of cells and cell products is associated with bone homeostasis and the renewal of bone following injury. The coupled interactions among cells provide the power behind sculpting of bone, sustaining form, and ensuring functionality. Local and systemic regulatory molecules (e.g. growth factors, hormones) direct cellular interactions through autocrine, paracrine, and hormonal pathways. Recently, genes for a class of osteogenic regulatory molecules have been cloned, and gene product expression has enabled investigators to assess safety and efficacy in animal studies. The molecules are known as bone morphogenetic proteins (BMPs). Therapeutic applications of BMPs depend on a carrier system. A carrier could spatially and temporally localize BMP for regional needs and be custom-tailored for acute craniofacial applications or for recalcitrant extremity non-unions. The poly(alpha-hydroxy acids) (PHAs) may be suitable for these applications. Therefore, the purposes of this paper are (i) to mention, briefly, basic concepts of the bone wound continuum and the possible therapeutic roles of BMPs; (ii) to outline several properties of selected PHAs relevant to bone regeneration dynamics; and (iii) to review selected preclinical studies with PHAs. PMID- 8624396 TI - Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry. AB - Materials with spatially resolved chemistries (i.e. patterned surfaces) have been used to guide and organize the position of mammalian cells in vitro. A common theme in guiding the spatial distribution of cells has been the use of patterned alkylsiloxanes, where one region contains an aminosilane and the other an alkylsilane. The regions of the aminosilane served as preferential sites for cell attachment and spreading, presumably dependent on the association between cell surface proteoglycans the positively charged amine. In this study, experiments were conducted with patterns of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS) to determine the kinetics of spatial organization of bone-derived cells, and whether initial attachment and spreading affected the rate of matrix mineralization (i.e. bone formation) in extended cultures. The bone cells required the presence of serum or preadsorption of serum proteins to the patterned EDS/DMS surface to organize according to the lithographically defined surface chemistry. Time-lapse video microscopy indicated that cells were randomly distributed over the EDS/DMS surface at the time of plating, but organized on the EDS regions within 30 min. When cultures were extended for 15 and 25 days, the matrix synthesized by the cells was preferentially mineralized on the EDS chemistry. These results demonstrate the ability of surface chemistry modifications to organize cells and form mineralized tissue in vitro. The methods employed should have general value to the engineering of tissues in vitro. PMID- 8624397 TI - Conditions which promote mineralization at the bone-implant interface: a model in vitro study. AB - This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue. PMID- 8624398 TI - Biochemical surface modification of Co-Cr-Mo. AB - Because of the limited mechanical properties of tissue substitutes formed by culturing cells on polymeric scaffolds, other approaches to tissue engineering must be explored for applications that require complete and immediate ability to bear weight, e.g. total joint replacements. Biochemical surface modification offers a way to partially regulate events at the bone-implant interface to obtain preferred tissue responses. Tresyl chloride, gamma-aminopropyltriethoxysilane (APS) and p-nitrophenyl chloroformate (p-NPC) immobilization schemes were used to couple a model enzyme, trypsin, on bulk samples of Co-Cr-Mo. For comparison, samples were simply adsorbed with protein. The three derivatization schemes resulted in different patterns and levels of activity. Tresyl chloride was not effective in immobilizing active enzyme on Co-Cr-Mo. Aqueous silanization with 12.5% APS resulted in optimal immobilized activity. Activity on samples derivatized with 0.65 mg p-NPC cm-2 was four to five times greater than that on samples simple adsorbed with enzyme or optimally derivatized with APS and was about eight times that on tresylated samples. This work demonstrates that, although different methods have different effectiveness, chemical derivatization can be used to alter the amount and/or stability of biomolecules immobilized on the surface of Co-Cr-Mo. PMID- 8624399 TI - Bone remodelling adjacent to intramedullary stems: an optimal structures approach. AB - The internal parameters in bone remodelling theories often are not clearly related to the bony structure which results from the simulations in which they are implemented. For a restricted class of bone remodelling theories, we have previously found a connection between overall structural optimization and the parameters within a continuum-level remodelling rule. In this study, we assess whether a simplified analytical formula based on structural optimization can predict the behaviour of a large-scale finite element bone remodelling simulation. The analytical formula predicts when bone will remain around an intramedullary implant. The predictions of the formula are borne out in the numerical results. This leads to a physical interpretation of one of the two parameters in the remodelling rule used. The results also show some characteristics which are clinically relevant. This study extends earlier results due to Huiskes for internal remodelling around intramedullary implants by using a different, numerically stable remodelling algorithm based on optimization. The study also shows a direct practical application of the optimizing remodelling theory the authors have developed previously. PMID- 8624400 TI - Tissue engineering scaffolds using superstructures. AB - Here, scaffolds as cell and tissue carriers are approached from an engineering point of view, emphasizing material superstructuring in the design of supports. Superstructure engineering provides optimal spatial and nutritional conditions for cell maintenance by the arrangement of structural elements (e.g. pores or fibres) so as to vary the order of cell to cell contact. This approach is illustrated in the design of several scaffolds: knitted fabrics as three dimensional superstructures for optimized osteosynthesis implants, a new injectable open porous implant system, an angiopolar non-degradable ceramic cell carrier, and an injectable or microsurgically implantable entangled carrier system. The implications for tissue engineering are discussed. PMID- 8624401 TI - Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. AB - This is a review of salient studies of sterilization, toxicity, biocompatibility, clinical applications and current work in the field of orthopaedics, using implants made of polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers. The intrinsic nature of these biomaterials renders them suitable for applications where temporally slow releases of bioactive agents in situ may be required. They are also desirable as fixation devices of bone, because they can virtually eliminate osteopenia associated with stress shielding or additional surgery. The majority of currently available sterilization techniques are not suitable for these thermoplastic materials and it may be desirable to develop new sterilization standards, which can account for the special character of PLA-PGA materials. Biocompatibility and toxicity studies suggest that, overall, PLA-PGA biomaterials may be suitable for orthopaedic applications, although certain problems, especially pertaining to reduction in cell proliferation, have been reported. Clinical applications are also promising, albeit not without problems usually associated with transient tissue inflammation. The future of these materials appears bright, especially in soft tissues. They may be used to address the exceedingly complex problem of osteochondral repair, but also as a means to enhance fixation and repair processes in tendons and ligaments. PMID- 8624402 TI - A novel phloem-specific gene is expressed preferentially in aerial portions of Vicia faba. AB - We have isolated a gene from bean (Vicia faba L.), called Vein1, that encodes a novel protein. The Vein1 cDNA was isolated as a result of a differential screen for genes that are expressed in leaves but not in the most common cell type, the mesophyll cell. Northern blot analysis revealed that Vein 1 transcripts are differentially expressed in the plant with expression in leaves, stems and sepals but not in petals, mesophyll cells or roots. In situ hybridization studies of stem and leaf sections indicate that the expression of Vein1 is localized to the phloem tissue. Interestingly, Vein1 was differentially expressed in stem tissue with the highest expression in the oldest internodes. The deduced Vein1 protein sequence does not share homology with any known protein sequences. The 17 kDa Vein1 protein is highly hydrophilic and contains a histidine-rich motif, where six out of seven amino acids are histidines. The function of Vein1 is unknown, although the expression patterns suggests that it may play a role in mature phloem tissue in the aerial parts of the plant. PMID- 8624403 TI - Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans. AB - The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H2O2 challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiation TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicated that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response. PMID- 8624404 TI - Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana. AB - Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the general phenylpropanoid pathway leading to the production of phenolic compounds with a significant range of biological function. A PAL gene we designated gPAL1, cloned from tobacco, consists of two exons separated by an intron of 1932 bp. Exon I, 398 bp, and exon II, 1747 bp, together encode a polypeptide of 715 amino acids. A putative TATA box and polyadenylation signal are found 144 bp upstream of the initiation codon and 193 bp downstream from the stop codon, respectively. Using various parts of gPAL1 as probes, genomic Southern blots indicated the presence of a small family of PAL genes in the tobacco genome that can be divided into two distinct subfamilies, one consisting of pal1 and pal2 and another of pal3 and pal4. Comparative genomic blot analysis of progenitor species (Nicotiana tomentosiformis and N. sylvestris) indicated that each species contains one PAL gene from each of the subfamilies, suggesting that pal1 and pal3 (or pal2 and pal4) diverged prior to the evolution of N. tabacum. Expression of the PAL gene family was examined using RNA gel blots. PAL transcript levels were significantly higher in flowers and roots than in leaves and stems of mature plants. PAL transcripts accumulate differentially during flower and leaf maturation in that mRNA levels decline during flower maturation but increase during leaf maturation. In leaves, PAL transcripts rapidly accumulated afer wounding. PMID- 8624405 TI - A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana. AB - Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5'-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues. PMID- 8624406 TI - A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. AB - We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5'-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2-3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark induced senescence of detached leaves or of leaves in planta resulted in a high level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency. PMID- 8624407 TI - Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. AB - Several cDNAs for mRNAs that change in abundance during tomato leaf senescence were isolated. In this paper we report molecular cloning and expression analysis of two cysteine proteases. SENU2 is identical to the cDNA C14 which encodes a cysteine protease previously shown to be expressed in response to extremes of temperature in tomato fruit [43]. SENU3 cDNA clone was 1.2 kb in length and hybridized to a transcript of 1.4 kb which suggested that the clone was not full length. The missing 5' end was isolated using rapid amplification of cDNA ends (RACE). Southern blot analysis of tomato genomic DNA indicates that SENU3 is encoded by a single or low copy gene. SENU3 was also shown to have significant homology with known cysteine proteases. These two senescence-associated cysteine proteases are also expressed during other developmental processes, including seed germination, consistent with a role in protein turnover. SENU2 and SENU3 mRNAs were detectable in young fully expanded leaves and increased in abundance with leaf age, reaching a maximum during the later stages of visible leaf senescence. Such a pattern of expression suggests that the onset of leaf senescence is a gradual event. Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescence is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented. PMID- 8624408 TI - Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles. AB - Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F1-ATPase beta-subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial beta-presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immunodetection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import. PMID- 8624409 TI - The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. AB - The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed. PMID- 8624410 TI - Microsequencing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5 phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts. AB - Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5 phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids. PMID- 8624412 TI - Hormonal regulation of S-adenosylmethionine synthase transcripts in pea ovaries. AB - Two cDNA clones coding for S-adenosyl-L-methionine synthase (SAMs, EC 2.5.1.6) have been isolated from a cDNA library of gibberellic acid-treated unpollinated pea ovaries. Both cDNAs were sequenced showing a high degree of identity but coding for different SAMs polypeptides. The presence of two SAMs genes in pea was further confirmed by Southern analysis. Expression of the SAMs genes in the pea plant was found at different levels in vegetative and reproductive tissues. We characterized the expression levels of SAMs genes during the development or senescence of pea ovaries. Northern analysis showed that transcription of SAMs genes in parthenocarpic fruits was upregulated by auxins in the same manner as in fruits from pollinated ovaries. In both pollinated and 2,4-dichlorophenoxyacetic acid-treated ovaries, and benzyladenine, although able to induce parthenocarpic development, did not affect SAMs mRNA levels. These data are consistent with an active participation of auxins in the upregulation of SAMs during fruit setting in pea and suggest that, at the molecular level, parthenocarpic development of pea ovaries is different for gibberellin- and cytokinin-treated ovaries than for auxin-induced parthenocarpic biosynthesis since treatment of the ovaries with aminoethoxyvinylglycine resulted in a delay of senescence and prevention of SAMs mRNA accumulation. A possible mechanism for hormonal regulation of SAMs during ovary development is discussed. PMID- 8624411 TI - Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening. AB - The cDNA which encodes lycopene cyclase, CrtL, was cloned from tomato (Lycopersicon esculentum cv. VF36) and tobacco (Nicotiana tabacum cv. Samsun NN) and functionally expressed in Escherichia coli. This enzyme converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene. The enzyme interacts with half of the carotenoid molecule and requires a double bond at the C-7,8 (or C-7,8') position. Inhibition in E. coli indicated that lycopene cyclase is the target site for the inhibitor MPTA, 2-(4 methylphenoxy)tri-ethylamine hydrochloride. The primary structure of lycopene cyclase in higher plants is significantly conserved with the enzyme from cyanobacteria but different from that of the non-photosynthetic bacteria Erwinia. mRNA of CrtL and Pds, which encodes phytoene desaturase, was measured in leaves, flowers and ripening fruits of tomato. In contrast to genes which encode enzymes of early steps in the carotenoid biosynthesis pathway, whose transcription increases during the 'breaker' stage of fruit ripening, the level of CrtL mRNA decreases at this stage. Hence, the accumulation of lycopene in tomato fruits is apparently due to a down-regulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis. PMID- 8624413 TI - cDNA cloning and gene expression of ascorbate oxidase in tobacco. AB - A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Nothern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant. PMID- 8624414 TI - Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana. AB - Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a lambda clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity. PMID- 8624416 TI - An automated preconcentration-derivatization system for the determination of cocaine and its metabolites in urine and illicit cocaine samples by gas chromatography/mass spectrometry. AB - Although gas chromatography/mass spectrometry (GC/MS) is the most powerful technique for analysing cocaine and its hydrolysis products (benzoylecgonine, ecgonine methyl ester and ecgonine) in urine, the sample pretreatment required is rather laborious and involves a large number of steps. This paper reports a continuous method for the separation (based on the retention properties of cocaine and its major metabolites on a reversed phase C18 column), preconcentration (evaporation under nitrogen) and derivatization (collection of the residue in a few microlitres of derivatizing reagent) of cocaine and its metabolites in urine samples for subsequent analysis by GC/MS. Calibration graphs are linear from 5 to 3000 ng/mL; the detection limits are 1 ng/mL (cocaine and ecgonine methyl ester), 20 ng/mL (benzoylecgonine) and 200 ng/mL (ecgonine). The method was applied to illicit cocaine samples and to urine from a drug addict. PMID- 8624415 TI - A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase. AB - The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter Ptrc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl-beta-D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase. PMID- 8624417 TI - Structural characterization and location of disulphide linkages of a potent vasodilatory peptide, recombinant maxadilan, by a multiple mass spectrometric approach. AB - A multiple mass spectrometric strategy using fast-atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) has been used to confirm the sequence and to locate the disulfide linkages of recombinant maxadilan (r maxadilan) (average molecular mass 7422.5 Da), a potent vasodilatory peptide from Lutzomyia longipalpis. MALDI measurements of intact r-maxadilan, its reduced form and its pyridylethylated form (p-maxadilan) indicated the presence of four Cys residues without major post-translational modifications. FAB and FAB-tandem mass spectrometry measurements of chymotryptic digests of p-maxadilan were sufficient to map the primary structure of p-maxadilan, though the complementary use of MALDI was necessary for complete mapping using Asp-N digestion due to a strong suppression observed in FAB. Assignment of the Cys-5-Cys-9 linkage was achieved by comparison of FAB mass spectra before and after reduction of tryptic digests of r-maxadilan. Since the molecular weight of the peptide fragment containing the Cys-18-Cys 55 linkage is more than 4000, MALDI measurement was indispensable for assignment of this linkage. The results fully support the value of the multiple mass spectrometric strategy in the structural characterization of peptides and proteins. PMID- 8624418 TI - Sherpa: a Macintosh-based expert system for the interpretation of electrospray ionization LC/MS and MS/MS data from protein digests. AB - A computer program, Sherpa, has been implemented to aid in the elucidation of electrospray ionization liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry data from protein digests. The program differs from existing analysis software in its ability to search concurrently against multiple protein sequences, to search and compare two LC/MS files concurrently, to search for glycosylated and phosphorylated peptides, and to give a simple evaluation of the quality of a match between the data and a prediction. PMID- 8624419 TI - Characterization of nitrosation products in cosmetics raw materials by liquid chromatography/mass spectrometry techniques. AB - Characterization of nitrosation products in cosmetics raw material samples has been accomplished by three liquid chromatography/mass spectrometry (LC/MS) techniques. Two of the techniques involved conventional methodologies of LC/MS and LC/tandem mass spectrometry, both of which can be used to detect and identify products formed under extreme-nitrosation conditions. The third technique utilized an on-line coupling of a photolysis reactor with the LC/MS, and it may be used as a rapid and specific means of screening for the presence of known and unknown N-nitrosamines, which may be carcinogenic. PMID- 8624420 TI - The credibility of the profession. PMID- 8624422 TI - The impact of different types of anesthesia on in vitro fertilization-embryo transfer treatment outcome. AB - OBJECTIVE: Our objective was to evaluate retrospectively the influence of different types of anesthesia on the outcome of ovum retrieval. METHODS: Sedation combined with local anesthesia was used on 120 occasions (Group I), epidural block in 139 ovum retrievals (Group II), and general anesthesia in 173 cycles (Group III). RESULTS: No differences were found in embryo yield or number or the quality of the embryo transferred. Of 99 pregnancies achieved, 66 live deliveries were recorded. Significantly lower clinical pregnancy rates were found in Group III (14.5%) compared with Group II (23.7%; P = 0.018) or Group I (25.8%; P = 0.0074). Highly significant differences were found in the delivery rates between Group III (8.7%), Group II (20.11%; P = 0.0017), and Group I (19.2%; P = 0.0043). CONCLUSION: The use of general anesthesia, especially nitrous oxide, for oocyte retrieval has an adverse effect on IVF outcome. This deleterious effect manifests itself only after embryo transfer and leads to lower pregnancy and delivery rates. PMID- 8624423 TI - Ovarian function in relation to the outcome of in vitro fertilization (IVF) treatment in regularly menstruating women with tubal infertility. AB - STUDY DESIGN: Ovarian endocrine function was evaluated in 53 regularly menstruating women, 27 to 38 years of age, who failed to conceive at least 2 years following reconstructive surgery for tubal infertility. Thirty apparently healthy women, 24 to 40 years of age with proven fertility, served as controls. Blood samples were obtained daily on cycle days 2-4 and 22-26 to assess FSH, E2, and P4 results. RESULTS: There was a tendency for women with more advanced tubal damage to be subjected to more extensive surgery. Based on extent of reconstructive surgery, the patients were divided into three groups. Group A (n = 29) had less extensive surgery, limited to the fallopian tubes, group B (n = 14) included patients with extended adhesiolysis, and group C (n = 10) comprised patients that had the most extensive reconstructive procedures involving ovarian surgery. Significantly highest FSH levels were found in group C (P < 0.001) compared to groups A and B in the early follicular phase. During the luteal phase, E2 levels were lower in groups B (P < 0.01) and C (P < 0.001) compared to group A. P4 levels were lower in group C compared to groups A and B. CONCLUSION: When the patients underwent IVF treatment higher grades of tubal damage, more extensive surgery and hormonal signs of ovarian insufficiency were highly related to treatment failure. PMID- 8624424 TI - Effect of coculture on subsequent survival and implantation of cryopreserved human embryos. AB - PURPOSE: This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously match controls. RESULTS: During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P < 0.05). CONCLUSION: Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation. PMID- 8624421 TI - Potential use of embryo coculture with human in vitro fertilization procedures. AB - PURPOSE: This review was designed to outline potential uses of an embryo co culture system in human assisted reproduction programs to improve embryo quality and pregnancy rates. RESULTS: The various cell types used in embryo co-culture were reviewed in addition to the use of co-culture for both animal and human embryos. Co-culture provides a method to enhance embryo development in an inadequate in vitro environment without compromising embryo quality. Human IVF laboratories have used various types of "helper cells" to improve rate of development, reduce cell fragmentation rate and in some instances increases pregnancy and implantation rates. CONCLUSION: In conjunction with several assisted reproduction procedures such as IVF, microsurgical fertilization, cryopreservation and genetic evaluation, co-culture may increase the number of viable embryos for replacement and improve pregnancy rates. PMID- 8624425 TI - Early first-trimester ultrasound provides a window through which the chorionicity of twins can be diagnosed in an in vitro fertilization (IVF) population. AB - PURPOSE: Early and accurate diagnosis of placentation alerts the obstetrician to potential clinical sequelae. The reproductive endocrinologist has a unique opportunity to sonographically evaluate the very early intrauterine pregnancy. We undertook this study to determine whether chorionicity could accurately be predicted using early first-trimester transvaginal ultrasound. RESULTS: Of 47 sets of twins conceived through our IVF-ET program, all underwent detailed transvaginal sonography. These findings were then compared with results of placental pathology examination, after birth. Transvaginal sonography was performed 41 days following embryo transfer. All 3 monochorionic placentas were correctly predicted by ultrasound, while the remaining 44 placentas were dichorionic. CONCLUSIONS: The significance of our findings lies both in our 100% accuracy in diagnosis and in the extremely early gestational age at which we were able to establish correctly the diagnosis of chorionicity. The errors in diagnosis made by previous investigators were often the result of not recognizing single placentae which were later histologically shown to be the result of placental fusion. The infertility specialist has a unique window of opportunity to evaluate placentation and should provide useful information regarding chorionicity to the obstetrical team. PMID- 8624426 TI - Comparison between a two-layer discontinuous Percoll gradient and swim-up for sperm preparation on normal and abnormal semen samples. AB - PURPOSE: This work was to compare the effects of Percoll gradient and swim-up treatments for sperm preparation on the percentage of progressive motility, recovery of motile sperm, removal of debris, percentage of normal forms according to strict criteria, and movement characteristics of sperm using computer-assisted velocity analysis. RESULTS: In total, 50 semen samples from 50 patients were tested and divided into two groups: a normal group (n = 27) with normal parameters and an abnormal group (n = 23) with abnormal parameters. The results in both the normal and abnormal groups revealed that the sperm concentration in the Percoll samples was significantly greater than that in the swim-up samples. Although the percentage of progressive motility was greater in the swim-up samples than in the Percoll samples, the number of motile sperm, reflecting the percentage of motile sperm recovery, was till greater in the Percoll samples. The debris of semen was equally removed by both methods and the percentage of normal forms was also similar in the samples treated according to these two procedures. Both curvilinear velocity (VCL) and straight-line velocity (VSL) of sperm were significantly greater in the swim-up samples than in the Percoll samples. Sperm from the swim-up procedure also showed a greater mean amplitude of lateral head displacement than that from the Percoll gradient procedure, but the distinction was insignificant. CONCLUSION: The Percoll gradient technique, by recovering more motile sperm, may be applied to prepare oligospermic samples. The swim-up method may become the standard choice to prepare normal semen which could obtain sufficiently motile sperm, due to its simplicity and recovered sperm with superior motility. PMID- 8624427 TI - An evaluation of the effect of pentoxifylline on sperm function and treatment outcome of male-factor infertility: a preliminary study. AB - OBJECTIVE: Our objective was to study the effect of pentoxifylline (PF) on fertilization rates in couples with previous failure of fertilization and male factor infertility and to determine the predictive value of conventional semen analysis parameters in selecting the couples who would benefit from the elective use of PF in IVF. DESIGN: This prospective controlled study was conducted in an assisted conception METHODS: Sixty-nine couples with previous failed IVF cycle, who had a low fertilization rate and/or male-factor infertility, were recruited to the study. Multiple follicular development was induced using the same protocol of human menopausal gonadotropin and gonadotropin releasing hormone analogue in both cycles. The oocytes were inseminated with spermatozoa treated with PF. The fertilization rates in the PF cycle were compared to the reference cycle based on semen analysis parameters and previous fertilization rates. RESULTS: In couples with male infertility, the fertilization rate improved significantly, from 17 to 50% in PF cycles (P < 0.001). A significant improvement in fertilization rate was also demonstrated in couples with previous poor fertilization, < 30% (P < 0.01), particularly in those with a very low fertilization rate, < 20% (P < 0.001). Although there was an overall improvement in fertilization rates in couples with male-factor infertility, there was no cutoff value in sperm motility that would make a significant difference in the impact of PF on fertilization rates. CONCLUSION: Couples with poor fertilization rates in vitro benefit with a significant improvement in fertilization by the elective use of PF. The improvement is most significant in couples with previous complete failure of fertilization and poor fertilization rates, < 30%. PMID- 8624429 TI - Comparison between nafarelin acetate and D-Trp6-LHRH for temporary pituitary suppression in in vitro fertilization (IVF) patients: a prospective crossover study. AB - PURPOSE: Nafarelin acetate is a new gonadotropin releasing (GnRH) agonist analogue with unique potency, intranasal administration, and convenient storage. Hence, nafarelin was considered as an alternative for temporary pituitary suppression in patients undergoing ovulation induction in IVF. A crossover treatment in a prospective study was performed including 40 women with bilateral obstructed tubes and normal ovarian function, treated in 80 ovulation induction cycles using the long protocol. Twenty patients used nafarelin acetate 600 micrograms/daily in their first cycle and received D-Trp6-LHRH, 0.5 mg/daily, in their following cycle. The other 20 women used decapeptyl in their cycle and received nafarelin in the second. RESULTS: Estradiol suppression was achieved by both D-Trp6-LHRH and nafarelin at equal time intervals. The average total number of ampoules (P = 0.0005) and the length of administration of hMG required for ovarian stimulation (P = 0.0002) and the time interval between GnRHa initiation to oocyte retrieval (P = 0.04) was significantly lower in nafarelin cycles. The number and the distribution between large and small follicles as well as the average number of oocytes retrieved did not differ between the two GnRH analogues. CONCLUSION: Our results demonstrate that nafarelin acetate is comparable to D-Trp6-LHRH for temporary pituitary suppression used for controlled ovarian stimulation in IVF patients. However, using nafarelin ovarian stimulation was achieved with few ampoules of hMG, administered for a shorter period of time, thus with a lesser cost. PMID- 8624428 TI - Successful use of pentoxifylline in male-factor infertility and previous failure of in vitro fertilization: a prospective randomized study. AB - OBJECTIVE: Our objective was to determine whether the use of pentoxifylline (PF) would improve the in vitro fertilization (IVF) rate and outcome in couples with male factor infertility and previous failure of fertilization in vitro. DESIGN: This prospective randomized controlled study was conducted in an assisted conception unit. MATERIALS AND METHODS: Forty-nine couples with previous failed fertilization in vitro attributable to male factor or male-factor infertility without previous IVF were recruited for the study. Controlled ovarian hyperstimulation was performed using a combination of gonadotropin releasing hormone agonist and human menopausal gonadotropin. Oocytes of the same grade and maturity were inseminated with spermatozoa treated with PF or control spermatozoa. A maximum of three embryos was replaced after 48 hr and all other embryos were cryopreserved. Pregnancy outcome was followed up and evidence of fetal or neonatal anomalies reported. RESULTS: A significantly higher fertilization rate occurred in the group where oocytes were inseminated with spermatozoa treated with PF compared with controls (56.3 versus 30.7%; P < 0.05). Fertilization occurred in 45 of the 49 cycles (92%). In seven cycles, only the oocytes that were inseminated with spermatozoa treated with PF fertilized, in contrast to only one cycle where the oocytes inseminated with control sperm fertilized (P < 0.05). Fifty-seven PF and 31 control embryos were replaced and 11 clinical pregnancies occurred. Three of the pregnancies occurred in the seven cycles in which only PF embryos were replaced, one in the single cycle where control embryos were replaced and seven from the 37 cycles in which both PF and control embryos were replaced. There was no evidence of congenital malformations in any of the offsprings resulting from this study. CONCLUSION: This study suggests that PF improves the fertilization rate and outcome in couples with male factor infertility and poor fertilization rates. This study does not suggest any increase in teratogenicity or evidence of congenital malformations in pregnancies following IVF cycles where PF was used. PMID- 8624431 TI - Effects of a gonadotropin releasing hormone agonist on oocyte maturation, fertilization, and embryonal development of mice. AB - PURPOSE: The effects of a GnRH agonist (GnRHa) on oocyte quality were investigated by assessing the influence of GnRHa on oocytes, and fertilized oocytes were examined in vivo and in vitro. Administration of gonadotropin in conjunction with GnRHa induced a significantly greater degree of germinal vesicle breakdown, significantly higher rates of in vitro fertilization, and significantly faster development of the oocytes than the pregnant mare serum gonadotropin alone RESULTS: The hatching-success rate in the GnRHa treated hypophysectomized mice was higher than in control mice. The rate of in vitro fertilization was also higher in oocytes cultured in the presence of low loses of GnRHa and these effects were reversed by a GnRH antagonist. CONCLUSION: Oocytes obtained following ovarian stimulation with GnRHa were of higher quality than control oocytes, and the efficacy of GnRHa may be due in part to its direct action on the ovary. PMID- 8624430 TI - Epidermal growth factor (EGF) receptor localization in cultured human granulosa lutein cells and the stimulation of progesterone production by EGF and transforming growth factor-alpha (TGF-alpha). AB - PURPOSE: The purpose of this study was to investigate the expression of EGF receptor (EGF-R) in human granulosa cells undergoing luteinization and progesterone production by these cells in response to EGF an TGF-alpha alone or in combination with luteinizing hormone (LH). Granulosa cells were obtained from IF patients following oocyte retrieval 34 to 36 hr post-hCG injection. EGF receptor was localized in cells by means of immunoperoxidase staining using a polyclonal primary antibody directed against the human EGF-R. To assess progesterone production, cells were seeded overnight, washed, and cultured with the growth factors +/- LH. Medium and treatments were changed every 24 hr for 3 days. RESULTS: Specific EGF-R staining was observed in the cultured cells compared to those incubated with antibody that was preabsorbed with a 10-fold excess of EGF. Basal progesterone accumulation per 24-hr period was stimulated dose dependently on each day of culture, by both EGF (up to 3.5-fold at 5 or 50 ng/ml) and TGF-alpha (up to 4-fold at 50 ng/ml). The addition of LH alone also stimulated progesterone accumulation daily, and this effect was further enhanced dose dependently by cotreatment with EGF or TGF-alpha. Furthermore, tyrphostin, an EGF-R-specific tyrosine kinase inhibitor, inhibited both basal and growth factor-stimulated progesterone production. CONCLUSION: These data suggest an EGF receptor-mediated physiological role for EGF and TGF-alpha in human luteal function involving an autocrine and/or a paracrine stimulation of progesterone production. PMID- 8624432 TI - Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development. AB - PROBLEM: The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture. METHOD: To determine the ability of mouse cumulus cell coculture in the presence of human follicular fluids to support preimplantation mouse Balb/cJ embryo development in vitro. RESULTS: Culture of preimplantation mouse Balb/cJ embryo's independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P < 0.01) improved blastocyst development and the total number of cells per blastocyst. CONCLUSION: Cumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro. PMID- 8624433 TI - Use of follicle-stimulating hormone (FSH) to increase the in vitro fertilization (IVF) efficiency of mice. AB - PURPOSE: The possibility of increasing the efficiency of an in vitro fertilization system (IVF) for Swiss OF1 mice was studied. The experimental protocol proposed analyzed the use of FSH as a superovulatory inducing hormone in comparison to traditional PMSG treatment. At the same time, the quality of IVF derived embryos was evaluated both in vitro, with culture in CZB medium and fixation in advanced stages of development, and in vivo, by transfer to female recipients. RESULTS: Treatment with FSH induced a much higher ovulation number compared PMSG (64.26 vs 33.85; P < 0.01). With this gonadotropin, IVF provided a positive tendency to normal fecundation (67.76 vs 64.72; P < 0.1) and a much lower index of abnormal division in embryos (10.57 vs 15.11; P < 0.05). The viability of embryos obtained from donors treated with hormones was similar, although differences did exist regarding embryo origin: those obtained following natural fertilization showed a higher developmental capacity from in vivo (P < 0.05) and in vivo (P < 0.05). CONCLUSIONS: We conclude that FSH is an improved superovulation alternative treatment in comparison to PMSG for IVF. It provides a higher number of embryos with the same in vitro and in vivo viability as those obtained from PMSG. PMID- 8624435 TI - Viability of embryos following second polar body removal in a mouse model. AB - PURPOSE: Our purpose was to evaluate the effect of the second polar body removal on the viability and development potential of the resulting embryos. METHOD: The second polar body was removed in 343 mouse oocytes and the development potential of the resulting embryos was compared in vitro to that in 223 intact mouse embryos and 222 controls which were placed under the same conditions as those micromanipulated. RESULTS: The proportion of morphologically normal blastocysts formed by polar body biopsied oocytes (70.8%) was not significantly different from that obtained from the control (81.1%) and intact (85%) oocytes. Cell counts of blastocysts obtained from the manipulated and non-manipulated oocytes were also not statistically different. CONCLUSION: The second polar body biopsy does not have a significant adverse effect on preimplantation development and may be applied for genetic evaluation of oocytes. PMID- 8624434 TI - Gonadotropin hyperstimulation influences the 35S-methionine metabolism of mouse preimplantation embryos. AB - The effects of gonadotropin stimulation on mouse embryo uptake and incorporation of 35S-methionine were studied. We found that the uptake of 35S-methionine was reduced in embryos of stimulated females in both the two-cell and the blastocyst developmental stage. The incorporation of 35S-methionine into protein was not statistically significantly different between the embryos of stimulated and those of unstimulated females. Qualitatively, protein synthesis was equal in both groups as determined with one-dimensional SDS-PAGE. The results are discussed and we conclude that mouse embryo viability in vivo is decreased by ovarian stimulation. PMID- 8624436 TI - Refrigerated storage of Ham's F10 culture medium in assisted reproduction programs. PMID- 8624437 TI - Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site. AB - The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha TIP showed that the TIPs are differentially expressed. PMID- 8624438 TI - Far-red light blocks greening of Arabidopsis seedlings via a phytochrome A mediated change in plastid development. AB - We have characterized a far-red-light response that induces a novel pathway for plastid development in Arabidopsis seedlings. This response results in the inability of cotyledons to green upon subsequent white light illumination, and the response is suppressed by exogenous sucrose. Studies with mutants showed that this far-red block of greening is phytochrome A dependent and requires an intact downstream signaling pathway in which FHY1 and FHY3 may be components but in which HY5 is not. This highlights a previously undefined branchpoint in the phytochrome signaling pathway. Ultrastructural analysis showed that the far-red block correlates with both the failure of plastids to accumulate prolamellar bodies and the formation of vesicles in the stroma. We present evidence that the far-red block of greening is the result of severe repression of protochlorophyllide reductase (POR) genes by far-red light coupled with irreversible plastid damage. This results in the temporal separation of phytochrome-mediated POR; repression from light-dependent protochlorophyllide reduction, two processes that normally occur in coordination in white light. PMID- 8624439 TI - Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. AB - Systemic acquired resistance is an important component of the disease resistance repertoire of plants. In this study, a novel synthetic chemical, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), was shown to induce acquired resistance in wheat. BTH protected wheat systemically against powdery mildew infection by affecting multiple steps in the life cycle of the pathogen. The onset of resistance was accompanied by the induction of a number of newly described wheat chemically induced (WCI) genes, including genes encoding a lipoxygenase and a sulfur-rich protein. With respect to both timing and effectiveness, a tight correlation existed between the onset of resistance and the induction of the WCI genes. Compared with other plant activators, such as 2,6 dichloroisonicotinic acid and salicylic acid, BTH was the most potent inducer of both resistance and gene induction. BTH is being developed commercially as a novel type of plant protection compound that works by inducing the plant's inherent disease resistance mechanisms. PMID- 8624440 TI - A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. AB - A novel transposon-tagging strategy designed to recover dominant gain-of-function alleles was performed with Arabidopsis by using a Dissociation element with a cauliflower mosaic virus 35S promoter transcribing outward over one terminus. Lines containing transposed copies of this transposon were screened for mutants, and a semidominant mutation affecting plant height, hypocotyl elongation, and fertility was recovered. The pleiotropic effects of this mutation appear to result from a general reduction in cell expansion, and some of the effects are similar to those caused by supplying exogenous ethylene or cytokinin to wild-type seedlings. In addition, the arrangement of cells in some organs such as the etiolated hypocotyl, is disorganized. The mutation was called tiny, and the affected gene was cloned by first using transposon sequences to isolate the mutant allele. The predicted protein product of the TINY gene shows strong homology with the DNA binding domain of a recently identified class of plant transcription factors. This domain, called the APETALA2 domain, was initially identified as a duplicated region within the APETALA2 gene of Arabidopsis and then as a conserved region between APETALA2 and the ethylene responsive element binding proteins of tobacco. In the mutant allele, the Dissociation element is inserted in the untranslated leader of the TINY gene, 36 bp from the ATG, and the mutant contains a novel transcript that initiates from the cauliflower mosaic virus 35S promoter within the transposon. This transcript is present in greater abundance than the wild-type TINY transcript; therefore, the semidominant tiny mutation most likely results from increased, or ectopic, expression of the gene. PMID- 8624441 TI - An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells. AB - Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane. PMID- 8624442 TI - Cloning and functional expression of a plant voltage-dependent chloride channel. AB - Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than 120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID- 8624443 TI - Autonomous transposition of the tobacco retrotransposon Tto1 in rice. AB - The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool. PMID- 8624446 TI - [Preliminary results after the transperitoneal laparoscopic treatment of the first 50 inguinal hernias]. AB - The authors are presenting the results of first 50 inguinal hernias treated laparoscopically trough the transperitoneal approach (39 men, 11 women, mean age- 42,5 years, 44 uncomplicated and 6 epiploon's incarceration). After the dissection of the hernia's pouch, the parietal defect was covered with Prolene meche in 15 cases, with Marlex in 10 cases and Plastex prosthesis in 25 cases, all of them without fixation. The prosthesis itself was covered with the parietal peritoneum in running suture. The immediate postoperative evolution was good, the patients being able to walk soon after being awake, and without any urinary problems (even in elder patients). The patients left the hospital 24-48 hours after surgery. They were followed-up 2 to 10 months, periodically. The results were good, except 4 patients: 1 case with port-site hematoma (in the right lower quadrant, necessitating open surgery). 2 patients with seromas (vulvar and scrotal) in case of section and abandoning the hernial pouch. 1 patient with prosthesis migration with precocious recidive (24 hours after the operation). We consider the transperitoneal approach as very good in the treatment of uncomplicated and not very large hernias. The easiest and safest technic is that using Prolene prosthesis. PMID- 8624445 TI - Methylation pattern of Activator transposase binding sites in maize endosperm. AB - The maize transposable element Activator (Ac) transposes after replication from only one of the two daughter chromatids. It has been suggested that DNA methylation in conjunction with methylation-sensitive transposase binding to DNA may control the association of Ac transposition and replication. We present here a detailed genomic sequencing analysis of the cytosine methylation patterns of the transposase binding sites within both Ac ends in the wx-m9::Ac allele, where Ac is inserted into the tenth exon of the Waxy gene. The Ac elements in wx-m9::Ac kernels exhibit intriguing methylation patterns and fall into two distinct groups. Approximately 50% of the elements are fully unmethylated at cytosine residues through the 256 nucleotides at the 5' end (the promoter end). The other half is partially methylated between Ac residues 27 and 92. In contrast, at the 3' end, all Ac molecules are heavily methylated between residues 4372 and 4554. The more internally located Ac sequences and the flanking Waxy DNA are unmethylated. Although most methylated cytosines in Ac are in the symmetrical CpG and CpNpG arrangements, nonsymmetrical cytosine methylation is also common in the hypermethylated regions of Ac. These results suggest a model in which differential activation of transposon ends by hemimethylation controls the chromatid selectivity of transposition and the association with replication. PMID- 8624444 TI - The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats. AB - Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID- 8624447 TI - [Pancreatoduodenectomy with pancreaticogastric anastomosis]. AB - There is not general agreement concerning the ideal way of preventing pancreatic leakage after duodenopancreatectomy. The aim of the present study is to present the experience of a three years period of performing a pancreatogastroanastomosis (PGA) after duodenopancreatectomy (DPC) in 12 consecutive patients. In 7 cases PGA was performed after closure of the gastric stump using a transverse incision of the posterior wall of the stomach. A total layer of nylon 9 points was doubled by an interrupted serocapsular suture. In one case only the seromuscular layers of the stomach were cut, adjusted to the size of the pancreatic section with a central hole for the duct of Wirsung. Four nylon 10 points were used to anastomose the latter to the gastric mucosa. PGA was performed in one layer interrupted suture between the pancreatic capsule and the seromuscular of the stomach. In 4 cases, after closure of the gastric stump an anterior gastrotomy was associated to the posterior incision of the gastric wall in order to perform PGA using an intragastric route. The postoperative follow-up showed a good evolution with only one anastomotic leakage that necessitated reintervention in the 10th day. PGA might be elective after DPC when appropriate dissection and mobilisation of the pancreas is possible. PMID- 8624448 TI - The practical research on the rectangular valve anastomosis to prevent regurgitation in effecting a radical cure of antral gastric carcinoma. AB - Having observed the drain through the stomach tube after the rectangular valve anastomosis for 58 cases in effecting a radical cure of antral gastric carcinoma. Determined the pH value, BAO and DGRI, duodenal-graphed and watched through gastroscope. We see that the rectangular valve anastomosis is an ideal method to prevent regurgitation in effecting a radical cure of antral gastric carcinoma. PMID- 8624449 TI - [The therapeutic options in central hydatid cysts of the right hepatic lobe]. AB - The authors of the article propose a method that is highly efficient and presents minimal risks in the treatment of hepatic hydatid cysts located in the central area of the right lobe. This "dangerous area" of the VIIIth segment generates the most delicate problems for the surgical treatment, due to limitation of therapeutic solutions and the highest rate of postoperative complications. The technique presented is that of external extraperitoneal transligamentous drainage introduced by D. Burlui in 1968. This technique should be considered as an alternative to lobectomies or wedge resections or to the direct external drainage of the remaining cavity, the latter being responsible for numerous complications. Considering the figures covering the past 35 years at Caritas Hospital, the authors note the decrease of corrective reinterventions from 27% to 10% after 1968. By associating cholecystectomy after 1983 they dropped to only 4%. The authors do not claim that the method should be universally applied but consider it obviously the choice in the treatment of hydatid cysts in the "dangerous area" in which other therapeutic methods are inappropriate (pericysto-digestive anastomosis) or excessive (right lobe resection), avoiding the problems of direct drainage. PMID- 8624450 TI - [Mechanical colonic anastomosis by triangulating]. PMID- 8624451 TI - [Our experience in using the air-bag technic in the treatment of burns of the hands]. PMID- 8624452 TI - [Amputation of the rectum via laparoscopy]. AB - We report the case of a 66-year-old woman with a bleeding adenocarcinoma of the lower thirty of the rectum. The laparoscopic procedure was initiated with mobilization of the sigmoid colon. The left ureter was identified as it crosses the left iliac vessels. The peritoneum along the right side of the rectosigmoid mesentery was transected. The superior rectal artery was divided utilizing extracorporeal ligatures. The anterior rectum was separated from the uterus, the pararectal tissue was mobilized, the middle rectal arteries were ligated and the posterior rectum was dissected from the presacral tissue. The perineal component of the procedure was simultaneously performed. The sigmoid loop was extracorporeally transected with a linear stapler. The proximal sigmoid end was exteriorized through the colostomy site (site of the left superior port). The distal sigmoid end was replaced in the abdominal cavity and was grasped through the perineal wound; the anus, rectum and sigmoid were removed through the perineal wound. The feasibility of this procedure has been well established. However, it seems to be an alternative for the classical abdominoperineal resection as treatment for adenocarcinoma of the lower rectum. His superiority has yet to be confirmed by future studies. PMID- 8624453 TI - [Establishing the diagnosis of the lesions and structuring the order of the therapeutic measures in a case of multiple trauma]. PMID- 8624454 TI - [A spontaneously ruptured primary hydatid cyst of the great epiploon. Hydatid peritonitis]. PMID- 8624455 TI - [The Shouldice procedure in the surgical treatment of inguinal hernias]. AB - The Shouldice repair gained a major importance in the treatment of inghinal hernias provided by the excellent results that had been obtained in the Shouldice Hospital on more than 200,000 cases. This study analyze prospectively 101 cases of inguinal hernias that had been treated using the Shouldice procedure with in a 3 year period, comparative with a control cohort of 102 cases of inguinal hernias treated using classic procedures (Bassini, Kimbarowski). The data analyze was performed using Kruskal-Wallis H test and t Student test. The immediately postoperative results have been favorable in 95% cases and the long term postoperative results have been very good in 99% cases (a unique recurrence in a multiple recidivated patient). The result of this study proves that Shouldice repair has advantages comparative with classic treatment: short postoperative hospitalization, low incidence of complication and recurrence. PMID- 8624457 TI - Bcl-2 expression and glucocorticoid-induced apoptosis of leukemic and lymphoma cells. AB - The lytic response of lymphoid cells to glucocorticoid hormones (GC) is prototypical of the induction of apoptosis: a special form of cellular demise for the removal of unwanted or redundant cells. Initiation and execution of a death programme are therefore major checkpoints in GC-sensitivity. Although Bcl-2 protein can prevent or delay apoptosis of lymphoma and leukemia cells, exposed to multiple cytotoxic agents, its antagonism of GC-induced apoptosis appears most critical in conferring resistance to corticosteroids. Moreover, Bcl-2 may modulate GC-signalling to apoptosis through its association with fundamental cellular processes such as energy state, Ca2+ homeostasis and transmembrane transport. However, this signalling pathway can also be interrupted by Bcl-2- independent mechanisms. This review discusses the various cellular and oncogenetic factors that control GC sensitivity of leukemia/lymphoma cells and proposes a hypothesis of how GC may induce a death programme, sensitive to blockade by Bcl-2. PMID- 8624456 TI - The clinical significance of residual disease in childhood acute lymphoblastic leukemia as detected by polymerase chain reaction amplification by antigen receptor gene sequences. AB - The polymerase chain reaction (PCR) has been applied to detect occult leukemia cells in children with acute lymphoblastic leukemia who are otherwise considered in complete remission by traditional morphological examination of bone marrow specimens. To determine whether PCR provides unique prognostic information of use for the clinical investigator, we reviewed the 20 clinical studies published to date. From this review, it is evident that discrepancies exist for the detection of residual disease for patients who remain in complete remission and for those who relapse. However, because of the fundamentally different approaches used to apply the PCR method to each of these studies, an entirely different interpretation can be reached when critical technical factors are considered. The combined data from the various studies suggest that a consistent pattern for residual disease disappearance over many months exists for patients who remain in extended complete remission and a pattern of residual disease persistence and reappearance preceding clinical findings exists for the majority of those who ultimately relapse in the bone marrow. PMID- 8624458 TI - Clinical implication of the integration patterns of human T-cell lymphotropic virus type I proviral DNA in adult T-cell leukemia/lymphoma. AB - In this review, we discuss the possible relationship between the clinical characteristics and the integration patterns of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA in patients with adult T-cell leukemia/ lymphoma (ATL). Some ATL patients show unusual integration patterns such as multiple or defective HTLV-I and have clinical characteristics unlike those of most ATL patients who have the characteristic integration pattern of one complete provirus. Multiple HTLV-I integrations can be detected as two or more bands using the standard Southern blotting method when the tumor cellular DNA is digested with an endonuclease that does not cleave within the provirus. This includes cases of one tumor cell clone carrying two or more copies of the provirus, or alternatively two or more cell clones, each carrying one copy of the provirus. The former group of patients always manifest severe dyspnea and hypoxemia with unusual organ infiltrations including the retina and muscle and an extremely aggressive clinical course. On the other hand, the latter group of patients have an indolent course with skin lesions or small T lymphocytes with cleaved or lobulated nuclei. A solitary defective HTLV-I in some ATL patients can be detected as one smaller band after digestion of cellular DNA with an endonuclease that does not cleave within the provirus. These patients generally have a favourable clinical course with small cleaved or bilobulated T lymphocytes without lymphadenopathy or skin lesions. These findings suggest that there are clinical implications for the integration patterns of HTLV-I and this may be one of the explanations for the heterogeneous behaviour of the disease. Such studies may provide information on the relationship between virus integration and the clinical manifestations and also improve our understanding of the pathogenesis of ATL. PMID- 8624459 TI - CD45 (leucocyte common antigen) expression in T and B lymphocyte subsets. AB - CD45 is the dominant tyrosine phosphatase in haematopoietic cells and can modulate the effects of many other signaling molecules by dephosphorylation. The extracellular portion of CD45 has considerable variability due to differential splicing and glycosylation. This may allow for interactions with a variety of ligands expressed on interacting cells or on the same cell surface. Monoclonal anti CD45 antibodies that are reactive with epitopes that result from differential splicing and glycosylation can distinguish between cell populations that differ in maturation and function. These reagents can be used in the immunophenotyping of hematopoietic malignancies as well as in immunodeficiencies and autoimmune diseases. Several studies have shown that different anti CD45 reagents have different activating or inhibiting effects in vitro on a variety of T and B cell activation events. There are some indications that anti CD45 reagents can also selectively modify lymphocyte function in vivo. Such applications could potentially allow for the selective upregulation and down regulation of lymphocyte functions in a variety of immunologically mediated diseases. PMID- 8624460 TI - New insights into the regulation of ICAM-1 gene expression. AB - Cell-cell adhesion is critical in the generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) is an inducible cell surface glycoprotein expressed at a low level on a subpopulation of hematopoietic cells, vascular endothelium, fibroblasts, and certain epithelial cells. However, its expression is dramatically increased at sites of inflammation, providing important means of regulating cell-cell interactions and thereby inflammatory responses. Inasmuch the modulation of ICAM-1 expression during inflammation by pharmacologic agents might be very attractive for medical treatment, the intracellular regulatory elements and signaling pathways underlying the inducible expression of ICAM-1 by proinflammatory cytokines remain largely unknown. In this review, a novel posttranscriptional regulation of ICAM-1 gene expression by two inflammatory mediators, interferon-gamma and phorbol myristate acetate, and the possible role of the serine/threonine phosphorylation pathway in the cycloheximide-induced ICAM-1 message stabilization are discussed in light of our current understanding of ICAM-1 gene regulation during an inflammatory response. PMID- 8624461 TI - Pulmonary involvement in lymphoma. AB - Intrathoracic involvement is common in both Hodgkin's disease (HD) and non Hodgkin's lymphoma (NHL). The most common manifestation is mediastinal lymphadenopathy. In HD, nodal involvement is by contiguity and usually involves the superior mediastinum, while the findings in NHL are more variable. Pulmonary parenchymal disease occurs in 38% of HD and 24% of NHL. In untreated HD, parenchymal involvement is invariably associated with mediastinal lymphadenopathy and often with widespread disease. Three distinct radiological patterns of pulmonary lymphoma are recognised: nodular, bronchovascular-lymphangitic and pneumonic-alveolar. Rarely lymphoma may be endobronchial. Pleural effusion occurs in 16% of lymphoma patients and is usually associated with disease elsewhere. It is frequently caused by lymphatic obstruction but may be due to direct pleural involvement by tumour. Chylothorax may occur in NHL but is unusual in HD. Diagnosis of intrathoracic lymphoma is by transbronchial or transthoracic biopsy or by needle aspiration of tissue or pleural fluid. The addition of immunostaining improves the diagnostic yield in equivocal cases. Treatment and prognosis vary depending on cell-type, location and extent of disease. PMID- 8624462 TI - Value of PCR analysis for long term survivors after allogeneic bone marrow transplant for chronic myelogenous leukemia: a comparative study. AB - At present, allogeneic bone marrow transplantation (BMT) seems to be the only curative treatment for patients with chronic myeloid leukemia (CML). The mechanisms of this therapeutic approach probably include anti-leukemic immune response or "graft-versus-leukemia" (GVL) reaction against leukemic cell clones, since even aggressive chemotherapy is not sufficient per se to cure this type of disease. In many patients, allogeneic BMT results in the disappearance of the Ph chromosome; this negativity does not exclude the presence of residual leukemic cells since sensitivity of cytogenetic analysis is low. The chimeric BCR-ABL gene in CML is a tumor-specific marker that is well suited to allowing detection of low numbers of residual leukemic cells through the extremely sensitive detection method of polymerase chain reaction (PCR). Using this method, 1 leukemic cell in 10(5) or 10(6) normal cells can be detected. Many studies have evaluated the clinical significance and predictive value of BCR-ABL gene rearrangement detected by PCR assay after BMT, most often on heterogeneous populations. Results from such studies have been conflicting. We have conducted a review of the literature, focussing on long-term survivors (> 3 years from BMT) of CML to determine the value of PCR in such patients (n = 183). With the consideration that such a population is obviously heterogenous, long-term PCR negativity strongly correlated with achieving a cure since no patient relapsed in this population (n = 117). Among the PCR positive patients (n = 66), only 5 (8%) relapsed, suggesting a poor prognostic value for a PCR+ test > 36 months post-transplant. Besides the potential clinical value of these data, they contribute to the understanding of the biology of the disease and may suggest a crucial role for the long term GVL effect of marrow transplantation. PMID- 8624463 TI - Chronic natural killer cell lymphocytosis. AB - Chronic proliferations of natural killer (NK) cells (CD3- CD16+) are identified initially by detecting large granular lymphocyte (LGL) excess in a peripheral blood smear and subsequent lymphocyte immunophenotyping by flow cytometry. A related disease, T-LGL leukemia, has an indolent clinical course with chronic neutropenia and a close association with rheumatoid arthritis. Herein are described the clinical presentation and long-term clinical course of patients with chronic NK cell lymphocytosis (CNKL). The majority of the 14 patients followed up for a median of 4 years presented with severe cytopenias or vasculitic syndromes that were responsive to immunosuppressive therapy. Other manifestations included fever and arthralgias. In general, the disease was nonprogressive and had a course similar to that of T-LGL leukemia. PMID- 8624464 TI - High dose cyclophosphamide, fractionated total body irradiation with involved field irradiation and autologous bone marrow transplantation in malignant lymphoma. AB - The objective of this clinical trial was to determine if radiation to areas of recurrence or bulky disease prior to total body irradiation (TBI) and chemotherapy followed by autologous bone marrow transplantation (ABMT) altered the site of relapse and/or prolonged survival. Forty-eight patients with recurrent or refractory malignant lymphoma were treated with high-dose cyclophosphamide and fractionated TBI followed by ABMT. Thirty-four patients were eligible to receive involved field radiation therapy (IF-RT) to sites of recurrence or bulky disease. The overall response rate in 46 evaluable patients was 89% with 33 complete remissions (CR) and 8 partial remissions (PR). In a multivariant analysis increasing LDH, decreased serum albumin, older age, and lack of sensitivity to prior chemotherapy were associated with poorer survival. There were 10 deaths due to treatment related complications, 8 died of pulmonary complications of whom 6 were in CR. Of 11 who had received IF-RT and subsequently relapsed, 4 recurred in or adjacent to the involved field. We conclude that intensive chemo-radiotherapy proved to be an effective salvage therapy for patients with recurrent malignant lymphoma, resulting in a projected actuarial 33% DFS at 5 years, but was associated with a high transplant-related mortality. PMID- 8624465 TI - High-dose therapy and autologous bone marrow transplantation in first complete or partial remission for poor prognosis Hodgkin's disease. AB - We report the experience of three French centres which evaluated high-dose therapy (HDT) as consolidation therapy for poor prognosis Hodgkin's disease (HD). From March 1986 to April 1990, 23 consecutive patients with poor prognosis stage IV HD underwent HDT followed by autologous bone marrow transplantation (ABMT) after achieving either complete remission (CR1) or good partial response (GPR1) (reduction mass> 75%). The median age was 31 years (range 18 to 55 years), 14 were male. All patients except one initially had at least 2 poor prognosis factors such as: systemic symptoms (n = 19), bulky tumor (n = 16), more than one extranodal site (n = 9), bone marrow involvement (n = 5), lymphocyte count < or = 1.10(9)/1 (n = 8) and biological stage B (n = 21). All patients had previously been treated with alternating MOPP/ABVD. Ten patients were in GPR1 and 13 in CR1 before transplant. The conditioning regimens were: CBV (n = 17), BEAM (n = 5), BEAC (n = 1) followed by bone marrow rescue. Radiotherapy was introduced just before the conditioning regimen for 6 patients or after ABMT for 5 patients. Nine of 10 patients in GPR1 achieved CR after ABMT but one died early of treatment related toxicity. Five of 22 patients who were in CR posttransplant, relapsed (3, 4, 4, 18, 36 months). Seventeen patients remain alive in continuous CR with a median follow-up of 60 months (range: 30-100 months). The overall survival (OS) and disease-free survival (DFS) projected at 5 years are 92% and 77% respectively. Consolidation by HDT and ABMT proved to be well tolerated. An international trial is currently underway to attempt to demonstrate a clear benefit on survival for this subset of poor prognosis HD patients. PMID- 8624466 TI - Isochromosome i(3q) and premature chromosome condensation are recurrent findings in chronic B-cell lymphocytosis with binucleated lymphocytes. AB - Chronic B lymphocytosis with binucleated lymphocytes (LWBL) is a recently described entity. The lymphocytosis is stable over years and atypical binucleated lymphocytes are detected on peripheral blood smears. Further investigation has shown a predominance in females, a polyclonal increase in serum IgM and HLA-DR7 expression in most cases. In almost all cases cytogenetic studies with classical culture conditions have not shown any abnormality. We describe 7 patients with LWBL associated with an abnormal karyotype. An additional i(3q) was found in 6 cases. Premature chromosome condensation (PCC) was detected in all 7 cases. As both abnormalities are rarely present in other benign or malignant proliferations, we suggest a strong correlation between LWBL and i(3q) and/or PCC. PMID- 8624467 TI - Chronic B-cell lymphocytosis with binucleated lymphocytes (LWBL): a review of 38 cases. AB - LWBL is characterized by persistent and stable lymphocytosis, seen in but not exclusively female patients, with binucleated lymphocytes evident on peripheral blood smears. There is a polyclonal increase of serum IgM and in all cases immunophenotyping revealed a polyclonal expansion of B-lymphocytes. Whether this syndrome represents a premalignant disease process or a true polyclonal lymphocytosis remains unsettled. The occasional reports of clonal Ig rearrangements in this disorder suggest that in a minority of cases the polyclonal expansion may be followed by the emergence of one predominant clone. The demonstration of a clinically, benign, clonal lymphoid population is well recognised in several T cell disorders such as in the syndrome of large granular lymphocytes expansion. The benign clinical course and the lack of biological evolution in the majority of cases suggest that the importance of recognition of this disorder lies in avoiding aggressive therapy in these cases. A careful follow-up of these patients is mandatory and prospective immunological and genetic studies performed at different stages of the disease may well clarify this issue, particularly since most patients were HLA-DR7 positive. PMID- 8624469 TI - Memory T cell subsets in B cell non-Hodgkin's lymphomas. AB - Memory T cells were quantitated by three color flow cytometry in cell suspensions from biopsy specimens of 34 B cell non-Hodgkin's lymphomas (NHL) and 10 benign lymphoid hyperplasias (BLH). CD3+CD45R0-CD45RA+ (naive), CD3+CD45R0+CDRA- (memory) and total CD3+ T cells were compared in BLH, low grade (LG), intermediate (IG) and high grade (HG) B cell NHL. Mean percentage +/- s.e. of CD45R0 + T cells for BLH, LG, IG and HG NHL were: 14.7 +/- 3.8, 11.2 +/- 3.5, 28.9 +/- 7.5 and 45 +/- 15, respectively. Mean percentage +/- s.e. of CD45RA+ T cells were: 10.2 +/- 2.6, 7.1 +/- 2.3, 5.4 +/- 1.4 and 3.5 +/- 1.2, respectively. Mean percentage +/- s.e. of total CD3+ T cells were: 42.5 +/- 11, 22.4 +/- 7.5, 39.3 +/- 10.1 and 62.7 +/- 22.2, respectively. Memory and total T cells progressively increased from LG toward IG and HG NHL while naive T cells simultaneously decreased. Although the differences in naive T cells were non significant, the differences in memory T cells were significant between LG and IG and LG and HG NHL (both p < .0100). We previously reported an increase in activated-cytotoxic T cells in IG -HG B cell NHL. We now report that this subset of lymphocytes has also acquired memory function. Future studies should determine if in vitro expansion and adoptive transfer of this subset of T cells results in tumor cytolysis. PMID- 8624468 TI - Complete nucleotide sequence of Ig V genes in three cases of Burkitt lymphoma associated with AIDS. AB - To investigate the role of polyclonal stimulation and antigen driven selection in the pathogenesis of acquired immunodeficiency syndrome (AIDS) related lymphomas, we studied the variable region nucleotide sequence of heavy (VH) and light (VL) chains expressed by 3 Burkitt lymphomas (BL) associated with HIV infection. Two cases expressed the VH3-30P1 gene with 88.6% and 86.7% homology when compared to their germinal counterpart, whereas the VH4-18 was rearranged in the third one (89% identity). All these genes displayed high numbers of mutations (27, 22, 28 respectively), predominating in CDR regions. The encoded light chain genes determined for cases 1 and 2 expressed the same V kappa I-018 gene. These results indicate that: 1) Although, it is difficult to address the issue of VH usage based on the limited number of cases studied, Burkitt's lymphoma associated with AIDS may use a restricted repertoire of Ig genes. 2) Mutations and/or replacements predominated in CDR regions, which might suggest the occurrence of an antigen driven selection process, at least in some AIDS associated lymphomas. However, the high ratio of mutations observed in framework (FW) regions also favors the possibility that the antigen selection process is associated with polyclonal B cell stimulation. PMID- 8624470 TI - Clinical characteristics of non-Hodgkin's lymphoma as a second primary tumor: a population-based survey. AB - Data from 29,845 patients with lymphomas, 981 of whom had lymphoma as a second primary tumor, registered in the Surveillance Epidemiology and End Results (SEER) program in the U.S.A. between 1973 and 1986 were analyzed. The characteristics of the 274 patients with lymphoma as a second tumor who had received chemotherapy and/or radiotherapy for their primary tumor (SEP PT) were compared with 675 patients with second primary lymphomas who had no prior treatment (SEP NT) and with patients with single lymphomas (SIP). Patients with SEP PT disease had a significantly higher percentage of intermediate and high-grade tumors (80%) compared to those with SEP NT or SIP tumors (73% and 72%, respectively). The survival of all patients with SIP tumors did not differ from those with SEP tumors, but the median survival of those with SEP PT disease was shorter than for SEP NT disease. This was most probably due to the high percentage of intermediate and high-grade lymphomas in the SEP PT group. This was statistically significant in the group treated by combined chemotherapy and radiotherapy but not in those treated by a single modality alone. These findings relating to a worse prognosis in patients with SEP PT lymphoma are in line with our previous observations of a poor survival in patients with other multiple primary tumors. PMID- 8624471 TI - Aggressive non-Hodgkin's lymphoma: long-term results of full dose CHOP chemotherapy, and analysis of prognostic determinant. AB - Prognostic factors for complete remission rate (CR), duration of remission and survival were analysed in a series of 150 patients with intermediate (diffuse large-cell) and high grade non-Hodgkin's lymphoma treated with full doses of CHOP over a 10 year period with a minimum follow-up of 5 years. The two factors which significantly predicted for CR were normal LDH level and absence of bone marrow involvement. For duration of CR, the only significant factor was disease bulk, while for duration of survival age, LDH and B-symptoms were all significant. 48 patients were alive and in CR at 7 years or more with a projected long term (10 years or >) overall survival of 55 percent. PMID- 8624472 TI - Survival of childhood acute lymphoblastic leukemia: results of therapy at Tata Memorial Hospital, Bombay, India. AB - The purpose of this study was to analyze the outcome of patients who completed therapy for acute lymphoblastic leukemia (ALL) and to study the role of an aggressive induction regimen in preventing post therapy relapses. Four hundred and twenty-two patients with ALL who completed therapy during the period 1975 1991 were followed. Two hundred and sixty patients received the aggressive MCP 841 protocol and 162 patients received various other less aggressive treatment regimens. Patients were followed with periodic examination and complete blood counts. The incidence of post therapy relapse was 27% in the less aggressive protocols and 15% in the MCP 841 protocol (p = 0.001). An higher percentage of relapses was seen in males (p = 0.05) and 89% relapses occurred within two years of stopping therapy. The relapse rate after 5 years of cessation of therapy was 0.59%. In conclusion, aggressive induction therapy is the most crucial factor in predicting relapses following cessation of therapy in ALL patients. However, relapses are unlikely to occur five years post therapy. PMID- 8624473 TI - Treatment of adult patients with acute lymphocytic leukemia in relapse. AB - Thirty-eight patients with a diagnosis of relapsed acute lymphocytic leukemia were accrued to a treatment program of reinduction therapy by the Eastern Cooperative Oncology Group (ECOG). A combination of mitoxantrone, etoposide (VP 16), and high-dose cytarabine (ARA-C) were administered over a five day period. Thirty-four patients were eligible for follow-up subsequent to treatment. Twenty seven patients were in first relapse and seven were in second relapse. Fifteen of the thirty-four patients treated were given two cycles of induction chemotherapy. The complete remission (CR) rate for the entire group treated was 17%. The median duration of the CR was 2.4 months and the estimated median survival for first relapse patients was 4.5 months and 5.0 months for second relapse patient group. There were five deaths attributable to toxicity associated with the chemotherapy. The study emphasizes the difficulty in achieving durable remissions in adult patients with relapsed ALL. PMID- 8624474 TI - Hematopoietic growth factor (G-CSF or GM-CSF) after salvage chemotherapy in refractory or relapsed adult de novo acute leukemia. AB - Nineteen adults with primary refractory or relapsed acute leukemia (12 ALL and 7 ANLL) were treated with an intensive salvage chemotherapy (intermediate-dose ara C, intermediate-dose methotrexate, vindesine, cyclophosphamide, mitoxantrone and prednisolone) followed by a hematopoietic growth factor (HGF), either granulocyte colony-stimulating factor (5 micrograms/kg) or granulocyte-macrophage colony stimulating factor (10 micrograms/kg). Both were given from the day after chemotherapy ended and until the neutrophil count rose above 1 X 10(9)/l for three consecutive days. Eleven patients (58%, 95% CI 33% to 82%) achieved complete remission, and 15 courses of salvage therapy were given to these complete responders. In a historical control group that did not receive HGF, 23 out of 38 patients (60%, 95% CI 44% to 77%) achieved complete remission, and 27 courses of therapy were delivered to complete responders. Treatment with a HGF accelerated the recovery of neutrophils to 0.5 X 10(9)/l significantly, shortening it from a mean of 28 to 22 days (p = .0002), with no effect on platelet recovery. There were no differences in the rates of documented and fatal infections, which were relatively high in both groups. In the patients with ANLL, there was no evidence that HGF accelerated leukemic regrowth. We conclude that HGF accelerates neutrophilic recovery following intensive salvage chemotherapy for acute leukemia, although no reduction in documented infections was found. Many factors, including the small patient sample, may have contributed to this latter finding. PMID- 8624475 TI - Autotransplantation of peripheral blood stem cells mobilized by G-CSF in hematological malignancies: evidence for rapid and long-term sustained hematopoietic reconstitution. AB - We assessed the ability of granulocyte colony-stimulating factor (G-CSF) to mobilize peripheral blood stem cells (PBSC), the efficacy of PBSC infusion in the rescue of hematopoiesis after high-dose chemotherapy in 22 adult patients with hematological malignancies, and the long-term quality of engraftment. Bolus subcutaneous injections of G-CSF (12 micrograms/kg/day) were administered for 6 days. A median of three leukaphereses resulted in the collection of a median of 5.45 X 10(8) mononuclear cells/kg, 4.52 X 10(6) CD34+ cells/kg, and 3.97 X 10(4) CFU-GM/kg. G-CSF (5 micrograms/kg daily) was administered after PBSC infusion until granulocyte recovery. The median time to attain a neutrophil level > 0.5 X 10(9)/L and a platelet count > 20 X 10(9)/ L was 11 days. The median follow-up in 17 survivors was 23.8 months. These patients have maintained a complete and stable graft and some of them with neoplastic recurrence tolerated further chemotherapy. These results confirm that mobilization of PBSC by G-CSF can be performed on an outpatient setting and used in heavily pretreated patients. G-CSF mobilized PBSC transplantation provides early and complete engraftment in most patients. PMID- 8624476 TI - Bone marrow involvement, in intensively treated patients with intermediate grade non-Hodgkin's lymphoma, is a risk factor for granulocytopenia and fever. AB - Intensive combination chemotherapy administered to patients with non-Hodgkin's lymphoma (NHL) is inevitably associated with neutropenia and fever. However, the subset of patients who are at increased risk for myelotoxicity has not been clearly identified as yet. We evaluated 58 patients with NHL in order to verify whether bone marrow involvement (BMI) at diagnosis is a risk factor for neutropenia and infection following intensive chemotherapy. Patients with bone marrow involvement had significantly lower neutrophil counts both at diagnosis and following chemotherapy. An higher percentage of patients with BMI developed neutropenia (< 1000/mm3) following chemotherapy. Furthermore events of febrile neutropenia were more frequently encountered in patients with BMI. We conclude that BMI in patients with NHL is a risk factor for chemotherapy induced neutropenia and fever. The role of colony stimulating factors, administered in an attempt to prevent this complication, should be explored. PMID- 8624477 TI - Fatal vascular leak syndrome with extensive hemorrhage, peripheral neuropathy and reactive erythrophagocytosis: an unusual complication of recombinant IL-3 therapy. AB - A 39-year-old patient with severe aplastic anemia (AA), resistant to therapy, received recombinant human IL-3 (rhIL-3) on a phase I/II trial. During treatment she developed disseminated skin lesions, suggestive of vasculitis, and severe progressive peripheral neuropathy culminating in complete paralysis. She died 25 days after beginning treatment from profuse bleeding. On autopsy, evidence of vascular leaks with widespread bleeding and extensive hemorrhagic involvement of peripheral nerves was found. An additional feature was massive reactive erythrophagocytosis in lymph nodes, spleen and bone marrow. The coincidence between rhIL-3 administration and the dramatic events suggest a causal relation. As a possible pathogenic mechanism, an rhIL-3 induced excessive stimulation of macrophages and production of secondary cytokines such as tumor necrosis factor (TNF) is suggested. TNF is considered as a major factor in the development of both a vascular leak and reactive erythrophagocytosis. This case report can be regarded as an example of the possible unusual pathologic phenomena we may expect to see in the near future with increasing use of growth factors. PMID- 8624478 TI - Progressive thrombosis after treatment of diffuse large cell non-Hodgkin's lymphoma and concomitant lupus anticoagulant. AB - We report a case of diffuse large cell non-Hodgkin's lymphoma with concomitant lupus anticoagulant at initial diagnosis. Progressive thrombosis occurred despite radiologically proven response of the lymphoma after chemotherapy treatment. Extraordinary bone scintigraphy with multiple "cold" lesions probably due to bone ischemia is described. PMID- 8624479 TI - Transformation of essential thrombocythaemia to T cell acute lymphoblastic leukaemia. AB - Leukaemic transformation of essential thrombocythaemia is a rare event and is usually associated with previous treatment with either alkylating agents or radioactive phosphorous. We describe a patient with essential thrombocythaemia who developed an acute leukaemia of T cell phenotype following hydroxyurea therapy. The T cell phenotype of the blasts suggests the target cell for leukaemic transformation was a pluripotential stem cell. PMID- 8624480 TI - Spontaneous regression of chemotherapy-refractory non-Hodgkin's lymphoma preceding the development of secondary leukaemia. AB - In this report a rare case of spontaneous regression of a long-standing chemotherapy resistant low grade non-Hodgkin's lymphoma (NHL) of follicular small cleaved cell type is described. Extensive bulky lymphadenopathy substantially resolved, 7 months before the diagnosis of acute myelomonocytic leukaemia was established, in the absence of further treatment. It is possible that in this case the mutagenic effects of alkylating agents given during the course of the NHL reprogrammed lineage commitment in an early progenitor cell, deviating potential B-lineage cells along the myelomonocytic pathway. PMID- 8624481 TI - Remission induction in acute myeloid leukemia by granulocyte colony- stimulating factor: differentiation or apoptosis? PMID- 8624482 TI - Endothelin-1 production and endothelin converting enzyme expression by guinea pig airway epithelial cells. AB - Significant amount of endothelin-1 (ET-1) production and endothelin converting enzyme (ECE) activity have been detected in cultured guinea pig airway epithelial cells. Subsequent screening of the cDNA library constructed from epithelial cells, a complete cDNA of guinea pig ECE and a partial cDNA encoding preproET-1 were newly isolated. The production of ET-1 from epithelial cells was influenced by some vasoactive or inflammatory mediators, especially LPS enhances ET-1 production. Northern blot analysis revealed that mRNA level of ET-1 was increased in the LPS stimulated epithelial cells, but the effect on the ECE mRNA expression was obscure. These data suggest that the ET-1 production in the airway epithelial cells is regulated by the level of ET-1 mRNA expression with a constitutive manner, and the level of ECE, which act at a key biosynthetic step of the conversion of big ET-1 to ET-2, seems not to make a major contribution to the regulation of ET-1 release. PMID- 8624483 TI - Purification and some properties of two phospholipases and a toxin from the venom of desert cobra (Walterinnesia aegyptia). AB - An acidic and a basic phospholipase A (PLA) and a toxin have been purified from the venom of W. aegyptia using a TSK G 3000 SW gel filtration column and a Mono Q ion-exchange column. Both phospholipases and the toxin were found to be in a homogeneous state on SDS-PAGE and their purity was verified by isoelectric focusing. The acidic PLA showed higher enzymatic activity and concentration in venom when compared to basic PLA. The acidic and the basic PLA showed 16.5 +/- 0.5 Kd and 14.5 +/- 0.5 Kd molecular weights respectively, while the toxin showed 12.0 +/- 0.5 Kd molecular weights on SDS-PAGE. The isoelectric points for the acidic and basic PLA were at pH 4.5 and 7.8 respectively. The toxin was focused in the basic region at pH 10. Both phospholipases constitute about 20% of the venom protein and were nontoxic. However, only basic PLA when mixed with the toxin reduced the time required by the toxin alone to kill the mice, while the toxin was highly lethal to mice and its LD50 was 0.4 micrograms/g of body weight of mouse. The toxin showed on phospholipase activity. PMID- 8624484 TI - Dimethyl sulfoxide rather than superoxide is the reactive species in horseradish peroxidase--KO2/dimethyl sulfoxide system. AB - It has been shown that dimethyl sulfoxide competes with the enzyme substrate towards horseradish peroxidase compound I, but probably not towards horseradish peroxidase compound II. Dimethyl sulfoxide may hinder the reaction of superoxide with horseradish peroxidase compound II by modifying heme center. In this connection dimethyl sulfoxide is not recommended as a solvent in catalytic studies of horseradish peroxidase. PMID- 8624485 TI - Vitamin D--related modification of enzyme activities in synaptosomes and mitochondria isolated from rat cerebral cortex. AB - We previously demonstrated that feeding rats the Steenbock and Black rickets inducing diet produces remarkable changes in the metabolic pattern of intestinal mucosa, kidney, liver, cerebral cortex and heart. We have now determined the levels of calcium, phosphorus and citrate in cerebral cortex and the activity of some enzymes in synaptosomes and cerebral cortex mitochondria of three rat groups: control (Group A), fed a vitamin D-deficient diet (Group B), fed a vitamin D-deficient diet and treated with 1,25-dihydroxyvitamin D3 (Group C). While calcium content increased in Groups B and C, phosphorus concentration increased only in Group C and citrate in Group B in comparison with control. The increase in acetylcholinesterase and citrate synthase registered in Group B was restored to control values by 1,25-dihydroxyvitamin D3 treatment, while, neither the decrease in cytochrome c oxidase, nor the increase in glucose-6-phosphate dehydrogenase, acid phosphatase and NADP+(-)isocitrate dehydrogenase observed in Group B were corrected by 1,25-dihydroxyvitamin D3 supply. Acyl phosphatase showed a remarkable increase in consequence of 1,25-dihydroxyvitamin D3 administration. PMID- 8624486 TI - In vitro inhibition of rat heart sarcoplasmic reticular membrane ATPase activities by amphotericin B and their reversal by fructose-1,6-diphosphate. AB - The effect of amphotericin B on rat heart sarcoplasmic reticular membrane Na(+) K+ and Ca2+ ATPase activities in vitro was investigated. Amphotericin B in selected concentrations of 100-1000 ng significantly inhibited the sarcoplasmic reticular membrane ATPase activities studied. Fructose-1,6-diphosphate (1000 microM concentration) completely reversed the inhibition of Ca2+ ATPase activity in particular, but failed to reverse that of Na+(-)K+ATPase activities at 1000 microM concentration. Fructose-1,6-diphosphate may afford some protection against 1000 ng amphotericin B-induced myocardial toxicity. These damages may vary depending upon the dose of amphotericin B used in experimental studies. PMID- 8624487 TI - High DNA-binding activity of transcription factor NF-kappa B in synovial membranes of patients with rheumatoid arthritis. AB - Our objective was to clarify the DNA-binding activity of transcription factor NF kappa B as related to cytokine induction in the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Synovial membranes were obtained during arthroplasty of the knee from 7 patients with RA and 4 patients with OA. Nuclear extracts obtained from the synovial membranes were examined by electrophoretic mobility shift assay to determine the DNA-binding activity of NF kappa B. Markedly high DNA-binding activity of NF-kappa B was detected in the synovial membranes of RA patients, while virtually no activity was observed in those of OA patients. These results suggest that the high induction of NF-kappa B in the nuclear extracts may be relatively specific to synovial membranes in RA. NF-kappa B may regulate the production of cytokines at the site of synovial inflammation. PMID- 8624488 TI - Changes in cell growth, cyclin/kinase, endogenous phosphoproteins and nm23 gene expression in human prostatic JCA-1 cells treated with modified citrus pectin. AB - Modified citrus pectin (MCP) added to the media of cultured androgen-independent human prostatic JCA-1 cells reduced cell growth and correspondingly [3H]thymidine incorporation into DNA, which was correlated with the down-regulation of cyclin B and p34cdc2 MCP also induced distinct increases in specific endogenous phosphoproteins, including a cAMP-stimulated 52,000 (52-kDa) protein. Since metastatis has been inversely correlated with nm23 gene expression in some cancer cells and was reportedly inhibited by MCP in a rat prostate model, we investigated steady state level changes in the nm23 protein in JCA-1 cells and found it to be unexpectedly suppressed as a result of exposure to MCP. PMID- 8624489 TI - Temperature induced interstrand crosslinks in cisplatin-DNA adducts detected by electrophoresis and UV spectrophotometer. AB - Thermal melting profiles of DNA samples complexed with the anti-tumor drug cisplatin exhibit significant hypochromicity at pre-melting temperatures while a reduction in total hyperchromicity is observed in the melting region. Densitometric analysis of agarose gel electrophoresis pattern of DNA-cisplatin adducts heated to different temperatures in the pre-melting region and frozen in their conformations reveals a gradual retardation of mobility as the temperature increases. We attribute these results to a temperature induced transition in the mode of binding of cisplatin to DNA from an initial intrastrand monofunctional binding to bifunctional interstrand crosslink formation which results in gradual bending of the helix. PMID- 8624490 TI - Pest sequences in EF-hand calcium-binding proteins. AB - Calcium binding proteins are subdivided into two major families: the EF-family and the Annexin family. The EF-hand family is distinguished by the characteristic helix-loop-helix motif which consists of two alpha-helices separated by a loop. The EF-hand Ca2+(-)binding protein family contain subfamilies rich in proline, glutamic acid, serine and threonine residues (called PEST sequences) and non PEST containing subfamilies. A few of the Ca2+(-)binding proteins contain KFERQ-like sequences which are thought to be possible signals for lysosomal degradation. Arginine pairs (RR) which have also been suggested to act as signals for proteolysis were found to be few or absent. However, most EF-hand proteins were found to possess lysine pairs (KK) which may also act as signals for proteolysis. PMID- 8624491 TI - Strain differences of liver aldehyde oxidase activity in rats. AB - The present study demonstrated a significant variation of liver aldehyde oxidase activity in twelve strains of rats. The highest activity was found with Sea:SD rats and the lowest one with WKA/Sea rats. When assayed using benzaldehyde as a substrate, the difference in the activity between the two strains was 63.5-fold. Little significant differences in Km values of the enzyme were observed among several strains. On the other hand, Western blot analysis for the enzyme suggested that the strains possess different quantities of the enzyme, which are roughly parallel to aldehyde oxidase activity observed in the strains. PMID- 8624492 TI - Buffering and activity coefficient of intracellular free magnesium concentration in human erythrocytes. AB - Human erythrocytes either untreated or Mg2+(-)loaded by means of A23187 in the presence of 3 or 12 mM MgCl2 were haemolysed by freezing and thawing. In the haemolysates the concentration of total Mg2+ (by atomic absorption spectrophotometry), free Mg2+ (by a Mg2+(-)sensitive electrode) and ATP were determined. The increase in the free Mg2+ concentration was also measured when titrating the haemolysates with MgCl2. The experiments yielded a total Mg2+ buffer capacity of 4.85mM with a Ka of 0.9 mM-1, indicating that 2,3 bisphosphoglycerate was the main Mg2+ buffer in haemolysates of erythrocytes which had been ATP-depleted during preparation. The activity coefficient of free Mg2+ in erythrocytes was the same as in the chloride-based calibration solutions. PMID- 8624493 TI - The interaction between "R/S domain" of rat 28S ribosomal RNA and ribosome inactivating proteins investigated by fusidic acid. AB - The interaction between "R/S domain" of rat 28S rRNA and ribosome-inactivating proteins (RIPs) has been studied by blocking the action site of RIPs on "R/S domain" of 28S rRNA with fusidic acid or S100. Fusidic acid alone could form stable complexes with rat ribosomes and block the action site of RNA N glycosidases. incubation of fusidic acid and S100 or GDP with ribosomes could also protect ribosomes from the action of ricin A-chain. However, different effect of fusidic acid, S100 an dGDP was observed when alpha-sarcin was used. Fusidic acid alone could not block the action site of alpha-sarcin. Fusidic acid together with the elongation factors in S100 could block the action site of alpha sarcin. These results are consistent with the previous report that ricin A-chain and alpha-sarcin recognized different conformation of "R/S domain" in rat 28S rRNA. PMID- 8624494 TI - Intrinsic mutagenicity and electrophilicity of 1-sulfooxy-3-methylcholanthrene: implications for metabolic activation of the carcinogen 3-methylcholanthrene. AB - Hydroxylation of a meso-anthracenic carbon atom with subsequent formation of a reactive ester bearing a good leaving group (e.g., sulfate) has been proposed as a possible biochemical mechanism responsible for DNA binding, mutagenicity and tumorigenicity of 3-methylcholanthrene, one of the most potent carcinogenic polycyclic aromatic hydrocarbons in experimental animals. In support of this supposition, the chemically synthesized sulfuric acid ester, 1-sulfooxy-3 methylcholanthrene (1-SMC) was directly mutagenic in bacteria and covalently bound to DNA without metabolic activation. The intrinsic mutagenicity of this reactive ester was significantly potentiated by addition of extra acetate or chloride anions to the media. Reduced glutathione and ascorbic acid protected against 1-SMC-induced mutagenesis. These findings suggest 1-SMC as a potential ultimate electrophilic and tumorigenic metabolite of 3-methylcholanthrene. PMID- 8624495 TI - Formation of inclusion bodies may be the key factor for the stability of expressed products in E. coli. AB - Des-B30 single-chain insulin gene was constructed from chemically synthesized DNA encoding human proinsulin by oligo-nucleotide induced deletion, cloned into the expression plasmid pBV220, and expressed in E. coli with a level of around 5%. The expressed product was in the form of inclusion bodies. After downstream processing, 45 mg of des-B30 single-chain insulin with a purity of up to 90% was obtained from 1 liter of high density fermentation medium. It is suggested that the fusion protein model to increase the stability of small proteins seems inadequate to some extent, the formation of inclusion bodies may be the key factor for the stability of some expressed products, large or small, in the cytoplasm of E. coli cells. PMID- 8624496 TI - What determines the antioxidant potential of yeast cells? AB - Reactivity which organic radicals was compared in yeast (Saccharomyces cerevisiae) strains defective in catalase and superoxide dismutase, and with a decreased level of glutathione. Yeast cell homogenates did not show considerable strain-related differences in the ability to scavenge nitroxide (TEMPO) stable free radicals and alkoxyl free radicals generated by decomposition of the free radical initiator AAPH. The "total antioxidant status" based on scavenging of ABTS free radicals showed a good correlation with the radiation resistance of the yeasts. These results point to the importance of other factors, apart from antioxidative enzymes and glutathione, in the determination of cellular resistance to ionizing radiation and other types of free-radical stress. PMID- 8624497 TI - An unknown hydrolase activity of human serum albumin: beta-lactamase activity. AB - The interaction of a chromogenic cephalosporin, 3-(2, 4 dinitrostyryl)-(6 R, 7 R) -7(2 thienylacetamido)-ceph 3-em-5-carboxylic acid) (nitrocefin) with human serum albumin was studied. Purified human albumin was showed to have a hydrolase activity, catalyzing the decomposition of the chromogenic cephalosporin. The dimeric and trimeric form of albumin also showed this hydrolase activity. The presence of the following amino acid residues at the catalytic site of albumin was demonstrated: tyrosyls 199 and 411, tryptophan 214, lysyls 195, 225 and 240, histidyl 146, also some argynyls were involucrated. PMID- 8624498 TI - Prostate specific antigen-alpha 2-macroglobulin complexes in prostate cancer patient sera. AB - Quantitative immunoblotting of prostate cancer patient sera revealed that most prostate specific antigen was in complexes with alpha 1-antichymotrypsin or alpha 2-macroglobulin with little of it being free antigen. Complexes of prostate specific antigen with these protease inhibitors in patient sera comigrated during electrophoresis with the respective purified complexes. Each complex was selectively removed from patient sera by absorption with specific antibodies. When prostate specific antigen was added to normal plasma, complexes with alpha 2 macroglobulin appeared first and after 1 hr, the distribution was approximately 40% free antigen, approximately 40% complexes with alpha 2-macroglobulin, and approximately 20% complexes with alpha 1-antichymotrypsin. These data show that prostate specific antigen reacts more readily with alpha 2-macroglobulin than with any other protease inhibitor in plasma and that the antigen complexes with alpha 2-macroglobulin in vivo in cancer patients. PMID- 8624499 TI - 1,1'-Ethylidenebis[L-tryptophan], an impurity in L-tryptophan associated with eosinophilia-myalgia syndrome, stimulates type I collagen gene expression in human fibroblasts in vitro. AB - Eosinophilia-myalgia syndrome (EMS), a recently described inflammatory disorder characterized by myalgia, peripheral eosinophilia, and multisystem inflammation is associated with L-tryptophan consumption. Fibrosis of various tissues due to excessive accumulation of type I collagen is a prominent late manifestation of the syndrome. 1,1'-Ethylidenebis[L-tryptophan] (EBT), an impurity distinct from L tryptophan found in case-associated lots, has been implicated in function in vitro. Incubation of confluent fibroblasts with EBT, but not its hydrolysis product 1-methyl-tetrahydro-beta-carboline-3-carboxylic acid, caused a dose dependent increase in collagen synthesis and in type I collagen mRNA levels independent of its effect on proliferation. In contrast, expression mRNA for fibronectin was not affected. These findings indicate that EBT stimulates type I collagen production by human fibroblast, and suggest that EBT may be involved in the development of fibrosis in EMS. PMID- 8624500 TI - Inhibition of Df-protease--induced kinin release by synthetic inhibitors. AB - A protease (Df-protease) from house dust mite (D. farinae) is closely associated with mite-induced allergy: Df-protease has a similar substrate specificity to blood coagulation factor XIIa and catalyzes the activation of kallikrein-kinin system in human plasma. With the purpose of prevention of kinin-formation in plasma by Df-protease, inhibition of Df-protease with synthetic inhibitors was tested in vivo and in vitro. Among the inhibitors, including amidine and guanidine derivatives, N-allyl-N-[4-(4-amidinophenoxycarbonyl)-alpha methylcinnamoyl++ +]glycine ethyl ester mesylate was the most effective to inhibit Df-protease with Ki = 9 x 10(-9) M and also to prevent kinin-release from Df-protease in human plasma. Enhancement of vascular permeability in guinea pigs caused by kinin-release was stoichiometrically suppressed by the inhibitor. PMID- 8624501 TI - Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells. AB - Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells. PMID- 8624502 TI - Nitrite-induced export of glutathione disulfide from erythrocytes. AB - Export of glutathione disulfide (GSSG) from human erythrocytes induced by several agents (H2O2, t-butyl hydroperoxide, phenazine methosulfate and NaNO2/) was compared. NaNO2 treatment was found optimal for induction of GSSG transport. GSSG was exported from NaNO2-treated erythrocytes mainly via the low-affinity kinetic component. The transport rate increased with increasing pH in the pH range of 6.5 8.0. The transport was inhibited by fluoride, vanadate and intracellular DNP-SG. On the basis of the obtained results, a possibility of existence of one common low-affinity transporter for glutathione S-conjugates and GSSG is discussed. PMID- 8624503 TI - Evaluation of antibody responses in Indian kala-azar by immunoblot. AB - When infected with Leishmania species, patients develop specific antibodies that constitute the basis of serodiagnosis. using Western blot analysis we studied the specificity of anti-leishmania donovani antibodies in patients with visceral leishmaniasis, healthy subjects living in an endemic and non-endemic areas, and patients of other infectious diseases like malaria, leprosy, tuberculosis and tropical splenomegaly. Sera from patients with kala-azar recognised numerous antigens that had a molecular weight of 150 KD, 145 KD, 120 KD, 92 KD, 87 KD, 72 KD, 65 KD, 56 KD, 50 KD, 40 KD, 26 KD, 21 KD, 14 KD, AND 12 KD. The 150, 145, 120, 92, 87, 81, 65, 25, 21, 14, and 12 KD antigens had the greatest specificity for kala-azar sera while the bands of molecular weights 72, 56, 50, and 40 KD were found to be cross reactive with sera of patients of other diseases. PMID- 8624504 TI - The comparative study of peroxidase activity and substrate binding properties of cytochrome P450 2B4 incorporated into phospholipid vesicles with the use of two different methods. AB - The comparative study of peroxidase activities and substrate binding properties of cytochrome p450 2B4 in reconstituted vesicles prepared with the use of two different techniques, and microsomal cytochrome P450 was carried out. The data obtained show that the two types of cytochrome P450 2B4-containing proteoliposomes do not differ substantially from each other with respect to H2O2- or cumene hydroperoxide-dependent substrate hydroxylation activities as well as substrate binding properties of hemoprotein reconstituted. However, some parameters measured with proteoliposomal cytochrome P450 are markedly different from those measured with microsomal hemoprotein, suggesting the existence of conformational differences between the molecules of these two cytochromes or the differences in the depth of their immersion into lipid bilayer. PMID- 8624505 TI - Peroxide complex of cytochrome bd: kinetics of generation and stability. AB - Hydrogen peroxide reacts with the isolated fully oxidized cytochrome bd from Escherichia coli bringing about spectral changes characterized by increased absorption at 680 nm, disappearance of a charge transfer band at 740 nm and a red shift in the Soret band. Only one type of spectral changes is observed throughout the entire range of H2O2 concentration studied, 5 - 5000 microM. The absorption changes are consistent with peroxide binding to heme d and do not show any evidence for reaction with heme b-595. The spectral response saturates at increased H2O2 concentration with apparent Kd of 30 microM and is reversed by catalase. Stopped-flow measurements show the reaction to be first order with respect to H2O2 with a second order rate constant Kon = 600M-1s-1. Decay of the H2O2-induced spectral changes upon addition of catalase (k approximately 0.001 s 1) is about 20-fold slower than expected for dissociation of peroxide from the complex with heme d assuming a simple reversible binding of H2O2 with Kd and Kon values give above (Koff = Kon). We suggest that the reaction of H2O2 with cytochrome bd may be in fact irreversible, the initial binding followed by a cleavage of the O-O bond and formation of the oxoferryl complex of heme d. Upon removal of excess peroxide, the oxoferryl compound could decay being reduced to the ferric state by endogenous reductants. PMID- 8624507 TI - E. coli D-glyceraldehyde-3-phosphate dehydrogenase modified by 2,3-butanedione: manifestation of a pairwise of non-equivalence of active centers. AB - Chemical modification of E. coli d-glyceraldehyde-3-phosphate dehydrogenase by an arginine-specific reagent, 2,3-butanedione, stabilized the tetrametric enzyme in an asymmetric state, with only two of the four active centers able to catalyze oxidative phosphorylation of D-glyceraldehyde-3-phosphate. The catalytically incompetent active centers retain the capacity of binding NAD+, forming charge transfer complex, and be alkylated by iodoacetamide. Analogous results have been previously obtained with the rabbit muscle D-glyceraldehyde dehydrogenase modified at a single arginine residue per subunit (Kuzminskaya, E.V., Asryants, R.A., and Nagradova, N.K. (1991) Biochim. Biophys. Acta 1075, 123-130), the only differences being inaccessibility of the catalytically incompetent pair of active centers to the alkylating reagent, on one hand, and lower residual activity exhibited by the functioning active centers (3-4%), on the other. In the case of E. coli enzyme, activity loss upon arginine modification never exceeded 80-82%. These results are consistent with the idea that the two enzymes share common principles of the protein design, but differ in the peculiarities of their active centers conformations. An improved method for D-glyceraldehyde-3-phosphate dehydrogenase purification from a wild type E. coli strain is described. PMID- 8624506 TI - Changes in enzyme levels in hypertensive heart tissue. AB - The levels of activity of some enzymes involved in oxidative metabolism have been determined in left ventricular tissue from spontaneously hypertensive rats compared with those in normotensive controls. Levels of pyruvate kinase were increased about 1.3 fold indicative of elevated glycolytic activity. Similarly, enhanced levels of lactate dehydrogenase were found, consistent with a requirement for increased oxidation of cytosolically-generated NADH. In addition a more active malate-aspartate shuttle, which in heart provides the major route for transfer of reducing equivalents to the mitochondria, was suggested by elevated levels of the cytosolic isoenzyme of aspartate aminotransferase; malate dehydrogenase did not increase but the activity of this enzyme is very high and unlikely to be rate-limiting in the shuttle. The levels of expression of mRNAs for three of these enzymes (pyruvate kinase, aspartate aminotransferase and malate dehydrogenase) were also determined and correlated well with the extent of change, if any, in the changes in enzymatic activity. Thus it seems that one response to development of hypertension in rats is an increase in expression of the genes for certain key enzymes involved in oxidative metabolism. PMID- 8624509 TI - Depth-dose distribution in bricks determined by thermoluminescence and by Monte Carlo calculation for external gamma-dose reconstruction. AB - The analysis of depth-dose distributions in bricks sampled from walls in areas with nuclear waste or accident contamination has the potential of providing information on the energy and source configuration of the gamma-radiation that had been incident on the brick. In this study, a brick from a mill facing a shallow water reservoir of the contaminated Techa river in the South Ural region is investigated. Thermoluminescence (TL) methods were used to measure the accumulated dose at several depths in the brick. The accidental external gamma dose is obtained by subtracting the natural radiation background dose from the total accumulated dose. In the first segment of the brick, at a depth of about 1.5 cm, the accident dose was found to be roughly 3.5 Gy. Monte-Carlo simulations of the photon transport from the reservoir bed contaminated with 137Cs were calculated for different depths in the brick. The calculations were made assuming different attenuating water levels. It is found that the depth-dose distribution determined by measurements corresponds to a water level between 20 and 50 cm. The results indicate that TL measurements combined with Monte-Carlo modelling calculations are highly promising for external gamma-dose reconstruction applications. PMID- 8624508 TI - Application of accelerator mass spectrometry (AMS) for high-sensitivity measurements of 14CO2 in long-term studies of fat metabolism. AB - Long-term measurements of 14C in CO2 expired after ingestion of 14C-labelled triolein were performed using accelerator mass spectrometry (AMS). About 30% of a given amount of 14C-labelled triolein was catabolized rapidly, while the remaining 70% had a very slow turnover. The study shows the potential of the AMS technique for the study of the long-term biokinetics of 14C-labelled pharmaceuticals. The AMS technique allows the administered activity to be reduced by several orders of magnitude without compromising the study. It may also allow studies of rare drug metabolites. PMID- 8624510 TI - Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2. AB - By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana, two dominant photomorphogenic mutants, shy1-1D and shy2-1D, for two genetic loci designated as SHY1 and SHY2 (suppressor of hy2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red-far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis. This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants. PMID- 8624511 TI - Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link site. AB - Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose 6) gel filtration assay of cross-linked extensin oligomers, a pl 4.6 extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross linking or CL-extensins and non-cross-linking or NCL-extensins. CL-extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated. However, the simplest motif common to CL-extensins but absent from NCL-extensins was Val Tyr-Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine. PMID- 8624512 TI - A sugar transporter from Medicago truncatula: altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations. AB - A cDNA clone encoding a hexose transporter has been isolated from a library prepared from Medicago truncatula roots colonized by the mycorrhizal fungus Glomus versiforme. The clone (Mtst1) represents a M. truncatula gene and expression studies in yeast indicate that the encoded protein transports glucose and fructose but not sucrose. Transcripts corresponding to Mtst1 are expressed in leaves, stems and roots of M. truncatula, with the highest levels of expression in roots. In the roots, Mtst1 transcripts were detected in two distinct locations; the phloem fiber cells of the vascular tissue, and the cells of the root tip. Mtst1 expression in the roots is regulated in response to colonization by G. versiforme; transcript levels increased two- to fourfold in both M. truncatula and M. sativa following colonization by G. versiforme but did not increase during the unsuccessful interaction between G. versiforme and a M. sativa myc- mutant, suggesting that the increase in Mtst1 transcripts in the successful mycorrhizal interaction is correlated with internal growth of the fungus and potentially with a functioning symbiosis. Mtst1 transcripts were also detected in the cortical cells of the mycorrhizal root, specifically in areas of the root that were highly colonized by the mycorrhizal fungus. Thus, the formation of a symbiotic association with a VA mycorrhizal fungus is accompanied by a change in the cell type-specific expression of a transporter that potentially functions to supply sugars to root cells critically involved in the symbiotic association. PMID- 8624513 TI - Regulation of a carotenoid biosynthesis gene promoter during plant development. AB - Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation. PMID- 8624514 TI - Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants. AB - Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB, that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F655, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F655. PMID- 8624515 TI - Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato. AB - The tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed. PMID- 8624516 TI - Coordinate transcriptional induction of myo-inositol metabolism during environmental stress. AB - The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10 fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences. PMID- 8624517 TI - Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis. AB - The maize abscisic acid (ABA) responsive gene rab28, has been shown to be ABA inducible in embryos and vegetative tissues. A polyclonal antiserum was raised against Rab28 protein. Using immunoblotting and immunoprecipitation, the antiserum specifically recognized a protein of about 30 kDa and pl 6 which is in close agreement with the molecular weight and pl predicted by the deduced amino acid sequence. The rab28 gene product accumulated during late embryogenesis. In vegetative tissues, dehydration stress induced rab28 gene expression both in the light and in the dark. The spatial and temporal pattern of rab28 mRNA expression during embryogenesis was investigated by in situ hybridization using digoxigenin labelled rab28 probes, and the immunochemical localization of Rab28 protein using anti-Rab28 antibodies. Expression of rab28 mRNA is restricted to provascular tissues in young embryos, and at later stages of development the most prevalent accumulation occurred in meristem and in the vascular elements of the plumule, root and scutellum. Using immunoelectron microscopy the Rab28 protein has been located in the nucleolus of different cell types. In light of these results the stress regulation of rab28 and a likely role for this protein during late embryogenesis are discussed. PMID- 8624518 TI - A protocol for obtaining embryogenic cell lines from Arabidopsis. AB - Embryogenic cell lines with lasting embryogenic potential can be obtained from somatic embryos induced directly from zygotic embryos of Arabidopsis thaliana, ecotype Columbia. The response to a critical concentration of auxin, which seems to be the all-important factor in the generation of embryogenic cell lines, is exhibited by somatic embryos but not by zygotic ones. The basis for this differential response remains obscure and will be discussed in relation to other systems. PMID- 8624519 TI - [Wermer syndrome associated with a bilateral renal tumor]. AB - Wermer's syndrome is a polyendocrinopathy characterized by neoplasia of the parathyroidis, pancreas and the pituitary. We report a case, unique in our knowledge, of an unusual association of a bilateral renal tumors and Wermer's syndrome. The originality of this case is: Bilateral renal blow. Therapeutic difficulties. This manifestation may represent a new manifestation of this pleiotropic syndrome. PMID- 8624520 TI - [Diagnostic value of Stamey's test in chronic prostatitis]. AB - OBJECTIVES: Analysis and technical details of Staomey's fractionated urine cultures, the reference method for the evaluation and bacteriological diagnosis of chronic bacterial prostatitis, but the application of which has been rarely described in the literature. METHOD: Stamey's test is based on comparative analysis of urine samples representative of the urethra, bladder and prostatic secretions obtained by prostatic massage. This method allows the demonstration of bacteria in the urine or prostatic secretions in the presence of bacterial prostatitis, in contra with non-bacterial prostatitis and prostatodynia. The sampling conditions require a sufficiently full bladder and the samples must be collected according to rigorously sterile procedure. The first step of the examination must not be preceded by urethral swabbing and the urine samples must have a well defined volume. Prostatic secretions are obtained by a prolonged massage of each lobe of the prostate gland. RESULTS: Bacterial prostatitis is confirmed by the presence of bacteria in the prostatic secretions and U3 in numbers largely exceeding the bacterial counts of the other samples. In the case of lower urinary tract infection, the test must be repeated after disinfection of the bladder urine. The pathogenic role of Gram positive bacteria is confirmed by isolation of a high bacterial count on several occasions. The pathogenic role of Ureaplasma urealyticum and Chlamydiae trachomatis is more controversial, while the role of Trichomonas vaginalis is unlikely and fungal prostatitis is very rare. Semen culture is less reliable than Stamey's test in the diagnosis of prostatitis. CONCLUSION: The diagnosis of chronic prostatitis is difficult due to the absence of typical clinical symptoms, specific ultrasonographic signs and the sometimes difficult interpretation of the culture results. Stamey's test is a reference diagnostic examination provided it is performed according to a rigorous methodology. PMID- 8624521 TI - [What to do when a Hautmann type cystoplasty "does not descend"?]. PMID- 8624522 TI - [Cadaver donor nephrectomy. Surgical technique in the context of multi-organ donation]. AB - 95% of renal transplantations performed in France use cadaver donor kidneys. Two donor nephrectomy techniques are proposed: a beating heart technique and an arrested heart technique. In the very great majority of cases (80%), donor nephrectomy can be performed during multi-organ removal, performed according to bioethical regulations: unrelated, anonymity between donor and recipient, security, traceability and evaluation. The urologist has a role to play at each step of organ donation, in which he is the main protagonist. The first steps are performed in close collaboration with the intensive care unit which established the diagnosis of brain death of the potential donor. The following steps, guided by the French transplant establishment, are the urologist's responsibility: He is responsible for abdominal exploration looking for a tumour or any other abnormality. A strictly aseptic technique is essential to prevent contamination of the organ. He is responsible for removing kidneys in such a way as to ensure the shortest possible warm ischaemia time, the best storage solution, and the best preservation of their anatomical structure. He must be familiar with the outcome of transplants in order to adapt his technique to the results of transplants. PMID- 8624524 TI - [Cooling shell in renal transplantation. Thermometric evaluation of a prototype]. AB - We have developed a cooling system for renal transplants designed to eliminate the second period of warm ischaemia corresponding to the vascular anastomosis phase of renal transplantation. This is an autonomous and independent system which forms a shell around the transplant. Following application of the system, cooling is achieved by refrigeration of a Multitherm sponge contained in the wall of the shell. The thermometric characteristics of a prototype were evaluated in vitro and in vivo in pigs. This system allows the kidney to be preserved at a temperature of less than 10 degrees C for 1 hour without inducing any risk of lesions of the renal surface. Human applications should be developed in the near future. PMID- 8624523 TI - [Hot flashes and hormonal treatment of prostate cancer]. AB - Androgen suppression in the context of the treatment of prostatic cancer is responsible for hot flashes in 75% of patients, which alter the quality of life to varying degrees depending on the patient. They constitute a source of major discomfort in 30 to 40% of patients. The pathophysiology of this effect is now known and involves: sex steroids, central opiates and intrahypothalamic catecholamines. The incidence of hot flashes appears to vary according to the type of hormonal treatment administered. The various treatments available are not equally effective. Non-hormonal treatments are of little value. Hormonal treatments: oestrogens and steroidal antiandrogens are the most effective. Progestogens also appear to be just as effective or even more effective than these other agents, with negligible adverse effects at the doses used in this indication. PMID- 8624525 TI - [Double kidney-total pancreas transplantation with bladder reimplantation. 25 cases]. AB - Twenty five double kidney-pancreas transplantations were performed according to the total pancreas transplantation technique with drainage of exocrine secretions into the bladder via a vesicoduodenostomy. 72% of kidney-pancreas grafts were functional at one year and 59% were functional at four years. The authors observed a slightly higher rejection rate (0.56 versus 0.34) and a higher incidence of urinary tract infection (60% versus 35%) following double pancreas and renal transplantation than after isolated renal transplantation. Complications were rare: two venous thromboses and two cases of urethritis requiring of rediversion of the duodenum into the intestine. These good results, comparable to those reported in the international registry, reflect the value of the pancreatic and renal transplantation technique using a total pancreas drained into the bladder. It would probably be preferable to transplant patients earlier, when chronic renal failure secondary to insulin-dependent diabetes induces end stage renal failure and the need for haemodialysis. PMID- 8624526 TI - [Combined renal-pancreas transplantation in the treatment for chronic renal insufficiency of diabetic origin . Results from the Pitie Urology Clinic]. AB - Between 1989 and 1995, 32 patients underwent combined kidney-pancreas transplantation for diabetic chronic renal failure. Only one of these patients received an isolated pancreas following cessation of function of a previously implanted segmental pancreas. The surgical technique always consisted of pure retroperitoneal transplantation into the right iliac fossa of a total pancreas transplant with duodenovesical anastomosis. The postoperative complications included one death on D10 from pulmonary vein thrombosis in a patient with sickle cell anaemia and early loss of the transplanted pancreas due to venous thrombosis. Nine patients underwent at least one surgical revision, due to a leaking duodenovesical anastomosis in 8 cases. With a mean follow-up of 33 +/- 20 months, the results demonstrate, apart from the early death indicated above, another death at 50 months of a patient who had lost his pancreas due to early venous thrombosis and who died with a functioning kidney. 23 of the 30 surviving patients have a functioning kidney and pancreas (79%), i.e. 74% of the total population of 32 patients. Loss of pancreatic function was surgical in two cases (one case of infection of the transplant site, one case of thrombosis), vascular in one case due to rupture of a mycotic aneurysm into the duodenum and immunological in three cases: two of these pancreases retained partial function allowing perfect blood glucose control with less than 10 units of ordinary insulin per day. Lastly, a perfectly functioning pancreas was removed 13 months after transplantation because of renal rejection not controlled by reinforced immunosuppression. Compared to the data of the international registry, these results demonstrate the value of the retroperitoneal approach used in this series and the improvement of the results obtained with increasing experience of the transplant team. PMID- 8624527 TI - [Metabolic investigation of calcium oxalate urinary lithiasis. Indicators of lithogenic risk: modalities, applications and perspectives]. AB - Despite exhaustive metabolic investigations, calcium oxalate urinary stones often remain idiopathic and, in the absence of any objective aetiopathogenic parameter, the prevention of recurrences and follow-up are particularly problematical. Various indicators have been elaborated to quantify, the lithogenic risk, based on various parameters potentially involved in the crystallization process. Their disparity illustrates the difficulty of achieving such an objective as well as the progress in fundamental concepts and their frequent technical complexity penalizes their practical application. The performances of these indices, except in the hands of the team which has proposed them, often lack reproducibility and paradoxical or even contradictory findings are frequent. They may simply reflect the variability of the lithogenic process or may reflect the predominant role of potent inhibitors, especially macromolecules. The metabolic investigation of calcium oxalate urinary stones is initially limited to detection of lithogenic diseases amenable to specific treatment. After repeating the basic laboratory tests, recurrences, especially when they are unexplained, require more complex investigations. At the present time, applications of lithogenic risk indices are essentially confined to the field of clinical and basic research. Among the various indicators available, the urinary citrate/urinary calcium ratio and Parks' index appear to be particularly promising. PMID- 8624528 TI - [General practitioners' approach to urination disorders in men over the age of 50. A survey of 250 physicians in Brittany]. AB - A questionnaire was set to 250 general practitioners in the Brittany region, to determine the impact of information campaigns concerning benign prostatic hyperplasia (BPH) and prostatic cancer on their everyday approach to disorders of micturition in men over the age of 50 years. 225 questionnaires were interpretable. 75% of general practitioners systematically investigated the presence of disorders of micturition. In the presence of such symptoms, 76% of general practitioners conducted further investigations to exclude prostatic cancer. 89% of general practitioners performed first-line digital rectal examination, and 34% of them systematically performed this examination once a year. The investigations most frequently performed after digital rectal examination concerned the state of urine (50%) but only 30% of general practitioners used reagent dip-sticks and 43% ordered urine cultures. Other investigations consisted of transrectal ultrasonography (31%) an PSA assay (26%). 3% of PSA assays were ordered before digital rectal examination. Renal and vesical ultrasonography occupied 5th place, although distension of the upper urinary apparatus was a source of concern for 51% of general practitioners. In the presence of symptomatic BPH, general practitioners readily prescribed medical treatment (96%). They assessed the results of this treatment on the course of disorders of micturition (95%), digital rectal examination (91%), and PSA (50%). General practitioners are familiar with disorders of micturition after the age of 50 years. However, 25% of them do not systematically question patients about these symptoms. Digital rectal examination is now performed more frequently, although systematic examination of the prostate is rare (34%). PSA assay is not the doctor's first priority (3rd place in the list of examinations). Two examinations are rarely used and should be developed: reagent dip-sticks (30%) and renal and vesical ultrasonography (5th place). Only 4% of general practitioners did not prescribe any treatment in the case of uncomplicated BPH, which is not in line with current recommendations. There is certainly a need for better information of general practitioners, but this information is only valid when a consensus has been reached among urologists. PMID- 8624529 TI - [Bladder suspension by retropubic endoscopy. Techniques and preliminary results (24 cases)]. AB - The authors propose an original technical based on the concept of bladder neck support by a sling (Goebell-Stoeckel) to treat urinary stress incontinence, without cystocele, in young women. The technical modifications concern: the incision: retropubic endoscopy facilitated by a dissection balloon and combined with a short vaginal incision. The use of synthetic material for the sling: expanded polytetrafluoroethylene (Gore-Tex) attached to Cooper's ligaments by a suture tied extracorporeally. The long-term objective is to achieve results comparable to those of open surgery with a lower morbidity. From 1992 to 1994, 24 patients were treated according to this technique by the same operator. The mean age was 48 years. In every case, this operation constituted the first procedure for incontinence, and only one patient had a history of previous pelvic surgery (Caesarean section). Incontinence was classified as stage 3 according to the Ingelmann Sundberg classification in 46% of cases. 35% of patients present uninhibited contractions, and 35% presented urethral hypotonia. Two intraoperative complications and immediate postoperative complications were only minor. The mean operating time was 2 hours 45 minutes. With experience, it gradually decreased, as did the hospital stay, which was an average of 4.3 days. With a short mean follow-up (1 year 7 days), the results were good in 71% of cases, satisfactory in 8% of cases (1 case of urgency, 1 case of persistent retention), with a failure in 5 cases (21%); 1 case of true incontinence confirmed by clinical examination and 4 cases of minor incontinence during occasional violent effort. PMID- 8624530 TI - [A single positive prostatic biopsy out of six systematic biopsies is not correlated with the intracapsular nature of the tumor on an individual level]. AB - OBJECTIVE: To evaluate whether or not a single positive prostatic biopsy out of six systematic ultrasound-guided biopsies, is reliably correlated manner with favourable histopathological features of the tumour on the radical prostatectomy (RP) specimen. MATERIALS AND METHODS: In a series of 158 patients undergoing RP for clinically localized prostatic cancer, 15.2% had only one positive biopsy out of 6 systematic biopsies. We compared the rates of capsular effraction (C+) and positive resection margins (RM+), assessed on the operative specimen, in this group of patients with a single positive biopsy (group 1) and in the group (group 2) diagnosed by more than one positive biopsy. The postoperative biological progression rate (P+), defined as an immediate or secondary postoperative elevation of PSA beyond 0.1 ng/ml by polyclonal assay, was also evaluated in the two groups. The Gleason score was evaluated and compared on biopsies and on RP specimens. RESULTS: 29.2 of cases were C+, 16.7% were RM+ and 26% were P+ in group 1, versus 70%, 46.5% and 49.5%, respectively, in group 2. All differences were statistically significant. All patients in group 1 with less than 10% of prostatic tissue invaded on the positive biopsy had stage P2, while all patients with 100% of the length of the biopsy invaded by tumour had stage P3. The Gleason score was accurately predicted by the positive biopsy in 39% of cases and was underestimated in 39% of cases. CONCLUSION: A single positive prostatic biopsy out of six systematic biopsies is a useful predictive factor of local extension, but, in the individual patient, does not guarantee favourable histopathological characteristics of the tumour, nor a favourable course of the disease. PMID- 8624531 TI - [Decisional criteria in the management of vesico-ureteral reflux in children with congenital neurogenic bladder]. AB - OBJECTIVES: To determine, in the particular case of neurogenic bladder, the best criteria for selection and grading of the modalities available for the treatment of vesicoureteric reflux, which is often associated. METHODS: 194 patients with congenital neurogenic bladder, including 76 cases with vesicoureteric reflux were retrospectively reviewed. The various treatments applied, their results and their complications were reviewed. Conclusions are drawn and proposals are made for the optimal management of vesicoureteric reflux in the particular context of congenital neurogenic bladder. RESULTS: This series included 8 grade I, 28 grade II, 49 grade III and 23 grade IV and V. In more than 50% of cases, reflux was detected on during routine assessment of congenital neurogenic bladder, demonstrating the value of this assessment (25% of cases of reflux were detected before the age of one year). Management initially consisted of vesical drainage, often by intermittent catheterization, from the age of 2 to 3 years and antibiotic prophylaxis. This treatment was sufficient to correct reflux in 9 cases and to control it in 16 cases (no urinary tract infection, no deterioration of the upper tract). An elective anti-reflux operation was decided in 69 cases of VUR (1 grade I, 15 grade II, 38 grade III, 16 grade IV or V). Cohen's technique was performed in 3 cases and endoscopic injection of PTFE was performed in 3 cases. Most of these refluxing bladders were hypertonic and/or presented an abnormally high closing pressure. The operative criteria were: symptomatic reflux; persistent reflux; renal deterioration; non-compliance with treatment. Several complications were observed and are described. CONCLUSION: Vesicoureteric reflux in the context of congenital neurogenic bladder plays a major role in the deterioration of renal function. These forms of reflux usually occur in hypertonic bladders. Urodynamic studies appear essential before deciding treatment. Intermittent catheterization is able to control or even correct a certain number of these cases of reflux and should be introduced by the age of 2 or 3 years. When surgery is required, Cohen's operation is the easiest to perform, but is associated with an increased complication rate. Injection of PTFE or macroplastic could constitute a good indication. PMID- 8624532 TI - [Experimental approach to reflex erection in rats: modeling and functional neuroanatomy of the involved nerve pathways]. AB - The neurophysiology of erection remains poorly elucidated, particularly at the spinal cord level. We studied variation of intracavernous pressure (ICP) in rats. Tactile stimulation of the prepuce in conscious rats induces the appearance of sequences of reflex erectile responses affecting the corpora cavernosa and/or corpus spongiosum and glans. During this test, implantation of a telemetric pressure transducer allowed us to record ICP increases occurring simultaneously to erectile responses. These increases were characterized by brief suprasystolic peaks preceded by an infrasystolic plateau. Each type of reflex erectile response was accompanied by a characteristic profile of ICP increase. Participation of the corpora cavernosa was demonstrated, particularly during erections of the glans. In anaesthetised rats, electrical stimulation of the dorsal nerve of the penis induces an increase of intracavernous pressure which tends to reach blood pressure values. This erectile response of the corpora cavernosa is abolished by proximal section of the dorsal nerve of the penis, markedly decreased by section of the homolateral pelvic nerve, and abolished after bilateral section of the two pelvic nerves. Striated muscle paralysis does not abolish the erectile response. These data help to define the neurophysiology of the reflex erection. In particular, they provide a clearer explanation of the spinal integration of reflex erection. PMID- 8624533 TI - [Ureterosigmoidostomy in adults: long term results]. AB - In 65 patients (pts) (130 renal units) with invasive bladder cancer treated at our institution from 1971 to 1992, ureterosigmoidostomy was performed. Early complications (< 3 month) occurred in 25 patients: 9 pts had a pyelonephritis, 3 pts underwent surgery for revision of the ureteral anastomosis because of leakage and 3 had abdominal wall revisions. 4 pts had a severe hyperchloremic metabolic acidosis, 2 pts had respiratory problems. Late complications observed were: 12 pts with pyelonephritis, 4 pts with ureteral stenosis and 9 pts with hyperchloremic metabolic acidosis. 5 patients were incontinent and 3 pts had a anastomotic colon cancer after 10, 12 and 17 years respectively. In 26/65 (40%) of patients with survival over 5 years continence and quality of life were evaluated by means of a questionnaire. 23/26 pts (88%) were continent during daytime and complete continence during the night was reported by 14/26 pts (54%). Quality of life was assessed in a global manner (family and social life, sexuality, comfort, travel and sport) and was rated as satisfactory in 24/26 pts (92%). 2 pts were unsatisfied due to diarrhea. In conclusion, good long-term functional results can be obtained with ureterosigmoidostomy with a careful follow-up. Our long-term results may serve as a basis for comparison with other more recently developed continent urinary diversions such as low pressure systems. PMID- 8624534 TI - [Antiandrogen withdrawal syndrome in the hormonal treatment of metastatic prostatic cancer in hormonal escape]. AB - A fall in the PSA level after stopping antiandrogens has been described at the stage of hormonal escape of prostatic cancer treated by complete androgen inhibition. The authors report a new case. The patient was offtially treated by pulpectomy and nitulamide for N+ prostatic carcinoma (PSA: 165 ng/ml). At the stage of hormonal escape, discontinuation of nitulamide induced a reduction of the PSA. Replacement of nitulamide by cytoproterone acetate was followed by a renewed increase of PSA, which again decreased after stomming cyproterone acetate. Three years later, the PSDA level was 3.5 ng/mg. This syndrome is probably due to mutation of the androgen receptor. In hormonal escape, suspension of all antiandrogens apart from LHRH analogues is recommended and can be followed by a temporary fall of PSA. No other antiandrogen must be administered in the place of the previous drug. PMID- 8624535 TI - [Nephronogenic nephroma: a benign kidney tumor in adults. Two case reports]. AB - The authors report 2 cases of a benign and uncommon type of renal tumour in adult, the nephronogenic nephroma. The pathology is discussed and compared to other groups of solid renal tumour. The confusion with malign tumour is usual. According to radiological investigations and pathological analysis, aspects of surgical treatment are presented and discussed. PMID- 8624536 TI - [The neurobiological bases of creativity]. PMID- 8624538 TI - [Vagovagal regulation of the gastrointestinal tract: from signal conduction to performance response]. PMID- 8624537 TI - [The cholinergic dependence of the cortical neuronal mechanism underlying the Pavlovian blinking conditioned reflex]. PMID- 8624539 TI - [The current level of the development of I. P. Pavlov's concept of digestion]. PMID- 8624540 TI - [The early studies of I. P. Pavlov (1874-1880): the innervation of the pancreas and salivary glands]. PMID- 8624541 TI - [The axon reflex: new views in an old area]. PMID- 8624542 TI - [Glutathione homeostasis: the cardinal physiological defense against oxygen toxicity induced by physical exercise]. PMID- 8624543 TI - [Pavlov's dog and Benzer's fly: the genetics of higher nervous activity]. PMID- 8624544 TI - [Brain substrates of the Pavlovian conditioning of discrete behavioral responses]. PMID- 8624545 TI - [I. P. Pavlov's Nobel Prize speech]. PMID- 8624546 TI - [The development of I. P. Pavlov's concepts of the food center]. PMID- 8624547 TI - [The temporal factors in behavioral response conditioning]. PMID- 8624548 TI - [The early studies of I. P. Pavlov: the neural regulation of the blood circulation (1875-1883)]. PMID- 8624549 TI - [The neural mechanisms of the autonomic responses evoked by somatic sensory stimulation]. PMID- 8624550 TI - [Food regulation of exocrine pancreatic secretion and bile secretion in swine]. PMID- 8624551 TI - [The role of subcortical structures in the conditioning process]. PMID- 8624552 TI - [From the psychic secretion of the digestive glands to the system of the neurosciences]. PMID- 8624553 TI - Influenza 1995/96. PMID- 8624554 TI - New insights into mechanisms of bacterial pathogenesis in periodontitis. AB - This review focuses on two topics that in the past decade have significantly enhanced our understanding of the mechanisms by which bacteria produce disease in humans: population structures of bacterial pathogens and tactics used by these bacteria to gain entry to mammalian cells. Studies in these areas have identified a number of virulence factors of some medically important pathogens. Very recent applications of this knowledge in efforts to understand mechanisms by which the most extensively studied "putative" periodontal pathogens, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, may contribute to the pathogenesis of periodontitis are presented. Knowledge of the population structures of these periodontopathic bacterial species and the mechanisms used by these bacteria to subvert host cell communication systems and functions will shape future approaches to diagnosis, prevention, and treatment of periodontal diseases. PMID- 8624555 TI - Surgical advances in the coverage of exposed roots. AB - A review of the past year's literature on the coverage of exposed roots reveals that surgical advances have centered on the use of membranes for guided tissue regeneration as well as surgical ingenuity combining subepithelial connective tissue with previously described root coverage techniques. In this review, the articles are critiqued, and their clinical significance is discussed. Surgical correction of exposed roots has become a remarkably successful and predictable procedure with either connective tissue or guided tissue regeneration. In most cases, connective tissue grafting for root coverage should be preferred to guided tissue regeneration, and the use of epithelialized palatal tissue (free gingival graft) for root coverage is a dated procedure. PMID- 8624556 TI - Evidence-based periodontal regenerative therapy. AB - Periodontal health care has progressed into a new era. It is distinguished by a rapidly expanding volume of literature, rapid influx of new technologies, and need for new skills to perform increasingly complex procedures. Correspondingly, practice management changes are required to adapt to the extensive follow-up care associated with some of these new treatments. This must be accomplished while also acknowledging the deepening concern for escalating costs and increased attention to the quality of care provided. As with most change, clinicians can fight it by continuing to rely on old ways of doing things and hope to keep these issues at bay, but history would say they are unlikely to succeed. Instead, clinicians can embrace these changes and adapt to them by adding new tools, such as evidence-based methodology, to their armamentarium. The evidence-based approach offers a "bridge" from science to clinical practice. It can strengthen the foundation by providing a framework for integrating patient preferences, scientific knowledge, clinical judgment, and personal experience. By adapting the way treatment decisions are made in daily practice to an evidence-based approach, clinicians can deliver the highest quality care to their patients and be in better control of their own destiny. These new challenges can be perceived as problems or as opportunities--it is a choice! PMID- 8624557 TI - Current status of periodontal microsurgery. AB - Periodontal microsurgery has proven to be an effective means of improving the predictability of gingival transplantation procedures used in treating recession. It also promises to make papillary reconstruction a realistic possibility in the hands of most periodontists. The use of microsurgical principles and techniques has been extended to flap reflection and suturing as an ideal means of having precise control of the gingiva without traumatizing the tissue by stretching, distorting, or tearing it. Microsurgical use requires training and practice using visual feedback rather than tactile feedback as is common in macrosurgery. It is an area of great interest and practical application for the growth and future of periodontics. PMID- 8624558 TI - Periodontal regeneration following surgical treatment. AB - Predictable and complete regeneration of lost periodontium remains an elusive goal, despite advances in surgical procedures and materials. Nevertheless, studies clearly demonstrate the potential for significant clinical improvements after regenerative therapy. Collectively, studies support the use of bone grafts and guided tissue regeneration (GTR) for the correction of intrabony and furcation defects. Results of several studies suggest the possibility of enhanced periodontal regeneration and enhanced stability following the use of combination techniques, such as GTR procedures with osseous grafts. Demineralized freeze dried bone allograft (DFDBA) remains the most widely used allogeneic graft material in periodontics. Recent evidence suggests that substantial variations exist in the osteoinductive potentials of available DFDBA material. The predictability and extent of periodontal regeneration are associated with defect morphology, compliance, plaque control, inflammation, bacterial colonization, and smoking. Long-term (3- to 5-year) studies suggest that improvements following periodontal regeneration remain stable provided that patients comply with oral hygiene regimens and regular supportive periodontal treatment. PMID- 8624559 TI - Barrier membranes in the treatment of periodontal defects. AB - Efficacious guided tissue regeneration for intrabony, furcation class II, and recession defects can be accomplished with both nonresorbable and bioresorbable barriers. The potential for regeneration of the periodontium is highly dependent on defect morphology and the availability of "progenitor cells." Many factors associated with surgical technique and barrier properties influence the regenerative outcome of guided tissue regeneration. Maintained flap coverage of the barrier minimizes epithelial down-growth as well as the risk of bacterial contamination of the barrier and the healing wound. Coverage of the newly regenerated tissues after removal of nonresorbable barriers is essential. The use of bioresorbable barriers eliminates a second operation for membrane removal and the associated potential trauma to the regenerating tissues. A stringent postoperative plaque control regimen is necessary during the healing period. The use of systemic antibiotics, prescribed concomitantly with insertion of the barriers has limited effect in controlling various pathogens and is therefore questionable. PMID- 8624560 TI - Polypeptide growth factors for periodontal regeneration. AB - The regeneration of periodontal attachment apparatus is particularly difficult to achieve, primarily because of the presence of many different kinds of tissue that must be restored to produce a functional unit. Traditional methods aimed at regenerating the periodontium have limited indications, and their results are not predictable. Recently, investigators have begun to understand the cellular processes necessary for repair and regeneration of periodontal tissues. Proteins called growth factors have been identified that coordinate these cellular events. The growth factors that may contribute to periodontal regeneration include platelet-derived growth factor, insulin-like growth factor, transforming growth factor-beta, and bone morphogenetic proteins. In vitro studies have demonstrated the positive effects of these factors on a number of cell types essential for periodontal regeneration. For instance, it has been shown that platelet-derived and insulin-like growth factors promote proliferation of osteoblasts an periodontal ligament cell-derived fibroblasts. Animal models have also been used to verify that growth factors can enhance regeneration in vivo following periodontal disease and as an adjunct to implant placement. In the future, human clinical trials will be required to identify the ideal growth factors, their proper doses, and the most suitable carrier system for them. PMID- 8624561 TI - Hypothetical considerations in the regenerative treatment of molar furcation defects. AB - Treatment of the molar furcation defect is a challenge to the dental professional. Responses of molar furcation sites to both closed and open surgical debridement have not been shown to result in significant clinical improvement or bone fill. Regenerative techniques using grafting materials, guided tissue regeneration with resorbable and nonresorbable membranes, coronally positioned flaps, and combination techniques are more frequently used to treat class II molar furcations. The clinical responses to these surgical approaches have shown varied results. This article reviews some of the studies on regeneration in furcation defects as well as some of the uniqueness and complexities of the furcation problem. The bone morphology differences in molar bifurcation and trifurcation defects are discussed, and a bone morphologic classification is presented in an effort to standardize future regenerative studies in class II furcations. PMID- 8624562 TI - Guided bone regeneration for dental implants. AB - The application of barrier membranes to promote bone regeneration was first described by Hurley et al. (J Bone Joint Surg 1959, 41A:1243-1254) in orthopedic research. However, the clinical potential of this membrane technique was recognized in the early 1980s for periodontal regeneration. Based on promising results in periodontology, researchers started to evaluate the potential of this technique--often called guided bone regeneration (GBR)--to regenerate bone defects in the alveolar process. This review describes the current knowledge of GBR in implant dentistry. Emphasis is placed on the scientific basis of GBR and the various surgical factors necessary to achieve predictable results with GBR procedures. In addition, unanswered questions that require future research are addressed including long-term success rates of dental implants placed in combination with barrier membranes, evaluation of resorbable membranes, and use of bone substitutes or growth factors to enhance bone regeneration in membrane protected defects. PMID- 8624563 TI - The sinus elevation procedure in endosseous implant therapy. AB - Since the initial application of sinus lift augmentation and implant placement in the mid-1970s, there have been some variations in technique used and graft material applied. Although most authors continue to use the lateral wall, Caldwell-Luc approach, less invasive procedures such as the osteotome procedure for sinus elevation, graft, and implant placement have been proposed. Many authors use iliac crest bone that is of both a cancellous and cortical nature. However, due to the high morbidity rate of this procedure, other donor sources are explored in this review. Good success rates are shown with intraoral bone and combinations of intraoral bone with both resorbable and nonresorbable materials. Varying results are reported with demineralized freeze-dried bone, although its use in combination with hydroxyapatite seems to improve its effectiveness. All reports of clinical success are relatively short term (6 years to 6 months). Medication recommendations and posttreatment prosthetic care are covered. PMID- 8624564 TI - Treatment of orofacial pain and temporomandibular disorders. AB - Recent advances and research in the etiology and clinical treatment of orofacial pain and temporomandibular disorders are reviewed. PMID- 8624565 TI - Effects of systemic diseases on the periodontium. AB - A number of systemic diseases have been associated with manifestations on the periodontal tissues, the nature and severity of which may vary considerably. Two main categories of systemic diseases are covered in this report: those that may influence the initiation, progression, and severity of plaque-induced periodontal disease, and those that affect the periodontal tissues regardless of the presence of periodontal (i.e., inflammatory) disease. The category of systemic diseases that alter the liability to plaque-related disease includes primary immunodeficiencies, AIDS, diabetes mellitus, congenital disorders, and Crohn's disease. The second category includes systemic diseases that affect mainly the gingivae, causing enlargement, ulceration, red and white lesions, and hyperpigmentation. PMID- 8624566 TI - Osteoporosis and its relationship to oral bone loss. AB - Osteoporosis, an age-related condition, is classified into primary and secondary types. Primary osteoporosis encompasses the postmenopausal and senile types; secondary osteoporosis occurs "secondary" to endocrine and renal diseases. Subjects affected by osteoporosis have an overall reduced bone mass and become highly susceptible to bone fractures. Dual energy x-ray absorptiometry is the method most often used to determine the risk for osteoporotic fractures. In the past decade, a number of studies have suggested a possible correlation between systemic osteoporosis and alveolar bone loss in periodontal disease pathogenesis. It appears that a clear correlation between periodontal health and the general mineral status of the skeleton is still lacking. This review addresses the pathogenesis of osteoporosis and emphasizes the multifactorial nature of bone loss. The current concepts in alveolar bone loss resulting from osteoporosis and its implications as a risk factor in periodontal disease development are also presented. PMID- 8624567 TI - Epidemiology of periodontal diseases. AB - This review summarizes findings from 46 reports selected from over 130 epidemiologic studies published in 1993 and 1994. Recent findings from longitudinal studies are consistent with the concept that periodontal destruction proceeds in random bursts at specific sites and demonstrate that some individuals have an elevated risk of attachment loss. These and other studies provide further evidence that cigarette smoking is a significant risk factor. New studies have quantified the risk of tooth loss due to periodontal disease, and there is better knowledge about HIV-associated periodontal diseases. Familial studies suggest that both environmental and genetic factors contribute to individual variations in etiologic factors (such as plaque) and periodontal diseases. Several studies propose an intriguing link between periodontal diseases and coronary heart disease, which may be mediated through risk factors common to both diseases, and as a direct consequence of the contribution of periodontal bacteria and their products to atherosclerosis. PMID- 8624569 TI - Early-onset periodontitis. AB - The group of periodontal diseases known as the early-onset periodontal diseases are defined by the age of onset of periodontal destruction, distribution of lesions, association of disease with specific microbial infections, and identification of characteristic alterations, in the host response. Significant progress has recently been made in our understanding of the etiology of juvenile and rapidly progressive periodontitis. Considerable evidence points to a familial pattern of disease; both localized and generalized forms of disease may be observed in the same family. The exact mode of inheritance remains unclear, and disease may be the result of a complex interplay between genetically determined alterations of the host response and a specific bacterial challenge. Both neutrophil function and immunoglobulin response are altered and appear to be characteristic of an immunologic hyperresponsiveness. Bacterial colonization by Actinobacillus actinomycetemcomitans (serotype b) and/or Porphyromonas gingivalis appears to be the primary initiator of disease. Evidence suggests that lesion distribution may be a function of the nature of the infecting agent and the characteristics of the immune response. PMID- 8624568 TI - Bone cell interactions and regulation by inflammatory mediators. AB - The loss of alveolar bone tissue associated with periodontal disease appears to be related to local factors that alter the physiologic balance between bone formation and resorption. Physiologic bone metabolism requires the coupled activities of bone resorption and bone formation. Recent investigations of this coupling phenomenon have been directed at understanding the regulatory mechanisms involved in osteoblast-osteoclast interactions. These regulatory mechanisms utilize multiple cytokines, which function in context with numerous other factors, including cell-cell interactions, extracellular matrix molecules, and levels of differentiation. Local alterations of these factors may disrupt the physiologic balance in bone metabolism and lead to the pathologic loss of alveolar bone. Future strategies for altering bone loss found in the periodontal diseases will be directed at modulating the balance of bone resorption and bone formation at the local site. PMID- 8624570 TI - Radiographic diagnosis of periodontal disease progression. AB - This review covers papers on the use of radiographs to assist in the assessment of periodontal disease progression, choice of treatment, and evaluation of treatment outcome, both in practice and clinical research. It reviews the latest papers on radiographic prescription strategies, the average quality of radiographs in daily practice, the potential of new developments, and the clinical applications of digital subtraction. The review ends with some interesting reports on the effect of smoking on the periodontal tissues, the risk of periodontal disease progression to teeth with periapical pathology, remodeling characteristics after guided tissue regeneration, and the effect of implant placement on the periodontium of neighboring teeth, all of which include conventional radiographic evaluations as major parameters in the protocols. In general, there is a striking discrepancy between the quality of radiographs taken in daily practice and the valuable information that could be gained from properly applied radiographic procedures. Furthermore, improved education in this field is urgently needed to raise the quality of radiographic diagnosis to acceptable standards. PMID- 8624571 TI - Identifying losing sites at periodontal reevaluation. AB - Our present method for clinical evaluation of marginal periodontal tissues involves examination for bleeding and suppuration and the measurement of probing depth. There is little doubt that this method provides useful guidelines for evaluation of the overall periodontal status of dentitions. On an individual-site basis, however, it has limitations in the identification of locations prone to disease progression. So far, there is no reliably clinical, microbiologic, or other method to predict or identify sites with disease activity (i.e., ongoing loss of attachment). Therefore, there are no clear guidelines for distinguishing between active and arrested lesions. A treatment resulting in a plaque-free mouth with nonbleeding and shallow probing depths throughout the dentition will reduce the risk of residual, active disease. Such a result, although desirable, is not realistic for many patients. Therefore, the clinician is faced with the delicate task of evaluating sites with various grades of involvement using current clinical methods of examination. PMID- 8624572 TI - The status, future, and problems of oral antiseptics. AB - Caries and periodontal disease, the most widespread oral diseases, are commonly treated with various oral antiseptics. These antiseptics are derived from different chemical categories and have different mechanisms of action. Even though the properties of an ideal oral antiseptic have been identified, its formulation has proven elusive. Recent studies with chlorhexidine, triclosan, an amine and stannous fluoride rinse, and essential oil rinse, and others are discussed. Development of novel antiseptic products continues. The hope for the future is that now antiseptic products will treat oral disease better and oral health will improve. PMID- 8624574 TI - Mucogingival considerations for the orthodontic patient. AB - Recession of the gingival margin during or subsequent to orthodontic therapy may occur as either pseudorecession or true recession. Assessment of both predisposing and possible precipitating factors will assist in evaluating the need for surgical intervention. Unfortunately, several parameters cannot be evaluated by objective measures. The clinician must address both objective and subjective criteria. Factors predisposing a site to recession are elucidated by evaluating the anatomy of the area, including the width of keratinized and attached gingiva, the facial-to-lingual dimension of both soft tissue and alveolus, and the position and angulation of the teeth in question. Marginal inflammation must be controlled to define the mucogingival junction necessary for these anatomic evaluations. Precipitating factors relate to the amount of trauma imposed on the marginal tissues. A definition of trauma would vary from direct trauma such as laceration to plaque-derived inflammation. Treatment that affects either trauma or the anatomy could enhance or reduce the potential for recession. Because the situation is dynamic, multiple evaluations are needed. If an area is to be observed, the presence of adequate donor and recipient sites for root coverage grafting techniques should be assessed to determine the potential for successful grafting in the event recession occurs. PMID- 8624573 TI - Matrix metalloproteinases and their inhibition in periodontal treatment. AB - Matrix metalloproteinases (MMPs), produced by both infiltrating and resident cells of the periodontium, play a role in physiologic (e.g., tooth eruption) and pathologic (e.g., periodontitis) events. The evidence for the role of MMPs in periodontal destruction has accumulated over three and a half decades, and it is now recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. This understanding has stimulated the search for a number of synthetic inhibitors that could be used as potential therapeutic agents. Tetracycline analogues, as proteinase inhibitors, are currently closer to being used clinically than any other agents in periodontal therapy. Other MMP inhibitors, such as Batimastat (British Bio-technology, Oxford, UK), are currently being tested clinically for inhibition of cancer metastasis and other diseases. Multiple mechanisms of MMP inhibition by tetracycline analogues have been proposed. The nonantimicrobial chemically modified tetracyclines are potent inhibitors of MMPs, preventing collagen breakdown and alveolar bone loss in animal models of periodontitis. In addition, nonantimicrobial low doses of the commercially available semisynthetic tetracycline doxycycline, have been used in human clinical trails to reduce MMP activity in the gingival crevicular fluid and gingival tissues, with a resultant reduction in pocket depth and attachment loss. Of particular interest, periodontal research on the nonantimicrobial properties of tetracycline has generated new therapeutic approaches to a variety of medical disorders characterized by excess MMP activity. PMID- 8624576 TI - Expression of the monoclonal antibody HECA-452 defined E-selectin ligands in Langerhans cell histiocytosis. AB - The cutaneous lymphocyte associated antigen (CLA) recognized by the monoclonal antibody (moAb) HECA-452 plays a major role in the homing of lymphocyte subpopulations to the skin by binding to E-selectin on dermal microvessels. The factors responsible for the immigration of Langerhans cells (LC) and their precursors into the skin are still unknown, but because normal resting LC are also capable of expressing CLA, the antigen was proposed as a candidate LC-homing structure. To gain insight into these mechanisms, the expression of HECA-452 on neoplastic LC within and outside the skin was investigated in paraffin-embedded sections from 44 patients with localized and disseminated forms of Langerhans cell histiocytosis (LCH) presenting with proliferating cells positive for CD45, CD1a, S100 and HLA-DR. Irrespective of the clinical presentation or the type of organ involved, HECA-452 positive LC were detected in all biopsies tested (range 5->90%). The most prominent HECA-452 reactivity was observed in skin lesions and in areas with accumulations of eosinophilic granulocytes. Our data provide evidence for heterogeneous expression of sLex/sLea structures in various stages of activated and/or differentiated LCH cells. Remarkably, CLA-antigen expression on LCH-cells was not restricted to cutaneous sites. In view of recent findings on the expression of HECA-452 on resting epidermal LC, our data are compatible with the view that local cytokine production by keratinocytes or cells from the surrounding infiltrate induce and/or modulate CLA expression on LC in both cutaneous and extra-cutaneous sites. PMID- 8624577 TI - Antigastric autoantibodies in Helicobacter pylori gastritis: prevalence, in-situ binding sites and clues for clinical relevance. AB - Colonization of human gastric mucosa with Helicobacter pylori leads to chronic active gastritis and induces the occurrence of an acquired mucosa-associated lymphoid tissue (MALT) in the stomach. This remodelling of the gastric mucosa together with chronic antigen persistence may induce autoimmune reactions. The aim of this study was to investigate humoral autoimmune reactions to human gastric mucosa in H. pylori gastritis and their clinical relevance. Sera from patients with dyspeptic symptoms were tested for presence of IgG immunoglobulins against H. pylori. Gastric infection with H. pylori and alterations of gastric mucosa were demonstrated by histological examination of gastric biopsy specimens. All sera were tested for reactivity against human gastric mucosa by immunohistochemistry. Two different in-situ binding sites of antigastric autoantibodies were observed. Binding to canalicular structures within parietal cells was significantly correlated with antibodies to H. pylori, elevated basal gastrin levels and atrophy of gastric corpus glands. Our data indicate that autoimmune reactions to antigens in the human gastric mucosa occur in H. pylori gastritis and that they may play a role in the pathogenesis of the disease. PMID- 8624579 TI - Expression of vimentin, cytokeratin, and desmin in Sertoli cells of human fetal, cryptorchid, and tumour-adjacent testicular tissue. AB - The intermediate filament of mature human Sertoli cells is vimentin. A co expression of vimentin together with cytokeratin has been demonstrated in Sertoli cells during embryonal development and under pathologic conditions in adult testes. We analysed the presence of vimentin, cytokeratin, and desmin in Sertoli cells of fetal testes (n=20), in seminiferous tubules of cryptorchid testes (n=10) and adjacent to testicular germ cell tumours (n=47) using specific monoclonal antibodies and single and double-labelling immunohistochemistry. During embryonal development prominent cytokeratin expression disappears after the 20th week of gestation. Interestingly, we also found desmin in immature intratubular Sertoli cells between weeks 11 and 14. In adult cryptorchid testes and in peritumour tubules, desmin was also prominently present in Sertoli cells in the vast majority of the cases investigated, as well as vimentin and cytokeratin co-expression. This first description of desmin immunoreactivity may shed some light on the ontogeny of human Sertoli cells and demonstrates that this cell type is able to express three types of intermediate filaments in a complex manner. PMID- 8624578 TI - Prognostic relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in gastric cancer. AB - Expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was evaluated in 125 surgically resected gastric cancers by immunohistochemical analysis. Tissue was stained immunohistochemically with a monoclonal antibody against human uPA and monoclonal antibodies against human PAI-1 and PAI-2. In addition, DNA ploidy patterns were determined by cytofluorometer after staining with propidium iodide. We found that 82 (66%) of the 125 gastric cancers expressed uPA as diffuse cytoplasmic staining, as intensely outlined luminal borders. PAI-1 expression was observed in 62 (50%) of 125 gastric cancer as a fine, diffuse and granular pattern in the cytoplasm. PAI-2 expression was observed in 65 (52%) of the 125 gastric cancers as a diffuse cytoplasmic staining. uPA-positive tumours showed a higher incidence of infiltration, lymph node metastasis and peritoneal dissemination that uPA-negative ones. Patients with uPA-positive tumours proved to have a significantly poorer prognosis than those with negative ones. PAI-1 negative tumours showed a higher incidence of liver metastasis and carried a poorer prognosis than PAI-1-positive ones. There was no significant correlation between uPA or PAI-1 expression and DNA ploidy patterns. Conversely, there was no significant relationship between PAI-2 expression and clinicopathological parameters and prognosis. According to the expression of uPA and PAI-1 status, groups of 19 uPA(-)/PAI-1(-), 44 uPA(+)/PAI-1(-), 23 uPA(-)/PAI-1(+) and 39 uPA(+)/PAI-1(+) were subdivided. Tumours with uPA(+)/PAI-1(-) had a significantly higher incidence of liver metastasis, lymph node metastasis and serosal invasion than the other groups of tumours. Patients with uPA(+)/PAI-1(-) tumours had a significantly poorer prognosis than those with uPA(-)/PAI-1(+) tumours. These results indicate that uPA expression is a useful biological prognostic indicator, and that uPA expression is a useful biological prognostic indicator, and that uPA and PAI-1 may play an important part in the tumour progression and metastasis in gastric cancer. PMID- 8624575 TI - The role of the microcirculation in multiple organ dysfunction syndrome (MODS): a review and perspective. AB - Major advances in intensive care medicine during the past two decades have altered the spectrum of disease encountered by intensive care physicians, anaesthesiologists, traumatologists and pathologists. One of the most important manifestations of severe trauma or infections is the multiple organ dysfunction syndrome (MODS), a life-threatening condition that often ends in multiple organ failure (MOF) and death. Evidence gathered from clinical and morphological observations in humans, taken together with experimental animal studies and a vast accumulation of in vitro data, clearly indicate that the microcirculation lies at the centre of this complex process, which results in peripheral vascular insufficiency, inadequate oxygen delivery to vital organs, and hence, severe organ dysfunction. The multifunctional nature of the endothelium makes it a prime candidate for study of the pathomechanisms of MODS. This paper reviews the evidence for the hypothesis that the microcirculation, and in particular its endothelial component, has a central role in the pathogenesis of MODS. The evidence is reviewed principally from the standpoints of classical morbid anatomy and cell pathobiology. PMID- 8624580 TI - Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy. AB - Viral infection, especially by enteroviruses, has been considered to be the most common cause of myocarditis, which may progress to dilated cardiomyopathy (DCM). Although the mechanism of progression remains uncertain, a cytokine-associated injury of myocytes has been proposed. Using reverse transcriptase polymerase chain reaction (RT-PCR), we examined the expression of interleukin 1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and the presence of enteroviral genomic RNA in endomyocardial biopsy tissues obtained from patients with myocarditis and DCM. We examined endomyocardial biopsy tissues obtained from 6 patients with myocarditis, 21 with DCM and 15 with non-infectious cardiac diseases as controls. In patients with myocarditis, endomyocardial biopsy was performed twice at an interval of 1 month to 8 years after the onset of myocarditis. We used RT-PCR to detect IL-1 beta, IL-6, IL-8 and TNF-alpha genes expression and nested RT-PCR (nRT-PCR) to detect enteroviral genomic RNA. IL-1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in 100% (6/6) and enteroviral genomic RNA in 67% (4/6) of myocarditis patients at the first biopsy. At the second biopsy, IL-1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in none, 50% (3/6), 67% (4/6) and 67% (4/6), respectively, and enteroviral genomic RNA in 67% (4/6). Four patients with myocarditis, in whom IL-8 and TNF-alpha genes and enteroviral genomic RNA were detected, progressed to DCM at the second biopsy. IL 1 beta, IL-6, IL-8 and TNF-alpha genes were expressed in none, 24% (5/21), 38% (8/21), 57% (12/21) of DCM patients, respectively. Enteroviral genomic RNA was detected in 43% (9/21) of DCM. Neither cytokine expression nor enteroviral genomic RNA were detected in the controls. the high incidence of cytokines, especially IL-6, IL-8 and TNF-alpha, expression in myocarditis and DCM, which might be induced by enteroviral infection, suggests that cytokines play an important role in myocytic damage leading to DCM. PMID- 8624581 TI - Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60. AB - Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 or Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process. PMID- 8624583 TI - Hepatoxicity of ricin, saporin or a saporin immunotoxin: xanthine oxidase activity in rat liver and blood serum. AB - Male Wistar rats each received an i.p injection of the ribosome-inactivating proteins ricin or saporin, or a Ber-H2 (anti-CD30)-saporin immunotoxin at a dose corresponding to three times the LD50 calculated for mice. Animals were killed 24, 48 or 72 h after treatment. Histological examination showed hepatic necrosis in all treated animals, although the sinusoidal lining was affected only in ricin poisoned rats. The activities of xanthine dehydrogenase (D-form) and oxidase (O form) were determined spectrophotometrically in liver and serum samples. In ricin treated animals the liver enzyme was progressively converted from the D- to the O form, which accounted for more than 60% of total activity after 48 h of poisoning, whilst no change in the xanthine oxidase activity was found in the serum. In the liver of rats treated with free or Ber-H2-conjugated saporin, the D form was more than 75%, as in normal animals. In the same animals the serum xanthine oxidase activity was up to three-fold control values. The determination of serum xanthine oxidase may prove helpful in the evaluation of liver damage in patients treated with immunotoxins. It may become a diagnostic tool for the differential diagnosis of liver diseases. PMID- 8624584 TI - Ectopic adrenocorticotropin syndrome associated with undifferentiated carcinoma of the colon showing multidirectional neuroendocrine, exocrine, and squamous differentiation. AB - We report a rare case of ectopic ACTH syndrome associated with undifferentiated carcinoma of the ascending colon. A 62-year-old woman developed hypokalaemia and metabolic alkalosis associated with markedly elevated serum cortisol and plasma ACTH levels. High-dose dexamethasone (8 mg/day) did not suppress increased urinary 17-hydroxycorticosteroid and 17-keto-steroid excretion. Barium enema and abdominal computerised tomography showed a Borrmann II type tumour in the ascending colon, multiple metastatic nodules in the liver and bilateral enlargement of the adrenal glands. Histological examination of the resected primary colon cancer and metastatic liver tumour showed undifferentiated carcinoma with areas of distinct neuroendocrine, exocrine, and squamous differentiation. ACTH production by the tumour was confirmed by radioimmunoassay and immunohistochemistry. This is a unique case report of carcinoma of the colon with distinct multidirectional differentiation causing ectopic ACTH syndrome. PMID- 8624582 TI - Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique. AB - Morphological studies using immersion or perfusion fixation methods do not reveal the ultrastructure of functioning kidneys with normal circulation. A simple apparatus was developed for freezing the kidneys in vivo without stopping the blood supply, and the ultrastructure of the glomerular capillary loops was examined under different haemodynamic conditions. Mouse kidneys were frozen under normal blood flow conditions; others were frozen in the same way after ligation of the abdominal aorta at a point caudal to the renal arteries. They were then processed for the freeze-substitution or deep-etching method. Good ultrastructural preservation was obtained within about 5 microM depth from the frozen tissue surface. Functioning glomeruli with normal blood flow possessed open capillary lumens, different shapes of foot processes and atypical basement membranes with low density. Moreover, heterogeneity in width between foot processes was identified on the replica membranes. Under the acute conditions used to increase blood supply into the kidneys, the spaces between the flat foot processes became more widely dilated and the basement membrane was seen to be three-layered. The ultrastructure of glomeruli in functioning kidneys has been demonstrated for the first time by this "in vivo cryotechnique." PMID- 8624585 TI - Ectopic prostatic tissue in the spleen. AB - Ectopic prostatic tissue was found in the spleen in a 49-year-old white man who died of wide-spread malignant mesothelioma. The prostatic origin of the tissue was affirmed by positive immunohistochemical staining for prostatic specific antigen and prostatic acid phosphatase. PMID- 8624586 TI - Immunohistochemical expression of metallothioneins in colorectal adenocarcinoma. PMID- 8624587 TI - [Treatment of pelvic endometriosis with goserelin, an LHRH agonist]. AB - OBJECTIVE: To determine the safety and effectiveness of ZOLADEX depot (goserelin) in the therapy of endometriosis (study No. 9393CZ/0001). MATERIAL AND METHODS: A multicentric prospective open study of 60 women with endometriosis. R-AFS score of pelvic endomemtriosis was established in patients suffering from pelvalgia, dyspareunia and dysmenorrhoea during pre-treatment laparoscopy. The effect of Zoladex depot applied subcutaneously were evaluated by gynaecological and laboratory examinations, including assessment of subjective complaints every four weeks in the course of six months. Laparoscopic check-up was carried out immediately after terminating the therapy. RESULTS: (1) The average total score of subjective complaints decreased by 90% after 24 weeks of treatment. (2) Treatment was successful in 67% patients, i.e. laparoscopic examination showed a decrease of the R-AFS score by at least 50%. In 28% treated women visible endometrial foci disappeared completely. (3) The most frequent side-effects of therapy are associated with hypoestrogenic effects of GnRH agonists. Vasomotor instability, flushes and night hyperidrosis occurred in 93% and 90% treated women, respectively. CONCLUSION: Goserelin acetate is a synthetic GnRH agonist suitable for safe and effective therapy of pelvic endometriosis. The administration of 3.6 mg goserelin every day for 24 weeks reduced markedly the size of endomerial foci and painful symptoms. PMID- 8624588 TI - [The effect of diazepam and flumazenil on pulsatile secretion of prolactin and LH in women]. AB - The authors examined the effect of diazepam and flumazenil on prolactin and LH secretion in women. The experiment lasted 12 hours during which specimens of venous blood were collected for assessment of LH and prolactin after 10-minute intervals. After 5 hours to a group of 8 women (4 in the luteal and 4 in the follicular stage of the cycle) flumazenil was administered--3 x 10 mg i.v. after 30 minute intervals, and to a group of 7 women (3 in the follicular and 4 in the luteal stage of the cycle) diazepam was administered--10 mg i.v. after 30-minute intervals. Flumazenil administration did not affect the LH or prolactin secretion. Diazepam caused in 3 of 4 women in the luteal phase of the cycle a significant rise of the prolactin secretion (p < 0.001), in the remaining women in the luteal phase and 3 women in the follicular stage a rise of prolactin was recorded but it did not reach statistical significance. The LH level was significantly influenced (i.e. decreased) in one of 7 women. PMID- 8624589 TI - [Present trends in surgical treatment of endometrial carcinoma]. AB - Contemporary trends in the treatment of endometrial carcinoma are in favour of radical surgery, in particular when prognostic factors signalize possible failure of standard surgical treatment. When selecting a suitable operation it is important to respect the patient's general health status, age and anatomical conditions. High-standard evaluation of prognostic factors, proper surgical technique and tactics are further basic prerequisites of successful treatment. PMID- 8624590 TI - [Immunologic factors in the serum and peritoneal fluid in women after laparoscopy. I. Nonspecific factors]. AB - The authors examined by diagnostic laparoscopy and immunologically 180 patients in 1993-1995. They assessed in serum and peritoneal fluid IgG, IgA, IgM, haptoglobulin, A2-macroglobulin, A1-antitrypsin, transferrin, C4- and C3 complement, orosomucoid, A2AP-glycoprotein, albumin, prealbumin and hemopexin. The authors found that some individual pathological results as regards non specific immunity supplement the clinical diagnosis, in particular in endomeriosis, multiple adhesions, polycystic ovaries and chronic anovulation. PMID- 8624591 TI - [Sex behavior of women seeking hormonal contraception and women seeking induced abortion]. AB - Using an anonymous questionnaire, the authors assessed the sexual and contraceptive behaviour of 130 applicants for induced abortions and 330 applicants for hormonal contraception. Between the two groups these main differences were found: applicants for abortion used less frequently effective contraception and used more frequently unreliable contraceptive methods. This difference was obvious during the first intercourse as well as during later life. The use of contraception in this group is more limited also as compared with the nationwide average. In the case-history of applicants for abortion previous abortions were more frequent, as well as prostitute behaviour and the prevalence of venereal diseases. As regards heterosexual behaviour the applicants for abortion and applicants for hormonal contraception did not differ substantially. According to the authors the differences between the examined groups depend more on personality traits than sexuological characteristics. PMID- 8624592 TI - [The first pregnancy in the Czech Republic achieved by oocyte fertilization using ICSI--intracytoplasmic sperm injection]. AB - ICSI--an intracytoplasmic sperm injection can fertilize an oocyte in vitro with one sperm, using micromanipulation. ICSI was done on an inverted Diaphot 300 microscope with Hoffmann modulation contrast and Narishige micromanipulators. Between March and May 1995 23 ICSI cycles were performed. Of 166 oocytes (7.2 per cycle) 22% were degenerated or immature and 6% damaged during ICSI. 45% of the injected eggs were fertilized. Embryos were transferred in 65% couples. Three pregnancies resulted (20%/ET), all are viable. ICSI is suitable in cases of severe oligospermia, asthenospermia and teratospermia, immunology infertility and low cleavage rate of oocytes in the first IVF cycle. PMID- 8624593 TI - [Results and complications of intrauterine transfusion in fetuses with and without hydrops]. AB - The group comprises 50 intrauterine transfusions in 15 patients with a severe form of foetal erythroblastosis. The authors describe the results and complications in hydropic and non-hydropic foetuses, diagnosed by ultrasound before transfusion treatment was started. In the group of non-hydropic foetuses the success rate of transfusion treatment was 90.1% and 10 neonates were born with a mean weight of 2030 g. The mean gestation period was 34 weeks. From four hydropic foetuses it proved possible to save only one, the success rate in this group was only 25%. The mean weight of hydropic foetuses was 1650 g and the gestation period at delivery 29 weeks. The rate of Caesarean sections in both groups was as high as 80%. Serious complications included thrombosis of the umbilicus, thromboembolism of the a. axillaris in a hydropic neonate and acute hypoxia of the foetus after transfusion. Four of six serious complications were recorded in hydropic foetuses. PMID- 8624594 TI - [Changes in the position of the urethra and bladder neck during pregnancy and after delivery]. AB - It is estimated that some 30-50% women suffer occasionally from urinary incontinence. At the end of pregnancy these complaints are more marked. In the author's group of 44 women 55% suffered from stress incontinence. The psychosocial impact of impaired function of the lower urinary pathways in women on different spheres of the patient's life is so serious that it calls for a qualified approach of a specialist. The increased mobility of the UV junction in incontinent women has been described by several authors [6, 12, 13]. It was also proved in the author's investigation closely before delivery and 3-5 days after delivery. Six weeks after delivery no differences were found in the mobility of the UV junction in continent and incontinent women. If the difference of the gamma angle (formed by the axis of the symphysis and the connecting line between the UV junction and the lower margin of the symphysis) during contraction of the muscles of the pelvic floor and Valsalva's manoeuvre (intraabdominal pressure raised by 30 cm H2O) is more than 30 degrees during the 40th week of gestation or 3-5 days after delivery (when this mobility is even greater), then the woman is liable to develop the stress type of incontinence and the authors recommend to use Kolpexin after the puerperium for exercise and strengthening of the muscles of the pelvic floor. Changes in the length of line p and angle beta are not statistically significant and cannot assess the disposition for development of urinary incontinence. PMID- 8624595 TI - [Management errors in the expulsion stage of labor]. PMID- 8624596 TI - [Is it necessary to be concerned about coagulation disorders in women using combined oral contraceptives?]. PMID- 8624597 TI - [The third priority in perinatal care]. PMID- 8624598 TI - [Nifuratel and nystatin combination therapy in vulvovaginitis of mixed etiology in children and adolescents]. PMID- 8624600 TI - [How successful is treatment of sterility using IVF and ET in reality?]. PMID- 8624599 TI - [Significant teratogenicity of angiotensin converting enzyme inhibitors]. PMID- 8624601 TI - [Maternal mortality in the Czech Republic in 1994]. PMID- 8624602 TI - [The role of prenatal diagnosis on the decreasing incidence of congenital defects in the pediatric population in the Czech Republic 1988-1994]. PMID- 8624603 TI - [Assisted hatching--a useful micromanipulation technic in women after repeated failure of embryo transfer]. AB - One reason for the low implantation rate after IVF/ET is the limiting ability of blastocysts to escape from the zona pellucida, and of nidation. This procedure can be assisted by the creation of an artificial opening in the zona pellucida before the transfer (assisted hatching). An artificial slot in the zona of 58 embryos (20 patients) was created mechanically by using Leitz Fluovert microscope and Narishige micromanipulators. From 20 embryo transfers 6 clinical pregnancies resulted (30.0% clin. preg. rate). In 4 cases women with three or more unsuccessful transfers were involved. This method should be used in classical IVF, specially in cases of repeated high quality embryo transfers which failed. PMID- 8624604 TI - [Cryopreservation of embryos with the Cryogenetic apparatus]. AB - For cryopreservation of human supernumerary embryos in our IVF programme we use a "Cryogenetic" computerised freezer. 1,2-propandiol and sucrose were used as cryoprotectant in the slow freezing/thawing protocol. In 20 transferred cycles we achieved 3 pregnancies. One is proceeds. Although the number of described cycles is low, the results are comparable with other centres. Our centre is the second one in the Czech Republic, which published data on a pregnancy after the transfer of cryopreserved embryos. Our Cryogenetic freezer is a high quality freezer suitable for gamete and embryo freezing. PMID- 8624605 TI - Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa. AB - Spectacular success has recently been made towards elucidation of the molecular basis of various forms of epidermolysis bullosa (EB), a group of heritable blistering skin diseases. The information derived from these studies has already had a profound impact in terms of precise diagnosis and classification, early prenatal prediction of the phenotype and genetic counseling in families at risk for recurrence. This review highlights recent progress made in defining the molecular basis of junctional and dystrophic forms of EB and the genotype/phenotype relationships established from these studies. Extensive molecular studies, such as the ones captured in this review, form a foundation for the rational design of gene therapies to counteract these conditions in the future. PMID- 8624606 TI - Expression of adhesion molecules and their ligands in contact allergy. AB - Sequential biopsies from skin lesions induced by nickel sulphate and sodium lauryl sulphate, respectively, were investigated with respect to expression of extracellular matrix proteins and adhesion molecules on lymphocytes, endothelial cells, and keratinocytes. The majority of the infiltrating lymphocytes expressed VLA-4, LFA-1, CD44 and ICAM-1, a variable fraction expressed Leu-8 and VLA-5, and few or no cells were positive for VLA-1, VLA-2 and VLA-6. Noteworthy, was that the infiltrating cells showed a substantial amount of fibronectin but relatively small or negligible presence of laminin, collagen type IV, IgG, IgA, IgM, and albumin. The fibronectin was associated with cell bodies as well as the area surrounding infiltrating cells. The number of infiltrating cells was larger in biopsies from nickel-sulphate induced lesions and the infiltrates contained more fibronectin than biopsies from lesions induced by sodium lauryl sulphate. However, at the single-cell level, the expression of VLA antigens, LFA-1, CD44 and ICAM-1 was similar in both groups. The endothelial cells of skin biopsies from nickel-sulphate-induced lesions showed a stronger expression of VCAM-1, ELAM 1 and ICAM-1 compared to biopsies from sodium lauryl sulphate-induced lesions. In the biopsies from nickel sulphate-induced lesions, the keratinocytes showed a tendency to less VLA-6 expression. These results suggest that fibronectin plays a role in lymphocyte extravasation or extravascular lymphocyte migration. PMID- 8624607 TI - The induction of the alpha-1-adrenoceptor signal transduction system on human melanocytes. AB - Human melanocytes established in MCDB-153 culture medium do not express alpha 1-, beta 1-, beta-2 adrenoceptors without extracellular stimulation. The addition of 50 x 10-9 M norepinephrine to the medium causes a time-dependent induction of alpha-1-adrenoceptors with 4.278 receptors/melanocyte after 24 h. Under the same experimental conditions, the dendricity of melanocytes as well as melanogenesis was unaffected over 60 h. Since keratinocytes hold the full capacity for catecholamine biosynthesis but melanocytes lack this system, the secretion of catecholamines from keratinocytes appears to be of critical importance to the alpha-1-adrenoceptor in melanocytes, underlining the symbiosis of both cells in the epidermal unit. PMID- 8624608 TI - All-trans retinoic acid inhibits binding of 1,25-dihydroxy-vitamin D3 to the vitamin D receptor in cultured human keratinocytes. AB - Psoriasis is characterized by hyperproliferation and impared differentiation of epidermal keratinocytes (KCs). Psoriasis can be treated with derivatives of retinoic acid (RA) and vitamin D3. Analogues of vitamin D3 are able to inhibit proliferation and stimulate differentiation of KCs. In contrast, RA inhibits terminal differentiation of KCs. Interactions are known to occur between RA and vitamin D3 signalling pathways. The purpose of the present study was to determine the effect of all-trans RA on the binding of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) to the vitamin D3 receptor (VDR) of cultured human KCs. Cultured KCs from normal adults were incubated with or without RA (10-9-10-7M) for 4-24 h. Cells were then harvested, homogenized and ultrasonicated. The extracted protein was incubated with 3H-1,25 (OH)2D3 (0.015-1.0 nM) with or without 250-fold excess nonradioactive 1,25 (OH)2D3 for 24 h and specific binding was determined by use of the dextran coated charcoal binding assay. Western blot analysis utilizing the monoclonal antibody 9A7 gamma to VDR was performed on protein extracted from the KCs. The bands resulting from Western blot analysis were visualized by enhanced chemiluminescence. From Scatchard analysis it was found that KCs bind 1,25 (OH)2D3 with high affinity (Kd = 0.175 nM). This binding was dose and time dependently inhibited by RA (60% inhibition at 10-7 M after 24 h of incubation). By Western blot analysis, RA had no effect on the amount of protein extracted from KCs at any of the RA concentrations tested. In conclusion, these results show that binding of vitamin D3 to its receptor of human KCs can be inhibited markedly by RA without effecting the amount of protein. These results are in contrast to results with other cell types in which RA upregulates binding of 1,25 (OH)2D3 to the VDR. Because interaction between retinoids and vitamin D3 may occur at different levels during signal transduction, it is not possible to predict from our results whether RA will inhibit the effects of vitamin D3 in vivo. PMID- 8624609 TI - Modulation of Ia+ Langerhans cell numbers in vivo by cultured epidermis derived supernatants and by GM-CSF. AB - This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC. PMID- 8624611 TI - Improved method for rhesus monkey or rat skin preparation and cryosectioning for topical or transdermal skin distribution studies. AB - A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6mm punch biopsies from freshly excised, full-thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap-freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap-frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 mu m cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure. PMID- 8624610 TI - Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro. AB - Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha melanocyte stimulating hormone either inhibit or enhance in a dose-dependent fashion an interleukin-4 (IL-4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose- and donor-dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B-cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin-derived peptides such as alpha-, beta-, and gamma-endorphins as well as by the opioid binding pentapeptide Leu-enkephalin. Furthermore the neuropeptide substance P accentuated an IL-4 or an IL-4 and anti-CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL-4 induced IgE response. PMID- 8624612 TI - Lipoperoxidase activity of Pityrosporum: characterisation of by-products and possible role in pityriasis versicolor. AB - Modification of pigmentation and damage of melanocytes are characteristic features of skin colonisation of Pityrosporum orbiculare hyphae in pityriasis versicolor (PV). The yeast is lipophylic and lipid-dependent, capable of oxidising unsaturated lipid components of skin surface, i.e., unsaturated fatty acids, cholesterol and squalene (SQ). The oxidation of unsaturated fatty acids gives rise to dicarboxylic acids (DA) which behave, in vitro, as competitive inhibitors of tyrosinase. In this work, we further investigate the oxidase activity of Pityrosporum in vitro, by evaluating (a) the generation of lipoperoxides in cultures supplemented with fatty acids at various degrees of unsaturation; (b) the mechanism of SQ oxidation; (c) the chemical characteristics of some by-products of lipoperoxidation; (d) the formation of peroxisomes in fungal cells. In cultures supplemented with the saturated palmitic acid (C16:0) and monounsaturated oleic acid (C18:1 n-9), low amounts of lipoperoxides were detected by a spectrophotometric test, whereas in cultures supplemented with di unsaturated linoleic acid (C18:2 n-6), significant concentrations were found. Gas chromatography-mass spectrometry analyses showed the generation of linoleic acid hydroperoxides both in Pityrosporum cultures and following incubation of acetone powder of the fungus with the unsaturated fatty acid, indicating the presence of a lipoxygenase activity in the fungus. In cultures supplemented with linoleic acid plus SQ, and increase of lipoperoxide generation was observed and trans trans farnesal and squalene epoxides have been identified. Electron microscopic examinations have evidenced peroxisomes in cells grown in the presence of linoleic acid, whereas they were not detected in cultures supplemented with oleic acid and palmitic acid. The metabolic activities of peroxisomes, through the formation of hydrogen peroxide and the subsequent generation of hydroxyl radicals, may account for the peroxidation of SQ, which is not a substrate of lipoxygenase. Following these results, we propose a mechanism for DA generation by Pityrosporum metabolism and hypothesize that the lipoperoxidation process induced by lipoxygenase activity of the fungus may be the key to understanding the clinical appearance of skin manifestation of PV. PMID- 8624613 TI - Decreased expression of alpha 2 beta 1 integrin in scleroderma fibroblasts. AB - Systemic scleroderma (SSc) is a complex connective tissue disorder of unknown etiology. In early stages of the disease, fibroblasts are activated to produce large amounts of collagen with subsequent fibrosis. Collagen metabolism of fibroblasts is modulated by their contact with the extracellular matrix (ECM), which involves distinct receptors on the cell surface, mainly belonging to the integrins. We investigated the expression of collagen receptor alpha 2 beta 1 in SSc and normal fibroblasts, since this receptor has been shown to be utilized by fibroblasts for adhesion to and reorganization of collagen I. 9 strains of scleroderma fibroblasts grown as monolayer cultures were first analyzed with respect to their collagen I expression. 6 of these strains were similar to controls "low" producers) and 3 strains showed up to 2-3 x higher levels of collage I mRNA expression ("high" producers). Northern hybridization using a cDNA probe specific for the alpha 2 integrin subunit revealed a decrease of the corresponding mRNA in SSc fibroblasts as compared to controls (75% versus 100%). "High" collagen producing cell strains displayed the lowest values for alpha 2 integrin mRNA. The decrease of alpha 2 integrin subunit expression at the mRNA level in selected fibroblasts was further substantiated by radioimmunoprecipitation using specific mAbs directed against alpha 2 integrin subunit. No significant changes in beta 1 integrin expression could be observed - neither at mRNA nor at the protein level. Our data indicate a correlation between excessive synthesis of collagen and low levels of alpha 2 integrin subunit expression in SSc fibroblasts. Further experiments should clarify whether this observation is a phenomenon specific for scleroderma or whether it reflects an "activated" state of fibroblasts. PMID- 8624614 TI - Orthopaedic medicine--the remit of the rheumatologist? PMID- 8624615 TI - Antibodies against retinal S-antigen in patients with juvenile chronic arthritis associated uveitis. PMID- 8624616 TI - Familial presence of primary cryofibrinogenaemia, a report of three cases. PMID- 8624617 TI - Sulphasalazine-induced systemic lupus erythematosus in a patient with rheumatoid arthritis. PMID- 8624618 TI - Corticosteroids in the management of rheumatoid arthritis. PMID- 8624619 TI - Increased expression of interferon (IFN)-gamma together with IFN-gamma receptor in the rheumatoid synovial membrane compared with synovium of patients with osteoarthritis. AB - Data concerning the presence of T-cell-derived cytokines in the rheumatic joint are conflicting, challenging the hypothesis that rheumatoid arthritis (RA) is a T cell-mediated disease. In this study synovial tissue specimens of 11 patients with RA and eight patients with osteoarthritis (OA) were stained for interferon gamma (IFN-gamma) and its receptor. The level of expression of IFN-gamma was compared with that in tissue specimens of delayed-type hypersensitivity (DTH) reactions of the skin and of chronic tonsillitis. Furthermore, the percentage of T-lymphocytes which stained positive for IFN-gamma was determined using double staining techniques. IFN-gamma and its receptor were detected in all patients with RA and in 7/8 and 3/8, respectively, of patients with OA. Expression of IFN gamma (P<0.02) and IFN-gamma receptor (P<0.01) in synovial tissue of patients with RA was more abundant compared with that in patients with OA. Although IFN gamma could be detected in RA synovial tissue, the level of expression was less when compared with DTH reactions of the skin and tonsillitis. The percentage of CD3+ cells being positive for IFN-gamma was approximately 1% in RA, whereas in DTH reactions of the skin it was >90% and in tonsillitis approximately 30%. We conclude that the presence of IFN-gamma and its receptor in RA synovial tissue suggests a role for this cytokine in the ongoing immunological reaction of the inflamed joint. PMID- 8624620 TI - Differential expression of the costimulatory molecules B7.1 (CD80) and B7.2 (CD86) in rheumatoid synovial tissue. AB - CD4+ T-lymphocytes require two signals to become activated--antigen receptor (TcR) occupancy and an antigen-presenting cell (APC)-derived costimulus. The latter may be provided by B7.1 (CD80) or B7.2 (CD86) on APC interacting with CD28 on T-cells. We have studied the expression of these costimulatory molecules in rheumatoid and osteoarthritic synovial membrane. Very few B7.1-positive cells were seen in synovial tissue from either established or early rheumatoid disease, or in rheumatoid arthritis (RA) or osteoarthritis (OA) synovia at arthroplasty. In contrast, B7.2 was readily detected in rheumatoid synovia, predominantly in the lining layer, in a pattern of expression that corresponded to the presence of CD68-positive macrophages. Only occasional B7.2-positive cells were seen in OA synovia. The presence of B7.2 but the relative lack of expression of B7.1 may be partly responsible for the observations of 'frustrated' T-cell activation or T cell hyporesponsiveness in the rheumatoid synovium. PMID- 8624621 TI - Antineutrophil cytoplasmic antibodies in rheumatoid arthritis patients. AB - We determined the occurrence of antineutrophil cytoplasmic antibodies (ANCAs) and their specificities in 77 rheumatoid arthritis (RA) patients and compared them with 25 patients with psoriatic arthritis (Pso), 19 with drug-induced lupus erythematosus (DI-LE) and 11 with systemic lupus erythematosus (SLE). Thirty-two percent of RA patients had positive indirect immunofluorescence (IIF) stains (P or atypical ANCA). Twenty-nine per cent of patients with rheumatoid vasculitis (RAV), 48% with long-standing RA (LSRA) and 20% with early RA (Ely RA) had positive ANCAs compared with 4% of Pso patients, 47% of DI-LE patients and 45% of SLE patients. Western blotting (with polymorphonuclear cell extracts or alpha granules) and alpha-granule enzyme-linked immunosorbent assay (ELISA) yielded variable results and proved unhelpful for characterizing the specificities of ANCAs. ELISAs based on commercial purified lactoferrin (LF), myeloperoxidase (MPO), human elastase (HLE) and cathepsin G (CG) showed that anti-HLE antibody was the most prevalent (14%) antibody in RA, followed by anti-MPO antibody and anti-LF antibody (10% each). Statistical analysis of antibody prevalence by clinical presentation showed that LSRA patients were more likely to have anti-HLE antibody and that DI-LE patients were more likely to have anti-CG antibody compared with the other patient groups. In lupus patients serial ELISA titration of ANCAs (LF and MPO) was found to be reliable for predicting the outcome. The overall incidence of ANCAs in RA patients was 33% by IIF. PMID- 8624622 TI - Secondary amyloidosis in patients with rheumatoid arthritis: diagnostic and prognostic value of gastroduodenal biopsy. AB - Upper gastrointestinal endoscopy was performed in patients with rheumatoid arthritis (RA) during the period 1989-1991, and biopsy specimens were obtained from the stomach and from the duodenum for examining amyloid deposits. Among 407 patients, gastrointestinal amyloidosis was confirmed in 54 (13.3%). Twenty-two patients were regarded as having slight amyloid deposits, while 32 patients were categorized as having marked amyloid deposits. The incidence of clinical manifestations suggestive of systemic amyloidosis was more frequent in the marked deposits group than in the slight deposits group (47% vs 14%, P<0.05). Among the patients who died of manifestations associated with amyloidosis, the survival period following endoscopy was shorter in the marked deposits group than in the slight deposits group. These findings suggest that gastroduodenal biopsies may be useful for diagnosing secondary amyloidosis and that the degree of amyloid deposits seems to be correlated with the clinical manifestations of RA. PMID- 8624623 TI - Michael Mason Prize Essay 1995. Complement, immune complexes and systemic lupus erythematosus. PMID- 8624624 TI - Quantitative assessment of synovial inflammation by dynamic gadolinium-enhanced magnetic resonance imaging. A study of the effect of intra-articular methylprednisolone on the rate of early synovial enhancement. AB - The effect of temporary inflammatory suppression on synovial membrane enhancement, as determined by dynamic and static gadolinium-DTPA enhanced magnetic resonance imaging (MRI), was studied. MRI of 18 arthritic knees was performed before and 1, 7, 30 and 180 days after intra-articular methylprednisolone injection until clinical relapse. MRI of another six knees was performed twice within 2-4 days in order to assess interobserver and inter-MRI variation. The rate of early enhancement of the entire synovial membrane of a pre selected central sagittal slice (REEsyn,tot), determined by dynamic T1-weighted FLASH MRI, decreased in all knees within the first post-treatment week and remained low during remission, although gradually increasing. In cases of clinical relapse, REEsyn,tot increased to pre-treatment levels. The interobserver plus inter-MRI variation was maximally 27%. The predictive values of a REEsyn,tot below/above 1.0%/s were 0.94 and 0.91, respectively, with respect to the absence/presence of clinical synovitis. Evaluation of small synovial areas revealed marked regional heterogeneity. Static spin echo MRI was not informative. The study indicates that the rate of early synovial enhancement reflects synovial inflammatory activity. Subclinical changes may be revealed. Evaluation of large synovial areas increases reproducibility and reduces the effect of regional heterogeneity. Dynamic MRI may prove a clinically useful measure of synovial inflammation. PMID- 8624625 TI - Compliance monitoring of NSAID drug therapy in ankylosing spondylitis, experiences with an electronic monitoring device. AB - We concluded a randomized, controlled trial to compare once-daily 20 mg piroxicam versus once-daily 20 mg tenoxicam in ankylosing spondylitis. We recorded patients' dosing histories with electronic monitors for an average of 225 days (range 55-379) in 34 recipients of piroxicam and 31 recipients of tenoxicam. Dosing histories with the two agents were similar and are combined. Patients took 81% of prescribed doses; 78% once daily (as prescribed) and 3% as two or more daily doses. On 19% of all monitored days, there was no record of a dose being taken; 68% were single no-dose days, the rest (32%) being 2 to >10 consecutive no dose days. In 3% of monitored days, extra doses were evidently taken, 88% as twice daily and 12% as three or more doses. Only 22% of all patients (14/65) strictly complied with the regimen: one dose daily every day. The remainder alternated between no-dose days and extra-dose days. We found no correlation between patient compliance and improvement in reported pain or morning stiffness. PMID- 8624626 TI - The Bath Ankylosing Spondylitis Patient Global Score (BAS-G). AB - In the absence of an ideal objective measure for assessing ankylosing spondylitis (AS), self-administered measures of disease activity (the Bath Ankylosing Spondylitis Disease Activity Index, BASDAI) and function (the Bath Ankylosing Spondylitis Functional Index, BASFI) have been developed, in addition to an objective measure of spinal mobility (the Bath Ankylosing Spondylitis Metrology Index, BASMI). However, a more global assessment is also desirable. We report on the design and validation of a global measure (the Bath Ankylosing Spondylitis Patient Global Score, BAS-G) which reflects the effect of AS on the patient's well-being. A pilot study was performed to select the most appropriate wording for BAS-G. Using 392 patients with AS, BAS-G's construct and predictive validity and test-retest reliability were assessed. Correlations between BAS-G and BASDAI/BASFI were calculated, and multiple regression was used to examine the significant correlates. The distribution of the responses covered the whole scale. As predicted, BAS-G correlated best with BASDAI (r=0.73), followed by BASFI (r=0.54). The best fitting regression equation included these scales as well as patients' gender and current age. One week and 6 month scores were significantly different (P<0.001). Construct validity was good: BAS-G correlated more strongly with each component of BASDAI and BASFI than with BASMI or with gender. Predictive validity was satisfactory: there was an improvement (mean=29%) in in-patient BAS-G scores over a 2 week treatment period (P<0.001). Test-retest reliability was excellent (1 week r=0.84, 6 months r=0.93). BAS-G correlates well with both BASDAI and BASFI, suggesting that disease activity and functional ability play a major role in patients' well-being, whereas metrology does not. The score is sensitive to change, reliable, and meets face, predictive and construct validity criteria. PMID- 8624627 TI - A case of primary Sjogren's syndrome, complicated by cryoglobulinaemic glomerulonephritis, pericardial and pleural effusions. AB - Primary Sjogren's syndrome (SS1) complicated by glomerulonephritis is rare and is usually associated with the presence of cryoglobulins. Cryoglobulinaemic glomerulonephritis as described in type II essential mixed cryoglobulinaemia, characterized by the presence of deposits of cryoglobgulins within the glomerular capillary lumen, occurring in SS1 has previously been reported only once in the literature. Our case was also complicated by pleural and pericardial effusions. We discuss the treatment options available for the renal lesion. PMID- 8624628 TI - Infertility may sometimes be associated with NSAID consumption. AB - Non-steroidal anti-inflammatory drugs are widely used in the treatment of inflammatory joint diseases. Many patients suffering from these disorders are young women during their childbearing years. We report three cases of infertility where the cause may have been NSAID-induced 'luteinized unruptured follicle' syndrome. This phenomenon is well recognized in obstetric circles, and we would like to bring it to the attention of rheumatologists since it is not documented in the rheumatological literature. PMID- 8624629 TI - Future expectations: adolescents with rheumatic diseases and their transition into adulthood. PMID- 8624630 TI - Young adults with arthritis: meeting their transitional needs. PMID- 8624631 TI - Correlates of disablement in polyarticular juvenile chronic arthritis--a cross sectional study. AB - To assess the impact of disease on the functional outcome of patients with polyarticular juvenile chronic arthritis (JCA), the relationship between impairments and functional limitations was studied. Therefore, variables from the impairment domain were correlated with variables of the functional limitation domain and outcome variables were analysed for differences as a result of inflammatory disease, rheumatoid factor (RF), disease duration and age at onset. Twenty-three patients with polyarticular JCA were subjected to auxologic evaluation, a laboratory check, radiographic evaluation, joint count on tenderness and swelling, joint mobility/deformity examination, functional assessment of skills, health assessment and psychosocial evaluation. Inflammatory disease parameters, like CRP, ESR, thrombocytosis and leucocytosis, were increased in 6/23 patients. The parameters of the impairment domain, like joint tenderness and swelling, showed mild outcome, while parameters of the functional limitation domain showed more severe outcome. Generally, perceived competence was found to be normal. A clinically relevant number of patients (10/13) showed low scores on the activity factor of the Child Behaviour Check List (CBCL). A significant relationship was found between inflammatory disease variables and functional limitation outcome. RF seropositivity was not a good outcome predictor. Disease duration and age of onset showed no significant difference in the outcome of the domains. Significant correlation was found between the parental report of the Childhood Health Assessment Questionnaire (CHAQ) and all impairment parameters. Joint swelling showed a significant relationship with CHAQ and Juvenile Arthritis Functional Assessment Report (JAFAR). Disability outcome did not correlate with functional limitation. In general, children with polyarticular JCA function rather well when using a multidomain evaluation approach. Compensatory and adaptational mechanisms might contribute to the poor correlation between impairment and functional limitation parameters. Laboratory evaluation of inflammatory disease, a joint count of swollen joints and parent's report of the child's health status related best in our study. PMID- 8624632 TI - Polymyositis-associated overlap syndromes. PMID- 8624633 TI - The diagnosis of gout and CPPD crystal arthropathy. PMID- 8624634 TI - Bone mass measurement and bone metabolism in rheumatoid arthritis: a review. AB - The involvement of bone in rheumatoid arthritis (RA) is well recognized, and hand bone densitometry appears to be a promising new technique to monitor disease progression by assessing serial changes in hand bone mass in patients with RA. New biochemical markers of bone formation (i.e. osteocalcin) show contradictory results in different studies, although markers of bone resorption (i.e. urinary collagen cross-links) have shown significant increase in patients with RA. Bone histomorphometric studies suggest that the periarticular osteopenia in RA could be related to increased bone turnover locally, whereas generalized osteoporosis could be due to a global negative remodelling balance. The important factors implicated in the pathogenesis of the bone loss are circulating cytokines [e.g. tumour necrosis factor alpha (TNF alpha), interleukin (IL) 1 and IL6] produced by the inflammatory process, use of oral corticosteroids (in the dose of > or = 5 mg) and reduced mobility due to functional impairment. Apart from this underlying osteoporosis, patients with RA have an increased risk of falls secondary to functional impairment and there is an increased risk of fractures in patients with RA. Very few studies are presently available looking at the therapeutic measures to prevent osteoporosis in RA. Future drug trials on the treatment of RA should include bone mass measurement, especially of the hand, as one of the outcome measures. PMID- 8624635 TI - Characterization of E-selectin expression, leucocyte traffic and clinical sequelae in urate crystal-induced inflammation: an insight into gout. AB - The self-limiting response to urate crystals allows the exploration of events involved in both the onset and resolution of gout. Using i.v. injected radiolabelled anti-E-selectin monoclonal antibody 1.2b6 together with differentially radiolabelled neutrophils, mononuclear cells and albumin, we have characterized the expression of E-selectin in relation to leucocyte traffic, microvascular permeability and clinical sequelae following intracutaneous injection of monosodium urate crystals. We found that the inflammatory response in this model involved several distinct phases. First, E-selectin expression increased over 2-6 h in the context of increases in neutrophil and mononuclear cell accumulation, and albumin leakage. Secondly, leucocyte accumulation rapidly declined despite persisting E-selectin expression. Thirdly, E-selectin expression peaked at approximately 8 h and then fell despite an increase in clinically detectable erythema and induration. Lastly, these clinical manifestations of inflammation resolved despite the continued presence of urate crystals in the tissues. The further dissection of mechanisms regulating these phases will lead to a better understanding of events in both the pathogenesis and resolution of gout. Of broader significance, this inflammatory model may yield information about the protective events that underly resolution of inflammation, and provide insights into factors which determine chronicity. PMID- 8624636 TI - Agalactosyl IgG in aggregates from the rheumatoid joint. AB - It has been postulated that agalactosyl immunoglobulin G (IgG) self-associates to form pathological aggregates in the rheumatoid joint. To examine this hypothesis, IgG aggregates from synovial fluid (SF) of 22 patients with RA were prepared by precipitation with polyethylene glycol (PEG) 6000. The PEG precipitates and SFs were reduced with 2-mercaptoethanol (2ME) and bound to protein G. This procedure isolated the IgG in the PEG precipitates from other contaminating glycosylated proteins. The levels of galactose and N-acetylglucosamine (GlcNAc) residues present on the reduced IgG were quantified by their ability to bind the lectins Ricinus communis (RCA)120 and Bandeiraea simplicifolia (BS) II. Proportionally less galactose (expressed as a ratio of bound RCA120 to BS II) was present on the IgG from the PEG precipitates than on the IgG in the paired SF (P = 0.001). However, in many cases more RCA120 as well as BS II bound to IgG from PEG precipitates than from the corresponding SF. It is considered that agalactosyl IgG occurs preferentially in RA SF PEG precipitates and that this IgG may also exhibit increased Fab glycosylation. PMID- 8624637 TI - In vitro effects of methotrexate on human articular cartilage and bone-derived osteoblasts. AB - Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover and interleukin 1 (IL-1)- and RA mononuclear cell (RA-MNC)-induced cartilage damage in human articular cartilage tissue cultures, and the effects of MTX on basal and RA-MNC influenced proliferation and differentiation of osteoblasts in cultures of human bone-derived osteoblasts. MTX exerted no direct effect on cartilage nor did MTX influence IL-1- or RA-MNC-induced cartilage damage, despite strong suppression of basal as well as mitogen- and antigen-induced RA-MNC proliferation. MTX induced strong inhibition of osteoblast proliferation, but did not significantly interfere with osteoblast differentiation (i.e. alkaline phosphatase activity). RA-MNC-enhanced proliferation and differentiation of osteoblasts were abolished by MTX. These results suggest that if MTX is able to induce retardation of radiological progression in RA, this is not based on an initial direct effect of MTX on cartilage as measured by PG turnover, nor on an initial inhibition of IL-1 or RA-MNC-induced cartilage damage. However, longstanding MTX-induced inhibition of RA-MNC proliferation may lead to reduction of the catabolic activity involved in cartilage destruction. On the other hand, long-term inhibition of osteoblast proliferation may eventually lead to decreased bone formation and osteopenia. Whether this will turn out to be a problem of clinical importance in the treatment of RA has to be established. PMID- 8624638 TI - Anterior pituitary function in patients with newly diagnosed rheumatoid arthritis. AB - Hormonal dysfunction involving the hypothalamic-pituitary-adrenal (HPA) axis, prolactin (PRL) secretion and sex hormone status has been supposed to contribute to the development or persistence of rheumatoid arthritis (RA). In addition, a reduced number of glucocorticoid receptors on circulating lymphocytes has been found in patients with RA. However, so far most studies have been performed in pre-treated patients. A combined test for total anterior pituitary reserve was performed in 10 patients with newly diagnosed untreated RA. Before and after stimulation with the respective hypothalamic releasing hormones, RA patients showed no difference in plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol, prolactin (PRL) and thyroid-stimulating hormone (TSH) when compared to healthy controls. In contrast, the growth hormone (GH) response to growth hormone-releasing hormone (GHRH) was blunted in RA patients. The hypothalamic-pituitary-thyroid/gonadal and adrenal axes seem to be unaltered in RA. However, if one considers the presence of chronic inflammation, normal plasma ACTH and cortisol concentrations must be considered as inappropriately low. The observed blunted GH release could be mediated by cytokines (e.g. IL-1), which are known to be elevated in RA. PMID- 8624639 TI - Endothelin-1 expression in serum and bronchoalveolar lavage from patients with active Behcet's disease. AB - In view of the pulmonary manifestations in Behcet's disease (BD), we investigated the production of endothelin-1 (ET-1) by the respiratory tract in active BD. A group of 10 patients with active BD with pulmonary manifestations and 10 control subjects were studied. Immunoreactive ET-1 was measured in BAL and in serum by RIA. All the BD patients exhibited higher BAL ET-1 levels than controls. ET-1 expression may contribute to the functional and morphological abnormalities of the vasculature associate with BD. PMID- 8624640 TI - Prediction of diagnosis in acute and subacute oligoarthritis of unknown origin. AB - A total of 146 consecutive patients between 18 and 60 yr of age with oligoarthritis of unknown origin (< or = 6 active joints, < or = 8 weeks duration) were examined by a variety of clinical, laboratory and microbiological investigations, and followed longitudinally for 24 weeks. Reactive arthritis was diagnosed in 46 patients (19 induced by Chlamydia trachomatis, 27 by enterobacteria), 62 had undifferentiated arthritis, eight other inflammatory arthritic diseases, 15 acute sarcoid arthritis and 15 non-inflammatory joint diseases. Group differences were found for many baseline variables, but with considerable overlap between the groups. A set of four clinical and laboratory variables (elevated CRP, genitourinary symptoms, metatarsophalangeal joint involvement. HLA B27) could predict reactive arthritis with a sensitivity of 69.2% and a specificity of 93.5%. A wide range of clinical and laboratory examinations are required to determine the final diagnosis in oligoarthritis, but individual and sets of clinical and laboratory measures may give helpful clues for the correct diagnosis. PMID- 8624641 TI - Comparison of methotrexate with placebo in the treatment of systemic sclerosis: a 24 week randomized double-blind trial, followed by a 24 week observational trial. AB - In this study, methotrexate (MTX) was compared with placebo in the treatment of systemic sclerosis (scleroderma, SSc) in a 24 week randomized double-blind trial, followed by an observational trial of 24 weeks duration. Twenty-nine scleroderma patients were allocated to receive weekly injections of either 15 mg MTX or placebo. Patients who responded favourably after 24 weeks continued with the same regimen for a further 24 weeks; those who showed a poor response on placebo were allocated to further treatment with 15 mg MTX weekly, and those who responded poorly to treatment with 15 mg MTX had their doses increased to 25 mg. A favourable response was defined as an improvement of total skin score (TSS) by > or = 30%, of single breath diffusion capacity (DLCO) by > or = 15%, or of the score on a visual analogue scale of general well-being (VAS) by > or = 30%, provided that such improvements were not accompanied by persistent digital ulcerations or worsening of DLCO > or = 15%. Seventeen patients were allocated to MTX treatment and 12 to treatment with placebo. After 24 weeks, a significantly larger number of patients receiving MTX (n = 8, 53%) who completed the first 24 weeks of the study had responded favourably compared to patients receiving placebo (n = 1, 10%, P = 0.03). Comparison of separate variables between the two treatment groups by intention-to-treat analysis at week 24 showed improvement in the MTX group of TSS (P = 0.06) and creatinine clearance (P = 0.07). At week 48, 13 patients received MTX from the start of the study and nine during 24 weeks. From these 22 patients, 15(68%) responded favourably and compared with the start of the study they showed significant improvement of TSS (P = 0.04), VAS (P = 0.02), grip strength of the right hand (P = 0.02) and ESR (P = 0.01). Although the number of patients enrolled in this study is small, these results suggest that in a group of patients with active systemic sclerosis, low-dose MTX seems to be more effective than placebo according to pre-defined response criteria. PMID- 8624642 TI - Radiological outcome in ankylosing spondylitis: use of the Stoke Ankylosing Spondylitis Spine Score (SASSS). AB - We investigated the role of the Stoke Ankylosing Spondylitis Spine Score (SASSS) in a longitudinal cohort study of 53 patients with AS, assessed 9 yr apart, and its relation to clinical, functional and laboratory measures of disease activity and outcome. We also examined the long-term predictive value of quantitative sacroiliac scintigraphy (QSS). SASSS correlated closely with clinical measures, including occiput-wall distance (OWD) (P < 0.001) and modified Schober (P < 0.001). QSS was significantly correlated with final X-ray score (P < 0.05). SASSS changed significantly (P < 0.001) over the study period; two patterns of change in spine score were discernible, one with little change in radiological score and the other showing marked progression. The SASSS is a useful, valid score, which correlates with clinical outcomes measures and which has identified specific patterns of radiographic progression in AS. PMID- 8624643 TI - Microangiopathic haemolytic anaemia and systemic vasculitis. AB - Two cases of systemic vasculitis complicated by microangiopathic haemolytic anaemia (MAHA) are described: this association has not previously been reported. Both patients had atypical presentations of their primary disease, one with parotitis and one with a Guillain-Barre syndrome. Other causes of MAHA were excluded and a possible link with macromolecular von Willebrands factor is speculated upon. PMID- 8624644 TI - Letter from Kuwait. PMID- 8624645 TI - Quality of care and patient satisfaction. PMID- 8624646 TI - Comparison of a community clinic with a hospital out-patient clinic in rheumatology. AB - Community clinics are increasingly advocated as a way of delivering specialist health care. In this study, a hospital-based clinic is compared to a rotating community clinic in terms of descriptive consultation data, patient satisfaction, case mix and cost. Among other things, community clinics were more local (1.6 vs 4.9 miles), involved less waiting for the first appointment (47 vs 27% seen within 1 month), provided longer consultation times (49 vs 31% 20 min appointments) and produced more satisfactory consultations (82 vs 52% said their questions were always answered). Consultation data, however, showed that less patients were seen in the community clinic (8.6 vs 14.1 patients/doctor/clinic), with a higher old/new ratio (6.04 vs 3.96) and the cost per patient was higher (15.93 pounds vs 10.35 pounds). No differences were found in the case-mix data. PMID- 8624647 TI - The clinical need for an acute rheumatology referral service. AB - Should rheumatologists provide an acute referral service for general practitioners (GPs) and other clinical units? Is it cost effective? We prospectively studied acute referrals to one unit over 10 months, recording their source, diagnosis, management and outcome. Current rheumatology patients and cases only needing telephone advice were excluded. There were 253 referrals: 82 from GPs, nine from Accident and Emergency, and 162 from other hospital units. Their diagnoses comprised connective tissue diseases (22), back pain (46), inflammatory arthritis (59), osteoarthritis (22), paediatric cases (11), soft tissue problems (41) and 52 other disorders. Thirty-two needed active treatment within 24 h (classified as emergencies); examples included cerebral lupus, vasculitic pulmonary haemorrhage, retroperitoneal lymphoma with sacral plexus compression, temporal arteritis with reduced visual acuity and acute monoarthritis. All needed immediate therapy; only one died. Most (176 cases) were less urgent and needed advice in 48 h. Examples included osteoporotic vertebral collapse and acute rheumatoid disease. Forty-five could have been seen routinely; examples included lateral epicondylitis and adhesive capsulitis. The service required 1 day per week of medical staff time at an average cost of 45 pounds per case. We concluded that an acute rheumatology service is needed; it can be provided within the working day and is cost effective. PMID- 8624648 TI - Report on the Hypermobility Special Interest Group, British Society for Rheumatology meeting in Glasgow, April 1995. PMID- 8624649 TI - Report of Special Interests Group held at the British Society of Rheumatology meeting in Glasgow, April 1995. PMID- 8624650 TI - Undifferentiated seronegative spondyloarthropathy in females. PMID- 8624651 TI - Lymphatic function in inflammatory arthritis. PMID- 8624652 TI - Flatfeet in pregnancy. PMID- 8624653 TI - CD69 on synovial T cells in rheumatoid arthritis correlates with disease activity. PMID- 8624654 TI - Re: Audit of shared care between hospital and general practice in the management of rheumatoid arthritis. PMID- 8624655 TI - Leg veins and stretch marks. Have they seen the light? PMID- 8624656 TI - Photothermal sclerosis of leg veins. ESC Medical Systems, LTD Photoderm VL Cooperative Study Group. AB - BACKGROUND: The flashlamp-pumped pulse dye laser at 585 nm has previously been reported to be somewhat effective in treating leg telangiectasia with diameters less than 0.2 mm. A pulsed photothermal devise (intense pulsed light source [IPLS]) has been developed to treat leg veins ranging in size from 0.1 to 3 mm in diameter. Vessel necrosis occurs from an intense pulsed light that penetrates through the skin and is absorbed by the blood vessels based on the principle of selective photothermolysis. OBJECTIVE: To determine the effectiveness of this novel device on treating leg veins. METHODS: One hundred fifty-nine patients with 369 lesions were treated with the IPLS in a multicenter trial. RESULTS: Clearance of 75-100% was achieved in 79% of treated lesions. Better than 50% clearance was achieved in 94% of completed cases. A very low rate of adverse effects occurred. CONCLUSION: The IPLS is a safe and effective modality to treat leg veins ranging in size from 0.1 to 3 mm in diameter. PMID- 8624657 TI - Treatment of stretch marks with the 585-nm flashlamp-pumped pulsed dye laser. AB - BACKGROUND: Striae, or stretch marks, are very common skin disorders that do not impair bodily function, but are of considerable cosmetic concern to many patients. Traditionally, treatment options have been very limited. This study examines the results of treating striae using the 585-nm pulsed dye laser. Stimulation of a variety of wound healing processes has been attributed to low energy laser therapy. Clinically, improvement of hypertrophic and erythematous scars with the 585-nm pulsed dye laser at energy densities of 6-7 J/cm2 is well established. Since striae are dermal scars, evaluation of this same therapy to treat striae was undertaken. OBJECTIVE: To evaluate the effectiveness of the 585 nm flashlamp-pumped pulse dye laser in treating cutaneous striae. METHODS: Thirty nine striae were treated with four treatment protocols. These treated striae were compared with untreated striae controls in the same patient. The patients ages ranged from 23 to 52 years, with an average age of 36 years. The average age of the treated striae prior to initial treatment was 14 years (range, 8 months to 32 years). Treatment parameters included spot sizes of 7 and 10 mm and fluences of 2.0, 2.5, 3.0, and 4.0 J/cm2. Response to therapy was evaluated through clinical grading, sequential photography, and optical profilometry at a blinded laboratory. Skin biopsies were also examined with light microscopy from two of the 39 striae that were treated. RESULTS: Subjectively, striae appeared to return toward the appearance of normal skin with all protocols. However, the protocol with 10-mm spot size using 3.0 J/cm2 fluence improved the appearance of striae better than the other treatment protocols. Objectively, shadow profilometry revealed that all treatment protocols reduced skin shadowing in striae. This result corresponds with surface patterns of striae returning to that closely resembling adjacent normal skin surface patterns. Histologically, using hematoxylin and eosin stains as well as elastin strains, striae treated with a low fluence pulsed dye laser treatment protocol regained normal appearing elastin content when compared with normal (non-striae) skin adjacent to the treated striae. CONCLUSION: Treatment with the 585-nm pulsed dye laser at low energy densities was shown to improve the appearance of striae. Apparent increased dermal elastin was also observed 8 weeks posttherapy and possibly contributed to the improvement seen in the study patients. PMID- 8624658 TI - Porokeratosis and cutaneous malignancy. A review. AB - BACKGROUND: Porokeratosis is a disorder of epidermal keratinization manifested clinically by a raised border and histologically by a cornoid lamella. While cutaneous malignancy has been reported to arise in porokeratosis, the risk remains unknown. In the past it has been associated with a history of radiation therapy. OBJECTIVE: The purpose of this study is to estimate the frequency of cutaneous malignancy arising in porokeratosis and define those patients who are at highest risk. METHODS: All cases in the English language literature in the last 30 years were reviewed. RESULTS: Twenty-one (7.5%) of 281 cases reported revealed a malignancy arising within porokeratosis. Large lesions, those of long standing duration, and the linear type were at greatest risk. Radiation therapy was an infrequently identified risk factor. CONCLUSION: Porokeratosis is a premalignant condition, with certain groups of patients at greatest risk for malignant transformation. PMID- 8624659 TI - A histopathological comparison of "char-free" carbon dioxide lasers. AB - BACKGROUND: New "char-free" carbon dioxide lasers are capable of precise tissue vaporization with minimal residual thermal damage. These lasers operate either by producing high energy, rapid pulses or by scanning a highly focused continuous mode beam. OBJECTIVE: To determine the depth of ablation and the depth of residual thermal injury produced with one to three passes of the pulsed and scanned systems. METHODS: The distal ends of preauricular donor skin from 12 patients requiring full-thickness skin grafts following Mohs micrographic surgery were treated with zero to three passes of each of the lasers, and fixed for histopathological analysis. RESULTS: The three lasers tested produced vaporization of thin (20-50 microns) layers of tissue and narrow (20-150 microns) zones of thermal injury following one, two, or three passes on intact skin. CONCLUSION: The pulsed and scanned technologies are capable of producing "char free" tissue ablation with minimal residual thermal damage. PMID- 8624660 TI - Basal cell carcinoma on the neck. AB - BACKGROUND: In our Mohs surgery practice, a large number of basal cell carcinomas (BCCs) occurring on the neck were noted to be of the superficial type. OBJECTIVE: Our purpose was to examine a series of consecutive cases of BCC on the neck. METHODS: We reviewed all cases of BCC on the neck that were treated in our Mohs surgery unit from 1988 to 1993. Permanent histologic sections of the BCCs, obtained by excisional debulking of the tumors, were examined and the BCCs were typed histologically. Each histologic type was correlated with the patient's age, race, sex, its location on the neck, and its status as either a primary or recurrent lesion. RESULTS: In total, 97 BCCs on the neck from 93 patients were examined. All patients were Caucasians with an average age of 62.7 years. A peak incidence in the fifth decade occurred in males while this peak occurred in the eighth decade for females. Males outnumbered females 3.4:1. The type and incidence of each BCC was studied with the following results: superficial (38.1%), mixed-superficial (30.0%), nodular (15.5%), infiltrative (7.2%), morpheaform (5.1%), adenoid (2.1%), keratotic (1.0%), and metatypical (1.0%). Fifty-one percent of the tumors were primary and 49% were recurrent. The most common location on the neck was the skin overlying the superior aspect of the sternocleidomastoid muscles. CONCLUSION: BCCs occurring on the neck were most commonly of the superficial type. PMID- 8624661 TI - Consecutive wide and long single hair-bearing transposition flaps in combination with hair transplantation for the management of slot formation after alopecia reducing surgery. AB - BACKGROUND: Because the Frechet-style triple hair-bearing transposition flaps use up a great deal of scalp laxity from both occipitoparietal areas, it is virtually impossible to perform simultaneous donor harvesting for hair transplantation. The flaps in this technique are also prone to tip necroses due to their narrow width and pointed tips. This article presents a sequential protocol that dramatically and easily treats the slot, while simultaneously allowing hair transplantation to be performed to the frontal area. OBJECTIVE: To present a simple method that combines hair transplantation with consecutive wide and long single hair-bearing transposition flaps for the management of slot formation after alopecia reducing surgery. METHOD: A wide and long single hair-bearing transposition flap is first performed. The area directly inferior and contralateral to the side from where the superior flap is transposed is then used for donor harvesting. This method is repeated with each progressive hair grafting session until the desired result is accomplished. RESULTS: In the over 100 cases performed by the author to date, the results have been consistently excellent with complications nonexistent thus far. CONCLUSION: Using consecutive wide and long single hair-bearing transposition flaps is a safer, more aesthetically pleasing, and easier way to manage slot formation when compared with the triple flap technique. Besides these advantages, it affords the surgeon the opportunity to work on the frontal hairline immediately after the flap procedure is performed. PMID- 8624662 TI - Recollagenation of acne scars. AB - BACKGROUND: Depressed acne scars remain a difficult problem to correct with present methods. OBJECTIVE: A new process to restore the dermal thickness destroyed by acne inflammation, called recollagenation, was developed. METHODS: After an intradermal pocket was created at each depressed site, an implant of freeze-dried, irradiated, human cadaver fascia lata was inserted through a needle hole to elevate the depressed epidermis. Selected sites were retreated to establish the desired dermal topography. RESULTS: Initially, local inflammation created an elevated phlegmon at the treatment site, which then progressively leveled over the subsequent 4-10 weeks. Ultimately, a predictable improvement in skin contour occurred in the majority of depressions. CONCLUSION: Recollagenation is an effective method to repair acne lesions. PMID- 8624663 TI - The intrinsic antimicrobial activity of selected sclerosing agents in sclerotherapy. AB - BACKGROUND: Detergent sclerosing agents may have intrinsic antimicrobial properties. In addition, they may have synergistic effects with other antibiotics such as penicillin. They may induce suppression of intrinsic resistance to penicillin in Staphylococcus aureus. OBJECTIVE: It is in this setting that the present study was carried out in order to determine the degree of suppression of resistance to methicillin and oxacillin in S. aureus by two detergent sclerosing solutions. METHODS: Four strains of S. aureus including a quality control strain were isolated. The minimal inhibitory concentration (MIC) of Sotradecol 1.0% and Polidocanol 0.5% were determined in Mueller Hinton Broth. These dilutions were subsequently seeded with 10(5) organisms of the strain of S. aureus being tested. Serial dilutions of penicillin were made and then the sclerosing agents were added in the appropriate dilutions. RESULTS: Sotradecol 1.0% produced a MIC of 1/64 in two strains of S. aureus and 1/128 in two other variant strains. Polidocanol 0.5% produced a MIC of 1/64 against two strains of S. aureus and an MIC of 1/8 and 1/4 with two other variant strains. In addition, in three of the four S. aureus strains both sclerosing agents had synergistic activity with penicillin and augmented its activity approximately 16-fold. CONCLUSION: This study presents the first successful modification in which detergent sclerosing solutions influence methicillin resistance in a Staphylococcal species. This points out a new potential therapeutic indication for this class of agents. PMID- 8624664 TI - Stockings and the prevention of recurrent venous ulcers. AB - BACKGROUND: Cost-effective therapy that heals ulcers rapidly and prevents recurrence would significantly impact patient care and the health system. OBJECTIVE: To evaluate compression stockings for treatment of venous ulcerations and prevention of recurrent ulceration; to analyze patient compliance; and to evaluate cost of compression stocking therapy. METHODS: Stocking therapy healed venous ulcers in 53 patients. The effect of continued stocking use on ulcer recurrence rate and treatment costs was evaluated. RESULTS: Twenty-five patients had good stocking usage; one developed recurrence (4%). Twenty-eight patients had bad or none usage; 22 had at least one recurrence (79%). Bad/none usage was associated with 31 of 32 (97%) recurrent ulcerations; good usage was associated with 52 of 58 (90%) nonrecurrent ulcers. Cost was a major reason for noncompliance. CONCLUSIONS: Continued stocking use after ulcer healing will prevent most recurrences and will provide a significant cost saving to the nation's health care budget. PMID- 8624665 TI - Thrombotic complications of varicose veins. A literature review of the role of superficial venous thrombosis. AB - BACKGROUND: Some recent publications have emphasized the risk (up to 25%) of deep venous thrombosis (DVT) coexisting with a clinical evidence of superficial venous thrombosis (SVT). However, most papers on this topic are old and do not consider the use of the duplex scanning. OBJECTIVE: To determine what the spontaneous risk is of venous thrombosis and emboli in varicose patients, in the superficial veins, and in the deep veins; what the risk is of extension or coexistence between superficial and deep thrombosis; and whether the treatments of varicose disease are responsible for thrombosis? METHODS: Review of the literature. RESULTS: The frequency of venous thrombosis appears to be increased in patients with varicose disease. CONCLUSIONS: In all cases of clinical SVT a duplex scan examination of both deep and superficial veins is necessary in order to provide a complete diagnosis. The treatment of SVTs depends on the situation and the size of the thrombi. In case of associated DVT, the most important treatment is of the DVT. The interest of heparin or low molecular weight heparin (therapeutic doses) is proved for patients with coexisting DVT, and thought so for ascending SVT. Interest and doses have not been stated in other cases. SVT must be considered as a risk factor of DVT and treated from this point of view. Biological analysis and a complete check-up are mandatory in cases of varicose thrombosis in young patients and in cases of recurrence. PMID- 8624666 TI - Duplex ultrasound examination of the acutely painful and swollen leg. AB - OBJECTIVE: The aim of this study was to assess the value of duplex ultrasound examination in establishing diagnosis in acutely swollen and painful lower limbs, and to examine how often the diagnosis of superficial and/or deep venous thrombosis can be made in the symptomatic legs. METHODS: Venous duplex scan examination was performed on 188 extremities of 180 ambulatory outpatients. The examination included testing patency and competency of the deep and superficial veins. The ultrasound study was also extended to finding soft tissue abnormalities. RESULTS: Of the 188 lower limbs venous thrombosis was found on 82 occasions: 29 legs contained deep venous thrombosis only; 23 legs presented with superficial thrombophlebitis as a sole venous pathology; and in 30 cases superficial and deep venous thrombosis coexisted. In those 106 limbs where venous thrombosis was not present the most frequent positive ultrasound findings were incompetent leg veins or soft tissue masses (Baker's cyst and calf haematoma). CONCLUSION: The variety of pathological processes related to the lower limb veins and the extravascular tissue suggest that routine, "detailed" ultrasound scanning would be indicated in all acutely symptomatic legs with a suspected venous disorder. The important diagnostic information so gained provides guidance to the most appropriate treatment strategy. PMID- 8624667 TI - The treatment of the splitting nail with phenol alcohol partial nail matricectomy. AB - BACKGROUND: Split nails are a common problem originating in focal thinning of the nail matrix. OBJECTIVE: To determine if cosmetically acceptable results could be achieved utilizing phenol alcohol partial nail matricectomy in patients with split nails. METHODS: After proper anesthesia, the nail lateral to the split is avulsed followed by destruction of the corresponding portion of the nail matrix to prevent the avulsed nail from regrowing. RESULTS: Phenol alcohol partial nail matricectomy has proven to be a simple, effective, reproducible treatment with excellent cosmetic results for laterally located split nails. CONCLUSION: A simple nail procedure, widely used for ingrown nails, can be easily adapted for the definitive treatment of split nails. PMID- 8624669 TI - VIIIth French Congress of Rheumatology. Paris, France, November 13-15, 1995. Proceedings and abstracts. PMID- 8624668 TI - Low-dose glucocorticoid therapy in rheumatoid arthritis. PMID- 8624670 TI - Apoptosis: relevance to rheumatology. PMID- 8624671 TI - Exercise-induced lower leg pain. PMID- 8624672 TI - Strategy for identifying primary malignancies with inaugural bone metastases. AB - The diagnosis of primary tumors with inaugural bone metastases is a serious and difficult problem commonly encountered in rheumatology. Helpful information can be derived from the radiologic appearance of the metastases, history of the patient, clinical findings, chest film, standard laboratory tests, imaging studies, serum marker assays, and histologic findings. Based on our personal experience and on previously published data, we have developed a decision tree aimed at enhancing the efficacy of the diagnostic process while ensuring optimal patient comfort and containing costs. PMID- 8624673 TI - Encephalomyelitis in primary hypogammaglobulinaemia. AB - The neurological features of 13 patients with primary hypogammaglobulinaemia are described. Seven patients had X-linked agammaglobulinaemia (XLA) and six had common variable immunodeficiency (CVID). Three clinical pictures emerged: (i) a progressive myelopathy (one case); (ii) a myelopathy progressing to an encephalopathy (four cases); (iii) a pure encephalopathy (eight cases). In four patients the encephalopathy was temporarily reversible; the relationship of this to immunoglobulin therapy is unclear. Additional features occurred in some patients. Three had retinopathy interpreted as retinitis pigmentosa, in one of whom the retinopathy resolved. Two patients had a sensori-neural hearing loss and three had features of dermatomyositis; a variable pleocytosis was found in the CSF of nine patients. Imaging revealed atrophic changes in the cerebral hemispheres in eight cases. Ten patients have died, 1-11 years after the onset of the CNS manifestations, and in four autopsies were obtained. Two patients had encephalopathy, one with XLA had evidence of end-stage encephalitis and the other with CVID had a multi-focal leucoencephalopathy. The other two with XLA had leptomeningitis without evidence of encephalitis. Enteroviral infection is probably an important cause of neurological disease in these patients as CSF from seven patients was either positive by polymerase chain reaction (PCR) or by culture for enteroviruses. Other possible mechanisms are discussed. PMID- 8624674 TI - Deactivation of human visual cortex during involuntary ocular oscillations. A PET activation study. AB - Prompted by the observation of decreased glucose metabolism in the striate and extrastriate visual cortex in a patient with opsoclonus, we studied the influence of involuntary eye movements on visual cortex activity. Repeated measurements of cerebral blood flow (CBF) by PET were performed in 12 healthy volunteers using H2(15)O-bolus technique after ear canal irrigation with ice cold or warm (44 degrees C) water with the subjects eyes closed. In addition to blood flow increases in areas involved in central vestibular processing, statistical subtraction analysis revealed a nearly symmetrical, bilateral, highly significant decrease in the occipital cortex covering Brodmann areas 17, 18, and 19 after ice water stimulation of either ears. Region of interest analysis revealed in all subjects a mean decrease in regional CBF (rCBF) of 12.8% (range 4.6-21.0%) in these areas. A similar but less pronounced effect (mean rCBF decrease in visual cortex 4.8%, range 1.1-11.5%) was observed after warm water irrigation. The observations suggest that deactivation of the visual cortex is induced by involuntary ocular oscillations. This deactivation is not dependent on changes of the retinal input (eyes closed). The physiological significance of this hitherto unknown phenomenon may be the protection from inadequate visual input (oscillopsia) during involuntary ocular oscillations. PMID- 8624675 TI - Identification of famous faces and famous names in early Alzheimer's disease. Relationship to anterograde episodic and general semantic memory. AB - We assessed remote memory in 33 patients with dementia of Alzheimer's type (DAT) with Mini-Mental State Examination (MMSE) scores between 17 and 30, and 30 matched controls using a Famous Faces Test and Famous Names Test designed to assess face recognition, identification and naming, and name recognition and identification, respectively, together with a range of anterograde episodic and semantic memory tests. Patients with DAT were impaired on all components of the remote memory tests, i.e. famous face recognition, identification and naming, and famous name recognition and identification. There was also evidence of a modest temporal gradient, with relatively greater impairment of more recent memory, which may be artefactual resulting from the very insidious onset of their anterograde amnesia. In contrast to the uniform impairment of anterograde memory, there was considerable heterogeneity in performance on remote memory. Although the DAT patient group's performance on remote memory measures was impaired with respect to controls, some patients had significant impairment on all measures, whereas others had intact remote memory. Overall, there was only a weak correlation between dementia severity and remote memory, and no correlation between performance on the Faces and Names tests and measures of anterograde memory. At a cognitive level, the deficit in face and name processing in DAT involved recognition, identification and naming. This would suggest that so called 'face and name recognition units', semantic knowledge of famous persons and post-semantic processing are all affected by the disease. There was also supporting evidence for the concept that recognition of famous faces and names both draw on common sources. Similar results were found for face and name identification. This suggests that face and name recognition units are closely linked, and that identification of a face or name accesses the same central pool of semantic knowledge regarding the famous person. Performance on famous names tests correlated, to a limited degree, with that on general semantic tests, suggesting that knowledge of famous people, at least as accessed by names, is associated with general semantic memory. By contrast, no correlation was found between performance on the famous faces and on other general semantic tasks. PMID- 8624676 TI - The fractionation of remote memory. Evidence from a longitudinal study of dementia of Alzheimer type. AB - We studied remote memory, both autobiographical and public, longitudinally over a 1-year period in 24 patients with dementia of Alzheimer type (DAT) and 30 matched controls. Although both public and autobiographical memory were impaired in DAT, public memory deteriorated longitudinally, while autobiographical memory did not. These data support the hypothesis that remote memory may be fractionated and that one important dichotomy is autobiographical memory versus famous person knowledge. A cognitive analysis of famous face and name processing showed evidence of progressive breakdown in the identification of famous faces and names, with preservation of face and name recognition, and face naming. The declining performance on identification appeared to be due primarily to loss of semantic knowledge regarding famous persons, while retrieval a deficit contributed more significantly to the proper name anomia which was over and above the semantic deficit in DAT. PMID- 8624677 TI - Is developmental dyslexia a disconnection syndrome? Evidence from PET scanning. AB - A rhyming and short-term memory task with visually presented letters were used to study brain activity in five compensated adult developmental dyslexics. Their only cognitive difficulty was in phonological processing, manifest in a wide range of tasks including spoonerisms, phonemic fluency and digit naming speed. PET scans showed that for the dyslexics, a subset only of the brain regions normally involved in phonological processing was activated: Broca's area during the rhyming task, temporo-parietal cortex during the short- term memory task. In contrast to normal controls these areas were not activated in concert. Furthermore the left insula was never activated. We propose that the defective phonological system of these dyslexics is due to weak connectivity between anterior and posterior language areas. This could be due to a dysfunctional left insula which may normally act as an anatomical bridge between Broca's area, superior temporal and inferior parietal cortex. The independent activation of the posterior and anterior speech areas in dyslexics supports the notion that representations of unsegmented and segmented phonology are functionally and anatomically separate. PMID- 8624678 TI - Noun and verb retrieval by normal subjects. Studies with PET. AB - PET activation studies identify significant local changes in regional cerebral blood flow (rCBF) in contrasts of behavioural tasks with control states, and these local changes identify net changes in local synaptic activity. A number of studies on word retrieval have all demonstrated left frontal (dorsolateral and medial) involvement in the task. However, there have been differences in the responses observed in the left temporal lobe, with variously a deactivation (significant decrease in rCBF), no response and an activation (significant increase in rCBF). In the four studies described here, we have examined word (verbs and nouns) retrieval contrasted with a number of different control states. The studies confirmed extensive activation of the left dorsolateral prefrontal cortex and, medially, the anterior cingulate cortex and the supplementary motor area (SMA). Activations of the left posterior temporal lobe and the inferior parietal lobe were consistently demonstrated when word retrieval was contrasted with a rest state. Contrasts with other single word tasks controlled out the activation in the perisylvian part of the left posterior temporal lobe, suggesting a role for this region in lexical processing. The left inferolateral temporal cortex and the posterior part of the inferior parietal lobe were only activated by word retrieval, particularly verbs. It is proposed that these activated regions reflect access to semantic fields. PMID- 8624679 TI - An analysis of clinical seizure patterns and their localizing value in frontal and temporal lobe epilepsies. AB - The differentiation of frontal lobe epilepsy (FLE) and temporal lobe epilepsy (TLE) is a clinical problem of major theoretical and practical importance. Current electroclinical classification is based on retrospective studies of highly selected patients. When applied to the presurgical evaluation of patients, it has poor specificity. The current study adopts a different and prospective approach to the analysis of ictal clinical manifestations and their value in differentiating FLE and TLE. Two hundred and fifty-two patients with partial epilepsy were selected according to criteria of focal abnormality and imaging, ictal EEG or interictal EEG or highly focal clinical pattern. A witnessed seizure description was obtained for each of their habitual seizures and the sequence of manifestations encoded and entered into a statistical cluster analysis to form a clinical classification of the 352 seizures identified, which comprised 14 clinical groups. Neuroimaging abnormalities were measured, using a template technique, and graded 0-3 according to extent of involvement of each region in the lesion, using standard anatomical divisions. A chi 2 analysis of lesion location against seizure type was performed to assess the strength of association of seizure types with specific cerebral regions. The distribution of interictal EEG spikes and ictal EEG onsets were assessed qualitatively. An independent analysis was also performed, comparing clinical seizure manifestations associated with lesions restricted to either frontal or temporal lobes. Of the 14 clinical groups, four were predominantly related to temporal lobe abnormalities: fear/olfactory/gustatory; absence with no focal symptoms; experiential and visual. Within these groups, 45 out of 58 lesional cases involved the temporal lobes (P<<0.001). A minority of seizures in these groups were associated with frontal lesions and these seizures were significantly more likely to involve version/posturing, without an intervening absence phase, than the temporal cases (P<0.001). Two groups were related to perirolandic abnormalities; somatosensory and Jacksonian clonic with 22 out of 24 lesional cases involving this region (P<0.001). Two other groups were related to the frontal lobes; version/posturing and motor agitation. Early focal tonic activity or head turning were associated with lateral premotor lesions (P<0.001) and ictal and interictal EEG showed strong frontal predominance. Seizures characterized by general motor agitation were associated with lesions of the orbitofrontal (eight out of thirteen cases) and frontopolar (six out of thirteen cases) cortices (P<0.001). Location of interictal EEG spikes and ictal EEG onsets were generally consistent with lesion sites and where there were discrepancies, EEG localization tended to be more diffuse than lesion localization, rather than frankly discordant. Analysis of manifestations associated with pure frontal and pure temporal lesions supported the results of the cluster analysis and also showed a significant association of oro-alimentary automatisms with temporal lobe abnormalities. There were no consistent differences between groups with different localizations in terms of seizure frequency or other characteristics of seizure timing, although very high seizure frequencies were seen more often in association with frontal lesions. Only one combination of different seizure types in the same patient occurred with statistical significance: absence and generalized motor seizures and pseudo generalized epilepsy. The results of this study suggest that relatively few seizures can be localized reliably on clinical grounds and that even in those seizure types where there is a statistically significant association with specific cortical areas, an important minority do not share the same associations. Analysis of the seizure evolution as well as initial symptoms may be of value in localizing some cases, but even here wide variation occurs... PMID- 8624680 TI - Quantification of cortical atrophy in a case of progressive fluent aphasia. AB - A patient with a rapidly developing fluent progressive aphasia was tested prospectively up to the time of death and examined neuropathologically. Severe impairment in accessing semantic skills with substantially intact phonological, syntactic and discourse skills was found. Some social behavioural difficulties were also noted. This case presented a unique opportunity to relate this significant language impairment to the pattern of neurodegeneration, a difficult task in most neuropathological studies of severe end-stage dementia. A detailed neuropathological examination revealed focal atrophy with neuronal loss without neuronal inclusions (Pick bodies, Lewy bodies, neurofibrillary tangles or senile plaques) or neuronal changes (shrinkage or swelling). In addition, spongiform degeneration (confined to layer two of the cortex) and gliosis were detected at atrophic sites. To establish the amount of tissue loss and pathology associated with the focal language deficit, volume analyses were performed and compared with two age- and sex-matched, neurologically normal controls. Both the left and right angular gyri and Brodmann's area 37 showed marked volume reduction compared with controls. The predominant language impairment seen in this case is likely to reflect these marked changes in the posterior parieto-temporal areas. The milder unilateral atrophy was concentrated in the right temporal lobe as well as the right hemisphere homologue of Broca's area. Recent work suggests a relationship between such unilateral changes and the social behavioural difficulties which were noted in this case. The hippocampus and other gyri such as the supramarginal gyrus showed no volume loss compared with controls correlating with the relative preservation of other language skills. PMID- 8624682 TI - Incidence of multiple sclerosis in More and Romsdal, Norway from 1950 to 1991. An age-period-cohort analysis. AB - We have examined the time trends in multiple sclerosis incidence over the past four decades from 1950 to 1991 in More and Romsdal County, Norway. Incidence rates by year of birth, year of onset and year of diagnosis according to sex, age and clinical course were studied. All patients with multiple sclerosis diagnosed by August 1992 were included, giving a total of 419 patients (171 men, 248 women) with onset of multiple sclerosis in the period 1950-91. Mean age at onset was 33.2 years, and mean age at diagnosis was 39.2 years. The incidence rate by year of onset increased from 2.87 per 100 000 in the period 1950-54 (men 3.06, women 2.67) to 5.57 per 100 000 in 1985-91 (men 3.75, women 7.94) (P<0.001). The incidence was particularly high in the period from 1975 to 1985. Major period- or cohort-effects in incidence, apart from the general increasing trend, could not be identified by an age-period-cohort analysis. PMID- 8624681 TI - Non-spatial attention disorders in patients with frontal or posterior brain damage. AB - The few studies that have looked at attention in patients with brain damage suggest a prominent role for the frontal lobe in nonspatial attentional control. However, the studies usually focus on one variety of attention and do not address the nature of the alteration of attention. In addition, the behavioural consequences of nonspatial attentional deficit remain unknown. The aim of this study was to evaluate the role of focal brain damage on divided and focused attention and the relationship between attention disorders and behavioural changes. The study group consisted of patients with lesions of the prefrontal and posterior cortices and control subjects. The assessment of attention used reaction time tests that evaluated the ability to divide attention between two sources (detection tests) and to focus attention on one source (Go/No-Go Tests). The response retardation of the 'frontal' group became more pronounced as the number of sources to be monitored increased, suggesting the presence of a deficit of divided attention. Focused attention deficit was demonstrated in the 'frontal' group by the more frequent responses to irrelevant stimuli on Go/No-Go Tests. Both focused and divided attention deficits were prominent when the lesion included the left dorsolateral prefrontal cortex and the caudate nucleus. Selective deficit of divided or focused attention was shown in a few patients. Finally, the clinically assessed distractibility was related to disorders of divided and focused attention. This study provides additional evidence for the prominent role of the frontal lobe in nonspatial attention regulation and shows that it also operates in elementary perceptuomotor processes. The relationship between distractibility and attention indexes supports the idea that attention disorders may have a functional counterpart that is clinically assessable. The demonstration of selective deficit of divided or focused attention suggests that nonspatial attention depends upon different mechanisms and that it is not an undifferentiated general purpose mechanism. Further studies addressing the nature of the interactions between attentional mechanisms and other cognitive processes are required. PMID- 8624683 TI - Augmented expression of tumour necrosis factor-alpha and lymphotoxin in mononuclear cells in multiple sclerosis and optic neuritis. AB - The involvement of proinflammatory cytokines tumour necrosis factor-alpha (TNF alpha) and lymphotoxin (LT) in multiple sclerosis is suggested by the parallel occurrence of these proinflammatory cytokines in acute and chronic active multiple sclerosis brain lesions. We describe the use of in situ hybridization with radiolabelled cDNA oligonucleotide probes to detect and enumerate TNF-alpha and LT mRNA expressing mononuclear cells without culture, and after culture in the presence of myelin basic protein (MBP), control antigens or without antigen. Compared with patients with aseptic meningo-encephalitis, non-inflammatory neurological diseases and healthy controls, the multiple sclerosis patients had elevated numbers of TNF-alpha and LT mRNA expressing mononuclear cells in blood when enumerated without previous culture, and also after culture with MBP. The MBP-induced upregulation of TNF-alpha and LT was major histocompatibility complex (MHC) class II molecule dependent. Tumour necrosis factor-alpha mRNA expressing mononuclear cells were further enriched in the multiple sclerosis patients' CSF. Positive correlations were observed in multiple sclerosis between TNF-alpha and LT mRNA expressing blood mononuclear cells, MBP-reactive TNF-alpha and LT mRNA expressing cells, and TNF-alpha and interferon-gamma (INF-gamma) mRNA expressing mononuclear cells. Upregulation of TNF-alpha correlated positively with exacerbation, enhanced disability and the secondary progressive phase of multiple sclerosis. Patients with optic neuritis, in many instances representing very early multiple sclerosis, had TNF-alpha and LT positive blood mononuclear cells that were elevated to the same extent as patients with clinically definite multiple sclerosis. The findings support the hypothesis that TNF-alpha and LT play a harmful role in the development of multiple sclerosis and suggest that TNF alpha could be useful as a disease activity marker in multiple sclerosis. PMID- 8624684 TI - Transient increase in symptoms associated with cytokine release in patients with multiple sclerosis. AB - Fourteen patients with multiple sclerosis were treated with the humanized monoclonal antibody CAMPATH-1H which targets the CD52 antigen present on all lymphocytes and some monocytes; four also received anti-CD4 antibody. Lymphopaenia developed rapidly and was sustained for at least 1 year. In 12 patients, the first infusion of antibody was characterized by significant exacerbation or re- awakening of pre-existing symptoms lasting several hours. These clinical effects of antibody treatment correlated with increased levels of circulating cytokines. Peak levels of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma occurred at 2 h, whereas the rise in interleukin-6 (IL-6) was significantly delayed and peaked at 4 h after starting antibody treatment. There was a decline in CH50, indicating complement activation. The neurological symptoms could not be attributed directly to pyrexia and were not provoked (in one patient) by an artificial rise in temperature. In the remaining two patients, a single pre-treatment with intravenous methylprednisolone (500 mg) prevented both the transient increase in neurological symptoms and the cytokine release. Our results, involving 14 intensively studied patients treated with humanized monoclonal antibodies, suggested that soluble immune mediators contribute to symptom production in multiple sclerosis; the mechanism remains uncertain but, on the available evidence, we favour the interpretation that cytokines directly affect conduction through partially demyelinated pathways. PMID- 8624685 TI - In vivo MRI and its histological correlates in acute adoptive transfer experimental allergic encephalomyelitis. Quantification of inflammation and oedema. AB - In vivo proton MRI was carried out on a 7 Tesla system at 2-3 day intervals over 10 days in rats with adoptive transfer experimental allergic encephalomyelitis (AT-EAE), an animal model of some aspects of multiple sclerosis. In order to assess the integrity of the blood-brain barrier (BBB), MRI was performed by acquiring quantitative MR-relaxation time T1 images of the AT-EAE rat brain before and after i.v. injection gadolinium-diethylene triaminepentaacetic acid (Gd-DTPA) using an ultrafast MRI technique. The MRI findings were compared with the immunohistochemical stain of T cells, macrophages and albumin and, in addition, apoptosis of T cells was assessed using in situ nick translation (ISNT). Prior to injection of Gd-DTPA, an increase of T1 times in the brain of the AT-EAE rats was observed, which paralleled the time course of albumin in histological sections. These were MRI findings observed well before the onset of major cellular infiltration and before the onset of clinical signs. After i.v. injection of Gd-DTPA the observed decrease of T1 times paralleled macrophage activation, and less closely T-cell infiltration. Our results provide evidence that using MRI, it is possible to assess quantitatively the breach of the BBB and to distinguish in vivo between two components of the early phase of the lesion, inflammatory infiltrates and vasogenic oedema. PMID- 8624686 TI - Central activation of the trigeminovascular pathway in the cat is inhibited by dihydroergotamine. A c-Fos and electrophysiological study. AB - Recent studies have delineated a clear role for the trigeminal innervation of pain-sensitive intracranial structures in the pathophysiology of migraine. The development of new compounds for the treatment of the acute attack of migraine has led to a greater understanding of serotonin (5-hydroxytryptamine; 5HT) receptor diversity. The ergot alkaloids have been used in the treatment of acute attacks of migraine for many years and parenteral administration of dihydroergotamine (DHE) can be a useful treatment strategy. In this study, the question of a possible central site of action of DHE is considered using both anatomical and physiological approaches. The c-Fos method has been used to map functional activation of central neurons in response to stimulation of the superior sagittal sinus (SSS) in the cat. This structure has been used as it refers pain to the ophthalmic division of the trigeminal nerve in humans, and in cats induces changes in neuropeptides and cranial blood flow similar to those seen in migraine. In addition, the temporal aspects of the effect of DHE have been studied by making extracellular recordings from cells in the most caudal aspect of the trigeminal nuclear complex. Stimulation of the SSS results in Fos expression in the superfical laminae of the trigeminal nucleus caudalis and in the dorsal horn of C1 and C2. This activation is blocked by a clinically relevant dose of DHE. Similarly, cells can be recorded in this region that respond to SSS stimulation. This linked cellular activity can be inhibited by the same intravenous dose of DHE. Together, these studies show that DHE can inhibit activity in central trigeminal neurons. Since the sinus and its nerve supply are directly stimulated, the peripheral nerve/vessel innervation is bypassed and this inhibition cannot have happened at any other site. These data imply that drugs acting at the central trigeminal neurons may have a role in the treatment of acute attacks of migraine. PMID- 8624687 TI - Sensory pathophysiology in chronic acquired demyelinating neuropathy. AB - Pathophysiological changes in sensory fibres in chronic acquired demyelinating neuropathy (CADP) are poorly understood, and it is not known to what extent sensory loss may be due to axonal loss or to conduction block. Motor and sensory nerve condition were studied in 18 patients with CADP to delineate abnormalities in the compound sensory action potential (CSAP) recorded proximally along the limb. To distinguish small CSAPs from noise, near-nerve needle electrodes and electronic averaging were used. In all, 58 motor and 78 sensory nerves in the upper and lower limbs were studied, and in 29 nerves, motor and sensory conduction was compared over the same proximal and distal segments of the upper limbs. The proximal/distal amplitude ratio (P/D ratio) of the compound muscle action potential (CMAP) was reduced in 76% of the nerves compared with only 21% of the CSAPs. The amplitudes of CMAPs evoked and of CSAPs recorded distally were reduced to the same extent. The prolongation of the distal motor latency (DML) was linearly related to the reduction in amplitude of the CMAP whereas reduction of the distal sensory conduction velocity (SCVd) mainly occurred if the amplitude of the CSAP was reduced more than 70%. The proximal motor nerve conduction velocity (MCVp) was reduced by 40-50%, twice as much as the reduction in distal MCV (MCVd) (calculated from the reciprocal DML), and related to the reduction in the P/D ratio of the CMAP. The proximal SCV (SCVp) decreased approximately 20%, similar to the reduction in SCVd and out of proportion to the marked reduction of the MCVp. The results suggest different pathophysiological changes in sensory and motor fibres in CADP. Thus, nerve fibre loss could account for most of the abnormal parameters in sensory conduction, whereas demyelination was the dominating cause of motor nerve dysfunction. PMID- 8624688 TI - Regeneration through a long nerve graft used in the correction of facial palsy. A qualitative and quantitative study. AB - A surgical technique has been developed for the correction of established unilateral palsy in man. A long (20 cm or more) sural nerve graft is anastomosed to a facial nerve branch on the unaffected side and its distal end left lying free in the cheek of the affected side. After regeneration times of 5.5-14.5 months, the distal end of the graft is joined to a free (pectoralis minor) muscle graft. In due course the muscle graft contracts in unison with the unaffected side giving near normal symmetry to facial movements. In 30 cases (ages 6-52 years) qualitative and quantitative examination was made of the distal end of grafts taken at the time of joining the graft to the muscle. Total axon counts, myelinated plus non-myelinated, confirmed abundant regeneration when compared with total axons in the supplying facial nerve; myelinated fibres remained small (mean diameter 2.5 micrometers) over the range of regeneration times. Quantitation included non-myelinated axons because they probably have the potential to become myelinated once the nerve is functional. Numbers of regenerating axons were not correlated with age, nor with regeneration time. Lack of a distal connection did not appear to lead to secondary degeneration of the regenerated myelinated fibres. These were maintained in an 'immature' state for many months. This observation is of practical interest since it has been suggested that delayed connection to the distal target may have a deleterious effect on the outcome of the procedure. PMID- 8624689 TI - Interjoint coordination during pointing movements is disrupted in spastic hemiparesis. AB - Approaches to the rehabilitation of movement in spastic hemiparetic patients depend on knowledge of the underlying mechanisms of movement deficits. The goals of this study were to characterize end-point trajectories and interjoint coordination of arm pointing movements to different targets on a horizontal planar surface and to correlate disruptions in motor control in the affected arm of hemiparetic subjects with the level of spasticity and the degree of functional impairment measured clinically. Arm movements were studied in six normal and 10 hemiparetic subjects. Data from the affected arms of hemiparetic subjects were compared with those from their non-affected arms and to data from the arms of normal subjects. Subjects were seated in front of a horizontal surface adjusted to the height of the sternal notch with the trunk stabilized. They made planar arm reaching movements (20 and 40 cm) to four different targets located directly in front of them and in the ipsilateral and contralateral workspace. Kinematic data from the finger, wrist, elbow and shoulder were recorded with a three dimensional optical tracking system. Results showed that movement amplitudes were lower and movement times were significantly prolonged in the affected arms. Although trajectories were marked by deviations from smooth straight lines and characterized by increased dispersion and segmentation, even those subjects with the most severe spasticity could reach into all parts of the workspace with both their affected and non-affected arms. This indicated that movement planning in terms of extrapersonal space was unaffected in these subjects. On the other hand, the interjoint coordination of movements made into or out of the typical extensor or flexor synergies was equally disrupted. These findings suggest a bi-level control organization of pointing movements in both normal and hemiparetic subjects: the level of trajectory planning in extrapersonal space and the level specifying interjoint coordination according to the trajectory plan. Deficits in motor performance in stroke patients may be associated with problems at the second control level. This implies some strategies for the rehabilitation of stroke patients with motor disorders. Treatment aimed at improving arm function should be oriented toward restoring the normal sensorimotor relationships between the joints. We also found that while clinical spasticity scores were correlated with some aspects of motor performance, they provided little information about the movement deficit itself. PMID- 8624690 TI - Juvenile limb-girdle muscular dystrophy. Clinical, histopathological and genetic data from a small community living in the Reunion Island. AB - A series of patients affected by a muscular dystrophy, similar to the original description of a juvenile scapulo-humeral form by Erb in 1884 and fitting with the criteria used to define limb-girdle muscular dystrophies, was discovered in a small community living in the southern part of Reunion Island in the Indian Ocean. A detailed clinical analysis was conducted over 5 years on a cohort of 20 patients. This community presented a high degree of consanguinity as it was segregated from the majority of the island population for more than a century. In previous molecular genetic studies, the disease locus has been mapped to chromosome 15p. Mutations were recently identified in a gene located in this region encoding for muscle-specific calcium activated neutral protease (CANP3). Clinical, pathological, genetic and complete identification of the mutations are presented here, establishing, for the first time, precise clinico-genetic correlations in this form of autosomal recessive, juvenile, limb-girdle muscular dystrophy (LGMD). PMID- 8624691 TI - Abnormalities of the balance between inhibition and excitation in the motor cortex of patients with cortical myoclonus. AB - Patients with cortical myoclonus may have purely focal or multifocal jerks, or they may have additional bilateral or generalized jerks, suggesting the spread of excitatory myoclonic activity between the cerebral hemispheres and across the sensorimotor cortex. The factors contributing to this spread of activity were investigated in 10 patients with multifocal cortical myoclonus and eight patients with multifocal and bilateral or generalized cortical myoclonus. The two groups were termed 'non- spreaders' and 'spreaders' respectively. Eight of the patients were also epileptic. Motor thresholds to single transcranial magnetic shocks at rest were higher in 'non- spreaders' (median 88%, range 45-100% of stimulator output) than either 'spreaders' (50%, range 26-90%, P=0.023) or health controls (38%, range 28-53%, P<0.001). This pathological elevation in motor threshold was not simply an effect of treatment with antiepileptic drugs. Paired transcranial magnetic stimuli were used to investigate ipsilateral cortico-cortical and transcallosal inhibition, There was less (MANOVA, P<0.05) ipsilateral inhibition at interstimulus intervals (ISIs) of 1-6 ms in 'spreaders' (mean 107+/-SEM 23% of control) compared with 'non- spreaders' (75+/-15%) or healthy subjects (59+/ 10%). There was also less (P<0.05) transcallosal inhibition across inhibitory timings (10, 12 and 14 ms) in the 'spreaders' (98+/-6% of control) compared with the 'non-spreaders' (64+/-8%) or healthy subjects (59+/-6%). There was no relationship between ipsilateral cortico-cortical and transcallosal inhibition and the presence or absence of epilepsy, although non-epileptic patients did have higher motor thresholds (median 85%, range 32-100% of stimulator output) than either epileptic patients (50%, range 26-90%, P<0.001) or healthy controls (38%, range 28-53%, P=0.002). Abnormalities in ipsilateral and transcallosal inhibition appear to facilitate the spread of the cortical myoclonic activity responsible for bilateral and generalized jerks. However, these abnormalities in inhibition do not play a major role in the development of generalized seizures in patients with cortical myoclonus. PMID- 8624692 TI - The basal ganglia and apraxia. AB - Ever since Liepmann's original descriptions at the beginning of the century apraxia has usually been attributed to damage confined to the cerebral cortex and/or cortico-cortical connecting pathways. However, there have been suggestions that apraxia can be due to deep subcortical lesions, which raises the question as to whether damage to the basal ganglia or thalamus can cause apraxia. We therefore analysed 82 cases of such 'deep' apraxias reported in the literature. These reports consisted of a small number (n=9) of cases studied neuropathologically, and a much larger group (n=73) in which CT or MRI was used to identify the size and extent of the lesion. The reports were subdivided into (i) those with small isolated lesions which involved nuclei of the basal ganglia or thalamus only, and not extending to involve periventricular or peristriatal white matter; (ii) those with large lesions which involved two or more of the nuclei, or one or more of these deep structures plus damage to closely adjacent areas including the internal capsule, periventricular or peristriatal white matter; and (iii) lesions sparing basal ganglia and thalamus but involving adjacent white matter. The main conclusions to be drawn from this meta-analysis are that lesions confined to the basal ganglia (putamen, caudate nucleus and globus pallidus) rarely, if ever, cause apraxia. Lesions affecting the lenticular nucleus or putamen nearly always intruded into the adjacent lateral white matter to involve association fibres, in particular those of the superior longitudinal fasciculus and frontostriatal connections. Apraxia occurred with deep lesions of the basal ganglia apparently sparing white matter in only eight out of the 82 cases. Apraxia was most commonly seen when there were lesions in the lenticular nucleus or putamen (58 out of 72 cases) with additional involvement of capsular, and particularly of periventricular or peristriatal, white matter. Lesions of the globus pallidus (no cases) or caudate nucleus (three cases) rarely caused apraxia. The caudate lesions also had white matter involvement. Indeed, involvement of periventricular white matter alone caused apraxia. The vast majority of cases described with apraxia associated with deep lesions were in the left, dominant hemisphere. Ideomotor apraxia was described in most reports (72 out of 82 cases). Orofacial apraxia was less common (37 cases), usually with ideomotor apraxia. Ideational apraxia was rare (five cases), all with ideomotor apraxia. Apraxia was either bilateral or involved the left hand if there was a right hemiparesis, in those cases where descriptions were available. Lesions of the thalamus can sometimes cause apraxia (26 cases), even if there is no apparent involvement of white matter (12 cases). Small lesions confined to the thalamus can also sometimes cause apraxia (eight cases). The role of the thalamus in higher order motor control and apraxia remains to be determined. It is suggested that the term limb-kinetic apraxia should be retained to describe motor deficits in planning 'what to do', 'how to do it' and 'when to do it'; decisions which appear to involve activation of a complex distributed network of dorsolateral prefrontal cortex, supplementary motor areas, anterior cingulate regions and lateral premotor cortex. Such deficits need to be quantified. If they are present in patients with basal ganglia disease, over and above classical akinesia, bradykinesia and hypokinesia, then such patients could be said to exhibit limb kinetic apraxia. PMID- 8624693 TI - Long-term outcome of unilaterally transplanted parkinsonian patients. I. Clinical approach. AB - Five patients with Parkinson's disease, unilaterally transplanted with foetal mesencephalic cells into putamen (n=1) or putamen and caudate (n=4), were followed throughout a period of 15-36 months after surgery, according to the recommendations of the core assessment programme for intracerebral transplantations (CAPIT). All these patients exhibited an increase in the fluorodopa uptake in the grafted putamen, which was most significant in the first and last patient of the series. Long-term bilateral improvement of skilled hand movements was observed, starting between the third and sixth month after grafting, and confirmed by the statistical analysis of CAPIT timed tests. A mild to moderate effect on the amount of 'off' time and 'on-off' fluctuations was observed, whereas, apart from one case, no other clear effect on gait, walking and speech was found. One patient included in the study, already suffering slight cognitive impairment, clearly exhibited progression of a dementia process after surgery. Daily living activities were clearly improved in only one of the other four patients. At the end of the study period, all patients needed L-dopa therapy at a similar or higher dose than before grafting, but, in most of them, other dopaminergic drugs were reduced or stopped. All patients exhibited bilateral dyskinesias before grafting that were greatly decreased in intensity a few months after surgery. Delayed asymmetrical dyskinesias, occurring on the side displaying the better motor improvement, i.e. contralateral to the graft, were observed in three patients. These results suggest that neural transplants may influence two central mechanisms involved in motor function and the onset of dyskinesias. These effects are likely to occur through complex interactions with the post-synaptic dopaminergic receptors. The occurrence of dyskinesias might simply reflect increased presynaptic storage and release of dopamine. Alternatively, it might, in part, represent some other long-term deleterious effect of the graft. Since PET-scan data indicate that the reinnervation obtained is sub-optimal, it will be of interest to obtain a larger and denser reinnervation of the host striatum and to try, thereafter, to reduce the dose of L-dopa. PMID- 8624694 TI - The accuracy and precision of timing of self-paced, repetitive movements in subjects with Parkinson's disease. AB - In separate experiments, we studied the temporal accuracy and precision of self paced, repetitive finger-tapping in two groups of 12 patients with Parkinson's disease and a group of 12 controls matched to the patients with respect to age and general cognitive state. One group (I) of patients was studied initially following 12-15 h abstinence from normal levodopa medication ('off') and again, subsequently, approximately 1 h after ingestion of a single normal dose ('on'). A second group (II) of patients, each of whom had bilaterally asymmetrical neurological signs, was tested using 'worse' and 'better' hands separately. Within each session, subjects were tested repeatedly on a tapping task during which they were required to produce a regular series of self-timed inter-tap intervals, the target duration (550 ms) of which had been established previously during an initial period of tapping in synchrony with the beats of a regular metronome. We employed Wing and Kristofferson's (1973) model of control of motor timing to partition the total variance (TV) about the mean inter-response interval (IRI) produced during the self-paced phase of each run into separate components ['clock' variance (CV) and 'motor-delay' variance (MDV)] attributable to hypothetical 'clock' and 'motor-implementation' processes. Although the mean self-paced IRI of parkinsonian patients was generally shorter than that of controls, only during the 'on' medication condition (Group I) was it significantly so. By comparison with control values, and those observed during the 'on' medication condition, values of TV, CV and MDV in Group I were all significantly higher when subjects were 'off' medication. During the 'on' medication condition, only CV was significantly higher than the control value. In Group II, values of TV, CV and MDV associated with use of the 'worse' hand were all significantly higher than both control values and those associated with use of the 'better' hand. Values of these variables when subjects used the 'better' hand did not, however, differ significantly from control values. The theoretical import of these results is discussed in the light of several important procedural, statistical and computational issues and we conclude that TV, CV, and MDV may all vary significantly as a function of the efficacy of dopaminergic transmission in the basal ganglia. PMID- 8624695 TI - Cortical inhibition in Parkinson's disease. A study with paired magnetic stimulation. AB - The activity of motor cortical inhibitory circuits was studied with paired transcranial magnetic stimuli in 16 patients with Parkinson's disease 'off' therapy, five patients 'off' and 'on' therapy, and 11 normal subjects. Paired stimuli were delivered at short (3-20 ms) as well as long (100-250 ms) intervals during slight voluntary contraction. The intensity of the conditioning stimulus was subthreshold (80%) at short, and suprathreshold (150%) at long intervals. In addition, the silent period following a single magnetic shock given at 150% of threshold was measured. With short interstimulus intervals, no significant difference between patients and normal subjects could be detected. With long interstimulus intervals, the test response was significantly more inhibited in patients than in normal subjects. Although the cortical silent period was found to be slightly shorter, the recovery of motor evoked potentials was incomplete in patients with Parkinson's disease. This alteration could be partially reverted in dopaminergic therapy. In conclusion, the responsiveness of motor cortices to suprathreshold magnetic stimuli delivered after the end of the silent period is impaired in patients with Parkinson's disease, possibly due to prolonged activity in intracortical inhibitory circuits. The positive effect of L-dopa suggests that dopaminergic modulation of cortical activity, most probably at basal ganglia level, is involved in the pathogenesis of this phenomenon. PMID- 8624696 TI - Directional bias of initial visual exploration. A symptom of neglect in Parkinson's disease. AB - In the present study, side preferences in spontaneous visual exploration were assessed systematically in 27 patients with idiopathic Parkinson's disease (IPD) and 17 age-matched controls. Assessment of initial visual exploration asymmetry (IVE) was based on the exploration of texture arrays requiring attentive oculomotor scanning. As shown in a previous study, most healthy subjects exhibit a marked asymmetry of IVE with a strong left-sided bias when assessed by this paradigm, while most neglect patients initiate exploration in the right half of the arrays. Standard assessments for symptoms of neglect (line bisection, line cancellation and double simultaneous stimulation) were performed as reference tests. In the IVE task 65% of normal controls and 69% of patients with predominantly right-sided IPD started exploration in the left half of the arrays. By contrast only 14% of patients with predominantly left-sided disease showed a leftward IVE. The majority shows an ambiguous (21%) or rightward (64%) directional bias for initial exploration and thus a behaviour that corresponds to the IVE abnormalities found in neglect patients. No abnormalities were found in the standard neglect tests in any of the groups. The atypical IVE in patients with predominantly left-sided Parkinson's disease should be interpreted in the context of recent concepts of attention postulating that a bias in early spontaneous orientation directed to the ipsilesional hemifield reflects a mild and residual manifestation of hemineglect. Since this subtle orientational bias is less subject to compensation than more conspicuous clinical signs of neglect, sensitivity is higher in IVE testing than in conventional neglect assessments in chronic disorders with subclinical neglect. The present findings contribute a new aspect to the complex picture of cognitive and visuospatial abnormalities in Parkinson's disease. Furthermore our results extend previous knowledge on the mechanisms of neglect and the role of dopamine in the mediation of attention. PMID- 8624697 TI - Changes in cortical activity during mental rotation. A mapping study using functional MRI. AB - Mental imagery is an important cognitive method for problem solving, and the mental rotation of complex objects, as originally described by Shepard and Metzler (1971), is among the best studied mental imagery tasks. Functional MRI was used to observe focal changes in blood flow in the brains of 10 healthy volunteers performing a mental rotation task. On each trial, subjects viewed a pair of perspective drawings of three-dimensional shapes, mentally rotated one into congruence with the other, and then determined whether the two forms were identical or mirror-images. The control task, which we have called the 'comparison' condition, was identical except that both members of each pair appeared at the same orientation, and hence the same encoding, comparison and decision processes were used but mental rotation was not required. These tasks were interleaved with a baseline 'fixation' condition, in which the subjects viewed a crosshair. Technically adequate studies were obtained in eight of the 10 subjects. Areas of increased signal were identified according to sulcal landmarks and are described in terms of the Brodmann's area (BA) definitions that correspond according to the atlas of Talaraich and Tournoux. When the rotation task was contrasted with the comparison condition, all subjects showed consistent foci of activation in BAs 7a and 7b (sometimes spreading to BA 40): 88% had increased signal in middle frontal gyrus (BA 8) and 75% showed extrastriate activation, including particularly BAs 39 and 19, in a position consistent with area V5/human MT as localized by functional and histological assays. In more than half of the subjects, hand somatosensory cortex (3-1-2) was engaged, and in 50% of subjects there was elevated signal in BA 18. In frontal cortex, activation was above threshold in half the subjects in BAs 9 and/or 46 (dorsolateral prefrontal cortex). Some (four out of eight) subjects also showed signal increases in BAs 44 and/or 46. Premotor cortex (BA 6) was active in half of the subjects during the rotation task. There was little evidence for lateralization of the cortical activity or of engagement of motor cortex. These data are consistent with the hypothesis that mental rotation engages cortical areas involved in tracking moving objects and encoding spatial relations, as well as the more general understanding that mental imagery engages the same, or similar, neural imagery as direct perception. PMID- 8624698 TI - Cortical stimulation induces Fos expression in striatal neurons via NMDA glutamate and dopamine receptors. AB - Cortical electrical stimulation has been shown to induce dense and widespread Fos expression throughout the ipsilateral and contralateral striatum. This raises interest for studying the mechanisms underlying the regulation of striatal neuron activity by cortical afferents, and for elucidating the interactions with other systems. However, the receptors mediating cortical-stimulation-induced expression of Fos in striatal neurons have not been identified. This was studied in the work reported here by stimulating the cortex after administration of glutamate or dopamine receptor antagonists, or after 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopaminergic system. Pretreatment with the non-competitive N-methyl D-aspartate (NMDA) glutamate receptor antagonist MK-801 led to a marked reduction in the stimulation-induced density of Fos-immunoreactive nuclei in both the medial (about 80% reduction) and lateral (about 50-60% reduction) striatum. Preadministration of the D1-selective dopamine antagonist SCH-23390 alone or in combination with the D2-selective dopamine antagonist eticlopride led to a reduction in the stimulation-induced density of Fos-positive nuclei of about 60 65% in the lateral striatum, but no significant change in the medial region. The effects of 6-OHDA lesion were less pronounced, and the stimulation-induced density of Fos-immunoreactive nuclei decreased by only about 25% in the lateral region. These results indicate that both dopamine and NMDA glutamate receptors are involved in the induction of Fos by cortical stimulation, and support the hypothesis that cortex-dopamine interactions in the lateral striatum may be functionally different from those in the medial striatum. PMID- 8624699 TI - Estimation of oxytocin mRNA in the human paraventricular nucleus in AIDS by means of quantitative in situ hybridization. AB - Recently, a 40% reduction in the total of oxytocin immunoreactive neurons of the paraventricular nucleus of the hypothalamus in AIDS patients was reported. In the present study, we determined whether this decrease is associated with a diminished amount of oxytocin mRNA. We used in situ hybridization combined with densitometric image analysis for the quantitative assessment of oxytocin gene expression in the paraventricular nucleus of a group of AIDS patients (n=10) and a carefully matched control group (n=8). We found no significant difference (P=0.08) in the amount of oxytocin mRNA per total paraventricular nucleus between the two groups. In addition, no significant differences were found in the part of the volume of the paraventricular nucleus that was occupied by hybridized cells (P=0.12) or in the mean signal density (P=0.08). The findings do not support the hypothesis that the extensive decrease in oxytocin immunoreactive neurons of the paraventricular nucleus in AIDS is associated with a decrease in total oxytocin mRNA content in this nucleus. The data are compatible with the suggestion that in AIDS the biosynthesis of oxytocin is changed in an unknown way at the (post)transcriptional level. PMID- 8624701 TI - Parallel metabolic and feeding responses to lateral hypothalamic stimulation. AB - Energy metabolism and food intake effect each other. Many studies demonstrated that the lateral hypothalamus (LH) participates in the control of both feeding behavior and energy metabolism. We assessed the effect of a bipolar near threshold feeding eliciting electrical stimulation of the LH first on the feeding response and then on the background metabolism (metabolism free from the part due to locomotion) and respiratory quotient (RQ), on Wistar male rats, during either the light or the dark phase of the nycthemeron. LH stimulation resulted in a delayed and rather long increase in background metabolism that was more dramatic during the light phase. This metabolic response paralleled the delayed feeding response (more than 10 min) we obtained during behavioral tests. The increase in energy production was sustained by release of energy substrates from the endogenous stores, and this showed a circadian dependence. Mainly carbohydrates (rise in RQ) were used during the light phase stimulation whereas lipids (decrease in RQ) were released in the dark phase. PMID- 8624700 TI - Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist. AB - Bilateral intranigral administration of the selective NK-1 tachykinin receptor agonist [AcArg6, Sar9, Met(O2)11]SP6-11 (0-1 nmol total bilateral dose) selectively induced grooming in rats. This response was blocked by concurrent intranigral administration of the NK-1 tachykinin receptor antagonist RP 67580 (2 nmol), but not by NK-2 (L-659,877) or NK-3 ([Trp7, beta-Ala8]NKA4-10) antagonists. Pretreatment with systemic opioid (naloxone 1.5 mg/kg) and D1 dopamine (SCH 23390 100 micrograms/kg) receptor antagonists also attenuated tachykinin-induced grooming, which was unaffected by D2 dopamine (sulpiride 30 mg/kg) or 5-HT2A+C (ritanserin 2 mg/kg) antagonists. Grooming induced by intranigral [AcArg6, Sar9, Met(O2)11]SP6-11 was also attenuated by bilateral 6 hydroxydopamine lesions of the substantia nigra. These findings indicate that grooming induced by intranigral tachykinins reflects activation of NK-1 receptors and is dependent upon endogenous dopamine and consequent selective stimulation of D1 dopamine receptors. PMID- 8624702 TI - [14C]2-deoxyglucose uptake in the brain of the ring dove (Streptopelia risoria). I. Prolactin-induced uptake. AB - Quantitative [14C]2-deoxyglucose (2DG) autoradiography was used to identify areas of the ring dove brain involved in the expression of incubation behavior. Compared with non-breeding controls, 2DG uptake was increased in birds of both sexes during late incubation in all areas of the fore, mid- and hind-brain examined. This increase occurred irrespective of whether the birds were sitting on their eggs at the time of 2DG administration. A similar pattern of 2DG uptake into the brain was observed in non-breeding females treated with 30 I.U. ovine prolactin (i.p.) twice daily for 5 days. It is concluded that there is a generalised increase in neural activity in the brain of doves during late incubation which may be dependent on increased concentrations of plasma prolactin. PMID- 8624703 TI - Spatial and sub-cellular localization of the membrane cytoskeleton-associated protein alpha-adducin in the rat brain. AB - Studies on the identification and characterization of constituents of rat brain synaptic junctions have lead to the isolation of cDNA clones encoding segments of alpha-adducin. These and other studies suggest that adducin, a protein involved in promoting the assembly of actin and spectrin filaments at the plasma membrane, may play a role in dynamic assembly-disassembly processes underlying synaptic plasticity. In order to verify that brain alpha-adducin is indeed a constituent of synaptic structures, we have generated monoclonal antibodies against epitopes in the C-terminal region of alpha-adducin and have determined its spatial and sub cellular distribution in postnatal day-30 rat brain. Alpha-adducin is found to be highly enriched in regions with high synapse densities of the hippocampus, corpus striatum, cerebral cortex and cerebellum. Immuno-electron microscopic analysis of peroxidase stained sections of the hippocampus and the cerebellum revealed that alpha-adducin is localized at distinct sub-cellular structures. In the CA1 and CA3 regions of the hippocampus alpha-adducin immunoreactivity is found in a distinct subset of dendrites and dendritic spines. In the molecular layer of the cerebellum, a distinct fraction of pre-synaptic terminals of parallel fiber terminals is labeled. In both cases the majority of synaptic structures does not contain adducin. Significant immunoreactivity is also detected in processes of glial cells both in the hippocampus and the cerebellum. PMID- 8624704 TI - [14C]2-deoxyglucose uptake in the brain of the ring dove (Streptopelia risoria). II. Differential uptake at the onset of incubation. AB - Brain areas involved in the expression of incubation behaviour were identified in male ring dove at the onset of incubation using quantitative [14C]2-deoxyglucose (2DG) autoradiography. The uptake of 2DG in twenty-eight areas found in the hypothalamus and thalamus, and six areas in the forebrain were compared in control non-breeding and incubating birds. Although 2DG utilisation varied significantly between brain areas, significant differences between non-breeding males and incubating males were observed only in 4 of them. These were the nucleus tuberis, nucleus preopticus medialis, nucleus ovoidalis and paleostriatum primitivum. The uptake of 2DG was increased at the onset of incubation in the nucleus tuberis, nucleus preopticus medialis and nucleus ovoidalis and decrease in the paleostriatum primitivum. These findings are consistent with the hypothesis that these brain areas play a role in the initiation of incubation behaviour. PMID- 8624705 TI - GABAA-receptor subunit composition in the circadian timing system. AB - In the present study, the distribution of GABAA-receptor alpha 1-, alpha 2-, alpha 3-, alpha 5-, beta 2.3- and gamma 2-subunits were localized immunohistochemically with subunit specific antibodies in the rat circadian timing system (CTS). The areas examined include the principal circadian pacemaker, the suprachiasmatic nucleus (SCN), and areas that receive important SCN input including the intergeniculate leaflet (IGL), subparaventricular zone (SPVZ), paraventricular hypothalamic nucleus (PVH), the retrochiasmatic area (RCh) and the paraventricular nucleus of the thalamus (PVT). The SCN has an unusual pattern with immunoreactivity for the alpha 2-, alpha 3-, alpha 5-, and gamma 2-subunits but not for the commonly expressed alpha 1- and beta 2.3 subunits. In all of the areas receiving SCN efferent input (SPVZ, PVH, RCh, PVT and IGL), staining is present either for all six subunits or for the three common subunits, alpha 1-, beta 2.3-, and gamma 2. There is some evidence for a differential distribution of subunits at the cellular level. The alpha 2-, and beta 2.3-subunits are predominantly expressed in neuropil, the alpha 3-, alpha 5- and gamma 2-subunits are predominantly expressed over perikarya and the alpha 1 subunit is expressed over both neuropil and perikarya in the areas in which subunit immunoreactivity is found. The demonstration of this regional and cellular expression of GABAA-receptor subunits should contribute to our understanding of GABAergic neurotransmission in the CTS. PMID- 8624706 TI - Retrograde axonal transport of the alpha subunit of the GTP-binding protein Gz to the nucleus of sensory neurons. AB - Nerve cells are exquisitely sensitive to target tissue derived factors and the discovery that nerve growth factor could be retrogradely transported in axons suggested that the physical translocation of proteins along the axon could be a mechanism to convey this signal. This message is not due to the neurotrophic factor itself but rather due to second messengers generated by interaction with receptors. We have previously demonstrated the retrograde axonal transport of the alpha subunits of two putative second messenger molecules Gi and Gz. We have investigated more thoroughly the transport of the alpha subunit of Gz (Gz alpha) and in order to be more certain that the immunoreactivity seen is due to Gz alpha, we have made antibodies to peptides from both the N- and C-terminal regions of Gz alpha, which recognise the same 41 kDa band on Western blots of brain and sciatic nerve extracts. This band is eliminated when the antibodies are previously incubated with the specific peptide to which they were made. Using these antibodies for immunohistochemical localisation for Gz alpha, we now report that the GTP-binding protein Gz, is not only retrogradely transported in axons but that it translocates to the neuronal nucleus. Furthermore, the levels seen in the nuclear compartment decline after axotomy or ligation of the mice under ether anaesthetic, suggesting it is the retrogradely transported Gz alpha that is accumulating in the nucleus after activation at the nerve terminal. PMID- 8624707 TI - Cutaneous nociceptive facilitation of Ib heteronymous pathways to lower limb motoneurones in humans. AB - The effects of tonic pain stimulation on heteronymous Ib pathways from the gastrocnemius medialis (GM) to the soleus (Sol) and to the quadriceps (Q) muscles were investigated in four healthy human subjects. Tonic pain stimulation was performed by subcutaneous injection of 0.5 mg levo-ascorbic acid or vitamin C (L LAS) in a volume of 0.5 ml on the dorsal surface of the ipsilateral foot. The mean curve of L-AS-induced pain sensation showed a steep rising phase reaching maximum intensity at 2-3 min, followed by a slow decay phase lasting about 15-20 min. Between about 5 and 20 min after injection, there was evidence of pure pain stimulation due to chemical activation of free nerve endings. During this interval, significant potentiation of Ib inhibition from GM to both Sol and Q motoneurones was observed. The time-course of these Ib heteronymous changes paralleled that of subjective pain sensation. These findings demonstrate that nociceptive discharge modifies the gain of Ib heteronymous effects in humans. Since the man function of these Ib pathways is to coordinate activity of muscles operating at different joints, it is suggested that nociceptive input may change muscle synergies by selecting specific subpopulations of Ib interneurones, thus contributing to establish appropriate adaptive motor strategies. PMID- 8624708 TI - Simultaneous activation of spinal antiopioid system (neuropeptide FF) and pain facilitatory circuitry by stimulation of opioid receptors in rats. AB - Neuropeptide FF (NPFF) is a mammalian FMRFamide-like octapeptide with antiopioid properties that inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, a series of three experiments was carried out to investigate the interactions between opioid receptor stimulation and antiopioid systems. First, by using in vitro superfusion system with rat spinal cord slices, we showed that morphine stimulated NPFF release in a dose-dependent manner. The stimulating effect which was observed with morphine concentrations as low as 100 fM reached a maximum at 0.1 nM, then decreased and was ineffective at 10 microM. The morphine-induced release of NPFF was abolished by naloxone (1 microM) but unaltered by tetrodotoxin. Second, by an in vivo approach, we showed that a single heroin administration (2.5 mg/kg, s.c.) elicited in 30 min a drastic drop (38%) in spinal NPFF content. In a third experiment, we evaluated the capacity of naloxone in revealing an antiopioid component associated with opioid receptor stimulation. The administration of naloxone (1 mg/kg, s.c..) 25 min following that of heroin (2.5 mg/kg, s.c.) not only abolished the heroin induced increase of tail-flick latency, but also lowered it under the basal value by 30%. These results indicate that opioid receptor stimulation activates both pain inhibitory and pain facilitatory systems in which NPFF may play a significant role and that opiate-induced analgesia is always partly masked. PMID- 8624710 TI - Response of newborn and adult sheep to pyrogens: relation between fever and brain eicosanoid changes. AB - We investigated whether the weak febrile response to pyrogens in newborns is due to a diminished activation of the putative pyrogen mediator, prostaglandin (PG)E2. Indwelling cannulas in the third ventricle of lambs (age, 5-31 days) and adult ewes were used to collect cerebrospinal fluid (CSF) for radioimmunoassay of PGE2. Intravenous (i.v.) endotoxin caused a smaller increase in body temperature but a larger increase in CSF PGE2 in lambs compared to adults. PGE2 by intracarotid infusion raised body temperature in 5 of 7 trials in 3 lambs and in 4 of 4 trials in 1 adult. Endotoxin given intracerebroventricularly (i.c.v.) induced a rise in temperature and CSF PGE2 in the lamb but, in the adult, these responses were delayed and smaller. Interleukin-1 i.c.v. and PGE2 i.c.v. were weak pyretic agents at both ages. We conclude that the lamb's diminished febrile response to endotoxin i.v. is not caused by a lesser rise in CSF PGE2, rather it may be due, at least in part, to reduced responsiveness to this putative mediator. Regardless of age, the sheep differs from other species in that pyrogen/PGE2 coupling occurs primarily at a site in brain that is better accessible from blood than CSF. PMID- 8624709 TI - Effects of MK801 on Fos expression in the rat brainstem after unilateral labyrinthectomy. AB - Unilateral labyrinthectomy (UL) causes ocular and postural asymmetries, which disappear over time in the processes of equilibrium recovery known as vestibular compensation. It has been reported that N-methyl-D-aspartate (NMDA) receptors are involved in vestibular compensation. In the present study, in order to elucidate the NMDA receptor-mediated neural circuit responsible for the development of vestibular compensation, we used Fos expression as a marker of neural activation and examined the effects of MK801, a specific antagonist of NMDA receptors, on UL induced Fos expression in the rat brainstem. After UL, Fos-like immunoreactive ( LIR) neurons were observed in the ipsilateral medial vestibular nucleus (ipsi MVe), the contralateral prepositus hypoglossal nucleus (contra-PrH) and the contralateral inferior olive beta subnucleus (contra-IOb). Fos-LIR neurons gradually disappeared in the processes of vestibular compensation. It is suggested that the activation of the ipsi-MVe, the contra-PrH and the contra-IOb neurons after UL are the initial event of vestibular compensation. Intraperitoneal injection of MK801 in the processes of vestibular compensation caused reappearance of UL-induced behavioral deficits. During the decompensation induced by MK801, Fos-LIR neurons appeared in the contra-MVe, the ipsi-PrH and the bilateral-IOB. It is suggested that the contra-MVe, the ipsi-PrH and the bilateral-IOb neurons are inhibited by glutamatergic synapses driving inhibitory neurons via NMDA receptors in the processes of vestibular compensation and that disinhibition of these nuclei induced by MK801 causes decompensation. However, MK801 caused neither Fos expression nor behavioral decompensation after vestibular compensation is accomplished. All these findings that the NMDA receptor-mediated inhibitory modulation in the central vestibular system plays an important role for the initial processes of the development of vestibular compensation. PMID- 8624711 TI - General, mu and kappa opioid antagonists in the nucleus accumbens alter food intake under deprivation, glucoprivic and palatable conditions. AB - Ventricular microinjection studies found that whereas mu (beta-funaltrexamine, B FNA), mu1 (naloxonazine) and kappa (nor-binaltorphamine, Nor-BNI) opioid receptor antagonists, but not delta antagonists, reduce deprivation-induced intake, kappa and mu, but not mu1 or delta antagonists reduce both 2-deoxy-D-glucose (2DG) hyperphagia and sucrose intake. Since opioid agonists stimulate spontaneous food intake in the accumbens, the present study examined whether administration of either naltrexone, B-FNA or Nor-BNI in the accumbens altered intake under deprivation (24 h), glucoprivic (2DG: 500 mg/kg, i.p.) or palatable sucrose (10%) conditions. Naloxonazine's effects in the accumbens were also evaluated for deprivation-induced intake. Deprivation-induced intake was significantly decreased over 4 h by naltrexone (5-20 micrograms, 44%), B-FNA (1-4 micrograms, 55%) and Nor-BNI (4 micrograms, 31%) but not naloxonazine (10 micrograms) in the accumbens. 2DG hyperphagia was significantly decreased by naltrexone (10-20 microgram, 79%), B-FNA (1-4 micrograms, 100%) and NOR-BNI (104 micrograms, 75%) in the accumbens. Sucrose intake was significantly decreased by naltrexone (50 micrograms, 27%) and B-FNA (1-4 micrograms, 37%), but not NOR-BNI in the accumbens. These data suggest that mu receptors, and particularly the mu2 binding site in the accumbens are responsible for the opioid modulation of these forms of intake in this nucleus, and that this control may be acting upon the amount of intake per se. PMID- 8624712 TI - Prejunctional modulation of nitroxidergic nerve function in canine cerebral arteries. AB - Modulation by acetylcholine, VIP, clonidine, omega-conotoxin and Mg2+ of the relaxant response to electrical and chemical stimulation of nitroxidergic nerves, in which nitric oxide (NO) acts as a neurotransmitter, was investigated in isolated canine cerebral arteries. Acetylcholine attenuated the response, the inhibition being reversed by atropine; however, physostigmine failed to reduce the response. VIP in submaximal doses did not alter the neurally induced relaxation. The same was true with clonidine, morphine and naloxane. Treatment with omega-conotoxin depressed the response to electrical nerve stimulation but did not influence the nicotine-induced relaxation. Mg2+ inhibited the relaxation caused by nerve stimulation and Ca2+ reversed the response. It is concluded that activation of prejunctional muscarinic receptors seems to inhibit the synthesis of release of NO from nerve terminals but endogenous acetylcholine from cholinergic nerve does not play a role in inhibiting the nitroxidergic nerve function. Prejunctional VIP, alpha 2, adrenergic and opioid receptors are not likely to participate in the regulation of nerve function. Ca2+ responsible for NO synthase activation would be produced into nerve terminals via N-type Ca2+ channels when electrically stimulated and via non-N-, non-L-type channels when stimulated by nicotine. Mg2+ and Ca2+ counteract in the neurally induced relaxation, although the underlying mechanism was not determined. PMID- 8624714 TI - Effects of micromolar and nanomolar calcium concentrations on non-synaptic bursting in the hippocampal slice. AB - To determine how [Ca2+]0 affects non-synaptic epileptogenesis in the CAI area of hippocampal slices, we compared the extracellularly recorded hyperactivity induced by ACSF containing either micromolar ('low-Ca2+, LC-ACSF) or nanomolar concentrations of Ca2+ ('zero-Ca2+, ZC-ACSF). Both solutions effectively blocked chemical synaptic transmission but spontaneous bursts developed more quickly and consistently in ZC-ACSF and were longer in duration and more frequent than those recorded in LC-ACSF Antidromically evoked bursts were less epileptiform, i.e., they exhibited fewer population spikes (PSs), in ZC-ACSF. Increasing [Mg2+]0 or decreasing [K+]0 suppressed spontaneous LC-ACSF bursting but only decreased the intensity and frequency of bursting in ZC-ACSF. Either manipulation increased the epileptiform nature of the antidromically evoked field potential, thereby mimicking the effect of increasing [Ca2+]0 from nanomolar to micromolar levels. Bath application of 250-500 microM GABA commonly arrested spontaneous bursting in LC-ACSF. In ZC-ACSF, GABA decreased the burst frequency but paradoxically superimposed high amplitude PSs on each burst. These effects were reversed by the GABAA receptor antagonists bicuculline methiodide or picrotoxin (50-100 microM). These results indicate that simply lowering [Ca2+]0 from micromolar to nanomolar concentrations increases the burst propensity and intensity of the CA1 population and can dramatically alter responses to pharmacological agents. PMID- 8624713 TI - Effects of intracerebroventricular administration of pituitary adenylate cyclase activating polypeptide (PACAP) on the motor activity and reserpine-induced hypothermia in murines. AB - We investigated the effects of i.c.v. administration of pituitary adenylate cyclase-activating polypeptide (PACAP) on the spontaneous motor activity and reserpine-induced hypothermia in murines. The administration of PACAP (1 or 2 nmol) caused a dose-dependent increase in both spontaneous motor activity and rearing behavior in the rat. The peptide (0.1 or 0.2 nmol) counteracted reserpine induced hypothermia in a dose-dependent manner in mice. On the other hand, i.c.v. injection of vasoactive intestinal polypeptide, which is structurally similar to PACAP, at a dose similar to that of PACAP (2 nmol in rats, 0.2 nmol n mice) did not show a significant effect on either behavior or body temperature. Therefore, the stimulating effect of PACAP observed here may be mediated by PACAP-specific (type I) receptors. PACAP was more potent and longer-lasting than a known potent stimulating peptide, thyrotropin-releasing hormone, in both stimulating motor activity and counteracting reserpine-induced hypothermia. Results of the present study, in combination with those of previous studies identifying endogenous PACAP in the brain, suggest that PACAP may play a important role in the CNS as a stimulant in regulating motor activity and body temperature. PMID- 8624716 TI - Graded action potentials generated by neurons in rat hypothalamic slices. AB - Preoptic neurons in rat hypothalamic slices were investigated with tight-seal whole-cell recording techniques. The main aim was to investigate the ability to generate graded, stimulus-dependent impulses. In response to rectangular current pulses, all cells generated impulses with an amplitude that to some degree depended on the stimulus strength. Stronger current steps induced impulses of larger amplitude. In 50% of the cells, a systematic variation of impulse amplitude of more than 10 mV (up to 40 mV) was recorded, implying a clear deviation from the 'all-or-nothing' principle. A clear variation in amplitude of spontaneous impulses was also recorded, in the whole-cell mode as well as from intact cells in the cell-attached mode. PMID- 8624715 TI - Optical recording from cerebellar Purkinje cells using intracellularly injected voltage-sensitive dyes. AB - We evaluated several techniques for their ability to record membrane potential changes with voltage-sensitive dyes introduced into CNS neurons in the brain slice preparation. Using a probe designed for intracellular application, JPW1114, we found that iontophoresis or pressure pulses could not push the lipophilic dye through electrodes whose resistance was sufficiently high to produce good electrical recordings in cerebellar Purkinje neurons. However, properly selected patch electrodes could introduce the dye into the cell and still give good electrical records. Using this technique we recorded depolarizing and hyperpolarizing transients and climbing fiber responses using either a single photodiode or a fast, cooled CCD camera. While these results are promising, there are still problems due to the slow diffusion of the dye in the dendrites and a low sensitivity which requires signal averaging to acquire traces with a good signal to noise ratio. PMID- 8624717 TI - Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus. AB - We studied messenger RNA (mRNA) expressions of receptors for glucocorticoid (GR), thyroid hormone (TR), androgen (AR), and estrogen (ER) and their changes with age in the hippocampal subregions in postmortem human brain. In situ hybridization was done with biotin-labeled antisense synthetic oligonucleotide probes. About 80% or more of the pyramidal neurons in the hippocampal subregions expressed mRNAs for individual receptors in the brains of subjects younger than 65. The ratio of mRNA-containing neuron density to total neuron density significantly decreased with age for GR in CA1 and CA3, and for AR in CA1. Non-significant trends in the reduction with age in the ratio of ER mRNA-containing neurons in CA1 and the ratio of GR mRNA-containing neurons in the hilus also were found. Age related reductions in nuclear receptor protein mRNA expression in neurons in the hippocampal subfields may be important in the impairments of cognition, emotion, and responses to acute stress in the aged. PMID- 8624718 TI - Ultrastructural localization of delta-opioid receptor and Met5-enkephalin immunoreactivity in rat insular cortex. AB - The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the delta-opioid receptor (DOR) and the endogenous opioid ligand, Met5 enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex. PMID- 8624719 TI - Neurotrophin-4/5 postpones the death of injured spinal motoneurons in newborn rats. AB - We have compared the short- and long-term effects of Neurotrophin-4/5 (NT-4/5) on the survival of spinal motoneurons in neonatal rats. To this aim, the sciatic nerve was cut in newborn rats and NT-4/5 or buffer were applied to the proximal nerve stem in a gelfoam as well as by daily local injection. The number of motoneurons or ventral root fibers was determined 4, 11, and 14 days later. Four days after received NT-4/5 lost only 19% + 2.7% (540 + 81). No systemic effect on the contralateral motoneurons was observed. Surprisingly, 11 and 14 days following the operation, the loss of motoneurons and of ventral root fibers was the same in buffer and NT-4/5 treated animal despite the daily treatment. Thus, although NT-4/5 is a potent survival factor for motoneurons in culture and can transiently prevent the death of motoneurons in vivo, it can not, under these experimental conditions, permanently rescue lesioned motoneurons. These results indicate that other trophic factors or combinations of factors may be required to permanently prevent the death of spinal motoneurons and that the short term survival promoting effects of neurotropic factors in motoneuron lesion models can not be used to predict their therapeutic potential in chronic neurodegenerative disorders. PMID- 8624720 TI - Identification of a 140 kDa protein of rat presynaptic terminal membranes encompassing the active zones. AB - A polyclonal antiserum raised against the carboxy-terminal 17 amino acids of the rat p185c-neu (anct) reacted with a 140 kDa polypeptide in membranes of synaptosome fractions from neocortex and hippocampus of 11-day-old and adult rats. The same antiserum reacted with a 185 kDa polypeptide in microsome membranes from rat pheochromocytoma cells (PC12). By light microscopic immunocytochemistry, the anct antibodies against the 140 kDa protein were localized in the neuropile of brain, cerebellum and spinal cord of 11-day-old and adult rats. Especially prominent staining was obtained in the CA2-CA3 zones of the hippocampus, and in the substantia gelatinosa in the spinal cord. The finely granular and diffuse pattern of the immunostain was consistent with synaptic localizations. Interestingly, antibodies against the entire endodomain of p185c neu (a-Bacneu) were localized in granular structures, probably representing axo somatic and axo-dendritic synapses, on a subset of pyramidal neurons of the CA3 zone. By immunoelectron terminals in the giant mossy fiber type in the CA3 and CA4 regions. The immunolocalization of the anct antibodies was restricted in segments of the presynaptic membrane facing the synaptic cleft which include the active zone. The identify and function of the 140 kDa membrane protein of rat brain presynaptic terminals, detected by the anct antibodies, is unknown. The 140 kDa protein may be related to p185c-neu, a tyrosine kinase, or to other known or unknown kinases. PMID- 8624721 TI - The opposing effects of interleukin -1 beta microinjected into the preoptic hypothalamus and the ventromedial hypothalamus on nociceptive behavior in rats. AB - The effects of microinjections of recombinant human interleukin-1 beta (rhIL-1 beta) into the hypothalamus and neighboring basal forebrain on nociceptive behavior were studied using a hot-plate test in rats. The microinjection of rhIL 1 beta at doses between 5 pg/kg and 50 pg/kg into the medial part of the preoptic area (MPO) reduced the paw-withdrawal latency. The maximal reduction was obtained 30 min after the injection of rhIL-1 beta at 20 pg/kg. RhIL-1 beta (20 pg/kg) induced hyperalgesia was completely blocked by the simultaneous injection of IL-1 receptor antagonist (IL-1ra, 20 ng/kg), Na salicylate (200 ng/kg) or alpha melanocyte-stimulating hormone alpha-MSH, 20 ng/kg). The intra-MPO injection of rhIL-1 beta at doses of less than 5 pg/kg or more than 50 pg/kg (up to 2 ng/kg) into the paraventricular nucleus, the lateral hypothalamic area and the septal nucleus had no effect on nociception. The microinjection rhIL-1 beta (20 pg/kg-50 pg/kg) into the ventromedial hypothalamus produced a prolongation of the paw withdrawal latency. A maximal prolongation was obtained 10 min after the injection of rhIL-1 beta at 50 pg/kg. This reaction was also blocked by the simultaneous injection of IL-1ra (50 ng/kg) and Na salicylate (500 ng/kg). These findings indicate that IL-1 beta in the MPO and the VMH produces hyperalgesia and analgesia, respectively, while, in addition, both effects are mediated by IL-1 receptors and the synthesis of prostaglandins. PMID- 8624722 TI - The effect of a unilateral inflammation at the rat's ankle joint on the expression of preprotachykinin-A mRNA and preprosomatostatin mRNA in dorsal root ganglion cells--a study using non-radioactive in situ hybridization. AB - In rats with an acute (2 days) and chronic (20 days) unilateral ankle joint inflammation (induced by Freund's complete adjuvant), the proportion of dorsal root ganglion cells containing preprotachykinin-A mRNA or preprosomatostatin mRNA was determined using non-radioactive in situ hybridization. At the acute stage of inflammation, the proportion of neurons containing preprotachykinin-A mRNA was similar to that in control rats. At the chronic stage, the proportion of neurons expressing preprotachykinin-A mRNA was significantly higher on the inflamed side than on the contralateral side. The proportion of dorsal root ganglion cells containing preprosomatostatin mRNA did not change. These data suggest that inflammation influences the synthesis of substance P but not of somatostatin in afferent neurons. PMID- 8624723 TI - Developmental immunohistochemistry of the 22 kDa peroxisomal membrane protein in the human brain. AB - We studied immunohistochemically the 22 kDa peroxisomal membrane protein (PMP 22). In the control brain, the immunopositive neurons for PMP 22 appeared 2-3 weeks earlier than those for peroxisomal enzymes. PMP 22-immunopositive glial cells appeared at 26-27 gestational weeks, increased with gestational age, and were rarely recognized after 1 year of age. In the patients with Zellweger syndrome, PMP 22-immunoreactivity was recognized in neurons, glias, liver and kidney cells. PMID- 8624725 TI - Small neurons in the vestibular nerve root project to the marginal shell of the anteroventral cochlear nucleus in the cat. AB - We injected biotinylated dextran amine (BDA) into marginal shell regions of the anteroventral cochlear nucleus (AVCN) of the cat. These injections led to retrograde labeling of cells including small cells (median some area = 111 micron2, equivalent diameter = 11.9 microns) in the vestibular nerve root (VNR), just ventral to an anterior part of the AVCN. This is an unexpected new finding. The cells were scattered among BDA-labeled fibers and were oriented parallel to the course of the VNR fibers. We suggest that the small neurons of the VNR might serve as second-order vestibular neurons conveying information from vestibular end organs to the cochlear nucleus (CN) and/or act as interneurons between the olivocochlear fibers in the VNR and the CN. PMID- 8624724 TI - NMDA receptors mediate heat shock protein induction in the mouse brain following administration of the ibotenic acid analogue AMAA. AB - Expression of inducible heat shock protein-70 (HSP-70) and hsp-70 mRNA were studied in the adult mouse brain following systemic administration of the ibotenic acid analogue (+/-)-2-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMAA), which is a potent N-methyl-D-aspartate (NMDA) agonist. At the dose of 20 mg/kg, AMAA produced excitatory behaviours in adult mice but overt convulsions were not seen. This treatment did not result in any detectable morphological brain damage at 4 days following administration. At 2.5 h and 5 h following treatment induction of hsp-70 mRNA expression was found in the pyramidal cell layers of CA1 and, to a lesser extent, CA3 fields of hippocampal Ammon's horn, amygdala, olfactory lobes, tenia tecta, hypothalamic nuclei and a superficial layer of cingulate, frontal and retrosplenial cortices. The presence of HSP-70 was detected by immunochemistry at 24 h following drug administration in those regions previously showing hsp-70 mRNA induction. AMAA-induced hsp-70 mRNA expression was prevented by pre-treatment with the non-competitive NMDA antagonist MK-801. These results suggest that NMDA receptors are involved in the stress response induced by AMAA. PMID- 8624726 TI - Metabotropic glutamate antagonist, MCPG, treatment of traumatic brain injury in rats. AB - The metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl-4 carboxyphenylglycine (MCPG) was administered into the left lateral ventricle 5 min prior to fluid percussion traumatic brain injury (TBI) in the rat. A single 5.0 microliters ventricular infusion of the active isomer. (+)-MCPG (0.2 mumol), significantly reduced beam walking motor deficits on days 1-5 after injury and learning/memory deficits measured on days 11-15 after injury. Neither a lower dose of (+)-MCPG (0.2 mumol) affected behavioral outcome. (+)-MCPG (0.2 mumol) did not affect systemic hemodynamic responses to injury. These results suggest that TBI induced activation of mGluRs contributes to behavioral morbidity and that blockade of certain mGluR subtypes (mGluR1, mGluR5 and/or mGluR2) may reduce these pathophysiological responses. PMID- 8624727 TI - The effects of vision and task complexity on Hoffmann reflex gain. AB - Previous research demonstrates modulation of the Hoffmann reflex amplitude and gain during changes in environmental conditions. H-reflex gain (defined in this study as the ratio of H-reflex amplitude to average soleus background EMG) is considered a functional measure of reflex modulation. In this study the effects of manipulating visual input and surface stability were to investigated in 17 subjects under four experimental conditions: (1) vision-stable surface, (2) no vision-stable surface, (3) vision-unstable surface, and (4) no vision-unstable surface. In each condition, subjects performed fifteen trials of a single leg stance for 7 s. The H-reflex was electrically elicited at the end of each trial by delivering a 1 ms square wave stimulation to the tibial nerve in the popliteal online for each trial (sampling rate = 2 kHz). An analysis of variance revealed significant decreases in H-reflex gain for the visual (F1.16 = 4.71, P < 0.05) and, surface conditions (F1.16 = 7.67, P < 0.05), however there was no interaction (F1.16 = 0.48, P < 0.05), between these variables. These results suggest that supraspinal mechanisms, possibly presynaptic inhibition, modulate H reflex gain across environmental conditions. We conclude that visual and possibly cutaneous inputs were responsible for driving presynaptic inhibition and thus decreasing H-reflex gain. PMID- 8624728 TI - Developmental changes of ganglioside expressions in postnatal rat cerebellar cortex. AB - We previously described the differential distribution of gangliosides in adult rat brain as detected by specific antibodies. We report here the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse monoclonal antibodies (mAbs). Eleven mAbs that specifically recognize each ganglioside were used. Our study revealed that the expression of each ganglioside changed dramatically during the development. GD3 and O-Ac-GD3 were expressed intensely in the external granular layer at 1, 5, and 10 days, whereas GD2 was firstly detected in the internal granular layer at 5 days and GD1b WAS diffusely detected throughout all layers of the cerebellar cortex at early postnatal days. GD2 and GD1b were more intensely expressed in the granular layer at 20, 30, and 80 days, suggesting that premature granule cells expressed GD3 and its derivative, O-Ac-GD3, whereas mature granule cells express GD2 and GD1B intensely. On the other hand, GM1 was exclusively detected in the external granular layer and the molecular layer at 1 and 5 days. The staining sites spread gradually from these outer layers into the internal granular layer and the white matter after 10 days. The positive cells in the external granular layer and the molecular layer appeared to be Bergmann glial cells and their radially ascending cytoplasmic processes. The intensity of the staining in these specialized astroglial cells decreased gradually during postnatal days. In contrast, the expression of GQ1b was very faint at birth, but gradually increased during the development and was detected intensely in the internal granular layer, particularly in the cerebellar glomeruli in adulthood, suggesting that GQ1b expression may be associated with synapse-related structures. The developmental changes of the expression of other gangliosides were also recognized in the postnatal rat cerebellum. These results suggest that specific gangliosides may play an important role in regulating the early events responsible for the orderly formation of the cerebellar cortex. PMID- 8624729 TI - Involvement of cyclic AMP at the level of the nucleus reticularis pontis caudalis in the acoustic startle response. AB - Rats were implanted with cannulas in the nucleus reticularis pontis caudalis (PnC), an obligatory part of the neural pathway that mediates the acoustic startle reflex. Following at least 1 week of recovery, rats were tested for acoustic startle amplitude before or after infusion of compounds known to alter the second messenger, adenosine cyclic 3', 5'-monophosphate (cAMP). Local infusion into the PnC of the cAMP analog, 8-bromo cAMP (0.125-1.0 micrograms), increased the amplitude of the acoustic startle response in a dose-dependent manner. In addition, local infusion of a phosphodiesterase inhibitor, rolipram (10 micrograms) or the water soluble adenylate cyclase activator, forskolin-DHA (2.5 micrograms), produced a significant enhancement of startle amplitude. These effects probably resulted from intracellular actions because cAMP itself, which does not readily penetrate lipid membranes, had no effect. Moreover, the effects seemed somewhat specific because the precursor of cAMP, ATP or 8-bromo cGMP, also failed to alter startle at doses where 8 bromo-cAMP did. The fact that a phosphodiesterase inhibitor elevated startle suggests that cAMP serves to tonically elevate startle at this level of the pathway. Hence, treatments that either increase (fear, sensitization) or decrease (habituation, pre-pulse inhibition) startle at the level of the PnC may do so via release of neurotransmitters either positively or negatively coupled to cAMP, which in turn may alter either sound evoked transmitter release, excitability of PnC neurons or both. PMID- 8624731 TI - Selective transport of blood-borne interleukin-1 alpha into the posterior division of the septum of the mouse brain. AB - Film autoradiography was used to demonstrate the transport and sites of accumulation of blood-borne radioiodinated interleukin-1 alpha (II-1 alpha) and other cytokines into the brain after intravenous administration. [125 I]Il-1 alpha, [125I]Il-1 beta, [125I]interleukin-1 receptor antagonist (II-1ra), and [125I]tumor necrosis factor-alpha (TNF alpha) labeled the choroid plexus and the capillary network 30 min after injection into the blood, suggesting that these areas may serve as sites of blood-to-brain transport. [125I]Il-1alpha, but not [125I]Il-1beta, [125I]Il-1ra, [125I]TNF alpha, or [125I]interleukin-2 (Il-2), was also found localized to the caudal region of the septal nuclei. Only unlabeled II 1 alpha was able to inhibit this accumulation. These findings provide further evidence for the passage of select cytokines across the blood-brain barrier (BBB) and are the first to identify a target site within the central nervous system (CNS) for a transported cytokine. PMID- 8624730 TI - A pharmacological study of the modulation of neuronal and behavioural nociceptive responses in the rat trigeminal region. AB - Electrical stimulation of the brain, particularly in the periventricular grey areas, caused long-lasting increases in behavioural escape thresholds to heating and mechanical stimuli applied to the facial region of the rat. The brain stimulation selectively suppressed responses to noxious stimuli. Responses to non noxious stimuli, evoked by low threshold brush, were unaffected. The same animals that were studied in the behavioural tests were then anaesthetized with urethane and the inhibitory effect of the same brain stimulation was studied in single neurones recorded in the caudal trigeminal nucleus. A clear correlation (rs = 0.63) emerged between degree of behavioural antinociception and the amount of inhibition seen in nociceptive neurones. In addition the mean duration of the inhibition (6 min) was similar to the mean duration of the antinociceptive effect (7.3 min). Other classes of non-nociceptive neurones were unaffected by the stimulation. The neurones were also studied using iontophoretically applied monoamine candidates for the inhibitory neurotransmitter noradrenaline (NA) and 5 hydroxytryptamine (5-HT). The profile of the effects of NA most closely fitted that of the inhibitory neurotransmitter. This profile was expressed in terms of depression and excitation of different classes of neurones, and by the duration of effects. The depressants effects could be antagonized by iontophoretic idazoxan. In addition clonidine induced long-lasting depression of firing. 5-HT was more likely than NA to excite nociceptive neurones and to depress non nociceptive neurons. Only NA consistently elevated thermal response thresholds in a similar manner to that produced by brain stimulation. These results provide some support for the hypothesis that selective descending inhibition of nociceptive responses in neurones of the rat caudal trigeminal nucleus is mediated by NA, possibly by an action at alpha2-adrenoceptors. PMID- 8624732 TI - The cloned mu, delta and kappa receptors and their endogenous ligands: evidence for two opioid peptide recognition cores. AB - The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind to the mu, delta and kappa receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned mu, delta and kappa receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met enkephalin are selective for delta, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to micro, delta and kappa. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptor types. While dynorphin A peptides and alpha- and beta-neoendorphin have a preference for kappa, they also bind the cloned delta and mu receptors. Our findings do not easily fit a simple 'message address' model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Try-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for mu and delta but not kappa and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for kappa. PMID- 8624733 TI - Effect of repeated methamphetamine administrations on dopamine and glutamate efflux in rat prefrontal cortex. AB - Pretreatment with psychostimulants such as methamphetamine (METH) results in augmented mesostriatal dopamine transmission upon a challenge administration of the drug. This effect can be blocked by dopamine antagonists and excitatory amino acid antagonists. However, no direct comparisons have been made with respect to the effects of a low-dose pretreatment regimen of METH on impulse and transporter mediated dopamine release or to what extent glutamate release is altered by a pretreatment regimen with METH. The purpose of this study was to examine dopamine and glutamate efflux in the prefrontal cortex and striatum in rats pretreated with METH following either high potassium (80 microM) infusion or after a systemic injection of a low dose of METH. Extracellular dopamine and glutamate concentrations in the prefrontal cortex and striatum were measured in vivo by microdialysis. Potassium infusion increased extracellular dopamine and glutamate concentrations to a greater extent in the prefrontal cortex than in the striatum of METH-pretreated rats compared to saline-pretreated controls. A low dose METH challenge significantly increased extracellular dopamine but not glutamate concentrations in both prefrontal cortex and striatum of all animals. Moreover, the acute METH-induced increased in cortical dopamine efflux was significantly greater in rats pretreated with METH. Overall, these data are the first evidence that repeated METH administrations can enhance cortical glutamate efflux and indicate that a low dose pretreatment regimen of METH enhances dopamine transmission in the prefrontal cortex through both transporter and depolarization induced mechanisms. PMID- 8624735 TI - [Methylene blue as an endocrine modulator: interactions with thyroid hormones]. AB - Methylene blue (MB) is a thiazine dye used in the treatment of methemoglobinemia. Since it was shown to scavenge free radicals, it is now being examined clinically in reperfusion syndrome and septic shock. We tested methylene blue in a series of experimental endocrine situations, in which this scavenging effect could play a role. Indeed, we observed that MB partly inhibited the increase in adenohypophyseal weight and cAMP and prolactin levels after the administration of estrogens in male rats. MB also inhibited the increase of another scavenger of free radicals, the metalloenzyme ceruloplasmin in the blood of estrogenized rats. MB also inhibited the stimulation of bone mass after estradiol in male rats. In this respect, it behaved as an antiestrogen. In the bones, MB also prevented the increase of bone minerals induced by estradiol. MB also produced a decrease in adenohypophyseal ascorbic acid content an potentiated the effect of estradiol in the same direction. Surprisingly, blood thyroxine levels increased consistently in rats after MB treatment. An interaction of MB with thyroid hormone synthesis and/or actions thus should be evaluated. This idea is supported by our observation of an antagonistic action of MB in carbimazole-induced rise in thyroid weight and decrease in thyroxine blood levels. (Ref. 14.). PMID- 8624734 TI - [The effect of neonatal nutrition on lipolysis and catecholamine binding in rat adipocytes]. AB - Underfeeding or overfeeding of rats during the suckling period, realized by the adjustment of the number of sucklings per litter, are followed by important changes in the function of endocrine glands and hormonal stimulation of lipolysis in adipocytes. For explanation of causes of the differences in the stimulation of lipolysis the changes of beta-receptors in adipocytes were studied in animals with different neonatal nutrition. The number of suckling pups in litter was adjusted to 4, 8, 14 on the second postnatal day. After weaning (at the age of 30 500 days) the size of adipocytes, basal and norepinephrine stimulated lipolysis, the binding of 3H-dihydroalprenolol in adipocytes were determined. In neonatally underfed animals, in comparison to overfed, those a decrease in the size of adipocytes, a diminution of basal lipolysis and lower stimulation of lipolysis by norepinephrine were observed. The decrease in binding capacity (at the age of 30 60 days) and of affinity of beta-receptors (at the age of 60 to 360 days) were found in neonatally overfed rats. These results showed that the changes of beta adrenergic receptors in adipocytes of animals with neonatal nutritions have a role in the alteration of the stimulation of lipolysis in adipocytes. (Tab. 2, Fig. 4, Ref. 29.). PMID- 8624737 TI - [The clinical significance of natriuretic hormones]. AB - Out of all until now discovered natriuretic factors it is still the atrial natriuretic factor (ANF) which is the most significant with its diuretic, natriuretic and vasodilatory effects. Its effect is antagonistic to sodium retention factors. The increase of its levels in arterial hypertension is more of secondary character, but according to some authors the functional deficit of ANF secretion can be applied also primarily in the development and maintenance of high blood pressure. ANF levels represent a good marker of the clinical severeness and are of prognostic value. Increased levels were detected also in cases of renal failure and partially in hepatic cirrhosis. Natriuretic hormone, in comparison to ANF, is a natriuretic and vasoconstrictive substance, the effect of which is based on the mechanism of sodium pump inhibition. Chemically the main candidate is represented by endogenous ouabain, or a digitalis-like activity. It increases physiologically due to the expansion of extracellular fluid during gravidity and in newborn. Its pathological increase is brought about by some forms of essential hypertension and in the diseases associated with fluid retention and edema development. Cirrhosis of the liver can reflect both the degree of sodium retention and haemodilution, as well as the severeness of hepatic lesion. (Tab. 2, Ref. 30.). PMID- 8624736 TI - [Glutamate neurotransmission, stress and hormone secretion]. AB - Glutamate neurotransmission has been investigated in relation to several physiological processes (learning, memory) as well as to neurodegenerative and other disorders. Little attention has been paid to its involvement in neuroendocrine response during stress. Penetration of excitatory amino acids from blood to the brain is limited by the blood-brain barrier. As a consequence, several toxic effects but also bioavailability for therapeutic purposes are reduced. A free access to circulating glutamate is possible only in brain structures lacking the blood-brain barrier or under conditions of its increased permeability. Excitatory amino acids were shown to stimulate the pituitary hormone release, though the mechanism of their action is still not fully understood. Stress exposure in experimental animals induced specific changes in mRNA levels coding the glutamate receptor subunits in the hippocampus and hypothalamus. The results obtained with the use of glutamate receptor antagonists indicate that a number of specific receptor subtypes contribute to the stimulation of ACTH release during stress. The authors provided also data on the role of NMDA receptors in the control of catecholamine release, particularly in stress-induced secretion of epinephrine. These results were the first piece of evidence on the involvement of endogenous excitatory amino acids in neuroendocrine activation during stress. Neurotoxic effects of glutamate in animals are well described, especially after its administration in the neonatal period. In men, glutamate toxicity and its use as a food additive are a continuous subject of discussions. The authors found an increase in plasma cortisol and norepinephrine, but not epinephrine and prolactin, in response to the administration of a high dose of glutamate. It cannot be excluded that these effects might be induced even by lower doses in situations with increased vulnerability to glutamate action (age, individual variability). (Tab. 1, Fig. 6, Ref. 44.). PMID- 8624738 TI - [Steroidal sex hormones and insensitivity syndromes]. AB - BACKGROUND: Today more than one hundred mutations are known of the gene for androgen receptor (AR) and merely one mutation of that for estrogen. The current trend in research has included the completion of the list of receptor defects and seeking the coincidence with their variable clinical manifestations. AIM: The aim of the study is to review the current state of the research on the defects of androgen and estrogen receptors and to draw attention to the clinical manifestations of the resistance to the sexual steroid hormones and currently to present own contributions in this problem. METHODS: Diagnostic application of the androgen resistance dynamic test residing in investigation of the SHBG level after androgen or anabolics administration. Molecular-biological analysis of AR defects with Phe 878 Ser mutation. CONCLUSIONS: Perspective acknowledgement of androgen and estrogen receptor defects allows a more precise diagnosis statement and perhaps also therapy of various forms of resistance to steroid sexual hormones. It can contribute to the understanding of some subsequent pathological processes dependent on the expression of androgens and estrogens. (Tab. 1, Ref. 20.). PMID- 8624739 TI - [Cushing's syndrome is still a potentially fatal disease]. AB - The paper summarizes the studies documenting the outlasting of increased cardiovascular mortality in patients with Cushing's syndrome even after the hypercortisolism resolution. Despite the hypercortisolism resolution the mortality during the subsequent period of life is up to four-fold higher than in the comparable population as a whole. The pathogenesis of this cardiovascular risk is based on arterial hypertension, steroid cardiomyopathy and hyperlipidaemia with subsequent atherosclerosis. The post mortem material bears a parallel of the extent of atheromatous changes and duration of hypercorticolism. The prevention has to its disposition two mutually complementing means. The first is represented by clinical screening of hypercorticolism which enables an early recognition and therapy of the Cushing's syndrome. The successive measure resides in increased attention to the cardiovascular system even after hypercorticolism resolution. (Tab. 1, Fig. 1, Ref. 25.). PMID- 8624741 TI - [The present and future of providing iodinated salt to the population]. AB - Iodine content was estimated in 401 samples of table salt (out of which 365 from the distribution network and 101 from households). The values of 85.5% of samples from the distribution network were in normal range (i.e. 25 +/- 10 mg KI/kg) while 9.8% of samples showed the values below that range. The samples from households showed lower values, only 70.2% being in normal range and 28.6% below that which was 3 times more than in those from the distribution network. Such difference may be explained by the loss of iodine from the salt during the storage due to the effects of moisture, temperature and light. However, less satisfactory iodine levels were found in the salt imported from Poland (9 of 28 samples below the normal range, i.e. 32%), Austria (5 from 9 samples below normal range, i.e. 55%) and in the sea salt (7 from 11 samples below normal range, i.e. 63%), while satisfactory values were found in the salt from Germany (all 5 samples examined were in normal range). From 1126 samples from the distribution network examined during 1981-1986 were 63.6 in normal range, while 88.4% out of 612 samples examined during the period 1987-1990 hat their iodine levels in normal range. Present data show satisfactory iodine content in the salt which justifies the assumption of satisfactory iodine intake in the population of Slovakia. (Tab. 2, Ref. 6.). PMID- 8624740 TI - [The effect of insufficient iodine intake on the size and function of the thyroid gland]. AB - BACKGROUND: The supply of iodine indisputably affects the function of the thyroid gland. It is not clear, however, as to what extent a moderate iodopenia affects common thyroid parameters. OBJECTIVES, STARTING POINT AND MAIN PURPOSE: The aim of the study is to evaluate the influence of iodopenia (ioduria below 50 micrograms of iodine per litre of urine) on the values of T4, T3, TSH, the ankle jerk time and sonographically ascertained thyroid gland volume in randomly selected sample of Czech population from 4 regions. METHODS: Ioduria was assessed by means of Sandel-Kolthoff method, total thyroxine, triiodothyronine and thyreotropic hormone by immunoluminiscence method; the volume of the thyroid gland sonographically according to Gutekunst and the ankle-jerk reflex by electromagnetic record method. RESULTS: Iodopenia affects a majority of investigated parameters in dependence on age. T4 is significantly higher in iodopenic adults, T3 is significantly higher in iodopenic children, TSH is significantly higher in iodopenic adults and the ankle-jerk time is prolonged in iodopenic adults. The statistical significance of the effect of iodopenia on the volume of thyroid gland was not proved. CONCLUSION: Iodopenia of a mediate degree affects commonly used parameters of the thyroid gland function in dependence on age and other factors which are to be more precisely analyzed using a larger sample. The statistical significance of the effect of iodopenia on the volume of thyroid gland was not proved. (Tab. 1, Fig. 5, Ref. 13.). PMID- 8624742 TI - [Thin-needle aspiration biopsy in the preoperative diagnosis of non-toxic nodular goiter]. AB - The ascertainment of diagnostic reliability of fine needle aspiration biopsy in preoperative examination of non-toxic goitres is performed by comparing its results with the findings of the definitive histologic examination. The set of patients did not include those with the cytological finding of Hurtle tumour for the impossibility to distinguish between adenomas and carcinomas from oxyphilic cells by means of cytologic examination. Aspirates were stained by May, Grunwald and Giemsa method. Histologic results were gained from 185 patients. Cytologic findings were considered as benign in 95 cases (however 3 cases of cancer were revealed by the histologist--false negativity). In 90 cases, atypia, findings of suspectivity, or diagnosis of malignity were described, however the histologist evaluated 49 cases of goitres as being benign (false positivity). Totally 44 cases of malignant tumours were histologically diagnosed. Diagnostic sensitivity in our set of patients was 0.97 and diagnostic specificity was 0.46. In accordance with the available literature, fine needle cytodiagnosis is considered to represent an appropriate screening method in the process of decision as to whether conservative or surgical therapy with histologic verification in patients with non-toxic nodular goitres is to be indicated. (Tab. 1, Ref. 6.). PMID- 8624743 TI - [Chronological changes in the representation of papillary and follicular carcinomas in our patient group 1960-1993]. AB - BACKGROUND: Differentiated carcinoma of the thyroid gland is globally changing its character and occurrence frequency. METHODS: Retrospective analysis of patients sent to the Clinic of Nuclear Medicine in Prague, Motole in order to undergo the radioactive iodine therapy during the period from 1960 to 1993 in comparison to the set of patients of the 1950-1959 period. MAIN RESULTS: A conspicuous change took place in the proportion of papillary and follicular carcinomas. The previous predominance of follicular carcinoma has turned to the predominance of papillary carcinoma, the occurrence of which is increasing. The authors see the causes not only in introduction of iodidation which decreases the occurrence of more aggressive forms of differentiated carcinoma, but also in radioactive fall, as well as in the change of diagnostic procedures. CONCLUSIONS: The occurrence of differentiated carcinoma is so far increasing, but in the recent years the increase of more aggressive forms of the disease (follicular carcinoma) has ceased and an expressive increase of less aggressive papillary carcinoma is continuing. (Tab. 1, Ref. 14.). PMID- 8624744 TI - [The effectiveness of iodine prophylaxis of endemic goiter in Slovakia from the viewpoint of physical and ultrasonographic examinations of the thyroid gland]. AB - Being aimed at the effectivity of iodine prophylaxis of endemic goitre the authors examined 2946 children and adolescents at the age of 6-18 years during the period from 1989 to 1995 in 6 regions of Slovakia. The thyroid glands were examined physically and ultrasonographically. The authors have found out that the occurrence rate of diffuse goitre is acceptable and it is not meeting the criteria of endemic goitre. No case of nodular goitre was revealed. Similarly to the relative frequency of diffuse goitre, neither the ultrasonographically measured volumes of thyroid glands differed among various areas. They are comparable with the volumes detected in countries with a sufficient iodine supply. The use of criteria for iodine deficiency evaluation recommended by WHO (goitre occurrence, thyroid gland volume, ioduria) leads the authors to consider the current prophylaxis of endemic goitre as being effective and successful. (Tab. 5, Ref. 20.). PMID- 8624745 TI - [Functional morphology of the spleen]. AB - The spleen was till the first half of the twentieth century considered to represent an organ of no special significance. Nowadays its clearance function and position in the immunity system is thoroughly investigated. Informations on its cellular composition and compartments are sufficient. The problems coinciding with circulation in the spleen are solved. The study of interactions of T and B lymphocytes regarding the development of humoral immunity do not provide clear results. Unclear remain also the relations of extracellular and cellular structures. The localisation of the spleen in blood circulation and large amount of migrating lymphocytes justify its significant position in the protection of host against infections spreading haematogenously. It is not inevitable for survival, but its removal increases the risk of unmanageable infections carried by bacteria with polysaccharide capsules. The microenvironment in the spleen enables intercellular relations and determines the development of immune reactions. Microcirculation enables it to function as a biological filter without the necessity of specialized filtrating structures being present. In consequence of blood filtration everything leaks into the extravasal space. Subsequently all components normally constituting the blood return back into the blood within vessels. That is why no special structures are formed in order to filter individual components from the blood. All components that should not be present in the blood are excluded extravasally. The substances which are not let through the wall of sinuses into the blood, remain in the marginal zone and in the red pulp. The macrophages of the marginal zone and red pulp destroy and degrade the unrequired substances. (Ref. 26). PMID- 8624746 TI - [Diagnosis and treatment of erectile dysfunctions]. AB - OBJECTIVES: Erectile dysfunction (ED) is the most frequent form of male sexual dysfunctions. The incidence is not known. Approximately 10% of men suffer from ED. Aging is concomitted by an exponential increase in the occurrence of erectile dysfunctions. Some groups of men (suffering from diabetes, alcoholism, ICHS, renal diseases) yield several times higher values of their occurrence. In general, ED (formerly impotentio coeundi) can be defined as an inability to achieve erection and maintain it for an appropriately long period enabling sexual intercourse. ETIOPATHOGENESIS: The etiology includes psychological, neurological, arterial, sinusoidal, venous and hormonal factors which participate in erectile function. ED arises predominantly in consequence of impaired haemodynamics of the penis. It develops either due to insufficient blood inflow into the erectile bodies or due to their excessive drainage. The traumatic lesions of penile vessels are less frequent. Drugs and medicaments play a significant role among etiologic factors causing erectile dysfunctions. DIAGNOSIS: No regulations and principles are generally valid in coincidence with examination of patients with erectile dysfunction. The signs of neurologic, hormonal and vascular defects are detected. An objective method of examination of erectile capacity of the penis resides in the monitoring of penile nocturnal tumescensions. The examination of arterial system of the penis provides information on the perfusion pressure and blood flow in the cavernous arteries. The vascular structures of the penis and their functions can be evaluated by means of duplex ultrasonography and flowmetry. The quality to arterial supply to erectile bodies is evaluated by dynamic examination of arteries prior to and after intracavernous application of vasoactive substances. The functional state and size of venous leak from the cavernous bodies are evaluated by dynamic cavernosometry. THERAPY: The application of vasoactive substances meant a significant contribution to the diagnosis establishment and therapy of erectile dysfunctions. They have a common feature which resides in their myorelaxation effect and thus in their ability to induce erection by means of relaxation of the smooth muscles of cavernous bodies. Intracavernous application of vasoactive substances is the mostly spread means of therapy of erectile dysfunction as a whole, frequently with no regard to the etiology. Surgical therapy of vasculogenous impotence is based on knowledge concerning the impact of vascular lesions on the haemodynamics of erectile function of the penis. the revascularisation operations are aimed at the regeneration of normal haemodynamic conditions in the penis. CONCLUSION: Sexual life is currently considered as a proportion of life of individuals. The physicians therefore have to seek ways of how to help the patients who due to acquired erectile dysfunctions are handicapped in the establishment of family harmony and sexual life. (Ref. 31). PMID- 8624748 TI - Night and day differences in the food-intake of laboratory rats Wistar and Koletsky strains. AB - BACKGROUND: Rats have an extraordinary expressive circadian organization and prefer the dark as the optimal condition of its life environment. During darkness, the rhythm of its several parameters reaches the acrophase. MAIN PURPOSE: The aim was to establish the differences in the level of food-intake of laboratory rats of the Wistar strain and in those of the spontaneously obese rats of the Koletsky strain by means of ad libitum feeding under natural light-dark conditions. METHODS: The levels of day and night food consumptions in both strains of rats, Wistar and the spontaneously obese Koletsky strain, were studied within the period from January 10th to 20th, (males), and from April 16 to 26, 1990, (females). The animals were fed by Larsen's diet under natural light conditions. The data were evaluated by means of the t-test. RESULTS: It was detected that during the period between 5 o'clock PM and 6 o'clock AM the male rats of the Wistar strain consumed 80% and the female rats 79% of their total daily amount of food. Food consumption of the Koletsky rats during the period from 5 o'clock PM to 6 o'clock AM in males reached the value of 72% and in females 74%. CONCLUSIONS: There were detected significant differences in circadian food-intake in both strains. Besides the nocturnal preference in food intake of Wistar rats, which is in accordance with the data gained from literature, a similar food-intake with expressive diurnal-nocturnal differences exists also in the spontaneously obese Koletsky rats. Daily food consumption in these male rats was significantly higher when compared to the Wistar strain. (Tab. 1, Fig. 1, Ref. 5.). PMID- 8624747 TI - [Procedure for multiorgan procurement (heart, liver, kidneys)]. AB - A significant growth in transplantation activity has been recorded in the past decade, namely in transplantations of the heart and liver. The numbers of transplantations of the lungs, and of the combination of pancreas with kidneys have increased to a smaller extent. The number of transplanted kidneys has become stabilized during the past several years. The development of organ transplantations led to a change of conception in the procurement of cadaveric organs from the until now performed procurement of a single organ to multiorgan procurements where in addition to the kidneys at least one other organ is procured. In countries with the highest number of multiorgan procurements the latter form as many as 80% of the total number of cadaveric procurements. Since January 1, 1995 a principle has been officially recognized in the Slovak Republic where an agreement with organ donation is presumed. Procurements of organs do not take place in cases when the donors have made during their lives a written or any other kind of declaration disapproving any intervention into their corporal integrity. Organs are also not procured in cases of foreigners, persons under legal sentence and those whose identities cannot be proved. The authors describe the technique of multiorgan procurement (heart, liver, kidneys) as well as contraindications to procurements of individual organs. (Fig. 2, Tab. 1, Ref. 12.). PMID- 8624749 TI - [Reconstructive operations in our clinical data]. AB - The authors present the results of reconstructive vascular operations performed during the period from 1990 to 1994. According to the criteria of the European Vascular Surgery Council the lowest patency was recorded in the distal type of femoropopliteal bypasses in the lower extremities. They emphasize the necessity to apply the "Graft Surveillance Programme" in vascular consultation centre. (Tab. 2, Ref. 7.). PMID- 8624750 TI - [Selective vulnerability of neuronal injury after experimental heart arrest]. AB - The study was aimed at the analysis of vulnerability of the olfactory bulb neurons in dogs after experimental heart arrest lasting for 15 minutes and recirculation lasting for 1 hour. By means of the Nauta degenerative neurohistologic method the reactions of individual types of nerve cells in the olfactory bulb were investigated. Nauta-positive granules were observed in the cytoplasm of the mitral and tufted cells of the olfactory bulb, which are of dopaminergic character. The granulations were present in the cellular areas which are rich in Nissl substance. The granular and short-axon cells of GABA-ergic character which contain a small amount of Nissl granules lack the Nauta-positive granules. Similarly, the Nauta-positive granules were absent in the axon hillock of mitral cells which under normal conditions do not contain the Nissl substance. These results justify the conclusion that ischemia lasting for 15 minutes and one hour recirculation primarily affect the excitatory neurons rich in the Nissl substance. (Fig. 4, Ref. 16.). PMID- 8624751 TI - [A mechanical heart valve prosthesis with a spherical shape and open center]. AB - A mechanical valve prosthesis is described with central blood flow. The generations I and II of the mechanical valve obstruct the blood flow and cause turbulence with undesirable consequences. The opening in the centre of Simkovic Bolf valve preserves the laminar blood flow, reduces the trauma of blood and myocardium stress. Our new valve represents a mechanical valve prosthesis of generation III. (Fig. 5, Ref. 16.). PMID- 8624752 TI - [Postoperative analgesia with epidural administration of a combination of tramadol and clonidine]. AB - Postoperative analgesia inhibits the stress cascade with negative effects on whole organism. Therefore the spectrum of drugs used for soothing postoperative pain quickly widens. The epidural route appears as being logical, since due to the direct effect in the transmission and processing of pain it suffices with a lower dosage. The authors refer to a group of 30 patients postoperatively treated by a combination of tramadol and Clonidine administrated by means of an epidural catheter. 26 patients evaluated the induced analgesia as excellent or sufficient. (Tab. 3, Ref. 4). PMID- 8624754 TI - [References to the article by Prof. V. Zikmund: Problems with the limitations of medicine]. PMID- 8624753 TI - [Variations in histologic and ultrastructural findings in renal glomerular biopsies]. AB - On the basis of 506 renal biopsies performed within a six-year period (237 of which were evaluated electron-microscopically) the authors discovered that in 11 cases the diagnosis based on electron microscopic examination had to be corrected or supplemented by the diagnostic statement of membranous glomerulonephritis. These cases included 1 boy (HBsAg positive), 2 men and 8 women were middle aged. 5 women were treated due to polyarthritis. All cases when examined light microscopically yielded a pattern evaluated as normal, or as minute abnormalities of glomeruli. Half-thick sections were not sufficient for the diagnosis. 3 cases of biopsies yielded a variously developed chronic tubulointerstitial nephritis, two cases were accompanied with polyarthritis. On the basis of the latter the authors consider the electron microscopic examination as indicated: 1. in cases when the diagnosis statement is not achieved by other examinations, 2. findings are in the range of the so-called variable norm, 3. in all cases of minute abnormalities in glomeruli detected without electron microscopy, 4. the impact of any of effective drugs cannot be excluded, 5. the diagnostic conclusion based on renal biopsy and the clinical-laboratory pattern of the disease are inconsistent. The authors assume that especially the fourth and fifth groups are going to be gradually supplemented and made more precise. (Fig. 1, Ref. 5.). PMID- 8624755 TI - Examining the molecular genetics of HTLV-I with an infectious molecular clone of the virus and permissive cell culture systems. AB - Infectious molecular clones of HTLV-I proviruses have only recently been reported. The long wait for such provirus clones reflects the difficulties inherent in propagating HTLV-I in vitro, and thus a rigorous demonstration of infectivity has awaited improved cell culture systems and sensitive detection techniques for HTLV-I. An intact HTLV-I provirus, originating from an American ATL patient, was subcloned into a plasmid vector and was designated pCS-HTLV. Transient transfections of mammalian cells with pCS-HTLV resulted in the synthesis of viral proteins and mRNAs which were assembled into virions that had physical and morphological characteristics typical of HTLV-I particles. The ability of these virus particles to infect cells, replicate, and produce infectious progeny was demonstrated initially in short term, cell-free infection assays by monitoring the expression of specific viral mRNAs. These studies have been extended in cell culture systems that support continuous virus production. Primary T-lymphocytes have been infected either with cell-free supernatant fluids from, or by coculture with, cells transiently transfected with pCS-HTLV, giving rise to continuous, IL-2-dependent cell lines that have been in culture for >1 year. Furthermore, fetal rhesus lung cells (FRhL) were shown to be permissive for HTLV-I replication and sustained virus expression after infection with pCS-HTLV. Continuous FRhL cell lines now have been established that express various HTLV-I proviruses and mutants. These provirus clones and cell lines provide us with the means to address long-standing questions dealing with the biology of HTLV-I. PMID- 8624756 TI - High levels of anti-HIV-1 envelope antibodies in cerebrospinal fluid as compared to serum from patients with AIDS dementia complex. AB - The antibody response to the HIV-1 envelope protein has not been well characterized in patients with AIDS dementia complex (ADC). We evaluated the frequency of antibodies against the HIV-1 envelope in cerebrospinal fluid (CSF) and serum from 21 persons with ADC and 10 symptom-free HIV-1-positive subjects using Western immunoblot with reducing and nonreducing buffer and radioimmunoprecipitation (RIP) analysis. RIP analysis revealed anti-envelope antibodies in all sera tested. Higher anti-envelope levels were observed in CSF than in serum of 12 of 21 ADC patients and only 1 of 10 symptom-free subjects (two-sided Fisher exact test, p < 0.05). All persons with moderate to severe ADC had higher anti-envelope levels in CSF than in sera (p < 0.005). CSF anti-gp120 antibodies were not as readily detected by Western blot analysis even under nonreduced conditions, suggesting that they are directed to conformational epitopes. Higher CSF anti-envelope antibodies appear to be more common in patients with ADC than in symptom-free HIV-1-positive subjects. This antibody pattern may serve as a marker for ADC and its progression. PMID- 8624757 TI - The mutation frequency of feline immunodeficiency virus enhanced by 3'-azido-3' deoxythymidine. AB - We have developed a host range system to measure the mutation frequency of feline immunodeficiency virus (FIV), the feline homologue of human immunodeficiency virus type 1 (HIV-1). When wild-type FIV was grown in the presence of a known mutagen, 5-bromo-2'-deoxyuridine (BUdR), a dose-dependent increase of host range mutants was detected. Using this system, we have evaluated the effects of antiviral drugs upon the mutation frequency of FIV. Subinhibitory concentrations of 3'-azido-3'-deoxythymidine (AZT), the most common antiviral drug used in AIDS chemotherapy, increased the mutation frequency of FIV in a dose-dependent manner. Two other antivirals, 2',3'-dideoxyinosine (ddI) and 2'3'-dideoxycytidine (ddC), did not show this effect. PMID- 8624758 TI - Use of a quantitative cytomegalovirus (CMV) antigenemia test in evaluating HIV+ patients with and without CMV disease. AB - Cytomegalovirus (CMV) infection remains a life-threatening infection in patients with HIV disease. A rapid, quantitative diagnostic technique is needed to adi in the diagnosis of CMV disease. This study was undertaken to evaluate the CMV antigenemia test in patients with HIV disease who are at risk for CMV disease. The study included 22 patients who underwent ophthalmologic exams or selected diagnostic techniques in whom CMV cultures and CMV antigenemia tests were performed. All of 11 patients with CMV disease had positive CMV antigenemia assays [range, 48-1,000 positive cells/2 x 10(5) peripheral blood leukocytes (PBL)], and 10 were also CMV viremic. There was no clinical evidence of CMV disease in 11 patients, including seven in whom the CMV antigenemia assay was negative and who remained without evidence of CMV disease after a median follow up of 159 days. Four patients had low antigenemia levels. Of these four, two subsequently developed CMV retinitis. In conclusion, a positive CMV antigenemia result with > or = 48 positive cells/2 x 10(5) PBL correlated with concurrent CMV disease. The CMV antigenemia test appears to be a valuable tool for the rapid diagnosis of CMV disease in HIV-infected individuals. PMID- 8624760 TI - Impact of missing data due to dropouts on estimates of the treatment effect in a randomized trial of antiretroviral therapy for HIV-infected individuals. Canadian HIV Trials Network A002 Study Group. AB - PURPOSE: To evaluate the impact of missing data due to nonrandom dropout on estimates of the effect of treatment on the CD4 count in a clinical trial of antiretroviral therapy for HIV infected individuals. METHODS: The effect of treatment on CD4 counts in a recent study of continued ZDV versus ddI in HIV infected individuals was estimated from the observed data and after imputing missing CD4 counts for patients who dropped out of the study. Imputation methods studied were (a) carrying forward the last observed CD4 count, (b) predicting missing CD4 counts from regression models, and (c) assuming that CD4 counts of patients who dropped out declined at a rate of 100 cells per year. RESULTS: Of the 245 patients enrolled in the study, 52% completed the planned 48 weeks of follow-up. Patients with lower CD4 counts were more likely to drop out of the study (RR = 1.77; p = 0.0001). Patients receiving ZDV had a greater tendency to drop out than patients receiving ddI (p = 0.07). Mean CD4 counts calculated after imputing missing data were lower than those obtained from the observed data at all follow-up times for both treatment groups. Imputing CD4 counts with regression models yielded higher estimates of the effect of treatment than were obtained using the observed data. CONCLUSION: Missing outcome data due to dropouts can result in an underestimation of the treatment effect and overly optimistic statements about the outcome of participants on both treatment arms due to the selective dropout of participants with lower or decreasing CD4 counts. When there are significant dropout rates in randomized trials, imputation is a useful technique to assess the range of plausible values of the treatment effect. PMID- 8624759 TI - Patterns of opportunistic infections in patients with HIV infection. AB - The pattern of the development of opportunistic infections (OIs) in HIV-infected patients was evaluated, based on a cohort of 1,530 patients enrolled in two AIDS Clinical Trials Group anti-retroviral studies. We quantified the increase in risk of OIs associated with the occurrence of a previous OI. This assessment was based on the observed event rates of the more common AIDS-defining OIs: Pneumocystis carinii pneumonia (PCP), Mycobacterium avium complex (MAC), cytomegalovirus (CMV), and a systemic mycosis. Additionally, for each OI, we assessed the relative risks associated with a history of prior OIs, changes in CD4 levels, and baseline prognostic factors. We found that the occurrence of each of these OIs increased the risk of subsequent OIs, even after adjusting for the CD4 count. Specifically, the occurrence of PCP significantly increased the risk of MAC and CMV, and somewhat increased the risk of systemic mycoses. Diagnosis with MAC was associated with an increased risk of subsequent CMV, whereas the occurrence of CMV increased the risk of MAC. Finally, once patients were diagnosed with a systemic mycosis, they were at a somewhat increased risk of subsequently developing MAC or CMV. Although current practice for determining the timing and initiation of prophylactic therapies relies chiefly on CD4 count, the occurrence of specific AIDS-defining OIs in patients with HIV infection should also be taken into account in making decisions regarding prophylaxis strategies. PMID- 8624761 TI - Off-label drug use in human immunodeficiency virus disease. AB - We wished to determine the extent to which drugs used to treat HIV disease and its clinical manifestations are prescribed for conditions other than those listed on the U.S. Food and Drug Administration's approved drug label, how such "off label" use varies by patient characteristics and type of HIV-related medical condition, and the extent to which physicians alter the way they treat HIV related conditions because of reimbursement problems associated with off-label drug use. We surveyed 1,530 primary care providers for people with HIV disease between February and May 1993. A three-part survey instrument was used to obtain data on the drugs prescribed for the last three patients with HIV disease treated by the provider, the preferred choice of therapy for 32 specific HIV-related conditions, and the extent to which providers faced reimbursement problems regarding the use of drugs for off-label indications. Three drug compendia were used as cited sources of off-label drug uses. In all, 387 (32%) evaluable surveys were returned, yielding data on 1,148 patients. The majority (81%) of patients received at least one drug off-label, and almost half (40%) of all reported drug therapy was off-label. Most off-label drug use was for treatment and prevention of HIV-related opportunistic infections, which frequently represented the community standard of practice (e.g., trimethoprim/sulfamethoxazole for prevention of Pneumocystis carinii pneumonia), or the de facto standard of practice when no licensed therapies were available (e.g., drugs for treatment of Mycobacterium avium complex, MAC). More than 75% of off-label usage was cited in at least one of the three authoritative medical compendia. The use of drugs for off-label indications in HIV care is common and frequently represents community standards of care. Reliance on drug compendia for support of off-label drug use accounts for the majority of such uses, although many legitimate off-label uses may not be included because of compendia publication lag. The prevalence of off label drug use in routine clinical practice and the development of newer and more costly drugs for treatment of HIV and its medical complications argues for the articulation of an explicit national reimbursement policy for off-label uses of prescription drugs so that medically appropriate therapies will be available to those with insurance in a rational, consistent way. PMID- 8624762 TI - Evolution of zidovudine resistance-associated genotypes in human immunodeficiency virus type 1-infected patients. AB - Substantial differences have been described in the response of individual patients to zidovudine (ZDV) therapy, both in the clinical impact and in virus load. Genotypic changes associated with the appearance of drug resistance may also be different or occur at different rates. We have obtained the nucleotide sequence of the RT domain of individual HIV-1 genomes extracted from 10 plasma and peripheral blood mononuclear cell (PBMC) samples donated by two haemophiliac patients before, during, and after long-term ZDV therapy. Although the plasma virus load was similar throughout, the order and timing of appearance of resistance-associated substitutions differed in the two patients. In patient p74, K70R appeared after 4 months, T215Y at 5.5 months, and M41L at 13 months. In p87, K70R also appeared at 4 months, but T215Y and K219Q were not observed until 18 months and M41L not at all. Much greater sequence change overall occurred in p74. The evolution of the viral population in that patient was dominated by the unique appearance of T215Y and subsequently M41L, with all sequences from the last time point being descended by a single path from the pretreatment samples. However, in p87, several different lineages of RT sequences were found to persist throughout treatment. We propose that these differences in outcome may be determined by differences in genetic background at sites other than the five generally considered to be associated with ZDV sensitivity. PMID- 8624764 TI - Opportunities for targeting publicly funded human immunodeficiency virus counseling and testing. AB - We wished to identify opportunities for improving the yield of positive HIV test results from federally funded HIV counseling and testing programs. We reviewed client records from 1992 and 1993 for targeting opportunities at the site level based on site type (i.e., family planning clinic) and the seropositivity in the past and at the client level based on the client's history of a past negative test, demographics, and risk history. We studied 1,281,606 records from 1992. The number of tests and opportunities for site-level targeting varied by project area. Seropositivity varied by site type, but the best predictor of seropositivity was seropositivity at that site in the past. Of 1,102 sites with <1% of tests positive in 1992, only five had >3% positive in 1993. Sites with no positive tests in 1992 performed 99,468 tests in 1993, and only 292 (0.3%) were positive. Clients with a past negative test had a slightly lower seropositivity (1.5%) than clients with no previous test (2.0%). In sites with a low (0.1-2.0%) seropositivity, clients with no transmission risk by history were unlikely to be infected (0.8% for black men). However, in sites with high (> or =5%) seropositivity, clients without risk were often infected (5.7% for black men). Opportunities for targeting were identified. They vary considerably by project area and testing site. These opportunities for targeting should be considered by sites as AIDS prevention strategies evolve. PMID- 8624763 TI - Temporal and geographical trends of anti-HIV-1 antibodies screening among newborns in Italy, 1990-1993. Italian Collaborative Study Group for HIV Prevalence in Newborns. AB - To describe the dynamic of HIV-1 prevalence in Italian childbearing women and to estimate the future incidence of pediatric AIDS due to vertical transmission, dried-blood specimens collected from a consecutive sample of newborns in all Italian regions were examined for the presence of anti-HIV-1 antibodies (HIV-Ab) after the routine neonatal screening program was completed. Of 555,722 blood samples collected and examined for HIV-Ab between 1990 and 1993, 550 (0.099%) were positive. Nationwide, the HIV seroprevalence decreased between 1990 and 1992 (0.124% in 1990, 0.100% in 1991, 0.085% in 1992), and increased, as compared with that in the previous year, in 1993 (0.096%). In an univariate analysis, HIV seroprevalence was positively was positively associated with being born in regions having higher AIDS cumulative incidence and in metropolitan areas, but negatively associated with year of delivery. In a multiple logistic regression analysis, only the AIDS cumulative incidence level of the delivery area and being born in a metropolitan area remained independently associated with HIV seroprevalence. Our results show significant geographical variation in the spread of HIV infection among childbearing women in Italy and provide useful indications to target prevention and care strategies for HIV-infected women and their children and to estimate the potential impact of implementing programs aimed at preventing vertical transmission of HIV infection. PMID- 8624765 TI - Micronutrient profiles in HIV-1-infected heterosexual adults. AB - There is compelling evidence that micronutrients can profoundly affect immunity. We surveyed vitamin supplement use and circulating concentrations of 22 nutrients and glutathione in 64 HIV-1 seropositive men and women and 33 seronegative controls participating in a study of heterosexual HIV-1 transmission. We assayed antioxidants (vitamins A, C, and E; total carotenes), vitamins B6 and B12, folate, thiamin, niacin, biotin, riboflavin, pantothenic acid, free and total choline and carnitine, biopterin, inositol, copper, zinc, selenium, and magnesium. HIV-infected patients had lower mean circulating concentrations of magnesium (p < 0.0001), total carotenes (p = 0.009), total choline (p = 0.002), and glutathione (p = 0.045), and higher concentrations of niacin (p < 0.0001) than controls. Fifty-nine percent of HIV+ patients had low concentrations of magnesium, compared with 9% of controls (p < 0.0001). These abnormal concentrations were unrelated to stage of disease. Participants who took vitamin supplements had consistently fewer low concentrations of antioxidants, across HIV infection status and disease stage strata (p = 0.0006). Nevertheless, 29% of the HIV+ patients taking supplemental vitamins had subnormal levels of one or more antioxidants. The frequent occurrence of abnormal micronutrient nutriture, as found in these HIV+ subjects, may contribute to disease pathogenesis. The low magnesium concentrations may be particularly relevant to HIV-related symptoms of fatigue, lethargy, and impaired mentation. PMID- 8624766 TI - HIV and infections of similar transmission patterns in a drug injectors community of Santos, Brazil. AB - To study the prevalence of HIV and infections with related transmission patterns, we interviewed and obtained blood samples from 220 injecting drug users (IDUs), sampled by snowballing, from the city of Santos in the state of Sao Paulo, Brazil, where the estimated number of IDUs (10,000) comprises approximately 2% of the entire population. Seroprevalence of HIV, hepatitis B and C, syphilis, and HTLV (1 and 2) was assessed and compared with that in 197 blood donors from the same city, matched for age and gender. Risk behavior related to HIV was assessed by a standard questionnaire applied to the IDU sample. Univariate and multivariate analyses of the risk factors were performed. Seroprevalences found were 62% for HIV, 75% for HCV, 75% for HBV, 34% for syphilis, and 25% for HTLV (1 and 2) among IDUs, which compare with 0.0%, 2%, 23%, 12%, and 1% for blood donors, respectively. The risk for parenterally transmitted infections in this IDU community was higher than that for sexually transmitted infections (odds ratio for syphilis, 3.57; hepatitis B, 10.0; and hepatitis C, 100). The results of the mutivariate risk analysis showed that daily rate of ID use >5 times/day (OR = 6.73), not changing behavior to avoid AIDS (OR= 3.28), ID use >15 days/month (OR = 2.72), and ID use in the last 2 months (OR = 2.23) were the risk behaviors significantly associated with HIV infection. PMID- 8624767 TI - Campylobacter spp. bacteremia in AIDS patients. PMID- 8624768 TI - IL-6 and b-FGF overproductions precede development of AIDS-associated Kaposi's sarcoma. PMID- 8624769 TI - Prevalence of and risk factors for HTLV-I and HTLV-II infection among patients at a hospital in the South Bronx, New York. PMID- 8624770 TI - White blood cell count and the risk of coronary heart disease and all-cause mortality in elderly men. AB - Because the importance of established risk factors for coronary heart disease (CHD) is unclear in older people, the associations of white blood cell (WBC) count with the risk for CHD and all-cause mortality were investigated in an elderly cohort that was followed up for 5 years. In 1985, complete information on the risk factors of interest was available for 884 randomly selected men, aged 64 to 84 years, from the Dutch town of Zutphen (participation rate, 74%). Relative risks (RRs) for each 10(9)/L increase in WBC count were obtained for the 5-year incidence of and mortality from CHD and all causes. RRs were adjusted for age, body mass index, systolic blood pressure, total and high density lipoprotein cholesterol levels, and cigarette smoking habit. The WBC count was 6.7 +/- 1.8 X 10(9)/L (means +/- SD) at baseline. An increased WBC count was independently associated with mortality due to CHD, and the RR amounted to 1.32 (95% confidence interval [95% CI], 1.15 to 1.51). For the incidence of CHD the RR was 1.14 (95% CI, 0.98 to 1.32). These associations were observed regardless of cigarette smoking habit. Regarding all-cause mortality, the RR amounted to 1.25 (95% CI, 1.17 to 1.35). This association was especially noticeable among former smokers and those who had never smoked. In conclusion, during 5 years of follow-up WBC count predicted CHD and all-cause mortality in elderly men, independent of the conventional risk factors for CHD. PMID- 8624771 TI - Dose-response relationship between ischemic heart disease mortality and long-term arsenic exposure. AB - The cardiovascular effects of inorganic arsenic have been documented, but the dose-response relationship between ischemic heart disease (ISHD) and long-term arsenic exposure remains to be elucidated. Mortality rates from ISHD among residents in 60 villages of the area in Taiwan with endemic arseniasis from 1973 through 1986 were analyzed to examine their association with arsenic concentration in drinking water. Based on 1 355 915 person-years and 217 ISHD deaths, the cumulative ISHD mortalities from birth to age 79 years were 3.4%, 3.5%, 4.7%, and 6.6%, respectively, for residents who lived in villages in which the median arsenic concentrations in drinking water were <0.1, 0.1 to 0.34, 0.35 to 0.59, and > or = 0.6 mg/L. A cohort of 263 patients affected with blackfoot disease (BFD), a unique arsenic-related peripheral vascular disease, and 2293 non BFD residents in the endemic area of arseniasis were recruited and followed up for an average period of 5.0 years. There was a monotonous biological gradient relationship between cumulative arsenic exposure through drinking artesian well water and ISHD mortality. The relative risks were 2.5, 4.0 and 6.5, respectively, for those who had a cumulative arsenic exposure of 0.1 to 9.9, 10.0 to 19.9, and > or = 20.0 mg/L-years compared with those without the arsenic exposure after adjustment for age, sex, cigarette smoking, body mass index, serum cholesterol and triglyceride levels, and disease status for hypertension and diabetes through proportional-hazards regression analysis. BFD patients were found to have a significantly higher ISHD mortality that non-BFD residents, showing a multivariate-adjusted relative risk of 2.5 (95% CI, 1.1 to 5.4). PMID- 8624772 TI - Gemfibrozil treatment of combined hyperlipoproteinemia. No improvement of fibrinolysis despite marked reduction of plasma triglyceride levels. AB - Hypertriglyceridemia is linked to impaired fibrinolytic function, and lipid lowering treatment with fibric acid derivatives could hypothetically improve fibrinolysis in this condition. We therefore conducted a double-blind, placebo controlled, crossover study of gemfibrozil treatment on fibrinolytic function in 21 men with combined hyperlipoproteinemia. Measurements were performed at rest and during mental stress and after venous occlusion. The patients had clearly disturbed fibrinolytic function, with elevated plasminogen activator inhibitor-1 (PAI-1) activity at rest ( approximately 25 U/mL; reference, <15 U/mL). Gemfibrozil reduced plasma total and VLDL cholesterol as well as all triglyceride fractions, whereas HDL cholesterol increased (P <.001 for all). Total triglyceride levels were reduced by 57 +/- 4% (from 5.3 to 2.1 mmol/L). Fasting serum insulin levels were not altered by gemfibrozil treatment. Plasma levels of PAI-1 activity and tissue-type plasminogen activator (TPA) activity or antigen were unaffected by gemfibrozil treatment both at rest and during the provocations. The levels of D-dimer, plasmin/antiplasmin complex, and fibrinogen were also uninfluenced by gemfibrozil treatment. Mental stress elevated plasma TPA (P=.0036) and lowered PAI-1 (P=.0012) activity during placebo but not gemfibrozil treatment (P=.28 and P=.17, respectively), but treatment effects did not differ by ANOVA on delta values (ie, stress minus rest). Venous occlusion reduced PAI-1 activity, whereas TPA and plasmin/antiplasmin complex increased during both treatments. Thus, gemfibrozil treatment did not improve fibrinolysis or lower fibrinogen levels in men with combined hyperlipoproteinemia despite marked reduction of plasma triglyceride levels. It seems unlikely that improved fibrinolysis explains the primary preventive effect of gemfibrozil. PMID- 8624773 TI - Hypercholesterolemia with cholesterol-enriched LDL and normal levels of LDL apolipoprotein B. Effects of the step I diet and bile acid sequestrants on the cholesterol content of LDL. AB - One form of hypercholesterolemia is characterized by high levels of LDL cholesterol and normal levels of LDL-apolipoprotein (apo) B. The reason for hypercholesterolemia, therefore, is enrichment of LDL particles with cholesterol. We have reported previously that about one third of patients with primary moderate hypercholesterolemia have this lipoprotein pattern and have no apparent abnormality in LDL-apo B metabolism. The current study was designed to determine whether the combination of the Step I Diet (30% of total calories as fat, <10% saturated fatty acids, and <300 mg per day cholesterol) with or without cholestyramine therapy will correct the hypercholesterolemia in patients of this type. Ten hypercholesterolemic men of this type were identified and recruited into the study. Their LDL cholesterol levels were > or = 160 mg/dL and LDL-apo B levels were <120 mg/dL (LDL cholesterol/apo B ratio >1.60). For patient selection, subjects were challenged with a high fat diet (40% of total calories as fat, 18% saturated fatty acids, and 400 mg per day cholesterol) for 6 weeks to confirm persistence of a high LDL cholesterol/apo B ratio. Thereafter, they were started on a Step I Diet, and lipoprotein analyses were repeated. Finally, cholestyramine (16 g per day) was added to the Step I Diet. The Step I Diet alone significantly reduced the LDL cholesterol/apo B ratios and produced a trend toward lowering LDL cholesterol levels. Cholestyramine therapy further reduced LDL cholesterol levels and maintained a normal LDL cholesterol/apo B ratio. The present investigation thus confirms the existence of a form of moderate hypercholesterolemia that arises from a defect in LDL composition. In addition, it demonstrates that the combination of Step I Diet and cholestyramine therapy corrects this defect and normalizes LDL levels and LDL composition. PMID- 8624776 TI - Inhibitors of fibrinolysis are elevated in atherosclerotic plaque. AB - The proteins of the fibrinolytic system have been examined in the human normal and atherosclerotic arterial wall by immunohistochemical techniques and by quantitative immunoassay of extracts. The concentration of plasminogen activator inhibitor-1 (PAI-1) increased significantly during the progression from normal vessels to fatty streaks to the developed atherosclerotic plaque. Staining for PAI-1 was strongly positive, particularly in the areas adjacent to the plaque. In these areas, PAI-1 appeared to colocalized with its binding protein vitronectin. Alpha2-antiplasmin (alpha2-AP) was present in the aorta at even higher concentrations than PAI-1; a small but significant increase was seen in some atherosclerotic compared with normal vessel walls. Tissue plasminogen activator (TPA) showed the opposite trend, being lowest in lesions with plaque. Thus, higher concentrations of the two principal inhibitors of fibrinolysis, PAI-1 and alpha2-AP, together with lower levels of TPA, are characteristic of advanced atheromatous lesions. Alteration in the balance of the fibrinolytic system, favoring its inhibition, may predispose to the development or maintenance of atherosclerotic plaque. PMID- 8624775 TI - Oxidized lipids in the diet accelerate the development of fatty streaks in cholesterol-fed rabbits. AB - Studies have indicated that oxidized lipoproteins may play a role in atherosclerosis. We have recently demonstrated that the levels of oxidized lipoproteins in the circulation can be directly correlated to the quantity of oxidized lipids in the diet. The present study tested the hypothesis that dietary oxidized lipids accelerate the development of atherosclerosis. For 12 to 14 weeks, 36 male New Zealand White rabbits were fed a low-cholesterol (0.25%) diet containing either 5% unoxidized corn oil (control diet) or 5% oxidized corn oil (oxidized-lipid diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of the cholesterol in the beta-migrating very low density lipoprotein (beta-VLDL) fraction. Beta-VLDL from control animals contained 3.86+/- 0.57 versus 9.07 +/- 2.14 nmol conjugated dienes per micromol cholesterol (P<.05) in rabbits fed the oxidized-lipid diet. No difference in oxidized lipid levels was detected in LDL. Most important, feeding a diet rich in oxidized-lipid resulted in a 100% increase in fatty streak lesions in the aorta. Additionally, rabbits that were fed the oxidized-lipid++ diet had a >100% increase in total cholesterol in the pulmonary artery that was primarily due to an increase in cholesteryl ester. Oxidized lipids are frequently present in the typical US diet, and our results suggest that consumption of these foods may be an important risk factor for atherosclerosis. PMID- 8624774 TI - Apolipoproteins B, (a), and E accumulate in the morphologically early lesion of 'degenerative' valvular aortic stenosis. AB - Nonrheumatic aortic stenosis of trileaflet aortic valves has been considered to be a "degenerative" process, but the early lesion of aortic stenosis contains the chronic inflammatory cells, macrophages and T lymphocytes. Because lipoprotein deposition is prominent in atherosclerosis, another chronic inflammatory process, this study examined whether lipoproteins accumulate in aortic valve lesions. Immunohistochemical studies were performed to detect apolipoprotein (apo) B, apo(a), apoE, macrophages, and alpha-actin-expressing cells on 18 trileaflet aortic valves that ranged from normal to stenotic. All three apolipoproteins were detected in early through end-stage lesions of aortic stenosis but not in histologically normal regions. Comparison with oil red O staining suggested that most of the extracellular neutral lipid in these valves was associated with either plasma-derived or locally produced apolipoproteins. Thus, in early through end-stage aortic valve lesions, apolipoproteins accumulate and are associated with the majority of extracellular valve lipid. These results are consistent with the hypothesis that lipoprotein accumulation in the aortic valve contributes to pathogenesis of aortic stenosis. PMID- 8624777 TI - Smoking impairs the activity of endothelial nitric oxide synthase in saphenous vein. AB - Smoking impairs the endothelium-dependent relaxation of arteries and veins, with the maximum relaxation in response to the calcium ionophore A23187 of saphenous vein rings being reduced from 53 +/- 4% in nonsmokers to 27 +/- 5% in smokers. We have investigated whether this endothelial dysfunction was attributable to altered activity or concentration of nitric oxide synthase (NOS). The concentration of NOS in saphenous vein endothelium, determined by Western blotting and immunohistochemistry, was not different in nonsmokers and smokers. Nitrite production from vein strips stimulated with A23187 was higher in nonsmokers (median 23.6 nmol.cm-2.h-1) than smokers (median 3.3 nmol.cm-2.h-1), P=.001, this difference being abolished when vein strips were preincubated in the presence of NG-monomethyl-L-arginine. Organ chamber studies to monitor the endothelium-dependent relaxation of vein rings in response to A23187 showed that preincubation of rings from smokers with either L-arginine (3mmol/L) or superoxide dismutase (250 U/mL) did not improve the maximum relaxation. In contrast, preincubation of vein rings from smokers with 20 micromol/L tetrahydrobiopterin increased the maximum relaxation from 27 +/- 5% to 51 +/- 6%, P=.01. Preincubation of vein from smokers with tetrahydrobiopterin also significantly increased nitrite and cGMP production in response to stimulation with A23187. The impaired endothelium-dependent relaxation of saphenous vein rings from smokers appears to be caused by a reduction in the activity of endothelial NOS that is attributable to an inadequate supply of the coenzyme tetrahydrobiopterin. PMID- 8624779 TI - Evidence that implicates the parathyroid hormone-related peptide in vascular stenosis. Increased gene expression in the intima of injured carotid arteries and human restenotic coronary lesions. AB - Proliferation of vascular smooth muscle cells (VSMCs) is considered to be one key event underlying the pathophysiology of restenosis after angioplasty. The parathyroid hormone-related peptide (PTHrP) and its receptor, a local autocrine and paracrine regulator of cellular growth in a variety of normal cell types, have been reported in the vicinity of VSMCs. To investigate how PTHrP might be involved in the process of neointimal formation after balloon angioplasty, we examined PTHrP expression in balloon-denuded rat carotid arteries and human coronary arteries that had been retrieved by directional atherectomy. In rat carotid arteries, the RNase protection assay and in situ hybridization demonstrated that PTHrP mRNA expression increased fourfold to sixfold 1 to 7 days after denudation and continued for 28 days, coincident with downregulation of PTH/PTHrP receptor mRNA expression. In situ hybridization and immunohistochemistry revealed that PTHrP expression in balloon-denuded carotid arteries was mainly localized to the neointima. To confirm the involvement of the PTHrP in human coronary artery restenotic lesions, immunohistochemical analysis of human coronary atherectomy specimens (23 primary and 10 restenotic lesions) was then performed. The number of intimal cells that expressed PTHrP protein was significantly higher in restenotic (407 +/- 53 cells/mm2; range, 143 to 739) than in stable angina (50 +/- 12 cells/mm2; range, 18 to 132; P<.05) or unstable angina (129 +/- 16 cells/mm2; range, 21 to 232; P<.05) specimens. These data demonstrate that PTHrP gene expression in VSMCs markedly increases during neointimal formation, supporting the hypothesis that PTHrP may play an important role in vascular stenosis as a regulator of VSMC proliferation. PMID- 8624778 TI - Triple drug immunosuppression significantly reduces aortic allograft arteriosclerosis in the rat. AB - We evaluated the effect of triple drug immunosuppression (cyclosporine A 10 mg . kg(-1) . d(-1), methylprednisolone 0.5 mg . kg(-1) . d(-1) on the development of allograft arteriosclerosis (chronic rejection). The recipients of rat aortic allografts from the DA (AG-B4,RT1a) to the WF (AG-B2,RT1u) strain were either treated with triple drug immunosuppression (n=23) or left untreated (n=23) and used as controls. The grafts were removed 7, 14, 30, 90, and 180 days after transplantation, and vascular wall changes were evaluated by quantitative histology, [3H]thymidine autoradiography, and immunohistochemistry. Nonimmunosuppressed aortic allografts developed progressive arteriosclerotic alterations 1 to 6 months after transplantation that were virtually identical to those observed during chronic rejection in human cardiac allografts. Linear regression analysis revealed that triple drug immunosuppression with clinically relevant dosages of drugs significantly reduced intimal thickening (r=.69 versus r=.88, P<.05). Concomitantly, there was a marked reduction in the number of inflammatory cells (P<.01) and their rate of proliferation (P<.025) in the allograft adventitia during the period of acute inflammation (30 days after transplantation). Immunohistochemistry revealed that the number of helper T cells (W3/25) and monocyte/macrophages (OX42) but not cytotoxic T cells (OX8) or natural killer cells (3.2.3) was significantly (P<.05) reduced. The number of adventitial cells expressing interleukin-2 receptor (CD25) (P<.05), MHC class II (OX6) (P<.05) and leukocyte function-associated antigen-1 alpha-chain (CD11a) (P<.025) was also significantly reduced at 30 days. Triple drug immunosuppression downregulated the induction of MHC class II and intercellular adhesion molecule-1 on the graft endothelium but had no significant effect on the number of subendothelial inflammatory cells. In addition, [3H]thymidine autoradiography demonstrated that triple drug immunosuppression significantly reduced the rate of cell proliferation in the media, composed of smooth-muscle cells, 30 and 90 days after transplantation. Thus, triple drug immunosuppression efficiently reduced the development of allograft arteriosclerosis by down-regulating the inflammatory response and the level of immune++ activation in the allograft adventitia during the acute rejection period, resulting in diminished intimal thickening of the graft in the long run. These results support the concept that allograft arteriosclerosis is due to or at least initiated by immune injury of the graft. PMID- 8624780 TI - Beta ig-h3, a transforming growth factor-beta-inducible gene, is overexpressed in atherosclerotic and restenotic human vascular lesions. AB - Transforming growth factor-beta (TGF-beta) plays an important role in vascular lesion formation and possibly the renarrowing process ("restenosis") that occurs after balloon angioplasty. Secreted in a latent form by most cells, TFG-beta requires enzymatic conversion before it is biologically active. TGF-beta inducible gene h3 (beta ig-h3) is a novel molecule that is induced when cells are treated with TGF-beta1. This study examined the expression of beta ig-h3 in normal and diseased human vascular tissue. To determine the expression pattern of beta ig-h3 in human arteries, immunocytochemistry was performed on tissue sections from (1) normal internal mammary arteries, (2) the proximal left anterior descending coronary artery (with minimal intimal thickening) of 15 patients aged 18 to 40 years, (3) primary and restenotic coronary lesions from 7 patients, and (4) fresh directional atherectomy tissue from 11 patients. A polyclonal antibody consistently immunodetected beta ig-h3 protein in endothelial cells of all vascular tissue. In normal coronary arteries of young individuals, beta ig-h3 protein was absent from the intima and media but was found in the subendothelial smooth muscle cells of some arteries with modest intimal thickening. In diseased arteries beta ig-h3 protein was more abundant in the intima than the media. Restenotic coronary lesions tended to show higher levels of immunodetectable beta ig-h3 protein, especially in areas of dense fibrous connective tissue. Beta ig-h3 protein was immunodetected in the cytoplasm of plaque macrophages as well as smooth muscle and endothelial cells. By using in situ hybridization on fresh directional atherectomy specimens, we found beta ig h3 mRNA to be overexpressed by plaque macrophages and smooth muscle cells. Nondiseased human internal mammary arteries also expressed beta ig-h3 mRNA in endothelial cells but not in the smooth muscle cells of the normal intima and media. These results document the expression of beta ig-h3 in diseased human arterial tissue and support the hypothesis that active TGF-beta plays a role in atherogenesis and restenosis. PMID- 8624782 TI - Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low density lipoproteins. AB - A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2. PMID- 8624781 TI - Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. AB - Native LDL (n-LDL) increased human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL treated ECs bound nearly threefold more phorbol myristate acetate (PMA) stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti-cellular adhesion molecule-1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL-treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule-1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis. PMID- 8624783 TI - Lysophosphatidylcholine potentiates the mitogenic activity of modified LDL for human monocyte-derived macrophages. AB - The growth of murine peritoneal macrophages is induced by oxidized LDL (Ox-LDL), and lysophosphatidylcholine (lysoPC) plays an important role in its mitogenic activity. In the present++ study, Ox-LDL-induced macrophage growth was examined with human monocyte-derived macrophages. The cell growth of human macrophages was significantly induced by Ox-LDL but not by acetylated LDL (acetyl-LDL). The treatment of acetyl-LDL with phospholipase A2, however, led to a marked increase in its mitogenic activity, with a concomitant conversion of 75% of its phospholipids to lysoPC. The growth-stimulating activity became positive only when both acetyl-LDL and lysoPC were coincubated, although neither of them exhibited cell growth-promoting activity. These results suggest that Ox-LDL could stimulate the growth of human monocyte-derived macrophages, and lysoPC may play an essential role in the mitogenic activity of Ox-LDL. PMID- 8624784 TI - Acyl-coenzyme A:cholesterol O-acyltransferase is not identical to liver microsomal carboxylesterase. AB - Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein. PMID- 8624785 TI - A mathematical model for predicting trends in carbon monoxide emissions and exposures on urban arterial highways. AB - The roadway is one of the most important microenvironments for human exposure to carbon monoxide (CO). To evaluate long-term changes in pollutant exposure due to in-transit activities, a mathematical model has been developed to predict average daily vehicular emissions on highways. By utilizing measurements that are specific for a given location and year (e.g., traffic counts, fleet composition), this model can predict emissions for a specific roadway during various time periods of interest, allowing examination of long-term trends in human exposure to CO. For an arterial highway in northern California, this model predicts that CO emissions should have declined by 58% between 1980 and 1991, which agrees fairly well with field measurements of human exposure taken along that roadway during those two years. An additional reduction of up to 60% in CO emissions is predicted to occur between 1991 and 2002, due solely to the continued replacement of older cars with newer, cleaner vehicles. PMID- 8624787 TI - Non-isotopic vs isotopic immunoassay: is white wine "alternative" to the red? PMID- 8624786 TI - How alternative are immunoassay systems employing non-radioisotopic labels? A comparative appraisal of their main analytical characteristics. AB - The immunoassay is one of the most sensitive and reliable analytical techniques available in the clinical laboratory. The original label for immunoassays was radioisotopes, and these methods, radioimmunoassay (RIA) and immunoradiometric assay (IRMA) are still the reference methods, because of invulnerability of the radioactive emission with respect to environmental interference. Labels other than radioisotopes have been tested for use in immunoassay to improve the sensitivity and reliability and to avoid some of the disadvantages of radioisotopic techniques. New labels have continued to be developed (Horseradish peroxidase-HPR-, pyrophosphatase, luciferases, pyrodopirazines, europium cryptates, porphirins, phosphors) and new label detection methods have been set up (e.g. chemiluminescence assay, thermometric assay, NADP+ and FADP- based coupled assay). New immunoassay strategies such as simultaneous multianalyte automated test have been developed and the reliability of the assays has in some cases caused division among researchers about the choice between the radioisotopic immunoassay or the non-radioisotopic immunoassay, as considerable effort and investment had been devoted to the search for more sensitive and practicable tests than the classic RIA-IRMA methods. The evolution of immunoassays (Monoclonal Antibodies, non-radioactive tracers, automation) has produced systems which allow a large number of laboratories to determine a great number of analytes with very good practicability. The availability of fully automated systems has generated the opinion that analytical performance of immunoassays can be considered similar to that of many traditional parameters of clinical chemistry. This conclusion seems however too optimistic, in fact data collected from interlaboratory studies demonstrate that problems concerning the analytical reliability of the measurements still remain not completely solved. In the authors' opinion, this opposition between immunological assay based on isotopic or non-isotopic labels is misleading, because each assay (whether it uses isotopic, enzymatic, fluorimetric or luminescent labels) has its own analytical characteristics and performance. For this reason the term "alternative", used to indicate all non-isotopic assays as a unique class of tests, should be abandoned. From a theoretical point of view the choice should not be between isotopic and non isotopic techniques. For each analyte to be tested, it is advisable to use the immunological assay that suits the requirements of the laboratory, irrespective of type of label. From a practical point of view, the choice should be based on the analytical performance and on the characteristics of each assay, on its cost and the type of instrumentation available in the laboratory, and on the experience and the knowledge of the laboratory personnel. PMID- 8624788 TI - Identification of coronary artery disease by 99mTc-MIBI myocardial perfusion single photon emission computed tomography: evaluation using Diamond's method and segmental score system. AB - Three hundred and eighty-one consecutive patients referred to our Nuclear Medicine Service from the Cardiology Department during a period of two years have been studied to evaluate Coronary Artery Disease (CAP) using 99mTc-MIBI single photon emission computed tomography (SPECT). The sensitivity and specificity for detecting CAD in the whole group of patients, referred (n = 161) or not (n = 220) for coronary angiography, were calculated following Diamond's method, and were found to be 87% and 92%, respectively. Diamond's method estimates these values from those obtained in the subset of patients with angiography referral (sensitivity 91% and specificity 85%) and the pattern of SPECT responses of both groups (69% and 52%, respectively). A new segmental score system was used to evaluate the location and extent of CAD in the catheterized group. The sensitivity for detecting individual artery disease was 69% and specificity was 81%. The sensitivity in patients with single, double and triple-vessel disease was 90%, 96% and 89%, respectively. In the subgroup of patients without infarction (n = 66) the sensitivity and specificity were 76% (19/25) and 90% (37/41). PMID- 8624789 TI - Postoperative infections after neurosurgery and cardiosurgery: the value of labelled white blood cells. AB - Post surgical-infections in neurosurgery and cardiosurgery are infrequent, but potentially fatal complications. The aim of this study was to compare the utility of 99mTc-HMPAO white blood cells scintigraphy (WBCS) with traditional diagnostic approaches in post-surgical complications, in order to obtain timely demonstration of a current infection. We studied 23 patients with a suspicion of infection after major cardiosurgery or neurosurgery. Planar imaging was performed at 4 and 20 hours after injection of autologous white blood cells labelled with 99mTc-HMPAO. Eight patients underwent CT scan, but only in one case did CT findings lead to a clear definition of a bulky inflammation process of the chest. WBCS identified one or more sites of focal increased uptake of the radiopharmaceutical in 6 patients: five of these patients were scheduled for a "second look" surgical operation that confirmed the sites and extention of the primary infection, thus confirming the presence of an abscess. In 3 cases WBCS showed only a weak increase of focal uptake and in 14 cases there was no evidence of abnormal uptake. The absence of deep infections was confirmed at surgery or at clinical follow-up. Thus WBCS seems to be useful in evaluating patients with the clinical suspicion of infective complications after surgery. PMID- 8624790 TI - Relationship between physical activity level and bone mineral density in two groups of female athletes. AB - In order to evaluate the possible relationships among hormonal status, physical activity and bone density, we carried out a study on two groups of female athletes engaged in different levels of physical activity. We measured the following hormones: luteinizing hormone (LH), folliculo-stimulating hormone (FSH), 17-beta-oestradiol (E2), progesterone (PRG), prolactin (HPRL), estrone (E1), thyreo-stimulating hormone (TSH), free thyroxine (FT4), and the markers of phosphate-calcium metabolism: calcitonine (CT), parathormone (PTH), and osteocalcine (BGP). We also measured bone mineral density (BMD). All of these variables were related to the amount of work performed during training. The groups were defined as follows: medium workload (group M, n = 10) and heavy workload (group H, n = 20), engaged in 10 and 18 hours of weekly training at 35 and 60 average percent of VO2max, respectively. All of the hormones and the markers of calcium-phosphate metabolism studied were normal; BMD was also normal for all subjects except for two sisters in group M with reduced BMD. The group H athletes with regular menstrual cycles were found to have an upper limit normal BMD. From these data we conclude that in regularly menstruating athletes an increase in BMD induced by heavy physical activity is evident, while in dysmenorrhoeic athletes the effect of physical activity compensates, to some extent, for the hypothetical bone mineral reduction possibly caused by the hormonal imbalance. PMID- 8624791 TI - Tissue polypeptide specific antigen (tps) and cytokeratin 19 fragment (CYFRA 21.1) immunoradiometric assay in non small cell lung cancer evaluation. AB - The aim of our work was the evaluation of the immunoradiometric assay (IRMA) of two cytokeratinic markers, TPS and CYFRA 21.1, in clinical setting on non small cell lung cancer (NSCLC). Serum samples were obtained from 148 untreated NSCLC patients, 60 patients with non malignant lung diseases and 100 healthy subjects: TPS and CYFRA 21.1 serum levels were assayed by IRMA methods. Diagnostic performance of the markers was evaluated and the TPS and CYFRA 21.1 distribution analysed according to some different clinical and biological variables as histological subtypes, stage and survival time by using the Mann-Whitney "U" test. Sensitivity, specificity and accuracy were 0.54 (80/148), 0.47 (28/60), 0.52 (108/208) and 0.73 (108/148), 0.74 (44/60), and 0.73 (152/208) for TPS and CYFRA 21.1 respectively. CYFRA 21.1 demonstrate a higher sensitivity than TPS in all stages of the disease and in the spinocellular and adenocarcinoma histological subtypes while TPS sensibility is higher in large cell carcinoma. The CYFRA 21.1 specificity is better than TPS probably by reason of its preferential distribution in respiratory epithelium. Both markers serum levels differ significantly between Stage I-II and IV and between Stage I-II-IIIa and IIIb-IV but neither TPS nor CYFRA 21.1 can discriminate Stage IIIa from IIIb. No significant differences were found in the serum expression of the markers by the different histological subtypes. A value of both markers less than the selected cut-off is related to a longer survival of the patients apart from therapy (p < 0.05). Our conclusion supports similar behaviour of these markers in NSCLC and indicates CYFRA 21.1 as the more needed biochemical index to evaluate NSCLC patients. PMID- 8624792 TI - Effect of chlorpromazine as a sensitizer of rat brain on radiation-induced AMP and adenosine metabolism. AB - The role of AMP and adenosine was investigated in the radiosensitization of normal brain tissues by chlorpromazine. Their metabolism was evaluated by estimating the levels of 5'-nucleotidase and adenosine deaminase activity in the brains of rats treated with chlorpromazine alone or chlorpromazine and irradiation. The extent of lipid peroxidation, measured in terms of the lipid peroxidase enzyme formed, increased with chlorpromazine treatment and irradiation. Chlorpromazine treatment was found to decrease AMP and adenosine metabolism, as shown by a marked reduction in the level of 5'-nucleotidase and ADA activity which was accompanied by a marked curtailment in the DNA, RNA and protein contents of the brain. Chlorpromazine was also found to increase the radiation-induced activity of acid phosphatase, indicating its action on the lysosomal activity of the brain cells. In the present study a low dose of chlorpromazine, i.e. 17 mg/kg body weight, was found to be more effective than a high dose of 34 mg/kg. The results of this study suggest that chlorpromazine probably sensitizes normal brain tissues to radiation by inhibiting AMP and adenosine metabolism via a hydroxy-radical induced decrease in DNA, RNA and protein metabolism with a concomitant increase in lysosomal activity. PMID- 8624793 TI - Synthesis and evaluation of a new bifunctional chelating agent for the preparation of radioimmunoconjugates. AB - The conjugation of radiometals to monoclonal antibodies results in agents for radioimmunoimaging and other medical applications. Due to remarkable ability to form stable metal complexes with a great number of metal ions in different oxidation states, polyaminocarboxylate chelates are useful tools for this purpose. Bifunctional chelators that can hold radiometals with high stability under physiological conditions are essential to avoid radiation damage to non target organs. We have synthesized a new bifunctional chelate 2-(p-aminobenzyl) 1,3-propylenediamine-N,N,N',N'-tetraacetic acid by a simple method and studied the rate of loss of radioactivity from the radiolabeled (111In, 90Y) chelates to serum proteins in human serum at 37 degrees C. The relative stability constant of this new bifunctional chelate was found to be very similar to the underivated form. This chelate was conjugated to murine monoclonal antibody (B43) and immunoreactivity of the conjugated was determined by competitive binding analysis, which showed no significant change in its immunological activity. Biodistribution of the 111In radioconjugate was examined in conventional Balb/c and tumor-bearing (-OVCAR-3) athymic Balb/c mice. PMID- 8624794 TI - A case of bronchial carcinoid: diagnosis and follow-up with 111In-DTPA octreotide. AB - Scintigraphy with radiolabelled analogue of somatostatin is highly sensitive in detecting carcinoid tumors especially if performed with Single Photon Computed Tomography (SPECT). In this report we describe our experience with 111In-DTPA Octreotide in a female patient affected by a small asymptomatic intrabronchial carcinoid demonstrated by CT scan and bronchial endoscopy performed after recurrent left pneumonias. Planar views and SPECT images, using 111In-DTPA Octreotide, were collected before and four hours after the first endoscopic laser resection. All groups of SPECT images were positive in the left parahilar region but at a different degree. Scans performed after resection showed a low degree of uptake which was considered to be probably secondary to local swelling; CT scan was negative. Follow up endoscopic biopsy repeated at six months, showed a relapse always in the same site; CT scan of the thorax was again negative. 111In DTPA-Octreotide images obtained at twelve months were positive always in the left parahilar region, CT scan was negative but another biopsy was not possible. Therefore it was suspected a relapse of the carcinoid which was probably growing only through the bronchial wall without spreading towards the bronchial lumen and/or the lung parenchima. In this occasion, it was also thought that images collected four hours after resection could be positive not only for swelling but for a relapse as well. In every scintigraphic session, SPECT images presented higher quality than planar. This case suggests that 111In-DTPA-Octreotide SPECT is a non-invasive diagnostic technique which could be applied as a follow-up tool especially to patients with no-secreting carcinoid neoplasm and/or with negative or doubtful endoscopic and radiological investigations. PMID- 8624795 TI - Cancer statistics for African Americans, 1996. AB - Although cancer remains a major public health burden for African Americans, progress is being achieved. For both genders, stomach cancer mortality and mortality related to Hodgkin's disease showed large decreases over the past 30 years. Among African-American females, large decreases in cancer mortality occurred for nonmelanoma skin cancers, rectal cancers, and cervical and other uterine cancers. Tobacco use continues to decline among African Americans and, at present, is significantly lower among African-American youths than among their white counterparts. Despite these successes, additional work remains. Increased patient education regarding self-examinations and improved access to cancer screening are necessary to reduce the high percentage of cancers diagnosed at late stages among African Americans. Improved screening ultimately would increase survival and decrease cancer mortality. Some research has suggested that the increased morbidity and mortality in African Americans are related to poverty, lower education, and inadequate access to care as opposed to inherent racial characteristics. A recent study of black-white differences according to stage at diagnosis of breast cancer confirms some of these factors but also suggests that multiple factors may explain these differences, including mammograms, having a breast examination by a physician, and a history of patient delay. Such observations point to the importance of enacting broad social policies and establishing support mechanisms to diminish the impact of cancer in the African American community. PMID- 8624797 TI - Treatment of head and neck cancer. PMID- 8624796 TI - Early detection of oral cancers. PMID- 8624798 TI - The prevention of occupational cancer. AB - Occupational exposures to carcinogens are widespread and can result in tragic consequences for exposed workers. Clinicians are in a unique position to identify associations between workplace exposures and human malignancy, and virtually all occupational carcinogens have first been recognized by astute clinicians. Further, occupational cancers are usually preventable, and clinicians can be very effective in triggering preventive activities by industry, unions, and public authorities. PMID- 8624799 TI - Cancer and the workplace. AB - The study of occupationally related cancers has played an important role in the control of cancer in general. The very nature of the workplace allows for studies that can help establish a relationship between an environmental exposure and cancer. Once carcinogens are identified, strategies can be developed to prevent disease. The article reviews procedures for identifying, evaluating, and categorizing human carcinogens; provides an overview of the basic epidemiologic strategies that have been used to study cancer and the workplace; and outlines the clinical evaluation of occupational exposures. PMID- 8624800 TI - Bladder cancer, 1996. AB - In superficial Ta or T1 tumors intravesical chemotherapy can eradicate existing carcinoma in one third to one half of patients and reduce tumor recurrence by 12 to 15 percent, on average. Superficial bladder cancer is remarkably sensitive to immunotherapy, particularly BCG. The use of BCG eradicates about two thirds of papillary carcinoma and nearly 90 percent of carcinoma in situ and reduces tumor recurrence by an average of 40 percent. Data now suggest that BCG immunotherapy reduces long-term tumor recurrence, disease progression, and mortality. The proclivity for tumor recurrence makes superficial bladder cancer an ideal malignancy for the evaluation of chemoprevention, and preliminary data suggest that high doses of vitamins may also reduce tumor recurrence. In locally advanced T2b to T4, N0 or N1, M0 bladder cancer, substantial clinical responses can be achieved if chemotherapy is used prior to surgical resection of muscle-invasive tumor (neoadjuvant treatment). Controlled trials are necessary to ascertain whether neoadjuvant chemotherapy improves survival. The use of CMV prior to and concomitant with bladder irradiation is also encouraging, but will require randomized trials to clarify its role in the treatment of invasive, nonmetastatic cancer. Finally, trials suggest benefit for chemotherapy used adjuvantly (after cystectomy) for muscle-invasive bladder cancer. However, further investigation is necessary to clarify and confirm the role of chemotherapy in this setting before it can be recommended routinely for patients. In metastatic disease, chemotherapy with CMV or M-VAC with surgical resection of residual masses can produce a cohort of long-term survivors with advanced bladder cancer. How to increase this small but important population of patients is a question for further research. PMID- 8624801 TI - Life and death in germinal centers (redux). PMID- 8624802 TI - IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain. AB - IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL 2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences. PMID- 8624803 TI - Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor. AB - IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R. PMID- 8624804 TI - Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. AB - Allelic exclusion at the IgH locus was examined in B lineage cells of wild-type mice and mice unable to express the surrogate light chain molecule lambda 5 using a single-cell PCR approach. By analyzing B precursor cells containing two VHDHJH rearrangements, we found that in wild-type animals, cells are allelically excluded as soon as mu chains are expressed. Furthermore, we provide evidence that in cells expressing D mu proteins VH-->DHJH rearrangement is inhibited. In contrast, in the absence of lambda 5 protein, B precursor cells were allelically "included", indicating that allelic exclusion at the IgH locus requires expression of the pre-B cell receptor either containing a mu chain or a D mu chain. However, although mu chain double-producing B precursor cells are generated in lambda 5-deficient mice, such cells were not detected among surface immunoglobulin positive B cells. PMID- 8624805 TI - Assembly of the truncated immunoglobulin heavy chain D mu into antigen receptor like complexes in pre-B cells but not in B cells. AB - Rearrangements of the IgH locus with JH joined to reading frame 2 of DH are greatly underrepresented in B cells. These rearrangements encode the truncated heavy chain D mu. In pre-B cells, we found D mu protein expressed on the cell surface and assembled into a complex with surrogate light chains, Ig alpha, and Ig beta. Cross-linking of either mu m- or D mu m- containing pre-B cell receptors triggered signal transduction reactions. In contrast, when expressed in mature B cell lines, D mu was not detected on the cell surface and did not efficiently bind kappa immunoglobulin light chains, but did associate with Ig alpha and Ig beta. These results characterize the interactions of D mu chain with other components of the B cell antigen receptor complex and suggest possible mechanisms by which D mu expression may interfere with B cell development. PMID- 8624806 TI - The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER. AB - Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum. PMID- 8624808 TI - The C-C chemokine MCP-1 differentially modulates the avidity of beta 1 and beta 2 integrins on T lymphocytes. AB - The ability of chemokines, particularly MCP-1, to induce integrin-dependent binding of T lymphocytes to endothelial adhesion molecules or extracellular matrix (ECM) components was examined. MCP-1 induced significant adhesion to fibronectin (FN) and to endothelial-secreted ECM but not to purified ICAM-1 or VCAM-1, or to activated endothelium. The MCP-1-induced binding of T lymphocytes to FN was rapid, dose dependent, and resulted from activation of both VLA-4 and VLA-5. Like MCP-1, the chemokines RANTES and MIP-1 beta induced T lymphocyte binding to FN, but not to ICAM-1. We suggest therefore, that these T lymphocyte chemokines may be most important, not in initiating integrin-dependent firm adhesion of T lymphocytes to the vascular wall, but rather, in subsequent adhesive interactions during migration into tissue. PMID- 8624807 TI - Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression. AB - CIITA activates the expression of multiple genes involved in antigen presentation and it is believed to be required for both constitutive and IFN gamma-inducible expression of these genes. To understand the role of CIITA in vivo, we have used gene targeting to generate mice that lack CIITA. CIITA-deficient (-/-) mice do not express conventional MHC class II molecules on the surface of splenic B cells and dendritic cells. In addition, macrophages resident in the peritoneal cavity do not express MHC class II molecules upon IFN gamma stimulation nor do somatic tissues of mice injected with IFN gamma, in contrast with wild-type mice. The levels of Ii and H-2M gene transcripts are substantially decreased but absent in CIITA (-/-) mice. The transcription of nonconventional MHC class II genes is, however not affected by CIITA deficiency. A subset of thymic epithelial cells express MHC class II molecules. Nonetheless, very few mature CD4 T cells are present in the periphery of CIITA (-/-) mice despite MHC class II expression in the thymus. Consequently, CIITA(-/-) mice are impaired in T-dependent antigen responses and MHC class II-mediated allogeneic responses. PMID- 8624809 TI - Monoclonal T cells identified in early NOD islet infiltrates. AB - To examine the hypothesis that a single initiating antigen was recognized by a monoclonal T cell population leading to subsequent inflammatory insulitis in non obese (NOD) mouse islets, we examined the T cell receptor TCR V beta repertoire of islet-infiltrating T cells in very young (2-week-old) NOD mice. In independent experiments, we repeatedly identified one monoclonal TCR V-beta 8.2 gene product expressed by T lymphocytes infiltrating the islets of NOD mice at 2 weeks of age. The resultant inflammatory response quickly obscures the monoclonal nature of the initiating event. These data suggest that autoimmune diabetes in NOD mice may be initiated by recognition of a single autoantigen. PMID- 8624810 TI - ICE family proteases: mediators of all apoptotic cell death? PMID- 8624811 TI - An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. AB - The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix. PMID- 8624812 TI - Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53. AB - The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes. PMID- 8624813 TI - HLA-DM is localized to conventional and unconventional MHC class II-containing endocytic compartments. AB - HLA-DM molecules remove invariant (Ii) chain peptides from newly synthesized MHC class II complexes. Their localization may thus delineate compartments, e.g., MIIC, specialized for loading peptides onto class II molecules. In murine A20 B cells, however, DM is not restricted to specialized endosomal class II-containing vesicles (CIIV). Although DM was found in CIIV, it was also found throughout the endocytic pathway, principally in lysosomes devoid of class II molecules. In human lymphoblasts, HLA-DM was found in structures indistinguishable from late endosomes or lysosomes, although in these cells the lysosomes contained MHC class II molecules. Thus, the distribution of HLA-DM does not necessarily identify specialized class II compartments. Many "MIIC" may represent conventional lysosomes that accumulate MHC class II and HLA-DM in a number of cell types. PMID- 8624814 TI - Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation. AB - Human tonsillar B cells were separated into naive IgD+CD38-CD23- (Bm1) and IgD+CD38-CD23 (Bm2), germinal center IgD-CD38+CD23- centroblasts (Bm3) and IgD CD38+CD77- centrocytes (Bm4) and memory IgD-CD38- (Bm5) subsets. Previous IgVH sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile I gamma, I alpha, and I epsilon expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated with centroblasts (Bm3). Finally, the high level of 5'S gamma-S mu 3' DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching. PMID- 8624815 TI - Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. AB - Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens. PMID- 8624816 TI - Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis infection. AB - To study the role of CD40 ligand (CD40L) in the host immune responses against intracellular pathogens, we infected CD40L knockout (CD40L-/-) mice with Leishmania amazonensis. Although wild-type mice were susceptible to infection and developed progressive ulcerative lesions, tissue parasite burdens in CD40L-/- mice were significantly higher. This heightened susceptibility to infection was associated with an impaired T cell and macrophage activation and altered inflammatory response, as reflected by low levels of IFN gamma, lymphotoxin-tumor necrosis factor (LT-TNF), and nitric oxide (NO) production. Furthermore, CD40L-/- mice failed to generate a protective immune response after immunization. These results indicate an essential role of cognate CD40-CD40L interactions in the generation of cellular immune responses against an intracellular parasite. PMID- 8624818 TI - CD40 ligand is required for protective cell-mediated immunity to Leishmania major. AB - The CD40-CD40 ligand (CD40L) signaling process is a pivotal component of multiple immunoregulatory pathways. Although the role that CD40L plays in humoral immune responses is fairly well defined, its function(s) in cell-mediated responses in vivo has not been established. We investigated this issue by assessing the course of Leishmania major infection in CD40L knockout (CD40LKO) mice that were generated on a resistant background. In response to parasite challenge, CD40LKO mice developed ulcerating cutaneous lesions and failed to mount a vigorous Th1 like response. The impaired Th1-like response appears to be related to a defect in the ability of CD40LKO T cells to induce the production of IL-12 from macrophages. Treatment with exogenous IL-12 prevented disease progression in CD40LKO mice, and administration of recombinant CD40L provided partial protection against infection. Thus, a protective cell-mediated immune response to L. major appears to be dependent upon CD40L-induced IL-12 secretion by antigen-presenting cells. PMID- 8624817 TI - Protective role of CD40 in Leishmania major infection at two distinct phases of cell-mediated immunity. AB - CD40-deficient mice are susceptible to Leishmania major infection while their wild-type littermates can resolve the infection. Upon stimulation with L. major antigens, draining lymph node T cells of the mutant mice and the susceptible mice, BALB/c, secrete comparable amounts of IL-4. The mutant mice produce less IFN gamma than wild-type mice. The expression of IL-12 p40 mRNA was significantly lower in L. major antigen-stimulated cells of mutant mice than those of wild-type or BALB/c mice. In normal mice, engagement of CD40 activates macrophages to a leishmanicidal state in vitro in the presence of IFN gamma. The results suggest that the CD40-CD40 ligand interaction plays an important role in two critical steps of cell-mediated immunity to L. major infection: the generation of a Th1 response and activation of macrophages to a leishmanicidal state. PMID- 8624819 TI - Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. AB - During B lymphocyte development, pro-B cells that fail to rearrange an immunoglobulin heavy (IgH) chain allele productively are thought to undergo developmental arrest and death, but because these cells are short-lived in vivo they are not well characterized. Transgenic mice expressing the apoptosis regulatory gene bcl-xL in the B lineage developed large expansions of pro-B cells in bone marrow. V(D)J rearrangements in the expanded populations were nearly all nonproductive, and DJH rearrangements were enriched for joints in DH reading frame 2 and for aberrant joints with extensive DH or JH deletions. Thus, the death of pro-B cells with failed immunoglobulin rearrangements occurs by apoptosis, and bcl-xL can deliver a strong survival signal at the pro-B stage. This analysis also demonstrated that immunoglobulin gene rearrangement is less precise than previously appreciated. PMID- 8624820 TI - Asymmetric redundancy in CD4 silencer function. AB - We and others have defined a transcriptional silencer critical for the proper expression of the CD4 gene at all stages of T cell development. In this report, we use biochemical techniques to identify three different factor-binding sites within the CD4 silencer, denoted sites I, II, and III. Using transgenic analyses, we determine that although all three factor-binding sites are important for silencer activity, there is significant redundancy in that the presence of either site II alone, or the combination of sites I and III permits silencer function. Thus, our data indicate that the mechanism of function of the CD4 silencer is extremely complex. Further biochemical analyses indicate that the factor binding to site II has the same sequence specificity as a factor binding to an E box site in the CD4 enhancer; thus, a member of the bHLH factor family may be important in mediating silencer function. PMID- 8624821 TI - Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. AB - Interleukin-4 (IL-4) stimulation of cells leads to the activation of multiple signaling pathways, one of which involves Stat6. We have generated Stat6 deficient mice by gene targeting in embryonic stem cells to determine the role of this transcription factor in mediating the biologic functions of IL-4. IL-4 induced increases in the cell surface expression of both MHC class II antigens and IL-4 receptor are completely abrogated, and lymphocytes from Stat6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6 deficient T lymphocytes fail to differentiate into Th2 cells in response to either IL-4 or Il-13. These results demonstrate that, despite the existence of multiple signaling pathways activated by IL-4, Stat6 is essential for mediating responses to IL-4 lymphocytes. PMID- 8624823 TI - Transcription factors 2: helix-loop-helix. PMID- 8624824 TI - Actin-binding proteins 1: spectrin superfamily. PMID- 8624822 TI - The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. AB - Using cells from TCR transgenic mice that do or do not express Fas, we show that there are two mechanistically distinct forms of apoptosis in CD4+ T cells. Naive T cells undergo apoptosis if cultured in the absence of antigen or costimulation. This form of programmed cell death (PCD) is not dependent on Fas, and is prevented by CD28-mediated signals, which lead to the secretion of growth factors and the expression of survival genes, such as bcl-xL. Recently activated T cells undergo apoptotic death upon repeated stimulation. This activation-induced cell death (AICD) is mediated by Fas, but is independent of costimulation and is not prevented by IL-2 or bcl-xL. Finally, we show that peripheral tolerance may be induced in vivo independent of Fas-mediated cell death. PMID- 8624825 TI - FDA investigates injury reported from "homemade" laser. PMID- 8624826 TI - Automated in situ keratomileusis for myopia. AB - BACKGROUND: In situ keratomileusis is a rapidly changing refractive corneal surgical procedure for the correction of myopia. We report here the results of a retrospective study of in situ keratomileusis with the refractive cut made with a microkeratome. The technique represents an intermediate stage of development that used a sutured corneal disc, an early automated microkeratome, and no reoperations for residual refractive error. METHODS: We carried out a retrospective chart review of the first 107 consecutive eyes (73 patients) that received in situ keratomileusis with an automated microkeratome (Ruiz corneal shaper) at the El Maghraby Hospital in Jeddah, Saudi Arabia between November, 1991 and February, 1993. Sixty-three eyes (59%) were followed for a mean of 1 year. Manifest refraction measured by an ophthalmologist or optometrist was the major response variable. Surgery followed the Ruiz nomogram, which specified an anterior corneal disc approximately 7.2 mm in diameter and 160 microns in thickness and a plano in situ refractive cut a mean of 4.2 mm in diameter and 93 microns in thickness. RESULTS: The mean baseline spherical equivalent of the manifest refraction was -11.97 +/- 5.30 diopters (D) (range, -3.75 to -28.00 D). At a mean of 1 year after surgery, the mean refraction was -1.86 +/- 3.13 D (range, -13.25 to +4.63 D); 13 eyes (21%) had a refraction within +/- 0.50 D and 22 (35%) within +/- 1.00 D. Undercorrection of more than -1.00 D was present in 36 eyes (57%). At baseline, 48 of 63 eyes (75%) could see 20/40 or better with spectacle correction. At 1 year, 13 of 63 eyes (21%) could see 20/40 or better uncorrected. To measure the stability of refraction in 61 eyes, the mean refraction at 1 month of +0.62 +/- 2.43 D was compared to that at 1 year after surgery (mean, -1.41 +/- 3.10 D), indicating a loss of effect in the direction of increasing myopia. Nine eyes (14%) lost two to five lines of spectacle-corrected visual acuity. Two eyes had central epithelial implantation plaques in the lamellar bed that required removal. CONCLUSION: In situ keratomileusis using an automated corneal shaper and sutured corneal discs at an intermediate stage of development produced less than desirable refractive and visual acuity results. PMID- 8624827 TI - Wound healing following intrastromal photorefractive keratectomy with the Nd:YLF picosecond laser in the cat. AB - BACKGROUND: We studied the histopathology of the stromal wound healing response in the cat cornea following intrastromal photorefractive keratectomy (IPRK) with the Nd:YLF picosecond laser. METHODS: Intrastromal PRK was performed in the anterior stroma of cat corneas with the Nd:YLF picosecond laser. The cats were sacrificed at predetermined intervals ranging from immediately to 6 months postoperatively. Effects of the laser treatment on the epithelium, Bowman's layer, stroma, and the endothelium were evaluated using light and scanning electron microscopy. No anti-inflammatory agents were used. RESULTS: Intrastromal PRK resulted in no perceptible damage to the corneal epithelium or Bowman's layer either acutely or at 6 months. The corneal stroma showed multiple cavitations immediately after intrastromal PRK, which collapsed over several hours, followed by thinning of the cornea over 2 weeks. At 1 month, the stromal collagen was abnormal with surrounding hypercellularity. The endothelium showed no injury, acutely or at 6 months. No thermal effects on stromal collagen were observed at 6 months, and disruption of the lamellar pattern was not apparent after the cavitation bubbles were reabsorbed. CONCLUSION: Intrastromal PRK can effectively remove stromal tissue without acute damage to the adjacent lamellae, epithelium, or endothelium. There is a transient cellular wound healing response associated with a transient stromal collagen abnormality at 2 weeks to 1 month, which was not apparent 2 months after the procedure. PMID- 8624828 TI - Ultrasound biomicroscopy of intrastromal photorefractive keratectomy with the Nd:YLF picosecond laser. AB - PURPOSE: To evaluate corneal morphology in vivo following intrastromal photorefractive keratectomy (IPRK) with the Nd:YLF picosecond laser, using the ultrasound biomicroscope. MATERIALS AND METHODS: Myopic intrastromal PRK was performed in the anterior stroma of cat corneas with the neodymium:yttrium lithium fluoride (Nd:YLF) picosecond laser. Periodic examination of the treated corneas up to 6 months postoperative was performed with high resolution ultrasound biomicroscopy. Corneal thickness, depth of the laser patterns from the corneal surface, alignment of the laser treatment, extent of stromal opacification until resolution, and contour of the corneal surface were measured with the ultrasound biomicroscope throughout follow up. RESULTS: The treated corneas were thickened on ultrasonic biomicroscopy measurement immediately after intrastromal PRK and revealed a densely echogenic shadow parallel to the corneal surface at the treatment site. There was increased stromal echogenicity denoting mild edema. The echoes decreased over time and appeared thinner at 2 months compared to the preoperative and immediate postoperative measurements. At 6-month follow up, the treated corneas were echolucent on ultrasonic biomicroscopy. CONCLUSION: The ultrasound biomicroscope might become a helpful tool in assessing accuracy of treatment parameters of intrastromal PRK as well as in monitoring the response of the cornea to treatment. PMID- 8624829 TI - Oblique versus vertical-horizontal incision orientation in four-incision radial keratotomy. AB - BACKGROUND: Four-incision radial keratotomy is used for the correction of mild to moderate myopia. The traditional orientation of incisions was naturally on the horizontal and vertical meridians. We undertook a comparative study of oblique versus traditional four-incision radial keratotomy (orthogonal orientation) to show the advantage of an oblique incision orientation. METHODS: A prospective study of two groups of 21 eyes was conducted: an orthogonal group with vertical horizontal orientation of the four incisions, and an oblique group with four obliquely oriented incisions. Spatial contrast sensitivity, glare, and night vision tests were performed and results compared. The follow up period was 3 months. RESULTS: Glare and contrast sensitivity were less affected with oblique orientation of incisions. There was no significant difference regarding night vision effects. We observed a decrease in induced astigmatism with oblique orientation. CONCLUSION: The location of horizontal incisions within the palpebral fissure and the unequal lengths of horizontal and vertical incisions are responsible for ocular alterations. An oblique orientation is preferable to avoid glare, starburst, and induced astigmatism. PMID- 8624830 TI - Effect of corticosteroids on rabbits corneal keratocytes after photorefractive keratectomy. AB - BACKGROUND: To determine the corticosteroid effect on the activity and repopulation of keratocytes after photorefractive keratectomy (PRK). METHODS: A 193-nm excimer laser (VISX Twenty/Twenty) created a central ablation depth of 22 microns (diameter:5 nm) on 22 corneas of 16 albino rabbits. Two ablated eyes were examined 6 hours following PRK. Twelve eyes received no postoperative corticosteroids and eight were treated with topical fluoromethalone for 3 months. Corneas were examined 1, 3, 6, and 12 months after PRK by immunofluorescence and transmission electron microscopy. RESULTS: Corticosteroids reduced haze (p=0.02), but all corneas (treated or untreated) cleared 6 months after PRK. Keratocytes were absent from the anterior 100 microns of the stroma 6 hours after PRK. However, the number and activity of keratocytes were significantly greater in this area in untreated corneas at 1 month and then gradually decreased. By 6 and 12 months, the number of keratocytes approached controls. Treated corneas had fewer keratocytes than either controls or untreated eyes (p<0.01) and by 3 months, a subepithelial acellular zone of 30 to 50 microns thickness appeared and persisted until at last 12 months after PRK. CONCLUSIONS: Corticosteroids have a transient effect in reducing haze and seem to inhibit keratocyte movement, leading to an acellular subepithelial region beneath the ablated area. PMID- 8624831 TI - Do three-piece PMMA IOLs rotate after implantation in the capsular bag? AB - BACKGROUND: With the current interest in toric intraocular lenses (IOLs), it is critical to establish whether these implants rotate following cataract surgery. If there is continual rotation of the implant with capsular bag contraction, then the orientation of the astigmatic correction would be unstable and thus not clinically useful. METHODS: Twenty-eight eyes were followed for up to 6 months postoperatively. These consecutive eyes were selected on the basis of demonstrating adequate dilation following surgery, such that the points of haptic to optic insertion could be visualized with the slit-lamp microscope. Sequential photographs and comparisons of the orientation of the lenses postoperatively were performed. RESULTS: With the exception of one lens implant, none of the 28 IOLs demonstrated significant rotation. Of the 16 eyes followed for 6 months, none demonstrated rotation of greater than 4 degrees, which was within experimental error. CONCLUSIONS: Continued rotation of PMMA IOLs following implantation in the capsular bag seldom occurs. Thus, the concept of toric IOLs appears to be quite usable. PMID- 8624832 TI - Combined wedge resection and relaxing incisions for astigmatism after penetrating keratoplasty. AB - BACKGROUND: Videokeratography may provide information for surgical correction of astigmatism after penetrating keratoplasty. We used a combination of wedge resection and relaxing incisions to treat high refractive astigmatism after penetrating keratoplasty. METHODS: Videokeratography using the normalized scale of the Topographic Modeling System was used as a guide in determining the location and the length of incisions and resections. Nine eyes were treated with both relaxing incisions and a wedge resection. All patients had more than 3.00 diopters (D) of refractive astigmatism. All patients were intolerant of spectacles or contact lenses. The depth of the corneal relaxing incisions was constant at 0.5 mm and the width of the corneal wedge resections was constant at 0.75 mm. RESULTS: The relaxing incisions produced flattening of the steeper meridian and the wedge resection produced steepening of the flatter meridian. The average preoperative keratometric astigmatism was 7.44 D (range, 3.50 to 11.00 D) and the average refractive astigmatism was 5.56 D (range, 4.00 to 8.00 D). The average preoperative spherical equivalent was 0.08 D (range, -7.00 to 4.25 D). Postoperatively, the average keratometric astigmatism was 2.97 D (range, 1.00 to 5.00 D) and the average refractive astigmatism was 2.58 D (range, 0.00 to 5.00 D). The average postoperative spherical equivalent refraction was -0.32 D. CONCLUSIONS: Combined corneal wedge resection and relaxing incisions appears to be effective in reducing high refractive astigmatism following corneal transplantation. PMID- 8624833 TI - Retrospective comparison of photorefractive keratectomy and radial keratotomy. AB - BACKGROUND: The efficacy and predictability of photorefractive keratectomy and radial keratotomy become increasingly relevant. This retrospective study compares one surgeon's experience with photorefractive keratectomy and radial keratotomy over a 3-year period from 1990 to 1993. METHODS: Photorefractive keratectomy was performed on 103 eyes of 76 patients that met the inclusion criteria for the phase IIb, phase III, and phototherapeutic keratectomy studies as delineated by the United States Food and Drug Administration. Radial keratotomy was performed on 117 eyes of 81 patients with up to 9.00 diopters (D) of myopia. RESULTS: In the photorefractive keratectomy group, 83% of the eyes achieved uncorrected visual acuity of at least 20/40; 37% saw 20/20; 88% had a refraction within 1.00 D of emmetropia, and 63% within 0.50 D of emmetropia. For the radial keratotomy group, 85% of the eyes achieved an uncorrected visual acuity of 20/40 or better; 27% saw 20/20; 88% had a refraction within 1.00 D of emmetropia; and 55% within 0.50 D of emmetropia. There were no serious complications, and only one single eye in each of the photorefractive keratectomy and radial keratotomy groups lost two lines or more of spectacle-corrected visual acuity. CONCLUSION: Photorefractive keratectomy and radial keratotomy are both effective procedures, and result in similar refractive outcomes for myopia of -1.00 to -9.00 D. PMID- 8624834 TI - Long-term stability of refraction after intrastromal suture correction of hyperopia following radial keratotomy. AB - BACKGROUND: Our first series of nine cases using circular and interrupted sutures for hyperopia from overcorrected radial keratotomy was reported 2 years ago. Additional cases and long-term follow up with this technique are presented here. METHODS: Thirteen eyes of 12 patients with symptomatic hyperopia underwent corneal suturing with pursestring and interrupted sutures and were followed for 3 years. There were six original overcorrections and seven overcorrections after reoperation. The mean preradial keratotomy refractive spherical equivalent refraction was -3.85 +/- 1.40 RESULTS: The mean preoperative refractive spherical equivalent was +2.55 +/- 0.93 D (range +1.125 to +5.375 D). The mean follow up after the suturing procedure was 40 +/- 14.3 months with a range of 10 to 72 months. The change in refraction following suturing procedures averaged -1.84 D, resulting in an overall correction of 72% of the overcorrection. The mean increase of postoperative central keratometric power was 1.42 +/- 0.95 D. All eyes reduction of hyperopia. Uncorrected visual acuity was improved from 0.367 +/ 0.11 (20+60) preoperatively to 0.712 +/- 0.25 (20/30+) postoperatively. The average improvement was 3.3 Snellen lines. using the visual function score to assess the effectiveness of the procedure, we found 85% (11/13) of eyes obtained excellent or good results and 15% appeared fair or poor. In the 3 years since the study began, eight eyes had follow-up data from the previous study and the present study. During this period, an average change of refraction of only -0.19 D was observed. A refractive change less than 0.50 D was observed in 62.5% of the eyes, and all of the eyes had a refractive change within 1.00 D. There were no new complications and no loss of effect during the mean 43 months of follow up for those patients. CONCLUSION: Based on the 3-year follow up, we concluded that intrastromal pursestring and interrupted techniques are stable, safe, and effect procedures for correcting hyperopia following radial keratotomy, especially for less than +3.00 D. PMID- 8624835 TI - Intraocular lens power calculation for eyes after refractive keratotomy. AB - BACKGROUND: Calculating the intraocular lens (IOL) power for an eye that has previously had refractive keratotomy is a problem because of the difficulty of accurately measuring the central power of the cornea using standard keratometers. METHODS: Three methods are proposed to better estimate this parameter. The clinical history method involves subtracting the change in myopia induced by the refractive keratotomy from the average corneal power measured before the keratotomy. The contact lens method determines the difference between the manifest refraction with and without a plano hard contact lens of known base curve and subtracts this difference from that base curve. Videokeratography measures the central corneal power inside the approximately 3-mm zone measured by keratometry, and therefore gives a more accurate power to use in IOL calculation formulas, especially with newer software applications becoming available. RESULTS: Published reports have demonstrated that standard keratometers do not accurately measure corneal power after refractive keratotomy and that regression formulas are less accurate than modern third-generation theoretic formulas for eyes that have flatter corneas from refractive surgery. CONCLUSION: For eyes that have had refractive surgery, the corneal power derived from clinical history, contact lens refraction, or videokeratography should be used in a third generation theoretic formula (Hoffer Q, Holladay, SRK/T) to calculate the intraocular lens power used during cataract surgery. PMID- 8624836 TI - Bilateral penetrating autokeratoplasty. AB - BACKGROUND: Corneal transplantation is an important modality for treatment of a variety of degenerative, congenital, and traumatic disorders. Despite great advances in surgical techniques, postoperative care, and eye banking over the past 20 years, corneal graft rejection continues to be a substantial risk to successful outcome. METHODS: We describe a series of three cases in which the clear cornea from a blind eye was used to replace an opaque cornea on a fellow eye with known visual potential and the abnormal cornea was transplanted on the blind eye. Mean follow up was 74 months (range, 66 to 84 months). All three patients had concurrent glaucoma at presentation, and one had a highly vascularized cornea. RESULTS: Spectacle-corrected vision improved in all cases postoperatively. However, one patient had a non-immune graft failure 58 months after surgery. CONCLUSIONS: The use of autologous tissue avoids the risk of allograft rejection after penetrating keratoplasty and eliminates the need for long-term postoperative corticosteroids which may exacerbate pre-existing glaucoma. In selected patients, autokeratoplasty can be a useful technique, especially if heavy corneal vascularization or pre-existing glaucoma is present. PMID- 8624837 TI - Epikeratoplasty for myopia: 2-year results and a proposed nomogram. AB - BACKGROUND: Epikeratoplasty for myopia is a relatively safe and reversible surgical technique that can result in large refractive corrections. However, it is also claimed that long-term stability and predictability are not satisfactory due to refractive regression and undercorrection. METHODS: In an effort to improve predictability and stability of epikeratoplasty for myopia, we followed 24 cases for 2 years and isolated factors affecting the postoperative refractive results. RESULTS: We found that better refractive results were achieved at 24 months postoperatively for cases in which the power of preoperative myopia was lower. Our investigations also disclosed no significant correlation between the surgical outcome at 24 months and patients' sex, age, and preoperative keratometric readings. CONCLUSION: On the basis of our observations, we propose a new nomogram where higher powers of minus keratolenses, compared to the traditional prescription chart, should be selected for those with high preoperative myopia to result in improved long-term results with epikeratoplasty for myopia. This nomogram has not yet been clinically tested. PMID- 8624839 TI - The role of protein kinases in adaptational growth of the heart. AB - The ventricular myocyte is a terminally-differentiated cell that can no longer undergo cell division. In response to a variety of stimuli, including exposure to endothelin-1, phenylephrine or mechanical stretch, the myocyte increases its size and its complement of organized myofibrils. These adaptational changes during myocyte hypertrophy are accompanied by distinct changes in gene expression. The signalling cascades that initiate these changes are currently under intensive investigation. Many hypertrophic agonists activate protein kinase C (PKC). Transfection of ventricular myocytes with constitutively-active PKC isoforms initiates the changes in gene expression typical of the hypertrophic response. Similarly, the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway can be activated by a variety of hypertrophic agents. Transfection of ventricular myocytes with components of this pathway has demonstrated that MAPK is essential for the changes in gene expression associated with the development of hypertrophy. However a Ras-dependent, but Raf-independent, pathway may regulate the organization of the contractile apparatus. Other protein kinases, such as ribosomal S6 kinases, p90RSK or p70/p85S6K, which are poorly characterized in the ventricular myocyte, may also regulate changes in gene expression. Further research is required to investigate cross-talk between these signal transduction pathways so that the spatial and temporal relationships that integrate the multiple signaling events leading to the adaptational growth of the ventricular myocyte may be understood. PMID- 8624840 TI - Incorporation of 2-deoxy-D-glucose into glycogen. Implications for measurement of tissue-specific glucose uptake and utilisation. AB - Estimation of glucose uptake in vivo using 2-deoxy-D-[2,6-3H]glucose (2DG) relies upon the assumption that the phosphorylated form, 2-deoxy-D-2,6-3H]glucose 6 phosphate (2DGP), cannot be further metabolised. We aimed to determine whether this assumption leads to underestimation of glucose uptake due to the incorporation of 2DGP into glycogen. Rats were infused with [U-14C]glucose and 2 [3H]DG, and the incorporation into glycogen was measured. These were compared to the accumulation of 2-[3H]DGP in heart, liver, muscle, white adipose tissue and brown adipose tissue. 2DG was incorporated into glycogen in an insulin-dependent manner (e.g. in soleus, at basal, physiological and supraphysiological insulin concentrations, glycogen synthesis rates from 2DG were 17.81 +/- 3.07, 64.47 +/- 7.47 and 203.23 +/- 44.52 nmol glycogen incorporated/g min-1, respectively). The rate of glycogen synthesis from 2-[3H]DG was identical to that for [U-14C]glucose in all tissues studied except for heart and brown adipose tissue (e.g. in soleus at physiological insulin concentration, 2-[3H]DG incorporation was 64.47 +/- 7.47 and [U-14C]glucose incorporation was 61.87 +/- 7.56 nmol glucose/g min-1). Furthermore, the proportion of 2DG incorporated into glycogen was significant with respect to total glucose uptake at all plasma insulin concentrations (10.7% +/- 0.9, 14.0 +/- 1.9 and 25.6% +/- 5.6 at basal, physiological and supraphysiological insulin concentrations, respectively). 2DG was metabolised to glycogen in all tissues studied causing an underestimation of the rate of glucose uptake by measurement of 2DGP accumulation alone. In addition, use of 2DG could provide a method for assessing the rate of direct glycogen synthesis in the rat. PMID- 8624838 TI - In vivo evaluation of a collagen corneal allograft derived from rabbit dermis. AB - BACKGROUND: This study evaluates epithelialization, clarity, intraocular inflammation, and fibroblast ingrowth of a collagen corneal allograft derived from rabbit dermis. METHODS: Dermal collagen fibers were dispersed intact and chemically modified to make them soluble. The allografts consisted of a fibrous, opaque peripheral zone and a central clear area. Twelve New Zealand white rabbits underwent penetrating keratoplasty with implantation of an allograft. The grafts were evaluated daily for clarify, anterior segment inflammation, and extent of reepithelialization with a slit-lamp microscope. RESULTS: Epithelialization of the fibrous peripheral zone of the graft ranged from 0% to 90%. The central clear area did not epithelialize in any of the animals. Fibroblasts migrated into the peripheral zone in all eyes. Complications included ulceration of the central clear area in two eyes. There was no ulceration or leakage at the graft-host interface and no synechia, fibrous, or inflammatory retrocorneal membranes in any of the eyes. CONCLUSIONS: Our study is the first to describe the method of modifying dermal type I collagen into a clear corneal allograft. Survival of the corneal collagen allograft beyond 1 month may be limited by several factors including lack of epithelialization of the central clear area. PMID- 8624841 TI - Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice. AB - Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1. PMID- 8624842 TI - Subcellular distribution of phosphorylase kinase in rat brain. Association of the enzyme with mitochondria and membranes. AB - The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues. PMID- 8624843 TI - Blood cardiolipin in haemodialysis patients. Its implication in the biological action of platelet-activating factor. AB - Bovine heart cardiolipin specifically inhibits platelet aggregation induced by PAF in vitro. In the past we have reported that patients with primary glomerulonephritis have increased PAF levels in plasma (Iatrou et al., 1995b). In this work we investigate the existence of cardiolipin in the blood of end-stage renal patients due to primary glomerulonephritis and we study its implication in the biological study of PAF. Lipids from blood samples of end-stage renal patients were extracted, fractionated onto silicic acid column and onto High Performance Liquid Chromatography (HPLC) cation exchange column. PAF fraction was removed and phospholipids were separated from the rest lipid fraction with current counter distribution and furthermore fractionated onto HPLC silica column. The results show: 1. cardiolipin is present in the blood of end-stage renal patients. 2. Blood cardiolipin specifically inhibits PAF-induced aggregation in washed rabbit platelets. 3. Scatchard plot analysis of PAF binding, in the presence of unlabelled PAF and in the presence of cardiolipin, shows that rabbit platelets possess two different types of binding sites. One of which is saturable and of high affinity, kD = 0.103 +/- 0.03 nM (SEM, n = 3) with 337 +/- 94 binding sites per platelet for PAF and kD = 0.087 +/- 0.02 nM with 371 +/- 92.7 binding sites per platelet for cardiolipin while the other one has almost infinite binding capacity. 4. Blood cardiolipin competes [3H]PAF binding in rabbit platelets. This work shows that cardiolipin exists in the blood of end stage renal patients and specifically inhibits PAF-induced aggregation as well as PAF binding in rabbit platelets. The possible implication of the biological actions of cardiolipin in the anticardiolipin-antiphospholipid syndrome is also discussed. PMID- 8624844 TI - Complex assembly of calgranulins A and B, two S100-like calcium-binding proteins from pig granulocytes. AB - Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo. PMID- 8624845 TI - The biological activity of acetylated sphingosylphosphorylcholine derivatives. AB - Taking into account their stereochemical similarity with 1-O-alkyl-2-acetyl-sn glycerol-3-phosphorylcholine (PAF), which is known to exhibit a diverse spectrum of biological actions, including platelet aggregating and glycogenolytic activity, the acetylated derivatives of sphingosylphosphorylcholine and sphingomyelin were synthesized and tested for their ability to promote washed rabbit platelet aggregation and glycogenolysis in Tetrahymena pyriformis cells. Sphingomyelin (SPM) and sphingosylphosphorylcholine (SPC) were subjected to acetylation, following chromatographic purification and physicochemical characterization. The synthesized compounds N-acetyl, O-acetyl SPC (NAc, OAc SPC), N-acetyl SPC (NAc SPC) and O-acetyl SPM (OAc, SPM) were tested for their biological activity in the washed rabbit platelets and washed Tetrahymena pyriformis cell systems. These derivatives induced [3H]serotonin and ATP release and a monophasic aggregation of washed rabbit platelets in the concentration range 10(-8)-10(-6) M. However, authentic PAF-induced washed rabbit platelet aggregation was inhibited at higher concentrations of the acetylated sphingophospholipid compounds ( > 10(-6) M) and by the PAF-specific receptor(s) antagonists kadsurenone and triazolam. Platelet desensitization and crossed desensitization experiments with authentic PAF and the acetylated sphingophospholipids, suggested interaction with the same receptor(s) as PAF and different from the ADP or thrombin receptor. The involvement of calmodulin and protein kinase C in the biological activity of the prepared analogues was also demonstrated. Besides their action on rabbit platelets, the PAF-like activity of the acetylated sphingophospholipids was also demonstrated in a platelet independent system, by showing that they stimulate glycogenolysis in washed Tetrahymena pyriformis cells. These observations indicate that a new class of compounds with PAF-like activity were synthesized but their role in the cellular metabolism remains to be shown. The existence of acetylated sphingophospholipids with PAF-like activity has so far been reported only in Urtica dioica. PMID- 8624846 TI - Characterization of ATP-diphosphohydrolase from rat mammary gland. AB - ATP-diphosphohydrolase (or apyrase) hydrolyses nucleoside di- and triphosphates in the presence of millimolar concentration of divalent cations. It is insensitive towards sulfhydryl and aliphatic hydroxyl-selective reagents and to specific inhibitors of ATPases. We present further evidence that ATPase and ADPase activities present in rat mammary gland correspond to apyrase. Two kinetic approaches have been employed, competition plot and chemical modification with group-selective reagents. The M(r) of these activities was determined by 60Co radiation-inactivation. The kinetic approaches employed, competition plot (which discriminate whether competitive reactions occur at the same site) and chemical modification, point to the presence of a single protein which hydrolyses ATP and ADP. The similar M(r) values of ATPase and ADPase activities also support this proposal. ATPase and ADPase activities of mammary gland show a similar sensitivity or insensitivity towards several chemical modifiers. These results suggest that this enzyme is ATP-diphosphohydrolase, also known as apyrase. The results obtained are compared with the ones obtained by us and other authors with the enzyme isolated from other sources. PMID- 8624847 TI - Cloning, expression and genomic organization of human placental protein disulfide isomerase (previously identified as phospholipase C alpha). AB - Phosphoinositol-specific Phospholipase C plays an important role in transducing receptor generated signals to the rest of the cell. A cDNA encoding a phospholipase has been described (Bennett et al., 1988, Nature 334, 268-270). However it is probable that this cDNA in fact encodes a protein disulfide isomerase. Since the original work suggested that this enzyme was important in the reproductive tract we sort to clone, sequence, express and characterize the recombinant protein isolated from the placenta. We have cloned and sequenced the cDNA encoding the human homolog of this cDNA from human placenta, although the mRNA was widespread in the female reproductive tract. We have transiently expressed it in both COS cells and also 1BR fibroblasts. Cell lysates were assayed for increased phospholipase activity and protein disulfide activity. We describe the entire cDNA sequence which is highly conserved between species. We have also cloned a portion of the genomic gene and described the intron/exon boundaries. In vitro translation of this cDNA showed that it encoded a protein of 61 kD with a cleavable signal peptide. Transient expression showed the protein produced had no phospholipase activity but did show protein disulfide isomerase activity. The expression work shows that this cDNA indeed encodes a protein disulfide isomerase and not a phospholipase. The nucleotide sequence shows marked conservation of the coding and regulatory regions which may suggest that this enzyme has evolved to perform a highly specialized function. PMID- 8624848 TI - Ergocalciferol and cycloheximide in vivo stimulate protein kinase C of intestinal crypt cells. AB - Previous reports have suggested that 1,25-dihydroxychole-calciferol regulated cellular differentiation via its effects on protein kinase C activity. This study examined the in vivo effects of ergocalciferol on the activity of protein kinase C, and whether the differentiation of crypt intestinal cells is dependent on the activation of this enzyme. Ergocalciferol in saline was injected intramuscularly into rats and the animals sacrificed 24 hr after fasting. Protein kinase C specific activity was determined from the rate of incorporation of 32p-ATP into protamine. Injections of 60 micrograms ergocalciferol/200 g of body wt, raised protein kinase C specific activity to 59818 +/- 4010 (SEM, n = 5) cpm 32p protamine/min/mg cell protein, compared with a control of 46173 +/- 4612 (P < 0.0005). Optimal specific activities were seen within 72 hr of injection. Administration of 120 micrograms ergocalciferol/200 g of body wt, raised the concentrations of serum calcium to 9.8 and 10.4 mg/dl following the intramuscular injection by 24 and 72 hr, respectively, compared with a control of 7.7 mg/dl. Actinomycin D (intramuscular, 100 micrograms/200 g of body wt) together with ergocalciferol (120 micrograms/200 g of body wt) reduced protein kinase C activity by 51% 24 hr after injection. Cycloheximide blocked the activation, but when injected alone stimulated endogenous protein kinase C activity by 34% 24 hr injection. The study shows activation of crypt protein kinase C by ergocalciferol. The inhibition of activation by actinomycin D and cycloheximide suggests the involvement of both transcriptional and translational processes in this activation. PMID- 8624849 TI - Partial purification and characterization of two phosvitin phosphatases from rat brain. AB - The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160 220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain. PMID- 8624850 TI - Fusion genes and their hybrid proteins in human leukemias and lymphomas. PMID- 8624851 TI - Mammalian G1 cyclins and cell cycle progression. AB - Since their discovery in 1991, the D- and E-type cyclins, their associated catalytic subunits, and the molecules that regulate their kinase activity have been subjects of intense scrutiny by a rapidly expanding group of investigators. Part of the excitement stems from an emerging realization that these molecules define a final common pathway for growth factor-induced and antiproliferative signals during the cell cycle. As such, their study is helping to describe the biochemical pathways that determine the basis of late GI phase control, much as studies of growth factors, receptors, cytoplasmic signaling molecules, and responding transcription factors helped to define temporally earlier steps in the mitogenic process. In retrospect, it is neither surprising that there is a functional interplay between these cell cycle regulators and previously recognized tumor suppressor gene products (p53 and pRB) that are themselves engaged in GI phase checkpoint control, nor is it surprising that genetic perturbations affecting the functions of these genes contribute to cancer. It seems equally likely that further elucidation of the functions of these molecules will help to better explain such diverse processes as differentiation and apoptosis, which cooperatively depend on the cell cycle machinery for their proper execution. PMID- 8624852 TI - Heterogeneity of hematopoietic stem cells: implications for clinical applications. PMID- 8624853 TI - Immortalized neural stem cells: insights into development; prospects for gene therapy and repair. PMID- 8624854 TI - Fate of DNA targeted to hepatocytes by asialoglycoprotein polylysine conjugates. AB - The data indicate that partial hepatectomy results in a decrease in degradation of targeted DNA. This appears to be due to an inhibition of the endocytotic pathway shortly after hepatectomy and is associated with the accumulation of targeted DNA within a population of light endosomal vesicles. It is likely that these vesicles serve as a reservoir from which targeted DNA gradually escapes and can be found in the nucleus. The DNA targeted to liver is capable of expressing the marker gene, and the DNA in the vesicles is transfection competent, suggesting that a substantial portion is intact. Overall, the receptor-mediated delivery system is highly efficient in transporting foreign DNA to liver cells. Partial hepatectomy and the ensuing cellular events provide a means of inhibiting the degradative portion of the endocytotic pathway. Pharmacological agents that can mimic the cellular processes that occur after partial hepatectomy may be useful in increasing the duration of foreign gene expression without the trauma of surgery. PMID- 8624855 TI - Folate, epithelial dysplasia and colon cancer. PMID- 8624856 TI - The expanded trinucleotide repeat in Kennedy's disease. PMID- 8624857 TI - Analysis of the CAG repeat and gene product in spinocerebellar ataxia type 1. PMID- 8624858 TI - An animal model of bullous pemphigoid: what can it teach us? AB - BP is an organ-specific autoimmune disease characterized by blistering of the skin due to a detachment of the epidermis from the underlying connective tissue layer and by the production of autoantibodies directed against the cutaneous basement membrane zone. A 180-kD epidermal protein, BP180, has been identified as one of the major antigenic targets of these autoantibodies and, interestingly, has been localized to the epidermal hemidesmosome, a cellular structure involved in anchoring basal epithelial cells to the basement membrane. An immunodominant and potentially pathogenic epitope associated with BP has recently been mapped by our laboratory to a site on the extracellular domain of the human BP180 antigen. This antigenic site, designated the MCW-1 epitope, has been shown to be recognized by the majority of sera from patients with either BP or HG, a pregnancy-associated subepidermal bullous disease. Unfortunately, the MCW-1 epitope is absent from the murine BP180 molecule; therefore, human autoantibodies directed against this site could not be tested directly for pathogenicity using the conventional passive transfer mouse model. As an alternative approach, rabbit antibodies were prepared against the segment of murine BP180 that is homologous with the MCW-1 epitope region and were tested for pathogenicity by passive transfer experiments. Remarkably, neonatal mice injected with these antibodies developed a subepidermal blistering disease that faithfully reproduced all of the key immunopathological features of BP and HG--deposition of lgG and complement at the dermal-epidermal junction, inflammatory infiltration of the upper dermis, and frank, subepidermal blistering. These results strongly suggest that the autoimmune response against the human BP180 MCW-1 epitope is relevant in the pathogenesis of blister formation in BP and HG patients. This animal model is currently being used to further dissect the pathophysiology of these autoimmune disorders. PMID- 8624859 TI - Calcitonin gene-related peptide modulates Langerhans cell antigen-presenting function. PMID- 8624860 TI - Premature termination codon mutations in the type VII collagen gene (COL7A1) underlie severe recessive dystrophic epidermolysis bullosa. PMID- 8624861 TI - The Gorlin syndrome gene: a tumor suppressor active in basal cell carcinogenesis and embryonic development. PMID- 8624862 TI - Multistage skin carcinogenesis in transgenic mice. PMID- 8624863 TI - Pathophysiology and time course of silent myocardial ischaemia during mental stress. PMID- 8624864 TI - Extent of oxidative modification of low density lipoprotein determines the degree of cytotoxicity to human coronary artery cells. AB - OBJECTIVE: To assess whether the extent of LDL oxidation influences its cytotoxic effects, thus contributing to its atherogenic potential. DESIGN AND SETTING: The effects of native and modified LDL on cultured human coronary artery smooth muscle cells (SMC) and endothelial cells (ECs) were investigated. MAIN OUTCOME MEASURES: Four indices of cytotoxicity were studied: (i) chromium-51 release; (ii) 5-bromo-2'-deoxyuridine (BrDUrd) uptake; (iii) morphological appearance; and (iv) EC migration. RESULTS: (i) Minimally modified (mm) LDL (400 micrograms/ml) causes significant 51Cr release; the cytotoxic effect was significantly greater for copper oxidised (ox) LDL (400 micrograms/ml). Native LDL had no effect. (ii) BrDUrd uptake studies showed significant inhibition of cell proliferation by 100 micrograms/ml of oxLDL and to a lesser extent by mmLDL; native LDL had no effect. (iii) Morphological appearance was not altered by native LDL. Changes in cell morphology were induced by mmLDL (400 micrograms/ml), and were more pronounced with oxLDL in concentrations of > or = 200 micrograms/ml. (iv) EC migration was significantly inhibited by oxLDL (100 micrograms/ml), but not by native or mmLDL. CONCLUSION: The extent of oxidation of LDL determined its cytotoxicity to coronary artery cells. Native LDL had no cytotoxic effect. In contrast, oxLDL and to a lesser extent mmLDL caused cytotoxicity at concentrations to which cells in vivo might be exposed. This may contribute to the atherogenicity of modified LDL by enhancing cellular injury and inflammation, and by inhibiting re endothelialisation of areas of coronary artery damaged during the atherogenic process. PMID- 8624866 TI - Induction of ventricular fibrillation predicts sudden death in patients treated with amiodarone because of ventricular tachyarrhythmias after a myocardial infarction. AB - OBJECTIVE: To examine the value of programmed electrical stimulation of the heart in predicting sudden death in patients receiving amiodarone to treat ventricular tachyarrhythmias after myocardial infarction. DESIGN: Consecutive patients; retrospective study. SETTING: Referral centre for cardiology, academic hospital. PATIENTS: 106 patients with ventricular tachycardia (n = 77) or ventricular fibrillation (n = 29) late after myocardial infarction. INTERVENTIONS: Programmed electrical stimulation was performed while on amiodarone treatment for at least one month. MEASUREMENTS AND MAIN RESULTS: In 80/106 patients either ventricular fibrillation (n = 15) or sustained monomorphic ventricular tachycardia (n = 65) was induced. After a mean follow up of 50 (SD 40) months (1-144), 11 patients died suddenly and two used their implantable cardioverter debfibrillator. By multivariate analysis two predictors for sudden death were found: (1) inducibility of ventricular fibrillation under amiodarone treatment (P << 0.001), and (2) a left ventricular ejection fraction of < 40% (P < 0.05). The survival rate at one, two, three, and five years was 70%, 62%, 62%, and 40% respectively for patients in whom ventricular fibrillation was induced, and 98%, 96%, 94%, 94% for patients with induced sustained monomorphic ventricular tachycardia. Where there was no sustained arrhythmia, five year survival was 100%. CONCLUSIONS: In patients receiving amiodarone because of life threatening ventricular arrhythmias after myocardial infarction, inducibility of ventricular fibrillation, but not of sustained monomorphic ventricular tachycardia, indicates a high risk of sudden death. PMID- 8624865 TI - Significance of perfusion of the infarct related coronary artery for susceptibility to ventricular tachyarrhythmias in patients with previous myocardial infarction. AB - OBJECTIVE: To study the significance of perfusion of the infarct related coronary artery for susceptibility to ventricular tachyarrhythmias in patients with a remote myocardial infarction. SETTING: Tertiary referral cardiac centre. METHODS: Angiographic filling of the infarct related artery was assessed in a consecutive series of 85 patients with different susceptibilities to ventricular tachyarrhythmias after previous (> 3 months) Q wave myocardial infarction: 30 patients had a history of cardiac arrest (n = 16) or sustained ventricular tachycardia (n = 14), and sustained ventricular tachyarrhythmia was inducible in these by programmed electrical stimulation (arrhythmia group); 47 patients had no clinical arrhythmic events and no inducible ventricular tachyarrhythmias during programmed ventricular stimulation (control group). Eight patients without a history of any arrhythmic events were inducible into ventricular tachycardia. RESULTS: The patients in the arrhythmia group were older (63 (SD 8) years) than the control patients (59 (6) years, P < 0.05), and had larger left ventricular volumes in cineangiography (P < 0.01), but ejection fraction, severity of left ventricular wall motion abnormalities, previous thrombolytic therapy, and time from previous infarction did not differ between the groups. Patients with susceptibility to ventricular tachyarrhythmias more often had a totally occluded infarct related artery on angiography (77%) than patients without arrhythmia susceptibility (21%) (P < 0.001), and complete collateral filling of the infarct artery in cases without complete anterograde filling was less common in the arrhythmia group than in the control group (P < 0.001). Patients without a history of malignant arrhythmia but with inducible ventricular tachyarrhythmia also had no or poor perfusion of the infarct artery more often than the patients without inducible arrhythmia (P < 0.001). Logistic multiple regression showed that no or poor anterograde or collateral filling of the infarct related artery was the most powerful predictor of susceptibility to ventricular tachyarrhythmias (P < 0.001). Left ventricular size and function were not independently related to arrhythmic susceptibility. CONCLUSIONS: No or poor angiographic filling of the infarct related artery is closely associated with susceptibility to ventricular tachyarrhythmias late after acute myocardial infarction, suggesting that perfusion of the infarct artery will modify favourably the electrophysiological substrate of the infarct scar independently of the myocardial salvage achieved by early reperfusion. PMID- 8624867 TI - Regional distribution and kinetics of [18F]6-flurodopamine as a measure of cardiac sympathetic activity in humans. AB - OBJECTIVES: To determine whether an increase in cardiac sympathetic activity produced by exercise or sublingual glyceryl trinitrate causes an increased rate of loss of fluorine-18 from the myocardium after intravenous [18F]6 fluorodopamine ([18F]F-DA) in normal volunteers. In addition, to determine the contribution of non-specific uptake of [18F]F-DA in the myocardium in patients with recent heart transplant. PROTOCOL: [18F]F was prepared by direct electrophilic fluorination of dopamine. Nine healthy volunteers each received 1.85 x 10(8) Bq (168-250 micrograms) [18F]F-DA over a period of 3 min and were scanned for 2 h in an ECAT 953/31 tomograph. Three controls were scanned before and after vigorous cycle exercise and two were scanned before and after sublingual glyceryl trinitrate. In addition, two patients (1 and 2 years post heart transplant) underwent a myocardial perfusion study with ammonia labelled with nitrogen-13 followed by an [18F]F-DA study. RESULTS: There was intense uniform uptake of [18F]F-DA throughout the myocardium in the healthy volunteers. The time course of 18F in the myocardium under resting conditions fitted a biexponential function with mean half-times of 8.0 and 109 min. Vigorous exercise produced a three to fivefold increase in the rate of loss of 18F compared with that when resting. After glyceryl trinitrate, one control had a profound reduction in blood pressure (23%) and twofold increase in the rate of loss of myocardial 18F. The other control had no physiologically significant change in blood pressure, heart rate, or rate of loss of myocardial 18F. Uptake of [18F]F DA in the two posttransplant patients was confined to a small anterobasal region adjacent to the atrioventricular groove, while blood flow, as measured with [13N] ammonia, was uniformly distributed throughout the myocardium. Partial reinnervation of the myocardium was confirmed by the presence of distinct low frequency spectral peaks of the heart rate power spectrum in both patients. CONCLUSIONS: These results suggest that the uptake of [18F]F-DA reflects the distribution of cardiac sympathetic innervation and that the rate of loss of 18F from the myocardium partially reflects spill over of noradrenaline. The technique may be useful in investigating various cardiac conditions in which the sympathetic system is compromised. PMID- 8624868 TI - Prolapse of an aortic dissection flap imaged by transoesophageal echocardiography. PMID- 8624870 TI - Exercise response after cardiac transplantation: correlation with sympathetic reinnervation. AB - OBJECTIVE: To investigate the relation between sympathetic efferent reinnervation and chronotropic competence during exercise testing after cardiac transplantation. PATIENTS: Twenty five long-term cardiac transplant recipients and 11 normal controls. SETTING: Regional cardiothoracic centre. METHODS: Intracoronary tyramine was given to the transplant recipients and the per cent heart rate change measured. Exercise tests were performed in patients and controls according to the chronotropic assessment exercise protocol, and the per cent heart rate reserve measured at peak exercise and 6 min afterwards to estimate the recovery rate. RESULTS: The mean (SD) percentage heart rate change after intracoronary tyramine was 15.7 (15.4). Heart rate reserve achieved at peak exercise was 68.3 (20.6)% compared with 102.7 (9.3)% in the controls (P < 0.001). Heart rate recovery at 6 min was 41.7 (20.1)% compared with 79.5 (9.0)% in the controls (P < 0.001). Total workload was 69.0 (33.0) METS.min compared with 117.2 (41.9) METS.min in the controls (P < 0.01). There was a positive correlation between heart rate reserve achieved at peak exercise and response to tyramine (r = 0.66, P < 0.01), between heart rate recovery and response to tyramine (r = 0.69, P < 0.001), and between total workload and response to tyramine (r = 0.63, P = 0.04). CONCLUSION: Functional sympathetic efferent reinnervation of the sinus node occurred in some patients after transplantation, and was associated with improved heart rate response during and recovery after exercise, as well as with increased total workload. PMID- 8624869 TI - Circadian variation of left ventricular diastolic function in healthy people. AB - AIM: To assess whether left ventricular function shows circadian variation in healthy people. SUBJECTS AND METHODS: 10 healthy men (7) and women (3) aged 35-50 underwent M mode echocardiography of the left ventricle and Doppler velocimetry of transmitral flow at 4 h intervals over 24 h. The participants were in hospital over the study period and their diet, meal times, and sleeping hours were standardised as far as possible. MEASUREMENTS: Heart rate, blood pressure, left ventricular and atrial diameters, fractional shortening, peak early and late transmitral velocities, time from the second heart sound to the early diastolic velocity peak (relaxation time), isovolumic relaxation period, acceleration and deceleration of the early transmitral flow, atrial filling fraction. RESULTS: A circadian rhythm was observed in heart rate and blood pressure, but neither the left ventricular diameters and systolic function nor the left atrial size showed statistically significant diurnal trends. The relaxation time (mean (SD)) measured 144 (16) ms at 2 pm, 144 (21) ms at 6 pm, 149 (22) ms at 10 pm, 168 (23) ms at 2 am, 174 (28) ms at 6 am, and 151 (21) ms at 10 am (P = 0.009). Diurnal rhythms were seen also in the isovolumic relaxation period (P = 0.003) and in the acceleration of the early diastolic transmitral flow (P = 0.037); the lowest and highest values of flow acceleration were observed during the nocturnal and daytime hours, respectively. CONCLUSIONS: The Doppler indices of left ventricular filling in healthy people show diurnal changes suggestive of a circadian rhythm in the rate of left ventricular relaxation. The most likely underlying mechanism is the day-night cycle in sympathoadrenal activity. PMID- 8624871 TI - DDD pacing in hypertrophic cardiomyopathy: a multicentre clinical experience. AB - BACKGROUND: DDD pacing has been advocated as an effective treatment for drug refractory obstructive hypertrophic cardiomyopathy. This study reports the outcome of pacing in 56 patients with refractory symptoms referred to four tertiary centres. METHODS: Core data on symptoms, drug burden, and left ventricular outflow tract gradient were recorded. Patients underwent a temporary pacing study with optimisation of the atrioventricular (AV) delay for greatest gradient reduction without haemodynamic compromise. Patients were assessed after implantation in terms of changes in symptoms, drug load, and outflow tract gradient. RESULTS: 56 patients underwent pacing assessment. The mean (SD) left ventricular outflow tract gradient before pacing was 78 (31) mm Hg. At temporary study the mean (SD) left ventricular outflow tract gradient was 38 (24) mm Hg with a median (range) optimised sensed AV delay of 65 (25-125) ms. Fifty three patients were implanted and followed up for a mean (SD) of 11 (11) months. The median (range) programmed sensed AV delay was 60 (31-200) ms. Left ventricular outflow tract gradient at follow up was 36 (25) mm Hg. Forty four patients had improved functional class. Although a correlation (r = 0.69) was shown between acute and chronic left ventricular outflow tract gradient reduction, there was no correlation between magnitude of gradient reduction and functional improvement, and no appreciable change in pharmacological burden. CONCLUSION: This series confirms symptomatic improvement after DDD pacing in hypertrophic cardiomyopathy. There remains, however, a discrepancy between perceived symptomatic benefit and modest objective improvement. Furthermore, the optimal outcome has been achieved only with continued pharmacological treatment. Current methods of temporary evaluation do not predict functional outcome which seems to be independent of the magnitude of gradient reduction. PMID- 8624872 TI - Myocardial beta adrenoceptors and left ventricular function in hypertrophic cardiomyopathy. AB - OBJECTIVE: To assess the relation between left ventricular function and myocardial beta adrenoceptor density. METHODS: 17 patients with hypertrophic cardiomyopathy, six with and 11 without heart failure, were studied. Left ventricular function was assessed by echocardiography, and myocardial beta adrenoceptors by positron emission tomography. Patient data were compared with those obtained in normal controls. RESULTS: Myocardial beta adrenoceptor density in the 17 patients was 7.00 (SD 1.90) pmol/g v 11.50 (2.18) pmol/g in normal controls (P < 0.01). beta Adrenoceptor density in the six patients with left ventricular failure was 5.61 (0.88) pmol/g v 7.71 (1.86) pmol/g in the 11 patients with normal ventricular function (P < 0.05), and there was a significant correlation (r = 0.52; P < 0.05) between left ventricular fractional shortening and myocardial beta adrenoceptor density. A positive correlation (r = 0.51; P < 0.05) was also found between myocardial beta adrenoceptor density and the E/A transmitral flow ratio, an index of left ventricular diastolic function. CONCLUSIONS: There is myocardial beta adrenoceptor downregulation in patients with hypertrophic cardiomyopathy with or without signs of heart failure. PMID- 8624873 TI - Haemodynamic and energetic properties of stunned myocardium in rabbit hearts. AB - OBJECTIVE: To amplify the description of myocardial stunning. DESIGN: Control versus 30 min after a 20 min no flow ischaemia. EXPERIMENTAL ANIMALS: 15 isolated rabbit hearts perfused with erythrocyte suspension. MAIN OUTCOME MEASURES: Left ventricular systolic function in terms of aortic flow, peak systolic pressure (LVPmax), dP/dtmax, and the end systolic pressure-volume relation (ESPVR); early relaxation from dP/dtmin and rate of left ventricular pressure decay (tau). Passive properties: ventricular and myocardial stiffness. Coronary resistance from coronary blood flow and perfusion pressure. Total myocardial oxygen consumption (MVo2tot). Total mechanical energy via pressure-volume area (PVA). Contractile efficiency (Econ) and MVo2 of the unloaded contracting heart (MVo2unl). External mechanical efficiency (Eext) from stroke work and MVo2tot. RESULTS: Systolic variables in stunned myocardium were significantly decreased (mean (SD)): aortic flow: 38 (13) v 9 (11) ml/min; LVPmax: 112 (19) v 74 (18) mm Hg; dP/dtmax: 1475 (400) v 1075 (275) mm Hg/s. ESPVR was not significantly decreased, at 138 (73) v 125 (58) mm Hg/ml, but the volume axis intercept was shifted rightward: 0.30 (0.37) v 0.65 (0.25) ml. Likewise, early relaxation was impaired: dP/dtmin (-1275 (250) v -975 (250) mm Hg/s) and tau (37 (7) v 46 (10) ms). LVPed was significantly decreased at 19 (12) v 12 (7) mm Hg, and both the ventricular (end diastolic pressure-volume relation) and the myocardial stiffness (constant k) were increased by 75% and 31%, respectively. Coronary resistance increased non-significantly from 0.83 (0.31) to 1.04 (0.41) mm Hg/(ml/min/100 g). Decreases in PVA (570 (280) v 270 (200) mm Hg.ml/100 g), MVo2tot (40 (9) v 34 (8) microliters/beat/100 g), and MVo2unl (26 (9) v 22 (6) microliters/beat/100 g) did not reach significance, in contrast to significant decreases in Econ (31 (18) v 14 (7)%) and Eext (0.75 (0.29) v 0.18 (0.25) arbitrary units). CONCLUSIONS: Ventricular systolic function is decreased after brief episodes of ischaemia. The decrease in diastolic function probably amplifies the systolic deterioration during myocardial stunning. Passive diastolic properties are also changed, shown by increases in both ventricular and myocardial stiffness. The increase in coronary resistance indicates stunning at the vascular level which could limit oxygen supply. With maintained MVo2tot during stunning, external efficiency is decreased. Possible candidates for this metabolic stunning are inadequate excitation-contraction coupling and disturbed O2 utilisation by the contractile apparatus. PMID- 8624875 TI - Fate of the native aorta after repair of acute type A dissection: a magnetic resonance imaging study. AB - OBJECTIVE: To determine late patency of the aortic false lumen and propensity for aneurysm formation after repair of type A dissection. DESIGN: Retrospective follow up study. SETTING: Regional cardiac surgical unit. PATIENTS: 28 patients after repair of type A dissection. METHODS: Magnetic resonance imaging (MRI) was performed between 6 weeks and 12 months after operation. RESULTS: A patent distal false lumen with demonstrable blood flow was found in 22 patients (78%). Only six patients had complete obliteration of the false lumen by thrombus. The conduct of operation did not influence this. Nine patients (32%) showed aneurysmal dilatation of the false lumen and three had a repeat operation. CONCLUSIONS: So called "successful repair" of aortic dissection does not obliterate the distal false lumen. MRI is a safe and effective radiological procedure for determining patency and dilatation in the false lumen. Patients with type A dissection with blood flow in the false lumen should be studied every 6 months to look for aneurysmal dilatation. PMID- 8624874 TI - Amiodarone in congestive heart failure: unravelling the GESICA and CHF-STAT differences. PMID- 8624876 TI - Pericardial effusion in patients with cancer: outcome with contemporary management strategies. AB - OBJECTIVE: To investigate the clinical presentation and current management strategies of pericardial effusion in patients with malignancy. DESIGN: Retrospective single centre, consecutive observational study. SETTING: University hospital. PATIENTS: 93 consecutive patients with a past or present diagnosis of cancer and a pericardial effusion, including 50 with a pericardial effusion > 1 cm. RESULTS: Of the 50 patients with pericardial effusions > 1 cm, most had stage 4 cancer (64%), were symptomatic at the time of presentation (74%), and had right atrial collapse (74%). Twenty patients were treated conservatively (without pericardiocentesis) and were less symptomatic (55% v 87%, P = 0.012), had smaller pericardial effusions (1.5 (0.4) v 1.8 (0.5), P = 0.02), and less frequent clinical (10% v 40%, P = 0.02) and echocardiographic evidence of tamponade (40% v 97%, P < 0.001) than the 30 patients treated invasively with initial pericardiocentesis (n = 29) or pericardial window placement (n = 1). Pericardial tamponade requiring repeat pericardiocentesis occurred in 18 (62%) of 29 patients after a median of 7 days. In contrast, only four (20%) of 20 patients in the conservative group progressed to frank clinical tamponade and required pericardiocentesis (P = 0.005 v invasive group). The overall median survival was 2 months with a survival rate at 48 months of 26%. Survival, duration of hospital stay, and hospital charges were similar with both strategies. By multivariable analysis, the absence of symptoms was the only independent predictor of long-term survival (relative hazards ratio = 3.2, P = 0.05). Survival was similar in the 43 patients with cancer and pericardial effusions of < or = 1 cm. CONCLUSION: Asymptomatic patients with cancer and pericardial effusion can be managed conservatively with close follow up. In patients with symptoms or clinical cardiac tamponade, pericardiocentesis provides relief of symptoms but does not improve survival and has a high recurrence rate. Surgical pericardial windows or possibly percutaneous balloon pericardiotomy should be used for recurrences and should be considered for initial treatment. PMID- 8624877 TI - Palliative Mustard operation for transposition of the great arteries: late results after 15-20 years. AB - OBJECTIVE: To assess the clinical, functional state, and complications late (15 20 years) after palliative Mustard operation. DESIGN: Examination and evaluation of all patients presenting in adolescence and adult life after palliative Mustard operation for transposition of the great arteries and pulmonary vascular disease. SETTING: Grown-up Congenital Heart Unit specialised in the care of adolescents and adults with congenital heart disease, designated as having "quaternary" status within a tertiary referral centre for cardiac diseases. PATIENTS AND METHODS: Database searched for patients referred after palliative Mustard for classic transposition of the great arteries. Ten patients aged 18-31 (mean (SD) 25.9 (5.2)) years with a palliative Mustard operation performed at age 1.7-15 (mean (SD) 9 (4.6)) years were fully evaluated by echocardiography, exercise testing, Holter monitoring, and magnetic resonance imaging or radionuclide ventriculography, or both 15-20 years later. RESULTS: One patient died aged 25 years with biventricular failure (ability index 3/4), haemoptysis, and atrial flutter, eight were well (ability index 2), and one disabled (ability index 3). Arterial oxygen saturation at rest was 85-98% (mean (SD) 93.8 (4)%) decreasing to 59-87% (mean 77 (9.5)%) after limited exercise. Symptomatic arrhythmias occurred in four patients, atrial flutter being the most common, and two had sinus node dysfunction. Significant tricuspid regurgitation occurred in four patients. CONCLUSION: Patients with palliative Mustard operation have a low incidence of symptomatic ventricular dysfunction and despite exercise limitation by hypoxia, continue to live active, near normal lives until their thirties. PMID- 8624878 TI - Bidirectional superior cavopulmonary anastomosis: how young is too young? AB - OBJECTIVE: To define the lowest age at which the bidirectional superior cavopulmonary anastomosis can safely be used in infants with complex congenital heart defects. DESIGN: A retrospective analysis of clinical, echocardiographic, haemodynamic, and angiographic data in four consecutive patients undergoing bidirectional superior cavopulmonary anastomosis below the age of 2 months. PATIENTS: Between November 1990 and September 1993, four infants less than 8 weeks of age (3, 4, 6, and 7 weeks) underwent bidirectional superior cavopulmonary anastomosis as a primary palliation for complex heart disease. The indication for early surgical intervention was progression of cyanosis (n = 3) and high pulmonary blood flow causing heart failure (n = 1). In two infants with tricuspid atresia, surgery was performed through a right thoracotomy without the use of cardiopulmonary bypass. In one infant with double inlet left ventricle and discordant ventriculoarterial connection, cavopulmonary anastomosis was combined with an arterial switch procedure. The final infant had double inlet left ventricle with pulmonary atresia; the central pulmonary arteries were virtually discontinuous and each branch was supplied by a separate arterial duct. The central pulmonary arteries were reconstructed using the subaortic innominate vein. Temporary prostacyclin infusion was used in three patients in the immediate postoperative period. RESULTS: Early postoperative extubation (5, 7, and 48 h) was successful in three patients. The youngest child required ligation of the ductus arteriosus four days later because of severe upper body oedema. The postoperative course was complicated by prolonged effusions in two patients. All were alive and well 14-48 months postoperatively, with satisfactory systemic saturations (80-87%) and haemodynamic indices. CONCLUSIONS: This limited experience challenges the widely held belief that the bidirectional superior cavopulmonary anastomosis cannot be used as a primary palliation for complex heart disease in early infancy. A wider experience is required to determine the safety and indications for this approach. PMID- 8624879 TI - Combining coronary angiography and angioplasty. PMID- 8624880 TI - Large left ventricular thrombus in a patient with Friedreich's ataxia. PMID- 8624881 TI - Transcatheter closure of secundum atrial septal defects with the atrial septal defect occlusion system (ASDOS): initial experience in children. AB - OBJECTIVE: To report initial experiences with transcatheter occlusion of atrial septal defects using a new occlusion device. SUBJECTS: 10 children aged 1.1 to 14.9 years. INCLUSION CRITERIA: Patients with a body weight above 10 kg, normal pulmonary resistance and an indication for surgical closure of a secundum atrial septal defect, a residual tissue rim of interatrial septum surrounding the defect of more than 5 mm, and a maximum defect diameter of 20 mm. METHODS: The defects were closed by a transcatheter device (ASDOS) consisting of two umbrellas which are introduced over a guidewire loop. Both umbrellas consist of a central body and five arms formed from preshaped nitinol wire covered with a thin polyurethane patch. The central body of the distal umbrella contains a thread, the proximal umbrella contains a bolt. The two umbrellas are connected by screwing the bolt on the thread using a screwdriver catheter. RESULTS: The implantation was performed under echocardiographic guidance; in six of 10 patients, transoesophageal echocardiography was necessary. The "stretched" diameter of the defect evaluated during balloon sizing ranged from 10 to 20 mm, and the pulmonary to systemic blood flow ratio from 1.5:1 to 2.8:1. Transcatheter closure was successfully performed in 9/10 patients using devices with a diameter of 25 mm to 40 mm. No severe complications occurred. However, in one patient with a pre-existing prolonged PR interval brief periods of second and third degree atrioventricular block occurred after the implantation but normalised within 3 d. During a follow up period of 21 to 29 weeks no device embolisation, thromboembolic complications, fractures of the implanted device, atrial perforations, pericardial effusions, obstructions of systemic or pulmonary veins, atrioventricular valve dysfunction, or other complications occurred. CONCLUSIONS: The new device is a promising transcatheter approach for the occlusion of secundum atrial septal defects in children. However, further evaluation and long term data are needed before this transcatheter technique can be recommended. PMID- 8624882 TI - Cardiac rehabilitation in the United Kingdom: guidelines and audit standards. National Institute for Nursing, the British Cardiac Society and the Royal College of Physicians of London. AB - This paper summarises a multidisciplinary workshop convened to prepare clinical guidelines and audit standards in cardiac rehabilitation in the United Kingdom. The workshop developed a three element model of the rehabilitation process and identified needs relating to medical and psychosocial care and the potential contributions of exercise, education, secondary prevention, and vocational advice. Draft clinical standards are proposed as a basis for locally developed guidelines and further research. PMID- 8624884 TI - Congenital absence of aortic and pulmonary valve in a fetus with severe heart failure. AB - A case of congenital absence of both aortic and pulmonary valves with severe heart failure detected prenatally by cross-sectional and pulsed and colour Doppler echocardiography is reported in small for gestational age male fetus in 17th week of gestation. Additional double outlet right ventricle, hypoplastic left ventricle, and ventricular septal defect, as well as multiple extracardiac anomalies, were found by prenatal echocardiographic investigation and confirmed by necropsy examination. Retrograde diastolic Doppler waveforms retrieved from pulmonary artery, aorta, and umbilical arteries revealed massive insufficiency throughout both the great arteries, which eliminated diastolic placental perfusion, documented by absent anterograde diastolic flow in the umbilical vein. These prenatal echocardiographic findings may contribute to an understanding of the mechanism of rapid and progressive heart failure and growth retardation in the fetus. Severe cardiac failure may explain why congenital aplasia of both the aortic and the pulmonary valves has not been described postnatally, and only two fetal cases revealed by necropsy have been published. PMID- 8624883 TI - Restrictive and hypertrophic cardiomyopathies in Noonan syndrome: the overlap syndromes. AB - A woman with Noonan syndrome had clinical and haemodynamic features of restrictive cardiomyopathy. There was no ventricular hypertrophy on echocardiography but myocardial biopsies showed myocyte hypertrophy without pathological disarray. This case illustrates the overlap of the cardiac phenotypes of Noonan syndrome, restrictive cardiomyopathy, and hypertrophic cardiomyopathy. PMID- 8624885 TI - Association of multiple human papillomavirus types with vulvar neoplasias. AB - OBJECTIVE: The purpose of this study was to clarify the association of multiple genital human papillomavirus (HPV) types with vulvar neoplasias. METHODS: We examined vulvar neoplasias by the polymerase chain reaction (PCR) method, which can detect almost all genital HPV types sensitively (L1-PCR). RESULTS: Eight HPV types (types 6, 11, 16, 18, 52, 53, 56, and 68) were detected in 52 of 53 (98.1%) vulvar condylomas; 2 HPV types (types 16 and 18) were detected in all 6 Grade-3 vulvar intraepithelial neoplasias; and 5 HPV types (types 6, 16, 18, 51, and 56) were detected in 8 of 11 (72.7%) invasive vulvar carcinomas. Detection of HPV types 52, 53, 56, and 68 in vulvar condylomas and detection of HPV types 51 and 56 in vulvar carcinomas are the first examples ever reported. CONCLUSION: This study suggests that more HPV types are associated with the generation of vulvar neoplasias than was thought to be the case up to now. PMID- 8624886 TI - Prevalence of hemoglobin S and beta-thalassemia in northern Jordan. AB - OBJECTIVE: The aim of this study was to determine the incidence of HbS and beta thalassemia in the Northern Jordan. STUDY DESIGN: The investigation targeted 3 areas of Northern Jordan. Blood samples from 2,290 volunteers were evaluated to determine the prevalence of HbS and beta-thalassemia by preparing a hemolysate for cellulose acetate electrophoresis. An additional 568 newborn samples were collected from the umbilical and analyzed for the presence of HbS using electrophoresis. RESULTS: The overall prevalence of HbS and beta-thalassemia was 4.45% and 5.93%, respectively. The incidence of sickle cell trait in the newborn sample was 3.17%. The prevalence of both HbS and beta-thalassemia was higher in the Al-Gor area in comparison to Ajloun and Irbid. CONCLUSION: A better understanding of the demographics of these diseases has the potential to aid in the more efficient utilization of health care resources and improved planning and provision of health care services. PMID- 8624887 TI - Sublingual nifedipine compared with intravenous hydrallazine in the acute treatment of severe hypertension in pregnancy: potential for use in rural practice. AB - OBJECTIVES: The purposes of this study were to compare the efficacy of sublingual nifedipine with intravenous hydrallazine in the control of acute hypertension of pregnancy and to make a preliminary assessment whether sublingual nifedipine could be recommended for use by midwives faced with severe hypertension in pregnancy in a rural setting. METHODS: Subjects were 200 consecutive patients admitted to Kuala Tereng-ganu General Hospital, Malaysia with severe hypertension in pregnancy between August 1989 and June 1990. Admission criteria were an ongoing viable pregnancy more than 28 weeks and diastolic blood pressure (DBP) more than 120 mmHg. The patients were randomly divided into 2 groups. In group I, sublingual nifedipine 5 mg was administered and repeated after 15 minutes if DBP > 120 mmHg; and in group II hydrallazine 5 mg was intravenously injected and repeated after 15 minutes if DBP > 120 mmHg. Both groups were put on hydrallazine infusion if DBP > 120 mmHg after 30 minutes. The Chi-square test was used for analysis with significance at p < 0.05. RESULTS: There was no statistical difference in the efficacy of therapy for decreasing blood pressure between the 2 groups. The groups were comparable by age, parity, gestational age at presentation, birth weight of infants, incidence of postpartum haemorrhage and fetal distress. Caesarian section rates were similar. In the observational studies on nurses administering the drugs, no significant difficulties were observed. CONCLUSION: Sublingual nifedipine was comparable to IV hydrallazine in the treatment of acute hypertension of pregnancy. Nurses were able to administer lingual nifedipine without difficulty. PMID- 8624888 TI - Mixed gonadal dysgenesis with gonadoblastoma in a female with Y chromosome: case report. AB - A young female with primary amenorrhea and male karyotype (46/XY) is reported. Physical examination and clinical investigation of the patient suggested mixed gonadal dysgenesis (MGD). After gonadectomy, a tumor was found on the left side and histologically diagnosed gonadoblastoma. Postoperative decreased level of testosterone and estradiol reflected the activities of the tumor. PMID- 8624889 TI - A case of vulvar schwannoma. AB - Schwannoma is generally a solitary, nodular, benign tumor of a peripheral nerve sheath, and it rarely affects the external female genitalia. Its most-common locations are the head, neck, upper and lower extremities, posterior mediastinum, and retroperitoneum. We treated a 29-year-old woman with a vulvar schwannoma. A tumor of 2.5 cm in diameter was resected for treatment and biopsy. It was a capsulate tumor with a smooth surface, and was elastic in consistency. A histological examination revealed a swirling pattern with some palisading of spindle-shaped cells in H.E. stains. Immunohistochemical studies showed the tumor to be positive for S-100 protein and negative for desmin. As a result, the mass proved to be schwannoma originating from the vulva. PMID- 8624890 TI - Gonadoblastoma in patient with Turner's syndrome. AB - A 15-year-old phenotypic female referred for the investigation of primary ammenorrhea, was found to have a 45,XO karyotype and an ovarian cyst. She demonstrated some of the features of Turner's syndrome, as well as virilization. On laparotomy, she was found to have bilateral gonadoblastomas. She was treated with total abdominal hysterectomy and bilateral salpingo-oophorectomy. Subsequently, repeated chromosomal analysis detected the presence of Y chromosomes, which was confirmed by the use of polymerase chain reaction (PCR). The difficulties encountered in searching for the Y chromosome in patients with gonadoblastoma are discussed. Prophylactic gonadectomy is suggested in those cases associated with atypical features. PMID- 8624891 TI - Fetal and neonatal haemodilution associated with multiple placental chorioangioma: case report. AB - A pregnancy with polyhydramnios and abnormal antepartum fetal heart rate pattern was found to have multiple placental haemangiomas. Multiple placental haemangiomas can give rise to fetal cardiac failure due to a hyperdynamic circulation or fetal anaemia either due to haemodilution or possibly destruction of blood cells in the chorioangioma. Whether fluid restriction with or without diuretics or blood transfusion is the correct form of treatment of neonatal cardiac failure in such a case is discussed. PMID- 8624892 TI - Landry-Guillain-Barre-Strohl syndrome in pregnancy. AB - We report here a case of Landry-Guillain-Barre-Strohl syndrome (LGBSs) in pregnancy. The patient had rubella viral infection, and the patient's inspiratory ability rapidly weakened. In the end, aggressive plasmapheresis prevented the need for respiratory support and the inherent risk it poses in terms of maternal morbidity and mortality. PMID- 8624893 TI - Ovarian mucinous cystadenocarcinoma with sarcoma-like mural nodules. AB - We present a rare case of a 31-year-old woman with a mucinous ovarian tumor with sarcoma-like mural nodules. The epithelial elements consisted of a mixture of benign, borderline-malignant, and mucinous adenocarcinoma; the sarcoma-like mural nodules consisted of pleomorphic and epulis-like cells. The lesion was resected for an ovarian tumor. In an immunohistochemical study, these cells stained for lysozyme and vimentin. The patient received 2 courses of cisplatin (100 mg/m2). There was no evidence of disease at a second-look laparotomy after 1 year, and none upon examination 6 years after the initial operation. The mural nodules might be of non-epithelial origin. The prognosis of patients with this rare lesion appears favorable, according to this and previous reports. PMID- 8624894 TI - Triple test for prenatal detection of Edwards syndrome (trisomy 18). AB - OBJECTIVE(S): To demonstrate the limitation of complete reliance on computer generated interpretations and to highlight the need for understanding of pregnancy-related biochemistry when offering prenatal screening. METHODS: Four cases of cytogenetically confirmed trisomy 18 pregnancies are presented. All four cases underwent prenatal screening (Triple Test-AFP, uE3, t beta-hCG) at midgestation and risk assessment by the alpha algorithm. RESULTS: All four cases of trisomy 18 were assessed as being at low risk for DS and/or open NTD. Although marker levels were not consistent with either of these clinical situations, they were indicative of a compromised pregnancy. Circulating levels of trophoblast derived antigens (uE3, t beta-hCG) were depressed (< or = 0.5 MoM) in all four cases. Further investigations (ultrasonography, amniocentesis) confirmed a trisomy 18 fetus. CONCLUSIONS: Risk assessment by computer based algorithms relies on maternal factors and specific DS/NTD marker profiles. Aberrant marker profiles are not distinguished from normal. Therefore, it is essential that prenatal screening is offered only by those competent in pregnancy biochemistry and able to identify these abnormal situations. PMID- 8624895 TI - In vitro fertilization of in vitro matured oocytes obtained from the follicles without hCG exposure for prevention of severe ovarian hyperstimulation syndrome: a case report. AB - We aspirated ovarian follicles of an infertile patient who encountered a great risk of ovarian hyperstimulation (OHSS) during her ovulation-induction cycle, which had been stimulated with gonadotropins, in order to avoid OHSS without interrupting the treatment. If this action were to yield a considerable number of immature oocytes, some of which were to achieve in vitro maturation and subsequent fertilization, this method could be an alternative to conventional ovulation-induction methods for patients who repeatedly present imminent signs of hyperstimulation. However, the number of oocytes collected from the patient was disappointingly low, even though a large number of follicles were thoroughly aspirated. One of these oocytes was matured in vitro in a medium containing 30% follicular fluid and granulosa cells of mature follicles, fertilized, cryopreserved, and transferred in a later artificial cycle; but pregnancy was not achieved. A low number of recovered oocytes from patients in this situation might be a factor that greatly limits the clinical applications of this method. PMID- 8624896 TI - Clinical utility of calculated creatinine clearance as a guide to chemotherapy in patients with gynecologic malignancies. AB - OBJECTIVE: To examine the relationship between calculated and measured creatinine clearance, and also to evaluate the clinical utility of calculated creatinine clearance before administration of chemotherapy in patients with gynecologic malignancies. METHODS: In 38 patients with gynecologic malignancies, a total-69 paired creatinine clearance measured by 24-hour urinary creatinine clearance and calculated were compared. RESULTS: The significant correlation (r = 0.597, p < 0.0001) was observed between creatinine clearances determined by both methods. There were 26 (37.7%) 24-hour urine collections. The correlation between two methods improved when these inaccurate samples were excluded (r = 0.876, p < 0.0001). In addition, if a creatinine clearance of 50 ml/min is set as a threshold for dose modification, only 2.9% of the patients would have received reduced doses inappropriately, by using the calculated creatinine clearance alone. CONCLUSION: These results suggest that in patients with gynecologic malignancies, a calculated creatinine clearance prior to chemotherapy is sufficient for evaluation of renal function and measured creatinine clearance would be determined only when calculated creatinine clearance is below the threshold for dose modification. PMID- 8624897 TI - Short protocol of gonadotropin releasing hormone agonist administration gave better results in long protocol poor-responders in IVF-ET. AB - OBJECTIVE: To determine the effectiveness of a short protocol (SP) of gonadotropin-releasing hormone agonist (GnRH-a) administration in patients who already had undergone at least one in vitro fertilization (IVF) and embryo transfer (ET) treatment cycle with the long protocol (LP) and had shown poor results. METHODS: One hundred and twelve patients were studied. The E2 levels, the number of preovulatory follicles, the number of oocytes retrieved and fertilized, the number of embryos cleaved, the fertilization rate and the number of embryos transferred were calculated. RESULTS: The values for the mentioned parameters were significantly higher in the SP than those in the LP. So were the pregnancy rates per cycle and per ET (23.2% and 24.1%, respectively) in the SP significantly (p < 0.0001) higher than those in the LP (12.5% and 13.5%, respectively). CONCLUSION: In cases who showed poor results to the LP, instead of repeating the same protocol the SP may be substituted. This strategy may not only improve the results, but also decrease the total cost of the IVF-ET treatment. PMID- 8624898 TI - A comparative study of outcome of laparoscopic salpingo-oophorectomy versus open salpingo-oophorectomy. AB - OBJECTIVE: To determine the effectiveness and appropriateness of laparoscopic salpingo-oophorectomy (LSO) versus open salpingo-oophorectomy (OSO) in developing country. METHOD: A clinical study with historical control consisted of 31 LSO cases of benign ovarian cysts were compared with 41 OSO cases matched by diagnosis and difficulty. RESULTS: The operative time of the LSO cases was higher (p < 0.001), but the morbidity was comparable (p = 0.22). The postoperative hospital stay of the LSO was shorter (p < 0.001). Patients who had LSO revealed meaningfully decreased postoperative use of analgesic (p < 0.05). The time to full recovery for LSO was shorter (p < 0.001), but the hospital charges were higher (p < 0.001). CONCLUSIONS: LSO is considerable safe and effective alternative to OSO but incurs higher hospital charges. The overall cost effectiveness of LSO, especially in the developing countries, need further evaluation. PMID- 8624899 TI - Two cases of twisted fetal ovarian cysts. AB - Two twisted fetal ovarian cysts were detected antenatally by routine ultrasonographic examination. Serial changes of twisted ovarian cysts can be monitored by ultrasonic observation. If signs of torsion appear, obstetricians should consider prompt delivery in order to preserve the patient's fertility. PMID- 8624900 TI - Ascitic positive cytology and intraperitoneal metastasis in ovarian dysgerminoma. AB - OBJECTIVES: To verify the pattern of the spread of tumors in ovarian dysgerminoma, with special reference to intraperitoneal metastasis, and to assess clinicopathologic factors for predicting tumor extension. METHODS: Detailed data regarding ascitic cytology, macroscopical findings at surgery, and the histopathology of the surgical specimens were retrospectively reviewed in 12 patients with dysgerminoma who were treated at Kyoto University Hospital. The relationships between the tumor extension and the period of symptoms, the serum lactic dehydrogenase (LDH) level, and the operative findings also were analyzed. RESULTS: Ascitic cytology revealed a high incidence of positivity in 6 of the 10 (60%) cases examined. Extraovarian metastases were present in 4 of the 6 (67%) cases with positive cytology, and in 1 of the 4 (25%) cases with negative cytology. Intraperitoneal metastatic nodules were detected in 5 of the 12 (42%) patients either by inspection during surgery or by postsurgical histological examination. In addition, these metastatic lesions were 5 or fewer in number and 7 mm or less in diameter, except in 1 patient with widespread disease. The presence or absence of extraovarian spread of the tumor was not significantly correlated with the period of symptom, the serum LDH level, the size of the primary tumor, or the volume of the ascitic fluid. CONCLUSION: The incidence of intraperitoneal spillage and/or metastases of dysgerminoma cells might be higher than previously reported. These findings indicate the importance of ascitic cytology and careful inspection at the time of operation, as well as the rationale of postsurgical chemotherapy for dysgerminoma of an apparently early stage. PMID- 8624901 TI - The imbalance of plasminogen activators and inhibitor in preeclampsia. AB - OBJECTIVE: To evaluate the role of plasminogen activators (tPA uPA) and inhibitor (PAI-1) in the pathogenesis of preeclampsia. STUDY DESIGN: tPA uPA and PAI-1 antigens were measured in amniotic fluid, maternal plasma, and placental homogenates in normal pregnancy by ELISA method and compared with that of preeclampsia. RESULTS: In normal pregnancy, tPA, uPA and PAI-1 levels increase as the gestation advance, but the increment of PAI-1 in amniotic fluid (28.3-fold) is larger than that of tPA and uPA (1.8-fold, 8.5-fold) (p < 0.001). Decidua homogenates contained larger amount of PAI-1 than villi. Whereas, villi had higher levels of tPA uPA than decidua (p < 0.01). In preeclampsia, the significantly higher levels of PAI-1 were observed in AF and decidua tissue as compared to the normals (p < 0.01), and the increment of PAI-1 in preeclampsia is larger than that of tPA, uPA. CONCLUSIONS: The elevated PAI-1 level is associated with the preeclampsia and the imbalance between the plasminogen activators (tPA, uPA) and the inhibitor (PAI-1) might be involved in the pathogenesis of preeclampsia. PMID- 8624902 TI - Use of intravenous adenosine triphosphate (ATP) to terminate supraventricular tachycardia in a pregnant woman with Wolff-Parkinson-White syndrome. PMID- 8624903 TI - Free amino acids in the hemolymph of the cockroach, Blaberus discoidalis. AB - The concentrations of free amino acids in the hemolymph of adult males of the cockroach Blaberus discoidalis were determined by automated amino acid analysis. Eighteen of the twenty standard amino acids as well as two nonstandard amino acids (hydroxyproline and ornithine) were found. Aspartic acid and cysteine were not present. Proline levels were up to 100-fold higher than those of any other amino acid. Total amino acid concentration was comparable to those reported for most other cockroaches. The reported amino acid profile formed the basis for an organ culture medium which sustained metabolic viability of isolated B. discoidalis neuroendocrine glands and brain-gland complexes for at least 24 hr. PMID- 8624904 TI - Adrenergic and cholinergic response of ventricular muscle from the turtle, Trachemys (Pseudemys) scripta. AB - Muscle strips were dissected from the ventricle of the turtle, Trachemys (Pseudemys) scripta. Individual strips were placed in a muscle bath, set at a resting tension of three grams and electrically paced at 24 min(-1) for a control period of 1 hr. Cumulative dose-response curves were constructed using the adrenergic agonist, epinephrine (Epi) and the cholinergic agonist, metacholine (MCh). The administration of Epi to the muscle bath resulted in a dose-dependent increase in active tension development. The maximum tension developed was 40% above the control values and the concentration producing a half-maximal response (EC50) was 7.5 x 10(-7) M. The positive inotropic effect of Epi was diminished following the administration 1 x 10(-7) M propranolol. This reduced the maximum active tension by over 80% at the highest dose of Epi. In addition, the EC50 was increased to 8.9 x 10(-6) M. The administration of MCh resulted in a dose dependent decrease in active tension development. The maximum decrease in active tension was 35% below the control values and the EC50 was 2.5 x 10(-6) M. The negative inotropic effect of MCh was diminished by the application of 1 x 10(-6) M atropine, with the EC50 increasing to 2.5 x 10(-4) M. These findings show that the isolated, in vitro turtle ventricle responds to adrenergic and cholinergic agonist. The results of this study support the hypothesis that the ventricular function, in reptiles, is controlled by both subdivisions of the autonomic nervous system. PMID- 8624905 TI - Species differences in hepatic bile acid uptake: comparative evaluation of taurocholate and tauroursodeoxycholate extraction in rat and rabbit. AB - Dose-response curves for taurocholate and tauroursodeoxycholate were performed in rat and rabbit livers to get more insight into species differences in the hepatic bile acid uptake. The bile acids showed saturation kinetics in both animals, the Vmax in rat being higher than in rabbit and the Km being lower in the rat than in the rabbit for both the bile acids, with no significant difference in the hepatic cells morphometric parameters. Therefore, it is possible that differences in the kinetic parameters are related to the number and, to a lesser extent, to the affinity of the transporters on the sinusoidal plasma membranes. PMID- 8624906 TI - Immune rainbow trout serum has an anti-proliferation effect on isolated mouse T cells: possible autoimmune downregulatory implications. AB - Multiple sclerosis is generally believed to involve an autoimmune syndrome in which affected individuals experience degradation of the myelin sheath in the central nervous system. In this study, rainbow trout were immunized with myelin basic protein and myelin in an attempt to induce a myelin-specific autoimmune response using the experimentally induced autoimmune response in Lewis rats as a model. Subsequent to immunization, rainbow trout antibodies to myelin basic protein were detected via an ELISA. Although clinical signs of nerve dysfunction were never apparent, disease due to demyelinization cannot be ruled out until histological studies are performed. It is shown that both rainbow trout immune and normal sera exert an inhibitory affect on the mitogenic stimulation of isolated mouse splenic T-cells. This in vitro inhibitory activity, which is clearly greater in immune serum, is hypothesized to be derived from a nonspecific antiinflammatory factor. While the identity of the factor(s) is not yet known, likely possibilities are discussed. PMID- 8624907 TI - A mammary epithelial cell line is transiently stimulated towards milk lipid synthesis by lactogenic treatments. AB - A subcloned mouse mammary epithelial cell line (COMMA-D/MME) was cultured on Engelbreth-Holm-Swarm (EHS) tumor cell matrix with lactogenic hormones (insulin, cortisol, prolactin) to stimulate differentiation and challenged with growth factors to interpret the relationship between the signals for growth and differentiation. After 21 days of pretreatment to promote differentiation, cells were capable of growth, but were always less responsive than cells that did not receive lactogenic pretreatment. Although the cells failed to express beta-casein mRNA, droplets of neutral lipids were present in the cell cytoplasm regardless of treatment. Lactogenic hormones induced the appearance of larger droplets that occurred in intracytoplasmic lumens and lipid synthesis rates were initially increased. However, the glycerol incorporation pattern of these lipids only reached 53% of the changes expected for lactating tissue. Furthermore, because secretion of the lipid was inhibited, the accumulation eventually inhibited synthetic capacity. The cellular expression of acetyl Co-A carboxylase message was increased by growth, but not by lactogenic treatments. It is concluded that the COMMA-D/MME are capable of partial and transient differentiation to synthesize milk lipids, but are inhibited by the accumulation of material due to inability to secrete. PMID- 8624908 TI - Tissue deposition rates in relation to muscle fibre and fat cell characteristics in lean female pigs (Sus scrofa) following treatment with porcine growth hormone (pGH). AB - Two experiments were conducted to examine the effect of porcine growth hormone (pGH) on performance, carcase composition, muscle and fat deposition rates, muscle fibre characteristics, and fat cell volume in pigs. In the first experiment, ten pairs of littermates were treated with vehicle (saline buffer) or 80 micrograms pGH per kg live weight per day for 42 days starting at 50 kg live weight. In the second experiment, twelve pairs of littermates were untreated or treated with 3 mg pGH per day from approximately 56 kg live weight to slaughter at approximately 103 kg live weight. All pigs were fed ad libitum. In Experiments 1 and 2, respectively, feed intake decreased (10 and 11%) and the feed/grain ratio improved (8 and 13%), while daily gain was unaltered. There was an increase in deposition rates of muscle (11 and 22%), skin (27 and 23%), and bone (15% in both), and a decrease in deposition rates of intermuscular (48 and 24%) and subcutaneous (82 and 50%) fat. This resulted in a change in carcase composition towards more muscle (5 and 9%), bone (6 and 4%), and skin (18 and 12%), and less intermuscular (30 and 16%) and subcutaneous fat (51 and 32%). The increased muscle mass was due to enhanced hypertrophy of all muscle fibre types, while pGH did not affect the frequency of the different muscle fibre types. The reduction in subcutaneous fat was reflected in a similar reduction in fat cell volume. In contrast to the majority of pigs used in pGH experiments, the genotype used in the present experiments did not respond with respect to daily gain following pGH treatment. Furthermore, the increase in muscle deposition was rather low compared to results reported in pigs of other genotypes. These data together with published data on the cross-sectional area of muscle fibres indicate that genotypes with relatively large muscle fibres are less responsive to pGH treatment than genotypes with relatively small muscle fibres. PMID- 8624909 TI - Scaling of organ subunits in adult mammals and birds: a model. AB - Members of one class of organs--including kidney and lung--consist chiefly of repeating units, or subunits, similar in size and shape. Across species, both the number and size of repeating units may increase with increasing organ size. A simple model is proposed, relating the scaling of unit-size and unit-number to that of organ volume. The model makes three structural assumptions, the crucial one, biologically speaking, being that the numerical density of repeating units scales as does organ surface-to-volume ratio. Data were collected from the literature bearing on the number, diameter, total surface area and total volume of such repeating units (i.e., alveoli, air capillaries, renal tubules and glomeruli), for avian and mammalian lung and for mammalian kidney, each as a function of organ size. These data, after log-log transformation, were submitted to standard linear least squares regression analysis. The resultant slopes for nine different regression lines are in good agreement with the model predictions. This finding suggests, surprisingly, that organ scale-up, at least for selected organs, expressed in terms of repeating units, as a function of organ volume, in mammals and birds, and conceivably in other phyla, may be based on a small number of elementary structural principles. PMID- 8624910 TI - [Phospholipase A2 in schizophrenia]. AB - The objective of the present work was to evaluate on the basis of the authors' data and data in the literature the role of phospholipase A2 in the pathogenesis of schizophrenia. The authors submit available literature pertaining to the problem as well as their own experimental findings. Based on the submitted facts, it cannot be states that phospholipase A2 is a specific marker of schizophrenia. PMID- 8624911 TI - [Educational status and sexual development in adolescent girls]. AB - By means of interviews two sexologists examined in 1986-1994 771 Czech girls aged 16-18 years during their stay in the spa of Frantiskovy Lazne. Up to the end of 1989 the authors studied the sexual development of 158 girls apprentices and 231 girl students. After the "Velvet Revolution" 159 apprentices and 223 students were examined. Comparison of these sub-groups revealed that the sexual development of students was highly significantly retaried, as compared with the apprentices; this applied to the period before and after 1989. This difference is interpreted mainly by the higher education, expressed by the sum of years of school attendance of both parents of the examined girls and a higher standard of the education process in secondary schools as compared with schools for apprentices. PMID- 8624912 TI - [Patient satisfaction with treatment at the Psychiatric Hospital in Brno]. AB - The objective of the research was to assess the satisfaction with treatment among patients of the mental sanatorium by an anonymous enquiry. In the enquiry 301 patients with different diagnoses participated, incl. some who had protective treatment. The investigation was made in open and closed wards. The majority of patients, reported a relatively high general satisfaction with treatment and the care provided by health professionals and a low grade of satisfaction as regards information on the drugs they took, on their health status and with opportunities how to spend their leisure time. PMID- 8624913 TI - [Demonology in old medical manuscripts]. AB - Demonology, i.e. the faith in the existence of mental diseases caused by demons was a medical problem from the 16th to the beginning of the 18th century. Two types of mental diseases were differentiated: those caused by natural causes and those caused by demonic obsession with attempts of clinical differentiation of the two groups. Among obsessions most frequently hallucinatory and hallucinotic mental diseases were included (in particular with demonic contents), mental disorders with a queer, unusual and antisocial behaviour, conditions of extreme unrest, vomiting of queer things, prediction of the future, "talking foreign languages" the subjects had not learned etc. The way to overcome demonological prejudices was an attempt to clear the obsessed of guilt, to prove that they are the victims of the devil whom they are unable to fight because of their impaired fantasy and because the devil is found of melancholic juice. Therefore gradually the idea was enforced that the obsessed should receive somatic treatment (in addition to magic and liturgical) which rids the obsessed of black bile. Demonology and witchcraft (which was revived during the renaissance along with demonology) are two different phenomena (witchcraft was a social phenomenon) which were frequently confounded or considered identical. This is why some mentally ill people were summoned in court and tried as sorcerers and witches. PMID- 8624914 TI - [Peripeteia in the development of new antidepressive agents in the early 1990s]. PMID- 8624915 TI - [Anorexics in the treatment of eating disorders]. PMID- 8624916 TI - [Development of electroconvulsive therapy]. PMID- 8624917 TI - [Treatment of lymphostasis of the lower limbs]. PMID- 8624918 TI - [Classification and strategy of treatment in diabetic angiopathies of the lower limbs]. PMID- 8624920 TI - [Vascular surgery aspects of the treatment of limb phlegmons in patients with drug dependence]. PMID- 8624919 TI - [Tactics of surgical treatment of post-injection lesions of blood vessels in patients with drug dependence]. PMID- 8624921 TI - [Significance of cerebral hemodynamics signs based on Doppler ultrasonography, choice of therapeutic tactics and prognostication of the course of acute disorders of cerebral circulation]. PMID- 8624922 TI - [Clinico-immunological comparisons at a distant period after craniocerebral trauma]. PMID- 8624923 TI - [Etiology and pathogenesis of abdominal aortic aneurysms]. PMID- 8624924 TI - [Treatment of atherosclerotic occlusion of blood vessels]. PMID- 8624925 TI - [Acute pelvic phlebothrombosis simulating acute appendicitis]. PMID- 8624926 TI - [Fibrolipoma of the spermatic cord simulating irreducible inguinal hernia]. PMID- 8624927 TI - [Errors in the diagnosis and treatment of melanoma of the finger in a patient with chronic leukemia]. PMID- 8624928 TI - [Difficulties in the diagnosis of skin melanoblastoma]. PMID- 8624929 TI - [Metastasis of rectal cancer into the appendix in association with acute destructive appendicitis]. PMID- 8624930 TI - [Method of reamputation of the limb at the thigh level]. PMID- 8624931 TI - [Guide of tubes in intubation]. PMID- 8624932 TI - [Suture technique]. PMID- 8624933 TI - [Method of surgical intervention in movable left kidney]. PMID- 8624934 TI - [Assembled puncture needle]. PMID- 8624935 TI - [Sparing method in the removal of a fishhook ]. PMID- 8624936 TI - [Surgical laminated hook-dilator]. PMID- 8624937 TI - [Experience in reuse of removed electric cardiac stimulator and endocardial electrodes]. PMID- 8624938 TI - [Complex approach to the treatment of suppurative lesions of the foot in patients with diabetes mellitus]. PMID- 8624939 TI - [Diagnosis and treatment of lesions of the subclavian artery]. PMID- 8624940 TI - [Use of lipochromin in the treatment of trophic ulcers and wounds not healing for protracted time]. PMID- 8624941 TI - [Double transplantation of internal thoracic arteries in severe coronary artery disease and atherosclerosis of the ascending aorta and aortic arch]. PMID- 8624942 TI - [Thromboembolism of the pulmonary artery in a newborn infant]. PMID- 8624943 TI - [A rare case of performance of simultaneous surgery in the reconstruction the aorto-iliac arterial segment in a patient with critical ischemia of the lower limbs]. PMID- 8624944 TI - [A case of crush syndrome]. PMID- 8624945 TI - [Case of successful treatment of gunshot wound of the neck with injury of the carotid artery bifurcation]. PMID- 8624946 TI - [Angiosarcoma of the cavity of the pelvis minor]. PMID- 8624947 TI - [Oleogranuloma of male genital organs]. PMID- 8624948 TI - [Absence of the appendix]. PMID- 8624949 TI - [Foreign body of the abdominal cavity]. PMID- 8624950 TI - [Long term results of the surgical treatment of patients with cancer of the stomach and duodenum at the early stage]. PMID- 8624951 TI - [Menetrier's disease]. PMID- 8624952 TI - [Dermal autograft in surgery of postoperative abdominal hernia]. PMID- 8624953 TI - [Successful treatment of melanoblastoma in a child]. PMID- 8624954 TI - [An unusual case of small-large intestine invagination simulating cancer of the sigmoid]. PMID- 8624955 TI - [Case of cervix uteri cancer metastasizing into soft tissues]. PMID- 8624956 TI - [Surgical treatment of chronic venous insufficiency of lower limbs complicated by trophic disorders]. PMID- 8624957 TI - [Criteria of choice of the method in correction of unsound valves of the deep veins in patients with varicose veins]. PMID- 8624959 TI - A theory of the visual motion coding in the primary visual cortex. AB - This paper demonstrates that much of visual motion coding in the primary visual cortex can be understood from a theory of efficient motion coding in a multiscale representation. The theory predicts that cortical cells can have a spectrum of directional indices, be tuned to different directions of motion, and have spatiotemporally separable or inseparable receptive fields (RF). The predictions also include the following correlations between motion coding and spatial, chromatic, and stereo codings: the preferred speed is greater when the cell receptive field size is larger, the color channel prefers lower speed than the luminance channel, and both the optimal speeds and the preferred directions of motion can be different for inputs from different eyes to the same neuron. These predictions agree with experimental observations. In addition, this theory makes predictions that have not been experimentally investigated systematically and provides a testing ground for an efficient multiscale coding framework. These predictions are as follows: (1) if nearby cortical cells of a given preferred orientation and scale prefer opposite directions of motion and have a quadrature RF phase relationship with each other, then they will have the same directional index, (2) a single neuron can have different optimal motion speeds for opposite motion directions of monocular stimuli, and (3) a neuron's ocular dominance may change with motion direction if the neuron prefers opposite directions for inputs from different eyes. PMID- 8624958 TI - Stable encoding of large finite-state automata in recurrent neural networks with sigmoid discriminants. AB - We propose an algorithm for encoding deterministic finite-state automata (DFAs) in second-order recurrent neural networks with sigmoidal discriminant function and we prove that the languages accepted by the constructed network and the DFA are identical. The desired finite-state network dynamics is achieved by programming a small subset of all weights. A worst case analysis reveals a relationship between the weight strength and the maximum allowed network size, which guarantees finite-state behavior of the constructed network. We illustrate the method by encoding random DFAs with 10, 100, and 1000 states. While the theory predicts that the weight strength scales with the DFA size, we find empirically the weight strength to be almost constant for all the random DFAs. These results can be explained by noting that the generated DFAs represent average cases. We empirically demonstrate the existence of extreme DFAs for which the weight strength scales with DFA size. PMID- 8624960 TI - Alignment of coexisting cortical maps in a motor control model. AB - How do multiple feature maps that coexist in the same region of cerebral cortex align with each other? We hypothesize that such alignment is governed by temporal correlations: features in one map that are temporally correlated with those in another come to occupy the same spatial locations in cortex over time. To examine the feasibility of this hypothesis and to establish some of its detailed implications, we studied a multilayered, closed-loop computational model of primary sensorimotor cortex. A simulated arm moving in three dimensions formed the external environment for the model cortical regions. Coexisting proprioceptive and motor maps formed and generally aligned in a fashion consistent with the temporal correlation hypothesis. For example, in simulated proprioceptive sensory cortex the map of elements responding strongly to stretch of a particular muscle matched the map of tension sensitivity in antagonist muscles. In simulated primary motor cortex the map of elements responding strongly to increased tension in specific muscles matched the map of output elements for the same muscles. These computational results suggest specific experimental measurements that can support or refute the temporal correlation hypothesis for map alignments. PMID- 8624961 TI - Analog versus discrete neural networks. AB - We show that neural networks with three-times continuously differentiable activation functions are capable of computing a certain family of n-bit boolean functions with two gates, whereas networks composed of binary threshold functions require at least omega(log n) gates. Thus, for a large class of activation functions, analog neural networks can be more powerful than discrete neural networks, even when computing Boolean functions. PMID- 8624962 TI - Using neural networks to model conditional multivariate densities. AB - Neural network outputs are interpreted as parameters of statistical distributions. This allows us to fit conditional distributions in which the parameters depend on the inputs to the network. We exploit this in modeling multivariate data, including the univariate case, in which there may be input dependent (e.g., time-dependent) correlations between output components. This provides a novel way of modeling conditional correlation that extends existing techniques for determining input-dependent (local) error bars. PMID- 8624963 TI - Engineering multiversion neural-net systems. AB - In this paper we address the problem of constructing reliable neural-net implementations, given the assumption that any particular implementation will not be totally correct. The approach taken in this paper is to organize the inevitable errors so as to minimize their impact in the context of a multiversion system, i.e., the system functionality is reproduced in multiple versions, which together will constitute the neural-net system. The unique characteristics of neural computing are exploited in order to engineer reliable systems in the form of diverse, multiversion systems that are used together with a "decision strategy" (such as majority vote). Theoretical notions of "methodological diversity" contributing to the improvement of system performance are implemented and tested. An important aspect of the engineering of an optimal system is to overproduce the components and then choose an optimal subset. Three general techniques for choosing final system components are implemented and evaluated. Several different approaches to the effective engineering of complex multiversion systems designs are realized and evaluated to determine overall reliability as well as reliability of the overall system in comparison to the lesser reliability of component substructures. PMID- 8624964 TI - Outcome measurement in cardiac and pulmonary rehabilitation. AACVPR Outcomes Committee. American Association of Cardiovascular and Pulmonary Rehabilitation. PMID- 8624965 TI - National pulmonary rehabilitation survey. Update. PMID- 8624966 TI - Inpatient cardiac rehabilitation. Patient education implementation and documentation. PMID- 8624967 TI - Fighting fear of carbohydrates. PMID- 8624968 TI - Adherence in cardiac and pulmonary rehabilitation. PMID- 8624969 TI - The effect of moderate exercise training on oxygen uptake post-aortic/mitral valve surgery. AB - BACKGROUND: This study determined the response of aortic and/or mitral valve replacement/reconstruction (AVR/MVR) surgery patients to a 3-month exercise rehabilitation program (ERP) of moderate intensity, frequency, and duration that commenced approximately 9 weeks post-operatively. METHODS: Based on geographic proximity and availability of transportation to attend ERP classes, 29 experimental subjects were enrolled in the ERP and 20 control subjects received standard care that did not include the ERP, but did not prohibit activity/exercise. Exercise tolerance was determined from estimated oxygen uptake (VO2) during exercise tolerance testing (GXT) before and after standard care or the ERP. RESULTS: VO2 at the maximum stage of the GXT increased significantly (P < or = 05) for the experimental (4.89 +/- 5.07 mL/kg/min) and control (5.11 +/- 4.48 mL/kg/min) groups. No significant between-group differences were noted in VO2 at the maximum stage of the exercise testing or at the target heart rate (HR). Furthermore, reported exercise levels of subjects in both groups were comparable and sufficient for training effects to occur. CONCLUSION: Alternate strategies to improve exercise tolerance such as home-based rehabilitation programs should be investigated for relatively healthy aortic and/or mitral valve surgical patients. PMID- 8624970 TI - Association of physical fitness and transient myocardial ischemia in patients with coronary artery disease. AB - PURPOSE: To examine the relationship between physical fitness and transient myocardial ischemia (TMI) in the laboratory and during daily life, in a sample of coronary patients with a recent positive exercise test. METHODS: 47 patients with coronary disease (CAD) participated in laboratory mental and exercise stress testing and 48-hour outpatient Holter monitoring after being withdrawn from ant ischemic medications. During laboratory testing, left ventricular performance was assessed by radionuclide ventriculography. Physical fitness was assessed by measurement of oxygen consumption during symptom-limited exercise treadmill testing. RESULTS: Higher physical fitness subjects were less likely to exhibit wall-motion abnormalities in response to mental stress (P < .05), and to exercise stress (P < .05) testing. They also had smaller decreases in left ventricular ejection fraction (LVEF) in response to mental stress than less fit subjects (P .056), and exhibited a mean increase in LVEF during exercise, compared to the less fit subjects who exhibited a decrease in LVEF (P < .02). Moreover, higher fit subjects exhibited fewer episodes of TMI (P < .04), for a shorter duration (P < .04), and less total ischemic burden (P < .04) during Holter monitoring. CONCLUSION: These findings suggest that within a population of CAD patients, higher levels of physical fitness are associated with less TMI assessed in the laboratory and during routine activities of daily living. PMID- 8624971 TI - Prevalence and effects of nonpharmacologic treatment of "isolated" low-HDL cholesterol in patients with coronary artery disease. AB - PURPOSE: To determine the the prevalence of isolated low high-density lipoprotein cholesterol and its response to hygienic therapy in coronary patients after major cardiac events. METHODS: Data before and after phase II cardiac rehabilitation and exercise training were assessed in 275 consecutive patients from two institutions. All patients had known coronary artery disease and underwent a 12 week program of aggressive hygienic therapy. The prevalence of isolated low high density lipoprotein cholesterol in this cohort was determined and its response to vigorous nonpharmacologic therapy was assessed and compared with that of other coronary patients undergoing similar therapy. RESULTS: At baseline, 113 (41%) had low high-density lipoprotein cholesterol, including 70 (25%) with isolated low high-density lipoprotein cholesterol. In contrast, 147 (53%) had "high-risk" low density lipoprotein-cholesterol > or = 130 mg/dL. In the subgroup with isolated low high-density lipoprotein cholesterol, improvements occurred in body mass index, metabolic equivalents, high-density lipoprotein cholesterol, and low density lipoprotein/high-density lipoprotein cholesterol. Compared with 205 patients without isolated low high-density lipoprotein cholesterol, this subgroup had much greater improvements in high-density lipoprotein cholesterol (+17% versus +2%; P < .001) and low-density lipoprotein/high-density lipoprotein cholesterol (-11% versus -6%; P < .02), with similar improvements in most other major coronary artery disease risk factors. CONCLUSION: These results suggest that isolated low high-density lipoprotein cholesterol is prevalent in patients with coronary artery disease and is sensitive to aggressive hygienic interventions. PMID- 8624973 TI - Trends in the utilization of noninvasive cardiac diagnostic tests in Ontario from fiscal year 1989/90 to 1992/93. AB - OBJECTIVE: To describe pattern of utilization of noninvasive cardiac diagnostic test in Ontario. DESIGN: Retrospective analysis using Ontario Health Insurance Plan (OHIP) administrative data. SETTING: Ambulatory care settings in Ontario. MAIN OUTCOME MEASURES: First, the volume of services and expenditures on electrocardiograms (ECG), ambulatory ECG, radionuclide angiocardiograms (RNA), echocardiograms, exercise stress tests (EST), and myocardial perfusion scintigrams from 1989/90 to 1992/93; second, the number and specialty of physicians performing these tests. MAIN RESULTS: Ontario spent $119 million on noninvasive diagnostic cardiology test in 1992/93, representing 2.67% of total OHIP expenditures. Expenditures on these procedures grew by 49.3 % over the four year period, exceeding the overall OHIP growth rate, and was most rapid for nuclear cardiology and echocardiography. Changing demographics accounted fpr only a minor portion of expenditure growth. Second, age-adjusted utilization rates for EST, myocardial perfusion scintigraphy and RNA were higher for men, but sex differences tended to diminish over time. Third, utilization rates differed markedly by geographic region, and variations were greatest for nuclear medicine studies. Geographic variations tended to be attenuated over time. There was also wide variation in the frequency with which physicians performed Doppler studies with two-dimensional echocardiography. CONCLUSIONS: The use of noninvasive cardiac diagnostic test has grown rapidly in recent years. This growth may have been influenced by practice guidelines, by greater diffusion of, and access to, newer technology and by more testing in women. Wide regional variations suggest that clearer practice guidelines are needed concerning the appropriate use of noninvasive cardiac diagnostic investigations. PMID- 8624972 TI - Cell biology of valvular interstitial cells. AB - OBJECTIVE: To review the cell biology of the interstitial cells of heart valves. While studying the function of interstitial cells of heart valves, the authors observed that interstitial cells from porcine mitral valve have mixed phenotypic expression. DATA SOURCE: English-language literature from 1985-95 that discussed the cell biology of heart valve interstitial cells, including their structural and functional characteristics. RESULTS: Morphologically, heart valve interstitial cells have characteristics of both fibroblasts and smooth muscle cells. It is not clear, however, whether these cells represent a single (myofibroblast) cell type or whether there are two populations of cells within the valve. This review summarizes reports of the structural and functional characteristics of heart valve interstitial cells including ultrastructure, cell junctions, smooth muscle cell markers, cell growth, role in wound repair and contractility. CONCLUSIONS: Vascular interstitial cells play an important role in both normal and diseased valves. Our understanding of the heterogeneity of valvular interstitial cells is incomplete; however, this heterogeneity allows for multiple functions to be carried out including cell-cell communication, matrix secretion, wound repair and contractility. Future studies are needed to focus on the regulation of cellular heterogeneity as it relates to valve structure and function in health and disease. PMID- 8624974 TI - Trends and variations in the use of vascular surgery in Ontario. AB - OBJECTIVE: To measure changes over time in overall surgical rates and geographic rate variations for three major vascular procedures (abdominal aortic aneurysm repairs, peripheral vascular disease procedures and carotid endarterectomies). BACKGROUND: There is little research literature on population-based usage profiles of vascular procedures. The three procedures profiled were all subject to marked shifts in evidence or surgical opinions, raising the issue of the interplay between temporal trends and geographic variations in their use. METHODS: Based on Ontario's hospital discharge abstracts and census data, population-based usage rates were calculated by site of patient residence from 1981 to 1991. Extent of rate variation was summarized with the coefficient of variation, systematic component of variation and the adjusted-likelihood ratio chi2. Spearman rank correlations were also calculated to assess stability of county rankings for each procedure. RESULTS: The overall rates of peripheral vascular procedures and repair for abdominal aortic aneurysms fell 24% and increased 42% respectively. The overall rate of carotid endarterectomies dropped from 46/100,000 in 1981 to 20/100,000 in 1989, but by 1991 had increased to 37/100,000. Through the decade measures of variation fell minimally for all three procedures. CONCLUSION: Overall use of vascular procedures shifted in apparent response to new research evidence and technologies. Despite marked changes in surgical rates, the extent of geographic variation was stable, suggesting that differing factors influence overall surgical rates and geographic rate variations. Audit at the local level using primary clinical data is needed to understand why disparities in use persist. PMID- 8624975 TI - Left atrial enlargement in healthy obese: prevalence and relation to left ventricular mass and diastolic function. AB - BACKGROUND: Left atrial (LA) enlargement has been reported in the obese. However, its prevalence in the healthy obese, clinical correlates and relation to left ventricular (LV) mass and diastolic function have been little investigated. METHODS: Thirty-five consecutive, healthy, normotensive obese (body mass index greater than 28, mean +/- SD 34.2 +/- 2.3 kg/m2) and 35 nonobese subjects (body mass index 24.6 +/- 2.3 kg/m2) comparable in age and sex underwent echocardiographic measurements of LA posteroanterior (parasternal view), mediolateral and superoinferior (apical views) dimensions, aortic root diameter, LV mass and Doppler assessment of LV diastolic function. LA enlargement was defined as a posteroanterior dimension greater than 40 mm. A ratio of LA posteroanterior dimension to aortic root diameter greater than 1.4 was used as an index for disproportionate LA enlargement. RESULTS: LA enlargement was more frequent in the obese than in the nonobese (37% versus 6%, P<0.0001). Similarity, disproportionate LA enlargement was more frequent in the obese (34% versus 6%, P<0.0001). LA posteroanterior dimension correlated well with body mass index (r=0.52, P<0.0001) and LV mass (r=0.56, P<0.0001), and weakly with blood pressure (r=0.28, P<0.02). There was no significant correlation with LV diastolic function, age or sex. In multivariate analysis (multiple r=0.61, P<0.0001), LA posteroanterior dimension correlated significantly only with mass (P<0.005), and the association with body mass index and blood pressure became nonsignificant. Similar results were obtained when LA posteroanterior dimension was replaced with mediolateral or superoinferior dimensions. CONCLUSIONS: LA enlargement is frequent in the normotensive, otherwise healthy obese and correlates well with LV mass. It is not mediated through impairment of LV diastolic function, and likely reflects a physiological adaptation of the heart to the obese state. Further studies are needed to determine whether LA enlargement in the obese is associated with adverse long term outcome. PMID- 8624976 TI - Serum insulin, IGF-I,IGF-II and growth hormone, and left ventricular mass in noninsulin-dependent mellitus. AB - OBJECTIVE: To investigate whether serum insulin, insulin-like-growth factor I (IGF-I),insulin-like-growth factor II (IGF-II) and growth hormone (GH)--each of which is a known trophic factor in vitro for the cardiomyocyte, and levels of which can be altered in noninsulin-dependent diabetes mellitus (NIDDM)--related of left ventricular (LV) mass in patients with NIDDM. DESIGN: Observational study. SETTING: University teaching hospital. PATIENTS AND METHODS: Patients with NIDDM without signs, symptoms or past history of hypertension, ischemic or valvular heart disease, or heart failure were recruited from the diabetes clinic of an university hospital. Fasting patients had blood drawn for determination of serum insulin, IGF-I, IGF-II and GH by radioimmunoassay. Height, weight, and blood pressure were recorded. An electrocardiogram was obtained and echocardiography were performed for calculation of LV mass. RESULTS: Patients' average age was 54.7 +/-1.6 years, and duration of NIDDM was 9.3 +/- 1.2 years. LV mass was 113.7 +/- 8.9 g/m2 in men (n=13) and 104.1 +/- 10.8 g/m2 in women (n=10). Serum insulin was 25.3 +/- ng/mL, IGF-I was 255 +/- 15 ng/mL, IGF-II was 0.62 +/- 0.05 microg/mL and GH was 5.4 +/- 0.5 ng/mL. There were no significant differences in LV mass among patients with serum insulin, IGF-I, IGF-II or GH in the upper compared with the lower 50th percentile. There were no significant differences in serum insulin, IGF-I, IGF-II or GH among patients in the upper compared with the lower 50th percentile for LV mass. Standardization of LV mass for body size, either by height or body surface area, did not alter the relationships. The correlation coefficients from linear least squares regression analysis between these hormones and LV mass were low (r<0.003), suggesting that even much larger sample sizes might not alter these findings. CONCLUSIONS: These data suggest that circulating total serum concentrations of growth factor (insulin, IGF-I, IGF-II and GH) are not determinants of LV mass in nonhypertensive patients with NIDDM. PMID- 8624977 TI - Sociodemographic variation in the prevalence of cardiovascular disease. AB - OBJECTIVE: To estimate the prevalence and social correlates of cardiovascular disease. DESIGN: Population-based cross-sectional survey. Survey data were obtained through a standardized home interview and a clinic visit by trained nurses. The question sequence of the London School of Hygiene (the Rose Questionnaire) was used to identify the presence of definite angina, possible infarction, definite infarction, intermittent claudication and stroke. SETTING: Eight urban communities and rural areas in Saskatchewan in 1990. PARTICIPANTS: A probability sample of 2167 noninstitutionalized men and women aged 18 to 74 years who participated in the Saskatchewan Heart Health Survey. MAIN OUTCOME MEASURES: Prevalence of cardiovascular diseases. RESULTS: Among men, the prevalence of definite angina increased gradually with age from 1.7% (95% CI 0.6% to 2.7%) in the 18 to 34 year group, 3.8% (1.3% to 6.0%) in the 35 to 54 year group to 4.8% (2.8% to 8.3%) in the 55 to 74 year group, while the prevalence among women ranged from 2.5% (1.2 to 3.7%), 4.0% (1.6% to 6.5%) to 7.1% (5.1% to 11.6%) in these same age groups. The prevalence of possible angina, definite infarction, possible infarction and intermittent claudication increased with age as well, being higher in men than in women. Generally, the conditions were more prevalent among those with less education, lower income and those who were unemployed. CONCLUSIONS: These findings indicate that there is sociodemographic inequality in the prevalence of these manifestations of cardiovascular disease, and there is a need for in-depth qualitative research into causal factors in this relationship and for targeted programs of health promotion. PMID- 8624979 TI - Traumatic tricuspid insufficiency: a case followed for 32 years, with a note on early sources. AB - A healthy young man suffered nonpenetrating chest trauma in an automobile accident in August 1962, sustaining tricuspid valve disruption and insufficiency, a rare event. Clinical diagnosis was confirmed by cardiac catherization, but valve replacement did not take place for 10 years. Since initial valve replacement he has had two further operations to deal with valve malfunction resulting in recurrent tricuspid stenosis. He had been followed, in various hospitals, for more than 32 years. Traumatic tricuspid insufficiency has become more common with the rise of car accidents and steering wheel trauma. It is characterized by the subtlety of presentation in its mainly young male victims, although it may be suspected from simple bedside examination, and by the frequently long delay between injury and overt clinical problem. The unusual history of the earliest clinical descriptions is reviewed. PMID- 8624978 TI - Serial changes of left atrial thrombus in mitral stenosis: transesophageal echocardiographic studies in candidates for balloon mitral commissurotomy. AB - BACKGROUND: Little is known concerning therapeutic modification of left atrial thrombus in mitral stenosis and its formation after successful balloon valvuloplasty (percutaneous transvenous mitral commissurotomy [PTMC]). OBJECTIVE: To test the roles of the clinical use of anticoagulant therapy and PTMC, serial changes in left atrial thrombus were examined by transesophageal echocardiography in 80 patients with mitral stenosis. RESULTS: No difference in embolic history, New York Heart Association functional class, cardiac rhythm, left atrial size or mitral valve area was found between patients with (n=23) and without (n=57) left atrial thrombus. Diuretic use was the prevalent factor for left atrial thrombi, which were dissolved completely in seven (30.4%) and partially in eight (34.7%) of the patients by adequate warfarin. However, after PTMC (n=53), newly formed thrombus was observed in four of 51 (7.8%) patients, which correlated with left atrial size (at least 50mm), atrial fibrillation and poorly controlled warfarin (thrombo test: 63.8 +/- 18.8% versus 35.9 +/- 17.7%, P<0.005). The newly formed thrombi were dissolved within several weeks. CONCLUSION: Left atrial thrombus in symptomatic mitral stenosis exhibits elusive characteristics and should be strictly monitored even after successful PTMC by transesophageal echocardiography in order to achieve adequate anticoagulation. PMID- 8624980 TI - Cardiac compression and decompensation due to hiatus hernia. AB - A seventy-seven year old man with long standing hiatus hernia, coronary artery disease and pulmonary fibrosis developed left atrial compression from incarceration of a paraesophageal hernia. PMID- 8624981 TI - Congenital complete atrioventricular block: asymptomatic at 38 years. AB - Congenital complete atrioventricular block is quite rare, with usually a benign clinical evolution. However, because the first manifestation can be sudden death, it is important to evaluate patients and to establish clear prognostic criteria. A case of complete congenital atrioventricular block in a 38-year-old woman is presented to illustrate these criteria. PMID- 8624982 TI - Transvenous closure of patent ductus arteriosus in a sick 2780g infant. AB - A three-and-a-half-month-old 2780 g critically ill infant had successful transvenous coil occlusion of a ductus arteriosus. At six-and-a-half months of age the infant died, and autopsy showed coverage of the coil on the aortic aspect and no lumenal narrowing. It was concluded that transvenous coil occlusion of the patent ductus arteriosus in small infants is possible and that th venous route is preferred to be arterial route. PMID- 8624983 TI - An intracardiac mass presenting as atrial fibrillation in a patient with testicular carcinoma. AB - A 20-year-old male with known testicular carcinoma presented with atrial fibrillation. Investigations, which revealed a pulmonary metastasis with invasion of the left atrium, are presented along with a review of the current literature. PMID- 8624984 TI - Quality provisional restorations: a must for successful restorative dentistry. PMID- 8624986 TI - Sedation for outpatient dental procedures. AB - Pain and anxiety in the dental setting prevent many patients from seeking needed treatment. As a result, various techniques of anesthesia or sedation have been developed over the last 150 years to overcome this problem. Both the historic evolution of sedation and the use of several currently popular techniques are described in this article. Also discussed is the balance between individual patient responses to drugs, dosages, and rate of administration. Currently used definitions of pharmacologic depression of consciousness are detailed, as are contemporaneous training requirements. Finally, unexpected, rare, and catastrophic events that can occur with sedation are briefly discussed. PMID- 8624985 TI - Treating periodontal diseases with tetracycline-impregnated fibers: data and controversies. AB - This article addresses the use of tetracycline-impregnated fibers in the treatment of periodontal diseases. Clinical and microbiologic data are reviewed to provide pragmatic guidelines for fiber use. In addition, controversial issues associated with the labeling of Actisite, the potential of developing antibiotic resistant strains, and the benefits of systemic vs local drug delivery are discussed. Ultimately, the article concludes that tetracycline-impregnated fibers can be used as an adjunct to scaling and root planing in certain patients at sites that do not respond to conventional therapy. PMID- 8624987 TI - Issues in oral health policy--managed care. PMID- 8624988 TI - Bibliographic retrieval in dentistry--MEDLINE and the Dentists' Silverplatter: Part 2. PMID- 8624989 TI - Odontogenic tumors that mimic a dentigerous cyst. AB - Hyperplastic follicular tissue and a dentigerous cyst are the two most common entities associated with the crown of an impacted tooth. However, other diagnostic possibilities, including odontogenic tumors should also be considered in the differential diagnosis of a pericoronal radiolucency. This article presents three cases of radiolucent lesions that were histologically diagnosed as an odontogenic tumor, and stresses the importance of histological evaluation of all pericoronal radiolucencies. PMID- 8624990 TI - The ERA implant-supported overdenture. AB - Extracoronal Resilient Attachment (ERA) implant-supported overdentures, Class I, Division 1 or Division 3 prostheses, provide an exceptionally stable and retentive prosthetic design for edentulous patients with osseointegrated implants. The ERA System uses either an individual female implant abutment or plastic female patterns that can be incorporated into a bar. The male portion of the attachment is a nylon piece of varying retentive quality. The attachment is resilient, stable, and can be easily serviced. PMID- 8624991 TI - Ectodermal dysplasia: literature review and a case report. AB - Ectodermal dysplasia is a group of rare inherited disorders that affect various tissues of ectodermal origin. The mode of inheritance varies among the different disorders. The most common form of ectodermal dysplasia is hypohidrotic ectodermal dysplasia (HED), which most severely affects the hair, nails, teeth, and skin. Men are more often and more severely affected than women. However, transmission is from the female carrier, who usually appears normal and unaffected. Dental treatment for these patients varies on an individual basis. This article illustrates a case of overdentures in a 7-year-old boy with HED. PMID- 8624992 TI - Unemployment: cause or effect? PMID- 8624993 TI - Project on role of allied health care professions needs online input. PMID- 8624994 TI - Regulated analgesics and pain control. PMID- 8624995 TI - Regulated analgesics and pain control. PMID- 8624996 TI - Regulated analgesics and pain control. PMID- 8624997 TI - Regulated analgesics and pain control. PMID- 8624998 TI - The effect of health care reform on academic medicine in Canada. Editorial Committee of the Canadian Institute for Academic Medicine. AB - Although Canadian health care reform has constrained costs and improved efficiency, it has had a profound and mixed effect on Canadian academic medicine. Teaching hospitals have been reduced in number and size, and in patient programs have shifted to ambulatory and community settings. Specialized care programs are now multi-institutional and multidisciplinary. Furthermore, the influence of regional planning bodies has grown markedly. Although these changes have likely improved clinical service, their impact on the quality of clinical education is uncertain. Within the academic clinical department, recruitment of young faculty has been greatly complicated by constraints on licensing, billing numbers, fee for-service income and research funding. The departmental practice plan based on university funds and fee-for-service income is being replaced by less favourable funding arrangements. However, emphasis on multidisciplinary programs has rendered these departments more flexible in structure. The future of Canadian academic medicine depends on an effective alliance with government. Academia and government must agree, particularly on human-resource requirements, research objectives and the delivery of clinical and academic programs in regional and community settings. The establishment of focal points for academic health sciences planning within academic health sciences centres and within governments would assist in these developments. Finally, government and the academic health sciences sector must work together to remove the current impediments to the recruitment of highly qualified young faculty. PMID- 8625001 TI - Preventive therapies: weighing the pros and cons. AB - The author comments on three issues raised by Dr. Kenneth G. Marshall in his series on the benefits and harms of preventive therapies, which begins in this issue on page 1493. First, because the method by which the results of clinical trials are presented markedly affects the perception of those results some measure of absolute benefit and harm must be used when the results of clinical trials are presented. Second, there is increasing interest in decision aids as a means of helping patients to understand evidence and make therapeutic choices. It is important that these aids undergo rigorous testing before they are adopted for common use. Third, evidence-based clinical practice guidelines are a welcome resource for busy clinicians. However, physicians and patients should bear in mind that interpretations of the available evidence can vary, leading to different conclusions about the appropriateness of preventive therapies. PMID- 8624999 TI - Prevention. How much harm? How much benefit? 1. Influence of reporting methods on perception of benefits. AB - Before a physician or a patient can decide whether a preventive program is worth while, each must understand the nature and degree of its benefits and the frequency and magnitude of its adverse effects. Preventive interventions can be divided into two major categories: those with infrequent or minor adverse effects and those with adverse effects that are frequent or serious. Accident prevention, avoidance of high-risk behaviour and healthy lifestyle choices such as breast feeding and moderate exercise are associated with few adverse consequences. By contrast, screening populations for disease, risk classification for the purpose of selective preventive interventions, dietary intervention and prophylactic drug treatment may lead to more frequent and serious adverse effects. When assessing whether the benefits of a preventive intervention outweigh the harm, one must be aware that the methods used to report benefits of clinical trials may distort the reader's perception of their magnitude. The relative reduction of morbidity or mortality rate often grossly exaggerates benefits and should never be used as a basis for clinical decision making. More realistic ways of recording benefits are the absolute reduction of morbidity or mortality rate, the number of patients that need to be treated to avoid one adverse event, and the total cohort mortality rate. PMID- 8625000 TI - Cost of allogeneic and autologous blood transfusion in Canada. Canadian Cost of Transfusion Study Group. AB - OBJECTIVE: To determine the cost, from a societal perspective, of blood transfusion in Canada. STUDY DESIGN: Cost-structure analysis. SETTING: Data were collected from eight hospitals and from six blood centres operated by the Canadian Red Cross Society in four provinces. OUTCOME MEASURES: Costs associated with four stages of transfusion-- collection, production, distribution and delivery--in 1933 were assessed. Costs were divided into the following categories; personnel, purchases, external services, overhead, donors' time, patients' time (for autologous transfusion), wastage and infection. RESULTS: The mean overall cost of a transfusion performed on an inpatient basis was $210 per unit of red blood cells for an allogeneic transfusion and $338 per unit of blood for an autologous transfusion. The mean cost of an allogeneic transfusion performed on an outpatient basis was $280 per unit of red blood cells. CONCLUSION: The costs determined in this study can be used in future studies comparing the cost-effectiveness of allogeneic transfusion with that of alternative methods. PMID- 8625002 TI - Signposts to therapy: recent advances in inflammatory bowel disease research. AB - Research on ulcerative colitis and Crohn disease, the two conditions grouped under the heading of inflammatory bowel disease (IBD), is improving our understanding of how the intestine functions at the cellular level in health and disease. Researchers in the McGill Inflammatory Bowel Disease Research Program are studying the factors that affect the transport of nutrients, salt and water across cell membranes in the intestinal epithelium and investigating the cellular mechanisms of diarrhea. Their main interest is in how the intestine adapts in response to inflammation. Their findings promise to yield new targets for the pharmacologic and dietary management of IBD. PMID- 8625003 TI - Anaphylaxis: statement on initial management in nonhospital settings. Laboratory Centre for Disease Control. PMID- 8625004 TI - Dr. Gachet. PMID- 8625005 TI - Plans to run private facility in public hospital draw some strong opposition in Alberta. AB - Proposals to create private wings catering mainly to American patients at some public hospitals in Alberta have attracted strong opposition. Physicians at the Leduc General Hospital near Edmonton voted unanimously against a proposal to create a private wing at their hospital. Proponents say such moves would simply provide additional funding for Canada's cash-strapped health care system. PMID- 8625006 TI - Twelve weeks of coronary artery disease. AB - Osler said the components of the student spirit are humility, confidence, pride and hope. In this retrospective look at 12 weeks spent on a cardiology ward, medical student Paul Fedak recounts his experiences concerning a patient diagnosed with terminal coronary artery disease. PMID- 8625007 TI - Fighting the odds in the UK. AB - Caroline Richmond reports on miscellaneous winners and losers from the health care scene in the United Kingdom. The winners include a young patient who is holding her own against formidable medical odds after receiving heroic treatment for leukemia, and the country's osteopaths, who have won the right to compile a statutory register. The losers are the venerable St. Bartholomew's Hospital, which appears to have lost its battle to stay open, and a 32-year-old man who almost made it to medical school by posing as a teenager. PMID- 8625008 TI - New college data may shed light on issue of sexual abuse by physicians. AB - During a recent conference on physician health that was cosponsored by the CMA and American Medical Association, physicians learned that there had been an increase in the number of reported cases of abuse of patients by physicians in Ontario. The increase occurred after new legislation made the reporting of suspected sexual abuse mandatory in the province in 1994. Dr. Laurel Dempsey said there was "a considerable body of opinion" at the College of Physicians and Surgeons of Ontario that there would be a backlog of cases waiting to be reported once the new law took effect. However, it won't be possible to tell if this backlog actually exists until data have been gathered for at least a few more years. PMID- 8625009 TI - FPs can play important role in treatment of infertile patients, counsellors say. AB - Family physicians can do a great deal more to help patients face infertility assessment and treatment, but they must first recognize how their patients may be emotionally and physically affected by the diagnosis, a therapist told a recent workshop in Toronto. Strategies used by medical counsellors can be adapted by FPs and specialists to help patients address infertility issues. PMID- 8625010 TI - Study of new mothers looks at language and cultural barriers facing immigrant women. PMID- 8625011 TI - Catch-up immunization programs will eliminate measles threat for most schoolchildren. AB - The threat of measles will be eliminated for 72% of Canadian schoolchildren this spring as seven provinces/territories establish routine two-dose schedules for measles-mumps-rubella vaccination, including five jurisdictions that are undertaking universal catch-up immunization programs. The initiatives move the country closer to its commitment to eradicate measles by the year 2000. PMID- 8625012 TI - Breast-feeding still faces many roadblocks, national survey finds. AB - Although breast-feeding receives strong support from physicians, recent focus groups conducted for Health Canada found that it still faces roadblocks because some new mothers find it too embarrassing. In some cases, their male partners oppose breast-feeding. The solution appears to be more and better education provided very early in pregnancy. There is also a need to "spell out explicitly" the role male partners can play in supporting breast-feeding. PMID- 8625013 TI - Canadian MDs gone south dispute view of US medicine. PMID- 8625014 TI - Canadian MDs gone south dispute view of US medicine. PMID- 8625015 TI - How important is maturity in medical school entry? PMID- 8625016 TI - How important is maturity in medical school entry? PMID- 8625017 TI - Modifying prescribing of regulated analgesics. PMID- 8625018 TI - Modifying prescribing of regulated analgesics. PMID- 8625019 TI - Modifying prescribing of regulated analgesics. PMID- 8625020 TI - New guidelines on fluoride supplementation for children. PMID- 8625021 TI - Strategies for improving prescribing practice. AB - Drug therapy is an integral component of modern medical care, and practising physicians are faced with the difficult task of keeping up with rapid changes in pharmacologic treatments. Recent evidence indicates that prescribing practice is often inconsistent with criteria for safety and effectiveness. Surveys indicate that community-based physicians are not satisfied with current sources of information on prescription drugs. The dissemination of printed material alone does not lead to improved prescribing practice, but specific education and feedback strategies can. To improve prescribing practice in Canada we need to systematically evaluate strategies to change prescribing behaviour, to design quality assurance programs based on proven strategies and to develop collaboration and cooperation among providers, manufacturers, governments and the public. PMID- 8625022 TI - A curriculum on physician-patient sexual misconduct and teacher-learner mistreatment. Part 2: Teaching method. AB - Medical educators have become increasingly aware of the need for health care professionals to receive more training about the causes and consequences of physician-patient sexual misconduct and teacher-learner mistreatment and harassment. A curriculum in use at the University of Toronto includes a didactic component, consisting of lectures, and an experiential component, consisting of a workshop. This article concerns how, by discussing case vignettes designed to illustrate salient points, the participants have an opportunity to consider their responses in actual clinical and teaching situations. Evaluation of the course by 373 participants shows that the curriculum is considered acceptable and is likely to be of benefit. Of the course participants, 54% (15/28) of those attending the course for faculty and 39% (133/345) of those at subsequent courses stated that they would change their clinical and teaching practices in positive ways as a result of attending. A further 38% (130/345) stated that they already practised in a manner congruent with the model discussed. PMID- 8625023 TI - Prevalence of HIV infection among pregnant women in Newfoundland. AB - OBJECTIVE: To determine the prevalence of HIV infection among pregnant women in Newfoundland. DESIGN: Anonymous unlinked seroprevalence study. SETTING: Newfoundland. PATIENTS: A total of 14911 women receiving prenatal care or undergoing an abortion, representing nearly all pregnancies in Newfoundland from Nov. 1, 1991, to Oct. 31, 1993. OUTCOME MEASURES: HIV antibody status, as determined by enzyme immunoassay of leftover serum samples (initially obtained for routine screening) and confirmation of reactive samples by the Western blot technique, health region of residence, and age group. RESULTS: Of the 14911 serum samples 13 were positive for HIV, for an overall crude prevalence rate of 1 per 1147 or 8.7 per 10000 pregnant women (95% confidence interval [CI] 4.7 to 14.9). Seven of the positive samples were from women residing in the Eastern Health Region of the province, for a crude prevalence rate of 1 per 376 or 26.6 per 10000 pregnant women (95% CI 10.7 to 54.8) for that region. All women found to be HIV positive were 15 to 29 years of age, the peak prevalence (20.8 per 10000 pregnant women [95% CI 9.5 to 39.4]) was observed among those 20 to 24 years. CONCLUSIONS: The overall prevalence rate of 8.7 per 10 000 pregnant women in Newfoundland is the highest provincial rate recorded among those from similar studies in Canada. Although it may be concluded that there are an estimated 125 HIV-positive women of childbearing age in Newfoundland (95% CI 67 to 213), the age-adjusted estimate is 84 (95% CI 36 to 131). This study provides an independent confirmation of an outbreak of HIV infection among women in the Eastern Health Region of the province. PMID- 8625024 TI - Career changes among Saskatchewan physicians. AB - OBJECTIVE: To determine how often Saskatchewan physicians changed career paths during medical training and practice. DESIGN: Population survey (mailed questionnaire). SETTING: Saskatchewan. PARTICIPANTS: All 1077 active members of the Saskatchewan Medical Association were sent a questionnaire; 493 (45.8%) responded. OUTCOME MEASURES: Long-term career goal or plan in next-to-last year of undergraduate medical school, probable choice of career if forced to choose at that time, and number of physicians who changed their field of training or practice at any time since graduation. RESULTS: In all, 57.8% (237/410) of the respondents were currently practising in a field different from that planned in their next-to-last year of medical school, 63.5% (275/436) were not practising in the field they would have chosen if forced to at that time, and 42.9% (211/492) had changed their field of training or practice at some time since graduation. Older physicians, those who graduated outside of Canada and specialists were the most likely to have changed career paths, family physicians, and those who graduated in Saskatchewan were the least likely to have changed. CONCLUSION: The current system of postgraduate training in Canada does not permit career changes of the sort made by most of the practising Saskatchewan physicians in the survey sample. The implications of this new system are as yet unknown but require careful monitoring. PMID- 8625025 TI - Recent trends in pediatric Haemophilus influenzae type B infections in Canada. Immunization Monitoring Program, Active (IMPACT) of the Canadian Paediatric Society and the Laboratory Centre for Disease Control. AB - OBJECTIVE: To describe changes in the number of cases of Haemophilus influenzae type b (Hib) infections among Canadian children before and after the introductory phases of Hib vaccination. DESIGN: Multicentre case series. SETTING: All 10 pediatric tertiary care centres across Canada participating in the Immunization Monitoring Program, Active (IMPACT) of the Canadian Paediatric Society and the Laboratory Centre for Disease control. PATIENTS: Children with a Hib infection admitted to any of the participating hospitals from 1985 to 1994. Annual case totals from 1985 to 1990 were determined from records of hospital laboratories or coded discharge diagnoses, or both. From 1991 to 1994 intensive case surveillance was conducted on the wards in addition to thorough record searches as above. OUTCOME MEASURES: Estimated annual case totals for 1985-90. For 1991-94 intensive surveillance for quarterly case totals, yearly age distribution of cases, and proportion of recent cases that represent vaccination failures or missed opportunities to prevent infection. RESULTS: The total number of Hib cases from 1985 to 1990 was 2095; from 1991 to 1994, there were 326 laboratory-confirmed cases and 15 probably cases supported by Hib antigen detection. The annual number of cases declined from an estimated 485 in 1985 to 24 in 1994, a decrease of 95.1%. The steepest interannual decrease (63.7%) occurred between 1992 and 1993, following the introduction of infant-based vaccination programs across Canada. The number of Hib cases involving children most at risk (those 6 to 18 months old) decreased from 78 in 1991 to 4 in 1994. Of the 24 cases in 1994, 6 were categorized as preventable, 1 was fatal, and 8 were vaccine failures (2 of which involved currently used vaccines). CONCLUSION: The prevalence of Hib infections reported by the IMPACT centres has declined greatly since the introduction of vaccination programs. However, deaths and complications continue to occur, attesting to the need to vaccinate all eligible infants and children against this virulent pathogen. PMID- 8625026 TI - Changes in career plans during medical training and practice: it's time to look ahead and act. AB - Major changes in physician-resource policies and in the structuring of medical licensure requirements in the past decade have resulted in a less flexible system with respect to both choosing and changing a career path in medicine. The survey results reported by Drs. Susan Shaw, Gordon Goplen and Donald S. Houston in this issue (see pages 1035 to 1038) indicate that a high percentage of physicians now practising in Saskatchewan changed their career plans after graduation. The author argues that this finding points to the need to reexamine the transition from undergraduate to postgraduate medical education. The present system needs to be made more flexible so that medical students can gain sufficient clinical experience before deciding on an area of practice and to give practising physicians who want to change specialties the option of retraining. PMID- 8625027 TI - US ad uses lure of prompt treatment to entice Canadians needing joint replacement. AB - A Virginia hospital has used newspaper advertisements to solicit Ontario patients who are waiting for hip- or knee-replacement surgery. The ads promote the medical services of US orthopedic surgeons and call attention to exasperatingly long waiting lists for the same surgery in Canada. "Pain doesn't wait," they state. "Neither should you." The hospital says it has been receiving more than 100 calls a week inquiring about the procedure, which costs $15000 (US). PMID- 8625029 TI - Changes in health care system reflected in way medical students are chosen, Queen's alumni learn. AB - Society expects today's physicians to be expert clinicians, effective communicators, resource managers and health care advocates, all rolled into one. This is changing both medical school curricula and the way medical students are selected. During a homecoming weekend at Queen's University, faculty brought medical alumni up to date on the selection process. "I remember writing a little not saying that I wanted to come into medicine and sending it off with my transcript, and that was it," noted Dr. Robert Madsley, the vice-dean. Today, the situation is much different. PMID- 8625028 TI - Pursuing conception: a physician's experience with in-vitro fertilization. AB - Infertility is a common problem. Approximately one in seven North American couples will experience it, either by being unable to conceive after a year of trying or by experiencing recurrent miscarriages. A family physician outlines her experiences when being treated for infertility by in-vitro fertilization and embryo transfer. PMID- 8625030 TI - Visions of our health care future: is a parallel private system the answer? AB - In this time of spending restraint, arguments for and against a two-tier medical system are common. Proponents say governments can no longer afford to supply all the health care we want and Canadians should have the right to purchase it, just as they purchases other services and commodities. Opponents fear that administrative costs will rise greatly if this happens, the best physicians will leave the public system and public support for medicare will erode. For this article, Charlotte Gray sought opinions on whether a parallel private system is a good option for Canadians to consider. PMID- 8625031 TI - Prison became second home for psychiatrist (George Scott). AB - Retired prison psychiatrist George Scott recalls his career working in Canada's penal system, including his peacemaking role in a hostage-taking incident and his work with Steven Truscott. Life "inside" is dangerous for guards, inmates, staff and psychiatrists, he says, but he never regretted his decision to devote his career to studying criminal behaviour. PMID- 8625032 TI - Scottish MDs benefit from database model developed in Saskatchewan. AB - A data-tracking system developed in Saskatchewan has helped health authorities in Scotland identify some deficiencies in the way certain drugs are prescribed and used. A researcher who published survey results in a local medical journal said the findings have helped doctors improve prescribing practices and identified a need to educate patients about the way antidepressant drugs work. PMID- 8625033 TI - The roles and functions of hospital-based ethics committees. AB - Ethics committees ar becoming much more visible on the Canadian health care scene. They range from research-ethics committees that decide whether research projects are ethically sound to case-oriented committees that look at particular issues and give advice. Eike-Henner Kluge says that ethics committees are useful tools, but only when they are appropriately constituted and function in a professional manner. Otherwise, he warns, they become either useless or a liability. PMID- 8625034 TI - The relative survival rate. PMID- 8625035 TI - Basaloid-squamous carcinoma of the nasopharynx. An Epstein-Barr virus-associated neoplasm compared with morphologically identical tumors occurring in other sites. AB - BACKGROUND: Basaloid-squamous carcinoma is a newly characterized, highly aggressive neoplasm occurring mostly in the base of tongue, hypopharynx, larynx, and esophagus. Its occurrence in the nasopharynx is rare. METHODS: The clinicopathologic features of three cases of basaloid-squamous carcinoma of the nasopharynx are described and were studied for the presence of Epstein-Barr virus (EBV) by in situ hybridization for EBV-encoded small nuclear RNA (EBER). For comparison, basaloid-squamous carcinomas occurring in other sites also were studied for the presence of EBV. RESULTS: EBER was detected in all 3 cases of basaloid-squamous carcinoma occurring in the nasopharynx, but in none of the 13 cases from other sites including the esophagus, larynx, pharynx, hypopharynx, and nasal cavity. The nasopharyngeal basaloid-squamous carcinomas occurred in two male and one female patients with an age range of 48-70 years. The serum immunoglobulin A against the EBV-viral capsid antigen was elevated in all three cases. Two patients developed cervical lymph node involvement during the course of the disease. All three patients were treated by radiotherapy and survived for longer than 34 months compared with the average reported median survival of approximately 2 years for basaloid-squamous carcinomas occurring in the usual sites. CONCLUSION: Based on this limited study, basaloid-squamous carcinoma occurring in the nasopharynx appears to be an EBV-associated neoplasm, whereas the same tumor occurring in other sites is not. The prognosis is potentially better for patients with nasopharyngeal basaloid-squamous carcinoma, which appears to be pathogenetically and biologically more related to the much more common nasopharyngeal undifferentiated carcinoma. PMID- 8625036 TI - Epirubicin, cisplatin, and continuous infusion 5-fluorouracil is an active and safe regimen for patients with advanced gastric cancer. An Italian Group for the Study of Digestive Tract Cancer (GISCAD) report. AB - BACKGROUND: A Phase II confirmatory multicenter trial was performed to evaluate a combination of epirubicin, cisplatin, and continuous infusion 5-fluorouracil (ECF) in treating patients with advanced gastric cancer. METHODS: Fifty-three patients with locally advanced (n = 7) or metastatic (n = 46) gastric cancer received a dose of epirubicin (50 mg/m2) and cisplatin (60 mg/m2) intravenously every 21 days for eight cycles with 5-fluorouracil (200 mg/m2/day) by intravenous continuous infusion for 21 consecutive weeks, administered through a central line using an external pump. RESULTS: Eight complete responses and 22 partial responses (response rate = 56%, 95% confidence interval +/- 13) were achieved. Twelve patients had stable disease. The median duration of response was 10 months (range, 3-21 months), and the median survival for all the patients was 9+ months (range, 2-28 months). Overall toxicity, which was primarily hematologic, was mild with only three patients requiring hospitalization for neutropenic fever. No death due to toxicity occurred. CONCLUSIONS: This study found that the ECF regimen is substantially active in treating patients with advanced gastric cancer and has a favorable pattern of toxicity. This schedule clearly deserves randomized comparative trials for palliation of metastatic disease and for adjuvant purposes. PMID- 8625037 TI - Expression of Thomsen-Friedenreich-related antigens in primary and metastatic colorectal carcinomas. A reevaluation. AB - BACKGROUND: Expression of the pancarcinoma Thomsen-Friedenreich (TF) carbohydrate antigen or, more correctly, hapten, in colorectal carcinomas is not generally agreed on. Furthermore, its suggested role in liver metastasis so far has not been substantiated by direct immunohistochemical evidence. METHODS: Cryostat sections from 52 primary tumors (20 with adjacent transitional mucosa), 22 liver metastases of colorectal carcinomas, and 17 samples of normal mucosae were examined immunohistologically with a panel of at least two monoclonal antibodies (mAbs) each to TF, to its precursor, Tn, and to sialosyl-Tn, among them two newly developed anti-TF mAbs. RESULTS: Of the primary colorectal carcinomas, 60% expressed TF. Staining was more intense with TF-alpha/beta-reactive than with exclusively TF-alpha- or TF-beta-reactive mAbs. Normal and transitional mucosae were negative. Liver metastases were positive for TF in a significantly higher percentage of cases (91%) than primary carcinomas. Patients with TF-positive primary tumors had a significantly higher risk to develop liver metastases compared with patients with TF-negative tumors (57% vs. 14%, respectively). Tn and sialosyl-Tn were expressed concomitantly in most primary (85%) and metastatic (95%) colorectal carcinomas. These antigens also were detected in transitional mucosae (Tn in 25%, sialosyl-Tn in 55% of cases). Normal mucosae were negative. CONCLUSIONS: These results prove unequivocally the presence of exposed TF epitopes in a majority of colorectal carcinomas in which both anomers of TF are expressed. These data further suggest that TF favors liver metastasis and that its expression in primary colorectal carcinomas is a significant risk factor for the development of liver metastasis. PMID- 8625038 TI - Randomized phase III study of 5-fluorouracil plus high dose folinic acid versus 5 fluorouracil plus folinic acid plus methyl-lomustine for patients with advanced colorectal cancer. AB - BACKGROUND: Metastatic colorectal cancer is generally incurable. The most active regimen available, 5-fluorouracil (5-FU) and folinic acid (Leucovorin), produces response rates of approximately 25% to 30%. Methyl-lomustine is a nitrosourea with modest activity against colorectal cancer. A randomized trial was undertaken to evaluate the impact the addition of methyl-lomustine would have on response, duration of survival, and survival rates in patients with advanced colorectal cancer. METHODS: The methyl-lomustine/5-FU/Leucovorin (MFL) regimen consisted of methyl-lomustine (110 mg/m2), administered on Day 1 of each 8-week cycle with six weekly boluses of 5-FU (600 mg/m2), and Leucovorin (500 mg/m2). The FL treatment arm consisted of the administration of 5-FU and Leucovorin as described above. Patients were evaluated for response and toxicity after each 8-week cycle. RESULTS: Of 319 patients included in this trial, 297 (93.1%) had disease evaluable for response and toxicity: 145 received MFL, and 152 received FL. In this trial, 526 courses of MFL and 529 courses of FL were administered. Methyl lomustine/5-FU/Leucovorin treatment resulted in 4 complete and 30 partial responses (response rate, 21.9%), and FL treatment resulted in 9 complete and 33 partial responses (response rate, 26.4%). There was no significant difference in median survival duration between patients in the two arms (MFL = 48 weeks, FL = 51 weeks). However, MFL was significantly more toxic with greater myelosuppression than was FL (Grade 3-4 neutropenia: MFL = 56 patients, FL = 27 patients, P < 0.001; Grade 3-4 thrombocytopenia: MFL = 49 patients, FL = 2 patients, P < 0.001; Grade 3-4 anemia: MFL = 15 patients, FL = 6 patients, P < 0.001; and more prolonged median duration of granulocytopenia: MFL = 9 days, FL = 7 days, P < 0.001; and thrombocytopenia: MFL = 14 days, FL = 7.5 days, P < 0.001). CONCLUSION: Because the addition of methyl-lomustine in the MFL schedule markedly increased the toxicity of the regimen and because the FL regimen was as effective as MFL, the authors recommend that Leucovorin and 5-FU remain the treatment choice for treating patients with metastatic colorectal cancer. PMID- 8625040 TI - Prognostic evaluation of DNA flow cytometric and histopathologic parameters of colorectal cancer. AB - BACKGROUND: The clinical value of DNA flow cytometry of colorectal cancer is unclear. The purpose of this retrospective study was to evaluate the relationship between tumor flow cytometry, histopathologic parameters, and survival. METHODS: Flow cytometry was performed on paraffin embedded specimens from 653 patients who had surgery from 1980 to 1983. RESULTS: Aneuploidy was associated with distal tumor, perineural invasion, desmoplastic reaction, and failure to secrete mucin. TNM Stage I tumors were more frequently diploid than were more advanced tumors (71% vs. 41%). An abnormal DNA content had a marginal impact on survival as evaluated by univariate analysis (69% vs. 61% 10-year survival rate, P = 0.06). Multivariate analysis revealed that significant predictors of outcome were lymph node metastasis (95% confidence interval of relative risks of death from recurrent disease, 1.50-2.92), rectal cancer (1.22-2.19), absence of lymphocytic infiltration (1.20-2.17), invasion through bowel wall (1.17-3.13), lymphatic vessel invasion outside bowel wall (1.05-2.69), perineural invasion (1.15-3.19), and male gender (1.00-1.79). CONCLUSIONS: These findings suggest that ploidy is associated with some histopathologic parameters, but flow cytometry does not correlate with long term survival of patients with colorectal carcinoma. PMID- 8625039 TI - Characterization of malignant colon tumors with 31P nuclear magnetic resonance phospholipid and phosphatic metabolite profiles. AB - BACKGROUND: To further characterize selected pathologic features on a biochemical level, the authors analyzed the nuclear magnetic resonance metabolite and phospholipid spectra of 30 malignant colon tumors using 31P magnetic resonance spectroscopy. METHODS: Eleven individual generic phospholipids were identified in the spectra of 17 phospholipid extracts, and 31 individual phosphatic metabolites were identified in the spectra of 13 perchloric acid extracts. The metabolites and lipids were quantified for statistical intergroup comparisons based on tumor stage, lymph node status, differentiation, mucin production, blood vessel invasion (BVI), and lymphatic vessel invasion (LVI). RESULTS: Significant elevations in the relative concentration of alpha-glycerol phosphate were noted when comparing AJCC tumor classification (T3 vs. T2, 0.92 +/- 0.14 vs. 0.46 +/- 0.11, P < 0.009), tumor differentiation (moderately vs. well differentiated, 0.92 +/- 0.14 vs. 0.46 +/- 0.11, P < 0.009), and BVI (presence vs. absence, 1.03 +/- 0.04 vs. 0.68 +/- 0.10, P < 0.028) by elastic tissue stain. Among the tissue phospholipids analyzed, the relative concentration of a choline phospholipid was significantly different when comparing moderately and poorly differentiated tumors (6.26 +/- 0.56 vs. 3.29 +/- 0.30, P < 0.001), T2 and T3 tumors (3.90 +/- 0.45 vs. 6.31 +/- 0.56, P < 0.009), and mucin-positive vs. mucin-negative tumors (4.46 +/- 0.56 vs. 6.83 +/- 0.76, P < 0.028). Differences in lymph node status of the cases analyzed in this study (lymph node positive vs. lymph node negative) were noted for tumors with elevated levels of sphingomyelin (8.13 +/- 0.40 vs. 6.88 +/- 0.16, P < 0.02), diminished levels of phosphatidylinositol (5.25 +/- 0.27 vs. 6.38 +/- 0.34, P < 0.02), elevated levels of beta-glycerol phosphate (5.30 +/- 0.70 vs. 1.20 +/- 0.06, P < 0.05), and elevated levels of glycerol 3 phosphoserine (0.48 +/- 0.01 vs. 0.23 +/- 0.02, P < 0.002). CONCLUSIONS: The characteristic differences in the phospholipid and intermediate phosphate metabolite profiles identified through magnetic resonance spectroscopic and histopathologic analysis may provide important information regarding the nature of tumor and cell membrane metabolism. Differences in these profiles may identify markers useful for biologic behavior, provide prognostic information, and characterize the impact of the pathologic features of colon cancer. PMID- 8625041 TI - Results of combined modality therapy for patients with anal cancer (E7283). An Eastern Cooperative Oncology Group study. AB - BACKGROUND: This prospective study assessed combined modality therapy of patients with International Union Against Cancer classification T1-4 N0 M0 anal cancer. METHODS: Protocol therapy consisted of a dose of 4000 cGy to the pelvis, anus, and perineum, followed by a 1000-1300 cGy boost. Infusions of 5-fluorouracil and mitomycin-C were administered when radiation therapy began. A second infusion of 5-fluorouracil was administered 28 days later. Biopsy was performed 6-8 weeks after completion of treatment. Positive biopsy findings resulted in abdominal perineal resection. RESULTS: Survival at 7 years for 50 eligible patients was 58%. White patients and those with favorable performance status had significantly better survival. Of the 46 patients evaluable for response, 34 had a complete response, 11 had a partial response, and 1 had no response. Seven-year survival for partial responders was 53%. Freedom from locoregional progression was 80% at 7 years. CONCLUSION: Treatment with a combination of chemotherapy and radiation therapy is effective for patients with anal cancer. The investigation of methods of improving therapy is warranted. PMID- 8625042 TI - Treatment of small hepatocellular carcinoma with percutaneous ethanol injection. Analysis of prognostic factors in 105 Western patients. AB - BACKGROUND: Percutaneous ethanol injection (PEI) has been used in the Far East for treating small, unresectable hepatocellular carcinoma (HCC). To clarify when treatment with PEI may be best indicated for Western patients with HCC, the authors performed a retrospective analysis of the clinicopathologic factors influencing prognosis. METHODS: From December 1987 to August 1994, 105 patients with cirrhosis with HCC received PEI as the sole anticancer treatment. Eighty-two patients had uninodular tumors smaller than 5 cm, and 23 patients had multiple lesions (2-4) smaller than or equal to 3 cm each. All patients were in Child-Pugh class A (n = 64) or B (n = 41). Survival was analyzed according to patient- and tumor-related factors by means of the Kaplan-Meier method. RESULTS: The estimated survival rates of all 105 patients were 96% at 1 year, 86% at 2 years, 68% at 3 years, 51% at 4 years, 32% at 5 years, and 24% at 6 years. Survival was not affected by sex, age, etiology of cirrhosis, or hepatitis B surface antigen or anti-hepatitis C virus positivity, but depended on Child-Pugh class (P = 0.006) and presence of ascites (P = 0.009). Patients with a pretreatment alpha fetoprotein level of 200 ng/ml or less had a better prognosis than patients with an alpha-fetoprotein level higher than 200 ng/ml (P = 0.007). Patients with unmodular HCC of 3 cm or less had significantly better long term survival (P = 0.04) than patients with uninodular HCC of 3.1-5 cm or with multinodular tumors. Tumor grade according to Edmondson and Steiner and tumor volume, in contrast, did not significantly influence prognosis (P > 0.1). CONCLUSIONS: For Western patients with HCC treated with PEI, the prognosis was highly dependent on the severity of the underlying cirrhosis. Treatment with PEI is best indicated for patients with uninodular tumors of 3 cm or less in greatest dimension and an alpha-fetoprotein level lower than 200 ng/ml. PMID- 8625043 TI - Risk factors for gallbladder cancer. An international collaborative case-control study. AB - BACKGROUND: Gallbladder cancer has an unusual geographic and demographic distribution, suggesting many possible etiologies. METHODS: A case-control study was undertaken at four hospitals in La Paz, Bolivia, and at one hospital in Mexico City, Mexico. Eighty-four patients with newly diagnosed, histologically confirmed gallbladder cancer were compared with 126 control subjects without stones and with 264 control subjects with cholelithiasis or choledocholithiasis without cancer. All study subjects underwent abdominal surgery. Study subjects were interviewed regarding demographic characteristics, medical history, family history, diet, and exposure to agents presumed to be risk factors for biliary cancer. RESULTS: Virtually all subjects in Mexico were judged to be mestizos (i.e., persons of mixed ancestry) In contrast, race was a very strong risk factor for gallbladder cancer in Bolivia. Relative to mestizos who spoke neither language, the odds ratio (95% confidence interval [CI]) for cases versus control subjects without stones for those who spoke Aymara well was 15.9 (CI, 1.9-179), whereas it was 1.4 (CI, 0.2-8.2) for those who spoke Quechua well. An increased risk was also noted for elevated maximum body mass index (P = 0.03), family history of gallstones (odds ratio [OR] = 3.6 [CI, 1.3-11.4]), and physician diagnosed typhoid (OR = 12.7 [CI, 1.5-598]). An increased risk was also seen with elevated maximum body mass index; compared with those with a body mass index less than 24 kg/m2, those with an index of 24-25 kg/m2, 26-28 kg/m2, and greater than 28 kg/m2 had odds ratios of 1.6 (CI, 0.4-7.6), 1.3 (CI, 0.3-5.6), and 2.6 (CI, 0.5-18.6), respectively (asymptotic test for trend, P = 0.03). Finally, a number of associations were noted with certain dietary and cooking habits. CONCLUSIONS: Patients with gallbladder cancer differed from control subjects in race, body mass, physician-diagnosed typhoid, and certain dietary patterns. These findings may provide useful clues to the pathogenesis of gallbladder cancer, but given the number of analyses performed, additional cases need to be studied. PMID- 8625044 TI - Flow cytometric DNA analysis and lysosomal cathepsins B and L in locally advanced laryngeal cancer. Relationship with clinicopathologic parameters and prognostic significance. AB - BACKGROUND: The traditional factors of locally advanced laryngeal squamous cell carcinoma (LSCC) have limited predictive value for the identification of high risk patients. Therefore, it is extremely important to define prognostic factors that identify the more aggressive types. Reliable and reproducible prognostic indicators are being investigated to help clinicians identify high risk groups and address more rational treatment. METHODS: Flow cytometric DNA ploidy and S phase fraction (SPF) measurements were performed on frozen tumor tissues from a consecutive series of 71 patients with Stage III and IV LSCC: Lysosomal cathepsin B and L activity levels were determined biochemically in matched paired sets of tumor tissue and normal mucosa samples. RESULTS: By univariate analysis, lymph node positivity, poor histologic differentiation, DNA aneuploidy, high SPF, and high tumor/mucosa ratio of cathepsin B activity were significantly related to risk of relapse, whereas only DNA aneuploidy and high SPF proved to be significantly related to risk of death. Multivariate analysis showed that high histologic grade and high SPF values (> 15.1%) were independent prognostic factors related to risk of relapse (relative risk [RR] = 3.54; 95% confidence limits [CL] = 1.05-12.0; and RR = 4.22; CL = 1.54-11.6, respectively), whereas only high SPF was related to risk of death (RR = 3.63; CL = 1.17-11.3). CONCLUSIONS: S-phase fraction is an independent predictor of relapse free and overall survival in patients with locally advanced LSCC. On the basis of these findings, SPF should be used in addition to other established prognostic factors to refine the prognostic assessment of these patients further. More studies are needed for a better evaluation of the prognostic significance of DNA ploidy and that of lysosomal cysteine proteinases in these tumors. PMID- 8625045 TI - Microsatellite instability in keratoacanthoma. AB - BACKGROUND: Tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) and from a subset of patients with the related Muir-Torre syndrome (MTS) exhibit a novel type of genomic instability known as microsatellite instability (MIN). In general, this form of genomic instability results from mutations that inactivate DNA mismatch repair genes. The detection of MIN in a keratoacanthoma (KA) from a patient with MTS suggested that defective mismatch repair may play a role in the pathogenesis of these neoplasms. METHODS: Randomly selected paraffin embedded KA from 53 patients and paraffin embedded tumors from an additional 12 patients diagnosed with KA and colorectal carcinoma were examined for MIN at six loci. In addition, several KA were examined for mutations within the hMSH2 gene. RESULTS: Six of the 53 randomly selected KAs had MIN at two or more loci. One of these six patients had HNPCC, whereas another had MTS. Two patients with KAs lacking MIN had colon tumors that exhibited widespread MIN, and one of these patients had MTS. Three of the 12 additional patients diagnosed with a KA and a colorectal carcinoma had at least one tumor that had MIN at two or more loci, and one of these patients had HNPCC: A 2-base pair somatic deletion in exon 3 of the hMSH2 gene was identified in one of the MIN+ KAs. CONCLUSIONS: Defective mismatch repair appears to play a role in the process of tumorigenesis in some KAs. Microsatellite instability in a KA or the cooccurrence of a colorectal carcinoma and a KA in a patient suggests that the patient may have either HNPCC or its phenotypic variant MTS. PMID- 8625046 TI - The prognostic value of lymphatic and blood vessel invasion in operable breast cancer. AB - BACKGROUND: This study assessed the prognostic effect of lymphatic and blood vessel invasion (LVI and BVI) on survival in a retrospective sample of 1408 patients with breast cancer. METHODS: Survival analysis was evaluated by univariate (Kaplan-Meier product limit method and log rank test) and multivariate (Cox model) analysis. Correlations among variables were studied by contingency tables and statistical significance was evaluated by chi-square test. RESULTS: Lymphatic vessel invasion was present in 34.2% of cases, and BVI in 4.2%. Lymphatic vessel invasion correlated with BVI (P < 0.0001), and both were correlated with metastatic axillary lymph nodes and increasing tumor size and grade; BVI was sporadic (only 10 cases) among lymph node negative patients. Although LVI was more frequent among premenopausal patients and those with ductal carcinomas, BVI was unrelated to menopausal status and tumor type. Lymphatic vessel invasion and BVI were associated with poor survival by univariate analysis (P < 0.0001). By multivariate analysis, relative risk of death was significantly increased when LVI was present both in the whole series as well as in the lymph node negative and lymph node positive subgroups; the prognostic role of LVI was independent of menopausal and lymph node status, tumor size, tumor grade, or adjuvant treatment. In the lymph node negative sample, LVI had strong prognostic power. In the lymph node positive sample, the prognostic role of LVI was also independent of the number of lymph nodes with metastases. Blood vessel invasion had no prognostic role in any subgroup. CONCLUSION: The prevalence of BVI was particularly low in this study, and the question of its possible prognostic role for patients with breast cancer should be assessed with methods that amplify its detection. LVI is a strong prognostic factor for operable patients with breast cancer. In lymph node negative patients, LVI is a predictor of poor prognosis for those who are consequently candidates for adjuvant therapy. Similarly, in lymph node positive patients, LVI is a predictor for a high risk of death for those who are candidates for highly intensive adjuvant strategies. PMID- 8625047 TI - Progression of fibroadenoma to phyllodes tumor demonstrated by clonal analysis. AB - BACKGROUND: The histogeneses of fibroadenoma and phyllodes tumor of the breast appear to be closely related, but it is still unclear whether fibroadenoma can progress directly to phyllodes tumor. METHODS: This issue was studied by conducting clonal analysis of fibroadenoma and phyllodes tumors that were obtained sequentially from the same patient. One patient developed local recurrence of phyllodes tumor twice, and the other two patients each developed a phyllodes tumor after excision of a primary fibroadenoma. The method for clonal analysis was based on trinucleotide repeat polymorphism of the X chromosome linked androgen receptor (AR) gene and on random inactivation of the gene by methylation. RESULTS: Clonal analysis revealed that all the three primary fibroadenomas were monoclonal and all four recurrent phyllodes tumors were also monoclonal in origin. In addition, the same allele of the AR gene was inactivated in fibroadenoma and phyllodes tumor(s) in each patient. The probability that phyllodes tumors of different origin happen to inactivate the same allele of the AR gene as fibroadenomas in every case is quite low. Rather, it is more reasonable to assume that the phyllodes tumor has the same origin as fibroadenoma. CONCLUSIONS: These results identified monoclonal fibroadenomas that can progress to phyllodes tumors. PMID- 8625049 TI - Nuclear morphometry for improved prediction of the prognosis of human bladder carcinoma. AB - BACKGROUND: Histologic grade and clinical stage generally are used for estimating the prognosis of bladder carcinoma. However, both methods have been reported to have a rather low reproducibility and to be unsatisfactory for predicting the recurrence and progression of superficial bladder carcinoma. Recently, nuclear morphometry was used to quantitate the malignant potential of cancer cells in a more objective and reproducible manner. The authors quantitatively analyzed the malignant potential of bladder carcinoma at initial presentation using a combination of several nuclear morphometric variables. METHODS: The subjects were 156 patients with previously untreated bladder carcinoma. Three morphometric variables were measured in each subject: the mean nuclear volume (MNV), the nuclear roundness factor (NRF), and the variation of nuclear area (VNA). RESULTS: Univariate analysis showed that MNV and NRF were significant prognostic indicators for survival (MNV, P < 0.0001; NRF, P = 0.008). In addition, MNV was a prognostic indicator for tumor recurrence (P = 0.001), whereas MNV and NRF were prognostic indicators for invasive progression (MNV, P = 0.02; NRF, P = 0.009). For accurate prediction of the prognosis of patients with bladder carcinoma, a prognostic score, a recurrence score, and a progression score were designed using the coefficients of MNV and NRF in a proportional hazards model. The prognostic score clearly divided the patients into two different groups with 5-year survival rates of 88% and 64% (P = 0.0002). In addition, patients with superficial bladder carcinoma and a low recurrence score had a significantly higher 5-year recurrence free rate than those with a high recurrence score (40% vs. 23%, P = 0.0004), and the 5-year progression free rate of patients with a low progression score was significantly higher than that of those with a high progression score (98% vs. 73%, P = 0.0006). CONCLUSIONS: These findings suggest that nuclear morphometry is a reliable technique with which to identify prognostic indicators for human bladder carcinoma. A combination of several nuclear morphometric variables provides a more accurate indication of prognosis than any single parameter. PMID- 8625048 TI - Prognostic value of immunohistochemically detected p53 expression in vulvar carcinoma. AB - BACKGROUND: Overexpression of the p53 protein has been reported to correlate with poor prognosis in several types of tumors. To the authors' knowledge, there are no studies concerning the prognostic value of p53 protein overexpression in squamous cell vulvar carcinoma. METHODS: Twenty-five cases of squamous cell carcinoma of the vulva with International Federation of Gynecology and Obstetrics (FIGO) Stage I-II were examined for p53 protein overexpression using immunohistochemistry. The correlation of p53 protein overexpression with clinical stage, histologic grade, and overall survival was investigated. Follow-up ranged from 36 to 120 months. RESULTS: Clinical stage and histologic grade did not correlate with p53 protein overexpression. p53 protein overexpression was associated with poorer overall survival (log rank: P < 0.05). CONCLUSION: Immunohistochemically detected p53 protein overexpression is significantly correlated with a reduced overall survival rate for patients with vulvar carcinoma. PMID- 8625050 TI - Platinum analogue combination chemotherapy: cisplatin, carboplatin, and methotrexate in patients with metastatic urothelial tract tumors. A phase II trial with evaluation of prognostic factors. AB - BACKGROUND: Cisplatin is one of the single drugs that has shown the best documented effect in treating patients with locally recurrent or metastatic urothelial cancer. To the authors' knowledge, the effect of the combination of different platinum analogues in treating transitional cell carcinoma has not been evaluated previously neither experimentally nor in clinical studies. METHODS: A Phase II trial of carboplatin (200 mg/m2), cisplatin (100 mg/m2), and methotrexate (250 mg/m2) with folinic acid rescue every 3 weeks was performed on 55 previously untreated patients with metastatic or locally recurrent urothelial cell carcinoma. RESULTS: A response (complete response and partial response) was achieved in 21 of 51 evaluable patients (41%; 95% confidence limits, 28-56%). Twelve patients had no change, whereas 18 had progressive disease. Eight patients (16%) achieved a complete response, and most of these survived more than 2 years. No patient with poor performance (performance status score > or = 2) or bone metastases achieved a complete response. The median survival for all patients was 8.4 months. Multivariate survival analyses showed that performance status and alkaline phosphatase levels were significant prognostic factors for survival. CONCLUSION: Combination therapy with cisplatin, carboplatin, and methotrexate is feasible but offers no advantage over other combinations with cisplatin and methotrexate in treating metastatic urothelial cell cancer. It is important to select patients for treatment carefully, and further studies of prognostic factors in these patients are warranted. PMID- 8625051 TI - Patients with malignant meningitis presenting with neuropsychiatric manifestations. AB - BACKGROUND: Malignant meningitis is often thought of as a late event in the course of cancer. At one time, it was thought to be rare, but it has been recognized more frequently in recent times. Clinical suspicion of malignant meningitis is prompted by neurologic symptoms and signs in patients at risk for this oncologic complication. Neuropsychiatric symptoms previously were not considered as presenting symptoms of malignant meningitis. METHODS: Three patients with cancer with no neurologic symptoms were examined for malignant meningitis based on neuropsychiatric symptoms. Cerebrospinal fluid was examined for malignant cells to confirm the diagnosis of malignant meningitis. RESULTS: The clinical presentation of malignant meningitis for three patients was neuropsychiatric. None of the patients had delirium during their initial presentation. CONCLUSIONS: It is important that clinicians recognize that psychiatric symptoms without neurologic findings may indicate malignant meningitis and that malignant meningitis needs to be included in the differential diagnosis of neuropsychiatric disorders in patients with cancer. PMID- 8625052 TI - Proliferating cell nuclear antigen and Ki-67 immunohistochemistry of oligodendrogliomas with special reference to prognosis. AB - BACKGROUND: The biologic behavior of oligodendrogliomas is somewhat unpredictable. A supplementary prognostic factor is, therefore, desirable. METHODS: Thirty-two pure supratentorial oligodendrogliomas were investigated using proliferating cell nuclear antigen (PCNA) and Ki-67 immunohistochemical analyses. The correlation of PCNA and Ki-67 labeling index (LI) with prognosis were studied, and the correlation of LI with clinical data was evaluated. RESULTS: The PCNA LI had a range of 0-17% (mean, 5.27%; standard deviation [SD] = 4.65), and the Ki-67 LI had a range of 0-29% (mean, 4.19%; SD = 5.66). In general, the PCNA LI seemed to be higher than the Ki-67 LI. The mean survival time was 4.4 years, and 5- and 10-year survival rates were 38% and 19%, respectively. Ki-67 and PCNA staining indicated that patients with a high LI (> 3% and > 4%, respectively) had a significantly higher mortality, with mean survival time of 23.5 months and 26.2 months, respectively. No significant correlation between LI (or survival) and tumor size, cerebral localization, radiation, resection/biopsy, sex, age, or cytologic atypia was found. CONCLUSIONS: The use of Ki-67 and PCNA LI higher than 3% and 4%, respectively, appears reliable as prognostic factors when investigating pure supratentorial oligodendrogliomas. PMID- 8625053 TI - Prolactin-secreting pituitary carcinoma with implants in the cheek pouch and metastases to the ovaries. A case report and literature review. AB - BACKGROUND: Prolactin-secreting pituitary carcinomas are uncommon, locally destructive neoplasms that rarely metastasize outside the central nervous system. The authors report a case of a prolactin-secreting tumor that initially presented as the empty sella syndrome. Two recurrences along transsphenoidal surgery tracts in cheek pouches were followed by distant metastases later in the abdomen and pelvis. Only 10 previous cases of either extracranial or intracranial metastases from prolactin-secreting pituitary carcinomas have been reported. No metastases below the diaphragm have been reported previously. METHODS: The patient's cheek pouch implants, lymph node metastases, ovarian metastases, and uterine metastases were studied with prolactin-specific immunohistochemistry. RESULTS: Long term treatment with bromocriptine, several debulking surgeries, extensive local radiation therapy (external beam and proton beam), and cytotoxic chemotherapy had little impact. Tamoxifen, however, may have slowed tumor growth. CONCLUSION: Tamoxifen may have efficacy in the treatment of prolactin-secreting pituitary carcinomas. PMID- 8625054 TI - Oral granisetron with or without methylprednisolone versus metoclopramide plus methylprednisolone in the management of delayed nausea and vomiting induced by cisplatin-based chemotherapy. A prospective randomized trial. AB - BACKGROUND: A single-institution, randomized open trial was prospectively performed to compare orally administered granisetron with or without intramuscularly administered methylprednisolone to metoclopramide plus methylprednisolone in the prevention of delayed nausea and vomiting induced by cisplatin-based chemotherapy. The effects of antiemetic treatments were evaluated from days 2 to 5 of the first cycle after cisplatin administration among patients who had never before received chemotherapy. METHODS: All patients were treated with chemotherapeutic regimens containing cisplatin greater than or equal to 80 mg/m2 and received antiemetic therapy with granisetron 3 mg intravenously for the control of acute emesis. Patients who responded to treatment during the first 24 hours were randomized to receive (1) metoclopramide (0.5 mg/kg) intramuscularly three times daily plus methylprednisolone (125 mg) intramuscularly once a day or (2) granisetron (1 mg) orally twice daily or (3) oral granisetron (1 mg) orally plus methylprednisolone (125 mg) intramuscularly from days 2 to 5. RESULTS: Of the patients treated with metoclopramide plus methylprednisolone (n = 92), 53% had complete protection from delayed emesis, 16% a major response, 15% a minor response, and 15% no response. Of the patients treated with granisetron alone (n = 84), 33% had a complete response, 21% a major response, 23% a minor response, and 21% no response. In the patients treated with orally administered granisetron plus intramuscularly administered methylprednisolone (n = 86), 47% had a complete response, 17% a major response, 23% a minor response, and 13% no response. These differences reached statistical significance only when the complete response rate achieved in the metoclopramide plus methylprednisolone group was compared with that recorded in the oral granisetron group (P = 0.012). Moreover, the metoclopramide plus methylprednisolone and the orally administered granisetron plus corticosteroid arms were superior to the orally administered granisetron alone arm in preventing nausea (P < 0.038 and P < 0.002, respectively). No extrapyramidal side effects were noted for the granisetron alone and the granisetron plus methylprednisolone arms, whereas 6% of patients treated with metoclopramide had extrapyramidal adverse effects. Headache was recorded in 8% of patients treated with granisetron alone, in 9% treated with granisetron plus methylprednisolone, and in 3% treated with metoclopramide plus methylprednisolone. CONCLUSIONS: These data suggest that orally administered granisetron with or without methylprednisolone may be given safely to patients with cancer as prophylactic therapy against delayed emesis after high dose cisplatin therapy. Orally administered granisetron alone was less active than a standard combination of metoclopramide plus methylprednisolone. However, the addition of corticosteroid to orally administered granisetron improved the control of delayed emesis. The efficacy of the combination of metoclopramide plus methylprednisolone and oral granisetron with or without methylprednisolone against delayed emesis still is not entirely satisfactory. PMID- 8625056 TI - Cutaneous melanoma and atypical Spitz tumors in childhood. AB - BACKGROUND: Malignant melanoma in childhood is rare. As a result, the biology and natural history of melanoma in this age group is still poorly understood. Although the majority of Spitz nevi are benign regardless of atypical features, a particular problem is the continued confusion of Spitz nevi with atypical features with melanoma and the lack of specific criteria for their distinction. The latter discrimination is perhaps not so difficult when Spitz nevi are minimally atypical; however, the greater the atypia, the more challenging is this discrimination. METHODS: All cases of malignant melanoma referred to Children's Hospital (Boston, MA) and to one of the authors were examined during the period of 1959-1995. Criteria for inclusion in the study included: (1) age up to 15 years; (2) availability of microscopic slides; and (3) availability of demographic data. RESULTS: There were 11 males and 12 females, ranging in age from 2 to 15 years (mean age, 9.4 years). Histopathologically, the 23 tumors were categorized into four subgroups: (1) small cell melanoma (5); (2) adult-like melanoma (6); (3) Spitz-like melanoma (3), and (4) atypical Spitz tumors (9). The small cell melanomas were notable for localization to the scalp, significant thickness, and fatal outcome. The adult-like melanomas resembled typical tumors occurring in adults. The one fatal Spitz-like melanoma was located on the neck of a 14-year-old male. Two tumors in this group metastasized to regional lymph nodes, but were not associated with further aggressive disease on follow-up despite treatment with surgical excision only. The atypical Spitz tumors were characterized by significant thickness and abnormal features including prominent cellularity and mitotic activity. CONCLUSIONS: Anatomic site and cell type may be important prognostic factors in addition to tumor thickness for childhood melanoma, but these tumors require further study. In addition, the biologic potential of atypical Spitz tumors has not been characterized sufficiently. PMID- 8625055 TI - Isolated metastases to peripheral nerves. Report of five cases involving the brachial plexus. AB - BACKGROUND: Primary true isolated metastasis in a peripheral nerve trunk is considered a rare phenomenon. Several routes of tumor invasion of the peripheral nervous system from different types of cancer and in various anatomic locations have been described in the literature; however, the brachial plexus is an uncommon site of such blood-born metastases. METHODS: Five patients with severe brachial plexus neuropathy without obvious external signs of tumor masses and no detectable regional lymph node involvement underwent exploration of the plexus. RESULTS: Isolated metastatic deposits were identified inside the nerve trunks. Radiation therapy to the area of the lesion provided good symptomatic relief to all patients and local arrest of the disease to four of five. CONCLUSIONS: Despite its rich vascularity, the Peripheral nerve has relative resistance to metastatic spread because there are blood-nerve barriers. This barrier should be investigated further in a tumor model. PMID- 8625057 TI - Magnetic resonance imaging and magnetic resonance angiography in long term survivors of acute lymphoblastic leukemia treated with cranial irradiation. AB - BACKGROUND: Successful treatment of acute lymphoblastic leukemia (ALL) has resulted in an increasing number of patients whose disease is cured. This treatment includes cranial irradiation as prophylaxis against central nervous system relapse. The late effects of irradiation are well documented, but their incidence is unknown. The authors investigated the late effects of this treatment modality further by scanning 35 long term survivors of ALL who received cranial irradiation. METHODS: Thirty-five survivors of ALL with no known complication of treatment were included in this study. They were examined with magnetic resonance imaging (MRI) of the brain and magnetic resonance angiography (MRA) of the circle of Willis. A control group of 24 patients who were cured of other childhood malignancies without exposure to cranial irradiation also were scanned. RESULTS: Fifteen of 35 (43%) abnormalities were found in the study group versus 4/24 (17%) in the control group. Excluding minor atrophic changes that are known to be produced by irradiation and chemotherapy, there were 9/35 (26%) abnormalities in the study group and 1/24 (4%) in the control group (P < 0.05). These abnormalities included three tumors, a meningioma, a paranasal sinus rhabdomyosarcoma, and an anaplastic astrocytoma. In addition, there were two cases of large vessel vasculopathy, two small cystic infarcts, one diffuse white matter abnormality, and one cryptic vascular malformation. The abnormal control patient had a cerebellar infarct. CONCLUSION: Complications of cranial irradiation in the treatment of ALL appear to be more frequent than currently are appreciated. That these complications include tumors that are potentially treatable suggests that screening may be valuable for these patients. PMID- 8625058 TI - Nonrandom chromosome breakpoints at Xq26 and 2q33 characterize cemento-ossifying fibromas of the orbit. AB - BACKGROUND: Cytogenetic reports of histologically benign fibroosseous lesions are rare, with only nine previously reported cases. None of these previous studies revealed consistent numerical or structural chromosome aberrations, and to the authors' knowledge, no karyotypic abnormalities in cemento-ossifying fibromas of the orbit have been reported. METHODS: Short term in situ culture and Giesma-band chromosome methods were used to analyze three cementifying fibromas of the orbit: one from a 13-year-old African American male, one from a 14-year-old Hispanic male, and one from a 17-year-old white male. RESULTS: Cytogenetic findings in these three cases revealed the presence of identical chromosomal breakpoints occurring in all three tumors at bands Xq26 and 2q33. Two of the tumors showed an identical t(X;2)(q26;q33) reciprocal translocation as the sole abnormality. The third tumor revealed an interstitial insertion of bands 2q24.2q33 into Xq26 as the sole abnormality. CONCLUSIONS: The authors described new nonrandom breakpoints in fibroosseous lesions of the orbit, which can result from at least two different types of structural chromosomal aberrations. The identification of recurring breakpoints at Xq26 and 2q33 provides a new cytogenetic tumor marker for the identification of this tumor subtype. The sublocalization of breakpoints in this tumor should provide important information for the precise localization and characterization of genes involved in the histiogenesis of these lesions. PMID- 8625059 TI - Second malignant neoplasms in long-term survivors of childhood rhabdomyosarcoma. AB - BACKGROUND: The occurrence of second malignant neoplasms (SMNs) in successfully treated pediatric patients with cancer has been an area of increasing concern because survival of these patients has improved with intensification of therapy. Therefore, the incidence of SMNs in long term survivors of childhood rhabdomyosarcoma (RMS) was studied. METHODS: From 1970 to 1989, 210 newly diagnosed patients (median age, 9.7 years; range, 1 month to 27 years) with RMS were treated at Memorial Sloan-Kettering Cancer Center (New York, NY). Multimodality treatment included chemotherapy, surgery, and radiotherapy, when indicated. There were 130 long term survivors (> 2 years off therapy) with a median follow-up of 9 years (range, 2-20 years). The cumulative dose of each chemotherapeutic agent and the radiation doses each patient received were reviewed. Statistical analysis was performed by comparison with the Connecticut Tumor Registry data. RESULTS: Seven patients developed a SMN, including three with acute nonlymphoblastic leukemia (ANLL) and four with solid tumors. Acute nonlymphoblastic leukemia developed a median of 4.5 years after diagnosis. Of the solid tumors, 3 developed within the radiation field at a median of 10 years after diagnosis, whereas the fourth occurred 9.3 years after initial diagnosis in a patient who did not receive radiotherapy. All seven patients with SMNs received total dactinomycin doses higher than the median (9.6 mg/M2) for the group. All three patients with ANLL received total cyclophosphamide doses higher than the median (16.8 g/M2). Moreover, six of the seven patients received a dose of radiotherapy greater than 4000 cGy. The standardized incidence ratio was: 17.07 (95% confidence interval, 6.68-35.18; P < 0.0001). CONCLUSIONS: Multimodality therapy has improved long term survival for patients with childhood RMS. The combination of high dose radiotherapy and chemotherapy appears to increase the risk for developing a second malignancy. PMID- 8625060 TI - Introduction to the U.S.--Italy Workshop on the Molecular Genetics of Cancer. PMID- 8625061 TI - Molecular genetics of cancer. Oncogenes and tumor suppressor genes. PMID- 8625062 TI - Molecular genetics of cancer. Tumor invasion and angiogenesis. PMID- 8625063 TI - Molecular genetics of cancer. Gene therapy and other novel therapeutic approaches. PMID- 8625064 TI - Colorectal cancer genetics. Closing the gap between genotype and phenotype. PMID- 8625065 TI - Acute lymphoblastic leukemia. A comprehensive review with emphasis on biology and therapy. AB - BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. It is now curable in 60-70% of children. Most of the current understanding of the biology and treatment of ALL originates from studies of children. In adults, although much progress has been achieved, ALL is curable in only 20-35% of patients. METHODS: A review of the biology and treatment of ALL from the English literature was performed. RESULTS: Immunophenotypic and cytogenetic analyses of ALL have contributed to a more rational classification of ALL. These analyses have identified subgroups with poor prognosis or with different therapeutic requirements. Overall, 60-70% of adults with ALL have poor prognostic features, including older age, a high leukocyte count, non-T-cell immunophenotype, Ph-positive genotype, and longer time to achieve a complete remission. These patients have a cure rate of 20-25%, whereas those without these risk factors, have a 60-70% probability of survival. The use of more intensive induction regimens with growth factor support may improve survival rates. Also, intensive consolidation-intensification may improve survival rates. Most patients benefit from maintenance therapy, but the dose schedule must be optimized. Central nervous system (CNS) prophylaxis is beneficial, particularly for patients with a high risk for CNS relapse and when introduced early during induction of remission. Patients with high risk characteristics may benefit from allogeneic bone marrow transplantation (BMT) during first remission, and all other patients may benefit from it during first or subsequent relapse. Autologous BMT may be a valuable option for poor compliant patients. CONCLUSIONS: Although the prognosis of patients with ALL has improved markedly during the past decades, newer strategies, including more dose-intensive therapy, the search for new drugs, and more target-specific therapy, are needed to improve the current cure rates. PMID- 8625066 TI - Mechanisms mediating cancer cachexia. AB - BACKGROUND: Cancer cachexia encompasses a wide range of metabolic, hormonal, and cytokine-related abnormalities that result in a wasting syndrome possibly accounting for up to 30% of cancer-related deaths. METHODS: A literature search was performed to review those pathways of metabolic interference involved in cancer cachexia. RESULTS: An elevated basal metabolic rate and increased energy expenditure combined with systemic catabolism of muscle and adipose tissue are the predominant manifestations of the metabolic and physiologic perturbations noted in this pathologic state. CONCLUSIONS: To date, although some of the cachexia-related metabolic abnormalities have been elucidated, there has been little success in relation to therapeutic manipulation of these pathways. This review evaluates current knowledge relating to cancer cachexia and cautions against generalizations concerning treatment regimens. PMID- 8625067 TI - Attenuated familial adenomatous polyposis (AFAP). A phenotypically and genotypically distinctive variant of FAP. AB - BACKGROUND: The usual manifestation of familial adenomatous polyposis (FAP) is hundreds or thousands of colonic adenomas. The authors previously described a colon cancer-prone syndrome characterized by fewer adenomas (1-100), most located in the proximal colon, and upper gastrointestinal lesions, particularly fundic gland polyps and duodenal adenomas. The colonic adenomas are often flat rather than polypoid, a feature emphasized in earlier reports with the term "hereditary flat adenoma syndrome." The syndrome has an autosomal dominant pattern of inheritance and is linked to the adenomatous polyposis coli (APC) locus at 5q. METHODS: This is a descriptive study based on one family that was followed for more than a decade. Total cell RNA was isolated from cultured lymphoblasts, and an in vitro protein synthesis assay was used to detect APC mutations. Sixteen individuals whose APC mutation status was known had sequential endoscopic evaluations. Five patients were given one or more courses of sulindac. RESULTS: There was perfect concordance between clinical affected status and an APC mutation. All affected members generated a 16-kDa polypeptide from the mutant allele, consistent with a 2-base pair deletion at the extreme 5' end of the APC gene. Sixteen mutation-positive individuals underwent upper gastrointestinal endoscopy and colonoscopy; 13 had colonic adenomas, with the number visualized at any one examination ranging from 1 to greater than 50. Upper gastrointestinal examination revealed fundic gland polyps in 15, gastric or duodenal adenomas in 4, and periampullary carcinoma in 1. CONCLUSION: AFAP is a phenotypically distinctive syndrome, differing from classic FAP by having fewer colonic adenomas that tend to be proximally distributed and flat rather than polypoid. The position of the APC germline mutation appears to allow for the molecular differentiation between FAP and the attenuated variant in that the extreme 5' APC mutations are associated with the latter. PMID- 8625068 TI - Characterization of focal hepatic tumors. Value of two-phase scanning with spiral computed tomography. AB - BACKGROUND: Spiral computed tomography (CT) allows imaging of the liver during the peak contrast material levels due to the capability of fast data acquisition. The objective of this study was to evaluate the usefulness of two-phase spiral CT in the differential diagnosis of focal hepatic tumors. METHODS: One hundred two patients who had hepatic tumors (211 nodules; 149 hepatocellular carcinomas [HCCs], 36 metastases, and 26 hemangiomas) underwent two-phase spiral CT with 10 mm collimation at 10 mm/second table speed and 120 mL of contrast material injected at the rate of 3 mL/second. Computed tomography images of the hepatic arterial phase and late (equilibrium) phase were obtained at 35-second and 180 second delays, respectively. The enhancement patterns of tumors were divided into six types and were compared with the attenuation of surrounding liver parenchyma: totally high, peripherally high, centrally high, mixed, iso, and low. RESULTS: The common enhancement patterns of HCC in two-phase spiral CT were totally high in the arterial phase and low (n = 63, 42%) or iso (n = 28, 19%) in the late phase. Metastasis showed peripherally nonnodular high attenuation (n = 9, 25%) or low attenuation (n = 9, 25%) in the arterial phase and low attenuation in the late phase, followed by totally high attenuation in the arterial phase and iso in the late phase (n = 6, 17%). Hemangiomas showed totally or peripherally nodular enhancement in the arterial and late phases (n = 23, 89%). In distinguishing hemangiomas from malignant tumors, totally high or peripherally nodular high attenuation in the late phase was the most useful contrast enhancement pattern (96% of hemangioma vs. 0% of malignant tumors). In distinguishing HCCs from metastases, a combination of contrast enhancement pattern of totally high attenuation in the arterial phase and low in the late phase was the most useful contrast enhancement pattern (42% of HCCs vs. 0% of metastases). The predictability of differentiation between hemangiomas and malignant tumors and between HCCs and metastases was 99% and 90% with spiral CT, respectively. CONCLUSIONS: Two-phase spiral CT is useful in the differential diagnosis of focal hepatic tumors with evaluation of contrast enhancement patterns. PMID- 8625069 TI - Prognostic significance of pathologic features of hepatocellular carcinoma. A multivariate analysis of 278 patients. AB - BACKGROUND: In patients with hepatocellular carcinoma, surgical resection may offer a chance of cure. However, tumor recurrence is not infrequent after resection. METHODS: To identify the pathologic factors that are of prognostic significance and predictive value in tumor recurrence, the authors studied 278 patients (243 men, 35 women) who had hepatectomy for hepatocellular carcinoma. Disease free and actuarial survival were correlated with 20 pathologic parameters of the resected specimens using multivariate analysis. RESULTS: The median follow up period was 23.6 months. The overall disease free survival rates at 1, 3 and 5 years were 42%, 23%, and 17%, respectively, and the overall actuarial survival rates for the corresponding time periods were 70%, 39%, and 28%, respectively. The results indicated that tumor encapsulation (P = 0.004) and heavy intratumor inflammatory infiltrates (P = 0.003) were independent favorable factors related to tumor recurrence. Negative resection margins (P = 0.001) and heavy intratumor inflammatory infiltrates (P = 0.003) were independent favorable factors correlated with survival. CONCLUSIONS: From this analysis, it was determined that detailed histologic examination of resected specimens of hepatocellular carcinoma is important in assessing long term prognosis and stratification of patients for treatment. PMID- 8625070 TI - Clinicopathologic spectrum of resected extraductal mass-forming intrahepatic cholangiocarcinoma. AB - BACKGROUND: The mode of tumor growth of intrahepatic cholangiocarcinoma (CC) varies considerably from patient to patient. This study describes the clinicopathologic variety of the extraductal mass-forming type of CC. METHODS: Patients with CC characterized by an extraductal mass (n = 26) who underwent hepatectomy from 1976 through 1992 were clinicopathologically classified into three types: Type I (n = 7), no biliary stricture; Type II (n = 13), biliary stricture without jaundice; and Type III (n = 6), biliary stricture with jaundice. RESULTS: Type I included three patients with microductular-trabecular arrangement and behavior reminiscent of hepatocellular carcinoma (high association with chronic liver disease, mild positivity for alpha-fetoprotein [AFP], no lymph node metastasis, but frequent intrahepatic metastasis), in contrast to the other typical cholangiocarcinoma. Hepatolithiasis was associated only with Type II CC: The serum positivity for AFP and carcinoembryonic antigen was much higher in Type I CC, whereas positivity of CA 19-9 was highest in Type III. Involvement of the portal vein, hepatic artery, or hepatic duct was most frequent in Type III CC, which necessitated resection of the extrahepatic bile duct and hepatectomy. CONCLUSION: The clinicopathologic behavior of intrahepatic CC differs considerably according to the presence or absence of stricture of the biliary tree. Thus, CC without biliary stricture behaves more like hepatocellular carcinoma, whereas CC with biliary stricture is more like hilar or extrahepatic bile duct carcinoma. PMID- 8625071 TI - Prognostic significance of p53 and ras p21 expression in nonsmall cell lung cancer. AB - BACKGROUND: Alterations of the p53 gene are one of the most common genetic changes in various types of cancer, including lung cancer. Abnormalities in the ras genes, including point mutations and overexpression, are another common feature in the molecular biology of lung cancer and are associated with a poorer prognosis. The authors' purpose was to determine expression of the mutated p53 gene in nonsmall cell lung cancer (NSCLC) specimens that were studied for expression of ras p21 and to document whether altered p53 expression was also an important factor for survival. METHODS: Ninety-six patients with NSCLC underwent surgical resection between 1977 and 1985, 63 of whom received postoperative combination chemotherapy. None received radiation therapy. Tumor specimens were analyzed for altered p53 expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between p53 expression and survival. RESULTS: Fifty-six (58%) of 96 tumor specimens showed altered p53 expression, and 91 patients were analyzed for survival. Altered p53 expression did not correlate with clinicopathologic characteristics except for postsurgical pathologic tumor (pT) classification. The patients with altered p53 expression survived for a significantly shorter period after surgery than those without p53 expression, including all patients who underwent resection and potentially curative resection (P = 0.02 and P = 0.048, respectively, generalized Wilcoxon test). Multivariate analysis showed independent prognostic significance for altered p53 expression (hazard ratio [HR] = 1.72, P = 0.04) and surgical cure (HR = 4.69, P < 0.001). The combined analysis of mutated p53 and ras p21 expression in the same tumor specimens revealed that patients with p53- and ras p21-negative tumors survived the longest among those with different p53 and ras p21 features (P = 0.005, generalized Wilcoxon test). CONCLUSION: Altered p53 expression is a significant and independent negative prognostic factor for patients with surgically resected NSCLC: Combined immunohistochemical analysis of mutated p53 and ras p21 expression can divide patients with NSCLC into more accurate prognostic groups. If the current findings can be confirmed in larger prospective studies, combined immunohistochemical analysis of mutated p53 and ras p21 expression can be a useful clinical tool for stratifying patients with NSCLC into accurate prognostic groups and for identifying the population with a different risk of recurrence. PMID- 8625072 TI - Blood and lymphatic vessel invasion as prognostic factors for patients with primary resected nonsmall cell carcinoma of the lung with intrapulmonary metastases. AB - BACKGROUND: The new classification of intrapulmonary metastases of lung cancer was proposed by the American Joint Committee on Cancer; however, the prognostic factors are heterogeneous and not yet fully clarified. In this study, the authors evaluated the prognostic factors for and the possible routes of intrapulmonary metastases. METHODS: The factors influencing the prognosis of primary resected nonsmall cell lung carcinomas with intrapulmonary metastasis in the resected specimens were evaluated according to the Cox proportional hazards model using a total of 66 nonsmall cell lung carcinomas. The possible routes of tumor spread via the blood or lymphatic vessels also were evaluated. RESULTS: The overall 5 year survival rate was 26.1%, and the statistical analysis of survival curves revealed a significant difference with regard to N classification (P = 0.042), site of intrapulmonary metastasis (P = 0.012), blood vessel invasion (P = 0.0046), and lymphatic vessel invasion (P = 0.0267); there were no significant differences in relation to age, sex, histology, differentiation, T classification, tumor size, stage, number of intrapulmonary metastases, or size of intrapulmonary metastasis. Multivariate analysis according to the Cox proportional hazards model identified a significant correlation between survival and blood vessel invasion (P = 0.044) and lymphatic vessel invasion (P = 0.042), suggesting independent prognostic significance. The correlation between site of intrapulmonary metastasis and the ratio of blood or lymphatic vessel invasion showed a significantly lower ratio of blood vessel invasion in cases with intrapulmonary metastases at sites central to the primary lesion or in different segment(s) compared with those in cases with intrapulmonary metastases at sites peripheral to the primary lesion or in ipsilateral different lobe(s), suggesting a possible lymphatic vessel route of tumor spread. CONCLUSION: Blood vessel and lymphatic vessel invasion are important clinical factors in evaluating prognosis and the route of tumor spread in primary resected nonsmall cell carcinoma with intrapulmonary metastasis. PMID- 8625073 TI - Langerhans' cell histiocytosis (histiocytosis X) of bone. A clinicopathologic analysis of 263 pediatric and adult cases. AB - BACKGROUND: Langerhans' cell histiocytosis (LCH) of bone is a disorder of histiocytic proliferation with variable and often unpredictable behavior. METHOD: The authors evaluated the clinical and pathologic features of 263 patients (172 children, 91 adults) with biopsy-proven LCH examined during an 80-year period at the Mayo Clinic. Only patients with bone involvement pathologically and/or radiographically were included in the study. Clinical follow-up was available for 245 patients and ranged from 3 months to 50 years (mean, 12 years; median, 10 years). Chi-square tests were used to determine associations between age, gender, extent of osseous involvement, visceral disease, and pathologic features. Survival analyses were performed by univariate and multivariate Cox regression methods. RESULTS: Age at presentation ranged from 2 months to 71 years with a clear predominance in children. The most common presenting complaint was pain, often worse at night. The skull was the most frequent osseous site in children and adults. Diabetes insipidus was documented in 40 patients. Forty-four children developed skeletal recurrence and/or new bone lesions, 19 of whom had diabetes insipidus. Fourteen children and 3 adults died either directly or indirectly from LCH. One adult patient developed systemic amyloidosis. All but two of these pediatric patients were 3 years of age or younger at presentation. All children with hepatosplenomegaly (7 patients) and/or thrombocytopenia (9 patients) died. Nine of the 14 children who died presented initially with three or more bone lesions. CONCLUSIONS: The clinical behavior of LCH of bone is often unpredictable; however, young age at diagnosis, hepatosplenomegaly, thrombocytopenia, and polyostotic (> or = 3 bones involved) disease are associated with a poor prognosis (P < 0.005). Recrudescence in children, but not in adults, strongly correlates with the presence of diabetes insipidus (P < 0.0005). PMID- 8625074 TI - Phase II study of oral etoposide for patients with advanced breast cancer. AB - BACKGROUND: The objective of this study was to define the activity and toxicity of etoposide for patients with previously treated metastatic breast cancer. METHODS: Thirty patients with measurable metastatic breast cancer who progressed after prior chemotherapy, hormonal therapy, or both, either as adjuvant therapy or for metastatic disease, were enrolled. There were 26 patients evaluable for response and 30 evaluable for toxicity. Treatment consisted of oral etoposide at a dose of 50 mg/m2/day administered for 21 consecutive days. Doses were modified depending on toxicity. Patients were evaluable for response after at least one cycle of therapy. RESULTS: One complete response and four partial responses were observed. The overall objective response rate was 19% (5/26). Six patients had stable disease. Toxicity consisted mainly of Grade 4 neutropenia for 24% and Grade 4 thrombocytopenia for 13% of patients. There was one death due to neutropenic sepsis. CONCLUSION: Oral etoposide is an effective, easily administered outpatient regimen with minimal toxicities. Etoposide is effective for patients with pretreated metastatic breast cancer. PMID- 8625075 TI - Effects of a very low fat, high fiber diet on serum hormones and menstrual function. Implications for breast cancer prevention. AB - BACKGROUND: Low fat, high fiber dietary interventions that decrease blood estrogen levels may reduce breast cancer risk. Asian women consuming their traditional low fat, high fiber diets have lower blood estrogen levels before and after menopause and lower rates of breast cancer compared with Western women. The current controlled feeding study of premenopausal women was designed to determine the effects of a very low fat (10% of calories) and high fiber (35-45 g/day) diet on blood estrogen levels and menstrual function. METHOD: Twelve healthy premenopausal women with regular ovulatory cycles were followed for 3 months. Subjects consumed a diet providing 30% of their energy from fat and 15-25 g of dietary fiber per day for 1 month, and they consumed a very low fat, high fiber and libitum diet providing 10% of their energy from fat and 25-35 g of dietary fiber per day for 2 months. RESULTS: At the end of the second month of the very low fat, high fiber diet, there was a significant reduction in serum estrone and estradiol levels during the early follicular and late luteal phases. There were no significant changes observed in serum estrone sulfate, sex hormone binding globulin, or progesterone. Despite a significant decrease in serum estradiol and estrone levels after 2 months of a very low fat, high fiber diet, there was no interference with ovulation or the magnitude of the mid-cycle leuteinizing hormone surge. Small changes in menstrual cycle length of up to 3 days were not ruled out due to the small sample size of the study. CONCLUSIONS: A very low fat, high fiber diet in healthy premenopausal women can reduce estradiol and estrone levels without affecting ovulation, thereby providing a rationale for the prevention of breast cancer through a very low fat, high fiber diet. PMID- 8625076 TI - Delaying the initiation of intact breast irradiation for patients with lymph node positive breast cancer increases the risk of local recurrence. AB - BACKGROUND: The impact of delaying irradiation to the intact breast for patients receiving chemotherapy for lymph node positive breast cancer is controversial. METHODS: From 1974 to 1989, 474 patients underwent lumpectomy and intact breast irradiation for early stage invasive breast cancer. Chemotherapy was administered to 84 patients (1 patient with bilateral breast cancer) because of positive axillary lymph nodes. Time from definitive breast surgery (lumpectomy or reexcision) to the initiation of breast irradiation was 21-314 days, with a median of 124 days. Forty-two patients began receiving radiation therapy before 120 days (early) and 42 more than 120 days after surgery (delayed). In the early group, cyclophosphamide/methotrexate/5-fluorouracil (CMF) was administered to 32 patients, doxorubicin, cyclophosphamide or cyclophosphamide, doxorubicin, 5 fluorouracil (AC or CAF) to 6 patients, and other regimens to 4 patients; in the delayed group, CMF was given to 29 patients, CAF to 12 patients, and L-PAM/5 fluorouracil to 1 patient. RESULTS: Median follow-up was 62 months. There was one breast recurrence in the early group, compared with six in the patients receiving delayed irradiation. The actuarial relapse rates for these groups at 5 years were 2% and 14%, respectively (P = 0.05). Survival and distant disease free survival were not significantly different between the two groups. CONCLUSIONS: Delays in the initiation of irradiation are associated with increased risk of relapse in the breast. When possible, the interval between definitive breast surgery (lumpectomy or reexcision) and the initiation of radiation therapy should be fewer than 120 days. PMID- 8625077 TI - Comparison of p53 gene abnormalities in bilateral and unilateral breast cancer. AB - BACKGROUND: The results of recent studies have suggested that p53 gene abnormalities are associated with carcinogenesis in several neoplasms. It is believed that bilateral breast carcinomas develop as a result of a different carcinogenetic mechanism and genetic environment from those of unilateral lesions. METHODS: p53 Gene abnormalities in bilateral primary breast cancer were detected by polymerase chain reaction-single strand comformation polymorphism (PCR-SSCP) analysis. A total of 76 paraffin embedded tissue specimens from 38 patients with bilateral primary breast cancer were examined, and 62 patients with unilateral breast cancer were analyzed as control subjects. The bilateral tumors were defined as primary, based on clinical parameters and the presence of an intraductal component. There were 13 patients with synchronous bilateral breast cancer and 25 with metachronous bilateral breast cancer. RESULTS: p53 Gene abnormalities were detected in 50% of the bilateral and 25.8% of the unilateral cases, and the difference was significant (P < 0.01, chi-square test). Abnormalities were detected in 56% of the metachronous cases, representing a much higher incidence than that of the unilateral cases (P < 0.001, chi-square test). The incidence of p53 gene abnormalities in the first and second tumors of the metachronous cases was 44% and 68%, respectively. The percentage of patients with a p53 gene abnormality and positive family history was higher for those with bilateral than with unilateral breast cancer (P < 0.01, chi-square test). CONCLUSION: These findings indicate that the genetic changes and mechanism of carcinogenesis in bilateral and unilateral breast cancer are different. PMID- 8625078 TI - Application of quantitative analysis to biologic profile evaluation in breast cancer. AB - BACKGROUND: The biologic profile of 907 infiltrating breast carcinomas was determined considering estrogen receptor (ER) and progesterone receptor (PR), proliferation index (PI) and c-erbB-2/Neu expression. The relationship with pathologic parameters (lymph node status, size, histotype) were studied by a multivariate analysis. The clinical prognostic power of biologic profile also was evaluated for 265 patients. METHODS: In 907 infiltrating breast carcinomas, the quantitation of ER, PR, an PI was obtained with an image analysis system (CAS 200, Becton Dickinson Cell Analysis Systems, San Jose, CA); Neu was evaluated semiquantitatively. A clinical study of 265 patients was performed (median follow up, 42.5 months). RESULTS: Seventy-seven percent of tumors were ER-positive, 70% were PR-positive, 58% had a high PI, and 35% were Neu-positive. The overall analysis indicated a direct correlation between ER and PR (Spearmans' rho [rs] = 0.47, P < 0.001) and an inverse correlation between PI and ER (rs = -0.39, P < 0.001), PI and PR (rs = -0.32, P < 0.001), Neu and ER (rs = -0.20, P < 0.001), and Neu and PR (rs = -0.21, P < 0.001). Cluster analysis, performed based on the biologic profile (ER, PR, PI, c-erbB-2/Neu expression), identified two final groups of tumors with different pathologic features. This study showed a longer relapse free interval for patients with ER- and PR- positive tumors (P = 0.016 and P = 0.007) and low PI and Neu-negative tumors (P < 0.001 and P = 0.047). CONCLUSIONS: These results stress the importance of the biologic profile for defining tumor behavior and patient management, leading to integration of, and eventually the substitution for, the actual staging system. PMID- 8625079 TI - Cervical cancer survival in a high risk urban population. AB - BACKGROUND: Cervical cancer remains an important public health problem, particularly for the urban minority population. To the authors' knowledge, determinants of cervical cancer survival have not been studied in this high risk population. METHODS: This study included all 158 women diagnosed and treated for invasive cervical cancer from January 1, 1986, through December 31, 1992, at the Grady Memorial Hospital and Clinics (Atlanta, GA). Medical records were abstracted to determine age at diagnosis, race, International Federation of Gynecology and Obstetrics (FIGO) clinical stage, treatment, and survival. Pathologic material was reviewed to confirm the diagnosis. RESULTS: Most patients (80%) were African American, and the stage distribution was similar for African American and white patients. Sixty-six (42%) had FIGO Stage I disease; 50%, Stage II or III; and 8%, Stage IV. Four-year actuarial survival differed significantly according to clinical stage (Ia = 94%, Ib = 79%, II = 39%, III = 26%, IV = 0%). Overall survival was lower for patients with glandular carcinomas than for those with squamous cell carcinomas (26% vs. 55%, P = 0.09). This difference was almost entirely due to increased mortality in patients with Stage Ib adenocarcinomas (53% vs. 88% for squamous cell carcinoma, Stage Ib, P = 0.03). CONCLUSIONS: The major prognostic markers for cervical cancer survival in this high risk patient population were clinical stage and histology, factors identical to those identified for other populations. PMID- 8625080 TI - Comparative analysis of the sensitivity of endometrial cancer cells to epidermal growth factor and steroid hormones. AB - BACKGROUND: Epidermal growth factor (EGF) and EGF-regulated processes play an important role in steroid signal transduction. Comparative analysis of EGF and steroid receptor expression and the sensitivity of early stages of proliferation induction, such as activation of phospholipid turnover to EGF and steroids, may provide a useful new approach to characterizing the sensitivity of endometrial cancer to hormone therapy. METHODS: Progesterone (PR), estradiol (ER), and EGF receptor (EGFR) content was measured by radioligand competitive methods in surgically excised tumors from 26 patients with primary endometrial cancer. In short term cell cultures isolated from 11 of these tumors, the influence of a 10 minute treatment with 10(-8)M EGF either alone or combined with 10(-8)M progesterone on 32P-incorporation into phospholipids was studied. Phospholipids were fractionated by thin-layer chromatography and were located by autoradiography, and quantification of the labeled compounds was made by densitometric scanning of the autoradiograms. RESULTS: Epidermal growth factor receptor was found in 15 of 26 (58%) endometrial cancer samples. Eighty-two percent of the tumors studied contained PR, and 81% contained ER. No significant correlations were revealed between EGFR and ER/PR status or concentration. Epidermal growth factor stimulated 32P-incorporation by more than 120% of the control level in five of seven EGFR-positive and in one of four EGFR-negative endometrial cancer samples. An inverse relationship was revealed between EGFR content and the percentage of EGF-induced stimulation of phospholipid turnover in endometrial cancer cells (r = -0.6; P = 0.15) and between EGFR content in EGFR positive samples and the extent of progesterone suppression of EGF-induced turnover (r = -0.77; P = 0.04). CONCLUSIONS: Determination of EGF sensitivity on a receptor and a functional level may provide important additional information about the hormonal sensitivity of endometrial cancer. PMID- 8625081 TI - Distant metastasis after radical prostatectomy in patients without an elevated serum prostate specific antigen level. AB - BACKGROUND: Serum prostate specific antigen (PSA) is a sensitive indicator of prostate cancer recurrence after radical prostatectomy. Prostate cancer rarely recurs after radical surgery without PSA elevation. Of the few patients noted in the literature who had a recurrence of cancer without PSA elevation, all had local recurrence alone, except for one, who had bone metastases. METHODS: In the authors' series of 628 patients, PSA was the first indicator of recurrence in all but 2 (2.6%) of 77 patients with clinical T1-T3NxM0 classification prostate cancer. RESULTS: Two of our patients, despite having undetectable PSA levels, had distant recurrence, including one with multiple visceral (lung and brain) metastases. CONCLUSIONS: These two cases demonstrate that although uncommon, prostate cancer can recur and metastasize after radical prostatectomy without an increase in the serum PSA level. PMID- 8625082 TI - Survival after radical retropubic prostatectomy of men with clinically localized high grade carcinoma of the prostate. AB - BACKGROUND: This study was performed to evaluate the efficacy of radical prostatectomy for men with clinically localized, poorly differentiated (Gleason score > or = 7) prostate cancer and to characterize further the prognostic significance of traditional pathologic variables. The effectiveness of adjuvant radiotherapy was assessed in a subpopulation of men for whom the pathologic assessment suggested a high risk of persistent disease. METHODS: Two hundred thirty-eight consecutive men, 74 of whom had clinically localized, poorly differentiated carcinoma, were followed for a median of 6.2 and 5.1 years, respectively. The disease specific outcomes were derived from a non-prostate specific antigen (PSA) screened population. RESULTS: The 5-year disease specific survival (DSS) for 52 men with a clinically localized Gleason score of 7 and for 22 men with a Gleason score greater than or equal to 8 carcinoma was 92% and 79%, respectively. The 5-year likelihood of having an undetectable PSA level was 50% for those with a Gleason score of 7 and 38% for those with a Gleason score greater than or equal to 8. Gleason score was the most powerful pathologic predictor of disease progression and survival. Pathologic stage was significantly associated with disease progression for carcinomas with Gleason scores less than 7 but was found to be less predictive of progression for carcinomas with Gleason scores greater than or equal to 7. Adjuvant radiotherapy provided a significantly reduced risk of PSA-detectable progression (P = 0.02, relative risk = 0.56, 95% CI: 0.34, 0.92); however, radiotherapy had no significant impact on DSS. CONCLUSIONS: Long term DSS is possible in a non-PSA screened series of men with poorly differentiated prostate cancer treated by radical prostatectomy. These results compare favorably with alternative treatment strategies, although they do illustrate a continued need to develop more effective adjuvant therapies for men with poorly differentiated prostate cancer. PMID- 8625083 TI - Prognostic value of nuclear grade of renal cell carcinoma. AB - BACKGROUND: The authors assessed the interest and the value of Fuhrman's nuclear grade as a possible prognostic factor for renal cell carcinoma (RCC). METHODS: An 11-year retrospective study of 190 patients with RCC treated by radical nephrectomy was performed. The distribution by grade was: Grade I, 54 patients; Grade II, 58; Grade III, 58; and Grade IV, 20. The distribution of the patients by tumor stage according to the TNM15 classification was: pT1, 56 patients; pT2, 41; pT3a, 55; pT3b, 25; pT3c + pT3d + pT4b, 5; and pT4a, 8. Significant correlations with other prognostic parameters were noted. Survival curves by grade were evaluated by the Kaplan-Meier method. RESULTS: Nuclear grade was correlated with tumor stage (P = 0.0001), synchronous metastases (P = 0.003), lymph node involvement (P = 0.0001), renal vein involvement (P = 0.0001), tumor size (P = 0.0001), and perirenal fat involvement (P = 0.001). No correlation was found between nuclear grade and tumor multicentricity (P = 0.14) and cell type (P = 0.2). Nuclear grade was an effective parameter in predicting development of distant metastases after nephrectomy. Among the 54 patients who presented with Grade I tumors, only one tumor did metastasize during the 5-year follow-up, whereas 17% of the Grade III and 30% of the Grade IV tumors metastasized. The 5 year actuarial survival rates of the patients with Grade I, II, III, and IV tumors was 76%, 72%, 51%, and 35%, respectively. The comparison of the survival curves by grade showed a statistically significant difference between the curves when Grade I and II tumors were compared with Grade III and IV tumors (P = 0.001). CONCLUSION: In this study, nuclear grade was found to have prognostic significance and seems to be an important criterion when considering the outcome of patients with RCC. PMID- 8625084 TI - Helium-neon laser effects on conditioning-induced oral mucositis in bone marrow transplantation patients. AB - BACKGROUND: Oral mucositis is a common complication of bone marrow transplantation (BMT) conditioning therapy. Sequelae consist of increased risk for infection, moderate to severe pain, compromised oral function, and bleeding. This study investigated helium-neon laser treatment for prevention of conditioning-induced oral mucositis in BMT patients. Patterns and severity of mucositis for specific conditioning drug regimens also were analyzed. METHODS: Twenty patients received laser radiation to their oral mucosa, either left or right of midline. The contralateral side was sham-treated and served as a control. Mucositis severity was scored independently by two modified versions of the Oral Mucositis Index Scale (OMI-A and OMI-B) and the Eastern Cooperative Oncology Group (ECOG) Oral Toxicity Scale; pain severity was scored by subjects on a visual analogue scale (VAS). Cumulative scores were analyzed for differences between the laser-treated and sham-treated sides. RESULTS: Oral mucositis and pain scores were significantly lower for the treated versus the untreated side by OMI-A and B (P < 0.005) and VAS (P = 0.027) criteria, respectively. Ulcerative lesions occurred in all patients bilaterally; severity increased until Day +6, and lesions resolved by Day +21. Mucositis was more severe for patients conditioned with busulfan/carboplatin/thiotepa than for patients conditioned with busulfan/cyclophosphamide/etoposide. CONCLUSIONS: Helium-neon laser treatment was well-tolerated and reduced the severity of conditioning-induced oral mucositis in BMT patients. PMID- 8625085 TI - A prospective evaluation of ifosfamide-related nephrotoxicity in children and young adults. AB - BACKGROUND: Ifosfamide has been associated with proximal renal tubular dysfunction resembling Fanconi-like syndrome and leading to rickets in young children. The characteristic manifestations of this nephrotoxicity include phosphaturia and hypophosphatemia, glycosuria, aminoaciduria, renal tubular acidosis, and urinary loss of low molecular weight serum proteins. However, the relationship between acute ifosfamide nephrotoxicity, which is frequently subclinical, and long term renal damage is unclear. In this prospective study, the laboratory features of ifosfamide-induced acute nephrotoxicity were characterized further and correlated with the development of chronic nephropathy. METHODS: The renal function of newly diagnosed children and young adults with high risk sarcomas was followed during therapy with a high dose ifosfamide containing regimen. Serum and urine were collected regularly immediately before and after 5-day cycles of ifosfamide throughout treatment for determination of the fractional excretion of electrolytes (sodium, potassium, phosphate, magnesium, calcium) and glucose and urinary excretion of amino acids and beta 2 microglobulin. RESULTS: Significant changes in the renal threshold of phosphate excretion, the fractional excretion of calcium and glucose, and the urinary excretion of beta 2-microglobulin were observed when comparing pretreatment values with those at the end of a 5-day treatment cycle. The median renal threshold of phosphate excretion decreased from 1.22 to 0.82 mmol/L (P < 0.0001). The median fractional excretions of calcium and glucose increased from 1.05% to 1.68% (P < 0.0001) and 0.05% to 0.08% (P = 0.0006), respectively. Beta 2 microglobulin excretion increased by 70-fold from 0.02 to 1.42 mg/mmol (P < 0.0001). Except for glucose and beta 2-microglobulin excretion, renal parameters returned to baseline before the next ifosfamide treatment cycle. Acute aminoaciduria was observed in 21 of 23 patients. Chronic nephrotoxicity, as defined by the development of a Fanconi-like syndrome or chronic tubular electrolyte loss requiring oral supplementation, developed in the three patients with the highest urinary excretion of beta 2-microglobulin after ifosfamide therapy. CONCLUSIONS: Prospectively, high dose ifosfamide was associated with a 4% incidence of Fanconi-like syndrome; however, evidence of acute reversible subclinical nephrotoxicity was observed for all patients. Severe beta 2 microglobulinuria appeared to be a prognostic laboratory indicator for the development of chronic nephrotoxicity. PMID- 8625086 TI - Characterization of extracellular matrix-degrading proteinase and its inhibitor in gynecologic cancer tissues with clinically different metastatic form. AB - BACKGROUND: The authors conducted a comparison study of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-1 (TIMP-1) activities in clinically different metastatic types of ovarian cancer, cervical cancer, and endometrial cancer tissues. METHODS: Gelatinase activity in culture medium obtained from each cancer tissue was detected by zymography and was quantitated by densitometer. Tissue inhibitor of metalloproteinase-1 activity was measured in culture medium by the human TIMP-1 enzyme immunoassay kit. RESULTS: Six dominant gelatinases were detected in ovarian, cervical, and endometrial cancers: 200-kDa; 130-kDa; 92-kDa (MMP-9); 83-kDa, which is an active form of 92-kDa gelatinase; 72 kDa (MMP-2); and 66-kDa gelatinase, which is an active form of 72-kDa gelatinase. The 92-kDa and 72-kDa gelatinolytic bands were present in all samples. The expression rates of 200-, 130-, and 83-kDa gelatinase in endometrial cancer and cervical cancer tissues were higher than that observed in ovarian cancer tissue. Densitometric analysis showed that the 92-kDa/72-kDa ratio was significantly higher in cervical cancer tissue than in ovarian and endometrial cancer tissues (P < 0.05), and the 66-kDa/72-kDa ratio was significantly higher in endometrial cancer tissue than in ovarian cancer tissue (P < 0.01). Tissue inhibitor of metalloproteinase-1 activity was significantly lower in cervical cancer tissue than in ovarian and endometrial cancer tissues (P < 0.01). CONCLUSIONS: These results reflect the difference of metastatic forms and are indicative of the possibility of the strong relationship to MMP activity in the invasion and metastasis of cervical cancer and endometrial cancer, compared with those of ovarian cancer. PMID- 8625087 TI - Management of primary central nervous system lymphoma for the patient with acquired immunodeficiency syndrome. Confronting a clinical catch-22. PMID- 8625088 TI - Monitoring versus blunting styles of coping with cancer influence the information patients want and need about their disease. Implications for cancer screening and management. AB - BACKGROUND: Two main psychologic coping styles for dealing with cancer and other health threats have been identified: monitoring (attending to) or blunting (avoiding) potentially threatening information. This article reviews results and implications from this research relevant to cancer screening and management. METHODS: The Monitor-Blunter Style Scale has been used extensively to assess and categorize patients with regard to these coping styles to predict their differential responses to various cancer-related screening and management regimens. RESULTS: Patients characterized by a monitoring coping style generally are more concerned and distressed about their cancer risk, experience greater treatment side effects, are more knowledgeable about their medical situation, and are less satisfied with and more demanding about the psychosocial aspects of their care. They also prefer a more passive role in clinical decision making, are more adherent to medical recommendations, and manifest greater psychologic morbidity in response to cancer-related threats. CONCLUSIONS: Patients fare better (psychologically, behaviorally, and physiologically) when the information they receive about their medical condition is tailored to their own coping styles: generally those with a monitoring style tend to do better when given more information, and those with a blunting style do better with less information. However, patients with a monitoring style who are pessimistic about their future or who face long term, intensely threatening, and uncontrollable medical situations may require not just more information, but also, more emotional support to help them deal with their disease. PMID- 8625089 TI - Prognosis of early esophageal cancer. Comparison between adeno- and squamous cell carcinoma. AB - BACKGROUND: The purpose of this study was to compare the prognosis of patients with T1 squamous cell carcinoma (SCC) with those with T1 adenocarcinoma of the esophagus and to explain prognostic differences by an analysis of clinicopathologic characteristics. METHODS: Seventy-seven patients with early esophageal cancer who underwent esophagectomy and lymphadenectomy from 1982 to 1993 were included in the study. Clinical and histopathologic characteristics, patterns of lymph node metastasis, results of surgery, and long term prognosis of 47 patients with SCC were compared with 30 patients with adenocarcinoma; a multivariate analysis of various prognostic factors was performed. RESULTS: The groups with adenocarcinoma and SCC were comparable regarding age, postoperative 90-day mortality (6.6% vs. 8.5%), infiltration of submucosa (74.5% vs. 80%), and rate of lymph node metastasis (17% vs. 16.6%). Cancer limited to the mucosa was not associated with lymph node metastasis in either group, whereas submucosal spread showed lymph node involvement in 21% of patients with adenocarcinoma and 26% of those with SCC. The 5-year survival rate of patients with complete tumor removal was superior for those with adenocarcinoma (82.5%) compared with those with SCC (59.2%) (P < 0.03). Multivariate analysis indicated that the histopathologic type (adenocarcinoma vs. SCC) was the only independent prognostic factor. The unfavorable prognosis of patients with T1 SCC was due to a higher recurrence rate and the more frequent development of second primary tumors (21% vs. 0%). CONCLUSIONS: The prognosis of patients with early esophageal cancer depends on the histologic tumor type. Patients with T1 SCC should be examined for another primary cancer before surgery and during follow-up. PMID- 8625090 TI - Growth factor expression and proliferation kinetics in periampullary neoplasms in familial adenomatous polyposis. AB - BACKGROUND: Patients with familial adenomatous polyposis develop periampullary adenomas at a high rate. However, little is known regarding the factors that control the growth, the natural history, or the malignant potential of these tumors. METHODS: In this study, the authors systematically evaluated the expression of the intestinal peptide growth factor, transforming growth factor alpha (TGF-alpha), and its corresponding receptor, epidermal growth factor receptor (EGF-R), in 49 periampullary adenomas and 6 periampullary carcinomas from 29 patients. Tumor proliferation rates were evaluated with the MIB-1 antibody. RESULTS: All periampullary adenomas and carcinomas (100%) had TGF-alpha expression, whereas 63% of adenomas and 67% of carcinomas expressed EGF-R. The extent of TGF-alpha expression was greater in carcinomas compared with adenomas and increased progressively in adenomas relative to the degree of dysplasia and villous architecture of these lesions. The extent of EGF-R expression correlated only with the degree of dysplasia in adenomas. With regard to proliferation kinetics, higher MIB-1 labeling indices were observed in adenomas that were larger, more severely dysplastic, and villous. Transforming growth factor-alpha, and to a lesser extent, EGF-R expression, correlated directly with the MIB-1 labeling indices. CONCLUSIONS: These results support the adenoma-carcinoma sequence in the progression of malignancy in the duodenum in familial adenomatous polyposis, suggesting a possible involvement for TGF-alpha and EGF-R expression in this process. PMID- 8625091 TI - Flow cytometric identification of cell surface markers on cultured human colonic cell lines using monoclonal antibodies. AB - BACKGROUND: The expression of tumor-associated cell surface antigens is a reflection of the state of cell differentiation of tumor cells in culture. METHOD: Monoclonal antibodies (MoAbs) against the tumor-associated antigens carcinoembryonic antigen (CEA) and CA19-9 and the extracellular matrix protein CD44 were used to label the cell surface of human colonic cells in culture. The binding of each antibody to its respective antigen was measured by fluorescence activated flow cytometry and expressed as a percentage of positive cells. RESULTS: The human colon adenocarcinoma cell (HCAC) line, LS-180, showed strong binding with CEA (81%), CA 19-9 (87%), and CD44 (83%). LS-174t cells, a trypsinized variant of LS-180 cells, showed less binding with CEA (66%) and CA 19 9 (49%), but no binding with CD44. With cells from HCAC line HT-29, antigen expression was highly variable for CEA (13% +/- 18) and CD44 (31% +/- 35) but was consistently positive for CA19-9 (33% +/- 13). The expression of CEA in the Caco 2 cell line was weak (24%), whereas there was no expression of CA19-9 and CD44. Normal human colon fibroblast cells (CCD-18Co) did not recognize the monoclonal antibodies to CEA or CA 19-9, but were strongly positive with the CD44 antibody (97%). CONCLUSIONS: These results support the concept that the expression of the tumor associated markers CEA and CA19-9 and the cell surface marker CD44 on human colonic cell lines varies with the degree of cellular differentiation. Carcinoembryonic antigen and/or CA19-9 were expressed in all four human colon adenocarcinoma cell lines, but not in the normal colon fibroblast cells (CCD 18Co). Using these two MoAbs appeared to be a more reliable measure of the state of differentiation of human colon adenocarcinoma cells. PMID- 8625092 TI - Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme linked immunosorbent assay. AB - BACKGROUND: Point mutations of the ras protooncogene, primarily within codons 12 and 13, are commonly identified in colorectal carcinomas and large adenomas. Despite data suggesting that ras genotyping may have clinical significance with respect to colorectal cancer screening and prognosis, more widespread use has been limited because of the lack of a suitable assay system. The principal objective of this study was to assess the feasibility and validity of a qualitative enzyme-linked immunosorbent assay (ELISA) for detecting the four most common ras mutations in human colorectal tumors at the protein (p21ras) level. METHODS: Tissue homogenates (11-121 micrograms) from endoscopically or surgically resected colorectal adenomas, carcinomas, and normal mucosae were evaluated by a commercially available ELISA (Oncogene Science, Inc. Cambridge, MA) for mutant p21ras containing arginine position 12 (arg12), valine position 12 (val12), aspartate position 12 (asp12), and aspartate position 13 (asp13) amino acid substitutions. Portions of the same tissue from an initial series of 27 specimens also were subjected to mutant-enriched polymerase chain reaction (PCR) and/or PCR amplification with subsequent DNA sequence analysis to validate the ELISA data. RESULTS: Forty-seven adenomas, 9 carcinomas, and 14 normal mucosae were assayed. Mutations were identified in 16 (34%) of the adenomas (7-asp12, 7-val12, 2 asp13), 3 (33%) of the carcinomas (1-asp12, 1-arg12, 1-asp13), and none of the normal mucosae by ELISA: Polymerase Chain Reaction and DNA sequencing analyses demonstrated identical results for 21 of the 23 (91%) and 14 of 16 (88%) homogenates tested, respectively. The ELISA demonstrated an overall sensitivity of 80-86%, specificity of 90-92%, positive predictive value of 86-100%, and negative predictive value of 86-91%. CONCLUSIONS: The ELISA is a feasible and valid approach for identifying p21ras mutations in human colorectal adenomas and carcinomas. PMID- 8625093 TI - Hepatic cryosurgery in treating colorectal metastases. AB - BACKGROUND: The purpose of this study was to determine the effectiveness of cryosurgery as an adjunct to resection in treating patients with hepatic metastases from colorectal adenocarcinoma. METHODS: Forty-seven patients with documented metastases limited to the liver from colorectal adenocarcinoma were treated with cryosurgery with or without resection from November 1987 to February 1992 and were followed until February 1994. Intraoperative ultrasound was used to map the lesions and place the cryoprobes. Each lesion was frozen to -196 degrees centigrade for 15 minutes, thawed for 10 minutes, and frozen again for 15 minutes. Follow-up computed tomographic scans were obtained before hospital discharge and 6 months and 1 year after cryosurgery. Carcinoembryonic antigen levels were obtained monthly. RESULTS: Thirty-one males and 16 females, with a median age of 63 years, were treated. The median hospital stay was 10 days, and follow-up ranged from 24 to 57 months, with a median follow-up of 26 months. The actual survival at 24 months was 62%. Eleven percent of these patients had no evidence of disease at a median follow-up of 30 months. Complications included myoglobinuria, coagulopathy, pleural effusions, and bile duct injuries. Two patients (4%) died because of multisystem organ failure with irreversible coagulopathies. CONCLUSIONS: Cryosurgical ablation increases the number of patients with liver metastases who potentially can become disease free. However, the effect on overall survival will require a longer follow-up period. PMID- 8625094 TI - Detection efficiency of colorectal carcinoma recurrence using technetium pertechnetate-anti-carcinoembryonic antigen monoclonal antibody BW 431/26. AB - BACKGROUND: A new anti-carcinoembryonic antigen (CEA) antibody, BW 431/26 (Scintimun, Behring-Werke, Marburg, Germany), labeled with technetium pertechnetate (Tc-99m), is an intact immunoglobulin G1 monoclonal antibody that has been used to image colorectal cancer (CRC). This report is part of a prospective multicenter clinical trial initiated by the International Atomic Energy Agency to evaluate the role of this antibody in radioimmunoimaging of patients with suspected disease recurrence. METHODS: A group of 31 consecutive patients underwent radioimmunoimaging with Tc-99m-BW 431/26 after resection of their primary CRC. Patient referral was based on either a persistent rise in serum CEA levels of unknown origin and/or questionable findings by other imaging studies. Whole-body planar scans and single photon emission computed tomography scans of selected body regions (e.g., chest, abdomen) were performed up to 24 hours after the intravenous antibody injection. Pathologic antibody concentration localizations by radioimmunoimaging were correlated with surgical, clinical, and other imaging modality findings to validate the accuracy of radioimmunoimaging in detecting CRC recurrence. RESULTS: A total of 75 detected tumoral lesions was evaluated: 26 of 75 were of known origin (36%), and 49 of 75 were of unknown origin (65%). There were four true-negative lesions, one false-negative lesion, and no false-positive lesions; all others were true-positive lesions. Sensitivity was 96.8%, specificity 100%, and accuracy 98.6%. The study was easy to perform, without untoward side effects on patients after antibody administration. CONCLUSIONS: Anti-CEA antibody radioimmunoimaging is a highly reliable diagnostic procedure in detecting CRC recurrence and is useful especially for the diagnosis of patients with rising CEA blood levels of unknown origin, thereby significantly affecting patient management. Radioimmunoimaging should become part of the diagnostic workup of patients suspected of having CRC recurrence. PMID- 8625095 TI - p53 expression in dedifferentiated chondrosarcoma. AB - BACKGROUND: p53 acts as a tumor suppressor gene because of to its negative control of the cell cycle and its central role in programmed cell death. It frequently is mutated, as observed in a variety of human neoplasms. The mutations inhibit tumor-suppressor activities of p53, which may gain a new function of tumor promotion. In this study, p53 was investigated in various components of dedifferentiated chondrosarcoma and correlated with their proliferative activities. METHODS: Immunohistochemical assays for p53, Ki-67, and proliferating cell nuclear antigen (PCNA) were used in a series of eight dedifferentiated chondrosarcomas of bone. The cartilaginous component was low grade (Grade I-II) in five cases. It was predominantly low grade with foci of a high grade (Grade III) chondrosarcoma in the remaining three cases. The noncartilaginous (dedifferentiated) high grade component consisted of malignant fibrous histiocytoma in five cases and osteosarcoma in three. RESULTS: Regardless of the histological type, diffuse strong nuclear staining for p53 occurred in the high grade noncartilaginous component of all eight of the tumors. The low grade cartilaginous component of six cases was negative for p53, with focal weak staining in the two remaining cases. The high grade cartilaginous component showed strong positive staining for this protein in all three cases. Ki-67 and PCNA expression were similar to that of p53. CONCLUSIONS: The percentage of p53 positive staining roughly was parallel to the proliferating fraction of cells in various components of dedifferentiated chondrosarcoma. Moreover, p53 overexpression was consistently present in the high grade noncartilaginous (dedifferentiated) component of the tumor and was accompanied by increased proliferative activity. PMID- 8625096 TI - Impaired cell-mediated immunity of apparently normal patients who had multiple skin cancers. AB - BACKGROUND: Investigations of the immune status of patients with skin cancer have had conflicting results, possibly because uniform groups of patients were not studied. Patients who had multiple skin cancers (three or more) were studied to determine whether they had impaired cell-mediated immunity (CMI). METHODS: Thirty four patients and 34 matched control subjects were studied who were younger than 60 years, were not immunosuppressed, and were free from internal cancer. Cell mediated immunity was tested by the cutaneous reaction to recall antigens (Multitest CMI, Pasteur Merieux, Lyon, France) and the estimation of lymphocyte levels. RESULTS: Patients had significantly lower Multitest CMI scores (P < 0.0005), lower lymphocyte counts (P < 0.02), and were more likely to have a first degree relative with skin cancer (P < 0.002). Multitest CMI scores decreased with the number of skin cancers removed (P < 0.0006) and were a significant predictor for CD4 (P < 0.04) and CD8 cell counts (P < 0.02). CONCLUSIONS: Patients who had multiple skin cancers had impaired CMI, and the degree of impairment correlated with the number of skin cancers removed. PMID- 8625097 TI - Central nervous system progression of metastatic breast cancer in patients treated with paclitaxel. AB - BACKGROUND: The possibility of tumor sanctuary sites in the central nervous system (CNS) of patients receiving paclitaxel has been suggested by laboratory data identifying low concentrations of drug in the brain and cerebrospinal fluid (CSF) of rats. METHODS: The pattern of disease progression in patients with metastatic breast cancer who had an initial response to paclitaxel treatment in five Phase II trials at the Memorial Sloan-Kettering Cancer Center was reviewed. RESULTS: Of 152 patients, 53 had a partial or complete response, and 25 had a minor response. Of the 78 patients who responded to paclitaxel, 52 had subsequent disease progression, 22 changed treatments before progression occurred (as specified by the protocol and/or to receive high dose consolidation chemotherapy), 2 stopped receiving treatment because of toxicity, 1 continued receiving treatment, and 1 died with no evidence of disease progression. Six of the 52 patients who progressed after initially responding to paclitaxel had isolated CNS progression while maintaining their systemic response (leptomeningeal metastasis in three, brain metastases in two, brain and leptomeningeal metastases in one). One patient had CNS progression (brain metastases) associated with other systemic sites of progression. All patients with CNS disease developed neurologic symptoms, prompting neurologic evaluation; one had only a mild headache, which was not recognized until evaluation for paclitaxel-related peripheral neuropathy. CONCLUSIONS: These data suggest that the CNS, and particularly the CSF, is an important sanctuary site for patients with metastatic breast cancer receiving paclitaxel. PMID- 8625098 TI - Conditional survival of 56,268 patients with breast cancer. AB - BACKGROUND: Survival rates calculated from the date of diagnosis may not be predictive of future outcome for patients who have already survived several years after diagnosis. Conditional survival rates are more informative clinically because they take into account survival after diagnosis. METHODS: Conditional relative survival rates were calculated by the life-table method using data from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. RESULTS: Survival rates up to 8 years for patients having survived 1, 2, 3, 4 or 5 years after diagnosis are presented by stage of disease for 56,268 women who were diagnosed as having invasive breast cancer from 1983 to 1987. CONCLUSIONS: Women with Stage IV breast cancer had a better survival as they moved further in time from their diagnosis. Survival rates did not improve for those with Stage I and II disease regardless of the number of years they survived after diagnosis. PMID- 8625099 TI - Timing of weight gain and breast cancer risk. AB - BACKGROUND: Obesity and body-fat distribution have been associated with the incidence of breast and endometrial cancers. It may be critical to determine if the timing of weight gain during periods of hormonal change, such as menarche, pregnancy, or menopause, has different biologic effects, especially secondary to differences in the localization of body fat during these periods. The objective of the current study was to determine if excess weight in any particular decade of life or the timing of weight gain was more significant relative to breast cancer risk. METHODS: Anthropometric, medical, and hormonal histories were obtained from 218 consecutively recruited, newly diagnosed patients with breast cancer admitted to the H. Lee Moffitt Cancer Center and Research Institute (Tampa, FL) and 436 control subjects, matched in a two to one ratio for age and menopausal status. RESULTS: A weight gain of 15 pounds or more was observed for 63.8% of the patients compared with 49.3% control subjects (P = 0.0006) from age 30 to current age. Similarly, more than 48% of cases gained more than 15 pounds from ages 16 to 30 compared with 37% (P = 0.01) of the control population. Although weight gain from age 16 to adulthood was significantly higher in patients with breast cancer at each decade when compared with control subjects, a significant and independent association between weight at age 30 (P < 0.0001) and risk of breast cancer was noted. CONCLUSION: Women who progressively gain weight from puberty to adulthood, and specifically in the third decade of life, should be considered at a higher risk for developing breast cancer. PMID- 8625100 TI - Chromosome abnormalities in bilateral breast carcinomas. Cytogenetic evaluation of the clonal origin of multiple primary tumors. AB - BACKGROUND: Although acquired somatic mutations presumably are crucial in carcinogenesis, nothing is known about the chromosome aberrations of bilateral breast carcinomas. METHODS: Eighteen specimens from 16 bilateral carcinomas were analyzed cytogenetically. The banding analysis was supplemented with fluorescence in situ hybridization with painting probes. RESULTS: In two cases, the finding of the same clonal abnormalities in samples from both breasts indicated that the bilaterality had arisen through a metastatic process. In the remaining cases, the absence of similarities between the two sides indicated an independent origin of the two carcinomas. Also, in multifocal lesions within the same breast, examples were found both of karyotypically related and unrelated clones. Altogether, multiple clones without similarities were detected in nine specimens, sometimes together with other, karyotypically related clones. There was no indication that bilateral carcinomas of the breast are cytogenetically different from unilateral ones. The following chromosomal abnormalities were recurrent: der(1;16)(q10;p10), del(1)(q11-n12), del(1)(q42), and del(3)(p12-n13p14-n21). CONCLUSIONS: Bilateral breast carcinomas have the same cytogenetic aberrations, including evidence of polyclonality, as unilateral carcinomas. The majority apparently arise independently, but some result from a metastasis from one breast to the other. In this sense, bilateral breast carcinomas are similar to multifocal breast cancer in general, of which bilateral tumors may represent a special case. PMID- 8625101 TI - The importance of the lumpectomy surgical margin status in long-term results of breast conservation. AB - BACKGROUND: The impact of the surgical margin status on long-term local control rates for breast cancer in women treated with lumpectomy and radiation therapy is unclear. METHODS: The records of 289 women with 303 invasive breast cancers who were treated with lumpectomy and radiation therapy from 1972 to 1992 were reviewed. The surgical margin was classified as positive (transecting the inked margin), close (less than or equal to 2 mm from the margin), negative, or indeterminate, based on the initial biopsy findings and reexcision specimens, as appropriate. Various clinical and pathologic factors were analyzed as potential prognostic factors for local recurrence in addition to the margin status, including T classification, N classification, age, histologic features, and use of adjuvant therapy. The mean follow-up was 6.25 years. RESULTS: The actuarial probability of freedom from local recurrence for the entire group of patients at 5 and 10 years was 94% and 87%, respectively. The actuarial probability of local control at 10 years was 98% for those patients with negative surgical margins versus 82% for all others (P = 0.007). The local control rate at 10 years was 97% for patients who underwent reexcision and 84% for those who did not. Reexcision appears to convey a local control benefit for those patients with close, indeterminate, or positive initial margins, when negative final margins are attained (P = 0.0001). Final margin status was the most significant determinant of local recurrence rates in univariate analysis. By multivariate analysis, the final margin status and use of adjuvant chemotherapy were significant prognostic factors. CONCLUSIONS: The attainment of negative surgical margins, initially or at the time of reexcision, is the most significant predictor of local control after breast-conserving treatment with lumpectomy and radiation therapy. PMID- 8625102 TI - Ethnic differences in risk and prognostic factors for breast cancer. AB - BACKGROUND: Poor survival among African American patients with breast cancer has been attributed to low socioeconomic status and lack of access to health care. However, Hispanics of equivalent socioeconomic status and health care access exhibit much higher survival rates, almost comparable to whites. This suggests that biologic differences play a role in differences in breast cancer survival in addition to socioeconomic and health care access factors. METHODS: The authors studied clinical and molecular differences between patients with breast cancer of different ethnicity to determine biologic explanations for the observed differences in survival. Consecutive patients scheduled for breast biopsies were identified preoperatively and were interviewed. Blood was withdrawn for serum marker measurements, and tumor specimens collected at frozen section diagnosis were analyzed by flow cytometry, hormone receptor concentration, tumor grade, and Ki-67 nuclear antigen, HER-2/neu, and epidermal growth factor oncoprotein expression. RESULTS: Age, age at menarche, number of lymph nodes with metastasis, estrogen and progesterone receptor levels, ploidy status, S-phase, Ki-67, HER 2/neu expression, tumor grade, epidermal growth factor receptor expression, lipid associated sialic acid (LASA), and carcinoembryonic antigen level were not significantly related to ethnicity. African Americans presented at a significantly more advanced stage and with significantly larger tumors. They were significantly heavier and had a significantly higher mean Quetelet's index and a significantly higher number of pregnancies and number of live births. Whites and Hispanics were significantly older at menopause. CONCLUSIONS: The molecular indices associated with breast cancer prognosis do not differ significantly among whites, African Americans, and Hispanics, suggesting that the reported differences in survival among these groups are not due to biologic differences in breast cancer among ethnic groups. PMID- 8625103 TI - Better breast cancer survival for postmenopausal women who are less overweight and eat less fat. The Iowa Women's Health Study. AB - BACKGROUND: The authors sought to determine whether prediagnosis obesity, body fat distribution, and dietary intake of fats, antioxidants, and fiber may be related to survival after the diagnosis of breast cancer. METHODS: The mortality rates of 698 postmenopausal patients with unilateral breast cancer in a large cohort study were analyzed. Body-mass index, waist-to-hip ratio, and food frequency data were collected by questionnaire within 6 years before breast cancer was diagnosed. RESULTS: Adjusted for age, women in the highest tertile of body mass index had a 1.9-fold higher risk (95% confidence interval = 1.0-3.7) of dying after breast cancer than those in the lowest tertile; adjusted for other prognostic variables (age, smoking, education level, extent of breast cancer, and tumor size), this relative risk was 1.5 (95% CI = 0.7 to 2.9). Waist-to-hip ratio was not related to risk of dying nor was intake of fiber or several dietary antioxidants. Independent of other prognostic variables, risk of death after breast cancer was statistically significantly elevated, with a relative risk greater than 2.0 for the highest tertiles of total fat, saturated fat, and monounsaturated fat intake, expressed as grams per day. An adjustment for energy intake, which also was associated positively with fatality, weakened these associations somewhat. CONCLUSIONS: Although clinical trials are required, these findings support the hypothesis that a high fat intake is associated with reduced survival of postmenopausal women with breast cancer and suggest that women with breast cancer should consider limiting their intake of fat. PMID- 8625105 TI - Overexpression or mutation of the p53 tumor suppressor gene does not occur in malignant ovarian germ cell tumors. AB - BACKGROUND: The p53 tumor suppressor gene has been well studied in epithelial ovarian cancers. However, little is known of the expression of this gene in ovarian germ cell tumors. The authors attempted to investigate whether p53 alterations occurred in this group of tumors. METHODS: Twenty-two patients with malignant ovarian germ cell tumors were included in this study. Immunohistochemical staining for p53 was performed on paraffin embedded tissue of each case. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene was performed on 9 of the 22 tumors where genomic DNAs were obtained from the frozen tissue samples. Three tumors that revealed focal p53 positivity by immunostaining were studied further with direct DNA sequencing. RESULTS: Overexpression of p53 was not observed in all of the 22 ovarian germ cell tumors; only 3 were found to have nuclear staining in a small fraction of the malignant cells (< 5% in 1 immature teratoma, 5-10% in 2 yolk-sac tumors). Among the nine frozen tumors subjected to single-strand conformation polymorphism analysis, none revealed p53 mutation in exons 4-9. There was no p53 mutation detected by DNA sequencing of the three tumors with focal immunoreactivity. CONCLUSIONS: Alterations of the p53 tumor suppressor gene may not be associated with the pathogenesis of ovarian germ cell tumors. Instead, genetic changes such as inactivation of other tumor suppressor genes and/or activation of some protooncogenes need to be studied to determine the genetic mechanisms of the tumor development. PMID- 8625104 TI - A prospective study of reproductive factors and risk of epithelial ovarian cancer. AB - BACKGROUND: Parity and long term use of oral contraceptives have been associated consistently with a decreased risk of ovarian cancer. However, previous reports of the relationship of other reproductive factors (time since first use or last use of oral contraceptives, age at menarche or menopause, age at first birth) with ovarian cancer have been inconsistent. METHODS: The authors studied these relationships in the Nurses' Health Study, a prospective cohort study of 121,700 female registered nurses aged 30-55 years in 1976 when the study began. From 1976 to 1988, 260 cases of confirmed epithelial ovarian cancer occurred among 1.2 million person-years of follow-up. RESULTS: A statistically significant inverse association was observed between parity and ovarian cancer risk (relative risk [RR] = 0.84; 95% confidence interval [CI] = 0.77-0.91 per pregnancy); age at first birth was not associated independently with risk. In age-adjusted analyses, a significant inverse association was noted between long term use of oral contraceptives and ovarian cancer, which was no longer significant after controlling for other ovarian cancer risk factors (RR with > or = 5 years' use: 0.65; 95% CI = 0.40-1.05). After control for duration of use, a weak nonsignificant inverse association was observed with time since first oral contraceptive use and no independent effect of time since last use. Neither age at menarche nor age at menopause was associated significantly with ovarian cancer risk. CONCLUSIONS: In this large prospective study, parity was the only reproductive factor that had a substantial independent association with ovarian cancer. Long term oral contraceptive use also appeared to have an inverse relationship with ovarian cancer, although this association was of borderline significance (P = 0.11) after adjustment for other risk factors. PMID- 8625106 TI - Significance of conventional and new prognostic factors for locally confined renal cell carcinoma. AB - BACKGROUND: The prognosis of patients with locally confined renal cell carcinoma is variable. To improve the prognostic knowledge and select patients at high risk, additional prognostic parameters are needed. METHODS: The significance with respect to survival and tumor recurrence of "classic" and "new" prognostic parameters has been examined by following 41 patients with locally confined renal cell carcinoma after nephrectomy (mean follow-up, 5.2 years). The significance of histologic grade, tumor stage, Ki-67 index, proliferating cell nuclear antigen index, 3H-thymidine labeling index, tumor ploidy status, and tumor growth after xenotransplantation into nude mice (GAX range) was tested using the Kaplan-Meier plots by the log rank test or Tarone's test and also by the Cox multiple hazard regression analysis. RESULTS: Tumor stage (P < 0.0025), histologic grade (P < 0.005), Ki-67 index (P < 0.006), and GAX range (P < 0.00004) were found to be significant prognostic parameters for survival and tumor recurrence using single factor analysis. Applying the multivariate analysis, the combination of the "new" factors, GAX range and Ki-67 index, resulted in even a higher prognostic relevance than the combination of the "classic" prognostic factors, tumor stage and histologic grade. The calculated prognostic index based on the results of the Cox analysis, which, except for stage and grade, included the Ki-67 index, was shown to be highly correlated with survival (P = 0.00002) and tumor recurrence (P = 0.0004). Its prognostic validity was studied with the receiver operating characteristics procedure and was found to be considerably superior to that of the two conventional prognosticators. CONCLUSIONS: The additional determination of the Ki-67 labeling index increases the prognostic assessment of patients with locally confined renal cell carcinoma. PMID- 8625108 TI - The expression of the MDM2 gene, a p53 binding protein, in thyroid carcinogenesis. AB - BACKGROUND: The authors previously found p53 mutations in 24% of malignant thyroid tumors, representing a wide stating spectrum. Overexpression of MDM2, most often due to gene amplification, has been suggested to be an additional mechanism for abrogation of the p53 function. In the current study, MDM2 gene expression and amplification were examined in a randomly selected subset of these tumors to explore the possibility that wild-type p53 may be inactivated by complexing with MDM2 in specimens without p53 mutations. METHODS: MDM2 gene expression and amplification were studied by Northern and Southern blot analysis, respectively. Twenty-two thyroid tumors were included: 16 papillary carcinomas, 1 follicular carcinoma, 3 anaplastic carcinomas, and 2 multinodular goiters (adenomatous goiters). RESULTS: A two- to threefold increase in MDM2 expression in 4 of 20 thyroid carcinomas was found. It was noteworthy that all of these four samples harbored p53 mutations. The association between increased MDM2 expression and p53 mutation was statistically significant (P < 0.005). No evidence of MDM2 gene amplification or rearrangement accounting for such an increase in MDM2 expression was found. CONCLUSIONS: Genetic and/or environmental factors contributing to random p53 mutations also may cause increased MDM2 expression. Given the moderate increase in MDM2 expression without associated genetic alterations such as gene amplification and rearrangement, MDM2 may not play any significant role in the development and progression of thyroid carcinoma. PMID- 8625107 TI - Central pathology review in clinical trials for patients with malignant glioma. A Report of Radiation Therapy Oncology Group 83-02. AB - BACKGROUND: Confounding biologic factors, including histologic grade, may influence the outcome of adult patients with malignant gliomas more than may modifications in therapeutic approach. Any clinical trial design for malignant gliomas in adults must account for such biologic factors, including the accurate identification of the two histologic subgroups astrocytoma with anaplastic foci (AAF) or glioblastoma multiforme (GBM), which are associated with distinctly different survival outcomes. This paper examines the need for a central pathology review before entry of patients in cooperative group clinical trials stratified by histologic grade. METHODS: Pathology slides from Radiation Therapy Oncology Group (RTOG) trial 83-02, a randomized Phase II study of hyperfractionated and accelerated hyperfractionated radiation therapy and carmustine for malignant gliomas, provided 747 analyzable cases, with 680 (91%) available for central pathology review. This review was performed by a single pathologist according to RTOG/Eastern Cooperative Oncology Group histopathologic criteria. The kappa statistic was used to measure agreement between the institutional and central classification of AAF and GBM. The influence of misclassification was examined using computer simulation of varying clinical trial sizes (n = 25, 50, or 200). The effect on the statistical power of trials (n = 200) with varying mixtures of AAF and GBM tumors was investigated using computer simulations. RESULTS: Of 159 tumors classified as AAF by institutional pathology review, only 66% (105) were classified as AAF (AAF/AAF) by central review, and 54 of these cases (34%) were classified as GBM (GBM/AAF), whereas 96% (501) of 521 institutionally classified as GBM (GBM/GBM) were similarly classified by central review. Computer simulations demonstrated a 59% underestimation in the median survival (1.82 vs. 4.49 years) for trials of patients with institutionally defined AAF compared to patients with centrally defined AAF in studies of 200 patients, resulting from the addition of poor prognosis of GBM in the trial. Misclassification can also substantially reduce the statistical power of a clinical trial. In one of the simulation studies, statistical power was reduced from 65% to 14% if 50% of the patients were to receive an inaccurate histologic classification. Even greater losses in power are possible in many plausible clinical settings. CONCLUSIONS: This examination of a central versus an institutional pathology review demonstrates a low level of agreement on AAF classification and a high level of concordance on GBM classification. The results indicate the need to adjust sample size for trials of both AAF and GBM tumors to have adequate statistical power. A central pathology review remains essential for trial entry for patients with AAF and could be omitted for trials enrolling patients with GBM only. PMID- 8625109 TI - Cutaneous angiosarcoma of the head and neck. A therapeutic dilemma. AB - BACKGROUND: Because of a tendency for diffuse, clinically undetectable local spread, cutaneous angiosarcoma is difficult to treat with surgery alone. Radiation is a rational treatment modality for this disease, because a wide region of dermis can be treated, whereas the underlying normal tissues are spared. METHODS: The authors retrospectively studied 14 patients with dermal angiosarcoma of the head and neck who were treated with electron-beam radiation from 1970 to 1989. Primary tumors were located in the scalp and forehead (11 patients) and in the upper face (3 patients). Eleven patients presented with multiple foci of disease. Three patients were treated with radiotherapy alone; the other 11 were treated with chemotherapy (10 patients) and/or surgery (7 patients). Surgical excisions were limited procedures for patients whose disease readily could be encompassed; total scalp resections were not performed. Patients were irradiated with a multiple-field electron-beam technique. Six patients presented postoperatively for radiotherapy with no macroscopic disease in the treatment field and were given a median dose of 60 Gy (range, 50-66 Gy) over a median of 40 days (range, 37-43 days). Eight patients were irradiated with clinically evident disease; doses ranged from 55 to 75 Gy over a median of 44 days (range, 33-66 days). RESULTS: Five of the six patients irradiated without clinically detectable disease were controlled in the treatment field, but only two are currently disease free. Of the eight patients irradiated with macroscopic tumor, initial disease recurrence occurred in the radiation field in two patients and at the radiation field margin in three patients. The actuarial 5-year control rates above the clavicles for patients irradiated with and without clinical disease were 24% and 40%, respectively (P = 0.03). The 5-year actuarial incidence of distant metastases for all patients was 63%. The 5-year actuarial survival rate for patients irradiated with and without clinical disease was 13% and 50%, respectively (P = .04). CONCLUSIONS: Radiation is an effective modality for treating local disease, especially when used after surgical resection of macroscopic tumor. Our current strategy is to resect clinically evident tumor in patients presenting with focal, limited disease, and to follow this resection with moderate dose, very wide-field radiation. The survival outcome for patients presenting with diffuse multifocal disease is bleak, but some patients can be controlled infield with radiation. There must be continued efforts to develop effective systemic therapy. PMID- 8625110 TI - Additional experience with empiric radiotherapy for presumed human immunodeficiency virus-associated primary central nervous system lymphoma. AB - BACKGROUND: In light of the steadily improving capability to treat opportunistic infections, the authors reviewed their recent experience with short course empiric radiotherapy for the treatment of human immunodeficiency virus (HIV) associated presumed central nervous system (CNS) lymphoma. METHODS: Medical records were reviewed of 32 previously unreported HIV-infected patients who had computed tomography and/or magnetic resonance imaging findings consistent with lymphoma, whose lesions had failed to respond to antitoxoplasmosis therapy and therefore subsequently treated with empiric radiotherapy to the cranium and meninges, nearly always 3000 cGy in 10 fractions. RESULTS: The majority of patients were in poor general condition (median Karnofsky score = 50) when radiotherapy was initiated. Fifty percent improved during or after radiation. Median survival was 2.1 months. CONCLUSIONS: Despite progress made in the past several years in the treatment of opportunistic infections and brief clinical response to radiotherapy, patients with acquired immunodeficiency syndrome who have a presumed diagnosis of CNS lymphoma continue to have extremely poor survival. Early biopsy in patients with lesions that fail to respond to empiric antitoxoplasmosis treatment or with lesions radiographically most consistent with lymphoma may improve outcome. PMID- 8625111 TI - Entry and evaluation of elderly patients in European Organization for Research and Treatment of Cancer (EORTC) new-drug-development studies. AB - BACKGROUND: This study aimed to determine the extent to which elderly patients (> or = 65 years) currently are being entered in Phase II single-agent studies, and if advanced age is associated with increased toxicity. METHODS: This analysis was based on the age distribution of 2344 patients with various solid tumors entering 16 Phase II EORTC single-agent trials from January 1983 to February 1992. RESULTS: Of the study group, 22% were 65 years or older and 8% were 70 years or older. Delay in dose administration and dose reduction were significantly higher for elderly patients compared with younger patients (P < 0.05). When adjusting for previous chemotherapy pretreatment, no difference between elderly and nonelderly patients was noted in the frequency of grade 3 and 4 hematological toxicity, nausea, vomiting, and diarrhea. A significant higher frequency of episodes of severe oral toxicity (P < 0.05) and alopecia were observed for elderly patients, but a higher proportion (P < 0.05) of elderly patients received drugs for which stomatitis was an established side effect. No significant difference in the frequency of complete plus partial responses was observed between older and younger patients (9% versus 7%). Treatment discontinuation significantly increased with age, ranging from 24% for those younger than 50 years to 33% among elderly patients (x for trend = 12.83, P < 0.001). There was not evidence that excess toxicity was an obstacle to treatment continuation for elderly patients, whereas treatment discontinuation did occur for the older age group more frequently because of loss to follow-up and treatment refusal. CONCLUSION: Selected elderly patients can and should enter new-drug-development protocols without an increased risk of more severe or frequent side effects. A priori exclusion based on an arbitrary chronologic age limit no longer should occur. PMID- 8625112 TI - Mitoxantrone is effective in treating childhood T-cell lymphoma/T-cell acute lymphoblastic leukemia. AB - BACKGROUND: T-cell malignancies in childhood are highly malignant diseases usually treated with intensive multidrug chemotherapy. In these regimens, anthracyclines, which are considerably cardiotoxic, are used. Mitoxantrone is structurally related to the anthracyclines, but it is considered to be less cardiotoxic. Therefore, the effectiveness of mitoxantrone in treating childhood T cell malignancies was studied. METHODS: Fourteen newly diagnosed children with T cell malignancies (12 T-cell non-Hodgkin's lymphoma, 2 T-cell acute lymphoblastic leukemia) initially were treated with one bolus injection of mitoxantrone, 12 mg/m2 intravenously, 1 week before standard induction therapy was begun. The effect of mitoxantrone was evaluated at Day 8, just before standard induction therapy was begun. RESULTS: Of 12 evaluable patients, 11 had significant positive responses with regard to the size of the primary tumor, the size of the involved peripheral lymph nodes, and the presence of lymphoblasts in the peripheral blood. The toxicity of mitoxantrone was mild. CONCLUSIONS: Mitoxantrone is effective in treating childhood T-cell malignancies. Its efficacy needs to be compared with the anthracyclines in a future randomized study. PMID- 8625114 TI - Prognostic significance of the MIB-1 proliferation index for patients with squamous cell carcinoma of the esophagus. AB - BACKGROUND: A number of studies have indicated that the ki-67 proliferation index is of important prognostic significance for a variety of neoplasias. It was the aim of this study to investigate whether any correlation exits between the MIB-1 proliferation index and various clinicopathologic parameters in squamous cell carcinomas of the esophagus from 72 patients (20 women: median age, 64 years; range, 45-79 years; and 52 men: median age, 61 years; range, 43-77 years). METHODS: Proliferative activity was determined using an immunohistochemical method with monoclonal antibody MIB-1 (ABC method), for tumor samples obtained from individuals who underwent esophagectomy in the period from 1983 to 1991. The percentage proliferation index (PI) was calculated as the number of positive cells divided by the total number of cells examined. Thirty-nine patients (54%) died of recurrence of esophageal cancer, with a median survival span of 15 months (range, 1-58 months). Thirty-three patients (46%) were still alive at the time of this study; their median follow up was 57 months (range, 40-98 months). RESULTS: Significant differences between proliferative index values were recorded for the following parameters: survival rate, P < 0.0001; presence of lymph node metastasis, P < 0.05; size of the primary esophageal lesion, P < 0.01; proliferation pattern of the tumor, P < 0.01; and age of the patients, P < 0.05. No correlation was found regarding histologic differentiation, clinical stage, location of the lesion, intraepithelial cancerous spread, lymphatic and blood vessel invasion, and sex of the patients. CONCLUSION: The MIB-1 proliferation index may be a powerful prognostic marker for patients with squamous cell carcinomas of the esophagus. PMID- 8625113 TI - Progress in reducing nausea and emesis. Comparisons of ondansetron (Zofran), granisetron (Kytril), and tropisetron (Navoban). AB - BACKGROUND: Nausea and vomiting are the most distressing side effects associated with the administration of chemotherapy for neoplastic diseases. Nausea, in particular, often had been ignored in studies of chemotherapy side effects. Recently, progress has been made in the control of chemotherapy-induced nausea and vomiting, due, in part, to a better understanding of the physiologic mechanisms involved. METHODS: This paper reviews recent advances in the control of emesis, focusing on pharmacologic treatments. RESULTS: The efficacy and safety of the serotonin (5-HT3) receptor antagonists granisetron, ondansetron, and tropisetron in the control of acute and delayed emesis and emesis induced by repeat-cycle chemotherapy are summarized. Although differences in study design and definitions of response criteria have made it difficult to compare the studies that have evaluated these three agents, the overall body of literature supports several clinical findings. CONCLUSIONS: (1) The 5HT3 antiemetic agents have been shown to be clinically more effective in the control of nausea and emesis than previously used agents. (2) No one of the three has demonstrated consistently greater efficacy. (3) Efficacy appears to be more pronounced for cisplatin-containing regimens than for moderate or less emetogenic chemotherapy regimens. (4) Effectiveness of the 5HT3 agents appears to be less for delayed nausea and emesis than for acute symptoms. Potential control of anticipatory nausea and emesis has not been investigated. (5) Control over nausea appears to be significantly less than control over emesis. In the studies in which it has been measured, nausea control remains incomplete for approximately half the patients given 5HT3 agents. (6) The efficacy of the agents appears to diminish across repeated days and, perhaps, across repeated chemotherapy cycles. (7) The addition of a steroid such as dexamethasone increases the efficacy of both 5HT3 and other antiemetic agents. This effect also seems to apply to delayed nausea and emesis. PMID- 8625116 TI - Gastric epithelial dysplasia. How clinicopathologic background relates to management. AB - BACKGROUND: Gastric epithelial dysplasia (GED) in metaplastic mucosa is considered the most advanced preinvasive lesion in the multistep morphogenesis of intestinal-type gastric cancer (GC). The rate of GED's evolution into GC is still under debate and probably is related to pathologic and clinical parameters other than the dysplasia itself. The aim of this study was to evaluate whether clinical aspects (sex and age) and/or morphologic variables (GED grade, coexisting atrophic gastritis) are relevant to the outcome of dysplasia, with a view toward initiating the establishment of a rational follow-up protocol for practical GED management. METHODS: Ninety-three patients harboring GED (G1: 56, G2:34, G3:18) were followed for more than 12 months according to a previously-agreed protocol. Regression, progression, or evolution into GC were detected for each grade of GED. Multivariate analysis was used to check the independence of clinical and pathologic variables in the progression of GED into more severe dysplastic lesions and/or as risk factors for evolution into GC. RESULTS: Age, male sex, GED grade and grade of coexisting atrophic gastritis proved independent risk factors for GED progression, with no significant interactions. Only GED grade (G2 and G3) was significantly associated with carcinomatous evolution. In G1-GED, age and the grade of coexisting atrophy proved to be independent risk factors for carcinomatous evolution. CONCLUSIONS: In G1-GED, more stringent follow-up should be recommended for older patients with coexisting high grade atrophic gastritis; stringent follow-up is always mandatory for G2-GED; and a surgical approach is justified in G3-GED. PMID- 8625118 TI - Distal spread of rectal cancer and optimal distal margin of resection for sphincter-preserving surgery. AB - BACKGROUND: Ascertaining the optimal distal margin of resection in sphincter preserving surgery has become an important problem. This study was designed to examine distal rectal spread of rectal carcinoma and to determine the optimal distal margin of resection for sphincter-preserving surgery. METHODS: Six hundred ten consecutive specimens of resected rectal carcinomas were analyzed retrospectively and pathologically. RESULTS: Sixty-one patients (10%) had distal spread. In patients who underwent curative surgery, distal spread was observed in only 3.8% (19/505). Distal spread was not found in patients with Stage I disease (0/150), according to the International Union Against Cancer stage. Only 1.2% (2/162) of patients with Stage II disease and 5.1% (10/195) with Stage III disease had slight spread but this was confined within a 1 cm length. Most patients with distal spread had a lower survival rate and died of distant metastasis rather than local recurrence, even after curative surgery. CONCLUSIONS: Distal spread seems to be an important risk factor for distant metastasis. Distal margin of resection of 1 cm may be appropriate clearance for most rectal cancers. PMID- 8625115 TI - Cysteine and serine proteases in gastric cancer. AB - BACKGROUND: Cysteine proteases (cathepsin B [CATB] and cathepsin L [CATL]), the serine protease urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) are thought to play an important part in cancer invasion and metastasis. The aims of this study were to measure CATB, CATL, UPA, and PAI-1 in gastric cancer (GC) and normal mucosa distant from the tumor (NORM); to evaluate whether tissue levels are related to tumor stage, grade, or histotype; to assess their prognostic relevance; and to examine UPA and PAI-1 expression immunohistochemically. METHODS: Gastric cancer and NORM samples were obtained from 25 patients with gastric cancer patients undergoing surgery (17 males, 8 females; mean age, 62 years; range, 31-84 years). Antigen concentrations were measured using the enzyme-linked immunosorbent assay method. Immunohistochemistry was performed using monoclonal UPA and PAI-1 antibodies. RESULTS: Significantly higher antigen levels were found: (1) in GC vs. NORM (CATB, CATL, UPA, PAI-1) tissues; (2) in GC with versus without metastasis (CATB, CATL, UPA); (3) in poorly or moderately versus well differentiated GC; and (4) in diffuse versus intestinal-type GC (CATB, CATL). Urokinase-type plasminogen activator, PAI-1 and CATB levels had a significant prognostic impact. Cancer and stromal cells, showed immunoreactivity to anti-UPA and anti-PAI-1 antibodies. CONCLUSIONS: These results confirm the important role of CATB, CATL, UPA and PAI 1 in gastric cancer progression. Higher levels are detected in GC with metastases, poorer differentiation, and diffuse histotype, thus identifying patients with a worse prognosis. PMID- 8625117 TI - Five-year prospective study of DNA tumor ploidy and colorectal cancer survival. AB - BACKGROUND: Retrospective studies have suggested that DNA tumor content (ploidy) has a significant effect on survival. This group has reported, prospectively, that among patients who had colorectal resections for carcinoma, the 2-year tumor recurrence rate was significantly greater for patients with aneuploid tumor than for those with diploid tumors. This paper reports the 5-year survival rates of this cohort of patients. METHODS: Three hundred sixty-three patients who had colorectal resections for cancer between November, 1982, and March, 1988, were studied prospectively. The DNA tumor ploidy was measured from fresh and paraffin embedded tissues. These patients were followed regularly in a dedicated colorectal clinic for a minimum of 5 years or until death. Of the 363 patients studied, 2 were lost to follow-up. RESULTS: Forty percent of the tumors were diploid, the remainder aneuploid. The 5-year survival for patients who had curative resections was 76% for those with diploid tumors compared with 64% for aneuploid tumors (P = 0.05; Mantel-Cox, 3.7). On further analysis, the survival benefit conferred by a diploid tumor appeared to be confined to those with Stage B tumors. There was no relation between ploidy and sex, age of patient, stage, histologic grade, or site of tumor. CONCLUSIONS: Ploidy is a useful objective measurement of the aggressiveness of Stage B tumors. Patients with aneuploid Stage B tumors have a poor prognosis; this group may benefit from adjuvant therapy. PMID- 8625119 TI - Surgical treatment for patients with pulmonary metastases after resection of primary colorectal carcinoma. AB - BACKGROUND: The optimum treatment for patients with pulmonary metastases from colorectal carcinoma is still controversial. METHODS: To evaluate the results of pulmonary resection in 22 patients with pulmonary metastases after resection of primary colorectal carcinoma, patients' characteristics were examined retrospectively. Eighteen patients had a solitary metastatic lesion and four had two lesions. RESULTS: Postthoracotomy recurrence occurred in 17 of the 22 patients (77.3%), and showed no correlation with the type of pulmonary resection. In 11 patients with a solitary lesion of less than 3.0 cm in greatest dimension, the 3- and 5-year postthoracotomy survival rates (PTSR) were 71.6% and 36.8%, respectively. Six of these patients received two or more thoracotomies for pulmonary recurrence. The 3-year PTSR for seven patients with a solitary lesion greater than or equal to 3.0 cm was 19.0%, and for the four patients with two lesions, 25.0%. A significant difference was noted in the survival rate according to both number and size of the metastatic lesions (P < 0.05). CONCLUSIONS: The number and size of metastatic lesions appear to be important prognostic determinants. Repeated thoracotomy can prolong survival for selected patients. PMID- 8625121 TI - Long term results of a phase I/II study of aggressive chemotherapy and sequential upper and lower hemibody radiation for patients with extensive stage small cell lung cancer. AB - BACKGROUND: A Phase I/II study of an aggressive six-drug chemotherapy regimen followed by the use of sequential hemibody radiation therapy as a possible non cross-resistant systemic treatment was undertaken for patients with extensive stage small cell lung cancer. METHODS: The 20 enrolled patients received 7 cycles of cyclophosphamide-based chemotherapy. The first cycle consisted of cyclophosphamide, doxorubicin, etoposide, vincristine, and lomustine. Subsequent cycles used a regimen of doxorubicin alternating with cisplatin. Thoracic radiation was delivered in a split-course fashion during the first week of chemotherapy cycles 5 and 6 (2000 cGy in five fractions during each week). Prophylactic cranial radiation was delivered in a split-course fashion during the first week of chemotherapy cycles 2 and 3 (1700 cGy in 5 fractions during each week). After the 7 cycles, patients received 600 cGy upper hemibody radiation followed by 800 cGy lower hemibody radiation. RESULTS: Nineteen of 20 patients were evaluable for toxicity and response to treatment. Hematologic toxicity accounted for treatment delays or decreased doses in 16 of 19 patients. Thirteen patients completed the initial 7 cycles; progressive disease was the only reason for discontinuing treatment. Two patients had fatal hematologic complications after lower hemibody radiation. Three patients had severe or greater peripheral neurologic toxicity, two had severe central neurologic toxicity, and one had severe cardiac toxicity. Of 19 patients, 9 achieved a complete response; median survival was 11.5 months. Five-year progression free survival and 5-year overall survival were 27% and 16%, respectively. CONCLUSIONS: This aggressive regimen is feasible for patients with extensive stage small cell lung cancer; however, hematologic-related mortality after lower hemibody radiation suggests that future investigations should be initiated at lower initial doses of lower hemibody radiation. Long term survival of the patients suggests that sequential hemibody radiation treatment warrants further investigation. PMID- 8625120 TI - Clinicopathologic features and prognosis of resected hepatocellular carcinomas of varied sizes with special reference to proliferating cell nuclear antigen. AB - BACKGROUND: Proliferating cell nuclear antigen (PCNA) is an intranuclear protein that is linked closely to the cell cycle. An immunohistochemical study was performed on the expression of PCNA in various sized hepatocellular carcinomas (HCCs) to determine the relation between the proliferative activity of cancer cells and prognosis. METHODS: One hundred forty-one neoplasms resected from 141 patients who underwent hepatic surgery for HCC at Kyushu University hospital from April, 1991, to July, 1993, were studied immunohistochemically using monoclonal antibody for PCNA (PC10), and analyzed for a possible correlation between PCNA labeling index (PCNA-LI) and the prognosis of patients with HCC. RESULTS: Proliferating cell nuclear antigen reactive cancer cells were observed throughout the HCC. The PCNA-LI ranged from 1.2%-91.6%, with a mean of 37.7%. The high PCNA LI (> 37.7) group showed a significantly higher incidence of tumor thrombus in the portal vein, higher Edmondson's Grade, and a higher recurrence rate than the low PCNA-LI (< 37.7) group. Hepatocellular Carcinomas were divided into three groups according to tumor size. Based on the clinicopathologic findings, in small (< 30 mm) HCCs, the high PCNA-LI (> 29.0) group showed a significant higher Edmondson's grade and a higher recurrence rate than the low PCNA-LI (< 29.0) group. In medium (30-60 mm) HCCs, the high PCNA-LI (> 36.1) group showed a significantly higher recurrence rate than the low PCNA-LI (< 36.1) group, although there was no difference in the pathologic findings between the high (> 59.4) and low (< 59.4) PCNA-LI groups. CONCLUSIONS: Proliferating cell nuclear antigen labeling index is closely related to cell differentiation and the prognosis of HCC. Furthermore, PCNA-LI was found to be useful in predicting the intrahepatic spread and prognosis of small (< 30 mm) and medium (30-60 mm) but not large (> 60 mm) HCCs. PMID- 8625122 TI - Primary lymphoepithelioma-like carcinoma of the lung. A clinicopathologic study of 11 cases. AB - BACKGROUND: Lymphoepithelioma-like carcinoma (LELC), best known to occur in the nasopharynx, can arise in a variety of sites, such as the salivary gland, thymus, lung, stomach, and skin. Primary LELC of the lung is very rare, with only limited information in the literature. METHODS: The clinicopathologic features of 11 patients with pulmonary LELC collected from two regional hospitals in Hong Kong are described. RESULTS: The patients, all Chinese, were aged 38 to 73 years (median, 54 years), with equal sex incidence. Two of the 8 patients were smokers. Four presented with coin lesions incidentally discovered on chest X-ray, five with cough and blood-stained sputum, and two with pleural effusion. The tumor formed a discrete (9 patients) or an ill-defined (1 patient) nodule in the lung, or, rarely, showed extensive bilateral pulmonary involvement (1 patient). The major bronchi were not involved except in 1 patient. Three patients had lymph node metastasis at presentation; two of them had bone metastasis, one at presentation and one after 9 months. The tumors had pushing margins, and grew in the form of anastomosing islands and sheets, comprising syncytial-appearing large cells with vesicular nuclei and prominent nucleoli. They were infiltrated by an appreciable number of small lymphocytes and plasma cells. Intratumoral amyloid globules were found in one tumor. In five patients, the tumor showed intraepithelial growth within the small bronchi; this could represent either the in-situ phase of the tumor or pagetoid spread into the bronchial epithelium. The neoplastic cells of all patients harbored Epstein-Barr virus (EBV) as demonstrated by in situ hybridization for EBV-encoded small nuclear RNAs. All eight Asian patients with pulmonary LELC previously reported in the literature similarly have been EBV-positive, whereas the four reported Caucasian patients all have been EBV-negative. CONCLUSION: Lymphoepithelioma-like carcinoma of lung occurring in Asians is an EBV-associated neoplasm; it also appears to occur at a higher frequency in Asians than Caucasians. It usually presents as a solitary subpleural nodule, and there is no strong association with cigarette smoking. Most patients have early stage disease at presentation. From the limited available data, the behavior of LELC of lung is highly variable, ranging from apparent curability by excision (particularly for localized disease) to highly aggressive, extensive disease at presentation. PMID- 8625123 TI - Disease-related hypocholesterolemia in patients with hairy cell leukemia. Positive correlation with spleen size but not with tumor cell burden or low density lipoprotein receptor activity. AB - BACKGROUND: Hypocholesterolemia is common in patients with various malignant diseases, and may be a risk factor for the development or a consequence of the tumor, by different possible mechanisms. METHODS: Serum lipids were analyzed in 66 patients with symptomatic hairy cell leukemia (HCL) before and repeatedly after treatment with cladribine. Low density lipoprotein (LDL) receptor activity of hairy cells from 12 patients was analyzed. RESULTS: The median pretreatment serum cholesterol was 4.78 mmol/l. Total cholesterol, LDL cholesterol, and triglycerides were inversely correlated with the spleen size, but not with other markers of tumor burden. High density lipoprotein (HDL) cholesterol correlated to serum beta 2-microglobulin. Anemia and hypocholesterolemia developed synchronously before diagnosis in one patient. After cladribine therapy, there was a highly significant increase in all serum lipids. Low density lipoprotein receptor activity of HCL cells was elevated in only one of 12 patients; this patient had high serum cholesterol. Hypocholesterolemia predicted posttreatment neutropenic fever. CONCLUSION: Hypocholesterolemia is a common disease-related finding in HCL, which is not caused by an increased LDL receptor activity of leukemia cells, but related to spleen size, predicts posttreatment fever, and is completely reverted after successful treatment of leukemia. PMID- 8625124 TI - Papillary serous carcinoma of the peritoneum in women. A clinicopathologic and immunohistochemical study. AB - BACKGROUND: Papillary serous carcinoma of the peritoneum (PSCP) is a primary peritoneal tumor in women that histologically resembles papillary serous carcinoma of the ovary (PSCO). Recognition of PSCP as an entity is controversial, as is the histogenesis, histopathologic differential diagnosis, and treatment. METHODS: Ten cases of PSCP retrieved from the pathology files of 4 hospitals in Kyoto and Nara, Japan, were studied clinicopathologically and immunohistochemically. RESULTS: Patient ages at presentation ranged from 40 to 74 years (median, 56 years). All patients were Asian (Japanese). None of the patients had a history of asbestos exposure. Most of the patients had abdominal swelling, ascites with positive cytology, and elevated serum CA125. At surgery, omental tumors with dissemination to the abdominal and pelvic peritoneum were found in all patients. The histology was similar to that of Grade 2 to 3 PSCO. Immunohistochemical studies using a panel of monoclonal antibodies against carbohydrates showed that Lewis Y is a good marker, in addition to S-100, placental alkaline phosphatase, CA125, and CD15 for separating PSCP from malignant mesothelioma (MM). With cytoreductive surgery and cisplatin-based combination chemotherapy and in some cases adoptive immunotherapy and radiation, a median survival of 27 months and a 5-year survival rate of 27% were attained. One patient with Grade 3 tumor has survived for more than 6 years after surgery. CONCLUSIONS: (1) Papillary serous carcinoma of the peritoneum is a definite clinicopathologic entity; (2) immunohistochemistry is a useful tool for distinguishing PSCP from MM; (3) cytoreductive surgery and cisplatin-based combination chemotherapy with other adjunct therapies such as immunotherapy and radiation may improve patient survival in PSCP. PMID- 8625125 TI - Malignant pleural mesothelioma after radiation therapy for breast cancer. A report of two additional patients. AB - BACKGROUND: Malignant pleural mesotheliomas (MPMs) are rare tumors that usually have a fatal outcome. The association of these tumors with asbestos exposure is well established. Induction of malignant mesothelioma by nonasbestos-related causes also has been reported in the literature, although the number of documented cases is extremely small. Two additional patients with malignant pleural mesothelioma many years after radiotherapy for breast cancer are reported. METHODS: The observations as reported in the literature on the involvement of radiation in the development of MPMs are reviewed and compared with the authors' clinical experience. In a retrospective random review, 1000 patients who received thoracic irradiation at M. D. Anderson Cancer Center were studied for histologic and radiographic evidence of MPM. The selection criteria included the development of a unilateral pleural effusion years after successful treatment with thoracic irradiation for a proven malignancy. Patients with a history compatible with asbestos exposure were excluded from the review. RESULTS: There have been only three previous cases of documented MPM associated with thoracic irradiation reported in the English literature. A review of the experience at our institution has demonstrated three patients with radiation induced MPM. One patient has been reported elsewhere. Details of the other two patients are discussed in this paper. CONCLUSIONS: Nonasbestos-related causes of MPMs are rare. The additional two patients lend added support to the association between thoracic irradiation and the development of MPM. The development of a unilateral pleural effusion occurring years after successful treatment of a proven malignancy with thoracic irradiation should alert the clinician to the possibility of MPM. PMID- 8625126 TI - Second primary cancer after treatment for cervical cancer. An international cancer registries study. AB - BACKGROUND: The pattern of second cancers after treatment for cervical cancer provides important information on the risk of radiation-induced malignancies. Large numbers of women survive many years and can be studied for late effects. METHODS: Incident second cancers in 86,193 patients with cervical cancer reported to 13 population-based cancer registries in 5 countries were evaluated to estimate the risk of second cancer among very long term survivors. RESULTS: Overall, 7543 second cancers were observed versus 6015 cancers expected based on population rates (observed/expected = 1.2). Lung cancer accounted for nearly half of the excess cancers. Among the 49,828 women treated with radiation, 3750 survived 30 or more years and a two-fold risk of cancers of heavily irradiated organs was seen. Most of the excess cancers were of the rectum, vagina, vulva, ovary, and bladder. Patterns of risk over time since treatment were consistent with a radiation etiology. Significant increases of nonchronic lymphocytic leukemia and cancers of the bone and kidney were also linked to radiotherapy. Women treated surgically were also at significant risk of second cancers, in all likelihood related to cigarette smoking and risk factors similar to those of cervical cancer. CONCLUSIONS: Curative therapy for cervical cancer results in large numbers of long term survivors who develop second cancers very late in life. Radiation is an important cause of this increase and there is no evidence that risk returns to normal levels. PMID- 8625128 TI - Early detection of prostate cancer. Results of a prostate specific antigen-based detection program in Japan. AB - BACKGROUND: Prostate cancer increasingly is becoming a medical problem in Japan. To maximize the chance for cure, extensive effort is being made to detect prostate cancer while confined to the gland. METHODS: Determination was made of serum prostate specific antigen (PSA) in the early detection of prostate cancer in an Asian male population aged 55 years or older. Recommendation for biopsy was based solely on this parameter when its value exceeded 2.0 ng/ml in IMx immunoenzymetric assay. RESULTS: Of 1189 men, 150 (12.6%) had elevated values. The proportion of males with serum PSA greater than 4.0 ng/ml was only 3.4%. Of the 99 males who underwent ultrasound-guided biopsies, 16 cancers were detected, a cancer detection rate of 1.3%. Most of these (81.3%) were clinically localized cancers. Seven patients with cancer had serum PSA levels below 4.0 ng/ml including 5 less than 3.0 ng/ml. These tumors would have been overlooked if the conventional cutoff levels of 4.0 ng/ml had been applied. Radical prostatectomy was conducted on 13 patients. All had histologic features of clinically significant cancer and 69.2% were pathologically confined within the gland. CONCLUSIONS: Elderly Asian males appear to have lower serum PSA than their Western counterparts. The optimal cutoff of serum PSA for early detection should be examined further in Asian male populations. Differences in the incidence of prostate cancer between ethnic groups may have been overestimated in previous studies owing in part to unawareness by the physician and general public. Though the frequent use of more sophisticated diagnostic modalities will likely disclose greater numbers of prostate cancers in Japan, whether early detection reduces mortality by minimizing risk of death from cancer remains a point to be determined. PMID- 8625127 TI - Antitumor activity of suramin in hormone-refractory prostate cancer controlling for hydrocortisone treatment and flutamide withdrawal as potentially confounding variables. AB - BACKGROUND: A prospective Phase II clinical trial was conducted to assess the clinical activity of a pharmacokinetically guided suramin regimen in patients who had documented progression of metastatic prostate cancer after hydrocortisone plus antecedent or simultaneous withdrawal of flutamide. METHODS: Fifty-four patients whose disease had progressed after castration and flutamide administration were enrolled on this trial. The study was divided into two parts. Initially, 52 patients received hydrocortisone (30 mg/day) and for those patients receiving flutamide, at study entry (34 patients) flutamide was simultaneously discontinued. Forty-three patients whose disease progressed on hydrocortisone received suramin for 6-8 weeks. Six patients who progressed on hydrocortisone became ineligible for suramin due to clinical deterioration, four patients are still responding to hydrocortisone at more than 1 year, and one patient elected to postpone initiation of suramin. Suramin was given as intermittent infusions at fixed doses on days 1-5 and thereafter dosing was guided by adaptive control with feedback to maintain plasma suramin concentrations between 300-175 micrograms/ml. Antitumor activity was assessed by prostate specific antigen (PSA) decline and soft-tissue disease response. RESULTS: Ten patients (19%; 95% CI, 9.6%-32.5%) responded to hydrocortisone therapy with either a 50% or greater PSA decline for at least 4 weeks (9 patients) and/or a partial response of measurable soft-tissue disease (2 patients). Five of these patients (10%) demonstrated a 80% or greater PSA decline. All responders to hydrocortisone had simultaneous flutamide withdrawal, and had been receiving flutamide as part of initial combined androgen blockade. Seven of 37 evaluable patients (19%; 95% CI, 8.0%-35.2%) responded to suramin with a 50% or greater decline in PSA for 4 weeks or longer. One patient (3%) had a 80% or greater decline in PSA. There were no soft-tissue disease responses to suramin. The median time to progression was 1.9 months for hydrocortisone therapy and 2.6 months for suramin therapy. The median survival for all patients was 14.6 months. CONCLUSION: Suramin has antitumor activity in metastatic prostate carcinoma independent of the therapeutic effect of hydrocortisone administration or flutamide withdrawal. The role of prior flutamide withdrawal and hydrocortisone replacement should be taken into account in future studies of suramin. PMID- 8625129 TI - Urinary beta-glucuronidase activity as an initial screening test for urinary tract malignancy in high risk patients. Comparison with conventional urine cytologic evaluation. AB - BACKGROUND: Routine urine cytology is not particularly useful as a screening test for urinary tract malignancy in the general population, due to its low detection rate. Bladder, ureteral, and pelvic lavage and flow cytometry increased the test sensitivity but could be applied only to a limited number of patients. A simple, sensitive screening test is needed. METHODS: Two hundred eighty-two urine samples from 146 patients from the Urology Tumor Clinic patients during their initial visits were subject to cytologic evaluation and measurement of the activity of endogenous beta-glucuronidase, followed by confirmation procedures, including cystoscopy with biopsy, ultrasonography, radiography and/or computed tomography. RESULTS: Among 146 patients, 32 had confirmed transitional cell carcinoma and 14 renal cell carcinoma. The urinary beta-glucuronidase activity was higher in patients with cancer than in those without cancer in the Urology Service and in 80 normal healthy control subjects. The accuracy of the urine enzyme diagnostic system measured by the receiver-operating characteristic (ROC) plot was 98% or higher based on the number of patients. The upper threshold value determined by ROC analysis was 1.7 nmol/min/mumol creatinine. At this threshold value, the sensitivities of the enzyme assay for transitional cell carcinoma, renal cell carcinoma, and all cancers were, respectively, 94%, 98%, and 95%. These values were much higher than the corresponding sensitivities of urine cytology: 41%, 0%, and 22%. CONCLUSION: Urinary beta-glucuronidase is a much more sensitive test to screen for urinary tract malignancy than routine urine cytology in high risk patients, provided that a positive test is followed by diagnostic procedures for confirmation. PMID- 8625131 TI - Detection of polyomaviral DNA sequences in normal and adenomatous human pituitary tissues using the polymerase chain reaction. AB - BACKGROUND: Tumor viruses are known to have a role in the pathogenesis of many types of benign and malignant human tumors. The possible roles of these viruses in the development of human pituitary tumors have not been investigated. METHODS: The polymerase chain reaction was used to screen human pituitary tumors for human papillomaviral (HPV) and Polyomaviral DNA sequences. Sets of consensus primers, which are capable of amplifying HPV Types 16, 18, and 33 and polyomavirus BK, JC, and SV40, were used in these experiments. RESULTS: Amplification products were not detected using HPV consensus primers in 30 tumors. Twenty-six of 30 tumors demonstrated an amplification product with polyomaviral primers that hybridized to SV40 and BK internal probes and was confirmed to be SV40 in one tumor by direct sequencing. Ten normal postmortem pituitary samples then were examined similarly with Polyomaviral consensus primers; 8 of 10 normal samples demonstrated a similar amplification product that also hybridized with SV40 and BK internal probes by Southern blotting. Polyomaviral DNA sequences in normal and tumor samples were not present at levels detectable by genomic Southern blotting. Expressed viral protein (large T antigen) was not demonstrated in positive samples by Western blot analysis. CONCLUSIONS: These findings, that polyomaviral DNA sequences are detectable at low levels in certain normal tissues, are in agreement with those of other groups and, to the authors' knowledge, serve as the first report of polyomaviral latency in human pituitary tissue. A role for polyomaviruses in pituitary tumorigenesis could not be established in this analysis. PMID- 8625132 TI - Severe, irreversible renal failure after ifosfamide treatment. A clinicopathologic report of two patients. AB - BACKGROUND: Chronic renal failure has been described only rarely in patients treated with the alkylating agent ifosfamide, which is known to cause renal tubular dysfunction and acute renal failure, and the associated histopathologic features have not been well characterized. METHODS: This report describes the clinical course and renal histopathologic features in two patients in whom irreversible renal failure occurred requiring permanent dialysis after treatment with ifosfamide. RESULTS: Irreversible renal failure developed in a 60-year-old man with malignant fibrohistiocytoma, requiring chronic dialysis within several months after he received two cycles of ifosfamide in a cumulative dose of 28 g/m2. The second patient, a 53-year-old man with osteogenic sarcoma, received two cycles of ifosfamide with a cumulative dose of 26 g/m2, after initial therapy with cisplatin and doxorubicin. His renal function worsened over the next 11 months, at which time permanent dialysis was initiated. In neither patient were other causes of renal failure apparent. Renal biopsies in both patients showed diffuse tubular epithelial damage with degenerative and regenerative epithelial changes, diffuse interstitial fibrosis, and arterial and arteriolar sclerosis. CONCLUSIONS: Irreversible severe renal failure, which appears due to nephrotoxic damage of renal tubular epithelium and/or the renal microvasculature may develop after treatment with ifosfamide. Neither large cumulative doses of ifosfamide nor prior cisplatin treatment are necessary for this toxicity to occur. Because a rising serum creatinine may develop months after completion of treatment with ifosfamide, renal function should be monitored closely both during and after ifosfamide treatment. PMID- 8625130 TI - Analysis of RET protooncogene point mutations distinguishes heritable from nonheritable medullary thyroid carcinomas. AB - BACKGROUND: The distinction of sporadic from inherited medullary thyroid carcinomas (MTCs) is of clinical importance because of the differences in prognosis, and the need for family screening for genetic counseling required in the latter. Germline mutations in the RET protooncogene are associated with multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Somatic point mutations in the same gene have been identified in a subset of sporadically occurring medullary thyroid carcinomas. METHODS: A nonisotopic polymerase chain reaction-(PCR) based single strand conformation polymorphism (SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from 32 formaldehyde fixed and paraffin embedded MTC specimens and normal tissue or blood of the same patient for point mutations in RET exons 10, 11, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR-products using an automated DNA-sequencer. Results were compared with the disease phenotype, clinical findings, and follow up. RESULTS: Six different missense germline mutations were identified at cysteine residues 618, 630, and 634 of the cysteine-rich extracellular RET domain encoded by exons 10 and 11 in all patients with FMTC and MEN 2A. The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a 634 Cys-->Arg mutation was associated with parathyroid disease in three patients. A germline Met-->Thr point mutation at codon 918 of the RET tyrosine kinase domain was identified in all three patients with MEN 2B. Two patients with clinically sporadic MTCs and negative family history exhibited a RET germline mutation at codon 634, indicating the presence of an nonpredicted inherited MTC. Furthermore, one patient had a 618 Cys-->Ser mutation in the tumor and nontumorous thyroid DNA but not in blood DNA, indicating a mosaic mutation affecting thyroid tissue but not blood cells. Tumor specific (somatic) Met-->Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs. The remaining eight sporadic MTCs lacked mutations in all three RET exons tested. CONCLUSIONS: This study demonstrates that (1) the molecular methods are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B but also to distinguish heritable from nonheritable MTCs using archival tissue specimens, and (2) that more MTCs than clinically expected are heritable, indicating the need for genetic analysis of all patients with MTC. PMID- 8625133 TI - Cryotherapy treatment of patients with hepatic metastases from neuroendocrine tumors. AB - BACKGROUND: Liver metastases from neuroendocrine tumors often present with disabling symptoms due to syndromes of hormonal excess. A locally destructive technique such as hepatic cryotherapy not only alleviates symptoms but may improve survival in this group of patients. METHODS: Six patients with metastatic neuroendocrine tumors were treated with hepatic cryotherapy. Four patients were symptomatic and three of these had elevated tumor markers from ectopic hormone production. RESULTS: All patients are alive and asymptomatic, with a median follow-up of 24 months (range, 6 months to 6 years). All have had a complete radiologic response. All with elevated preoperative markers have had a greater than 89% decrease in tumor markers. Coagulopathy occurred in two patients necessitating additional surgery, but there was no other morbidity attributable to the cryotherapy. CONCLUSION: To the authors' knowledge, this study demonstrates for the first time that hepatic cryotherapy offers supportive treatment for patients with neuroendocrine tumors metastatic to the liver. Cryotherapy alleviates symptoms and may improve survival. PMID- 8625134 TI - Granulocyte-macrophage-colony stimulating factor for prevention of neutropenia and infections in children and adolescents with solid tumors. Results of a prospective randomized study. AB - BACKGROUND: Chemotherapy is an essential modality of curative strategies in pediatric oncology. Dose and dose intensity are, above all, restricted by the myelosuppressive effects of cytotoxic drugs. Neutropenia constitutes an important risk of morbidity and mortality. Granulocyte-macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor that increases the number of circulating neutrophils as demonstrated in adults. METHODS: A prospective randomized study of the effects of GM-CSF was performed with 11 patients who were treated for solid tumors and received GM-CSF for 2 weeks starting 48 hours after completion of chemotherapy. Forty-two intraindividual identical chemotherapy courses with and 42 without GM-CSF were compared. The monitoring program included the surveillance of the hematological reconstitution and the number and duration of infectious episodes. RESULTS: The average nadir of the absolute neutrophil count (ANC) with GM-CSF was higher than without GM-CSF. The average number of days with an ANC below 500/microliters was significantly reduced by GM-CSF. Fewer infectious episodes were observed among those who received GM-CSF therapy. Erythropoiesis was not significantly influenced by GM-CSF, whereas patients with GM-CSF therapy showed a longer thrombocytopenia without requiring more platelet transfusions. Rashes developed in two patients. CONCLUSIONS: In children and adolescents undergoing intensive chemotherapy for solid tumors, GM-CSF reduces neutropenia and infectious episodes at the cost of mild thrombocytopenia. PMID- 8625135 TI - Diamond-Blackfan anemia and malignancy. A case report and a review of the literature. AB - BACKGROUND: Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome involving the erythropoietic lineage. A preleukemic predisposition has been suggested but not proven. METHODS: The development of Hodgkin's disease in a 15-year-old boy with DBA, in remission for 13 years after cessation of steroid therapy, is described. Review of the literature revealed 11 other cases of malignancy (10 hematologic) in DBA. RESULTS: This patient, together with those described in the literature, shows that the incidence of hematological malignancy in DBA is increased (2.5% of all reported cases of DBA). Treatment was successful in three, including one after allogeneic bone marrow transplantation and our patient in whom recurrence of DBA complicated treatment. CONCLUSIONS: The incidence of hematologic malignancies in patients with DBA is increased. Treatment can be successful but may be complicated by recurrence of DBA. Bone marrow transplantation should be considered for patients with a suitable donor as part of treatment of patients with DBA and hematologic malignancy. PMID- 8625136 TI - Carboxypeptidase-G2 rescue in a patient with high dose methotrexate-induced nephrotoxicity. AB - BACKGROUND: High dose methotrexate (HDMTX) induced renal failure is a medical emergency, as methotrexate (MTX) is primarily eliminated by renal excretion. High doses of leucovorin (LV) do not necessarily prevent toxicity in the presence of sustained elevated plasma MTX concentrations. The bacterial enzyme carboxypeptidase-G2 (CPDG2) hydrolyzes MTX into inactive metabolites and has been demonstrated to lower plasma MTX concentrations to nontoxic levels rapidly in the nonhuman primate after HDMTX infusion. Therefore, CPDG2 was evaluated as a rescue agent in a patient with acute renal dysfunction secondary to HDMTX: METHODS: A 16 year old patient with osteosarcoma experienced acute renal dysfunction after HDMTX administration, which resulted in markedly elevated and sustained plasma MTX concentrations. She received three doses of CPDG2 on the fifth day after HDMTX: Plasma MTX concentrations were determined before and after CPDG2 administration. RESULTS: The plasma MTX concentrations decreased from 60 to 1.2 microM within 15 minutes after the first dose of CPDG2. No rebound increase in plasma MTX concentrations or adverse reactions to the enzyme were observed. The patient developed only mild mucositis. Serum creatinine at the time of CPDG2 administration was 5 mg/dl and returned to normal within 7 weeks of enzyme administration. CONCLUSIONS: Carboxypeptidase-G2 rapidly, markedly, and persistently lowered plasma MTX concentrations to a level that could be rescued safely with LV. Based on the experience with this patient and on preclinical studies in nonhuman primates, CPDG2 appears to be more effective than hemodialysis or hemoperfusion, and may prove beneficial for patients at risk for life-threatening toxicity secondary to delayed excretion of MTX. PMID- 8625137 TI - Nasopharyngeal lymphoid tissue masses in patients with human immunodeficiency virus-1. PMID- 8625138 TI - 5-fluorouracil/leucovorin/interferon alpha-2a in patients with advanced colorectal cancer. PMID- 8625139 TI - Alternated approach with local irradiation and combination chemotherapy including cisplatin or carboplatin plus epirubicin and etoposide in intermediate stage non small cell lung cancer. PMID- 8625141 TI - The detection of breast carcinoma micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. PMID- 8625140 TI - Reproductive history and prognosis in patients with operable breast cancer. PMID- 8625142 TI - Ondansetron versus granisetron in the prevention of chemotherapy-induced nausea and vomiting. PMID- 8625143 TI - Polymorphic reticulosis is a neoplasm of large granular lymphocytes with CD3+ phenotype. PMID- 8625144 TI - The National Cancer Data Base report on colorectal cancer. PMID- 8625145 TI - Human cyclin D1 oncogene and esophageal squamous cell carcinoma. AB - BACKGROUND: Oncogene activation and tumor suppressor gene inactivation have been implicated in the genetic basis of esophageal squamous cell carcinoma (ESCC). Cyclin D1, an oncogene that has a critical role in G1 progression of the cell cycle, has been observed to be amplified in carcinomas of the breast and head and neck, and translocated in parathyroid adenomas and centrocytic lymphomas. METHODS: Established ESCC cell lines were assayed for cyclin D1 amplification and overexpression by Southern, Northern, and Western blot analyses. In addition, cyclin D1 overexpression was determined in primary tumors and adjacent normal mucosa by differential polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: The authors observed that approximately 50% of ESCC cell lines with cyclin D1 DNA amplification also had RNA and protein overexpression. Related genes, cyclin D2 and D3, were not amplified or overexpressed in these cell lines with rare exception. The cyclin D1 protein was able to associate with the cell cycle-dependent kinases, cdk4 and cdk6, but not always with proliferating cell nuclear antigen in selected cell lines tested, representing a novel finding. In addition, approximately 50% of primary tumors had cyclin D1 overexpression that was not present in adjacent normal mucosa. Cyclin D1 overexpression based on PCR correlated with enhanced cyclin D1 protein nuclear staining in malignant cells. CONCLUSION: Cyclin D1 is amplified and overexpressed in ESCC and may be important in its molecular pathogenesis. PMID- 8625146 TI - Apoptosis of stomach carcinoma cells induced by a human monoclonal antibody. AB - BACKGROUND: Although stomach carcinoma is estimated to be one of the most frequent cancers worldwide, still little is known about the immunity of patients with stomach cancer. Humoral tumor immunity has been studied by isolating B cells of patients with cancer to characterize the activity of such antibodies on tumor cells. Apoptosis, programmed cell death, explains the suicide of cells by fragmentation of DNA, cell shrinkage and dilation of endoplasmatic reticulum, followed by cell fragmentation and formation of membrane vesicles called apoptotic bodies. Apoptosis serves to remove unwanted, damaged, or dangerous, e.g., precancerous cells. METHODS: The human monoclonal antibody SC-1 was isolated from a patient with a signet ring cell carcinoma of the stomach by fusion of spleen lymphocytes to the heteromyeloma SPM4-0. The antibody was tested for growth-inhibiting effects in vitro in soft agar assays, in 3-H thymidine uptake experiments, and in a mitochondrial enzymatic activity assay. In vivo intraperitoneal tumor growth was investigated in nu-nu mice. RESULTS: The immunoglobulin M (lambda) antibody identifies a molecule with a molecular weight of approximately 49 kilodaltons in stomach carcinoma cells. No reactivity was observed with carcinomas of other origins, melanomas, lymphomas, or normal tissue. When tested in vitro, the antibody inhibited tumor cell growth in cell culture and soft agar. In vivo growth of stomach carcinoma cells in nu-nu mice was reduced when the antibody was injected after the tumor cells. Ultrastructural and functional studies revealed that the SC-1 antibody induced apoptosis of tumor cells. CONCLUSION: The human monoclonal antibody SC-1 described here inhibited growth of stomach carcinoma cells in vitro and in vivo by inducing apoptosis, and may, therefore, be valuable for treating patients with stomach carcinoma. PMID- 8625148 TI - Survival after curative resection of lymph node negative colorectal carcinoma. A prospective study of 910 patients. AB - BACKGROUND: Approximately half of all patients treated for colorectal carcinoma by bowel resection have neither lymph node metastases nor known residual tumor (clinicopathologic Stages A and B). The aim of this study was to compare the survival of these patients with that of the general population and to explain any significant difference. METHODS: Prospectively collected data recorded for 910 patients from one institution during a period of 21.5 years were used in the analysis. Patient follow-up ranged from 6 months to 21.5 years. The "Survival" procedure, developed by the Finnish Cancer Registry, was used to compare the observed survival of patients with their expected survival, based on age- and sex matched data from the population of New South Wales. Survival analysis was performed by the Kaplan-Meier method. Multivariate models were examined using Cox proportional hazards regression. RESULTS: Males with tumor spread beyond the muscularis propria (Stage B) was the only group with significantly poorer survival than expected. The reduced survival in this group was due to the effects of four clinical variables (cardiovascular complication, permanent stoma, urgent operation, respiratory complication) and one pathologic variable (direct spread involving a free serosal surface) acting independently. CONCLUSION: The survival of patients with clinicopathologic Stages A or B tumors closely matched their expected survival as predicted from the general population. Males with Stage B tumors were the only exception and their significantly reduced survival was largely due to clinical, as distinct from pathologic factors. These findings suggest that the risk of occult metastases is low for patients with Stages A and B tumors using this classification. PMID- 8625147 TI - A phase II trial of weekly high dose continuous infusion 5-fluorouracil plus oral leucovorin in patients with advanced colorectal cancer. The Spanish Cooperative Group for Gastrointestinal Tumor Therapy (TTD). AB - BACKGROUND: In a previous Phase II trial, the authors showed that a weekly continuous infusion of 5-fluorouracil (5-FU) at a dose of 3.5 g/m2 for 48 hours is an active treatment for advanced colorectal cancer. The overall response rate was 38.5%, and the median survival was 12 months. These data were comparable to those achieved by biochemical modulation of 5-FU with leucovorin. To study the modulation of this weekly, high dose, continuous infusion 5-FU with oral leucovorin, a new Phase II trial was planned. METHODS: From December 1991 to July 1992, 43 previously untreated patients with measurable advanced colorectal cancer were included in a multicenter study. They received on an outpatient basis 5-FU at a weekly dose of 3 g/m2 by continuous infusion for 48 hours until progression or toxicity. Oral leucovorin (60 mg every 6 hours) also was given during the infusion of 5-FU. RESULTS: Patients received a median dose intensity of 5-FU of 2.2 g/m2/week (range, 0.76-3 g/m2/week). One complete response and 11 partial responses were observed. The overall response rate was 29% (95% confidence interval [CI], 16-45%). Median time to progression was 7 months, and the median survival was 15 months. World Health Organization Grades 3 and 4 diarrhea were observed in 19 (45%) and 6 (14%) patients, respectively. Grade 3 mucositis also was observed in 10 (24%) patients, and Grade 4 mucositis was observed in 1. Grade 3 nausea and vomiting were reported in seven (17%) patients. Grade 3 hand-foot syndrome was detected in only two (2.5%) patients. No leukopenia or thrombocytopenia was observed. CONCLUSIONS: Oral leucovorin modulation of a weekly 48-hour infusion of 5-FU at a dose of 3 g/m2 of leucovorin is a toxic regimen, always requiring dose reduction, with diarrhea and mucositis as the main limiting toxicities. Its antitumor activity does not seem superior to that observed with a weekly 48-hour infusion of 5-FU alone at a dose of 3.5 g/m2. PMID- 8625149 TI - Octreotide reduces the kinetic index, proliferating cell nuclear antigen-maximum proliferative index, in patients with colorectal cancer. AB - BACKGROUND: Somatostatin has been shown to inhibit in vitro and xenograft growth of human colon cancer. The kinetic index, proliferating cell nuclear antigen (PCNA), has previously been used to measure the effects of manipulation of growth of normal rectal epithelium. METHODS: Twenty-five patients with distal colorectal cancer were considered for entry in a presurgical study of Sandostatin (Sandoz, East Hanover, NJ) 1 mg every 8 hours. Biopsies were performed pretreatment, during treatment (14 days), and day of surgical resection (2 days off treatment). A control series of 16 patients underwent endoscopic and subsequent surgical biopsy. A kinetic index was created called PCNA-maximum proliferative index (PCNA MPI), which was reproducible within one biopsy and between two separate biopsies. Multiple biopsies were taken from the growing edge of tumors, the most cellular and best-stained fields selected, and the highest 6 of 10 separate counted fields were used to produce PCNA-MPI. RESULTS: A significant decline in PCNA-MPI was observed in 6 of the 10 treated patients for whom all three biopsies were available, followed by a significant elevation on withdrawal of treatment. Changes in PCNA-MPI in the control group were less frequent and smaller. CONCLUSIONS: Sandostatin causes a reduction in PCNA-MPI in patients with human colorectal cancer. PMID- 8625150 TI - Comparison of tumor pathology with duration of survival of North American patients with hepatocellular carcinoma. AB - BACKGROUND: Several of the large studies addressing prognosis and survival from hepatocellular carcinoma (HCC) have been from Europe and Asia, but only a few have emanated from North America. Survival statistics from other parts of the world may not be applicable to the North American population because of etiologic, demographic, cultural, lifestyle, and intangible differences. The current study investigated the survival experience and histologic correlates of North American patients with HCC and compared findings with similar studies from North America and other parts of the world. METHODS: One thousand sixty-three patients examined during a 14-year period, met inclusion criteria for this study. Each patient was placed in one of three categories based on the duration of survival from date of diagnosis of HCC. Each tumor was examined histologically and classified according to World Health Organization criteria. Patient's sex and age at diagnosis were obtained from case records. Survival analyses and comparisons were performed using appropriate methods. Variables were tested for association using chi-square tests and randomization tests as appropriate. RESULTS: Age, sex, tumor growth pattern, Edmondson and Steiner's nuclear grades, mitotic index, and presence or absence of tumor giant cells or portal venous invasion, were found to have statistically significant (P < 0.05) relationships with the duration of patient survival. Significantly better survival was found to be associated with female sex, low nuclear grade, low mitotic index, age at diagnosis younger than 50 years, absence of giant cells, and absence of portal venous invasion. CONCLUSIONS: Certain histopathologic features of HCC may be useful for predicting patient survival and, thus, for empiric prognostication of these patients. PMID- 8625151 TI - Pancreas cancer as a second primary malignancy. A population-based study. AB - BACKGROUND: The study of second primary malignancies may give clues to the etiology of various cancers. Little is known about risk factors for pancreatic carcinoma; therefore, its occurrence as a second primary malignancy was investigated. METHODS: Data from the Surveillance, Epidemiology, and End-Results (SEER) program were used for the period from January 1, 1973 through December 31, 1990. Person-years of follow-up for various cancer sites were calculated, excluding the initial 6 months after diagnosis, and were multiplied times the age and sex-specific incidence rates for pancreas cancer to calculate the expected number of second primary pancreas cancer cases. The observed number of cases was divided by the expected number to estimate the relative risk (RR) of pancreas cancer as a second primary cancer, and 95% confidence limits were calculated. RESULTS: The risk of second primary cancer was elevated after lung cancer for men (RR 1.3, 95% CI 1.0-1.6) and women (RR 2.5, 95% CI 1.9-3.2). An elevation in risk also was found after head and neck cancer in women (RR 1.8, 95% CI 1.2-2.5) and bladder cancer in women (RR 1.5, 95% CI 1.1-2.0), but not in men. Other significant elevations were found after prostate cancer (RR 1.2, 95% CI 1.1-1.3), and a decreased risk was found after lymphoma in men (RR 0.2, 95% CI 0.0-0.8). CONCLUSIONS: Second primary pancreas cancer is increased after tobacco-related malignancies, particularly in females, supporting the role of cigarette smoking as a risk factor for pancreas cancer and suggesting a stronger effect of cigarette smoking for women. The elevation in risk after prostate cancer and the decreased risk after lymphoma in males need to be confirmed in other data sets. PMID- 8625152 TI - Randomized trials of radiotherapy alone versus combined chemotherapy and radiotherapy in stages IIIa and IIIb nonsmall cell lung cancer. A meta-analysis. AB - BACKGROUND: For patients with Stage III nonsmall cell lung cancer (NSCLC), radiation is the standard treatment, but survival remains poor. The authors performed a meta-analysis study using clinical trials that evaluated combined radiotherapy plus chemotherapy versus radiotherapy alone in patients with Stages IIIa and IIIb NSCLC: METHODS: For the meta-analysis study and the point estimates, essential data were extracted directly from published reports. RESULTS: Survival probabilities at 1, 2, 3, and 5 years, as estimated from published survival curves, were considered as the endpoints of interest. For survival at 3 and 5 years, the point estimates and the confidence intervals were used. Quality scoring of the studies also was performed. Fourteen trials were selected, comprising 1887 patients in the meta-analysis. For the cisplatin-based group, the estimated pooled odds ratio of death at 1 and 2 years was 0.76 (0.6 0.9 CI) and 0.70 (0.5-0.9 CI), with a reduction in mortality of 24% and 30%, respectively. For the noncisplatin-based group, the estimated pooled odds ratio at 1 and 2 years was 1.05 (0.7-1.5 CI) and 0.82 (0.5-1.3 CI), with a reduction in mortality of 5% and 18%, respectively. However, no significant differences were found between the percentage of survival and the CI at 3 and 5 years using the point estimates. CONCLUSIONS: These results favor combined cisplatin-based chemotherapy and radiotherapy, although it was not so at 3 and 5 years of survival. These data must, however, be considered in the light of their clinical relevance and of the balance between quality of life, toxicity, and costs of chemotherapy. PMID- 8625153 TI - Significant reduction of medical costs by differentiation therapy with all-trans retinoic acid during remission induction of newly diagnosed patients with acute promyelocytic leukemia. The Japan Adult Leukemia Study Group. AB - BACKGROUND: Differentiation therapy with all-trans retinoic acid (RA) induces more than 80% complete remission with the least complications in patients with acute promyelocytic leukemia (APL). The authors studied the cost-benefit of this new treatment modality compared with conventional chemotherapy in newly diagnosed patients with APL. METHODS: Costs were calculated retrospectively through the monthly bills of each patient to national health insurance and were compared between 36 patients treated with conventional chemotherapy in the AML-87 or AML 89 studies and 40 patients treated with all-trans RA alone or with all-trans RA plus low dose chemotherapy in the AML-92 study of the Japan Adult Leukemia Study Group who were treated at 22 university and cancer hospitals in Japan. RESULTS: Average medical costs except for antileukemic drugs during 2 months after admission were 4,164,026 yen (approximately US$46,300) in the chemotherapy group and 2,906,825 yen (approximately US$32,300) in the all-trans RA group (P < 0.0001). The difference resulted mainly from the costs of antibiotics and of platelet and erythrocyte transfusions. Average costs of antibiotics were 650,494 yen (approximately US$7,200) and 349,138 yen (approximately US$3,900), respectively (P < 0.0002), and those of platelet and erythrocyte transfusions were 1,016,190 yen (approximately US$11,300) and 633,444 yen (approximately US$7,000), respectively (P < 0.0020). CONCLUSION: During the remission induction therapy of newly diagnosed APL, all-trans RA significantly reduced medical costs. all-trans RA should be incorporated into the front-line therapy of patients with APL for medical and economic reasons. PMID- 8625154 TI - Transformation of chronic lymphocytic leukemia to lymphoma of true histiocytic type. AB - BACKGROUND: Chronic lymphocytic leukemia (CLL) may evolve into large cell lymphoma (Richter's syndrome), prolymphocytic leukemia, acute lymphoblastic leukemia, and myeloma. METHODS: A patient with CLL that transformed into a lymphoma of true histiocytic type is described, and the literature on the association of these two disorders is reviewed. RESULTS: Lymphomas of true histiocytic type developing as an aggressive terminal phase of CLL previously have been reported in nine patients. Fever and rapidly increasing lymphadenopathy and splenomegaly were the most common signs and symptoms. As with de novo lymphoma of true histiocytic type, extranodal involvement of the soft tissue, gastrointestinal tract, kidneys, bone marrow, liver, and lungs was documented among the 10 patients with lymphoma of true histiocytic type transformed from CLL. The median interval between diagnosis of CLL and the evolution to lymphoma of true histiocytic type was 25.5 months. Patients with lymphomas of true histiocytic type after CLL were treated with fludarabine and various other combination chemotherapy regimens with only short-lived responses. The median time to death after transformation was only 33 days (range, 10 days to 5 months). CONCLUSION: Lymphomas of true histiocytic type appear to represent an additional, though uncommon, form of transformation in CLL. Although their presentation is reminiscent of other intermediate to high grade lymphomas, they can be distinguished based on their morphologic and immunophenotypic features. In the patients described in this study to date, transformation of CLL to lymphomas of true histiocytic type is a poor prognostic sign, with survival generally of only days to weeks. PMID- 8625156 TI - Malignant breast masses detected only by ultrasound. A retrospective review. AB - BACKGROUND: The authors attempted to appraise the ability of high resolution, real-time ultrasound to find malignant breast masses that are nonpalpable and undetectable by high quality mammography in women with radiographically dense breasts, who were referred because of palpable or mammographically detected lesions. METHODS: The records of breast ultrasound examinations of 12,706 women were retrospectively reviewed. All lesions were classified according to clinical and mammographic status as palpable or nonpalpable and as visible or nonvisible, respectively. Solid masses were sampled percutaneously by fine needle aspiration biopsy (FNAB) using ultrasound guidance and either were excised surgically or followed by sequential imaging. RESULTS: There were 1575 solid masses detected sonographically that were nonpalpable and nonvisible by mammography; percutaneous biopsies (FNABs) were performed on 279 of these. Cytologic interpretation was definitely malignant in 22, suspicious in 18 (6 confirmed cancers), and benign in 183 (no false negatives). Surgery confirmed malignancy in 44 of the 1575 solid masses (2.8%), including 16 in patients with multifocal cancers. CONCLUSIONS: Ultrasound can detect unsuspected, mammographically occult cancers in radiographically dense breasts and can alter treatment planning when a second cancer is found in a breast that otherwise was considered appropriate for conservative surgery. The authors recommend that any solid lesion detected incidentally during breast sonography either should be biopsied percutaneously under ultrasound guidance and/or closely followed with sequential scans. PMID- 8625155 TI - Prognostic implication of the p53 protein and Ki-67 antigen immunohistochemistry in malignant fibrous histiocytoma. AB - BACKGROUND: Mounting evidence indicates that p53 regulates cell growth and abnormal p53 immunophenotypic expression is associated with an unfavorable prognosis for patients with some types of carcinoma. The prognostic significance of p53 overexpression in malignant fibrous histiocytomas (MFHs) of soft tissue has not yet been elucidated. METHODS: Expressions of p53 protein and Ki-67 antigen in 54 primary MFHs of soft tissue were investigated immunohistochemically and indexed quantitatively by counting the number of immunoreactive nuclei versus the total neoplastic nuclei in the representative fields of each tumor to evaluate their prognostic implications and interrelations with other clinicopathologic parameters. RESULTS: The percentages (labeling indices [LIs]) of p53 and Ki-67-immunoreactive nuclei versus the total neoplastic nuclei were 0.1-93.2% (mean +/- standard deviation [SD], 40.6% +/- 21.8%) and 5.3-90.8% (mean +/- SD, 42.7% +/- 29.4%), respectively. The Ki-67 LI correlated with histologic grade (P = 0.01498), primary tumor size (P = 0.04985), disease free interval (reverse correlation, P = 0.00776), and recurrence and metastasis (P = 0.00360). The p53 LI correlated with primary tumor size (P = 0.00431) but did not show any significant correlation with histologic grade, Ki-67 LI, primary tumor size, disease free interval, or recurrence and metastasis. Other significant correlations included histologic grade and disease free interval (P = 0.00010), primary tumor size and disease free interval (reverse correlation, P = 0.00869), histologic grade and recurrence (P = 0.02714), and primary tumor size and primary tumor location (P = 0.00028). In the grouped survival analysis, patients with recurrence or metastasis or with tumors of larger size (> or = 7 cm), high histologic grade, or higher Ki-67 LI (> or = 25%) had a significantly reduced survival (P < 0.05). The different p53 immunohistochemical expression and the different histologic types did not reflect different cumulative survival (P > 0.05). Regression analysis revealed that the primary tumor size and histologic grade, but not Ki-67 or p53 LIs, were independent statistical variables for prognostication. CONCLUSIONS: These results indicate that (1) primary tumor size and histologic grade are two important prognostic factors, (2) Ki-67 LI should be used in adjunct with other main prognostic factors for patients with MFHs, and (3) nuclear p53 overexpression in MFHs of soft tissue is a comparatively common event that has no prognostic implication. PMID- 8625157 TI - Social support and survival among women with breast cancer. AB - BACKGROUND: Two recently reported randomized trials, one among patients with advanced breast cancer and the other among patients with early stage melanoma, suggested that social support may affect survival favorably. This study assesses relationships of social support indicators with 7-year survival among women diagnosed with localized or regional stage breast cancer. METHODS: All newly diagnosed patients with surgically treated localized or regional disease in seven Quebec City hospitals in 1984 were considered for this analysis. Among 235 eligible patients, 224 (95%) participated in a home interview 3 months after surgery. This interview provided information on the use of confidants in the 3 months after surgery. Data on disease and treatment characteristics were abstracted from patients' medical records. RESULTS: Compared with women who used no confidant in the 3 months after surgery, the hazard ratio for the 7-year period was 0.61 (95% confidence interval [CI], 0.33-1.12) among those who had used at least one confidant, 0.54 (95% CI, 0.28-1.06) in women who used two or more types of confidant, and 0.51 (95% CI, 0.22-1.18) among those whose confidants included either physician or nurse. These results were adjusted for age, presence of invaded axillary lymph nodes, adjuvant radiotherapy, and adjuvant systemic therapy (hormone or chemotherapy). CONCLUSION: These results support the view that social support may be associated with longer survival among women with localized or regional stage breast cancer. PMID- 8625159 TI - Leukemia after irradiation for endometrial cancer in Ontario. AB - BACKGROUND: In human studies, the risk of leukemia after ionizing radiation has been found to be increased more often than for any other cancer. It is useful to study patients with cancer treated with radiation because exposure can be measured accurately, follow-up may be long, and often a comparable and sizable nonexposed group exists. Women with endometrial cancer represent an excellent population for study because they meet these Developed Leukemia After Endometrial. METHODS: A population-based matched case-control study, nested among all patients with endometrial cancer diagnosed in Ontario, was undertaken to describe the relationship between radiation therapy and leukemia risk. Among 13,843 subjects treated from 1964 to 1987 who survived at least 1 year, 47 confirmed cases of leukemia were identified. Four control subjects were matched to each patient based on age, calendar year of diagnosis, and length of survival free of a second neoplasm. Medical records were abstracted, and radiation dose administered to active bone marrow was determined by dosimetry. RESULTS: An elevated risk of all leukemias other than chronic lymphocytic leukemia was observed, but only within the first 10 years after endometrial cancer treatment (odds ratio 12.0; 90% confidence interval 2.8-52.1). There was insufficient statistical evidence that risk was influenced by dose or type of radiation therapy. Nor was there any evidence that risk was influenced by age at endometrial cancer diagnosis or by calendar period at diagnosis. CONCLUSIONS: There is an increased risk of leukemia associated with radiation therapy for patients with endometrial cancer, but only within the first 10 years after treatment. PMID- 8625158 TI - Cytokeratin subtypes and involucrin in squamous cell carcinoma of the vulva. An immunohistochemical study of 41 cases. AB - BACKGROUND: Histologic grade seems to be of limited prognostic significance in patients with vulvar carcinoma. However, the study of cytokeratin expression is of potential interest because it allows a more precise evaluation of the degree of squamous differentiation. This study was conducted to investigate whether differences in cytokeratin expression exist between normal vulvar epithelium and vulvar carcinoma and whether these differences are prognostically significant. METHODS: The expression of several differentiation markers, i.e., cytokeratin (CK) 10, CK 13, and involucrin, was studied in samples of 41 vulvar carcinomas. The expression of CK 8, 10, 13, and 14 was compared with CK expression in normal vulvar epithelium and was correlated with tumor grade and tumor growth pattern. Tumor growth pattern was considered type A if infiltrating tumor cell nests showed a layer of small, basaloid cells bordering the surrounding mesenchymal tissue and was considered type B if this was not the case. Prognosis was based on whether disease recurred or not. RESULTS: Sixteen patients had disease recurrence. No prognostic significance of tumor grade was found. Tumor growth pattern was prognostically significant: in patients with a type A tumor, recurrence was observed less often than in patients with a type B tumor (P = 0.03). Cytokeratin 14, typical for basal cells of normal vulvar epithelium, was expressed in all tumors, whereas CK 8 was not expressed in any tumor. A relationship between tumor growth pattern and the concordant expression of differentiation markers was observed: in 55% of type A tumors and in none of type B tumors, concordant expression of CK 10, CK 13, and involucrin was found. CONCLUSION: The expression of differentiation markers in vulvar carcinoma is related strongly to the tumor growth pattern, and this pattern is prognostically significant. PMID- 8625160 TI - Loss of heterozygosity and genomic instability in synchronous endometrioid tumors of the ovary and endometrium. AB - BACKGROUND: The unknown etiology of endometrioid carcinomas of the ovary and the relatively high frequency of a concomitant carcinoma of the endometrium in these patients warrants study of such tumors. The aim of this study was to identify the genetic alterations involved in endometrioid ovarian cancer development, and to determine whether primary tumors of the endometrium and synchronous primary endometrioid tumors of the ovary could be distinguished based on differing patterns of genetic alterations. The distinction of metastatic carcinoma of the ovary from other synchronous primary tumors is often difficult but has important therapeutic and prognostic implications. METHODS: This study examined the genetic alterations at 28 polymorphic DNA markers in the DNA of tumors of 17 patients with endometrioid carcinoma of the ovary, including 5 nonmetastatic ovarian tumors, 5 ovarian tumors metastatic to the uterus, and 7 endometrioid ovarian tumors with a synchronous primary endometrial tumor. RESULTS: Chromosomes 17 and 22 were found to be the most common sites of loss of heterozygosity (LOH) in the 17 patients studied. Loss of heterozygosity on chromosome 17 was associated with advanced stage ovarian tumors. In 96% of LOH events in the metastatic tumors, LOH was observed in the primary tumor and in the metastatic site. Conversely, in four of seven synchronous tumors in which LOH was observed, LOH was confined to the ovarian tumor. Genomic instability was identified in two of seven patients with synchronously occurring tumors that did not demonstrate LOH. A positive family history was noted for these two patients. CONCLUSIONS: A lack of shared genetic alterations and in synchronously occurring endometrial and endometrioid ovarian tumors indicates independent developmental pathways for these tumors. Loss of heterozygosity on chromosome 17 in endometrioid ovarian carcinoma may indicate transition to a more aggressive tumor. PMID- 8625161 TI - Absence of Epstein-Barr virus in penile carcinoma. A study of 42 cases using in situ hybridization. AB - BACKGROUND: The relationship between Epstein-Barr virus (EBV) and carcinomas of the lower female genital tract has been the focus of some studies in recent years. However, the association between EBV and penile carcinoma never has been investigated. The aim of this study was to identify the possible role of EBV in penile carcinoma of Chinese patients. METHODS: Formalin fixed, paraffin embedded tissue of 42 cases of penile carcinoma (including 7 verrucous, 14 well differentiated, 15 moderately differentiated, and 6 poorly differentiated squamous cell carcinomas), 6 penile warts, and 9 normal penile samples were analyzed for EBV using in situ hybridization for EBV-encoded RNA (EBER). RESULTS: Epstein-Barr virus was only found in a few lymphocytes adjacent to the tumor epithelium in 12% (5 cases) of penile carcinoma. Conversely, the tumor cells, penile warts, and normal penile tissue were negative for EBV. CONCLUSIONS: The results suggest that EBV does not play a major role in the etiology of penile carcinoma. PMID- 8625162 TI - Sensitivity of frozen section examination of pelvic lymph nodes for metastatic prostate carcinoma. AB - BACKGROUND: A Total Quality Improvement review of frozen section diagnoses yielded four sequential false-negative frozen section diagnoses in lymph nodes in the evaluation of metastases from prostate carcinoma. Because these results appeared to differ from the department's overall frozen section experience, frozen section diagnoses of pelvic lymph nodes in patients with prostate carcinoma were reviewed to benchmark the department's performance. METHODS: All 220 pelvic lymph node frozen section samples from 110 patients with prostate carcinoma from January, 1986 to July, 1993 at Bridgeport Hospital (BH) were reviewed, and the department's frozen section diagnostic efficiency was compared with: (1) all BH frozen section procedures performed during the same period; (2) pelvic lymph node frozen section analysis and (3) imaging techniques for prostate carcinoma from other institutions found in MEDLARS literature searches from 1973 to 1993. RESULTS: Frozen section diagnostic efficiency for patients with prostate carcinoma from BH was 93.6% (sensitivity 63.2%, specificity 100%) compared with (1) 98% for all BH frozen section analyses (sensitivity 99%, specificity 98%); (2) 90.1% for MEDLARS search pelvic lymph node frozen section prostate carcinoma analyses (sensitivity 66.5%, specificity 100%); and (3) 88% for MEDLARS search magnetic resonance imaging (MRI) pelvic lymph node prostate carcinoma (sensitivity 41%, specificity 97%). CONCLUSIONS: Frozen section diagnosis of pelvic lymph nodes approaches the overall diagnostic efficiency of frozen section analysis and is more sensitive for the diagnosis of prostate carcinoma in pelvic lymph nodes than is MRI because more than half of the metastases are smaller than the 1.0-cm resolution limit of the MRI. False-negative frozen section diagnoses (67% sensitivity) occur because of errors in sampling microscopic metastases. PMID- 8625163 TI - Papillary renal tumors. Morphologic, cytochemical, and genotypic features. AB - BACKGROUND: Papillary renal tumors lack alterations of chromosome 3 and show trisomy of chromosomes 7 and 17, genotypic features distinct from nonpapillary carcinomas. METHODS: The authors examined 39 papillary renal neoplasms to identify morphologic features allowing distinction of high grade from low grade tumors. Twenty-nine papillary tumors and 13 nonpapillary tumors were examined for the presence of trisomy of chromosome 7 using fluorescence in situ hybridization. Data recorded included tumor size, stage, grade, architectural pattern, and the presence of glycogen, foam cells, and iron. RESULTS: Nineteen tumors were classified as low grade and 20 as high grade. The high grade tumors more often formed tall papillae with solid and tubular areas and had more intracellular glycogen, whereas the low grade tumors were more often trabecular. There was no significant difference in tumor size or iron deposition. High grade tumors were of higher stage. Foam cells more commonly were noted in low grade tumors. Sixty seven percent of low grade, 43% of high grade, and none of the nonpapillary tumors showed trisomy of chromosome 7. Metastases developed only in patients with high grade papillary tumors (10/19, 7 within 2 years of diagnosis), all of whom died of disease. CONCLUSIONS: Papillary renal carcinomas with high nuclear grade are more likely to behave in an aggressive fashion, whereas those with low nuclear grade may be associated with longer disease free survival. Furthermore, trisomy of chromosome 7 can be identified by fluorescence in situ hybridization and is useful in differentiating true papillary from nonpapillary renal neoplasms. PMID- 8625165 TI - Acute rhabdomyolysis after concurrent administration of interleukin-2, interferon alfa, and chemotherapy for metastatic melanoma. AB - BACKGROUND: Acute rhabdomyolysis has been described to occur only rarely after systemic administration of cancer chemotherapy drugs, such as cytarabine and 5 azafluorene. A single case of rhabdomyolysis after treatment with alfa-interferon recently was reported, but to the authors' knowledge, there have been no published cases of clinically relevant rhabdomyolysis after systemic therapy with the other agents. METHODS: The case of a 28-year-old woman with metastatic melanoma and no known history of neuromuscular disorders who developed severe myalgia followed by acute, extensive rhabdomyolysis with multiorgan failure after concurrent administration of systemic biologic therapy and chemotherapy consisting of alfa-interferon, interleukin-2, cisplatin, vinblastine, and dacarbazine is described. RESULTS: The patient sustained considerable morbidity requiring hemodialysis and respiratory support but eventually recovered. Review of the literature revealed no reported cases of acute rhabdomyolysis after the systemic administration of these agents with the exception of alfa-interferon. CONCLUSION: Acute rhabdomyolysis should be considered when evaluating patients receiving similar biochemotherapy regimens, particularly for those regimens that are alfa-interferon-based and for patients who develop myalgia along with evidence of multiorgan failure. PMID- 8625164 TI - Molecular analysis at the NF1 locus in astrocytic brain tumors. AB - BACKGROUND: Patients with neurofibromatosis type 1 (NF1) are at increased risk for developing malignant neural crest tumors and juvenile myeloid leukemia. Although the normal allele of the NF1 tumor-suppressor gene is frequently deleted in some of the malignant tumors that arise in patients with NF1, the role of NF1 alterations in the sporadic forms of these cancers is unclear. METHODS: A series of intragenic sequence polymorphisms was used to investigate lymphocyte and tumor DNA samples from 22 adults with high grade malignant gliomas for loss of heterozygosity (LOH) at NF1. In addition, an assay based on the polymerase chain reaction was used to screen these tumors for point mutations at codon 1423. RESULTS: One recurrent anaplastic astrocytoma showed LOH within NF1 but not with a flanking marker located near the gene. Of 21 informative tumors, none showed point mutations affecting codon 1423 of NF1. CONCLUSION: These data suggest that LOH at NF1 is uncommon in sporadic high grade astrocytoma, and codon 1423 is not a "hot spot" for activating point mutations in these tumors. PMID- 8625166 TI - all-trans retinoic acid for treating germ cell tumors. In vitro activity and results of a phase II trial. AB - BACKGROUND: Germ cell tumors (GCTs) are characterized by a capacity to differentiate in vivo and in vitro. The authors' previous work highlighted the finding that retinoid-mediated GCT differentiation in vitro correlates with expression of the retinoic acid receptor-gamma. METHODS: Six human GCT cell lines were studied to determine whether their growth and differentiation were linked to the retinoic acid response pathway. The maturation and growth inhibitory responses to all-trans retinoic acid (RA) were studied as well as the expression of retinoic acid receptor-gamma (RAR-gamma). The clinical antitumor activity of RA was studied in a Phase II trial of RA in patients with chemotherapy-refractory GCTs. RESULTS: The RA response pathway was correlated with the control of growth and maturation in three of the six GCT cell lines studied. In one GCT cell line, RA induced RAR-gamma expression and terminal differentiation. In a second, there was high basal expression of RAR-gamma and poor basal proliferative potential. A third cell line showed a more mature basal phenotype than other cell lines studied and marked growth inhibition when treated with RA. Sixteen patients were treated with RA for an average of 7 weeks in the clinical trial. No complete or partial responses were noted. CONCLUSION: A Phase II clinical trial of RA failed to show significant clinical antitumor activity in patients with chemotherapy refractory GCTs. However in vitro data suggest that the control of growth and maturation in some GCT cell lines involve the RA signaling pathway. Further studies are warranted to define the role that other retinoids with receptor selectivity or more favorable pharmacologic properties may play in the maturation or antitumor responses of GCTs. PMID- 8625167 TI - The use of polyethylene glycol-modified interleukin-2 (PEG-IL-2) in the treatment of patients with metastatic renal cell carcinoma and melanoma. A phase I study and a randomized prospective study comparing IL-2 alone versus IL-2 combined with PEG-IL-2. AB - BACKGROUND: Conjugation of polyethylene glycol to recombinant human interleukin-2 (IL-2) results in a compound, polyethylene glycol-modified IL-2 (PEG-IL-2) that retains the in vitro and in vivo activity of IL-2, but exhibits a markedly prolonged circulating half-life. In mice, one dose of PEG-IL-2 results in tumor regression comparable to that achieved with multiple bolus doses of IL-2. Based on these preclinical studies, a Phase I study with PEG-IL-2 was undertaken in patients with metastatic renal cell carcinoma (RCC) and melanoma. Then, to exploit the higher peak levels attained with IL-2 and the improved trough levels of PEG-IL-2, a combination regimen beginning with bolus IL-2 followed by low dose maintenance PEG-IL-2 was devised and tested for efficacy. METHODS: Patients with measurable metastatic melanoma or RCC were entered in a Phase I dose escalation trial of PEG-IL-2 given once a week by intravenous bolus. This trial then was repeated using a twice-a-week schedule. After determining the maximum tolerated dose (MTD) and a safe outpatient regimen for PEG-IL-2, a hybrid regimen combining an initial high dose IL-2 cycle followed by chronic maintenance with weekly PEG IL-2 was devised. This regimen was compared with a standard regimen consisting of two cycles of high dose unconjugated IL-2 in a randomized prospective clinical trial. RESULTS: When given by intravenous bolus once a week, the MTD of PEG-IL-2 was 12 million IU/m2, with toxicities similar to those of unconjugated IL-2. A twice-a-week schedule was less well tolerated. Of 28 patients given 32 treatment courses at varied dose levels, there were two partial responses and one minor response. A total of 124 patients with metastatic melanoma or RCC were randomized to either standard unconjugated IL-2 therapy or the hybrid regimen. There was no treatment-related mortality in either arm, and the use of PEG-IL-2 resulted in a significant decrease in the need for intensive-care-unit care. The response rates (partial response and complete response) for patients with RCC and melanoma were 19% and 15%, respectively, for IL-2 alone and 17% and 11%, respectively, for the IL-2 and PEG-IL-2 combination. CONCLUSIONS: The combination of high dose IL-2 followed by PEG-IL-2 is a well tolerated regimen with significant activity against RCC and melanoma, but it shows no significant increase in antitumor activity compared with high dose IL-2 alone. PMID- 8625168 TI - Unfavorable DNA ploidy and Ha-ras p21 findings in neuroblastomas detected through mass screening. AB - BACKGROUND: Urinary mass screening has been available for 6-month-old infants throughout Japan since 1985. It is still controversial as to whether the program contributes to the detection of unfavorable neuroblastomas destined to present clinically when a patient reaches an older age. DNA diploidy and tetraploidy, low expression of Ha-ras p21, and an amplified N-myc gene status relate to an unfavorable prognosis. The authors examined these biologic indicators in neuroblastomas detected by urinary mass screening. PATIENTS AND METHODS: Seventy eight neuroblastomas detected by mass screening were studied for DNA ploidy using DNA flow cytometry, Ha-ras p21 expression using immunostaining, and N-myc gene copy number using slot-blot or Southern blot hybridization methods. RESULTS: Of 73 tumors with analyzable DNA flow cytometric results, 18 (24.7%) had diploidy (n = 7) or tetraploidy (n = 11). Twenty-eight (40.0%) of 70 tumors examined showed low-to-absent expression of Ha-ras p21. DNA diploid and tetraploid status correlated significantly with the low-to-absent expression of Ha-ras p21 (P = 0.00021). Fourteen (20.0%) of the 70 patients had both of these two unfavorable prognostic markers. N-myc amplification was not detected in 41 of 41 tumors studied. All 78 patients were alive 8-92 months after completion of treatment. CONCLUSIONS: At least 20.0% of neuroblastomas detected by mass screening have unfavorable biologic prognostic markers. These patients may benefit from early detection and immediate treatment. However, the biologic features associated with a poor prognosis are not predictive of poor outcome in individual patients, and, therefore, should not be used to justify more intensive therapies. PMID- 8625169 TI - Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer. AB - BACKGROUND: Matrix metalloproteinases (MMPs), especially gelatinase A and gelatinase B (GLB), are believed to be important components of the metastatic process. Tissue Inhibitors of Metalloproteinases (TIMPs) form complexes with MMPs and inhibit cancer dissemination. After local secretion, MMPs and their complexes with TIMPs leach into the blood stream where their concentration can be measured, thereby serving as surrogate markers of disease. Elevated plasma gelatinase B levels have been detected in gastrointestinal cancer and breast cancer. The goal of this study was to determine whether plasma GLB:TIMP complexes also are increased in cancer and whether these tests have potential use as prognostic tumor markers. METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed to measure the plasma concentration of GLB:TIMP complexes in patients with cancer. Correlation between ELISA results and clinical outcome was sought. RESULTS: Plasma GLB:TIMP complexes were significantly increased in patients with gastrointestinal cancer and gynecologic cancer, but not in patients with breast cancer. When results from plasma GLB:TIMP complexes and plasma GLB assays were combined (GLB/complexes), abnormal levels of one or both assays were found in 36% and 65% of patients with gastrointestinal and gynecologic cancer, respectively. In Stage IV gastrointestinal cancer, patient survival was shorter (P < 0.001) in the group with increased plasma GLB/complexes than for those with normal plasma levels (4 months vs. 20 months, respectively). CONCLUSIONS: The assay of plasma gelatinase B and GLB:TIMP complexes may be clinically useful in predicting survival in subsets of patients with cancer. The possibility of using these assays in early stage cancer to predict metastasis should be studied. PMID- 8625170 TI - What is the etiology of human brain tumors? A report on the first Lebow conference. AB - Although the conference could not provide a definitive etiologic explanation for the observed increase in incidence of human brain tumors, particularly among the elderly, nevertheless, this interdisciplinary gathering of prominent scientists and clinicians proved invaluable in identifying new avenues of research. Clearly, the puzzle of what causes brain tumors is highly complex, involving links to ionizing/electromagnetic radiation, familial, and dietary factors. The Lebow Conference provided an important scientific framework upon which to build further research studies, and its influence will be felt for years to come. PMID- 8625172 TI - The relationship of p53 expression to the prognosis of 418 patients with gastric carcinoma. AB - BACKGROUND: Mutations of the p53 gene belong to the most common genetic alterations in human cancer that have been implicated in tumorigenesis and tumor progression. Although p53 expression appears to be correlated with prognosis in patients with breast cancer and some other types of cancer, its prognostic role in gastric cancer is still uncertain. In the present study, therefore, the prognostic impact of p53 expression was evaluated in 418 patients with curatively resected gastric carcinomas without residual tumor (RO-resection). METHODS: Tumor sections of 418 RO-resected gastric carcinomas were stained with the monoclonal antibody DO-1 after microwave processing. p53 expression was statistically compared with clinico-pathologic features and postoperative survival. RESULTS: p53 expression was detected in 57.5% of all tumors, with strong inter- and intratumor heterogeneity. No immunoreactivity was observed in the normal mucosa adjacent to the tumor. Statistically, no significant correlation between p53 overexpression and depth of invasion, lymph node involvement, or grade of tumor differentiation was detected. The correlations with blood vessel invasion (P = 0.049) and lymphatic vessel invasion (P = 0.047) were of marginal significance. Survival analysis revealed no significant impact of p53 expression on survival, the 5-year survival rates were 40.6% +/- 7.8% for patients with p53 positive tumors and 49.2% +/- 8.9% for patients with p53 negative tumors (P = 0.43). Furthermore, no correlation with survival was found when four different levels of p53 expression were analyzed (P = 0.51). p53 expression had no influence on survival, in either the lymph node positive or negative groups. CONCLUSIONS: The data suggest that the immunohistochemical detection of p53 expression is at present not a predictor of outcome of patients with gastric cancer or to identify subgroups of patients who may be at higher risk. PMID- 8625171 TI - Phase II study of 5-fluorouracil and folinic acid in the treatment of patients with advanced gastric cancer. A Southwest Oncology Group study. AB - BACKGROUND: The biochemical modulation of 5-fluorouracil (5-FU) by the reduced folate folinic acid (FA) in the treatment of patients with advanced gastric cancer was examined. METHODS: The Southwest Oncology Group performed parallel randomized Phase II trials of two schedules of 5-FU and FA in 80 patients with advanced gastric cancer. Of 76 analyzable patients, 36 were randomized to receive bolus FA (200 mg/m2, days 1-4) along with continuous infusion 5-FU (1000 mg/m2, days 1-4) and 40 were randomized to receive bolus FA (200 mg/m2, days 1-5) before the bolus 5-FU (375 mg/m2, days 1-5). RESULTS: There were three (8%) partial responses (95% confidence interval [CI] 2%-22%) on the continuous infusion arm. The bolus arm had two (5%) complete responses and six (15%) partial responses for an overall response rate of 20% (95% CI 9%-36%). The median duration of response was 4.6 months for the infusion patients and 16.6 months for the bolus patients. Survival was poor, with median survival of 5 months on both regimens. Gastrointestinal toxicity was substantial, with Grade 3 mucositis observed in 36% of patients on the continuous infusion regimen versus only 10% of patients on the bolus regimen. Grade 3 or higher hematologic toxicity occurred more often in the bolus arm than in the continuous infusion arm (28% vs. 14%, respectively). Two toxic deaths occurred, one related to sepsis the other secondary to coronary insufficiency. CONCLUSIONS: Biochemical modulation of 5-FU by FA using the dose and schedules tested has only modest activity in the treatment of advanced gastric cancer. PMID- 8625173 TI - Quantitative lectin-histochemical and immunohistochemical studies on the occurrence of alpha(2,3)- and alpha(2,6)-linked sialic acid residues in colorectal carcinomas. Relation to clinicopathologic features. AB - BACKGROUND: Enhanced sialylation has been considered important for the metastatic growth of colorectal carcinomas. Using sequence- and sialic acid-specific lectins and a monoclonal antibody, the tumor-associated expression of alpha(2,3)- and alpha(2,6)-sialylated oligosaccharides was investigated. The study was designed to examine whether a random increase of sialylation or the expression of oligosaccharides carrying distinct sialic acid residues affect the biology of colorectal carcinomas. METHODS: Using computerized image analysis, formalin fixed and paraffin wax embedded specimens from 152 primary colorectal carcinomas were subjected to a quantitative analysis of the occurrence of sialoglycoconjugates detected by the maackia amurensis agglutinin (MAA: specific for alpha(2,3)-linked sialic acid residues), sambucus nigra agglutinin (SNA: specific for alpha(2,6) linked sialic acid residues), and the monoclonal antibody B72.3 (MAB B72.3: specific for alpha(2,6)-N-acetyl-galactosamine-1-O-Ser/Thre). The data obtained by quantitating lectin/immunohistochemistry were related to morphologic and clinical parameters. RESULTS: Alpha(2,3)-linked sialic acid residues increased from Stage I to Stage II tumors but decreased in advanced carcinomas. Alpha(2,6) sialylated glycoconjugates did not show any association with local tumor growth (depth of invasion). However, metastatic tumor growth was accompanied by a significant increase of alpha(2,6)-sialylated carbohydrate sequences. Univariate survival analysis revealed that the expression of SNA- and MAB B72.3-defined reactivity displayed an inverse relation to 5-year survival. Although more advanced tumor stage was associated with poor 5-year survival, tumors below the cutoff points for SNA- and MAB B72.3-defined reactivity indicated a better prognosis than the neoplasms above the cutoff points. In contrast, the expression of alpha(2,3)-linked sialic acid residues as detected by MAA had no significant effect on survival. Multivariate regression analysis revealed that SNA reactivity, followed by tumor stage and the MAB B72.3-defined antigen reactivity were independent prognostic variables predicting overall survival, whereas MAA reactivity, sex, age, histologic differentiation, and tumor grade had no independent prognostic value. The simultaneous expression of both sialyl-Tn- and SNA-reactivity determined tumors of high risk patients within the different tumor stages. CONCLUSIONS: Sequence-specific sialylation is associated with altered biologic behavior of colorectal carcinomas. PMID- 8625174 TI - Nourishment of hepatocellular carcinoma cells through the portal blood flow with and without transcatheter arterial embolization. AB - BACKGROUND: Although transcatheter arterial embolization (TAE) for hepatocellular carcinoma (HCC) is very effective, local recurrence is not so rare. The reason is thought to be related to portal blood flow. The changes in the nourishing vessels in HCC after TAE were examined from the viewpoint of cell kinetics with direct reference to intracellular transportation of biochemical substrates, namely 5 bromo-2'-deoxyuridine (BrdU), an analogue of thymidine. METHODS: To examine the cell kinetics of carcinoma cells, BrdU was infused intraoperatively in 23 patients with HCC (12 without TAE and 11 post-TAE patients) directly into the portal branch feeding the region to be resected. Specimens were prepared for immunohistochemical staining using BrdU monoclonal antibodies and analyzed using the labeling index (LI). The distribution of the labeled S-phase cell was also examined. RESULTS: The LI in post-TAE patients was about six times higher than patients without TAE, with a significant difference at P < 0.001. Labeled cells were distributed not only peripherally but in the central part of the tumor, although the number was extremely small. CONCLUSIONS: Some HCC cells, although small in number, are nourished by the portal blood flow directly throughout the viable tumor mass, which may act as the main blood supplier during the period after TAE. For greater local control of HCC, complete resection of HCC and/or the use of chemotherapy via not only the hepatic artery but also the portal blood flow are beneficial. PMID- 8625175 TI - Prospective study of chemoprevention of hepatocellular carcinoma with Sho-saiko to (TJ-9). AB - BACKGROUND: Most hepatocellular carcinomas (HCC) arise in patients with cirrhosis, in whom its incidence is high. The prevention of HCC in patients with cirrhosis is important. METHODS: A prospective, randomized, nonblind controlled study was performed to evaluate the preventive effect of Sho-saiko-to (TJ-9) on HCC development. TJ-9 is a Chinese herbal medicine that contains crude extracts of seven herbs; it has antitumor effects in experimental animals. Two hundred sixty patients with cirrhosis were randomly assigned to two groups, matched for age, sex, presence of hepatitis B surface antigen, and the severity of liver damage. The patients in the trial group were given TJ-9 at a daily oral dose of 7.5 g in addition to the conventional drugs given to the control patients. The patients were prospectively monitored for 60 months and the cumulative incidence of HCC and the survival rate in the two groups were calculated. RESULTS: The cumulative incidence curve for 5 years of the trial group was lower than that of the control group (P = 0.071). For the patients without HBs antigen, the difference was significant (P = 0.024). The survival curve for 5 years of the trial group was higher than that of the control group (P = 0.053). For the patients without HBs antigen, the difference was significant (P = 0.043). CONCLUSIONS: TJ-9 helped to prevent the development of HCC in patients with cirrhosis, particularly in patients without HBs antigen. PMID- 8625176 TI - Effectiveness of the cytologic examination of pure pancreatic juice in the diagnosis of early neoplasia of the pancreas. AB - BACKGROUND: The early diagnosis of pancreatic carcinoma through the use of traditional radiographic or ultrasonographic methods is extremely difficult. METHODS: To detect an early and potentially curable cancer of the pancreas, endoscopic retrograde pancreatography (ERP) and aspiration cytology of pure pancreatic juice were performed in 295 consecutive patients who had symptoms or findings that suggested pancreatic disease but in whom there was neither a pancreatic mass nor ductal stenosis. RESULTS: Positive cytologic results were obtained in 12 patients (4%). With the aid of intraoperative cytodiagnosis, all 12 early neoplasms of the pancreas were successfully resected. Of these 12 resected specimens, 4 were adenocarcinoma with minimal invasion, 3 were in situ adenocarcinoma and 5 were marked atypia. All 12 patients were alive with no evidence of recurrence for an average of 32 months after surgery. The 283 patients who had negative ERP-cytology results were observed, but no further cases of pancreatic cancer were found. CONCLUSIONS: Because ERP-cytology is simple to perform, safe, and reliable, it is useful in the diagnosis of patients who have early neoplasm of the pancreas. PMID- 8625177 TI - The role of squamous cell carcinoma antigen in the management of laryngeal and hypopharyngeal cancer. AB - BACKGROUND: The efficacy of squamous cell carcinoma antigen (SCC-Ag) in laryngeal cancer to predict those patients who will relapse after primary treatment (surgery or radiotherapy) and its utility to detect relapses early and thereby increase salvage rates and cure were assessed. METHODS: Sixty healthy donors and 168 patients with laryngeal cancer were included in this prospective trial. Squamous cell carcinoma antigen was measured at diagnosis in all patients, 24 hours and 1 week after surgery in 113 patients and every 10 Gy of administered dose and 2 weeks after treatment in 49 patients primarily referred to radiotherapy. The marker was determined every 3-6 months during follow-up. All patients who relapsed had SCC-Ag studies before and after salvage treatment. RESULTS: The selected cut-off value was 1.5 ngr/ml (mean value in control group, 0.65 + 2 standard deviation [0.38]). Seventy-eight percent of patients with cancer had elevated SCC-Ag values at diagnosis. Squamous cell carcinoma antigen was statistically related to TNM categories (T, P < 0.04; N, P < 0.05; Stage, P < 0.01). Seventy-five percent of those patients with previously elevated pretreatment values normalized after treatment. Incomplete surgical resection (P < 0.0001) or persistence of the disease after radiotherapy (P < 0.01) were related to high posttreatment values. Squamous cell carcinoma antigen was elevated in 88% of the patients who relapsed. In 55% of the recurrences, SCC-Ag was elevated 3 months before pathologic confirmation of relapse. Salvage by surgery or radiotherapy was effective in 70% of the patients. Squamous cell carcinoma antigen posttreatment values were the most important factor in predicting disease free survival (DFS) (P < 0.0001) and overall survival (P < 0.03). CONCLUSIONS: Squamous cell carcinoma antigen is an excellent marker of residual disease after primary treatment that can lead to the addition of other therapeutic procedures (surgery and postoperative radiotherapy). The absence of posttreatment SCC-Ag is the best predictor of DFS, its presence detects recurrence in early stages, permitting salvage of an increased proportion of patients primarily referred for palliative treatment. PMID- 8625178 TI - The treatment of patients with recurrent brain metastases. A retrospective analysis of 109 patients with nonsmall cell lung cancer. AB - BACKGROUND: Brain metastases represent a major source of morbidity in patients with cancer. METHODS: Treatment outcomes were analyzed retrospectively in 214 patients with brain metastases from nonsmall cell lung cancer (NSCLC) who underwent resection at Memorial Hospital (New York, NY) between January, 1976, and December, 1990. RESULTS: The study group included 109 patients (51%) with symptomatic recurrent brain tumors (median, 5.0 months after complete resection). Recurrence in the brain was at the original site in 62% of patients and at other sites in 38%. The median survival (MS) was 11.3 months in the recurrence group compared with 9.5 months (P < 0.5) in the nonrecurrence group (N = 105). Thirty two patients had further surgery after recurrence; their median relapse time was 5.2 months. In these patients, survival (MS, 15.0 months) calculated from the time of their first operation, was significantly different (P < 0.001) from that of patients who did not undergo a second procedure (N = 77) (MS, 10.0 months). In the 32 patients who underwent reoperation, MS from the time of the second operation was 10 months, whereas the median interval from the first operation was 5 months (average, 5.7 months). Eight of these 32 patients had a third operation, after a median relapse time of 4 months; their MS was 42 months. There was a significant difference (P < 0.02) between the MS of 39 patients synchronously diagnosed with lung cancer and brain metastasis (MS, 9.0 months) and 70 metachronously diagnosed patients (MS, 14.6 months). Women (N = 55) survived longer than men (N = 54) (14.4 months vs. 9.7 months, P < 0.01). Univariate analysis showed that histology, disease stage, and completeness of resection of the primary tumor also affected survival (P < 0.02, P < 0.014, and P < 0.001, respectively). Although no significant difference was found between survival of patients with recurrence in the supratentorial space and patients with recurrence in the posterior fossa (MS, 11.4 months vs. 11.2 months, P < 0.13), no one from the latter subgroup survived 3 years. CONCLUSIONS: If technically feasible, further surgery is effective in prolonging the survival of patients with NSCLC who have recurring brain metastases. PMID- 8625179 TI - Oral combination antiemetics in patients with small cell lung cancer receiving cisplatin or cyclophosphamide plus doxorubicin. AB - BACKGROUND: Intravenous antiemetic combinations containing a 5-HT3 receptor antagonist (like metoclopramide, ondansetron, or granisetron) with dexamethasone have become the standard therapy for the treatment of acute chemotherapy-induced vomiting. Intravenous antiemetics, however, can be more costly and take more time to prepare and deliver, and therefore are not preferred for home, outpatient, or office use. The objective of this study was to determine the antiemetic activity and safety of the oral combination antiemetic regimen of metoclopramide, dexamethasone, and diphenhydramine in patients with small cell lung cancer receiving standard outpatient chemotherapy programs. METHODS: Fifty-two patients receiving initial cisplatin (60 mg/m2) or cyclophosphamide (600-1500 mg/m2) plus doxorubicin (30-45 mg/m2) received an oral regimen of metoclopramide (3 mg/kg x 2 then 2 mg/kg x 2 or 4 doses), dexamethasone (20 mg) and diphenhydramine (50 mg x 2 or 3 doses) (oral MDD), beginning 30 minutes before chemotherapy. RESULTS: Vomiting was prevented in 15 of 21 (76%) patients (95% confidence interval [CI], 53%-92%) receiving cisplatin and 21 of 31 (71%) individuals (95% CI, 52%-86%) given cyclophosphamide plus doxorubicin. Adverse effects were mild and transient and included sedation, loose stools, akathisia, and hiccoughs. CONCLUSIONS: The oral MDD antiemetic regimen prevented acute emesis in 73% of the patients entered and was well tolerated in this population of patients with small cell lung cancer. PMID- 8625181 TI - Stage I nonsmall cell lung cancer. A multivariate analysis of treatment methods and patterns of recurrence. AB - BACKGROUND: Nonsmall cell lung cancer (NSCLC) has become the leading cause of cancer-related deaths in women and men in the United States, with more than 157,000 estimated deaths in 1995. Surgical resection remains the mainstay of therapy in Stage I and II disease. However, local and distant recurrence account for the disappointing survival rates after resection. Appropriate selection of surgical procedures and effective use of adjuvant therapies will depend upon the elucidation of prognostic factors that predict for recurrence. METHODS: A detailed analysis was undertaken to evaluate surgical therapy and to define risk factors associated with recurrence and cancer death in 289 consecutive patients with NSCLC who were diagnosed, resected and followed at the Duke University Medical Center from January 1, 1980, until December 31, 1988. These patients had no evidence of metastases on head and chest/abdominal computed tomograms and radionuclide bone scans before resection. Resected specimens from these patients pathologic verification of Stage I disease. Follow-up was complete in all cases through 8/1/94 (median, 61 months). Variables analyzed included age, sex, smoking history, presenting signs and symptoms, operative procedure, histopathology, hospital course including complications, and the time and location of any recurrence or cancer death. RESULTS: The 30-day mortality rate was 5 of 289 (1.7%), with minor and major morbidity rates of 17% and 9%, respectively. Statistical comparison of lobectomy (193) wedge resection (75) and pneumonectomy (21) revealed significantly (P < 0.04) smaller tumors (T1), more comorbidity, and fewer complications for wedge resection patients. A trend (P < 0.09) toward an increased rate of local/regional recurrence and no difference in survival was also observed for wedge resection. One hundred five patients died of cancer (13 month median time to recurrence) for an actual 5-year survival of 63%. Significant univariate predictors of early recurrence and decreased survival (P < 0.01) were: male sex, the presence of symptoms, hemoptysis, chest pain, type of cough, tumor size in cm and by T-classification, visceral pleural invasion, high mitotic index, and vascular invasion. Significant (P < 0.05) multivariate independent variables for early recurrence and cancer death were the presence of symptoms, vascular invasion, pleural invasion, high mitotic index, and tumor size greater than 3 cm. CONCLUSION: Current surgical therapy for stage I NSCLC has an acceptable morbidity and mortality rate. The current data also stratify patients with Stage I NSCLC into high and low risk populations that can be used in future randomized trials of adjuvant therapy. PMID- 8625180 TI - Adjuvant radiotherapy versus combined sequential chemotherapy followed by radiotherapy in the treatment of resected nonsmall cell lung carcinoma. A randomized trial of 267 patients. GETCB (Groupe d'Etude et de Traitement des Cancers Bronchiques). AB - BACKGROUND: The effect of adjuvant chemotherapy after resection of nonsmall cell lung cancer (NSCLC) remains an unresolved question. METHODS: From October, 1982, to November, 1986, 267 patients with resected NSCLC were included in a randomized trial. The adjuvant allocated treatments were either postoperative radiotherapy, 60 Gy in 6 weeks (radiotherapy group = 129 patients), or three courses of postoperative COPAC (cyclophosphamide, doxorubicin, cisplatin, vincristine, lomustine) chemotherapy followed by a similar radiotherapy schedule (chemotherapy/radiotherapy group = 138 patients). RESULTS: The sex ratio (M:F) was 19/1; mean age was 57 +/- 9 years. According to postoperative staging, 8 patients were Stage I, 70 were Stage II, and 189 were Stage III. The histologic type was squamous cell carcinoma in 175 patients, adenocarcinoma in 57, and large cell carcinoma in 35. The minimum follow-up was 6 years. Four patients were lost to follow-up. Death was recorded in 233 patients. No significant difference was observed in terms of disease free interval (P = 0.47, log-rank test), or overall survival (P = 0.68, log-rank test). With respect to the first site of relapse, distant metastasis occurred more frequently in the radiotherapy group (P = 0.09, log-rank test) whereas local relapse occurred similarly in both groups (P = 0.27). An interaction was observed between lymph node involvement and treatment in terms of overall survival. CONCLUSIONS: The COPAC chemotherapy as postoperative treatment failed to improve overall survival in patients with resected NSCLC receiving postoperative radiotherapy but decreased the pattern of metastatic progression, mainly in the N2 patients. PMID- 8625183 TI - Axillary lymph node metastases of bronchogenic carcinoma. AB - BACKGROUND: Metastasis of bronchogenic carcinoma to axillary lymph nodes is rare. The pathways and possible significance of axillary lymph node metastasis from bronchogenic carcinoma were investigated. METHODS: Seventeen patients with probable axillary lymph node metastases from bronchogenic carcinoma were identified by computed tomography. There were 15 nonsmall cell lung cancers and 2 small cell lung cancers. Axillary lymph node metastasis was proven by biopsy in six cases. Metastases were presumed because of an increase in the size of axillary lymph nodes compared with prior studies in six patients and enlarged axillary lymph nodes associated with biopsy-proven ipsilateral supraclavicular lymph node metastasis in five patients. RESULTS: Four of 10 right-sided lung cancers had ipsilateral and six had contralateral axillary lymph node metastases. Six of seven left-sided cancers had ipsilateral and one had contralateral axillary lymph node metastases. Patients with ipsilateral lymph node disease had chest wall involvement and/or supraclavicular and mediastinal lymph node metastases. All seven patients with contralateral axillary lymph node metastases had supraclavicular and/or mediastinal lymph node metastases. CONCLUSION: Bronchogenic carcinoma may involve ipsilateral axillary lymph nodes via either chest wall invasion or retrograde spread from supraclavicular lymph nodes. Contralateral axillary lymph node involvement requires involvement of contralateral mediastinal and supraclavicular lymph nodes with retrograde spread to the axillary lymph nodes. PMID- 8625182 TI - Objective malignancy grading of squamous cell carcinoma of the lung. Stereologic estimates of mean nuclear size are of prognostic value, independent of clinical stage of disease. AB - BACKGROUND: The prognostic value of quantitative histopathologic parameters was evaluated in 55 consecutively treated patients with operable lung carcinoma of squamous (N = 39) and mixed, adenosquamous (N = 16) cell type. Patients alive were followed for at least 12 years. METHODS: Using a projection microscope and a simple test system in fields of vision systematically selected from the whole tumor area of one routine section, five quantitative histopathologic variables were estimated: the mean nuclear volume, the mean nuclear profile area, the density of nuclear profiles, the volume fraction of nuclei to tissue, and the number of mitotic profiles per 10(3) nuclear profiles. For each patient, information was recorded regarding sex, age at diagnosis, and clinical stage of disease. RESULTS: Single-factor analyses showed that a favorable prognosis was associated with early clinical stages (Stages I and II) and young age (P < or = 0.03), and that females tended to do better than males (P = 0.09). Estimates of the mean nuclear volume were of prognostic significance (P = 0.02), small nuclei being associated with the worst prognosis. In a multivariate Cox analysis, clinical stage, age, and mean nuclear volume were found to be parameters of significant, independent prognostic value. CONCLUSIONS: The present feasibility study indicates that estimates of the mean nuclear volume are of prognostic value, independent of the clinical stage of disease. This quantitative histopathologic variable is highly reproducible and easily obtained using an unbiased stereologic method. Thus, the mean nuclear volume may be a parameter of future importance in the clinical management of patients with carcinoma of the lung. PMID- 8625184 TI - Clinical value of pretreatment serum Cyfra 21-1, tissue polypeptide antigen, and squamous cell carcinoma antigen levels in patients with cervical cancer. AB - BACKGROUND: The clinical value of pretreatment serum concentrations of cytokeratin 19 fragments, measured by Cyfra 21-1, was compared with tissue polypeptide antigen (TPA) and squamous cell carcinoma antigen (SCC-Ag) in 78 patients with squamous cell cervical cancer. METHODS: Serum levels were compared with tumor stage, size, lymph node status, parametrial involvement, and prognostic data. The clinical performance of the different tests was evaluated by their receiver operating characteristic (ROC) curves. RESULTS: Serum levels of all markers were related significantly to tumor stage and size. Elevated serum levels of these markers were not found to be predictive for the presence of lymph node metastases. In contrast, a positive relation was found between quantitative serum Cyfra 21-1, TPA, and SCC-Ag levels and the presence of either lymph node metastases or parametrial involvement (i.e., extracervical disease). An elevated, i.e. positive, serum Cyfra 21-1 level was related significantly to the presence of extracervical disease (P = 0.020). The clinical performance of each serum marker in predicting lymph node metastases or parametrial involvement appeared to be similar as expressed by their ROC curves. In the univariate analysis, Cyfra 21 1, TPA, and SCC-Ag showed prognostic value with respect to disease free interval and survival. Elevated serum levels were associated with a poor prognosis. However, after adjusting for tumor stage and size, none of these markers remained statistically significant. CONCLUSIONS: Cyfra 21-1 may be of additional value in assessing stage of disease, tumor size, and the presence of extracervical disease in patients with cervical cancer. Determining its value during follow-up warrants further study. PMID- 8625185 TI - Phase I/II study of neoadjuvant intraarterial chemotherapy with mitomycin-C, vincristine, and cisplatin in patients with stage IIb bulky cervical carcinoma. AB - BACKGROUND: Stage IIb bulky cervical carcinomas have been considered difficult to treat successfully by radiation and/or surgery, compared with smaller lesions. This study was designed to evaluate the efficacy of neoadjuvant pelvic intraarterial chemotherapy (IAC) and to determine the optimal dosage of cisplatin for reducing tumor volume in these patients. METHODS: Twenty-one previously untreated patients with primary cervical carcinoma of more than 4 cm in greatest dimension and parametrial invasion were included in this study. Pelvic IAC was administered using a combination of mitomycin-C, 10 mg/m2; vincristine, 1 mg/m2; and cisplatin, 50 mg/m2 (MVC; group 1, 8 patients) or 75 mg/m2 (group 2, 13 patients). Tumor volumes were measured three-dimensionally by magnetic resonance imaging (MRI) before and after three courses of IAC. Clinical responses were evaluated with gynecologic examination and MRI; pathologic responses were evaluated with histologic examinations of surgical specimens. RESULTS: The mean volume reduction rate (74.2% vs. 97.2% in groups 1 and 2, respectively, P = 0.0022), the clinical complete response rate (0% vs. 69.2%, P = 0.0033), and the pathologic complete response rate (0% vs. 46.2%, P = 0.0445) were significantly higher in group 2. Type III radical hysterectomy was possible in 19 patients (90.5%). Toxicities of grades 2-3 (World Health Organization criteria) were nausea and/or vomiting (38.1%), leukopenia (33.3%), and fever (14.2%). CONCLUSIONS: These preliminary results suggest that neoadjuvant pelvic IAC with MVC (especially with cisplatin at a dose of 75 mg/m2) is effective in reducing tumor volume, increasing the clinical and pathologic complete response rate, and improving the operability in most patients with Stage IIb bulky cervical carcinoma, generally considered inoperable. PMID- 8625186 TI - Phase III randomized trial of interleukin-2 with or without lymphokine-activated killer cells in the treatment of patients with advanced renal cell carcinoma. AB - BACKGROUND: Treatment with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative therapeutic benefit of the addition of LAK to IL-2 was unknown. METHODS: A randomized Phase III trial was conducted in patients with RCC comparing continuous intravenous infusion (CI) IL-2 alone with CI IL-2 plus LAK. Interleukin-2 was administered at 3 x 10(6) U/m2/day on days 1 5, 13-17, 21-24, and 28-31. Patients on the LAK treatment arm underwent leukapheresis on days 8-10 and LAK cell reinfusion on days 13-15. The results are reported with long-term follow-up. The published experience with IL-2 alone or with the addition of LAK was investigated in a quantitative literature survey. The response proportions were studied by schedule (high dose bolus, moderate dose, low dose) and by concomitant administration of LAK. RESULTS: Seventy-one patients were treated, 36 on the IL-2 arm and 35 on the IL-2 plus LAK arm. Four patients (6%) had major responses (two complete, two partial). The median survival of all patients was 13 months (95% confidence interval [CI], 9-18 months). There were no differences between treatment arms with regard to response (P = 0.61) and survival (P = 0.67). More patients on the LAK arm experienced pulmonary toxicity (P = 0.008). The overall weighted response proportion was 16% (95% CI, 8%-24%) for the 39 published series of 1291 patients treated with IL-2. The 95% confidence intervals for response proportion overlapped when compared by schedule and by administration of LAK. CONCLUSIONS: The dose and schedule of IL-2 used in this study resulted in a low level of antitumor activity and the addition of LAK did not improve the response rate against RCC. Given the infrequent, but reproducible, responses with IL-2 and interferon-based regimens, continued investigation of these agents is warranted as is the study of new cytokines. Alternative treatment strategies should be studied in RCC and new agents and treatment regimens that appear promising in Phase II studies must be studied in randomized trials. PMID- 8625187 TI - Radical cystectomy for high risk patients with superficial bladder cancer in the era of orthotopic urinary reconstruction. AB - BACKGROUND: Many patients with aggressive clinically superficial bladder cancer ultimately die of their cancer, due in part to resistance to undergo radical cystectomy. Radical cystectomy is usually curative for patients with pathologic superficial tumors. Orthotopic urinary reconstruction has lessened the morbidity and lifestyle changes in patients after cystectomy, and may increase patient acceptance of cystectomy as therapy for high risk superficial tumors. METHODS: A retrospective analysis was performed on 182 patients with clinically superficial bladder cancer (Ta, Tis, T1) who had radical cystectomy between 1971 and 1989, to determine the incidence of pathologic upstaging and to assess overall survival, cause-specific survival, and recurrence free survival. Indications for cystectomy included failure of intravesical chemo- or immunotherapy, high grade lamina propria invasive tumors, presence of bladder diverticulae, spread of superficial tumors into the prostatic urethra, and endoscopically uncontrollable tumors. RESULTS: Pathologic upstaging to muscle-invasive or metastatic tumors occurred in 34% of patients, and of these, only half remained organ confined. Metastases were present in 8% of patients at the time of cystectomy. Mucosa-confined tumors were upstaged only 19% of the time, whereas tumors demonstrating lamina propria invasion (T1) were upstaged 40% of the time. Pathologic upstaging to muscle invasive or metastatic disease was significantly associated with a decreased probability of survival (P = 0.042). With a median follow-up of 7.2 years, overall survival was estimated to be 86%, 72%, and 47%, and recurrence free survival 90%, 83%, and 77% at 2, 5, and 10 years, respectively. CONCLUSIONS: Pathologic upstaging to muscle-invasive or metastatic tumors occurs in one third of highly selected patients with clinically superficial bladder cancer who have had radical cystectomy, half of whom have extravesical disease. Survival is significantly decreased in this group of upstaged patients. With the alternative of orthotopic urinary diversion available to most men and women requiring cystectomy, radical cystectomy should be considered a viable alternative to continued conservative measures for selected patients with aggressive superficial bladder tumors. PMID- 8625188 TI - Improved long term survival after intracavitary interleukin-2 and lymphokine activated killer cells for adults with recurrent malignant glioma. AB - BACKGROUND: The median survival for adults with glioblastoma multiforme (GBM) is 12 months, despite surgery, radiation, and chemotherapy. Regimens using interleukin-2 (IL-2) plus lymphokine-activated killer (LAK) cells have been beneficial against systemic cancers, albeit with significant toxicity. METHODS: Nineteen adults with recurrent malignant glioma (5 GBMs, and 4 anaplastic astrocytomas (AA)), Karnofsky performance status 60 or greater, were treated with intracavitary autologous LAK cells plus IL-2 after reoperation. Lymphokine activated killer cells and IL-2 were given on day 1, and IL-2 alone was given 5 times during a 2-week cycle. This cycle was repeated at 2 weeks to constitute one 6-week course of therapy. Each two-cycle course of treatment was repeated at 3 month intervals for patients with stable disease or response to therapy. At the conclusion of immunotherapy, all patients were offered chemotherapy, generally carmustine or procarbazine, including responders. Corticosteroids were strictly limited during immunotherapy. Sequential reservoir aspirates were obtained for microbiologic and cytologic analyses. RESULTS: The maximal tolerated dose for a 12-dose course of therapy was 1.2 million international units (MIU) per dose. Dose-limiting, cumulative IL-2-related central nervous system (CNS) toxicity was observed at 2.4 MIU per dose. Three responses were confirmed by computed tomography scan during therapy: one complete response (CR) (1 AA), and two partial responses (PR) (2 GBM); as well as a significant increase in GBM survival. One additional CR (GBM) was observed at 17 months. The median survival for immunotherapy patients with GBM was 53 weeks after reoperation (N = 15) (mean, 87.9 +/- 21.4 weeks, standard error for the mean), with 8 of 15 surviving more than 1 year (53%). The median survival for 18 contemporary patients with GBM reoperated and treated with chemotherapy was 25.5 weeks (mean, 27.4 +/- 3.7 weeks), with 1/18 alive at 1 year (> 6%). Six of the 15 patients with GBM had additional surgery or biopsy, and chemotherapy after immunotherapy. The contribution of subsequent chemotherapy to survival cannot be discounted. CONCLUSIONS: Lymphokine-activated killer cells and IL-2 can be administered safely within the CNS resulting in improved long term survival in patients with recurrent glioblastoma. Increased survival was associated with significant biologic changes characterized by a regional eosinophilia, and extensive lymphocytic infiltration. A prospective randomized clinical trial is warranted. PMID- 8625189 TI - Chromosomal abnormalities involving 11q13 are associated with poor prognosis in patients with squamous cell carcinoma of the head and neck. AB - BACKGROUND: In individual patients with squamous cell carcinoma of the head and neck (SCCHN), established prognostic factors do not satisfactorily predict clinical outcome. Although the karyotype is an independent prognostic factor in certain hematologic malignancies and solid tumors, no data have been reported concerning the possible relationship between chromosomal abnormalities and clinical outcome in patients with SCCHN: METHODS: In 116 cases of primary SCCHN, short term cultures were analyzed cytogenetically during 1987 through 1991, the karyotypes were divided into four groups: k1, normal (n = 35); k2, numeric changes only (n = 31); k3, simple structural abnormalities (n = 27); and k4, complex karyotypes (n = 23). The patients were followed for at least 18 months after diagnosis or until death. RESULTS: The 2-year survival rate was lower in the k4 subgroup (35%) than in the k1, k2, and k3 subgroups taken together (61%), both in the series as a whole (P = 0.02), and in the largest tumor site subgroup, laryngeal squamous cell carcinoma (n = 32), (P = 0.04). The most prevalent breakpoint was in chromosome band 11q13, occurring in 11 tumors, 10 of which belonged to the k4-subgroup. The 2-year survival rate was lower for patients with 11q13 rearrangements (20%) than for those without (60%), both in the series as a whole (P = 0.001), and in the k4-subgroup (P = 0.02). CONCLUSIONS: The results suggest that in SCCHN the presence of a complex karyotype is associated with poor prognosis, particularly when 11q13 rearrangements are present. PMID- 8625190 TI - Multiple cycles of high dose chemotherapy supported by hematopoietic progenitor cells as treatment for patients with advanced malignancies. AB - BACKGROUND: Retrospective studies suggest that dose intensity is an important determinant of outcome in the treatment of patients with a variety of malignant diseases such as breast cancer, ovarian cancer, and lymphoma. Unfortunately, these results have not been clearly substantiated in prospective randomized trials. One problem with these studies may be that the degree of dose escalation is not sufficient to result in an improved outcome because the chemotherapy doses are limited by hematopoietic toxicity. In an attempt to deliver more dose intensive therapy, the feasibility of the administration of multiple cycles of high dose chemotherapy with hematopoietic progenitor cell and growth factor support was investigated in patients with advanced malignancies. METHODS: Nineteen patients with metastatic breast cancer and six patients with refractory non-Hodgkin's lymphoma were initially treated with etoposide (VP-16) (2 gm/m2) and granulocyte-colony stimulating factor (G-CSF). Peripheral blood hematopoietic progenitor cells were collected by leukapheresis and cryopreserved as the patients' leukocyte counts recovered from the nadir induced by VP-16. Patients were then treated with four cycles of mitoxantrone (18 mg/m2), thiotepa (150-200 mg/m2) and cyclophosphamide (4500-5000 mg/m2) as a 48-72 hour continuous infusion followed by infusion of one-quarter of their progenitor cells 48 hours later. All patients also received G-CSF (5 micrograms/kg/day) until engraftment. RESULTS: A total of 88 of a planned 100 cycles of therapy were administered to these 25 patients. The median time to recovery of an absolute neutrophil count of 500/microliters or greater was 13-14 days (range, 7-18 days) and time to recovery of a platelet count of 20,000/microliters or greater was 13-14 days (range, 7-16 days) after the initiation of each cycle of chemotherapy. The median number of platelet transfusions required after each cycle was 2-3 (range, 0-18 transfusions) and the number of erythrocyte transfusions was 4 (range, 0-10). The most common toxicity was diarrhea. Prophylactic intravenous antibiotics were administered to avoid fever with neutropenia. Two patients developed interstitial pneumonitis and one patient died. One heavily pretreated patient failed to engraft after the first cycle. Reversible veno-occlusive disease of the liver developed in one patient after the fourth cycle of therapy. Four patients progressed while on therapy. Eight patients were disease free and 13 patients had a partial response or had a positive bone scan as the only evidence of disease at the completion of therapy. Seven patients, two with lymphoma and five with breast cancer (28%), remain progression free with a median follow-up of 24.7 months (range, 17-28 months). CONCLUSIONS: Support with hematopoietic progenitor cells and growth factors allows the timely administration of repetitive cycles of high dose chemotherapy, resulting in a significant increase in dose intensity with acceptable toxicity. PMID- 8625191 TI - Myelodysplasia. The clinical spectrum of 51 patients. AB - BACKGROUND: Over a 15 year period, the authors followed 51 male patients with myelodysplastic syndromes whose clinical findings, laboratory data, and evolution demonstrated a wide spectrum of disease. METHODS: The following characteristics were assessed: age at diagnosis, risk factors, clinical presentation, laboratory features, category of myelodysplasia, leukemic conversion, and overall survival. RESULTS: The clinical manifestations included hemolytic episodes in two patients, antibody-mediated thrombopenia in one, marked marrow fibrosis in two; thrombocytosis in three, and simultaneous lymphoproliferative disorders in two. There were 21 patients whose marrow was either normo- or hypocellular. Six patients presented with single cytopenia but not anemia. There were six instances of overlapping of the French-American-British classification. Eighteen patients progressed to acute leukemia and 1 to chronic myelomonocytic leukemia. CONCLUSIONS: These observations indicate that patients with myelodysplastic syndromes may have single cytopenia without anemia that progresses to acute leukemia and may, rarely, evolve into chronic myelomonocytic leukemia. The clinical aspects of these syndromes may include autoimmune phenomena and myeloproliferative features. PMID- 8625192 TI - Risk of neoplastic and other diseases among people with heterozygosity for hereditary hemochromatosis. AB - BACKGROUND: Increased body iron stores have been associated with risk of heart attack in men and risk of cancer and colonic adenoma in both sexes. Because heterozygous carriers of hereditary hemochromatosis (HH) have on the average increased iron stores compared with noncarriers of the HH gene and comprise as much as 15% of the American population, disease risk in HH heterozygotes was investigated. METHODS: A community-based cohort was defined, in which the exposure variable was heterozygosity for HH. Heterozygotes were identified by mailing individuals homozygous for HH questionnaires concerning the health histories of their parents (predominantly heterozygotes or exposed). Spouses of the HH homozygotes were asked to complete accompanying questionnaires concerning their parents (unexposed). The frequencies for exposed and unexposed, age adjusted relative risks (RR), and 95% confidence intervals (CI), of cancer, heart disease, and stroke as causes of death as well as the cumulative incidence of heart attack, diabetes, stroke, hypertension, colonic adenoma, and cancers of the lung, colorectum, breast, cervix, pancreas, stomach, and blood were estimated. RESULTS: Data were available for 1950 HH heterozygotes and 1656 unexposed subjects. Elevated RR were observed in HH heterozygotes in males for diabetes (RR, 1.16; 95% CI, 1.01-1.33), colorectal cancer (RR, 1.28; CI, 1.07-1.53), and hematologic malignancy (RR, 1.30; CI, 1.30-1.63), for colonic adenoma in females (RR, 1.29; CI, 1.08-1.53) and males (RR, 1.24; CI, 1.05-1.46), and for stomach cancer in females (RR, 1.37; CI, 1.04-1.79). CONCLUSIONS: Heterozygosity for HH is associated with increased risk for colorectal neoplasia, diabetes, hematologic malignancy, and gastric cancer. No increased risk of heart disease, cancer death, or cancers of the lung, breast, or cervix were demonstrated. PMID- 8625193 TI - Evaluation of oncology registry follow-up methods. AB - BACKGROUND: The accuracy and completeness of oncology (tumor) registry patient follow-up information directly affects the validity of the oncology registry system for determining outcomes, as a quality assurance measure, and for research activities. At this institution, if a hospital-based patient encounter has not been identified during the previous year, a follow-up letter is sent to the attending physician to obtain current information about the patient. Difficulties using this method include (1) correct identification of the appropriate physician (2) constraints on physician and staff time to provide information, and (3) accuracy, currentness, and completeness of information provided. METHODS: A study was conducted to compare the accuracy and completeness of the oncology registry patient follow-up information obtained from three sources: responses from the patient, responses from the physician, and affiliated clinic chart reviews. Seven hundred ninety-one patient and physician letters were sent during a 2-month period, and 122 clinic charts were reviewed. RESULTS: Physicians responded more frequently than patients (82% vs. 58%) (P < 0.003). From the responses received, requested information regarding recurrence, treatment, quality of survival, and survival was obtained most frequently from patients. However, it was difficult to ascertain information about cancer status from patient responses. Patients supplied more current follow-up information than physicians or the clinic chart reviews. CONCLUSIONS: A follow-up system that delivers accurate and complete information while maintaining efficiency is a critical aspect of an oncology registry. Requesting follow-up information from the patient before physician contact or chart review allows for more current information while maintaining accuracy regarding cancer recurrence, treatment, quality of survival, and survival. Consequently, staff time required for obtaining follow-up information from physicians and/or chart reviews is lessened and costs decreased. PMID- 8625194 TI - Indolent course of advanced neuroblastoma in children older than 6 years at diagnosis. AB - BACKGROUND: An early observation suggests that children older than 6 years of age at diagnosis of neuroblastoma constitute a favorable prognostic group. METHODS: Kaplan-Meier plots of survival of all such patients diagnosed at the Children's Hospital of Pittsburgh 1975-1992 were compared with curves of concurrently treated patients with Stage IV disease who were 1-6 years of age at diagnosis ("younger patients"). Known prognostic features, including stage and primary site of disease, pattern of metastases, histopathology, MYCN gene amplification, and urinary catecholamine metabolite ratios, were reviewed. RESULTS: Of 17 children diagnosed after the age of 6 years ("older patients"), 13 patients had Evans' Stage IV disease and 4 had Stage III disease. The median survival was 3.24 years (range, 0.63-15.04 years) for the entire cohort and 3.07 years for those children with Stage IV disease. This compared with a median survival of 1.05 years in 34 concurrent younger patients (P < 0.01). In most cases, disease in these older patients was characterized by a short-lived complete or partial remission followed by aggressive recurrent disease that was partially and only transiently chemo- or radiosensitive. Only 3 patients (2 with Stage IV disease) are in continuous complete remission at 3, 5 10/12, and 14 1/2 years from diagnosis. Although poor prognostic factors were common, including the presence of bony metastases (12/17), biopsy material from pretreatment tumor specimens demonstrated a single MYCN gene copy number in all patients and favorable histology in 15 of 16 samples. CONCLUSION: Older children with neuroblastoma have a more indolent course than do younger patients, a finding that appears to be related to favorable histology and the absence of MYCN amplification. Examination of larger numbers of such patients from cooperative groups should lead to a better understanding of what appears to be a subset of pediatric patients with neuroblastoma who may benefit from specifically tailored treatment protocols. PMID- 8625195 TI - Successful treatment of invasive thoracopulmonary mucormycosis in a patient with acute lymphoblastic leukemia. AB - BACKGROUND: Pulmonary mucormycosis associated with hematologic malignancy is an uncommon, but important opportunistic fungal pneumonia that is usually a fatal infection. Only a few survivors of pulmonary mucormycosis have been reported. METHODS: A case report of invasive thoracopulmonary mucormycosis during remission induction therapy for acute lymphoblastic leukemia and a review of the literature are presented. RESULTS: The fungal lesions extended to both lungs, the left ribs, and intercostal muscles. Percutaneous needle biopsy and immunostaining of the fungal hyphae established the diagnosis of thoracopulmonary mucormycosis. The patient was treated with granulocyte-colony stimulating factor (G-CSF) and intravenous amphotericin B for 9 weeks and the lesions in the right lung disappeared. Left pneumonectomy and partial resection of the chest wall were later performed. The left lung was grossly necrotic and contained a large cavity and bronchopulmonary fistula. Thereafter, antileukemic therapy was resumed and completed without recurrence of mucormycosis or leukemia. CONCLUSIONS: In the management of mucormycosis, the addition of G-CSF to the conventional treatment may substantially improve outcome. PMID- 8625196 TI - Characteristics of follicular tumors and nonneoplastic thyroid lesions in children and adolescents exposed to radiation as a result of the Chernobyl disaster. AB - BACKGROUND: In addition to the previously reported increase in incidence of thyroid carcinomas in Belarussian children after the Chernobyl disaster in April, 1986, benign thyroid lesions were also found to be increased in the exposed population. METHODS: A total of 60 follicular neoplasms and benign nonneoplastic thyroid lesions arising after the Chernobyl disaster in children and adolescents of 7 to 18 years of age were studied. RESULTS: The primary diagnoses in this series were follicular carcinoma in 1 (2%) case, follicular adenoma in 9 (15%), cystic adenomatoid nodule with papillae in 18 (30%), multinodular goiter in 18 (30%), diffuse hyperplasia in 2 (3%), diffuse hyperplasia with atypia and nodularity in 5 (8%), lymphocytic thyroiditis in 6 (10%), and thyroid cyst in 1 patient (2%). Additional histologic changes in thyroid glands from these patients were similar to those reported after radiation exposure, and included perifollicular fibrosis (72%), focal epithelial hyperplasia (73%), colloid accumulation (47%), follicular atrophy (33%), and cellular atypia (25%). Vascular abnormalities were found more often (75%) than previously reported in the thyroid gland after irradiation, and had a somewhat different appearance. They affected primarily medium-size arteries and were characterized by damage of the internal elastic lamina in addition to intimal fibrosis. CONCLUSIONS: The first case of thyroid follicular carcinoma in the exposed Belarussian children was diagnosed after a latent period of 6.5 years, as compared with 4 years of minimal latency for post-Chernobyl papillary carcinomas. Among benign thyroid lesions, cystic adenomatoid nodules of papillary type and diffuse hyperplasia with cellular atypia and nodularity seem to be commonly associated with radiation exposure to the thyroid gland. PMID- 8625197 TI - Incidence of cancer among patients with systemic sclerosis. AB - BACKGROUND: To determine cancer risk among patients with systemic sclerosis and localized scleroderma, a population-based retrospective cohort study was performed. Patients in Sweden with a discharge diagnosis of systemic sclerosis or localized scleroderma were obtained from the computerized database of hospital discharge diagnoses for the years 1965-1983. Nine hundred seventeen patients with systemic sclerosis and 102 with localized scleroderma were identified. METHODS: Using record linkage analysis with data from the Swedish National Cancer Registry, standardized incidence ratios (SIR)s (the ratio of observed to expected incidence) were calculated for specific cancer sites. RESULTS: The SIR for developing cancer in the cohort with systemic sclerosis was 1.5 (95% CI, 1.2 1.9). For specific cancer sites, risks were elevated for lung cancer (SIR, 4.9; 95% CI, 2.8-8.1), nonmelanoma skin cancers (SIR, 4.2; 95% CI, 1.4-9.8), and primary liver cancer (SIR, 3.3; 95% CI, 1.1-7.6). There was a suggestive increase in hematopoietic cancers (SIR, 2.3; 95% CI, 0.9-4.8). In contrast, cancer risks in the similarly ascertained cohort with localized scleroderma were no different from those of the general population. CONCLUSIONS: This study confirms earlier reports of an association between systemic sclerosis and an increased risk of cancer. Specific tumor sites correspond to the sites commonly affected by fibrosis such as the lung and skin. PMID- 8625198 TI - Etiologic factors and clinical presentation of hepatocellular carcinoma: differences between cirrhotic and noncirrhotic Italian patients. PMID- 8625199 TI - Tumor angiogenesis in human lung adenocarcinoma. PMID- 8625200 TI - Peripheral neurotoxicity of taxol in patients previously treated with cisplatin. PMID- 8625201 TI - Thrombosis of tibial arteries in a patient receiving tamoxifen therapy. AB - BACKGROUND: Tamoxifen has been used extensively as adjuvant therapy in the treatment of pre- and post-menopausal patients with breast cancer. One of its known complications is venous thromboembolism. However, arterial thrombosis has been reported rarely. METHODS: A 49-year-old patient with breast cancer had had a total mastectomy 3 years earlier. She was receiving tamoxifen therapy when she developed a sudden onset of pain and numbness of the left foot and calf. An arteriogram showed thrombosis of her tibial arteries. RESULTS: This thrombosis was lysed successfully with urokinase therapy, and tamoxifen therapy was discontinued. At follow-up 4 months later, the patient had normal circulation to both legs. CONCLUSIONS: Patients receiving tamoxifen should be monitored closely for the development of venous or arterial thromboembolism. PMID- 8625202 TI - Proliferative rate by S-phase measurement may affect cure of breast carcinoma. AB - BACKGROUND: Standard, nonparametric statistical methods measure the interaction of covariates with survival rate or relative risk. Conversely, parametric methods measure the interaction of covariates with the two cardinal features of malignant potential: the likelihood of cure and the median time to relapse among uncured patients. METHODS: The authors performed parametric analysis on data from 810 patients with breast cancer using relapse as the survival end point. Prognostic covariates included lymph node status, tumor size, patient age, nuclear size, S phase by thymidine or bromodeoxy-uridine labeling, and type of adjuvant therapy (chemotherapy, radiation, or hormone therapy). Also included was the cross product term (labeling index X chemotherapy). RESULTS: Multivariate analysis revealed that: likelihood of cure was associated positively with labeling index X chemotherapy and associated negatively with lymph node status, tumor size, and patient age; and time to relapse was associated negatively with node status, nuclear size, and labeling index. CONCLUSION: The associations of labeling index and chemotherapy with the clinical course suggest that rapidly dividing tumors have a high likelihood of cure, especially with adjuvant chemotherapy, but those not cured may have early relapse. PMID- 8625203 TI - Multimodal treatment with neoadjuvant intraarterial chemotherapy and radical surgery in patients with stage IIIB-IVA cervical cancer. A preliminary study. AB - BACKGROUND: The purpose of this study was to determine the role of neoadjuvant intraarterial chemotherapy (NIC) in patients with advanced cervical cancer. METHODS: From June 1989 to December 1993, 36 consecutive patients with International Federation of Gynecology and Obstetrics Stage IIIB-IVA cervical cancer were admitted to the study. Treatment consisted of a bilateral infusion in the internal iliac artery of bleomycin (2.5 mg), doxorubicin (10 mg), and cisplatin (20 mg) for five courses after a 4-day rest period. RESULTS: Twenty nine (80.5%) patients received 100% of the programmed chemotherapeutic dose. The major toxic effects, according to World Health Organization criteria, were hematologic (19.4%, Grade 3 or 4), renal (2.8%, Grade 2), and gastrointestinal (61.1%, Grade 1 or 2). Only four patients (11.1%) had scanty bleeding around the site of catheter insertion. Neoadjuvant intraarterial chemotherapy induced responses in 33 of the 36 patients (5 complete, 28 partial; overall response rate, 91.7%), thus permitting radical surgery in all of these cases. Pretreatment characteristics were analyzed for response to NIC. None of the clinical parameters studied were related to chemoresponsiveness. A lower than expected incidence of lymph node metastases was detected (33.3%). Ten (30.3%) of the 33 patients who underwent surgery had disease recurrence. Lymph node status and pathologic parametrial involvement were significant prognostic factors for recurrence. The 5-year estimated survival for patients with a complete response, partial response, and stable disease was 100%, 36.2%, and 0%, respectively (P < 0.001). Clinical stage (P = 0.003) and response to NIC (P < 0.001) were significant prognostic factors in the overall estimated survival. The 5-year actuarial survival for patients with Stage IIIB and IVA disease was 66.7% and 0%, respectively. CONCLUSION: These results suggest that NIC should be considered as a means of achieving prompt local control before surgery and/or radiotherapy. PMID- 8625204 TI - Ovarian dysplasia in epithelial inclusion cysts. A morphometric approach using neural networks. AB - BACKGROUND: Ovarian dysplasia has been described in the ovarian surface epithelium by histologic and morphometric studies. This study evaluates ovarian dysplasia in epithelial inclusion cysts adjacent to overt carcinoma and also incidentally found in ovaries removed for nonneoplastic diseases, including oophorectomies for family history of ovarian cancer, using an artificial neural network. METHODS: Histologic sections from 37 ovaries of which 26 were diagnosed with dysplasia in epithelial inclusion cysts (10 adjacent to carcinoma and 16 incidental) and 11 with benign epithelial inclusion cysts were evaluated by tracing nuclear profiles and assessing measures of nuclear area, shape, and texture. These sections were analyzed using artificial neural networks and also statistically using the Kruskal-Wallis test with the Dunn procedure to compare the morphologic similarity of dysplasia found incidentally in inclusion cysts unrelated to carcinoma from that in inclusion cysts adjacent to carcinoma. RESULTS: Neither statistical nor artificial neural network analysis was able to distinguish between incidental and adjacent dysplasia. Both types differed significantly from the control cases. CONCLUSIONS: Neural networks are powerful classification tools when applied to multiple variables extracted from individual cases. In this study, they helped to substantiate the similarity between dysplasia found incidentally and that adjacent to ovarian carcinoma. Because dysplasia represents a potential precancerous lesion, its incidental finding may help identify patients at risk for developing ovarian carcinoma. PMID- 8625205 TI - Carcinosarcoma of the prostate. Report of 21 cases. AB - BACKGROUND: Carcinosarcoma of the prostate is an unusual malignancy characterized by adenocarcinoma intimately admixed with sarcoma. METHODS: The authors reviewed the clinical and pathologic findings of 21 cases of carcinosarcoma of the prostate from the files of the Mayo Clinic. RESULTS: Patient age ranged from 50 to 89 years (mean, 68 years). Ten patients (48%) had a previous diagnosis of prostatic acinar adenocarcinoma 2-73 months (mean, 33 months) before the diagnosis of carcinosarcoma, 8 of whom had received androgen deprivation therapy and/or radiation therapy in the interim. The Gleason score of adenocarcinoma ranged from 7 to 10 (mean, 9; median, 9). The sarcomas consisted of osteosarcoma (13), leiomyosarcoma (5), fibrosarcoma (1), malignant fibrous histiocytoma (1), and rhabdomyosarcoma (1), and the grade of the sarcoma component ranged from 2 to 4 (mean, 4; median, 4). Adenocarcinoma was the dominant histologic pattern in 7 cases and sarcoma in 14. Immunohistochemical staining revealed cytoplasmic reactivity for prostate specific antigen or keratin in 16 of 16 cases in the adenocarcinoma component. The sarcoma component was positive for vimentin in 16 of 16 cases, actin in 8 of 16 (focal in 2), S-100 protein in 2 of 16, and desmin in 1 of 16. At the time of diagnosis of carcinosarcoma, 11 patients had metastases, including 4 with metastatic adenocarcinoma before the diagnosis of carcinosarcoma. Treatment varied, and nonsurgical therapy was ineffective. Sites of metastases included lung (10 cases), bone (7), brain (4), liver (1), and peritoneum (1). Follow-up ranged from 1 to 107 months after the diagnosis of carcinosarcoma (mean, 34 months; median, 10 months). Mean time to tumor progression was 23 months (range, 1-96 months; median, 18 months). Eighteen patients died of carcinosarcoma from 2 to 107 months (mean, 34 months; median, 9.5 months) after diagnosis. Five-year survival was 41%, and 7-year survival was 14%. Progression and survival were not affected by histologic pattern. CONCLUSIONS: Carcinosarcoma of the prostate is an aggressive malignancy, regardless of histologic type. PMID- 8625206 TI - Galactosylgloboside expression in seminoma. Inverse correlation with metastatic potential. AB - BACKGROUND: Altered glycosylation is a common phenotype expressed in essentially all types of human cancer and has been found to be correlated closely with the invasive and metastatic properties of a given tumor. Because there was no prognostic information concerning aberrant glycosylation of seminoma, the authors studied this topic. METHODS: Glycosphingolipid (GSL) composition of orchiectomy samples of seminoma were analyzed systematically. GSL patterns from seminoma samples of the following three groups were compared after a 44-month postoperative period: Stage I disease with no evidence of metastasis during the 44-month postoperative period, Stage I with metastatic relapse during this period, and Stage II with retroperitoneal lymph node metastasis. Unknown GSLs detected were analyzed chemically by 1H-nuclear magnetic resonance spectroscopy and mass spectrometry. RESULTS: All nonmetastatic seminomas (n = 12) contained a GSL band that was identified as galactosylgloboside (Gb5; Gal beta 1-->3GalNAc beta 1-->3Gal alpha 1-->4 Gal beta 1-->4Glc beta 1-->1Cer). All metastatic seminomas (n = 5) lacked this GSL, although the sample sizes were admittedly small. CONCLUSION: Only the presence or absence of galactosylgloboside (Gb5), but of no other GSL or gangliosides, clearly correlated with metastatic potential in patients with seminoma. This observation is useful in the estimation of prognosis of patients with seminoma, especially those with Stage I disease. PMID- 8625208 TI - S-phase fraction by the labeling index as a predictive factor for progression and survival in low grade non-Hodgkin's lymphoma. AB - BACKGROUND: The purpose of this study was to learn if the bromodeoxyuridine labeling index (LI), a measure of the S-phase fraction, is an independent prognostic factor for overall survival (OS) for patients with newly diagnosed low grade non-Hodgkin's lymphoma (NHL). In addition, the ability of the LI to predict time to progression (TTP) in a group of patients observed without therapy after initial diagnosis was determined. METHODS: Patients eligible for this study had biopsy proven low grade NHL, adequate tissue to perform the LI, and were previously untreated. The bromodeoxyuridine LI was performed on fresh biopsy samples using a slide-based immunofluorescence procedure. RESULTS: One-hundred twelve patients were followed prospectively for OS, and 50 of these patients who initially were observed without therapy were eligible for an analysis of TTP. The LI (< or = 1% vs. > 1%) and presence of "B" symptoms were significant univariate prognostic factors for survival (P values of 0.004 and < 0.001, respectively). In a multivariate analysis, the LI and symptoms retained independent prognostic significance, whereas disease stage, histologic subtype, and age did not. In the group who were observed after diagnosis, the LI was not an independent predictor of TTP. CONCLUSIONS: The LI at initial diagnosis is an independent prognostic factor for OS of patients with low grade NHL, but it does not help choose patients for observation without therapy. Measurements of the LI should be considered as part of the on-study evaluation of patients entering cooperative group trials evaluating new therapies for this group of lymphomas. PMID- 8625207 TI - Head and neck liposarcoma. AB - BACKGROUND: Liposarcoma of the head and neck region represents approximately 1% of head and neck sarcomas. Therefore, there are few data on the natural history, presentation, treatment, and prognosis of this neoplasm. METHODS: This study is a report of data from 76 patients with head and neck liposarcoma of whom 4 were treated at The Royal Marsden Hospital during the past 50 years. RESULTS: The median age of patient presentation was the seventh decade (range, 6 months-86 years), and 65% of the patients were male. The commonest site of presentation was the neck (28%), followed by the larynx (20%) and pharynx (18%). Sixty-two percent of tumors were low grade (well differentiated and myxoid), and 38% were high grade (pleomorphic and round cell). The principal determinant of outcome was histologic grade. Five-year survival by life-table analysis was 67% overall and varied with tumor type as follows: well differentiated 100%, myxoid 73%, pleomorphic 42%, and round cell 0%. Site appears to have had some influence on prognosis. Oral liposarcoma had a poor prognosis with a 5-year survival of 50%, despite the low grade of all tumors; however, the 5-year survival for laryngeal (89%) and head (83%) liposarcoma was considerably better. Tumor size did not affect prognosis. The mainstay of treatment was surgical excision, used alone in 70% of the cases. Radiotherapy was used with other treatments in 25% of the cases. Prognosis was best for patients treated with surgery only (5-year survival, 83%), compared with those receiving surgery plus radiotherapy (5-year survival, 63%), chemotherapy (5-year survival, 33%), and radiotherapy alone (5 year survival, 0%). CONCLUSIONS: Liposarcoma rarely involves the head and neck region. The prognosis for patients with this disease appears to be better than for those with liposarcoma arising elsewhere, particularly in the retroperitoneum. Prognosis is principally dependent on histologic grade. Complete surgical excision provides the most effective treatment. PMID- 8625209 TI - Mediastinal needle biopsy. A 15-year experience with 139 cases. AB - BACKGROUND: Radiologically guided needle biopsy and cytologic evaluation provide a reliable method of diagnosis for planning definitive therapy of patients with mediastinal lesions. MATERIALS AND METHODS: In this retrospective study of one of the largest series from a single institution, 141 consecutive mediastinal needle biopsies from 139 patients were reviewed during a 15-year period. RESULTS: Adequate material was obtained with a diagnosis achieved in 128 cases (92%). Of these, 33 cases (26%) had benign diagnoses; the remaining 95 (74%) had malignant diagnoses, including 81 carcinomas, 3 sarcomas, 8 lymphoproliferative lesions, 2 malignant germ cell tumors, and 1 malignant thymoma. All benign cases were diagnostically confirmed, and 94 of 95 malignant cases were classified correctly. The only discrepancy that occurred involved a malignant lymphoma diagnosed as a malignant germ cell tumor. Of the 13 inadequate samples, the major category included a nodular sclerosis variant of Hodgkin's disease (4 cases), 1 case of thymoma, 1 case of tuberculous lymphadenitis, and 7 cases for which no follow-up data were available. CONCLUSION: Needle biopsy is reaffirmed as a reliable and sensitive diagnostic tool for mediastinal lesions, with an overall cytologic diagnostic accuracy of 99% with adequate material. Sclerotic lesions may pose a limitation to this technique and require generous sampling before a more invasive diagnostic procedure is undertaken. PMID- 8625210 TI - Radiation recall dermatitis and pneumonitis in a patient treated with paclitaxel. AB - BACKGROUND: Radiation recall refers to a tissue reaction produced by a chemotherapeutic agent in a previously irradiated field that would not occur in a nonirradiated field. A number of agents have been reported to cause radiation recall. Recently, there have been case reports of recall dermatitis from paclitaxel treatment. METHODS: A patient with metastatic lung cancer received palliative radiation to her mediastinum and ribs. Because of disease progression, she subsequently received paclitaxel. RESULTS: After paclitaxel administration, the patient became acutely dyspneic. A subsequent chest X-ray revealed a parenchymal opacity in a region that corresponded with the patient's radiation portal. She also developed a severe skin reaction in the previously treated electron field. CONCLUSIONS: This is one of few reported cases of recall dermatitis from paclitaxel and is also suggestive of recall pneumonitis, a phenomenon previously unreported to the authors' knowledge. Given paclitaxel's ability to function as a radiosensitizer, this response is not unexpected. As the frequency of paclitaxel administration increases, its potential as a radiation sensitizer and radiation recall should be considered. PMID- 8625211 TI - Classification of rhabdomyosarcomas and related sarcomas. Pathologic aspects and proposal for a new classification--an Intergroup Rhabdomyosarcoma Study. AB - BACKGROUND: There is a need to develop a single prognostically significant classification of rhabdomyosarcomas (RMS) and other related tumors of children, adolescents, and young adults which would be a current guide for their diagnosis, allow valid comparison of outcomes between protocols carried out anywhere in the world, and should enhance recognition of prognostic subsets. METHOD: Sixteen pathologists from eight pathology groups, representing six countries and several cooperative groups, classified by four histopathologic classification schemes 800 representative tumors of the 999 eligible cases treated on Intergroup Rhabdomyosarcoma Study II. Each tumor was classified according to each of the four systems by each of the pathologists. In addition, two independent subsamples of 200 of the 800 patients were reviewed according to the new system, so that 343 distinct patients were reviewed once, and 57 of these twice. RESULTS: A study of the survival rates of all subtypes in the sample of 800 patients led to the formation of a new system. This was tested on two independent subsets of 200 of the original cases and found to be reproducible and predictive of outcome by univariate analysis. A multivariate analysis of the 343 patients classified according to the new system indicated that a survival model including pathologic classification and known prognostic factors of primary site, clinical group, and tumor size was significantly better at predicting survival than a model with only the known prognostic factors. CONCLUSION: This new classification, termed International Classification of Rhabdomyosarcoma (ICR) by the authors, was reproducible and predictive of outcome among patients with differing histologies treated uniformly on the Intergroup Rhabdomyosarcoma II protocols. We believe it should be utilized by all pathologists and cooperative groups to classify rhabdomyosarcomas in order to provide comparability among and within multi institutional studies. PMID- 8625213 TI - The National Cancer Data Base report on ovarian cancer. American College of Surgeons Commission on Cancer and the American Cancer Society. AB - BACKGROUND: Reports generated from the National Cancer Data Base (NCDB), a joint project of the American College of Surgeons Commission of Cancer and the American Cancer Society, have described trends in demographics, stage, treatment patterns, and survival for a variety of cancers. In this report, the most current (1991) data for ovarian cancer are presented and include some comparisons with 1985/1986 data. METHODS: Three calls for data from hospital registries across the United States have yielded 17,114 ovarian cancer cases for 1985, 1986, and 1991 combined. These data represent approximately 23%, 23%, and 43%, respectively, of the annual number of cases of ovarian cancer in the United States for those years. RESULTS: One-fourth of the reported cases of ovarian cancer were diagnosed in women less than 50 years of age. Younger patients (< 40 years) were more likely to have received conservative therapy (unilateral oophorectomy), consistent with their high prevalence (59%) of Stage I disease. The number of patients reported with an unknown American Joint Committee on Cancer (AJCC) stage decreased from 49% in 1985/1986 to 17% in 1991, although the distribution within stages was unchanged. Increases in important staging procedures were reported in 1991, with threefold increase in the proportion of debulking procedures and a 50% increase in omentectomies accompanying hysterectomy compared with 1985/1986. More advanced disease was reported for those of older age, lower income, African Americans, and patients in smaller hospitals. Relative 5-year survival rates were 74% for patients with Stage I disease, 58% for Stage II, 30% for Stage III, and 19% for Stage IV. Asians and Hispanics presented with a relatively high rate of Stage I-II disease (45%) compared with non-Hispanic whites and African Americans (38% and 33%, respectively). Hispanics presented with the most favorable Stage I/IV ratio (1.5) and had an overall 5-year survival of 50% compared with 41% and 37% for non-Hispanic whites and African Americans (Stage I/IV ratios of 1.0 and 0.7, respectively). There was little difference reported in the use of multimodality treatment between 1985/1986 and 1991. CONCLUSIONS: A trend toward more complete surgery with full surgical/pathologic staging was observed in 1991, but there was not yet evidence to indicate significant improvements in ovarian cancer survival compared with published figures during the past 10-15 years. Important ethnic and demographic differences in type of surgery and survival were noted but could not be differentiated from differences in tumor stage. PMID- 8625212 TI - trk A gene expression in neuroblastoma. The clinical significance of an immunohistochemical study. AB - BACKGROUND: Neuroblastomas display a spectrum of morphologic and cytologic features of neural cells, and the prognosis of patients with these tumors varies widely. Expression of trk A in these tumors, as documented by Northern blot analysis, is associated with a favorable prognosis. To examine the expression of trk A at the cellular level in individual tumors and apply the results to routine clinical use, the authors designed this immunohistochemical study using an antibody with a predetermined specificity on formalin fixed, paraffin embedded tumor sections. METHODS: Expression of trk A and Ha-ras genes in 105 neuroblastomas was examined by avidin-biotin-complex immunoperoxidase staining. N myc gene amplification was examined in 81 of the tumors by Southern blot analysis. RESULTS: Immunohistochemical expression in tumors correlated strongly with a favorable prognosis for trk A expression (P < 0.0001) and for Ha-ras expression (P < 0.0001). N-myc amplification was found in neuroblastomas with low expression of trk A and of Ha-ras genes. Kaplan-Meier analysis resulted in a favorable outcome associated with high trk A expression and no N-myc amplification, and a poor outcome associated with low trk A expression and demonstrable N-myc amplification (P < 0.0001). Univariate analysis showed that immunohistochemical expression of trk A at the time of diagnosis was a powerful predictor of the patient's prognosis, as were N-myc amplification and Ha-ras expression. trk A expression even correlated significantly with prognosis when the analysis was restricted to Stages III and IV tumors. CONCLUSIONS: Immunohistochemical detection of the trk A gene product in tumor cells is strongly predictive of a favorable prognosis for patients with neuroblastomas. The coexpression of trk A and Ha-ras genes with clinical behavior of the tumor may indicate close linkage of these genes in the nerve growth factor signal transduction system. Prognostic evaluation at diagnosis based on such molecular and genetic information should be important clinically. PMID- 8625214 TI - The National Cancer Data Base report on prostate cancer. American College of Surgeons Commission on Cancer and the American Cancer Society. AB - BACKGROUND: Previous Commission on Cancer data from the National Cancer Data Base (NCDB) examined time trends in disease stage, treatment patterns, and survival for patients with selected cancers. The most current (1992) data for prostate cancer are described in this Communication. METHODS: Calls for data yielded a total of 52,597 prostate cancer reports for 1986/1987 and 101,903 for 1992 from hospital cancer registries across the United States. RESULTS: Data were received for 154,500 patients with prostate cancer. Prostate cancer is detected increasingly at localized stages and among younger men. African American men have a continuing pattern of more advanced disease at diagnosis. Selection of prostatectomy as the primary treatment has increased, and its use varies by region and several patient and hospital characteristics. Selection of radiation treatment has increased to a lesser degree. The 5-year survival for patients diagnosed in 1986/1987 was 60%. Outcomes varied by stage, age, and race. CONCLUSION: The NCDB provides valuable information concerning patterns and trends in prostate cancer care in the United States. The data show that prostate cancer detection and treatment have changed markedly in recent years. These changes appear to be related to the increased use of early detection measures. The improvements in prostate cancer detection and trends in treatment have not affected the population evenly, with African American men having more advanced disease and lower survival. Continued monitoring of these important trends is needed. PMID- 8625215 TI - Early detection of esophageal cancer by chromoendoscopy. PMID- 8625216 TI - Prognostic value of histopathologic parameters of esophageal squamous cell carcinoma. AB - BACKGROUND: The grading of squamous cell carcinoma (SCC) of the esophagus as proposed by the World Health Organization (WHO) has not yet proved to be prognostically significant. Therefore, the prognostic impact of various histologic parameters was investigated and compared with that of the WHO grading. METHODS: Hematoxylin and eosin-stained tumor samples from 138 patients with SCC of the esophagus who underwent potentially curative resection (no residual tumor or distant metastases) were evaluated for the following histologic parameters: degree of keratinization, nuclear polymorphism, pattern of invasion, mitotic activity, and inflammatory response. The prognostic impact of these parameters was analyzed by univariate and multivariate survival analyses. RESULTS: In the univariate analysis, the inflammatory response (P = 0.0006), pattern of invasion (P = 0.0011), and nuclear polymorphism (P = 0.0161) were the only parameters that correlated with survival. However, in a multivariate survival analysis including these parameters, only pattern of invasion (P = 0.0010) and inflammatory response (P = 0.0076) were prognostically significant. Based on these results, a new prognostic score system was defined that correlated significantly with survival in the univariate survival analysis (P = 0.0002). In contrast, the WHO histologic grade was not prognostically significant. In the multivariate Cox regression analysis, the new prognostic score system proved to be an independent prognostic parameter (P = 0.0062), ranking next to pT classification (P = 0.0001) and pN classification (P = 0.0014). CONCLUSIONS: For SCC of the esophagus, histologic grading based on pattern of invasion and inflammatory response had an independent prognostic impact, whereas the grading system proposed by the WHO had no significant prognostic value. PMID- 8625217 TI - Successful screening for early esophageal cancer in alcoholics using endoscopy and mucosa iodine staining. AB - BACKGROUND: Epidemiologic studies have provided evidence that alcohol abuse is an important risk factor for esophageal carcinoma. However, no systematic screening program has been established yet in the early detection of esophageal cancer in high risk populations of heavy drinkers. METHODS: A cohort of 629 male alcoholics (54 +/- 8 years old) were consecutively and systematically screened by endoscopy combined with iodine staining and targeted biopsy at the National Institute on Alcoholism (Kanagawa, Japan). For mucosal carcinomas, endoscopic esophageal mucosal resection (EEMR) was used to serve confirmatory diagnostic and therapeutic purposes. RESULTS: Iodine-unstained lesions, distinctly demarcated, white, and 5 mm or larger in greatest dimension, were observed on the esophageal wall in 162 patients (25.8%). Thirty-six such unstained lesions in 21 of 629 patients, with an unexpectedly high rate of 3.3%, turned out to be squamous cell carcinomas of the superficial type. According to some established criteria, EEMR was performed in 17 of these patients, 3 of whom were given additional irradiation. Esophagectomy was performed in two patients, chemotherapy combined with irradiation in one, whereas still another was followed endoscopically. The cancer invasion was confined within the epithelium in eight patients, to the proper mucosal layer in nine, and to the submucosa in four. Multiple logistic regression revealed that the risks for distinct iodine-unstained lesions and superficial esophageal carcinoma increased independently for users of stronger alcoholic beverages, i.e., whiskey or shochu (odds ratio [OR] = 1.47 and 2.94, respectively) compared with lighter beverages, i.e., sake or beer and 30+ cigarettes/day (OR = 1.68 and 3.85, respectively). CONCLUSION: Routine application of this program for these high risk individuals yielded an unusually high rate of detection of esophageal carcinoma. PMID- 8625218 TI - Surgical strategy for patients with gastric carcinoma with submucosal invasion. A multivariate analysis. AB - BACKGROUND: Early gastric cancer can be treated by endoscopic excision or simple wedge surgical resection. Standard gastrectomy often is advised if submucosal invasion is found, even though only 15-25% of these patients have lymph node metastases. In this study, the risk of lymph node involvement was examined by multivariate analysis to develop a simple discriminant function for surgical decision making in this setting. METHODS: The authors determined factors significantly correlated with lymph node involvement in a retrospective review of 196 patients with gastric adenocarcinoma invading into, but not beyond, the submucosa. Depth and horizontal spread of cancer in the submucosa were evaluated in addition to ordinary pathologic factors. Discriminant analysis for lymph node involvement was performed using explanatory variables chosen from the results of the univariate analyses. RESULTS: Lymph node involvement correlated significantly with larger tumor size; greater dimension of submucosal invasion; deeper submucosal invasion; gross appearance of Type I, IIc + III or IIa + IIc; and severity of vessel invasion. Of the variables, the amount of lymphatic invasion, macroscopic appearance, and maximum dimension of submucosal infiltration were selected as effective predictors of lymph node involvement according to discriminant analysis. A correct discrimination of 74.8% was obtained with a linear discriminant function using these variables. Lymph node involvement was observed in 50% of the cases with a discriminant score less than -1 and in 25% of those with a score between -1 and 0, whereas no lymph node involvement was observed in those with a score greater than 2. CONCLUSIONS: Discriminant function as used in this study provided a useful criterion for additional surgery for patients with gastric carcinoma invading the submucosa who were treated initially by localized excision. Prophylactic lymph node dissection may not be necessary for a discriminant score greater than 2, whereas extended lymphadenectomy would be recommended for a score less than -1. PMID- 8625219 TI - E-cadherin and urokinase-type plasminogen activator tissue status in gastric carcinoma. AB - BACKGROUND: E-cadherin (ECD) is known to be an invasion suppressor gene, and urokinase-type plasminogen activator (uPA) plays a central role in infiltration of solid cancers. METHODS: To elucidate the relationship between expression of these factors and metastasis in patients with gastric cancer, the authors examined immunohistochemically a combination analysis of uPA and E-cadherin expression in 98 primary tumors, and the results were correlated with several parameters related to metastasis. RESULTS: Among 125 tumors, 42 (34%) were evaluated as having E-cadherin expression (E-cadherin-positive), and the other 83 (66%) were defined as having reduced E-cadherin expression (E-cadherin-negative). uPA immunoreactivity was observed in 82 tumors (66%). There were four subtypes of patterns of uPA and E-cadherin expression: 22 uPA-negative/E-cadherin-positive, 17 uPA-negative/E-cadherin-negative, 21 uPA-positive/E-cadherin-positive, and 65 uPA-positive/E-cadherin-negative, uPA overexpression and reduced E-cadherin expression were associated with lymph node metastasis, vessel invasion, serosal involvement, and poor prognosis. In addition, uPA-positive/E-cadherin-negative tumors were associated significantly with large tumors, positive serosal invasion, lymph node involvement, and poor prognosis. Patients with uPA positive/E-cadherin-negative expression had the poorest prognoses, compared with the three other groups of patients uPA-positive/E-cadherin-negative tumors had a fourfold relative risk of death when compared with uPA-negative/E-cadherin positive tumors. A Cox proportional hazard model projected lymph node status as the strongest of the prognostic variables followed by DNA ploidy patterns and uPA/E-cadherin tissue status. CONCLUSIONS: These results indicate that immunohistochemical combination analysis of uPA and E-cadherin expression may be a powerful aid in evaluating metastatic potential or the prognosis of patients with gastric cancer. PMID- 8625221 TI - A phase III evaluation of a somatostatin analogue (octreotide) in the treatment of patients with asymptomatic advanced colon carcinoma. North Central Cancer Treatment Group and the Mayo Clinic. AB - BACKGROUND: The purpose of this study was to determine by randomized, controlled, double-blind evaluation whether therapy with the somatostatin analogue, octreotide, would delay tumor progression and improve survival of patients with metastatic colorectal carcinomas who were ambulatory with no significant symptoms. METHODS: Two hundred sixty patients with an Eastern Cooperative Oncology Group performance status of 0 or 1 and without symptoms related to colon cancer were randomized to receive 150 micrograms of octreotide subcutaneously three times daily or, initially, no treatment. After 91 patients were entered in the double-blind study, saline placebo injections were used for patients in the control arm. RESULTS: The randomization culminated in balanced assignment of patients with respect to disease site(s), presence or absence of measurable or evaluable disease, and interval from diagnosis of metastasis to protocol entry. Steatorrhea and diarrhea, usually mildly severe, resulted more often from treatment than from the placebo. The major end points were time to progression and survival. Curves for both parameters overlapped in the blind and open trial segments. CONCLUSION: Octreotide at a dose of 150 micrograms given three times daily is not effective therapy for patients with advanced asymptomatic colon carcinoma. PMID- 8625220 TI - Expression of alpha-3/4-monofucosylated polylactosaminoglycan epitope, as defined by monoclonal antibody FW6, is a marker of the colorectal adenoma-carcinoma sequence. AB - BACKGROUND: The expression of a distinct alpha-3/4-monofucosylated polylactosaminoglycan epitope, which is detected by monoclonal antibody FW6, was investigated by comparative immunohistochemical analysis of colorectal tissue specimens exhibiting different grades of premalignant and malignant transformation. The presence of this peculiar epitope was compared with different lewis type 2 blood group antigens. METHODS: Paraffin embedded specimens from 8 hyperplastic polyps, 46 adenomas, 27 colorectal carcinomas, and 10 corresponding liver metastases were studied. Staining reactions included monoclonal antibodies FW6, AM-3 (anti-sialosyl-Le(x)), LeuM1 (anti-Le(x)), and 12-4LE (anti-Le(y)) in a standard peroxidase-antiperoxidase method. RESULTS: Hyperplastic polyps were not reactive with FW6 or LeuM1, but showed a slight binding of AM-3 and 12-4LE in some cases. Approximately two-thirds of the adenomatous polyps displayed a pronounced staining activity by AM-3, and approximately half of them revealed FW6, LeuM1, and 12-4LE binding. Only the expression of the FW6 (P < 0.005) epitope correlated with the presence of severe dysplasia. All antibodies were more or less reactive with colorectal carcinomas and their liver metastases, and some showed correlating binding patterns. CONCLUSIONS: FW6 revealed a high specificity for adenomas with areas of severe epithelial dysplasia. Because this monoclonal antibody also detects the great majority of carcinomas, it is reasonable to postulate that the alpha-3/4-monofucosylated polylactosaminoglycan epitope is an important marker for malignant transformation in the colorectal adenoma-carcinoma sequence. PMID- 8625222 TI - The influence of local tumor ulceration on the effectiveness of endocavitary radiation for patients with early rectal carcinoma. AB - BACKGROUND: Endocavitary radiation therapy is an alternative to surgical therapy for some early rectal carcinomas. Careful patient selection is necessary to ensure good results. The purpose of this study was to examine the authors' experience with endocavitary radiation at their institution from 1984 to 1991 to determine which factors were associated with treatment failure to provide for better future patient selection. METHODS: Thirty-two patients with carcinoma of the rectum, not apparently involving the muscle wall, underwent 75-120 Gy of endocavitary radiation as potentially curative therapy. Treatment was given as a series of 2-4 doses of 30 Gy at three weekly intervals. Twenty-two patients had polypoid tumors, 5 sessile, and 5 ulcerated. RESULTS: After a mean follow-up of 43 months (range, 6-103 months), 4 of 5 patients (80%) with ulcerated tumors developed local recurrences, compared with only 4 of 27 (15%) with sessile or polypoid lesions. Not only was the incidence of local recurrence greater for patients with ulcerated tumors (P = 0.009), but the time to recurrence was shorter also (P = 0.0001). Tumor size, anterior or posterior location, and dose of radiation received did not affect the rate of recurrence. CONCLUSIONS: These results indicate that superficial polypoid and sessile rectal tumors can be managed successfully with endocavitary radiation. Ulcerating tumors are likely to recur locally within a short time and therefore should be considered for surgical treatment initially. PMID- 8625223 TI - Osteosarcoma versus malignant fibrous histiocytoma of bone in patients older than 40 years. A clinicopathologic and immunohistochemical analysis with special reference to malignant fibrous histiocytoma-like osteosarcoma. AB - BACKGROUND: It is often difficult to discriminate between osteosarcoma and malignant fibrous histiocytoma (MFH) of bone, especially in older patients because of the clinical similarities, including the lytic radiologic appearance. A histologic analysis of MFH-like osteosarcoma, which closely resembles MFH of bone both clinically and radiologically, has not yet been conducted thoroughly, and therefore this issue remains controversial. METHODS: Using clinicopathologic and immunohistochemical techniques, the authors studied 24 cases of osteosarcoma arising in patients older than 40 years of age and compared them with 20 cases of MFH of bone from similarly aged patients. RESULTS: Radiography revealed that 68.2% of the osteosarcoma cases were predominantly lytic, whereas all cases of MFH of bone showed either a predominantly or purely lytic pattern. Histologically, osteosarcoma was subclassified as conventional osteoblastic (54.2%), MFH-like (29.2%) containing various amounts of tumor osteoid and/or bone in each of the cases, and conventional fibroblastic (4.2%), whereas all the cases of MFH of bone had a storiform-pleomorphic pattern. Immunohistochemically, no overexpression of p53 protein was found in MFH-like osteosarcoma, whereas it tended to occur more frequently in osteoblastic osteosarcoma (66.7%) and MFH of bone (50.0%). The Ki-67 labeling index was significantly lower in MFH-like osteosarcoma than in MFH of bone. The 5-year survival rate was 18.2% in patients with osteoblastic osteosarcoma, 66.7% in patients with MFH-like osteosarcoma, and 21.5% in patients with MFH of bone. A significant difference in the survival curve was observed between osteoblastic and MFH-like osteosarcoma. CONCLUSIONS: It is proposed that MFH-like osteosarcoma, which shows characteristically different clinical and histologic features from that of conventional osteosarcoma, thus may be considered a variant of osteosarcoma. PMID- 8625224 TI - Adult acute lymphoblastic leukemia at relapse. Cytogenetic, immunophenotypic, and molecular changes. AB - BACKGROUND: There have been published reports on cytogenetic, immunophenotypic, and molecular changes at relapse in childhood acute lymphoblastic leukemia (ALL) including lineage switch and secondary leukemia. There are limited data, however, on the cytogenetic, immunophenotypic, and molecular parameters of adult ALL at relapse. Because, as in children, the cytogenetic and/or immunophenotypic changes observed in adult ALL at relapse may have prognostic significance, the authors investigated the significance of such changes. METHODS: Fifty-three patients with relapsed adult ALL for whom cytogenetic, immunophenotypic, and/or molecular analyses were performed at diagnosis and at relapse were studied. Changes in any of the parameters at relapse were correlated with total survival and survival from the time of relapse. RESULTS: Of the 32 patients for whom cytogenetic studies were performed at relapse, 21 (66%) showed clonal cytogenetic changes, 40% of which were clonal evolution. None of these cases, however, showed two entirely different abnormal karyotypes at diagnosis and at relapse. The immunophenotypes showed occasional gain or loss of one or two surface markers, and the molecular genetic configurations for JH, JK, and the T-cell receptor beta were stable throughout the evolution of the disease. Patients with clonal evolution had a shorter overall survival than the rest of the group (P = 0.02). This difference, however, was not significant with respect to survival measured from the time of relapse. CONCLUSIONS: The most frequent changes in the biologic profile of adult ALL at relapse are shifts in the karyotype, with or without clonal evolution. Clonal evolution detected at relapse is associated with a higher frequency of unfavorable karyotypes at diagnosis and with a worse overall prognosis. However, survival from the time of relapse is similar in patients with and without clonal evolution. PMID- 8625225 TI - The breakpoint cluster region site in patients with Philadelphia chromosome positive chronic myelogenous leukemia. Clinical, laboratory, and prognostic correlations. AB - BACKGROUND: The breakpoint site of the breakpoint cluster region (bcr) has been correlated with patient characteristics, with the disease phase, and with the prognosis of patients with chronic myelogenous leukemia (CML), but the findings remain controversial. METHODS: Appropriate restriction enzymes and the 3' and universal probes were used to map the breakpoint site by Southern blot analysis into 5' and 3' breakpoints and a breakpoint in zone 3 (or fragment 2) in 362 patients in different phases of CML (238 in early chronic phase, 69 in late chronic phase, 31 in accelerated phase, and 24 in blastic phase). Standard statistical methods were used to evaluate differences in characteristics and in prognosis by the breakpoint site. RESULTS: No correlation was noted between CML phases and breakpoint site. Among patients in the early chronic phase, thrombocytosis was significantly associated with the 3' breakpoint site (P = 0.02), whereas peripheral basophilia occurred more frequently with the 5' breakpoint site (P = 0.05). Other patient and disease characteristics were similar in frequency among the breakpoint-site subgroups. There was no difference in response to alpha-interferon therapy (186 patients treated) by the breakpoint site. Survival, dated from either referral to the authors' institution or from diagnosis, was not significantly different among patients with early chronic phase CML by the breakpoint site. However, patients with a 3' deletion tended to have a shorter survival. CONCLUSION: Determination of the breakpoint site by Southern blot analysis does not help to predict prognosis of patients with CML. PMID- 8625226 TI - Thrombospondin and transforming growth factor-beta 1 increase expression of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in human MDA-MB-231 breast cancer cells. AB - BACKGROUND: Thrombospondin is a high molecular weight adhesive glycoprotein that has been shown to function in mechanisms of tumor progression. The authors' previous studies have shown that thrombospondin promotes human lung carcinoma invasion by up-regulation of the plasminogen activator system through a mechanism involving the activation of transforming growth factor-beta 1 (TGF-beta 1). In this study, a similar thrombospondin-mediated mechanism operative in breast carcinoma cells is described. METHODS: The effect of thrombospondin and TGF-beta 1 on the capacity of a line of breast carcinoma cells to activate plasminogen was measured as well as the physiologic consequences of these activities on cell adhesion and proliferation. Plasminogen activation was assessed by measuring the plasmin activity and plasminogen activator inhibitor-1 (PAI-1) levels in cell conditioned media and the cell-associated urokinase-type plasminogen activator (uPA) levels. RESULTS: Treatment of MDA-MB-231 breast carcinoma cells with either thrombospondin or TGF-beta 1 caused increased secretion of PAI-1 with a concomitant decrease in plasmin activity, whereas cell-associated uPA expression was increased with respect to controls. Thrombospondin (40 micrograms/ml) or TGF beta 1 (5 ng/ml) stimulated the cells to secrete 5.5- and 6.7-fold more PAI-1 than controls, respectively, and caused decreased plasmin activity in the cell culture medium. Conversely, either thrombospondin (40 micrograms/ml) or TGF-beta 1 (5 ng/ml) caused the cells to express 4.55- and 5.38-fold more uPA than controls, respectively. Thrombospondin and TGF-beta 1 induced a more flattened and spread appearance in the cells with no effect on proliferation. These effects could be reversed with antibodies to either thrombospondin or TGF-beta 1 and were not due to contamination of thrombospondin with active TGF-beta 1. CONCLUSIONS: Thrombospondin and TGF-beta 1 function similarly to increase cell-associated uPA and cell-secreted PAI-1. These data suggest that thrombospondin may not only function as an adhesive molecule, but through a mechanism involving the activation of TGF-beta 1, may modulate cell surface protease expression. In addition, these observations suggest that thrombospondin and TGF-beta 1 could promote metastasis by increasing uPA-mediated cell invasion, whereas through the action of PAI-1, also protect blood-born tumor emboli from destruction by host fibrinolytic enzymes. PMID- 8625227 TI - Salivary duct carcinoma. Clinicopathologic and immunohistochemical review of 26 cases. AB - BACKGROUND: Salivary duct carcinoma (SDC) is a high grade aggressive malignancy of the major salivary glands. Clinical and pathologic features that may be predictive of survival are not well delineated. The microscopic features of SDC are remarkably similar to those of mammary ductal carcinoma, raising the question of whether these tumors share antigenic or hormonal features. METHODS: We reviewed the clinical and pathologic characteristics of 26 cases of SDC treated at the Mayo Clinic from 1960 to 1989. Immunoperoxidase studies and flow cytometry were performed in 25 and 24 cases, respectively. RESULTS: The study population consisted of 22 men and 4 women (mean age, 66 years). The parotid gland was involved in 23 patients and the submaxillary gland in 3. Five of 24 tumors studied were diploid (21%), and 19 (79%) were nondiploid. Nine tumors (35%) recurred locally and 16 (62%) metastasized distantly; 20 patients (77%) died of disease at a mean interval of 3 years after diagnosis. Female sex was the only significant negative prognostic factor analyzed, but positive nodal status approached significance. Paraffin-section immunostaining showed positive reactions for epithelial membrane antigen (100%), keratin (AE1/AE3) (88%), alpha lactalbumin (88%), GCDFP-15 (76%), and carcinoembryonic antigen (72%); S-100 protein was rarely detected (4%). Stains for estrogen receptor were uniformly negative, but one tumor was positive for progesterone receptors. CONCLUSIONS: The prognosis for SDC is dismal, and clinically useful prognostic factors were not found. Our results do not confirm hormonal concordance between SDC and breast carcinoma. PMID- 8625228 TI - Expression of EBER1 in primary and metastatic nasopharyngeal carcinoma tissues using in situ hybridization. A correlation with WHO histologic subtypes. AB - BACKGROUND: The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) is well documented. Previous studies reported abundant expression of EBER1 in primary NPC and tumors metastatic to lymph nodes. However, a large series of case studies correlating World Health Organization (WHO) histologic subtypes with EBER1 is needed. METHODS: The authors applied the EBER1 in situ hybridization to investigate the expression of EBER1 in 140 primary NPCs, 11 metastatic tumors to lymph nodes, 6 metastatic tumors to bone marrow, and 2 metastatic tumors to the liver. All 19 metastatic tumors had paired specimens from their primary NPCs for comparison. The in situ hybridization method was performed on paraffin embedded tissues by using polymerase chain reaction derived, digoxigenin-labelled EBER1 DNA probes. RESULTS: The EBER1 signal was identified in nuclei of malignant epithelial cells in 135 of 140 (96.4%) primary NPCs, including 4 of 5 (80%) WHO-I histologic subtypes, 71 of 73 (97.3%) WHO-II histologic subtypes, and 60 of 62 (96.8%) WHO-III histologic subtypes (P > 0.05). However, the positive hybridization signal in WHO-I NPC was less in proportion to malignant cells, usually limited to basal cells, than in other histologic types of NPC. In 10 of 11 specimens with metastases to the lymph nodes, hybridization was always limited to the malignant cells and not associated with lymphocytes. All 10 paired primary NPCs also demonstrated positive EBER1 hybridization. Only one paired specimen showed negative EBER1 in primary NPC and a metastases to a lymph node. Eight distant metastases, all EBER1-positive in their primary NPC, also demonstrated positive EBER1 hybridization signals in the malignant cells. The proportion of EBER1 positivity in metastatic NPC is higher than that in primary lesions as observed in paired specimens from the same patient. CONCLUSIONS: Because of abundant expression of EBER1 in primary NPC as well as in metastatic malignant cells, it is recommended that EBER1 in situ hybridization be performed on routinely processed specimens whenever NPC is suspected. PMID- 8625229 TI - Biologic characteristics of esophageal epithelial dysplasia assessed by proliferating cell nuclear antigen. AB - BACKGROUND: Proliferating cell nuclear antigen (PCNA) correlates with the cell proliferative state. Using a PCNA specific monoclonal antibody, various malignant neoplasms have been assessed for cell proliferation and malignant potential. However, there are few studies regarding the expression of PCNA in esophageal dysplasia. METHODS: PCNA expression was immunohistochemically determined in resected specimens from 27 patients with superficial esophageal carcinoma that contained areas with various degrees of dysplasia, carcinoma in situ (CIS), and carcinoma invading the mucosal layer. The authors estimated the cell proliferative activity and biologic characteristics of the basal and parabasal cells of these esophageal epithelial disorders. RESULTS: The degrees of PCNA labeling index (PCNA LI) of the basal layers were lower than those of the parabasal layers in normal epithelium and dysplastic lesions, respectively, whereas no difference in CIS was recognized between the two layers. The proportion of PCNA LI in the basal layer of moderate dysplasia was lower than that in the basal layer of severe dysplasia. Conversely, there were no statistical differences between severe dysplasia and CIS in both basal and parabasal layers. In terms of lymphocytic infiltration beneath the dysplasia, there was a close relationship between the degree of lymphocytic infiltration and the PCNA index of the basal and parabasal layers. CONCLUSIONS: PCNA expression may be a useful marker to reflect the biologic characteristics of esophageal dysplasia, suggesting that a high grade of dysplasia is as serious a lesion as esophageal carcinoma. PMID- 8625230 TI - The combination of cisplatin, doxorubicin, and mitomycin (PAM) compared with the FAM regimen in treating advanced gastric carcinoma. A phase II randomized trial of the Italian Oncology Group for Clinical Research. AB - BACKGROUND: In a randomized Phase II study, the authors evaluated the activity and toxicity of the new cisplatin, doxorubicin, and mitomycin C (PAM) combination, that includes cisplatin (P) instead of 5-fluorouracil as in the 5 fluorouracil, doxorubicin, and mitomycin C (FAM) combination, in patients with advanced gastric carcinoma. FAM was utilized as a control treatment arm. METHODS: Fifty eligible patients were assigned to the FAM (5-fluorouracil 600 mg/m2 intravenous (i.v.) on Days 1, 8, 29, 36; doxorubicin 30 mg/m2 i.v. on Days 1 and 29; mitomycin C 10 mg/m2 i.v. on Day 1; every 8 weeks) and 52 to the PAM combination (cisplatin 60 mg/m2 i.v. on Days 1 and 29; doxorubicin 30 mg/m2 i.v. on Days 1 and 29; mitomycin C 10 mg/m2 i.v. on Day 1; every 8 weeks). All eligible patients were included in the evaluation of response, toxicity and survival. RESULTS: The PAM combination complete response (CR) rate was 8%, and the CR plus partial response (PR) rate was 21% (95% confidence interval [CI] from 10% to 32%). The median time to progression, duration of response, and duration of survival were 15, 26, and 29 weeks, respectively. The FAM combination CR rate was 2% and the CR plus PR rate was 26% (95% CI from 14% to 38%). The median time to progression, duration of response, and duration of survival were 17, 27, and 23 weeks, respectively. Hematologic and nonhematologic toxicity were mild with both regimens. CONCLUSIONS: This study shows that this new combination, that does not include 5-fluorouracil, is active in patients with advanced gastric carcinoma. Since treatment with 5-fluorouracil alone is still considered the standard according to some authors, the PAM combination may be included among the sequential clinical options before or after treatment with 5-fluorouracil alone. PMID- 8625231 TI - Incidence and treatment of periampullary duodenal cancer in the U.S. veteran patient population. AB - BACKGROUND: Because fewer than 1000 cases of primary adenocarcinoma of the duodenum have been reported, earlier series are limited by local referral patterns and the long periods of time needed for retrospective reviews. METHODS: This study reports the outcomes of preiampullary duodenal adenocarcinoma treatments in all hospitals of the Department of Veterans Affairs from 1987 through 1991, using computer and tumor registry records. Patients were grouped by their most aggressive treatment (resection > operative bypass > percutaneous biliary intubation) and survival calculated from the date of this procedure. RESULTS: Of 2185 patients with periampullary cancers (1753 pancreatic, 432 other periampullary), 85 were duodenal and thus comprised only 4% of periampullary tumors. Thirty-four duodenal cancers were resected, 44 bypassed, and 7 had biliary intubation, with 30-day mortality rates of 6%, 18%, and 0%, respectively. Mean survival exceeded 1 year in all groups, and resection resulted in a significant increase in mean survival (784 vs. 438 days for nonresection, P = 0.01). The projected 5-year survival rate after resection was 23%. Mean survival after resection of 9 Stage I-II cancers was 668 days, but was similar after 5 resections with nodal or other metastases. Similarly, survival did not correlate with cancer stage in 13 palliated patients. CONCLUSIONS: This large study of patients with duodenal cancer provides a unique perspective of disease prevalence and response to surgical treatment. Prolonged survival is common with any treatment, but the longest survivals were after resection. PMID- 8625232 TI - Apoptosis and immunohistochemical bcl-2 expression in colorectal adenomas and carcinomas. Aspects of carcinogenesis and prognostic significance. AB - BACKGROUND: The bcl-2 oncoprotein confers a survival advantage to cells by inhibiting programmed cell death (PCD) or apoptosis. Overexpression of bcl-2 probably plays a role in colorectal carcinogenesis. The aims of our study were to determine bcl-2 expression and PCD index in colorectal adenomas and carcinomas in correlation with p53 expression, Ki-67 index, and histopathology, and to test their prognostic significance in patients with colorectal carcinomas. METHODS: Immunohistologic staining for bcl-2 (MoAb clone 124), the proliferation associated Ki-67 antigen (MoAb MIB1), and p53 (MoAb DO1) was performed on archival material from 44 colorectal adenomas and 95 adenocarcinomas (TNM classifications pT2 and -3, pN0, and M0). The PCD was visualized by enzymatic detection of DNA fragmentation. RESULTS: bcl-2 was expressed in 86% of the adenomas and 67% of the carcinomas. Mean PCD and Ki-67 rates were 1.7 +/- 0.14% and 35 +/- 13% in adenomas and 1.9 +/- 0.15% and 28 +/- 14% in carcinomas, respectively. In carcinomas, bcl-2 expression was correlated with a low PCD index (< 1.5%; P = 0.005). Furthermore, a high Ki-67 index (> or = 25%) was associated with a high PCD index (> or = 1.5%; P < 0.0001). p53 accumulation was seen in 16% of adenomas and in 42% of carcinomas, and did not correlate with bcl-2 expression or PCD index. In the univariate analyses, significantly longer disease free survival intervals were observed in three groups: all patients with bcl-2 positive carcinomas (P < 0.05); the subgroup of carcinomas with bcl-2 expression and low PCD index (P = 0.037); and the subgroup of bcl-2-positive and p53 negative carcinomas (P = 0.021). In the multivariate analysis, however, only tumor stage and p53 expression were independent risk factors for prognosis. CONCLUSIONS: Our data indicate that bcl-2 expression is characteristic of the early phase of colorectal carcinogenesis. Its physiologic function as an inhibitor of PCD is preserved in most colorectal carcinomas, whereas p53 is apparently not involved in the regulation of PCD in colorectal neoplasias. bcl-2 expression in colorectal carcinomas is associated with a better clinical course. This correlation became even more evident in the subgroups of patients with carcinomas that also had low PCD index or lacked p53 immunoreactivity. PMID- 8625233 TI - Clinical implications of microsatellite instability in colorectal cancers. AB - BACKGROUND: Microsatellite instability (MI) has been reported in some sporadic colon tumors and in cases of hereditary nonpolyposis colorectal cancer (HNPCC). The criteria for HNPCC have not been fully defined, and clinical criteria are used to identify as many HNPCC patients as possible. To clarify the conformity of these criteria with the identification of eligible HNPCC cases, we analyzed MI in HNPCC patients diagnosed using clinical criteria. METHODS: Genomic DNA was extracted from surgical specimens of 56 colorectal cancers, including 36 from patients diagnosed with HNPCC using the clinical criteria. We analyzed four microsatellite loci using 32P-labeled primers. RESULTS: Among HNPCC patients diagnosed using clinical criteria, patients who were positive for MI accounted for 62% of Group A (a confirmed group) and 35% of Group B (a high risk group); only 5% of randomly selected colorectal cancer patients (Group C), were positive for MI. Furthermore, MI-positive tumors were found in patients who had a tendency for tumors to involve the right side of the colon, an association with cancers in other organs, a lower incidence of p53 protein positivity, and a higher proportion of poorly differentiated cancers. CONCLUSIONS: The presence of MI, in concert with modified clinical criteria, may identify legitimate cases of HNPCC in patients who might otherwise be excluded by the minimum criteria. PMID- 8625234 TI - Cytogenetic analyses of hepatocellular carcinoma by in situ hybridization with a chromosome-specific DNA probe. AB - BACKGROUND: Numerical chromosome analysis has been established in solid tumors by using in situ hybridization (ISH) with a chromosome-specific probe. We analyzed human hepatocellular carcinoma (HCC) by ISH for chromosome 17 and investigated the correlation of its copy number with histologic malignancy, proliferative activity, p53 mutation, and DNA ploidy. METHODS: Chromosome 17 was hybridized with a pericentromere-specific DNA probe directly on the tumor cells isolated from paraffin blocks of 25 surgically resected HCCs. Proliferative activity was measured by Ki-67 immunohistochemistry, p53 mutation was analyzed by p53 immunohistochemistry, and DNA ploidy was estimated by cytofluorometry. RESULTS: Forty-four percent of the 25 HCCs showed numerical abnormality of chromosome 17. Many disomic cases had a less malignant histology, whereas many polysomic cases had a more malignant histology. The Ki-67 positive index of polysomic cases was higher than that of disomic cases. In 22 cases (88.0%), the copy number of chromosome 17 was well matched with DNA ploidy. However, the numerical abnormality of chromosome 17 did not show a significant correlation with p53 mutation. Two of four HCCs that showed histologic heterogeneity were also heterogenous on ploidy pattern and the copy number of chromosome 17. Conversely, there was one case in which only ISH could demonstrate heterogeneity, although the other features exhibited homogeneity. CONCLUSIONS: Numerical chromosome abnormalities correlated with the increase of histologic malignancy proliferative activity, and DNA ploidy. Moreover, ISH analysis was useful in assessing the intratumoral heterogeneity in HCC, especially when current methods failed to detect it. Thus, ISH provides information on important biologic features, such as malignant potential and intratumoral heterogeneity, in HCC. PMID- 8625235 TI - Histologic changes in small cell lung carcinoma after treatment. AB - BACKGROUND: Small cell lung carcinoma (SCLC) has been divided into three subtypes: pure SCLC, mixed small cell/large cell carcinoma (mixed SC/LC), and combined SCLC. Patients with mixed SC/LC show a worse prognosis than those with pure SCLC. METHODS: Persistence of histologic subtype in SCLC in the primary sites during the course of treatment or in the different organs at autopsy was examined. For this purpose, biopsy or cytologic specimens before chemotherapy, and autopsy specimens from 175 patients with SCLC were reviewed. They included 147 (84%) men and 28 (16%) women with an age range of 29-83 (median, 65) years. RESULTS: The frequency of mixed SC/LC in the primary sites was statistically higher in autopsy (14.3%) than that in biopsy or cytology specimens (8.6%) (P < 0.05). At autopsy, involved organs were categorized into two groups according to frequency of appearance of mixed SC/LC, i.e., a higher frequency group, including the liver (31 of 85; 36.4%), adrenal gland (15 of 56; 26.8%), brain (6 of 9; 66.7%), and extrathoracic lymph nodes (17 of 59; 28.8%) and a lower frequency group, including the lung (metastatic sites) (12 of 102; 11.8%), pleura (8 of 74; 10.8%), and intrathoracic lymph nodes (12 of 94; 12.8%). The difference in frequency between these two groups was statistically significant (P < 0.05). CONCLUSIONS: These findings suggest that primary pure SCLC can progress to mixed SC/LC with an increased potential for distant metastasis. PMID- 8625236 TI - DNA ploidy in nonmelanoma skin cancer. AB - BACKGROUND: Patients with head and neck squamous cell carcinoma die of locoregional recurrence and those with basal cell carcinoma suffer cosmetic and functional changes from its treatment. Prognostic factors are based upon tumor characteristics and host factors. Flow cytometry can assist with prognostic characterization of nonmelanoma skin cancer. METHODS: Specimens from 40 sequential patients with head and neck nonmelanoma skin cancers were prospectively obtained at the time of surgery. The patients were followed for four years for local recurrence and metastasis to regional lymph nodes. Samples were prepared from frozen specimens using a modification of the Vindelov procedure. RESULTS: DNA aneuploidy or tetraploidy and histology of well differentiated squamous cell carcinoma were significantly associated with metastasis to regional lymph nodes. Direct extension of tumor below the adipose tissue was associated with an S-phase greater than 4.1 and a proliferative fraction greater than 5.5. No tumors recurred at the site of surgical resection. CONCLUSIONS: The measurement of DNA ploidy of well differentiated squamous cell carcinoma, and proliferative capacity or S-phase of both basal cell and squamous cell carcinomas assists in predicting the biologic proclivity for locoregional invasion or metastasis of nonmelanoma skin cancer. Identification of aggressive tumors at the time of surgery may offer the opportunity for prevention of lethal metastasis by using adjunctive therapy. PMID- 8625238 TI - Body mass and breast cancer. Relationship between method of detection and stage of disease. AB - BACKGROUND: Obesity is associated with advanced stage breast cancer at diagnosis and a poorer prognosis. Stage of breast cancer at diagnosis is also strongly influenced by the method of cancer detection. The objective of this study was to determine the relationship between body mass index (BMI) and breast cancer disease stage, taking into account the method of cancer detection (i.e., self detection, screening mammography, and clinical breast examination [CBE]). METHODS: From 1988 to 1990, 2863 patients with invasive breast cancer were identified through a statewide, population-based, cancer reporting system and were interviewed as part of a larger study of breast cancer etiology. Stage of disease was classified as either localized or nonlocalized (regional and distant disease combined). The relation between BMI and disease stage was examined by using multiple logistic regression adjusting for age, education, race, year of diagnosis, and prior mammography use. RESULTS: Thirty-eight percent (1092 of 2863) of the women had nonlocalized breast cancer. A strong dose-response relationship was observed between increased BMI and the likelihood of nonlocalized disease (P < 0.001). However, this association was present only among the 55% of women (1585 of 2863) who self-detected their tumors. The odds ratios for nonlocalized cancer increased from 1.0 for the lowest quintile of BMI to 1.3, 1.6, 1.7, and 1.8 for the second through fifth quintiles, respectively, for this group. CONCLUSIONS: Greater body mass was associated with nonlocalized breast cancer; however, this association was restricted to women who detected their own cancer. No association was found between BMI and stage of disease among cases detected by either mammography or CBE. PMID- 8625237 TI - Sequential assessment of multidrug resistance phenotype and measurement of S phase fraction as predictive markers of breast cancer response to neoadjuvant chemotherapy. AB - BACKGROUND: The authors examined the relevance of S-phase fraction (SPF) and multidrug resistance (MDR) phenotype as predictive tests of breast cancer response in a series of patients treated by conventional doses of neoadjuvant chemotherapy with (FAC) or without (FTC) doxorubicin. METHODS: Fine needle samplings of tumors were used to measure SPF by flow cytometry before treatment (Day 0), and to assess the MDR phenotype using semiquantified reverse transcriptase polymerase chain reaction and immunocytochemistry, before and after (Days 8 and 28) the first cycle of chemotherapy. RESULTS: Measurement of SPF before treatment was significantly associated with clinical response, but sequential assessment of MDR phenotype identified three groups of tumors with distinct outcomes: (1) tumors with a positive and constant expression of MDR1, in which prediction of resistance was restricted to patients treated by FAC; (2) tumors without any detectable expression, in which resistance to FAC or FTC treatments was rarely observed; and (3) tumors with an early (Day 8) acquired or increased MDR1 gene expression, which were always resistant to therapy to both treatment regimens. These results were confirmed at the protein level. CONCLUSIONS: Sequential assessment of MDR phenotype is a relevant tool for monitoring breast cancer response in neoadjuvant chemotherapy. PMID- 8625239 TI - Racial differences in survival of female breast cancer in the Detroit metropolitan area. AB - BACKGROUND: In the United States, breast cancer survival is worse among African American women compared with white women. This difference in survival is likely due to several factors, including tumor biology and/or access to care. In this analysis, we evaluated the effects of sociodemographic and clinical variables on differences in breast cancer survival among African-American and white women. METHODS: The study population included 10,502 women (82% white, 18% African American), diagnosed between 1988 and 1992 and identified through the Metropolitan Detroit Cancer Surveillance System, a member of the Surveillance, Epidemiology and End-Results (SEER) Program. Cox proportional hazards regression was used to estimate the relative risk of death comparing African-American women with white women after controlling for variables believed to influence survival. RESULTS: The mean age at diagnosis was 61 years and average length of follow-up was 34 months (range, 1-78 months). African-American women were more likely to present with regional or distant disease (45%) than were white women (37%). Although white women had better survival than African-American women during the first 4 years postdiagnosis (P < 0.0001), there were no significant differences in survival by race for women who lived longer than 4 years (P = 0.64). There was a significant interaction between age and race. The unadjusted relative risk of dying for African-American women compared with white women was 2.35 (95% confidence interval [CI], 1.88-2.93) for women younger than 50 years of age, and was 1.66 (95% CI, 1.46-1.88) for women age 50 years or older. After controlling for age, tumor size, stage, histologic grade, census-derived socioeconomic status, and residency training status, the relative risk was 1.68 (95% CI, 1.27 2.24) for women younger than 50 years of age and 1.33 (95% CI, 1.13-1.56) for women age 50 years and older. Adjustment for marital status, hospital size, and the proportion of Medicaid or Medicare discharges had no further effect on the relative risk. CONCLUSIONS: Known factors that predict survival differences between African-American and white women are more prevalent among women younger than age 50. PMID- 8625240 TI - Evaluation of chromosome aneuploidy in tissue sections of preinvasive breast carcinomas using interphase cytogenetics. AB - BACKGROUND: Little is known about cellular level genetic alterations in preinvasive breast lesions, particularly lobular carcinoma in situ. METHODS: We employed fluorescence in situ hybridization (FISH) using pericentromeric (alpha satellite) probes to assess numerical alterations of chromosomes 1, 7, 8, 16, 17, and X in deparaffinized archival tissue sections of 9 lobular carcinomas in situ (LCIS), 10 ductal carcinomas in situ (DCIS), and a spectrum of proliferative lesions (including 3 ductal hyperplasias, 1 adenosis, 1 radial scar, and 2 atypical hyperplasias). Three of the LCIS lesions and five of the DCIS lesions were from patients who had a concurrent invasive neoplasm as a component of the tumor. RESULTS: None of the proliferative lesions exhibited detectable chromosome gains, and only 1 showed evidence of signal loss consistent with monosomy (chromosome 7 in the adenosis lesion). Six LCIS patients (67%) displayed evidence of monosomy, with involvement of chromosome 17 in 6 of 6 patients, chromosome 8 in 2 of 6 patients, and chromosome 7 in 2 of 6 patients. Two LCIS patients, each of whom had a concurrent invasive neoplasm, exhibited signal gains consistent with trisomy for chromosomes 1 and 8 (1 patient each). Chromosome aneuploidies were observed in 7 of 10 (70%) DCIS patients, including 2 of 5 patients (40%) without concurrent invasive neoplasm and 5 of 5 patients (100%) with concurrent invasive neoplasm. The pattern of numerical chromosome alteration in DCIS included two patients with losses only, 2 patients with gains only, and 3 patients with both gains and losses (i.e., involving different chromosomes). Chromosome 17 aneuploidy was observed in all DCIS and all LCIS patients who exhibited abnormalities; however, DCIS patients showed more frequent aneuploidies for chromosomes X and 16 (0 LCIS patients vs. 4 DCIS patients with each). CONCLUSIONS: Distinctive pathologic subsets of preinvasive breast neoplasia have divergent patterns of genetic instability. Foci of residual in situ neoplasia that accompany invasive disease may have a greater degree of genetic instability than neoplasms that lack progression to invasive phenotype. PMID- 8625241 TI - Stepwise abnormality of sex steroid hormone receptors, tumor suppressor gene products (p53 and Rb), and cyclin E in uterine endometrioid carcinoma. AB - BACKGROUND: In the normal cell cycle, the appropriate interaction of factors such as tumor suppressor gene products (retinoblastoma susceptibility [Rb], p53) and cyclins is essential. Abnormalities in the interaction of these factors may result in malignant transformation of the cell. Malignant transformation of the endometrium, which is believed to be a sex steroid-dependent tumor, probably involves a process of uncoupling of these factors and sex steroid hormone receptors. This study is designed to test this hypothesis. METHODS: Fifty-six patients whose pathology slides contained either normal or hyperplastic endometrium adjacent to endometrial carcinoma were selected. Immunohistochemical staining of serial paraffin sections was performed using antibodies to estrogen receptors (ER) and progesterone receptors (PR), p53, and Rb, as well as cyclin E. RESULTS: The normal and hyperplastic endometria adjacent to carcinoma showed positive staining for ER and PR and negative staining for p53. Of 56 carcinomas, 39 (69.6%) showed homogeneous positive staining for ER and PR and negative staining for p53, whereas the remaining 17 carcinomas (30.4%) contained varied distributions of ER- and PR-negative cells, and p53-positive cells (6 were negative or focally positive for ER/PR and diffusely-positive for p53, 4 were regionally-positive for ER/PR and regionally-positive for p53, and 7 were diffusely positive for ER/PR and focally-positive for p53). The p53-positive cells corresponded to those that stained negatively for ER/PR: This topographic inverse relationship between ER/PR expression and p53 expression also correlated with the staining intensities. Furthermore, the cells with weak or negative staining for p53 had a tendency to stain positively for Rb and weakly positive for cyclin E, whereas the cancer cells with definite positivity for p53 tended to stain either weakly or negatively for Rb and definitely positive for cyclin E. The cells showing diffusely positive for p53 were present significantly in clinical Stage III and pathologic Grade G3. CONCLUSIONS: In the development of endometrial carcinoma, stepwise abnormalities of sex steroid receptors, tumor suppressor gene products, and cyclins apparently exist, and may correlate with the progression of the malignant process. PMID- 8625242 TI - Expression of heat shock proteins HSP70 and HSP90 in endometrial carcinomas. Correlation with clinicopathology, sex steroid receptor status, and p53 protein expression. AB - BACKGROUND: It has been suggested that heat shock proteins HSP70 and HSP90 are involved in the functional modulation of sex steroid receptors and are expressed in normal endometrium. However, little is known about the expression of HSP70 and HSP90 in endometrial carcinomas. METHODS: The immunohistochemical reactivity of monoclonal antibodies against HSP70 and HSP90 was examined in 42 endometrial carcinomas, and the presence or absence of correlation with the clinicopathologic features, sex steroid receptor status, and p53 protein expression was analyzed. RESULTS: Expression of HSP70 was found in 52% of endometrial carcinomas and was correlated with nonendometrioid histology (P < 0.05), a poorly differentiated state (P < 0.01), p53 protein expression (P < 0.01), and absence of sex steroid receptors (P < 0.001) in the tumor. By contrast, strong expression of HSP90 was observed in 29% of endometrial carcinomas, and occurred more frequently in well differentiated carcinomas that were positive for sex steroid receptors. CONCLUSIONS: Both HSP70 and HSP90 are significantly correlated with the histology and the sex steroid receptor status of endometrial carcinomas. Expression of HSP70 may be associated with a loss of sex steroid receptors in either nonendometrioid or poorly differentiated carcinoma of the endometrium, which frequently exhibits p53 protein expression. Conversely, strong expression of HSP90 may indicate high levels of sex steroid receptors in the tumor cells. PMID- 8625243 TI - Torsion of intraabdominal testicular tumors. A case report. AB - BACKGROUND: Torsion of an intraabdominal testicular tumor is a rare event. We report a new case and review the literature. METHODS: A review of the literature was carried out in order to identify reported cases of an intraabdominal testicular tumor undergoing torsion of its vascular pedicle. Data on histologic type, common presenting signs and symptoms, radiographic studies, and adjuvant therapy were tabulated. RESULTS: There are 36 reports of an intraabdominal testicular tumor presenting with torsion. The most common histologic types, in descending order, were seminoma, sarcoma, teratoma, embryonal carcinoma, and choriocarcinoma. Common presenting signs and symptoms included pain and/or tenderness, fever, nausea and/or vomiting, and a palpable mass. Most seminoma patients received adjuvant radiation therapy whereas one patient with choriocarcinoma received adjuvant chemotherapy. CONCLUSIONS: Although a rare event, the diagnosis of torsion of an intraabdominal testicular tumor should be considered in any patient presenting with an acute abdomen and a history of cryptorchidism. The presence of a mass may raise the index of suspicion. Preoperative investigation with computed tomography scanning may aid surgical planning. PMID- 8625244 TI - Comparison between a cisplatin-containing regimen and a carboplatin-containing regimen for recurrent or metastatic bladder cancer patients. A randomized phase II study. AB - BACKGROUND: The aim of this randomized Phase II study was to compare the efficacy and toxicity of a cisplatin-containing regimen with a carboplatin-containing regimen for patients with recurrent or metastatic bladder cancer. METHODS: Fifty seven patients with recurrent or metastatic bladder cancer were randomized to receive M-VEC treatment (methotrexate, vinblastine, epirubicin, and cisplatin) (n = 29) or M-VECa treatment (methotrexate, vinblastine, epirubicin, and carboplatin) (n = 28). The chemotherapy was scheduled at 28-day intervals. Recombinant granulocyte-colony stimulating factors were administered daily when the absolute neutrophil count fell below 1000/mm3. The development of ototoxicity was evaluated by measuring auditory brain stem response. RESULTS: Of the 57 entered patients, 55 were evaluable for response and toxicity. The overall clinical response rate was 71% (with 25% complete responses) in the M-VEC group and 41% (with 11% complete responses) in the M-VECa group (P = 0.04). M-VEC chemotherapy was associated with more pronounced side effects. There was a statistically significant difference between M-VEC and M-VECa in terms of gastrointestinal toxicity (P = 0.04), nephrotoxicity (P = 0.03), and neurotoxicity (P = 0.02) during Cycle 3 of chemotherapy. Leukopenia and neutropenia were worse in the M-VECa arm, but not significantly so (P = 0.4). Ototoxicity was only detected in one of seven examined M-VEC patients after two cycles of chemotherapy. CONCLUSIONS: M-VECa has a low level of gastrointestinal, renal, neurologic, and otologic toxicity, but is apparently less effective than M VEC in the treatment of recurrent or metastatic bladder cancer. However, a larger, randomized Phase III trial is needed to confirm these results. PMID- 8625245 TI - Distinctive immunohistochemical profiles of small heat shock proteins (heat shock protein 27 and alpha B-crystallin) in human brain tumors. AB - BACKGROUND: Recent studies have described alpha B-crystallin as a member of the small heat shock protein (HSP) family, and the expressions of alpha-crystallin related small heat shock proteins, namely HSP27 and alpha B-crystallin, in the brain appear to be regulated in a similar way by various stress conditions. METHODS: A comparative immunohistochemical analysis was performed on 198 human brain tumors to examine the expressions of HSP27 and alpha B-crystallin. RESULTS: Positive staining with HSP27 was frequently observed in schwannomas, craniopharyngiomas, epidermoid cysts, and metastatic tumors to the brain. The immunopositivity of HSP27 was relatively low in tumors originating from neuroepithelium as well as in meningiomas; however, a statistically significantly higher percentage of HSP27-positive cells was noted in their anaplastic counterparts, such as glioblastomas, anaplastic oligodendrogliomas, anaplastic ependymomas, and anaplastic meningiomas (P < 0.005). Conversely, a positive immunoexpression of alpha B-crystallin was frequently observed among astrocytic tumors, schwannomas, hemangioblastomas, and chordomas. CONCLUSIONS: The immunohistochemical expression of HSP27 and alpha B-crystallin differed among histologic types of tumors. Furthermore, the immunopositivity of HSP27, which was considered to play a role not only in drug resistance but also in the regulation of cell proliferation, increased in proportion to the anaplasia of the tumors. PMID- 8625246 TI - Microvessel density is a prognostic indicator for patients with astroglial brain tumors. AB - BACKGROUND: Microvessel density in tumors, a measure of angiogenesis, has been shown to be a prognostic indicator that correlates with an increased risk of metastasis in various epithelial cancers and with overall and relapse free survival in patients with breast cancer. Astrocytic brain tumors, particularly malignant astrocytomas, are recognized to be highly vascular tumors with potent angiogenic activity. However, the prognostic significance of microvessel density in these tumors is not known. METHODS: Sections from formalin fixed paraffin embedded tumor tissue from 93 unselected adult patients with supratentorial astrocytic brain tumors were immunostained for factor VIII-related antigen in order to highlight microvessel endothelial cells. Microvessels were counted at 200x and 400x magnification. Microvessel density was graded as 1+ to 4+ on 1 low power field, without knowledge of clinical outcome. Microvessel count and microvessel grade were correlated with postoperative survival using the Cox proportional hazards regression model. The prognostic significance of microvessel count and grade were also compared with established prognostic indicators, including patient age, Karnofsky performance status, and tumor histology using multivariate analyses. RESULTS: Both microvessel grade and microvessel count correlated significantly with postoperative survival by univariate analysis in both previously untreated and treated patients. Patients with tumors containing a microvessel Grade of 3+ or 4+ had significantly shorter survival time than patients with a microvessel Grade of 1+ or 2+ (P = 0.0022). Likewise, patients with microvessel counts of 70 or greater had significantly shorter survival than those with microvessel counts of fewer than 70 (P = 0.041). Patient age, Karnofsky performance status, tumor histology, and extent of resection were also correlated with survival by univariate analysis. Microvessel count was further shown to be an independent prognostic indicator by multivariate analyses. There were correlations between microvessel density and patient age and between microvessel density and astrocytic tumor grade. CONCLUSIONS: These findings support the importance of microvessel density as a prognostic indicator of postoperative survival of patients with astroglial brain tumors. Regional tumor heterogeneity may limit the use of these techniques for routine pathologic examination. PMID- 8625248 TI - Radiation therapy for glottic cancer using 6-MV photons. AB - BACKGROUND: It has been recommended that cobalt-60 or 4-MV photons be used when treating glottic cancer with radiation therapy. Underdosing may occur when using higher energy photons, particularly when the anterior commissure is involved. The authors report their experience using higher energy photons (6 MV) for the treatment of glottic cancer. METHODS: Between January 1975 and July 1991, 73 patients with Tis, T1, T2, or T3 glottic tumors underwent radiation therapy with curative intent. Cobalt-60 or 4-MV photons were used to treat the cancers of 30 patients, and 6-MV photons were used to treat 43 patients. Clinical records were reviewed retrospectively to determine patterns of treatment failure, cancer deaths, and local tumor control in the 43 patients receiving treatment with 6-MV photons. Patients were followed until death or for a minimum of two years. RESULTS: Treatment failures were: local recurrence, one patient; local recurrence and distant metastasis, one patient; delayed neck metastasis, two patients; and delayed neck metastasis with distant metastasis, one patient. Three patients who had treatment failure are alive and free of cancer after salvage treatment. Two patients died of neck and distant metastases. The 2-year initial local tumor control rate was 94.8%. CONCLUSIONS: Glottic cancer can be treated successfully with 6-MV photons. Local tumor control is similar to that reported using cobalt 60 or 4-MV photons. PMID- 8625247 TI - Prognostic significance of Ki-67 labeling indices obtained using MIB-1 monoclonal antibody in patients with supratentorial astrocytomas. AB - BACKGROUND: Identification of the prognosis of patients with gliomas is important for selecting and evaluating the effectiveness of treatment. The aim of this study was to evaluate the Ki-67 labeling index (LI) using the newly generated MIB 1 monoclonal antibody (MoAb) as a prognostic indicator for patients with astrocytomas. METHODS: Ki-67 immunohistochemistry was performed on paraffin sections to estimate the growth potential of 72 supratentorial astrocytomas using the MIB-1 MoAb after hydrated autoclaving treatment. Multivariate analysis using the Cox proportional hazard stepwise model was used to evaluate the influence of Ki-67 LI, as well as other prognostic factors, on the duration of survival of patients with supratentorial astrocytomas. RESULTS: The mean Ki-67 LI was 3.8% (+/- 2.7%; standard deviation [SD]) in Grade 2 gliomas (n = 19), 18.4% (+/- 9.7% SD) in Grade 3 gliomas (n = 25), and 31.6% (+/- 12.9% SD) in Grade 4 gliomas (n = 28). Multivariate survival analysis showed that histologic grade, Ki-67 LI, and Karnofsky performance status (KPS) score before and after treatment were independent, statistically significant prognostic factors for patients with all grades of astrocytomas. In high grade lesions, Ki-67 LI, tumor location (superficial vs. deep), and KPS score after treatment were significant prognostic variables, whereas histologic grade did not meet the significance level for entry into the stepwise model. CONCLUSIONS: These results indicate that the Ki-67 LI obtained using MIB-1 MoAb is an important and practical tool for estimating biologic behavior of gliomas, as well as for predicting survival. PMID- 8625249 TI - Cerebral demyelination syndrome in a patient treated with 5-fluorouracil and levamisole. The use of thallium SPECT imaging to assist in noninvasive diagnosis- a case report. AB - BACKGROUND: The use of 5-fluorouracil (5-FU) and levamisole in patients with Stage III adenocarcinoma of the colon has now become standard. There have been several reports of a multifocal cerebral demyelination syndrome following 5-FU and levamisole administration. METHODS: We describe a patient who developed focal neurologic symptoms while being treated with levamisole and 5-FU in whom the diagnosis of central nervous system (CNS) metastases was considered. RESULTS: A magnetic resonance imaging (MRI) scan showed a diffuse, multifocal white matter process. Diagnostic evaluation did not support a diagnosis of CNS metastasis. 201Thallium chloride single photon emission computed tomography (SPECT) study was cold. A stereotactic brain biopsy disclosed demyelination but not tumor. The patient had complete functional resolution of symptoms with 1 month of dexamethasone therapy, although follow-up MRI scans have shown persistent abnormality on T2-weighted images. CONCLUSIONS: In patients receiving 5-FU and levamisole who develop focal neurologic symptoms with an abnormal MRI scan, the diagnosis of CNS metastasis should not be made without a thorough diagnostic evaluation. We suggest the use of 201thallium chloride SPECT imaging to support the diagnosis of multifocal leukoencephalopathy related to 5-FU and levamisole. In atypical cases, a stereotactic brain biopsy may be required for confirmation. PMID- 8625250 TI - Validation of a multidimensional evaluation scale for use in elderly cancer patients. AB - BACKGROUND: Although aging is one of the most important risk factors for cancer, elderly patients tend to be excluded from cancer clinical trials, only on the basis of chronologic age. Performance Status (PS) has been used widely to select adult patients for entry into clinical trials, but it does not include a comprehensive evaluation of various age-related factors in the elderly. This study was designed to assess the reliability and validity of a multidimensional geriatric assessment protocol for elderly patients with cancer. METHODS: Thirty consecutive elderly patients (> or = 65 years), diagnosed with hematologic neoplasia or solid tumors and undergoing chemotherapy or radiotherapy, were given a specifically structured multidimensional questionnaire (MACE) three times during one week by two different physicians. MACE was intended to collect information on demographics, socioeconomic status, cognitive status, depression, physical performance, disability, and tumor characteristics. In parallel with MACE, information was collected by means of the Sickness Impact Profile (SIP). RESULTS: Both for inter-rater and test-retest reliability, the values of the intraclass correlation coefficient (ICC) were generally higher than 0.7. Disability, cognitive status, depressive symptoms, and the number of days spent in bed sick in the last two weeks were markedly correlated with the global, physical, and social SIP score. Disability alone explained 70% of the variance in the SIP global score, 83% of the variance in the SIP physical score, and 45% of the variance in the SIP psychosocial score. MACE proved to be applicable in a reasonable amount of time (around 30 minutes) for a medical oncology ward. CONCLUSIONS: These data indicate that this structured evaluation of functional status is feasible and reliable. MACE is therefore proposed as a clinical research tool to avoid arbitrary decisions on patient selection for enrollment in clinical trials, to favor uniform monitoring of treatment, and to allow a better comparison of results. PMID- 8625251 TI - Clinical efficacy of octreotide in the treatment of metastatic neuroendocrine tumors. A study by the Italian Trials in Medical Oncology Group. AB - BACKGROUND: The unsatisfactory control of neuroendocrine tumor growth with chemotherapy and/or interferon (IFN-2a) stimulated us to investigate the role of the somatostatin analogue octreotide (SMS 201.995), which is reported to be highly effective in controlling carcinoid syndrome symptoms. Octreotide has been used in a wide range of doses, and it was postulated that higher doses might lead to an objective response. METHODS: The aim of the present multicenter Phase II study was to determine the safety and efficacy of SMS 201.995 in controlling carcinoids and other neuroendocrine tumors. Fifty-eight patients were treated subcutaneously with 2 sequential doses of the drug (Sandostatina, Sandoz, Inc., S.b.A. Pharmaceuticals, Basel, Switzerland). The first 23 patients received 500 micrograms 3 times a day and the remaining 35 patients received 1000 micrograms 3 times a day. The treatment was continued until the tumor progressed. RESULTS: All of the patients were adequately treated and evaluated. The predominant histotype was carcinoid, although there were instances of medullary thyroid carcinoma, pancreatic islet cell tumors, and Merkel cell carcinoma. Carcinoid syndrome was documented in 16 patients and abnormal urinary 5-hydroxyindoloacetic acid excretion in 15. The median treatment duration was 5 months (range, 2-31 months). The responses were evaluated in three categories: tumor regression for tumor growth control, symptom response, and biochemical response. There was an effect on tumor growth in two patients with carcinoids. Symptomatic control was achieved in 73% of patients and a biochemical response in 77% of patients. In twenty-seven patients, the disease stabilized for at least 6 months (range, 6-32+). The median survival time for all patients was 22 months (range, 1-32+). CONCLUSIONS: In terms of tumor regression, octreotide is disappointing (partial response: 3%); symptomatic response and biochemical control are satisfactory. These data confirm that somatostatin analogues are comparable to interferons in the treatment of carcinoid syndrome, although other efforts are necessary to control tumor regression. PMID- 8625252 TI - Disseminated neuroblastoma in the nude rat. A xenograft model of human malignancy. AB - BACKGROUND: The overall survival of children with neuroblastoma remains less than 30% due to disease dissemination at the time of diagnosis. An animal model of neuroblastoma, with characteristics similar to those observed clinically in children, would be beneficial to investigations into the diverse biology of this tumor. The purpose of this study was (1) to develop a model of disseminated neuroblastoma in the nude rat by intracardiac injection of neuroblastoma cells derived from cell lines with different N-myc copy numbers; (2) to investigate the effect of age on tumor growth and dissemination; and (3) to assess progression of disease radiologically and correlate with autopsy findings. METHODS: Nude rats (n = 38), 5-13 weeks of age, underwent intracardiac injection of the human neuroblastoma cell lines IMR-32 with amplified N-myc oncogene and SKNSH with 1 N myc copy. The animals were observed for at least six weeks for the development of tumor. Twelve rodents injected with IMR-32 cells underwent imaging studies including magnetic resonance imaging (MRI), skeletal radiographs, and indium 111(IN-111)-diethylenetriamine penta-acetic acid-D-Phe1-octreotide scintigraphy. Autopsies with standardized microscopic examinations were performed on all animals. RESULTS: Most of the nude rats (95%) developed neuroblastoma following intracardiac injection of neoplastic cells. Disseminated tumor was evident in 66% of animals. Anatomic sites of neuroblastoma growth were similar to those observed clinically in children, including adrenal glands, paraspinal ganglia, bone, bone marrow, and skin, but no tumor was identified in the liver. Disseminated disease occurred in more animals injected with IMR-32 (78% of animals) than with SKNSH cells (34% of animals) (P < 0.05). Tumor spread appeared to be age dependent; only rodents 5-8 weeks old at the time of injection developed disseminated disease when compared with animals 9 weeks of age or older (P < 0.0001). Radioreceptor scintigraphy demonstrated only pericardial tumor; MRI identified pericardial, adrenal gland, and subcutaneous neoplasms; only skeletal radiographs detected neuroblastoma in cortical bone. CONCLUSIONS: (1) Following intracardiac injection of human neuroblastoma cell lines into nude rats, a xenograft model of disseminated disease develops that closely parallels the malignant process in children. (2) Tumor dissemination is associated with the cell line that demonstrates N-myc amplification and with young age of the recipient at the time of injection. (3) Tumor growth and dissemination may be assessed radiologically. (4) This model of human malignancy may offer an opportunity to investigate the pathophysiologic mechanisms underlying tumor development and dissemination in advanced stage neuroblastoma. PMID- 8625253 TI - More detailed characterization of some of the HL60 karyotypic features by fluorescence in situ hybridization. AB - We performed a focused chromosome analysis on the HL60 cell line by multicolor fluorescence in situ hybridization (FISH), using probes for the unequivocal identification of specific chromosome regions and subregions. The purpose of this karyotypic re-evaluation was to confirm and to characterize in more detail chromosome rearrangements already identified by means of classic cytogenetic approaches and recurrently detected from the initial establishment of the cell line. The observations reported may help reassess the potential of the HL60 cell line in understanding the molecular events underlying the non-random karyotype alterations associated with acute myeloid leukemias (AML). PMID- 8625254 TI - High incidence of a second BCR-ABL fusion in chronic myeloid leukemia revealed by interphase cytogenetic analysis on blood and bone marrow smears. AB - The t(9;22)(q34;q11) is the single most common chromosomal abnormality in leukemias. Recently, dual-color fluorescence in situ hybridization (FISH) protocols for the detection of the BCR-ABL fusion, which is the molecular counterpart of this translocation, have been described. In the present study, we analyzed blood or bone marrow smears of 46 patients (34 with chronic myeloid leukemia [CML] and 12 with acute lymphoblastic leukemia [ALL]) for the presence of a BCR-ABL fusion. On these clinical routine samples, hybridization was performed with high efficiency and the BCR-ABL fusion was detected reliably. This series includes one case with a Philadelphia chromosome (Ph) on banding analysis and negative reverse transcriptase polymerase chain reaction (RT-PCR) results. Surprisingly, in 13 of the 34 CML patients (4 of 17 patients with chronic phase and 9 of 17 patients with blast crisis), and in 1 of the 12 ALL patients, an additional BCR-ABL fusion was diagnosed in 4% to 72.5% of interphase cells. In 10 of these 14 patients, banding data are available; only in two cases was the additional Ph detected by metaphase analysis. The data from this interphase cytogenetic analysis indicate that an additional Ph occurs more frequently than would be assumed based on banding analysis. PMID- 8625255 TI - Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and "chromosome instability" syndromes. AB - DNA topoisomerase II is involved in DNA topologic changes through the formation of a cleavable complex. This is stabilized by the antitumor drug VP16, which results in DNA breakage, aberrant recombination, and cell death. In this work, we compare the chromosomal damage induced by VP16 with that induced by bleomycin (BLM) in lymphoblasts from patients affected by the chromosome breakage syndromes ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Bloom syndrome (BS), and by the progeroid syndromes Werner (WS) and Cockayne (CS). Patients affected by AT, XP, BS, and WS have a greatly enhanced risk of developing cancer. The results show that AF and WS cells are hypersensitive to VP16, as revealed in the higher proportion of metaphases showing exchange figures and more than two breaks. All lines except AT and one CS line showed normal sensitivity to BLM. Our data on the sensitivity to VP16 of all these mutant cells underline the fact that VP16 damage is amplified only in cells that have abnormal illegitimate recombination (i.e., AT and WS). PMID- 8625257 TI - Detection of numerical chromosome abnormalities by FISH in childhood acute lymphoblastic leukemia. AB - Fluorescence in situ hybridization (FISH) was performed on interphase bone marrow cells to study numerical chromosome abnormalities in childhood acute lymphoblastic leukemia (ALL). Ten patients were selected for this study on the basis of having an extra chromosome 6 in the abnormal clone of the bone marrow at diagnosis. The numerical changes that were detected by FISH with a chromosome 6 specific alpha-satellite DNA probe correlated well with the cytogenetic and clinical data in all patients. Three hybridization signals were seen in 43.8- 83.0% of interphase cells in the specimens with a hyperdiploid karyotype. The diagnostic bone marrow sample of the patient with a tetraploid karyotype revealed four signals in 67.0% of cells. Two signals were detected in the majority of the cells in the three nonleukemic control bone marrow samples (97.0--97.7%), assessing the cut-off value of about 1% for trisomy 6. This study demonstrates that FISH analysis is a useful and sensitive tool to screen for the presence of extra chromosomes in interphase cells and is important clinically for evaluating the achievement and maintenance of remission in hyperdiploid childhood ALL. However, to detect structural chromosome aberrations which carry important diagnostic and prognostic information, as seen in our patient with the translocation t(1;19) at diagnosis but not at relapse, conventional cytogenetic analysis should be performed both at diagnosis and at relapse. PMID- 8625256 TI - Increased tubulin acetylation accompanies reversion to stable ploidy in vincristine-resistant CCRF-CEM cells. AB - The T-cell leukemia line CCRF-CEM is unstable with respect to ploidy, whereas a vincristine-resistant subline, CEM/VCR R, maintains a stable pseudodiploid karyotype. Ploidy change in the parental cells requires the involvement of two cell cycle lesions. The first, in mitosis, prevents cell division after S-phase. The second, in G1, allows a cell with 4N DNA content to re-enter S-phase. We examined differences in expression of tubulin, a major component of the mitotic spindle and the cellular target for vincristine, between the two cell lines. Levels of the beta III isotype were decreased and levels of acetylated alpha tubulin, a marker for microtubule stability, were increased in the CEM/VCR R cells relative to the parental line, which suggests that the CEM/VCR R cells have a more stable mitotic spindle. Both cell lines exhibit some level of constitutive expression of p53 and c-myc. Constitutive expression of and mutant p53 would contribute to the failure of these cells to recognise G1 checkpoints. Therefore, G1 checkpoint failure and the intrinsically less stable mitotic spindle in the CCRF-CEM cells may contribute to the observed ploidy instability. Conversely, the presence of markers of microtubule stability in the CEM/VCR R cells would predispose them to maintain their ploidy. PMID- 8625258 TI - Mutagen sensitivity in humans. A comparison between two nomenclature systems for recording chromatid breaks. AB - Currently, there are two systems in use for recording chromatid breaks, either spontaneously occurring or induced by mutagens: The International System for Human Cytogenetic Nomenclature (ISCN) and the Chatham Barrs Inn Conference (CBIC) recommendation. The former system considers that a chromatid break is recognized only when the chromatid fragment is displaced to the other side of its sister chromatid, while all others, regardless of the distance between the two broken ends, are called chromatid gaps. The CBIC system recognizes a chromatid break when the intervening achromatic segment is longer than or equal to the diameter of the chromatid, whether the fragment is displaced or not. Minor lesions are called chromatid gaps. We conducted experiments using bleomycin treatment of human cells (primary cultures or lymphoblastoid cell lines) and read the chromatid lesions both ways. We conclude that the CBIC system appears to have more direct biologic relevance than the ISCN system. PMID- 8625259 TI - Analysis of 3p allelic loss in papillary and nonpapillary renal cell carcinomas. Correlation with tumor karyotypes. AB - Nonpapillary renal cell carcinomas (RCCs) are characterized by deletions of the short arm of chromosome 3 (3p) and papillary RCCs by increased numbers of selected chromosomes. Although recent molecular genetic studies have reported some papillary RCCs to show loss of heterozygosity (LOH) on 3p, a 3p deletion has not been demonstrated in a papillary RCC by karyotype analysis. To investigate this apparent discrepancy between molecular methods and chromosomal changes in the genetic evaluation of RCC, a series of 13 papillary and nonpapillary RCCs was investigated for 3p LOH by PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis and for 3p and 3q LOH by microsatellite analysis. Karyotypes were obtained in six cases. Loss of 3p but not of 3q alleles was found in 8 of 10 nonpapillary RCCs. The region of overlapping deletion was 3p14--p21, and in six cases the deletion involved 3pter loci. One papillary RCC displayed 3p and 3q LOH, but the tumor had two morphologically normal chromosomes 3 and several trisomies. This indicated that nondisjunction of a chromosome from one parent compensated a whole chromosome loss from the other parent during tumor development. LOH in this papillary RCC constituted a reduction of chromosome 3 alleles to homozygosity, but the karyotype change, consisting of an increased number of whole chromosomes and an absence of a structural chromosome 3 abnormality, is regarded as being more characteristic of papillary than nonpapillary RCC. PMID- 8625260 TI - Karyotype evolution of the clonal rat liver cell line CL 52 during progression in vitro and in vivo. AB - Liver cell line CL 52, derived from a diethylnitrosamine-treated rat at the stage of preneoplasia/early neoplasia, had an inconspicuous 2n karyotype when analyzed 6 months after in vitro propagation. Malignant progression was accompanied by cytogenetic alterations of chromosomes 1, 3, and 11. In addition, trisomy of chromosomes 4, 6, and 7 led to a significant reduction of the tumor latency period after retransplantation. During 4 years of cytogenetic observation, the once clonal 2n population showed a characteristic karyotype evolution: loss of diploidy, occurrence of polyploid sidelines, deletions followed by unbalanced rearrangements, clonal diversification, and selection of the in vitro most rapidly growing or in vivo most malignant cell type. The karyotype alterations in the four sublines of CL 52 are discussed with special reference to oncogenesis related genes assigned to the involved rat chromosomes 1, 3, 11, 12, 4, 6, 7, 10. The observed karyotype evolution of this cell line exemplifies genetic/ chromosome instability of carcinogen-induced preneoplastic/early neoplastic liver cells and provides a tool for analyzing, under controlled conditions, stage dependent sequences of molecular genetic alterations in liver carcinogenesis. PMID- 8625261 TI - Structural aberrations of chromosome 6 in three uterine smooth muscle tumors. AB - Clonal karyotypic alterations of chromosome 6 in three uterine smooth muscle tumors are reported. In all cases an apparently identical breakpoint on the short arm of chromosome 6 was found. Two cases displayed the histologic features of cell-rich myomas with severe nuclear atypia but no clear evidence for malignancy. The remaining case was a primary uterine leiomyosarcoma of an 80-year-old patient showing an apparently balanced reciprocal chromosomal translocation, t(1;6)(p32 33;p21.3), as the sole karyotypic abnormality. This type of aberration has not been reported before in leiomyosarcomas. Because of the nuclear atypia in the other myomas with a breakpoint involving the short arm of chromosome 6 we feel that this cytogenetically recognizable but rare subgroup of uterine smooth muscle tumors warrants a careful clinical follow-up. PMID- 8625262 TI - Significance of clonal chromosome aberrations in breast fibroadenomas. AB - Despite the high frequency of fibroadenomas of the breast, cytogenetic results are relatively limited. We describe our cytogenetic findings in 30 fibroadenomas. Of these, three showed clonal chromosome abnormalities, i.e., 46,XX,der(6)t(1;6)(q25;p21.3); 48,XX,del(6)(q21),r(11)(?),der(14)t(6;14)(q21;q32),+2mar; and 47,XX,+5. PMID- 8625263 TI - Unusual and unrelated polyclonality in a case of myeloid disease. AB - A case of myelofibrosis with myeloid metaplasia was found to have five cytogenetically unrelated clones in the marrow, four of these clones being abnormal. We present the hypothesis that the hematologic disease in this patient was caused by a genetic event not discernible microscopically (cytogenetically) and that the distinctive abnormal clones may have been generated at different anatomic sites. PMID- 8625264 TI - Metastatic extraosseous Ewing tumor. Association of the additional translocation der(16)t(1;16) with the variant EWS/ERG rearrangement in a case of cytogenetically inconspicuous chromosome 22. AB - In Ewing sarcoma and related tumors, recently referred to as the Ewing tumors (ET), t(11;22)(q24q12) and its molecular genetic equivalent, the EWS/FLI-1 rearrangement, characterize approximately 85% of cases, while variant aberrations are rare. A second nonrandom aberration in ET is the unbalanced t(1;16) accompanying the t(11;22) in roughly 17% of cases. We present a 17-year-old man with estraosseous ET and multiple metastases, in whom the only cytogenetically detectable chromosomal aberration was der (16)t(1;16)(q12;q11.2). This finding was confirmed by fluorescence in situ hybridization (FISH). Using the RT-PCR technique, a variant EWS/ERG fusion transcript was noted, resulting from a t(21;22) chromosomal rearrangement which recently demonstrated in roughly 10% of ET. However, data on possible biologic differences in EWS/FLI-1 versus EWS/ERG expressing ET are as yet unavailable. This is the first reported combination of t(1;16) with the EWS/ERG rearrangement. A possible significance of this finding for Ewing tumor progression is discussed. PMID- 8625265 TI - Chromosome abnormalities in primary endometrioid ovarian carcinoma. AB - Specific and recurrent chromosome abnormalities may occur in regions of the genome that are involved in the conversion of normal cells to those with tumorigenic potential. Ovarian cancer is the primary cause of death among patients with gynecologic malignancies. We performed cytogenetic analysis in a subgroup of epithelial ovarian tumors, the endometrioid tumors, which are histologically indistinguishable from endometrial carcinoma of the uterus. We studied 10 endometrioid tumors to determine the degree of cytogenetic similarity between these two carcinomas. Six of 10 endometrioid tumors showed a near triploid modal number, and one had a tetraploid modal number. Eight of the 10 contained structural chromosome abnormalities, of which the most frequent were 1p - (5 tumors), 6q-- (4 tumors), 19q+ (4 tumors), and chromosome 3 rearrangements (4 tumors). These cytogenetic results resemble those reported for papillary ovarian tumors and differ from those of endometrial carcinoma of the uterus. We conclude that despite the histologic similarities between the endometrioid and endometrial carcinomas, the genetic abnormalities in the genesis of these tumors differ significantly. PMID- 8625266 TI - Rearrangement of one RAR-alpha gene in an acute promyelocytic leukemia case with t(15;17) and t(6;17) involving chromosomes 17 band q21. AB - Acute promyelocytic leukemia (APL) is a specific type of acute myelogenous leukemia characterized by a typical morphology and by a translocation between chromosomes 15 and 17, t(15;17)(q22;q21). Because this translocation is not found in other subtypes of acute myelogenous leukemias, it has become an important marker in the diagnosis and treatment of APL. Here we report a case with apparent absence of t(15;17) cells by cytogenetic techniques at diagnosis. At relapse, a metaphase FISH analysis (not dependent on chromosomal quality), followed by G banding, was performed. t(15;17) was detected in 60% of the metaphases. In 30% of metaphases, an additional t(6;17) was detected involving the other chromosome 17 at band q21. In parallel, a molecular study (using the Southern blotting technique) was carried out and a single molecular rearrangement of the RAR-alpha gene was detected implying that RAR-alpha was not rearranged in the t(6;17) translocation. PMID- 8625267 TI - Multiple chromosome rearrangements in a fibrosarcoma. AB - Clonal chromosome changes of a complex nature were found in a case of adult fibrosarcoma. Few studies on this tumor have been published, all showing complex changes without characteristic anomalies. PMID- 8625268 TI - der(16)t(1;16)(q21;q13) as a secondary change in alveolar rhabdomyosarcoma. A case report and review of the literature. AB - Alveolar rhabdomyosarcoma is an aggressive childhood tumor that exhibits muscle cell differentiation. Cytogenetically, it is characterized by t(2;13)(q35;q14); no consistent secondary abnormalities have been reported. Cytogenetic analysis of bone marrow in a case of alveolar rhabdomyosarcoma revealed t(2;13)(q35;q14) and der(16)t(1;16)(q21;q13). The present case and a review of the literature suggest that up to 11% of these tumors possess der(16)t(1;16)(q21;q13). This is similar to the incidence observed in the Ewing family of tumors, where unbalanced der(16)t(1;16) translocations, resulting in partial trisomy of 1q, are regarded as a consistent secondary cytogenetic change. PMID- 8625269 TI - Translocation (2;3)(p13;q26) in two cases of myeloid malignancies. Acute myeloblastic leukemia (M2) and blastic phase of chronic myeloid leukemia. AB - Two cases of myeloid leukemias (acute [AML M2] and chronic [CMC]), blastic crisis, with identical t(2;3)(p13;q26) are described. These cases had some peculiarities: no significant decrease of blood thrombocyte count in the AML patient and high increase of blood thrombocyte count during blastic phase in the CML patient; dysplastic megakaryocytes in bone marrow and unfavorable course of the disease; and short remission (3 months) in AML and short chronic phase (8 months) in CML. Clinical and morphologic findings in patients with t(2;3)(p13;q26) resembled those in cases with 3q21q26 syndrome or with other chromosome rearrangements involving 3q21 or 3q26. PMID- 8625270 TI - Translocation (21;22)(q22;q12) in a Ewing sarcoma. New case characterized by cytogenetic analysis and fluorescence in situ hybridization of a fresh tumor. PMID- 8625271 TI - Fluorescence in situ hybridization-based approaches for detection of 12p overrepresentation, in particular i(12p), in cell lines of human testicular germ cell tumors of adults. AB - Overrepresentation of the short arm of chromosome 12 is frequently detected in human testicular germ cell tumors of adolescents and adults (TGCT). This overrepresentation mostly results from the formation of an isochromosome: i(12p). Whether the overrepresentation consistently involves the complete 12p arm including the centromere is still unclear. We studied five TGCT-derived cell lines (NT2, 2102Ep, H12.1, NCCIT, and S2), combining conventional chromosome banding, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) to investigate the suitability of each of these techniques to detect aberrations involving chromosome 12. Karyotyping showed one or more i(12p)s in NT2, 2102Ep, H12.1, and S2. However, FISH with a centromere-specific probe (p alpha 12H8), a 12p "paint" and a 12p11.2--p12.1 region-specific probe yeast artificial chromosome (YAC)#5 and CGH could not confirm the presence of an i(12p) in S2. Additional randomly distributed 12p sequences were detected by FISH in H12.1, NCCIT, and S2. In most of these cases, (a part of) the centromere was included. All overrepresented 12p regions, except for those in S2, showed hybridization with YAC#5. CGH showed increased copy numbers of the complete 12p arm in the cell lines with one or more i(12p)s but no overrepresentation was noted in the cell lines without i(12p). In metaphase spreads, the centromeric block of the i(12p)s differed in size as compared with those of normal chromosomes 12. This was rarely noted in interphase nuclei. A decrease in size of the centromeric block in 2102Ep and H12.1 caused a weak FISH signal, which was difficult to detect, especially in interphase nuclei. The ratio between p alpha 12H8- and YAC#5-derived signals reflected the presence or absence of one or more i(12p)s. Our results indicate that double FISH with a centromere- and a 12p specific probe can be used to detect 12p overrepresentation [including i(12p)] in TGCT both in metaphase spreads and interphase nuclei. CGH confirmed the relative overrepresentation of 12p sequences as detected by FISH and showed that in these cell lines the complete 12p was involved. PMID- 8625272 TI - Receptors characterization of intraperitoneally N-nitroso-N-methylurea-induced mammary tumors in rats. AB - Mammary adenocarcinomas induces in female Sprague-Dawley rats by three intraperitoneal injections of N-nitroso-N-methylurea were studied in order to characterize their estrogen (ER), progesterone (PgR), prolactin (PRLR) and epidermal growth factor (EGFR) receptors. All samples evaluated showed the presence of ER and PgR in the cytosol fraction and PRLR amd EGFR in the membrane fraction. Q (fmol/mg) and K(d) (nM) values were as follows: ER, 56 +/- 11 and 0.5 +/- 0.1; PgR, 109 +/- 25 and 2.2 +/- 0.5 and PRLR, 335 +/- 75 and 0.5 +/- 0.2, respectively. In all tumors studied, two specific sites were found for EGFR, one with Q(1) = 22 +/- 9 and K(d1) = 0.6 +/- 0.3, and the other with Q(2) = 125 +/- 33 and K(d2) = 2.1 +/- 0.5. Receptor content was found to be independent of tumor histopathological variety. Displacement index (DI) with estradiol and tamoxifen of [I(3)H]E2-ER binding showed great heterogeneity, with values ranging from 0.01 to1.54. No correlation between ER content and DI values was found. Antiestrogenic binding sites were not found in the microsomal fraction of ten mammary tumors examined. Proliferation of this experimental mammary tumor may be regulated by a complex interaction of steroid and polypeptide hormones, as well as growth factors. PMID- 8625273 TI - Immunohistochemical localization of extracellular matrix proteins in human glioma, both in vivo and in vitro. AB - Expression of type IV collagen, fibronectin and laminin in various types of primary human brain tumor sections and normal brain tissue sections as well as cultured glioma cell lines was examined by an immunofluorescence technique. Type IV collagen, fibronectin, and laminin were mainly localized to the basement membrane of the vasculature in glioblastoma, anaplastic astrocytoma, low grade glioma, and in normal brain. However, positive staining for all the extracellular matrix (ECM) components tested was found only in glioblastoma sections both in the cells and in the ECM. In all other tumor types and in normal brain tissue, the cells did not stain for any of the ECM components. Four glioblastoma cell lines and autologous ECM synthesized by respective glioblastoma cell lines also showed positive staining for type IV collagen, fibronectin and laminin in vitro. These results suggest that glioblastoma cells both in vitro and in vivo express the extracellular matrix components that are involved in the regulation of tumor cell invasion. PMID- 8625274 TI - Wild type p53 reduces the size of tumors caused by bovine leukemia virus-infected cells. AB - Bat lung (BAT(2)CL6) cells infected with bovine leukemia virus (BLV) cause malignant tumors in nude mice that after 6 weeks subcutaneous growth, have an average volume of 0.3m(3). Uninfected bat lung cells (Tb 1 Lu) produce small benign neoplasms that average 0.003 cm(3). BAT(2)CL6 cells were transfected in vitro with expression vectors that produce wild type human or mutant p53. Production of human p53 in transfected BAT(2)CL6 cells was confirmed by immunoprecipitation of p53 and by immunohistochemical staining using anti-human p53 monoclonal antibodies. BAT(2)CL6 cells transfected with wild type p53 produced tumors in nude mice averaging 0.03 cm(3) whereas cells transfected with mutant p53 yielded tumors averaging 0.3cm(3). BAT(2)CL6 cell tumors after 1 week subcutaneous growth were transfected in situ with the wild type p53 gene. At 6 weeks tumor volume of in situ transfected tumors was similar to those resulting from cells transfected in vitro. Histopathologic examination and immunochemical staining of tumors produced in nude mice after wild type p53 treatment showed no significant differences when compared to tumors produced by untreated BAT(2)CL6 cells. Therefore, it is likely that the tumors produced by p53 treated-cells arose from cells that escaped transfection. The reduction of tumor size by restoration of wild type 53 may prove to be a useful therapy for BLV-induced tumors. PMID- 8625275 TI - Hormone dependent and independent mammary tumor development form N-methyl-N nitrosourea-treated rat mammary epithelial cell xenografts in the nude mouse: multiple pathways and H-ras activation. AB - A nude mouse mammary fat pad xenograft system was developed to examine hormone dependent and independent mammary tumorigenesis and progression from N-methyl-N nitrosourea (MNU)-induced hyperplastic lesions. Ninety-one percent of transplanted mammary tumors grew, with an orthotopic preference, and maintained their hormone dependence, histopathology, and H-ras mutation frequency. Grafted mammary epithelial cells, from MNU-treated rats, developed normal; and hyperplastic outgrowths, representative of those found in the rat mammary gland after MNU-treatment. Hyperplasias developed into neoplasias that were both hormone dependent and independent. We demonstrate that hormone independent tumors can develop directly either from lobuloalveolar or ductal hyperplasias or from hormone dependent tumors. H-ras mutation was detected in mammary preneoplasias (4 lines) before they developed into tumors and was associated with an elevated tumorigenic potential. Our observations suggest that there are multiple histopathogenic pathways in the development and progression to hormone independent rat mammary tumors. PMID- 8625276 TI - Potential advantages in therapeutic index of OK-432 ointment to abdominal skin over systemic applied in a mouse model. AB - A mixture of OK-432 powder and an ointment base, lanolin, was applied to mouse abdominal skin. After application of the mixture of 5 KE OK-432 and 0.2g lanolin, the leucocyte count and IL-b content in the abdominal cavity increases to up to 3.6 x 10(6) cells and 1.4 ng, respectively. INF-gamma content in the cavity after application of a mixture of 10 KE OK-432 and 0.2 g lanolin also increases, although not after application of the 5 KE OH-432 mixture of dorsum subcutaneous injection of a saline solution of 5 KE OK-432. These results indicate that an application of OK-432 mixed in lanolin to the mouse abdomen is able to exert an influence on the immunomodulatory effect in the abdominal cavity. The concentrations of IL-6 in serum after applying the ointment mixture, and subcutaneous of intraperitoneal injection of the saline solution of 5 KE OK-432, were <20, 370, and 430 pg/ml, respectively. After application of the ointment mixture of 5 KE OK-432, serum IL-6 did not increase. IL-6 levels in the abdominal cavity after 5 KE OK-432 ointment of subcutaneous injection increases to up to 1.4 and 2.1 ng/cavity. From these results, the immunomodulatory systemic response of OK-432 brought about by applying OK-432 ointment to the abdominal skin is lower than that brought about by injecting OK-432 subcutaneously. Therefore, it is thought that application of OK-432 ointment might be most suitable for potentiating local immune response. PMID- 8625277 TI - Thalidomide promotes metastasis of prostate adenocarcinoma cells (PA-III) in L-W rats. AB - Two contradictory actions have been ascribed to thalidomide relative to tumor metastasis: immunosuppression and anti-angiogenesis. The latter effect was determined with basic fibroblast growth factor in a rabbit cornea micropocket assay system. The prostate adenocarcinoma (PA-III) transplanted tumor line in Lobund-Wistar (L-W) rats produces a tumor at the subcutaneous implant site from which tumor cells metastasize uniformly only through lymphatic channels through the heart to the lungs in which secondary tumors develop. L-W rats were implanted with PA-III cells and administered, by gavage, thalidomide (50 mg/kg body wt per day) in corn oil. Control rats with PA-III cells were administered corn oil. Autopsy examinations on day 30 revealed that the thalidomide-treated rats developed more metastatic tumor foci in the lungs than in the controls. PMID- 8625278 TI - Glucosylceramide synthetase inhibitor, D-threo-1-phenyl-2-decanoylamino-3 morpholino-1-propanol exhibits a novel decarcinogenic activity against Shope carcinoma cells. AB - The glucosylceramide synthetase inhibitor, D-threo-1-phenyl-2-decanoylamino-3 morpholino-1-propanol (D-PDMP) was tested to determine whether it could exhibit anti-tumor activity against two different Shope carcinoma cell lines. The cell growth was suppressed in a dose-dependent manner in the presence of D-PDMP. This supression seem to be accounted for by prolongation of the lag phase and this phenomenon was especially marked in the undifferentiated cell line. The growth suppression was also partly explained by direct inhibition of cell proliferation because the suppression was released by removing the agent from the medium. The treated cells became morphologically differentiated with lower density at confluence and regained contact inhibition in flask culture. Colony-forming ability in soft agar, which has been reported to be closely correlated with tumorigenicity, was also inhibited dose-dependently in the presence of D-PDMP. These results suggested that D-PDMP could exhibit a novel decarcinogenic activity against Shope carcinoma cells. PMID- 8625279 TI - Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation. AB - We hypothesized that malignant transformation of normal mucosa of the upper aerodigestive tract to squamous cell carcinoma of the head and neck (SCCHN) might be associated with altered CK2 activity in the chromatin compartment of these tumors. We measured CK2 activity in the cytosol and chromatin of 7 surgical specimens of SCCHN, and 5 specimens of normal oropharyngeal mucosa from non smokers/non-drinkers. CK2 activity in SCCHN tumors was significantly elevated in both the nuclear chromatin (P < 0.0005) and cytosolic (P <0.04) compartments relative to normal mucosa. These data suggest that activation of dysregulation of the chromatin-associated CK2 signal may play a role in the pathobiology od SCCHN. PMID- 8625280 TI - Potent tumorigenicity of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene in the neonatal B6C3F (1) male mouse. AB - The tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA, and environmental contaminant, and 7 bromobenz[a]anthracene (7-Br-BA) was determines in the male B6C3F(1) newborn mouse. Mice receiving 7-Cl-BA and 7-Br-BA by i.p. injections at a dose of 1600 nmol per mouse on 1, 8, and 15 days after birth developed 92 and 96% hepatocellular adenomas, and 100 and 83% hepatocellular carcinoma, respectively. Metabolism by liver microsomes of 15-day-old mice each produced the corresponding trans-3,4-dihydrodiol. Analysis by (32)P-postlabeling/HPLC indicated the presence of DNA adducts derived from 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol. Our results indicate that both 7-Cl-BA and 7 Br-BA are potent carcinogens and that bay-region diol epoxides are the ultimate metabolites that lead to DNA adduct formation and tumor initiation. PMID- 8625281 TI - Apoptosis amd metastasis: a superior resistance of metastatic cancer cells to programmed cell death. AB - We studied the response to different external signals leading to apoptosis of several poorly and highly metastatic cell lines employing a murine B16 melanoma experimental metastasis model. We found that highly metastatic cells exhibit a superior survival ability and resistance to apoptosis compared to poorly metastatic cells which would give the former an obvious selective growth advantage during tumor progression. Our results indicate that there is a genetic link between aggressive metastatic phenotype and resistance to apoptosis. PMID- 8625282 TI - Down-regulation of protein kinase C in murine splenocytes: a potential mechanism for 2-acetylaminofluorene-mediated immunosuppression. AB - 2-Acetylaminofluorene (AAF), an arylamide carcinogen, inhibited in a dose dependent manner mouse spleen cell proliferation in response to lipopolysaccharide (LPS). The objective of the present studies was to investigate the effects of AAF on protein kinase C (PKC) activation, an enzyme required for LPS-induced splenocytes proliferation. After treatment with 50 microM AAF for 18 h, PKC activity in the cytosolic fraction decreases by 50% from the control level, and splenocytes lost 30% of total PKC activity. Furthermore, as determined by the electrophoretic mobility shift assay, AAF inhibited the binding activity of the transcription factor complex, NF-KB, whose LPS-mediated induction is dependent on PKC activation in murine splenocytes. These results strongly suggest that LPS-mediated signaling in spleen cells is interrupted by AAF early in the signal transduction pathway, at a point proximal to the activation of PKC. PMID- 8625283 TI - Histopathologic effects of tamoxifen on the uterine epithelium of breast cancer patients: analysis by menopausal status. AB - We evaluated the histopathologic changes of the uterine epithelium in 73 breast cancer patients with tamoxifen stratified by menopausal status. Clinicopathologic data at the time of breast cancer diagnosis and endometrial sampling were analyzed and compared with 122 breast cancer patients not receiving the drug. The incidence of endocervical and/or endometrial polyps was increased in tamoxifen treated postmenopausal patients compared with untreated patients, 43% (25 of 58) and 24% (16 of 68), respectively (odds ratio=2.46, P=0.02). In contrast, there was no increase in polyps in premenopausal tamoxifen-treated patients. This finding suggests that the effects of tamoxifen on the endometrium may vary with menopausal status. PMID- 8625284 TI - In vitro and in vivo (32)P-postlabeling analysis of 4-vinyl-1-cyclohexene (butadiene dimer) diepoxide-DNA adducts. AB - 4-Vinyl-1-cyclohexene diepoxide (VCD), and industrial chemical, and its parent compound, 4-vinyl-1-cyclohexene (VCH), are potential health hazards, a they destroy oocytes in follicles in rodents. VCD is also a skin carcinogen at the site of application in both female and male mice and rats and after gavage, induces ovarian tumors in mice and forestomach tumors in rats. A (32)P postlabeling assay was developed for the detection and measurement of VCD-DNA adducts. VCD, a direct-acting carcinogen, was reacted with DNA in vitro, as well as through mouse skin painting for 3 days with different doses of VCD. (32)P Labeled adducts were separated by polyethyleneimine (PEI)-cellulose TLC and detected by screen-enhanced autoradiography. Comparable adduct profiles were obtained in vitro and in vivo. At higher doses (36-225 micro mol/mouse), adduct levels in vivo showed a linear dose response, while there was no difference between 14 and 36 micro mol/mouse. The limit of detection was estimated to be 1-3 adducts in 10(8) DNA nucleotides. The results show that VCD exposure gives rise to (presumably pre-mutagenic) DNA adducts in vivo and that (32)P-postlabeling can be applied to biomonitoring of VCD exposure. PMID- 8625285 TI - Inhibitory potential of pregnancy and lactation on mammary carcinogenesis induced by a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in Sprague-Dawley rats. AB - Effects of pregnancy and lactation on a food-derived carcinogen 2-amino-1-methyl 6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were investigated in female Sprague-Dawley rats. When rats were administrated PhIP in the diet (100 ppm) for 15 weeks, palpable subcutaneous tumors were first detected at week 24 in three nulliparous rats, while tumor latency was apparently increased in animals undergoing delivery and weaning during the PhIP administration period. However, since the incidence of palpable tumors was not consistently increased in the nulliparous animals, possibly due to the short PhIP treatment, and the fact that spontaneous tumors developed in parous animals after week 44, the final incidences and multiplicities of histopathologically confirmed adenocarcinomas at week 48 were not significantly different between the two groups. These findings art partly supported by the epidemiological evidence that breast cancer is more prevalent in nulliparous than in parous women. PMID- 8625286 TI - Evaluation of the potential carcinogenic activity of Senna and Cascara glycosides for the rat colon. AB - Anthraquinone glycosides of Senna and Cascara were investigated for their ability to induce aberrant crypt foci (ACF) in the rat colon mucosa, which are considered putative preneoplastic lesions. Dietary exposure to high doses of these glycosides for 56 successive days did not cause the appearance of ACF or increase in incidence of ACF induced by 1,2-dimethyl-hydrazine (DMH). However, in rats treated with both DMH and the highest dose of glycosides, the average number of aberrant crypts per focus, considered a consistent predictor of tumor outcome, was higher than in rats given DMH alone. These findings suggest that Senna and Cascara glycoside might behave as weak promoters in rat colon carcinogenesis. PMID- 8625287 TI - Lack of p16/CDKN2 alterations in thyroid carcinomas. AB - Exons 1-3 of the p16/CDKN2 gene and exons 4-9 of the p53 gene were screened for mutations by single-strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified DNA from human primary thyroid carcinomas and thyroid carcinoma cell lines. The samples included 21 papillary carcinomas, 2 undifferentiated carcinomas, 1 follicular carcinoma, 1 medullary carcinoma and 2 cell lines originating from thyroid undifferentiated carcinomas. No homozygous deletions and mutations in the p16/CDKN2 were observed in any of the primary tumors or cell lined. In contrast, one of the two undifferentiated carcinomas an both cell lines demonstrated point mutations in the p53 gene. These results that p16/CDKN2 gene alteration is not required for malignant transformation in the thyroid, while p53 gene mutations may play a role in the progression from differentiated to undifferentiated carcinoma. PMID- 8625288 TI - Studies of the activities of lysosomal enzymes in serum and buccal pouch tissue of hamsters during 7,12-dimethylbenz[a]anthracene-induced carcinogenesis. AB - Alterations in the activities of certain lysosomal enzymes such as beta-D galactosidase, beta-D-glucosidase, beta-D-glucuronidase, alpha-L-fucosidase, N acetyl-beta-D-glucosaminidase, cathepsins B and D were studied in serum and tissue homogenates of buccal mucosa of hamsters treated with 0.5%, 7,12 dimethylbenz[a]anthracene (DMBA) in liquid paraffin. Among the enzymes studied, the activities of beta-D-galactosidase and N-acetyl-beta-D-glucosaminidase showed significant elevation both in serum and tissue homogenates fro papilloma onwards and the elevations were progressive with the development of carcinomas. The elevations in the activities of alpha-D-fucosidase and cathepsin D were found to be significant from papillomatous tissue onwards whereas in serum they showed higher activities only in carcinoma stages. The activities of beta-D-glucosidase, beta-D-glucuronidase and cathepsin B in both serum and in tissue homogenate were elevated markedly only in carcinoma stages. It is suggested that beta-D galactosidase and N-acetyl-beta-D-glucosaminidase may be used as diagnostic markers for premalignant and malignant lesions of oral mucosa. PMID- 8625289 TI - The preventive and therapeutic potential of the squalene-containing compound, Roidex, on tumor promotion and regression. AB - Recent scientific evidence has shown free radicals or reactive oxygen species (ROS) to play an important role in the initiation and progression of cancer. Many radical scavengers have also been found to help reduce the attacks by these ROS. Interestingly, the ROS scavengers that have been investigated are naturally occurring compounds such as vitamins C and E. Roidex is a formulation of squalene, vitamin e, and aloe vera. It was our goal to investigate whether Roidex was able to prevent the development of chemically induced cancer and to cause regression of any tumors already formed in a mouse skin model. In the prevention study, skin tumors were initiated in 50 female CD-1 mice with 7,12 dimethylbenz[a]-anthracene (DMBA) and promoted with 12-O-tetradecanoylphorbol-13 acetate (TPA). The mice were treated with either mineral oil, 5% squalene, or Roidex. At the end of the prevention study, there was a 33.34% incidence to tumors (multiplicity of 1.40) in the mineral oil-treatment group, 26.67% (multiplicity of 0.467) in the 5% squalene and Roidex groups, respectively. The tumor regression study involved the selection of mice with tumors and possible regression of these tumors with Roidex treatment. There was a regression of 33.34% of the tumors in the Roidex-treated group (39 tumors to 26 tumors) compared to the non-treated group whose tumors regressed only 3.44% (29 tumors to 28 tumors). PMID- 8625290 TI - Levels of myc, fos, Ha-ras, met and hepatocyte growth factor mRNA during regenerative cell proliferation in female mouse liver and male rat kidney after a cytotoxic dose of chloroform. AB - Chloroform is a liver carcinogen in mice and a kidney carcinogen in rats. It is thought to act through a non-genotoxic-cytotoxic mode of action. Changes in expression of growth control genes accompanying chloroform-induced cytolethality and regeneration may play a part in the development of chloroform-induced tumors. In this experiment, we examined the levels of the myc, fos, Ha-ras, met and hepatocyte growth factor mRNA in livers of female B6C3F(1) mice and kidneys of male F-344 rats to detect changes in gene expression following a single, cytotoxic gavage dose of chloroform in corn oil. Poly A+ RNA was purified from homogenates of livers of mice treated with 350mg/kg chloroform and kidneys of rats treated with 180 mg/kg chloroform and used for Northern blot analysis. Livers of female mice showed large transient increases in levels of myc and fos mRNA while levels of Ha-ras, met and the hepatocyte growth factor gene mRNA remained near control levels. In the male rat kidney, levels of myc mRNA increased after treatement, while levels of mRNA of all other genes examined remained near control levels. This pattern of gene expression is consistent with that induced by other cytotoxic carcinogens and suggest that alteration of the myc and fos genes could be involved in the regenerative cell proliferation that ultimately could play a role in chloroform-induced tumors. PMID- 8625291 TI - Possible roles of tumor-associated carbohydrate antigens. AB - Recent progress in the studies on the roles of carbohydrates has brought about critical discoveries, which allow us to have working hypotheses for understanding the roles of tumor-associated carbohydrate antigens. In this report, I focus my description on three different aspects of this progress. I discuss: (a) the immunological response to oligosaccharides aberrantly expressed under pathological conditions; (b) possible roles of carbohydrate-dependent cell adhesion during tumor metastasis; and (c) the roles of carbohydrates in modulating the functions of proteins that attach those carbohydrates. These three areas in the roles of carbohydrates will likely be the target for continuous research efforts to reveal the roles of tumor-associated carbohydrates. PMID- 8625292 TI - Enhanced tumor suppressor gene therapy via replication-deficient adenovirus vectors expressing an N-terminal truncated retinoblastoma protein. AB - The preclinical studies presented here demonstrate that treatment of human non small cell lung carcinoma and bladder carcinoma cells by a recombinant adenovirus vector, AdCMVpRB94, expressing the N-terminal truncated retinoblastoma (RB) protein (pRB94) completely suppressed the tumorigenicity of the treated tumor cells in nude mice. Furthermore, gene therapy of established human RB- and RB+ bladder xenograft cancers in nude mice by AdCMVpRB94 resulted in regression of the treated tumors. Of note, although both the full-length and the truncated forms of the RB protein, when overexpressed in tumor cells via replication deficient adenovirus vectors, were capable of suppression of tumor growth, the pRB94 was evidently more potent than the full-length RB protein. PMID- 8625293 TI - Repair Defect in p21 WAF1/CIP1 -/- human cancer cells. AB - p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cisplatinum-damaged cytomegalovirus-driven beta-galactosidase reporter DNA into tumor cells revealed a significant decrease (2-5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2-3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2-3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage. PMID- 8625294 TI - Expression of the cisplatin resistance phenotype in a human ovarian carcinoma cell line segregates with chromosomes 11 and 16. AB - Many mechanisms have been proposed to explain cisplatin resistance, suggesting that this phenomenon is multifactorial. In an attempt to define the chromosome(s) responsible for cisplatin resistance in the human ovarian carcinoma 2008/C13* cell lines, somatic cell hybrids were obtained following fusion of the cisplatin resistant 2008/C13* cells with an A9 rodent fibroblast cell line. The hybrids were then analyzed for segregation of the human chromosomes with the drug resistant phenotype. Chromosomes 11 and 16 were present in all of the resistant somatic cell hybrids, with the highest concordance for chromosome 16. The role of both of these chromosomes was further established with microcell hybrids. Microcell hybrids of A9 cells with chromosome 16 from the 2008/C13* cells did not exhibit cisplatin resistance, but the presence of a normal chromosome 11 with chromosome 16 (derived from cisplatin-resistant 2008/C13* cells and not from the cisplatin-sensitive 2008 cells) resulted in increased resistance to cisplatin. In addition, loss of chromosome 11 from a resistant somatic cell hybrid resulted in the hybrid becoming sensitive to cisplatin, implicating this chromosome in maintaining the resistant phenotype. The results demonstrate that resistance to cisplatin is a dominant trait in the 2008/C13* human ovarian cells, and both chromosomes 11 and 16 are required for its expression. PMID- 8625296 TI - Radiation with 1 Gy prevents the activation of the mitotic inducers mitosis promoting factor (MPF) and cdc25-C in HeLa cells. AB - The mechanism of the transient G2 arrest induced by small doses of ionizing radiation involves the failure to activate the correctly formed pre-mitosis promoting factor (MPF) complex of cyclin B and p34cdc2 by dephosphorylation at Tyr15 of the latter, as recent studies of other laboratories have indicated. Similar data were obtained with the G2 arrest-inducing agents epidermal growth factor and the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (H. Barth and V. Kinzel, Exp. Cell Res., 212: 383-388, 1994, and H. Barth and V. Kinzel, J. Cell. Physiol., 162: 44-51, 1995). To differentiate the radiation consequences in synchronized HeLa cells from those of 12-0-tetradecanoylphorbol-13-acetate and epidermal growth factor, experiments with a very small dose (1 Gy) have been carried out in cells close to the G2-M border and, for comparison, in mitotic cells. We show that in addition to the failure of p34cdc2 dephosphorylation at Tyr15, radiation with 1 Gy also prevents the activation of the phosphatase cdc25 C, the enzyme catalyzing the MPF activation. In contrast, irradiation of mitotic cells with 1 Gy did not influence that fraction of either MPF or cdc25-C already activated. Moreover, the gain in MPM-2 antigenicity of cdc25-C, usually indicative of an activating phosphorylation, is shown to be prevented by 1 Gy. The data indicate that the initiation of the proposed autocatalytic loop between MPF and cdc25-C becomes interrupted by radiation, but they give no hint at which point. PMID- 8625295 TI - Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer. AB - Utilizing tissue microdissection and PCR techniques, we have examined 35 prostate tumors paired with normal tissues from the same patients for allelic loss at 24 polymorphic loci spanning chromosome 10. Twenty-five tumors (71%) were deleted for at least one chromosome 10 locus. Of the total 35 tumors, 6 (17%) were deleted for 10p loci only, 5 (14%) for 10q loci only, and 14 (40%) were deleted for both 10p and 10q loci. The common region of deletion on 10p included loci D10S211-D10S89-D10S111. Fluorescence in situ hybridization of yeast artificial chromosome probes encompassing these loci demonstrated that the 10p region of deletion maps to 10p11.2. Losses involving 10p loci alone were most common in localized (5/14, 36%) and least common in metastatic (0/8) tumors. The common region of deletion on 10q included loci D10S219-D10S215, consistent with the major region of deletion recently defined for prostate tumors on 10q. Losses involving 10q loci alone were lowest in localized and locally invasive tumors (1/14 and 2/12, respectively) and highest in tumors metastatic to regional lymph nodes (2/8). These results suggest that 10p losses may define less invasive tumors, whereas 10q losses may play a role in the progression to more advanced tumor states in the prostate. Furthermore, this is the first report of allelic loss of a defined region on 10p potentially harboring tumor suppressor gene loci in human prostate cancer. PMID- 8625297 TI - Differential growth inhibition by the aspirin metabolite salicylate in human colorectal tumor cell lines: enhanced apoptosis in carcinoma and in vitro transformed adenoma relative to adenoma relative to adenoma cell lines. AB - Regular aspirin intake may reduce the risk of colorectal cancer by 50%. However, the mechanism of this chemopreventive effect is not known. The effect of the aspirin metabolite salicylate on the growth of human colorectal tumor cell lines was determined. Salicylate showed dose-dependent inhibitory effects on all of the cell lines (IC50, 1.65 +/- 0.36 to 7.38 +/- 1.08 mM), yet carcinoma and in vitro transformed adenoma cell lines were more sensitive than adenoma cell lines. Salicylate caused all cell lines to accumulate in G0-G1 and induced apoptosis in carcinoma and in vitro-transformed adenoma cell lines but not in all adenoma cell lines. In those adenoma lines that did show salicylate-induced apoptosis, the extent was considerably less than that in the more transformed cell lines. The ability of salicylate to induce cell cycle arrest and apoptosis and, in particular, the increased sensitivity of cells at later stages of neoplastic progression may be mechanistically important in the chemopreventive action of aspirin toward colorectal cancer. PMID- 8625298 TI - 13-cis-retinoic acid with alpha-2a-interferon enhances radiation cytotoxicity in head and neck squamous cell carcinoma in vitro. AB - The treatment of locally advanced squamous cell carcinomas of the head and neck presents a challenge for oncologists. Radiation therapy alone fails to control many of these tumors. Chemotherapy added to radiation therapy has not clearly demonstrated an improvement in survival in the majority of trials reported to date. In this study, we have evaluated whether IFN-alpha-2a and/or 13-cis retinoic acid (RA) enhance radiation cytotoxicity in a head and neck squamous cell carcinoma cell line (FaDu). Using a clonogenic cell survival assay, IFN alpha-2a (1000 units/ml) or RA (1 microM) alone did not significantly enhance radiation cytotoxicity. The combination of the two agents, however, significantly increased the cytotoxicity of radiation against FaDu cells. The calculated survival fraction at 2 Gy was decreased from 0.649 with radiation alone to 0.477 when combined with the other two agents (P = 0.016), and the MID was decreased from 3.318 to 2.499 Gy (P = 0.028). A Phase I clinical trial to combine IFN-alpha 2a and/or RA in patients with unresectable head and neck cancer has been initiated. PMID- 8625299 TI - Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells. AB - We examined the anti-invasive activity of ursolic acid (UA) on the highly metastatic HT1080 human fibrosarcoma cell line. UA reduced tumor cell invasion through a reconstituted basement membrane in a transwell chamber. A significant down-regulation of matrix metalloproteinase-9 [MMP-9; Mr 92,000 gelatinase/type IV collagenase (gelatinase B)] by UA was detected by Northern blot analysis. However, MMP-2 [Mr 72,000 gelatinase/type IV collagenase (gelatinase A)] and membrane-type MMP were constantly expressed, and the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 also was not changed after 3 and 6 days of treatment with UA. Quantitative gelatin-based zymography confirmed a markedly reduced expression of MMP-9 but not MMP-2 after treatment with UA. To confirm the UA-induced down-regulation of MMP-9 expression, we constructed a secreted alkaline phosphatase (SEAP) reporter vector including MMP-9 promoter. After transfection of MMP-9/SEAP reporter vector into HT1080 cells, reduced SEAP activity was detected after treatment with UA. These results suggest that down regulation of MMP-9 contributes to the anti-invasive activity of UA in HT1080 cells. PMID- 8625300 TI - Microsatellite alterations in bronchial and sputum specimens of lung cancer patients. AB - Early diagnosis of lung cancer based on conventional screening procedures has been unable thus far to decrease lung cancer mortality. We explored the possibility of using microsatellite instability in the detection and screening of early phases of lung carcinogenesis. We studied tumor, histopathologically normal bronchial mucosa, and cytological specimens of 51 lung cancer patients for the presence of clonal variations at microsatellite polymorphisms. Microsatellite alterations were found in tumor, normal bronchial mucosa and cytological specimens of 25 of 51 (49%) of the patients. The detection of microsatellite alterations in histopathologically normal bronchial specimens and cytological clinical samples with minimal atypia suggests a possible application of this genetic marker in early diagnosis of precancerous lesions and lung cancer. PMID- 8625301 TI - A truncated hMSH2 transcript occurs as a common variant in the population: implications for genetic diagnosis. AB - Germline mutations of the hMSH2 gene are responsible for many cases of hereditary nonpolyposis colorectal cancer. While screening for hMSH2 gene mutations in hereditary nonpolyposis colorectal cancer kindreds, we observed that a previously reported germline mutation is in fact a common, alternatively spliced variant in the population. Using RT-PCR and the protein truncation test, the hMSH2 exon 13 deletion variant was found in more than 90% of individuals. The exon 13 deletion transcript was only present in lymphocyte RNA, no abnormalities were detected in genomic DNA flanking exon 13, and the deletion transcript is apparently not translated. These findings highlight further that caution should be exercised in providing genetic risk assessment on the basis of currently used germline mutation detection strategies. PMID- 8625302 TI - BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines. AB - Malignant gliomas aggressively invade the surrounding normal brain, whereas brain metastases of nonglial tumors do not. The invasive behavior of gliomas may be mediated by tissue- or tumor-specific extracellular proteins. mRNA for the brain specific extracellular brain enriched hyaluronan-binding protein (BEHAB) is not detectable in normal adult human cortex or in any nonglioma tumor examined. BEHAB is consistently expressed in surgical samples of glioma (n = 27). Glioma cell lines maintained under standard cell culture conditions or grown as s.c. tumors do not express BEHAB. When grown as intracranial grafts, glioma cell lines that invade the brain express BEHAB, whereas noninvasive cell lines do not. BEHAB is a unique and selective marker for glioma and may play a role in tumor invasion. PMID- 8625303 TI - Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. AB - Mutations or loss of both alleles of the von Hippel-Lindau (VHL) tumor suppressor gene has been documented in sporadic renal cell carcinomas and neoplasms that arise in individuals having the VHL syndrome. The well-vascularized phenotype of tumors that form in VHL disease let us consider vascular endothelial growth factor (VEGF) as a mediator of tumor growth in VHL disease. Human renal carcinoma cells that either lacked endogenous wild-type VHL or were transfected with an inactive mutant VHL showed deregulated expression of VEGF on the mRNA and protein level that was reverted by introduction of wild-type VHL. Stimulation of proliferation of endothelial cells by conditioned medium of cells expressing mutant VHL was almost abolished by neutralizing the VEGF. In contrast, expression of basic fibroblast growth factor and of c-myc proto-oncogene was not affected by VHL. Our data suggest VEGF as the key tumor angiogenesis factor in VHL disease. PMID- 8625304 TI - Integrin signaling to NF-kappa B in monocytic leukemia cells is blocked by activated oncogenes. AB - Integrin-mediated signals play an important but poorly understood role in regulating the growth and behavior of tumor cells. In monocytes and monocytic leukemia cells, integrin-mediated adhesion results in a strong induction of a set of immediate early genes that are characteristic of monocytic differentiation and contain consensus NF-kappa B elements in their 5' regulatory regions. To investigate the role of integrin signaling in control of differentiation in a human monocytic leukemia cell line, THP-1 cells were transiently transfected with an NF-kappa B driven CAT reporter gene. Adhesion to fibronectin or cross-linking of beta1 integrins resulted in an NF-kappa B-dependent induction of CAT activity. To evaluate whether integrin signaling in this system intersects with the Ras signal transduction cascade, THP-1 cells were cotransfected with the NF-kappa B reporter and with plasmids that direct the synthesis of normal or mutant forms of Ras or Raf. We found that Ras or Raf dominant negative mutants did not inhibit integrin-mediated activation of the NF-kappa B-driven reporter. However, cotransfection with activated Ras, or with several other cytoplasmic oncogenes, blocked this process. This suggests that in monocytic leukemia cells, an antagonism exists between the mitogenic signals provided by oncogenes and the signals generated by integrin ligation. This antagonism may play an important role in regulating the balance between proliferation and differentiation in monocytic leukemias. PMID- 8625305 TI - Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention. AB - Oltipraz (OPZ) is currently being considered for human use to protect against aflatoxin B1 (AFB)-induced hepatocarcinogenesis based on its proven protective effect in rats. The effectiveness of this treatment presumes that orthologous cytochrome P450 and glutathione S-transferase (GST) isozymes metabolize AFB in humans as they do in rats. In this study, alterations in the expression of multiple forms of cytochrome P450 and GST were evaluated after treatment with OPZ, as well as other known P450 inducers, including 3-methylcholanthrene, pregnenolone-16alpha-carbonitrile, and ciprofibrate. Evidence is presented that the male-specific rat CYP 3A2, an orthologue of human CYP 3A4, may be primarily responsible for AFB activation in rat liver at both high and low AFB substrate concentrations. The CYP 1A2 enzyme does not appear to play a role in AFB activation in rat liver at any substrate concentration, whereas the major human P450 enzyme capable of activating AFB at a low substrate concentration has been identified as CYP 1A2. Surprisingly, we found that the CYP 1A2 steady-state mRNA level and the CYP 1A2-associated methoxyresorufin-O-demethylase activity were induced approximately 3- and 2-fold, respectively, by OPZ in rat liver. However, because CYP 1A2 does not appear to participate in AFB activation, induction of CYP 1A2 may be insignificant for AFB-induced hepatocarcinogenesis in rat models. In the rat, a heterodimeric alpha class GST enzyme containing the Yc2 subunit is the only polypeptide characterized to date in this species with high catalytic activity for the conjugation of activated AFB with glutathione. The GST Yc2 steady-state mRNA level was induced 5-fold by OPZ treatment. This induction was mirrored by significant increases in both the corresponding protein level and AFB 8,9-epoxide-conjugating enzyme activity, which may contribute significantly to protection against AFB-induced carcinogenesis in the rat. Investigations from this and other laboratories have not revealed any evidence for a Yc2-like GST isozyme with high AFB-8,9-epoxide-conjugating activity in human liver. We have also been unable to demonstrate that the two major human alpha class GST isozymes, A1-1 and A2-2, purified from bacteria expressing the corresponding cDNAs, exhibit any significant AFB-8,9-epoxide-conjugating activity. Our results suggest that humans may not be protected to the same extent as rats against AFB induced hepatocarcinogenesis by treatment with OPZ and that further investigations are needed to establish the usefulness of OPZ for protection against human exposure to AFB. PMID- 8625306 TI - Effect of amount and types of dietary fat on intestinal bacterial 7 alpha dehydroxylase and phosphatidylinositol-specific phospholipase C and colonic mucosal diacylglycerol kinase and PKC activities during stages of colon tumor promotion. AB - It is evident from many studies that the effect of dietary fat on colon tumor promotion depends not only on the amount of fat but especially on fatty acid composition. Animal model studies have shown that diets which are high in omega-6 fatty acids increase colon tumor promotion, whereas diets rich in omega-3 fatty acids have no such enhancing effect. The mechanisms by which the high fat content of the diet promotes colon carcinogenesis may include the production of secondary bile acids in the colon and the modulation of colonic luminal bacterial 7 alpha dehydroxylase that is involved in generating secondary bile acids, phosphatidylinositol-specific phospholipase C (PI-PLC), and mucosal PI-PLC, as well as diacylglycerol (DAG) kinase and protein kinase C (PKC). In the present study, we investigated the effect of high-fat diets that are rich in omega-3 and omega-6 fatty acids on cecal bacterial 7 alpha-dehydroxylase and PI-PLC, fecal secondary bile acids, and colonic mucosal DAG kinase and PKC activities during different stages of colon carcinogenesis in male F344 rats. At 5 weeks of age, groups of animals were fed a low-fat diet containing 5% corn oil (LFCO). Beginning at 7 weeks of age, all animals, except those intended as vehicle controls, received azoxymethane (AOM) s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body weight. Vehicle-treated groups received s.c. injections of normal saline. One day after the second AOM or saline treatment, the experimental groups of animals were transferred to a high-fat diet containing 23.5% corn oil (HFCO) or 20.5% fish oil + 3% corn oil (HFFO). One group continued on the LFCO diet. Animals were sacrificed at weeks 1, 12, and 36 after the AOM or saline treatment. Colonic mucosa were harvested at weeks 1, 12, or 36, and the colonic tumor tissues were examined for PKC and DAG kinase activities. Contents of the cecum were analyzed for bacterial 7 alpha-dehydroxylase and PI-PLC activities. Stool samples collected at week 12 were analyzed for bile acids. High corn oil content of the diet significantly increased the cecal bacterial 7 alpha dehydroxylase and PI-PLC activities as compared to the diets with high fish oil or low corn oil content. Animals fed the HFCO diet excreted higher levels of secondary bile acids, such as deoxycholic acid and lithocholic acid, than those fed the LFCO or HFFO diets. Carcinogen treatment significantly enhanced the activities of DAG kinase and total membrane PKC activities in colonic mucosa compared to saline treatment in all dietary groups. Animals treated with saline or AOM and fed HFCO showed increased levels of DAG kinase and membrane PKC activities in the colonic mucosa when compared to LFCO and HFFO groups. DAG kinase and membrane PKC activities were higher in colon tumors than in the surrounding colonic mucosa, and also increased levels of these enzyme activities were found in the HFCO diet group. These results indicate that the modifying effect of dietary fat on colonic bacterial enzymes, secondary bile acids, colonic mucosal and tumor DAG kinase, and PKC that may play a role in colon carcinogenesis depends on the types and amount of fat given. The colon tumor enhancing effect of a HFCO diet in contrast to the high dietary fish oil may be, in part, explained on the basis of its modulating effect on these bacterial and colonic mucosal enzymes and colonic secondary bile acids relevant to colon tumor promotion. PMID- 8625307 TI - Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines. AB - The expression and stability of the estrogen receptor (ER) is the result of a complex process that is modulated by estrogens and antiestrogens. Regulation of the steady-state ER mRNA and protein levels in breast cancer cells appears to be the result of either of two distinct regulatory mechanisms. Estrogen exposure causes a rapid down-regulation of the steady-state level of ER mRNA and protein in model I regulation, as exemplified by the MCF-7:WS8 cell line. Conversely, in model II regulation, as observed in the T47D:A18 cell line, estrogen exposure causes an increase in the steady-state ER mRNA level and a maintenance of the ER protein level. In both these cell lines, the nonsteroidal antiestrogen 4 hydroxytamoxifen has little effect on the mRNA level but causes a net accumulation of the ER protein over time. In contrast, the pure antiestrogen ICI 182,780 causes a dramatic reduction of the ER protein in both the MCF-7:WS8 and T47D:A18 cell lines. This loss has little effect upon the ER mRNA level in the MCF-7:WS8 cells but leads to a decline in the ER mRNA in the T47D:Al8 cells. The estrogen-independent MCF-7:2A cell line, which has adapted to growth in estrogen free media, expresses two forms of the ER, a wild-type Mr66,000 ER and a mutant Mr77,000 ER (ER77). ER77 is the product of a genomic rearrangement resulting in a tandem duplication of exons 6 and 7 (J. J. Pink et al, Nucleic Acids Res., 24:962 969,1996). This exon duplication has abolished ligand binding by this protein. Here we demonstrate that the loss of ligand binding has eliminated the effects of 4-OHT and ICI 182,780 on the steady-state ER77 protein level. However, in the MCF 7:2A cells, antiestrogens affect the wild-type ER protein in the same manner as observed in the MCF-7:WS8 and T47D:A18 cells. Estrogen regulates the ER mRNA and wild-type ER and ER77 proteins in the MCF-7:2A cells in the same manner as observed in the MCF-7:WS8 cells. Interestingly, treatment of the MCF-7:2A cells with ICI 182,780 causes a slight increase in ER mRNA, which is reflected in a net increase in the ER77 protein but a dramatic decrease in the wild-type ER. The models presented here describe the response of two human breast cancer cell lines in short-term studies. These distinct regulation pathways are predictive of the response of these cell lines to long-term estrogen deprivation. This study illustrates two alternative regulation pathways that are present in ER-positive, estrogen-dependent breast cancer cells. This variable response highlights the diversity of responses potentially present in the heterogeneous cell populations of clinically observed breast cancer. PMID- 8625308 TI - Adjunctive treatment of murine neuroblastoma with 6-hydroxydopamine and Tempol. AB - Currently available therapy for disseminated neuroblastoma affords only a 5-20% 5 year survival rate. We have attempted to design targeted chemotherapy for this disease by exploiting the dopamine uptake system on neuroblastoma cells. 6 Hydroxydopamine (6OHDA) is a neurotransmitter analogue, which generates cytolytic oxygen radicals in neuroblastoma cells that take it up. It is, however, predictably, systemically toxic, because of its spontaneous oxidation. Its toxicity is particularly severe in the sympathetic nervous system, because this tissue selectively concentrates dopamine and its analogues. Lowering the dose of 6OHDA below toxic levels prohibitively compromises its antitumor effect. To avoid both the systemic and sympathetic nervous system toxicity yet retain the antitumor efficacy of 6OHDA, we have used the antioxidant Tempol adjunctively with 6OHDA. Administration of Tempol (250 mg/kg, i.p.) 10 min prior to administration of toxic doses of 6OHDA (350 or 400 mg/kg, i.p.) resulted in a decrease in the mortality rate, sympathetic nervous system impairment, and activity impairment compared with those seen with 6OHDA alone. Tumor weights from mice administered saline or Tempol alone were 3.6 +/- 1.9 and 2.9 +/- 0.7 g, respectively. In contrast, mice administered Tempol followed by 6OHDA had an average tumor weight of 0.7 +/- 0.3 g. Tumor incidence was also reduced from 80 100% to 40%. Studies performed using electron spin resonance spectroscopy suggest that Tempol acts in this system by reacting directly with both the 6OHDA radical and, in the presence of iron, its oxidation product, the hydroxyl radical. PMID- 8625309 TI - Retroviral transfer of deoxycytidine kinase into tumor cell lines enhances nucleoside toxicity. AB - Deoxycytidine kinase (dCK) phosphorylates a number of nucleoside analogues that are useful in the treatment of various malignancies. Although the level of dCK activity in malignant cells is thought to correlate with chemotherapeutic response, no direct data are available to support this assumption. We have tested this hypothesis by infecting three tumor cell lines, MCF-7, HT-29, and H1437, with the retroviral vector LNPO containing either dCK or LacZ cDNA and measuring the corresponding sensitivity to nucleoside analogues. DCK activity was increased by 1.7-, 2.3-, and 16-fold in MCF-7, HT-29, and H1437 cells, respectively. Northern and Western blots demonstrated a similar increase in mRNA and protein levels. As a result of dCK expression, MCF-7 cells demonstrated a 2.5-fold increase in drug sensitivity to 1-beta-D-arabinofuranosylcytosine (AraC) and 2 chloro-2'-deoxyadenosine (CdA). HT-29 cells had a 7-fold increase in sensitivity to AraC, CdA, and 2-fluoro-9-beta-D-arabinofuranosyladenine, whereas H1437 cells demonstrated a 20- to 106-fold increase. For all three drugs, there was a linear relationship between dCK activity in clonally derived cell lines and IC50s. These data demonstrate a direct effect of dCK activity on drug sensitivity in cell lines. Because many tumors have relatively low levels of dCK, it is possible that dCK gene transfer will be a useful adjunct to the treatment of these malignancies. PMID- 8625310 TI - Drug-induced down-regulation of topoisomerase I in human epidermoid cancer cells resistant to saintopin and camptothecins. AB - The anticancer agent saintopin induces DNA cleavage mediated by both topoisomerase (topo) I and topo II in vitro through stabilization of the reversible enzyme-DNA cleavable complex. We established saintopin-resistant cell lines (KB/STP-1 and KB/STP-2) from human epidermoid cancer KB cells by stepwise exposure to increasing doses of the drug. KB/STP-1 and KB/STP-2 cells showed 12- and 44-fold increases, respectively, in resistance to saintopin relative to that of KB cells. Both saintopin-resistant cell lines showed only small reductions in sensitivity to the topo II inhibitor etoposide but developed marked cross resistance to the topo I-targeting camptothecin derivative CPT-11 [(4s)-4,11 diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbony loxy] dione hydrochloride trihydrate] and its active form, SN-38 (7-ethyl-10-hydroxycamptothecin). In contrast, both KB/STP-1 and KB/STP-2 cells showed increased collateral sensitivity to cisplatin, a nitrosourea derivative, mitomycin C, and UV light. The protein concentration, activity, and mRNA abundance of both topo I and topo II were similar in KB/STP-1, KB/STP-2, and the parental KB cells. There were no significant changes in the drug-stabilized topo-DNA cleavable complex formation in KB and KB/STP-2 cells. Two point mutations were detected in topo I cDNA from KB/STP-2 cells, but these were also present in KB cells. Topo I mRNA abundance decreased markedly immediately after exposure of KB/STP-2 cells to saintopin; no such effects were apparent in KB cells. In contrast, topo II mRNA was not markedly affected by saintopin in either KB or KB/STP-2 cells. Treatment with CPT 11 or SN-38 also induced a markedly greater and more persistent reduction in topo I mRNA abundance in KB/STP-2 cells than in KB cells. Etoposide had no marked effect on topo I mRNA abundance in either KB/STP-2 or KB cells. Topo I mRNA was highly unstable in KB/STP-2 cells in comparison to KB cells when incubated with saintopin. This novel regulation of topo I mRNA by topo I-targeting agents could be associated with acquirement of drug resistance to saintopin or SN-38/CPT-11 in KB/STP-2 cells. PMID- 8625311 TI - Photodynamic therapy-mediated oxidative stress can induce expression of heat shock proteins. AB - Photodynamic therapy (PDT) is an experimental cancer therapy inducing tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. A previous report indicates that oxidative stress induced by hydrogen peroxide or menadione activates the heat shock transcription factor in mouse cells but does not result in either increased transcription or translation of heat shock proteins (HSPs). Our study documents that photosensitizer-mediated oxidative stress can activate the heat shock factor as well as increase HSP-70 mRNA and protein levels in mouse RIF-1 cells. The cellular heat shock response after PDT varied for the different photosensitizers being examined. Treatments using either a chlorin (mono-L aspartyl chlorin-e6)- or purpurin (tin etio-purpurin)-based sensitizer induced HSP-70 expression, whereas identical photosensitization conditions with a porphyrin (Photofrin)-based sensitizer failed to induce a cellular HSP response. These sensitizers, which generate singlet oxygen as the primary oxidant during photosensitization, were used in experiments under isoeffective treatment conditions. HSP-70 expression after photosensitization was associated with the concomitant induction of thermotolerance in PDT-treated cells. Interestingly, reverse transcription-PCR demonstrated that in vivo PDT treatments of RIF-1 tumors induce expression of HSP-70 for all photosensitizers including Photofrin. These results indicate that photosensitizer-generated singlet oxygen exposure can induce in vitro and in vivo HSP-70 expression, and that specific subcellular targets of PDT (which can differ for various sensitizers) are determinants for HSP-70 activation after oxidative stress. PMID- 8625312 TI - Improved immunotherapy of a recombinant carcinoembryonic antigen vaccinia vaccine when given in combination with interleukin-2. AB - Interleukin-2 (IL-2) has been an effective immune modulator in several active specific immunotherapy experimental protocols using either viral or oncolysate based vaccines. In this report, data indicate that IL-2 administration can appreciably augment the therapeutic effect of a single immunization of a recombinant vaccinia virus-carcinoembryonic antigen (rV-CEA) vaccine using a CEA expressing syngeneic experimental murine model system. A single rV-CEA immunization of C57BL/6 mice bearing palpable CEA-positive colon adenocarcinoma tumors results in complete tumor regression in approximately 20% of the mice. The addition of a course of low-dose IL-2 results in complete tumor regression in 60 70% of the mice. Moreover, the combination of rV-CEA and IL-2 induces systemic immunity, which protects those tumor-free mice from subsequent rechallenge with the CEA-expressing tumor cells. No such tumor regression or protection was observed in those mice immunized with the wild-type vaccinia vaccine (V-Wyeth) alone or with IL-2 administration alone. Cellular immune assays revealed that the addition of IL-2 to rV-CEA immunization significantly increased the CEA-specific T-cell proliferative responses as well as the cytolytic T-cell responses when compared with rV-CEA immunization alone. The enhanced CEA-specific immune response, coupled with the improved experimental therapeutic outcome following IL 2 administration, suggests that treatment with that cytokine may effectively substitute for multiple rV-CEA immunizations in active-specific immunotherapy clinical protocols directed at CEA-expressing tumors. PMID- 8625313 TI - Dual recognition of a human cytotoxic T-cell clone for melanoma antigens. AB - It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tumor antigens in MHC restricted and MHC-unrestricted manners. We have established two alpha beta CTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2 restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CD8, and alpha beta T-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single alpha beta TCR. Both CTL clones contained the same Valpha and Vbeta gene segments although they carried different Jalpha and Jbeta gene segments. Taken together, these results confirm that CTL clones that carry a single alpha beta TCR recognize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo. PMID- 8625314 TI - Mechanisms for the involvement of DNA methylation in colon carcinogenesis. AB - C --> T transitions at CpG sites are the most prevalent mutations found in the p53 tumor suppressor gene in human colon tumors and in the germline (Li-Fraumeni syndrome). All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the majority of these mutations are caused by spontaneous hydrolytic deamination of this base to thymine. We have previously reported that bacterial methyltransferases induce transition mutations at CpG sites by increasing the deamination rate of C --> U when the concentration of the methyl group donor S-adenosylmethionine (AdoMet) drops below its Km, suggesting an alternative mechanism to create these mutations. Unrepaired uracil pairs with adenine during replication, completing the C --> T transition mutation. To determine whether this mechanism could contribute to the development of human colon cancer, we examined the level of DNA (cytosine-5)-methyltransferase (MTase) expression, the concentration of AdoMet, and the activity of uracil-DNA glycosylase in human colon tissues, and searched for the presence of mutations in the MTase gene. Using reverse transcription-PCR methods, we found that average MTase mRNA expression levels were only 3.7-fold elevated in tumor tissues compared with surrounding normal mucosa from the same patient. Also, no mutations were found in conserved regions of the gene in 10 tumors sequenced. High performance liquid chromatographic analysis of extracts from the same tissues showed that AdoMet concentrations were not reduced below the Km value for the mammalian enzyme, and the concentration ratio of AdoMet:S-adenosylhomocysteine, the breakdown product of AdoMet and the competitive MTase inhibitor, did not differ significantly. Finally, extracts from the tumor tissue efficiently removed uracil from DNA. Therefore, biochemical conditions favoring a mutagenic pathway of C --> U --> T were not found in a target tissue known to undergo a high rate of C --> T transitions at CpG sites. PMID- 8625315 TI - Different genetic susceptibility to aberrant crypts and colon adenomas in mice. AB - Aberrant crypt foci (ACFs) are the earliest identifiable epithelial lesions thought to precede the development of a subset of colon tumors. To assess their predictive value to adenoma development, we have tested in mice whether the development of ACFs and adenomas is controlled by the same genes. Therefore we used the CcS/Dem series of recombinant congenic strains, in which the effect of multiple susceptibility genes might be studied separately. We investigated susceptibility to ACFs in nine CcS/Dem strains and their parental strains, BALB/cHeA and STS/A, 4 weeks after s.c. injection of 1,2-dimethylhydrazine (20 mg/kg body weight). For the strains BALB/cHeA, STS/A, and CcS-19, we also examined the number of ACFs 2, 8, and 12 weeks after treatment. Susceptibility to adenomas was measured as the number of adenomas 6 weeks after 26 weekly s.c. injections of 1,2-dimethylhydrazine (15 mg/kg body weight). The nine CcS/Dem strains, the BALB/ cHeA strain, and the STS/A strain exhibited different patterns of susceptibility to ACFs and adenomas, demonstrating that different subsets of susceptibility genes are involved. Therefore, in evaluating the role of ACFs as a predictive marker for adenoma development, genetic factors must be taken into account. PMID- 8625316 TI - Ras-interacting domain of Ral GDP dissociation stimulator like (RGL) reverses v Ras-induced transformation and Raf-1 activation in NIH3T3 cells. AB - Ral GDP dissociation stimulator (RalGDS) and RalGDS like (RGL) are putative effector proteins of Ras and contain the Ras-interacting domain (RID) at their C terminal regions. v-Ras is known to activate c-fos promoter/enhancer and Raf-1 and to transform NIH3T3 cells. It is also known that v-Raf activates c-fos promoter/enhancer and transforms NIH3T3 cells. In this study, we examined the effect of RID on the phenotype of the cells transformed by v-Ras and v-Raf. Overexpression of RID greatly reduced cell growth in low serum, colony-forming activity in soft agar, c-fos promoter/enhancer activity, and Raf-1 activity of v Ras-transformed cells. However, overexpression of RID did not affect the phenotype of v-Raf-transformed cells. These results clearly indicate that RID of RGL specifically binds to Ras in mammalian cells, that it blocks the signal from Ras to Raf-1, and that it reverses v-Ras-induced malignant phenotype. It has been reported that Ras-binding domains of Raf-1 and neurofibromatosis type 1 (NF1) reverse v-Ras-induced malignant phenotype. Since there is no homology in primary structures of RGL, Raf-1, and NF1, there may be a similarity of secondary or tertiary structure among RID of RGL and Ras-binding domains of Raf-1 and NF1, and the structure might be useful for developing a potential medicine for human cancers caused by Ras. PMID- 8625318 TI - p27/Kip1 mutation found in breast cancer. AB - The p27/Kip1 protein belongs to the recently identified family of proteins called cyclin-dependent kinase inhibitors. These proteins play an important role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Since cyclin-dependent kinase inhibitors can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine whether p27 alterations may be involved in tumorigenesis, we examined its mutational status in 36 primary breast carcinomas and 9 breast cancer cell lines using PCR-single-strand conformational polymorphism, direct DNA sequencing, and Southern blot analysis. Southern blot analysis showed no homozygous deletions of the p27 gene in either the clinical samples or cell lines. Two point mutations were found in primary tumors. One represents a previously undescribed polymorphism at codon 142; another is a nonsense mutation at codon 104. The latter mutation was absent in the normal matched control sample, and, in addition, it was accompanied with the loss of heterozygosity (LOH) of a microsatellite marker in the vicinity of the p27 gene on chromosome 12p13. These data indicate that p27 mutations are a rare event in breast cancer, but may play an important role in the development of a minority of these cancers. Furthermore, LOH analysis of the 12p13 locus revealed that an additional four of six matched DNA samples had LOH at 12p13 but did not have an alteration of the p27 gene, suggesting that another tumor suppressor gene is located on the short arm of human chromosome 12 which may be frequently involved in the pathogenesis of breast cancers. PMID- 8625317 TI - Increased frequency of specific genomic deletions resulting from in vitro malathion exposure. AB - Malathion is a widely used pesticide with high potential for human exposure. Epidemiological studies suggest that individuals with chronic environmental exposures to pesticides have increased risks of various hematological malignancies. The genotoxic data to date have been somewhat inconclusive with regard to malathion exposure. We have used a cell cloning assay to study the genotoxicity of in vitro exposure of human T lymphocytes to malathion. We exposed cells in G0 to doses of malathion ranging from 10 to 600 microg/ml. Mutant frequencies of treated samples showed both intra- and interindividual variability and, in some cases, slight significant increases over the controls. Molecular analysis of hprt mutants resulting from both in vitro and an in vivo malathion exposure was performed by genomic multiplex PCR. In seven in vitro experiments (using cells from four different individuals) and one experiment on an individual exposed in vivo, one or more independent mutant(s) containing a partial deletion of exon 3 have been isolated from each individual. In five of the seven mutants, the deleted regions overlap extensively, revealing an area within exon 3 exceptionally prone to deletions upon exposure to malathion, This work provides the first evidence of an association between malathion exposure and specific mutations in human T lymphocytes. Additional work is necessary to determine the underlying molecular mechanism for these deletions and how this may relate to agricultural workers' increased risk of cancer. PMID- 8625319 TI - Silencing of p16/CDKN2 expression in human gliomas by methylation and chromatin condensation. AB - The product of the p16/CDKN2 locus, p16ink4, negatively regulates the cell cycle through binding and inactivation of cyclin-dependent kinases (CDKs) 4 and 6. This locus is frequently targeted for deletion in cell lines and primary tumor tissues. In gliomas, although up to 50% do not have detectable expression of p16/CDKN2 protein or mRNA, often the gene is wild type in sequence. Here, we tested the hypothesis that transcriptional repression of p16/CDKN2 in gliomas may be mediated by aberrant methylation of the CpG island, which is in the 5' region of the locus. Partial rather than complete p16/CDKN2 methylation was detected in 24% (10 of 42) of the gliomas, regardless of tumor grade, but was not observed in normal brain (0 of 10). We tested whether this partial methylation could inhibit expression in a human tumor cell line in which suppressed p16/CDKN2 expression was associated with both methylation and tightly compacted chromatin around the p16/CDKN2 promoter. Exposure of these cells to 5-aza-2-deoxycytidine resulted in a dramatic increase in promoter accessibility and induction of p16/CDKN2 expression, indicating that chromatin structure, CpG island methylation, and p16/CDKN2 expression are intimately associated. Taken together, these data suggest that methylation occurs in only a subset of cells within gliomas and that the methylation-associated inactivation of p16/CDKN2 expression observed in many common human cancers may mechanistically result from structural changes in the chromatin containing the p16/CDKN2 locus. PMID- 8625320 TI - Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21. AB - To investigate the possible involvement of a tumor suppressor gene(s) on chromosome 8 in prostatic neoplasms, we performed a comprehensive loss of heterozygosity (LOH) study on 99 tumors from 97 prostate cancer patients. One of the carcinomas was a lymph node metastasis; the other 98 were primary carcinomas. Pure populations of carcinoma cells and normal epithelia were procured by tissue microdissection. Two separate tumor foci were obtained from each of two patients. Microsatellite markers from 25 loci on the short arm and one locus on the long arm of chromosome 8 were used for PCR-based LOH analysis on matched normal and tumor DNA samples. The overall LOH on 8p in this study was 85.9% (85 of 99) of carcinomas. The loss was highest at markers D8S133, D8S136, NEFL, and D8S137 (62,72, 64, and 75%, respectively), which are located at 8p12-21. Seventy-nine of 99 tumors exhibited loss in at least one of these four loci. In contrast, LOH at 8p22 was much lower: 17,18,18, and 19% at D8S549, D8S602, D8S254, and D8S261, respectively, with 25 of 99 tumors showing deletion in one or more of the four loci. All but 5 tumors with deletions in this more distal region had at least one retained locus between the 8p22 deletion and a more proximal region of loss at 8p12-21; 1 tumor had loss at 8p22 but not 8p12-21. This suggests there may be two distinct regions of loss and, therefore, two tumor suppressor genes on this chromosomal arm. The loss on 8p12-21 showed little or no correlation with grade or stage of disease. PMID- 8625321 TI - Identification of the lysyl oxidase gene as target of the antioncogenic transcription factor, IRF-1, and its possible role in tumor suppression. AB - The transcriptional activator IFN regulatory factor 1 (IRF-1) and its antagonistic repressor IRF-2 are regulators of the IFN system. IRF-1 also manifests tumor suppressive activity, and its inactivation could contribute to the development of human hematopoietic malignancies. Here, we report the identification of the lysyl oxidase gene as a target gene of IRF-1. An IRF response element was identified in the lysyl oxidase gene promoter. We also demonstrate that the transformed phenotype of ras-expressing embryonic fibroblasts with a null mutation in the IRF-1 allele could be suppressed by the expression of the lysyl oxidase cDNA, implicating its potential role in tumor suppression. Thus, the regulation of the lysyl oxidase gene by IRF-1 could contribute to the multistep process of malignant transformation. PMID- 8625322 TI - Elevated expression of Bcl-X and reduced Bak in primary colorectal adenocarcinomas. AB - Expression of several members of the BCL-2 family of genes was investigated by immunohistochemical methods in 30 primary colorectal adenocarcinomas and 24 adenomatous polyps. When compared to the intensity observed in adjacent normal mucosal epithelial cells, the intensity of Bcl-X immunostaining was elevated in 18 of 30 (60%) carcinomas (P = 0.0001) and 12 of 24 (50%) adenomatous polyps (P = 0.0001). Immunoblot analysis of five pairs of tumors and adjacent normal colonic tissue indicated marked elevations in the relative levels of the anti-apoptotic Bcl-XL, protein in all cases. In contrast to the increased Bcl-X expression, the intensity of Bcl-2 immunostaining was greater than that of normal colonic mucosa in only 3 of 30 (10%) carcinomas and, in fact, was lower than that of adjacent normal epithelia] cells in 25 (83%) cases (P = 0.0001). Furthermore, the percentage of Bcl-2 immunopositive cells was generally lower in carcinomas than in adenomas (mean +/- SE, 44 +/- 6% versus 73 +/- 5%, respectively; P = 0.001) and in moderately or poorly differentiated tumors than in well-differentiated tumors (39 +/- 6% versus 70 +/- 11%, respectively; P = 0.045). In addition, the proportion of tumors in which the Bcl-2 immunointensity was more than or equal to that of normal colonic mucosa was significantly lower in carcinomas than adenomas (5 of 30 versus 15 of 24, respectively; P < 0.001), suggesting that decreases in Bcl-2 expression represent a later event associated with the progression of colorectal cancers. When compared to that of normal adjacent colonic epithelium, the intensity of Mcl-1 immunostaining was reduced in 20 of 30 (67%) of carcinomas (P = 0.0001) compared to only 1 of 24 adenomas, suggesting that decreases in Mcl 1 expression represent a later event associated with progression from a benign to a malignant phenotype or with transition to a less-differentiated state, because most of the carcinomas evaluated here (25 of 30; 83%) were not well differentiated. The intensity of immunostaining for the pro-apoptotic protein Bak was reduced compared to that of normal mucosal epithelial cells in 27 of 30 (90%) carcinomas and 22 of 24 (92%) adenomas, suggesting that reductions in Bak expression occur early in colorectal tumor progression (P = 0.0001). In contrast, the intensity of immunostaining for the pro-apoptotic protein Bax was not significantly altered in carcinomas; compared to that of normal colonic mucosa, Bax immunointensity was reduced in only 7 of 30 (23%) carcinomas and 3 of 24 (13%) adenomas, and the percentage of Bax immunopositive cells was also not significantly different in any of the histological subgroups. Taken together, these results suggest that expression of Bcl-XL is increased in undifferentiated primary colorectal cancers, often with accompanying reciprocal decreases in the anti-apoptotic proteins Bcl-2 and Mcl-1 and the pro-apoptotic protein Bak, whereas Bax expression is relatively constant. Thus, a shift from expression of the anti-apoptotic proteins Bcl-2 and Mcl-1 to the Bcl-XL protein may occur during progression of colorectal tumors. PMID- 8625323 TI - Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. AB - Urokinase plasminogen activator (uPA) and its receptor are key components of a cell surface proteolytic cascade used by tumor cells and capillary endothelial cells for basement membrane invasion, a process required for metastasis and angiogenesis. We have cloned, expressed, and purified the epidermal growth factor like domain of murine uPA alone and fused it to the Fc portion of human IgG as high-affinity murine urokinase receptor antagonists. These molecules are potent inhibitors of murine urokinase binding to its receptor and inhibit angiogenesis in an in vitro model of capillary tube formation in fibrin gels. In vivo, basic fibroblast growth factor-induced neovascularization and B16 melanoma growth in syngeneic mice are also substantially suppressed by these molecules. Coupled with previous studies showing inhibition of metastasis, these findings suggest that urokinase receptor antagonists may be useful therapeutically as inhibitors of tumor progression. PMID- 8625324 TI - Multidrug resistance and malignancy in human osteosarcoma. AB - In osteosarcoma, resistance to chemotherapy and metastatic spread are the most important mechanisms responsible for the failure of current multimodal therapeutic programs. We have shown previously that overexpression of the MDR1 gene product P-glycoprotein is the most important predictor of an adverse clinical course in patients with osteosarcoma. treated with chemotherapy. In this study, we analyzed the relationship between P-glycoprotein expression and local aggressiveness and systemic dissemination of multidrug-resistant (MDR) human osteosarcoma cells. Compared to parental sensitive cells, MDR cells showed a decreased tumorigenicity,and metastatic ability in athymic mice, together with a reduced migratory and invasive ability and a lower homotypic adhesion ability in vitro, suggesting that P-glycoprotein overexpression is associated with a less malignant phenotype. These experimental observations were confirmed by clinical data. In fact, the time of appearance of lung metastases in a series of osteosarcoma patients treated with chemotherapy was significantly shorter in the group of cases with no expression of P-glycoprotein in the primary lesion compared to the group with P-glycoprotein overexpression. Moreover, the incidence of P-glycoprotein overexpression was found to be higher among patients with localized disease at the clinical onset than in patients with evidence of metastasis at the time of diagnosis. These data indicate that, in osteosarcoma, the MDR phenotype is not associated with a more aggressive behavior both in vitro and in clinical settings, suggesting that the previously shown association of the MDR phenotype with a worse outcome in osteosarcoma is not related to a higher metastatic ability of cells with P-glycoprotein overexpression but is more likely due to their lack of responsiveness to cytotoxic drugs. PMID- 8625325 TI - Introduction of basic fibroblast growth factor gene into mouse renal cell carcinoma cell line enhances its metastatic potential. AB - To clarify the role of basic fibroblast growth factor (FGF-2) in the malignant progression of renal cell carcinoma, we transfected the FGF-2 gene, which lacks the typical signal sequence, into RenCa, a mouse renal cell carcinoma cell line that does not express FGF-2 mRNA. In an in vitro tumor cell invasion assay, the FGF-2-transfected cell lines (RenCa/F) exhibited 3- to 4-fold higher invasive potential than either the parental RenCa (RenCa/P) or the vector-only transfected cell line (RenCa/C). Zymography showed a marked increase in matrix metalloproteinase 2 (MMP-2) production in the culture supernatants of RenCa/F. Furthermore, when injected i.v. or into the renal subcapsule in syngeneic mice, RenCa/F formed more than 10 times as many metastatic nodules in the lung as did RenCa/P and RenCa/C. Metastases to the liver and mesenteric lymph nodes were observed only after the injection of RenCa/F into the renal subcapsule. In contrast, there was no significant difference in either cell proliferation in vitro or tumor growth in vivo among RenCa sublines. These results suggest that if it is overexpressed, endogenous native FGF-2 plays an important role in the invasion and metastasis of renal cell carcinoma, probably through the production of MMP-2. PMID- 8625326 TI - Effect of overexpression of the small heat shock protein HSP27 on the heat and drug sensitivities of human testis tumor cells. AB - In contrast to most metastatic cancers, testicular germ cell tumors are cured in more than 80% of patients using cisplatin-based combination chemotherapy. Testis tumor cells in vitro retain their sensitivity to chemotherapeutic drugs, radiation, and other stresses, such as heat shock. Having shown that this is associated with low constitutive levels of heat shock protein (HSP) 27, we determined the effect of overexpression of HSP27 on the heat and drug sensitivities of a human testis tumor cell line, 833K. Cells were cotransfected with plasmids containing a neomycin resistance gene and the full-length human HSP27 gene, and four clones that overexpressed HSP27 by factors of 3.7-38.3-fold compared with the parental cells were selected. The overexpressing cells were more resistant to heat shock, cisplatin, and doxorubicin, and this was associated with modest increases (17-30%) in population doubling times and a small reduction in the number of S-phase cells. These results suggest that the low constitutive levels of HSP27 in testis tumor cells may contribute to the sensitivity of testicular germ cell tumors to chemotherapy, and that targeting HSP27 may improve response rates in other types of cancer. PMID- 8625327 TI - Tumor necrosis factor alpha interferes with the cell cycle of normal and papillomavirus-immortalized human keratinocytes. AB - We have shown that normal and human papillomavirus (HPV) type 16 immortalized human foreskin keratinocytes are growth inhibited by tumor necrosis factor alpha (TNF-alpha), whereas HPV-18- and SV40-immortalized keratinocytes are resistant to this cytokine (1). In this report, we investigated the expression of mitotic regulatory proteins, such as cyclin A, cyclin B, and p34cdc2. After exposure to TNF-alpha, normal and HPV-16-immortalized cells exhibited a dramatic decrease in the expression of these proteins. In contrast, no alteration in the levels of these proteins was observed after treatment of the resistant cell lines, as well as two HPV-positive cervical carcinoma cell lines. Expression of cyclin E does not seem to be modulated by TNF-alpha in any of the cells tested. On the other hand, cyclin D1, expression is slightly increased in normal keratinocytes and in the HPV-16-immortalized cells, whereas no alteration was observed in the HPV-18 transfected cells. The phosphorylation state of pRb correlated with cell growth; sensitive cells, which accumulate in G0-G1, after exposure to TNF-alpha, exhibited an accumulation of hypophosphorylated pRb, whereas no effect on pRb phosphorylation was observed for HPV-18-immortalized cells. These results clearly correlate with TNF-alpha-induced growth arrest in G0-G1. PMID- 8625329 TI - Chronic lymphocytic leukemia. PMID- 8625328 TI - Augmentation of the therapeutic activity of lometrexol -(6-R)5,10 dideazatetrahydrofolate- by oral folic acid. AB - Recent clinical trials with lometrexol [(6R)-5,10-dideazatetrahydrofolate] have revealed a level of toxicity in humans that was not predicted on the basis of previous in vivo preclinical studies. Because standard laboratory animal diets contain high levels of folic acid relative to human folate intake, the toxicity and therapeutic activity of lometrexol was studied in mice under conditions of restricted dietary folate intake. Remarkably, the lethality of this drug increased by three orders of magnitude in mildly folate-deficient mice, mimicking the unexpected toxicity seen in humans. Lometrexol had limited therapeutic activity in folate-deficient mice bearing the C3H mammary adenocarcinoma, compared with the substantial therapeutic index for treatment of this tumor in animals on standard diet. When folic acid was administered p.o. to mice that were mildly folate deficient, antitumor activity was again observed at nontoxic doses of lometrexol, and the range of lometrexol doses that allowed safe therapeutic use of this drug increased at higher dietary folate intake. At a fixed dose of lometrexol, the antitumor effects in animals were dependent on the level of dietary folate and went through a distinct optimum. Excessively high folate intake reversed the antitumor effects of lometrexol. Optimization of the folic acid content in the diet and of the lometrexol dosage are predicted to have substantial impact on the clinical activity of this class of drugs. PMID- 8625330 TI - Clinical, toxicological and pharmacological aspects of gemcitabine. PMID- 8625331 TI - Hemolytic anemia and cancer. PMID- 8625332 TI - The role of the occupational therapist in oncology. PMID- 8625333 TI - Estimating the cost-effectiveness of flutamide in metastatic cancer. PMID- 8625334 TI - Advantages of laparoscopic palliative surgery in upper GI tract cancer. PMID- 8625335 TI - Palliative treatment for advanced gastrointestinal cancer: is response a suitable end-point? PMID- 8625336 TI - Asthenia: an important symptom in cancer patients. PMID- 8625337 TI - The use of the Hospital Anxiety and Depression Scale (HADS) and the EORTC QLQ-C30 questionnaires to screen for treatable unmet needs in patients attending routinely for radiotherapy. PMID- 8625338 TI - Assessment of values, utilities and preferences in cancer patients. PMID- 8625339 TI - Capsules and suppositories of methadone for patients on high-dose opioids for cancer pain: clinical and economic considerations. AB - The dose ratio, analgesic efficacy, toxicity and cost of methadone for cancer pain were evaluated in a retrospective review of 50 consecutive patients treated on a Palliative Care Unit. Patients were switched from hydromorphone 267.7 +/- 178.8 mg sc per day to custom-made capsules (24) or suppositories (26) of methadone for reasons of uncontrolled pain (24), toxicity (8), both (15) or other (2). The change in opioid occurred over 2.5 +/- 3.6 days, with another 4.4 +/- 3.7 days required to reach stable methadone dose. The ratio of stable methadone dose to final hydromorphone dose was 1.07 +/- 0.9 (oral) and 1.88 +/- 1.27 (rectal) (p = 0.01). Visual analogue scores for pain intensity (0-100 mm) declined from 50.8 +/- 22 to 40 +/- 20 (p = 0.01). The most frequent toxicities were constipation (46), sedation (42) and nausea (18). Six patients developed respiratory depression. Total cost of treatment was Canadian $116.77 +/- 157.17 for methadone capsules and Canadian $105.34 +/- 146.35 for methadone suppositories, vs Canadian $3450.51 +/- 5098.58 (p = 0.0001) for hydromorphone parenteral solution and Canadian $1801.21 +/- 2661.52 (p = 0.0001) for hydromorphone powder. It is concluded that methadone is an effective and inexpensive alternative in patients receiving high-dose opioids for cancer pain, at dose ratios much lower than recommended in the literature. The incidence of serious toxicities suggests that methadone should only be initiated in an adequately monitored setting by pain management experts. PMID- 8625340 TI - Patient-controlled analgesia (PCA) in the domiciliary care of tumour patients. AB - Patient-controlled analgesia (PCA) was administered in the domiciliary environment in 143 pre-terminally and terminally ill tumour patients suffering either from excruciating chronic pain or severe chronic/acute complex pain that could not be relieved adequately by oral analgesia. Morphine solutions were infused subcutaneously in concentrations between 1% and 3%. The intravenous route was preferred in patients with indwelling catheters or those susceptible to inflammatory skin reactions at the infusion site. After initial dose adjustment, lasting 2-3 days, the morphine amounts infused by PCA reached a median of 93 mg day(-1) (range 12-464 mg day(-1)). The median was 28% lower than the median dose administered orally. A total of 84% of patients utilized the option of bolus self administration. The median percentage administered via the bolus mode amounted to 5.3% of the total requirements. During the course of treatment, morphine requirements increased by a median of 2.3 mg day(-1) (range -29 +52 mg day(-1)). Most patients were treated continuously in the home care setting until death, the median duration being 27 days (range 1-437 days). The terminal morphine demands reached a median of 188 mg day(-1) (range 15-1008 mg day(-1)). PCA turned out to be safe and effective, attaining excellent results in 95 (66%) patients and satisfactory pain relief in 43 (30%). PCA proved to be insufficient in five (4%) cases. Side-effects were mild: constipation, fatigue, nausea and local inflammatory skin reactions occurred in 9%. Thus, with support from an experienced mobile nursing team, PCA can be safely administered in the terminal domiciliary care of tumour patients. PCA is superior to oral analgesia, especially in the treatment of severe oscillating pain. PCA provides adequate pain control in about 96% of patients who are poorly responsive to oral opioids. PMID- 8625341 TI - Factors influencing Swedish doctors' decision-making in the care of incompetent elderly patients. PMID- 8625342 TI - The delivery of palliative care. PMID- 8625343 TI - The medical mind of the Middle Ages in eleventh century Mediterranea. PMID- 8625344 TI - Perspectives of palliative medicine. PMID- 8625345 TI - Dimensions of quality control in oncology. PMID- 8625346 TI - Patient assessment in palliative cancer care. PMID- 8625347 TI - Treatment intention in hospitalized cancer patients in oncological wards in Norway: a national survey. AB - Clinicians often do not agree whether a treatment is given with a palliative or curative intent. A common clear definition does not exist. This study has assessed the usefulness of dividing the goal of treatment into three distinct categories: curative treatment; palliative, symptom preventive treatment; and palliative, symptom relieving treatment. In a cross-sectional study among all cancer centres in Norway, a total of 629 patients were included into the study. Of these patients, 60% received palliative treatment, with an equal distribution between symptom preventive and symptom relieving. The definitions were found easy to use by the physicians. It gave important information of differences between cancer diagnosis with respect to the number of patients receiving palliative treatment. In order to refine the classification system, the authors will, in future studies, include a fourth category, life prolonging treatment, which is located between curative and palliative treatment with respect to treatment intensity. PMID- 8625349 TI - Clinical trials vs socio-economic trials. PMID- 8625348 TI - Quality assurance by specification and achievement of goals in palliative cancer treatment. AB - As the goals of palliative cancer treatments have not always been clearly specified, this paper describes how frequently the goals of palliative cancer treatment can be specified according to a given definition and how frequently those specified goals can be achieved. The clinical problems of 171 cancer patients were discussed in the Interdisciplinary Oncologic Conference (IOC) of the Cancer Centre University of Ulm (CCUU) and recommendations concerning further diagnostic treatments and/or therapy were provided. These recommendations had been documented and analysed retrospectively. The goals were classified as either cure or palliation or further investigation. If the goal was palliation, it was investigated whether or not the goal was specified as either alleviation of existing problems or prevention of impending problems. The achievement of the specified goals was assessed. Palliation was the goal of treatment in 119 (71%) of the 168 evaluable recommendations. In 83 of the 119 cases (70%), immediate treatment was recommended. The goal was specified in 57 (69%) of the 83 recommendations and could be realized in 24 of 57 specified cases (42%). Patients in this group survived longer (p < 0.01) than patients in whom the goals could not be achieved. Impending problems could be prevented more often (p = 0.001) in 14 out of 18 cases, while existing problems could be alleviated in only 10 out of 34 cases. It is concluded that specification of the goals of palliation is necessary because it is impossible to decide if a goal of treatment could be achieved or not unless the goal of treatment has been defined (as existing/impending problem). The prevention of impending problems could be investigated in prospectively controlled clinical trials. PMID- 8625350 TI - Response-shift bias: a challenge to the assessment of patients' quality of life in cancer clinical trials. PMID- 8625352 TI - Rationale for the timing of health-related quality-of-life (HQL) assessments in oncological palliative therapy. PMID- 8625351 TI - On the receiving end. VI. Which dimensions of quality-of-life scores carry prognostic information? PMID- 8625353 TI - Practical aspects of quality-of-life measurement: design and feasibility study of the quality-of-life recorder and the standardized measurement of quality of life in an outpatient clinic. PMID- 8625354 TI - Randomized trial of group psychosocial support in metastatic breast cancer: the BEST study. Breast-Expressive Supportive Therapy study. PMID- 8625355 TI - The Rural Cancer Outreach Program: clinical and financial analysis of palliative and curative care for an underserved population. PMID- 8625356 TI - Molecular and crystal structure of N-(2-deoxy-D-aldohexos-2-yl)-glycines (Heyns compounds). AB - Heyns compounds, 2-carboxymethylamino-2-deoxy-D-glucose (1), -mannose (2), and galactose (3), were prepared by N-carboxymethylation of the corresponding hexosamines and 1 was also prepared via the reaction of D-fructose with glycine. Both 1 and 3 crystallize from aqueous solutions as zwitterions in the alpha pyranose form and in the 4C1 conformation. Crystalline 1 is nearly isostructural to N-acetylglucosamine, forming stacks of molecules with infinite chains of homodromic hydrogen bonds along the stacks. For both 1 and 3, all hydroxyl, ammonium, and carboxyl groups are involved in intermolecular hydrogen-bonding, and an intramolecular hydrogen bond in 3 is formed via interaction of the ammonium and carboxyl groups. 1H and 13C NMR spectra (D2O solutions) indicate that all of the compounds are conformationally unstable, and that the major form present in D2O solution at 25 degrees C is the 4C1 alpha-pyranose form, with the 4C1 beta-pyranose form present in lesser amounts. In addition, for solutions of 2 and 3, considerable amounts of alpha- and beta-furanose forms are present and exist in conformations favorable for a cis-relationship between the carboxymethylammonium and anomeric hydroxyl groups. PMID- 8625357 TI - Conformational analysis of two glycoproteins: a Monte Carlo simulated annealing approach using a soft-sphere potential. AB - The Monte Carlo simulated annealing method was effectively used to predict the three-dimensional structure of the carbohydrate part of two glycoproteins: 1 vsg and 2 fbj from a protein data bank, utilizing a soft-sphere potential. The result was compared both to the crystal structure and to the structure of the corresponding isolated oligosaccharide structure simulated using an ECEPP/2 force field. A good agreement with crystal structure was reached. The interaction with the protein environment was found to significantly influence the structure of the carbohydrate moiety. PMID- 8625358 TI - Synthesis of 1',6'-disubstituted sucroses and their behavior as glucosyl donors for a microbial alpha-glucosyltransferase. AB - Versatile 6'-chloro-6'-deoxy-1'-substituted sucrose derivatives were synthesized in search of an optimum donor substrate for the intermolecular transglucosylation with the alpha-glucosyltransferase from Protaminobacter rubrum. Two substituents at the C-1' and C-6' positions of sucrose were introduced utilizing the distinct reactivity of the corresponding sulfonates. Methyl beta-D-arabinofuranoside was most efficiently glucosylated with the 1'-deoxy derivative 5. Hydroxyl and fluoro groups at C-1' show a tendency to enhance the intramolecular transglucosylations, giving 3-O-(alpha-D-glucopyranosyl)-D-fructose derivatives. PMID- 8625359 TI - Metal-saccharide chemistry and biology: saccharide complexes of zinc and their effect on metallothionein synthesis in mice. AB - Monosaccharide (D-Fru, D-Gal, D-Glc, D-Xyl, and D-Rib) and disaccharide (Mal) complexes of Zn2+ were synthesized using different precursors and isolated in the solid state. These were found to be anionic with a Zn-to-saccharide ratio of 1:1 and 2:1 for monosaccharide and disaccharide complexes, respectively. Electrochemical behaviour in aqueous solution was studied by extensive cyclic voltammetric studies in the pH range 3.7-10.3. The effect of subcutaneously injected Zn-D-Fru, Zn-D-Gal and Zn-D-Glc complexes on the metallothionein synthesis in mice was found to be significant in the liver, but not in the brain. PMID- 8625360 TI - Rheological studies of the interaction of mucins with alginate and polyacrylate. AB - The associative interaction of purified ovine and porcine submaxillary mucins (OSM and PSM) with sodium alginate was evaluated by comparing the rheological properties of mixtures against those of pure alginate and mucin in dilute, semi dilute, and concentrated solutions. These systems were investigated as models for the interaction of mucin with the extracellular alginate produced by Pseudomonas aeruginosa. In dilute solution, evidence for such interaction cannot be obtained because aggregate species exist both in the OSM-alginate mixtures as well as in pure OSM. However, in the semi-dilute regime, mixtures containing a higher proportion of mucin show systematically higher viscosities than those predicted by simple additivity. In concentrated solutions containing higher proportions of mucin, an enhanced elastic response is observed. These results demonstrate a substantial binding interaction of mucins with alginate. This property is not observed in mixtures containing a high proportion of alginate, suggesting that mucins possess relatively low numbers of interacting sites. Introduction of 3 mM Ca2+ ions to all mucin-alginate mixtures enhances the elasticity due to gelation of alginate. Finally, comparison of the rheological properties of PSM-alginate mixtures with those of PSM-polyacrylate mixtures indicates that the binding strength of alginate to mucin is significantly weaker than that of polyacrylate. PMID- 8625361 TI - Disregulation in TH1 and TH2 subsets of CD4+ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression. AB - Recent theories have established that, during an ongoing immune response, the lymphokines produced by TH1 and TH2 subsets of CD4+ T cells are critical to the effectiveness of that response. In vivo and in vitro studies have demonstrated that the type of environmental cytokines plays a determinant role in directing the development of naive T cells into TH1 or TH2 effector cells. Disregulated expansion of one or other subset may contribute to the development of certain diseases. To establish whether a similar situation might exist in the cells of the peripheral blood (PBMC) of colorectal cancer patients, we have performed immunological studies on a group of patients and a group of healthy subjects. We examined the interleukin-2 (IL-2), interferon gamma (IFNgamma), IL-4, IL-6 and tumour necrosis factor alpha levels in serum; the production of IL-4 and IL-2, with and without activating agents, by PBMC, tumour-draining lymph node lymphocytes and tumour cells; and the proliferative response of PBMC to IL-2, IL 4 and anti-CD3 monoclonal antibody (anti-CD3), which were variously combined. The data of the present study lead us to hypothesize that, because of suppressive effects probably due to environmental IL-4, in the peripheral blood of patients there seems to be a disregulation in the functionality of TH1 and TH2 subsets of CD4+ T cells, with an expansion in TH2 and a malfunction in TH1 cells. Moreover it seems that this disregulation increases with as the disease progresses through the stages, suggesting that it can be directly implicated in the mechanisms that allow the tumour to locate and progress in the host. PMID- 8625362 TI - High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats. AB - NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly by not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAB 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3(bright) cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3(bright) cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. PMID- 8625363 TI - Degradation of radioiodinated B cell monoclonal antibodies: inhibition via a FCgamma-receptor-II-mediated mechanism and by drugs. AB - Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37 degrees C, 20%-25% of initially cell-bound (125)I-CD19 mAb and (125)I-CD22 mAb was degraded in B cells, whereas almost no degredation occurred after binding of (125)I-CD20 mAb. For B cells expressing Fcgamma receptor II (FcgammaRII), isotype-dependent degradation was noted as the CD19 IgGl mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with (125)I-CD19 mAb or of CD20 with (125)I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcgammaRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant b cells and consequently result in an enhanced therapeutic effect in vivo. PMID- 8625364 TI - Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor alpha and interferon gamma treatment. AB - Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i.p.) TNFalpha and IFNgamma once weekly over 3-8 weeks in order to evaluate the effect of locoregionally administered TNFalpha/IFNgamma on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E2 values in ascites were measured immediately before and 24 h after each TNFalpha/IFNgamma infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i.p. TNFalpha/IFNgamma application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFalpha/IFNgamma treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i.p. TNFalpha/IFNgamma therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. PMID- 8625365 TI - Effects of N(g)-methyl-L-arginine, an inhibitor of nitric oxide synthesis, on interleukin-2-induced capillary leakage and antitumor responses in healthy and tumor-bearing mice. AB - We tested whether treatment with an inhibitor of nitric oxide synthesis (Ng methyl-L-arginine, MeArg) can ameliorate interleukin-2(IL-2)-therapy-induced capillary leak syndrome in healthy or tumor-bearing mice without compromising the antitumor effects of IL-2 therapy. Healthy or C3-L5-mammary-adenocarcinoma bearing C3H/HeJ mice were treated with one or two rounds of various doses of IL-2 (ten injections, i. p., every 8 h) or MeArg (ten injections s. c., every 8 h) or their combination. In an additional experiment, MeArg was given chronically in the drinking water, rather than s. c. to healthy mice subjected to one round of therapy as above. Mice were killed 1 h after their last IL-2 injection to measure the water content of the lungs and pleural cavities (markers of capillary leakage), NO production (given by NO2- and NO3- levels in the serum and pleural effusion), as well as the effect of therapies on the primary tumor size and number of spontaneous lung metastatic nodules. Results revealed that all doses of IL-2 (7500-35000 Cetus U/injection), as well as both rounds of IL-2 therapy, caused capillary leakage. However, no pleural effusion was seen after the second round in any of the IL-2-treated groups. MeArg therapy, given subcutaneously (5 20 mgkg(-1) injection(-1) in healthy and 20 mgkg(-1) injection(-1) in tumor bearing mice), did not ameliorate IL-2-induced capillary leakage in either group of mice, and did not compromise antitumor effects of IL-2. However, subcutaneous MeArg therapy alone reduced the growth of the primary tumors, the occurrence of lung metastases and the amount of tumor-induced pulmonary edema. When MeArg therapy was given orally (1 mg/ml drinking water), a substantial drop in NO production, as well as reduction in capillary leakage was noted in IL-2-treated healthy mice. These findings suggest that NO inhibitors could be a valuable adjunct to IL-2 therapy of cancer and infectious diseases. PMID- 8625366 TI - Phenotypic, functional and molecular analysis of lymphocytes associated with bladder cancer. AB - Bladder-washing-derived lymphocytes (BWDL) from 67 patients with bladder cancer were studied. The large majority of samples contained a pure population of T lymphocytes, whereas B and NK cells were absent. A comparative analysis of bladder lymphocytes and peripheral blood lymphocytes (PBL), collected in parallel, showed that BWDL significantly differed from PBL. In vitro cultures of bladder lymphocytes were attempted on 21 samples but in vitro expansion was only possible on six patients treated with bacillus Calmette-Guerin (BCG). This finding indicates that BWDL are characterized by a severe proliferative defect. Nevertheless, the addition of BCG on bladder lymphocytes expanded in vitro enhanced their proliferation, suggesting that this population is sensitized against BCG antigen(s). The analysis of T cell receptor restriction patterns showed that bladder lymphocytes from patients under BCG treatment were oligoclonal. A possible explanation for the efficiency of the immune response and good clinical outcome in patients treated with BCG could be found in the high homology between some BCG antigens and human heat-shock proteins, which are overexpressed in transformed cells. PMID- 8625367 TI - Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells. AB - The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug:mAb ratios of 7:1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 microM and 1 microM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients. PMID- 8625368 TI - Inhibition of rat C6 glioblastoma tumor growth by expression of insulin-like growth factor I receptor antisense mRNA. AB - The expression of insulin-like growth factor I receptor (IGF-IR) antisense mRNA inhibits the growth of C6 rat glioblastoma cells both in vitro and in vivo [Cancer Res (1994) 54:2218]. Moreover, the injection of C6 cells expressing an antisense mRNA to the IGF-IR into syngeneic rats prevents subsequent wild-type tumorigenesis and induces regression of established tumors. For the study of immune function in syngeneic rats, C6 cells expressing either IGF-IR sense or IGF IR antisense mRNA were injected and splenic lymphocyte function analyzed in vitro after 2 weeks. Cytotoxic, CD8+ lymphocytes from animals injected with IGF-IR antisense cells, but not from those treated with IGF-IR sense cells, proliferated in vitro in response to wild-type C6 cells. Wild-type C6 cells or IGF-IR-sense RNA-expressing cells rapidly formed tumors upon subcutaneous injection into athymic nude mice. IGF-IR antisense cells were weakly tumorigenic, exhibiting a six- to tenfold increase in tumor latency. Injection of IGF-IR antisense C6 cells mildly delayed the development of wild-type tumors, and did not induce the regression of established wild-type C6 tumors in athymic nude mice. Thus, these findings demonstrate the stimulation of a cellular immune response in rats following the injection of IGF-IR antisense cells. However, studies of athymic nude mice indicate that expression of IGF-IR antisense mRNA also inhibits C6 cells tumorigenicity by additional mechanisms. PMID- 8625369 TI - T cell receptor Vbeta repertoire of tumour-infiltrating lymphocytes in oral squamous-cell carcinoma. AB - Oral squamous cell carcinoma (OSCC) are frequently infiltrated by tumour infiltrating lymphocytes (TILs) which may demonstrate specific anti-tumour cytotoxicity. We have used a panel of family-specific primers in reverse transcriptase (RT)-polymerase chain reactions (PCR) to determine the TCR Vbeta repertoire of TILs in the primary and metastatic tumours in patients with OSCC. We observed a diverse and heterogenous usage of TCR Vbeta genes by TILs in the primary tumours, a scenario not unlike normal oral mucosal lymphocytes. In one patient with a longstanding history of OSCC, the repertoire was restricted. Restricted TCR Vbeta gene usage also occurred in TILs of metastatic tumours. Within individual patients, discrepancies of TCR Vbeta gene usage were also observed between TILs in the primary OSCC and those in metastatic lymph nodes. Work is ongoing to determine the clonality and functional significance of these TILs. PMID- 8625371 TI - [Pathologic gambling]. AB - The author presents a review on pathological gambling. Similarly as in other addictive diseases, early therapeutic intervention is important. The latter may include: 1: Evaluation of the problem 2. Recommendation that the subject should avoid places where the gambling is pursued. He should not have larger financial sums on him. 3. Recommendations pertaining to lifestyle and prevention of excessive stress. 4. Handling of printed material (the author mentions the text issued to his patients). In the paper therapeutic procedures are described, incl. the author's experience such as the foundation of the group of Gamblers anonymous. Prevention is also considered. It is important that gambling should be less readily available and the demand for it should be smaller. PMID- 8625370 TI - Interleukin-12 increases bispecific-antibody-mediated natural killer cell cytotoxicity against human tumors. AB - The combination of CD16/CD30 bispecific monoclonal antibodies (bi-mAb) and unstimulated human resting natural killer (NK) cells can cure about 50% of mice with severe combined immunodeficiency (SCID) bearing subcutaneously growing established Hodgkin's lymphoma. As interleukin-2 (IL-2) and IL-12 have been shown to increase NK cell activity, we tested the capacity of these cytokines to increase bi-mAb-mediated NK cell cytotoxicity against two types of human tumors (Hodgkin's disease and colorectal carcinoma). Unstimulated NK cells needed a three- to five-times higher antibody concentration than cytokine-stimulated NK cells to exert similar levels of bi-mAb-mediated cytotoxicity. The augmented tumor cell lysis was achieved with IL-12 at considerably lower concentrations than with IL-2 and was associated with a significantly increased bi-mAb-mediated intracellular Ca2+ mobilization. The efficiency of IL-12 in this setting together with its low toxicity make it the ideal candidate for a combination therapy with NK-cell-activating bi-mAb in human tumors that are resistant to standard treatment. PMID- 8625372 TI - [Sexual development of Czech girls before and after November 1989]. AB - BACKGROUND: It was revealed that sexual development and behaviour of adolescents, in particular girls, reflect rapidly and immediately changes in the social environment. The objective of the present investigation was to assess whether changes of the psychosexual development of girls occurred, i.e. girls examined in 1986 to 1989 and those examined after "velvet revolution" between 1990 to 1994. METHODS AND RESULTS: Using a interview based on a uniform examination pattern comprising 78 items, two sexologists examined in 1986 to 1994 the sexual development and behaviour of 771 Czech girls aged 16-18 years. This number comprised 317 apprentices and 454 secondary school students. The sexological examination was made in Frantiskovy Lazne where the probands were sent for preventive treatment, mostly after appendectomies. Comparison of the two sub groups revealed that the apprentices and students reported changes as regards the motivation of the first sexual intercourse. After November 1989 there was a decline in the group "complied" and a marked increase of the number in the category "desired herself". It was surprising that the psychosexual development of girls after the "velvet revolution" was rather retarded than hastened. The most marked change was recorded in the category "intercourse with several partners" -a drop by 12% (p < 0.03). CONCLUSIONS: The findings that despite a markedly increased motivation for the first intercourse in the examined girls no changes were recorded in the girls in some items which characterize the development of sex life, is surprising. PMID- 8625373 TI - [Postmenopausal osteoporosis. Treatment with calcitonin and a diet rich in collagen proteins]. AB - BACKGROUND: Calcitonin has an important role in the treatment of post-menopausal osteoporosis. The authors investigated the effect of calcitonin administration or calcitonin administration supplement with a diet rich in collagen proteins on markers of bone metabolism. METHODS AND RESULTS: A group of 108 patients with postmenopausal osteoporosis (BMD lower than 80% of the BMD in premenopausal women) was treated with Calsynar (Rhoune Poulenc-Rorer), 100 u., i.m. twice a week for 24 weeks. Forty-nine of these women took an oral collagen hydrolysate, 10 g per day, for the same period of time. Before and after termination of treatment clinical and laboratory tests were made, X-ray examination of the LS spine and the right forearm, single-photon osteometry of the right forearm and urinary excretion of pyridinoline (UPD), deoxypyridinoline (UDPD) and hydroxyproline (Uhyp) was assessed. As a result of treatment the BMD values increased only insignificantly (by 1.8%) the UPD values declined (to 62.51%) and those of UDPD (to 70.4%), as compared with basal values. The statistical significance is at the 1% level. When collagen proteins were administered concurrently, the decline was more marked (to 56.22% and 56.1% resp.), and as compared with the calcitonin treated group (to 67.73% and 82.30% resp.); the difference is significant at the 5% level. The decline of UPD and UDPD values persisted also three months after discontinued treatment; in patients on the diet with collagen hydrolysate practically no rise of these indicators occurred (54.02% and 56.66% resp.). CONCLUSIONS: a) administration of 100 u. calcitonin twice a week for 24 weeks led to a decline of excretion indicators of bone collagen breakdown products, b) the effect of treatment must be monitored using these indicators, c) oral administration of collagen proteins enhanced and prolonged the effect of calcitonin. PMID- 8625374 TI - [Renal vasculitis and glomerulonephritis with anti-neutrophil cytoplasmic antibody positivity]. AB - BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) define pathogenetically related group of renal vasculitides and glomerulonephritides mostly with serious prognosis. If unrecognized, these life-threatening diseases may cause loss of independent renal function and other dangerous extrarenal complications (e. g. pulmonary haemorrhage). We concentrated on the diagnosis, treatment and log-term follow-up of these patients. METHODS AND RESULTS: Renal biopsy was performed in 46 ANCA-positive patients. Age and sex distribution, type of ANCA, organ involvement, renal biopsy findings, renal function and effect of therapy were analyzed in these patients. Twenty three patients suffered from renal vasculitis, most commonly Wegener's granulomatosis (14 patients) and microscopic polyarthritis (7 patients). IgA nephropathy (7 patients) and idiopathic necrotizing/crescentic glomerulonephritis (8 patients) prevailed in patients with limited renal involvement. Renal morphology and function was most seriously impaired in patients with Wegener's granulomatosis. Immunosuppressive treatment was able to control the activity of the disease with the negativization of ANCA and improvement or stabilization of renal function in more than 90% of patients.CONCLUSIONS. ANCA-positive renal vasculitis and glomerulonephritis is relatively common. Clinical signs of extrarenal involvement are present in about 50% of patients with ANCA-positive glomerulonephritis. Rapidly introduced immunosuppressive treatment effectively controls renal and extrarenal manifestations of the disease. PMID- 8625375 TI - [The role of dietary plant polyphenols in health maintenance]. AB - Plant polyphenols are typical representatives of natural substances in foods of plant origin, in particular in fruit, tea and vegetables. They display multiple structure-conditioned interactions with various biomolecules. Their general property is the antioxidant and chelating effect and modulation of activity of various enzyme systems. In the diet they act mostly as health promoting factors during various chemical and physical stresses of the organism. They are antiatherogenic and anticarcinogenic, on the principle of inhibition of oxidative destruction of various oxylabel biological structures, inhibition of processes of bioactivation of carcinogens, blocking LDL oxidation and stimulating the activity of antioxidant and detoxication enzymes. Some of them have mutagenic properties in genotoxicity tests (quercetin, colours of red wine, phenolic acids of coffee), however, the results of animal experiments and epidemiological studies do not confirm the risk of neoplastic disease in subjects with a normal intake of these substances. The biological activity of plant polyphenols is part of the activity of other chemopreventive dietary constituents and essential nutrients. In this complex polyphenols act in many ways on the principle of synergism. The use of the health promoting properties of polyphenols isolate from plants and their administration in a pure state is not foreseen. However, under conditions it is desirable to increase the consumption of foods which are important sources of these substances. PMID- 8625376 TI - [Thrombocyte concentrates prepared from the buffy coat. A modified method]. AB - Since 1992 in the Institute of Haematology and Blood Transfusion thrombocyte concentrates are prepared from buffy coat by the method of Pietersz. The advantages of the above procedure include in particular improvement of the quality of thrombocyte concentrates with regard to their low contamination with leucocytes and erythrocytes. Contemporary resuspended erythrocyte concentrates are devoid of the buffy coat and have a very low leucocyte content. Administration of preparations of this type probably reduces the risk of alloimmunization and leads to a reduced incidence of post-transfusion febrile reactions in recipients. The objective of the submitted work was the implementation and evaluation or several centrifugation procedures for preparation of thrombocyte concentrates from buffy coat with the aim to increase the yield of thrombocytes and to obtain thrombocyte concentrates with a higher number of thrombocytes. From the four mentioned centrifuging procedures for the thrombocyte preparation it is apparent that centrifuging whole blood at 3500 g for 11 minutes with slow acceleration and subsequent spinning of the buffy coat at 555 g for 3 minutes leads to the highest thrombocyte content per transfusion unit of the preparation. Also leucocyte and erythrocyte values present in the preparation were favourable, close to recommendations of the European Council as regards the quality of thrombocyte concentrates. PMID- 8625377 TI - [Unacceptable responsibility. The role of the physician in health care economics]. PMID- 8625378 TI - [Transgenic animals in medicine]. AB - Transgenic animals are an experimental model of human diseases and enable us to study pathophysiological mechanisms, the interaction between genetic environmental factors and new therapeutic approaches incl. Their long-term effect. By using various methods it introduces into the genome of the experimental organism alien genes which become expressed. Mutated genes are introduced as well as the regulation sequence of expression of various genes, the sequence disrupting a certain gene or gene construction. Investigations of transgenic animals, whose production is rapidly increasing, are used in all spheres of medicine and biology. The author presents some examples from the sphere of oncology, cardiovascular, pulmonary, inflammatory and immunological diseases, human reproduction and early ontogenetic development, neuropsychiatric diseases and toxicology. PMID- 8625379 TI - [Changes in hemostasis and fibrinolysis in gestational diabetes]. AB - BACKGROUND: The most serious complication of diabetes is the progressive development of vascular changes in which impaired hemocoagulation and fibrinolysis participate. The latter were investigated in diabetes type 1 and 2, but les is known about them in gestational diabetes (GDM). The objective of the submitted work was to assess wither these disorders occur also during GDM and to compare the assessed changes of haemostasis and fibrinolysis with findings in a) non-pregnant healthy controls (n = 58), b) healthy pregnant women (n = 41) and c) groups of pregnant women with impaired haemostasis during gestation/gestational hemorrhage (n = 15), preeclampsia (n = 22), varicosities (n = 15) and dead foetus syndrome (n = 16), but normal carbohydrate metabolism. The changes in GDM were moreover compared with changes found in diabetes type 1 and 2. METHODS AND RESULTS: In pregnant women with GDM (n = 29) which was diagnosed according to WHO criteria the following parameters were examined: number of thrombocytes, APTT, fibrinogen-Fbg (according to Clauss), euglobulin fibrinolysis-ECLT, t-PA concentration, PAI-I (Coaliza, Kabi) and by microturbidimetry the concentration of plasma proteins/orosomucoid (ORM), alpha-1-antitrypsin (A1AT), prealbumin (PREA), transferrin (TRF) and alpha-2-macroglobulin (A2M). In patients with GDM a high Fbg level was found (4.51 +/- 0.98 g/l, p<0.01) not only as compared with Fbg in non-pregnant women (2.42 +/- 0.40 g/l), Fbg in healthy pregnant women (3.63 +/- 0.70 g/l) but also Fg in other patient groups with a pathological pregnancy. In pregnant women with GDM a reduced fibrinolytic activity - ECLT (464 +/- 98 min., p<0.01) was observed as compared with the finding in non-pregnant women (273 +/- 98 min.) but also in healthy pregnant women (303 +/- 106 min.). Another important deviation as compared with findings in healthy pregnant women in GDM is the reduced value of two proteinase inhibitors: A2M (2.04 +/- 0.59 g/l vs. 2.89 +/- 0.90 g/l, p < 0.01) and A1AT (2.98 +/- 0.80 g/l vs. 3.96 +/- 0.85 g/l, p < 0.01). The rise of t-PA (Ag), PAI-1 (Ag), fibrinogen and reduction of fibrinolytic activity (longer ECLT) made the changes the haemostasis and fibrinolysis in GDM closer to findings in DM type 2 than type 1. CONCLUSIONS: In GDM a higher thrombophilia was found (higher Fbg, longer ECLT) than in other groups of pregnant women. Another pathological finding is the reduced A2M level (proteinase inhibitor but also of PDGF and interleukins) and A1AT (inhibitor of leucocytic proteinases). The authors assume that these deviations favour the development of possible vascular changes in GDM and possibly also diabetic foetopathy (reduced A2M). PMID- 8625380 TI - [Treatment of chronic viral hepatitis B with interferon alfa. Experience of the last 3 years]. AB - BACKGROUND: interferon alpha is nowadays the drug of choice in the treatment of chronic viral hepatitis. The objective of the present work was to assess the effectiveness of interferon alpha in the treatment of chronic active hepatitis B in our population and to test whether the known "prognostic factors" of successful treatment are applicable in our patients. METHODS AND RESULTS: In 1991 1994 a total of 25 patients were treated (15 men, 10 women, mean age 46.1 years, range 18-69 years) suffering from chronic active hepatitis B (HBsAg and HBeAG positive) with interferon alpha 2b (Intron A, Schering-Plough International ). Seroconversion to anti-HBe and the drop of serum aminotransferase activity was achieved in 15 patients (60%). Only in one patient within one year after termination of treatment a relapse developed. In the group of successfully treated patients the activity of serum aminotransferases before treatment was significantly higher. The mean duration of the disease was surprisingly longer than in the group where treatment failed (106 vs. 62 months), but this difference was not statistically significant. Only in two successfully treated patients during the second and third month of treatment acute exacerbation of the inflammation occurred and manifestations of hepatic insufficiency with subsequent slow improvement developed. In the remainder the activities of serum aminotransferases declined slowly to normal values. PMID- 8625381 TI - [The first case of Crigler-Najjar syndrome in the Czech Republic]. AB - BACKGROUND: Crigler-Najjar syndrome is a rare disease due to a congenital deficiency of bilirubin UDP glucuronosyl transferase in the liver tissue. It is characterised by high levels of unconjugated bilirubin in plasma through the whole life. The aim of the study was to confirm the clinical diagnosis of the first Crigler-Najjar syndrome case in our country. METHODS AND RESULTS: 34 years old Gypsy women was admitted to our GI clinic for clinical examination before scheduled cholecystectomy. The high plasmatic level of unconjugated bilirubin was found and therefore the diagnosis of Crigler-Najjar syndrome was anticipated. The diagnosis was based on the chromatographic analysis of biliary bile pigments. The amount of diconjugates was considerable decreased. In addition, the molecular analysis of DNA isolated from peripheral blood leukocyte was performed to confirm our conclusions. Our patients was found to be homozygous for a nucleotide shift in the unique exon of bilirubin UDP glucuronosyl transferase 1, substituting guanine into an adenine at position 211. PMID- 8625382 TI - [Renal failure caused by rhabdomyolysis induced by hypokalemia in Conn's syndrome]. AB - The authors submit a case-history of a patient who developed renal failure as a result of acute rhabdomyolysis induced by severe hypokalaemia in Conn's syndrome. The authors describe the diagnosis of the disease, its course and discuss the relationship of hypokalaemia, rhabdomyolysis and acute renal failure. PMID- 8625383 TI - [Suicide from the aspect of medical history]. PMID- 8625384 TI - [The discovery of hepatitis G virus]. AB - Some 20% of cases of posttransfusion and sporadic hepatitis non-A, non-B are anti HCV negative. In 1995 it proved possible in collaboration of Genelabs with Boehringer Co. Mannheim to identify a new RNA virus which causes acute and chronic hepatitis in humans and tamarins. The genome of the virus contains some 2900 amino acids, and as to its structure, it resembles flaviviruses. It was described as hepatitis G virus (HGV). It differs from the hepatitis C, virus as it has only a 26% homology of amino acids. It is transmitted through blood during transfusion along with other parenteral routes of infection. Risk groups comprise i.v. drug addicts, blood donors and patients with thalassaemia and repeated blood transfusions. HGV can infect the liver as an independent virus or along with the virus of hepatitis B or C (dual infection). As to clinical aspects, hepatitis G is very mild and not associated with jaundice. Some patients develop chronic hepatitis. About half the patients infected with HGV have only a slightly raised transaminase activity, the remainder have normal liver enzymes. As compared with hepatitis C, the mean transaminase activity is one half. It can be diagnosed by assessment of HGV RNA by means of PCR. In the USA the prevalence of HGV RNA in blood donors with normal ALT activity is 1.7% and in donors with increased ALT activity 1.5%. The virus is sensitive to interferon, after treatment the serum concentration of HGV RNA declines rapidly but after withdrawal of treatment the values return to pre-treatment levels. This is the first report on the newly discovered hepatitis G virus. PMID- 8625385 TI - Cytoplasmic annulate lamellae in cultured cells: composition, distribution, and mitotic behavior. AB - We have used antibodies against different proteins of the nuclear pore complex to identify cytoplasmic annulate lamellae by immunocytochemistry at the light- and electron-microscopic level in various lines of cultured mammalian cells. Although annulate lamellae (AL) are seldom observed in some cell lines, they occur in large numbers in other lineages, also with cell-type-specific size distributions. AL are especially abundant and prominent in African green monkey kidney epithelial cells of line RC37 and bovine mammary gland epithelial, cells of line BMGE. We have studied the distribution of AL in relation to other organelles by using double-label immunofluorescence microscopy and have demonstrated a significant relationship between AL and rough endoplasmic reticulum. We have further shown that AL are disassembled during prophase and reassembled at the end of mitosis, almost concomitantly with the disassembly and reassembly of the nuclear envelope. Cultured mammalian cells that are rich in AL can therefore be used as suitable models for studies of the biogenesis of these lamellae, which can conveniently be detected by immunofluorescence microscopy. PMID- 8625386 TI - The spatio-temporal pattern of photoreceptor degeneration in the aged rd/rd mouse retina. AB - Photoreceptor degeneration in the retina of the rd/rd (retinal degeneration) mice has been studied using immunocytochemistry with antisera against cone- and rod opsin. The rd/rd mice exhibited different regional specific rates of degeneration for rods and cones. As early as postnatal day 25, cells labelled with the rod opsin and cone-opsin antisera disappeared preferently from the central retina. Whereas in the inferior half of the retina, degeneration subsequently proceeded towards the periphery, this did not occur in the dorsal hemisphere. By the age of 100 days, many cells immunoreactive for the cone-opsin antiserum and a few cells immunoreactive for the rod-opsin antiserum were located in an area of the dorsal retina. The ventral retina lacked labelled elements at this age. Finally, rd/rd mice at one year or 600 days of age contained a similar number of cone-opsin immunopositive cells (approximately 2000-2800 cells), occupying almost the same area in the retina as that found at 100 days of age. A photoreceptor candidate for the entrainment of non-visual photoreception probably remains in the cone population in aged rd/rd mice. PMID- 8625387 TI - Complex synaptic arrangements in the rat suprachiasmatic nucleus: a possible basis for the "Zeitgeber" and non-synaptic synchronization of neuronal activity. AB - A special type of complex synaptic arrangement occurs in the ventro-lateral portion of the rat suprachiasmatic nucleus. These arrangements are polycentric, with about equal numbers of pre- and postsynaptic elements. Because of an incomplete astroglial covering, these synaptic complexes are connected with each other and form a continuous reticulum or sponge-like system throughout the ventro lateral region of the nucleus. In two partially reconstructed complex synaptic arrangements, boutons from retinal afferents could be seen to make up the majority of presynaptic elements. They form asymmetric and symmetric synaptic appositions with dendritic elements. Non-optic axo-dendritic synapses of unknown origin with asymmetric and symmetric appositions and dendro-dendritic synapses with symmetric appositions are also seen in complex synaptic arrangements. Within complex synaptic arrangements, dendrites often run in bundles, with some dendrites spiralling around others. Membranes of neighbouring dendrites are closely apposed. These interdendritic appositions are possibly ephapses and may, together with intersomatic contacts, mediate non-synaptic synchronization of neuronal activity in the suprachiasmatic nucleus, as described by other authors. The activity of optic and non-optic synapses in complex synaptic arrangements over a 24 h period may also produce an integrated response that influences the circadian rhythm of neuronal activity in this nucleus. PMID- 8625388 TI - Morphology and projections of neurons in Remak's nerve of the domestic fowl revealed by intracellular injection of biocytin. AB - Micro-injections of biocytin were made into neurons in whole-mount preparations of Remak's nerve of the domestic fowl to visualise the morphology and projections of Remak's neurons. Remak's neurons were classified into four distinct morphological types. Remak type-I and -II neurons had a morphology resembling that of mammalian sympathetic neurons. Type-I neurons (found only in juxta jejunal ganglia) had numerous primary dendritic processes (8-14) with large dendritic fields and extensive dendritic arborizations. Type-II neurons had 2-9 primary dendritic processes, large dendritic fields and sparse dendritic branching. These were found in similar numbers in juxta-rectal and juxta-jejunal ganglia. Remak type-III neurons were the most numerous cell type of juxta-rectal ganglia. They had small cell somata and short dendritic processes that branched infrequently. Remak type-IV neurons (found only in juxta-jejunal ganglia) had a morphology resembling that of invertebrate neurons in that they possessed a prominent long tapering axon from which most of the numerous long dendritic processes emerged. In juxta-jejunal ganglia, all type-IV and most type-I, -II and -III neurons projected orally, whereas axons of juxta-rectal neurons (types II and III) projected either orally or aborally, or projected directly into a lateral nerve bundle supplying the gut. These regional differences in neuron types and axonal projections suggest that different neural circuits exist between Remak's nerve and the small and large intestine. PMID- 8625389 TI - Mineralization during matrix-vesicle-mediated mantle dentine formation in molars of albino rats: a microanalytical and ultrastructural study. AB - The purpose of this study was to elucidate the mineralization process of mantle dentine by ultrastructural and element-analytical investigation of matrix vesicles and successive stages. Upper second molars of albino rats were cryofixed and embedded in resin after freeze drying. Semithin dry sections were prepared for analyzing the calcium and phosphorus concentrations in the mineralized matrix vesicles or noduli, larger mineralized islands, and the mantle dentine. For ultrastructural studies, it was necessary to reduce section contact with hydrous fluids to a minimum in order to avoid preparation artifacts. The first mineral deposits were recognized as dot-like formations both in the interior of matrix vesicles and in association with the inner vesicle membrane. This indicated the existence of mineral nucleating sites located both at the inner membrane and at calcium-phosphate-binding macromolecules in the interior of the matrix vesicles. A significantly higher mineral content was found in mineralized matrix vesicles than in the mineralized extravesicular regions of the mineralized islands, suggesting the existence of a rapidly and densely mineralized matrix in the matrix vesicles. A significant increase in mineral content per volume proceeding from the mineralized islands to mantle dentine suggested a further increase in the density of mineral. PMID- 8625391 TI - Immunocytochemistry by electron spectroscopic imaging using a homogeneously boronated peptide. AB - A linear all-L-oligopeptide containing five carboranyl amino acids (corresponding to 50 boron atoms) was synthesized and specifically attached to the free thiol group of monovalent antibody fragments F(ab)'. The boronated immunoreagent was used for the direct post-embedding detection of somatotrophic hormone in ultrathin sections of porcine pituitary embedded in Spurr resin. The specific boron-labelling of secretory vesicles in somatotrophs was detected by electron spectroscopic imaging and confirmed by conventional immunogold labelling run in parallel. In comparison with immunogold, boron-labelled F(ab)'-fragments showed higher tagging frequencies, as was expected; the small uncharged immunoreagents have an elongated shape and carry the antigen-combining structure and the detection tag at opposite ends, thus allowing for high spatial resolution in electron spectroscopic imaging. PMID- 8625392 TI - Structural and functional differences between prolactin cells from the inner and outer zones of the male rat anterior pituitary. AB - The aim of this study was to compare the ultrastructure of prolactin cells in the inner and outer zones of the male rat anterior pituitary, and to relate their morphological features to their secretory activity, by means of standard and ultrastructural reverse hemolytic plaque assays (RHPA). The immuno ultrastructural study showed that in the inner pituitary small-granulated cells represented 52% of the prolactin cells, there being only 5% with large granules, whereas the prolactin cells with large granules accounted for 52% in the outer zone, with only 7% being small-granulated. Percentages of cells with intermediate sized granules were 43% and 41%, respectively. Analysis of RHPA data revealed that, under basal conditions, prolactin cells secreted more actively in the inner zone than in the outer zone. Stimulation with thyrotropin-releasing hormone or KCl treatment increased the percentage of secretors and the sizes of hemolytic plaques in both zones. However, in response to thyrotropin-releasing hormone, the increase in number of secretors was always higher in the outer zone, whereas the enlargement of plaque sizes was greater for the "inner" cells. These findings are in favor of the small-granulated cells, which predominate in the inner zone, being in a stage of active secretion and responsiveness. PMID- 8625393 TI - Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) forebrain before and after upstream migration. AB - Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) was examined before and after upstream migration by an immunocytochemical technique with a specific antiserum to salmon gonadotropin releasing hormone and an in situ hybridization technique with an oligonucleotide encoding salmon gonadotropin-releasing hormone precursor (pro-salmon gonadotropin releasing hormone). In the forebrain (olfactory nerve, olfactory bulb, telencephalon, and preoptic area), salmon gonadotropin-releasing hormone immunoreactive neurons and neurons showing signals for pro-salmon gonadotropin releasing hormone mRNA were compared between fish from the coastal sea and those from the spawning ground. Neurons in the dorsal region of the olfactory nerve and in the ventral region of the transitional area between olfactory nerve and olfactory bulb showed strong salmon gonadotropin-releasing-hormone immunoreactivity and strong hybridization signals in fish from the coastal sea, but these activities and signals were not observed or were decreased in number in fish from the spawning ground. The neurons in the olfactory bulb, telencephalon, and preoptic area consistently revealed salmon gonadotropin-releasing-hormone immunoreactivity and hybridization signals, and the hybridization signals of salmon gonadotropin-releasing hormone in the telencephalon and the preoptic area were stronger in fish from the spawning ground than in those from the coastal sea. These findings suggest that salmon gonadotropin-releasing-hormone neurons in the olfactory nerve and the transitional area between olfactory nerve and olfactory bulb have different patterns of hormone production than those in the telencephalon and the preoptic area. PMID- 8625390 TI - Heterogeneity and migration-related zonation of K(+)-ATPase activities in the oxyntic cell lineage of adult cattle. AB - Activities of the H+, K(+)-ATPase and the Na+, K+, ATPase have been localized in the morphologically heterogeneous oxyntic cell lineage of adult bovine abomasal mucosa, by means of K(+)-dependent paranitrophenylphosphatase (K(+)-pNPPase) histochemistry. At the light- and electron-microscopic level, only members of the mature oxyntic cell population within the oxyntic glandular base exhibit strong enzyme activity. Superficial oxyntic cells of the proximal isthmus and deep pit, arising from the upward migration of precursor cells and commonly supposed to have a high capacity of secreting acid, show weak or no enzyme activity. This is also true of the immature and pre-oxyntic cells of the generative zone. Global enzyme activity varies among the mature glandular oxyntic cell population. Ultracytochemically, strong H+, K(+)-ATPase (ouabain-insensitive K(+)-pNPPase) activity is associated with the apical plasmalemmal and expanded canalicular membrane in contrast to Na+, K(+)-ATPase (ouabain-sensitive K(+)-pNPPase) activity, which is localized on the basolateral plasmalemmal folds. In both cases, histochemical deposition is confined to the cytoplasmic aspect of the membranes. These results suggest a functional zonation and position-dependent heterogeneity of the oxyntic cell lineage related to the bidirectional mode of migration of pre-oxyntic cells during physiological cell renewal. Functional heterogeneity within the mature glandular oxyntic cell population is in accordance with the continuous mode of gastric acid secretion in cattle. PMID- 8625394 TI - Immunocytochemical mapping of a C-terminus anti-peptide antibody to the GABA receptor subunit, RDL in the nervous system in Drosophila melanogaster. AB - An antibody raised against a peptide based on the C-terminal derived amino acid sequence from a cloned Drosophila melanogaster (fruit fly) gene, Rdl (resistant to dieldrin), was used to investigate localization of a GABA receptor subunit in adult male D. melanogaster. Many regions in the brain and thoracic ganglia were stained with this antibody. For example, staining was detected in the medulla, lobula and lobular plate optic neurpiles. Also stained were the antennal lobe glomeruli, the ellipsoid body of the central complex and the mushroom bodies. These results suggest possible roles for an RDL-like GABA receptor subunit in the processing of olfactory, visual and mechanosensory information in the nervous system of D. melanogaster. PMID- 8625395 TI - Structure and chemical coding of human, canine and opossum gallbladder ganglia. AB - Immunohistochemistry and cholinesterase histochemistry were used to evaluate the structure and neurotransmitter content of the ganglionated plexuses of the human, canine, and opossum (Monodelphis domestica) gallbladders. In each species, the ganglionated plexus consisted of small (mean approximately 4 neurons/ganglion), irregularly dispersed ganglia that were interconnected by bundles of nerve fibers. The density of ganglia was about ten-fold higher in the opossum than in the human or the dog. Immunostaining for choline acetyltransferase (ChAT) was accomplished in the human, dog, opossum, and the guinea pig where all neurons were found to express ChAT-immunoreactivity. In the human, immunoreactivities for vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were the most abundant followed by substance P (SP). In the dog, immunoreactivity for galanin (GAL) was the strongest, followed closely by VIP and then by SP. NPY immunoreactive neurons were not observed in the dog, but immunoreactive nerve fibers were seen in the perivascular plexus. In the opossum, immunoreactivity for GAL was the most intense and abundant followed by SP, which was followed by VIP. NPY-immunoreactivity in the opossum was limited to scarce perivascular nerve fibers. Immunoreactivity for calcitonin-gene-related peptide (CGRP) was not observed in neuronal somata, but CGRP/SP-immunoreactive nerve fibers were a feature of each species studied. These findings, along with previously published work on the guinea pig, indicate that it is likely that all gallbladder neurons are cholinergic, and that VIP, SP, and NPY and/or GAL are commonly expressed in gallbladder neurons. PMID- 8625396 TI - A morphometric analysis of exocytosis in KCl-stimulated bovine chromaffin cells. AB - Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data. PMID- 8625397 TI - Development of T-lymphocyte subpopulations in the postnatal chicken oviduct. AB - Postnatal development of t-cell subpopulations in the oviduct was investigated in the Dekalb strain of the White Leghorn chicken by using an immunohistochemical method. T-lymphocytes first infiltrated the oviduct at 5 weeks. The number of T cells peaked at 15 weeks in the magnum, isthmus and uterus, and at 19 weeks in the infundibulum and vagina. The epithelium of the oviduct contained both granular and agranular lymphocytes. TcR1+ cells were predominant in the epithelium, whereas TcR2+ cells were more numerous than TcR1+ cells in the lamina propria. TcR3+ cells were absent from the epithelium and were not numerous in the lamina propria. CT8+ cells, equivalent to CD8+ cells in mammals, were located both in the epithelium and in the lamina propria. The relative frequency of T cell subpopulations was found to be higher in the vaginal part than in the other parts of the oviduct. These results suggest that the postnatal developmental changes of T-cell subpopulations depend on different anatomical regions of the oviduct and on the age of the chicken. PMID- 8625398 TI - Thymocyte-directed enhancement of apoptosis via soluble factor(s) derived from a cortical and a medullary thymic epithelial cell line. AB - Apoptosis of murine thymocytes was examined either in intact fetal thymus lobes or in thymus cell suspensions, both cultured alone or in the presence of either a cortical (TEC 1.4) or a medullary (TEC 2.3) thymic epithelial cell line. Both TECs induced a pronounced increase of apoptosis in 24-h cultivated single thymus cell suspensions but not in spleen or bone marrow cell cultures. Co-culture of thymocytes with murine fibroblasts did not enhance apoptosis of the thymus cells. A similar enhancement of thymocyte apoptosis was observed with dialysed culture supernatants derived from both TEC lines, the active component(s) having a molecular weight of > 30 kDa. In contrast, the cortical TEC 1.4 had a pronounced apoptosis inducing effect on intact fetal thymus lobes cultivated for six days, whereas the medullary TEC 2.3 had only a marginal influence. TEC 1.4 also induced a significant alteration in the ratio of CD4+CD8+ to CD4-CD8- cells. It is concluded that both the cortical and medullary epithelial cell lines are able to induce thymocyte apoptosis but that a large proportion of the cells within the intact thymus stroma is refractory to the respective signal(s) of the medullary epithelial cell line. PMID- 8625399 TI - Endogenous opioid peptides in parasympathetic, sympathetic and sensory nerves in the guinea-pig heart. AB - Research has suggested that exogenous opioid substances can have direct effects on cardiac muscle or influence neurotransmitter release via presynaptic modulation of neuronal inputs to the heart. In the present study, multiple labelling immunohistochemistry was employed to determine the distribution of endogenous opioid peptides within the guinea-pig heart. Approximately 40% of cardiac ganglion cells contained immunoreactivity for dynorphin A (1-8), dynorphin A (1-17) and dynorphin B whilst 20% displayed leu-enkephalin immunoreactivity. Different populations of opioid-containing ganglion cells were identified according to the co-existence of opioid immunoreactivity with immunoreactivity for somatostatin and neuropeptide Y. Immunoreactivity for prodynorphin-derived peptides was observed in many sympathetic axons in the heart and was also observed, though to a lesser extent, in sensory axons. Leu enkephalin immunoreactivity was observed in occasional sympathetic and sensory axons. No immunoreactivity was observed for met-enkephalin-arg-gly-leu or for beta-endorphin. These results demonstrate that prodynorphin-derived peptides are present in parasympathetic, sympathetic and sensory nerves within the heart, but suggest that only the prodynorphin gene is expressed in guinea-pig cardiac nerves. This study has shown that endogenous opioid peptides are well placed to regulate cardiac function via both autonomic and sensory pathways. PMID- 8625400 TI - Expression of heat-shock protein 47 in mouse liver. AB - Expression of heat-shock protein 47 in intact and fibrotic liver and in hepatic constituent cells was investigated in mice. Immunohistochemical study of intact liver and Western blot analysis of the protein from isolated liver cells revealed that stellate cells and smooth muscle cells of interlobular vessels, but not hepatocytes, Kupffer cells, or endothelial cells, expressed heat-shock protein 47. The protein was found in both vitamin-A-storing stellate cells and myofibroblast-like cells. The amount of the protein in cultured stellate cells was reduced by dexamethasone but was not regulated by quercetin, transforming growth factor beta, interferon gamma, or retinoic acid. In CCl4-treated or bile duct-ligated mouse liver, the number of cells positive for heat-shock protein 47 markedly increased in the centrilobular area or around the periportal area, respectively, and the level of heat-shock protein 47 also increased. PMID- 8625401 TI - Mother and daughter are doing fine: asymmetric cell division in yeast. PMID- 8625402 TI - Die and let live: eliminating dangerous lymphocytes. PMID- 8625403 TI - Clustering membrane proteins: It's all coming together with the PSD-95/SAP90 protein family. PMID- 8625404 TI - Neuronal growth and death: order and disorder in the axoplasm. PMID- 8625405 TI - Heterochronic genes control cell cycle progress and developmental competence of C. elegans vulva precursor cells. AB - Heterochronic genes control the timing of vulval development in the C. elegans hermaphrodite. lin-14 or lin-28 loss-of-function mutations cause the vulval precursor cells (VPCs) to enter S phase and to divide one larval stage earlier than in the wild type. A precocious vulva is formed by essentially normal cell lineage patterns, governed by the same intercellular signals as in the wild type. Mutations that prevent the normal developmental down-regulation of lin-14, activity delay or block VPC division and prevent vulval differentiation. A genetic pathway that includes lin-4, lin-14, and lin-28 controls when VPCs complete G1 and also controls when VPCs acquire the competence to respond to the intercellular patterning signals and express vulval fates. PMID- 8625406 TI - Regulation of the Drosophila protein timeless suggests a mechanism for resetting the circadian clock by light. AB - Circadian behavioral rhythms in Drosophila depend on the appropriate regulation of at least two genes, period (per) and timeless (tim). Previous studies demonstrated that levels of PER and TIM RNA cycle with the same phase and that the PER and TIM proteins interact directly. Here we show the cyclic expression of TIM protein in adult heads and report that it lags behind peak levels of TIM RNA by several hours. We alsoshow that nuclear expression of TIM depends upon the expression of PER protein. Finally, we report that the expression of TIM, but not PER, is rapidly reduced by light, suggesting that TIM mediates light-induced resetting of the circadian clock. Since both PER and TIM RNA are unaffected by light treatment, the effects of light on TIM appear to be posttranscriptional. PMID- 8625407 TI - Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins. AB - Certain cell types give rise to progeny that adopt different patterns of gene expression in the absence of any differences in their environment. Cells of budding yeast give birth to mother and daughter cells that differ in that only mother cells express the HO endonuclease gene and thereby switch mating types. We describe the identification of five genes, called SHE1-SHE5, that encode cytoplasmic proteins required for mother-specific HO expression. She1p, which is identical to the minimyosin Myo4p, and She3p are not, however, mother-specific proteins. On the contrary, they accumulate in growing buds. She proteins might be required for the transport of factors that promote HO repression from the mother cell into its bud. In an accompanying paper, we show that SHE genes are needed for the accumulation in daughter nuclei of Ash1p, a repressor of HO. PMID- 8625408 TI - Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells. AB - Cell division in haploid yeast gives rise to a "mother" cell capable of mating type switching and a "daughter" cell that is not. Switching is initiated by the HO endonuclease, whose gene is only transcribed in cells that have previously given birth to a bud (mother cells). HO expression depends on a minimyosin, She1p/Myo4p, which accumulates preferentially in growing buds. We describe a gene, ASH1, that is necessary to repress HO in daughters. ASH1 encodes a zinc finger protein whose preferential accumulation in daughter cell nuclei at the end of anaphase depends on She1p/Myo4p. The greater abundance of Ash1p in daughter cells is responsible for restricting HO expression to mother cells. PMID- 8625409 TI - Identification of asymmetrically localized determinant, Ash1p, required for lineage-specific transcription of the yeast HO gene. AB - S. cerevisiae cells exhibit asymmetric determination of cell fate. Cell division yields a mother cell, which is competent to transcribe the HO gene and switch mating type, and a daughter cell, which is not. We have isolated a mutant in which daughters transcribe HO and switch mating type. This mutation defines the ASH1 gene (asymmetric synthesis of HO). Deletion and overexpression of ASH1 cause reciprocal cell fate transformations: im ash1delta strains, daughters switch mating type as efficiently as mothers. Conversely, overexpression of ASH1 inhibits switching in mother cells. Ash1p has a zinc finger motif related to those of GATA transcriptional regulators. Ash1p is localized to the daughter nucleus in cells that have undergone nuclear division. Thus, Ash1p is a cell fate determinant that is asymmetrically localized to the daughter nucleus where it inhibits HO transcription. PMID- 8625410 TI - Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. AB - The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization. PMID- 8625411 TI - The making of a compound leaf: genetic manipulation of leaf architecture in tomato. AB - The most distinctive morphogenetic feature of leaves is their being either simple or compound. To study the basis for this dichotomy, we have exploited the maize homeobox-containing Knotted-1 (Kn1) gene in conjunction with mutations that alter the tomato compound leaf. We show that misexpression of Kn1 confers different phenotypes on simple and compound leaves. Up to 2000 leaflets, organized in compound reiterated units, are formed in tomato leaves expressing Kn1. In contrast, Kn1 induces leaf malformations but fails to elicit leaf ramification in plants with inherent simple leaves such as Arabidopsis or in tomato mutant plants with simple leaves. Moreover, the tomato Kn1 ortholog, unlike that of Arabidopsis, is expressed in the leaf primordia. Presumably, the two alternative leaf forms are conditioned by different developmental programs in the primary appendage that is common to all types of leaves. PMID- 8625412 TI - Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. AB - NMDA (N-methyl-D-aspartate) receptors are excitatory neurotransmitter receptors in the brain critical for synaptic plasticity and neuronal development. These receptors are Ca2+-permeable glutamate-gated ion channels whose physiological properties are regulated by intracellular Ca2+. We report here the purification of a 20 kDa protein identified as calmodulin that interacts with the NR1 subunit of the NMDA receptor. Calmodulin binding to the NR1 subunit is Ca2+ dependent and occurs with homomeric NR1 complexes, heteromeric NR1/NR2 subunit complexes, and NMDA receptors from brain. Furthermore, calmodulin binding to NR1 causes a 4-fold reduction in NMDA channel open probability. These results demonstrate that NMDA receptor function can be regulated by direct binding of calmodulin to the NR1 subunit, and suggest a possible mechanism for activity-dependent feedback inhibition and Ca2+-dependent inactivation of NMDA receptors. PMID- 8625413 TI - Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. AB - Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes. PMID- 8625414 TI - The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. AB - Human cytomegalovirus (HCMV) down-regulates expression of MHC class I products by selective proteolysis. A single HCMV gene, US11, which encodes an endoplasmic reticulum (ER) resident type-I transmembrane glycoprotein, is sufficient to cause this effect. In US11+cells, MHC class I molecules are core-glycosylated and therefore inserted into the ER. They are degraded with a half-time of less than 1 min. A full length breakdown intermediate that has lost the single N-linked glycan in an N-glycanase-catalyzed reaction transiently accumulates in cells exposed to the protease inhibitors LLnL, Cbz-LLL, and lactacystin, identifying the proteasome as a key protease. Subcellular fractionation experiments show this intermediate to be cytosolic. Thus, US11 dislocates newly synthesized class I molecules from the ER to the cytosol, where they are acted upon by an N-glycanase and the proteasome. PMID- 8625415 TI - TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74. AB - Some TAF subunits of transcription factor TFIID play a pivotal role in transcriptional activation by mediating protein-protein interactions, whereas other TAFs direct promoter selectivity via protein-DNA recognition. Here, we report that purified recombinant TAFII250 is a protein serine kinase that selectively phosphotylates RAP74 but not other basal transcription factors or common phosphoacceptor proteins. The phosphorylation of RAP74 also occurs in the context of the complete TFIID complex. Deletion analysis revealed that TAFII250 contains two distinct kinase domains each capable of autophosphorylation. However, both the N- and C-terminal kinase domains of TAFII250 are required for efficient transphosphorylation of RAP74 on serine residues. These findings suggest that the targeted phosphorylation of RAP74 by TAFII250 may provide a mechanism for signaling between components within the initiation complex to regulate transcription. PMID- 8625416 TI - Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin binding protein, EBNA1, bound to DNA. AB - The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction. PMID- 8625417 TI - A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT AC) intron in vitro. AB - A minor class of introns with noncanonical splice (AT-AC) and branch site sequences exists in metazoan protein coding genes. We have established a HeLa cell in vitro system that accurately splices a pre-mRNA substrate containing such an intron from the human P120 gene. Splicing occurs via a lariat intermediate whose branch site A residue is predicted to bulge from a duplex formed with the low abundance U12 small nuclear ribonucleoprotein (snRNP), which we confirm by psoralen cross-linking. Native gel electrophoresis reveals that U11, U12, and U5 snRNPs assemble onto the P120 pre-mRNA to form splicing complexes. Inhibition of P120 splicing by 2'-O-methyl oligonucleotides complementary to U12 or U5 demonstrates that U12 and U5 snRNPs perform essential roles in the AT-AC spliceosome. PMID- 8625418 TI - The gene for a subunit of an ABC-type heme transporter is transcribed together with the gene for subunit 6 of NADH dehydrogenase in rice mitochondria. AB - We previously identified a chloroplast-derived (ct-derived) sequence of 32 base pairs (bp) in rice mitochondrial DNA that includes a part (30 bp; psitrnI) of a gene for isoleucine tRNA (CAU) of the chloroplast. Analyzing the ct-derived psitrnI, we found that an open reading frame (orf240), which was homologous to the gene for a subunit of an ATP-binding cassette-type (ABC-type) heme transporter, namely helC, of Rhodobacter capsulatus, and a gene for subunit 6 of NADH dehydrogenase (nad6) were located upstream of and downstream from the ct derived psitrnI, respectively. Northern-blot hybridization and analysis by reverse transcription-polymerase chain reaction (RT-PCR) revealed that both orf240 and nad6 were co-transcribed in rice mitochondria. An analysis of PCR amplified fragments of the region of orf240/nad6 from the DNA of some Gramineae suggests that the arrangement of orf240/nad6 was generated in the mitochondrial genome of the genus Oryza during evolution after its divergence from the other Gramineae. Most of the transcripts of orf240 are edited, with a change from cytidine to uridine, at 35 positions. Editing of the RNA changes 33 amino-acid residues among the 240 encoded amino-acid residues, suggesting that the orf240 gene is functional in rice mitochondria. PMID- 8625419 TI - Multiple initiation sites for transcription of a gene for subunit 1 of F1-ATPase (atp1) in rice mitochondria. AB - We identified the sites for the initiation of transcription of a gene for subunit 1 of F1-ATPase (atp1) in rice mitochondrial DNA. Capping and ribonuclease protection experiments in vitro, together with primer extension analysis, demonstrated that there were at least eight transcription initiation sites upstream of atp1. One initiation site, expressed most actively, was flanked by a sequence identical to the consensus promotor motif of rice mitochondrial genes. However, the sequences surrounding the other seven initiation sites exhibited no similarity to the consensus sequence. PMID- 8625420 TI - Sex-biased heteroplasmy and mitochondrial DNA inheritance in the mussel Mytilus galloprovincialis Lmk. AB - An exceptional mode of mtDNA inheritance involving separate maternal and paternal transmission routes has been reported recently in the mussel Mytilus edulis. This mode of inheritance provides an explanation for the high levels of heteroplasmy for two highly diverged genomes observed in males of this species. Here we provide evidence for a similar pattern of heteroplasmy in Atlantic and Mediterranean forms of the related mussel M. galloprovincialis. The results support the hypothesis that this mode of mtDNA inheritance has an ancient origin. In addition, the detection of some heteroplasmic females suggests preferential, rather than exclusive, transmission within male and female lines of descent. We also present evidence that the two highly diverged genomes display a parallel split between the Atlantic and Mediterranean forms, consistent with neutral evolution. PMID- 8625422 TI - Overexpression of ADH1 confers hyper-resistance to formaldehyde in Saccharomyces cerevisiae. AB - In an attempt to clone genes involved in resistance to formaldehyde we have screened a genomic library based on the episomal plasmid YEp24 for the ability to increase resistance to formaldehyde in a wild-type strain. In addition to SFA, the gene encoding the formaldehyde dehydrogenase Adh5, an enzyme most potent in formaldehyde de-toxification, we isolated a second plasmid that conferred a less pronounced but significant hyper-resistance to formaldehyde. Its passenger DNA contained the gene ADH1, encoding alcohol dehydrogenase 1 (EC 1.1.1.1), which could be shown to be responsible for the observed hyper-resistance phenotype. Construction of an adh1-0 mutant revealed that yeast lacking a functional ADH1 gene is sensitive to formaldehyde. While glutathione is essential for Adh5 mediated formaldehyde de-toxification, Adh1 reduced formaldehyde best in the absence of this thiol compound. Evidence is presented that formaldehyde is a substrate for Adh1 in vivo and in vitro and that its cellular de-toxification employs a reductive step that may yield methanol. PMID- 8625421 TI - Electron microscopic investigation of mitochondrial DNA from Chenopodium album (L.). AB - DNA molecules from mitochondria of whole plants and a suspension culture of Chenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0. 3-183 kb represented 23-26% of all mtDNA molecules in the preparations from the suspension culture and 13-15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rolling-circle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants. PMID- 8625423 TI - Aromatic amino-acid biosynthesis in Candida albicans: identification of the ARO4 gene encoding a second DAHP synthase. AB - The primary step in the aromatic amino-acid biosynthetic pathway in Saccharomyces cerevisiae is catalyzed by two redundant isozymes of 3-deoxy-d arabinoheptulosonate-7-phosphate (DAHP) synthase, either of which alone is sufficient to permit growth on synthetic complete media lacking aromatic acids (SC-Aro). The activity of one isozyme (encoded by the ARO3 gene) is feedback inhibited by phenylalanine, whereas the activity of the other isozyme (encoded by the ARO4 gene) is feedback-inhibited by tyrosine. Transcription of both genes is controlled by GCN4. We previously cloned the ARO3 gene from the opportunistic pathogen Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine; and (2) its expression is induced in response to amino-acid deprivation, consistent with the presence of two putative GCN4-responsive promoter elements (Pereira and Livi 1993, 1995). To determine whether other DAHP synthases exist in C. albicans, we have constructed a homozygous aro3-deletion mutant strain. Such a mutant was found to be phenotypically Aro+, i. e., capable of normal growth on SC Aro media, suggesting the presence of at least one additional isozyme. To confirm this result, a 222-bp DNA fragment was amplified by the polymerase chain reaction (PCR) from genomic DNA prepared from the homozygous aro3-deletion mutant, using a degenerate primer based on a conserved N-terminal region of Aro3p plus a degenerate comeback primer encoding a conserved region of the protein that lies within the deleted portion of the gene. The nucleotide sequence of this PCR fragment predicts a 74-amino acid DAHP synthase-related protein which shows strong homology to Aro3p from S. cerevisiae and C. albicans, but even greater homology (78% identity) to S. cerevisiae Aro4p. We conclude that cells of C. albicans contain a second Aro4p-related DAHP synthase. PMID- 8625425 TI - pH sensitivity of Schizosaccharomyces pombe: effect on the cellular phenotype associated with lacZ gene expression. AB - We report on a series of experiments in Schizosaccharomyces pombe to detect the blue-colour colony phenotype associated with expression of the Escherichia coli lacZ gene. Increasing the pH in solid minimal medium to optimize blue colony colour revealed a pH-sensitive phenotype in auxotrophic strains requiring uracil and leucine as external supplements. This phenotype was observed among common S. pombe stock strains, 5-fluoroorotic acid (5-FOA)selected strains, and random genetic segregants. Growth of prototrophic S. pombe strains 972 and 975 or the adenine auxotrophic strain NCYC 1860 were unaffected by an increase in external pH. Analysis of genetic segregants from three independent crosses indicated that a single auxotrophic marker (ura4- or leu1-32) was sufficient for yeast cell growth inhibition when the medium pH was increased above 6.6. In contrast, growth of a Saccharomyces cerevisiae strain isogenic to AH22, requiring uracil, leucine and histidine, was unaffected by changes in the pH of the medium. These observations suggest that uptake of uracil and leucine into S. pombe cells is compromised by alterations in external pH. Our results have implications for detection of the lacZ gene-encoded bluecolour colony phenotype in S. pombe, which is optimized by growth in the presence of 5-bromo-4-chloro-3-indolyl- "beta"-D galactoside (Xgal) at pH 7.0. We discuss the conditions under which this blue colour phenotype can be routinely observed in S. pombe. PMID- 8625424 TI - The 3-phosphoglycerate kinase gene of the yeast Yarrowia lipolytica de-represses on gluconeogenic substrates. AB - We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeast Yarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs. It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that of Aspergillus nidulans (76% identity). The expression of the Y. lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones. Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol. PMID- 8625426 TI - Evidence for sltA1 as a salt-sensitive allele of the arginase gene (agaA) in the ascomycete Aspergillus nidulans. AB - Strains of Aspergillus nidulans carrying the sltA1 mutation, conferring sensitivity to KCl and NaCl, also showed an arginine-sensitive phenotype whereby concentrations of the L-amino acid at or above 10 mM were toxic to growth. Sexual progeny of a cross between a sltA1 mutant and a wild-type strain showed a co segregation of salt and arginine sensitivity. Similarly, revertants to salt tolerance showed a loss of arginine sensitivity as did sltA1 strains that were transformed with a cosmid carrying the putative sltA1+ wild-type allele. In addition, arginine sensitivity could be relieved by L-ornithine. It is suggested that sltA1 is a salt-sensitive allele of the arginase gene (agaA). PMID- 8625427 TI - Identification and cloning of a mobile transposon from Aspergillus niger var. awamori. AB - Aspergillus niger var. awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of the niaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in both A. niger var. awamori and A. niger. A single copy of Vader was detected from only one of the two laboratory strains of A. nidulans which were also examined. Insertion of the Vader element into the niaD gene of A. niger var. awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements at niaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. Mutants niaD392 and niaD436 reverted at a frequency of 9x10(-3) and 4x10(-2), respectively. Two of the mutants, niaD587 and niaD410, reverted at a lower frequency of 6x10(-4). PMID- 8625428 TI - Expression of an Erwinia pectate lyase in three species of Aspergillus. AB - Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes. PMID- 8625429 TI - Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum. AB - Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum. The efficiency of transformation of P. chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. The AMA1 region works as an origin of replication in P. chrysogenum and A. nidulans but not apparently in Acremonium chrysogenum. PMID- 8625431 TI - Molecular karyotype of the phytopathogenic fungus Sclerotinia sclerotiorum. AB - Molecular techniques have been used to characterize different field isolates of Sclerotinia sclerotiorum, an ubiquitous phytopathogen. Chromosomal DNA resolved by pulsed-field gel electrophoresis (PFGE) revealed that S. sclerotiorum contains at least 16 chromosomes ranging from 1.5 Mb to 4.0 Mb. The size of the haploid genome was estimated to be 43.5 Mb. Six field isolates with different levels of virulence on sunflower germlings or green beans were differentiated by random amplification of polymorphic DNA (RAPD), and analysed by clamped homogeneous electric field electrophoresis. This analysis revealed few chromosome-length polymorphisms among these strains. Chromosomal DNA hybridization indicated that the endopolygalacturonase-encoding pg1 gene is localized on the smallest chromosome of all the strains, whereas the ribosomal DNA mapped to different sized chromosomes. The less-aggressive strain was characterized by the presence of a supernumary small band, presumably consisting of dsRNA. In contrast to numerous other phytopathogenic fungi, this study reveals a strong karyotypic stability among the strains of S. sclerotiorum which may be preserved by the sexual mode of reproduction of this species PMID- 8625432 TI - Influence on isometric muscle contraction during shoulder abduction by changing occlusal situation. AB - Many studies have been performed on the mandibular orthopedic repositioning appliance (MORA) as an athletic performance enhancer since the late 1970s. The concept that changing the mandibular position would increase the strength and improve the performance of subjects has been a source of controversy among various researchers. The strength of shoulder abduction and electronic activities of six muscle groups in the upper body were tested on seven subjects in this study. The normalized data for four different situations: rest position, MVC (Maximum Voluntary Contraction) in an intercuspal position, MVC with placebo, and MVC with MORA in supported rest position were analysed. Statistical analysis demonstrated that the MVC with MORA was significantly stronger than the rest position for upper appendage strength. The electronic activities of all muscle groups were significantly greater with MORA than those in rest position, and some of them were significantly greater with MORA than with either MVC or placebo situation. THe results suggested that the muscle activities of the upper appendage were increased by biting MORA in the supported rest position during the shoulder abduction performance. PMID- 8625430 TI - Endoglucanase II (EGII) of Penicillium janthinellum: cDNA sequence, heterologous expression and promotor analysis. AB - The cDNA coding for the endoglucanase EGII of P. janthinellum was cloned and sequenced. The open reading frame comprises 1230 nucleotides and the deduced amino-acid sequence shows an overall homology of 63% with the T. reesei egl2. The cellulose-binding domain of EGII represents a typical member of the A family of cellulases. The egl2 gene is only induced by cellulose or cellobiose and not by sophorose. A promotor fragment including 1 kb was cloned and sequenced. Three major transcription startpoints were identified. Five motifs matching the binding site of the carbon-catabolite repressor CREA of A. nidulans were detected. Their potential implication in repression was analyzed by bandshift assays. PMID- 8625433 TI - Analysis on decay rate of vibration following impact to human dry skull with and without mouthguards. AB - The modal analysis on vibration in the human dry skull with and without mouthguards was performed in order to clarify which elasticity modulus of the material and what form of the mouthguard are best. Nine different mouthguards were prepared for this study from three materials with different elasticity moduli amd three different forms: Soft MOLTENO (registered) mouthguards of long, middle, and short forms. Regular MOLENTO (registered) mouthguards of long, middle and short forms, and Hard MOLENTO(registered) mouthguards of long, middle and short forms. Thirty-five measurement points were established on the skull. The main measurement system was composed of a 512-D Vibration Generator, LV-1300 Laser-Doppler Vibrometer, CF-6400 FFT Analyzer, PC-9821Xn personal computer, and Vibrant PC modal analysis software. The decay rate was calculated to evaluate effectiveness of the absorption of vibration of the skull with and without mouthguards when stress was put on the skull. The results showed that he skull fitted with the long and middle mouthguards made of regular MOLTENO (registered) had a higher decay rate than the skull w the mouthguard, and that the material properties of the mouthguards influenced the absorption of the skull vibration more intensively that did the forms of the mouthguards. PMID- 8625434 TI - Characterization and canine bone tissue reaction of hydroxyapatite-coated titanium using thermal decomposition method. AB - Hydroxyapatite (HA)-coating by plasma spraying technique, which has been the common method used for HA coating, is not useful for coating the surfaces of porous metal implants because of difficulty in producing a homogeneous HA coating layer less than 30 micrometers. A new coating method, thermal decomposition, has been developed to obtain a dense coating layer with a thickness of 5 micrometer. In this study, the phase composition, surface morphology and adhesive strength between the coating layer and titanium substrate were evaluated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and tear-off testing, respectively. The biocompatibility of the implant prepared using this coating technique was compared with that of implants obtained using the plasma spraying technique or uncoated titanium following implantation into canine femurs. Bonding strength between the bone and implant was evaluated by pull-out testing. New bone formation was observed by light microscopy and SEM. The dense coating layer obtained by thermal decomposition ,method was found to be composed of well crystallized HA on XRD and SEM, and exhibited high adhesive strength. The biocompatibility of the samples obtained using this coating method was superior to that of the uncoated titanium implants and comparable to that obtained using the plasma spraying technique. The thin coating layer obtained using the thermal decomposition method should be useful for coating porous implants. PMID- 8625435 TI - Nature and nurture: possibilities for cancer control. AB - Nature and nurture interact and in most cases it is impossible to specify quantitatively the contribution of either to the causation of a disease. Only rarely is either neutral in the sense that the other accounts for all the variation in risk. It seems unlikely that more than a few per cent of all cancers will be accounted for by inherited susceptibility with a high penetrance and a neutral environment. Small variations in susceptibility may, however, be associated with different genetic alleles that will facilitate focused measures of prevention and perhaps provide a lead to causation. For the practical control of most cancers we must intervene by treatment or prevention. Knowledge of the variation in the incidence of cancers suggests that age-specific incidence rates could be reduced by 80-90%, half by the application of existing knowledge. The possibilities of control by prevention are discussed under the headings of tobacco smoke, alcohol, infection, diet, physical activity, reproduction, medicines and medical procedures, and occupation and pollution. In conclusion, attention is drawn to eight types of cancer that have become more common in the UK in the last 25 years, some of which we do not know how to prevent and which require urgent research. PMID- 8625436 TI - Convergence of three steroid receptor pathways in the mediation of nongenotoxic hepatocarcinogenesis. AB - The mechanisms by which peroxisome proliferators are able to regulate metabolic processes such as fat metabolism, while at the same time creating an environment for the development of hepatocellular carcinomas, is a central issue in the non genotoxic carcinogenesis field. The convergence of two members of the steroid receptor family (peroxisome proliferator-activated receptor, PPAR; and retinoid X receptor, RXR) has provided strong support for an oxidative stress component in this carcinogenesis process, but has yet to define clearly a pathway for the classical tumor promotion events associated with peroxisome proliferation. The findings presented here integrate a third member of the steroid receptor family into this process and suggest a novel autocrine loop and mechanism for creating both oxidative stress and tumor promotion. A central regulatory component in this pathway is farnesol which has recently been shown to induce transcription mediated by the steroid receptor family member, farnesoid X receptor (FXR). In this report, it is clearly demonstrated that farnesol can also upregulate the transcriptional events of PPAR, but most likely through a different farnesoid metabolite, resulting in the regulation of an entirely different set of genetic components. Deregulation of the activities of these receptors offers a provocative mechanism for explaining the hepatocarcinogenic effects of peroxisome proliferators in chronically treated rodents. PMID- 8625437 TI - Strain dependent effects of sex hormones on hepatocarcinogenesis in mice. AB - In order to study the interaction of genetic and hormonal factors during murine hepatocarcinogenesis, we compared the number of liver tumors induced by treatment of 12-day-old mice with N,N-diethylnitrosamine (DEN) (0.05 mumol/g body wt) in intact mice and animals gonadectomized at 8 weeks of age from the three inbred strains, C3H/HeJ (C3H), C57BL/6J (B6), and C57BR/cdJ (BR). At 50 weeks of age, the mean liver tumor multiplicity in intact BR females was 28 +/- 13, while that for intact female C3H and B6 mice was 1.4 +/- 4.7 and 0.5 +/- 1.0, respectively. In ovariectomized mice, the yield of liver tumors was approximately 8-fold higher than in intact C3H (10.3 +/- 7.5) and B6 (4.1 +/- 6.6) females. Only a slight increase (35 +/- 14) was seen in ovariectomized BR females compared to intact BR females. Castration resulted in lower mean tumor multiplicities at 32 weeks of age in the males of all three strains. Intact male C3H, B6, and BR mice had mean liver tumor multiplicities of 61 +/- 34, 7.4 +/- 13, and 26 +/- 18, respectively, while the mean tumor multiplicities in castrated C3H, B6, and BR mice were 24 +/- 14, 0.5 +/- 0.9, and 6.1 +/- 10 tumors per mouse, respectively. The apparent rate of growth of glucose-6-phosphatase-deficient, preneoplastic foci in DEN-treated BR females was significantly higher than in B6 females. The growth rates of hepatic foci in BR and B6 males were similar but foci in BR males were 5-fold more numerous than in B6 males. The high sensitivity of BR females may be due, at least in part, to the failure of ovarian hormones to inhibit the growth of preneoplastic foci and the subsequent development of liver tumors. Since BR males had a larger number of hepatic foci, it is likely that androgens increase the rate of focus formation in BR males. PMID- 8625438 TI - Growth modulation of hepatocytes and rat liver epithelial cells (WB-F344) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AB - Modulation of DNA synthesis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in primary cultures of hepatocytes and in rat liver epithelial cells (WB F344) to develop models for studies on the interactions between the activated Ah receptor and cellular growth control. In hepatocytes TCDD either positively or negatively modulated EGF-stimulated DNA synthesis. In the presence of ethinylestradiol 10(-12) M TCDD moderately increased EGF-stimulated DNA synthesis (approximately 30%). In contrast, 10(-9) M TCDD in the absence of ethinylestradiol decreased DNA synthesis (approximately 30%). Analysis of variance revealed that the TCDD effects were highly significant. The response of 'early genes' of the jun/fos family and the corresponding proteins was also studied under these two conditions. In agreement with the DNA synthesis data, the level of c-Jun was increased or decreased in nuclear extracts. Furthermore, DNA binding of Jun/Fos proteins, including c-Jun and Fra-1, was decreased under conditions of mitoinhibition, while the level of Fra-1 in nuclear extracts was increased. In WB-F344 cells TCDD treatment for 44 h increased DNA synthesis 2- to 3-fold in comparison with controls, based on measuring [3H]thymidine incorporation into DNA or on determining the nuclear labeling index with bromodeoxyuridine. This effect is probably due to inhibition of high density growth arrest by TCDD. The proposed cellular models may be useful to elucidate the interactions between the activated Ah receptor and signaling pathways of growth homeostasis. PMID- 8625439 TI - VP16 hypersensitivity and increased faulty recombination in ataxia telangiectasia lymphocytes characterized by the tandem translocation t(14;14)(q11;q32). AB - Ataxia telangiectasia (AT) patients show variable degrees of immunodeficiency and a higher than normal predisposition to lymphoid malignancies. AT cells are characterized by spontaneous chromosome instability resulting in chromosome breakage and in non random chromosome rearrangements. Sequential cytogenetic studies on T-lymphocytes from an AT patient showed the progressive development of a clone bearing a tandem translocation t(14;14)(q11;q32). The abnormal clone had spontaneous chromosome rearrangements. Compared to non clonal cells, the abnormal clone displayed a higher frequency of spontaneous chromosome rearrangements. In only the clonal cells we observed two particular and predominant rearrangements: isodicentric chromosomes and telomeric associations which may derive from faulty recombination. Chromosome instability induced by the etoposide VP16, a DNA topoisomerase II inhibitor, was evaluated in terms of chromosome breakage and SCE frequency. T-lymphocytes from the AT patient showed hypersensitivity to VP16 significantly higher than normal T-lymphocytes. The chromosome instability induced by VP16 is significantly higher in clonal than in non clonal cells, whilst the chromosome instability induced by the radiomimetic drug bleomycin is not significantly different in the two AT lymphocyte subpopulations. The different spontaneous chromosome instability in clonal and non clonal cells together with their different behavior after treatment with only VP16, suggest that clonal cells bearing the tandem translocation could have increased faulty recombination. Given the presence of translocations t(14;14)(q11;q32) in T prolymphocytic leukemias and T-cell tumors of non AT patients, our findings suggest that VP16 could be considered an antineoplastic treatment particularly indicated in these patients. PMID- 8625440 TI - Inhibition of 7,12-dimethylbenz[a]anthracene-induced lung tumorigenesis in A/J mice by food restriction is reversed by adrenalectomy. AB - Prior work has demonstrated that food restriction of mice markedly suppresses 12 O-tetradecanoylphorbol-13-acetate (TPA) promotion of skin papillomas and adrenalectomy prior to initiating food restriction completely reverses the tumor inhibitory effect of underfeeding. In the present experiment the effect of food restriction, with or without prior adrenalectomy, on 7,12 dimethylbenz[a]anthracene (DMBA)-induced lung tumor development in A/J mice was explored. Food restriction (27%), beginning 3 weeks after a single oral dose of 0.5 mg DMBA and continued for the duration of the experiment (14 weeks), significantly inhibited lung adenoma development, whereas adrenalectomy 2 weeks before initiating food restriction abolished the tumor inhibitory effect of underfeeding and also enhanced tumor development in the ad libitum fed mice. Plasma corticosterone levels were significantly elevated in food-restricted A/J mice, whereas plasma dehydroepiandrosterone (DHEA) levels showed no apparent change. These studies suggest that adrenal gland secretory products may play a general role in the tumor preventive effect of food restriction in laboratory mice. PMID- 8625441 TI - Mutations and oxidative DNA damage in phage M13mp2 exposed to N-nitrosomorpholine plus near-ultraviolet light. AB - Previously we reported that a direct-acting mutagen can be formed from N nitrosomorpholine (NMOR) on exposure to near-ultraviolet light (UVA, 320-400 nm). We have now studied the spectrum of mutations caused by NMOR plus UVA. M13mp2 phages suspended in a sodium phosphate buffer were treated with NMOR under UVA irradiation and Escherichia coli NR9099 was then infected with the phage. Mutations induced in the phage DNA lacZ alpha region were analyzed. The majority (approximately 50%) of the induced sequence changes were G to T transversions. This suggested that modifications in guanine residues were responsible for these transversions. We explored the formation of 7,8-dihydro-8-oxodeoxyguanosine (8 oxodG) in the DNA. When the phage were treated with NMOR plus UVA, 8-oxodG/dG in DNA increased up to 12-fold over the value in untreated control. When a mutM deficient mutant of E. coli CSH50 was used as the host, the mutation level was higher than that observed with CSH50. We conclude that 8-oxodG may be involved in mutations induced by NMOR plus UVA. PMID- 8625442 TI - Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts. AB - Suppressed expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), characterized as the Mer- phenotype, occurs only in malignant or transformed cell lines. To investigate the relationship between the transformation process and loss of MGMT expression, we derived 20 cloned lines of IMR90 normal fibroblasts transfected with the plasmid pSV3neo expressing the SV40 large-T antigen. Of the five lines that were grown until crisis phase, four emerged as continuously proliferating immortal lines. Of these, only one retained MGMT, the other three having become Mer-. In every case the loss of MGMT coincided with the final phase of immortalization following crisis. Because these were cloned cell lines it is clear that the phenotypic change to Mer- is not merely due to selection of a Mer- cell from the initial population, but must involve a cellular change in MGMT regulation. It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression. In addition, we have shown that, consistent with previous observations, both hypermethylation in promoter sequences and hypomethylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression. These studies also illustrate the utility of these new cloned cell lines for characterizing molecular events associated with transformation and immortalization. PMID- 8625443 TI - Enhancement of BALB/c 3T3 cells transformation by 1,2-dibromoethane promoting effect. AB - Two of the most representative halogenated aliphatic hydrocarbons, 1,2 dibromoethane and 1,1,2,2-tetrachloroethane, were tested in the two-stage cell transformation model for analysing the promoting ability. Both of these compounds had previously been found to exert genotoxic effects, probably acting as moderate initiators. BALB/c 3T3 cells were initiated with subtransforming doses of N methyl-N-nitro-N-nitrosoguanidine or 3-methylcholanthrene and then exposed to a chronic treatment with different non-transforming dosages of the two haloalkanes. 1,1,2,2-Tetrachloroethane did not exert any promoting activity in that system. By contrast, significant promoting effects by 1,2-dibromoethane were observed both in cells treated with N-methyl-N-nitro-N-nitrosoguanidine and in cells treated with 3-methylcholanthrene. Promotion of the transformation process initiated with 3-methylcholanthrene was detectable when confluent cells in the chemical-treated plates were replated in the level-II amplification test. This experimental procedure allowed cells to perform further rounds of replications and transformed foci to became detectable. Results gave evidence for a promoting role of 1,2 dibromoethane in multistep carcinogenesis, probably responsible for the higher oncogenic ability of this compound with respect to 1,1,2,2-tetrachloroethane. PMID- 8625444 TI - Azoxymethane enhances ligand-induced activation of EGF receptor tyrosine kinase in the colonic mucosa of rats. AB - Recent observations suggest that transforming growth factor alpha (TGF-alpha), which binds to the epidermal growth factor (EGF) receptor (EGFR), may induce neoplastic growth of the colonic mucosa through an autocrine mechanism. To assess the functional role of TGF-alpha in colonic carcinogenesis the present investigation examines the changes in TGF-alpha-and EGF-induced activation of intrinsic tyrosine kinase (Tyr-k) activity of EGFR in the colonic mucosa of rats after administration of the colonic carcinogen azoxymethane (AOM; 20 mg/kg body wt). Five days after a single injection of AOM to 4- to 5-month old rats proliferative activity (as assessed by 5-bromo-2'-deoxyuridine immunoreactivity) in the colonic mucosa was increased by approximately 700% over the corresponding saline-injected controls. This was accompanied by: (i) a marked rise in autophosphorylation of a number of mucosal proteins, including one with a M(r) of 170 kDa, a molecular mass that corresponds to EGFR; (ii) a 110-130% increase in basal EGFR Tyr-k activity. Despite this rise in basal EGFR Tyr-k activity, exposure of isolated colonocytes or detergent-solubilized colonic mucosa from AOM treated animals to either 1 x 10(-8) M TGF-alpha or EGF caused a further 90-160% increase in EGFR Tyr-k activity over the corresponding basal levels. In contrast, bombesin produced no apparent change in EGFR Tyr-k activity. We conclude that increased ligand-induced activation of EGFR Tyr-k may be an important event for development of the hyperproliferative state associated with induction of colorectal neoplasia. PMID- 8625445 TI - Apoptotic and anti-proliferative effects of fumonisin B1 in human keratinocytes, fibroblasts, esophageal epithelial cells and hepatoma cells. AB - Fumonisin B1 is associated with various animal and human carcinomas and toxicoses, including leukoencephalomalacia, hepatocarcinoma, pulmonary edema and esophageal carcinoma. We have examined the cellular effects of fumonisin B1 in vitro using cellular model systems relevant to potential human target tissues. Although fumonisin B1 has been described as a mitogen in Swiss 3T3 cells based on stimulation of [3H]thymidine incorporation, in the current work it was found that fumonisin B1 inhibited incorporation of [3H]thymidine by cultured neonatal human keratinocytes and HepG2 human hepatocarcinoma cells at 10(-7) and 10(-4) M respectively. Fumonisin B1 also inhibited clonal expansion of normal human keratinocytes and HET-1A human esophageal epithelial cells at 10(-5) M and growth in mass culture of normal human fibroblasts at 10(-7) M. The clonogenicity of normal human keratinocytes decreased to 45.5% of controls after exposure to 10( 4) M fumonisin B1 for 2 days. However, no differences in the cell cycle distribution of cultured keratinocytes was noted after exposure to 10(-5) M fumonisin B1 for 40 h. The viability of normal human keratinocytes and HET-1A cells decreased as a result of fumonisin B1 treatment, as determined by a fluorescein diacetate/propidium iodide flow cytometric cell viability assay. Fumonisin B1-treated keratinocytes released nucleosomal DNA fragments into the medium 2-3 days after exposure to 10(-4) M fumonisin B1 and increased DNA strand breaks were detected in attached keratinocytes exposed to 0-10(-4) M fumonisin B1 using a terminal deoxynucleotidyl transferase-based immunochemical assay system. Furthermore, fumonisin B1-treated keratinocytes and HET-1A cells developed morphological features consistent with apoptosis, as determined by phase contrast microscopy, fluorescent microscopy of acridine orange stained cells and electron microscopy. These results are consistent with the occurrence of fumonisin B1 mediated apoptosis in vitro. PMID- 8625446 TI - Dual phenotypic expression of hepatocytes and bile ductular markers in developing and preneoplastic rat liver. AB - This study supports the existence of a pluripotent liver stem cell population which has the potential to differentiate into hepatocytes and bile ductular cells. We compared the expression of hepatocyte-specific and bile ductular specific markers in fetal and preneoplastic rat liver. L-pyruvate kinase (L-PK) and alpha glutathione S-transferase (GST) were used as adult hepatocyte-specific markers, while cytokeratin 19 (CK19) was used as a bile ductular-specific marker. pi GST and M2-pyruvate kinase (M2-PK), which are fetal hepatocyte-specific and expressed at high levels in the oval and duct-like cells, were also used. We characterized fetal liver derived from 13-21 days of gestation (E13-E21). pi GST was detected in the E18 hepatoblasts, which form the intrahepatic bile ducts, while CK19 was detected at E19. Some of these cells express alpha GST and L-PK from E19 to E21. Oval, duct-like and bile ductular cells in rats treated with a choline-deficient diet containing 0.07% ethionine (CDE diet) for up to 8 weeks were characterized by double immunocytochemistry. L-PK and alpha GST are absent from bile ductular cells in the normal adult liver and up to 3 weeks of CDE treatment. After 4-5 weeks on CDE treatment, the majority of bile ductular cells express L-PK, while at 6 weeks some co-express L-PK and alpha GST. There are two populations of oval cells, a major population expressing only the fetal hepatocyte markers, while a minor population expresses the fetal hepatocyte, adult hepatocyte and bile ductular markers. There are at least three different duct-like cell populations which co-express different markers and have characteristics of fetal hepatocytes at sequential stages of differentiation. One population co-expresses pi GST and M2-PK and is similar to fetal hepatocytes derived from E13-E14 fetuses. The second expresses the two fetal markers and L PK, and this reflects characteristics of E15 hepatocytes. The third expresses pi GST, M2-PK, L-PK and alpha GST which is characteristic of E16-E19 hepatocytes. Upon withdrawal of the CDE diet, autoradiography using tritiated thymidine shows that oval and duct-like cells differentiate into hepatocytes. This study demonstrates that oval and duct-like cells express both hepatocytic and bile ductular markers, and have the capacity to differentiate into hepatocytes, characteristics similar to hepatoblasts in the developing rat liver. PMID- 8625447 TI - Analysis of a germ line polymorphism of the p53 gene in lung cancer patients; discrete results with smoking history. AB - The p53 tumor suppressor gene is often mutated in various human cancers and a common polymorphism is known at codon 72 of exon 4, with two alleles encoding either arginine (CGC) or proline (CCC). Association of this polymorphism with any human cancer susceptibility has yet to be clarified. We have conducted a case control study in Japan on the distribution of the three genotypes with 191 lung cancer patients, 152 control patients with non-cancerous pulmonary diseases and 115 colorectal cancer patients. The genotypes were examined by PCR using DNA samples from peripheral blood lymphocytes. Frequency distributions of the three genotypes were quite comparable with each other among groups, with allelic frequencies of approximately 60% for arginine and 40% for proline. The genotypic frequencies in lung cancer patients, however, were largely different between smokers and non-smokers (chi 2 = 13.5, df = 2, P < 0.001). Compared with the control and colorectal cancer patients a significant difference in genotypic frequency was observed only in non-smoker lung cancers (chi 2 = 10.9, df = 2, P < 0.01), with an excess of Arg/Arg homozygotes and a deficit of Arg/Pro heterozygotes. Our present data suggest that the p53 polymorphism affects the risk of lung cancer unrelated to smoking. PMID- 8625448 TI - Structure-function relationships of the dietary anticarcinogen ellagic acid. AB - Ellagic acid is a complex planar molecule which demonstrates a variety of anticarcinogenic activities. Ellagic acid has been shown to inhibit the CYP1A1 dependent activation of benzo[a]pyrene; to bind to and detoxify the diolepoxide of benzo[a]pyrene; to bind to DNA and reduce the formation of O6-methylguanine by methylating carcinogens; and to induce the phase II detoxification enzymes glutathione S-transferase Ya and NAD(P)H:quinone reductase. Chemical analogs of ellagic acid were synthesized to examine the relationship between the hydroxyl and lactone groups of the ellagic acid molecule and its different anticarcinogenic activities. These studies demonstrated that both the 3-hydroxyl and the 4-hydroxyl groups were required for ellagic acid to directly detoxify the diolepoxide of benzo[a]pyrene, while only the 4-hydroxyl groups were necessary for ellagic acid to inhibit CYP1A1-dependent benzo[a]pyrene hydroxylase activity. Induction of glutathione S-transferase Ya and NAD(P):quinone reductase required the lactone groups of ellagic acid, but the hydroxyl groups were not required for the induction of these phase II enzymes. In addition, the lactone groups, but not the hydroxyl groups, were required for the analogs to reduce the carcinogen induced formation of O6-methylguanine. Thus, different portions of the ellagic acid molecule are responsible for its different putative anticarcinogenic activities. PMID- 8625449 TI - Molecular effects of genistein on estrogen receptor mediated pathways. AB - Genistein, a component of soy products, may play a role in the prevention of breast and prostate cancer. However, little is known about the molecular mechanisms involved. In the present study, we examined the effects of genistein on the estrogen receptor positive human breast cancer cell line MCF-7. We observed that genistein stimulated estrogen-responsive pS2 mRNA expression at concentrations as low as 10(-8) M and these effects can be inhibited by tamoxifen. We also showed that genistein competed with [3H]estradiol binding to the estrogen receptor with 50% inhibition at 5 x 10(-7) M. Thus, the estrogenic effect of genistein would appear to be a result of an interaction with the estrogen receptor. The effect of genistein on growth of MCF-7 cells was also examined. Genistein produced a concentration-dependent effect on the growth of MCF-7 cells. At lower concentrations (10(-8)-10(-6) M) genistein stimulated growth, but at higher concentrations (> 10(-5) M) genistein inhibited growth. The effects of genistein on growth at lower concentrations appeared to be via the estrogen receptor pathway, while the effects at higher concentrations were independent of the estrogen receptor. We also found that genistein, though estrogenic, can interfere with the effects of estradiol. In addition, prolonged exposure to genistein resulted in a decrease in estrogen receptor mRNA level as well as a decreased response to stimulation by estradiol. PMID- 8625450 TI - CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane. AB - The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats against 9,10-dimethyl-1,2 benz[a]anthracene tumorigenesis, thought to be due, at least in part, to induction of phase II detoxification. We investigated the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible for activation of several carcinogens, including dialkylnitrosamines. Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown to be a potent competitive inhibitor of CYP2E1 with a Ki of 37.0 +/- 4.5 microM. In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA). Sulforaphane at concentrations of > 0.8 microM inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone. Unscheduled DNA synthesis induced by NDMA (33.5 microM) in mouse hepatocytes was inhibited in a dose dependent manner by sulforaphane at 0.064-20 microM. Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 micrograms/plate), a direct acting mutagen, in the Salmonella assay. It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of Salmonella employed and cytotoxic only at high concentrations (> or = 0.5 mM). These findings suggest that inhibition of CYP2E1 by sulforaphane may offer chemoprotection against carcinogenic substrates of this enzyme. PMID- 8625451 TI - Mutagenic specificities and adduct distributions for 7 bromomethylbenz[a]anthracenes. AB - Mutation induction in the supF gene of the plasmid pS189 by 7 bromomethylbenz[a]anthracene and 7-bromomethyl-12-methylbenz[a]anthracene was examined. The former compound was substantially more mutagenic than the latter but a much greater proportion of the total mutations were located at mutation hotspots for the 12-methyl derivative. The overall correlation between sites of mutation and sites of polymerase arrest (an indicator of adduct formation) through the supF gene was poor. Although these bromocompounds should form only a single guanine adduct (unlike dihydrodiol epoxides that form both cis and trans adducts) more than one mutational change was found at a given site, although the predominant base substitution was G-->T for either compound. PMID- 8625452 TI - A subnecrogenic dose of diethylnitrosamine is able to initiate hepatocarcinogenesis in the rat when coupled with fasting/refeeding. AB - Caloric restriction causes a generalized decrease in growth rate and has been repeatedly associated with an inhibitory effect on cancer development in several systems. In contrast, exposure to complete fasting followed by refeeding is a metabolic condition associated with increased cell turnover in different organs, including the liver. The present study examines whether such condition is able to sustain the induction of initiated hepatocytes following a subnecrogenic dose of diethylnitrosamine (DENA). Male Fisher-344 rats were fasted for 4 days and 1 day after refeeding they were given a single dose of DENA (20 or 200 mg/kg body wt, i.p.). Negative and positive control groups were fed ad libitum and injected with 20 and 200 mg/kg of DENA, respectively. One week later all animals were subjected to the resistant hepatocyte model for the selection of hepatocyte nodules and they were killed 2 weeks thereafter. Results indicated the presence of gamma glutamyltransferase (GGT) positive foci and nodules (38 +/- 7/cm2) in rats regularly fed and given 200 mg/kg of DENA, while virtually no focal lesions (< 1/cm2) were found in the group receiving 20 mg/kg of DENA and fed throughout the experiment. However, a significant number of GGT positive foci/nodules (14 +/- 7) also developed in rats exposed to fasting and given 20 mg/kg of DENA 24 h after refeeding. No evidence of hepatocellular necrosis was found in the latter group following DENA administration. No effect of fasting was observed when rats received 200 mg/kg of DENA. It is concluded that fasting/refeeding provides conditions which are able to sustain initiation in rat liver by a subnecrogenic dose of a carcinogen. These findings are in contrast with the commonly reported inhibitory effect of chronic food restriction on various stages of carcinogenesis, including initiation. PMID- 8625453 TI - Effect of selective and non-selective muscarinic blockade on baclofen inhibition of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats. AB - The effects of baclofen, a gamma-amino-n-butyric acid receptor B agonist, on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine and how its effects are influenced by selective (M1) and non-selective (M1 and M2) pharmacological blockade of muscarinic receptors were investigated in inbred Wistar rats. Rats were given s.c. injections of 8 mg/kg body wt baclofen with and without 0.5 mg/kg body wt atropine (non-selective M1 and M2 muscarinic receptor antagonist) or 1.0 mg/kg body wt pirenzepine (selective M1 muscarinic receptor antagonist) every other day after a 25 week carcinogen treatment. At week 52 baclofen significantly decreased the incidence of gastric cancers. Concomitant treatment with atropine significantly attenuated the inhibition by baclofen of gastric carcinogenesis, but combined use with pirenzepine had no significant effect on the inhibition by baclofen of gastric carcinogenesis. Baclofen also significantly decreased the labeling index of the antral mucosa. Baclofen plus atropine attenuated the decrease in the labeling index of the antral mucosa due to baclofen, but baclofen plus pirenzepine had no significant effect on the labeling index. These results suggest that the inhibition of gastric carcinogenesis by baclofen is mediated through muscarinic receptors and M2 receptors, but not M1 receptors, are involved in this response. PMID- 8625455 TI - High yields of K-ras mutations in intraductal papillary mucinous tumors and invasive adenocarcinomas induced by N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine in the pancreas of female Syrian hamsters. AB - Ductal adenocarcinoma, the most common form of pancreatic cancer in humans, is associated with activation of the K-ras oncogene in approximately 90% of cases. In contrast, K-ras mutations are found in < 50% of the relatively rare intraductal papillary mucinous tumor (IPMT), which arises in the main pancreatic ducts. Since both adenocarcinomas and IPMTs are believed to arise from ductal cells and progress through similar sequences of morphological changes (i.e. flat hyperplasia, papillary hyperplasia, atypia and carcinoma in situ), it is clear that such progression may not always necessitate activation of the ras oncogene. Experimentally ductal adenocarcinomas of the pancreas can be induced in the hamster model by a brief treatment with N-nitroso(2-hydroxypropyl)(2 oxopropyl)amine (HPOP), while IPMTs can be induced by a combined treatment with HPOP and orotic acid (OA) in an initiation/promotion schedule. Since animals are exposed to the carcinogen only once, initiated normal epithelium is expected to give rise to a wide spectrum of neoplastic and preneoplastic lesions, progression of which will depend on the extent of mutagenesis induced at initiation in the targeted cells. In order to investigate the role of K-ras in progression of IPMTs as compared with adenocarcinomas we have examined the presence of K-ras mutations in the above two types of experimentally induced pancreatic cancers, as well as in associated and preneoplastic lesions. K-ras mutations at codons 12, 13 and 61 were determined by a designed restriction fragment length polymorphism method using mismatched nested primers in 77 neoplastic and preneoplastic foci microdissected from 20 pancreases. Mutations were found in all foci of atypical hyperplasia, in carcinomas in situ and invasive cancer, whether such lesions originated in lobular tissue or in the main pancreatic duct. Mutations were also found in papillary hyperplasia and flat hyperplasia in small ducts and also in the main duct at high frequency. With one exception, all ras mutations were G-->A transitions at the second base of codon 12. Mutations were occasionally accompanied by excessive presence of the mutant ras allele or loss of the wild type ras allele, events that were more frequent in atypical hyperplasia (5/17), carcinomas in situ (5/14), IPMTs (2/5) and invasive adenocarcinomas (2/5) than in flat hyperplasia (0/6) or papillary hyperplasia (2/18). Our results demonstrate that: (i) K-ras mutations, predominantly G-->A transitions, are present in all experimentally induced hamster tumors; (ii) the incidence of K-ras mutations in IPMTs is lower in humans than in the hamster model; (iii) advanced lesions in both adenocarcinomas and IPMTs were frequently associated with an excess of the mutant over the wild-type K-ras allele. It is likely that both adenocarcinomas and IPMTs induced chemically in the hamster model arise by mechanisms which involve early activation of K-ras. Such a mechanism seems to be applicable only in a fraction of human IPMTs. PMID- 8625454 TI - A new Salmonella typhimurium NM5004 strain expressing rat glutathione S transferase 5-5: use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system. AB - The Escherichia coli mu operon was subcloned into a pKK233-2 vector containing rat glutathione S-transferase (GST) 5-5 cDNA and the plasmid thus obtained was introduced into Salmonella typhimurium TA1535. The newly developed strain S.typhimurium NM5004, was found to have 52-fold greater GST activity than the original umu strain S.typhimurium TA1535/pSK1002. We compared sensitivities of these two tester strains, NM5004 and TA1535/pSK1002, for induction of umuC gene expression with several dihaloalkanes which are activated or inactivated by GST 5 5 activity. The induction of umuC gene expression by these chemicals was monitored by measuring the cellular beta-galactosidase activity produced by umuC"lacZ fusion gene in these two tester strains. Ethylene dibromide, 1-bromo-2 chloroethane, 1,2-dichloroethane, and methylene dichloride induced umuC gene expression more strongly in the NM5004 strain than the original strain. 4 Nitroquinoline 1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine were found to induce umuC gene expression to similar extents in both strains. In the case of 1 nitropyrene and 2-nitrofluorene, however, NM5004 strain showed weaker umuC gene expression responses than the original TA1535/pSK1002 strain. 1,2-Epoxy-3-(4' nitrophenoxy)propane, a known substrate for GST 5-5, was found to inhibit umuC induction caused by 1-bromo-2-chloroethane. These results indicate that this new tester NM5004 strain expressing a mammalian GST theta class enzyme may be useful for studies of environmental chemicals proposed to be activated or inactivated by GST activity. PMID- 8625456 TI - Ciprofibrate represses alpha 2u-globulin expression in liver and inhibits d limonene nephrotoxicity. AB - Peroxisome proliferators are a class of compounds which induce hepatomegaly and peroxisome proliferation in liver parenchymal cells. One of the earliest known effects of peroxisome proliferators is the rapid transcriptional activation of the genes responsible for the peroxisomal beta-oxidation system in liver. Long term administration of these chemicals to rats and mice results in the development of hepatocellular carcinomas. Here we report that mRNA for alpha 2u globulin, a rodent male specific protein, is markedly reduced or undetectable by Northern blot analysis of total RNA in the livers of rats treated with ciprofibrate. This was further confirmed by immunoblot analysis using antibodies against alpha 2u-globulin. Nevertheless, immunohistochemical staining and in situ hybridization showed respectively the presence of a few cells that contained alpha 2u-globulin protein and its mRNA. The alpha 2u-globulin mRNA reappeared in the liver 2 weeks following the cessation of ciprofibrate treatment. Feeding of ciprofibrate for two weeks followed by simultaneous feeding of ciprofibrate and a nephrotoxic chemical d-limonene for 5 weeks revealed that ciprofibrate prevented the renal accumulation of alpha 2u-globulin and the nephrotoxicity associated with the binding of d-limonene with alpha 2u-globulin. PMID- 8625457 TI - Generation of DNA base modification following treatment of cultured murine keratinocytes with benzoyl peroxide. AB - Benzoyl peroxide (BzPO) is a free radical generating compound that acts as a tumor promoter and progressor in mouse skin. BzPO is cleaved in the presence of copper to produce benzoyloxyl and phenyl radicals. Treatment of mutation reporter plasmids with BzPO and copper yields predominantly single-strand breaks and G-->T transversion mutations. To explore the role of base modifications in the possible mammalian mutagenicity of BzPO the formation of 8-hydroxy-2'-deoxyguanosine (8 OHdG) within the DNA of cultured murine keratinocytes was investigated. Treatment with 10 microM BzPO produced a maximum 3-fold increase in levels of 8-OHdG versus vehicle controls within 1-2 h, with significant levels of 8-OHdG persisting 6 h after initial exposure to BzPO. Pretreatment with the copper chelator bathocuproine disulfonic acid reduced the levels of 8-OHdG generated by BzPO to near background. However, treatment with the iron chelator desferal did not. The stable metabolic product of BzPO benzoic acid was ineffective in producing 8 OHdG. Depletion of cellular glutathione with L-buthionine-(S,R)-sulfoximine increased the amount of BzPO-generated 8-OHdG, while supplementation with glutathione monoethyl ester reduced the number of 8-OHdG molecules formed. Collectively, these results suggest that BzPO at non-cytotoxic concentrations undergoes copper-dependent activation to a reactive product to generate 8-OHdG within cultured murine keratinocytes. PMID- 8625458 TI - Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme. AB - The Escherichia coli MutY gene was cloned into a modified pET-11 plasmid which was then transfected into an E.coli HMS174 host for overproduction of the MutY mismatch repair (MR) enzyme. Approximately 30-50% of the total cellular protein in the transformed HMS174 cells was isopropyl-beta-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS-PAGE gel following electrophoresis. The MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chromatography followed by gel filtration chromatography. The purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and then the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activity was DNA sequence-dependent. The reliability of the assay in determining the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymerase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/A or C/A for cleavage by the MutY MR enzyme. The assay results revealed the site and nature of known G:C<-->T:A mutations. In addition, a previously unknown G:C to T:A mutation, which was misread in the sequencing analysis of a tumor DNA preparation, was identified by this assay. PMID- 8625459 TI - Influence of the antioxidant N-acetylcysteine and its metabolites on damage induced by bleomycin in PM2 bacteriophage DNA. AB - Bleomycin is considered to be a useful model compound for studying environmental carcinogenesis, due to its broad spectrum of DNA damaging properties. In addition, bleomycin is a useful antitumor drug because of its cytotoxic properties. To investigate the influence of the antioxidant N-acetylcysteine and its metabolites glutathione and cysteine on bleomycin-induced DNA damage and more importantly to gain insight into the biological relevance of such damage, PM2 DNA was exposed to Cu(2+)-bleomycin in the presence and absence of the thiols N acetylcysteine, glutathione and cysteine. It was found that the presence of these thiols led to a considerable enhancement of bleomycin-induced single- and double strand breaks and a concomitant decrease in the biological activity of PM2 DNA in a dose-dependent way. A similar observation was made when ascorbic acid was used. Bleomycin showed no DNA damaging activity when PM2 DNA was pretreated with the strong Fe ion chelator desferal and its activity was strongly inhibited by the addition of Cu2+ ions or under hypoxic (N2) conditions. Cu(2+)-bleomycin under our conditions is not active by itself, but most probably after binding to DNA exchanges Cu2+ for Fe3+ bound to DNA. Fe(3+)-bleomycin is then reduced to Fe(2+) bleomycin, a process potentiated by the added antioxidants, and subsequently activated by O2. The contribution to biological inactivation of bleomycin alone or in the presence of ascorbic acid is only approximately 15%. The contribution to lethality in the presence of thiols is higher. These results indicate that ascorbic acid only enhances the DNA damaging properties of bleomycin, whereas the thiol compounds in addition influence the type of DNA damage. The remainder of the biological inactivation is probably caused by double damage, such as single strand breaks with closely opposed alkali-labile sites or base damage. PMID- 8625460 TI - The use of initiated cells as a test system for the detection of inhibitors of gap junctional intercellular communication. AB - The effects of five non-mutagenic carcinogens--Aroclor 1260, benzoyl peroxide (BP), phenobarbital (PB), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,1' (2,2,2-trichloroethylidene)bis[4-chlorobenzene] (DDT)--on gap junctional intercellular communication (GJIC) were tested in a cell line consisting of initiated cells (3PC). Four agents suspected of tumor promotion activity--o anisidine, clofibrate, L-ethionone and d-limonene--were also tested for their effects on GJIC. Finally sodium fluoride (NaF), whose carcinogenic property is still unclear, was tested for its effects on GJIC in the 3PC cell line. Four of the five selected tumor promoters (Aroclor 1260, BP, DDT and TPA) decreased GJIC between these initiated epidermal cells. The four non-mutagenic carcinogens with tumor-promoting activity in vivo (o-anisidine, clofibrate, L-ethionine and d limonene) all inhibited GJIC, whereas NaF had no effect. Seven compounds (o anisidine, Aroclor 1260, BP, DDT, L-ethionine, d-limonene and TPA) had a dose dependent as well as time-dependent inhibitory effect on GJIC. Under the experimental conditions used, clofibrate showed only a dose-related inhibition of GJIC. PB showed no inhibitory effect on GJIC in the 3PC cell line. In order to determine the role of biotransformation in the tumor-promoting activity of PB, its effect on GJIC was also examined in the presence of an Aroclor 1254-induced rat liver homogenate (S9 mix) and in the hepatoma cell line HepG2. In the presence of rat liver homogenate PB decreased GJIC in the 3PC cell line, whereas in the HepG2 cells PB showed a time- and dose-dependent inhibitory effect. To study the potential differences in susceptibility of cells representing different stages in the process of tumor formation, the effect of the selected tumor promoters on GJIC was also investigated in primary mouse keratinocytes and in a mouse skin carcinoma-derived cell line (CA3/7). Primary keratinocytes were sometimes more (BP and clofibrate) and sometimes less sensitive (ethionine and limonene) for inhibitory effects on GJIC compared to the effects in the cell line 3PC. Except for TPA and anisidin, GJIC between the CA3/7 cells was less affected by the selected agents compared to the 3PC cell line. These results show that, during the process of tumor formation the susceptibility of cells to inhibition of GJIC by tumor promoters is variable. Overall the CA3/7 cells are less sensitive compared to 3PC cells. The susceptibility of primary keratinocytes is variable compared to 3PC cells, depending on the agent used. These results also show that GJIC is a valid parameter for testing the tumor-promoting activity of compounds. Finally, this study demonstrates that mouse keratinocyte cell lines could serve as an in vitro model for the detection of non-mutagenic carcinogens with diverse target organs in vivo. For this use the cell line consisting of initiated cells (3PC) is more sensitive than the carcinoma-derived cell line CA3/7. PMID- 8625461 TI - Chemically induced lung and forestomach neoplasias in transgenic mice carry mutant forms of the human c-Ha-ras transgene. AB - Susceptibility to lung carcinogens and genetic changes in neoplastic lesions were investigated in transgenic mice carrying a human hybrid c-Ha-ras gene, encoding a prototype p21 gene product. Nine-week-old male and female transgenic mice and non transgenic littermates were injected i.p. with 6-nitrochrysene (6NC) three times biweekly or administered urethane in their drinking water for 3 weeks. Control mice were given dimethylsulfoxide (DMSO), the solvent for 6NC, alone. The incidences of lung adenocarcinomas were four out of seven female (57%) transgenic mice treated with 6NC and three out of three males (100%) and three out of three females (100%) receiving urethane. No adenocarcinomas were observed in control animals or non-transgenic mice. Adenomas developed in all treated groups, but the incidence and multiplicity were higher in transgenic animals than in their non transgenic counterparts. In the 6NC-treated group, forestomach papillomas and squamous cell carcinomas were also observed in both male (25 and 50%) and female (56 and 33%) transgenic mice. PCR-SSCP and DNA sequence analysis of these induced lesions revealed point mutations at codon 61 of transgenic human c-Has-ras, from CAG (Gln) to CTG (Leu) or CAG (Gln) to AAG (Lyn) in lung hyperplasias (two out of three), an adenoma (one out of two), adenocarcinomas (five out of seven) and forestomach squamous cell carcinomas (four out of five). Mutations were not observed in forestomach papillomas. No changes in mouse Ha-ras or Ki-ras were found in any lesions. Furthermore, p21 overexpression was not evident in lung or forestomach tumors on immunohistochemical analysis. These findings indicate a high sensitivity to lung carcinogens in transgenic mice carrying the human c-Ha ras gene and that this might be effected by mutational activation. PMID- 8625462 TI - Observation and prevention of an artefactual formation of oxidized DNA bases and nucleosides in the GC-EIMS method. AB - Gas chromatography coupled to electron impact mass spectrometry (GC-EIMS) analysis following hydrolysis of DNA is a widely used assay for the detection of oxidized nucleobases and nucleosides. However, evidence was recently provided for an oxidation of guanine residues of hydrolysed DNA during the silylation prior to GC-EIMS analysis. This reaction accounts for the overestimation of the yield of 8 oxo-7,8-dihydroguanine by GC-EIMS. In the present work, we showed that adenine, cytosine, thymine and thymidine also give rise to oxidized derivatives during the derivatization. This was inferred from the measurement of the amount of 5 formyluracil, 5-hydroxymethyluracil, 5-hydroxycytosine (5-OHCyt), 8-oxo-7,8 dihydroadenine (8-OxoAde) and 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd) in a series of experiments based on the use of purified bases and nucleosides. Isotopically labelled oxidized bases and 5-HMdUrd were used as internal standards to control the quantitative aspect of the silylation reaction. Support for an artefactual oxidation of hydrolysed DNA was provided by the comparison of the amount of 8-OxoAde and 5-OHCyt detected within native and gamma-irradiated DNA by HPLC-EC and GC-EIMS. To prevent the artefactual formation of oxidized bases during the silylation, an approach based on an HPLC prepurification was developed to remove the precursors of the oxidized bases measured in the DNA sample. The HPLC/GC-EIMS assay was successfully applied to the quantitation of 8-OxoAde and 5 OHCyt in calf thymus DNA. In addition, the detection of the dose-dependent formation of 5-HMdUrd in isolated DNA exposed to ionizing radiation was achieved using the same approach. PMID- 8625463 TI - Relationship between induction of mammary tumors and change of testicular functions in male rats following gamma-ray irradiation and/or diethylstilbestrol. AB - Male Wistar-MS (W/MS), Fisher-344 (F-344) and Sprague-Dawley (SD) rats were divided into four groups including a control group implanted with a cholesterol pellet. Rats in the three experimental groups were treated with gamma-ray irradiation (260 cGyt) alone, diethylstilbestrol (DES) pellet implantation alone or both irradiation and DES, and all rats were observed for detection of mammary tumors for 1 year. Morphologically, well-developed mammary glands were observed in the SD rats at ages corresponding to the time of irradiation. But, the mammary glands in the W/MS and F-344 rats showed a lower degree of differentiation than those in the SD rats. No mammary tumor developed spontaneously in the W/MS and F 344 strains of rats during the experimental period. The rats administered both DES and irradiation showed significantly increased incidence of mammary tumors compared with the control, the incidence being 80.9% in the SD rats, 35.0% in the W/MS rats, and 9.4% in the F-344 rats, respectively. The incidence of tumor in the SD rats treated with irradiation alone and with DES alone was 9.5% and 14.3%, respectively, but no tumor development was observed in the F-344 rats treated with either irradiation alone or DES alone or in the W/MS rats treated with DES alone. The magnitude of the decrease of testicular weight in the SD rats implanted with DES after irradiation (to 70% of the control weight) was slightly less marked than that in either the W/MS (35%) or F-344 (16%) rats. The testicular atrophy showed a correlation with the accessory sex organ weight at the end of the experiment, serum testosterone concentration, and incidence of mammary tumors. Following administration of DES pellets after the irradiation, the activity of delta 5-3 beta- and of 17 beta-hydroxysteroid dehydrogenase in the testes showed the order F-344 < W/MS = SD and F-344 = W/MS < SD, respectively. Compared with the control, the irradiated F-344 rats implanted with DES pellets showed hypertrophied pituitary glands (10.7-fold, P < 0.01) as well as increased serum prolactin concentration (21.4-fold, P < 0.01). Of the three strains treated with both irradiation and DES, the F-344 rats showed the highest concentration of serum prolactin but the lowest incidence of mammary tumors. Our results suggest that W/MS, F-344 and SD male rats have differing susceptibilities for the induction of mammary tumor following irradiation. We discuss the relationship between testicular and pituitary functions and male mammary tumorigenesis. PMID- 8625465 TI - Inhibitory effects of d-limonene on the development of colonic aberrant crypt foci induced by azoxymethane in F344 rats. AB - The modifying effect of the monoterpenoid d-limonene in drinking water on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of d-limonene intake on ornithine decarboxylase (ODC) activity and on the silver stained nucleolar organizer region protein (AgNOR) count in the colonic mucosa were also estimated. Animals were given 3 weeks s.c. injections of AOM (15 mg/kg body wt) to induce ACF. These rats were treated with or without 0.5% d-limonene in the drinking water, starting 1 week before the first dosing with AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity and AgNOR count/nucleus in the colon. In rats given AOM and d-limonene the frequencies of ACF and aberrant crypts/colon, and aberrant crypts/focus were significantly decreased compared with those of rats given AOM alone (P < 0.01, P < 0.001 and P < 0.001 respectively). Number of AgNOR counts/nucleus of rats treated with AOM and d-limonene was significantly smaller than that of rats treated with AOM alone (P < 0.001). These results suggest that the monoterpenoid d-limonene might be a chemopreventive agent for colonic carcinogenesis in rats. PMID- 8625464 TI - Nickel(II) induces alterations in EGF- and TGF-beta 1-mediated growth control during malignant transformation of human kidney epithelial cells. AB - We have previously described immortalization of normal human kidney epithelial cells by nickel(II) and the subsequent tumorigenic conversion by v-Ha-ras transfection. We report here that nickel(II) induces alterations in growth regulatory control. Normal human kidney epithelial cells (NHKE) were growth inhibited by transforming growth factor beta 1 (TGF-beta 1). This effect was abrogated in both the immortalized (IHKE) and transformed (THKE) cells. NHKE expressed approximately 4700 high-affinity binding sites/cell for TGF-beta 1. IHKE and THKE showed reduced binding of 47% and 44% relative to NHKE respectively. On the other hand, expression of epidermal growth factor (EGF) receptors was elevated in IHKE (260%) and THKE (236%) relative to NHKE, which expressed 1.5 x 10(5) receptors/cell. Preincubation of IHKE and THKE with TGF beta 1 resulted in reduced EGF binding, whereas this binding was unaltered in NHKE. Exposure of human kidney epithelial cells to EGF led to tyrosine phosphorylation of the EGF receptor and other cellular proteins in the mol. wt range from 42 to > 300 kDa. The level of receptor phosphorylation induced by EGF reflected receptor expression. Tyrosine phosphorylated proteins appear to be identical in all three cell lines, and reach phosphorylation maxima independently of EGF receptor expression. These studies indicate that nickel carcinogenesis may involve changes in sets of genes important in normal growth regulation. PMID- 8625466 TI - Piroxicam-induced regression of azoxymethane-induced aberrant crypt foci and prevention of colon cancer in rats. AB - Piroxicam has been shown to prevent azoxymethane (AOM)-induced aberrant crypt foci and colon cancer in rats. In this communication we evaluate whether piroxicam can also cause regression of precancerous lesions identified as aberrant crypt foci, thus preventing the occurrence of cancer. Male Fischer-344 rats were administered 0.125 g/kg piroxicam in their diet starting either 1 week prior to or 12 weeks after a single subcutaneous injection of AOM (30 mg/kg body wt). The yield of aberrant crypt foci and of colon adenomas and adenocarcinomas was determined at 5, 12, 27 and 37 weeks after administering the AOM respectively. When piroxicam was administered starting 1 week prior to AOM the yield of aberrant crypt foci at the three initial time points was reduced. When the administration of piroxicam was delayed until 12 weeks after AOM the yield of aberrant crypt foci was reduced from 53.8 +/- 8.1 foci/colon at 12 weeks to 11.1 +/- 2.0 at 27 weeks. At 37 weeks after administering AOM the yield of colon tumors was 0.59 +/- 0.11 tumors/animal, while in rats administered piroxicam beginning either 1 week prior to or 12 weeks after AOM the yield was similarly reduced to 0.14 +/- 0.07 and 0.17 +/- 0.07 tumors/animal respectively. Thus piroxicam was demonstrated not only to prevent, but also to cause regression of aberrant crypt foci, both of which were associated with the prevention of colon tumors. PMID- 8625467 TI - Regulated expression of APE apurinic endonuclease mRNA during wound healing in porcine epidermis. AB - Abasic (AP) sites in DNA are cytotoxic and mutagenic and their repair is initiated by AP endonucleases. The major AP endonuclease of mammalian cells is encoded by the APE gene. Ape protein has also been proposed to modulate the activity of some transcription factors independently of its AP endonuclease activity. We investigated whether APE expression is coordinated with cell division, which could diminish mutagenesis. The level of APE mRNA was followed during wound healing in porcine epidermis, in which surgical wounding prompts rapid cell proliferation followed by a differentiation program to regenerate normal skin. In situ hybridization with a probe from human APE cDNA revealed strongly decreased expression in rapidly proliferating migrating cells during the first 1-3 days following wounding, succeeded by sharply increased APE expression that exceeded the pre-wounding levels by days 9-17. These changes were not observed in the surrounding undamaged tissue. In contrast to the foregoing in vivo results, APE expression in cultured primary human fibroblasts (IMR90) or myeloid leukemia cells (K562) was not coordinated with cell division. This biphasic APE expression during wound healing could relate to transcription factor regulation or it could allow unhindered DNA synthesis or prepare the developing epidermis to handle DNA damage. However, if transient under-expression of APE encoded repair enzyme does occur, it might render regenerating skin especially vulnerable to mutagenesis during the cell proliferation phase. PMID- 8625468 TI - Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine(PhIP) at the Chinese hamsters hprt locus. AB - The mutagenic 'fingerprint' of the cooked food carcinogen 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine (PhIP) was determined in a Chinese hamster cell line genetically engineered to express human CYP1A2 (XEMh1A2-MZ). The parental Chinese hamster V79 and XEMh1A2-MZ cells were exposed to PhIP at various concentrations for 24h. There was a dose-dependent increase in frequency of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus only in the metabolically competent XEMh1A2-MZ cells. The mutant frequency ranged from 25 to 90 X 10(-6) with final concentrations of 2.5 to 100 microM PhIP compared to 8 X 10(-6) in the solvent controls and the V79MZ cells. The molecular nature of the PhIP-induced mutations in XEMh1A2-MZ cells was determined by examining DNA sequence modifications at the hprt locus in forty five 6-thioguanine resistant (6 TGr) mutant clones. Single base substitutions predominantly GC-->TA transversions, were the major class of PhIP-induced mutation. However, a -1 frameshift 'hotspot' in a 5'-GGGA sequence was also observed. With the exception of a compound modification, all of the PhIP-induced mutations involved G.C base pairs. This is consistent with the previously observed PhIP-induced mutations in cultured mammalian cells and 32P-postlabelling experiments that show PhIP adducts to the guanine base and that major adduct is at the C8 position. Furthermore, nearly all of these mutations involved guanine bases on the non-transcribed strand which is possibly indicative of preferential repair of PhIP adducts from the transcribed strand. Nearest neighbor analysis of induced base substitutions indicates a preference for 5' guanine and 3' adenine. These data effectively define a mutation 'fingerprint' for PhIP, which may provide the basis for definitive studies on the role of PhIP in diet associated cancers such as tumours of colon. It is, therefore, intriguing that in their recent report of mutation in tumours of the colon induced by PhIP in male rate Kakiuchi et al. (Proc. Natl Acad. Sci. USA, 92, 910-914) report that four out of eight tumors had identical mutation of the tumour suppressor gene apc which is comprised of a -1 G frameshift in a 5'-GGGA sequence. PMID- 8625469 TI - Mutation in the p53 tumor suppressor gene in rat esophageal papillomas induced by N-nitrosomethylbenzylamine. AB - Mutations in the p53 tumor suppressor gene have been implicated in the pathogenesis of a wide variety of human neoplasms. The location and types of p53 gene mutation can reflect exposure of humans to certain types carcinogenic agents. Much less is known about the role of p53 mutational inactivation in rodent tumors. Using both 'Hot' (radioactive) and 'Cold' (non-radioactive) single strand conformation polymorphism (SSCP) analyses, the present study of analyzed exons 5-8 and the exon-intron junction of the p53 gene from rat esophageal papillomas induces by N- nitrosomethylbenzylamine (NMBA) for mutations. Nine of 30 (30%) esophageal papillomas contained SSCP mobility shifts, principally within exons 5 and 7. These positive SSCP findings were further validated by direct DNA sequencing analysis. Eight of the nine mutations were G:C-->A:T transitions in codons 131, 149, 153, 242 (2), 243, 248, and the 5 end of intron 7. None of these G:C-->A:T mutations occurred at the CpG sites. The other mutation was a frameshift mutation in codon 176. The G:C-A:T transitions observed in this study are consistent with the documented formation of O(6)-methylguanine adducts in DNA N-nitroso compounds. These results suggest that point mutations of the p53 gene are involved in the development of approximately one-third of NMBA-induced rat esophageal papillomas. 'Hot' and 'Cold' SSCP methods were equally sensitive for the identification of mutations in the rat p53 gene. PMID- 8625470 TI - Transformation of immortalized woodchuck hepatic cell lines with the c-Ha-ras proto-oncogene. AB - Woodchuck hepatocytes were immortalized with the simian virus 40 T antigen (SV40 T-ag) oncogene and utilized in an oncogenic transformation assay. Transfection of these cell lines with an activated C-Ha-ras oncogene (EJ6.6) resulted in the transformation of cells to a phenotype characterized by anchorage-independent growth in soft agar. Colonies of transformed cells were subcloned and up to 80% were positive for oncoprotein expression detected by immunoblot and Northern blot procedures. When compared with the parental cell lines, ras-transformed derivatives were altered both morphologically and in growth rate. The tumorigenic potential of c-Ha-ras transformed cells was demonstrated in severe combined immunodeficient (SCID) mice. There was a latency period of 1 to 4 weeks before tumors were detectable and a period of 7 weeks was required for tumors to reach a diameter of 1 cm. Histologically, tumors derived from cell lines fully transformed by the SV40 T-ag had the appearance of well-differentiated hepatocellular carcinoma (HCC) while tumors derived from c-Ha-ras transformed cell lines had the appearance of poorly differentiated HCC. The capacity to induce oncogenic transformation events in immortalized woodchuck hepatic cell lines should provide the opportunity to study the cooperative effects of hepadnaviral genes in hepatocarcinogenesis in vitro. PMID- 8625471 TI - Inter- and intracellular heterogeneity of O6-alkylguanine-DNA alkyltransferase expression in human brain tumors: possible significance in nitrosourea therapy. AB - The inter- and intracellular distribution of the DNA repair protein O6 alkylguanine-DNA alkyltransferase (ATase) may be an important factor in the sensitivity or resistance of tumours to treatment with certain alkylating agents, including the methyltriazenes and nitrosoureas. In order to examine this issue 26 human brain tumour sections (23 high grade gliomas and three low grade gliomas) were examined for ATase expression by immunohistochemistry using a rabbit anti human ATase polyclonal antibody. Positive staining, seen as fine black granules mainly confined to the nucleus, was observed in all the glioma sections examined. There was marked cellular heterogeneity, ranging from cells completely devoid of staining to cells with very intense staining. Semi-quantitatively, in the 23 high grade gliomas examined six had 1+ staining, seven had 2+ staining and 10 had 3+ staining, whereas all three low grade gliomas had 1+ staining. These results are in contrast to published reports showing that approximately 35% of human brain tumour-derived cell lines and xenografts had very low levels of ATase activity and suggest that the complete lack of ATase is not a common occurrence in high grade glioma. PMID- 8625472 TI - Excision of DNA adducts of nitrogen mustards by bacterial and mammalian 3 methyladenine-DNA glycosylases. AB - Nitrogen mustards are among the DNA alkylating agents most widely used in chemotherapy. The homogeneous Escherichia coli AlkA protein (3-methyladenine-DNA glycosylase II) is shown to excise damaged guanine and adenine bases from DNA modified by mechlorethamine, uracil mustard, phenylalanine mustard and chlorambucil, and less efficiently acridine mustard adducts. Homogeneous recombinant human and rat 3-methyladenine-DNA glycosylases excise adducts formed by nitrogen mustards less efficiently than the AlkA protein. In addition to the in vitro excision of adducts, the AlkA protein eliminates cytotoxic mechlorethamine adducts from DNA in vivo. PMID- 8625473 TI - Relationship between the adaptive response to oxidants and stable menadione resistance in Chinese hamster ovary cell lines. AB - To study the genetic changes that generate resistance to oxidants in mammalian cells, we isolated cell lines that are resistant to the naphthoquinone, menadione, from a Chinese hamster ovary cell line (CHO-K1). Corss-resistance to other oxidants (H2O2 and sodium arsenite) was observed. The IC50 of menadione (measured using a clonogenic assay) was 7.8-fold greater for one menadione resistant cell line (MRc40) than for CHO-K1. Acquisition of resistance was associated with elevations of 2- and 3.2-fold in the low molecular weight thiols, glutathione and cysteine, respectively. Further, characterization demonstrated significant changes in the expression of enzymes associated with the oxidative stress response and with protection against oxidizing agents. The expressions of glutathione S-transferase pi (GST pi), glutathione peroxidase (GPX) and heme oxygenase mRNAs were all increased. Accompanying these changes the enzyme activity of GST pi, GPX and gamma-glutamyl transpeptidase (gamma-GT) were also elevated. Interestingly, in a revertant cell line heme oxygenase overexpression approached wild-type levels. Intriguingly, similar changes in gene expression seen in the menadione-resistant cells were also observed in wild-type cells following transient oxidative stress, indicating that the observed changes in the resistant line may be due to the immortalization of a normally transient adaptive stress response. PMID- 8625474 TI - Chromosomal mechanisms in murine radiation acute myeloid leukaemogenesis. AB - Chromosome 2 abnormalities, particularly interstitial deletions, characterize murine radiation-induced acute myeloid leukaemias (AMLs). Here, G-band analyses in CBA/H mice of early (1-6 month) post 3 Gy X-irradiation events in bone marrow cells in vivo and of karyotype evolution in one unusual AML are presented. The early event analysis showed that all irradiated animals carry chromosome 2 abnormalities, that chromosome 2 abnormalities are more frequent than expected and that interstitial deletions are more common in chromosome 2 than in the remainder of the genome. On presentation AML case N122 carried a t(2;11) terminal translocation which, with passaging, evolved into a del2(C3F3). Therefore, two pathways in leukaemogenesis might exist, one deletion-driven, the other terminal translocation-driven involving interstitial genes and terminal genes respectively of chromosome 2. As all irradiated individuals carried chromosome 2 abnormalities, the formation of these aberrations does not determine individual leukaemogenic sensitivity as only 20-25% of animals would be expected to develop AML. Similar lines of argument suggest that chromosome 2 abnormalities are necessary but not sufficient for radiation leukaemogenesis in CBA/H nor are they rate limiting in leukaemogenesis. PMID- 8625475 TI - Induction of heme oxygenase in mammalian cells by mineral fibers: distinctive effect of reactive oxygen species. AB - Exponentially growing human-hamster hybrid [AL] cells treated with a 40 micrograms/ml (8 micrograms/cm2) dose of UICC standard reference chrysotile fibers induced heme oxygenase (HO) protein with a maximum expression level at 8 h post-treatment. While the constitutive HO expression was detectable in non treated AL cells, the protein level was increased approximately 4.5-fold in fiber treated cells. The induction was dose-dependent at fiber concentration between 2.5 micrograms/ml (0.5 microgram/cm2) and 40 micrograms/ml (8 micrograms/cm2) with the induced HO concentrated mostly in the cytoplasm as shown by immunostaining. Several other types of mineral fibers examined including crocidolites, tremolites, and erionites also induced HO synthesis with varying degree of efficiency. In general, chrysotile and crocidolite were more efficient inducers of HO than tremolite and erionite when compared at fiber doses that resulted in approximately 50% survival (LD50) level. The effects of antioxidant enzymes on HO induction were examined by concurrent treatment of fiber-exposed cultures with SOD and catalase. Although addition of superoxide dismutase (SOD) and catalase inhibited HO induction in a dose-dependent manner, they offered no protection on fiber-mediated clonogenic toxicity in the same population of treated cells. These results suggest that reactive oxygen species (ROS) produced by asbestos fibers play an essential role in the induction of HO and that different mineral fibers, when applied at equitoxic doses, often result in different oxidative stress status as determined by the induction of HO proteins. PMID- 8625476 TI - Diallyl disulfide induces apoptosis of human colon tumor cells. AB - The present studies compared the effects of various oil-soluble compounds containing allyl and disulfide groups on the proliferation of cultured human colon tumor cells (HCT-15). Diallyl disulfide (DADS) was more effective in inhibiting the growth of HCT-15 cells than isomolar concentrations of S-allyl cysteine, dipropyl disulfide (DPDS), allyl chloride, allyl glycidyl ether and allyl alcohol. These studies clearly demonstrate the importance of both the diallyl and the disulfide groups in DADS. Treatment of HCT-15 cells with 100 microM DADS increased the intracellular calcium levels by 40%, while DPDS caused only a 12% increase in intracellular calcium. Exposure to 100 microM DADS or more, but not DPDS, caused the cells to undergo apoptosis as determined by morphological changes and DNA fragmentation. A positive correlation (r=0.944) was found between DADS-induced DNA fragmentation and its ability to increase intracellular free calcium levels. The widespread effectiveness of DADS was evident by its ability to inhibit the growth of human colon (HCT-15), skin (SK MEL-2) and lung (A549) tumor cell lines. PMID- 8625477 TI - A non-bile duct origin for intestinal crypt-like ducts with periductular fibrosis induced in livers of F344 rats by chloroform inhalation. AB - To evaluate the toxic effects of prolonged exposure to chloroform vapors, female and male F344 rats were exposed to 0, 2, 10, 30, 90 and 300 p.p.m. chloroform by inhalation for 7 or 5 days/week for up to 13 weeks. The purpose of this study was to characterize a lesion that occurred in the livers of rats in the 300 p.p.m. exposure groups. Atypical glandular structures lined by intestinal-like epithelium and surrounded by dense connective tissue occurred in the livers of rats exposed to strongly hepatotoxic atmospheric concentrations of chloroform. Bile duct bromodeoxyuridine labeling indices as well as observations of the locations of the early lesions at the 3 and 6 week time points indicate that these lesions arose from a population of cells remote from the bile ducts. We refer to these lesions as intestinal crypt-like ducts with periductular fibrosis to distinguish them from true cholangiofibrosis. Here, intestinal crypt-like ducts with periductular fibrosis were seen only in rats exposed to 300 p.p.m. chloroform, and the multiplicity and severity of the lesions were greater in the right liver lobe. The lesion only occurred in association with liver necrosis and dramatic increases in hepatocyte labeling indices, while labeling indices in bile ducts in the same animals were not significantly different from controls. There was a treatment-related increase of transforming growth factor-alpha immunoreactivity in hepatocytes, bile duct epithelium, bile canaliculi and oval cells, and an increase in transforming growth factor-beta immunoreactivity in hepatocytes, bile duct epithelium and intestinal crypt-like ducts. Thus, intestinal crypt-like ducts with periductular fibrosis appeared to develop from a population of cells unrelated to bile ducts. Also, they occurred only in animals exposed to chloroform concentrations that induced significant hepatocyte necrosis and regenerative cell proliferation and were associated with increased growth factor expression or uptake. PMID- 8625478 TI - MARCKS functions as a novel growth suppressor in cells of melanocyte origin. AB - Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-Kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compound with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transformed melanocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinoma. PMID- 8625479 TI - Faulty DNA polymerase delta/epsilon-mediated excision repair in response to gamma radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome. AB - Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53 function. In this study we have examined the ability of these p53-deficient strains to carry out the long patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to 60Co gamma radiation or far ultraviolet (UV) (chiefly 254 nm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (apc) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline sucrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both apc and araC decreased the level of UV induced UDS by approximately 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne's syndrome. Together these results imply a critical role for wild type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes. PMID- 8625480 TI - Thapsigargin, a weak skin tumor promoter, alters the growth and differentiation of mouse keratinocytes in culture. AB - Thapsigargin (Tg), applied twice weekly to the backs of CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene, promoted papillomas on the skin of approximately 50% of the mice. Tg alone induced papillomas in 10% of the mice. Although Tg and 12-O-tetradecanoylphorbol-13-acetate (TPA) are synergistic in a keratinocyte co culture assay for promotion, skin tumor promotion by TPA was inhibited by co treatment with Tg. Treatment of cultured keratinocytes with non-toxic doses of Tg increased the level of intracellular free Ca(2+)-induced stratification. Tg blocked expression of the spinous layer differentiation marker keratin 1 (K1), while inducing cornification, a marker of differentiation in the granular layer/stratum corneum. In medium with 1.4 mM Ca2+, Tg prolonged keratinocyte proliferation and selected foci of monolayer cells. A Tg-independent cell line, TK-1, was developed from a single dish in which foci continued to expand after Tg removal. Grafting TK-1 cells on to the backs of nude mice as part of a reconstituted skin resulted in the development of dysplastic papillomas with a high rate of progression to squamous cell carcinomas. PMID- 8625481 TI - Differences in the levels and pattern of DNA-adduct labelling in human cell lines MCL-5 and CCRF, proficient or deficient in carcinogen-metabolism, treated in vitro with bile from familial adenomatous polyposis patients and from unaffected controls. AB - In patients with familial adenomatous polyposis (FAP), duodenal adenomas cluster around the ampulla and their distribution closely resembles mucosal exposure to bile, suggesting a role for bile in their development. Previous studies using 32P postlabeling to detect DNA adducts, have provided evidence to support this hypothesis. We have now investigated the role of metabolic activation in influencing the levels and patterns of adduct formation by incubating precolectomy gallbladder bile from FAP patients and bile from unaffected controls with human lymphoblastoid cell lines that are metabolically proficient (MCL-5), or deficient (CCRF). 32P-Postlabeling assays showed that MCL-5 cells (genetically engineered to express five human cytochromes P450 and microsomal epoxide hydrolase) formed characteristic adduct spots with benz[a]pyrene, benzo[g]chrysene, 7,12-dimethylbenz[a]anthracene, benzidine, sterigmatocystin and 3-methylcholanthrene, whereas CCRF cells did not. Accordingly, we assayed the ability of bile from FAP patients and controls to form DNA adducts in MCl-5 and in CCRF cells. Relative adduct labelling (RAL) in MCL-5 cells treated with FAP bile (12 patients, median 10, range 1-74) was significantly higher than in cells treated with control bile (12 patients, median 4, range 0-9; P = 0.0007) as was RAL for the two major adduct spots. These two major adduct spots were not observed when bile was incubated with CCRF cells. The adduct spots in CCRF DNA appeared in positions similar to some of the minor adduct spots produced by bile in MCL-5 DNA and to some of the adduct spots seen previously when bile was incubated with salmon sperm DNA in vitro. RAL for CCRF cells incubated with FAP bile (seven patients, median 23.0, range 0-49) was significantly higher than in cells treated with control bile (seven patients, median 2.0, range 0-26; P = 0.0034). These results indicate that the bile obtained from FAP and control patients contains adduct-forming substances, some of which are direct acting and some of which require metabolic activation. In both cell lines, FAP bile produced significantly higher adduct labelling than control bile, adding to the evidence that bile can induce DNA damage in vitro and plays a role in neoplastic development in the FAP foregut. PMID- 8625482 TI - Molecular mechanisms of toxic effects of fotemustine in rat hepatocytes and subcellular rat liver fractions. AB - Fotemustine is a clinically used DNA-alkylating 2-chloro-ethyl-substituted N nitrosourea, which sometimes shows signs of haematotoxicity and reversible liver and renal toxicity as toxic side-effects. Mechanistic data on these side-effects are scarce and incomplete. In this study, firstly the cytotoxicity of fotemustine in freshly isolated rat hepatocytes was investigated and secondly the metabolism of fotemustine and possible mechanisms involved in the observed cytotoxicity. Fotemustine caused concentration- and time-dependent cytotoxic effects in rat hepatocytes. Extensive GSH-depletion and formation of GSSG were first observed, followed by lipid peroxidation and finally by cell death measured as LDH-leakage. 2-Chloroethyl analogues of fotemustine, which in contrast to fotemustine have no carbamoylating potency, were not toxic to rat hepatocytes. The data suggest that the cytotoxicity of fotemustine is resulting from its reactive decomposition product, DEP-isocyanate. GSH-conjugation of DEP-isocyanate was shown to protect against the cytotoxicity of fotemustine, however, only temporary and not completely. Synthetical DEP-SG, the GSH-conjugate of DEP-isocyanate, was also toxic to rat hepatocytes, albeit to a significantly lesser extent than fotemustine. In rat liver microsomes, no fotemustine-induced LPO was observed, suggesting that reactive decomposition products of fotemustine do not directly cause peroxidation of cellular membranes. Fotemustine did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase, GSSG reductase and GSH S-transferases. Thus, direct effects on these antioxidant enzymes are not likely to explain the cytotoxic effects of fotemustine in hepatocytes. In conclusion, it is proposed that the cytotoxicity of fotemustine in rat hepatocytes is caused by rapid and extensive depletion of GSH by DEP isocyanate, a reactive decomposition product of fotemustine, consequently hampering the endogenous protection against its own toxicity. Knowledge of molecular mechanisms of the cytotoxicity of fotemustine may contribute to a more rational design of selective protection against toxic side-effects which occur upon therapy of patients with fotemustine. PMID- 8625483 TI - Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine. AB - The mutagenic, carcinogenic and cytotoxic activity of dacarbazine, a drug employed in cancer chemotherapy, may be related to the induction in DNA of O6 methylguanine (O6-meG), a quantitatively minor but biologically important lesion. In the present study the kinetics of O6-meG formation and repair in blood leukocyte DNA were examined in 20 Hodgkins lymphoma patients treated i.v. with 180 +/- 13 (mean +/- SD) mg/m2 dacarbazine and compared with those observed in various tissues of rodents treated with different doses of the drug. In Hodgkin's lymphoma patients adduct levels reached a value of 0.27 +/- 0.14 fmol/microgram DNA 2 h after dacarbazine administration, while the rate of subsequent loss suggested an adduct half-life of < or = 30 h. Measurement of adduct levels in the same individuals after successive courses of treatment spaced 3 weeks apart (up to 10 treatment courses) demonstrated a consistent individual response and statistical analysis of variance confirmed that intra-individual variation in adduct accumulation after a given dose of dacarbazine accounted for only 5% of the total variance observed. In contrast, inter-individual variation accounted for 70% of the observed variance, with adduct levels 2 h after drug treatment varying approximately 7.5-fold among adduct-positive individuals. No significant depletion of lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) occurred after patient treatment with dacarbazine. No significant relationship between adduct levels and clinical response to treatment was observed. In rats treated with single or multiple doses of dacarbazine causing varying degrees of AGT depletion the highest levels of O6-meG were seen in the liver, followed by the lymph nodes, bone marrow and blood leukocytes, which showed up to approximately 2-fold lower levels. A similar tissue distribution was also observed in mice and in a single rabbit. These observations suggest that O6-meG levels assayed in blood leukocytes of therapeutically treated humans reflect those present in the -lymph nodes (target tissue for chemotherapy) and the bone marrow (target tissue for leukaemogenesis) and may be utilized as a measure of the drug dose reaching these tissues. The quantitative data reported in this study show that under conditions of no depletion of AGT O6-meG accumulates in blood leukocyte DNA of humans at a rate similar to that observed in rats, suggesting that human susceptibility to any O6-meG-mediated genotoxic effects of dacarbazine may be comparable with that of the rat. PMID- 8625484 TI - Relationship between p53 mutation incidence in oral cavity squamous cell carcinomas and patient tobacco use. AB - It is well-established that a high incidence of p53 mutations exist in oral cavity squamous cell carcinomas (OCSCCs). To determine whether p53 mutations are etiologically associated with OCSCC development or are associated with exposure to specific carcinogens, we have analyzed the conserved regions of the p53 gene (exons 5-9) in 48 OCSCCs obtained from patients with varied tobacco and alcohol use histories by polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) and DNA sequencing analysis. Thirty-eight percent (18/48) of the OCSCCs exhibited a mutation in exons 5-9 of the p53 gene. There was a significantly higher incidence of p53 mutations in OCSCCs from tobacco users (predominantly cigarette smokers) compared to those who had never used tobacco. No increase in the incidence of p53 mutation was observed in tobacco users who drank alcohol. G to A transitions and deletions were the predominant mutations observed in OCSCCs from tobacco users. No specific pattern of mutation was observed in OCSCCs from those subjects who had never used tobacco. These data suggest that a history of tobacco use was associated with a high incidence of p53 mutations in patients with OCSCC and that tobacco carcinogens include a specific pattern of mutations in oral cavity tissue in vivo. PMID- 8625485 TI - Distribution of 7-alkyl-2'-deoxyguanosine adduct levels in human lung. AB - Human lung tissue is frequently studied as a target organ for DNA damage from carcinogen-DNA adducts. In order to assess the distribution of carcinogen-DNA adducts in human lung, we measured 7-methyl-2'-deoxyguanosine-3'-monophosphate (7 methyl-dGp), 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyl-dGp) and 4 hydroxy-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in different lobes. The first two result from exposure to N-nitrosamines, including tobacco-specific nitrosamines, and the latter only from tobacco-specific nitrosamines. Using a chemically-specific 32P-postlabeling assay for 7-alkyl-2'-deoxyguanosines, adducts were measured in eight separate lung segments of ten autopsy donors. 7 Methyl-dGp levels were detected in all eighty samples (range from 0.3 to 11.5 adducts/10(7) dGp; mean 2.5 +/- 2.3 adducts/10(7) dGp). 7-Ethyl-dGp were detected in all but five of the samples (range from <0.1 to 7.1 adducts/10(7) dGp; mean 1.6 +/- 1.7 adducts/10(7) dGp). 7-Methyl-dGp levels were approximately 1.5-fold higher than 7-ethyl-dGp levels, and they were positively correlated with each other in most individuals. There was no consistent pattern of adduct distribution in the different lobar segments. Most individuals, especially those with the lowest levels, had similar levels among the lobes, while those with the highest levels had a widely variable pattern ranging as much as ten-fold. Moreover 7 methyl-dGp and 7-ethyl-dGp levels in all people showed a highly significant inter individual variation (P = 0.0001). The levels of 7-alkyl-2'-deoxyguanosine among individuals could not be explained by differences in tobacco exposure (measured by serum cotinine), onset of death, gender, age, race, blood ethanol, or ventilation and perfusion variability. In an effort to corroborate 7-alkyl-2' deoxyguanosine adducts variability among lobes or individuals, we sought to determine a correlation with HPB-releasing DNA adducts as an independent marker of tobacco exposure. However, this tobacco- specific carcinogen-DNA adduct could not be detected in four individuals tested (detection limit: 0.3 adducts per 10(7) dGp). Based upon the lack of 7-alkyl-2'-deoxyguanosine discernible adduct patterns, no conclusions could be drawn regarding a potential relationship to lobar cancer incidence. The results indicate that in studies of carcinogen-DNA adducts, such as 7-alkyl-dGp in human lungs, for most individuals a random lung sample would be representative of other parts of the lungs. Some individuals however might be misclassified due to highly variable 7-alkyl-dGp levels. PMID- 8625486 TI - Effects of dietary 1,4-phenylenebis(methylene)selenocyanate on 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation in lung and liver of A/J mice and F344 rats. AB - 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) was tested for its ability to inhibit DNA adduct formation induced by the tobacco-specific N-nitrosamine 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the liver and lung of A/J mice and F344 rats. Dietary p-XSC, providing a dose of 5 p.p.m. selenium, significantly inhibited the formation of 7-methylguanine (7-mGua) induced by a single i.p. injection of 10 mumol of NNK(12.8% inhibition at 4 h and 19.9% at 96 h) and O6-methylguanine (O6-mGua) (16.5% at 4 h and 34.8% at 96 h) in the liver of A/J mice. Dietary supplements of p-XSC providing 15 p.p.m. of selenium reduced the levels of 7-mGua by 17.3% (4 h) and 33.6% (96 h). The formation of O6-mGua was inhibited by 69.5% (4th) and 73.8 (96h). In A/J mouse lung DNA the most significant reduction was observed in levels of O6-mGua. Dietary p-XSC at 5 p.p.m. as selenium inhibited the formation of this adduct by 73.1% (4 h). Ninety six hours after NNK injection, and at both time points with p-XSC providing 15 p.p.m. selenium, O6-mGua was not detected. Although levels of 7- mGua in mouse lung DNA were also reduced, this was significant only 4 h after carcinogen administration. In general, selenite at a5 p.p.m. as selenium had no significant effect on the levels of these lesions; however, it inhibited O6-mGua in the liver only 4 h after NNK administration. These effects may explain why there is chemopreventive activity for p-XSC, but not for selenite, in NNK-induced lung carcinogenesis in A/J mice. Moreover, these findings raised our interest in determining the potential chemopreventive activity of p-XSC against NNK-induced lung adenocarcinomas in male F344 rats by first determining its effects on NNK induced DNA methylation in the lungs of rats. Diet supplemented with 10 p.p.m. selenium as p-XSC did indeed inhibit the formation of adducts in pulmonary DNA of F344 rats treated with four consecutive injections of 81 mg/kg of NNK. Statistically significant inhibition of O6-mGua formation was observed 4 h after carcinogen treatment in both pulmonary (49.1% inhibition) and hepatic (39.8%) DNA. Statistically significant inhibition of 7-mGua formation was also measured in lung DNA isolated 24 h after the last NNK injection (45.0%) and in liver DNA 4 h after carcinogen treatment (31.8%). These results suggest that p-XSC would also inhibit induction of lung adenocarcinoma in male F344 rats by NNK. PMID- 8625487 TI - The essential role of the functional group in alkyl isothiocyanates for inhibition of tobacco nitrosamine-induced lung tumorigenesis. AB - The importance of the isothiocyanate group in alkyl isothiocyanate for inhibition of tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK)-induce lung tumorigenesis was examined in A/J mice. Our previous structure activity relationship study of isothiocyanates showed that 1-dodecyl isothiocyanate [CH3(CH2)11NCS], a simple alkyl isothiocyanate, is a potent inhibitor of NNK-induced lung tumorigenesis. It was chosen for this study due to its structural features and potency. A single dose of 1-dodecyl isothiocyanate given by gavage at 1 micromol/mouse 2 h prior to NNK administration completely inhibited lung tumorigenesis, while removal of the isothiocyanate group or replacing it with a hydroxyl group abolished the inhibitory activity. These results demonstrate that the isothiocyanate functional group is critical for the inhibitory activity of isothiocyanates in NNK-induced lung tumorigenesis. To gain more insights into the relationship of in vivo inhibition of tumorigenesis with the cytochrome P-450 enzyme inhibitory activity, the effects of these compounds on metabolism of NNK in mouse lung microsomes were studied. 1-Dodecyl isothiocyanate inhibited all three known oxidative pathways of NNK metabolism, with a stronger inhibitory activity toward NNK N-oxidation (IC50 430 nM) and keto alcohol formation (IC50 500 nM) than keto aldehyde formation (IC50 13,000 nM). 1 Dodecanol had a similar selectivity in inhibition of these metabolic pathways, but was less potent than 1-dodecyl isothiocyanate. Dodecane showed little or no inhibitory activity in the same concentration range. These results indicate that the isothiocyanate group of 1-dodecyl isothiocyanate is important for inhibition of NNK-induced lung tumorigenesis and also for effective inhibition of cytochrome P-450 enzymes involved in NNK oxidation. PMID- 8625488 TI - Inhibitory effects of caffeic acid phenethyl ester (CAPE) on 12-O tetradecanoylphorbol-13-acetate-induced tumor promotion in mouse skin and the synthesis of DNA, RNA and protein in HeLa cells. AB - Topical application of caffeic acid phenethyl ester (CAPE), a constituent of the propolis of honeybee hives, to the backs of CD-1 mice previously initiated with 7,12-dimethylbenz[a]anthracene (DMBA) inhibited 12-O-tetradecanoylphorbol-13 acetate (TPA)-induced tumor promotion and the formation of 5-hydroxymethyl-2' deoxyuridine (HMdU) in epidermal DNA. Topical application of 5 nmol TPA twice weekly for 20 weeks to mice previously initiated with 200 nmol of DMBA resulted in 18.8 skin papillomas per mouse. Topical application of 1, 10, 100 or 3000 nmol of CAPE together with 5 nmol of TPA twice a week for 20 weeks inhibited the number of skin papillomas per mouse by 24, 30, 45 or 70%, respectively, and tumor size per mouse was decreased by 42, 66, 53 or 74%, respectively. Topical application of 5 nmol of TPA twice weekly for 20 weeks to mice previously initiated with DMBA produced an average of 12.6 HMdU residues per 10(4) normal bases in epidermal DNA. Topical application of 1, 10, 100 or 3000 nmol of CAPE with 5 nmol of TPA twice weekly for 20 weeks to DMBA-initiated mice decreased the level of HMdU in epidermal DNA by 40-93%. The in vitro addition of 1.25, 2.5, 5, 10 or 20 microM CAPE to cultured HeLa cells inhibited the synthesis of DNA by 32, 44, 66, 79 or 95%, respectively, the synthesis of RNA was inhibited by 39, 43, 58, 64 or 75%, respectively, and the synthesis of protein was inhibited by 29, 30, 37, 32 or 47%, respectively. The results indicate a potent inhibitory effect of CAPE on TPA-induced tumor promotion and TPA-induced formation of HMdU in DNA of mouse skin as well as an inhibitory effect of CAPE on the synthesis of DNA, RNA and protein in culture HeLa cells. PMID- 8625489 TI - Exposure to dimethylarsinic acid, a main metabolite of inorganic arsenics, strongly promotes tumorigenesis initiated by 4-nitroquinoline 1-oxide in the lungs of mice. AB - The effect of dimethylarsenics on the pulmonary tumorigenesis initiated by 4 nitroquinoline 1-oxide (4NQO) in mice was examined. The exposure of mice to dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenics in mammals, resulted in not only promotion but also progression of the tumorigenic process in the lungs of mice administered 4NQO. Furthermore, dimethylarsenics influenced the differentiation process in lung tumorigenesis by 4NQO. These results may pave the way for the elucidation of lung carcinogenesis caused by arsenics. PMID- 8625490 TI - Low expression of the Lsp1 gene in early mouse T-lymphomas induced by N-methyl-N nitrosourea. AB - The mouse Lsp1 gene encodes a 330 amino acid intracellular F-actin binding protein. Previously we showed that the mouse and human Lsp1 genes are expressed in normal B-cells and T-cells, including Thy1+ thymocytes and in normal macrophages and neutrophils. No or little LSP1 RNA and protein was found in a series of transformed mouse and human T-lymphoma cell lines, although normal antigen and lymphokine dependent T-cell lines expressed the Lsp1 gene. Here we show by Northern analysis that three mature antigen independent T-cell lines (CTLL-2, HT-2 and 532.10) which grow in the presence of lymphokine only, do not express LSP1 RNA, while mature resting and activated lymph node T-cells express high levels of LSP1 RNA. To determine whether the down-regulation of LSP1 expression is an early event which occurs in vivo in the tumor, rather than as a consequence of in vitro propagation of T-lymphoma cells, we analyzed LSP1 expression in primary T-lymphomas induced in AKR/J or AKR/J x BALB/cJ mice by a single intraperitoneal (i.p.) injection with 75 mg N-methyl-N-nitrosourea (MNU) per kg body-weight. Two- color Fluorescence Activated Cell Sorter (FACS) analysis showed that all tumors had an immature CD4+/CD8+ double positive (DP) phenotype. Many tumors contained a substantial population of CD4+ single positive (SP) cells. Since these cells may be infiltrating lymphocytes which can be expected to express the Lsp1 gene at a high level these tumors were not included in our analysis. Nineteen tumors were analyzed for Lsp1 gene expression and 13 contained reduced levels of LSP1 RNA, ranging from 4% to 44% of those found in age-matched normal thymus. Six tumors showed either no or only a small reduction in LSP1 RNA. These tumors had developed later than those expressing low levels of LSP1. The level of LSP1 RNA in tumors developing <110 days after injection of MNU was 19.1% +/- 5.2 (mean +/- SEM), while the level of LSP1 in later tumors was 78.4% +/- 13.0 (P = 0.004). Similar data were obtained when the expression of LSP1 protein was analyzed. These findings extend our previous data in several ways. First, they suggest a correlation between the down-regulation of LSP1 expression and abnormal regulation of growth or survival of immature and mature T-lymphocytes. Second, they show that down-regulation of the Lsp1 gene in transformed T-cells is not the result of prolonged in vitro culture, but occurs in the majority of primary tumors. Third, they show that there are two classes of T-lymphoma, which differ in their expression of LSP1 RNA and that the down-regulation of LSP1 is specifically associated with the early appearance of T-lymphoma after injection with MNU. This strongly suggests that the absence or reduced expression of LSP1 contributed to the transformation process and argues against the possibility that loss of LSP1 expression is a mere inconsequential epigenetic event. PMID- 8625491 TI - Overexpression of transforming growth factor-alpha and epidermal growth factor receptor, but not epidermal growth factor, in exocrine pancreatic tumours in hamsters. AB - Using immunohistochemistry, Northern blotting and a semi-quantitative PCR technique, epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha) and epidermal growth factor receptor (EGFR) expression were studied in the pancreas of N-nitrosobis(2-oxopropyl)-amine (BOP)-treated hamsters. After initiation pancreatic carcinogenesis was modulated by a high fat diet or by injections with the cholecystokinin analogue caerulein. Autopsies were performed 6 and 12 months after the last injection with BOP. Immunohistochemistry revealed a weak expression of TGF-alpha in nomal acinar cells and a stronger expression in ductular and centro-acinar cells. Over-expression of TGF-alpha was observed in advanced putative preneoplastic lesions (classified as borderline lesions) and in ductular adenocarcinomas. EGFR immunoreactivity was present only in ductular adenocarcinomas. EGF peptide expression was observed both in acinar and ductular normal and tumorous cells and the level of expression did not change significantly during carcinogenesis. Moreover, the post-initiation treatments did not cause differences in EGF, TGF-alpha or EGFR peptide or mRNA levels, except for a significantly lower expression of TGF-alpha mRNA in hamsters fed a high fat diet when compared with those fed a low fat diet. TGF-alpha mRNA levels increased, whereas EGF mRNA levels decreased significantly in total pancreatic homogenates of BOP-treated hamsters in comparison with untreated controls. Also, in ductular adenocarcinomas TGF-alpha and EGFR (but not EGF) mRNA levels were significantly higher than in normal pancreatic homogenates. In pancreatic homogenates obtained 6 months after the last BOP injection, these differences were less pronounced in comparison with those obtained after 12 months. The present results indicate that TGF-alpha (but not EGF) might act in a paracrine or autocrine manner in pancreatic tumours in BOP-treated hamsters via simultaneously expressed EGFR. However, TGF-alpha, EGF and EGFR do not seem to be involved in the modulating effects of a high fat diet or caerulein treatment on pancreatic carcinogenesis in BOP-treated hamsters. PMID- 8625492 TI - Determination of 8-hydroxydeoxyguanosine in human cells under oxygen-free conditions. AB - To establish an accurate 8-hydroxydeoxyguanosine (8OHdG) determination system, we examined two potential factors causing experimental error in 8OHdG determination. First, we examined the efficiency of the enzymatic digestion of DNA, that could cause misestimation of 8OHdG. Second, since we considered that the oxygen molecules in atmosphere and in reagents were the main factor contributing to the experimental errors, we carried out the 8OHdG determination under oxygen-free conditions and compared the 8OHdG value with that determined by the methods under ambient atmosphere. The calf thymus DNA was sufficiently digested in the condition we used and the yields of dG were constant, even when the DNA was damaged with H2O2 (80 mM) and UV irradiation. By carrying out the DNA extraction manually, instead of using the DNA extractor, we could reduce the additional 8OHdG formation during sample processing. No trend was found in the difference between the 8OHdG values determined under oxygen-free conditions and under ambient atmosphere. However, when the 8OHdG values were compared in samples with asbestos, the value determined under oxygen-free conditions was significantly lower than that determined under ambient atmosphere. These findings suggest that the removal of oxygen molecules was effective in reducing accidental ROS generation by impurities in the sample, which could cause the additional 8OHdG formation, and that the oxygen-free system made the determination of 8OHdG reproducible and more accurate than before. When the oxygen-free system was applied to human leukocytes, the system showed good reproducibility (r = 0.535, P < 0.001), even though the 8OHdG level was low. With the system, we could detect a significant difference between 8OHdG in polymorphonuclear leukocytes (0.241 +/- 0.129) and mononuclear leukocytes (0.188 +/- 0.126, P < 0.01). PMID- 8625493 TI - Effects of dietary broccoli on human in vivo drug metabolizing enzymes: evaluation of caffeine, oestrone and chlorzoxazone metabolism. AB - Ingestion of cruciferous vegetables may prevent chemically induced carcinogenesis by their influence on specific cytochrome P450 enzymes (CYP) and phase II drug metabolizing enzymes in humans and rodents. Thus CYP enzymes are involved in transformation of procarcinogens, mutagens, steroid hormones and a large variety of other endogenous and exogenous components. In order to learn more about the influence of cruciferous vegetables on drug metabolizing enzymes in man two CYP enzymes previously suggested to be induced by vegetables were selected in an in vivo experiment in humans. Sixteen healthy non-smoking subjects, two females and 14 males, were exposed to three different types of diets and afterwards assayed for CYP1A2 catalysed caffeine metabolites and for CYP2E1 catalysed 6 hydroxylation of chlorzoxazone. Further, 2-hydroxyoestrone:16 alpha-hydroxylation ratios were determined in urine by means of a monoclonal antibody-based enzyme immunoassay. The three dietary periods were: (A) a customary home diet; (B) a 6 day standard diet avoiding well-known dietary inducers and inhibitors of CYP; (C) a 12 day dietary supplement to the standard diet of 500 g/day broccoli. The average 6-hydroxychlorzoxazone:chlorzoxazone ratio decreased by 21% (P < 0.05) after diet B compared with diet A in a 2 h plasma sample after ingestion of 500 mg chlorzoxazone. The ratio increased by 19% after diet C, however, this was not statistically significant. The caffeine metabolic ratio (CMR) was determined in urine 6 h after ingestion of 100 mg caffeine. The mean CMR increased by 5.5% when changing from diet A to diet B. When shifting to diet C the mean CMR increased a further 19% (P < 0.0005). The average 2-hydroxyoestrone:16 alpha-hydroxyoestrone ratio decreased by 1.3% when comparing diet A with diet B. Daily broccoli intake increased the ratio by 29.5% (P < 0.05). A low correlation of CMR with the 2 hydroxyoestrone:16 alpha-hydroxyoestrone ratio indicates that human CYP1A2 and other CYP enzymes involved in oestrone 2-hydroxylation are induced by dietary broccoli. On the other hand, the catalytic activity of CYP2E1 is not affected to the same degree by dietary broccoli. PMID- 8625494 TI - 7-Alkylguanine adducts of styrene oxide determined by 32P-postlabeling in DNA and human embryonal lung fibroblasts (HEL). AB - The modified 32P-postlabeling method was used for the detection of N-7-(2-hydroxy phenylethyl) guanine adducts in DNA and human embryonal lung (HEL) cells treated in vitro with styrene oxide (SO). The total recovery of 7-alkylguanine adducts of styrene oxide in DNA analysed by 32P-postlabeling assay was 4.1 +/- 0.6%. The disappearance of 7-alkyldeoxyguanosine monophosphate adducts from SO-modified DNA at 37 degrees C showed a half-life of 19 h. The levels of 7-alkylguanine DNA adducts and single-strand breaks (SSBs) in DNA were determined in HEL cells treated with SO for 3 and 18 h. In the 3 h treatment there was a concentration dependent increase of both 7-alkylguanine adducts and SSBs in DNA (r = 0.98, P = 0.012 and r = 0.99, P = 0.003, respectively). We found a significant correlation between 7-alkylguanine DNA adducts and SSBs in DNA (r = 0.98, P = 0.011). In HEL cells approximately 3-fold higher levels of both 7-alkylguanine DNA adducts and SSB in DNA were found after the SO (100 microM) treatment for 3 h than after the treatment for 18 h. A significant concentration-dependent increase was found only for SSB ( r = 0.95, P = 0.024) in the 18 h treatment with SO. There was no significant correlation between 7-alkylguanine adducts and SSBs (r = 0.72, P = 0.15). Our data suggest relatively fast removal of both 7-alkylguanine adducts of SO and DNA SSBs. PMID- 8625495 TI - Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl isothiocyanate. AB - 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific nitrosamine in animals and has been suggested to play a role in human tobacco related cancers. Our previous study demonstrated that cytochrome P450 (P450) 1A2 catalyzes the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) (an alpha-hydroxylation product) from NNK in human liver microsomes. Phenethyl isothiocyanate (PEITC) inhibits NNK tumorigenesis by blocking the activation of NNK. The purpose of the present study was to elucidate the mechanism of inhibition of P450 1A2-catalyzed NNK activation by PEITC. Human P450 1A2 was expressed in Escherichia coli and purified to homogeneity. In a reconstituted system, P450 1A2 catalyzed the formation of keto alcohol and 4-oxo-1-(3-pyridyl) 1-butanone (keto aldehyde) from NNK, with the keto alcohol being the major metabolite. The apparent Km and Vmax values for keto alcohol formation was 380 microM and 1.7 nmol/min/nmol P450, respectively. For the tobacco-specific nitrosamine N-nitrosonornicotine (NNN), P450 1A2 catalyzed the formation of the derived 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid),4-oxo-4-(3 pyridyl)butyric acid (keto acid) and keto alcohol. In comparison to NNK, NNN had a lower rate of oxidation with P450 1A2. PEITC decreased the formation of the NNK derived keto alcohol in a concentration-dependent manner, with an IC50 value of 0.14 microM. PEITC was a competitive inhibitor of P450 1A2, exhibiting a Ki value of 0.18 microM. Preincubation of PEITC with NADPH in the reconstituted system resulted in a further decrease (25%) in the catalytic activity of P450 1A2, suggesting that there is a slow metabolism-dependent inhibition of P450 1A2 by PEITC. The formation of keto aldehyde and keto alcohol was inhibited by PEITC in human liver microsomes with IC50 values of 9.5 and 4.6 microM respectively. Methoxyresorufin O-dealkylase activity, a marker for P450 1A2, was decreased by PEITC in a concentration-dependent manner, with an IC50 of 0.34 microM. The results suggest that PEITC itself is a potent inhibitor of P450 1A2 and that a metabolite(s) of PEITC can also inhibit P450 1A2. We conclude that PEITC may be an effective inhibitor of the carcinogenicity or toxicity of chemicals that are activated by P450 1A2. PMID- 8625496 TI - Involvement of glutathione in induction of c-jun proto-oncogene by methylmethanesulfonate in NIH 3T3 cells. AB - The c-jun proto-oncogene plays a vital role in the carcinogenic process. Although numerous works have extensively investigated the induction mechanisms of c-jun by UV, hydrogen peroxide or 12-O-tetradecanoylphorbol-13-acetate, the mechanism induced by alkylating agents has received little attention. In this study, NIH 3T3 cells were exposed to methylmethanesulfonate (MMS), revealing that the agent clearly induced c-jun expression with a peak at 2 h. Pretreatment of cells with various kinase inhibitors, e.g. H7, genistein, herbimycin A and tyrphostin, did not show any significant effects on the MMS-induced c-jun expression. Benzamide, an inhibitor of poly(ADP)-ribosylation, enhanced the MMS-induced DNA breakages, but did not potentiate that agent which elicited c-jun expression. Another experiment showed that this agent transfected and overexpressed an activated v-H ras gene in NIH 3T3 cells, which became more resistant to MMS-induced DNA damage but expressed the same level of c-jun transcript as compared with NIH 3T3 cells in response to MMS. If intracellular glutathione (GSH) was completely depleted by buthionine sulfoximine (BSO), the MMS-elicited c-jun expression was blocked. Subsequently, re-elevating intracellular GSH by washing off BSO caused the expression of c-jun by MMS to increase proportionately. Based on these findings, we can conclude that the mechanism by which MMS induced c-jun expression does not occur through activation of protein tyrosine kinases or initiation of DNA damage, but is closely related to the intracellular GSH. PMID- 8625497 TI - The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes. AB - Ellagic acid (EA), a naturally occurring plant polyphenol possesses broad chemoprotective properties. Dietary EA has been shown to reduce the incidence of N-2-fluorenylacetamide-induced hepatocarcinogenesis in rats and N nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumors. In this study changes in the expression and activities of specific rat hepatic and esophageal mucosal cytochromes P450 (P450) and phase II enzymes following dietary EA treatment were investigated. Liver and esophageal mucosal microsomes and cytosol were prepared from three groups of Fisher 344 rats which were fed an AIN-76 diet containing no EA or 0.4 or 4.0 g/kg EA for 23 days. In the liver total P450 content decreased by up to 25% and P450 2E1-catalyzed p-nitrophenol hydroxylation decreased by 15%. No changes were observed in P450 1A1, 2B1 or 3A1/2 expression or activities or cytochrome b5 activity. P450 reductase activity decreased by up to 28%. Microsomal epoxide hydrolase (mEH) expression decreased by up to 85% after EA treatment, but mEH activities did not change. The hepatic phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone reductase [NAD-(P)H:QR] and UDP glucuronosyltransferase (UDPGT) activities increased by up to 26, 17 and 75% respectively. Assays for specific forms of GST indicated marked increases in the activities of isozymes 2-2 (190%), 4-4 (150%) and 5-5 (82%). In the rat esophageal mucosa only P450 1A1 could be detected by Western blot analysis and androstendione was the only P450 metabolite of testosterone detectable. However, there were no differences in the expression of P450 1A1, the formation of androstendione or NAD(P)H:QR activities between control and EA-fed rats in the esophagus. Although there was no significant decrease in overall GST activity, as measured with 1-chloro-2,4-dinitrobenzene (CDNB), there was a significant decrease in the activity of the 2-2 isozyme (66% of control). In vitro incubations showed that EA at a concentration of 100 microM inhibited P450 2E1, 1A1 and 2B1 activities by 87, 55 and 18% respectively, but did not affect 3A1/2 activity. Using standard steady-state kinetic analyses, EA was shown to be a potent non-competitive inhibitor of both liver microsomal ethoxyresorufin O deethylase and p-nitrophenol hydroxylase activities, with apparent Ki values of approximately 55 and 14 microM respectively. In conclusion, these results demonstrate that EA causes a decrease in total hepatic P450 with a significant effect on hepatic P450 2E1, increases some hepatic phase II enzyme activities [GST, NAD-(P)H:QR and UDPGT] and decreases hepatic mEH expression. It also inhibits the catalytic activity of some P450 isozymes in vitro. Thus the chemoprotective effect of EA against various chemically induced cancers may involve decreases in the rates of metabolism of these carcinogens by phase I enzymes, due to both direct inhibition of catalytic activity and modulation of gene expression, in addition to effects on the expression of phase II enzymes, thereby enhancing the ability of the target tissues to detoxify the reactive intermediates. PMID- 8625498 TI - Conformational studies of depurinating DNA adducts from syn-dibenzo[a,l]pyrene diolepoxide. AB - One of the major DNA adducts from the extremely potent aromatic carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is the depurinating adduct syn-DB[a,l]P diolepoxide 14-N7Ade. Low-temperature fluorescence spectra of this adduct (and related derivaties bound to N3-adenine and N7-guanine) showed two distinct (0,0) origin bands with different excited-state vibrational frequencies, as measured by means of fluorescence line narrowing spectroscopy. The relative intensity of the two origin bands was solvent dependent. The hypothesis that this phenomenon could be due to a conformational equilibrium was tested using molecular mechanics, dynamical simulations and semi-empirical quantum-mechanical calculations. The hydrolyzed metabolite DB[a,l]P tetraol was also studied for comparison. It was found that the syn-DB[a,l]P diolepoxide-14-N7Ade adduct is formed via trans addition to the epoxide. Exploration of the conformational space indeed produced two potential energy minima; both corresponding to structures in which the aromatic ring system is severely distorted. In conformation I the proximity of the distal ring forces the adenine base into a pseudo-axial position and the cyclohexenyl ring adopts a half-boat structure. In conformation II the distal ring is bent in the opposite direction, allowing the cyclohexenyl ring to adopt a half-chair structure with the base in a pseudo-equatorial position, partially stacked over the distal ring. THe difference in (0,0) transition energy calculated for the two conformers agrees very well with the spectroscopic data, and the relative orientations of the hydrogens bound to the cyclohexenyl ring in the major (half-boat) conformation I are in full agreement with the experimentally observed proton NMR coupling constants. PMID- 8625499 TI - Identification of the cytochrome P450 isozymes involved in the metabolism of N nitrosodipropyl-,N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine. AB - The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates. PMID- 8625500 TI - O6-ethylguanine and O6-benzylguanine incorporated site-specifically in codon 12 of the rat H-ras gene induce semi-targeted as well as targeted mutations in Rat4 cells. AB - To examine the miscoding properties of modified guanine residues bearing increasingly bulky O6-substituents, Rat4 cells, grown in the presence of O6 benzylguanine to deplete the DNA repair protein O6-alkylguanine-DNA alkyltransferase, were transfected with plasmids carrying H-ras genes in which O6 methyl, O6-ethyl- and O6-benzylguanine were substituted for the first, second or both the first and second guanine residues of codon 12 (GGA). DNA from isolated transformed colonies was amplified by PCR and directly sequenced by high temperature manual and automated methods. The results show that O6-ethylguanine and O6-benzylguanine induced semi-targeted as well as targeted mutations, in contrast to O6-methylguanine, which induced only targeted mutations. When incorporated in place of the first guanine of H-ras codon 12, the targeted mutations induced by all these modified guanines were exclusively G-->A transitions. When incorporated at the second position of codon 12, O6 benzylguanine induced G-->A, G-->T and G-->C mutations. O6-Ethylguanine at the second position induced chiefly G-->A transitions, and O6-methylguanine induced G ->A transitions exclusively. Semi-targeted mutations were strictly G-->A at the base 3' to a position 1 adduct or 5' to a position 2 adduct. The mechanism for induction of targeted mutations probably involves decreasing preference to thymidine incorporation opposite an O6-modified guanine as the size of the O6 substituent increases, while the mechanism for non-targeted mutations may be related to abasic site formation or to translesion synthesis which might be made error-prone by obstructive DNA lesions in this context. PMID- 8625501 TI - Neoplastic transformation and DNA-binding of 4,4'-methylenebis(2-chloroaniline) in SV40-immortalized human uroepithelial cell lines. AB - The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4-4'-methylenebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho toluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (PBQ) and N-hydroxy-4 aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40 immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as the N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorob enz yl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4 amino-3-chlorot oluene (pdApACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen. PMID- 8625502 TI - Carcinogenicity, DNA adduct formation and K-ras activation by 7H dibenzo[c,g]carbazole in strain A/J mouse lung. AB - N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmental pollutants formed during the combustion of organic materials. 7-H Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung, liver and skin. We undertook these studies to determine whether tissue specificity for DBC lung carcinogenicity in the strain A/J mouse is mirrored by formation of DBC-DNA adducts in lung tissue and whether these adducts are consistent with mutation patterns in the K-ras gene. Strain A/J mice were given a single i.p. injection of DBC at doses of 0, 5, 10, 20 or 40 mg/kg and levels of DNA adducts in the lung were monitored by 32P-postlabeling on days 1, 3, 5, 7, 14 and 21. The remaining animals were sacrificed 8 months after DBC treatment and lung tumor multiplicity and K-ras mutation patterns in the tumors were determined. The lung tumor response to DBC was dose related, with an average of 4.7 +/- 1.2 tumors/mouse at 5 mg/kg and 48.1 +/- 5.5 tumors/mouse at 40 mg/kg. As many as seven DBC-DNA adducts were observed in the lung. DNA binding levels in the lung were highest at 40 mg/kg, with maximum binding at 5-7 days. At lower dose levels the maximum binding to DNA decreased and shifted to earlier time points. The DBC-DNA adduct in the lung with the highest level of binding at all dose levels was DBC-DNA adduct 3. The majority of DBC-induced mutations in the K-ras gene in the lung were A-->T (80%) transversions in the third base of codon 61, a mutation that has not been previously observed in chemically induced lung tumors in strain A/J mice. PMID- 8625503 TI - Infrequent alterations of the p53 gene in rat skin cancers induced by ionizing radiation. AB - Radiation carcinogenesis almost certainly involves multiple genetic alterations. Identification of such genetic alterations would provide information to help understand better the molecular mechanism of radiation carcinogenesis. The energy released by ionizing radiation has the potential to produce DNA strand breaks, major gene deletions or rearrangements, and other base damages. Alterations of the p53 gene, a common tumour suppressor gene altered in human cancers, were examined in radiation-induced rat skin cancers. Genomic DNA from a total of 33 rat skin cancers induced by ionizing radiation was examined by Southern blot hybridization for abnormal restriction fragment patterns in the p53 gene. An abnormal p53 restriction pattern was found in one of 16 cancers induced by electron radiation and in one of nine cancers induced by neon ions. The genomic DNA from representative cancers, including the two with an abnormal restriction pattern, was further examined by polymerase chain reaction amplification and direct sequencing in exons 5-8 of the p53 gene. The results showed that one restriction fragment length polymorphism (RFLP)-positive cancer induced by electron radiation had a partial gene deletion which was defined approximately between exons 2-8, while none of the other cancers showed sequence changes. Our results indicate that the alterations in the critical binding region of the p53 gene are infrequent in rat skin cancers induced by either electron or neon ion radiation. PMID- 8625504 TI - Inhibitory effect of dietary flavonol quercetin on 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis. AB - The inhibitory effect of dietary supplementation with flavonol quercetin on 7,12 dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis was investigated. Dietary quercetin inhibited the incidence of both papillomas and tumors induced by DMBA. The fluorescence spectra of papillomas and tumors showed different prominent maxima and a characteristic peak around 620-630 nm, which could be attributed to the accumulation of porphyrin compounds. Further, the fluorescence intensities at 630 nm (FI630nm) were elevated, whereas the ratio FI530nm/FI630nm was decreased in DMBA-induced lesions. Quercetin treatment significantly decreased FI630nm and increased the ratio FI520nm/FI630nm when compared with DMBA-induced lesions. It is therefore evident that quercetin has an inhibitory effect on DMBA-induced carcinogenesis and further studies will throw more light on its use as a chemopreventive agent against oral cancer. PMID- 8625505 TI - Glutathione S-transferase GSTT1 genotypes and susceptibility to cancer: studies of interactions with GSTM1 in lung, oral, gastric and colorectal cancers. AB - Allelism in glutathione S-transferase GSTM1 and GSTT1 has been suggested as a risk factor in various cancers. Accordingly, we describe a group of case-control studies carried out to identify associations between GSTT1 genotypes and susceptibility to lung, oral, gastric and colorectal cancers. The frequencies of the putatively high risk GSTT1 null genotype were not increased in the lung, oral or gastric cancer cases compared with controls but the frequency of this genotype was significantly increased (P = 0.0011, odds ratio = 1.88) in the colorectal cancer cases. No significant interactions between the GSTT1 and GSTM1 null genotypes types were identified in the cancer groups studied. Indeed, no significant associations between GSTM1 genotypes and susceptibility were identified though further evidence was obtained that the protective effect of GSTM1*A and GSTM1*B is not equal. The data complement studies showing that GSTT1 null is associated with an increased susceptibility to total ulcerative colitis and suggests that this enzyme is important in the detoxification of unidentified xenobiotics in the large intestine. PMID- 8625506 TI - AG8 cells, which are highly resistant to hydrogen peroxide, display collateral sensitivity to the combination of hydrogen peroxide and L-histidine. AB - The results obtained in the present study indicate that AG8 cells, which are highly resistant to H2O2, are not cross-resistant to the combination of H2O2/L histidine. In fact, once the influence of elevated catalase on the AG8 pheno-type has been circumvented (by treatment of AG8 cells with aminotriazole), AG8 cells display essentially no cross-resistance to the H2O2/L-histidine cocktail while retaining considerable resistance to H2O2 alone (when compared to wild-type AA8 cells). Although H2O2 alone dose not produce DNA double strand breaks (DSBs), this type of lesion was readily detected upon exposure of sensitive or resistant cells to the oxidant in the presence of the amino acid. Interestingly, similar levels of DNA DSBs were detected in AA8 and catalase-depleted AG8 cells. An excellent correlation was found when the cytotoxicity and the level of DNA DSBs obtained in sensitive and resistant cells (with normal or reduced catalase levels) challenged with the cocktail H2O2/L-histidine were compared. This would suggest that DSBs produced on a per cell basis always result in an equal level of toxicity, regardless of the cell type (resistant versus sensitive cell line), the lethality of each of these cell lines being dependent on the number of induced DSBs. In conclusion, the results presented here provide further evidence in support of the hypothesis that cell killing elicited by the combination of H2O2/L histidine involves a mechanism distinct from that following treatment with H2O2 alone. The fact that H2O2-resistant AG8 cells, which are not cross-resistant to agents promoting cell death via DNA DSB-induction, display collateral sensitivity to the cocktail H2O2/L-histidine, strongly suggests that cell killing triggered by this treatment is mediated by DNA double strand breakage. PMID- 8625507 TI - Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro. AB - Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas. PMID- 8625508 TI - Anti-endothelial cell antibodies: only for scientists or for clinicians too? PMID- 8625509 TI - Dynamics of HIV-1 replication following influenza vaccination of HIV+ individuals. AB - Levels of HIV-1 have been reported to increase in peripheral blood after influenza vaccination of HIV+ individuals. In this study we have evaluated the dynamics of these changes. Ten HIV-1+ individuals classified in revised CDC clinical categories B and C as well as five seronegative healthy controls were vaccinated with the recommended influenza strains. HIV viral RNA and proviral DNA were sequentially quantified in serum and blood lymphocytes, respectively. Nine of the 10 HIV+ individuals had an increase in the frequency of infected CD4 cells 2 weeks after influenza vaccination. Individuals with low viral load had a rapid increase in viraemia and a small increase in frequency of infected cells in peripheral blood. In contrast, individuals with high viral load had a small drop in viraemia followed by a significant rise in the rate of infected cells. The observed change may resemble those taking place during intercurrent infections in HIV+ individuals. The effects of the relative increases in infectious virus after the transient viraemic phase should be further investigated to evaluate potential risks of vaccination. PMID- 8625510 TI - IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans. AB - We examined the role of IL-12 in host resistance to Cryptococcus neoformans using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with viable yeast cells. Mice untreated with IL-12 allowed an uncontrolled multiplication of yeast cells in the lung with infiltrations of few inflammatory cells, and a cryptococcal dissemination to the brain and meningitis by 3 weeks, resulting in death of all animals within 4-6 weeks. IL-12, when administered from the day of tracheal infection for 7 days, induced a marked infiltration of inflammatory cells, consisting mostly of mononuclear cells, and significantly reduced the number of viable yeast cells in the lung. The treatment suppressed brain dissemination, as shown by a marked reduction of yeast cells in the brain and prevention of meningitis. These effects resulted in a significant increase in the survival rate of infected mice. In contrast, late administration of IL-12 commencing on day 7 after instillation of yeast cells failed to protect the mice against infection with C. neoformans. In further experiments, early administration of IL-12 markedly induced interferon gamma (IFN-gamma) mRNA in the lungs of infected mice, while no IFN-gamma mRNA was detected without this treatment. Our results indicate that IL-12 is effective when administered in the early period of pulmonary cryptococcal infection. PMID- 8625512 TI - A malaria parasite toxin associated with Plasmodium vivax paroxysms. AB - We have previously demonstrated a correlation between clinical paroxysms in Plasmodium vivax malarial infections and the appearance in patients' plasma of factors that kill blood stage parasites (gametocytes). This activity was, as previously shown, dependent on the presence in paroxysm plasma of tumour necrosis factor-alpha (TNF-alpha), which acts in conjunction with other 'complementary' factors. Here we have identified a parasite component which is essential for this activity and functions as a 'complementary' factor together with TNF, and a third component of unknown origin. The P. vivax parasite component present in paroxysm plasma can be substituted with a blood-stage schizont extract of either P. vivax or P. falciparum. This was demonstrated by restoring the parasite-killing activity to post-paroxysm plasma (from which it was absent) with the addition of the extracts together with TNF. The active materials in these extracts, however, are different from the natural components in P. vivax paroxysm plasma, i.e. while the schizont extracts are immunologically cross-reactive between species, the activity of the natural P. vivax toxin(s) in patients' plasma is neutralized only by the homologous antisera. Plasmodium falciparum infections have neither distinct paroxysms nor parasite-killing activity in plasma. The pronounced paroxysms of P. vivax infections may thus be due in part of a species-specific toxin(s). PMID- 8625511 TI - Macrophage nitric oxide synthase (NOS) activation by Nocardia opaca fractions and 15- and 56-kD isolated antigens. AB - The Gram-positive bacterium, Nocardia opaca, is a source of substances with adjuvant effect, ability to stimulate macrophages and natural killer cells for enhanced cytotoxity and cytokine production and B lymphocytes for polyclonal immunoglobulin secretion. We determined the immunogenicity of isolated N. opaca fractions and prepared MoAbs against immunogenic water-soluble mitogen (NWSM). Two main proteins of molecular mass 15 and 56 kD were detected in western blot analysis and isolated by affinity chromatography using anti-NWSM MoAb B7/7. Both these isolated nocardial antigens were found to stimulate mouse peritoneal macrophage NOS. The effect of 5 micrograms NWSM was comparable to that of 5 micrograms lipopolysaccharide (LPS) or 20 U of interferon-gamma (IFN-gamma) added to cell cultures. The MoAb B7/7 decreased No2- production induced by NWSM or by isolated nocardial antigens, but did not significantly influence the production elicited by LPS or IFN-gamma. On the other hand, NOS activation by NWSM was not affected by anti-IFN-gamma MoAb. The possible independent pathway for IFN-gamma and NWSM macrophage activation is discussed. PMID- 8625513 TI - Beneficial effect of Salmonella typhimurium infection and of immunoglobulins from S. typhimurium-infected mice on the autoimmune disease of (NZB x NZW) F1 mice. AB - Various infections can precede or aggravate autoimmune diseases. Yet a beneficial effect of infection has also been described an various mechanisms have been postulated to explain this effect. The aim of this study was to examine the hypothesis that infection can have an immunoregulatory effect on the autoimmune process via the increased production of natural polyreactive antibodies. The effect of Salmonella typhimurium infection on the lupus-like disease of (NZB x NZW)F1 (B/W) mice was therefore studied. The effect of IgM and IgG preparations isolated from the serum of S. typhimurium-infected C57B1/6 and CBA mice on the autoimmune disease of B/W mice was also tested. C57B1/6 and CBA mice were chosen because they are respectively genetically susceptible and resistant to S. typhimurium infection and they differ in their antibody response during the early phase of infection. CBA mice can mount a specific anti-bacterium antibody response, whereas C57B1/6 mice present increased production of polyreactive antibodies. The infection effect was evaluated on several disease parameters, i.e. survival, incidence of high grade proteinuria and serum IgM and IgG antibody activity directed against a panel of autoantigens. Our main findings were: (i) infection of B/W mice with an attenuated strain of S. typhimurium delayed the course of the autoimmune disease when performed before the appearance of autoimmune symptoms; and (ii) IgM and IgG preparations from S. typhimurium infected C57B1/6 mice had a similar effect whereas the IgM and IgG preparations from infected CBA mice, as well as from normal C57B1/6 and CBA mice, were ineffective. These results suggest that S. typhimurium infection can beneficially influence the development of the autoimmune disease of B/W mice. The immunoregulatory effect of the infection seems to be related at least partially, to the increase of a particular population of antibodies, the polyreactive antibodies. PMID- 8625514 TI - Detection of nucleosome-IgG immune complexes in ascites from mice transplanted with anti-DNA antibody-secreting hybridomas and in plasma from MRL-lpr/lpr mice. AB - Autoantibodies directed against chromatin components characterize lupus diseases. Immune complexes made of these autoantibodies bound to nucleosomes released from dead cells could play some pathogenic role. The aims of this study were to investigate if nucleosome-IgG complexes could contaminate IgG anti-DNA MoAb preparations, and if such complexes circulate in lupus diseases. A new method was set up using preformed nucleosome-IgG complexes. Complexes were adsorbed onto microplate through Fc binding and nucleosomal DNA was detected by internal incorporation of labelled nucleotide. Using this method, high amounts of complexes were found in ascites from mice transplanted with anti-DNA antibody secreting hybridomas. In some ascites, nucleosome was found to be strongly associated with the MoAb, confirming that nucleosome-IgG complexes could contaminate monoclonal autoantibody preparations. In MRL-lpr/lpr mice, nucleosome IgG complexes were detected at 16-24 weeks of age at a time when kidney lesions are rapidly worsening, raising the question of their pathogenic significance. PMID- 8625515 TI - Autoantibodies to the collagenous region of C1q occur in three strains of lupus prone mice. AB - We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid-phase ligand, in the presence of 1 M NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q-CLR, as the solid-phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (rsp = 0.73, P less than 0.001), and (ii) human Clq-CLR alone (rsp = 0.86, P = 0.001). Antibodies to Clq were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus-prone mice. Median (range) anti-C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16-66), 21 (17-39) and 19 (15-27) EU, respectively. The median anti-Clq antibody level in MRL/lpr mice aged 5 months was 76 (35-142) EU, significantly higher than that at 3 months (U = 558, P less than 0.0005). Median anti-C1q antibody level in NZB/W mice at 8 months was 37 (13 74) EU and in BXSB mice at 11 months was 62 (31-231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P less than 0.001, respectively). This is the first demonstration of anti-C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus-prone mice are under evaluation. PMID- 8625516 TI - Successful induction of severe destructive arthritis by the transfer of in vitro activated synovial fluid T cells from patients with rheumatoid arthritis (RA) in severe combined immunodeficient (SCID) mice. AB - In order to investigate the role of pathogenic T cells in RA, the establishment of an RA model using patients' T cells is thought to be essential. In this study, multiple and severe destructive arthritis was established by transferring in vitro-stimulated synovial fluid T (SFT) cells from patients with RA through simultaneous injection into knee joint and peritoneal cavity of SCID mice without causing xenogeneic graft-versus-host disease (GVHD). Neither the transfer of unstimulated SFT cells nor sole i.p. injection was sufficient to induce severe arthritis. Interestingly, in contrast with SFT cells, in vitro-activated peripheral blood lymphocytes from RA patients failed to trigger such arthritis, suggesting that pathogenic T cells might be concentrated in synovial fluid of RA patients. This, the first severe arthritis model mimicking RA induced by RA patients' T cells, is expected to provide important information about RA pathogenesis and a possible therapeutic approach. PMID- 8625518 TI - Fc gamma RIIa polymorphism in systemic lupus erythematosus (SLE): no association with disease. AB - An allotypic variant of Fc gamma RIIa, Fc gamma RIIa-HR (Fc gamma RIIa-R131), has been shown in vitro to reduce the capacity of phagocytic cells to bind and internalize IgG-containing immune complexes. Our aim was to determine whether this allotypic variant was associated with susceptibility to SLE and the development of lupus nephritis, as previous studies have suggested. Fc gamma RIIA genotype analysis was performed by amplification refractory mutation system polymerase chain reaction (ARMS-PCR) in 215 Caucasoid, 70 Afro-Caribbean, and 46 Chinese patients with SLE, and in 259,77 and 49 ethnically matched controls, respectively. Distribution of Fc gamma RIIa genotypes between the patients and ethnically matched controls was not significantly different in the three populations studied. No association between the Fc gamma RIIa-HR allotype and nephritis was found. Our results suggest that the Fc gamma RIIa-HR allotype is not a major factor predisposing to the development of SLE, or to lupus nephritis. PMID- 8625517 TI - Structural differences between the human and mouse 52-kD Ro autoantigens associated with poorly conserved autoantibody activity across species. AB - Anti-nuclear autoantibodies found in human autoimmune diseases frequently cross react with homologous autoantigens in distant species, supporting the notion that autoantibodies target conserved functional domains. However, the 52-kD Ro(SS-A) protein is an exception, in that human autoantibodies are not known to recognize any equivalent antigen in the cells of rodents and other non-primate species. To understand this lack of cross-reactivity we have isolated cDNAs encoding the mouse 52-kD Ro molecule. The cDNA encoding mouse 52-kD Ro revealed an open reading frame of 470 amino acids, with 70% sequence identity to the human 52-kD Ro antigen. The putative leucine-zipper and zinc-finger motifs present in human Ro52 were conserved in the mouse protein. Recombinant mouse 52-kD Ro protein reacted with human autoantibodies by ELISA and immunoblot, but with approximately 10-fold lower reactivity than recombinant human 52-kD Ro protein under the same conditions. Detection of both human and mouse 52-kD Ro by immunoblot was dependent on antigen concentration which was limiting in the cell equivalents generally used in immunoblot assays. Differential chaotropic disruption of antibody binding suggested a lower avidity of human autoantibody binding to the mouse 52-kD Ro protein compared with the human antigen. Thus the poor reactivity of native mouse 52-kD Ro with human autoantibodies is associated with species divergence diffusely distributed throughout the primary structure of the 52-kD Ro molecule. PMID- 8625519 TI - Affinity-purified cardiolipin-binding antibodies show heterogeneity in their binding to oxidized low-density lipoprotein. AB - Antiphospholipid antibodies in autoimmune sera have been shown to react with a complex of phospholipids (cardiolipin) and a plasma phospholipid-binding protein, beta 2-glycoprotein I (apolipoprotein H). The binding of these antibodies was inhibited by oxidized low-density lipoprotein (LDL) in sera from patients with systemic lupus erythematosus (SLE), suggesting cross-reactivity between antiphospholipid antibodies and antibodies binding to oxidized LDL. We purified antiphospholipid antibodies by cardiolipin-polyacrylamide column from seven SLE sera and studied the reactivity of eluted fractions with cardiolipin-beta 2 glycoprotein I complex and oxidized LDL (malondialdehyde-conjugated LDL) in solid phase enzyme immunoassay. In four sera the binding of IgG antibodies to cardiolipin-beta 2-glycoprotein I complex and to oxidized LDL appeared in the same fractions, whereas in three sera reactivities against cardiolipin and oxidized LDL were observed, at least in part, in separate fractions. The binding to solid-phase cardiolipin was dependent on the presence of exogenous beta 2 glycoprotein I in all fractions. Our findings show that antiphospholipid antibodies are heterogeneous in their binding to oxidized LDL, indicating that these two antibodies may have different subspecificities. Some eluted fractions reacted only with oxidized LDL, and did not show binding to cardiolipin-beta 2 glycoprotein I complex, suggesting that the lipid part in the antigenic complex might be responsible for the cross-reactivity of these antibodies. Accordingly, the biological functions of antibodies against phospholipid-beta 2-glycoprotein I complex and antibodies against oxidized LDL may also be different. PMID- 8625520 TI - L-selectin in patients with common variable immunodeficiency (CVID): a comparative study with normal individuals. AB - L-selectin in one of the key members of the selectin family of adhesion molecules and initiates leucocyte attachment to specialized high endothelial venules. The shed form, which retains functional activity, can be detected in biological fluids and is increased in diseases of many kinds. In the present study, we investigated L-selectin expression on leucocytes and measured the soluble form in the plasma of healthy individuals and patients with CVID. A significant loss of L selectin expression is found on CVID B cells, which is marked by the presence of a substantial population of L-selectin-negative B cells in the peripheral blood of some CVID patients. On CD4+ T cells, the loss in L-selectin expression affects mostly the CD45RO+ population. Peripheral blood leucocytes other than lymphocytes express L-selectin molecule normally. Moreover, soluble L-selectin was detected in significantly increased levels in CVID plasma compared with healthy controls. Our data suggest that the loss of L-selectin expressed by lymphocytes may be due to increased or aberrant lymphocyte activation in CVID patients who remain immunodeficient, and down-regulation of L-selectin from these lymphocytes may significantly contribute to the elevated levels of soluble L-selectin in the plasma, which may in turn affect further lymphocyte trafficking. PMID- 8625522 TI - Distribution of activated complement, C3b, and its degraded fragments, iC3b/C3dg, in the colonic mucosa of ulcerative colitis (UC). AB - The third component of complement (C3) is central to both the classical and alternative pathways in complement activation. In this study, involvement of C3 activation in the mucosal injury of UC was investigated. We examined the distribution of activated (C3b) and degraded fragments (iC3b/C3dg) of C3, terminal complement complex (TCC), and complement regulatory proteins in normal and diseased colonic mucosa including UC and other types of colitis using immunohistochemical techniques at the level of light and electron microscopy. While C3b and iC3b/C3dg staining was negligible in the normal mucosa, iC3b/C3dg and, to a lesser extent, C3b were deposited in UC mucosa along the epithelial basement membrane. The deposition was enhanced in relation to the severity of mucosal inflammation (C3b, P less than 0.05; iC3b/C3dg, P less than 0.01). Epithelial deposition of TCC was not observed in most UC mucosa. Immunoelectron microscopy showed that C3b and iC3b/C3dg were distributed mainly along the epithelial basement membrane and the underlying connective tissue in a granular, studded manner, and weakly present along the basolateral surface of epithelial cells. These C3 fragments were also deposited in inflammatory control mucosa such as ischaemic and infectious colitis. Our findings suggest that deposition of the C3 fragments occurs in inflamed colonic mucosa of diverse etiologies, including UC, but to define a role of the deposition in the development of mucosal injury in UC awaits direct study. PMID- 8625521 TI - The distribution of dividing T cells throughout the intestinal wall in inflammatory bowel disease (IBD). AB - T cell hypersensitivity has been implicated in the tissue damage in Crohn's disease (CD). All studies to date have examined mucosal T cells, although much of the tissue damage occurs in the submucosa and muscle layers. The aim of this work was to study T cell proliferation throughout the intestinal wall in children with IBD. Surgical resection material from 19 children with CD (10 ileal, 10 colonic), seven with ulcerative colitis (UC), and 12 normal controls was studied. The distribution of dividing T cells was investigated by double-immunohistochemistry using Ki67 to identify proliferating cells, and CD3 to identify T cells. In ileal and colonic lamina propria virtually no Ki67+, CD3+ cells were seen in control, UC or CD tissue. In contrast, there were significantly more Ki67+, CD3+ cells within the lymphoid follicles of ileal and colonic CD than in the follicles in UC and controls. Increased numbers of Ki67+, CD3+ cells were present in the submucosa, muscle layers (M) and serosa in Crohn's ileitis and colitis compared with the lamina propria (LP), although only in the muscle of the colon was the difference statistically significant (LP, 0.4% (0-1%); M, 1.6% (0-5.2%); P = 0.03). Pooling data from ileal and colonic CD, however, did show significantly increased Ki67+, CD3+ cells in both serosa and muscle layers compared with the LP. Dividing T cells have been identified in the deeper layers of the gut wall in CD. These may contribute to the fibrosis and muscle hyperplasia characteristic of the condition. PMID- 8625523 TI - Effect of intrajejunal elemental diet perfusion on local secretion of soluble CD4 and CD8. AB - The effects of nutrients on the mucosal immune system are poorly understood. The aim of this work was to study the cellular mucosal immune response to intrajejunal perfusion of an elemental diet (ED) or a control (C) electrolyte solution by measuring jejunal secretion of soluble CD4 (sCD4) and sCD8. sCD4 and sCD8 are markers of helper/inducer and suppressor/cytotoxic regulatory functions of T cells, respectively. A four lumen tube with a proximal occluding balloon at the angle of Treitz was used for jejunal perfusion in seven healthy volunteers (mean age 23 years). The length of the test segment was 40 cm. The jejunum was successively perfused with C for 80 min and then with ED containing 21.3 g/l of free amino acids and 104.2 g/l of oligosaccharides for 100 min. Jejunal fluid and serum concentrations of sCD4 and sCD8 were measured and their jejunal outputs calculated. When compared with C perfusion, jejunal perfusion with the ED resulted in a significant increase of sCD8 but not sCD4 jejunal secretion rates. sCD8 jejunal values increased early after ED perfusion and stayed at roughly the same level during the perfusion. Serum concentrations of sCD4 and sCD8 were not modified during ED perfusion. These data support the hypothesis that ED suppresses the immunologic tone of the gut, which could explain its beneficial effect in the management of intestinal inflammatory disease. PMID- 8625524 TI - Experimental T cell-mediated inflammatory reactions in the murine oral mucosa. II. Immunohistochemical characterization of resident and infiltrating cells. AB - The nature and phenotype of infiltrating cells in DTH-like reactions elicited in the murine oral mucosa have been examined by routine histological and immunohistochemical procedures. During the first few hours that followed buccal challenge with the contact sensitizer oxazolone, a discrete lymphocytic reaction was disclosed in the oral mucosa of animals previously sensitized at skin sites, but was absent in animals that had been sensitized at buccal sites. The early lymphocytic reaction in the oral mucosa of skin-sensitized animals preceded the emergence of CD11+ polymorphonuclear cells (PMN) which was most prominent 8 h after hapten challenge and invaded the whole thickness of the oral epithelium. The PMN rapidly disappeared by 24 h. In contrast, early PMN infiltration was virtually absent in specimens from animals similarly challenged but that had been sensitized at local buccal sites. Irrespective of site of initial sensitization, inflammatory reactions developed in the oral mucosa, being maximal by 24 h. At that stage, CD11+ macrophages were the predominant cell type. Both CD4 and CD8 T lymphocytes were scattered in both lamina propria and epithelium, and their numbers were raised throughout the time period studied (2-168 h). IL-2 receptor expression was maximal 16 h post-challenge, and was paralleled by increased DNA synthesis in CD4+ and CD8+ cells, as demonstrated by paired immunohistoautoradiography. Focal accumulations of mononuclear cells containing IL-2 producing cells were readily detected as early as 2-3 h following local challenge with hapten in animals primed at skin but not at buccal sites. Maximal IL-2 staining was detected at 24 h irrespective of initial sensitization site. Interferon-gamma-producing cells were detected at 8 h post-challenge and remained increased during the first 24 h. MHC class II expression was detected on few oral mucosa cells during the first 4 h following hapten challenge, being mainly confined to dendritic-like cells. Consistent with increased numbers of macrophages, MHC class II expression was most intense in specimens obtained 8-24 h after hapten challenge. Thereafter, MHC class II expression was still observed in specimens obtained as late as 72 h, but was essentially associated with patches of basal keratinocytes. Taken together, these observations support the notion that the murine oral mucosa can serve as the site of expression of locally or remotely induced DTH reactions, but also indicate that the site of initial sensitization can profoundly affect the cellular composition of inflammatory reactions subsequent to local buccal challenge. PMID- 8625525 TI - Circulating IgA, IgG, and IgM class antibody against Haemophilus parainfluenzae antigens in patients with IgA nephropathy. AB - We previously demonstrated a close relationship between the outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and IgA nephropathy (IgAN). Our objective was to clarify the relationship among IgA, IgG, and IgM class antibody against OMHP in the sera of 44 patients with IgAN and 62 patients with other glomerular diseases (OGD) by ELISA. Patients with IgAN showed a significantly higher level of IgA antibodies (P less than 0.0005) and IgG antibodies (P less than 0.001) against OHMP, than did patients with OGD. Positive correlations were observed between IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against OMHP in the sera of patients with IgAN. Immunoblotting showed that IgA, IgG, or IgM antibodies against OMHP in the sera of all patients with IgAN bound to components of OMHP. Amino acid sequences of three components of OMHP recognized by the sera from patients with IgAN revealed homology with those reported for outer membrane protein (OMP) P6 precursor, OMP P5, and P2 porin protein of H. influenzae. Results suggest that patients with IgAN have glomerular deposits of OMP P6 precursor, OMP P5, or P2 porin protein of HP, and a specific increase in the production of IgA antibodies against OMHP via polyclonal activation against these, with switching of production from one isotype to another, e.g. from IgM to IgA. PMID- 8625526 TI - Isolation of cDNAs encoding immunogenic regions of gp330, the autoantigen involved in Heymann nephritis. AB - Active Heymann nephritis is an organ-specific autoimmune disease of the rat kidney, characterized by the formation of immune complexes located subepithelially in the glomerulus. The T cell-mediated humoral immune response is directed to gp330, a large renal epithelial glycoprotein which is expressed both in the proximal tubule and on glomerular podocytes. In this study polyclonal rabbit antibodies raised against affinity-purified rat gp330 were used to screen a lambda-gt11 expression library of the rat kidney. One cDNA clone that was recognized by the antibodies coded for a 2.7-kb protein that is not described in the sequence database of GenBank/EMBL. Two other groups of cDNA clones were identified that displayed similarity with several members of the low-density lipoprotein (LDL)-receptor gene family to which gp330 belongs. By comparison with the gp330-cDNA sequence, these two clones could be mapped to two remote areas on the extracellular domain of gp330. The antigenicity of these two areas is in accordance with their location in highly hydrophilic regions on the extracellular domain of gp330. The cDNA clones described in this study may represent two main immunodominant regions on rat gp330. PMID- 8625527 TI - Elevation of plasma-soluble tumour necrosis factor receptors (TNF-R) in sarcoidosis. AB - Two types of receptor for tumour necrosis factor-alpha (TNF-R), the 55-kD receptor (TNF-RI) and the 75-kD receptor (TNF-RII), have been identified. Soluble TNF-RI (sTNF-RI) and soluble TNF-RII (sTNF-RII) can be measured in culture supernatants and biological fluids, and the role of sTNF-R has been suggested. In the present study, we measured plasma sTNF-RI and sTNF-RII levels in 19 patients with active sarcoidosis by ELISA in order to assess the state of both types of receptors in this disease. Both plasma sTNF-RI and sTNF-RII levels in patients with active sarcoidosis were significantly higher than those in normal control subjects. A longitudinal evaluation of plasma sTNF-RI and sTNF-RII levels showed that the magnitude of changes in sTNF-RII was closely related with the clinical course of sarcoidosis. These results suggest that plasma sTNF-RII levels may be useful parameters for monitoring the clinical course of sarcoidosis as well as markers for identifying disease activity. PMID- 8625528 TI - Lidocaine in bronchoalveolar lavage fluid (BALF) is an inhibitor of eosinophil active cytokines. AB - Eosinophils and eosinophil granule proteins may play an important role in the pathogenesis of asthma. BALF from 40 patients with symptomatic asthma were analysed for cytokine activity by the eosinophil survival assay. BALF from 15 patients showed increased survival activity. Survival activities in BALF from four of these patients were almost completely blocked by anti-IL-5 MoAb, and the remaining activities were blocked by anti-granulocyte-macrophage colony stimulating factor (GM-CSF), anti-IL-3 antibody, or both. Surprisingly, BALF samples from the other 25 patients decreased eosinophil viabilities below the levels of medium control. The inhibitory factor in these BALF was of low molecular weight, was heat-stable, was largely overcome by excess exogenously added cytokines, and was positively correlated with the concentrations of lidocaine in the BALF. Lidocaine itself inhibited eosinophil survival at concentrations less than those present in the BALF. These findings indicate that lidocaine is an inhibitor of cytokines in the eosinophil survival assay, and they suggest the need for caution in analyses of BALF containing lidocaine or other local anaesthetics. PMID- 8625529 TI - Dose- and time-dependent activation of rat alveolar macrophages by glucocorticoids. AB - Effects of glucocorticoids on immune functions are generally thought to be suppressive and anti-inflammatory. However, most reports dealing with this issue describe effects of long-term treatment with high doses of glucocorticoids on immune functions. In the present study we have investigated both dose and timing effects of exposure of alveolar macrophages with dexamethasone on lipopolysaccharide (LPS)-induced IL-1 beta and nitric oxide secretion. For this purpose, alveolar macrophages were preincubated with various doses of dexamethasone during varying intervals, followed by stimulation of the cells with endotoxin, either in the absence or presence of dexamethasone. Subsequently, the effects of this treatment of IL-1 beta and nitric oxide secretion were measured. It was shown that both short-term incubation of alveolar macrophages with high doses of dexamethasone and long-term incubation with low doses of dexamethasone lead to enhanced nitric oxide and enhanced IL-1 beta secretion upon subsequent stimulation of the cells with LPS. In contrast, long-term incubation of alveolar macrophages with high-dose dexamethasone leads to decreased IL-1 beta and nitric oxide secretion upon subsequent stimulation. Thus, it is concluded that the effects of dexamethasone on rat alveolar macrophages are both time- and dose dependent. It is therefore argued that effects of glucocorticoids on immune functions are not a priori suppressive, but that both dose and timing effects should be taken into account. PMID- 8625530 TI - The allergenic properties of fresh and preserved Hevea brasiliensis latex protein preparations. AB - The allergenic properties of the proteins of two lyophilized fractions of fresh natural rubber latex obtained by ultracentrifugation, the C serum and the sedimented bottom or lutoid fraction, have been compared with those of the serum proteins of two samples of high ammonia latex (HAL) [A]HALS obtained from HAL stored for more than 1 year, and [M]HALS derived from HAL stored for 6 weeks before ultracentrifugation and lyophilization. The most potent source of allergenic polypeptides both for skin prick testing of latex-sensitive patients and for immunoblots of their blood serum was the lutoid fraction of fresh latex. Skin prick tests and immunoblots of patients' sera showed that the allergenicity of the ammoniated latex decreased during storage. Skin prick tests using fractions of [A]HALS, C serum and lutoid proteins obtained after passage through a Sephacryl S300 column showed that the components of all three preparations which eluted in the largest volumes were almost equally effective in provoking the largest number of responses. Immunoblots of the sera of 43 latex-sensitive individuals showed that the majority (66%) of sera of the adult allergic patients reacted with a polypeptide of 19 kD. No characteristic pattern of binding latex polypeptides could be recognized in the sera from patients who were also asthmatic or from those who had an anaphylactic response to latex proteins. PMID- 8625531 TI - T cell receptor (TCR) repertoire and function of human epidermal T cells: restricted TCR V alpha-V beta genes are utilized by T cells residing in the lesional epidermis in fixed drug eruption. AB - Human epidermis contains a phenotypically heterogeneous population of T cells. No information however, is available regarding the TCR repertoire of these T cells and their relevant physiologic and pathologic functions in vivo. To this end, T cells were prepared from the lesional epidermis in two patients with fixed drug eruption (FDE) and their phenotype, function and TCR repertoire were examined in parallel. Both epidermal T cells, termed FDE-1 and -2 cells, respectively, expressed alpha beta TCR, but displayed some phenotypic heterogeneity. These T cells were induced to display cytolytic activity by ligation of the CD3/TCR-alpha beta complex. Comparative analyses of TCR V alpha and V beta expression in the epidermal T cells and the paired peripheral blood lymphocytes (PBL) by quantitative polymerase chain reaction (PCR) demonstrated that the epidermal T cells, but not the paired PBL, utilized a very limited range of V alpha and V beta genes. These results indicate that some expansion or preferential migration of epidermal T cells that recognize a restricted set of antigens expressed within the epidermis could occur in situ following ingestion of the causative drug. The persistence of these epidermal T cells in FDE lesions suggests their pathologic role in a drug-induced flare. PMID- 8625533 TI - Idiotype-induced T cell stimulation requires antigen presentation in association with HLA-DR molecules. AB - An important and yet unresolved question concerns the mode of T cell recognition of idiotypic epitopes on immunoglobulin molecules in humans. Results from murine and human studies show that some idiotype-specific T cells recognize conformational epitopes on immunoglobulin, and such T cells are not MHC restricted. In the present study T cell stimulation induced by idiotypic determinants on the autologous monoclonal IgG (M-components) from patients with monoclonal gammopathies was studied. In parallel, T cell stimulation in response to a conventional antigen, purified protein derivative, was also examined. It is shown that, as with conventional antigen, idiotype-induced T cell stimulation requires the presence of antigen-presenting cells (APC; monocytes and/or B cells), and is MHC class II (DR)-restricted. B cells, but not monocytes, can present idiotypic determinants to T cells at very low antigen concentrations, while monocytes do so only when antigen is present at high concentrations. Antigen processing and presentation is abrogated by treatment of APC with chloroquine. In conclusion, our study demonstrates that human idiotype-specific T cells recognize processed idiotypic determinants presented by MHC class II (HLA DR) molecules on APC, and that B cells require about 1000-fold less antigen that monocytes. PMID- 8625532 TI - Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM CSF) antibodies in carcinoma patients following GM-CSF combination therapy. AB - In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy. PMID- 8625534 TI - Human natural killer (NK) cells present staphylococcal enterotoxin B (SEB) to T lymphocytes. AB - Superantigen-mediated T cell activation requires the participation of antigen presenting cells (APC). Once superantigen has bound class II MHC molecules on the surface of APC, it then can interact with the T cell receptor to induce T cell activation. Superantigen-mediated T lymphocyte activation, along with its consequent cytokine production is thought to be the basis for the pathophysiology of conditions such as toxic shock syndrome, Kawasaki's disease and possibly rheumatoid arthritis. We examined the role of CD56+ NK lymphocytes in the interaction between superantigens and T lymphocytes. First, we found that a subpopulation of CD56+ cells freshly isolated from human peripheral blood expressed class II MHC molecules. The amount of HLA-DR expression varied between individuals, ranging from 9.3% to 37.7%. CD56+ (NK) cells were purified from the peripheral blood by cell sorting and were tested for their ability to support SEB mediated T cell activation as assessed by surface expression of IL-2 receptor alpha-chain (CD25) on CD3+ lymphocytes. We observed that when enriched T cells were incubated with SEB in the presence of NK cells, there was a significant up regulation of CD25 expression of the T cells. When HLA-DR+ cells were removed from sorted CD56+ populations, the remaining HLA-DR- NK cells were unable to support SEB-mediated T cell activation. Also, SEB up-regulated the expression of HLA-DR on CD56+ cells in peripheral blood mononuclear cell (PBMC) populations after 24 h of incubation, implying that the ability of NK cells to function as superantigen-presenting cells is up-regulated by superantigens themselves. Together, these data demonstrate for the first time that human CD56+ HLA-DR+ NK cells can function as superantigen-presenting cells, and imply that NK cells may be involved in the activation of non-specific T cell reactivity during early host defences against superantigen-elaborating microorganisms in vivo. Furthermore, the physical linkage of NK cells and T cells by the interaction of superantigen with HLA class II molecules and T cell receptors, respectively, may lead to NK cell activation and augmented lytic potential, helping to clear the body of superantigen-elaborating microorganisms. PMID- 8625536 TI - Intravenous immune globulin: mechanisms of action and model disease states. Proceedings of a symposium. Geneva, Switzerland, 9-10 June 1995. PMID- 8625535 TI - Down-modulation of mycobacterial-induced IL-1 beta production in human mononuclear cells by IL-4. AB - Tuberculosis is characterized by a cellular immune response mediated by various cytokines, including IL-1 beta released by stimulated mononuclear cells. It is now well established that IL-I beta plays an important role in local and systemic inflammatory response in tuberculosis. Here we have demonstrated, for the first time, that addition to IL-4 to human mononuclear cells obtained from 11 healthy bacille Calmette-Guerin (BCG)-vaccinated donors reduced BCG-induced production of IL-1 beta by 91.46 +/- 2.2%. This inhibitory effect was found highly significant (P less than 0.001) and was dose-dependent. The effect of IL-4 on the secretion of IL-1 beta was specific, since a complete reversion was obtained with a neutralizing MoAb to human IL-4. In addition, this inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in response to IL-4. Interestingly, the induction of IL 1 beta was regulated by IL-4, at least in part, by direct mechanism mediated through the extracellular domain of the 130-kD IL-4 receptor, as demonstrated by incubation of the mononuclear cells with the neutralizing anti-IL-4 receptor MoAb. Finally, a significant down-regulation of IL-1 beta secretion was observed in hsp70-stimulated mononuclear cells cultured with IL-4. Further experimental work is needed to establish the relevance of IL-4 in human mycobacterial infection in vivo. However, an understanding of the mechanisms that control IL-1 beta secretion in human mycobacterial infections is essential to understand the pathogenesis of tuberculosis. PMID- 8625537 TI - Intravenous immune globulin affects cytokine production in T lymphocytes and monocytes/macrophages. AB - Immune globulin for intravenous use (IVIG) has been used in many inflammatory conditions due to its immunomodulatory potential. The effector mechanisms are incompletely understood. This study dealt with the effects of IVIG on cytokine production in vitro. Cytokine synthesis was identified at the single-cell level using cytokine-specific MAb and indirect immunocytochemical techniques. Peripheral blood mononuclear cells (PBMC) were stimulated for 96 h by immobilized anti-CD3 MAb or by a combination of a protein kinase C activator (PMA) and a calcium ionophore (ionomycin). The addition of IVIG (6 mg/ml) caused a marked inhibition of proliferation and blast transformation despite unaffected cell survival. Anti-CD3-stimulated cultures containing IVIG exhibited a significant inhibition of production of T-cell derived lymphokines IL-2, IL-10, TNF-beta, IFN gamma and TNF-alpha (made by both monocytes and T cells), while synthesis of the monokine IL-8 was significantly increased. The expression of IL-2 receptors was significantly suppressed. Similar but transient inhibition of most T-cell products (IL-2, IL-3, IL-4, IL-5, IL-10, TNF-beta and GM-CSF) was noted in the PMA/ionomycin-containing cultures. In contrast, no effects were found on IFN gamma or TNF-alpha production. The superantigen streptococcal pyrogenic exotoxin A (SPE-A) induced vigorous cell activation and extensive cytokine synthesis. IVIG was added either at the beginning or 24 h after the initiation of cultures in order to elucidate the importance of direct toxin-neutralization. Addition of IVIG from the beginning of cultures induced a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular of IFN-gamma and TNF-beta. Supplementation with IVIG 24 h after initiation of cultures also led to a significant decrease in lymphokine synthesis. Monokine production (IL-1 alpha, IL-1 beta, IL-1ra, IL-6 and IL-8) was either unaffected or even increased. These two facts argue against direct antigen neutralization as being the only mechanism at work. However, in IVIG-exposed PBMC stimulated with LPS, IL-6 production was significantly reduced. A significant upregulation of IL-1ra was noticed in unstimulated PBMC cultured with IVIG. The results in all the experiments did not indicate a cytotoxic effect by IVIG on cell survival and the production of certain cytokines were unaffected. Instead, the authors believe that the results suggest a previously little examined functional link where the humoral immune response may have direct immunoregulatory effects on the cellular immune system. PMID- 8625539 TI - Parvovirus infection and its treatment. AB - B19 parvovirus is an important pathogen in man. Acute infection produces fifth disease (erythema infectiosum) in normal individuals, transient aplastic crisis in the patient with haemolysis, and pure red cell aplasia in the immunologically incompetent host. Fetal infection can lead to hydrops fetalis. The target cell of the virus is the marrow erythroid progenitor. The immune response to the virus is largely humoral and directed against limited numbers of epitopes. Persistent infection is due to failure to produce neutralizing antibodies. Because viral infection is prevalent in the population, therapeutic immune globulin preparations are a good source of anti-B19 antibodies. IgG administration can lead to cure of anaemia in the congenitally immunodeficient patient and to its amelioration in AIDS patients with persistent parvovirus infection. PMID- 8625540 TI - Mechanisms of action of intravenous immune globulin in immune-mediated diseases. AB - Intravenous immune globulin (IVIG) exhibits a number of immunomodulatory properties that are mediated by the Fe portion of IgG and by the spectrum of variable (V) regions contained in the immune globulin preparations. Five predominant and non-exclusive mechanisms of action have been proposed to account for the immunomodulatory effects of IVIG in immune-mediated diseases: (i) functional blockade of Fc receptors on splenic macrophages; (ii) inhibition of complement-mediated damage, an effect that is dependent on the ability of IgG to bind C3b and C4b and thus reduce the number of activated complement fragments that may deposit on target surfaces of complement activation; (iii) modulation of the production of cytokines and cytokine antagonists; (iv) neutralization of circulating autoantibodies by complementary (e.g. anti-idiotypic) antibodies in IVIG, a mechanism that accounts for the rapid decrease in titre of circulating autoantibodies that is often observed within hours following the infusion of IVIG; (v) selection of immune repertoires, a complex set of effects that may be observed in individuals receiving IVIG far beyond the half-life of the infused immunoglobulin and that is directly relevant to the ability of IVIG to, for example, suppress autoantibody-producing clones in patients with antibody mediated autoimmune disease and modulate graft versus host disease (GVHD). IVIG has been shown to downregulate or activate B-cell clones expressing surface IgG that is complementary (anti-idiotypic) to V regions of antibodies present in IVIG. IVIG has been shown also to interact with surface molecules of T cells that are essential to immune regulation, such as the alpha beta TCR, CD5, CD4, non polymorphic determinants of MHC class I molecules and adhesion molecules of T and B cells. The complex interactions of IVIG with functional molecules of cells of the immune system are relevant to its therapeutic effects in T cell- as well as B cell-mediated diseases and indeed, to our understanding of the physiological role of normal IgG and antibody networks in controlling autoreactivity in healthy individuals. PMID- 8625538 TI - Modulation of complement-mediated immune damage by intravenous immune globulin. AB - High-dose intravenous immune globulin (IVIG) exerts a beneficial effect in a variety of immune disorders. One possible underlying mechanism of this effect could be interference with the complement system. This conclusion was based on the results obtained in animal models of complement-mediated pathology, in vitro complement assays and studies on related human diseases. Clearance of IgM sensitized erythrocytes was specifically suppressed by IVIG treatment. The same therapy prevented pulmonary endothelial cell lesions, the hallmark of Forssman shock, in 75% of animals. All control animals, either untreated or injected with control reagents, died within minutes following induction of Forssman shock. In vitro uptake of C3b and C4b complement fragments onto corpusculate immune complexes was significantly inhibited by IVIG. Studies that involved patients suffering from disorders with pathogenesis similar to animal models of complement mediated immune injury fully supported the hypothesis that IVIG interacts with activated complement components and prevents their deposition on target cells. The author's results suggest that IVIG can be an effective modulator of inappropriate complement attack. PMID- 8625541 TI - Infectious risk of plasma donations: relationship to safety of intravenous immune globulins. AB - Although intravenous immune globulin (IVIG) preparations may be derived from tens of thousands of donations, the residual risk of viral contamination of recovered plasma appears remarkably low, at least for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Consequently, most pools are unlikely to contain these viruses although the situation is not as well understood for US-derived source plasma. In addition, routine preparation methods are likely to eliminate infectivity from the relatively fragile HIV. Current plasma screening measures are generally designed to maintain reasonable titres of anti-HBs in pools, perhaps contributing to the absence of HBV infection. The situation with respect to hepatitis C virus (HCV) is not well understood and it appears that sensitive screening for anti-HCV may have had an impact on the safety of at least one IVIG preparation. The implementation of effective viral inactivation procedures is expected to eliminate any further risk of HCV infection from IVIG preparations. PMID- 8625542 TI - The viral safety of intravenous immune globulin. AB - The viral safety of intravenous immune globulin (IVIG) preparations has been investigated since 1983 when it was discovered that non-A, non-B hepatitis (NANBH) could be transmitted by an experimental IVIG preparation. Recently, it has been demonstrated that the virus causing NANBH is the hepatitis C virus (HCV). A number of subsequent episodes of HCV transmission by IVIG have been reported, but not all the factors that have led to this transmission are clearly understood. However, based on two episodes of HCV transmission by anti-D immune globulin (formulated for intravenous administration), it appears that cold ethanol fractionation is important in ensuring viral safety because both of the implicated anti-D immune globulin preparations were manufactured without cold ethanol fractionation. Other HCV transmission episodes have been associated with chromatography (particularly DEAE-Sephadex chromatography) as a separation step carried out to further purify IgG, after cold ethanol fractionation, and it is possible that such a procedure has only a marginal partitioning capacity for infective HCV virions. The role of anti-HCV screening in improving the viral safety of IVIG preparations remains unclear, but a recent transmission episode by a previously safe IVIG preparation suggests that the absence of anti-HCV antibodies during plasma fractionation may affect the partitioning characteristics of HCV and may also cause a loss of neutralizing antibody against HCV. All of the IVIG preparations associated with HCV transmission have been formulated as freeze-dried preparations and this may have been important in stabilizing HCV during the period prior to administration to patients. No other viruses appear to have been transmitted by IVIG preparations, but prior to seroconversion, HCV-infected plasma donors may continue to contaminate plasma pools used for the manufacture of blood products, despite anti-HCV screening, and additional viral inactivation steps such as incubation at pH4 or solvent detergent treatment should be incorporated into the production process of all IVIG preparations. PMID- 8625543 TI - Immunomodulation in allogeneic marrow transplantation: use of intravenous immune globulin to suppress acute graft-versus-host disease. AB - Intravenous immune globulin (IVIG) has been used with success to prevent and treat infection in patients with immunodeficiencies of humoral immune function. More recently, IVIG has been shown to modulate immune responses and to treat successfully several autoimmune disease. Initial trials in bone marrow transplant recipients were aimed at the prevention of cytomegalovirus (CMV) disease. Although most studies showed such a benefit, CMV prophylaxis is now commonly provided by use of CMV-negative blood products or ganciclovir prophylaxis in, respectively, CMV-seronegative and CMV-seropositive patients. Acute and chronic graft-versus-host disease (GVHD) and associated infections remain critical barriers to the wider application of allogeneic blood or marrow transplantation, especially among patients given HLA-disparate grafts. To date, four controlled clinical trials have shown a significant reduction in acute GVHD in patients given IVIG (500-1000 mg/kg) weekly until 90-120 days post-transplant. In addition, bacterial infection in the early transplant period appears reduced. Meta-analysis of controlled trials reporting acute GVHD endpoints in 379 patients found a relative risk of acute GVHD of 0.68 (95% CI, 0.45-1.02) in IVIG recipients compared to untreated controls. Overall mortality reported in 809 patients in these trials showed an odds ratio of mortality of 0.74 (0.55-0.99) in IVIG recipients compared to controls (values < 1.0 suggest that IVIG was effective in preventing the event). More recently it has been shown that in the absence of hypogammaglobulinaemia, IVIG (500 mg/kg) given monthly from day 90 to 360 did not reduce the incidence of chronic GVHD or late complications. Future research is aimed at characterizing the mechanism of action of suppression of acute GVHD with weekly IVIG prophylaxis and the role of IVIG prophylaxis in patients receiving unrelated marrow grafts. PMID- 8625544 TI - Kawasaki syndrome: immunomodulatory benefit and potential toxin neutralization by intravenous immune globulin. AB - Kawasaki Syndrome (KS) is an acute multi-system vasculitis of infancy and early childhood associated with the development of coronary artery abnormalities. The prevalence of cardiovascular abnormalities can be significantly reduced by treating patients during the first 10 days of illness with high-dose intravenous immune globulin (IVIG). Despite the widely held belief that KS is caused by an infectious agent, the aetiology of this illness remains controversial. Recent immunological and microbiological studies suggest a potential role for staphylococcal and streptococcal toxins (superantigens) in the pathogenesis of KS. Confirmation of these findings could result in more effective diagnostic and therapeutic approaches for the treatment of this common cause of acquired heart disease in children. PMID- 8625545 TI - Clinical benefits and immunopathological correlates of intravenous immune globulin in the treatment of inflammatory myopathies. AB - High-dose intravenous immune globulin (IVIG) is emerging as a promising therapy for patients with inflammatory myopathies who have become unresponsive to, or cannot tolerate, conventional therapies. In a double-blind, placebo-controlled study, using objective criteria for improvement, IVIG demonstrated moderate to dramatic improvement in 75% of the patients with dermatomyositis. Preliminary results from a controlled study in inclusion-body myositis show that IVIG may also exert a mild benefit, but only in a small number of patients and in certain muscle groups. In some patients with polymyositis, IVIG is reported to be of benefit but controlled studies have not yet been completed. Immunocytochemical, immunological and in vitro studies on the patients' repeated muscle biopsies and follow-up sera showed that IVIG exerts its action in inflammatory myopathies by: (i) inhibiting myotoxic cytokines, such as TNF-alpha and IL-1; (ii) blockade of Fc receptors on endomysial macrophages interfering with Fc receptor-mediated phagocytosis; and (iii) inhibiting the uptake of C3 and intercepting the formation and deposition of membranolytic attack complex on the endomysial capillaries. PMID- 8625546 TI - Intravenous immune globulin: an alternative therapy in steroid-dependent allergic diseases. AB - A fundamental feature of asthma is abnormal airway function, now recognized to result from both acute and chronic inflammatory changes. Central to the development of these inflammatory changes may be the activation of T cells and the release of pro-inflammatory cytokines. In the skin, a similar cascade of events may underlie the pathogenesis of atopic dermatitis. Asthma and atopic dermatitis often share several features that may be important in their pathogenesis: T-cell infiltration of the tissues, elevated IgE levels, and a history of known triggers associated with positive immediate skin-test reactions. In both diseases, administration of intravenous immune globulin (IVIG) on a regular basis appears to reduce the need for systemic corticosteroids, reduce symptoms and for asthmatics, reduce hospitalization costs. Although the mechanism of action of IVIG in these disorders remains to be defined, it may be exhibiting significant anti-inflammatory activity. IVIG may be a potent alternative in the treatment of severe, steroid-dependent allergic disorders, reducing steroid dependency. PMID- 8625547 TI - Intravenous immune globulin in multiple sclerosis: clinical and neuroradiological results and implications for possible mechanisms of action. AB - Extensive evidence exists indicating that immunoregulatory mechanisms are involved in the pathogenesis of Multiple Sclerosis (MS). Several possible mechanisms by which intravenous immune globulin (IVIG) modulates the course of the disease are related to limiting the inflammatory process and repairing the damage by enhancing remyelination. Presently, the evidence for the effect of IVIG in MS is based on the results of small open trials, some of which have been encouraging. In the current study, the positive impact of IVIG treatment on arresting disease progression was evident by decreased brain Magnetic Resonance Imaging (MRI) scores of the lesion area. In an effort to extend these findings, the authors initiated a multicentre, prospective, randomized, double-blind, placebo-controlled study. The trial was designed to compare the efficacy of IVIG treatment with placebo in relapsing-remitting patients (ages 20-55 years) with definite MS, disease duration of 2-10 years and frequency of exacerbations 1 3/year during the 2 years prior to the study. Patients were examined monthly and brain MRI studies scheduled at entry, and after the first and second years of the trial. The primary endpoints included the number of acute exacerbations and neurological disability. The secondary endpoints included change in the MRI lesion burden, evaluated by the number and area of lesions. The trial ended in June 1995. PMID- 8625548 TI - Intravenous immune globulin in the treatment of intractable childhood epilepsy. AB - Many clinical and experimental data strongly support the role of immune mechanisms in the pathogenesis of childhood epilepsy. Following Pechadre's first observations with intramuscular immune globulin (IMIG), intravenous immune globulin (IVIG) has been employed in some forms of intractable childhood epilepsy (ICE), mainly in West syndrome (WS) and Lennox Gastaut syndrome (LGS), with good results. So far, 373 children suffering from ICE have been treated in 29 studies and 174 have responded favourably. Although these studies are heterogeneous and controls are lacking, most authors report similar responsiveness ranging from 30% to 50%. Several mechanisms have been suggested to account for the efficacy of IVIG in ICE including antiviral effect, substitutive therapy in patients with concomitant humoral immunodeficiency, idiotype-anti-idiotype interaction or a neuromodulant effect. To better define the real efficacy of IVIG in ICE in paediatric patients, a randomized, multicenter, double-blind clinical trial was started in 1993, including only patients suffering from WS and LGS. To date, only one double-blind trial had been carried out (with both adult and paediatric patients); it showed a clear trend in favour of IVIG treatment but lacked statistical significance, perhaps because of the small and heterogeneous sample. Controlled multicentre studies on well-defined populations are needed and patients with WS and LGS are probably the best candidates. PMID- 8625549 TI - New treatment strategies for systemic vasculitis: the role of intravenous immune globulin therapy. AB - Intravenous immune globulin (IVIG) is now under evaluation for the treatment of patients with the forms of systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA). Although IVIG may produce effects by a variety of mechanisms, including control of T-cell function, interference with cytokine action, and Fc receptor blockade, it is the regulation of autoantibody production by B cells, through idiotypic-anti-idiotypic reactions, that makes this treatment attractive for patients with systemic vasculitis. The author and others have shown the importance of ANCA idiotypic-anti-idiotypic reactions in vitro and demonstrated that these could be influenced by anti-idiotypic determinants present in IVIG. Thus F(ab')2 fragments of IVIG could block ANCA binding to antigen, in a dose-dependent fashion. The degree of inhibition was variable, ranging up to 100% for the ANCA-containing sera of certain patients. Similar inhibitory activity could be found in the sera of patients in remission after treatment, as well as in occasional patients whose disease remitted spontaneously, without drugs being used. Thus, IVIG appears to be particularly valuable in the management of vasculitis in the elderly, the very young and for pregnant women, as well as for those at any age who are vulnerable to infection. PMID- 8625550 TI - Supplemental immune globulins in sepsis: a critical appraisal. AB - For 'the total population of patients with sepsis, sepsis syndrome or SIRS', the question of whether intravenous immune globulin (IVIG) reduces mortality is neither proved nor disproved. For the sepsis subgroups 'postoperative sepsis with a sepsis score more than 19' and 'endotoxaemic, early septic shock', a significant reduction in mortality by IVIG has been documented in a single, placebo-controlled, small trial of each subgroup; subsequent studies are needed for confirmation. The incidence of some severe infections in defined 'patients at risk' and 'operations at risk' is lowered by IVIG prophylaxis. Postoperative APACHE II-score identification of high-risk cardiac surgery patients prone to sepsis and severe SIRS may represent one approach to optimize individual, early therapy. Applying this concept to immune globulin treatment in a pilot study, the administration of IgG-IVIG and IgGMA-IVIG yielded similar results. PMID- 8625551 TI - Intravenous immune globulin therapy of lupus nephritis: use of pathogenic anti DNA-reactive IgG. AB - The authors have recently shown that antibodies with anti-idiotype (Id) specificity to pathogenic Ids of lupus nephritis may occasionally occur in several intravenous immune globulin (IVIG) preparations because they are present in healthy donors and the healthy relatives of SLE patients. In the present study, the authors purified these anti-Ids and treated two SLE patients with nephritis in parallel with conventional high-dose IVIG management with a commercial preparation (IVIG 6) in three controls for two months. Because pathogenic Ids of anti-DNA molecules, such as both 8.12 and F4 Ids, show a cationic mobility in isoelectric focusing, a commercial preparation of IVIG (11) was absorbed on a Sepharose column coupled with DC-305-39 myeloma protein, namely an 8.12+ and F4+ cationic IgG. Infusion of the eluate (EL-11) induced a prompt resolution of proteinuria levels and an evident decrease of serum levels of anti DNA antibodies in both patients, whereas in the three controls, proteinuria and anti-DNA antibodies were scarcely reduced. In addition, plasma levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha were also significantly influenced by both treatments. The mean values of both cytokines increased significantly after 1 h and then progressively declined over the next 48 h. It was of interest, however, that the increased TNF-alpha in the two EL-11-treated patients was significantly lower than in the three controls. The data suggest that reduction of active lupus nephritis by enriched specific anti-Id molecules is the result of two (or perhaps more) mechanisms: suppression of pathogenic idiotypes at the cellular level and improvement in the mesangium of the secretion of anti-inflammatory cytokines, such as IL-6, whose defective function is related to the autoimmune disorder. PMID- 8625552 TI - Immunobiology of visceral leishmaniasis. PMID- 8625553 TI - Procainamide-induced agranulocytosis differs serologically and clinically from procainamide-induced lupus. AB - Agranulocytosis is a well recognized but uncommon complication of procainamide (PA) therapy, whereas a lupus-like syndrome occurs in approximately 20% of patients treated chronically with PA. In order to gain insight into the immunopathogenic relationships among these conditions, we compared the humoral immune abnormalities in these patient groups as well as in asymptomatic PA treated patients. A relatively uniform profile of IgM but not IgG autoantibody reactivity with a set of chromatin-related antigens was observed in eight elderly men who developed agranulocytosis after treatment with PA. In contrast PA-induced lupus patients had predominant reactivity with [(H2A-H2B)-DNA] in both IgM and IgG classes. Five of eight patients with agranulocytosis had elevated levels of neutrophil-reactive IgG which appeared to be due to immune complexes based on Fc gamma receptor blocking studies. However, 12 of 15 patients with PA-induced lupus, none of whom had neutropenia, had similar levels of neutrophil-reactive IgG, suggesting that this reactivity was not causally related to agranulocytosis. Agranulocytosis developed after less than 3 months treatment with PA in six of eight patients. This time course was similar to that seen in 77 PA-induced agranulocytosis patients reported in the literature plus 127 patients reported to the U.S. Food and Drug Administration in whom 90% developed agranulocytosis within 3 months of starting PA. In contrast, the mode duration of treatment with PA before lupus-like symptoms develop is 10-12 months. These findings, together with the different profiles of autoantibodies and clinical presentations, suggest that agranulocytosis arises from a different mechanism than that underlying PA induced lupus. PMID- 8625555 TI - Preferential activation of CD4+V beta 5.2/5.3+ intestinal intraepithelial lymphocytes in the inflamed lesions of Crohn's disease. AB - To analyze the nature of intestinal intraepithelial lymphocytes (iIELs) in inflammatory intestinal disease, we established T cell lines of iIELs isolated from endoscopic biopsied ileal and colonic mucosa of Crohn's disease patients. Seven T cell lines from the inflamed terminal ileum of 13 patients, but none of 16 T cell lines from normal terminal ileum, have shown a deviation of T cell receptor variable region gene usage and were enriched in the proportion of CD4+V beta 5.2/5.3+ cells. CD4+V beta 5.2/5.3+ cells in the T cell lines were not increased after stimulation with purified protein derivatives or 65-kDa heat shock protein but significantly increased after stimulation with staphylococcal enterotoxins C1 and D. Those cells showed increased cytolytic activity against target cells cross-linked by anti-V beta 5.2/5.3 and produced a large amount of interferon-gamma. These results indicated that CD4+V beta 5.2/5.3+ iIELs were preferentially activated in the inflamed lesions of Crohn's disease and may play a possible role in the triggering and progression of human inflammatory intestinal disease. PMID- 8625554 TI - The occurrence of serum autoantibodies against enolase in cancer-associated retinopathy. AB - Cancer-associated retinopathy (CAR) is an uncommon paraneoplastic disease in which degeneration of the retina occurs as a remote effect of cancer in a distant part of the body. Immunoreactivity of sera from CAR patients and controls have been analyzed. Immunostaining of human retinal proteins showed that a soluble protein of Mr approximately 46 kDa (p46) is labeled by antibodies from several CAR patients with various types of cancer (lung, breast, bladder, prostate, salivary gland, and gastrointestinal tract cancer and chronic lymphocytic leukemia). These sera did not show reactivity with the 23-kDa protein previously associated with CAR. To identify and further characterize p46, the retinal protein was purified to homogeneity by anion-exchange chromatography and preparative gel electrophoresis. Protein sequence analysis of the peptides from p46 revealed a high homology with human enolase, an important glycolytic enzyme. Although enolase has been previously identified as a product of several types of tumors, and enolase activity has been detected in the sera of some cancer patients, the existence of autoantibodies directed to enolase has not been described. This is the first report of the presence of serum antibodies to retinal enolase in the patients with cancer and the CAR syndrome. When antibodies of specific isotypes (IgG, IgM, and IgA) were measured, IgG1 isotype was dominant. The significance of these antibodies for the disease process is under investigation. PMID- 8625556 TI - Expression and function of monocyte chemoattractant protein-1 in experimental nephrotic syndrome. AB - This study investigates the expression and function of monocyte chemoattractant protein-1 (MCP-1) in rats with aminonucleoside nephrosis induced by a single intraperitoneal injection of puromycin aminonucleoside (PAN). On Day 7, PAN treated rats had a sixfold increase in renal MCP-1 messenger (m)RNA levels and a twofold increase in interleukin-1 beta mRNA levels. During the course of PAN nephrosis, most of the de novo MCP-1 protein resembled protein droplets that were prominent in glomeruli between Days 3 and 14 and weaker but visible in tubules between Days 5 and 10. In addition, occasional tubules showed a cytoplasmic staining pattern for MCP-1. Two studies evaluated the effect of MCP-1 neutralization on renal monocyte recruitment. In the first study, PAN-treated rats were treated with affinity-purified MCP-1-neutralizing rabbit IgG on Days 0, 1, 3, and 5; kidneys were harvested on Day 7. There was no difference in the mean number of interstitial macrophages [119 +/- 28 vs 88 +/- 9 ED-1+ cells/1000 tubulointerstitial (TI) cells; 106 +/- 28 vs 119 +/- 33 Ia+ cells/1000 TI cells] or intraglomerular macrophages [2.0 +/- 0.9 vs 1.7 +/- 0.5 ED-1+ cells/glomerular cross section (gcs); 1.2 +/- 0.3 vs 1.1 +/- 0.4 Ia+ cells/gcs] compared with nephrotic rats treated with nonimmune rabbit IgG. In the second study, a group of PAN-treated rats was treated with MCP-1-neutralizing IgG administered continuously by an intraperitoneal miniosmotic pump for 7 days and was compared with a control group treated in an identical fashion with PAN and nonimmune IgG. On Day 7 there was no difference in the mean number of interstitial macrophages (55 +/- 45 vs 67 +/- 16 ED-1+ and 70 +/- 63 vs 61 +/- 13 Ia+ cells/1000 TI cells) and intraglomerular macrophages (1.0 +/- 0.4 vs 1.6 +/- 0.9 ED-1+ and 0.6 +/- 0.1 vs 1.1 +/- 0.7 Ia+ cells/gcs). The results of this study suggest that although MCP-1 gene and protein expression are increased in the kidneys of rats with aminonucleoside nephrosis, MCP-1 does not appear to play an essential role in early renal monocyte recruitment in this model. PMID- 8625557 TI - V gene sequences of human anti-ssDNA antibodies secreted by lupus-derived CD5 negative B cell hybridomas. AB - V gene sequences encoding two lupus-derived human monoclonal IgMk anti-ssDNA antibodies (2F7 and 1A6) and CD5 mRNA expression by the corresponding hybridomas were investigated. Both antibodies displayed V gene sequences nearly in germline configuration compared with their putative germline counterparts. It appeared that 2F7 used hv3019b9/HUD-3/JH6 and 12La/Jk2, while 1A6 utilized HHG19/D31-HUD 3/JH2 and Humkv328h5/Jk1. Assessment of R/S mutation ratios suggested that 2F7 and 1A6 have not undergone the antigen-driven somatic mutation. The HCDR3 featuring arginine appeared to be important in determining the anti-ssDNA specificity. CD5 mRNA was negative in both hybridomas. Since 2F7 was previously shown to be monospecific and of high affinity, these results provide the molecular basis of such unique immunochemical characteristics of the IgM anti ssDNA antibody. Germline V genes and N sequences may be selected to confer such anti-ssDNA specificity during V gene rearrangement, which might involve CD5 negative B cells. PMID- 8625558 TI - Markedly elevated serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a potentially useful laboratory marker. AB - In an attempt to find a potentially useful serum marker in rheumatoid arthritis (RA) which reflects underlying pathogenic mechanisms, we measured the circulating levels of matrix-degrading metalloproteinase-9 (MMP-9), also termed gelatinase B, in sera and synovial fluid (SF) from patients with RA and also quantitated the deposition and local synthesis of MMP-9 in RA synovium. Clinical samples, subjected to gelatin substrate zymography, antigenic immunoassay, and a quantitative substrate degradation assay, revealed elevated 92- and 72-kDa proenzyme forms of MMP-9 and MMP-2 in RA sera and SF compared with healthy controls. Immunostaining on fresh RA synovial specimens revealed MMP-9 within vascular walls in fibroblast-like cells and macrophages; mRNA synthesis was detected using reverse transcriptase in situ PCR. In summary, MMP-9 levels are substantially elevated in the sera and SF from patients with RA. The RA synovium is a source of this MMP-9 production, with abundant mRNA and protein observed within several different type of rheumatoid synovial cells. PMID- 8625559 TI - Cultured Kaposi's sarcoma tumor cells fail to stimulate T cell proliferation. AB - Prior to the AIDS epidemic, Kaposi's sarcoma (KS) was a rare neoplasm. However, in the context of immunosuppression, cutaneous KS lesions more frequently develop and express various surface molecules recognized by T cells such as intercellular adhesion molecule-1 (ICAM-1; CD54) and HLA-DR. The KS tumor cells are thought to arise locally from endothelial cells via a transdifferentiation process. To determine if KS tumor cells can stimulate resting T cell proliferation, we asked whether the tumor cells express the critically important T cell costimulatory molecules B7-1 (CD-80) and B7-2 (CD-86). In contrast to cytokine-activated endothelial cells, which were induced to express B7-1, but not B7-2 and could function in bacteria-derived superantigen-driven T cell proliferation, four different KS tumor cell lines failed to express either B7-1 or B7-2 and were unable to stimulate allogeneic T cell proliferation upon addition of bacteria derived superantigen. These results suggest that KS tumor cells behave differently in their response to cytokines compared with endothelial cells and may be able to evade the local immune response by not expressing costimulatory molecules necessary for T cell proliferation. PMID- 8625560 TI - Induction of thyroid autoantibodies in naive mice by idiotypic manipulation. AB - Previously, we have shown that it is possible to induce, in naive mice, systemic autoimmune diseases (e.g., antiphospholipid syndrome and systemic lupus erythematosus) by idiotypic manipulation. In the present study we expanded our experience to examine whether idiotypic manipulation could be utilized to induce organ-specific autoantibodies and a disease mediated by cellular mechanisms, namely, experimental autoimmune thyroiditis. Fifteen BALB/c mice were immunized with a monoclonal mouse anti-human thyroglobulin (hTg) antibody; controls were immunized with an irrelevant mouse IgG. The mice immunized with anti-hTg antibody developed, 6 weeks after immunization, autoantibodies to human thyroglobulin, but not to dsDNA, cardiolipin, or myeloperoxidase. The presence of specific autoantibodies was associated with low production of thyroid hormones, and during a follow-up of 20 weeks the mice did not develop characteristic histological signs of thyroiditis. We conclude that idiotypic manipulation can induce anti thyroglobulin autoantibodies. PMID- 8625561 TI - Murine bispecific antibody 1A10 directed to human transferrin receptor and a 42 kDa tumor-associated glycoprotein. AB - Previously, we observed that bispecific antibodies ("antigen forks") that bound to certain pairs of different tumor surface antigens could inhibit cell growth. The chemically linked heteroconjugate of MAb 454A12 (murine IgG1 recognizing human transferrin receptor) and 317G5 (murine IgG1 recognizing a 42-kDa tumor associated glycoprotein) was particularly inhibitory toward human colorectal cancer cell lines, and the iron-chelating agent deferoxamine was found to augment inhibition of tumor cell growth by this antigen fork. Further experiments revealed that an antigen fork constructed by linking Fab' fragments instead of whole antibodies retained activity, which led us to construct a fork-secreting hybrid hybridoma. Hybridoma 454A12 was fused with hybridoma 34F2 (murine IgG1 with the same specificity as 317G5). Hybrid hybridomas whose supernatants blocked binding of both 454A12 and 34F2 probes were further tested for the ability to block growth of SW948 human colorectal cancer cells in an MTT growth assay, and were chosen for subcloning. Ascites produced by clone 1A10 was purified by affinity and cation exchange chromatography. Purified 1A10 bispecific antibody showed growth inhibitory activity comparable to that of a chemically linked heteroconjugate of its parental antibodies 34F2 and 454A12. Adding deferoxamine greatly enhanced the inhibitory activity of 1A10 and effectively prevented regrowth of tumor cells in vitro. By heterologously crosslinking two antigens that are coexpressed on many tumor cells, this bispecific antibody is able to inhibit tumor growth with enhanced selectivity. PMID- 8625562 TI - CD8 T-cells are not essential for the induction of "low-dose" oral tolerance. AB - To examine whether CD8 T-cells are essential for the induction of "low-dose" oral tolerance, we tested animals deficient in CD8 T-cells, i.e., Lewis rats in which CD8 cells were eliminated by injections of specific antibodies and beta 2m(-/-) mice in which the CD8 cells are scarce and poorly functional. Oral tolerance was induced in the rats by repeated feedings with the uveitogenic retinal S-antigen, while in the mice the fed antigen was ovalbumin. Feeding reduced in both species the specific cellular immune response, measured by the lymphocyte proliferation assay. In the rats, this treatment also inhibited the development of the inflammatory eye disease, experimental autoimmune uveoretinitis. In addition, the levels of specific antibodies in the fed animals were moderately lower than those in their controls. Both the CD8-depleted rats and the beta 2m(-/-) mice resembled their normal controls in demonstrating reduced immune responses following feeding with the corresponding antigen. This observation thus indicates that CD8 T-cells are not essential for the induction of low-dose oral tolerance. PMID- 8625563 TI - A synthetic peptide corresponding to the second extracellular loop of the human M2 acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 months in rabbits. AB - We have previously shown the presence of functional autoantibodies against the second extracellular loop of the human M2 acetylcholine receptor as an autoimmune epitope in 39% of patients with idiopathic dilated cardiomyopathy (DCM). To see if this autoimmune DCM can be experimentally reproduced, a synthetic peptide corresponding to the above autoimmune epitope was used as an autoantigen to immunize rabbits monthly for 6 months. Affinity-purified antibodies were able to display the negative chronotropic effects on neonatal rat cardiomyocytes in culture. The density of the M2 acetylcholine receptors was increased in the immunized rabbits compared to controls but the receptors showed only low-affinity binding sites for the agonist carbachol. Ultrastructural changes such as moderate sarcolemma alterations, mitochondrial swelling, disruption of cristae, and formation of myelin-like structures in the myocardial cells of rabbits immunized for 6 months suggest an immune-induced cardiotoxicity. With only a limited infiltration of inflammatory cells, these results point toward a pathophysiological role of anti-M2 acetylcholine receptor autoantibodies. PMID- 8625564 TI - The effects of pregnancy on autoimmune diseases. AB - Despite a now reasonable large body of developed information, critical gaps are apparent. Pregnancy is the only naturally occurring event in which an individual is exposed to nonself HLA. How the HLA mismatched fetus escapes rejection remains a biologic enigma. More definitive evidence is needed to determine if TH2 bias occurs in normal human pregnancy and how this impacts on established autoimmune diseases. With regard to specific autoantibody-associated placental and fetal disease, why aren't all pregnancies affected when the putative maternal antibodies are present? Future studies of maternal-fetal immunology, inclusive of information on cytokine regulation, should yield insights not only into the maintenance of normal pregnancy but also into the disproportionate increase of autoimmune diseases in females and the variable course of these diseases during pregnancy. PMID- 8625565 TI - Shoulder instability in athletes. The Asian perspective. AB - Recently, there has been an upsurge of interest among the orthopaedic sports medicine community in Asia on this subject of athletic shoulder instability. This is particularly prominent in countries such as Japan, Korea, Taiwan, and Hong Kong, where sports requiring throwing and overhead use of the upper limb are popular. In this review, the classification of the condition is analyzed with a specific account of the rotator interval injury. A systematic plan of clinical assessment is presented highlighting the specific regional considerations in clinical practice and cost-effectiveness of the health care systems in Asia. The management of athletic shoulder instability depends on patient attitude and cultural background. In Asia, the popular choice of Bankart repair with capsular shift procedure is presented with special reference to the unique experience of multidirectional and habitual posterior instability. Arthroscopic surgery is becoming popular in Asia, and the indications and limitations of the technique in the management of shoulder instability are discussed. PMID- 8625566 TI - Scoliosis in China. A general review. AB - In modern China the investigation and treatment of scoliosis began in the late 1970s. The incidence of scoliosis in the Chinese population of the Sichuan province was 0.064%, and the incidence of >10 degrees for Beijing students was 1.04%. The surgical treatment of scoliosis for 2122 patients reported in the Chinese literature was reviewed by the authors. In this study, the female to male ratio was 1.3:1, the incidence of idiopathic scoliosis was 67.7%, and the incidence of congenital scoliosis was 18.1%. The overall result showed that the correction rate after surgery in the coronal plane was approximately 50% using the Harrington, Luque, Dwyer, or Cotrel-Dubousset instrumentation techniques. However, it would be increased by >60% if a partial vertebrectomy was carefully done and the soft tissue in the concave side was released completely. The postoperative complications were approximately 20% with 0.24% of major spinal cord injury. Biologic and anatomic research of the spine has been done so that the quality of surgical implants can be improved in the future. PMID- 8625567 TI - Use of the phrenic nerve for brachial plexus reconstruction. AB - To examine the clinical effectiveness and safety of phrenic nerve neurotization for brachial plexus reconstruction, the authors retrospectively analyzed the surgically treated cases within the period between August 1970 and March 1990. There was a total of 180 patients who sustained brachial plexus injuries and had phrenic nerve transfer. The phrenic nerve was identified and traced distally to give the longest possible length and sectioned. The proximal stump was coapted to the distal segment of the musculocutaneous nerve, either directly or through a nerve graft. Sixty-five patients who were seen in followup for >2 years were studied. The time taken for the return of a muscle power rating of 3 (M3) in the biceps muscle ranged from 3 to 30 months; the average time was 9.5 months. Of the patients, 84.6% regained biceps power to M3 and greater strength. Only 1 patient had a transient respiratory problem after surgery. Pulmonary function tests showed decreased pulmonary capacities within 1 year of operation, improving toward 2 years. Thus, it is concluded that phrenic nerve neurotization can be accepted as a sound option for the restoration of biceps function in brachial plexus injury. PMID- 8625568 TI - Congenital anomalies of the hand. The Asian perspective. AB - To have a better understanding of teratogenic mechanisms of congenital absence of digits, ulnar and radial deficiencies, cleft hand, and symbrachydactyly were analyzed in clinical cases. The same anomalies were induced in rat fetuses by busulfan, and their characteristics were investigated. The formation process of longitudinal deficiency also was observed histologically. There seemed to be 4 teratogenic mechanisms of congenital absence of digits. Ulnar and radial deficiencies have the same clinical features, and the cause of these deficiencies is related closely to a deficit of mesenchymal cells in the limb bud caused by the impairment before the formation of the limb bud. Cleft hand, central polydactyly, and osseous syndactyly were induced by the same treatment at the same developmental stage in rats. Cleft hand formation process from osseous syndactylies and central polydactylies was supposed. The teratogenic mechanism of cleft hand seemed to be failure of induction of digital rays in the hand plate. The sequence of anomalies from brachysyndactyly, or the atypical cleft hand, to the transverse deficiency can be regarded as equivalent to the category of bony dysplasia of the hand. Congenital constriction ring syndrome does appear after the formation of the digital rays. PMID- 8625569 TI - Pott's Paraplegia--67 cases. AB - Sixty-seven patients were treated for Pott's paraplegia: 58 were adults and 9 were children. Sixty-four patients had active disease, and 3 had healed disease. All patients had triple chemotherapy with or without decompression surgery. Thirteen patients, including 9 children, were treated conservatively, whereas 54 patients who met the selection criteria for surgery were treated surgically. Fifty-two patients had anterior radical decompression surgery, and for 14 of them, anterior surgery was preceded by posterior instrumental stabilization surgery. Two patients with healed disease had posterior decompressive corpectomy. There was functional recovery in 60 (89.6%) patients, including 13 who had active disease that was treated conservatively. In 47 of the 54 surgically treated patients there was neurologic recovery, and 2 of these recovered incompletely with some residual spasticity. In the remaining 7 patients, there was no recovery. It took 2 to 6 months for recovery for the patients with conservative treatment, whereas it took <2 months for the patients with anterior decompression. The patients who had the combined 2-stage procedure could be mobilized earlier after neurologic recovery than could the patients having the anterior radical surgery and the conservatively treated patients. It was proven that paraplegia of active disease can be treated successfully by conservative or surgical means and that paraplegia caused by healing of fibrosis in the severely deformed spine was difficult to treat successfully, even with radical surgery. PMID- 8625570 TI - Cervical myelopathy in the Japanese. AB - Surgeries (1155 cases) for cervical myelopathy in a northeastern prefecture (population, 2.26 million) and surrounding areas were reviewed. The annual operation rate per 100,000 residents in the prefecture was 5.7. Most of the patients were in their sixth or seventh decade of life (27% each), but the annual operation rate per 100,000 people of each decade of age was the highest in the eighth decade (16.5 per 100,000 people). At the largest spine center, 41% of 306 patients had a preoperative disease period of more than 1 year, and 65% had severe disabilities. Anterior and posterior compression were about equally chosen. The former, mainly indicated for younger adults and single- or 2-level spinal cord compression, led to better functional improvement. Laminoplasty (93%) was predominant over laminectomy. Forty percent of the patients had developmental stenosis; 48%, dynamic stenosis; 27%, disc herniation; 11%, segmental ossification of the posterior longitudinal ligament; 9%, continuous ossification of the posterior longitudinal ligament; 8%, posterior spur; and 4%, calcification of the ligamentum flavum. Fifty-two percent had more than 1 of these spinal diseases. PMID- 8625571 TI - Femoral head reconstruction and revascularization. Treatment for ischemic necrosis. AB - There is no solution to ischemic necrosis of the femoral head, a condition that leads to collapse, rapid degenerative changes, and subluxation of the hip joint. Core decompression, osteotomy, and various means of femoral head reconstruction have been tried, but the results have been mixed. When ischemic necrosis has led to cartilage indentation and marked collapse, any means of reconstructing the femoral head could be too late. However, if the collapse is not severe, the logical treatment may be the removal of the dead bone and restoration of the bony contents with chip grafts and a vascularized bone strut. A new method of treatment using a vascular pedicled iliac crest strut graft has been used for 13 years. Followup assessments 4 to 12 years later have shown good results with the early involvements, whereas the late collapsed cases showed good control of pain but unsatisfactory maintenance of femoral head integrity. Thus, the technique is recommended for restoring and maintaining femoral head integrity for Stage 3 and early Stage 4 of the disease. PMID- 8625572 TI - Multicenter trial of modified Gamma nail in East Asia. AB - The mismatch of the standard Gamma nail in the Chinese femora led to the modification of its femoral shaft component based on the anthropometric study of 28 pairs of Chinese femora. A multicenter clinical trial was done in 6 countries and regions in East Asia. Three hundred forty-nine fractures were treated and studied. More than 60% of the fractures were unstable by Evans classification. The average age of the patients was 73.6 years. The male to female ratio was 2:3. The mean followup period was 11.7 months. The result of the trial showed that the intraoperative complication rate was 7.7%, and complications primarily were related to faulty operative technique. There were 7.2% postoperative complications related to the fractures that again decreased toward the later stage of the trial. Functionally, there was progressive improvement of the hip score within the followup period. The results show that the modified Gamma nail gave better results in the treatment of geriatric trochanteric fractures by minimizing reaming, thereby reducing complications related to the implants. PMID- 8625573 TI - Clubfeet in congenital annular constricting bands. AB - Thirty-five children who had congenital annular constricting bands (Streeter's dysplasia) with associated clubfeet seen by the author from September 1989 to March 1992 were reviewed with respect to quality of the clubfeet,the relationship of the bands with the clubfeet, possible prenatal factors, and treatment results. It was noted that all the clubfeet were rigid even if there were no constricting bands in the leg with the clubfoot. The clubfeet responded poorly to casting (6% success) and 77% required surgical correction. Z-plasties of deep bands were done before clubfoot correction. There was a high incidence of abnormal pregnancies with attempts at abortion in 66% of the cases, suggesting intrauterine insult early in the pregnancy as a factor causing both the clubfeet and the Streeter's dysplasia. PMID- 8625574 TI - Infection of vascularized fibular grafts. AB - Between June 1978 and July 1991, 64 limb reconstructions were done using vascularized fibular grafts. Of these, 10 became infected. Five grafts were proven viable, whereas the other 5 were proven nonviable based on the survival of the skin in the composite osteocutaneous graft and from bone scans, angiograms, and biopsies. A distinct difference in the radiologic manifestation and clinical course of the infection was noted between the viable grafts and nonviable grafts. The viable grafts showed radiologic changes of osteomyelitis that were localized, and the graft incorporated, healed with antibiotics, and exhibited graft hypertrophy. In the nonviable grafts, the radiologic changes were extensive, evidence that resorption of the grafts had resulted. This suggests that, because of the poor prognosis associated with infection of the nonviable vascularized fibular grafts, the infected grafts should be removed early to minimize the morbidity and to shorten the protracted course associated with infection. With the infected viable grafts, efforts at salvaging the graft with multiple debridements and systemic antibiotics were rewarding. PMID- 8625575 TI - Bone banking in Thailand. A 10-year experience (1984-1994). AB - The tissue bank of the Bangkok Biomaterial Center was initiated in Thailand in 1984, and the first 10 years of progress are summarized. Three hundred ninety seven cadavers and sixteen living donors provided a total of 13,676 bone and soft tissue allografts. They were used by 134 surgeons in 44 hospitals. A total of 1178 patients were surgically treated (581 males and 486 females). There were 240 cases of deep-frozen bone allografts, 600 cases of freeze-dried bone allografts, and 338 cases of soft tissue allografts. The results for 1035 cases (95.3%) were normal, whereas the results for 51 cases (4.33%) showed complications. PMID- 8625576 TI - Total knee arthroplasty in tuberculous arthritis. AB - Sixteen cases of tuberculosis of the knee treated by total knee arthroplasty with followup of 3.4 to 11 years were reviewed. Eight cases were treated with antituberculous chemotherapy for 2 to 20 months before and 1 year after the arthroplasty. Another 8 cases were not diagnosed primarily and therefore received only postoperative antituberculous chemotherapy. Five cases had a recurrence of tuberculosis. Four of these 5 cases did not receive preoperative antituberculous treatment. The infection in 1 case was controlled satisfactorily with chemotherapy alone, and for the other 3 cases, chemotherapy was supported by surgical debridements. The last case of recurrent infection occurred in a patient who had received long- standing corticosteroid therapy. He required an excisional arthroplasty to control the infection. The mean functional knee score was 30.5 points before surgery and 82.6 points at the time of the last followup. These results suggest that arthroplasty may be formed for knees badly damaged with tuberculous infection, and good results may be expected for those patients who have received effective antituberculous chemotherapy both before and after surgery. There is a substantial risk of reactivation of tuberculous infection for those patients not treated before surgery or for patients dependent on corticosteroids. PMID- 8625577 TI - Repair of chronic distal biceps brachii tendon rupture using free autogenous semitendinosus tendon. AB - Distal biceps brachii tendon ruptures occur much less frequently than do their proximal counterparts. Distal tendon ruptures usually are associated with considerable function deficits and may require surgical treatment. Repair of chronic distal biceps brachii ruptures are extremely unusual. A free autogenous semitendinosus tendon graft was used to reconstruct the distal biceps tendon by reattaching the graft to the radial tuberosity via a 2-incision technique in a patient with symptoms and a chronic injury. PMID- 8625578 TI - Unusual cause of ulnar nerve palsy. AB - An ulnar nerve palsy occurring as the result of an elbow dislocation is well recognized. An unusual case of an elbow dislocation with concomitant radial head fracture and ulnar nerve palsy caused by the radial head lying in the medial epicondylar groove is presented. PMID- 8625579 TI - Posterolateral fusion for radicular pain in isthmic spondylolisthesis. AB - Reported is the outcome for 25 patients in whom spondylolisthesis with radicular pain was treated by posterolateral fusion alone (Group A). These outcomes are compared with those obtained in 23 other patients with the same symptomatology and spondylolisthesis treated by root release and posterolateral fusion (Group B). Most patients had Grade I or II isthmic spondylolisthesis. Results were assessed functionally and radiographically with an average followup of 32 months. Posterolateral fusion in situ provided excellent or good results in 88% of patients according to the modified classification of Stauffer and Coventry. In Group A, radicular pain at exertion disappeared in 92% of patients, and radicular pain at rest disappeared in 88%. In Group B, radicular pain at exertion disappeared in 65% of patients, and radicular pain at rest disappeared in 70%. There was no significant statistical difference between the 2 groups. Resection of the loose lamina and root decompression do not seem to be mandatory. The overall fusion rate was 81%. Instrumentation in case of instability and the use of allografts are advised. PMID- 8625580 TI - Orthopedic problems in Asia/Pacific region. PMID- 8625581 TI - Internal fixation for the transforaminal sacral fracture. AB - The mechanical stability of alternate forms of internal fixation of the transforaminal sacral fracture were compared. A transforaminal sacral fracture was made in each of 6 fresh-frozen cadaveric pelvic specimens. Implants compared for fixation included: a single and 2 fully threaded iliosacral screws inserted through the posterior ilium and anchored into the first sacral vertebral body both with and without the addition of a posterior tension band plate; and 2 transiliac bars inserted through the posterior tubercles. The femora of each specimen were potted and fixed to the table of a materials tester. The pelvis was restrained only from flexing and extending, and a compressive load was applied through the lumbar spine, representing a standing loading condition. Flexion of the sacrum and displacement at the fracture site were measured during loading. Although creation of the injury increased motion considerably, there was no measurable increase in stability provided by any of the implants or combination of implants in this model when an anatomic reduction was obtained. PMID- 8625582 TI - A clinical study of the natural remodeling of burst fractures of the lumbar spine. AB - Surgical decompression and fixation are considered by many to be the preferred treatment for burst fractures of the lumbar spine, regardless of neurologic deficit. For 6 patients with burst fracture of the lumbar spine but without neurologic deficit, computed tomography scans revealed >50% encroachment of the spinal canal. All 6 patients were treated conservatively, and during the followup period (range, 6-49 months), the narrowing of the spinal canal decreased progressively because of resorption of the fragments and natural remodeling. The presence of a neurologic deficit should be the primary indication for surgery in patients with a burst fracture of the lumbar spine. PMID- 8625583 TI - Radiographic course after acetabuloplasty and femoral osteotomy in hip dysplasia. AB - The radiographic course of 101 hips with residual dysplasia treated with roof plasty combined with intertrochanteric varus derotation osteotomy using the osteotomy wedge as a roof graft after Mittelmeier were reviewed. The average followup period was 8.8 years. The acetabular angle was improved by an average of 16 degrees (postoperative mean, 19 degrees; average at followup, 18 degrees). The center edge angle also was improved by 16 degrees and was stable at 25 degrees average at followup. The neck shaft angle, abnormal in 70% of hips preoperatively, was reduced by the varus osteotomies to a mean of 111 degrees and showed a spontaneous postoperative increase to normal values of an average of 129 degrees. There was no correlation of the postoperative of the neck shaft angle to patient age, preoperative valgus extension, correction angle, or length of followup. In nearly all cases, an almost anatomic joint shape was achieved. With a complication rate of only 1%, especially regarding the rate of necroses of the femoral head, the presented surgical technique can be recommended as highly effective, reliable, and safe for the treatment of congenital hip dislocation. PMID- 8625584 TI - Anthropometric measurements and body proportions among Chinese children. AB - There are significant racial differences in body proportions. Such data are not readily available for Chinese children. This article reports a cross section study of body proportions of 2193 Hong Kong Chinese children, ages 4 to 16 years, with equal gender distribution. Standing height, sitting height, and arm span were measured with standard equipment and methodology. Lower segment height was calculated as the difference between standing height and sitting height. Statistical analysis of the results showed a high linear correlation of the standing height with arm span, sitting height, and lower segment height, with a correlation coefficient ranging from 0.965 to 0.983 for both genders. When expressed as the ratio of standing height to arm span, the value was relatively constant and changed only linearly from 1.03 to 1 in girls and 1.03 to 0.98 in boys ages 4 to 16 years. The ratio of sitting height to lower segment height varied from a mean of 1.4 to 1.14 in boys and 1.36 to 1.18 in girls ages 4 to 16 years. The Chinese children were found to have a proportional limb segmental length relative to the trunk that differed significantly from the proportionally longer limbs in whites and blacks. PMID- 8625586 TI - Dega acetabuloplasty combined with intertrochanteric osteotomies. AB - Between 1973 and 1984, 70 modified Dega acetabuloplasties done simultaneously with intertrochanteric osteotomies in 51 patients with developmental dysplasia of the hip were reviewed for long-term results and analyzed for causes of failure. Mean patient age at operation was 2.9 years (range, 8 months-8 years), and mean followup time was 15.2 years (range, 10-19 years). Topical investigations were based on clinical criteria of the Severin classification and radiologic criteria of the Commission for the Study of Hip Dysplasia of the German Society of Orthopaedics and Traumatology. The review of clinical documents and investigations very good and good results in 80% of the hips. The Trendelenburg sign was a useful clinical indicator for problematic cases. Measurements on radiographs were classified into deviation grades from the normal range. Values of the acetabulum and the acetabulum to head relation were normal or slightly abnormal in >80% of hips. Measurements of the femoral head and neck were in the low normal range. The lowest percentage of normal values was in 24 cases with coxa valga after acetabuloplasty combined with derotational varus osteotomy. Poor clinical and radiologic outcome usually was the result of avascular necrosis of the femoral head or recurring valgus deformity after derotational varus osteotomy. The rate of avascular necrosis as a consequence of the operation was 5.7%. The risk of avascular necrosis caused by derotational varus osteotomy is lower than the risk of later development of deformities in the proximal femur. PMID- 8625585 TI - Core decompression for osteonecrosis of the femoral head. AB - A retrospective++ review of core decompression of the femoral head for treatment of osteonecrosis was done. Cause of osteonecrosis, radiographic stage and progression, complications, and clinical results were evaluated. The study was based on 54 hips in 45 patients (98.2% followup rate). All patients reported pain preoperatively. Thirty-five hips (30 patients) were considered to have failed. Of these, 26 hips (23 patients) underwent total hip arthroplasty. The remaining 9 hips (7 patients) had little or no relief of pain and no improvement in function, but had not undergone total hip arthroplasty at last followup. The average time to failure was 11.1 months (2-34 months). Nineteen hips (16 patients) were considered successful. Fifteen hips (12 patients) were graded good to excellent and 4 hips (4 patients) were graded fair in terms of clinical results with an average followup of 47.5 months (12.4-95.7 months). The mean preoperative Hospital for Special Surgery hip score improved from 24.6 points (range, 18-38 points) to 34.2 points (range, 20-40 points). There were 2 intertrochanteric femur fractures in this group (5 and 6 weeks postoperatively). The overall success rate of core decompression in this series was 35.2% (19 of 54 hips, 45 patients). The results of core decompression in this study were poor in general and had an unpredictable effect on disease progression. PMID- 8625587 TI - Functional loss after total knee arthroplasty for poliomyelitis. AB - Recurvatum deformity secondary to poliomyelitis was corrected during total knee arthroplasty. Modular distal augmentation of the femoral component was successful in achieving and maintaining correction. This case also shows the potential functional disadvantages of correcting recurvatum in patients with quadriceps weakness. PMID- 8625588 TI - Forefoot pressures during walking in feet afficted with hallux valgus. AB - Forefoot pressures during walking in feet with hallux valgus were recorded using pressure-sensitive film, and the relationships between deformities and foot pressures were analyzed. There were 32 female subjects comprising 50 feet with hallux valgus, of which 20 feet underwent surgery. As in normal feet, the pressure patterns of feet with hallux valgus were varied: peak pressures were under the first metatarsal head, under the second and/or third metatarsal heads, and first, second, and/or third metatarsal heads. Peak pressures of hallux valgus feet were larger in each type than those of the normal foot. In feet with hallux valgus showing peak pressures under the first metatarsal heads, the hallux valgus angle and the intermetatarsal angle were larger than those in feet showing peak pressures on the second and/or third metatarsal heads. After surgery, peak pressures were under the second and/or third metatarsal heads and they decreased. PMID- 8625589 TI - Split posterior tibial tendon transfer for varus deformity of hindfoot. AB - Sixteen patients (23 feet) who underwent split posterior tibial tendon transfers were evaluated. The patients were seen on a followup basis for a minimum of 1 year postoperatively. The causes were spastic cerebral palsy in 13 feet, spastic athetoid cerebral palsy in 3 feet, hydrocephalus in 3 feet, and other diseases in 4 feet. The indication for surgery was varus deformity during the stance phase of gait and increased varus deformity during the swing phase of gait because of spasticity of the posterior tibial muscle. Heel cord lengthening was done on 17 feet. Preoperative and postoperative gaits were evaluated while the patients were walking. Axial radiographs of the calcaneus and the tibia were taken of all patients while they were weightbearing. There were 15 excellent, 6 good, and 2 poor results. The poor ratings were assigned to patients who had recurrence of varus deformity; there were no cases of overcorrection. Split posterior tibial tendon transfer was effective for treating spastic varus deformity of the hind part of the foot. This treatment also could be considered for a patient with spastic-athetoid cerebral palsy, if the deformity was determined to be caused by overactivity of the tibialis posterior muscle. PMID- 8625590 TI - Osteoneogenesis and its influencing factors during treatment with the Ilizarov method. AB - The radiographs of 57 patients who had undergone 58 callus distractions and 13 epiphyseal distractions were evaluated. During the course of treatment, the density of the distraction areas on radiographs was measured in total and along the medial, lateral, ventral, and dorsal margins using a digital radiograph processing system. The densities of the whole distraction length were correlated to the following parameters: age of patient, start of distraction after corticotomy, mean distraction speed, mean amount of weight load during the period of distraction and consolidation, location of corticotomy (distal femoral metaphysis versus the proximal tibial one), and diclofenac medication. Except for the location of corticotomy and the diclofenac medication, the density was influenced by all these parameters. Age of the patient and weight load were the most important parameters. Patients with leg shortening caused by poliomyelitis and a patient with a shortened leg after amniotic strangulation showed a slower rise of the density trend curve on radiographs than the other patients. When comparing the different regional density curves, a significant gradual density decrease could be observed from the medial to the lateral side in the femur, from lateral to medial in the tibia, and from the dorsal to the ventral side in both bones. The respective differences between lateral and medial density, and between dorsal and ventral density, were significantly higher in cases of callus distraction than in cases in which epiphyseal distraction had been used. The amount of bone regeneration varied regionally primarily because of the inhomogeneous soft tissue covering of the bone and the impairment of its local blood supply by the surgical exposure for the corticotomy. The clinical relevancy of the various parameters for osteoneogenesis is discussed in this article. PMID- 8625591 TI - Axial computed tomography of pilon fractures. AB - Computed tomography (CT) is used in the preoperative planning of many fractures. It is particularly helpful in anatomic regions with complex anatomy and in fractures with complicated patterns. The authors evaluated the use of CT scans in the preoperative planning of pilon fractures. Twenty-two patients were studied with plain radiographs and with CT scans. The fracture pattern, number of fragments, comminution, impaction, and location of the major fracture line were recorded. The CT scan revealed an increased number of fragments in 12 patients, increased impaction in 6 patients, and increased comminution in 11 patients. The operative plan was changed in 14 (64%) patients, and additional information was gained in 18 (82%) patients. PMID- 8625592 TI - Heterogeneous survival rates for isolated skeletal metastases from melanoma. AB - In this study, the authors examine the survivorship of individuals who constitute the small subset of patients with Stage IV melanoma who present with their first and only detectable metastasis to the skeleton. One thousand two hundred six patients were identified with primary melanoma at the authors' institution since 1962. There were 14 patients with isolated (solitary) skeletal metastases. Survival was calculated by Kaplan-Meier technique. The survival rate for any individual with an isolated metastasis to the axial skeleton was 0. Three of 8 patients with melanoma metastases to the appendicular skeleton are alive at 18, 25, and 52 months after Stage IV detection. A statistically significant prolonged latency period (initial diagnosis to Stage IV) in the appendicular group compared with the axial group contributed to their significant survival rate advantage. The melanoma literature strongly supports the complete resection of soft tissue metastatic foci. These data support this concept and extend it to include isolated skeletal metastases. The mechanisms by which axial and appendicular skeletal metastases occur are significantly different. This difference manifests itself as a survival advantage for the appendicular group and warrants an aggressive surgical approach for these individuals. PMID- 8625593 TI - Osteosarcoma and Ewing's sarcoma in a retinoblastoma patient. AB - Retinoblastoma, though uncommon, frequently is associated with other second malignant neoplasms, especially bone sarcomas. These second neoplasms can occur in sporadic cases of retinoblastoma, though they more commonly are associated with genetic forms of the disease. This is the first known report of 2 histologically distinct bone sarcomas in a patient with retinoblastoma. PMID- 8625594 TI - Langerhans' cell histiocytosis of the spine in children. AB - Between May 1972 and December 1991, 58 children were seen in the authors' institution for treatment of Langerhans' cell histiocytosis. Of these, 15 children (7 males, 8 females) had biopsy-proven Langerhans' cell histiocytosis with vertebral involvement. The average age at presentation was 6.4 years (range, 0.2-13.3 years). At presentation, 8 patients had involvement of a single vertebra and 4 had involvement of multiple vertebrae. Nine patients had extraspinal skeletal involvement. Two patients had visceral involvement. Currently, 13 patients have had >2 years followup (average, 8.9 years; range, 2-21 years). None of these patients had clinical evidence of disease at the latest encounter. This group of patients with Langerhans' cell histiocytosis with vertebral involvement has fared well. Most patients have had involvement of multiple vertebrae and many have had extraspinal bony involvement as well. There was a great deal of variability in the extent of collapse and reconstitution of vertebral height, and the amount of reconstitution did not correlate with patient age. PMID- 8625595 TI - The blood supply to the greater trochanter. AB - The blood supply to the greater trochanter was investigated in adult rabbits using a hydrogen washout technique. The control blood flow rate of the greater trochanter averaged 13.8 ml/minute per 100 g of tissue. Stripping of the origin of the vastus lateralis muscle did not cause significant decrease of blood flow rate. Blood flow rate was significantly decreased to 10.4 ml/minute per 100 g of tissue after an anterior trochanteric slide osteotomy. Surgical dissection of the proximal soft tissues including the gluteus medius and minimus tendons from the greater trochanter caused significant decrease of blood flow rate to 4.72 ml/minute per 100 g of tissue. The addition of surgical dissection of the proximal soft tissues to anterior trochanteric slide caused a complete arrest of blood flow in the greater trochanter. These results indicate that the greater trochanter receives >2/3 of its blood supply for the extraosseous vascular system in a rabbit model. PMID- 8625596 TI - The Ilizarov external fixator for treatment of deformities in Paget's disease. AB - Paget's disease of bone may cause progressive bowing of affected femurs and tibiae. Fractures or osteotomies in these patients technically are difficult to treat and may lead to delayed union or nonunion. The authors report their early results with the use of the Ilizarov external fixator to affix 3 corrective closing wedge osteotomies, 1 fracture, and 1 delayed union. Deformities were corrected fully during the surgery, with an option to correct remaining deformation progressively during the rest of treatment. Compression of the healing bone was applied to enhance osseous healing, when necessary. Three dimensional control is possible, and the stability of the fixation is excellent in this structurally weak bone. Bony union was achieved without complications in all patients within 4 months. PMID- 8625597 TI - A case of recurrent Caffey's disease treated with naproxen. AB - Caffey's disease (infantile cortical hyperostosis) is considered to be a benign self-limiting disease of uncertain etiology that typically appears in early infancy. The following case of Caffey's disease in a young girl is significant from 2 standpoints. First, the course of the disease is atypical because of its severe recurrent nature. The patient experienced 6 highly symptomatic episodes of cortical hyperostosis involving multiple new sites and previously healed lesions. These recurrent episodes persisted into her fourth year of life. In general, the reported cases of late recurrence of Caffey's disease describe patients with minimal symptoms and mild bony involvement. The second area of significance involves the successful use of naproxen to control the symptoms and arrest the progression of the patient's disease. Reports of hyperostosis in patients receiving prostaglandin E to maintain a patent ductus arteriosus suggest that prostaglandins may play a role in the etiology of Caffey's disease. Given this evidence, it seems plausible that there may be a therapeutic role for prostaglandin inhibitors in the successful treatment of Caffey's disease. The patient in this case had immediate and complete resolution of her symptoms while receiving naproxen with no recurrent bone formation while being treated with antiinflammatory drugs. PMID- 8625598 TI - Thumb duplication at the metacarpophalangeal joint. Management and a new classification. AB - Duplication at the metacarpophalangeal joint level (Wassel Type IV) is the most common type thumb duplication (polydactyly), accounting for 50%. It can be subdivided into 4 types: (A) hypoplastic (12%), (B) ulnar deviated (64%), (C) divergent (15%), and (D) convergent (complex) (9%). Type D has the most complex bone and soft tissue anomalies and is most likely to have residual deformities. A surgical protocol emphasizing early operation within 1 year of age, 1 stage osteotomy, meticulous collateral ligament repair, and abductor pollicis brevis tendon reattachment is presented. Twenty-one cases with an average followup of 5.25 years were reviewed. The cosmetic and functional results were satisfactory. Of a score system of 20 points, the mean score was 17.9. All except 1 patient had a stable metacarpophalangeal joint. Fifteen patients had a good range of movement, and 17 patients are satisfied with the outcome of surgery. Scar hypertrophy occurred in 2 patients. PMID- 8625599 TI - Structural and functional changes in the canine shoulder after cessation of immobilization. AB - The functional and structural consequences of remobilization of the glenohumeral joint after 12 weeks of immobilization were studied in 10 beagle dogs. The dogs were grouped in pairs: 2 were used as controls, and in 8 others 1 forelimb was immobilized. At the end of 12 weeks, 2 dogs in the experimental group were euthanized, as were the 2 dogs used as controls. The cast was removed from the other 6 dogs, and the remobilized animals were euthanized in pairs at the end of 4, 8, and 12 weeks. It was found that after 12 weeks of immobilization, the passive range of motion was markedly impaired, intraarticular pressure was raised during movements, and the filling volume of the joint cavity was reduced. Histologically, the capsule showed synovial lining hyperplasia and vascular proliferation in the wall. Immunohistochemical labeling for collagen Types I and III failed to show an increase of fibrous collagens in the capsular wall. Both the functional and structural changes were unaltered after 4 weeks of remobilization, but after 8 weeks they began to reverse, and they returned to normal levels after 12 weeks. Both functional and structural changes after 12 weeks of immobilization of the uninjured glenohumeral joint are reversible by remobilization. The collagen composition of the capsule seems unrelated to the degree of capsular contraction that occurs during 12 weeks of immobilization and subsequent remobilization. PMID- 8625600 TI - Drug inhibition of the macrophage response to metal wear particles in vitro. AB - The wear particles of cobalt chrome alloy and titanium alloy have been implicated as a cause of aseptic loosening of prostheses. It is thought that their ability to induce either cell death or the release of mediators that induce bone resorption contributes to this loosening. This study was designed to test the hypothesis that these adverse biologic effects are due to wear particle corrosion at low pH after they have been phagocytosed by macrophages. Cobalt chrome alloy and titanium alloy particles of similar size and concentration to those found in the tissues surrounding failed prostheses were added to cultured rodent peritoneal macrophages. Treatment of macrophages with drugs that prevent a drop in pH within phagosomes significantly reduced the toxicity of phagocytosed cobalt chrome alloy particles. The same drugs also reduced the levels of prostaglandin E2 and interleukin-6 release induced by phagocytosed titanium alloy particles. When both types of particles were incubated at a low pH, similar to that encountered by phagocytosed particles, soluble products were released that induced the same effects as the particles themselves. These results show that enhanced corrosion of wear particles by phagocytic cells may contribute significantly to the adverse biologic effects of wear particles and identify drug therapies that may be investigated further. PMID- 8625601 TI - Anterior shoulder instability. Current review. AB - During the past several decades, the treatment of anterior shoulder instability has evolved substantially. Treatment initially was based on conservative management with rehabilitation protocols for shoulder girdle strengthening. Functionally disabling instability and recurrent dislocation precluded the acceptance of conservative management as the definitive treatment for all patients. Attention then was turned to open repair strategies. Despite generally positive results, a significant number of complications became evident. With time, however, techniques improved and surgical complications were lessened. As shoulder arthroscopy was more widely practiced, the surgical reconstruction of instability became significantly less invasive. A high learning curve has been associated with the new arthroscopic techniques, and higher redislocation rates than those of open procedures have limited their acceptance by many orthopaedic surgeons. This review article examines the anatomy and pathology of anterior shoulder dislocations, and critically reviews the actual laboratory and clinical data supporting use of these various treatment options. Only through careful examination of well-controlled scientific studies can clinicians devise their own treatment protocol for symptomatic anterior instability of the shoulder. PMID- 8625602 TI - Ankle pain and swelling in a 24-year-old woman. PMID- 8625603 TI - The natural history of ultra high molecular weight polyethylene. PMID- 8625604 TI - The integration of modern and traditional Chinese medicine in the treatment of fractures. A simple method of treatment for fractures of the shafts of both forearm bones. 1963. PMID- 8625605 TI - Geometric mismatch of the Gamma nail to the Chinese femur. AB - The geometric mismatch of the femoral component of the Gamma nail to the Chinese femora resulted in intraoperative complications. To provide scientific data for modification of the implant, 28 pairs of normal Chinese femora were studied with the 3-dimensional reconstruction from the computed tomography scan data. Measurements were taken from the reconstituted drawings, and the anthropometric data were applied in the modification of the implant. This study is the first report on the application of Chinese anthropometric data on the design of a trauma implant. It is hoped that a larger scale of the study will provide a more comprehensive data base for wider application to orthopaedic implant design in the future. PMID- 8625606 TI - Osteosarcoma in Hong Kong. AB - Since 1984, the authors have treated 82 Chinese patients with sarcoma of bone. Of these patients, 58 had high grade osteosarcomas in the extremities. Of the 41 patients available for examination, the age range was 8 to 59 years, with a median of 17 years. Treatment consisted of preoperative and postoperative neoadjuvant chemotherapy, limb salvage surgery, or amputation. The overall survival rate was 62.9% at 2 years and 43% at 5 years. No significant difference was found in survival between the amputation and limb salvage groups, although local control was much better in the amputation group. There seemed to be better survival for patients who had a good response to preoperative chemotherapy, although the data did not reach statistical significance. PMID- 8625607 TI - Giant cell tumor of bone. AB - Giant cell tumor is a locally aggressive tumor with a high recurrence rate if not completely excised. The condition is more common among Asians than whites. During a 10-year period 44 patients, ranging in age from 12 to 51 years, were treated. The most common sites were the proximal femur and the distal radius. The aim of treatment was to excise the tumor completely. Because the tumor usually occurred at the juxtaarticular region of the bone, difficult reconstruction was expected. The authors' method of choice for filling large defects resulting from resection was to use a large vascularized bone graft from the iliac crest to enhance bone to bone corporation. The recurrence rate was low (4.5%), with only 2 recurrences: 1 at the proximal femur and the other at the proximal tibia. The functional results were highly satisfactory, with excellent stability of the joint, although in the case of the distal radius there was some degree of subluxation of the wrist joint. By applying microsurgical technique for the reconstruction, one was able to be more aggressive with excision of this locally aggressive tumor while maintaining excellent functional results. In the case of the knee, as long as 1 tibial condyle was intact, reconstruction of the other condyle using a vascularized iliac crest block maintained the joint integrity perfectly or created a pseudojoint component that was perfectly stable. PMID- 8625608 TI - The epidemiology of osteoporosis. The oriental perspective in a world context. AB - In the past 3 decades, the incidence of osteoporotic hip fracture increased in Hong Kong Chinese by 2-fold to reach an incidence of approximately 10 per 1000 in women and men who are 70 years of age or older. It has been projected that 50% of all hip fractures in the world will occur in Asia by the next century. The bone mineral density of Asian populations is comparable to that of whites after adjusting for height and weight. Physical inactivity, a low dietary calcium intake, and falls have been found to be major risk factors for hip fractures in Asia. Other risk factors are cigarette smoking, heavy alcohol consumption, and multiparity. Population strategies to increase physical activity and calcium intake and to prevent falls in Oriental populations will be effective in the prevention of osteoporosis and hip fracture because of the high attributable risk for these factors. PMID- 8625609 TI - Body measurements, bone mass, and fractures. Does the East differ from the West? AB - There are population differences in hip fractures between the East and West. The hip fracture incidence in Asians is approximately 40% to 50% of that in whites in the United States. There also are differences in body measurements and bone mass between the population groups. This article discusses the role of body measurements in relation to bone mass and bone loss at different stages of life and interrelationship of these variables with fracture risks. PMID- 8625610 TI - The epidemiology of hip osteoarthritis and rheumatoid arthritis in the Orient. AB - The prevalence of hip osteoarthritis and rheumatoid arthritis has been found to be lower in Chinese, Japanese, and other Asian populations than in whites. The low prevalence of osteoarthritis hip in the Chinese and Japanese populations cannot be explained by the rarity of hip dysplasia, which is as prevalent in those two populations as in whites. As in other populations, rheumatoid arthritis in the Chinese is associated with the shared epitope of the third hypervariable region of the DR gene. However, the low prevalence of rheumatoid arthritis in the Chinese cannot be accounted for entirely by genetic factors. PMID- 8625611 TI - The osteoporotic spine. AB - With an aging population, osteoporotic vertebral collapse is an increasingly common condition in both the Western and Eastern hemispheres. This review looked into the situation in Hong Kong with the aim of highlighting the major differences in data from the Western. Between 1989 and 1994, 497 patients with osteoporotic vertebral collapse, aged 65 to 94 years old, were admitted under the authors' care, with a female to male ratio of 5:1. More than 1/3 of the patients had fracture involving 1 single vertebra. The mean hospital stay was 5 days, with an additional 6 to 23 days in a convalescent hospital. Complication of spinal cord compression is uncommon and occurred in only 10 patients (2%) in this series who were treated mostly with anterior decompression and iliac crest graft with varying degrees of neurologic recovery. Despite the general benign nature of the problem, the high morbidity and the long hospital stays undoubtedly are significant drains on health care resources. PMID- 8625612 TI - Athletic knee injuries. Similarities and differences between Asian and Western experience. AB - The pattern and prevalence of knee injuries among athletes are different in Asia, Europe, and North America because of factors such as the different sporting cultures of the regions and the type of favorite sports. Sampling and data bias and the variable levels of diffusion of sports in the different parts of the world also are responsible for the variation. In this review, the knee emerges as the most commonly injured part of the body; such injuries present significant problems of mechanical block or instability that may compromise athletic performance. Sports specific injuries are delineated, and specific groups, such as children and adolescents, female, and master athletes, are reviewed in light of the characteristic patterns of knee injuries. Differences in the patients' attitude, health care systems, and consultations of traditional medical practitioners produce major differences in management styles. However, it is encouraging to find a concerted effort on all continents in the search for a better understanding of knee injuries and a more satisfactory coordination between clinical and basic science research in this field. PMID- 8625613 TI - Control of growth hormone synthesis. AB - A large body of research, primarily in the rodent and human species, has elucidated many of the details regarding the control of GH synthesis and release. Cell type-specific transcriptional control has been identified as the main mechanism of the somatotroph-specific expression of GH. The recent detailed analysis in rodents and humans of a highly specific transcriptional activator protein, PIT-1, has opened several new areas of study. This is especially true for research in the farm animal species, where PIT-1 has been cloned and its binding elements on the GH gene are being investigated in a number of economically important species. Genetic and biochemical analyses of PIT-1 and other GH regulators have shown the central role of PIT-1 not only in the cell autonomous stimulation of GH gene transcription, but also in the participation of PIT-1 in the response at the GH gene to exogenous hormones such as RA and TH. PIT 1 has been implicated in the proliferative development of the pituitary itself, in the maintenance of anterior pituitary cell types once cell types are defined, and in the mechanism by which the hypothalamic signal for GH release is transduced. However, PIT-1 by itself does not activate the GH gene, so that additional unknown factors exist that need to be identified to fully understand the cell type-specific activation of the GH gene. In addition, GH gene regulatory elements acting through well-characterized systems such as TH have seemingly different effects; the specific context of the regulatory elements relative to the promoter elements appear to be crucial. These contextual details of GH gene regulation are not well understood for any species and need to be further studied to be able to make predictions for particular elements and regulatory mechanisms across species. The regulation of the pulsatile secretion of GH by GHRH and SRIH is reasonably well understood after the cloning and analysis of the two releasing factors and their receptors. Modification or manipulation of the pathways involved in the regulation of GH secretion is a potential means of enhancing the lean tissue growth of meat animals. However, further understanding of the systems controlling the in vivo release of GH is needed before such manipulations are likely to be productive. Several other research questions regarding the control of GH expression and release remain to be answered. What is the biochemical connection between exogenous signal transduction (i.e., GRH/GHRH-R, TR, ER, RAR) and PIT-1 at the GH gene? Are there additional coactivators or repressors of GH that respond to cAMP levels? Do ubiquitous regulatory factors such as GHF-3 and Zn-15, identified thus far only in the rat, exist in humans or livestock? Zn-15 is expected to be found in many mammalian species, because its recognition sequence between the PIT-1 binding sites is highly conserved across mammals (Figure 2). What is the mechanism causing GH levels to drop during aging? Does PIT-1 expression decrease during the lifespan of animals? Is it possible to increase GH gene expression within target tissues by directing the expression of PIT-1 to these tissues via transgenesis, or are other factors limiting in peripheral tissues so that the lack of PIT-1 expression is not the deciding factor? Finally, is there genetic variation in the expression of GHRH and/or SRIH or in their respective receptors? These questions are relevant to and could be investigated in several of the livestock species. PMID- 8625614 TI - Interspecies variations of corticosteroid-binding globulin parameters. AB - In mammalian plasma, cortisol binds to a specific alpha 1-glycoprotein: corticosteroid-binding globulin (CBG). In this study, we measured the protein binding of cortisol by equilibrium dialysis in seven species in which plasma cortisol concentrations varied from 0.02 to 0.05 (ewe, dog, cow) to 0.1 to 0.6 (horse, human, cynomolgus monkey) to reach 1.6 microM (squirrel monkey). No binding of cortisol to CBG was discernible in plasma from squirrel monkey. In all other species examined, we showed that the CBG maximal capacity (Bmax) was 3 (1.7 to 5.2) times more than the plasma cortisol levels, with cow, dog, ewe exhibiting the lowest and cynomolgus monkey exhibiting the highest values. We also noted the existence of a linear relationship between Bmax and the corresponding dissociation constant (Kd), Bmax being systematically 10 (8.5 to 11.8) times more than Kd. The low binding affinity of cortisol assigned to albumin did not differ between species. The free (6 to 14%), CBG-bound (67 to 87%), and albumin-bound (7 to 19%) cortisol fractions calculated from the estimated binding parameters and measured plasma cortisol concentrations were similar within species, except for squirrel monkey, in which half of the cortisol was albumin bound, and the other half remained protein free. Our most appealing finding was that in most species, as much as 68% of plasma CBG remained free of cortisol under physiologic conditions. These results are discussed with respect to the theories concerning the role of CBG in plasma transport and the local delivery of cortisol and free CBG as a proper hormone. PMID- 8625615 TI - Characterization of growth hormone-binding protein in cattle plasma: prolactin binding activity and 24-hour profile. AB - The purpose of this study was to characterize circulating growth hormone-binding proteins (GHBP) and prolactin-binding proteins (PRLBP) in cattle blood plasma. In particular, the 24-hr profile of these molecules was investigated. The preincubation of bull plasma with iodinated bovine growth hormone (bGH) or bovine prolactin (bPRL), followed by gel filtration chromatography (Superdex 200; 1.6 x 60 cm column), resulted in the formation of essentially two complexes. The majority of [125I]bPRL eluted with the first one (M(r) approximately 600 kDa), whereas [125I]bGH mainly appeared in the second one (M(r) approximately 70 kDa). The fractions corresponding to these two peaks were analyzed by western ligand blotting (WLB), under reducing conditions. WLB revealed, respectively, 190-, 56-, 52-, and 28-kDa bands for the first peak and only 52- and 28-kDa bands for the second one. The nature of the 600-kDa peak is at present undetermined, but the 70 kDa one was previously identified as high-affinity GHBP. Displacement studies demonstrated that bGH and bPRL were both able to bind to this GHBP, because the bGH- and bPRL-binding activities of this protein could be saturated by an excess of either of these two hormones. This was indirectly confirmed by the close correlation (r = 0.615; P = 0.0001; n = 155) observed between plasma bGH- and bPRL-binding activities, because this correlation could suggest that both ligands are bound to the same proteins. The temporal concentrations of plasma GHBP were measured in samples collected at 20-min intervals for 24 hr from 8 young bulls. The evaluation of GHBP was realized by WLB, followed by densitometric analysis. Some fluctuations were observed, but these were not correlated with bGH release, even with a +/- 2-hr lag period. In summary, we found that bovine high-affinity GHBP binds not only bGH, but also bPRL. A second type of protein, of higher molecular weight, also binds these two hormones, but further investigations are needed to determine its nature. Finally, GHBP concentrations in cattle blood plasma apparently show fluctuations over a 24-hr period, but no correlation was found between these fluctuations and plasma growth hormone concentrations. PMID- 8625616 TI - GABAergic inhibition of the pituitary release of adrenocorticotropin and alpha melanotropin is impaired in dogs with hepatic encephalopathy. AB - gamma-Aminobutyric acid (GABA) is the principal depressant neurotransmitter system, but its possible role in the regulation of the hypothalamic-pituitary adrenocortical (HPA) axis has not yet been investigated in the dog. Moreover, GABA is one of the factors underlying the syndrome of hepatic encephalopathy (HE), and in dogs with HE, the regulation of the HPA axis is deranged. We have therefore investigated the role of the GABA system in the regulation of the HPC system in 10 healthy dogs and 10 dogs with HE due to congenital portosystemic shunts. The effect of an intravenous injection of the GABA antagonist bicuculline on the release of adrenocorticotropin (ACTH), alpha-melanotropin (MSH), and cortisol was measured in plasma. In healthy dogs, a dose of 1.0 mg/kg caused a marked release of ACTH, MSH, and cortisol, but doses of 0.001 to 0.5 mg/kg produced an inconsistent or no response. The high release of MSH after bicuculline administration indicated that the effect of GABA was predominantly in the neurointermediate lobe of the pituitary. In order to investigate whether the effect of GABA was exerted in the pituitary or at a suprapituitary level, the effect of incubation with GABA on basal and corticotropin-releasing hormone induced ACTH release was measured in primary cultures of anterior and neurointermediate lobe cells from healthy dogs, and no response was observed. We conclude that in healthy dogs, GABA inhibits the release of ACTH and MSH from the neurointermediate lobe of the pituitary at a suprapituitary level. In dogs with HE, 1.0 mg/kg of bicuculline caused virtually no stimulation of the secretion of ACTH, MSH, or cortisol, indicating deranged GABAergic neurotransmission in HE. This may be explained by an increased GABA tone that prevents the effect of the antagonist. Such a high GABA tone associated with HE has been documented in several other species. Dogs with HE had significantly increased basal levels of ACTH, MSH, and cortisol in plasma, and their cortisol:creatinine ratios in 24-hr urine samples (63 +/- 14.10(-6)) were higher than those of healthy dogs (9 +/- 2.10(-6)). An increased basal HPA activity in dogs with HE is not in agreement with augmented GABAergic inhibition, but this contradiction may be explained by the predominance of effects of dopaminergic disinhibition that has been reported in such dogs. PMID- 8625617 TI - Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes. AB - The effects of progesterone (P4) on follicular growth and fertility in ewes were examined. In Experiment 1, 22 ewes received either one or three packets of P4 (5 g/packet) or an empty packet subcutaneously (sc) from Days 5 to 15 of the estrous cycle (estrus = Day 0). On Day 6, P4-treated ewes received 12.5 mg of prostaglandin F2 alpha. Follicles > or = 3 mm in diameter were observed via transrectal ultrasonography daily from Day 4 through estrus, corpora lutea (CL) were observed 5 to 7 d after estrus. Ewes with low (LOW; < or = 1 ng/ml; n = 5), intermediate (MED; >1 and <2 ng/ml; n = 10), or normal (NOR; > or =2 ng/ml; n = 7) P4 in jugular plasma on Days 7 through 15 differed in follicular development. The largest follicle at estrus was larger in ewes with LOW vs. MED and NOR P4 (7.8 +/- 0.3 vs. 6.9 +/- 0.2 mm; P < 0.05). Treatments differed in proportions of multiple-ovulating ewes, in which the oldest ovulatory follicle was first observed before Day 10 (LOW: 3 of 3, MED: 6 of 10, NOR: 0 of 5, respectively; P < 0.05). Estradiol was higher early in the treatment period in LOW ewes than in MED and NOR ewes (day x treatment; P < 0.05). In Experiment 2, ewes received 5 mg of P4 in corn oil (low progesterone [LP]; n = 51) or 2 ml of corn oil (CON; n = 49) sc every 12 hr on Days 6 through 14 of the estrous cycle before mating. LP ewes received 15 mg of prostaglandin F2 alpha on Day 6. Mean serum P4 on Days 7 through 15 was 0.6 +/- 0.1 ng/ml in LP and 1.9 +/- 0.1 ng/ml in CON ewes. Eleven LP and 12 CON ewes were scanned daily from Day 4 through mating, and in all ewes (n = 93), CL were counted 10 d after mating and embryos were counted at 25, 40, and 60 d of gestation. In multiple-ovulating ewes, day of cycle of appearance was earlier for the oldest (Day 6.1 +/- 0.8 vs. 10.4 +/- 0.8) but not second oldest (Day 11.7 +/- 1.0 vs. 12.2 +/- 0.9) ovulatory follicles in LP compared with CON ewes. The conception rate was lower in LP (72%) than in CON ewes (98%; P < 0.01). However, numbers of CL 10 d after mating, and in pregnant ewes, numbers of embryos 25 d after mating and lambs born, did not differ with treatment. In summary, low P4 increased the size of the largest follicles and the age of the oldest ovulatory follicles. Embryos resulting from the ovulation of older and younger follicles in the same ewe did not differ in their ability to survive. PMID- 8625618 TI - Aspartate and glutamate modulation of growth hormone secretion in the pig: possible site of action. AB - The influence of excitatory amino acids (EAA) on growth hormone (GH) secretion and the possible site of action was investigated in the pig. In Experiment (Exp) I three replicates were conducted with 30 prepuberal gilts, 130 d of age and averaging 70.6 +/- 1.3 kg body weight (BW). Six gilts each received intravenously (i.v.) 0, 50, 100, or 150 mg/kg BW of aspartate (ASP) or glutamate (GLU) in saline. Blood samples were collected every 15 min for 2 hr before and 3 hr after treatment. In Exp II, mature ovariectomized gilts (163 +/- 10 kg BW) that had been immunized against growth hormone releasing factor (GRF) conjugated to human serum albumin (GRFi; n = 4) or against human serum albumin alone (HSAi; n = 5) received 150 mg/kg BW ASP or GLU i.v. in a 2 x 2 factorial arrangement of treatments, which was then repeated in a crossover design. One week later, all animals received 10 mg/kg N-methyl-D,L-aspartate (NMA; EAA agonist) in saline i.v. Blood samples were collected as described above. In Exp III, cultures of anterior pituitary cells from market-weight (averaging 105 kg BW) gilts were studied. On Day 4 of culture, cells (10(5) seeded/well) were challenged with 10( 8), 10(-6), or 10(-4) M ASP or GLU, 10(-6) M [Ala15]-human GRF (1-29)-NH2, or the EAA antagonist, 2-amino-5-phosphonopentanoic acid (10(-4) M; AP5), alone or in combination with ASP or GLU. In Exp I, all doses of ASP and the 100- and 150-mg doses of GLU increased (P < 0.05) GH secretion when compared with Time 0. However, serum GH concentrations were higher (P < 0.01) after 150 mg/kg of ASP when compared with those after 150 mg/kg of GLU. In Exp II, serum GH concentrations increased (P < 0.05) in HSAi but not in GRFi pigs (averaging 1.2 +/- 0.2 ng/ml before and 8.2 +/- 0.7, 6.3 +/- 0.5, and 9.2 +/- 0.5 ng/ml by 15 min after ASP, GLU, and NMA, respectively). In Exp III, relative to controls (40 +/- 6 ng/ml), GH increased (P < 0.05)1.6-, 1.9-, and 1.9-fold and 1.7-, 1.8-, and 2.0-fold after 10(-8), 10(-6), and 10(-4) M ASP and GLU, respectively. The EAA receptor antagonist AP5 failed to prevent the GH response to ASP or GLU, except for 10(-8) M ASP. In summary, ASP is a more potent secretagogue of GH secretion than is GLU in vivo, whereas each is equipotent in vitro. Because no stimulation of GH by EAA was observed in GRFi pigs and no specific dose-response effect of EAA was found in vitro, it may be concluded that modulation by EAA is mediated primarily at the level of the hypothalamus or higher brain centers. PMID- 8625619 TI - Physiological responses to repeated endotoxin challenge are selectively affected by recombinant bovine somatotropin administration to calves. AB - The study determined 1) whether the pretreatment of calves with recombinant bovine somatotropin (bST, sometribove) would alter the change in packed cell volume (PCV), rectal temperature (RT), and the plasma concentrations of Ca2+, Fe2+, glucose (G), urea N (PUN), nonesterified free fatty acids (NEFA), albumin (ALB), and blood cell populations after endotoxin challenge (EC) and 2) whether the natural development of physiologic tolerance to repeated EC was affected by bST. Twelve steer beef calves were assigned to either control (-bST) or +bST treatment in equal numbers. Calves were injected intramuscularly with either HCO3(-)-buffered saline or bST (0.1 mg/kg) daily for 5 d. On Day 6, the first EC was administered (Escherichia coli, 055:B5, 0.2 microgram/kg, intravenous bolus in pyrogen-free saline). Saline or bST injections were continued from Day 7 up to the repeat of EC on Day 11. RT and PCV were measured hourly through 12 and 6 hr, respectively. Jugular blood was obtained at 0, +1, 2, 3, 4, 6, 8, 12, and 24 hr relative to each EC. bST had no effect on the increase in RT, the hyperglycemic phase of the G response, the biphasic change in Fe2+, or increases in NEFA and PUN. PCV increased after each EC only in -bST. The mean decrease in G during the hypoglycemic phase was less in +bST. Hypocalcemic responses were significantly less in +bST. ALB concentrations decreased after each challenge; the response was unaffected by bST. CD2+, CD4+, and CD8+ T-lymphocyte populations were unaffected by bST and EC. Overall, the magnitude of change in all plasma variables was less after the second EC compared with the first, either in terms of magnitude or duration. The data suggest that the treatment of calves with bST diminishes the magnitude of hypoglycemic, hypocalcemic, and PCV changes after EC and does not compromise fever response, changes in blood cell populations, or tolerance to repeated EC. PMID- 8625620 TI - Clinical molecular genetics and critical care medicine. PMID- 8625621 TI - Adjunctive ventilation with tracheal gas insufflation--good vibrations? PMID- 8625622 TI - Occult, occult auto-PEEP in status asthmaticus. PMID- 8625623 TI - A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis. AB - OBJECTIVES: To determine the allele frequency and genotype distribution of a bi allelic tumor necrosis factor (TNF) gene polymorphism and plasma TNF-alpha concentrations in postoperative intensive care unit (ICU) patients suffering from severe sepsis. DESIGN: Prospective, consecutive entry study of patients with severe sepsis in a postoperative ICU. SETTING: University hospital. PATIENTS: Forty patients with diagnosis of severe sepsis, admitted to the ICU between June 1993 and December 1994. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A 782 basepairs fragment of genomic DNA, including the polymorphic site of the restriction enzyme Ncol within the TNF locus, was amplified by means of polymerase chain reaction. The genotype of each patient was determined after Ncol digestion of the amplified product and subsequent agarose gel electrophoresis. Reading the size of the resulting DNA bands from the agarose gel demonstrated the genotype, as defined by the two alleles TNFB1 and TNFB2. Serial blood samples were drawn every sixth hour during the first 48 hrs and every 12th hour thereafter, for < or = 96 hrs after diagnosis. TNF-alpha plasma concentrations were detected by an enzyme-linked immunosorbent assay. Assessment of organ dysfunction was performed by calculating a Multiple Organ Failure score. The overall allele frequency (TNFB1 0.35; TNFB2 0.65) and genotype distribution (TNFB1 homozygotes 10%; TNFB1/TNFB2 heterozygotes 48%; TNFB2 homozygotes 42%) in 40 patients with severe sepsis were comparable with those values found in normal individuals. Development of multiple organ failure occurred in 33 (82.5%) of 40 patients, whereas 23 (57.5%) of 40 patients did not survive. In contrast to the overall allele frequency, nonsurvivors showed a significantly higher prevalence of the allele TNFB2(p < .005). Patients homozygous for the allele TNFB2 demonstrated a higher mortality rate than heterozygous (TNFB1/TNFB2) patients (p = .0022). In addition, patients with TNFB2 homozygotes displayed higher circulating TNF-alpha concentrations as well as higher Multiple Organ Failure scores compared with heterozygous (TNFB1/TNFB2) patients. CONCLUSIONS: The bi allelic Ncol polymorphism within the TNF locus is a genomic marker for patients with increased TNF-alpha response and poor prognosis in severe sepsis. The amount of TNF released in situations of severe infection and sepsis appears to be influenced genetically. TNFB2 homozygous individuals displaying increased circulating TNF plasma concentrations combined with high mortality rate may be included in future studies testing anti-TNF strategies in severe sepsis. PMID- 8625624 TI - Influence of different volume therapies and pentoxifylline infusion on circulating soluble adhesion molecules in critically ill patients. AB - OBJECTIVE: To study the influence of long-term volume therapy with different solutions and continuous administration of pentoxifylline on plasma concentrations of circulating adhesion molecules. DESIGN: Prospective, randomized study. SETTING: A surgical intensive care unit (ICU) of a university hospital. PATIENTS: Forty-two patients with sepsis secondary to major surgery were included. INTERVENTIONS: The patients were randomly separated into three groups. In group 1 (n=14), volume therapy was exclusively carried out with 10% low molecular-weight hydroxyethyl starch solution. In group 2 (n=14), patients exclusively received 20% human albumin for volume therapy. In group 3 (n=14), volume therapy was not defined and pentoxifylline was continuously given (1.4 mg/kg/hr iv). MEASUREMENTS AND MAIN RESULTS: From arterial blood samples, plasma concentrations of soluble adhesion molecules (endothelial leukocyte adhesion molecule-1 [soluble ELAM-1], intercellular adhesion molecule-1 [soluble ICAM-1], vascular cell adhesion molecule-1 [soluble VCAM-1], and soluble granule membrane protein-140) were serially measured on the day of admission to the ICU ("baseline" value) and during the next 5 days at noon. No significant differences between the three groups were found for macrohemodynamics. Soluble ELAM-1, soluble ICAM-1 and soluble VCAM-1 plasma concentrations were markedly higher than normal values at baseline in all groups. In the hydroxyethyl starch group, soluble ELAM-1 plasma concentration decreased to the normal range, whereas it further increased in the human albumin group and was almost unchanged in the pentoxifylline group. During the study period, soluble ICAM-1 and soluble VCAM-1 plasma concentrations remained unchanged in the hydroxyethyl starch group. However, these concentrations increased in the other groups. Soluble granule membrane protein-140 increased significantly only in the human albumin group (483 +/- 103 to 683 +/- 94 ng/mL). In the hydroxyethal starch-treated patients, Acute Physiology and Chronic Health Evaluation II score decreased significantly (from 24.3 +/- 3.4 at baseline to 17.0 +/- 3.3 at the end the study period). Only PaO2/FIO2 was correlated to plasma concentrations of adhesion molecules. CONCLUSIONS: Sepsis is associated with markedly increased plasma concentrations of adhesion molecules, indicating endothelial activation or damage. By long-term volume therapy with hydroxyethal starch solution, these concentrations remained unchanged or even decreased, whereas in patients in whom human albumin was infused or pentoxifylline was given continuously, plasma concentration of soluble adhesion molecules further increased. PMID- 8625625 TI - Plasma concentrations of cytokines, their soluble receptors, and antioxidant vitamins can predict the development of multiple organ failure in patients at risk. AB - OBJECTIVES: The aims of this study were: a) to evaluate plasma concentrations of cytokines and their soluble receptors, as well as antioxidant substances in patients at high risk of developing multiple organ failure; b) to investigate early change: and c) to examine the possible prognostic value of these elements. DESIGN: Prospective analysis. SETTING: Surgical intensive care unit (ICU) of a university hospital. PATIENTS: sixteen patients at risk for multiple organ failure. MEASUREMENTS AND MAIN RESULTS: Ten patients developed multiple organ failure and five of them died. Whereas tumor necrosis factor-alpha (TNF-alpha) plasma concentrations were only borderline higher in patients developing multiple organ failure, TNF-soluble receptors 55 and 75 were significantly increased during all ICU days compared with patients not going into organ failure. Interleukin-6 plasma concentrations were higher in patients developing multiple organ failure during the first 2 days after ICU admission. The antioxidant vitamin C was significantly decreased in patients going into multiple organ failure during all ICU days. Other biochemical markers of antioxidant activity, such as vitamin E, copper, and zinc plasma concentrations, did not differ between the two groups. CONCLUSIONS: Our data suggest that there is a marked increase in anti-TNF activity and a decrease of antioxidant defense in patients at risk of developing multiple organ failure. The predictive value of plasma concentrations of circulating TNF-soluble receptors and vitamin C in this type of patient needs further evaluation. PMID- 8625626 TI - Acute hemodynamic and neurohoromonal effects of furosemide in critically ill pediatrics patients. AB - OBJECTIVES: To study the acute hemodynamic effects of furosemide in critically ill pediatrics patients, the temporal relationship between hemodynamic changes and changes in neuroendocrine axis, and the temporal relationship between hemodynamic changes and urine output. DESIGN: Prospective study. SETTING: Pediatric intensive care unit in a tertiary care university center. PATIENTS: Fourteen critically ill pediatric patients who clinically required diuretic therapy. INTERVENTIONS: Before and after furosemide administration, hemodynamic and neurohormonal measurements were taken. MEASUREMENTS AND MAIN RESULTS: Hemodynamic and neurohormonal responses to acute diuretic therapy were measured in 14 pediatric patients treated with furosemide (1 mg/kg/dose). Cardiac index deteriorated by 10 mins after drug administration (-9.4+/-3.9%, p<.05) and was associated with an increase in systemic vascular resistance (17.1+/-4.8%, p<.05). There was a subsequent increase in cardiac index (20+/-4.9%, p<.05) at 30 mins, with a decrease in systemic vascular resistance (-11.5+/-5.2%, p<.05). These hemodynamic changes were associated with marked increases in renin and norepinephrine concentrations and an increase in urinary prostaglandin release. The hemodynamic and neurohormonal effects had their onset before maximum diuresis. CONCLUSION: Intravenous furosemide administration in acutely ill pediatric patients results in an acute but transient deterioration in cardiac function that appears to parallel the neuroendocrine changes rather than the acute diuresis. PMID- 8625627 TI - Lack of effects of recombinant growth hormone on muscle function in patients requiring prolonged mechanical ventilation: a prospective, randomized, controlled study. AB - OBJECTIVE: To evaluate the benefit of recombinant human growth hormone administration on muscle strength and duration of weaning in critically ill patients undergoing prolonged mechanical ventilation. DESIGN: Prospective, randomized, controlled, single-blind study. SETTING: Intensive care unit. PATIENTS: Twenty patients requiring > or = 7 days of mechanical ventilation for acute respiratory failure. INTERVENTION: Random assignment to receive either 0.43 IU (approximately 0.14 mg) recombinant growth hormone/kg body weight/day (treated group), or saline (nontreated group) for 12 days. MEASUREMENTS AND MAIN RESULTS: Nutritional support was guided by indirect calorimetry. Cumulative nitrogen balance was positive throughout the study period in the treated group 17.3 (44.9 +/- 17.3[SEM] g/12 days) vs. the nontreated group (-65.8 +/- 11.8 g/12 days) (p<.0001). Despite similar initial plasma concentrations, recombinant growth hormone supplementation resulted in marked increases in growth hormone, insulin like growth factor-1, and insulin concentrations (p<.05, .02, and .0001, respectively, vs. nontreated group). Body impedance determined net fat-free mass increased in the treated group (0.8 +/- 0.6 kg) vs. the nontreated group (-1.1 +/ O.5 kg) (p<.03). Initial peripheral muscle function, assessed by computer controlled electrical stimulation of the adductor pollicis, was similarly lower in treated and nontreated groups than sex and age-matched normal controls, and decreased further during the study period. Arterial blood gases, cumulative total mechanical ventilation time, and number of hrs/day of mechanical ventilation during weaning were similar in both patient groups. Only three of the ten patients in each group were weaned from mechanical ventilation by day 12. CONCLUSIONS: Daily administration of recombinant growth hormone in mechanically ventilated patients with acute respiratory failure promotes a marked nitrogen retention. However, this reaction is accompanied neither by an improvement in muscle strength nor by a shorter duration of ventilatory supports. PMID- 8625628 TI - Therapy of alcohol withdrawal syndrome in intensive care unit patients following trauma: results of a prospective, randomized trial. AB - OBJECTIVES: To assess the effect of three different alcohol withdrawal therapy regimens in traumatized chronic alcoholic patients with respect to the duration of mechanical ventilation and the frequency of pneumonia and cardiac disorders during their intensive care unit (ICU) stay. DESIGN: A prospective, randomized, blinded, controlled clinical trial. SETTING: A university hospital ICU. PATIENTS: Multiple-injured alcohol-dependent patients (n=180) transferred to the ICU after admission to the emergency room and operative management. A total of 180 patients were included in the study; however, 21 patients were excluded from the study after assignment. INTERVENTIONS: Patients who developed actual alcohol withdrawal syndrome were randomized to one of the following treatment regimens: flunitrazepam/clonidine (n=54); chlormethiazole/haloperidol (n=50); or flunitrazepam/haloperidol (n=55). The need for administration of medication was determined, using a validated measure of the severity of alcohol withdrawal (Revised Clinical Institute Withdrawal Assessment for Alcohol Scale). MEASUREMENTS AND MAIN RESULTS: The duration of mechanical ventilation and major intercurrent complications, such as pneumonia, sepsis, cardiac disorders, bleeding disorders, and death, were documented. Patients did not differ significantly between groups regarding age, Revised Trauma and Injury Severity Score and Acute Physiology and Chronic Health Evaluation II score on admission. In all except four patients in the flunitrazepam/clonidine group, who continued to hallucinate, the Revised Clinical Institute Withdrawal Assessment for Alcohol Scale decreased to <20 after initiation of therapy. ICU stay did not significantly differ between groups (p=.1669). However, mechanical ventilation was significantly prolonged in the chlormethiazole/haloperidol group (p=.0315) due to an increased frequency of pneumonia (p=.0414). Cardiac complications were significantly (p=.0047) increased in the flunitrazepam/clonidine group. CONCLUSIONS: There was some advantage in the flunitrazepam/clonidine regimen with respect to pneumonia and the necessity for mechanical ventilation. However, four (7%) patients had to be excluded from the study due to ongoing hallucinations during therapy. Also, cardiac complications were increased in this group. Thus, flunitrazepam/haloperidol should be preferred in patients with cardiac or pulmonary risk. Further studies are required to determine which therapy should be considered. PMID- 8625630 TI - Nasal bridle revisited: an improvement in the technique to prevent unintentional removal of small-bore nasoenteric feeding tubes. AB - OBJECTIVE: To demonstrate the efficacy and safety of an updated version of the nasal "bridle," which is used to prevent the accidental removal of small-bore nasoenteric feeding tubes. DESIGN: A descriptive study. SETTING: Surgical intensive care unit in a tertiary care hospital. PATIENTS: Twenty-six critically ill patients without nasotracheal tubes or facial trauma or fractures who received enteral nutrition and either had removed or were at risk for removing their properly positioned nasoenteric feeding tubes. INTERVENTIONS: A length of one-eighth inch (3.2 mm) umbilical tape is looped around the nasal septum and vomer by serially attaching the ends of the umbilical tape to a suction catheter, passing the catheter through the nostrils, into the oropharynx, and retrieving the ends from the oropharynx. The properly positioned umbilical tape loops into one nostril around the vomer, and out the other nostril. The feeding tube is then anchored to the umbilical tape with a central venous catheter fastener clamp. MEASUREMENTS AND RESULTS: Communicative patients denied discomfort, and there were no episodes of bleeding, infection, sinusitis, or nasal septal trauma caused by the umbilical tape bridle. Five patients had the bridle in place >30 days. There were only two cases in which the bridle failed to prevent removal of a feeding tube. One of these cases occurred because the fastener clamp anchor failed, but this patient had had the same bridle and feeding tube for 170 consecutive days. CONCLUSIONS: An umbilical tape bridle with a central venous catheter fastener clamp anchor is a safe and effective method to prevent the accidental removal of nasoenteric feeding tubes in critically ill patients. We recommend its use in confused or uncooperative patients, or when the risk of unintentional feeding tube removal is high. PMID- 8625629 TI - Perioperative plasma concentrations of tumor necrosis factor-alpha and interleukin-6 in infected patients. AB - OBJECTIVE: To characterize the sequential plasma concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) and their relationship with the clinical outcome in patients with intra-abdominal infection who underwent surgical intervention. DESIGN: A prospective, comparative study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: Fifteen patients with surgically proved intra-abdominal infection were included as the infected group. The comparative noninfected group consisted of ten patients who underwent major abdominal surgery without infection. INTERVENTIONS: Blood samples were obtained from the indwelling arterial catheter before induction of general anesthesia, and 1, 1.5, 2, 3, 4, 6, and 24 hrs after skin incision. MEASUREMENTS AND MAIN RESULTS: Plasma cytokine concentrations were measured using radioimmunoassay. The hemodynamic and physiologic parameters were recorded for comparison with cytokine concentrations. In the noninfected group, the TNF-alpha concentration was very low throughout the observation period, and the IL-6 concentration increased 4 hrs after skin incision. The infected group had significantly higher TNF-alpha and IL-6 concentrations than the noninfected group. The TNF-alpha concentration increased from 129.2 +/- 46.4 to 1196.0 +/- 445.8 pg/mL and the IL-6 concentration increased from 54.2 +/- 24.3 to 560.3 +/- 187.5 pg/mL 2 hrs after skin incision in the infected group. The postoperative APACHE II score correlated significantly with both peak IL-6 (r2=.39) and peak TNF-alpha (r2=.32) concentrations. CONCLUSIONS: Both TNF-alpha and IL-6 concentrations increased significantly after surgical intervention in patients with intra-abdominal infection. The pulse increase in TNF-alpha concentration and the persistent increase in IL-6 concentration were related to the poor postoperative clinical condition in infected patients. PMID- 8625631 TI - Organ donor potential and performance: size and nature of the organ donor shortfall. AB - OBJECTIVES: To estimate the potential for solid organ donation; to identify modifiable reasons for nondonation. DESIGN: Retrospective medical records review. SETTING: Sixty-nine acute care hospitals in four geographic areas of the United States in 1990, and a stratified random sample of 89 hospitals in three of the same areas and 33 of the same hospitals in 1993. PATIENTS: PATIENTS < or = 70 yrs of age who were brain dead and medically suitable for donation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Standard forms were used to record patient demographic and hospital information. Reasons for nondonation were coded as "not identified," "family not asked," "consent denied," or "other." The main outcome measures were rate of donation and rates of nonidentification, not asking, and nonconsent. Organ donation occurred among 33% (299/916) of medically suitable cases identified in 1990 (95% confidence interval 30% to 36%). Ninety-four potential donors were not identified, 156 were not asked, 326 families denied consent, and 41 potential donors were categorized as "other," including patients who had suffered a cardiac arrest, and medical examiner prohibition of donation. In the 1993 study, organ donation occurred in an estimated 33% of suitable cases. In 1990, rates of donation were highest among patients <50 yrs of age, patients who died of traumatic causes, and non-Hispanic white patients. Logistic regression showed lower odds of donation for African American patients (odds ratio 0.38, 95% confidence interval 0.23 to 0.63) independent of potentially confounding hospital and patient variables (p=.0001). Donation rates did not vary by hospital size or type. CONCLUSIONS: Despite legal and policy initiatives, only one third of potential donors became donors in 1990, with similar results in 1993. Extrapolating the 1990 findings to the United States suggests a pool of 13,700 medically suitable donors per year. Prospective identification and requesting donation in all suitable potential donor cases could lead to 1,800 additional donors per year. PMID- 8625632 TI - Pharmacokinetics of reconstituted human high-density lipoprotein in pigs after hemorrhagic shock with resuscitation. AB - OBJECTIVES: Reconstituted human high-density lipoprotein (HDL) can inhibit lipopolysaccharide effects in vivo. The major objectives of this study were to characterize the pharmacokinetics of reconstituted HDL in a stressed large-animal model and to provide preclinical tolerance information in support of use of reconstituted HDL in humans. DESIGN: A randomized, blinded, placebo-controlled trial where each animal received either reconstituted human HDL at a dose of 100 mg/kg (apolipoprotein A-I) or placebo, immediately after hemorrhagic shock and resuscitation. SETTING: Animal laboratory. SUBJECTS: Twelve immature female swine (18 to 25 kg) were studied. INTERVENTIONS: Six to 8 days before shock and study drug administration, animals were anesthesized and catheters were placed in the external jugular vein and abdominal aorta. These catheters were secured to the dorsal surface. On the day of shock, the animals were sedated (alpha-chloralose) and 50 mL/kg of arterial blood was removed over 0.5 hr. One half hour after blood removal, shed blood was infused, which was immediately followed by study drug (reconstituted HDL or placebo), and then by 1 L of lactated Ringer's solution. MEASUREMENTS AND MAIN RESULTS: Physiologic (arterial blood pressure, heart rate, respiratory rate) and laboratory (serum chemistries, hematologic and coagulation studies, and blood gases) measurements were determined intermittently for 96 hrs after the induction of shock. Blood was collected intermittently for 48 hrs after shock for assay of apolipoprotein A-I and phosphatidylcholine in plasma. Reconstituted HDL was well tolerated and did not appear to alter the physiologic responses to shock and resuscitation. HDL transient increase in aspartate aminotransferase concentration was noted in the reconstituted group but this increase normalized by 24 hrs after drug administration. Mean apolipoprotein A-I pharmacokinetic parameters were as follows: half-life 24.5+/-5.3 (SD) hrs; clearance 41.9+/-10 mL/hr; and volume of distribution 1.39+/-0.08 L. The apparent mean half-life of phosphatidylcholine was 5.4+/-0.8 hrs. CONCLUSIONS: Reconstituted human HDL was well tolerated in animals that had undergone hemorrhagic shock with resuscitation. The apolipoprotein component of reconstituted HDL had a relatively long half-life, with distribution limited to the vascular space. These findings support the investigational use of this product in humans. PMID- 8625633 TI - Small-volume resuscitation restores hemorrhage-induced microcirculatory disorders in rat pancreas. AB - OBJECTIVES: Pancreatic hypoxia/ischemia, as a consequence of shock-induced microcirculatory failure, is considered a causative factor in the initiation and/or progression of pancreatic tissue injury. The aim of this study was to compare the effects of "small volume resuscitation" with conventional isovolemic colloid and hypervolemic crystalloid resuscitation on pancreatic microcirculation after hemorrhagic shock. DESIGN: Randomized, controlled intervention trial. SETTING: University laboratory. SUBJECTS: Twenty-three male Sprague-Dawley rats anesthetized with a-chloralose mechanically and ventilated. INTERVENTIONS: Rats subjected to 1 hr of hemorrhagic shock (mean arterial pressure of 40 mm Hg) were resuscitated with lactated Ringer's solution (four-fold shed volume/20 mins), 10% hydroxyethyl starch (shed volume/5 mins), or 7.2% sodium chloride-10% hydroxyethyl starch (10% shed volume/2 mins). MEASUREMENTS AND MAIN RESULTS: The microcirculation of pancreatic acinar tissue was studied by means of intravital fluorescence microscopy and laser Doppler flowmetry. At 1 hr after resuscitation, mean arterial pressure, pancreatic capillary erythrocyte velocity, and erythrocyte flux were found to be significantly increased when compared with those values in the shock state. However, mean arterial pressure, pancreatic capillary erythrocyte velocity, and erythrocyte flux did not completely return to preshock values, regardless of the type of fluid used for resuscitation. At 15 mins and 1 hr after resuscitation, shock-induced capillary perfusion failure (reduction of functional capillary density) was restored to 91% to 94% of baseline values in all groups. Pancreatic capillary narrowing, indicating microvascular endothelial cell swelling, was abolished by resuscitation with both isotonic hydroxyethyl starch and hypertonic hydroxyethyl starch (p<.05 vs. lactated Ringer's solution). CONCLUSIONS: Despite replacement of only 10% of actual blood loss, small-volume resuscitation with hypertonic hydroxyethyl starch is as effective as the ten-fold volume of isotonic hydroxyethyl starch and, due to prevention of microvascular endothelial cell swelling, superior to the 40-fold volume of isotonic lactated Ringer's solution in regard to restoration of the shock-induced microcirculatory disturbances of rat pancreatic acinar tissue. PMID- 8625634 TI - Chest vibration redistributes intra-airway CO2 during tracheal insufflation in ventilatory failure. AB - OBJECTIVE: To determine if high-frequency external chest wall vibration added to low flow intratracheal fresh gas insufflation alters the intra-airway CO2 distribution and the resistance to CO2 transport from the lungs. DESIGN: Prospective study. SETTING: Experimental laboratory. SUBJECTS: Six adult anesthesized and paralyzed mongrel dogs (mean weight 24.3+/- 4.4 kg). INTERVENTIONS: Dogs were ventilated by three methods: a) intermittent positive pressure ventilation; b) intermittent positive pressure ventilation with tracheal insufflation of fresh gas (FIO2 of 0.4) flowing at 0.15 L/kg/min through a catheter positioned at the carina; and c) intermittent positive pressure ventilation with tracheal insufflation and with external high-frequency chest wall vibration of the dependent hemithorax. MEASUREMENTS AND MAIN RESULTS: We measured arterial blood gas values as an index of global gas exchange, and intrapulmonary airway CO2 concentrations as an index of local gas exchange. Intra airway CO2 concentrations along the axis of the airways were measured via a sampling catheter. Airway axial concentration profiles were constructed and resistances to gas transport were calculated from the measured data. Vibration increased intraluminal CO2 concentrations from 1.1% to 2.5% mouthward of the insufflation catheter tip. Peak resistance to CO2 transport decreased by 65% during vibration relative to the insufflation-only value. Vibration displaced peak transport resistance from second- to fourth-generation airways. CONCLUSIONS: Global gas exchange improves during ventilation by chest wall vibration with low flow insufflation. Local gas exchange in the central airways is also improved due to increased intraluminal mixing and CO2 elimination. This ventilation technique may confer therapeutic advantages over conventional mechanical ventilation in the treatment of ventilatory failure. PMID- 8625635 TI - Tracheal gas insufflation combined with high-frequency oscillatory ventilation. AB - OBJECTIVES: To determine the efficacy of tracheal insufflation delivered by two different catheter designs on CO2 elimination when used in conjunction with high frequency oscillatory ventilation. DESIGN: A nonrandomized before and after trial. Each animal served as his own control. SUBJECTS: Ten mongrel dogs weighing 20.9 +/- 1.9 kg. Four animals were assigned to a normal lung group and six animals underwent lung injury by large volume saline lavage. INTERVENTION: Permissive hypercapnia was allowed to occur by selecting oscillator settings that would lead to alveolar hypoventilation. Proximal mean airway pressure was kept constant. Tracheal gas was insufflated at 1 cm above the carina for 30 min periods at gas flows of 5 to 15 L/min. MEASUREMENTS AND MAIN RESULTS: Carinal pressure, hemodynamic parameters (cardiac output, mean arterial pressure, pulmonary artery occlusion pressure), and gas exchange parameters (PaCO2, PaO2, PaO2/FIO2, shunt fraction, D O2) were measured. For the normal dogs, at catheter flow of 15 L/min; the forward thrust catheter increased carinal pressure and Pao2/FIO2 BY 30% (p<.003) and 105% (p<.005), respectively. The forward thrust catheter reduced Paco2 by 40% (p<.04). The reverse thrust catheter increased PaO2/FIO2 by 102% (p<.001) and decreased pressure and PaCO2 by 44% (p<.001) and 34% (p<.003), respectively. For the injured dogs, at catheter flow rate of 15 L/min, the forward thrust catheter increased carinal pressure, PaO2, and PaO2/FIO2 by 6% (p<.001), 23% (p<.001), and 24% (p<.02), respectively. The forward thrust catheter reduced PaCO2 by 29% (p<.002). The reverse thrust catheter increased PaO2 and PaO2/FIO2 both by 11% (p<.02) and reduced carinal pressure and PaCO2 by 23% (p<.001) and 18% (p<.002), respectively. CONCLUSIONS: Tracheal gas insufflation is capable of improving oxygenation and ventilation in acute lung injury when combined with high-frequency oscillatory ventilation. The addition of this second gas flow at the level of the carina raises or lowers distal airway pressure, the magnitude of which is dependent on the direction and rate of gas flow. The beneficial effects of tracheal gas insufflation may be tempered by the long-term effects of altering distal airway pressure; lowering distal airway pressure may lead to atelectasis, whereas raising distal airway pressure may lead to an auto-positive end-expiratory pressure (auto-PEEP) effect. PMID- 8625636 TI - Perfluorocarbon-associated gas exchange improves oxygenation, lung mechanics, and survival in a model of adult respiratory distress syndrome. AB - OBJECTIVE: To compare the effectiveness of perfluorocarbon-associated gas exchange to volume controlled positive pressure breathing in supporting gas exchange, lung mechanics, and survival in an acute lung injury model. DESIGN: A prospective, randomized study. SETTING: A university medical school laboratory approved for animal research. SUBJECTS: Neonatal piglets. INTERVENTIONS: Eighteen piglets were randomized to receive perfluorcarbon-associated gas exchange with perflubron (n=10) or volume controlled continuous positive pressure breathing (n=8) after acute lung injury was induced by oleic acid infusion (0.15 mL/kg iv). MEASUREMENTS AND MAIN RESULTS: Arterial and venous blood gases, hemodynamics, and lung mechanics were measured every 15 mins during a 3-hr study period. All animals developed a metabolic and a respiratory acidosis during the infusion of oleic acid. Following randomization, the volume controlled positive pressure breathing group developed a profound acidosis (p<.05), while pH did not change in the perfluorocarbon-associated gas exchange group. Within 15 mins of initiating perfluorocarbon-associated gas exchange, oxygenation increased from a PaO2 of 52 +/- 12 torr (6.92 +/- 1.60 kPa) to 151 +/- 93 torr (20.0 +/- 12.4 kPa) and continued to improve throughout the study (p<.05). Animals that received volume controlled positive pressure breathing remained hypoxic with no appreciable change in PaO2. Although both groups developed hypercarbia during oleic acid infusion, PaCO2, steadily increased over time in the control group (p<.01). Static lung compliance significantly increased postrandomization (60 mins) in the animals supported by perflurocarbon-associated gas exchange (p<.05), whereas it remained unchanged over time in the volume controlled positive pressure breathing group. However, survival was significantly higher in the perfluorocarbon associated gas exchange group with eight (80%) of ten animals surviving the entire study period. Only two (25%) of the eight animals in the volume controlled positive pressure breathing group were alive at the end of the study period (log rank statistic, p=.013). CONCLUSIONS: Perflurocarbon-associated gas exchange enhanced gas exchange, pulmonary mechanics, and survival in this model of acute lung injury. PMID- 8625637 TI - Perfluorocarbon-associated gas exchange in normal and acid-injured large sheep. AB - OBJECTIVES: We hypothesized that a) perfluorocarbon-associated gas exchange could be accomplished in normal large sheep; b) the determinants of gas exchange would be similar during perfluorocarbon-associated gas exchange and conventional gas ventilation; c)in large animals with lung injury, perfluorocarbon-associated gas exchange could be used to enhance gas exchange without adverse effects on hemodynamics; and d) the large animal with lung injury could be supported with an FIO2 of <1.0 during perfluorocarbon-associated gas exchange. DESIGN: Prospective, observational animal study and prospective randomized, controlled animal study. SETTING: An animal laboratory in a university setting. SUBJECTS: Thirty adult ewes. MEASUREMENT AND MAIN RESULTS: Five normal ewes (61.0 +/- 4.0 kg) underwent perfluorocarbon-associated gas exchange to ascertain the effects of tidal volume, end-inspiratory pressure, and positive end-expiratory pressure (PEEP) on oxygenation. Respiratory rate, tidal volume, and minute ventilation were studied to determine their effects on CO2 clearance. Sheep, weighing 58.9 +/- 8.3 kg, had lung injury induced by instilling 2 mL/kg of 0.05 Normal hydrochloric acid into the trachea. Five minutes after injury, PEEP was increased to 10 cm H2O. Ten minutes after injury, sheep with Pao2 values of <100 torr (<13.3 kPa) were randomized to continue gas ventilation (control, n=9) or to institute perfluorocarbon-associated gas exchange (n=9) by instilling 1.6 L of unoxygenated perflubron into the trachea and resuming gas ventilation. Blood gas and hemodynamic measurements were obtained throughout the 4-hr study. Both tidal volume and end-inspiratory pressure influenced oxygenation in normal sheep during perfluorocarbon-associated gas exchange. Minute ventilation determined CO2 clearance during perfluorocarbon-associated gas exchange in normal sheep. After acid aspiration lung injury, perfluorocarbon-associated gas exchange increased PaO2 and reduced intrapulmonary shunt fraction. Hypoxia and intrapulmonary shunting were unabated after injury in control animals. Hemodynamics were not influenced by the institution of perfluorocarbon-associated gas exchange. CONCLUSIONS: Tidal volume and end-inspiratory pressure directly influence oxygenation during perfluorocarbon-associated gas exchange in large animals. Minute ventilation influences clearance of CO2. In adult sheep with acid aspiration lung injury, perfluorocarbon-associated gas exchange at an FIO2 of <1.0 supports oxygenation and improves intrapulmonary shunting, without adverse hemodynamic effects, when compared with conventional gas ventilation. PMID- 8625638 TI - Effects of nitric oxide synthase inhibition on the cardiovascular response to low output shock. AB - OBJECTIVE: To study the role of nitric oxide in the cardiovascular response to a model of a low output syndrome. DESIGN: Prospective animal study. SETTING: Animal research laboratory. SUBJECTS: Sheep anesthetized with pentobarbital, mechanically ventilated, and monitored with pulmonary arterial and peripheral arterial catheters. INTERVENTIONS: A low output state was induced by inflating a balloon-tip catheter placed in the right atrium. Cardiac index was maintained at 1 L/min/m2 throughout the experiment in three groups of sheep: a) control (n=6) b)LNNA group (pretreated with the nitric oxide synthase inhibitor N omega-nitro-L arginine [LNNA, 100 mg/kg, iv bolus, n=6); and c) dexamethasone group (pretreated with dexamethasone (6 mg/kg, intravenous bolus, n=6). Dexamethasone is an inhibitor of the induction of nitric oxide synthase. LNNA or dexamethasone were administered 15 mins before inducing the low output state. MEASUREMENTS AND MAIN RESULTS: Hemodynamic and oxygen transport variables, and plasma lactate and pyruvate concentrations, were measured at baseline and during the next 3 hrs. For a comparable decrease in cardiac index and oxygen delivery in all groups, the LNNA group had less hypotension and a more marked increase in systemic vascular resistance as compared with the control group. Oxygen consumption and oxygen extraction were higher in the LNNA group as compared with the control group at 30 and 60 mins. Plasma lactate concentration increased significantly less in the LNNA group than in the control and the dexamethasone groups during the observation period. CONCLUSIONS: Inhibition of nitric oxide synthesis during a severe low output state in sheep is associated with a better hemodynamic response, as evidenced by a greater vasoconstriction, and signs of less marked tissue hypoxia. It is likely that inhibition of nitric oxide synthesis in this model leads to an imbalance between the tonic relaxing action of nitric oxide and the influences of vasoconstrictor agents. PMID- 8625639 TI - Aerosolized and instilled surfactant therapies for acute lung injury caused by intratracheal endotoxin in rats. AB - OBJECTIVE: To compare the effects of surfactant replacement by aerosol inhalation and bolus instillation on acute lung injury caused by the intratracheal injection of endotoxin in rats. DESIGN: Prospective, randomized study. SETTING: University Laboratory. SUBJECTS: Male Wistar rats weighing 368 +/- 31 (SD) g. INTERVENTIONS: Escherichia coli endotoxin (57 +/- 20 mg/kg) was injected into the tracheas of 36 anesthetized and mechanically ventilated rats (FIO of 1.0). When the PaO2 had decreased to <200 torr (<26.7 kPa), the rats were randomly assigned to one of three groups: a control group (n=12)given no material; a bolus group (n=12) given a modified natural surfactant suspension (100 mg/kg in 2.0 mL/kg saline) by bolus instillation into the trachea; and an aerosol group (n=12) given surfactant aerosolized with an ultra-sonic nebulizer for 60 mins. MEASUREMENTS AND MAIN RESULTS: Bolus instillation transiently decreased the mean blood pressure by approximately 30%. However, aerosol inhalation did not. The PaO2 values of the control group remained <90 torr (<12.0 kPa) until the end of the experiment (180 mins). In contrast, the PaO2 of the bolus group increased to 387 +/- 134 torr (51.6 +/- 17.9 kPa; p<.05 vs. other groups) 15 mins after surfactant replacement, and remained at approximately 400 torr (approximately 53.3 kPa) throughout the experiment. The PaO2 values of the aerosol group increased slowly, peaked at 240 +/- 109 torr (32.0 +/- 14.5 kPa; p<.05 vs. the control group) 60 mins after the start of surfactant replacement, and remained at approximately 200 torr (approximately 26.7 kPa). CONCLUSIONS: Bolus instillation was superior to aerosol inhalation concerning maximum efficacy, the rapid onset of therapeutic effects, and the necessary dose of surfactant. However, aerosol that does not cause hypotension may be of use in the treatment of adult respiratory distress syndrome in patients with circulatory instability. PMID- 8625640 TI - Iced temperature injectate for thermodilution cardiac output determination causes minimal effects on cardiodynamics. AB - OBJECTIVES: Controversy exists regarding the ideal injectate temperature for measuring cardiac output. Iced temperature injectate gives a higher signal/noise ratio and less variability in the measured cardiac output. Thus, less volume and fewer measurements are required. Advocates of room temperature injectate have suggested that iced temperature injectate may perturb cardiodynamics. This concern has remained largely untested. To help resolve this controversy, we examined the effects of 5 mL iced injectate (0 degrees to 4 degrees) infusions on cardiodynamics. DESIGN: Prospective, randomized, controlled study. SETTING: A critical care research laboratory. SUBJECTS: Five domestic pigs, weighing between 20 to 25 kg. INTERVENTIONS: Under barbiturate anesthesia, pigs underwent placement of a) a thermodilution catheter in the right internal jugular vein; b) a right carotid artery catheter for mean arterial pressure; and c) sonomicrometry crystals for dynamic measurements of left ventricular dimensions. Calculations were made of end-systolic and end-diastolic left ventricular volume and ejection fraction. Six cardiac output measurements were performed in each pig. Data were obtained at baseline (just before iced temperature injectate infusion) and every 3 sec for 9 secs. MEASUREMENTS AND MAIN RESULTS: The only significant effect seen with iced temperature injectate infusion was a small, transient decrease in heart rate (-5.9 +/- 1.1 beats/min from a baseline heart rate of 144.8 +/- 20.6 beats/min). Indices of preload, contractile function, and dynamic cardiac geometry were unaffected. CONCLUSIONS: Iced temperature injectate used in clinically relevant volumes causes transient negative chronotropic effects, but reservations regarding other perturbations of cardiodynamics are unfounded. Thus, the use of iced temperature injectate for cardiac output determination is still a viable alternative to room temperature injectate use, especially when a larger signal/noise ratio is required. PMID- 8625641 TI - Effects of availability of patient-related charges on practice patterns and cost containment in the pediatric intensive care unit. AB - OBJECTIVE: To investigate the effects of the availability of daily patient related charges to healthcare providers on practice patterns and cost containment in the pediatric intensive care unit (ICU) setting. DESIGN: Prospective, nonrandomized, controlled trial. SETTING: Pediatric ICU. PATIENTS: All patients admitted to the pediatric ICU during the study period. This number included a prospective control group (n=325) and an intervention group (n=273). These 598 patients spent 2,274 patient days in the pediatric ICU. INTERVENTIONS: The daily itemized patient charges related to diagnostic studies ordered in the pediatric ICU were made available to healthcare providers during the intervention period of the study. MEASUREMENTS AND MAIN RESULTS: Information was collected prospectively on patients in the control group before the intervention period. This information included data on demographics, daily severity of illness measures, daily resource consumption, intensity of nursing and medical interventions, and daily patient related charges. Outcome information on survival and length of pediatric ICU stay was also collected. The same data were collected prospectively during the intervention period of the study. Measurements on quality assurance and morbidity were made to ensure that there was no compromise in patient care. There were no significant differences in patient demographics and diagnoses between the control and intervention groups. There was a reduction in the average daily laboratory (16.7%), radiology (9.1%) computerized axial tomography (8.5%), and pharmacy (25.1%) charges in the intervention group as compared with controls. The decreases in laboratory and pharmacy charges were statistically significant (p<.0001). The decreases in laboratory and pharmacy charges remained significant even after adjustment for severity of illness. CONCLUSIONS: The availability of patient-related charges to healthcare providers can result in changes in practice patterns, producing a decrease of patient charges and an improvement in cost containment in the pediatric ICU. PMID- 8625642 TI - Accuracy of pulse oximetry in hypothermic neonates and infants undergoing cardiac surgery. AB - OBJECTIVES: To assess the accuracy of pulse oximetry under hypothermic conditions in neonates and infants undergoing cardiac surgery, and to assess the effect of probe site as well as probe site skin temperature on the reliability of pulse oximetry. DESIGN: Prospective Study. SETTING: Cardiac operating room and intensive care unit of children's hospital. PATIENTS: Twenty-five infants <3 months of age undergoing cardiac surgery with cardiopulmonary bypass. INTERVENTIONS: Pulse oximeter readings (Sp O2) from probes placed on the hand and foot were recorded at various skin temperatures and compared with hemoximeter oxygen saturations (Sa O2) obtained on simultaneously drawn arterial blood samples. Core temperature, arterial pressure and vasodilator use were recorded simultaneously. MEASUREMENTS AND MAIN RESULTS: Pulse oximetry bias (Sp O2 - Sa O2) increased to an unacceptable range (>+3% or <-3%) in 45.5% of the readings at foot probe site temperatures of <27 degrees. Pulse oximetry bias was within an acceptable range in 94.7% of the readings at temperatures >29 degrees. There was no significant difference between oximeter readings obtained from two probe sites (hand and foot). Administration of phenoxybenzamine improved the accuracy of pulse oximetry in ten infants at skin temperature of <27 degrees. CONCLUSIONS: Pulse oximetry readings in small infants are likely to be unreliable at skin temperatures of <27 degrees, irrespective of probe site. Intravenous phenoxybenzamine appeared to improve the accuracy of pulse oximetry at low temperatures. PMID- 8625643 TI - Gastric tonometry: precision and reliability are improved by a phosphate buffered solution. AB - OBJECTIVE: To compare a phosphate buffered solution with normal saline as tonometric fluid in intramucosal PCO2 measurement in humans. DESIGN: Prospective, unblinded comparison. SETTING: Postsurgical critical care unit of a university hospital. PATIENTS: Six septic patients. INTERVENTIONS: Two tonometric probes were positioned in the gastric lumen in each patient. One tube was used for conventional tonometry (saline-filled balloon), while phosphate buffered solution was instilled into the second tube. MEASUREMENTS AND MAIN RESULTS: PCO2 was determined with three blood gas analyzers (ABL 2 [Radiometer, Copenhagen, Denmark], Corning 288 [Ciba Corning Diagnostics GmbH, Neuss, Germany], and StatProfile 9 Plus [Nova Biomedical, Waltham, MA]). Eight parallel PCO2 measurements per patient were evaluated, yielding a total of 48 measurements with each tonometric solution. Intrainstrumental comparison of the PCO2 determinations demonstrated an increase of 12.3 +/- 9.9% for ABL 2, 3.10 +/- 12.9% for Ciba Corning 288, and 101.2 +/- 31.5% for StatProfile 9 Plus with the phosphate buffered solution. The PCO2 values were decreased by the following amounts when the three instruments were compared, using the saline method: 14.2 +/- 8.2% (Ciba Corning 288 vs. ABL 2); 40.7 +/- 9.9% (StatProfile 9 Plus vs. ABL 2); and 30.9 +/ 9.35% (StatProfile 9 Plus vs. Ciba Corning 288). The difference in PCO2 determination, resulting from the different instrument designs, were significant between the three blood gas analyzers (p<.001). In addition, the variance of the intramucosal PCO2 values was significant between blood gas analyzers (p<.001) with normal saline as tonometric solution, but not with phosphate buffered solution. The coefficients of determination between PCO2 values in saline and phosphate buffered solution were r2=.85 for ABL 2, r2=.81 for Ciba Corning 288, and r2=.74 for StatProfile 9 Plus. When all 48 PCO2 values were analyzed, the interinstrumental coefficients of determination within a method for saline (and for phosphate buffered solution in parenthesis) were:r2=.83 (.92) between ABL 2 and Ciba Corning 288, r2=.72 (.92) between ABL 2 and StatProfile 9 Plus, and r2=.81 (.98) between Ciba Corning 288 and StatProfile 9 Plus. CONCLUSIONS: A considerable instrumental bias in PCO2 analysis is observed when saline is used as tonometric fluid in gastric tonometry, thus preventing a reliable determination of intramucosal pH. The present in vivo data show that the accuracy and reliability of intramucosal pH measurement can be improved by the use of phosphate buffered solution as tonometric fluid. PMID- 8625644 TI - Maximizing oxygen delivery in critically ill patients: a methodologic appraisal of the evidence. AB - OBJECTIVE: To systemically review the effect of interventions designed to achieve supraphysiologic values of cardiac index, oxygen delivery (DO2), and oxygen consumption (VO2) in critically ill patients. DATA SOURCES: Computerized bibliographic search of published research, citation review of relevant articles, and contact with primary investigators. STUDY SELECTION: We included all randomized clinical trials of adult intensive care unit (ICU) patients that evaluated interventions (fluids, inotropes, and vasoactive drugs) designed to achieve supraphysiologic values of cardiac index, DO2, and/or VO2. Independent review of 64 articles identified seven relevant studies of 1,016 patients. DATA EXTRACTION: We abstracted data on the population, interventions, outcomes, and methodologic quality of the studies by duplicate independent review. Agreement was high (weighed kappa 0.73); differences were resolved by consensus. DATA SYNTHESIS: Targeting therapy to achieve supraphysiologic end points in critically ill patients is associated with a nonstatistically significant trend toward decreased mortality rates (relative risk 0.86, 95% confidence intervals 0.62 to 1.20). For the two studies in which supraphysiologic goals were initiated preoperatively, the relative risk was 0.20 (95% confidence intervals 0.07 to 0.55). This value differed significantly from the combined estimate of the remaining studies, in which the intervention was started after ICU admission (relative risk 0.98, 95% confidence intervals 0.79 to 1.22; p<.01). However, there are several methodologic problems with the primary studies. In no trials were caregivers or outcome assessors blinded to treatment allocation. Only three of seven trials analyzed patients according to the group to which they were allocated. None adequately controlled for cointerventions, and there was considerable crossover between groups (patients in the control group achieved the goals of the intervention group and vice versa). CONCLUSIONS: Interventions designed to achieve supraphysiologic goals of cardiac index, DO2, and VO2 did not significantly reduce mortality rates in all critically ill patients. However, there may be a benefit in those patients in which the therapy is initiated preoperatively. Methodologic limitations weaken the inferences that can be drawn from these studies and preclude any evidence-based clinical recommendations. PMID- 8625645 TI - Is it time to reposition vasopressors and inotropes in sepsis? AB - OBJECTIVES: To review the literature on the current use of vasopressors and inotropes in patients with sepsis and sepsis syndrome with respect to the choice of agent, therapeutic end points, and safe and effective doses to be used. To examine the available evidence that supports or refutes goal-directed therapy toward supranormal oxygen transport in optimizing the outcome of critically ill sepsis syndrome patients. DATA SOURCES: All pertinent English and French articles dealing with hemodynamic support with selected vasopressors and inotropic agents in human sepsis and sepsis syndrome retrieved from a computerized MEDLINE search from 1985 to 1994. STUDY SELECTION: Clinical studies with norepinephrine, epinephrine, phenylephrine, dopamine, and dobutamine in sepsis syndrome were considered if goal-directed therapy with oxygen transport variables was utilized. Emphasis was placed on prospective, randomized, controlled comparative trials. However, open-label, observational, and comparative studies, or case series, were also evaluated when limited data were available. DATA EXTRACTION: From the selected studies, information was obtained regarding patient population, dosing regimen, type of therapeutic goals or end points (hemodynamic, or normal vs. supranormal oxygen transport variables) and outcome data (e.g., achievement of goals, resolution of the episode, mortality rate, and development of end-organ dysfunction). DATA SYNTHESIS: When used in larger than usual doses, epinephrine, norepinephrine, and phenylephrine uniformly increased hemodynamic values. Epinephrine may increase oxygen transport values more reliably than norepinephrine. Dobutamine doses in the range of 2.5 to 6 microgram/kg/min increase oxygen transport variables and hemodynamics to predetermined goals in only 30% to 70% of patients. Larger infusion rates offer no further benefits. CONCLUSIONS: Insufficient evidence exists to support goal-directed therapy with vasopressors and inotropes in the treatment of sepsis syndrome. No definitive recommendations can be made about the superiority of a vasopressor or inotropic agent due to the lack of data. However, it may be that evaluation of vasopressors earlier in sepsis syndrome will yield more promising results. Large, comparative, controlled trials assessing mortality rate and development of multiple organ system dysfunction are needed. PMID- 8625646 TI - Ascites and its effects upon respiratory muscle loading and work of breathing. PMID- 8625647 TI - Low measured auto-positive end-expiratory pressure during mechanical ventilation of patients with severe asthma: hidden auto-positive end-expiratory pressure. AB - OBJECTIVE: To describe the occurrence of low measured auto-end-expiratory pressure (auto-PEEP) during mechanical ventilation of patients severe asthma. DESIGN: Observational clinical study. SETTING: Medical intensive care unit of a university-affiliated county hospital. PATIENTS: Four mechanically ventilated patients with severe asthma who had low measured auto-PEEP despite marked increase in both peak and plateau airway pressures. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Peak pressure, plateau pressure, and auto-PEEP were measured at an early time point, when airflow obstruction was most severe, and again at a later time after clinical improvement. Auto-PEEP was measured by the method of end-expiratory airway occlusion. From the early to the late point, there was a marked decrease in peak pressure (76 +/- 7 to 53 +/- 6 cm H2O; p<.001) and in plateau pressure (28 +/- 2 to 18 +/- 3 cm H2O; p<.001), but only minimal change in auto-PEEP (5 +/- 3 to 4 +/- 3 cm H2O). The difference between plateau pressure and auto-PEEP decreased between the early and late time points (23 +/- 1 to 14 +/- 1 cm H2O; p<.01), even though tidal volume was larger at the late time point. In three patients, low auto-PEEP and a large difference between plateau pressure and auto-PEEP was only seen after expiratory time was prolonged. In these three patients, prolongation of expiratory time resulted in a large decrease in measured auto-PEEP (14 +/- 4 to 5 +/- 4 cm H2O), but a much smaller change in plateau pressure (31 +/- 3 to 29 +/- 3 cm H2O). CONCLUSIONS: We conclude that measured auto-PEEP may underestimate end-expiratory alveolar pressure in severe asthma, and that marked pulmonary hyperinflation may be present despite low measured auto-PEEP, especially at low respiratory rates. This phenomenon may be due to widespread airway closure that prevents accurate assessment of alveolar pressure at end-expiration. PMID- 8625648 TI - Cardiac arrest and sodium bicarbonate. PMID- 8625649 TI - Prolonged studies of perfluorocarbon associated gas exchange. PMID- 8625650 TI - Adjuvant treatments for serious infections. PMID- 8625651 TI - Respiratory infections. An opportunity for integrated disease management. PMID- 8625652 TI - Observance of long-term oxygen therapy at home. PMID- 8625653 TI - A perspective on radiation therapy for lung allograft rejection. PMID- 8625654 TI - Discordance of tumor diagnosis for bronchial biopsies. PMID- 8625655 TI - Angina-like chest pain. Heart, esophagus, or something else? PMID- 8625656 TI - OA for OSA. PMID- 8625657 TI - Differentiating total work of breathing into its component parts. Essential for appropriate interpretation. PMID- 8625658 TI - Long-term oxygen therapy at home. Compliance with medical prescription and effective use of therapy. ANTADIR Working Group on Oxygen Therapy. Association Nationale de Traitement a Domicile des Insuffisants Respiratories. AB - STUDY OBJECTIVE: Daily duration of oxygen administration is an important factor in the effectiveness of long-term oxygen therapy (LTOT) for hypoxic chronic pulmonary disease. We have assessed the daily use of oxygen therapy in 930 patients with COPD and examined factors associated with the effective use of this treatment. METHODS: Objective daily duration of oxygen use over a 3-month period was prospectively measured using the counter clock of the oxygen concentrators or by weighing the liquid oxygen container at each delivery. A questionnaire was filled in by an independent investigator asking about home situation, lifestyle, and whether oxygen therapy was used during all domestic and outside activities. In addition, prescribing physicians were asked about the duration and modalities of oxygen prescribed in each case. RESULTS: The patients had been receiving LTOT for 36 +/- 24 months and had hypoxemia (PaO2 = 56 +/- 9 mm Hg), hypercapnia (PaCO2 = 47 +/- 8 mm Hg), and severe airflow obstruction (FEV1/VC = 42 +/- 14%). The mean duration of oxygen treatment prescribed was 16 +/- 3 h/d. The mean duration of oxygen therapy achieved was 14.5 +/- 5 h, but only 45% of the patients achieved oxygen therapy for 15 h or more per day. Patients with effective use of LTOT, ie receiving oxygen therapy for at least 15 h/d, were significantly more hypoxic (PaO2 = 54 +/- 9 vs 57 +/- 9 mm Hg; p < 0.001), more hypercapnic (PaCO2 = 48 +/- 8 vs 46 +/- 7 mm Hg; p < 0.005), and also more obstructed (FEV1/VC = 39.5 +/- 13 vs 45 +/- 14%; p < 0.001) than the rest of the patients under treatment. Other factors associated with effective use of oxygen were (1) initial prescription for 15 h or more per day, (2) supplementary education on oxygen therapy by a nurse or physiotherapist, (3) cessation of smoking, (4) use of oxygen in all domestic situations (toilet, meals, leisure, etc.), and (5) absence of side effects from oxygen treatment. CONCLUSIONS: Attention by the prescribing physicians to such factors could optimize oxygen prescription and constitute goals for education of patients. PMID- 8625659 TI - Comparison of oxygen saturation by pulse oximetry and co-oximetry during exercise testing in patients with COPD. AB - INTRODUCTION: Measurement of oxygen saturation by pulse oximetry (SpO2) is frequently performed during exercise testing of patients with COPD to monitor for hypoxemia. The purpose of this study was to assess the accuracy and precision of pulse oximetry during exercise. We hypothesized that the SpO2 would more closely reflect oxygen saturation as measured by co-oximetry (SaO2) when it was corrected for carboxyhemoglobin (COHb). We also hypothesized that SpO2 would more closely reflect SaO2 when the pulse rate by oximeter was equivalent to the heart rate by ECG. Finally, we hypothesized that SpO2 would be a better measure of SaO2 at maximal workloads than at rest or submaximal workloads. METHODS: Eight white men with severe COPD (mean +/- SD FEV1, 0.91 +/- 0.37) underwent progressive, symptom limited exercise testing by cycle ergometry. SaO2 was measured from arterial blood at each workload using a co-oximeter. SpO2 and pulse rate were obtained by a pulse oximeter (Ohmeda 3700). Heart rate was continuously monitored by ECG. RESULTS: Reliable oximetric values as determined by a dicrotic notch in each waveform and adequate signal intensity were obtained in all eight patients. SpO2 was a moderately accurate measure of SaO2 (bias, 1.7%; precision, 2.9). The bias actually increased (4.1%) when SpO2 was corrected for COHb. Accuracy of SpO2 was not improved when pulse rate by oximetry and heart rate by ECG were equivalent, nor was the accuracy improved at maximal workloads relative to submaximal workloads during the exercise test. CONCLUSION: Oxygen saturation as measured by pulse oximetry (SpO2) in patients with COPD undergoing exercise testing is not sufficiently accurate to replace SaO2 as the gold standard for oxygen saturation. PMID- 8625660 TI - Effects of long-term treatment with corticosteroids in COPD. AB - STUDY OBJECTIVE: To determine the effectiveness of treatment with corticosteroids in patients with COPD. METHODS: In this study, we investigated the effect of a 2 year treatment with corticosteroids on clinical symptoms and the decline of lung function in 58 nonallergic patients with COPD. Subjects were treated in a double blind, randomized, placebo-controlled, parallel way with inhaled budesonide (bud), 1,600 micrograms/d; inhaled budesonide, 1,600 micrograms/d, plus oral prednisolone, 5 micrograms/d (bud + pred); or placebo (plac). Clinical assessment (history, physical examination, and spirometry) was carried out every 2 months. The rate of decline in FEV1 was assessed by calculating individual regression co efficients from linear regression of FEV1 on time for each subject. RESULTS: Eleven patients dropped out. The number of withdrawals due to pulmonary problems was significantly higher in the plac group (n = 5 out of 18) than in the actively treated groups (n = 2 out of 40). Treatment with corticosteroids significantly reduced pulmonary symptoms. Median decline of FEV1 was 60 mL/yr in the plac group, 40 mL/yr in the bud + pred group, and 30 mL/yr in the bud group. Variation was large and differences were not statistically significant. No treatment effect was found on frequency or duration of exacerbations, possibly because of the high number of withdrawals due to pulmonary deterioration in the plac group. Treatment with a combination of inhaled plus oral corticosteroids was not more effective than inhaled corticosteroids alone. Morning plasma cortisol levels remained within the normal range in all three groups. CONCLUSIONS: Our study shows beneficial effects of long-term daily treatment with inhaled corticosteroids in patients with COPD with regard to symptoms and drop out due to pulmonary problems. Lung function decline tends to decrease during treatment with inhaled corticosteroids. The observed effects are limited but warrant further studies on the effectiveness of corticosteroids in larger numbers of patients with COPD. PMID- 8625661 TI - Spirometry and dyspnea in patients with COPD. When small differences mean little. AB - OBJECTIVE: To determine when a difference in FEV1 is sufficiently large to be associated with a noticeable difference in dyspnea symptoms for patients with chronic lung disease. DESIGN: Cross-sectional analysis of 15 groups (n = 112 patients, 832 contrasts). SETTING: Respiratory rehabilitation program. PATIENTS: Patients with COPD (mean FEV1 = 35% predicted). MEASURES: Patients' perspectives assessed through subjective comparison ratings of dyspnea and of overall health. Relation between the FEV1 and patients' perspectives determined the smallest difference in spirometry that was associated with a noticeable difference in patients' symptoms. RESULTS: The FEV1 was moderately correlated with patients' ratings of dyspnea (r = 0.29; 95% confidence interval (CI), 0.22 to 0.35). In contrast, the FEV1 was minimally correlated with patients' ratings of overall health (r = 0.10; 95% CI, 0.03 to 0.17). The FEV1 needed to differ by 4% predicted for the average patient to stop rating his or her dyspnea as "about the same" and start rating his or her dyspnea as either "a little bit better" or "a little bit worse" relative to other patients (95% CI, 1.5 to 6.5). This was equivalent to the average patient's FEV1 increasing by 112 mL (starting from 975 mL and ending at 1,087 mL). CONCLUSIONS: Some statistically significant differences in the FEV1 are so small that they may not represent important differences in symptoms for the average patient with severe COPD; an awareness of the smallest difference in FEV1 that is noticeable to patients can help clinicians interpret the effectiveness of symptomatic treatments. PMID- 8625662 TI - Dyspnea ratings for prescribing exercise intensity in patients with COPD. AB - STUDY OBJECTIVE: We tested the hypothesis that patients with COPD can use dyspnea ratings obtained from a prior graded exercise test as a target to reliably produce specific exercise intensities. DESIGN: Four visits over a 7-week period. SETTING: Pulmonary function and cardiorespiratory exercise laboratory at a university hospital. PATIENTS: Fifteen symptomatic patients with stable COPD. Age was 68 +/- 7 (mean +/- SD) years. FEV1 was 1.12 +/- 0.22 L (45 +/- 8% predicted). INTERVENTIONS: At each visit, patients estimated the heaviness of weights to evaluate their magnitude estimation of a nonrespiratory task; after pulmonary function testing was completed, patients were tested on the cycle ergometer. At estimation trial 1 (day 0), patients estimated the intensity of dyspnea using the 0 to 10 category-ratio scale during an incremental exercise test. Estimation trial 2 (day 5 to 7) was the same as the previous trial. At production trials 1 (day 10 to 14) and 2 (day 40 to 44), patients were instructed to produce specific intensities of dyspnea (ie, dyspnea targets) at 50% and at anaerobic threshold (AT) or 80% of peak oxygen consumption (Vo2) as calculated from results at estimation trial 2. MEASUREMENTS AND RESULTS: Lung function was stable at all visits. Dyspnea ratings were 1.8 +/- 0.9 (range, 1 to 3) at 50% of peak Vo2 and 5.5 +/- 1.5 (range, 4 to 8) at AT/80% of peak Vo2 (17.0 +/- 3.4 mL/kg/min) at estimation trial 2. The individual percent changes in Vo2 at the lower dyspnea target were 12 +/- 19% and 11 +/- 19% for production trials 1 and 2, respectively, compared with estimation trial 2. At the higher dyspnea target, the corresponding individual percent changes in Vo2 were -4 +/- 9% and -7 +/- 11%, respectively. For all 15 patients, there were borderline statistical differences for the Vo2 values at the lower (p = 0.04 and p = 0.07) and at the higher (p = 0.04 for each production trial) dyspnea targets for production trials 1 and 2 compared with estimation trial 2. Two patients showed 50% or greater variability in the calculated exponent for magnitude estimation of weights. In a subgroup analysis of the 13 patients with reproducible magnitude estimation of the heaviness of weights, there were no significant differences in Vo2 for the two production trials compared with estimation trial 2 at both exercise intensities. CONCLUSIONS: Dyspnea ratings obtained from an incremental exercise test can be used as a target for patients with COPD to regulate/monitor the intensity of exercise training. The ability of patients with COPD to achieve a desired Vo2 based on an individual dyspnea target was generally more accurate at the higher exercise level (AT/80% of peak Vo2) compared with the lower intensity (50% of peak Vo2). Acceptable accuracy was maintained over a 5-week time period. PMID- 8625663 TI - Severe osteoporosis before and after lung transplantation. AB - STUDY OBJECTIVE: Osteoporosis and/or fractures have been reported as a complication of kidney, heart, liver, bone marrow, and, most recently, lung transplantation (LTx). We measured bone mineral density (BMD) before and after LTx to determine the frequency and severity of preoperative and postoperative osteoporosis. PATIENTS AND METHODS: We conducted a cross-sectional study of BMD in 100 patients with various diagnoses before (n = 55) and after (n = 45) LTx. RESULTS: Age-matched mean spine and femoral BMDs were severely depressed before and after LTx placing 45% of the pre-LTx and 73% of the post-LTx patients at or below the fracture threshold (2 SDs below the mean). As expected, patients with cystic fibrosis had lower (p < 0.05) mean spine and femoral BMD than patients with COPD or other lung diseases both before and after LTx. Nevertheless, considerable osteoporosis was present in the patients with COPD and other lung diseases before and after LTx. The best predictors of pre- and post-LTxZ scores were body mass index (r = 0.56 to 0.59, p < 0.001) and cumulative steroid dose/per kilogram (r = 0.49-0.51, p < 0.001), respectively. Osteoporosis has resulted in 3 symptomatic fractures before and 12 after LTx. CONCLUSIONS: Osteoporosis appears to be widespread in patients with end-stage respiratory failure before LTx and LTx, while often life-saving, has an adverse impact on BMD. As patients live longer after LTx, osteoporosis-related fractures may compromise quality of life by causing pain and debilitation. PMID- 8625665 TI - Pleural effusions following lung transplantation. Time course, characteristics, and clinical implications. AB - The time course and characteristics of ipsilateral pleural effusion in nine consecutive single lung transplant recipients are described and compared with those of six patients who underwent other cardiothoracic operations. Ipsilateral pleural fluid occurs in all lung transplant recipients, beginning immediately following transplantation and continuing for up to 9 days. Pleural fluid immediately after lung transplantation is bloody, exudative, and neutrophil predominant, which is similar to the characteristics of pleural fluid following other cardiothoracic surgery. Pleural fluid cellularity, lactate dehydrogenase, and total protein content decrease rapidly over the first week in lung transplant recipients. The percentage of neutrophils decreases from 90 to 50% by day 7. Pleural fluid output in lung transplant recipients declines steadily during the first week and is minimal by day 9. Pleural fluid output declines more rapidly in patients who have undergone cardiothoracic surgery than in the lung transplant recipients. An early rise in pleural fluid output may reflect the development of posttransplant pulmonary edema. We conclude that it is unnecessary to analyze pleural fluid after lung transplantation if the pleural fluid output is decreasing and the clinical course is appropriate. PMID- 8625664 TI - Total lymphoid irradiation for refractory acute rejection in heart-lung and lung allografts. AB - Persistent or recurrent acute allograft rejection (AR) refractory to high-dose steroid therapy can adversely affect long-term outcomes of heart-lung (HLT), bilateral-lung (BLT), and single-lung (SLT) transplantations. The use of total lymphoid irradiation (TLI) for the management of refractory acute AR in six transplant recipients (two men, four women; mean age, 29.8 +/- 3.8 years) is detailed. There are two HLT (primary pulmonary hypertension [PPH], cystic fibrosis [CF]), 1 BLT (pulmonary hypertension postventricular septal defect repair), and 3 SLT (sarcoid, PPH, congenital heart disease with atrial septal defect) recipients. Refractory AR is defined as persistent rejection unresponsive to high-dose steroid therapy in all cases. The BLT and SLT recipients had at least two moderate and one mild AR events per patient. The HLT recipients had at least two moderate acute heart and one severe and one mild asynchronous acute lung rejection events per patient. A total of 800 cGy of total lymphoid irradiation (TLI) was administered over a 5-week period. Mild and transient leukopenia was the only observed side effect. The patient with PPH received TLI 313 days after HLT for recurrent AR at another institution and died of ARDS 4 weeks after completing TLI. The patient with CF received TLI 707 days after HLT and died 457 days after TLI of severe obliterative bronchiolitis (OB) with multiorgan failure. The patient with BLT received TLI 176 days after transplant and died 372 days after TLI of respiratory failure related to severe rejection. One patient with SLT received TLI 78 days after transplant and died 679 days after TLI of severe acute AR. The two remaining patients with SLTs have been free from acute AR for more than 4 years. The patient with sarcoidosis received TLI 37 days after SLT following a clinical rejection event and two severe acute AR events. He is alive with normal lung function 5 years later. The patient with PPH received TLI 108 days after SLT following three moderate acute AR events and is alive with stable OB 4 years later. These limited preliminary results suggest that TLI has merit for the treatment of intractable acute AR following HLT and lung transplantation. PMID- 8625666 TI - A randomized, double-blind, placebo-controlled study of lorazepam as premedication for bronchoscopy. AB - To our knowledge, no study has clearly demonstrated the advantage of sedative premedication for bronchoscopy. In a double-blind study, we evaluated the efficacy of oral lorazepam as premedication for bronchoscopy. One hundred patients were randomly assigned to receive placebo (group A) or lorazepam (2 mg) (group B) approximately 1.5 h before bronchoscopy. Immediately after the procedure and the following day, a questionnaire addressing the patient's perception of the procedure was administered. Specifically, subjects were asked to grade the bronchoscopy as very easy, easy, difficult, or very difficult to tolerate and if they would agree to a second bronchoscopy if believed necessary. In addition, their recollection of the procedure was graded as clear, indistinct, or not at all. No difference was found between the two groups for age, duration of the bronchoscopy, and the answers to the questionnaire administered immediately after the procedure. Most patients from both groups found their level of sedation adequate. On the following day, however, group B reported with lower frequency that the technique was difficult or very difficult (38.0% vs 65.3% for group A; p < 0.005) and that they would be less reluctant to a repeated bronchoscopy (30.0% vs 57.1% for group A; p < 0.015). Moreover, their recollection of the procedure was now less precise than for those who had received the placebo (p < 0.005). This suggests that the difference observed between the two groups at 24 h was related to the amnesic effect of lorazepam. We conclude that lorazepam, by improving patient's perception of the bronchoscopy, is a useful premedication and may facilitate patient's investigation when a second bronchoscopy becomes necessary. PMID- 8625667 TI - Cell type accuracy of bronchial biopsy specimens in primary lung cancer. AB - STUDY OBJECTIVES: To evaluate the diagnostic accuracy of bronchial biopsy (BB) specimens in establishing the specific cell type in primary lung cancer (LC), and to study the influence of several factors on this accuracy. SETTING: Tertiary health-care center. PATIENTS: One hundred forty-six patients with LC diagnosed by BB specimens who underwent thoracotomy (T). MEASUREMENTS: We have studied the specific LC cell type observed in the BB specimen and compared it with the T specimen (reference diagnosis). Age, location and type of bronchial lesion, number and size of the biopsy fragments, tumoral size, sample necrosis, degree of cell differentiation, tumoral stage, pathologist's experience, and the presence of other diagnostic tests with the same cell type were analyzed to assess their influence on the concordance between the two diagnoses. RESULTS: The overall concordance between BB and T histologic diagnosis was 0.70 (kappa coefficient [K]). Of the different histologic types, the worst result was obtained in large cell carcinoma (LLC) (K, 0.49). Squamous carcinoma and adenocarcinoma gave similar results (0.74 and 0.77, respectively), while small cell lung cancer (SCLC) only reached a value of 0.60. The degree of cell differentiation, the absence of necrosis, and presence of other preoperative diagnoses were the variables that most influenced the histologic accuracy of BB specimens. Therefore, the probability of BB accuracy was 2.7, 7.7, and 25 times higher in well-differentiated, than in poorly differentiated, moderately differentiated, or undifferentiated carcinomas; 5.2 times higher when there was no necrosis in the sample; and 7.43 higher when there was another preoperative diagnosis. CONCLUSIONS: The histologic results of BB must be examined carefully, especially in cellular subtypes like LLC. The absence of differentiation and presence of necrosis in BB samples were the factors that require the greatest caution in ascertaining the cell type. When they are involved and also in all cases in which identifying the specific cell type has important implications, we prefer to classify the patients as having SCLC or non-small cell lung cancer, and then reclassify them later after using a second diagnostic technique. PMID- 8625668 TI - Comparison of quantitative cultures to semiquantitative loop cultures of bronchoscopic protected specimen brush samples. AB - STUDY OBJECTIVE: To compare quantitative growth from a calibrated loop to growth from the standard serial dilution technique of culturing bronchoscopic protected specimen brush (PSB) samples and to determine the effect of refrigeration of the PSB sample on subsequent quantitative growth. DESIGN: Laboratory stock cultures were sampled with a PSB and cultured by both standard 100-fold serial dilution as well as 1:100 mL and 1:1,000 mL calibrated loops. Stock cultures were also sampled with a PSB and growth before and after refrigeration for 24 h at 4 degrees C (both serial dilution and calibrated loops) was compared. Clinical PSB samples from seven patients suspected of having lower respiratory tract infections were cultured by both techniques as well. SETTING: Clinical research laboratory and teaching hospital. PATIENTS AND INTERVENTIONS: PSB samples from inpatients and outpatients who had clinically indicated bronchoscopy. No interventions. MEASUREMENTS AND RESULTS: Quantitative growth from the 1:1,000 mL calibrated loop was within 1 log10 of growth from the serial dilution technique for 20 of 21 organisms, including 2 yeasts. Except for Haemophilus influenzae (known to be cold intolerant), there were no important differences in growth of bacteria before and after 24 h at 4 degrees C. For quantitative bacterial growth, there was a significant correlation between serial dilution and the 1:1,000 mL loop cultures (r=0.86, p < 0.0001). CONCLUSION: In this study, quantitative growth from a single 1:1,000 mL loop culture of PSB samples was comparable to growth from the standard serial dilution technique. Our results also suggest that overnight refrigeration of PSB samples may be possible in certain clinical situations. PMID- 8625669 TI - Prevalence of esophageal disorders in patients with chest pain newly referred to the cardiologist. AB - STUDY OBJECTIVE: The prevalence of esophageal disorders (dysmotility and/or gastroesophageal reflux) in patients with chest pain newly referred to a cardiologic clinic is unknown. The aims of our study were to investigate the prevalence of esophageal abnormalities in these patients and to assess the value of medical history in predicting the origin of the patient's chest pain. DESIGN: We evaluated 28 consecutive patients who were newly referred to the cardiologist because of angina-like chest pain. Patients with evidence of severe myocardial ischemia were excluded. Cardiologic evaluation included medical history, physical examination, ECG, and exercise testing; further cardiologic workup was carried out only when considered necessary. Gastroenterologic evaluation consisted of medical history, esophageal manometry, endoscopy, and 24-h ambulatory monitoring of esophageal pH and pressure. MEASUREMENTS AND RESULTS: In five patients a diagnosis of ischemic coronary artery disease was made. In only two of these five patients, the cardiologic history strongly suggested a cardiac origin of the pain. Twelve patients had a pathologic 24-h pH profile, four of whom also had reflux esophagitis. Ten patients had symptomatic reflux. In only three of these ten patients, the history was judged to be indicative of an esophageal origin of the chest pain. No motility disorders were found. CONCLUSIONS: Thirty-six percent of the patients with chest pain newly referred to a cardiologic out-patient clinic have symptomatic gastroesophageal reflux. Neither cardiologic nor gastroenterologic history data have a high predictive value with respect to the origin of the chest pain. PMID- 8625670 TI - Effects of hypercapnia on hemodynamic, inotropic, lusitropic, and electrophysiologic indices in humans. AB - STUDY OBJECTIVE: The inotropic, lusitropic, and electrophysiologic effects of acute hypercapnia in humans are not known. Although the effects of hypercapnia on the systemic circulation have been well documented, there is still some debate as to whether hypercapnia causes true pulmonary vasoconstriction in vivo. We have therefore evaluated the effects of acute hypercapnia on these cardiac indices and the interaction of hypercapnia with the systemic and pulmonary vascular beds in humans. PARTICIPANTS AND INTERVENTIONS: Eight healthy male volunteers were studied using Doppler echocardiography. After resting for at least 30 min to achieve baseline hemodynamic parameters (T(0)), they were rendered hypercapnic to achieve an end-tidal carbon dioxide (CO2) of 7 kPa for 30 min by breathing a variable mixture of CO2/air (T1). They were restudied after 30 min recovery breathing air (T2). Hemodynamic, diastolic, and systolic flow parameters, QT dispersion (maximum-minimum QT interval measured in a 12-lead ECG), and venous blood samples for plasma renin activity (PRA), angiotensin II (ANG II), and aldosterone (ALDO) were measured at each time point. RESULTS: Hypercapnia compared with placebo significantly increased mean pulmonary artery pressure 14 +/- 1 vs 9 +/- 1 mm Hg and pulmonary vascular resistance 171 +/- 17 vs 129 +/- 17 dyne.s.cm-5, respectively. Heart rate, stroke volume, cardiac output, and mean arterial BP were increased by hypercapnia. Indexes of systolic function, namely peak aortic velocity and aortic mean and peak acceleration, were unaffected by hypercapnia. Similarly, hypercapnia had no effect on lusitropic indexes reflected by its lack of effect on isovolumic relaxation time, mitral E-wave deceleration time, and mitral E/A wave ratio. Hypercapnia was found to significantly increase both QTc interval and QT dispersion: 428 +/- 8 vs 411 +/- 3 ms and 48 +/- 2 vs 33 +/- 4 ms, respectively. There was no significant effect of hypercapnia on PRA, ANG II, or ALDO. CONCLUSION: Thus, acute hypercapnia appears to have no adverse inotropic or lusitropic effects on cardiac function, although repolarization abnormalities, reflected by an increase in QT dispersion, and its effects on pulmonary vasoconstriction may have important sequelae in man. PMID- 8625672 TI - Bilateral and unilateral use of internal thoracic artery for myocardial revascularization. Comparison of extubation outcome and duration of hospital stay. AB - The left internal thoracic artery is usually used as arterial conduit for myocardial revascularization; however, there is an increasing popularity of bilateral use of this artery for grafting. We examined 180 patients with both types of arterial conduits to answer whether bilateral use of the internal thoracic artery makes the difference in postoperative extubation outcome and duration of hospital stay in comparison to the unilateral use of this conduit. Ninety-three patients with bilateral conduit and 87 patients with unilateral conduit, with comparable age, cardiopulmonary bypass, and aortic cross-clamp time have been studied. On the basis of statistical and retrospective analysis, we conclude that bilateral use of internal thoracic artery for myocardial revascularization prolongs required postoperative respiratory support (12.0 h against 7.6 h) not affecting the duration of the mean hospital stay. PMID- 8625671 TI - Preoperative prediction of postoperative respiratory outcome. Coronary artery bypass grafting. AB - OBJECTIVE: The hypothesis that traditionally defined preoperative risk factors predict prolonged mechanical ventilation after coronary artery bypass graft surgery (CABG) was tested in our cohort. The predictive power of these factors was quantified, and specific patient subsets destined for prolonged mechanical ventilation after CABG surgery were defined. DESIGN: Five hundred thirteen consecutive patients undergoing CABG were prospectively evaluated. Preoperative pulmonary evaluation included clinical historic data, standard spirometry, and arterial blood gas. Preoperative cardiac parameters included clinical parameters and left ventricular function assessment. Nonthoracic organ (renal, endocrine, pancreas, liver) function was assessed. SETTING: University-based, tertiary referral center. INTERVENTIONS: None (observational only). OUTCOMES MEASURED: Duration of mechanical ventilation, duration of surgical ICU stay, and mortality. RESULTS: Multivariate regression analyses revealed that for the patient undergoing routine elective surgery and the patient undergoing urgent surgery, prolonged mechanical ventilation and death were rare events (8.3% and 2.0%, respectively). The combination of reduced left ventricular ejection fraction and the presence of selected preexisting comorbid conditions (clinical congestive heart failure, angina, current smoking, diabetes) served as modest risk factors for prolonged mechanical ventilation; their absence strongly predicted an uncomplicated postoperative respiratory course. No pulmonary diagnosis, mechanical lung function, or blood gas parameter substantially contributed to predicting adverse outcome. Classification and regression tree subgroup analysis refined specific factors important in specific subgroups. CONCLUSION: With the exception of left ventricular ejection fraction, no preoperative factors emerge as good predictors across all subgroups. This series suggests that pulmonary diagnosis, lung mechanics, and blood gas parameters do not offer the clinician global rules in predicting postoperative respiratory outcome, nor should they be used as exclusion crteria for CABG surgery. PMID- 8625673 TI - Thoracoscopic management of malignant pleural effusions. AB - Malignant pleural effusion is a common condition and often presents a challenge for treatment. We report our experience from a single institution with the use of video-assisted thoracoscopic surgery (VATS) in the management of malignant effusions. From September 1992 to April 1995, 69 patients (31 men, 38 women; age range, 38 to 76 years) underwent diagnosis and/or treatment of malignant effusions; these included 46 pleural biopsies, 34 talc insufflations, and 16 limited decortications. There was no mortality and there were no intraoperative complications. Postoperative complications occurred in seven patients (10%). Specific histologic diagnoses were obtained in all but 6 patients (87%). Malignant effusion was confirmed in 25 of 46 cases (54%). Thoracoscopic talc insufflation with or without additional decortication was successful in 32 of 34 cases (94%) in controlling recurrence of effusion after a mean follow-up of 6 months among the survivors (22 patients died during the follow-up period without effusion reaccumulation). We conclude that VATS not only provides an accurate diagnosis but also allows effective therapeutic procedures to be performed for malignant effusions that are associated with an acceptable morbidity. PMID- 8625674 TI - Treatment of malignant mesothelioma with methotrexate and vinblastine, with or without platinum chemotherapy. AB - STUDY OBJECTIVE: To determine the efficacy of methotrexate, vinblastine, and platinum chemotherapy in patients with diffuse unresectable malignant mesothelioma. DESIGN: Patients with histologically confirmed malignant mesothelioma were evaluated for treatment with methotrexate, vinblastine, and cisplatin chemotherapy. If the patient had preexisting hearing loss or neuropathy, or was significantly disabled (eg, spending greater than half of the day in bed or a chair), cisplatin therapy was withheld. SETTING: All patients were initially evaluated at the University of Washington Medical Center and received chemotherapy at the University of Washington or in the community. INTERVENTIONS: Between 1990 and 1994, 17 patients received this chemotherapy. Ten patients received cisplatin, 100 mg/m2 IV on day 1, methotrexate, 30 mg/m2 IV on days 8, 15, and 22, and vinblastine, 3 mg/m2 IV on days 8, 15, and 22, in 28-day cycles. One patient had carboplatin substituted for cisplatin due to preexisting hearing loss. Six patients received weekly methotrexate and vinblastine at the same doses without platinum. MEASUREMENTS AND RESULTS: Nine of the 17 (53%; 95% confidence interval [CI], 28 to 77%) patients responded, including two complete remissions, two partial remissions, and five regressions. Median time to progression is 8 months. The median survival time for all patients is 14 months. Projected 2-year survival is 35% (95% CI, 12 to 60%). CONCLUSIONS: Although the number of the patients in this study is small, the response rate and projected 2 year survival of 35% are better than those typically reported for unresectable malignant mesothelioma. Further investigation is warranted in confirmatory trials. PMID- 8625675 TI - Legionella species community-acquired pneumonia. A review of 56 hospitalized adult patients. AB - BACKGROUND: In a prospective study, Legionella species (Lsp) was identified as the causative agent in 56 (16.2%) of 346 adult patients hospitalized over the course of 1 year with community-acquired pneumonia (CAP), in the Soroka Medical Center, Beer-Sheva, Israel. OBJECTIVE: To characterize patients with Lsp CAP in our study. METHODS: The diagnosis of infection with Lsp was based on serologic testing of antibodies using the indirect immunofluorescent method. RESULTS: In 35 (62.5%) of the patients, at least one other etiologic agent for CAP was identified in addition to Lsp. The patient population was relatively young, with relatively low rates of chronic comorbidity, and a broad spectrum of disease severity compared with previously published studies. No single epidemiologic, clinical, laboratory, or radiographic characteristic differentiated between Lsp CAP and other CAP patients in our study. Three patients (5.4%) who were not treated with erythromycin died. However, in contrast, nine patients who were treated with beta-lactam antibiotics recovered completely. CONCLUSIONS: Lsp is a common cause of CAP in our region, usually as a coinfection with another causative agent. Lsp CAP, which cannot be characterized on the basis of clinical, routine laboratory, or radiographic data, requires specific microbiologic or serologic diagnosis. Treatment with erythromycin appears to be important to reduce mortality from this disease, but in a significant number of patients, the disease may be self-limited. PMID- 8625676 TI - Aerosolized pentamidine as alternative primary prophylaxis against Pneumocystis carinii pneumonia in adult hepatic and renal transplant recipients. AB - STUDY OBJECTIVE: To examine the safety and efficacy of aerosolized pentamidine (AP) as alternative primary prophylaxis against Pneumocystis carinii pneumonia (PCP) in adult liver and kidney transplant recipients. DESIGN: Retrospective review of medical records. SETTING: Tertiary care urban teaching hospital with active liver and kidney transplant programs. PATIENTS: Adult liver and kidney transplant recipients intolerant of trimethoprim-sulfamethoxazole (TMP-SMX) therapy and referred to the AP clinic between June 1991 and December 1994. INTERVENTIONS: Each patient received monthly AP, 300 mg, delivered by a nebulizer (Respirgard-II), preceded by inhaled albuterol, 180 micrograms. During the period of follow-up, information related to side effects of AP and incidence of PCP was recorded. RESULTS: A total of 35 patients were identified, 18 liver and 17 kidney transplant recipients. Fourteen patients received AP as initial prophylaxis because of prior sensitivity to TMP-SMX. In another 19 patients, initial TMP-SMX therapy was discontinued for leukopenia (5), elevated liver function test values (4), rash (3), nausea (2), renal failure (2), seizure (2), and thrombocytopenia (1). In addition, two patients received AP in the setting of organ rejection. Liver transplant recipients received AP for an average of 4.28 +/- 1.6 months, and renal transplant recipients received AP for an average of 5.71 +/- 4.3 months. Adverse effects of AP included bronchospasm (two), dyspnea (one), cough (one), and nausea (one). AP therapy was discontinued in only one patient due to severe bronchospasm. There were no cases of PCP in the 35 patients receiving AP. CONCLUSIONS: These observations suggest that AP is well tolerated and may be an effective alternative for PCP prophylaxis in adult liver and kidney transplant recipients intolerant to TMP-SMX therapy. PMID- 8625677 TI - Evaluation of heliox in children hospitalized with acute severe asthma. A randomized crossover trial. AB - STUDY OBJECTIVE: To determine whether breathing a blend of 70% helium:30% oxygen (heliox) would improve pulmonary function, decrease clinical score, and improve the sensation of dyspnea in children hospitalized with acute severe asthma. DESIGN: Prospective, randomized, double-blind, crossover study. SETTING: The inpatient pediatric service of a military, tertiary care, teaching hospital. PATIENTS: Children 5 to 18 years who required hospital admission for treatment of acute asthma. INTERVENTIONS: All patients received 5 mg of nebulized albuterol every 1 to 4 h, with a dose given within 30 min of the start of the study, and IV administered methylprednisolone. Patients breathed heliox and a 30% oxygen enriched air mixture for 15 min each in random order. MEASUREMENTS AND RESULTS: Clinical score, dyspnea score, oxygen saturation, heart rate, and respiratory rate, followed by FVC, FEV1, peak expiratory flow rate (PEFR), and, mean midexpiratory flow rate (FEF25-75) were obtained at study entry, 15 min after breathing the first gas mixture (heliox or air per randomization), 15 min after breathing the second mixture, and again 15 min after stopping the second gas mixture (study end values). Eleven children were enrolled, and all completed the study. There were no significant differences between study entry and study end spirometric values. Using the paired t test, we found no significant differences between mean values (SD) of FEV1 and FVC obtained while breathing heliox vs air; FEV1-heliox, 53% (18%) of the predicted value; FEV1-air, 52% (16%) of the predicted value (p = 0.36); FVC-heliox, 69% (22%) of the predicted value; and FVC air, 70% (21%) of the predicted value (p = 0.50). The differences in values for PEFSR and FEF25-75 while breathing heliox vs air were small but did reach statistical significance in favor of heliox: PEFR-heliox, 56% (20%) of the predicted value; PEFR-air, 50% (16%) of the predicted value (p = 0.04); FEF25-75 heliox, 32% (13%) of the predicted value; and FEF25-75-heliox, 29% (11%) of the predicted value (p = 0.006). Heliox had no effect on either clinical or dyspnea scores. CONCLUSION: The short-term inhalation of heliox did not benefit this group of children hospitalized with acute, severe asthma. PMID- 8625678 TI - Habitual coughing and its associations with asthma, anxiety, and gastroesophageal reflux. AB - STUDY OBJECTIVE: Coughing was studied in relation to different disorders and objective variables indicative of airway inflammation. SETTING: A random sample of 800 persons, aged 20 to 44 years, was chosen from a larger cohort of participants in the European Community Respiratory Health Survey in Uppsala Sweden; of these, 623 participated. This sample was enriched with 201 individuals who reported asthma-related symptoms or the use of asthma medication. METHODS: The study comprised a structured interview, including questions about habitual (productive and nonproductive) and nocturnal coughing and spirometry, methacholine challenge, peak flow diary, skin prick tests, and measurements of blood eosinophil count and serum eosinophil cationic protein (S-ECP). RESULTS: A significant positive correlation was found between productive coughing and asthma (adjusted odds ratios [OR] = 2.0), allergic rhinitis (OR = 1.9), gastroesophageal reflux (OR = 4.4), smoking (OR = 1.9), and anxiety (OR = 1.8), while nonproductive coughing was related to female gender (OR = 1.8) and anxiety (OR = 1.7). Nocturnal coughing was positively correlated to female gender (OR = 1.8), smoking (OR = 1.9), and asthma (OR = 2.2). Bronchial hyperresponsiveness was positively related to productive coughing (p < 0.001), nonproductive coughing, and nocturnal coughing (p < 0.05). S-ECP was significantly higher in individuals with nonproductive coughing compared with subjects without habitual coughing (p < 0.01). CONCLUSIONS: We conclude that habitual coughing has a significant association with different disease categories. PMID- 8625679 TI - A randomized crossover study of an oral appliance vs nasal-continuous positive airway pressure in the treatment of mild-moderate obstructive sleep apnea. AB - STUDY OBJECTIVE: To compare efficacy, side effects, patient compliance, and preference between oral appliance (OA) therapy and nasal-continuous positive airway pressure (N-CPAP) therapy. DESIGN: Randomized, prospective, crossover study. SETTING: University hospital and tertiary sleep referral center. PATIENTS: Twenty-seven unselected patients with mild-moderate obstructive sleep apnea (OSA). INTERVENTIONS: There was a 2-week wash-in and a 2-week wash-out period, and 2 x 4-month treatment periods (OA and N-CPAP). Efficacy, side effects, compliance, and preference were evaluated by a questionnaire and home sleep monitoring. MEASUREMENTS AND RESULTS: Two patients dropped out early in the study and treatment results are presented on the remaining 25 patients. The apnea/hypopnea index was lower with N-CPAP (3.5 +/- 1.6) (mean +/- SD) than with the OA (9.7 +/- 7.3) (p < 0.05). Twelve of the 25 patients who used the OA (48%) were treatment successes (reduction of apnea/hypopnea to <10/h and relief of symptoms), 6 (24%) were compliance failures (unable or unwilling to use the treatment), and 7 (28%) were treatment failures (failure to reduce apnea/hypopnea index to <10/h and/or failure to relieve symptoms). Four people refused to use N CPAP after using the OA. Thirteen of the 21 patients who used N-CPAP were overall treatment successes (62%), 8 were compliance failures (38%), and there were no treatment failures. Side effects were more common and the patients were less satisfied with N-CPAP (p < 0.005). Seven patients were treatment successes with both treatments, six of these patients preferred OA, and one preferred N-CPAP as a long-term treatment. CONCLUSIONS: We conclude that OA is an effective treatment in some patients with mild-moderate OSA and is associated with fewer side effects and greater patient satisfaction than N-CPAP. PMID- 8625681 TI - Averaged and time-gated spectral analysis of respiratory sounds. Repeatability of spectral parameters in healthy men and in patients with fibrosing alveolitis. AB - STUDY OBJECTIVE: To obtain a basis for assessment of changes in breath sound spectra in patients with pulmonary diseases, short-term and day-to-day repeatability of spectral parameters was studied. DESIGN: Breath sounds were recorded simultaneously from the trachea and from the chest twice at an interval of 15 min (short-term repeatability) and of 1 to 3 days (day-to-day repeatability). During recordings, air flow at the mouth was controlled, the target inspiratory and expiratory peak flow being 1.25 L/s. Inspiratory and expiratory breath sound spectra were averaged over 7 to 10 successive respiratory cycles. The repeatability of sound intensity (RMS), frequency of maximum intensity (Fmax), and median frequency (F50) was analyzed with analysis of variance. PARTICIPANTS: Short-term repeatability was studied in 10 healthy nonsmoking men (age 25 to 44 years), and day-to-day repeatability was studied in 10 healthy nonsmoking men (age 23 to 41 years) and in 12 patients with clinically stable fibrosing alveolitis (age 35 to 82 years). RESULTS: Short-term coefficient of variation (CoV) of Fmax and F50 was 2.6 to 6.7% when recorded from the chest, and 6.2 to 8.7% when recorded from the trachea. Day-to-day CoV of Fmax and F50 in healthy subjects was 4.7 to 8.5% and 5.0 to 8.7% recorded from the chest or from the trachea, respectively. Inspiratory day-to-day variation in those parameters was higher in patients with fibrosing alveolitis. CoV of RMS was high, ranging from 18 to 47% in different subject groups and sampling situations. CONCLUSIONS: Repeatability of F50 of averaged flow-controlled lung sound spectra is good both in healthy subjects and in patients with fibrosing alveolitis. Thus, F50 of respiratory sound spectra may be useful in monitoring of changes induced by respiratory diseases and interventions. These results emphasize the importance of standardization of recording conditions and of analyzing techniques. PMID- 8625680 TI - Serum KL-6 for the evaluation of active pneumonitis in pulmonary sarcoidosis. AB - KL-6, a mucinous high-molecular weight glycoprotein, expressed on type II pneumonocytes, is elevated in the serum of patients with active interstitial pneumonia. Forty-seven patients with histologically confirmed sarcoidosis were analyzed by chest radiography, CT, 67Ga scintigraphy, BAL fluid (BALF), and serum KL-6. Serum KL-6 level was significantly elevated in radiographic type II (945 +/ 725 U/mL; n = 13; p < 0.01) and type III (1,179 +/- 1,320 U/mL; n = 9; p < 0.01), as compared with type 0 (333 +/- 173 U/mL; n = 10) and type I (430 +/- 225 U/mL; n = 15). Serum KL-6 level was significantly elevated in patients with CT findings of irregular small opacities, ground-glass opacities, and thickened bronchovascular bundle (p < 0.01), as compared to those without these findings. Serum KL-6 level was significantly elevated in patients with positive pulmonary accumulation in 67Ga scintigraphy (1,108 +/- 1,044; n = 20; p < 0.001) as compared to those without accumulation (390 +/- 206; n = 27). Serum KL-6 level was elevated in patients with a higher CD4+/CD8+ ratio (> or =3) in BALF, as compared to those with lower ratios. These results suggest that serum KL-6 is a useful marker of sarcoidosis activity. PMID- 8625684 TI - Volemic status influences the response of plasma atrial natriuretic factor to positive airway pressure. AB - STUDY OBJECTIVE; To evaluate interactive effects of volemic status and positive end-expiratory pressure (PEEP) on the plasma levels of atrial natriuretic factor (ANF) in assist-controlled mechanical ventilation (MV). DESIGN: Three successive protocols applied in randomized order to each participant. SETTING: Clinical investigation laboratory. PARTICIPANTS: Twenty-one young, healthy adults. INTERVENTIONS: The three protocols were as follows: (1) MV+PEEP, normovolemia; (2) MV+PEEP, hypervolemia; and (3) spontaneous breathing (SB), hypervolemia. In protocols 1 and 2, a preliminary period of SB lasting 2 h was followed by MV alone (0.5 h), MV+20 cm H2O PEEP (1 h), and a recovery period of SB (1.5 h). Hypervolemia was induced by the continuous i.v. infusion of 3 L of 0.9% NaCl in 5 h (protocols 2 and 3). MEASUREMENTS AND RESULTS: Heart rate, BP, and the plasma levels of immunoreactive ANF and catecholamines were measured serially. During hypervolemia, ANF significantly decreased when PEEP was added to MV (protocol 2: from 31.1 +/- 2.7 to 20.7 +/- 1.5 fmol/mL; p < 0.01). This did not occur in normovolemia (protocol 1: from 20.0 +/- to 16.7 +/- 1.2 fmol/mL; p = NS). The different effects of MV+PEEP in normovolemia and hypervolemia were not related to differences in circulating catecholamine levels. CONCLUSIONS: These results demonstrate for the first time (to our knowledge) that volemic status modulates the response of plasma ANF to PEEP in humans. The role of ANF in the water and salt retention induced by MV with PEEP might be limited to hypervolemic conditions. PMID- 8625683 TI - Hemodynamic effects of i.v. milrinone lactate in pediatric patients with septic shock. A prospective, double-blinded, randomized, placebo-controlled, interventional study. AB - STUDY OBJECTIVE: To determine the hemodynamic effects of i.v. milrinone lactate in pediatric patients with nonhyperdynamic septic shock. Specifically we tested the hypothesis that i.v. milrinone would increase cardiac index by 20% and decrease systemic vascular resistance index by 20% during a 2-h study period. DESIGN: Prospective, double-blinded, randomized, placebo-controlled, descriptive, interventional study. SETTING: Twenty-six-bed pediatric ICU at Children's Medical Center of Dallas and a 10-bed pediatric trauma ICU at Parkland Memorial Hospital. PATIENTS/PARTICIPANTS: Twelve patients (age range, 9 months to 15 years) with nonhyperdynamic septic shock despite administration of catecholamines (cardiac index [CI] normal [3.5 to 5.5 L/min/m2] or low [< or =3.5 L/min/m2]; systemic vascular resistance index [SVRI] normal [800 to 1,600 dyne.s.cm5/m2] or high [> or =1,600 dyne.s.cm5/m2]; and pulmonary capillary wedge pressure [PCWP] normal [8 to 12 mm Hg] or higher) with clinical signs of poor perfusion were enrolled, randomized, and treated in a blinded fashion with i.v. milrinone and placebo. INTERVENTIONS: Patients were randomized into two groups. Group A received a loading dose of 50 micrograms/kg i.v. of milrinone followed by a continuous i.v. infusion of 0.5 microgram/kg/min while group B received an equal volume loading dose and continuous infusion of placebo. After 2 h, group A received an equal volume loading dose followed by a continuous infusion of placebo while the milrinone infusion continued, while group B received a 50 micrograms/kg loading dose of milrinone followed by a continuous infusion of 0.5 microgram/kg/min while the placebo infusion remained. Outcome variable were measured at baseline, 0.5, 1.0, 2.0, 2.5, 3.0, and 4.0 h. Echocardiographic measurements were taken at baseline, hour 2, and hour 4 in all subjects. No changes in other inotropic or mechanical ventilatory support were allowed during the study period. MEASUREMENTS AND MAIN RESULTS: Milrinone significantly increased CI, stroke volume index (SVI), right and left ventricular stroke work index, and oxygen delivery (Do2) at 0.5, 1.0, and 2.0 h postloading dose (p < 0.05) while significantly decreasing SVRI, pulmonary vascular resistance index, and mean pulmonary arterial pressure at 0.5, 1.0, and 2.0 h postloading dose (p < 0.05). No clinically or statistically significant changes in heart rate, systolic and diastolic BP, mean systemic arterial pressure, or PCWP were observed during milrinone treatment compared to placebo. CONCLUSIONS: CI, SVI, and Do2 significantly increased while SVRI significantly decreased when compared to placebo after i.v. administration of milrinone to pediatric patients with nonhyperdynamic septic shock. No adverse effects were observed. In a volume-resuscitated pediatric patient with septic shock, when administered in addition to catecholamines, milrinone will improve cardiovascular function. PMID- 8625685 TI - Mechanism of relief of tachypnea during pressure support ventilation. AB - Pressure support ventilation (PSV) provides a range of ventilatory support from partial respiratory muscle unloading, where inspiratory work is shared between the patient and the mechanical ventilator, to total respiratory muscle unloading, where inspiratory work is performed solely by the ventilator. This study is designed to determine if minimizing work fully accounts for relief of tachypnea during PSV. We examined respiratory parameters over a range of PSV that includes the crossover from partial to total respiratory muscle unloading. Eight studies were obtained on seven intubated patients in respiratory failure. Ventilation, occlusion pressure (P0.1), and patient inspiratory work (WOBinsp) were measured while PSV was varied. In all patients, WOBinsp decreased as PSV increased. The level of PSV where WOBinsp was minimized was identified; this marked the crossover from partial to total respiratory muscle unloading. Frequency decreased with increasing PSV but remained elevated (range, 22 to 38 breaths/min) at the crossover. Frequency was normalized only at PSV levels 131 to 193% of the levels of pressure at the crossover. Tidal volume (VT) changed little during partial support and averaged 5.9 mL/kg at the crossover. VT increased only on PSV providing total unloading. Six of seven patients exhibited increasing static compliance with increasing VT suggesting alveolar recruitment. P0.1 tracked WOBinsp over the entire range of PSV (r = 0.95, p < 0.001). The normalization of frequency observed above the crossover coincided with increasing VT rather than decreasing work. These observations suggest that reflexes resulting from increased VT and/or alveolar recruitment may have contributed to the normalization of frequency. PMID- 8625686 TI - Local increase in polymorphonuclear leukocyte elastase is associated with tumor invasiveness in non-small cell lung cancer. AB - The production of tumor cell proteases is implicated in tumor cell invasion and metastasis. To determine whether lung cancer cells can produce polymorphonuclear leukocyte elastase (PMN-E), we measured the concentration of immunoreactive (ir) PMN-E in the conditioned medium of seven lines of non-small cell lung cancer cells, EBC-1, LC-1sq, LK-2, A-549, PC-3, RERF-LC-MS, and RERF-LC-OK, and three normal lung epithelial cell lines, CCD-8Lu, WI-1003, and LL-24, by using a recently developed enzyme immunoassay (EIA). We measured the concentration of ir PMN-E in extracts of 40 non-small cell lung cancers, and evaluated its association with the clinicopathologic findings in these patients. The ir-PMN-E level in the culture medium increased with time in six of the seven lines of lung cancer cells; the exception was PC-3. No detectable ir-PMN-E was secreted into the culture medium of the three lines of normal lung epithelial cells. The ir-PMN E was detected in tissue extracts from 34 to 40 specimens at concentrations ranging from 0.11 to 15.5 micrograms/100 mg of protein. When 40 specimens of lung cancer were categorized by clinical stage of disease, the ir-PMN-E concentration was significantly higher in stage IIIB vs stages I, II, or IIIA. Similarly, the ir-PMN-E concentration was significantly higher in stage IIIA than in stage I. Evaluation of correlations between the ir-PMN-E concentration and patient characteristics showed that the ir-PMN-E level was significantly higher in T3 and T4 tumors than in T1 or T2 tumors. Analysis of prognostic factors in a group of 101 patients with non-small cell lung cancer demonstrated that those with high ir PMN-E had a significantly shorter overall survival vs those with a low ir-PMN-E at the cutoff point of 3.5 micrograms/100 mg of protein. Multivariate analysis showed that ir-PMN-E was a significant porgnostic factor for early death (hazard ratio, 4.04; 95% confidence interval, 1.65 to 9.95) (p = 0.005), suggesting it was an independent marker for prognosis. Results suggest that the local production of PMN-E may be involved in the tumor invasion associated with a poor prognosis in patients with non-small cell lung cancer. PMID- 8625682 TI - Pharmacodynamics and pharmacokinetics of milrinone administration to increase oxygen delivery in critically ill patients. AB - OBJECTIVES: The positive inotropic and vasodilator actions of phosphodiesterase (PDE) inhibitor drugs may offer therapeutic alternatives to beta-agonists in critically ill patients. We hypothesized that milrinone administration would increase cardiac index (CI) and oxygen delivery (Do2) in ICU patients, and that a pharmacokinetic model previously developed in cardiac surgery patients may be used to predict milrinone plasma concentrations in a medical-surgical ICU population. SETTING: ICU in two tertiary-care, university medical centers. DESIGN AND INTERVENTIONS: A prospective, open-label, multicenter, dose-escalating study in three successive groups of eight ICU patients who received a 10-min loading dose of milrinone (25 micrograms/kg [LOW], 50 micrograms/kg [MED], and 75 micrograms/kg [HIGH]). In addition, all patients then received a milrinone infusion of 0.5 microgram/kg/min for 1 h. MEASUREMENTS: Hemodynamic measurements included heart rate (HR); mean arterial, pulmonary artery, central venous, and pulmonary artery occlusion pressures; and thermodilution cardiac output. Oxygen transport indexes included arterial and venous blood oxygen tensions to determine Do2 and oxygen consumption (Vo2). Data were analyzed by univariate repeated measures analysis of covariance, with baseline values utilized as covariate regressors. RESULTS: Twenty-four adult ICU patients 20 to 84 years of age completed the study. The three groups did not differ, except that the patients in the MED group were significantly older (67 +/- 4 years, mean +/- SEM) compared with either the patients in the LOW (48 +/- 7 years) or HIGH (47 +/- 6 years) group. While HR did not change in the LOW group (90 +/- 4 to 93 +/- 3 beats/min), HR increased significantly in the HIGH group (94 +/- 5 to 112 +/- 8 beats/min) (baseline to 60 min infusion time points). All milrinone doses increased both CI and Do2. At the end of the 10-min loading dose, CI increased 0.3 L/min/m2 in the LOW group, 1.1 L/min/m2 in the MED group, and 0.9 L/min/m2 in the HIGH group. Do2 increased 8% in the LOW group, 33% in the MED group, and 23% in the HIGH group, similar to the changes in CI. Mixed venous oxygen saturation increased 3 to 5% during the 10-min loading dose of milrinone. During this same time period, mean arterial pressure decreased 6 to 16% and pulmonary artery pressures decreased 9 to 15%. Peak plasma milrinone concentrations increased as a function of the loading dose (159 +/- 9 ng/mL in the LOW group, 302 +/- 33 ng/ml in the MED group, and 411 +/- 45 ng/mL in the HIGH group). However, milrinone concentrations were similar in all three groups after the 1-h infusion; 113 +/- 14 ng/ml (LOW), 147 +/- 22 ng/mL (MED), and 119 +/- 14 ng/ml (HIGH). In all patients with final plasma milrinone concentrations greater than 100 ng/mL (15/23), the CI increased by at least 0.4 L/min/m2 (range, 0.4 to 1.8 L/min/m2). CONCLUSIONS: Our study confirms that a milrinone loading dose of 50 micrograms/kg/min followed by an infusion of 0.5 microgram/kg/min achieves adequate plasma concentrations of 100 ng/mL or greater, which significantly increases both CI and Do2. In addition, a previously established pharmacokinetic model of milrinone disposition is confirmed in this mixed ICU population. PMID- 8625687 TI - Evaluation of a new thin-walled endotracheal tube for use in children. AB - Conventional endotracheal tubes have high intrinsic resistive properties due to their high outer-to-inner diameter ratio. This has significant disadvantages in the treatment of the small neonatal or pediatric patient as work of breathing increases with decreasing internal radius. Diagnostic and therapeutic procedures, including suctioning, may be very difficult in patients with small endotracheal tubes. We therefore measured airway resistance and pressure differential during simulated mechanical ventilation using proximal and distal endotracheal tube flow transducers. Conventional and new, ultrathin-walled endotracheal tubes reinforced with flat stainless steel or a novel, crush-proof nickel-titanium alloy were compared using fixed ventilator settings. Ventilation through the ultrathin walled tubes resulted in a significantly reduced airway resistance (p < or = 0.01). These new ultrathin-walled endotracheal tubes showed flow characteristics typical of much larger conventional endotracheal tubes: the 3.2-mm internal diameter had an airway resistance (Raw) of 36, while a standard 2.5-mm internal diameter endotracheal tube had a Raw of 146. Both endotracheal tubes have identical external diameters of 3.6 mm. We conclude that ultrathin-walled endotracheal tubes could have a significant role in the treatment of the ventilated child by facilitating interactive ventilation and maintenance of airway patency and may make procedures such as fiberoptic endoscopy and intrapulmonary ventilation using reverse-thrust catheters possible in the small child. PMID- 8625689 TI - Treatment of obstructive sleep apnea. A review. AB - Treatment of obstructive sleep apnea (OSA) has developed over the last 25 years from tracheostomy to a variety of options, including weight loss, nasal continuous positive airway pressure (N-CPAP), pharyngeal surgery, and medications. None of these options is definitive or curative, except possibly weight loss. The most widely prescribed treatment is N-CPAP, but recently published studies using objective measurement of patient compliance show less than ideal compliance. Attempts have been made to design pharyngeal surgery according to the site of upper airway collapse or narrowing, as identified by various techniques in wakefulness. How representative these studies are of upper airway physiology in sleep is questionable. Recent studies have shown improved surgical success in correcting OSA. However, disturbing data are available in a limited number of patients that demonstrate worsening of the OSA months after a favorable response to surgery. More studies assessing the long-term outcome of pharyngeal surgery are needed. Several pharmacologic agents have been used to treat OSA. Results with any particular agent are not better than with N-CPAP or surgery. However, studies of subgroups of patients with OSA in which a particular pharmacologic agent may be specifically indicated, such as thyroxine in hypothyroidism, have not been conducted (to our knowledge). An algorithm for the approach to treatment recommendations is presented. Basic to this algorithm is an objective presentation of therapeutic options to the patient with OSA and a respect for the patient's preferences. PMID- 8625688 TI - Effects of steroids on the lung accumulation of neutrophil and monocyte in rabbits with endotoxemia. AB - STUDY OBJECTIVE: To determine the effects of steroids on the lung accumulation of polymorphonuclear leukocytes (PMNs) and monocytes in rabbits with endotoxemia. DESIGN: A prospective, randomized, controlled animal trial. SETTING: Surgical research laboratory, Keio University School of Medicine. SUBJECTS: Twenty-four female Japanese white rabbits. INTERVENTIONS: Rabbits with endotoxemia were pretreated with steroids sufficient to inhibit the production of tumor necrosis factor (TNF) and to prevent the fall of BP. MEASUREMENTS AND RESULTS: The circulating leukocyte counts and the leukocyte accumulation in the lung were evaluated. Endotoxin caused a rapid decrease in circulating PMNs and monocytes followed by an 8-fold greater accumulation of PMNs (p < 0.001) and a 6.5-fold greater accumulation of monocytes (p < 0.05). Steroids failed to inhibit this initial drop of PMNs and monocytes. However, steroids inhibited the PMN accumulation in the lung by 50% (p < 0.05), without inhibiting the monocyte accumulation in the lung. CONCLUSIONS: These findings suggest that the lung accumulation of PMNs is dependent on TNF or other inflammatory mediators that are inhibited by steroids while the lung accumulation of monocyte is not. PMID- 8625690 TI - Cost-effectiveness in clinical cardiology. Part 1: Coronary artery disease and congestive heart failure. PMID- 8625691 TI - Bronchoprovocation tests in the diagnosis of isocyanate-induced asthma. AB - Over the past 25 years, investigators have continued to improve on the approach to providing nonirritant exposures for the accurate diagnosis of isocyanate induced asthma. Although the technology used in testing has become more sophisticated and may be fairly considered the domain of the bioengineer, the chemist, and the industrial hygienist, the requirements of the physician have remained unchanged. The physician must observe the level of exposure closely and monitor the worker's symptoms and lung function. Direct physician involvement in the testing procedure remains critical to the worker's safety and for the accurate diagnosis of isocyanate-induced asthma. PMID- 8625692 TI - Pneumonia with an enlarged cardiac silhouette. PMID- 8625693 TI - A chest radiograph showing abnormal mediastinal contour. PMID- 8625694 TI - Nonproductive cough, dyspnea, malaise, and night sweats in a 47-year-old woman. PMID- 8625695 TI - Pulmonary veno-occlusive disease in an adult following bone marrow transplantation. Case report and review of the literature. AB - Pulmonary veno-occlusive disease (PVOD) was diagnosed in an adult following chemotherapy and bone marrow transplantation (BMT) for acute lymphoblastic leukemia. A medical literature review showed only three previous reports of PVOD following BMT occurring in children but no prior cases in adults. PMID- 8625696 TI - Twiddler's syndrome complicating a transvenous defibrillator lead system. AB - Twiddler's syndrome is a rare complication seen in patients with implanted pacemakers or defibrillators. The condition typically presents with device malfunction and occurs when the patient either consciously or unconsciously twists and rotates the implanted device in its pocket, resulting in torsion and dislodgement of the implanted lead. A case of twiddler's syndrome involving a transvenous defibrillation lead and an abdominally implanted defibrillator is described. This is the first report of this complication with this particular lead. The patient in this report was a middle-aged obese diabetic woman who presented 7 months after defibrillator implantation with device noncapture and intermittent nonsensing. Review of the literature reveals that the majority of patients with this complication are middle-aged obese women with a defibrillator pocket that exceeds the size of the defibrillator. Treatment measures are discussed both for the patient with this complication and for the patient at increased risk for its occurrence. PMID- 8625697 TI - Pheochromocytoma associated with clinical and echocardiographic features simulating hypertrophic obstructive cardiomyopathy. AB - Left ventricular hypertrophy simulating hypertrophic obstructive cardiomyopathy is a rare complication of pheochromocytoma. In this report, two cases of pheochromocytoma with this complication are described. Successful tumor removal in both cases led to relief of symptoms, normalization of BP, regression of abnormal clinical features, normalization of the ECGs, but only partial regression of the echocardiographic features despite prolonged follow-up of 24 and 32 months, respectively. PMID- 8625698 TI - Azathioprine as a steroid-sparing agent in radiation pneumonitis. AB - Radiation therapy is commonly used for treatment of neoplastic disease involving the thorax. Treatment complications include radiation pneumonitis that may require therapy with corticosteroids which possess significant side effects. We report the use of azathioprine as a steroid-sparing agent in a patient with severe radiation pneumonitis and steroid-induced myopathy. PMID- 8625699 TI - Atrial myxoma mimicking systemic disease. Profile modified by flurbiprofen administration. AB - A patient had a left atrial myxoma which was modified by flurbiprofen administration. The diagnosis was made 42 months after the first symptoms appeared. Flurbiprofen may have reduced interleukin-6 secretion by the tumor, leading to a delayed diagnosis. PMID- 8625700 TI - Unusual complications after embolization of a pulmonary arteriovenous malformation. AB - A pulmonary arteriovenous malformation was embolized in a patient with hereditary hemorrhagic telangiectasia. Several unusual complications, including early deflation of a detachable balloon, migration of a coil, and development of severe pulmonary hypertension, occurred. Pulmonary hypertension was attributed to a coexistent left-to-right shunt caused by a large hepatic arteriovenous malformation. PMID- 8625701 TI - Descending necrotizing mediastinitis causing pleuroesophageal fistula. Successful treatment by combined transcervical and pleural drainage. AB - Descending necrotizing mediastinitis (DNM) develops as a complication of an oropharyngeal infection and can be life-threatening. Aggressive therapy is generally advised; usually, treatment consists of cervicomediastinal and transthoracic drainage combined with broad-spectrum antimicrobial therapy, especially when the necrotizing process extends below the level of the fourth thoracic vertebra. A rare case of DNM secondary to a retropharyngeal abscess with fistula to both pleural cavities and to the hypopharynx is reported. The patient was successfully treated by cervicomediastinal surgical drainage and percutaneous drainage of both pleural cavities. In our opinion, even complicated DNM can be treated without aggressive surgery if the patient is in good condition. PMID- 8625702 TI - Evaluating work of breathing measurements in weaning. PMID- 8625703 TI - Lung health in sawmill workers exposed to pine and spruce. PMID- 8625704 TI - Smear negative, culture positive AFB bronchial washings. Infection control implications. PMID- 8625705 TI - That's no lady. PMID- 8625706 TI - Magnesium saga. PMID- 8625707 TI - Isoniazid safety. PMID- 8625708 TI - Relationship between the 6-min walk test and maximal oxygen consumption. PMID- 8625709 TI - Hepatocyte growth factor and idiopathic pulmonary fibrosis. PMID- 8625711 TI - Goodbye to ABCD resuscitation? PMID- 8625710 TI - Cardiac troponins in the diagnosis of myocardial contusion. PMID- 8625712 TI - Kids speak out on adoption: a multiage bookwriting group for adopted children with special needs. AB - This article describes the conceptualization and development of the Saturday Club for Adopted Kids (a four-session, multiage, bookwriting group for adopted children and young people) and the benefits of offering such a project-oriented group in which adoptees can tell their stories and share their experiences in an environment that supports their recovery from early trauma and offers them a way to transform the meaning of their personal losses into social action to help others. Club participants produced a book, Kids Speak Out on Adoption, based on their experiences, perceptions, and feelings. PMID- 8625713 TI - Mexican-American and Anglo-American children's responsiveness to a theory centered AIDS education program. AB - Third, fifth, and seventh graders, most of them Mexican-American, were exposed to an empirically based and culturally sensitive AIDS curriculum designed to replace their intuitive theories with a coherent, scientific account of the causal processes that lead from risk behavior to AIDS symptomatology. Compared to students in control classes, experimental students knew more about AIDS risk factors and AIDS generally, displayed more conceptual understanding of the causes of AIDS and flu, and were more willing to interact with people who have AIDS (although not less worried about AIDS) at posttest and typically at follow-up 10 11 months later. The findings point to the potential value of adopting an intuitive theories approach in assessing and modifying children's concepts of health and illness and suggest, contrary to Piagetian formulations, that even relatively young children can, with appropriate instruction, grasp scientific theories of disease. PMID- 8625715 TI - Does schooling buffer the effects of early risk? AB - The data presented here come from a 20-year study conducted in a rural area of Guatemala. Data on early biological indicators, graduated parameters of social structure, and preschool cognition were combined into a risk scale and analyzed in relation to primary school grade attainment and adolescent psychoeducational test performance. Similar to empirical relations reported in industrialized countries, performance declined as the number of risk factors to which a subject was exposed increased. More important, primary education was observed to buffer the effects of early risk for a subset of subjects. Subjects at high risk who stayed in school performed significantly better than subjects with similar levels of risk who completed fewer than 4 years of primary school. PMID- 8625714 TI - The younger sisters of childbearing adolescents: their attitudes, expectations, and behaviors. AB - To examine the precursors of the disproportionately high rates of early childbearing among the younger sisters of adolescent mothers, this study compared the attitudes, expectations, and behaviors of early adolescent girls (M age = 12.93) who had an adolescent childbearing sister (n = 75) to those of early adolescent girls who had only adolescent nonchildbearing sisters (n = 348). Results indicated that the younger sisters of childbearing adolescents were consistently different from the younger sisters of nonchildbearing adolescents on key characteristics known to be correlated with early sexual activity and adolescent childbearing: that is, they were more accepting of nonmarital adolescent childbearing, perceived younger ages for typical life-course transitions (best age to get married, have first child), had more pessimistic school and career expectations, and were more likely to have engaged in problem behaviors (smoke cigarettes, skip school). These younger sister characteristics were associated with a nonvirgin sexual status in the current sample and with high closeness and high rivalry with the childbearing sister but could not be accounted for by such within-family experiences as subjects' mothers' permissiveness or lack of mother-daughter communication. Findings suggest the mechanisms by which the younger sisters of childbearing teens themselves become vulnerable to early parenthood. PMID- 8625716 TI - The onset and cross-temporal patterning of sexual intercourse in middle adolescence: prospective relations with behavioral and emotional problems. AB - This study examined precursors and correlates of sexual intercourse patterns (e.g., earlier/later onset, persistence across time) among tenth and eleventh graders. Data regarding intercourse were collected for lifetime and for 6-month periods at 4 occasions of measurement over a 2-year span. Significant differences in behavioral (e.g., delinquency) and emotional (e.g., depression) problems were indicated via repeated-measures MANOVA analyses. Significant interactions among intercourse patterns, gender, and time on emotional and behavioral problems indicated synergistic relations. Planned comparisons indicated that the transition to the onset of sexual intercourse was associated with increases in delinquency and a slower rate of increase for school grades. Earlier onset and a more persistent pattern of sexual intercourse were associated with more childhood problem behaviors, earlier alcohol use, and higher levels of preadolescent antisocial behavior. PMID- 8625717 TI - Predicting the timing of first sexual intercourse for at-risk adolescent males. AB - Event history analysis was used to test a developmental model of the timing of first sexual intercourse in the Oregon Youth Study sample of adolescent males at risk for delinquency. The event history models spanned grades 7-12 with yearly multimethod, multiagent measures. A 3-step mediational model of predictors was tested, including contextual and process factors and boys' characteristics. Predictors included one-time measures of socioeconomic status, parental antisocial behavior, and time-varying measures of parental transitions, parental monitoring, deviant peer association, and the boys' antisocial/delinquent behavior, substance use, physical maturation, academic achievement, and anxiety. As predicted, antisocial/delinquent behavior and substance use along with early physical maturity and parental transitions predicted early onset of sexual intercourse. Anxiety was related to delay of first intercourse. Results and intervention implications are discussed in terms of the developmental findings. PMID- 8625718 TI - A model of the effects of perceived parent and peer support on adolescent false self behavior. AB - A model linking 3 perceived support variables, namely, level of support, quality of support (unconditional or conditional), and hope about future support, to false self behavior (acting in ways that are not the "real me") was hypothesized. Both parent and peer support were examined. The best fitting model for the parent and peer data revealed that perceived quality and level of parent support predict hope about future parent support, which in turn predicts false self behavior. Adolescents' motives for engaging in false self behavior were also examined. Those whose reported motives were hypothesized to be the most clinically debilitating (devaluation of the self) reported the most negative outcomes (depressed affect, low self-worth, hopelessness, and less knowledge of the true self). In contrast, adolescents citing the developmentally normative motive of role experimentation reported the most positive affect, highest self-worth, greatest hopefulness, and most knowledge of true self. Those reporting that they engaged in false self behavior to please, impress, or win the approval of parents and peers had intermediate scores on the depression, self-worth, hope, and knowledge of true self measures. Discussion focused on the potential causes and consequences of false self behavior. PMID- 8625719 TI - Adolescent girls' relationships with mothers and best friends. AB - The present study examined factors associated with harmony in adolescent girls' relationships with their mothers and their best friends. A framework was proposed in which relationship harmony was expected to be related to individual characteristics of each partner and the match between the individual characteristics of each partner. 60 adolescent girls, their mothers, and their best friends participated in self-report and observational tasks. Harmonious mother-daughter partners (vs. disharmonious ones) had more similar needs, felt their needs were better met, perceived their partners as more socially skilled, and had more similar interests. Harmonious friends (vs. disharmonious ones) had more similar needs, and target adolescents perceived partners to be more socially skilled and better at meeting their needs. Observational ratings of attunement, positive affect, and power negotiation were greater in harmonious relationships with both mothers and friends. Discussion focuses on the value of a common framework for studying different relationships. PMID- 8625720 TI - Ethnic differences in children's intelligence test scores: role of economic deprivation, home environment, and maternal characteristics. AB - We examine differences in intelligence test scores of black and white 5-year olds. The Infant Health and Development Program data set includes 483 low birthweight premature children who were assessed with the Wechsler Preschool and Primary Scale of Intelligence. These children had been followed from birth, with data on neighborhood and family poverty, family structure, family resources, maternal characteristics, and home environment collected over the first 5 years of life. Black children's IQ scores were 1 SD lower than those of white children. Adjustments for ethnic differences in poverty reduced the ethnic differential by 52%. Adjustments for maternal education and whether the head of household was female did not reduce the ethnic difference further. However, differences in home environment reduced the ethnic differential by an additional 28%. Adjustments for economic and social differences in the lives of black and white children all but eliminate differences in the IQ scores between these two groups. PMID- 8625722 TI - Peer relationships and emotional well-being of youngsters with sickle cell disease. AB - Comparisons with measures of peer relationships and emotional well-being were made between youngsters with sickle cell disease (SCD) and same-classroom comparison peers. Relative to the comparison subjects, females with SCD were perceived by peers as being less sociable and less well accepted; males with SCD were perceived as being less aggressive than comparison peers. For both males and females with SCD, no other differences were identified on numerous measures of emotional well-being. None of the multiple measures of illness severity were significantly related to measures of psychological adjustment. The common side effects of SCD, chronic fatigue and small physical size, may divert males with the illness from manifesting difficulties related to aggressive behavior with peers. For females with the illness, the common side effects of the illness may hinder the development of normal social relationships. Despite chronic exposure to numerous stressful life events associated with SCD, the youngsters with the illness were remarkably similar to comparison peers, showing evidence of considerable hardiness. PMID- 8625721 TI - Genetic and environmental influences on temperament in middle childhood: analyses of teacher and tester ratings. AB - Parent ratings of temperament in infancy and childhood yield evidence for genetic influence in twin studies but not in adoption studies. The present study used the sibling adoption design to investigate teacher and tester ratings of temperament in middle childhood. When each child was 7 years old, ratings on the Colorado Childhood Temperament Inventory were obtained from a teacher and tester for more than 50 pairs each of adoptive and nonadoptive siblings in the Colorado Adoption Project. Significant genetic influence emerged for both teacher and tester ratings of Activity, for tester ratings of Sociability, and for teacher ratings of Emotionality. Results obtained from bivariate genetic analysis suggest that the modest covariance between teacher and tester ratings of Activity is entirely mediated genetically. Except for teacher ratings of Attention Span, evidence of shared family environment was nonsignificant, despite the power of the sibling adoption design to detect it. PMID- 8625723 TI - The peer relations of preschool children with communication disorders. AB - The peer-related social interactions of preschool-age children with communication disorders were compared to those of normally developing chronological age-mates. All children were previously unacquainted with one another and participated in a series of short-term play groups. Differences between the 2 groups emerged primarily in terms of overall social activity, as children with communication disorders engaged in fewer positive social interactions and conversed with peers less often during non-play activities. Children with communication disorders also were less successful in their social bids and appeared to be less directive with their peers. However, both groups of children exhibited similar patterns of socially competent interactions including the ability to sustain play (group play), to minimize conflict, to join others in ongoing activities, and to respond appropriately to the social bids of others. Based on peer sociometric ratings, both groups of children were equally accepted. These general patterns of similarities and differences were found in settings in which play groups consisted of all children with communication disorders (specialized settings) as well as in settings in which the play groups included both children with communication disorders and normally developing children (mainstreamed settings). However, even during the relatively brief acquaintanceship process, an analysis of peer preference patterns revealed that children with communication disorders in mainstreamed settings were less socially integrated in the play groups than normally developing children. The potential for additional difficulties in peer interactions for children with communication disorders when children become more familiar with one another and play becomes more intricate was discussed in light of interaction patterns formed during the short-term play groups. PMID- 8625724 TI - Inhibitory control in young children and its role in emerging internalization. AB - We examined inhibitory control as a quality of temperament that contributes to internalization. Children were assessed twice, at 26-41 months (N = 103) and at 43-56 months (N = 99), on repeated occasions, in multiple observational contexts and using parental reports. Comprehensive behavioral batteries incorporating multiple tasks were designed to measure inhibitory control at toddler and preschool age. They had good internal consistencies, corresponded with maternal ratings, and were developmentally sensitive. Individual children's performance was significantly correlated across both assessments, indicating stable individual differences. Girls surpassed boys at both ages. Children's internalization was observed while they were alone with prohibited objects, with a mundane chore, playing games that occasioned cheating, being induced to violate standards of conduct, and assessed using maternal reports. Inhibitory control was significantly associated with internalization, both contemporaneously and as a predictor in the longitudinal sense. The implications for considering children's temperament as a significant, yet often neglected contributor to developing internalization are discussed. PMID- 8625725 TI - Behavioral inhibition and stress reactivity: the moderating role of attachment security. AB - The role of the mother-toddler attachment relationship in moderating the relations between behavioral inhibition and changes in salivary cortisol levels in response to novel events was examined in 77 18-month-olds. Behavioral inhibition was determined by observing toddler inhibition of approach to several novel events. Attachment security to mother was assessed using the Ainsworth Strange Situation. Changes in salivary cortisol were used to index activity of the stress-sensitive hypothalamic-pituitary-adrenocortical (HPA) system. In addition, toddler coping behaviors and the behaviors used by mothers to help toddlers manage novel events were examined. Elevations in cortisol were found only for inhibited toddlers in insecure attachment relationships. Mothers in these relationships appeared to interfere with their toddlers' coping efforts. These results are discussed in the context of a coping model of the relations between temperament and stress reactivity. PMID- 8625726 TI - Behavioral and physiological antecedents of inhibited and uninhibited behavior. AB - 4-month-old infants were specifically selected for patterns of affective and motoric reactivity that were hypothesized to be associated with later inhibited and uninhibited behavior. Infants were classified as high on motor activity and negative affect, high on motor activity and positive affect, or low on motor activity and affect. Brain electrical activity was assessed in these infants at 9 months of age, and behavior toward novelty was observed at 14 months of age. Infants who were high on motor activity and negative affect exhibited greater right frontal EEG activation at 9 months of age and inhibited behavior at 14 months of age. Infants classified as high motor/high positive at 4 months of age exhibited uninhibited behavior at 14 months of age. No relations were found between frontal asymmetry at 9 months of age and inhibited behavior at 14 months of age. However, greater activation in both the left and right frontal hemispheres was associated with higher inhibition scores at 14 months of age. These findings are discussed in terms of the role that affective and physiological reactivity may play in the development of social behavior during toddlerhood. PMID- 8625727 TI - Associations among attachment classifications of mothers, fathers, and their infants. AB - Associations are reported among classifications of Adult Attachment Interviews (AAIs) obtained from expectant parents and subsequent classifications of their infants in the Strange Situation Procedure (SSP). Mothers' AAIs predicted infant mother SSPs (chi 2 = 41.87, N = 96, df = 9, p < or = .0001), and fathers' AAIs predicted infant-father SSPs (chi 2 = 18.94, N = 90, df = 6, p < or = .005). Associations between parents' AAIs and infant-parent SSPs were lessened by the failure to predict the insecure-resistant pattern with mother and the absence of this pattern with father. Counter to expectation, infant-father SSPs were associated with infant-mother SSPs (chi 2 = 3.78, N = 90, df = 1, p < or = .05), which could not be accounted for in terms of an overlap between parental AAIs. A secondary analysis of the data suggested that this dependency effect of SSPs may be explained by the influence of maternal AAIs upon child-father SSPs. Results are discussed in terms of intergenerational and relationship-specific influences upon attachment during infancy, the possible influence of infant temperament, and the relative influence of mother and father upon the child's evolving representations of attachments within the family. PMID- 8625728 TI - Trouble in the second year: three questions about family interaction. AB - 3 questions regarding family interaction in the second year of life are addressed in this report on 69 families rearing firstborn sons. Question 1 concerns the identification, via cluster analysis, of families having difficulty managing their child, using codings of narrative records of family interaction when children were 15 and 21 months of age. Parents in families identified as "troubled" at each age tried to control their toddlers most often, were least likely to rely upon control-plus-guidance management strategies, had children who defied them most frequently, and experienced the greatest escalation of negative affect in these control encounters. Families identified as "troubled" at both 15 and 21 months had children who received the highest "externalizing" problem scores at 18 months and mothers who experienced the most daily hassles during the second year. Question 2 concerns the antecedents of "trouble in the second year." Discriminant function analyses indicated that membership in the groups of families that appeared troubled at both ages of measurement (n = 15), at only one age (n = 28), or never (n = 26) could be reliably predicted (hit rate = 71%) using a set of 9 measurements of parent personality, child emotionality/temperament, marital quality, work-family relations, and social support, suggested by Belsky's model of the determinants of parenting, and social class. Question 3 concerns the proposition that extensive nonmaternal care in the first year is a risk factor for troubled family functioning in the second year. As hypothesized, prediction analysis showed that families at moderate and high contextual risk (based on 10 antecedent variables pertaining to Question 2), were significantly more likely to experience trouble in the second year when children experienced 20 or more hours per week of nonmaternal care in their first year, and these results could not be attributed to "selection effects." PMID- 8625729 TI - And baby makes four: predictors of attachment security among preschool-age firstborns during the transition to siblinghood. AB - The present study examined preschool-age firstborns' adjustment to siblinghood, as indexed by security of firstborn-mother attachment, in a sample of 194 2 Parent families. Security of firstborn attachment decreased significantly after a secondborn's birth, but the size of the decrease was smaller among firstborns under 24 months relative to 2-5-year-olds. Mothers' marital harmony and affective involvement with firstborns predicted firstborn security before and after the baby's birth, whereas mothers' psychiatric symptoms predicted firstborn security only after the birth. Post-hoc analyses of select subgroups revealed that mothers of firstborns with high security scores before the newborn's birth, regardless of whether scores remained high or dropped after the birth, showed higher levels of psychosocial and behavioral functioning than did mothers of firstborns with consistently low security scores at both time points. However, substantial drops in firstborn security after a secondborn's birth were associated with higher maternal psychiatric symptom scores both prior to and following the birth. Results suggest that quality of firstborn adjustment to siblinghood can be predicted from both structural and familial aspects of the firstborn environment. PMID- 8625730 TI - Using mothers versus trained observers in assessing children's secure base behavior: theoretical and methodological considerations. AB - The Attachment Q-Set (AQS) has emerged as a psychometrically sound method for assessing young children's secure base behavior in the home. However, considerable disagreement exists about whether mothers versus trained observers should be used as AQS sorters. The present study examined associations between mothers' and trained observers' AQS sorts for preschoolers, and assessed mother observer concordance in relation to observers' confidence about how representative the behavioral samples they witnessed were of the domain of AQS items. Mothers with careful training and supervision on the AQS system completed AQS sorts with regard to their children's current behavior, and the same children were assessed with the AQS during a 2-3 hour visit 1-2 weeks later by trained, "blind" observers. Trained observers provided a confidence rating regarding the degree to which the samples of behavior observed were representative of the universe of AQS items. Mothers' and observers' sorts were significantly intercorrelated; however, observer sorts converged with mother sorts as observers' confidence ratings increased. Results are discussed in relation to circumstances that affect mother-observer reliability with the AQS and to factors that should be weighed when considering whether to use mothers versus trained observers as sorters. PMID- 8625731 TI - Quality of center child care and infant cognitive and language development. AB - The relations between quality of center-based child care and infant cognitive and language development were examined in a sample of 79 African-American 12-month old infants. Both structural and process measures of quality of child care were collected through interviews with the center director and observation of the infant classroom. Results indicated that quality of infant care positively correlated with scores on standardized assessments of cognitive development (Bayley Scales of Infant Development), language development (Sequenced Inventory of Communication Development), and communication skills (Communication and Symbolic Behavior Scales). In addition, quality of care in child care centers and at home was positively related. Analyses that adjusted for this association between quality of care at home and in child care suggested that the process measure of quality of child care independently related to the infant's cognitive development, and one structural measure, the infant-adult ratio, independently related to the infant's overall communication skills. Neither child nor family factors was found to moderate the association between child care quality and infant development. These findings, in conjunction with the growing child care literature, suggest that researchers and policymakers should focus on how quality of child care can be improved to enhance, not impair, infant development. PMID- 8625732 TI - Response modality affects human infant delayed-response performance. AB - Delayed response performance was assessed in 120 7-, 9-, and 11-month-old infants with correct response defined as either retrieval of a hidden object or gaze toward the location where the object was hidden. Performance improved with age, was above chance for each age group in each condition, and was more often correct with the gaze response. When direction of gaze and reach differed, direction of gaze was more likely to be correct. Infants in the reach condition were more likely to fail to reverse a previously correct response (i.e., to make the A-not B error). Perseverative responding occurred frequently and was more likely in the reach than the gaze condition. This effect emerged primarily in the context of an incorrect response, which suggests modality-specific sensitivity to the effect of priming rather than reinforcement. Many infants showed strong side biases, and there was a tendency for more reaches to the left but gazes to the right. In a second experiment, 12 5-month-olds gazed toward the correct location more frequently than would be expected by chance but failed to reverse a previously correct response more often than older infants. These findings indicate that response modality has a significant effect on delayed-response performance. PMID- 8625733 TI - A comparison of young children's understanding of contradictory representations in pretense, memory, and belief. AB - The present study examined the nature of young children's understanding of various mental representations. 3- and 4-year-olds were presented with story protagonists who held mental representations (beliefs, pretenses, and memories) that contradicted reality. Subjects chose 1 of 2 alternate "thought pictures" (depicting either the mental representation or reality) that reflected the mental state. While 4-year-olds performed relatively well on all scenario types, 3-year olds chose the correct thought picture significantly more often for pretense and memory scenarios than for false belief scenarios. These results suggest that young children conceptualize pretense as involving mental representations, and that they have more difficulty understanding contradictory mental representations that purport to correspond to reality. PMID- 8625734 TI - Knowing about guessing and guessing about knowing: preschoolers' understanding of indeterminacy. AB - In this article we investigate preschool children's understanding of indeterminacy by examining their ability to distinguish between determinate situations--in which the available evidence eliminates all uncertainty about an outcome--and indeterminate situations--in which it does not. We argue that a full understanding of indeterminacy requires the coordination of 3 processes: search, evaluation, and mapping. We describe 3 experiments aimed at discovering the extent to which these processes, each of which has been implicated in previous accounts of indeterminate reasoning, are developed in preschoolers and the extent to which different children organize the processes into different strategies. Experiment 1 examines 5-year-olds' performance on 1- versus 2-solution problems having different configurations of irrelevant information. Experiments 2 and 3 extend the possible sources of indeterminacy from 2 to 4 and vary the amount of consistent, inconsistent, and to-be-discovered evidence. Our results show that 4- and 5-year-old children readily give "Can tell" responses to determinate problems, as well as "Can't tell" responses when they think that the evidence warrants such a response. In addition, we report 2 new findings: (a) different children use different strategies to process determinate evidence, and these strategies, in turn, predict their performance on indeterminate problems; (b) evidence patterns in which a single positive instance is contrasted with 1 or more negative or unknown instances are particularly difficult to resolve. Many children use a decision rule--the Positive Capture rule--that produces consistent errors on this type of problem. PMID- 8625735 TI - Transcription and masking of mRNA in germ cells: involvement of Y-box proteins. AB - Gametogenesis is directed by various specialized genetic mechanisms which, to a considerable extent, apply to the production of both eggs and sperm and have been conserved across a wide spectrum of eukaryotic organisms. Two key aspects which are discussed here are: germ-cell-specific gene transcription; and translational repression (masking) of mRNA accumulated in oocytes and spermatocytes/spermatids. Together, these two processes conspire to deliver often large amounts of essential proteins at the appropriate stages of development. It is perhaps not surprising that recent evidence points to a functional link between transcription activation and translation repression, both processes being determined in the nucleus and involving common components. One set of components which has been studied recently are members of the Y-box family of regulatory proteins. Most information of the involvement of Y-box proteins in germ cell development comes from studies on amphibian oocytes and mammalian spermatids. In these cells, Y-box proteins have been detected as major components of both maternal and paternal mRNP particles and have been shown to be instrumental in the masking process. Y box proteins are also implicated in the regulation of several germ-cell-specific genes. Possible connections between these processes are discussed. PMID- 8625736 TI - A recA-like gene in Drosophila melanogaster that is expressed at high levels in female but not male meiotic tissues. AB - The RecA protein is the central enzyme in prokaryotic recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination. The RecA-like proteins Rad51 and Dmc1 of yeast are both required for meiotic recombination and the former is also necessary for repair of double-strand breaks in vegetative cells. Genes encoding Rad51 homologs have been isolated recently from several higher eukaryotes. This paper describes the isolation and molecular characterization of a genomic DNA fragment from Drosophila melanogaster containing the coding sequence for a RecA like protein. This protein exhibits strong sequence homology with the Rad51 proteins of budding yeast, fission yeast, chickens, mouse and humans, and slightly less (but still strong) homology with yeast Dmc1. Both in situ hybridization and Southern analysis indicate that the Rad51 gene is present only once per genome in Drosophila (at 99D on chromosome arm 3R). However, there are at least three other fragments that cross-hybridize strongly at low stringency. RNA blotting analysis detects a single transcript of about 1.35 kb that is present throughout development at low levels. Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis. Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males. PMID- 8625738 TI - In situ footprinting of chicken histone H5 gene in mature and immature erythrocytes reveals common factor-binding sites. AB - In vitro DNAase I footprinting and gel mobility shift assays have shown that the activities of several nuclear factors (GATA-1, Sp1) that bind to the promoter and downstream enhancer regions of the chicken histone H5 gene are reduced in mature erythrocytes relative to those in immature erythrocytes. In this study we investigated site occupancy in the promoter and downstream enhancer regions of the H5 gene in mature and immature erythrocytes. The ligation-mediated polymerase chain reaction was used to detect DNAase I footprints generated in situ. Most of the sites that bound to Sp1 and/or Sp1-like proteins and GATA-1 in the promoter and enhancer were occupied in situ in mature and immature erythrocytes. However, the level of protection at Sp1/Sp-1-like binding sites in the H5 enhancer region of mature erythroid cells was generally less than that observed for immature cells, suggesting that for any given mature cell not all of the Sp1/Sp1-like binding sites are occupied. Nevertheless, the results of this study suggest that the enhancer and promoter of the H5 gene in mature erythrocytes should be functional, agreeing with nuclear run-on studies showing transcriptional activity of the H5 gene in mature permeabilized cells. PMID- 8625737 TI - The chromatin of the Saccharomyces cerevisiae centromere shows cell-type specific changes. AB - We have analysed the centromeric chromatin from chromosome XIV of Saccharomyces cerevisiae at different stages of mitosis with the help of mutants of the cell division cycle. The pattern of centromeric chromatin in cells arrested using cdc20-1, tub2-401 and cdc15-1 alleles was indistinguishable from that of vegetatively growing cells, indicating that the centromeric complex is constitutively present during mitosis and possibly throughout the entire cell cycle. In contrast chromatin isolated from G0 cells and spores exhibited distinct differences in centromeric chromatin probably due to structural rearrangements of the centromeric complex. In particular the alterations found in spores are indicative of an inactive centromeric complex. The differences in centromeric chromatin in spores do not reflect a general reorganisation of the chromatin in this cell type, as the chromatin structure of the PHO3/PHO5 locus in spores was found to be identical to that in vegetative cells under repressed conditions. Thus the structural analysis of the centromere in different cell types provides evidence about the requirement of CEN DNA/protein complexes in different cell types and in different stages of the cell cycle. PMID- 8625739 TI - Mapping of replication initiation sites in the mouse ribosomal gene cluster. AB - We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found. PMID- 8625740 TI - DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues of Drosophila melanogaster. AB - Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformed Drosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatic hsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatic hsp26 gene. In contrast, the heterochromatic hsp26 transgenes are present in approximately the same copy number as the endogenous euchromatic hsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV. PMID- 8625741 TI - DNA methylation of the X chromosomes of the human female: an in situ semi quantitative analysis. AB - We present an in situ semi-quantitative analysis of the global DNA methylation of the X chromosomes of the human female using antibodies raised against 5 methylcytosine. The antibodies were revealed by immunofluorescence. Images were recorded by a CCD camera and the difference in intensity of fluorescence between active (early replicating) and inactive (late-replicating) X chromosomes was measured. Global hypomethylation of the late-replicating X chromosomal DNA was observed in three cases of fibroblast primary cultures that were characterized by numerical and structural aberrations of the X chromosomes [46,X,ter rea(X;X), 48,XXXX and 46, X,t(X;15)]. In these cases, the difference between early and late replicating X chromosomes was significantly greater than the intra-metaphasic variations, measured for a pair of autosomes, that result from experimental procedures. In cells with normal karyotypes, the differences between the two X chromosomes were in the range of experimental variation. These results demonstrated that late replication and facultative heterochromatinization of the inactive X are two processes that are not related to global hypermethylation of the DNA. PMID- 8625742 TI - Long-term biochemical and virological response to natural interferon-alpha in patients with chronic hepatitis C. AB - To determine the long-term response to interferon-alpha, 134 patients with chronic hepatitis C were followed for more than a year after therapy. Follow-up was stopped for 14 patients and 43 patients received retreatment. The remaining 77 patients were followed for 26-46 months, and 39 of them achieved long-term sustained alanine aminotransferase (ALT) normality. This normality was achieved in 35/38 short-term sustained responders, which was significantly higher (P < 0.001) than in the short-term relapsers (2/38) and the short-term nonresponders (2/44). Hepatitis C virus (HCV) RNA remained negative in 27 (73%) of 37 patients in the long-term sustained ALT normality group. However, only one (1.6%) of 63 long-term responders was negative for HCV RNA. The results of HCV RNA testing for the long-term period agreed with those at three to six months after therapy. Serum ALT levels during the first six months after therapy and HCV RNA testing at three to six months after therapy are important for predicting long-term sustained ALT normality and HCV RNA negativity. PMID- 8625743 TI - Amount and composition of bone minerals in chronic liver disease. AB - Alterations in bone mineral are a common complication of chronic liver disease. The aim of the current study was to assess bone mineral status in patients with chronic liver disease not treated with corticosteroids and to investigate any possible correlation with the histological stage of liver disease. Bone mineral status in 27 patient with chronic active hepatitis, and 17 with active cirrhosis was compared to that of matched controls. Partial body neutron activation analysis was applied for measuring hand bone phosphorus, single-photon absorptiometry for measuring forearm bone mineral content, and dual-energy x-ray absorptiometry for measuring spinal bone mineral density. These noninvasive measurements were supplemented with data obtained by high resolution radiography and biochemistry. Decreased metacarpal cortical thickness was found in five patients, all in the cirrhotic group. In addition, both mean intact parathyroid hormone and 25-hydroxyvitamin D levels were reduced in this group of patients. The mean values of the quantities assessed by the in vivo techniques in patients in the early stages of the hepatic disease did not differ statistically from those of matched normal controls. On the contrary, these quantities were reduced by 9% in the patients at the late stages relative to controls. In conclusion, only the late stages of liver disease are associated with an increased risk of fractures. PMID- 8625744 TI - Role of protein kinase A in human hepatocyte DNA synthesis. AB - The cellular mechanisms associated with the replicative response of hepatocytes to growth factor simulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [3H] thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogens-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis. PMID- 8625745 TI - Inhibition of myofibroblastic transformation of cultured rat hepatic stellate cells by methylxanthines and dibutyryl cAMP. AB - Stellate cells isolated from rat liver and cultured on uncoated plastic plates in serum-containing medium started proliferating and transforming to myofibroblastic cells. However, stellate cells did not proliferate when cultured in the presence of 3-isobutyl-1-methylxanthine or dibutyryl cAMP (dBcAMP). These substances significantly reduced [3h] thymidine incorporation of the proliferating cells. Morphologically, stellate cells cultured in the presence of 3-isobutyl-1 methylxanthine or dibutyryl cAMP kept well-developed processes and lipid droplets while untreated cells exhibited myofibroblastic characteristics. Western blot analysis and immunocytochemical studies revealed that 3-isobutyl-1-methylxanthine and dBcAMP suppressed the expression of alpha-smooth muscle actin in stellate cells. 3-isobutyl-1-methylxanthine increased the cellular levels of cAMP from a basal value of 0.7 +/- 0.1 to 8.5 +/- 1.7 pmol/well in stellate cells. Thus, 3 isobutyl-1-methylxanthine and dBcAMP inhibit the myofibroblastic transformation of stellate cells in vitro in some cAMP-related mechanism. PMID- 8625746 TI - Role of platelet-activating factor in pathogenesis of galactosamine lipopolysaccharide-induced liver injury. AB - In an attempt to clarify the role of platelet-activating factor (PAF) in the pathogenesis of hepatic injury induced by galactosamine (GalN) plus lipopolysaccharide (LPS), effects of WEB 2086 (PAF receptor antagonist) on hepatic injury in vivo as well as on neutrophil adherence to hepatic endothelial cells in vitro have been investigated, as we have recently clarified the role of neutrophils in this experimental model of hepatic injury. Although an enhanced serum TNF-alpha level after GalN-LPS administration was not reduced by WEB 2086, hepatic injury and hepatic neutrophil accumulation in the liver after GalN-LPS administration were attenuated by WEB 2086. An in vitro study revealed that an enhanced neutrophil adhesion to hepatic endothelial cells by stimulation with the sera that were collected from the GalN-LPS-treated rats, was reduced in the presence of WEB 2086 in a dose-dependent manner. In addition, LPS, TNF-alpha, and PAF were found to enhance the neutrophil adherence to hepatic endothelial cells, which was reduced in the presence of WEB 2086. These results suggest that PAF play an important role in the GalN-LPS induced hepatic injury and that PAF receptor antagonist reduces the neutrophil adherence to hepatic endothelial cells in the liver. PMID- 8625747 TI - Impaired synthesis of retinol-binding protein and transthyretin in rat liver with bile duct obstruction. AB - To gain further insight into the protein metabolism in bile duct-obstruction, we examined the synthesis of retinol-binding protein (RBP) and transthyretin (TTR) in rats with common bile duct-ligation. In these rats, liver and plasma levels of RBP and TTR decreased markedly, whereas liver retinoid contents remained unchanged. Although there appeared no decrease in the total amount of RBP or TTR mRNA expressed in the liver, the subcellular distribution of these mRNAs changed from the membrane-bound polysome fraction to the membrane-unbound polysome fraction. This abnormal distribution recovered rapidly after biliary drainage, resulting in the subsequent recovery of the plasma RBP and TTR levels. These observations suggest that cholestasis inhibits the synthesis and secretion of RBP and TTR by disrupting the binding of their mRNAs to membrane-bound polysomes. Plasma levels of RBP and TTR might be sensitive indicators of the recovery of protein synthesis after biliary drainage in patients with obstructive biliary disorders. PMID- 8625748 TI - Ischemia- and reperfusion-related injury in pancreatitis. PMID- 8625749 TI - Ischemia-reperfusion-induced pancreatic microvascular injury. An intravital fluorescence microscopic study in rats. AB - With the concept that ischemia-reperfusion injury may contribute to the pathogenesis of acute pancreatitis, we have quantitatively analyzed the pancreatic microcirculation of rats during postischemic reperfusion using intravital fluorescence microscopy. Ischemia to the pancreas of Sprague-Dawley rats (N = 7) was induced by clamping the arteriae gastroduodenalis, lienalis, gastrica sinistra, and gastricae breves for 60 min followed by 120 min of reperfusion. Ischemic conditions were verified by measurement of microvascular hemoglobin oxygenation using reflection spectrophotometry (n = 9). Postischemic reperfusion was characterized by a significant (P < 0.05) reduction of functional capillary density to approximately 69% of baseline (no reflow). Reperfusion induced inflammatory response was reflected by a marked increase (100-fold; P < 0.01) of the number of permanently adherent leukocytes in postcapillary venules (reflow paradox). Postischemic reperfusion was further associated with increased serum lipase activities, and histomorphological analysis revealed alterations, similar as known in acute interstitial pancreatitis, ie, neutrophil infiltration, interstitial edema, and hemorrhagic lesions. We, therefore, conclude that ischemia-reperfusion- associated events, ie, no reflow and reflow paradox, may be considered as trigger mechanisms in the manifestation of distinct types of acute pancreatitis, in particular posttransplant pancreatitis. PMID- 8625750 TI - Delayed hemorrhage following endoscopic retrograde sphincterotomy for choledocholithiasis. AB - To define the clinical significance of delayed postsphincterotomy hemorrhage, we reviewed 476 consecutive ERCP procedures performed over a three-year period. Of 250 patients who underwent endoscopic sphincterotomy (ES), five (2%) developed postprocedure hemorrhage, two of whom had immediate, self-limited bleeding that resolved after endoscopic injection of epinephrine and did not require transfusion. The other three had delayed hemorrhage characterized by: onset 20-48 hr after the procedure, melena without hematemesis as the index clinical manifestation of bleeding, and atraumatic balloon extraction of common duct stones. Transfusion of 2-6 units of packed erythrocytes was necessary in each and one patient required surgical hemostasis. Delayed hemorrhage following ERS is an important, frequently severe complication to remember when contemplating performing ERS as an outpatient procedure. PMID- 8625752 TI - Gallbladder cancer. A comparative study among clinicopathologic features, AgNORs, and DNA content analysis. AB - A comparative study among clinicopathologic features, silver-stained nucleolar organizer region associated proteins (AgNORs), and DNA content analysis in 76 patients with gallbladder cancer was performed. The AgNOR count, AgNOR area, and the ration of AgNOR area to nuclear area were significantly higher in patients with a low grade of histological differentiation and deep invasion into the gallbladder wall. Moreover, these parameters were higher in cases with lymph node involvement and distant metastases. DNA ploidy pattern and clinicopathologic features showed to statistical significance. Our results demonstrate that AgNOR parameters are useful indicators to evaluate the malignant behavior of gallbladder cancer. Furthermore, the AgNOR count together with the depth of the neoplastic invasion and lymph node metastases proved to be independent prognostic factors for survival in patients with gallbladder cancer. PMID- 8625751 TI - Gastric antrectomy with selective gastric vagotomy does not influence gallbladder motility during interdigestive and postprandial periods. AB - Fasting gastrointestinal motility and gallbladder motility during the interdigestive state and in the postprandial period was studied in eight patients who were operated for ulcer disease with an antrectomy and selective gastric vagotomy. Nocturnal motility recording revealed all three phases of the migrating motor complex (MMC) in all but one patient, where no phase III activity was recorded. In the rest of the patients 3-10 events with phase III activity were recorded. At scintigraphy ([75Se]HCAT) a cyclic gallbladder filling and emptying in relation to the MMC cycle was found. Episodes with emptying were confined to phase II and a total of 13 episodes with a median duration of 25 min (range 10-70 min) were observed. A median of 10.7% (6.1-17.7%) of the gallbladder contents was emptied. In a control group of eight healthy young men the values were 13.5 min (9-36 min) and 6.9% (3.7-31.1%), respectively. These differences were not significant. During the postprandial period, a lag period in gallbladder emptying of median 15 min (5-20 min) was observed when food ingestion took place during phase I of the MMC. Thereafter a gradual emptying occurred with a rate of 0.95% min (0.71-1.15%/min). In a control group of healthy young males, the lag period was 13.5 min (9-22.5 min) and the emptying rate 0.61%/min (0.08-0.77%/min). When food ingestion occurred during phase II of the MMC, the lag period of gallbladder emptying in the patient group was median 0 min (0-5 min) and the emptying rate was 0.77%/min (0.33-0.86%/min). The values in the control group were 0 min (-9 to 13.5 min) and 0.76%/min (0.54-2.25%/min), respectively. These differences between the patients and controls were not significant. In conclusion, antrectomy and selective gastric vagotomy do not influence fasting gastrointestinal motility or gallbladder motility during the interdigestive state or in the postprandial period. PMID- 8625753 TI - Ascites, pleural, and pericardial effusions in acute pancreatitis. A prospective study of incidence, natural history, and prognostic role. AB - Ascites and pleural and pericardial effusions can be observed during acute pancreatitis. The aims of this study were to evaluate their incidence, natural history, and prognostic role in patients with acute pancreatitis. One hundred patients consecutively admitted with a diagnosis of acute pancreatitis were prospectively submitted to abdominal, pleural, and cardiac ultrasonography at admission and during follow-up. Ascites was found in 18 patients, pleural effusion in 20, and pericardial effusion in 17. Twenty-four patients of this series had severe pancreatitis; three of them died. All effusions disappeared spontaneously in patients who survived pancreatitis up to two months after dismissal. At multivariate analysis ascites and pleural effusion were demonstrated to be accurate independent predictors of severity. The respective odds ratios were 5.9 [95% confidence interval (CI), 1.5-23.0%) and 8.6 (95% CI, 2.3-32.5%). Furthermore the presence of pleural effusion, ascites, and pericardial effusion were associated with an increased incidence of pseudocyst during follow-up. Ascites and pleural and pericardial effusions are frequent during acute pancreatitis. Pleural effusion and ascites are accurate predictors of severity in these patients. PMID- 8625754 TI - Plasma immunoreactive cationic trypsin(ogen) pattern in reserpinized rat model of cystic fibrosis. Resemblance to humans. AB - Plasma immunoreactive cationic trypsin (ogen) is elevated in cystic fibrosis during early infancy, before exocrine pancreatic insufficiency is fully developed. The recently developed cystic fibrosis mouse model carrying a mutated gene presents only minor pathologic findings in the pancreas. However, the reserpinized rat model shows cystic fibrosis-like defects in various exocrine glands, including the exocrine pancreas. Plasma immunoreactive cationic trypsin (ogen) has not been studied yet in this model. The present study explored the plasma immunoreactive cationic trypsin (ogen) pattern and possible mechanisms in this rat model. Plasma immunoreactive cationic trypsin (ogen) (RIA), pancreatic juice volume, protein, and trypsin, and pancreas weight were determined in rats treated with reserpine (0.5 mg/kg/day subcutaneously) for four or seven days, following cerulein stimulation (5 micrograms/kg/dose intraperitoneally), versus pair-fed controls. The first of four consecutive 30 min periods revealed peak values in all parameters. Four-day reserpine-treated rats demonstrated significantly higher plasma immunoreactive cationic trypsin (ogen) levels (167.3 +/- 12.8 vs 88.9 +/- 6.1 ng/ml; P < 0.0001) with similar values of pancreatic juice trypsin (8.2 +/- 2.4 vs 6.6 +/- 1.8 units/mg protein; P = NS) and volume (5.6 +/- 1.3 vs 4.2 +/- 1.6 mg/min/g pancreas; P = NS), compared to controls. Rats treated with reserpine for seven days revealed significantly lower values of plasma immunoreactive cationic trypsin (ogen) (39.2 +/- 8.4 vs 66.8 +/- 4.9 ng/ml; P < 0.001), pancreatic juice trypsin (1.9 +/- 0.3 vs 3.2 +/- 0.9 units/mg protein; P < 0.001) and volume (1.6 +/- 0.7 vs 3.1 +/- 0.6 mg/min/g pancreas; P < 0.001) compared to controls. We conclude that the reserpinized rat model resembles human cystic fibrosis as to elevated plasma immunoreactive cationic trypsin (ogen) before exocrine pancreatic insufficiency is fully developed. Since exocrine pancreatic volume secretion is intact at this stage, the mechanism of elevated plasma immunoreactive cationic trypsin is probably not due to ductular obstruction. We suggest that this model be studied further in order to investigate other possible mechanisms. PMID- 8625755 TI - Clinical value of duodenojejunal manometry. Its usefulness in diagnosis and management of patients with gastrointestinal symptoms. AB - The records of all patients who had duodenojejunal manometry (DJM) from 1989 to 1995 were retrospectively reviewed. We evaluated the main symptoms of the patients, the indication for the study, its result, and the impact on therapy and management. One hundred sixteen patients out of 154 were included in the study, of whom 96 were women and 20 were men, with a mean age of 41.2 years. Twenty-five had perfused tube studies, and 91 had prolonged ambulatory recordings. Forty-one patients were referred for evaluation of abdominal pain, 34 for chronic constipation, 24 for nausea and vomiting, 8 for pseudoobstruction, and the remaining 9 for other reasons. All patients had appropriate endoscopic, radiographic, or scintigraphic studies prior to manometry. Forty-seven (40.5%) had abnormal manometry: 20 of 41 (48.8%) for abdominal pain, 7 of 34 (20.6%) for chronic constipation, 10 of 24 (41.7%) for nausea and vomiting, 5 of 8 (62.5%) for pseudoobstruction, and 5 of 9 (55.6%) for the miscellaneous group. The various subgroups did not have specific patterns of motor abnormalities. In 22 patients (18.9%) manometry helped in the choice of therapy: in 15 patients by affecting surgical approach, particularly in the constipation group, and in 7 patients by affecting feeding options and prokinetic agents. Detection of motor abnormalities was helpful in patients with severe symptoms thought to have functional disease even when no specific therapy was rendered. Thus, DJM was abnormal in 2/5 patients referred for evaluation of suspected motility disorders. It directly affected therapy in approximately 1/5 patients, particularly in those with constipation. It is helpful in the management of patients even when specific therapy is not rendered, particularly in those with abdominal pain. The modest impact on specific therapy is related to limited availability of effective prokinetic drugs and the limited specificity and predictive value of tests results. PMID- 8625756 TI - Gastric myoelectric activity changes following open abdominal surgery in humans. AB - We examined the postoperative changes in fasting gastric myoelectric activity in 11 patients undergoing nongastric surgery (colon surgery) via celiotomy. Recordings were performed on postoperative days (POD) 1, 2, 3, 5, and 7+ (7-35) for 1-1.5 hr after overnight fasting. Patients had placement of bipolar seromuscular recording electrodes on the proximal (N = 9) and distal (N = 11) antrum at the time of surgery. Data were analyzed visually and analysis of variance or tests of proportion were used for statistical analysis. Although there was a trend of decreasing slow wave frequency from POD 1 to 7+ in the proximal and distal antrum, no significant differences were observed in slow wave amplitude or in the percentage of slow waves with spike activity between postoperative day. In a few of the patients, several types of gastric dysrhythmias were infrequently observed. We conclude that certain parameters of fasting gastric myoelectric activity do not change sufficiently following open abdominal surgery to adequately reflect clinical recovery from postoperative ileus. PMID- 8625757 TI - Cisapride in neurologically impaired children with chronic constipation. AB - The efficacy of cisapride as a treatment for chronic constipation in children with severe brain damage was studied in 20 children. Each subject was randomly assigned to double-blind treatment with either cisapride (N = 10) or placebo (N = 10) for 12 weeks. Stool habits, total gastrointestinal transit time, colonic segmental transit times, and anorectal motility were evaluated in all children before and at the end of the treatment period. Although cisapride significantly (P < 0.05) increased stool frequency from baseline to week 12 and no significant change was documented in the placebo group, the mean change in stool frequency per week from baseline to 12 week was not significantly different between the two treatment groups. The use of laxatives or suppositories was significantly (P < 0.05) decreased by cisapride, but remained unchanged in the placebo group. Furthermore, cisapride significantly (P < 0.05) reduced rectal compliance but had no effect on total gastrointestinal transit time and colonic segmental transit times. In summary, in neurologically impaired children with chronic constipation, cisapride increased bowel frequency but did not alter the delay in total and segmental gastrointestinal transit times. PMID- 8625758 TI - Small bowel transplantation. A life-saving option for selected patients with intestinal failure. AB - Thirty-seven patients were listed for small bowel transplantation; 16 were transplanted and 15 died while waiting for a donor. Cyclosporine (N = 6) or tacrolimus (N = 10) were used for immune suppression. Graft rejection rates were lower in the combined liver/small bowel grafts than the isolated intestinal transplants (1/7 vs 5/7; P < 0.01) All of the cyclosporine group have died; the median survival was 25.7 months with two patients living more than five years. The tacrolimus group had fewer infections and a shorter hospital stay. All but two are alive with a median survival of 13 months. Seven of eight long-term survivors are off intravenous feedings. We conclude that small bowel transplantation is a life-saving option for patients with intestinal failure who cannot be maintained on total parenteral nutrition. PMID- 8625759 TI - Jejunoileal transplantation. Effects on characteristics of canine jejunal motor activity in vivo. AB - This study was designed to determine if extrinsic innervation and intrinsic neural continuity with the duodenum (neuroenteric physiologic pathways disrupted during intestinal transplantation) modulate the characteristics of interdigestive motor activity in the canine small bowel. Five dogs served as neurally intact controls (group 1) and 10 dogs (group 2) underwent a model of jejunal autotransplantation involving in situ neural isolation of the jejunoileum. Fasting duodenal and jejunal motor activity was recorded on-line to a microcomputer using closely spaced duodenal and jejunal manometry catheters. Characteristics of global motor patterns, the migrating motor complex (MMC), and local motor patterns, including individual contractions and grouped clustered contractions, were determined. Neural isolation of the jejunoileum disrupted coordination of duodenal and jejunal phase III activity, increased the variability of cycling of the MMC, decreased the period of the jejunal MMC, and increased motility indices in the neurally isolated jejunum. In contrast, single pressure waves and clustered contractions in the neurally isolated jejunum were not altered significantly in incidence or direction, distance, or velocity of spread. In situ neural isolation of the jejunoileum leads to temporal dissociation of the MMC between the transplanted segment (jejunum) and the duodenum but does not appear to alter markedly the characteristics of local contractile activity as measured by individual or grouped contractions. The occurrence of interdigestive jejunal motor patterns and the local organization of individual and grouped small intestinal contractions are not controlled by extrinsic innervation or intrinsic neural continuity with the duodenum. PMID- 8625760 TI - Effects of somatostatin on luminal transit and absorption of nutrients in the proximal gut of minipigs. AB - A discrepancy exists on the effects of somatostatin on the absorption of nutrients: in humans, absorption was found to be reduced, whereas in rats no effects were observed. However, intestinal absorption might be influenced by the transit rate of contents. This was not considered in previous studies. Therefore, we investigated simultaneously the effects of somatostatin on the absorption of nutrients and on luminal transit. In five minipigs (44-62 kg), a 150-cm segment of the proximal jejunum was temporarily isolated by two cannulas and perfused with an oligomer diet (60% carbohydrate, 18% protein, and 22% fat). The perfusion rate was 2 kcal/min. Flow rate and mean transit time were determined by markers (Cr-EDTA and Cu-EDTA). Somatostatin was infused intravenously at rates of 0.5, 1.25, 2.5, and 5 micrograms/kg/hr. In control experiments saline was administered intravenously. Somatostatin dose-dependently diminished flow rate of luminal contents and increased the transit time. At the largest dose of somatostatin (5 micrograms/kg/hr) flow rate was reduced by 50% compared with control infusion of saline (1.0 +/- 0.4 vs 2.0 +/- 0.05 ml/min, P < 0.05), and transit time was increased 3.6-fold (39.8 +/- 4.7 vs 11.2 +/- 4.9 min; P < 0.05). Somatostatin also dose-dependently enhanced the absorption of nutrients and energy. However, the increase in absorption was small compared with the effects on flow rate and transit time. At the largest dose (5 micrograms/kg/hr) absorption of energy, carbohydrate, protein, and fat was enhanced only by 9.7%, 7.0%, 5.2%, and 15.3%, respectively (49.9 vs 40.2%, 50.9 vs 43.9%, 67.3 vs 62.1%, and 30.1 vs 14.8% during saline infusion; P < 0.05). Results indicate that the major effects of somatostatin consist in a marked reduction of flow rate and a delay of luminal transit. The small increase in absorption was caused by the delay in transit and the prolonged contact of the nutrients with the mucosa. Therefore, in absorption studies, effects on transit need to be considered. PMID- 8625761 TI - Intraepithelial lymphocytes in colon have similar properties to intraepithelial lymphocytes in small intestine and hepatic intermediate TCR cells. AB - Recently, properties of intraepithelial lymphocytes (IEL) in the colon (C-IEL) have been analyzed in comparison with those of IEL in the small intestine (SI IEL). We compared the properties of C-IEL with those of SI-IEL and hepatic intermediate TCR cells, two other types of extrathymic T cells. C-IEL and intermediate TCR cells contain many NK1+T cells, although SI-IEL contain few. V gamma and V delta usage of C-IEL was the same as that SI-IEL, and that of intermediate TCR cells was different. C-IEL responded to Con A while SI-IEL did not. As to adhesion molecules, C-IEL include both extrathymic and thymus originated type T cells. With age, TCR- alpha beta(+) CD4+ CD8+ cells do not increase among C-IEL but do increase among SI-IEL. IL-2R beta(+) or CD4- CD8- C IEL increase as observed in the liver. These results indicate that these organ specific T cells have different roles at their respective sites and that they may be of different lineages. PMID- 8625763 TI - Exercise-induced asthma. Is gastroesophageal reflux a factor? AB - An acid-induced cholinergic esophagobronchial reflex has been described whereby acid refluxing into the esophagus causes bronchospasm. Reports of exertional gastroesophageal acid reflux prompted us to study the possibility that exercise induced asthma (EIA) could be related to gastroesophageal reflux (GER). Following an overnight fast, 10 athletes with a history of EIA (nine men, one woman; mean age 31) were studied. Continuous monitoring of intraesophageal pH and motility, ECG, and arterial oxygen saturation was done. After baseline monitoring at rest for 15 min, subjects underwent treadmill exercise for 10 min followed by continuous monitoring for 30 min after exercise. Spirometry was done at baseline prior to exercise, then repeated every 5 min after exercise for 30 min. Two subjects were retested at a later date following a standard test meal. All 10 subjects demonstrated a decrease in FEV1 in response to exercise, but only half met criteria for EIA. Although 60% (6/10) showed some evidence GER, only three subjects demonstrated a pathologic degree of GER. In the two subjects retested postprandially, change in FEV1 was no different in one and improved in the other despite worsening of GER in both. There was no significant correlations between GER and EIA (P = 0.2). EIA correlated inversely with amplitude of esophageal contractions (P = 0.029) and was directly related to the percentage of multipeaked contractions and the duration of peristaltic contractions (P = 0.08). EIA is not associated with exertional GER. PMID- 8625762 TI - Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in Lewis rat. AB - A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg (P8720), was used to define the relationship between the kallikrein-kinin (K-K) system and acute intestinal inflammation induced by bacterial peptidoglycan-polysaccharide (PG APS) in Lewis rats. Group I received human serum albumin (HSA) intramurally in the intestine and was treated with HSA. Group II received PG-APS and was treated with P8720. Group III received PG-APS and was treated with HSA. P8720 attenuated the decrease of high-molecular-weight kininogen and factor XI activity (group II vs group III, P < 0.01). P8720 therapy significantly but modestly decreased acute intestinal inflammation measured by gross gut score (P < 0.01) and more dramatically reduced the tissue myeloperoxidase activity (P < 0.05), a measure of granulocyte recruitment, in group II compared with group III. We conclude that the K-K system is directly involved in the pathogenesis of the acute phase of experimental acute inflammation. A specific inhibitor may modulate inflammatory bowel disease. PMID- 8625764 TI - Relationship between postprandial esophageal acid exposure and meal volume and fat content. AB - The effect of meal volume and fat content on gastroesophageal reflux was investigated in 20 asymptomatic healthy subjects. In each subject, intraesophageal pH monitoring was performed during a 3-hr postprandial period (PP) in the same position (supine or upright) on two successive days. On day 1, 500-ml low- and high-fat meals were ingested and, on day 2, an 800-ml low-fat meal was ingested. The acid exposure time was assessed as the percentage of time with a pH < 4.0. The acid exposure time in subjects in the upright position was significantly longer in the 800-ml group than in the 500-ml group for the entire PP (2.7 +/- 1.5%; mean +/- SE, 0.7 +/- 0.4%; P < 0.05). Of subjects in the supine position, the high-fat group showed significantly longer acid exposure time than the low-fat group both for the entire PP (7.6 +/- 3.0%, 0.7 +/- 0.5%; p < 0.05) and for the second hour (P < 0.05). We have demonstrated that differences in the meal volume and fat content influence gastroesophageal reflux in healthy asymptomatic subjects and that this influence varies with the position. PMID- 8625765 TI - Predictors for frequent esophageal dilations of benign peptic strictures. AB - Recurrence of esophageal peptic stricture necessitating repeated dilation treatments remains a problem for many patients despite optimal acid suppressive therapy. The factors associated with frequent relapses are poorly understood. We studied retrospectively a population of 58 patients with benign peptic strictures and dysphagia treated by esophageal dilation and followed for 66.5 +/- 6.7 months. Data was collected for age, sex, heartburn, weight loss, esophagitis, Barrett's esophagus, number of dilation treatments during the first year of follow-up, frequency and number of subsequent dilation treatments, type of dilator used, and history of other concurrent treatments. Patients who lacked heartburn (P = 0.007) or who reported a history of weight loss (P = 0.006) at the time of their initial presentation required more frequent dilations during the first year of follow-up. The mean number of dilations in year 1 was 6.2 +/- 0.9 for patients lacking heartburn versus 3.2 +/- 0.5 for patients with heartburn (P = 0.004), and 9.0 +/- 1.8 for patients who reported weight loss versus 4.1 +/- 0.5 (P = 0.006) for those who did not. The patients requiring frequent treatment during their first year also required frequent subsequent dilations because of stricture recurrence (P < 0.0001). We did not demonstrate any relationship between the other factors studied and treatment frequency. These observations suggest that patients who require frequent retreatment for recurrent peptic stricture are more likely to provide a history of weight loss and less likely to complain of heartburn at initial presentation. The pattern of frequent repeat dilation for recurrent peptic strictures is established during the first year of follow-up. PMID- 8625766 TI - Histological predictors of active Helicobacter pylori infection. AB - Helicobacter pylori is a common cause of gastritis. No single test is 100% accurate for H. pylori diagnosis. In order to determine whether the presence of typical histological features of H. pylori gastritis may yield diagnostic information, we compared antral histology by H & E stain to the presence of organisms as detected on Thiazine stain of antral specimens, CLOtest, urea breath test and anti-H. pylori serology in 50 consecutive patients. Patients were diagnosed as having active H. pylori infection if at least two of these tests were positive. Patients with only one test positive (N = 5) were considered indeterminate for H. pylori and were excluded, resulting in 19 patients (42%) being classified as H. pylori positive. All slides were reviewed by a single blinded expert gastrointestinal pathologist and graded 0 (none) to 3 (severe) for the presence of acute (polymorphonuclear cells) or chronic (lymphocytes, monocytes, plasma cells) inflammation, lymphoid aggregates, and intestinal metaplasia. Active infection was associated with the presence of both acute and chronic inflammation (P < 0.0001) but not lymphoid aggregates (P = 0.09) or intestinal metaplasia (P = 0.10). The best positive predictors of infection were the presence of any acute inflammation (PPV = 86%) and the combination of any acute and chronic inflammation (PPV = 92%). The best negative predictor was absence of chronic inflammation (NPV = 100%). The presence of moderate to severe (grade 2 or 3) acute or chronic inflammation were each 100% predictive of infection. Moderate to severe chronic inflammation had both 100% sensitivity and specificity for active H. pylori infection, while moderate to severe acute inflammation was only 26% sensitive but 100% specific. The presence of any acute, or the combination of acute and chronic gastritis, is predictive of active H. pylori infection. Moreover, the predictive value correlates closely with the severity of the inflammation. The absence of chronic inflammatory cells rules out active H. pylori infection. These findings support the use of H & E histology determined features in conjunction with other tests to diagnose H. pylori infection. PMID- 8625767 TI - Profound increase of Helicobacter pylori urease activity in gastric antral mucosa at low pH. AB - The effect of pH on H. pylori urease activity in its ecological niche was studied in gastric antral biopsy specimens. Specimens were incubated in 10 mmol/liter urea solutions at pH range 3.3-8.2. Activity of urease was studied by measuring production of ammonia and change in pH of the solutions. Urease activity was reduced at pH 8.2 (1424 +/- 218 mumol/liter) but decreasing initial pH to neutral and acidic values resulted in significant maximal 6.5-fold increase in ammonia production (9491 +/- 1073 mumol/liter, P < 0.0005), which considerably raised the pH of the test solutions. Peak urease activity was between pH 5.0 and 7.0. In contrast to specimens incubated initially at pH 8.0, reincubation of washed specimens from solutions with initial pH 7.0 showed eightfold decreased urease activity. It is concluded that urease activity is markedly pH dependent with pH optima below the physiological mucosal surface pH. Furthermore, availability of urease is limited. Thus, an impaired gastric mucosal integrity allowing back diffusion of hydrogen ions may release urease activity, which might further weaken the mucus barrier and damage the gastric epithelium. PMID- 8625768 TI - Helicobacter pylori in promotion of gastric carcinogenesis. AB - Gastric atrophy and intestinal metaplasia are considered the earliest phenotypic changes in the cascade of events leading from normal mucosa to intestinal-type gastric cancer, and epidemiological evidence links Helicobacter pylori to gastric epithelial malignancies. To evaluate any causal relationship between bacterial infection and atrophic metaplastic lesions, gastric pathology was histologically and histochemically evaluated in 267 consecutive, nonulcerous, untreated subjects, with attention given the phenotypes of intestinal metaplasia. The prevalence of Helicobacter pylori infection was 61%. Intestinal metaplasia (particularly types II and III) was significantly associated with both Helicobacter pylori detection (chi 2 LR: P < 0.002) and increasing age (chi 2 LR: P < 0.002). Using logistic regression analysis, the development of intestinal metaplasia proved more significantly linked with Helicobacter pylori infection [odds ratio = 4.55 (95% confidence interval: 1.51-13.7)], than with age [odds ratio = 1.03 (95% CI: 1.01-1.06)], with no interaction. In conclusion, Helicobacter pylori can be considered among the major causal agents of mucosal lesions involved in the multistep process of gastric carcinogenesis, justifying any attempt to eradicate this bacterial infection. PMID- 8625769 TI - Cold restraint stress-induced gastric mucosal dysfunction. Role of nitric oxide. AB - The objectives of this study were to determine the cold restraint stress-induced changes in gastric mucosal permeability and to assess whether nitric oxide synthesis inhibition affects gastric mucosal integrity after cold-restraint administration. Cold-restraint stress caused multiple gastric lesions in 90% of animals. The lesion index was found to be 3.87 +/- 0.97 mm. Gastric mucosal permeability to the [51CR]EDTA molecule was significantly elevated in the cold restraint group compared to control. In order to evaluate the role of nitric oxide in cold restraint stress-induced gastropathy, L-arginine analog NG-nitro-L arginine methyl ester (L-NAME) was given as a bolus (10 mg/kg, intravenously) and infused at a rate of 2 mg/ml/hr for 2 hr after cold-restraint administration. L NAME greatly exacerbated gastric mucosal dysfunction associated with cold restraint stress. D-NAME, the biologically inactive enantiomer, did not enhance mucosal dysfunction, whereas L-arginine, the substrate for nitric oxide, reversed the effect of L-NAME. In an additional group of experiments, effects of cold restraint stress and L-NAME on net transmucosal fluid flux as well as tissue myeloperoxidase activity (MPO) were assessed. Cold-restraint stress administration significantly reduced the absorptive capacity of stomach, whereas L-NAME treatment did not affect the stress-induced alterations on net fluid absorption. Furthermore, L-NAME treatment did not affect the cold restraint stress-induced changes in tissue MPO activity. Our results suggest that gastric barrier function is altered after cold-restraint stress and nitric oxide production is important in minimizing mucosal barrier dysfunction associated with cold-restraint stress administration. Our results also indicate that L-NAME induced alterations on mucosal permeability are not related to net transmucosal fluid flux and tissue neutrophils. PMID- 8625770 TI - Metabolic base production and mucosal vulnerability during acid inhibition in a mammalian stomach in vitro. AB - Acid inhibition increases gastric mucosal susceptibility to damage by luminal acid. This might be due to reduced metabolic CO2 and bicarbonate whereas, during normal acid, secretion cytoprotective CO2/HCO3- production parallels acid production. Metabolic activity and mucosal damage caused by luminal acid perfusion was determined in an in vitro mouse stomach, with and without acid inhibition, and at 0%, 1%, or 5% serosal CO2 supply. Without acid inhibition there was no mucosal damage at any level of serosal CO2/HCO3- supply. Acid inhibition reduced metabolic CO2 production by 29% (P < 0.004) and resulted in microscopic damage to 55% of the mucosal area and perforation in four of five stomachs (P < 0.05). Although, 1% CO2 supply completely replaced the reduction in metabolic CO2, it did not protect against mucosal damage. Overreplacement by 5% serosal CO2/HCO3- was required to prevent damage. There was no correlation between luminal CO2/HCO3- output and mucosal damage. The protection by endogenous or exogenous CO2/HCO3- appears to act intracellularly rather than by intragastric or intercellular neutralization. PMID- 8625771 TI - Histamine H2-receptor antagonists stimulate proliferation but not migration of human gastric mucosal cells in vitro. AB - Gastric mucosal cell migration and proliferation are crucial events in the repair of gastric mucosal erosions. This study was designed to test the hypothesis that the H2 blockers roxatidine and ranitidine might stimulate migration and proliferation of gastric mucous cells derived from a human well-differentiated gastric adenocarcinoma cell line (MKN 28 cells) in vitro, in conditions independent of systemic factors and of acid inhibition. Confluent monolayers of MKN 28 cells were wounded with a razor blade and were then incubated with roxatidine or ranitidine. The number of cells migrating to the damaged area was determined 24 hr later. Cell proliferation was assessed by means of [3H] thymidine uptake and cell counts after incubation with roxatidine or ranitidine. Neither H2 antagonist significantly stimulated cell migration. On the other hand, cell proliferation was dose-dependently and significantly enhanced by incubation with roxatidine and ranitidine. Exogenous administration of TGF-alpha significantly stimulated MKN 28 cell division. However, incubation with roxatidine or ranitidine did not increase the steady-state mRNA expression of TGF alpha or EGFR as assessed by northern blot analysis. Based on these in vitro findings, we postulate that the ulcer healing effect of these H2 antagonists in vivo might be due in part to stimulation of gastric mucosal cell proliferation. PMID- 8625772 TI - Effect of long-term sucralfate ingestion on antral and fundic epithelial proliferation in the rat. AB - Sucralfate accelerates the healing of chronic gastric ulcers, but its mechanism is not well understood. We studied the effect of long-term administration of sucralfate on gastric epithelial proliferation in the rat by means of tritiated thymidine autoradiography. Rats were treated perorally with 500 mg/kg sucralfate once a day. After 28 days, rats were injected with tritiated thymidine 1 microCi/g body weight and sacrificed 1 hr later. Autoradiographs from antral and fundic mucosae were prepared and a number of proliferative measurements were made. Long-term sucralfate administration produced an increase in tritiated thymidine labeling of epithelial cells and expansion of the proliferative zone in antral mucosa. These results indicate that long-term sucralfate ingestion stimulates gastric antral epithelial proliferation in the rat. In light of the fact that chronic gastric ulcers are usually located in the antral region in humans, this enhanced epithelial proliferation may contribute to the beneficial effect of sucralfate in accelerating the healing of gastric ulcers. PMID- 8625773 TI - Effects of experimental hemosiderosis on intestinal morphology, permeability, and tissue iron content. AB - Effects of iron overload on intestinal function and structure are unknown and were, therefore, investigated. Sprague-Dawley rats were randomized into an iron overloaded group, which received a single subcutaneous injection of 1.2 g/kg elemental iron-dextran complex, and placebo-treated pair-fed controls. Animals were studied after a 10-month observation period. Intestinal permeability was assessed by measuring the urinary excretion of lactulose, rhamnose, and mannitol after oral administration. In addition, tissue nonheme iron content was measured, and histologic examination and morphometric measurements were carried out. The chronic iron-overloaded group showed a significant increase in intestine tissue iron content and stainable iron in the submucosa and muscularis propria and adipose tissue of the small intestine and lamina propria and muscularis mucosa of the large intestine. There was a significant decrease in the crypt depths without discernible change in the intestine permeability to any of the markers used. In addition, the iron-overloaded animals showed a significant number of iron-laden cells, which primarily consisted of macrophages, fibroblasts, myocytes, and adipocytes. In contrast, no iron-laden cells were present in tissues obtained from the normal control group. Thus, chronic experimental iron overload in rats leads to significant morphologic, but no permeability, alterations of the alimentary tract. PMID- 8625774 TI - Hepatic iron contents and response to interferon-alpha in patients with chronic hepatitis C. Relationship to genotypes of hepatitis C virus. AB - Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 +/- 100 vs 875 +/- 110 micrograms/g liver, P < 0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection. PMID- 8625776 TI - Acute respiratory distress syndrome. AB - The acute respiratory distress syndrome (ARDS) is a serious and complex clinical problem that often threatens the lives of patients. Emerging clinical data suggest that the survival of patients with this disorder may have improved during the last two decades, presumably because of advances in supportive medical care. Among the supportive therapies used to treat patients with ARDS, none is more complex than mechanical ventilation. New strategies for administering mechanical ventilation to patients with ARDS may reduce the occurrence of iatrogenic volotrauma and oxygen toxicity, accounting in part for the recently observed improvements in patient survival. Prevention and cure of ARDS have remained elusive goals because of the lack of specific therapies directed against the known pathogenic factors. Ongoing investigations are aimed at identifying specific therapies to interrupt the mechanisms of inflammation and lung injury responsible for this syndrome. Until such therapies become available, clinicians caring for patients with ARDS should attempt to minimize additional morbidity and mortality resulting from nosocomial infections and iatrogenic injuries. PMID- 8625775 TI - Changes in serum levels of metalloproteinases and their inhibitors by treatment of chronic hepatitis C with interferon. AB - We treated 18 patients with chronic hepatitis C by recombinant interferon-alpha (6 MIU for 24 weeks). In seven patients, serum aminotransferase levels declined to normal (responders). To evaluate the effect of interferon on matrix metalloproteinases (MMPs) and their inhibitors, namely tissue inhibitors of metalloproteinases (TIMPs), the serum levels of these enzymes were determined by enzyme immunoassay (EIA) using a specific monoclonal antibody. In responders, there was a tendency, but not a significant one, towards either an increase in serum MMP 1 levels or a decrease in serum TIMP 1 levels. In contrast, in nonresponders, both a significant decrease in MMP 1 and MMP 3 and a significant increase in TIMP 1 were observed. The number of cases of either increase in serum MMP levels or decreased in serum TIMP levels was significantly larger in responders than in nonresponders. Furthermore, the ratio of MMP 1 to TIMP 1 significantly increased in responders, suggesting that the balance between matrix formation and degradation in hepatic fibrosis tended to move toward degradation. These data indicate that interferon may exert a beneficial effect on hepatic fibrosis in parallel with improvement of aminotransferase activity. PMID- 8625777 TI - [Pidotimod causes apoptotic cell death and inhibits the proliferative activity of YAC-1 tumor cells in vitro]. PMID- 8625779 TI - [Stimulation of B-cell immunity in the nude mice by pidotimod: immunohistochemical stucy of various lymphoid organs]. PMID- 8625778 TI - [Potentiation of the resistance to viral and bacterial infections after pidotimod administration in mice]. PMID- 8625780 TI - [Effect of pidotimod on phagocytosis and intracellular killing of Staphylococcus aureus by human circulating polymorphonuclear neutrophils and alveolar macrophages]. PMID- 8625781 TI - [Effect of pidotimod on the function of the human immune system: in vitro and ex vivo study]. PMID- 8625783 TI - [Levels of interleukin-2 )IL-2 and tumor necrosis factor alpha (TNF-alpha) mRNA in aged rat spleen under physiological conditions and in septic shock: possible role of pidotimod in gene expression modulation]. PMID- 8625782 TI - [Pidotimod, a new biological response modifier, is able to partially prevent immune response changes related to surgical stress]. PMID- 8625784 TI - [Long term course over 10 years after balloon dilatation in stable and unstable angina pectoris]. AB - OBJECTIVE: To assess the cardiac status of patients ten years after percutaneous transluminal coronary artery angioplasty (PTCA). PATIENTS AND METHODS: Data of 534 patients (436 men, 98 women; mean age 53.2 +/- 8 years) in whom a PTCA had been performed between 1983 and 1986 were analysed, based on a questionnaire answered 121 +/- 11 months after the initial procedure. At the time of PTCA 184 patients (35%) had unstable angina, 350 (65%) stable angina. RESULTS: 116 patients (63%) with unstable angina and 164 (47%) with stable angina had at least one cardiac event after the initial PTCA (repeat PTCA, bypass operation, myocardial infarction, death). None of these events occurred in 68 patients (37%) with unstable or in 186 (53%) with stable angina (P < 0.001). After 10 years 196 of the 302 surviving patients with stable angina (65%) and 104 of the 153 surviving patients with unstable angina (68%) were symptom-free. CONCLUSIONS: Ten year follow-up after PTCA has shown that cardiac events are significantly more frequent in patients who had had unstable angina than in those with stable angina. This difference already develops in the first year post-PTCA, with no increase later. Symptoms are lastingly improved after 10 years in both groups of patients. PMID- 8625785 TI - [Left-sided hydronephrosis as the first sign of Crohn disease]. AB - HISTORY AND CLINICAL FINDINGS: A previously well 29-year-old man was referred to the emergency department because of acute left lower abdominal pain without intestinal symptoms. Physical examination was unremarkable except for moderate suprapubic pain on pressure. INVESTIGATIONS: There was evidence of a urinary infection. Erythrocyte sedimentation rate was increased (48 mm/h) and C-reactive protein elevated to 42 mg/l. There was mild leucocytosis (10000/microliter). Abdominal sonography revealed an enlarged left renal pelvis with hydroureter and possible prevesical stone, findings confirmed at excretion urography. TREATMENT AND COURSE: He was discharged with the suspected diagnosis of ureteric stone and given co-trimoxazole, diclofenac and hyoscine butylbromide. After discontinuing medication the same symptoms developed two weeks later. Cystoscopy and retrograde pyelography were unremarkable and it was assumed the stone had been passed. The patient was again discharged on the above medication. When it was discontinued the same symptoms recurred and he was again admitted. Computed tomography, selective small-intestine radiology, coloscopy and biopsy indicated Crohn's disease with hydronephrosis as complication. Treatment trial with prednisone and mesalazine brought about only transient improvement so that an ileocaecal resection and ureterolysis were performed. The patient has since been free of symptoms. CONCLUSION: If there are urological complications, chronic inflammatory intestinal disease should be included in the differential diagnosis even in the absence of any intestinal symptoms. PMID- 8625786 TI - [Miliary tuberculosis and silicosis with predominantly cerebral symptoms]. AB - HISTORY AND CLINICAL FINDINGS: About 10 weeks before admission to hospital a 73 year-old woman developed a fever of up to 40 degrees C for three days. She then had subfebrile temperature for several weeks with some rises to 39 degrees C. She was known to have type II a diabetes mellitus and pulmonary silicosis, having worked in a porcelain and ceramic factory for many years. Before admission her cerebral functions were rapidly deteriorating, especially short-term memory. This was followed by increasing paraplegia of the legs with inability to walk. She finally had urinary and faecal incontinence and swallowing difficulties with tendency to aspiration, which necessitated hospitalisation. INVESTIGATIONS: Both lactate dehydrogenase (339 U/l) and C-reactive protein (112 mg/l) were elevated; the platelet count was low (73000/microliters). Cerebrospinal fluid was unremarkable, as was computed tomography of the skull. But magnetic resonance imaging revealed multiple spotty lesions with low contrast-medium uptake throughout the brain, pointing to a disseminated bacterial or mycotic infection. 3 days later the chest-ray showed small nodular soft shadows in the lungs, and lung functions had decreased. Mycobacteria were found in the urine and liver biopsy showed granulomatous hepatitis, establishing the diagnosis of miliary tuberculosis in the presence of silicosis. TREATMENT AND COURSE: Tuberculostatic treatment was instituted with four drugs (pyrazinamide, ethambutol, isoniazid and streptomycin. After 6 weeks the patient was again able to walk and continent of urine during the day. All cerebral functions gradually improved. CONCLUSION: Miliary tuberculosis should be included in the differential diagnosis of ill defined feverish disease, especially in the elderly. PMID- 8625787 TI - [Diagnosis of pulmonary embolism]. PMID- 8625789 TI - [Documentation of occurrence of testing in medical examinations. Decision of the Hessian administrative court of 8 August 1995]. PMID- 8625788 TI - [Chronic myeloproliferative diseases. New knowledge about well known diseases]. PMID- 8625790 TI - [Wanting a child in Hashimoto thyroiditis with hypothyreosis]. PMID- 8625791 TI - [Quality of life after cancer-caused rectal resection]. PMID- 8625792 TI - Two distinct roles for Ras in a developmentally regulated cell migration. AB - Receptor tyrosine kinases have been shown to promote cell movement in a variety of systems. The Ras protein, a well-documented downstream effector for receptor tyrosine kinases, may contribute to receptor tyrosine kinase-mediated motility. In the present study, we have examined the role of Ras in the migration of a small subset of follicle cells, known as the border cells, during Drosophila oogenesis. A dominant-negative Ras protein inhibited cell migration when expressed specifically in border cells during the period when these cells normally migrate. When expressed prior to migration, dominant-negative Ras promoted premature initiation of migration. Conversely, expression of constitutively active Ras prior to migration resulted in a significant delay in the initiation step. Furthermore, the defect in initiation of border cell migration found in slbo1, a mutation at the locus that encodes Drosophila C/EBP, was largely rescued by reducing Ras activity in border cells prior to migration. Taken together, these observations indicate that Ras activity plays two distinct roles in the border cells: (1) reduction in Ras activity promotes the initiation of that migration process and (2) Ras activity is required during border cell migration. We further examined the possible involvement of two downstream effectors of Ras in border cell migration. Raf activity was dispensable to border cell migration while reduced Ral activity inhibited initiation. We therefore suggest that Ras plays a critical role in the dynamic regulation of border cell migration via a Raf-independent pathway. PMID- 8625793 TI - Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. AB - We have recently isolated a novel cytokine, cardiotrophin-1 (CT-1), from an in vitro embryonic stem cell system of cardiogenesis that can activate embryonic markers in neonatal rat cardiac myocytes. CT-1 is a new member of the interleukin 6 (IL-6)/leukemia inhibitory factor (LIF) cytokines, which activate downstream signals via gp130-dependent pathways. To define the developmental pattern of expression of CT-1 during murine embryogenesis, we have developed antibodies directed against a CT-1 fusion protein. As assessed by immunolocalization, CT-1 is predominantly expressed in the early mouse embryonic heart tube (E8.5-10.5). In the heart, CT-1 is primarily expressed in myocardial cells, and not in endocardial cushion or outflow tract tissues. After E12.5, CT-1 expression is found in other tissues, including skeletal, liver and dorsal root ganglia. Given the effects of a related family member (ciliary neurotrophic factor, CNTF) on neuronal cell survival, we studied the ability of CT-1 to promote cardiac myocyte survival and proliferation in vitro. Both CT-1 and LIF, which share the same receptors, dramatically promote neonatal cardiac myocyte survival, while IL-6 and CNTF are without effect. A cell proliferation assay documents that CT-1 provokes an approximate 2-fold increase in embryonic cardiac myocyte proliferation. Thus, CT-1 may play an autocrine role during cardiac chamber growth and morphogenesis by promoting the survival and proliferation of immature myocytes, most likely via gp130-dependent signaling pathways. PMID- 8625794 TI - Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. AB - Newly formed somites or unsegmented paraxial mesoderm (UPM) have been cultured either in isolation or with adjacent structures to investigate the influence of these tissues on myogenic differentiation in mammals. The extent of differentiation was easily and accurately quantified by counting the number of beta-galactosidase-positive cells, since mesodermal tissues had been isolated from transgenic mice that carry the n-lacZ gene under the transcriptional control of a myosin light chain promoter, restricting expression to striated muscle. The results obtained showed that axial structures are necessary to promote differentiation of paraxial mesoderm, in agreement with previous observations. However, it also appeared that the influence of axial structures could be replaced by dorsolateral tissues, adjacent to the paraxial mesoderm. To elucidate which of these tissues exerts this positive effect, we cultured the paraxial mesoderm with a variety of adjacent structures, either adherent to the mesoderm or recombined in vitro. The results of these experiments indicated that the dorsal ectoderm exerts a positive influence on myogenesis but only if left in physical proximity to it. In contrast, lateral mesoderm delays the positive effect of the ectoderm (and has no effect on its own) suggesting that this tissue produces an inhibitory signal. To investigate whether axial structures and dorsal ectoderm induce myogenesis through common or separate pathways, we dissected the medial half of the unsegmented paraxial mesoderm and cultured it with the adjacent neural tube. We also cultured the lateral half of the unsegmented paraxial mesoderm with adjacent ectoderm. The induction of the myogenic regulatory factors myf-5 and MyoD was monitored by double staining of cultured cells with antibodies against MyoD and beta-galactosidase since the tissues were isolated from mouse embryos that carry n-lacZ targeted to the myf-5 gene, so that myf-5 expressing cells could be easily identified by either histochemical or immunocytochemical staining for beta-galactosidase. After 1 day in culture myogenic cells from the medial half expressed myf-5 but not MyoD, while myogenic cells from the lateral half expressed MyoD but not myf-5. By the next day in vitro, however, most myogenic cells expressed both gene products. These data suggest that the neural tube activates myogenesis in the medial half of paraxial mesoderm through a myf-5-dependent pathway, while the dorsal ectoderm activates myogenesis through a MyoD-dependent pathway. The possible developmental significance of these observations is discussed and a model of myogenic determination in mammals is proposed. PMID- 8625795 TI - Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors. AB - Pancreatic organogenesis has been a classic example of epitheliomesenchymal interactions. The nature of this interaction, and the way in which endocrine, acinar and ductal cell lineages are generated from the embryonic foregut has not been determined. It has generally been thought that mesenchyme is necessary for all aspects of pancreatic development. In addition islets have been thought to derive, at least in part, from ducts. We microdissected 11-day embryonic mouse pancreas and developed several culture systems for assays of differentiation: (i) on transparent filters; (ii) suspended in a collagen I gel; (iii) suspended in a basement membrane rich gel; (iv) under the renal capsule of an adult mouse. Epithelia were grown either with or without mesenchyme, and then assayed histologically and immunohistochemically. Epithelium with its mesenchyme (growth systems i-iv) always grew into fully differentiated pancreas (acinar, endocrine, adn ductal elements). In the basement membrane-rich gel, epithelium without mesenchyme formed ductal structures. Under the renal capsule of the adult mouse the epithelium without mesenchyme exclusively formed clusters of mature islets. These latter results represent the first demonstration of pure islets grown from early pancreatic precursor cells. In addition, these islets seemed not to have originated from ducts. We propose that the default path for growth of embryonic pancreatic epithelium is to form islets. In the presence of basement membrane constituents, however, the pancreatic analage epithelium appears to be programmed to form ducts. Mesenchyme seems not to be required for all aspects of pancreatic development, but rather only for the formation of acinar structures. In addition, the islets seem to form from early embryonic epithelium (which only express non acinar genes). This formation occurs without any specific embryonic signals, and without any clear duct or acinus formation. PMID- 8625796 TI - Functional cooperation between the non-paralogous genes Hoxa-10 and Hoxd-11 in the developing forelimb and axial skeleton. AB - The Abdominal B-related Hoxa-10 gene displays similar expression patterns in the differentiating forelimbs and hindlimbs of the mouse, with preferential expression around the humeral and femoral cartilages and more diffuse expression in distal regions. We found that a targeted disruption of Hoxa-10 has almost no effect in the forelimbs, while it affects the proximal hindlimb skeleton. The alterations were located along the dorsolateral side of the femur (labium laterale), with an enlargement and distal shift of the third trochanter, a misshapen lateral knee sesamoid, a supernumerary 'ligament' connecting these structures and an occasional duplication of the femoral trochlea. Some Hoxa-10-/- mutant mice developed severe degenerative alterations of the knee articulation upon ageing. Viable Hoxa-10/Hoxd-11 double mutant mice were produced by genetic intercrosses. The compound mutation resulted in synergistic forelimb phenotypic alterations, consisting of: (i) an exacerbation of Hoxd-11-/- phenotypic traits in the carpal and digital region, e.g. more pronounced truncations of the ulna styloid, pyramidal and pisiform bones and of some metacarpal and phalangeal bones and (ii) marked alterations in a more proximal region which is nearly unaffected in Hoxd-11-/- single mutants; the entire radius and ulna were truncated and thickened, with deformations of the ulna proximal extremity. Thus, functional redundancy can occur even between non-paralogous Abdominal B-related Hox genes. The double Hoxa-10/Hoxd-11 mutation also conferred full penetrance to the sacral and caudal vertebrae transformations which are approximately 50% penetrant in Hoxd-11-/- single mutants, revealing that functional cooperation can also occur between non-paralogous Hox gene products in axial skeleton patterning. PMID- 8625797 TI - Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial skeleton patterning. AB - Using gene targeting, we have produced mice with a disruption of Hoxa-9 or Hoxd 9, two paralogous Abdominal B-related genes. During embryogenesis, these genes are expressed in limb buds and along the vertebral axis with anterior expression boundaries at the level of prevertebra #20 for Hoxa-9 and #23 for Hoxd-9. Skeletal analysis revealed homeotic transformations corresponding to anteriorisations of vertebrae #21 to #25 (L1 to L5) in the lumbar region of Hoxa 9-/- mutants; vertebrae #23 to #25 (L3 to L5) in the lumbar region together with vertebrae #28, #30 and #31 (S2, S4 and Ca1) in the sacrum and tail were anteriorized in Hoxd-9-/- mutants. Thus, anteriorisation of vertebrae #23 to #25 were common to both phenotypes. Subtle forelimb (but not hindlimb) defects, corresponding to a reduction of the humerus length and malformation of its deltoid crest, were also observed in Hoxd-9-/-, but not in Hoxa-9-/-, mutant mice. By intercrosses between these two lines of mutant mice, we have produced Hoxa-9/Hoxd-9 double mutants which exhibit synergistic limb and axial malformations consisting of: (i) an increase of penetrance and expressivity of abnormalities present in the single mutants, and (ii) novel limb alterations at the level of the forelimb stylopod and additional axial skeleton transformations. These observations demonstrate that the two paralogous genes Hoxa-9 and Hoxd-9 have both specific and redundant functions in lumbosacral axial skeleton patterning and in limb morphogenesis at the stylopodal level. Taken all together, the present and previously reported results show that disruption of different Hox genes can produce similar vertebral transformations, thus supporting a combinatorial code model for specification of vertebral identity by Hox genes. PMID- 8625798 TI - Interactions between rhombomeres modulate Krox-20 and follistatin expression in the chick embryo hindbrain. AB - The rhombomeres of the embryonic hindbrain display compartment properties, including cell lineage restriction, genetic definition and modular anatomical phenotype. Consistent with the idea that rhombomeres are autonomous developmental units, previous studies have shown that certain aspects of rhombomere phenotype are determined early, at the time of rhombomere formation. By contrast, the apoptotic depletion of neural crest from rhombomeres 3 and 5 is due to an interaction with their neighbouring rhombomeres, involving the signalling molecule Bmp4. In this paper, we have examined whether inter-rhombomere interactions control further aspects of rhombomere phenotype. We find that the expression of Krox-20 and the repression of follistatin in r3 is dependent upon neighbour interaction, whereas these genes are expressed autonomously in r5. We further demonstrate that modulation of Krox-20 and follistatin expression is not dependent on Bmp4, indicating the existence of multiple pathways of interaction between adjacent rhombomeres. We also show that, although some phenotypic aspects of r3 are controlled by neighbour interactions, the axial identity of the segment is intrinsically determined. PMID- 8625799 TI - The tangled-1 mutation alters cell division orientations throughout maize leaf development without altering leaf shape. AB - It is often assumed that in plants, where the relative positions of cells are fixed by cell walls, division orientations are critical for the generation of organ shapes. However, an alternative perspective is that the generation of shape may be controlled at a regional level independently from the initial orientations of new cell walls. In support of this latter view, we describe here a recessive mutation of maize, tangled-1 (tan-1), that causes cells to divide in abnormal orientations throughout leaf development without altering overall leaf shape. In normal plants, leaf cells divide either transversely or longitudinally relative to the mother cell axis; transverse division are associated with leaf elongation and longitudinal divisions with leaf widening. In tan-l mutant leaves, cells in all tissue layers at a wide range of developmental stages divide transversely at normal frequencies, but longitudinal divisions are largely substituted by a variety of aberrantly oriented divisions in which the new cell wall is crooked or curved. Mutant leaves grow more slowly than normal, but their overall shapes are normal at all stages of their growth. These observations demonstrate that the generation of maize leaf shape does not depend on the precise spatial control of cell division, and support the general view that mechanisms independent from the control of cell division orientations are involved in the generation of shape during plant development. PMID- 8625800 TI - Prenatal and postnatal requirements of NT-3 for sympathetic neuroblast survival and innervation of specific targets. AB - Postnatal homozygous neurotrophin-3 mutant mice display a loss of about half the sympathetic superior cervical ganglion (SCG) neurons (Ernfors, P., Lee, K.-F., Kucera, J. and Jaenisch, R. (1994a) Cell 77, 503-512; Farinas, I., Jones, K. R., Backus, C., Wang, X. Y. and Reichardt, L. F. (1994) Nature 369, 658-661). We found that this loss is caused by excessive apoptosis of sympathetic neuroblasts leading to a failure to generate a normal number of neurons during neurogenesis. NT-3 was also found to be required postnatally. In Nt-3-/- mice, sympathetic fibers failed to invade pineal gland and external ear postnatally; whereas other targets of the external and internal carotid nerves, including the submandibular gland and the iris, displayed a normal complement of sympathetic innervation. Sympathetic fibers of mice carrying one functional copy of the Nt-3 gene (Nt-3+/- mice) invaded the pineal gland, but failed to branch and form a ground plexus. Cultured neonatal sympathetic neurons responded to NT-3 by neurite outgrowth and mRNA upregulation of the NT-3 receptor, trkC. Exogenously administered NT-3 promoted sympathetic growth and rescued the sympathetic target deficit of the mutant mice. We conclude that NT-3 is required for the survival of sympathetic neuroblasts during neurogenesis and for sympathetic innervation and branching in specific targets after birth. PMID- 8625801 TI - Regulative interactions in zebrafish neural crest. AB - Zebrafish trunk neural crest cells that migrate at different times have different fates: early-migrating crest cells produce dorsal root ganglion neurons as well as glia and pigment cells, while late-migrating crest cells produce only non neuronal derivatives. When presumptive early-migrating crest cells were individually transplanted into hosts such that they migrated late, they retained the ability to generate neurons. In contrast, late-migrating crest cells transplanted under the same conditions never generated neurons. These results suggest that, prior to migration, neural crest cells have intrinsic biases in the types of derivatives they will produce. Transplantation of presumptive early migrating crest cells does not result in production of dorsal root ganglion neurons under all conditions suggesting that these cells require appropriate environmental factors to express these intrinsic biases. When early-migrating crest cells are ablated, late-migrating crest cells gain the ability to produce neurons, even when they migrate on their normal schedule. Interactions among neural crest cells may thus regulate the types of derivatives neural crest cells produce, by establishing or maintaining intrinsic differences between individual cells. PMID- 8625802 TI - A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. AB - The Sox gene family consists of a large number of embryonically expressed genes related via the possession of a 79-amino-acid DNA-binding domain known as the HMG box. Partial clones for the first three Sox genes (al-a3) were isolated by homology to the HMG box of the testis-determining gene Sry and are now termed Sox 1, Sox-2 and Sox-3, Sox-3 is highly conserved amongst mammalian species and is located on the X chromosome. This has led to the proposal that Sry evolved from Sox-3. We present the cloning and sequencing of Sox-1, Sox-2 and Sox-3 from the mouse and show that Sox-3 is most closely relate to Sry. We also confirm that mouse Sox-3 is located on the X chromosome between Hprt and Dmd. Analysis of the distribution of Sox-3 RNA shows that its main site of expression is in the developing central nervous system, suggesting a role for Sox-3 in neural development. Moreover, we demonstrate that Sox-3, as well as Sox-1 and Sox-2, are expressed in the urogenital ridge and that their protein products are able to bind the same DNA sequence motif as Sry in vitro, but with different affinities. These observations prompt discussion of an evolutionary link between the genes and support the model that Sry has evolved from Sox-3. However our findings imply that if this is true, then Sry has undergone concomitant changes resulting in loss of CNS expression and altered DNA-binding properties. PMID- 8625803 TI - Maternal mRNA encoding the orphan steroid receptor SpCOUP-TF is localized in sea urchin eggs. AB - The SpCOUP-TF gene is a highly conserved sea urchin homologue of the vertebrate COUP-TFs and the Drosophila seven up subfamily of transcription factors, which are members of the orphan steroid hormone receptors. Whole-mount in situ hybridization experiments, using three sea urchin species, detect the maternal SpCOUP-TF mRNA deposited unevenly in the oocytes, mature eggs and the blastomeres of the early embryo. The localization pattern indicates that, in all three sea urchin species, the maternal SpCOUP-TF mRNA is placed in the egg in a fixed position relative to the embryonic axes, i.e. lateral to the animal-vegetal and at 45 degree angle to the oral-aboral (ventral-dorsal) axis. The embryonic expression of the SpCOUP-TF gene is spatially restricted in the oral ectoderm of the early embryo and, at later stages, in the cells of the ciliated band, the neurogenic cell lineage of the sea urchin embryo. The SpCOUP-TF mRNA, the first sea urchin maternal mRNA encoding a transcription factor that is specifically localized with respect to both embryonic axes in the egg, could be involved in early specification events in the sea urchin embryo. PMID- 8625804 TI - eagle, a member of the steroid receptor gene superfamily, is expressed in a subset of neuroblasts and regulates the fate of their putative progeny in the Drosophila CNS. AB - We isolated and characterized the eagle gene, encoding a member of the steroid receptor superfamily in Drosophila. In the central nervous system eagle RNA was expressed in a limited number of cells. During stages 10 and 11, eagle RNA expression was observed in four neuroblasts, NB2-4, NB3-3, NB6-4 and NB7-3. Except for NB6-4, eagle RNA expression reached a maximum at the very beginning of expression or in the period of neuroblast delamination. Weak eagle RNA expression was also observed in a few putative progeny of NB7-3 during stages, late 11 and 12. All eagle RNA in abdominal segments disappeared at stage 13. Using an eagle kinesin-lacZ fusion gene as a reporter, the division, migration, and axonogenesis in eagle-positive cells and their derivatives were examined. At stage 14, several types of neural or glial cells were detected which include EG and EW interneurons joining to the anterior and posterior commissures, respectively. Lack of eagle expression caused altered axonogenesis in an appreciable fraction of eagle Kinesin-LacZ-positive neurons. Some EG cells failed to acquire the neural fate or underwent an extremely delayed differentiation, while EW neurons produced neurites in abnormal directions, suggesting that eagle may play a critical role in development of the progeny of eagle-positive neuroblasts. PMID- 8625805 TI - Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity. AB - Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554-566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R. L., Laufer, E. and Tabin, C. J. (1993) Cell 25, 1401-1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994 Development 120, 3339-3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARalpha). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process. PMID- 8625807 TI - Granule cell specification in the developing mouse brain as defined by expression of the zinc finger transcription factor RU49. AB - The creation of specific neuronal cell types within the developing brain is a critical and unsolved biological problem. Precedent from invertebrate development, and from vertebrate myogenesis and lymphogenesis, has established that cell specification often involves transcription factors that are expressed throughout the differentiation of a given cell type. In this study, we have identified in Zn2+ finger transcription factor RU49 as a definitive marker for the cerebellar granule neuron lineage. Thus, RU49 is expressed in the earliest granule cell progenitors at the rhombic lip as they separate from the ventricular zone of the neural tube to generate a secondary proliferative matrix, and it continues to be expressed in differentiating and mature granule neurons. Proliferating granule cell progenitors isolated from the rhombic lip at E14 or from the external germinal layer at P6 continue to express RU49 in vitro. Both the olfactory bulb and dentate gyrus granule cell lineages also express this factor as they are generated with the developing brain. RU49 binds a novel bipartite DNA-binding element in a manner consistent with chemical rules governing the DNA-binding specificity of this class of transcription factor. The novel biochemical properties of RU49 and its restricted expression within the three lineages of CNS granule neurons suggest that RU49 may play a critical role in their specification. Furthermore, these results raise the interesting possibility that the generation of these three neuronal populations to form displaced germinative zones within the developing brain may reflect their use of a common developmental mechanism involving RU49. PMID- 8625806 TI - Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20. AB - The hindbrain is a segmented structure divided into repeating metameric units termed rhombomeres (r). The Hox family, vertebrate homologs of the Drosophila HOM C homeotic selector genes, are expressed in rhombomere-restricted patterns and are believed to participate in regulating segmental identities. Krox-20, a zinc finger gene, has a highly conserved pattern of expression in r3 and r5 and is functionally required for their maintenance in mouse embryos. Krox-20 has been shown to directly regulate the Hoxb-2 gene and we wanted to determine if it was involved in regulating multiple Hox genes as a part of its functional role. Hoxa 2 is the only known paralog of Hoxb-2, and we examined the patterns of expression of the mouse Hoxa-2 gene with particular focus on r3 and r5 in wild type and Krox 20-/- mutant embryos. There was a clear loss of expression in r3, which indicated that Hoxa-2 was downstream of Krox-20. Using transgenic analysis with E. coli lacZ reporter genes we have identified and mapped an r3/r5 enhancer in the 5' flanking region of the Hoxa-2 gene. Deletion analysis narrowed this region to an 809 bp Bg/II fragment, and in vitro binding and competition assays with bacterially expressed Krox-20 protein identified two sites within the enhancer. Mutation of these Krox-20 sites in the regulatory region specifically abolished r3/r5 activity, but did not affect neural crest and mesodermal components. This indicated that the two Krox-20 sites are required in vivo for enhancer function. Furthermore, ectopic expression of Krox-20 in r4 was able to transactivate the Hoxa 2/lacZ reporter in this rhombomere. Together our findings suggest that Krox 20 directly participates in the transcriptional regulation of Hoxa-2 during hindbrain segmentation, and is responsible for the upregulation of the r3 and r5 domains of expression of both vertebrate group 2 Hox paralogs. Therefore, the segmental phenotypes in the Krox-20 mutants are likely to reflect the role of Krox-20 in directly regulating multiple Hox genes. PMID- 8625808 TI - REGA-1 is a GPI-linked member of the immunoglobulin superfamily present on restricted regions of sheath cell processes in grasshopper. AB - REGA-1 is a glycoprotein localized to sheath cell processes in the developing CNS when NBs are producing progeny and neurons are maturing and extending processes. It is also present on a subset of muscles and on the lumenal surface of the ectoderm in the embryonic appendages when pioneer neurons are growing into the CNS. REGA-1 is associated with the extracellular side of the cell membrane by a glycosyl-phosphatidylinositol linkage. We have identified a cDNA clone encoding REGA-1 using a sequence from purified protein. Sequence analysis defines REGA-1 as a novel member of the immunoglobulin superfamily containing three immunoglobulin domains and one fibronectin type III repeat. Each Ig domain has distinct sequence characteristics that suggest discrete functions. REGA-1 is similar to other Ig superfamily members involved in cell adhesion events and neurite outgrowth. PMID- 8625809 TI - Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development. AB - Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, we have examined a collection of Drosophila female-sterile mutants for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of bicoid and Toll mRNAs. Cortex embryos contain a bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly(A)) tail. Injection of polyadenylated bicoid mRNA into cortex embryos allows translation demonstrating that insufficient polyadenylation prevents endogenous bicoid mRNA translation. In contrast nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. Cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development. PMID- 8625810 TI - Hikaru genki protein is secreted into synaptic clefts from an early stage of synapse formation in Drosophila. AB - The development of neural circuits is regulated by a large number of factors that are localized at distinct neural sites. We report here the localization of one of these factors, hikaru genki (hig) protein, at synaptic clefts in the pupal and adult nervous systems of Drosophila. In hig mutants, unusually frequent bursting activity of the muscles and abnormal motor behavior during the adult stage suggest the misfunction of neuromuscular circuitry. Our immunohistochemical analyses revealed that hig protein, produced by neurons, is secreted from the presynaptic terminals into the spaces between the presynaptic and postsynaptic terminals. In addition, we have found that the localization of this protein in the synaptic spaces temporally correlates with its functional requirement during a critical period that occurs in the middle stage of pupal formation, a period when a number of dendrite and axon growth cones meet to form synapses. These findings indicate that hig protein functions in the formation of functional neural circuits from the early stages of synapse formation. PMID- 8625811 TI - Unequal cleavage in leech embryos: zygotic transcription is required for correct spindle orientation in subset of early blastomeres. AB - Leech embryos undergo invariant sequences of equal and unequal cell divisions to give rise to identifiable progeny cells. While many of the early cleavages are under maternal control, the divisions of a subset of early blastomeres (the large cells of the D' lineage) are perturbed after the inhibition of zygotic transcription. Analysis of the different types of cells produced in embryos injected with the transcriptional inhibitor, alpha-amanitin, revealed that the symmetry of cell division is perturbed in these large D'-derived cells during this early period of development. These cells, which would normally undergo a series of equal and unequal cleavages, always undergo equal cleavages after the inhibition of zygotic transcription. It appears that zygotically transcribed gene product(s) are required in the large cells of the D' lineage to orient the mitotic spindles properly for these unequal cell cleavages. PMID- 8625812 TI - Regulative capacity of the archenteron during gastrulation in the sea urchin. AB - Gastrulation in the sea urchin involves an extensive rearrangement of cells of the archenteron giving rise to secondary mesenchyme at the archenteron tip followed by the foregut, midgut and hindgut. To examine the regulative capacity of this structure, pieces of the archenteron were removed or transplanted at different stages of gastrulation. After removal of any or all parts of the archenteron, the remaining veg 1 and /or veg 2 tissue regulated to replace the missing parts. Endoderm transplanted to ectopic positions also regulated to that new position in the archenteron. This ability to replace or regulate endoderm did not decline until after full elongation of the archenteron was completed. When replacement occurred, the new gut was smaller relative to the remaining embryo but the recognizable morphology of the archenteron was re-established. Long after the archenteron reveals territorial specification through expression of specific markers, the endodermal cells remain capable of being respecified to other gut regions. Thus, for much of gastrulation, the gut is conditionally specified. We propose that this regulative ability requires extensive and continuous short range communication between cells of the archenteron in order to reorganize the tissues and position the boundaries of this structure even after experimental alterations. PMID- 8625813 TI - Autonomous and nonautonomous Notch functions for embryonic muscle and epidermis development in Drosophila. AB - The Notch (N) gene encodes a cell signaling protein that mediates neuronal and epidermal determination in Drosophila embryos. N also regulates several aspects of myogenic development; embryos lacking N function have too many muscle founder cells and fail to properly differentiate somatic muscle. To identify cell autonomous requirements for Notch function during muscle development, we expressed a Notch minigene in the mesoderm, but not in the ectoderm, of amorphic N-embryos. In these embryos, muscle founder hypertrophy is rescued, indicating that Notch is autonomously required by mesoderm cells to regulate the proper number of muscle founders. However, somatic muscle differentiation is only partially normalized, suggesting that Notch is also required in the ectoderm for proper muscle development. Additionally, mesodermal expression of Notch partially rescues epidermal development in overlying neurogenic ectoderm. This is unexpected, since previous studies suggest that Notch is autonomously required by proneural ectoderm cells for epidermal development. Mesodermal expression of a truncated Notch protein lacking the extracellular domain does not rescue ventral epidermis, suggesting that the extra-cellular domain of Notch can non autonomously rescue epidermal development across germ layers. PMID- 8625814 TI - Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene. AB - The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution. PMID- 8625815 TI - Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor. AB - Previous studies have shown that heparin-binding epidermal growth factor (EGF) like growth factor (HB-EGF) mRNA is synthesized in the mouse uterine luminal epithelium temporally, just prior to implantation, and spatially, only at the site of blastocyst apposition (Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K. and Dey, S. K. (1994) Development 120, 1071-1083). HB-EGF is synthesized as a transmembrane protein (HB-EGF TM) that can be processed to release the soluble growth factor. An antibody that cross-reacts only with the transmembrane form detected HB-EGF TM in uterine luminal epithelium in a spatial manner similar to that of HB-EGF mRNA. HB-EGF TM is a juxtacrine growth factor that mediates cell-cell contact. To ascertain if HB EGF TM could be an adhesion factor for blastocysts, a mouse cell line synthesizing human HB-EGF TM was co-cultured with mouse blastocysts. Cells synthesizing HB-EGF TM adhered to day-4 mouse blastocysts more extensively than parental cells or cells synthesizing a constitutively secreted form of HB-EGF. Adhesion of cells synthesizing HB-EGF TM to blastocysts was inhibited by excess recombinant HB-EGF but less so by TGF-alpha. Adhesion was also inhibited by the synthetic peptide P21 corresponding to the HB-EGF heparin binding domain, and by incubating the blastocysts with heparinase. In addition, adhesion to delayed implanting dormant blastocysts, which lack EGF receptor (EGFR), was diminished relative to normal blastocysts. These results suggested that adhesion between blastocysts and cells synthesizing HB-EGF TM was mediated via interaction with both blastocyst EGFR and heparan sulfate proteoglycan (HSPG). It was concluded that HB-EGF TM, which is synthesized exclusively in the luminal epithelium at the site of blastocyst apposition, and which is a juxtacrine adhesion factor for blastocysts, could be one of the mediators of blastocyst adhesion to the uterus in the process of implantation. PMID- 8625816 TI - 6B4 proteoglycan/phosphacan is a repulsive substratum but promotes morphological differentiation of cortical neurons. AB - 6B4 proteoglycan/phosphacan is one of the major phosphate-buffered saline-soluble chondroitin sulfate proteoglycans of the brain. Recently, this molecule has been demonstrated to be an extracellular variant of the proteoglycan-type protein tyrosine phosphatase, PTPzeta (RPTPbeta). The influence of the 6B4 proteoglycan, adsorbed onto the substratum, on cell adhesion and neurite outgrowth was studied using dissociated neurons from the cerebral cortex and thalamus. 6B4 proteoglycan adsorbed onto plastic tissue culture dishes did not support neuronal cell adhesion, but rather exerted repulsive effects on cortical and thalamic neurons. When neurons were densely seeded on patterned substrata consisting of a grid-like structure of alternating poly-L-lysine and 6B4 proteoglycan-coated poly-L-lysine domains, they were concentrated on the poly-L-lysine domains. However, 6B4 proteoglycan did not retard the differentiation of neurons but rather promoted neurite outgrowth and development of the dendrites of cortical neurons, when neurons were sparsely seeded on poly-L-lysine-conditioned coverslips continuously coated with 6B4 proteoglycan. This effect of 6B4 proteoglycan on the neurite extension of cortical neurons was apparent even on coverslips co-coated with fibronectin or tenascin. By contrast, the neurite extension of thalamic neurons was not modified by 6B4 proteoglycan. Chondroitinase ABC or keratanase digestion of 6B4 proteoglycan did not affect its neurite outgrowth promoting activity, but a polyclonal antibody against 6B4 proteoglycan completely suppressed this activity, suggesting that a protein moiety is responsible for the activity. 6B4 proteoglycan transiently promoted tyrosine phosphorylation of an 85x10(3) Mr protein in the cortical neurons, which correlated with the induction of neurite outgrowth. These results suggest that 6B4 proteoglycan/phosphacan modulates morphogenesis and differentiation of neurons dependent on its spatiotemporal distribution and the cell types in the brain. PMID- 8625817 TI - A critical period for the specification of motor pools in the chick lumbosacral spinal cord. AB - When 3-4 segments of the chick lumbosacral neural tube are reversed in the anterior-posterior axis at stage 15 (embryonic day 2.5), the spinal cord develops with a reversed organization of motoneurons projecting to individual muscles in the limb (C. Lance-Jones and L. Landmesser (1980) J. Physiol. 302, 581-602). This finding indicated that motoneuron precursors or components of their local environment were specified with respect to target by stage 15. To identify the timing of this event, we have now assessed motoneuron projections after equivalent neural tube reversals at earlier stages of development. Lumbosacral neural tube segments 1-3 (+/- one segment cranial or caudal) were reversed in the anterior-posterior axis at stages 13 and 14 (embryonic day 2). The locations of motoneurons innervating two thigh muscles, the sartorius and femorotibialis, were mapped via retrograde horseradish peroxidase labeling at stages 35-36 (embryonic days 9-10). In a sample of embryos, counts were made of the total number of motoneurons in the lateral motor columns of reversed segments. The majority of motoneurons projecting to the sartorius and femorotibialis were in a normal position within the spinal cord. Segmental differences in motor column size were also similar to normal. These observations indicate that positional cues external to the LS neural tube can affect motoneuron commitment and number at stages 13 14. Since these observations stand in contrast to results following stage 15 reversals, we conclude that regional differences related to motoneuron target identity are normally specified or stabilized within the anterior LS neural tube between stages 14 and 15. To examine the role of the notochord in this process, neural tube reversals were performed at stages 13-14 as described above, except that the underlying notochord was also reversed. Projections to the sartorius and femorotibialis muscles did not differ significantly from those in embryos with neural tube reversals alone, indicating that the notochord is not the source of cues for target identity at stages 13-14. PMID- 8625818 TI - The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell migration. AB - The assembly of the nervous system in the nematode C. elegans requires the directed migrations of cells and growth comes along the anteroposterior and dorsoventral body axis. We show here that the gene vab-8 is essential for most posteriorly directed migrations of cells and growth cones. Mutations in vab-8 disrupt fourteen of seventeen posteriorly directed migrations, but only two of seventeen anteriorly directed and dorsoventral migrations. For two types of neurons that extend axons both anteriorly and posteriorly, vab-8 mutations disrupt only the growth of the posteriorly directed axon. vab-8 encodes two genetic activities that function in the guidance of different migrations. Our results suggest that most posteriorly directed cell and growth cone migrations are guided by a common mechanism involving the vab-8 gene. PMID- 8625819 TI - Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark- and light-grown Arabidopsis seedlings. AB - Plant morphogenesis is dependent on a tight control of cell division and expansion. Cell elongation during post-embryonic hypocotyl growth is under the control of a light-regulated developmental switch. Light is generally believed to exert its effects on hypocotyl elongation through a phytochrome-and blue-light receptor-mediated inhibitory action on a so far unknown cell elongation mechanism. We describe here a new class of allelic mutants in Arabidopsis, at the locus PROCUSTE1 (prc1-1 to -4), which have a hypocotyl elongation defect specifically associated with the dark-grown development program. Normal hypocotyl elongation is restored in plants grown in white, blue or red light. In agreement with this, the constitutive photomorphogenic mutation cop1-6, which induces a de etiolated phenotype in the dark, is epistatic to prc1-2 for the hypocotyl phenotype. Epistasis analyses in red and blue light respectively, indicate that phytochrome B but not the blue light receptor HY4, is required for the switch from PRC1-dependent to PRC1-independent elongation. The conditional hypocotyl growth defect is associated with a deformation of the hypocotyl surface due to an uncontrolled swelling of epidermal, cortical or endodermal cells, suggesting a defect in the structure of the expanding cell wall. A similar phenotype was observed in elongating roots, which was however, independent of the light conditions. The aerial part of mature mutant plants grown in the light was indistinguishable from the wild type. prc1 mutants provide a means of distinguishing, for the first time, two genetic pathways regulating hypocotyl cell elongation respectively in dark- and light-grown seedlings, whereby light not only inhibits hypocotyl growth, but also activates a PRC1-independent cell elongation program. PMID- 8625820 TI - Bax promotes neuronal survival and antagonises the survival effects of neurotrophic factors. AB - Bcl-2, Bcl-x and Bax are members fo a family of cytoplasmic proteins that influence cell survival. Whereas increased expression of Bcl-2 or Bcl-x promotes cell survival following withdrawal of survival factors, increased expression of Bax is thought to suppress survival. To investigate the potential roles of these proteins in regulating the survival of developing neurons, we compared the effects of overexpressing these proteins in embryonic neurons deprived of different neurotrophic factors in vitro. Surprisingly, overexpression of Bax rescued populations of sensory neurons deprived of nerve growth factor, as did overexpression of Bcl-2 and two Bcl-x variants, Bcl-XL and Bcl-Xbeta. Bax also enhanced the survival of ciliary neurons deprived of ciliary neurotrophic factor, although this effect was short-lived. Whereas Bcl-2 overexpression did not affect the survival response of neurons to neurotrophic factors, Bax overexpression partially inhibited the action of neurotrophic factors. Co-injection of Bcl-2 and Bax expression vectors promoted the survival of neurotrophic factor-deprived neurons if either was in excess, but failed to rescue neurons if they injected at a 1:1 ratio. Our findings demonstrate that Bax can promote the survival of neurotrophic factor-deprived neurons and that its effect on survival is dominant to that of neurotrophic factors. Our results also argue that the relative amounts of Bcl-2 and Bax are critical in regulating neuronal survival. PMID- 8625821 TI - An extracellular matrix molecule that is selectively expressed during development is important for gastrulation in the sea urchin embryo. AB - The extracellular matrix is important in the regulation of many cellular events of early development including migration, shape change, proliferation and gene expression. In the sea urchin embryo, disruption of the extracellular matrix results in selective defects in each of these events during gastrulation. Here we describe a new molecule of the extracellular matrix in Lytechinus variegatus, referred to as ECM 18, that has several important features. First, antibody interference of ECM 18 results in a profound but reversible inhibition of primary mesenchyme cell organization and endoderm morphogenesis during gastrulation. Second, during gastrulation, ECM 18 mRNA accumulates to highest levels in the invaginating endoderm and the ECM 18 protein deposited in the basal lamina surrounding the archenteron as well as in other areas of the blastocoel wall. Immunolocalization by fluorescence and electron microscopy demonstrates the selective accumulation of ECM 18 in the extracellular matrix. Third, although the mRNA encoding ECM 18 is present throughout development, the protein accumulates only during gastrulation. ECM 18 protein is not detected in eggs or early embryos and analysis of polysome-associated mRNA suggests that at least part of the translational regulation of ECM 18 is at the level of ECM 18 mRNA-polysome formation. Finally, sequence analysis of ECM 18 shows that the protein contains a repeat sequence with a conserved cysteine motif, suggestive of involvement in protein-protein interactions. Thus, ECM 18 appears to be important in mediating select morphogenetic changes during gastrulation and the pattern of its expression in the embryo is unique among the extracellular matrix molecules known in this embryo. PMID- 8625822 TI - The expression of trkB and p75 and the role of BDNF in the developing neuromuscular system of the chick embryo. AB - The neurotrophin, brain-derived neurotrophic factor, prevents motoneuron cell death during the normal development of the chick embryo. Brain-derived neurotrophic factor is a ligand for the low-affinity NGF receptor, p75, and for the high-affinity neurotrophin receptor, trkB. If motoneurons respond directly to brain-derived neurotrophic factor then they must possess at least one, and possibly both, of these receptors during the period of naturally occurring cell death. Histological sections from the lumbar region of chick embryos were probed for the presence of trkB and p75 mRNA using digoxigenin-labeled anti-sense RNA probes. p75 mRNA was present in spinal cord motoneurons at stages of development that correlate with motoneuron cell death. Immunohistochemical localization also revealed that p75 protein was present in motoneurons, primarily along the ventral roots and developing intramuscular nerves. In contrast trkB mRNA was not present in chick motoneurons until after the process of cell death was underway. The timing of trkB expression suggested that some motoneurons, i.e., those that die prior to the onset of trkB expression, may be insensitive to brain-derived neurotrophic factor. This was confirmed by comparing the number of surviving motoneurons following different in vivo treatment paradigms. The evidence indicates that motoneurons undergo a temporal shift in sensitivity to brain derived neurotrophic factor. PMID- 8625823 TI - A group of genes required for maintenance of the amnioserosa tissue in Drosophila. AB - The amnioserosa is an extraembryonic, epithelial tissue that covers the dorsal side of the Drosophila embryo. The initial development of the amnioserosa is controlled by the dorsoventral patterning genes. Here we show that a group of genes, which we refer to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, we examined amnioserosa development in the ush-group mutants: u-shaped (ush), hindsight (hnt), serpent (srp) and tail-up (tup). Our results show that the amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. We demonstrate that this cell loss is a consequence of programmed cell death (apoptosis) in ush, hnt and srp, but not in tup. We discuss the role of the ush-group genes in maintaining the amnioserosa's viability. We also discuss a possible role for the amnioserosa in germ-band retraction in light of these mutants' unretracted phenotype. PMID- 8625824 TI - The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. AB - We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth. PMID- 8625825 TI - Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. AB - We have shown previously by means of quail/chick transplantations that external and visceral organs, i.e., somatopleural and splanchnopleural derivatives, acquire their endothelial network through different mechanisms, namely immigration (termed angiogenesis) versus in situ emergence of precursors (or vasculogenesis). We have traced the distribution of QH1-positive cells in chick hosts after replacement of the last somites by quail somites (orthotopic grafts) or lateral plate mesoderm (heterotopic grafts). The results lead to the conclusion that the embryo becomes vascularized by endothelial precursors from two distinct regions, splanchnopleural mesoderm and paraxial mesoderm. The territories respectively vascularized are complementary, precursors from the paraxial mesoderm occupy the body wall and kidney, i.e., they settle along with the other paraxial mesoderm derivatives and colonize the somatopleure. The precursors from the two origins have distinct recognition and potentialities properties: endothelial precursors of paraxial origin are barred from vascularizing visceral organs and from integrating into the floor of the aorta, and are never associated with hemopoiesis; splanchnopleural mesoderm grafted in the place of somites, gives off endothelial cells to body wall and kidney but also visceral organs. It gives rise to hemopoietic precursors in addition to endothelial cells. PMID- 8625826 TI - lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H). AB - The homologous receptors LIN-12 and GLP-1 mediate diverse cell-signaling events during development of the nematode Caenorhabditis elegans. These two receptors appear to be functionally interchangeable and have sequence similarity to Drosophila Notch. Here we focus on a molecular analysis of the lag-1 gene (lin-12 -and glp-1), which plays a central role in LIN-12 and GLP-1-mediated signal transduction. We find that the predicted LAG-1 protein is homologous to two DNA binding proteins: human C Promoter Binding Factor (CBF1) and Drosophila Suppressor of Hairless (Su(H)). Furthermore, we show that LAG-1 binds specifically to the DNA sequence RTGGGAA, previously identified as a CBF-1/Su(H) binding site. Finally, we report that the 5' flanking regions and first introns of the lin-12, glp-1 and lag-1 genes are enriched for potential LAG-1-binding sites. We propose that LAG-1 is a transcriptional regulator that serves as a primary link between the LIN-12 and GLP-1 receptors and downstream target genes in C. elegans. In addition, we propose that LAG-1 may be a key component of a positive feedback loop that amplifies activity of the LIN-12/GLP-1 pathway. PMID- 8625827 TI - Retinoic acid signaling is required during early chick limb development. AB - In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity. PMID- 8625828 TI - Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. AB - The tracheal (respiratory) system of Drosophila melanogaster is a branched network of epithelial tubes that ramifies throughout the body and transports oxygen to the tissues. It forms by a series of sequential branching events in each hemisegment from T2 to A8. Here we present a cellular and initial genetic analysis of the branching process. We show that although branching is sequential it is not iterative. The three levels of branching that we distinguish involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. We further show that the breathless FGF receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching and that the pointed ETS-domain transcription factor is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy. PMID- 8625829 TI - The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. AB - We have previously shown that mice carrying a null mutation in the homeobox gene ipf1, now renamed to pdx1, selectively lack a pancreas. To elucidate the level at which PDX1 is required during the development of the pancreas, we have in this study analyzed the early stages of pancreas ontogeny in PDX-/- mice. These analyses have revealed that the early inductive events leading to the formation of the pancreatic buds and the appearance of the early insulin and glucagon cells occur in the PDX1-deficient embryos. However, the subsequent morphogenesis of the pancreatic epithelium and the progression of differentiation of the endocrine cells are arrested in the pdx1-/- embryos. In contrast, the pancreatic mesenchyme grows and develops, both morphologically and functionally, independently of the epithelium. We also show that the pancreatic epithelium in the pdx1 mutants is unable to respond to the mesenchymal-derived signal(s) which normally promote pancreatic morphogenesis. Together these data provide evidence that PDX-1 acts cell autonomously and that the lack of a pancreas in the pdx1-/- mice is due to a defect in the pancreatic epithelium. PMID- 8625830 TI - The chinless mutation and neural crest cell interactions in zebrafish jaw development. AB - During vertebrate development, neural crest cells are thought to pattern many aspects of head organization, including the segmented skeleton and musculature of the jaw and gills. Here we describe mutations at the gene chinless, chn, that disrupt the skeletal fates of neural crest cells in the head of the zebrafish and their interactions with muscle precursors. chn mutants lack neural-crest-derived cartilage and mesoderm-derived muscles in all seven pharyngeal arches. Fate mapping and gene expression studies demonstrate the presence of both undifferentiated cartilage and muscle precursors in mutants. However, chn blocks differentiation directly in neural crest, and not in mesoderm, as revealed by mosaic analyses. Neural crest cells taken from wild-type donor embryos can form cartilage when transplanted into chn mutant hosts and rescue some of the patterning defects of mutant pharyngeal arches. In these cases, cartilage only forms if neural crest is transplanted at least one hour before its migration, suggesting that interactions occur transiently in early jaw precursors. In contrast, transplanted cells in paraxial mesoderm behave according to the host genotype; mutant cells form jaw muscles in a wild-type environment. These results suggest that chn is required for the development of pharyngeal cartilages from cranial neural crest cells and subsequent crest signals that pattern mesodermally derived myocytes. PMID- 8625831 TI - Effects of GGF/neuregulins on neuronal survival and neurite outgrowth correlate with erbB2/neu expression in developing rat retina. AB - We have examined the expression of neuregulin and its putative receptors, erbB2/neu, erbB3 and erbB4/tyro2 during retinal development, and tested several potential functions of this class of molecules in dissociated rat retinal cell cultures. At least one form of neuregulin is expressed in the retina, from the earliest stages of retinal development examined; in addition, all three of the known receptors are expressed by retinal neurons in a developmentally regulated manner. When added to cultures of embryonic or neonatal rat retinal cells, neuregulin (rhGGF2) promotes survival and neurite extension from retinal neurons in a dose-dependent manner. These results indicate that in addition to their well described effects on glia, the neuregulins also have direct effects on central nervous system neurons. PMID- 8625832 TI - Contrasting effects of protein synthesis inhibition and of cyclic AMP on apoptosis in the developing retina. AB - The role of protein synthesis in apoptosis was investigated in the retina of developing rats. In the neonatal retina, a ganglion cell layer, containing neurons with long, centrally projecting axons, is separated from an immature neuroblastic layer by a plexiform layer. This trilaminar pattern subsequently evolves to five alternating cell and plexiform layers that constitute the mature retina and a wave of programmed neuron death sweeps through the layers. Apoptosis due to axon damage was found in ganglion cells of retinal explants within 2 days in vitro and was prevented by inhibition of protein synthesis. Simultaneously, protein synthesis blockade induced apoptosis among the undamaged cells of the neuroblastic layer, which could be selectively prevented by an increase in intracellular cyclic AMP. Both the prevention and the induction of apoptosis among ganglion cells or neuroblastic cells, respectively, occurred after inhibition of protein synthesis in vivo. The results show the coexistence of two mechanisms of apoptosis within the organized retinal tissue. One mechanism is triggered in ganglion cells by direct damage and depends on the synthesis of proteins acting as positive modulators of apoptosis. A distinct, latent mechanism is found among immature neuroblasts and may be repressed by continuously synthesized negative modulators, or by an increase in intracellular cyclic AMP. PMID- 8625833 TI - Analysis of Hox gene expression in the chick limb bud. AB - The vertebrate Hox genes have been shown to be important for patterning the primary and secondary axes of the developing vertebrate embryo. The function of these genes along the primary axis of the embryo has been generally interpreted in the context of positional specification and homeotic transformation of axial structures. The way in which these genes are expressed and function during the development of the secondary axes, particularly the limb, is less clear. In order to provide a reference for understanding the role of the Hox genes in limb patterning, we isolated clones of 23 Hox genes expressed during limb development, characterized their expression patterns and analyzed their regulation by the signalling centers which pattern the limb. The expression patterns of the Abd-B related Hoxa and Hoxd genes have previously been partially characterized; however, our study reveals that these genes are expressed in patterns more dynamic and complex than generally appreciated, only transiently approximating simple, concentric, nested domains. Detailed analysis of these patterns suggests that the expression of each of the Hoxa and Hoxd genes is regulated in up to three independent phases. Each of these phases appears to be associated with the specification and patterning of one of the proximodistal segments of the limb (upper arm, lower arm and hand). Interestingly, in the last of these phases, the expression of the Hoxd genes violates the general rule of spatial and temporal colinearity of Hox gene expression with gene order along the chromosome. In contrast to the Abd-B-related Hoxa and Hoxd genes, which are expressed in both the fore and hind limbs, different sets of Hoxc genes are expressed in the two limbs. There is a correlation between the relative position of these genes along the chromosome and the axial level of the limb bud in which they are expressed. The more 3' genes are expressed in the fore limb bud while the 5' genes are expressed in the hind limb bud; intermediate genes are transcribed in both limbs. However, there is no clear correlation between the relative position of the genes along the chromosome and their expression domains within the limb. With the exception of Hoxc-11, which is transcribed in a posterior portion of the hind limb, Hoxc gene expression is restricted to the anterior/proximal portion of the limb bud. Importantly, comparison of the distributions of Hoxc-6 RNA and protein products reveals posttranscriptional regulation of this gene, suggesting that caution must be exercised in interpreting the functional significance of the RNA distribution of any of the vertebrate Hox genes. To understand the genesis of the complex patterns of Hox gene expression in the limb bud, we examined the propagation of Hox gene expression relative to cell proliferation. We find that shifts in Hox gene expression cannot be attributed to passive expansion due to cell proliferation. Rather, phase-specific Hox gene expression patterns appear to result from a context-dependent response of the limb mesoderm to Sonic hedgehog. Sonic hedgehog (the patterning signal from the Zone of Polarizing Activity) is known to be able to activate Hoxd gene expression in the limb. Although we find that Sonic hedgehog is capable of initiating and polarizing Hoxd gene expression during both of the latter two phases of Hox gene expression, the specific patterns induced are not determined by the signal, but depend upon the temporal context of the mesoderm receiving the signal. Misexpression of Sonic hedgehog also reveals that Hoxb-9, which is normally excluded from the posterior mesenchyme of the leg, is negatively regulated by Sonic hedgehog and that Hoxc 11, which is expressed in the posterior portion of the leg, is not affected by Sonic hedgehog and hence is not required to pattern the skeletal elements of the lower leg. PMID- 8625834 TI - Specification of the anteroposterior axis in Caenorhabditis elegans. AB - Anteroposterior asymmetries are apparent in C elegans development before the first cell division. Here we identify the cue that specifies the anteroposterior axis, and investigate how this cue is interpreted to generate initial asymmetry. In C. elegans, the sperm normally enters the egg in an invariant position. We have found that causing fertilisation to occur in the abnormal end of the egg completely reverses the orientation of the anteroposterior axis, but gives otherwise normal development. This result suggests that a component of the sperm normally specifies the anteroposterior axis. We have found that a cytoplasmic rearrangement in the uncleaved zygote is directed by the sperm, suggesting a mechanism by which the sperm may specify the axis. The results additionally reveal that the C elegans oocyte is constructed with no axis prespecified in the form of asymmetrically localised cytoplasmic determinants. PMID- 8625835 TI - Notochord signals control the transcriptional cascade of myogenic bHLH genes in somites of quail embryos. AB - Microsurgical, tissue grafting and in situ hybridization techniques have been used to investigate the role of the neural tube and notochord in the control of the myogenic bHLH genes, QmyoD, Qmyf5, Qmyogenin and the cardiac alpha-actin gene, during somite formation in stage 12 quail embryos. Our results reveal that signals from the axial neural tube/notochord complex control both the activation and the maintenance of expression of QmyoD and Qmyf5 in myotomal progenitor cells during the period immediately following somite formation and prior to myotome differentiation. QmyoD and Qmyf5 expression becomes independent of axial signals during myotome differentiation when somites activate expression of Qmyogenin and alpha-actin. Ablation studies reveal that the notochord controls QmyoD activation and the initiation of the transcriptional cascade of myogenic bHLH genes as epithelial somites condense from segmental plate mesoderm. The dorsal medial neural tube then contributes to the maintenance of myogenic bHLH gene expression in newly formed somites. Notochord grafts can activate ectopic QmyoD expression during somite formation, establishing that the notochord is a necessary and sufficient source of diffusible signals to initiate QmyoD expression. Myogenic bHLH gene expression is localized to dorsal medial cells of the somite by inhibitory signals produced by the lateral plate and ventral neural tube. Signaling models for the activation and maintenance of myogenic gene expression and the determination of myotomal muscle in somites are discussed. PMID- 8625836 TI - Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein. AB - While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. PMID- 8625837 TI - Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. AB - Morphogenesis is characterized by orchestrated changes in the shape and position of individual cells. Many of these movements are thought to be powered by motor proteins. However, in metazoans, it is often difficult to match specific motors with the movements they drive. The nonmuscle myosin II heavy chain (MHC encoded by zipper is required for cell sheet movements in Drosophila embryos. To determine if myosin II is required for other processes, we examined the phenotypes of strong and weak larval lethal mutations in spaghetti squash (sqh), which encodes the nonmuscle myosin II regulatory light chain (RLC). sqh mutants can be rescued to adulthood by daily induction of a sqh cDNA transgene driven by the hsp70 promoter. By transiently ceasing induction of the cDNA, we depleted RLC at specific times during development. When RLC is transiently depleted in larvae, the resulting adult phenotypes demonstrate that RLC is required in a stage specific fashion for proper development of eye and leg imaginal discs. When RLC is depleted in adult females, oogenesis is reversibly disrupted. Without RLC induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping). Finally, we show that in sqh mutant tissues, MHC is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail-binding protein p127. This suggests that sqh mutant phenotypes are chiefly caused by sequestration of myosin into inactive aggregates. These results show that myosin II is responsible for a surprisingly diverse array of cell shape changes throughout development. PMID- 8625838 TI - A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton. AB - Segment identity in both invertebrates and vertebrates is conferred by spatially restricted distribution of homeotic gene products. In Drosophila, the expression of Homeobox genes during embryogenesis is initially induced by segmentation gene products and then maintained by Polycomb group and Trithorax group gene products. Polycomb group gene homologs are conserved in vertebrates. Murine mel-18 and closely related bmi-1 are homologous to posterior sex combs and suppressor two of zeste. Mel-18 protein mediates a transcriptional repression via direct binding to specific DNA sequences. To gain further insight into the function of Mel-18, we have inactivated the mel-18 locus by homologous recombination. Mice lacking mel 18 survive to birth and die around 4 weeks after birth after exhibiting strong growth retardation. Similar to the Drosophila posterior sex combs mutant, posterior transformations of the axial skeleton were reproducibly observed in mel 18 mutants. The homeotic transformations were correlated with ectopic expression of Homeobox cluster genes along the anteroposterior axis in the developing paraxial mesoderm. Surprisingly, mel-18-deficient phenotypes are reminiscent of bmi-1 mutants. These results indicate that the vertebrate Polycomb group genes mel-18 and bmi-1, like Drosophila Polycomb group gene products, might play a crucial role in maintaining the silent state of Homeobox gene expression during paraxial mesoderm development. PMID- 8625839 TI - Fates and migratory routes of primitive streak cells in the chick embryo. AB - We have used carbocyanine dyes to fate map the primitive streak in the early chick embryo, from stages 3+ (mid-primitive streak) to 9 (8 somites). We show that presumptive notochord, foregut and medial somite do not originate solely from Hensen's node, but also from the anterior primitive streak. At early stages (4- and 4), there is no correlation between specific anteroposterior levels of the primitive streak and the final position of their descendants in the notochord. We describe in detail the contribution of specific levels of the primitive streak to the medial and lateral halves of the somites. To understand how the descendants of labelled cells reach their destinations in different tissues, we have followed the movement of labelled cells during their emigration from the primitive streak in living embryos, and find that cells destined to different structures follow defined pathways of movement, even if they arise from similar positions in the streak. Somite and notochord precursors migrate anteriorly within the streak and pass through different portions of the node; this provides an explanation for the segregation of notochord and somite territories in the node. PMID- 8625840 TI - Pathways for inflorescence and floral induction in Antirrhinum. AB - The presentation of flowers on a modified stem, the inflorescence, requires the integration of several aspects of meristem behaviour. In Antirrhinum, the inflorescence can be distinguished by its flowers, hairy stem, modified leaves, short internodes and spiral phyllotaxy. We show, by a combination of physiological, genetical and morphological analysis, that the various aspects of the inflorescence are controlled by three pathways. The first pathway, depends on expression of the floricaula gene, and is rapidly and discretely induced by exposure to long daylength. Activation of this pathway occurs in very young axillary meristems, resulting in a floral identity. In addition, the length of subtending leaves and hairiness of the stem are partially modified. The second pathway affects leaf size, internode length, and stem hairiness, but does not confer floral meristem identity. This pathway is induced by long daylength, but not as rapidly or discretely as the floricaula-dependent pathway. The third pathway controls the switch in phyllotaxy from decussate to spiral and is activated independently of daylength. The coordination of these three programmes ensures that apical and axillary meristem behaviour is integrated. PMID- 8625841 TI - Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos. AB - Injection of RNA encoding BMP-4 into the early Xenopus embryo suppresses formation of dorsal and anterior cell types. To understand this phenomenon, it is necessary to know the stage at which BMP-4 acts. In this paper, we present three lines of evidence showing that BMP-4 misexpression has no effect on the initial steps of mesoderm induction, either dorsal or ventral, but instead causes ventralization during gastrulation. Firstly, activation of organizer-specific genes such as goosecoid, Xnot, pintallavis and noggin occurs normally in embryos injected with BMP-4 RNA, but transcript levels are then rapidly down-regulated as gastrulation proceeds. Similarly, BMP-4 does not affect the initial activation of goosecoid by activin in animal caps, but expression then declines precipitously. Secondly, embryos made ventral by injection with BMP-4 RNA cannot be rescued by grafts of Spemann's organizer at gastrula stages. Such embryos therefore differ from those made ventral by UV-irradiation, where the defect occurs early and rescue can be effected by the organizer. Finally, the dorsalizing effects of the organizer, and of the candidate dorsalizing signal noggin, both of which exert their effects during gastrulation, can be counteracted by BMP-4. Together, these experiments demonstrate that BMP-4 can act during gastrulation both to promote ventral mesoderm differentiation and to attenuate dorsalizing signals derived from the organizer. PMID- 8625842 TI - The TGF-beta signaling pathway is essential for Drosophila oogenesis. AB - We examine roles of signaling by secreted ligands of the TGF-beta family during Drosophila oogenesis. One family member, the DPP ligand encoded by the decapentaplegic (dpp) gene, is required for patterning of anterior eggshell structures. This requirement presumably reflects the expression pattern of dpp in an anterior subset of somatic follicle cells: the centripetally migrating and the nurse cell-associated follicle cells. Similar requirements are also revealed by mutations in the saxophone (sax)-encoded receptor, consistent with the idea that DPP signaling is, at least in part, mediated by the SAX receptor. A loss of germline sax function results in a block in oogenesis associated with egg chamber degeneration and a failure of the transfer of nurse cell contents to the oocyte, indicating that TGF-beta signaling is required for these events. Some phenotypes of sax mutations during oogenesis suggest that SAX responds to at least one other TGF-beta ligand as well in the posterior follicle cells. PMID- 8625843 TI - The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. AB - The CLAVATA (CLV1 and CLV3) and SHOOT MERISTEMLESS (STM) genes specifically regulate shoot meristem development in Arabidopsis. CLV and STH appear to have opposite functions: c1v1 and Clv3 mutants accumulate excess undifferentiated cells in the shoot and floral meristem, while stm mutants fail to form the undifferentiated cells of the shoot meristem during embryonic development. We have identified a weak allele of stm (stm-2) that reveals STM is not only required for the establish- ment of the shoot meristem, but is also required for the continued maintenance of undifferentiated cells in the shoot meristem and for proper proliferation of cells in the floral meristem. We have found evidence of genetic interactions between the CLV and STM loci. clv1 and c1v3 mutations partially suppressed the stm-1 and stm-2 phenotypes, and were capable of suppression in a dominant fashion. clv stm double mutants and plants homozygous for stm but heterozygous for clv, while still lacking an embryonic shoot meristem, exhibited greatly enhanced postembryonic shoot and floral meristem development. Although stm phenotypes are recessive, stm mutations dominantly suppressed clv homozygous and heterozygous phenotypes. These results indicate that the stm phenotype is sensitive to the levels of CLV activity, while the clv phenotype is sensitive to the level of STM activity. We propose that these genes play related but opposing roles in the regulation of cell division and/or cell differentiation in shoot and floral meristems. PMID- 8625844 TI - Functional domains in the Deformed protein. AB - A chimeric protein consisting of Deformed with a substituted Abdominal-B homeodomain (Dfd/Abd-B) is used to identify protein domains outside the homeodomain that are required for regulatory activity in vivo. A series of deletion proteins were generated based on regions showing amino acid composition similar to known regulatory domains. Each mutant protein can influence regulation of homeotic genes in a manner distinct from the intact protein. Activity was also tested using promoter elements from empty spiracles and Distal-less, two genes known to be directly regulated by Abdominal-B. Removal of the acidic region and the C-tail region convert the chimera from a strong activator to a repressor of the Distal-less element, but had comparatively little effect on the activation of the empty spiracles element. Constructs without a third domain, the N domain, fail to show any regulatory activity. The N domain is the only domain of the Dfd/Abd-B protein which exhibits significant activation activity when fused to a heterologous DNA binding domain. Our results suggest transcriptional activity of the N domain can be modulated by the acidic and C-tail domains. PMID- 8625845 TI - Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. AB - For genetic analysis of mechanisms of leaf morphogenesis, we chose Arabidopsis thaliana (L.) Heynh. as a model for leaf development in dicotyledonous plants. Leaves of the angustifolia mutant were the same length as but narrower and thicker than wild-type leaves. The total number of cells in leaf blades of angustifolia plants was the same as in the wild type. At the cellular level in the angustifolia mutant it was found that the cells were smaller in the leaf width direction and larger in the leaf-thickness direction than in wild type, revealing the function of the ANGUSTIFOLIA gene, which is to control leaf morphology by regulating polarity-specific cell elongation. The existence of similar genes that regulate leaf development in the length direction was, therefore, predicted. Three loci and several alleles associated with short-leaved mutants were newly isolated as rotundifolia mutants. The rotundifolia3 mutant had the same number of cells as the wild type, with reduced cell elongation in the leaf-length direction. The features of the angustifolia rotundifolia3 double mutant indicated that ANGUSTIFOLIA and ROTUNDIFOLIA3 genes act independently. We propose that leaf expansion in Arabidopsis involves at least two independent developmental processes: width development and length development, with the ANGUSTIFOLIA and ROTUNDIFOLIA3 genes playing different polarity-specific roles in cell elongation. PMID- 8625846 TI - The mec-8 gene of C. elegans encodes a protein with two RNA recognition motifs and regulates alternative splicing of unc-52 transcripts. AB - Mutations in the mec-8 gene of Caenorhabditis elegans were previously shown to affect the functions of body wall muscle and mechanosensory and chemosensory neurons. Mutations in mec-8 also strongly enhance the mutant phenotype of specific mutations in unc-52, a gene that encodes, via alternative splicing of pre-mRNA, a set of basement membrane proteins, homologs of perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis and cuticle. We have cloned mec-8 and found that it encodes a protein with two RNA recognition motifs, characteristic of RNA binding proteins. We have used reverse transcription-PCR and RNase protection experiments to show that mec 8 regulates the accumulation of a specific subset of alternatively spliced unc-52 transcripts. We have also shown with antibodies to UNC-52 that mec-8 affects the abundance of a subset of UNC-52 isoforms. We propose that mec-8 encodes a trans acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and one or more additional genes that affect mechanosensory and chemosensory neuron function. PMID- 8625847 TI - Surface tensions of embryonic tissues predict their mutual envelopment behavior. AB - During embryonic development, certain tissues stream to their destinations by liquidlike spreading movements. According to the 'differential adhesion hypothesis', these movements are guided by cell-adhesion-generated tissue surface tensions (sigmas), operating in the same manner as surface tensions do in the mutual spreading behavior of immiscible liquids, among which the liquid of lower surface tension is always the one that spreads over its partner. In order to conduct a direct physical test of the 'differential adhesion hypothesis', we have measured the sigmas of aggregates of five chick embryonic tissues, using a parallel plate compression apparatus specifically designed for this purpose, and compared the measured values with these tissues' mutual spreading behaviors. We show that aggregates of each of these tissues behave for a time as elasticoviscous liquids with characteristic surface tension values. Chick embryonic limb bud mesoderm (sigma = 20.1 dyne/cm) is enveloped by pigmented epithelium (sigma = 12.6 dyne/cm) which, in turn, is enveloped by heart (sigma = 8.5 dyne/cm) which, in turn, is enveloped by liver (sigma = 4.6 dyne/cm) which, in turn, is enveloped by neural retina (sigma = 1.6 dyne/cm). Thus, as predicted, the tissues' surface tension values fall in the precise sequence required to account for their mutual envelopment behavior. PMID- 8625848 TI - The Drosophila Polycomb group gene Sex comb on midleg (Scm) encodes a zinc finger protein with similarity to polyhomeotic protein. AB - The Sex comb on midleg (Scm) gene is a member of the Polycomb group (PcG) of genes in Drosophila melanogaster. The PcG genes encode transcriptional repressors required for proper spatial expression of homeotic genes. We report the isolation of new Scm mutations and the molecular characterization of the Scm gene. Scm mRNA is expressed maternally, at peak levels in early embryos and then at lower levels throughout the remainder of development. Scm encodes a putative zinc finger protein of 877 amino acids. Scm protein is similar to polyhomeotic, another member of the PcG, both in the zinc finger region and in a separate C-terminal domain of 60 amino acids, which we term the SPM domain. Sequence analysis of an Scm mutant allele suggests a functional requirement for the SPM domain. Scm protein also bears homology in multiple domains to a mouse protein, Rae-28 (Nomura, M., Takihara, Y. and Shimada, K. (1994) Differentiation 57,39-50) and to a fly tumor suppressor protein, the product of the lethal(3)malignant brain tumor gene (Wismar, J. et al., (1995) Mech. Dev. 53, 141-154). Possible functional relationships among these proteins and potential biochemical roles for Scm protein in PcG repression are discussed. PMID- 8625849 TI - aubergine enhances oskar translation in the Drosophila ovary. AB - Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. In this report, we describe the role of aubergine in oskar translation. Previously, aubergine has been implicated in dorsoventral patterning, as eggs from aubergine mutant mothers are ventralized and seldom fertilized (Schupbach, T. and Wieschaus, E. (1991) Genetics 129, 1119-1136). We have isolated two new alleles of aubergine in a novel genetic screen and have shown that aubergine is also required for posterior body patterning, as the small fraction of eggs from aubergine- mothers that are fertilized develop into embryos which lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either oskar mRNA or protein, the level of oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance oskar translation. While aubergine-dependence is conferred upon oskar mRNA by sequences in the oskar 3' UTR, aubergine may influence oskar translation through an interaction with sequences upstream of the oskar 3' UTR. PMID- 8625851 TI - A C. elegans Hox gene switches on, off, on and off again to regulate proliferation, differentiation and morphogenesis. AB - Hox genes establish body pattern throughout the animal kingdom, but the role these genes play at the cellular level to modify and shape parts of the body remains a mystery. We find that the C. elegans Antennapedia homolog, mab-5, sequentially programs many independent events within individual cell lineages. In one body region, mab-5 first switches ON in a lineage to stimulate proliferation, then OFF to specify epidermal structures, then ON in just one branch of the lineage to promote neuroblast formation, and finally OFF to permit proper sense organ morphology. In a neighboring lineage, continuous mab-5 expression leads to a different pattern of development. Thus, this Hox gene achieves much of its power to diversify the anteroposterior axis through fine spatiotemporal differences in expression coupled with a changing pattern of cellular response. PMID- 8625850 TI - A functional homologue of goosecoid in Drosophila. AB - We have cloned a Drosophila homologue (D-gsc) of the vertebrate homeobox gene goosecoid (gsc). In the Gsc proteins, the pressure for conservation has been imposed on the homeodomain, the functional domain of the protein: sequence homology is limited to the homeodomain (78% identity) and to a short stretch of 7 aminoacids also found in other homeoproteins such as Engrailed. Despite this weak homology, D-gsc is able to mimic gsc function in a Xenopus assay, as shown by its ability to rescue the axis development of a UV-irradiated embryo. Moreover, our data suggest that the position of insect and vertebrate gsc homologues within a regulatory network has also been conserved: D-gsc expression is controlled by decapentaplegic, orthodenticle, sloppy-paired and tailless whose homologues control gsc expression (for BMP4 and Otx-2), or are expressed at the right time and the right place (for XFKH1/Pintallavis and Tlx) to be interacting with gsc during vertebrate development. However, the pattern of D-gsc expression in ectodermal cells of the nervous system and foregut cannot easily be reconciled with that of vertebrate gsc mesodermal expression, suggesting that its precise developmental function might have diverged. Still, this comparison of domains of expression and functions among Gsc proteins could shed light on a common origin of gut formation and/or on basic cellular processes. The identification of gsc target genes and/or other genes involved in similar developmental processes will allow the definition of the precise phylogenetic relationship among Gsc proteins. PMID- 8625852 TI - Alternative RNA splicing that determines agrin activity regulates binding to heparin and alpha-dystroglycan. AB - Agrin is a component of the extracellular matrix that regulates aspects of neuromuscular junction differentiation. Identification of agrin-binding proteins has lead to the suggestion that alpha-dystroglycan is a muscle cell surface proteoglycan that mediates agrin activity. To further test this hypothesis, we have compared the ability of differentially active agrin isoforms to interact with a model component of proteoglycans, heparin, as well as with the putative proteoglycan alpha-dystroglycan. We demonstrate that an alternately spliced exon (encoding the sequence lysine, serine, arginine, lysine: Y site) is necessary for agrin-heparin interactions. We also show that alternate splicing at another site (Z site) dramatically affects interaction of alpha-dystroglycan with agrin. We propose a model in which multiple distinct domains of agrin interact with both protein and sugar moieties of alpha-dystroglycan. The isoform-specific binding of agrin to alpha-dystroglycan is consistent with a functional role for this interaction during synaptogenesis. PMID- 8625853 TI - Emerging and re-emerging global microbial threats. PMID- 8625854 TI - Yellow fever: an emerging threat for Kenya and other east African countries. AB - Yellow fever (YF) is a well known disease that had plagued the tropics relentlessly until an effective vaccine was developed. Although the yellow fever vaccine is relatively affordable and one dose protects for over ten years, its use has predominantly been for known endemic areas of the world and international travellers. Eastern and southern African states, have hitherto been free of epidemic yellow fever, hence routine YF vaccination is not a policy in these countries. The sudden emergence of YF in the Rift Valley in Kenya in 1992-1993, introduces new dimensions into the challenges of YF to eastern and southern African states. Isolation of a virus deemed to be native of the area is discussed in this article in the context of YF policy issues confronting the region. A case has been argued for the establishment of a network of active surveillance systems in the region backed by adequate laboratory YF expertise locally, regionally, and internationally. PMID- 8625855 TI - Human immunodeficiency virus infection and AIDS in east Africa: challenges and possibilities for prevention and control. AB - Human immunodeficiency infection and AIDS are a major recent microbial infection in east Africa with serious health and socioeconomic impacts in the region. At present HIV infection and AIDS account for more than 50% of adult medical admissions into some of the national and provincial hospitals as well as for 10 15% of paediatric admissions. AIDS is also at present the commonest cause of death among those aged 15-45 years. Tuberculosis, a closely associated disease to HIV infection, has increased more than three fold in some countries in the region. The prevalence of HIV infection currently ranges from 10-30% among adults in urban areas and from less than 1% to 25% in adults in rural areas; since this prevalence is still rising, the full impact of the AIDS problem in east Africa is yet to be realised. This is different from the situation in many developed countries where AIDS is no longer a priority health issue and where peak prevalences of the infection have been reached. The differences in HIV prevalences between east Africa and developed countries are due to poverty, ignorance, high prevalence of other STDs and associated cultural and traditional practices which prevail and facilitate HIV transmission in the region. While more than 80% of HIV infection in east Africa is transmitted through heterosexual intercourse, 5-15% of cases are perinatally transmitted and the remaining cases are transmitted through blood and blood products. While a lot of scientific advances have been made in immunopathology of AIDS, diagnostics and in social behavioural studies, we are still a long way towards getting curative therapy and or effective preventive vaccines. Recent discovery that use of zidovudine can significantly reduce perinatal HIV transmission is an additional breakthrough. While knowledge and tools for preventing HIV transmission are available in the world, prospects for AIDS control in east Africa appear gloomy unless major efforts are made in the reduction of poverty, ignorance and in the control of other common sexually transmitted diseases. PMID- 8625857 TI - Threat of Marburg and Ebola viral haemorrhagic fevers in Africa. AB - Marburg and Ebola viruses are members of the filovirus family that can be regarded as recently emerged. These viruses have caused sporadic outbreaks of fatal haemorrhagic disease in Africa, Europe and recently in the USA. The case fatality rates rank among the highest ranging from 33-80%. The mode of transmission of these viruses are clearly through close contact with blood and body fluids. Disease outbreaks have been amplified in hospital situations with poor blood precautions. In villages disease has been amplified through contamination with blood and fluids during nursing the sick and burial rituals. The source of the viruses has eluded discovery and new theories regarding the nature of these viruses are being entertained. The threat of new outbreaks in Africa is real since serological evidence of the presence of the virus has been documented in Kenya, Sudan, Zaire, Zimbabwe, Gabon, Cote-d'Ivoire and Gabon. PMID- 8625856 TI - Human immunodeficiency virus and AIDS in Uganda. AB - HIV-1 infection, initially described as "slim disease", was first recognized in Uganda in 1982, and is now a predominant health problem. Approximately 1.5 million Ugandans are now infected, largely through heterosexual transmission. In many areas half of adult deaths are now caused by HIV. Seroprevalence rates in urban antenatal clinics have been dropping in the last several years, as have rates in young adults in two rural community cohorts where the epidemic is long established. Tuberculosis cases and admissions have increased dramatically. Among the clinical manifestations of HIV in Uganda, epidemic Kaposi sarcoma, cryptococcal meningitis, suspected toxoplasmosis and cardiomyopathy, as well as atypical or extrapulmonary tuberculosis are seen with increasing frequency. Mother to child transmission of HIV accounts for about 10% of total cases, with a transmission rate of 26% in two studies. Epidemiological and clinical research programs are well developed in Uganda, especially in areas of tuberculosis, maternal and paediatric HIV infection and sexually transmitted infections. Societal openness, a multisectoral approach by the government and innovative programmes, including large-scale HIV testing and counselling and the pioneering work of The AIDS Support Organization (TASO), distinguish the Ugandan response to the epidemic. PMID- 8625858 TI - Possible contributing factors to the paucity of yellow fever epidemics in the Ashanti region of Ghana, west Africa. AB - Yellow fever virus vectors identified in the Ashanti region of Ghana included Aedes aegypti, Aedes africanus, Aedes luteocephalus and Aedes vittatus. Other mosquito species, unrelated to yellow fever transmission, identified in this study included Culex tigripes, Culex thalassius, Culex decens, Culex tarsalis, Anopheles gambiae, Anopheles stephansi and Toxorynchites brevipalpis. Factors generally known to influence yellow fever transmission were also studied in the Ashanti region. These included Aedes mosquito larval indices, biting or man contact rates, rainfall, relative humidity and duration of sunshine. Calculated values for these factors were found to be far below internationally accepted threshold values, due, perhaps, to the vast distribution, resilience and preferential predatory propensity of the larvae of T. brevipalpis, a mosquito species we found exclusively in the Ashanti region of Ghana, for A. aegypti larvae. Other predators of mosquito larvae encountered included Notonecta (Nepa species), Hydromerta, Culex tigripes, Belostoma and Lispa. The observed paucity of yellow fever outbreaks in the Ashanti region of Ghana may, in the main, be due to the preponderance resilience and selective predatory propensity and preference of T. brevipalpis for A. aegypti larvae. Furthermore, the observed presence of other predators which prey on A. aegypti larvae in the study areas, the low larval indices and the low man-vector contact rates recorded as well as the high prevalence of Group B antibodies found in the blood of the population of this region may also be contributory to the paucity of yellow fever outbreaks in the Ashanti region. PMID- 8625859 TI - Rabies: the emergence of a microbial threat. AB - Since 1912 when rabies was first confirmed in Kenya, the disease has largely existed in varying degrees of occurrence. Spatial and temporal distribution of cases of animal rabies are well documented. Records of human cases have not been as detailed as the veterinary cases. The principal animal reservoir for rabies has been the dog. Save for the 1960s, rabies outbreaks occurred in the country for most part of this century. The 1980's witnessed a dramatic upward swing in the number of cases reported annually. Over the years an enzootic pattern covering most parts of the country emerged. This article reviews available data showing rabies as an emerging microbial threat in Kenya. PMID- 8625860 TI - Meningococcal meningitis. AB - Meningococcal meningitis has been recognised as serious problem for almost 200 years. In Africa the disease occurs in epidemics periodically during the hot and dry weather in the "meningitis belt" and in east Africa, which is outside this belt the epidemics tend to occur during the cold and dry months. The infection is mainly transmitted from person to person by nasopharyngeal carriers in crowded places like refugee camps and army barracks. The rural/urban migration, the basic structural conditions of housing in squatter settlements and slums together with an overcrowded transport system have also contributed to the transmission of meningococcal meningitis. The earlier treatment of meningococcal meningitis was by the way of repeated CSF drainage. The first important advance in the treatment was intrathecal injection of antimeningococcal serum. A major break through in the treatment was the introduction of sulphonamides which was the preferred treatment until emergence of resistance to sulphonamides in mid 1960's. Penicillin remains the drug of choice currently. Mass immunisation of selected communities using polyvalent A and C polysaccharide vaccine is a useful control measure. Chemoprophylaxis is generally not recommended during epidemics. Given the current population densities and rural/urban migration together with financial constraints, future epidemic in Kenya may be more explosive unless strict surveillance programmes are maintained. PMID- 8625861 TI - Cholera: its story in Africa with special reference to Kenya and other east African countries. AB - Cholera is an acute often fatal infection whose aetiology, epidemiology, clinical presentation, management, prevention and control are well understood. However, the disease has recently re-emerged as a serious microbial threat due to environmental pressures, increased international travel, population migrations and the emergence of new strains of "old" pathogens. This paper emphasizes, the significance of putting into place quick surveillance methods backed by laboratory services and the importance of creating a rapid response capacity and mechanics to outbreaks. PMID- 8625862 TI - Animal reservoirs of leishmaniasis in Marigat, Baringo District, Kenya. AB - A total of 1128 rodents belonging to seven genera were examined for leishmanial parasites over a period of sixteen months. Parasites were isolated from 36 (12.5%) Tatera robusta, 3 (0.5%) Arvicanthis niloticus, and 2 (0.8%) Mastomys natalensis. All isolates were characterised by isoenzyme analysis using nine enzymes. The enzymes examined were: malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), mannose phosphate isomerase (MPI), malic enzyme (ME) and 6 phosphogluconate dehydrogenase (6PGD). The enzyme profiles from these isolates were compared with those from Leishmania reference strains and also with isolates of Leishmania major from man and sandfly, P. duboscqci from the same area. All the isolates except one from a Mastomys were identified as L. major. The isolate from Mastomys was trypanosome-like and remains unidentified. The results in this study show that Tatera robusta is the main reservoir of cutaneous leishmaniasis in Baringo District. None of the animals trapped were found infected with Leishmania donovani suggesting that rodents do not play a role in the transmission of visceral leishmaniasis in this area. PMID- 8625863 TI - Phlebotomine sandflies (Diptera:, Psychodidae): their importance and some aspects of control. PMID- 8625864 TI - Some emerging issues on the malaria problem in Kenya. AB - Malaria in Kenya has been characterised by marked variability in its epidemiology, partly reflecting the obstacles and progress made in the control strategies. The impact of anti-vector activities in the 1970s and before have been observed for variable lengths of time afterwards. Malaria has re-emerged in areas previously with little or no transmission. The recovery of infective Anopheles gambiae vector in higher altitudes affirms the potential for transmission in areas where epidemics have been known to occur. Morbidity and mortality patterns in the otherwise endemic lowlands have become increasingly severe, an observation which would be attributed to the increasing inefficacy of chloroquine. Efforts to promote personal protection suffer substantial setbacks in sustainability inspite of apparent acceptability. There are indications that the mosquito vector susceptibility to permethrin and other insecticides will now require continual monitoring in order to detect development of significant resistance. In this communication, we review some emergent issues in malaria transmission in Kenya and the potential for control as adduced from historical and contemporary perspectives. PMID- 8625865 TI - Outbreak of louse-borne relapsing fever in Jimma, south western Ethiopia. AB - Some epidemiological aspects of louse-borne relapsing fever during the epidemic of 1991 in Ethiopia are described. There was male preponderance and the age ranged from two months to 58 years (mean, sd = 18.05,11.19). There was no sex differential in children. Students, soldiers and jobless contributed the majority. There were two peaks (August and March) in the seasonal pattern. About two-thirds was infested with lice and significant associations were found among adults, soldiers, ethnicity, illiterate persons and family size above seven. The household attack rate was 15.1%, the attack being higher among children. Promotion of preventive measures are recommended. PMID- 8625866 TI - Prevalence of enteropathogens in stools of rural Maasai children under five years of age in the Maasailand region of the Kenyan Rift Valley. AB - Stool samples were collected during August 1994 from seventy rural Maasai children under the age of five years who were living in the Maasailand region of the Kenyan Rift Valley. Microbiological analysis was carried out on these samples to identify which intestinal pathogens were present among the infant population of the Maasai. Of the samples studied 54% were pathogen positive. The most common pathogen isolated was Giardia lamblia which was detected in 31% of the samples. Other pathogens that were detected include: Entamoeba histolytica (23%), Enteropathogenic Escherichia coli (13%), Strongyloides stercoralis (4%), Blastocystis hominis (3%) and Cryptosporidium sp (3%). Although all samples were screened for Campylobacter and rotavirus, neither pathogen was detected. Water samples were taken from all the water sources in the study area and analysed microbiologically. Results showed that all the sources were contaminated with the faecal E. coli whose populations ranged from 14 CFU/100 ml to greater than 1800 CFU/100ml. PMID- 8625867 TI - Chimeric synthetic peptides as antigens for detection of antibodies to HIV-1 and HIV-2. AB - Two chimeric peptides incorporating immunodominant sequences from both HIV-1 (LGIWGCSGKLICTT) and HIV-2 (LNSWGCAFRQVCHT) were synthesized. Peptides KS1-KS2 and KS2-KS1 represented sequences from the two viruses in both possible orders, separated by two glycine residues as spacers. These peptides were evaluated as antigens in an ELISA using a panel of specimens derived from HIV-1 (n = 25) and HIV-2 (n = 25) infected individuals and seronegative people (n = 38). The results were compared to plates coated with individual peptides KS1 and KS2 and to plates coated with two peptides (KS1 and KS2) together. Data demonstrated that individually, KS1 and KS2, are good antigens and can detect antibodies to their respective viruses quite efficiently. However, when coated together, their ability to detect antibodies to both HIV-1 and HIV-2 was reduced, as evidenced by a decrease in OD values obtained. The chimeric peptides KS1-KS2 and KS2-KS1 detected antibodies to HIV-1 and HIV-2; however, their sensitivity of detection was variable and dependent upon the order of their sequence. For both peptides, antibodies directed to the C-terminal portion were detected with higher sensitivity than those directed to the N-terminal part of the peptides. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. Such chimeric antigens may be very useful for simultaneous detection of antibodies to HIV-1 and HIV-2. PMID- 8625868 TI - Antibiotic sensitivity pattern of prevalent bacterial pathogens in Gondar, Ethiopia. AB - The prevalence and sensitivity pattern of common bacterial isolates from clinical specimens processed over one year in the bacteriology laboratory of a teaching hospital in north-west Ethiopia was investigated. Staphylococcus aureus, Escherichia coli and other enteric Gram-negative rods were the predominant pathogens cultured. Klebsiella species were responsible for a nosocomial outbreak among children in the year. The majority of the strains, irrespective of genera, were resistant to tetracycline (> 60%), co-trimoxazole (> 55%) and chloramphenicol (> 45%). Resistance to ampicillin was seen in > 60% of isolates other than S. aureus. Sensitivity to gentamicin was high (> 89%) among S. aureus, E. coli and Pseudomonas strains. Isolates of Klebsiella, Enterobacter and Proteus were the least sensitive to the aminoglycoside. A multiplicity of antibiograms and predominance of certain multiresistant strains was observed for the prevalent species. Comparison made with reports from elsewhere in Ethiopia indicates that resistance to the commonly available (and cheaper) broad-spectrum antibiotics is a nationwide problem. A suggestion is made to enforce rational drug use before potent antibiotics are introduced under prescriber pressure. PMID- 8625869 TI - Microsporidium in AIDS patients: a perspective. AB - Microsporidia are ubiquitous, obligate intracellular protozoan parasites increasingly detected as opportunistic pathogens in AIDS patients. These parasites have been associated with chronic diarrhoea, hepatitis, cholangitis, pancreatitis, enteritis, keratoconjunctivitis, and peritonitis in either homosexuals or heterosexuals. Optimum diagnostic and therapeutic measures of these pathogens still elude both clinicians and researchers. Further study is required to elucidate the exact prevalence and clinical characteristics of microsporidia. PMID- 8625870 TI - Susceptibility of Ethiopian bulinid snails to Schistosoma haematobium from Somalia. AB - Susceptibility of four Ethiopian bulinid snails to a Somalian strain of S. haematobium was tested. Bulinus abyssinicus was highly susceptible and lowland B. africanus was partially susceptible while B. truncatus and B. forskalii were refractory to the parasite. It is suggested that Ethiopian refugees returning from Somalia and/or Somalian refugees entering Ethiopia should be screened and treated for S. haematobium before they are allowed to work/resettle in development areas where B. abyssinicus and B. africanus are known or ecologically suspected to occur. PMID- 8625871 TI - Pattern of postoperative pyrexia in Khartoum. AB - The pattern of postoperative pyrexia in Khartoum was prospectively studied in 260 patients who underwent a variety of surgical operations. Ninety four patients (36.1%) developed postoperative pyrexia. The commonest causes of pyrexia encountered were wound sepsis (10%), malaria (9.6%) and respiratory tract infection (7.3%). Less frequent causes were urinary tract infection, thrombophlebitis, intra-abdominal sepsis and deep vein thrombosis. In 14.6% of the patients, the cause of pyrexia was undetermined. The risk factors for postoperative pyrexia were the patient's age, diabetes mellitus, obesity, preoperative chest infection, smoking, duration of surgery, operator's surgical experience and urethral catheterisation. The postoperative pyrexia was associated with 7.4% mortality rate which was due to intra-abdominal sepsis and pulmonary embolism. The incidence of postoperative pyrexia can be minimised by adequate preoperative preparation, meticulous surgical technique and good postoperative care. PMID- 8625872 TI - Components of EMG symmetry and variability in parkinsonian and healthy elderly gait. AB - Variability and bilateral symmetry of EMG gait-cycle profiles were studied in parkinsonian and healthy elderly subjects in the gastrocnemius, tibialis anterior, and vastus lateralis muscles. Components reflecting shape and timing were defined by the magnitude and phase of the cross-correlation function between individual stride profiles and the latency corrected ensemble average (LCEA) (variability), and between bilateral LCEAs (symmetry). Statistical significance was set at a confidence level of 0.01 reflecting a Bonferroni adjustment due to multiple measures. Parkinsonian gait was significantly different from the healthy elderly in several measures: increased shape variability and asymmetry in the gastrocnemius and tibialis anterior muscles, and reduced timing variability in the gastrocnemius. A portion of the parkinsonian group participated in a 3 week therapy program where they walked to rhythmic auditory stimulation. Gait parameters shifted toward healthy elderly values in each measure where population differences were found. Significant changes were observed in decreased tibialis anterior shape variability and asymmetry, and gastrocnemius shape variability. Strong trends were also observed in increased gastrocnemius timing variability and reduced bilateral asymmetry. In addition to the expected decreased in variability and asymmetry of healthy elderly, increased timing variability in the gastrocnemius was associated with a more normal gait, possibly reflecting feedback adaptability of muscle activity which may be useful in generating stable locomotion. PMID- 8625873 TI - Cortical modulation of spinal excitability: an F-wave study. AB - F-waves are known to be highly sensitive to changes in the excitatory state of the spinal cord. This paper describes the effects of subthreshold transcranial magnetic stimulation on the F-waves evoked in hand and foot muscles. In the abductor pollicis brevis muscle, the F-wave was significantly enhanced when the cortical stimulus was given with a delay corresponding approximately to the expected time of collision, i.e., the difference between the mean latency of the F-wave and the mean latency of the motor evoked potential. A second, usually larger facilitatory phase follows the first phase after 2-3 msec, and later peaks of enhancement often occurred. After the enhancement periods, a significant inhibition of the F-wave was usually observed. In the extensor digitorum brevis muscle, the first facilitatory phase was observed some milliseconds earlier than expected in 4 of 5 subjects, and the inhibitory phase was less pronounced. We argue that the sequential arrival of I-waves at the spinal segment could be responsible for the changes observed in the F-wave recorded from the small hand muscle. In the foot muscle, anatomical and technical factors could contribute to the generation of a D-wave. The strong inhibition observed in the F-wave recorded from the hand muscle is likely to be due to the arrival on alpha-motoneurons of inhibitory postsynaptic potentials (IPSPs) generated by the cortical stimulus. Our data show that the F-wave ia a probe for changes in the spinal cord excitatory state. PMID- 8625874 TI - Sympathetic skin responses from the limbs and the genitalia: normative study and contribution to the evaluation of neurourological disorders. AB - The autonomic innervation of the limbs and the genitalia was studied by means of electrical stimulation of the median nerve at the wrist and the dorsal nerve of the penis/clitoris and recording of sympathetic skin responses from the hand, the foot, and the perineum. In males, the response was also recorded from the penile shaft following median nerve stimulation. Normative data were obtained from 40 healthy volunteers (20 men and 20 women). The clinical contribution of these tests was investigated in a series of 42 patients referred to our laboratory for urinary and/or sexual disorders. Sympathetic skin responses are helpful in the evaluation of sexual impotence and of disorders affecting the pelvic floor as well as of well-known neuropathies. PMID- 8625875 TI - Abnormal excitability of the corticospinal pathway in patients with amyotrophic lateral sclerosis: a single motor unit study using transcranial magnetic stimulation. AB - The pathophysiology of corticospinal tract degeneration in amyotrophic lateral sclerosis (ALS) was investigated by studying the effect of transcranial magnetic stimulation on discharge characteristics of single motor units during voluntary activation. The motor units were recorded from the first dorsal interosseus muscles of 12 patients with ALS, 14 healthy subjects, 12 patients with upper motor neuron lesions and 9 with pure lower motor neuron diseases. More than 100 magnetic stimuli were delivered over the scalp during minimal muscle contraction. The occurrence of motor unit discharges was plotted in a peristimulus time histogram. An increase in discharge probability at latencies of 20-30 msec, that represents monosynaptic activation (primary peak) was found in normal units. Motor units from ALS patients with short disease durations had significantly increased discharge probabilities in the primary peak (P < 0.001). Motor units from 4 ALS patients with upper motor neuron signs showed double primary peaks: an initial synchronized peak followed by a dispersed peak. The latter was ascribed to a slow corticospinal pathway, which remains undetected or is functionally insignificant in healthy subjects. We conclude that the excitabilities of the surviving corticospinal tract pathways are abnormally increased in ALS, especially in the early stage. PMID- 8625876 TI - Pre-symptomatic neurological involvement in Behcet's disease: the diagnostic role of magnetic transcranial stimulation. AB - Twenty Behcet's disease (BD) patients with or without neurologic signs and symptoms were studied by means of magnetic transcranial stimulation. The most frequent abnormalities were an increase of motor evoked potential latency and a delay of central motor conduction time (CMCT). In 5 out of 9 BD patients with pyramidal signs central motor conduction changes were present. Furthermore, 2 out of 7 patients without neurologic signs and symptoms showed an increased threshold and a prolonged CMCT. The data obtained suggest that this technique is useful not only to confirm the neurologic impairment but also to reveal subclinical or pre symptomatic central motor involvement in BD subjects. PMID- 8625877 TI - Corticospinal direct response to transcranial magnetic stimulation in humans. AB - The corticospinal motor evoked potential (MEP) response to transcranial magnetic stimulation of the motor cortex was investigated in comparison with the direct (D) response to electrical stimulation of the exposed motor cortex from the spinal epidural space in 7 neurologically normal patients during brain tumor surgery. The D response during operation was obtained by transcranial magnetic stimulation of the scalp over the areas of the cerebral motor cortex, the hand or arm areas. The magnetic induced D response showed a conduction velocity of 50.5 72.7 m/sec and was resistant to anesthesia and unaffected by muscle relaxants and tolerant to high frequency (500 Hz) paired magnetic stimulus, and the latencies of magnetic MEPs corresponded to those with direct electrical stimulation. Thus, recordings of the D response by transcranial magnetic stimulation are useful for not only identifying the location of the motor cortex during intracranial surgery but also for non-invasive recording of pyramidal tract activity during extracranial surgery under general anesthesia. PMID- 8625878 TI - Effect of transcranial magnetic stimulation over the cerebellum on the excitability of human motor cortex. AB - There have been conflicting reports over whether it is possible to stimulate the human cerebellum through the intact scalp using transcranial magnetic stimulation. Here we attempt to clarify the situation in normal subjects by comparing the various methods which have been used. EMG responses evoked by magnetic stimulation over the motor cortex could be suppressed by a prior magnetic stimulus over the cerebellum but the onset latency of the effect varied according to the type of magnetic coil used. Inhibition began at a latency which ranged from 5 to 9 msec in different subjects if conditioning stimuli were given through a flat figure-of-eight coil held horizontally over the basal occiput. The effect lasted a further 6-10 msec. With a larger double cone coil, held vertically over the basal occiput, inhibition began earlier and at a more constant latency of 5 msec. It lasted only 3 msec. Stimulation of the C6/7 nerve roots in the brachial plexus with either an electrical or magnetic stimulus also could suppress EMG responses evoked by cortical stimulation. This began at a conditioning-test interval of 7 or 8 msec and lasted for some 5 msec. We suggest that two types of motor cortical suppression may be elicited from stimulation over the posterior neck/skull: a cerebellar effect starting at 5 msec, and a peripheral nerve effect starting later at 7/8 msec. Stimulation with a horizontal large figure-of-eight coil may produce a mixture of effects because the lower wing of the coil overlaps the posterior neck and can activate peripheral nerve fibres in the brachial plexus. PMID- 8625880 TI - Advances in fingerprint detection. AB - Since the first conviction using fingerprints as evidence in 1902, the detection of fingerprints at crime scenes has become a key weapon in the identification and apprehension of criminals. Almost a century after Edward Henry published The Classification and Use of Fingerprints in 1898, a new magnetic flake technology has been purpose-designed for scene-of-crime investigations. PMID- 8625879 TI - Apomorphine can increase cutaneous inhibition of motor activity in Parkinson's disease. AB - We studied the effect of non-nociceptive ipsilateral digital stimulation on EMG recorded from a small hand muscle before and after the administration of subcutaneous apomorphine in 6 patients with Parkinson's disease. All were receiving the drug to control ?on-off? fluctuations in motor performance. Averaged rectified EMG was recorded from tonically contracted abductor pollicis brevis (APB) following index finger stimulation using a brief stimulus train. In 5 patients motor evoked potentials (MEPs) were also recorded from APB during tonic contraction. A conditioning stimulus train was applied to the index finger at intervals between 15 and 65 msec prior to the transcranial magnetic stimulus. After apomorphine administration the patient group showed a significant increase in both EMG and MEP inhibition induced by digital stimulation. In patients with Parkinson's disease who have marked motor fluctuations, the inhibitory response of upper limb motor neurones to low level digital cutaneous stimulation can be altered by dopamine agonists. PMID- 8625881 TI - Tell-tale teeth: abrasion from the traditional clay pipe. AB - The long story of the once very widespread clay pipe has a minor but significant odontological aspect: some clay-pipe smokers may be characterized by a distinctive abrasion pattern of the teeth. The marks in question are of special interest to the archaeologist; attention is here directed particularly to the Scandinavian experience. PMID- 8625882 TI - An OAF by any other name. PMID- 8625883 TI - Oncostatin-M: a new bone active cytokine that activates osteoblasts and inhibits bone resorption. AB - Osteoblasts and their precursors respond to specific cytokines, growth factors, and hormones. One facet of this response includes the secretion of additional cytokines, some of which are part of the circuitry involved in the regulation of osteoblast and osteoclast function. Therefore, understanding which cytokines are able to activate osteoblastic cells and the consequences of that activation are central to understanding normal and pathologic bone remodeling. Oncostatin M (OSM) is a glycoprotein belonging to a new subfamily of cytokines related by sequence and structural homology and the use of the signal transducing receptor component gp130. Osteoblastic cells secrete and respond to leukemia-inhibiting factor (LIF) both in vitro and in vivo, suggesting that LIF is an autocrine regulatory factor. OSM is closely related to LIF, and therefore we hypothesized that OSM should regulate the function of cells in the osteoblastic lineage. Primary neonatal murine or fetal rat calvarial osteoblastic cultures were treated with OSM or LIF and a series of biochemical and biological parameters were determined. In these cultures, OSM induced proliferation, collagen synthesis, and interleukin-6 secretion, whereas it inhibited alkaline phosphatase activity. Bone resorption was also inhibited by OSM. These data represent the first report of OSM's effects on bone cell function and indicate that, like some other members of the LIF/interleukin-6 subfamily, OSM has potent bone regulatory activity. PMID- 8625884 TI - Activation of the JAK-STAT signal transduction pathway by oncostatin-M cultured human and mouse osteoblastic cells. AB - Oncastatin M (OSM) is one member of the leukemia inhibitory factor/interleukin-6 family of cytokines that has been shown to be a growth regulatory molecule. In osteoblastic cultures, OSM causes marked phenotypic changes and the enhanced secretion of interleukin-6. In this study, we have shown that stimulation of murine and human osteoblastic cultures and a human osteosarcoma cell line with OSM resulted in the tyrosine phosphorylation of a number of cellular proteins including members of both the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) family of signaling proteins. The JAKs, a family of intracellular kinases, and the STATs, a family of transcription factors, have both previously been shown to be tyrosine phosphorylated and activated in response to various cytokines, interferons, and growth factors in cells of non-skeletal origin. Using three different sources of cells of the osteoblast lineage, we demonstrate that OSM induces a rapid but transient tyrosine phosphorylation of the three JAK family members tested, JAK1, JAK2 and Tyk2. In addition, two members of the STAT family, Stat1alpha and Stat3, are tyrosine phosphorylated in osteoblastic cells in culture in response to OSM. OSM activation of this pathway in cells of the osteoblast lineage will result in the transcription of specific genes that ultimately may be associated with osteoblast function. PMID- 8625885 TI - Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats. AB - Glucose availability controls reproductive activity through modulation of LH secretion. The aim of the present study was to determine whether the glucoprivic suppression is potentiated by gonadal steroids and if glucoprivic suppression of pulsatile LH release is sexually differentiated. Pulsatile LH secretion was examined in rats after peripheral (jugular) administration of the competitive inhibitor of glycolysis, 2-deoxyglucose (2DG). Fourteen days after gonadectomy, blood samples were collected every 6 min for 3 h. One hour after the onset of sampling, 2DG was administered peripherally (200, 400, or 800 mg/kg BW, iv), and food intake was determined after 2DG injection in gonadectomized males and females in the presence or absence of sex steroids (testosterone or estradiol). To test the ability of the pituitary to produce LH under glucoprivic conditions, LHRH was injected every 30 min for 2.5 h in ovariectomized (OVX) rats 30 min after treatment with 400 mg/kg 2DG. At all peripheral doses of 2DG in females and at the middle and high doses of 2DG in males, mean plasma LH and LH pulse frequency decreased (P < 0.05) in the presence of steroids. However, in the absence of sex steroids, the lowest dose in females and the middle dose in males were not effective. Pituitary function appeared normal, because increases in mean plasma LH in response to the exogenous LHRH occurred in OVX rats treated with the middle dose of 2DG. Food intake significantly (P < 0.05) increased after 2DG injection in all groups except estrogen-treated OVX females at the low and high doses of 2DG. These findings suggest that glucoprivic suppression of LH pulses is potentiated by gonadal steroids in both sexes. Moreover, the hypothalamo hypophyseal axis of the female rat seems to be more sensitive to the decreased glucose availability induced by 2DG than that of the male. PMID- 8625886 TI - Suppression of luteinizing hormone pulses by restriction of glucose availability is mediated by sensors in the brain stem. AB - The availability of metabolic fuels such as glucose is known to influence reproductive function. Peripheral administration of 2-deoxyglucose (2DG), a competitive inhibitor of glycolysis, inhibits pulsatile LH secretion in the rat and growth-retarded lamb. We hypothesized that such glucoprivic suppression of LH secretion is mediated by the lower brain stem, because studies of both ingestive and reproductive behavior implicate lower brain stem structures, such as the area postrema, as a site that is sensitive to glucose availability. In the present study, the effect of a 2DG infusion, targeted to the fourth ventricle, on pulsatile LH secretion was examined in male rats. The males were castrated or castrated and immediately implanted with testosterone. Blood samples were collected through an indwelling atrial cannula every 6 min for 4 h for LH determination. After the first hour of blood sampling, 2DG (4 or 40 mg/kg) was infused into the fourth ventricle at a flow rate of 0.2 microliter/min through a cannula that had been stereotaxically implanted 1 week before sampling. The high dose of 2DG (40 mg/kg), but not the low dose (4 mg/kg), suppressed pulsatile LH secretion and increased food intake in both castrated and testosterone-treated castrated rats. LH secretion and food intake were not affected by the infusion of xylose (40 mg/kg) as an isoosmotic control. The site specificity of the 2DG treatment was confirmed by histological examination after an isovolumetric infusion of dye (0.2 microliter/min). These results suggest that glucose availability could influence LH secretion as well as feeding through a central sensor in the lower brain stem and are consistent with the idea that the area postrema might be an important glucosensor involved in the modulation of LH secretion. PMID- 8625887 TI - Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol. AB - Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5 fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor. PMID- 8625888 TI - Steroidogenic factor-1 as a positive regulator of rat granulosa cell differentiation and a negative regulator of mitosis. AB - Ovarian follicles contain small nonaromatase-expressing and large aromatase expressing granulosa cells (GCs). The present studies were designed to determine whether small GCs can differentiate into large GCs and/or express aromatase. Additional studies were conducted to assess the role of steroidogenic factor-1 (SF-1), an orphan nuclear receptor, in regulating GC differentiation and proliferation. For these studies, small GCs were isolated from immature rats by Percoll gradient centrifugation and cultured for up to 48 h with FSH and/or 8 bromo-cAMP (8-br-cAMP). FSH/8-br-cAMP induced a 2-fold increase in SF-1 messenger RNA levels within 4 h. This increase was maintained throughout the culture period. By 24 h culture, FSH/8-br-cAMP increased the percentage of large GCs. It was not until 48 h of culture with FSH and 8-br-cAMP that aromatase expression increased. This increase was detected by both Western blot and quantitative immunocytochemistry. 8-br-cAMP alone did not promote GC differentiation. Small GCs were then cultured with FSH/8-br-cAMP in the presence or absence of an antisense oligonucleotide complementary to the putative SF-1 ligand-binding site (SF-1 AS). As a control, small GCs were cultured with FSH/8-br-cAMP and an 18-mer nonsense oligonucleotide (SF-1 NS). The SF-1 AS, but not the SF-1 NS, prevented FSH/8-br-cAMP from increasing 1) SF-1 messenger RNA levels, 2) transcription of a SF-1(x2) promoter/luciferase construct, 3) GC size, and 4) aromatase expression. In a third series of experiments, small GCs were cultured for 24 h in 1) control media supplemented with 2) a mitogen, phorbol ester [12-O-tetraphorbol acetate (TPA)], 3) FSH/8-br-cAMP, or 4) both. TPA increased the number of GCs by 51 +/- 9%. FSH/8-br-cAMP completely blocked TPA-induced mitosis. When small GCs were cultured with FSH/cAMP, TPA, and SF-1 AS, the number of GCs increased by 50 +/- 7%. This increase was not observed with SF-1 NS. Taken together, these data demonstrate that SF-1 is expressed in both small and large GCs, and enhanced SF-1 expression is part of the molecular mechanism associated with GC differentiation. Interestingly, SF-1 not only regulates differentiation, but also inhibits TPA induced GC mitosis. PMID- 8625889 TI - N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner. AB - Previous studies have shown that both progesterone and cell contact inhibit granulosa cell (GC) apoptosis in vitro. Since the progesterone concentration associated with aggregated GCs may be higher than that of single GCs, experiments were conducted to differentiate progesterone's action from that of cell contact. For these studies, GCs were isolated from immature rats. Large GCs were collected after Percoll gradient centrifugation and placed in serum-free culture for 24 h. These studies confirmed that the rate of apoptosis was 2-3 times higher for single GCs than for aggregated GCs. This relationship was observed in the presence of aminoglutethimide, where progesterone concentrations were 3 ng/ml or less. A dose-response studied revealed that a minimum of 100 ng/ml progesterone were required to suppress apoptosis of single GCs. In addition, a single cell contact was shown to be sufficient to suppress apoptosis, with a small nonsteroidogenic GC being as effective as a large steroidogenic GC. Taken together, these data support the concept that cell contact blocks apoptosis in a progesterone-independent manner. GC contact is due to the presence of gap and adhesion-type junctions. To assess which, if either, of these junctions is involved in mediating the antiapoptotic action of cell contact, cocultures were set-up between GCs and R2C cells. Contact with R2C cells inhibits GC apoptosis, but does not result in the formation of functional gap junctions. This demonstrates that gap junctions are not essential to maintain GC viability. Adhesion-type junctions result from a homophilic binding of N-cadherin, which is expressed by both GCs and R2C cells. When this binding is inhibited by treatment with either an antibody to N-cadherin or a synthetic N-cadherin peptide, cell aggregation is attenuated. For those cells that form cell contacts in the presence of these N-cadherin-binding inhibitors, the percentage of apoptotic cells is increased compared to that in controls. These observations suggest that homophilic binding of N-cadherin molecules on the surface membranes of adjacent GCs initiates a signal transduction cascade that ultimately inhibits apoptosis. PMID- 8625890 TI - Angiotensin II induces ovulation and oocyte maturation in rabbit ovaries via the AT2 receptor subtype. AB - The present study was undertaken to investigate the role of angiotensin II (Ang II) in ovulation and ovarian steroidogenesis and prostaglandin (PG) production via the Ang II receptors in rabbit ovaries. In in vitro perfused rabbit ovaries, PD123319, a selective nonpeptide antagonist for AT2 receptors, reduced the Ang II induced ovulation in a dose-dependent manner, whereas CV-11974, a selective nonpeptide antagonist for AT1 receptor, did not affect the Ang II-induced ovulation. Ang II also significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes in the absence of gonadotropin. The addition of PD123319 at 10 (-6) M to the perfusate significantly inhibited the Ang II induced oocyte maturation. Ang II did not stimulate the production of progesterone by perfused rabbit ovaries but significantly stimulated the production of estradiol (E2) and PGs. When PD123319 at 10(-6) M was added to the perfusate 30 min before the onset of Ang II administration, the Ang II-stimulated production of E2 and PGs was significantly blocked. Saralasin, a peptide analog of Ang II, inhibited the specific binding of [125I] iodo-[Sar1, Ile8] Ang II to rabbit ovarian membranes in a concentration-dependent manner, yielding an inhibitory constant (IC50) value of 1.58 x 10(-9) M. PD123319 and CV-11974 also inhibited the binding of [125I]iodo-[Sar1, Ile8] Ang II; however, PD123319 and CV 11974 were 15 and 40 times less potent than saralasin, respectively. Autoradiographic study revealed that an intense localization of Ang II receptors in the rabbit ovaries was present in the granulosa cell layers and the stroma of the preovulatory follicles. AT2 receptors were predominantly located in granulosa cells, whereas AT1 receptors were more concentrated in the stroma and thecal cell layers. In summary, Ang II induced ovulation and oocyte maturation and stimulated the production of E2 and PG by perfused rabbit ovary in vitro via the AT2 receptor. Thus, locally produced Ang II may be part of a novel intraovarian paracrine or autocrine control mechanism during the ovulatory process. PMID- 8625891 TI - Persistent changes in corticotropin-releasing factor neuronal systems induced by maternal deprivation. AB - There is considerable evidence that CRF-containing neurons integrate the endocrine, autonomic, immune, and behavioral responses to stress. In this study we examined long term effects of early stress on developing hypothalamic and extrahypothalamic CRF neural systems in the rat brain and subsequent responses to stress in the adult. Specifically, we sought to determine whether adult male rats previously isolated for 6 h daily during postnatal days 2-20 react in a biochemically distinct manner to a mild foot shock stress compared to controls. Four treatment groups were examined: nondeprived (NDEP)/no shock, NDEP/shock, deprived (DEP)/no shock, and DEP/shock. Compared to the NDEP group, DEP rats exhibited an increase in both basal and stress-induced ACTH concentrations. Moreover, DEP rats exhibited a 125% increase in immunoreactive CRF concentrations in the median eminence and a reduction in the density of CRF receptor binding in the anterior pituitary compared to those in all NDEP rats. Alterations in extrahypothalamic CRF systems were also apparent in DEP vs. NDEP animals, with an observed 59% increase in the number of CRF receptor-binding sites in the raphe nucleus and an 86% increase in immunoreactive CRF concentrations in the parabrachial nucleus. These results indicate that maternal deprivation before weaning in male rats produces effects on CRF neural systems in both the central nervous system and pituitary that are apparent several months later and are probably associated with persistent alterations in behavioral response in adult rats. PMID- 8625892 TI - Islet acid glucan-1,4-alpha-glucosidase: a putative key enzyme in nutrient stimulated insulin secretion. AB - Little attention has been paid to a possible relationship between lysosomal function and stimulation of secretory processes in endocrine cells. The last few years it has become increasingly evident that the secretion of insulin from the pancreatic beta-cell is the result of a very complex cascade of events, the details of which are far from elucidated and indeed may include the participation of the lysosomal system. We report here, with a combined in vitro and in vivo approach, that selective inhibition of islet lysosomal glycogenolytic acid glucan 1,4-alpha-glucosidase activity by the long-acting 1-deoxynojirimycin derivative emiglitate induces a profound suppression of nutrient-induced insulin release. In islet homogenate emiglitate strongly and dose-dependently inhibited the activity of acid glucan-1,4-alpha-glucosidase (EC50 approximately 10(-6) M) without affecting other classical lysosomal enzyme activities. The emiglitate-induced inhibition curve for glucose-stimulated insulin secretion from isolated islets was remarkably similar to the inhibition curve for acid glucan-1,4-alpha glucosidase. Moreover, insulin release stimulated by the nonglucose nutrient secretagogues, leucine, and alpha-ketoisocaproic acid (KIC) was totally suppressed by emiglitate. In contrast, receptor activated insulin secretion induced by the insulinotropic hormone cholecystokinin (CCK-8) was unaffected by the drug. Further, parenteral pretreatment of mice with emiglitate markedly suppressed the insulin secretory response to an iv injection of glucose or KIC, whereas the response to an iv injection of CCK-8 was unaffected. In accordance with this, islets isolated from emiglitate-treated mice showed a reduced activity of acid glucan-1,4-alpha-glucosidase and, moreover, such islets incubated in vitro, secreted less insulin in response to glucose than did control islets. Finally, pretreatment of mice with purified fungal acid glucan-1,4-alpha glucosidase, enzyme replacement, brought about a markedly increased insulin secretory response after an iv injection of KIC, whereas the insulin response after CCK-8 injection was unaffected. Taken together with previous observations, the present data strongly suggest that islet lysosomal acid alpha glucosidehydrolases are involved in the multifactorial process of nutrient induced insulin secretion. The existence of hitherto unresolved and complex interactions between different beta-cell organelles in the insulin secretory processes should be thoroughly reevaluated. PMID- 8625893 TI - RU486 on an estrogen background blocks the rise in serum follicle-stimulating hormone induced by antiserum to inhibin or ovariectomy. AB - We used passive immunization with an antiserum to the alpha-subunit of inhibin (anti-I) or acute ovariectomy to investigate the relationship between serum inhibin levels and FSH secretion in the presence of the progesterone/glucocorticoid antagonist RU486. We demonstrated previously that 1) anti-I administered at 1700 h causes serum FSH to rise on the morning of estrus, even in the presence of a GnRH antagonist, when the two treatments are delivered on proestrus; and that 2) RU486 given on proestrus (1230 h), a time when serum estradiol levels are high, not only blocks the natural secondary FSH surge, but also suppresses the anti-I-induced rise in serum FSH on the morning of estrus. We have now extended our studies of the relationship between inhibin and RU486 to investigate treatment with RU486 and anti-I on a different day of the cycle, estrus, when serum estradiol levels are low. When both RU486 and anti-I were given on estrus (1230 and 1700 h, respectively), RU486 failed to block the anti-I induced rise in serum FSH on the next morning of metestrus, in contrast to the blockade seen with RU486 treatment on the day of proestrus. However, pretreatment with estradiol benzoate (50 microgram) on the evening of proestrus, before the RU486 and anti-I treatment on estrus, caused RU486 to suppress the effects of anti-I on serum FSH, as it does when given on proestrus. We then repeated the study, using ovariectomy on proestrus or estrus (1700 h) to raise serum FSH, and assessed the effects of RU486 treatment at proestrus and estrus and estradiol benzoate treatment on proestrus. Our results indicate that treatment with RU486 can block the postovariectomy rise in serum FSH only in the presence of high circulating estradiol levels. We conclude that the inhibitory action of RU486 on FSH secretion after a fall in serum inhibin depends on a precedent estradiol background, probably due to induction of progesterone receptors by estradiol. PMID- 8625894 TI - Differential coexpression of genes encoding prothyrotropin-releasing hormone (pro TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: implications for differential processing of pro-TRH. AB - Pro-TRH is cleaved at paired basic residues to yield five copies of TRH and cryptic peptides. Recent studies have shown that the prohormone convertases, PC1 and PC2, can process pro-TRH correctly. To determine whether these two enzymes could play a role in pro-TRH processing in vivo, the regional and cellular colocalization of pro-TRH messenger RNA (mRNA) with the mRNAs encoding the prohormone convertases PC1 and PC2 was examined in rat brain, using in situ hybridization histochemistry. Differential regional distribution of pro-TRH mRNA with PC1 and/or PC2 mRNA was found in several brain regions. For example, in the olfactory regions, there was coexpression of pro-TRH mRNA in the glomerular layer with PC2 mRNA, but not PC1 mRNA, whereas in the tenia tecta, coexpression of pro TRH and PC1 mRNAs was evident, but PC2 mRNA was absent. Pro-TRH mRNA in the paraventricular nucleus was coexpressed with both PC1 and PC2 mRNAs, whereas the basal lateral hypothalamus showed coexistence of pro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Interestingly, pro-TRH was expressed in the thalamic reticular nucleus, but neither PC1 nor PC2 was detectable in this region. Cellular colocalization studies using double in situ hybridization histochemistry showed the presence of PC2 mRNA in the pro-TRH neurons of the olfactory glomerular layer and basal lateral hypothalamus, and PC1 mRNA in the pro-TRH neurons in the paraventricular nucleus. These results suggest that PC1 and PC2 are enzyme candidates for the processing of pro-TRH in vivo. Moreover, the differential distribution of PC1 and PC2 mRNAs with pro-TRH mRNA may be responsible for the differential processing of this prohormone in the central nervous system. The absence of PC1 and PC2 mRNAs in certain TRH neurons raises the possibility that prohormone convertases other than PC1 and PC2 may be involved in the processing of brain pro-TRH. PMID- 8625895 TI - Pretreatment with bovine growth hormone is as effective as treatment during metabolic stress to reduce catabolism in fasted lambs. AB - The effects of recombinant bovine GH (rbGH) treatment on the insulin-like growth factor (IGF) axis and protein metabolism during fasting induced metabolic stress were evaluated in young lambs. To explore whether rbGH pretreatment alone might offer a degree of protection against nutritional stress, we compared the effects of rbGH given only before or during the fasting-induced metabolic stress with that given over the whole period. The animals were fed ad libitum for 5 days (well fed phase) and then fasted for 70 h (fasted phase). The rbGH was administered during either the well fed and the fasted phase (G-G), only during the well fed phase (G-S), or only during the fasted phase (S-G), and the effects were compared with those of saline treatment throughout both phases (S-S; n = 7/group). The rate of net protein catabolism, analyzed on the final day of the study, was reduced (P < 0.001) to a similar degree in all rbGH-treated groups compared with that in the S-S group. rbGH pretreatment was as effective as rbGH administered during the catabolic phase. Plasma IGF-I was increased (P < 0.001) in the well fed phase by rbGH treatment and decreased in the fasted phase in all groups. The rbGH treatment during the fasted phase resulted in a smaller fall in plasma IGF-I levels than saline treatment (P < 0.05, G-G vs G-S and S-G vs. S-S), but no difference was observed in the specific binding of [125I]ovine GH to the hepatic membranes from animals of the different groups. There was a negative correlation between net protein catabolism and plasma IGF-I levels (r = -0.48; P < 0.01) and specific binding of [125I]ovine GH to hepatic membranes (r = -0.56; P < 0.001). Plasma IGF-II levels were decreased by rbGH treatment during the well fed phase, but the responses to treatment during the fasted phase were variable, suggesting that plasma IGF-II is regulated in a different manner than plasma IGF I. The fasting-induced fall (P < 0.05) in plasma concentrations of IGF-binding protein (IGFBP)-3 was reduced with rbGH treatment, and plasma concentrations IGFBP-2 were altered in an inverse manner. This study suggests that fasting induced GH resistance can be alleviated by rbGH treatment independent of whether treatment is commenced before or after the onset of catabolic stress. Our observation of prolonged anticatabolic action of prophylactic rbGH treatment supports the proposal that prophylactic use of GH may reduce the degree of catabolism associated with subsequent interventions and, thus, improve clinical outcome. PMID- 8625896 TI - Expression and regulation of neuropeptide Y messenger ribonucleic acid in cultured immature rat Leydig and Sertoli cells. AB - Neuropeptide Y (NPY) potentiates the release of gonadotropins from the pituitary in response to GnRH in the hypothalamus and modulates reproductive function. In the present study, we showed that 1) specific organs in the male rat reproductive tract express NPY messenger RNA (mRNA); 2) the multifactorial regulation of NPY mRNA in rat Leydig and Sertoli cells is temporally and hormonally regulated in vitro; 3) both Sertoli cell factor(s) and germ cell factor(s) potentiated to stimulate NPY gene levels in Leydig cells; and 4) intense NPY immunoreactivity was detected in cultured Leydig cells. Using the RT-PCR method, we found that Leydig cells, Sertoli cells, epididymis, and vas deferens expressed NPY mRNA, whereas germ cells, seminal vesicle, and prostate did not. Northern blot analyses showed that NPY mRNA was not expressed in freshly isolated immature Leydig cells, but that NPY mRNA levels were increased by the addition of LH, cytokines such as interleukin-1 alpha and -1 beta, forskolin, or phorbol 13-myristate 12-acetate. Npy mRNA levels in immature Sertoli cells were also increased by FSH. In addition, a germ cell factor(s) secreted from pachytene spermatocytes or round spermatids purified by centrifugal elutriation as well as a Sertoli cell factor(s) stimulated by FSH increased NPY gene levels in Leydig cells. Immunocytochemical analyses showed that the immunostaining was more marked in Leydig cells than in Sertoli cells in vitro. These findings indicate that testicular NPY gene expression is induced in Leydig cells or Sertoli cells by gonadotropins or cytokines within the testes, and that factors secreted from Sertoli cells or germ cells affect NPY gene expression in Leydig cells in vitro. Our findings suggest that NPY expressed in the reproductive system may modulate reproductive function as well as that in the nervous system. PMID- 8625897 TI - Impaired estrogen action in the uterus of insulin-like growth factor binding protein-1 transgenic mice. AB - Insulin-like growth factor-I (IGF-I) has been implicated as autocrine/paracrine mediator of estrogen action in the rodent uterus. Here, we examined the effects of 17-beta estradiol (E2), epidermal growth factor (EGF), and IGF-I on DNA synthesis in the uterus of ovariectomized (ovex) transgenic mice (Tg), which overexpress rat insulin-like growth factor binding protein-1 (IGFBP-1). Litter size was significantly reduced in Tg mice compared with wild-type mice. Immunohistochemical studies localized the expression of the transgene to the luminal and glandular epithelium. In addition, rat IGFBP-1 immunoreactivity was present in luminal secretions. E2-induced uterine DNA synthesis as measured by methyl-3H thymidine incorporation, was significantly reduced in ovex Tg mice; 4.77 +/- 0.59 and 4.97 +/- 0.53 for Tg strains 57C and 277A, respectively, compared with 8.65 +/- 0.73 fmol/microgram of DNA for Wt mice. Similarly, uterine weight after three daily injections of E2 was reduced in Tg mice compared with Wt mice; 2.85 +/- 0.39 vs. 4.23 +/- 0.26 mg/g BW, P < 0.01. Semiquantitative RT-PCR assays were used to demonstrate changes in uterine IGF-I messenger RNA (mRNA) and EGF mRNA abundance after administration of E2. An approximately 3-fold increase in IGF-I mRNA abundance was seen 6 h after E2 in both Tg and Wt mice. Over the same time course, little change was seen in EGF mRNA levels, which were similar in Tg and Wt mice. After 3 days of E2 treatment, an increase in EGF mRNA was apparent in Wt mice but not in Tg mice. The uterine DNA response to both IGF-I and EGF was significantly attenuated in Tg mice compared with Wt mice. The data reported here together with previous reports of E2 regulation of IGF-I and IGFBP 1 expression in uterus support the hypothesis that the IGF-I is a mediator of estrogen action in the uterus. In addition attenuation of the EGF response in the uterine tissue of Tg mice suggests that this response is also mediated, in part, by IGF-I. PMID- 8625898 TI - Testosterone is required for gonadotropin-releasing hormone stimulation of luteinizing hormone-beta messenger ribonucleic acid expression in female rats. AB - Pulsatile GnRH stimulates the synthesis and secretion of LH and FSH in both male and female rats. In the male rat, exogenous GnRH pulses increase alpha, LH and FSH beta messenger RNAs (mRNAs) 3-fold within 24 h. In contrast, the results of recent in vivo and in vitro studies have shown that GnRH stimulates an increase in alpha and FSH beta mRNAs, but not LHbeta. However, during the estrous cycle, LHbeta mRNA increases during the GnRH-induced LH surge on proestrus afternoon. This increase in LHbeta mRNA appears to be coincident with a transient rise in serum testosterone (T). Therefore, the present study was conducted to determine whether T has a role in facilitating GnRH stimulation of LHbeta mRNA expression. In the first group of studies, adult female rats were ovariectomized, and T implants were inserted sc 7 days before the study (serum T, 1.86 ng/ml). Animals received iv pulses of GnRH (25 ng; 30-min interval) for 6-24 h (saline pulses to controls). The data showed that in the presence of T, GnRH stimulated a significant increase in LHbeta (as well as alpha and FSH beta) mRNAs within 6 h (P < 0.05 vs. saline-pulsed controls). Other results revealed that T treatment was critical to the stimulatory effect of GnRH on LH beta mRNA. A second group of studies examined the time course and dose effects of T on LH beta mRNA expression. Maximal LH beta mRNA responses to GnRH (3-fold increase vs. saline controls; P < 0.05) were seen after pretreatment with the lowest dose of T examined (serum T, 0.42 ng/ml), which is similar to T concentrations on proestrus. Higher doses of T suppressed LH release, as well as LH mRNA responses to GnRH. The T-induced LHbeta mRNA response to pulsatile GnRH was seen within 24 h of exposure to T and was the result of an androgenic action, as similar results were observed in rats that received dihydrotestosterone. These findings suggest that T is required to facilitate GnRH stimulation of LHbeta mRNA in the female rat. Moreover, in the presence of the concentrations of T seen on proestrus, LHbeta mRNA increases within 6 h, which is similar to the time course seen during the LH surge. Thus, the present results also suggest that the combined effects of the rise in serum T and increased GnRH secretion induce the rapid rise in LHbeta mRNA expression on the afternoon of proestrus. PMID- 8625899 TI - Detection of extended distribution of beta2-thyroid hormone receptor messenger ribonucleic acid (RNA) in adult rat brain using complementary RNA in situ hybridization histochemistry. AB - In situ hybridization histochemistry using a complementary RNA probe, directed at the entire length of the thyroid hormone receptor beta2 (TRbeta2)-specific region (-56 to 495 bp), was used to evaluate expression of TRbeta2 messenger RNA (mRNA) in coronal sections of adult rat brain. An extended distribution and intense expression of TRbeta2 mRNA was found in several regions of rat brain, including regions where TRbeta2 mRNA had not been previously identified using PCR or in situ hybridization histochemistry. These areas included hippocampus (dentate gyrus and C1, -2, and -3), cerebral cortex (predominantly layer 3), arcuate nucleus, median eminence, medial geniculate nucleus, tegmental bundle, medial and lateral lemniscus, Purkinje layer of the cerebellum, and several brain stem nuclei. In conclusion, we have developed a highly sensitive and specific method to demonstrate TRbeta2 mRNA expression in adult rat brain. The present findings are in agreement with immunoreactive TRbeta2 studies of rat brain and argue against the presence of an unidentified T3-binding protein to explain the previous discordant results of TRbeta2 mRNA and protein studies. In addition, the specificity of distribution of TR beta2 mRNA to certain brain nuclei, particularly those involved in hearing, implies a specific functional role of this receptor subtype and provides a physiological basis to understand the effects of hypothyroidism on brain development. PMID- 8625900 TI - Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone. AB - The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein. PMID- 8625901 TI - Constitutively active G(S) alpha-subunits stimulate Pit-1 promoter activity via a protein kinase A-mediated pathway acting through deoxyribonucleic acid binding sites both for Pit-1 and for adenosine 3',5'-monophosphate response element binding protein. AB - Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cell-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response to active alpha(S) subunits contributes to the formation of these adenomas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimulates expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promoter chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yielded, respectively, reduced or no stimulation of CAT activity. A dominant inhibitor of protein kinase A (PKA), RAB, blocked almost completely either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that PKA is required for the action of Q227L-alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 binding site reduced but did not eliminate either FSK or Q227L-alpha(S) stimulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alpha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduction in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that one or both of the Pit-1 promoter adenosine 3'5'-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L alpha(S). The observation that S133A-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated site PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter. PMID- 8625902 TI - Biphasic developmental expression of adrenocorticotropin receptor messenger ribonucleic acid levels in the baboon fetal adrenal gland. AB - We have previously shown an estrogen-dependent developmental regulation of placental oxidation of cortisol to cortisone that results in enhanced fetal pituitary ACTH production and the induction of steroidogenic enzymes in and de novo cortisol production by the fetal adrenal in the second half of baboon pregnancy. However, it is not known whether the receptor for ACTH is simultaneously generated at this time in development to provide a mechanism for mediating the tropic action of ACTH on steroidogenesis in the primate fetal adrenal gland. Therefore, in the present study we determined the levels of ACTH receptor messenger RNA (mRNA) and correlated ACTH receptor expression with appearance of the mRNA for delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD), the enzyme protein that signals functional maturation of the definitive cortical zone in the baboon fetal adrenal. A baboon ACTH receptor complementary DNA was cloned and hybridized with polyadenylated RNA isolated from baboon (Papio anubis) fetal adrenals obtained in early (days 58-64; RNA from seven baboon fetuses pooled to yield three samples), mid-(days 99-103; RNA from five baboons pooled to yield four samples), and late (days 165-168; RNA of four individual baboon fetuses) gestation (term = 184 days). Expression of the primary 3.4-kilobase ACTH receptor mRNA transcript, determined by Northern blot and expressed as a ratio of beta-actin mRNA, was minimal early in gestation (mean +/- SE, 0.11 +/- 0.05 arbitrary densitometric units). However, fetal adrenal ACTH receptor mRNA levels increased (P < 0.001, by ANOVA) approximately 13-fold to 1.41 +/- 0.16 at midgestation, then declined by 70% (P < 0.001) to 0.41 +/- 0.10 in late gestation. To determine whether the decrease in ACTH receptor expression by the fetal adrenal in the second half of pregnancy reflected programmed cell death, the integrity of genomic DNA was assessed by 32P-labeled DNA gel electrophoresis and in situ DNA end labeling. Because DNA oligonucleosomes and apoptotic DNA strand breaks characteristic of apoptosis were absent in the adrenal glands of fetal baboons, the decline in ACTH receptor mRNA levels in the fetal adrenal did not seem to reflect programmed cell death. Expression of the single 2.0-kilobase mRNA transcript for 3betaHSD, an enzyme localized specifically in the definitive zone of the fetal adrenal, was minimal in early (0.01 +/- 0.00 arbitrary units) and mid- (0.10 +/- 0.01) gestation. However, 3betaHSD mRNA levels were markedly increased late in gestation to a value (1.38 =/- 0.34) approximately 13-fold greater (P < 0.001) than that in midgestation. These findings indicate that there was a biphasic monomodal developmental expression of the ACTH receptor in the baboon fetal adrenal, which contrasted with the progressive increase in adrenal weight, 3betaHSD expression, and de novo cortisol production previously determined. Because the fetal adrenal is comprised mainly of the fetal cortical zone throughout gestation, the decrease in ACTH receptor expression between mid- and late gestation seems to occur primarily in the latter zone and may signal a selective decline in tropic responsivity of and delta5-C19-steroid, e.g. dehydroepiandrosterone, biosynthesis within the baboon fetal adrenal gland. PMID- 8625903 TI - Testosterone regulates gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs. AB - As the GnRH-induced secretion of gonadotropins is critically dependent upon an increase in the intracellular calcium ion concentration ([Ca2+]i) and modulated by gonadal factors, the effects of gonadal steroids on the pattern of calcium mobilization in single gonadotrophs of the male rat were examined using the fluorescent Ca2+ indicator fura-2/AM. In cells from intact rats, low concentrations of GnRH induce repetitive oscillations in [Ca2+]i, whereas spike plateau responses are observed at higher concentrations in single gonadotrophs. After castration, there was a significant change in the relationship between the GnRH concentration and the changes in [Ca2+]i. Increasing concentrations of GnRH (to 1 micron) generate fewer spike-plateau responses in gonadotrophs from castrate rats, with oscillatory responses predominating. This change develops with time after castration, with the proportion of cells oscillating in response to 100 nM GnRH peaking by 7 days. This effect of castration on GnRH-induced [Ca2+]i signals was reversed by treatment with testosterone propionate (100 microgram/100 g BW-day). Castration-induced decreases in serum testosterone, seminal vesicle, and prostate weights and increases in serum LH concentration were also corrected by testosterone propionate treatment. These findings demonstrate that testosterone regulates GnRH-stimulated Ca2+ signals in gonadotrophs and suggest that gonadal steroids exert a regulatory role in the secretion of gonadotropins at the level of Ca2+ mobilization. PMID- 8625904 TI - Effect of endothelin-1 on bovine luteal cell function: role in prostaglandin F2alpha-induced antisteroidogenic action. AB - Endothelin-1 (ET-1) a vasoactive peptide, is synthesized and secreted by endothelial cells. In the bovine corpus luteum (CL), endothelial cells constitute a major proportion (53.5%) of total CL cells. This study was designed to examine the effects of ET-1 on bovine luteal cell functions and its involvement in the action of PGF2alpha. To better define the cells implicated in this process, we used CL slices, whole CL-derived cells, and steroidogenic large (LLC) and small (SLC) luteal-like cells. High affinity binding sites for ET-1 (K(d), approximately 0.3 x 10(-9)) were present in both steroidogenic luteal cells. The binding affinity of ET-1 was 3 orders of magnitude higher than that of ET-3, and a selective ETA receptor antagonist (BQ123) competed similarly to ET-1, suggesting the presence of ETA receptors. The lack of effect of ET-3 on CL derived cells further supported this conclusion. Both basal progesterone secretion and bovine LH (5 ng/ml)-stimulated progesterone secretion from CL derived cells were significantly inhibited by ET-1 in a dose-dependent manner, whereas preincubation of these cells with ETA receptor antagonist prevented the inhibitory effect of added ET-1. Incubation of LLC with 10(-8) M ET-1 inhibited their progesterone secretion (114.8 vs. 176.7 ng/10(5) cells-20 h; P < 0.05). On the other hand, ET-1 did not affect progesterone production from SLC despite the presence of ET-binding sites. PGF2alpha only inhibited LH-stimulated progesterone secretion by luteal slices. This antisteroidogenic effect of PGF2alpha could be prevented by the addition of a selective ETA receptor antagonist. Luteal tissue and microvascular endothelial cells isolated from bovine CL produced ET-1; in contrast, the peptide was undetectable in the culture medium or in cell extracts of either LLC or SLC. These data support the concept that ET-1 may play a paracrine regulatory role in bovine luteal function and propose a novel role for this peptide in mediating PGF2alpha-induced luteal regression. PMID- 8625905 TI - Involvement of tyrosine phosphorylation in the regulation of 5'-deiodinases in FRTL-5 rat thyroid cells and rat astrocytes. AB - Recent studies suggest that protein tyrosine phosphorylation may play a role in the regulation of thyroid growth and function. In the present study, we used genistein, a specific inhibitor of tyrosine phosphorylation, to determine if tyrosine phosphorylation is involved in the regulation of type I 5'-deiodinase (5'D-I) expression in FRTL-5 cells and type II 5'-deiodinase (5'D-II) in rat astrocytes. Incubation of FRTL-5 cells with genistein (100 microM) for 3 days had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived cells, incubation of FRTL-5 cells with genistein (100 microM) resulted in a modest, but not significant, decrease in 5'D-I activity. Incubation of FRTL-5 cells with TSH (100 microU/ml), Bu2cAMP (0.5 mM) or forskolin (1 microM) resulted in marked increases in 5'D-I activity. In the presence of genistein (100 microns), however, the TSH, Bu2cAMP and forskolin-induced increases in 5'D-I activity were completely inhibited. In Bu2cAMP-stimulated FRTL-5 cells, incubation with genistein (1, 10, and 100 microM) resulted in a dose-dependent decrease in 5'D-I activity, with 100 microns genistein completely blocking the Bu2cAMP-induced increase in 5'D-I activity. Similarly, we found that in FRTL-5 cells, genistein (100 microns) completely blocked the Bu2cAMP-induced increase in 5'D-I messenger RNA (mRNA) levels, DNA synthesis as assessed by [3H]thymidine incorporation, and the T3-induced increase in 5'D-I activity. To determine if addition of genistein to FRTL-5 cells resulted in a general inhibition of Bu2cAMP induced responses, we examined its effect on the Bu2cAMP-induced increase in c fos mRNA levels. Bu2cAMP-induced c-fos mRNA levels were not affected by the treatment of cells with genistein (100 microM). We then examined the effect of genistein on the Bu2cAMP and hydrocortisone-induced 5'D-II activity in cultured rat astrocytes. Genistein (100 microM) had no effect on cell viability as assessed by trypan blue exclusion. In serum deprived astrocytes, addition of Bu2cAMP (1 mM) and hydrocortisone (100 nM) resulted in a 110-fold increase in 5'D II activity. Addition of genistein (100 microM) to stimulated astrocytes completely blocked the Bu2cAMP and hydrocortisone-induced increase in 5'D-II activity. The present data suggest that tyrosine phosphorylation dephosphorylation may play an important role in the regulation of thyroid hormone deiodination and action in the thyroid and brain. PMID- 8625906 TI - Regulation of hypothalamic neuropeptide-Y neurons by growth hormone in the rat. AB - GH is thought to exert a short-loop feedback action on the hypothalamic somatostatin- and GH-releasing hormone (GHRH)-containing neurons. The direct actions of GH are mediated through GH receptors. In the male rat, few GHRH containing neurons in the arcuate nucleus (ARC) appear to express the GH receptor messenger RNA (mRNA); however, some unidentified neurons near GHRH neurons do. Recent evidence suggests that neuropeptide-Y (NPY)-containing neurons, which are located near GHRH neurons in the ARC, are targets for GH action because treatment of rats with GH induces c-fos expression in these cells. We conducted two experiments to test the hypothesis that GH acts on NPY neurons in the ARC. First, we performed double-label in situ hybridization to determine whether NPY neurons in the ARC express GH receptor mRNA. Second, we investigated the possibility that GH regulates NPY mRNA expression by using in situ hybridization to compare ARC NPY mRNA levels among groups of normal (n = 7), hypophysectomized (n = 7), and hypophysectomized/rGH-treated (1.5 mg rat GH over 3 days; n = 6) rats. We found that most of the NPY-containing neurons in the ARC expressed GH receptor mRNA, whereas hypothalamic NPY neurons residing outside of the ARC did not. Furthermore, hypophysectomy significantly decreased NPY mRNA levels, and GH treatment restored the levels to those of the intact animals. We conclude that GH regulates the activity of NPY neurons in the ARC by a direct action on GH receptors that are expressed by NPY neurons. Whether the action of GH on NPY neurons in the ARC is related to the feedback control of GH secretion or some other physiological function remains to be determined. PMID- 8625907 TI - Developmental regulation of pituitary growth hormone-releasing hormone receptor gene expression in the rat. AB - The mechanisms governing age-dependent patterns of GH secretion are not well understood. Studies have shown that pituitaries of fetal and neonatal mammals are highly responsive to the stimulatory effect of GH-releasing hormone (GHRH) compared to those of mature mammals. Differential pituitary responsiveness to GHRH may, therefore, contribute to the elevated circulating GH concentrations characteristic of the perinatal period and the subsequent decline in circulating GH late in life. Age-dependent expression of GHRH-receptor (GHRH-R) would provide a cellular mechanism to direct differential somatotroph responsiveness to GHRH. Therefore, we determined the ontogeny of rat GHRH-R gene expression. We studied rats at ages that correspond to major changes in circulating GH levels: embryonic day 19.5 (of 21.5-day gestation period); postnatal days 2, 12, 30, 45, and 70; and 1 yr of age. We found that GHRH-R messenger RNA (mRNA) expression was markedly age dependent (P < 0.0003). The concentration of GHRH-R mRNA was highest on day 19.5 of gestation, the earliest age studied, and declined during the perinatal period to reach a nadir at 12 days of age. GHRH-R mRNA levels increased at 30 days of age, at time corresponding to the onset of sexual maturation, and then declined later in life. In addition, we assessed the expression of GH and Pit-1 mRNAs in pituitaries of the same age groups; we found age-dependent patterns for these transcripts that did not parallel the ontogenic pattern observed for GHRH-R mRNA. Together, these data indicate that expression of rat pituitary GHRH-R mRNA is developmentally regulated and suggest that maturation of GHRH-R may be an important determinant of somatotroph function during early development. PMID- 8625908 TI - Glycoprotein hormone alpha-subunit functions synergistically with progesterone to stimulate differentiation of cultured human endometrial stromal cells to decidualized cells: a novel role for free alpha-subunit in reproduction. AB - Glycoprotein hormone-free alpha subunit is secreted by the pituitary throughout the menstrual cycle and by the placenta during pregnancy. We showed previously that free alpha subunit stimulated PRL secretion from term pregnancy decidual cells, suggesting a function for free alpha in pregnancy. However, no role has been ascribed to free alpha in the normal menstrual cycle. Using an in vitro model, we examined the role of alpha subunit in regulating human endometrial stromal cell differentiation (decidualization). PRL and insulin-like growth factor binding protein-1 (IGFBP-1), specific decidual secretory products, were used as markers of decidualization. We found that alpha subunit acted synergistically with progesterone (P) to induce more rapid decidualization with higher output (2- to 6-fold) of PRL and IGFBP-1, compared with P alone (P < 0.01). The effect of alpha was dose dependent, with stimulatory activity starting at 0.05 ng/ml and reaching maximal levels at 1-2 ng/ml. These levels correspond to serum concentrations of free alpha found during the luteal phase of the menstrual cycle when endometrial decidualization occurs in vivo. These findings demonstrate new biological activity for alpha subunit in the regulation of human endometrial decidualization and indicate that free alpha plays a role in human reproduction. Furthermore, demonstration of potential bioactivities of free alpha subunit has important implications for understanding normal endocrine function and various pathological conditions. PMID- 8625910 TI - Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein. AB - PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts. PMID- 8625909 TI - Characterization of SNARE protein expression in beta cell lines and pancreatic islets. AB - Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6 f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t) membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis. PMID- 8625911 TI - Effect of 1alpha-vitamin D3 and estrogen therapy on cortical bone mechanical properties in the ovariectomized rat model. AB - It is well documented that both bone mass and size of ovariectomized rats can be increased by 1alpha-vitamin D3 therapy. The repercussion of this therapy on bone mechanical competence is far less clear. Therefore, the objective of this study was to examine the mechanical properties of the shaft femur in ovariectomized rats (3 months old) receiving estrogen (0.25 mg/kg-week) and /or 1alpha-vitamin D3 (0.5 microgram/kg-day). The medication was given during 6 months starting immediately after ovariectomy or starting 3 months later. Torsional testing was performed from which the parameters strength, stiffness, maximum angular displacement, and energy-absorbing capacity (toughness) were derived. Multiple regression models were generated to estimate the relative importance of the therapies on bone mechanical properties. Bone stiffness increased with age. Ovariectomy improved bone mechanical parameters until 6 months postovariectomy, whereas estrogen treatment resulted in similar mechanical properties as those in intact age-matched controls. A significant improvement of all mechanical parameters was observed after 1alpha-vitamin D3 therapy. The combined therapy of 1alpha-vitamin D3 and estrogen was less effective than 1alpha-vitamin D3 alone, but better than estrogen therapy alone, suggesting interactive effects between both therapies. We conclude that 1alpha-vitamin D3 treatment of ovariectomized rats improves bone mechanical competence, which might be partially related to alterations in both bone mass and size. PMID- 8625912 TI - Regulation of peptide YY homeostasis by gastric acid and gastrin. AB - Peptide YY (PYY) is a gut hormone localized primarily in the distal bowel. Because circulating PYY inhibits gastric acid secretion, we investigated the effects of gastric acid secretion and gastrin on gene expression and secretion of PYY. In conscious dogs, PYY release in response to oral food was inhibited (P < 0.05) by pharmacologic inhibition of gastric acid secretion (omeprazole, famotidine). In rats, omeprazole treatment resulted in a significant elevation in serum gastrin concentrations and a simultaneous decrease in PYY messenger RNA (mRNA) and peptide levels in the colon; administration of a gastrin receptor antagonist (L365, 260) prevented the inhibitory actions of omeprazole on colonic PYY mRNA levels. In athymic-nude mice, implantation of a human gastrinoma resulted in an elevation of serum gastrin concentrations and a concomitant depression of colonic PYY mRNA levels. We conclude that endogenous gastric acid secretion up-regulates PYY release and PYY mRNA expression. Circulating gastrin acts to down-regulate PYY release and PYY mRNA expression. This study provides evidence that foregut functions (i.e., gastric acid secretion and gastrin release) exert control over an antiacid signal (e.g. PYY release) emanating from the hindgut. PMID- 8625913 TI - Carbohydrate and protein determinants are involved in thyroglobulin recognition by FRTL 5 cells. AB - To avoid premature lysosomal degradation, thyrocytes have a system able to recycle internalized immature thyroglobulin molecules (Tg) to the follicular lumen via the Golgi apparatus. It has been shown that this quality control system depends on recognition of exposed N-acetylglucosamine (GlcNAc) determinants (Miquelis et al., J Cell Biol, 1993, 123, 1695) present on immature Tg (Bastiani et al., 1995, Endocrinology, 1995, 136, 4204). However, the same in vitro kinetics studies also showed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinants might also be involved in binding. For this purpose, we studied binding of Tg to FRTL 5 cells, a widely available TSH-dependent cell line and found that binding to plasma membranes occurred at acidic pH in the presence of calcium, i.e. under conditions previously reported for binding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, binding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Simplificiola II affinity column analysis indicated that GlcNAc-bearing Tg were preferentially bound and N glycanase treatment of Tg inhibited interaction. Ovomucoid, GlcNAc-BSA, and porcine Tg oligosaccharides did not inhibit binding, indicating that carbohydrates were not the sole determinants for binding. The fact that pronase digestion of Tg totally abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation that porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with the binding of both human and porcine Tg. Based on these findings we conclude that, besides the involvement of GlcNAc-bearing oligosaccharides, Tg receptors form a stable bond with peptide determinants. PMID- 8625915 TI - Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. AB - Accumulation of DNA is essential for muscle growth, yet mechanisms of androgen induced DNA accretion in skeletal muscle are unclear. The purpose of this study was to determine whether androgen receptors (AR) are present in cultured skeletal muscle satellite cells and myotubes and examine the effects of testosterone on satellite cell proliferation and differentiation. Immunoblot analysis using polyclonal AR antibodies (PG-21) revealed an immunoreactive AR protein of approximately 107 kDa in porcine satellite cells and myotubes. Immunocytochemical AR staining was confined to the nuclei of satellite cells, myotubes, and muscle derived fibroblasts. Administration of 10(-7) M testosterone to satellite cells, myotubes, and muscle-derived fibroblasts increased immunoreactive AR. In satellite cells and myotubes, AR increased incrementally after 6, 12, and 24 h of exposure to testosterone. Testosterone (10(-10) - 10(-6) M), alone or in combination with insulin-like growth factor I, basic fibroblast growth factor, or platelet-derived growth factor-BB, had no effect (P > 0.01) on porcine satellite cell proliferation, and testosterone pretreatment for 24 h did not alter the subsequent responsiveness of cells to these growth factors. Satellite cell differentiation was depressed (20-30%) on days 2-4 of treatment with 10(-7) M testosterone. This effect was not reversible within 48 h after treatment withdrawal and replacement with control medium. These data indicate that satellite cells are direct targets for androgen action, and testosterone administration increases immunoreactive AR protein and reduces differentiation of porcine satellite cells in vitro. PMID- 8625914 TI - Transcriptional regulation of the insulin-like growth factor-I receptor gene: evidence for protein kinase C-dependent and -independent pathways. AB - An important mechanism whereby growth factors stimulate vascular smooth muscle cell proliferation is by increasing insulin-like growth factor (IGF)-I receptor binding. To characterize the mechanisms involved, we studied transcription of the IGF-I receptor gene in rat aortic smooth muscle cells. Angiotensin II (100 nM) and basic fibroblast growth factor (5 ng/ml) caused a marked increase in IGF-I receptor messenger RNA (mRNA) levels, peaking at 3 h (215 +/- 16.8% and 85 +/- 7.4% above control, respectively). Nuclear run-on assays indicated that angiotensin II and fibroblast growth factor stimulated IGF-I receptor gene transcription by 2.1- and 2.5-fold, respectively. Down-regulation of protein kinase C, a serine/threonine kinase that is important in growth factor-activated signal transduction, completely inhibited fibroblast growth factor- but not angiotensin II-mediated up-regulation of IGF-I receptor mRNA. The protein kinase C inhibitors chelerythrine (3 microns), calphostin C (100 nM), and staurosporine (10 nM) also blocked fibroblast growth factor but not angiotensin II induction of IGF-I receptor mRNA. Thus, angiotensin II and fibroblast growth factor transcriptionally regulate the IGF-I receptor gene by protein kinase C independent and -dependent pathways, respectively. In view of our prior data indicating that IGF-I receptor density is a critical determinant of vascular smooth muscle cell growth, our findings have particular relevance to understanding mechanisms whereby growth factors regulate vascular proliferation in vivo. PMID- 8625916 TI - Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone. AB - During the secretory phase of the human menstrual cycle, the endometrium is minimally responsive to the estrogens secreted from the ovaries. Conjugation of beta-estradiol (E2) with sulfate is thought to be an important mechanism in the regulation of the levels of active E2 in endometrial tissue. Estrogen sulfation is reportedly increased during the secretory phase in response to the high levels of progesterone secreted by the ovaries. Estrogen sulfotransferase (hEST), a distinct form of human cytosolic sulfotransferase (ST) with an affinity for E2 and estrone at low nanomolar concentrations, has recently been cloned and expressed in mammalian cells and in bacteria (J Steroid Biochem Mol Biol 52:529, 1995). At least two other forms of human cytosolic ST, dehydroepiandrosterone ST (hDHEA-ST) and the phenol-sulfating form of phenol-ST (hP-PST), also conjugate estrogens but at micromolar concentrations. This report describes the specific induction of hEST in human Ishikawa endometrial adenocarcinoma cells by progesterone as a model for the increases in estrogen sulfation observed in women during the secretory phase of the menstrual cycle. Treatment of Ishikawa cells with 10 microns progesterone for 48 h resulted in a 7-fold increase in the sulfation of 20 nM E2. The sulfation of selective substrates for human dehydroepiandrosterone sulfotransferase (hDHEA-ST) and the two forms of phenol sulfotransferase (hP-PST, hM-PST) were not affected by treatment with progesterone. The levels of immunoreactive hEST and hEST mRNA in the Ishikawa cells were both increased by progesterone, whereas the levels of immunoreactive hDHEA-ST, hP-PST, and hM-PST were not altered. hEST activity was not induced by treatment of Ishikawa cells with varying concentrations of E2, testosterone, or cortisol. The induction of hEST by progesterone was inhibited by RU-486, indicating that progesterone is acting via the progesterone receptor. These results indicate that progesterone is capable of specifically inducing hEST and estrogen sulfation in human Ishikawa adenocarcinoma cells and suggest a mechanism for increasing estrogen sulfation in the endometrium during the secretory phase of the menstrual cycle. PMID- 8625917 TI - Isolation and characterization of human pituitary chorionic gonadotropin. AB - Human CG (hCG) is the hormone associated with the maintenance of pregnancy. Although three related glycoprotein hormones, LH, hFSH, and hTSH, are secreted by the pituitary, HCG is the only one of this family of glycoprotein hormones that is produced by the placenta in primates to maintain the steroid hormone secretions of the corpus luteum. Although hCG is not considered to be a pituitary hormone, hCG-like immunoreactive materials have been reported in pituitary tissue, blood, and urine from healthy nonpregnant individuals for 2 decades, but it was never isolated. We now report the purification and characterization of pituitary hCG from acetone-preserved human pituitary glands. After gell permeation chromatography, the fractions containing hCG molecules were pooled and purified by immunoaffinity chromatography using antibodies to the COOH-terminal region of hCGbeta. Amino acid analyses, amino-terminal sequence analyses, as well as mass spectrometric studies gave similar results for both pituitary hCG and hCG purified from the urine of pregnant women. Analyses for sulfate and sialic acid contents demonstrated that pituitary hCG contained both sulfate and sialic acid. In vitro biological activity of pituitary hCG indicated that it was 50% as active as hCG purified from the urine of pregnant women in cAMP assays. PMID- 8625918 TI - Functional melatonin receptors in human prostate epithelial cells. AB - Melatonin, secreted nocturnally by the pineal gland, affects gonadal growth and pubertal development in rodents and, presumably, in humans. Recently, we have found, using 125I-labeled melatonin as a probe, specific melatonin binding sites in the human benign prostate tissue; these sites were primarily associated with the microsomal fraction of the epithelial cells. In the present study, we have explored 125I-melatonin binding sites in human benign prostate epithelial cells in culture and investigated the effects of melatonin on growth and viability of these cells. 125I-melatonin bound to the prostate cells with high (K(d) = 68 pM) affinity. Competition experiments revealed that specific binding was inhibited by subnanomolar concentrations of melatonin and 2-iodomelatonin, whereas serotonin and 5-methoxytryptamine reduced the binding only partially. Melatonin (10 pM-10 nM) inhibited the incorporation of 3H-thymidine and 3H-uridine into the prostate epithelial cells in a dose-dependent manner. Inhibition was transient, and the incorporation recovered to control levels within less than 24 h. Protein synthesis as measured by the incorporation of 35S-methionine into cell proteins decayed to minimal levels about 2 h after addition of melatonin, and its recovery was slower compared with that of 3H-thymidine or 3H-uridine incorporation. Melatonin treatment (1 nM) for 2-7 days inhibited cell growth and markedly increased the percentage of non-viable cells in culture, measured by the trypan blue exclusion assay. The results demonstrate high affinity melatonin receptors in the human benign prostate epithelial cells, which may affect cell growth and viability. PMID- 8625919 TI - Neuroprotective effect of insulin-like growth factor I in immortalized hypothalamic cells. AB - The neuroprotective action of insulin-like growth factor I (IGF-I) was tested in immortalized hypothalamic GT1-7 cells exposed to reduced glutathione depleting agents, which cause oxidative stress and cell death. The extent of cell survival was assessed by either using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide cytotoxicity assay or counting at the fluorescence microscope GT1-7 cells prelabeled with fluorescent dyes selective for viable and dead cells. Treatments with buthionine sulfoximine (500 microns), diethylmaleate (1 mM), and ethacrynic acid (200 microns) caused diffuse GT1-7 cell death (40 60%). Exposure of the same cells to IGF-I (either before or concomitant to the toxic agent, depending on the drug used) significantly prevented neuronal death. This effect was rapid, concentration-dependent, maximal at concentrations of 25 50 ng/ml, and mimicked by IGF-II, fibroblast growth factor, and the potent antioxidant idebenone. In contrast, IGF-I, as well as idebenone, were completely ineffective in antagonizing the toxic effect produced by different concentrations of menadione. In conclusion, the present data demonstrate a protective role for IGF-I against glutathione depleting agents-induced damage in GT1-7 cells suggesting an antioxidant action of this growth factor in hypothalamic neurons. PMID- 8625920 TI - Changes in the gene expression of a protein with the cdc10/SWI6 motif, V-1, during rat follicular development and corpus luteum formation. AB - We examined the gene expression of V-1, a novel soluble protein with the cdc10/SWI6 motif, in pseudopregnant rat ovaries. Northern blot analysis on days 1, 5, and 11 of pseudopregnancy revealed an approximately 2-fold increase in the V-1 messenger RNA (mRNA) expression level on day 5 to that on day 1, and no significant change was observed between those on day 5 and day 11. An injection of human CG on days 5 further increased th V-1 mRNA level to about 1.6-fold of that of the untreated control. Western blot analysis showed higher V-1 protein expression on days 5 and 11 of pseudopregnancy than that on day 1. In situ hybridization and immunohistochemistry with ovaries on day 3 of pseudopregnancy showed that luteal cells of corpora lutea and also cells of the coexisting follicles including oocytes express V-1 mRNA and the protein, with apparent rank order of the expression: oocytes > luteal cells > follicular cells >> atretic follicular cells including oocytes. These data indicate the dynamic change in the V-1 gene expression in the ovarian steroidogenic cells and oocytes and suggest potential roles of the V-1 protein in ovarian functions including corpus luteum formation and folliculogenesis. PMID- 8625921 TI - Calcium currents in a pituitary cell line (AtT-20): differential roles in stimulus-secretion coupling. AB - The purpose of the present investigation was to identify voltage-dependent calcium channel subtypes that control the release of ACTH in AtT-20 cells, a clonal mouse pituitary cell line. Using the perforated patch-clamp technique, we identified dihydropyridine (nimodipine)-, omega-Agatoxin IVA-, and omega Conotoxin MVIIC-sensitive calcium currents. No omega-Conotoxin GVIA-sensitive currents are present in these cells. There also existed a considerable resistant component to the recorded inward current that was inhibited by cadmium, a nonselective calcium channel antagonist. Using RIA, we examined the contributions of each of the pharmacologically distinct calcium channel populations to CRH- or potassium chloride (KCI)-stimulated release of ACTH at various time intervals (10 sec to 60 min). We found that nimodipine markedly inhibited ACTH release at all intervals tested, whereas omega-Agatoxin IVA, omega-Conotoxin MVIIC, and omega Conotoxin GVIA had no significant effect. Moreover, the inhibition by nimodipine was comparable to that seen after cadmium application, and the effects of these two antagonists were not additive. These data suggest that although AtT-20 cells possess dihydropyridine-, omega-Agatoxin IVA-, and omega-Conotoxin MVIIC sensitive calcium channels as well as a considerable toxin-resistant current, only the dihydropyridine-sensitive calcium channels appear to be coupled to CRH- or KCI-induced ACTH release. PMID- 8625922 TI - Enhancement of thyroid hormone receptor isoform specificity by insertion of a distant half-site into a thyroid hormone response element. AB - The two isoforms of thyroid hormone receptor (TR), alpha and beta, are highly homologous, except in the amino-terminal domain. Specific physiological roles for the receptor isoforms have not yet been defined. In transient transfection assays, TRalpha is twice as potent a thyroid hormone (T3)-dependent transactivator as TRbeta on a number of thyroid hormone response elements (TREs). Using chimeras of TRalpha and -beta, we have determined that the higher transactivation by TRalpha requires the entire ligand-binding domain. The amino terminal and DNA-binding domains of the two isoforms are interchangeable. These studies were facilitated by the use of a synthetic TRE composed of a direct repeat separated by 4 bp which also included a third half-site 19 bp 3' to this on the opposite strand. In the presence of T3, this TRE confers a 5-fold higher response to TRalpha than to TRbeta, but there is no difference in expression without T3. Functional studies indicate that all three half-sites are needed for the increased responsiveness to TRalpha, but gel shift analyses show no striking differences in the ratios of TRalpha to TRbeta binding compared to other wild type TREs. These results suggest that important functional differences are present in the ligand-binding domain of TRalpha and -beta despite their high homology. PMID- 8625923 TI - Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. AB - Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin 1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions. PMID- 8625925 TI - The N-methyl-D-aspartate-mediated inhibitory control of gonadotropin-releasing hormone release in the hypothalamus of the adult male guinea pig is expressed through opioidergic systems. AB - Previously, we reported on a dual role of N-methyl-D-aspartate (NMDA) receptor mediated neurotransmission in the control of GnRH secretion from the medial basal hypothalamus (MBH) of the adult male guinea pig, with a predominantly inhibitory action in the intact animal, which is reversed to a facilitatory role by orchidectomy. In the present study we examined the hypothesis that endogenous opioids are involved in the NMDA receptor-mediated inhibition of GnRH release. A static incubation system was used to test the effects of excitatory amino acid agonists and an excitatory amino acid antagonist, alone or in the presence of either the opiate agonist morphine or the mu-receptor antagonist naloxone, on in vitro GnRH release from the isolated MBH of intact, orchidectomized, or sham operated guinea pigs. GnRH output from the MBH of intact guinea pigs was markedly suppressed in the presence of the NMDA-specific receptor agonist, N-methyl-D,L aspartic acid (NMA; 50 mM), whereas NMDA-specific receptor blockade with D,L amino-5-phosphonovaleric acid (AP-5; 1 mM) resulted in a pronounced facilitation of GnRH release, as did exposure to the non-NMDA-specific receptor agonist, kainic acid (50 mM). Opioidergic blockade with naloxone (1 mM) caused a reversal of the responses to NMA and AP-5, with exposure to these compounds this time resulting in clear facilitation and inhibition, respectively. The stimulatory action of kainic acid, on the other hand, remained unaffected by the presence of naloxone. Morphine inhibited basal GnRH output and also annulled the stimulatory effect of AP-5 on GnRH secretion. The results obtained from MBHs of sham-operated guinea pigs were identical to those seen for the intact animals, with naloxone effectively increasing baseline GnRH release and reversing the inhibitory effect of NMA and stimulatory action of AP-5 on GnRH secretion to a facilitation and inhibition, respectively. On the other hand, NMA caused a marked stimulation, whereas AP-5 produced a significant inhibition of GnRH release from the MBHs of orchidectomized guinea pigs; neither of these effects was altered by the presence of naloxone, which, moreover, had only a marginal effect on basal GnRH output in this group of animals. In conclusion, our present data provide evidence to support the view that the primary inhibitory action of NMDA receptor-mediated neurotransmission on GnRH release in the MBH of the intact male guinea pig is the result of activation of opioidergic systems and that a marked reduction of opioid tone after orchidectomy brings a facilitatory NMDA receptor-mediated system to the fore. On the other hand, non-NMDA-specific kainate receptor-mediated facilitation of GnRH, previously shown to be unaffected by gonadal status, appears to be also independent from opioidergic modulation. PMID- 8625924 TI - Molecular cloning of cytochrome P450 aromatase complementary deoxyribonucleic acid from periimplantation porcine and equine blastocysts identifies multiple novel 5'-untranslated exons expressed in embryos, endometrium, and placenta. AB - To facilitate studies on the molecular mechanisms involved in the unique temporal expression of the P450 aromatase gene in porcine periimplantation embryos, a complementary DNA (cDNA) library was prepared from day 12 porcine embryo messenger RNAs (mRNAs) and screened with a cDNA fragment encoding the amino terminus of human aromatase. Two size variants of nearly full-length cDNA clones (designated clones 33F and 34B; 2470 and 2588 bp, respectively) were isolated and sequenced. Clones 33F and 34B encoded identical aromatase proteins of 503 amino acids, but differed in size due to alternative polyadenylation signal usage for the corresponding mRNAs. Using the 5'-rapid amplification of cDNA ends procedure, two classes of cDNA clones that contained distinct putative exon 1 sequences (E1A and E1B, respectively), but were otherwise identical in exon 2 and 3 sequences, were isolated. The E1A DNA sequence was identical to the 5'-end specified by clones 33F and 34B, except for an additional 23 nucleotides. The E1A and E1B exons showed no significant homology with each other or with the aromatase cDNA sequences from other species, including human. Expression of E1A-containing aromatase mRNA was higher than that of E1B in day 12 blastocysts, although both mRNAs were expressed at low levels in porcine endometrium and placenta of early pregnancy and in ovary at periestrus. To determine whether the E1A or E1B sequence was common to aromatase mRNAs in other steroidogenic preimplantation embryos, aromatase cDNA clones spanning exons 1-3 were isolated from equine embryos by a combination of RT-PCR and 5'-rapid amplification of cDNA ends. Equine embryonic exon 1 cDNA sequence had no homology with corresponding regions in aromatase transcripts of pigs or other species; in contrast, exon 2 and 3 cDNA sequences predicted an amino acid sequence with significant homology to aromatase. Results from the present study demonstrate alternative splicing of a novel first exon(s) in P450 aromatase gene transcripts in porcine preimplantation embryos and in endometrium and placenta of pregnancy. The lack of homology among equine, porcine, human, and bovine aromatase 5'-untranslated exons may indicate divergence in the corresponding regulatory motifs of these regions among mammalian species. These observations suggest the potential significance of activation of the E1A promoter and/or splicing of novel aromatase 5'-exons in the transient production of estrogens by periimplantation blastocysts. PMID- 8625926 TI - Immunocytochemical localization and regulation of connexin43 in the adult rat epididymis. AB - Connexin43 (Cx43) is one of the most predominant gap junction proteins found in vivo, and although present in the testis, it has not been examined in the epididymis. Immunocytochemistry using an anti-Cx43 antibody revealed a punctate immunoperoxidase reaction at the apical margins between adjacent epithelial cells of the efferent ducts. In the epididymis, punctate reactive sites were observed at the base of the epithelium between basal and principal cells. There was no staining between adjacent principal cells at their apical or lateral margins. Cx43 immunostaining was also seen between myoid cells surrounding the tubules but only in the cauda epididymidis. Using a Cx43 cDNA probe, Northern blot analysis of total cellular RNA revealed a single hybridizing band of approximately 3.0 kb in all regions of the epididymis. Throughout the epididymis of bilaterally orchidectomized rats at 7 or 14 days, an immunoperoxidase reaction for Cx43 persisted at the base of the epithelium between principal and basal cells. However, in the initial segment only, immunolocalization of Cx43 was also observed apically between adjacent principal cells. This apical staining was lost in rats that received testosterone replacement. Myoid cells in the cauda epididymidis of control rats expressed Cx43, however, orchidectomized rats did not express Cx43 in this cell layer. Western blots revealed the presence of a major protein band at 43 kDa corresponding to unphosphorylated Cx43 as well as Cx43 species at 44 and 46 kDa, which were more prominent in orchidectomized rats. Together these data represent one of the first examples of Cx43 gap junctions between heterologous cell types (i.e. principal and basal cells). Moreover, Cx43 expression in myoid cells of the cauda epididymidis is androgen-dependent and in the initial segment of the epididymis only, the intracellular targeting of Cx43 towards the principal-principal cell interface under normal conditions is regulated by androgens. PMID- 8625927 TI - Translocation of glucokinase in pancreatic beta-cells during acute and chronic hyperglycemia. AB - Glucokinase (GK) plays a key role in the regulation of glucose-induced insulin secretion, and questions have been raised about its relationship to the glucose transporter GLUT2 and its function in diabetes. This study examined the location of immunostained GK and GLUT2 in beta-cells using confocal microscopy. On double stained sections from pancreases of normal fed rats, GLUT2 Texas Red staining was restricted to the plasma membrane, and GK fluorescein isothiocyanate staining was found in a limited area of cytoplasm that was perinuclear with slight extension toward the apical pole. The GK staining occupied 8.6 +/- 1.7% of total cytoplasmic area and was almost never adjacent to the GLUT2 staining of the plasma membrane. To determine whether the GK staining pattern is altered by metabolic perturbation, normal rats were made acutely hyperglycemic with iv glucose injections; after 20 min the GK staining changed from being localized to become diffusely distributed throughout the cytoplasm. To examine the influence of chronic hyperglycemia, rats were subjected to 90% partial pancreatectomy (Px), which produced glucose levels of 10.9-20.8 mM. When studied 6 or 14 days after Px, those rats with glucose levels greater than 17.7 mM had an altered GK staining pattern that was variable; in some beta-cells GK staining was diffuse and in others the localized staining pattern was preserved. GLUT2 staining was reduced overall, but variability between cells was observed, unlike the more uniform reductions seen with hyperglycemia of longer duration. Other rats received islet transplants to prevent hyperglycemia after Px; their GK and GLUT2 staining patterns were normal. These findings indicate that GK is translocated in association with acute and chronic hyperglycemia. The translocation of this key enzyme for glucose recognition by beta-cells may lead to altered rates of insulin secretion during acute perturbations of fuel provision and in the diabetic state. PMID- 8625928 TI - Differential expression of the full-length and truncated forms of the epidermal growth factor receptor in the preimplantation mouse uterus and blastocyst. AB - The present investigation examined the differential expression of the full-length (fl) and the truncated (tr) forms of the epidermal growth factor receptor (EGFr) in the preimplantation mouse uterus and blastocyst. Northern blot hybridization using a complementary RNA probe specific to the full-length form (EGFr-fl) detected a 6.5-kb transcript, whereas that of the truncated form (EGFr-tr) detected a 2.7-kb transcript in the preimplantation mouse uterus on days 1 and 4 of pregnancy (day 1 = vaginal plug). In situ hybridization using these probes detected the EGFr-fl transcripts only in the stroma and myometrium, but not in the epithelium, whereas EGFr-tr transcripts were detected in all major uterine cell-types. To confirm the results of in situ hybridization, RT-PCR was performed on RNA isolated from separated uterine cell-types on day 4 of pregnancy using sequence specific primers for the two forms of the receptor. The results concur that the EGFr-tr transcript is expressed in the epithelium, stroma and myometrium, whereas that of the EGFr-fl transcript is not expressed in the epithelium. In the preimplantation blastocyst, RT-PCR detected the EGFr-fl messenger RNA, but not the EGFr-tr messenger RNA. These results suggest that the blastocyst, not the uterine epithelium, is the target for EGF family of growth factors in embryo-uterine interaction during implantation. PMID- 8625929 TI - Localization of binding sites in the central nervous system for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db) C57BL/6J mice. AB - Leptin (OB protein) fused to the FLAG epitope and a kinase recognition site was expressed in bacteria, immunopurified, and phosphorylated using [gamma-(33)P] ATP. The resulting probe was used to characterize the distribution of leptin binding sites within brain sections of normal, ob/ob, and db/db C57BL/6J male mice. Leptin binding sites were found in leptomeninges and choroid plexus. Leptin binding within the choroid plexus is slightly elevated in ob/ob mice when compared to normal males (p<0.05). Binding of leptin by the choroid plexus of db/db male mice is lower than in normal males (p<0.05), but normally distributed. Based on the association and dissociation rates of leptin binding on tissue sections, we estimate the K(D) of the choroid plexus site at 0.25X10(-9) M. From our results, we hypothesize that the binding of leptin to its site may cause the release or transport of uncharacterized factor(s) into the cerebral spinal fluid (CSF) to affect neuronal populations controlling feeding and metabolism. PMID- 8625931 TI - Experiments on proliferation of normal human breast tissue in nude mice do not show that progesterone does not stimulate breast cells. PMID- 8625930 TI - Radiommunological measurement of leptin in plasma of obese and diabetic human subjects. AB - Studies in mice have identified the ob gene product, leptin, as a signaling factor regulating body weight homeostasis and energy balance. Defective production of the encoded protein may be one of the causes for the development of obesity. Using a high affinity antibody, that in immunohistochemical studies specifically stained human adipocytes, a radioimmunoassay was established and leptin immunoreactivity was quantified in plasma of lean and obese human subjects. Chromatographic analysis suggested that the immunoreactive material in plasma is identical to that found in extracts from human fat and represent a protein with a molecular size of approximately 16 kD. Fasting levels were measured in plasma of 75 lean and obese human subjects (body mass index (BMI) 17.7 - 87.3). The mean concentration of leptin in plasma of lean subjects (BMI < or = 28) was 69.3 +/- 36.9 fmol/ml plasma (mean +/- SD, n=27). The highest concentration measured in obese was 533.3 fmol/ml plasma. The levels showed a strong positive correlation with BMI (r=0.77, p<0.001). A subgroup of diabetic patients did not significantly differ in their leptin plasma levels from non diabetic subjects with similar BMI. PMID- 8625932 TI - Urinary catecholamine levels in children with and without a history of dentofacial injuries. AB - This study was conducted to investigate the existence of any differences in the urinary catecholamine levels in children with and without a history of dentofacial injuries. Three hundred fourteen children, boys and girls, aged 6 to 8 years were included in this study. A questionnaire was distributed to the parents to collect information about the child's history of dentofacial injuries. A 24-h urine sample was collected for each subject and a representative sample (25 ml) was analyzed by the HPLC technique to assay the catecholamine content. Of the examined children 68 (43 boys and 25 girls) had a history of dentofacial injury and constituted the experimental group. A control group of 68 children (43 boys and 25 girls) without a history of dentofacial injury was matched by age and sex. The Student t-test was used to identify any differences in urinary catecholamine levels between the two groups. The 95% probability was used. The results showed statistically significant differences in the mean values of epinephrine, while the differences in norepinephrine and dopamine were not significant. This study suggested that children with emotionally stressful states run a greater risk of having dentofacial injuries. PMID- 8625933 TI - Clinical investigation of traumatic injuries of permanent incisors in Izmir, Turkey. AB - 470 injured teeth of 370 patients who consulted the Dental Clinic of Ege University, Izmir, Turkey for examination or treatment between 1981-1993 were evaluated. Information concerning sex, age of patients at the time of injury, cause of trauma, number of injured teeth, type of tooth and type of trauma were recorded. More boys suffered traumatic injuries (64.8%) than girls (35.2%). Patients aged 11-15 years old exhibited the highest number of injuries (34.4%) followed by the 6-10 years old group (24.5%). Most injuries involved one tooth (60%) and maxillary central incisors were the most often affected teeth (66.2%). The leading cause of injury was undefined falls (45.1%). At the initial examination, cases seen after a long posttraumatic period showed more complications than those presented within a short time period. Educational programs about the importance of dental trauma, the benefits of immediate attendance and conservation of avulsed and fractured teeth would be very helpful for patients. Additionally improving the knowledge of the dental practitioner about trauma would be another important point in solving the problem. PMID- 8625934 TI - Adhesion of a glass ionomer cement to human radicular dentine. AB - The adhesion of cements to root canal surfaces is a crucial factor for strengthening non-vital teeth--weakened due to extensive loss of tooth structure- by cemented posts. The aim of this study was to determine the tensile strength of a glass ionomer cement (Ketac-Cem) on root canal walls following pretreatment with conditioners. Upon cleaning and shaping, 56 straight root canals--divided into seven groups--were conditioned with one of the following solutions: NaOCl (1%) + EDTA (20%), H3PO4 (37%), HNO3 (2.5%), citric acid (6%), polyacrylic acid (10% and 20%) and NaCl (0.9%) as control. Standardized dentine cylinders were prepared out of the coronal half of each root perpendicular to the root axis and subsequently split. The exposed root canal areas were coated with Ketac-Cem. Using an universal testing machine a tensile force was applied to Ketac-Cem up to fracture. Pretreatment with EDTA-NaOCl provided the strongest bond strength (2.2 MPa). The median values for the other conditioning solutions ranged from 1.2 to 1.9 MPa. The significantly weakest bond (0.5 MPa) was recorded for NaCl. The elimination of the smear layer appeared to be an essential factor in order to improve the adhesion. PMID- 8625935 TI - Nd:YAG laser irradiation in root canal disinfection. AB - The purpose of this study was to assess the efficacy of Nd:YAG laser irradiation in disinfecting the root canal system. Seventy-five teeth were uniformly instrumented and sterilized, and their root canals were infected for 60 min with an overnight culture of Enterococcus faecalis in Tryptic Soy Broth. The teeth were divided into 6 groups: 1--non-infected controls; 2--infected controls; 3- infected, laser treated; 4--infected, laser dummy (dye only); 5--infected, laser dummy (dye and air-water spray); 6--infected, NaOCl-treated (reference group). After treatment, the root canals were dried and dentinal shavings removed for a post-treatment culture. Quantitative analysis of bacteria surviving the various treatments were performed and the bacterial counts for each group were compared. SEM analysis of teeth split longitudinally was used to illustrate the effect of treatment on the smear layer and on surface bacteria. Nd:YAG laser irradiation (group 3) significantly reduced the number of bacteria while NaOCl irrigation (group 6) effectively disinfected the canals. PMID- 8625936 TI - Evaluation of sealing ability, pH and flow rate of three calcium hydroxide-based sealers. AB - Three endodontic sealers containing calcium hydroxide (Sealapex, Sealer 26 and Apexit) and a zinc oxide-eugenol cement (Grossman's sealer) were examined for sealing ability, release of hydroxyl ions and flow rate. Dye penetration test was used to assess the sealing ability of the endodontic sealers. The pH test was accomplished to evaluate the release of hydroxyl ions from sealers and the pH readings were done at intervals of 30 min, 60 min and 7 days. In the flow test the sealers were placed between two glass slabs and a weight of 500 g was placed on the top of the glass. The diameters of the formed discs were recorded. The results revealed no statistically significant difference between apical seal provided by the four sealers. All of the calcium hydroxide based sealers alkalinized the surrounding medium. Sealer 26 cement presented significantly superior flow rate when compared with the other cements. These findings indicated that endodontic sealers containing calcium hydroxide presented satisfactory physicochemical properties when compared with a zinc oxide-eugenol sealer commonly used in endodontics. PMID- 8625937 TI - Endodontic pathogens: propagation of infection through patent dentinal tubules in traumatized monkey teeth. AB - Periapical pathology indicating endodontic infection, when present in marginal periodontitis-affected teeth, has recently been shown to be an aggravating factor in progression of marginal destruction. This has been associated with patency of dentinal tubules in the tooth cervix, an area normally devoid of cementum following periodontal therapy. These studies are, however hampered by that only circumstantial evidence such as presence of periapical destruction have been applied as criteria of endodontic infection. The purpose of the present investigation was to study to what extent a predefined selection of endodontic pathogens inoculated in the root canal can influence periodontal pathology and healing in areas of the root covered by or devoid of cementum, using root resorption as a histomorphometric marker. Exposed dentine surfaces, in the present study showed significantly larger areas of resorption in infected roots compared to non-infected roots, while cementum surfaces showed an almost identical distribution of tissue reactions regardless of root canal infection or not. It was concluded that endodontic pathogens or their products were not able to penetrate the cementum barrier. The significantly larger areas of resorption on exposed dentine surfaces in infected roots compared to non-infected roots indicated that endodontic pathogens or their products could spread through dentinal tubules to a root surface void of cementum. Extrapolated to the marginal situation this indicated that endodontic pathogens in the root canal might be able to aggravate marginal infection in areas of root devoid of cementum. PMID- 8625938 TI - A retrospective clinical and radiologic study of teeth re-implanted following traumatic avulsion. AB - Re-implantation is the recommended therapeutic procedure following traumatic exarticulation of teeth though its long-term prognosis remains controversial. The purpose of the following study was to evaluate the periodontal healing of 33 reimplanted incisors lost after trauma. The sample, which included 24 upper and 9 lower incisors, was divided in two groups: 15 teeth were reimplanted within 1 hour (=Group A) and 21 teeth after 3 hours or more (=Group B). The reimplanted teeth were followed for different radiographic evaluation periods up to 5 years (mean=2-9 years). The results showed a high rate of periodontal healing in Group A (66.7%), while Group B demonstrated a high percentage (83.3%) of both inflammatory and replacement resorption. Thus, even if the whole sample was prevented from drying before reimplantation, the teeth were affected by different rates of root resorption. Among the various prognostic factors suggested by the literature, bacterial contamination during extra-alveolar storage seemed the most critical. PMID- 8625939 TI - Dentists' views on cervical hypersensitivity and their knowledge of its treatment. AB - A random sample of 400 Dutch general practitioners was asked to complete a questionnaire dealing with the prevalence, conditions and therapies of cervical hypersensitivity of their patients. According to the 259 responding dentists an average of 10% of their patients suffered from moderate cervical pain. The estimated mode for severe pain was 1%. More than two thirds of the dentists reported inadequate brushing of the teeth to cause the hypersensitivity, about one half acknowledged periodontal causes, well over one quarter mentioned the involvement of dietary acids, and about one sixth implicated psychosomatic factors. Other causal factors were mentioned less often. Of the therapies available, the home-care methods appeared to be promoted most often, i.e. brushing with therapeutic toothpastes (77%), improvement of oral hygiene (51%) and local self-application (with finger) of a therapeutic toothpaste or fluoride preparations before the night (41%). Well over 50% reported to apply occasionally bondings/varnishes and 28% said to make sometimes cervical fillings. Other therapeutic possibilities were not, or very seldom, used. PMID- 8625941 TI - A new concept of education and knowledge retrieval through network systems. AB - Recent epidemiological investigations reveal increasing figures of prevalence of dental traumatology; however, basic and clinical research is rather scarce in the scientific literature. The development of computer technologies and the advent of communication pathways through network systems offer increased opportunities to disseminate theoretical and practical data on dento-facial traumatisms. A new server launched on Internet, the global network system, is described and proposed to allow better recognition and information sharing through both practitioners and institutions in the field of dental traumatology. PMID- 8625940 TI - In vitro evaluation of the cytotoxicity of calcium hydroxide-based root canal sealers. AB - The cytotoxicity of three calcium hydroxide-containing root canal sealers (Sealapex, CRCS and Apexit) was tested by using L929 and BHK 21/C13 cells. After setting for 24 h, the sealers were covered with cell suspension. Cytotoxicity was determined by a quantitative technique at 24 h, 48 h and 72 h. All the sealers were found to be cytotoxic. Sealapex showed the highest cytotoxicity, causing a significant decrease in cell density. CRCS was less toxic than Sealapex and more toxic than Apexit. Apexit proved to be the least toxic material. PMID- 8625942 TI - In vivo genotoxicity of dichloroacetic acid: evaluation with the mouse peripheral blood micronucleus assay and the single cell gel assay. AB - Chlorination is a widely used method for disinfection of drinking water supplies. Reaction of chlorine with naturally present organic compounds can result in toxic by-products. One major disinfection by-product from the chlorination of drinking water is dichloroacetic acid (DCA). This chemical has been shown to be carcinogenic in rodents, yet little genotoxicity data are available to assess the possible role of DNA and/or chromosomal damage in this process. We have used the peripheral blood erythrocyte micronucleus (MN) assay and the alkaline single cell gel electrophoresis (SCG) technique to investigate the in vivo genotoxicity of DCA in bone marrow and blood leukocytes, respectively. The MN assay detects chromosome breakage and/or malsegregation, while the SCG assay detects DNA damage (e.g., single strand breaks, alkali-labile sites, crosslinking). Mice were exposed to this compound in drinking water, available ad libitum, for up to 31 weeks. Our results show a small but statistically significant dose-related increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) after subchronic exposure to DCA for 9 days. In addition, at the highest dose of DCA tested (3.5 g/l), a small but significant increase in the frequency of micronucleated normochromatic erythrocytes (NCE) was detected following exposure for > or = 10 weeks. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage. Based on the lack of any difference observed in the proportion of kinetochore-positive micronuclei between the treated and control animals, we interpret MN as arising from clastogenic events. The SCG technique suggested the presence of DNA crosslinking in blood leukocytes in mice exposed to 3.5 g/l DCA for 28 days. These data provide evidence that DCA may be an extremely weak inducer of chromosome damage when provided to mice in drinking water under conditions which lead to increased levels of tumors. PMID- 8625943 TI - Cell cycle traverse in AHH-1 tk +/- human lymphoblastoid cells exposed to the chromosomal mutagen, m-amsa. AB - AHH-1 tk +/- cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (GO/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of subdiploid peak, consistent with cells undergoing apoptosis. PMID- 8625944 TI - Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8 diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, and 3-nitrobenzo[a]pyrene trans-7,8- diol-anti-9,10-epoxide. AB - Both 1- and 3-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites,trans-7,8-dihydroxy anti-9,10-epoxy-7,8,9,10-epoxy -7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1 nitro-BaP-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3 nitrobenzo[a]- pyrene (3-nitro-BaPDE), together with trans-7,8-dihydroxy-anti-9, 10-ep- oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10(6) clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP DE mutants resulted from simple base pair substitution, with the predominant mutation being G-->T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3' purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5'-GG-3', 5'-GGA-3', and 5'-TGGA-3', while the proportions of 1-nitro-BaP-DE mutants in these contexts were often lower. The results indicate that nitro substitution at C1 or C3 of BaP-DE reduces mutational potency in CHO cells and appears to have only subtle effects upon the mutational pattern in the hprt gene. PMID- 8625945 TI - Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II) AB - Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eukaryotic cells to low concentrations of lead (II) or mercury (II). There have been conflicting reports concerning the mutagenic potential of these heavy metals, and there have not been any studies performed to determine the molecular mechanism(s) by which these metals are mutagenic. The Chinese hamster ovary cell line, AS52, contains a stably integrated single functional copy of the Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene confer resistance to 6-thioguanine (TG). There was little effect on viability, as measured by relative cloning efficiency, of AS52 cells exposed to lead (II) or mercury (II) up to concentrations of 0.5 microM and 0.3 microM, respectively. However, higher concentrations of the metals caused a significant increase in cell death. There was also a dose-dependent increase in the isolation of mutants resistant to TG in treated cells when compared to non-treated controls. Concentrations of the metals as low as 0.1 microM caused a significant increase in the number of mutants resistant to TG when compared to the number of spontaneous mutants obtained in nontreated controls. While the molecular mechanism(s) by which lead and mercury (II) are genotoxic is unknown, the results of this study demonstrate that low concentrations of lead (II) and mercury (II) are mutagenic in eukaryotic cells. PMID- 8625946 TI - TCR beta PCR from crude preparations for restriction digest or sequencing. AB - T cell specificity is determined by the combinatorial association of specific variable (V), diversity (D), and junctional (J) regions. Clones of T cells (clonality) can occur, in the blood or in tissue, after proliferation of activated T cells. Determining clonality in mutation assays is necessary to distinguish between mutants and mutational events. We have developed a novel approach to determine clonality among T cell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplified by use of a consensus primer, beginning from a cell pellet of 2,000-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta chain PCR product can also be directly sequenced, allowing simple and easy identification of Vbeta and CDR3 sequence from a small number of cells. The utility of this method is demonstrated by PCR, restriction digest, and sequencing of the TCR beta cDNA from eight T cell clones isolated from 2 individuals. A clone of three identical isolates (one 3-mer) and a clone of two identical isolates (one 2-mer) were determined from restriction digests using two different enzymes. This new method is an easier and more rapid way of determining clonality than traditional methods, e.g., Southern blotting. PMID- 8625947 TI - Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay. AB - Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D0 value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D0 values= 0.12-0.60 Gy. In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkali-labile sites was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stomal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as "comets" by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. PMID- 8625948 TI - Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae. AB - The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr3) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strain at higher levels than in the insecticide-sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B1 to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7,12 dimethylbenz[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp P-2), were metabolized in the transformed strain to cytotoxic products. PMID- 8625949 TI - Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2 acetoxyacetylaminofluorene. AB - Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant-activated m phenylenediamine (mPDA) or with direct-acting 2-acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non-toxic concentrations of plant-activated mPDA and paraoxon a 10-fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant-activated mPDA product required that O-acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon-dependent mutagenic synergy was observed using the direct-acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8 DNP6 regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p nitrophenol). PMID- 8625950 TI - Effects of the nitro-group on the mutagenicity and toxicity of some benzamines. AB - The Ames Salmonella/microsomal assay was employed to test the mutagenicity of some benzamines (aniline, and o- and p-phenylenediamine) and their nitro derivatives (p-nitroaniline, 2-nitro-p-phenylenediamine, 3- and 4-nitro-o phenylenediamine), using strains TA98 and TA100 and their nitroreductase deficient mutants, TA98NR and TA100NR, in the presence and absence of rat S9 mix. The addition of the nitro-group to benzamine molecules converted them into direct mutagens. Furthermore, the position of the nitro-group affected their mutagenic activities. Cytotoxicity testing with Chinese hamster ovary cells (CHO-K1) showed that the presence of the nitro-group in these compounds had no specific effect on toxicity. The test compounds all showed a dose-related increase in inducing chromosomal aberrations in CHO cells. However, the presence of the nitro-group did not affect potency in inducing chromosomal aberrations. Compounds containing the nitro-group had higher initial oxidation potentials and dipole moments (mu) than their nonnitro-containing counterparts. The mutagenicity and toxicity of these compounds were not related to physico-chemical properties, including oxidation potential, energy difference (deltaE) between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), ionization potential (I.P.), and mu. PMID- 8625952 TI - Monitoring hprt mutant frequency over time in T-lymphocytes of people accidentally exposed to high doses of ionizing radiation. AB - Modern technologies have provided the opportunity to monitor mutations in people in vivo. The subjects of this study were accidentally exposed to 137Cesium in a radiological accident that occurred in September 1987 in Goiania, Brazil, during which more than 150 people received doses greater than 0.1 Gy and as high as 7 Gy. The objective of this study was to determine how long the hprt mutant T-cells in the peripheral blood contribute to mutant frequency by examining the time course of the T-lymphocyte response to ionizing radiation. This report describes the results obtained over a period of 2.3 to 4.5 years subsequent to the accident, from 11 subjects with doses ranging from 1 to 7 Gy, and from nine control subjects selected from the same population. The mean In MF (+/- SE) of the control group was 2.5 (+/- 0.2) + In10(-6). The exposed group had a significantly increased mutant frequency; the mean In MF (+/- SE) were 3.3 (+/- 0.3) + In10(-6), 2.8 (+/- 0.2) + In10(-6), and 2.3 (+/- 0.2) + In10(-6), in the years 1990-1992 respectively. Based on the decline of mutant frequency and using Buckton's models [Buckton et al. (1967): Nature 214:470-473], we demonstrated that mutant T-cells have a short-term memory with a half-life of 2.1 years. This relatively short half-life limits the effective use of the hprt assay as the method of choice to monitor past exposure. The data also demonstrate a positive correlation with age, and an inverse correlation with plating efficiency. PMID- 8625951 TI - Influence of cytotoxicity on test results in the L5178Y TK+/- mouse lymphoma assay. PMID- 8625953 TI - Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells. AB - The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN-) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. We also applied the assay to exfoliated bladder cells of 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 micrograms As/L) and 18 matched controls (average level, 16 micrograms As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN-) and those containing whole chromosomes (MN+) both increased (1.65-fold, P = 0.07, and 1.37 fold, P = 0.15, respectively), suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN- frequency increased 2.06-fold (P = 0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN- and MN+ were positively associated with urinary arsenic and its metabolites. However, the association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can now be determined in epithelial tissues. PMID- 8625954 TI - Products of ozonized arachidonic acid potentiate the formation of DNA single strand breaks in cultured human lung cells. AB - In this study we examined the potential for environmental levels of ozone (03) to degrade arachidonic acid (AA), a polyunsaturated fatty acid abundantly present in the lung, into products that can produce DNA single strand breaks (ssb) in cultured human lung cells. Human lung fibroblasts were incubated with 60 microM AA that had been previously exposed to and degraded by 0.4 ppm 03 (1 hr.) Incubation of the cells with 03-exposed AA (but not with vehicle alone) for 1 hr at 4 degrees C and 37 degrees C produced 555 and 245 rad-equivalents of DNA ssb, respectively, as determined by the DNA alkaline elution technique. These breaks were completely eliminated when the ozonized AA solution was incubated with catalase prior to cell treatment, indicating that h202 was solely responsible for damaging DNA. Superoxide dismutase bovine serum albumin, or heat-inactivated catalase showed little, if any, inhibitory activity. The H202 content of the ozonized AA (31 +/- 4 microM) could account for only about 40% of the observed breaks. Potentiation of the H202-induced DNA ssb persisted after removal of the carbonyl substances by chromatographic procedures, suggesting that the non carbonyl component of ozonized AA was the responsible component for inducing augmentation of the observed increases in DNA ssb. Ozonized AA also induced DNA ssb in cultures of the human bronchial epithelial cell line BEAS-2B. Again, these breaks were shown to exceed levels that could be attributed to the presence of H202 alone. These results indicate that products of ozonized AA can interact to potentiate DNA ssb in human lung cells. PMID- 8625955 TI - Detection of DNA-crosslinking agents with the alkaline comet assay. AB - The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37 degrees C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed. PMID- 8625956 TI - Unexpected genetic toxicity to rodents of the N',N'-dimethyl analogues of MNU and ENU. AB - Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU [N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity. Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the NH2 group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained. PMID- 8625957 TI - Study of the forces of stabilizing complexes between chlorophylls and heterocyclic amine mutagens. AB - Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, forms molecular complexes with heterocyclic amine mutagens in vitro. In a previous study [Dashwood and Guo (1993): Environ Mol Mutagen, 22:164-171], we observed an inverse correlation between the binding constants of several mutagen-CHL complexes and the antimutagenic potency of CHL in the Salmonella assay. The present investigation utilized molecular mechanics methods of energy minimization and spectrophotometric titration to examine structural features of chlorophylls, chlorins, and porphyrins that might be important for complex formation with heterocyclic amines. The exocyclic amine group of the mutagen aligned consistently with acid groups in CHL, suggesting that H-bond or electrostatic interactions facilitate complex formation. Replacement of the exocyclic amine with a nitro group abrogated this specific orientation and raised the minimized energies of the complexes. No relationship was found between complex strength and the specific positions of amine or methyl groups on the mutagen. However, the presence of methyl groups increased the minimized energies and lowered the binding constants of the complexes, perhaps due to partial disruption of pi-pi interaction by steric effects. All of the compounds examined, including chlorophyll a, required the presence of pi-pi interactions to form stable complexes with the heterocyclic amines. In general, the present results were in agreement with the inhibitory potency of each compound in the Salmonella assay, and they provide further support for the hypothesis that chlorophylls in the diet might act as interceptor molecules of food-borne carcinogens and mutagens. PMID- 8625958 TI - The somatic white-ivory eye spot test does not detect the same spectrum of genotoxic events as the wing somatic mutation and recombination test in Drosophila melanogaster. AB - A groups of six chemical compounds was tested in parallel in two different somatic genotoxicity assays in Drosophila melanogaster, the wing somatic mutation and recombination test (SMART) and the white-ivory eye spot test. The wing spot test makes use of the wing cell markers multiple wing hairs (mwh) and flare (flr) and detects both mitotic recombination and various types of mutational events. The white-ivory eye spot test makes use of the white-ivory (wi) quadruplication and detects the somatic reversion of the recessive eye color mutation wi to the wild-type (w+). Three- or two-day-old larvae were fed chronically with the compounds ethylnitrosourea (ENU), N-nitrosopyrrolidine (NNP), caffeine (CAF), chromium (VI) oxide (CRO), potassium chromate (POC), and 2,4 dichlorophenoxyacetic acid (2,4-D). All six compounds are genotoxic to various degrees in the wing spot test. The percentage of the genotoxic activity that is due to mitotic recombination was between 84% and 91% for the hexavalent chromium compounds CRO and POC and about 68% for 2,4-D. In contrast, ENU and NNP showed only 46% and 25% recombinagenic activity, respectively. In the white-ivory eye spot test, the three compounds (CRO, POC, and 2,4-D) with high recombinagenic activity and CAF were clearly nongenotoxic, whereas only ENU and NNP gave a positive response. From these results, it is concluded that the spectrum of genotoxic events detected by the two assays is different. In particular, the white-ivory eye spot test appears not to detect mitotic recombination the way the wing spot test does. PMID- 8625959 TI - Spiral Salmonella assay: validation against the standard pour-plate assay. AB - The spiral Ames assay, an automated approach to bacterial mutagenicity testing which simplifies the test procedure and reduces the amount of drug required to generate mutagenic dose-response information, has been evaluated and validated for routine screening. The spiral plater delivers the Salmonella bacteria, exogenous metabolic activation system and drug to the surface of a rotating agar plate one on top of another in such a way that a uniform density of bacteria is exposed to a logarithmically decreasing volume of drug. Following an incubation of 48 hr at 37 degrees C, the plates are scanned by a laser counter, and the data are subjected to a computerized analysis. Petri plates of 15 cm diameter were used to provide a concentration range of about 250-fold per plate. The Salmonella were concentrated 20-fold to increase sensitivity. Thirty-eight compounds from a variety of chemical classes, including both pharmaceuticals and known mutagens of moderate to strong potency, were tested in both the spiral and the standard pour plate assays. There was overall test agreement on positive or negative results for 82% of the compounds tested. When only the results from strains TA98 plus TA100 were considered, the agreement was 87%. When positive results were obtained, the fold increase over vehicle control was on average twice as great for the spiral assay compared to the pour-plate assay. It was concluded that the two assay procedures generally provided comparable results, with the spiral assay being somewhat more sensitive in terms of dose-response than the pour-plate assay. PMID- 8625960 TI - Speculations on the rodent carcinogenicity of 30 chemicals currently under evaluation in rat and mouse bioassays organised by the U.S. National Toxicology Program. AB - As a contribution to the second round of rodent carcinogenesis prediction organised by the U.S. National Institute of Environmental Health Sciences (NIEHS), speculative predictions for 30 chemicals currently under evaluation in U.S. National Toxicology Program (NTP) rodent bioassays are presented. Core chemical data received from NIEHS were supplemented by relevant information from commercially available scientific databases to provide input for reasoned assessment. For each chemical, carcinogenesis by a genotoxic or nongenotoxic mechanism or noncarcinogenesis is predicted; species-and target organ-specific predictions are also presented, together with arbitrary indices of confidence in each such prediction. In all or nearly all cases, an element of informed speculation was a necessary part of the prediction process, but the rationale for each decision is briefly described. It is predicted that ten chemicals will prove noncarcinogenic, five will be carcinogenic to mice only, and 15 will induce tumours in both species. PMID- 8625961 TI - CD4 and CD8 expression and T cell antigen receptor gene rearrangement in early intrathymic precursor cells. AB - The earliest T precursor population in the adult mouse thymus, considered to have the surface phenotype CD4lo8-3-44+25-Thy-1lo c-kit+ (termed the low CD4 precursor), has been shown to have the capacity to produce B cells and dendritic cells, as well as T cells, and to have the T cell antigen receptor (TCR) C beta gene region in germ-line configuration. Because of evidence that this precursor population may have low levels of CD8 as well as CD4 on the cell surface, it was isolated, stained for surface CD4 and CD8 and assayed for the expression of messenger RNA (mRNA) for CD4 and CD8 by the reverse transcriptase polymerase chain reaction (RT-PCR). The low CD4 precursors gave definite, moderate levels of staining for both CD8 and CD4, in contrast to downstream precursors which showed only marginal staining and so could be considered as genuine CD4-8-3- triple negatives. The low CD4 precursor expressed a significant level of mRNA for CD4, indicating that the surface CD4 was produced by these cells. However, the low CD4 precursor did not express a detectable level of mRNA for CD8, suggesting that the surface CD8 was acquired from other cells. Since the low CD4 precursor population was found already to express mRNA for enzymes involved in TCR gene rearrangement, including in this study terminal deoxynucleotidyl transferase (TdT), a PCR procedure was used to assay early precursors for D-J rearrangements at the TCR beta gene locus. However, the low CD4 precursor had the TCR beta D-J genes in germ-line configuration, D-J gene rearrangements being first detected several stages downstream in the CD3-4-8-44-25-+ precursor population. We conclude that a transient synthesis of CD4, but not of CD8, characterize these early thymus precursors. Although they have initiated synthesis of some recombination associated enzymes, full commitment to the T lineage and TCR gene rearrangement is a later event. PMID- 8625962 TI - Characterization of mouse CD6 with novel monoclonal antibodies which enhance the allogeneic mixed leukocyte reaction. AB - Human CD6 is a cell surface protein expressed by thymocytes, mature T cells, a subset of B cells and certain cells of the brain. On human T cells, CD6 has been shown to act as a co-stimulatory molecule which modulates T cell receptor (TCR) mediated T cell activation. To study further the recently identified mouse CD6 (mCD6), we generated and characterized a set of anti-mCD6 mAb. Anti-mCD6 mAb recognizing the mCD6 scavenger receptor cysteine-rich (SRCR) extracellular domains 1 and 3 were identified. mAb against SRCR domain 3, but not domain 1, inhibited the interaction of CD6 with a recently identified ligand, activated leukocyte cell adhesion molecule (ALCAM). Immunohistochemical analysis indicated that mCD6 expression was largely localized to the T cell areas of lymphoid tissue and, as previously reported in the human, CD6 was also expressed by neurons. CD6 was highly expressed on mouse T cells isolated from the spleen, lymph node and thymus as demonstrated by two-color immunofluorescence analysis. The CD4+ and CD8+ cells in these lymphoid compartments expressed similar levels of CD6. Immunoprecipitation studies showed that mouse thymocytes predominantly express a CD6 isoform of approximately 130 kDa, while splenocytes predominantly express a CD6 isoform of approximately 100 kDa. Anti-mCD6 mAb enhanced allogeneic mixed leukocyte reactions (MLR), indicating that CD6-ALCAM interactions may regulate the proliferative capacity of T cells. PMID- 8625963 TI - Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro. AB - The thymus is surrounded by a thin layer of mesenchyme and the epithelial mesenchymal interaction is known to be essential for the thymus development. To clarify the roles of mesenchyme in the thymus lobule formation that occurs around embryonic days 14-15 in vivo, we set up a three-dimensional organ culture system. The epithelium of embryonic day 14 thymic primordium was separated from the mesenchyme and cultured in Matrigel (reconstituted basement membrane). Addition of the mesenchyme to a chamber separated by a membrane filter induced the lobule formation of the thymic epithelium in vitro. We found that epidermal growth factor (EGF) can replace the mesenchyme for lobulation of the embryonic thymus in vitro. Among other growth factors tested, only transforming growth factor (TGF) alpha was as effective as EGF, in agreement with the fact that EGF and TGF-alpha bind to the same receptor. These results suggest that EGF or its family members may be involved in morphogenesis and differentiation of the thymus gland epithelium, although we cannot exclude the possibility that other unknown factors are required in vivo. PMID- 8625964 TI - Expression of high- and low-affinity receptors for C3a on the human mast cell line, HMC-1. AB - The proteolytic cleavage product of complement component 3, (C3a), is like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC-1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC-1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors of C3a in HMC-1 cells couple in part to Bordetella pertussis toxin-sensitive G-proteins and, therefore, appear to belong to the family of serpentine receptors that require G-proteins for signal transduction. HMC-1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I-C3a with high-(Kd1 = 2.1 4.8 nM) or low-affinity (Kd2 = 30-150 nM), and both receptors are expressed at high level: 3 x 10(5)-6 x 10(5) C3aR1/cell and 5 x 10(5)-2.3 x 10(6) C3aR2/cell. Results from cross-linking experiments with 125I-C3a fully agree with the presence of two different classes of C3a receptors in HMC-1 cells. Two membrane proteins with apparent molecular masses of 54-61 kDa (p57) and 86-107 kDa (p97) could be covalently modified with 125I-C3a, and this cross-linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL-8, NAP-2, GRO alpha, ENA-78, IP10, PF4, MCP-1, 2 and 3, RANTES, MIP-1 alpha, MIP-1 beta and I309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC-1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC-1 cell line an excellent choice for the cloning of the receptor genes. PMID- 8625965 TI - CD31/PECAM-1-driven chemokine-independent transmigration of human T lymphocytes. AB - We assessed the relative contribution of CD31/PECAM-1 (platelet-endothelial cell adhesion molecule-1) to T lymphocyte transmigration by the use of transfected murine fibroblasts stably expressing either the human CD31/PECAM-1 or the intercellular adhesion molecule-1 (CD54/ICAM-1). Unlike CD54/ICAM-1, CD31/PECAM-1 supported migration of activated T cells in the absence of chemokines: most of the migrating lymphocytes were CD31+ and displayed a phenotype corresponding to the naive subpopulation (LFA-1 dull and CD45RA+). Migration of activated T lymphocytes through CD54/ICAM-1+ transfected monolayers could be induced by creating a chemotactic gradient with the chemokine monocyte chemotactic protein 1, and the migrating cells mainly displayed a memory phenotype (LFA-1 bright CD45RO+) under these conditions. Furthermore, we found that in transfected cells CD54/ICAM-1 is uniformly distributed along the apical surface of the cells, while CD31/PECAM-1 is concentrated at the intercellular junctions, suggesting the existence of a haptotactic gradient (i.e. a gradient of substrate- or cell-bound molecules) responsible for T cell migration. This was also confirmed by the finding that monolayers of murine fibroblasts transfected with a CD31/PECAM-1 mutant lacking the cytoplasmic domain (CD31/PECAM-1-delta cyto), which has a reduced tendency to localize at cell-cell contact areas, supported efficient adhesion but were unable to induce migration of activated T cells unless a chemotactic gradient was created. We propose that in lymphocytes, homophilic CD31/PECAM-1 adhesion may be primarily involved in transmigration of naive T cells and that its role is complementary to that of CD54/ICAM-1. PMID- 8625966 TI - Unique role of thyroxine in T cell recognition of a pathogenic peptide in experimental autoimmune thyroiditis. AB - We have previously demonstrated the importance of iodination and the requirement of the thyroxine residues in thyroglobulin (Tg) for the stimulation of two clonotypically distinct murine T cell hybridomas reactive against human and mouse Tg. We are now able to show that these T cell hybridomas only recognize an 11 residue peptide containing a thyroxine structure that has iodine at two positions on each ring. This iodination state is critical for recognition by these hybridomas as a peptide containing de-iodinated thyroxine is nonstimulatory. Furthermore we have demonstrated that a peptide lacking the thyroxine residue or containing de-iodinated thyroxine cannot block the recognition of the thyroxine containing peptide. We suggest that in our system the thyroxine residue is involved in binding to major histocompatibility complex (MHC) class II molecules. We have also been able to show that the thyroxine residue is available for contact by the T cell receptor (TCR) as recognition of the peptide/H-2A(k) complex is blockable by an antibody directed against thyroxine. Using substituted peptides, we have been able partially to define the residues within the peptide that are critical for recognition of the 11-residue peptide by our hybridomas. From our data, we suggest that the thyroxine residue may bind the MHC and TCR, while the residues identified in the peptide backbone as important for the stimulation of the hybridomas may bind only the TCR. PMID- 8625967 TI - Cytotoxic T lymphocytes to Plasmodium falciparum epitopes in an area of intense and perennial transmission in Tanzania. AB - Studies in The Gambia have provided indirect evidence that cytotoxic T lymphocytes (CTL) play a protective role against malaria in humans and recently, using allele-specific HLA class I peptide motifs, several peptide epitopes for CTL in four pre-erythrocytic Plasmodium falciparum antigens have been identified in naturally exposed Gambians. However, CTL levels were low, suggesting that boosting these low levels by immunization might provide substantial protection. In the Kilombero valley of Tanzania, malaria transmission is holoendemic and 300 times more intense than in The Gambia. We report here that several of the epitopes identified in The Gambia are also recognized in naturally exposed, partially immune Tanzanian adults and that levels of CTL are similar to or slightly higher than in Gambian subjects, despite the much higher inoculation rate. We report a new HLA-A2.1-restricted epitope from the thrombospondin-related anonymous protein (TRAP) and we demonstrate that peptide epitopes in TRAP are naturally processed for recognition by CTL from naturally exposed humans. The common allele of a variable HLA-B7-restricted epitope in the circumsporozoite protein behaved as an altered peptide ligand (APL) with respect to CTL cognate for a rarer allelic variant of this epitope, suggesting that APL antagonism may occur in natural CTL responses to P. falciparum. The moderate levels of CTL observed, even in this area of intense malaria transmission, points to the need to assess candidate vaccines aimed at increasing CTL levels. PMID- 8625969 TI - Murine epidermal Langerhans cells do not express inducible nitric oxide synthase. AB - In Leishmania-infected macrophages (M phi), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to M phi, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and M phi display the same leishmanicidal effector mechanism. Unlike M phi, stimulation of unselected epidermal cells with interferon gamma/lipopolysaccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in M phi as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated M phi and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO independent mechanism appears to be responsible for the control of parasite replication in LC. PMID- 8625968 TI - CTX, a novel molecule specifically expressed on the surface of cortical thymocytes in Xenopus. AB - CTX, a novel developmentally regulated type-I transmembrane protein is expressed specifically by a large fraction of cortical thymocytes in the amphibian Xenopus. This apparently monomeric 55-kDa glycoprotein is composed of two immunoglobulin domains, one variable (V) and one constant (C2 type), followed by a transmembrane and a 64-amino acid cytoplasmic domain. The first immunoglobulin domain is a V-J segment that is generated without gene rearrangement. In the genome, the V and C2 domains are both encoded by two half-domain exons. Two CTX loci are found per haploid genome, and they exhibit sequence differences with a high replacing/silent ratio in the CDR1-like region of the V domain, suggesting that these differences were selected. The cytoplasmic domain contains a motif that is highly conserved evolutionarily in several types of proteins, including adenylyl cyclases. Based on its unique tissue distribution, the variability of its V region and the motif of its cytoplasmic domain, CTX is a candidate for a new type of specific molecule involved in thymocyte selection. PMID- 8625970 TI - One cause for the apparent inability of human T cell clones to function as professional superantigen-presenting cells is autoactivation. AB - Human T cell clones (TCC) are antigen-presenting cells (APC) able to present peptides and superantigens (SAg) and to process and present intact proteins. TCC express major histocompatibility complex (MHC) class II antigens and molecules involved in the accessory signal delivery, such as B7.1 and B7.2/B70. Notwithstanding these observations, the role of professional APC has been often denied to T cells because anergy of responder T cells rather than proliferation has been observed following the TCC presentation in the absence of added professional APC. Here, we show that upon stimulation with free SAg, TCC undergo proliferative responses followed, after a 1-week culture, by an SAg-dependent unresponsiveness to T cell receptor (TCR)-mediated stimuli, but not to interleukin-2. The anergy induced by the SAg can not be prevented by the addition of autologous Epstein-Barr virus (EBV)-transformed B cells, indicating that the induction of anergy occurs also in the presence of conventional APC. Conversely, if the TCC are stimulated by SAg-prepulsed irradiated APC, either EBV and TCC, the induction of anergy is not observed. After a 1-week culture, in fact, TCC stimulated with APC-bound SAg responded to TCR-mediated stimuli, irrespective of the APC (EBV or TCC) used for the SAg presentation. Stimulation of TCC with free SAg in a semisolid medium that prevents T-T cell contacts resulted in an activation followed by a state of anergy, suggesting that anergy is the consequence of SAg recognition at the single T cell level. These data indicate that the anergy observed in TCC upon a 1-week culture in the presence of soluble SAg is not the result of an inherent inability of TCC to act as professional APC. Rather the phenomenon depends on the presence of soluble SAg, leading to T cell autostimulation. PMID- 8625971 TI - T cell receptor-gamma, delta-expressing fetal mouse thymocytes are generated without T cell receptor V beta selection. AB - We investigated whether fetal mouse T cell receptor (TCR) gamma delta cells have been subjected to so-called TCR beta selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis to TCR gamma delta cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCR gamma delta cells were CD2+ suggesting an origin at a post CD25 stage. Molecular analysis of TCR beta rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of V beta 6 and V beta 8 to J beta 2 rearrangements day-15 fetal TCR gamma delta showed extensive TCR beta rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJ beta 2 fragments showed that the majority (58%) were rearranged out of frame . Taken together, these phenotypic and molecular analyses suggest that fetal TCR gamma delta cells have not been subject to TCR beta selection. PMID- 8625972 TI - B cell early response gene expression coupled to B cell receptor, CD40 and interleukin-4 receptor co-stimulation: evidence for a role of the egr-2/krox 20 transcription factor in B cell proliferation. AB - B lymphocytes are activated following antigen stimulation of the B cell receptor but require co-stimulation with accessory molecules provided by interleukin (IL) 4/CD40 ligand for cell cycle progression and proliferation. By analyzing a panel of 11 early response genes induced by cross-linking of surface immunoglobulin, we show that CD40 signaling alone induces only 2 genes, c-myc together with an anonymous gene, 3L3, and that these are distinct from the set of genes induced in response to IL-4. Co-stimulation with the proliferative combination of anti-mu, IL-4 + CD40 signaling led to a fourfold enhancement of egr-2/krox 20 expression over that seen with anti-mu alone. Egr-2 expression/activity was selectively inhibited by the immunosuppressive drug cyclosporin A, and antisense oligonucleotide blockade of Egr-2 activity elicited a dose-dependent inhibition of B cell proliferation. Taken together, these observations show that the early gene regulatory programs coupled to different surface receptors on B cells are largely distinct from each other, but that certain genes, exemplified by egr-2, may represent a point of convergence in the integration of different signaling pathways into the B cell proliferative response. PMID- 8625974 TI - Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1). AB - The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170-180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly upregulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocytes adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin. PMID- 8625973 TI - CD31-triggered rearrangement of the actin cytoskeleton in human natural killer cells. AB - In this report, we analyze whether CD31, also known as platelet-endothelial cell adhesion molecule-1 (PECAM-1), can transduce an outside-in signal in human natural killer (NK) lymphocytes in vitro. We show that CD31, but not HLA class-I cross-linking triggers an outside-in transmembrane signal in NK lymphocytes, mediating cell spreading and cytoskeletal rearrangement. These phenomena are Mg2+, but not Ca2+ dependent, suggesting that signal transduction elicited by CD31 cross-linking may involve an associated integrin. Two possible candidates would be alpha v and alpha L, whose function is known to depend on Mg2+. However, the CD31-induced cytoskeletal rearrangement was not reduced by the use of alpha v or alpha L-specific F(ab')2, suggesting that CD31 could transduce a signal by itself or by association with a still-undefined integrin. Moreover, talin, but not vinculin or tubulin, appears to co-localize with actin microfilaments in the membrane ruffles of NK cells that undergo cytoskeleton rearrangement following CD31 cross-linking. Both spreading and cytoskeletal rearrangement appear to be regulated by intracellular cyclic-3',5'-adenosine monophosphate (cAMP). Indeed, the activator of the adenylyl cyclase, forskolin, inhibited cell spreading and cytoskeletal rearrangement induced by CD31 cross-linking. This phenomenon was also observed using the membrane-permeants cAMP analog Sp adenosine-3', 5' cyclic monophosphothioate (Sp-cAMPS), but not its inactive isomer Rp-cAMPS. Likewise, adhesion of NK lymphocytes to NIH/3T3 murine fibroblasts transfected with the cDNA encoding human CD31 was blocked by increasing intracellular cAMPS levels. We suggest that intracellular cAMP may be involved in CD31-mediated signal transduction, and may regulate NK-endothelial cell adhesion and possibly, the tissue localization of NK cells. PMID- 8625975 TI - Time course analysis of alpha+ beta+ T cell clones during normal pregnancy. AB - During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen-specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase-polymerase chain reaction for T cell receptor beta chain gene and subsequently analyzed the PCR product by single-strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post-conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen-specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy. PMID- 8625976 TI - Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-kappa B. AB - Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)- 1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and N-tosyl-Phe chloromethylketone, blocked IL-1 beta induction of VCAM-1 and ICAM-1 promoter driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-kappa B in response to IL-1 beta stimulation. In contrast, norLEU did not prevent IL-1 beta-induced nuclear translocation of NF-kappa B. The effects of norLEU were specific because it did not inhibit the IL-1 beta induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. PMID- 8625978 TI - Permissive recognition during positive selection. AB - In the periphery alpha beta T lymphocytes recognize antigens in conjunction with major histocompatibility complex (MHC) molecules. In the thymus immature T cells are positively selected on MHC molecules in the apparent absence of cognate peptides. Thus, at different developmental stages a T cell responds to different epitopes, yet uses the identical alpha beta T cell antigen receptor (TcR). To explain this paradox it has been hypothesized that during positive selection immature T cells see peptides/ligands unique to the thymus, are selected by specific antagonists related to their cognate peptides, or are driven by lowered affinity thresholds of their TcR. Though different in detail, these theories rely on defined peptides uniquely matched to select certain TcR. However, we find that in a TcR-transgenic (TcR(trans +)) mouse severely limiting the diversity of peptides does not impair positive selection. We show that many unrelated peptides, including some naturally occurring on the cell surface, induce maturation of CD4-CD8+TcR(high) thymocytes. The same peptides when presented in conjunction with the selecting MHC molecule, are not recognized by peripheral T cells expressing the same TcR(trans). Therefore, these findings point to a promiscuous rather than discriminate recognition mode used by immature T cells. PMID- 8625977 TI - Increase of intracellular calcium is the essential signal for the expression of CD40 ligand. AB - CD40 ligand (CD40L) is present on activated but not on resting T cells. In contrast to the activation markers CD25 and CD71, a strong CD40L expression could be induced by calcium ionophore alone but not by phorbol 12-myristate 13-acetate (PMA). Ionomycin induced a very early mRNA and protein surface expression of CD40L within the first 2 h, whereas CD25 and CD71 did not appear earlier than 6 h after stimulation. The mitogens phytohemagglutinin and concanavalin A induced little CD40L, but together with PMA, a markedly increased CD40L expression was observed. In T cells stimulated with immobilized anti-CD3, co-stimulation with anti-CD28 or PMA induced an earlier and higher maximal CD40L expression. CD40L expression of purified T cells was higher and more prolonged compared to that of T cells in unseparated peripheral blood mononuclear cells. We conclude that the expression of CD40L on T cells is profoundly different from other early activation markers with regard to signal requirements, kinetics and the role of accessory cells in the system. PMID- 8625979 TI - Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand-dependent mechanism: CTLA4-Ig protects V beta 8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B. AB - Co-stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co-stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing V beta 8+, which represent approximately 30% of all CD4+ T cells. In litter-mate controls of CTLA4-Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of V beta 8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of V beta 8 T cells occurs in CTLA4-Ig transgenic mice. However, in contrast to normal mice, the remaining V beta 8+ T cells from CTLA4-Ig transgenic mice are not anergic and remain responsive to superantigen in vitro. PMID- 8625980 TI - A third interferon-gamma-induced subunit exchange in the 20S proteasome. AB - The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation. PMID- 8625981 TI - Translatable immunoglobulin germ-line transcript. AB - During B cell differentiation, the functional genes encoding immunoglobulin (Ig) heavy (H) and light (L) chains are generated by two rearrangement processes--VDJ rearrangement generates the exon encoding the Ig variable (V) regions, and the class switch reconstructs a rearranged IgH gene by exchanging the segment encoding the constant (C) region, which determines the Ig class. Both types of rearrangement are preceded by transcripts originating from a transcriptional start site 5' of the I exon, which is then spliced to the C exons. These germ line transcripts, which are thought to be necessary for the initiation of both types of rearrangement, are said to be sterile. We demonstrate here that the mu germ-line transcript is translatable into a polypeptide chain, to which we assign the symbol psi. Thus, protein products of these transcripts might be part of or signal to the recombinases that catalyze Ig gene rearrangement. PMID- 8625982 TI - Expression levels of stress protein gp96 are not limiting for major histocompatibility complex class I-restricted antigen presentation. AB - Immunization of mice with gp96 induces CTL with specificity for proteins that are expressed in the cells from which gp96 was isolated (Arnold et al., J. Exp. Med. 1995. 182: 885, Udono et al., Proc. Natl. Acad. Sci. USA 1994. 91: 3077). Recently, it has been shown that gp96 from cells transfected with vesicular stomatitis virus (VSV) nucleocapsid protein as well as gp96 loaded in vitro with peptides containing an epitope of this protein are taken up by phagocytic cells which obtain thereby the capacity for stimulating VSV-specific cytotoxic T lymphocytes (Suto and Srivastava, Science 1995. 269: 1585). The immunization experiments together with the peptide transfer from gp96/peptide complexes to major histocompatibility complex (MHC) class I molecules of phagocytic cells are consistent with the hypothesis that the endoplasmic reticulum-resident protein gp96 plays a crucial role in the antigen presentation of a cell (Srivastava et al., Immunogenetics 1994. 29: 93). To examine the involvement of gp96 in class I restricted antigen presentation, we reduced gp96 RNA and protein levels by transfecting P13.1 cells with a vector containing part of gp96 cDNA in antisense orientation to the promotor. We found that antisense clones expressing strongly reduced levels of gp96 mRNA and gp96 protein show normal levels of MHC class I molecules on the cell surface and are recognized by T cells to the same extent as wild-type cells. Thus, our results show that normal levels of gp96 expression in a cell are not limiting for class I-restricted antigen presentation. PMID- 8625983 TI - Expression and function of B7-1 and B7-2 in hapten-induced contact sensitivity. AB - We analyzed the expression and function of the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) during contact sensitivity reactions induced by the hapten 2,4-dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7-2. In contrast, following challenge with DNFB, expression of B7-2 is up-regulated in both epidermis and dermis. Importantly, B7-1 is induced later and at lower levels compared to B7-2. Intravenous injections of anti-B7-2 mAb, but not anti-B7-1 mAb partially inhibit the hapten-induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti-B7-2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7-2 is important for successful antigen priming. PMID- 8625984 TI - Regions of RAG1 protein critical for V(D)J recombination. AB - The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutation for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conversed sequence. We show here that this "dispensable" region of RAG1 is not necessary for coding joint formation or recombination of an integrated substrate, and this portion is not functionally redundant with the "dispensable" region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene. PMID- 8625985 TI - Tracing interactions of thymocytes with individual stromal cell partners. AB - Cellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation most--if not all--stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substitution of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro environments is rate limiting for positive selection, and that in positive selection each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing system for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma-derived MHC class II determinants. When MHC class II-deficient precursors are placed in H-2b x k F1 environments (where all stromal cells co-express H-2b and H-2k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H-2b or H-2k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others. PMID- 8625986 TI - Distribution of alpha 4 beta 7 and alpha E beta 7 integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes. AB - Beta 7 is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of beta 7. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express alpha 4 beta 7 and alpha E beta 7 using mAb against alpha E, beta 7 and mAb DATK32 which recognizes a combinatiorial epitope on alpha 4 beta 7+ thymocytes have a mature phenotype: TcR+, CD11a(hi)CD44(hi)HSA(dull). Small subsets of double-negative CD4-CD8-, single-positive CD4+ and CD8+ thymocytes express beta 7, while double-positive CD4+CD8+ thymocytes are beta 7-. However, two integrins alpha E beta 7 and alpha 4 beta 7 recognized by anti-beta 7 are not expressed on an identical subpopulation of thymocytes, as alpha E beta 7+ alpha 4 beta 7-, alpha E beta 7 + alpha 4 beta 7+ and alpha E beta 7- alpha 4 beta 7+ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of alpha E beta 7 but little alpha 4 beta 7. In the spleen, Peyer's patches and lymph nodes, alpha 4 beta 7 is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4+ and CD8+ lymphocytes, express high levels of beta 7 in the form of alpha 4 beta 7 and alpha E beta 7, although, as observed with lymphocytes, not all alpha 4 beta 7 hi CD4+ lymphocytes expressed alpha 4 beta 7. The population of alpha 4 beta 7 hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4+ lymphocyte population, which can be further subdivided on the basis of alpha E beta 7, L-selectin and alpha 4 expression. Therefore, memory CD4+ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties. PMID- 8625988 TI - The expansion and selection of T cell receptor alpha beta intestinal intraepithelial T cell clones. AB - In conventional mice, the T cell receptor (TCR) alpha beta+ CD8 alpha alpha+ and CD8 alpha beta+ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Ep. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8 alpha alpha and CD8 alpha beta TCR alpha beta+ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCR alpha beta+ T cells than conventional mice. The TCR beta repertoire of the CD8 alpha alpha and the CD8 alpha beta IEL populations of germ free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show the intestinal microflora is not responsible for the repertoire oligoclonality of TCR alpha beta+ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8 alpha alpha and the CD8 alpha beta TCR alpha beta IEL clones have a heterogeneous size pattern, with clones containing from 3 x 10(3) cells up to 1.2 x 10(6) cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCR alpha beta IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium. PMID- 8625987 TI - The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co-expressed during B cell development. AB - The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16 17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre B cell receptor complexes. PMID- 8625989 TI - Differential regulation of human T cell responsiveness by mucosal versus blood monocytes. AB - Human intestinal T lymphocytes are constantly exposed to a large number of foreign antigens without developing a systemic immune response. One crucial mechanisms leading to this intestinal hyporesponsiveness is based on impaired signal transduction through the T cell receptor/CD3 complex in lamina propria T lymphocytes (LP-T). In this study, we addressed the question whether a lack of co stimulatory/progression signals might also contribute to LP-T hyporesponsiveness. To this end, isolated human monocyte populations from the intestinal lamina propria were obtained and their phenotypes as well as their capacity to promote T cell activation studied. Here, we demonstrate that lamina propria macrophages (LP MO), in contrast to peripheral blood monocytes (PB-MO), do not support proliferation of either LP-T or PB-T. This may be due to the low expression of ligands (CD54, CD58, CD80) for the T cell accessory receptors CD11/18, CD2 and CD28/CTLA-4 on mucosal macrophages. Thus, down-regulation of both recognition/competence and co-stimulatory/progression signals contribute to intestinal hypo- or unresponsiveness. PMID- 8625990 TI - Specificity of an HLA-DRB1*0401-restricted T cell response to type II collagen. AB - A panel of HLA-DRB1*0401-restricted CD4+ mouse T cell hybridomas specific for bovine type II collagen were generated from transgenic mice expressing the human HLA-DRA1*0101/-DRB1*0401 and CD4 molecules. The vast majority recognized a single peptide determinant corresponding to residues 261-273 (CII 261-273). This determinant was rapidly defined by the use of a predictive algorithm for peptide binding to DRB1*0401. CII 261-273 is conserved in bovine and human type II collagen and overlaps with an important I-A q - restricted T cell determinant in mice with collagen-induced arthritis. This study demonstrates how HLA-DR and human CD4-transgenic mice can be used to identify a T cell epitope in a potential or candidate autoantigen. PMID- 8625991 TI - Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. AB - There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD). In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions. In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ). We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e. are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e. are immune when exposed to bacterial sonicates derived from the heterologous intestine of syngenic littermates (BsH). Furthermore, we demonstrate that both local and systemic tolerance to BsA is broken in a murine model of chronic intestinal inflammation induced by the hapten reagent 2, 4, 6-trinitrobenzene sulfonic acid (TNBS), which mimics several important characteristics of Crohn's disease. Tolerance to BsA was restored and TNBS-induced colitis was abrogated in mice systemically treated with interleukin (IL)-10 or antibodies to IL-12. Treatment specifically restored tolerance to BsA, but did not suppress proliferation to BsH. In summary, we here report a new model for the study of immunity and tolerance towards bacterial products. Our data suggest that tolerance to BsA is an important protective mechanism and that restoration of tolerance intestinal flora by IL-10 and antibodies to IL-12 may be of potential therapeutic utility in patients with inflammatory bowel disease. PMID- 8625993 TI - Proliferative recruitment of intestinal intraepithelial lymphocytes after microbial colonization of germ-free mice. AB - Intraepithelial lymphocytes (IEL), particularly alpha beta TCR-bearing IEL (alpha beta-IEL), dramatically increase in number after microbial colonization of formerly germ-free (ex-GF) mice (Umesaki et al., Immunology 1993. 79: 32). In this study, the kinetics of expansion of IEL after microbial colonization in ex GF mice were investigated by the bromodeoxyuridine (BrdUrd) continuous labeling method. In GF mice, gamma delta- and alpha beta-IEL were gradually labeled with BrdUrd, reaching approximately 30% and 15% labeling, respectively, after 10 days of continuous BrdUrd labeling. In conventional (CV) mice, the percentage of BrdUrd-labeled alpha beta-IEL was a little higher than that for gamma delta-IEL. The maximal labeling for alpha beta-IEL and gamma delta-IEL reached 50-60% and 40%, respectively, in 10 days. In the case of conventionalized ex-GF mice, continuous labeling was started 11 days after microbial colonization of GF mice because alpha beta-IEL outnumbered gamma delta-IEL during this period. In this case, 75% of alpha beta-IEL and 67% of gamma delta-IEL were labeled with BrdUrd in 10 days. On the other hand, the apparent half lives of alpha beta-IEL and gamma delta-IEL were 10 and 20 days, respectively in CV mice. These results strongly suggest that the number of IEL, particularly alpha beta-IEL, increases after microbial colonization through recruitment into the cell cycle of a large proportion of IEL or their immediate precursors. The difference in the relative BrdUrd-labeling rate of alpha beta-IEL to that of gamma delta-IEL between germ free and conventionalized mice indicates a preferential increase in the alpha beta-IEL subset during the course of conventionalization. PMID- 8625992 TI - A new Ig-superfamily member, molluscan defence molecule (MDM) from Lymnaea stagnalis, is down-regulated during parasitosis. AB - To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to approximately 21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper function of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling on non-self recognition. PMID- 8625994 TI - Recognition of altered self major histocompatibility complex molecules modulated by specific peptide interactions. AB - Antigen-specific and major histocompatibility complex (MHC)-restricted recognition by the T cell receptor involves multiple structural contacts over a large molecular surface area. Using a human T cell clone specific for a rubella viral peptide restricted by subsets of HLA DR4 molecules, we identified structurally diverse combinations of peptide-MHC complexes which were functionally equivalent to T cell recognition. Presentation of the rubella derived peptide on DR4 molecules with an E-74 polymorphism triggered T cell recognition, as did presentation of a single amino acid-substituted peptide in the context of DR4 molecule which lacked the E-74 site. Peptide binding and molecular modeling analysis indicates the structural and functional complementarity of T cell recognition for a specific amino acid side chain, whether contributed by the peptide or by the MHC molecule. PMID- 8625995 TI - Non-obese diabetic mice hemizygous at the T cell receptor alpha locus are susceptible to diabetes and sialitis. AB - To test the hypothesis that T cells carrying two T cell receptor (TCR) alpha chains play a role in autoimmunity, we backcrossed the non-obese diabetic (NOD) strain with one carrying a TCR alpha gene disrupted by homologous recombination. Mice carrying one copy of the disrupted gene are incapable of generating T cells carrying two cell surface TCR alpha chains. Our early results suggested that either dual TCR alpha T cells play a role in insulin-dependent diabetes mellitus (IDDM) induction in NOD mice or that a locus co-segregating with the disrupted TCR alpha locus protected mice from diabetes induction. From the analysis both of mice in which the region co-segregating with the disrupted TCR alpha locus is minimized and of the F1 offspring of NOD mice with the 129 strain (TCR alpha hemizygous mice), the apparent protective effect of the absence of dual TCR alpha T cells is lost; thus, such cells do not appear to play a critical role in autoimmune disease in NOD mice. PMID- 8625996 TI - T lymphocytes from the normal human peritoneum contain high frequencies of Th2 type CD8+ T cells. AB - The majority of peritoneal T lymphocytes have been shown to be CD8+ and to co express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)-4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8+ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)-gamma and exhibited high alloantigen-specific cytolytic activity, some clones secreted IL-4 and IL-5 but no detectable IFN-gamma. These Th2-type CD8+ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8+ T cell may occur in different immune compartments. PMID- 8625997 TI - Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double-positive thymocytes. AB - The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40 T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double -positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of the specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes. PMID- 8625998 TI - Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario. AB - This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA gene showed that 151 (35.2%) strains were positive for the eaeA gene. All isolates (9-21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7-34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle. PMID- 8625999 TI - Characteristics of gonococci isolated from men with urethritis in Dubai. AB - Neisseria gonorrhoeae were collected from men attending out-patient clinics in Dubai. The susceptibility to a range of therapeutic antibiotics and their auxotype and serotype was determined. The plasmid content of all penicillinase producing strains was also analysed. Thirty-six strains of N. gonorrhoeae were isolated from specimens collected from 79 patients over a 24-day period. Of the 36 isolates, 9(25%) were penicillinase-producing N. gonorrhoeae (PPNG) and 15 (42%) were chromosomally resistant N. gonorrhoeae (CMRNG). CMRNG exhibited higher levels of resistance to cefuroxime, chloramphenicol, tetracycline and erythromycin than PPNG. All isolates were susceptible to ceftriaxone and spectinomycin. Three (8%) isolates showed reduced susceptibility (MIC, > or = 0.25 mg/l) to ciprofloxacin. Six isolates of PPNG carried the 4.4 MD and three the 3.2 MD penicillinase encoding plasmid. The total gonococcal population was phenotypically diverse, with 12 serovars, 6 auxotypes and 21 A/S classes. Gonorrhoea was found to be a major cause of urethritis in Dubai and the strains exhibited high levels of resistance to penicillin. PMID- 8626001 TI - Association between clinical presentation, biogroups and virulence attributes of Yersinia enterocolitica strains in human diarrhoeal disease. AB - Traditionally the enteric pathogen Yersinia enterocolitica has been differentiated into biogroups. Despite being considered as non-pathogenic, biogroup 1A isolates have constituted a sizeable fraction of strains from patients with gastroenteritis in many reports. To establish a potential clinical significance for biogroup 1A isolates of Y. enterocolitica, clinical disease in patients with gastroenteritis excreting such isolates was compared with symptoms among patients found infected with pathogenic biogroups. Clinical data and isolates of 66 patients from whom Y. enterocolitica had been isolated by direct plating were available for study. There was an association between patient age below 3 years and infection with 'pathogenic' Y. enterocolitica. The severity of gastroenteritis and other symptoms, however, did not depend on the biogroup, or the presence of the virulence plasmid in the yersinia strain isolated from the patients. Strains belonging to biogroup 1A of Y. enterocolitica showed two clusters of ribotypes, one of which encompassed most isolates recovered from humans, the other being associated with environmental isolates. This might indicate the existence of human-adapted and potentially pathogenic strains among biogroup 1A of Y. enterocolitica. PMID- 8626000 TI - Differing antibody responses to Haemophilus influenzae type b after meningitis or epiglottitis. AB - Two common forms of invasive disease due to Haemophilus influenzae type b (Hib) are epiglottitis and meningitis. It is not known why some children develop epiglottitis and others meningitis. To examine the hypothesis that epiglottitis occurs in children who may have been previously exposed to Hib, and who would therefore exhibit a more vigorous antibody response in convalescence, we measured levels of antibody to Hib capsule in 92 children. Geometric mean convalescent phase IgG, IgA, IgM and total antibody levels were significantly higher in 45 children with epiglottitis than in 47 with meningitis, even after adjustment for age differences (mean total antibody, 95% confidence intervals: meningitis 0.38, 0.34-0.43; epiglottitis: 2.25, 2.0-2.54 micrograms/ml). However, contrary to previous reports, a poor antibody response was only observed in a minority of children with meningitis; the antibody response of the majority was indistinguishable from that observed in children with epiglottitis. PMID- 8626002 TI - Micromorphometrical analysis of rodent related (SPF) and unrelated (human) gut microbial flora in germfree mice by digital image processing. AB - Digital image processing (DIP) of bacterial smears is a new method of analysing the composition of the gut microbial flora. This method provides the opportunity to compare and evaluate differences in the complex highly concentrated anaerobic fraction of gut microbial flora, based on micromorphological differences. There is ample evidence that this fraction can be characterized as related or unrelated to the host organism by its immunogenicity. In this study germfree ND2 mice were associated with either related (rodent) SPF microflora (SPF-MF) or unrelated human MF (HUM-MF). DIP analysis was performed on original SPF-MF and HUM-MF and on the faeces of ex-germfree mice 4 weeks after association. The micromorphological pattern of highly concentrated anaerobic bacteria in faeces of HUM-MF associated ex-germfree mice was significantly different from SPF-MF associated counterparts with regard to the scores for elongation (P < 0.01) and morphological variety (P < 0.05). Moreover, gross morphological variability was present between individual HUM-MF associated mice but not between individual SPF MF associated animals. No differences were found between original SPF and HUM-MF. The data are discussed with regard to differences in the presence of (non )immunogenic bacteria and the ability for related and unrelated flora to colonize the murine gut. This study provides evidence that murine host specificity of microbial flora may not only be reflected in the number of non-immunogenic bacteria but also in the micromorphological pattern of highly concentrated anaerobic bacteria in faeces measured by DIP analysis. PMID- 8626003 TI - Genetic characterization of Mycobacterium avium isolates recovered from humans and animals in Australia. AB - Genetic relationships amongst 115 mainly Australian isolates of Mycobacterium avium were assessed using multilocus enzyme electrophoresis (MEE). The isolates were divided into 58 electrophoretic types (ETs), with a mean genetic diversity of 0.29. Isolates from humans were closely related to but distinct from those cultured from birds, whilst some porcine isolates belonged to the same ETs as certain human isolates. Pulsed field gel electrophoresis (PFGE) was used to differentiate related isolates, and those from birds and some from other animals, including pigs, were distinguished from the human isolates. The results of MEE and PFGE suggested that certain strains of M. avium may be transmitted between birds and pigs, but there was no clear evidence of transmission to humans. The serovar of the M. avium isolates was not obviously related to their ET assignment or their PFGE type. PMID- 8626004 TI - Impact of influenza and respiratory syncytial virus on mortality in England and Wales from January 1975 to December 1990. AB - The effects of influenza A and B and RSV on mortality in England and Wales were assessed by regression analysis for the period 1975-90. Morbidity data from sentinel practices were used to calculate 4-weekly rates of aggregated upper respiratory tract infections (URTI); PHLS laboratory reports were used as indices of infection, and 4-weekly death rates from all causes, excluding childbirths, were used to study relationships with mortality. Deaths correlated strongly with influenza A and B reports, temperature, and interactions between aggregated URTI and temperature, and RSV outbreaks and temperature. Estimates of 'seasonal' 4 weekly mortality associated with URTI were made by substituting into primary regression models the mean of annual trough consultation rates for aggregated URTI and baseline values for RSV and influenza. Peak 4-weekly mortality associated with URTIs was estimated at c. 24000 and c. 28000 during combined influenza and RSV epidemics of 1975-6 and 1989-90 respectively. Secondary regression analysis was carried out with the estimated 'seasonal' 4-weekly deaths associated with URTI as dependent variable and laboratory data as regressors. Estimated excess mortality associated with influenza was considerable even during years without major epidemics. Overall during the 15 winters the estimated mortality associated with RSV was 60-80% more than that associated with influenza. The modelling permits only a crude estimate of RSV associated mortality. None the less it suggests that RSV is an important cause of winter mortality. PMID- 8626005 TI - Measles immunity and response to revaccination among secondary school children in Cumbria. AB - The prevalence of antibody to measles virus in 759 children aged 11-18 years attending a secondary school in Cumbria was measured using a salivary IgG antibody capture assay. Serum IgG antibody levels were measured using a plaque reduction neutralization assay in subjects whose saliva was antibody negative. Vaccination histories were obtained from the child health computer and general practice record. A total of 662 pupils (87% of those tested) had detectable measles-specific IgG in saliva. Of the remaining 97, 82 provided blood samples and 29 had serum neutralizing antibody levels above 200 mIU/ml. Afer adjusting for non-participation rates, the proportion considered non-immune (no IgG in saliva and < or = 200 mIU/ml in serum) was 9% overall, ranging from 6% in vaccinated children to 20% in unvaccinated children. Measles-mumps-rubella vaccine was given to 50 children of whom 38 provided post-vaccination serum and 32 saliva samples. Thirty (79%) had a fourfold or greater rise in serum neutralizing antibody and 28 (88%) developed IgG antibody in saliva. Half of the children considered non-immune by antibody testing would have been overlooked in a selective vaccination programme targeted at those without a history of prior vaccination. A programme targeted at all school children should substantially reduce the proportion non-immune since a primary or booster response was achieved in three quarters of previously vaccinated children with low antibody levels and in all unvaccinated children. While it is feasible to screen a school-sized population for immunity to measles relatively quickly using a salivary IgG assay, a simple inexpensive field assay would need to be developed before salivary screening and selective vaccination could substitute for universal vaccination of populations at risk of measles outbreaks. The salivary IgG assay provided a sensitive measure of a booster response to vaccination. PMID- 8626007 TI - An outbreak of cholera from food served on an international aircraft. AB - In February 1992, an outbreak of cholera occurred among persons who had flown on a commercial airline flight from South America to Los Angeles. This study was conducted to determine the magnitude and the cause of the outbreak. Passengers were interviewed and laboratory specimens were collected to determine the magnitude of the outbreak. A case-control study was performed to determine the vehicle of infection. Seventy-five of the 336 passengers in the United States had cholera; 10 were hospitalized and one died. Cold seafood salad, served between Lima, Peru and Los Angeles, California was the vehicle of infection (odds ratio, 11.6; 95% confidence interval, 3.3-44.5). This was the largest airline-associated outbreak of cholera ever reported and demonstrates the potential for airline associated spread of cholera from epidemic areas to other parts of the world. Physicians should obtain a travel history and consider cholera in patients with diarrhoea who have travelled from cholera-affected countries. This outbreak also highlights the risks associated with eating cold foods prepared in cholera affected countries. PMID- 8626006 TI - The transmission dynamics of hepatitis B in the UK: a mathematical model for evaluating costs and effectiveness of immunization programmes. AB - Complex hepatitis B (HBV) epidemiology makes it difficult to evaluate and compare effectiveness of different immunization policies. A method for doing so is presented using a mathematical model of HBV transmission dynamics which can represent universal infant and adolescent vaccination strategies and those targeted at genito-urinary (GU) clinic attenders and infants born to infectious mothers. Model structure, epidemiological underpinning, and parameterization, are described. Data from the UK National Survey of Sexual Attitudes and Lifestyles is used to define patterns of sexual activity and GU clinic attendance; data deficiencies are discussed, in particular that of UK seroprevalence of HBV markers stratified by age, sex, and risk factors. General model predictions of endemic HBV marker prevalence in homosexual and heterosexual populations seem consistent with published UK data. The simulations exhibit non-linearities in the impact of different vaccination strategies. Estimated number of carriers prevented per vaccine dose for each strategy provides a measure of costs and benefits, varying temporally over the course of a programme, and with level of vaccine coverage. Screening before vaccination markedly increases payback per dose in homosexuals but not in heterosexuals; mass infant vaccination gives the poorest effectiveness ratio and vaccination of infants after antenatal screening the best; in general, increasing vaccine coverage yields lower pay-back per dose. The model provides a useful framework for evaluating costs and benefits of immunization programmes, but for precise quantitative comparison more UK epidemiological data is urgently needed. PMID- 8626008 TI - Relative abundance of enterovirus serotypes in sewage differs from that in patients: clinical and epidemiological implications. AB - One thousand one hundred and sixty-one non-polio enterovirus strains, isolated during regular screening of Finnish sewage specimens, were analysed for serotype distribution seasonally through 20 years, and the findings were compared with similar data based on 1681 clinical isolates. Coxsackievirus B4 (CBV-4), CBV-5, echovirus 11 (EV-11), EV-6, CBV-2 and CBV-3 were the most common serotypes in sewage, whilst CBV-5, EV-11, coxsackievirus A9, EV-22, CBV-3 and EV-30 were the most common clinical isolates. Reasons for the differences are not known but several explanations are possible. Seasonal variation of enterovirus occurrence in both sources showed an expected peak in the autumn with a trough in the spring. The occurrence of enteroviruses was closely correlated with monthly recordings of mean relative humidity. A further observation concerning the clinical specimens in Finland was the relative excess of some serotypes, such as echovirus 22 and coxsackievirus A9, and paucity of others, for instance, echoviruses 4 and 9, when compared to published data from other countries. This is consistent with the idea of geographically restricted circulation of enteroviruses. PMID- 8626010 TI - A Xenopus gene, Xbr-1, defines a novel class of homeobox genes and is expressed in the dorsal ciliary margin of the eye. AB - We have isolated Xbr-1, a novel Xenopus homeobox gene that defines a new class of homeobox genes, distantly related to the Drosophila BarH1 and BarH2 genes. Xbr-1 is predominantly expressed in the developing Xenopus eye, starting as early as the neural plate stage. At early stages of eye development XBR-1 is expressed in the eye vesicle dorsally, in the tissue between the prospective neural retina and pigment epithelium. Later, in the optic cup, it is restricted to the prospective dorsal ciliary margin, an area containing progenitor cells at the edge of retina. Comparing the expression of Xbr-1 in the eye to that of X-Notch-1 suggests that Xbr-1 marks a unique population of pluripotent stem cells within the dorsal ciliary margin. The expression of Xbr-1 and BMP-4 in the dorsal ciliary margin is largely coincident, suggesting that the two molecules interact. We propose that Xbr-1 and BMP-4 may play a role in the development of progenitor cells on the dorsal half of the eye and that they may be involved in the establishment of polarity of the eye along the dorsoventral axis. PMID- 8626009 TI - A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis. AB - Desmin, the muscle-specific intermediate filament protein is one of the earliest known myogenic makers both in heart and in somites. We have previously shown that high levels of desmin expression in the skeletal cell line C2C12 are due to a distal enhancer, which contains a muscle-specific factor-(2MEF2) binding site, adjacent to an E box, the binding site of the myogenic Helix-Loop-Helix (mHLH) regulators. We have further shown that MEF2C, a myocyte restricted member of the MEF2 family and all four mHLH factors can bind to their corresponding sites and through a cooperation with a second proximal E box can transactivate the desmin promoter. To study the significance of these regulatory elements in vivo, we have generated transgenic mice with desmin-lacZ reporters, intact or mutated at the MEF2 and E box of the enhancer. We show that the cis-acting DNA sequences within the 1-kb 5' flanking region of the mouse desmin gene are sufficient to direct appropriate temporal transcription both in heart and in skeletal muscle during mouse embryogenesis. Mutation at the MEF2 site completely suppressed transcription of the linked lacZ transgene in both developing heart and somites of the embryos. Mutation of the E box only suppressed activation in skeletal muscle precursors (somites and limb buds) but not in cardiac muscle. These data demonstrate that the MEF2 site is indispensable for the desmin enhancer function both in heart and in skeletal muscle. In addition, MEF2 cooperation with the mHLH regulators is absolutely necessary for proper transcriptional activity during embryonic skeletal muscle development. PMID- 8626011 TI - Analysis of regulatory elements of the developmentally controlled chorion s15 promoter in transgenic Drosophila. AB - The Drosophila s15 chorion gene is expressed only in the follicular epithelium surrounding the developing oocyte, with tight quantitative control and a very narrow temporal specificity. We have used germ-line transformation analysis to conduct an extensive mutational dissection of its promoter between -189 and -39 bp relative to the transcriptional start site. Quantitative control and temporal specificity are disrupted by several of these mutations. The results suggest that this 150-bp DNA region encompasses many positive and negative, at least partially degenerate, cis-regulatory elements, which are involved in specifying the highly precise expression pattern of the s15 gene during development. PMID- 8626012 TI - Synaptic integrins in developing, adult, and mutant muscle: selective association of alpha1, alpha7A, and alpha7B integrins with the neuromuscular junction. AB - Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution. PMID- 8626013 TI - The role of interleukin-4 in the regulation of mouse primordial germ cell numbers. AB - Interleukin-4 (IL-4), a pleiotropic cytokine, stimulates a dose-dependent increase in the number of mouse primordial germ cells in culture. Results from bromodeoxyuridine incorporation assays suggest that IL-4 acts as a survival factor rather than as a mitogen for primordial germ cells in this system. Studies on the embryonic expression patterns of IL-4 and its receptors, using RT-PCR and ELISA, show that IL-4 and its receptors are present at the correct time and place to influence PGC numbers in vivo. PMID- 8626014 TI - Myf-5(m1)/Myf-6(m1) compound heterozygous mouse mutants down-regulate Myf-5 expression and exert rib defects: evidence for long-range cis effects on Myf-5 transcription. AB - Myf-6 and Myf-5, two members of the family of muscle-specific regulatory genes, are located less than 10 kb apart in the mouse and human genomes. We have shown recently that homozygous mutant mice carrying a pgk-neo-cassette in the first exon of the Myf-6 gene display minor alterations of skeletal musculature but develop a severe rib defect, most likely due to a drastic down-regulation of Myf 5 expression. The mechanism by which the Myf-6 mutation affects the Myf-5 gene is unknown. In order to determine whether Myf-5 transcription is inhibited by the Myf-6 mutation in cis or in trans, we generated compound heterozygous mice carrying inactivated Myf-5 and Myf-6 alleles on different chromosomes. Here, we demonstrate that double-heterozygous mutants exhibit truncated ribs and severe depression of Myf-5 transcription, a phenotype similar to the previously described homozygous Myf-6 mutant mice. These results indicate that the Myf-6 mutation inhibits Myf-5 gene expression by a long-range cis effect. PMID- 8626015 TI - Purification of AV-1, a position-specific molecule, and the effects of its antiserum on chick limb pattern formation. AB - AV-1 protein is a membrane-localized glycoprotein which is expressed in a stage- and position-specific manner during limb development and is a candidate pattern formation molecule. In this study, we have purified the AV-1 protein using an affinity column coupled with the anti-AV-1 monoclonal antibody. Using the purified protein, we produced a rat anti-AV-1 antiserum. Characterization of the antiserum by immunoblotting and immunostaining showed highly specific reactivity which was identical to that of the anti-AV-1 monoclonal antibody. The rat anti-AV 1 antiserum was injected into the vitelline vein of stage 20 chick embryos and these embryos were allowed to develop further to examine the effect of the antibody on limb pattern. The injected embryos developed deformed limbs. Many of the abnormalities in limb cartilage pattern were related to the normal pattern of the AV-1 expression, suggesting an involvement of the AV-1 protein in the process of limb pattern formation. PMID- 8626016 TI - Tip-localized calcium entry fluctuates during pollen tube growth. AB - Studies have been conducted on the dynamics of Ca2+ entry in pollen tubes using ratiometric ion imaging to measure the intracellular gradient and an ion selective vibrating electrode to detect the extracellular influx. A steep tip focused gradient occurs in all species examined, including Lilium longiflorum, Nicotiana sylvestris, and Tradescantia virginiana. Anlaysis of Lilium pollen tubes loaded with dextran conjugated fura-2 reveals that the gradient derives from Ca2+ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. Since the apical membrane is continually swept to the flanks during tube elongation, either Ca2+ channels are specifically retained at the extreme apex or, as seems more likely, the Ca2+ channels which were active at the tip rapidly inactivate, as new ones are inserted during vesicle fusion. Ratiometric imaging further indicates that the high point of the gradient fluctuates in magnitude from 0.75 to above 3 microM, during measuring intervals of 60 sec, with the elevated points being correlated with an increased rate of tube growth. Independent analysis of the growth at 2- to 3-sec intervals reveals that the rates can fluctuate more than threefold; tubes longer than 700 mu m exhibit oscillations with a period of 23 sec, while tubes shorter than 700 mu m display erratic fluctuations. Inhibition of pollen tube growth caused by mild temperature shock or caffeine (1.5 to 3.0 mM) is correlated with the dissipation of the tip-focused gradient and the Ca2+ influx. Recovery from both treatments is denoted by a global swelling of the pollen tube tip, concomitant with a high transient entry of Ca2+ in the tip. The location of the highest Ca2+ domain within the tip region defines the point from which normal cylindrical elongation will proceed. PMID- 8626017 TI - Overexpression of the homeobox gene Xnot-2 leads to notochord formation in Xenopus. AB - Xnot-2 is a homeobox gene expressed in Spemann's organizer. Here we present evidence that microinjection of synthetic Xnot-2 mRNA leads to the formation of notochord. Microinjection into the dorsal side of the Xenopus embryo results in greatly expanded notochords. Nearby somitic and prechordal mesoderm becomes recruited into these enlarged notochords, which also affect CNS patterning. Two early genes expressed in the developing notochord, chd and XFKH-1, are activated by Xnot-2. We conclude that gain-of-function of Xnot-2 promotes notochord formation. PMID- 8626018 TI - Cellular interactions mediated by the homeotic PISTILLATA gene determine cell fate in the Arabidopsis flower. AB - Flowers develop from the coordinated division and differentiation of cells derived from the shoot apical meristem. By inducing chromosomal deletions in individual shoot apical meristem cells, we have generated Arabidopsis plants that are genetically mosaic for the homeotic PISTILLATA gene. Flowers bearing wild type PISTILLATA epidermal tissue and mutant pistillata internal tissues are phenotypically normal. Based on this non-cell-autonomy, we suggest that PISTILLATA controls the production of a substance involved in cell-cell communication between the outer and inner tissue layers of the flower. These mosaic flowers were also used to assess the relative contributions of meristematic cells to the developing floral organs. These observations indicate that meristematic cells have discrete but somewhat variable contributions to the Arabidopsis flower. We have used these results to construct a fate map of the Arabidopsis floral primordium. PMID- 8626019 TI - Xenopus laevis egg jelly coats consist of small diffusible proteins bound to a complex system of structurally stable networks composed of high-molecular-weight glycoconjugates. AB - The extracellular matrix surrounding Xenopus laevis eggs includes three morphologically distinct jelly layers designated J1, J2, and J3 from the innermost to outermost. Previously, using the quick-freeze, deep-etch, rotary shadow technique, we found that each layer has a unique fibrillogranular ultrastructure. In this study, we show that the fibrillar network is composed of high-molecular-weight glycoconjugates, while the globular material consists of low-molecular-weight proteins some of which are released into the aqueous medium. Analysis by SDS-PAGE and differential staining of individually dissected jelly layers shows that both J1 and J2 contain three high-molecular-weight, acidic, Alcian blue-straining components (450, 630, and 900 kDa), while J3 contains two high-molecular-weight components that strain with PAS but not with Alcian blue. Each jelly layer also contains low-molecular-weight proteins from 75 to 250 kDa that do not stain with PAS or Alcian blue. Chromatography of whole egg jelly on a Sephacryl 500 column resulted in isolation of the major Alcian blue staining band (630 kDa) which eluted first, and two PAS staining bands which eluted second. Rotary-shadowing demonstrated that these high-molecular-weight glycoconjugates are long and branched, suggesting that they are major constituents of the jelly fiber network. SDS-PAGE analysis shows that these networks are stable for at least 16 hr after eggs are oviposited. In contrast, the low-molecular-weight globular proteins which constitute 30% of the total jelly protein are steadily released into the surrounding medium. PMID- 8626020 TI - SpRunt-1, a new member of the runt domain family of transcription factors, is a positive regulator of the aboral ectoderm-specific CyIIIA gene in sea urchin embryos. AB - In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt 1. These results are discussed with reference to known features of the runt domain family of transcription factors. PMID- 8626021 TI - Targeted disruption in the mouse Hoxc-4 locus results in axial skeleton homeosis and malformation of the xiphoid process. AB - Hoxc-4 is a mouse homeobox gene located at the 3' end of the HoxC cluster. Of the HoxC genes, Hoxc-4 is expressed in the most anterior regions of the central nervous system and prevertebral column. To investigate its role in mouse development, we have generated Hoxc-4 mutant mice by gene targeting. Mice homozygous for the Hoxc-4 mutation are viable and fertile. Analysis of the skeletal system of homozygous mutants revealed various abnormalities in the cervical and thoracic regions. The most frequent abnormality was a partial posterior homeotic transformation of the seventh cervical vertebra. Less frequently, anterior transformations of the third and eighth thoracic vertebrae were observed. Furthermore, the xiphoid process of the sternum was malformed such that it had an aperture or a fissure. Although Hoxc-4 is expressed abundantly in the central nervous system, no obvious defects were observed. These results suggest that Hoxc-4 is required for specifying cervical and thoracic vertebral identity. PMID- 8626022 TI - inscuteable, a neural precursor gene of Drosophila, encodes a candidate for a cytoskeleton adaptor protein. AB - In Drosophila, neural precursor genes are expressed in neural progenitor cells as the neuroblasts and sense organ mother cells. These genes are thought to control the fate and/or behavior of neural progenitor cells once their fate decision has been made. We have isolated and characterized a novel neural precursor gene, inscuteable, whose expression is coincident with sites of cell shape changes or cell and tissue movement in the embryo, e.g., neuroblasts, trachea, Malphigian tubules, and in pupal wing epithelia. The Inscuteable protein is localized to the apical submembranous surface of neuroblasts and other cell types and shows certain features common to a family of putative cytoskeletal associated proteins. The inscuteable mutant phenotype, together with these other observations, suggests a possible role for the protein in cytoskeleton organization. PMID- 8626023 TI - A cell-cycle phase-associated cell-type choice mechanism monitors the cell cycle rather than using an independent timer. AB - Upon starvation, cells of the simple eukaryote Dictyostelium discoideum aggregate and differentiate into several cell types. Two main cell types are prestalk and prespore, which later usually become stalk and spore cells. The differentiation is plastic, and several factors can alter cell-type ratios. Two mechanisms have been proposed to regulate the initial cell type. We and others have proposed that cell type is initially determined by cell-cycle phase at the time of starvation: prestalk cells are derived from cells which, are the time of starvation, happen to be in a roughly 2-hr-long sector of the cell cycle which overlaps S and early G2 and that certain extracellular factors are then used to maintain the proper prestalk:prespore ratio and to control later stages of development such as the prestalk-to-stalk conversion. To examine the relationship between initial cell type choice and the cell cycle, and how this 2-hr-long sector is generated, we increased the length of S phase by mild treatments of cells with DNA-synthesis inhibitors. When the fraction of the cell cycle occupied by S phase is increased and the cells are then starved, the prestalk:prespore ratio increases. This increase was observed using two markers for prestalk cells, CP2 and ecmA::lacZ. In addition, there is a close correlation between the fraction of the cell cycle occupied by S phase and the prestalk:prespore ratio, irrespective of total cell cycle length. These results validate the hypothesis that the initial choice of cell type is determined by cell-cycle phase at the time of starvation, and indicate that the cell-type choice mechanism monitors the cell cycle rather than using an independent 2-hr-long timer started at the beginning of S phase. PMID- 8626024 TI - Dorsal mesoderm has a dual origin and forms by a novel mechanism in Hymenochirus, a relative of Xenopus. AB - The dorsal mesoderm in the frog Hymenochirus forms by a mechanism not previously described in any other vertebrate. Unlike its close relative, Xenopus laevis, in which the mesoderm derives entirely from the deep mesenchymal cells of the marginal zone, Hymenochirus has "surface mesoderm" originating in the involuting marginal zone epithelium. Fluorescently labeled grafts show stage-specific invasion of deep axial tissue by cells originally located in the surface layer. These cells participate in normal mesoderm development. In video recordings, the labeled surface area shrinks as surface cells invade the deep layer. Furthermore, the mechanism of surface mesoderm morphogenesis differs from that described in other amphibians. Scanning electron microscopy at several neurula stages indicates that prospective somite cells do not individually detach from their epithelial neighbors to ingress into the deep layer, as seen in other amphibians; instead, their basal ends adhere to the somitic mesoderm as a coherent layer, taking on somitic morphology while still a part of the archenteron lining. This novel morphogenetic process we dub "relamination." Prospective notochord cells individually spread on the ventral surface of the notochord, gradually ingressing from their epithelial neighbors, but by a mechansism involving active pulling and spreading by their invasive basal ends rather than depending on apical constriction as do the corresponding "bottle cells" in other amphibians. Lateral endoderm migrates dorsally, replacing the relaminating surface mesoderm and fusing at the dorsal midline of the archenteron. These processes demonstrate the diversity of morphogenesis at the cellular, and presumably the molecular, level and shed light on the evolution of morphogenetic mechanisms. PMID- 8626025 TI - The activity of D-raf in torso signal transduction is altered by serine substitution, N-terminal deletion, and membrane targeting. AB - The Raf family of serine/threonine kinases are essential components in many receptor tyrosine kinase-mediated signal transduction pathways. Here, we analyze the function of D-raf in the Torso (Tor) pathway required to specify cellular fates at the embryonic poles. Using mutant embryos lacking endogenous D-raf protein, we show that D-raf's serine/threonine kinase activity is essential for its role in Tor signal transduction and that human Raf-1 will substitute for D raf in this pathway. After Tor activation, D-raf becomes hyperphosphorylated. We identified two putative serine phosphorylation sites (S388 and S743) in SF9 cells and demonstrate that S743 or its phosphorylation is essential for D-raf function in embryos. Alanine substitution at S388, N-terminal truncation, or targeted membrane association permits transmission of the Torso signal by D-raf, but these D-raf molecules differ in their rescuing potential and relative biological activity. Membrane-targeted D-raftor4021 showed the highest level of activity, followed by alanine-substituted D-rafS388A and N-terminal-truncated D-raf delta 445. Since the activity profiles for these altered forms of D-raf are distinct, these findings indicate that each structural modification differentially affects the regulation and/or propagation of the Tor signal by these mutant D-raf proteins. PMID- 8626026 TI - Identification and characterization of a hunchback orthologue, Lzf2, and its expression during leech embryogenesis. AB - Lzf1 and Lzf2 are leech zinc finger (Lzf) genes that are shown to be orthologues to the Drosophila gap gene hunchback (hb). Neither in situ hybridization nor RT PCR detected Lzf1 transcripts in leech embryos or adults. Lzf2 expression was examined in leech embryos at various stages by in situ hybridization. Lzf2 is expressed continually throughout the early embryonic cleavage divisions, including the period during which the embryo forms stem cells that will give rise to the segmented tissues of the adult. At the time of segmental pattern formation, Lzf2 RNA is expressed uniformly along the length of the segmented trunk in both the ectodermal and mesodermal tissues. This is in contrast to the anteriorly restricted gradient of hb RNA shown to be critical to the normal anteroposterior (AP) patterning of the insect embryo. Thus, this leech orthologue of hb does not appear to play a comparable role in the patterning of the AP axis. In addition, Lzf2 is expressed during organogenesis in segmentally restricted patterns in the central nervous system, the gut, and epidermally derived structures. Lzf2 is the first hb orthologue to be characterized in detail outside of insects and its expression pattern suggests that hb may have acquired a gap gene function in arthropods or insects after their phyletic separation from the annelids. PMID- 8626027 TI - Cell-cell adhesion prevents mutant cells lacking myosin II from penetrating aggregation streams of Dictyostelium. AB - When a small number of fluorescently labeled myosin II mutant cells (mhcA-) are mixed with wild-type cells and development of the chimeras is observed by confocal microscopy, the mutant cells are localized to the edges of aggregation streams and mounds. Moreover, the mutant cells stick to wild-type cells and become distorted (Shelden and Knecht, 1995). Two independent adhesion mechanisms, Contact Sites A and Contact Sites B, function during the aggregation stage and either one or both might be responsible for excluding the myosin II null cells. We have mixed mhcA- cells with cells in which the appearance of Contact Sites B is delayed (strain TL72) as well as cells which lack Contact Sites A (strain GT10) and double mutants in which both adhesion mechanisms are affected (strain TL73). In all chimeras, the mhcA- cells were distorted by interactions with the adhesion mutant cells, indicating that it does not require significant adhesive interaction to distort the flaccid cortex of mhcA- cells mhcA- cells were excluded from streams composed of cells lacking either Contact Sites A or Contact Sites B but mixed randomly with cells lacking both adhesion systems. By 10 hr of development, cells of strain TL73 acquire Contact Sites B adhesion. If cells of this strain were mixed with labeled mhcA- cells, allowed to develop for 9 hr, and then dissociated before replating, the myosin II null cells were seen to be distorted and excluded from the reaggregates. Thus, the exclusion of mhcA- cells from streams can be accomplished by either Contact Sites A or B. When chimeras of labeled TL73 and wild-type cells were made, the TL73 cells were found to be randomly mixed into aggregation streams. This result indicates that adhesive sorting does not function during aggregation and so cannot account for the exclusion of mhcA- cells from streams. We hypothesize that the flaccid cortex of mhcA- cells cannot generate sufficient protrusive force to break the contacts between adhered cells in aggregation streams but can enter streams where the cells are weakly adherent. PMID- 8626028 TI - Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene. AB - Transforming growth factor-beta 1 (TGF-beta 1) influences the morphogenesis of many organs, regulating cell growth, differentiation, gene expression, extracellular matrix deposition, and angiogenesis. In order to assess the effects of TGF-beta 1 on lung development in vivo, transgenic mice were generated bearing a chimeric gene composed of human surfactant protein C (SP-C) gene promoter and the porcine TGF-beta 1 cDNA mutated to ensure constitutive activation of the TGF beta 1 peptide. Because of the perinatal loss related to the SP-C-TGF-beta 1 transgene, embryos bearing the transgene were obtained on Days 16 and 18.5 of gestation. TGF-beta 1 was selectively expressed in respiratory epithelial cells of the transgenic embryos. Body weight, length, and lung size were not altered in the transgenic embryos; however, lung morphogenesis of Day 18.5 transgenic mice was arrested in a late pseudoglandular stage of development, while that of their nontransgenic littermates was typical of the saccular stage. Lungs of transgenic mice on Day 16 contained fewer acinar buds than those of nontransgenic littermates. At both ages, epithelial cell differentiation, assessed by the expression of Clara cell secretory protein2 and pro-SP-C, was inhibited. While collagen III deposition was not affected by the transgene, collagen I expression was persistent in terminal airways of fd 18.5 transgenic lungs. The distribution of alpha-smooth muscle actin was markedly altered, being detected in the mesenchyme surrounding the distal leading edges of epithelial tubules in the SP-C TGF-beta 1 transgenic mice. Expression of TGF-beta 1 in the developing respiratory epithelium of transgenic mice arrested lung sacculation and epithelial cell differentiation in vivo, supporting the role of TGF-beta family members in lung morphogenesis and differentiation. PMID- 8626029 TI - Citral, an inhibitor of retinoic acid synthesis, modifies chick limb development. AB - Exogenously applied retinoic acid (RA) is known to affect cartilage pattern in developing and regenerating limbs. There are, however, few reports which analyze the participation of endogenous RA in limb pattern formation. Using an organ culture system, we attempted to reduce the concentration of endogenous RA in the developing chick wing buds by the treatment with citral (3,7-dimethyl-2,6 octadienal), an inhibitor of retinoic acid formation. After this treatment, the cultured wing buds were grafted to the stumps of host embryos. These citral treated limb buds frequently formed truncated cartilage elements and the defects were rescued by simultaneous treatment with an appropriate concentration of RA. These results suggest that endogenous RA plays a role in chick limb bud development. PMID- 8626030 TI - Epidermal fate map of the Arabidopsis shoot meristem. AB - The shoot meristem generates all of the aerial structures of an adult plant. It is organized in three layers which produce the epidermis (L1 layer) and the subepidermal layers (L2 and L3). The origin of adult structures has previously been fate mapped to the primary meristem for L2 and L3 tissues. In this work we constructed a fate map of L1 cells in the embryonic shoot meristem. Using the trichome mutation stichel as an epidermal marker, we analyzed 153 plants that included 178 sectors. Sectors on early leaves were found to be smaller and occurred more frequently than those on late leaves. Sectors on late leaves also appeared often to affect more than one leaf. In general, the width and extent of sectors were found to be variable rather than cell lineage-restricted. Our analysis allowed us to assign the most likely fates of L1 precursor cells within the embryonic shoot meristem. The results suggest that the meristem integrates growth dynamics and patterning of all three tissues. In contrast to this coordinated growth behavior of meristematic cells, we found a difference in the lineage restrictions between the L1 and the L2 for the formation of axillary buds. PMID- 8626031 TI - Overexpression of Xgsk-3 disrupts anterior ectodermal patterning in Xenopus. AB - The Xenopus homolog of glycogen synthase kinase-3, Xgsk-3, plays a major role in regulating the formation of the dorsal-ventral axis, most likely through effects on the mesoderm. To determine whether Xgsk-3 is involved in ectodermal patterning, Xgsk-3 was ectopically overexpressed in the presumptive ectoderm. This approach resulted in a dramatically expanded cement gland, which is due to early changes in cement gland specification at the anterior end of the embryo. Explant experiments were used to show that Xgsk-3 overexpression enhances the response of ectoderm to cement-gland-inducing signals from the mesoderm and to the intercellular signaling factor noggin. Expression of two other noggin inducible genes, Xotx2 and XANF-2, was also expanded in whole embryos, while the expression of the epidermal marker, Xgbx-2, was eliminated. These results suggest that Xgsk-3 may play a role in anterior ectodermal patterning as a component of an intracellular pathway that regulates the ectodermal responsiveness to endogenous inducing signals. PMID- 8626032 TI - Pigment patterns of larval salamanders (Ambystomatidae, Salamandridae): the role of the lateral line sensory system and the evolution of pattern-forming mechanisms. AB - In many species of salamanders, pigment cells derived from the neural crest give rise to a horizontal stripe pattern in hatchling larvae. A defining element of these horizontal stripe patterns is a region over the middle of the myotomes that is relatively free of melanophores. This study shows that formation of a "melanophore-free region" and horizontal stripe pattern in Ambystoma tigrinum tigrinum (family Ambystomatidae) correlates with the development of the trunk lateral line sensory system. Moreover, prevention of lateral line development results in greater densities of melanophores in the middle of the flank, essentially eliminating the melanophore-free region in this taxon. A phylogenetic survey also revealed that ablation of the lateral lines has qualitatively similar effects on melanophores in seven of eight additional taxa (Ambystomatidae: A. barbouri, A. maculatum, A. talpoideum; Salamandridae: Notophthalmus viridescens, Pleurodeles waltl, Taricha granulosa, T. rivularis). In Taricha torosa, however, a superficially similar melanophore-free region forms prior to lateral line development, and ablation of the lateral lines does not perturb the horizontal stripe pattern. Finally, heterospecific grafting experiments demonstrated that T. torosa lateral lines are competent to generate a melanophore-free region, and T. torosa melanophores are competent to respond to cues associated with the lateral lines. These results indicate that lateral line-dependent pattern-forming mechanisms are common and probably ancestral within the families Ambystomatidae and Salamandridae and suggest that these ancestral mechanisms have been retained in T. torosa as redundant, lateral line-dependent mechanisms for stripe formation have evolved. PMID- 8626033 TI - When neural crest and placodes collide: interactions between melanophores and the lateral lines that generate stripes in the salamander Ambystoma tigrinum tigrinum (Ambystomatidae). AB - A prominent element of the early larval pigment pattern in the salamander Ambystoma tigrinum tigrinum (family Ambystomatidae) is a horizontal stripe over the lateral surface of the myotomes where otherwise abundant, neural crest derived melanophores are not found. This study examines the formation of this "melanophore-free region". When the trunk lateral lines were ablated (by removing cranial lateral line placodes), the melanophore-free region did not form; instead, melanophores populated the middle of the flank and the distribution of yellow, neural chest-derived zanthophores was perturbed. Time-lapse videomicrography demonstrated that during normal development, the melanophore free region is established because melanophores retreat from the midbody lateral line primordium as it migrates caudally along the inner side of the epidermis. Melanophores do not repopulate the middle of the flank after primordium migration and heterochronic grafting experiments suggest that extracellular factors contribute to maintaining the melanophore-free region during these later stages. Finally, photographic series, microsurgical manipulations, electron microscopy, and staining for molecules of the extracellular matrix (peanut agglutinin-binding components, tenascin, chondroitin sulfate proteoglycans, fibronectin, laminin) suggest that several factors contribute to establishing and maintaining the melanophore-free region, including steric effects of the lateral lines, interactions between melanophores and xanthophores, lateral line-dependent alterations of the subepidermal basement membrane, and a general elaboration of the extracellular matrix. Lateral line effects on melanophores are inferred to be a shared, ancestral feature of pigment pattern development for the families Ambystomatidae and Salamandridae (D.M. Parichy, Dev. Biol. 174, 265-282. 1996). The results of this study thus provide insights into a phylogenetically primitive mechanism for stripe formation, and a context for interpreting evolutionary innovations in pattern-forming mechanisms. PMID- 8626034 TI - Deletion of integrin alpha 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion. AB - Integrin alpha 1 is a receptor for laminin and collagen which is expressed widely and dynamically in embryogenesis and has been implicated in various developmental processes including establishment of the placenta and formation of the central and peripheral nervous system. In the adult it is the sole collagen receptor in smooth muscle and liver and is thought to be important for the stability of these tissues. We have generated a null allele of the alpha 1 gene in the germline of mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutation are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development. Embryonic fibroblasts derived from mutant animals are unable to spread on or migrate into substrata of collagen IV and are deficient in spreading on and migrating into laminin. Further in vitro analysis of cell spreading and migration suggests that alpha 1 beta 1 is not required for binding to collagen I and implicates a third receptor, possibly integrin alpha 3 beta 1, in collagen I binding. PMID- 8626035 TI - Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo. AB - Previous studies on the regulation of the segmentation gene even-skipped (eve) have centered on the transcription of stripe 2. Here, we characterize another enhancer module contained within the complex eve promoter that directs expression of stripes 3 and 7. This enhancer is approximately 500 bp in length and maps approximately 3.3 kb upstream of the transcription start site. The stripe 3 + 7 enhancer appears to be regulated by one or more ubiquitously distributed activators, including components of a JAK-Stat pathway. The two-stripe pattern results via multiple tiers of repressors which delimit this ubiquitous activation. The zinc finger repressor hunchback appears to be responsible for establishing the anterior border of stripe 3 and the posterior border of stripe 7. knirps, a member of the nuclear receptor family of transcription factors, appears to establish the posterior border of stripe 3 and the anterior border of stripe 7. Activator and repressor proteins bind in vitro to several sites within the enhancer. These findings suggest a general model for the regulation of segmentation stripes, whereby enhancers integrate positional information provided by broadly distributed activators and spatially restricted repressors. PMID- 8626036 TI - Changes in the segmental pattern of sensory neuron projections in the chick hindlimb under conditions of altered cell adhesion molecule function. AB - In the developing chick hindlimb, pathfinding by sensory axons is affected by their interactions with other axons entering the limb. Cell adhesion molecules (CAMs) expressed on the growing axons are likely to influence these interactions. Accordingly, to elucidate how CAMs affect sensory axon pathfinding, we injected antibodies that block the functions of NCAM, G4/L1, or N-cadherin into the hindlimb, starting at St. 25, when all motoneuron axons but only a few sensory axons had entered the plexus. In each case, the segmental pattern of projections was assessed 2-3 days later by retrogradely labeling individual peripheral nerves. With all perturbations, the gross anatomical nerve pattern developed normally; that is, some sensory axons formed cutaneous nerves while other sensory axons projected to muscles. However, the segmental pattern of sensory projections was changed when either G4/L1 or N-cadherin function was blocked in that fewer sensory axons crossed the anterior-posterior axis of the plexus. A likely reason for this effect is that anti-G4/L1 and anti-N-cadherin each decreased the amount of fasciculation and that sensory axons are less able to travel across the plexus when they are defasciculated. Anti-G4/L1 affected both cutaneous and muscle sensory projections while anti-N-cadherin affected cutaneous but not muscle sensory projections, in accord with known differences in the expression of these two CAMs on sensory and motoneuron axons. Although anti-NCAM did not appear to alter sensory projections, when polysialic acid (PSA) was enzymatically removed from NCAM, there was a marked increase in cutaneous projections from the most proximate DRG, although muscle sensory projections were unchanged. PSA removal may cause an increase in fasciculation that forces sensory axons to track along neighboring axons. Thus, without PSA, cutaneous axons project more in accord with the relative anterior-posterior positions they had as they entered the plexus. Taken together, these studies suggest that axonal fasciculation mediated by CAMs and regulated by PSA influences the ability of sensory growth cones to navigate through the plexus and project along the correct peripheral nerves. PMID- 8626037 TI - A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody. AB - Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation. PMID- 8626038 TI - Retinoic acid induces stage-specific repatterning of the rostral central nervous system. AB - We had previously reported that in gastrulating mouse embryos retinoic acid (RA) induces morphological as well molecular alterations strictly depending on the time of administration. In particular, embryos treated with RA at the mid-late streak stage share reduction of the rostral central nervous system (CNS) and increase of the hindbrain. In the same embryos, loss of the forebrain-expressed genes, such as Emx1, Emx2, and Dlx1, and rostral ectopic expression of the Hoxb-1 gene suggest an antero-posterior (A/P) ordered repatterning of the fore-, mid-, and hindbrain regions. Several genes, such as Pax-2, Wnt-1, En-2, and En-1, are involved in the establishment of midbrain and rostral hindbrain regional identities and boundaries. We report that these genes become coordinately anteriorized only in embryos treated with RA at the late streak stage. Moreover, in the hindbrain of the same embryos, at 8.5 days post coitum (dpc), Wnt-1 and Pax-2 are rostrally induced all along the neural plate. Considering that forebrain markers are repressed in embryos treated with RA at the same time, these findings strongly support the idea that RA administration at the late streak stage induces an ordered repatterning of the rostral CNS, possibly altering the A/P nature of mesendodermal inductive signals. PMID- 8626039 TI - Regulation of nuclear envelope assembly/disassembly by MAP kinase. AB - Mouse eggs arrested in metaphase II display high levels of cdc2/cyclin B1 and MAP protein kinase activities. Following fertilization there is a time-dependent decrease in the activity of each of these protein kinases. The decline in cdc2/cyclin B1 protein kinase correlates with the resumption of meiosis and the emission of the second polar body and precedes the decline in MAP kinase activity, which correlates temporally with the formation of the male and female pronuclear envelopes. These results suggest that high levels of MAP kinase activity are incompatible with the presence of a pronuclear envelope. To test this possibility, we expressed in mouse eggs a constitutively active form of MAP kinase kinase (MEK) whose only known target is p42/p44 MAP kinase. We show that following fertilization cdc2/cyclin B1 kinase activity declines and a second polar body is emitted. The endogenous MAP kinase remains active, however, and no pronuclear envelopes form. Thus, high levels of MAP kinase activity by itself in mouse eggs appear incompatible with the presence of a pronuclear envelope. PMID- 8626040 TI - Cardiovascular lesions and skeletal myopathy in mice lacking desmin. AB - In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues. PMID- 8626041 TI - Anatomical heterogeneity of the intestinal mucosa and cholesterol homeostasis. A review of studies with normal and genetically hypercholesterolemic (RICO) Rat. AB - Quantitative data on cholesterol movements in mucosa cell as a function of its localization in the small intestine have been obtained in normocholesterolemic (SW) or genetically hypercholesterolemic (RICO) rats. Bile cholesterol absorption is greater and more proximal than dietary cholesterol absorption, both taking place mainly in the top cells of the duodenum or the proximal jejunum. Esterification of cholesterol also takes place mainly in the villus cells, while cholesterol synthesis is predominantly carried out in the crypt cells of the proximal duodenum and distal ileum. Cholesterol exchanges, which replace half of the cell cholesterol through the cell life, can be estimated at 3-4%.h-1 between plasma and mucosa cells, according to its location, i.e. 12-25 micrograms.h-1 (per mg cell DNA). In comparison, the cholesterol HDL or LDL uptake appears to be very low (0.02-0.06 and 0.2-0.6 microgram.h-1, respectively). Compartmentalization of cholesterol metabolism in the enterocyte can be suggested by different experimental data. The turnover of newly synthesized cholesterol is about 2-fold lower than that of exogenous (dietary) cholesterol. PMID- 8626042 TI - Elevated liver isoenzymes of alkaline phosphatase and disease activity in patients with Crohn's disease. AB - Serum alkaline phosphatase (ALP) and its isoenzymes - liver (L-ALP), fast liver (F-ALP; also called high-molecular-weight ALP), bone (B-ALP) and intestinal (I ALP) - were measured by agarose electrophoresis after pretreatment of sera with neuraminidase in 100 patients with Crohn's disease (CD) and 100 age-matched healthy controls. Total ALP was increased in 25 patients whereas the isoenzymes F ALP, L-ALP, B-ALP, I-ALP were increased in 64, 84, 16 and 8%, respectively. But also in patients with CD and with normal ALP the isoenzymes were high in 39, 15, 1 and 1%, respectively. Total ALP, L-ALP and F-ALP correlated with gamma glutamyltransferase (GGT) (tau = 0.22, 0.20 and 0.26, respectively, p < 0.05), but F-ALP also correlated with parameters of disease activity such as Crohn's disease activity index (CDAI), orosomucoid (OM) and erythrocyte sedimentation rate (tau = 0.28, 0.23 and 0.37, respectively, p < 0.001). In 38 patients, ALP and isoenzymes were controlled within 6 months. In these patients there were correlations between changes of total ALP and F-ALP with changes of inflammatory activity such as CDAI (tau = 0.31, p = 0.07 and tau = 0.30, p = 0.01) or OM (tau = 0.26 and 0.25, p < 0.05). Therefore, ALP seems to be influenced by inflammatory activity mainly via F-ALP which could be a marker of disease activity. PMID- 8626043 TI - Changes in intestinal alpha-methyl-D-glucoside uptake due to pregnancy and lactation in rats. AB - Uptake of alpha-methyl-D-glucoside, a nonmetabolizable glucose analogue, by everted intestinal sleeves was studied in virgin, pregnant and lactating rats. The animals showed an increase in jejunal and ileal tissue mass, mucosal mass, nominal surface area, and enzymatic activities. No changes were observed in the carrier affinity throughout the breeding stages. Nevertheless there was a significant increase in glucose carrier density (Vmax) per unit of length of jejunum and ileum in pregnant and lactating animals. Integrating the results obtained, and increased overall ability to transport hexoses by nonspecific adaptation can be observed in breeding stages. PMID- 8626044 TI - Metabolism and influence of glycine-extended gastrin on gastric acid secretion in man. AB - Glycine-extracted gastrins are the immediate precursors of the bioactive carboxyamidated gastrins. The effect on gastric acid secretion and the pharmacokinetics of glycine-extended gastrin-17 were studied in 8 normal subjects. The elimination in plasma after bolus injection was biexponential, the half-lives being 4.1 +/- 0.2 and 21.8 +/- 0.9 min, and clearance and apparent volume of distribution being 7.9 +/- 0.6 ml/kg/min and 69.5 +/- 2.7 ml/kg, respectively. Infusion of the peptide at three consecutive dose rates did not stimulate gastric acid secretion, although plasma concentrations reached supraphysiological levels. Nor did glycine-extended gastrin-17 influence submaximal acid secretion induced by amidated gastrin-17. In contrast to amidated gastrins, the concentration of glycine-extended gastrins in peripheral venous plasma did not increase significantly after a meal. The postprandial rise in amidated gastrin was unaffected by concomitant infusion of glycine-extended gastrin-17. A reduction in glycine-extended gastrin-17 concentrations in plasma during constant-rate infusion of the peptide was observed after a protein meal (p < 0.05). This reduction was reflected by an increase in glycine-extended gastrin 17 is without immediate effect on gastric output in man. The postprandial increase in clearance might be due to increased splanchnic blood flow with subsequently increased peptide elimination. PMID- 8626045 TI - Carbon-14-urea breath test as a noninvasive method to monitor Helicobacter felis colonization in mice. AB - We have developed a 14C-urea breath test to follow the course of Helicobacter felis infection in mice. Peak 14CO2 production occurred approximately 8 min after substrate administration. The test values were compared to those from a rapid urease test and correlated with the presence of pathogens by histology. The sensitivity was 99%, specificity 91%, positive predictive value 95% and negative predictive value 99% when the assay was conducted in fasted mice. We conclude that in mice the breath test analysis is a useful noninvasive method for detecting the presence of H. felis or for evaluating therapeutic agents affecting growth or survival of the organism. PMID- 8626046 TI - Effect of teprenone on portal hypertensive gastric mucosa. AB - Teprenone (geranylgeranylacetone) is a gastric mucosal protective drug used clinically in Japan for treatment of gastric ulcers and gastritis. Its effect on portal hypertensive (PHT) gastric mucosa which has impaired defensive mechanisms is not known. In 20 PHT and 20 sham-operated rats, we studied the effects of teprenone or placebo on: (1) portal pressure; (2) gastric pH; (3) gastric mucosal blood flow using laser doppler flowmetry, and (4) hexosamine content in gastric mucosa. The gastric mucosal blood flow was significantly higher in the PHT + teprenone group than in the PHT + placebo group (463 +/- 75 and 381 +/- 82 perfusion units, respectively; p < 0.05). The hexosamine content was significantly lower in PHT rats than in sham-operated controls (12.6 +/- 2.3 vs. 14.3 +/- 2.2 micrograms/mg, respectively). Teprenone treatment significantly increased the gastric mucosal hexosamine concentration in both sham-operated and PHT rats (17.2 +/- 3.1 and 15.6 +/- 3.6 micrograms/mg, respectively). These effects of teprenone, combined with its known prostaglandin-stimulating action, suggest a potential role for this agent in the treatment of PHT gastric mucosal abnormalities. PMID- 8626047 TI - Prostaglandin-induced protection of cultured rat gastric cells against ethanol is inhibited by a microtubule inhibitor. AB - Prostaglandins reduce damage by noxious agents to isolated gastric cells. The mechanism remains unexplained. This study was done to examine involvement of microtubules with prostaglandin-mediated cell protection. Cultured mucus producing cells of rat gastric mucosa were used. Cultured cells were incubated with 16, 16-dimethyl-prostaglandin E2 (dmPGE2), colchicine, or vehicle alone in calcium-containing (1.3 mM) and calcium-free medium. The cells were then exposed to 8% ethanol. Cell integrity and damage were assessed by 51Cr release and in part by transmission electron microscopy (TEM). Specific 51Cr release was closely related to percentage change of necrotic cells assess by TEM. Cell damage caused by 8% ethanol was greater in medium-containing calcium than in calcium-free medium. dmPGE2 at 10(-5) M inhibited cell damage in calcium-containing medium. Colchicine itself at concentrations up to 10(-5) M did not affect cell integrity, but at 10(-5) M concentration potentiated damage caused by 8% ethanol in calcium containing medium. Pretreatment with 10(-5) M colchicine abolished the protective effect of dmPGE2. Microtubules may mediate protection by dmPGE2 of cultured gastric cells against ethanol damage. PMID- 8626048 TI - Effect of 10% ethanol and sofalcone on prostaglandin E2 content, mucus gel thickness, and experimental ulcers in the stomach of developing rats. AB - We studied the effects of a mild irritant, 10% ethanol, and sofalcone on the gastric mucosal defense mechanisms in newborn rats, comparing the effects to those seen in adult rats. The results indicated (1) that both mucosal prostaglandin E2 (PGE2) content and mucus gel layer thickness increased with age, (2) that sofalcone, but not 10% ethanol, increased mucosal PGE2 content and mucus gel thickness in 1- and 8-week-old rats, (3) that both sofalcone and 10% ethanol decreased mucosal damage induced by 50 or 75% ethanol in both age groups, and (4) that 10% ethanol, but not sofalcone, decreased ethanol-induced mucosal damage in rats pretreated with indomethacin. We concluded that 10% ethanol and sofalcone increase gastric mucosal defence mechanisms in newborn rats as in older rats. PMID- 8626049 TI - Adaptive gastric mucosal cytoprotection in rats: different modes of action by three mild irritants. AB - The mechanisms of adaptive mucosal cytoprotection by mild irritants were investigated in rats. In an ex vivo chamber preparation, application of 20% ethanol, 5% NaCl or 0.3 M HCl to the posterior side of the mucosa significantly protected that side of the stomach against mucosal damage caused by subsequent exposure to 100% ethanol, with contralateral transmission of protection to the anterior side by 20% ethanol and 0.3 M HCl. Atropine or lidocaine significantly reversed the cytoprotection of 20% ethanol. Bilateral vagotomy partially prevented the antilesion action of 20% ethanol, and completely prevented the action of 0.3 M HCl. However, the three mild irritants did not affect gastric mucosal blood flow, but restored the ion transport mechanism which was depressed by ethanol. It is therefore concluded that the three mild irritants have their own distinctive cytoprotective mechanisms against ethanol ulceration, which is predominantly not mediated by effects on the vascular system of the gastric mucosa. PMID- 8626050 TI - Effects of chronic nitric oxide synthase inhibition in cold-restraint and ethanol induced gastric mucosal damage in rats. AB - Gastric actions of Nw-nitro-1-arginine methyl ester (L-NAME) were investigated in rats, as this agent is a reliable nitric oxide synthase inhibitor L-NAME solutions were placed in subcutaneous osmotic minipumps which continuously released L-NAME at 0.1, 1.0, 10, or 40 mg/kg/day. L-NAME dose and time dependently enhanced stress-induced gastric ulceration but did not affect mucosal mast cell population. Ulcerogenic actions of L-NAME were reversed by L-arginine but not by D-arginine. Ten L-NAME treatment also enhanced the ethanol-induced gastric mucosal damage, depressed gastric mucosal blood flow but did not alter gastric mucus, secretory volume, or acid output. It is concluded that in the present models, chronic nitric oxide synthase inhibition enhanced ulcerogenesis by decreasing mucosal resistance due to reduced mucosal blood perfusion. This implicates nitric oxide as a mucosal defense factor which acts in part by maintaining mucosal blood flow. PMID- 8626051 TI - Cholecystokinin increases small intestinal motility and reduces enteric bacterial overgrowth and translocation in rats with surgically induced acute liver failure. AB - Enteric bacterial translocation into extraintestinal sites has been proposed as a potential route for bacterial infection in acute liver failure. Bacterial overgrowth in the intestine plays an important role in the etiology of bacterial translocation from the gut. The aim of the present study was to evaluate the influence of exogenous cholecystokinin (CCK) on intestinal transit time, enteric bacterial overgrowth and translocation in experimental acute liver failure induced by 90% hepatectomy. A delayed intestinal transit time was noted in animals subjected by subtotal hepatectomy after 1 h, followed by enteric bacterial overgrowth and translocation into the systemic circulation, mesenteric lymph nodes, and systemic organs at 2 and 4 h. Intravenous infusion of CCK stimulated intestinal transit time in animals with sham operation and subtotal hepatectomy, though a restoration to sham levels could not be obtained in the later. Moreover, enteric bacterial overgrowth and translocation were prevented in hepatectomized animals. The present data imply that impaired intestinal motility, followed by enteric bacterial overgrowth and translocation in experimental acute liver failure, could be prevented by CCK infusion. PMID- 8626052 TI - Secondary peristaltic contractions, like primary peristalsis, are preceded by inhibition in the human esophageal body. AB - Swallow-induced esophageal peristalsis is preceded by a wave of inhibition in the esophageal body. The aim of this study was to determine whether a similar wave of inhibition precedes secondary peristalsis. Primary and secondary peristalsis were studied in 6 healthy subjects. Inhibition was visualized as relaxation of an artificial high-pressure zone which was created in the esophageal body by the inflation of a small intraesophageal balloon. Secondary peristaltic contractions induced by injection of 10 ml of air in the proximal esophagus or secondary peristaltic contractions spontaneously occurring after repeated swallowing were preceded by inhibition similar to that observed in primary peristalsis (96.4 +/- 2.8%, 93.2 +/- 4.2% and 91.5 +/- 3.3%) respectively. It is concluded that physiologically triggered secondary peristaltic contractions are preceded by inhibition in the esophageal body. PMID- 8626053 TI - The Rz1 gene product of bacteriophage lambda is a lipoprotein localized in the outer membrane of Escherichia coli. AB - The Rz1 gene of bacteriophage lambda is located within the Rz1 lysis gene. It codes for the 6.5-kDa prolipoprotein (Rz1) which undergoes N-terminal signal sequence cleavage and post-translational lipid modification of the N-terminal Cys of the mature protein. Globomycin, the antibiotic which inhibits bacterial signal peptidase II, specific for prolipoproteins containing diacylglyceryl cysteine [Hayashi and Wu, J. Bioenerg. Biomembr. 22 (1990) 451-471] inhibits the N terminal sequence cleavage of the Rz1 precursor. The mature protein is rich in Pro, which constitutes 25% of its amino acids (aa). Using a computer-predicted, synthetic, 15-aa antigenic determinant of Rz1 polyclonal anti-Rz[46-60] antibodies, were obtained, and employed to localize Rz1 in bacterial fractions. In induced Escherichia coli lambda lysogens Rz1 was found almost exclusively in the outer membrane (OM). In a strain overproducing Rz1 from the pSB54 plasmid, it was distributed in all the fractions, OM, fraction A and inner membrane (IM). Expression of Rz1 from the pSB54 caused enlargement of fraction A, corresponding to the adhesion sites of OM and IM. Such an enlargement was previously observed in induced lambda lysogens, shortly before the onset of lysis. PMID- 8626054 TI - A gene-sized DNA molecule encoding heat-shock protein 70 in Oxytricha nova. AB - The gene-sized DNA molecule in the macronucleus (mac) of Oxytricha nova (On) encoding heat-shock protein 70 (Hsp70) was cloned and sequenced. It contains 2654 bp, including telomeres. It consists of a 394-bp A+T-rich 5' leader, a 1956-bp open reading frame (ORF) encoding a putative polypeptide of 651 amino acids (aa), and a 240-bp A+T-rich 3' trailer. The 5' leader contains two copies of the 13-bp heat-shock element (HSE) consensus sequence of other eukaryotes. On responds to heat and CdCl2 stress with a major increase in hsp70 transcripts. The presence of HSE in the nucleotide sequence of hsp70 and the stress-induced in hsp70 transcripts are the first evidence of conventional transcriptional regulation of a hypotrich gene-size DNA molecule. The ORF has large putative ATPase and polypeptide-binding domains with high aa identity to Hsp70 polypeptides of other eukaryotes. PMID- 8626055 TI - Two different macronuclear EF-1 alpha-encoding genes of the ciliate Euplotes crassus are very dissimilar in their sequences, copy numbers and transcriptional activities. AB - Genes (EFA) encoding the translation elongation factor EF-1 alpha (EFA) or the prokaryotic homolog EFTu frequently occur in multiple copies in the same organism. This has been interpreted either in terms of a potential of differential gene expression during different phases of development, or as gene dosage adaptation to the need of high-level production of the gene products. Since ciliates can differentially amplify their genes, the latter argument would lead to the expectation of only one EFA gene in the macronucleus. However, we have found two such genes which strongly differ in both copy number and codon usage. Both transcripts are detectable at very different levels. The expression of the genes takes place both in the vegetative and sexual phases, i.e.,during conjugation. PMID- 8626056 TI - The iron-containing superoxide dismutase-encoding gene from Chlamydomonas reinhardtii obtained by direct and inverse PCR. AB - The complete sequence of the Fe2+ -containing superoxide dismutase (Fe-SOD) encoding gene of Chlamydomonas reinhardtii was determined from genomic DNA by use of the direct (PCR) and inverse PCR (IPCR). It was confirmed by reverse transcription-PCR. Primers employed in the PCR represented oligodeoxyribonucleotides corresponding to conserved amino acid (aa) residues of Fe-SOD. From the sequence of direct PCR product, primers were designed for IPCR. All amplified fragments were cloned and sequenced. Analysis of Fe-SOD reveals that the open reading frame consists of 234 aa which show a high degree of homology to other Fe-SOD. The first 33 aa are predominantly either hydrophobic or basic, and may serve as the signal peptide for this chloroplastic protein. Potential transcription start points and eukaryotic control elements, as well as a polyadenylation site, were identified. PMID- 8626058 TI - Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31. AB - Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli sigma 70 and Bacillus subtilis sigma 43 showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding. PMID- 8626057 TI - Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii. AB - The genomic and complementary DNA sequences were determined for the unique actin encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin encoding gene has one intron in the 5'-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5'-upstream region contains four short stretches of sequence similar to the so-called 'tub box', a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism. PMID- 8626059 TI - Sequence and expression of an isocitrate dehydrogenase-encoding gene from a polycyclic aromatic hydrocarbon oxidizer, Sphingomonas yanoikuyae B1. AB - An 18.5-kb DNA fragment was cloned from Sphingomonas yanoikuyae (Sy) B1 (previously Beijerinckia B1). Analysis of a 4.3-kb sequence revealed an isocitrate dehydrogenase (Idh)-encoding gene (idhA), an unidentified open reading frame (ORF) and a partial glucosamine synthetase-encoding ORF (glmS). As in a number of bacteria, Tn7 insertion was found specifically at a site past the stop codon of glmS. The predicted 406-amino-acid sequence of IdhA shows, for the first time, an extensive sequence identity (66%) with an eukaryotic NADP+-specific Idh. The idhA gene was expressed in Escherichia coli. Identical restriction fragments carrying idhA were found in B1, Sy IFO15102 and Sy Q1 (formerly S. paucimobilis Q1), indicating a well-conserved idh gene. PMID- 8626061 TI - Creating seamless junctions independent of restriction sites in PCR cloning. AB - A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event. PMID- 8626060 TI - Negative transcriptional regulation of PH081 expression in Saccharomyces cerevisiae. AB - The PH081 gene product functions as an inhibitor of the cyclin-cyclin-dependent kinase pair, Pho80-Pho85, and is required for derepression of acid phosphatase encoding gene (PH05) expression. PH081 is the only known regulator of this system whose transcriptional expression is regulated by the level of inorganic phosphate. This effect is mediated by the gene products of the PH04 and PH02 (BAS2, GRF10) genes which act as transcription factors. Fine structural analysis of the PH081 promoter region has revealed the existence of a negative regulatory sequence (NRS). That is, removal of this element causes an approx. 4-fold increase in PH081 expression. The NRS functions in either orientation, but only when located downstream from activation sequences. Interestingly, this element shows significant homology to a sequence present in the promoter of the PH08 gene, encoding a phosphate-repressible alkaline phosphatase. An electrophoretic mobility shift assay (EMSA) using whole-cell extracts and a NRS-containing DNA fragment detects a protein which specifically binds to this element. PMID- 8626062 TI - A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase. AB - A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6). The RpoSp6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P(lac)) and operator (O(lac)) on the plasmid. Therefore, a controlled expression system for the target genes can easily be constructed in various host strains by co transformation of the system plasmid pFSP6 with other vector plasmids containing the genes linked to the SP6 promoters (P(SP6)). Using the lac gene linked to P(SP6) as a reporter, we evaluated the regulation of expression in this system in various host strains. Low-level expression of lac was detected in Escherichia coli harboring this expression system when RpoSP6 was uninduced, although very low activities of beta-galactosidase (beta-Gal) were observed which were independent of the presence of pFSP6. This basal level of beta-Gal activity was possibly derived, because the P(SP6) element has very weak activity for E. coli RNA polymerase (Rpo). These results showed that RpoSP6 seemed to be produced at very low levels in uninduced cells. Beta-GAl activity increased about 18-32-fold when the expression of rpoSP6 was induced, as compared with the beta-Gal activity when uninduced. The tight regulation of this system is superior to that of other known systems and it has a considerable advantage for gene expression in E. coli. PMID- 8626063 TI - Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menE gene from Escherichia coli. AB - In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster. One of these, the conversion of o succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate. Formation of the intermediate is mediated by OSB-CoA synthetase encoded by the menE locus known to be located either 5' of menB, or 3' of menC. A DNA fragment overlapping the 3' end of menC in shown by enzymatic complementation to elevate OSB-CoA synthetase activity. Nucleotide sequence analysis of the fragment identified a 1.355-kb open reading frame (ORF) which, when deleted at either the 5' or 3' end, failed to generate increased enzymatic activity. The ORF is preceded by a consensus ribosome-binding site, but no apparent sigma-70 promoter. An oppositely transcribed unidentified gene cluster follows the menE ORF. The region 5' of menB contains an an additional ORF of unknown function (orf241) and establishes the order of genes in the men cluster as menD, orf241, menB, menC and menE. All loci are transcribed counter-clockwise. PMID- 8626065 TI - Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase. AB - We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169 degrees on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3' part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identify. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue. PMID- 8626064 TI - The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production. AB - The production of phenazine (Ph) antibiotics in Pseudomonas aureofaciens (Pau) 30 84 is positively regulated by PhzR, a protein belonging to the LuxR family of transcriptional activators. We have now identified phzI, a second gene required for PH production. The product of phzI is a member of the LuxI family of N-acyl homoserine lactone (N-acyl-HSL) synthases. Inactivation of phzI results in the loss of Ph production in Pau 30-84. The presence of phzI in Escherichia coli is sufficient for the production of a diffusible signal which activates phzB expression in Pau 30-84 and traA expression in a N-acyl-HSL-dependent reporter strain of Agrobacterium tumefaciens. In addition, synthetic N-(3-oxohexanoyl)-L HSL induces phzB expression in Pau 30-84. These results suggest that Pau 30-84 produces a N-acyl-HSL signal that regulates Ph production, and that phzI plays a central role in this signaling pathway. PMID- 8626066 TI - Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis. AB - A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1-G2, respectively, exhibited higher extra-cellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of beta-galactosidase into the extracellular fraction. PMID- 8626067 TI - Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange. AB - We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty. PMID- 8626068 TI - FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria. AB - Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution. PMID- 8626069 TI - Characterization of genes encoding topoisomerase IV of Mycoplasma genitalium. AB - A type-II toposiomerase (Topo-IV) encoded by the parC and parE genes in Escherichia coli and Salmonella typhimurium is thought to be involved in cell septation and in the decatenation of newly replicated chromosomes. We have identified parC and parE homologs in the pleomorphic, wall-less organism Mycoplasma genitalium. Since the mechanics of cell septation in conventional eubacterial species is believed to be mediated by cell-wall constituents, there is no clear understanding of what coordinates that process in wall-less species. The presence of par genes in this bacterium, which has the smallest genome of any free-living organism, suggests that Topo-IV has been evolutionarily conserved because of an essential role in mediating cell division. PMID- 8626071 TI - The amino-acid sequence similarity of plant glutamate dehydrogenase to the extremophilic archaeal enzyme conforms to its stress-related function. AB - A cDNA clone encoding grapevine (Vitis vinifera L. cv Sultanina) NAD(H)-glutamate dehydrogenase (GDH) was isolated from a cDNA expression library by immunoscreening with a polyclonal antibody raised against grapevine GDH. Nucleotide sequence analysis revealed an open reading frame (ORF) encoding a precursor protein of 411 amino acids (aa) with a calculated molecular mass of 44.517 kDa. The deduced aa sequence showed relatively higher homology to GDH from archaebacteria species, than to those from eukaryotes and eubacteria. This resemblance indicated a functional and/or evolutionary relationship in this class of enzymes which might be relevant to the stress-related function of plant GDH. We have shown that the bacterially produced plant GDH was thermostable. PMID- 8626070 TI - An Archaea 5S rRNA analog is stably expressed in Escherichia coli. AB - Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment. PMID- 8626072 TI - Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants. AB - The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA 1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR. PMID- 8626073 TI - Cloning and and characterization of hydrophobins-encoding cDNAs from the ectomycorrhizal basdiomycete Pisolithus tinctorius. AB - Major alterations of fungal gene expression are induced by the development of ectomycorrhiza, a symbiosis between tree roots and filamentous fungi. Several cDNAs corresponding to highly expressed transcripts of the Basidomycete Pisolithus tinctorius (Pt) were isolated from symbiotic tissues. Two of these abundant transcripts (hydPt-1 and hydPt-2) encoded polypeptides belonging to the hydrophobin (Hyd) family, a group of small cysteine-rich fungal proteins involved im morphogenesis and plant-fungus interactions. As shown for other Hyd, the hydPt 1 and hydPt-2 mRNAs were barely detectable in mycelium grown in liquid culture and highly accumulated in aerial hyphae. In addition, these transcripts were also abundant in eucalyptus globulus-Pt ectomycorrhiza in early stages of differentiation, during the colonisation of roots. PMID- 8626074 TI - The chsA gene, encoding a class-I chitin synthase from Ampelomyces quisqualis. AB - Degenerate oligodeoxyribonucleotide primers, designed on the basis of conserved regions of the chitin synthase gene family, were used to amplify a fragment of the Ampelomyces quisqualis (Aq) chsA gene. Subsequently, the PCR product was used as a probe in order to identify and isolate genomic clones harboring the entire chsA gene. Aq chsA is 2786-nt long, has one intron and encodes a 910-amino-acid polypeptide belonging to the class-I chitin synthases. Low-stringency Southern hybridizations to Aq genomic DNA provided evidence for the presence of additional DNA fragments resembling chsA in the fungal genome, suggesting the presence of a multigene family of chitin synthases in Aq. PMID- 8626075 TI - Osteoporosis: using 'bone markers' for diagnosis and monitoring. AB - Bone is continuously resorbed and formed in the process of remodeling. Osteoporosis results when bone loss is sufficient to cause increased risk of fracture. High rates of bone turnover can be identified by measuring biochemical byproducts of resorption and formation. Collagen crosslinks and associated peptides measured in urine are newly recognized markers for bone resorption. Bone specific alkaline phosphatase and osteocalcin are serum markers for bone formation. The clinical utility of these new techniques is two-fold: to identify adults at high risk of osteoporosis so that preventive therapy can be instituted, and to provide noninvasive, sensitive tools for monitoring clinical course and effect of therapy. PMID- 8626076 TI - Type II diabetes: how to use the new oral medications. Interview by David B. Jack. AB - Several new oral drugs have been approved for the management of type II diabetes. Metformin is an "antihyperglycemic agent" that decreases hepatic glucose production and improves insulin sensitivity. It may be used as monotherapy or in combination with a sulfonylurea. Acarbose slows carbohydrate absorption after a meal, giving endogenous or injected insulin more time to respond to ingested glucose. Glimepiride is an insulin-sparing sulfonylurea with a once-daily dosing schedule. How might these medications fit into the primary care of older diabetics? PMID- 8626077 TI - Healthy People 2000: what progress toward better nutrition? AB - At the 1995 status review of the Healthy People 2000 nutrition objectives, progress was reported in several areas. These include dietary fat consumption, calcium intake, use of nutrition label information, incidence of coronary and stroke mortalities and blood pressure and cholesterol levels. However, a lack of progress was reported in the incidence of overweight in both men and women. Physicians can help their older patients achieve better health by providing assessments for under- or over-nutrition and education in age-appropriate dietary practices. PMID- 8626078 TI - Urinary incontinence: taking action against this 'silent epidemic'. Interview by Marc E. Weksler. PMID- 8626079 TI - Multiple pigmented spots on the face. PMID- 8626080 TI - A spineless attack on the AHCPR. Agency that is the backbone of science and medical cost containment is in jeopardy. PMID- 8626081 TI - Static fundus perimetry using the scanning laser ophthalmoscope with an automated threshold strategy. AB - PURPOSE: The purpose of this study was to develop software that allows the performance of routine static threshold perimetry using the scanning laser ophthalmoscope (SLO) and the comparison of the results with conventional computerized cupola perimetry. The original software does not allow performance of static threshold perimetry within a reasonable examination time. METHODS: Static perimetry was performed in random order on 50 healthy eyes using our SLO staircase threshold perimetry technique and the Octopus 500 (program 38). We compared the relative sensitivities for each of 25 corresponding visual field locations. RESULTS: Mean sensitivity in the SLO perimetry amounted to 32.7 dB (range 25-37 dB) while it was 28.7 dB in the Octopus. For all test locations the SLO showed higher dB values on average. The mean difference between both methods was 3.7 +/- 0.8 dB (range 1.4-5.8 dB) when the test locations at the blind spot were excluded (linear regression between the two methods: r = 0.843, P < 0.0001). The mean time interval between two stimulus presentations was 2.5 s with the SLO perimetry. CONCLUSION: With the Heidelberg software, automated static threshold perimetry using the SLO is possible within reasonably short examination times. The mean time interval between two test point presentations is about one tenth of that necessary using the original Rodenstock software. There is a systematic difference between SLO and Octopus fields of about 4 dB which was not very much influenced by the stimulus locations. PMID- 8626082 TI - Modelling series of visual fields to detect progression in normal-tension glaucoma. AB - BACKGROUND: Use of statistical modelling techniques to identify models that both describe glaucomatous sensitivity decay and allow predictions of future field status. METHOD: Twelve initially normal fellow eyes of untreated patients with confirmed normal tension glaucoma were studied. All had in excess of 15 Humphrey fields (mean follow-up 5.7 years). From this cohort individual field locations were selected for analysis if they demonstrated unequivocal deterioration at the final two fields. Forty-seven locations from five eyes satisfied this criterion and were analysed using curve-fitting software which automatically applies 221 different models to sensitivity (y) against time of follow up (x). Curve-fitting was then repeated on the first five fields, followed by projection to the date of the final field to generate a predicted threshold which was compared to the actual threshold. Competing models were therefore assessed on their performance at adequately fitting the data (R2) and their potential to predict future field status. RESULTS: Models that provide the best fit to the data were all complex polynomial expressions (median R2 0.93). Other simple expressions fitted fewer locations and exhibited lower R2 values. However, accuracy in predicting future deterioration was superior with these less complex models. In this group a linear expression demonstrated an adequate fit to the majority of the data and generated the most accurate predictions of future field status. CONCLUSIONS: A linear model of the pointwise sensitivity values against time of follow-up can provide a framework for detecting and forecasting glaucomatous field progression. Linear modelling allows the clinically important rate of sensitivity loss to be estimated. PMID- 8626083 TI - Measurement of facial growth in the human fetus. AB - BACKGROUND: The fetal face is clearly seen by ultrasonography: we considered measurement of certain orbitofacial parameters of interest in the human fetus in order to establish norms for facial development. METHODS: We included 108 "normal" fetuses ranging in age from 16.5 to 41 weeks of amenorrhea. The orbitofacial parameters studied were outer canthal distance, inner canthal distance, oropalpebral distance right side and left side, and palpebral fissure length side and left side. The ocular parameters studied were corneal horizontal diameter and axial length. The traditional anthropometric parameters of the fetus were determined by pathological examination: age, weight head circumference and height. A statistical study analyzed the different correlations and established linear regression equations for orbitofacial parameters as a function of age. Polynomial regression models were tested to the third degree as a function of age and the head circumference/II ratio. RESULTS: Results are given in six different age groups. We find excellent correlation between the different parameters. Statistically valid linear regression equations were established for orbitofacial parameters. Polynomial regression equations were compared to linear equations their correlation coefficient and standard error, but showed no greater validity. Skull growth is more rapid than facial growth, which itself is more rapid vertically than horizontally. CONCLUSION: This study establishes norms for the different orbitofacial parameters, in particular the oropalpebral distance, for which we found no bibliographic references. The general interest of these measures lies in the description of malformation syndromes. PMID- 8626084 TI - Explorative study of interleukin levels in the human cornea. AB - BACKGROUND: The presence of interleukins has been demonstrated in the cornea and other ocular tissues. Although pathogenic mechanisms are unknown, interleukins seem to be involved in inflammatory disorders of the cornea. The present study was undertaken to analyse concentrations of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) in human corneas with various clinical diagnoses. METHODS: Immediately after keratoplasty 127 explanted human corneas with various corneal diseases were snap frozen and cryosections were prepared for histological examination. Furthermore, the protein content was measured according to the method of Bradford and the concentration of IL-1 beta and IL-6 were determined using a specific immunosorbent test (ELISA). RESULTS: It was found that IL-1 beta and IL-6 level were clearly higher in corneas with ulcerations and distinct inflammatory signs. Lower levels of both interleukins were found in corneas with a weak expression of inflammatory signs. CONCLUSIONS: Keratitis, keratoconus with inflammatory signs, and ulcerating processes showed higher interleukin levels than corneas with non-inflammatory disorders like scar formation, corneal dystrophy and keratoconus. The results could show that, depending on the clinical diagnosis, the inflammatory status of the cornea may be evaluated by the interleukin levels determined in the corneal tissue. PMID- 8626085 TI - Epidemiologic characteristics of rhegmatogenous retinal detachment in Kumamoto, Japan. AB - BACKGROUND: The epidemiology of rhegmatogenous retinal detachment in Asians is not well known. We studied the epidemiologic characteristics of rhegmatogenous retinal detachment in Kumamoto, Japan. METHODS: The study was based on a retrospective chart review of hospital patients who were treated for primary rhegmatogenous retinal detachment in 1990. The data were collected from seven hospitals in the Kumamoto area. RESULTS: From a population of 1 840 000, 192 residents developed retinal detachment. The annual incidence was therefore 10.4 per 100 000 population (9.6 for males, 11.2 for females). The incidences of three types of detachment-nontraumatic phakic, aphakic, and blunt trauma--were 9.8, 0.5 and 0.2 per 100 000 population, respectively. In 109 of 180 patients (60.6%) with nontraumatic phakic detachment, retinal breaks were associated with lattice degeneration. In females, 14 of 106 nontraumatic phakic cases (13.2%) were secondary to macular holes. CONCLUSION: Compared with previously published studies from other countries, the incidence of detachments associated with lattice degeneration and macular hole was higher, while the incidences of aphakic detachment and detachment due to blunt trauma were lower in Japan. Racial factors and living habits may affect the development of retinal detachment. PMID- 8626086 TI - Precision of ultrasonic estimates of choroidal melanoma regression. AB - BACKGROUND: Important prognostic information may be gained from knowledge of the volume and, over time, the change in volume of intraocular tumors such as choroidal melanomas. METHODS: The precision and time consumption of three different ultrasonographic methods were evaluated. Seven choroidal melanomas were analysed after placement of a ruthenium plaque and again after tumor regression had occurred. Perpendicular ultrasound B-scans were printed and analysed by overlay grid counting or outline tracing of tumor structures to calculate the volume. These two methods, using only two perpendicular scans, were compared with a micro-computer-controlled ultrasonographic three-dimensional rotation scanning system, where each tumor was manually outlined in 20 revolving scan planes RESULTS: The three-dimensional volume scanning method was the most precise, but also the most demanding in hardware and time consumption. CONCLUSION: Increased precision and less observer-dependent estimation of shrinkage rate after radiotherapy is available at the cost of sophisticated equipment. PMID- 8626087 TI - Central retinal venous outflow pressure. AB - BACKGROUND: Does the venous collapse phenomenon provide the possibility of venous dynamometry? METHOD: A technical model is presented which allows analysis of the conditions of the collapse of the central retinal vein in vitro. The conditions of the venous collapse were analysed with regard to intraocular pressure, intravasal pressure in the outflow of the central retinal vein and the overall perfusion. For clinical measurements dynamometry of the venous collapse is performed parallel to the experimental setting. RESULTS: The experiment reveals identical results for venous outflow pressure measured by venous dynamometry and by intravasal pressure detector. Venous dynamometry in vivo means that we use the onset of the venous collapse phenomenon to register the pressure in the central retinal vein at the point where it leaves the eye. Using this technique, retroocular obstruction of the venous outflow may be assessed. The venous outflow pressure itself depends on the venous flow resistance, intracranial pressure and arterial perfusion pressure. Any disorder of these three parameters may be assessed when the absolute venous outflow pressure is registered. CONCLUSION: The venous collapse phenomenon enables us to determine the venous outflow pressure. Clinical applications have proven promising. PMID- 8626088 TI - Surgical risk factors for severe postoperative proliferative vitreoretinopathy (PVR) in retinal detachment with grade B PVR. AB - BACKGROUND: Previous studies have shown that grade B proliferate vitreoretinopathy (PVR) is a considerable risk factor for the development of severe postoperative PVR. We conducted a prospective study to elucidate which surgical procedures used in retinal detachment management may stimulate the PVR process in such eyes. MATERIALS AND METHODS: The study included 156 eyes of 152 consecutive patients with rhegmatogenous retinal detachment complicated by grade B PVR referred before any failed surgery and operated on between 1983 and 1993. The parameters evaluated by multivariate statistical analysis included the cumulative circumferential extent of the retinal tears, the extent of the scleral buckle, gas injection, vitrectomy, the method used for retinopexy, and the time of surgical management during the period of the study. RESULTS: The incidence of severe postoperative PVR was 25.8% in eyes managed with cryotreatment versus 2.2% in eyes managed with argon laser photocoagulation (P = 0.001). The rate of severe postoperative PVR was not influenced by the other surgical variables. CONCLUSION: We conclude that cryotherapy may be a risk factor for the development of severe postoperative PVR in retinal detachments associated with grade B PVR. PMID- 8626089 TI - Proliferating cell nuclear antigen: contradictory results regarding its presence in the lens. AB - BACKGROUND: Cell division of the normal lens is believed to occur in the germinative zone of the lens epithelium only. The expression of proliferating cell nuclear antigen (PCNA) at least in its insoluble form, is restricted to cells shortly after and during DNA synthesis. METHOD: To elucidate the location of the previously reported notable amounts of PCNA in lens epithelium, 6-micron sections of 10 New Zealand rabbit corneas were stained with specific antibodies. RESULTS: No PCNA-positive cells were detected in the lens epithelium, neither in the central-zone nor in the germinative zone. CONCLUSION: As the mitotic activity of lens epithelium is physiologically low (below 0.03% in adult rabbits), the likelihood of detecting mitotic figures in histologic sections is very limited. We hence conclude that PCNA may be expressed in the epithelia of the lens but is only occasionally visible in histological sections. Our most recent studies have confirmed this. The fixation process and the use of specific antibodies are decisive factors that may, when used correctly, allow specific localization PCNA that is involved in cell replication of lens epithelium. However, it has to be pointed out that the mitotic index of lens epithelium is age- and species related, and hence so is the expression of PCNA, which makes comparison with other studies more difficult. PMID- 8626090 TI - Reduced levels of gamma-crystallin transcripts during embryonic development of murine Cat2nop mutant lenses. AB - BACKGROUND: From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. METHODS: The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. RESULTS: The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. CONCLUSION: The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice. PMID- 8626091 TI - Early and late visual prognosis in solar retinopathy. AB - BACKGROUND: Solar retinopathy was observed in a total of 86 eyes of 58 patients following the solar eclipse over Turkey in April 1976. The visual prognosis and the presence of late complications were evaluated at the early and late periods. METHODS: Of the 58 patients, 34 (51 eyes) presented during the first week and came for follow-up examination in the succeeding week, also after 1, 3, 12, and 18 months. After that they were examined at yearly intervals (mean 4.2 years). Twenty-four patients (35 eyes) presented during the period between 1 and 11 years post-eclipse and were followed up for a mean period of 3.4 years. After a period of 15 years, all of the patients were invited for re-examination and nine patients (14 eyes) attended. RESULTS: The improvement in visual acuity was observed to have taken place mostly during the first 2 weeks to 1 month after the eclipse. Further improvement in visual acuity was not observed in any of the eyes after the 18-month examination. The improvement in visual acuity was more prominent and earlier in the eyes that had visual acuity of 0.2 or better initially. Only the eyes with initial visual acuity equal to or better than 0.4 had a chance to improve their acuity to 10/10. Having observed the 51 eyes for mean period of 4.2 years and the 35 eyes for 3.4 years, no change in visual acuity was observed. Among the total of 86 eyes, 9 were found to have pseudolamellar macular holes. CONCLUSION: Correlation was found between initial visual acuity and the funduscopic appearance after the 2nd week. Fluorescein angiography was not found to be a conclusive test in solar retinopathy. No late complications were observed. PMID- 8626092 TI - The pseudo-posterior limiting layer syndrome: a vitreoretinal heredodegeneration with autosomal dominant transmission. Graefe's Arch Clin Exp Ophthalmol (1994) 232:16-24. PMID- 8626093 TI - Is more always better? PMID- 8626094 TI - High complete response rate of concomitant chemoradiotherapy for locally advanced squamous cell carcinoma of the uterine cervix. AB - A prospective study with a newly designed schedule of concomitant chemoradiotherapy was initiated for 42 patients with previously untreated squamous cell carcinoma of the uterine cervix. Their ages ranged from 34 to 77 years, median 57 years. There were 13 FIGO stage IIB, 1 IIIA, 27 IIIB, and 1 IVA. Radiotherapy was administered using 1.8 Gy/day, 5 days a week, to the whole pelvis (50.4 Gy/28 fractions) with local boost if indicated. Intracavitary brachytherapy of 5 Gy for five times was delivered after 1-2 weeks of rest. The first 21 patients received concomitant chemotherapy of biweekly PEB regimen (100 mg/m2 etoposide + 50 mg/m2 cisplatin + 50 mg/m2 bleomycin) for two to three cycles during external irradiation. The chemotherapy for the latter 21 patients was modified to weekly PEBF (50 mg/m2 etoposide + 20 mg/m2 cisplatin + 10 mg/m2 bleomycin + 800 mg/m2 5-FU, mixed in normal saline, 24-hr continuous iv infusion) for five to six cycles. All except 1 patient achieved complete response (97.6%) and sustain so after a median follow-up time of 30 months. There were three relapses--one with persistent pelvic disease and two with distant metastasis. Two year overall survival and disease-free survival rates were 97.6 and 92.9%, respectively. Myelosuppression was moderate but fully recovered. Other acute toxicities were tolerated except for 1 patient who encountered grade IV radiation colitis with cecum perforation and required surgery. As to late morbidity, the incidence of radiation proctitis was high (21.4%) but of a mild degree, with 1 patient needing repeated transfusion. One patient developed chronic cystitis with an acontractile bladder. Our preliminary results show that concomitant chemoradiotherapy for advanced cervical carcinoma is both feasible and effective with acceptable toxicities. Further follow-up is mandatory to ensure whether this high complete response protocol will translate into long-term local control and survival. PMID- 8626095 TI - The prognostic implication of ascites in advanced-stage ovarian cancer. AB - Ovarian carcinoma accounts for greater than 50% of the gynecologic cancer deaths in the United States each year. One of the central reasons for this dismal outcome is that many patients present with advanced disease. In this series, a retrospective review of 130 patients with stage III and IV invasive epithelial ovarian carcinoma was performed to determine the prognostic significance of ascites. Patients were divided into two study groups based upon the presence or absence of ascites. Survival for the entire study group was 15%, but differed markedly when separated for the presence of ascites. In these patients, ascites was associated with a statistically decreased 5-year survival of 5% versus 45% without ascites (P = 0.0001). Individuals were found to be similar in each group when examined for age, height, weight, cell type, grade, and surgical and chemotherapeutic treatment modalities. More patients proportionately with stage IIIC disease had ascites than those without ascites (P = 0.0015). More of the individuals without ascites underwent second-look laparotomies and achieved a negative result than those with ascites (P = 0.04; P = 0.0038). We conclude that ascites in the presence of stage III and IV disease produces an almost uniformly fatal outcome. PMID- 8626096 TI - One-session management of cervical intraepithelial neoplasia: a solution for developing countries. AB - Six hundred thirty-nine patients with CIN on referral Pap were evaluated cytocolposcopically at the first visit and decided whether to be treated the same day or not. One hundred ninety-two patients (30%) were considered negative. Follow-up evidenced later appearance of CIN in five of them. One hundred fifty three (24%) were candidates for delayed treatment due to conditions contraindicating same-day treatment. Two hundred ninety-four patients (46%) were randomly allocated in LEEP (149) or excisional laser (145) arms, and treated the same day under local anesthesia. Both arms were comparable. There were three microinvasive carcinomas diagnosed in the surgical specimen. LEEP was faster and produced less bleeding than laser, although required a mean of four slices to remove the lesion. Arterial hypertension after anesthetic infiltration was detected in 26% of cases. Two intraoperative and two delayed bleeders required surgery. The size of lesion and surgical defect were larger than those reported in the literature. Margins were involved in 8 patients (2.7%). Only 4.7% (7/149) of patients randomized to LEEP and 3.4% (5/145) with excisional laser had persistent or recurrent CIN on follow-up. Factors predisposing to failure included depth of surgical defect, grade of lesion, and operator's expertise. With this approach, 69% of patients referred for cytology of CIN were adequately managed in the first visit, which contrasts to classical management that reaches the state of treatment in 30% of patients. LEEP appears to be faster, less costly, and requires less expertise. Its use in conjunction with adequate screening is recommended for developing countries. PMID- 8626097 TI - Vaginal clear cell adenocarcinoma in the United States. AB - We elected to examine available information from several sources to approximate the annual number of cases of vaginal adenocarcinoma in the United States for recent years. Data were obtained from the Registry of Hormonal Transplacental Carcinogenesis, the Surveillance, Epidemiology and End Results (SEER) program of the National Cancer Institute, the National Cancer Databank of the American College of Surgeons Commission on Cancer, and a survey of gynecologic oncologists practicing in the United States. In 1990 a total of 33 new cases and 11 recurrences were reported, while in 1991 23 new cases and 8 recurrences were reported. Neither SEER nor the Registry appear to provide adequate surveillance for this rare disease. Phase III clinical trials are not feasible, given the small number of patients. Statistically effective phase II one-armed studies to investigate new agents in the treatment of advanced or recurrent vaginal clear cell cancer may be possible. Effective mobilization of patients and physicians will be required for such trials to be completed in a timely manner. PMID- 8626098 TI - Multiorgan fatal chronic complications following radiation treatment for cancer of the cervix--results of fibroblast assay. AB - We report a case of severe chronic normal tissue injury following conventional irradiation in a 33-year-old patient with a strong family history of cancer. Her cultured fibroblasts exhibited a response to low-dose-rate irradiation similar to fibroblasts taken from a normal individual. For acute irradiation her fibroblasts showed a slight increased sensitivity to single doses compared to fibroblasts from a normal individual. This difference was much larger when fractionated irradiation was given. It is concluded that the hypersensitive response of this patient was due to the fractionated radiotherapy and not to the low-dose-rate brachytherapy. We believe that this is the first report of this form of cellular radiosensitivity which is mainly expressed in fractionated radiotherapy. PMID- 8626099 TI - Adenoid cystic carcinoma of Bartholin's gland: a review of the literature and report of a patient. AB - We report the case of a 77-year-old white female with adenoid cystic carcinoma of Bartholin's gland. To date only 45 cases of adenoid cystic carcinoma of Bartholin's gland have been reported in the world literature. The longest reported survival is 27 years. This patient was originally diagnosed 33 years ago and presented with her fourth recurrence. She was treated with radical surgery and did well for 6 months, but later died secondary to renal failure. Adenoid cystic carcinoma of the Bartholin's gland is a rare tumor of the vulva. When diagnosed the treatment should be tailored to the patient. When margins are found to be positive, adjuvant radiotherapy may prove to be beneficial. PMID- 8626100 TI - Treatment of resistant gestational choriocarcinoma with taxol: a report of two cases. AB - Two patients with resistant gestational choriocarcinoma were treated w with Taxol after extensive prior chemotherapy including EMACO and platinum-based salvage chemotherapy. Both patients showed significant responses to Taxol. One patient relapsed in the brain and liver after treatment and died. Taxol was combined with carboplatin in the other patient who achieved sustained clinical and hormonal remission after craniotomy and excision of resistant disease in the brain. PMID- 8626101 TI - HIV-related primary non-Hodgkin's lymphoma of the vulva. AB - We reported on a 25-year-old HIV-positive woman diagnosed with Ann Arbor Stage IEB primary extranodal immunoblastic lymphoma arising in the vulva. This is the first documented instance of an HIV-associated malignant lymphoma originating in the lower female genital tract, and only the 16th reported case of primary malignant lymphoma of the vulva. The nonspecific clinical presentation coupled with the unanticipated finding of lymphoma made diagnostic confirmation an arduous task, requiring weeks of persistence. The patient's disease was refractory to three courses of chemotherapy, but did respond to a brief palliative course of external beam irradiation prior to her demise 7 months after presentation. Our findings are presented, and 15 cases from the literature, the largest series to date, are discussed. PMID- 8626102 TI - Hyperglycemia secondary to megestrol acetate for endometrial neoplasia. AB - Two patients with endometrial neoplasia developed severe hyperglycemia secondary to megestrol acetate. Patients who receive progestational therapy with megestrol acetate for endometrial neoplasia should be monitored closely for potentially severe hyperglycemia, especially if they have a history of abnormal glucose metabolism. PMID- 8626103 TI - Malignant rhabdoid tumor of the uterine corpus. AB - Malignant rhabdoid tumor (MRT) was first described as a variant of Wilms' tumor but was subsequently found to be a highly malignant tumor composed of primitive cells that resemble rhabdomyoblasts. In the past decade, extrarenal MRTs were reported in different locations and organs throughout the body including the female genital tract. We here report an MRT that arose in the corpus uteri, the second such case reported. PMID- 8626104 TI - Metastatic cerebral choriocarcinoma coexistent with a viable pregnancy. AB - Gestational choriocarcinoma associated with a viable pregnancy is extremely rare, and such choriocarcinoma with metastases presenting during the pregnancy is even more exceptional. Metastases from this tumor may present in a variety of ways depending on the site of the metastatic lesion, and can be fatal before choriocarcinoma is even suspected. We describe a 32-year-old primigravida presenting at 31 weeks gestation with a grand mal seizure due to cerebral metastases secondary to gestational choriocarcinoma who was subsequently successfully treated with chemotherapy. To our knowledge this is the first reported case of choriocarcinoma presenting with signs due to cerebral metastases during pregnancy where both the mother and child survived. This paper emphasizes the need to consider metastatic choriocarcinoma in any gravid female who presents with unusual neurological signs because of the life-threatening nature of this potentially curable disease. PMID- 8626105 TI - Primary retroperitoneal mucinous cystadenocarcinoma of low malignant potential: a case report and literature review. AB - A case of primary retroperitoneal mucinous cystadenocarcinoma of low malignant potential in the presence of normal ovaries is reported. The precise etiology of these neoplasms has not been defined; however, they may arise from heterotopic ovarian tissue, monodermal teratomas, embryonal urogenital remnants, intestinal duplication, or coelomic metaplasia. Although minimal data exist to define the appropriate management, it seems reasonable to extrapolate from the treatment of analogous ovarian neoplasms. PMID- 8626107 TI - Primary lymphatic dissemination of malignant elements in a mature cystic ovarian teratoma. PMID- 8626106 TI - Krukenberg tumor complicated by pregnancy. AB - A 32-year-old female presented with a right ovarian mass, and the unilateral oophorectomy specimen revealed adenocarcinoma with signet-ring cells histology consistent with a Krukenberg tumor. The high-grade gastric primary adenocarcinoma was later identified, but by this time a viable 7-week fetus and left ovarian Krukenberg tumor were present. Second trimester palliative oophorectomy and partial gastrectomy were performed. The patient delivered a healthy infant by cesarean section and is now 36 months after the initial diagnosis of metastatic gastric carcinoma and remains asymptomatic and free of disease. The unexpectedly favorable clinical outcome suggests that a patient may benefit from resection of gastric adenocarcinoma metastatic to ovary when other sites of metastatic disease are not evident. PMID- 8626108 TI - Regarding a series of IB bulky cervical cancer treated with neoadjuvant chemotherapy (NACT) and radical hysterectomy and pelvic lymphadenectomy. PMID- 8626109 TI - Incidence of adenomyosis. PMID- 8626110 TI - Proliferating cell nuclear antigen in endometrial carcinoma: pretreatment identification of high-risk patients. AB - OBJECTIVE: The aim of our study was to retrospectively examine the proliferating cell nuclear antigen (PCNA) immunoreactivity of tumor cells in curettage specimens containing endometrioid adenocarcinoma and obtained immediately before definitive surgical staging. This PCNA index was compared with the one subsequently derived from surgical specimens and assessed as a function of histologic grade, depth of myometrial invasion, neoplastic nodal involvement, cervical spread, and progression-free survival in order to determine a new prognostic parameter valuable at the time of diagnosis. MATERIALS AND METHODS: A population of 79 patients with locally advanced (stage I and II) endometrioid carcinoma, who underwent both the preliminary diagnostic curettage and the subsequent definitive surgical management, selected from January 1986 to June 1993 at the Department of Gynecology and Obstetrics, Ancona University, was retrospectively recruited from our series of 99 endometrial carcinomas. The archival paraffin blocks from the curettage and uterine specimens were identified and assessed for histologic reexamination and PCNA immunostaining [PC10 monoclonal antibody (Dako, Denmark)]. RESULTS: After a median follow-up of 47 months, recurrences were detected in 7 cases, and the Kaplan-Meier disease-free survival curve estimated for the entire study group was 91%. The median PCNA index of the curettage specimens presented a good overlap with the PCNA immunostaining in corresponding uterine samples with a correlation coefficient of 0.4 (P=0.02). A PCNA index >/=30% in curettage specimen was predictive of deep myometrial invasion; of 35 patients with PCNA index > or = 30%, 29 (83%) had myometrial invasion > or = 50%. No significant relationship was observed with neoplastic cervical spread, and histologic differentiation. By Cox hazard analysis, the PCNA index evaluated on curettage specimens was significantly related to disease-free survival, with significant disease-free survival advantages for patients with PCNA <30% (P<0.001). CONCLUSION: Our findings suggest that the PCNA immunostaining has proved to be considerably promising for the risk assessment in locally advanced endometrial carcinoma. The PCNA index is an objective and reproducible parameter accruably valuable also before starting the treatment; in presence of a high PCNA index, the patients should be referred to gynecologic oncologists for appropriate management. PMID- 8626111 TI - The impact of diabetes mellitus on the toxicity of therapy for advanced ovarian cancer. AB - A retrospective review was undertaken to obtain more precise information about neurotoxicity, nephrotoxicity, and the effects of dexamethasone on the frequency and severity of hyperglycemia in diabetic patients with epithelial ovarian cancer treated with paclitaxel and/or cisplatin. Thirty-three patients were identified from 1254 patients over a 10-year period. In the cisplatin-treated patients, 21 of 24 (67%) had progression of neurological symptoms, three experienced grade 3 sensory neuropathy, and two had ototoxicity. Four patients had evidence of mild nephrotoxicity and two required a 50% dose reduction. In the group of patients treated with paclitaxel, 9 of the 18 (50%) had progression of symptoms, 2 to grade 3, and 2 had ototoxicity. No discontinuation of therapy due to neuropathy was required and no patient had evidence of drug-induced autonomic nervous system dysfunction. Hyperglycemia was frequently exacerbated, and 5 patients required treatment change, but no patient was hospitalized in relation to this. Our results indicate that the paclitaxel/cisplatin combination regimen or paclitaxel alone could be safely administered in diabetic patients at standard doses, with concurrent glucose and creatinine monitoring, as well as history of neurological symptoms and physical examination. PMID- 8626112 TI - Combination chemotherapy with hydroxyurea, dacarbazine (DTIC), and etoposide in the treatment of uterine leiomyosarcoma: a Gynecologic Oncology Group study. AB - Advanced or recurrent uterine leiomyosarcomas have traditionally been resistant to most chemotherapeutic regimens. Preliminary reports suggested the combination of hydroxyurea, dacarbazine (DTIC), and etoposide (VP-16) was sufficiently effective to warrant larger trials. In a Phase II trial undertaken by the Gynecologic Oncology Group, 39 patients with advanced or recurrent leiomyosarcoma were treated with 2 g of hydroxyurea, 700 mg/m2 of DTIC, and 300 mg/m2 of VP-16 in divided doses every 4 weeks. Thirty-eight patients were evaluable for response. Two patients experienced complete responses and five had partial responses for a total objective response rate of 18.4% (95% confidence interval: 7.7-34.3%). In general, therapy was well tolerated with moderate toxicity. Six of the seven responders had disease outside the pelvis. The combination of hydroxyurea, DTIC, and VP-16 exhibits moderate activity against uterine leiomyosarcoma. PMID- 8626113 TI - Adjuvant chemotherapy versus chemotherapy plus pelvic irradiation for high-risk cervical cancer patients after radical hysterectomy and pelvic lymphadenectomy (RH-PLND): a randomized phase III trial. AB - OBJECTIVE: To compare the clinical efficacy of adjuvant chemotherapy a lone vs chemotherapy plus whole pelvic radiation therapy (RT) on recurrence rates, patterns of recurrence, and survival of patients post-RH-PLND for cervical cancer at high risk for recurrence. METHODS: Prospective multicenter randomized Phase III trial. Patients with Stage IB-IIA cervical cancer undergoing RH-PLND were eligible. Risk factors include deep cervical invasion, tumor > or = 4 cm, parametrial involvement, nonsquamous histology, and/or pelvic lymph node metastasis. Chemotherapy consisted of cisplatin and bleomycin, alone or in combination with whole pelvic RT. Survival was determined by Kaplan-Meier estimate. RESULTS: Eighty-nine patients were entered from 1987 to 1994. Seventy five patients had a Stage IB cancer and 14 patients had Stage IIA. Twenty-five patients had > or = 3 risk factors. Forty-four patients received chemotherapy alone vs 45 patients treated with chemotherapy and RT. Nineteen patients had recurrences and 16 patients have died. Nine of 44 (20%) patients receiving chemo alone recurred compared to 10/45 (22%) patients receiving chemo and RT (P=ns). Patterns of recurrence were statistically similar between the two treatment arms, even among the subgroup of patients with > or = 3 risk factors. Both regimens were well tolerated. CONCLUSION: CT + RT did not prove a superior adjuvant therapy for patients at high risk of recurrence after RH-PLND for early cervical cancer in this limited trial. Recurrence rates and patterns of recurrences (local, regional, or distant) were not influenced by the addition of RT. PMID- 8626114 TI - All-trans-retinoic acid inhibits the proliferation of cell lines derived from human cervical neoplasia. AB - The purpose of this study was to determine whether all-trans-retinoic acid (RA) can inhibit the growth of cervical neoplastic cells by inducing differentiation or by increasing the secretion of transforming growth factor-beta (TGF-beta). Normal and HPV DNA-positive cervical cells (2 cell lines derived from cervical intraepithelial neoplasia (CIN), 2 HPV DNA-transfected cell lines, and 6 cervical carcinoma cell lines) were treated with RA (1 to 1000 nM), and both total viable cell count and [3H]thymidine incorporation were used to evaluate proliferation. In vitro differentiation was evaluated in organotypic (collagen gel raft) cultures with hematoxylin/eosin staining, and using specific immunostaining for fillagrin and cytokeratin 10. TGF-beta 1 and TGF-beta 2 secretion were measured with specific SELISAs. One-way analysis of variance and t tests were performed. RA causes a dose-dependent (P<0.05) growth arrest of comparable magnitude in normal ectocervical cells, in HPV DNA-transfected cell lines, in CIN-derived cell lines, and in four of six carcinoma cell lines. Endocervical cells and two carcinoma cell lines are unaffected. In vitro differentiation is decreased in CIN cells and is unchanged in carcinomas treated with RA as compared to control. Secretion of either TGF-beta 1 or TGF-beta 2 is significantly increased (P<0.05) in response to RA, both in RA-sensitive and in RA-resistant cells. RA induces growth inhibition in cervical neoplastic cell lines, including cervical carcinoma cells. This does not appear to be the result of increased differentiation or of increased TGF-beta secretion. PMID- 8626115 TI - Brain metastases from endometrial carcinoma. AB - Central nervous system (CNS) involvement by endometrial carcinoma is uncommon. Among 1069 patients registered for endometrial carcinoma at our institution between 1982 and 1994, 10 (0,9%) developed brain metastases. Median age at the time of CNS metastasis diagnosis was 59 years. Median interval between diagnosis of endometrial cancer and documentation of brain involvement was 26 months. Clinical manifestation of brain metastasis included headache (80%), motor weakness (50%), seizures (20%), confusion (10%), balance (10%), and visual disturbances (10%). All lesions (4 multiple, 6 single) were contrast enhancing on computed tomography (CT) scans, and were located in the cerebrum in seven cases, in the cerebellum in one case, and in both in two cases. The CNS was the only site of detectable disease in six patients with recurrent disease. Nine patients died and one is alive with disease 3 months after surgical resection of a single cerebral deposit. Median survival from diagnosis of brain metastases for the entire series was 1 month (range 1-83). Six patients receiving only steroids died within 1 month from the diagnosis. One patient received radiotherapy (survival, 3 months) and two underwent surgical resection of solitary metastasis followed by radiotherapy (survival = 28 and 83 months). Prognosis of patients with CNS metastases from endometrial carcinoma appears poor; however, in a selected group of patients early diagnosis followed by multimodal treatment may result in a palliation of the disease. PMID- 8626116 TI - Locally advanced cervical adenocarcinoma: is there a place for chemo-surgical treatment? AB - The increased frequency and poor prognosis of cervical adenocarcinoma call for new therapeutic strategies, especially in locally advanced disease. Combined neoadjuvant chemotherapy (NACT)-radical surgery (RS) has been investigated to assess its feasibility and the possible impact on disease outcome. Data were pooled from three consecutive trials on a total of 42 patients with FIGO Stage IB IIA >4 cm (9), IIB (19), and IIIB (14) cervical adenocarcinomas. NACT regimens consisted of cisplatin (P), bleomycin (B) and methotrexate, high-dose PB, and P and doxorubicin combinations for one to three cycles. Responding patients underwent RS while those still ineligible for RS underwent radiotherapy. Fisher and chi squared tests were used to detect significant factors affecting response to NACT. Cox multivariate regression analysis was used to evaluate parameters affecting response and survival. Medians and life tables were computed by the method of Kaplan and Meier. Median follow-up times were 56 (17-95) and 54 months (15-92) from enrollment and RS, respectively. NACT-induced toxicity was generally mild and did not compromise RS when indicated. The 33 (79%) responders underwent laparotomy, while the 9 nonresponders received radiotherapy. RS was feasible in 29 (69%) patients. Macroscopic intraperitoneal tumor (IPT) excluded abandoning RS in 4 cases. Mild to moderate RS-related complications were seen in 41% of cases with the same pattern as in the absence of any prior treatment. In patients undergoing RS, node metastasis and microscopic IPT were detected in 2 (7%) and 3 (10%) patients, respectively. The 5-year overall and disease-free survivals were 71% (100% IB-IIA and 84% IIB vs 36% IIIB; P = 0.001) and 88%, respectively. None of the nonresponders survived (median 10 months, 6-25), compared with an 84% 5 year survival of responders (P < 0.001). FIGO stage and parametrial involvement significantly predicted response to NACT which was the only independent variable affecting survival (P = 0.006). This retrospective study provided evidence of the chemosensitivity of locally advanced cervical adenocarcinoma and that chemoresponsiveness is the most potent predictor of cure, as demonstrated in squamous cell cervical cancer. Combined NACT and RS is a feasible treatment which seems to be able to improve the outcome of Stage IB-IIB cervical adenocarcinoma. Randomized trials comparing this new strategy with conventional treatments seem to be warranted. PMID- 8626117 TI - Overexpression of p53 is not a feature of ovarian granulosa cell tumors. AB - The p53 tumor suppressor gene has been extensively studied in various human tumors including epithelial ovarian cancers. However, little is known about the expression of this gene in ovarian granulosa cell tumors, the most common histologic type of sex cord-stromal tumors. We investigated whether overexpression of the p53 gene product occurs in this specific ovarian tumor. Nineteen patients with ovarian granulosa cell tumors were recruited in this study. Immunohistochemical staining for the p53 protein with monoclonal antibody PAb 1801 was performed in the paraffin-embedded tissue of each case to screen for p53 overexpression. Among the 19 ovarian granulosa cell tumors, there was only one well-differentiated tumor found to have nuclear immunoreactivity in a small fraction of tumor cells. Polymerase chain reaction--single-stranded conformation polymorphism was used to study the tumor showing focal p53 positivity, but no mobility shift was noted from exon 4 through exon 9 of the p53 gene. On the basis of this observation, we propose that alteration of the p53 tumor suppressor gene is not a common finding in ovarian granulosa cell tumors. PMID- 8626118 TI - Squamous cell carcinoma of the endometrium: a report of eight cases and a review of the literature. AB - BACKGROUND: Endometrial squamous cell carcinoma is extremely rare, with only 56 cases reported in the literature. METHODS: Six cases of endometrial squamous cell carcinoma were found in a review of 1182 cases of uterine corpus cancer treated at the Massachusetts General Hospital from 1975 to 1993. Two additional cases were seen in pathological consultation. The clinicopathological features of these 8 cases and the 56 reported cases were analyzed. RESULTS: The average age of the patients was 67 years; almost all of them were postmenopausal. The most frequent presenting symptom was vaginal bleeding. Chronic pyometra and nulliparity were predisposing factors. The average duration of symptoms before diagnosis was 11.5 months. Total abdominal hysterectomy with bilateral salpingo-oophorectomy was the primary treatment in 58 patients. Eighty percent of the patients with Stage I tumors survived; the median follow-up time was 32 months. The survival rate for patients with Stage III tumors was only 20%, and all 6 patients with Stage IV disease died. CONCLUSIONS: The preoperative diagnosis of endometrial squamous cell carcinoma may be difficult, since curettage specimens may show only highly differentiated squamous epithelium. The strong relationship between tumor stage and survival suggests that early diagnosis and treatment are imperative. PMID- 8626119 TI - Second primary cancers in a cohort of Israeli women with primary gynecologic malignancies. AB - Second cancer in a women with a primary gynecological malignancy could reflect a common etiology or sequealae of a potentially carcinogenic treatment. The aim of the present study was to determine the incidence rate of second primary cancer in a cohort of women with primary gynecological cancer and patient characteristics, namely age, diagnosis, type of treatment, and duration of follow-up. The study cohort comprised 925 Israeli Jewish women with histologically confirmed gynecologic malignancies. The file was linked by computer matching to the Israel Cancer Registry for identification of second primary cancers. Standardized incidence rates (SIRs) for site-specific as well as for all cancer were computed. A significant excess for subsequent leukemia among ovarian (SIR 10; 95% CI 1.1 36.1) and uterine corporal cancer patients (SIR 10.0; 95% CI 2.7-25.6) was found. The significant increase in leukemia was treatment-related and limited to patients treated with radiotherapy. Age older than 60 years constituted a significantly unfavorable factor. The estimated probability of developing a subsequent cancer at 175 months is 6.1%. Physicians should be aware of the possibility of a second primary malignancy when prescribing treatment and during follow-up. PMID- 8626120 TI - Differential diagnosis of adnexal masses with transvaginal sonography, color flow imaging, and serum CA 125 assay in pre- and postmenopausal women. AB - The aim of the present study was to compare the diagnostic accuracy of transvaginal sonography, color flow imaging, and serum CA 125 assay in pre- and postmenopausal women undergoing laparotomy for a clinical diagnosis of an adnexal mass. In 109 consecutive women, the morphology of the mass was evaluated with transvaginal sonography, the pulsatility index (PI) was computed on the arteries detected with color flow imaging, and blood samples were obtained for CA125 assay. Descriptive statistics were performed for the whole series and according to the menopausal status. The diagnostic accuracy of transvaginal sonography was significantly higher in premenopause than in postmenopause (97 versus 85%, P<0.05). In premenopause, the three tests showed a similar diagnostic accuracy (85, 82, and 79%, respectively). In conclusion, the addition of further tests besides transvaginal sonography is not warranted in premenopausal women with an adnexal mass, but they seem to be useful in postmenopause. PMID- 8626121 TI - The association of human papillomavirus type 16 E6 and E7 antibodies with stage of cervical cancer. AB - OBJECTIVE: Previous investigators have reported higher HPV type 16 antibody positivity among cervical cancer patients than among healthy women. The objective of this study was to determine the association of HPV 16 antibody levels with the stage of cervical cancer. METHODS: Pretreatment tumor biopsies and sera were obtained from 137 newly diagnosed cervical cancer patients residing in Mexico. Using peptide ELISA and radioimmunoprecipitation assay (RIPA), HPV 16 E6- and E7 specific antibodies were measured. RESULTS: By ELISA, elevated antibody titers to HPV 16 E6 and E7 were detected in 16.8 and 32.8% of the women, respectively. While sera positivity did not differ by disease stage, the mean absorbance in the E7-positive sera was 0.42, 0.62, 0.91, and 0.81 for stages I to IV, respectively. Using RIPA, anti-E6 and E7 positivity was demonstrated in 46.7 and 38.7% of the females, respectively. Although no difference across disease stage was detected for E6, increasing proportions of positivity to E7 with stage of disease was detected. The rates for increasing disease stage were 0.14, 0.37, 0.40, and 0.67. Sera from the 6-month postradiation follow-up examinations of a small group of patients demonstrated a statistically significant decrease in antibody positivity from pretreatment positivity to HPV 16 E6 (n = 14; P = 0.01) and HPV 16 E7 (n = 20; P = 0.0001) using ELISA. CONCLUSIONS: These data suggest that HPV 16 E7 antibody positivity may be associated with stage of cervical cancer. Such immune parameters may be applicable to disease staging, monitoring of recurrence and, perhaps, diagnosis. Further investigation into the relationship of HPV 16 E6 and E7 antibodies with stage of cervical cancer and response to therapy is warranted. PMID- 8626122 TI - Operative retroperitoneal ureteral catheterization for obstructive uropathy in primary locally advanced carcinoma of the cervix: description of a technique and experience. AB - Ten patients with obstructive uropathy from locally advanced carcinoma of the cervix underwent operative ureteral catheterization at the time of retroperitoneal paraaortic lymphadenectomy. This procedure allowed preservation of renal function and removal of percutaneous nephrostomies that had been placed. This reduced the risk of infection, need for continued nursing care, and patient discomfort associated with the percutaneous nephrostomy. PMID- 8626123 TI - Recurrence patterns in locally advanced cervical carcinoma: role of nodal status and 72-kDa metalloproteinase index. AB - OBJECTIVE: The aim of the study was to analyze the relationship between lymph nodal involvement and regional and/or distant recurrences in locally advanced squamous cervical carcinomas, and also evaluate tumor 72-kDa metalloproteinase, as a biologic parameter useful for understanding the mechanisms of disease relapse and prognosis. In particular, 72-kDa metalloproteinase is an enzyme that specifically cleaves type IV collagen and seems to play a critical role in tumor invasion and metastatic dissemination. METHODS: The medical records of 62 patients with FIGO (International Federation of Gynecology and Obstetrics) stage Ib and IIa squamous cervical carcinoma who underwent primary radical surgery with systematic pelvic and paraaortic lymphadenectomy and then were routinely followed were recruited from our series of 76 consecutive cases and reviewed. Fifty-four patients with complete clinicopathologic information were considered eligible for the study. All recurrences were defined as histologically and/or cytologically documented disease, following a minimum 3-month disease-free interval. Sites of recurrences were classified as distant, or regional to the pelvis. Immunostaining with 72-kDa metalloproteinase was performed on serial sections of tumors using avidin-biotin complex technique. Affinity-purified rabbit anti-72-kDa metalloproteinase antibody was used. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells (72-kDa metalloproteinase index). RESULTS: After a median follow-up of 38 months (range 9 71 months), 11 patients recurred with a 20% overall incidence. Seven patients (64%) recurred regionally, with side-wall infiltration in 2 cases, and 4 patients (36%) recurred distantly. By Cox hazard multivariate analysis, lymph nodal status was significantly related to disease-free survival (P = 0.01); in particular, all the patients with side-wall or distant recurrences had lymph nodal involvement. A significant relationship was also observed between tumor 72-kDA metalloproteinase immunostaining and disease-free survival (P = 0.02). The 72-kDA metalloproteinase index was significantly higher in patients who recurred than in patients with disease-free follow-up (P < 0.001); in particular, the highest values were detected in patients who recurred distantly. A relationship between 72-kDa metalloproteinase staining and nodal status was observed (P < 0.001). CONCLUSIONS: In conclusion, nodal status and the 72-kDa metalloproteinase index were two independent prognostic parameters, significantly related to recurrence risk and pattern of recurrence in locally advanced cervical carcinoma. Although they are independent prognostic parameters, a relationship between nodal involvement and 72-kDa metalloproteinase was observed. A model of tumor recurrence in which intrinsic tumor factors exert their negative influence directly or by contributing to the development of nodal metastases seems possible. PMID- 8626124 TI - Phase 1 trial of intraperitoneal AD-32 in gynecologic malignancies. AB - AD-32 (N-trifluoroacetyladriamycin-14-valerate), an analogue of doxorubicin, was examined for intraperitoneal (ip) administration in a phase 2 trial involving 25 patients with advanced gynecologic malignancies. At an AD-32 dose of 600 mg/m2, the limiting toxicity was grade 4 neutropenia (64% of patients), while severe abdominal pain was relatively uncommon (12%). Intraperitoneal AD-32 administration was associated with a 200-fold pharmacokinetic advantage for cavity exposure, compared to the systemic compartment. At the 600 mg/m2 dose level, 4 of 9 patients (44%) with ascites experienced control of malignant fluid reaccumulation. Based on the results of this phase 1 trial, further exploration of a possible role for the ip administration of AD-32 in individuals with gynecological malignancies appears indicated, particularly in patients with either small volume residual disease after initial systemic chemotherapy or in those with intractable ascites. PMID- 8626125 TI - Phase II trial of hydroxyurea, dacarbazine (DTIC), and etoposide (VP-16) in mixed mesodermal tumors of the uterus: a Gynecologic Oncology Group study. AB - OBJECTIVE: The purpose of this study was to evaluate the efficacy of this three drug regimen--hydroxyurea, dacarbazine (DTIC), and etoposide (VP-16)--in patients with advanced or recurrent mixed mesodermal tumors (MMT) of the uterus who had not undergone previous chemotherapy. The study was performed as a groupwide phase II study of the Gynecologic Oncology Group. STUDY DESIGN: Thirty-three evaluable patients received hydroxyurea 2 g in divided doses on Day 1, 700 mg/m2 DTIC and 100 mg/m2 VP-16 on Day 2, and VP-16 100 mg/m2 on Days 3 and 4. Thirty-two patients were evaluable for response. Twenty-six patients had previously undergone abdominal hysterectomy and 11 had received prior radiation therapy, for whom one dose level reduction of the first course was required. RESULTS: Two patients exhibited complete response and three patients showed partial responses for an overall response rate of 15.7% (95% confidence interval: 5.3-32.8%). Seventeen of 32 patients had stable disease on therapy. Toxicity was acceptable and there were no treatment-related deaths. CONCLUSION: This regimen reveals moderate activity in patients with advanced or recurrent MMT. PMID- 8626126 TI - Vascular endothelial growth factor expression is not regulated by estradiol or medroxyprogesterone acetate in endometrial carcinoma. AB - OBJECTIVE: To determine whether the expression of vascular endothelial growth factor (VEGF) is altered by treatment in an in vivo tumor with 17 beta-estradiol (E2) or medroxyprogesterone acetate (MPA). METHODS: A well-differentiated endometrial carcinoma tumor was isolated from a patient and explanted into the dorsal skin of ovariectomized nude mice, from which it was serially passaged in vivo. The explanted tumor retained all the properties of the original tumor, including estrogen and progesterone receptor expression and growth promotion and inhibition by E2 and MPA, respectively. The mice were treated with continuous E2 administration followed by treatment with either a single intramuscular administration of 2 mg MPA or weekly administrations of 2 mg MPA. Untreated tumor bearing mice served as controls. The tumors were harvested at 0 to 21 days from first MPA administration. RNA from the tumors was isolated and VEGF expression was determined by Northern analysis. RESULTS: VEGF was expressed in the absence of treatment with E2 or MPA, and expression was unaltered by continuous treatment with E2. Additional treatment with a single does of MPA did not alter expression at Days 1, 2, 3, 7, 14, and 21, and additional treatment with E2 or E2 + MPA. Regulation of VEGF expression is not a mechanism by which these hormones exert their growth effects on endometrial tumors. PMID- 8626127 TI - Molecular biology in the clinicopathologic assessment of endometrial carcinoma subtypes. PMID- 8626128 TI - p53 overexpression and bcl-2 persistence in endometrial carcinoma: comparison of papillary serous and endometrioid subtypes. AB - Forty-two cases, including 21 uterine papillary serous carcinomas (UPSC) and 21 age-, nuclear-grade-, and clinical-stage-matched uterine endometrioid carcinomas (UEC), were studied immunohistochemically for p53 and bcl-2 in archival paraffin embedded tissue. Compared to UEC (28.6% positive), UPSC (71.4% positive) had a significantly higher frequency of p53 overexpression (P = 0.005); furthermore, in a clinical-stage-matched fashion, a higher frequency of p53 overexpression was found in early-stage cases (P = 0.032), but not in late-stage cases. In a nuclear grade-matched comparison, no statistical difference in p53 overexpression was identified between the two subtypes, although UPSC had stronger p53 immunoreactivity than UEC. Of UPSC, no difference in p53 overexpression was detected between tumors of early and late stages; additionally, in 5 cases, there was an abrupt transition from nonstaining morphologically benign glands to uniformly positive p53 nuclear staining in regions of intraepithelial carcinoma. Conversely, in UEC, there was a significant difference in p53 immunostaining between tumors of early and late stages (P = 0.01); no case had an abrupt transition for p53 immunostaining. For bcl-2 immunostaining, UEC had a significantly higher immunohistochemical staining score than did UPSC (P = 0.0002). In general, the staining intensity of bcl-2 diminished progressively from proliferative phase and hyperplastic endometrium to UEC and then to UPSC, with 3 of 21 (14.3%) UPSC being negative. These results suggest that p53 alteration may be an early event in the development of UPSC and may be related to its clinical aggressiveness, while it is a late event in UEC. Early detection of p53 nuclear accumulation may help to identify precursor lesions of UPSC. bcl-2 persistence is frequently associated with endometrial carcinoma, and failure to inactivate bcl-2 expression probably is related to the development of endometrial carcinoma. PMID- 8626129 TI - In vivo gene therapy of ovarian cancer by adenovirus-mediated thymidine kinase gene transduction and ganciclovir administration. AB - Efficacy and toxicity of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene followed by administration of ganciclovir were studied in vivo. A human epithelial ovarian cancer animal model was established in nude mice using the serous ovarian adenocarcinoma cell line Ov-ca-2774. Intraperitoneal (ip) injection of 1 x 10(8) Ov-ca-2774 cells resulted in tumor growth and formation of malignant ascites in all 15 animals. In a prospective randomized experimental design mice were treated 1, 3, or 7 days after ip injection of 1 x 10(8) cells with ip injection of 2 x 10(8), 6.7 x 10(8), or 2 x 10(9) pfu ADV.RSV TK followed by administration of ganciclovir (10 microgram /ml, ip, bid) for 6 consecutive days. End points were survival and toxicity. Mice treated with GCV or HSV-TK alone died from 14.4 +/- 1.7 to 19.5 +/- 3.5 days after treatment as did untreated controls. No toxicity of ADV.RSV-TK was found up to 2 x 10(9) pfu (2 x 10(11) particles). The mice with the highest tumor burden treated with the lowest viral dose lived significantly longer than controls (P < 0.05). Median survival in all other groups of mice treated with ADV.RSV-TK plus GCV was even longer (P < 0.01). Treatment benefit was dependent on ADV/RSV-TK dose and tumor burden. Adenovirus-mediated thymidine kinase gene therapy is a realistic approach to ovarian cancer treatment that warrants investigation in the clinical setting. PMID- 8626130 TI - p53 allelotypes and enhanced detection of allelic loss in ovarian cancer: lack of correlation with familial and clinical factors. AB - To develop a mapping strategy for improved detection of p53 allelic loss associated with ovarian carcinoma, we utilized multiple intragenic polymorphisms and single-strand conformation polymorphism (SSCP) analysis. p53 allelotype distributions were defined for 80 ovarian cancer patients from germ-line DNA. All polymorphic sites studied had polymorphism information content (PIC) rates of greater than 0.25. Tumor loss of heterozygosity (LOH) was determined from informative polymorphisms and migratory shifts on SSCP screening of exons 5-9. Of the four polymorphisms analyzed, the intron 1 (alu) was the most informative (PIC = 0.66). A novel allele of 110 base pairs was found in 4.4% of our ovarian cancer cohort at this site. The intron 3 (16-base pair repeat) and intron 6 (MSP1) polymorphisms were in relative equilibrium; thus, we chose to use only the intron 3 polymorphic site in the mapping strategy. Family cancer history did not influence the allelotype distribution frequencies. Overall, 56 of 61 tumors (91.8%) were informative for allelic loss, and LOH was observed in 66.1 %. A reduction to homozygosity at the p53 locus did not correlate with familial or clinical factors. These observations are consistent with the multiple mechanisms by which p53 dysfunction can occur. PMID- 8626131 TI - Radiation therapy as exclusive treatment for medically inoperable patients with stage I and II endometrioid carcinoma with endometrium. AB - From 1975 to 1992, 54 patients with clinical Stage I and II endometrioid carcinoma of the endometrium, representing 3.5% of all such patients, were deemed medically inoperable and exclusively received radiation therapy. A cohort of 108 operable patients adjusted for age, clinical stage, and grade served as a control group. The 5-year actuarial cancer-specific survivals for patients with Stage I inoperable, Stage 11 inoperable, Stage I operable, and Stage II operable disease were 80, 85, 98, and 100%. The corresponding 5-year overall survival rates were 30, 24, 88, and 85%. Inoperable patients had a median disease-free interval of 36 months for clinical Stage I and 50 months for Stage II disease versus 74.5 and 77 months for the operable patients (P = 0.001). Inoperable patients with Stage I disease had a median survival of 37 months versus 50 months for Stage II (P = NS), with only 7 (13%) of these patients dying with endometrial cancer. Operable patients had a median survival of 75 and 79 months in Stage I and II, respectively, with 14 patients dying with endometrial carcinoma (13%). Stage I and II inoperable patients had significantly shorter survival than operable patients (P < 0.0001). More deaths from intercurrent disease occurred within the inoperable Stage I group than with the operable group (28 of 32 vs 3 of 15, P < 0.0001). Inoperable patients had a significantly shorter overall survival and more deaths due to intercurrent disease than operable patients (P < 0.0001). However, inoperable patients who did not die from intercurrent disease had a median 5-year survival which approaches that of operable patients. Our study demonstrates that exclusive radiation therapy is a well-tolerated and effective treatment for medically inoperable patients. PMID- 8626132 TI - Microinvasive cervical carcinoma and cervical intraepithelial neoplasia: biologic significance and clinical implications of 72-kDa metalloproteinase immunostaining. AB - OBJECTIVE: The immunohistochemical expression of 72-kDa metalloproteinase was evaluated in cervical intraepithelial neoplasia (CIN) and microinvasive carcinoma, with the aim to define a relationship between 72-kDa metalloproteinase expression and neoplastic invasiveness, useful to identify subsets of intraepithelial lesions with higher risk of progression. MATERIALS AND METHODS: Cervical bioptic samples were obtained consecutively from 54 women who attended our Colposcopic Service from January 1993 to July 1993 because of abnormal pap smear, suspicious for cervical dysplasia and/or human papillomavirus infection. After written consent, 29 women with CIN were included in the study. All women with CIN 3 lesion underwent conization; in 21 women with mild or moderate cervical dysplasia, we did not perform any medical or physical treatment but followed them longitudinally at close interval. After 12 months, the clinical evolution was classified as spontaneous remission, persistence, or progression depending on the absence or presence of lesion and/or HPV infection in colposcopy, histology, and polymerase chain reaction findings. In the study we also included surgical specimens from 10 women with microinvasive squamous carcinoma who underwent primary radical surgery. Seventy-two kilodalton metalloproteinase positivity was immunohistochemically stained on serial sections by using the avidin-biotin complex technique (Vector Laboratories, Burlingame, CA) and expressed as percentage of cells per 10(3) counted neoplastic cells. RESULTS: Cytoplasmatic positive 72-kDa metalloproteinase immunostaining was significantly higher in microinvasive cervical carcinomas than in CIN lesion (Student's t test; P < 0.001). Considering only cervical intraepithelial neoplasias, a significant increase in 72-kDa metalloproteinase immunostaining was observed with CIN degree increasing (one-way analysis of variance; P = 0.002). No correlation was found between 72-kDa metalloproteinase immunostaining and HPV infection and lesion size defined by quadrants of the cervix involved with colposcopically evident dysplasia. By analyzing 72-kDa metalloproteinase positivity, regressive dysplasia showed low values of 72-kDa metalloproteinase immunostaining (median 1.2%, range 0.5-1.8%), while persistent (median 2.6%, range 1.9-3.6%) and progressive lesions (median 4.6%, range 2.3-6.9%) presented a significantly higher positivity (one-way analysis of variance; P < 0.001). DISCUSSION: In conclusion, the 72-kDa metalloproteinase expression is related to invasive potential with a significant increase in staining positivity in microinvasive carcinomas; 72-kDa metalloproteinase is detectable in cervical dysplasia, and it is related to the severity of cellular atypia. A clinical implication of 72-kDa metalloproteinase immunostaining seems to be indicated, by analyzing the differences in 72-kDa metalloproteinase positivity rates between regressive and persistent or progressive disease. PMID- 8626133 TI - Image analysis of cellular DNA content in peritoneal fluid of patients with ovarian tumors of low malignant potential and invasive epithelial ovarian cancer. AB - Image analysis has rarely been used to quantitate the DNA content of intact cells derived from peritoneal fluid in patients with ovarian malignancy. An average of 118 (range 100-208) of the most atypical, visually selected Feulgen-stained cells in peritoneal fluid obtained from 46 patients undergoing primary cytoreductive surgery for histologically proven ovarian tumors of low malignant potential and truly invasive ovarian cancer were evaluated retrospectively using the SAMBA-4000 Image analysis system. The patients were stratified into 3 groups: 16 with ovarian tumors of low malignant potential (LMP), 14 with low-stage disease (LSD) (FIGO I and II), and 16 with advanced-stage (ASD) (FIGO III and IV). A pattern of high-degree aneuploidy with negative balance (means: LMP, 3.3; LSD, 20.5; ASD, 32.0), increased proliferative index (LMP, 11.2; LSD, 16.1; ASD, 13.9), and percentage of cells with DNA content greater than 5C (LMP, 6.7; LSD, 6.5; ASD, 9.5) was demonstrated in the peritoneal fluid of 8 of 16 patients with LMP (50%), 8 of 14 patients with LSD (57%), and 13 of 16 with ASD (81%). The median disease free interval for patients with invasive epithelial ovarian cancer with peritoneal DNA diploid tumor cells was 57 months and for those with DNA aneuploid tumor cells 28 months, while in patients with LMP it was 65 and 54 months, respectively. In total, 19 patients developed a recurrence (LMP, 2; LSD, 5; ASD, 12) of which 17 were shown to have DNA aneuploid cells in the peritoneal fluid. Multivariate analysis, however, did not identify aneuploid population in the fluid, ploidy balance, proliferation indices, or degree of hyperploidy as an independently significant variable for predicting recurrence. It did appear, however, that tumor cells in peritoneal fluid with a degree of hyperploidy greater than 8 had a strong correlation for development of recurrence, although not statistically significant. Interactive image analysis of tumor cells in peritoneal fluid proved to be a valuable adjunct to cytodiagnosis. Seven of 28 patients (25%) who were underdiagnosed by cytology alone (LMP, 2; LSD, 3; ASD, 2) were shown to have malignant cells in their peritoneal fluid, while 2 of 18 patients (11%) who were called positive by cytology (LMP, 1; LSD, 1) showed diploid pattern histograms and upon review were interpreted as reactive mesothelial cells. PMID- 8626134 TI - Human papillomavirus detection of endometrioid carcinoma with squamous differentiation of the uterine corpus. AB - Many studies have shown a link between human papillomavirus (HPV) and cervical carcinoma. However, studies on the association of HPV with endometrioid carcinoma of the corpus uteri are sparse and controversial. In this study, 33 formalin fixed, paraffin-embedded tissue samples of endometrioid carcinoma with squamous cell differentiation in grade 1 (adenoacanthoma) and 10 additional samples of endometrioid carcinoma with less squamous cell differentiation in grade 2 or 3 (adenosquamous carcinoma) were examined by the hybrid capture system for the presence of the 14 most common anogenital HPV types, consisting of low-risk HPV types 6, 11, 42, 43, and 44, and intermediate- and high-risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56. No evidence of high-risk HPV DNA types was found in any of these samples. The low- risk HPV DNA types were found in three samples and showed borderline results (+/-) in 6 samples by the hybrid capture system. The 43 samples were tested by dot blot hybridization with HPV probes 6/11, 16/18, and 31/33/35. Only 1 sample was positive for HPV 6/11. The results of this study did not indicate an association between HPV infection and endometrioid carcinoma with squamous cells, though the endometrial mucosa of the corpus uteri is anatomically connected to the endocervical epithelium, and in some cases HPV has been postulated to possibly cause squamous cell differentiation of the endometrium. Our findings are in accord with the concept that HPV infection leading to malignancy is highly site- and tissue-specific. In conclusion, the endometrium may not be a suitable host epithelium for HPV replication and maturation. PMID- 8626135 TI - Prothrombin fragment F1+2 and thrombin-antithrombin III complex (TAT) plasma levels in patients with gynecological cancer. AB - The pretreatment plasma levels of prothrombin fragment F1+2 and thrombin antithrombin III complex (TAT) were measured in 56 consecutive patients with gynecological cancer and in 33 apparently healthy volunteer nonpregnant women as control. Prothrombin fragment F1+2 concentrations were significantly elevated in the 18 patients with ovarian cancer (median, 3.2 nmol/ liter; range, 0.9-17.0 nmol/liter, P < 0.0001), in the 24 patients with cervical cancer (median, 1.7 nmol/liter; range, 0.6-15.0 nmol/ liters, P < 0.0001), and in the 14 patients with endometrial cancer (median, 1.5 nmol/liter; range, 0.6-3.0 nmol/liter, P = 0.036) when compared to controls (median, 1.0 nmol/liter; range, 0. 1 -2.1 nmol/ liter). Similarly, TAT levels were significantly higher in patients with ovarian cancer (median, 5.3 microgram /ml; range, 1.3-65.0 microgram/ml , P < 0.0001), cervical cancer (median, 3.8 microgram/ml; range, 2.1 - 11.0 microgram / ml, P < 0.0001), and endometrial cancer (median, 2.7 microgram/ml; range, 1.9-19.0 microgram /ml, P = 0.008) when compared to controls (median, 2.2 microgram/ml; range, 1.0-5.0 microgram/ml). Prothrombin fragment F1+2 levels correlated with TAT levels (r = 0.659, P < 0.0001). Among ovarian cancer patients, prothrombin fragment F1+2 and TAT concentrations correlated with FIGO stage (III-IV versus 1, P = 0.01 and P = 0.003, respectively) and CA 125 levels (P 0.02 and P < 0.0001, respectively). The present data confirmed the occurrence of hemostasis activation in patients with gynecological cancer, and in particular in those with ovarian cancer. PMID- 8626136 TI - Management of low-risk gestational trophoblastic tumors with etoposide (VP16) in patients resistant to methotrexate. AB - Careful selection and treatment monitoring of patients with gestational trophoblastic tumors (GTT) is critical because 20 - 50% of patients may develop a resistance and consequently require alternative chemotherapeutic agents. In our study we propose and demonstrate the efficacy of etoposide (VP16) as a second line drug. An average of 5 courses with VP16 were used to achieve a remission in 12 patients resistant to MTX with low-risk GTT. Toxicity was mild and no resistance to VP16 was observed. A follow-up of 66 months (range, 22-109) has been performed for the patients and all of them are now disease free. Two patients had a pregnancy, respectively, 3 and 4 years after treatment. The others did not desire any pregnancy. PMID- 8626137 TI - Final results of a phase II chemoradiation protocol for locally advanced cervical cancer: RTOG 85-15. AB - BACKGROUND: The lack of improved cure rates for advanced cervical cancer after three decades of megavoltage radiotherapy (RT) has prompted continued efforts in improved treatment delivery. Concurrent chemoradiation (CR) is one of the several avenues being explored to improve these results. METHODS: Sixty women with advanced cervical cancer (30 patients with unfavorable Stage IIB and 30 patients with Stages III and IVA) were treated with CR comprising of a combination of external and intracavitary RT delivering between 7000 to 7500 cGy total to point A and 5890 to 6015 cGy to point B along with one cycle of 5-FU and mitomycin C and a second cycle of 5-FU and cis-platinum. RESULTS: Grade 3 and 4 RT-related toxicities were 15 and 3%, respectively. Chemotherapy-related Grade 3 and 4 toxicities were 9 and 2%, respectively. The 5-year survival for unfavorable Stage IIB patients was 48%; for Stages III and IVA it was 39%. CONCLUSIONS: The toxicity of this particular CR regimen was acceptable and suggests that further qualitative and quantitative intensification of chemoradiation may be attempted. Retrospective comparisons with PCS studies and previous RTOG studies 79-20 and 80 05 suggest that this particular chemoradiation regimen may offer a modest survival advantage over RT alone for Stages III and IVA disease. A CR regimen with higher doses of radiotherapy and a greater number of active chemotherapeutic agents may yet result in acceptable toxicity and further improve cure rates in advanced and poor prognostic featured cervical cancer. PMID- 8626138 TI - Mitotic count, nuclear atypia, and immunohistochemical determination of Ki-67, c myc, p21-ras, c-erbB2, and p53 expression in granulosa cell tumors of the ovary: mitotic count and Ki-67 are indicators of poor prognosis. AB - In spite of extensive research, the behavior of granulosa cell ovarian tumors remains unpredictable and is complicated by the lack of prognostic factors in early-stage disease. Forty patients with granulosa cell tumors were identified from tumor registries and data were analyzed for patient outcome. Mitotic count and nuclear atypia were determined at time of histological review. Paraffin embedded archival tumor tissues from 32 of 40 patients were available, and immunohistochemical testing for Ki-67, c-myc, p21-ras, c-erbB2, and p53 was performed on archival tissues. Results were correlated with patients' outcome. Mitotic count and Ki-67 were found to be negatively associated with survival in granulosa cell tumors. Nuclear atypia, c-myc, p21-ras, c-erbB2, and p53 were not found to be of prognostic significance. PMID- 8626140 TI - Loop electrocautery excisional procedure: therapeutic effectiveness as an ablation and a conization equivalent. AB - This study was designed to compare the effectiveness of loop excision procedures as ablation and conization equivalents, and their use in recurrent cervical high grade dysplasia patients. We retrospectively reviewed patients undergoing loop procedures between January 1992 and April 1994. Conization equivalents were defined as procedures performed for unsatisfactory colposcopy, positive endocervical curettage, discordant cytology and histology, and cytology suggestive of invasion. Effectiveness in high-grade recurrent lesions was evaluated separately. Therapeutic failure was defined as any recurrence of high grade dysplasia. One hundred sixty-two patients underwent loop procedures, of which 123 (75.9%) had follow-up examinations and are included. Mean follow-up was 17.9 months. Seventy-six patients (61.8%) had the loop procedure performed as an ablation equivalent, 29 (23.6%) as a conization equivalent, and 18 (14.6%) for recurrent disease. Overall effectiveness was 82.1 % and for ablation and conization equivalent procedures was 82.9 and 82.8%, respectively. There was no significant difference between effectiveness when performed for initial therapy (82.9%) and for recurrent disease (72.2%) (P = 0.46). In conclusion, loop excision procedures are effective as ablation and conization equivalents and as treatment for recurrent high-grade dysplasia. PMID- 8626139 TI - Transfection of human ovarian cancer cells with the HER-2/neu receptor tyrosine kinase induces a selective increase in PTP-H1, PTP-1B, PTP-alpha expression. AB - Protein tyrosine phosphorylation is a key regulator of cell growth regulation and, when aberrant, is linked with the process of oncogenic transformation. Since tyrosine phosphatases (PTP) reverse phosphorylation mediated by tyrosine kinases (PTK), it has been hypothesized, but insufficiently studied to date, that PTPs function as tumor suppressors. Alternatively, PTPs may augment signal transduction by repriming substrates. To begin to assess the role of PTPs in ovarian cancer cell growth regulation, PTP partial cDNAs were cloned from the OVCA 433 ovarian cancer cell line using RT/PCR and degenerate oligonucleotide primers for the PTP consensus domain. Thirteen partial cDNAs were isolated, the sequences of which, when compared to GenBank, suggested that they were derived from PTP family members. These included PTP-alpha, PTP-gamma, LAR, PTP-delta, T cell PTP, PTP-2A(1D), PTP-1B, PTP-1C, PTP-H1, PTP-PEST, PTP-Gallus, and two isolates which potentially represent novel PTPs. Steady-state expression analyses revealed that PTP-1B expression was undetectable in normal epithelium but was expressed in four of five ovarian cancer cell lines. In contrast, the expression of two SH2-containing PTPs, PTP-1C and PTP-2A, was detected in the normal ovarian epithelium but lost from one or more ovarian cancer cell lines. Finally, PTP-1B, PTP-alpha, and PTP-H1 expression was increased following HER-2/neu transfection of ovarian cancer cell lines. These results suggest that both normal ovarian epithelial cells and ovarian cancer cells express multiple PTPs. Further, some PTPs are differentially expressed between normal ovarian epithelium and ovarian cancer cells. Intriguingly, the transfection and increased expression of the prognostically significant HER-2/neu PTK induced a selective increase in the expression of the PTP-alpha, PTP-1B, and PTP-H1 tyrosine phosphatases. PMID- 8626141 TI - p53 as a prognostic indicator in endometrial cancer. AB - BACKGROUND: One of the most common genetic alterations to occur in human cancers is an alteration of the p53 tumor suppressor gene. Although endometrial cancer is the most common gynecologic malignancy in the United States, any connection between it and p53 is just beginning to be explored. METHODS: Forty-six consecutively surgically treated patients with endometrial cancer had their p53 expression studied by immunoperoxidase staining and quantified by image analysis. RESULTS: Thirty-five patients had endometrioid adenocarcinomas, 3 had adenosquamous carcinomas, 3 had papillary serous carcinomas, 3 had clear cell carcinomas, and 2 had undifferentiated carcinomas. p53 expression ranged from 0.0 to 55.8% with a mean of 10.7% for the cohort. For the patients with endometrioid carcinomas, the mean p53 expression was 3.9%, while for those with more aggressive histologies it was 32.4% (P < 0.001). Sixteen of the 35 endometrioid tumors (45.7%) stained positive for p53, while 11 of the remaining 12 (91.2%) tumors with more aggressive histologies stained positive (P < 0.01). Increasing histologic grade correlated with an increasing p53 expression (P = 0.006). The percentage of patient tumors expressing p53 was found to be higher in FIGO stage II, III, and IV than in FIGO stage I cancer (P = 0.01). However, the mean p53 expression was not different between early (stage 1) and advanced (stage 11, III, and IV) cancers (P = 0.55). Utilizing recurrence as the endpoint for multivariate analysis, FIGO stage and p53 expression were the only independent prognostic indicators found. CONCLUSION: p53 expression is more common in more aggressive histologic subtypes than in endometrioid adenocarcinomas. It is an independent prognostic indicator of disease recurrence. PMID- 8626142 TI - Treatment of refractory ovarian cancer with 5-fluorouracil and leucovorin. AB - The objective of this study was to evaluate the activity of the combination of 5 fluorouracil (5-FU) and leucovorin in women with platinum-resistant recurrent or advanced ovarian cancer. A retrospective study was conducted of consecutive patients treated with weekly intravenous 5-FU and leucovorin, both at 500 mg/m2. Sixty-one cycles were administered to 28 women between 1989 and 1995. The median age of the patients was 58 years and median number of previous chemotherapy regimens was 2. No patient had a complete response. There was 1 partial response (PR) among 8 patients with measurable disease, and 4 PR among 20 women with evaluable disease; 2 had stable disease and 21 progressed on treatment for a response rate of 18% (95% confidence interval, 3-33%). Duration of response was 2 to 14 months, median 5 months. Survival from the start of 5-FU/leucovorin ranged from 1 to 78 months, median 5.5 months, and a mean of 10 +/- 15 months (mean +/- SD). The combination of 5-FU/leucovorin had minimal activity in this patient population. Only 2 women achieved resolution of their ascites and experienced a significant palliation. PMID- 8626143 TI - Malignant melanoma of the vulva FIGO stage I: Evaluation of prognostic factors in 43 patients with emphasis on DNA ploidy and surgical treatment. AB - Forty-three cases of malignant melanoma of the vulva FIGO stage I, primary treated from 1956 to 1987, have been reviewed. Initial surgery was local excision in 14 patients, vulvectomy in 14, and radical vulvectomy with inguinal lymph node dissection in 15. Recurrent disease was seen in 27 (63%) patients. The 5-year corrected survival was 63%; 10-year survival was 52%. Independent prognostic factors for disease-free and long-term survival were angioinvasion and DNA nondiploidy. Tumor thickness was of prognostic importance in univariate analysis, but did not obtain independent significance. Initial surgical modality did not influence long-term survival, but affected disease-free survival significantly. Local excision carried the greatest risk of local recurrence, but in some of these patients secondary surgery was successful. Because radical surgery did not improve long-term prognosis, wider local excision or vulvectomy seems to be the recommended surgical approach. PMID- 8626144 TI - Primary ovarian carcinoid tumors. AB - Primary ovarian carcinoid tumors were reviewed from Mayo Clinic and Colorado Tumor Registry data. A total of 17 patients with this diagnosis were identified. Histologic analysis of these carcinoid tumors revealed 9 (53%) were insular, 5 (29%) were trabecular, and 4 (26%) were strumal carcinoid with or without a mature dermoid component. There were 11 patients with stage I and 6 patients with stage III or IV disease at diagnosis. Carcinoid syndrome was found in 29% of patients. Pressure or pain with defecation was noted in 41% of cases. Recurrence of tumor occurred in 1 of 11 patients with suspected stage I disease 13 years after initial diagnosis. Overall survival was excellent in the 11 patients whose disease was confined to one ovary (100% 5-year survival), but only 1 of 6 patients survived (33% 5-year survival) if advanced stage at diagnosis. Systemic chemotherapy for advanced disease achieved a complete response in 1 patient and stable disease in another. Although rare, primary ovarian carcinoid tumors treated with surgery alone and found to be confined to the ovary can be expected to have an excellent overall outcome. PMID- 8626145 TI - Subcellular localization of accumulated p53 in ovarian cancer cells. AB - Inactivation of the tumor suppressor gene p53 is frequently associated with ovarian cancer. Accumulation of stabilized p53 protein is a common feature in this tumor type. Underlying mutations in the p53 core region can lead to loss of the normal conformational state or loss of residues necessary for DNA binding and transcriptional regulation. Five HPV-free ovarian cancer cell lines established in our laboratory with and without immunocytochemically detectable p53 expression were selected for the correlation of subcellular localization of aberrant p53 and the type of gene mutation. The expression level regarding staining intensity and proportion of cells accumulating p53 was characterized employing an immunoreactive score. Two cell lines with point missense mutations in the core region showed strong nuclear or nuclear plus cytoplasmic staining. One cell line with exclusive staining of the cytoplasm contained a deletion of the major nuclear localization signal. Among two cell lines without p53 accumulation, one contained a microdeletion resulting in a frame shift, the other carried the wild type sequence. The MDM2 oncogene was not amplified and its gene product was not overexpressed. In ovarian cancer, inactivated p53 can accumulate in both major cell compartments depending on the type of the underlying mutation. PMID- 8626146 TI - Malignant degeneration in leiomyomatosis peritonealis disseminata. AB - Leiomyomatosis peritonealis disseminata (LPD) is a rare clinico-pathologic entity typically observed in women of reproductive age. Its malignant degeneration has been reported in literature in only three cases. In this report we describe the clinical course of two more cases with proved malignant transformation of LPD; both cases were treated with combined chemotherapy. PMID- 8626147 TI - Luteinized thecoma with sclerosing peritonitis presenting as an acute abdomen. AB - The seventh reported case of luteinized thecoma with sclerosing peritonitis is described. Unlike the other reported patients with this condition, the patient presented with a short history of secondary amenorrhea, a subsequent acute abdomen, and an absence of abdominal distention and ascites. The tumor had undergone extensive hemorrhagic necrosis secondary to infarction, and the patient's amenorrhea may have been secondary to the release of steroid hormones from the necrotic cells. The presence of extensive necrosis and a brisk mitotic rate raised the suspicion of a malignant thecoma, but the finding of subserosal fibrosis in the biopsied omental tissue led to the diagnosis of a benign luteinized thecoma with sclerosing peritonitis. PMID- 8626149 TI - Metastatic clear cell eccrine hidradenocarcinoma of the vulva: survival after primary surgical resection. AB - A case of clear cell eccrine hidradenocarcinoma of the vulva metastatic to regional lymph nodes with long survival after surgical resection is presented. Like the only other case reported to date, this suggests that surgical therapy alone may be adequate, even when metastasis is present. PMID- 8626148 TI - Hepatic venoocclusive disease as a complication of whole abdominopelvic irradiation and treatment with the transjuglar intrahepatic portosystemic shunt: case report and literature review. AB - We report the novel use of the transjugular intrahepatic portosystemic shunt (TIPS) procedure for the treatment of intractable ascites due to hepatic venooclusive disease as a result of whole abdominopelvic radiotherapy. A patient with Stage III endometrioid carcinoma of the endometrium treated with postoperative whole abdominopelvic irradiation developed intractable ascites. Multiple paracenteses and computerized tomography were negative for recurrent carcinoma. Liver biopsy demonstrated hepatic venoocclusive disease, a rare complication of therapeutic radiation involving the liver. Successful relief of ascites and its adverse symptomology were achieved with the transjugular intrahepatic portosystemic shunt. Relevant literature regarding the pathogenesis, prognosis, and treatment of radiotherapy-related hepatic venoocclusive disease are reviewed. PMID- 8626150 TI - Pilomatricoma-like endometrioid adenosquamous carcinoma of the ovary with neuroendocrine differentiation. AB - A case of high-grade endometrioid adenosquamous carcinoma of the ovary showing pilomatricoma-like areas and neuroendocrine differentiation is presented. The pilomatricoma-like areas were strongly positive for cytokeratin and vimentin, whereas the poorly differentiated areas of the tumor were positive for cytokeratin, synaptophysin, and neuron-specific enolase. Review of the literature did not show any published report of adenosquamous carcinoma with pilomatricoma like areas either in the ovary or in the uterus. PMID- 8626151 TI - Transitional cell bladder carcinoma with presentation mimicking ovarian carcinoma. AB - In the case described here, the patient's initial presentation suggested ovarian carcinoma. She had recurrent ascites, a pelvic mass, elevated CA-125, and extensive peritoneal carcinomatosis with transitional cell histology. The presence of hematuria prompted a cystoscopy, which revealed the true site of origin to be the urinary bladder rather than ovaries. This presentation is extremely rare for bladder cancer. Since transitional cell tumors from the bladder have a much worse prognosis than those of ovarian origin, it is important to identify the primary site correctly. Therefore, cystoscopy is essential for patients with hematuria, and should be considered in cases of apparent primary peritoneal carcinoma with transitional cell histology. PMID- 8626152 TI - High levels of CA-125 in a case of a parasitic leiomyoma presenting as an abdominal mass. AB - A 32-year-old woman presented with increasing abdominal girth and discomfort secondary to a 18-week-size mass and a CA-125 level of 1539. She underwent an exploratory laparotomy and resection of a parasitic fibroid following which the CA-125 levels decreased and normalized within a month. A review of English literature indicates that association of raised CA-125 levels with fibroids is inconsistent and very modest and such high levels have not been previously reported. PMID- 8626153 TI - Use of health insurance in county-funded clinics: issues for health care reform. AB - This article determines to what extent people with and without health care insurance use public health care facilities and various health care services. A descriptive study analyzed the characteristics of 6,298 patients from four county funded primary health care clinics. The patients were overwhelmingly poor regardless of insurance status. High cost-sharing requirements, coupled with larger families and low wages, may be causing some people with insurance to use public clinics. Implications for social work policy and practice are discussed. PMID- 8626154 TI - Online help: cancer patients participate in a computer-mediated support group. AB - This study investigated a computer mediated support group of six breast cancer patients. For a three-month period, patients used home computers to connect to a computer bulletin board on which they read messages from and posted messages to each other. The patients had no difficulty learning to use the computer and used it an average of one hour a week. The patients discussed their medical conditions, shared personal concerns, and offered support. This online approach provided many features of traditional face-to-face support groups. PMID- 8626155 TI - NAFTA, American health, and Mexican health: they tie together. North Atlantic Free Trade Agreement. PMID- 8626157 TI - Screening for social and environmental problems in a VA primary care setting. AB - Social workers are in an ideal position to identify and treat social and environmental problems early in the continuum of care. Information on these problems will facilitate informed decision making on the development and reallocation of resources to better meet patients needs. This study assessed the social and environmental problems of 132 patients seen in a primary care clinic at a university-affiliated Veterans Affairs (VA) medical center. The most prevalent social problems were financial difficulties, personal stress, family problems, legal concerns, and employment concerns. When asked, nearly one-third of all respondents requested social work services or information about services related to their problems. The findings suggest a clear need for social work interventions in VA primary care clinics that focus on both psychosocial problems. PMID- 8626156 TI - Implementation of discharge plans for chronically ill elders discharged home. AB - Although discharge plans are viewed as the primary means to ensure that patients' needs will be met in the posthospital environment, little is known about the implementation of arranged care. This study addressed the extent to which discharge plans for elderly patients with congestive heart failure were implemented as planned, tested the consequences of implementation problems, and identified factors associated with implementation problems. For 40 percent of patients, one or more components of the discharge plan were not implemented as planned, with discrepancies more likely among low-income patients. Implementation discrepancies had negative consequences in terms of unmet needs, deficient quantity of help, and less than adequate care. Implications for hospital discharge planners and home health care are discussed. PMID- 8626158 TI - Anticipatory grief and AIDS: strategies for intervening with caregivers. AB - Anticipatory grief may have beneficial effects for caregivers of people with HIV infection or AIDS. However, the duration of the illness and the stigmatization and multiple losses associated with the disease may impede the caregiver's ability to effectively engage in the grief process. This article discusses the impact of these aspects of the disease on the anticipatory grief process and mourning tasks for caregivers at each stage of the illness. Intervention strategies developed to help the caregiver remain actively involved with the patient and simultaneously grieve losses and prepare for death are specified. PMID- 8626160 TI - Are mental illnesses biological diseases? Some public policy implications. PMID- 8626159 TI - The challenge of HIV prevention among high-risk adolescents. AB - This article reports findings from an exploratory study of HIV knowledge and risk behaviors among 60 teenagers and young men engaged in the street life of Hollywood, California. The sample was composed largely of youths of homosexual or bisexual orientation who were substance abusers, prostitutes, or both. The data suggest that although community-based education efforts may be associated with lower-risk behavior among this population, the overall risk profiles of these socially marginalized youths remained high. Inferences are drawn about the cofactors of risk that must be addressed and the education needed to enhance the health prospects of these youths. PMID- 8626161 TI - An empowerment-centered, church-based asthma education program for African American adults. PMID- 8626162 TI - Managing mental health: whose responsibility? PMID- 8626163 TI - Effects of ageism on individual and health care providers' responses to healthy aging. AB - Although misconceptions about the aging process have lessened over recent years, ageism is still having a detrimental effect on healthy aging. This article reviews the literature to support this contention and to demonstrate how stereotyping can affect the shape and nature of programs for elderly people. It is argued that for long-lasting change to occur, service providers need to directly target these negative attitudes in themselves, their professional institutions, their clients, and their communities. Suggestions are made for professional development, research, and program planning. PMID- 8626165 TI - Statement regarding early orthodontic treatment. Deutsche Gesellschaft fur Kieferorthopadie. PMID- 8626164 TI - Quo vadis? PMID- 8626166 TI - Bending and torquing accuracy of the bending art system (BAS). AB - With the bending art system (BAS) the computerized production of individual arch wires has become possible. The BAS consists of an intraoral camera, a computer program and a bending machine producing the archwire by consecutive bending and twisting procedures. This study examines the accuracy of the bending machine when using 0.016" x 0.016" and 0.016" x 0.022" steel wire of rectangular cross section. Bending angles ranging from 6 degrees to 54 degrees, and torsion angles ranging from 2 degrees to 35 degrees were tested; also the minimum distance between these individual operations was determined. The bent pieces of wire were analysed in a 3D-coordinate gauging system. The 0.016" x 0.016" steel wire showed a mean measuring error of 0.62 degree in bending procedures and of 0.72 degree in torsion procedures, whereas the 0.016" x 0.022" steel wire showed an error of 0.87 degree with edgewise bendings and of 0.86 degree with torsions. To ensure this accuracy a minimum distance of 0.5 mm to 0.7 mm, depending on which kind of bending combination is used, between bending and torsion is required. The error could be reduced even further if a more constant wire material and a more accurate calibration of the bending machine were used. All in all the precision of the bending machine meets the clinical requirements. PMID- 8626167 TI - Mechanical stretching of periodontal ligament fibroblasts--a study on cytoskeletal involvement. AB - To investigate molecular aspects of the mechanism(s) involved in orthodontic tooth movement, periodontal ligament fibroblasts (PDL) were isolated and grown on culture dishes with a flexible bottom. The cells were stimulated by stretching the bottom of the culture dishes and whole cell extracts were prepared and subjected to sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoresis, electrotransferred to nitrocellulose membrane and immunoprobed with antibodies specific for vimentin and alpha- and beta-tubulin. No apparent alterations in the expression profile of the above mentioned major cytoskeletal elements were evident after mechanical stretching. Moreover, immunofluorescence against the same proteins revealed no changes in their topographical organisation between stretched and unstretched cell cultures. PMID- 8626168 TI - Integration of three-dimensional cephalometry and 3D-skull models in combined orthodontic/surgical treatment planning. AB - In 15 adult patients with severe dentomaxillofacial deformities we integrated 3 dimensional cephalometry and 3D-model surgery with individually milled or stereolithographically built skull models in our combined orthodontic/surgical diagnosis and treatment planning. After the generation of contiguous axial CT scans the CT data sets were transferred to a commonly used computing system (IBM PC) to reconstruct 3D-images from any point of view. After the definition of measurement points, distances and angles at the skin and bone surface a 3 dimensional cephalometric analysis could be performed directly in the 3D-objects on the monitor. This allows a quantitative assessment of skeletal asymmetries. The transfer of CT data to life-size 3D-skull models and replacement of imprecise dental arches by dental casts different orthodontic and surgical treatment concepts could be evaluated. The 3D-model surgery represents a new quality of treatment prediction in the individual dentomaxillofacial morphology. The orthodontic set-up and 3D-model surgery permit a verification of the feasibility of the most suitable mobilization and placement of bone segments. The clinical treatment sequences indicated that the integration of 3-dimensional cephalometry and 3D-model surgery in patients with severe asymmetric dentomaxillofacial deformities allowed a higher precision of diagnosis and treatment planning. PMID- 8626170 TI - Mandibular distraction osteogenesis as first step in the early treatment of severe dysgnathia in childhood. AB - The sole orthodontic treatment of severe dysgnathias in childhood often leads to unsatisfactory results. On the other hand, standard surgical procedures are very difficult and due to their high risks not practicable in early childhood. The distraction osteogenesis enables us to correct hypoplastic mandibles, so that secondary malformations of the midfacial complex can be avoided. During the operation the hypoplastic site of the mandible is osteotomized behind the last visible tooth bud and a bidirectional distractor is inserted. Following the principles of Ilizarov the new callus is lengthened gradually until the required length of the mandible has been achieved. Out of a total sample of 27 patients 3 case reports of young children are presented. The new surgical concept describes new treatment perspectives. PMID- 8626169 TI - Analysis of forces and moments in arch guided molar protraction using Class I and Class II elastics. An in-vitro study. AB - The use of class I and II elastics in arch guided tooth movement of the lower molars belongs to the proven clinical methods to achieve space closure even though risks are present. The vertical force component of class II elastics tends to interact with the sagittal force and thus the vertical force may change the desired sagittal force and movement direction. The objective of the study presented here was to investigate friction behavior and the movement dynamics of the arch guided protraction of the lower first molar being acted on by differing class I and class II elastic band geometries. The influence of class I and class II elastics at different force levels (1 N and 2 N) were studied. The pattern of the force line varied in the area of angulation from 0 degree to 40 degrees relative to the arch plane. The orthodontic measurement and simulation system (OMSS) was employed to determine force loss due to friction and to analyze side effects. In the arch guided mesialization of the lower first molar, the vertical component of class II elastics induces a minor force loss in comparison with class I elastics. This holds, however, only for the lower 1 N force level. When employing class II bands at a greater force level and with increased angulation, relatively greater force loss and increased side effects, such as extrusion and mesial tipping of the first molar, occur. PMID- 8626171 TI - Profile changes following extraction vs. nonextraction orthodontic treatment in a pair of identical twins. AB - A pair of 11.5-year-old monozygotic twin boys--with a class I occlusion, deep bite and crowding in the lower arch--was presented for orthodontic treatment. One of them was treated in combination with extraction of 4 premolars while in the other the treatment was carried out without extractions. The superimposition of the pretreatment cephalometric tracings on the N-Pog line displayed minimal differences in the sagittal position of the incisors and no differences in the soft tissue profile. As a result of protrusion of the incisors in the nonextraction case and retrusion in the extraction case, a sagittal difference of 7.0 mm for the incision superius and 5.5 mm for the incision inferius was registered between the twins in relation to the N-Pog line at the end of treatment. However, the corresponding soft tissue differences were only 2.0 mm for the labrale superius and 3.2 mm for the labrale inferius and seemed to be reduced 1 year later. PMID- 8626172 TI - [Parkinson disease. Early diagnosis and combined therapy for better prognosis]. PMID- 8626173 TI - Gerodontology: now is the time for setting a health services research agenda. PMID- 8626174 TI - Comparison of the effect of the linseed extract Salinum and a methyl cellulose preparation on the symptoms of dry mouth. AB - The effect of a linseed extract Salinum and a sodium carboxymethyl cellulose preparation called MAS-84 was compared with regard to its effect on the symptoms of dry mouth. Twenty patients with xerostomia, who had been treated for cancer in the head and neck by radiation were recruited from the clinic for maxillofacial surgery, Malmo University Hospital. Following radiation treatment the salivation was severely reduced. The symptoms of a general feeling of a dry mouth, difficulties in chewing and swallowing, taste disturbances, problems with speech and mouth burning were registered on a subjective verbal rating scale. In addition plaque index and gingival bleeding were determined. The study design was crossover and performed single blind. The experimental period was 7 weeks. The patients were randomly divided into 2 groups. One group used Salinum and the other MAS-84 for 3 weeks. The fourth week was a wash out period and for the next three weeks the patients shifted preparation. Each of the preparations was used ad libitum. Registrations of the various parameters were undertaken on days 0, 7 and 21 of the respective period. At the initial examination all patients reported considerable disturbances from mouth-dryness. These symptoms were reduced in 15 patients during the Salinum period and in 9 during the MAS-84 period. The relief was significantly more pronounced during the use of Salinum compared to that during the use of the methyl cellulose preparation. On day 21 plaque and gingival bleeding were significantly reduced during the Salinum period but not during the MAS-84 period. The results of the present study confirm those of a previous pilot study and indicate that the linseed mucilage significantly reduced the symptoms of dry mouth. This effect increased with increasing time of saliva substitute use. The linseed mucilage Salinum appeared to be a suitable saliva replacement in mouth dry patients. PMID- 8626175 TI - Xerostomia in older adults: a longitudinal study. AB - Although xerostomia in older adults has received substantial research attention, there have been few longitudinal studies of non-patient populations. Consequently, little is known about the incidence or course of this condition among this group. This paper reports the results of a longitudinal study designed to address these issues. In 1989, data on xerostomia were collected from 907 randomly-selected community dwelling adults aged 50 years and over. Three years later, 611 (71% of those presumed to be alive) were followed-up and data on xerostomia collected again. At baseline, 15.5% of these 611 subjects reported xerostomia, while at follow-up this had risen to 29.5%. The majority of the latter (115/180) were incident cases, reporting xerostomia only at follow-up, while the remainder were chronic cases, reporting xerostomia at both baseline and follow-up. A crude estimate of the three-year incidence rate was 22.5%. In a logistic regression analysis, three baseline variables were associated with incidence; older subjects, those with one or more chronic medical conditions and those reporting their general health as poor were more likely to develop xerostomia. In a similar analysis, age was the only variable associated with chronicity, with older subjects more likely to be chronic cases. The data also suggest that the onset of xerostomia was associated with an increase in other oral symptoms and problems with eating, communication and social interaction. PMID- 8626176 TI - Tomographic measurements of age changes in the human parotid gland. AB - This study involved 48 subjects of both sexes with ages ranging from 22 years to 90 years. Computerised tomography was used to scan the right and left parotid gland. Gland mean density was calculated in Hounsfield units and regression graphs drawn. A significant fall in gland density was noted with subject age but no differences were noted between male and female subjects. A significant correlation was observed between the mean density of right and left glands in the same individuals. It is postulated that the fall in gland density is related to an increase in fibro-fatty tissue within the gland. This study provides evidence that age related changes occur in the human parotid gland from youth to old age. This work supports the earlier findings of Scott's who studied the superficial lobe of the parotid gland histologically and found an increase in adipose tissue in this area of the gland with age. PMID- 8626177 TI - Food for thought. Guidelines for putting oral health into the context of healthy eating for older people. British Dental Association Community Dental Services Group and the Gerodontic Study Group. PMID- 8626178 TI - The masseteric jaw-jerk reflex in older dentate subjects and edentulous denture wearers. AB - The aim of the present study was to investigate any variations in the jaw-jerk reflex in edentulous subjects wearing complete dentures, compared to an age and sex-matched dentate group. The reflex was elicited by chin taps in 22 older dentate subjects with mean age 61.3 years and in 22 denture wearers with mean age 63.1 years. Surface electromyographic recordings were obtained from the masseter muscle of the preferred chewing side during mandibular rest and at moderate clenching (40% of the individual maximum clenching masseteric EMG activity). A jaw-jerk reflex was recorded in all subjects at least once, and its occurrence during clenching was reduced compared to rest. The occurrence of the reflex was however increased in the denture wearers in both experimental conditions, while minor differences were observed in the values for latency, duration and amplitude between the two dental status groups. These results suggest that under the present experimental conditions the periodontal ligament receptors might inhibit reflex activity. Multiple sensory interactions are expected in denture wearing. However a particular source of sensory feedback is provided by the stimulation of mucosal receptors from the acrylic denture base. Since the occurrence of the jaw jerk at clench in the denture wearers was also reduced compared to the rest experiments, a potential inhibitory effect of the mucosal receptors can be speculated. According to the findings in the present study the loss of teeth and the rehabilitation with complete dentures do not severely disrupt the reflex activity investigated. PMID- 8626179 TI - An epidemiological survey of oral mucosal lesions among elderly Malaysians. AB - A house to house random survey on elderly subjects was undertaken in the District of Klang in Malaysia. The objective of this study was to investigate the prevalence of oral mucosal lesions (OML) among the elderly in this area. The primary units in the sampling frame were the Enumeration Blocks (EBs) as defined under the population census. All households of the selected EBs were considered as sampling units and members aged 60 and above were considered as respondents. There was a slight preponderance of females, with the Malays comprising the majority of the subjects. Of the 486 respondents, mean aged 69.1 +/- 7.3 yr, 111 had at least one oral mucosal lesion, a prevalence of 22.8%. A total of 145 lesions were detected. The prevalence of OML was highest among Indians and least among the Chinese. The most common finding was tongue lesions, recording a prevalence of 10.7%, followed by oral pigmentation (4.9%) and white lesions (4.3%). Denture related lesions were comparatively low at 2.5%. Two cases of oral cancer if representative would give a relatively high prevalence of 0.4%. PMID- 8626180 TI - Oral health status of a population of community-dwelling older Canadians. AB - A sample of 170 responsive residents of seniors housing centres in Winnipeg, Canada, were studied, (>65 years, mean 82 years), with the objectives of relating their dental state to their perception of need and uptake of service. Only 6% rated their oral health as poor, 46% reported a dental visit within the previous year and 68% felt they needed dental treatment. A lack of perceived need (88%) was the primary reason why dental care was not sought more frequently. Hygiene practices revealed that only 7% brushed < 1 time/day, 60% never flossed, 14% cleaned their dentures <1 time/day, and 42% slept with their dentures. Dental histories showed that examination (94%), prosthodontic treatment (76%), and restorative services (65%) were the most commonly sought treatments. Perceived dental needs included prosthodontic treatment (39%), periodontal/prophylactic treatment (10%), restorative treatment (9%) and pain relief (9%). Study subjects had 2.8 decayed teeth, a DMFT of 25.1, and a Root Caries Index of 38%. CPITN scores of 3 or 4 in at least one sextant were found in 80% of subjects. Of the 41% edentulous, all wore complete dentures but 15% of complete upper dentures and 51% of complete lower dentures fitted poorly. Of the partial dentures, 20% fitted poorly. Soft tissue anomalies were seen in 67% of subjects and 47% had TMJ anomalies. Overall, 77% of edentulous subjects and all dentate subjects required some dental treatment even though 46% had seen a dentist within the preceding year. It is concluded that appropriate management of such people needs further attention. PMID- 8626181 TI - Examiner agreement on caries detection and plaque accumulation during dental surveys of elders. AB - Indices used to evaluate plaque accumulation and coronal caries have been widely accepted in epidemiological studies, yet their reliability cannot be guaranteed. The aim of this study was to evaluate the reliability of clinical criteria used in coronal and root caries diagnosis and oral hygiene evaluation as applied in elders. Nineteen elderly subjects, 73 years old on average, were examined at a first appointment by two independent examiners. They were re-examined two weeks later. Plaque accumulation was evaluated using the Plaque Index (PI) and coronal and root caries were detected according to the WHO criteria and Fejerskov et al. (1991), respectively. Recurrent caries was recorded as recommended by WHO and by probing at the interface tooth-restoration. Inter- and intra-examiner agreement was evaluated using kappa statistics. The PI score showed good reliability except for examiner b, for whom a simplification of the 4-point scale in 3-point scale improved significantly the reliability. The prevalence of coronal caries was very low and intra- and inter-examiner agreement was poor. Most of the root caries lesions were covered by plaque and the kappa values indicated only poor agreement. Recurrent caries were found with good agreement using WHO criteria but the detection with the probe was not reliable. In conclusion, it seems that examiners should be trained carefully to maximise their reliability and that plaque should be removed to obtain reliable diagnoses of caries. Retraining and calibration may be necessary for surveys continuing over a long period. PMID- 8626182 TI - Ageing: physiology or pathology? PMID- 8626183 TI - A robust parametric estimator for single-trial movement related brain potentials. AB - Current estimators for single-trial evoked potentials (EP's) require a signal-to noise ratio (SNR) of 0 dB or better to obtain high quality estimations, yet many types of EP's suffer from substantially lower SNR's. This paper presents a robust evoked-potential-estimator (REPE) facilitating high quality estimations of single movement related EP's with a relatively low SNR. The estimator is based on a standard ARX model, enhanced to support estimation under poor SNR conditions. The REPE was tested successfully on a computer simulated data set giving reliable single-trial estimations for the low SNR range of around -20 dB. THe REPE was also applied to experimental data, producing clear single-trial estimations of movement related brain signals recorded in a classic scenario of self-paced finger tapping experiment. PMID- 8626184 TI - Nonlinear alignment and averaging for estimating the evoked potential. AB - This paper addresses the problems associated with averaging brain responses evoked through a repetitive application of an external stimulus. In order to improve the estimate of the evoked potential (EP) through signal averaging, a method which incorporates nonlinear alignment of the EP's into the averaging operation is developed. The method makes no prior assumptions about the properties of the EP or which response in the set best characterizes the EP to be estimated. The nonlinear alignment procedure is designed to pairwise generate optimally aligned EP's by backtracking along the optimal alignment path. The nonlinear alignment and averaging operations are systematically combined to develop methods to estimate the EP. Results from a series of experiments conducted on simulated and real sets of responses show that, through nonlinear alignment and averaging, the events in the EP's are preserved and the estimates of the EP are quite robust. PMID- 8626185 TI - Parametric signal restoration using artificial neural networks. AB - The problem of parametric signal restoration given its blurred/nonlinearly distorted version contaminated by additive noise is discussed. It is postulated that feedforward artificial neural networks can be used to find a solution to this problem. The proposed estimator does not require iterative calculations that are normally performed using numerical methods for signal parameter estimation. Thus high speed is the main advantage of this approach. A two-stage neural network-based estimator architecture is considered in which the vector of measurements is projected on the signal subspace and the resulting features form the input to a feedforward neural network. The effect of noise on the estimator performance is analyzed and compared to the least-squares technique. It is shown, for low and moderate noise levels, that the two estimators are similar to each other in terms of their noise performance, provided the neural network approximates the inverse mapping from the measurement space to the parameter space with a negligible error. However, if the neural network is trained on noisy signal observations, the proposed technique is superior to the least-squares estimate (LSE) model fitting. Numerical examples are presented to support the analytical results. Problems for future research are addressed. PMID- 8626186 TI - Direct speech feature estimation using an iterative EM algorithm for vocal fold pathology detection. AB - The focus of this study is to formulate a speech parameter estimation algorithm for analysis/detection of vocal fold pathology. The speech processing algorithm proposed estimates features necessary to formulate a stochastic model to characterize healthy and pathology conditions from speech recordings. The general idea is to separate speech components under healthy and assumed pathology conditions. This problem is addressed using an iterative maximum-likelihood (ML) estimation procedure, based on the estimation-maximization (EM) algorithm. A new feature for characterizing pathology, termed enhanced-spectral-pathology component (ESPC), is estimated and shown to vary consistently between healthy and pathology conditions. It is also shown that the mean-area-peak-value (MAPV) and the weighted-slope (WSLOPE) indexes, which are obtained from the ESPC estimate, are meaningful measures of speech pathology conditions. For classification purposes, a five-state hidden-Markov-model (HMM) recognizer was formulated, based on the MAPV, WSLOPE, and ESPC spectral features. A set of log Mel-frequency filter bank coefficients were used to parameterize the ESPC feature. An evaluation of the HMM-based classifier was performed using speech recordings from healthy and vocal fold cancer patients of sustained vowel sounds. It is shown that while both MAPV and WSLOPE are useful features for vocal fold pathology detection, superior performance was achieved using a finer spectral representation of ESPC (e.g., a detection rate of 88.7% for pathology and 92.8% for healthy condition). One main advantage of the proposed method is that it does not require direct estimation of the glottal flow waveform. Therefore, the limitation of the inability to characterize vocal fold pathology, due to incomplete glottal closure, is no longer an issue. The results suggest that general analysis of the ESPC feature can provide a quantitative, noninvasive approach for analysis, detection, and characterization of speech production under vocal fold pathology. PMID- 8626188 TI - The surface Laplacian of the potential: theory and application. AB - The use of the surface Laplacian of the potential (Ls) in bioelectricity is discussed. Different estimates of Ls, in particular the field measured by coaxial electrodes, are compared to that of the true Laplacian. A method to compute Ls on the surface of an inhomogeneous volume conductor of arbitrary shape resulting from assumed electrical sources in introduced. In two applications the sensitivity of the body surface Laplacian is carried to that of body surface potentials. This comparison is carried out for dipolar sources within the human brain as well as for distributed sources within the heart. PMID- 8626187 TI - The potential for Laplacian maps to solve the inverse problem of electrocardiography. AB - This paper presents a method to solve the inverse problem of electrocardiography using the Laplacian of the body surface potentials. The method presented is studied first using trade-off curves from a concentric spheres model representing a heart-torso system. Then a more conventional study is undertaken where a limited number of current dipoles are placed within the inner sphere and noise is added to the resulting potentials and Laplacians on the surface of the outer sphere. The result indicate that measurements of the outer surface Laplacian can more accurately reconstruct epicardial potentials than measurements of the outer surface potentials. The reconstructions are more accurate in that extrema are placed very close to their correct positions and multiple extrema and high potential gradients are recovered. Identical conclusions are observed in the presence of noise and even when the Laplacians are subject to greater noise than the potentials. PMID- 8626189 TI - Evaluation of inductively heated ferromagnetic alloy implants for therapeutic interstitial hyperthermia. AB - Ferromagnetic alloys heated by magnetic induction have been investigated as interstitial hyperthermia delivery implants for over a decade, utilizing low Curie temperatures to provide thermal self-regulation. The minimally invasive method is attractive for fractionated thermal treatment of tumors which are not easily heated by focused microwave or ultrasound techniques. Past analyses of ferromagnetic seeds by other authors depict poor experimental correlation with theoretical heating predictions. Improvements in computer hardware and commercially available finite element analysis software have simplified the analysis of inductively heated thermal seeds considerably. This manuscript examines end effects of finite length implants and nonlinear magnetic material properties to account for previous inconsistencies. Two alloys, Ni-28 wt% Cu (NiCu) and Pd-6.15 wt% Co (PdCo), were used for comparison of theoretical and experimental calorimetric results. Length to diameter (L/d) ratios of over 20 for cylindrical seeds are necessary for minimization of end effects. Magnetic properties tested for alloys of NiCu and PdCo illustrate considerable nonlinearity of these materials in field strength ranges used for induction heating. Field strength dependent magnetic permeabilities and calorimetric data illustrate that more detailed material information must be included to accurately estimate induction power loss for these implants. PMID- 8626191 TI - Reduced order Kalman filtering for the enhancement of respiratory sounds. AB - In the processing and analysis of respiratory sounds, heart sounds present the main source of interference. This paper is concerned with the problem of cancellation of the heart sounds using a reduced-order Kalman filter (ROKF). To facilitate the estimation of the respiratory sounds, an autoregressive (AR) model is fitted to heart signal information present in the segments of the acquired signal which are free of respiratory sounds. The state-space equations necessary for the ROKF are then established considering the respiratory sound as a colored additive process in the observation equation. This scheme does not require a time alignment procedure as with the adaptive filtering-based schemes. The scheme is applied to several synthesized signals with different signal-to-interference ratios (SIR) and the results are presented. PMID- 8626190 TI - Impedance imaging of lung ventilation: do we need to account for chest expansion? AB - Electrical impedance tomography (EIT) uses surface electrical measurements to image changes in the conductivity distribution within a medium. When used to measure lung ventilation, however, measurements depend both on conductivity changes in the thorax and on rib cage movement. Given that currently available reconstruction techniques assume that only conductivity changes are present, certain errors are introduced. A finite element model (FEM) is used to calculate the effect of chest expansion on the reconstructed conductivity images. Results indicate that thorax expansion accounts for up to 20% of the reconstructed image amplitude and introduces an artifact in the center of the image tending to "move" the reconstructed lungs closer together. Although this contribution varies depending on anatomical factors, it is relatively independent of inspiration depth. For certain applications in which one is only interested in changes in the level of physiological activity, the effect of the expansion can be neglected because it varies linearly with impedance changes. We conclude that chest expansion can contribute significantly to the conductivity images of lung ventilation and should be taken into account in the interpretation of these images. PMID- 8626192 TI - Classification of cardiac arrhythmias using fuzzy ARTMAP. AB - We have investigated the QRS complex, extracted from electrocardiogram (ECG) data, using fuzzy adaptive resonance theory mapping (ARTMAP) to classify cardiac arrhythmias. Two different conditions have been analyzed: normal and abnormal premature ventricular contraction (PVC). Based on MIT/BIH database annotations, cardiac beats for normal and abnormal QRS complexes were extracted from this database, scaled, and Hamming windowed, after bandpass filtering, to yield a sequence of 100 samples for each ORS segment. From each of these sequences, two linear predictive coding (LPC) coefficients were generated using Burg's maximum entropy method. The two LPC coefficients, along with the mean-square value of the QRS complex segment, were utilized as features for each condition to train and test a fuzzy ARTMAP neural network for classification of normal and abnormal PVC conditions. The test results show that the fuzzy ARTMAP neural network can classify cardiac arrhythmias with greater than 99% specificity and 97% sensitivity. PMID- 8626193 TI - Automated detection of the left ventricular region in gated nuclear cardiac imaging. AB - An approach to automated outlining the left ventricular contour and its bounded area in gated isotopic ventriculography is proposed. Its purpose is to determine the ejection fraction (EF), an important parameter for measuring cardiac function. The method uses a modified version of the fuzzy C-means (MFCM) algorithm and a labeling technique. The MFCM algorithm is applied to the end diastolic (ED) frame and then the (FCM) is applied to the remaining images in a "box" of interest. The MFCM generates a number of fuzzy clusters. Each cluster is a substructure of the heart (left ventricle,...). A cluster validity index to estimate the optimum clusters number present in image data point is used. This index takes account of the homogeneity in each cluster and is connected to the geometrical property of data set. The labeling is only performed to achieve the detection process in the ED frame. Since the left ventricle (LV) cluster has the greatest area of the cardiac images sequence in ED phase, a framing operation is performed to obtain, automatically, the "box" enclosing the LV cluster. THe EF assessed in 50 patients by the proposed method and a semi-automatic one, routinely used, are presented. A good correlation between the two methods EF values is obtained (R = 0.93). The LV contour found has been judged very satisfactory by a team of trained clinicians. PMID- 8626195 TI - Changes in endodontic status 1973-1993 among 35-year-olds in Oslo, Norway. AB - Epidemiological investigations from Scandinavia have documented high prevalence of apical periodontitis and inferior quality and results of endodontic treatment performed in general practice. The present investigation is part of a 20-year follow-up study on oral health in 35-year-old Oslo citizens focusing on endodontic conditions. The results indicate reduced prevalence of apical periodontitis and statistically significant improvement in endodontic status compared with results from a similar study performed in 1984. PMID- 8626194 TI - Diagnostic confidence and the accuracy of treatment decisions for radiopaque periapical lesions. AB - The periapical radiograph is a tool used in the diagnosis of periapical bone pathology. Radiographic diagnosis might therefore influence endodontic treatment planning and the quality and the cost of dental health care. The hypotheses were tested that (1) the subjective prevalence of a lesion would be associated with the accuracy with which it is diagnosed, (2) 'confident dentists' would be as accurate in their diagnosis as 'doubtful dentists' and (3) the diagnostic confidence of dentists for the diagnosis of periapical pathology would not influence their treatment planning. It was found that the probability of an appropriate treatment decision increased when dentists were relatively confident about a diagnosis. The results showed, furthermore, that the accuracy of diagnostic decisions increased when the dentist's confidence about a particular decision was high. Ways to increase diagnostic accuracy of radiopaque periapical pathology are proposed as a result of this study. PMID- 8626196 TI - Light and SEM observation of internal root resorption of a traumatized permanent central incisor. AB - A clinical case report is presented which illustrates internal root resorption of a traumatized tooth. Light and scanning electron microscope were used to further examine the defect. An explanation of the morphological structure of the resorption is discussed. PMID- 8626198 TI - The smear layer: a phenomenon in root canal therapy. AB - When the root canals are instrumented during endodontic therapy, a layer of material composed of dentine, remnants of pulp tissue and odontoblastic processes, and sometimes bacteria, is always formed on the canal walls. This layer has been called the smear layer. It has an amorphous, irregular and granular appearance under the scanning electron microscope. The advantages and disadvantages of the presence of smear layer, and whether it should be removed or not from the instrumented root canals, are still controversial. It has been shown that this layer is not a complete barrier to bacteria and it delays but does not abolish the action of endodontic disinfectants. Endodontic smear layer also acts as a physical barrier interfering with adhesion and penetration of sealers into dentinal tubules. In turn, it may affect the sealing efficiency of root canal obturation. When it is not removed, the durability of the apical and coronal seal should be evaluated over a long period. If smear layer is to be removed, EDTA and NaOCl solutions have been shown to be effective, among various irrigation solutions and techniques, including ultrasonics, that have been tested. Once this layer is removed, it should be borne in mind that there is a risk of reinfecting dentinal tubules if the seal fails. Further studies are needed to establish the clinical importance of the absence or presence of smear layer. PMID- 8626197 TI - Multidisciplinary aspects of root resection failure: a case report. AB - Creative approaches to tooth maintenance often include tooth resection and retention of one or more of the roots. Although this procedure is reasonably successful, failure of supportive endodontic, periodontic, and restorative management of the retained roots can jeopardize a successful outcome. The following is a case report evaluating multiple aetiologies contributing to root resection failure. PMID- 8626199 TI - An in vitro comparison of 10 radiographic methods for working length estimation. AB - The aim of this in vitro study was to assess the efficacy of 10 currently available methods of producing radiographic images, two conventional and eight rapid, in working length estimation. Thirty extracted teeth comprising 42 root canals were mounted in blocks of poly(methylmethacrylate) and access cavities prepared. A size 15 Hedstrom file was inserted into each root canal and sealed into position. Each tooth was imaged by 10 methods: combinations of conventional (D- and E- speed) film processed with conventional manual chemistry of two proprietary brands of rapid processing chemistry (Westone Rapid Dental and Kodak 'Rapid Access'), Super X30 packet processed film, Hanshin Hi-Fi and Nix NF45-100 films processed using their respective manufacturers' monobath solutions, and the Sens-A-Ray digital X-ray system. Comparisons of length of file visible were made between the D-speed films (conventionally processed using Kodak chemistry) and the nine other radiographic methods. No significant differences in percentage length of file seen were found between D-speed film processed with conventional chemistry and E-speed film processed with the same chemistry or between D-speed film processed with with conventional chemistry and six of the rapid imaging systems studied. The percentage length of the file visible was significantly less for Sens-A-Ray (P=0.02) and for Nix NF45-100 film (P<0.005) when compared with D speed film processed with conventional chemistry. This difference in file length is probably not of clinical importance for the Sens-A-Ray images but may be so for the Nix images. PMID- 8626200 TI - Reliability of reduced air pressure methods used to assess the apical seal. AB - The reliability of two commonly used methods of sealability testing, dye leakage under normal atmospheric pressure and dye leakage in a partial vacuum, was tested using a repeated studies design. Forty extracted teeth were instrumented and obturated using lateral condensation of gutta-percha and Sealapex sealer. One half of the teeth were placed in India ink under normal atmospheric pressure and the other half underwent air evacuation before immersion in the ink. After 24 h the teeth were removed from the ink and rendered transparent for linear leakage analysis. A second study, repeated within the exact parameters of the first, was performed approximately 1 month later. Mann-Whitney U tests indicated that there was no significant difference (P>0.05) between the air and vacuum groups or within each group (air or vacuum) between trials, indicating that the methodology is repeatable and that application of reduced air pressure did not enhance reliability. A large majority of the teeth placed under partial vacuum showed evidence of vacuum-induced artefacts that may negate the value of vacuum as a research tool. PMID- 8626201 TI - Isolation and characterization of the proteoglycans synthesized by adult human pulp fibroblasts in vitro. AB - The proteoglycans synthesized by fibroblasts derived from healthy human adult dental pulps have been isolated and characterized on the basis of their glycosaminoglycan content, molecular size and charge. The proteoglycans were identified by their labelling with [35S] sulphate and susceptibility to digestion by papain. The sulphated glycosaminoglycans associated with the proteoglycans were identified following specific enzymatic and chemical degradations as chondroitin sulphate, dermatan sulphate and heparan sulphate. Dermatan sulphate and chondroitin sulphate and heparan sulphate were the principal glycosaminoglycans associated with the cell layers. The proteoglycans could be fractionated on the basis of their charge and size into a number of heterogeneous pools. The principal proteoglycans isolated were small and contained either chondroitin sulphate or dermatan sulphate and most likely correspond to decorin and biglycan. Other molecules with features similar to versican and syndecan were also identified. PMID- 8626202 TI - Pulpotomy of carious vital teeth with periapical involvement. AB - Twenty-six permanent vital molars with carious pulp exposures and periapical involvement presenting as radiolucencies or radiopacities on radiographic examination, in patients aged between 10-24 years, were treated using an atraumatic surgical technique with calcium hydroxide alone. The healing was evaluated using clinical and radiographic criteria: absence of clinical symptoms, sensitivity of the radicular pulp, formation of a hard tissue barrier in the exposed area, resolution of periapical involvement and no intraradicular pathosis radiographically. Assessed by these criteria, successful results were achieved in 24 teeth. The observation period following pulpotomy treatment was 16-72 months. The favourable results of this study demonstrate that pulpotomy treatment in teeth with cariously exposed vital pulps and with periapical involvement may be an alternative treatment to root canal therapy. PMID- 8626203 TI - Spring Scientific Meeting of the British Endodontic Society: Abstracts and Posters. PMID- 8626204 TI - Ultrasonic preparation and obturation of root-end cavities. AB - This study evaluated the preparation of root-end cavities using an ultrasonically activated file, and the obturation of such cavities. In the first part, the root canals of 40 extracted teeth were prepared to size 40 and irrigated with NaOCl and EDTA. The root ends were resected and the teeth inoculated with Enterococcus faecalis, incubated for 10 days and divided into four groups: control; saline; irrigation; hand instrumentation performed via a retrograde approach up to size 50 using saline irrigant; ultrasonic instrumentation with prebent size 40 K-flex file inserted into a Piezon Master using saline irrigant. The teeth were fixed, sectioned longitudinally and viewed under scanning electron microscopy. Bacterial and smear layer scores were obtained at 1, 3, and 5 mm from the resected end. The bacterial scores for control and saline groups were similar; the scores for each instrumentation group were significantly lower than the control group (P<0.001). In addition, there were significantly fewer bacteria in the ultrasonic group compared with the hand instrument group (P<0.001). The smear layer scores for control and saline groups were similar, and significantly lower than in either instrumentation group (P<0.001). In the second part, root-end cavities were ultrasonically prepared in 20 extracted teeth. Ten cavities were filled with amalgam, and 10 with thermoplasticized gutta-percha and Grossman's sealer. After 24-h storage, the root ends were immersed in dye for 48 h. Cross-sectional slices of the obturated part of the root were evaluated using both light and confocal microscopy for dye leakage along the interface of filling material and dentine. There were no statistical differences between the leakage of amalgam and gutta percha root-end fillings, nor between the two methods of microscopy. PMID- 8626205 TI - Torsional and stiffness properties of nickel-titanium K files. AB - The purpose of this study was to compare stiffness and resistance to fracture of four brands of nickel titanium K files. Instruments of sizes 15 to 40 were tested according to ANSI/ADA Specification No. 28. Resistance to fracture was determined by twisting and measuring the maximum torque and angular deflection at failure. Stiffness was determined by measuring the moment required to bend the instrument 45 degrees. The permanent deformation angle remaining between the tip and the flutes of the instruments after bending ceased was also recorded. Nickel titanium K files satisfied and far exceeded specification standards for stiffness. They also satisfied and exceeded the standards for angular deflection at failure. They met or exceeded the maximum torque at failure standards in all sizes except for the size 40 of the Maillefer Niti, and the size 30 of the MacSpadden Niti. Nickel titanium K files presented a null permanent deformation angle. Clinical studies are required to evaluate the influence of low bending moment on other properties such as breakage and canal transportation. PMID- 8626206 TI - Studies into the microbial spectrum of apical periodontitis. AB - This study examined the variety of obligate and facultative anaerobic bacterial species recovered from cases of acute apical periodontitis. A total of 19 root canal samples and 24 periapical granuloma samples were taken from patients suffering pain and discomfort. Bacteria were identified by applying the following techniques: culturing on various media, Gram-staining and using commercially available biochemical test strips. In addition, Prevotella intermedia and Porphyromonas endodontalis were differentiated on a molecular genetic level using species-specific oligodeoxynucleotide probes. The most frequently identified bacteria were Prevotella intermedia, Bifidobacterium spp., Streptococcus sanguis, Streptococcus milleri-group and Bacteroides spp. Obligate anaerobes occurred at a rate of 82.3%, and the average number of isolates was 6.4 per sample. PMID- 8626207 TI - Long-term evaluation of endodontic and periodontal treatment. AB - One hundred and ninety-five teeth in 35 patients with periodontitis who had received both endodontic and periodontal treatment were evaluated 9 years after endodontic treatment and 8 years after periodontal treatment. Some 91.4% of cases were well maintained and 8.6% showed a deterioration in their periodontal condition. Twelve of the 195 teeth with endodontic treatment were lost, eight for periodontal reasons, three as a result of fracture and one because of caries, and the periodontal condition of 10 teeth had worsened. An apical lesion formed on one tooth. The results indicate that the risk of endodontic failure in this group of 195 teeth is very low, and that there is little risk of tooth loss for periodontal reasons, provided that the patients receive supportive periodontal treatment. PMID- 8626208 TI - Clinical diagnosis of internal resorption: an exception to the rule. AB - This paper describes a case in which the radiographic appearance of a resorptive lesion in a mandibular incisor tooth posed difficulty in the diagnosis of its origin. The lesion did not conform to the normally accepted criteria for diagnosis of internal resorption. Clinical examination did not support the diagnosis of external cervical resorption. Serial histological section of the extracted tooth subsequently confirmed the diagnosis of internal resorption. The criteria for differential diagnosis of resorptive lesions with smooth outlines in the cervical regions of teeth are discussed together with the rationale for their management. PMID- 8626209 TI - Factors associated with endodontic flare-ups: a prospective study. AB - The purpose of this prospective study was to assess the incidence of flare-ups (a severe problem requiring an unscheduled visit and treatment) among patients who received endodontic treatment by the two authors in their respective practices during a period of one year, and also to examine the correlation with pre operative and operative variables. The results showed an incidence of 1.58% for flare-ups from 1012 endodontically treated teeth. Statistical analysis using the chi-square test (P<0.05) indicated that flare-ups were found to be positively correlated with multiple appointments, retreatment cases, periradicular pain prior to treatment, presence of radiolucent lesions, and patients taking analgesic or anti-inflammatory drugs. In contrast, there was no correlation between flare-up, and age, sex, different arch/tooth groups and the status of the pulp. PMID- 8626211 TI - ["Sicilian gambit": basic principles and strategies of anti-arrhythmia therapy. Satellite symposium of the 15th Congress of the European Society for Cardiology, Barcelona, 30 August 1992]. PMID- 8626210 TI - The adaptation of mechanically softened gutta-percha to the canal walls in the presence or absence of smear layer: a scanning electron microscopic study. AB - The objective of this study was to compare the adaptation of mechanically softened gutta-percha to the root canal wall in the presence and absence of smear layer. The root canals of 20 freshly extracted human maxillary incisors were cleaned and shaped. Prior to obturation, 10 root canals were irrigated with 20 ml of 50% citric acid followed by 20 ml of 5.25% sodium hypochlorite. All canals were obturated with mechanical compaction of gutta-percha and AH-26 sealer. After 72 h, each tooth was fractured in half. Scanning electron microscopy demonstrated that the sealer had formed a continuous layer in contact with the canal walls, becoming progressively thinner towards the apex. The sealer penetrated into the dentinal tubules along with projections of gutta-percha only in those teeth without smear layer. PMID- 8626212 TI - [Fatty acids in the diet and their significance for serum lipids and atherosclerosis]. PMID- 8626213 TI - [Furth pneumological symposium. Nuremberg, 3. April 1993]. PMID- 8626216 TI - Pseudotumor cerebri as presenting syndrome of Addisonian crisis. AB - In a patient who developed clinical signs of intracranial hypertension, bilateral papilledema and diplopia, in association with mild hypotension, hyponatremia and hyperkalemia, the hypothesis of Addison's disease was raised and confirmed. Substitutional therapy led to complete recovery. The present paper represents the first report of pseudotumor cerebri as the only clinical sign of an Addisonian crisis. PMID- 8626215 TI - Age, sex and mental retardation related changes of brainstem auditory evoked potentials in Down's syndrome. AB - Brainstem auditory evoked potentials (BAEPs) were recorded in 51 Down's syndrome (DS) subjects and compared with those of 38 normal controls; the correlations between the BAEP measures and age, sex, and degree of mental retardation were then evaluated. The DS patients showed a significant reduction in wave V latency and amplitude and in I-III, III-V, and I-V interpeak intervals. An age-related shortening of the I-V interpeak interval found in DS patients was interpreted as being a result of changes in central inhibitory/excitatory mechanisms. In both groups, female subjects presented an I-V interval shorter than that of males but this difference was greater in the DS subjects than in the normal population. The DS patients with severe mental retardation showed significantly longer I-V interpeak intervals than those with moderate retardation; this could be due to the presence of additional central nervous system abnormalities. PMID- 8626218 TI - 4th Congress of the Italian Association of Neuro-Oncology. Siena, Certosa di Pontignano, September 7-9, 1995. Abstracts. PMID- 8626214 TI - Brain electrical activity (quantitative EEG and bit-mapping neurocognitive CNV components), psychometrics and clinical findings in presenile subjects with initial mild cognitive decline or probable Alzheimer-type dementia. AB - Clinical, neuropsychological and neuropsychophysiological data (Q-EEG, ERPs and CNV/RT activity) were obtained from 24 patients who had more or less severe presenile primary cognitive decline without depression, and compared with similar data from 10 age-matched healthy volunteers (mean age, 59.4 years). All of the patients (15 M and 9 F; mean age 59.6 years) were selected according to the DSM III-R, ICD-10 and NINCDS-ADRDA criteria and underwent CT and MRI scanning, in addition to a standard clinical examination, a battery of psychometric tests, spectral EEG, and bit-mapped CNV complex and RT to S2 analyses. Twelve of the 24 patients presented an initial presenile idiopathic cognitive decline (PICD) but did not wholly fulfil the clinical and neuropsychological criteria for primary dementia or for a diagnosis of probable AD; the remaining 12 patients showed characteristic clinical signs and symptoms of a very probable early stage of presenile Alzheimer-type dementia (PAD). ANOVA, correlational and discriminant analyses of the neuropsychological test scores, and the neurophysiological and RT to S2 data revealed 22 highest-ranked between-group discriminant factors (all with a significance level of p < 0.01). The conclusive discriminant analysis retained 13 of these factors as final canonical functions, and these showed a 97% grouping accuracy (33 of the 34 subjects examined); the same percentage of correct classifications was also achieved using only the 15 best indicators in the group of CNV/RT findings. Using both of these sets of highest-ranked discriminators, all of the normal subjects and all of the PAD patients were correctly classified; only 1 PICD patient was misclassified as normal when the first group of 13 factors was used, and another PICD patient was misclassified as PAD using the second group of 15 factors. Our findings suggest that, providing they are correctly performed and interpreted, these non-invasive techniques may be an important tool for identifying incipient stages of presenile Alzheimer-type dementia. PMID- 8626217 TI - Primary antiphospholipid syndrome. PMID- 8626219 TI - SINE sequences detect DNA fingerprints in salmonid fishes. AB - DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays. PMID- 8626220 TI - Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico. AB - RAPD-PCR polymorphisms at 57 presumptive loci were used to examine the breeding structure of the mosquito Aedes aegypti in Puerto Rico. Mosquitoes were sampled from 16 locations in six cities and samples were located in a nested spatial design to examine local patterns of gene flow. Allele frequencies were estimated assuming (1) that genomic regions amplified by RAPD-PCR segregate as dominant alleles, (2) that genotypes at RAPD loci are in Hardy-Weinberg proportions, (3) identity in state (iis) among dominant amplified alleles and (4) iis among null alleles. The average genic heterozygosity was 0.354, more than twice the level detected in earlier allozyme surveys. Nested analysis of variance indicated extensive genetic differentiation among locations within cities. Effective migration rates (Nm) among cities were estimated from FST assuming an island model of migration. Estimates of Nm ranged from 9.7 to 12.2 indicating a high dispersal rate. The large number of polymorphisms revealed by RAPD-PCR allowed the distribution of FST and linkage disequilibrium to be examined among loci and demonstrated that small samples inflate FST and linkage disequilibrium. No linkage disequilibrium maintained through epistasis was detected among alleles at the 57 loci. PMID- 8626222 TI - Testing for linkage disequilibrium in genotypic data using the Expectation Maximization algorithm. AB - We generalize an approach suggested by Hill (Heredity, 33, 229-239, 1974) for testing for significant association among alleles at two loci when only genotype and not haplotype frequencies are available. The principle is to use the Expectation-Maximization (EM) algorithm to resolve double heterozygotes into haplotypes and then apply a likelihood ratio test in order to determine whether the resolutions of haplotypes are significantly nonrandom, which is equivalent to testing whether there is statistically significant linkage disequilibrium between loci. The EM algorithm in this case relies on the assumption that genotype frequencies at each locus are in Hardy-Weinberg proportions. This method can accommodate X-linked loci and samples from haplodiploid species. We use three methods for testing significance of the likelihood ratio: the empirical distribution in a large number of randomized data sets, the X2 approximation for the distribution of likelihood ratios, and the Z2 test. The performance of each method is evaluated by applying it to simulated data sets and comparing the tail probability with the tail probability from Fisher's exact test applied to the actual haplotype data. For realistic sample sizes (50-150 individuals) all three methods perform well with two or three alleles per locus, but only the empirical distribution is adequate when there are five to eight alleles per locus, as is typical of hypervariable loci such as microsatellites. The method is applied to a data set of 32 microsatellite loci in a Finnish population and the results confirm the theoretical predictions. We conclude that with highly polymorphic loci, the EM algorithm does lead to a useful test for linkage disequilibrium, but that it is necessary to find the empirical distribution of likelihood ratios in order to perform a test of significance correctly. PMID- 8626221 TI - Evolutionary conservation and molecular characteristics of repetitive sequences of Drosophila koepferae. AB - Thirteen middle repetitive DNA clones obtained from the genome of Drosophila koepferae have been tested for their evolutionary conservation in the other seven species of the buzzatii and martensis clusters (repleta group). All but two of these clones exhibit qualitatively similar patterns of hybridization in the eight species. The average interspecific hybridization signal is 85 per cent of that found intraspecifically, ranging from 73 to 93 per cent. Partial sequencing of six of these clones has shown sequences related to the retrotransposon Gypsy, first characterized in D. melanogaster, as well as to the Anopheles gambiae LINE elements T1Ag and Q. A fragment of a hitherto unknown, short inverted repeat transposable element has also been found. The evolutionary conservation of repetitive D. koepferae sequences seems to be related to the high proportion of simple DNA and inactive mobile elements in the genome of this species. PMID- 8626223 TI - Genetic evidence for repression of somatic P element movements in Drosophila melanogaster consistent with a role for the KP element. AB - The P family of transposable elements in Drosophila melanogaster has, since its introduction into D. melanogaster populations in this century, diversified into a number of internally deleted forms. One of these, the KP element, is abundant in the genomes of flies from populations from Asia, Europe and Africa. There is some evidence that this sequence can act as a repressor of transposition. We have studied a mutation, signed(very weak) (snVW), in which a KP element is one of two P elements inserted into the 5' exon of the X-linked singed gene. These elements can be mobilized by a trans-acting dominant mutation, Mo, with a maternal effect, on the second chromosome. The rate of somatic reversion of snvw induced by Mo is reduced threefold if the mother herself possesses snvw on her X chromosomes. This implies that snvw may be responsible for a form of repression of P element movement. The cause of this effect may be related to transcription of the KP element in snvw. However, an effect of other genomic P elements in the repression of somatic reversion of snvw cannot be entirely excluded. PMID- 8626225 TI - Measuring selective effects of modifier gene polymorphisms on the Bare locus of Drosophila subobscura. AB - An attempt to quantify the effects of modifier gene polymorphisms on the operation of natural selection on a major locus has been carried out. The modifier system we have investigated is constituted by a set of polygenic modifier loci affecting the morphological expression of the Bare (Ba) bristle mutant of Drosophila subobscura. Ba is a dominant mutant that is lethal in homozygous condition and both the polygenic modifiers and Ba are located on the O chromosome of this species. Experimental populations were found with Ba/+ individuals and two different types of populations were started according to their modifier genetic background: populations with wild O chromosomes of either high or low modifier effect (cages H and L, respectively). Fitness estimates (total fitness, viability and fertility) for genotypes of the Ba locus were obtained under the two different modifier backgrounds. In the populations with high modifier background the total fitness of the Ba/+ heterozygote was very similar to that of the +/+ homozygote (fitness equal to 1). However, in cages with low modifier background a strong selection against the Ba/+ heterozygote was detected (average of total fitnesses over generations was 0.66 +/- 0.10), and fertility appears to be the fitness component responsible for this effect (mean fertility was 0.55 +/- 0.08). These findings demonstrate that modifier gene polymorphisms affecting the expression of the Ba mutant may be associated with large selective effects on the major locus. PMID- 8626224 TI - Phylogeny of the genus Hegeter (Tenebrionidae, Coleoptera) and its colonization of the Canary Islands deduced from Cytochrome Oxidase I mitochondrial DNA sequences. AB - The genus Hegeter comprises 23 species of darkling beetles (Tenebrionidae) endemic to the Macaronesian archipelagos, with 21 of them exclusive to the Canary Islands. We have sequenced 438 bp of the mitochondrial Cytochrome Oxidase I gene in 17 species (24 taxa) of Canarian Hegeter. Estimates of nucleotide composition, transition/transversion ratios and nucleotide change frequencies are very similar to those found in another tenebrionid Canarian genus Pimelia, indicating that similar molecular mechanisms are driving the sequence evolution. The sequence variation found allows phylogenetic analyses of the genus and the deduction of colonization patterns. These involve sequential island invasion with more rapid establishment and radiation than found in the related beetles of the genus Pimelia. PMID- 8626226 TI - Updated quality indicators for the Cooperative Cardiovascular Project. PMID- 8626227 TI - Marketing key to increased women applicants to COMs. PMID- 8626228 TI - Osteopathic medicine: an inherently natural approach to cholesterol reduction. PMID- 8626229 TI - Comparison of newborn circumcision pain to calcaneal heel puncture pain: is newborn circumcision pain control clinically warranted? AB - In newborns, elective male circumcision and calcaneal puncture for obtaining blood samples both cause pain. With elective male circumcision, dorsal penile nerve block (DPNB) is recommended for pain control, but no pain control is routinely recommended or used during calcaneal puncture. A prospective investigation was conducted to compare pain during elective circumcision (with and without DPNB) and calcaneal puncture to determine whether pain control should be used with the latter procedure. The study was conducted at a community hospital during a 4-month period. Two nurses observed newborn behavior during elective circumcision and routine calcaneal puncture and rated pain reactions by using a modified Gronigen Distress Scale. Ninety-one male newborns were circumcised without DPNB, and eight male newborns were circumcised with DPNB. Calcaneal puncture for blood samples was performed in 97 newborns (males and females). Pain scores during circumcision with and without DPNB were comparable with those during calcaneal puncture (2.1, 2.4, and 2.2, respectively). Pain control during circumcision may thus be considered, but caution should be taken that measures to prevent pain do not create new patient risk or additional pain. PMID- 8626230 TI - Chronic fatigue complaints in primary care: incidence and diagnostic patterns. AB - The complaint of chronic fatigue is ubiquitous in the primary care setting. Because of the nonspecific nature of chronic fatigue, practitioners do not focus on this complaint. Furthermore, most physicians use a problem-based approach. Such a prematurely narrowed focus could overlook the chronic fatigue complaint. Omissions in the data collection process would prove this oversight. Therefore, we postulated that a retrospective review of evaluations for chronic fatigue would demonstrate significant categorical deficiencies. These deficiencies would indicate a problem focus different than the chronic fatigue complaint itself. The authors reviewed the current literature to establish historical, physical, and laboratory findings pertinent to the evaluation of chronic fatigue. Six major categories and the associated data elements were identified for use in analyzing patient records. The patient records from the preceding 6 months were reviewed to find those containing a complaint of chronic fatigue. These records were analyzed to determine if a complete data set had been sought and if an associated diagnosis was made. A total of 425 consecutive charts from an academic family practice clinic were retrospectively reviewed; 9.9% (42) mentioned chronic fatigue. Physicians were lax in performing the mental status and physical examinations; taking the patient's psychiatric and sleep history, as well as the history of chief complaint; and ordering laboratory evaluations. The physician diagnoses included: depression (40.4%), nonspecific fatigue (35.7%), general medical disorders (16.6%), chronic fatigue syndrome (2.4%), fibromyalgia (2.4%), and sleep apnea (2.4%). From these data, the investigators conclude that the workup for chronic fatigue is often incomplete or lacks documentation. This oversight is likely due to a problem focus not directed at the chronic fatigue complaints. Also complicating the evaluation process are the multiple associated disorders, the prevalence of the complaint, and cost/benefit issues facing the primary care physician. PMID- 8626231 TI - Soft tissue sarcoma of the anterior abdominal wall: review of reconstruction techniques. AB - Tumors of the abdominal wall excised with inadequately wide margins have a high local recurrence rate. The surgeon's fear of iatrogenic defects of the abdominal wall should not outweigh the need for wide resection margins to prevent tumor recurrence when excising primary and secondary malignancies. Appropriate monobloc excision of abdominal wall malignancies can be satisfactorily accomplished through a wide array of modalities. The authors describe successful abdominal wall reconstruction with the use of polypropylene mesh after excision of a recurrent soft tissue sarcoma. They review various methods of abdominal wall reconstruction to assist the surgeon in choosing the appropriate reconstruction technique. PMID- 8626232 TI - Simultaneous bilateral facial palsy in pregnancy. AB - The risk of facial palsy for pregnant women is higher than for nonpregnant women. Most cases occur in the third trimester and the immediate postpartum period. Unilateral facial palsy is a commonly seen mononeuropathy. Bilateral facial palsy, however, is rare and is most often a sign of a systemic disease. The authors present the case of a pregnant woman who was first seen with left-sided facial paralysis. The paralysis then spread to the right side, resolved quickly after delivery, but appeared again on the right side 6 months later. The authors warn that bilateral facial palsy seen in pregnancy may be a sign of serious underlying disease. Differential diagnosis, possible etiologies, treatment, and prognosis are discussed. PMID- 8626233 TI - Spontaneous renocolic fistula: a rare occurrence associated with renal cell carcinoma. AB - A patient with abdominal discomfort, weight loss, and general weakness was discovered by way of ultrasound and computed tomography to have a tumor in the lower pole of the left kidney. At a second computed tomography examination, the mass had shrunken and air-fluid levels and contrast medium from previous examinations were evident, confirming a renocolic fistula. The patient underwent en bloc left radical nephrectomy with resection of the descending colon. Renal cell carcinoma was confirmed histologically. The patient has been free of disease for 4 years. Such fistulas are usually related to renal calculi or tuberculosis but, of late, more have been the result of iatrogenic trauma sustained during percutaneous renal procedures and lithotripsy. Use of computed tomography and other imaging methods should enable early detection of most renocolic fistulas, which usually are a late manifestation of advanced renal disease. PMID- 8626234 TI - New isochromophilones VII and VIII produced by Penicillium sp. FO-4164. AB - New isochromophilones VII and VIII were isolated from the culture broth of Penicillium sp. FO-4164. The structures were elucidated as 6H-2-benzopyran 6,8(7H)-dione, 5-chloro-3-(3',5'-dimethyl-1',3'-heptadienyl)-1,7,8a-trihydro-7, 8a-dihydroxy-7-methyl-7-acetate for isochromophilone VII and 6H-2-benzopyran-6 one,5-chloro-3-(3',5'-dimethyl-1', 3'-heptadienyl)-1,7,8,8a-tetrahydro-7,8 dihydroxy-7-methyl-8-acetate for isochromophilone VIII. Isochromophilones VII and VIII inhibited diacylglycerol acyltransferase activity with IC50 values of 20.0 and 127 microM and acyl-CoA: cholesterol acyltransferase activity with IC50 values of 24.5 and 47.0 microM, respectively. PMID- 8626235 TI - Antascomicins A, B, C, D and E. Novel FKBP12 binding compounds from a Micromonospora strain. AB - 5 novel ascomycin-like compounds, antascomicins A, B, C, D and E were isolated from a strain of Micromonospora. The antascomicins bind strongly to the FK506 binding protein FKBP12 and antagonize the immunosuppressive activity of FK506 and rapamycin. The strain description, fermentation, structure elucidation and biological activity of these compounds are described. PMID- 8626236 TI - BMS-182123, a fungal metabolite that inhibits the production of TNF-alpha by macrophages and monocytes. AB - A fungal metabolite, BMS-182123, which inhibited bacterial endotoxin-induced production of tumor necrosis factor (TNF-alpha) in murine macrophages and human peripheral blood monocytes (in vitro), was isolated from the culture broth of Penicillium chrysogenum strain V39673. The effective BMS-182123 concentration (IC50) resulting in 50% inhibition of lipopolysaccharide-induced TNF-alpha production in murine macrophages and human monocytes was 600 ng/ml and 4.0 microgram/ml, respectively. BMS-182123 suppressed the lipopolysaccharide-induced TNF-alpha promoter activity and did not affect the stability of posttranscriptional mRNA. Addition of hydrophobic resin, Amberlite XAD-8 (1%), to the fermentation enhanced the production of BMS-182123 by 5.5 fold. A total of 577 mg pure BMS-182123 was recovered from a 250-liter fermentation supplemented with 1% Amberlite XAD-8. PMID- 8626237 TI - Studies on cochleamycins, novel antitumor antibiotics. I. Taxonomy, production, isolation and biological activities. AB - Novel antitumor antibiotics cochleamycins A, A2, B and B2 (Fig. 1) were isolated from the culture broth of Streptomyces sp. DT136. They were purified by column chromatography on silica gel, reversed phase HPLC and then isolated as colorless powder. Cochleamycins showed growth inhibition against tumor cells in vitro. PMID- 8626238 TI - Studies on cochleamycins, novel antitumor antibiotics. II. Physico-chemical properties and structure determination. AB - The structure of cochleamycins A, A2, B and B2 (Fig. 1), novel antitumor antibiotics, were elucidated by NMR spectral analysis. Cochleamycins were found to possess novel carbocyclic skeletons. PMID- 8626239 TI - Studies on cochleamycins, novel antitumor antibiotics. III. Biosyntheses of cochleamycins: incorporation of 13C- and 2H-labeled compounds into cochleamycins. AB - Biosynthetic studies using 13C- and 2H-labeled compounds revealed that the carbon skeletons of cochleamycins A and B were derived from eight acetic acid units and one propionic acid unit with the introduction of an acetoxy group at C-10, which was replaced by an isobutyryl residue derived from valine in cochleamycins A2 and B2. PMID- 8626240 TI - Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity. AB - Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase. PMID- 8626241 TI - Karalicin, a new biologically active compound from Pseudomonas fluorescens/putida. I. Production, isolation, physico-chemical properties and structure elucidation. AB - An original compound, named karalicin, was isolated from a fermentation broth of the Pseudomonas fluorescens/putida strain SS-3 (CCM 4430). Production, physico chemical properties and structure elucidation are described. PMID- 8626242 TI - Karalicin, a new biologically active compound from Pseudomonas fluorescens/putida. II. Biological properties. AB - The biological activities of karalicin, a new product from the Pseudomonas fluorescens/putida strain SS-3 (CCM 4430) are described. It shows a weak, but specific and irreversible, antiviral activity on Herpes simplex viruses. It also presents some inhibitory activity on different species of yeasts. PMID- 8626243 TI - Xanthoquinodin B3, a new anticoccidial agent produced by Humicola sp. FO-888. AB - Xanthoquinodin B3, a new component of anticoccidial xanthoquinodins, which was detected in the culture broth of Humicola sp. FO-888, was isolated by heat treatment of the xanthoquinodins complex. Structural elucidation indicated that xanthoquinodin B3 has the same heterodimer of xanthone- and anthraquinone-derived monomers as other xanthoquinodins. Schizont formation of monensin-resistant Eimeria tenella in BHK-21 cells was inhibited by xanthoquinodin B3 at concentrations greater than 0.035 microM. PMID- 8626245 TI - Poststatin, a new inhibitor of prolyl endopeptidase. III. Optical resolution of 3 amino-2-hydroxyvaleric acid and absolute configuration of poststatin. AB - 3-Amino-2-hydroxyvaleric acid was prepared, and separated into its diastereomers. The relative stereochemistry was determined by 1H NMR in their oxazolidone derivatives. The threo-isomer was resolved by (S)-1-(1-naphthyl) ethylamine in the N-(p-methoxybenzyloxycarbonyl) derivative. The absolute configuration of (--) threo-3-(p-methoxybenzyloxycarbonyl)amino-2-hydroxyvaleric acid was confirmed to be 2R, 3S. The absolute configuration of 3-amino-2- oxovaleric acid in poststatin was confirmed to be S by comparison of the four stereoisomers of methyl N, O bis(3,5-dinitrobenzoyl)-3-amino-2-hydroxyvalerate derived from 3-amino-2 hydroxyvaleric acid and that derived from 3-amino-2-oxovaleryl moiety of poststatin by means of HPLC with chiral column. PMID- 8626244 TI - Further novel milbemycin antibiotics from Streptomyces sp. E225. Fermentation, isolation and structure elucidation. AB - Ten novel alpha and beta class milbemycins have been isolated and characterized from the Streptomyces sp. E225, which has previously been shown to produce four related milbemycins. Some of the metabolites contain new structural features including, VM48641 which possesses an alpha-methoxyl substituent at C-27, and VM48642 which contains a furan ring at the terminus of the C-26 side chain. Several of these new compounds were shown to possess potent anthelmintic activity. An analysis of NMR chemical shift trends in this series of metabolites is presented. PMID- 8626246 TI - Poststatin, a new inhibitor of prolyl endopeptidase. IV. The chemical synthesis of poststain. AB - Total synthesis of poststatin was achieved by both liquid phase and solid phase methods. In both methods, the (2R,3S)-3-amino-2-hydroxyvaleric acid moiety was incorporated into protected pentapeptides, and was oxidized to (S)-3-amino-2 oxovaleric acid (postine). Deprotection of the oxidized pentapeptides gave a specimen identical with natural poststatin in physico-chemical properties and prolyl endopeptidase inhibitory activity. PMID- 8626247 TI - Pyripyropenes, novel ACAT inhibitors produced by Aspergillus fumigatus. IV. Structure elucidation of pyripyropenes M to R. AB - Six new pyripyropenes, M to R, were isolated from the ethyl acetate extracts of the jar fermentation broth of Aspergillus fumigatus FO-1289-2501. Structural elucidation indicated that all the pyripyropenes have the same pyridino-alpha pyrone sesquiterpene core as pyripyropenes A to L. Among them pyripyropene M showed the most potent inhibition against acyl-CoA : cholesterol acyltransferase activity with an IC50 value of 3.80 microM in rat liver microsomes, but pyripyropenes N to R showed moderate inhibitory activity (IC50 11.0 approximately 78.0 microM). PMID- 8626248 TI - Himastatin, a new antitumor antibiotic from Streptomyces hygroscopicus. III. Structural elucidation. AB - The structure of the antitumor antibiotic himastatin was determined using a combination of spectroscopic and chemical degradation techniques. Himastatin is a unique dimeric cyclohexadepsipeptide joined through a biphenyl linkage between two oxidized tryptophan units. The gross structure of the dimer was established through degradative ozonolysis. Himastatin consists of D-valine, D-threonine, L leucine, L-alpha-hydroxyisovaleric acid, (3R,5R)-5-hydroxypiperazic acid, and (2R,3aR,8aR)-3a-hydroxyhexahydropyrrolo[2,3b]indole 2-carboxylic acid subunits. PMID- 8626249 TI - A neuronal cell protecting substance, 4-demethoxymichigazone, produced by Streptomyces halstedii. PMID- 8626250 TI - BE-40644, a new human thioredoxin system inhibitor isolated from Actinoplanes sp. A40644. PMID- 8626251 TI - Base decomposition of erythromycin A methoxime. PMID- 8626252 TI - Synthesis and antimetastatic activity of 6-trichloroacetamido and 6-guanidino analogues of siastatin B. PMID- 8626253 TI - Epoxyquinomicins A and B, new antibiotics from Amycolatopsis. PMID- 8626255 TI - Azole drug resistance in yeasts. PMID- 8626256 TI - Laboratory assessment of antibacterial activity of zwitterionic 7-methoxyimino cephalosporins. AB - Zwitterionic 7-methoxyimino cephalosporins (cefpirome, cefepime, cefclidin, DQ2556, FK037 and SCE2787) possess a variable substitution at C3 which contains a quarternary nitrogen. These cephalosporins display low affinities for Class I beta-lactamase and rapid penetration through the outer membrane of Gram-negative bacilli, so that an increased number of periplasmic beta-lactam molecules interact with PBP's per unit of time. As a consequence, the new zitterionic compounds remain active against some, but not all, ceftazidime-resistant Enterobacteriaceae producing high levels of Class I beta-lactamase or Bush type 2b beta-lactamases. Antipseudomonas activities are generally similar to that of ceftazidime except that cefclidin is more active. The new zwitterionic compounds, especially cefpirome and FK037, express greater antistaphylococcal potency than does ceftazidime. A variety of animal models including meningitis and endocarditis have confirmed the potential of these compounds in-vivo. On the basis of structural and antibacterial characteristics, the expression 'forth generation' is acceptable to describe the zwitterionic 7-methoxyimino cephalosporins. PMID- 8626254 TI - Carbazate as a glycine bioisostere in restricticin. PMID- 8626257 TI - Antimicrobial prescribing in patients on haemofiltration. AB - Continuous haemo(dia)filtration techniques as a means of extracorporeal renal replacement therapy are being used more and more, especially on intensive care units. The effect of intermittent haemodialysis on the pharmacokinetics of systemic antibiotics is well documented and advice is provided in the drug data sheets regarding dosage, timing and additional doses (post haemodialysis). Continuous haemofiltration significantly alters the handling of these same antibiotics compared with haemodialysis, such that if the advice given for 'haemodialysis' is used for patients on haemofiltration, under-dosing the patient may lead to sub-therapeutic antibiotic levels. The reasons for these differences are discussed and suggested dosage modifications are given for commonly used antimicrobials based on available published data. PMID- 8626258 TI - Tests for susceptibility of Streptococcus pneumoniae to cefdinir: proposed interpretive criteria and quality control parameters for both microdilution and disc diffusion methods. AB - Proposed quality control (QC) parameters for susceptibility testing of Streptococcus pneumoniae to cefdinir were developed following the procedure recommended by the National Committee for Clinical Laboratory Standards. The proposed QC MIC range for microdilution susceptibility testing of S. pneumoniae ATCC 49619 is 0.06-0.25 mg/L. The proposed QC limits for inhibitory zone diameters of S. pneumoniae ATCC 49619 around 5 micrograms cefdinir disks is 26-31 mm. We recommend the following for tentative interpretive criteria for determining the susceptibility of S. pneumoniae to cefdinir: susceptible, MIC < or = 0.5 mg/L or inhibition zone diameter > or = 23 mm; intermediate, MIC 1.0 mg/L or inhibition zone, 20-22 mm; resistant, MIC > or = 2.0 mg/L or inhibition zone diameter, < or = 19 mm for broth microdilution and disc diffusion tests, respectively. PMID- 8626259 TI - Itraconazole susceptibilities of fluconazole susceptible and resistant isolates of five Candida species. AB - The in-vitro susceptibilities of 1380 isolates of five Candida species were determined in order to establish whether isolates resistant to fluconazole were cross-resistant to itraconazole. IC50 values were determined by a broth microdilution method. 690 Candida albicans isolates, seven Candida glabrata isolates, seven Candida krusei isolates, 120 Candida parapsilosis isolates and 37 Candida tropicalis isolates were susceptible to both fluconazole (IC50 < or = 32 mg/L) and itraconazole (IC50 < or = 4 mg/L). Twenty eight of 160 C. albicans isolates (17.5%), 180 of 293 C. glabrata isolates (61.4%), six of 48 C. krusei isolates (12.5%), and 10 of 18 C. tropicalis isolates (55.5%) resistant to fluconazole (IC50 > or = 64 mg/L) were also resistant to itraconazole (IC50 > or = 8 mg/L). In contrast, drug-specific resistance to itraconazole was not observed in any of the isolates tested. However, the itraconazole IC50s for fluconazole susceptible isolates were lower than those for fluconazole resistant isolates, which suggests that patients who fail fluconazole treatment might require itraconazole at higher dosages than usual. PMID- 8626260 TI - Cytotoxicity of hamycin for Trichomonas vaginalis, HeLa and BHK-21. AB - Hamycin, a polyene antibiotic related to amphotericin B, has been used topically to treat fungal and protozoan infections in India. We assessed the cytotoxic activity of hamycin on nine metronidazole resistant or susceptible Trichomonas vaginalis strains isolated from symptomatic or asymptomatic patients. Cytotoxic activity of hamycin against two mammalian cell lines, BHK-21 and HeLa, was also determined. Tritiated thymidine pulse labeling after drug-washout and a recovery period was used to distinguish death of target cells from temporary static effects of the drug. Liposomal hamycin and hamycin formulated with dimethyl sulphoxide, deoxycholate or glycerin were compared. Although all hamycin preparations were trichomonacidal at approximately 1 mg/L, hamycin-dimethyl sulphoxide was stable for only 24 h, and hamycin glycerin was incompletely solubilized. Hamycin-deoxycholate remained a stable gel for 2 months at room temperature, but its activity was reduced four-fold when compared to the fresh preparation. The mammalian tissue culture cell lines HeLa and BHK-21 were killed by trichomonacidal concentrations of hamycin-deoxycholate. This cytotoxicity of hamycin for mammalian cells is a concern, but new forms of the drug are under development that may allow more widespread use of this drug. PMID- 8626261 TI - Nephrotoxicity, high frequency ototoxicity, efficacy and serum kinetics of once versus thrice daily dosing of netilmicin in patients with serious infections. AB - The effect of dosing regimen on nephrotoxicity, high frequency ototoxicity, efficacy and serum kinetics was studied in a prospective, randomised clinical study. Therapy was started with total daily doses of 6 mg/kg given once (od) or thrice (tid) daily to 56 and 57 patients, respectively. Subsequent doses were adjusted according to serum levels. No major differences in toxicity or efficacy were noticed between od and tid regimens: clinical failures occurred in two and two patients, four and five patients suffered from a decrease of > or = 20 dB at least unilaterally at one frequency between 8 and 18 kHz, six and seven patients had a > 25 mumol/L or > 25% increase in serum creatinine, respectively. Serum creatinine or creatinine clearance did not change significantly during either therapy. Major differences between the two study groups were limited to pharmacokinetic parameters. Od dosing resulted in higher peak (mean of 21.6 vs 7.2 mg/L) and lower trough levels (0.5 vs 1.4 mg/L). Half-lives of netilmicin determined between 1 and 8 h increased significantly during therapy with tid (from a mean of 2.75 to a mean of 3.33 h, P < 0.01) but not significantly with od (rise from 2.8 to 3.03 h). Much longer half-lives were determined between 8 and 24 h in the od group (mean of 5.7 h, P < 0.01). In conclusion, only minimal differences in toxicity and efficacy were observed. Their clinical relevance appears to be minimal. PMID- 8626262 TI - Tetracycline resistance determinants, Tet B and Tet M, detected in Pasteurella haemolytica and Pasteurella multocida from bovine herds. AB - Resistance to antibiotics has recently emerged in Pasteurella haemolytica and Pasteurella multocida isolated from bovine herds. Forty-two clinical strains resistant to antibiotics and isolated through a French national network from different origins were analysed for their resistance to tetracycline. The MICs of tetracycline ranged from 32-256 mg/L. The resistance determinants Tet B and Tet M were detected in two strains, in which they are probably chromosomal. PMID- 8626263 TI - Characterization of vancomycin resistance in Enterococcus durans. AB - During investigation of an outbreak of vancomycin resistant Enterococcus faecium in a paediatric hospital, an isolate of Enterococcus durans resistant to vancomycin, teicoplanin, ampicillin and highly resistant to gentamicin and streptomycin was found in the stools of a patient also colonized with a strain of E. faecium with the same resistance pattern. Minimal inhibitory concentrations of vancomycin and teicoplanin were 512 and 64 mg/mL, respectively. Resistance to vancomycin as well as high-level resistance to gentamicin was transferable to an E. faecium recipient strain. Both multiresistant E. faecium and E. durans isolates as well as the transconjugant presented only one plasmid. The vanA gene was detected and localized to the high molecular weight plasmid by DNA hybridization with a vanA gene probe. Growth in vancomycin resulted in induction of an approximately 40 kDa protein visible in membrane preparations from these cells. Genetic linkage between vancomycin and gentamicin resistance genes in the same plasmid is suggested. PMID- 8626264 TI - Inhibition of Helicobacter pylori growth by 4-hydroxy-2-alkyl-quinolines produced by Pseudomonas aeruginosa. AB - 4-Hydroxy-2-alkylquinolines produced by Pseudomonas aeruginosa can inhibit the growth of both metronidazole-sensitive and resistant strains of Helicobacter pylori in vitro. The MIC of one analogue, 4-hydroxy-2-heptylquinoline, was determined by agar plate incorporation as 0.1-0.5 mg/L which compares favourably with other anti-helicobacter agents. PMID- 8626265 TI - Susceptibility of Streptococcus pneumoniae strains isolated in Italy to penicillin and ten other antibiotics. AB - The susceptibility of 312 Streptococcus pneumoniae strains isolated during 1993 in Italy has been studied. Overall resistance to penicillin, erythromycin and co trimoxazole was 5.5, 6 and 18% respectively. The penicillin resistant isolates (nine displaying low- and eight high-level resistance) were further tested against cefotaxime, ceftriaxone, cefepime, cefodizime, ceftazidime, imipenem, meropenem and rifampicin. The low-level penicillin resistant strains were inhibited by all these drugs. Among the eight high-level penicillin resistant pneumococci, four were resistant to one or more cephalosporins. Imipenem and rifampicin remained effective against all isolates. In comparison with other European Mediterranean countries, penicillin resistance is a minor threat at present in Italy. PMID- 8626266 TI - In-vitro antimicrobial susceptibility of Yersinia enterocolitica isolates from stools of patients in The Netherlands from 1982-1991. AB - The MICs of 24 antimicrobial agents were determined for 335 strains of Yersinia enterocolitica isolated from faeces in the Netherlands during 1982-1991. The isolates belonged to biotypes 1A, 1B, 2, 3, 4 and to serotypes O3, O5.27, O6.3, O7.8, O8, O9. Almost all strains were susceptible to piperacillin, piperacillin/tazobactam, imipenem, all cephalosporins except cefazolin, the aminoglycosides, quinolones, co-trimoxazole, doxycycline and chloramphenicol but resistant to amoxycillin, co-amoxiclav and macrolides. No association was observed between susceptibility patterns, biotype or serotype nor were there marked changes in the susceptibility during the last decade. The agents traditionally used to treat human infection, including co-trimoxazole, doxycycline and chloramphenicol may remain drugs of first choice. PMID- 8626267 TI - Activity of biapenem (LJC 10627) against 51 imipenem-resistant bacteria and selection and characterisation of biapenem-resistant mutants. AB - For wild-type bacteria the activity of biapenem was similar to that of imipenem, but for 51 imipenem-resistant strains meropenem was more active than either. When penicillin-binding protein 2a (PBP 2a) was expressed in Staphylococcus aureus biapenem had reduced activity. Mutant bacteria with decreased susceptibility to biapenem were selected in agar. Most of the mutant Gram negative bacteria were unstable and readily reverted to susceptible. The mutant Proteus vulgaris and Pseudomonas aeruginosa lacked an outer membrane protein. Biapenem-resistant S. aureus could be selected only from MRSA. PMID- 8626268 TI - The effect of burn wound surgery and teicoplanin on the bactericidal activity of polymorphonuclear leucocytes against Staphylococcus aureus. AB - Polymorphonuclear leucocyte (PMN) function is suppressed for several weeks after burn injury, rendering patients susceptible to infection, commonly with Staphylococcus aureus. A study was performed to determine the effects of surgery to the burn wound and antimicrobial prophylaxis with teicoplanin on killing of S. aureus by PMNs taken from burn patients. The bactericidal rate was significantly reduced before surgery compared to controls (P < 0.01 Mann Whitney), but neither surgery nor teicoplanin had any significant additional effect on the bactericidal rate. The bactericidal rate of PMNs did not have any significant effect on clinical outcome following surgery. PMID- 8626269 TI - Conflicts of interest: the genesis of synthetic antimalarial agents in peace and war. AB - Malaria has had an enormous impact on human history, not least in times of war. The disease has been treatable by a natural remedy, quinine, since the 17th century, but the production of synthetic antimalarial agents was first achieved in Germany in the wake of the Great War of 1914-1918, in which malaria had caused immense problems. In the 1920s research workers in the Bayer laboratories of the IG Farbenindustrie consortium developed the 8-aminoquinoline plasmoquine (the forerunner of primaquine). They went on to develop the acridine dye, atebrin (mepacrine) and the 4-aminoquinolines, Resochin (developed at the end of the Second World War in America as chloroquine) and Sontochin. British attempts to match the advances achieved by the Germans were at first unproductive, partly because collaboration between academic and industrial organizations in the UK was beset by concerns over patent rights. However, with the outbreak of World War II, when supplies of antimalarials were scarce, ICI succeeded in the large-scale production of mepacrine (essential to prosecution of the war, particularly in the Far East) and also initiated a programme of collaborative research that eventually led to the discovery of proguanil (Paludrine); this, in its turn led to the diaminopyrimidine, pyrimethamine. A massive cooperative screening programme in the USA during World War II eventually bore fruit in the realization of the therapeutic potential of chloroquine, and in the later development of amodiaquine and primaquine. Some of this work also influenced the subsequent discovery of mefloquine and halofantrine at the Walter Reed Army Institute of Research. PMID- 8626270 TI - In-vitro activity of the fluoroquinolone trovafloxacin against penicillin susceptible and -resistant Streptococcus pneumoniae. PMID- 8626271 TI - In-vitro activity of trospectromycin against gram-positive cocci. PMID- 8626272 TI - In-vitro susceptibility to thirteen antibiotics of Pasteurella spp. and related bacteria isolated from humans. PMID- 8626273 TI - Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region. AB - A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization. PMID- 8626275 TI - Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti. AB - The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability. PMID- 8626274 TI - Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. AB - Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A. tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C. To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A. tumefaciens was tested at various temperatures. Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C. Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine. However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature. A. tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided. Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself. As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly. PMID- 8626276 TI - Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. AB - In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing. PMID- 8626277 TI - Identical transcriptional control of the divergently transcribed prtP and prtM genes that are required for proteinase production in lactococcus lactis SK11. AB - We have investigated transcriptional regulation of the divergently transcribed genes required for proteinase production (prtP and prtM) of Lactococcus lactis SK11. Their promoters partially overlap and are arranged in a face-to-face configuration. The medium-dependent activities of both prtP and prtM promoters were analyzed by quantitative primer extension studies and beta-glucuronidase assays with L. lactis MG1363 cells harboring transcriptional gene fusions of each promoter with the promoterless beta-glucuronidase gene (gusA) from Escherichia coli. High-level production of prtP- or prtM-specific mRNAs was found after the growth of cells in media with low peptide concentrations, while increases in peptide concentrations resulted in an approximately eightfold decrease in mRNA production. Furthermore, prtP and prtM promoters exhibited similar efficiencies under different growth conditions. Deletion analysis of the prt promoter region showed that all the information needed for full activity and regulation of the prtP and prtM promoters is retained within a 90-bp region which includes both transcription initiation sites. An inverted repeat sequence positioned around the prtP and prtM transcription initiation sites was disrupted by either deletion or insertion of a small DNA sequence to analyze their effects on the activities of both prtP and prtM promoters. The mutations affected the activities of these promoters only marginally at low peptide concentrations but resulted in 1.5- to 5 fold derepression at high peptide concentrations. These results indicate that the expression of both prtM and prtP genes is controlled in an identical manner via a control mechanism capable of repressing transcription initiation at high peptide concentrations. PMID- 8626278 TI - A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. AB - It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids. PMID- 8626279 TI - Cloning, sequencing, and expression of the gene encoding a large S-layer associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli. AB - The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface. PMID- 8626280 TI - Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20. AB - The lemA gene of Pseudomonas syringae pv. syringae encodes the sensor kinase of a bacterial two-component signal transduction system. Phenotypes that are lemA dependent in P. syringae include lesion formation on bean and production of extracellular protease and the antibiotic syringomycin. Recently, the gacA gene has been identified as encoding the response regulator of the lemA regulon. To identify additional components that interact with LemA, suppressors of a lemA mutation were sought. A locus was identified that, when present in multiple copies, restores extracellular protease production to a lemA insertion mutant of P. syringae pv. syringae. This locus was found to encode the P. syringae homologs of translation initiation factor IF3 and ribosomal proteins L20 and L35 of Escherichia coli and other bacteria. Deletion analysis and data from Western immunoblots with anti-IF3 antiserum suggest that protease restoration does not require IF3. Deletion of both the L35 and L20 genes resulted in loss of protease restoration, whereas disruption of either gene alone increased protease restoration. Our results suggest that overexpression of either L20 or L35 is sufficient for protease restoration. It is unclear how alteration of ribosomal protein expression compensates in this instance for loss of a transcriptional activator, but a regulatory role for L20 and L35 apart from their function in the ribosome may be indicated. PMID- 8626281 TI - Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli. AB - The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene have been investigated under different environmental conditions with single-copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase. ArcA and AppY activated transcription of the cyx-appA operon during entry into stationary phase and under anaerobic growth conditions. The expression of the cyx-appA operon was affected by the anaerobic energy metabolism. The presence of the electron acceptors nitrate and fumarate repressed the expression of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high level of expression of the operon was obtained in glucose medium supplemented with formate, in which E. coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented in this paper indicate a clear difference in the regulation of the cyx-appA operon and that of the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function under even more-oxygen-limiting conditions than cytochrome d oxidase. The expression of the appY gene is induced immediately by anaerobiosis, and this anaerobic induction is independent of Fnr, and AppY, but dependent on ArcA. The expression of the appY gene is not affected significantly by the anaerobic energy metabolism, i.e., fermentation versus anaerobic respiration. A model incorporating the anaerobic regulation of the appY gene and the two operons which are controlled by AppY, the hydrogenase 1 (hya) operon and the acid phosphatase (cyx-appA) operon, is presented. The expression of the appY gene is inversely correlated with the growth rate and is induced by phosphate starvation as well as during entry into stationary phase. During oxygen limiting conditions the stationary-phase induction is partially dependent on ArcA. The alternative sigma factor sigma S has limited influence on the transcription of the appY gene during entry into stationary phase and no effect on the induction by phosphate starvation. PMID- 8626282 TI - An autolysin ring associated with cell separation of Staphylococcus aureus. AB - atl is a newly discovered autolysin gene in Staphylococcus aureus. The gene product, ATL, is a unique, bifunctional protein that has an amidase domain and a glucosaminidase domain. It undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, a 59-kDa endo-beta-N acetylglucosaminidase and a 62-kDa N-acetylmuramyl-L-alanine amidase. It has been suggested that these enzymes are involved in the separation of daughter cells after cell division. We recently demonstrated that atl gene products are cell associated (unpublished data). The cell surface localization of the atl gene products was investigated by immunoelectron microscopy using anti-62-kDa N acetylmuramyl-L-alanine amidase or anti-51-kDa endo-beta-N-acetylglucosaminidase immunoglobulin G. Protein A-gold particles reacting with the antigen-antibody complex were found to form a ring structure on the cell surface at the septal region for the next cell division site. Electron microscopic examination of an ultrathin section of the preembedded sample revealed preferential distribution of the gold particles at the presumptive sites for cell separation where the new septa had not been completed. The distribution of the gold particles on the surface of protoplast cells and the association of the gold particles with fibrous materials extending from the cells suggested that some atl gene products were associated with a cellular component extending from the cell membrane, such as lipoteichoic acid. The formation of a ring structure of atl gene products may be required for efficient partitioning of daughter cells after cell division. PMID- 8626283 TI - Multiple glucan-binding proteins of Streptococcus sobrinus. AB - Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90 kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation. PMID- 8626284 TI - Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival. AB - Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems. PMID- 8626285 TI - Regulatory factors acting at the bacteriophage Mu middle promoter. AB - Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity. PMID- 8626286 TI - The Erwinia chrysanthemi pecT gene regulates pectinase gene expression. AB - A new type of Erwinia chrysanthemi mutant displaying a derepressed synthesis of pectate lyase was isolated. The gene mutated in these strains, pecT, encodes a 316-amino-acid protein with a size of 34,761 Da that belongs to the LysR family of transcriptional activators and presents 61% identity with the E. coli protein LrhA. PecT represses the expression of pectate lyase genes pelC, pelD, pelE, pelL, and kdgC, activates pelB, and has no effect on the expression of pelA or the pectin methylesterase genes pemA and pemB. PecT activiates its own expression. The mechanism by which PecT regulates pectate lyase synthesis is independent of that of the two characterized regulators of pectate lyase genes, KdgR and PecS. In contrast to most of the members of the LysR family, pecT is not transcribed in a direction opposite that of a gene that it regulates. pecT mutants are mucoid when grown on minimal medium plates and flocculate when grown in liquid minimal medium, unless leucine or alanine is added to the medium. Thus, pecT may regulate other functions in the bacterium. PMID- 8626287 TI - Cyclization reaction catalyzed by branching enzyme. AB - The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product. PMID- 8626288 TI - Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli. AB - The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine. Stimulation of rpoS translation and a change in the half-life of sigma(s) from 3 to 50 min both contribute to osmotic induction. Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for sigma(s) degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence. PMID- 8626289 TI - Analysis of CRP-CytR interactions at the Escherichia coli udp promoter. AB - Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators. PMID- 8626290 TI - Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA. AB - Two homologs of the outer membrane protein OmpA were identified in Aeromonas salmonicida by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and amino-terminal sequence analyses. An A. salmonicida genomic DNA library was constructed by using lambda GEM-11 and recombinant phage carrying both genes ompAI and ompAII) selected by immunoscreening. A 5.0-kb BamHI fragment containing the two genes in tandem was subcloned in pBluescript and used for further subcloning and sequencing of the genes. The encoded proteins (Mr = 33,564 and 32,536 for mature OmpAI and OmpAII, respectively) had only 64% identity with each other and otherwise had the highest level of homology to OmpA proteins from the members of the family Enterobacteriaceae. Based on the Escherichia coli OmpA model, an eight-stranded amphipathic beta-barrel model for the membrane assembly of the N-terminal half of OmpAI and OmpAII was predicted. Most variation between the two proteins was localized to the predicted surface loops and periplasmic turns, while the transmembrane strands and C-terminals domains were highly conserved. Expression of ompAI and ompAII separately in E. coli indicated that both genes could be independently transcribed from their own promoters and that both gene products were assembled into the E. coli outer membrane. A survey of different Aeromonas spp. by PCR revealed that possession of two tandem ompA genes was widespread among this genus. This is the first report of any bacterial species possessing two genes for homologs of this major outer membrane protein. PMID- 8626291 TI - The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development. AB - A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID- 8626293 TI - Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. AB - Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen. PMID- 8626292 TI - RNases involved in ribozyme degradation in Escherichia coli. AB - Hammerhead ribozymes are small catalytic RNA molecules that can be designed to specifically cleave other RNAs. These ribozymes have exhibited low efficiency when examined inside cells, perhaps in part because of their sensitivity to intracellular RNases. In an effort to better understand intracellular degradation of small, foreign RNAs and to develop more stable ribozymes, the ability of Escherichia coli RNase mutants to digest ribozymes was examined. In soluble extracts, most (80 to 90%) of the endonucleolytic activity was due to RNases I and I*, since degradative activity was inhibited by Mg2+ and by the rna-2 mutation. Degradation by exonucleolytic activities was temperature sensitive in extracts from an rna pnp rnb(Ts) triple mutant but not in extracts from an rna rnb(Ts) double mutant. Thus, the products of rnb and pnp, RNase II and polynucleotide phosphorylase, respectively, appear to be the major exonucleases that degrade hammerhead ribozymes. Examination of intracellular degradation revealed that RNases I and I* contributed to about half of the degradative activity as judged by comparison of the rate of ribozyme decay in wild-type and rna-2 mutant cells. Little additional effect was observed in rne(RNase E) and rnc (RNaseIII) mutants. Taken together, these data indicate that hammerhead ribozymes are digested largely by the degradative class of RNase (RNases I, I* and II and polynucleotide phosphorylase). PMID- 8626294 TI - The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress. AB - The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline. It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase). The bet genes are regulated by oxygen, choline, and osmotic stress. Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts. The transcripts were initiated 61 bp apart. Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (beta-galactosidase) operon fusions. Mutation of betI on plasmid F'2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline. Both promoters remained inducible by osmotic stress in a betI mutant background. On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent. The bet promoters were repressed by ArcA under anaerobic growth conditions. An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8. Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters. PMID- 8626295 TI - DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation. AB - The betT and betIBA genes govern glycine betaine synthesis from choline in Escherichia coli. In an accompanying paper we report that the betT and betI promoters are divergently organized and partially overlapping and that both are negatively regulated by BetI in response to choline. (T. Lamark, T.P. Rokenes, J. McDougall, and A.R. Strom, J. Bacteriol. 178:1655-1662, 1996). In this paper, we report that the in vivo synthesis rate of the BetI protein constituted only 10% of that of BetA and BetB dehydrogenase proteins, indicating the existence of a posttranscriptional control of the betIBA operon. A genetically modified BetI protein called BetI*, which carries 7 extra N-terminal amino acids, was purified as a glutathione S-transferase fusion protein. Gel mobility shift assays showed that BetI* formed a complex with a 41-bp DNA fragment containing the -10 and -35 regions of both promoters. Only one stable complex was detected with the 41-bp fragment and all larger promoter-containing fragments tested. In DNase I footprinting, BetI* protected a region of 21 nucleotides covering both the -35 boxes. Choline stimulated complex formation but did not change the binding site of BetI*. We conclude that in vivo BetI is bound to its operator in both repressed and induced cells and that BetI represents a new type of repressor. PMID- 8626296 TI - The organization of the outside end of transposon Tn5. AB - The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction. PMID- 8626297 TI - Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. AB - We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)] beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments. PMID- 8626298 TI - Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis. AB - Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner. PMID- 8626300 TI - Identification and overexpression in Escherichia coli of a Mycobacterium leprae gene, pon1, encoding a high-molecular-mass class A penicillin-binding protein, PBP1. AB - Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins. PMID- 8626299 TI - The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope. AB - Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/'phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low osmolarity medium. The P. putida DNA surrounding the mini-Tn5/'phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro. PMID- 8626301 TI - Mutations within the first LSGGQ motif of Ste6p cause defects in a-factor transport and mating in Saccharomyces cerevisiae. AB - Mating between the two haploid cell types (a and alpha) of the yeast Saccharomyces cerevisiae depends upon the efficient secretion and delivery of the a- and alpha-factor pheromones to their respective target cells. However, a quantitative correlation between the level of transported a-factor and mating efficiency has never been determined. a-Factor is transported by Ste6p, a member of the ATP-binding cassette (ABC) family of transporter proteins. In this study, several missense mutations were introduced in or near the conserved LSGGQ motif within the first nucleotide-binding domain of Ste6p. Quantitation of extracellular a-factor levels indicated that these mutations caused a broad range of a-factor transport defects, and those directly within the LSGGQ motif caused the most severe defects. Overall, we observed a strong correlation between the level of transported a-factor and the mating efficiency of these strains, consistent with the role of Ste6p as the a-factor transporter. The LSGGQ mutations did not cause either a significant alteration in the steady-state level of Ste6p or a detectable change in its subcellular localization. Thus, it appears that these mutations interfere with the ability of Ste6p to transport a-factor out of the MATa cell. The possible involvement of the LSGGQ motif in transporter function is consistent with the strong conservation of this sequence motif throughout the ABC transporter superfamily. PMID- 8626302 TI - Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of yopN of Yersinia spp. AB - Type III secretion functions in flagellar biosynthesis and in export of virulence factors from several animal pathogens, and for plant pathogens, it has been shown to be involved in the export of elicitors of the hypersensitive reaction. Typified by the Yop delivery system of Yersinia spp., type III secretion is sec independent and requires multiple components. Sequence analysis of an 11.5-kb region of the hrp gene cluster of Erwinia amylovora containing hrpI, a previously characterized type III gene, revealed a group of eight or more type III genes corresponding to the virB or lcrB (yscN-to-yscU) locus of Yersinia spp. A homolog of another Yop secretion gene, yscD, was found between hrpI and this group downstream. Immediately upstream of hrpI, a homolog of yopN was discovered. yopN is a putative sensor involved in host-cell-contact-triggered expression and transfer of protein, e.g., YopE, to the host cytoplasm. In-frame deletion mutagenesis of one of the type III genes, designated hrcT, was nonpolar and resulted in a Hrp- strain that produced but did not secrete harpin, an elicitor of the hypersensitive reaction that is also required for pathogenesis. Cladistic analysis of the HrpI (herein renamed HrcV) or LcrD protein family revealed two distinct groups for plant pathogens. The Yersinia protein grouped more closely with the plant pathogen homologs than with homologs from other animal pathogens; flagellar biosynthesis proteins grouped distinctly. A possible evolutionary history of type III secretion is presented, and the potential significance of the similarity between the harpin and Yop export systems is discussed, particularly with respect to a potential role for the YopN homolog in pathogenesis of plants. PMID- 8626303 TI - Structure of the core oligosaccharide in the serotype O8 lipopolysaccharide from Klebsiella pneumoniae. AB - Two classes of mutants with O-antigen-deficient lipopolysaccharides were isolated from the serotype O8 reference strain, belonging to Klebsiella pneumoniae subspecies ozaenae. These mutants were selected by resistance to bacteriophage KO1-2, which recognizes and lyses strains with lipopolysaccharide molecules containing the D-galactan II O antigen. Strain RFK-11 contains a defect in O antigen synthesis and has a complete core, including the attachment site for O antigen. This mutation is complemented by a plasmid carrying the rfb (O-antigen biosynthesis) gene cluster from the related K. pneumoniae serotype O1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide from strain RFK-9 has a mobility typical of deep-rough lipopolysaccharide. RFK-9 lipopolysaccharide lacks the attachment site for O antigen. Lipopolysaccharides from strains RFK-9 and RFK-11 were isolated, and their structures were determined by methylation analyses, muclear magnetic resonance spectroscopy, and mass spectroscopy. The deduced O8 core oligosaccharide includes the partial core structure reported for the K. pneumoniae subspecies pneumoniae serotype O1 lipopolysaccharide (M. Susskind, S. Muller-Leonnies, W. Nimmich, H. Brade, and O. Holst, Carbohydr. Res. 269:C1-7, 1995), consistent with the possibility of a conserved core structure within the species. The core oligosaccharide differs from those of the genera Salmonella and Escherichia by the absence of a hexose containing outer core, the lack of phosphate residues in the inner core, and the presence of galacturonic acid residues. PMID- 8626304 TI - Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4. AB - Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB. PMID- 8626305 TI - Transcriptional analysis of the 16s rRNA gene in Rickettsia prowazekii. AB - The control of rRNA synthesis in the etiological agent of epidemic typhus, Rickettsia prowazekii, a slowly growing obligate intracytoplasmic bacterium, was investigated. Transcription of the rickettsial 16S rRNA gene (rrs), of which there is only a single copy, was controlled by a single promoter region, and the site for the initiation of transcription (base A) was found 117 bp upstream of the rrs coding region for the mature product. The promoter region contained an Escherichia coli promoter-like sequence, TTGACA-N17-TATAAC, centered 139 bp upstream of the coding region for the mature product. To investigate whether transcription of the rickettsial rrs responds to amino acid starvation conditions, total RNA was isolated from R. prowazekii-infected mouse L929 cells with or without methionine starvation. The level of newly synthesized 16S rRNA precursors in R. prowazekii, as analyzed by ribonuclease protection assays, decreased significantly after methionine starvation for 6 h and then recovered within 12 h after the addition of methionine. The chemical half-lives of the 16S rRNA precursors in the methionine-starved rickettsiae did not differ significantly from those in the normal rickettsiae. These results suggest that R. prowazekii regulates transcription of the rrs in response to amino acid starvation conditions. PMID- 8626306 TI - Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer. AB - For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s( 1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms. PMID- 8626307 TI - Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics. AB - A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei, S. Altamura, R. Huber, K. O. Stetter, and P. Cammarano, J. offteriol. 170-4353-4360, 1988), Aquifex ribosomes are susceptible to all of the aminoglycosides tested (disubstituted 2-deoxystreptamines, monosubstituted 2-deoxystreptamines, sand streptidine compounds). The significance of this result in light of the Aquifex and Thermotoga placements in phylogenetic trees of molecular sequences is discussed. PMID- 8626308 TI - Topology of LcnD, a protein implicated in the transport of bacteriocins from Lactococcus lactis. AB - Four in-frame translational fusions to both the reporter proteins beta galactosidase and alkaline phosphatase support a topological model of LcnD, a protein implicated in the transport of several bacteriocins from Lactococcus lactis, in which the N-terminal part is located intracellularly and one transmembrane helix spans the cytoplasmic membrane. PMID- 8626309 TI - SurA assists the folding of Escherichia coli outer membrane proteins. AB - Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins. PMID- 8626310 TI - Importance of the E-46-D-160 polypeptide segment of the non-penicillin-binding module for the folding of the low-affinity, multimodular class B penicillin binding protein 5 of Enterococus hirae. AB - Compared with the other class B multimodular penicillin- binding proteins (PBPs), the low-affinity PBP5 responsible for penicillin resistance in Enterococcus hirae R40, has an extended non-penicillin-binding module because of the presence of an approximately 110-amino-acid E-46(-)D-160 insert downstream from the membrane anchor. Expression of pbp5 genes lacking various parts of the insert-encoding region gives rise to proteins that are inert in terms of penicillin binding, showing that during folding of the PBP, the insert plays a role in the acquisition of a correct penicillin-binding configuration by the G-364(-)Q-678 carboxy-terminal module. PMID- 8626311 TI - Heme synthesis in the rhizobium-legume symbiosis: a palette for bacterial and eukaryotic pigments. PMID- 8626312 TI - A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles. AB - A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide. PMID- 8626313 TI - Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. AB - The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Muller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola. PMID- 8626314 TI - Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. AB - Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K. Dobos, K. Swiderek, K.-H. Khoo, P. J. Brennan, and J. T. Belisle, Infect. Immun. 63:2846-2853, 1995). However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined. First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D [U-14C] glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar. Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S7, S18, S22, S29, and S41 among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions. Fast atom bombardment-mass spectrometry, N terminal amino acid sequencing, and alpha-mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit. Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography-mass spectrometry of oligosaccharides released by beta-elimination. Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone. Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-D-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)alpha-D-Manp, or alpha-D Manp(1--> 2)alpha-D-Manp(1-->2)alpha-D-Manp. This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins. PMID- 8626315 TI - Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. AB - The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively. Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator. We found that Rob (i) activates the transcription of zwf,fpr,fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase 1, (iv) is ambidextrous, i.e., requires the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer. Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E. coli genome encodes three genes with the same functional capacity. However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown. PMID- 8626316 TI - The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. AB - Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS. Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule. Primer extension analysis led to the identification of two promoters upstream of virR. Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C, theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level. However, the modes of regulation of these genes were shown to differ. The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent. The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent. In contrast, the alpha-toxin structural gene, p1c, had only one promoter, which was shown to be partially regulated by the virR and virS genes. Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites. It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence. PMID- 8626317 TI - Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae. AB - Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8 azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity. PMID- 8626318 TI - Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains. AB - The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp. PMID- 8626319 TI - Thiamine pyrophosphate (TPP) negatively regulates transcription of some thi genes of Salmonella typhimurium. AB - In Salmonella typhimurium, thiamine is a required nutrient that is synthesized de novo. Labeling studies have demonstrated probable precursors for both the 4-amino 5-hydroxymethyl-2-methylpyrimidine pyrophosphate moiety and the 4-methyl-5-(beta hydroxyethyl) thiazole monophosphate moiety. The isolation of thiamine auxotrophs with mutations in at least five different genetic loci is reported. The majority (22 of 25) of the mutants required only the thiazole moiety of thiamine to satisfy their growth requirement. Most (14 of 25) of the mutants were affected in the thi cluster at min 90 on the S. typhimurium genetic map. Data provided herein indicate that this cluster encodes an operon whose transcription is regulated by thiamine and suggest that thiamine pyrophosphate, or a molecule derived form it, is the effector molecule. Mutants with altered regulation of this operon were isolated, and we propose that they are defective in thiamine phosphate kinase, the product of the thiL gene. PMID- 8626320 TI - Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus. AB - omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403. We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or beta-galactosidase accumulation in cells containing Tn5 lac omega 4403. The putative transcriptional start site was mapped, and sequences centered near -10 and -35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli sigma70 RNA polymerase. However, deletions showed that an essential promoter element lies between -80 and -72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression. PMID- 8626321 TI - Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions. AB - A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species. PMID- 8626322 TI - Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis. AB - We have examined the effect of replacing umuDC with mucAB or rumAB on the mutagenic properties of a T-T cyclobutane dimer in an attempt to determine the molecular basis for the differences in UV-induced mutagenesis that are associated with these structurally and functionally related genes. A single-stranded vector carrying a site-specific T-T cis-syn cyclobutane dimer was transfected into a set of isogenic Escherichia coli delta umuDC strains harboring low-copy-number plasmids expressing UmuDC, MucAB, RumAB, or their genetically engineered and mutagenically active counterparts UmuD'C, MucA'B, and RumA'B, respectively. Although the overall mutation frequency was similar for all strains, the relative frequencies of the two classes of mutation induced by the T-T dimer varied according to the mutagenesis operon expressed. In umuDC strains, 3' T-->A mutations outnumbered 3' T-->C mutations, but the reverse was true for the mucAB and rumAB strains. We also found that the T-T dimer was bypassed with differing efficiencies in unirradiated cells expressing wild-type UmuDC, MucAB, and RumAB proteins. These differences can probably be attributed to the relative efficiency of the normal cellular posttranslational activation of UmuD, MucA, and RumA, respectively, since recombinant constructs expressing the mutagenically active UmuD'C, MucA'B, and RumA'B proteins all promoted similarly high levels of bypass in UV-irradiated cells. These results suggest that the UmuD'/UmuC complex and its homologs may differ in their relative abilities to promote elongation from T - T and T - G mismatched termini. Alternatively, they may differentially influence the efficiency with which these mismatches are edited or influence nucleotide insertion by the catalytic subunit of the DNA polymerase III. PMID- 8626323 TI - Cloning and analysis of sodC, encoding the copper-zinc superoxide dismutase of Escherichia coli. AB - Benov and Fridovich recently reported the existence of a copper- and zinc containing superoxide dismutase (CuZnSOD) in Escherichia coli (L. T. Benov and I. Fridovich, J. Biol. Chem. 269:25310-25314,1994). We have used the N-terminal protein sequence to isolate the gene encoding this enzyme. The gene, denoted sodC, is located at 37.1 min on the chromosome, adjacent to lhr and sodB. A monocistronic transcript of sodC accumulates only in stationary phase. The presence of a conventional leader sequence is consistent with physical data indicating that the E. coli enzyme, like other bacterial CuZnSODs, is secreted into the periplasm. Because superoxide cannot cross membranes, this localization indicates that the enzyme has evolved to defend periplasmic biomolecules against an extracytoplasmic superoxide source. Neither the source nor the target of the superoxide is known. Although once considered an exclusively eukaryotic enzyme, CuZnSOD has now been found in species that span three subdivisions of the purple bacteria. The bacterial CuZnSODs are more homologous to one another than to the eukaryotic enzymes, but active-site residues and structural motifs are clearly shared by both families of enzymes. The use of copper and an invariant disulfide bond suggest that the ancestral gene of present-day CuZnSODs evolved in an aerobic environment, long after the evolutionary split between the eukaryotes and the eubacteria. If so, a CuZnSOD gene must have been transferred laterally between members of these domains. The eukaryotic SODs most closely resemble that of Caulobacter crescentus, a relatively close descendant of the mitochondrial ancestor, suggesting that sodC may have entered the eukaryotes during the establishment of mitochondria. PMID- 8626324 TI - Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium. AB - Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels. The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log- or stationary-phase growth. The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary phase physiology. Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I. S. Lee, J. Lin, H. K. Hall, B. Bearson, and J. W. Foster, Mol. Microbiol. 17:155-167, 1995). The acid shock induction of these proteins appears to be the result of an acid shock induced increase in the level of sigmaS itself. We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+. MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W. H. Benjamin, Jr., and P. D. Hall, abstr. B-67, p. 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993). Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size. Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels. Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels. Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message. We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis. The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance. The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS dependent gene expression during pathogenesis. PMID- 8626325 TI - Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins. AB - One of the components of the RecA-LexA-controlled SOS response in Escherichia coli cells is an inducible error-prone DNA replication pathway that results in a substantial increase in the mutation rate. It is believed that error-prone DNA synthesis is performed by a multiprotein complex that is formed by UmuC, UmuD', RecA, and probably DNA polymerase III holoenzyme. It is postulated that the formation of such a complex requires specific interactions between these proteins. We have analyzed the specific protein-protein interactions between UmuC, UmuD, and UmuD' fusion proteins, using a Saccharomyces cerevisiae two hybrid system. In agreement with previous in vitro data, we have shown that UmuD and UmuD' are able to form both homodimers (UmuD-UmuD and UmuD'-UmuD') and a heterodimer (UmuD-UmuD'). Our data show that UmuC fusion protein is capable of interacting exclusively with UmuD' and not with UmuD. Thus, posttranslational processing of UmuD into UmuD' is a critical step in SOS mutagenesis, enabling only the latter protein to interact with UmuC. Our data seem to indicate that the integrity of the entire UmuC sequence is essential for UmuC-UmuD' heterotypic interaction. Finally, in our studies, we used three different UmuC mutant proteins: UmuC25, UmuC36, and UmuC104. We have found that UmuC25 and UmuC36 are not capable of associating with UmuD'. In contrast, UmuC104 protein interacts with UmuD' protein with an efficiency identical to that of the wild-type protein. We postulate that UmuC104 protein might be defective in interaction with another, unknown protein essential for the SOS mutagenesis pathway. PMID- 8626326 TI - PchR, a regulator of ferripyochelin receptor gene (fptA) expression in Pseudomonas aeruginosa, functions both as an activator and as a repressor. AB - The product of the pchR gene, an AraC-like regulatory protein, is required for production of the FptA ferric pyochelin receptor in response to iron limitation and pyochelin (D. E. Heinrichs and K. Poole, J. Bacteriol. 175:5882-5889, 1993). The influence of iron, pyochelin, PchR, and FptA on fptA and pchR gene expression was assessed with fptA-lacZ and pchR-lacZ transcriptional fusions. As was expected, the expression of fptA decreased dramatically following the inactivation of pchR by the insertion of an OmegaHg cartridge, although the effect (> 10-fold) was not as dramatic as that of pyochelin deficiency, which obviated fptA gene expression. Insertional inactivation of pchR in a pyochelin deficient (Pch-) background restored fptA expression to levels observed in the pyochelin-producing (Pch+) PchR- strain, suggesting that PchR represses fptA expression in the absence of pyochelin. Consistent with this, the cloned gene caused a five-fold decrease in the expression of the fptA-lacZ fusion in Escherichia coli. pchR gene expression was inducible by iron limitation, a result in agreement with the previous identification of a Fur box upstream of the gene, although the magnitude of the induction was less than that observed for fptA in response to iron limitation. Expression of pchR was effectively absent in a pyochelin-deficient strain, and insertional inactivation of pchR in a Pch+ or Pch background caused an increase in pchR gene expression. PchR, thus, negatively regulates its own expression. Two related heptameric sequences, CGAGGAA and CGTGGAT, were identified upstream of the putative -35 region of both fptA and pchR and may function as a binding site for PchR. Insertional inactivation of fptA caused a marked decrease in fptA expression in a Pch+ background and obviated the apparent repression of fptA expression in a Pch- background, reminiscent of the effect of a pchR mutation. The fptA mutant did not, however, exhibit a defect in pchR expression. Interestingly, fptA mutants were unable to grow in the presence of pyochelin, suggesting that FptA is the sole outer membrane receptor for ferric pyochelin. These data indicate that PchR functions as both an activator and a repressor in controlling the expression of fptA and pchR. The involvement of FptA in this control is unclear, although it may be important in mediating the pyochelin effect on fptA expression, possibly by modulating PchR activity. PMID- 8626327 TI - Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family. AB - The kinetics of glucose transport in a number of different mutants of Saccharomyces cerevisiae with multiple deletions in the glucose transporter gene family were determined. The deletions led to differences in maximal rate and affinity for glucose uptake by the cells, dependent on the growth conditions. At the same time, there were changes in glucose repression, as determined by expression of invertase activity. Only in the strain with genes HXT1-4 and SNF3 deleted but carrying HXT6/7 were glucose uptake kinetics and invertase activity independent of the presence or concentration of glucose in the growth medium. Some degree of glucose sensitivity was recovered if the SNF3 or HXT2 gene was present in the multiple-deletion background. It is hypothesized that during growth on glucose, both modulation of the kinetics of glucose uptake and derepression of invertase activity require the presence of more than one active gene of the glucose transporter family. PMID- 8626328 TI - C-terminal half of Salmonella enterica WbaP (RfbP) is the galactosyl-1-phosphate transferase domain catalyzing the first step of O-antigen synthesis. AB - We previously showed that the product of the wbaP gene of Salmonella enterica serovar Typhimurium has two functions: it is involved in the first step of O antigen synthesis (the galactosyltransferase [GT] function) and in a later step (the T function), first thought to be the flipping of the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic face to the periplasmic face of the cytoplasmic membrane. We now locate two wbaP(T) mutations within the first half of the wbaP gene by sequencing. Both mutants retain GT activity, although one was a frameshift mutation resulting in a stop codon 10 codons after the frameshift to give an open reading frame containing only 138 of the 476 codons in WbaP. We also show that there is a secondary translation starting within the wbaP gene resulting in the synthesis of a polypeptide with GT activity. These results indicate that the N- and C-terminal halves of WbaP are the T and GT functional domains, respectively. We now propose that the T block operates prior to the flippase function, probably at the release of undecaprenyl pyrophosphate-linked galactose from WbaP. PMID- 8626329 TI - Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus. AB - A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear. PMID- 8626331 TI - How to achieve constitutive expression of a gene within an inducible operon: the example of the nagC gene of Escherichia coli. AB - The nagC gene, encoding the NagC repressor/activator of the nag regulon, is part of the nagBACD operon. When the promoter-proximal nagB and nagA genes are induced 20- to 40-fold, the nagC gene is induced only two- to threefold. In addition to being transcribed as part of the polycistronic nagBACD mRNA, nagC is also expressed from two promoters located within the upstream nagA gene. These promoters are comparable in strength to the induced nagB promoter, resulting in a high basal level of the nagC mRNA. This means that when the nagBA genes are induced, there is a much smaller effect on the amount of nagC mRNA. The nagC gene is subject to low-level translation so that the amount of NagC protein is kept low despite the relatively high transcription levels. PMID- 8626333 TI - Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100. AB - Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5 trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself. PMID- 8626330 TI - Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis. AB - Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype. PMID- 8626332 TI - Transcriptional analysis of bglPH expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon catabolite repression. AB - In Bacillus subtilis, aryl-beta-glucosides such as salicin and arbutin are catabolized by the gene products of bglP and bglH, encoding an enzyme II of the phosphoenolpyruvate sugar-phosphotransferase system and a phospho-beta glucosidase, respectively. These two genes are transcribed from a single promoter. The presence of a transcript of about 4,000 nucleotides detected by Northern (RNA) blot analysis indicates that bglP and bglH are part of an operon. However, this transcript is only present when cells are grown in the presence of the inducing substrate, salicin. In the absence of the inducer, a transcript of about 110 nucleotides can be detected, suggesting that transcription terminates downstream of the promoter at a stable termination structure. Initiation of transcription is abolished in the presence of rapidly metabolized carbon sources. Catabolite repression of bglPH expression involves the trans-acting factors CcpA and HPr. In a ccpA mutant, transcription initiation is relieved from glucose repression. Furthermore, we report a catabolite responsive element-CcpA independent form of catabolite repression requiring the ribonucleic antiterminator-terminator region, which is the target of antitermination, and the wild-type HPr protein of the phosphotransferase system. Evidence that the antitermination protein LicT is a crucial element for this type of regulation is provided. PMID- 8626334 TI - Topology of the phenylalanine-specific permease of Escherichia coli. AB - The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved. PMID- 8626335 TI - NADPH-dependent reductive ortho dehalogenation of 2,4-dichlorobenzoic acid in Corynebacterium sepedonicum KZ-4 and Coryneform bacterium strainNTB-1 via 2,4 dichlorobenzoyl coenzyme A. AB - Corynebacterium sepedonicum KZ-4, described earlier as a strain capable of growth on 2,4-dichlorobenzoate (G.M. Zaitsev and Y.N. Karasevich, Mikrobiologiya 54:356 369, 1985), is known to metabolize this substrate via 4-hydroxybenzoate and protocatechuate, and evidence consistent with an initial reductive dechlorination step to form 4-chlorobenzoate was found in another coryneform bacterium, strain NTB-1 (W.J.J. van den Tweel, J.B. Kok, and J.A.M. de Bont, Appl. Environ. Microbiol. 53:810-815, 1987). 2-Chloro-4-fluorobenzoate was found to be converted stoichiometrically to 4-fluorobenzoate by resting cells of strain KZ-4, compatible with a reductive process. Experiments with cell extracts demonstrated that Mg - ATP and coenzyme A (CoA) were required to stimulate reductive dehalogenation, consistent with the intermediacy of 2-chloro-4-fluoro-benzoyl-CoA and 2,4-dichlorobenzoyl-CoA thioesters. 2,4-Dichlorobenzoyl-CoA was shown to be converted to 4-chlorobenzoyl-CoA in a novel NADPH-dependent reaction in extracts of both KZ-4 and NTB-1. In addition to the ligase and reductive dehalogenase activities, hydrolytic 4-chlorobenzoyl-CoA dehalogenase and thioesterase activities, 4-hydroxybenzoate 3-monooxygenase, and protocatechuate 3,4 dioxygenase activities were demonstrated to be present in the soluble fraction of KZ-4 extracts following ultracentrifugation. We propose that the pathway for 2,4 dichlorobenzoate catabolism in strains KZ-4 and NTB-1 involves formation of 2,4 dichlorobenzoyl-CoA, NADPH-dependent ortho dehalogenation yielding 4 chlorobenzoyl-CoA, hydrolytic removal of chlorine from the para position to generate 4-hydroxybenzoyl-CoA, hydrolysis to form 4-hydroxybenzoate, oxidation to yield protocatechuate, and oxidative ring cleavage. PMID- 8626337 TI - Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. AB - A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC 824 containing the putative hydrogenase gene (hydA) was cloned and sequenced. The 1,745-bp hydA encodes a 64,415-Da protein and presents strong identity with the [Fe] hydrogenase genes of Desulfovibrio and Clostridium species. The level of the putative hydA mRNA was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited continuous culture. These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level. Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon. Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions. The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases. PMID- 8626336 TI - The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis. AB - Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates. PMID- 8626338 TI - Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88. AB - Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications. They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11. We refer to these polysaccharides as sphingans, after the genus name. This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis. Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule negative mutants. The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined. The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88. Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen). The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans. Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster. The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88. PMID- 8626339 TI - Regulation by cyanate of the genes involved in carbon and nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942. AB - A mutant (M45) of the cyanobacterium Synechococcus sp. strain PCC 7942, which is defective in active transport of nitrate, was used for the studies of the nitrogen regulation of the genes involved in nitrate and CO2 assimilation. In a medium containing 30 mM nitrate as the nitrogen source, M45 grew under constant stress of nitrogen deficiency and accumulated a five-times-larger amount of the transcript of nirA, the gene for nitrite reductase, compared with nitrate-grown wild-type cells. By contrast, the level of the transcript of rbcL, the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was 40% of the wild-type level. Addition of ammonium to the culture of M45 abolished the accumulation of the nirA transcript and stimulated the accumulation of the rbcL transcript, showing that ammonium repressed and activated the transcription of nirA and rbcL, respectively. Glutamine, the initial product of ammonium fixation, also showed negative and positive effects on nirA and rbcL, respectively. One of the metabolites of glutamine, carbamoylphosphate, and its decomposition product, cyanate, were found to repress nirA and also to markedly activate rbcL. Cyanate negatively regulated another ammonium-repressible gene, glnA, but had no effect on the psbAI and rps1 genes. The effects of cyanate were not ascribable to the ammonium and CO, resulting from its decomposition. These findings suggested that cyanate may act as a regulator of the ammonium-responsive genes involved in carbon and nitrogen assimilation in the cyanobacterium. PMID- 8626341 TI - Transformation of Coxiella burnetii to ampicillin resistance. AB - A 5.8-kb chromosomal fragment isolated from Coxiella burnetii initiates plasmid replication in Escherichia coli and was characterized as an autonomous replication sequence, ars (M. Suhan, S.-Y. Chen, H.A. Thompson, T.A. Hoover, A. Hill, and J.C. Williams, J. Bacteriol. 176:5233-5243, 1994). In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(+)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding beta-lactamase. pSKO(+)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(+)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing beta-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (beta-lactamase) gene. PMID- 8626340 TI - Cloning, sequencing, and expression of ruvB and characterization of RuvB proteins from two distantly related thermophilic eubacteria. AB - The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expressed in Escherichia coli. Both thermostable RuvB proteins were purified to homogeneity. Like E. coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima (> or = 70 degrees C). In the absence of ATP, T. thermophilus RuvB protein bound to linear double-stranded DNA with a preference for the ends. Addition of ATP or gamma-S-ATP destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA. Expression of thermostable T. thermophilus RuvB protein in the E. coli ruvB recG mutant strain N3395 partially complemented the UV-sensitive phenotype, suggesting that T. thermophilus RuvB protein has a function similar to that of E. coli RuvB in vivo. PMID- 8626342 TI - Elevated mutation rate in mutT bacteria during starvation: evidence for DNA turnover? AB - The rate of appearance of prototrophic revertants when Escherichia coli tyrA14 (ochre) or trpA23 bacteria were incubated on plates lacking the required amino acid was greatly elevated when the organisms also carried a mutT mutation. One possible explanation for this result is that the amount of DNA replication or turnover under these conditions is much greater than has been previously recognized. PMID- 8626343 TI - Intracellular inducer Hg2+ concentration is rate determining for the expression of the mercury-resistance operon in cells. AB - Experiments involving mercury resistance mer operon-lacZ fusions, point mutations in the mercuric ion reductase merA gene, and transcomplementation have revealed that in Hg2+-resistant cells, the inducer Hg2+ concentration is rate determining for activation of transcription. mer operon expression is activated by the presence of nanomolar concentrations of Hg2+ in liquid media only when the mercuric ion reductase function is artificially inactivated in cells, whereas cells with active mercuric ion reductase require micromolar concentrations of Hg2+ for effective induction of the operon. PMID- 8626344 TI - Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression. AB - A binding site for integration host factor (IHF) was identified upstream of the aceBAK promoter. Under inducing conditions, IHF activates aceB::lacZ expression by opposing IclR repression. In contrast, IHF has little effect on aceB::lacZ expression under repressing conditions. The ability of IHF to relieve repression under inducing but not repressing conditions allows this protein to amplify the induction of aceBAK. PMID- 8626345 TI - Synonymous codon selection controls in vivo turnover and amount of mRNA in Escherichia coli bla and ompA genes. AB - A number of silent codon changes were made in two Escherichia coli genes. For the ompA gene, the replacement of seven consecutive frequently used codons with synonymous infrequently used codons reduced the ompA mRNA level and its half life. For the bla gene, the exchange of 24 codons for the most frequently used synonymous codons extended the bla mRNA half-life. A modification of ribosome traffic could account for these observations. PMID- 8626346 TI - Sensitivity of polyamine-deficient Saccharomyces cerevisiae to elevated temperatures. AB - Saccharomyces cerevisiae cells that cannot synthesize spermidine or spermine because of a deletion in the gene coding for S-adenosylmethionine decarboxylase are very sensitive to elevated temperatures when incubated in a polyamine deficient medium; i.e., growth is inhibited and the cells are killed. This sensitivity is very pronounced at 39 degrees C, but a moderate effect is noted even at 33 to 34 degrees C. These findings support findings from other studies from our laboratory on the importance of polyamines in protecting cell components against damage. The sensitivity of spermidine-deficient cells to the temperature 39 degrees C provides a useful method for screening for polyamine auxotrophs. PMID- 8626347 TI - Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli. PMID- 8626348 TI - Venlafaxine in adults with attention-deficit/hyperactivity disorder: an open clinical trial. AB - BACKGROUND: It is becoming commonly recognized that adults suffer from attention deficit/hyperactivity disorder (ADHD). Since medications used in the past of treat adults with ADHD may be ineffective or poorly tolerated by some patients, it is important to determine if newly available medications can safely ameliorate symptoms of ADHD in adults. METHOD: An open clinical trial was undertaken to examine whether venlafaxine was safe and effective in the treatment of adults with ADHD. Ten subjects who met DSM-IV criteria for ADHD were enrolled in this 8 week trial. Individuals were started on 37.5 mg of venlafaxine b.i.d. If moderate ADHD symptoms persisted at the end of Week 4, the dose of venlafaxine was increased to 75 mg b.i.d. Assessments of ADHD symptomatology included the ADHD Rating Scale, Self-Report Version (ARS) and the Clinical Global Impressions (CGI) scale. RESULTS: Nine patients completed the study. At the end of the study, 7 patients were receiving 37.5 mg b.i.d. of venlafaxine. Repeated measures ANOVA demonstrated that treatment with venlafaxine was associated with significant reductions in ADHD symptomatology (p < .02 for the ARS; p < .005 for the CGI). Of the 9 subjects who completed the trial, 7 were considered responders. Venlafaxine was well tolerated, and most patients experienced only mild side effects. CONCLUSION: Venlafaxine may be a promising agent for the treatment of ADHD in adults. Controlled clinical trials are needed to further examine this issue. PMID- 8626349 TI - Staging methods for the assessment of dementia: Perspectives. AB - Most dementias in old age are characterized by a progressive course with interindividual variability in pattern and rate of progression. Developing a system for staging such dementia poses a challenge in capturing this variability in a system that will afford comparisons among individuals and predictions of future change. Several core questions underlie the development of such systems: (1) Is there a definable order in which abilities are lost? (2) Which skills and functions should be considered essential for the staging of dementia and what is their relative weight? (3) Can the different skills be captured within one staging system? (4) How is the whole range of function captured, and are the differences between stages clearly defined? (5) Which populations can be rated with each staging system? The determination of this last question is based on understanding which other medical conditions may interfere with the course of dementia and how prior characteristics, such as education, affect ratings on specific scales for the staging of dementia. Several systems for staging dementia in older adults are described. These include the Clinical Dementia Rating, the Global Deterioration Scale/Brief Cognitive Rating Scale/Functional Assessment Staging System, the Six Clinical Phases of Cognitive Decline, the Hierarchic Dementia Scale, and the Functional Capacity Scale. Some aspects of the utility of these systems are reviewed, and the issues for further research are discussed. PMID- 8626350 TI - Asystole in electroconvulsive therapy: Report of four cases. AB - BACKGROUND: Asystole is an uncommon but potentially fatal complication of electroconvulsive therapy (ECT). Although the risk of asystole can be reduced with anticholinergic medications, the recent emphasis on new modifications of technique (first, the use of subconvulsive stimuli to titrate the seizure threshold, and second, pretreatment with intravenous beta-blockers) may increase the risk of asystole in ECT patients. METHOD: I present four new cases of asystole in ECT and outline a scheme for anticipating and preventing asystole. RESULTS: An episode of asystole did not prove to be an obstacle to further uncomplicated ECT. CONCLUSION: If risk factors contributing to asystole are reduced and adequate doses of intravenous atropine are on hand, a patient's ECT treatments need not be interrupted. PMID- 8626351 TI - Pharmacokinetic interactions and side effects resulting from concomitant administration of lithium and divalproex sodium. AB - BACKGROUND: Valproic acid is added to the lithium regimens of many patients with bipolar disorder, especially those with mania refractory to lithium treatment. METHOD: We evaluated the pharmacokinetic effects and safety of coadministration of lithium and valproate in 16 healthy volunteers in this randomized, placebo controlled, two-period (12 days each) crossover trial. Valproate or placebo was given twice daily. On Days 6-10, lithium was added. Blood samples drawn on Days 5 and 10 were analyzed for valproate by high-pressure liquid chromatography (HPLC) and for lithium by atomic absorption spectrophotometry. RESULTS: Lithium pharmacokinetics were unchanged by valproate, but valproate C(max), C(min), and AUC rose slightly during lithium coadministration. Adverse events did not change significantly. CONCLUSION: Concomitant administration of lithium and valproate appears to be safe in patients with bipolar disorder. PMID- 8626352 TI - A review of the effects of moderate alcohol intake on the treatment of anxiety and mood disorders. AB - BACKGROUND: There is no published review to help the clinician clarify the potential role of moderate ethanol consumption in patients being treated for anxiety and mood disorders. Product labels and textbook chapters routinely warn the individual against the consumption of ethanol when using prescription psychotropic drugs. A general understanding is that the reason for this recommendation is the potential for adverse synergistic effects or sedation and decreased psychomotor performance. What is overlooked by this emphasis on safety is the effect of alcohol use both on the underlying psychiatric disorder being treated and on the effectiveness of drug therapy. METHOD: We review the available literature on the interactions of ethanol with neurotransmitters and psychotropic medications and explore the clinical consequences of these interactions. RESULTS: Ethanol might affect anxiety and mood disorders by different mechanisms. Principal among these are the effects of ethanol on multiple neurotransmitter systems, which adapt in different ways to the acute and/or chronic presence of ethanol. Perturbations in the balance of CNS neurotransmitter systems may modify the acute clinical course of primary mood disorders and undermine the therapeutic response to psychotropic medications. Ethanol also modifies the clearance and disposition of psychotropic metabolites and interferes with their clinical effectiveness. Neurotransmitter responses may additionally be manifested clinically by rebound phenomena, akin to a subsyndromal withdrawal, which affect sleep and precipitate anxiety and mood symptoms. Recent alcohol use also may alter the subjective interpretation of the patient's "internal milieu," causing confusion and eliciting reactive psychopathology. CONCLUSION: While much research remains to be done, there is abundant evidence that patients with mood and anxiety disorders should abstain from even moderate ethanol use, as this adversely affects their clinical course and response to treatment. PMID- 8626354 TI - Drug-drug interactions. PMID- 8626353 TI - A pharmacoeconomic model of divalproex vs. lithium in the acute and prophylactic treatment of bipolar I disorder. AB - BACKGROUND: Divalproex and lithium are the two most rigorously studied pharmacologic treatments for acute mania in bipolar I disorder in randomized, controlled trials. The differences between the drugs in their time course of onset, predictors of response, and side effects have potentially important pharmacoeconomic implications. METHOD: Utilizing data from published studies, the University of Cincinnati Mania Project, and a consensus panel of psychiatrists, we developed a decision-analytic model to estimate the costs of treating patients with bipolar I disorder, acutely and prophylactically, for 1 year with divalproex or lithium. RESULTS: In the overall group of patients with bipolar I disorder, initial treatment with divalproex led to costs that were 9% lower than costs for initial treatment with lithium. Cost savings associated with divalproex were greatest for patients with mixed mania and rapid cycling, whereas cost savings for patients with classic mania were greater for lithium. CONCLUSION: According to the decision-analytic model developed in this study, divalproex, possibly because of a more rapid rate of antimanic activity associated with oral loading, is a less costly treatment than lithium in the acute and prophylactic treatment of patients with bipolar I disorder over 1 year. PMID- 8626355 TI - Fluoxetine associated paresthesias and alopecia in a woman who tolerated sertraline. PMID- 8626356 TI - Ketoconazole therapy for atypical depression. PMID- 8626357 TI - The effect of nefazodone on comorbid anxiety symptoms associated with depression: experience in family practice and psychiatric outpatient settings. AB - Retrospective analysis of two randomized, placebo-controlled trials evaluated the effectiveness of nefazodone in relieving depression-associated anxiety symptoms of patients with major depression in two practice settings. One study involved depressed patients (N = 138) treated in a family practice setting, the other, psychiatric clinic outpatients (N = 180). Nefazodone treatment was broadly effective across several measures of anxiety symptoms (HAM-D, HAM-A and SCL rating scales) in relieving depression-associated anxiety. The comparison drug, imipramine, was also found to be more effective than placebo treatment, although the treatment effect on depression-associated anxiety, as measured by several factors, was less than that seen with nefazodone. A subgroup of psychiatric clinic outpatients with comorbid major depression and panic disorder (N = 55) was identified by blinded record review. During nefazodone treatment, patients with panic disorder experienced marked global improvement compared with placebo treated patients, including relief of panic and phobic anxiety symptoms. Imipramine treatment was not significantly better than placebo for improvement in depression and anxiety ratings in this patient group. These results extend previous findings indicating that the new antidepressant nefazodone effectively relieves anxiety and depressive symptoms of patients with major depression. PMID- 8626358 TI - Responders to antidepressant drug treatment: a study comparing nefazodone, imipramine, and placebo in patients with major depression. AB - BACKGROUND: Nefazodone hydrochloride, an antidepressant that acts as a 5-HT2 antagonist and serotonin (5-HT) and norepinephrine uptake inhibitor, was evaluated in a double-blind, imipramine- and placebo-controlled study involving 128 patients with major depression. METHOD: Eligible patients were randomly assigned to receive placebo (2 to 6 capsules/day), imipramine (100 to 300 mg/day), or nefazodone (200 to 600 mg/day) for 8 weeks. The principal efficacy outcome measure assessed was the number of patients who experienced an adequate response during treatment. RESULTS: Based on global improvement (Clinical Global Impressions-Improvement), 67% of nefazodone-treated patients (p < or = .01) and 63% of imipramine-treated patients (p < or = .05) responded during 8 weeks of treatment, compared with 36% of placebo controls. Sixty-two percent of nefazodone treated, 53% of imipramine-treated, and 26% of placebo-treated patients had 17 item Hamilton Rating Scale for Depression (HAM-D-17) scores < or = 10 on completion of acute treatment. Nefazodone-treated patients had a lower incidence of premature treatment discontinuation and fewer dropouts for adverse events than the imipramine group. CONCLUSION: In a three arm comparison with imipramine and placebo, nefazodone had the greatest number of patients with major depression who responded to therapy. Nefazodone, a new antidepressant with novel pharmacology, is a well-tolerated, efficacious antidepressant. PMID- 8626359 TI - Nefazodone in the treatment of severe, melancholic, and recurrent depression. AB - The development of a new antidepressant medication is usually accompanied by a concern as to whether or not the compound will be sufficiently effective in clinically important subgroups of patients (e.g., depressed patients with increased severity of symptomatology, patients with melancholic features, and patients whose illness is recurrent). This paper describes results of a pooled analysis of four placebo-controlled studies included in the development program of the antidepressant nefazodone. These studies involved a total of 247 patients receiving nefazodone in a dose of up to 600 mg/day, 251 patients on placebo, and 166 patients receiving imipramine. For purposes of the analysis, patients were defined as being more severely depressed (Clinical Global Impressions scale [CGI] psychopathology score of at least markedly ill), having melancholia using DSM-III R criteria, or having recurrent major depression (using DSM-III-R criteria). Efficacy was assessed by improvement in the Hamilton Rating Scale for Depression (17 items; HAM-D-17) Total score and CGI scale. Nefazodone (mean dose at endpoint = 379 mg/day) was effective in the management of depressed patients with moderate or severe symptomatology, depressed patients with or without melancholic features, and patients with single or recurrent episodes of depression. PMID- 8626360 TI - Efficacy in long-term treatment of depression. AB - Available evidence suggests that antidepressants need to be continued on a long term basis after the acute response. Premature discontinuation soon after symptomatic response is associated with the return of depression (relapse) in many patients. One efficacy measure of an antidepressant required by many regulatory authorities prior to approval of the agent is the ability of the antidepressant to continue the acute response compared with a placebo control. The reference tricyclic antidepressants (TCAs) amitriptyline and imipramine both have been shown to be effective in this continuation phase, but there is surprisingly little evidence on the efficacy of the other TCAs. The serotonin selective reuptake inhibitors paroxetine, fluoxetine, sertraline, and citalopram, as well as nefazodone, a new antidepressant with a dual mechanism of action that is classified as a serotonin receptor modulator, are effective compared with placebo. Placebo appears to be a good comparator in assessing the long-term efficacy of antidepressants. For example, in an assessment of the long-term efficacy of citalopram, patients who responded while taking placebo and were continued on placebo treatment were compared with patients who responded to drug and were transferred to placebo. The relapse rates in both cases were similar, validating placebo as a useful control. Most studies of long-term efficacy use the discontinuation design in which patients are treated with an active drug until response is obtained and then are either continued on the active drug or switched to placebo in a random and blinded manner. Alternatively, studies with nefazodone have used the double-blind continuation design, which may be preferred because it avoids any confounding effects of the discontinuation of drug in a drug-responsive patient. For example, in acute studies, responders to treatment with nefazodone, imipramine, or placebo were continued on the same treatment under double-blind conditions for 1 year; both antidepressants were effective compared with placebo. This study design may be useful in providing an estimate of long-term efficacy that can be obtained relatively early in a drug development program. The length of treatment in the continuation phase of therapy is also of interest. The separation of drug and placebo efficacy is sharpest in the first 4 months, which is consistent with the recommendation that all treatment for depression should continue for a minimum of 4 to 6 months to prevent relapse after symptomatic response of the acute episode. After the continuation phase (relapse prevention), evolving evidence strongly indicates that antidepressant treatment should be continued in patients at risk for recurrence. Depending on the number of recurrences, lifelong prophylactic therapy may be warranted. PMID- 8626361 TI - The safety profile of nefazodone. AB - Comprehensive review of safety data from approximately 3500 patients who received nefazodone in premarketing clinical trials demonstrates the drug to be very well tolerated, with a favorable side effect profile compared with other antidepressant drugs. Nefazodone treatment was associated with fewer side effects than were the control drugs. The incidence of side effects was generally low, and treatment discontinuations for adverse effects were less frequent with nefazodone than with imipramine and comparable with fluoxetine. No late-appearing side effects or toxicity emerged during the long-term treatment (1 year or longer) of several hundred patients. There were no drug-related fatalities and no evidence that nefazodone caused specific organ toxicity, although some cardiovascular side effects were noted (e.g., asymptomatic reduced systolic blood pressure, asymptomatic sinus bradycardia). Experience in 488 elderly patients treated with nefazodone yielded no evidence of increased susceptibility of older patients to nefazodone-associated adverse experiences, including those pertaining to the cardiovascular system. However, treatment should be initiated at a reduced dose in elderly patients because of reduced hepatic clearance of nefazodone in this age group. Final dose range may be similar in healthy younger and older patients. Although nefazodone may interact with some other medications (e.g., increases at steady state in AUC: alprazolam, twofold; triazolam, fourfold), drug-drug interactions involving patients have been clinically minor. On the basis of the inhibition of cytochrome P450 3A4 isoenzyme by nefazodone in vitro, coadministration of terfenadine or astemizole with nefazodone is contraindicated because nefazodone can increase the plasma levels of these two drugs. Extensive clinical experience provides substantial evidence that nefazodone is an extremely safe and effective treatment for depression, with important advantages over existing therapies. Therapeutic benefits include a low incidence of clinically troublesome side effects and lack of unwanted psychic activation, sexual dysfunction, weight change, and the cardiotoxicity of other antidepressants. PMID- 8626362 TI - Tolerability and safety: essentials in antidepressant pharmacotherapy. AB - Current antidepressants achieve similar efficacy, with 60% to 80% of patients responding adequately. Clinical response is gradual, and differential response factors are difficult to discern. However, side effect profiles and toxicity vary substantially, so the choice of medication depends primarily on tolerability and safety. Dry mouth is prevalent with tricyclic antidepressants (TCAs), whereas nausea occurs more frequently with a serotonin selective reuptake inhibitor (SSRI). Long-term unwanted effects tend not to be a major problem, with a dropout rate of approximately 5% due to side effects. The relationship between suicidality and antidepressants remains under debate. Many TCAs are highly toxic in overdose whereas the SSRIs appear much safer. Nefazodone is a unique antidepressant with demonstrated efficacy. It is different from other antidepressants because of its two actions in the serotonin system, moderate serotonin selective reuptake blocking properties and direct 5-HT2 antagonism, which also can enhance 5-HT1 neurotransmission. The 5-HT2 antagonist properties may limit serotonin-mediated effects and, as a result, nefazodone may be more anxiolytic than other antidepressants. Nefazodone also moderately inhibits norepinephrine reuptake and blocks alpha 1-adrenergic receptors. The data base on the safety of nefazodone currently comprises approximately 3,500 patients from all research trials, which include controlled trials that allow comparisons of nefazodone treatment with several hundred patients taking TCAs or SSRIs and nearly 900 patients receiving placebo. The most frequent adverse experiences with nefazodone as compared with placebo treatment are nausea (21% vs. 14%), somnolence (19% vs. 13%), dry mouth (19% vs. 13%), dizziness (12% vs. 6%), constipation (11% vs. 7%), asthenia (11% vs. 6%), light-headedness (10% vs. 4%), and amblyopia (blurred vision; 6% vs. 3%). Approximately 12% of nefazodone treated patients dropped out because of adverse experiences, as compared with 7.4% on placebo, 10.4% on SSRIs, but 21.8% on imipramine after short-term exposure in placebo-controlled trials. Long-term safety data include nearly 1,300 patients; nefazodone was well tolerated. Nefazodone was evaluated in normal subjects by the author and was found to produce less impairment than imipramine and was less likely to interact with alcohol. In summary, nefazodone has a favorable adverse-event profile as compared with the TCAs and a rather different one from the SSRIs. It appears to be safe and well tolerated after both acute and long-term use. PMID- 8626363 TI - A multicenter double-blind comparison of nefazodone and paroxetine in the treatment of outpatients with moderate-to-severe depression. AB - BACKGROUND: The efficacy, safety, and tolerance of nefazodone and paroxetine in the treatment of depressed outpatients were compared in a randomized, double blind parallel group study at 20 centers in the United Kingdom and Republic of Ireland. METHOD: The study population comprised 206 outpatients meeting DSM-III-R criteria for a moderate-to-severe nonpsychotic major depressive episode. Patients considered to be at serious risk of suicide were excluded from participation in the study. After a drug-free baseline phase of 1 to 4 weeks, patients were randomly assigned to treatment with either nefazodone or paroxetine. Outcome measures for efficacy included the Clinical Global Impressions scales, Hamilton Rating Scale for Depression, Hamilton Rating Scale for Anxiety, Montgomery-Asberg Depression Rating Scale, and Patient Global Assessment scale. Tolerance and safety were assessed using spontaneously reported adverse events, vital signs, and laboratory investigations. RESULTS: There were no significant differences between the groups in clinical outcome. Analysis of the efficacy measures revealed a consistent and continuous improvement in both groups. A similar proportion of patients in each group discontinued treatment owing to adverse events: 15 (14%) in the nefazodone group and 13 (13%) in the paroxetine group. CONCLUSION: Nefazodone and paroxetine have similar efficacy and tolerability in the treatment of outpatients with major depression. PMID- 8626365 TI - Therapeutic dose range of nefazodone in the treatment of major depression. AB - The therapeutic dose range of nefazodone for treatment of major depression was examined in a series of placebo-controlled efficacy studies carried out during phase 2 and 3 premarketing clinical evaluation. Nefazodone is a new antidepressant drug with pharmacologic effects on both serotonin and norepinephrine neurotransmitters. The usual starting dose of nefazodone for depressed patients, unless they are being switched from a serotonin selective reuptake inhibitor (SSRI), is 100 mg. b.i.d. A lower starting dose is recommended for elderly patients or patients being treated with an SSRI. Following assessment of the patient's clinical response after the first week of therapy, the daily dose should be adjusted upward for most patients. In the efficacy studies, the majority of patients were being maintained on a dose of 300 to 500 mg daily at the end of the acute treatment period. The side effects of nefazodone most often related to dosage were sedation, nausea, and visual symptoms. Imipramine-treated patients, on the other hand, had a high incidence of dry mouth, constipation, and asthenia. In these studies, nefazodone was found to be effective and well tolerated by patients, the majority of whom were being maintained at a 300- to 500-mg/day dose, following an initial starting dose of 100 mg b.i.d. PMID- 8626364 TI - Nefazodone versus sertraline in outpatients with major depression: focus on efficacy, tolerability, and effects on sexual function and satisfaction. AB - BACKGROUND: The efficacy, tolerability, and effects on sexual function and satisfaction of nefazodone and sertraline were compared in a multicenter, randomized, double-blind, parallel-group study in outpatients with major depression. METHOD: One hundred sixty patients, 18 years of age or older, who met DSM-III-R criteria for single or recurrent nonpsychotic major depressive episodes were randomly assigned to 6 weeks of treatment with either nefazodone (100-600 mg/day) or sertraline (50-200 mg/day). Symptoms were assessed before and during treatment using the 17-item Hamilton Rating Scale for Depression (HAM-D-17), Clinical Global Impressions (CGI) Improvement scale, the CGI Severity of Illness scale, and a sexual function questionnaire. RESULTS: Of 143 patients evaluable for efficacy, 72 received sertraline and 71 received nefazodone. The mean modal daily dose at endpoint was 148 mg for sertraline and 456 mg for nefazodone. Analysis of efficacy measures (HAM-D-17 and CGI) showed consistent and comparable improvement in symptoms of depression for both treatment groups. Sertraline had negative effects on sexual function and satisfaction in both men and women, and nefazodone had no adverse effect on sexual well-being. Safety assessments based on adverse events, vital sign measurements, electrocardiographs, physical examinations, and clinical laboratory tests revealed no serious adverse events or organ toxicity associated with nefazodone or sertraline administration. CONCLUSION: Nefazodone and sertraline are well tolerated, and there was no statistically significant difference in their antidepressant activity. Sertraline treatment has negative effects on sexual function and performance in both sexes, while nefazodone has none. These findings may have clinical implications when choosing antidepressant therapy. PMID- 8626366 TI - Mania, psychosis, and antipsychotics. AB - Although psychotic symptoms often occur in the manic phase of bipolar disorder, psychotic mania has been and continues to be frequently misdiagnosed as schizophrenia or other psychotic illnesses. In addition, the role of antipsychotic agents in the acute and maintenance treatment of psychotic mania remains unclear. In this paper, we present an overview of studies of psychosis in mania. We also review studies of conventional and newer antipsychotic agents in the acute and maintenance treatment of psychotic mania to help clarify the role of these agents in treating this illness. PMID- 8626367 TI - Psychotic depression. AB - The presence of delusions or hallucinations in major depressive disorder indicates a severe form of that disorder. Compared with patients with nonpsychotic depression, those with psychotic depression have depressive symptoms that are individually more severe. They also are more likely to have hypothalamic pituitary-adrenal axis hyperactivity and increased ventricular-to-brain ratios. The short- and long-term outcomes for such patients are poor. Findings that this prognostic disadvantage may be permanent and that psychotic features recur at high rates in subsequent episodes indicate that these symptoms in major depressive disorder have a lifelong significance. Although monotherapy with antidepressants may be effective, recovery is more rapid when antidepressants are combined with antipsychotics. Electroconvulsive therapy is particularly effective for psychotic depression. The importance of combination therapy for prophylaxis is unknown. PMID- 8626368 TI - Delusional disorder: the recognition and management of paranoia. AB - Delusional disorder, the contemporary conceptualization of paranoia, is an uncommon condition characterized by the presence of one or more nonbizarre delusions and the relative absence of associated psychopathology. The delusions concern experiences that can conceivably occur in real life, such as being followed (persecutory type), having a disease (somatic type), being loved at a distance (erotomanic type), having an unfaithful sexual partner (jealous type), and possessing inflated worth, power, identity, or knowledge (grandiose type). The diagnosis requires at least 1 month's duration of the delusion; impact on functioning that is consistent with the delusion or its ramifications; generally normal appearance and behavior; and the exclusion of schizophrenia, mood disorder, substance-induced toxicity, and medical disease. Typically, patients are unaware of the psychiatric nature of the condition. They may present to internists, dermatologists, plastic surgeons, lawyers, or the police rather than to psychiatrists. Although the prevalence is low, delusional disorder is not rare. Age at onset is usually middle or late adulthood, and the course is variable. Familial transmission is suspected, and comorbidity (frequently mood disorders) may exist. Successful management is difficult and may include hospitalization, pharmacotherapy, and certain forms of psychotherapy. PMID- 8626369 TI - Management of late-life psychosis. AB - Since the average lifespan is becoming longer, the number of older patients with psychoses is expected to increase. Late-life schizophrenia is prototypical of these chronic psychotic disorders. Antipsychotic drugs are the most effective symptomatic treatment. Pharmacotherapy in older patients, however, is complicated by alterations in pharmacokinetics and pharmacodynamics. The risk of many adverse effects is considerably higher in the elderly. For example, we found the cumulative annual incidence of tardive dyskinesia among patients over age 45 to be 26%, which was five to six times greater than that reported in younger patients. Studies suggest that most patients with schizophrenia relapse without neuroleptic maintenance therapy, exemplifying the need for improved pharmacologic regimens. Data concerning the use of the newer serotonin-dopamine antagonists in patients with late-life psychoses are limited. Initial studies suggested that clozapine is efficacious, but its use is limited by side effects. Risperidone is also clinically beneficial and is generally well tolerated, but needs to be prescribed in lower doses than those recommended for younger adults. Antipsychotic use in the elderly should be accompanied by careful conservative dosing and close patient monitoring. PMID- 8626370 TI - Psychoses in children and adolescents: a review. AB - The differential diagnosis of psychoses in children and adolescents encompasses a wide range of disorders. The interpretation of psychotic symptoms in these patients must consider age, developmental level, symptomatology, and etiology for an appropriate DSM-IV diagnosis. Previous classifications of psychoses provided little information about the underlying processes or possible treatment. Clinical experience suggests that psychotic manifestations in young patients are influenced by developmental stage and that eliciting target symptoms from a young patient necessitates using and understanding the youth's language. Proper patient assessment demands interviews with family and collaborative sources, as well as endocrine, metabolic, neurologic, infectious, and toxicologic laboratory evaluations. Treatment involves interventions aimed at all spheres of life. The prognosis and outcome are generally poor in early-onset psychoses and are complicated by comorbidities. PMID- 8626371 TI - Management of schizophrenia. AB - The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for diagnosing schizophrenia require the presence of characteristic symptoms and disabilities, as well as the specific exclusion of other disorders with similar symptomatology. Once schizophrenia is diagnosed, the classification of schizophrenic symptoms as psychotic, disorganized, or negative can help clinicians predict treatment outcomes and individualize both pharmacologic and psychosocial treatment. Three treatment phases (acute, resolving, and maintenance) have been described; each has different medication strategies and goals. The introduction of novel antipsychotic agents, such as clozapine and risperidone, has enhanced the clinicians' ability to manage schizophrenic patients. Nonetheless, psychosocial treatment remains an important component of overall patient management. PMID- 8626372 TI - Structure and function of a novel voltage-gated, tetrodotoxin-resistant sodium channel specific to sensory neurons. AB - Small neurons of the dorsal root ganglia (DRG) are known to play an important role in nociceptive mechanisms. These neurons express two types of sodium current, which differ in their inactivation kinetics and sensitivity to tetrodotoxin. Here, we report the cloning of the alpha-subunit of a novel, voltage-gated sodium channel (PN3) from rat DRG. Functional expression in Xenopus oocytes showed that PN3 is a voltage-gated sodium channel with a depolarized activation potential, slow inactivation kinetics, and resistance to high concentrations of tetrodotoxin. In situ hybridization to rat DRG indicated that PN3 is expressed primarily in small sensory neurons of the peripheral nervous system. PMID- 8626373 TI - Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro. AB - Tyrosine phosphorylation has been implicated as a means by which neurite outgrowth is regulated. Because paxillin is a tyrosine-phosphorylated protein that may play a role in regulating cell morphology, we examined its expression in neuronal cells and how its tyrosine phosphorylation is related to neurite outgrowth. Paxillin was identified in several neuronal cell lines with an increased level upon differentiation. In SH-SY5Y cells, paxillin was localized along with actin filaments where processes extended from the cell body and in neuritic growth cones. Furthermore, paxillin was tyrosine-phosphorylated in SH SY5Y cells upon adhesion to laminin. Paxillin tyrosine phosphorylation paralleled that of focal adhesion kinase and occurred as cell spreading, and neurite formation was initiated. Colchicine blocked neurite outgrowth but had no effect on cell spreading or on paxillin or focal adhesion kinase tyrosine phosphorylation. In contrast, cytochalasin D eliminated neurite outgrowth, cell spreading, and the tyrosine phosphorylation of paxillin and focal adhesion kinase. These results show that paxillin is tyrosine-phosphorylated upon integrin ligand binding in neuronal cells. Our findings suggest that paxillin tyrosine phosphorylation is linked to a remodeling of the actin cytoskeleton that leads to cell spreading and neurite formation and thus a differentiated neuronal phenotype. PMID- 8626374 TI - Dominant negative stat3 mutant inhibits interleukin-6-induced Jak-STAT signal transduction. AB - Interleukin-6 (IL-6) induces tyrosine phosphorylation and activation of the latent transcription factor Stat3 in HepG2 cells. Mutation of Stat3 tyrosine 705 to phenylalanine (Y705F) inhibits IL-6-induced tyrosine phosphorylation of this Stat3 mutant in transfected HepG2 cells. In cotransfections of HepG2 cells, the Stat3 mutant Y705F causes a reduction of the tyrosine phosphorylation of wild type Stat3-FLAG. Moreover, Y705F inhibits the action of endogenous Stat3 in cotransfected cells, reducing IL-6 induction of a Stat3-responsive reporter construct. Y705F therefore acts as a dominant negative mutation of Stat3. PMID- 8626375 TI - Human native soluble CD40L is a biologically active trimer, processed inside microsomes. AB - CD40 ligand (CD40L) is a glycoprotein expressed on the surface of activated helper T cells, basophils, mast cells, and eosinophils. Binding of CD40L to its receptor CD40 on the B cell surface induces B cell proliferation, adhesion, and immunoglobulin class switching. We have identified soluble cleavage products of human CD40L in the supernatant of a stimulated human T cell clone. Subcellular fractionation experiments have shown that the transmembrane CD40L is processed inside the microsomes and that its cleavage is stimulation-dependent. The native human soluble CD40L is trimeric and, when used in conjunction with interleukin-4, induces B cell proliferation. PMID- 8626376 TI - A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT. AB - The Fas antigen (Apo1/CD95) is a transmembrane protein belonging to the nerve growth factor receptor family. It is expressed on a variety of cells, including activated T lymphocytes. Ligation of Fas with its natural ligand or with anti-Fas antibodies often results in the apoptotic death of the cell, making Fas an important mediator of down-regulating immune responses. The signal transduction pathways utilized by Fas are currently unknown, although tyrosine kinase activity has recently been strongly implicated. Here, we report that the tyrosine kinase p59fyn physically associates with Fas in Fas-sensitive cells. In addition, we show that activated T lymphocytes from fyn knockout mice exhibit elevated lifespans and reduced apoptosis in vitro compared to their normal counterparts. Furthermore, activated T lymphocytes from the fyn-deficient mice are less sensitive to killing by both anti-Fas antibody and Fas-ligand cytotoxic T cells. These results suggest that p59fyn plays an important role in Fas signal transduction. PMID- 8626377 TI - Cloned and expressed rat Ca2+-sensing receptor. AB - We have stably expressed cDNA for the rat brain Ca2+ sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor. PMID- 8626378 TI - Peroxynitrite inhibits glutamate transporter subtypes. AB - The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide (O2-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO- reduces selectively the Vmax of transport; its action is fast (reaching > or = 90% within 20 s), dose-dependent (50% inhibition at 50 microM), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO- with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO . H2O2 also inhibits reconstituted transport, and its action matches that of ONOO- on all respects; however, this is observed only with 5-10 mM H202 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO- and other oxidants. PMID- 8626379 TI - Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786. AB - We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation. PMID- 8626380 TI - Contrasting in vivo effects of murine and human apolipoprotein A-II. Role of monomer versus dimer. AB - The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously in transgenic mice overexpressing either human or murine apoA-II. These studies have shown differences between these two groups of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric human apoA-II (hA-IImon) in transgenic mice. Site-directed mutagenesis (Cys6 -> Ser) was used to generate 15 transgenic founder lines of hA-IImon mice, that contained plasma hA-IImon concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-IImon floated in the d < or = 1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels were not correlated with hA-IImon levels (r = -0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes, were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing human dimeric apoA II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II molecules and are not solely due to the fact that human apoA-II exists as a dimer. PMID- 8626381 TI - Positive regulation of cdc2 gene activity by protein phosphatase type 2A. AB - Several lines of evidence indicate that serine/threonine protein phosphatases may act as negative regulators of cellular growth. For example, treatment of cells with the tumor-promoter okadaic acid, an inhibitor of certain types of these phosphatases, resulted in the increased expression of several proto-oncogenes, indicating a negative role of the respective phosphatases in gene regulation. However, it was puzzling to find that okadaic acid-treated cells, even in the presence of highly expressed proto-oncogenes, did not proliferate, but were arrested at certain points of the cell cycle. To further analyze this discrepancy, we investigated the involvement of protein phosphatases in the control of other cell cycle regulatory genes, such as cdc2 which encodes an essential cell cycle regulatory kinase. We found that cdc2 gene expression was blocked by okadaic acid, but stimulated by protein phosphatase 2A. Protein phosphatase 2A is shown to be a positive regulator of cdc2 gene activity and to be required for cdc2 expression. Thus, our findings identify protein phosphatase 2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control. PMID- 8626382 TI - Drosophila RNA polymerase II mutants that affect transcription elongation. AB - We have examined the properties of two Drosophila RNA polymerase II mutants, C4 and S1, during elongation, pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid substitution in the largest and second largest subunits, respectively. Compared with wild type, C4 had a lower elongation rate and was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and NH4+ whereas the S1 mutant was less responsive to both. Differences between the two mutants also appeared during DmS-II-mediated transcript cleavage and pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at generating the final pattern of shortened transcripts. S1 and wild type were equal in the rate of extended DmS-II mediated transcript cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in elongation competent mode and that the C4 mutation may affect the movement of the polymerase. PMID- 8626383 TI - Characterization of sulfur-centered radical intermediates formed during the oxidation of thiols and sulfite by peroxynitrite. ESR-spin trapping and oxygen uptake studies. AB - Using a novel phosphorylated spin trap, 5-diethoxy-phosphoryl-5-methyl-1 pyrroline N-oxide (DEPMPO), an analog of the commonly used trap 5,5'-dimethyl-1 pyrroline N-oxide (DMPO), we have investigated the reactions of sulfur-centered radicals produced from the oxidation of thiols and sulfite by peroxynitrite. The predominant species trapped in all cases are the corresponding sulfur-centered radicals, i.e. glutathionyl radical (GS) from glutathione (GSH), N-acetyl-DL penicillamine thiyl radical (S-NAP) from N-acetyl-DL-penicillamine (NAP) and sulfate anion radical (SO3-) from sulfite. These radicals consume molecular oxygen forming either peroxyl or superoxide anion radicals. GS, S-NAP, and (SO3-) derived radicals react with ammonium formate to form the carbon dioxide anion radical (CO2-). Further support of spin adduct assignments and radical reactions are obtained from photolysis of S-nitrosoglutathione and S-nitroso-N-acetyl-DL penicillamine. We conclude that the direct reaction of peroxynitrite with thiols and sulfate forms thiyl and sulfate anion radicals, respectively, by a hydroxyl radical-independent mechanism. Pathological implications of thiyl radical formation and subsequent oxyradical-mediated chain reactions are discussed. Oxygen activation by thiyl radicals formed during peroxynitrite-mediated oxidation of glutathione may limit the effectiveness of GSH against peroxynitrite mediated toxicity in cellular systems. PMID- 8626384 TI - Inhibition of monocyte chemotaxis to C-C chemokines by antisense oligonucleotide for cytosolic phospholipase A2. AB - Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1 stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis. PMID- 8626385 TI - The cytoplasmic domain of alphaIIb beta3. A ternary complex of the integrin alpha and beta subunits and a divalent cation. AB - Peptides corresponding to the cytoplasmic tails of the alphaIIb (alphaIIb (985 1008)) and beta3 (beta3 (713-762)) subunits of the integrin receptor alphaIIb beta3 (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. alphaIIb (985-1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy. The binding of Tb3+ to alphaIIb (985-1008) was of high affinity (Kd = 8.8 +/- 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxy] terminus (alphaIIb (999 1008), PLEEDDEEGE). The affinity of this site for divalent cations was in the micromolar range, suggesting that this site would be constitutively occupied in the intracellular environment. Incubation of alphaIIb (999-1008) with beta3 (713 762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for alphaIIb (999-1008) in beta3 was mapped to beta3 (721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (alphaIIb (985-1008).beta3 (721-740)) and a ternary (Tb3+.alphaIIIb (999-1008).beta3 (721 740)) complex were detected by mass spectroscopy, but the distribution and intensity of the mass/charge peaks were distinct. These difference may reflect the involvement of distinct cation coordination sites and the formation of salt bridges in stabilizing the ternary complex. These data demonstrate the formation of a novel entity composed of the cytoplasmic tails of alphaIIb and beta3 and a cation which may constitute a functional intracellular domain. PMID- 8626387 TI - 4-Hydroxyhexenal is a potent inducer of the mitochondrial permeability transition. AB - Mitochondria undergo at least two types of structural alteration in response to various physiological and pathophysiological stimuli. One type is nonreversible and is associated with mitochondrial lysis. The second is reversible and appears to be associated with calcium-mediated activation of a specific inner mitochondrial membrane channel. The mechanisms underlying the induction of this second alteration, termed a mitochondrial permeability transition (PT), have been the subject of a great deal of recent research. Using rat liver mitochondria, our data demonstrate that calcium-mediated PT induction can be affected by the lipid peroxidation byproducts 4-hydroxynonenal and 4-hydroxyhexenal (HHE). 4 Hydroxynonenal appears inactive at concentrations <1 micromole but displays both stimulatory and inhibitory effects as part of a biphasic dose response between approximately 1 and 200 micromole. In contrast, HHE consistently enhances calcium mediated induction of the PT, even at femtomolar concentrations. The exquisite specificity and sensitivity of HHE led to further studies to examine the nature of this induction. Studies showing that HHE-mediated induction could be prevented by cyclosporin A confirmed PT involvement. Further studies showed that induction was dependent on both calcium and electron transport chain function. Pretreatment of the HHE with glutathione also prevented PT induction, but simultaneous addition of the thiol reagents dithiothreitol or glutathione, which often prevents PT induction, was ineffective, attesting to the effectiveness of HHE as an inducer. Together, these data provide a possible mechanistic explanation for the previously observed effects of lipid peroxidation on PT induction. PMID- 8626386 TI - Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalyzed proteolysis. AB - The intrinsic factor Xase complex (FXase) is comprised of a serine protease, FIXa, and a protein cofactor, FVIIIa, assembled on a phospholipid surface. Activity of FXase decays with time and reflects the lability of FVIIIa. Two mechanisms potentially contribute to this decay: (i) a weak affinity interaction between the FVIIIa A2 subunit and Al/A3-Cl-C2 dimer and (ii) FVIIIa inactivation resulting from FIXa-catalyzed proteolysis of the Al subunit. At low reactant concentrations (0.5 nm FVIIIa; 5 nm FIXa), FXase decay is governed by the inter FVIIIa subunit affinity and residual activity approaches a value consistent with this equilibrium, as judged by reactions containing exogenous A2 subunit. Analysis using a mutant form of FVIII (FVIIIR336I) possessing an altered FIXa cleavage site, showed similar rates of FXase decay (0.12 min(-1)) and confirmed the lack of contribution of proteolysis under these conditions. When the concentration of FIXa was increased 10-fold, the initial rate of decay of FXase containing native FVIIIa increased (0.82 min(-1)) and paralleled the rate of proteolysis of Al subunit. However, the rate of decay of FXase containing the FVIIIaR336I was reduced (0.048 min(-1)) consistent with the elevated concentration of FIXa stabilizing the labile subunit structure of the cofactor. Reconstitution of FVIII with FIXa-cleaved light chain showed that cleavage at the alternate FIXa site (A3 domain) was not inhibitory to FXase. The presence of substrate FX resulted in a 10-fold reduction in the rate of FIXa-catalyzed proteolysis of FVIIIa. These results suggest a model whereby decay of FXase results from both FVIIIa subunit dissociation and FIXa-catalyzed cleavage, dependent upon the relative concentration of reactants, with greater contribution of the former at low values and, in the absence of substrate, greater contribution of the latter at high values. PMID- 8626388 TI - In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in the activation of transcription factor NF-kappaB. AB - Transcription factor NF-kappaB is essential for the induction of nitric oxide synthase (NOS) II (iNOS) by bacterial lipopolysaccharide in murine macrophages (Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994) J. Biol. Chem. 269, 4705 4708). In 3T3 fibroblasts, agents other than cytokines are efficacious inducers of NOS II expression. In addition to cytokines such as interferon-gamma or tumor necrosis factor-alpha, protein kinase C-stimulating agents such as tetradecanoylphorbol-13-acetate, or cyclic AMP-elevating agents such as forskolin and 8-bromo-cAMP markedly increased NOS II mRNA (measured by Sl nuclease and RNase protection analyses), NOS II protein (determined by Western blotting), and NOS activity (measured by chemiluminescence detection of NO2-). Transforming growth factor-beta1 (which is an inhibitor of NOS II induction in other cell types) potentiated NOS II mRNA expression produced by all inducing agents listed, whereas dexamethasone, pyrrolidine dithiocarbamate and 3,4-dichloroisocoumarin (inhibitors of NF-kappaB activation) suppressed NOS II mRNA induction in response to all stimulants. In electrophoretic mobility shift assays, nuclear protein extracts from 3T3 cells stimulated with any of the inducing agents significantly slowed the migration of an NF-kappaB-binding oligonucleotide, whereas nuclear extracts from untreated control cells did not. These experiments indicate that NF kappaB is the key control element for the induction of NOS II in response to at least three different second messenger pathways in 3T3 cells. PMID- 8626389 TI - Substrate specificity of glycinamide ribonucleotide transformylase from chicken liver. AB - Several glycinamide ribonucleotide analogs have been prepared and evaluated as substrates and/or inhibitors of glycinamide ribonucleotide transformylase from chicken liver. The side chain modified analogs, in which the glycine side chain, R = CH2NH2, has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, are substrates, with V/K (relative intensity) of 2.4% and 16.3%, respectively. Several carbocyclic analogs of glycinamide ribonucleotide, including the phosphonate derivative of carbocyclic glycinamide ribonucleotide, did not serve as substrates, but were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values ranging from 7.4 to 23.6 times the Km for glycinamide ribonucleotide. However, the O-phosphonate analog of carbocyclic glycinamide ribonucleotide did support enzymatic activity, with V/K (relative intensity) of 0.8%. In addition, glycinamide ribonucleoside was neither a substrate for, nor an inhibitor of, glycinamide ribonucleotide transformylase. Furthermore, alpha-glycinamide ribonucleotide had no effect on enzyme activity. These studies have begun to define the structural features of the nucleotide substrate required to support enzymatic activity. PMID- 8626390 TI - Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue specific and cell cycle-regulated expression. AB - We describe the isolation and characterization of cDNAs encoding full-length human and murine cyclin G1 and a novel human homologue of this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels in skeletal muscle, ovary, and kidney. Following an initial up-regulation from early G1 to G1/S phase, cyclin G1 mRNA is constitutively expressed throughout the cell cycle in T and B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1 mRNA is maximal in early G1 phase and declines in cell cycle progression. Cyclin G1 levels parallel p53 expression in murine B lymphocytes; however, in several human Burkitt's lymphomas, murine lymphocytes treated with transforming growth factor-beta, early murine embryos, and several tissues of p53 null mice, cyclin G1 levels are either inverse of p53 levels or expressed independent of p53. The cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence identity and 53% amino acid sequence identity with cyclin G1, and like cyclin G1, exhibits closest sequence identity to the cyclin A family. Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a PEST-rich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA is differentially expressed compared to cyclin G1, the highest transcript levels seen in cerebellum, thymus, spleen, prostate, and kidney. In contrast to the constitutive expression of cyclin G1 in lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle with peak expression in late S phase. PMID- 8626391 TI - Lipoprotein association of human apolipoprotein E/A-I chimeras. Expression in transfected hepatoma cells. AB - Both apolipoprotein (apo) E and apoA-I are associated with lipoproteins, although with different particle classes. ApoE is associated with very low density lipoprotein (VLDL) and with the larger high density lipoprotein (HDL) subspecies, while apoA-I is found predominantly in association with most HDL subclasses. The genes encoding these proteins have a similar overall structure with the nucleotide sequences of the third and fourth exons coding for the mature protein. In an effort to understand the difference in lipoprotein association patterns of these two apoproteins, we have constructed and expressed chimeric apoproteins using cDNAs in which the third (n) and fourth (c) exons of human apoE and apoA-I are exchanged. McArdle rat hepatoma cells (McA-RH7777), which secrete VLDL- and HDL-like particles, were stably transfected with these cDNAs, and the cDNAs for human apoE and human apoA-I. Single spin NaBr gradient fractions of lipoprotein deficient serum-treated cell medium from transfected McA-RH7777 cells were analyzed. The distributions of transfected human apoE and apoA-I and endogenous rat apoE and apoA-I were compared with those of the chimeras. Among HDL subspecies, human apoE expressed by these cells is associated with particles of density 1.108 g/ml. Similarly, chimera apoA-InEc (exon 3 of apoA-I and exon 4 of apoE) is found in particles of density 1.111 g/ml. Human apoA-I, however, distributes in a broader range of particles with peak densities of 1.111 g/ml and 1.164 g/ml. The distribution of the complementary chimera, apoEnA-Ic, follows this same pattern, with peak particle densities of 1.098 and 1.137 g/ml. This is in contrast to the narrow distributions of endogenous rat apoE and apoA-I, which were found in particles of density 1.099 and 1.089 g/ml, respectively. When metabolically labeled medium was fractionated via gel filtration column chromatography, apoA-InEc was found to associate with the VLDL fractions; apoEnA Ic was absent from these same fractions. These results suggest that the fourth exon largely determines the distinctive lipoprotein distribution patterns of these two human apoproteins and that the human apoA-I fourth exon sequence may account for the polydisperse HDL pattern as observed by others in transgenic mice expressing human apoA-I. PMID- 8626392 TI - Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. AB - Numerous epidermal growth factor (EGF)-related peptide binding members of the ErbB family of receptor tyrosine kinases have been described. While several EGF agonists bind and activate ErbB-1/EGF receptor, neu differentiation factor (NDF) functions as a ligand for ErbB-3 and ErbB-4. However, it is currently unknown which specific subsets of ErbB receptors become activated in response to each of these ligands. The present study addresses this issue using the T47D breast tumor cell line, which expresses moderate levels of all the presently known ErbB receptors. We show that all the EGF agonists, but not NDF, stimulated tyrosine phosphorylation of ErbB-1. In contrast, all the EGF-related factors except amphiregulin were able to induce tyrosine phosphorylation of ErbB-2. The ability to induce tyrosine phosphorylation of ErbB-3 varied dramatically among the different EGF-related peptides. While EGF, transforming growth factor (TGF) alpha, and amphiregulin only had a moderate effect, NDF dramatically increased the ErbB-3 phosphotyrosine content. Most notably, heparin binding EGF-related growth factor (HB-EGF) and betacellulin (BTC) were more effective than other EGF agonists. Consequently, only NDF, HB-EGF, and BTC significantly stimulated association of phosphatidylinositol kinase activity with ErbB-3. Among the EGF agonists, HB-EGF induced a low level of ErbB-4 tyrosine phosphorylation, while BTC was as efficient as NDF in activating ErbB-4. The BTC activation of ErbB-4 appears to be independent of ErbB-1, as shown by pretreatment of cells with an antibody that inhibits binding of EGF agonists to ErbB-1. As a result of the differential activation of ErbB receptors, most of the EGF-related growth factors had distinguishable biological activities on cultured mammary epithelial cell lines. PMID- 8626393 TI - The C-terminal region of carboxypeptidase E involved in membrane binding is distinct from the region involved with intracellular routing. AB - Carboxypeptidase E (CPE) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. Previously, the C-terminal region of CPE has been shown to participate in the binding of the protein to membranes and to also contribute to the sorting of CPE into the regulated pathway. In this study, the role of the C-terminal region of CPE was further examined using several approaches. A series of CPE mutants with C-terminal deletions was expressed in the baculovirus system; constructs with a deletion of 14 or 23 residues were expressed at levels comparable to wild-type CPE. In contrast, deletion of 33 or more residues eliminated CPE activity, and the resulting protein was not secreted from the cells. Even though CPE mutants with a deletion of 14 or 23 residues were expressed normally, the resulting protein was mainly soluble, whereas approximately 55% of wild-type CPE was membrane associated. When expressed in AtT 20 cells, CPE with a deletion of 43 C-terminal amino acids was not secreted, whereas CPE with a deletion of 23 residues was secreted via the regulated pathway. Pulse-chase analysis revealed the protein with a deletion of 43 residues to be degraded in a non-acidic intracellular compartment. To investigate whether the C-terminal region of CPE can confer membrane binding and regulated pathway sorting to another protein, portions of the CPE C-terminal region were attached to the C terminus of albumin and the fusion proteins expressed in AtT-20 cells. Of the constructs examined, only the protein containing 51 amino acids of CPE was sorted to the regulated pathway, although with reduced efficiency compared to endogenous CPE. Although the C-terminal 14 amino acids of CPE are sufficient to target albumin to membranes, this fusion protein is not sorted into the regulated pathway. Taken together, these results indicate that the C-terminal 14 amino acids of CPE are important for membrane binding and that membrane binding and sorting require distinct signals. PMID- 8626394 TI - Phosphorylation of p105 PEST sequence via a redox-insensitive pathway up regulates processing of p50 NF-kappaB. AB - The p105 Rel protein has dual functions; it is the precursor of the p5O subunit of NF-kappaB, and it acts as an IkappaB-like inhibitor to retain other Rel subunits in the cytoplasm. We have investigated the posttranslational regulation of p105 following activation of Jurkat T cells and find that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-kappaB, is correlated with an increase in proteolytic processing to p5O. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, as a 68-amino acid C-terminal deletion abolishes the response to stimulation. When compounds that block Ikappa B alpha phosphorylation and degradation were tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro 3-tosyl-amido-7-amino-2-heptanone blocked inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol did not. Thus, while regulation of the p105 IkappaB resembles that of lkappaBa, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, it depends on a different, redox-insensitive, signaling pathway. PMID- 8626395 TI - TrkA mediates the nerve growth factor-induced intracellular calcium accumulation. AB - Regulation of the cytosolic free Ca2+ concentration by nerve growth factor was investigated in C6-2B glioma cells newly expressing the high affinity nerve growth factor receptor trkA, using Fura-2 fluorescence ratio imaging. In these cells, nerve growth factor (50 ng/ml) evoked a novel approximately 3-fold increase in cytosolic free Ca2+ concentration, while no measurable Ca2+ response was observed in wild type or mock-transfected cells lacking a functional trkA receptor. K-252a, a tyrosine kinase inhibitor which prevents nerve growth factor mediated responses in C6-2B cells expressing trkA, also blocked the rise in cytosolic free Ca2+ concentration by nerve growth factor. Moreover, basic fibroblast growth factor, which in these cells elicits biochemical changes similar to nerve growth factor, failed to affect cytosolic free Ca2+ concentration, further supporting the specificity of nerve growth factor/trkA receptor in mediating a Ca2+ response. While insensitive to chelation of extracellular Ca2+, the response was abolished following depletion of Ca2+ stores or blockade of intracellular Ca2+ release, providing strong evidence that intracellular Ca2+ is the main source for nerve growth factor-evoked cytosolic free Ca2+ concentration increase. Nerve growth factor increased the cytosolic free Ca2+ concentration also in NIH3T3 cells overexpressing trkA but devoid of p75 nerve growth factor receptor. Our data suggest that trkA but not p75 is required for nerve growth factor-evoked Ca2+ signaling. PMID- 8626396 TI - Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin. AB - Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg201-Arg218) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 region decrease the affinity shift to 1.1-3.2-fold. Lys 211 was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K21IN mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift. PMID- 8626397 TI - Purification and characterization of human cleavage factor Im involved in the 3' end processing of messenger RNA precursors. AB - Six different protein factors are required for the specific cleavage and polyadenylation of pre-mRNA in mammals. Whereas four of them have been purified and most of their components cloned, cleavage factor Im (CF Im) and cleavage factor IIm (CF IIm) remained poorly characterized. We report here the separation of CF Im from CF 11m and the purification of CF Im to near homogeneity. Three polypeptides of 68, 59, and 25 kDa copurify with CF Im activity. All three polypeptides can be UV cross-linked to a cleavage and polyadenylation substrate in the presence of a large excess of unspecific competitor RNA, but not to a splicing-only substrate. No additional protein factor is required for the binding of CF Im to pre-mRNA. Gel retardation experiments confirmed the results obtained by UV cross-linking. In addition, we could show that CF Im stabilizes the binding of the cleavage and polyadenylation specificity factor (CPSF) to pre-mRNA and that CPSF and CF Im together form a slower migrating complex with pre-mRNA than the single protein factors. Cleavage stimulation factor (CstF) and poly(A) polymerase (PAP) had no detectable effect on the binding of CF Im to pre-mRNA. Furthermore, the CstF-CPSF-RNA as well as the CstF-CPSF-PAP-RNA complex are supershifted and stabilized upon the addition of CF Im. PMID- 8626398 TI - Regulation by spermine of native inward rectifier K+ channels in RBL-1 cells. AB - Polyamines have been shown to participate in the rectification of cloned inwardly rectifying potassium channels, a class of potassium channel proteins that conducts inward current more readily than outward current. Here, basophil leukemia cells were used to determine the effects of polyamines on a native, inwardly rectifying potassium current. Rat basophil leukemia cells were cultured in the presence of two different polyamine biosynthesis inhibitors, and both the electrophysiological properties and the polyamine levels were monitored. Treatment with alpha-difluoromethylornithine, a specific ornithine decarboxylase inhibitor, resulted in no significant change of electrophysiological properties. In contrast, treatment with 5'-[(Z)-4-amino-2-butenyl]- methyl-amino-5' deoxyadenosine (MDL73811), an inhibitor of S-adenosylmethionine decarboxylase, resulted in increased outward currents through inwardly rectifying potassium channels while intracellular putrescine was markedly increased and spermidine and spermine levels were decreased. Fluctuations of intracellular polyamine concentrations as imposed by MDL73811 were directly translated in an altered cell excitability. Based on these results we conclude that the rectification properties of native inwardly rectifying potassium channels are largely controlled by intracellular spermine. PMID- 8626399 TI - Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin. AB - Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE. PMID- 8626400 TI - Somatostatin inhibits PC Cl3 thyroid cell proliferation through the modulation of phosphotyrosine activity. Impairment of the somatostatinergic effects by stable expression of E1A viral oncogene. AB - In this study, we report the effects of somatostatin on the proliferation of PC C13 thyroid cell line and the intracellular mechanisms involved. We also evaluated the possible alterations, induced by E1A oncogene transformation on the intracellular pathways mediating somatostatin inhibition of cell proliferation. We showed that somatostatin was able to powerfully inhibit insulin- and insulin + TSH-dependent cell proliferation by inducing a block in the G1/S progression in the cell cycle. These cytostatic effects were completely reverted by vanadate, suggesting that somatostatin may induce antiproliferative effects through the modulation of phosphotyrosine phosphatases. In the E1A-transformed cell line, somatostatin was completely ineffective. The lack of somatostatin inhibitory effects on cell proliferation were not due to alterations in the expression of somatostatin receptors, which were regularly expressed and coupled to adenylyl cyclase activity, but were dependent on an alteration in their coupling with the phosphotyrosine phosphatase. In fact, although in PC C13 cells somatostatin increased by 100% phosphotyrosine phosphatase activity, it was completely ineffective in E1A-expressing cells. In conclusion we demonstrated that somatostatin activates phosphotyrosine phosphatases in PC C13 thyroid cells to inhibit cell proliferation and that the stable expression of E1A oncogene in these cells completely abolishes this antiproliferative effect. PMID- 8626401 TI - Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action. AB - Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK. PMID- 8626402 TI - The chemistry and tumoricidal activity of nitric oxide/hydrogen peroxide and the implications to cell resistance/susceptibility. AB - The mechanism of cytotoxicity of the NO donor 3-morpholino-sydnonimine toward a human ovarian cancer cell line (OVCAR) was examined. It was found that the NO mediated loss of cell viability was dependent on both NO and hydrogen peroxide (H2O2). Somewhat surprisingly, superoxide (O2) and its reaction product with NO, peroxynitrite (-OONO), did not appear to be di- rectly involved in the observed NO-mediated cytotoxicity against this cancer cell line. The toxicity of NO/H2O2 may be due to the production of a potent oxidant formed via a trace metal-, H202 , and NO-dependent process. Because the combination of NO and H2O2 was found to be particularly cytotoxic, the effect of NO on cellular defense mechanisms involving H2O2 degradation was investigated. It was found that NO was able to inhibit catalase activity but had no effect on the activity of the glutathione peroxidase (GSHPx)-glutathione reductase system. It might therefore be expected that cells that utilize primarily the GSHPx-glutathione reductase system for degrading H2O2 would be somewhat resistant to the cytotoxic effects of NO. Consistent with this idea, it was found that ebselen, a compound with GSHPx-like activity, was able to protect cells against NO toxicity. Also, lowering endogenous GSHPx activity via selenium depletion resulted in an increased susceptibility of the target cells to NO-mediated toxicity. Thus, a possible NO/H2O2/metal-mediated mechanism for cellular toxicity is presented as well as a possible explanation for cell resistance/susceptibility to this NO-initiated process. PMID- 8626403 TI - G alpha 13 stimulates Na+-H+ exchange through distinct Cdc42-dependent and RhoA dependent pathways. AB - Activity of the ubiquitously expressed Na+-H+ exchanger subtype NHE1 is stimulated upon activation of receptor tyrosine kinases and G protein-coupled receptors. The intracellular signaling pathways mediating receptor regulation of the exchanger, however, are poorly understood. Using transient expression of dominant interfering and constitutively active alleles in CCL39 fibroblasts, we determined that the GTPases Ha-Ras and Galpha 13 stimulate NHE1 through distinct signaling cascades. Exchange activity stimulated by constitutively active RasV12 occurs through a Rafl- and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase (MEK)-dependent mechanism. Constitutively active Galpha 13QL, recently shown to stimulate the Jun kinase cascade, activates NHE1 through a Cdc42- and MEK kinase (MEKK1)-dependent mechanism that is independent of Rac1. Constitutively active Rac1V12 does stimulate NHE1 through a MEKK1 dependent mechanism, but dominant interfering Rac1N17 does not inhibit Galpha 13QL-mediated or constitutively active Cdc42V12-mediated stimulation of the exchanger. Conversely, Cdc42NI7 does not inhibit Rac1V12 activation of NHE1, suggesting that Rae I and Cdc42 independently regulate a MEKK1-dependent activation of the exchanger. Rapid (<10 min) stimulation of NHE1 with a Ga13/Gaz chimera also was inhibited by a kinase-inactive MEKK. Galpha 13QL, but not RasV12, also stimulates NHE1 through a RhoA-dependent pathway that is independent of MEKK, and microinjection of mutationally active Galpha 13 results in a Rho phenotype of increased stress fiber formation. These findings indicate a new target for Rho-like proteins: the regulation of H+ ex- change and intracellular pH. Our findings also suggest that a MEKK cascade diverges to regulate effectors other than transcription factors. PMID- 8626405 TI - Ubiquinol-cytochrome c oxidoreductase. The redox reactions of the bis-heme cytochrome b in ubiquinone-sufficient and ubiquinone-deficient systems. AB - Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol cytochrome c oxidoreductase), exerting their highest degree of inhibition at I mol each/mol of complex III monomer. Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents. (i) In SMP, reduction of hemes bH and bL by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each inhibitor alone allows reduced bH and bL to accumulate, indicating that each inhibits the reoxidation of these hemes. (E)-Methyl-3-methoxy-2-(4')-trans stilbenyl)acrylatc in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol. (ii) Reoxidation of reduced b, is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline N-oxide, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin). (iii) Reoxidation of reduced bH is also inhibited by any one of these reagents. These inhibitions are also incomplete, and reduced bL is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced bH. Heme bH can be reduced in SMP via cytochrome c, and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p phenylenediamine). Energization of SMP by the addition of ATP affords reduction of bL as well. Reverse electron transfer to bH and bL is inhibited partially by myxothiazol, much more by antimycin. Ascorbate + TMPD also reduce bH in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to aproximately 0.06. Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b, reduction by ascorbate + TMPD. These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis. PMID- 8626404 TI - Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation. AB - Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product. PMID- 8626406 TI - Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis. AB - We have recently shown that a four-amino acid epitope (VTIL) on the m2 muscarinic receptor (corresponding to Val385, Thr386, Ile389, and Leu390) is essential for Gi/o coupling specificity and Gi/o activation (Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646). Because this sequence element is thought to be located at the junction between the third intracellular loop and the sixth transmembrane helix (TM VI), we speculated that agonist binding to the m2 receptor protein results in conformational changes that enable the VTIL motif to interact with Gi/o proteins. To test the hypothesis that such structural changes might involve a relative movement of TM VI toward the cytoplasm, we created a series of mutant m2 muscarinic receptors in which one to four extra Ala residues were inserted into TM VI immediately after Leu390. Based on the geometry of an alpha-helix, such mutations are predicted to "push" the VTIL sequence away from the lipid bilayer. Consistent with our working hypothesis, second messenger assays with transfected COS-7 cells showed that all mutant m2 receptors containing extra Ala residues C terminal of Leu390 could activate the proper G proteins even in the absence of agonist. However, replacement of the VTIL motif in such constitutively active m2 receptors with the corresponding m3 muscarinic receptor sequence (AALS) or deletion of Ala391 from the wild type m2 receptor completely abolished G protein coupling. Interestingly, introduction of extra Ala residues C-terminal of the AALS motif in the m3 muscarinic receptor completely abolished functional activity. Mutant m2 and m3 receptors that contained extra Ala residues immediately N-terminal of the VTIL and AALS motif, respectively, displayed wild type-like coupling properties. Our data are consistent with a model in which agonist binding to the m2 muscarinic receptor leads to a relative movement of TM VI toward the cytoplasm, thus enabling the adjacent VTIL sequence to interact with the C terminus of Galpha(i/o) subunits. PMID- 8626407 TI - Tyrosine kinase activity modulates catalysis and translocation of cellular 5 lipoxygenase. AB - Tyrosine kinase activity, a determinant of Src homology domain interactions, has a prominent effect on cellular localization and catalysis by 5-lipoxygenase. Six separate inhibitors of tyrosine kinase each inhibited 5(S) hydroxyeicosatetraenoic acid formation by HL-60 cells stimulated with calcium ionophore, in the presence or absence of exogenous arachidonic acid substrate, indicating that they modulated cellular 5-lipoxygenase activity. The tyrosine kinase inhibitors also blocked the translocation of 5-lipoxygenase from cytosol to membranes during cellular activation, consistent with their effects on its catalytic activity. These results fit a model which postulates that Src homology domain interactions are a molecular determinant of the processes which coordinate the subcellular localization and functions of 5-lipoxygenase. In addition, we demonstrate that activated leukocytes contain two molecularly distinct forms of 5 lipoxygenase: a phosphorylated form and a nonphosphorylated form. In activated HL 60 cells the pool of phosphorylated 5-lipoxygenase accumulates in the nuclear fraction, not with the membrane or cytosolic fractions. The amount of phosphorylated 5-lipoxygenase is a small fraction of the total. Overall, equilibrium reactions involving the nuclear localizing sequence, the proline-rich SH3 binding motif, and the phosphorylation state of 5-lipoxygenase may each influence its partnership with other cellular proteins and any novel functions derived from such partnerships. PMID- 8626408 TI - Different conformations and site selectivity of HO-2-Co(III)-bleomycin A2 and Co (III)-bleomycin A2 bound to DNA oligomers. AB - Conformational properties of HO2(-)-Co(III)-bleomycin A2 (Form I) and Co(III) bleomycin (Form II) bound to DNA oligomers offering either principal cleavage site for the drug, d(GGAAGCTTCC)2 or d(AAACGTIT)2, have been studied by NMR methods. Form I binds in slow exchange to these oligomers. It retains most of its solution nuclear Overhauser effects (NOEs) upon binding to either oligomer. Pyrimidinyl methyl protons from the metal domain of the drug make an NOE connection with a G5 2-amino proton on DNA. The bithiazole intercalates between base pairs involving either C6 and T7 or T6 and T7 of the two DNA molecules, according to NOE connections between the bithiazole protons and protons from these bases and changes in the positions of their chemical shifts. Form II also retains most of its solution NOEs upon association with the first oligomer. However, in contrast to Form I it binds to DNA in fast exchange on the NMR time scale over the temperature range of 5-35 degrees C and does not break the degeneracy of the DNA proton chemical shifts. No intermolecular NOEs between Form II and the 10-mer have been detected. Likewise, the major perturbation in chemical shift of the histidine H2 and guanine G5 protons seen in Form I-DNA adducts is absent in Form II-DNA. The association constant of Form II with d(GGAAGCTTCC)2 in 20 mM HEPES buffer at pH 7.4 and 25 degrees C is 1.7 x 10(5) M( 1), and 1.0 mol of Form II bind per mol of 10-mer. PMID- 8626409 TI - Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin. AB - Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs. PMID- 8626410 TI - Photoactivated azido fatty acid irreversibly inhibits anion and proton transport through the mitochondrial uncoupling protein. AB - The protonophoretic function of uncoupling protein (UCP) is activated by fatty acids. According to the "docking site" hypothesis (Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the fatty acid binding site is identical with the anion channel of UCP. Skulachev (Skulachev, V. P. (1991) FEBS Lett. 294, 158-162) extended this hypothesis by suggesting that fatty acid anions are transported by UCP and that H+ are delivered by back-diffusion of the protonated fatty acid through the lipid bilayer. In this model, UCP does not transport H+ at all but rather enables fatty acids to act as cycling protonophores. New evidence supports this mechanism (Garlid, K. D., Orosz, D. E., Modriansky, M., Vassanelli, S., and Jezek, P. (1996) J. Biol. Chem. 271, 2615-2620). To help elucidate these hypotheses, we synthesized a photoreactive analog of dodecanoic acid, 12-(4-azido 2-nitrophenylamino)dodecanoic acid (AzDA), and studied its effect on transport in mitochondria and proteoliposomes. AzDA behaved in every respect like a typical fatty acid. In micromolar doses, AzDA activated H+ translocation and inhibited Cl and hexanesulfonate uniport through UCP. After UV light exposure, however, activation of H+ transport was inhibited, whereas inhibition of anion transport was preserved. These effects were irreversible. Photolabeling of mitochondria with [3H]AzDA resulted in a prominent 32 kDa band of UCP, and few other proteins were labeled. The results indicate that AzDA can be ligated to the protein at or near the docking site, causing irreversible inhibition of both H+ and anion transport. The finding that fatty acid-induced H+ transport disappears along with anion transport supports the fatty acid-protonophore mechanism of H+ transport by UCP. PMID- 8626411 TI - Molecular cloning and sequencing of the cytostatic G protein-activated protein kinase PAK I. AB - The serine/threonine protein kinase PAK I (p2l-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties. Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere. To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5'-terminus was obtained by polymerase chain reaction. The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3'-noncoding region of 2826 nucleotides. Clones with the same open reading frame but with 3'-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription. The amino acid sequence of PAK I shows high homology to the p2l-activated protein kinases from human placenta and rat brain and to yeast STE20. PAK I is activated by Cdc42(GTP). The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157). PMID- 8626412 TI - Evidence for the existence of both proteasomes and a novel high molecular weight peptidase in Entamoeba histolytica. AB - To screen for high molecular weight proteases in Entamoeba histolytica, we subjected a soluble amebal extract to density gradient centrifugation and tested the fractions for activity against the chymotryptic peptide substrate, Suc-leucyl leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide. Two peaks of activity, of approximately 11 and 20 S, were clearly separated. Based on SDS-electrophoretic pattern and immunoblot analysis, we ascribe the 20 S activity to proteasomes. The 11 S protein was purified from amebal homogenates by a series of chromatographic steps. As determined by molecular sieve chromatography and nondenaturing gel electrophoresis, the native complex had an apparent Mr of 385,000 +/- 10%. On SDS gels, the purified enzyme exhibited a single band of Mr 62,000 that yielded a single N-terminal sequence, indicating that the preparation was homogeneous and that the native complex consisted of six very similar or identical subunits. The enzyme preferred peptides with aromatic residues at the P1 position and had low but distinct activity toward azocasein. We conclude that the 11 S enzyme is a novel high molecular weight protease that is distinct from proteasomes. PMID- 8626413 TI - Negative interaction between the RelA(p65) subunit of NF-kappaB and the progesterone receptor. AB - Interactions between transcription factors are an important means of regulating gene transcription. The present study describes the mutual repression of two transcription factors, the RelA(p65) subunit of NF-kappaB and the progesterone receptor (PR). This trans-repression is shown to occur independent of PR isoform, reporter construct, or cell type used. Together with the demonstration of an interaction between PR and RelA in vitro, these findings suggest that the mutual repression is due to a direct interaction between these proteins. Furthermore, activation of NF-kappaB by tumor necrosis factor-alpha also results in repression of PR, while PR is able to repress tumor necrosis factor-alpha-induced NF-kappaB activity. Since NF-KB-regulating cytokine receptors are expressed in progesterone target tissues, like breast and endometrium, the mutual repression of PR and RelA could play an important role in a wide variety of physiological processes in these tissues, including maintenance of pregnancy, immunosuppression, and tumorigenesis. PMID- 8626414 TI - N-terminal mutants of chloroplast cytochrome f. Effect on redox reactions and growth in Chlamydomonas reinhardtII. AB - The N-terminal tyrosine of cytochrome f, which provides the sixth ligand to the heme group, has been changed by site-directed mutagenesis in Chlamydomonas reinhardtii to evaluate the role of this amino acid in assembly and function. The second and third residues, proline and valine, respectively, have also been mutated. Y1P is the only strain that did not grow photoautotrophically. The other strains show cytochrome b6f complex/photosystem I reaction center chlorophyll, photosystem I unit size and chlorophyll a+b/cell ratios comparable with wild-type cells. Rates of cytochrome f photooxidation in all strains were similar (t1/2 approximately = 300 microsec), whereas the rate of re-reduction sensitive to stigmatellin (at Eh = 0 mV, (where Eh is the ambient redox potential) for wild type, Y1W, Y1F, Y1S, P2V, and V3P had a tl/2 of 3, 4, 5, 9, 40, and 2 ms, respectively. Rates of oxygen evolution by whole cells of P2V, Y1F, and Y1S were 67, 80, and 80% of wild-type rates, respectively. At low light intensity, all competent strains had the same growth rate whereas at saturating intensities, only P2V showed a significant inhibition. These results are considered in relation to structure-function relationships in the cytochrome f molecule. PMID- 8626415 TI - Mutagenic analysis of platelet thromboxane receptor cysteines. Roles in ligand binding and receptor-effector coupling. AB - The human platelet thromboxane A2 receptor is a member of the G-protein-coupled superfamily of receptors. Previous pharmacologic studies examining the effects of biochemical reduction, oxidation, or sulfhydryl alkylation on thromboxane receptors have suggested a role for cysteines in determining receptor binding characteristics. To characterize the roles of individual cysteines, we employed site-directed mutagenesis to substitute serines for cysteines at seven positions throughout the human K562 thromboxane receptor and analyzed mutant receptor radioligand ([1S-(1alpha,2beta(5Z),3alpha- (1E,3S),4alpha]-7-[3-(3-hydroxy-4-(p iodophenoxy)-l-butenyl)-7-oxabicyclo-[2. 2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid position 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Introduction of serines for cysteines in the first (position 105) or the second (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond between these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitude calcium signal, suggesting the involvement of a free sulfhydryl group at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same region (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cysteines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete amino acid sequences in the third intracellular loop, especially cysteine 223, in thromboxane receptor effector coupling. PMID- 8626416 TI - A nascent secretory protein may traverse the ribosome/endoplasmic reticulum translocase complex as an extended chain. AB - We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase. The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly alpha-helical conformation in the translocase. PMID- 8626417 TI - Keratinocyte growth factor inhibits cross-linked envelope formation and nucleosomal fragmentation in cultured human keratinocytes. AB - Keratinocyte growth factor (KGF) exhibits paracrine action on numerous epithelia, including skin. We have found that cultures of normal human keratinocytes must attain confluence before KGF promotes an increase in cell number relative to untreated controls. In postconfluent cultures, treatment with KGF promoted tight packing of keratinocytes with a small basal cell morphology. Based on these observations, we hypothesized that KGF increased cell number in postconfluent cultures by affecting the ability of normal keratinocytes to undergo terminal differentiation and/or programmed cell death. In support of this hypothesis, keratinocytes treated with KGF produced fewer cross-linked envelopes and exhibited reduced membrane-associated transglutaminase activity relative to cells treated with epidermal growth factor or untreated controls. We also found that nucleosomal fragmentation was reduced in postconfluent cultures of KGF-treated keratinocytes. Furthermore, KGF-treated keratinocytes were more resistant to suspension-induced nucleosomal fragmentation than control or epidermal growth factor-treated cultures. Therefore, it appears that KGF modulates aspects of keratinocyte terminal differentiation which share features with programmed cell death. We propose that stromally-derived KGF may act as a paracrine survival factor in skin and perhaps other renewal tissues. PMID- 8626418 TI - Photolyase of Myxococcus xanthus, a Gram-negative eubacterium, is more similar to photolyases found in Archaea and "higher" eukaryotes than to photolyases of other eubacteria. AB - We report the identification of the gene encoding a DNA photolyase (phrA) from the Gram-negative eubacterium Myxococcus xanthus. The deduced amino acid sequence of M. xanthus photolyase indicates that the protein contains 401 amino acids (Mr 45,071). By comparison of the amino acid and DNA sequences with those of other known photolyases, it has been found that it is more similar to the deduced amino acid sequences of the photolyases of "higher" eukaryotes than to the photolyases of other eubacteria. Recombinant plasmids carrying M. xanthus phrA rescue the photoreactivation activity of an irradiated strain of Escherichia coli with a deletion in phrA. This rescue is light-dependent. PMID- 8626419 TI - Identification and charaterization of the second retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter. AB - A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5' deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA. PMID- 8626420 TI - Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin. AB - Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3 phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3 K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3 Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated. PMID- 8626421 TI - Nuclear factor I as a potential regulator during postembryonic organ development. AB - Nuclear factor I (NFI) family members are transcription factors that are believed to also participate in DNA replication. We have cloned two Xenopus laevis NFIs that are up-regulated by thyroid hormone. They are 84-95% identical to their counterparts in birds and mammals. In contrast, the two Xenopus NFIs are much less homologous to each other, sharing only 58% homology, which largely resides in the DNA binding domain at the amino terminus. However, both NFIs can bind to a consensus NFI binding site and activate the transcription of a promoter bearing the site. Northern blot reveals that both NFI genes are regulated in tissue- and developmental stage-dependent manners. They are first activated, independently of thyroid hormone, to low levels at stages 23/24, around the onset of larval organogenesis. After stage 54, their mRNA levels are dramatically upregulated by endogenous thyroid hormone, and high levels of their expression correlate with organ-specific metamorphosis. Furthermore, gel mobility shift assay indicates that the NFI proteins are present in different organs and that their levels are regulated similarly to the mRNA levels. These results strongly suggest that NFIs play important roles during postembryonic organ development, in contrast to the general belief that NFIs are ubiquitous factors. PMID- 8626422 TI - Two functional thioredoxins containg redox-senesitive vicinal dithiols from the Chlamydomonas outer dynein arm. AB - We describe here the molecular cloning and analysis of the Mr 14,000 and 16,000 outer arm dynein light chains (DLCs) from Chlamydomonas flagella. Within the outer arm, the Mr 14,000 DLC apparently is associated with the intermediate chains at the base of the soluble dynein particle; the Mr 16,000 DLC interacts directly with the a dynein heavy chain. Sequence analysis indicates that both molecules are novel members of the thioredoxin superfamily and share approximately 30% sequence identity with thioredoxin from Penicillium. Both DLCs have a perfect copy of the thioredoxin active site (WCGPCK); the Mr 16,000 DLC also contains the canonical P-loop motif (AX4GKS). There is a single gene for both DLCs within Chlamydomonas and only single messages that were upregulated more than 10-fold upon deflagellation were observed on Northern blots. Both recombinant DLCs were specifically eluted from a phenylarsine oxide matrix with beta-mercaptoethanol indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction. Furthermore, we demonstrate that outer (but not inner) arm dynein may he purified on the basis of its affinity for phenylarsine oxide suggesting that the predicted redox-sensitive vicinal dithiols exist within the native complex. PMID- 8626423 TI - Amino acid conservation in animal glucokinases. Identification of residues implicated in the interaction with the regulatory protein. AB - To delineate the regions of liver glucokinase that are involved in the binding of its regulatory protein and have therefore been conserved throughout evolution, we have cloned the cDNA of the Xenopus laevis enzyme. It contains an open reading frame of 1374 nucleotides and encodes a protein of 458 amino acids, which displays 78 and 79% overall identity to rat and human liver glucokinases, respectively. The conserved regions are predicted to be present mainly in the small domain and the hinge region of glueokinase, and the nonconserved regions in the large domain of the enzyme. We constructed five mutants of Xenopus glucokinase by replacing sets of 2-5 glucokinase-specific residues with their counterparts in the C-terminal half of rat hexokinase I. The affinity for the regulatory protein was not markedly changed for mutants B, D, and E despite a decreased affinity for glucose in mutants B and D. Two other mutants (A and C) were 9- and 250-fold less sensitive to the rat regulator and 40- and 770-fold less sensitive to the Xenopus regulator, respectively, but presented a normal affinity for glucose. The double mutant (A-C) was completely insensitive to inhibition by the regulatory protein. A control mutant (F), obtained by replacing 3 residues that were not conserved in all glucokinases, had a normal affinity for glucose and for the regulatory protein. The property of glucokinase to be inhibited by palmitoyl-CoA was not affected by the mutations described. It is concluded that His-141 to Leu-144, which are located close to the tip of the small domain, as well as Glu-51 and Glu-52, which are present in the large domain of the enzyme close to the hinge region, or nearby residues participate in the binding of the regulatory protein. PMID- 8626424 TI - Developmental expression of a tandemly repeated, proline-and glutamine-rich amino acid motif on hyphal surfaces on Candida albicans. AB - cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS. The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues. The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris. The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues. The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms. These studies demonstrate that the bud hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces. The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins. PMID- 8626425 TI - Bcl-x(S) anatagonizes the protective effects of Bcl-x(L). AB - Bcl-x, a member of the Bcl-2 family, has two alternatively spliced forms, Bcl x(L) and Bcl-x(S). Bcl-x(L), like Bcl-2, is able to protect cells from a wide variety of apoptotic stimuli. Bcl-x(S), as a result of alternative splicing, lacks 63 amino acids that comprise the region of greatest amino acid identity between Bcl-x(L) and Bcl-2. These amino acids contain the highly conserved BH1 and BH2 regions, which have been used to define the Bcl-2 family. We show that both Bel-x(L) and Bcl-x(S) are able to regulate cell survival in a dose-dependent fashion. Bcl-x(L) is able to increase the cellular apoptotic threshold and is able to form stable complexes with Bax both in vitro and in vivo. In contrast, Bcl-x(S) can effectively inhibit the protective effects of Bcl-x(L) following growth factor withdrawal and chemotherapeutic drug treatment. However, compared with Bax, Bcl-x(S) binds to Bcl-x(L) weakly when assessed by in vitro binding assays. Coimmunoprecipitation from mammalian cells demonstrates that Bcl-x(S) does not show an observable ability to form heterodimers with other Bcl-2 family members. In addition, overexpression of Bel-x(S) does not alter the ability of Bax to heterodimerize with Bcl-x(L) in vivo. Thus, Bcl-x(S) does not appear to function by competitively disrupting the formation of dimers composed of other Bcl-2 family members. This suggests that Bcl-x(S) can enhance cellular sensitivity to apoptosis via a mechanism of action distinct from other Bc1-2 family members that promote apoptosis. PMID- 8626426 TI - Effects of conserved residues on the regulation of rabbit muscle pyruvate kinase. AB - A cDNA encoding the complete rabbit muscle pyruvate kinase isozyme (RMPK) was cloned using the method of rapid amplification of cDNA ends. The sequence encodes a polypeptide chain of 530 amino acids which differs in three amino acid residues from a sequence reported by Larsen et al. (Larsen, T.M., Laughlin, T., Holden, H.M., Rayment, L, and Reed, G.H. (1994) Biochemistry 33, 6301-6309). Glu233 Gln234 and Ala400 were identified instead of Asp233-Glu234 and Ser400, respectively. The recombinant RMPK was overexpressed in the Escherichia coli JM 105 cells. Purified recombinant pyruvate kinase displayed identical physical and enzymatic properties as the authentic enzyme. Three point mutants of RMPK were constructed using site-directed mutagenesis. Like the wild type RMPK, sedimentation, and CD spectroscopic studies show that purified RI 19C and T340M are tetrameric proteins with similar secondary and tertiary structures. Mutant R119C enzyme exhibits 0.6% of the value of k(cat) and an order of magnitude decrease in the apparent affinity for ADP as compared to the wild type PK. The overall response to inhibitor and activator, Phe and FBP, respectively, were not affected by the R119C mutation. The T340M mutant enzyme is only half as active as the wild type PK. T340M is more susceptible to inhibition by Phe but apparently is not responsive to the activator FBP. The kinetic behavior of the Q377K mutant enzyme is in between that of the R119C and T340M mutants exhibiting 5% of the wild type enzymatic activity and an enhanced sensitivity to the inhibitor, Phe, while maintaining the same responsiveness to FBP and apparent affinities for substrates. The significant decrease in activity in all three mutants mimics the exact consequences of the same mutations in human erythrocyte PK from hemolytic anemia patients. Thus, this study demonstrates not only the effects of these conserved residues in the regulatory properties of mammalian PK. but also that the observed effects are most likely applicable to all isozymic forms of PK. PMID- 8626427 TI - The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation. AB - Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192- >Ala. In support of the importance of Ser188, binding of phenylephrine (meta hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292- >Leu mutation. A simple interpretation of these findings is that the meta hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR. PMID- 8626428 TI - SOS phosphorylation and disassociation of the Grb2-SOS complex by the ERK and JNK signaling pathways. AB - Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin stimulated feedback uncoupling of the Grb2- SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex. PMID- 8626429 TI - Physical and functional interaction of Nef with Lck. HIV-1 Nef-induced T-cell signaling defects. AB - The nef gene is unique to the primate lentiviruses and encodes a cytoplasmic membrane-associated protein that affects T-cell signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell signaling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lek tyrosine kinase both in vitro and in vivo. The molecular basis for this interaction was analyzed. We show that cell-derived Nef is precipitated in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely participants in this interaction. The precipitation of Nef by the Lck recombinant proteins was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase nor phospholipase Cgamma SH2 domains coprecipitated Nef from T-cells. Finally, depressed Lck kinase activity resulted from the presence of Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal Lck-mediated signaling events. These results provide a molecular basis for the Nef-induced T-cell signaling defect and its role in AIDS pathogenesis. PMID- 8626430 TI - Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies. AB - P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress. PMID- 8626431 TI - Molecular cloning and expression of the Modulatory subunit of the cyclic nucleotide-gated cation channel. AB - The cDNA of three variants of a cyclic nucleotide-gated (CNG) channel modulatory subunit (CNG4c-CNG4e) has been cloned. CNG4c, CNG4d, and CNG4e differ slightly from each other within an amino-terminal sequence that was originally reported as part of the bovine retinal glutamic acid-rich protein (GARP). The core region of CNG4 is homologous to the second subunit of the human rod photoreceptor channel (hRCNC2b), suggesting that both proteins are alternatively spliced products of the bovine and human homologue of the same gene. CNG4 transcripts are present in retina, testis, kidney, heart, and brain. Expression of CNG4 in HEK293 cells did not lead to detectable currents. Coexpression of CNG4 with the principal subunit of the bovine testis CNG channel (CNG3) resulted in currents which differed in several aspects from that induced by CNG3 alone. The heterooligomeric CNG3/CNG4 and the homooligomeric CNG3 channels were modified by Ca2+-calmodulin and some calmodulin antagonists. The results suggest that CNG4 forms functional heterooligomeric channels with CNG3 in vitro and probably also in intact tissues. PMID- 8626432 TI - Expression and function of ryanodine receptors in nonexcitable cells. AB - We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses. PMID- 8626433 TI - The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency. AB - N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency. PMID- 8626434 TI - In vitro activity of hepatitis C virus protease NS3 purified from recombinant Baculovirus-infected Sf9 cells. AB - A recombinant Baculovirus expression system was used for the production of a 20 kDa protein encompassing the hepatitis C virus NS3 protease domain. The protein was purified to apparent homogeneity after detergent extraction of cell homogenates. It was shown to be a monomer in solution and to cleave the in vitro translated precursor proteins NS4A-NS4B and NS5A-NS5B, but not the NS4B-NS5A or the NS3-NS4A precursors. The enzyme also cleaved a 20-mer peptide corresponding to the NS4A-NS4B junction with kcat/Km = 174 m(-1) s(-1). Peptides harboring NS4A sequences comprising amino acids 21-54 (Pep4A21-54) and 21-34 (Pep4A21-34) were found to induce an up to 2.8-fold acceleration of cleavage. Kinetic analysis revealed that this acceleration was due to an increase in kcat whereas no significant effect on Km could be detected. Pep4A21-54 was also an absolute requirement for cleavage of in vitro translated NS4B-NS5A by the purified protease. From these data we conclude that: (i) the purified protease domain shows substrate specificity and cleavage requirements similar to those previously reported on the basis of transfection experiments, (ii) activation of the purified protease by the NS4A co-factor can be mimicked by synthetic peptide analogs, and (iii) a central hydrophobic region of NS4A with a minimum core of 14 amino acids is responsible for the interaction with NS3. PMID- 8626435 TI - Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. AB - The respiratory burst oxidase is responsible for superoxide (O2) production by phagocytes and B lymphocytes. This multicomponent enzyme is dormant in resting cells but is activated on exposure of the cells to an appropriate stimulus. Upon activation, several serine residues on the cytosolic oxidase subunit p47phox become phosphorylated. Using two-dimensional tryptic phosphopeptide mapping, we studied the phosphorylation of p47phox in 32Pi-loaded Epstein-Barr virus transformed B lymphoblasts expressing wild type p47phox or any of several P47phox Ser -> Ala mutants. We were able to identify the labeled peptides from wild type p47phox as those contain- ing Ser303/304 Ser315, Ser320, Ser328 and/or Ser359/370, and Ser345/348 ; no 32P-labeled Ser310-containing peptide was found. When purified p47phox, was phosphorylated in vitro by various protein kinases, varying phosphopeptide patterns were observed. Protein kinase C phosphorylated all the peptides except the one containing Ser345/348; protein kinase A phosphorylated the peptide containing Ser320 and one or both of the peptides containing Ser328 and Ser359/370; while mitogen-activated protein kinase phophorylated only the peptide containing Ser345/348. These findings suggest that these three kinases play distinct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47phox. PMID- 8626436 TI - Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. AB - Transcription of the prostate-specific antigen (PSA) gene is androgen regulated. The PSA promoter contains at position -170 the sequence AGAACAgcaAGTGCT, which is closely related to the ARE (androgen response element) consensus sequence GGTACAnnnTGTTCT. This sequence is a high affinity androgen receptor (AR) binding site and acts as a functional ARE in transfected LNCaP cells. A 35-base pair segment starting at -400 (ARR: androgen response region; GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTG) cooperates with the ARE in androgen induction of the PSA promoter. A construct with three ARR copies linked to a minimal PSA promoter showed a strong (104-fold) androgen induced activity. The ARR was also able to confer androgen responsiveness to a minimal thymidine kinase promoter. Both AR binding and transcriptional activity resided in a 20-base pair ARR subfragment: CAGGGATCAGGGAGTCTCAC (2S). Mutational analysis indicated that the sequence GGATCAgggAGTCTC in the 2S fragment is a functionally active, low affinity AR binding site. Like AR, the glucocorticoid receptor was able to stimulate PSA promoter activity. Both the ARE and ARR are involved in dexamethasone regulation of the PSA promoter. Both the AR and glucocorticoid receptor were 20-100-fold more active on ARR-PSA and ARR-thymidine kinase promoter constructs in LNCaP cells than in other cell types (COS, HeLa, Hep3B, and T47D cells), indicating (prostate) cell specificity. PMID- 8626437 TI - Physical and functional interactions between Lyn and p34cdc2 kinases in irradiated human B-cell precursors. AB - Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function. PMID- 8626438 TI - Specificity of coupling of muscarinic receptor isoforms to a novel chick inward rectifying acetylcholine-sensitive K+ channel. AB - The G-protein-gated inward-rectifying K+ channel GIRK1 has been demonstrated in heart and brain. These tissues also both express the M2, M3, and M4, muscarinic acetylcholine receptors (mAChR) (Gadbut, A.P., and Galper, J.B. (1994),J. Biol. Chem. 269,25823-25829). Only the M2 mAChR has been demonstrated to couple to GIRK1 (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 264, 802-806). In this study we determined the specificity of coupling of the M3 and M4 mAChR to a new GIRK1 cloned from a chick brain cDNA library. This clone codes for a 492-amino acid protein that is 93% identical to rat GIRK1 and is expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laetis oocytes co-expression of GIRK1 with either the chick M2 or M4 mAChR gave carbamylcholine (10 microm)-stimulated K+ currents of 308 +/-26 nA and 298 +/-29 nA, respectively, which were both Ba2+- and pertussis toxin-sensitive. Activation of the M3 receptor produced 2382 +/-478 nA of current which was insensitive to Ba2+ and pertussis toxin, but was 85% inhabitable by the Cl channel blocker 5 nitro-2-(3-phenylpropylamino)benzoic acid (10-20 microm) consistent with coupling to an endogenous Ca2+-activated Cl- channel via a phosphatidylinositol-dependent mechanism. Co-expression of the cardiac inward rectifier CIR with chick M2 or M4 mAChR and GIRK1 increased currents more than 10-fold, but had no effect on specificity of coupling. These data demonstrate a new function for the M4 mAChR and a high degree of specificity for coupling of each receptor subtype to GIRK1. PMID- 8626439 TI - Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. AB - A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase. PMID- 8626441 TI - Identification of a major protein kinase C-binding protein and substrate in rat embryo fibroblasts. Decreased expression in transformed cells. AB - We have used an interaction cloning strategy to isolate cDNAs for sequences that interact with protein kinase C (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268,6858-6861). In this paper, we report a novel sequence, clone 72, isolated according to this method. Clone 72 has a 4.8 kilobase pair open reading frame; antibodies to clone 72 recognize a >200-kDa protein in cell and tissue extracts. Clone 72 message and protein are detected in a variety of tissues. Immunoprecipitation studies demonstrate that clone 72 is the major >200-kDa binding protein described previously in REF52 fibroblasts (Hyatt, S. L., Liao, L., Aderem, A., Nairn, A., and Jaken, S. (1994) Cell Growth & Differ. 5, 495-502). Expression of clone 72 message and protein are decreased in progressively transformed REF52 cells. Since clone 72 is both a protein kinase C (PKC)-binding protein and substrate, decreased levels of clone 72 may influence both the subcellular location of endogenous PKCs as well as signaling events associated with clone 72 phosphorylation. Our results emphasize that the role of PKCs in carcinogenesis may involve several factors, including the quantity and location of the PKCs isozymes and their downstream targets. PMID- 8626440 TI - Regulation of lysyl oxidase by basic fibroblast growth factor in osteoblastic MC3T3-E1 cells. AB - Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking. A cross-linked collagenous extracellular matrix is required for bone formation. This study investigated whether lysyl oxidase, like its type I collagen substrate, is down-regulated by basic fibroblast growth factor (bFGF) in osteoblastic MC3T3-E1 cells and determined the degree of post transcriptional control. Steady-state lysyl oxidase mRNA levels decreased to 30% of control after 24 h of treatment with 1 and 10 nm bFGF. This regulation was time-dependent. COL1A1 mRNA levels declined to less than 10% of control after 24 h of bFGF treatment. Media lysyl oxidase activity decreased consistent with steady-state mRNA changes in cultures that were refed after 24 h of growth factor treatment. Interestingly, treatment of MC3T3-E1 cells with 0.01-0.1 nm bFGF for 24 h and treatment with 1 nm bFGF for up to 12 h resulted in a modest stimulation of lysyl oxidase gene expression and enzyme activity. At least 50% of the down regulation of lysyl oxidase was shown to be posttranscriptional. New protein synthesis was not required for the down-regulation by bFGF, but cycloheximide did increase constitutive lysyl oxidase mRNA levels 2.5-fold. We conclude that lysyl oxidase and COL1A1 are regulated similarly by bFGF in these osteoblastic cells, consistent with the in vivo effects of this growth factor on bone collagen metabolism. PMID- 8626442 TI - Reinitiation of protein translocation across the endoplasmic reticulum membrane for the topogenesis of multispanning membrane proteins. AB - The reinitiation of the translocation of the growing nascent chain across the endoplasmic reticulum membrane is essential for the topogenesis of multispanning membrane proteins. We investigated the requirements for the reinitiation process using model proteins in which systematically designed sequences were inserted after two preceding topogenic sequences, namely the N-terminal signal sequence (S) and stop transfer sequence (St). The model proteins were translated in vitro in the presence of rough microsomes, and the final topology of the proteins in the microsomal membrane was examined by proteolytic digestion. The structural requirements for S and the reinitiation sequence (R) overlapped to some extent, but substantial differences were noticed. When St and R were separated by a short cytoplasmic segment (58 amino acids), the efficiency of the reinitiation was not affected by the concentration of the signal recognition particle (SRP) in the translation system, even though the sequence inserted as R was an SRP-dependent signal sequence. However, when the cytoplasmic segment was longer (100 amino acids), the reinitiation efficiency was reduced, and the SRP improved the overall efficiency as well as impaired the accessibility of the processing site after the R to the signal peptidase. PMID- 8626443 TI - Different glycosylation requirements for the synthesis of enzymatically active angiotensin-converting enzyme in mammalian cells and yeast. AB - For facilitating crystallization and structural studies of the testicular isozyme of angiotensin-converting enzyme (ACE,), we attempted the production of enzymatically active ACET proteins which are unglycosylated or underglycosylated. Expression in Escherichia coli of the rabbit ACET cDNA resulted in the synthesis of an unglycosylated but inactive protein. Similarly, unglycosylated ACET synthesized in HeLa cells, by using a cDNA in which all five potential N glycosylation sites had been mutated, was inactive and rapidly degraded. Several ACET variants carrying mutations in one or more of the potential N-glycosylation sites were used to examine the role of glycosylation at specific sites on ACET synthesis, transport to the cell surface, cleavage processing, and enzyme activity. These experiments demonstrated that allowing glycosylation only at the first or the second site, as counted from the NH2 terminus, was sufficient for normal synthesis and processing of active ACET. In contrast, ACETg3, which had only the third glycosylation site available, was unglycosylated, enzymatically inactive and rapidly degraded. N-Glycosylated ACET could also be produced in yeast. Surprisingly, the mutant ACETg3 was synthesized, N-glycosylated, and properly transported in yeast. Wild type and mutant ACE proteins were cleavage secreted from yeast and enzymatically active. PMID- 8626444 TI - Identification, characterization, and comparison of the calmodulin-binding domains of the endothelial and inducible nitric oxide synthases. AB - The calmodulin (CaM)-binding regions in bovine endothelial nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) are identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and NOS 501-532 produce a (Ca2+)-dependent, electrophoretic mobility shift of CaM on 4 M urea gels. The two peptides are also potent inhibitors of the CaM-mediated activation of neuronal nitric oxide synthase and have dissociation constants for CaM binding of 4.0 and 1.5 nM respectively. Substitution of eNOS and iNOS CaM-binding domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baculovirus/Sf9 insect cell system. Replacement of aligned NOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both (Ca2+)- and CaM-dependent. Replacement of aligned eNOS sequence with NOS 501-532 creates a functional, chimeric eNOS that is CaM-independent but that remains (Ca2+)-dependent. Specific amino acid residues critical for CaM binding by eNOS are also identified in this study as Phe-498, Lys-499, and Leu-511 in the bovine eNOS sequence. PMID- 8626446 TI - A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import. AB - The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest. PMID- 8626445 TI - Acidification of serotonin-containing secretory vesicles induced by a plasma membrane calcium receptor. AB - Parafollicular (PF) cells secrete 5-hydroxytryptamine in response to increased extracellular Ca2+ ([Ca2+]e). This stimulus causes Cl- channels in PF secretory vesicles to open, leading to vesicle acidification. PF cells express a plasmalemmal heptahelical receptor (CaR) that binds Ca2+, Gd3+, and Ba2+. We now report that the CaR mediates vesicle acidification. Ca2+, Gd3+, and Ba2+ induced vesicle acidification, which was independent of channel-mediated Ca2+ entry. Agonist-induced vesicle acidification was blocked by pertussis toxin, inhibitors of phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl cyclase, or protein kinase G. PF cells contained NO synthase immunoreactivity, and vesicles were acidified by NO donors and dibutyryl cGMP. [Ca2+]e, and Gd3+ mobilized thapsigargin-sensitive internal Ca2+ stores. [35S]G alpha i and [35S]G alpha q were immunoprecipitated from PF membranes incubated with agonists in the presence of [35S]adenosine 5'-O-(thiotriphosphate). Labeling of G alpha i but not G alpha q was antagonized by pertussis toxin. Vesicles acidified in response to activation of protein kinase C; however, protein kinase C inhibition blocked calcium channel- but not CaR-dependent acidification. We propose the following signal transduction pathway: CaR -> Gi -> phosphatidylinositol-phospholipase C -> inositol 1,4,5-trisphosphate -> [Ca2+]i -> Ca2+/calmodulin -> NO synthase -> NO > guanylyl cyclase -> cGMP -> protein kinase G -> opens vesicular Cl- channel. PMID- 8626447 TI - Reconstitution of B cell antigen receptor-induced signaling events in a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. AB - B cell antigen receptor (BCR) cross-linking activates both Src family and Syk tyrosine kinases, resulting in increased cellular protein-tyrosine phosphorylation and activation of several downstream signaling enzymes. To define the role of Syk in these events, we expressed the BCR in the AtT20 mouse pituitary cell line. These nonlymphoid cells endogenously expressed the Src family kinase Fyn but not Syk. Anti-IgM stimulation of these cells failed to induce most of the signaling events that occur in B cells. BCR-expressing AtT20 transfectants were generated that also expressed Syk. Syk expression reconstituted several signaling events upon anti-IgM stimulation, including Syk phosphorylation and association with the BCR, tyrosine phosphorylation of numerous proteins including Shc, and activation of mitogen-activated protein kinase. In contrast, Syk expression did not reconstitute anti-IgM-induced inositol phosphate production. A catalytically inactive Syk mutant could associate with the BCR and become tyrosine phosphorylated but could not reconstitute downstream signaling events. Expression of the Src family kinase Lck instead of Syk also did not reconstitute signaling. Thus, wild type Syk was required to reconstitute several BCR-induced signaling events but was not sufficient to couple the BCR to the phosphoinositide signaling pathway. PMID- 8626449 TI - Substrate specificity of the dolichol phosphate mannose: glucosaminyl phosphatidylinositol alpha1-4-mannosyltransferase of the glycosylphosphatidylinositol biosynthetic pathway of African trypanosomes. AB - The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN alpha 1-6-D-myo-inositol-1-PO4-sn 1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcN alpha 1-6-D-myo-inositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C5 and C15) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PI alpha 1-4-mannosyltransferase. PMID- 8626448 TI - Activity of the distal positive element of the peripherin gene is dependent on proteins binding to an Ets-like recognition site and a novel inverted repeat site. AB - The peripherin gene, encoding a neuron-specific intermediate filament protein, is transcriptionally induced when PC12 cells begin to terminally differentiate into neurons in response to nerve growth factor. Previously we identified two regulatory sequences of the peripherin gene: a proximal negative element (centered at -173), which prevents peripherin expression in undifferentiated PC12 cells, and a distal positive region (-2660 to -2308) necessary for full induction of peripherin in differentiated PC12 cells (Thompson, M., Lee, E. Lawe, D., Gizang-Ginsberg, E., and Ziff, E. (1992) Mol. Cell. Biol. 12,2501-2513). Here we define a distal positive element (DPE, -2445 to -2337) within the distal positive region. Methylation interference footprinting of the DPE identified DNA-protein contact points at a novel inverted repeat sequence (AACCACTGGTT) and an Ets-like recognition sequence (CAGGAG). Functional analysis using site-directed mutagenesis demonstrates that both sites are necessary for the activity of the DPE. In addition, ternary complex formation at the DPE is dependent on both sites. Antibody competition assays confirm that an Ets family member participates in the DNA-protein complex. We have indirect evidence that the inverted repeat binding protein and the Ets-related protein interact directly with each other. Finally, we demonstrate that the DPE is constitutively active and that neuron specific regulation of peripherin expression may be due to interaction with distal and proximal negative regulatory elements. PMID- 8626450 TI - Specific interaction of topoisomerase II beta and the CD3 epsilon chain of the T cell receptor complex. AB - T cell antigen receptor (TCR)-CD3 complex is composed of six different subunits: TCR alpha and TCR beta and CD3 gamma, CD3 delta, CD3 epsilon, and CD3 eta. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 epsilon with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3 epsilon. One of these clones was found to encode topoisomerase II beta (topoII beta). The binding region of CD3 epsilon is located within the N terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the gamma chain of Fc receptors (FcR gamma) but not in the CD33 eta chain, and indeed, FcR gamma but not CD3 eta bound to topoII beta. The binding region of topoII beta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 epsilon might affect the function of topoII beta. Although topoII beta is localized mainly in the nucleus and CD3E is a membrane protein, we demonstrated the presence of CD3 epsilon in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoII beta and CD3E from the nuclear fraction of T cells. The possible function of this interaction is discussed. PMID- 8626451 TI - Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis. AB - Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation. PMID- 8626452 TI - The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. AB - Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, and p38, which are thought to carry out unique functions within cells. We demonstrate that ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr, and the dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the in vivo inactivation of ERK or of ERK and JNK, respectively. Here we characterize a new MAP kinase phosphatase, MKP-2, that is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate and is expressed in a variety of nonhematopoietic tissues as well. We show that the in vivo substrate specificities of individual phosphatases are unique. PAC1, MKP-2, and MKP-1 recognize ERK and p38, ERK and JNK, and ERK, p38, and JNK, respectively. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2 (D319N), analogous to the Drosophila sevenmaker gain of-function mutation, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo. PMID- 8626453 TI - Retinoic acid down-regulation of fibronectin and retinoic acid receptor alpha proteins in NIH-3T3 cells. Blocks of this response by ras transformation. AB - All-trans-retinoic acid (RA) markedly reduced the level of intracellular fibronectin (FN) in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Pulse/chase experiments indicated that RA affects FN biosynthesis rather than its turnover rate. Steady state levels of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, suggesting that RA acts at the translational level. Similar effects were observed in other fibroblasts. In NIH 3T3 cells, RA had distinct effects on different receptors; it down-modulated retinoic acid receptor (RAR) a protein and transcript levels, it up-regulated RAR beta transcripts, and it had no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene down-modulated RAR expression and abolished responsiveness to RA. We identified the retinoid signal transduction pathways responsible for the effects of RA on FN and RAR alpha proteins by the use of the retinoid X receptor-selective compound, SR11237, by stable over expression of a truncated form of the RAR alpha gene, RAR alpha 403 with strong RAR dominant negative activity, and by overexpression of RAR alpha. We conclude that: 1) RA-dependent FN down-modulation is mediated by RARs, 2) retinoid X receptors mediate the observed reduction of RAR alpha by RA, and 3) the block of RA responsiveness in Ha-ras cells cannot be overcome by overexpression of RAR alpha. These studies have defined fibronectin and RAR alpha as targets of RA in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action. PMID- 8626454 TI - The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. AB - The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515 amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, D. J. (1994) J. Biol. Chem. 269, 22853 22857). Direct comparisons between S. cerevisiae strain DTY167, harboring a deletion of the YCF1 gene, and the isogenic wild type strain, DTY165, demonstrate that YCF1 is required for increased resistance to the toxic effects of the exogenous glutathione S-conjugate precursor, 1-chloro-2,4-di-nitrobenzene, as well as cadmium. Whereas membrane vesicles isolated from DTY165 cells contain two major pathways for transport of the model compound S-(2,4 dinitrophenyl)glutathione (DNP-GS), an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway, the corresponding membrane fraction from DTY167 cells is more than 90% impaired for MgATP-dependent, uncoupler insensitive DNP-GS transport. Of the two DNP-GS transport pathways identified, only the MgATP-dependent, uncoupler-insensive pathway is subject to inhibition by glutathione disulfide, vanadate, verapamil, and vinblastine. The capacity for MgATP-dependent, uncoupler-insensitive conjugate transport in vitro strictly copurifies with the acuolar membrane fraction. Intact DTY165 cells, but not DTY167 cells, mediate vacuolar accumulation of the quorescent glutathione conjugate, monochlorobimane-GS. Introduction of plasmid borne, epitope-tagged gene encoding functional YCF1 into DTY167 cells alleviates the 1-chloro-2,4 dinitrobenzene-hypersensitive phenotype concomitant with restoration of the capacity of vacuolar membrane vesicles isolated from these cells for MgATP dependent, uncoupler-insensitive DNP-GS transport. On the basis of these findings, the YCF1 gene of S. cerevisiae is inferred to encode an MgATP energized, uncoupler-insensitive vacuolar glutathione S-conjugate transporter. The energy requirements, kinetics, substrate specificity, and inhibitor profile of YCF1-mediated transport demonstrate that the vacuolar glutathione conjugate pump of yeast bears a strong mechanistic resemblance to the MRP1-encoded transporter of mammalian cells and the cognate, but as yet molecularly undefined, function of plant cells. PMID- 8626455 TI - Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. AB - Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noneaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in non-caveolar plasma membrane. 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide. PMID- 8626456 TI - Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and alpha 1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein. AB - The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)-thrombin, heparin cofactor II (HCII)-thrombin, and alpha1-antitrypsin (alpha1AT)-trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII-125I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs. PMID- 8626457 TI - In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system. AB - Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54 independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4 fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter. PMID- 8626459 TI - Phosphorylation of the transit sequence of chloroplast precursor proteins. AB - A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step, which could lead to a specific sorting and productive translocation in plant cells. PMID- 8626458 TI - Cloning and characterization of the Neurospora crassa cyt-5 gene. A nuclear-coded mitochondrial RNA polymerase with a polyglutamine repeat. AB - The Neurospora crassa mutants, cyt-5-1 and cyt-5-4 have a cytochrome b- and aa3 deficient phenotype, suggesting that they result from a deficiency in a nuclear coded component of the mitochondrial gene expression apparatus (Bertrand, H., Nargang, F. E., Colllins, R. A., and Zagozeski, C. A. (1977) Mol. Gen. Genet. 153,247-257). The complementing wild-type gene has been cloned and and shown to encode a protein with significant sequence similarity to Saccharomyces cerevisiae mitochondrial RNA polymerase and bacteriophage RNA polymerases. There are remarkable differences between the N. crassa protein and its yeast homologue, including a region of very little homology near the N termini of the two gene products. The cyt-5 gene encodes a stretch of polyglutamine in this region of uniquesequence. In addition, an acidic insertion (86 amino acids, of which 24 are Asp or Glu and 10 are Arg or Lys) is present near the C terminus of the cyt-5 gene product. Transcript levels of the cytochrome b and cytochrome oxidase subunit III genes are severely reduced in cyt-5 mutants, suggesting a likely mechanism for the cytochrome-deficient phenotype. In contrast, mitochondrial rRNAs accumulate to nearly normal levels in cyt-5 mutants. However, mitochondrial rRNA levels are not indicative of the rate of transcription of the corresponding genes, since crude lysates of mitochondria from cyt-5 mutants exhibit greatly reduced transcriptional activity with a 19 S rRNA promoter. The cyt-5 gene is flanked by at least one gene whose product also may be involved in mitochondrial function. PMID- 8626460 TI - Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double stranded DNA antibodies. AB - Nucleic acid specificity was tested for two monoclonal anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound double-stranded oligonucleotides with a preference for dA-dT over dG-dC base pairs and did not bind single-stranded oligonucleotides. Distamycin A, an antibiotic that binds to the minor groove, inhibited 2C10 binding of double-stranded DNA, suggesting that this antibody interacts with dA dT base pairs in the minor groove. Antibody H241 binding was previously shown to have a dG-dC preference and to involve both major and minor grooves. In attempted footprinting assays, both 2C10 and H241 markedly en- hanced rather than protected against cleavage of DNA by hydroxyl radical-generating systems. With 2C10, this enhancement effect was observed only when hydroxyl radical generation was associated with oxidation of Fe(II). In contrast, H241 enhancement occurred in the presence of H2O2 and ascorbate or UV light irradiation and did not depend on added metal ion. The enhancement sites were related to the antibody binding specificities. The oligonucleotide 5'-AAAATATATATTT-3' was a much more effective inhibitor of the 2C10 enhancement than of the H241 effect, whereas the oligonucleotide 5'-GGGGCGCGCGCCC-3' was a much more effective inhibitor of the H241 enhancement. In addition, the enhanced cleavage occurred preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich regions with H241. These findings raise the possibility that anti-DNA autoantibodies could enhance DNA damage in inflammatory lesions in which hydroxyl radicals are generated. PMID- 8626461 TI - Construction of a high affinity zinc switch in the kappa-opioid receptor. AB - Very limited structural information is available concerning the superfamily of G protein-coupled receptors with their seven-transmembrane segments. Recently a non peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family. PMID- 8626462 TI - Cloning and expression of a novel human brain Na+ channel. AB - We have cloned a novel cDNA from human brain which encodes a non-voltage dependent Na+ channel (BNC1). BNC1 has some sequence similarity (24-28%) with a new channel family that includes subunits of the mammalian epithelial Na+ channel, the Caenorhabditis elegans degenerins, and the Helix aspersa FMRF-amide gated Na+ channel. Like other family members it is inhibited by amiloride. However, its predicted structure differs from other family members, its discrimination between Na+ and Li+ is different, and in contrast to other mammalian family members, coexpression with other cloned subunits of the family does not increase current. BNC1 has a unique pattern of expression with transcripts detected only in adult human brain and in spinal cord. Thus, BNC1 is the first cloned member of a new subfamily of mammalian Na+ channels. The function of BNC1 as a non-voltage-gated Na+ channel in human brain suggests it may play a novel role in neurotransmission. PMID- 8626463 TI - Rac-dependent and -independent pathways mediate growth factor-induced Ca2+ influx. AB - We report that expressing interfering mutants of the small Ras-related GTPase Rac, using either recombinant vaccinia virus or stable DNA transfection, eliminates epidermal growth factor-induced Ca2+ signaling, without affecting Ca2+ mobilization or influx from G protein-coupled receptors. Platelet-derived growth factor-dependent Ca2+ influx, however, is only partly sensitive to dominant negative Rac proteins. Thus, whereas epidermal growth factor-induced Ca2+ influx is completely mediated by Rac proteins, platelet-derived growth factor-induced Ca2+ influx involves Rac-dependent and -independent signaling pathways. PMID- 8626464 TI - Cellular phosphorylation of anti-HIV nucleosides. Role of nucleoside diphosphate kinase. AB - Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates. Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catalysis. PMID- 8626465 TI - Calreticulin binding affinity for glycosylated laminin. AB - Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface. PMID- 8626466 TI - Molecular cloning and expression of the cDNAs encoding human and yeast mevalonate pyrophosphate decarboxylase. AB - The importance of lowering serum cholesterol levels for the prevention of cardiovascular disease has been well documented. Because mevalonate pyrophosphate decarboxylase is a unique enzyme in the cholesterol biosynthetic pathway it is a potential therapeutic target for the treatment of hypercholesterolemia and other diseases. For this reason we cloned and expressed the cDNA for the human enzyme. We also cloned and expressed the yeast homolog using the human enzyme's similarity to a previously unidentified and incomplete genomic sequence. Northern blot analysis revealed a transcript of approximately 2 kilobases in a variety of human tissues. The recombinant human enzyme is a homodimer of 43-kDa subunits with a specific activity of 2.4 units/mg. Computer searches for similarity with known sequences showed that mevalonate pyrophosphate decarboxylase has little similarity to other proteins. PMID- 8626467 TI - Identification of the repressor subdomain within the signal reception module of the prokaryotic enhancer-binding protein XylR of Pseudomonas putida. AB - In the presence of m-xylene, the protein XylR encoded by the TOL plasmid of Pseudomonas putida, activates the final sigma54-dependent promoter Pu. Early activation stages involve the release of the intramolecular repression caused by the signal reception N-terminal (A domain) of XylR on the central module of the protein. A genetic approach has been followed to locate the specific segment within A domain of XylR that is directly responsible for its down-regulation in the absence of inducer, as compared to that involved in effector (m-xylene) binding. For this, a reporter Escherichia coli strain carrying a monocopy transcriptional fusion of Pu to lacZ was transformed with a collection of plasmids encoding equivalent truncated varieties of XylR, consisting of nested and internal deletions throughout the entire A domain. Examination of the resulting phenotypes allowed the assignment of the A domain region near the central activation domain, as the portion of the protein responsible for the specific repression of XylR activity in the absence of m-xylene. PMID- 8626468 TI - Posttranslational regulation of a Leishmania HEXXH metalloprotease (gp63). The effects of site-specific mutagenesis of catalytic, zinc binding, N-glycosylation, and glycosyl phosphatidylinositol addition sites on N-terminal end cleavage, intracellular stability, and extracellular exit. AB - Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition. Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient variant, which produced gp63 approximately 20-fold less than wild-type cells. The selective conditions chosen fully restored this deficiency in transfectants with the wild-type gene. Under these conditions, all transfectants were found comparable in both the plasmid copy number per cell and elevation of gp63 transcripts. Mutant and wild type products in the transfectants were then compared quantitatively and qualitatively by specific immunologic and protease assays. The results indicate the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. The propeptide of the inactive mutant products was cleaved, suggestive of a non-intramolecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Substitution of Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp63 as a functional protease is regulated by these posttranslational modification pathways. PMID- 8626469 TI - Involvement of the Saccharomyces cerevisiae HDF1 gene in DNA double-strand break repair and recombination. AB - The HDF1 protein of Saccharomyces cerevisiae shares biochemical properties and structural homology with the 70-kDa subunit of the human autoantigen Ku. The Ku protein, a heterodimer composed of a 70-kDa subunit and an 80-kDa subunit, has been identified as the regulatory subunit of the DNA-dependent protein kinase. This enzyme has recently been shown to be involved in DNA repair and recombination processes in mammalian cells. Here we show that hdf1-disrupted S. cerevisiae strains are strongly sensitive toward the radiomimetic antibiotic bleomycin. In addition, mating-type switching and rates of spontaneous mitotic recombination are strongly reduced. This phenotype is similar to that of mammalian cells lacking components of the DNA-dependent protein kinase holoenzyme, suggesting that HDF1 participates in and exerts equivalent functions in S. cerevisiae. PMID- 8626470 TI - Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation by the nonsterol mevalonate metabolite farnesol in vivo. AB - We have previously reported that degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the isoprenoid pathway leading to cholesterol production, can be accelerated in cultured cells by the addition of farnesyl compounds, which are thought to mimic a natural, nonsterol mevalonate metabolite(s). In this paper we report accelerated reductase degradation by the addition of farnesol, a natural product of mevalonate metabolism, to intact cells. We demonstrate that this regulation is physiologically meaningful, shown by its blockage by several inhibitory conditions that are known to block the degradation induced by mevalonate addition. We further show that intracellular farnesol levels increase significantly after mevalonate addition. Based on these results, we conclude that farnesol is a nonsterol, mevalonate-derived product that plays a role in accelerated reductase degradation. Our conclusion is in agreement with a previous report (Correll, C. C., Ng, L., and Edwards, P. A. (1994) J. Biol. Chem. 269, 17390-17393), in which an in vitro system was used to study the effect of farnesol on reductase degradation. However, the apparent stimulation of degradation in vitro appears to be due to nonphysiological processes. Our findings demonstrate that in vitro, farnesol causes reductase to become detergent insoluble and thus lost from immunoprecipitation experiments, yielding apparent degradation. We further show that another resident endoplasmic reticulum protein, calnexin, similarly gives the appearance of protein degradation after farnesol addition in vitro. However, after the addition of farnesol to cells in vivo, calnexin remains stable, whereas reductase is degraded, providing further evidence that the in vivo effects of farnesol are physiologically meaningful and specific for reductase, whereas the in vitro effects are not. PMID- 8626471 TI - Novel properties of L-type polypeptide subunits in mouse ferritin molecules. AB - Properties of the L- and H-type polypeptide subunits forming ferritin 24-mer molecules in mice were investigated, using the products of in vitro transcription and translation from the two cloned genes, and recombinant ferritin molecules (H24L0 or H0L24) produced by transformation in Escherichia coli. Several different conditions for analytical electrophoresis reproducibly show that the relative migration position of the two mouse ferritin subunits is reversed from that reported for ferritin H- and L-subunits in all other mammals; since mouse and human H-polypeptides almost co-migrate, this unusual relative mobility is due largely to novel properties of the murine L-subunit. This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin. Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected. In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit. However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data. PMID- 8626472 TI - Application of biochemical systems theory to metabolism in human red blood cells. Signal propagation and accuracy of representation. AB - Human erythrocytes are among the simplest of cells. Many of their enzymes have been characterized kinetically using steady-state methods in vitro, and several investigators have assembled this kinetic information into mathematical models of the integrated system. However, despite its relative simplicity, the integrated behavior of erythrocyte metabolism is still complex and not well understood. Errors will inevitably be encountered in any such model because of this complexity; thus, the construction of an integrative model must be considered an iterative process of assessment and refinement. In a previous study, we selected a recent model of erythrocyte metabolism as our starting point and took it through three stages of model assessment and refinement using systematic strategies provided by biochemical systems theory. At each stage deficiencies were diagnosed, putative remedies were identified, and modifications consistent with existing experimental evidence were incorporated into the working model. In this paper we address two issues: the propagation of biochemical signals within the metabolic network, and the accuracy of kinetic representation. The analysis of signal propagation reveals the importance of glutathione peroxidase, transaldolase, and the concentration of total glutathione in determining systemic behavior. It also reveals a highly amplified diversion of flux between the pathways of pentose phosphate and nucleotide metabolism. In determining the range of accurate representation based on alternative kinetic formalisms we discovered large discrepancies. These were identified with the behavior of the model represented within the Michaelis-Menten formalism. This model fails to exhibit a nominal steady state when the activity of glutathione peroxidase is decreased by as little as 9%. Our current understanding, as embodied in this working model, is in need of further refinement, and the results presented in this paper suggest areas of the model where such effort might profitably be concentrated. PMID- 8626473 TI - DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo. AB - The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the microsomal enzyme cytochrome P4501A1 by increasing the transcription rate of the CYP1A1 gene. Induction requires two basic helix-loop-helix proteins, the ligand binding aromatic hydrocarbon receptor (AhR) and its heterodimerization partner, the AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer induces transcription by binding to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene. The basic regions of AhR and Arnt are crucial for DRE binding. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mobility shift assay) and induction of CYP1A1 transcription (determined in vivo by genetic complementation of AhR-defective and Arnt-defective mouse hepatoma cells, using an RNase protection assay to measure mRNA accumulation). Our findings reveal the amino acids in the basic regions of AhR/Arnt that are important for both DRE binding and induction of transcription. This information provides biological background for the interpretation of structural (e.g. crystallographic) studies of the interactions between AhR/Arnt and the DRE. Our findings also indicate that the in vitro behavior of the mutants does not consistently predict their functional activity in vivo. Thus, genetic complementation constitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function. PMID- 8626475 TI - Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization. AB - We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling. PMID- 8626474 TI - Mutations of two adjacent amino acids generate inactive and constitutively active forms of the human platelet-activating factor receptor. AB - We have mutated two residues, Ala230 and Leu231, in the C-terminal portion of the third intracellular loop of the human platelet-activating factor (PAF) receptor into Glu230 and Arg231, respectively. The Leu231 --> Arg231 substitution led to two major modifications: 1) increased constitutive activity of the PAF receptor resulting in agonist-independent production of inositol phosphates and 2) increased affinity of the receptor for binding PAF (agonist) but not WEB2086 (antagonist). The L231R mutant was able to adopt at least two conformations: (i) a higher affinity state than the corresponding state of the wild-type receptor (WT), dependent on G protein coupling, and (ii) a low affinity state, higher than the one for the uncoupled WT receptor. The Ala230 --> Glu230 substitution also resulted in two major modifications: 1) unresponsiveness in terms of phosphatidylinositol hydrolysis in response to PAF and 2) a marked decrease in affinity of the receptor for binding the agonist but not the antagonist. Competition binding studies of transient receptor expression in COS-7 cells and the inability of guanosine 5 -O-(3-thiotriphosphate) to modulate the decrease in affinity of a stable A230E mutant in Chinese hamster ovary cells suggest an inherent low affinity conformation for this mutant. Alternatively, mutation of Ala230 to Gln230 suggested that the residue 230 has a fundamental effect on receptor affinity and its charge is determinant in G protein coupling of the PAF receptor. In this report, we show that substitution of two immediately adjacent residues of the PAF receptor, Ala230 and Leu231, surprisingly leads to an inactive and a constitutively active phenotype, respectively. These results further support the concept of constitutively active G protein-coupled receptors as adopting ''active'' state conformations similar to those induced by agonist binding to WT receptors. PMID- 8626476 TI - The environment of the lipoxygenase iron binding site explored with novel hydroxypyridinone iron chelators. AB - The mechanisms of lipoxygenase inhibition by iron chelators have been investigated in human neutrophils and in isolated soybean lipoxygenase. Their Fe(III)-containing active sites have been targeted by synthesizing novel bidentate chelators from the hydroxypyridinone family sufficiently small to gain access through the hydrophobic channels of lipoxygenase. In stimulated human neutrophils, release of [3H]arachidonate-labeled eicosanoids is dependent on the lipid solubility of hydroxypyridinones, but larger hexadentate chelators have no effect on this or on total cellular leukotriene B4 production. Lipophilic hydroxypyridinones inhibit 5-lipoxygenase at equivalent concentrations to the established inhibitor, piriprost, and show additional but minor anti phospholipase A2 activity. Soybean 15-lipoxygenase inhibition is also dependent on the lipid solubility and coordination structure of chelators. Inhibition is associated with the formation of chelate-iron complexes, which are removed by dialysis without restoration of enzyme activity. Only after adding back iron is activity restored. Electron paramagnetic resonance studies show the removal of the iron center signal (g = 6) is concomitant with formation of Fe(III)-chelator complexes, identical in spectral shape and g value to 3:1 hydroxypyridinone Fe(III) complexes. Removal of iron is not the only mechanism by which hydroxypyridinones can inhibit lipoxygenase in intact cells, however, as a lipophilic non-iron-binding hydroxypyridinone, which shows no inhibition of the soybean lipoxygenase activity, partially inhibits 5-lipoxygenase in intact neutrophils without inhibiting neutrophil phospholipase A2. PMID- 8626477 TI - Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum. AB - Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe4S4]2+/1+ cluster (C-center) that serves as the COoxidation site and a standard [Fe4S4]2+/1+ cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in electron paramagnetic resonance (EPR) and Mossbauer studies. In this report, EPR-redox titrations demonstrate that the fully oxidized, diamagnetic form of the C-center (Cox) undergoes a one-electron reduction to the Cred1 state (gav = 1.87) with a midpoint potential of -110 mV. The reduction of Cox to Cred1 is shown to coincide with the reduction of an [Fe4S4]2+/1+ cluster in redox-titration experiments monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe4S4]2+/1+ clusters, does not exhibit EPR-active states or reduced Fe4S4 clusters at potentials more positive than -350 mV. PMID- 8626478 TI - Identification of novel Pax-2 binding sites by chromatin precipitation. AB - The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci. PMID- 8626479 TI - A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments. AB - Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells. PMID- 8626480 TI - Oncogenic Neu/ErbB-2 increases ets, AP-1, and NF-kappaB-dependent gene expression, and inhibiting ets activation blocks Neu-mediated cellular transformation. AB - Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers. PMID- 8626481 TI - Regulation of phospholipase C-beta1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. AB - The phospholipase C-beta1 (PLC-beta1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant m1 muscarinic cholinergic receptor, Gq, and 2-4 mol % [3H]phosphatidylinositol 4,5-bisphosphate. In this system, the muscarinic agonist carbachol stimulated steady-state PLC activity up to 90-fold in the presence of GTP. Both GTP and agonist were required for PLC activation, which was observed at physiological levels of Ca2+ (10-100 nM). PLC-beta1 is also a GTPase-activating protein for Gq. It accelerated steady-state GTPase activity up to 60-fold in the presence of carbachol, which alone stimulated activity 6-10-fold, and increased the rate of hydrolysis of Gq-bound GTP by at least 100-fold. Despite this rapid hydrolysis of Gq-bound GTP, the receptor maintained >10% of the total Gq in the active GTP-bound form by catalyzing GTP binding at a rate of at least 20-25 min 1, approximately 10-fold faster than previously described. These and other kinetic data indicate that the receptor and PLC-beta1 coordinately regulate the amplitude of the PLC signal and the rates of signal initiation and termination. They also suggest a mechanism in which the receptor, Gq, and PLC form a three protein complex in the presence of agonist and GTP (stable over multiple GTPase cycles) that is responsible for PLC signaling. PMID- 8626482 TI - p21ras signaling is necessary but not sufficient to mediate neurotrophin induction of calcium channels in PC12 cells. AB - Nerve growth factor and basic fibroblast growth factor bind to and activate receptor tyrosine kinases, causing sequential signaling via the p21ras/extracellular signal-regulated kinase pathway. The necessity and sufficiency of this signaling pathway in transducing neuronal differentiation have been tested in the PC12 cell model. Although necessary for morphological changes, the sufficiency of p21ras-mediated signaling in these events has come into question. We report that growth factor induction of voltage-gated calcium channels, a hallmark of physiological differentiation, also requires p21ras mediated signaling, but cannot be driven by p21ras activation alone. Thus, constitutive expression of the dominant negative N17ras mutant blocks growth factor-induced increases in Omega-conotoxin GVIA-sensitive, nimodipine-sensitive, and Omega-conotoxin GVIA/nimodipine-resistant calcium currents, but it does not block sodium current induction. However, manipulations that produce sustained activation of the p21ras signaling pathway and the neurite extension characteristic of morphological differentiation fail to increase calcium channel current densities. These results indicate the existence of distinct signaling requirements for morphological and physiological differentiation and further emphasize the importance of p21ras-independent signaling pathways in growth factor-mediated neuronal development. PMID- 8626483 TI - Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase. AB - Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states. PMID- 8626484 TI - Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. Cloning, heterologous expression, and localization. AB - The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina. PMID- 8626485 TI - Engineering a mineralocorticoid- to a glucocorticoid-synthesizing cytochrome P450. AB - Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam. PMID- 8626486 TI - Scorpion toxins affecting sodium current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels. AB - Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind alpha- and alpha-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, for the first time, the interaction of different scorpion neurotoxins, all affecting sodium current inactivation and toxic to mammals, with alpha-scorpion toxin receptor sites on both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled alpha-scorpion toxins AaH II and LqhalphaIT, the most active alpha-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The alpha-mammal scorpion toxins and the anti-insect Lqh alphaIT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with alpha scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh alphaIT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which is very similar on insect and rat brain sodium channels, in spite of the structural and pharmacological differences between them. The sea anemone toxin ATX II is also suggested to bind within this cluster. PMID- 8626487 TI - Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. AB - The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. We have examined the characteristics of a Mal- mutant of malE corresponding to the double substitution Gly32 --> Asp/Ile33 --> Pro, MalE31, previously obtained by random mutagenesis. In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm. The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full maltose binding affinity (Kd= 9 x 10(-7) M), suggesting that its folded structure is similar to that of the wild-type protein. Unfolding/refolding experiments show that MalE31 is less stable (-5. 5 kcal/mol) than the wild-type protein (-9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates. In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein. PMID- 8626488 TI - Transcriptional regulation of squalene epoxidase by sterols and inhibitors in HeLa cells. AB - Regulation of squalene epoxidase (SE) gene expression was studied in comparison with those of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and low density lipoprotein (LDL) receptor. An increased expression of SE mRNA and protein content in mouse L929 cells grown in 10% lipoprotein-deficient fetal bovine serum (LPDS) for 48 h was found by performing immunoblot and Northern blot analyses when compared with the culture in the presence of fetal bovine serum (FBS). The same results in mRNA levels were seen using human cell lines HepG2, HeLa, and Chang liver cells. The increase of SE mRNA in HeLa cells grown in LPDS was preventable in a dose-dependent manner by feeding cells with 25 hydroxycholesterol or cholesterol. When an SE inhibitor, NB-598, was fed to HeLa cells grown in LPDS, it caused further increases in mRNA levels of SE, HMG-CoA reductase, and LDL receptor. In contrast, NB-598 had no effect on the message levels of these genes when fed to HeLa cells grown in FBS. These results suggest that sterol produced endogenously can also regulate SE expression at the level of transcription. PMID- 8626489 TI - Direct association of STAT3 with the IFNAR-1 chain of the human type I interferon receptor. AB - Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalpha-dependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFN alpha but in which other components of the JAK-STAT pathway are activated by IFNalpha. PMID- 8626490 TI - Calcium ion modulation of meizothrombin autolysis at Arg55-Asp56 and catalytic activity. AB - When a recombinant variant of prothrombin with the cleavage site mutations R155A, R271A, and R284A (rMZ) is exposed to either prothrombinase or ecarin, a form of meizothrombin (rMZa) is generated that is stable for weeks in the presence of Ca2+ (Cote, H. C. F., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., and MacGillivray, R. T. A. (1994) J. Biol. Chem. 269, 11374-11380). In the absence of Ca2+ however, rMZa is rapidly cleaved within a disulfide bonded loop in the F1 domain at Arg55 in the sequence RTPR downward arrowDKL, yielding a molecule with 3 chains joined by two disulfide bonds (rMZa*). Cleavage kinetics are first order regardless of the rMZa concentration, indicating an intramolecular cleavage. This cleavage does not occur at Ca2+ concentrations in excess of 1.0 mM. To assess the role of the F1 domain in rMZa activity, another variant lacking the R155A mutation (rMZdesF1) was expressed, which when activated yields meizothrombin lacking the F1 domain (rMZdesF1a). Rates of hydrolysis of the tripeptide substrate S2238 by rMZa or rMZa* increase from 60% to 90% that of recombinant thrombin as Ca2+, Mg2+, or Mn2+ concentrations are varied from 0 to 10 mM. Km and kcat values for rMZa in the absence and presence of 5 mM Ca2+ are 1.9 and 2.2 microM and 65 and 105 s-1. TAME esterase activity of rMZa also increases with 5 mM Ca2+. No such metal ion-dependent effects are obtained with either thrombin or rMZdesF1a. Fibrinogen clotting activities, relative to that of thrombin, increase in a manner analogous to those obtained with small substrates, for rMZa and rMZa* but not rMZdesF1a. Complexes of the active site probe dansylarginine N-(3-ethyl-1,5-pentanediyl)amide with rMZa and rMZa*, but not thrombin or rMZdesF1a exhibit large cation-dependent decreases in fluorescence intensity, suggesting that metal ion binding in the F1 domain alters the environment of the probe at the active site. These results indicate that in the absence of divalent cations, the activity of rMZa is inhibited, perhaps by obstruction of the active site by the F1 domain, and that Ca2+ binding to the F1 domain modulates the properties of not only the F1 domain but also the protease domain. PMID- 8626491 TI - The alpha-isoform of the CCAAT/enhancer-binding protein is required for mediating cAMP responsiveness of the phosphoenolpyruvate carboxykinase promoter in hepatoma cells. AB - The gene coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is expressed in all gluconeogenic tissues, but stimulation of its rate of transcription by cAMP is robust only in liver. Evidence has accumulated which suggests that a liver-enriched transcription factor, likely a member of the CCAAT/enhancer binding protein (C/EBP) family, is required along with other ubiquitously expressed transcription factors to mediate this liver-specific response to cAMP. In this study, we examined the ability of C/EBP to participate in the cAMP-mediated activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in hepatoma cells. Expression of a dominant repressor of C/EBP in hepatoma cells significantly inhibited the protein kinase A-stimulated transcription of the PEPCK promoter, suggesting that a C/EBP family member was required for maximal transcriptional activation by protein kinase A. To provide additional support for this hypothesis, we prepared GAL4 fusion proteins containing C/EBP domains. Both C/EBPalpha and C/EBPbeta GAL4 fusion proteins were capable of stimulating transcription from promoters containing binding sites for the DNA-binding domain of GAL4. However, only the GAL4-C/EBPalpha fusion protein demonstrated the ability to synergize with the other transcription factors bound to the PEPCK promoter which are required to mediate cAMP responsiveness. The DNA binding domain of C/EBPalpha was not required for this activity in hepatoma cells, although in non-hepatoma cells the basic region leucine zipper domain appeared to inhibit the ability of C/EBPalpha to participate in mediating cAMP responsiveness. These results suggest that the liver-specific nature of the cAMP responsiveness of the PEPCK promoter involves the recruitment of C/EBPalpha to the cAMP response unit. PMID- 8626492 TI - Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells. AB - In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process. PMID- 8626493 TI - Adenovirus E1A proteins regulate phosphoenolpyruvate carboxykinase gene transcription through multiple mechanisms. AB - Recently, Kalvakolanu et al. (Kalvakolanu, D. V. R., Liu, J., Hanson, R. W., Harter, M. L., and Sen, G. C. (1992) J. Biol. Chem. 267, 2530-2536) showed that E1A inhibited the basal and cAMP-stimulated transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). This inhibition was mediated by the conserved region 1 (CR1) domain of E1A, which has been shown by other laboratories to bind to the cellular transcriptional adaptor proteins, p300 and cAMP response element binding protein (CREB)-binding protein (CBP). The PEPCK gene promoter contains a functional cAMP-response element, through which CREB and, therefore, CBP modulate transcription, and a consensus p300 DNA binding sequence is also present in a distal protein binding site of the promoter. We hypothesized that E1A might inhibit PEPCK gene transcription by binding to p300 and/or CBP. Surprisingly, we found that E1A consistently stimulated basal transcription from the PEPCK promoter in transfection assays in adenovirus (Ad) infected HepG2 hepatoma cells or E1A-expressing, stably transfected 3T3 fibroblasts and nuclear run-on assays in Ad-infected H4IIE hepatoma cells. E1A also enhanced the stimulation of PEPCK gene transcription by Bt2cAMP. In transfection assays, wild type Ad5 expressing both 243R and 289R forms of E1A or a mutant virus expressing the 289R form alone stimulated transcription from the PEPCK promoter by approximately 5-fold 20 h postinfection. However, no stimulation was observed in cells infected with a virus expressing either the 243R protein alone or a 289R protein from which conserved region 3 (CR3) was mutated. Mutation or deletion of CR1 of E1A had no significant effect on transcription from the PEPCK promoter. Mutations within conserved region 2 (CR2) of E1A that inhibit the binding of E1A to the retinoblastoma gene product (pRb) further enhanced the stimulation of transcription from the PEPCK promoter by 2 3 fold compared with wild type E1A. These findings suggested that the normal function of pRb is to stimulate PEPCK gene transcription, and that this process is inhibited by the binding of E1A to pRb. This hypothesis was confirmed by overexpressing pRb in HepG2 cells, which stimulated transcription from the PEPCK promoter. Our findings indicate that Ad E1A regulates PEPCK gene transcription through a stimulatory mechanism involving CR3, and by attenuating a stimulatory effect of pRb through CR2. PMID- 8626494 TI - Inhibition of tumor necrosis factor-induced p42/p44 mitogen-activated protein kinase activation by sodium salicylate. AB - Tumor necrosis factor (TNF) activates both p42 and p44 mitogen-activated protein kinases (MAPK) in human FS-4 fibroblasts, cells for which TNF is mitogenic. We now show that TNF activates p42 MAPK in two cell lines whose growth is inhibited by TNF. A mutant TNF that binds only to the p55 TNF receptor (TNFR) produced a similar degree of activation as wild-type TNF in FS-4 fibroblasts, indicating that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The upstream intracellular signals that couple the TNFR to MAPK activation are still poorly defined. We now show that neither phorbol ester-sensitive protein kinase C nor Gialpha link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to down-regulate protein kinase C or pretreatment with pertussis toxin to block Gialpha does not inhibit p42/p44 MAPK activation by TNF. To further analyze MAPK activation in FS-4 cells, we compared p42/p44 MAPK activation by TNF and epidermal growth factor (EGF). While tyrosine phosphorylation of p42/p44 MAPK was detected almost immediately (30 s) after stimulating cells with EGF, TNF-induced tyrosine phosphorylation was detected only after a more prolonged time interval (initially detected at 5 min and peaking at 15-30 min). In addition, the anti-inflammatory drug sodium salicylate, previously demonstrated to inhibit NF- kappaB activation by TNF, blocked the activation of p42/p44 MAPK in response to TNF but not in response to EGF. These findings demonstrate that the TNF and EGF receptors utilize distinct signaling molecules to couple to MAPK activation. Elucidation of the mechanism whereby sodium salicylate blocks TNF-induced p42/p44 MAPK activation may help to clarify TNF-activated signaling pathways. PMID- 8626495 TI - Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis. AB - The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo containing enzyme. PMID- 8626496 TI - Kinetics and crystallographic analysis of human glutathione reductase in complex with a xanthene inhibitor. AB - We have determined the crystal structure of a complex between the noncompetitive inhibitor (Kis = 27 microM, Kii = 48 microM with respect to oxidized glutathione (GSSG) and Kis = 144 microM, Kii = 176 microM with respect to NADPH) 6-hydroxy-3 oxo-3H-xanthene-9-propionic acid (XAN) and human glutathione reductase (hGR). The structure, refined to an R-factor of 0.158 at 2.0 A resolution, reveals XAN bound in the large cavity present at the hGR dimer interface where it does not overlap the glutathione binding site. The inhibitor binding causes extensive local structural changes that primarily involve amino acid residues from a 30-residue alpha-helix that lines the cavity and contributes to the active site of hGR. Despite the lack of physical overlap of XAN with the GSSG binding site, no GSSG binding is seen in soaks carried out with high XAN and GSSG concentrations, suggesting that some subtle interaction between the sites exists. An earlier crystallographic analysis on the complex between hGR and 3,7-diamino-2,8-dimethyl 5-phenyl-phenazinium chloride (safranin) showed that safranin bound at this same site. We have found that safranin also inhibits hGR in a noncompetitive fashion, but it binds about 16 times less tightly (Kis = 453 microM, Kii = 586 microM with respect to GSSG) than XAN and does not preclude the binding of GSSG in the crystal. Although in structure-based drug design competitive inhibitors are usually targetted, XAN's binding to a well defined site that is unique to glutathione reductase suggests that noncompetitive inhibitors could also serve as lead compounds for structure-based drug design, in particular as components of chimeric inhibitors. PMID- 8626497 TI - Constitutive insulin-like growth factor-II expression interferes with the enterocyte-like differentiation of CaCo-2 cells. AB - In this study we have examined the role of insulin-like growth factor-II (IGF-II) in the differentiation of the CaCo-2 human colon carcinoma cell line. We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 cells. IGF II expression is high in proliferating, undifferentiated CaCo-2 cells and markedly decreases when cells become confluent and start to differentiate. To evaluate whether differentiation of CaCo-2 cells depends on an IGF-II related pathway, we treated cells with a blocking antibody to the IGF-I receptor that mediates most IGF-II biological effects. Treatment of preconfluent CaCo-2 cells with this antibody decreased by 40% autonomous cell proliferation and induced differentiation as shown by an increase in sucrase isomaltase activity and apolipoprotein A-I (apoA-I) mRNA levels. To examine the significance of autocrine IGF-II production in CaCo-2 cell differentiation, we generated stable CaCo-2 cell lines that constitutively express rat IGF-II under the control of a Rous sarcoma virus promoter. Sustained expression of IGF-II resulted in: (a) increased proliferative rate; (b) high IGF-I receptor number, even after reaching confluence; (c) increased capability of anchorage-independent growth; (d) inhibition of the expression of apoA-I and SI mRNAs. Analysis of several independent IGF-II-transfected clones showed an inverse correlation between IGF II mRNA levels and expression of the differentiation markers, the cells expressing the higher levels of the transfected IGF-II being the less differentiated ones. Our data suggest that perturbation of IGF-II-mediated cell proliferation interferes with the enterocyte-like differentiation pathway of CaCo 2 cells. PMID- 8626498 TI - The 5'-untranslated region of the N-methyl-D-aspartate receptor NR2A subunit controls efficiency of translation. AB - The N-methyl-D-aspartate (NMDA) receptor plays a central role in such phenomena as long term potentiation and excitotoxicity. This importance in defining both function and viability suggests that neurons must carefully control their expression of NMDA receptors. Whereas the NR1 subunit of the NMDA receptor is ubiquitously transcribed throughout the brain, transcription of NR2 subunits is spatially and temporally controlled. Since heteromeric assembly of both subunits is required for efficient functional expression, post-transcriptional modification of either subunit would affect NMDA receptor activity. Here it is demonstrated that the 5'-untranslated region (5'-UTR) of the NR2A subunit severely restricts its protein translation in both Xenopus oocytes and in an in vitro translation system. Mutational analysis of the 5'-UTR implicates secondary structure as the major translational impediment, while the five alternate start codons play minor roles. An important biological role for the 5'-UTR of NR2A is further suggested by the unusually high level of sequence conservation between species. In contrast, the 5'-UTR of NR1 does not inhibit translation and is not consrved. Taken together, these findings suggest a mechanism for modulation of NMDA receptor activity through the control of translational efficiency of a single subunit. PMID- 8626499 TI - Elements within the first 17 amino acids of human osteonectin are responsible for binding to type V collagen. AB - The region in human osteonectin (ON) responsible for binding to type V collagen has been identified as the first 17 NH2-terminal residues. This conclusion is based upon binding studies with deletion mutants of ON produced in Escherichia coli, in which parts of the first 17 amino acids have been removed. Wild-type ON from E. coli and mammalian cell-derived nonglycosylated ON bind identically to type V collagen and at least twice as effectively as mammalian cell-derived N glycosylated ON. In previous studies, it was shown that N-glycosylation at residue 99 significantly reduces the capacity of ON to bind to type V collagen. Results reported in this communication demonstrate that the actual binding site on ON for type V collagen is distal from the site of N-glycosylation in terms of amino acid sequence but may be proximal in the folded, fully glycosylated, three dimensional structure. Consistent with this conclusion is the ability of a synthetic peptide consisting of amino acids 1-17 to specifically inhibit the binding of ON to type V collagen. PMID- 8626500 TI - Dual regulation of a chimeric plant serine/threonine kinase by calcium and calcium/calmodulin. AB - A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca2+ binding domain was recently cloned from plants (Patil, S., Takezawa, D., and Poovaiah, B. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4797-4801). The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca2+/calmodulin-dependent manner. The calmodulin-binding region of CCaMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of 32P/mol) that is stimulated 3.4-fold by Ca2+ (0.339 mol of 32P/mol), while calmodulin inhibits Ca2+-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca2+-stimulated autophosphorylation activity but retained Ca2+/calmodulin-dependent protein kinase activity at a reduced level. Ca2+-dependent mobility shift assays using E. coli-expressed protein from residues 358 520 revealed that Ca2+ binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca2+-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca2+/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its Ca2+-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca2+ and Ca2+/calmodulin. The presence of a visinin like Ca2+-binding domain in CCaMK adds an additional Ca2+-sensing mechanism not previously known to exist in the Ca2+/calmodulin-mediated signaling cascade in plants. PMID- 8626501 TI - Expression and insulin-regulated distribution of caveolin in skeletal muscle. Caveolin does not colocalize with GLUT4 in intracellular membranes. AB - Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells. Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle. Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle. PMID- 8626502 TI - Diethylstilbestrol-DNA interaction studied by Fourier transform infrared and Raman spectroscopy. AB - The interaction of diethylstilbestrol (DES) with calf thymus DNA was investigated at physiological pH with drug/DNA (phosphate) molar ratios (r) of 1:40, 1:20, 1:10, 1:4, 1:2, and 1. Fourier transform infrared and laser Raman difference spectroscopy were used to establish correlations between spectral changes and drug binding mode, sequence selectivity, DNA conformation, and structural properties of DES.DNA complexes in aqueous solution. Spectroscopic results indicated that DES is a weak intercalator with affinity for A-T-rich regions. It is also a groove binder with a major interaction with the thymine O-2 atom. At low drug concentration (r = 1:40), the A-T-rich region is the main target of drug intercalation, while at a higher drug content (r > 1:5), external binding to the G-C bases also occurs with a partial helix destabilization. Evidence for this comes from the spectral alterations of the A-T vibrational frequencies at 1661 cm 1 (Raman) and 1663 and 1609 cm-1 (IR) and of the G-C vibrations at 1581 and 1491 cm-1 (Raman) and 1717 and 1492 cm-1 (IR). Drug intercalation leads to a major reduction of B-DNA structure in favor of A-DNA. PMID- 8626503 TI - Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. AB - The neuronal-specific Elav-like proteins (HuD, Hel-N, and HuC) contain three RNP type concensus motifs and bind to AU-rich elements. We have identified and cloned a fourth member of this family (HuR) that is expressed in a wide variety of cell types. The purified recombinant protein binds avidly to the AU-rich element in c fos and interleukin-3 mRNAs. In the case of the c-fos AU-rich element, HuR binds to a core element of 27 nucleotides that contain AUUUA, AUUUUA, and AUUUUUA motifs. Mutational analysis has shown that all three AU motifs are required for maximal binding. PMID- 8626504 TI - Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase. AB - Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate. In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin. PMID- 8626506 TI - Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons. AB - Previous studies have demonstrated that multiple agents that promote survival of PC12 cells and sympathetic neurons deprived of trophic support also block cell cycle progression. Presently, we address whether inhibition of cell cycle-related cyclin-dependent kinases (CDKs) prevents neuronal cell death. We show that two distinct CDK inhibitors, flavopiridol and olomoucine, suppress the death of neuronal PC12 cells and sympathetic neurons. In addition, we demonstrate that inhibitor concentrations required to promote survival correlate with their ability to inhibit proliferation. Promotion of survival, however, does not correlate with inhibition of extracellular signal-regulated kinase or c-Jun kinase activities or with interference with the activation of c-Jun kinase that accompanies serum/nerve growth factor deprivation. In contrast to their actions on nerve growth factor-differentiated PC12 cells, the CDK inhibitors do not prevent the death of proliferation-competent PC12 cells and, in fact, promote their cell death. These findings support the hypothesis that post-mitotic neuronal cells die after removal of trophic support due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner. We speculate that cycling PC12 cells are not saved by these agents due to a signaling conflict between an inherent oncogenic signal and the inhibition of CDK activity. PMID- 8626505 TI - Human tenascin-R. Complete primary structure, pre-mRNA alternative splicing and gene localization on chromosome 1q23-q24. AB - We have established the primary structure of human tenascin-R (TN-R), a component of the extracellular matrix of the central nervous system, by sequencing cDNA clones which cover its complete coding region. The deduced amino acid sequence of human TN-R (1358 amino acids) showed a homology to chicken and rat TN-R of 75 and 93%, respectively. By reverse transcriptase-polymerase chain reaction we have studied the existence of TN-R isoforms generated by pre-mRNA alternative splicing in various human astrocytomas and meningiomas. Our findings demonstrate the existence of a human isoform in which one fibronectin-like repeat is omitted. Northern blot analysis of the poly(A)-rich RNA from different tissues showed two mRNAs having sizes of about 10 and 11 kilobases. Using DNA from a panel of human hamster and human-mouse somatic cell hybrids and by fluorescence in situ hybridization, we have assigned the gene for human TN-R to the region 1q23-q24. The mouse mutation loop-tail (Lp), which has been proposed as a model for human neural tube defects, maps to region of mouse chromosome 1 syntenic with human 1q23-q24. PMID- 8626507 TI - Identification of the peptides that stimulate the phosphoinositide hydrolysis in lymphocyte cell lines from peptide libraries. AB - Peptides which stimulate the formation of inositol phosphates (InoPs) in lymphocyte cell lines were identified by screening synthetic peptide libraries composed of random sequences of hexapeptides. The peptides containing the consensus sequence XKYX(P/V)M were found to be most active in the phospholipase C (PLC)-mediated formation of InoPs in a human B myeloma cell line, U266. The peptides also stimulated the phosphoinositide hydrolysis and the release of [Ca2+]i in HL60 and U937 cell lines. On the other hand, these peptides showed no effect in the following cell lines: NIH3T3, PC12, Daudi, Sp2, Jurkat, H9, Molt-4, SupT-1, K562, and RBL-2H3. The result suggests the possibility that the peptides may have cell type specificity. Experiments with one of the active peptides, WKYMVM-NH2 showed that its action mimics the effect of AlF4- which is a G-protein activator in the InoPs generation, and pertussis toxin partially blocked the InoPs accumulation and [Ca2+]i release induced by the peptide in the U266 cells. Binding assays with the peptide labeled with 125I showed that U266 cells have a saturable number of binding sites for the peptide. Taken together, these results suggest that the peptides could activate PLC-mediated signal transduction via a pertussis toxin-sensitive G-protein coupled receptor in certain cell types. PMID- 8626508 TI - Structure and expression of the Chlorobium vibrioforme hemB gene and characterization of its encoded enzyme, porphobilinogen synthase. AB - Plasmids containing DNA from the green photosynthetic bacterium Chlorobium vibrioforme complement a heme-requiring Escherichia coli hemB mutant that is deficient in porphobilinogen (PBG) synthase activity. PBG synthase activity was detected in extract of complemented cells but not in that of cells transformed with control plasmid. The sequence of the C. vibrioforme hemB gene predicts a HemB protein that contains 328 amino acids, has a molecular weight of 36,407, and is 53% identical to the homologous proteins of Synechocystis sp. PCC 6301 and Rhodobacter capsulatus. The response of C. vibrioforme PBG synthase to divalent metals is unlike that of any previously described PBG synthase; Mg2+ stimulates but is not required for activity, and Zn2+ neither stimulates nor is required. This response correlates with predicted sequences of two putative variable metal binding regions of C. vibrioforme HemB. The C. vibrioforme hemB open reading frame begins 1585 bases downstream from the end of the hemD open reading frame and is transcribed in the same direction as hemA, hemC, and hemD. However, hemB is not part of the same transcription unit as these genes, and the hemB transcript is approximately the same size as the hemB gene alone. Between hemD and hemB there is an intervening open reading frame that is oriented in the opposite direction and encodes a protein with a predicted amino acid sequence significantly similar to that of inositol monophosphatase, an enzyme that is not involved in tetrapyrrole biosynthesis. The gene order within hem gene clusters is highly conserved in phylogenetically diverse prokaryotic organisms. This conservation suggests that there are functional constraints on the relative order of the hem genes. PMID- 8626509 TI - Functional expression and signaling properties of cloned human parathyroid hormone receptor in Xenopus oocytes. Evidence for a novel signaling pathway. AB - Expression of human parathyroid hormone receptor (hPTHR) was obtained in Xenopus oocytes. Receptor function was detected by hormone stimulation of endogenous Ca2+ activated Cl- current. This current was blocked by injected, but not by extracellular, EGTA, confirming that the hPTHR activates cytosolic Ca2+ signaling pathways. PTH responses were acutely desensitized but were regained in 6 12 h. Injection of cAMP or analogues had no effect on either responsiveness or desensitization to hPTH. The hPTH response was more sluggish than seen with serotonin 5-hydroxytryptamine (5-HT2C) receptor. In oocytes co-expressing both hPTHR and 5-HT2C receptors, homologous desensitization was seen, but cross desensitization was not observed. Injection of inositol 1,4,5-trisphosphate (InsP3) elicited a fast inward current similar to that induced by serotonin, and complete cross-desensitization occurred between the InsP3 and 5-HT2C responses. Desensitization by hPTH did not affect responses to either InsP3 or serotonin, but cells desensitized to injected InsP3 still responded strongly to PTH. Oocytes did not respond to either cADPR or NAADP+, but NADP+ and analogues were found to be potent inhibitors of PTH signaling. We suggest that PTH cytosolic Ca2+ signaling in oocytes either involves a novel signaling system or proceeds through a Ca2+ compartment whose responsiveness is regulated in a novel way. PMID- 8626510 TI - Substrate specificity of glycinamide ribonucleotide synthetase from chicken liver. AB - Several analogs of glycinamide ribonucleotide and phosphoribosylamine have been prepared and evaluated as substrates for glycinamide ribonucleotide synthetase purified from chicken liver. Glycinamide ribonucleotide analogs include side chain modifications wherein the glycine side chain (R = CH2NH2) has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, ribose ring replacement by chiral cyclopentane and cyclopentene derivatives, and phosphate replacement by phosphonates. All of these, with the exception of the O-phosphonate, served as substrates for the reverse enzymatic reaction, with Vmax values comparable to that obtained with glycinamide ribonucleotide, although the Km values ranged from 21 to 118 times the Km for glycinamide ribonucleotide. Analogs of phosphoribosylamine examined as substrates for the forward reaction consist of chiral derivatives of cyclopentane and cyclopentene and a chiral carbocyclic phosphonate. These also served as substrates, with Km values ranging from 5 to 23 times the Km for phosphoribosylamine and with diminished Vmax values. These studies have begun to define the structural features of the nucleotide substrate necessary to support enzymatic activity. Sarcosine (N-methylglycine) and beta-alanine were also accepted as substrates, albeit with reduced affinity compared with glycine. PMID- 8626511 TI - The differential effects of the Gly-60 to Ala mutation on the interaction of H Ras p21 with different downstream targets. AB - We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H Ras. The significance of our findings is discussed in the light of the GTP induced conformational change model. PMID- 8626512 TI - Identification of plectin as a substrate of p34cdc2 kinase and mapping of a single phosphorylation site. AB - Plectin is an in vitro substrate for various kinases present in cell lysates from mitotic and interphase Chinese hamster ovary cells. Sensitivity of plectin kinase activity to the inhibitor olomoucine, and two-dimensional tryptic peptide mapping of plectin phosphorylated by various kinase preparations suggested that the major plectin kinase activity in mitotic extracts is related to the cell cycle regulator kinase p34cdc2. Bacterial expression of various truncated plectin mutant proteins comprising different domains of the molecule and their phosphorylation by purified p34cdc2kinase revealed that the target site of this kinase resided within plectin's C-terminal globular domain. Among the subdomains of the C-terminal region (six repeats and a short tail sequence), only repeat 6 and the tail were phosphorylated by p34cdc2 kinase. As shown by two-dimensional phosphopeptide mapping, repeat 6, but not the tail, contained a mitosis-specific phosphorylation site targeted by p34cdc2 kinase in intact plectin molecules. By performing site-directed mutagenesis of a potential p34cdc2 recognition sequence motif within the repeat 6 domain, threonine 4542 was identified as the major target for the kinase. Protein kinase A, phosphorylating plectin also within repeat 6, targeted sites that were clearly different from those of p34cdc2 kinase. PMID- 8626513 TI - Observations on the binding of four anti-carbohydrate monoclonal antibodies to their homologous ligands. AB - The binding of four monoclonal immunoglobulins, two with specificity for beta(1- >6)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(1-->6)-linked d-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnKa and 1/T, where Ka is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaGu, is virtually independent of T, and the DeltaSu is small when compared with DeltaGu. The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-d-galactopyranoside and the methyl glycoside of beta(1-->6)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans). PMID- 8626514 TI - Plasminogen activator inhibitor-1 and vitronectin promote the cellular clearance of thrombin by low density lipoprotein receptor-related proteins 1 and 2. AB - Thrombin is a multifunctional protein that has both proteinase and growth factor like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha2 macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN). In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, 125I thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-proteinase inhibitor, or d phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of 125I-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP 2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury. PMID- 8626515 TI - Identification of a 79-kDa heparin-binding fibroblast growth factor (FGF) receptor in rat hepatocytes and its correlation with the different growth responses to FGF-1 between hepatocyte subpopulations. AB - We reported previously that the potency of heparin-binding fibroblast growth factor-1 (FGF-1) as a mitogen for rat hepatocytes in primary culture is as high as that of epidermal growth factor (EGF) and hepatocyte growth factor. To gain insight into the pathophysiological significance of FGF-1 in hepatocyte growth, we analyzed the cooperative mitogenicity of FGF-1 and EGF. Results from a nuclear labeling assay using [3H]thymidine suggest that most hepatocytes in primary culture consist of two cell populations that differ in response to FGF-1; one is an FGF-1-responsive cell population, and the other is an EGF-responsive (but not FGF-1-responsive) cell population. On the other hand, autoradiographic analysis of 125I-FGF-1 binding demonstrated that high affinity FGF receptors were homogeneously distributed on the surface of all hepatocytes. Cross-linking 125I FGF-1 to the nonstimulated hepatocyte surface indicated that the high affinity FGF receptors comprise two FGF receptors that differ in molecular mass (128 and 79 kDa). Furthermore, the 79-kDa receptor was preferentially down-regulated when the hepatocytes were stimulated with EGF or hepatocyte growth factor. These data suggest that the abundant expression of the 79-kDa FGF receptor on some populations of hepatocytes is involved in their lack of response to FGF-1. The 128- and 79-kDa FGF receptors were assigned as FGFR2 using an antibody specific to the ectodomain of FGFR2, whereas the 79-kDa receptor was not reactive to the antibody against the carboxyl terminus of FGFR2. This 79-kDa FGF receptor was not tyrosine-phosphorylated in response to FGF-1 stimulation, while the 128-kDa FGF receptor was recognized by anti-phosphotyrosine antibody under the same conditions. Also, the heterodimer of 79- and 128-kDa FGF receptors was less tyrosine-phosphorylated than the homodimer of 128-kDa FGF receptors. These data suggest that the 79-kDa FGF receptor inhibits the function of the 128-kDa FGF receptor through their heterodimerization. Thus, we surmise that the difference in response to FGF-1 between the cell populations of normal rat hepatocytes was caused by the different levels of the 79-kDa FGF receptor in each cell population. PMID- 8626516 TI - Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket. AB - To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1 18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity. PMID- 8626517 TI - Structure and polymorphism of HIV-1 third variable loops. AB - The third variable (V3) loop of HIV-1 surface glycoprotein, gp120, has been the target of neutralizing antibodies. However, sequence variation inside the V3 loop diminishes its effectiveness as a potential vaccine against HIV-1. The elusive nature of the V3 loop structure prompted us to carry out a systematic study on different isolates in an attempt to identify a common structural motif in the V3 loop regardless of the amino acid sequence variability. We have previously determined the structural features of two V3 loops: V3 Thailand and V3 MN. In this paper, we present the structure of two other variants: V3 Haiti and V3 RF. Our results show that similar secondary structures are observed in all the four V3 loops: a GPG(R/K/Q) crest in the center of the neutralizing domain, two extended regions flanking the central crest, and a helical region in the C terminal domain. For the Haitian V3 loop, we also show how the conserved structural features are masked through a conformational switch encoded in the amino acid sequences on the C-terminal side of the GPGK crest. PMID- 8626518 TI - Autoregulatory loop in the regulation of the mammalian ftz-f1 gene. AB - The mammalian ftz-f1 (mftz-f1) gene encoding Ad4BP/SF-1 has been demonstrated to be essential for the development of adrenal and gonadal glands. In a previous study, we identified an E box as the transcriptional element in the 5'-upstream region of the rat mftz-f1 gene. In the present study, we found a steroidogenic cell-specific transcriptional element in the first intron of the gene. Gel mobility shift and DNase I footprint analyses clearly revealed that Ad4BP itself binds to the element (Ad4 site). This finding was further supported by the positive effect of an Ad4BP expression vector on the transcription and by the significant decrease in the transcription caused by nucleotide substitutions within the Ad4 site. Similar loss was also caused by substitutions in the E box, indicating that the two elements are essential for the full transcriptional activity of the gene. DNase I hypersensitivity assays revealed that the chromatin structure around the Ad4 site and the E box was "open up" in the adrenal glands and Y-1 cells, whereas "closed down" in the liver. These observations indicated that the mftz-f1 gene is controlled by the autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissues differentiated. PMID- 8626520 TI - DTEF-1, a novel member of the transcription enhancer factor-1 (TEF-1) multigene family. AB - M-CAT motifs mediate muscle-specific transcriptional activity via interaction with binding factors that are antigenically and biochemically related to vertebrate transcription enhancer factor-1 (TEF-1), a member of the TEA/ATTS domain family of transcription factors. M-CAT binding activities present in cardiac and skeletal muscle tissues cannot be fully accounted for by existing cloned isoforms of TEF-1. TEF-1-related cDNAs isolated from heart libraries indicate that at least three classes of TEF-1-related cDNAs are expressed in these and other tissues. One class are homologues of the human TEF-1 originally cloned from HeLa cells (Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M., and Chambon, P. (1991) Cell 65, 551-568). A second class represents homologues of the avian TEF-1-related gene previously isolated (Stewart, A. F., Larkin, S. B., Farrance, I. K., Mar, J. H., Hall, D. E., and Ordahl, C. P. (1994) J. Biol. Chem. 269, 3147-3150). The third class consists of a novel, divergent TEF-1 cDNA, named DTEF-1, and its preliminary characterization is described here. Two isoforms of DTEF-1 (DTEF-1A and DTEF-1B) were isolated as 1.9-kilobase pair clones with putative open reading frames of 433 and 432 amino acids whose differences are attributable to alternative splicing at the C terminus of the TEA DNA binding domain. Cardiac muscle contains high levels of DTEF-1 transcripts, but unexpectedly low levels are detected in skeletal muscle. DTEF-1 transcripts are present at intermediate levels in gizzard and lung, and at low levels in kidney. DTEF-1A is a sequence-specific M-CAT-binding factor. The distinct spatial pattern of expression, and unusual amino acid sequence in its DNA binding domain, may indicate a particular role for DTEF-1 in cell-specific gene regulation. Recent work also suggests that at least one more TEF-1-related gene exists in vertebrates. We propose a naming system for the four TEF-1 gene family members identified to date that preserves existing nomenclature and provides a means for extending that nomenclature as additional family members may be identified. PMID- 8626519 TI - Expression of the alpha(1,3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands. AB - Lymphocyte homing to lymph nodes and Peyer's patches is mediated, in part, by adhesive interactions between L-selectin expressed by lymphocytes and L-selectin ligands displayed at the surface of the cuboidal endothelial cells lining the post-capillary venules within lymphoid aggregates. Candidate terminal oligosaccharide structures thought to be essential for effective L-selectin ligand activity include a sulfated derivative of the sialyl Lewis x tetrasaccharide. Cell type-specific synthesis of this oligosaccharide is presumed to require one or more alpha(1,3)fucosyltransferases, operating upon common 3' sialylated and/or sulfated N-acetyllactosamine-type precursors. The identity of the alpha(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin ligands has not been defined. We report here the molecular cloning and characterization of a murine alpha(1,3)fucosyltransferase locus whose expression pattern correlates with expression of high affinity ligands for L-selectin. In situ hybridization and immunohistochemical analyses demonstrate that this cDNA and its cognate alpha(1,3)fucosyltransferase are expressed in endothelial cells lining the high endothelial venules of peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. These expression patterns correlate precisely with the expression pattern of L-selectin ligands identified with a chimeric L selectin/IgM immunohistochemical probe and by the high endothelial venule reactive monoclonal antibody MECA-79. Transcripts corresponding to this cDNA are also detected in isolated bone marrow cells, a source rich in the surface localized ligands for E- and P-selectins. Sequence and functional analyses indicate that this murine enzyme corresponds to the human Fuc-TVII locus. These observations suggest that Fuc-TVII participates in the generation of alpha(1,3)fucosylated ligands for L-selectin and provide further evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes. PMID- 8626521 TI - The role of transcription enhancer factor-1 (TEF-1) related proteins in the formation of M-CAT binding complexes in muscle and non-muscle tissues. AB - M-CAT sites are required for the activity of many promoters in cardiac and skeletal muscle. M-CAT binding activity is muscle-enriched, but is found in many tissues and is immunologically related to the HeLa transcription enhancer factor 1 (TEF-1). TEF-1-related cDNAs (RTEF-1) have been cloned from chick heart. RTEF-1 mRNA is muscle-enriched, consistent with a role for RTEF-1 in the regulation of muscle-specific gene expression. Here, we have examined the tissue distribution of TEF-1-related proteins and of M-CAT binding activity by Western analysis and mobility shift polyacrylamide gel electrophoresis. TEF-1-related proteins of 57, 54 and 52 kDa were found in most tissues with the highest levels in muscle tissues. All of these TEF-1-related proteins bound M-CAT DNA and the 57- and 54 kDa TEF-1-related polypeptides were phosphorylated. Proteolytic digestion mapping showed that the 54-kDa TEF-1-related polypeptide is encoded by a different gene than the 52- and 57-kDa TEF-1-related polypeptides. A comparison of the migration and proteolytic digestion of the 54-kDa TEF-1-related polypeptide with proteins encoded by the cloned RTEF-1 cDNAs showed that the 54-kDa TEF-1-related polypeptide is encoded by RTEF-1A. High resolution mobility shift polyacrylamide gel electrophoresis showed multiple M-CAT binding activities in tissues. All of these activities contained TEF-1-related proteins. One protein-M-CAT DNA complex was muscle-enriched and was up-regulated upon differentiation of a skeletal muscle cell line. This complex contained the 54-kDa TEF-1-related polypeptide. Therefore, RTEF1-A protein is a component of a muscle-enriched transcription complex that forms on M-CAT sites and may play a key role in the regulation of transcription in muscle. PMID- 8626522 TI - Induction of apoptosis and potentiation of ceramide-mediated cytotoxicity by sphingoid bases in human myeloid leukemia cells. AB - Prior studies demonstrated that ceramide promotes apoptotic cell death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77), and that this lethal process is potently suppressed by diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L., Gewirtz, D. A., Kolesnick, R. N., and Grant, S. (1994) J. Biol. Chem. 269, 31685-31692). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0. 001 10 microM) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations (30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3'-incision in the absence of XPF ERCC1. Kinetic experiments revealed that the 3'-incision precedes the 5' incision. Consistent with the kinetic data, uncoupled 5'-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease. PMID- 8626525 TI - Epidermal growth factor receptor targeting prevents uncoupling of the Grb2-SOS complex. AB - Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex. PMID- 8626526 TI - Interleukin-1 beta-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F. AB - The aim of this investigation was to study the putative involvement of lipid second messengers, protein kinases, and transcription factors in interleukin-1 beta (IL-1beta)-induced signal transduction in insulin-producing cells. For this purpose, insulin-producing RINm5F cells were exposed to IL-1beta (25 units/ml), and the ceramide, ceramide 1-phosphate, sphingomyelin, diacylglycerol, and phosphatidic acid contents of the cells were subsequently determined. It was found that IL-1beta induced a transient increase (2-5 min) in ceramide and diacylglycerol, which was not paralleled by an increase in ceramide 1-phosphate and phosphatidic acid. A rapid decrease in the sphingomyelin content of the cells was, however, observed. The cell-permeable ceramide analogue N-acetylsphingosine and the phorbol ester phorbol 12-myristate 13-acetate (PMA) both induced the phosphorylation and increased the activities of the protein kinase JNK1 and the transcription factor ATF2. These effects were, however, not as pronounced as those induced by IL-1beta. The DNA binding activity of transcription factors in nuclear extracts was determined using the electrophoretic mobility shift assay method. Transcription factor binding to the ATF/cAMP-responsive element consensus sequence was increased 4-5-fold by acetylsphingosine, PMA, or IL-1beta, whereas binding to the CCAAT/enhancer-binding protein and AP-1 elements was found to be only slightly stimulated by these three agents. Binding to the NF-kappaB element was strongly induced by IL-1beta, but not by acetylsphingosine or PMA. Finally, acetylsphingosine and PMA did not mimic the nitric oxide-inducing effects of IL 1beta. It is concluded that IL-1beta-stimulated formation of ceramide and diacylglycerol may contribute to JNK1 and ATF2 transcription factor activation, which may be a necessary (but not sufficient) step in beta-cell nitric-oxide synthase induction. PMID- 8626527 TI - Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites. AB - The retinoblastoma tumor suppressor protein, RB, contains at least three distinct protein binding domains. The A/B pocket binds proteins with the LXCXE motif, the C pocket binds the nuclear c-Abl tyrosine kinase, and the large A/B pocket binds the transcription factor E2F. Dissociation of RB from its targets is observed as RB becomes phosphorylated during G1/S progression. There are 16 Cdk consensus phosphorylation sites in RB. It was previously unknown whether the many phosphorylation sites had redundant or distinct functions in the regulation of RB. Using RB mutant proteins lacking specific phosphorylation sites, we show that each of the binding domains is inhibited by different sites. Thr-821/826 phosphorylation is required to inhibit the binding to LXCXE containing proteins. Mutation of these two sites does not interfere with the hyperphosphorylation of RB. However, this phosphorylated mutant retains the ability to bind T-Ag, E7, and Elf-1, all of which contain the LXCXE motif. In contrast, Ser-807/811 phosphorylation is required to disrupt c-Abl binding. Mutation of Ser-807/811 and Thr-821/826 does not abolish the regulation of E2F binding. Taken together, these results show that the protein binding domains of RB are each regulated by distinct Cdk phosphorylation sites. PMID- 8626528 TI - Nuclear appearance of a factor that binds the CD28 response element within the interleukin-2 enhancer correlates with interleukin-2 production. AB - Activation of T lymphocytes requires the combined signaling of the T cell receptor and costimulatory molecules such as CD28. The ability of T cells to produce interleukin-2 (IL-2) is a critical control point in T lymphocyte activation. The IL-2 enhancer contains a functional motif named CD28 response element (CD28RE) that serves a role as a target for mitogenic T cell activation signals. The CD28RE sequence reveals similarity to the consensus kappaB binding motif. Here we demonstrate that CD28RE binds an inducible protein with a molecular mass of approximately 35 kDa called nuclear factor of mitogenic activated T cells (NF-MATp35) that is clearly different from the known NF- kappaB/Rel family members. Induction of NF-MATp35 was shown to depend on de novo protein synthesis and was restricted to T cells that received a mitogenic combination of T cell stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell stimulation did not result in appearance of NF-MATp35. These results indicate that mitogenic combinations of T cell activation signals are integrated at the level of NF-MATp35 induction. Similar to its effect on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35. Taken together, these data demonstrate that the nuclear appearance of NF-MATp35 shows excellent correlation with IL-2 production, which is a unique characteristic among nuclear factors implicated in the control of IL-2 gene expression. PMID- 8626529 TI - Molecular characterization of trans-Golgi p230. A human peripheral membrane protein encoded by a gene on chromosome 6p12-22 contains extensive coiled-coil alpha-helical domains and a granin motif. AB - Using autoantibodies from a Sjogren's syndrome patient, we have previously identified a 230-kDa peripheral membrane protein associated with the cytosolic face of the trans-Golgi (Kooy, J., Toh, B. H., Pettitt, J. M., Erlich, R. and Gleeson, P. A. (1992) J. Biol. Chem. 267, 20255-20263). Here we report the molecular cloning and sequence analysis of human p230 and the localization of its gene to chromosome 6p12 22. Partial cDNA clones, isolated from a HeLa cell cDNA library using autoantibodies, were used to obtain additional cDNAs, which together span 7695 base pairs (bp). The p230 mRNA is approximately 7.7 kilobases. Two alternatively spliced mRNAs for p230 were detected. These differed by 21- and 63-bp insertions in the 3'-sequence, resulting in differences in amino acid sequence at the carboxyl terminus. The predicted 261-kDa protein is highly hydrophilic with 17-20% homology with many proteins containing coiled-coil domains. Apart from two proline-rich regions (amino acids 1-117 and 239-270), p230 contains a very high frequency of heptad repeats, characteristic of alpha helices that form dimeric coiled-coil structures. p230 also includes the sequence ESLALEELEL (amino acids 538-546), a motif found in the granin family of acidic proteins present in secretory granules of neuroendocrine cells. This is the first report of a cytosolic Golgi protein containing a granin motif. The structural characteristics of p230 indicate that it may play a role in vesicular transport from the trans-Golgi. PMID- 8626530 TI - Involvement of ErbB2 in the signaling pathway leading to cell cycle progression from a truncated epidermal growth factor receptor lacking the C-terminal autophosphorylation sites. AB - To investigate the mechanisms underlying the enhanced mitogenic activity of the truncated epidermal growth factor receptor (EGFR) lacking the C-terminal autophosphorylation sites (Delta973-EGFR), we studied the intracellular signaling pathways in NR6 cells expressing human wild type EGFR and Delta973-EGFR. Microinjection of dominant/negative p21ras(N17) completely inhibited EGF-induced DNA synthesis in both cell types. EGF stimulated Shc phosphorylation as well as the formation of wild type EGFR.Shc complexes. In contrast, EGF stimulated Shc phosphorylation without formation of Delta973-EGFR.Shc complexes. Tyrosine phosphorylated Shc formed complexes with Grb2.Sos, and microinjection of anti-Shc antibody and Shc-SH2 GST fusion protein inhibited EGF stimulation of DNA synthesis in both cell lines. EGF markedly increased ErbB2 tyrosine phosphorylation in wild type EGFR cells. In Delta973-EGFR cells, ErbB2 was tyrosine phosphorylated in the basal state and EGFR stimulated further phosphorylation of ErbB2. In addition to ErbB2, additional proteins were tyrosine phosphorylated in Delta973-EGFR cells, mostly in the molecular mass range of 120 170 kDa. Taken together with our findings indicating coupling of ErbB2 to Shc, these data suggest the importance of an alternative signaling pathway in Delta973 EGFR cells mediated by the formation of heterodimeric structures between the truncated EGFR and ErbB2, followed by coupling through Shc to Grb2.Sos and the p21ras pathway, ultimately leading to mitogenesis. PMID- 8626531 TI - Phorbol ester inhibits the phosphorylation of the retinoblastoma protein without suppressing cyclin D-associated kinase in vascular smooth muscle cells. AB - To elucidate the role of protein kinase C in vascular smooth muscle cell proliferation, we examined the effects of phorbol 12-myristate 13-acetate (PMA) on G1 events in human arterial cells. About 15 h after G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, [3H]thymidine incorporation started. PMA (10 nM) inhibited the incorporation over 90% when added earlier than 3 h after stimulation, but had no effect when added 12 h or later. PMA inhibited the phosphorylation of the retinoblastoma protein (pRb), which normally began at about 9 h. PMA did not inhibit the gene expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of which began at 0-3 h. However, PMA reduced the expression of cyclins E and A, which usually began at 3 9 h and about 15 h, respectively. PMA inhibited the histone H1 kinase activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit the pRb kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable from 0-3 h. These results suggested that the PMA-induced inhibition of pRb phosphorylation is not mediated by suppressing cyclin D-associated kinase(s) including Cdk4, but involves the suppression of Cdk2 activity that results from the reduced expression of cyclins E and A. PMID- 8626532 TI - Partial reactions catalyzed by protein components of the acetyl-CoA decarbonylase synthase enzyme complex from Methanosarcina barkeri. AB - In methanogens, the acetyl-CoA decarbonylase synthase (ACDS) complex, which has five different subunits, catalyzes synthesis and cleavage of acetyl-CoA according to the reaction: CO2 + 2H+ + 2e- + CH3-H4SPt + CoA <--> acetyl-CoA + H4SPt + H2O, where H4SPt and CH3-H4SPt are tetrahydrosarcinapterin and N5-methyl tetrahydrosarcinapterin, respectively. We have dissociated the ACDS complex into three protein components by limited proteolytic digestion. Catalysis of acetyl CoA synthesis was lost in parallel with the loss of the intact beta subunit; however, no decrease in activity was detected in any of three partial reactions found to be catalyzed by distinct protein components of the proteolyzed ACDS complex: (a) CO dehydrogenase, catalyzed by the alpha epsilon component, (b) CH3 H4pteridine:cob(I)amide-protein methyltransferase, catalyzed by the intact gamma subunit and fragments of the delta subunit, and (c) acetyltransferase, catalyzed by a truncated form of the beta subunit. The results indicated that the beta subunit is responsible for binding CoA and acetyl-CoA and suggested that acetyl enzyme formation occurs on the beta subunit. A value of 5.5 x [H+]-1 M-1 was determined for the equilibrium constant of the following reaction at pH 7.5 and 25 degrees C: CH3-H4SPt + cob(I)amide-protein + H+ <--> H4SPt + CH3-cob(III)amide protein. PMID- 8626533 TI - Translational initiation factor eIF-4E. A link between cardiac load and protein synthesis. AB - To define the coupling mechanism between cardiac load and the rate of protein synthesis, changes in the extent of eIF-4E phosphorylation were measured after imposition of a load. Electrically stimulated contraction of adult feline cardiocytes increased eIF-4E phosphorylation to 34% after 4 h, as compared with 8% phosphorylation in quiescent controls. However, eIF-4E phosphorylation did not increase upon electrical stimulation in the presence of 7.5 mM 2,3-butanedione monoxime, an inhibitor of actin-myosin cross-bridge cycling and active tension development. Treatment of adult cardiocytes with either 0.1 microM insulin or 0.1 microM phorbol 12-myristate 13-acetate increased eIF-4E phosphorylation to 23 and 64%, respectively, but these increases were not blocked by 2,3-butanedione monoxime. In canine models of acute hemodynamic overload in vivo, eIF-4E phosphorylation increased to 23% in response to left ventricular pressure overload as compared with 7% phosphorylation in controls. Acute volume overload had no effect on eIF-4E phosphorylation. These changes in eIF-4E phosphorylation account for differences in anabolic responses to acute pressure versus acute volume overload. These data suggest that eIF-4E phosphorylation is a mechanism by which increased cardiac load is coupled to accelerated rates of protein synthesis. PMID- 8626534 TI - A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins. AB - Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has been shown to interact with the HIV 1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors. Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex. PMID- 8626535 TI - Human apolipoprotein E receptor 2. A novel lipoprotein receptor of the low density lipoprotein receptor family predominantly expressed in brain. AB - Isolation and characterization of a human cDNA demonstrated a novel lipoprotein receptor designated apolipoprotein E receptor 2 (apoER2). The new receptor consists of five functional domains resembling the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) receptors. LDL receptor deficient Chinese hamster ovary cells expressing human apoER2 bound apoE rich beta-migrating VLDL with high affinity and internalized. LDL was bound with much lower affinity to these cells. The 4.5- and 8.5-kb mRNAs for the receptor were most highly expressed in human brain and placenta. In rabbit tissues, multiple species of the mRNA with 4, 4.5, 5.5, 8.5, and 11 kb were detected most intensely in brain and testis and, to a much lesser extent, in ovary, but were undetectable in other tissues. In rat adrenal pheochromocytoma PC12 cells, the receptor mRNA was induced by treatment of the cells with nerve growth factor. The receptor transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex as revealed by in situ hybridization histochemistry. In the cerebellar cortex, the receptor transcripts were densely deposited in Purkinje cell somata. PMID- 8626536 TI - The role of the four Ca2+ binding sites of troponin C in the regulation of skeletal muscle contraction. AB - In order to study the role of the Ca2+-specific sites (I and II) and the high affinity Ca2+-Mg2+ sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca2+ binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca2+ binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca2+ binding loop with Ala. The binding of these mutated proteins to TnC depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH2-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca2+-dependent force. This supports earlier findings that the low affinity Ca2+ binding sites (I and II) in TnC are responsible for the Ca2+ dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca2+ binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH2- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca2+ sensitivity of force development. PMID- 8626537 TI - Interaction between ryanodine and neomycin binding sites on Ca2+ release channel from skeletal muscle sarcoplasmic reticulum. AB - Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876 15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus. PMID- 8626538 TI - Molecular cloning of a novel diacylglycerol kinase isozyme with a pleckstrin homology domain and a C-terminal tail similar to those of the EPH family of protein-tyrosine kinases. AB - A fourth member of the diacylglycerol kinase (DGK) gene family termed DGK delta was cloned from the human testis cDNA library. The cDNA sequence contains an open reading frame of 3,507 nucleotides encoding a putative DGK protein of 130,006 Da. Interestingly, the new DGK isozyme contains a pleckstrin homology domain found in a number of proteins involved in signal transduction. Furthermore, the C-terminal tail of this isozyme is very similar to those of the EPH family of receptor tyrosine kinases. The primary structure of the delta-isozyme also has two cysteine-rich zinc finger-like structures (C3 region) and the C-terminal C4 region, both of which have been commonly found in the three isozymes previously cloned (DGKs alpha, beta and gamma). However, DGK delta lacks the EF-hand motifs (C2) and contains a long Glu- and Ser-rich insertion (317 residues), which divides the C4 region into two portions. Taken together, these structural features of DGK delta indicate that this isozyme belongs to a DGK subfamily distinct from that consisting of DGKs alpha, beta, and gamma. Increased DGK activity without marked preference to arachidonoyl type of diacylglycerol was detected in the particulate fraction of COS-7 cells expressing the transfected DGKdelta cDNA. The enzyme activity was independent of phosphatidylserine, which is a common activator for the previously sequenced DGKs. Northern blot analysis showed that the DGK delta mRNA (approximately 6.3 kilobases) is most abundant in human skeletal muscle but undetectable in the brain, thymus, and retina. This expression pattern is different from those of the previously cloned DGKs. Our results show that the DGK gene family consists of at least two subfamilies consisting of enzymes with distinct structural characteristics and that each cell type probably expresses its own characteristic repertoire of DGKs whose functions may be regulated through different signal transduction pathways. PMID- 8626539 TI - Binding specificity and modulation of the ApoA-I promoter activity by homo- and heterodimers of nuclear receptors. AB - Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand dependent nuclear receptors retinoic X receptor (RXRalpha), RXRalpha/RARalpha, and RXRalpha/T3Rbeta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRalpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T3R beta heterodimers repressed transcription to 60% of control, in the presence of T3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXRalpha homodimers, RXRalpha/RARalpha, and RXRalpha/T3Rbeta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXRalpha homodimers and RXRalpha/T3Rbeta heterodimers is associated with ligand-dependent activation by 9 cis-RA or repression by T3. Upon deletion or mutation of repeat 1, homodimeric binding of RXRalpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXRalpha heterodimers with repeat 2 are associated with low levels of ligand-independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene. PMID- 8626540 TI - Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system. AB - Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP B can function as sulfatide/globotriaosylceramide activator. PMID- 8626541 TI - Phosphotyrosine-independent binding of SHC to the NPLH sequence of murine protein tyrosine phosphatase-PEST. Evidence for extended phosphotyrosine binding/phosphotyrosine interaction domain recognition specificity. AB - The phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domain of the proto-oncoprotein p52SHC binds to an NPXpY consensus sequence found in several growth factor receptors (Kavanaugh, W. M., Turck, C. W., and Williams, L. T. (1994) Science 268, 1177-1179). The amino-terminal region of p52SHC, which includes the PTB/PI domain, has been previously shown to associate with protein tyrosine phosphatase-PEST (PTP-PEST) in vivo (Habib, T. , Herrera, R., and Decker, S. J. (1994) J. Biol. Chem. 269, 25243-25246). We report here the detailed mapping of this interaction in a murine context using glutathione S transferase fusion protein binding studies and peptide competition assays. We show that the interaction between murine SHC and murine PTP-PEST is mediated through the PTB/PI domain of murine SHC and an NPLH sequence found in the carboxyl terminus of murine PTP-PEST. Since this interaction is not dependent on the presence of a tyrosine-phosphorylated residue in the target sequence, this reveals that the PTB/PI domain of SHC can recognize both tyrosine-phosphorylated sequences and non-tyrosine-based recognition motifs. PMID- 8626542 TI - Phospholipid composition dependence of Ca2+-dependent phospholipid binding to the C2A domain of synaptotagmin IV. AB - Synaptotagmins I and II are Ca2+- and phospholipid-binding proteins of synaptic vesicles that may function as Ca2+ receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca2+-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca2+-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC50 values of approximately 5 and 120 microM free Ca2+ and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC50 of 0.3-1 microM free Ca2+; Hill coefficient of 3 3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca2+-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca2+ binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca2+ sensor for neurotransmitter release. PMID- 8626543 TI - Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G. AB - We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells. PMID- 8626544 TI - Degradation of amyloid beta-protein by a serine protease-alpha2-macroglobulin complex. AB - Progressive cerebral deposition of the amyloid beta-peptide (Abeta) is an early and constant feature of Alzheimer's disease. Abeta is derived by proteolysis from the beta-amyloid precursor protein. beta-Amyloid precursor protein processing and the generation of Abeta have been extensively characterized, but little is known about the mechanisms of degradation of this potentially neurotoxic peptide. We identified and purified a proteolytic activity in culture medium that can degrade secreted Abeta but not larger proteins in the medium. Detection of the activity in conditioned medium required the presence of fetal bovine serum and the passage of the cells with a pancreatic trypsin preparation. Its inhibitor profile showed that the activity was a serine protease other than trypsin or chymotrypsin. The protease occurs as a stable approximately 700-kDa complex with the inhibitor, alpha2-macroglobulin (alpha2M), that retains activity against small substrates such as Abeta. Its NH2-terminal sequence suggests that the protease is previously unidentified. Our results indicate that the Abeta-degrading protease we have detected is a non-trypsin component of a pancreatic trypsin preparation or else derives from a zymogen in serum that is activated by a protease in the latter preparation. Because Abeta-bearing plaques in Alzheimer's disease brain contain both alpha2M and receptors of alpha2M-protease complexes, the same or a similar alpha2M-protease complex could arise in vivo and play a role in Abeta clearance. PMID- 8626546 TI - Oxidized low density lipoprotein reduces thrombomodulin transcription in cultured human endothelial cells through degradation of the lipoprotein in lysosomes. AB - Oxidized low density lipoprotein (LDL), a potent atherogenic lipoprotein, has been shown to cause the alteration of various endothelial functions. We have examined the effect of oxidized LDL on the cofactor activity for thrombin dependent protein C activation and expression of thrombomodulin (TM), a cell surface antithrombotic glycoprotein, on cultured human umbilical vein endothelial cells. Oxidized LDL prepared by irradiation of LDL with 254-nm ultraviolet light did not directly affect the cofactor activity of isolated TM. Exposure of the cells to oxidized LDL (25-200 microg/ml), but not native LDL and acetylated LDL, reduced TM cofactor activity in parallel with its antigen levels on the cell surface in an oxidation-, concentration- and time-dependent manner. TM mRNA levels were reduced prior to decrease in TM antigen levels and were 50% of the control levels at 3.0 h after treatment of the cells with oxidized LDL. The apparent half-life time (t1/2 = 2.8 h) of TM mRNA in the oxidized LDL-treated cells, however, did not significantly differ from that (t1/2 = 2.6 h) in the control cells when the cells were coincubated with 5,6-dichloro-1-beta-D ribofuranosylbenzimidazole, a transcriptional inhibitor. Treatment of the cells with bafilomycin A1, an inhibitor for the proton pump of the lysosomes, inhibited intracellular degradation of the LDL and prevented down-regulations of the mRNA and the cell surface TM antigen levels caused by oxidized LDL. The inhibitor molecule in oxidized LDL was shown to be a lipid; organic solvent extracts (300 mg/ml cholesterol, an equivalent concentration with lipids in 200 microg/ml oxidized LDL) of oxidized LDL inhibited expression of TM antigen to nearly the same extent as the oxidized LDL, although water extracts did not affect TM expression on the cells. These results suggested that down-regulation of TM on endothelial cells exposed to oxidized LDL resulted from inhibition of its transcription mediated by lysosomal degradation of oxidized LDL and that a lipid component in the LDL could be an active species. A decrease in TM expression on the surface of endothelial cells may contribute to promote thrombosis in atherosclerotic lesions. PMID- 8626545 TI - Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes. AB - In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene. PMID- 8626547 TI - C-terminal trimerization, but not N-terminal trimerization, of the reovirus cell attachment protein Is a posttranslational and Hsp70/ATP-dependent process. AB - The C-terminal globular head of the lollipop-shaped final sigma1 protein of reovirus is responsible for interaction with the host cell receptor. Like the N terminal fibrous tail, it has its own trimerization domain. Whereas N-terminal trimerization (formation of a triple alpha-helical coiled coil) occurs at the level of polysomes (i.e. cotranslationally) and is ATP-independent, C-terminal trimerization is a posttranslational event that requires ATP. Coprecipitation experiments using anti-Hsp70 antibodies and truncated final sigma1 proteins synthesized in vitro revealed that only regions downstream of the N-terminal alpha-helical coiled coil were associated with Hsp70. Hsp70 was also found to be associated with nascent final sigma1 chains on polysomes as well as with immature postribosomal final sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini). These latter structures were true intermediates in the final sigma1 biogenetic pathway since they could be chased into mature final sigma1 trimers with the release of Hsp70. Thus, unlike N terminal trimerization, C-terminal trimerization is Hsp70- and ATP-dependent. The involvement of two mechanistically distinct oligomerization events for the same molecule, one cotranslational and one posttranslational, may represent a common approach to the generation of oligomeric proteins in the cytosol. PMID- 8626548 TI - Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells. AB - Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf 1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo. PMID- 8626549 TI - Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer. AB - We have developed a lipidic gene transfer vector, LPDII, where DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and then entrapped into folate targeted pH-sensitive anionic liposomes composed of dioleoyl phosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate/folate-polyethlene glycol-DOPE (6:4:0.01 mol/mol) via charge interaction. LPDII transfection of KB cells, a cell line overexpressing the tumor marker folate receptor, was affected by both the lipid to DNA ratio and the lipid composition. At low lipid to DNA ratios (e.g. 4 and 6), LPDII particles were positively charged; transfection and cellular uptake levels were independent of the folate receptor and did not require a pH-sensitive lipid composition. Meanwhile, transfection and uptake of negatively charged LPDII particles, i.e. those with high lipid to DNA ratios (e.g. 10 and 12), were folate receptor-dependent and required a pH-sensitive lipid composition. The transfection activity of LPDII was lost when the inverted cone-shaped DOPE was replaced by dioleoyl phosphatidylcholine. LPDII particles with lipid to DNA ratios of 4, 6, 10, and 12 were approximately 20-30 times more active than DNA.3-beta-[N-(N',N'-dimethylethane)carbamoyl]cholesterol cationic liposome complexes in KB cells and were much less cytotoxic. On the sucrose gradient, LPDII particles had a migration rate in between those of the free DNA and the DNA.polylysine complex. An electron micrograph of LPDII showed a structure of spherical particles with a positively stained core enclosed in a lipidic envelope with a mean diameter of 74 +/- 14 nm. This novel gene transfer vector may potentially be useful in gene therapy for tumor-specific delivery. PMID- 8626550 TI - Identification of mitogen-activated protein (MAP) kinase-activated protein kinase 3, a novel substrate of CSBP p38 MAP kinase. AB - CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase 3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo. PMID- 8626551 TI - Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway. AB - In both cell culture based model systems and in the failing human heart, beta adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3' untranslated region mRNA. PMID- 8626552 TI - The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G. AB - A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit. [14C]N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid. A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening. Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16 kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned. In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels. The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to the Vma10p protein, which behaved like a V0 subunit, the M. sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase. We designate this new V1 component subunit G. PMID- 8626553 TI - Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. AB - The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs. PMID- 8626554 TI - Computer simulation of the triosephosphate isomerase catalyzed reaction. AB - A major challenge for theoretical simulation methods is the calculation of enzymic reaction rates directly from the three-dimensional protein structure together with some idea of the chemical reaction mechanism. Here, we report the evaluation of a complete free energy profile for all the elementary steps of the triosephosphate isomerase catalyzed reaction using such an approach. The results are compatible with available experimental data and also suggest which of the possible reaction intermediates is kinetically observable. In addition to previously identified catalytic residues, the simulations show that a crystallographically observed active site water molecule plays an important role during catalysis and an intersubunit interaction that could explain the low activity of the monomeric enzyme is also observed. The calculations clearly demonstrate the important catalytic effects associated with stabilization of charged high energy intermediates and reduction of reorganization energy, which are likely to be general principles of enzyme catalyzed charge transfer and separation reactions. PMID- 8626555 TI - Reduction of BiP levels decreases heterologous protein secretion in Saccharomyces cerevisiae. AB - Increased levels of the endoplasmic reticulum-resident protein folding chaperone BiP would be expected to either increase protein secretory capacity by improved solubilization of folding precursors or decrease secretory capacity by binding and retaining misfolded proteins. To address this question, the relationship between BiP levels and heterologous secretion in yeast was determined. A yeast strain was constructed in which BiP expression is tunable from 5 to 250% of wild type levels, and this strain was used to explore the effect of varying BiP level on overall secretion of three heterologous proteins: human granulocyte colony stimulating factor, Schizosaccharomyces pombe acid phosphatase, and bovine pancreatic trypsin inhibitor. For all three proteins examined, reduction in BiP expression below wild-type level diminished overall secretion, whereas 5-fold BiP overexpression from a constitutive glycolytic promoter did not substantially increase or decrease secretion titers. These results are consistent with a positive role for BiP in promoting membrane translocation and solubilization of folding precursors but are inconsistent with a negative role in proofreading and improper retention of heterologous secreted proteins. PMID- 8626556 TI - Growth-associated protein-43 (GAP-43) facilitates peptide hormone secretion in mouse anterior pituitary AtT-20 cells. AB - The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP 43-expressing AtT-20 cells without a significant change in Ca2+ influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43. PMID- 8626557 TI - Sugar binding to Na+/glucose cotransporters is determined by the carboxyl terminal half of the protein. AB - d-Glucose is absorbed across the proximal tubule of the kidney by two Na+/glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na+:glucose coupling ratio of 1, a K0.5 for sugar of approximately 2 mM, and a K0.5 for Na+ of approximately 1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K0.5 for sugar and Na+ of approximately 0.2 and 5 mM, respectively. We have constructed a chimeric protein consisting of amino acids 1-380 of porcine SGLT2 and amino acids 381-662 of porcine SGLT1. The chimera was expressed in Xenopus oocytes, and steady-state kinetics were characterized by a two-electrode voltage-clamp. The K0.5 for alpha methyl-d-glucopyranoside (0.2 mM) was similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2 transports poorly D-galactose and excludes 3-O-methylglucose. The apparent K0.5Na was 3.5 mM (at -150 mV), and the Hill coefficient ranged between 0.8 and 1.5. We conclude that recognition/transport of organic substrate is mediated by interactions distal to amino acid 380, while cation binding is determined by interactions arising from the amino- and carboxyl-terminal halves of the transporters. Surprisingly, the chimera transported alpha-phenyl derivatives of D glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and beta-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K0.5 for phlorizin was 5 microM). Thus, the pocket for organic substrate binding is increased from 10 x 5 x 5 (A) for SGLT1 to 11 x 18 x 5 (A) for the chimera. PMID- 8626558 TI - Assessment of the ATP binding properties of Hsp90. AB - Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90. PMID- 8626559 TI - The ntpJ gene in the Enterococcus hirae ntp operon encodes a component of KtrII potassium transport system functionally independent of vacuolar Na+-ATPase. AB - The ntpJ gene, the tail end in the vacuolar type Na+-ATPase (ntp) operon of Enterococcus hirae, encodes a putative 49-kDa hydrophobic protein resembling K+ transporter protein in Saccharomyces cerevisiae (Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044). Northern blotting experiment revealed that the ntpJ gene was transcribed as a cistron in the ntp operon. We constructed an Enterococcus strain in which the ntpJ gene was disrupted by cassette mutagenesis with erythromycin resistance gene. The growth of this mutant was normal at low pH. However, the mutant did not grow at high pH in K+-limited medium (less than 1 mM), while the wild type strain grew well; the internal K+ concentration of this mutant was as low as 7% of that of the wild type strain, suggesting that the K+ accumulation at high pH was inactivated by disruption of the ntpJ gene. Potassium uptake activity via the KtrII system, which had been proposed as the proton potential independent, Na+-ATPase-coupled system working at high pH (Kakinuma, Y., and Harold, F. M. (1985) J. Biol. Chem. 260, 2086-2091), was missing in this mutant strain. However, this mutant retained as high activities of Na+-ATPase and Na+ pumping as the wild type strain. From these results, we conclude that the NtpJ is a membraneous component of the KtrII K+ uptake system but not a functional subunit of vacuolar Na+-ATPase complex; the interplay between the KtrII system and the Na+-ATPase was discussed. PMID- 8626560 TI - Intracellular polymerization of the serpin plasminogen activator inhibitor type 2. AB - Plasminogen activator inhibitor type 2 (PAI-2) is synthesized in two molecular forms: an intracellular, nonglycosylated form and an extracellular, glycosylated form. The bitopological distribution of PAI-2 is caused by an inefficient internal secretion signal. In addition, the secretion efficiency of PAI-2 seems to differ, depending on the cell type, differentiation state, and culture conditions. In recombinant cell clones designed for the synthesis of the secreted form of PAI-2, the fraction of secreted PAI-2 decreased with increasing expression levels. Subcellular fractionation of cell clones with higher expression levels revealed that PAI-2 accumulating in the cell was mainly associated with the organelles of the secretory pathway. Electrophoresis under nondenaturating conditions revealed that the PAI-2 retained at higher expression levels was mainly polymerized. Polymers of PAI-2 were also detected in cytosolic extracts prepared from human placenta and phorbol ester-stimulated U 937 cells, indicating that intracellular polymerization of PAI-2 may occur in the cytosols of cells that normally express PAI-2 under physiological conditions. When purified PAI-2 or cellular extracts were incubated at 37 degrees C for 24 h most of the PAI-2 protein was found to polymerize. Polymer formation was prevented by the addition of synthetic peptides with sequences corresponding to residues P2 to P14 in the reactive center loop of PAI-2 and antithrombin. These synthetic peptides also caused dissociation of prepolymerized purified PAI-2 and PAI-2 polymers in cellular extracts. Incubation with unrelated peptides of the same size had no effect on polymer formation or dissociation of preformed polymers, indicating that polymerization of PAI-2 occurs by the loop-sheet mechanism. Taken together, our data suggest that the wild-type form of PAI-2, like some natural pathological genetic variants of alpha1-antitrypsin, antithrombin, and C1 inhibitor readily polymerizes intracellularly and that polymerization may lead to a reduced secretion efficiency. PMID- 8626561 TI - Association between mitogen-activated protein kinase and the zeta chain of the T cell receptor (TcR) with the SH2,3 domain of p56lck. Differential regulation by TcR cross-linking. AB - A number of protein-tyrosine kinases have been shown to be important in T cell activation. One such kinase, Lck, has been demonstrated genetically to be essential for T cell receptor (TcR) signaling, and the SH2 and SH3 (src homology 2 and 3) domains of Lck have been shown to be indispensable for T cell activation. We have sought substrates with which the SH2,3 domain would interact following T cell activation, using fusion proteins containing the Lck SH2 and SH3 domains linked to glutathione S-transferase. We demonstrate that the SH2,3 region interacts specifically and directly with numerous tyrosine-phosphorylated molecules following TcR cross-linking, including constitutively with mitogen activated protein kinase (MAPK)/extracellular-regulated kinase and inducibly with the zeta chain of the TcR. The interaction with MAPK/extracellular-regulated kinase was via the SH3 domain. The interaction with the tyrosine-phosphorylated zeta chain, while phosphotyrosine-dependent, required both the SH3 and SH2 domains. These interactions were specific as molecules known to be tyrosine phosphorylated following TcR cross-linking, phospholipase C-gamma1 and Fyn, were not bound. Thus, we suggest that during TcR signaling, Lck interacts with numerous molecules, including MAPK and TcR-zeta, via its SH2,3 domain. The interaction with MAPK would place Lck in a position to be involved in the complex resulting in the activation of MAPK. In addition, the binding of Lck to the tyrosine-phosphorylated zeta chain of the TcR would serve to strengthen the interaction of the associated CD4 and the TcR complex, leading to increased avidity for the antigen-major histocompatibility protein complex. PMID- 8626562 TI - Glycine betaine fluxes in Lactobacillus plantarum during osmostasis and hyper- and hypo-osmotic shock. AB - Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor. The primary response of Lactobacillus plantarum to an osmotic upshock involves the accumulation of compatible solutes such as glycine betaine, proline, and glutamate. We have studied the osmotic regulation of glycine betaine transport in L. plantarum by measuring the overall and unidirectional rates of glycine betaine uptake and exit at osmostasis, and under conditions of osmotic upshock and downshock. At steady state conditions, a basal flux of glycine betaine (but no net uptake or efflux) is observed that amounts to about 20% of the rate of "activated"' uptake (uptake at high osmolarity). No direct exchange of 14C-labeled glycine betaine in the medium for unlabeled glycine betaine in the cytoplasm was observed in glucose metabolizing and resting cells, indicating that a separate glycine betaine efflux system is responsible for the exit of glycine betaine. Upon osmotic upshock, the uptake system for glycine betaine is rapidly activated (within seconds), whereas the basal efflux is inhibited. These two responses account for a rapid accumulation of glycine betaine until osmostasis is reached. Upon osmotic downshock, glycine betaine is rapidly released by the cells in a process that has two kinetic components, i.e. one with a half-life of less than 2 s which is unaffected by the metabolic status of the cells, the other with a half-life of 4 5 min in glucose-metabolizing cells which is dependent on internal pH or a related parameter. We speculate that the former activity corresponds to a stretch activated channel, whereas the latter may be facilitated by a carrier protein. Glycine betaine uptake is strongly inhibited immediately after an osmotic downshock, but slowly recovers in time. These studies demonstrate that in L. plantarum osmostasis is maintained through positive and negative regulation of both glycine betaine uptake and efflux, of which activation of uptake upon osmotic upshock and activation of a "channel-like" activity upon osmotic downshock are quantitatively most important. PMID- 8626563 TI - In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. AB - Continuous replication of human immunodeficiency virus type 1 requires the expression of the regulatory protein Rev, which binds to the Rev response element (RRE) and up-regulates the cytoplasmic appearance of singly spliced and unspliced mRNA species. It has been demonstrated that the murine protein YL2 interacts with Rev in vivo and modulates the activity of Rev (Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850-3856). Here we show that the YL2 human homologue, the p32 protein, which co-purifies with alternative splicing factor ASF/SF2, interacts directly with the basic domain of Rev in vitro and that the Rev-p32 complex is resistant to high concentrations of salt or nonionic detergent. Protein footprinting data suggest that Rev interacts specifically with amino acids within the 196-208 region of p32. An analysis of the ternary complex, formed among p32, Rev, and RRE RNA, shows that Rev can bridge the association of p32 and RRE. Furthermore, we demonstrate that exogenously added p32 specifically relieves the inhibition of splicing in vitro exerted by the basic domain of Rev. Our data are consistent with a model in which p32 functions as a link between Rev and the cellular splicing apparatus. PMID- 8626564 TI - A stereospecific myo-inositol/D-chiro-inositol transporter in HepG2 liver cells. Identification with D-chiro-[3-3H]inositol. AB - D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian glycosylphosphatidylinositol protein anchors and inositol phosphoglycans possessing insulin-like bioactivity. In order to generate a probe for metabolic studies, D-chiro-[3-3H]inositol was synthesized by selective reduction of D-chiro 3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-3H]Inositol was taken up by HepG2 human liver cells through a saturable and stereospecific pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. Dd-Glucose, but not L-glucose, competed for D-chiro-[3 3H]inositol uptake over glucose concentrations of 4-28 mM. Maximum transport capacity was 717 pmol/mg cell protein/3 h with a Km value of 348 microM. Uptake was reduced by 76% when sodium was eliminated from the medium and by 94% when the experiment was performed at 0 degrees C. The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol. PMID- 8626565 TI - The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. AB - Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor. PMID- 8626566 TI - Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis. AB - Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E) GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP. PMID- 8626567 TI - Adenosine deaminase inhibition prevents free radical-mediated injury in the postischemic heart. AB - In the presence of its substrates hypoxanthine and xanthine, xanthine oxidase generates oxygen free radicals that cause postischemic injury. Recently, it has been demonstrated that the burst of xanthine oxidase-mediated free radical generation in the reperfused heart is triggered by a large increase in substrate formation, which occurs secondary to the degradation of adenine nucleotides during ischemia. It is not known, however, whether blocking this substrate formation is sufficient to prevent radical generation and functional injury. Therefore, studies were performed in isolated rat hearts in which xanthine oxidase substrate formation was blocked with the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and measurements of contractile function and free radical generation were performed. Chromatographic measurements of the intracellular adenine nucleotide pool showed that preischemic administration of EHNA blocked postischemic hypoxanthine, xanthine, and inosine formation. Electron paramagnetic resonance spin trapping measurements of free radical generation showed that inhibition of adenosine deaminase with EHNA blocked free radical generation and that it also increased the recovery of contractile function by more than 2-fold. Exogenous infusion of hypoxanthine and xanthine totally reversed the protective effects of EHNA. These results demonstrate that blockade of xanthine oxidase substrate formation by adenosine deaminase inhibition can prevent free radical generation and contractile dysfunction in the postischemic heart. PMID- 8626568 TI - Interaction of nucleotides with the NAD(H)-binding domain of the proton translocating transhydrogenase of Rhodospirillum rubrum. AB - Transhydrogenase catalyzes the reduction of NADP+ by NADH coupled to the translocation of protons across a membrane. The polypeptide composition of the enzyme in Rhodospirillum rubrum is unique in that the NAD(H)-binding domain (called Ths) exists as a separate polypeptide. Ths was expressed in Escherichia coli and purified. The binding of nucleotide substrates and analogues to Ths was examined by one-dimensional proton nuclear magnetic resonance (NMR) spectroscopy and by measuring the quenching of fluorescence of its lone Trp residue. NADH and reduced acetylpyridine adenine dinucleotide bound tightly to Ths, whereas NAD+, oxidized acetylpyridine adenine dinucleotide, deamino-NADH, 5'-AMP and adenosine bound less tightly. Reduced nicotinamide mononucleotide, NADPH and 2'-AMP bound only very weakly to Ths. The difference in the binding affinity between NADH and NAD+ indicates that there may be an energy requirement for the transfer of reducing equivalents into this site in the complete enzyme under physiological conditions. Earlier results had revealed a mobile loop at the surface of Ths (Diggle, C., Cotton, N. P. J., Grimley, R. L., Quirk, P. G., Thomas, C. M., and Jackson, J. B. (1995) Eur. J. Biochem. 232, 315-326); the loop loses mobility when Ths binds nucleotide; the reaction involves two steps. This was more clearly evident, even for tight-binding nucleotides, when experiments were carried out at higher temperatures (37 degrees C), where the resonances of the mobile loop were substantially narrower. The binding of adenosine was sufficient to initiate loop closure; the presence of a reduced nicotinamide moiety in the dinucleotide apparently serves to tighten the binding. Two-dimensional 1H NMR spectroscopy of the Ths-5'-AMP complex revealed nuclear Overhauser effect interactions between protons of amino acid residues in the mobile loop (including those in a Tyr residue) and the nucleotide. This suggests that, in the complex, the loop has closed down to within 0.5 nm of the nucleotide. PMID- 8626569 TI - Mutation of Tyr235 in the NAD(H)-binding subunit of the proton-translocating nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum affects the conformational dynamics of a mobile loop and lowers the catalytic activity of the enzyme. AB - The Tyr residue in the mobile loop region of the soluble, domain I polypeptide (called Ths) of the proton-translocating transhydrogenase from Rhodospirillum rubrum has been substituted by Asn and by Phe. The recombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity. The two well defined resonances at 6.82 and 7.12ppm, observed in the one-dimensional proton NMR spectrum of wild-type protein, and previously attributed to the Tyr residue, were absent in both mutants. In the Tyr235 --> Phe mutant Ths, they were replaced by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue. In both mutants, narrow resonances attributable to Met residues (and in the Tyr235 --> Phe mutant, resonances attributable to Ala residues) were shifted relative to the wild type, but other features in the NMR spectra were unaffected. The conformational dynamics of the mobile loop closure in response to nucleotide binding by the protein were altered in the two mutants. The fluorescence emission from Trp72 was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH. The mutant Ths proteins bound to chromatophore membranes depleted of their native Ths with undiminished affinity. In these reconstituted systems, the Km values for thio-NADP+ and NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the kcat values were decreased about 2-fold. In reverse transhydrogenation, the Kmvalues for NADPH were slightly decreased in the mutants relative to wild-type, but those for acetyl pyridine adenine dinucleotide were increased about 10- and 13-fold, respectively, and the kcat values were decreased about 2- and 5-fold, respectively, in the Tyr235 --> Phe and Tyr235 --> Asn mutants. It is concluded that Tyr235 may contribute to the process of nucleotide binding and that substitution of this residue prevents proper functioning of the mobile loop in catalysis. PMID- 8626570 TI - NK-lysin, a disulfide-containing effector peptide of T-lymphocytes, is reduced and inactivated by human thioredoxin reductase. Implication for a protective mechanism against NK-lysin cytotoxicity. AB - The cytotoxic and antibacterial polypeptide NK-lysin has a molecular mass of approximately 9 kDa and contains three disulfide bonds. The activity was highly dependent on intact disulfides, because the bactericidal effect on Escherichia coli and the cytolytic effect on human 3B6 lymphocytes was inhibited when NK lysin was treated with dithiothreitol prior to incubation with the cells. NK lysin was a direct substrate for human or calf thymus thioredoxin reductase and preincubation of the peptide with mammalian thioredoxin reductase, and NADPH abolished its antibacterial and cytolytic activities. The addition of human thioredoxin further enhanced the inhibitory effect of thioredoxin reductase and NADPH. In contrast, e. coli thioredoxin reductase showed no direct disulfide reductase activity with NK-lysin in agreement with previous data showing large differences in structure and substrate specificity between the mammalian and E. coli enzymes. NK-lysin is the first identified macromolecular disulfide substrate for human thioredoxin reductase apart from human thioredoxin. When 3B6 cells were incubated with NADPH, thioredoxin, and thioredoxin reductase prior to addition of NK-lysin, cytotoxicity was markedly reduced. These data suggest that thioredoxin reductase inactivates NK-lysin and provides a mechanism by which the cytotoxic activity of NK-lysin is regulated. PMID- 8626571 TI - Phosphorylation of Vif and its role in HIV-1 replication. AB - Vif is a 23-kDa protein encoded by human immunodeficiency virus, type 1 (HIV-1) which is important for virion infectivity. Here, we describe the phosphorylation of HIV-1 Vif and its role in HIV-1 replication. In vivo studies demonstrated that Vif is highly phosphorylated on serine and threonine residues. To identify phosphorylation sites and characterize the Vif kinase(s), Vif was expressed in Escherichia coli and purified for use as a substrate in in vitro kinase assays. The purified Vif protein was phosphorylated in vitro on serine and threonine residues by a kinase(s) present in both cytosol and membrane fractions. Phosphorylation of Vif was stimulated by phorbol 12-myristate 13-acetate and inhibited by staurosporine and hypericin, a drug with potent anti-HIV activity. The Vif kinase(s) was resistant to inhibitors of protein kinase C, cAMP-dependent kinase, and cGMP-dependent kinase, suggesting that it is distinct from these enzymes. To identify the phosphorylation sites, 32P-labeled Vif was digested by V8 protease and the peptides were resolved by reverse-phase high performance liquid chromatography. Radioactive peptide sequencing identified three phosphorylation sites within the C terminus, Ser144, Thr155, and Thr188. Two dimensional tryptic phosphopeptide mapping indicated that these sites are also phosphorylated in vivo. Both Ser144 and Thr188 are contained in the recognition motifs (R/KXXS*/T* and R/KXXXS*/T*) used by serine/threonine protein kinases such as cGMP-dependent kinase and PKC. Ser144 is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins. Mutation of Ser144 to alanine resulted in loss of Vif activity and >90% inhibition of HIV-1 replication. These studies suggest that phosphorylation of Vif by a serine/threonine protein kinase(s) plays an important role in regulating HIV-1 replication and infectivity. PMID- 8626572 TI - Stability of the heme-globin linkage in alphabeta dimers and isolated chains of human hemoglobin. A study of the heme transfer reaction from the immobilized proteins to albumin. AB - The stability of the heme-globin linkage in alphabeta dimers and in the isolated chains of human hemoglobin has been probed by studying the transfer of heme from the proteins immobilized onto CNBr-activated Sepharose 4B to human albumin. The kinetic and equilibrium features of the reaction have been measured spectrophotometrically given the stability of the heme donors and the ease with which heme donor and acceptor can be separated. Isolated alpha and beta chains transfer heme to albumin at similar rates (1 6 x 10(-2) s-1 at pH 9.0 and 20 degrees C) in the ferrous CO-bound and in the ferric state. In alpha beta dimers the heme-globin linkage is strengthened considerably, albeit to a different extent in the ferrous CO-bound and ferric met-aquo derivatives. Only in the latter heme is lost at a measurable rate, 0.065 +/- 0.011 x 10(-2) s-1 for alpha heme and 2.8 +/- 0.6 x 10(-2) s-1 for beta heme at pH 9.0 and 20 degrees C, which is very close to the rate measured with soluble met-aquo-hemoglobin at micromolar concentrations. These results indicate that in human hemoglobin the heme-globin linkage in the alpha chains is stabilized by interactions between unlike chains at the alpha1 beta1 interface, whereas heme binding to the beta chains is stabilized by interactions at the alpha1beta2 interface. These long range factors have to be taken into account in addition to the local factors at the heme pocket when evaluating the effect of point mutation and chemical modification. PMID- 8626573 TI - Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease. AB - PIM1 protease in mitochondria belongs to a conserved family of ATP-dependent proteases, which includes the Escherichia coli Lon protease. Yeast cells lacking PIM1 are largely defective in degrading misfolded proteins in the mitochondrial matrix, are respiratory deficient, and lose integrity of mitochondrial DNA. In order to analyze whether E. coli Lon protease is functionally equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a mitochondrial targeting sequence fused onto the Lon protease. In these cells, the fusion protein was expressed and imported into mitochondria, and the targeting sequence was removed. In the absence of PIM1 protease, the E. coli Lon protease mediated the degradation of misfolded proteins in the matrix space in cooperation with the mitochondrial hsp70 system. These cells maintained the integrity of the mitochondrial genome and the respiratory function at 30 degrees C but not at 37 degrees C. Stabilization of mitochondrial DNA in Deltapim1 cells depended on protein degradation by the E. coli Lon protease, as a proteolytically inactive Lon variant was not capable of substituting for a loss of PIM1 protease. These results demonstrate functional conservation of Lon-like proteases from prokaryotes to eukaryotes and shed new light on the role of Lon-like proteases in mitochondrial biogenesis. PMID- 8626575 TI - Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation. AB - The lethal toxin (LT) from Clostridium sordellii belongs to the family of large clostridial cytotoxins causing morphological alterations in cultured cell lines accompanied by destruction of the actin cytoskeleton. C. sordellii LT exhibits 90% homology to Clostridium difficile toxin B, which has been recently identified as a monoglucosyltransferase (Just, I., Selzer, J., Wilm, M., von Eichel Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). We report here that LT too is a glucosyltransferase, which uses UDP-glucose as cosubstrate to modify low molecular mass GTPases. LT selectively modifies Rac and Ras, whereas the substrate specificity of toxin B is confined to the Rho subfamily proteins Rho, Rac, and Cdc42, which participate in the regulation of the actin cytoskeleton. In Rac, both toxin B and LT share the same acceptor amino acid, threonine 35. Glucosylation of Ras by LT results in inhibition of the epidermal growth factor-stimulated p42/p44 MAP-kinase signal pathway. LT is the first bacterial toxin to inactivate Ras in intact cells. PMID- 8626574 TI - Involvement of G protein-coupled receptor kinase 5 in homologous desensitization of the thyrotropin receptor. AB - Homologous desensitization of G protein-coupled receptors involves agonist dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs). To identify GRK(s) that play a role in homologous desensitization of the thyrotropin (TSH) receptor, thyroid cDNA was amplified by polymerase chain reaction using degenerate oligonucleotide primers from highly conserved regions in GRK family. GRK5 is found in the predominant isoform expressed in the thyroid. Rat GRK5 cDNA was then isolated, which encodes a 590-amino acid protein with 95% homology to human and bovine homologs. Northern blot identified GRK5 mRNA of approximately 3, 8, and 10 kilobases with highest expression levels in lung > heart, kidney, colon > thyroid. In functional studies using a normal rat thyroid FRTL5 cells, overexpression of GRK5 by transfecting the plasmid capable of expressing the sense GRK5 RNA suppressed basal cAMP levels and augmented the extent of TSH receptor desensitization, whereas suppression of endogenous GRK5 expression by transfecting the antisense GRK5 construct increased basal cAMP levels and attenuated the extent of receptor desensitization. Although exogenously overexpressed GRK6 also enhanced TSH receptor desensitization, we conclude that GRK5, the predominant GRK isoform in the thyroid, appears to be mainly involved in homologous desensitization of the TSH receptor. PMID- 8626576 TI - Neurotrophin-3 increases intracellular calcium in a rat insulin-secreting cell line through its action on a functional TrkC receptor. AB - Pancreatic beta cells and neuronal cells show a large number of similarities. For example, functional receptors for nerve growth factor are present in beta cells. Here we investigate whether TrkC, a neuronal high affinity receptor for neurotrophin-3, is expressed in the insulin-secreting cell line INS-1. We demonstrate the expression in INS-1 cells of mRNAs coding for TrkC identical in size to those found in the brain. As in neuronal cells, different alternatively spliced forms of TrkC mRNA, differing by the insertion of an alternative exon in their kinase domain, were expressed in INS-1 cells. TrkC protein is also expressed in INS-1 cells and is functional. Indeed, when INS-1 cells were treated with neurotrophin-3, TrkC became phosphorylated on tyrosine residues, and the expression of early response genes was induced. This activation of the receptor was paralleled by a rapid and transient increase in cytosolic free calcium due to an influx of extracellular calcium. Functional receptors for NT-3 are thus expressed in INS-1 cells. This cell line provides a new model for the study of NT 3 signal transduction and should be useful in the understanding of the role of neurotrophins in insulin-secreting cells. PMID- 8626577 TI - Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition. AB - In the present study we examine the role of transmembrane aromatic residues of the delta-opioid receptor in ligand recognition. Three-dimensional computer modeling of the receptor allowed to identify an aromatic pocket within the helices bundle which spans transmembrane domains (Tms) III to VII and consists of tyrosine, phenylalanine, and tryptophan residues. Their contribution to opioid binding was assessed by single amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm IV), F218A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII). Scatchard analysis shows that mutant receptors, transfected into COS cells, are expressed at levels comparable with that of the wild-type receptor. Binding properties of a set of representative opioids were examined. Mutations at position 129 most dramatically affected the binding of all tested ligands (up to 430-fold decrease of deltorphin II binding at Y129A), with distinct implication of the hydroxyl group and the aromatic ring, depending on the ligand under study. Affinity of most ligands was also reduced at Y308F mutant (up to 10-fold). Tryptophan residues seemed implicated in the recognition of specific ligand classes, with reduced binding for endogenous peptides at W173A mutant (up to 40 fold) and for nonselective alkaloids at W274A mutant (up to 65-fold). Phenylalanine residues in Tm V appeared poorly involved in opioid binding as compared with other aromatic amino acids examined. Generally, the binding of highly selective delta ligands (TIPPpsi, naltrindole, and BW373U86) was weakly modified by these mutations. Noticeably, TIPPpsi binding was enhanced at W274A receptor by 5-fold. Conclusions from our study are: (i) aromatic amino acid residues identified by the model contribute to ligand recognition, with a preponderant role of Y129; (ii) these residues, which are conserved across opioid receptor subtypes, may be part of a general opioid binding domain; (iii) each ligand-receptor interaction is unique, as demonstrated by the specific binding pattern observed for each tested opioid compound. PMID- 8626578 TI - Oxidative stress increases production of beta-amyloid precursor protein and beta amyloid (Abeta) in mammalian lenses, and Abeta has toxic effects on lens epithelial cells. AB - Many amyloid diseases are characterized by protein aggregations linked to oxidative stress. Such diseases including those of the brain, muscle, and blood vessels exhibit plaques containing beta-amyloid (Abeta). Here we demonstrate that Alzheimer's precursor protein (betaAPP) and A beta are present at low levels in normal lenses and increase in intact cultured monkey lenses treated with H2O2 or UV radiation (known cataractogenic agents), and with phorbol 12-myristate 13 acetate. AP-1 factor binding, shown by others to up-regulate betaAPP expression, increased in the monkey lenses treated with H2O2, UV radiation, or phorbol 12 myristate 13-acetate and paralleled the increase in betaAPP expression. Rat lenses exposed to oxidative stress showed increased betaAPP in the anterior epithelium and cortex. Incubation of cultured rabbit lens N/N1003A epithelial cells with Abeta induced inclusions and vacuoles and was cytotoxic. Abeta cross reacting protein was readily detected in the cortex of a cataractous human lens. Our data show that betaAPP and Abeta increase in mammalian lenses as part of a response to H2O2 or UV radiation and suggest that they may contribute to the mechanism by which oxidative damage leads to lens opacification. PMID- 8626579 TI - Functional malleability of the carboxyl-terminal tail in protein kinase A. AB - The catalytic (C) subunit of protein kinase A (PKA) is regarded as a framework for the protein kinase family. Its sequence is composed of a conserved core (residues 40 300) between two segments at the amino and carboxyl termini of the protein. Since the various protein kinases differ in their specificity, it seems reasonable to assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of acidic amino acids (328DDYEEEE334) at the carboxyl terminal "tail" of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three complementary lines of evidence: (i) on a conformation-sensitive cleavage of the C subunit by a kinase-splitting membranal proteinase that specifically recognizes this cluster, to demonstrate the occurrence in solution of "open" (cleavable) and "closed" (noncleavable) conformations of the C subunit; (ii) on analysis of the three-dimensional structures of the open and closed conformations of the C subunit, showing an approximately 7-A movement of the phenolic hydroxyl of Tyr330 to reach (in the closed conformation) an approximately 3-A distance from the nitrogen atoms of the Arg residue at position p-3 of the PKA consensus sequence; and (iii) on single site mutations of the C subunit (e.g. Y330A) that show a significant contribution of Tyr330 to the Km of PKA for its substrates/inhibitors and to its catalytic efficacy (Vmax/Km). PMID- 8626580 TI - A novel Ca2+-binding protein, p22, is required for constitutive membrane traffic. AB - We have identified a novel protein, p22, required for "constitutive" exocytic membrane traffic. p22 belongs to the EF-hand superfamily of Ca2+-binding proteins and shows extensive similarity to the regulatory subunit of protein phosphatase 2B, calcineurin B. p22 is a cytosolic N-myristoylated protein that undergoes conformational changes upon binding of Ca2+. Antibodies against a p22 peptide block the targeting/fusion of transcytotic vesicles with the apical plasma membrane, but recombinant wild-type p22 overcomes that inhibition. Nonmyristoylated p22, or p22 incapable of undergoing Ca2+-induced conformational changes, cannot reverse the antibody-mediated inhibition. The data suggest that p22 may act by transducing cellular Ca2+ signals to downstream effectors. p22 is ubiquitously expressed, and we propose that its function is required for membrane trafficking events common to many cells. PMID- 8626581 TI - Transforming growth factors beta1, beta2, and beta3 and their receptors are differentially regulated during normal and impaired wound healing. AB - A series of studies has shown that application of transforming growth factor beta (TGF-beta) to a wound has a beneficial effect, especially in animals with wound healing disorders. In this study we have investigated the regulation of TGF beta1, beta2, and beta3 and their receptors during the repair process. We found a large induction of all three TGF-beta isoforms and also of TGF-beta types I and II receptors, although the time course of induction and the absolute expression levels were different for these genes. Furthermore, each TGF-beta isoform had distinct sites of expression in the wound. Systemic treatment with glucocorticoids significantly altered the expression levels of TGF-betas and TGF beta receptors. Whereas expression of TGF-beta1, TGF-beta2, and TGF-beta type II receptor was suppressed by glucocorticoids in normal and wounded skin, expression of TGF-beta3 and TGF-beta receptor type I mRNA was stimulated. These findings provide an explanation for the beneficial effect of exogenous TGF-beta in the treatment of impaired wound healing in glucocorticoid-treated animals. Furthermore, they suggest that a disturbed balance between the levels of the three TGF- beta isoforms and their receptors might underlie the wound healing defect seen in glucocorticoid-treated animals. PMID- 8626582 TI - Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells. AB - Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation. PMID- 8626583 TI - Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. AB - Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3 kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS 1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane. PMID- 8626584 TI - Suppression of apoptosis by dominant negative mutants of cyclin-dependent protein kinases. AB - In many cell types, position in the cell cycle appears to play a role in determining susceptibility to apoptosis (programmed cell death), and expression of various cyclins and activation of cyclin-dependent kinases (CDKs) have been shown to correlate with the onset of apoptosis in a number of experimental systems. To assess the role of CDK-mediated cell cycle events in apoptosis, we have expressed CDK dominant negative mutants in human HeLa cells. Dominant negative mutants of CDC2, CDK2, and CDK3 each suppressed apoptosis induced by both staurosporine and tumor necrosis factor alpha, whereas a dominant negative mutant of CDK5 was without effect. Like CDC2 and CDK2, CDK3 was shown to form a complex with cyclin A in vivo. CDK5 did not bind cyclin A to any detectable extent. Overexpression of wild type CDC2, CDK2, CDK3, or cyclin A (but not cyclin B) markedly elevated the incidence of apoptosis in BCL-2+ cells, which otherwise fail to respond to these agents. These results help identify cell cycle events that are also important for efficient apoptosis. PMID- 8626585 TI - Identification of a new outwardly rectifying Cl- channel that belongs to a subfamily of the ClC Cl- channels. AB - A new outwardly rectifying Cl- channels (ORCC) that belongs to ClC Cl- channel family has been identified from rat kidney and designated as ClC-5. ClC-5 cDNA encodes a polypeptide of 746 amino acids, which is indicated by hydrophobicity analysis to have structural features that are common of the ClC family. However, the amino acid sequence was weakly homologous to those of other ClC Cl- channels except for ClC-3, which we recently identified as a Ca2+-sensitive ORCC. Northern blot analysis of rat tissues showed that ClC-5 mRNA was predominantly expressed in the kidney and colon. To characterize the functional properties of ClC-5 by whole cell patch-clamp technique, we established the stably transfected CHO-K1 cell line using intranuclear microinjection technique. The transfected cells induced outwardly rectifying and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitive Cl- currents on whole cell configuration. Following the identification of two highly homologous ORCCs, ClC-3 and ClC-5, a new subfamily encoding ORCC has emerged in the ClC family. Furthermore, ClC-5 was almost identical to a partial sequence of human cDNA that is related to Dent's disease. The molecular structure and functional properties of ClC-5 will provide an important insight into ORCCs and the pathogenesis of Dent's disease. PMID- 8626586 TI - Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. AB - Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo. PMID- 8626587 TI - Binding of granzyme B in the nucleus of target cells. Recognition of an 80 kilodalton protein. AB - Granzyme B (cytotoxic cell proteinase 1) is a serine proteinase that has been implicated in cytotoxic T lymphocyte-induced apoptosis. In order to understand how granzyme B is involved in mechanisms of target cell destruction, characterization and identification of substrates are required. We have developed an in situ binding assay using permeabilized cells and recombinant granzyme B that allows us to visualize potential substrates after immunostaining with anti granzyme B antiserum. Confocal laser scanning microscopy and immunoelectron microscopic analyses demonstrate that granzyme B recognizes a nuclear substrate. The labeling pattern observed corresponds with regions of positive staining with uranyl acetate which binds to heterochromatin in the nucleus. Positive labeling of target cells with granzyme B is dependent on the presence of a catalytically active proteinase, since an inactive proenzyme form of granzyme B fails to give rise to any binding in the target cells. Far-Western blotting and immunoprecipitation of subcellular fractions of target cells have shown that the putative substrate of catalytically active granzyme B is an 80-kDa nuclear protein. Minor cytosolic bands of 50 and 94 kDa are also observed. A cytoplasmic band of 69 kDa is detected by both active and zymogen forms of granzyme B. PMID- 8626588 TI - Molecular cloning and characterization of a novel human diacylglycerol kinase zeta. AB - Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3 diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols. PMID- 8626589 TI - Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates. AB - Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK epsilon, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition. PMID- 8626590 TI - Multiple and tissue-specific promoter control of gonadal and non-gonadal prolactin receptor gene expression. AB - Prolactin receptors (PRLRs) are widely expressed, and multiple mRNA transcripts encoding PRLRs are present in prolactin target tissues. The molecular basis for the control of the PRLR gene expression is currently unknown. Analyses of the 5' untranslated regions of PRLR mRNAs expressed in gonadal and non-gonadal tissues and their genomic organization revealed three alternative first exons designated as E11, E12, and E13. Each of these exons is alternatively spliced to a common noncoding exon (exon 2, nucleotides -115 to -56) that precedes the third exon containing the translation initiation codon. Alternative utilization of exons E11, E12, and E13, as well as alternative splicing of exon 2, generates multiple 5'-untranslated regions in PRLR transcripts. These alternative first exons (E11, E12, and E13) were found to be utilized in a tissue-specific manner in vivo. E11 is predominantly expressed in the ovary, E12 is specifically expressed in the liver, and E13 is expressed as a predominant form in the Leydig cell and as a minor form in the ovary and liver. Genomic 5'-flanking regions containing the three putative PRLR gene promoters (PI, PII, and PIII) that initiate the transcription of E11, E12, and E13, respectively, were identified. E11 was found to initiate from a single site at -549, E12 from multiple sites at -405, -461, and -506, and E13 from two major sites at -340 and -351. These findings indicate that multiple promoters control transcription of the PRLR gene and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues. PMID- 8626591 TI - Cloning and sequence analysis of genes coding for paramecium secretory granule (trichocyst) proteins. A unique protein fold for a family of polypeptides with different primary structures. AB - The architecturally complex secretory granules of Paramecium, known as trichocysts, have two unusual and seemingly contradictory features: their protein contents have crystalline organization (Sperling, L., Tardieu, A., and Gulik Krzywicki, T. (1987) J. Cell Biol. 105, 1649-1662), yet these proteins are a heterogeneous set of molecules encoded by a large multigene family (Madeddu, L., Gautier, M.-C., Vayssie, L., Houari, A., and Sperling, L. (1995) Mol. Biol. Cell 6, 649-659). We present here the first complete sequences of three genes coding for three different precursors of the trichocyst crystalline matrix proteins. The deduced protein sequences indicate that each precursor gives rise to two of the mature polypeptides found in the crystalline trichocyst matrix. Analysis of putative processing sites suggests that a series of reactions, some of which may involve a novel endopeptidase, are involved in their proteolytic maturation. Each of the 6 mature polypeptides contains heptad segments. Characterization of the heptad segments leads us to propose that the mature polypeptides that compose the crystalline trichocyst matrix, despite their different primary structures, all share a unique protein fold, probably a 4 alpha-helical antiparallel bundle. PMID- 8626592 TI - Drosophila neurocalcin, a fatty acylated, Ca2+-binding protein that associates with membranes and inhibits in vitro phosphorylation of bovine rhodopsin. AB - Neurocalcins belong to a family of neuronal specific EF hand Ca2+-binding proteins defined by recoverin. Previously, we reported the cloning and initial characterization of neurocalcin in Drosophila melanogaster (Teng, D. H.-F., Chen, C.-K., and Hurley, J. B. (1994) J. Biol. Chem. 269, 31900-31907). We showed that the Drosophila neurocalcin protein (DrosNCa) is expressed in neurons and that bacterially expressed recombinant DrosNCa (rDrosNCa) can be myristoylated. Here, we present two lines of evidence that DrosNCa is fatty acylated in vivo. First, the mobility of affinity-purified native DrosNCa on two-dimensional gel electrophoresis is identical to that of myristoylated rDrosNCa and distinct from that of nonacylated rDrosNCa. Second, the membrane binding properties of native DrosNCa are similar to those of myristoylated rDrosNCa; both of these proteins bind to membranes at 0.2 mM Ca2+, whereas nonacylated rDrosNCa always remains soluble. It has been shown that recoverin inhibits the phosphorylation of rhodopsin when Ca2+ is present (Kawamura et al., 1993) and that a dependent recoverin/rhodopsin kinase interaction underlies the inhibitory effect of recoverin (Chen et al., 1995). Given the similarities between recoverin and neurocalcin, we examined the effect of DrosNCa on rhodopsin phosphorylation. We find that rDrosNCa is capable of inhibiting bovine rhodopsin phosphorylation in vitro in a Ca2+-dependent manner. The inhibitory activity of rDrosNCa is enhanced by myristoylation, and the potency of its effect is similar to that of recoverin. Two other related EF hand proteins, guanylate cyclase-activating protein-2 and calmodulin, are only poor inhibitors in these phosphorylation assays. in vitro inhibition of rhodopsin phosphorylation therefore appears to be an assayable property of a subset of recoverin-like proteins. PMID- 8626593 TI - Rapid identification of yeast proteins on two-dimensional gels. AB - This work describes a rapid and sensitive technique for the identification of Saccharomyces cerevisiae proteins on two-dimensional gels based on the determination of their amino acid ratios. Specific double labeling with 3H and 14C or 35S-labeled amino acids, chosen among those that are specifically incorporated into proteins without interconversion, allowed an accurate measurement of different amino acid ratios for 200 proteins. A computer program was developed to screen a yeast data base containing 1700 protein sequences and to identify proteins matching the measured Mr, pI, and amino acid ratios. The method, tested with 45 reference proteins, allowed 79 new identifications corresponding to abundant proteins belonging to a few functional families. Some protein spots correspond to homologs of mammalian proteins or to uncharacterized open reading frames. Remarkably, among identified proteins of similar abundance, the organellar proteins have a markedly lower codon usage bias than the cytosolic ones. The double labeling technique is particularly suited to the analysis, on a single two-dimensional gel, of the influence of physiological or genetic changes on yeast protein content. PMID- 8626594 TI - Discordant expression of osteoblast markers in MC3T3-E1 cells that synthesize a high turnover matrix. AB - To examine the autocrine effects that an organizing extracellular matrix has on osteoblast precursors, we created MC3T3-E1 cell lines that stably expressed pro alpha1(I) collagen chains with a truncated triple helical domain. Cells that had incorporated the pro-alpha1(I) expression plasmid (pMG155) expressed shortened pro-alpha1(I) transcripts at high levels and efficiently secreted the expression gene products into culture media. Those cells lost over 30% of newly deposited collagenous matrix compared with virtually no loss in control cultures, and media from the abnormal cells had qualitative differences in matrix metalloprotinase production. Electron micrographs strongly suggested that type I collagen molecules containing the truncated pro-alpha1(I) chains dramatically interfered with collagen fibrillogenesis in newly forming osteoblast matrix. Abnormal collagen fibrillogenesis was also associated with altered characteristics of cellular differentiation in that abnormal cells displayed a delayed and attenuated increase in alkaline phosphatase activity. Surprisingly, synthesis of osteocalcin was more than 5-fold higher than control cultures. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for up-regulation of alkaline phosphatase activity. However, in the presence of rapid turnover of osteoblast matrix, osteocalcin gene expression may be up-regulated in response to local signals by an unknown mechanism. PMID- 8626595 TI - Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of ApoB-containing lipoproteins. AB - Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells. PMID- 8626596 TI - Characterization of AMP-activated protein kinase beta and gamma subunits. Assembly of the heterotrimeric complex in vitro. AB - There is growing evidence that mammalian AMP-activated protein kinase (AMPK) plays a role in protecting cells from stresses that cause ATP depletion by switching off ATP-consuming biosynthetic pathways. The active form of AMPK from rat liver exists as a heterotrimeric complex and we have previously shown that the catalytic subunit is structurally and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae. Here we describe the isolation and characterization of the two other polypeptides, termed AMPKbeta and AMPKgamma, that together with the catalytic subunit (AMPKalpha) form the active kinase complex in mammalian liver. Sequence analysis of cDNA clones encoding these subunits reveals that they are related to yeast proteins that interact with SNF1, providing further evidence that the regulation and function of AMPK and SNF1 have been conserved throughout evolution. The amino acid sequence of the beta subunit is most closely related to SIP2 (35% identity), while the amino acid sequence of the gamma subunit is 35% identical with SNF4. We show that both AMPKbeta and AMPKgamma mRNA and protein are expressed widely in rat tissues. We show that AMPKbeta interacts with both AMPKalpha and AMPKgamma in vitro, whereas AMPKalpha does not interact with AMPKgamma under the same conditions. These results suggest that AMPKbeta mediates the association of the heterotrimeric AMPK complex in vitro, and will facilitate future studies aimed at investigating the regulation of AMPK in vivo. PMID- 8626597 TI - In vivo assembly of the tau-complex of the DNA polymerase III holoenzyme expressed from a five-gene artificial operon. Cleavage of the tau-complex to form a mixed gamma-tau-complex by the OmpT protease. AB - A plasmid was constructed that encodes all five subunits of the Escherichia coli tau-complex on a single artificially constructed operon under the control of an inducible promoter. The proteins tau, delta, delta , chi, and psi overproduced from this artificial operon assemble efficiently in vivo, providing an efficient source of homogeneous tau-complex. The gamma subunit is a truncated form of tau that is produced by a translational frameshift. When protein expression was induced in bacterial strains containing the outer membrane protein T (OmpT) protease, tau was proteolyzed after lysis to a gamma-like protein, gammaP, and a peptide, C-tau, corresponding to the C terminus of tau. N-terminal sequencing of C-tau revealed a cleavage site between two lysines at positions 429 and 430 of tau. The deduced sequence of gammaP is, therefore, only two amino acids shorter than natural gamma. The proteolysis by OmpT was also shown directly by using purified OmpT and tau-complex in an in vitro reaction. A gammaP-complex and a mixed tau-gammaP-complex were purified from ompT+ cells. When the tau-complex proteins were overexpressed in ompT- bacteria, intact tau-complex lacking gammaP could be purified. PMID- 8626598 TI - Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae. AB - Previously we showed that interleukin 1 beta stimulates the conversion of sphingomyelin to ceramide in the caveolae fraction of normal human fibroblasts. The ceramide, in turn, blocked platelet-derived growth factor (PDGF) stimulated DNA synthesis. We now present evidence that the PDGF receptor initiates signal transduction from caveolae. Cell fractionation and immunocytochemistry show caveolae to be the principal location of PDGF receptors at the cell surface. Multiple caveolae proteins acquire phosphotyrosine when PDGF binds to its receptor, but the hormone appears to have little effect on the tyrosine phosphorylation of non-caveolae membrane proteins. Five proteins known to interact with the phosphorylated receptor were found to be highly enriched in caveolae membrane. PDGF caused the concentration of three of these proteins to significantly increase in the caveolae fraction. Finally, PDGF stimulated the association of a 190-kDa phosphoprotein with the caveolae marker protein, caveolin. Therefore, ceramide may modulate PDGF receptor function directly in caveolae. PMID- 8626599 TI - Chicken guanylate-binding protein. Conservation of GTPase activity and induction by cytokines. AB - To gain further insights into the cytokine network of birds, we used polymerase chain reaction technology to clone a cDNA that codes for a chicken homolog of the interferon-induced guanylate-binding proteins (GBPs). In its N-terminal moiety, the 64-kDa chicken GBP contains two sequence blocks of 100 and 19 amino acids, respectively, that are about 70% identical to mammalian GBPs. The first region includes two motifs of the canonical GTP-binding consensus element. The other parts of chicken GBP are poorly conserved, except for a CAAX motif at the extreme C terminus which might signal isoprenylation. Like mammalian GBPs, recombinant chicken GBP specifically bound to agarose-immobilized guanine nucleotides and hydrolyzed GTP to both GDP and GMP. Regulation by interferons was also conserved: chicken GBP RNA was barely detectable in uninduced chicken cells. Low GBP RNA levels were found in cells treated with type I interferon, whereas very high levels were observed in cells treated with supernatant of a chicken T cell line that secretes a gamma-interferon-like activity. Together with recent phylogenetic studies of interferon genes, these results suggest that in spite of low sequence conservation, the various components of the avian interferon system are functionally well conserved. PMID- 8626600 TI - Chymotryptic digestion of the cytoplasmic domain of the beta subunit of Na/K ATPase alters kinetics of occlusion of Rb+ ions. AB - This paper demonstrates that specific chymotryptic digestion of the cytoplasmic domain of the beta subunit of Na/K-ATPase leads to changes in the kinetics of occlusion of Rb+ ions. The experiments utilize extensively trypsinized Na/K ATPase, "19-kDa membranes," which lack cytoplasmic loops of the alpha subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containing transmembrane segments and extracellular loops are intact. The beta subunit is partially split into NH2- and COOH-terminal fragments of 16 and approximately 50 kDa, respectively. Cation occlusion and ouabain binding are preserved. The 19-kDa membranes were incubated, at 37 degrees C, with a selection of proteases, in the presence of Rb+ ions. In these conditions, only alpha-chymotrypsin destroyed the ability to occlude Rb+ ions. This process was associated with truncation of the 16-kDa fragment of the beta subunit in two stages. In the first stage, chymotrypsin removed 10 residues from the 16-kDa fragment to form a 15-kDa fragment (NH2-terminal Ile15) and 4 or 6 residues from the NH2 terminus of the alpha subunit fragment beginning at Asp68. In these membranes Rb+ occlusion was still intact at 37 degrees C. Strikingly, however, deocclusion of two Rb+ ions, which is characteristically biphasic in 19-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb+ ions compared with 19-kDa membranes; and, consistent with the lower Rb+ affinity, Rb+ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb+ occlusion. In the second stage, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH2-terminal Leu24), followed by thermal destabilization of Rb+ occlusion even at high concentrations of Rb+ ions. Eventually, the thermally inactivated complex of fragments of alpha and beta subunits was digested to the limit peptides. The results suggest that the cytoplasmic domain of the beta subunit interacts with that of the alpha subunit, possibly with residues leading into the first transmembrane segment, and controls access of Rb+ ions into or out of the occlusion sites. PMID- 8626601 TI - Alpha1,3-L-fucosyltransferase expression in developing human myeloid cells. Antigenic, enzymatic, and mRNA analyses. AB - In an attempt to correlate the cell surface expression of Lex and sialyl-Lex structures in immature and mature myeloid cells with the genes expressing alpha1,3-fucosyltransferase(s) we have examined: 1) the properties of the cellular alpha1,3-fucosyltransferases and the mRNA transcripts corresponding to the five cloned genes, Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, and Fuc-TVII, in mature granulocytes and in the myeloid cell line HL-60, before and after dimethyl sulfoxide-induced differentiation and 2) the properties of the alpha1,3 fucosyltransferases expressed in COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNAs. The previously shown increase in cell surface expression of sialyl-Lex on differentiation of HL-60 cells (Skacel P. O., Edwards A. J., Harrison C. T., and Watkins W. M. (1991) Blood 78, 1452-1460) is accompanied by a sharp fall in expression of Fuc-TIV mRNA and a persistence of expression of Fuc-TVII mRNA. The properties of the alpha1,3-fucosyltransferase expressed in COS-7 cells transfected with Fuc-TIV are consistent with this being the major gene responsible for the expression of Lex in the immature myeloid cells. In Northern blot analyses, no transcripts of Fuc-TIII, Fuc-TV, or Fuc-TVI were detected in total RNA from mature granulocytes or mRNA from HL-60 cells before or after differentiation. In total RNA from mature granulocytes, Fuc-TIV transcripts were only faintly visible, whereas Fuc-TVII transcripts were quite definitely expressed. The specificity properties of Fuc-TVII expressed in COS-7 cells are consistent with this gene being the major candidate alpha1, 3 fucosyltransferase controlling the expression of sialyl-Lex on mature cells. However, Lex continues to be expressed on the surface of mature granulocytes and cell extracts retain the capacity to transfer fucose to non-sialylated acceptor substrates. The question therefore remains as to whether these properties result from the weakly expressed Fuc-TIV gene or whether another alpha1, 3 fucosyltransferase gene remains to be identified. PMID- 8626602 TI - Angiotensin II controls p21ras activity via pp60c-src. AB - Angiotensin II is the major effector peptide of the renin-angiotensin system, and it exerts its physiologic functions via a G protein-coupled cell surface receptor called AT1. We found that in rat aortic smooth muscle cells, angiotensin II stimulated the formation of Ras-GTP, Ras-Raf-1 complex formation, and the tyrosine phosphorylation of two important Ras GTPase-activating proteins (GAPs), p120 Ras-GAP and p190 Rho-GAP. Electroporation of anti-pp60c-src antibody into cultured, adherent smooth muscle cells blocked the angiotensin II stimulation of Ras-GAP and Rho-GAP tyrosine phosphorylation. In contrast electroporation of antibodies against c-Yes or c-Fyn had no effect. Anti-pp60c-src antibody also blocked angiotensin II-stimulated Ras activation and Ras-Raf-1 complex formation. These data strongly suggest that a G protein-coupled receptor such as the AT1 receptor can activate the Ras protein cascade via the tyrosine kinase pp60c-src. PMID- 8626604 TI - MCC, a cytoplasmic protein that blocks cell cycle progression from the G0/G1 to S phase. AB - The MCC gene was isolated from the human chromosome 5q21 by positional cloning and was found to be mutated in several colorectal tumors. In this study, we prepared specific antibodies and detected the MCC gene product as a cytoplasmic 100-kDa phosphoprotein in mouse NIH3T3 cells. Immunoelectron microscopic analysis showed that the MCC protein is associated with the plasma membrane and membrane organelles in mouse intestinal epithelial cells and neuronal cells. The amount of the MCC protein remained constant during the cell cycle progression of NIH3T3 cells, while its phosphorylation state changed markedly in a cell cycle-dependent manner, being weakly phosphorylated in the G0/G1 and highly phosphorylated during the G1 to S transition. Overexpression of the MCC protein blocked the serum induced cell cycle transition from the G1 to S phase, whereas a mutant MCC, initially identified in a colorectal tumor, did not exhibit this activity. These results suggest that the MCC protein may play a role in the signaling pathway negatively regulating cell cycle progression. PMID- 8626603 TI - Phosphatidic acid generation through interleukin 2 (IL-2)-induced alpha diacylglycerol kinase activation is an essential step in IL-2-mediated lymphocyte proliferation. AB - Proliferation of T lymphocytes is triggered by the interaction of interleukin 2 (IL-2) with its high affinity specific receptor that is expressed on the cell surface following T lymphocyte activation. Significant advances have recently been made in identifying the multiple signals that follow IL-2 receptor occupancy, although the exact mechanism responsible for IL-2-induced proliferation remains an enigma. It has been shown previously that unique species of phosphatidic acid are rapidly produced in vivo following IL-2 binding. It was then suggested that, in contrast to other eukaryotic growth factor systems, phosphatidic acid was at least in part generated through IL-2-induced diacylglycerol (DG) kinase activation. In the present study we demonstrate IL-2 dependent activation of the alpha isoform of DG kinase. Confocal microscopy studies reveal that the enzyme is located in the cytosol and nuclei of resting T cells. Interleukin 2 stimulation induces translocation of the enzyme to the perinuclear region. Furthermore, our results indicate that inhibition of the alpha isoform of DG kinase has a profound effect on IL-2-induced T cell growth. Studies on cell cycle distribution demonstrate that the inhibition of IL-2 induced phosphatidic acid production induces arrest in late G1 phase of IL-2 dependent cells. Altogether, these results link previous observations of interleukin 2 and phosphatidic acid production to activation of an specific isoform of DG kinase and suggest that activation of this enzyme is part of a novel signaling cascade that utilizes phosphatidic acid as an effector molecule. PMID- 8626605 TI - Assembly and function of a cytosolic form of NADH-specific isocitrate dehydrogenase in yeast. AB - Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a rate-limiting step in the tricarboxylic acid cycle. Yeast isocitrate dehydrogenase is an octomer composed of two subunits (IDH1 and IDH2) encoded by different genes and possessing independent mitochondrial targeting presequences. Oligonucleotide directed mutagenesis was used to remove the presequences from each gene and from both genes carried on centromere-based expression plasmids. Effects on cellular localization were examined in a yeast strain containing chromosomal disruptions of IDH1 and IDH2 loci. Each subunit was found to be dependent upon its presequence for mitochondrial localization, and the subunits are independently imported into mitochondria under most growth conditions. Furthermore, an active holoenzyme can be assembled in the cytosol and this ''cytosolic'' form of isocitrate dehydrogenase can reverse the acetate- growth phenotype characteristic of the DeltaIDH1/ DeltaIDH2 disruption strain, indicating functional replacement of the mitochondrial enzyme. However, transformants containing plasmids lacking either the IDH1 or IDH2 presequence coding regions were unexpectedly found to be capable of growth on acetate medium. Further investigation demonstrated that cellular localization of the IDH1 subunit can be biased by this stringent growth pressure. PMID- 8626606 TI - Photolabile amiloride derivatives as cation site probes of the Na,K-ATPase. AB - Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5 position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4' nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization. PMID- 8626607 TI - Alternate strand DNA triple helix-mediated inhibition of HIV-1 U5 long terminal repeat integration in vitro. AB - Integration of the human immunodeficiency virus (HIV) DNA into the host genome is an obligatory process in the replicative life cycle of the virus. This event is mediated in vitro by integrase, a viral protein which binds to specific sequences located on both extremities of the DNA long terminal repeats (LTRs). These sites are highly conserved in all HIV genomes and thus provide potential targets for the selective inhibition of integration. The integrase-binding site located on the HIV-1 U5 LTR end contains two adjacent purine tracts on opposite strands, 5' . . . GGAAAATCTCT-3'/3'-CCTTTTAGAGA . . . 5', in parallel orientations. A single strand oligonucleotide 5'-GGTTTTTGTGT-3' was designed to associate with these tracts via its ability to form a continuous alternate strand DNA triplex. Under neutral pH and physiological temperature, the oligonucleotide, tagged with an intercalator chromophore oxazolopyridocarbazole, formed a stable triplex with the target DNA. The occurrence of this unusual triplex was demonstrated by both DNase I footprinting and electron microscopy. The triplex inhibits the two steps of the integrase-mediated reactions, namely, the endonucleolytic cleavage of the dinucleotide 5'-GT-3' from the 3' end of the integration substrate and the integration of the substrate into the heterologous target DNA. The midpoints for both inhibition reactions were observed at oligonucleotide concentrations of 50 100 nM. We believe that these results open new possibilities for the specific targeting of viral DNA LTR ends with the view of inhibiting integration under physiological conditions. PMID- 8626608 TI - The ligand recognition specificity of beta3 integrins. AB - AlphaIIbbeta3 (platelet membrane glycoprotein IIb-IIIa) and alphavbeta3 are members of the beta3 subfamily of integrin adhesion receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD), originally described by Scarborough et al. (Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol. Chem. 268, 1066-1073) has been employed as a high affinity ligand for alphaIIbbeta3 to examine the specificity of the beta3 integrins. cHarGD interacted with high affinity with purified alphaIIbbeta3 (Kd = 10 nM) or with platelets (Kd = 120 nM). While cHarGD was specific for alphaIIbbeta3 in the presence of Ca2+, it bound to both beta3 integrins in the presence of Mn2+. Barbourin, a snake venom disintegrin containing a reactive KGD sequence, remained alphaIIbbeta3-specific, even in the presence of Mn2+. cHarGD became cross-linked to a site in beta3 of alphaIIb beta3, which is distinct from that of RGD peptides. These results allow identification of at least four classes of beta3 ligands: Class I, represented by RGD peptides and vitronectin, react similarly with alphaIIbbeta3 and alphavbeta3; Class II, represented by cHarGD, gamma-chain peptides and fibrinogen, react with both receptors in the presence of Mn2+ but only with alphaIIbbeta3 in the presence of Ca2+; Class III, represented by barbourin, are alphaIIbbeta3-specific under all cation conditions; Class IV, represented by osteopontin, bind primarily to alphavbeta3. PMID- 8626609 TI - Partial inhibition of Na+/K+-ATPase by ouabain induces the Ca2+-dependent expressions of early-response genes in cardiac myocytes. AB - Exposure of neonatal rat cardiac myocytes to ouabain concentrations that caused partial inhibition of Na+/K+-ATPase but no loss of viability, increased c-fos and c-jun mRNAs and the transcription factor AP-1. The increased mRNAs were proportional to the extent of inhibition of Na+/K+-ATPase and the resulting rise in steady state intracellular Ca2+ concentration. The rapid and sustained increase of c-fos mRNA was shown to be due to increased transcriptional rate. Induction of c-fos by ouabain was prevented when either extracellular or intracellular Ca2+ was lowered and was attenuated by pretreatment of myocytes with a phorbol ester under conditions known to down-regulate protein kinase C. Exposure to ouabain for 24-48 h also increased total transcriptional activity and protein content of myocytes. The findings suggest that the same signal responsible for the positive inotropic action of ouabain, i.e. net influx of Ca2+ caused by partial inhibition of Na+/K+-ATPase, also initiates the rapid protein kinase C-dependent inductions of the early-response genes, the subsequent regulations of other cardiac genes by the resulting transcription factors, and stimulation of myocyte growth. Whether these hitherto unrecognized effects of cardiac glycosides are obtained in the intact heart and their relevance to the therapeutic uses of these drugs remain to be determined. PMID- 8626610 TI - Regulated cleavage of sterol regulatory element binding proteins requires sequences on both sides of the endoplasmic reticulum membrane. AB - Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are attached to the endoplasmic reticulum (ER) and nuclear envelope by a hairpin domain consisting of two transmembrane regions connected by a short lumenal loop of approximately 30 hydrophilic amino acids. In sterol-depleted cells, a protease cleaves the protein in the region of the first transmembrane domain, releasing an NH2-terminal fragment of approximately 500 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. In sterol-overloaded cells, proteolysis does not occur, and transcription is repressed. Through mutational analysis in transfected cells, we identify two segments of SREBPs that are required for proteolysis, one on either side of the ER membrane. An arginine in the lumenal loop is essential. A tetrapeptide sequence (DRSR) on the cytosolic face adjacent to the first transmembrane domain is also required for maximal cleavage. Both of these elements are conserved in the human and hamster versions of SREBP-1 and SREBP-2. Sterol-mediated suppression of cleavage of SREBP-1 was found to be dependent on the extreme COOH-terminal region (residue 1034 to the COOH terminus), which exists in two forms as a result of alternative splicing. The form encoded by the "a" class exons (exons 18a and 19a) undergoes sterol-regulated cleavage. The form encoded by the "c" class exons (18c and 19c) is cleaved less efficiently and is not suppressed by sterols. These studies were made possible through use of a vector that achieves low level expression of epitope-tagged SREBPs under control of the relatively weak thymidine kinase promoter from herpes simplex virus. In contrast to SREBPs overproduced by high level expression vectors, the SREBPs produced at low levels were subject to the same regulated cleavage pattern as the endogenous SREBPs. These results indicate that sterol-regulated proteolysis of SREBPs is a complex process, requiring sequences on both sides of the ER membrane. PMID- 8626611 TI - Positive effect of overexpressed protein-tyrosine phosphatase PTP1C on mitogen activated signaling in 293 cells. AB - PTP1C, an SH2 domain-containing protein-tyrosine phosphatase, is predominantly expressed in hematopoietic cells, in which it negatively regulates cellular signaling. However, this enzyme is also expressed in many non-hematopoietic cells. We demonstrate here that in non-hematopoietic 293 cells, overexpression of a catalytically inactive mutant of PTP1C strongly suppressed the stimulatory effects of the epidermal growth factor or serum on cell proliferation, early gene transcription, and DNA synthesis. Similarly, the phosphorylation of the mitogen activated protein kinase and mitogen-activated protein kinase kinase activity was markedly inhibited by overexpression of mutant PTP1C. The inhibitory effect of mutant PTP1C was overcome by cotransfection with wild-type PTP1C, but not with the structurally related PTP2C. Furthermore, expression of the mutant phosphatase resulted in hyperphosphorylation on tyrosine of a 95-kDa protein that was co immunoprecipitated with the mutant, but not with the wild-type protein. These results suggest that, unlike in hematopoietic cells, PTP1C in 293 cells plays a positive role in epidermal growth factor- or serum-activated mitogenesis. Thus, PTP1C participates in multiple signaling pathways, where the enzyme, depending on its target molecules, may function as either a positive or negative mediator. PMID- 8626612 TI - Visualizing a specific contact in the HIV-1 Tat protein fragment and trans activation responsive region RNA complex by photocross-linking. AB - Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a stem-loop structure containing two helical stem regions separated by a trinucleotide bulge. The Tat protein contains a basic RNA-binding region (amino acids 49-57) located in the carboxyl-terminal half of the protein, and peptides containing this basic domain of Tat protein can bind TAR RNA with high affinities. We synthesized a 31-amino acid Tat fragment (amino acids 42-72) containing the basic region and part of flanking regulatory core domain that formed a specific complex with TAR RNA. Upon UV irradiation (254 nm), this Tat fragment cross-linked covalently with TAR RNA. Sites of cross-links were determined on both the TAR RNA and Tat protein fragment by RNA and protein sequencing, respectively. These results revealed that guanosine 26 of TAR RNA was cross-linked with tyrosine 47 of the Tat peptide. Our results provide the first physical evidence for a direct amino acid-base contact in Tat-TAR complex. Recently, orientation of the Tat-(42-72) was determined in our laboratory by psoralen.Tat-(42-72) conjugate (Wang, Z., and Rana, T. M. (1995) J. Am. Chem. Soc. 117, 5438-5444). On the basis of our findings, we suggest a model in which Tat binds to TAR RNA by inserting the basic recognition sequence into the major groove with an orientation where lysine 41 in the core domain of Tat contacts the lower stem and Tyr47 is close to G26 of TAR RNA. The knowledge of the orientation of Tat and details of other interactions with TAR RNA in Tat-TAR complex has significant implications for understanding gene regulation in HIV-1. PMID- 8626613 TI - Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase. AB - The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself. Three-hybrid data indicate that the Vma4p self-interaction is stabilized by both Vma1p and Vma2p. Native gel electrophoresis reveals numerous partial complexes not previously described. In addition to a large stable cytoplasmic complex seen in wild-type, Deltavma3 and Deltavma5 strains, we see partial complexes in the Deltavma4 and Deltavma7 strains. All larger complexes are lost in the Deltavma1, Deltavma2, and Deltavma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V1 complex. PMID- 8626614 TI - Suppression of the human erythropoietin gene expression by the TR2 orphan receptor, a member of the steroid receptor superfamily. AB - A DNA response element, TR2RE-EPO (5'-TCTGACCTCTCGACCTAC-3') has been identified in the 3-minimal hypoxia-inducible enhancer of the human erythropoietin gene for the TR2 orphan receptor, an androgen-repressed transcription factor and a member of the steroid/thyroid hormone receptor superfamily. Electrophoretic mobility shift assay showed a specific binding with high affinity (Kd = 0.14 nM) between the TR2 orphan receptor and the TR2RE-EPO. Our data further indicated that this specific binding is not due to the homo-dimerization of the TR2 orphan receptor. In addition, reporter gene expression using chloramphenicol acetyltransferase assay demonstrated that the TR2 orphan receptor may suppress the expression of the chloramphenicol acetyltransferase activities via the TR2RE-EPO in the hypoxic/normoxic human hepatoma HepG2 cells. Finally, our in situ hybridization data also indicated that the TR2 orphan receptor and the erythropoietin transcripts can be co-expressed in mouse kidney and liver. Together, our data suggest that the human erythropoietin gene could represent the first human target gene regulated directly by the human TR2 orphan receptor. PMID- 8626615 TI - Functionally active recombinant alpha and beta chain-peptide complexes of human major histocompatibility class II molecules. AB - Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (alphabeta) glycoproteins that display processed antigens to T cell receptors (TCRs) of CD4-positive T cells. The present study describes that individual recombinant alpha and beta chains of human MHC class II molecules lacking the transmembrane region (alpha-Tm and beta-Tm) are capable of binding antigenic peptide and that these complexes of chain-peptide are recognized by TCRs to induce antigen-specific apoptosis in restricted T cells. The alpha-Tm and the beta-Tm of human HLA-DR2 (DRB5*0101) were cloned, expressed in Escherichia coli, and purified in large scale by conventional chromatographic methods. The in vitro binding of an immunodominant epitope from the myelin basic protein (MBP-(83 102)Y83) to purified DR2 alpha-Tm and DR2 beta-Tm was demonstrated with biotinylated and fluoresceinated MBP-(83-102)Y83 peptide. The specificity of the MBP-(83-102)Y83 peptide binding to both DR2 alpha-Tm and DR2 beta-Tm was demonstrated in a competitive peptide binding assay. When exposed to a transformed T cell clone (SS8T) restricted to DR2(DRB5*0101) and MBP-(84-102) peptide, complexes of DR2 alpha-Tm and DR2 beta-Tm with MBP-(83-102)Y83 peptide were able to specifically recognize TCRs as measured by the increase in gamma interferon (gamma-IFN) cytokine. Such recognition of TCRs by soluble alpha-MBP (83-102)Y83 and beta-MBP-(83-102)Y83 complexes led to the induction of antigen specific apoptosis in SS8T cells as measured by double fluorescence flow cytometry and electron microscopy. These results provide the first evidence that soluble complexes of antigenic peptide and individual chains of human MHC class II molecules lacking the transmembrane region can recognize TCRs and induce antigen-specific apoptosis in T cells. Since activated CD4-positive T cells are involved in pathogenesis of various autoimmune diseases, the apoptosis triggered by individual soluble chain-peptide complexes has significant potential for eliminating autoreactive T cells. PMID- 8626616 TI - Post-translational modification of human brain type I inositol-1,4,5 trisphosphate 5-phosphatase by farnesylation. AB - In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP3 5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP3. Activity of this enzyme could be measured in both soluble and particulate fractions of tissue homogenates. The protein sequence showed a putative C terminal isoprenylation site (CVVQ). In this study, two mutants have been generated. The first mutant (C409S) has a serine replacing a cysteine at position 409 of the wild-type enzyme. The second mutant (K407D1) is a deletion mutant that lacks the last five C-terminal amino acids. These constructs were individually expressed by transfection in COS-7 cells. Western blot analysis of wild-type transfected cells indicated that both soluble and particulate fractions had a 43 kDa immunoreactive band, with a higher proportion of the original homogenate associated with the particulate part. On the contrary, when the two mutated constructs were transfected in COS-7 cells, the phosphatase was predominantly soluble. Confocal immunofluorescence studies showed the wild-type enzyme to be present on the cell surface of transfected COS-7 cells and in subcellular compartments around the nucleus. This was not observed for the two mutants, where uniform immunofluorescence labeling was observed throughout the cytosol. Recombinant type I InsP3 5-phosphatase expressed in Escherichia coli was a substrate of purified farnesyltransferase. Altogether, the data therefore suggest a direct participation of Cys-409 in a C-terminally anchored InsP3 5-phosphatase by farnesylation. PMID- 8626618 TI - Expression cloning and molecular characterization of HAS protein, a eukaryotic hyaluronan synthase. AB - We developed a mammalian transient expression system to isolate cDNA clones that determine hyaluronan expression. HAS-, a mouse mammary carcinoma mutant cell line, which is defective in hyaluronan synthase activity, was first established and used as a recipient for the expression cloning. One cloned cDNA that overcame the deficiency was isolated. The cDNA termed HAS contains an open reading frame of 1749 base pairs encoding a new protein of 583 amino acids. Homology analysis of the amino acid sequence suggests that HAS protein is related to streptococcal hyaluronan synthase and also to Xenopus laevis DG42 protein that was found to be homologous to bacterial hyaluronan synthase. Expression of HAS cDNA in HAS- cells complemented not only their mutant phenotypes such as deficient hyaluronan-matrix deposition but also hyaluronan synthase activity itself. Therefore, HAS cDNA is responsible for the activity of the hyaluronan synthase, a key enzyme of hyaluronan synthesis in eukaryotic cells. PMID- 8626617 TI - Glucose regulates in vivo glucose-6-phosphatase gene expression in the liver of diabetic rats. AB - Overproduction of glucose by the liver is the major cause of fasting hyperglycemia in both insulin-dependent and non-insulin-dependent diabetes mellitus. The distal enzymatic step in the process of glucose output is catalyzed by the glucose-6-phosphatase complex. We show here that 90% partially pancreatectomized diabetic rats have a >5-fold increase in the messenger RNA and a 3-4-fold increase in the protein level of the catalytic subunit of glucose-6 phosphatase in the liver. Normalization of the plasma glucose concentration in diabetic rats with either insulin or the glycosuric agent phlorizin normalized the hepatic glucose-6-phosphatase messenger RNA and protein within approximately 8 h. Conversely, phlorizin failed to decrease hepatic glucose-6-phosphatase gene expression in diabetic rats when the fall in the plasma glucose concentration was prevented by glucose infusion. These data indicate that in vivo gene expression of glucose-6-phosphatase in the diabetic liver is regulated by glucose independently from insulin, and thus prolonged hyperglycemia may result in overproduction of glucose via increased expression of this protein. PMID- 8626619 TI - Nuclear orphan receptor as a repressor of glucocorticoid receptor transcriptional activity. AB - Nuclear orphan receptors belong to the superfamily of ligand-activated transcription factors that show a close structural relationship and sequence homology. Ligands and functions of most of the orphan receptors have not yet been identified. The first nuclear orphan receptors that were cloned displayed a high degree of amino acid identity with the human estrogen receptor and were termed estrogen receptor-related (ERR) 1 and 2. In the present study, we show that ERR2 functions as a potent repressor of transcriptional activity mediated by the glucocorticoid receptor (GR). Transient transfection of different cell lines with a steroid-responsive reporter plasmid and receptor expression plasmids revealed that transcriptional activity mediated by GR in response to agonists was strongly suppressed by coexpression of ERR2. The orphan receptor displayed no promoter activity when expressed without GR. The inhibitory activity of ERR2 is cell specific and also receptor-specific because transactivation mediated by the progesterone receptor is unaffected by ERR2. Our observations provide evidence that the nuclear orphan receptor ERR2 acts as an endogenous modulator of GR transcriptional activity. PMID- 8626620 TI - Regulation of insulin-like growth factor-I expression in mouse preadipocyte Ob1771 cells. AB - In mouse preadipocyte Ob1771 cells, transcription of the insulin-like growth factor-I (IGF-I) gene was stimulated by growth hormone (GH), and IGF-I protein combined with GH in medium was required for their differentiation to adipocytes. During induction of the differentiation, the intracellular expression of each class of IGF-I mRNA was analyzed by reverse transcriptase-polymerase chain reaction. When the cells were cultured in the presence of GH, the class 1del. IGF I mRNA was a major molecular species among IGF-I mRNAs. In the presence of both GH and IGF-I, the splicing pattern of IGF-I mRNA changed from class 1del. to class 1. Moreover, as detected by Western blotting, the IGF-I protein was present in cells and in the medium only when the cells were cultured in the presence of both GH and IGF-I. We found that IGF-I secreted from Ob1771 cells could act in an autocrine/paracrine fashion and induce the differentiation of other Ob1771 cells. It was demonstrated that the translation efficiency of class 1 mRNA was higher than that of class 1del. mRNA in vitro. These results suggested that stimulation with exogenous IGF-I in the presence of GH was required for the production of class 1 IGF-I mRNA and that the production of the IGF-I protein was activated by increasing the translation efficiency through shifting the splicing pattern of IGF-I mRNA from class 1del. to class 1. Exogenous IGF-I triggered the differentiation by initiating the synthesis of endogenous IGF-I. PMID- 8626621 TI - Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48. AB - A site-specific C to U editing reaction modifies nuclear apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the mammalian small intestine. This reaction is mediated by a multicomponent enzyme complex, which contains a catalytic subunit, Apobec-1. We have used gene targeting to disrupt mouse apobec 1 in order to establish its requisite importance in apoB mRNA editing and also, in view of its widespread tissue distribution in rodents, as a preliminary indication of other potential roles. Both heterozygous (apobec-1+/-) and homozygous (apobec-1-/-) gene-targeted mice appear healthy and fertile with no alterations in serum cholesterol or triglyceride concentrations. The apobec-1+/- mice demonstrated reduced levels of hepatic apoB mRNA editing. By contrast, levels of small intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1+/- animals, suggesting that Apobec-1 is expressed in limited quantities in the liver but not in the small intestine. The apobec-1-/- mice lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB mRNA in all tissues, and contained no apoB48 in their serum, demonstrating that there is no functional duplication of this gene. PMID- 8626622 TI - Inhibition of p38 mitogen-activated protein kinase by insulin in cultured fetal neurons. AB - Insulin supports the survival and differentiation of many types of fetal neurons. To determine if mitogen-activated protein (MAP) kinases play a role in mediating the neurotrophic actions of insulin, we identified the MAP kinases present in fetal chick forebrain neurons and examined their regulation by insulin. Cell extracts were fractionated on Mono Q columns, and phosphotransferase activity was measured using myelin basic protein as the substrate. In control neurons, four peaks of MAP kinase activity were resolved. Peaks I, II, and IV were identified by immunoblotting as c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 MAP kinase, respectively. Neurons treated with insulin showed a dramatic decrease, 80-90%, in p38 MAP kinase activity without significant changes in the other MAP kinase activities. Insulin decreased the phosphotyrosine content of p38 MAP kinase with maximal effects observed within 5 min. Pretreatment of neurons with sodium orthovanadate blocked the ability of insulin to inhibit the tyrosine phosphorylation and activity of p38 MAP kinase, suggesting that activation of a tyrosine or dual specific phosphatase is necessary for the inhibition of p38 MAP kinase by insulin. Since p38 MAP kinase has been recently implicated in neuronal cell apoptosis, negative regulation of this kinase by insulin may be critical for the neurotrophic actions of insulin. PMID- 8626623 TI - Sphingomyelinase and ceramide suppress insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1. AB - The sphingomyelin pathway is a newly described signal transduction pathway mediating the action of several cytokines including tumor necrosis factor-alpha (TNF). TNF was recently shown to interfere with insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1). In this work we examined the possible effect of direct activation of the sphingomyelin pathway on insulin-induced tyrosine phosphorylation of IRS-1. Incubation of the insulin sensitive rat hepatoma Fao cells with bacterial sphingomyelinase (SMase) that causes membrane hydrolysis of sphingomyelin led to a time- and dose-dependent decrease in insulin-induced tyrosine phosphorylation of IRS-1. The effect was apparent after 10 min of incubation and with a dose of 10 milliunits/ml SMase. It was not associated with a decrease in insulin receptor autophosphorylation. In addition, SMase treatment interrupted the association of the 85-kDa catalytic subunit of phosphatidylinositol 3-kinase with IRS-1. A similar impact on IRS-1 tyrosine phosphorylation was observed after addition of cell-permeable ceramide analogs (C2 and C6). Comparable changes in IRS-1 tyrosine phosphorylation and electrophoretic mobility were found after exposure of cells to either TNF, SMase, or ceramide. Our findings suggest that TNF may utilize the sphingomyelin pathway in its effect on the insulin-stimulated tyrosine phosphorylation of IRS-1. PMID- 8626624 TI - CD39 is an ecto-(Ca2+,Mg2+)-apyrase. AB - CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker of Epstein Barr virus (EBV) transformed B cells. In this report, we show that an ecto-(Ca2+,Mg2+)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells. PMID- 8626626 TI - Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m [125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site. AB - Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3 trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein. PMID- 8626625 TI - (-)-Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor mediated inhibition of adenylyl cyclase. AB - (-)-Delta9-Tetrahydrocannabinol ((-)-Delta9-THC) is the major active psychotropic component of the marijuana plant, Cannabis sativa. The membrane proteins that have been found to bind this material or its derivatives have been called the cannabinoid receptors. Two GTP-binding protein-coupled cannabinoid receptors have been cloned. CB1 or the neuronal cannabinoid receptor is found mostly in neuronal cells and tissues while CB2 or the peripheral cannabinoid receptor has been detected in spleen and in several cells of the immune system. It has previously been shown that activation of CB1 or CB2 receptors by cannabinoid agonists inhibits adenylyl cyclase activity. Utilizing Chinese hamster ovary cells and COS cells transfected with the cannabinoid receptors we report that (-)-Delta9-THC binds to both receptors with similar affinity. However, in contrast to its capacity to serve as an agonist for the CB1 receptor, (-)-Delta9-THC was only able to induce a very slight inhibition of adenylyl cyclase at the CB2 receptor. Morever, (-)-Delta9-THC antagonizes the agonist-induced inhibition of adenylyl cyclase mediated by CB2. Therefore, we conclude that (-)-Delta9-THC constitutes a weak antagonist for the CB2 receptor. PMID- 8626627 TI - Synergistic activation of transcription by the mutant and wild-type minimal transcriptional activation domain of VP16. AB - VP16 activates transcription by stimulating initiation, and for this function the aromatic residue at position 442 within its activation domain is critical. Recent studies have suggested that VP16 also stimulates transcriptional elongation. It has been shown that VP16 can activate transcription tethered downstream of the transcription start site to RNA. Here, we analyze the synergistic activation features of hybrid VP16 fusion proteins when tethered simultaneously to RNA downstream of the start site and to DNA upstream of a promoter in order to investigate its role in postinitiation control of transcription. Upon targeting the VP16 activation domain simultaneously to both DNA and RNA, high levels of transcriptional synergism is observed. Importantly, a transcription-defective VP16 minimal activation domain (amino acids 413-453) mutated at critical residue 442 (phenylalanine --> proline) maintained synergism, when bound to RNA, with the DNA-bound wild-type VP16 minimal activation domain. Targeting of this "functionally defective" VP16 minimal activation domain via RNA and an intact activation domain via DNA allowed us to uncover a postinitiation activity for VP16 not previously detected in DNA targeting studies. We suggest that, in addition to stimulating initiation, VP16 also acts at a postinitiation step involving residues other than the critical residue at position 442 within the same 41-amino acid minimal activation domain (amino acids 413-453) required for initiation. PMID- 8626629 TI - Posttranslational modifications in the C-terminal tail of axonemal tubulin from sea urchin sperm. AB - After proteolytic digestion of sperm tubulin from sea urchin Paracentrotus lividus, C-terminal peptides were isolated by chromatographic separations. The peptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. About 70% of the isolated C-terminal peptides were unmodified. The remaining modified peptides have undergone a combination of numerous posttranslational modifications generating significant heterogeneity of sperm tubulin. alpha-Tubulin is modified by detyrosylation, release of the penultimate glutamate, polyglutamylation, and polyglycylation. Glycylation and glutamylation can coexist within one alpha tubulin isoform. beta-Tubulin undergoes polyglycylation but was not observed to be polyglutamylated. The number of units posttranslationally added reaches 11 and 12 glycyl units on beta- and alpha-tubulin, respectively. This is different from the polyglycylation of axonemal tubulin in Paramecium cilia where up to 40 added glycyl units were observed both on alpha- and beta-tubulin. PMID- 8626628 TI - Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/H+ exchange. AB - In terminally differentiated ileal villus Na+-absorptive cells, epidermal growth factor (EGF) stimulates NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors, and as we confirm here, a brush border tyrosine kinase. In the present study we show that brush border phosphatidylinositol 3-kinase (PI 3-kinase) is involved in EGF stimulation of NaCl absorption and brush border Na+/H+ exchange. In rabbit ileum studied with the Ussing chamber-voltage clamp technique, EGF stimulation of active NaCl absorption is inhibited by the selective PI 3-kinase inhibitor wortmannin. PI 3 kinase, a largely cytosolic enzyme, translocates specifically to the brush border of ileal absorptive cells following EGF treatment. This translocation occurs as early as 1 min after EGF treatment and remains increased at the brush border for at least 15 min. EGF also causes a rapid (1 min) and large (4-5-fold) increase in brush border PI 3-kinase activity. Involvement of PI 3-kinase activity in intestinal Na+ absorption is established further by studies done in the human colon cancer cell line, Caco-2, stably transfected with the intestinal brush border isoform of the Na+/H+ exchanger, NHE3 (Caco-2/NHE3 cells). Brush border Na+/H+ exchange activity was measured using the pH-sensitive fluorescent dye 2'7' bis(carboxyethyl)5-(6)-carboxyfluorescein. EGF added to the basolateral surface but not apical surface of Caco-2/NHE3 cells increased brush border Na+/H+ exchange activity. The EGF-induced increase in brush border Na+/H+ exchange activity was completely abolished in cells pretreated with wortmannin. EGF treatment caused increased tyrosine phosphorylation of PI 3-kinase in both ileal brush border membranes and Caco-2/NHE3 cells, suggesting that a tyrosine kinase upstream of the PI 3-kinase is involved in the EGF effects on Na+ absorption. In conclusion, the present study provides evidence in two separate intestinal models, the ileum and a human colon cancer cell line, that PI 3-kinase is an intermediate in EGF stimulation of intestinal Na+ absorption. PMID- 8626631 TI - AP-1/jun is required for early Xenopus development and mediates mesoderm induction by fibroblast growth factor but not by activin. AB - In Xenopus, normal mesoderm formation depends on signaling through the fibroblast growth factor (FGF) tyrosine kinase receptor. An important signaling pathway from receptor tyrosine kinases involves Ras/Raf/MAP kinase. However, the downstream pathway that occurs in the nucleus to finally trigger gene expression for mesoderm formation remains unknown. We report here that a high level of activator protein-1 (AP-1)-dependent transcriptional activity is detected during the early development of Xenopus embryos. Injection of a dominant negative mutant jun (DNM jun or TAM67) RNA into the two-cell stage embryos inhibited endogenous AP-1 activity and blocked normal embryonic development with severe posterior truncation in tadpoles. The inhibition of AP-1 activity and the phenotypic change induced by TAM67 was rescued by co-injection of wild-type c-jun RNA, but not by the control beta-galactosidase RNA. The FGF-stimulated mesoderm induction was markedly inhibited in animal cap explants from the embryos injected with TAM67. Activin induction of mesoderm, on the other hand, was normal in the embryos injected with TAM67 RNA. These findings suggest that AP-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and the AP-1/Jun is a key signaling molecule in the development of posterior structure. PMID- 8626630 TI - Selective uptake of cytosolic, peroxisomal, and plasma membrane proteins into the yeast lysosome for degradation. AB - When glucose-starved cells are replenished with glucose, the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the yeast lysosome (vacuole) for degradation. The glucose-induced targeting of FBPase to the vacuole for degradation occurs in cells grown under a variety of metabolic conditions. Immunoelectron microscopic studies demonstrate that the uptake of FBPase by the vacuole is mediated in part by an autophagic process. FBPase can be found on the vacuolar membrane and also at the sites of membrane invaginations. Furthermore, FBPase is associated with different forms of vesicles, which are induced to accumulate inside the vacuole. We have identified peroxisomes as the organelles that are delivered to the vacuole for degradation when cells are replenished with glucose. Ultrastructural studies indicate that peroxisomes are engulfed by the vacuole by an autophagic process, leading to the destruction of whole organelles in the vacuole. Furthermore, the galactose transporter (Gal2p) is also delivered from the plasma membrane to the vacuole for degradation in response to glucose. Gal2p is delivered to the vacuole through the endocytic pathway, as mutants defective in receptor-mediated endocytosis fail to degrade Gal2p in response to glucose. PMID- 8626632 TI - A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance regulator gene. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells. PMID- 8626633 TI - Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases. AB - Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid. PMID- 8626634 TI - Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CD11a I domain. AB - Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. We recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM 3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp182 and Ser184 and was not present in CD11b or CD11c. Although mutation of Asp182 and Ser184 failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile126 and Asn129) recently implicated in the ICAM-3 binding site. Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM 3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1. PMID- 8626635 TI - Effects of diabetes mellitus on hepatocyte nuclear factor 1 decrease albumin gene transcription. AB - We have previously reported that albumin gene transcription is reduced in diabetes mellitus (DM). The present study explored the mechanism by which albumin gene transcription is down-regulated in DM. Deletional studies and displacement of factors binding to site B of the albumin promoter indicated that the repressive effects of DM are mediated by nuclear factors binding to this site. Since hepatocyte nuclear factor 1 (HNF1) activates albumin promoter activity and is the predominant factor binding to site B, we examined HNF1. The abundance and binding activity of HNF1 were reduced in hepatonuclear extracts from diabetic compared to control rats. However, HNF1 mRNA levels were unchanged, suggesting that the effect of DM on HNF1 is at the post-transcriptional level. Extracts from diabetic animals also contained another protein, distinct from HNF1 and vHNF1, which bound to site B in gel retardation studies. In summary, our studies demonstrate that the reduced abundance and binding activity of HNF1 correlates with decreased albumin gene transcription in DM. PMID- 8626636 TI - SH2 domain function is essential for the role of the Lck tyrosine kinase in T cell receptor signal transduction. AB - Tyrosine kinase activity is required for signal transduction through the T cell antigen receptor (TCR). The Src family tyrosine kinase Lck appears to play a key role in the initiation of TCR signaling events. We have investigated the role of the phosphotyrosine-binding Src homology-2 (SH2), domain of Lck in TCR signaling. Lck containing a mutation in the phosphotyrosine binding pocket of the SH2 domain was expressed in an Lck-deficient cell line. We found that, in contrast to wild type Lck, the SH2 domain mutant was unable to restore even the earliest TCR mediated signaling events. To investigate the role of the Lck SH2 domain, we examined the association of tyrosine phosphoproteins with Lck. The predominant associated phosphoprotein was the ZAP-70 tyrosine kinase, which has also been implicated in the initiation of TCR signaling. In addition, the zeta subunit of the T cell receptor was found to weakly associate with Lck. Further analysis indicated that the SH2 domain of Lck can directly recognize both ZAP-70 and zeta in immunoprecipitates from TCR-stimulated cells. Our findings demonstrate that the SH2 domain of Lck is essential for the initiation of signaling events following TCR stimulation probably as a result of its ability to mediate an interaction between Lck and the ZAP-70 tyrosine kinase and/or the zeta subunit of the T cell receptor. PMID- 8626637 TI - The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. AB - Nepsilon-(Carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined nonenzymatic glycation and oxidation (glycoxidation) reactions. We now report that CML is also formed during metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of protein. During copper catalyzed oxidation in vitro, the CML content of low density lipoprotein increased in concert with conjugated dienes but was independent of the presence of the Amadori compound, fructoselysine, on the protein. CML was also formed in a time-dependent manner in RNase incubated under aerobic conditions in phosphate buffer containing arachidonate or linoleate; only trace amounts of CML were formed from oleate. After 6 days of incubation the yield of CML in RNase from arachidonate was approximately 0.7 mmol/mol lysine compared with only 0.03 mmol/mol lysine for protein incubated under the same conditions with glucose. Glyoxal, a known precursor of CML, was also formed during incubation of RNase with arachidonate. These results suggest that lipid peroxidation, as well as glycoxidation, may be an important source of CML in tissue proteins in vivo and that CML may be a general marker of oxidative stress and long term damage to protein in aging, atherosclerosis, and diabetes. PMID- 8626638 TI - The active species of plasma membrane Ca2+-ATPase are a dimer and a monomer calmodulin complex. AB - The purified plasma membrane Ca2+-ATPase is fully activated through the enzyme concentration-dependent self-association at physiologically relevant Ca2+ concentrations (Kosk-Kosicka, D., and Bzdega, T. (1988) J. Biol. Chem. 263, 18184 18189; Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). We have previously shown that the Ca2+-ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. Presently, we have succeeded in determining the molecular mass of the two active enzyme species by equilibrium ultracentrifugation. For the calmodulin-dependent species, the molecular mass is 170 +/- 30 kDa, which is consistent with predominantly monomeric Ca2+-ATPase with bound calmodulin. The molecular mass of calmodulin-independent oligomers is 260 +/- 34 kDa, indicating that they are dimers. Results of experiments performed under different calcium and potassium concentrations and in the presence of dextran that causes molecular crowding verify a strict Ca2+ requirement of the dimerization process. We conclude that the active species of the Ca2+-ATPase are a monomer-calmodulin complex and a dimer. PMID- 8626639 TI - Roles of light chains in the activity and conformation of smooth muscle myosin. AB - The 20-kDa regulatory (LC20) and 17-kDa essential (LC17) light chain subunits could be removed from porcine aorta smooth muscle myosin by the use of trifluoperazine and ammonium chloride. The isolated heavy chain rebound both light chains, resulting in the restoration of native properties. Experiments on reconstitution of the isolated heavy chain with LC17 and/or LC20 showed that both light chains were required for folding into the 10 S conformation and thus for the phosphorylation-dependent filament formation of smooth muscle myosin. However, LC17 was not essential for the phosphorylation-dependent regulation of actin-activated ATPase activity and superprecipitation but was required for full regulation. LC17 and phosphorylated LC20 were found to act as activators, and dephosphorylated LC20 was found to act as a repressor of the motor activities of smooth muscle myosin. PMID- 8626640 TI - Function of the duplicated IIB domain and oligomeric structure of the fructose permease of Escherichia coli. AB - The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated IIB domain. The protein therefore consists of three distinct domains, B', B, and C (N-terminal to C-terminal), joined by flexible linkers and is thus designated FruB'BC. The N-terminal B' domain was removed using molecular genetic techniques, and the truncated Enzyme II (FruBC) was characterized relative to the wild-type enzyme both in vivo and in vitro. In vivo, FruBC exhibited depressed fermentation characteristics at low fructose concentrations. [14C]Fructose uptake measurements revealed reduced rates only when the permease was rate-limiting for transport. In vitro, FruBC exhibited a 10-fold lower affinity for its phosphoryl donating protein, the IIA-FPr diphosphoryl transfer protein (DTP), than was observed with the wild-type enzyme, and the maximal velocity of fructose phosphorylation was 7-fold depressed. Because the fructose-1 phosphate:[14C]fructose transphosphorylation reaction appeared normal, we conclude that the loss of the B' domain primarily affected phosphoryl transfer between the IIA and IIB domains of the permease. A mutant FruBC derivative with cysteine 112 replaced by serine (C112S FruBC) was inactive as a phosphoryl carrier and a sugar transport protein. Expression of the plasmid-encoded mutant protein inhibited the in vivo activity of the chromosomally encoded wild-type fructose permease, but it did not observably affect the activities of the mannitol or glucitol PTS permeases or of non-PTS sugar permeases. Further, the presence of the detergent extracted mutant protein inhibited the activity of the detergent solubilized wild-type or FruBC enzyme. In contrast, the wild-type FruB BC permease was apparently epistatic over the truncated FruBC permease in vivo. The experiments reported 1) show that the B' domain of the fructose permease functions to facilitate phosphoryl transfer between DTP and the permease, 2) establish the essentiality of cysteine 112 in the B domain of the permease, 3) provide evidence that a functional fructose permease consists of an oligomer in which both IIB domains must be active for the enzyme to catalyze normal rates of phosphoryl transfer and transport, 4) suggest that a single B' domain in the oligomeric Enzyme II is sufficient to allow high efficiency phosphoryl transfer between the IIA domain of DTP and the IIB domain of the permease, and 5) show that the B' domain is not important for oligomerization. PMID- 8626641 TI - Characterization of a defense complex consisting of interleukin 1 and phenol oxidase from the hemolymph of the tobacco hornworm, Manduca sexta. AB - Hemolymph of fifth instar Manduca sexta larvae collected under non-sterile conditions exhibited the presence of a novel high molecular weight protein complex, which was absent from the hemolymph collected aseptically. The high molecular weight complex consisted of, at least prophenol oxidase, phenol oxidase, and an interleukin 1-like molecule, thereby demonstrating the generation of this complex as a consequence of a host defense response. While the native phenol oxidase and the interleukin 1-like molecule possessed molecular weights of about 80,000 and 17,000, respectively, the complex had a molecular weight of about 400,000. Apart from prophenol oxidase, phenol oxidase, and interleukin 1, dopachrome isomerase and other, as of yet unidentified, proteins may be part of the complex as judged by the presence of additional bands observed during SDS polyacrylamide gel electrophoresis. The significance of the assembly of this defense complex for insect host defense strategies is discussed. PMID- 8626642 TI - Raf and mitogen-activated protein kinase regulate stellate cell collagen gene expression. AB - Hepatic stellate cells become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of hepatic stellate cell collagen gene expression is a central pathogenetic step during the development of cirrhosis. The cytoplasmic Raf and mitogen-activated protein (MAPK) kinases were found to differentially regulate alpha I(I) collagen gene expression in activated stellate cells. This suggests an unappreciated branch point exists between Raf and MAPK. A MAPK-stimulatory signal was mapped to the most proximal NF-1 and Sp-1 binding domains of the 5' untranslated region of the collagen gene. A Raf-inhibitory signal was mapped to a further upstream binding domain involving a novel 60-kDa DNA-binding protein (p60). The cell-specific expression and induction of p60 in stellate cells during the early stages of hepatic fibrogenesis in vivo suggest a central role for this pathway during liver injury and stellate cell activation. PMID- 8626643 TI - Purification of an RNA polymerase II transcript release factor from Drosophila. AB - Factor 2 was previously identified in Drosophila Kc cell nuclear extract (KcN) as an activity suppressing the appearance of long transcripts (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). A 154-kDa protein with factor 2 activity was purified to apparent homogeneity from KcN. An immobilized template assay indicated that factor 2 caused the release of transcripts by RNA polymerase II in an ATP-dependent manner. Some early elongation complexes were resistant to factor 2 action but became sensitive after treatment with 1 M KCl. In the absence of factor 2, transcription complexes still exhibited a low degree of processivity suggesting that factor 2 was only partially responsible for abortive elongation. PMID- 8626644 TI - Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease. AB - XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junction cutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNA-binding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease. PMID- 8626645 TI - Identification of a domain within the carboxyl-terminal region of the beta platelet-derived growth factor (PDGF) receptor that mediates the high transforming activity of PDGF. AB - We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR. PMID- 8626646 TI - Induction of calmodulin kinase IV by the thyroid hormone during the development of rat brain. AB - This communication reports the specific induction of calmodulin kinase IV by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3) in a time- and concentration dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system, which can grow and differentiate under chemically defined conditions. The induction of the enzyme that can be observed both on the mRNA and on the protein level is T3-specific, i.e. it cannot be induced by retinoic acid or reverse T3, and can be inhibited on both the transcriptional and the translational level by adding to the culture medium actinomycin D or cycloheximide, respectively. The earliest detection of calmodulin kinase IV in the fetal brain tissue of the rat is at days E16/E17, both on the mRNA as well as on the protein level. This is the first report in which a second messenger-dependent kinase involved in the control of cell regulatory processes is itself controlled by a primary messenger, the thyroid hormone. PMID- 8626647 TI - The myeloid leukemia-associated protein SET is a potent inhibitor of protein phosphatase 2A. AB - Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis. PMID- 8626648 TI - Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin alpha subunit. AB - Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps. PMID- 8626649 TI - Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common beta 1 chain. AB - The very late activation antigens (VLA) or beta 1 integrins mediate cell attachment to different extracellular matrix proteins and intercellular adhesions. The ligand binding activity of these adhesion receptors is not constitutive and can be regulated by temperature, presence of extracellular divalent cations, stimulatory monoclonal antibodies (mAbs), and cellular activation. We have generated three novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes on the common beta 1 subunit (CD29) of VLA integrins whose expression correlates with the ligand binding activity of these heterodimeric glycoproteins. This correlation has been demonstrated for several integrin heterodimers in different cell systems using a variety of extracellular and intracellular stimuli for integrin activation. Thus, the presence of micromolar concentrations of extracellular Mn2+, preincubation with the activating anti-beta 1 mAb TS2/16, and cell treatment with phorbol esters or calcium ionophores, induced the expression of the HUTS beta 1 epitopes on T lymphoblasts. Using a panel of human-mouse beta 1 chimeric molecules, we have mapped these epitopes to the 355-425 sequence of the beta 1 polypeptide. This segment represents therefore a novel regulatory region of beta 1 that is exposed upon integrin activation. Interestingly, binding of HUTS mAbs to partially activated VLA integrins results in maximal activation of these adhesion receptors and enhancement of cell adhesion to beta 1 integrin ligands collagen, laminin, and fibronectin. PMID- 8626651 TI - Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization. AB - The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized. PMID- 8626650 TI - Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors. AB - While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs. PMID- 8626653 TI - Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor. AB - This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates. PMID- 8626652 TI - Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity. AB - PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown the PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells. PMID- 8626654 TI - Alpha-tocopherol as a reductant for Cu(II) in human lipoproteins. Triggering role in the initiation of lipoprotein oxidation. AB - Initiation of lipid peroxidation by Cu(II) requires reduction of Cu(II) to Cu(I) as a first step. It is unclear, however, whether this reaction occurs in the course of lipoprotein oxidation. It is also unknown which reductant, if any, can drive the reduction of Cu(II) in this case. We found that Cu(II) was rapidly reduced to Cu(I) by all major human lipoproteins (high, low, and very low density lipoproteins (HDL, LDL, and VLDL), and chylomicrons). Cu(II)-reducing activity was associated with a lipid moiety of the lipoproteins. The rates of Cu(II) reduction by different lipoproteins were similar when the lipoproteins were adjusted to similar alpha-tocopherol concentrations. Enriching lipoproteins with alpha-tocopherol considerably increased the rate of CU(II) reduction. CU(II) reduction by alpha-tocopherol-deficient LDL isolated from a patient with familial inherited vitamin E deficiency was found to occur much slower in comparison with LDL isolated from a donor with a normal plasma level of alpha-tocopherol. Initial rate of CU(II) reduction by alpha-tocopherol-deficient LDL was found to be zero. Enriching LDL with ubiquinol-10 to concentrations close to those of alpha tocopherol did not influence the reaction rate. When LDL was treated with ebselen to eliminate preformed lipid hydroperoxides, the reaction rate was also not changed significantly. CU(II) reduction was accompanied by a consumption of lipoprotein alpha-tocopherol and accumulation of conjugated dienes in the samples. Increasing alpha-tocopherol content in lipoproteins slightly decreased the rate of conjugated diene accumulation in LDL and HDL and considerably increased it in VLDL. The results suggest that alpha-tocopherol plays a triggering role in the lipoprotein oxidation by CU(II), providing its initial step as follows: alpha TocH + CU(II) --> alpha Toc. + Cu(I) + H+. This reaction appears to diminish or totally eliminate the antioxidative activity of alpha tocopherol in the course of lipoprotein oxidation. PMID- 8626655 TI - Regulation of yeast CTP synthetase activity by protein kinase C. AB - CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity. PMID- 8626656 TI - Activation of the factor VIIIa-factor IXa enzyme complex of blood coagulation by membranes containing phosphatidyl-L-serine. AB - Factor IXa, a serine protease of blood coagulation, functions at least 100,000 times more efficiently when bound to factor VIIIa on a phospholipid membrane than when free in solution. We have utilized the catalytic activity of the factor VIIIa-factor IXa complex to report the effect of phospholipid membranes on binding of factor IXa to factor VIIIa and on enzymatic cleavage of the product. The apparent affinity of factor IXa for factor VIIIa was 10-fold lower in the absence of phospholipid membranes with a KD of 46 nM versus 4.3 nM with phospholipid membranes. The Km for activation of factor X by the factor VIIIa factor IXa complex was 1700 nM in solution, 70-fold higher than the value of 28 nM when bound to membranes containing phosphatidyl-L-serine, phosphatidylethanolamine, and phosphatidylcholine at a ratio of 4:20:76. The largest effect of phosphatidyl-L-serine-containing membranes on the factor VIIIa factor IXa complex was the accelerated rate of peptide bond cleavage, with the k(cat) increased by 1,500-fold from 0.022 to 33 min-1. Membranes in which phosphatidyl-L-serine was replaced by phosphatidyl-D-serine, phosphatidic acid, or phosphatidylglycerol were at least 10-fold less effective for enhancing the k(cat). Thus, while membranes containing phosphatidyl-L-serine enhance condensation of the enzyme with its cofactor and substrate, their largest effect is activation of the assembled factor VIIIa-factor IXa enzyme complex. PMID- 8626657 TI - A single histidine is required for activity of cytochrome c peroxidase from Paracoccus denitrificans. AB - The diheme cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN- bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state. PMID- 8626658 TI - Structural requirements of heparin binding to Chlamydia trachomatis. AB - Heparin is a functional and structural analog of the Chlamydia trachomatis heparan sulfate-like attachment ligand that mediates infectivity by bridging chlamydiae to eukaryotic cells. The binding of heparin to the Chlamydia organism's surface was characterized by a direct binding assay. Although for two C. trachomatis biovars the binding by heparin was saturable, trachoma biovar organisms bound twice the amount of heparin than lymphogranuloma venereum biovar organisms. To prove the structural nature of the heparan sulfate-like ligand interactions, a range of heparin-derived oligosaccharides and sulfation-modified species of heparin were compared for their ability to compete with [3H]heparin for binding to chlamydial organisms and for inhibition of chlamydial attachment and infection of eukaryotic host cells. The assays revealed that a decasaccharide was the minimal chain length required to effectively bind C. trachomatis organisms, compete with the host cell receptor and rescue infectivity. In addition, a moderately sulfated adhesin analog, N-desulfated, N-acetylated heparin, was able to compete with chlamydial organisms for host cell receptors, whereas this derivative could not compete with [3H]heparin for binding to chlamydial organisms. These results indicate that the specificity of the eukaryotic cell receptor and the chlamydial surface acceptor differ in their fine structure requirements of ligand binding, and that the size and sulfation density of the heparan sulfate-like ligand each contribute to its ability to bind and bridge chlamydiae to eukaryotic cells. PMID- 8626659 TI - Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins. AB - PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies. PMID- 8626660 TI - Ca2+ mobilizing action of sphingosine in Jurkat human leukemia T cells. Evidence that sphingosine releases Ca2+ from inositol trisphosphate- and phosphatidic acid sensitive intracellular stores through a mechanism independent of inositol trisphosphate. AB - Effects of sphingosine on Ca2+ mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with an ED50 of around 8 microM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca2+]i, which declined to the resting level in the absence of extracellular Ca2+. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca2+]i induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca2+]i induced by OKT3 in the presence of Ca2+. OKT3 increased IP3 formation, but sphingosine did not affect the level of IP3 by itself nor did it cause IP3 formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP3 released Ca2+ from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca2+]i and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1 phosphate slightly activated DG kinase without elevation of [Ca2+]i. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response to [Ca2+]i to sphingosine. Phosphatidic acid (PA) elevated [Ca2+]i, after which it declined to a resting level even in the presence of extracellular Ca2+. In accordance with this, PA did not stimulate 45Ca2+ uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca2+]i induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca2+. Under similar conditions, pretreatment with PA affected an elevation of [Ca2+]i induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca2+]i. In permeabilized Jurkat cells, the addition of IP3 did not release Ca2+, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca2+ ATPase inhibitor, abolished the rises of [Ca2+]i induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca2+. The present results suggest that at least two kinds of intracellular Ca2+ stores exist in Jurkat cells, both of which are IP3- and PA-sensitive, and that sphingosine mobilizes Ca2+ from both stores in an IP3-independent manner. Furthermore, the IP3- but not the PA-sensitive intracellular Ca2+ store seems to regulate Ca2+ entry induced by sphingosine. PMID- 8626661 TI - T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex. AB - After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini. PMID- 8626662 TI - Multiple forms of phospholipase D inhibitor from rat brain cytosol. Purification and characterization of heat-labile form. AB - Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP2). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70 degrees C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP2, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP2-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles. PMID- 8626663 TI - Hydrolysis of platelet vitronectin by calpain. AB - Vitronectin (Vn) is not only a major adhesive glycoprotein present in platelets but also regulates proteolytic enzyme cascades, including the blood coagulation, fibrinolytic, and complement systems. In human platelet lysates prepared by freeze-thawing or by the addition of nonionic detergent, the Vn antigen content was drastically reduced in comparison with lysates prepared in the presence of SDS, suggesting that Vn is hydrolyzed by platelet-associated enzymes. Exogenously added purified human Vn and Vn present in plasma were also cleaved by these enzyme systems. Degradation was mediated by a nonsecreted or membrane-associated protease system that was inhibited by E-64, EDTA, and leupeptin but not inhibitors of serine and aspartic proteases, suggesting an involvement of calcium dependent cysteine proteases. Consistently, calpastatin inhibited the hydrolysis of Vn, suggesting that Vn is a substrate for calpain. This was confirmed in a purified system. Vn was cleaved by calpains I and II in a dose- and time dependent manner, resulting in defined Vn fragments with similar electrophoretic mobility in comparison with those detected in platelet lysates. Functional characterization of the calpain-hydrolyzed Vn revealed that while the type 1 plasminogen activator inhibitor binding activity was unchanged, the heparin and cell binding functions were destroyed. These results suggest that calpains released upon platelet membrane damage or upon tissue injury and necrosis differentially regulate functional domains of the Vn molecule. PMID- 8626664 TI - The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb. AB - The protooncogene for Spi-1/PU.1 is an Ets-related transcription factor overexpressed during Friend erythroleukemia. The molecular basis by which Spi 1/PU.1 is involved in the erythroleukemic process remains to be elucidated. By using an immobilized protein binding assay, we have identified a 55-kDa protein as a putative partner of Spi-1/PU.1 protein. Microsequence analysis revealed that this 55-kDa protein was p54nrb (nuclear RNA-binding protein, 54 kDa) a RNA binding protein highly similar to the splicing factor PSF (polypyrimidine tract binding protein-associated splicing factor). In this paper, we show that Spi 1/PU.1 impedes the binding of p54nrb to RNA and alters the splicing process in vitro. Moreover, we present evidence that the transcriptional factor Spi-1/PU.1, unlike other Ets proteins, is able to bind RNA. Altogether, these results raise the intriguing possibility that the functional interference observed between Spi 1/PU.1 and RNA-binding proteins might represent a novel mechanism in malignant erythropoiesis. PMID- 8626665 TI - Transcription factor IIA mutations show activator-specific defects and reveal a IIA function distinct from stimulation of TBP-DNA binding. AB - The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and mediates transcriptional activation by distinct classes of activators. To elucidate the function of TFIIA in transcriptional activation, point mutants were created in the human TFIIA-gamma subunit at positions conserved with the yeast homologue. We have identified a class of TFIIA mutants that stimulate TBP DNA binding (T-A complex) but fail to support transcriptional activation by several different activators, suggesting that these mutants are defective in their ability to facilitate an activation step subsequent to TBP promoter binding. Point mutations of the hydrophobic core of conserved residues from 65 to 74 resulted in various activation-defective phenotypes. These residues were found to be important for TFIIA gamma-gamma interactions, suggesting that gamma-gamma interactions are critical for TFIIA function as a coactivator. A subset of these TFIIA-gamma mutations disrupted transcriptional activation by all activators tested, except for the Epstein-Barr virus-encoded Zta protein. The gamma Y65F, gamma W72A, and gamma W72F mutants mediate Zta activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation. The gamma W72A mutant failed to stimulate TFIID-DNA binding (D-A complex) but was able to form a complex with TFIID and DNA in the presence of Zta (Z-D-A complex). Thus, the ability of Zta to activate transcription with gamma W72A appears to result from a unique ability to form the stable Z-D-A complex with this mutant. Our results show that different activators utilize the general factor TFIIA in unique ways and that TFIIA contributes transcription activation functions in addition to the facilitation of TBP-DNA binding. PMID- 8626666 TI - A single point mutation in epsilon-COP results in temperature-sensitive, lethal defects in membrane transport in a Chinese hamster ovary cell mutant. AB - At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary cell conditionally lethal, temperature-sensitive (ts) mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type epsilon-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF cells of a point mutation in the epsilon-COP gene, Glu251 to Lys251, which prevents the corresponding cDNA from correcting the defects in transfected ldlF cells and the immunochemical analysis of the synthesis, structure, and stability of epsilon-COP. At the permissive temperature (34 degrees C), the steady state level of ts-epsilon-COP in ldlF cells was about half that of epsilon-COP in wild-type Chinese hamster ovary cells and the isoelectric point of ts-epsilon-COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts-epsilon-COP was temperature sensitive (t1/2 > 6 h at 34 degrees C and approximately 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in ldlF cells after incubation at 39.5 degrees C for > 6h. The steady state levels in ldlF cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in epsilon-COP that causes instability at 39.5 degrees C is responsible for all of the temperature-sensitive defects in ldlF cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. ldlF cells should be useful for the future analysis of the structure and function of epsilon-COP, the assembly of COPs into coatomers, and the participation of coatomers in intracellular membrane transport. PMID- 8626668 TI - Neuronal nitric-oxide synthase-mu, an alternatively spliced isoform expressed in differentiated skeletal muscle. AB - Nitric oxide (NO) functions as a molecular mediator in numerous processes in cellular development and physiology. Differential expression and regulation of a family of three NO synthase (NOS) gene products help achieve this diversity of action. Previous studies identify post-translational modification and interaction of NOS with specific protein targets as tissue-specific modes of regulation. Here, we show that alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle as its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family. PMID- 8626667 TI - Negative regulatory element involved in the hormonal regulation of GlcNAc-1-P transferase gene in mouse mammary gland. AB - The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. The involvement of cisacting elements in regulating the expression of the mouse GPT (mGPT) gene was investigated by transient transfections of various GPT promoter/luciferase (Luc) constructs into primary mouse mammary epithelial cells. A series of 5'-deletions of the GPT promoter identified a distal negative regulatory region (base pairs 1057 to -968) and deletion of this region results in enhanced hormonal induction (approximately 7-fold) with no effect on basal promoter activity. Electrophoretic mobility shift assays (EMSA) performed with nuclear extracts from different developmental stages of mouse mammary gland demonstrated that the binding activity of the nuclear proteins to the distal negative regulatory region was predominant in virgin stage as compared with pregnant and lactating stages. EMSA performed with nuclear extracts from virgin explants showed that the binding activity was markedly decreased after cultivation with the combination of the three lactogenic hormones. DNase I footprinting analysis identified two pentamer direct repeat motifs, AGGAA and GAAAC, within the negative regulatory region. EMSA competition experiments showed that mutations within the direct repeats failed to compete for binding of the nuclear proteins to labeled wild type oligonucleotide. Transcription from the promoter containing the mutated direct repeats was increased greatly, consistent with the conclusion that these motifs functions in vivo to repress GPT gene expression. These data suggest the importance of the negative regulatory region in hormonal control of mGPT gene expression in mammary gland. PMID- 8626669 TI - D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis. AB - Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein. PMID- 8626670 TI - Evidence that syntaxin 1A is involved in storage in the secretory pathway. AB - Syntaxin 1A is a nervous system-specific protein thought to function during the late steps of the regulated secretory pathway by mediating the docking of secretory vesicles with the plasma membrane. We have examined the effects of transiently overexpressing syntaxin 1A on protein secretion in constitutively secreting cell lines that do not normally express the protein. Syntaxin 1A showed the constitutive release of marker proteins human growth hormone (hGH) and vesicular stomatitis virus glycoprotein from COS-1 cells, increasing the intracellular half-life of human growth hormone from 90 min to 18 h. A similar effect was observed in HEK 293 cells. Immunofluorescence microscopy revealed that these secretory proteins were concentrated in the periphery of the cell. The effect was specific for the full-length neuronal protein. Neither a syntaxin 1A variant which lacks a membrane attachment domain nor syntaxin 2 caused the cells to retain human growth hormone. The effect of syntaxin 1A was partially reversed by incubating the cells with botulinum type C1 neurotoxin, which specifically cleaves syntaxin 1A. Release of human growth hormone from syntaxin 1A-expressing cells was maintained during a blockade of protein synthesis, suggesting that the hormone was being released from a pool of stored vesicles which accumulated before the addition of cycloheximide. The existence of a post-Golgi storage compartment in syntaxin 1A-expressing cells was confirmed using brefeldin A to collapse the Golgi stacks in both HEK 293 and COS-1 cells. Brefeldin A rapidly blocked growth hormone release in control cultures while having no effect on release in cells expressing syntaxin 1A. Reducing the temperature to 19 degrees C, which inhibits transport from the trans-Golgi network, also inhibited hGH secretion from cells without syntaxin 1A but had little effect on hGH secretion from cells with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released. PMID- 8626671 TI - Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. AB - Okadaic acid has been described previously as being a negative regulator of insulin signaling, as it inhibits insulin stimulation of glucose transport. In addition, this drug induces on insulin receptor substrate-1 (IRS-1) a decrease in tyrosine phosphorylation, concomitantly with an increase in serine/threonine phosphorylation. The present work was aimed at the identification of the serine/threonine residues that, upon phosphorylation, might be involved in modulating insulin signaling. To this end, we studied double-point mutants of IRS 1, in which serines 612/632 and 662/731 were replaced with alanine. These are four plausible sites of phosphorylation by mitogen-activated protein kinases and are in the immediate proximity of tyrosine residues, which are potential sites of interaction with phosphatidylinositol 3-kinase Src homology 2 domains. Using transient expression in 293 EBNA cells, we demonstrate that serines 612, 632, 662, and 731 and mitogen-activated protein kinases are not involved in the okadaic acid effect on IRS-1. Rather, these serines appear to play a role in modulating basal and insulin-stimulated IRS-1 tyrosine phosphorylation, association of IRS-1, with p85, and phosphatidylinositol 3-kinase activity in the IRS-1.p85 immune complex, since mutation of these sites enhances these events. Our findings suggest the existence of an IRS-1 desensitization mechanism resulting from serine/threonine phosphorylation, occurring at least on serines 612, 632, 662, and 731. PMID- 8626672 TI - Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu271 Ala277 of domain 3 in the heavy chain of high molecular weight kininogen. AB - The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM. A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292. Peptides Phe279 Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity. A heptapeptide Leu271-Ala277 inhibited thrombin induced aggregation of platelets with an IC50 of 65 microM. The effect is specific for the activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets. PMID- 8626673 TI - A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. AB - The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins. PMID- 8626674 TI - Anti-metatype antibody stabilization of Fv 4-4-20 variable domain dynamics. AB - Anti-metatype (anti-Met) antibodies are immunoglobulins that specifically recognize and stabilize antibodies in their liganded or metatypic state, but lack specificity for either the hapten or the unliganded antibody. Autologous anti-Met antibodies were previously observed in vivo, suggesting that a metatypic autoantibody response could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from circulation. The first elicited anti-Met antibodies were against the fluorescein-liganded high affinity murine anti-fluorescein monoclonal antibody 4-4-20. The fluorescein-hapten system has proved to be an invaluable tool for both the recognition and characterization of the metatypic response by utilization of its spectral properties. In this investigation, hydrostatic pressure measurements, in conjunction with fluorescence spectroscopy, were performed on the recombinant Fv derivative (Fv 4 4-20) of the high affinity anti-fluorescein monoclonal antibody 4-4-20 complexed to anti-Met antibodies to study the influence of anti-Met antibodies of Fv 4-4-20 intervariable domain interactions. Anti-Met antibodies bound to liganded Fv 4-4 20 were observed to cause a change in the fluorescence properties of fluorescein that was not observed when anti-Met antibodies were bound to the liganded parent immunoglobulin. The variation of these spectral properties upon addition of anti Met antibodies was shown to be correlated with dissociation of the variable domains in Fv 4-4-20 in response to its interaction with the anti-Met antibody. The ability to cause variable domain dissociation was dependent on whether monoclonal or polyclonal anti-Met antibodies were bound to the metatype. A model was proposed that elucidated the interaction of anti-Met antibodies, polyclonal and monoclonal, with variable domains of the primary anti-antigen antibody. PMID- 8626675 TI - Differential modulation of G1 cyclins and the Cdk inhibitor p27kip1 by platelet derived growth factor and plasma factors in density-arrested fibroblasts. AB - Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G1/S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27kip1. Exposure of PDGF, which stimulates cell cycle entry but not progression through G1, induces the formation of cyclin D1-Cdk4 complexes that bind p27kip1 and titrate the pool of Kip1 available to inhibit Cdk2. In addition, PDGF stimulates a moderate transient reduction in the abundance of p27kip1 protein. However, limited expression of cyclin E and cyclin A is observed after PDGF treatment, and in the absence of PPP, p27 levels are sufficient to bind and inactivate existing cyclin-Cdk complexes. Although plasma does not significantly increase the proportion of Kip1 bound to cyclin D1-Cdk4, stimulation of PDGF treated cells with plasma does overcome the threshold inhibition of p27kip1 by further increasing the expression of cyclins E and A and decreasing the amount of Kip1 over a prolonged time period. Our results indicate that the distinct mitogenic activities of PDGF and PPP differentially influence the activation of cyclin E- and cyclin A-associated kinases that ultimately regulate entry into S phase. PMID- 8626676 TI - Site of covalent labeling by a photoreactive batrachotoxin derivative near transmembrane segment IS6 of the sodium channel alpha subunit. AB - The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site. PMID- 8626677 TI - Suppression of glycolysis is associated with an increase in glucose cycling in hepatocytes from diabetic rats. AB - Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes. PMID- 8626678 TI - The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen activated protein kinase pathway. AB - The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O (thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1. PMID- 8626679 TI - Interleukin-1 beta and tumor necrosis factor-alpha stimulate the cat-2 gene of the L-arginine transporter in cultured vascular smooth muscle cells. AB - The production of nitric oxide (NO) from L-arginine by nitric oxide synthase (NOS) in cytokine-stimulated vascular smooth muscle cells (VSMC) is thought to play an important role in the pathophysiology of several vascular disease states including septic shock. This study examines the relationship between cytokine stimulated NO production and L-arginine transport in cultured VSMC. Cultured VSMC from rat aorta were stimulated with interleukin-1 beta, tumor necrosis factor alpha, and/or angiotensin II (Ang II); and the accumulation of nitrite, a stable product of NO metabolism, in the culture media and the rates of net L-arginine uptake were measured. Interleukin-1 beta and tumor necrosis factor-alpha, alone or in combination, stimulated both the uptake of L-arginine and the accumulation of nitrite in the culture media in a dose-dependent manner. Inhibition of NOS activity by substituted analogues of L-arginine had no effect on cytokine stimulated L-arginine transport. Ang II in the presence of cytokines up-regulated L-arginine transport while inhibiting nitrite accumulation. Two forms of the L arginine transporter, cat-1b and cat-2, are expressed in VSMC. Northern analysis revealed that the cytokine-stimulated increase in L-arginine transport coincided with increased levels of cat-2 mRNA. In contrast, cat-1b does not appear to be regulated by cytokines at the mRNA level, although significant increases in response to Ang II were observed. These results show that, while cytokines can stimulate both NOS activity and L-arginine uptake, NO production is not required to signal the increase in L-arginine transport. Furthermore, Ang II and cytokine stimulation of L-arginine uptake involves the differential regulation of the cationic amino acid transporter (cat) genes. PMID- 8626680 TI - The role of Phe-92 in the Ca(2+)-induced conformational transition in the C terminal domain of calmodulin. AB - Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92. PMID- 8626682 TI - Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli. AB - Oxidative folding of recombinant human stem cell factor (rhSCF) produced in Escherichia coli was investigated in vitro. Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography. Both I-1 and I-2 contain a native-like disulfide bond, Cys4-Cys89 and Cys43-Cys138, respectively, and I-3 forms a mispaired disulfide, Cys43-Cys89. These forms appear to reach steady state equilibrium and are important folding intermediates. I-1 was found to be the prominent intermediate that directly folds into native rhSCF (N); and the thermodynamically less stable I-2 favors rearrangment into I-1. I-3 may serve as an intermediate for disulfide rearrangment between I-1 and I-2. I-4 and I-5, which are disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF folding. Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by high performance liquid chromatography, proceed with the remaining oxidative folding process after reconstitution. Iodoacetate-trapped I-1 and I-2 contain low alpha-helical content and some tertiary structure, while I-3 and reduced rhSCF have little ordered structure. Gel filtration/light-scattering experiments indicate that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating that rhSCF dimerization precedes formation of disulfide bonds. I-1, I-2, I-3, and the C43,138A analog lacking Cys43-Cys138 bond are not biologically active or exhibit significantly lower activity. The two disulfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biological activities. PMID- 8626681 TI - Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine. AB - Rate constants for the interaction of fatty acids (FA) with fatty acid binding proteins (FABP) from adipocyte (A-FABP), heart (H-FABP), and intestine (I-FABP) were determined by using stopped-flow fluorometry and ADIFAB, the fluorescent probe of free fatty acids (FFA), or a new FFA probe, ADIFAB2, constructed by derivatizing with acrylodan the Leu72 --> Ala mutant of I-FABP. ADIFAB2, because its binding affinities are about 10-fold greater than ADIFAB, was found to be more accurate for monitoring the kinetics of the higher affinity reactions. On- (kappa on) and off- (kappa off) rate constants were determined as a function of temperature. Our results reveal that in all cases the FA-FABP equilibrium is achieved within 2 s at 37 degrees C and within 20 s at 10 degrees C. Off-rate constants varied by about 10-fold among the different underivatized FABPs; kappa off values were smallest for H-FABP and largest for A-FABP, while kappa on values for these proteins generally varied by less than 2-fold. The results show that the previously reported larger affinities of I- and H-FABPs as compared to A-FABP are primarily a reflection of larger kappa on values for I-FABP and smaller kappa off values for H-FABP. Eyring transition state theory was used to evaluate the activation thermodynamic parameters for both on- and off-reactions and the results show that in virtually all cases the rate-limiting steps are predominantly enthalpic. Activation free energies for binding to ADIFAB are generally composed of about 8 kcal/mol unfavorable enthalpy and about a 1 kcal/mol favorable entropic contribution. For the underivatized FABPs the activation free energies are all about 7 +/- 0.3 kcal/mol, suggesting that the transition state for entering or leaving the binding site involves a common protein structural change. We suggest that entering or leaving the FABP binding cavity involves similar mechanisms for all 3 FABPs and may involve amino acid residues located within the portal regions of these proteins. PMID- 8626683 TI - Isolation and characterization of a disulfide-linked human stem cell factor dimer. Biochemical, biophysical, and biological comparison to the noncovalently held dimer. AB - Distinct from the noncovalently linked recombinant human stem call factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide linked dimer. PMID- 8626684 TI - Nitric oxide attenuates vascular smooth muscle cell activation by interferon gamma. The role of constitutive NF-kappa B activity. AB - Atherogenesis involves cellular immune responses and altered vascular smooth muscle cell (SMC) function. Cytokines such as interleukin (IL)-1 alpha and interferon-gamma (IFN-gamma) may contribute to this process by activating SMC. To determine whether the anti-atherogenic mediator, nitric oxide (.NO), can modulate cytokine-induced SMC activation, we investigated the effects of various .NO generating compounds on the expression of intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). Induction of ICAM-1 expression by IL-1 alpha and VCAM-1 expression by IFN-gamma was attenuated by .NO donors but not by cGMP analogues. Nuclear run-on assays and transfection studies using various VCAM 1 promoter constructs linked to the chloramphenicol acetyl-transferase reporter gene showed that .NO repressed IFN-gamma-induced VCAM-1 gene transcription, in part, through inhibition of nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assay revealed that SMC possess basal constitutive NF-kappa B activity, which was augmented by treatment with IL-1 alpha. In contrast, IFN gamma induced and activated interferon regulatory factor (IRF)-1 but had little effect on basal constitutive NF-kappa B activity. .NO donors had no inhibitory effect on IRF-1 activation but did inhibit basal and IL-1 alpha-stimulated NF kappa B activation. These findings suggest that the induction of ICAM-1 and VCAM 1 expression requires NF-kappa B activation and that .NO attenuates IFN-gamma induced VCAM-1 expression primarily by inhibiting basal constitutive NF-kappa B activity in SMC. PMID- 8626685 TI - A cDNA encoding the calcitonin gene-related peptide type 1 receptor. AB - Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological effects including potent vasodilator activity. We report here the cloning of a complementary DNA (cDNA) encoding a human CGRP1 receptor, which shares significant peptide sequence homology with the human calcitonin receptor, a member of the G-protein-coupled receptor superfamily. Northern blot analysis revealed that the messenger RNA for this receptor is predominantly expressed in the lung and heart. In situ studies showed specific localization of the receptor mRNA to alveolar cells in the lung and to cardiac myocytes in the heart. Stable expression of the cDNA in human embryonic kidney 293 (HEK 293) cells produced specific, high affinity binding sites for CGRP that displayed pharmacological and functional properties very similar to native human CGRP1 receptor. Exposure of these cells to CGRP resulted in a 60-fold increase in cAMP production, which was inhibited in a competitive manner by the CGRP1 receptor antagonist, CGRP-(8-37). PMID- 8626686 TI - Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts. AB - Recent studies indicate that insulin-like growth factor-II (IGF-II) acts as an autocrine differentiation factor for skeletal myoblasts in culture. IGF-II mRNA and protein are induced as early events in muscle differentiation, and the rate and extent of IGF-II secretion correlate with both biochemical and morphological differentiation. Here we show that IGF-II also functions as an essential survival factor during the transition from proliferating to differentiating myoblasts. Stably transfected C2 muscle cell lines were established in which a mouse IGF-II cDNA was expressed in the antisense orientation relative to the constitutively active Moloney sarcoma virus promoter. IGF-II antisense cells proliferated normally in growth medium containing 20% serum but underwent rapid death when placed in low serum differentiation medium. Death was accompanied by characteristic markers of apoptosis with more than 90% of cells showing DNA fragmentation within 12-16 h. Myoblast death was prevented by IGF-I, des [1-3] IGF-I, IGF-II, and insulin with a dose potency consistent with activation of the IGF-I receptor; death also could be blocked by the protein synthesis inhibitor, cycloheximide. Exogenous IGFs additionally stimulated passage through a single cell cycle and subsequently induced terminal differentiation. Cell survival and cell cycle progression also were enhanced by fibroblast growth factor-2 and platelet-derived growth factor-bb, but these peptides did not promote differentiation. Our results define a novel system for studying apoptotic cell death and its prevention by growth factors, underscore the importance of IGF action in minimizing inappropriate cell death, and indicate that shared signal transduction pathways may mediate myoblast survival in vitro. PMID- 8626687 TI - APP-BP1, a novel protein that binds to the carboxyl-terminal region of the amyloid precursor protein. AB - beta-Amyloid protein precursors (APPs, 695-770 amino acids) are the source of the 39-43 amino acid beta-amyloid (A beta) peptides that comprise diffuse and fibrillar deposits in the cerebral cortex and vasculature of Alzheimer's disease brains. A beta is thought to play a role in the pathogenesis of Alzheimer's disease, and, hence, considerable effort has been invested in defining the means by which A beta is generated from the APPs. Knowledge of the normal function of the APPs is sure to provide insights into the genesis and pathological persistence of A beta in Alzheimer's disease. APP is a cell surface protein with a large extracellular amino-terminal domain, a single transmembrane segment, and a short cytoplasmic tail. Its location and structural features characteristic of a receptor for signal transduction led us to search for potential effector proteins capable of binding and interacting with its cytoplasmic domain. Here, we report the cloning of a cDNA encoding one such protein. This ubiquitously expressed 59-kDa APP-binding protein, called APP-BP1, is 61% similar to a protein encoded by the Arabidopsis AXR1 gene, required for normal response to the hormone auxin, and is a relative of the ubiquitin activating enzyme E1. PMID- 8626688 TI - Overexpression of Ha-ras selectively in adipose tissue of transgenic mice. Evidence for enhanced sensitivity to insulin. AB - To determine the role of Ras-dependent signaling pathways in adipocyte function, we created transgenic mice that overexpress Ha-ras in adipocytes using the aP2 fatty acid-binding protein promoter/enhancer ligated to the human genomic ras sequence. ras mRNA was increased 8-17-fold and Ras protein 4-5-fold in white and brown fat, with no overexpression in other tissues. The subcellular distribution of overexpressed Ras paralleled that of endogenous Ras. [U-14C]Glucose uptake into isolated adipocytes was increased approximately 2-fold in the absence of insulin, and the ED50 for insulin was reduced 70%, with minimal effect on maximally stimulated glucose transport. Expression of Glut4 protein was unaltered in transgenic adipocytes, but photoaffinity labeling of transporters in intact cells with [3H]2-N-[4-(1-azi-Z,Z,Z-trifluoroethyl)benzoyl]-1,3-bis-(D-mann os-4- yloxy)-2-propylamine revealed 1.7-2.6-fold more cell-surface Glut 4 in the absence of insulin and at half-maximal insulin concentration (0.3 nM) compared with nontransgenic adipocytes. With maximal insulin concentration (80 nM), cell surface Glut4 in nontransgenic and transgenic adipocytes was similar. Glut1 expression and basal cell-surface Glut1 were increased 2-2.9-fold in adipocytes of transgenic mice. However, Glut1 was much less abundant than Glut4, making its contribution to transport negligible. These in vitro changes were accompanied by in vivo alterations including increased glucose tolerance, decreased plasma insulin levels, and decreased adipose mass. We conclude that ras overexpression in adipocytes leads to a partial translocation of Glut4 in the absence of insulin and enhanced Glut4 translocation at physiological insulin concentration, but no effect with maximally stimulating insulin concentrations. PMID- 8626689 TI - Molecular cloning of the gene for human leukotriene C4 synthase. Organization, nucleotide sequence, and chromosomal localization to 5q35. AB - Leukotriene C4 (LTC4) synthase catalyzes the conjugation of LTA4 with reduced GSH to form LTC4, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC4 synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S transferase super-family. Genomic cloning from a P1 library now reveals that the gene for LTC4 synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC4 synthase align identically with those of FLAP; however, the small size of the LTC4 synthase gene contrasts with the > 31 kilobase pair size reported for FLAP. Confirmation of the LTC4 synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A)+ RNA from culture derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5' of the ATG translation start site. The 5'-flanking region revealed a GC-rich promoter sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5' from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5' and 3' oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC4 synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma. PMID- 8626690 TI - Inter-alpha-trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface. AB - Inter-alpha-trypsin inhibitor (ITI), a human serum protease inhibitor of molecular mass 240 kDa which may release physiological derivatives, has been shown to interact with hyaluronic acid (HA), resulting in pericellular matrix stabilization (Chen, L., Mao, S.J.T., McLean, L. R., Powers, R. W., and Larsen, W. J. (1994) J. Biol. Chem. 269, 28282-28287). The purpose of this study is to determine whether ITI binding to tumor cell surface is mediated by urinary trypsin inhibitor (UTI)-receptor or cell-associated hyaluronic acid (HA). We demonstrated specific complex formation of the heavy (H) chains of ITI with HA. Binding of the H-chains of ITI to immobilized HA was detected and quantified using colorimetric immunoassays. Binding was time-, temperature-, and concentration-dependent. However, UTI and HI-8 (the carboxyl terminus of UTI) failed to bind to immobilized HA. ITI bound to HA remained functional protease inhibiting activity. After incubation of SMT-cc1 cells with purified biotinylated ITI, biotinylated ITI is bound to the cells, dissociated, and gives rise to the H chains and UTI on the cell surface. The cell surface receptor-bound UTI derived from ITI may be the result of the limited proteolysis on the cell surface. In the cells treated with hyaluronidase, bound H-chains disappeared from the surface of the cells, while most of the cell surface ITI derivatives was present in deglycosylated UTI (28 kDa). It is suggested that the binding of ITI to the cell surface is mediated by HA on the cells. This was confirmed by the fact that the hyaluronidase-treated cells can abolish the ITI binding. The cell surface UTI formation was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and eglin C, suggesting that elastase-like enzyme(s) may be responsible for the UTI formation. Preincubation of the cells with UTI did not decrease in exogenously added ITI on the cell surface. A model for cell surface UTI formation is proposed in which ITI binding to cells from serum used for the culture is followed by the limited proteolysis by trace amounts of active serine proteases, to form cell-surface receptor-bound UTI and the H-chains intercalated into cell surface HA. This process is subject to regulation of cell-associated UTI and of stabilization of pericellular matrix. PMID- 8626691 TI - Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro neurotensin. AB - The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a > 80% reduction of pro NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro NT/NN with brain and intestinal phenotype, respectively. PMID- 8626692 TI - Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors. AB - The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor alpha precursor (proTGF-alpha) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-alpha, cell adhesion receptor L-selectin, interleukin 6 receptor alpha subunit, beta-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene. PMID- 8626694 TI - Induction of c-Erb A-AP-1 interactions and c-Erb A transcriptional activity in myoblasts by RXR. Consequences for muscle differentiation. AB - We have previously shown that c-Erb A and v-Erb A display a cell-specific activity in avian myoblasts. In this work, we have compared the molecular basis of thyroid hormone action in HeLa cells and in QM7 myoblasts. The transcriptional activity of c-Erb A alpha 1 through a palindromic thyroid hormone response element (TRE) was similar in both cell types. However, c-Erb A did not activate gene transcription through a direct repeat sequence (DR) 4 TRE in myoblasts in contrast to results obtained in HeLa cells. Moreover, whereas retinoic acid receptor-AP-1 interactions were functional in both cell types, thyroid hormone receptor (T3R)-AP-1 interactions were only functional in HeLa cells. Using electrophoretic mobility shift assays, functional tests, and Northern blot experiments, we observed that RXR isoforms are not expressed in proliferating myoblasts. Expression of RXR gamma in these cells did not influence T3R transcriptional activity through a palindromic TRE but induced such an activity through a DR4 TRE. Moreover, it restored c-Erb A-AP-1 functionality in QM7 myoblasts and enhanced the myogenic influence of T3. We also observed that c-Jun overexpression in proliferating QM7 cells restored T3R transcriptional activity through a DR4 TRE. Therefore, alternative mechanisms are involved in the induction of T3R transcriptional activity according to the cell status (proliferation: c-Jun; differentiation: RXR). In addition we provide the first evidence that RXR is required to allow inhibition of AP-1 activity by ligand activated T3R. Lastly, we demonstrate the importance of RXR in the regulation of myoblast differentiation by T3. PMID- 8626693 TI - Yeast proteins related to the p40/laminin receptor precursor are essential components of the 40 S ribosomal subunit. AB - We report here the isolation of two genes from the yeast, Saccharomyces cerevisiae, that encode proteins closely related to mammalian p40/laminin receptor precursors (LRPs). The yeast genes, designated YST1 and YST2, encode proteins with over 95% amino acid sequence identity with one another and over 60% identity with the human p40/laminin receptor precursor. The Yst/p40/37-LRP proteins are also more distantly related to the S2 family of ribosomal proteins. Analysis of the distribution of Yst1 tagged with the c-myc epitope revealed that the Yst proteins are components of the 40 S ribosomal subunit. Disruption of either YST1 or YST2 causes a significant reduction in growth rate, while disruption of both genes is lethal. Compared to wild type, polysome profiles in strains lacking either YST1 or YST2 show a pronounced shift from larger to smaller polysomes. This shift is accompanied by a substantial increase in free 60 S subunits and reduced levels of 40 S subunits. We conclude that the Yst proteins are required for translation and contribute to the assembly and/or stability of the 40 S ribosomal subunit. PMID- 8626695 TI - Requirement for the carboxyl-terminal domain of Saccharomyces cerevisiae carbamoyl-phosphate synthetase. AB - The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a 45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2 subunit accepting the ammonia moiety cleaved from glutamine, binding all of the remaining substrates and carrying out all of the other catalytic events. CPA2 is composed of two apparently duplicated amino acid sequences involved in binding the two ATP molecules needed for carbamoyl phosphate synthesis and a carboxyl-terminal domain which appears to be less tightly folded than the remainder of the protein. Using deletion mutagenesis, we have established that essentially all of the carboxyl-terminal domain of CPA2 is required for catalytic function and that even small truncations lead to significant changes in the CPA2 conformation. In addition, we have demonstrated that the C-terminal region of CPA2 can be expressed as an autonomously folded unit which is stabilized by specific interactions with the remainder of CPA2. We also made the unexpected finding that, even when ammonia is used as the substrate and there is no catalytic role for CPA1, interaction with CPA1 led to an increase in the Vmax of CPA2 in crude extracts. PMID- 8626696 TI - T-cell activation leads to rapid stimulation of translation initiation factor eIF2B and inactivation of glycogen synthase kinase-3. AB - Mitogenic stimulation of T-lymphocytes causes a rapid activation or protein synthesis, which reflects in part increased expression of many translation components. Their levels, however, rise more slowly than the rate of protein synthesis, indicating an enhancement of the efficiency of their utilization. Initiation factor eIF2B catalyzes a key regulatory step in the initiation of translation, and we have therefore studied its activity following T-cell activation. eIF2B activity rises quickly, increasing as early as 5 min after cell stimulation. This initial phase is followed by an additional slow but substantial increase in eIF2B activity. The level of eIF2B subunits did not change over the initial rapid phase but did increase at later time points. Northern analysis revealed that levels of eIF2B mRNA only rose during the later phase. The rapid activation of EIF2B following mitogenic stimulation of T-cells is therefore mediated by factors other than its own concentration. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase-3 (GSK-3), the activity of which rapidly decreases following T-cell activation. Since phosphorylation of eIF2B by GSK-3 appears to inhibit nucleotide exchange in vitro, this provides a potential mechanism by which eIF2B may be activated. PMID- 8626697 TI - C-terminal mutations that alter the turnover number for 3-O-methylglucose transport by GLUT1 and GLUT4. AB - Turnover numbers for 3-O-methylglucose transport by the homologous glucose transporters GLUT1 and GLUT4 were compared to those for truncated and chimeric transporters expressed in Xenopus oocytes to assess potential regulatory properties of the C-terminal domain. The ability of high intracellular sugar concentrations to increase the turnover number for sugar entry ("accelerated exchange") by GLUT1 and not by GLUT4 was maintained in oocytes. Replacing the GLUT1 C terminus with that of GLUT4 stimulated turnover 1.6-fold, but abolished accelerated exchange. Thus, the GLUT1 C terminus permits accelerated exchange by GLUT1, but in doing so must interact with other GLUT1 specific sequences since the GLUT4ctrm1 chimera did not exhibit this kinetic property. Removal of 38 C terminal amino acids from GLUT4 reduced its turnover number by 40%, whereas removing only 20 residues or replacing its C terminus with that of GLUT1 increased its turnover number 3.5-3.9 fold. Therefore, using mechanisms independent of those which alter transporter targeting to the plasma membrane, C terminal mutations in either GLUT1 or GLUT4 can activate transport normally restricted by the native C-terminal domain. These results implicate the C termini as targets of physiological factors, which through covalent modification or direct binding might alter C-terminal interactions to regulate intrinsic GLUT1 and GLUT4 transporter activity. PMID- 8626698 TI - Increased expression of protein kinase C beta activates ERK3. AB - In a prior study, we have shown that stable transfection of expression plasmids for protein kinases C beta 1 (PKC beta 1) or PKC beta 2 into differentiated colon cancer cells led to elevated levels of PKC beta 1 or PKC beta 2 protein and PKC beta kinase activities in the transfectants, without altering PKC alpha levels. PKC gamma is not found in these cells, so the major modulation was in PKC beta. PKC beta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKC beta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKC beta 1 and each of two PKC beta 2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKC beta 1, PKC beta 2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a > 10 fold increase in ERK3 activity in each PKC beta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKC beta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKC beta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKC beta 1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKC beta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKC beta 1 or PKC beta 2. PMID- 8626699 TI - Cloning and characterization of MEK6, a novel member of the mitogen-activated protein kinase kinase cascade. AB - Mitogen-activated protein kinases are members of a conserved cascade of kinases involved in many signal transduction pathways. They stimulate phosphorylation of transcription factors in response to extracellular signals such as growth factors, cytokines, ultraviolet light, and stress-inducing agents. A novel mitogen-activated protein kinase kinase, MEK6, was cloned and characterized. The complete MEK6 cDNA was isolated by polymerase chain reaction. It encodes a 334 amino acid protein with 82% identity to MKK3. MEK6 is highly expressed in skeletal muscle like many other members of this family, but in contrast to MKK3 its expression in leukocytes is very low. MEK6 is a member of the p38 kinase cascade and efficiently phosphorylates p38 but not c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) family members in direct kinase assays. Coupled kinase assays demonstrated that MEK6 induces phosphorylation of ATF2 by p38 but does not phosphorylate ATF2 directly. MEK6 is strongly activated by UV, anisomycin, and osmotic shock but not by phorbol esters, nerve growth factor, and epidermal growth factor. This separates MEK6 from the ERK subgroup of protein kinases. MEK6 is only a poor substrate for MEKK, a mitogen-activated protein kinase kinase kinase that efficiently phosphorylates the related family member JNKK. PMID- 8626700 TI - Purification and stabilization of transcriptionally active glucocorticoid receptor. AB - A major obstacle to the purification of glucocorticoid receptor (GR) is the very high nonspecific surface adsorption of this protein. This phenomenon is a property of the GR itself and does not reflect overall protein concentration or buffer conditions. We have observed that the zwitterionic detergent 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) is unique in its ability to stabilize the receptor and largely eliminate loss to nonspecific adsorption. We have coupled this observation with a two-step purification method that allows efficient purification and stabilization of transcriptionally active glucocorticoid receptor. For this procedure, the GR first undergoes a major purification by anion exchange chromatography following hormone binding and on column receptor transformation. Second, the GR is resolved to homogeneity utilizing a hydrophobic interaction chromatography step which consists of a 2.5 M to 0 M NaCl gradient elution of contaminating proteins followed by displacement of GR by CHAPS. GR at both stages of purification was able to activate transcription from the glucocorticoid response element containing the promoter region of the long terminal repeat of the mouse mammary tumor virus. This simple and efficient methodology should be of a considerable advantage for studies of the biology of the active, full-length GR. PMID- 8626701 TI - Molecular characterization of the di-leucine-based internalization motif of the T cell receptor. AB - Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinases. We have recently shown that both phosphorylation of Ser-126 and the presence of the di-leucine sequence Leu-131 and Leu-132 in CD3 gamma are required for protein kinase C (PKC)-mediated TCR down-regulation. To identify additional residues required for PKC-mediated phosphorylation of CD3 gamma and for TCR down regulation, an alanine scanning of CD3 gamma was done. Mutations of Arg-124, Ser 126, Lys-128, and Gln-129 inhibited both phosphorylation and TCR down-regulation, whereas mutation of Asp-127 only inhibited down-regulation. Further analyses demonstrated a discrepancy between the ability to be phosphorylated on CD3 gamma and to down-regulate the TCR in several transfectants. Phosphorylation was not as strictly dependent on the nature and position of the phosphoacceptor group and basic residues as were the subsequent steps involved in TCR down-regulation. Our results suggest that PKC-mediated TCR down-regulation may be regarded as a two step process. 1) Recognition and phosphorylation of CD3 gamma by PKC. In this process Arg-124, Ser-126, Lys-128, and Gln-129 are important. 2) Recognition of phosphorylated CD3 gamma by molecules involved in receptor internalization. In this process Ser(P)-126, Asp-127, Leu-131, and Leu-132 are important. PMID- 8626702 TI - Differential mu opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters. AB - mu opiate receptors, the principal sites for opiate analgesia and reward, can display compensatory responses to opiate agonist drug administration. Agonist induced K+ channel responses mediated by these receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked receptors. We used C-terminally directed anti mu receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the mu receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this mu opiate receptor protein was enhanced approximately 5-fold by treatment with the mu agonist morphine. The time course and dose-response relationships between mu receptor phosphorylation and agonist induced desensitization display interesting parallels. Phosphorylation of mu opiate receptor protein is also enhanced approximately 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13 acetate on mu receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to mu receptor phosphorylation events that may include mechanisms involved in mu receptor desensitization. PMID- 8626703 TI - The Spo0A protein of Bacillus subtilis inhibits transcription of the abrB gene without preventing binding of the polymerase to the promoter. AB - Repression of transcription of the abrB gene is essential to expression of many of the postexponential genes in Bacillus. The repression is due to the activity of the response regulator protein Spo0A. We have used in vitro transcription and DNase I and hydroxyl radical footprinting to explore the mechanism of transcription inhibition. Spo0A binds to specific DNA sequences (0A boxes), and two such boxes are found downstream of the tandem promoters for the abrB gene. The data indicate that both RNA polymerase and Spo0A bind simultaneously to a DNA fragment containing the promoters and the 0A boxes. The Spo0A prevents the polymerase from inducing DNA strand denaturation at the promoter for the abrB gene. PMID- 8626704 TI - Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation. AB - Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation. PMID- 8626705 TI - A delayed-early response nuclear gene encoding MRPL12, the mitochondrial homologue to the bacterial translational regulator L7/L12 protein. AB - We have characterized a new delayed-early response mRNA encoding a 21-kDa product (MRPL12) that accumulates during the G1 phase of growth-stimulated cells. MRPL12 is the mammalian homologue to chloroplastic and bacterial L12 ribosomal proteins. Immunofluorescence microscopy and cell fractionation indicate a predominant mitochondrial localization in various mammalian cell lines. The NH2-terminal 49 amino acids are necessary and sufficient to target the protein within the mitochondria and are probably cleaved off during import. MRPL12 proteins associated in vitro and cofractionate with ribosomal structures, as is the case for prokaryotic L12 proteins. Expression of a dominant inhibitory truncated protein leads to a severe reduction in cell growth by inhibiting mitochondrial ATP production. MRPL12 is the first mammalian mitochondrial ribosomal protein to be characterized. PMID- 8626706 TI - Insulin-like growth factor expression in human cancer cell lines. AB - The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis. PMID- 8626707 TI - Growth factor dependence of progression through G1 and S phases of adult rat hepatocytes in vitro. Evidence of a mitogen restriction point in mid-late G1. AB - Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes. PMID- 8626708 TI - Isolation and characterization of a folate receptor-directed metalloprotease from human placenta. AB - Glycosyl-phosphatidylinositol-anchored hydrophobic placental folate receptors (PFRs), which have an important functional role in maternal-to-fetal transplacental folate transport, can be converted to soluble hydrophilic forms by a placental metalloprotease. Using a Triton X-114 temperature-induced phase separation assay to monitor enzyme-mediated conversion of radiolabeled hydrophobic PFR into hydrophilic PFR, a metalloenzyme was isolated to apparent homogeneity from Triton X-114-solubilized human placenta using concanavalin A Sepharose and reverse-phase high performance liquid chromatography (HPLC) as major purification steps. The purified hydrophobic enzyme eluted as a single protein peak on reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis revealed a single 63,000 M(r) species, which was reduced to 58,000 M(r) following deglycosylation, findings comparable with amino acid analysis (M(r) approximately 59,000). The metalloenzyme was activated by Mg2+, Zn2+, Mn2+, and Ca2+, optimally at physiologic pH; it also exhibited EDTA-sensitive endoproteolytic cleavage of [3H]leucine-labeled full-length nascent PFR polypeptide generated in vitro in the absence of microsomes. Rabbit polyclonal anti-metalloprotease antiserum specifically immunoprecipitated 125I-metalloprotease and recognized cross reacting moieties on plasma membranes of normal human hematopoietic progenitor cells and human cervical carcinoma cells, both of which also express FR. PMID- 8626709 TI - Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells. AB - Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0 2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells. PMID- 8626710 TI - Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif. AB - An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing. PMID- 8626711 TI - Sequence divergence associated with species-specific splicing of the nonmuscle beta-tropomyosin alternative exon. AB - Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons. PMID- 8626712 TI - Purification and properties of HuD, a neuronal RNA-binding protein. AB - HuD is a human neuronal specific RNA-binding protein. In this study we have purified HuD and examined its RNA binding properties in detail. HuD binds to mRNAs that contain an AU-rich element with high affinity. In the case of the c fos AU-rich element, HuD binds to a 27-nucleotide core element comprising AUUUA, AUUUUA, and AUUUUUA motifs. Mutation in any two of these motifs abrogates binding. HuD contains two tandem RNA recognition motifs (RRM), a basic domain, and a third RRM. Deletion analysis has shown that only the first and second RRMs are essential for RNA binding. Thus, these specific RNA binding properties support the idea that the HuD regulates gene expression at the posttranscriptional level. PMID- 8626713 TI - Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186. AB - The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186. These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium. Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process. The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution. PMID- 8626714 TI - DNA binding by the coliphage 186 repressor protein CI. AB - The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B). Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR. Mutations which cause virulence reduce CI binding to p(R). The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR. The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A'). A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R). Thus, CI appears to recognize two distinct DNA sequences. CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions. A high order CI multimer appears to be the active DNA binding species, even at single operators. PMID- 8626715 TI - Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes. AB - Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway. PMID- 8626717 TI - Lyn and Fgr protein-tyrosine kinases prevent apoptosis during retinoic acid induced granulocytic differentiation of HL-60 cells. AB - The human promyelocytic leukemia cell line HL-60 can be induced to differentiate toward neutrophils and subsequently die via apoptosis in vitro. In this paper, we investigated the roles of protein-tyrosine kinases (PTKs) in retinoic acid (RA) induced granulocytic differentiation of HL-60 cells. Accompanying the RA-induced differentiation, activities of src family PTKs Lyn and Fgr became detected and reached a plateau 2 days after the stimulation. The immunoblotting using anti phosphotyrosine antibody (PY-20) showed that the proteins of 56 and 53 kDa were predominantly tyrosine-phosphorylated at day 2. Adsorption and immunoprecipitation of the cell lysate by specific antibodies evidenced that these phosphotyrosine-containing proteins are Lyn and Fgr PTKs. The degree of both activities and tyrosine phosphorylation of these PTKs was reduced to be minimal at day 5 when the HL-60 cells start to die by apoptosis. The inhibitors of PTKs, herbimycin A and genistein, were demonstrated to cause premature cell death of HL-60 cells in the presence of RA. The death was the consequence of an apoptotic process. The Ra-treated HL-60 cells, when incubated with specific c-lyn or c-fgr antisense oligodeoxynucleotide, also underwent premature death at day 2. These data implicate that Lyn and Fgr PTKs prevent programmed cell death to promote granulocytic differentiation of HL-60 cells. PMID- 8626716 TI - Molecular cloning and characterization of a newly identified member of the cadherin family, PB-cadherin. AB - We have isolated cDNA clones encoding novel proteins belonging to the cadherin family. These novel proteins are encoded by two distinct mRNA species generated by alternative splicing from a single gene, and based on preferential expression in the pituitary gland and brain, we named it PB-cadherin. One mRNA species encodes long type PB-cadherin composed of 803 amino acid residues with a longer cytoplasmic domain, whereas the other species encodes short-type PB-cadherin composed of 694 amino acid residues with a shorter cytoplasmic domain. Both long and short type PB-cadherin contain five repeats of a cadherin motif in the extracellular domain, the transmembrane domain, and the cytoplasmic domain, and the deduced amino acid sequences have a 30% homology to those of E-, N-, and P cadherins. Although the primary structure of N-terminal amino acids is identical between long and short type PB-cadherin, the following structures in the cytoplasmic regions are completely different. The long type PB-cadherin but not the short type contains the putative catenin-binding domain. When these two distinct forms of PB-cadherins were stably expressed in L cells, L cells expressing long type PB-cadherin or short type PB-cadherin both acquired a Ca2+ dependent cell adhesion property, thereby indicating that both types of PB cadherin are responsible for Ca2+-dependent cell adhesion. Persistent expression of PB-cadherin mRNA was found in the brain of rat embryos at least from embryonic day 15 to the postnatal period. In situ localization of PB-cadherin mRNA in the adult rat brain indicated that PB-cadherin mRNA is expressed in the inner granular layer of the olfactory bulb, Purkinje cell layer of the cerebellum, and in the pineal gland. PB-cadherin may play an important role in morphogenesis and tissue formation in neural and non-neural cells for the development and maintenance of the brain and neuroendocrine organs by regulating cell-cell adhesion. PMID- 8626718 TI - Substrate recognition by recombinant serine collagenase 1 from Uca pugilator. AB - Uca pugilator serine collagenase 1 was cloned and sequenced from a fiddler crab hepatopancreas cDNA library. A full-length sequence encodes a 270-amino acid pre pro-enzyme highly identical in structure to the chymotrypsin family of serine proteases. The zymogen form of the enzyme was expressed in Saccharomyces cerevisiae as a fusion with the alpha-factor signal sequence under control of the alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase promoter. Upon activation with trypsin, the recombinant collagenase possesses collagenolytic properties identical to those of the enzyme isolated from the crab hepatopancreas. The collagenase substrate binding pocket recognizes a wide range of basic, hydrophobic, and neutral polar residues. beta-Branched and acidic amino acids are poor substrates. Acylation is rate-limiting for collagenase versus peptidyl amides, rather than deacylation, as for trypsin and chymotrypsin. Correlations relating substrate volume and hydrophobicity to catalysis were found for collagenase and compared to those for chymotrypsin and elastase. Relative enzyme efficiencies on single amino acid versus tetrapeptide amide substrates show that collagenase derives less catalytic efficiency from binding of the primary substrate residue than trypsin or chymotrypsin, but compensates in binding of the extended peptidyl residues. Serine collagenase 1 is a novel member of the chymotrypsin protease family, by virtue of its amino acid sequence and multifunctional active site. PMID- 8626719 TI - Distinct domains in ribosomal protein L5 mediate 5 S rRNA binding and nucleolar localization. AB - Ribosomal protein L5, a 34-kDa large ribosomal subunit protein, binds to 5 S rRNA and has been implicated in the intracellular transport of 5 S rRNA. By immunofluorescence microscopy, L5 is detected mostly in the nucleolus with a fainter signal in the nucleoplasm, and it is known to also be a component of large ribosomal subunits in the cytoplasm. 5 S rRNA is transcribed in the nucleoplasm, and L5 is thought to play an important role in delivering 5 S rRNA to the nucleolus. Using RNA-binding assays and transfection experiments, we have delineated the domains within L5 that confer its 5 S rRNA binding activity and that localize it to the nucleolus. We found that the amino-terminal 93 amino acids are necessary and sufficient to bind 5 S rRNA in vitro, while the carboxyl terminal half of the protein, comprising amino acids 151-296, serves to localize the protein to the nucleolus. L5, therefore, has a modular domain structure reminiscent of other RNA transport proteins where one region of the molecule serves to bind RNA while another determines subcellular localization. PMID- 8626720 TI - Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol. AB - Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s). PMID- 8626721 TI - The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology. AB - The glycoprotein (GP) Ib-IX complex is one of the major platelet membrane glycoproteins. Its extracellular domain binds von Willebrand factor at a site of injury, an interaction that leads to activation of intracellular pathways. Its intracellular domain associates tightly with the platelet cytoskeleton through actin-binding protein. The goal of the present study was to investigate the role of the cytoplasmic domain of the GP Ib-IX complex and its interaction with the cytoskeleton. Cultured cells were transfected with the cDNAs coding for GP Ib(beta), GP IX, and full-length or truncated forms of GP Ib(alpha). Western blots of detergent-insoluble fractions of Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to Ser-590 from the cytoplasmic domain of GP IB(alpha) abolished the interaction of the entire GP Ib-IX complex with the cytoskeleton. Truncated GP Ib(alpha) that was unable to associate with the cytoskeleton retained its ability to associate with GP Ib(beta), to be inserted into the membrane, and to bind von Willebrand factor. Cells expressing GP Ib(alpha) changed their shape following adhesion to immobilized von Willebrand factor. Cells expressing truncated GP Ib(alpha) also changed their shape following adhesion but showed a very different morphology as compared to cells expressing full-length GP Ib(alpha). These results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganizations, that the cytoplasmic domain of GP Ib(alpha) regulates these reorganizations, and that the cytoplasmic domain of GP Ib(alpha) is absolutely required for attachment of the GP Ib-IX complex to the cytoskeleton. PMID- 8626722 TI - Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi. AB - Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6 mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II. PMID- 8626723 TI - Interaction between the phosphotyrosine binding domain of Shc and the insulin receptor is required for Shc phosphorylation by insulin in vivo. AB - Stimulation of the insulin receptor (IR) results in tyrosine phosphorylation of the intermediate molecules insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NPX-Tyr(P) motif in the juxtamembrane region of the IR is required for tyrosine phosphorylation of these molecules by the IR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domains, which bind phosphotyrosine in the context of an NPX Tyr(P) motif. To further clarify the role of the Shc PTB/PI domain, we identified a mutation in this domain that abrogated binding of Shc to the IR in vitro. Interestingly, this mutation completely abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, we identified specific amino acids on the IR that are required for the IR to stimulate Shc but not IRS-1 phosphorylation in vivo. As with the PTB/PI domain Shc mutant, the ability of these mutant receptors to phosphorylate Shc correlates with the binding of the PTB/PI domain of Shc to similar sequences in vitro. These findings support a model in which binding of the PTB/PI domain of Shc directly to the NPX Tyr(P) motif on the IR mediates Shc phosphorylation by insulin. PMID- 8626724 TI - Signaling from G protein-coupled receptors to c-Jun kinase involves beta gamma subunits of heterotrimeric G proteins acting on a Ras and Rac1-dependent pathway. AB - Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S. (1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of alphas, alphai2, alphaq, or alpha13 G protein alpha subunits. In contrast, overexpression of Gbetagamma subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves betagamma subunits of heterotrimeric G proteins, acting on a Ras and Rac1 dependent pathway. PMID- 8626725 TI - IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologues T1/ST2 and IL-1R AcP. AB - A novel member of the interleukin-1 receptor family has been cloned by polymerase chain reaction using degenerate oligonucleotide primers derived from regions of sequence conservation, using as template a yeast artificial chromosome known to contain both interleukin-1 (IL-1) receptors and T1/ST2. The new receptor, called IL-1 receptor-related protein or IL-1Rrp, fails to bind any of the known IL-1 ligands. A chimeric receptor, in which the IL-1Rrp cytoplasmic domain is fused to the extracellular and transmembrane regions of the IL-1 receptor, responds to IL 1 following transfection into COS cells by activation of NFkappaB and induction of IL-8 promoter function. PMID- 8626726 TI - Genomic structure and promoter analysis of the human obese gene. AB - The human gene encoding the homolog of the mouse obese (ob) gene was isolated and partially characterized. The human ob gene consists of three exons and two introns and spans about 18 kilobase pairs (kb), encoding a 3.5-kb cDNA. A 3-kb 5' flanking region of the gene was cloned and transient transfection assay with luciferase reporter confirmed the promoter activity in differentiated F442-A adipocytes. Potential regulatory elements are discussed in this report. PMID- 8626727 TI - Selective G protein coupling by C-C chemokine receptors. AB - The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through G beta gamma released from the Gi proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the Gq class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the Gq class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the Gi Gbetagamma-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs. PMID- 8626728 TI - Stoichiometry of 2',5'-oligoadenylate-induced dimerization of ribonuclease L. A sedimentation equilibrium study. AB - Ribonuclease L is an endoribonuclease that is activated by binding of 2',5' linked oligoadenylates. Activation of ribonuclease L also induces dimerization. Here, we demonstrate using equilibrium sedimentation that dimerization requires the binding of one 5'-monophosphate 2',5'-(adenosine)3 molecule per ribonuclease L monomer. No dimerization was observed in the absence of activator up to a protein concentration of 18 microM, indicating that unliganded enzyme is unable to dimerize or the association is very weak. In parallel with dimerization, enzymatic activity is also maximized at a 1:1 activator: ribonuclease L stoichiometry. The same stoichiometry for dimerization is observed using a nonphosphorylated activator 2'-5'-(adenosine)3. Adenosine triphosphate or RNA oligonucleotide substrates do not induce dimerization. The observed stoichiometry supports a model for ribonuclease L dimerization in which activator binds to monomer, which subsequently dimerizes. PMID- 8626729 TI - Activated protein C-catalyzed proteolysis of factor VIIIa alters its interactions within factor Xase. AB - Factor VIIIa, the cofactor for the factor IXa-dependent conversion of factor X to factor Xa, is proteolytically inactivated by activated protein C (APC). APC cleaves at two sites in factor VIIIa, Arg336, near the C terminus of the A1 subunit; and Arg562, bisecting the A2 subunit (Fay, P., Smudzin, T., and Walker, F. (1991) J. Biol. Chem. 266, 20139-20145). Factor VIIIa increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa (Fl-FFR-FIXa; Kd = 42.4 nM), whereas cleavage of factor VIIIa by APC eliminated this property. Isolation of the APC-cleaved A1/A3-C1-C2 dimer (A1336/A3-C1-C2), and the fragments derived from cleaved A2 subunit (A2N/A2C), permitted dissection of the roles of individual cleavages in cofactor inactivation. Intact A1/A3-C1-C2 dimer increased Fl-FFR-FIXa anisotropy and bound factor X in a solid phase assay, while these activities were absent in the A1336/A3-C1-C2. However, the residues removed by this cleavage, Met337 Arg372, did not directly participate in these functions since neither a synthetic peptide to this sequence nor an anti-peptide polyclonal antibody blocked these activities using intact dimer. CD spectral analysis of the intact and truncated dimers indicated reduced alpha and/or beta content in the latter. The A1/A3-C1-C2 dimer plus A2 subunit reconstitutes cofactor activity and produced a factor VIIIa-like effect on the anisotropy of Fl-FFR-FIXa. However, when A2 was replaced by the A2N/A2C fragments, the resulting fluorescence signal was equivalent to that observed with the dimer alone. These results indicate that APC inactivates the cofactor at two levels within the intrinsic factor Xase complex. Cleavage of either subunit modulates the factor IXa active site, suggesting an essential synergy of interactive sites in factor VIIIa. Furthermore, cleavage of the A1 site alters the conformation of a factor X binding site within that subunit, thereby reducing the affinity of cofactor for substrate. PMID- 8626730 TI - Scavenging of nitrogen dioxide, thiyl, and sulfonyl free radicals by the nutritional antioxidant beta-carotene. AB - Mechanisms of free radical scavenging by the nutritional antioxidant beta carotene have been investigated by pulse radiolysis. Free radicals, which can initiate the chain of lipid peroxidation, including nitrogen dioxide (NO2.), thiyl (RS.), and sulfonyl (RSO2.) radicals, are rapidly scavenged by beta carotene. Absolute rate constant k[NO2. + beta-carotene] = (1.1 +/- 0.1) x 10(8) m-1 s-1 and for the glutathione thiyl radical k[GS. + beta-carotene] = (2.2 +/- 0.1) x 10(8) m-1 s-1 have been determined. The mechanisms however are mutually exclusive, the former involving electron transfer to generate the radical-cation [ beta-carotene]+. and the latter by radical-addition to generate an adduct radical [RS... beta-carotene].. Rate constants for thiyl radical-addition reactions vary from 10(6) to 10(9) m-1 s-1 and correlate with the lipophilicity of the thiyl radical under study. Sulfonyl radicals undergo both electron abstraction, [ beta-carotene]+. and radical-addition, [RSO2... beta-carotene]. in an approximate 3:1 ratio. The beta-carotene radical-cation and adduct-radicals are highly resonance stabilized and undergo slow bimolecular decay to non-radical products. These carotenoid-derived radicals react differently with oxygen, a factor which is expected to influence the antioxidant activity of beta-carotene within tissues of varying oxygen tension in vivo. PMID- 8626731 TI - Formation of parallel and antiparallel coiled-coils controlled by the relative positions of alanine residues in the hydrophobic core. AB - The orientation of alpha-helical chains in two-stranded coiled-coils has been shown to be determined by the presence of favorable interchain electrostatic interactions. In this study, we used de novo designed 35-residue peptides to show that when interchain electrostatic interactions are not a factor in coiled-coil formation, the relative positions of Ala residues in the middle heptad can control the parallel or antiparallel orientation of alpha-helical chains in coiled-coils. The peptides formed four-stranded coiled-coils where the helices are either all-parallel or all-antiparallel with respect to their nearest neighbor. The common structural element in these four-stranded coiled-coils is an alternating pair of Ala and Leu residues (Ala-Leu-Ala-Leu) in each of the two planes in the middle heptad. These results indicate that both the relative positions of the Ala residues in the hydrophobic core and the interchain electrostatic interactions between charged residues in the e and g positions should be considered in designing coiled-coils with the desired number of strands in the multiple-stranded assembly. These design elements are also important in orienting functional groups or domains attached to the terminals ends of a coiled coil carrier. PMID- 8626732 TI - Characterization of a melanosomal transport system in murine melanocytes mediating entry of the melanogenic substrate tyrosine. AB - In this study, we identify a transport system for tyrosine, the initial precursor of melanin synthesis, in the melanosomes of murine melanocytes. Melanosomes preloaded with tyrosine demonstrated countertransport of 10 microM [3H]tyrosine, indicating carrier-mediated transport. Melanosomal tyrosine transport was saturable, with an apparent Km for tyrosine transport of 54 microM and a maximal velocity of 15 pmol of tyrosine/unit of hexosaminidase/min. Transport was temperature-dependent (Ea = 7.5 kcal/mol) and showed stereospecificity for the l isomer of tyrosine. Aromatic, neutral hydrophobic compounds (such as tryptophan and phenylalanine), as well as the small, bulky neutral amino acids (such as leucine, isoleucine, and methionine) competed for tyrosine transport. Tyrosine transport was inhibited by the classical system L analogue, 2 aminobicyclo[2.2.1]heptane-2-carboxylic acid and by monoiodotyrosine, but not by cystine, lysine, glutamic acid, or 2-(methylamino)-isobutyric acid. Tyrosine transport showed no dependence on Na+ or K+, and did not require an acidic environment or the availability of free thiols. These results demonstrate the existence of a neutral amino acid carrier in murine melanocyte melanosomes which resembles the rat thyroid FRTL-5 lysosomal system h. This transport system is critical to the function of the melanosome since tyrosine is the essential substrate required for the synthesis of the pigment melanin. PMID- 8626733 TI - The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. AB - The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions. PMID- 8626734 TI - Infrared and EPR studies on cyanide binding to the heme-copper binuclear center of cytochrome bo-type ubiquinol oxidase from Escherichia coli. Release of a CuB cyano complex in the partially reduced state. AB - Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding. PMID- 8626735 TI - Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis. AB - The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC* and hydrogen peroxide saturates at high peroxide concentrations (Km = 11. 8 mm). The data are consistent with a two-step mechanism involving the formation of an HRP-H2O2 intermediate (k = 1.1 x 10(4) m-1 s-1) whose conversion to compound I is rate-limiting (k = 142 s-1) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H2O2 to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 microM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC* was stable and reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent Km for guaiacol, p-cresol, and 2, 2'-azinobis(3 ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC* and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I. PMID- 8626736 TI - Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein. AB - An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllof, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex. PMID- 8626737 TI - Human enteric defensins. Gene structure and developmental expression. AB - Paneth cells, secretory epithelial cells of the small intestinal crypts, are proposed to contribute to local host defense. Both mouse and human Paneth cells express a collection of antimicrobial proteins, including members of a family of antimicrobial peptides named defensins. In this study, data from an anchored polymerase chain reaction (PCR) strategy suggest that only two defensin mRNA isoforms are expressed in the human small intestine, far fewer than the number expressed in the mouse. The two isoforms detected by this PCR approach were human defensin family members, HD-5 and HD-6. The gene encoding HD-6 was cloned and characterized. HD-6 has a genomic organization similar to HD-5, and the two genes have a striking pattern of sequence similarity localized chiefly in their proximal 5'-flanking regions. Analysis of human fetal RNA by reverse transcriptase-PCR detected enteric defensin HD-5 mRNA at 13.5 weeks of gestation in the small intestine and the colon, but by 17 weeks HD-5 was restricted to the small intestine. HD-6 mRNA was detectable at 13.5-17 weeks of gestation in the small intestine but not in the colon. This pattern of expression coincides with the previously described appearance of Paneth cells as determined by ultrastructural approaches. Northern analysis of total RNA from small intestine revealed quantifiable enteric defensin mRNA in five samples from 19 24 weeks of gestation at levels approximately 40-250-fold less than those observed in the adult, with HD-5 mRNA levels greater than those of HD-6 in all samples. In situ hybridization analysis localized expression of enteric defensin mRNA to Paneth cells at 24 weeks of gestation, as is seen in the newborn term infant and the adult. Consistent with earlier morphological studies, the ratio of Paneth cell number per crypt was reduced in samples at 24 weeks of gestation compared with the adult, and this lower cell number partially accounts for the lower defensin mRNA levels as determined by Northern analysis. Low levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms. PMID- 8626738 TI - Expression of the rat testis-specific histone H1t gene in transgenic mice. One kilobase of 5'-flanking sequence mediates correct expression of a lacZ fusion gene. AB - H1t is synthesized in mid to late pachytene spermatocytes of the male germ line and is the only tissue-specific member of the mammalian H1 histone family. As a step toward identifying DNA sequences that confer its tissue-specific expression, we have produced transgenic mice containing the intact rat H1t gene as well as a H1t-lacZ fusion gene. Transgenic mice carrying a 6.8-kilobase fragment of rat genomic DNA encompassing the H1t gene expressed rat H1t at high levels in the testis and in no other organ examined. H1t fragments truncated to within 141 base pairs (bp) of the gene in the 5' direction or within 837 bp in the 3' direction retained testis specificity. Expression of rat H1t protein was also evident in the testes of the transgenic mice, and in some lines the level of rat H1t exceeded that of the mouse protein. The stage of spermatogenesis of transgene expression was assessed by following appearance of transgenic mRNA in developing mice and by immunohistochemistry using an antiserum to rat H1t. In lines from three different constructs, expression was restricted to germinal cells, although in two strongly expressing lines the transgenes were expressed somewhat prematurely in preleptotene spermatocytes. An H1t(-948/+71)-lacZ fusion was also expressed specifically in the spermatocytes and round spermatids of a transgenic line, confirming that sequences sufficient for correct tissue and developmental expression lie within this 1,019-bp segment of the gene. PMID- 8626739 TI - Covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein requires import into mitochondria, presequence removal, and folding. AB - Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate. PMID- 8626740 TI - A requirement for matrix processing peptidase but not for mitochondrial chaperonin in the covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein. AB - Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial heterotetramer containing a flavoprotein subunit with an 8alpha N(3)-histidyl-linked FAD cofactor. The covalent linkage of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein precursor in mitochondrial matrix fractions. Flavoprotein modification does not depend on translocation across a membrane, but it does require proteolytic processing by the mitochondrial processing peptidase prior to flavin attachment. Since ATP depletion, N-ethylmaleimide, or proteinase treatments of matrix fractions inhibit flavoprotein modification, at least one additional matrix protein component appears to be required. Having previously suggested that the flavoprotein begins folding before FAD attachment occurs, we tested whether the mitochondrial chaperonin, heat shock protein 60, might be necessary. Co-immunoprecipitation of the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matrix fractions does not inhibit FAD attachment. Nonprotein components are also required for flavoprotein modification. In addition to ATP, effector molecules such as succinate, fumarate, or malate also stimulate modification. Together, these results suggest that FAD addition is an early event in succinate dehydrogenase assembly. PMID- 8626741 TI - Oligomeric regulation of gastric H+,K+-ATPase. AB - The H+,K+-ATPase of intact gastric vesicles has two Km values for ATP hydrolysis, 7 and 80 microM. Irradiation of vesicles with ultraviolet light in the presence of 1 mM ATP resulted in K+-ATPase activity that shows only the low affinity ATP binding. The irradiation stimulated or inhibited proton uptake rate compared with control vesicles at high or low ATP concentrations, respectively. The relation between proton uptake rate and K+-ATPase activity at different ATP concentrations was linear with irradiated vesicles and nonlinear with control vesicles. These results indicate that hydrolysis at the high affinity ATP binding site regulates the energy-transport coupling in negative and positive manners at high and low ATP concentrations, respectively. The complete inhibition of K+-ATPase by a specific proton pump inhibitor E3810 (rabeprazole) (2-([4-(3-methoxypropoxy)-3 methylpyridin-2-yl]methylsulf i nyl)-1H-benzimidazole sodium salt) occurred when E3810 bound to half of the alpha-subunit of H+,K+-ATPase in unirradiated vesicles at both 200 and 10 microM ATP, whereas the complete inhibition of proton uptake occurred when E3810 bound to half or a quarter of the alpha-subunit at 200 or 10 microM ATP, respectively. These results suggest that dimeric interaction between the alpha-subunits is necessary for the enzyme activity at all ATP concentrations and that dimeric or tetrameric interaction is necessary for proton transport at high or low ATP concentrations, respectively. PMID- 8626742 TI - Thr353, located within the COOH-terminal tail of the delta opiate receptor, is involved in receptor down-regulation. AB - Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist mediated down-regulation of the delta opiate receptor. PMID- 8626743 TI - Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. AB - Ultracentrifugation and graded molecular sieving, as well as a sensitive sandwich enzyme-linked immunosorbent assay were used to isolate and quantitate the amounts of water-soluble oligomers of beta amyloid (Abeta) peptides N-40 and N-42 in cerebral cortex of normal and Alzheimer disease (AD) brains. AD brains contained 6-fold more water-soluble Abeta (wsAbeta) than control brains. The majority of water-soluble peptides in most AD cases was A beta N-42, representing 12 times the amount found in control brains. The wsAbeta was present in the form of monomers and oligomers ranging from less than 10 kDa to greater than 100 kDa. The amount of wsAbeta N-42 in AD brains is about 50 times greater than the level of soluble Abeta N-42 found in the CSF of AD patients. This disparity may be due to the rapid association of wsAbeta N-42 into fibrillar deposits and/or to the integrity of the anatomical barriers which separate the two extracellular spaces. In this paper, we consider soluble any form of Abeta which has not yet polymerized into its insoluble, filamentous form. This includes both the newly synthesized forms of Abeta and those peptides which may be loosely attached to insoluble filaments but which can, nevertheless, still be considered soluble. It has been previously shown that, once it has aggregated into its filamentous form, the Abeta peptides are resistant to disaggregation and degradation by a number of denaturing agents and aqueous buffers containing proteolytic enzymes. Therefore, it is likely that the water-soluble Abeta peptides we quantified are precursors to its insoluble, filamentous form. Consequently, reducing the levels of soluble Abeta in AD brains could have profound effects on AD pathophysiology. PMID- 8626744 TI - Ricin A chain fused to a chloroplast-targeting signal is unfolded on the chloroplast surface prior to import across the envelope membranes. AB - The initial stages of chloroplast protein import involve the binding of precursor proteins to surface-bound receptors prior to translocation across the envelope membranes in a partially folded conformation. We have analyzed the unfolding process by examining the conformation of a construct, comprising the presequence of a chloroplast protein linked to ricin A chain, before and after binding to the chloroplast surface. We show that the presequence is highly susceptible to proteolysis in solution, probably reflecting a lack of tertiary structure, whereas the A chain passenger protein is resistant to extremely high concentrations of protease, unless deliberately unfolded using denaturant. The A chain moiety is furthermore active, indicating that the presence of the presequence does not prevent formation of a tightly folded, native state. In contrast, receptor-bound p33KRA (fusion protein comprising the 33-kDa presequence plus 22 residues of mature protein, linked to the A chain of ricin) is quantitatively digested by protease concentrations that have little effect on the A chain in solution. We conclude that protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors. PMID- 8626745 TI - Selective inhibition of Abeta fibril formation. AB - We describe here an inhibitor of in vitro fibril formation, hexadecyl-N methylpiperidinium (HMP) bromide, which is selective for the Alzheimer's disease peptide Abeta. At 10 microM, its IC50 for inhibiting Abeta aggregation at pH 5.8, HMP bromide does not inhibit fibril formation by other amyloidogenic polypeptides nor does it affect the folding stability of the beta-sheet-rich immunoglobulin VL domain REI. In addition, small structural modifications of HMP bromide reduce or eliminate its ability to inhibit pH 5.8 aggregation of Abeta. These indications of specificity, plus the ability of the molecule to inhibit A beta aggregation at concentrations almost an order of magnitude below its critical micelle concentration, suggest a mechanism of inhibition other than micellar solubilization of Abeta. HMP bromide is required in approximately a 1:1 stoichiometry for effective inhibition at pH 5.8. Although stoichiometric amounts of HMP bromide with respect to total Abeta inhibit Abeta fibril formation at pH 7.4, the molecule is incapable, at lower concentrations, of blocking the seeding of fibril formation by small amounts of added Abeta fibrils. The results suggest the existence of a binding surface on A beta capable of binding amphipathic molecules such as HMP bromide and which, when occupied, precludes assembly of A beta into amyloid fibrils. Molecules that bind to this site with high specificity may prove to be useful therapeutic agents for preventing or retarding the cerebral amyloid plaque formation implicated in Alzheimer's disease pathology. PMID- 8626746 TI - Identification of inhibitors of melittin using nonsupport-bound combinatorial libraries. AB - A strategy has been developed for the identification of inhibitors of toxins or regulatory proteins. This approach is based on blocking the access of such proteins to their biological targets during their solution transport. This approach uses the strength of nonsupport-bound synthetic combinatorial libraries (SCLs) for the study of acceptor-ligand interactions. A non-receptor assisted toxin, melittin, was selected for the present study to illustrate this application of the SCL approach. Hexapeptide SCLs were assayed for their ability to inhibit the cytolytic activity of melittin toward bacterial and erythrocyte cells. Over 20 inhibitory hexapeptides were identified following the screening and deconvolution processes from millions of sequences. The identified inhibitory peptides appeared to interact directly with melittin. These interactions appear to decrease melittin's ability to undergo lipid- and/or polysaccharide-induced conformational changes, and are demonstrated by fluorescence and circular dichroism spectroscopy. PMID- 8626747 TI - Catalytic properties of lipopolysaccharide (LPS) binding protein. Transfer of LPS to soluble CD14. AB - Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS.sCD14 complexes to HDL particles. To characterize the first of these two reactions, LPS covalently derivatized with the fluorophore, boron dipyrromethene difluoride (BODIPY), was used to monitor LBP-catalyzed movement of LPS in real time. The fluorescence efficiency of micelles of BODIPY-LPS was low but was strongly increased upon dissolution in detergent or upon binding to sCD14. Spontaneous binding of BODIPY-LPS to sCD14 was very slow but was accelerated by substoichiometric concentration of LBP, and the rate of binding was measured under a variety of conditions. LBP-catalyzed transfer was first order with respect to both sCD14 and LPS concentration, and the apparent Km values were 1 approximately 2 microg/ml for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP was approximately 150 molecules of LPS min-1 LBP-1. LBP alone caused a small but measurable increase in the fluorescence of BODIPY-LPS, suggesting that it bound LPS aggregates but did not readily remove LPS monomers. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14. These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate. PMID- 8626748 TI - Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prosequences. AB - Distinct antifreeze polypeptides (AFP) were isolated from the skin of the winter flounder, Pleuronectes americanus, by gel filtration and reverse phase high performance liquid chromatography. In parallel, several cDNA clones were isolated from a skin cDNA library using a liver AFP cDNA probe. Both protein and DNA sequence analyses indicate that flounder skin contains several distinct but homologous alanine-rich AFPs. Although the skin type AFPs contain 11 similar amino acid repeats found in the secretory liver type AFPs, the skin type AFPs are mature polypeptides lacking both the signal and prosequences, indicating that they may function intracellularly. The skin type AFP is significantly less active in thermal hysteretic activity than the liver type AFP. Genomic Southern analysis indicates that like the liver type AFP genes, there are multiple copies (30-40 copies) of skin type AFP. Although the liver type AFP genes are specifically expressed in the liver and to a lesser extent in intestine, the skin type AFP genes are expressed in all tissues examined including the liver and abundantly in exterior tissues, i.e. skin, scales, fin, and gills, suggesting an important protecting role in these exterior tissues. PMID- 8626749 TI - 13C and 31P NMR investigation of effect of 6-aminonicotinamide on metabolism of RIF-1 tumor cells in vitro. AB - The effect of 6-aminonicotinamide on the metabolism of RIF-1 tumor cells was investigated using 13C and 31P NMR spectroscopy. 6-Aminonicotinamide can be metabolized to 6-amino-NAD(P), a competitive inhibitor of NAD(P)-requiring processes. 40 microM 6-aminonicotinamide led to an inhibition of 6 phosphogluconate dehydrogenase and an accumulation of 6-phosphogluconate. A subsequent accumulation of the 6-phosphogluconate precursor 6-phosphoglucono delta-lactone was observed in the 13C NMR spectrum. These metabolites were shown to be intracellular, although a small amount of leakage of 6-phosphoglucono-delta lactone occurred. The intracellular concentrations of 6-phosphogluconate and 6 phosphoglucono-delta-lactone were 1.9 +/- 0.8 micromol/108 cells (+/-1 standard deviation) and 0.8 +/- 0.4 micromol/10(8) cells, respectively, after 15 h. Glucose utilization and lactate production were significantly inhibited by 6 aminonicotinamide (both p < 0.05), indicating inhibition of glycolysis. 31P NMR data showed that phosphocreatine was significantly depleted in cells exposed to 6 aminonicotinamide (p < 0.05). Exposure of RIF-1 cells to 6-aminonicotinamide prior to 3- or 6-Gy x-irradiation induced a supra-additive cell kill, indicating that 6-aminonicotinamide is acting as a radiosensitizer. There was no effect of 6 aminonicotinamide alone or when the drug was given postradiation, suggesting that its mechanism of action may be by inhibition of radiation-induced repair. PMID- 8626750 TI - Functionalized protein-like structures from conformationally defined synthetic combinatorial libraries. AB - An approach is described for the de novo design of protein-like structures in which synthetic combinatorial libraries (SCLs) were incorporated into an amphipathic alpha-helical scaffold (an 18-mer sequence made up of leucine and lysine residues) to generate conformationally defined SCLs. In particular, the SCLs in which the "combinatorialized" positions were on the hydrophilic face showed an alpha-helical conformation in mild buffer. These SCLs were used to generate context-independent but position-dependent scales of alpha-helical propensity for the L-amino acids. These scales were then used to design highly alpha-helical peptides that self-associated in mild buffer. The same approach was also found to permit the identification of conformation-dependent decarboxylation catalysts. PMID- 8626751 TI - Localization of granzyme B in the nucleus. A putative role in the mechanism of cytotoxic lymphocyte-mediated apoptosis. AB - One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis. PMID- 8626752 TI - Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation. AB - A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins. PMID- 8626753 TI - Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. AB - The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, H2O2 increased the mRNA expression of MAPK dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by H2O2. H2O2-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N 17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to H2O2 toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury. PMID- 8626754 TI - Reduction of quinonoid dihydrobiopterin to tetrahydrobiopterin by nitric oxide synthase. AB - Rat cerebellar nitric oxide synthase (NOS) purified from transfected human kidney cells catalyzes an NADPHdependent reduction of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). Reduction of qBH2 at 25 microM proceeds at a rate that is comparable with that of the overall reaction (citrulline synthesis) and requires calcium ions and calmodulin for optimal activity; NADH has only 10% of the activity of NADPH. The reduction rate with the quinonoid form of 6 methyldihydropterin is approximately twice that with qBH2. 7,8-Dihydrobiopterin had negligible activity. Neither 7,8-dihydrobiopterin nor BH4 affected the rate of qBH2 reduction. Reduction is inhibited by the flavoprotein inhibitor diphenyleneiodonium, whereas inhibitors of electron transfer through heme (7 nitroindazole and N-nitroarginine) stimulated the rate to a small extent. Methotrexate, which inhibits a variety of enzymes catalyzing dihydrobiopterin reduction, did not inhibit. These studies provide the first demonstration of the reduction of qBH2 to BH4 by NOS and indicate that the reduction is catalyzed by the flavoprotein "diaphorase" activity of NOS. This activity is located on the reductase (C-terminal) domain, whereas the high affinity BH4 site involved in NOS activation is located on the oxygenase (N-terminal) domain. The possible significance of this reduction of qBH2 to the essential role of BH4 in NOS is discussed. PMID- 8626755 TI - Activation of a recombinant petunia glutamate decarboxylase by calcium/calmodulin or by a monoclonal antibody which recognizes the calmodulin binding domain. AB - To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity. PMID- 8626756 TI - Functional expression of the multidrug resistance-associated protein in the yeast Saccharomyces cerevisiae. AB - The multidrug resistance-associated protein (MRP) is a member of the ATP binding cassette superfamily of transporters which includes the mammalian P-glycoproteins (P-gp) family. In order to facilitate the biochemical and genetic analyses of MRP, we have expressed human MRP in the yeast Saccharomyces cerevisiae and have compared its functional properties to those of the mouse Mdr3 P-gp isoform. Expression of both MRP and Mdr3 in the anthracycline hypersensitive mutant VASY2563 restored cellular resistance to Adriamycin in this mutant. MRP and Mdr3 expression produced pleiotropic effects on drug resistance in this mutant, as corresponding VASY2563 transformants also acquired resistance to the anti-fungal agent FK506 and to the K+/H+ ionophore valinomycin. The appearance of increased cellular resistance to the toxic effect of Adriamycin (ADM) in MRP and Mdr3 transformants was concomitant with a reduced intracellular accumulation of [14C]ADM in spheroplasts prepared from these cells. Moreover, MRP and Mdr3, but not control spheroplasts, could mediate a time-dependent reduction in the overall cell-associated [14C]ADM from preloaded cells, suggesting the presence of an active ADM transport mechanism in MRP and Mdr3 transformants. Finally, human MRP was found to complement the biological activity of the yeast peptide pheromone transporter Ste6 and partially restored mating in a sterile ste6 null mutant. These findings suggest that despite their relatively low level of structural homology, MRP and P-gp share similar functional aspects, since both proteins can mediate transport of chemotherapeutic drugs and the a mating peptide pheromone in yeast. PMID- 8626757 TI - Binding of mitochondrial presequences to yeast cytosolic heat shock protein 70 depends on the amphiphilicity of the presequence. AB - The interactions between a yeast cytosolic hsp70, Ssa1p, and various synthetic peptides, including mitochondrial presequences, have been studied. The interactions were monitored both indirectly, by measuring the effects of the presequences on the ATPase activity and oligomeric state of the enzyme, and directly, by measuring the increased steady-state fluorescence polarization of fluorescent derivatives of the presequences as they bind to Ssa1p. The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-dependent manner. The presequences are also able to stimulate the ATPase activity of the enzyme at similar concentrations. Quantification of the binding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. The most amphiphilic presequences, as measured by retention times on reversed-phase high pressure liquid chromatography or surface activity in lipid monolayers, had the highest affinity for Ssa1p. The least amphiphilic presequences, which had previously been shown to be ineffective as mitochondrial targeting sequences, had relatively low affinity for Ssa1p. The results show that Ssa1p interacts with a broad range of amino acid sequences and that the strength of these interactions is related to the physical properties of the sequence. That the physical properties recognized by Ssa1p are identical to those necessary for the targeting function of mitochondrial presequences suggests that Ssa1p may interact with mitochondrial precursor proteins in the cytosol. The interactions may serve a variety of purposes: the maintenance of precursors in translocation-competent forms, the prevention of improper association of precursors with non mitochondrial membranes, and the delivery of precursors to the mitochondrial surface. PMID- 8626758 TI - Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3. AB - The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively. PMID- 8626759 TI - Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin. AB - During exposure to ozone, the methionine and aromatic amino acid residues of Escherichia coli glutamine synthetase (GS) and bovine serum albumin (BSA) are oxidized rapidly in the order Met > Trp > Tyr approximately His > Phe. The loss of His is matched by a nearly equivalent formation of aspartate or of a derivative that is converted to aspartic acid upon acid hydrolysis. Conversion of His to aspartate was confirmed by showing that the oxidation of E. coli protein in which all His residues were uniformly labeled with 14C gave rise to 14C labeled aspartic acid in 80% yield and also by the demonstration that His residues in the tripeptides Ala-His-Ala or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The oxidation of BSA and GS led to formation, respectively, of 11 and 3.3 eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit. Although BSA and GS contain nearly identical amounts of each kind of aromatic amino acid residues, oxidation of these residues in BSA was about 1.5 2.0 times faster than in GS indicating that the susceptibility to oxidation is dependent on the primary, secondary, tertiary, and quaternary structure of the protein. PMID- 8626760 TI - A pH-sensitive yeast outward rectifier K+ channel with two pore domains and novel gating properties. AB - YORK is a newly cloned K+ channel from yeast. Unlike all other cloned K+ channels, it has two pore domains instead of one. It displays eight transmembrane segments arranged like a covalent assembly of a Shaker-type voltage-dependent K+ channel (without S4 transmembrane segments) with an inward rectifier K+ channel. When expressed in Xenopus oocytes, YORK does not pass inward currents; it conducts only K+-selective outward currents. However, the mechanism responsible for this strict outward rectification is unusual. Like inward rectifiers, its activation potential threshold closely follows the K+ equilibrium potential. Unlike inward rectifiers, the rectification is not due to a voltage-dependent Mg2+ block. The blocking element is probably intrinsic to the YORK protein itself. YORK activity is decreased at acidic internal pH, with a pKa of 6.5. Pharmacological and regulation properties were analyzed. Ba2+ ions and quinine block YORK currents through high and low affinity sites, while tetraethylammonium displays only one affinity for blocking. Activation of protein kinase C indirectly produces an increase of the current, while protein kinase A activation has no effect. PMID- 8626761 TI - Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion. AB - Alzheimer's disease amyloid consists of amyloid beta-peptides (Abeta) derived from the larger precursor amyloid precursor protein (APP). Non-amyloidogenic APP processing involves regulated cleavage within the Abeta domain followed by secretion of the ectodomain (APPs). APPs secretion can be stimulated by muscarinic acetylcholine receptors coupled to phospholipases and kinases. To determine whether other receptor classes can regulate APP processing, we examined the relation between serotonin receptors and APPs secretion. Serotonin increased APPs release 3-4-fold in 3T3 cells stably overexpressing 5-HT2aR or 5-HT2cR. The increase was dose-dependent and was blocked by serotoninergic antagonists. Phorbol esters also increased APPs secretion, but neither kinase inhibitors nor down-regulation of PKC blocked the serotonin-induced increase in APPs secretion. Thus PKC is not necessary to stimulate APPs secretion. Phospholipase A2 (PLA2) inhibitors blocked the 5-HT2aR-mediated increase in APPs secretion, suggesting a role of PLA2 in coupling 5-HT2aR to APP processing. In contrast, coupling of 5 HT2cR to APPs secretion involved both PKC and PLA2. Serotonin also stimulated the release of the APLP2 ectodomain, suggesting that additional members of the APP multigene family are processed via similar regulated pathways. Inasmuch as generation of APPs precludes the formation of amyloidogenic derivatives, serotonin receptors provide a novel pharmacological target to reduce these derivatives in Alzheimer's disease. PMID- 8626762 TI - In vitro and in vivo inactivation of transcobalamin II receptor by its antiserum. AB - Rabbits injected with pure human placental transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, transcobalamin II, in their plasma. Exogenously added transcobalamin II [57Co]cyanocobalamin bound very poorly (2-5%) to the affected rabbit liver, kidney, and intestinal total or intestinal basolateral membrane extracts relative to the binding by membrane extracts from normal rabbit tissues. The activity was restored to normal values following a wash of affected rabbit tissue membranes with pH 3 buffer containing 200 mM potassium thiocyanate. Immunoblot analysis of normal and affected rabbit kidney and liver total membranes revealed similar amounts of 124-kDa TC II-R dimer protein. The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II [57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl dependent enzymes. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes. Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and thus cause severe Cbl deficiency and the noted failure to thrive. PMID- 8626763 TI - Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA. AB - A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA. PMID- 8626764 TI - Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S nitrosylation and subsequent NADH attachment. AB - Nitric oxide (NO)-related activity has been associated with an NAD+-dependent modification of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the mechanism by which NO effects covalent attachment of nucleotide and its role in regulation of enzyme activity are controversial. Recent studies have shown that S-nitrosylation of GAPDH (Cys149) initiates subsequent modification by the pyridinium cofactor. Here we show that NADH rather than NAD+ is the preferred substrate. Transnitrosation from active site S nitrosothiol to the reduced nicotinamide ring system appears to facilitate protein thiolate attack on the enzyme-bound cofactor. This results in attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme inhibition, whereas attachment of NADH accounts for irreversible enzyme inactivation. S-Nitrosylation may serve to protect GAPDH from oxidant inactivation in settings of cytokine overproduction and to regulate glycolysis. NADH attachment is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis. PMID- 8626765 TI - Identification of a region required for subtype-specific agonist-induced sequestration of the m2 muscarinic acetylcholine receptor. AB - When the m1 and m2 muscarinic acetylcholine receptors are transiently expressed in JEG-3 cells, the m2, but not the m1, receptor undergoes agonist-induced sequestration. Both receptors exhibit internalization when expressed in Y1 cells. These results suggest that the m1 and m2 receptors use distinct cellular mechanisms or pathways for agonist-induced internalization and that JEG-3 cells are deficient in the mechanism or pathway used by the m1 receptor. Transfection experiments with chimeric receptors indicate that the specificity for agonist induced internalization for the m2 receptor lies in the carboxyl-terminal fifth of the receptor. The intracellular carboxyl-terminal tail of the m2 receptor is neither sufficient nor required for the m2-specific sequestration. Site-directed mutagenesis demonstrates that two amino acids in the carboxyl-terminal end of the third cytoplasmic loop of the m2 receptor are required for sequestration in JEG-3 cells. In addition, the sixth transmembrane domain, which is adjacent to this cytoplasmic domain, is also required. Thus, m2-specific agonist-induced sequestration requires sequences both in the carboxyl-terminal end of the third cytoplasmic loop and the adjacent transmembrane domain. PMID- 8626766 TI - Asp333, Asp495, and His523 form the catalytic triad of rat soluble epoxide hydrolase. AB - On the basis of the sequence similarity between mammalian epoxide hydrolases and bacterial haloalkane dehalogenase reported earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch, F., and Hammock, B. D. (1994) FEBS Lett. 338, 251-256; Beetham, J. K., Grant, D., Arand, M., Garbarino, J., Kiyosue, T., Pinot, F., Oesch, F., Belknap, W. R., Shinozaki, K., and hammock, B. D. (1995) DNA Cell. Biol. 14, 61-71) we selected candidate amino acid residues for the putative catalytic triad of the rat soluble epoxide hydrolase. The predicted amino acid residues were exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA, followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were analyzed for their epoxide hydrolase activity toward the model substrate trans-stilbene oxide. In case of impaired catalytic activity of a given mutant, the structural integrity of the recombinant enzyme protein was assessed either by its ability to covalently bind the substrate trans-stilbene oxide or by affinity purification on benzyl thio Sepharose, using the soluble epoxide hydrolase-specific competitive inhibitor 4 fluorochalcone oxide to release the bound enzyme from the affinity matrix. Of the mutants under investigation, only those with changes in the positions Asp333, Asp495, and His523 were completely inactive toward the model substrate trans stilbene oxide while retaining the proper protein fold. These amino acids were exactly those previously predicted by sequence alignment. Exchange of the amino acid residues flanking the catalytic nucleophile Asp333 significantly changed the kinetic properties of the enzyme. Mutation of His332 to Gln had no apparent effect on the Km but led to a heavily reduced Vmax (5% that of the wild type) of the mutant enzyme, while the exchange of Trp334 against Phe strongly increased the Km (7-fold) and also moderately enhanced the Vmax (2-fold) of the corresponding mutant. Mutation of Trp540 apparently had a strong effect on the protein conformation. PMID- 8626767 TI - Characterization of ERK1 activation site mutants and the effect on recognition by MEK1 and MEK2. AB - To discern MEK1 and MEK2 specificity for their substrate, extracellular signal regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants could be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 had greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg 208 or Thr-207 to an alanine residue significantly altered the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increased the phosphorylation of Tyr-204 approximately 4-fold yet almost completely eliminated the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine resulted in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK. PMID- 8626768 TI - Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon. AB - Tropomyosins are highly conserved, coiled-coil actin binding proteins found in most eukaryotic cells. Striated and smooth muscle alpha-tropomyosins differ by the regions encoded by exons 2 and 9. Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated tropomyosin requires troponin, found only in striated muscle, for strong actin binding. The residues encoded by exon 9 cause these differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10153 10157). We mapped the functional domains encoded by the alpha-tropomyosin exon 9a (striated muscle-specific) and 9d (constitutively expressed), by measuring actin binding and regulation of the actomyosin MgATPase by tropomyosin exon 9 chimeras and truncation mutants expressed in E. coli. We have shown that: 1) the carboxyl terminal nine residues define the actin affinity of unacetylated tropomyosin; 2) in the presence of Ca2+, the entire exon 9a is required for troponin to promote fully high affinity actin binding; 3) the first 18 residues encoded by exon 9a are critical for the interaction of troponin with tropomyosin on the thin filament, even in the absence of Ca2+. The results give new insight into the structural requirements of tropomyosin for thin filament assembly and regulatory function. PMID- 8626769 TI - The influence of sphingomyelin on the structure and function of reconstituted high density lipoproteins. AB - The effect of sphingomyelin (SPM) on the structure and function of discoidal and spherical reconstituted high density lipoproteins (rHDL) has been studied. Three preparations of discoidal rHDL with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of 99.6/0. 0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were prepared by cholate dialysis. SPM did not affect discoidal rHDL size or surface charge. Esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT) was inhibited in the SPM-containing discoidal rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio 99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL) and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t1/2 = 0.8 h) and spherical particles with a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3. 6/24.9/1.0 were formed. Depleting the spherical rHDL of SPM head groups by incubation with sphingomyelinase increased the negative charge on the surface, but did not change their size. Cholesteryl ester transfer protein (CETP)-mediated transfers of cholesteryl esters and triglyceride between spherical rHDL and Intralipid were not affected by SPM head group depletion. The effect of SPM on rHDL structure was assessed spectroscopically. SPM increased POPC acyl chain and head group packing in the discoidal rHDL. When the spherical rHDL were depleted of SPM head groups, POPC acyl chain packing order decreased, but head group packing order was not affected. SPM inhibited the lipid-water interfacial hydration of discoidal rHDL. This parameter was not affected when the spherical rHDL were depleted of SPM head groups. The SPM molecule and the SPM head group, respectively, inhibited the unfolding of apoA-I in discoidal and spherical rHDL. It is concluded that (i) SPM influences the structure of discoidal and spherical rHDL, (ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the SPM head group does not affect CETP-mediated lipid transfers into or out of spherical rHDL. PMID- 8626770 TI - An 11-amino acid sequence from c-met initiates epithelial chemotaxis via phosphatidylinositol 3-kinase and phospholipase C. AB - Interaction of hepatocyte growth factor with its high affinity receptor c-met initiates a cascade of intracellular events leading to epithelial motility. An 11 amino acid sequence from the c-met receptor has been found to cause cell transformation in transfected fibroblasts (Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., Dalla, Z. P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M.(1994) Cell 77, 261-271). We inserted this sequence into a mutant platelet derived growth factor receptor (F5) to determine if this region of c-met can initiate cell motility and which signaling pathways it activates. The platelet derived growth factor (PDGF) receptor/c-met hybrid (F5 met) initiated PDGF dependent chemotaxis in renal epithelial cells (8.0 +/- 2.3 versus 70.5 +/- 4.8 cells/mm2), while the parental construct, F5, did not. Addition of PDGF to cells expressing F5 met caused activation of the phosphatidylinositol (PI) 3-kinase (control 2.0 +/- 0.8, +PDGF 17.1 +/- 5.1, n = 3, p < 0.05) and phospholipase C (control 478.5 +/- 67 dpm/well, +PDGF 1049.3 +/- 93, n = 4, p = 0.003), while neither pathway was activated in cells expressing F5. The chemotactic response of F5 met was inhibited by both the PI 3-kinase inhibitor wortmannin and the phospholipase C inhibitor U-71322. Selective activation of the PI 3-kinase utilizing a PDGF receptor mutant (F3) containing the native high affinity PI 3 kinase binding site also resulted in PDGF stimulated chemotaxis, although less than that generated by the c-met sequence. These findings demonstrate that the 11 amino acid sequence from c-met initiates epithelial motility via coincident activation of the PI 3-kinase and phospholipase C and that selective activation of the PI 3-kinase can initiate a partial chemotactic response. PMID- 8626771 TI - The requirement of reductant for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase. AB - A source of reductant is routinely added to the in vitro iron-molybdenum cofactor (FeMo-co) synthesis assay, although a requirement for reductant has not been established. This report demonstrates that the addition of reductant to the in vitro FeMo-co synthesis system is not required when Azotobacter vinelandii cell free extract is prepared in buffer that lacks added reductant. The addition of reductant is required, however, if the A. vinelandii cell-free extract is chemically oxidized prior to addition to the assay. These results might suggest that extracts of A. vinelandii contain a physiological source of reductant that functions in the in vitro synthesis of FeMo-co. It is possible that the proteins required for FeMo-co biosynthesis (e.g. NIFNE and dinitrogenase reductase) are at the appropriate redox state to function in the in vitro reaction in the extract that is free of added reductant but not in the chemically oxidized extract. It is also possible that dinitrogenase reductase and/or NIFNE (both Fe-S proteins required for FeMo-co synthesis) might catalyze the reductant-dependent reaction for FeMo-co synthesis. Dithionite, Ti(III) citrate, and NADH are able to serve as the source of reductant for in vitro FeMo-co biosynthesis. PMID- 8626772 TI - Global conformational transitions in Escherichia coli primary replicative helicase DnaB protein induced by ATP, ADP, and single-stranded DNA binding. Multiple conformational states of the helicase hexamer. AB - The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is s20,w = 11.9 +/- 0.2 compared to the sedimentation coefficient s20,w = 10.5 +/- 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 +/- 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-depsilonA(pepsilonA)19, s20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of approximately 4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant approximately 3 orders of magnitude weaker than in the presence of the ATP analog (tense state). PMID- 8626773 TI - The cell cycle in polyploid megakaryocytes is associated with reduced activity of cyclin B1-dependent cdc2 kinase. AB - The platelet precursor, the megakaryocyte, matures to a polyploid cell as a result of DNA replication in the absence of mitosis (endomitosis). The factors controlling endomitosis are accessible to analysis in our megakaryocytic cell line, MegT, generated by targeted expression of temperature-sensitive simian virus 40 large T antigen to megakaryocytes of transgenic mice. We aimed to define whether endomitosis consists of a continuous phase of DNA synthesis (S) or of S phases interrupted by gaps. Analysis of the cell cycle in MegT cells revealed that, upon inactivation of large T antigen, the cells shifted from a mitotic cell cycle to an endomitotic cell cycle consisting of S/Gap phases. The level of the G1/S cyclin, cyclin A, as well as of the G1 phase cyclin, cyclin D3, were elevated at the onset of DNA synthesis, either in MegT cells undergoing a mitotic cell cycle or during endomitosis. In contrast, the level of the mitotic cyclin, cyclin B1, cycled in cells displaying a mitotic cell cycle while not detectable during endomitosis. Comparable levels of the mitotic kinase protein, Cdc2, were detected during the mitotic cell cycle or during endomitosis; however, cyclin B1 dependent Cdc2 kinase activity was largely abolished in the polyploid cells. Fibroblasts immortalized with the same heat-labile oncogene do not display reduced levels of cyclin B1 upon shifting to high temperature nor do they become polyploid, indicating that reduced levels of cyclin B1 is a property of megakaryocytes and not of the T-antigen mutant. We conclude that cellular programming during endoreduplication in megakaryocytes is associated with reduced levels of cyclin B1. PMID- 8626774 TI - The sequence of human betaB1-crystallin cDNA allows mass spectrometric detection of betaB1 protein missing portions of its N-terminal extension. AB - The sequence of human betaB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact betaB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of betaB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human betaB1 to the cleavage occurring in bovine betaB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human betaB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of betaB1 amoung species. Results from animal models suggest that cleavage of the N terminal extension of betaB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded betaB1 crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed betaB1-crystallins become water-insoluble is relatively slow in humans. PMID- 8626777 TI - Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene. AB - The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity. PMID- 8626775 TI - Insulin-like growth factor-I (IGF-I) regulates IGF-binding protein-5 synthesis through transcriptional activation of the gene in aortic smooth muscle cells. AB - Previous studies have shown that porcine aortic smooth muscle cells (SMCs) secrete two insulin-like growth factor-binding proteins (IGFBP), IGFBP-2 and -4, and that these IGFBPs modulate IGF-I-stimulated SMC proliferation and migration. In this study we demonstrate that porcine SMCs express IGFBP-5 mRNA and synthesize and secrete the protein. In this cell type, the biosynthesis of IGFBP 5 is up-regulated by IGF-I. This increase in IGFBP-5 synthesis is accompanied by an increase in the steady-state mRNA levels. The induction of IGFBP-5 mRNA by IGF I is time- and dose-dependent and requires de novo protein synthesis. IGF-II and insulin also increase IGFBP-5 mRNA levels at high doses. An IGF-I analog with normal affinity for the IGF-I receptor but reduced affinity for IGFBPs evokes a similar increase. Another analog that binds to IGFBPs but not to the receptor has no effect, indicating that this effect of IGF-I is mediated through the IGF-I receptor. The IGF-I-induced IGFBP-5 gene expression is cell type-specific because IGF-I had no such effect in other cell types examined. Nuclear run-on assays revealed that IGF-I increased transcription rate of the IGFBP-5 gene, while IGF-I did not change the IGFBP-5 mRNA stability. Furthermore, the IGFBP-5 promoter was 3.5-fold more active in directing expression of the luciferase reporter gene in IGF-I-treated aortic SMCs as compared to control cells, whereas the luciferase activity remained the same in control- and IGF-I-treated fibroblasts. These results suggest that IGF-I up-regulates IGFBP-5 synthesis by transcriptionally activating the IGFBP-5 gene in aortic SMCs. PMID- 8626776 TI - Structure-function analysis of P-selectin-sialyl LewisX binding interactions. Mutagenic alteration of ligand binding specificity. AB - P-selectin is a vascular cell adhesion molecule that is expressed on the surface of platelets and endothelial cells in response to inflammatory stimuli. It is believed to aid in the binding and recruitment of leukocytes to inflamed tissue. P-selectin adhesion to leukocytes is mediated by the amino-terminal lectin domain that binds the sialyl LewisX (sLeX) carbohydrate (Neu5Acalpha2-3Galbeta1 4(Fucalpha1-3)GlcNAc). Neither the three-dimensional structure of P-selectin nor the protein-carbohydrate interactions that mediate the binding of P-selectin to the sLeX carbohydrate have been determined. The most closely related protein for which a ligand-bound three-dimensional structure has been resolved is the rat mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134). Using the known binding interactions that occur between the rat mannose-binding protein and its ligand (oligomannose) as a template, we have used site-directed mutagenesis to substitute Ala-77 with lysine. This substitution changed P-selectin-carbohydrate binding specificity from sLeX to oligomannose. Further substitution altered the binding preference from mannose to galactose in a predictable manner. These results indicate that P selectin binds sLeX in a shallow cleft that is similar to the mannose-binding protein saccharide-binding cleft. Additionally, we present an extensive mutagenic analysis of P-selectin Lys-113, a residue that has previously been implicated in P-selectin binding to both sLeX and 3-sulfated galactosylceramide (sulfatide). Our analysis demonstrates that Lys-113 is probably not involved in P-selectin binding to either sulfatide or sLeX. Functionally, it appears that P-selectin has retained a conserved carbohydrate and calcium coordination site that enables it to bind carbohydrate in a manner that is quite similar to that which has been determined for the rat mannose-binding protein. PMID- 8626778 TI - Human skeletal muscle nebulin sequence encodes a blueprint for thin filament architecture. Sequence motifs and affinity profiles of tandem repeats and terminal SH3. AB - Analysis of deduced protein sequence and structural motifs of approximately 5500 residues of human fetal skeletal muscle nebulin reveals the design principles of this giant multifunctional protein in the sarcomere. The bulk of the sequence is constructed of approximately 150 tandem copies of approximately 35-residue modules that can be classified into seven types. The majority of these modules form 20 super-repeats, with each super-repeat containing a 7-module set (one of each type in the same order). These super-repeats are further divided into eight segments: with six segments containing adjacent, highly homologous super-repeats, one single repeat segment consisting of 8 nebulin modules of the same type, and a non-repeat segment terminating with a SH3 domain at the C terminus. The interactions of actin, tropomyosin, troponin, and calmodulin with nebulin fragments consisting of either repeating modules or the SH3 domain support its role as a giant actin-binding cofilament of the composite thin filament. Such affinity profiles also suggest that nebulin may bind to tropomyosin and troponin to form a composite calcium-linked regulatory complex on the thin filament. The modular construction, super-repeat structure, and segmental organization of nebulin sequence appear to encode thin filament length, periodicity, insertion, and sarcomere proportion in the resting muscle. PMID- 8626779 TI - Tau protects beta in the leading-strand polymerase complex at the replication fork. AB - Replication forks formed in the absence of the tau subunit of the DNA polymerase III holoenzyme produce shorter leading and lagging strands than when tau is present. We show that one reason for this is that in the absence of tau, but in the presence of the gamma-complex, leading-strand synthesis is no longer highly processive. In the absence of tau, the size of the leading strand becomes proportional to the concentration of beta and inversely proportional to the concentration of the gamma-complex. In addition, the beta in the leading-strand complex is no longer resistant to challenge by either anti-beta antibodies or poly(dA):oligo(dT). Thus, tau is required to cement a processive leading-strand complex, presumably by preventing removal of beta catalyzed by the gamma-complex. PMID- 8626780 TI - MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. AB - MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases. PMID- 8626781 TI - Purification of nuclear proteins from human HeLa cells that bind specifically to the unstable tandem repeat (CGG)n in the human FMR1 gene. AB - Autonomous expansions of trinucleotide repeats with the general structure 5' d(CNG)n-3' are associated with several human genetic diseases. We have characterized nuclear proteins binding to the unstable 5'-d(CGG)n-3' repeat. Its expansion in the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. Electrophoretic mobility shift assays using nuclear extracts from several human and other mammalian cell lines and from primary human cells demonstrated specific binding to double stranded DNA fragments containing only a 5'-d(CGG)17-3' repeat or the repeat and flanking genomic sequences of the human FMR1 gene. Protein binding was inhibited by complete methylation of the trinucleotide repeat. The complex formed with crude nuclear extract apparently did not contain the human transcription factor Sp1 that binds to a characteristic GC-rich sequence. A 20-kDa protein involved in specific binding to the double-stranded 5'-d(CGG)17-3' repeat was purified from HeLa nuclear extracts by DNA affinity chromatography. PMID- 8626782 TI - The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats. AB - Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity. PMID- 8626783 TI - Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1. AB - Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner. PMID- 8626784 TI - hCDC47, a human member of the MCM family. Dissociation of the nucleus-bound form during S phase. AB - hCDC47 is a putative human homologue of yeast CDC47 and a member of the MCM protein family, which has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. In the present study, we performed an initial characterization of hCDC47. We found that hCDC47 protein was present in the nucleus of cultured human cells in two different forms; one extractable by a non-ionic detergent and the other resistant to such extraction and tightly associated with the nucleus. The levels of the nucleus-bound form gradually diminished during S phase progression, although the total amount of nuclear hCDC47 protein remained relatively constant, suggesting that the nucleus-bound form becomes dissociated from the nuclear structure during DNA replication. This behavior of hCDC47 protein is very similar to that of other mammalian MCM proteins reported recently. We also found that expression of hCDC47 mRNA was repressed in quiescent cells but was induced at the late G1 to S phase by growth factor stimulation. Together, these findings indicate that hCDC47 protein together with other MCM proteins participates in the regulation of mammalian DNA replication. PMID- 8626785 TI - Identification and characterization of a versatile retinoid response element (retinoic acid receptor response element-retinoid X receptor response element) in the mouse tissue transglutaminase gene promoter. AB - Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site. PMID- 8626786 TI - Regulation of membrane-bound phospholipase D by protein kinase C in HL60 cells. Synergistic action of small GTP-binding protein RhoA. AB - In HL60 cells, the membrane-bound phospholipase D (PLD) was stimulated by 4beta phorbol 12-myristate 13-acetate (PMA) in the presence of the cytosolic fraction from HL60 cells or rat brain. The cytosolic factor for this PMA-induced PLD activation was subjected to purification from rat brain by sequential chromatographies. The PLD stimulating activity was found in protein kinase C (PKC) fraction containing alpha, betaI, betaII, and gamma isozymes. PKC isozymes were further separated by hydroxylapatite chromatography. PKCalpha and - beta, but not gamma, isozymes were found to activate membrane-bound PLD. PKCalpha was much more effective than PKCbeta for PLD activation. Millimolar concentrations of MgATP were required for the PKC-mediated PLD activation in HL60 membranes. MgATP is utilized to maintain the levels of phosphatidylinositol 4,5-bisphosphate (PIP2) under these assay conditions. The PKC-mediated PLD activation was completely inhibited by neomycin, a high affinity ligand for PIP2, and this suppression was recovered by the addition of exogenous PIP2. Thus, these results suggest that PIP2 is supposed to play a key role in PKC-mediated PLD activity in HL60 membranes. Furthermore, PKCalpha-mediated PLD activation was potentiated by the addition of recombinant RhoA protein in the presence of guanosine 5'-O-(3 thiotriphosphate) (GTPgammaS). The results obtained here indicate that PKCalpha and RhoA (GTP form) exert a synergistic action in the membrane-bound PLD activation in HL60 cells. PMID- 8626787 TI - Structure and cellular distribution of mouse brain testican. Association with the postsynaptic area of hippocampus pyramidal cells. AB - The complete deduced primary structure of mouse brain testican has been established from cDNA cloning. The cDNA encodes a polypeptide of 442 amino acids belonging to the proteoglycan family. The mouse brain testican core protein is 95% identical to its human testicular counterpart. In situ hybridization investigations revealed that mouse testican mRNA is mainly present in a subpopulation of pyramidal neurons localized in the CA3 area of the hippocampus. An immunocytochemical approach, with antibodies directed against an overexpressed chimeric antigen, produced in bacterial systems, showed that testican is associated with the postsynaptic region of these pyramidal neurons. Testican includes several putative functional domains related to extracellular or pericellular proteins associated with binding and/or regulatory functions. On the basis of its structural organization and its occurrence in postsynaptic areas, this proteoglycan might contribute to various neuronal mechanisms in the central nervous system. PMID- 8626788 TI - Cyclic AMP and chloride-dependent regulation of the apical constitutive secretory pathway in colonic epithelial cells. AB - Epithelial cells of the colonic crypt engage in cAMP-mediated fluid and electrolyte secretion. In addition to participating in electrolyte transport, colonic crypt cells also synthesize and secrete a number of proteins and peptides that play a crucial role in mucosal homeostasis. In the present study we show that cAMP regulates not only electrolyte secretion but also polarized protein secretion in a tissue culture model of colonic crypt cells. We found that apical but not basolateral protein secretion was stimulated by a physiological activator of the cAMP pathway, vasoactive intestinal peptide, as well as by a cell-permeant analogue of cAMP (8-(4-chlorophenylthio)cAMP) at concentrations as low as 12.5 microM. Based on several criteria, we determined that the regulation of protein secretion by cAMP in HT29-CL19A cells occurs via stimulation of constitutive membrane traffic from the trans-Golgi network (TGN) to the apical cell surface. In addition, the regulation of apical protein secretion by cAMP was Cl--dependent with cAMP inhibiting rather than stimulating secretion in Cl--depleted cells. The locus of cAMP action on the secretory pathway is at least in part at the level of the TGN, where it stimulates the sialylation of alpha1-antitrypsin (i.e. one of the identified secretory proteins) in addition to the traffic of secretory proteins from the TGN to the apical cell surface. We propose that a cyclic AMP and Cl--dependent regulation of TGN acidification could modulate both sialylation and secretory vesicle budding at the TGN. PMID- 8626789 TI - The GA-binding protein can serve as both an activator and repressor of ribosomal protein gene transcription. AB - The GA-binding protein (GABP), a heterodimeric transcription factor with widespread tissue distribution, has been found to be a strong positive regulator of several ribosomal protein (rp)-encoding genes. In such genes, e.g. the mouse rpL30 gene, the GABP-binding sites are located 40-80 base pairs upstream of the transcriptional start point. Potential GABP-binding sites are present in the promoters of numerous other rp genes, not only in upstream regions, but also in the immediate vicinity of the transcriptional start point. The mouse rpS16 gene is an example of the latter type. We demonstrate here that GABP binds to the rpS16 initiation region, and in so doing down-regulates rpS16 transcription both in vivo and in vitro. Supplementation of cell-free extracts with GABP inhibits transcription on rpS16 templates while concomitantly stimulating transcription on rpL30 templates. The repressive and stimulatory effects, which were proportional to the amount of GABP added, required both the GABP alpha subunit and either a beta1 or beta2 subunit. Mutations of the rpS16 GABP-binding sites that abolish binding increased rpS16 promoter activity in vivo and in vitro, whereas mutations that strengthen GABP binding caused a reduction in promoter activity. The binding of GABP to the rpS16 initiation region does not significantly affect the positioning of the transcriptional start points. Taken together with earlier studies, these new findings indicate that GABP can have a dual role as repressor or activator of rp gene transcription. PMID- 8626791 TI - Expression of rat cathepsin S in phagocytic cells. AB - Cysteine lysosomal proteases are essential for turnover of intracellular and extracellular proteins. These enzymes are strongly implicated in normal and pathological processes involving tissue remodeling. Among the cysteine proteases, cathepsin S seems to be best suited for such a process since it retains most of its enzymatic activity at neutral pH. In situ hybridization analyses of the adult rat brain, spleen, and lung reveal that cathepsin S mRNA is preferentially expressed in cells of mononuclear-phagocytic origin. After entorhinal cortex lesion of adult rat brain (a paradigm for neuronal degeneration and reactive synaptogenesis), cathepsin S mRNA is dramatically increased in activated microglia in the deafferented dentate gyrus and in macrophages at the wound site, suggesting a role in lesion-induced tissue remodeling. This possibility is further supported by the finding that cathepsin S degrades a number of extracellular matrix molecules at neutral pH and by the finding that inflammatory mediators stimulate its secretion from the microglia and macrophages. These data suggest that cathepsin S is an important player in degenerative disorders associated with the cells of the mononuclear phagocytic system. PMID- 8626790 TI - Lecithin:cholesterol acyltransferase overexpression generates hyperalpha lipoproteinemia and a nonatherogenic lipoprotein pattern in transgenic rabbits. AB - Cholesterol esterification within plasma lipoprotein particles is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact of the overexpression of this enzyme on plasma concentrations of the different plasma lipoproteins in an animal model expressing cholesteryl ester transfer protein was evaluated by generating rabbits expressing human LCAT. A 6.2-kilobase human genomic DNA construct was injected into the pronuclei of rabbit embryos. Of the 1002 embryos that were injected, 3 founder rabbits were characterized that expressed the human LCAT gene. As in mice and humans, the principal sites of mRNA expression in these rabbits is in the liver and brain, indicating that the regulatory elements required for tissue-specific expression among these species are similar. The alpha-LCAT activity correlated with the number of copies of LCAT that integrated into the rabbit DNA. Compared with controls, the high expressor LCAT-transgenic rabbits total and high density lipoprotein (HDL) cholesterol concentrations were increased 1.5-2.5-fold with a 3.1-fold increase in the plasma cholesterol esterification rate. Analysis of the plasma lipoproteins by fast protein liquid chromatography indicates that these changes reflected an increased concentration of apolipoprotein E-enriched, HDL1-sized particles, whereas atherogenic apolipoprotein B particles disappeared from the plasma. The concentrations of plasma HDL cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT activity (r = 0.94; p < 0.001) in the transgenic rabbits. These results indicate that overexpression of LCAT in the presence of cholesteryl ester transfer protein leads to both hyperalpha lipoproteinemia and reduced concentrations of atherogenic lipoproteins. PMID- 8626792 TI - Regulation of acetylcholinesterase expression during neuronal differentiation. AB - We have examined the developmental expression of acetylcholinesterase (AChE) during the process of neuronal differentiation from a pluripotent stem cell. P19 embryonic carcinoma cells form embryoid bodies, which, when cultured with retinoic acid, are induced to differentiate into neurons and glia. No AChE activity is present in the undifferentiated stem cells, and mRNA protection analyses do not detect AChE mRNA. Commitment to a neuronal differentiation pathway results in increased levels of AChE mRNA, production of a tetrameric form of the enzyme, and secretion of AChE into the culture medium. Concomitant with subsequent morphological differentiation into neurons, enzyme secretion diminishes and AChE becomes largely tethered to the neuronal cell membranes. The enzyme is attached to the cell surface as a globular tetramer. Its hydrodynamic properties are consistent with association through a noncatalytic hydrophobic subunit rather than anchorage by a glycophospholipid tail. No change in the rate of transcription of the Ache gene was detected during the course of differentiation, suggesting that the gene is actively transcribed at very early stages of development. Results suggest that stabilization of a labile mRNA governs the increase in AChE mRNA and gene product. The studies presented indicate that an early event in neuronal differentiation is the stabilization of the mRNA leading to expression of a secreted form of AChE. A subsequent step associated with neurite outgrowth results in a transition from secretion of the tetrameric enzyme to its localization on the cell membrane. PMID- 8626793 TI - The serotonin 1a receptor gene contains a TATA-less promoter that responds to MAZ and Sp1. AB - The structure and function of the 5'-flanking region of the mouse and human serotonin 1a receptor gene have been analyzed by RNA 5' end mapping, DNA-protein interaction, and transient expression assays. A large number of mRNA 5' termini, detected by mapping 5' ends from mouse brain RNA, were found dispersed over a region of about 700 base pairs flanking the receptor coding sequence. Consistent with the apparently heterogeneous pattern of transcription initiation, the flanking DNA sequence lacked typical TATA box elements and was rich in guanine and cytosine. The mouse and human 5'-flanking sequences were 63% homologus and similarly organized. A guanine-cytosine-rich DNA sequence motif related to the sequence 5'-GGGG(C/A)GGGG-3' was repeated within the 5'-flanking region and located at or near several mRNA 5' ends. This DNA sequence motif bound to proteins in a crude HeLa cell nuclear extract. A cDNA encoding a protein that interacts with this sequence was cloned and found to be the MAZ (Pur-1, Zif87) protein. The interaction between MAZ and the receptor gene 5'-flanking region proximal to the protein coding sequence was examined by DNase I footprinting, and four sites of MAZ interaction were identified. Three of the four MAZ binding sites also were shown to interact with transcription factor Sp1. Overproduction of MAZ or Sp1 in transient transfection assays increased expression directed by the human 5'-flanking sequence, although MAZ was substantially more effective. This result suggests that MAZ and Sp1 both participate in regulating expression from the serotonin 1a receptor gene promoter, and it raises the possibility that MAZ may act at a variety of promoters through the guanosine-cytosine-rich sequences generally thought to serve as binding sites for the Sp1 family of transcription factors. Analysis of one of the guanosine-cytosine-rich DNA sequences also revealed that it can serve as a transcription initiator sequence in vitro. This initiator sequence differs from previously characterized initiators and may represent a new class of this transcriptional control sequence. PMID- 8626794 TI - Full-length sequence, localization, and chromosomal mapping of ameloblastin. A novel tooth-specific gene. AB - We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization. PMID- 8626795 TI - Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. AB - Amyloid precursor protein (APP) and cholesterol metabolism are genetically linked to Alzheimer's disease, the latter through apolipoprotein E, a lipid and cholesterol transport protein. We have examined the hypothesis that the processing of APP is disrupted by elevated cholesterol, which is known to modulate the activity of several transmembrane proteins. In the current study, cholesterol, solubilized by methyl- beta-cyclodextrin or ethanol, was added to the culture media of APP 751 stably transfected HEK 293 cells. Radiolabeled APP and APPsol (the soluble N-terminal derivative following alpha-secretase cleavage) were precipitated from lysates and conditioned media of stably transfected HEK 293 cells; the relative levels were determined by quantitative densitometry following separation by SDS-polyacrylamide gel electrophoresis. The data show that cholesterol, solubilized by methyl-beta-cyclodextrin, greatly reduced the levels of APPsol. Low doses of ethanol-solubilized cholesterol similarly caused a dramatic reduction of APPsol. By contrast, levels of APP holoprotein remained the same or increased. The large decrease seen in APPsol production was not due to nonspecific inhibition of secretion because several secreted proteins increased in level. Cholesterol, which impedes membrane fluidity, may lower APPsol production by impeding the interaction of the substrate with its protease(s). If APPsol were to function trophically, as suggested by other studies, the current conclusion suggests that changes in cellular cholesterol levels in Alzheimer's disease could contribute to neuronal degeneration by decreasing the production of APPsol. PMID- 8626797 TI - Identification of a region of p53 that confers lability. AB - Degradation provides one means for controlling the cellular level of the p53 tumor suppressor. Here we have determined a structural element of p53 required for degradation. To create a substrate amenable to in vitro analysis of proteolysis, we appended to p53 the N terminus of antizyme, a protein that binds to and induces degradation of mammalian ornithine decarboxylase (ODC). We found using deletion analysis that an element within amino acids 100-150 is required for degradation of the fusion protein. A monoclonal antibody (PAb246) that binds close to this region prevents the degradation induced by human papillomavirus 16 E6 protein. Furthermore, we found that amino acids 100-150 of p53 can function as an independent domain to induce Trypanosoma brucei ODC, a stable protein, to be degraded in vivo or, by cooperating with an antizyme binding domain of ODC, to confer polyamine-dependent regulation. PMID- 8626796 TI - The N terminus of antizyme promotes degradation of heterologous proteins. AB - Regulated degradation of ornithine decarboxylase (ODC) is mediated by its association with the inducible protein antizyme. The N terminus of antizyme (NAZ), although unneeded for the interaction with ODC, must be present to induce degradation. We report here that covalently grafting NAZ to ODC confers lability that normally results from the non-covalent association of native antizyme and ODC. To determine whether NAZ could act similarly as a modular functional domain when grafted to other proteins, we fused it to a region of cyclin B (amino acids 13-90) capable of undergoing degradation or to cyclin B (amino acids 13-59), which is not subject to degradation. The association with NAZ made both NAZ cyclin B13-90 and NAZ-cyclin B13-59 unstable. Furthermore, NAZ and cyclin B 13-59 were together able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex bound to the 26 S protease but not the 20 S proteasome, consistent with the observation that ODC degradation is mediated by the 26 S protease. The association was shown to be independent of NAZ, suggesting that NAZ does not act as a recognition signal. PMID- 8626798 TI - Estrogen induces the assembly of a multiprotein messenger ribonucleoprotein complex on the 3'-untranslated region of chicken apolipoprotein II mRNA. AB - UV cross-linking was used to identify estrogen-induced hepatocyte proteins that bind to apoII mRNA. Probes spanning the entire message revealed the presence of eight estrogen-induced proteins cross-linked to the 3'-untranslated region (UTR), but not to the coding region or the 5'-UTR. Two estrogen-induced proteins of 132 and 50 kDa were either absent or barely detectable in control animals, whereas six additional proteins of 93, 83, 74, 65, 58, and 45 kDa were clearly present in control animals and increased 2-5-fold by estrogen. A similar profile of estrogen induced proteins was seen with the 3'-UTRs of the estrogen-regulated mRNAs for apoB and vitellogenin II, but not with the 3'-UTRs of the non-estrogen-regulated mRNAs for apoA-I and glyceraldehyde-phosphate dehydrogenase. These findings indicate that the estrogen-induced proteins discriminate among mRNAs and suggest that they interact selectively with the family of estrogen-regulated mRNAs. The estrogen-induced proteins are found in the cytoplasmic fraction of liver extracts, and a subset of them are also found in adrenal glands, testes, heart, brain, and kidneys, but they are estrogen-induced only in the liver. Deletion analysis defined a 150-nucleotide region of the apoII 3'-UTR that is necessary for maximal binding of the estrogen-induced proteins. An internal deletion of endonucleolytic cleavage sites previously identified within the apoII 3'-UTR selectively reduced the binding of the 58-kDa protein. These findings reveal remarkable complexity in estrogen-stimulated protein-RNA interactions within the 3'-UTRs of estrogen-regulated mRNAs. These proteins may participate in the mRNA degradation process or in other aspects of cytoplasmic mRNA metabolism that accompany estrogen-stimulated vitellogenesis. PMID- 8626799 TI - Distinct mechanisms contribute to stringent substrate specificity of tissue-type plasminogen activator. AB - Tissue-type plasminogen activator (t-PA) has evolved to optimize cleavage of plasminogen (Plg) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2 subsites of t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly selective cleavage of protein fusions. Surprisingly, t PA cleaves this engineered protein substrate with a Km that is reduced 950-fold relative to the Km for hydrolysis of the same target sequence within a peptide. This reduction of Km suggests that binding is facilitated by interactions between protein substrate and protease that are distant from the P4-P2' residues. We use this kinetic data to derive a model in which several distinct mechanisms contribute to the remarkable specificity of t-PA. PMID- 8626800 TI - A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. AB - In Drosophila melanogaster, the frizzled gene plays an essential role in the development of tissue polarity as assessed by the orientation of cuticular structures. Through a combination of random cDNA sequencing, degenerate polymerase chain reaction amplification, and low stringency hybridization we have identified six novel frizzled homologues from mammals, at least 11 from zebrafish, several from chicken and sea urchin, and one from Caenorhabditis elegans. The complete deduced amino acid sequences of the mammalian and nematode homologues share with the Drosophila frizzled protein a conserved amino-terminal cysteine-rich domain and seven putative transmembrane segments. Each of the mammalian homologues is expressed in a distinctive set of tissues in the adult, and at least three are expressed during embryogenesis. As hypothesized for the Drosophila frizzled protein, the frizzled homologues are likely to act as transmembrane receptors for as yet unidentified ligands. These observations predict the existence of a family of signal transduction pathways that are homologous to the pathway that determines tissue polarity in Drosophila. PMID- 8626801 TI - A transient precursor of the HIV-1 protease. Isolation, characterization, and kinetics of maturation. AB - Recently, the mechanism of autoprocessing of the protease (PR) of the human immunodeficiency virus type 1 from the model polyprotein, MBP-DeltaTF-PR DeltaPol, which contains the protease linked to short native flanking sequences (DeltaTF and DeltaPol) fused to the maltose binding protein (MBP) of Escherichia coli, was reported (Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina, D. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7970-7974). According to this mechanism, intramolecular cleavage of the N-terminal strands of the dimeric MBP-DeltaTF-PR-DeltaPol protein leads to the formation of the PR DeltaPol intermediate, which is subsequently converted to the mature protease by cleavage of the C-terminal strands. We now report the purification and characterization of the PR-DeltaPol intermediate and the kinetics of its processing to the mature protease. Unlike the MBP-DeltaTF-PR-DeltaPol precursor, PR-DeltaPol has proteolytic activity similar to that of the mature enzyme at pH 5.0. The pH rate profile for kcat/Km is similar to that of the mature protease above pH 4.0. Although the PR-DeltaPol is more sensitive than the mature protease toward denaturing reagents, both the enzymatic activity and the intrinsic fluorescence of PR-DeltaPol are linearly dependent on the protein concentration, indicating that the protein is largely in its dimeric form above 10 nM. In contrast to the first-order kinetics observed for the proteolytic reaction at the N terminus of the protease, the proteolytic reaction at the C terminus of the protease is second order in protein concentration. These results are discussed in terms of a mechanism in which the C-terminally located DeltaPol peptide chains are cleaved intermolecularly to release the mature protease. PMID- 8626802 TI - Differential activation of lung-specific genes by two forkhead proteins, FREAC-1 and FREAC-2. AB - We describe the cDNA sequences for two human transcription factors, Forkhead RElated ACtivator (FREAC)-1 and -2, that belong to the forkhead family of eukaryotic DNA binding proteins. FREAC-1 and -2 are encoded by distinct genes, are almost identical within their DNA binding domains and in the COOH termini, but are otherwise divergent. Cotransfections with a reporter carrying FREAC binding sites showed that both proteins are transcriptional activators, and deletions located the activation domains to the COOH-terminal side of the forkhead domains. Expression of FREAC-1 and FREAC-2 is restricted to lung and placenta. We show that the promoters of genes for lung-specific proteins such as pulmonary surfactant proteins A, B, and C (SPA, SPB, and SPC) and the Clara cell 10-kDa protein (CC10) contain potential binding sites for FREAC-1 and FREAC-2. DNaseI footprinting verified that FREAC proteins bind to the predicted sites in the CC10 and SPB promoters. While an SPB promoter construct could be transactivated by both FREAC-1 and FREAC-2, CC10 was only activated by FREAC-1. Efficient activation of CC10 by FREAC-1 is shown to be specific for a lung cell line with Clara cell characteristics (H441) and to involve a region of the FREAC 1 protein unable to activate in other cell types. PMID- 8626804 TI - High frequency and error-prone DNA recombination in ataxia telangiectasia cell lines. AB - The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40 transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA. PMID- 8626803 TI - Regulation of a sodium channel-associated G-protein by aldosterone. AB - The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. This channel is regulated, in part, by G-proteins, but it is not known if this mechanism is regulated by aldosterone. We report that aldosterone stimulates the expression of the 41-kDa alphai3 subunit of the heterotrimeric GTP-binding proteins in A-6 cells. Both mRNA and the total amount of this protein are increased by aldosterone. The G-protein is palmitoylated in response to the steroid, and the newly synthesized subunit is found to co-localize with the sodium channel. Aldosterone stimulation of sodium transport is significantly inhibited by inhibition of palmitoylation. These results suggest that aldosterone regulates sodium channel activity in epithelia through stimulation of the expression and post-translational targeting of a channel regulatory G-protein subunit. PMID- 8626805 TI - Regulation of phospholipase D by protein kinase C is synergistic with ADP ribosylation factor and independent of protein kinase activity. AB - Phospholipase D (PLD) which was partially purified from membranes of porcine brain could be stimulated by multiple cytosolic components; these included ADP ribosylation factor (Arf) and RhoA, which required guanine nucleotides for activity, and an unidentified factor which activated the enzyme in a nucleotide independent manner (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950). Here, we report purification of the latter factor, its identification as the alpha isoform of protein kinase C (PKCalpha), and characterization of its regulation of PLD activity. Stimulation of PLD by purified PKCalpha or recombinant PKCalpha (rPKCalpha) occurred in the absence of any nucleotide and required activators such as Ca2+ or phorbol ester. This action was synergistic with stimulation of PLD evoked by either Arf or RhoA. Dephosphorylation of rPKC alpha with protein phosphatase 1 or 2A resulted in a loss of its kinase activity, but had little effect on its ability to stimulate PLD either alone or in conjunction with Arf. Staurosporine inhibited the kinase activity of PKCalpha without affecting activation of PLD. Finally, gel filtration of PKCalpha that had been cleaved with trypsin demonstrated that stimulatory activity for PLD coeluted with the regulatory domain of the enzyme. These data indicate that PKC may regulate signaling events through direct molecular interaction with downstream effectors as well as through its well characterized catalytic modification of proteins by phosphorylation. PMID- 8626806 TI - Modulation of endothelial cell adhesion by hevin, an acidic protein associated with high endothelial venules. AB - High endothelial venules (HEV) are specialized plump postcapillary venules in lymphoid tissues that support high levels of lymphocyte extravasation from the blood. We have recently identified a novel human transcript, expressed to high levels in HEV, that encodes a secreted, acidic protein closely related to the anti-adhesive extracellular matrix protein known as BM-40, osteonectin, and SPARC (secreted protein acidic and rich in cysteine). Here, we show that this protein, designated hevin, is associated with basal, lateral, and apical surfaces of HEV cells, and unlike MECA-79 antigen, is not expressed on the underlying basement membrane. In contrast to fibronectin or other adhesive extracellular matrix proteins, purified hevin does not support endothelial cell adhesion in vitro. Moreover, addition of soluble exogenous hevin inhibits attachment and spreading of endothelial cells on fibronectin substrates. Hevin-treated cells do not form focal adhesions and exhibit a rounded morphology. Together, these results suggest that hevin is an abundant extracellular protein that modulates high endothelial cell adhesion to the basement membrane. PMID- 8626807 TI - Deletions of portions of the extracellular loops of the lutropin/choriogonadotropin receptor decrease the binding affinity for ovine luteinizing hormone, but not human choriogonadotropin, by preventing the formation of mature cell surface receptor. AB - The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor which binds either human choriogonadotropin (hCG) or lutropin (luteinizing hormone, LH) and, therefore, plays a central role in reproductive physiology. In addition to the seven transmembrane helices, three extracellular loops, three intracellular loops, and a cytoplasmic tail characteristic of all G protein-coupled receptors, the rLHR also contains a relatively large N-terminal extracellular domain. Since high affinity hormone binding occurs to this N terminal extracellular domain and since G proteins are activated by intracellular regions of the receptor, it has been hypothesized that upon hormone binding a portion of the hormone or the receptor's extracellular domain might interact with the receptor's extracellular loops and/or transmembrane helices, thus evoking an intracellular conformational change. To explore this possibility, we prepared and characterized several mutants of the rLHR in which portions of the extracellular loops were deleted. Ultimately, it was not possible to examine the signal transduction properties of the mutants because all but one mutant were retained intracellularly. Although the intracellularly retained mutants must be somewhat misfolded, all were found to bind hCG with high affinity if the cells were first solubilized in detergent. However, the binding of oLH to the detergent solubilized mutants was altered. Thus, whereas the wild-type rLHR bound oLH with two apparent affinities, the solubilized deletion mutants bound oLH with only one apparent affinity. Although these data could be interpreted to suggest that an ovine LH (oLH) binding site on the extracellular loops of the rLHR was deleted, data shown argue against this hypothesis. Rather, the results presented suggest that the two apparent affinities of the wild-type rLHR for oLH represent the binding affinities of two populations of rLHR where the mature, cell surface form binds oLH with a higher affinity than the immature, intracellular form. Furthermore, we show that mutations of the rLHR which cause intracellular retention of the receptor result in a decrease from two to one apparent binding sites for oLH due to the absence of the high affinity oLH binding component contributed by the mature cell surface receptor. Therefore, whereas hCG cannot discriminate between the mature cell surface wild-type receptor and an intracellularly retained rLHR mutant, oLH can make this discrimination, thus suggesting a conformational difference between the two forms of the receptor. PMID- 8626808 TI - Tissue-specific expression of the nonneuronal promoter of the aromatic L-amino acid decarboxylase gene is regulated by hepatocyte nuclear factor 1. AB - The rat aromatic l-amino acid decarboxylase (AADC) gene contains alternative promoters which direct expression of neuronal and nonneuronal mRNAs that differ only in their 5'-untranslated regions (UTRs). We have analyzed the expression of the nonneuronal promoter of the rat AADC gene in the kidney epithelial cell line LLC-PK1 and in cells which do not express the nonneuronal form of AADC by transient transfection. These studies revealed that the first 1.1 kilobases of the nonneuronal promoter, including the nonneuronal-specific 5'-UTR (Exon 1), contains sufficient information to direct tissue-specific expression. Serial deletions of this promoter localized the cis-active element to a region between 52 and -28 base pairs upstream of the nonneuronal transcription start site. An A/T-rich sequence, within this region which we have termed KL-1, was found to bind a kidney and liver-specific factor by DNase footprint analysis and was capable of directing tissue-specific expression from a heterologous promoter. Moreover, when the KL-1 sequence was mutated in the context of the entire promoter sequence, all transcriptional activity was abolished. DNA sequence comparison revealed that the KL-1 fragment is highly homologous to the binding site for hepatocyte nuclear factor-1 (HNF-1). Mobility shift studies utilizing an antibody to HNF-1 demonstrated binding of HNF-1 to the KL-1 fragment and cotransfection of HNF-1 cDNA into cells which do not express the nonneuronal form of AADC resulted in activation of transfected AADC nonneuronal promoter constructs. These results strongly suggest that the transcription factor which regulates the tissue-specific expression of the nonneuronal form of AADC mRNA is HNF-1. PMID- 8626809 TI - Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro. AB - Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF 2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF 2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha. PMID- 8626810 TI - Shedding of human thyrotropin receptor ectodomain. Involvement of a matrix metalloprotease. AB - The thyrotropin (TSH) receptor in human thyroid glands has been shown to be cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggested the shedding of the ectodomain. Indeed we observed such a shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. The shedding was increased by inhibitors of endocytosis, recycling, and lysosomal degradation, suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters, whereas forskolin and 8-bromo-cyclic AMP were without effect. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism. The shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the receptor into alpha and beta subunits and reduction of the disulfide bridge(s). The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease. This shedding mechanism may be responsible for the presence of soluble TSH receptor alpha subunit in human circulation. PMID- 8626811 TI - Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. AB - The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen. PMID- 8626812 TI - The proximal promoter of the human transglutaminase 3 gene. Stratified squamous epithelial-specific expression in cultured cells is mediated by binding of Sp1 and ets transcription factors to a proximal promoter element. AB - The transglutaminase 3 enzyme is expressed during the late stages of the terminal differentiation of the epidermis and in certain cell types of the hair follicle. The enzyme is thought to be critically involved in the cross-linking of structural proteins and in the formation of the cornified cell envelope, thereby contributing to rigid structures that play vital roles in shape determination and/or barrier functions. To explore the mechanisms regulating the expression of the transglutaminase 3 gene (TGM3), 3.0 kilobase pairs of sequences upstream from the transcription start site were assessed for their ability to control the expression of a chloramphenicol acetyltransferase reporter gene. Deletion analyses in transiently transfected epidermal keratinocytes defined sequences between -126 and -91 as the proximal promoter region of the gene, and which can confer epithelial-specific expression to the TGM3 gene in vitro. Mutation and DNA protein binding analyses indicated that a complex interaction between adjacent Sp1- and ets-like recognition motifs with their cognate binding factors is required for the function of the TGM3 promoter. As these TGM3 sequences can confer promoter/enhancer activity to reporter genes at a level comparable to the powerful SV40 promoter, they may be useful for gene therapy in keratinocytes. PMID- 8626813 TI - Clinical review 80: Management of hyperthyroidism and hypothyroidism in the pregnant woman. PMID- 8626814 TI - Mutations of the growth hormone receptor--widening the search. PMID- 8626815 TI - A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein. AB - Laron syndrome (LS) is a severe autosomal recessive form of GH resistance resulting from molecular defects in the GH receptor (GHR). Affected individuals have extreme short stature and a typical facial phenotype. The point mutations in the GHR gene identified in this condition have until now been confined to the region encoding the extracellular domain of the receptor. We report here the first homozygous point mutation within the intracellular domain of the GHR in two LS cousins distinguishable from classical LS patients only by the presence of elevated GH-binding protein (GHBP) in their serum. A G to C transversion at the vital - 1 position in the splice donor site of exon 8 disrupts normal splicing, resulting in the complete skipping of exon 8, producing a mutant GHR protein lacking transmembrane and intracellular domains. We predict that this mutant protein would not be anchored in the cell membrane and would be measurable in the circulation as GHBP, hence explaining the phenotype of severe GH resistance combined with elevated circulating GHBP. PMID- 8626816 TI - Eternal vigilance--mortality in children with growth hormone deficiency. PMID- 8626817 TI - Mortality in Canadian children with growth hormone (GH) deficiency receiving GH therapy 1967-1992. The Canadian Growth Hormone Advisory Committee. AB - The objective of this study was to determine the risk of death and potential for prevention of mortality in a large population of children with growth hormone deficiency (GHD). The Canadian GH Advisory Committee registry was initiated in 1967 to include all persons in Canada treated with pituitary-derived GH (1967 1985). Since 1985, the registry has been maintained for continuous surveillance of those treated with biosynthetic GH. Thirty-seven children have died out of a total of 1366 children treated for GHD in the 25 years up to December 31, 1992. Individual cases were reviewed for circumstances before death and autopsy information. The likelihood of individual deaths being caused by potentially preventable endocrine causes was graded on a scale of 1-5. Survival curves were analyzed for the children with idiopathic GHD and craniopharyngioma. Age- and sex specific mortality rates for children with idiopathic GHD were compared with those of the general population. The overall crude mortality rate was 2.7%. The most frequent cause of mortality was tumor recurrence (11/37). A surprisingly high proportion of deaths (9/37) were caused by the preventable endocrine complications of adrenal crisis and hypoglycemia. Children with idiopathic GHD receiving GH therapy had similar age- and sex-specific mortality rates compared with general population rates, except in a high-risk subgroup of males diagnosed with GHD before 2 yr of age. The highest mortality occurred in children with GHD secondary to craniopharyngioma. We concluded that preventable sudden deaths caused by adrenal crisis continue to occur in children with hypopituitarism. A high level of vigilance must be maintained in this population. PMID- 8626818 TI - Calcium, parathyroid function, bone and aging. PMID- 8626819 TI - Role of calcium intake in modulating age-related increases in parathyroid function and bone resorption. AB - Serum parathyroid hormone (PTH) and bone resorption increase in elderly women and contribute to age-related bone loss. Whether these abnormalities are caused by calcium deficiency resulting from age-related decreases in absorption and renal conservation is unclear. We studied 28 normal elderly women (mean +/- SD, age 69.3 +/- 2.7 yr) who were maintained for 3 yr on usual calcium intake levels (20.4 +/- 7.2 mmol/day [815 +/- 289 mg/day]; n = 15) (known as the usual calcium group) or high calcium intake levels (60.4 +/- 6.5 mmol/day [2414+/260 mg/day]; n = 13) (known as the high calcium group) and a reference group of 12 normal young adult women (age 30.1 +/- 4.4 yr), whose calcium intake was 23.0 +/- 4.8 mmol/day (918 +/- 193 mg/day) (known as the young group). Serum PTH was measured every 2 h, and urinary excretion of deoxypyridinoline (Dpd), a new marker for bone resorption, was measured in 4 h collections. Parathyroid gland secretory capacity was assessed during induced hypocalcemia. The mean 24 h serum PTH was 40% lower (P < 0.001), and the mean 24 h urinary Dpd was 35% lower (P < 0.005) in the high than in the usual calcium group. Mean parathyroid gland secretory capacity also was 47% lower (P < 0.005) in the high calcium group than in the usual calcium group. However, the usual calcium group had a mean 24 h serum PTH level that was 70% higher (P < 0.001) and a mean 24 h urinary Dpd level that was 30% higher (P < 0.005) than the young group, whereas the high calcium group was indistinguishable from the young group. Thus, failure of elderly women to increase their calcium intake to offset age-related increases in calcium requirement contributes substantially to their development of increased parathyroid activity and increased bone resorption, whereas a high calcium intake can reverse both abnormalities. PMID- 8626820 TI - Safety of recombinant deoxyribonucleic acid-derived growth hormone: The National Cooperative Growth Study experience. AB - The National Cooperative Growth Study has monitored the safety of recombinant human GH (rhGH) since 1985. Data have been collected from more than 19,000 children representing over 47,000 patient-years of rhGH treatment. Children receiving GH for renal disease were more likely to develop problems such as intracranial hypertension than those with GH deficiency (P < 0.01). Children with idiopathic short stature were less likely to develop slipped capital femoral epiphysis than those with GH deficiency or Turner's syndrome (P < 0.01). There was no evidence of an increased recurrence of leukemia or central nervous system tumors. There were 3 new cases of leukemia in children without known risk factors for developing leukemia and 5 cases in children with known risk factors. Growth deceleration associated with high affinity, high capacity antibodies to GH was found in only 2 of 5039 subjects tested (0.04%). Major adverse events in association with rhGH treatment have been rare, and preexisting medical conditions such as renal insufficiency may affect their frequency. PMID- 8626821 TI - Transsphenoidal pituitary resection for preoperative diagnosis of prolactin secreting pituitary adenoma in women: long term follow-up. AB - The long term efficacy and safety of transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenomas in a large series of women have not been described. Four hundred and nine consecutive women at this university tertiary referral center undergoing transsphenoidal resection for preoperative diagnosis of PRL-secreting pituitary adenoma were followed for a minimum of 4 yr. The objective was to determine the efficacy and morbidity of this procedure and to identify features correlating with the resolution of hyperprolactinemia. Outcome measures included referral, preoperative, surgical, postoperative hospitalization, and long-term follow-up information, including recent PRL concentration. Follow-up was ascertained in 83% of patients who were followed for a mean of 9.2 yr. Recurrence of hyperprolactinemia occurred in 47% of total patients, but in only 16% with a single surgical procedure, histological diagnosis of prolactinoma, and postoperative PRL concentration of 5 ng/mL or less. The best single predictor of cure was postoperative day 1 PRL concentration of 5 ng/mL or less. Eighty-eight percent of women desiring conception conceived within 1 yr. Glucocorticoid-dependent hypopituitarism occurred in 23% of patients undergoing postoperative radiotherapy. There was no operative mortality. Operative morbidity was low. Our experience demonstrates that women undergoing transsphenoidal surgery for diagnosis of PRL-secreting adenoma form a heterogeneous patient population. The best long term results are achieved in the pure prolactinoma group, for whom transsphenoidal resection is generally safe and effective. PMID- 8626822 TI - Body composition derived from whole body counting of potassium in growth hormone deficient adults: a possible low intracellular potassium concentration. AB - The validity of total body potassium (TBK) measurement in estimating fat mass and fat-free mass (FFM) in GHD adults was assessed by comparison with the reference technique of dual energy x-ray absorptiometry (DEXA). The TBK and FFM values determined by DEXA were used to calculate the potassium concentration per kg FFM in GH-deficient (GHD) adults and compared with standard values for normal subjects of 59.6 mmol for females and 66.4 mmol for males. There were considerable differences between predicted and measured TBK values for both males (3972 vs. 3577 mmol; P < 0.001) and females (2526 vs. 2277 mmol; P < 0.001). Similarly, the estimation of FFM and fat mass by TBK measurement was significantly inaccurate for both sexes compared to values determined by DEXA. These discrepancies may be accounted for by the lower calculated potassium concentrations compared with standard values for both males (56.2 vs. 66.4 mmol; P < 0.001) and females (53.1 vs. 59.6 mmol; P < 0.001). These observations suggest that caution should be exercised in the interpretation of TBK in GHD adults, and the reduced potassium concentrations would alleviate inaccuracies in the estimation of body composition. Secondly, the decreased intracellular potassium concentration of GHD adults may account for the decreased muscle strength and ease of fatigueability seen in GHD adults. PMID- 8626823 TI - Diagnostic accuracy of 131I scanning with recombinant human thyrotropin versus thyroid hormone withdrawal in a patient with metastatic thyroid carcinoma and hypopituitarism. AB - The diagnostic and therapeutic use of radioactive iodine in patients with thyroid cancer requires a sufficient serum concentration of thyrotropin (TSH) for efficient thyroid tissue uptake of radioiodine. Recombinant human TSH (rhTSH) is a promising new agent, which appears to facilitate radioiodine scanning with similar efficacy to thyroid hormone withdrawal without the immunologic side effects of bovine TSH (bTSH) administration. Patients with thyroid cancer and concomitant secondary hypothyroidism are particularly difficult to treat because of their inability to elevate endogenous TSH and the limitations of bTSH administration. We describe a patient with metastatic thyroid carcinoma and secondary hypothyroidism with metastases visible only after administration of rhTSH previously unappreciated on thyroid hormone withdrawal scans. This patient exemplifies the usefulness of rhTSH administration before radioactive iodine for this group of patients. PMID- 8626824 TI - Low dehydroepiandrosterone sulfate (DHEA-S) level is not a good predictor of hormonal activity in nonselected patients with incidentally detected adrenal tumors. AB - To assess its differential diagnostic value, dehydroepiandrosterone sulfate (DHEA S) was measured in a nonselected cohort of 84 patients with incidentally detected adrenal tumors (incidentaloma). Of the 38 histologically confirmed cases, 6 of 12 patients with primary or metastatic malignant tumor of the adrenals and 7 of 14 patients with benign cortical adenoma had low DHEA-S levels. Thus, the sensitivity, specificity, and predictive value of a low DHEA-S level to indicate a benign adrenal tumor were 0.35, 0.50, and 0.60, and the values to indicate a cortical adenoma were 0.50, 0.67, and 0.47, respectively. Of the 14 cases of histologically confirmed benign cortical adenoma, 10 had signs of hormonal activity, but DHEA-S was suppressed in only 7 cases. Thus, the sensitivity, specificity, and predictive value of a low DHEA-S level to indicate clinically significant hormonal activity of a benign cortical adenoma were 0.60, 0.75, and 0.86, respectively. For comparison, 5 of 5 males and 2 of 5 females with metastatic carcinomatosis, but without involvement of the adrenals, also had low DHEA-S levels. The data clearly show that in nonselected cases of incidentaloma a suppressed DHEA-S level is not a good predictor of hormonal activity and that DHEA-S measurement may be valuable only after having ascertained the cortical origin and benign feature of the tumor. PMID- 8626825 TI - 5 alpha-reductase-2 gene mutations in the Dominican Republic. AB - Male pseudohermaphroditism due to 5 alpha-reductase deficiency was clinically and biochemically described in a large Dominican kindred of 23 families with 38 affected subjects in 1974. Recently, the 5 alpha-reductase-2 gene defect in the large Dominican kindred was found to be due to a single base substitution of thymidine (TGG) for cytosine (CGG) on exon 5 of the 5 alpha-reductase-2 gene, causing a tryptophan replacement of arginine at amino acid 246 (R246W) of the enzyme. In the present report, affected subjects from four additional Dominican families were studied to determine whether they carried the same 5 alpha reductase-2 gene defect as the large kindred, suggesting a common ancestry for the gene defect within this small country. Using single strand conformational polymorphism and DNA sequencing, two other mutations of the 5 alpha-reductase-2 gene were found in affected subjects from two of the four families. A point mutation on exon 2 of the 5 alpha-reductase-2 gene, in which substitution of adenine (GAC) for guanine (GGC) caused an aspartic acid replacement of glycine at amino acid 115 (G115D), was demonstrated in one of these families, and a substitution of adenine (AGT) for guanine (GGT) on exon 3 causing a serine replacement for glycine at amino acid 183 (G183S) was detected in the other family. Affected subjects from the two remaining families demonstrated the same exon 5 mutation of the 5 alpha-reductase-2 gene as previously detected in the large Dominican kindred. The phenotypic and biochemical characteristics of the male pseudohermaphrodites were similar regardless of the genetic defect, except that one affected subject (C-VI-2) with the same exon 5 mutation as the large Dominican kindred had much more facial and body hair. Thus, the identification of multiple mutations in the 5 alpha-reductase-2 gene in male pseudohermaphrodites from the Dominican Republic demonstrates a lack of common ancestry, as had been previously postulated. PMID- 8626826 TI - Cyclin D1/PRAD1 expression in parathyroid adenomas: an immunohistochemical study. AB - The cyclin D1 (PRAD1) oncogene is rearranged with the PTH gene and is transcriptionally activated in a subset of parathyroid adenomas. Because of heterogeneity in rearrangement breakpoints, the true percentage of adenomas with cyclin D1 deregulation is unknown. Overexpression of the cyclin D1 protein in parathyroid adenomas appears to be a unifying consequence of all cyclin D1 gene rearrangements and can, therefore, be examined to more comprehensively identify adenomas in which cyclin D1 is pathogenetically important. We studied cyclin D1 expression in 65 parathyroid adenomas (from 64 patients), 51 normal parathyroid glands (from the same patients), and 4 parathyroid carcinoma specimens (from 3 patients) using a microwave-enhanced immunohistochemical method and affinity purified cyclin D1 polyclonal antiserum. When available, data on adenoma mass, intact PTH level, and concurrent serum calcium level were also collected. Twelve of the 65 adenomas (18%) showed diffuse nuclear staining of approximately 30-70% of the tumor cells. All 51 normal glands were negative, except 1 gland that showed scattered cells ( < 10%) with positive nuclear staining. In addition, scattered positive cells were seen in the compressed rim of histologically normal parathyroid tissue surrounding 2 adenomas that were cyclin D1 negative. No significant differences in adenoma mass, intact PTH levels, or concurrent calcium levels were found between positive and negative tumors. Two of 4 parathyroid carcinoma specimens from 2 of 3 patients showed strong nuclear staining for cyclin D1. Overexpression of the cyclin D1 oncogene in 18% of our cases, due to the cyclin D1/PTH translocation and/or other mechanisms, suggests that overexpressed cyclin D1 plays a role in the pathogenesis of a much larger proportion of parathyroid adenomas than previously appreciated. Cyclin D1 overexpression is a feature of typical parathyroid adenomas and is not confined to unusually large, symptom-causing adenomas as had been suggested by early DNA studies. Although only three patients with parathyroid carcinoma were studied, two of the patients' tumors stained for cyclin D1, raising the possibility that the frequency of cyclin D1 overexpression may be even greater in carcinomas. Cyclin D1 overexpression appears to highlight a central pathway in parathyroid neoplasia. PMID- 8626828 TI - Discrete characteristics of antibodies raised against thyrotropin receptor related peptides whose sequences are not conserved in the luteinizing hormone/chorionic gonadotropin receptor. AB - In order to identify the specific regions in the human TSH receptor for TSAb and thyroid stimulation-blocking antibody (TSBAb), we produced rabbit antibodies raised against several peptides of the extracellular domain of the human TSH receptor, where sequences are not conserved in the LH/CG receptor, and measured the TSAb activity and TSBAb activity of those antibodies using Chinese hamster ovary cells expressing human TSH receptors. Only antisera from rabbits that were immunized with a peptide of amino acid 32-56, including the small insertion near the N-terminal end of the extracellular domain, showed apparent TSAb activities and have been shown to be significantly precipitated by IgG of patients with Graves' disease. TSAb activity positively correlated with the antibody titers against the peptide in those rabbits. In contrast, antisera from rabbits immunized with a peptide of amino acid 352-378, including a part of the large insertion near the C-terminal end of the extracellular domain, showed the obvious TSBAb activities. TSBAb activity also positively correlated with the degree of antibody titers against the peptide in those rabbits. Moreover, this peptide was significantly immunoprecipitated by the IgG from hypothyroid patients who had TSBAb, and the immunoprecipitation of this peptide positively correlated with TSBAb activities. These results suggest that the epitope responsible for TSAb is quite different from that for TSBAb in the extracellular domain of the human TSH receptor. PMID- 8626827 TI - Fracture after cardiac transplantation: a prospective longitudinal study. AB - Cardiac transplantation is associated with increased prevalence of vertebral fractures, but the natural history of and risk factors for fracture after this life-saving procedure are unclear. We evaluated 47 patients (34 men and 13 postmenopausal women) before transplantation with spinal radiographs, determination of bone density by dual energy x-ray absorptiometry, and measurement of biochemical indexes of mineral metabolism. During the first year after transplantation, incident fractures were documented radiographically. Associations among demographic characteristics, bone density, biochemistries, and fracture risk were evaluated with logistic regression analysis. Despite supplementation with elemental calcium (1000 mg/day) and vitamin D (400 IU/day), 17 patients (7 women and 10 men) sustained a total of 34 fractures. Most fractures involved the spine, and 85% of the patients who experienced fracture did so within 6 months of transplantation. Fifty-four percent of the women and 29% of the men experienced fracture. Femoral neck bone mineral density was significantly lower in women who experienced fracture than in those who did not (0.604 +/- 0.11 vs. 0.760 +/- 0.12 g/cm2; P < 0.04), but did not differ in men according to fracture outcome. The amount of bone loss at the femoral neck by 6 months after transplantation was significantly greater in men with fracture than in men without fracture (12.0 +/- 6.4% vs. 6.8 +/- 5.3%; P < 0.04), but did not differ in women according to fracture outcome. Pretransplant 1,25 dihydroxyvitamin D levels were significantly lower (25 +/- 9 vs. 39 +/- 17 pg/mL; P < 0.007) and intact PTH levels tended to be higher in men who did not experience fracture (37 +/- 15 vs. 69 +/- 46 pg/mL; P < 0.06). Individual pretransplant bone density measurements demonstrated substantial overlap between patients who did and did not experience fracture, and normal bone density did not necessarily protect against fracture after transplantation. We conclude that fractures are a common and early complication of cardiac transplantation. No pretransplant measurement has yet been identified that reliably predicts fracture after transplantation in the individual patient. PMID- 8626829 TI - Sex steroid modulation of AT2 receptors in human myometrium. AB - In contrast to the abundant expression of the AT2 subtype of angiotensin II (AII) receptors during fetal development, AT2 receptor in adult life is expressed in few tissues. We now report studies on the presence and hormonal regulation of AT2 receptor in human pregnant and nonpregnant myometrium obtained from a large study population (n = 50). AT2 receptor subtypes have been characterized using self- and cross-competition curves among [125I]CGP42112A (a selective AT2 ligand), [125I](Sar1,Ile8)AII (a unselective antagonist), the corresponding unlabeled ligands, and several peptidic and nonpeptidic analogs with different affinities for the AT1 and AT2 receptor subtypes. We found that the human nonpregnant uterus expresses almost exclusively the AT2 subtype, and that [125I]CGP42112A is a selective probe to study human AT2 receptor. By using [125I]CGP42112A, we demonstrated that the density of AT2 receptor in human myometrium is dramatically affected by the hormonal milieu. Indeed, in the estrogen-dominant uterus of normal cycling women in the proliferative phase and that of perimenopausal women with anovulatory cycles, the density of binding sites was very high [1565 +/- 246 fmol/mg protein (n = 11) and 2176 +/- 429 (n = 7), respectively]. The concomitant presence of progestogens blunted the estrogen effect [term pregnancy, 61 +/- 12 fmol/mg protein (n = 5); secretive phase of the cycle, 453 +/- 154 (n = 10); combined oral contraceptive, 243 +/- 74 fmol/mg protein (n = 6)]. Very low concentrations of binding sites are also present in the sex steroid-deprived uterus of postmenopausal women (100 +/- 12 fmol/mg protein; n = 8) and the uterus of fertile women chronically treated with GnRH agonists (199 +/- 100 fmol/mg protein; n = 3). Hence, these data confirm the presence of AT2 receptors in human uterus and indicate their regulation by sex steroids. PMID- 8626830 TI - Epitopes for thyroid-stimulating antibodies in Graves' sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment. AB - To evaluate the extent and clinical relevance of epitope heterogeneity for stimulating TSH receptor antibodies (TSHRAbs), we measured the activity of IgG preparations from 66 untreated patients with Graves' disease using Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR and two TSHR chimeras with residues 9-165 (Mc1 + 2) or 90-165 (Mc2) substituted by equivalent residues of the LH/CG receptor. IgG from 68% of patients lose all of the stimulating TSHRAb activity with the chimeras; IgG from 27% lose most of the activity. Thus, we show that 95% of patients have stimulating TSHRAbs that require epitopes on the N-terminal portion of the extracellular domain of the TSHR and demonstrate the importance of epitopes within residues 90-165 for the first time. Heterogeneous epitope distribution, residual activity with one or both chimeras, i.e. with epitopes other than on the N-terminus of the TSHR, occurred in 21 patients (group A). Forty-five patients with homogeneous epitope distribution (group B) had stimulating TSHRAbs that depended only on epitopes on the N-terminus of the TSHR. Patients in group A were more likely to become euthyroid during antithyroid drug therapy and to do so more quickly than group B patients. The CHO-human TSHR cell system described herein appears to be as effective as the FRTL-5 rat thyroid system in stimulating TSHRAb detection; however, the two systems appear to measure different antibody populations in about 30% of cases. Further, stimulating TSHRAb activities measured in the FRTL-5 system tend to correlate better with goiter size and 99mTc pertechnetate uptake, whereas stimulating activities measured in the CHO-human TSHR/chimera system correlate better with free T4 and T3 levels. PMID- 8626831 TI - Vasopressin receptor expression and mutation analysis in corticotropin-secreting tumors. AB - Vasopressin is an important regulator of hypothalamo-pituitary-adrenal axis activation, primarily acting through the V3 receptor (V3R). Many patients with ACTH-secreting pituitary adenomas, but not normal individuals, respond to desmopressin, a relatively V2-specific vasopressin agonist, with increased ACTH and cortisol levels. We have searched for mutations of the V3R gene in ACTH secreting pituitary adenomas and one ectopic ACTH-secreting tumor. No abnormalities were found in 12 tumors studied by PCR-single strand conformation polymorphism (PCR-SSCP) analysis. We then verified by RT-PCR whether the response to desmopressin was due to overexpression of the V3R or abnormal expression of the V2R in the pituitary tumor. We found that the V2R gene was expressed in a number of corticotroph tumors and in the ACTH-secreting ectopic tumor, and that the V3R gene appears to be overexpressed in these tumors. We conclude that V3R mutations are unlikely to be present in the ACTH-secreting tumors we examined, but that the V2R gene is expressed in the majority of the samples tested, and the V3R is expressed in all of these tumors. We speculate that the response to the desmopressin test observed in patients with Cushing's disease may be due to abnormal expression of V3R or V2R in ACTH-secreting tumors. PMID- 8626832 TI - Aspirin inhibition of naloxone-induced luteinizing hormone secretion in man. AB - It is well known that endogenous opioid peptides exert a tonic inhibitory control on GnRH release, leading to the inhibition of LH secretion, whereas eicosanoids, particularly prostaglandin E2(PGE2), stimulate GnRH output. Furthermore, in vitro studies suggest the existence of an interaction between these two regulatory systems in animals. The present work was designed to evaluate the acute effect of the prostaglandin blocker aspirin on plasma LH response to the opiate antagonist naloxone or GnRH in normal volunteers in a placebo-controlled, single-blind study. To exclude a hypothetical action of aspirin directly at the testis level, plasma testosterone concentrations were monitored during basal sampling after acetylsalicylic acid ingestion, whereas the efficacy of the drug as a prostaglandin blocker was tested by the determination of seminal PGE2 levels. Aspirin pretreatment significantly lowered seminal PGE2 levels (from 86 +/- 5 before to 11 +/- 2 micrograms/mL [corrected] after drug administration; P < 0.001) without affecting testosterone concentrations. Moreover, the drug induced a significant reduction of LH response to naloxone, assessed as the mean integrated area under the curve, from 1666.5 +/- 116 to 1197.5 +/- 98 mUI/mL per min (P < 0.05), whereas it did not influence GnRH-induced LH release. We conclude that the effective cycloxygenase blockade inhibits the stimulatory activity of naloxone on LH release, suggesting that the inhibitory tone of opioids on GnRH secretion may be caused by the block of hypothalamic prostaglandin biosynthesis with consequent inhibition of PGE2-induced GnRH secretion. PMID- 8626833 TI - Congenital adrenal hyperplasia presenting as massive adrenal incidentalomas in the sixth decade of life: report of two patients with 21-hydroxylase deficiency. AB - Divergent recommendations exist regarding the evaluation of adrenal incidentalomas. Recent data have indicated a prevalence of adrenal tumors of 71% in nonclassical congenital adrenal hyperplasia (CAH) and unmasked heterozygotes. These data expand the differential diagnosis of such incidental tumors and substantially modify the approach to their evaluation. We present two patients, female pseudohermaphrodites with the simple virilizing form of CAH and 21 hydroxylase deficiency, who functioned successfully as married phenotypic males. Both came to medical attention in the sixth decade by virtue of massive adrenal incidentalomas encountered in the evaluation of recurrent urinary tract infections. Each had a 46, XX karyotype, no palpable testes, and markedly elevated baseline levels of 17-hydroxyprogesterone (17-OH Prog) of 6086 ng/dL and 6750 ng/dL. Both responded appropriately to dexamethasone suppression with reduction of 17-OH Prog, androgens and, in the second patient, ACTH to normal or near normal levels. Histologic and autopsy examination of the first patient's tumor and computed tomographic characteristics of the second revealed a benign adenoma and myelolipoma respectively. We extend and confirm previous recommendations that CAH be included in the differential diagnosis of adrenal incidentaloma and that baseline 17-OH Prog. levels be obtained, with ACTH stimulation if necessary, to diagnose the presence of nonclassical CAH. PMID- 8626834 TI - Mutations of the ret protooncogene in German multiple endocrine neoplasia families: relation between genotype and phenotype. German Medullary Thyroid Carcinoma Study Group. AB - It has been suggested that not only the position but also the nature of the mutations of the ret protooncogene strongly correlate with the clinical manifestation of the multiple endocrine neoplasm type 2 (MEN 2) syndrome. In particular, individuals with a Cys634-Arg substitution should have a greater risk of developing parathyroid disease. We, therefore, analyzed 94 unrelated families from Germany with inherited medullary thyroid carcinoma (MTC) for mutation of the ret protooncogene. In all but 1 of 59 families with MEN 2A, germline mutations in the extracellular domain of the ret protein were found. Some 81% of the MEN 2A mutations affected codon 634. Phenotype-genotype correlations suggested that the prevalence of pheochromocytoma and hyperparathyroidism is significantly higher in families with codon 634 mutations, but there was no correlation with the nature of the mutation. In all but 1 of 27 familial MTC (FMTC) families, mutations were detected in 1 of 4 cysteines in the extracellular domain of the ret protooncogene. Half of the FMTC mutations affected codon 634. Mutations outside of codon 634 occurred more often in FMTC families than in MEN 2A families. In all but 1 of 8 MEN 2B patients, de novo mutations in codon 918 were found. These data confirm the preferential localization of MEN 2-associated mutations and the correlation between disease phenotype and the position of the ret mutation, but there was no correlation between the occurrence of hyperparathyroidism or pheochromocytoma and the nature of the mutation. PMID- 8626835 TI - A primary, lateral-cervical medullary thyroid carcinoma: a case report. AB - Medullary thyroid carcinoma (MTC) arises from the parafollicular cells of the thyroid and occurs in a sporadic or in an inherited form. We present a case of an aberrant MTC in a patient with a functioning thyroid gland. At surgical dissection, the thyroid was present in its anatomical site with a nodule in the upper one third of the right lobe. A mass was also found in a lateral-cervical position distinct from the thyroid gland. Histological examination showed the mass to be the primary MTC, whereas the thyroid nodule was a follicular adenoma. Analysis of DNA extracted from the MTC, from the adenoma, and from peripheral blood revealed a mutation within exon 16 of the RET proto-oncogene only in the DNA from the tumor. The reported case represents a sporadic MTC in an aberrant localization, probably originating from a developmental abnormality of the primordial C cells. This event might have occurred during the migration and/or differentiation of the C cells and might be related to, or caused by, the mutated RET proto-oncogene. PMID- 8626836 TI - A new type of familial central diabetes insipidus caused by a single base substitution in the neurophysin II coding region of the vasopressin gene. AB - We studied the genetic basis of familial neurohypophyseal diabetes insipidus in a Japanese family. The members had polyuria and a deficiency of plasma vasopressin (AVP). Polymerase chain reaction (PCR) amplified exons of the AVP-neurophysin-II gene were subcloned and sequenced. Exons 1 and 3 were normal, but nucleotide 1884 Guanine (G) in exon 2 was substituted with Thymine (T), which induced a substitution of glycine (Gly) for valine (Val). To examine the presence of this mutation in the affected subjects, we designed two mutated primers. One of them induced a new endonuclease restriction site in the PCR fragments from normal, and the other induced a new endonuclease restriction site from patients with the mutation. DNA fragments from two affected members of this family were amplified with this primer, and the PCR products were digested by endonuclease and resolved by electrophoresis. The results indicated that these subjects had both normal and mutant alleles, indicating that the mutation was heterozygous. We concluded that this mutation caused neurohypophyseal diabetes insipidus in this family. PMID- 8626838 TI - Ontogeny of pulsatile gonadotropin releasing hormone secretion from midchildhood, through puberty, to adulthood in the human male: a study using deconvolution analysis and an ultrasensitive immunofluorometric assay. AB - The ontogeny of gonadotropin releasing hormone pulse generator activity underlying pubertal development in the human male is incompletely defined because of the limitations of assay sensitivity in measurements and the inaccuracies attendant upon the analyses of pulsatile secretion of circulating gonadotropins. Using an ultrasensitive immunofluorometric assay (DELFIA) to measure plasma LH and deconvolution analysis to depict LH secretory characteristics, we compared nocturnal (2000-0800 h) pulsatile LH secretion cross-sectionally in 16 boys in midchildhood (mean +/- SD age 6.6 +/- 0.3 yr), 8 prepubertal boys (12.0 +/- 0.3 yr), 8 early pubertal boys (14.3 +/- 0.4 yr), and in 8 young fertile adult men (32.6 +/- 1.6 yr) as an indirect in vivo assessment of hypothalamic GnRH pulse generator activity over the entire span of pubertal development in the human male. We confirmed that sleep-entrained GnRH/LH burst secretory activity was present in midchildhood. The first increase in sleep-entrained GnRH/LH secretion occurred some 2 yr before the clinical onset of puberty. From midchildhood to sexual maturity, LH production rate increased 39-fold. However, GnRH/LH pulse frequency showed only a relatively small (1.8-fold) increment from midchildhood to the clinical onset of puberty, with no subsequent changes to continuing development towards adulthood. Thus 91.7% of the increment in LH plasma concentration from childhood to sexual maturity could be accounted for by an amplification of a pre-existing ultradian rhythm of secretion with a steadily and markedly increasing mass of LH secreted per burst. The duration of secretory burst and apparent half-life of plasma LH disappearance remained constant from midchildhood, through puberty, to adulthood. The nyctohemeral rhythm-and sleep associated LH/GnRH secretion was eventually lost in young adulthood. We conclude that the onset of puberty in man is heralded by the reawakening of a partially quiescent GnRH pulse generator. This predominantly involves an amplification of a pre-existing pattern of hypothalamic GnRH secretion leading to a major augmentation of the total quantity of LH molecules released per burst. The almost two-fold increment in GnRH pulse frequency contributed synergistically to the pubertal process, before the clinical onset of puberty, possibly by enhancing gonadotropic sensitivity to increase the mass of LH produced per burst. The relative constancy of GnRH pulse frequency in the gonad-intact hypothalamic pituitary-testicular axis from pubertal onset to adulthood implies that testicular steroidal feedback plays a role in restraining the burst frequency of the GnRH pulse generator during pubertal development and adulthood. PMID- 8626837 TI - Quantitative genetic analyses of insulin-like growth factor I (IGF-I), IGF binding protein-1, and insulin levels in middle-aged and elderly twins. AB - With the use of quantitative genetic models, the relative importance of genetic and environmental influences on serum levels of insulin-like growth factor I (IGF I), IGF-binding protein-1 (IGFBP-1), and insulin was evaluated in 248 pairs of middle-aged and elderly Swedish twins reared apart and reared together. Heritability estimates (the relative influence of genetic effects) were 48% for insulin, 63% for IGF-I, and 36% for IGFBP-1. There was no indication of differences in heritability estimates for IGF-I, IGFBP-1, and insulin across age and gender groups. Nonshared environmental influences, unique to individuals, explained the remaining variance in the measures. The genetic influences on IGF-I levels were independent of the genetic influences on insulin and IGFBP-1 levels. However, a small, but significant, proportion of the genetic variation in IGFBP-1 was in common with genetic influences for insulin. Furthermore, genetic effects explained 36% of the phenotypic correlation between IGFBP-1 and insulin, whereas the phenotypic associations between IGF-I and both IGFBP-1 and insulin were entirely attributable to environmental effects. Finally, the phenotypic association between IGF-I and IGFBP-1 was mediated wholly by environmental influences in common with insulin. PMID- 8626839 TI - Outcome of a four-year randomized study of daily versus three times weekly somatropin treatment in prepubertal naive growth hormone-deficient children. Genentech Study Group. AB - A comparison was made of the growth responses of prepubertal naive GH-deficient children who were randomly assigned to receive 0.3 mg/kg.week recombinant human GH administered either daily (QD) or three times weekly (TIW) over 4 yr. The effects of the two regimens on annual growth velocity, change in height SD score, bone maturation, and age at onset of puberty are presented as the mean +/- SD. During each of the 4 yr, the annual growth velocity was significantly greater in the QD vs. TIW group. At 48 months, the mean total gain in height was 9.7 cm greater in the QD group (38.4 +/- 5.5) than that in the TIW group 28.7 +/- 3.2; P = 0.0002). The mean height SD score at the end of each year was significantly greater in the QD group. After 4 yr, the total gain in height SD score was 3.2 +/ 1.2 in the QD group compared to 1.5 +/- 0.5 in the TIW group (P = 0.0003). The height SD score at 4 yr was 0.2 in the QD group (pretreatment, -2.9) compared to 1.4 in the TIW group (pretreatment, -2.9). After 4 yr of rhGH treatment, the increment in bone age was similar in the QD (4.9 +/- 1.0 yr) and TIW (4.8 +/- 1.1 yr) groups. The change in height age minus the change in bone age was more favorable in the QD (1.2 +/- 0.8 yr) than in the TIW (0.0 +/- 0.9 yr) group (P = 0.003). The mean age at onset of puberty in boys was the same in the QD (13.2 yr) and TIW (13.0 yr) groups (P = 0.71), and the mean bone age at the start of puberty was also similar (11.5 in QD and 11.3 in TIW groups; P = 0.66). The advantages of QD rhGH treatment in prepubertal GH-deficient children after 4 yr were additional gains of 1.7 height SD score and 9.7 cm in height over those treated with the TIW regimen (P = 0.0002). PMID- 8626840 TI - Paget's disease is associated with changes in the immunohistochemical distribution of noncollagenous matrix proteins in bone. AB - Paget's disease of bone is characterized histologically by abnormal architecture of bone matrix. Extensive areas of woven bone and numerous scalloped cement lines occur as a result of increased irregular remodeling. Noncollagenous proteins (NCP) play an important role in the organization and mineralization of bone matrix and promote distinct cell-matrix interactions necessary for normal remodeling. To gain insight into the pathological changes in the biochemical composition of Pagetic bone, the distribution of NCPs in the calcified matrix of bone from patients with known Paget's disease was compared to that of bone from normal healthy volunteers. Undecalcified plastic-embedded sections of bone were stained immunohistochemically using antibodies generated against several NCPs. In Pagetic and normal bone a similar distribution of osteopontin was observed at cement (reversal) lines, whereas significant differences were observed in the distribution of osteopontin in the matrix immediately adjacent to Haversian canals, where initial osteoclast recruitment and attachment occur. The differences in osteopontin distribution appeared to be related to the state and severity of the disease. Site-specific differences in the distribution of osteonectin, osteocalcin, and decorin were also observed between normal bone and cortical and periosteal de novo Pagetic bone, whereas the distribution of other matrix proteins, such as biglycan, was unchanged. We conclude that these site specific changes in the biochemical distribution of NCPs in Pagetic bone probably reflect abnormal production and/or incorporation during bone remodeling and may lead to disorganized matrix assembly and mineralization as well as have profound effects on bone cell functions. PMID- 8626841 TI - Influence of some biological indexes on sex hormone-binding globulin and androgen levels in aging or obese males. AB - Several aspects of the regulation of androgen secretion and plasma levels in males remain controversial. Among these, we cite the problem of whether the age related decrease in testosterone (T) levels is an intrinsic aging phenomenon or is a sequel of previous illness, the mechanisms underlying the increase in sex hormone-binding globulin (SHBG)-binding capacity in aging men and the supranormal capacity observed immediately after a weight-reducing diet, and the role of insulin in the age-associated decrease in dehydroepiandrosterone (sulfate) [DHEA (DHEAS)] levels. To gain further insight into these issues, we investigated the influence of age, smoking, body mass index (BMI), serum albumin, insulin, GH, and insulin-like growth factor I (IGF-I) levels, respectively, on androgen levels and SHBG-binding capacity in a nonobese healthy population (n = 250) as well as in an obese population (n = 50) before and after weight loss. The influence of GH supplementation on SHBG, DHEAS, DHEA, and insulin levels was studied in a small group of men (n = 8) with isolated GH deficiency. In nonobese healthy men, age was inversely correlated with serum levels of all androgens studied (although total T levels stayed relatively stable until age 55 yr) as well as with albumin, GH, and IGF-I levels and positively correlated with BMI, insulin levels, and SHBG binding capacity. Nevertheless, SHBG levels were significantly negatively correlated with insulin levels (P < 0.001) as well as with mean 24-h GH and IGF-I levels. Among possible confounding factors affecting (free) T [(FT)] levels in healthy men, smoking appeared to be accompanied by higher (F)T levels than those in nonsmokers. BMI increased with age, but although BMI was negatively correlated with T, FT, and SHBG, respectively, the age-dependent decrease in T levels persisted after correction for BMI. Data not corrected for BMI may, nevertheless, overestimate the age-associated decrease in T levels. The albumin concentration decreased with age, and if FT is the feedback regulator of plasma T levels, albumin concentration might be a codeterminant (although, evidently, less important than SHBG) of T levels and contribute to the age-associated decrease in T levels. In any case, albumin concentration is a codeterminant of DHEAS concentration. T, DHEA, and DHEAS levels were significantly correlated, but this correlation disappeared after controlling for age; hence, there is no evidence for an adrenal-gonadal interaction in men. In obese men, T, FT, and SHBG levels were significantly lower than those in the nonobese men and inversely correlated with BMI; DHEAS levels were slightly lower than those in the nonobese controls, but no significant correlation between DHEA or DHEAS, and insulin levels was observed. After a weight-reducing, protein-rich diet, resulting in a mean weight loss of +/- 15 kg, SHBG-binding capacity increased to normal values notwithstanding the fact that the subjects were still obese and that the insulin levels remained higher than those in the nonobese controls. Considering that after weight loss, GH and IGF-I levels remained lower than those in the nonobese controls, that adult men with isolated GH deficiency presented with higher SHBG levels than normal controls, which decreased to normal levels during GH substitution, and that elderly men have elevated SHBG levels notwithstanding high insulin levels, we suggest that the low GH and/or IGF-I levels might play a role in the elevated SHBG levels observed in both elderly males and obese men after a weight-reducing diet. As weight loss did not influence DHEAS levels notwithstanding an important decrease in insulin levels, our data do not support a role of insulin in the regulation of plasma DHEAS levels. PMID- 8626842 TI - A (R80Q) mutation in 17 beta-hydroxysteroid dehydrogenase type 3 gene among Arabs of Israel is associated with pseudohermaphroditism in males and normal asymptomatic females. AB - Four isozymes of steroid 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) encoded by different loci catalyze the reversible conversion of androstenedione to testosterone and that of estrone to estradiol. The 17 beta HSD type 3 (17 beta HSD3) isozyme is encoded by the 17 beta HSD3 gene on chromosome 9q22 and expressed only in testes. Inherited defects in the 17 beta HSD3 isozyme cause a form of male pseudohermaphroditism that is rare within the general population, but frequent among a highly inbred Arab population in the Gaza strip. A point mutation in exon 3, codon 80 of the 17 beta HSD3 gene, R80Q, caused by a single base substitution from CGG to CAG was identified in both alleles of 24 individuals from 9 extended Arab families from Gaza, Jerusalem, and Lod-Ramle. Twenty-one homozygotes were male pseudohermaphrodites (46,XY) with testicular 17 beta HSD3 deficiency, born with either female-looking external genitalia or various degrees of genital ambiguity. If not reassigned in infancy, they were reared as females until puberty, when marked virilization occurred, often leading to the spontaneous adoption of a male gender role. In contrast, the 3 homozygote females (46,XX) were asymptomatic, had normal internal and external genitalia and normal sexual development, and revealed no biochemical evidence of 17 beta HSD3 deficiency. The molecular pattern in these families is compatible with an autosomal recessive mode of inheritance that is sex dependent. PMID- 8626843 TI - Pharmacokinetics and metabolism of a permeation-enhanced testosterone transdermal system in hypogonadal men: influence of application site- -a clinical research center study. AB - As part of a phase III multicenter study, the pharmacokinetics and metabolism of a permeation-enhanced testosterone (T) transdermal (TTD) system and the influence of application site were investigated in 34 hypogonadal men (21-65 yr of age). After an 8-week androgen washout period, two TTD systems were applied to the back for 24 h. Serum concentrations of total T, bioavailable testosterone (BT), dihydrotestosterone (DHT), and estradiol (E2) increased from hypogonadal levels into the respective normal physiological ranges and declined to baseline levels within 24 h after system removal. Peak concentrations occurred approximately 8 h after application for T and BT and at 13 h for DHT and E2. The baseline subtracted time-average steady state concentrations (C'ss) for T and BT were 18.1 +/- 7.49 (+/- SD) and 9.08 +/- 3.99 nmol/L, respectively. DHT/T and E2/T ratios, derived from the C'ss values, were 0.063 +/- 0.018 and 0.0033 +/- 0.0018, comparable to the precursor-product conversion ratios reported in healthy men. The estimated half-lives of each hormone were: T, 1.29 +/- 0.71 h; BT, 1.21 +/- 0.75 h; DHT, 2.83 +/- 0.97 h; and E2, 3.53 +/- 1.93 h. The influence of application site was then evaluated by applying two TTD systems for 24 h to the abdomen, back, chest, shin, thigh, or upper arm, according to a sequential cross over design. Hormone profiles were qualitatively similar at each site, but C'ss values showed significant differences (by ANOVA, P < 0.0001). Based on the BT levels, the rank ordering of the sites were: back > thigh > upper arm > abdomen > chest > shin. DHT/T and E2/T ratios showed negligible site to site variation and were comparable to the results from the initial study. Estimates of T input, based on hormone levels and analysis of the systems used, averaged 4-5 mg/day for the abdomen, back, thigh, and upper arm and were lower and more variable for the chest and shin. Individual C'ss values for T and BT increased linearly with the T input rates (derived from used system analysis) across all studies (n = 235; r = 0.564 for T and r = 0.754 for BT). From these data, T and BT clearance rates were estimated for each patient, averaging 1248 +/- 518 and 2435 +/- 778 L/day, respectively. T clearance rates were proportional to the BT/T ratio (nonsex hormone-binding globulin-bound fraction). On the basis of these studies, the optimal sites of TTD system application were identified as the back, thigh, upper arm, and abdomen PMID- 8626844 TI - Prolactinomas in a large kindred with multiple endocrine neoplasia type 1: clinical features and inheritance pattern. AB - Multiple endocrine neoplasia type 1 (MEN 1) is associated with neoplasia and hyperfunction of the parathyroid and pituitary glands, pancreatic islet cells, and neuroendocrine cells of the gut. The inheritance pattern is autosomal dominant, and the underlying genetic defect is situated at chromosome 11q13. The MEN 1 gene behaves as a defective copy of a normally constitutive tumor suppressor gene. Development of the MEN 1 phenotype, however, is a multistep and multifactorial process. The Tasman 1 genealogy is the largest MEN 1 pedigree detected to date. Thus far, 90 related members with MEN 1 have been screened for evidence of prolactinoma. Prolactinomas were found in 18 patients (20%). Prolactinomas were not evenly distributed in the genealogy; in 2 branches of the overall genealogy prolactinomas were present in 50% or more of MEN 1-affected members. The familial distribution of prolactinomas in these branches was consistent with an autosomal dominant mode of inheritance. In the remainder of the pedigree, prolactinomas were uncommon and did not display this inheritance pattern. This pedigree represents one of the largest published MEN 1 genealogies in which the risk of developing prolactinoma follows an autosomal dominant pattern of transmission. It is the first to demonstrate an inheritance pattern for prolactinomas acting in addition to, yet distinct from, the inheritance of the underlying MEN 1 gene defect. These findings are consistent with the existence of an undefined second genetic defect involved in the pathogenesis of prolactinoma in MEN 1. PMID- 8626845 TI - Insulin secretion and resistance in nondiabetic Mexican Americans and non Hispanic whites with a parental history of diabetes. AB - Both insulin resistance and decreased insulin secretion have been hypothesized as precursors of noninsulin-dependent diabetes mellitus (NIDDM). However, there are few data on insulin resistance and secretion in relationship to parental history of diabetes in ethnic groups at different risks for NIDDM. We examined the relationship of fasting insulin (as a marker of insulin resistance) and the ratio of the 30-min change in insulin to the 30-min change in glucose (delta I30/delta G30) during an oral glucose tolerance test (as a marker of insulin secretion) in relation to parental history of diabetes in 1672 nondiabetic Mexican Americans and 894 nondiabetic non-Hispanic whites. A parental history of diabetes was associated with increased fasting insulin concentrations. The association between decreased insulin secretion and parental history of diabetes was not significant. However, when insulin resistance and insulin secretion were included in the same regression model, both fasting insulin (odds ratio = 1.80; 95% confidence interval = 1.17, 2.76) and decreased insulin secretion (odds ratio = 0.70, 95% confidence interval = 0.52, 0.95) were significantly associated with a parental history of diabetes. These results were little changed after adjustment for obesity, body fat distribution, and glucose tolerance. These results were similar in both Mexican Americans and non-Hispanic whites and in both subjects with impaired and those with normal glucose tolerance. A parental history of diabetes in both Mexican Americans and non-Hispanic whites is associated with both increased fasting insulin and decreased delta I30/delta G30. These data suggest that both increased insulin resistance and early decreased insulin secretion in response to an oral glucose challenge may be important factors in the pathogenesis of NIDDM in populations at high and low risk of NIDDM. Our results also emphasize the importance of adjusting for insulin resistance in evaluating insulin secretion. PMID- 8626846 TI - Effect of adrenocorticotropin stimulation on the synthesis of 19-noraldosterone in man. AB - The hormone, 19-noraldosterone, which was recently shown to be synthesized and produced in the human adrenal gland, exhibits potent mineralocorticoid and hypertensinogenic activities. This hormone is controlled in part by the renin angiotensin system. We studied the effects of ACTH stimulation on the synthesis of 19-noraldosterone in vitro and in six normal men. 19-Noraldosterone was measured by a specific RIA after the urine extract or incubation medium was purified by high performance liquid chromatography. The 24-h urinary excretion of 19-noraldosterone increased approximately 4-fold during the administration of ACTH (40 U, injected im twice daily for 3 days). Virtually identical responses were observed with aldosterone, 18-hydroxycorticosterone, 18,19 dihydroxycorticosterone, and 18-hydroxy-19-norcorticosterone. Glomerulosa cells isolated from human adrenals were incubated with angiotensin II (10(-7), 10(-8), and 10(-9) mol/L) or ACTH (10(-8), 10(-9), and 10(-10) mol/L). Angiotensin II and ACTH increased the production of 19-noraldosterone dose-dependently from isolated glomerulosa cells. The secretion of aldosterone, 18-hydroxycorticosterone, 18,19 dihydroxycorticosterone, and 18-hydroxy-19-norcorticosterone in response to angiotensin II and ACTH was identical to that of 19-noraldosterone. These observations suggest that 19-noraldosterone is stimulated by the renin angiotensin system as well as ACTH. PMID- 8626847 TI - Insulin-like growth factor-I (IGF-I) and IGF-binding proteins in children with nephrotic syndrome. AB - Growth failure appears to be a major problem for nephrotic children who fail to respond to steroid therapy. Recently altered serum insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) profiles are reported in renal failure and glomerulonephritis. In this study, the serum IGFBP profile was evaluated by Western ligand blot and RIA in 22 patients with the nephrotic syndrome. Serum IGFBP-3 was decreased, whereas IGFBP-2 was increased in most patients with the nephrotic syndrome. The mean serum IGFBP-3 level was 2123 +/- 531 ng/mL in active states and was increased to a normal level (3593 +/- 407 ng/mL) in remission states. We also measured serum IGF-I by RIA. The serum concentration of IGF-I (mean +/- SD) was 67.4 +/- 23.2 ng/mL in active states and was increased to 127.1 +/- 21.8 ng/mL in remission states, but was still lower than that in control subjects (180.4 +/- 15.8 ng/mL). IGF-I and IGFBP-3 levels were not correlated with primary renal diseases or the amount of proteinuria. For serum IGF-IGFBP complexes, 150-kDa complexes were significantly decreased in patients with the nephrotic syndrome compared with those in control subjects. In urine from nephrotic syndrome patients, 150- and 50-kDa complexes were found, whereas these complexes did not exist in the urine of control subjects. We speculate that low serum IGF-I and IGFBP-3 levels would be partially due to the increased urinary losses of serum IGF-IGFBP complexes, especially that of 150 kDa, and these changes may contribute to growth failure in persistent nephrotic syndrome. PMID- 8626848 TI - Cellular distribution of prolactin receptors in human digestive tissues. AB - In the present study we analyzed the expression of prolactin receptors (PRLR) in human digestive tissues by immunohistochemistry. PRLR immunoreactivity was primarily localized in the cytoplasm. However, in some organs (liver and salivary glands) a nuclear positivity was also found. The liver was used as control and showed a diffuse immunostaining in the parenchymal cells. In the gastrointestinal tract, PRLR immunoreactivity was observed in the mucosa, muscularis layer, and nervous plexuses. The more intense immunostaining in the mucosa of the different segments was as follows: esophagus, superficial layers of the stratified squamous epithelium and mucous glands; stomach, parietal cells; small intestine, absorptive and Paneth cells; and colon, surface epithelium and superficial half of the crypts of Lieberkuhn. In the salivary glands, immunoreactivity was strong in the mucous tubules, moderate in the ducts, and weak in the serous cells. Endocrine pancreas showed a more intense immunoreactivity than the pancreatic acini. By serial sections of the islets of Langerhans we showed that immunostaining was confined to B cells. These findings demonstrate the widespread distribution of PRLR in human digestive tissues and its localization both in cytoplasms and nuclei. PMID- 8626849 TI - Short-term night-shift working mimics the pituitary-adrenocortical dysfunction in chronic fatigue syndrome. AB - The purpose of this study was to determine whether a short period (5 days) of night-shift work affected the pituitary-adrenal responses to CRH. Ten nurses (8 female and 2 male; age 28.1 +/- 1.7 yr: mean +/- SEM) working at the Royal Liverpool University Hospital, and who regularly undertook periods of night and day shift work were enrolled. Measurements were made of basal ACTH and cortisol concentrations, and their responses to iv ovine CRH (1 microgram.kg-1). Basal ACTH concentrations were higher during the night shift than during the day shift (12.9 +/- 5.1 pmol.L-1 vs. 4.7 +/- 1.2 pmol.L-1, P < 0.01) whereas cortisol concentrations were lower (551 +/- 48 nmol.L - 1 vs. 871 +/- 132 nmol.L - 1, P < 0.01). After CRH injection, ACTH concentrations remained consistently higher during the night shift, but the integrated increase in ACTH concentration was lower (P < 0.05) than during the day shift. Conversely, the increase in cortisol concentration was greater during the night shift than the day shift (283 +/- 53 nmol.L-1 vs. 134 +/- 41 nmol.L-1, P < 0.05). We conclude that the pituitary adrenal responses to CRH are markedly disrupted after only 5 days of nighttime work. These abnormalities mimic those previously observed in patients with chronic fatigue syndrome. Neuroendocrine abnormalities reported to be characteristic of chronic fatigue syndrome may be merely the consequence of disrupted sleep and social routine. PMID- 8626850 TI - Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison's disease, and premature ovarian failure. AB - Autoantibodies to steroidogenic enzymes, steroid 17 alpha-hydroxylase (17 alpha OH), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroid 21 hydroxylase (21-OH), were measured using specific and sensitive immunoprecipitation assays (IPAs) in patients with various forms of autoimmune adrenal disease. Autoantibodies to 17 alpha-OH were detected in 6 of 11 (55%) patients with autoimmune polyglandular syndrome (APS) type I, 8 of 24 (33%) patients with APS type II, 11 of 56 (20%) patients with adrenal cortex antibody (ACA; measured by immunofluorescence)-positive patients without Addison's disease, and only 3 of 64 (5%) patients with Addison's disease. Autoantibodies to P450scc were found at a prevalence similar to those to 17 alpha-OH: in 5 of 11 (45%) APS type I patients, 10 of 24 (42%) APS type II patients, 11 of 56 (20%) ACA-positive patients without Addison's disease, and only 6 of 64 (9%) patients of the Addison disease group. Autoantibodies to 21-OH were found in a majority of patients with APS type I (7 of 11;64%), APS type II (23 of 24; 96%), Addison's disease (41 of 64; 64%), and ACA-positive patients without Addison's disease (48 of 56; 86%). All sera that were positive for 17 alpha-OH or P450scc were also positive for 21-OH autoantibodies, except in 1 case. There was good agreement between the presence of ACA measured by immunofluorescence and 21-OH antibodies measured by IPA in all patient groups studied, and this indicates that 21-OH is a major autoantigen in adrenal autoimmune disease regardless of whether the disease presents as isolated Addison's disease or APS type I or type II. Autoantibodies to 17 alpha-OH and P450scc appeared to be the major components of the steroid producing cell antibodies measured by immunofluorescence. No autoantibodies to 21 OH, 17 alpha-OH, or P450scc were detected in 17 sera from patients with premature ovarian failure without evidence of adrenal autoimmunity (as judged by immunofluorescence studies), except for 1 serum in which low levels of 17 alpha OH antibodies were found. Overall, our studies indicate that 35S-labeled 17 alpha OH, P450scc, and 21-OH can be used successfully in IPAs for their respective autoantibodies. Assays such as these may well be valuable in the immunological assessment of patients at risk for or suspected of adrenal autoimmunity. PMID- 8626851 TI - Daytime plasma melatonin levels in male hypogonadism. AB - It has previously been shown that increased nocturnal melatonin (MT) secretion exists in male patients with hypogonadotropic hypogonadism. However, little is known about the effects of gonadotropin and testosterone (T) treatment on early morning plasma MT levels in male hypogonadism. Also, the impact of gonadal steroids on plasma MT levels is an open question. We, therefore, determined early morning plasma MT levels at the same hour before and 3 months after treatment in 21 patients with idiopathic hypogonadotropic hypogonadism (IHH), 10 patients with primary hypogonadism, and 11 male controls. Plasma FSH, LH, PRL, T, and estradiol levels were also determined before and 3 months after treatment. Patients with IHH were treated with hCG/human menopausal gonadotropin, whereas patients with primary hypogonadism received T treatment. Short term treatments did not achieve normal T levels, although significant increases in T were observed in both groups. Plasma MT levels were measured by a RIA with a sensitivity of 10.7 pmol/L. Mean plasma MT levels before treatment were significantly higher in IHH (41.8 +/- 24.4 pmol/L) compared with those in the controls (21.7 +/- 10.8 pmol/L; P < 0.05). However, a slight, but not significant, increase in MT (34.2 +/- 21.1 pmol/L) was found in primary hypogonadism. Mean MT levels did not change significantly 3 months after the initiation of gonadotropin (41.7 +/- 22.8 pmol/L) or T (28.4 +/- 12.6 pmol/L) treatment in either IHH or primary hypogonadism, although a tendency for MT to decrease was observed in both groups. No correlation was found between MT and circulating FSH, LH, PRL, and gonadal steroids either before or after therapy. We conclude that male patients with IHH have increased early morning MT levels, although the pathophysiological mechanism is not clear. Furthermore, our study demonstrated that mean plasma MT levels are not influenced by short term gonadotropin or T treatment in male hypogonadism, although a longer time effect of gonadotropins or T treatment may not be excluded. The lack of correlation between plasma MT and circulating gonadal steroids before and after treatment suggests that there is no classic feedback regulation between the pineal gland and the testes. PMID- 8626852 TI - Pharmacokinetics of melatonin in human sexual maturation. AB - To determine whether melatonin pharmacokinetics change during puberty, we infused melatonin iv in 9 prepubertal, 8 pubertal, and 16 adult subjects and measured melatonin in serum and saliva, and 6-hydroxymelatonin sulfate in urine. A pilot study of 3 adult males showed dose linearity, absence of saturation kinetics, and unaltered metabolism and urinary excretion for doses of 0.1, 0.5, and 5.0 micrograms/kg. All other subjects received 0.5 microgram/kg melatonin. The results of pharmacokinetic parameters calculated from serum melatonin showed no significant gender differences in adults. However, developmental differences were significant between prepubertal children and adults for terminal elimination rate constant (1.08 +/- 0.25 vs. 0.89 +/- 0.11 h-1), elimination half-life (0.67 +/- 0.12 vs. 0.79 +/- 0.10 h), and area under the concentration-time curve (250.9 +/- 91.8 vs. 376.9 +/- 154.3 (pg/mL).h, respectively). At all time points melatonin levels were higher in serum than in saliva, and the ratio between serum and salivary melatonin varied up to 55-fold within and between individuals. Results based on salivary melatonin showed significant differences between prepubertal children and adults for the terminal elimination rate constant (1.90 +/- 0.95 vs. 1.06 +/- 0.28 h-1). The described group differences in pharmacokinetic parameters suggest that prepubertal children metabolize melatonin faster than adults. The inconsistent ratio between serum and salivary melatonin calls for caution in the use of salivary melatonin for pharmacokinetic studies or to infer pineal function. The present findings, suggestive of faster melatonin metabolism in prepubertal children, combined with the known decline of serum melatonin with age and higher excretion rate of the metabolite in prepubertal children lead us to conclude that the prepubertal pineal gland has a higher melatonin secretion rate than the adult gland. PMID- 8626853 TI - High-dose growth hormone treatment of short children born small for gestational age. AB - The effect of GH administration was evaluated over 2 yr in 50 short, prepubertal, non-GH deficient children born small for gestational age, who had been randomly allocated to a group receiving no treatment or daily sc GH treatment at a dose of 0.2 or 0.3 IU/kg. At the start of the study, mean age was 5.2 yr, bone age was 4.0 yr, height SDS was -3.5, height velocity SDS was -0.8, weight SDS was -2.7, and body mass index SDS was -1.9. Catch-up growth was observed in none of the untreated and all of the treated children. The response to GH treatment included a near doubling of growth velocity and of weight gain and a mean height increment of more than 2 SDS. GH treatment was associated with a distinct acceleration of bone maturation. The differences between the growth responses evoked by the two GH doses were minor. The prepubertal GH-induced catch-up growth was associated with elevated serum concentrations of insulin, insulin-like growth factor-I, insulin-like growth factor binding protein-3, and osteocalcin, whereas insulin like growth factor-II levels remained unaltered. GH treatment was well tolerated. In conclusion, high-dose GH administration over 2 yr is emerging as a potential therapy to increase the short stature that results from insufficient catch-up growth in young children born small for gestational age. The long-term impact of this approach remains to be delineated. PMID- 8626854 TI - Interleukin-6: a potential mediator of the massive osteolysis in patients with Gorham-Stout disease. AB - Gorham-Stout disease (GSD) or massive osteolysis, is an extremely rare osteolytic condition that involves extensive locally aggressive resorption of bone. The etiology and pathophysiology are unknown, and the role of the osteoclast in GSD is unclear. We studied a patient with GSD who had massive resorption of his mandible, which extended to his maxilla, zygoma, right parietal region, and cranium. To investigate the cause of the extensive resorption, we tested the effects of the patient's serum, sampled early in the course of treatment and later after the osteolysis was stabilized, on the formation of osteoclast-like multinucleated cells (MNC) in cultures of normal human marrow. GSD serum (10%, vol/vol) markedly increased the number of MNC formed in these cultures compared to that in normal serum as well as stimulated the formation of resorption pits by these MNC on dentine slices. GSD serum, collected after further therapy, did not enhance the number of MNC formed in marrow cultures compared to that in normal serum. Elevated levels of interleukin-6 (IL-6) were detected in the earlier GSD serum that were 7 times the upper limit of the normal range, and after further treatment, IL-6 levels fell to one quarter the pretreatment value. The levels of IL-1 beta, tumor necrosis factor-alpha, transforming growth factor-alpha, PTH, and PTH-related peptide in pretreatment GSD serum were not increased. Moreover, the addition of neutralizing antibodies to IL-6 to the normal human bone marrow cultures effectively blocked the increase in MNC formation induced by active GSD serum. These data suggest that bone resorption in GSD patients is due to enhanced osteoclast activity, and that IL-6 may play a role in the increased bone resorption in GSD. PMID- 8626855 TI - Constitutive activation of the Gs alpha protein-adenylate cyclase pathway may not be sufficient to generate toxic thyroid adenomas. AB - In toxic thyroid adenomas, mutations in the TSH receptor (TSH-R) gene or the gene encoding the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs alpha) have been demonstrated to constitutively activate the cAMP cascade, which subsequently stimulates the growth and function of these tumors. However, the widely varying thyroid phenotypes in patients with TSH-R germline mutations, ranging from only slightly enlarged diffuse to multinodular goiters, suggest that additional mechanisms may be effective in the pathogenesis of toxic adenomas. We have investigated the levels of stimulatory and inhibitory G protein alpha subunits together with basal and TSH-stimulated adenylate cyclase (AC) activity in toxic adenomas with or without activating mutations and in nodular and extranodular tissues of a toxic goiter due to a germline mutation in the TSH-R gene. Augmented expression of Gs alpha protein was detected in all toxic adenomas, independent of the presence of mutations, and in the nodular tissue of the toxic goiter, but not in the nonnodular hyperplastic tissue of the toxic goiter with the mutated TSH-R. Analogously, the expression of the alpha-subunit of the inhibitory G protein (Gi alpha) was also increased in all adenomas and the nodular tissue of the goiter, but, again, not in the hyperplastic goiter tissue. Basal AC activity was high in all tissues with mutations, but was only slightly increased in adenomas without detected mutations. No correlation was detectable between basal or TSH-stimulated AC activity and the levels of Gs alpha and Gi alpha. Our data suggest that mutational activation of the cAMP cascade may not be sufficient to generate toxic nodules and adenomas, but far more complex mechanisms, including alterations of G protein signaling, may be effective in the pathogenesis of these tumors. PMID- 8626856 TI - The effect of Cushing's disease on bone mineral density, body composition, growth, and puberty: a report of an identical adolescent twin pair. AB - As endogenous Cushing's syndrome (CS) in children occurs during a critical developmental period, when the majority of peak bone mass is acquired, we hypothesized that children with CS might be at an increased risk of osteoporosis. To determine the effects of CS on bone density, bone metabolism, and growth, we studied a 15-yr-old female identical twin pair, one of whom had CS (twin A), and the other of whom was healthy (twin B). Before therapy for CS, twin A showed a severe loss of bone mineral density [BMD; -3.2SD at the lumbar spine (LS)] compared to twin B (-0.1 SD), which in twin A was associated with low serum osteocalcin levels and urinary pyridinium cross-link excretion. Cure of CS in twin A led to a marked increase in these bone markers, suggesting a state of active bone remodeling. After 27 months of follow-up, even though twin A's BMD improved significantly, it still remained abnormal [-1.9 SD at LS compared with that of twin B (0 SD)], suggesting that twin A continued to be at increased long term risk of osteoporosis. In addition, as a consequence of CS, twin A's final height was 21 cm less than that of her identical twin. We recommend that all children with CS should have BMD monitored after treatment to determine the long term risk of osteoporosis. PMID- 8626857 TI - Hypothalamic corticotropin-releasing hormone suppression during the postpartum period: implications for the increase in psychiatric manifestations at this time. AB - The third trimester of human pregnancy is characterized by a hyperactive hypothalamic-pituitary-adrenal axis, possibly driven by progressively increasing circulating levels of placental CRH and gradually decreasing levels of CRH binding protein. The postpartum period, on the other hand, is characterized by an increased vulnerability to psychiatric manifestations (postpartum "blues," depression, and psychosis), a phenomenon compatible with suppressed hypothalamic CRH secretion. To investigate the hypothesis that the postpartum period is associated with suppression of hypothalamic CRH secretion, we studied prospectively 17 healthy euthymic women (mean +/- SE age, 32.0 +/- 1.1 yr) with no prior history of depression, starting at the 20th week of gestation. Psychometric testing was performed monthly during pregnancy and postpartum on day 2 and weeks 2, 3, 6, 8, 12, 16, and 20, whereas serial ovine (o) CRH tests were performed postpartum at 3, 6, and 12 weeks. While pregnant, all 17 subjects remained euthymic; in the postpartum period, 7 women developed the "blues," and 1 developed depression. Overall, the mean plasma ACTH response to an iv bolus of 1 microgram/kg oCRH was markedly blunted at 3 and 6 weeks, but normal at 12 weeks postpartum, whereas the mean plasma cortisol response was at the upper limit of normal at all 3 times. These data are compatible with a suppressed hypothalamic CRH neuron that gradually returns to normal while hypertropic adrenal cortexes are progressively down-sizing. When the postpartum ACTH responses to oCRH were analyzed separately for the euthymic women and the women who had the "blues" or depression, the blunting of ACTH was significantly more severe and long lasting in the latter group; this was observed at all 3 times of testing. We conclude that there is central suppression of hypothalamic CRH secretion in the postpartum, which might explain the increased vulnerability to the affective disorders observed during this period. The suppressed ACTH response to oCRH might serve as a biochemical marker of the postpartum "blues" or depression. PMID- 8626858 TI - Multimerization of thyroglobulin (TG) during extracellular storage: isolation of highly cross-linked TG from human thyroids. AB - Thyroglobulin (TG) is the major soluble protein of the thyroid and is known to be extracellularly stored for future liberation of thyroid hormones. We have developed techniques for the isolation of an insoluble storage form of human TG present in the follicle lumen. The application of these techniques yielded insoluble and translucent colloid globules varying in size (50-500 microns) and shape and consisting primarily of densely packed TG. Intact colloid globules exhibited the imprints of the apical cell surfaces of thyrocytes that had surrounded the colloid globules in situ. Hence, in size and surface morphology, isolated colloid globules represent authentic lumenal content. Based on the total protein of single colloid globules and their volume, an average protein concentration of 590 mg/mL was calculated. The presence of protein disulfide isomerase in colloid globules and in the secretory product of cultured thyrocytes suggests its involvement in the extracellular multimerization of human TG. Native colloid globules increased their volume considerably upon reduction of disulfide bonds; they were completely dissolved by treatment with dithiothreitol and SDS. The results show that part of extracellular human TG undergoes multimerization, primarily by the formation of intermolecular disulfide bonds, thus allowing the storage of TG at excessively high, previously unknown, concentrations. PMID- 8626859 TI - Insulin-like growth factor I (IGF-I) and IGF-binding protein-3 concentrations compared to stimulated and night growth hormone in the evaluation of short children--a clinical research center study. AB - To evaluate the relative usefulness of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) in screening for GH status, GH stimulation (arginine-insulin/L-DOPA) tests and overnight GH studies (every 20 min sampling) were performed in 104 healthy short children (32 girls), aged 3-16 yr (height, 1.8 or more SD). IGFBP-3 had no advantage over IGF-I in screening sensitivity or specificity. IGF-I correlated with mean nighttime GH. Both IGF-I and IGFBP-3 correlated with peak stimulated GH. To identify more than 90% of children with GH deficiency (GHD) and borderline GHD, the mean values for age for IGF-I and IGFBP 3 were required as the cut-off criterion. However, at this criterion, 70% or more of idiopathic short stature (ISS) children would have to undergo testing to identify 90% of GHD or borderline GHD. More stringent criteria (-1.0, -1.64, and 2.0 SD) were more specific, but lost sensitivity. A practical application is suggested. Screening use of IGF-I with criterion of -1.0 SD would identify a subgroup that includes 88% of GHD, 71% of borderline GHD, and 46% of ISS. Both IGF-I and IGF-BP-3 higher than -1.0 SD would accurately identify 68% of ISS as not needing GH testing. Evaluation of growth velocity would identify the remaining children requiring definitive testing. Thus, combined screening for GHD using both IGF-I and IGFBP-3 has no better sensitivity than either test alone. However, such combined screening will improve the specificity and thus decrease the number of normal but short children who might otherwise undergo unnecessary testing. PMID- 8626860 TI - Antiprogestin and/or gonadotropin-releasing hormone agonist for endometriosis treatment and bone maintenance: a 1-year primate study. AB - The fact that RU 486 curtailed estrogen-induced endometrial proliferation in primates and relieved pelvic pain in women with endometriosis is the reason for continuing research on antiprogestins. Thirty-two adult female cynomolgus monkeys demonstrating menstrual regularity had surgery for the induction of endometriosis. After lesion staging, four treatment groups (n = 8), each of 1-yr duration, were made. Group I received combination/sequential therapy with depot GnRH agonist (GnRH-a) for 3 months, followed by weekly RU 486 for 9 months. Group II received weekly RU 486, group III received monthly GnRH-a, and group IV served as a vehicle control. A staging laparotomy was performed every 3 months to assess the area of peritoneal endometriosis (square centimeters) and the thickness of in situ endometrium. Bone density was measured serially by dual x-ray absorptiometry. Serum was collection weekly. Mean (+/- SE) serum estradiol levels were lower after GnRH-a (77.1 +/- 2.6 pmol/L) than after RU 486 (231 +/- 12 pmol/L) treatment and lower than those in untreated cycling controls (231 +/- 13 pmol/L). GnRH-a produced significant atrophy of endometriotic plaques within 3 months of therapy; this lesion reduction was sustained with RU 486. Both GnRH-a and RU 486 alone produced profound thinning of ectopic and eutopic endometrium throughout 1 yr of continuous therapy. Bone density decreased significantly after 6 months of GnRH-a alone (P < 0.05), without significant changes in the other groups. After RU 486 treatment, there were no significant changes in testosterone, androstenedione, sex hormone-binding globulin, or cortisol. Like GnRH-a, long term antiprogestin therapy produced a reduction in the volume of pelvic endometriotic lesions as well as atrophy of in situ endometrium; however, RU 486 allowed maintenance of tonic ovarian estradiol secretion, suggesting that efficacious endometriosis therapy can be sustained long term without the sequelae of hypoestrogenism, specifically bone density loss. PMID- 8626861 TI - A 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid, is increased in diabetics with incipient and early renal disease. AB - Earlier studies in diabetic animal models or ex vivo from diabetics suggest a deficiency in prostacyclin (PGI2) production and an increase in an alternate arachidonic acid metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), which stimulates angiogenesis, mitogenesis, and inhibits renin secretion. We studied the urinary excretion rate of 6-keto-PGF1 alpha (a stable metabolite of PGI2) and 12-HETE in controls and 42 noninsulin-dependent diabetes mellitus (NIDDM) patients with normal renal function and those with micro- or macroalbuminuria/hyporeninemic hypoaldosteronism (HH). The 2 eicosanoids were measured in urine using previously described high pressure liquid chromatography and RIA methods. Normal subjects and patients with NIDDM and microalbuminuria were infused with low dose calcium infusions that stimulate prostacyclin production in normal subjects. The PGI2 excretion rate of NIDDM patients with normal renal function was not different from that of controls (143 +/- 17 vs. 118 +/- 34 ng/g creatinine), but was reduced in those with microalbuminuria (75 +/- 10) and in macroalbuminuria patients (48 +/- 7; P < 0.01). In contrast, 12-HETE was increased in diabetics with normal renal function as well as in those with micro- or macroalbuminuria patients (69 +/- 18 vs. 250 +/- 62 vs. 226 +/- 60 and 404 +/- 131 ng/g creatinine; P < 0.01). Calcium did not stimulate PGI2, but increased 12-HETE in diabetics with microalbuminuria in contrast to levels in normal subjects. HH patients excreted less PGI2 (as previously reported), but had increased 12-HETE. HETE/PGI2 ratios further demonstrated these changes in the various groups. In a nondiabetic hypertensive microalbuminuria group, 12-HETE excretion was normal (73 +/- 28 ng/g creatinine). We conclude that the lipoxygenase product 12-HETE is increased early in the diabetic process, whereas PGI2 production is progressively impaired in NIDDM. These changes may play a role in the vascular disease of diabetes and partially explain the HH syndrome. PMID- 8626863 TI - The inhibitory effects of growth hormone-releasing hormone (GHRH)-antagonist on GHRH, L-dopa, and clonidine-induced GH secretion in normal subjects. AB - The relative inhibitory potency of GHRH-Antagonist (GHRH-Ant) to GHRH(1-44)NH2 and mechanism of L-dopa- or clonidine-induced GH release were studied in seven normal subjects using GHRH-Ant. One hundred micrograms of GHRH-Ant (iv for 75 min) did not inhibit plasma GH responses to bolus injection of 100 micrograms and 10 micrograms GHRH or simultaneous infusion of 5 micrograms GHRH (iv for 75 min). However, 200 micrograms GHRH-Ant (iv for 75 min) significantly inhibited GH release, which was induced by simultaneous infusion of 5 micrograms GHRH. Although 100 micrograms GHRH-Ant could not significantly inhibit L-dopa-induced GH release, 200 micrograms GHRH-Ant almost completely inhibited the response. Similarly, the same dose of GHRH-Ant markedly inhibited the GH-releasing activity of clonidine. It is concluded that the inhibitory potency of GHRH-Ant on GHRH(1 44)NH2 is relatively weak (about 1/60 in molar base), and that L-dopa- or clonidine-induced GH release seems to be mediated by the release of hypothalamic GHRH. PMID- 8626862 TI - Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age. AB - Reproductive aging in women (a physiological decline in the function of the hypothalamic-pituitary-ovarian axis) is an infrequently investigated and poorly understood biological phenomenon. Although menstrual irregularity and anovulation are known to precede the menopause, normal women in their fifth decade experience a profound decrease in fertility while still experiencing regular menstrual cycles. To further our understanding of the physiological changes associated with reproductive aging, this study examined the spontaneous development and function of ovarian follicles in normal women, aged 40-45 yr. The subjects were women (n = 21), aged 40-45 yr, who had regular 25- to 35-day ovulatory menstrual cycles, were not infertile, had no medical problems, and met specific criteria for weight, diet, and exercise. The controls were normal women (n = 20), age 20-25 yr, who met the same criteria. The subjects were monitored with daily hormone measurements [LH, FSH, estradiol (E), progesterone (P), and inhibin] and pelvic sonograms from day 1 of their study cycle until the dominant ovarian follicle reached a mean diameter of 15 mm and/or a serum E level of 550 pmol/L or higher was attained. At that time, 10,000 U hCG were given, and a transvaginal sonographic follicle aspiration was performed 32 h later. The follicular fluid (FF) was collected, stored frozen at -70 C, and later analyzed for E, P, testosterone (T), androstenedione, inhibin, insulin-like growth factor I (IGF-I), and IGF-II. The number of cycle days to aspiration was lower (11.6 vs. 15.6 days; P < 0.001) and the early follicular phase mean FSH and mean E levels were higher (9.3 vs. 6.6 mIU/mL and 305 vs. 160 pmol/L; P < 0.01) in the older (O) group compared to the younger group. There was a strong trend toward higher FF mean E (2280 vs. 1931 nmol/L) and lower FF mean T (978 vs. 2361 pmol/L) levels in group O. The E/T ratio was significantly higher (5253 vs. 2408; P < 0.03) in group O. In group O, the mean FF P levels were increased as well (25.1 vs. 18.8 micromol/L; P < 0.01). The serum mean IGF-I (153 vs. 226 ng/mL; P < 0.001) and FF mean IGF-I (113 vs. 158 ng/mL; P < 0.02) levels were significantly decreased in group O. There were no differences between groups in serum or FF IGF-II or inhibin levels. Whether reproductive aging is an intrinsic ovarian process or the ovary is simply responding to exogenous influences, the ovary in general and its follicles in particular are the primary site of the effects of aging. Ovarian follicles in older ovulatory women have some unique features: 1) the follicles are the same size as those in younger women, but form more rapidly; 2) secretion of E and inhibin is not compromised; 3) the concentrations of steroids in the FF are indicative of a healthier follicle, i.e. increased P levels and higher estrogen to androgen ratio; and 4) serum and FF levels of IGF-I are decreased, but there are no differences in IGF-II levels. PMID- 8626864 TI - Time kinetics of the endocrine response to acute psychological stress. AB - A first-time parachute jump was chosen as a model to evaluate the endocrine response to acute psychological stress. In 43 inexperienced tandem parachutists, blood was drawn continuously from 2 h before to 1 h after the jump and analyzed at 10-min intervals for plasma concentrations of epinephrine (E), norepinephrine (NE), cortisol, GH, PRL, and TSH. In addition, heart rate was recorded throughout the experiment. There was a significant increase in heart rate and E concentrations during the jump itself. NE, cortisol, GH, PRL, and TSH peaked with a latency of 10-20 min. Apart from cortisol and TSH concentrations, which were still elevated 1 h after the stress event, plasma levels of the other endocrine variables normalized within 1 h following the jump. Statistically significant cross-correlations could be observed between E and NE (r = 0.60, no time lag) and between E and PRL (r = 0.58, 10-min time lag) only. Even in a very homogenous group of subjects and under well-controlled conditions, endocrine responses to acute psychological stress show considerable variations. PMID- 8626865 TI - A variant of adenomatous goiter with characteristic histology and possible hereditary thyroglobulin abnormality. AB - A variant type of adenomatous goiter was identified in 24 of 2160 patients with adenomatous goiter who underwent thyroidectomy. The characteristics of the thyroid gland in these 24 patients included large goiter, small follicles, scant colloid, and columnar follicular cells containing yellow-green granules on hematoxylin-eosin staining. The thyroid gland was slightly orange-red, and electron microscopic examination showed abundant lysosomes with colloid droplets. When comparing the features of this group with those of 24 patients with common adenomatous goiter, the incidence of familial predisposition to thyroid diseases in the former group was higher. The age at the time of detection of goiter was lower, i.e. 17 +/- 15 vs. 44 +/- 17 yr (P < 0.001, variant type vs. common type), the serum total T4 concentrations were lower (84 +/- 21 vs. 103 +/- 18 nmol/L; P < 0.01), and the serum TSH concentrations were higher (2.4 +/- 2.1 vs. 1.0 +/- 0.9 mU/L; P < 0.01). Thyroid radioiodine uptake was remarkably increased (49 +/- 22 vs. 16 +/- 9%; P < 0.001), and lower levels of serum thyroglobulin were noted (33 +/- 51 vs. 484 +/- 603 micrograms/L; P < 0.01). The thyroglobulin content was low in the thyroid gland studied. The data suggest that the etiology of this variant type of goiter is a hereditary abnormality in thyroglobulin synthesis, and this type of goiter may be distinguished from common adenomatous goiter by the characteristic morphology of the thyroid gland in addition to clinical findings. PMID- 8626866 TI - Sex steroid regulation of insulin-like growth factor system gene expression and proliferation in primate myometrium. AB - To investigate the role of locally produced insulin-like growth factors (IGFs) in sex steroid-induced growth in the primate uterus, ovariectomized rhesus monkeys were treated with placebo (control), estradiol (E2) alone, or E2 plus progesterone (P4). After 2 weeks, uteri were removed, and serial thin uterine sections were analyzed by in situ hybridization for IGF-I, IGF-II, and IGF-I and II receptor messenger ribonucleic acids (mRNAs) and by immunocytochemistry for the cell proliferation-specific antigen Ki-67. IGF-I and IGF-II and both IGF receptor mRNAs are coexpressed by smooth muscle cells, supporting the possibility of autocrine/paracrine IGF action in stimulating myometrial growth. IGF-I mRNA is barely detected in control myometrium, is significantly increased by E2 treatment, and is augmented even more by the combination of E2 and P4 treatment, whereas little change is noted in myometrial IGF-II or IGF-I receptor mRNA levels. Ki-67-positive myometrial nuclei are also significantly increased by E2 and are augmented even more by E2 plus P4 treatment, with a correlation between local IGF-I mRNA concentration and local Ki-67-positive cell count of r = 0.891 (P = 0.003). These data provide direct experimental evidence for regulation of IGF-I gene expression by sex steroids in the primate uterus in vivo and implicate local IGF-I action in both estrogen- and P4-induced myometrial growth. PMID- 8626867 TI - The degree/rapidity of the metabolic deterioration following interruption of a continuous subcutaneous insulin infusion is influenced by the prevailing blood glucose Level. AB - This study aims at investigating the influence of the prevailing blood glucose level on the metabolic deterioration that follows a nocturnal interruption of a continuous sc insulin infusion (CSII). Fifteen CSII-treated, C-peptide negative, diabetic patients have been studied CSII was interrupted from 2300 h to 0500 h. Blood was collected hourly from 2200 h to 0600 h. According to blood glucose (BG) levels at 2300 h, patients were classified as hypoglycemic (BG between 1.5 and 2.5 mmol/L, n = 5), normoglycemic (BG between 4.0 and 8.0 mmol/L, n = 5), or hyperglycemic (BG between 9.0 and 15.0 mmol/L, n = 5). At 2300 h, BG (mean +/- SEM) was 1.9 +/- 0.1, 6.2 +/- 0.7 and 11.2 +/- 1.0 mmol/L, respectively. After 6 h of CSII interruption, BG increased to 13.5 +/- 1.3, 14.1 +/- 1.2, and 19.4 +/- 1.2 mmol/L, respectively. At 2300 h, plasma 3-OH-butyrate levels were similar in the three groups (around 150 micromol/L). At 0500 h, significantly higher values were obtained for hyperglycemic (1460 +/- 127 micromol/L) than for normoglycemic (868 +/- 150 micromol/L) or hypoglycemic (837 +/- 80 micromol/L) patients. Enhanced lipolysis in initially hyperglycemic patients may contribute to accelerated ketogenesis and metabolic degradation. In conclusion, the metabolic deterioration that follows CSII interruption is influenced by the initial metabolic situation. Hypoglycemic patients deteriorate more rapidly, and hyperglycemic patients suffer a more important degradation. The latter are prone to rapid ketoacidosis if accidental CSII interruption occurs. PMID- 8626868 TI - Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. AB - Polycystic ovary syndrome (PCOS) is a common disorder characterized by chronic anovulation and infertility, hyperandrogenaemia, and frequently insulin resistance. This study investigated whether mutations in the insulin receptor gene could explain the insulin resistance in subjects with PCOS. From a total of 108 women with PCOS, a subgroup of 24 were selected on the criteria of being in the upper quartile for insulin resistance as assessed by fasting serum insulin, insulin area under the curve following 75 g oral glucose tolerance test, and endogenous glucose disposal as a measure of insulin sensitivity. An additional five normal women were also investigated. The entire coding region of the insulin receptor gene, comprising of 22 exons, was amplified by the PCR using genomic DNA and then subjected to single-stranded conformation polymorphism (SSCP) analysis to screen for single-base DNA sequence changes. DNA sequencing revealed that SSCP variants were detected in regions encompassing exons 3, 6-8, 11, 13, 15, 17, and 22. SSCP variants in regions of exons 3, 6, 7, 11, 15 and 22 were caused by nucleotide substitutions within intronic regions flanking the exon. The considerable variation seen in the 5' intron of exon 3 was found to be caused by variation in the number of (ATTT, 8-11) and (TC, 10-13) short sequence repeats. SSCP variants in exons 8 (Asp519, Ala523), 13 (Asn 838), and 17 (Tyr984, His1058) were caused by known silent polymorphisms. Southern blotting experiments excluded major gene deletions, insertions, or rearrangements. We conclude that insulin resistance in subjects with PCOS is not commonly a consequence of missense or nonsense mutations in the insulin receptor gene. PMID- 8626869 TI - A novel substitution (Leu707Arg) in exon 4 of the androgen receptor gene causes complete androgen resistance. AB - A wide spectrum of androgen receptor (AR) gene mutations has been reported in complete androgen insensitivity syndromes. The molecular basis of androgen resistance was investigated in a female newborn with complete testicular feminization. Sequencing identified a point mutation in exon 4 responsible for a leucine (CTG) to arginine (CGG) replacement at codon 707. This novel mutation is located in the amino-terminal part of the ligand-binding domain of the AR. To determine the functional properties of the mutated AR and to establish the correlation with the clinical phenotype of androgen resistance, the mutation was reproduced in AR wild-type complementary DNA, and the plasmid was transfected into AR-free mammalian cells. In vitro studies showed that the mutant AR was functionally defective as an androgen-binding molecule. Electrophoretic mobility shift assay revealed that the binding of mutated AR to DNA was reduced. Finally, the mutant was unable to induce the transcriptional activation of androgen responsive reporter gene. This amino acid defect in the primary sequence probably involves the rupture of hydropathicity in a region that is conserved among members of the steroid receptor subfamily. Our data substantiate the major contribution of leucine 707 to normal AR function and demonstrate that its substitution by an arginine caused the complete androgen insensitivity in this patient. Our findings also contribute to the elaboration of the structure function map of the AR based on naturally occurring mutations. PMID- 8626870 TI - Predominant expression of 5 alpha-reductase type 1 in pubic skin from normal subjects and hirsute patients. AB - Dihydrotestosterone (DHT), the 5 alpha-reduced metabolite of testosterone, is the active molecule triggering androgen action, and 5 alpha-reductase (5 alpha-R), the enzyme converting testosterone to DHT, is a key step in this mechanism. Skin, like prostate, is a DHT- dependent tissue. Our laboratory demonstrated, many years ago, that 5 alpha-R in external genitalia was not regulated by androgens, whereas it was androgen dependent in public skin. As two genes, 5 alpha-R types 1 and 2, encoding for 5 alpha-R enzymes have been recently cloned, we undertook the present study to determine whether the two enzymes we had postulated on the basis of regulation studies were coincident with the cloned isoforms. The expression of the two isoforms was studied in genital and pubic skin fibroblasts from normal men, normal women, and hirsute patients. Messenger ribonucleic acid analysis, using Northern blot and RT-PCR techniques, indicated that both 5 alpha-R1 and -2 messenger ribonucleic acids are expressed in genital skin as well as in public skin fibroblasts. In contrast, studies using specific inhibitors of 5 alpha-R1 (LY306089) and 5 alpha-R2 (finasteride) showed that 5 alpha-R2 is predominant in pubic skin of normal men, normal women, and hirsute patients. These data raise the question of the possible use of specific 5 alpha-R1 inhibitors in the treatment of idiopathic hirsutism. PMID- 8626871 TI - The decrease in growth hormone (GH) response after repeated stimulation with GH releasing hormone is partly caused by an elevation of somatostatin tonus. AB - Repeated injection of GHRH leads to a decrease in the GH response in normal subjects. Arginine (Arg) stimulates GH secretion by suppression of hypothalamic somatostatin. To confirm these findings, eight normal men were examined in a series of five settings: test 1 (GHRH/GHRH-TRH), 100 micrograms GHRH injected iv, followed by 100 micrograms GHRH, iv, after 120 min and 200 micrograms TRH, iv, after 150 min; test 2 (GHRH/Arg-TRH), like test 1, but instead of the second GHRH injection, a 30 g Arg infusion over 30 min; test 3 (GHRH/GHRH-Arg-TRH), like test 1, but additionally a 30 g Arg infusion after 120 min; test 4 (GHRH-Arg-TRH), iv GHRH and Arg infusion initially, followed by iv TRH after 30 min; and test 5 (TRH), 200 micrograms TRH, iv, at 0 min. For statistical evaluation, the area under the GH curve (AUC) from 0-120 min was compared with the AUC from 120-240 min. The GH response to the second administration of GHRH was significantly lower (P < 0.02) than the first increase [AUC, 0.5 +/- 0.01 min.mg/L (mean +/- SE) vs. 1.2 +/- 0.3]. No significant differences were found between the GH responses to either GHRH or Arg alone (AUC, 0.9 +/- 0.2 min.mg/L vs. 0.9 +/- 0.2). A larger GH increase (P < 0.02) was seen after GHRH-Arg compared to GHRH alone (AUC, 1.9 +/- 0.4 min.mg/L vs. 1.2 +/- 0.3). The GH response (P < 0.02) to GHRH-Arg stimulation was lower after previous GHRH injection than after GHRH-Arg stimulation alone (AUC, 1.9 +/- 0.4 min.mg/L vs. 3.5 +/- 0.9). There was a statistically significant difference between the TRH-stimulated TSH response in test 4 compared to that in test 5. We could show that decreasing GH responses to repeated GHRH can be avoided by a combined stimulation with GHRH/Arg. These findings suggest that the decreased GH response to a second GHRH bolus may be partly due to an elevated hypothalamic somatostatin secretion, which can be suppressed by Arg. The lower GH response to GHRH-Arg stimulation after a previous GHRH bolus suggests, furthermore, that the readily available GH pool in the human pituitary may be limited. PMID- 8626872 TI - Growth hormone treatment increases cytochrome P450-mediated antipyrine clearance in man. AB - We used the antipyrine clearance test (APC) to examine the effect of growth hormone (GH) therapy on hepatic cytochrome P450 (CYP) enzyme activity. Eleven GH deficient adults were randomized to receive GH or placebo for 6 months, all subjects subsequently received GH. Before treatment, APC was below the normal range in six subjects. We found an increase in APC in the subjects randomized to receive GH compared to those on placebo (median change +0.14 ml/min/kg [range + 0.04 to + 0.20]vs -0.04 ml/min/kg [range -0.07 to + 0.04], p = 0.011). The stimulatory effect of GH on drug metabolism was confirmed by the data for 3 months GH treatment in all 11 subjects, with APC increasing from 0.33 ml/min/kg (range 0.22 to 0.69) to 0.50 ml/min/kg (range 0.27 to 0.83), p = 0.018). These data indicate that GH modulates hepatic CYP activity. This has important clinical implications, as the hepatic metabolism of drugs and hormones may be altered in patients undergoing GH therapy. PMID- 8626873 TI - A homologous radioimmunoassay for rat insulin-like growth factor-I (IGF-I): implications for studies of human IGF-I physiology. AB - We report the development of a unique radioimmunoassay (RIA) for rat insulin-like growth factor-I (rIGF-I) which does not recognize human IGF-I (hIGF-I). The rIGF RIA uses a specific anti-rIGF-I antiserum with rIGF-I standards and radioligand. Rat sera were extracted by either an abbreviated acid/ethanol procedure or by acid-chromatography with virtually identical results. Assay sensitivity is congruent to 0.5 ng/tube and inter- and intra-assay coefficients of variation were 3.4-7.6% and 4.8-9.2%, respectively. Comparisons of the rIGF-I RIA with a typical heterologous RIA shows that the latter underestimates rIGF-I levels congruent to of 3-fold. Sera from rats treated with hIGF-I were also studied using the rIGF-I RIA and a hIGF-I- specific immunoradiometric assay (IRMA), and results indicate poor correlation between the actual total IGF-I levels (rIGF-I RIA + hIGF-IRMA) and the heterologous RIA estimates. The availability of both rIGF-I and hIGF-I specific immunoassays facilitates more precise studies of hIGF I in the rat model. PMID- 8626874 TI - Age-related activation of the tyrosine kinase receptor protooncogenes RET and NTRK1 in papillary thyroid carcinoma. AB - Oncogenic rearrangements of RET and NTRK1 proto-oncogenes are frequently detected in papillary thyroid carcinomas. Several studies have shown an association between ionizing radiation and development of this tumor type. In addition in vitro irradiation of tumor cell lines induced rearrangements of RET similar to those observed in human papillary thyroid carcinomas. These two observations could be related to the reported increased incidence of papillary thyroid carcinomas in children living in contaminated areas around Chernobyl, given that it has been demonstrated that about 60% of them presents a RET oncogenic activation. However, this high frequency of RET positivity in radiation exposed children does not rule out the possibility that age could also play a role in the development of RET positive tumors. To assess this possibility we looked for a relationship between the presence of RET and NTRK1 oncogenic rearrangements and age at surgery in a sample of 92 consecutive patients. Our results show that, in papillary thyroid carcinoma, the frequency of RET and NTRK1 activation is significantly higher in the group of patients aged 4-30 years, thas supporting the concept that age could be contributing to this thyroid specific carcinogenic process. PMID- 8626875 TI - Contemporary use of and future roles for heparin in antithrombotic therapy. AB - Although heparin therapy is an established component of the prevention and treatment of thromboembolic disease, recent advances have resulted in improvements in the clinical use of this agent. Studies have shown that weight based dosing influences significantly both the time to reach a therapeutic intensity of anticoagulation and the incidence of thromboembolic recurrence. It is now considered the standard of care. A growing understanding of the variability among activated partial thromboplastin time (aPTT) reagents and the influence of these differences on aPTT outcomes has led to the use of reagent specific therapeutic ranges for heparin monitoring. Many practitioners now choose to adjust the therapeutic range to correspond to heparin serum concentrations of 0.2-0.4 U/mL rather than the more common practice of prolonging aPTT to 1.5-2.5 times the mean normal aPTT. Pharmaceutical companies have developed low molecular weight heparins to minimize adverse effects associated with unfractionated heparin. More specific thrombin inhibitors are also under investigation with the aim of improving clinical outcomes in coronary syndromes now treated with heparin. Low molecular weight heparins or specific thrombin inhibitors are unlikely to replace unfractionated heparin in the near future. Therefore, optimum dosing and appropriate monitoring of heparin are critically important in the management of thromboembolic disease. PMID- 8626876 TI - Ceftizoxime use in trauma celiotomy: pharmacokinetics and patient outcomes. AB - Seriously injured patients undergo vigorous resuscitation upon arrival at the emergency department and through the immediate perioperative period. Although resuscitation leads to volume loading and fluid shifts, drug dosing and dosing intervals are often not altered to account for changes in total body volume or circulatory volume. To address this, a prospective study of pharmacokinetics of ceftizoxime in 53 injured adults who underwent emergency celiotomy was conducted. Further, the relationship between serum ceftizoxime concentrations and infectious outcomes was evaluated. Per protocol, injured adults undergoing emergency celiotomy received prophylactic ceftizoxime treatment according to standard dosing regimens. Of the patients, 6 (11.5%) experienced postoperative infections and had lower peak serum ceftizoxime levels in the recovery room than patients not experiencing infection. For severely injured adults with extensive blood loss or undergoing lengthy operations requiring rigorous volume resuscitation, doses of ceftizoxime, and indeed all antibiotics, may need to be increased beyond conventional standards to minimize infectious complications. PMID- 8626877 TI - Effects of amlodipine on 24-hour ambulatory blood pressure profiles, electrocardiographic monitoring, and left ventricular mass and function in black patients with very severe hypertension. AB - In a 3-month, open-label study, 54 consecutive black patients with very severe hypertension were treated with amlodipine. Very severe hypertension was defined as an average sitting diastolic blood pressure (BP) > or = 115 mmHg and < or = 140 mmHg as a mean of 10 readings over a 30-minute period using an automatic BP measuring device and a mean 24-hour diastolic ambulatory blood pressure (ABP) > or = 110 mmHg and < or = 140 mmHg). Serial changes in 24-hour ABP and electrocardiographic monitoring, left ventricular (LV) mass index, and LV systolic function were evaluated. Mean 24-hour ABP was reduced from 181 +/- 14/119 +/- 6 to 140 +/- 15/92 +/- 9 mmHg at 3 months (P < 0.0001). Target BP (mean 24-hour diastolic ABP < 90 mmHg) was achieved in 35% of the patients. The reduction in BP was sustained for 24 hours after drug administration. Simultaneous BP measurements using the automatic BP measuring device were significantly different from the ABP measurements before and after treatment, suggesting a marked "white coat" pressor effect. At baseline, frequent or complex ventricular arrhythmias (> 30 ventricular extrasystoles per hour, ventricular couplets) were present in 2 (4%) patients, with no significant change after treatment. Left ventricular mass index regressed from 140 +/- 50 to 111 +/- 30 g/m2 at 3 months (P < 0.03); LV performance was not adversely affected. Adverse effects were few and tended to disappear during the treatment period. All of the clinical laboratory parameters tested remained unchanged. In this group of patients, treatment with amlodipine showed a marked and sustained antihypertensive action as demonstrated by 24-hour ABP monitoring, and was well tolerated and associated with LV mass regression without adverse effect on systolic cardiac function. Further, a low rate of complex ventricular arrhythmias was documented. PMID- 8626878 TI - Comparison of amlodipine and benazepril monotherapy to amlodipine plus benazepril in patients with systemic hypertension: a randomized, double-blind, placebo controlled, parallel-group study. The Benazepril/Amlodipine Study Group. AB - A single-blind, run-in, randomized, double-blind, parallel-group, placebo controlled comparison trial was conducted to assess the safety and efficacy of low-dose amlodipine 2.5 mg daily, low-dose benazepril 10 mg daily, and the combination of the two drugs at the same doses used once daily in patients (n = 401) with mild to moderate (stages I and II) systemic hypertension. Both monotherapy regimens were shown to significantly reduce both systolic and diastolic blood pressure compared with baseline placebo values, and the combination regimen was shown to be superior in lowering systolic and diastolic blood pressure when compared with either of the monotherapy regimens. The combination therapy also resulted in a greater percentage of patients having successful clinical response in mean sitting diastolic blood pressure. The amlodipine and benazepril regimen was also shown to be associated with a similar incidence of adverse experiences as the active monotherapy or placebo regimens, although the group given combination therapy appeared to have a lower incidence of edema than the group given amlodipine alone. Low-dose amlodipine (2.5 mg) plus benazepril (10 mg) provides greater blood-pressure-lowering efficacy than either monotherapy, and has an excellent safety profile. PMID- 8626879 TI - Comparison of venodilatory effect of amrinone and theophylline in human subjects. AB - Amrinone, a positive inotropic agent, is a selective phosphodiesterase III inhibitor and exerts vasodilatory effect on venous vessels. The present study was undertaken to compare the venodilatory effect of amrinone and theophylline, a nonselective phosphodiesterase inhibitor, in eight healthy male subjects. In a randomized crossover design, one of these drugs was infused into the dorsal hand vein preconstricted by phenylephrine and its diameter was measured using a linear variable differential transformer. The value of maximum vasodilation for amrinone (mean +/- standard deviation, 106% +/- 17%) was similar to that for theophylline (mean +/- standard deviation, 108% +/- 14%). However, the infusion rate of amrinone needed to induce 50% of maximum vasodilation was significantly less than that of theophylline (25 +/- 15 micrograms/minute vs. 192 +/- 87 micrograms/minute, respectively; P < 0.01). These findings suggest that the venodilatory activity of amrinone is more potent than that of theophylline in human subjects. PMID- 8626880 TI - Diurnal variation in the pharmacokinetics of nizatidine in healthy volunteers and in patients with peptic ulcer disease. AB - Six healthy volunteers and six patients with asymptomatic duodenal ulcer disease received placebo or 300 mg nizatidine once at night or twice daily (morning and evening) for a week in a random, cross-over fashion. Steady-state serum nizatidine concentrations and gastric pH were measured over a 24-hour period. No significant differences in the pharmacokinetic indices were observed between the healthy volunteers and patients with duodenal ulcer disease. In patients with duodenal ulcers, significantly lower peak serum concentrations, longer half-life (t1/2) and larger volume of distribution (Vd) were observed after the night doses compared with the daytime doses. The diurnal variation in drug kinetics between the nighttime and daytime doses in the twice daily regimen may be caused by a slower absorption rate, paralleled with a higher extent of distribution. Despite lower serum nizatidine concentrations, gastric pH was higher in the evening than in the daytime; it is speculated that this was due to a time-dependent enhanced distribution of the H2-receptor blocker into the site of action. PMID- 8626881 TI - Pharmacokinetics of verapamil and norverapamil enantiomers after administration of immediate and controlled-release formulations to humans:evidence suggesting input-rate determined stereoselectivity. AB - Verapamil is a racemic calcium channel-blocking drug that undergoes extensive hepatic first-pass metabolism to an active metabolite, norverapamil. The enantiomers of verapamil and norverapamil have differing negative inotropic, chronotropic, and dromotropic activities and differing effects on vascular smooth muscles; the S-enantiomers having greater activity. It is hypothesized that the R/S concentration ratio of verapamil enantiomers may be input-rate dependent. The pharmacokinetics of verapamil and norverapamil enantiomers were studied in 11 young, healthy male and female volunteers after oral administration of 80 mg immediate-release (IR) verapamil every 8 hours, and a 240 mg dose once daily of controlled-release (CR) formulation on two separate occasions. Both dosage regimens were continued for 1 week with a minimum 1-week period between the two drug treatments. After the last dose of each regimen, plasma samples were collected over the period corresponding to the dosing interval. Enantiomer concentrations were determined using a microwave-facilitated precolumn derivatization with high performance liquid chromatographic quantification. Stereospecific assay revealed that: (1) stereoselective R- and S-enantiomer disposition occurred regardless of formulation administered; (2) a trend of R:S concentration ratios of verapamil differed between the two formulations; and (3) fluctuations between Cmax and Cmin values of the two formulations were statistically different over respective dosing intervals (greater fluctuation after CR administration). Using nonstereospecific data analyses, however, the pharmacokinetic parameters for verapamil and norverapamil were similar for both formulations over a 24-hour period. We suggest that kinetic differences can be attributed to differences in release rates of drug from the tablet matrices. The relative bioavailabilities of verapamil and norverapamil from the two products may, therefore, be subject to input rate-dependent processes. PMID- 8626882 TI - The effect of food, time of dosing, and body position on the pharmacokinetics and pharmacodynamics of verapamil and norverapamil. AB - To evaluate the influence of food, time of dosing, and body position on the steady-state pharmacokinetics of an osmotically controlled formulation of verapamil (COER-verapamil), each of 29 healthy men received one tablet a day at specified times in an open-label, multiple-dose, four-period, crossover study. The verapamil tablets were administered in a randomized, balanced, crossover design: 240 mg at 8:00 AM on an empty stomach, subjects remaining ambulatory; 240 mg at 8:00 AM on an empty stomach, subjects remaining supine for 8 hours; 240 mg at 10:00 PM with a standardized meal, subjects remaining supine for 8 hours; and 240 mg at 10:00 PM on an empty stomach, subjects remaining supine for 8 hours. Plasma verapamil concentrations were measured at steady state over the dosing interval. Steady-state plasma concentrations were achieved by the fourth administration of the drug. Neither food nor posture had any effect on the pharmacokinetics of verapamil or norverapamil, or on hemodynamic measurements. Time of dosing did affect the rate of appearance and elimination of verapamil, but had no effect on the extent of verapamil absorption, norverapamil appearance, or hemodynamic measurements. PMID- 8626883 TI - Kidney function and age are both predictors of pharmacokinetics of metformin. AB - The effects of renal impairment and age on the pharmacokinetics of metformin were evaluated. The subjects, including 6 young, 12 elderly, and 3 middle-age healthy adults and 15 adults with various degrees of chronic renal impairment (CRI) each were given a single, 850-mg metformin HCl tablet. Multiple whole blood, plasma, and urine samples were collected and analyzed for metformin levels using a high performance liquid chromatography (HPLC) method. In healthy elderly individuals, the plasma and whole blood clearance/absolute bioavailability values [CL/F and (CL/F)b], and corresponding renal clearance values (CLR and CLR,b) of metformin were 35-40% lower than the respective values in healthy young individuals. These two groups did not differ significantly with respect to volume of distribution (Vd), time to peak concentration (tmax), and parameters related to metformin's appearance in the urine. In the moderate and severe CRI groups, all clearance values were 74-78% lower than in the healthy young/middle-age group, and all other evaluable pharmacokinetic parameters (with the exception of tmax) differed significantly in this group. In the mild CRI group, clearance values of metformin, which were 23-33% lower than in the young/middle-age group, were the only parameters that differed significantly. Based on a regression analysis of the combined data, both creatinine clearance (CL*cr; corrected for body surface area) and age are predictors of metformin clearance, with the following model best fitting the data: CL/F [or (CL/F)b, CLR, CLR,b] = alpha + beta.CL*cr + gamma.CL*cr.age. Metformin should not be used in patients with moderate and severe CRI, or in patients with mild, but not absolutely stable, renal impairment. The initial and maximum doses in elderly patients and patients with stable mild CRI should be lowered to approximately one third that given to the general (i.e., patients without non-insulin-dependent diabetes) population. PMID- 8626884 TI - Review of limited systemic absorption of orlistat, a lipase inhibitor, in healthy human volunteers. AB - Orlistat, a lipase inhibitor, acts locally in the gastrointestinal tract. Systemic absorption is not required for its efficacy, but knowledge of the extent of its systemic absorption is important for its safe use in obese patients, the intended target population. Pharmacokinetic screening was carried out by monitoring plasma concentrations of unchanged orlistat in 25 phase 1 studies (including two mass balance studies) in normal and obese healthy volunteers. The results of these studies indicate an extremely low degree of systemic absorption for orlistat when administered with a hypocaloric, well-balanced diet with 20% to 30% of calories derived from fat (50-80 gm). To further characterize the pharmacokinetics and excretion pathways of orlistat, two mass balance studies using 14C-labeled orlistat were conducted. After oral dosing of radiolabeled orlistat with a fatty meal (28-30 gm fat), almost the entire dose was recovered from fecal samples; little was found in plasma and urine. It is concluded that systemic absorption of orlistat is negligible; at a clinically efficacious dose level, orlistat is unlikely to produce systemic lipase inhibition. PMID- 8626885 TI - Pharmacokinetic and pharmacodynamic evaluation during coadministration of nefazodone and propranolol in healthy men. AB - Potential interactions between nefazodone (200 mg every 12 hours) and propranolol (40 mg every 12 hours) were assessed in 18 healthy male volunteers in an open label, randomized, three-way crossover study. The nature, frequency, and severity of adverse events during coadministration of nefazodone and propranolol were similar to those observed with either treatment alone. There were no clinically significant effects on vital signs, electrocardiographic results, or laboratory parameters. With coadministration, the maximum peak concentration (Cmax) and area under the concentration-time curve over the dosing interval (AUC tau) of propranolol decreased 29% and 14%, respectively; Cmax and AUC tau of 4-hydroxy propranolol decreased 15% and 21%, respectively. Despite decreased plasma concentrations of the beta-antagonists, the reduction in exercise-induced tachycardia and post-exercise double product was slightly greater with coadministration than with propranolol alone. Administration of nefazodone alone did not significantly affect either pharmacologic parameter. The pharmacokinetics of nefazodone and its metabolites were largely unaffected during coadministration. Coadministration of propranolol and nefazodone results in modest pharmacokinetic inequivalencies, but no clinically significant alterations of the pharmacodynamics of propranolol. PMID- 8626886 TI - Mosaic pattern of hepatocellular carcinoma: histologic basis for a characteristic CT appearance. AB - PURPOSE: The mosaic pattern is a characteristic CT appearance for hepatocellular carcinoma (HCC). This study was designed to assess the tissue composition responsible for the CT mosaic pattern. METHOD: Gross and whole-mount histologic sections of 10 HCC tumors from eight patients were prepared at identical levels as preoperative CT sections. CT features of the mosaic tumor pattern were spatially registered with the corresponding pathologic sections. RESULTS: CT of mosaic HCC demonstrated enhancing nodules (9/10), low attenuation areas (9/10), and internal septa (3/10). Spatial registration of CT and microscopic sections showed that enhancing tissue was viable tumor in nine of nine. Low attenuation areas were either necrotic (4/9) or of mixed tissue (5/9), including areas of necrosis, fibrosis, and hemorrhage. CONCLUSION: The variable tissue composition of HCC accounts for the mosaic CT pattern. In most patients, enhancing nodules indicate viable tumor cells, and low attenuation areas represent necrosis, fibrosis, or hemorrhage. PMID- 8626887 TI - Hepatic perfusion abnormalities during treatment with hepatic arterial infusion chemotherapy: value of CT arteriography using an implantable port system. AB - PURPOSE: The purpose of this study was to evaluate CT arteriography (CTA) using an implantable port system in the detection of perfusion abnormalities occurring during hepatic arterial infusion chemotherapy (HAIC). METHOD: In 51 patients with unresectable primary and metastatic liver tumors, who had implanted port systems for HAIC, CTA examinations through the infusion pump were performed. When perfusion abnormalities were found, selective angiography and/or digital subtraction angiography using the implantable port system were performed to determine the etiology. RESULTS: Forty-nine perfusion abnormalities were detected in 32 patients. Intrahepatic hypoperfusion was found in 24 cases. Of 11 patients in whom correction of the hypoperfusion was attempted, it was successful in 10. Of 13 patients in whom correction was not attempted, 6 patients showed progressive disease in nonperfused areas. Intrahepatic hyperperfusion was found in 14 cases, which showed no subsequent complication. Extrahepatic perfusion was found in 11 cases. CONCLUSION: We consider CTA to be useful in detecting perfusion abnormalities that may compromise HAIC. PMID- 8626888 TI - Differentiation between multiple liver hemangiomas and liver metastases of gastrinomas: value of enhanced MRI. AB - PURPOSE: Hepatic metastases of neuroendocrine tumors are known to mimic hemangiomas on nonenhanced SE MR sequences. The usefulness of MR examination with gadolinium injection to identify lesions was prospectively evaluated. METHOD: Nine patients with multiple liver metastases of gastrinomas were compared with six patients showing multiple liver hemangiomas. Patients underwent unenhanced T2 weighted SE, T1-weighted SE, and FLASH sequences, followed by enhanced sequential FLASH sequences and a 5 min delayed T1-weighted SE sequence. RESULTS: On T2 weighted SE sequence, all hemangiomas displayed the same typical morphology as a sharply defined, homogeneous, high signal intensity lesion, but this pattern was also observed for some or all of the lesions in seven of nine patients with gastrinoma metastases. Dynamic FLASH sequences were accurate for lesions larger than 2 cm, hemangiomas displaying a nodular peripheral enhancement with centripetal filling in, and metastases displaying either an initial homogeneous or a regular peripheral enhancement. Precise assessment of lesions smaller than 2 cm remained equivocal. Delayed T1-weighted. SE sequence (performed at least 5 min after Gd-chelate injection) was the most accurate technique to identify metastases by showing hypo- or isointensity signal, whereas all hemangiomas were hyperintense. CONCLUSION: Postcontrast delayed T1-weighted sequence is the primary technique to differentiate equivocal cases of hemangiomas from metastases of gastrinoma. PMID- 8626889 TI - Focal hepatic fatty infiltration in the posterior edge of the medial segment associated with aberrant gastric venous drainage: CT, US, and MR findings. AB - OBJECTIVE: The purpose of this study is to demonstrate the relation between focal hepatic fatty infiltration and aberrant gastric venous drainage (AGVD) in the posterior edge of the medial segment (PEMS) of the liver. MATERIALS AND METHODS: We present two cases of focal hepatic fatty infiltration with AGVD in the PEMS and discuss their imaging features. RESULTS: In both cases the focal fatty infiltration areas were hyperechoic on sonography, hypodense on CT, and hyperintense on T1-weighted MRI. Computed tomography during arterial portography (CTAP) showed nodular perfusion defects corresponding to the areas in both cases, and early enhancement of the area was observed with dynamic MRI in one case. Although the findings on CTAP and dynamic MRI suggested a neoplastic nature for the lesions, focal fatty infiltration was confirmed with surgical resection in one case and with imaging follow-up in the other. Aberrant gastric venous drainage into the area was demonstrated on arteriography in both cases. CONCLUSION: The variation in blood supply caused by AGVD may play an important role in fatty metabolism in the PEMS of the liver and may influence imaging features. PMID- 8626890 TI - Focally spared area of fatty liver caused by arterioportal shunt. AB - We describe a case with a focally spared area in fatty liver caused by arterioportal shunt. Furthermore, we discuss the cause of the focally spared area related to a localized dilution or reduction in portal blood flow. PMID- 8626891 TI - FDG-PET evaluation of indeterminate pancreatic masses. AB - PURPOSE: The purpose of this study was to assess the ability of PET with 2 [18F]fluoro-2-deoxy-D-glucose (FDG) to differentiate benign from malignant pancreatic masses in patients with indeterminate findings on CT. METHOD: We performed FDG-PET on 12 patients with indeterminate mass lesions and 2 patients with CT findings typical for malignancy. Eight were found to have pancreatic carcinoma and six had benign lesions. The final diagnosis was histopathologically confirmed in all patients but two with a presumed diagnosis of focal pancreatitis based on stable clinical follow-up for at least 12 months. Lesion uptake of FDG was evaluated qualitatively and semiquantitatively by determination of the standardized uptake value (SUV) RESULTS: With use of a 2.5 cutoff value for SUV, all eight malignant and four of six benign lesions were correctly categorized. Qualitative evaluation gave the same results. The two false-positive lesions had elevated SUV values of 3.4 and 3.8, respectively. CONCLUSION: Our results indicate that FDG-PET has potential value for assessing patients with CT findings that are indeterminate for pancreatic carcinoma. FDG-PET may obviate invasive diagnostic procedures in many patients with benign disease. PMID- 8626892 TI - CT and US findings of pancreatoblastoma. AB - PURPOSE: Our goal was to evaluate US and CT findings of pancreatoblastoma. METHOD: Three US and four CT scans before surgery and one follow-up CT after surgery were reviewed in four patients (two female, two male) with pathologically proven pancreatoblastoma. The mean age of the patients was 4 years (range 2-5 years). The masses were analyzed for origin, US and CT architecture, presence of calcification, enhancement pattern, and metastatic spread. RESULTS: In two patients, the tumors arose from the pancreatic head and in one patient from the pancreatic tail. The mean largest diameter of the masses was 10 cm, ranging from 5 to 14 cm. Sonography obtained from three cases showed mixed echogenic solid mass. On the CT scan, all tumors were huge, lobulated masses with heterogeneous attenuation. One tumor contained numerous foci of calcification. On the enhanced CT scan, three tumors showed multiloculated appearance by enhancing internal septations. There was no evidence of metastasis to distant organ or abdominal lymph node in any case. CONCLUSION: We suggest that the most common US finding of pancreatoblastoma is a mixed echogenic, solid mass inseparable from the pancreas, and the most common CT finding is a relatively well defined, lobulated, huge mass with multiloculated appearance by enhancing septae in or near the lesser sac. PMID- 8626893 TI - Salmonella- and Shigella-induced ileitis: CT findings in four patients. AB - PURPOSE: The purpose of this study is to describe and illustrate the CT appearance of four cases of acute terminal ileitis induced by nontyphoidal Salmonella and Shigella infection and to review the radiographic and endoscopic findings of these entities. METHOD: The medical records, CT examinations, and small bowel examinations of three patients with Salmonella ileitis and one patient with Shigella ileitis were retrospectively reviewed. CT examinations were done in four patients, colonoscopy in three patients, and small bowel examinations in two patients. Stool cultures established the diagnosis of nontyphoidal Salmonella enteritis in three patients and Shigella enteritis in one patient. The patients' symptoms and clinical findings resolved promptly following supportive therapy and appropriate antibiotic therapy. RESULTS: CT showed slight circumferential and homogeneous thickening of the terminal ileum over a segment of 10-15 cm in patients with Salmonella ileitis. Associated mild thickening of the wall of the colon was present in addition. Small bowel examination performed in one patient revealed a spastic terminal ileum with thickened mucosal folds. Colonoscopy revealed acute colitis involving the colon diffusely in one case, but sparing the distal 50 cm of the colon in one case. CT showed more pronounced thickening of the terminal ileum and a target configuration in the patient with Shigella ileitis. Small bowel examination revealed narrowing, irregular contour, several large nodular defects (thumbprinting), and a severely ulcerated mucosa affecting the terminal ileum. Colonoscopy revealed a normal colon and large ulcerations with fibropurulent exudate in the terminal ileum. CONCLUSION: In patients with severe Salmonella or Shigella infections or persistent and/or confusing clinical presentations, CT can play a complementary but important role in the initial diagnostic evaluation. It avoids clinical mismanagement, circumvents unnecessary invasive procedures, and contributes to the efficient workup and therapy in this group of individuals. PMID- 8626894 TI - Delayed enhancement of the bowel wall: a new CT sign of small bowel strangulation. AB - PURPOSE: To report a new CT sign of small bowel strangulation. MATERIALS AND METHODS: Two cases of small bowel obstruction evaluated by spiral CT with intravenous contrast injection. RESULTS: In both cases we observed an absence of parietal enhancement of the diseased bowel loop, visible on early scans only. This feature was accompanied by signs of bowel obstruction. The two patients had a segment of necrotic ileum resected. CONCLUSION: Delayed enhancement of a bowel loop in cases of small bowel obstruction should suggest the diagnosis of strangulation and lead to rapid surgical treatment. PMID- 8626895 TI - Case report. CT and ultrasound of gastric and duodenal duplications. AB - We present the radiological findings of gastric and duodenal duplications in four adults, in whom abdominal ultrasound, endoscopic ultrasound (EUS), and CT were primarily used for diagnosis. The diagnosis was surgically confirmed in all cases. Preoperative diagnosis of duplications was possible with ultrasound in three patients, in whom CT showed a nonspecific cystic structure. Ultrasound demonstrated a pathognomonic multilayered wall appearance suggestive of a digestive origin, including an echogenic inner mucosal layer and a hypoechoic muscular layer, better appreciated using EUS in one patient. In one case, digestive origin was confirmed by direct visualization of a peristaltic activity within the cystic wall after water ingestion. In the last patient, a non-specific heterogeneous mainly solid mass of the esophagogastric junction was found to be an adenocarcinoma arising from a duplication on the histological analysis of the surgical specimen. PMID- 8626896 TI - Imaging findings of mucinous type of cholangiocellular carcinoma. AB - OBJECTIVE: To demonstrate the imaging findings seen in two cases of mucinous type of cholangiocellular carcinoma of the liver. MATERIALS AND METHODS: The CT and MR findings of two patients with pathologically proven mucinous type of cholangiocellular carcinoma were analyzed. RESULTS: One of them showed calcification, both cases had no enhancing on the arterial dominant phase of dynamic CT, but showed enhancement in the periphery and internal septations on the equilibrium phase. The internal signal intensity was homogenous, and extremely hypo- and hyperintense on T1- and T2-weighted images, respectively. CONCLUSION: Imaging findings were unique reflecting the characteristic pathological features that cancer cell nests are suspended in a large mucinous lake, and the specific diagnosis was considered to be possible by the integrated imaging diagnosis. PMID- 8626897 TI - Case report. Small bowel obstruction due to phytobezoar formation within Meckel diverticulum: CT findings. AB - Intestinal obstruction due to a phytobezoar within a Meckel diverticulum is exceedingly rare, with only seven reported cases in the surgical literature. The most important precipitating factor is the ingestion of agents high in fiber and cellulose. Small bowel obstruction in all but one case was due to retrograde propagation of the bezoar into the small bowel lumen. We report the clinical and CT findings in such a patient following a vegetarian diet. PMID- 8626898 TI - CT and MR findings of Krukenberg tumors: comparison with primary ovarian tumors. AB - PURPOSE: The purposes of this study were to evaluate the CT and MR findings of Krukenberg tumors and to compare them with those of primary ovarian tumors. METHOD: This study included 20 patients with Krukenberg tumors and 65 patients with various primary ovarian tumors. CT/MR/both imaging studies were available in 15/1/4 patients with Krukenberg tumor and 31/10/24 patients with primary ovarian tumors, respectively. Imaging findings of the tumors were categorized into three subgroups: a solid mass with intratumoral cysts, a solid mass without intratumoral cysts, and a predominantly cystic mass. RESULTS: Among 32 Krukenberg tumors (bilateral in 12 patients), 22 were solid masses with intratumoral cysts, in 14 of which the wall of the intratumoral cysts showed apparently strong contrast enhancement on CT and/or MRI. Six Krukenberg tumors were solid masses without intratumoral cysts, and four were predominantly cystic masses. Imaging findings of 88 primary ovarian tumors (bilateral in 23 patients) were 5 solid masses with intratumoral cysts, 27 solid masses without intratumoral cysts, and 56 predominantly cystic masses. None of the five primary ovarian tumors with solid mass with intratumoral cysts demonstrated apparently strong contrast enhancement of the cyst wall. CONCLUSION: Krukenberg tumor should be suspected when one sees solid ovarian tumors containing well demarcated intratumoral cystic lesions, especially if the walls of those cysts demonstrate apparently strong contrast enhancement. PMID- 8626899 TI - Computed tomographic findings of Bellini duct carcinoma of the kidney. AB - OBJECTIVE: To analyze CT findings of Bellini duct carcinoma, a rare variant of renal cell carcinoma. MATERIALS AND METHODS: The CT findings of five cases of Bellini duct carcinoma were reviewed and the findings were recorded. RESULTS: In all cases the affected kidneys maintained the normal outer contours. In four cases the renal masses protruded into the central sinuses. Contrast enhancement was minimal in four cases. CONCLUSION: Bellini duct carcinoma should be suspected in cases with these CT findings. PMID- 8626900 TI - Case Report. CT and US features of renal matrix stones with calcified center. AB - We report imaging findings of renal matrix stones in a patient with congenital ureteropelvic junction obstruction associated with urinary tract infection. The stones were composed of a calcified center and a non-calcified peripheral matrix that was slightly high attenuated on CT and nonshadowing on US. PMID- 8626901 TI - Case report. Adrenocortical oncocytoma: CT and MRI findings. PMID- 8626902 TI - Case report. Inflammatory pseudotumor in the retrorectal space: CT and MR appearance. AB - We report a case of an inflammatory pseudotumor of the retrorectal space, with CT and MRI findings. The imaging findings in this case are consistent with those reported from other anatomic sites, demonstrating the aggressive nature of this histologically benign but frequently invasive mass. Recent discussions in the literature advise close imaging follow-up after surgical excision, particularly in cases in which incomplete resection is deemed preferable to a more mutilating radical resection. PMID- 8626903 TI - Case report. Antenatal diagnosis of chorioangioma of the placenta: MR features. AB - We report a case of chorioangioma of the placenta, in which MR findings were useful in establishing the antenatal diagnosis. Polyhydramnios and a placental tumor that was 5 cm in size were visualized. The tumor had relatively high signal intensities on proton density imaging and T2-weighted imaging and showed partially high intensity signal rims on T1-weighted imaging, especially when using a breath-holding technique. Magnetic resonance imaging has an important role in detection and diagnosis of these lesions, particularly the larger tumors, so that appropriate steps can be taken to deal with the complications that may accompany this tumor. PMID- 8626904 TI - MR of Toxoplasma encephalitis: signal characteristics on T2-weighted images and pathologic correlation. AB - PURPOSE: Our goal was to determine if there are any T2-weighted MR signal characteristics of Toxoplasma encephalitis that might be useful in diagnosis and/or in gauging the effectiveness of medical therapy. METHOD: We retrospectively analyzed the MR, CT, thallium-201 SPECT brain scans, and medical records of 27 patients with medically proven (26) and biopsy proven (1) Toxoplasma encephalitis, supplemented by autopsy findings in 4 additional patients, 2 of whom had postmortem MR correlation. The neuropathologic literature was also reviewed. RESULTS: Among the 27 patients, we discovered three distinct imaging patterns. Ten (37%) patients had predominantly T2-weighted hyperintense lesions and had been on medical therapy an average of 3 days (excluding one outlier). Ten (37%) patients had T2-weighted isointense lesions and had received medical therapy an average of 61 days. Seven (26%) patients had lesions with mixed signal on T2-weighted images and had been on treatment an average of 6 days. Analysis of autopsy material from the four additional patients revealed the presence of organizing abscesses in three and necrotizing encephalitis in one, while the patient who had a brain biopsy demonstrated both types of pathologic lesions. In both cases having postmortem MRI, organizing abscesses appeared isointense to hypointense on T2-weighted images. CONCLUSION: There is a definite variation in the appearance of lesions of Toxoplasma encephalitis on T2-weighted images that precludes a definitive diagnosis based on signal characteristics alone. Pathologically, our data suggest that T2-weighted hyperintensity correlates with necrotizing encephalitis and T2-weighted isointensity with organizing abscesses. Furthermore, in patients on medical therapy the T2-weighted MR appearance may be a transition from hyperintensity to isointensity as a function of a positive response to antibiotic treatment, indicating that the signal change might be used to gauge the effectiveness of medical therapy. PMID- 8626905 TI - MR-PET image coregistration for quantitation of striatal dopamine D2 receptors. AB - PURPOSE: Our goal was to assess the utility of MR-PET image coregistration to quantify dopamine D2 receptors in striatum. METHOD: Twenty-nine normal subjects were investigated with PET and [11C]raclopride and with MRI. D2 receptors were quantified using the ratio of the distribution volume in striatum to that in cerebellum. Measures obtained using regions selected directly from the PET images were compared with those obtained from MR images and then projected to coregistered PET images. RESULTS: There were no differences between measures selected from the PET images (3.9 +/- 0.5) and those from the MR images (3.9 +/- 0.65). The values for these two measures were significantly correlated and corresponded to r = 0.9, p < 0.0001. CONCLUSION: Regions of interest selected directly from PET images, where there is a large contrast between the region of interest and background, as for the case of dopamine D2 ligands, are almost identical to those obtained from coregistered MR images. PMID- 8626906 TI - Detection of CSF flow in the ventriculo-Peritoneal shunt using MRI. AB - OBJECTIVE: A noninvasive test for shunt function would be beneficial in the management of patients with CSF shunting. We present our method to diagnose shunt function using MRI. MATERIALS AND METHODS: We performed two experiments. In the first we simultaneously performed MRI of two tubes, one with water flowing inside at various flow speeds and the other with static water inside, and compared the signal intensity inside each lumen. In the second we took four MR images of the actual shunt tubes in three patients at different states of shunt function, each having a tube containing static water pasted side by side with the actual subcutaneous shunt tube. RESULTS: In the first experiment we could detect the enhancement effect of slow flow. The minimal detectable flow speed using this method was approximately 1.7 ml/h. The result obtained in actual patients matched the clinical status of the shunt function. CONCLUSION: This method would be useful as a qualitative test for shunt function. PMID- 8626907 TI - Multiple sclerosis of the spinal cord: magnetic resonance appearance. AB - OBJECTIVE: To determine the MR appearance of spinal cord multiple sclerosis (MS) plaques in patients presenting with myelopathy by using a high-field (1.5 T) imager. MATERIALS AND METHODS: We studied 119 patients who underwent high-field (1.5 T) MR studies of the spinal cord for evaluation of myelopathy. All 119 patients were thought to have possible findings of spinal cord MS at the time of the MRI interpretation. RESULTS: Sixty-four plaques were studied in 47 patients with clinically definite MS and adequate quality MRI. Of these patients 68% had a single spinal cord plaque, 19% had two plaques, and 13% had three or more plaques. Sixty-two percent of the plaques occurred in the cervical spinal cord and most frequently involved the posterior (41%) and lateral (25%) aspects of the spinal cord. None of the 64 lesions involved the entire thickness of the spinal cord. The lesion length varied from 2 to 60 mm, with 84% of the lesions < 15 mm in length. The spinal cord diameter was unchanged in 84% of plaques, enlarged at the level of the lesion in 14%, and atrophic in 2%. Just over half (55%) of the plaques enhanced with intravenously administered gadolinium. Of the patients who received synchronous head and spinal cord examinations on the same day, 24% had normal findings on the MR study of the head. Follow-up spinal cord studies were available in nine patients. New lesions developed in two patients, while previously described lesions resolved. In three patients only new lesions developed. In four patients no change occurred in the existing number of cord plaques. CONCLUSION: Spinal cord demyelinating plaques present as well circumscribed foci of increased T2 signal that asymmetrically involve the spinal cord parenchyma. Knowledge of their usual appearance may prevent unnecessary biopsy. An MR examination of the head may confirm the imaging suggestion of spinal cord demyelinating disease, because up to 76% of patients have abnormal intracranial findings. In the remaining 24% of cases in which the clinical diagnosis is not certain and MR findings in the head are negative, a follow-up spinal cord study is recommended, because these lesions evolve and change over time. PMID- 8626908 TI - Lingual mandibular bony defects: CT in the buccolingual plane. AB - OBJECTIVE: Our goal was to record the appearance of lingular mandibular bony defects (LMBD) on CT imaging of the mandible in the buccolingual plane. MATERIALS AND METHODS: During the CT evaluation of patients planning to undergo dental implant surgery, five cases of LMBD were found. Axial 1.2 x 1.00 mm overlapping CT sections of the mandible and the maxilla were obtained. Then with use of specific software (DentaCT; Elscint), panoramic and cross-sectional (buccolingual) images of the mandible and maxilla were reformatted. RESULTS: Five cases of posterior LMBD were identified; one patient had both a posterior as well as the much rarer anterior LMBD. All cases were incidental findings and all were asymptomatic. CT features of LMBD were displayed in axial, panoramic, and buccolingual planes. The characteristic opening of the bony defect in the lingual aspect of the mandible was clearly displayed on the axial as well as the buccolingual images; however, this key feature was not manifest on the panoramic images of the mandible. CONCLUSION: CT features of LMBD in the buccolingual plane are added to the known radiologic description of this entity. PMID- 8626909 TI - Observations on two cases of apparent submandibular gland cysts in HIV positive patients: MR and CT findings. AB - OBJECTIVE: To present two cases of probable lymphoepithelial cysts of the submandibular glands in patients who were human immunodeficiency virus (HIV) positive and who also had lymphoepithelial cysts of the parotid glands. MATERIALS AND METHODS: Computed tomography and MRI of two HIV positive patients with lymphoepithelial cysts of the parotid glands and cysts in the submandibular glands were correlated with the histories and the possible presence of other known causes of submandibular gland multiple cysts. RESULTS: Because of the present treatment philosophy regarding HIV positive patients with major salivary gland cysts, surgical resection of these glands was not performed. All other known causes of multiple submandibular gland cysts were excluded by either history or laboratory data. CONCLUSION: Computed tomography and MRI on two patients with known HIV infection and bilateral parotid lymphoepithelial cysts are presented. Both patients also had bilateral multiple submandibular gland cysts and no evidence of obstructive glandular disease, autoimmune disease, or other organ system cysts. These cases of presumed submandibular gland lymphoepithelial cysts are rare in the literature. They are presented in the hope that other radiologists will be stimulated to document the occurrence of this entity. PMID- 8626910 TI - Pseudodynamic imaging of the temporomandibular joint: SE versus GE sequences. AB - Pseudodynamic MR imaging of the temporomandibular joints (TMJs) has been used for the evaluation of the functional aspects of the TMJs. To evaluate the value of T1 weighted spin-echo (SE) and gradient-echo (GE) techniques, both techniques were performed in 9 asymptomatic (mean 25.7 years, 22-23 years), and 25 symptomatic (mean 44.9 years, 20-71 years) subjects with signs and symptoms of internal derangement or osteoarthrosis of the TMJs. The imaging time for the SE (180 ms/15 ms/110 degrees; repetition time/echo time/flip angle) and GE (fast low angle shot; FLASH, 90 ms/12 ms/40 degrees) sequences was 27 and 28 s, respectively. In asymptomatic and symptomatic subjects, the confidence of the identification of the meniscal position was better on SE than GE images (3.6 +/- 0.6 vs. 2.9 +/- 0.9, p < 0.01, 3.2 +/- 0.8 vs. 2.8 +/- 0.8, p < 0.05), respectively and the sizes of the menisci were bigger on SE than GE images. The delineation of the condylar cortex was better on GE than SE images. For pseudodynamic imaging display of the TMJs, the SE images might be better than GE images to provide the stable recognition of the menisci. PMID- 8626911 TI - Ferric ammonium citrate-cellulose paste for opacification of the esophageal lumen on MRI. AB - OBJECTIVE: A new ferric ammonium citrate-cellulose mixture for use in MRI of the esophagus was evaluated for its ability to opacify the esophageal lumen. MATERIALS AND METHODS: Thirty-two patients with esophageal disorders and ten patients with normal esophagus undergoing MRI at 1.5 T were given approximately 100 ml of the newly developed high-viscosity esophageal contrast preparation. Moreover, six of the patients with esophageal cancer were subjected to a second examination after radiation therapy. A total of 48 MR imagings were performed. RESULTS: Of the patients examined, successful esophageal opacification, graded as excellent, was obtained in 84.2, 78.9, and 57.9%, of the sagittal, axial, and coronal images, respectively. In cases of extrinsic disease involving the esophagus the contrast medium administration allowed the easy differentiation of the esophagus from adjacent mass lesions and proved very useful in identifying displacement and compression. In cases of esophageal carcinoma the contrast medium administration assisted in the measurement of wall thickness and length of the lesion as well as in the identification of the site of origin of the tumor. CONCLUSION: The results indicate that this product effectively opacifies the esophageal lumen in the majority of patients. We found that it is easy to use, is well tolerated, and does not produce artifacts. PMID- 8626912 TI - High level cross of the esophagus with the descending aorta in scoliosis: CT study. AB - OBJECTIVE: The esophagus occasionally crosses the descending aorta at an unusually high level (3-5 cm inferior to the carina) in right-sided scoliosis. The purpose of this study was to analyze the mechanism of this finding. MATERIALS AND METHODS: We prospectively evaluated thoracic CT scans in 30 patients with right-sided scoliosis. We assessed the alterations in the positions of the esophagus and the descending aorta by the thoracic deformity. RESULTS: The descending aorta followed the scoliotic curve of the spine in 26 (87%) patients. The esophagus followed the scoliotic curve of the spine in 14 (47%) patients and did not in 16 (53%). The anteroposterior diameter of the thorax in the former group was significantly smaller than that in the latter (p < 0.01). High level cross of both structures was identified in 14 (47%) patients, and all of them belonged to the group in which the esophagus did not follow the scoliotic curve of the spine. CONCLUSION: The unusual high level cross of the esophagus with the descending aorta occasionally seen in scoliosis is due to a difference in the positional alterations of the two structures resulting from the scoliosis. PMID- 8626913 TI - Case report. Chemical-shift MRI of exogenous lipoid pneumonia. AB - Exogenous lipoid pneumonia results from the aspiration or inhalation of fatty substances, such as mineral oil found in laxatives or nasal medications containing liquid paraffin. We present standard and lipid-sensitive (Chemical shift) MR findings in a patient with histologically confirmed lipoid pneumonia. The loss of signal intensity in an area of airspace disease on opposed-phase imaging was considered specific for the presence of lipid. PMID- 8626914 TI - CT of disseminated plasmacytoma in an AIDS patient. AB - It is well known that acquired immunodeficiency syndrome (AIDS) is associated with increased risk of neoplasms, particularly Kaposi sarcoma and non-Hodgkin lymphoma. There have been several recent reports in the literature describing plasma cell tumors in AIDS patients. We report the imaging findings in a case of widely disseminated plasmacytoma in a patient with known AIDS. PMID- 8626915 TI - Hydatid disease of the chest wall. PMID- 8626917 TI - Case report. MR and CT appearance of cardiac hemangioma. AB - We present a case of cardiac hemangioma in a symptomatic patient. MR and CT each have specific characteristics that should make one consider including or excluding this in the differential diagnosis of a cardiac tumor. PMID- 8626916 TI - Measurement of myocardial wall thickening from PET/SPECT images: comparison of two methods. AB - PURPOSE: We compared two methods for measuring myocardial wall thickening from nuclear medicine perfusion scans. The first method uses the percent change in peak activity, and the second method models a profile measured across the myocardium. METHOD: Mathematical simulations of the myocardium were used. In addition, images with PET or SPECT resolution were created from real MR images. Known amounts of noise were then added. RESULTS: The percent peak thickening (% PT) is nonlinear with true percent thickening, especially for PET resolutions [7 mm full width at half-maximum (FWHM)]. For the peak method, low levels of noise (10%) introduced an error of 8%PT for PET and of 16%PT for SPECT. Additional smoothing reduced these errors. For the fitted model, at 10% noise, the error in thickening was large: 2.3 mm for PET and 7.8 mm for SPECT. CONCLUSION: The fitted model works well only with good resolution and low noise (e.g., 7 mm FWHM and 10%). The peak method is also sensitive to noise, especially for poorer resolutions. Additional smoothing gives more reliable results for the peak method but not the fitted method. The peak method is therefore the more generally reliable, but even this method may only allow classification of myocardial thickening into broad categories. PMID- 8626918 TI - Case report. Congenital left ventricular aneurysm diagnosed by spiral CT angiography. AB - We report a rare case of congenital left ventricular aneurysm, diagnosed by spiral CT angiography. Despite 1 s time acquisition, spiral CT, with adequate acquisition parameters and bolus injection of contrast medium, produced sufficiently good images to permit visualization of the aneurysm. Subsequently, reconstructions (shaded surface display and multiplanar reformation) were performed to demonstrate the relationship of the aneurysm with the remainder of the left ventricle, the wide neck of the aneurysm, and the absence of contractility, therein permitting differentiation from a congenital diverticulum. PMID- 8626919 TI - Pictorial essay. MRI of the glenoid labrum with gross anatomic correlation. AB - The objective of this pictorial essay is to illustrate the magnetic resonance image (MRI) appearance of the glenoid labrum and the perilabral structures with and without instillation of intraarticular contrast material. Ten cadaveric shoulder specimens underwent axial MRI using various MR pulse sequences. The shoulders then were transversely sectioned, and the gross morphology of the labrum and perilabral structures was evaluated and correlated with the MR images. The contrast-enhanced sequences allowed for improved anatomic visualization of the structures evaluated. PMID- 8626920 TI - Artifacts and thresholding in X-ray CT of a cortical bone and titanium composite. AB - PURPOSE: X-Ray CT has the potential to provide precise and accurate data from which the mechanical properties of bone can be calculated. Such data would be useful in understanding the response of bone tissue to implants. Various artifacts can, however, degrade accuracy of the data. The aim of this study was to measure the artifactual errors produced in CT of a simulated femoral bone titanium composite and to propose a method to correct for them. METHOD: A composite phantom that simulates cortical bone and a titanium implant was designed and constructed. The phantom was scanned and the image data were analyzed over a range of thresholds with image analysis software developed for this study. The outer (OD) and inner (ID) diameter and the CT number of the cortical bone, with and without titanium, were measured over a range of cortical thicknesses. RESULTS: While ID can be accurately measured by choosing the proper threshold (800 HU), OD, even at optimal threshold, will be underestimated by approximately 2%. If a proper threshold is selected, CT number can also be accurately determined. Errors of up to 5%, however, are produced by titanium unless corrected by proper threshold selection. CONCLUSION: Intramedullary titanium is not a deterrent to obtaining accurate measurements of cortical bone dimensions and properties. Proper choice of thresholds for image analysis of CT scan data can yield accuracy and precision of < or = 2%. PMID- 8626921 TI - Aunt Minnie's corner. Meniscal cyst. PMID- 8626922 TI - Keynote address: the health care environment. PMID- 8626923 TI - Development of the "Dean's Dilemma" case study. PMID- 8626924 TI - Projective analysis of the "Dean's Dilemma" case study. PMID- 8626925 TI - Case study reaction paper. PMID- 8626926 TI - Case study reaction paper. PMID- 8626927 TI - Case study reaction paper. PMID- 8626928 TI - Dean's forum: is managed care necessary for dental schools to survive? PMID- 8626929 TI - Purchasers and payers: who's driving the market I? PMID- 8626930 TI - Purchaser and payers: who's driving the market II? PMID- 8626931 TI - Information demands: the managed dental care industry's response. PMID- 8626932 TI - Lessons from medicine: an economist's perspective. PMID- 8626933 TI - The future of academic dentistry in a changing health care environment. PMID- 8626934 TI - Constructing successful managed care dental plans. PMID- 8626935 TI - An introduction to activity-based costing. PMID- 8626936 TI - Beginning to understand contract analysis. PMID- 8626937 TI - Reflections. PMID- 8626938 TI - A composite view of cardiac rupture in the United States National Registry of Myocardial Infarction. AB - OBJECTIVES: This study was done to determine the incidence, timing and prevalence as a cause of death from cardiac rupture in patients with acute myocardial infarction. BACKGROUND: Several clinical trials and overview analyses have suggested that the survival benefit conferred by thrombolytic therapy may be offset by a paradoxic increase in early deaths from cardiac rupture. METHODS: Demographic, procedural and outcome data from patients with acute myocardial infarction were collected at 1,073 United States hospitals collaborating in the United States National Registry of Myocardial Infarction. RESULTS: Among the 350,755 patients enrolled, 122,243 received thrombolytic therapy. In-hospital mortality for the overall patient population, those not treated with thrombolytics (n = 228,512) and those given thrombolytics were 10.4%, 12.9% and 5.9%, respectively (p<0.001). Cardiogenic shock was the most common cause of death in each patient group. Although the incidence of cardiac rupture was low (<1.0%), it was responsible for 7.3%, 6.1% and 12.1%, respectively, of in hospital deaths (p<0.001). Death from rupture occurred earlier in patients given thrombolytic therapy, with a clustering of events within 24 h of drug administration. Despite the early risk, death rates were comparatively low in thrombolytic-treated patients on each of the first 30 days. By multivariable analysis, thrombolytics, prior myocardial infarction, advancing age, female gender and intravenous beta-blocker use were independently associated with cardiac rupture. CONCLUSIONS: This large registry experience, including over 350,000 patients with myocardial infarction, suggests that thrombolytic therapy accelerates cardiac rupture, typically to within 24 to 48 h of treatment. The possibility that rupture represents an early hemorrhagic complication of thrombolytic therapy should be investigated. PMID- 8626939 TI - Late assessment of thrombolytic efficacy (LATE) study: prognosis in patients with non-Q wave myocardial infarction. (LATE Study Investigators) AB - OBJECTIVES: We examined the impact of thrombolytic therapy and the prognosis of patients with non-Q wave myocardial infarction in a randomized placebo-controlled trial known as the Late Assessment of Thrombolytic Efficacy (LATE) study. BACKGROUND: Patients with non-Q wave as compared with Q wave myocardial infarction in the era before thrombolytic therapy were traditionally thought to have a higher rate of reinfarction and death between hospital discharge and 1 year such that the overall prognosis for outcome at 1 year was similar in the two groups. METHODS: The study patients began treatment with either recombinant tissue-type plasminogen activator (rt-PA) or matching placebo, 6 to 24 h after the onset of chest pain. Post hoc analysis of mortality and reinfarction was carried out by comparing rt-PA and placebo in various subsets of patients based on the presenting electrocardiogram (ECG) and the evolution of the ECG with respect to the development of Q waves. RESULTS: Among 5,711 participants, 4,759 had a confirmed myocardial infarction, including 1,309 classified as having a non Q wave infarction at hospital discharge. Irrespective of treatment assignment, all patients with non-Q wave versus Q wave infarction had a lower 1-year mortality rate (13.3% vs. 17.1%, p = 0.001) and a similar 1-year reinfarction rate (8.6% vs. 7.9%, p = 0.7). Of the 4,759 patients with confirmed myocardial infarction, 2,973 presented with ST segment elevation or bundle branch block, 528 with ST depression and 1,258 with neither ST elevation nor depression. No overall benefit from rt-PA versus placebo with respect to mortality rate at 1 year was seen among patients presenting with ST elevation (21.2% vs. 22.4%, p = 0.5 [90% power to detect 20% relative difference]). Patients with ST elevation who were treated with rt-PA versus placebo <3 h after hospital admission had a lower mortality rate at 1 year (15.8% vs. 19.6%, p = 0.028) than did those treated after 3 h (17.6% vs. 13.0%, p = 0.055). Patients presenting initially with ST depression >2 mm had significant benefit from treatment with rt-PA with respect to 1 year mortality rate (20.1% vs. 31.9%, p = 0.006). CONCLUSIONS: Patients with non-Q wave myocardial infarction constitute a heterogeneous group of patients. Although the observations presented here are limited by post hoc analysis, it is apparent that patients classified as having a non-Q wave infarction after thrombolytic therapy have a better prognosis than do those given placebo. Late admission of thrombolytic therapy (after 6 h) may also be beneficial in patients presenting with ST depression >2 mm and confirmed myocardial infarction. These hypotheses require prospective testing in a larger number of patients. PMID- 8626940 TI - Non-Q wave and ST segment depression myocardial infarction: is there a role for thrombolytic therapy? PMID- 8626941 TI - Characteristics and mortality outcomes of thrombolysis trial participants and nonparticipants: a population-based comparison. AB - OBJECTIVES: This study was done to compare characteristics and outcomes of patients with acute myocardial infarction participating in two thrombolysis trials with those of nontrial patients at study hospitals and external hospitals. BACKGROUND: Preferential recruitment of lower risk patients into randomized trials of thrombolysis has been suggested by earlier studies. However, to date there has not been a definitive population-based comparison of characteristics and outcomes for thrombolysis trial participants and nonparticipants. METHODS: Population-based data on hospital admissions and mortality from acute myocardial infarction for all hospitals in Ontario from 1989 to 1992 were linked to data on trial participants in two distinct thrombolysis studies (GUSTO I and LATE). Included were 1,304 patients entered into GUSTO, 12,657 nonparticipants at GUSTO hospitals, 249 patients entered into LATE, 5,997 nonparticipants at LATE hospitals and 12,299 patients at external hospitals. The main outcomes were differences in age, gender, comorbidity scores, coronary revascularization and survival to hospital discharge. RESULTS: Patients in both GUSTO and LATE were significantly more likely to be <70 years old (odds ratio [OR] 2.8 and 3.2, respectively), to be male (OR 2.0 and 2.1, respectively), to have low comorbidity scores (OR 2.0 and 2.3, respectively) and, for GUSTO alone, to undergo coronary revascularization (OR 2.4). Nontrial patients were similar between trial hospitals and external hospitals. In-hospital mortality rates for GUSTO and LATE patients were lower (6.9% and 6.6%, respectively) than for nonparticipants at study hospitals (16.8% and 19.7%, respectively; p<0.001 for both comparisons). Survival to hospital discharge remained higher among GUSTO (OR 1.9) and LATE patients (OR 2.0) than nonparticipants at study hospitals even after adjustment for age, gender, revascularization and comorbidity scores. CONCLUSIONS: Compared with nontrial patients, thrombolysis trial participants are younger, more often male, undergo more revascularization and have less comorbid disease. Even after adjustment for these factors, participants have a survival advantage over nonparticipants that is larger than expected from thrombolysis alone. These findings are not attributable to inferior care or skewed populations at hospitals that did not join these major trials. Further study of these selection biases may guide future trial design and deepen our understanding of why thrombolytics have been underused for high risk patients in routine practice. PMID- 8626942 TI - Decreased prevalence of late potentials with mechanical versus thrombolysis induced reperfusion in acute myocardial infarction. AB - OBJECTIVES: We sought to evaluate the influence of the method used to achieve early coronary reperfusion (i.e., intravenous thrombolysis or percutaneous transluminal coronary angioplasty) on the prevalence of late potentials after acute myocardial infarction. BACKGROUND: After myocardial infarction, late potentials are associated with an increased risk of ventricular tachyarrhythmia and sudden death. Although their prevalence is lower in patients with coronary reperfusion, the influence of the method used to achieve reperfusion remains debated. METHODS: We retrospectively analyzed 109 patients with acute myocardial infarction who were treated within 6 h of symptom onset and had angiographically proved early reperfusion. A signal-averaged electrocardiogram was recorded > or = 5 days after infarction. RESULTS: Reperfusion was successfully achieved by intravenous thrombolysis alone in 37 patients (34%), by "rescue" coronary angioplasty in 26 (24%) and by primary angioplasty in 46 (42%). There was no significant difference between groups in terms of gender ratio, infarct location, time to admission or to reperfusion, peak creatine kinase value or left ventricular ejection fraction. The prevalence of late potentials was similar in the two groups in which patency was achieved by primary and rescue coronary angioplasty (17.4% and 7.7%, respectively [p=NS]) but higher in patients who had successful thrombolysis (35.1%, p < 0.05). Multivariate analysis showed that the use of thrombolysis instead of angioplasty as the reperfusion method was the only variable significantly associated with the presence of late potentials. CONCLUSION: This study suggests that after acute myocardial infarction the prevalence of late potentials is lower when reperfusion is achieved by angioplasty (either primary or as a rescue procedure after failed thrombolysis) than by thrombolysis. PMID- 8626943 TI - Optimal intensity of oral anticoagulant therapy after myocardial infarction. AB - OBJECTIVES: This study attempted to determine the optimal intensity of anticoagulant therapy in patients after myocardial infarction. BACKGROUND: Treatment with oral anticoagulant therapy entails a delicate balance between over (risk of bleeding) and under-anticoagulation (risk of thromboemboli). The optimal intensity required to prevent the occurrence of either event (bleeding or thromboembolic) is not known. METHODS: A method was used to determine the optimal intensity of anticoagulant therapy by calculating incidence rates for either event associated with a specific international normalized ratio. The numerator included events occurring at given international normalized ratios, and the denominator comprised the total observation time. RESULTS: The study population included 3,404 myocardial infarction patients enrolled in the ASPECT (Anticoagulants in the Secondary Prevention of Events in Coronary Thrombosis) trial. Total treatment was 6,918 patient-years. Major bleeding occurred in 57 patients (0.8/100 patient-years), and thromboembolic complications in 397 (5.7/100 patient-years). The incidence of the combined outcome (bleeding or thromboembolic complications) with international normalized ratio <2 was 8.0/100 patient-years (283 events in 3,559 patient-years), with international normalized ratios between 2 and 3, 3.9/100 patient-years (33 events in 838 patient-years); 3.2/100 patient-years (57 events in 1,775 patient-years) for international normalized ratios between 3 and 4; 6.6/100 patient-years (37 events in 564 patient-years) for international normalized ratios between 4 and 5; and 7.7/100 patient-years (14 events in 182 patient-years) for international normalized ratios >5. After adjustment for achieved international normalized ratio levels, significant predictors were higher levels of systolic blood pressure and age. CONCLUSIONS: If equal weight is given to hemorrhagic and thromboembolic complications, these results suggest that the optimal intensity of long-term anticoagulant therapy for myocardial infarction patients lies between 2.0 and 4.0 international normalized ratio, with a trend to suggest an optimal intensity of 3.0 to 4.0. PMID- 8626944 TI - Coronary artery bypass grafting without cardiopulmonary bypass and without interruption of native coronary flow using a novel anastomosis site restraining device ("Octopus"). AB - OBJECTIVE: This study assessed the feasibility of coronary artery bypass grafting on the beating heart without interruption of native coronary blood flow using a novel anastomosis site restraining device. BACKGROUND: Recently, an end-to-side bypass technique was described that does not require interruption of flow in the recipient artery. METHODS: By means of a suction device ("Octopus"), in 31 pigs the epicardium was grasped and immobilized through an arm contraption fixed to the operating table. In the first 15 consecutive pigs (study I), the two dimensional motion of an epicardial beacon was monitored. In 16 subsequent pigs (study II), an internal mammary artery was grafted under the microscope in two steps to a proximal coronary artery segment, without cardiopulmonary bypass. First, the internal mammary artery was sutured end-to-side to the outside of the coronary artery. Secondly, an orifice was punched in the partitioning coronary wall by an excimer laser catheter introduced through a temporary side-branch of the internal mammary artery. RESULTS: Study II: During 43 suction periods in four anastomosis areas, immobilization was achieved for 15 to 169 min (>30 h in total) in 13 open- and 9 closed-chest procedures without hemodynamic deterioration. The area circumscribed by the edges of the beacon trajectory (area in which the anastomosis is to be tracked) was reduced from 73.0 +/- 43.0 mm(2) (mean +/- SD) to 1.3 +/- 0.5 mm(2) (p<0.001) in the open-chest and to 0.2 +/- 0.2 mm(2) in the closed-chest procedure. At 6 weeks, no myocardial or coronary suction lesions were found. Study II: Nonocclusive anastomosis surgery required 25 +/- 3 min. No leakage, serious arrhythmias, graft closure or hemodynamic deterioration occurred during the procedure or for 2 h after ligating the coronary artery proximally. At 6 weeks, all seven grafts were patent. CONCLUSIONS: Coronary bypass on the beating heart without interruption of coronary flow is feasible. In both open- and in closed-chest procedures, the "Octopus" reduced anastomosis site motion to about 1 X 1 mm without adverse consequences. PMID- 8626945 TI - Managed delay for coronary artery bypass graft surgery: the experience at one Canadian center. AB - OBJECTIVES: This study sought to assess the impact of delaying coronary artery bypass surgery at one Canadian academic tertiary referral center. BACKGROUND: Universal access to medical services in Canada comes at the expense of waiting lists whose impact has been incompletely assessed. METHODS: A prospective, observational study of all residents of Nova Scotia and Prince Edward Island accepted for bypass surgery between 1 April 1992 and 31 October 1992 was undertaken to determine 1) whether triage guidelines were being followed; and 2) the incidence of cardiac death, nonfatal myocardial infarction and worsening symptoms associated with delayed operation. The analysis had 90% power to detect a mortality rate of > or = 3% (alpha 0.05). RESULTS: Of 423 patients referred, 35% were triaged as urgent, 9.7% as semiurgent A, 39% as semiurgent B and 16.3% as elective, with no age or gender bias identified. Operation occurred at < or = 1 week in 25%, < or = 1 month in 47%, and >6 months in 1.4%. There were no nonfatal myocardial infarctions, but five cardiac deaths occurred (1.2%). Of 275 patients not initially classified as urgent, 12.4% required reclassification to higher priorities because of worsening symptoms; none had perioperative myocardial infarction or died. One in four patients queued longer than target waiting times. Only 4% of patients considered prioritization on the basis of medical need unfair, but 64% experienced at least moderate anxiety. CONCLUSION: This triage system equitably stratified patients to a queue. Deaths were rare and could not be attributed to the triage process. Patients with worsening clinical status were safely accommodated with earlier waiting times, but concerns remain regarding excessive waiting times and patient anxiety. PMID- 8626946 TI - Repeated coronary artery occlusions during routine balloon angioplasty do not induce myocardial preconditioning in humans. AB - OBJECTIVES: The purpose of the present study was to assess whether brief, repeated coronary artery occlusions during balloon angioplasty induce a myocardial ischemic protective effect. BACKGROUND: In animals, brief coronary artery occlusions preceding a more prolonged occlusion result in reduced infarct size. Whether myocardial protection against ischemia could also occur in humans during angioplasty remains controversial. METHODS: Thirteen patients with a proximal left anterior descending coronary artery stenosis with no angiographic collateral circulation underwent percutaneous transluminal coronary artery balloon angioplasty. Three 120-s balloon inflations separated by a 5-min equilibration period were performed. For each inflation, intracoronary ST segment modifications, septal wall thickening (M-mode echocardiography), left ventricular pressures and time derivatives were measured at baseline and at 30, 60 and 90 s after balloon inflation and 120 s after balloon deflation. RESULTS: Intracoronary electrocardiographic analysis showed that the time course of the maximal ST segment elevation was identical at each inflation, as were wall motion changes assessed by the decrease in septal wall thickening. For the first and last inflations, peak positive dP/dt decreased significantly by 13 +/- 9% (mean +/- SD) and 14 +/- 13%, whereas peak negative dP/dt increased by 23 +/- 15% and 22 +/ 10%, respectively (all p < 0.01 from baseline values). The relaxation time constant, tau, was altered similarly during the different inflations, from 44 +/- 6 to 74 +/- 13 ms and from 57 +/- 13 to 77 +/- 13 ms (all p < 0.001) for the first and last inflations, respectively. Left ventricular end-diastolic pressure increased to the same level after each inflation. In contrast to other hemodynamic variables, tau and left ventricular end-diastolic pressure did not return to baseline values in between the inflations, which may be due to myocardial stunning. CONCLUSIONS: In patients with proximal left anterior descending coronary artery stenosis and no evidence of collateral circulation, brief periods of ischemia, such as those used during routine coronary balloon angioplasty, do not provide any protection against myocardial ischemia. PMID- 8626948 TI - Another step toward resolving the contrast controversy. PMID- 8626947 TI - A randomized trial of low osmolar ionic versus nonionic contrast media in patients with myocardial infarction or unstable angina undergoing percutaneous transluminal coronary angioplasty. AB - OBJECTIVES: The purpose of this study was to determine prospectively whether the differences in anticoagulant and antiplatelet effects of ionic and nonionic contrast media after angiographic or clinical outcomes in patients with unstable ischemic syndromes undergoing percutaneous transluminal coronary angioplasty. BACKGROUND: The interaction of platelets and thrombin with the endothelium of injured vessels contributes to thrombosis and restenosis after coronary angioplasty. Case reports and retrospective observations have reported an increased risk of thrombosis with the use of nonionic contrast media. METHODS: A total of 211 patients with acute myocardial infarction or unstable angina undergoing coronary angioplasty were randomized to receive nonionic or ionic low osmolar contrast media. Coronary angiograms were assessed by a technician blinded to the study contrast media, and clinical events were monitored by an independent nurse for 1 month. RESULTS: Patients receiving the ionic media were significantly less likely to experience decreased blood flow during the procedure (8.1% vs. 17.8%, p = 0.04). After the angioplasty, residual stenosis, vessel patency, the incidence of moderate to large thrombi and use of adjunctive thrombolytic therapy were similar between the two groups. However, patients receiving ionic media had fewer recurrent ischemic events requiring repeat catheterization (3.0% vs. 11.4%, p = 0.02) and repeat angioplasty during the initial hospital stay (1.0% vs. 5.8%, p = 0.06). One month after angioplasty, patients receiving ionic contrast media reported significantly fewer symptoms of any angina (8.5 vs. 20.0%, p = 0.04) or of angina at rest (1.4% vs. 11.8%, p = 0.01) and a reduced need for subsequent bypass surgery (0% vs. 5.9%, p = 0.04), compared with patients receiving the nonionic media. CONCLUSIONS: These findings demonstrate that in patients with unstable ischemic syndromes undergoing coronary angioplasty, the use of ionic low osmolar contrast media reduces the risk of ischemic complications acutely and at 1 month after the procedure. Therefore, low osmolar ionic contrast media should be strongly considered when performing interventions in patients with unstable angina or myocardial infarction. PMID- 8626949 TI - Mechanisms and immediate and long-term results of adjunct directional coronary atherectomy after rotational atherectomy. AB - OBJECTIVES: The purpose of this study was to confirm the mechanisms and the immediate and long-term results of rotational atherectomy and adjunct directional coronary atherectomy. BACKGROUND: Rotational atherectomy is best suited for treating calcific stenoses, but the ability of rotational atherectomy alone to optimize lumen dimensions in large vessels is limited; this is only partly improved by adjunct balloon angioplasty. METHODS: We treated 165 lesions in 163 patients by use of rotational atherectomy and adjunct directional coronary atherectomy. Quantitative angiography and intravascular ultrasound were used for lesion analysis. A matched comparison with 208 lesions treated with rotational atherectomy and adjunct coronary angioplasty was performed. Patients were then followed up for at least 9 months, and target-lesion revascularization was assessed. RESULTS: In the 61 lesions imaged sequentially, lumen area increased from 1.7 +/- 0.8 (mean +/- 1 SD) to 3.9 +/- 1.1 mm(2) after rotational atherectomy, owing to a decrease in plaque plus media area from 16.8 +/- 5.0 to 15.2 +/- 5.2 mm(2) (both p < 0.0001). After adjunct directional coronary atherectomy, lumen area increased even more to 6.7 +/- 2.0 mm(2) (vs. 5.1 +/- 1.4 mm(2) after adjunct coronary angioplasty, p < 0.0001) as a result of both vessel expansion (18.8 +/ 5.3 to 20.8 +/- 5.7 mm(2)) and additional plaque removal (to 14.1 +/- 5.0 mm(2), all p < 0.0001). The total arcs of calcium decreased from 207 +/- 107 degrees to 166 +/- 93 degrees after rotational atherectomy and to 145 +/- 87 degrees after directional coronary atherectomy. Overall, procedural success was 96%, and final diameter stenosis was 15 +/- 17%. Target-lesion revascularization was 23%. The only independent predictor of target-lesion revascularization was a larger overall atherectomy index (84% vs. 59%, p = 0.048). CONCLUSIONS: There is a synergistic relationship between rotational atherectomy and directional coronary atherectomy in the treatment of calcific lesions. The immediate results show a high procedural success--lumen dimensions were larger and late target-lesion revascularization was lower in lesions treated with rotational atherectomy and directional coronary atherectomy than in those treated with rotational atherectomy and adjunct balloon angioplasty. PMID- 8626950 TI - Repeat interventions as a long-term treatment strategy in the management of progressive coronary artery disease. AB - OBJECTIVES: This study investigates whether repeat coronary interventions, applied over an extended time period, can successfully curtail the progression of ischemic symptoms and angiographic lumen narrowing. BACKGROUND: Coronary artery disease is a chronic and generally progressive disorder, and potential treatment strategies should be examined and compared with this chronicity in mind. Percutaneous interventional revascularization procedures could theoretically be useful in controlling progression of the disease through repeated use as new coronary lesions arise. However, the outcome of this long-term management concept has not previously been subjected to detailed investigation. METHODS: From a consecutive series of 4,357 interventional cardiac procedures, 544 patients were identified who received two or more interventions during the 13-year study period. These patients were categorized into one of three groups: restenosis (repeat interventions limited to the same target segment, n = 261), new stenosis (all repeat interventions directed to stenoses not previously treated, n = 155) or both (repeat interventions directed both to the same and to different target lesions, n = 128). RESULTS: Two to five procedures were performed per patient; the time period (mean +/- SD) separating each procedure was significantly less (p < 0.0001) for the restenosis group (4.2 +/- 2.3 months) than for the new stenosis (24.2 +/- 23.5 months) or the "both" groups (11.4 +/- 11.0 months). Despite the need for repeat procedures, the severity of angina (mean New York Heart Association functional class 1.6 +/- 0.9) after 6.2 +/- 2.3 years of follow-up was substantially better than before the initial procedure (mean functional class 3.2 +/- 0.8), with a similar magnitude of change found in all three groups. This long-term functional improvement was mirrored by a corresponding anatomic improvement, with the mean number of diseased vessels remaining constant at the time of each procedure (1.5 +/- 0.7, 1.5 +/- 0.7 and 1.6 +/- 0.7, respectively, for the first, second and third procedures, p = NS). The restenosis and the new stenosis groups also demonstrated statistically similar annual rates of mortality (1.9% vs. 1.8%) and coronary surgery (2.3% vs. 2.6%), although the restenosis group had a lower rate of infarction (1.4% vs. 3.2%, p = 0.002). CONCLUSIONS: Repeat interventional treatment of newly acquired stenoses provides a rational approach for the long-term management of chronic coronary artery disease. In addition to yielding a favorable late outcome, the use of this strategy can result in sustained functional improvement and can check the progression of clinically significant stenoses. PMID- 8626951 TI - Prospective, randomized trial of prolonged intracoronary urokinase infusion for chronic total occlusions in native coronary arteries. AB - OBJECTIVES: The purpose of this study was to determine the safety and efficacy of three dosing regimens of intracoronary urokinase for facilitated angioplasty of chronic total native coronary artery occlusions. BACKGROUND: Percutaneous transluminal coronary angioplasty of chronically occluded (>3 months) native coronary arteries is associated with low initial success secondary to an inability to pass the guide wire beyond the occlusion. METHODS: Patients were enrolled if a chronic total occlusion >3 months old could not be crossed with standard angioplasty equipment. Of the 101 patients enrolled, 41 had successful guide wire passage and were excluded from urokinase treatment. The remaining 60 patients were randomized to receive one of three intracoronary dosing regimens of urokinase over 8 h (group A = 0.8 million U; group B = 1.6 million U; group C = 3.2 million U), and angioplasty was again attempted after completion of the urokinase infusion in 58 patients. RESULTS: Coronary angioplasty was successful in 32 patients (53%) (group A 52%, group B 50%, group C 59%, p = 0.86). This study had a 90% power to detect at least a 50% difference between dosing groups at alpha 0.05. Bleeding complications requiring blood transfusion did not differ significantly among the dosing groups (A 0%, B 15%, C 6%, p = 0.14), although major bleeding episodes were less common in group A (p < 0.05). There were no major procedural or in-hospital complications. Angiographic follow-up in 69% of the patients with successful angioplasty revealed target vessel patency in 91% but an angiographic restenosis rate of 59%. CONCLUSIONS: A prolonged supraselective intracoronary infusion of urokinase can be safely administered and may facilitate angioplasty of chronic total occlusions. Lower doses of urokinase are equally effective and result in fewer bleeding complications than do higher dosage regimens. Vessel patency is frequently maintained, but restenosis remains a problem. PMID- 8626952 TI - Improved Doppler detection of proximal left anterior descending coronary artery stenosis after intravenous injection of a lung-crossing contrast agent: a transesophageal Doppler echocardiographic study. AB - OBJECTIVE: This study was designed to verify the usefulness of transesophageal Doppler recording of blood flow velocity in the proximal left anterior descending coronary artery, after a peripheral injection of a lung-crossing contrast agent (SHU 508A), in detecting and locating a hemodynamically significant stenosis (vessel narrowing > or = 50%) affecting this portion of the vessel. BACKGROUND: Transesophageal Doppler echocardiography has a limited diagnostic impact on the evaluation of proximal left anterior descending coronary artery stenoses. Peripheral injection of SHU 508A, a lung-crossing contrast agent enhancing Doppler signal to noise ratio in coronary arteries, may allow recording of localized disturbed blood flow velocity at the stenosis site even in the absence of a clear B-mode visualization of the vessel. METHODS: Transesophageal Doppler echocardiography, before and after echo contrast injection, was performed in 31 patients who underwent coronary angiography. Using color Doppler as a guide, pulsed wave Doppler recording of blood flow velocity in the left anterior descending coronary artery was attempted to detect a localized increase in blood flow velocity. B-mode evaluation of the vessel was also performed. RESULTS: Angiography showed a significant proximal left anterior descending coronary artery stenosis in 16 patients (group 1) and no stenosis in 15 patients (group 2). In 15 of 16 group 1 patients, Doppler after contrast injection revealed a localized velocity increase of at least 50% of the reference value; mean (+/-SD) percent increase in velocity was 150 +/- 89% (range 367% to 0%). In group 2 Doppler after contrast injection revealed a mild localized increase in velocity in four patients and no increase in velocity in the remaining 11 patients; mean (+/-SD) percent increase in velocity was 5 +/- 7% (range 21% to 0%, p < 0.001 vs. percent increase in group 1). When a percent velocity increase > or = 50% of the reference value was considered a positive criterion for detecting significant stenosis, the sensitivity and specificity were 92% and 100% respectively. The sensitivity of the evaluation before contrast injection or considering B-mode imaging alone was much lower (25% and 19%, respectively, p < 0.001 vs. evaluation after contrast injection). In addition, color Doppler after contrast injection correctly located the stenosis along the vessel, as compared with angiography. CONCLUSIONS: Blood flow evaluation of the proximal left anterior descending coronary artery by transesophageal Doppler echocardiography after contrast injection is a feasible and reliable method for detecting and locating significant stenoses affecting this part of the vessel and is an improvement over the traditional ultrasound approach. PMID- 8626953 TI - Combined low dose dipyridamole-dobutamine stress echocardiography to identify myocardial viability. AB - OBJECTIVES: We sought to evaluate the effects of combined administration of infra low dose dipyridamole and low dose dobutamine on assessment of myocardial viability. BACKGROUND: Low dose pharmacologic stress echocardiography with either dobutamine or dipyridamole infusion has been proposed for the recognition of myocardial viability. METHODS: Thirty-four patients with rest wall motion dyssynergy by two-dimensional echocardiography and with angiographically proved coronary artery disease underwent in combination with two-dimensional echocardiographic monitoring: 1) low dose (5 to 10 microgram/kg per min over 3 min) dobutamine infusion; 2) infra-low dose (0.28 mg/kg over 4 min) dipyridamole infusion; 3) combination of infra-low dose dipyridamole infusion immediately followed by low dose dobutamine infusion (combined dipyridamole-dobutamine). RESULTS: Follow-up rest echocardiography was available in 30 patients. After revascularization, 82 segments showed a contractile improvement of > or = 1 grade, whereas 63 segments remained unchanged. The sensitivity of dobutamine, dipyridamole and combined dipyridamole-dobutamine for predicting recovery was 72% (95% confidence interval [CI] 60.9% to 81.3%), 67% (CI 55.8% to 77%) and 94% (CI 86.3% to 97.9%), respectively. The specificity of dipyridamole, dobutamine and combined dipyridamole-dobutamine was 95% (CI 86.7% to 99%), 92% (CI 82.4% to 97.3%) and 89% (CI 78.4% to 95.4%), respectively. The accuracy of the dobutamine, dipyridamole and combined dipyridamole-dobutamine test was 80%, 79% and 92%, respectively (combined dipyridamole-dobutamine vs. dobutamine, p < 0.05; combined dipyridamole-dobutamine vs. dipyridamole, p < 0.01). CONCLUSIONS: Infra-low dose dipyridamole added to low dose dobutamine recruits an inotropic reserve in asynergic segments that were nonresponders after either dobutamine or dipyridamole alone and destined to recover after revascularization. PMID- 8626954 TI - Dobutamine-induced wall motion abnormalities: correlations with myocardial fractional flow reserve and quantitative coronary angiography. AB - OBJECTIVES: This study evaluated both the relation between dobutamine-induced wall motion abnormalities and the physiologic and morphologic features of epicardial coronary artery stenoses and the impact of the extent of the area at risk on the sensitivity of dobutamine echocardiography. BACKGROUND: The accuracy of dobutamine echocardiography has traditionally been assessed by comparing results with stenosis geometry. Myocardial fractional flow reserve is a functional index of coronary stenosis severity that takes into account both antero-grade and collateral flow and may therefore be a more appropriate standard for comparison. METHODS: Seventy-five patients with normal left ventricular function, good echocardiographic images and an isolated coronary stenosis underwent, within 6 h, dobutamine echocardiography, quantitative coronary angiography and intracoronary pressure measurements. Myocardial fractional flow reserve was calculated as the ratio of mean hyperemic distal coronary to aortic pressure. RESULTS: The degree of dobutamine-induced dyssynergy correlated significantly with percent diameter stenosis (r = 0.68), area stenosis (r = 0.68) and minimal lumen diameter (r = -0.60) and markedly better with myocardial fractional flow reserve (r = -0.77). However, marked dispersion of the individual data was observed. The sensitivity of dobutamine echocardiography in detecting lesions with a minimal lumen diameter < or = 1 mm and diameter stenosis > or = 50% was 83% and 80%, respectively. All but one patient with a myocardial fractional flow reserve >0.75 had a normal stress test result. Among patients with a myocardial fractional flow reserve < or = 0.75, the sensitivity of dobutamine echocardiography was significantly lower for lesions in vessels with a reference diameter < or = 2.6 mm than for lesions in larger vessels (58% vs. 90%, p = 0.008). CONCLUSIONS: 1) The magnitude of wall motion abnormalities induced by dobutamine infusion correlates with angiographic and, more closely, with functional indexes of stenosis severity, even though a wide scatter is observed. 2) In patients with a functionally significant stenosis, the amount of myocardium at risk is a critical determinant of the accuracy of dobutamine echocardiography. PMID- 8626955 TI - Prognostic value of heart rate adjustment of exercise-induced ST segment depression in the multiple risk factor intervention trial. AB - OBJECTIVES: We sought to assess the effect of heart rate adjustment of ST segment depression on risk stratification for the prediction of death from coronary artery disease. BACKGROUND: Standard analysis of the ST segment response to exercise based on a fixed magnitude of horizontal or downsloping ST segment depression has demonstrated only limited diagnostic sensitivity for the detection of coronary artery disease and has variable test performance in predicting coronary artery disease mortality. Heart rate adjustment of the magnitude of ST segment depression has been proposed as an alternative approach to increase the diagnostic and prognostic accuracy of the exercise electrocardiogram (ECG). METHODS: Exercise ECGs were performed in 5,940 men from the Usual Care Group of the Multiple Risk Factor Intervention Trial at entry into the study. An abnormal ST segment response to exercise was defined according to standard criteria as > or = 100 micro V of additional horizontal or downsloping ST segment depression at peak exercise. The ST segment/heart rate index was calculated by dividing the change in ST segment depression from rest to peak exercise by the exercise induced change in heart rate. An abnormal ST segment/heart rate index was defined as >1.60 micro V/beats per min. RESULTS: After a mean follow-up of 7 years there were 109 coronary artery disease deaths. Using a Cox proportional hazards model, a positive exercise ECG by standard criteria was not predictive of coronary mortality (age-adjusted relative risk [RR] 1.5, 95% confidence interval [CI] 0.6 to 3.6, p = 0.39). In contrast, an abnormal ST segment/heart rate index significantly increased the risk of death from coronary artery disease (age adjusted RR 4.1, 95% CI 2.7 to 6.0, p < 0.0001). Excess risk of death was confined to the highest quintile of ST segment/heart rate index values, and within this quintile, risk was directly related to the magnitude of test abnormality. After multivariate adjustment for age, diastolic blood pressure, serum cholesterol and cigarettes smoked per day, the ST segment/heart rate index remained a significant independent predictor of coronary death (RR 3.6, 95% CI 2.4 to 5.4, p < 0.001). CONCLUSIONS: Simple heart rate adjustment of the magnitude of ST segment depression improves the prediction of death from coronary artery disease in relatively high risk, asymptomatic men. These findings strongly support the use of heart rate-adjusted indexes of ST segment depression to improve the predictive value of the exercise ECG. PMID- 8626956 TI - Basal release of endothelium-derived nitric oxide at site of spasm in patients with variant angina. AB - OBJECTIVES: The aim of this study was to investigate the basal release of nitric oxide at spastic sites in patients with variant angina. BACKGROUND: We previously reported that endothelium-dependent dilator responses to acetylcholine, substance P and bradykinin are preserved at the site of coronary artery spasm. However, it is not known whether the basal release of endothelium-derived nitric oxide is altered at the spastic site. METHODS: The effects of intracoronary N(G) monomethyl-L-arginine (L-NMMA, an inhibitor of nitric oxide synthesis) at cumulative doses of 50, 100 and 200 micromol on basal coronary artery tone were investigated in eight patients with variant angina and normal coronary angiograms and in eight control subjects. The lumen diameters of large epicardial coronary arteries were assessed by quantitative coronary arteriography. RESULTS: Coronary spasm was provoked by the intracoronary administration of acetylcholine in all patients with variant angina. L-NMMA did not alter the arterial pressure and heart rate but significantly decreased the coronary artery diameter at spastic and nonspastic sites. Constrictive responses to L-NMMA were significantly greater (p < 0.01) at the spastic site (constriction by 200 micromol, 22+/-7%, mean +/- SD) than at the nonspastic site (10+/-7%). Constrictive responses to L-NMMA at the nonspastic site in patients with variant angina were comparable to those in the control subjects. CONCLUSIONS: These findings support the hypothesis that the basal release of nitric oxide may not be decreased at the spastic site in patients with variant angina. PMID- 8626957 TI - A new noninvasive method of diagnosing vasospastic angina based on dilation response of the left main coronary artery to nitroglycerin as measured by echocardiography. AB - OBJECTIVES: The purpose of the present study was to evaluate the feasibility of diagnosing vasospastic angina based on coronary artery tone as assessed by M-mode echocardiographic measurement of the dilation response of the left main coronary artery to nitroglycerin. BACKGROUND: The definite diagnosis of vasospastic angina is done by a coronary spasm provocative test using ergonovine maleate or acetylcholine during cardiac catheterization. Current noninvasive, nonpharmacologic diagnostic methods are not sensitive enough for the diagnosis of vasospastic angina. METHODS: Thirty-eight patients who had an angiographically normal left main trunk were studied. These patients were classified into four groups based on the presence or absence of more than 50% stenosis in the coronary arteries except for the left main trunk and the results of the acetylcholine or ergonovine provocative test. At 7 a.m. and at noon on the same day, the left main trunk diameter was measured by M-mode echocardiography before and after sublingual administration of nitroglycerin (0.3 mg), and its present dilation was calculated to assess coronary artery tone. RESULTS: The percent dilation of the left main trunk diameter induced by sublingual nitroglycerin at 7 a.m. and at noon was 22.4 +/- 4.7% (mean +/- SD) and 18.1 +/- 4.0% in 11 patients with vasospastic angina and without coronary stenosis, 14.9 +/- 7.1% and 11.2 +/- 6.9% in 9 patients with vasospastic angina and coronary stenosis, 6.1 +/- 3.5% and 7.0 +/- 5.1% in 8 patients without vasospastic angina but with coronary stenosis and 8.1 +/- 5.6% and 7.8 +/- 5.7% in 10 control subjects. The percent dilation at 7 a.m. was significantly greater in the vasospastic angina without coronary stenosis group than in the remaining three groups, and in the vasospastic angina groups, the percent dilation at 7 a.m. was significantly greater than that at noon. When percent dilation at 7 a.m. exceeding 15% was defined as positive for the diagnosis of vasospastic angina, the sensitivity was 80% and the specificity 94%. CONCLUSIONS: Basal tone of the left main trunk is elevated in the early morning in vasospastic angina. Dilation of the left main trunk diameter exceeding 15% induced by sublingual nitroglycerin in the early morning as measured by M mode echocardiography is a highly sensitive and specific criterion for the diagnosis of vasospastic angina. PMID- 8626958 TI - Impaired glucose tolerance with late hypersecretion of insulin during oral glucose tolerance test in patients with vasospastic angina. AB - OBJECTIVES: This study tested whether patients with vasospastic angina have impaired glucose tolerance or impaired insulin response. BACKGROUND: Hyperinsulinemia has been demonstrated in patients with coronary artery disease and syndrome X. METHODS: We performed an oral glucose tolerance test (75 g) in 30 patients with vasospastic angina in whom severe coronary vasospasm was induced by acetylcholine and in a matched group of 30 patients with atypical chest pain in whom no significant vasospasm was induced. The responses of insulin and glucose were compared between the two groups. No subjects had overt diabetes mellitus, hypertension, dyslipidemia, obesity or angiographically detected significant baseline coronary stenosis. Venous blood samples were taken during fasting and at 30, 60, 120 and 180 min after glucose load to obtain plasma glucose and immunoreactive insulin levels. RESULTS: Impaired glucose tolerance was detected in the 19 (63%) of 30 patients with vasospastic angina and in none of 30 patients with atypical chest pain (p < 0.001). The immunoreactive insulin levels at 60 and 120 min as well as the interval to peak insulin level were significantly greater in patients with vasospastic angina (p < 0.001). Among patients with vasospastic angina, those with acetylcholine-induced multivessel coronary vasospasm showed a significantly higher sum of insulin concentrations than those with single-vessel spasm (p < 0.01). During induction of coronary spasm, 10 patients with vasospastic angina presented ventricular arrhythmias. The sum of insulin concentrations was significantly greater in patients with than in those without ventricular arrhythmias. CONCLUSIONS: Patients with vasospastic angina exhibited a high incidence of impaired glucose tolerance and delayed and significantly higher insulin responses. These findings suggest that impaired glucose tolerance with late hypersecretion of insulin may contribute to the pathogenesis of severe coronary vasospasm. PMID- 8626959 TI - Platelets from patients with diabetes mellitus have impaired ability to mediate vasodilation. AB - OBJECTIVES: The purpose of this study was to examine vasomotor responses mediated by platelets from patients with diabetes mellitus. BACKGROUND: Diabetes mellitus is associated with increased cardiovascular morbidity and mortality, which in part may be due to a variety of abnormalities reported in diabetic platelets. However, the effects of diabetic platelets on vasomotor tone have not been characterized. METHODS: We compared platelet-mediated vasodilation elicited by platelets isolated from 30 healthy volunteers and 29 patients with diabetes mellitus as they were perfused through a preconstricted normal rabbit carotid artery. RESULTS: Platelets from the diabetic patients mediated an impaired dilatory response in comparison with normal platelets: 2.7 +/- 2% versus 15.8 +/- 3.4% (p < 0.001) and 4.1 +/- 2.7% versus 32.7 +/- 3.3% (p < 0.001) (mean +/- SEM) increase in vessel diameter, for 5 X 10(7) and 1 X 10(8) platelets per milliliter perfused, respectively. The degree of impairment was similar for type I (insulin dependent) and type II (non-insulin-dependent) diabetes mellitus. Normal platelets incubated in high D-glucose concentrations lost their ability to mediate dilation in a concentration-dependent and time-dependent manner. This was not true for incubation of normal platelets in high concentrations of L-glucose or insulin. However, there was not a significant correlation between glucose control in the diabetic patients and the ability of their platelets to mediate vasodilation. CONCLUSIONS: Platelets from patients with diabetes mellitus have an impaired ability to mediate vasodilation. This impairment appears to be mediated by high glucose concentration. Further work is needed to elucidate the mechanisms for this abnormality in diabetic platelets. PMID- 8626961 TI - Cellular telephones and pacemakers: urgent call or wrong number? PMID- 8626960 TI - Intermittent pacemaker dysfunction caused by digital mobile telephones. AB - OBJECTIVES: This study was designed to evaluate possible interactions between digital mobile telephones and implanted pacemakers. BACKGROUND: Electromagnetic fields may interfere with normal pacemaker function. Development of bipolar sensing leads and modern noise filtering techniques have lessened this problem. However, it remains unclear whether these features also protect from high frequency noise arising from digital cellular phones. METHODS: In 39 patients with an implanted pacemaker (14 dual-chamber [DDD], 8 atrial-synchronized ventricular-inhibited [VDD(R)] and 17 ventricular-inhibited [VVI(R)] pacemakers), four mobile phones with different levels of power output (2 and 8 W) were tested in the standby, dialing and operating mode. During continuous electrocardiographic monitoring, 672 tests were performed in each mode with the phones positioned over the pulse generator, the atrial and the ventricular electrode tip. The tests were carried out at different sensitivity settings and, where possible, in the unipolar and bipolar pacing modes as well. RESULTS: In 7 (18%) of 39 patients, a reproducible interference was induced during 26 (3.9%) of 672 tests with the operating phones in close proximity (<10 cm) to the pacemaker. In 22 dual-chamber (14 DDD, 8 VDD) pacemakers, atrial triggering occurred in 7 (2.8%) of 248 and ventricular inhibition in 5 (2.8%) of 176 tests. In 17 VVI(R) systems, pacemaker inhibition was induced in 14 (5.6%) of 248 tests. Interference was more likely to occur at higher power output of the phone and at maximal sensitivity of the pacemakers (maximal vs. nominal sensitivity, 6% vs. 1.8% positive test results, p = 0.009). When the bipolar and unipolar pacing modes were compared in the same patients, ventricular inhibition was induced only in the unipolar mode (12.5% positive test results, p = 0.0003). CONCLUSION: Digital mobile phones in close proximity to implanted pacemakers may cause intermittent pacemaker dysfunction with inappropriate ventricular tracking and potentially dangerous pacemaker inhibition. PMID- 8626962 TI - Value of analysis of ST segment changes during tachycardia in determining type of narrow QRS complex tachycardia. AB - OBJECTIVES: Repolarization changes during narrow QRS complex tachycardia were analyzed to differentiate the tachycardia mechanism and to guide the preliminary location of the accessory pathway. BACKGROUND: Noninvasive determination of the mechanism of tachycardia is becoming increasingly important in view of the role of catheter ablation techniques for the cure of supraventricular tachycardia. METHODS: We analyzed 159 12-lead electrocardiograms during narrow QRS complex tachycardia to evaluate 1) the tachycardia cycle; and 2) ST segment depression or T wave inversion, or both. Each patient underwent a complete electrophysiologic evaluation. RESULTS: There were 13 atrial tachycardias, 57 atrioventricular (AV) node reentrant tachycardias and 89 AV reciprocating tachycardias. The mean RR cycle did not differ among types of tachycardia. ST segment depression >2 mm or T wave inversion, or both, was present more often in AV reciprocating tachycardia (57%) than in AV node tachycardia (25%). The magnitude of ST segment depression was greater in AV reciprocating tachycardia than in AV node tachycardia (mean +/- SD 1.3 +/- 1.6 vs. 0.7 +/- 0.8 mm, p < 0.005). In AV reciprocating tachycardia distinct patterns of repolarization changes and P wave configuration were associated with different sites of the accessory pathway. CONCLUSIONS: The presence of ST segment depression >2 mm or T wave inversion, or both, during narrow QRS complex tachycardia suggests that AV reentry using an accessory pathway is the mechanism of the tachycardia. The phenomenon may be the consequence of a distinct pattern of retrograde atrial activation. Analysis of repolarization changes can guide preliminary localization of the accessory pathway even in the absence of ventricular preexcitation. PMID- 8626963 TI - Improvement of exercise capacity with treatment of Cheyne-Stokes respiration in patients with congestive heart failure. AB - OBJECTIVES: The aim of this study was to determine the impact of nasal nocturnal oxygen therapy on respiration, sleep, exercise capacity, cognitive function and daytime symptoms in patients with congestive heart failure and Cheyne-Stokes respiration. BACKGROUND: Cheyne-Stokes respiration is common in patients with congestive heart failure and is associated with significant nocturnal oxygen desaturation and sleep disruption with arousals. Oxygen desaturations and arousals cause an increase in pulmonary artery pressure and sympathoneural activity and therefore may reduce exercise capacity. Oxygen is an effective treatment of Cheyne-Stokes respiration and should improve exercise capacity in these patients. METHODS: The study was designed as a randomized crossover, double blind, placebo-controlled trial: 22 patients were assigned to 1 week each of nocturnal oxygen and room air. After each week, polysomnography, maximal bicycle exercise with expiratory gas analysis and trail-making test were performed, and a health assessment chart was completed. RESULTS: Nocturnal oxygen significantly reduced the duration of Cheyne-Stokes respiration (162 +/- 142 vs. 88 +/- 105 min [mean +/- SD]; p < 0.005). Sleep improved as evidenced by less stage 1 sleep and fewer arousals (20 +/- 13 vs. 15 +/- 9/h total sleep time; p < 0.05) as well as more stage 2 and slow-wave sleep; nocturnal oxygen saturation also improved. Peak oxygen consumption during exercise testing increased after oxygen treatment (835 +/- 395 vs. 960 +/- 389 ml/min; p < 0.05). Cognitive function evaluated by the trail-making test improved, but daytime symptoms in the health assessment chart did not improve significantly. CONCLUSIONS: Successful treatment of Cheyne-Stokes respiration with nocturnal nasal oxygen improves not only sleep, but also exercise tolerance and cognitive function in patients with congestive heart failure. PMID- 8626964 TI - Hypertrophic obstructive cardiomyopathy: preoperative echocardiographic predictors of outcome after septal myectomy. AB - OBJECTIVES: The purpose of this study was to determine whether two-dimensional and Doppler echocardiography are predictive of clinical outcome in patients with hypertrophic obstructive cardiomyopathy who undergo septal myectomy. BACKGROUND: Surgical myectomy provides excellent relief of symptoms in most patients with hypertrophic obstructive cardiomyopathy who are severely symptomatic despite medical therapy. There is a subset of patients who will remain symptomatic even after operation. Because comprehensive two-dimensional and Doppler echocardiography can define the range of anatomic and associated pathophysiologic abnormalities, it was hypothesized that preoperative echocardiographic variables may be predictive of clinical outcome after septal myectomy. METHODS: The clinical, electrocardiographic (ECG), echocardiographic and surgical data of 47 adult patients with hypertrophic cardiomyopathy who underwent isolated septal myectomy from 1986 to 1992 were analyzed. Specific symptoms were evaluated both preoperatively and at 1 year postoperatively. Electrocardiography and echocardiography were performed preoperatively and postoperatively. The ECG and echocardiographic variables were analyzed to determine whether any were predictive of residual symptoms 1 year postoperatively. RESULTS: The mean [+/-SD] age of the patients was 47 +/- 15 years. All were New York Heart Association functional class III or IV. Dyspnea was present in all 47 patients and was severe in 70%. Most patients experienced symptomatic improvement at 1 year; dyspnea persisted in 26 patients (55%). The preoperative echocardiographic variables of asymmetric hypertrophy, severe systolic anterior motion of the mitral leaflet(s) and prolonged isovolumetric relaxation time were independent predictors of mild or no residual dyspnea postoperatively. CONCLUSIONS: This initial study shows that the preoperative echocardiographic variables of asymmetric hypertrophy, severe systolic anterior motion of the mitral leaflet(s) and prolonged isovolumetric relaxation time can identify patients who are most likely to benefit from septal myectomy. PMID- 8626965 TI - Improved myocardial contrast with second harmonic transient ultrasound response imaging in humans using intravenous perfluorocarbon-exposed sonicated dextrose albumin. AB - OBJECTIVES: The objectives of this study were to determine whether a new method of ultrasound imaging (transient response imaging) could improve the myocardial contrast after intravenous injections of perfluorocarbon-exposed sonicated dextrose albumin microbubble contrast medium in humans. BACKGROUND: We have shown in animals that very low doses of intravenous contrast medium can produce transient but significantly better myocardial contrast when diagnostic ultrasound pulses are interrupted (delivered only once per cardiac cycle) instead of conventional 25- to 30-Hz frame rate imaging. METHODS: In 14 patients with normal rest wall motion, the peak myocardial contrast produced by transient response imaging was compared with that produced by conventional harmonic ultrasound imaging after injections of low doses (0.0025 to 0.01 ml/kg) of intravenous contrast medium. All studies were performed with second harmonic imaging (2.0 to 2.5 MHz-transmitted frequency). Blood pressure, oxygen saturation, respiratory rate and pulse were monitored before and after each injection. RESULTS: The intravenous contrast medium in the doses given produced no hemodynamic changes and no significant side effects in any patients. Overall, the mean (+/-SD) anterior and posterior myocardial contrast produced was significantly greater with transient response imaging than with conventional harmonic ultrasound imaging (anterior: 37 +/- 20 U transient response imaging vs. 18 +/- 14 U conventional harmonic imaging; posterior: 17 +/- 14 U transient response imaging vs. 5 +/- 5 U conventional; p< 0.01). With the sample size of 14 patients, the study had 80% power to detect a true difference of 18 U for anterior myocardial contrast and 90% power to detect a difference of 12 U for posterior contrast. Visually evident anterior or apical myocardial contrast was observed in 14 of 15 patients with transient response imaging but in only 7 patients with conventional harmonic imaging. Posterior or basal myocardial contrast was evident in 10 patients with transient response imaging but in only 1 patient with conventional harmonic imaging. CONCLUSIONS: Transient response imaging produces significantly better myocardial contrast than conventional harmonic imaging in humans and can be produced safely with minute quantities of intravenous perfluorocarbon. PMID- 8626966 TI - Three-dimensional echocardiography of normal and pathologic mitral valve: a comparison with two-dimensional transesophageal echocardiography. AB - OBJECTIVES: This study was done to ascertain whether three-dimensional echocardiography can facilitate the diagnosis of mitral valve abnormalities. BACKGROUND: The value of the additional information provided by three-dimensional echocardiography compared with two-dimensional multiplane transesophageal echocardiography for evaluation of the mitral valve apparatus has not been assessed. METHODS: Thirty patients with a variety of mitral valve pathologies (stenosis in 8, insufficiency in 12, prostheses in 10) and 20 subjects with a normal mitral valve were studied. Images were acquired using the rotational technique (ever 2 degrees), with electrocardiographic and respiratory gating. From the three-dimensional data sets, cut planes were selected and presented in both two-dimensional format (anyplane echocardiography) and volume-rendered dynamic display. The data were compared with the original multiplane two dimensional images. Different features of the mitral valve apparatus were defined and graded by three observers for clarity of visualization and confidence of interpretation as 1) inadequate, 2) sufficient, or 3) excellent. RESULTS: All the techniques provided good visualization of the mitral valve (mean global scores +/ SD for multiplane, anyplane and volume-rendered echocardiography were 2.22 +/- 0.34, 2.24 +/- 0.26 and 2.30 +/- 0.25, respectively). With volume-rendered echocardiography, the mitral valve apparatus was scored higher in pathologic than in normal conditions (2.38 +/- 0.24 vs. 2.16 +/- 0.21, p < 0.002). The spatial relationships between the mitral valve and other structures, leaflet mobility, commissures and orifice were scored higher by volume-rendered echocardiography. Prostheses were evaluated equally well by the three methods. Multiplane and anyplane echocardiography were superior for the evaluation of leaflet thickness, subvalvular apparatus and annulus. CONCLUSIONS: Transesophageal three-dimensional echocardiography facilitates imaging of some features of the mitral valve apparatus and provides additional information for comprehensive assessment of mitral valve abnormalities. PMID- 8626967 TI - Three-dimensional reconstruction of color Doppler flow convergence regions and regurgitant jets: an in vitro quantitative study. AB - OBJECTIVES: This study sought to investigate the applicability of a current implementation of a three-dimensional echocardiographic reconstruction method for color Doppler flow convergence and regurgitant jet imaging. BACKGROUND: Evaluation of regurgitant flow events, such as flow convergences or regurgitant jets, using two-dimensional imaging ultrasound color flow Doppler systems may not be robust enough to characterize these spatially complex events. METHODS: We studied two in vitro models using steady flow to optimize results. In the first constant-flow model, two different orifices were each mounted to produce flow convergences and free jets--a circular orifice and a rectangular orifice with orifice area of 0.24 cm(2). In another flow model, steady flows through a circular orifice were directed toward a curved surrounding wall to produce wall adherent jets. Video composite data of color Doppler flow images from both free jet and wall jet models were reconstructed and analyzed after computer-controlled 180 degrees rotational acquisition using a TomTec computer. RESULTS: For the free jet model there was an excellent relation between actual flow rates and three dimensional regurgitant jet volumes for both circular and rectangular orifices (r = 0.99 and r = 0.98, respectively). However, the rectangular orifice produced larger jet volumes than the circular orifice, even at the same flow rates (p < 0.0001). Calculated flow rates by the hemispheric model using one axial measurement of the flow convergence isovelocity surface from two-dimensional color flow images underestimated actual flow rate by 35% for the circular orifice and by 44% for the rectangular orifice, whereas a hemielliptic method implemented using three axial measurements of the flow convergence zone derived using three dimensional reconstruction correlated well with and underestimated actual flow rate to a lesser degree (22% for the circular orifice, 32% for the rectangular orifice). In the wall jet model, the jets were flattened against and spread along the wall and had reduced regurgitant jet volumes compared with free jets (p < 0.01). CONCLUSIONS: Three-dimensional reconstruction of flow imaged by color Doppler may add quantitative spatial information to aid computation methods that have been used for evaluating valvular regurgitation, especially where they related to complex geometric flow events. PMID- 8626968 TI - Second harmonic imaging of an intravenously administered echocardiographic contrast agent: Visualization of coronary arteries and measurement of coronary blood flow. AB - OBJECTIVES: This study sought to evaluate the potential of second harmonic contrast echocardiography to assess coronary vasculature. BACKGROUND: Newer transpulmonary ultrasound contrast agents capable of resonance phenomena detected by harmonic imaging may theoretically be able to demonstrate blood flow in the myocardium. METHODS: Transthoracic B-mode images and Doppler were obtained using a prototype second harmonic ultrasound system after femoral vein injection of AF0145 (10 to 40 mg) in 13 closed chest dogs (mean weight 25.6 kg). Coronary Doppler flow was simultaneously invasively measured using an intracoronary flow wire and visually compared with transthoracic Doppler flow. "Noninvasive" coronary vasodilator reserve was determined by measuring the ratio of the Doppler time velocity integral after adenosine to the baseline value and compared with the "invasive" intracoronary determination. RESULTS: Harmonic imaging showed heterogeneous opacification of the myocardium characterized by linear branching structures consistent with intramyocardial coronary arteries, which were not clearly visible during conventional ultrasound imaging. In nine dogs, transthoracic Doppler was performed, and characteristic coronary Doppler flow was observed, identical to the simultaneously observed intracoronary Doppler flow. Intracoronary adenosine (120 to 150 microgram) equally increased intracoronary and transthoracic Doppler flow velocities. The calculated "noninvasive" and "invasive" coronary vasodilator reserve ratios were similar ([mean +/- SD] 3.3 +/ 1.0 and 3.6 +/- 1.2, p = NS), with excellent correlation (r = 0.95, p = 0.0012). CONCLUSIONS: These findings indicate that noninvasive assessment of intramyocardial coronary vasculature and measurement of coronary blood flow reserve are possible using second harmonic contrast echocardiography. PMID- 8626969 TI - Triggered activity as a mechanism for inherited ventricular arrhythmias in German shepherd Dogs. AB - OBJECTIVES: This study sought to determine whether early afterdepolarization induced triggered activity is responsible for the initiation of ventricular arrhythmias in dogs with an inherited predisposition to sudden death. BACKGROUND: We have identified a colony of German shepherd dogs that display inherited ventricular ectopic activity and sudden cardiac death. The arrhythmias in these animals are pause dependent but are not associated with a prolonged QT interval, suggesting that they might be initiated by early afterdepolarization-induced triggered activity in Purkinje fibers. METHODS: Cardiac Purkinje fibers obtained from dogs that either did or did not exhibit ventricular tachyarrhythmias at the time of study were superfused in vitro with normal Tyrode solution (extracellular potassium ion concentration 4 mmol/liter) and were studied using standard microelectrode techniques. RESULTS: Early afterdepolarizations and triggered activity occurred spontaneously in Purkinje fibers obtained from affected dogs (n = 7) but not in fibers obtained from unaffected dogs (n = 13). Exit conduction block of triggered responses occurred to varying degrees within the Purkinje fiber but not at the Purkinje-muscle junction. Overdrive pacing suppressed triggered activity. The reemergence of triggered activity after cessation of pacing was both time and rate dependent. Triggered activity in fibers obtained from affected dogs was potentiated by phenylephrine and epinephrine and was suppressed by isoproterenol. Triggered activity was not induced by phenylephrine or epinephrine in fibers obtained from unaffected dogs. CONCLUSIONS: These results support the hypothesis that early afterdepolarization-induced triggered activity in Purkinje fibers is responsible for the initiation of ventricular arrhythmias in this canine model of inherited sudden death. PMID- 8626970 TI - Mechanism of antiarrhythmic drug-induced changes in defibrillation threshold: role of potassium and sodium channel conductance. AB - OBJECTIVES: We sought to determine which ion current predominantly affects defibrillation outcomes by using specific pharmacologic probes (lidocaine [a sodium channel blocking agent] and cesium [an outward potassium channel blocking agent]) in 26 swine. BACKGROUND: The effect of a drug on sodium or potassium channel conductance, or both, may affect defibrillation threshold values. However, it is unknown which ion channel predominates. METHODS: Each pig was randomly assigned to one of four treatment groups with two treatment phases: group 1 = placebo (D5W) in treatment phase I followed by placebo plus cesium in treatment phase II (n = 6); group 2 = lidocaine followed by lidocaine plus placebo (n = 7); group 3 = lidocaine followed by lidocaine plus cesium (n = 7); group 4 = placebo followed by placebo plus placebo (n = 6). Defibrillation threshold values and electrocardiographic measurements were obtained at baseline and at treatment phases I and II. RESULTS: Lidocaine increased defibrillation threshold values from baseline by 71% in group 2 (p = 0.02) and by 92% in group 3 (p < 0.01). There were no changes in defibrillation threshold values from baseline to D5W in groups 1 and 4. When D5W was added to lidocaine in group 2 and D5W in group 4, there were no significant changes in defibrillation threshold values. However, when cesium was added to lidocaine in group 3, the elevated defibrillation threshold values (mean +/- SD) returned to baseline values (from 15.7 +/- 3.46 to 7.55 +/- 3.19 J, p < 0.01). Cesium added to D5W in group 1 also significantly reduced defibrillation threshold values from 7.10 +/- 1.27 to 4.14 +/- 1.75 J (p < 0.01). The effect of cesium on defibrillation threshold values was similar between groups 1 and 3, regardless of lidocaine, such that these values were reduced by 40 +/- 14% and 51 +/- 18%, respectively (p = 0.28). CONCLUSIONS: Cesium, through potassium blockade, reverses lidocaine-induced elevation in defibrillation threshold values. The magnitude of defibrillation threshold reduction when cesium was added to lidocaine was similar to the defibrillation threshold reduction when cesium was added to placebo. Thus, inhibiting outward potassium conductance and prolonging repolarization decreases defibrillation threshold values independent of sodium channel blockade. PMID- 8626971 TI - Continued unauthorized use of FACC. PMID- 8626972 TI - President's page: convocation address. PMID- 8626973 TI - Dual-chamber pacing in dilated cardiomyopathy: insufficient sample size, heterogeneous population and inappropriate end point may lead to erroneous conclusions. PMID- 8626974 TI - Diagnosis and management of fetal cardiac tumors. PMID- 8626975 TI - Assessing the significance of preinfarction angina. PMID- 8626976 TI - Indications for catheter ablation in infants and small children with reentrant supraventricular tachycardia. PMID- 8626977 TI - Are the drug names Coumadin and Warfarin interchangeable? PMID- 8626978 TI - Pain and dermal reaction caused by injected glycerin in immunotherapy solutions. AB - BACKGROUND: Fifty percent glycerin preserves immunotherapy solution potency for at least 3 years but must be diluted before injection to reduce glycerin-induced pain and inflammation. We studied pain, erythema, induration, and bruises caused by glycerin (0% to 30%). METHODS: In 15 healthy subjects we compared, in double blind fashion, pain scores, injection site erythema, induration, and bruising caused by subcutaneous injections in randomized order of 0.1, 0.5, and 1 ml of glycerin 0%, 10%, 20%, and 30%. RESULTS: Injection volume did not significantly influence pain scores from diluent alone (0% glycerin) (p greater than 0.1). Pain scores of subjects given glycerin (0.1 to 1 ml, 10% to 30%) increased significantly as both injection volume (p less than 0.001) and glycerin concentration (p less than 0.001) increased. Pain scores correlated with total glycerin dose administered (volume x concentration) (rs = 0.67, p less than 0.0005) but varied widely, from minimal to severe, in those given the same dose. Injection site erythema, induration, and bruising occurred in some subjects with significant positive correlations between total glycerin dose and both frequency and diameters of erythema and induration. However, these dermal reactions were of trivial clinical importance. CONCLUSION: Injected glycerin produces pain that is proportional to total injected dose of glycerin, but individual variation in perceived discomfort is substantial. Total glycerin doses of less than 0.05 ml rarely produce clinically important pain. PMID- 8626979 TI - Measurement of airborne mite allergen exposure in individual subjects. AB - To evaluate the extent of personal exposure to airborne mite allergens, subjects were asked to carry a personal air sampler when in their houses. The level of Der 1 allergen trapped by the sampler was measured with a highly sensitive immunoassay. There were great variations in airborne Der 1 exposure in each subject. When used bedding was replaced with new allergen-free bedding, we detected a decrease in the allergen level. The use of new bedding seems to be an effective measure for reducing airborne mite allergen exposure. PMID- 8626980 TI - Positive nasal challenge responses to Blomia tropicalis. AB - BACKGROUND: Blomia tropicalis, a dust mite commonly found in subtropical and tropical environments, is the fourth most common mite in the United States. Thirty-eight percent of 167 consecutive subjects evaluated for allergic respiratory symptoms in the Tampa Bay area had positive skin test responses to B. tropicalis. METHODS: Nasal challenges were performed in 19 subjects with allergic rhinitis; 12 had positive skin test responses to B. tropicalis (group I), and seven had negative skin test responses to B. tropicalis but positive skin test responses to Dermatophagoides pteronyssinus and D. farinae (group II). Subjects were challenged with a normal saline control solution and increasing concentrations of a 1 mg/ml in-house extract of B. tropicalis (1:125,000 vol/vol, 1:25,000 vol/vol, 1:5000 vol/vol, 1:1000 vol/vol, and 1:200 vol/vol). Inspiratory nasal airway resistance (was measured every 5 minutes for 15 minutes by posterior rhinomanometry at 50 Pa after each nasal challenge. RESULTS: Nasal challenge response was considered positive and stopped when the mean inspiratory nasal airway resistance was greater than twice the mean inspiratory nasal airway resistance after the normal saline challenge. Ten of 12 (83%) subjects in group I and none of seven (0%) subjects in group II had positive nasal challenge responses to B. tropicalis (p = 0.0024). CONCLUSION: B. tropicalis is allergenic and should be considered as a cause of allergic rhinitis when evaluating a patient who lives in an area where it is endemic. PMID- 8626981 TI - A randomized, double-blind, placebo-controlled, controlled antigen delivery study of the onset of action of aerosolized triamcinolone acetonide nasal spray in subjects with ragweed-induced allergic rhinitis. AB - BACKGROUND: Clinically apparent relief of nasal symptoms of allergic rhinitis is generally recognized to occur within 3 days to 1 week when intranasal corticosteroids are used. OBJECTIVE: A study was designed to evaluate the onset of action of triamcinolone acetonide (TA) in patients with ragweed-induced allergic rhinitis with an environmental exposure unit (EEU). METHODS: Eighty-five adults with ragweed-induced allergic rhinitis were primed with ragweed allergen in the EEU. Symptoms were recorded during a baseline exposure in the EEU, and subjects were randomized with a 5:1 ratio to receive either TA 400 micrograms (n = 71) or its propellant (n = 14). Subjects received study medication for 7 days under supervision in the morning and returned to the EEU in the evening for ragweed allergen challenge and symptom assessment. Clinically apparent onset of action was defined as a 25% decrease in symptom scores from baseline. RESULTS: A mean reduction in nasal congestion from baseline of greater than 25% (onset of action) was observed in the TA group, but not in the placebo group, by 10 hours. This was also observed for itching of the nose or palate and a combined measure of symptoms. In addition, the proportion of subjects with less nasal congestion after 1 day of treatment was greater in the TA group (41%) than in the placebo group (7%) (p less than 0.05). CONCLUSION: The unexpected early relief of symptoms observed in the TA group and, to a lesser extent in the placebo group, has important clinical implications in the treatment of allergic rhinitis. PMID- 8626982 TI - Hymenoptera sting challenge of 348 patients: relation to subsequent field stings. AB - BACKGROUND: Patients with a history of a serious anaphylactic reaction after a Hymenoptera sting are usually given venom immunotherapy. Because the natural history of Hymenoptera sting anaphylaxis is often of a declining severity, there is a chance of overtreatment. OBJECTIVE: Identification of patients at risk for a future anaphylactic reaction may reduce the number of patients who need venom immunotherapy. METHODS: We investigated the relation between the grade of hypersensitivity to an in-hospital sting challenge and the reaction to a subsequent accidental field sting. From 1982 through 1992, 348 patients with mild or no symptoms after a sting challenge were not given venom immunotherapy. All patients were asked by letter whether they had experienced a subsequent field sting. In case of a sting, the severity of the reaction was further evaluated. RESULTS: Information could be obtained from 327 patients: 129 had been re-stung, and 110 of them had only had a local reaction. Thirteen patients had experienced mild systemic symptoms, and six patients had experienced serious manifestations. In two of the latter group hypotension was observed. CONCLUSION: In 95% of patients with a previous anaphylactic reaction, the result of the in-hospital sting challenge provided a good prediction of tolerance to a subsequent Hymenoptera field sting. PMID- 8626983 TI - Induction of atopic dermatitis by inhalation of house dust mite. AB - BACKGROUND: The pathogenetic role of house dust mite in atopic dermatitis remains controversial. Recent studies have shown that intensive epicutaneous contact of house dust mite allergen with premanipulated skin may induce dermatitis. It is, however, uncertain whether such conditions are met during natural contact with house dust mite. In the past, allergen inhalation has been suggested to induce exacerbation of atopic dermatitis. The aim of this study was to investigate whether dermatitis could be induced in patients with atopic dermatitis by inhalation of house dust mite. METHODS: Twenty patients with atopic dermatitis underwent bronchial provocations with house dust mite. Challenge tests were performed with four concentrations of a standardized house dust mite extract in a double-blind, randomized, placebo-controlled fashion. Spirometry was performed, and FEV1 was measured before and after each challenge dose. Changes in severity or localization of itching or erythema were recorded. RESULTS: In nine of 20 patients with atopic dermatitis bronchial challenge with house dust mite induced unequivocal skin symptoms after 1.5 to 17 hours. Pruritic erythematous lesions on noninvolved sites together with exacerbations of existing lesions were seen in three patients. Three patients had an exacerbation only, and three other patients had new lesions only. In eight of nine patients with house dust mite inhalation induced dermatitis, skin symptoms were preceded by an early bronchial reaction. All patients with house dust mite-induced dermatitis had a history of asthma, and as a group they had a higher mean blood total IgE level compared with the "negative skin responders." One patient had pruritic erythema on the placebo challenge day, without a preceding bronchoconstrictive reaction. The number of patients who had a skin response on the house dust mite challenge day was significantly higher than the number of patients who had a skin response on the placebo day (p = 0.011 [Prescott's test]). CONCLUSIONS: The respiratory route may be relevant in the induction and exacerbation of dermatitis in a subset of patients with atopic dermatitis who have early bronchial reactions after house dust mite inhalation, a history of asthma, and an elevated blood total IgE level. Furthermore, these findings suggest a possible causal relationship between bronchial reactions and skin reactions. PMID- 8626985 TI - Influence of allergen avoidance on the eosinophil phase of airway inflammation in children with allergic asthma. AB - BACKGROUND: Exposure to relevant allergens causes an increase in bronchial hyperresponsiveness, as well as an inflammatory reaction at the site of the bronchial mucosa in patients with asthma. OBJECTIVE: The purpose of this study was to determine whether antigen avoidance can exert an antiinflammatory effect on the eosinophil phase of airway inflammation in children with asthma. METHODS: The level of bronchial hyperreactivity and the percentage of eosinophils in sputum samples obtained by inhalation of hypertonic saline solution, were evaluated in a group of asthmatic children allergic to house dust mite before and after a period of antigen avoidance in an Alpine environment (1756 m). RESULTS: At the end of the avoidance period PC20 increased from a median value (lower and upper quartile: Q1, Q3) of 1.17 (0.74, 4.75) to 3.5 (1.18, 8.87) mg/ml (p = 0.02), and eosinophil percentage in the sputum decreased from a median value (Q1, Q3) of 14.02 (3.34, 28.24) to 2.08 (0, 7.4) (p less than 0.01). CONCLUSION: A 3 month period of antigen avoidance can significantly reduce the eosinophil phase of airway inflammation, along with bronchial hyperresponsiveness, in patients with asthma. PMID- 8626984 TI - Evaluation of household dust mite exposure and levels of specific IgE and IgG antibodies in asthmatic patients enrolled in a trial of immunotherapy. AB - BACKGROUND: Monitoring the response to immunotherapy entails understanding exposure to relevant allergens. For the major indoor allergens, this requires sampling of dust from the patient's house. The objectives of this study were to measure indoor allergen levels during a controlled trial of dust mite immunotherapy for asthma and to relate these results to serum antibody levels. METHODS: Eighty-eight asthmatic patients with mite allergy from seven geographic areas in the United States were enrolled in and completed a course of immunotherapy with Dermatophagoides extract or placebo control. Sensitization was evaluated by quantitative measurements of IgG and IgE antibodies. Dust samples were assayed for group I mite (Der p 1 and Der f 1), cat (Fel d 1), and cockroach (Bla g 1) allergens by monoclonal antibody-based ELISA. RESULTS: Over the 4 years of the study, each of the houses had at least one sample that contained more than 2 micrograms of group I mite allergen per gram of dust. Mean mite allergen levels, however, varied over a wide range, from 0.2 microgram/gm or less to more than 50 micrograms/gm. IgE antibodies to mite were present in sera from 78% of the patients, whereas IgE antibodies to cat and cockroach allergens were found in sera from 34% and 11% of patients, respectively. Sixty-four percent of the patients had exposure and sensitization to mite, whereas the comparable figure for each of the other allergens was 5%. CONCLUSIONS: Examination of the results suggested that allergen exposure, relative to a trial of immunotherapy, could be expressed as (1) the maximum level found in the house, (2) the percentage of sites having greater than 2 micrograms/gm, or (3) the mean value at the site with the maximum level. This report provides a background for evaluating the clinical results of immunotherapy in these patients and a model for the way in which sensitization and exposure should be monitored in studies of this kind. PMID- 8626986 TI - Trial of a "credit card" asthma self-management plan in a high-risk group of patients with asthma. AB - BACKGROUND: The "credit card" asthma self-management plan provides the adult asthmatic patient with simple guidelines for the self-management of asthma, which are based on the self-assessment of peak expiratory flow rate recordings and symptoms. OBJECTIVE: The study was a trial of the clinical efficacy of the credit card plan in a high-risk group of asthmatic patients. METHODS: In this "before and-after" trial, patients discharged from the emergency department of Wellington Hospital, after treatment for severe asthma were invited to attend a series of hospital outpatient clinics at which the credit card plan was introduced. Questionnaires were used to compare markers of asthma morbidity, requirement for emergency medical care, and medication use during the 6-month period before and after intervention with the credit card plan. RESULTS: Of the 30 patients with asthma who attended the first outpatient clinic, 26 (17 women and 9 men) completed the program. In these 26 participants, there was a reduction in both morbidity and requirement for acute medical services: specifically, the proportion waking with asthma more than once a week decreased from 65% to 23% (p = 0.005) and the proportion visiting the emergency department for treatment of severe asthma decreased from 58% to 15% (p = 0.004). The patients attending the clinics commented favorably on the plan, in particular on its usefulness as an educational tool for monitoring and treating their asthma. CONCLUSIONS: Although the interpretation of this study is limited by the lack of a randomized control group, the findings are consistent with other evidence that the credit card asthma self-management plan can be an effective and acceptable system for improving asthma care in a high-risk group of adult patients with asthma. PMID- 8626987 TI - Effect of acetazolamide on cough induced by low-chloride-ion solutions in normal subjects: comparison with furosemide. AB - BACKGROUND: The antitussive activity of inhaled furosemide has been attributed to its blocking effect on the Na(+)-2Cl(-)-K+ cotransporter. It is likely that the antitussive activity of inhaled diuretics is more complex because amiloride, a diuretic that has no effect on the Na(+)-2Cl(-)-K+ cotransporter, also shows a significant effect against cough induced by low-chloride-ion solutions. Apart from pharmacokinetics of inhaled diuretics, this activity could also depend on the inhibition of carbonic anhydrase. OBJECTIVES: We therefore studied the effect of inhaled acetazolamide, a selective inhibitor of carbonic anhydrase activity, on cough induced by the inhalation of different chloride ion solutions in a group of normal subjects. This was compared with the antitussive effect of furosemide. In addition, we attempted to determine whether the effect of acetazolamide is dose-dependent. METHODS: Cough challenge consisted of consecutive inhalations of four solutions having decreasing concentrations of chloride ions (150, 75, 37.5 and 0 mmol/L). Nine normal subjects underwent the cough challenge 5 minutes after the inhalation of saline placebo, acetazolamide (500 mg), and furosemide (30 mg) according to a randomized, double-blind study design. A group of six subjects were challenged according to the same procedure and study design, after the inhalation of saline placebo and of two doses of acetazolamide (250 mg and 500 mg). RESULTS: Inhaled acetazolamide significantly reduced cough response to 37.5 and 0 mmol/L chloride solutions compared with placebo (p less than 0.015 and p less than 0.015, respectively). Furosemide showed a similar protective effect (p less than 0.015 and p less than 0.025, respectively). Acetazolamide provided a significantly better protective effect than furosemide (p less than 0.025 and p less than 0.015, respectively). The antitussive activity of the two doses of acetazolamide was not statistically different. CONCLUSION: These results demonstrate that inhaled acetazolamide, a selective inhibitor of carbonic anhydrase, attenuates cough induced by low-chloride-ion solutions in normal subjects. At the applied doses, its antitussive activity is slightly greater than furosemide. This finding suggests that the inhibition of carbonic anhydrase activity is likely involved in modulating changes caused by absence of a chloride ion in the airway microenvironment of human beings. PMID- 8626988 TI - Skin testing with recombinant allergens rBet v 1 and birch profilin, rBet v 2: diagnostic value for birch pollen and associated allergies. AB - OBJECTIVE: This study assesses the value of two recombinant birch allergens for diagnosis of patients sensitized to birch pollen with or without associated food allergy. METHODS: Fifty-one patients with positive skin test responses to Betulaceae and seven nonallergic control subjects were investigated; specific IgE antibodies were evaluated by specific immunoassay and blot immunodetection. RESULTS: Among 51 patients, 47 reacted to rBet v 1 and 10 to rBet v 2. Seven patients reacted to both recombinant allergens. In skin prick tests we found a correlation between the wheal produced by the commercial birch extract and the wheal produced by rBet v 1. Among 47 patients with positive test responses to rBet v 1, 83% had IgE binding to the Bet v 1 protein as determined by immunoblotting. Among 10 patients sensitized to rBet v 2, six had IgE binding to Bet v 2. Eleven patients with negative results, as determined by immunoblotting, had low levels of birch IgE in the sera (less than 10 kU/L) and low concentrations of IgE to rBet v 1 or rBet v 2 in ELISA. The nonallergic control subjects (n = 7) did not react to rBet v 1 or rBet v 2 in skin prick tests, nor did they have detectable amounts of specific IgE to rBet v 1 or rBet v 2. Histamine release tests confirmed sensitization to Bet v 1 in two patients with discordant results; for Bet v 2, one patient had positive results only at a high concentration, and one had results that remained negative. Thirty-four patients had birch pollinosis, and all reacted to rBet v 1. Patients who were monosensitized to birch never reacted to rBet v 2. Sensitization to rBet v 2 was only found in patients who reacted to other pollens (mainly grass). Twenty-nine patients demonstrated allergy to apples, cherries, or hazelnuts; and all reacted to rBet v 1. Among 11 patients with allergy to Umbelliferae, only three reacted to rBet v 2. CONCLUSIONS: Use of the two recombinant allergens (rBet v 1 and rBet v 2) always permits the diagnosis of birch sensitization. Sensitization to rBet v 1 is specific for birch and Rosaceae allergies, whereas sensitization to birch profilin, Bet v 2, is encountered in multisensitized subjects and is not always related to Umbelliferae allergy. PMID- 8626989 TI - Identification of IL-5 and RANTES as the major eosinophil chemoattractants in the asthmatic lung. AB - The study was carried out to identify those molecules that are important in vivo in the attraction of eosinophil granulocytes to the lungs of patients with asthma. Asthmatic patients with birch pollen allergy had lavages performed before and during the pollen season, and the chemotactic activity of the bronchoalveolar lavage fluid was tested against normal eosinophils. The activity was significantly increased during the pollen season as compared with the activity before the pollen season (p less than 0.01). Neutralizing antibodies to IL-2, IL 5 and IL-8, leukemia inhibitory factor, and to RANTES were added to the bronchoalveolar lavage fluid. Antibodies to IL-5 and RANTES, but not to IL-2 and IL-8 or leukemia inhibitory factor, significantly inhibited the chemotactic activity for eosinophils (p less than 0.001). It is concluded that IL-5 and RANTES are important chemoattractants in the lungs of patients with allergic asthma. The effect of IL-5 may be that of a cofactor to the chemotactic molecules, of which RANTES may be one of the most important in allergic asthma. PMID- 8626990 TI - Comparison of latex-specific IgE binding among nonammoniated latex, ammoniated latex, and latex glove allergenic extracts by ELISA and immunoblot inhibition. AB - BACKGROUND: Nonammoniated latex, ammoniated latex, and latex glove extracts have been used as source materials for the preparation of allergenic extracts for the diagnosis of latex allergy. These materials showed different patterns of protein bands and immunoreactive bands. However, their IgE-reactive repertoires were not compared. OBJECTIVE: The goals of this study were to compare the IgE reactivity and to define the common IgE-reactive epitopes among three latex allergenic extracts. METHODS: Two serum pools were obtained from adults and children with latex allergy to evaluate the IgE reactivity among three latex extracts. IgE reactivity and IgE-reactive proteins were compared by inhibition ELISA and inhibition immunoblot methods, respectively. RESULTS: In this study inhibition curves were similar for nonammoniated latex and ammoniated latex but were different when the latex glove extracts were used. Several protein bands of ammoniated latex and latex glove extracts could not be inhibited by the nonammoniated latex. The ammoniated latex and latex glove extracts were able to remove all the latex-specific IgE from the serum. CONCLUSION: The IgE-reactive proteins differ among different latex extracts. Ammoniated latex and latex glove extracts contain more complete immunoreactive repertoires for detecting IgE antibodies. Our study provides useful information for selecting the latex extract. PMID- 8626991 TI - Interleukin-4 and low-affinity receptor for IgE on B cells in peripheral blood of patients with atopic bronchial asthma. AB - BACKGROUND: A greater frequency of type 2 helper cells producing IL-4 without interferon-gamma is thought to be responsible for the elevated IgE in serum of atopic subjects. However, the proportion of B cells responding to IL-4 by an increased synthesis of IgE is also higher in atopic subjects than in nonatopic subjects. OBJECTIVE: Important questions are whether the elevated IgE in atopic subjects is due to overproduction of IL-4 by T cells, the enhanced sensitivity of B cells to IL-4, or both and whether functional alterations of T and B cells are related to the development of allergic diseases. METHODS: Spontaneous and IL-4 induced CD23 expression on B cells was examined to evaluate the response of B cells to IL-4, and production of IL-4 by concanavalin-A-stimulated peripheral blood mononuclear cells (PBMCs) was measured to evaluate the T-cell function in nonatopic normal subjects, atopic normal subjects, and patients with symptomatic bronchial asthma. RESULTS: IL-4-induced expression of CD23 on B cells was greater in normal atopic subjects and atopic patients with bronchial asthma than in normal nonatopic subjects. IL-4 generated by concanavalin A-stimulated PBMCs was also higher in normal atopic subjects and atopic patients with bronchial asthma than in normal non-atopic subjects. The expression of CD23 on B cells and IL-4 generation by concanavalin-A-stimulated PBMCs were not different between normal atopic subjects and atopic patients with bronchial asthma. CONCLUSIONS: Both B cell and T-cell functions are enhanced in atopic subjects. However, neither enhanced B-cell nor T-cell function is a hallmark in development of allergic diseases. PMID- 8626992 TI - A new murine model of allergic conjunctivitis and effectiveness of nedocromil sodium. AB - BACKGROUND: Allergic conjunctivitis is the most common atopic disease affecting the eye. To study the pathophysiology and effectiveness of antiallergic drugs, it is necessary to develop animal models that closely mimic human allergic conjunctivitis. OBJECTIVE: The study was performed to develop an experimental murine model of ocular allergic conjunctivitis to an airborne allergen. METHODS: SWR/J mice were divided into the following groups: group 1, untreated, experimental; group 2, phosphate-buffered saline-treated; group 3, nedocromil sodium-treated; and group 4, unmanipulated controls. Groups 1, 2, and 3 were exposed to ragweed by topical contact with the nasal and conjunctival mucosae. Allergic conjunctivitis was evaluated by scoring of clinical signs, serum IgE levels, and histologic findings. RESULTS: Mice exposed to ragweed had clinicopathologic signs of allergic conjunctivitis and specific anti-ragweed IgE. Allergic conjunctivitis was modulated by nedocromil sodium. Treated mice had fewer clinical signs of allergy, lower levels of ragweed-specific IgE, reduction of conjunctival eosinophil infiltration, decrease in the number of intact and degranulating mast cells, and reduction of cytokine release. CONCLUSION: This is the first report of a murine model of allergic conjunctivitis to an airborne allergen that can be used to study the disease pathophysiology and its response to treatment. PMID- 8626993 TI - Mechanisms of inhibition of IgE synthesis by nedocromil sodium: nedocromil sodium inhibits deletional switch recombination in human B cells. AB - IgE synthesis requires IL-4 and a T cell-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. Nedocromil sodium (NS), an effective prophylactic agent in asthma, inhibits IgE synthesis by human B cells. In this report we examined the mechanisms of this inhibition. NS targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in highly purified B cells (greater than 98% CD19+). NS had no effect on the induction of epsilon-germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu --> S epsilon deletional switch recombination. The effect of NS was not specific for CD40 because it inhibited IgE synthesis in B cells stimulated with hydrocortisone plus IL-4. Moreover, the effect of NS was not specific for IgE because it inhibited CD40/IL-4-driven IgG4 synthesis by B cells sorted for lack of surface expression of IgG4. NS caused only modest inhibition of spontaneous IgE synthesis by B cells from patients with hyper-IgE syndrome, suggesting that it has little effect on B cells that have already undergone isotype switching. These results strongly suggest that NS inhibits IgE isotype switching by inhibiting deletional switch recombination and that NS has a novel potential mechanism for the prevention of asthma and other allergic diseases. PMID- 8626994 TI - Anti-IgE-induced accumulation of leukocytes, mediators, and albumin in skin chamber fluid from healthy and atopic subjects. AB - The aim of this study was to examine potential differences between healthy and atopic subjects with regard to IgE-mediated cutaneous inflammation. For this purpose, we analyzed histamine, tryptase, leukotriene B4, albumin, eosinophils, and total leukocytes in skin chamber fluid after challenge with anti-human IgE. We also measured gross skin reactivity (wheal, flare, and late-phase reactions), circulating IgE, and eosinophils, as well as the state of eosinophil activation. It was found that despite having more circulating IgE, the skin responsiveness of the atopic subjects did not differ significantly from that of the nonatopic subjects with respect to mediator release, albumin extravasation, or total recruitment of leukocytes. Moreover, the sizes of anti-IgE-induced wheal, flare, and late-phase reactions were very similar in the two groups. On the other hand, significant recruitment of eosinophils during the IgE-mediated reaction was more or less restricted to the atopic group. Yet the recruited eosinophils, of which the majority was in an early state of activation before degranulation, did not seem to contribute significantly to the IgE-mediated delayed skin edema. Furthermore, the eosinophil count in anti-IgE chambers of the atopic subjects did not correlate with any of the other parameters monitored. Thus because the anti IgE-induced recruitment of eosinophils appeared to be unrelated to factors such as the number of peripheral blood eosinophils, the degree of mast cell activation, the intensity of inflammatory skin changes, and the level of circulating IgE, it is apparent that the mechanisms for and pathophysiologic role of IgE-mediated dermal eosinophil accumulation in atopic subjects require further investigation. PMID- 8626996 TI - Dust mites at high altitude in a tropical climate. PMID- 8626995 TI - Increased prevalence of elevated serum IgE and IgG4 antibodies in students over a decade. PMID- 8626997 TI - Anaphylaxis to 6-alpha-methylprednisolone in an eight-year-old child. PMID- 8626998 TI - Successful desensitization for treatment of a fixed drug eruption to allopurinol. PMID- 8626999 TI - Distribution of beta1 integrin during development of chick tarsometatarsal skin in vivo and in vitro. AB - To determine the role of beta1 integrin in chick tarsometatarsal skin development, we examined the localization of the beta1 integrin immunohistochemically in vivo and in vitro by light and electron microscopy. Beta1 integrin was present over the entire cell surface of undifferentiated epidermis at early stages (Days 5, 9, 13). Marked changes in the localization of beta1 integrin occurred during epidermal keratinization and stratification, i.e., expression of beta1 integrin decreased in the superficial and intermediate cell layers from Day 13 to Day 17. After 17 days in vivo, when keratinization of the epidermis was completed, the distribution of beta1 integrin was confined to the basal layer of the epidermis in a pericellular distribution. During all stages examined, fibroblasts in the dermis were also stained. Immunoelectron microscopic study revealed that beta1 integrin was located on the plasma membrane of keratinocytes and dermal fibroblasts. The change in beta1 integrin localization that occurred in vivo could be reproduced in cultures of developing skin in which keratinization (differentiation) or mucous metaplasia (transdifferentiation) had been induced in vitro by hydrocortisone or retinol treatment, respectively. A monoclonal antibody against beta1 integrin caused striking changes in the epidermal keratinization process and in the basement membrane structure in vitro, i.e., inhibition of keratinization and detachment of the basement membrane from the basal surface of the epidermis. These results indicate that beta1 integrin plays an important role in cell-cell and cell-extracellular matrix interactions, which are important for epidermal development of the tarsometatarsal skin. PMID- 8627000 TI - A method of quantitative autoradiography for the spatial localization of proteoglycan synthesis rates in cartilage. AB - Incorporation of [35S]-sulfate into cartilage tissue indicates the synthesis of aggrecan, the large aggregating proteoglycan (PG) that endows cartilage with resistance to compression. Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfate but does not provide information on the spatial location of synthesis within the tissue. Such spatially specific information is necessary to determine which cell populations respond to diffusible factors and to correlate local mechanical events (e.g., deformation, interstitial fluid stress) to cellular biosynthetic responses. The aim of this study was to develop and characterize a liquid emulsion autoradiography technique, including an automated grain counting procedure, to derive spatial profiles of aggrecan synthesis rates in cartilage. We chose mature 10-14-month old bovine humeral head articular cartilage as a model system and applied a liquid emulsion autoradiography technique to [35S]-sulfate-labeled, resin embedded, and semithin-sectioned tissue explants. High-magnification light microscopy color images were captured on a computer. Automated image analysis for grain number determination included a color thresholding procedure to discriminate grains from the lightly stained structural image and computation of the average area of a single grain from each image. Determination of grain number, whether originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a single grain in the same image. This procedure largely eliminated the effects of variations of microscope light intensity, camera performance, image focus, section stain intensity, and thresholding on the resulting grain numbers. By altering the specific activity of the medium radiolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive content in the underlying section. By cutting specimens in half and performing liquid scintillation counting on one half and autoradiography on the other half, we found that each disintegration occurring in the section during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58). Therefore, counted grain numbers can be directly converted to incorporated sulfate, largely reflecting the synthesis of the PG aggrecan. As an example of calculated intratissue profiles of aggrecan synthesis rates, we found for the mature bovine tissue in serum-free medium that aggrecan synthesis increases monotonically from the articular surface to the radial zone by as much as tenfold. PMID- 8627001 TI - Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage. AB - We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite. PMID- 8627002 TI - Organ distribution of aminopeptidase A and dipeptidyl peptidase IV in normal mice. AB - The hydrolases aminopeptidase A and dipeptidyl peptidase IV, both present in the kidney on the brush borders of the proximal tubule epithelial cells and podocytes, are involved in the induction of experimental membranous glomerulonephritis in the mouse. However, little is known about their (co)distribution in other tissues and their function in health and disease. A detailed insight into the localization of these two enzymes is a prerequisite to elucidation of their function. Therefore, we investigated the presence and co distribution of aminopeptidase A and dipeptidyl peptidase IV by immunohistology with two different rat monoclonal antibodies, the specificity of which was determined by an immunodepletion technique. In addition, the molecular weight of the hydrolases; was analyzed by SDS-PAGE after isolation by solid-phase immunoprecipitation from glomeruli, renal brush borders, and thymus. Both hydrolases showed different molecular weights in renal corpuscle, renal brush borders, and thymic cells. A widespread organ distribution of the two hydrolases was observed, with co-localization in kidney, liver, small intestine, thymus, brain, spleen, and lymph nodes, either on the same cells or on different cells in the same organ. This distribution and partial co-localization suggests that the two hydrolases, acting either alone or in concert, have a role in many diverse biological processes. PMID- 8627003 TI - Localization of endothelial NOS at the basal microtubule membrane in ciliated epithelium of rat lung. AB - Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and iNOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, Week 1, Week 2, and in adult rats. By electron microscopy, eNOS immunoreactivity was confirmed within ciliated epithelium and was shown to be associated with the basal microtubule membrane of the cilia. Nonciliated cells were not stained. Type II epithelial cells also contain eNOS immunoreactivity, which is primarily associated with rough endoplasmic reticulum, and free ribosomes. However, macrophages in the lungs lacked eNOS immunoreactivity. This study demonstrated that eNOS was postnatally expressed in rat bronchial ciliated epithelium. The localization of eNOS at the basal membrane of ciliary microtubules suggests that eNOS may be involved in the function of epithelial cilia, consistent with previous physiological studies. PMID- 8627004 TI - Mapping of corticotropic cells in the normal human pituitary. AB - We accomplished the first mapping of corticotropic cells in the whole human adult pituitary. Corticotropic cells were identified by immunocytochemistry (ICC) and quantified by image analysis on 12 pituitaries obtained from people who had died suddenly. An overall view of each pituitary was given by 15-21 sections (mean 18 sections) at 300-micron intervals on six slides. Each section was systematically treated by indirect immunoperoxidase using an anti-ACTH[17-39] polyclonal antiserum. All the measures were done with a x 6.3 objective lens, each field (0. 5 mm2) being considered as the unit area. The mean pituitary density (surface of labeled cells/total surface) of corticotropic cells (9.5 +/- 3.0% per 0. 5 mm2) is significantly higher in men (11.5 +/- 5.1%) than in women (7.0 +/- 1.3%). This difference is due to an inverse relationship between the corticotropic cell density and the weight of the pituitary, which is higher in women than in men. The mean diameter of corticotropic cells is 14.9 micron and their total number per pituitary is approximately 10(7) cells. We confirmed that the spatial distribution of corticotropic cells is nonuniform: they are mainly distributed in the anteromedian part of the anterior lobe. In addition, our results demonstrated that the inferior part of the pituitary contained three times more corticotropic cells than the superior part (mean density 18.0% vs 6.0%) and the anterior part twice as many as the posterior part (mean density 12.3% vs 6.8%). On the horizontal plane, the pituitary was divided into eight zones, in which the mean of area was 2.5-21.0%. The maximal cell density may reach 40-60%. The use of this map should help the pathologist to recognize if there is corticotropic hyperplasia in a small pituitary fragment surgically removed from a patient with Cushing's disease. On the basis of this study, we put forward some criteria for diagnosing corticotropic hyperplasia. PMID- 8627005 TI - Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus. AB - To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal. PMID- 8627007 TI - Ultrastructural immunogold localization of vimentin and S-100 protein in guinea pig pars tuberalis. AB - Ultrastructural features of vimentin- and S-100 protein-positive cells in guinea pig pars tuberalis were examined by immunoelectron microscopy with the postembedding immunogold method. Interstitial cells that exhibited elongated cytoplasm and partly surrounded the specific secretory cells were filled with intermediate filament bundles on which immunogold particles for vimentin were densely located. Small colloid-containing follicles were occasionally distributed in the cranial region surrounding the median eminence. The follicular cells lining the luminal cavities contained considerable amounts of intermediate filaments immunoreactive for vimentin. Some specific secretory cells displayed various amounts of intermediate filaments immunoreactive for vimentin, which were scattered throughout the cytoplasm or concentrated around the nucleus. The specific secretory cells in which many parallel profiles of rough endoplasmic reticulum (RER) or a large number of mitochondria were dispersed throughout cytoplasm, i.e., being highly metabolically active, were devoid of intermediate filaments. The ventrocaudal region in continuity with the pars distalis was occupied by cells that were packed with vesicular inclusions, considered to be dilated cisternae of RER, and frequently formed colloid-containing follicles. Immunoreactivity for S-100 protein was located exclusively in this novel cell type. Immunogold particles for S-100 protein were distributed in the cytoplasmic matrix and were also associated with the membrane of vesicular inclusions. Gold particles were also detected on the tight junctions, desmosomes, and fibril materials at the apical cell regions of the colloid-containing follicles. The finding of two separate populations of cells containing vimentin and S-100 protein, respectively, supports the idea of functional separation in the pars tuberalis. PMID- 8627006 TI - Differential distribution of S-100 protein and vimentin in the hypophyseal pars tuberalis of the guinea pig. AB - In the hypophyseal pars tuberalis of guinea pigs, I examined immunohistochemical localization and development of vimentin and S-100 protein in comparison with those of the pars distalis. In the pars distalis, almost all folliculostellate cells expressed intense immunoreactivity for vimentin. A subpopulation of vimentin-immunoreactive folliculostellate cells was also immunoreactive for S-100 protein. During fetal development, vimentin-immunoreactive cells appeared at mid gestation in the pars distalis and became numerous at late stages, whereas only a few S-100 immunoreactive cells were observed even at late stages. In the pars tuberalis, a large number of vimentin-immunoreactive cells were distributed in the cranial region surrounding the median eminence and the dorsocaudal region surrounding the infundibular stalk. These cells, however, were sparse in the ventrocaudal region in continuity with the pars distalis. Conversely, dense distribution of S-100 immunoreactive cells was restricted in the ventrocaudal region. Vimentin-immunoreactive cells were elongated and were mostly distributed as solitary cells, whereas S-100 immunoreactive cells were gathered in large or small cell groups and frequently formed colloid-containing follicles. During fetal development, large cell groups immunoreactive for S-100 protein were detected at late stages in the ventrocaudal region. Therefore, in the pars tuberalis S-100 immunoreactive cells are distinct from the vimentin cells and do not correspond to the folliculostellate cells of the pars distalis. PMID- 8627008 TI - Histochemistry to detect Helix pomatia lectin binding in breast cancer: methodology makes a difference. AB - A number of studies have shown that altered cellular glycosylation, as detected by binding of Helix pomatia lectin to paraffin sections, is associated with metastatic disease and consequent poor patient prognosis in breast and other cancers. In a 24-year retrospective study, sections of 373 primary breast cancers were stained for binding of the lectin using two different histochemical techniques: a direct method (using peroxidase-conjugated lectin) and an indirect method (using native, unconjugated lectin). Similar percentages of cases were positive (79%) and negative (21%) for lectin binding with either technique, but there was enormous inconsistency when individual cases were examined. A total of 38/373 (10.2%) cases that were negative by the indirect method were positive by the direct method, and 37/373 (9.9%) cases that were negative by the direct method were positive by the indirect method. Life tables calculated for lectin staining vs nonstaining cases showed a very strong correlation between lectin binding and long-term survival (p < 0.0001) when staining was performed by the indirect method, but only very weak correlation with prognosis (p < 0.03, borderline significance) when the direct technique was employed. SDS-PAGE revealed that there were differences in breast cancer glycoproteins recognized by native lectin and peroxidase-conjugated lectin immobilized on Sepharose 4B affinity beads. Helix pomatia lectin binding appears to be an intriguing and potentially valuable marker of biological behavior in breast cancer. This study emphasizes the importance of selecting an appropriate immunohistochemical technique for its visualization. PMID- 8627010 TI - Electron microscopic demonstration of intracelluar promethazine accumulation sites by a precipitation technique: application to the cerebellar cortex of the mouse. AB - A method is described that allows electron microscopic identification of the phenothiazine neuroleptic promethazine after supravital intracardiac injection of high drug concentrations (greater than or equal to 3 %). The cerebellar cortex of the mouse was used for the investigation. This procedure is based on simultaneous fixation of drug and tissue by immersion in a paraformaldehyde-glutaraldehyde solution with the addition of phosphomolybdic acid. The electron microscopic investigation revealed that the drug could easily be identified as an electron dense precipitate. Subpopulations of neurons exhibited a higher affinity for the drug than others, but no preference for any nerve cell type was detected. Closer study showed that promethazine accumulated primarily within the cytoplasm, where it was bound to the endoplasmic reticulum. Furthermore, promethazine storage was observed within mitochondria and vesicular structures. Drug binding was also seen in the regions of synapses but without any predilection for these structures. Because this precipitation technique also appears to be well-suited for the ultrastructural identification of other drugs, it offers multiple possibilities for application. PMID- 8627009 TI - An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization. AB - Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5. PMID- 8627011 TI - Presidential Address: 33rd annual meeting of the Infectious Disease Society of America: secret wishes and selfish desires. PMID- 8627012 TI - Detection of ganciclovir resistance mutations quantitation of cytomegalovirus (CMV) DNA in leukocytes of patients with fatal disseminated CMV disease. AB - Cytomegalovirus (CMV) UL97 mutations associated with ganciclovir resistance at codons 460, 594, and 595 were detected by polymerase chain reaction (PCR) followed by restriction enzyme analysis in CMV blood isolates and directly in polymorphonuclear leukocyte (PMNL) DNA extracts of 4 subjects who died of progressive disseminated CMV disease due to ganciclovir-resistant CMV strains. The CMV DNA load was also serially determined in leukocyte fractions of these patients using a quantitative-competitive PCR assay. There was excellent concordance between specific UL97 mutations in blood culture isolates and those detected in PMNL fractions for all patients. Emergence of such UL97 mutations during ganciclovir therapy was associated with an increasing CMV DNA burden in leukocytes of the 2 patients with AIDS but not in the 2 subjects with chronic lymphocytic leukemia. Rapid molecular strategies, including detection of common CMV UL97 mutations and CMV DNA quantitation, can be used directly in leukocytes of immunocompromised subjects with CMV disease to monitor antiviral therapy. PMID- 8627013 TI - Epstein-Barr Virus DNA recombination and loss in sporadic Burkitt's lymphoma. AB - Epstein-Barr virus (EBV) antigens in tumor tissue define associations of virus with human malignancies and provide clues as to mechanisms of viral oncogenesis. In Burkitt's lymphoma, EBV markers are absent from 85% of sporadic cases and 4% of endemic (African) cases, raising questions as to the exact role EBV in the disease. Standard screening criteria may be insufficient to determine the EBV status of all tumors. One of 9 tumors from American patients expressed EBV nuclear antigen 1 (EBNA1) and contained standard episomal EBV DNA, making this series consistent with the 15% EBV association traditionally ascribed to sporadic Burkitt's lymphoma. Surprisingly, 3 tumors without detectable EBNA1 contained partial EBV genomes. Identification of defective, integrated viral DNA in some tumors indicates greater involvement of virus in sporadic Burkitt's lymphoma than previously documented and suggests a process of viral DNA rearrangement and loss during malignant progression most consistent with an initiating role for EBV in tumorigenesis. PMID- 8627014 TI - Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease. AB - Nucleic acid vaccinations with plasmids pWW65, containing the sequence for herpes simplex type 2 (HSV-2) gD2, and pRSVnt, lacking the gD sequence, were studied. Groups of mice were immunized with pWW65 alone, pWW65 plus 1,25-dihydroxyvitamin D3 (D3), or pRSVnt. Clinical disease (vaginitis), serum and vaginal washing antibody levels, and vaginal washing virus titers were measured intravaginal HSV 2 challenge. No animals (0/10) in the pWW65 + D3 group, 6/10 animals in the pWW65 group, and 10/10 animals in the pRSVnt group developed severe disease by postchallenge day 13 (P<.001, P=.04 vs. pRSVnt). Virus titers in vaginal washings were significantly reduced in the pWW65 and pWW65+D3 groups versus the pRSVnt group (P<.001). Increasing levels of serum anti-gD2 antibodies were measured 2 and 6 days after challenge among animals in the pWW65 and pWW65+D3 groups but not among animals in the pRSVnt group. Vaccinations with a plasmid containing the gD2 gene are immunogenic and provide some protection from HSV-2-induced disease. PMID- 8627015 TI - Human herpesvirus 8 is present in the lymphoid system of healthy persons and can reactivate in the course of AIDS. AB - In this study the prevalence of human herpesvirus (HHV) 8 DNA was determined in biopsies from persons with lymphoproliferative disorders and in peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-seronegative and HIV-infected persons. The results show that HHV-8 is present in 10% of biopsies from HIV-seronegative persons: HHV-8 is detected with similar prevalence values in HIV-infected patients with lymphoproliferative diseases, but the virus load is higher. HHV-8 was also found in PBMC. The presence of monoclonal Epstein-Barr virus (EBV) genomes in malignant lymphoproliferations was only infrequently associated with HHV-8 infection. Therefore, HHV-8 is fairly common in the population, and the lymphoid system could represent a reservoir of latently infected cells from which the virus may reactivate in conditions of immunodepression; furthermore, HHV-8 and EBV do not seem to act in conjunction in lymphomagenesis. PMID- 8627016 TI - Increased human immunodeficiency virus type 1 replication in human peripheral blood mononuclear cells induced by ethanol: potential immunopathogenic mechanisms. AB - Previous studies have shown that alcohol ingestion significantly increases human immunodeficiency virus type 1 (HIV-1) replication in peripheral blood mononuclear cells (PBMC) isolated and infected with HIV-1 in vitro. Whether the increased replication of HIV-1 observed after alcohol ingestion was due to unknown factors released from the gastrointestinal tract during alcohol ingestion or to certain metabolites produced by intestinal flora that degraded alcohol was investigated. In addition, cellular mechanisms involved in the increased replication of HIV-1 after alcohol exposure were evaluated. Twelve healthy HIV-1-seronegative subjects abstained from alcoholic beverages for >10 days. Nine were infused with 500 mg/kg ethanol (7.5% at 20 ml/kg/h) in saline, whereas 3 were infused with saline alone. Compared with saline-infused subjects, ethanol-infused subjects' PBMC exhibited significantly increased replication of HIV-1 when infected in vitro, which was associated with increased inhibition of CD8+ T lymphocytes' function by alcohol. PMID- 8627017 TI - Interleukin-12 decreases human immunodeficiency virus type 1 replication in human macrophage cultures reconstituted with autologous peripheral blood mononuclear cells. AB - In vitro interactions between interleukin (IL)-12, interferon (IFN)-gamma, and human immunodeficiency virus (HIV) type 1 infection in human macrophages were examined. Macrophages were infected with HIV-1 and cocultured with autologous monocyte-depleted peripheral blood mononuclear cells (PBMC). The addition of autologous PBMC to HIV-1-infected macrophages resulted in an expansive increase in reverse transcriptase (RT) activity; however, when both autologous PBMC and IL 12 were added, RT activity decreased (75%-90%) and high levels of IFN-gamma (9-16 ng/mL) were detected. The addition of anti-IFN-gamma antibodies blocked the IL-12 induced decrease in RT activity. Surprisingly, exogenous IL-12 added to HIV infected macrophage cultures without autologous lymphocytes resulted in a 50%-60% reduction in RT activity and no detectable increase in IFN-gamma. The addition of anti-IFN-gamma did not inhibit this IL-12-mediated effect. These results suggest that IL-12 is capable of indirectly down-regulating HIV proliferation in macrophage cultures reconstituted with autologous PBMC and of directly suppressing HIV replication in purified macrophage cultures. PMID- 8627018 TI - CD4 masking during human immunodeficiency virus type 1 infection, quantified on peripheral blood lymphocytes, is a potential marker of disease progression. AB - In human immunodeficiency virus type 1 (HIV-1)-infected adults, the proportion of gp120-free CD4 molecules on the surface of T lymphocytes was measured by double epitope EIA and expressed as a CD epitope concentration ratio. In 51% of these patients (n=81), CD4 T cells showed a significant decrease (up to 100%) in the accessibility of the CD4 epitope in the D1 domain remained accessible. Of interest, a significant increase in the CD4 gp120 binding site concentration, without a change in T cell counts, was observed within 10 days after initiation of zidovudine treatment. Furthermore, CD4 masking by gp120 was associated with a poor clinical patient status. The assessment of the CD4 epitope concentration ratio is proposed as a surrogate marker of disease progression in HIV-1-infected patients. PMID- 8627019 TI - The amount of early p24 antigenemia and not the time of first detection of virus predicts the clinical outcome of infants vertically infected with human immunodeficiency virus. AB - Twenty-three children vertically infected with human immunodeficiency virus type 1 (HIV-1) were studied for viremia during the first days of life. Nine had HIV-1 infection within the first week (early); 14 had HIV-1 first detected by day 11-90 (late). The groups had similar incidence and time of onset of symptomatic HIV-1 infection and survival. CD4 T cell percentages, rates of CD4 T cell attrition, quantitative cell-associated viremia, and p24 antigen concentrations were comparable. Children with peak antigen concentrations >100 pg/mL during the first 6 months (5 early, 6 late) fared worse than those with lower p24 levels. Thus, HIV-1-infected infants with detectable virus in the first few days of life do not have a worse prognosis than infants whose virus is detectable only later. Elevated p24 antigenemia during the first 6 months of life correlates strongly with poor clinical outcome and is independent of the time virus was first detected. PMID- 8627020 TI - Differences in the incidence of hepatitis B and human immunodeficiency virus infections among injecting drug users. AB - Both hepatitis B virus (HBV) and human immunodeficiency virus (HIV) type 1 seroconversions have been considered as outcome measures to evaluate the effectiveness of needle exchange programs. To assess the relationships between incident HBV and HIV infections among injecting drug users (IDUs), seroconversions were prospectively studied among a cohort of 240 HBV- and HIV- seronegative IDUs. The incidence of HBV seroconversion declined from 24.41/100 person-years in 1988 to 0 seroconversions in 1992. In contrast, HIV seroconversion rates varied little from the overall rate of 3.29/100 person years. HBV seroconversion predicted subsequent HIV seroconversion among male IDUs (relative incidence [RI]= 4.23) but not among female IDUs (RI=0.86). Because of different transmission dynamics, HBV seroconversion probably has limited utility as a surrogate outcome measure for incident HIV. However, HBV seroconversion itself is an appropriate and important outcome measure for evaluation of prevention programs among IDUs. PMID- 8627021 TI - Evaluation of morbidity among human T lymphotropic virus type 1 carriers in Miyazaki, Japan. AB - Morbidity associated with human T lymphotropic virus type I (HTLV-I) infection was investigated in a Japanese population within an area in which HTLV-I infection is endemic. Of 1824 subjects enrolled in the Miyazaki Cohort Study between November 1984 and May 1991, 500 (27.4%) were seropositive for HTLV-I antibodies. As expected from previous studies, HTLV-I positively appeared to be associated with baseline history of anemia (adjusted odds ratio [OR]= 1.3; 95% confidence interval [CI]= 0.99-1.7) and kidney disease (OR=1.6; 95% CI= 0.91 2.9); a positive association also was noted for asthma in men (OR=3.4; 95% CI=1.2 9.8). Unanticipated findings included a relationship between HTLV-I infection and cardiac disease history (OR=1.4; 95% CI=0.94-2.2; HTLV-I carriers also were more likely to have an abnormal electrocardiogram at baseline (OR=1.5; 95% CI=1.2 1.9). Furthermore, an apparent protective effect for ulcers (OR=0.62; 95% CI=0.40 0.95) and diabetes (OR=0.49;95% CI=0.22-1.1) was observed. HTLV-I infection may modify the risk of specific disease outcomes by altering host immune function. PMID- 8627022 TI - A simian strain of hepatitis A virus, AGM-27, functions as an attenuated vaccine for chimpanzees. AB - The AGM-27 strain OF hepatitis A virus (HAV) was originally isolated from an African green monkey with hepatitis and appears to represent a true simian strain. The virus caused acute hepatitis after intravenous inoculation into African green monkeys, rhesus monkeys, and marmosets. Cynomolgus monkeys inoculated with the virus did not develop hepatitis, probably because of prior exposure to HAV. Chimpanzees inoculated with a high dose of the virus did not develop signs of hepatitis, although the virus replicated and the animals seroconverted. Marmosets and chimpanzees convalescent from infection with the AGM 27 strain of HAV were rechallenged with the virulent HM-175 strain of human HAV. They were partially or totally protected from disease. PMID- 8627023 TI - Treatment of parainfluenza virus type 3 bronchiolitis and pneumonia in a cotton rat model using topical antibody and glucocorticosteroid. AB - Treatment of parainfluenza virus type 3 bronchiolitis and pneumonia in the cotton rat using topical IgG cleared infectious virus within 24 h but did not reduce pulmonary pathology. Treatment with topical triamcinolone acetonide dramatically reduced pathology but increased virus titers >10-fold. Combined therapy, however, demonstrated both favorable effects. Animals treated 3,4, and 5 days after infection showed a rebound of lung lesions to levels of untreated animals without reappearance of virus, but treatment through 8 days resulted in virus-free lungs without rebound of lesions. There was no difference in response to rechallenge 21 days after the original infection between treated and untreated animals. Combined antiviral and antiinflammatory therapy looks promising for viral bronchiolitis and pneumonia, but further questions relating to mechanism of action and to range of infections that can be treated in this fashion will need to be answered in animal models. PMID- 8627024 TI - Recall of original serologic response after challenge with homologous and heterologous Chlamydia trachomatis serovars. AB - Chlamydia trachomatis serovar-specific major outer membrane protein (MOMP) antigens are important targets of immune neutralization in vitro, and natural immunity to infection is associated with serovar specificity. Reinfection, often by different serovars, plays an essential role in chlamydial disease pathogenesis. By use of a murine model, the anamnestic serologic response was characterized following priming and challenge inoculations using 6 different serovars. The serologic response was evaluated using synthetic peptides representing MOMP variant segments (VS) 1,2,3, and 4 antigenic same serovar resulted in serologic responses to homologous VS1 peptides. After challenge with a different serovar, anti-VS1 serologic responses were often elicited with specificity to both the priming serovar and the challenge serovar. The recall of serologic response to the original serovar was typically dependent upon the antigenic relationship of the 2 serovars. PMID- 8627025 TI - Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure. AB - Severe group A streptococcal infections associated with early onset shock and multiorgan failure define the streptococcal toxic shock syndrome. In the United States, group A streptococcal strains most commonly isolated are M types 1 and 3, which produce pyrogenic exotoxin type A. The role of tumor necrosis factor (TNF) alpha and the dynamics of cardiovascular and laboratory abnormalities were investigated in a baboon model of group A Streptococcal bacteremia that mimics human Streptococcal toxic shock syndrome. Profound hypotension, leukopenia, metabolic acidosis, renal impairment, thrombocytopenia, and disseminated coagulopathy developed within 3 h after intravenous infusion of M type 3, pyrogenic exotoxin A-producing group A streptococci. Serum TNF-alpha peaked at 3 h and returned to baseline by 10 h. Mortality was 100%. Anti-TNF-alpha monoclonal antibody treatment markedly improved mean arterial blood pressure, tissue perfusion, and survival, suggesting that TNF-alpha plays an important role in the induction of shock and organ failure in group A streptococcal bacteremia. PMID- 8627026 TI - Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin. AB - Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice. After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders. High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern. Oral LT given with TT induced TT-specific response patterns identical to LT-B. Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response. Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types. PMID- 8627027 TI - The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae. AB - Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males. PMID- 8627028 TI - The excessive complement activation in fulminant meningococcal septicemia is predominantly caused by alternative pathway activation. AB - The relative contribution of the classical and alternative pathways in complement activation was quantified in 20 patients with systemic meningococcal disease. The activation products C4bc, C4bd, and Bb, indicating classical and alternative pathway activation, were measured with neoepitope-specific EIAs. Ten patients with persistent septic shock had significantly higher levels of Bb (P<.001), but not of C4bc (P=.43), than did 10 patients without septic shock. The Bb levels were significantly correlated with C3 activation products (C3bc; r= .72, P=.002), terminal SC5b-9 complement complex (TCC; r=.89, P<.001), and plasma lipopolysaccharides (LPS; r=.69, P= .01). There was no such association for C4bc versus C3bc, TCC, or LPS. Serially collected samples demonstrated activation of both pathways in patients with or without shock. Intervention strategies to stop the massive complement activation in fulminant meningococcal septicemia should include therapeutic principles that inhibit the alternative pathway. PMID- 8627029 TI - Ochrobactrum anthropi meningitis in pediatric pericardial allograft transplant recipients. AB - An epidemiologic investigation was done after 3 patients contracted Ochrobactrum anthropi meningitis at one hospital in October 1994. Neurosurgical patients with pericardial tissue implants were at greater risk of infection than other neurosurgical patients (3/14 vs. 0/566; P<.001). Cultures of implants removed from 2 case-patients, an implant at implantation, a nonimplanted pericardial tissue, and an unwrapped but unopened bottle of Hank's balanced salt solution (HBSS) grew O. anthropi. Patient and tissue isolates had identical genotypes; the isolate from the HBSS bottle had a unique genotype. Culture samples from an unopened HBSS bottle and from pericardial tissue grew Pseudomonas stutzeri of the same genotype; however, no P. stutzeri infections were detected. The investigation documented intrinsic P. stutzeri contamination of HBSS. O. anthropi contamination of tissues occurred during processing, possibly due to extrinsic contamination of HBSS. Active surveillance is needed to detect infection in patients receiving transplanted tissues, and rigorous infection control practice are necessary during tissue harvesting and processing to ensure sterility. PMID- 8627030 TI - Function of soluble CD14 in serum from patients with septic shock. AB - Soluble CD14 (sCD14) mediates lipopolysaccharide (LPS) activation of epithelial cells in vitro and may thereby be harmful in sepsis. sCD14 function was analyzed in sera from 62 patients with septic shock and compared with data from appropriate controls. sCD14 function was measured as sCD14-dependent LPS-induced interleukin (IL)-8 release in the SW620 epithelial cell line. In these cells, IL 8 production correlated with LPS concentration and the amount of sCD14. The effect of natural recombinant sCD14 was maximal at 100 ng/mL and blocked by anti CD14 antibodies. Patient and control sera (0.5% final concentration) promoted induction of IL-8 by 100 ng/mL LPS in SW620 cells. In sepsis patients (highest serum sCD14), values were significantly higher than in the other groups. The LPS induced IL-8 response was blocked by anti-CD14 and correlated with the serum CD14 level in sepsis patients. Thus, sCD14 could play a pathogenic role in sepsis. PMID- 8627031 TI - Polymerase chain reaction--based sequence-specific oligonucleotide hybridization analysis of HLA class II antigens in pulmonary tuberculosis: relevance to chemotherapy and disease severity. AB - HLA antigens were studied by serology and polymerase chain reaction-based sequence-specific oligonucleotide hybridization techniques in 153 patients with pulmonary tuberculosis (PTB) and 289 healthy controls. HLA-DR2 was present more frequently in PTB patients than in controls (51% vs. 36.3%; corrected P[Pc]=.029, relative risk [RR] = 1.8). The DR2 association was stronger in patients in the drug-failure group (n=56; Pc=.000012, RR=3.7) than in healthy controls and in patients in the drug-responder group. No significant deviation was observed in HLA allelic frequencies in various patient groups, as determined by radiographs of lung lesions. Molecular subtyping of DR2 revealed that the bulk of the allele was DRB1*1501 and DRB1*1502 in patients and controls. There was no skewing of the frequency of these molecular subtypes of DR2 in patients, suggesting that the whole DR2 molecule or its closely linked gene(s) may be involved in governing patient susceptibility to PTB and, particularly, development of the severe drug resistant form of the disease. PMID- 8627032 TI - Treatment of Mycobacterium avium complex infection: do the results of in vitro susceptibility tests predict therapeutic outcome in humans? AB - The ability of various in vitro methods of antibiotic susceptibility testing to predict therapeutic outcome in patients infected with Mycobacterium avium complex (MAC) was evaluated. Pretreatment bloodstream MAC isolates from 38 patients with AIDS, previously treated in a randomized fashion with either ethambutol, rifampin, or clofazimine, were tested by three conventional methods using broth or agar, as well as by cocultivation with macrophages. The results obtained with each method were compared with the quantitatively determined bacteriologic response to the administration of the single agent in humans. None of the conventional in vitro susceptibility methods was predictive of therapeutic outcome, while the results of cocultivation with macrophages were of moderate predictive value. The positive predictive value of a response in humans based on a response in macrophages (defined by > or = to 1.0 log reduction in baseline colony counts after 5 days of treatment) was 74%. The negative predictive value was 82%. PMID- 8627033 TI - Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. AB - Growth-subinhibitory nonlytic doses of cilofungin (lipopeptide antibiotic affecting (1,3)-beta-D-glucan synthesis) inhibited the incorporation of 46- to 48 kDa glucan-associated (46K) protein into the growing cell wall of Candida albicans. The purified 46K protein constituent strongly reacted with a monoclonal antibody against enolase, a major cytoplasmic enzyme of the fungus. In addition, two internal fragments of 12- and 15-amino acid residues from a tryptic digest of 46K protein showed 100% identity with amino acids in positions 34-45 and 66-80 of enolase. By immunoelectron microscopy with polyclonal and monoclonal anti-enolase antibodies, the 46K protein was clearly detected in the inner layers of the fungal cell wall. Thus, consistent with the proposed immunogenic and diagnostic roles of enolase in candidiasis, biochemical, immunochemical, and ultrastructural evidence strongly suggest that the cilofungin-susceptible 46K protein is a cell wall-associated form of this enzyme. PMID- 8627034 TI - Antimalarial drugs reduce cytoadherence and rosetting Plasmodium falciparum. AB - The in vivo and in vitro effects of antimalarials on cytoadherence and rosette formation were studied in 17 patients with severe and 46 with uncomplicated falciparum malaria. Cytoadherence was increased in severe malaria (P<.001). Artesunate and artemether were more potent than quinine in inhibiting both adherence properties. Artesunate was the most rapidly acting drug tested, producing >50% inhibition of both cytoadherence and rosetting in vivo and in vitro within 2 hr of drug exposure. Exposure to quinine for > or = to 4 h in vivo reduced rosetting by >50%, but not cytoadherence. Quinine did not reduce cytoadherence or rosetting significantly in vitro with exposure times of < or = to 8 h. These results suggest that artemisinin derivatives are more effective than quinine in preventing pathologic processes in parasitized erythrocytes that contribute to microvascular obstruction in severe malaria. PMID- 8627035 TI - Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis. AB - The abundance of macrophages in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL) lesions and differences in the composition of T cell subsets indicate involvement of cell-specific chemotaxis processes. The expression of macrophage chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and -1 beta, RANTES (regulated on activation, normal T cell expressed and secreted), I-309, and interleukin-8 were investigated in lesions of patients with LCL or DCL. In LCL, high levels of MCP-1 and moderate levels of MIP 1 alpha were detected. In DCL, MCP-1 expression was significantly lower and MIP-1 alpha expression was predominant. All other chemokines investigated were minimally expressed or absent. These findings suggest that MCP-1 and MIP-alpha are responsible for the recruitment of macrophages and T cells in cutaneous leishmaniasis. The results show that self-healing LCL is associated with higher levels of MCP-1, which may stimulate macrophage microbicidal mechanisms, and nonhealing DCL is associated with higher levels of MIP-alpha. PMID- 8627036 TI - Sequence variation of latent membrane protein-1 of Epstein-Barr virus strains associated with hairy leukoplakia. AB - The Epstein-Barr virus (EBV) latent membrane protein (LMP)-1 is expressed in hairy leukoplakia (HL), but data on LMP-1 sequence variation of HL isolates are limited. Variation in the LMP-1 repeat region and presence of a 30-nt deletion were studied using DNA scrapings from 28 HL lesions. cDNAs from 3 different HL isolates were sequenced, 2 from lymphocyte cell lines (LCLs) generated using HL biopsy material. The deletion was found in 16 (57%) of 28 HL scraping, and multiple repeat region variants were seen in 13 scrapings (46%). HL LMP sequence were similar to those described in nasopharyngeal cancer and lymphoma tissues, including two motifs of four amino acid changes relative to B95-8 upstream and downstream of the repeat region, respectively. Generation of LCLs using HL biopsy material confirmed the ability of HL EBV strains to infect and transform lymphocytes. PMID- 8627037 TI - Cervical abnormalities, human papillomavirus, and human immunodeficiency virus infections in women in Malawi. AB - Cervical lavage samples and Pap smears were obtained from 284 women in Malawi to evaluate the association between human papillomavirus (HPV) and human immunodeficiency virus (HIV) infections. Squamous intraepithelial lesions were present in 15% (17/16) of HIV-seropositive and 7% (11/152) of HIV-seronegative women (P=.05) and in 23% (19/83) of HPV polymerase chain reaction (PCR)-positive and 4% (6/156) of HPV PCR-negative women (P<.001). HPV DNA was detected in 23% of HIV-uninfected women but in 60% of HIV-infected women with <300 CD4 cells/mm(3) (P<.002). High-risk HPV types 16 and 18 constituted half of the identified types. HPV DNA in previously HPV-positive women was detected more often than in HIV seropositive abnormal cervical cytology than uninfected ones and were more likely to have persistent HPV infections. Early detection of HPV and regular monitoring of HIV-related cervical lesions may be important in HIV-infected women. PMID- 8627038 TI - Type-specific prevalence of human papillomavirus DNA among Jamaican colposcopy patients. AB - Human papillomavirus (HPV) types differ in their association with cervical cancer. Therefore, the types of HPV in precancerous lesions are important. In many regions with high cancer incidence, the HPV types in precancerous lesions have not been well studied. In Jamaica, a country that has high cervical cancer incidence, 174 colposcopy patients were tested for HPV DNA using polymerase chain reaction. HPV DNA detection was strongly related to presence and grade of cervical neoplasia (P<.001). Furthermore, severe neoplastic change was most highly associated with HPV DNA types also considered high-risk for severe neoplasia in other populations. HPV-45 DNA, a high-risk type uncommon in most previously tested countries, was detected in 12% of patients who had neoplasia. Thus, cervical neoplasia in Jamaica, as elsewhere, is linked to HPV. The high prevalence of HPV-45 was notable, and its relation to high cervical cancer incidence in Jamaica must be assessed. PMID- 8627039 TI - Cutaneous manifestations of human T cell leukemia virus type I infection in an experimental model. AB - Skin diseases ranging from infective dermatitis to cutaneous lymphoma have been associated with human T cell leukemia virus (HTLV) type I. A generalized exfoliative papillated dermatopathy occurred in a rabbit 20 months into a course of chronic HTLV-I infection. Biopsies revealed epidermotropic T cell infiltrates, including Sezary-like cells, that resulted in a pattern mimicking cutaneous T cell lymphoma. HTLV-I was isolated from affected skin, and virus expression was detected in cutaneous cultures. Sezary-like cells also occurred in circulation. Interleukin-2-independent lymphocyte cultures, established from blood exhibiting elevated CD8 T cell levels and CD25 expression, had polyclonal integration of provirus. The findings are similar to those in evolving adult T cell leukemia lymphoma and may represent a prelymphomatous change. The cutaneous lymphoproliferative lesion resulted from HTLV-I infection and further establishes the New Zealand White rabbit inoculated with the RH/K34 cell line as a suitable model for investigation of HTLV-I pathogenesis. PMID- 8627040 TI - Levels of hepatitis C virus in blood donors infected with different viral genotypes. International HCV Collaborative Study Group. AB - The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P<.001) but did not correlate with donor age (P=.06) or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment. PMID- 8627041 TI - Safety and immunogenicity of a combined live attenuated measles, mumps, rubella, and varicella vaccine (MMR(II)V) in healthy children. AB - An investigational tetravalent combined measles, mumps, rubella, and varicella vaccine and measles-mumps-rubella and varicella vaccines at separate injection sites given at the same visit were evaluated with respect to safety and cell mediated and humoral immune responses at 6 weeks and 1 year after vaccination. Varicella seroconversion rates and lymphocyte proliferation responses were 100% for both vaccine groups at 6 weeks and 1 year. However, the antibody titer to varicella was lower in the combined vaccine group at 6 weeks, but there was no statistical difference in cell-mediated immune responses. One-year geometric mean titers were not statistically different. Seroconversion rates for measles, mumps, and rubella were 100% for both vaccine at 6 weeks and 1 year. Long-term follow-up of these immune responses is planned. PMID- 8627042 TI - A novel nonpsychotropic cannabinoid, HU-211, in the treatment of experimental pneumococcal meningitis. AB - Typical features of pneumococcal meningitis have been demonstrated in rats inoculated with Streptococcus pneumoniae. HU-211, a novel noncompetitive N-methyl D-aspartate antagonist recently demonstrated to inhibit tumor necrosis factor alpha production under various conditions, improves recovery in some experimental models of brain injury. The present study tested the efficacy of HU-211 in combination with antimicrobial therapy in reducing brain damage in experimental pneumococcal meningitis. S. pneumoniae-infected rats were treated with saline alone, ceftriaxone alone, or with combination of ceftriaxone and HU-211 18 h after inoculation of the bacteria. Brain edema and blood-brain barrier impairment 48 h after infection were significantly (P<.05) reduced suggest that HU-211 when given concomitantly with antibiotics attenuates brain damage in the rat model of pneumococcal meningitis. PMID- 8627043 TI - Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu. AB - Recent studies have reported that Staphylococcus aureus small colony variants (SCVs) can cause highly persistent infections in humans and in cultured endothelial cells. To understand the process by which SCVs of S. aureus appear in subjects who have not received antibiotic treatment, bovine endothelial cells were coincubated with a wild S. aureus strain for 72 h in the presence of lysostaphin. Intracellular bacteria were harvested and screened for stable SCVs. Intracellular bacteria developed the SCV phenotype at a greater rate than control bacteria not exposed to endothelial cells: The intracellular induction rate was approximately 10(-3) versus a spontaneous rate of <10(-7). This observation suggest that SCVs are induced by the intracellular milieu and suggest a possible mechanism for the intriguing pathophysiology of tissue persistence of staphylococci. PMID- 8627044 TI - Augmented production of extracellular superoxide by blood isolates of Enterococcus faecalis. AB - To assess the frequency of extracellular superoxide (O-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19433), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25778), and Enterococcus gallinarum (ATCC 35038) produced extracellular O-2. Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O-2. Mean (+/-SE) rates of extracellular O-2 production in vitro by E . faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4+/-0.2, 1.9+/-0.2, 1.5+/-0.3 nmol of O-2 min(-1) 10(9) cfu(-1), respectively (P=.025, analysis of variance), suggesting an association between invasiveness and extracellular O-2 production. PMID- 8627045 TI - Defining inoculation conditions for the mouse model of ascending urinary tract infection that avoid immediate vesicoureteral reflux yet produce renal and bladder infection. AB - A satisfactory mouse model of ascending urinary tract infection (UTI) must avoid inoculation-induced vesicoureteral reflex (VUR) yet still produce kidney and bladder infection in a substantial proportion of mice. To define inoculation conditions that would satisfy both these conditions, mice were evaluated for VUR immediately after inoculation under a variety of conditions and were assessed for kidney and bladder infection 48 h after inoculation. Elimination of VUR required a slowed infusion rate, a reduced inoculum volume (25 microliter), and use of less traumatic methods for euthanasia and organ harvest. Bladder and kidney infection were highly prevalent at 48 h among mice inoculated under VUR-free conditions with either of 2 wild type Escherichia coli strains. Together with reports from other investigators, these findings indicate that satisfactory experimental conditions for the mouse model of ascending UTI can be defined empirically but may be laboratory-specific. PMID- 8627046 TI - Treatment of Myocardium avium complex infection: does the beige mouse model predict therapeutic outcome in humans? AB - To determine the predictive value of a standard murine model in the treatment of disseminated Myocardium avium complex (MAC) infection, beige mice were infected with MAC strains isolated from human immunodeficiency virus-infected patients and treated with the same antibiotic (ethambutol, clofazimine, or rifampin) that had been administered to the subject from whom that strain had been recovered. While ethambutol had the greatest bacteriologic efficacy in humans (mean decrease +/ SD, 1.0+/-0.5 log 10 cfu/mL of blood), clofazimine had the greatest bacteriostatic efficacy in mice (mean decrease +/- SD, 2.8 +/- 0.7 log(10) cfu/g of tissue). A linear correlation was not observed between bacteriostatic activity in mouse liver or spleen and the degree of bacteriologic response in humans (P > or = to .1). Odds ratios for a response in humans based on a bacteriologic response in mice were not significant for each agent (P > or = to .1, all cases). PMID- 8627047 TI - Identification by random amplification of polymorphic DNA of a common molecular type of Cryptococcus neoformans var. neoformans in patients with AIDS or other immunosuppressive conditions. AB - Sixty clinical isolates of Cryptococcus neoformans var. neoformans were analyzed by random amplification of polymorphic DNA (RAPD) using 12- to 22-mer primers in pairs. Five major profiles, which clearly distinguished between serotypes A (profiles I-III), AD (profile IV), and D (profile V), were identified. Forty-two of 58 serotype A isolates were assigned to profile I, 13 to profile II, and 3 to profile III. Profile I compromised 5 subtypes (profiles Ia-Ie), with 37 to 42 isolates in profile Ia. Twenty-seven of 28 isolates from patients with AIDS belonged to profile Ia (P<.001), as did 7 of 10 isolates from otherwise immunocompromised patients. Isolates from immunocompetent hosts were broadly distributed (profile I, 8 isolates; profile II, 10 isolates; profile III, 2 isolates). RAPD profiles were independent of body site and geographic origin of isolates. Isolates pairs from 3 patients produced identical profiles. A predominant genetic profile among serotype A strains from AIDS patients has not been reported previously. PMID- 8627048 TI - rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. AB - The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease. PMID- 8627049 TI - Cure of antimony-unresponsive Indian visceral leishmaniasis with amphotericin B lipid complex. AB - Twenty-one Indian patients with visceral leishmaniasis who did not respond to or relapsed after 28-60 days of pentavalent antimony therapy were treated with amphotericin B lipid complex (ABLC). Five infusions (3 mg/kg each) given every second day over 9 days (total dose, 15 mg/kg) resulted in a 100% apparent cure response. In 4 other patients who had not responded to antimony, apparent cure was also induced by ABLC given 3 mg/kg a day 5 consecutive days (total dose, 15 mg/kg). Fever and chills developed routinely during the initial 2-h infusions; these reactions were tolerated and diminished with successive infusions. Six months after treatment, all 25 patients were healthy, had parasite-free bone marrow aspirates, and were considered cured. ABLC is effective short-course therapy for kala-azar patients who do not respond to conventional antimony treatment. PMID- 8627050 TI - Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19-kDa C-terminal fragment of the merozoite surface antigen, PfMSP-1. AB - The development of an effective malaria vaccine depends upon identification of antigens that are targets of protective immune responses. An immunoepidemiologic approach has been used to investigate the relationship between antibody responses to a defined region of the major merozoite surface protein of Plasmodium falciparum (PfMSP-1 19) and resistance to clinical malaria in 2 populations of children from West Africa. After allowing for the confounding effects of age, antibodies to PfMSP-1 19 were shown the provide 40% protection against clinical malaria in children in Sierra Leone. In Gambian children, antibodies to one of the epidermal growth factor-like motifs of PfMSP-1 19 were strongly associated with resistance to both clinical malaria and high levels of parasitemia. PMID- 8627051 TI - High levels of spontaneous and parasite antigen-driven interleukin-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis. AB - To determine whether counterregulation by interleukin (IL)-10 plays a role in the generation or maintenance of the antigen-specific hyporesponsiveness seen in asymptomatic microfilaremic (MF) patients, parasite antigen (PAg)- and nonparasite antigen (NPAg)-driven IL-10 production by peripheral blood mononuclear cells (PBMC) was studied in 10 MF patients and in ll patients with chronic lymphatic pathology (CP). PBMC from MF patients spontaneously secreted 10 fold more IL-10 than did PBMC from patients with CP. PAg also induced significantly more IL-10 production by PBMC from CP patients. There was a negative correlation between PAg driven IL-10 production by PBMC and PAg-specific T cell proliferation in the MF group. IL-10 secretion by plastic adherent cells from MF persons was higher in response to PAg than NPAg, whereas IL-6 and tumor necrosis factor-alpha secretion were equivalent for PAg and NPAg, suggesting that PAg preferentially induces IL-10 secretion in these cells. Thus, PAg-induced IL 10 likely plays an important role in down-regulating antigen-specific proliferative responses in MF patients. PMID- 8627052 TI - Polymerase chain reaction-based assessment after macrofilaricidal therapy in Onchocerca volvulus infection. AB - A recently developed polymerase chain reaction (PCR)-based assay is significantly more sensitive than current methods for diagnosing Onchocerca volvulus infection, and it overcomes many difficulties in identifying active onchocerciasis. Since chemotherapy is widely used to treat onchocerciasis, the utility of PCR in assessing responses to treatment and in predicting recrudescence is important. Twenty-eight patients who had skin snips positive for microfilariae (Mf) were studied 120 days after receiving amocarzine, when each was negative for Mf: 16 (57%) were positive for O. volvulus DNA in the PCR-based assay. Of these, 14 (88%) were Mf positive when reassessed parasitologically on day 240, and all were Mf positive on day 365. Equally important was the finding that 12 patients had cleared both Mf and Mf DNA; only 1 was Mf positive at day 240. This suggest that the PCR-based assay provides a sensitive means assessing infection status after macrofilaricidal chemotherapy and is an early predictor of persons likely to have a recurrence of Mf. PMID- 8627053 TI - Pneumococcal vaccine response in human immunodeficiency virus-infected patients. PMID- 8627054 TI - Analysis of T helper cell response to glycoprotein H (gpUL75) of human cytomegalovirus: evidence for strain-specific T cell determinants. AB - The proliferative response of helper T cells against glycoprotein H (gH; gpUL75) of human cytomegalovirus (HCMV) was determined in T cell lines from 5 healthy HCMV-seropositive donors. A differential response in two lines was noted when gH from strain AD169 or Towne was used as antigen. T cell-reactive domains between aa 15 and 510 were identified using beta-galactosidase fusion proteins containing overlapping fragments of gH, and they were confirmed with synthetic peptides as stimulating antigen. T cell proliferation was observed with antigens containing aa 34-51, 111-142, 284-302, 324-342, and 454-510 of gH. None of the determinants stimulated all donors. The T cell epitope defined by aa 34-51 is located in close proximity to a strain-specific dominant B cell epitope; however, no strain dependence for this T cell determinant was observed. In contrast, the dominant T cell response against aa 284-302, which was observed in three T cell lines, was strain specific. PMID- 8627055 TI - Neutralization of human cytomegalovirus by human CD13-specific antibodies. AB - The mechanisms of inhibition of cytomegalovirus (CMV) infection by human CD13 (aminopeptidase N)-specific antibodies were studied. These antibodies protect CD13-negative and -positive cells from CMV infection only if incubated with the virus inoculum, suggesting they bind to CMV virions. The association of a CD13 like molecule with virions was further supported by the transfer of CD13 immunoreactivity to the surface of CD13-negative cells upon binding of CMV; the binding of CD13-specific antibodies directly to the surface of CMV virions; and the presence of anti-CD13 immunoreactive bands, including one with mobility similar to that of native cellular CD13 on immunoblots of proteins of purified CMV particles. Importantly, CD13-specific antibodies neutralize CMV in urine of neonates with congenital CMV, indicating that the CD13-like molecule is associated with natural CMV and not acquired in vitro. These studies demonstrate that a CD13-like molecule is associated with CMV particles and may be important in CMV pathogenesis. PMID- 8627056 TI - Time to detection of cytomegalovirus (CMV) DNA in blood leukocytes is a predictor for the development of CMV disease in CMV-seronegative recipients of allografts from CMV-seropositive donors following liver transplantation. AB - In 35 cytomegalovirus (CMV)-seronegative recipients of livers from CMV seropositive donors, 32 (91%) developed CMV infection and 24 of them (75%) experienced disease. Polymerase chain reaction for CMV DNA in leukocytes had the best positive and negative predictive values for the development of disease within 2 months from transplantation, and shell-vial or tube culture viremia was the best predictor thereafter. In patients who developed CMV disease, CMV DNA was first detected at 46 days (median; range, 13-128) after transplantation, significantly earlier than the 77 days (range, 46-174) for those who did not develop CMV disease (P = .02). By a semiquantitative method, the CMV DNA level in the first positive sample did not predict disease development. However, the maximum CMV DNA level during infection was significantly higher in patients who developed CMV disease. In CMV-seronegative recipients of livers from CMV seropositive donors, the time to DNA positivity following transplantation may predict disease progression and be useful as a guide for the initiation of preemptive therapy. PMID- 8627057 TI - Placental transfer and maternally acquired neonatal IgG immunity in human immunodeficiency virus infection. AB - Transplacental transfer of specific IgG antibodies was studied in 46 pairs of human immunodeficiency virus type 1 (HIV-1)-seropositive women and their neonates and in 53 pairs of healthy HIV-seronegative mothers and their newborns. Neonatal and maternal sera were assessed by nephelometry for total levels of serum IgG and by ELISA for IgG antibodies to herpes simplex virus (HSV), varicella-zoster virus (VZV), measles virus, tetanus toxoid, streptolysin O, and Streptococcus pneumoniae capsular antigens. Placental transfer of IgG antibodies to VZV, tetanus toxoid, measles, streptolysin O, and S. pneumoniae was decreased by maternal HIV infection. Maternal levels of total IgG had an independent effect on transfer of antibodies to HSV, VZV, measles, and S. pneumoniae. Neonatal antibody levels to tetanus toxoid, measles, and S. pneumoniae were significantly lower in the HIV group. Both maternal hypergammaglobulinemia and maternal HIV infection may contribute to these low antibody levels at birth and thus lead to early infection in this high-risk population. PMID- 8627058 TI - In vitro restoration of T cell immune function in human immunodeficiency virus positive persons: effects of interleukin (IL)-12 and anti-IL-10. AB - Cells from human immunodeficiency virus (HIV)-positive patients were evaluated for their in vitro responsiveness to recall antigen, alloantigen, and phytohemagglutinin (PHA) following the in vitro addition of interleukin (IL)-12 or anti-IL-10. Three-color flow cytometric analysis of CD4 and CD8 subsets was done to determine whether specific in vivo alterations in cell surface markers are associated with in vitro function changes. The results demonstrated a hierarchical response pattern to recall antigens versus alloantigen versus PHA, and these in vitro responses were associated with the number and activation status of CD4 cells. The in vitro addition of IL-12 or anti-IL-10 could restore antigen responses (HIV envelope peptides or influenza) in patients with 200-500 CD4 cells/microL; however, in patients with < 200 CD4 cells/microL, this improved response was limited to the influenza response. Studies of this nature may provide important insights into the role of cytokines in the natural history of HIV disease, and they suggest that immune therapy of this type may be most effective in patients who have more preserved immune systems. PMID- 8627059 TI - Changes in biologic phenotype of human immunodeficiency virus during treatment of patients with didanosine. AB - Progression to AIDS in patients harboring human immunodeficiency virus type-1 (HIV-1) isolates expressing a syncytium-inducing (SI) phenotype is faster than in those in whom the virus expresses a non-SI (NSI) phenotype. Zidovudine monotherapy does not appear to alter this outcome. To examine the role of didanosine (ddI) monotherapy in phenotype expression, HIV-1 isolates from 73 patients receiving ddI for up to 72 weeks were analyzed. After 12 weeks, the number of isolates expressing an NSI phenotype was 29% higher than at the start of treatment. Patients receiving high-dose ddI (375 mg twice daily) were significantly more likely to express the NSI phenotype at 12 weeks than patients who received low-dose ddI (100 mg twice daily), even after adjustment for phenotype and CD4 cell count at baseline, suggesting that ddI may be selective against the faster-replicating virus. ddI at 375 mg twice daily significantly increases the probability of an NSI phenotype over the short term in patients with advanced HIV disease. PMID- 8627060 TI - Randomized study of the tolerance and efficacy of high- versus low-dose zidovudine in human immunodeficiency virus-infected children with mild to moderate symptoms (AIDS Clinical Trials Group 128). Pediatric AIDS Clinical Trials Group. AB - The current dosage of zidovudine for children is 180 mg/m2 every 6 h. To investigate whether a lower dosage was equally effective, human immunodeficiency virus (HIV)-infected children (3 months to 12 years) with mild to moderate symptoms were randomly assigned to receive either high-dose (180 mg/m2/dose) or low-dose (90 mg/m2/dose) zidovudine (double-blind). Treatments were compared with respect to neuropsychologic function, survival, clinical and laboratory evidence of disease progression, and safety and tolerance. Four hundred twenty-six HIV infected children were enrolled; median time for receipt of study drug was 35 months. Zidovudine in either dose was well tolerated, with no difference in efficacy or tolerance by treatment group using any clinical or laboratory parameter. In children with mild to moderate disease, a reduction of zidovudine to 90 mg/m2/dose will result in substantial cost savings and should be the recommended dose. PMID- 8627061 TI - Phase I trial of interferon alfa-n3 in early-stage human immunodeficiency virus type 1 disease: evidence for drug safety, tolerance, and antiviral activity. AB - The safety and tolerance of interferon alfa-n3 (IFN-alpha n3) was tested in 20 adults with asymptomatic human immunodeficiency virus type 1 (HIV-1) infection (> 400 CD4 lymphocytes/mm3). IFN-alpha n3 was self-injected three times per week for 3-6 months: 5 patients received 1 mega-IU (MIU)/dose, 10 received 5 MIU/dose, and 5 escalated to their maximum tolerated dose. Subjects were evaluated every 2-4 weeks through 2 months after cessation of treatment. Neuropsychological tests were given at 3-month intervals. Markers of IFN activity, anti-IFN neutralizing antibodies, and antiviral response were measured monthly. IFN-alpha n3 was safe and well tolerated: influenza-like symptoms were uncommon, laboratory toxicity was minimal, no adverse neurobehavioral side effects were evident, and no patient developed neutralizing antibodies against IFN. IFN-alpha n3 induced IFN-specific biologic responses and dose-related antiviral activity against HIV-1. Subjects showed stabilization of CD4 cells for > 20 months. IFN-alpha n3 should be studied in combination with other antiretroviral agents and in persons with more advanced HIV-1 infection. PMID- 8627062 TI - The association of antibodies against human T cell lymphotropic virus type I (HTLV-I) pX gene mutant products with HTLV-I-associated myelopathy/tropical spastic paraparesis. AB - Antibodies specific for the products of the human T cell lymphotropic virus type I (HTLV-I) pX frame-shift mutants were studied by ELISA. The serum IgG antibodies to the synthetic peptide corresponding to one nucleotide insertion at position 7784 were significantly more common in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in HTLV-I carriers who had neither HAM/TSP nor adult T cell leukemia (39% vs. 5%). The seropositivities to the other synthetic peptides, which corresponded to the one nucleotide deletion at position 7475 and the internal deletion of nt 7754-7819 and nt 7853-7936, were rare. A genetic study confirmed the presence of the responsible mutation of the pX gene in peripheral blood mononuclear cells and central nervous system tissue from HTLV-I-infected subjects with and without HAM/TSP. These results suggest that HTLV-I pX frame-shift mutants are expressed in vivo in HTLV-I carriers; they also induce antibodies. especially in those with HAM/TSP. PMID- 8627063 TI - Passive and active immunotherapy for experimental pneumococcal pneumonia by polyvalent human immunoglobulin or F(ab')2 fragments administered intranasally. AB - Experimental pneumococcal pneumonia in leukopenic BALB/c mice enabled evaluation of passive immunotherapy with human polyvalent intravenous immune globulin (IVIG) given intravenously or intranasally and with F(ab')2 fragments administered intranasally. For intravenous and intranasal IVIG, the respective effective doses were < 5 but > 0.5 mg/kg and < 250 but > 2.5 micrograms/kg. For F(ab')2 fragments, the effective dose was < 500 but > 2.5 micrograms/kg. Assessment of the acquired immune responses of passively protected mice and convalescing controls 3 weeks after primary infection showed that antibody responses to whole bacteria were serotype-specific in all mice. Mice protected with IVIG and F(ab')2 fragments had more antibodies to pneumolysin than did controls. In addition, treated mice acquired greater resistance to reinfection than untreated survivors. Thus, local passive immunotherapy may be an effective means of treating pneumococcal pneumonia and may promote acquired resistance to reinfection. PMID- 8627064 TI - Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly glycopeptide-resistant enterococci. AB - Changes in fecal flora were evaluated in 22 healthy volunteers administered oral vancomycin or teicoplanin in 1989-1991 in Belgium. Evaluation of 5 colonies per subject revealed no glycopeptide-resistant enterococci in the predominant flora before glycopeptide administration; however, large numbers (mostly Enterococcus faecium) emerged by the end of the study in 14 (64%) of the subjects. Pediococci and lactobacilli also increased in number. In 1992, 40 healthy volunteers and 33 cancer patients were evaluated by plating stool samples directly onto selective media containing vancomycin; low numbers of vancomycin-resistant enterococci (< 50 cfu/g) were found in 11 (28%) of the 40 and 4 (12%) of the 33 samples, respectively. DNA restriction fragment length polymorphism analysis showed that most isolates were different, but all contained vanA in Tn1546-like elements. These results indicate that vanA and Tn1546-like elements were common in Belgium as early as 1989 and that community-based individuals in that location likely form a major reservoir for glycopeptide-resistant enterococci. PMID- 8627065 TI - Interaction of Haemophilus influenzae with the mammalian extracellular matrix. AB - The adhesiveness of 2 unencapsulated nonfimbriated strains of Haemophilus influenzae, 23459 and 23330, and the encapsulated fimbriated strain 770235 to extracellular matrix (ECM) and to its isolated components was studied, as was the potential of H. influenzae plasminogen receptors to enhance degradation of ECM and bacterial penetration through basement membrane. All strains exhibited efficient adhesiveness to reconstituted basement membrane and to ECM from cultured human endothelial cells. Strains 23459 and 23330 efficiently adhered to immobilized laminin, fibronectin, and various collagens. Strain 770235 adhered efficiently to fibronectin and type I and III collagens and with low efficiency to laminin. With all 3 strains, plasmin generated on H. influenzae plasminogen receptors degraded laminin and fibronectin as well as ECM from human endothelial cells. Plasmin bound on H. influenzae cells also potentiated penetration of bacteria through a basement membrane preparation reconstituted on membrane filters. These results give evidence for a role of ECM adherence and plasminogen activation in the spread of H. influenzae through tissue barriers. PMID- 8627066 TI - The relationship between plasminogen activator inhibitor-1 and proinflammatory and counterinflammatory mediators in children with meningococcal septic shock. AB - Proinflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin [IL] 6 and -8), counterinflammatory compounds (IL-10 and soluble TNF receptors p55 and p75 [sTNFR-55 and -75]), and hemostatic parameters were determined in 38 patients with meningococcal septic shock. Eleven patients (29%) died. Serum levels of pro- and counterinflammatory compounds and plasma levels of plasminogen activator inhibitor (PAI)-1 were significantly higher in nonsurvivors. The interval between appearance of petechiae and blood sampling was shorter in nonsurvivors than in survivors (3.6 +/- 2.4 vs. 6.1 +/- 3.3 h; P = 0.4). This interval correlated strongly with the levels of TNF-alpha, IL-6, -8, and -10, sTNFR-55 and -75, and PAI-1. However, with the exception of PAI-1, differences between concentrations of these mediators disappeared after adjustment for the interval. PAI-1 levels correlated with TNF-alpha concentrations (r = .75; P < .001) and were 1.9 (P = .01) times higher in nonsurvivors at a similar TNF-alpha concentration. Thus, an increased PAI-1 response to TNF-alpha may be associated with fatality, probably because of polymorphism of the PAI-1 gene. PMID- 8627067 TI - A noncovalent complex vaccine prepared with detoxified Escherichia coli J5 (Rc chemotype) lipopolysaccharide and Neisseria meningitidis Group B outer membrane protein produces protective antibodies against gram-negative bacteremia. AB - Earlier studies showed that purified IgG from sera of rabbits immunized with a boiled Escherichia coli J5 (Rc chemotype) whole cell vaccine protected neutropenic rats against gram-negative bacterial sepsis. In the present study, de O-acylated J5 lipopolysaccharide (J5 DLPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) elicited anti-J5 LPS antibodies in rabbits. IgG prepared from immune rabbit sera protected neutropenic rats against lethal challenge with Pseudomonas aeruginosa 12:4:4 (Fisher Devlin immunotype 6). Sixteen of 26 rats treated with the postimmune serum IgG were protected compared with none of 20 rats treated with the control rabbit serum IgG (P < .001). In vitro binding studies showed binding of anti-J5 IgG to several gram-negative bacteria. These results indicate that a subunit vaccine made of J5 DLPS as a noncovalent complex with GBOMP may protect against gram-negative bacteremia. PMID- 8627068 TI - Comparison of the effects of Shiga-like toxin 1 on cytokine- and butyrate-treated human umbilical and saphenous vein endothelial cells. AB - To examine the reported heterogeneity of endothelial cells to Shiga-like toxin 1 (Stx1), the responses of human umbilical (HUVEC) and saphenous (HSVEC) vein endothelial cells to cytokines, butyrate, and toxin were compared. Untreated HSVEC were generally more susceptible than were HUVEC to Stx1; pretreatment of either cell with lipopolysaccharide, interleukin-1 beta, or tumor necrosis factor alpha enhanced Stx1 toxicity. Dexamethasone alone increased total globotriaosylceramide (Gb3) content and toxin binding but inhibited cytokine enhanced cytotoxicity, whereas the differentiation agent, sodium butyrate, increased both Gb3 content and cytotoxicity responses to Stx1, most prominently in HSVEC. Stx1 toxicity directly correlated with the release of von Willebrand factor from HSVEC but not from HUVEC. Thus, HUVEC and HSVEC exhibit distinctive responses to Stx1, cytokines, and butyrate. This suggests the need for caution in extrapolating from in vitro studies utilizing one endothelial cell type to in vivo events during pathogenesis of Stx-mediated thrombotic microangiopathies. PMID- 8627069 TI - The interrelationship between cytotoxin-associated gene A, vacuolating cytotoxin, and Helicobacter pylori-related diseases. AB - The interrelationship between cytotoxin-associated gene A (CagA), vacuolating cytotoxin (VacA), and Helicobacter pylori-related diseases was investigated in 155 H. pylori-infected patients. Four (7%) of 60 subjects had mixed cagA+ and cagA- H. pylori infections. The H. pylori isolates from 98.3% of 121 patients with anti-CagA antibodies were cagA+. The occurrence of cagA+ H. pylori among 76 patients with peptic ulcer disease (PUD) was higher (93.4%) than among 79 patients with functional dyspepsia (FD; 64.6%) (odds ratio [OR] = 7.80; P < .001). VacA+ isolates were isolated from 56.6% of the PUD patients and 35.4% of the FD patients (OR = 2.37; P = .0132). For type I (cagA+VacA+) isolates, these numbers were 56.6% and 31.6%, respectively (P = .003). Only 4% of the 71 VacA+ isolates were cagA-. In addition, 37% of the patients with PUD were infected with cagA+VacA- H. pylori. Chi 2 results did not improve when VacA was entered into the model in the presence of cagA, indicating that only cagA is associated with PUD. PMID- 8627070 TI - The epidemiology of Vibrio infections in Florida, 1981-1993. AB - The epidemiology of 690 Vibrio infections reported in Florida during 1981-1993 is described. Most infections resulted in one of three clinical syndromes: gastroenteritis (51%), wound infections (24%), or primary septicemia (17%). Case fatality rates were 1% for gastroenteritis, 5% for wound infections, and 44% for primary septicemia. While gastroenteritis had little seasonal variation, 91% of primary septicemias and 86% of wound infections occurred from April through October, mostly due to the seasonality of Vibrio vulnificus and Vibrio parahaemolyticus infections. Infected wounds were largely a result of occupational activities around seawater. Some 68% of gastroenteritis cases and 83% of the primary septicemias were associated with raw oyster consumption. Preexisting liver disease was present in 48% of patients with primary septicemia and was associated with a fatal outcome in both wound infections (relative risk [RR], 28.3; 95% confidence interval [CI], 6.3-127.5; P < .0001) and primary septicemia (RR, 1.9; 95% CI, 1.2-3.1; P < .01). PMID- 8627071 TI - A flagella-less mutant of Borrelia burgdorferi as a live attenuated vaccine in the murine model of Lyme disease. AB - The immunogenicity and protective efficacy of an attenuated isolate of Borrelia burgdorferi, the agent of Lyme disease, were evaluated. An isogenic pair of isolates of strain HB19 differed in the expression of flagella; neither produced systemic infection or persisted in the skin of mice. In a comparison of intradermally administered live flagella-bearing and flagella-less cells, the flagella-less isolate was equal to or superior to the flagella-bearing wild type in eliciting growth-inhibiting antibodies and preventing infection in mice. In a comparison of live and killed flagella-less cells, immune responses to live cells were superior to those to killed cells, as assessed by ELISA, growth inhibition assay, and infectious challenge. The dose protecting 50% of mice was 10(3.8) and 10(5.2) for live and killed cells, respectively (P < .05). PMID- 8627072 TI - Multinucleated giant cell formation of swine microglia induced by Mycobacterium bovis. AB - Multinucleated giant cells (MGC) have been long recognized as a histopathologic feature of tuberculosis, yet little is known about the underlying mechanism of tubercle bacillus-induced formation of these fused macrophages. The main purpose of this study was to characterize cellular mechanisms involved in MGC formation of swine microglia, the resident macrophages of the brain, in cultures containing nonopsonized Mycobacterium bovis. Within 2 h of incubation, MGC were readily detected in these cultures by light and transmission electron microscopy. MGC formation was blocked by anti-CD14 and anti-CD18 antibodies and by thalidomide, a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha) production my microglia. Also, TNF-alpha alone induced MGC formation. These findings suggest that two microglial cell receptors, CD14 and a beta2 integrin, and the cytokine TNF-alpha participate in M. bovis-induced swine microglial MGC formation. PMID- 8627073 TI - Phagocytosis and intracellular killing of Candida albicans by macrophages exposed to myeloperoxidase. AB - Candida albicans is an opportunistic pathogen whose resurgence coincides with the rising number of AIDS patients. Neutrophils are known to be involved in the clearance of Candida infections; however, the role of macrophages in host defenses against this organism is not well understood. The present study was undertaken to examine an unrecognized interaction between neutrophils and macrophages resulting in enhanced killing of candidae in vitro. Murine peritoneal macrophages exposed to recombinant myeloperoxidase exhibited enhancement of the respiratory burst, increased phagocytosis, and a dose-dependent increase in intracellular killing of Candida species. Radical scavengers reduced the killing, indicating a role of reactive oxygen intermediates in the candidacidal activity observed. These data suggest that at the site of infection, myeloperoxidase released from neutrophils activates macrophages and induces microbicidal activity. PMID- 8627074 TI - Phase I study of amphotericin B colloidal dispersion for the treatment of invasive fungal infections after marrow transplant. AB - Amphotericin B colloidal dispersion (ABCD; Amphocil) was evaluated in a phase I dose-escalation study in 75 marrow transplant patients with invasive fungal infections (primarily Aspergillus or Candida species) to determine the toxicity profile, maximum tolerated dose, and clinical response. Escalating doses of 0.5 8.0 mg/kg in 0.5-mg/kg/patient increments were given up to 6 weeks. No infusion related toxicities were observed in 32% of the patients; 52% had grade 2 and 5% had grade 3 toxicity. No appreciable renal toxicity was observed at any dose level. The estimated maximum tolerated dose was 7.5 mg/kg, defined by rigors and chills and hypotension in 3 of 5 patients at 8.0 mg/kg. The complete or partial response rate across dose levels and infection types was 52%. For specific types of infections, 53% of patients with fungemia had complete responses, and 52% of patients with pneumonia had complete or partial responses. ABCD was safe at doses to 7.5 mg/kg and had tolerable-infusion-related toxicity and demonstrable antifungal activity. PMID- 8627075 TI - Treatment of experimental cryptococcal meningitis with fluconazole: impact of dose and addition of flucytosine on mycologic and pathophysiologic outcome. AB - Fluconazole is effective in the therapy of cryptococcal meningitis in patients with AIDS. The optimal dosage of fluconazole and the impact of combination with flucytosine are not known. In this study, rabbits with experimental cryptococcal meningitis were given fluconazole at low, intermediate, or high dose or in combination with a low or intermediate dose of flucytosine. Serial cerebrospinal fluid (CSF) examinations showed that all three doses of fluconazole and low-dose fluconazole in combination with intermediate-dose flucytosine were effective in reducing CSF cryptococcal titer, lactate, white blood cell count, and cryptococcal antigen (CRAG) titers. The intermediate and high doses of fluconazole reduced CSF fungal (P < .05) and CRAG (P < .001) titers earlier than low-dose fluconazole alone or in combination with flucytosine. Only the highest dose of fluconazole reduced brain edema after 7 days. In this model of cryptococcal meningitis, there was evidence of a dose response with fluconazole but no in vivo synergism with flucytosine. PMID- 8627076 TI - J774 murine macrophage-like cell interactions with Cryptococcus neoformans in the presence and absence of opsonins. AB - The interaction of Cryptococcus neoformans with the murine macrophage-like cell line J774.16 was studied in the presence and absence of monoclonal antibodies (MAbs) to the capsular polysaccharide glucuronoxylomannan (GXM). In the absence of MAb 2H1 to GXM, coincubation of J774.16 cells with C. neoformans reduced fungal colony-forming units in only 26.6% of 21 independent experiments. In the presence of MAb 2H1, coincubation of J774.16 cells with C. neoformans reduced fungal colony-forming units in > 95% of experiments. Comparison of the relative efficacy of two IgG1 MAbs revealed that the higher affinity MAb was more effective at low concentrations. Antibody-mediated reductions of C. neoformans colony-forming units by J774.16 cells occurred despite inhibition of nitric oxide synthase, addition of reactive oxygen intermediate scavengers, or the use of a superoxide-deficient J774 mutant line. PMID- 8627077 TI - Antibody response patterns against Schistosoma mansoni in a recently exposed community in Senegal. AB - Acquired immune resistance is believed to be largely responsible for age dependent infection and reinfection patterns in schistosomiasis. In a recently established but intense focus of Schistosoma mansoni in Senegal, the humoral immune response was studied in a random population sample of 289. Antibody levels of various isotypes to schistosome worm and egg antigens were determined by ELISA and related to egg counts (eggs per gram of feces [EPG]), age, and sex. Both IgG1 and IgG4 followed age-related patterns similar to egg counts and strongly correlated with EPG, even after allowing for age. Specific IgE levels increased slowly with age. The humoral immune response patterns in this recently infected population appeared to be largely similar to those in chronically infected communities. Thus far, the observations do not support the current hypothesis that age-related resistance to Schistosoma is determined by IgE-mediated protective immunity acquired during many years of exposure. PMID- 8627078 TI - Antischistosome IgG4 and IgE responses are affected differentially by chemotherapy in children versus adults. AB - Specific IgG4 and IgE responses and polyclonal cytokine profiles were studied in 110 Schistosoma haematobium-infected persons before and 5 weeks after chemotherapy. Pretreatment IgG4 responses to soluble egg antigen (SEA) correlated with intensity of infection. After chemotherapy, a significant decrease in egg output and circulating anodic antigen was associated with a substantial drop in the IgG4 response to SEA (IgG4-SEA) in adults and children, suggesting that egg laying is a major stimulus for IgG4-SEA. After chemotherapy, IgG4 and IgE to adult worm antigen and IgE to SEA increased in children but remained unchanged in adults. This indicates that the immunoregulatory mechanisms operative in S. haematobium-infected adults differ from those in infected children. The effect of treatment on cytokine profiles was determined following stimulation of whole blood with anti-CD3 antibodies. A significant decrease in interleukin-4 production after treatment indicated that reduction in helminth load may lead to a reduced number of Th2-type cells. PMID- 8627079 TI - Immunologic properties of Epstein-Barr virus-seronegative adults. AB - Epstein-Barr virus (EBV) seronegativity is rare in people > 20 years old. However, some persons remain EBV-seronegative for nearly their whole lives. The aim of this study was to examine properties of the immune system of EBV seronegative adults that could contribute to long-term EBV seronegativity. Therefore, differential blood cell counts and lymphocyte subpopulations were determined, and the production of interferon (INF)-alpha and -gamma and interleukin (IL)-6 and -2 in a whole blood assay was investigated. Whereas no differences in the distribution of lymphocyte subpopulations between EBV seronegative and -positive adults were found, a significant higher percentage of monocytes in EBV-seronegative adults was observed. Significantly more IFN-alpha and IL-6 were detected in culture supernatants of EBV-seronegative persons after stimulation with Newcastle disease virus. In contrast, no differences in the induction of the lymphokines IFN-gamma and IL-2 were seen. These data suggest that faster and higher production of IFN-alpha and IL-6 amy protect EBV seronegative adults against EBV infection. PMID- 8627080 TI - Effect of stavudine on human immunodeficiency virus type 1 virus load as measured by quantitative mononuclear cell culture, plasma RNA, and immune complex dissociated antigenemia. AB - The antiviral effect of stavudine (2', 3'-didehydro-3'-deoxythymidine) against human immunodeficiency virus (HIV) type 1 was measured in 15 HIV-infected patients at baseline and at weeks 4, 10, 22, 34, and 52 of therapy. Patients received 0.1, 0.5, 1.0, or 2.0 mg/kg/day of stavudine. At all time points examined during the 52 weeks of therapy, the median virus titers in peripheral blood mononuclear cells were decreased 1-2 logs, and median immune complex dissociated antigen levels were reduced 37%-67% compared with baseline values. Plasma RNA content measured by polymerase chain reaction was reduced approximately 0.5 log from baseline median values at both time points examined (weeks 10 and 52). These data demonstrate that stavudine has a substantial and durable antiviral effect. PMID- 8627081 TI - The seroepidemiology of Bordetella pertussis infections: a study of persons ages 1-65 years. AB - Although the incidence of Bordetella pertussis infections had decreased significantly since the introduction of widespread vaccination, an increase in the number of cases has occurred recently. In an attempt to define the seroepidemiology of pertussis in Nashville, antibody levels to pertussis toxin (PT) and filamentous hemagglutinin (FHA) were measured by ELISA in 585 serum samples from healthy 1- to 65-year-old subjects. Data were analyzed by smooth curve fitting and by comparison of trends in PT and FHA titers across age groups. The results demonstrated a peak in PT and FHA titers in the 4-6 year age group, concurrent with the administration of booster doses of diphtheria-tetanus toxoid pertussis vaccine. A second larger peak was noted in the 13-17 year age group. These data suggest that pertussis infection is frequent during the adolescent years. PMID- 8627082 TI - The public health impact of Lyme disease in Maryland. AB - The purpose of this study was to estimate the health burden imposed by Lyme disease (LD) in Maryland during 1992 and 1993. A cross-sectional 1-in-15 survey of physicians (total, 1200) in Maryland was conducted to estimate the incidence of diagnosed LD, presumptive cases of LD, patients with tick bites, and diagnostic tests ordered for LD. Results show that LD is underreported by 10- to 12-fold in Maryland, that 80% of cases are managed by primary care physicians, and that there is discordance between the actual clinical treatment of patients and the recommended approach. In addition, the much greater numbers of patients treated for presumptive LD, seen and given prophylaxis for tick bites, and having diagnostic tests indicate that real and perceived LD is a far greater public health problem and uses more medical resources than official surveillance data suggest. PMID- 8627083 TI - Staphylococcal food poisoning caused by imported canned mushrooms. AB - From February through April 1989, four outbreaks of staphylococcal food poisoning in the United States were associated with eating mushrooms canned in the People's Republic of China (PRC). In the four outbreaks, 99 persons who ate at a suspect facility developed gastrointestinal symptoms within 24 h, including 18 who were hospitalized. Illness was associated with eating mushrooms at a university cafeteria (relative risk [RR] = 53.0), a hospital cafeteria (RR = 13.8), a pizzeria (odds ratio [OR] = infinity), and a restaurant (OR = infinity) (all P < .0001). Staphylococcal enterotoxin A was found by ELISA in mushrooms at the sites of two outbreaks and in unopened cans from the three plants thought to have produced mushrooms implicated in outbreaks. These investigations led to multistate recalls and a US Food and Drug Administration order to restrict entry into the United States of all mushrooms produced in the PRC; until this action, the United States imported approximately 50 million pounds yearly. PMID- 8627084 TI - T lymphocytic and immature macrophage alveolitis in active pulmonary tuberculosis. AB - The phenotype of bronchoalveolar cells from 11 healthy subjects and from affected and unaffected lungs of 15 patients with pulmonary tuberculosis (PTB) was determined. An immature macrophage alveolitis was found in the affected lung and the unaffected lung versus controls as determined by morphology and peroxidase activity. T lymphocytic alveolitis also was found in the affected but not the unaffected tuberculous lung compared with healthy controls. The majority of alveolar lymphocytes in unaffected and affected PTB lungs were T cells expressing the alpha beta T cell receptor. Alveolar T cells from both unaffected and affected lungs were activated, as determined by increased expression of CD69 and HLA-DR. Interleukin-2 receptor (IL-2R alpha) expression was, however, unchanged on alveolar lymphocytes from affected lung and was decreased in the unaffected lung. Thus, activated T lymphocytes and immature macrophages in the tuberculous lung are basic to the local immunopathogenesis of PTB. PMID- 8627085 TI - Detection of coccidioidal antibodies by 33-kDa spherule antigen, Coccidioides EIA, and standard serologic tests in sera from patients evaluated for coccidioidomycosis. AB - During a 9-month study of patients being evaluated for coccidioidomycosis, 1 or more serum samples were obtained from 138 patients with an illness suggestive of recent infection. In this group, standard immunodiffusion tests of unconcentrated sera were positive for 25; 49 additional patients had at least 1 reactive test result by newer enzyme-linked serologic tests. At least 11 of these 49 patients had coccidioidomycosis as determined by culture or subsequent standard serologic tests. Patients with coccidioidomycosis identified only by newer tests had fewer or milder clinical abnormalities than did patients in whom the disease was detected by standard tests. For 31 other patients with illness of a chronic or undetermined duration, newer tests detected only 10 more than the 18 identified by standard tests, suggesting that later in the course of illness, standard testing gains in sensitivity for coccidioidal infection. PMID- 8627086 TI - Elevations in granulocyte-macrophage colony-stimulating factor and interleukin-5 levels precede posttreatment eosinophilia in onchocerciasis. AB - The eosinophil survival assay was used to quantitate cytokines in 17 serial serum samples from 10 patients treated for onchocerciasis with diethylcarbamazine. Eosinophils isolated from normal donors were cultured for 4 days in the presence of patients' sera, and cell viability was determined. Serum specimens from 9 of 10 patients enhanced eosinophil survival from 4.8% +/- 2.2% (mean +/- SE) before treatment to 50.0% +/- 6.4% after treatment. Survival enhancement activity peaked before posttreatment eosinophilia. Antibodies to interleukin (IL)-5, granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-3 were used to block cytokine activity in 22 serum samples. Antibodies to IL-5 blocked survival in 5 samples, antibodies to GM-CSF blocked survival in 6 samples, and a combination of antibodies to IL-5 and GM-CSF blocked survival in 8 additional samples. Overall, posttreatment sera from patients treated for onchocerciasis enhanced eosinophil survival; both GM-CSF and IL-5 may promote the posttreatment eosinophilia in filarial infection. PMID- 8627088 TI - Field evaluation of a polymerase chain reaction-based nonisotopic liquid hybridization assay for malaria diagnosis. AB - In a blind field evaluation of a nonisotopic liquid hybridization assay for detection of malaria parasites, 100 blood samples were tested from an area in which malaria is endemic; light microscopy was used as the reference test. Sensitivity, specificity, and positive and negative predictive values of the hybridization assay were 100%. One sample that was microscopy-negative and hybridization-positive was positive when reexamined. Another sample that was microscopy-positive and hybridization-negative was negative at reexamination. The detection limit of the test was > or = 0.0005% parasitemia. Four samples with mixed infections were misdiagnosed by microscopy as single-species infections. Four samples diagnosed as mixed infections by microscopy and single infection by the hybridization test had no evidence of a second Plasmodium species upon reexamination. The polymerase chain-reaction-based nonisotopic liquid hybridization assay was better than conventional light microscopy in detecting low-grade parasite infection and offers an exceptional advantage for detecting mixed infections. PMID- 8627089 TI - Coccoid forms of Helicobacter pylori. PMID- 8627087 TI - Big endothelin in patients with complicated Plasmodium falciparum malaria. AB - Plasma concentrations of big endothelin-1 were determined by ELISA in 18 patients with complicated Plasmodium falciparum malaria in Bangkok. Before therapy, elevated levels were recorded (21 +/- 12 vs. 2.9 +/- 1.1 pmol/L in age- and sex matched healthy subjects; P < .001). Even 7 days after therapy, elevated concentrations were seen (25 +/- 14 pmol/L). Plasma endothelin levels were correlated with levels of tumor necrosis factor-alpha (r = .632, P < .01), and a negative correlation with platelet counts was seen (r = .783, P < .005). No relation between plasma endothelin concentrations and parasitemia, fever, or other indices of severe infection (hypotension, renal, hepatic or pulmonary impairment, cerebral malaria) existed. During and after complicated malaria, increased levels of plasma endothelin could contribute to malarial pathology or reflect endothelial damage or both. PMID- 8627090 TI - Comparison of cotton and rayon/cotton tampons for efficacy of toxic shock syndrome toxin-1 production. PMID- 8627091 TI - Relative importance of oral versus intravenous vancomycin exposure in the development of vancomycin-resistant enterococci. PMID- 8627092 TI - Folinic acid supplements to pyrimethamine-sulfadiazine for Toxoplasma encephalitis are associated with better outcome. PMID- 8627093 TI - Evaluation and prediction of the facial appearance after surgical correction of mandibular hyperplasia. AB - The response of the entire facial soft tissue to correction of mandibular hyperplasia was evaluated in 25 patients by moire topography. Postoperative changes were quantitatively analyzed by a computer-aided superimposition technique. Posterior displacements occurred in the lower third of the face. A marked retraction was observed around the chin, but the changes gradually decreased in the coronal and posterior directions with distance from the chin. The upper lip was slightly reduced; however, no direct effect was found in the cheeks. The results of this study constitute a database that, if used in conjunction with an interactive imaging system, would allow surgeons to predict accurately the postoperative appearance of the patient. PMID- 8627094 TI - Experience with e-PTFE membrane application to bone grafting of cleft maxilla. AB - Previous clinical studies and animal experiments have demonstrated that the placement of expanded polytetrafluoroethylene (e-PTFE) membranes (GORE-TEX) may be valuable for bone regeneration in nonosteogenic areas. This study aimed to explore the application of this technique to bone grafting of wide alveolopalatal clefts. Ten patients with bilateral clefts were selected and during a 2-week period, all received autogenic cancellous iliac bone bilaterally. The membrane was placed nasally and orally on the larger cleft side and removed after 3-6 months. All patients have been followed for 14 months. Bone graft incorporation was successful except for one patient (membrane side), who was regrafted 1 year later. However, soft-tissue problems with membrane exposure occurred in the majority of patients, while on the nonmembrane side, healing was uneventful in all cases. Further research in membrane technology is necessary before this method can be accepted for cleft grafting. PMID- 8627095 TI - Overgrowth of a costochondral graft in an adult male. AB - This paper is the first report of overgrowth of a costochondral graft in an adult male. The case supports the notion that this graft partly exhibits features of a primary growth center with inherent potential for growth. PMID- 8627096 TI - Traumatic aneurysm of the maxillary artery: the role of interventional radiology. A report of two cases. AB - Two cases of post-traumatic aneurysm of the maxillary artery are described. The first patient was a 20-year-old man who sustained a Le Fort III type fracture in a road traffic accident. He experienced two episodes of significant maxillofacial haemorrhage, the first following admission and the second 5 days after initial reduction and fixation of his midfacial fractures. The second patient was a 23 year-old man with a bilateral cleft palate and extreme midfacial hypoplasia who underwent Le Fort I osteotomy. Significant bleeding commenced 3 h postoperatively and was not completely controlled by anterior and posterior nasal packing. Both the aneurysms were diagnosed on selective carotid angiography and successfully treated by embolization. PMID- 8627098 TI - A new appliance for condylar positioning (clamp system). AB - A new appliance for condylar positioning during sagittal split ramus osteotomy (SSRO) of the mandible is presented. The application procedure and our method of condylar repositioning are described. PMID- 8627097 TI - Anthelix-conchal reconstruction with postauricular "revolving door" island flap. AB - The postauricular "revolving door" island flap is commonly used to reconstruct the conchal area after ablative surgery. This procedure may also be used to reconstruct the anthelix-conchal area after surgical removal of a squamous cell carcinoma of the external ear. The technique is simple and safe, and it can be performed as a one-stage procedure; moreover, excellent aesthetic results can be achieved. PMID- 8627099 TI - Effectiveness of primary correction of traumatic telecanthus. AB - To assess the aesthetic and functional results of primary treatment of telecanthus in patients with naso-orbito-ethmoidal fractures, the results in 36 patients were evaluated in a retrospective study. Twenty had double-sided telecanthus: 13 required an indirect technique of canthopexy and seven a direct technique. Sixteen had unilateral telecanthus, of whom six were treated by the indirect and 10 by the direct technique. The intercanthal distance (ICD) was measured directly postoperatively and more than 12 months after reconstruction. The late ICD after application of the direct technique was nearly 3 mm smaller (ANOVA, P < or = 0.02, mean 34.3 mm) and yielded 2 mm less relapse (ANOVA, P < or = 0.02) as compared with the indirect technique. Delayed or late-primary treatment showed a significantly higher frequency of epiphora (chi-square test, P < or = 0.05). Early primary treatment of traumatic telecanthus produced the best aesthetic and functional result. PMID- 8627101 TI - Immunocytologic detection of isolated tumor cells in bone marrow of patients with squamous cell carcinomas of the head and neck region. AB - The well-documented specificity of anticytokeratin monoclonal antibodies for detection of epithelial micrometastatic cancer cells in bone marrow as a prognostic indicator inspired us to apply this approach to patients with squamous cell carcinomas (SSC) of the head and neck region. The sensitivity of the broad spectrum anticytokeratin monoclonal antibody (mAb) A45-B/B3 used for tumor cell detection was demonstrated by immunostaining of cryostat sections from the respective primary tumors. Analysis of 31 patients with SSC revealed A45-B/B3 positive cells in 10 cases (32.3%) at frequencies of 1-207 per 1 x 10(6) mononuclear cells. Most specimens displayed isolated tumor cells, while cell clusters were found in only two cases (6.5%). The present data suggest that hematogenous dissemination of cancer cells is more frequent than expected from clinicopathologic staging of patients with SSC of the head and neck region. PMID- 8627100 TI - Simplified split-bone technique for removal of impacted mandibular third molars. AB - A modification of the lingual split-bone technique is presented, based on an osteomucoperiosteal flap. The technique described is simple, and it requires less time and causes less tissue trauma than other accepted techniques. The bone exposure and bone loss are minimal. The complication rate is extremely low. PMID- 8627102 TI - Pitfalls in the treatment of delayed lymph-node metastases after control of small tongue carcinomas. AB - Between 1985 and 1992, 13 patients were treated for delayed lymph-node metastases that developed after obtaining control of primary lesions of the tongue. These lesions were treated primarily by radiotherapy or surgical resection; cervical metastases were treated mainly by delayed radical neck dissection (RND). Seven of the 13 patients survived with no evidence of recurrence. The other six patients developed tumors in the neck, and five of these patients died due to uncontrollable disease. In all but one patient, recurrence appeared between the site of the primary lesion and the region of RND, the oral floor and/or the parapharyngeal space. None of the patients with recurrence received radiotherapy in the area between the primary lesion and the site of RND. In contrast, there was no recurrence in patients who received external irradiation to the primary lesion and upper cervical lymph nodes. This review emphasizes the need to direct more attention to the area between the site of the primary lesion and the regional lymph nodes in patients receiving treatment for delayed metastases associated with small carcinomas of the tongue. PMID- 8627103 TI - Congenital teratoid cyst of the floor of the mouth--a case report. AB - A case of sublingual congenital teratoid cyst is reported and the recent literature is reviewed. Histopathologic study of the enucleated cyst revealed striated muscle, cartilage, fat, salivary gland tissue, and gastrointestinal epithelium in a cyst wall of dense fibrous tissue with a thin layer of ciliated columnar and partially stratified squamous epithelium. PMID- 8627104 TI - Spindle cell lipoma of the oral cavity. PMID- 8627105 TI - Evaluation of serum concentration of parathyroid hormone-related protein and its implication in hypercalcemia in squamous cell carcinoma of the head and neck. AB - Hypercalcemia is a common and serious complication associated with squamous cell carcinoma (SCC) and is considered to be caused by a tumor-derived factor, parathyroid hormone-related protein (PTHrP). However, the correlation between serum levels of calcium and PTHrP and the kinetics of PTHrP in SCC of the head and neck is unknown, because the behavior of the circulating form of PTHrP in patients has not been determined. In the present study, the PTHrP concentrations in serum samples from 54 patients (37 with SCC and 17 with benign tumors) were measured by a recently developed radioimmunoassay directed toward the C-terminal region of PTHrP, and the laboratory data including those calcium levels in corresponding samples were reviewed retrospectively. Results showed hypercalcemia in four patients with advanced cancer and in whom elevation of the serum PTHrP concentration was observed simultaneously. The regression analysis also revealed the linear relationship of the calcium level to the PTHrP concentration, but not to the concentration of phosphorus or creatinine, suggesting that monitoring of serum PTHrP level is useful for prediction of hypercalcemia associated with head and neck cancer. PMID- 8627106 TI - Lhermitte-Duclos disease associated with Cowden's disease. AB - The twelfth patient in the known literature with Lhermitte-Duclos disease (dysplastic gangliocytoma) occurring in association with Cowden's disease is described. The investigation, aetiology, and management of the case are discussed. PMID- 8627107 TI - An experimental study on the recovery of the lingual nerve after injury with or without repair. AB - The recovery of the mechanosensitive and thermosensitive afferent fibres in the lingual branch of the trigeminal nerve has been studied using electrophysiological techniques in cats after nerve section without repair or after section followed by nerve repair 12 weeks later. In the unrepaired group, recovery was permitted for 24 weeks, and after delayed repair there was a further recovery period of either 12 or 24 weeks. The characteristics of the regenerated fibres were then investigated, and data were also compared with those from normal control animals and from animals which had undergone immediate nerve repair. The results revealed only small differences between the repaired and unrepaired groups and it is concluded that delayed repair of a clean transection site results in only slightly better recovery than leaving the nerve unrepaired. It is also concluded that a 12-week delay before repair has little detrimental effect. PMID- 8627108 TI - Detection of Mycobacterium leprae in nerve lesions by the polymerase chain reaction. AB - A simple procedure is described for the detection of Mycobacterium leprae by the polymerase chain reaction in nerve biopsies sectioned with a cryostat and then treated with proteinase K. All samples from lepromatous leprosy patients and the majority of samples from paucibacillary cases yielded positive results. This approach may be useful for differentiating between leprosy and other inflammatory neuropathies. PMID- 8627109 TI - A survey of leprosy impairments and disabilities among patients treated by MDT in Burkina Faso. AB - Since 1990, Burkina Faso, a West African country, has carried out a national leprosy control program treating with WHO/MDT nearly 12,000 patients between 1990 and 1994. A sample survey of 600 cases among these patients showed that 29.8% were disabled cases. There was a predominance of males, older patients, the multibacillary form of leprosy, and former cases treated with dapsone before MDT. The actual rate increased 8.5% compared to the frequency of disabilities at detection (21.3%). The need for disability care was estimated, respectively, at 24.4% and 5% for primary and secondary grades of disability. These important needs were so great that the authors recommend the planning and initiation of a physical rehabilitation and disability prevention program in this country. PMID- 8627110 TI - Immunotherapy of far-advanced lepromatous leprosy patients with low-dose convit vaccine along with multidrug therapy (Calcutta trial). AB - This report describes a promising mode of treatment of lepromin-unresponsive, far advanced, lepromatous (LL) leprosy patients with antileprosy vaccines as an adjunct to multidrug therapy (MDT). The Trial Groups included 50 highly bacilliferous, lepromin-negative, untreated LL patients. They were given MDT for 2 years. Of them, 30 patients were administered a mixed antileprosy vaccine containing killed Mycobacterium leprae of human origin plus M. bovis BCG. The remaining 20 patients were given M. bovis BCG. Depending on the severity of lepromin unresponsiveness, they were given one to six inoculations at 3-month intervals. Another 20 similar LL patients were taken in the Control Group. They were given only MDT for 2 years. From the start of the study, all patients belonging to the Trial and Control Groups were followed every 3 months for clinical, bacteriological and immunological outcomes. Within 2 years all 50 patients of the Trial Groups and 19 of the 20 patients of the Control Group became clinically inactive and bacteriologically negative. However, the clinical cure and the falls of the bacterial and morphological indexes were much faster in those patients receiving the mixed vaccine therapy than in those patients who were given BCG plus MDT or only MDT. The immunological improvements in the patients of the Trial and Control Groups were assessed by: a) lepromin testing at the beginning of the study and at 3-month intervals and also by b) the in vitro leukocyte migration inhibition (LMI) test at both the beginning and end of the study. As the patients were given more and more vaccinations, the incidence of lepromin conversion increased, more so in the patients receiving the mixed vaccine. Thus, 63%, 15% and 5% of the patients became lepromin positive in those patients receiving the mixed vaccine, BCG, and MDT only, respectively. Lamentably, the vaccine-induced lepromin positivity was temporary and faded away within several months. At the beginning of the study, the LMI test against specific M. leprae antigen was negative in all patients of both the Trial and Control Groups. After the end of the chemo-immunotherapy schedule, the LMI test became positive in 50% and 20% of LL patients receiving the mixed vaccine and BCG, respectively. None of the Control Group could show LMI positivity after completion of the MDT schedule. These results show that treatment of LL patients with the mixed vaccine and MDT could quickly reverse the clinical course of the disease, remove immunologic anergy in some patients, and induce a rapid decrease in the bacterial load in them. PMID- 8627111 TI - Reduction of epidermal cell proliferation in skin lesions in lepromatous leprosy is greater than in indeterminate and tuberculoid leprosy lesions. AB - We have compared epidermal cell proliferation in skin biopsies from areas with lesions to contralateral areas without lesions in patients with indeterminate, tuberculoid and lepromatous leprosy. Cell proliferation was determined as the percentage of labeled cells in the basal and suprabasal epidermal layers, using autoradiographic preparations of skin biopsies taken 1 hr after a 3H-thymidine intradermal injection. We have found a significant reduction in epidermal cell proliferation in areas with lesions in the three groups of patients. The greatest reduction occurred in lepromatous patients. In lesions of patients with indeterminate or tuberculoid leprosy, the reduction was the same, and in both groups it was smaller than in lepromatous patients. In the areas without lesions, the index of labeled cells was similar to that of "normal" skin of nonleprosy patients. In the contralateral unaffected areas from leprosy patients and in "normal" skin from nonleprosy patients, as well as in affected areas from patients with indeterminate leprosy, epidermal cell labeling was greater in the suprabasal layer than in the basal layer. In lesions of lepromatous patients, cell labeling was greater in the basal layer than in the suprabasal layer. Our findings suggest that the reduction of epidermal cell proliferation in leprosy patients is restricted to the cell-mediated immune response, more intense in lepromatous leprosy. It does not seem to be related to denervation, which is greater in tuberculoid leprosy. PMID- 8627112 TI - Viability of Mycobacterium leprae in skin and peripheral nerves and persistence of nerve destruction in multibacillary patients after 2 years of multidrug therapy. AB - The pathological changes, bacterial load, and viability of Mycobacterium leprae in the skin and nerves of nine lepromatous leprosy patients who had undergone 2 years of multidrug therapy (MDT) were studied. M. leprae and varying amounts of their remnants were present in the nerves and skin of all but one patient. M. leprae isolated from skin biopsies of six patients and nerve biopsies of nine patients were inoculated into mouse foot pads. No growth was obtained from any one of them. During the electron-microscopic examination of three nerve biopsies, only one specimen showed a small number of solid-staining M. leprae. These findings would explain the low relapse rate in patients treated with 2 years of fix-duration MDT. Results of a long-term follow up of patients is awaited with interest. The possibility of nerve paralysis due to intraneural microreaction and fibrosis consequent to the continued presence of dead bacterial remnants should be seriously considered. PMID- 8627113 TI - Rapid village survey to determine the size of the leprosy problem in Khon Kaen Province, Thailand. AB - The rapid village survey (RVS) method has been developed as a simpler, less expensive alternative to random sample surveys for determining the prevalence of leprosy and was compared with a total population survey (TVS). In the RVS, the cluster population receives clear information about the disease, and those with symptoms are invited to be examined by the survey team. A list of household contacts and suspects was made and those on the list were actively traced. The registered population was 20,815; 10 new patients were found among the 2034 people self-reporting in the RVS, 0 among the household contacts and suspects, and an additional 2 new patients in the TVS. There were 12 registered patients among the sample population. The prevalence rate found by the RVS was 1.06 per 1000(95% CI = 0.49-1.63) and in the TVS 1.16 per 1000 (95% CI = 0.5-1.77). The man-days and costs of an RVS are considerably less than those for a TVS. It was concluded that the RVS is a valid replacement for the TVS as conducted in Khon Kaen Province, Thailand. The RVS can be applied under low-endemic conditions and could be carried out by the general health staff. PMID- 8627114 TI - Development of a SOD ELISA to determine the immunological relatedness among mycobacteria. AB - This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) system for the measurement of immunological distances (ImDs) of the superoxide dismutase (SOD) molecule among the cultivable mycobacteria, namely, Mycobacterium vaccae, M. phlei, M. smegmatis, M. avium, M. scrofulaceum, M. tuberculosis H37Ra, M. tuberculosis H37Rv, and M. bovis BCG, and M. leprae. SODs from cultivable mycobacteria were purified, antibodies were raised against these molecules, and ImDs between these anti-SOD antibodies and antigen (SODs) were determined by an immunoprecipitation technique standardized earlier and by the ELISA technique developed in this study. The ELISA system developed in this study showed higher sensitivity and consistent and reproducible ImDs among various mycobacteria, including pathogens such as M. tuberculosis, M. leprae and M. avium. These values were comparable with the values derived by the immunoprecipitation technique. Our ELISA technique appears to be a sensitive and rapidly reproducible method with the additional advantage of the stability of reagents, and holds promise in the taxonomy as well as in the development of diagnostics for leprosy and other mycobacterial infections. PMID- 8627116 TI - Climatic droplet keratopathy in leprosy. AB - An atypical gross form of climatic droplet keratopathy (CDK) occurring in the cornea of 24 eyes of 17 leprosy patients is described. The CDKs were situated in the lower outer quadrant of the cornea and were not associated with trauma, scars or vascularization. All patients, except one, belonged to the lepromatous group. Fifteen of the 24 eyes with this form of CDK had impaired corneal sensation. We suggest that this form of CDK is a distinctive condition found among lepromatous leprosy patients with possible infection and involvement of the corneal nerves. PMID- 8627115 TI - Immunological profiles of leprosy patients and healthy family contacts toward M. leprae antigens. AB - In this study, we measured simultaneously the in vitro and in vivo T lymphocyte reactivities and the antibody responses of leprosy patients and healthy family contacts (HFC) toward Mycobacterium leprae antigens. The in vitro lymphoproliferative response of the HFC to leprosin A was comparable to that of tuberculoid leprosy patients. However, their skin-test reactivity to Dharmendra lepromin was considerably higher compared to the in vitro response to leprosin A. A significant number of HFC failed to respond to M. leprae antigens, both in vitro and in vivo, and the unresponsiveness to either test was not related to the type of leprosy patients in the household. A marginal correlation was observed between the skin-test reactivity of HFC and the age of the individuals. Even though a significant proportion of HFC showed positive anti-PGL-I IgM levels, none showed a positive titer in the serum antibody competition test toward the M. leprae-specific epitope My2. A positive anti-PGL-I IgM response together with a negative lepromin skin-test reactivity showed a clear downward trend from the lepromatous pole toward the tuberculoid pole. A small number of HFC, all contacts of lepromatous patients, were lepromin skin-test negative with positive anti-PGL I IgM levels, but the majority among them showed T-cell reactivity to mycobacterial antigens in vitro. These results are discussed in relation to immunological correlates of the susceptibility to M. leprae infection. PMID- 8627118 TI - Regarding relapse after long-term follow up of multibacillary patients treated by WHO multidrug regimen. PMID- 8627117 TI - IgG subclass recognition pattern in leprosy: recognition of M. leprae antigens by IgG1 and IgG3 antibodies is distinct across the disease spectrum. AB - The recognition of Mycobacterium leprae antigens by IgG subclasses in patients with leprosy was investigated by electrophoresing M. leprae sonicate in SDS polyacrylamide gel and immunoblotting analysis. Serum pools were used from leprosy patients with either lepromatous (LL/BL) or tuberculoid (BT/TT) disease. A serum pool from healthy controls (EC) was used to determine the baseline antibody activity. To adjust for quantitative differences in antibodies across the disease spectrum, the LL/BL serum pool was used at a 1:200 dilution; the BT/TT serum pool, at 1:20 dilution. Monoclonal antibodies specific for each of the IgG subclasses were used as probes, with anti-mouse IgG conjugated to alkaline phosphatase as the revealing probe. IgG1 antibodies bound to several discrete bands in the range of 10-70 kDa in LL/BL patients, while BT/TT patients showed a more diffuse pattern with the strongest IgG1 antibody binding in the region of 25-40 kDa. Recognition with IgG2 was restricted to a region between 25 36 kDa (which also stained strongly for carbohydrates) in both LL/BL and BT/TT patients. Binding with IgG3 antibodies was more restricted than IgG1 antibodies in LL/BL sera with strong recognition restricted to 25 and 28 kDa. BT/TT sera showed strong binding with IgG3 antibodies in the region of 25-32 as well as 5-7 kDa. IgG4 antibodies showed weak binding to a 28-kDa in lepromatous patients only. The differences in IgG subclass recognition patterns and their implications are discussed. PMID- 8627119 TI - DNA extraction methods from Mycobacterium leprae and M. lepraemurium. PMID- 8627120 TI - Radiometric procedure for detecting a cultivable Mycobacterium in Mycobacterium leprae-infected armadillo tissue. PMID- 8627121 TI - A plea for routine use of fine-needle aspiration cytology in the diagnosis and follow up of leprosy. PMID- 8627122 TI - 1995 Damien-Dutton Award Recipient. PMID- 8627123 TI - Proximity effects for chromosome aberrations measured by FISH. AB - A Monte Carlo simulation computer program for radiation-produced chromosome aberrations, based on the breakage-and-reunion model, was extended to include proximity effects due to localization of chromosomes and limited range for break break interactions. Two adjustable parameters were used. One corresponds to total dose: the other determines proximity effects by specifying the number of 'interaction regions' in a cell nucleus. The use of additional adjustable parameters was avoided by assuming randomness of break induction and aberration production. FISH chromosome painting data were obtained from 1.9 Gy 60Co gamma rays-irradiated human lymphocytes. The data were compared with the computer simulation results, taking individual chromosome lengths into account. With about 13 interaction regions, agreement between the experiment and the simulation was good, even when detailed categories of damage were scored. An estimated average dsb-dsb interaction distance, based on 13 interaction regions, is about 1.3 micron. Monte Carlo methods give useful quantitative estimates of relative aberration yields, with a minimum of adjustable parameters and the theoretical assumptions, and indicated proximity effects. Computer simulation of FISH experiments can be adapted to any number of colours, any scoring criteria and any method of grouping aberrations into categories. Simulation allows systematic extrapolation of aberration data on painted chromosomes to whole-genome aberration frequencies. PMID- 8627124 TI - Classification of X-ray-induced Robertsonian fusion-like configurations in mouse splenocytes. AB - A characteristic karyotypic feature of mouse chromosomes is the presence of large blocks of heterochromatin in the vicinity of the centromeres. Breakage inside this centromeric heterochromatin might result in the formation of abnormal chromosomes, very similar to the metacentric chromosomes derived from Robertsonian fusion. X-rays are very efficient in inducing these Robertsonian fusion-like configurations (RLC) in cultured mouse splenocytes. Observed frequencies of these RLC increase in a linear-quadratic manner with dose. Two types of RLC were found. The first type (approximately 70% of induced RLC) has heterochromatin only in the middle of the chromosome and appears as a metacentric chromosome, whereas the other type (approximately 30%) has a heterochromatic block inside the chromosome arm (and has the appearance of a dicentric chromosome). Induced RLC are difficult to classify as either a stable or unstable aberration, based only on traditional cytogenetic techniques such as C-banding. Here we describe a cytogenetic approach to achieving better insight into the molecular organization of RLC. We utilized two-colour fluorescence in situ hybridization (FISH) using a combination of mouse minor satellite DNA probe and telomeric probe. Over 90% of RLC did not have detectable minor satellite arrays inside the interstitial heterochromatin. Consequently, most of the RLC of the first type should be classified as acentric fragments and those of the second type as translocations. The chance of inducing true Robertsonian fusions in mouse splenocytes by X-rays is < 2.5% based on the total RLC observed. PMID- 8627125 TI - Dose-response curves for simple and complex chromosome aberrations induced by X rays and detected using fluorescence in situ hybridization. AB - A primary human fibroblast cell-line was grown to confluence and X-irradiated at 2, 4 and 6 Gy. The resulting chromosome aberrations were detected in first division cells using a series of FISH assays in which either one or two chromosomes (nos. 1, 2, 4, 5, 7 and 13) and all centromeres were painted with distinct colours. Interchange aberrations were classed as simples if they appeared to originate from a break in each of two chromosomes (dicentric with fragment or reciprocal translocation), or complexes if their origins required three or more breaks in two or more chromosomes. Breaks not obviously connected with exchanges were also scored. The data were corrected to include paint patterns resulting from either incomplete or terminal exchanges. In addition we attempted to correct for the apparently simple exchanges which are actually derived from complex interactions (pseudosimples) using correction factors calculated by establishing the predominant complex families present at each dose. Power Law analysis of the corrected data showed a linear dose-response for simple exchanges and a dose-squared response for complex exchanges. Based upon this observation we suggest that simples result from lesions induced by the same radiation track and complexes arise from the interaction of lesions induced by separate tracks. PMID- 8627127 TI - Intrachromosomal localization of breakpoints induced by the restriction endonucleases AluI and BamHI in Chinese hamster ovary cells treated in S phase of the cell cycle. AB - The restriction endonucleases (REs) AluI and BamHI were electroporated into Chinese hamster ovary (CHO) cells during S phase of the cell cycle and breakpoints in G-banded metaphases were mapped to Giemsa-light or -dark bands or to band junctions. The majority of AluI- and BamHI-induced breakpoints were located in Giemsa-light bands. Both REs induced similar distributions of breakpoint clusters. The localization pattern of S phase-induced breakpoints in CHO cells is similar to the pattern of G1-induced breakpoints reported earlier. These data show that breakpoint localization for both REs is independent of the cell cycle stage (G1 or S) in which aberrations are induced and give further support to the hypothesis that nuclease hypersensitive regions (NHRs) associated with active genes play an important role in the distribution of breakpoints. PMID- 8627126 TI - Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridization. I. Dicentrics and translocations. AB - Chromosome translocations (stable aberrations) can persist longer during cellular proliferation than dicentrics (unstable aberrations). It is important to know the kinetics of the elimination of dicentrics and to what extent translocations persist in an in vivo cell population after irradiation. The female Swiss mouse were used to study the induction and persistence of dicentrics and translocations in splenocytes up to 112 days after exposure to 2 Gy whole-body X-irradiation. Metaphase spreads at different time intervals were analyzed by fluorescence in situ hybridization (FISH) using chromosome-specific DNA libraries for chromosomes 1, 11 and 13. The frequencies of dicentrics and translocations appear to be equal immediately after irradiation. Frequencies of dicentrics decreased exponentially with time according to the relationship D = ae(-kt). The rate of elimination was faster in the early period (days 0-14) than in the later period (> or = 14 days). The frequency of translocations was constant in the period 0-7 days and then decreased linearly or exponentially. For the whole period, the trend is highly significant. As mouse chromosome painting probes are becoming available and by using FISH, an in vivo mouse model for the analysis of translocations has become feasible. As translocations are involved in carcinogenesis and genetic disorders, risk estimation for induction of translocations by ionizing radiation can be made with greater confidence and extrapolated to the human situation. PMID- 8627128 TI - Heavy ion inactivation in Escherichia coli cells: experiment and theory. AB - Cross sections for inactivation of stationary cells of E. coli strains (B(s-1) and B/r) by heavy ions (Z = 8-92) with energies <15 MeV/u were determined from survival curves and compared with track structure calculations. Whereas for low energies, inactivation cross sections for both strains are similar, and thus do not reflect their difference in X-ray sensitivity, the ratios of cross sections of the two strains approach the ratio of X-rays sensitivities for heavy ions at high energies. The essential features of the experimental data are reproduced well by the track structure calculations, which do not use fitted values for the model parameters but independently determined values for the X-ray sensitivities and the size of the target structure. Quantitatively, the calculations underestimate the cross sections for heavier ions as well as their ratios at higher energies. Some possible reasons for these discrepancies are discussed. PMID- 8627129 TI - Radiosensitization of thymine by copper(II) and nickel(II) complexes of metronidazole. AB - Aqueous solutions of thymine (10(-3) mol dm(-3)) were irradiated with Co 60 gamma rays in the absence and presence of metronidazole as well as its Cu(II) and Ni(II) complexes (10(-4) mol dm(-3)) under different conditions. The yields for the loss of thymine and those for the formation of the products of radiolysis were determined by hplc. The degradation of thymine when compared with that in the absence of metronidazole was not significantly altered. However, it increased significantly when the radiolysis of thymine was carried out in the presence of the Cu(II) and Ni(II) complexes of metronidazole. All three compounds were found to promote the formation of thymine glycol although the increase was more significant for the metal complexes than for free metronidazole. The higher radiosensitizing efficiency observed with the metal complexes as compared with that for free metronidazole is due to their higher rate of oxidation of the transient thymine-OH radical adduct. The reduction of the sensitizers results in the formation of the nitro-anion radical which in the case of the Cu(II) complex undergoes intramolecular electron transfer to the metal centre leading to the formation of Cu(I). PMID- 8627130 TI - Repair of dGMP hydroxyl radical adducts by verbascoside via electron transfer: a pulse radiolysis study. AB - The repair activity of verbascoside (VER), isolated from Pedicularis spicata, towards the oxidizing hydroxyl radical adduct of dGMP and its reaction mechanism were studied using pulse radiolysis. Upon pulse radiolysis of nitrous oxide saturated aqueous solution of 2'-deoxyguanosine-5'-monophosphate (dGMP) and VER, it was found that the transient absorption spectrum of the hydroxyl adduct of dGMP decays with the formation of that of the phenoxyl radical of VER, several tens of microseconds after the electron pulse. From the formation kinetics of the phenoxyl radical of VER, the rate constant of the repair reaction was determined to be 1.12 x 10(9) dm(3) mol(-1) s(-1). PMID- 8627131 TI - Life span and tumours in the first-generation offspring of the gamma-irradiated male mouse. AB - C57BL/6 male mice were exposed to 3 Gy 60Co gamma-rays and mated with unirradiated females after 15 days to produce F1 progeny produced following irradiation of the spermatids. After weaning the offsprings were allowed to live their normal life span. The mean litter size of the irradiated group significantly decreased from 7.1 to 4.9 (p < 0.01), but the sex ratio was not altered by the irradiation. No significant differences in the survival curve and mean life-span between the irradiated and control groups were noted. The only radiation effect in tumour incidence was a decrease of histiocytic sarcoma in female offspring of irradiated males. Except for this, there were no significant differences between the irradiated group and the control group in the incidence or age distribution of tumours. PMID- 8627132 TI - Isolation and characterization of a novel Deinococcus radiodurans mutant abnormally susceptible to mutation induction by UV, gamma-ray, and mitomycin C. AB - We isolated and characterized a novel, radiation 'hypermutable' mutant of Deinococcus radiodurans. Compared with the wild-type strain D. radiodurans IR, this mutator strain, designated S101, exhibited sensitivity to UV light, gamma ray, mitomycin C, and N-methyl-N'-nitro-N-nitrosoguanidine. Spontaneous revertants of S101 that restored wild-type phenotype (non-mutability and resistance to these DNA-damaging agents) were also isolated. Furthermore, the increased susceptibility to DNA-damaging agents and mutability observed in S101 could be mimicked by treating D. radiodurans IR with Mn(II) ions. Our results suggest a putative new pathway of DNA repair in the extremely radioresistant bacterium D. radiodurans. PMID- 8627133 TI - Effects of 50 Hz magnetic field exposure on the rate of DNA synthesis by normal human fibroblasts. AB - Interest in the potential adverse biological effects of exposure to power frequency magnetic fields has centred on the possibility that these fields may influence tumour promotion, possibly by increasing the rate of cell proliferation. In order to investigate whether exposure to magnetic fields can indeed affect the rate of cell proliferation, normal human fibroblasts were serum starved overnight and then exposed to 50 Hz magnetic fields in a purpose-built facility. The rate of DNA synthesis was taken as a measure of cell proliferation, and was determined by following the incorporation of [3H]-thymidine into macromolecular material. The rate of DNA synthesis in exposed cells was compared with that in control cultures maintained in a standard CO2 incubator where they were exposed to background magnetic fields of < 200 nT. Positive controls were maintained in the same CO2 incubator, but were treated with human recombinant fibroblast growth factor to check that the cells were responsive to growth stimuli. Magnetic fields at 50 Hz and at a range of flux densities between 20 microT and 20 mT had no detectable effect on the rate of DNA synthesis by cells exposed for up to 30 h. PMID- 8627134 TI - Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. AB - We investigated the effects of acute (2-h) exposure to pulsed (2-micros pulse width, 500 pulses s(-1)) and continuous wave 2450-MHz radiofrequency electromagnetic radiation on DNA strand breaks in brain cells of rat. The spatial averaged power density of the radiation was 2mW/cm2, which produced a whole-body average-specific absorption rate of 1.2W/kg. Single- and double-strand DNA breaks in individual brain cells were measured at 4h post-exposure using a microgel electrophoresis assay. An increase in both types of DNA strand breaks was observed after exposure to either the pulsed or continuous-wave radiation, No significant difference was observed between the effects of the two forms of radiation. We speculate that these effects could result from a direct effect of radiofrequency electromagnetic energy on DNA molecules and/or impairment of DNA damage repair mechanisms in brain cells. Our data further support the results of earlier in vitro and in vivo studies showing effects of radiofrequency electromagnetic radiation on DNA. PMID- 8627135 TI - Residual postsurgical back pain. AB - Residual Postsurgical Back Pain (RPP) is a complex problem, involving considerable etiologic and diagnostic confusion. About two-thirds of all patients enrolled in chronic pain centers in the United States suffer from RPP. More than 50 billion dollars are spent on the diagnosis and treatment of back pain in this country. In most cases, the etiology of the patient's complaints is multifactorial. Treatment is difficult and frequently involves surgical as well as non-surgical modalities. Surgical treatment is of value in a carefully selected group of patients, either those in whom the original procedure failed to correct the underlying abnormality or those who show evidence of compression of neural elements, or instability of vertebral column. Because extensive co morbidity is often present, discrete operative intervention may not fully arrest the compete etiology of the patient's distress, and, indeed; may sometimes worsen the patient's complaints. Possible coexisting problems like degenerative disease or depression should be addressed in all patients prior to surgical intervention, so that the corrected variable is the preponderant cause of the patient's difficulty. PMID- 8627136 TI - Managed care and physician liability. PMID- 8627137 TI - A study of physicians, hospitals, and payers in the Atlanta health care market. PMID- 8627138 TI - Making a bad program worse: Clinton's attempt at Medicaid reform. PMID- 8627139 TI - The white coats are coming, the white coats are coming. PMID- 8627140 TI - Opting out of Medicare. One physician takes a stand. PMID- 8627141 TI - A wolf in sheep's clothing: the Clinton health reform plan. PMID- 8627142 TI - Managed health care costs more, not less. PMID- 8627143 TI - And the answer is ... "sorry, but being excluded from participation in a managed care plan probably isn't an antitrust violation". PMID- 8627144 TI - Nitric oxide loading of the salivary nitric-oxide-carrying hemoproteins (nitrophorins) in the blood-sucking bug Rhodnius prolixus. AB - The salivary glands of the blood-sucking bug Rhodnius prolixus are formed by a single layer of binucleated epithelial cells surrounded by a double layer of transversely oriented smooth muscle cells. The epithelial cells are rich in rough endoplasmic reticulum and mitochondria and have abundant microvillar projections towards the gland lumen. This cell layer surrounds a relatively large cavity where abundant secretory material is stored. Epithelial cells produce an intense and generalized NADPH diaphorase reaction, in contrast to other tissues such as brain, Malpighian tubules and skeletal muscle. Ultrastructural analysis of the osmiophilic reaction product indicates that it is localized within cytoplasmic vacuoles, a similar location to that of NADPH diaphorase (NO synthetase) activity in neuronal cells of vertebrates. Measurements of the time course of protein accumulation, NADPH diaphorase activity and the degree of nitrosylation of hemoproteins (nitrophorins) in the salivary glands of Rhodnius prolixus nymphs after a blood meal indicate that the nitrophorins are synthesized and accumulate when NO production is low (with a 25% loading of the nitrophorins during the fourth- to fifth-instar molt). NO loading of the nitrophorins increases to 90% after the molt, concomitant with a large increase in the salivary NADPH diaphorase activity. It is concluded that synthesis of NO occurs within the epithelial cells while the nitrophorins are stored extracellularly. It is hypothesized that the luminally oriented microvilli may serve as a diffusion bridge to direct intracellularly produced NO into the luminal cavity, where the nitrophorins are stored. PMID- 8627145 TI - Different neural pathways coordinate Drosophila flight initiations evoked by visual and olfactory stimuli. AB - To determine the role played by the giant fiber interneurons (GFs) in coordinating the jumping stages of visually elicited and olfactory-induced fight initiation we have recorded extracellularly from the cervical connective nerve during flight initiation. A spike is recorded from the cervical connective upon brain stimulation that has the same threshold as does activation of the tergotrochanteral muscle (TTM) and dorsal longitudinal muscles (DLMs). A consistent time interval occurs between the spike and activation of the TTM. Thus, the spike probably results from activity in the GFs. The time intervals between the spike and activation of the TTM during GF stimulation and visually elicited flight initiation are similar. These results suggest that the GFs coordinate the activation of the TTM and DLMs during the jumping stage of visually elicited flight initiation. A spike is also recorded from the cervical connective during olfactory-induced flight initiations, but its shape and the time interval between it and activation of the TTM is different from that observed during GF stimulation. Although some olfactory-induced flight initiations exhibit a pattern of muscle activation, olfactory-induced flight initiations exhibit a pattern of muscle activation indistinguishable from that evoked by GF stimulation, our results indicate that regardless of the pattern of muscle activation, olfactory-induced flight initiations are not coordinated by the GF circuit. The sterotypic sequence and timing of activation of TTM and DLMs characteristic of the GF pathway can, therefore, be evoked by neurons other than those constituting the GF pathway. PMID- 8627146 TI - A dorsal light reflex in a squid. AB - A dorsal light reflex is described in the squid Lolliguncula brevis. When illuminated from the side in visually homogeneous surroundings, a free-swimming squid rolls the dorsal side of its head and trunk 10-20 degrees towards the light. With the trunk restricted in a holder, the squid rolls its head 4-5 degrees towards the light; this reaction increases by about 50% when the statocysts are bilaterally removed and increases further when the neck receptor organ is also destroyed. The results indicate a multi-modal interaction of visual, statocyst and proprioceptive inputs during postural control. PMID- 8627147 TI - HLA-derived peptides which inhibit T cell function bind to members of the heat shock protein 70 family. AB - Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40. PMID- 8627149 TI - The inlAB locus mediates the entry of Listeria monocytogenes into hepatocytes in vivo. AB - The intracellular parasite Listeria monocytogenes is able to induce its internalization by cultured mammalian cells that are not normally phagocytic. This process requires the expression of the chromosomal locus inlAB. We studied the virulence of an inlAB mutant and of its parent in murine listeriosis. Irrespective of the route of inoculation, the inlAB mutant was severely attenuated for growth in the liver. The livers of mice inoculated with the inlAB mutant displayed much smaller infectious foci than the parent as early as 24 h after infection. Electron microscopy showed that these foci consisted of a few inflammatory cells, with few bacteria; bacteria were rarely found within hepatocytes. In contrast, foci in livers of mice inoculated with the parent consisted of islets of heavily infected hepatocytes that were infiltrated by numerous neutrophils; bacteria seemed intact within hepatocytes and damaged within neutrophils. A direct role of inlAB for the entry of L. monocytogenes into hepatocytes was confirmed in a cell infection system using the murine embryonic hepatocyte cell line TIB73. The inlAB mutant was approximately 20-fold less invasive in trans. The "invasion locus" inlAB contributes to protect L. monocytogenes from the host's innate defense mechanisms by promoting its entry into hepatocytes. PMID- 8627148 TI - Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope. AB - Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535-551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO-EBV; T cell clone 37 recognizes the TEC, whereas it is stimulated poorly by the TPO loaded to autologous peripheral blood mononuclear cells (PBMC). Clone 43, which fails to recognize the TPO-EBV, also fails to recognize the TEC, but is activated by exogenous TPO presented by autologous PBMC. These results show that exogenous versus endogenous processing in vivo generates a different TPO epitope repertoire, producing a "cryptic" epitope (epitope not always available for recognition). Our findings define a route by which human self-reactive T cells may escape thymic selection and become activated in vivo, thus possibly leading to autoimmunity. PMID- 8627150 TI - B and T cells are not required for the viable motheaten phenotype. AB - Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease. PMID- 8627152 TI - Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1. AB - The transcription factor human X-box binding protein 1 (hXBP-1) is a basic region leucine zipper protein implicated in the regulation of major histocompatibility complex class II gene expression as well as in exocrine gland and skeletal development. Multiple regulatory elements in the hXBP-1 promoter lie 3' to the transcription start site, including the hX2 site, whose core sequence is an AP-1 like element identical to the hXBP-1 target sequence in the HLA-DRA promoter. One complex identified by electrophoretic mobility shift assay (EMSA), complex 3, was previously shown to protect the hX2 site and more 3' bases. Sequence analysis now shows that this region contains a consensus binding site for transcription factor BSAP (B cell lineage-specific activator protein). Complex 3 and BSAP have identical cell-type specificities, as they are found only in pre-B and mature B cell lines. In EMSAs, BSAP antibody specifically recognized complex 3, and in vitro translated BSAP could bind to an hXBP promoter fragment. Cotransfections using an hXBP-1 reporter construct indicated that BSAP downregulates the hXBP-1 promoter. The highest levels of hXBP-1 mRNA were found when BSAP was not expressed, in pre-Pro-B cells and in plasma cell lines. In addition, hXBP-1 and BSAP levels were inversely correlated along the early stages of B cell development. In the regulation of the hXBP-1 promoter, a strong positive transcriptional influence at the hX2 site is opposed by the downregulatory actions of BSAP. PMID- 8627151 TI - bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice. AB - We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation. PMID- 8627153 TI - T cell-independent antibody-mediated clearance of polyoma virus in T cell deficient mice. AB - Polyomavirus (PyV) infection of SCID mice, which lack functional T and B cells, leads to a lethal acute myeloproliferative disease (AMD) and to high levels of virus replication in several organs by two wk after infection. This is in contrast to infection of T cell-deficient athymic nude mice, which are resistant to acute PyV-induced disease and poorly replicate the virus in their organs. This major difference in the virus load and in the outcome of PyV infection between SCID and nude mice suggested that an efficient, T cell-independent antiviral mechanism operates in T cell-deficient, PyV infected mice. To investigate this possibility, mice with different genetically engineered T and/or B cell deficiencies and SCID mice adoptively reconstituted with B and/or T cells were infected with PyV. The results indicated that the presence of B cells in the absence of T cells protected mice from the AMD, and this was accompanied by a major reduction of PyV in all organs tested. Sera from PyV-infected T cell receptor (TCR) alpha beta knockout or TCR alpha beta gamma delta knockout mice contained IgG2a antibodies to PyV. Sera or purified immunoglobulin fractions from PyV-infected TCR alpha beta knockout mice protected SCID mice from the PyV induced AMD. To our knowledge, this is the first report of an effective T cell independent antibody response clearing a virus and changing the outcome of infection from 100% mortality to 100% survival. PMID- 8627154 TI - T cell responses in calcineurin A alpha-deficient mice. AB - We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA. PMID- 8627157 TI - Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor. AB - P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor. PMID- 8627156 TI - Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand. AB - Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo. PMID- 8627158 TI - Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins. AB - Little is known about how mononuclear phagocytes (MP) are cleared from sites of inflammation as inflammatory lesions resolve. In this study, the possibility that MP could be cleared from tissues by migrating across endothelium in the basal to apical direction was investigated. In an in vitro model of a blood vessel wall consisting of human umbilical vein endothelial cells (HUVEC) grown on amniotic tissue, a majority of MP that initially transmigrated into the amnion later exited by migrating back across the endothelium in the basal to apical direction. MP that egressed from these cultures adhered to the apical surface of the endothelium or were found nonadherent in the medium above the endothelium. Egression of MP continued throughout the 4-d period examined, displaying higher than first order kinetics and a t(1/2) of approximately 24 h. These kinetics were decreased by increasing the volume of medium bathing the cultures, suggesting that a soluble factor(s) regulates the rate of egression. In contrast, the kinetics were accelerated by pretreating the endothelium with IL-1. The initial phase of this increased rate of egression was inhibited by antibodies to inter- cellular adhesion molecule 1 (ICAM-1) or CD18 by 100 and 71%, respectively. Immunostaining revealed that ICAM-1 was present on the apical and basal surfaces of umbilical vein endothelium in vitro and in situ. These data demonstrate that MP can traverse endothelium in the basal to apical direction, and lend insight into the mechanisms by which this process occurs. PMID- 8627155 TI - Fate of surrogate light chains in B lineage cells. AB - Biosynthesis of the immunoglobulin (Ig) receptor components and their assembly were examined in cell lines representative of early stages in human B lineage development. In pro-B cells, the nascent surrogate light chain proteins form a complex that transiently associates in the endoplasmic reticulum with a spectrum of unidentified proteins (40, 60, and 98 kD) and Bip, a heat shock protein family member. Lacking companion heavy chains, the surrogate light chains in pro-B cells do not associate with either the Ig(alpha) or Ig(beta) signal transduction units, undergo rapid degradation, and fail to reach the pro-B cell surface. In pre-B cells, by contrast, a significant portion of the surrogate light chain proteins associate with mu heavy chains, Ig(alpha), and Ig(beta) to form a stable receptor complex with a relatively long half-life. Early in this assembly process, Bip/GRP78, calnexin, GRP94, and a protein of approximately 17 kD differentially bind to the nascent mu heavy chains. The 17-kD intermediate is gradually replaced by the surrogate light chain protein complex, and the Ig(alpha) and Ig(beta) chains bind progressively to the mu heavy chains during the complex and relatively inefficient process of pre-B receptor assembly. The results suggest that, in humans, heavy chain association is essential for surrogate light chain survival and transport to the cell surface as an integral receptor component. PMID- 8627159 TI - Biochemical pathways of apoptosis: nicotinamide adenine dinucleotide-deficient cells are resistant to tumor necrosis factor or ultraviolet light activation of the 24-kD apoptotic protease and DNA fragmentation. AB - The function of nicotinamide adenine dinucleotide (NAD) and adenosine diphosphate (ADP) ribosylation reactions in the mechanism of apoptotic cell death is controversial, although one theory postulates an essential role for NAD depletion by poly-ADP-ribose polymerase. The present study examined the role of intracellular NAD in tumor necrosis factor (TNF) and ultraviolet (UV) light induced activation of the 24-kD apoptotic protease (AP24) leading to internucleosomal DNA fragmentation and death. Our results demonstrate that nutritional depletion of NAD to undetectable levels in two leukemia lines (U937 and HL-60) renders them completely resistant to apoptosis. This was attributed to a block in the activation of AP24 and subsequent DNA cleavage. Normal cells show an elevation of ADP-ribosyl transferase (ADPRT) in both the cytosol and nucleus after exposure to TNF, but before DNA fragmentation. ADPRT activity as well as cell death was suppressed by an inhibitor specific for mono-ADPRT. Nuclei from NAD-depleted cells were still sensitive to DNA fragmentation induced by exogenous AP24, indicating a selective function for NAD upstream of AP24 activation in the apoptotic pathway. We confirmed a requirement for intracellular NAD, activation of ADPRT, and subsequent NAD depletion during apoptosis in KG1a, YAC-1, and BW1547 leukemia cell lines. However, this mechanism is not universal, since BJAB and Jurkat leukemia cells underwent apoptosis normally, even in the absence of detectable intracellular NAD. We conclude that TNF or UV light-induced apoptotic cell death is not due to NAD depletion in some leukemia cell lines. Rather, NAD dependent reactions which may involve mono-ADPRT, function in signal transduction leading to activation of AP24, with subsequent DNA fragmentation and cell death. PMID- 8627160 TI - Neutralizing antibodies to human immunodeficiency virus type-1 gp120 induce envelope glycoprotein subunit dissociation. AB - The spectrum of the anti-human immunodeficiency virus (HIV) neutralizing immune response has been analyzed by the production and characterization of monoclonal antibodies (mAbs) to the viral envelope glycoproteins, gp41 and gp120. Little is known, however, about the neutralization mechanism of these antibodies. Here we show that the binding of a group of neutralizing mAbs that react with regions of the gp120 molecule associated with and including the V2 and V3 loops, the C4 domain and supporting structures, induce the dissociation of gp120 from gp41 on cells infected with the T cell line-adapted HIV-1 molecular clone Hx10. Similar to soluble receptor-induced dissociation of gp120 from gp41, the antibody-induced dissociation is dose- and time-dependent. By contrast, mAbs binding to discontinuous epitopes overlapping the CD4 binding site do not induce gp120 dissociation, implying that mAb induced conformational changes in gp120 are epitope specific, and that HIV neutralization probably involves several mechanisms. PMID- 8627161 TI - Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and beta(2)-microglobulin deficient BALB/c mice. AB - Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long-lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen-specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is amplified in BALB/c as compared to DBA/2, C3H, or C57BL/6 mice whereas tested. These findings support the hypothesis that continuous release of low amounts of protein antigens from pathogenic microorganisms may polarize the immune response toward a Th2 phenotype in susceptible mouse strains. PMID- 8627162 TI - LMP-associated proteolytic activities and TAP-dependent peptide transport for class 1 MHC molecules are suppressed in cell lines transformed by the highly oncogenic adenovirus 12. AB - Expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the in vivo oncogenicity of this virus. In primary embryonal fibroblasts (H 2b) that express transgenic swine class I antigen (PD1), Ad12-mediated transformation results in inhibition in transport of newly synthesized class I molecules, as well as significant reduction in transporter associated with antigen presentation (TAP) gene expression. In this report we show that reexpression of TAP molecules either by stable transfection of mouse TAP genes or by infection with recombinant vaccinia viruses expressing human TAP genes, only partially reconstitutes the expression and transport of the class I molecules. Further analysis of Ad12-transformed cells revealed that the expression of both LMP2 and LMP7, but not of other proteasome complex components, was downregulated, resulting in altered proteolytic activities of the 20S proteasomes. Reconstitution of both TAP and LMP expression resulted in complete restoration of PD1 cell surface expression and enhanced expression of the endogenous H-2D(b) molecules encoded by recombinant vaccinia viruses, in reconstituted Ad12 transformed cells, efficient transport of H-2 class I molecules could only be achieved by treatment of the cells with gamma-interferon. These data suggest that an additional factor(s) that is interferon-regulated plays a role in the biosynthetic pathway of the class I complex, and that its function is deficient in this cell system. Thus, Ad12 viral transformation appears to suppress the expression of multiple genes that are important for antigen processing and presentation, which allows such transformed cells to escape immune surveillance. This coordinate downregulation of immune response genes must likely occur through their use of common regulatory elements. PMID- 8627163 TI - Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. AB - Leishmania major promastigotes were found to avoid activation of mouse bone marrow-derived macrophages (BMM0) in vitro for production of cytokines that are typically induced during infection with other intracellular pathogens. Coexposure of BMM0 to the parasite and other microbial stimuli resulted in complete inhibition of interleukin (IL) 12 (p40) mRNA induction and IL-12 release. In contrast, mRNA and protein levels for IL-1(alpha), IL-1(beta), tumor necrosis factor (TNF) alpha, and inducible NO synthase (iNOS) were only partially reduced, and signals for IL-10 and monocyte chemoattractant protein (MCP-1/JE) were enhanced. The parasite could provide a detectable trigger for TNF-alpha and iNOS in BMM0 primed with interferon (IFN) gamma, but still failed to induce IL-12. Thus IL-12 induction is selectively impaired after infection, whereas activation pathways for other monokine responses remain relatively intact. Selective and complete inhibition of IL-12(p40) induction was observed using BMM0 from either genetically susceptible or resistant mouse strains, as well as IL-10 knockout mice, and was obtained using promastigotes from cutaneous, visceral, and lipophosphoglycan-deficient strains of Leishmania. The impaired production of the major physiological inducer of IFN-gamma is suggested to underlie the relatively prolonged interval of parasite intracellular survival and replication that is typically associate with leishmanial infections, including those producing self limiting disease. PMID- 8627164 TI - An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. AB - T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum. PMID- 8627165 TI - Unresponsiveness to a self-peptide of mouse lysozyme owing to hindrance of T cell receptor-major histocompatibility complex/peptide interaction caused by flanking epitopic residues. AB - A self-peptide containing amino acid residues 46-61 (NRGDQSTDYGIFQINSR) of mouse lysozyme (ML) (p46-61, which binds strongly to the A(k) molecule but does not bind to the E(k) molecule), can induce a strong proliferative T cell response in CBA/J mice (A[k], E[k]) but no response at all in B10.A(4R) and CBA/J mice. The critical residues within p46-59 are immunogenic in both B10.A(4R) and CBA/J mice. The critical residues within p46-61 reside between amino acid positions 51 and 59. T cells of B10.A(4R) mice primed with the truncated peptides in vivo cannot be restimulated by p46-61 in vitro. This suggests that T cell receptor (TCR) contact (epitopic) residue(s) flanking the minimal 51-59 determinant within p46 61 hinder the interaction of the p46-61/A(k) complex with the appropriate TCR(S), thereby causing a lack of proliferative T cell response in this mouse strain. Unlike B10.A(4R) mice, [B10.A(4R) x CBA/J]F1 mice responded vigorously to p46-61, suggesting that thymic APC of B10.A(4R) mice do not present a self ligand to T cells resulting in a p46-61-specific hole in the T cell repertoire in B10.A(4R) or the F1 mice. Moreover, APC from B10.A(4R) mice are capable of efficiently presenting p46-61 to peptide-specific T cell lines from CBA/J mice. The proliferative unresponsiveness of B10.A(4R) mice to p46-61 is not due to non major histocompatibility complex genes because B10.A mice (A[k], E[k]) respond well to p46-61. Interestingly, B10.A(4R) mice can raise a good proliferative response to p46-61 (R61A) (in which the arginine residue at position 61 (R61L/F/N/K), indicating that R61 was indeed responsible for hindering the interaction of p46-61 with the appropriate TCR. Finally, chimeric mice [B10.A(4R) ->B10.A] responded vigorously to p46-61, suggesting that thymic antigen presentation environment of the B10.A mouse was critical for development of a p46 61-reactive T cell repertoire. Thus, we provide experimental demonstration of a novel mechanism for unresponsiveness to a self peptide, p46-61, in the B10.A(4R) mouse owing to hindrance: in this system it is the interaction between the available TCR and the A(k)/p46-61 complex, which is hindered by epitopic residue(s) within p46-61. We argue that besides possessing T cells that are hindered by R61 of p46-61, CBA/J and B10.A mice have developed an additional subset of T cells bearing TCRs which are not hinderable by R61, presumably through positive selection with peptides derived from class II E(k), or class I D(k)/D(d) molecules. These results have important implications in self tolerance, shaping of the T cell repertoire, and in defining susceptibility to autoimmunity. PMID- 8627166 TI - CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C gamma(1) upon B cell activation. AB - Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C-gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC-gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2-terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22. PMID- 8627167 TI - Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism. AB - The idea that stem cells oscillate between a state of activity and dormancy, thereby giving rise to differentiating progeny either randomly or in orderly clonal succession, has important implications for understanding normal hematopoiesis and blood cell dyscrasias. The degree of clonal stability in individuals also has practical implications for the evaluation of clonal lymphomyeloproliferative diseases. To evaluate the clonality pattern of the different types of blood cells as a function of time we have validated the applicability, sensitivity, and reproducibility of a thermostable ligase reaction to detect transcripts of the G6PD allele on the active X-chromosome in normal heterozygous females. While the ratio of the two X-chromosome-derived allelic transcripts varied widely among hemopoietic tissues in a given individual, this allelic ratio was virtually identical in all types of mature myeloid and lymphoid cells. Longitudinal studies indicated constancy of the G6PD allelic ratio in blood cells over a 912-d period of observation in healthy females. The individual variability observed in this allelic ratio suggests that the progeny of a relatively small number of original embryonic hemopoietic stem cells, approximately eight, contribute to the sustained production of all types of blood cells in healthy individuals. PMID- 8627168 TI - Human vascular adhesion protein 1 (VAP-1) is a unique sialoglycoprotein that mediates carbohydrate-dependent binding of lymphocytes to endothelial cells. AB - The regulated interactions of leukocytes with vascular endothelial cells are crucial in controlling leukocyte traffic between blood and tissues. Vascular adhesion protein-1 (VAP-1) is a novel, human endothelial cell molecule that mediates tissue-selective lymphocyte binding. Two species (90 and 170 kD) of VAP 1 exist in lymphoid tissues. Glycosidase digestions revealed that the mature 170 kD form of VAP-1 expressed on the lumenal surfaces of vessels is a heavily sialylated glycoprotein. The sialic acids are indispensable for the function of VAP-1, since the desialylated form of VAP-1 no longer mediates lymphocyte binding. We also show that L-selectin is not required for binding of activated lymphocytes to VAP-1 under conditions of shear stress. The 90-kD form of VAP-1 was only seen in an organ culture model, and may represent a monomeric or proteolytic form of the larger species. These data indicate that L-selectin negative lymphocytes can bind to tonsillar venules via the VAP- 1-mediated pathway. Moreover, our findings extend the role of carbohydrate-mediated binding in lymphocyte-endothelial cell interactions beyond the known selectins. In conclusion, VAP-1 naturally exists as a 170-kD sialoglycoprotein that uses sialic acid residues to interact with its counter-receptors on lymphocytes under nonstatic conditions. PMID- 8627169 TI - Redirection of tumor metastasis by expression of E-selectin in vivo. AB - The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo. PMID- 8627170 TI - Impaired primary T cell responses in L-selectin-deficient mice. AB - L-selectin is a homing receptor that mediates the selective attachment of leukocytes to specialized high endothelial venules. To study the potential role of L-selectin in immune responses in intact mice, we generated L-selectin deficient mice by gene targeting. L-selectin-deficient mice are defective in cutaneous delayed-type hypersensitivity (DTH) responses when tested after conventional intervals of immunization (4 d). Primary T cell proliferative responses and cytokine production (interleukin [IL] 2, IL-4, and interferon gamma) were also compromised when tested after 5 d of immunization, indicating that L-selectin is important for the immune response to antigens. In contrast, after more prolonged immunization protocols (9 d), normal responses were observed, suggesting that L-selectin-independent compensatory mechanisms exist. Interestingly, humoral responses of L-selectin-deficient mice to keyhole limpet hemocyanin are indistinguishable from wild-type control mice, implying that L selectin plays no rate-limiting role in T cell help of B cell function. Thus, our results suggest that L-selectin plays an important role in the generation of primary T cell responses but may not be essential for humoral and memory T cell responses. L-selectin does not appear to be rate limiting for the events leading to antigen-driven neutrophil recruitment, since normal DTH responses are obtained at late time points after immunization. PMID- 8627171 TI - Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. AB - We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects. PMID- 8627172 TI - Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling. AB - Zeta-associated protein (ZAP)-70 is a cytoplasmic protein tyrosine required for T cell antigen receptor (TCR) signaling and development. Mutations in ZAP-70 result in severe combined immunodeficiency in humans. ZAP-70 interacts with the TCR by binding to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the invariant subunits of the TCR complex. Here we report that two ZAP-70 mutants devoid of kinase activity, generated either by a point mutation in the kinase domain to create an inactive kinase, or by truncation of the entire kinase domain (SH2[N+C]), functioned as dominant negative mutants to specifically suppress TCR-mediated activation of NFAT, a nuclear factor essential for inducible interleukin 2 gene expression. Biochemical studies with the SH2(N+C) mutant showed that it also blocked early TCR signaling events, such as p95vav tyrosine phosphorylation, extracellular signal-regulated kinase 2 activation, and the association of a number of tyrosine-phosphorylated proteins with growth factor receptor-binding protein 2 (GRB2). The inhibitory effects of the SH2(N+C) mutant revealed that it requires an intact phosphotyrosine-binding site in its COOH-terminal SH2 domain. Using a CD8-zeta chimeric receptor to analyze the interaction of the SH2(N+C) mutant with ITAMs of TCR-zeta, we found that this mutant was constitutively bound to the hyperphosphorylated CD8-zeta chimera. These results indicate that tyrosine phosphorylated ITAM is the target for the action of this dominant-negative mutant, suggesting that the assembly of a functional receptor signaling complex on ITAMs is a critical proximal TCR signaling event leading to downstream activation. PMID- 8627173 TI - Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae. AB - Heme uptake is a common means of iron and porphyrin acquisition by many pathogenic bacteria. The genus Haemophilus includes several important pathogenic bacterial species that characteristically require hemin-, protoporphyrin-, or heme-substituted proteins as essential growth factors under aerobic conditions. However, the mechanism of heme transport is not understood for Haemophilus. We have cloned a DNA fragment from H. influenzae that allows an Escherichia coli hemA mutant to employ exogenous hemin or protoporphyrin IX as sole sources of porphyrin. DNA sequencing of the cloned DNA fragment suggested that a previously characterized gene (hel) encoding an antigenic, outer membrane lipoprotein e(P4) was responsible for the complementation activity. Construction of hel insertion mutations in strain H. influenzae Rd demonstrated that hel is essential for growth under aerobic conditions but not under anaerobic conditions. The aerobic growth defect of hel mutants could be reversed by providing exogenous hemin in the presence of outer membrane. The analysis of hybrids between e(P4) and beta lactamase demonstrated that a domain of e(P4) near its NH2' terminus was required for its function in hemin use. Within this domain is a short amino acid sequence that displays similarity to H. influenzae hemin binding protein HbpA, hemin binding motifs present in eukaryotic transcription activator heme-activated protein, and the heme containing proteins hemoglobin (alpha-chain) and cytochrome C3, suggesting that this region may be involved in hemin binding and/or transport. PMID- 8627175 TI - Costimulation by CD48 and B7-1 induces immunity against poorly immunogenic tumors. AB - Genetic modification of many types of mouse tumors to express the B7-1 or B7-2 molecules, natural ligands for the T cell-costimulatory molecule CD28, increases their immunogenicity. However, even after transfection with the B7-1 and/or B7-2 genes, poorly immunogenic tumors fail to elicit and efficient immune response. We report here that two such tumors, the Ag104A sarcoma and the K1735-M2 melanoma, become immunogenic after transfection of the genes encoding murine B7-1 together with CD48, which is the natural ligand for CD2. Tumor-specific CD8+ cytotoxic T lymphocytes were readily generated and were effective for adoptive immunotherapy of metastasis induced by wild-type Ag104A sarcoma cells. A similar approach may be useful for developing therapy for other poorly immunogenic tumors, including those in humans. PMID- 8627174 TI - Aod2, the locus controlling development of atrophy in neonatal thymectomy-induced autoimmune ovarian dysgenesis, co-localizes with Il2, Fgfb, and Idd3. AB - In genetically susceptible strains of mice, such as A/J and (C57BL/6J x A/J)F1 hybrids, neonatal thymectomy-induced autoimmune ovarian dysgenesis (AOD) is characterized by the development of antiovarian autoantibodies, oophoritis, and atrophy. Temporally, atrophy may be observed during and after the regression of inflammatory infiltrates from the ovary. Histologically, lesions appear as areas devoid of ovarian follicles in all stages of development that have been replaced by luteinized interstitial cells. We report here the mapping of Aod2, the locus that controls this phenotype, to mouse chromosomes 3 within a region encoding Il2 and Fgfb. Most significant, however, is the co-localization of Aod2 to Idd3, a susceptibility gene that plays a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse. PMID- 8627176 TI - The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions. AB - Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions. PMID- 8627177 TI - Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. AB - Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production. PMID- 8627178 TI - Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft versus-host disease. AB - Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway. PMID- 8627179 TI - The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species. AB - A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species. PMID- 8627180 TI - T cell receptor-dependent cell death of T cell hybridomas mediated by the CD30 cytoplasmic domain in association with tumor necrosis factor receptor-associated factors. AB - CD30 is a member of the tumor necrosis factor superfamily and a surface marker for Hodgkin's disease. Normal activated T cells and several virally transformed T or B cell lines also show CD30 expression. The interaction of CD30 with its ligand induces cell death or proliferation, depending on the cell type. In this report we characterize the signals mediated by the intracellular domain of CD30 and show that, in combination with signal(s) transduced by the T cell receptor, the multimerization of CD30 cytoplasmic domain induces Fas(CD95)-independent cell death in T cell hybridomas. Deletion analysis shows that the COOH-terminal 66 amino acids of CD30 are required to induce cell death. Using the yeast two-hybrid system, we have identified that the same region of CD30 interacts with tumor necrosis factor receptor-associated factor (TRAF)1 and TRAF2. These results indicate that TRAF1 and/or TRAF2 play an important role in cell death in addition to their previously identified roles in cell proliferation. PMID- 8627181 TI - Physical and functional association of the cbl protooncogen product with an src family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor mediated signaling. AB - To identify novel signal transducers involved in signaling mediated by the Src family protein tyrosine kinases (PTKs), we used a yeast two-hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c-cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src family PTKs in the intracellular signaling mediated by the antigen receptors PMID- 8627182 TI - Deletion of the NH2-terminal residue converts monocyte chemotactic protein 1 from an activator of basophil mediator release to an eosinophil chemoattractant. AB - Chemotactic cytokines of the CC subfamily (CC chemokines) are considered as major mediators of allergic inflammation owing their actions on basophil and eosinophil leukocytes. The monocyte chemotactic protein (MCP) 1 is a potent inducer of mediator release from basophils but is inactive on eosinophils. To obtain information on the structural determinants of the activities of MCP-1, we have synthesized several NH2-terminally truncated analogues and tested their effects on basophils and eosinophils. Through deletion of the NH2-terminal residue, MCP 1(2-76) was obtained, which was a potent activator of eosinophils, as assessed by chemotaxis, cytosolic free Ca2+ changes, actin polymerization, and that induction of the respiratory burst. In contrast, the activity of MCP-1(2-76) on basophil leukocytes was dramatically decreased (50-fold) compared with that of full-length MCP-1. Deletion of the next residue led to total loss of activity on eosinophil and basophil leukocytes. Analogues with three or four residue deletions, MCP-1(4 76) and MCP-1(5-76), were again active on both cells, whereas all further truncation analogues, MCP-1(6-76) through MCP-1(10-76), were inactive. Thus, a minimal structural modification can change receptor and target cell selectivity of MCP-1. Our observations indicate that the recognition sites of CC chemokine receptors on eosinophils and basophils are similar, although they discriminate between MCP-1 and MCP-1(2-76) and suggest NH2-terminal processing as a potential mechanism for the regulation of CC chemokine activities. PMID- 8627183 TI - The efficiency of acute infection of CD4+ T cells is markedly enhanced in the setting of antigen-specific immune activation. AB - Human immunodeficiency virus (HIV) disease in sub-Saharan Africa generally differs from that observed in the United States and other developed countries in that the risk of seroconversion after exposure is greater and the rate of disease progression to AIDS and death is faster. One theory that could in part explain this difference is the increased state of immune activation associated with a relatively high rate of parasite infestation and other infections among inhabitants of these regions. Using a model based on the cellular microenvironment of lymphoid organs, the role of exposure to HIV during a state of antigen-specific immune activation was investigated. Dendritic cells and CD4+ T cells are the major cellular components of the paracortical region of lymphoid tissue, the primary site of HIV replication. We analyzed cocultures of HIV-pulsed dendritic cells that had matured in the presence of tetanus toxoid and CD4+ T cells before and after inducing an antigen-specific response by in vivo immunization with tetanus toxoid. During antigen-specific immune activation, 100 times less HIV was needed to initiate a productive infection. These findings provide a model system to further delineate the relationship between immune activation and the propagation of HIV infection and suggest a mechanism for the epidemiologic observations of an increased ease of developing HIV infection and faster progression for HIV disease in geographic areas where immune activation is prevalent. PMID- 8627184 TI - Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion. AB - The recent finding that CD40L on activated T cells induces interleukin (IL) 12 secretion in human peripheral blood monocytes in vitro suggests that the CD40L CD40 interaction may be of importance in the priming of T helper (Th) 1-type T cells. We therefore investigated the in vivo relevance of this interaction in an experimental model for a Th1-mediated disease, the hapten reagent (2,4,6 trinitrobenzene sulfonic acid [TNBS])-induced colitis. The administration of anti gp39 (CD40L) antibodies during the induction phase of the Th1 response prevented interferon gamma production by lamina propria CD4+ T cells and also clinical and histological evidence of disease. In contrast, the secretion of IL-4, a Th2-type cytokine, was increased after anti-gp39 treatment. In further studies we showed that the prevention of disease activity was caused by an inhibition of IL-12 secretion, as demonstrated by immunohistochemistry. In addition, the injection of recombinant IL-12 p70 heterodimer into TNBS + anti-gp39-treated mice reversed the effect of anti-gp39 and resulted in severe disease activity. When anti-gp39 was given after the disease was established, no effect on the disease activity was observed. In conclusion, we demonstrated that the CD40L-CD40 interaction is crucial for the in vivo priming of Th1 T cells via the stimulation of IL-12 secretion by antigen-presenting cells (APC). PMID- 8627185 TI - Two levels of help for B cell alloantibody production. AB - We have examined whether T cell stimulation by direct or indirect pathways contributes to alloantibody production by B cells after major histocompatibility complex (MHC)-disparate skin graft rejection in mice. Experiments were performed using normal mice, MHC class II-deficient mice, MHC class II-deficient mice with an intact peripheral CD4+ cell population (due to expression of class II antigens only on thymic epithelium), mice lacking the cytoplasmic tail of their MHC class II antigens, and mice depleted of CD4+ cells by anti-CD4 monoclonal antibody treatment. Depletion of recipient CD4+ cells reduced alloantibody production to barely detectable levels. Absence of donor MHC class II antigens did not affect the production of either immunoglobulin (Ig)M or IgG antibodies directed at class I alloantigens. Absence of recipient MHC class II antigens, however, led to production of only IgM but not IgG antibodies, even if the recipients had an intact CD4+ cell population. Absence of the cytoplasmic tail of the recipient's MHC class II antigens led to the production of slightly reduced amounts of IgG antibody. These findings indicate that (a) CD4+ cells are essential helper cells for B cell alloantibody production; (b) production of IgM alloantibody can occur with help from CD4+ cells, which recognize either donor class II antigens or modified recipient class II antigens; (c) isotype switching from IgM to IgG alloantibody requires help from CD4+ cells activated by antigens presented by recipient MHC class II molecules; and (d) the cytoplasmic domain of the recipient MHC class II molecules may be involved in the mechanism that leads to isotype switching by B cells. Thus, there are two levels of CD4-mediated help available for B cells responding to alloantigens: one (involving a noncognate interaction) can produce B cell activation, and a second (involving a cognate interaction) is required for differentiation and IgG alloantibody production. PMID- 8627187 TI - I'm dying of easy accessibility. PMID- 8627189 TI - Benzodiazepines in alcoholics. PMID- 8627188 TI - Medicating the elderly. PMID- 8627186 TI - Tumor necrosis factor alpha is a potent synergistic factor for the proliferation of primitive human hematopoietic progenitor cells and induces resistance to transforming growth factor beta but not to interferon gamma. AB - Since tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta have all been shown to be specific inhibitors of early human hematopoiesis, we wanted to investigate the interactions of these three cytokines on very primitive human adult bone marrow CD34++CD38- hematopoietic progenitor cells, using a pre-colony-forming cell (pre-CFC) assay, which detects the effects of these cytokines on the initial phases of the differentiation of these primitive progenitors, which are unresponsive to interleukin (IL) 3 alone. Surprisingly, TNF-alpha was a very potent stimulator of the proliferation of CD34++CD38- cells and was the most potent synergistic factor for the IL-3-induced proliferation of these cells of all cytokines tested (IL-1, IL-6, granulocyte colony-stimulating factor, kit ligand). TNF-alpha was the only cytokine that, as a single added factor, induced substantial proliferation in CD34++CD38- cells in the presence of IL-3, except for kit ligand, which induced very limited proliferation. TNF-alpha, moreover, induced a high degree of resistance to the inhibitory effects of TGF-beta in a dose-dependent way. The inhibitory effects of IFN-gamma, however, were not affected by the presence of TNF-alpha. We hypothesize that in situations of the hematopoietic stress, TNF-alpha may abrogate the inhibitory effect of ambient TGF-beta in the bone marrow microenvironment to allow primitive stem cells to proliferate and differentiate in response to an increased demand for mature blood cells. PMID- 8627190 TI - Sublingual vitamin B12. PMID- 8627191 TI - Soap to SNOCAMP. PMID- 8627192 TI - Managed care in the house of God. PMID- 8627193 TI - Effect of sugar on children. PMID- 8627194 TI - Asymptomatic severe carotid stenosis. PMID- 8627195 TI - Diagnosis of irritable bowel syndrome. PMID- 8627196 TI - Corticosteroid-induced bone loss. PMID- 8627197 TI - Antifungal pulse therapy for onychomycosis. PMID- 8627198 TI - Adolescents' use of levonorgestrel implants for contraception. PMID- 8627200 TI - Screening for abdominal aneurysm. PMID- 8627199 TI - Famciclovir for the treatment of herpes zoster. PMID- 8627201 TI - Influenza vaccine for healthy adults. PMID- 8627202 TI - Hope or experience? Clinical practice guidelines in family practice. PMID- 8627203 TI - Antibiotics and upper respiratory infection: do some folks think there is a cure for the common cold. AB - BACKGROUND: Symptomatic treatment is the only recommended therapy for the uncomplicated "common cold." The purpose of this study was to examine the use of antibiotics and other prescription medications for the common cold in a Medicaid population seen in ambulatory care settings. METHODS: A cross-sectional sample of Kentucky Medicaid claims from July 1, 1993, through June 30, 1994, was analyzed. Subjects were patients seen in an ambulatory setting for the common cold, defined as acute nasopharyngitis. A total of 1439 individuals were seen for 2171 separate outpatient and emergency department encounters for the common cold. Outpatient visits accounted for 99% (2144) of the encounters. RESULTS: Patients in 35% (752) of the encounters did not fill a prescription for medication, 6% (129) filled a prescription for an antihistamine or other symptomatic medication, and 60% (1290) filled a prescription for an antibiotic for the common cold. Nineteen different antibiotics, 54% of which were amoxicillin, were prescribed for the common cold. Less than 2% of the encounters had a secondary diagnosis of either acute sinusitis or otitis media. These encounters were not more likely than the total sample to receive antibiotics. Adults were more likely than children to receive an antibiotic (P<.001), and urban physicians were more likely than rural physicians to prescribe antibiotics (P=.02). A conservative estimate of the annual cost of antibiotic prescribing for the common cold in the United States was $37.5 million. CONCLUSIONS: A majority of persons receiving medical care for the common cold are given prescriptions for an unnecessary antibiotic. Unchecked, this practice may lead to greater antibiotic resistance and unnecessary use of health care resources. Future research should focus on the ability to institute behavioral changes for treatment of the common cold in both closed systems (eg, managed care) and open systems (eg, general community of physicians). PMID- 8627204 TI - Medicaid primary care services in New York State: partial capitation vs full capitation. AB - BACKGROUND: Forty-nine states have applied to the Health Care Financing Administration for waivers to allow special program development for Medicaid recipients. In an effort to identify issues relevant to making the transition of its entire Medicaid population into a capitation model, New York State has encouraged the development of partial capitation and full capitation models. This paper is a critical description analysis of a 1-year experience, utilizing data provided by the New York State Department of Social Services. METHODS: Data collected by the New York State Department of Social Services were used to compare the costs for matched cohorts enrolled in partial capitation programs in which the primary care physician is paid a monthly fee to provide ambulatory primary care for Medicaid recipients; and full capitation programs in which a health maintenance organization (HMO) or a hospital-based prepaid health services program (PHSP) is paid a more encompassing monthly fee to provide a larger range of services, including inpatient, outpatient, and specialty care. RESULTS: Partial capitation programs were reported to save the state 38% compared with a matched control group enrolled in traditional, fee-for-service Medicaid (P<.05), and offered greater savings than HMOs and PHSPs (P=NS). The HMOs and PHSPs saved the state 9.3% and 16.8%, respectively, compared with traditional enrollment. Quality measures and patient satisfaction for partial and full capitation programs were equivalent. CONCLUSIONS: These data suggest that New York State primary care physicians who participated in programs that reimburse a prepaid monthly fee for outpatient primary care services achieved savings comparable to those of HMOs. A partial capitation primary care model may offer an affordable and more flexible alternative to full-service HMOs in caring for Medicaid recipients, especially in communities with limited HMO penetration. PMID- 8627205 TI - Fluticasone propionate aerosol: efficacy in patients with mild to moderate asthma. Fluticasone Propionate Asthma Study Group. AB - BACKGROUND: This double-blind, randomized, parallel-group, placebo-controlled study investigated the efficacy and tolerability of fluticasone propionate aerosol (25, 50, or 100 mg bid for 12 weeks) administered as primary maintenance therapy to patients whose mild to moderate asthma was inadequately controlled by as-needed use of an inhaled beta-agonist. RESULTS: At all clinic visits, fluticasone propionate compared with placebo was associated with significant (P<.05) improvement in pulmonary function indexed by forced expiratory volume in 1 second (FEV1) as well as fewer night awakenings and less use of rescue albuterol. Values for patient-measured morning peak expiratory flow rates (PEFR) were significantly (P<.05) higher and the use of rescue albuterol was significantly (P<.05) lower beginning 3 to 5 days after initiation of therapy in the groups treated with fluticasone propionate, compared with the placebo group. Maximal improvement in FEV1 was achieved during the second week of treatment and maintained throughout the course of therapy. Differences among the three fluticasone propionate dosing groups for these efficacy measures were not statistically significant. The incidence of adverse events was similar across groups. CONCLUSIONS: These data indicate that fluticasone propionate aerosol is an effective and well-tolerated treatment for asthma and significantly improves pulmonary function within days of initiation of treatment in patients whose asthma is inadequately controlled with as-needed beta-agonists. PMID- 8627206 TI - The completion of advance directives in primary care. AB - BACKGROUND: The purpose of this study was to determine whether discussion about and distribution of advance directive forms in a rural, private primary health care office would increase the number of patients who complete and return advance directive forms. This study was also designed to identify individual characteristics of patients who complete advance directives compared with those who do not. METHODS: The sample consisted of 195 patients who ranged in age from 21 to 88 years and visited the primary care office during a 1-month period. Patients who met the inclusion criteria were asked to complete a brief questionnaire while waiting to see their primary care provider, either a physician or a nurse practitioner. The survey included questions about education, previous experience with illness, religion, contact with family members, and attitudes concerning death. The primary care provider then briefly discussed with each patient the advance directive and provided an advance directive form to be completed and returned. The form was short and easy to complete. RESULTS: The discussion about and distribution of advance directive forms in the primary care office results in a 45% return rate. Older patient age (P=.001), longer length of time in the practice (P=.039), less education (P=.025), and physician provider (gamma=.002) were associated with higher completion rates. The variables of provider and level of education were also influenced by older age. CONCLUSIONS: Discussion about and distribution of advance directive forms should be incorporated into primary office care for all adults. PMID- 8627207 TI - Use of the protein/creatinine ratio of a single voided urine specimen in the evaluation of suspected pregnancy-induced hypertension. AB - BACKGROUND: The use of a 24-hour urine collection to evaluate protein excretion in a woman with suspected pregnancy-induced hypertension (PIH) is cumbersome, time consuming, and subject to improper collection. Our purpose was to determine the correlation of the protein/creatinine ratio of a single voided urine specimen to the 24-hour urine collection for total protein in the range of zero to 1000 mg protein per 24 hours. METHODS: Single voided urine specimens and 24-hour total urine protein collections were ordered for 66 consecutive women admitted to an antepartum unit for suspected PIH. The correlation of the protein/creatinine ratio of the single voided specimen with the 24-hour urine protein excretion was calculated. RESULTS: Forty-one sets of data with a 24-hour urine protein less than 1000 mg per 24 hours were obtained. The correlation of the single voided protein/creatinine ratio to the 24-hour total protein was 0.80 (P<.001), with a regression equation of (protein/creatinine)=0.81X(24-hour protein)-0.011. No single protein/creatinine ratio cutoff was ideal to distinguish between significant and insignificant proteinuria; however, a ratio less than .15 efficiently ruled out significant proteinuria [corrected]. CONCLUSIONS: We conclude that the protein/creatinine ratio of a single voided urine specimen may have a role in the management of ambulatory women with suspected PIH, although further study is needed. The main potential benefit of this method is that in institutions where women with suspected PIH are hospitalized, women with insignificant proteinuria may be identified within a matter of hours and their follow-up care handled on an outpatient basis. PMID- 8627208 TI - The Papanicolaou smear: its value and limitations. AB - Although the Papanicolaou (Pap) smear is one of the most effective screening tests ever invented for a common cancer, it remains an imperfect test. The technical shortcomings of the Pap smear have been compounded by the general public's unrealistically high expectations of the test's accuracy, underestimations of the importance of regular smears, and the actions within the medico-legal system. To remedy some of the technical shortcomings, the Bethesda System, which better reflects our current knowledge about cervical neoplasia, has been proposed to replace the old Papanicolaou classification system. Although standardized cytologic criteria may reduce interobserver variability, the false negative rate of Pap smears is at least 5%, even in the best laboratories. No amount of training or experience with human observers can reduce the error rate to zero. Automated Pap screening holds the promise of higher sensitivity, but no instruments to date have been approved as a sole means of primary screening. The family physician can play a unique role in overcoming the limitations of the Pap smear by educating patients about the value and limitations of the test, instituting patient-specific treatment or follow-up of abnormal smears based on clinical and cytologic findings, and encouraging patients to get regular smears at intervals based on risk. PMID- 8627209 TI - Right mandible swelling of unknown origin. AB - Infantile cortical hyperostosis (ICH), or Caffey's disease, has a low prevalence, is not easily recognized clinically, and is seldom reported in the primary care literature. A case of infantile cortical hyperostosis of the right mandible of a newborn is reported. Multiple radiographic tests, as well as bone biopsy, were necessary to establish a definitive diagnosis. The most striking features of this disease are presented, including the clinical and physical presentation, radiographic characteristics, and pathology. PMID- 8627210 TI - Eosinophilic pleural effusion: is it always nondiagnostic? AB - The presence of eosinophils in the pleural effusion is generally considered nondiagnostic. It usually indicates that the patient has had a previous thoracentesis and that air or blood has come in contact with the effusion. Idiopathic acute eosinophilic pneumonia is characterized by acute onset of pulmonary symptoms with hypoxemia, pulmonary infiltrates, eosinophils in bronchoalveolar lavage fluid, and prompt response to steroid therapy. We report a patient who presented with symptoms of acute pneumonia in which the presence of increased eosinophils in the pleural effusion indicated eosinophilic pneumonia. PMID- 8627211 TI - Survival of family medicine in a corporate health care environment: lessons from the United Kingdom. AB - The preservation of small, independent family practices within a changed health care delivery environment is a goal worth pursuing. Properly organized, such practices would maintain the special fiduciary relationship between physicians and patients. Fundholding practices, which are a growing organizational form of general practice in the United Kingdom, are described as a model that could serve as the basis for an American alternative to corporate medicine. PMID- 8627213 TI - Managed care in the house of God. PMID- 8627212 TI - Managed care in the house of God. PMID- 8627214 TI - Testing for anergy. PMID- 8627215 TI - Troubled thyrotrophs. PMID- 8627216 TI - Hiccup. PMID- 8627217 TI - Do light-induced pH changes within the chloroplast drive turnip yellow mosaic virus assembly? AB - Turnip yellow mosaic virus (TYMV) induces gross morphological and biochemical changes in the chloroplasts of infected cells. Viral RNA is synthesized in vesicles formed by invagination of the outer chloroplast bilayer. Virion assembly occurs at the neck of these vesicles and requires illumination. Data collected over the last three decades are consistent with the hypothesis that light-induced generation of a low pH drives TYMV assembly within the intermembrane space of chloroplasts. In a low-pH environment, poly(C) regions within the genomic RNA of TYMV may interact to form tertiary structures, and the recognition of these structures by TYMV coat protein initiates virion assembly. PMID- 8627218 TI - Physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus. AB - A physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus (AfMNPV) DNA was constructed for restriction endonucleases EcoRI, HindIII, PstI and XhoI. The genome size was estimated to be 130 kbp. Ordering of the restriction fragments was accomplished by cross-blot hybridization, double digestion and DNA-DNA hybridization. The polyhedrin gene and homologous repeat (hr) regions were located by hybridization to the Autographa californica MNPV (AcMNPV) polyhedrin gene and hr4, respectively. Restriction pattern comparison and Southern blot analysis suggest that AfMNPV is closely related to AcMNPV. PMID- 8627219 TI - The hepatitis B virus X protein is a potent AMP kinase. AB - The hepatitis B virus X-protein (HBx) has been expressed in Escherichia coli both as an unfused protein and with an N-terminal hexaHis-containing fusion sequence. Both forms of HBx, after purification, displayed a potent AMP kinase activity, in which HBx phosphorylates AMP to ADP, using ATP as the exclusive phosphate donor. We also found that HBx has previously unreported GTPase and GTP-ADP nucleoside diphosphate kinase activities. PMID- 8627220 TI - Unusual activation of the integrated preS1 promoter of woodchuck hepatitis virus in a liver tumour. AB - We have analysed abnormal virus RNAs produced from integrated woodchuck hepatitis virus (WHV) sequences in two woodchuck liver tumours. Analysis of cDNA clones revealed that these transcripts consisted of rearranged, virus-specific RNAs encoding the WHV surface antigens. In one tumour, transcription was driven by the major preS2/S promoter and terminated at a cryptic poly(A) signal in the 5' end of the P gene, giving rise to a truncated version of the normal viral S message. In contrast, the integrated preS2/S promoter remained silent in the second tumour. The start sites of two abundant WHV transcripts encoding the large and middle surface proteins were localized about 100 bp upstream and 300 bp downstream of the preS1 translation initiation codon, corresponding to minor start sites of the normal surface protein mRNAs in chronically infected liver. Thus, the preS1 promoter, a weak promoter in episomal replicative forms of the virus, was activated in the integrated state in this tumour. Our results indicate that alternative usage of the preS1 or the preS2/S promoter in the integrated state may yield differential production of the three virus surface proteins in woodchuck liver tumours. PMID- 8627221 TI - Antigenicity of bovine papillomavirus type 1 (BPV-1) L1 virus-like particles compared with that of intact BPV-1 virions. AB - Virus-like-particles (VLPs) of various papillomavirus (PV) types have been produced by expressing recombinant L1 proteins in eukaryotic cells. Although VLPs have the same ultrastructural appearance as native virions and their immunogenicity appears to be similar, their antigenicity has not been carefully evaluated. For this reason, the antigenicity of intact bovine PV type 1 (BPV-1) virions was compared with that of BPV-1 recombinant L1 VLPs by ELISA using a well characterized panel of polyclonal and monoclonal antibodies generated against intact and denatured BPV-1 particles. The structural integrity of the authentic virions and recombinant VLPs was verified by electron microscopy. The specificity of antibodies raised against intact BPV-1 virions and their reactivity with VLPs revealed that the immunodominant, type-specific, conformational epitopes of intact virions were reproduced on VLPs. However, many monoclonal antibodies that define cross-reactive, non-conformational (linear) epitopes cryptic to the authentic BPV-1 virion tested positively when reacted with intact VLPs. One monoclonal antibody, which recognizes a BPV-1 and deer PV surface conformational epitope, did not react with VLPs. Therefore, although VLPs can be used to immunize animals against infection, the external exposure of broadly cross reactive epitopes of intact L1 VLPs suggests that the use of L1 VLPs in antigenicity studies such as serological screening should be done with caution. PMID- 8627222 TI - Sp1 is critical for basal and E2-transactivated transcription from the bovine papillomavirus type 1 P89 promoter. AB - The bovine papillomavirus type 1 (BPV-1) long control region (LCR) contains at least three consensus binding sites for the transcription factor Sp1 at nucleotides (nt) 7800, 7833 and 7854. A high basal-level P89 expression vector consisting of an origin-deleted LCR fused to the chloramphenicol acetyltransferase (CAT) gene was utilized to determine the role of these Sp1 sites in the regulation of transcription from the BPV-1 P89 promoter. The three Sp1 sites were capable of binding Sp1 in vitro. Mutation of these sites in the background of the origin-deleted LCR-CAT or a wild-type LCR-CAT construct resulted in decreased basal expression from P89. In addition, mutation of the Sp1 sites in the wild-type background caused a reduction in E2-transactivation potential. These data illustrate the importance of these Sp1 sites in regulating both basal and E2-transactivated P89 expression. PMID- 8627223 TI - Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system. AB - We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNA(ser) to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immunoblotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed. PMID- 8627224 TI - Induction of varicella-zoster virus-neutralizing antibodies in mice by co infection with recombinant vaccinia viruses expressing the gH or gL gene. AB - Recombinant vaccinia viruses (VV) expressing the varicella-zoster virus (VZV) glycoprotein H (gH) or glycoprotein L (gL) were constructed. The 94 kDa gH intermediate glycoprotein was synthesized in cell cultures infected with the VV gH recombinant, but only co-infection with both recombinants resulted in the synthesis of the fully processed 118 kDa gH molecule. The VV-expressed gH and gL formed a complex that displayed the conformational neutralization epitope detectable by means of human VZV gH-specific monoclonal antibody V3. Formation of this epitope was inhibited by tunicamycin but not by monensin. Simultaneous intraperitoneal inoculation of mice with high doses of both VV-gH and VV-gL viruses resulted in the development of VZV-neutralizing, complement-independent antibodies; these antibodies were not detected in mice infected solely with either the VV-gH or the VV-gL recombinant. PMID- 8627225 TI - Lack of MHC class I complex expression has no effect on spread and control of cytomegalovirus infection in vivo. AB - It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability of beta 2-microglobulin (beta 2m) and also the degree of peptide charging of the MHC class I heavy chain beta 2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in beta 2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8+ T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in beta 2m mutant mice. In addition, there was no indication of diminished virus propagation in beta 2m-/- embryonic fibroblasts. beta 2m-/- mice suffered from the lack of CD8+ T lymphocytes that was partially compensated for by the function of CD4+ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the beta 2m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection. PMID- 8627227 TI - Amino acids involved in distinguishing between monotypes of rotavirus G serotypes 2 and 4. AB - Neutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV 5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation. PMID- 8627226 TI - Epstein-Barr virus nuclear antigen 2 (EBNA2)-oestrogen receptor fusion proteins complement the EBNA2-deficient Epstein-Barr virus strain P3HR1 in transformation of primary B cells but suppress growth of human B cell lymphoma lines. AB - To develop a transformation system with a conditional Epstein-Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines. PMID- 8627228 TI - Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C. AB - The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction. PMID- 8627229 TI - Antibody and host cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif: a structural interpretation. AB - Foot-and-mouth disease virus (FMDV) of serotype C (isolate C-S8c1) was cleaved in situ by trypsin at the Arg-Gly-Asp (RGD) motif, which is involved both in attachment of FMDV to cells and in recognition of a major antigenic site (site A) by antibodies. Though 99.4% of the RGD moieties were cleaved, the virus remained infectious. A synthetic peptide which represented the sequence of the VP1 G-H loop of C-S8c1, including the RGD motif, greatly inhibited FMDV attachment to cells. The same peptide inhibited, very effectively and to the same extent (50% inhibition at about 1 microM), the infectivity of both intact and trypsin-treated virus. Replacement of Asp with Glu at the RGD motif abolished the inhibitory effects of the peptide. Thus, the RGD motif is involved in the infectivity of both intact and RGD-cleaved serotype C FMDV. Trypsin treatment did not affect the reactivity of the virus with some monoclonal antibodies (MAbs) directed to site A whose epitopes involve mainly residues contiguous to the cleaved bond, but diminished the reactivity with site A MAbs whose epitopes include the RGD sequence and flanking residues. However, high concentrations of any site A MAb tested neutralized close to 100% of the infectious trypsin-treated virus. We propose that, in spite of covalent cleavage, the high number of intramolecular non-covalent interactions observed within the G-H loop of FMDV C-S8c1 (complexed to antibody) may hold the RGD in a nearly correct conformation and allow--albeit with reduced affinity--antibody and cell receptor recognition of RGD-cleaved FMDV. PMID- 8627230 TI - Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA. AB - Foot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5' untranslated region (5'UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5'UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5'UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5'UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5'UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation. PMID- 8627231 TI - Limited genetic changes in the Sabin 1 strain of poliovirus occurring in the central nervous system of monkeys. AB - Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU-->AU or GU-->GC substitution at a complementary pair formed by nucleotides 480 and 525, U-->C substitution at nucleotide 612, and GU-->AU or GU-->GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral 'passengers', passively co-selected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88-->Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production. PMID- 8627232 TI - Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis. AB - The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination. PMID- 8627233 TI - Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11a) genetic groups. AB - Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279-9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups. PMID- 8627234 TI - Pathway of rubella virus infectious entry into Vero cells. AB - The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway. PMID- 8627235 TI - An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine. AB - The full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 micrograms per 10(6) cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 micrograms per 10(6) cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S' and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector. PMID- 8627236 TI - The JHM strain of mouse hepatitis virus induces a spike protein-specific Db restricted cytotoxic T cell response. AB - Cytotoxic T lymphocyte (CTL) activity specific for mouse hepatitis virus (MHV) JHM strain (JHMV or MHV-4) was examined using in vitro stimulated spleen cells derived from immunized C57BL/6 (H-2b) mice. Target cells infected with JHMV were specifically recognized; however, analysis of target cells expressing the virus structural proteins via recombinant vaccinia viruses showed no recognition of the viral nucleocapsid (N), membrane (M), small membrane (sM) or haemagglutinin esterase (HE) proteins. Only target cells expressing the virus spike (S) protein were recognized. Furthermore, the majority of CTL activity was restricted to target cells expressing the MHC class I Db molecules. Analysis of truncations and deletions of the S protein expressed by recombinant vaccinia viruses and peptide coated targets identified a single antigenic epitope, aa 510-518, conforming to the Db binding motif. These amino acids are contained within a domain deleted from a number of strains of mouse hepatitis virus, suggesting a role for immune pressure. To determine the potential for CTL specific for an epitope(s) within a non-structural protein, 24 CTL lines were established and characterized. No evidence for the induction of non-specific CTL activity or virus-specific CTL restricted to an epitope in a non-structural protein was obtained. These data indicate that the predominant CTL activity in JHMV-infected C57BL/6 mice is Db restricted and specific for a single epitope contained within aa 510-518 of the S protein. PMID- 8627237 TI - Identification of the sequences responsible for nuclear targeting of the V protein of human parainfluenza virus type 2. AB - In human parainfluenza virus type 2 (hPIV-2)-infected cells, anti-phosphoprotein (P)-specific monoclonal antibody (MAb) densely stained the perinuclear regions of infected cells throughout infection, indicating that the P protein was localized exclusively in the cell cytoplasm. By contrast, antigens recognized by MAbs directed against the P-V-common domain of hPIV-2 were located predominantly in the cytoplasm, but in some hPIV-2-infected cells they were also found in the nuclei, suggesting that a fraction of hPIV-2 V protein is localized there. hPIV-2 V protein expressed from a cDNA clone was localized in the nuclei of transfected cells. By using indirect immunofluorescence analyses, we examined the intracellular localization of various sequentially deleted V proteins, to determine the nuclear localization signals (NLS) of the V protein. Two noncontiguous regions in the V protein were required for nuclear localization and retention, since deletion of these regions [region I (aa 1-46) and region II (aa 175-196)] resulted in cytoplasmic localization. Both regions resulted in nuclear localization independently. A nucleoplasmin-like NLS was identified in region II but no consensus targeting sequence could be found in region I. When NP protein was co-expressed with V protein or the N-terminal fragment (aa 1-46) of V protein, a fraction of the NP protein was translocated into cell nuclei. PMID- 8627238 TI - Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites. AB - Nineteen yeast colonies secreting rabies virus glycoprotein (G) peptides immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54-494 of the G protein. These peptides, together with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 anti-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reacted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturation (WB-). This last category represents 98% of anti-rabies virus G MAbs. None of the WB- MAbs bound peptides. Of the 12 WB+ MAbs, one bound two peptides situated before the transmembrane domain of the protein and six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered 'immunodominant' if the peptide strategy had been used to study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had significant neutralizing activity; two were used for the selection of antigenic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both the peptide and escape mutant approaches. However, a few mutants had a substitution outside the peptide-delimited region, suggesting that remote mutation(s) could affect epitope accessibility in the native protein. PMID- 8627239 TI - The genomic structure of a new simian T-lymphotropic virus, STLV-PH969, differs from that of human T-lymphotropic virus types I and II. AB - A new simian T-lymphotropic virus, STLV-PH969, was recently isolated from a wild born Hamadryas baboon. Previous analysis had revealed that it differs sufficiently from the other HTLV/STLVs to be considered a new type, provisionally designated primate T-lymphotropic virus-L. Here we analyse a 3850 bp cDNA fragment spanning the 3' part of the STLV-PH969 genome. The fragment encodes three major proteins: Env, Tax and Rex. Sequence comparison and phylogenetic analysis indicate that in general STLV-PH969 tends to be more closely related to HTLV-II than to HTLV-I, although separate gene regions might have evolved under different constraints. Detailed comparison of the Env, Tax and Rex proteins among the HTLV-I, -II and STLV-PH969 prototypes reveals that the amino acid sequence of each protein shows a preferential conservation of functionally important domains. RNA-PCR on cytoplasmic messengers demonstrated splicing between a splice donor site immediately downstream of the env start codon, and two splice acceptor sites identified in the pX region. The predominant spliced messenger encodes both Tax and Rex. The other messenger potentially encodes a new viral protein from the proximal part of the pX region that is similar in amino acid composition to p12I and p10xI of HTLV-I and HTLV-II respectively. This genomic organization of the proximal pX region of STLV-PH969 is different from that found in HTLV-I and HTLV II. Therefore, the distinct classification of this virus can be justified, not only in terms of sequence divergence but also in terms of its different genomic structure. PMID- 8627240 TI - The three human T-lymphotropic virus type I subtypes arose from three geographically distinct simian reservoirs. AB - To investigate the origin of human T-lymphotropic virus I (HTLV-I), strains of diverse geographical origin were analysed. We sequenced the LTR and env genes of HTLV-I strains from Brazil, Central African Republic, Taiwan and Zaire, and the simian T-lymphotropic virus type I (STLV-I) strain PHSu1 from a baboon from the Sukhumi primate centre. We performed phylogenetic analyses using neighbour joining, parsimony and maximum likelihood methods. Three separate HTLV-I clusters were identified interspersed between STLV-I clusters. The Brazilian and the Taiwanese strains were within the first well-supported cluster containing all cosmopolitan HTLV-I strains flanked by west African STLV-I strains. The HTLV-I strains from Central African Republic and Zaire fell into a central African cluster close to the chimpanzee STLV-I isolates. The third well-supported cluster included all Melanesian HTLV-I strains and had Indonesian STLV-I strains as closest neighbours. Therefore, currently known HTLV-I strains represent three HTLV-I subtypes that most probably have originated from three geographically distinct interspecies transmission events. The highly divergent PHSu1, isolated from Papio hamadryas, was closely related to PCY-991, isolated from Papio cynocephalus, both from the Sukhumi primate centre. Both clustered together with Asian wild-caught rhesus macaque STLV-I strains suggesting recent interspecies transmission of virus from rhesus macaques to colony-bred African baboons at the Sukhumi primate centre. In the rooted env trees obtained using the STLV strain PH969 as an outgroup, the Asian strains branched off before the African strains, implying an Asian origin for HTLV/STLV type I based on presently available strains. PMID- 8627241 TI - Transfer of human T cell lymphotropic virus type I to human term trophoblast cells in vitro. AB - We studied the susceptibility of term placental trophoblast cells to in vitro infection with human T cell leukaemia/lymphotropic virus type I in order to provide further insight into the role of syncytiotrophoblast in transplacental passage of the virus. Pure villous trophoblast cultures were exposed to cell-free virus and the extent of infection was analysed by semiquantitative PCR assay to detect integrated proviral DNA. Four different primer pairs targeting the gag, pol, env and pX regions invariably revealed that virus sequences were present in amounts 10(2)-10(3) times less than in the reference cell line MT-2. Virus expression was studied at both the transcriptional and translational levels. Whereas doubly spliced mRNAs coding for the Tax and Rex regulatory proteins could be detected by RT-PCR no virus-specific proteins were found in the cells by immunoperoxidase staining. The present data lend support to the notion that the placental trophoblast may represent a barrier effectively protecting the fetal compartment from exposure to the virus. PMID- 8627242 TI - Characterization of the human endogenous retrovirus K proteinase. AB - The proteinase of the human endogenous retrovirus K (HERV-K) shows similarity to retrovirus aspartic proteinases. It is translated from a transcript composed of gag and prt. The proteinase was expressed either as full-length native protein or as truncated protein in Escherichia coli. Functional protein was demonstrated by its autocatalytic cleavage into an 18 kDa fragment recognized by a polyclonal antiserum. This autocatalytic cleavage was specifically inhibited by a human immunodeficiency virus type 1 proteinase inhibitor. The HERV-K proteinase expressed in E. coli was capable of cleaving HERV-K Gag translated in vitro. Major protein fragments of 39 and 30 kDa, and minor protein fragments of 26, 22 and 21 kDa were obtained. Similar fragments are also observed in the human teratocarcinoma cell line Tera1. Our data suggest that the HERV-K proteinase is functionally equivalent to other retrovirus proteinases and thus probably functions in the processing of Gag precursor protein. PMID- 8627243 TI - Purification, characterization, assembly and crystallization of assembled alfalfa mosaic virus coat protein expressed in Escherichia coli. AB - The coast protein of alfalfa mosaic virus (AMV) was cloned and expressed in Escherichia coli as a fusion protein containing a 37 amino acid extension with a (His)6 region for affinity purification. About half of the expressed recombinant coat protein (rCP) was soluble upon extraction and half was insoluble in inclusion bodies. Western blot analysis confirmed the identity of the rCP and protoplast infectivity assays indicated that the rCP was biologically active in an early event of AMV infection, called genome activation. The rCP assembled into T = 1 empty icosahedral particles, as described previously for native coat protein. Empty particles formed hexagonal crystals that diffracted X-rays to 5.5 A resolution. The crystals of trypsin-treated particles of rCP appear to be isomorphous with crystals of trypsin-treated particles of native coat protein, Spherical particles containing RNA assembled when the rCP was combined with in vitro transcripts of AMV RNA4, the smallest naturally encapsidated AMV RNA. Bacilliform particles that resembled native virions assembled when the rCP was combined with transcripts of RNA1, the largest genomic RNA. PMID- 8627244 TI - Nucleotide sequence analysis of RNA-5 of five isolates of beet necrotic yellow vein virus and the identity of a deletion mutant. AB - The nucleotide sequences of RNA-5 from two laboratory isolates (D-5 and D-6) and three field isolates (SH1, S43 and R83) of beet necrotic yellow vein virus (BNYVV) were determined. Isolates D-5 and D-6, derived from a D field culture during mechanical inoculation, contained RNA-5 of molecular size 1.4 kb and 1.0 kb, respectively. The sequences of D-5 SH1, S43 and R83 were found to be at least 98% identical and from 1342 to 1347 nucleotides in length, excluding the poly(A) tail. Each contained a single open reading frame (ORF) encoding a 228 amino acid protein with a molecular mass of 26189 Da (P26). The coding sequence was bordered by a long leader of 443 to 448 nucleotides and a 3'-terminal non-coding region of 215 nucleotides. In isolate D-6, containing the smaller approximately 1.0 kb RNA species referred to as RNA-5a, the ORF had undergone an internal deletion of 303 nucleotides. No sequence identity was found between RNA-5 and either RNA-3 or RNA 4, except for the 5'-terminal nine residues and for approximately the 3'-terminal 200 residues. Thus, the genome organization of BNYVV RNA-5 is very similar to that of RNA-3 and RNA-4, both of which are essential for survival of BNYVV in nature. Although RNA-5 is not essential, it may be associated with symptom expression of BNYVV. PMID- 8627245 TI - Genomic reassortment of barley mild mosaic virus: evidence for the involvement of RNA1 in pathogenicity. AB - A reverse transcription-polymerase chain reaction (RT-PCR) was developed for specific detection of RNA1 and RNA2 of two barely mild mosaic virus strains (BaMMV-Ka1 and BaMMV-Na1) and a barley yellow mosaic virus strain (BaYMV-II-1). Mechanical inoculation of barley cultivars with a mixture of BaMMV-Ka1 and BaMMV Na1, followed by RT-PCR to detect RNA components in infected plants, revealed that the RNAs of the two strains were exchangeable in vivo to generate all nine possible combinations containing at least one RNA1 and one RNA2. Infected plants with mixed or reassorted RNAs were selected and used as inocula for further analysis of cultivar reactions. The results demonstrate that the pathogenicity and symptomatology are determined solely by RNA1. In contrast, following inoculation with mixtures of BaYMV-II-1 and either BaMMV-Ka1 or BaMMV-Na1, no heterologous combinations of their RNAs were observed. PMID- 8627246 TI - Cooperative binding to nucleic acids by barley yellow mosaic bymovirus coat protein and characterization of a nucleic acid-binding domain. AB - The capacity of several coat protein (CP) mutants of a German isolate of barley yellow mosaic bymovirus (BaYMV) to bind of nucleic acids was studied in vitro. Recombinant CP, produced by overexpression in Escherichia coli, was purified from inclusion bodies and subsequently renatured. Binding to single-stranded (ss) RNA and ssDNA oligonucleotides was found to be cooperative and sequence non-specific. By deletion mutagenesis, several truncated CP derivatives were created and their nucleic acid-binding capacity was investigated in order to define a protein domain responsible for RNA- and DNA-binding. The nucleic acid-binding domain consists of a core which was located to an internal 23 amino acid peptide (aa 125 147) and an adjacent domain (aa 148-184) which stimulates binding. PMID- 8627247 TI - Proliferative T cell responses to human papillomavirus type 16 L1 peptides in patients with cervical dysplasia. AB - Human papillomavirus type 16 (HPV-16) can cause genital warts, cervical dysplasias and carcinoma of the cervix. Cell-mediated immunity is thought to be important in protection against the virus and in its elimination, but little is known about the mechanisms involved. In a cross-sectional study we have demonstrated proliferative T cell responses to peptides representing the HPV-16 L1 capsid protein (aa 199-409) in the peripheral blood of 63% of patients (n = 41) with histological evidence of cervical dysplasia and in 45% of healthy age matched controls (n = 11). This was achieved by generating short-term T cell lines (STLs) from each individual in vitro against a beta-galactosidase-HPV- 16 L1 (aa 199-409) fusion protein for 2 weeks, and then identifying the HPV epitopes they recognized with overlapping synthetic peptides (15-mers) spanning this region in 3 day specificity assays. Histological grading and HPV typing by PCR were performed on patients' cervical biopsies taken at the same clinical visit as the peripheral blood samples. An immunogenic region was identified between aa 311 345 in 73% of patients (18% in controls) who responded to HPV-16 L1 (aa 199-409). The number of responders to this region was significantly higher in patients with HPV-16-positive biopsies when compared to those with HPV-16-negative biopsies (P = 0.006), as was the number of responders to individual peptides 311-325 (NLASSNYFPTPSGSM; p = 0.04) and 321-335 (PSGSMVTSDAQIFNK; P = 0.004) representing this region. The mean level of response to each individual peptide was also higher in the patient group than the controls (P < 0.05). The most significant finding was that all patients with evidence of a current HPV-16 infection responded to one or more L1 peptides (P = 0.0004) and 92% had high grade cervical intraepithelial neoplasia (CIN III). We also found that the CIN III group was more likely to respond to any L1 peptide than either the atypical group (P = 0.04) or the controls (P = 0.05). Data from four individuals showed that the majority of peptide-specific STLs were CD4+ but some CD8+ STLs were also detected. PMID- 8627248 TI - Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment. AB - Several fowlpox virus (FPV) DNA fragments were selected by differential hybridization using cDNA of transcripts that were strongly transcribed early and/or later after infection of QT-35 cells. The EcoRI L fragment contained three strongly transcribed FPV genes: L1L, a late 1452 bp partial (amino end) ORF; L2R, an early/late 522 bp ORF; and L3R, a late 948 bp ORF. The protein products of L1L, L2R and L3R shared homology with the products of vaccinia virus (VV) genes H4L (RAP94), H5R (Ag35) and H6R (topoisomerase), respectively, suggesting a conservation of gene structure and order between VV and FPV. The 5' upstream non coding sequences of L1L and L3R were A + T rich and the sequence 5' TAAATG 3' overlapped the predicted translation start codon. Primer extension analysis of the L2R transcript mapped the transcriptional start sites of early and late mRNAs 14 nt downstream of a VV early promoter-like critical region sequence, AAAATTGAA AAAAAAA. A VV-like TAAAT late transcriptional element was present 20 nt upstream of the L2R ATG translational start codon. A plasmid with the putative early L2R promoter cloned upstream of the Newcastle disease virus haemagglutinin neuraminidase (HN) cDNA as a reporter gene was at least 6-fold more effective in generating HN MRNa than plasmids containing the P7.5 or P11 VV promoters in transient expression assays in FPV-infected CEF cells treated with cytosine arabinoside. The L2R promoter was also able to express an amount of HN mRNA equal to that expressed by the VV promoters late in infection. PMID- 8627249 TI - Identification and characterization of BICP27, an early protein of bovine herpesvirus 1 which may stimulate mRNA 3' processing. AB - Sequence analysis of the left genomic terminus of bovine herpesvirus 1 (BHV-1) revealed two convergently transcribed genes with 3' ends about 300 bp apart. The gene on the left is the previously described circ gene; that on the right was found to encode a protein of 400 amino acids which was designated BICP27 because of its homology to ICP27 (Vmw63) of herpes simplex virus 1 (HSV-1) and related proteins from other alpha- beta- and gammaherpesviruses. Rabbit antisera raised against a synthetic oligopeptide representing the amino terminus of the predicted polypeptide demonstrated the presence of BICP27 in the nuclei of infected cells by in situ immunoadsorbent assays. In Western immunoblots, BICP27 was detected as a 50 kDa BHV-1 specific protein expressed with early kinetics, in contrast to HSV 1 ICP27 which is an immediate early (IE) protein. A DNA fragment containing BICP27 coding sequences was inserted into a baculovirus genome. The recombinant BICP27 protein, identified by its reactivity with the antipeptide sera, exhibited the same electrophoretic mobility as BICP27 specified by BHV-1. Transient expression assays using target genes differing only in their poly(A) sites showed that BICP27, like its HSV-1 counterpart, may be involved in 3' processing of mRNA. PMID- 8627250 TI - Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells. AB - Murine gammaherpesvirus (MHV-68) causes an acute respiratory infection followed by a latent infection in B lymphocytes. In the first 2-3 weeks after infection mice develop a marked splenomegaly, where the spleen cell number increases by 2-3 fold. Cytofluorimetric analysis during splenomegaly revealed an increase in numbers of B lymphocytes and of both CD4+ and CD8+ T lymphocytes. The largest increase relative to uninfected spleens was in the CD8+ population. The number of latently infected cells in the spleen peaked at day 10 post-intraperitoneal infection, then declined to 1/10(6)-1/10(7) cells per spleen. Depletion of CD4+ T lymphocytes prevented the splenomegaly and greatly reduced the peak infective centre level, while having no effect on the long-term of latently infected cells. Given the similarity between MHV-68-induced splenomegaly and Epstein-Barr virus induced infectious mononucleosis, these data highlight the usefulness of MHV-68 as a mouse model for the study of gammaherpesvirus immunology and pathobiology. PMID- 8627251 TI - Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS) glycoprotein induces neutralizing antibodies and protects against experimental infection. AB - Two related glycoproteins (G and G(NS)) encoded in the bovine ephemeral fever virus (BEFV) genome were expressed from recombinant vaccinia viruses (rVV). Both proteins were detected in lysates of rVV-infected cells by labelling with D-[6 3H]glucosamine or by immuno-blotting. The recombinant G protein (mol. mass 79 kDa) appeared slightly smaller than the native G protein but reacted with monoclonal antibodies directed against all defined neutralizing antigenic sites (G1, G2, G3a, G3b and G4). The recombinant G(NS) protein (mol. mass 90kDa) was identical in size to the native G(NS) protein and failed to react by immuno fluorescence with anti-G protein monoclonal or poly-clonal antibodies. Antisera raised in rabbits against rVV-G or rVV-G(NS) both reacted strongly by immuno fluorescence and immuno-electron microscopy with BEFV-infected cells. The G protein was localized intracellularly in the endoplasmic reticulum/Golgi complex and at the cell surface associated with budding and mature virus particles. The G(NS) protein also localized intracellularly in the endoplasmic reticulum/Golgi complex; however, at the cell surface it was associated with amorphous structures and not with budding or mature virions. Rabbits vaccinated with rVV-G developed high levels of antibodies which neutralized BEFV grown in either mammalian or insect cells. Cattle vaccinated with rVV-G also produced neutralizing antibodies and were protected against experimental BEFV infection. In contrast, rVV-G(NS) vaccinated rabbits and cattle failed to produce neutralizing antibodies and, after challenge, BEFV was isolated from two-thirds of the vaccinated cattle. PMID- 8627252 TI - Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses. AB - The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/ 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses. PMID- 8627253 TI - A point mutation in the F1 subunit of human respiratory syncytial virus fusion glycoprotein blocks its cell surface transport at an early stage of the exocytic pathway. AB - Vaccinia virus recombinants expressing either wild-type or mutant forms of human respiratory syncytial (RS) virus (Long strain) fusion (F) glycoprotein were obtained. Proteolytic processing of the precursor, F0, and cell surface transport of the F glycoprotein were unaffected in the recombinants, except in those that contained the replacement Phe --> Ser at position 237 of the F1 subunit. In recombinants containing this mutation, either alone or in combination with others, the traffic of the F molecule was arrested at some intermediate step of its transport to the cell surface and, consequently, the endoproteolytic cleavage of the F0 precursor was inhibited. Immunofluorescence staining of infected cells and endoglycosidase H (Endo-H) sensitivity assays indicated that the arrest occurred before the mid-Golgi compartment. Dimerization and folding of the F protein were also affected by the Phe237 --> Ser substitution. Other amino acid replacements at positions 236 or 237 of the F1 subunit had various effects upon F0 maturation. These results are discussed in terms of the maturation requirements for the RS virus F molecule. PMID- 8627254 TI - Antigenic and genetic evolution of equine H3N8 influenza A viruses. AB - Evolution of equine influenza a H3N8 viruses was examined by antigenic and genetic analysis of a collection isolates from around the world. It was noted that antigenic and genetic variants of equine H3N8 viruses cocirculate, and in particular that variants currently circulating in Europe and the USA are distinguishable from one another both in terms of antigenic reactivity and genetic structure of the HA1 portion of the haemagglutinin (HA) molecule. Whilst the divergent evolution of American and European isolates may be due to geographical isolation of the two gene pools, some mixing is believed to occur as 'American-like' viruses have been isolated during outbreaks of equine influenza in the UK. The cocirculation of two antigenically and genetically distinct lineages of equine influenza H3N8 viruses has serious implications for vaccine strain selection. PMID- 8627255 TI - Evolution of the haemagglutinin-esterase gene of influenza C virus. AB - The nucleotide sequences of the haemagglutinin-esterase (HE) genes of 18 influenza C virus strains isolated in Japan during the period from 1964 to 1988 (11 published and 7 new sequences) were analysed to examine their evolutionary relationships. The phylogenetic tree constructed by the maximum parsimony method revealed the existence of four discrete lineages (I to IV), one of which (lineage III) may have died out in the late 1970s. Sequential evolution was demonstrated within seven strains of lineage I, which allowed estimation of an evolutionary rate of 0.49 x 10-3 nucleotide substitutions per site per year, a value corresponding to about one-ninth of the rates of human influenza A virus haemagglutinin genes. In the previously proposed immunodominant region on HE protein (positions 178 to 217), there was little or no amino acid sequence divergence among viruses on the same lineage although considerable divergence was seen among those on different lineages, raising the possibility that immune selection may not have played a significant role in the evolution of the glycoprotein, at least not after separation into lineages occurred. It was also found that the HE genes of the seven isolates obtained outside Japan during 1966 1983 could be each assigned to one of lineages I, II and IV, which suggests that influenza C virus is capable of spreading worldwide. PMID- 8627256 TI - Influenza C virus RNA is uniquely stabilized in a steady state during primary and secondary persistent infections. AB - The ability to establish persistent infections in vitro and in vivo has been illustrated for different human RNA viruses. However, little insight has been gained regarding the intracellular state of viral RNA species and the regulatory processes governing their long-term continuance. In this report, primary persistence of a variant of influenza C/Ann Arbor/1/50 virus in infected MDCK cells and secondary infections in human cell lines were investigated. Different PCR and staining techniques were applied for the description of low viral loads. The RNA pattern in primary persistence indicates that viral RNA synthesis is quantitatively linked to productive and non-productive phases, with negative strand RNA being present continuously. In single cells cultures, derived from the primary line, all clones tested were positive by nested PCR and Southern blot screening. This suggests that a true steady-state persistence of influenza C virus is established in each individual cell of the infected population. Secondary infection experiments, in terms of transfer of the persistent virus variant to different cell types, showed that a re-establishment of persistence can be accomplished in vitro. The stable persistent status remained reserved for distinct host cell lines. Hereby, vRNA is stably maintained in cell-type specific manner, whereas gene expression (e.g. HEF glycoprotein production) occurs in a variable fashion. These data point out novel characteristics in the understanding of influenza virus persistence. PMID- 8627257 TI - Khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from Microtus fortis trapped in far-east Russia. AB - Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR). PMID- 8627258 TI - Characterization of the nucleocapsid protein of Hantaan virus strain 76-118 using monoclonal antibodies. AB - We characterized the antigenic sites on the nucleocapsid protein (NP) of Hantaan virus (HTN) using 10 monoclonal antibodies (MAbs). At least seven antigenic sites were revealed by a competitive binding assay and divided into three partially overlapping antigenic regions (I, II and III). Regions I [amino acids (aa) 1 103], II (aa 104-204) and III (aa 205-402) were mapped on NP by examining the reactivity of truncated gene products. Those that corresponded to region I reacted with immune mouse serum, indicating that the region contained major linear epitopes as reported with Four corners virus (FCV) and Puumala virus (PUU) NP. At least one MAb to each region inhibited viral growth when they were introduced into cells by scrape-loading. In addition, they conferred protection from a lethal HTN challenge to newborn mice. A PEPSCAN assay localized the epitope of MAb E5/G6 between aa 166-175. Since E5/G6, which had the highest inhibitory effect both in cells and in mice, showed no virus neutralization activity by ordinary neutralization test, this region is suggested to be important for the virus growth after entry into the cells. PMID- 8627259 TI - Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: requirement for L3T4+ T cells. AB - The protective ability of cytotoxic T cells (CTL) raised in vitro against Japanese encephalitis virus (JEV) was examined by adoptive transfer experiments. Adoptive transfer of anti-JEV effectors by intracerebral (i.c.) but not by intraperitoneal (i.p.) or intravenous (i.v.) routes protected adult BALB/c mice against lethal i.c. JEV challenge. In contrast to adult mice, adoptive transfer of anti-JEV effectors into newborn (4-day-old)and suckling (8-14-day-old) mice did not confer protection. However, virus-induced death was delayed in suckling mice compared to newborn mice upon adoptive transfer. The specific reasons for lack of protection in newborn mice are not clear but virus load was found to be higher in newborn mice brains compared to those of adults and virus clearance was observed only in adult mice brains but not in newborn mice brains upon adoptive transfer. Specific depletion of Lyt 2.2+, L3T4+ or Thy-1+ T cell populations before adoptive transfer abrogated the protective ability of transferred effectors. However, when Lyt 2.2+ cell-depleted and L3T4+ cell-depleted effectors were mixed and transferred into adult mice the protective activity was retained, demonstrating that both Lyt 2.2+ and L3T4+ T cells are necessary to confer protection. Although the presence of L3T4+ T cells in adoptively transferred effector populations enhanced virus-specific serum neutralizing antibodies, the presence of neutralizing antibodies alone without Lyt 2.2+ cells are not sufficient to confer protection. PMID- 8627260 TI - The major echovirus group is genetically coherent and related to coxsackie B viruses. AB - In order to determine the overall molecular heterogeneity of echoviruses (EVs) we performed a genetic analysis of the prototype strains. Nucleotide and derived amino acid sequences from different genomic regions (5'UTR, capsid protein-coding and 3D polymerase genes) were used for molecular comparisons. On the basis of a comparison of partial amino acid sequences from the capsid protein VP2, all the sequenced EVs excluding EV22 and EV23 form a single cluster which is genetically homogeneous. All previously sequenced coxsackie B viruses (CBVs) and coxsackievirus A9 also belong to this same genetic cluster. Similar results were obtained when the 5'UTR or 3D polymerase gene sequences were used in comparisons. When amino acid sequences of the major capsid proteins of EV1 and EV16 were compared to those of previously sequenced enteroviruses, the length of the loops connecting the beta-sheets appeared to be relatively constant in the EV/CBV cluster. It can be concluded that EVs and CBVs have diverged relatively late in evolution. PMID- 8627261 TI - Recognition of foot-and-mouth disease virus and its capsid protein VP1 by bovine peripheral T lymphocytes. AB - The role of T cells in immunity to foot-and-mouth disease virus is still poorly defined compared to that of the humoral response. In this paper we describe a systematic, longitudinal study on the cellular recognition of FMDV and its subunit protein VP1 by bovine peripheral blood T lymphocytes. Multiple vaccination with a single virus serotype induced a serotype cross-reactive proliferative T cell repertoire that varied in magnitude between individual animals and with the serotype of the vaccine used. Primary proliferative T cell responses of vaccinated and acutely infected cattle were weak relative to the magnitude of responses determined for the same animals after boosting. In contrast, the level of circulating antibody produced after both primary and secondary exposure to virus was good. Phenotypic analysis of lymphocytes from vaccinated or infected cattle showed a small increase in CD8+ T cells after infection compared to vaccination. However, in general the profiles of circulating lymphocytes elicited were similar. Thus, we were not able to use proliferative or phenotypic analyses to distinguish between vaccinated and convalescent cattle. T cell recognition of VP1 by multiply-vaccinated cattle was serotype-specific implying that the cross-reactive responses observed with whole virus may be attributed to proteins other than VP1. In contrast to other studies, immunization with recombinant VP1 induced only low levels of neutralizing antibody and failed to elicit profound proliferative responses or protection ever after two immunizations. PMID- 8627262 TI - Depletion of Mac1-positive macrophages protects DBA/2 mice from encephalomyocarditis virus-induced myocarditis and diabetes. AB - DBA/2 mice treated with anti-Mac1 monoclonal antibody (MAb) failed to develop encephalomyocarditis virus (EMCV)-induced diabetes and myocarditis. Virus concentrations and the number of viral RNA-positive cells in the pancreas and heart were significantly reduced in mice treated with anti-Mac1 MAb. Mac1 positive macrophages seem to be involved in EMCV-induced disease and to affect the replication of EMCV in target organs. PMID- 8627263 TI - Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly. AB - Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information. PMID- 8627264 TI - Rapid selection for an N-linked oligosaccharide by monoclonal antibodies directed against the V3 loop of human immunodeficiency virus type 1. AB - The V3 loop of the human immunodeficiency virus (HIV) surface protein, gp 120, constitutes a principal neutralizing determinant. HIV strains lacking a naturally conserved N-linked oligosaccharide (at position 306) within the V3 loop are highly sensitive to neutralization. We subjected molecular clones of HIV(LAI) lacking this 306N-glycan to in vitro immune selection with MAbs directed against the V3 loop. In all, ten clones were characterized, and all proved resistant to V3-directed neutralization. Sequencing of the V3 loop revealed that six of the clones had become resistant at least partly by reacquisition of the 306N-glycan. Only two of the clones possessed mutations within the binding site of the antibody itself, while the two remaining clones did not display changes within the V3 loop itself. Thus, HIV strains lacking the 306N-glycan primarily develop resistance to V3-directed neutralization through acquisition of the specific oligosaccharide. This demonstrates that protein glycosylation can be a primary modifier of virus antigenicity of possible importance for the interaction of HIV with the host immune response. PMID- 8627265 TI - Neutralization sensitivity and accessibility of continuous B cell epitopes of the feline immunodeficiency virus envelope. AB - Antibodies elicited during natural infection of domestic cats by the feline immunodeficiency virus (FIV) recognize continuous epitopes in nine domains of the virus envelope glycoproteins. Whereas antibodies directed against the V3 envelope region can neutralize laboratory-adapted virus, neutralization of FIV has been shown to depend upon cellular substrate, and virus adaptation to laboratory cell lines may alter sensitivity to neutralizing antibodies. We therefore undertook a systematic analysis of the continuous B cell epitopes of the envelope of a primary FIV isolate, Wo. The capacity of feline antisera elicited against nine envelope domains to neutralize primary and laboratory-adapted virus was evaluated in feline peripheral blood mononuclear cells (PBMC). The laboratory-adapted strain Petaluma was used to compare neutralization in PBMC and Crandell feline kidney cells (CrFK). Antibodies specific for the V3 region neutralized both primary and laboratory-adapted virus whether residual infectivity was measured in CrFK or in feline PBMC. However, a large discrepancy in the efficiency of neutralization was observed in these ex vivo models of infection, perhaps reflecting diversity in the interaction between virus and different cellular targets. We also examined the accessibility of epitopes on the functional oligomeric envelope complex of FIV. Most of the epitopes were poorly exposed on native envelope glycoproteins at the surface of live infected cells. The most accessible domain was the only domain sensitive to neutralizing antibodies. These results suggest that inaccessibility on oligomeric envelope glycoproteins may frequently underlie the insensitivity of diverse lentivirus B cell epitopes to neutralization. PMID- 8627266 TI - Simian immunodeficiency virus infection in a patas monkey (Erythrocebus patas): evidence for cross-species transmission from African green monkeys (Cercopithecus aethiops sabaeus) in the wild. AB - Socio-ethological studies on troops of African green monkeys (AGMs) (Cercopithecus aethiops sabaeus) and patas monkeys (Erythrocebus patas) in Senegal have documented physical contacts between these two species. Elevated simian immunodeficiency virus (SIV) seroprevalence rates have been reported for the different AGM subspecies. We report here the extent to which patas monkeys are infected and compare the relatedness of the viruses isolated from theses two different species. Among the 85 AGMs and 54 patas monkeys studied, 47% of 7.5%, respectively, had antibodies that cross-reacted with HIV-2 envelope proteins. From two AGMs a virus was isolated. From the patas monkeys, virus isolation was generally not possible, but from one animal that was ill a virus designated pamG31 was amplified by PCR. In addition, for the two SIVagm isolates, an 830 bp region spanning the env and nef genes was amplified and sequenced. Comparisons of sequences from the env/nef region revealed 80% identity between pam G31 and SIVagm isolates from AGMs of the sabaeus subspecies, and 94% identity between the two SIVagm isolates. Phylogenetic analysis showed that pamG31 belongs to the SIVagm sabaeus subgroup. This is the first report of a lentiviral infection in a patas monkey. The close genetic relatedness between pamG31 and SIVagm sabaeus viruses is a strong argument in favour of cross-species transmission of SIV between AGMs and patas monkeys in the wild. For these reasons, we propose to refer to this patas virus as SIVagm-pamG31. PMID- 8627267 TI - Consistent risk group-associated differences in human immunodeficiency virus type 1 vpr, vpu and V3 sequences despite independent evolution. AB - Human immunodeficiency virus 1 type vpr, vpu and V3 sequences from 15 homosexual men and 19 intravenous drug users in the Amsterdam Cohort studies were analysed. Previously, we reported that V3 domains of viruses from drug users are distinguishable from those of homosexual men on the basis of two silent mutations. Phylogenetic analysis of vpr, vpu and V3 shows that differences in all three regions correlate with risk group. Two positions in both vpr and vpu were found to differ significantly between the risk groups. The distinguishing positions were confirmed for sequences from 11 Scottish and four German samples. The three regions show relatively independent evolution patterns; they resulted in different phylogenies, the only stable clustering being that based on the risk group distinction. Pairwise differences between sequences of the genes were moderately correlated (around 0.30). Surprisingly, when only silent changes are counted, the correlations dropped almost to zero, indicating that the evolution towards independence was more advanced in the silent than in the non-silent positions. This suggests that selection at the amino acid level is not the primary driving force for the independent evolutionary behaviour of the genes. Recombination, combined with restrictions on certain amino acids because of epistatic interactions between the genes, could be an alternative explanation of this phenomenon. PMID- 8627268 TI - Requirements for mouse mammary tumour virus internalization in mouse mammary epithelial cells. AB - Methylamine, a lysosomotropic alkalinizing agent, blocked mouse mammary tumour virus (MMTV) infection in normal mouse mammary epithelium, suggesting that internalization and acidification are necessary for cell penetration. This mechanism was further supported by the fact that intact MMTV induced the translocation of its cellular binding protein from the plasmalemma to the microsomes; however, isolated gp52, the MMTV envelope protein that binds this receptor, did not redistribute the binding protein. These data suggest that either another viral component, in addition to gp52, is needed for cell entry or that internalization requires receptor aggregation, which only the multivalent viral envelope can induce. PMID- 8627269 TI - Isolation and characterization of a member of the cysteine-rich gene family from Campoletis sonorensis polydnavirus. AB - The endoparasitic wasp Campoletis sonorensis injects a symbiotic polydnavirus into its host Heliothis virescens. Viral gene expression protects the wasp egg and larva from encapsulation by host haemocytes. Three related C. sonorensis polydnavirus (CsPDV) genes, which are expressed in parasitized H. virescens, have been previously isolated and grouped into a cysteine-rich gene family. In this report, a CsPDV gene encoding an abundant 1.4 kb mRNA expressed in parasitized insects was isolated and mapped to viral segment V (15.2 kb) by Southern blotting and PCR. The VHv1.4 cDNA is 1338 bp long and has an ORF that encodes 322 amino acids with two complete and one partial cysteine motifs. Similar to other characterized CsPDV cysteine motifs, the VHv1.4 motifs are also characterized by six cysteines at conserved positions and variable inter-cysteine amino acids. DNA sequence analyses show that the VHv1.4 gene shares regions of significant identity (73-97%) with the VHv1.1 gene, a member of the cysteine-rich gene family. The VHv1.4 and the VHv1.1 proteins are 62% identical overall; at the N termini including the signal peptide and the N-terminal cysteine motif the identity is greater (90%). Unlike other CsPDV cysteine-rich genes, the VHv1.4 cDNA has two conserved domains (77% identical in nucleotides, 55% identical in amino acids) that presumably result from the duplication of a portion of the gene. The VHv1.4 gene has four introns with splicing sites located at positions similar to VHv11.1 introns. Introns 2 and 3, located in the first and second domains respectively, have greater identity (97%) than the flanking exon sequences (77%). We propose, based on the evidence presented in this paper, that the VHv1.4 gene is a new member of the cysteine-rich polydnavirus gene family. PMID- 8627270 TI - Exposure to autoclaving or sodium hydroxide extends the dose-response curve of the 263K strain of scrapie agent in hamsters. AB - An analysis was made of incubation period data from experiments in which samples of brain-tissue infected with the 263K strain of scrapie agent were injected intracerebrally into hamsters following exposure of the tissue to autoclaving or sodium hydroxide. Where there was survival of infectivity, this often produced extended mean incubation periods compared with the maximal incubation periods in controls injected with untreated agent. These results confirmed that, after chemical or physical treatment, infectivity titre should not be calculated by comparing the incubation period from a single dilution-group against a standard dose-response curve for untreated agent. PMID- 8627271 TI - Personality characteristics and sexual functioning of 188 cross-dressing men. AB - The literature on cross-dressing men has been primarily limited to self identified patients at psychiatric clinics who are in distress. To understand the personality trait characteristics and sexual functioning of nonpatient cross dressers, 188 non-treatment-seeking male cross-dressers completed the NEO Personality Inventory (NEO-PI) and the Derogatis Sexual Functioning Inventory (DSFI). Respondents were classified as transvestites (TV; N = 83), transgenderists (TG; N = 61), or transsexuals (TS; N = 44) based on self-report and the nature of their cross-gender activities (e.g., use of female hormones, desire for sex reassignment, and amount of time spent in female role). These diagnostic groups did not differ on the five broad personality domains of the NEO PI, but TS men scored higher than TV and TG men on the Aesthetics facet scale of Openness to Experience (O). In terms of the DSFI scales, TS men reported lower sexual drive than TV and TG men, and TS and TG men exhibited greater psychiatric symptoms and feminine gender role, and poorer body image than TV men. Upon exclusion of a group of 49 respondents who previously sought treatment for psychological problems, no significant differences emerged among the three diagnostic groups on the NEO-PI domain and facet scales. Consideration of the DSFI scales showed that TS men experienced less sexual drive, more psychiatric symptoms, and a greater feminine gender role than TV or TG men. This study suggests that cross-dressers not seen for clinical reasons are virtually indistinguishable from non-cross-dressing men using a measure of personality traits, a sexual functioning inventory, and measures of psychological distress. These results emphasize the importance of using clinical significance criteria as required by DSM-IV guidelines before diagnosing men who cross-dress with an axis I disorder. PMID- 8627272 TI - Suicidal plans in patients with acute stroke. AB - The clinical correlates of suicidal thoughts were examined among a group of 301 patients with acute stroke. A total of 20 patients (6.6%) had suicidal thoughts. Previous history of stroke was associated with suicidal thoughts. Suicidal thoughts, however, were not related to severity of physical impairment. Most patients with suicidal thoughts met criteria for major depression. Other risk factors included younger age, poor social support, sensory deficit, and impaired cognitive function. Patients with suicidal plans had depression characterized by social withdrawal and brooding and self blame. Early identification of risk factors including the existence of depression may lead to effective therapeutic intervention. PMID- 8627273 TI - Neurological syndromes in factitious disorder. AB - Factitious disorder is characterized by the intentional feigning of physical or psychological signs and symptoms. The best known type of factitious disorder, Munchausen syndrome, is marked by a chronic unremitting course with repeated hospitalizations. The purpose of this study was to assess the frequency, psychopathological phenomenology, and diagnostic classification according to DSM III-R in patients with factitious disorder presenting as neurological syndromes. We prospectively included all patients who were hospitalized at our Department of Neurology, Freie Universitat Berlin, during a 1-year period. Five of 1538 (.3%) patients were diagnosed as having factitious disorder with feigning of neurological syndromes. Four presented with the classic variant, Munchausen syndrome. All patients had similar, characteristic psychopathological features including self-discharge, aggressive behavior, pseudologia phantastica, and hospital wandering. In these cases the additional diagnosis of personality disorder was made according to DSM-III-R criteria. We concluded that factitious disorder presenting with neurological syndromes may be more prevalent than generally assumed. Our findings confirm the idea of frequent coincidence of factitious and personality disorders. PMID- 8627274 TI - Intellectual functioning of inpatients with dissociative identity disorder and dissociative disorder not otherwise specified. Cognitive and neuropsychological aspects. AB - The intellectual functioning of 105 inpatients with multiple personality disorder and dissociative disorder not otherwise specified was assessed using the Wechsler Adult Intelligence Scale-Revised as part of a comprehensive research protocol. There were no significant intellectual differences between the groups on any major intelligence quotient summary score or any of the age-adjusted empirical factor scores. The anecdotal but widely accepted hypotheses that dissociative patients either have above average premorbid intelligence or that their current intellectual functioning is deleteriously affected by their fluctuant psychiatric disorder were not supported in this sample. A significant subsample of the multiple personality disorder group manifested abnormal interest scatter on the Wechsler Adult Intelligence Scale-Revised verbal subtests, and this variability was attributed to subtle neuropsychological deficits on the Memory/Distractibility factor. We speculate that dissociative patients might need to be evaluated for attention deficit disorder in addition to the range of dissociative symptoms in a comprehensive evaluation. PMID- 8627275 TI - Treatment of negative symptoms in schizophrenia and schizoaffective disorder by selegiline augmentation of antipsychotic medication. A pilot study examining the role of dopamine. AB - It has been suggested that schizophrenic negative symptoms may be manifestations of regionally deficient CNS dopaminergic activity. We sought to test this hypothesis by openly treating patients on chronic antipsychotic medication who showed prominent negative symptoms with low-dose selegiline (5 mg b.i.d.), a monoamine oxidase-B inhibitor that selectively enhances dopaminergic activity. Twenty-one patients meeting DSM-III-R criteria for chronic schizophrenia (N = 14) or schizoaffective disorder (N = 7) with prominent negative symptoms were studied. Subjects had been kept at their current antipsychotic and antiparkinsonian medication dose levels for at least a month before the study, which was continued unchanged throughout the trial. Over 6 weeks of selegiline treatment, a 34.7% reduction in negative symptoms was demonstrated on the Scale for the Assessment of Negative Symptoms. There were also reductions in depressive symptoms (21-item Hamilton Depression Scale dropped 36.8%) and extrapyramidal symptoms (Simpson-Angus Extrapyramidal Symptom Scale scores dropped 27.7%), but no change was observed in the severity of positive symptoms as measured by the Brief Psychiatric Rating Scale. Global clinical improvement was demonstrated, with mean Clinical Global Impressions Scale score rising 17.6%. These findings support the hypothesis that negative symptoms, as well as extrapyramidal symptoms and certain depressive symptoms, may be manifestations of regionally deficient dopaminergic activity. PMID- 8627276 TI - Dependency and alexithymia in psychiatric inpatients. AB - Research has demonstrated that alexithymic persons show a kind of defensive pseudonormality on psychological tests, minimizing problems and refusing to acknowledge psychological symptoms. The present study investigated whether alexithymic individuals are also unwilling to acknowledge the presence of a personality trait that is perceived by most adults as being undesirable (i.e., dependency). A mixed-sex sample of 372 psychiatric inpatients (209 women and 163 men) was screened for level of alexithymia; they completed self-report and projective measures of dependency. As predicted, there were significant negative correlations between alexithymia scores and scores on self-report measures of dependency in subjects of both sexes, but no significant relationships between subjects' alexithymia scores and their scores on the projective dependency measures. Apparently, alexithymic and nonalexithymic persons have comparable underlying dependency needs, but alexithymic persons are unwilling to acknowledge these needs on self-report tests. PMID- 8627278 TI - Abnormality ratings of the DSM-III-R personality disorder criteria for males vs. females. PMID- 8627277 TI - Current and lifetime psychiatric disorders among veterans with war zone-related posttraumatic stress disorder. AB - Previous research has found high rates of psychiatric disorders among veterans with war zone-related posttraumatic stress disorder (PTSD). However, many studies in this area are methodologically limited in ways that preclude unambiguous interpretation of their results. The purpose of this study was to address some of these limitations to clarify the relationship between war zone-related PTSD and other disorders. Participants were 311 male Vietnam theater veterans assessed at the National Center for PTSD at the Boston Veterans Affairs Medical Center. The Clinician-Administered PTSD Scale and the Structured Clinical Interview for DSM III-R were used to derive current and lifetime diagnoses of PTSD, other axis I disorders (mood, anxiety, substance use, psychotic, and somatoform disorders), and two axis II disorders (borderline and antisocial personality disorders only). Participants also completed several self-report measures of PTSD and general psychopathology. Relative to veterans without PTSD, veterans with PTSD had significantly higher rates of current major depression, bipolar disorder, panic disorder, and social phobia, as well as significantly higher rates of lifetime major depression, panic disorder, social phobia, and obsessive-compulsive disorder. In addition, veterans with PTSD scored significantly higher on all self report measures of PTSD and general psychopathology. These results provide further evidence that PTSD is associated with high rates of additional psychiatric disorders, particularly mood disorders and other anxiety disorders. The implications of these findings and suggestions about the direction of future research in this area are discussed. PMID- 8627279 TI - Suicide among Ethiopian Jews: a survey conducted by means of a psychological autopsy. PMID- 8627280 TI - Performance on the Wisconsin Card Sorting Test as a predictor of rehospitalization in schizophrenia. PMID- 8627281 TI - Drug-protein interactions: two-site binding of heterocyclic ligands to a monomeric hemoglobin. AB - The reactivity response of the heme proteins to the heterotropic effectors, purine, caffeine, theophylline, and (C2H5)4N+, have been examined. The heterotropic effectors influence the heme ligation affinities. The heme axial ligation of pyridine and pyrazole have not influenced the hemoglobin's affinity for caffeine and theophylline. The imidazole ligation indicates a mutual interaction between the heme active site and the noncoordinate binding site. PMID- 8627282 TI - Stable thiyl radicals in dried yeast Cu(I)6-thionein. AB - It was of interest to obtain long-lived thiyl radicals embedded in organic matrices. Solid thiol compounds including penicillamine, glutathione, and cysteine were UV irradiated under anaerobic conditions at 293 K for 60 min. The formed radicals were identified by electron paramagnetic resonance (EPR) (g = 2.0265 +/- 0.0015) at 293 K as thiyl radicals. The blue-colored radical species were subjected to reflection spectrometry (lambda max = 601 +/- 3 nm). The color and the EPR signal remained unchanged for six months. At the same time, the UV irradiation of lyophilisized yeast Cu(I)6-thionein generated stable EPR detectable thiyl was seen when the Cu(I)-thiolate was used. No EPR detectable thiyl radicals radicals at a g-value of 2.026 +/- 0.001. Unlike irradiated cysteine, a five times higher concentration of thiyl radicals were measured in the Cu(I)-thiolates of penicillamine, glutathione, and thiophenole, indicating that the hexanuclear copper arrangement in Cu(I)-thionein is most suitable for both the formation and stabilization of this sulfur radical species. PMID- 8627283 TI - Azide, cyanide, fluoride, imidazole and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a comparative study. AB - Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c. PMID- 8627284 TI - Tetrameric (G4) acetylcholinesterase: structure, localization, and physiological regulation. AB - Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G4 AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P-subunit). This study focuses primarily on current data involving the structure of the G4 AChE P subunit, the expression and regulation of G4 AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's disease. PMID- 8627285 TI - Differential regulation of GABA A receptor subunit mRNAs in rat cerebellar granule neurons: importance of environmental cues. AB - Levels of the GABA A receptor alpha1-, alpha6-, beta2-, beta3-, gamma2-, and delta-subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase-PCR was used to examine transcript expression in cultured granule neurons prepared at 2-day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the alpha1- and alpha6-subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the beta2-, beta3-, and gamma2-subunit mRNAs were constant in cultures prepared at postnatal days 2-6 but increased in those prepared at days 8-10. Third, the delta-subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only delta-subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are differentially regulated by multiple environmental cues. PMID- 8627286 TI - The cerebellum-enriched form of nuclear factor I is functionally different from ubiquitous nuclear factor I in glial-specific promoter regulation. AB - Nuclear factor I (NFI) binding sites are present in a wide range of brain specific gene enhancer and promoter sequences and appear to play a role in establishing cell type-specific expression within the CNS. The precise mechanisms used by various members of the NFI family of proteins to confer brain-specific expression are unclear. We have addressed this issue by comparing the transactivating capabilities of two forms of NFI in directing gliotropic expression from two different JC virus (JCV) promoter configurations. The JCV is an opportunistic pathogen of humans that causes lytic destruction of the oligodendrocytes and thus demyelination in immunocompromised patients. Our results show that the cerebellum-enriched form of NFI (NFI-A1) transactivates two gliotropic JCV early promoters to a greater extent than the ubiquitous form of NFI (NFI-C1). Activation by NFI-A1 was dramatically greater in glial than in nonglial cells. These results suggest that NFI proteins direct brain-specific expression through combinatorial interactions with cell specific coactivators and/or transcription factors that recognize adjacent sites within brain specific promoters. PMID- 8627288 TI - Thrombin attenuates neuronal cell death and modulates astrocyte reactivity induced by beta-amyloid in vitro. AB - beta-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that beta-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of beta-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of beta 1-42 and beta 25-35 peptides. In cultured astrocytes, the stellate morphology induced by beta 1-42 and beta 25-35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 microM. In contrast, thrombin potentiates rather than attenuates the beta-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of beta-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of beta amyloid and additional support for the role of thrombin as an important mediator of neuropathological events. PMID- 8627289 TI - Expression of glycine receptor subunits in glial cells of the rat spinal cord. AB - We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells. PMID- 8627287 TI - Characterization of the responses of Purkinje cells to neurotrophin treatment. AB - The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositve cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, approximately 200%, following BDNF, NT-4/5 or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c-fos. Immunocytochemical double labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT4/5 are not identical. PMID- 8627290 TI - Binding interactions of leukemia inhibitory factor and ciliary neurotrophic factor with the different subunits of their high affinity receptors. AB - Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low affinity LIF receptor. For CNTF, addition of a third subunit, or alpha subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high affinity LIF receptor, by adding a soluble form of the alpha CNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/alpha CNTF receptor bound to gp130 with an affinity of 3-5 x 10(-10)M, whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF. PMID- 8627291 TI - Glucocorticoids and nerve growth factor differentially modulate cell adhesion molecule L1 expression in PC12 cells. AB - The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels approximately 2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10(-7) M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland. PMID- 8627292 TI - Cytokine-regulated expression of platelet-derived growth factor gene and protein in cultured human astrocytes. AB - To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF, AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-beta 1 (TGF-beta 1) or tumor necrosis factor-alpha (TNF-alpha); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1beta (IL 1beta) by itself had little or no effect but synergized with TGF-beta 1 in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B- chains mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-beta 1, TNF-alpha, TNF-alpha/TGF-beta, or IL-1-beta/TGF-beta 1, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-beta 1, TNF-alpha, and IL-1 beta are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation. PMID- 8627293 TI - Staurosporine induces programmed cell death in embryonic neurons and activation of the ceramide pathway. AB - We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1-1.0 microM) and observed the meporphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N-Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [3H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 microM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death. PMID- 8627294 TI - Adenosine A2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells. AB - The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL 6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the levels of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A1 and A2 agonist 5'-(N ethylcarboxamido) adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A1-specific agonists R-phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A2a-specific agonist CGS-21860 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A2a-selective antagonists 8-(3-chlorostyryl) caffeine and KF17837 [(E)-8-(3,4 dimethoxystyryl)-1,3-dipropyl-7-methylxanthine] , which have also some antagonistic properties at A2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A1 antagonist 8 cyclopentyl-1,3 dipropylxanthine is much less potent. The results indicate that adenosine A2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells. PMID- 8627295 TI - Myelin gene expression in immortalized Schwann cells: relationship to cell density and proliferation. AB - Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions, [3H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P0 glycoprotein (P0) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and PO than sparse ones. PO mRNa and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact. PMID- 8627296 TI - Inhibition of Na+,K+-ATPase by ouabain opens calcium channels coupled to acetylcholine release in guinea pig myenteric plexus. AB - Ouabain, an Na+K+ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca2+ -independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [3H]ACh. Release of [3H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N type calcium channel blockage by the omega-conotoxins GVIA (selective N-type calcium channel blocker) and MVIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [3H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [3H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, omega-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [3H]ACh. Thus, extracellular Ca2+ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na+K+-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain. PMID- 8627297 TI - Characterization of excitatory amino acid modulation of dopamine release in the prefrontal cortex of conscious rats. AB - The effect of various classes of excitatory amino acid agonists on the release of dopamine in the medial prefrontal cortex (PFC) of awake rats was examined using intracerebral microdialysis. Local infusion of 20 muM alpha-amino-3-hydroxy-5 methylisoxazole-4-propionic acid (AMPA), through the microdialysis probe, produced a significant increase of more than twofold in extra-cellular levels of dopamine. Application of 100 microM AMPA increased these levels nearly 15 fold. The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50microM) blocked the increase in dopamine release produced by 20 microM AMPA. Local infusion of kainate at concentrations of 5 and 20 microM increased dopamine release by nearly 150 and 500%, respectively. Local application of CMQX (50 microM) before 20 microM kainate significantly attenuated the stimulatory effect of kainate on dopamine levels. In contrast to AMPA and kainate, infusion of N methyl-D-aspartate (NMDA) at 20 or 100 microM did not increase dopamine release. In fact, a trend toward a decrease in dopamine release was evident after 100 microM NMDA. The present study indicates that the in vivo release of dopamine in the PFC is facilitated by AMPA and kainate receptors. This modulation is more profound than that previously reported in basal ganglia. The lack of an excitatory effect of NMDA is in agreement with recent reports that the NMDA receptor may inhibit indirectly dopaminergic neurotransmission in the PFC. PMID- 8627298 TI - Characterization and localization of adrenal nicotinic acetylcholine receptors: evidence that mAb35-nicotinic receptors are the principal receptors mediating adrenal catecholamine secretion. AB - Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the alpha-subunit of muscle nAChRs and cross reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR-stimulated catecholamine release (IC50 of approximately 10nM). mAb35 had no effects on release stimulated through non-nAChR mechanisms. Unlike agonist-induced nAChR desensitization, the mAb35-induced reduction in nAChR-mediated secretion developed slowly. Although not immediately reversible, nAChR-stimulated release recovered after mAB35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was par tially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat alpha3 cDNA revealed two strong bands and two fainter bands: two higher-molecular-weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of 2.3kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on alpha 3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35-nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells. PMID- 8627299 TI - delta-Opioids stimulate inositol 1,4,5-trisphosphate formation, and so mobilize Ca2+ from intracellular stores, in undifferentiated NG108-15 cells. AB - delta-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108 15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [D-Pen 2,5] enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [D-Pen 2,5] enkephalin caused a dose-dependent (EC50= 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7+/-1.2 pmol/mg of protein with 1 microM, n=9) at 30 s and returned to basal levels (10.6+/-0.9 pmol/mg of protein, n=9) within 4-5 min. This response was fully naloxone (1 microM) reversible and pertussis toxin (100ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 microM) had no effect on the [D-Pen 2,5] enkephalin (1 microM) induced inositol 1,4,5-triphosphate response, and K+ (80mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 microM) enhanced, whereas acute expo sure to phorbol 12,13-dibutyrate (1 microM) abolished, the [D-Pen 2,5] enkephalin (0.1 microM)-induced inositol 1,4,5-triphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [D-Pen 2,5] Enkephalin also dose dependently (EC50 =2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 microM, n=5) at 30 s. This close temporal and dose response relationship strongly suggests that delta-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells. PMID- 8627300 TI - Role of external and internal calcium on heterocarrier-mediated transmitter release. AB - Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2-Bis(2-aminophenoxyl)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [3H] dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]-independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca2+. PMID- 8627301 TI - Visualization of D1 dopamine receptors on living nucleus accumbens neurons and their colocalization with D2 receptors. AB - To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine-N-(p aminophenethyl)-spiperone (NAPS) (10 nM), a derivative of the D2 antagonist spiperone, labels D2-like receptors on living nAcc neurons. We now show that rhodamine-Sch-23390 (30 nM), a derivative of the D1 antagonist, labels D1-like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (-) butaclamol, but blocked with (+)-butaclamol. We found that 52 +/- 7% of nAcc medium-sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1-positive and D1-negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine-Sch-23390 and then rhodamine-NAPS, we found that 38 +/- 6% of medium-sized neurons express both D1- and D2-like receptors, indicating that D1-D2 interactions may occur at the level of single postsynaptic neurons. PMID- 8627302 TI - Differential regulation of intracellular signaling systems by sodium fluoride in rat glioma cells. AB - We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca2+ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca2+-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration dependent manner. This enhancement was abolished by pretreatment with 100 microM BAPTA tetraacetoxymethal ester or in the presence of W-7 in a concentration dependent manner. N G-Monomethyl-L-arginine (NMMA), a competitive inhibitor or nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca2+ concentration ([Ca2+]i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca2+]i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca2+] was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca2+ was reduced by pretreating the cells with NaF. The reduction in Ca2+ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulated the cGMP generation. basal [Ca2+]i, and 5-HT2A receptor function in C6 glioma cells. PMID- 8627304 TI - Interleukin-1 beta uses common and distinct signaling pathways for induction of the interleukin-6 and tumor necrosis factor alpha genes in the human astrocytoma cell line U373. AB - Cytokines are involved in the etiology of different disorders of the CNS. For a better understanding of their pathogenic role, we analyzed signal transduction pathways mediating the interleukin (IL)-1 beta-induced synthesis of IL-6 and tumor necrosis factor alpha (TNF alpha) in the human astrocytoma cell line U373 MG. Both protein kinase C and reactive oxygen intermediates (ROIs) were involved in IL-6 and TNF alpha gene expression by IL-1 beta. In contrast, protein tyrosine kinases were only necessary for expression of the IL-6 gene. Whereas activation of protein kinase A was able to induce expression of the IL-6 gene, it did not induce TNF alpha gene expression and was not involved in IL-1 beta-induced IL-6 and TNF alpha gene expression. Activation of the transcription factor nuclear factor-kappa B by IL-1 beta involved ROIs, whereas the IL-1 beta-induced activation of the transcription factor AP-1 was mediated via protein kinase C. Our findings provide the basis for the development of specific drugs for the treatment of disorders of the CNS in which cytokines play a pathogenic role. PMID- 8627303 TI - Synaptic membrane G proteins are complexed with tubulin in situ. AB - The G proteins G S and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express G s alpha but not G i alpha 1, responded to the addition of tubulin with a stable activation of adenyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both G s and G i 1, responded to exogenous tubulin with a stable inhibition of adenyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and G i 1 and G s from detergent extracts of these membranes. Nearly 90% of the G s alpha from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction. PMID- 8627305 TI - In vivo expression of inducible nitric oxide synthase in cerebellar neurons. AB - In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS--both the neuronal and the endothelial types. The other class of NOS--the inducible NOS (iNOS)--is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-gamma) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults. PMID- 8627306 TI - Identification and determination of 3,4-dihydroxyphenylacetaldehyde, the dopamine metabolite in in vivo dialysate from rat striatum. AB - 3,4-Dihydroxyphenylacetic acid (DOPAC) is commonly considered to be the main dopamine (DA) metabolite produced by monoamine oxidase (MAO); however, the initial product of DA oxidation is 3,4-dihydroxyphenylacetaldehyde (DOPALD). Owing to technical difficulties in detecting DOPALD from a biological matrix, no studies have so far been performed to measure brain levels of this aldehyde in vivo. In this work, using transstriatal microdialysis in freely moving rats, we identified DOPALD by HPLC coupled to a coulometric detector. In chromatograms obtained from microdialysis samples, DOPALD appeared as a peak with a retention time coincident with that of the standards obtained via enzymatic and chemical synthesis. On the other hand, DOPALD was undetectable ex vivo from rat striatal homogenates. This discrepancy is probably due to the preferential extraneuronal localization together with the high reactivity of the aldehyde, which is rapidly removed by the dialysis probe, whereas the ex vivo procedure allows its condensation and enzymatic conversion. Measurement of DOPALD levels as a routine procedure might represent a reliable tool to evaluate DA oxidative metabolism directly, in vivo. Moreover, parallel detection of DOPALD and DOPAC levels in brain dialysate may make it possible to distinguish between the activity of MAO and aldehyde dehydrogenase. DOPALD, like many endogenous aldehydes, has been shown to be toxic to the cell in which it is formed. Therefore, in vivo measurement of DOPALD levels could highlight new aspects in the molecular mechanisms underlying both acute neurological insults and neurodegenerative diseases. PMID- 8627308 TI - Involvement of an intercellular adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats. AB - The intercellular adhesion molecule 1 (ICAM-1) plays an important role in immune responses by promoting infiltration of neutrophils into tissues; however, its implication in the secondary destructive pathomechanism after the initial mechanical injury to the spinal cord has not been clarified yet. This study was conducted to examine the role of ICAM-1 in this process after spinal cord injury (SCI) in rats. The expression of ICAM-1 mRNA was investigated by the reverse transcription-PCR method and the effect of monoclonal antibody (mAb) to ICAM-1 on SCI was evaluated by measuring various parameters. ICAM-1 mRNA expression correlated with the severity of injury and reached its maximum level 6 h after SCI. Intravenous injection of ICAM-1 mAb (1 mg/kg) 30 min after SCI reduced motor disturbance and enhanced recovery. Moreover, it significantly suppressed myeloperoxidase activity by 43.0% and spinal cord edema by 1.1% in the injured spinal cord tissue. The posttraumatic drop in spinal cord blood flow was also improved. These results suggest that ICAM-1 is deeply involved in the secondary self-destructive process after mechanical injury of the spinal cord and should be an effective target for developing a pharmacological treatment for SCI. PMID- 8627307 TI - Characterization of dp6troglycan-laminin interaction in peripheral nerve. AB - Dystoroglycan is encoded by a single gene and cleaved into two proteins, alpha and beta-dystroglycan, by posttranslational processing. The 120kDa peripheral nerve isoform of alpha-dystroglycan binds laminin-2 comprised of the alpha 2, beta 1, and gamma 1 chains. In congenital muscular dystrophy and dy mice deficient in laminin alpha 2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan- laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) alpha-dystroglycan is an extracellular peripheral membrane glycoprotein that links beta-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of alpha-dystroglycan in the interaction with laminin. PMID- 8627309 TI - Increased neural cell adhesion molecule in the CSF of patients with mood disorder. AB - Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder. PMID- 8627310 TI - Calcium influx into human neuroblastoma cells induces ALZ-50 immunoreactivity: involvement of calpain-mediated hydrolysis of protein kinase C. AB - Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system. PMID- 8627311 TI - Quantitative changes of amino acid distributions in the rat vestibular nuclear complex after unilateral vestibular ganglionectomy. AB - Changes of amino acid concentrations in the vestibular nuclear complex (VNC) during lesion-induced vestibular compensation were studied in rats after unilateral vestibular ganglionectomy. Distributions of 12 amino acids within the VNC were measured at 2,4,7, and 30 days after surgery, using microdissection of freeze-dried brain sections and HPLC. Glutamate decreased on the lesioned side in nearly all VNC regions. Changes were fully developed 2 days after lesion and persisted through 30 days. In some regions, glutamate decreased also on the unlesioned side, especially at longer survival times, so that bilateral asymmetries became reduced. Aspartate changes were similar to those of glutamate on either side. Lesion-induced glutamine asymmetry was usually opposite to that of glutamate. Although GABA concentration decreased at early survival times, it recovered at later times and sometimes increased in dorsal parts of lateral and medial nuclei. Taurine changes were similar to those of GABA in most regions. Glycine change was primarily limited to a bilateral decrease in the dorsal part of the lateral vestibular nucleus. Concentrations of other amino acids were much lower, but some showed postlesion changes. PMID- 8627312 TI - Mesenteric organ production, hepatic metabolism, and renal elimination of norepinephrine and its metabolites in humans. AB - This study used regional differences in plasma concentrations of norepinephrine and its metabolites to examine how production of the transmitter by sympathetic nerves, in particular, those innervating mesenteric organs, is integrated with metabolism by the liver and elimination by the kidneys. Higher concentrations of norepinephrine, its glycol metabolites 3,4-dihydroxyphenylglycol and 3-methoxy-4 hydroxyphenylglycol and their sulfate conjugates in portal venous than arterial plasma indicate substantial production of norepinephrine by mesenteric organs (15.5 nmol/min). Much lower concentrations of norepinephrine and its glycol metabolites in plasma leaving than entering the liver indicate their efficient hepatic removal (20 nmol/min). Higher concentrations of vanillylmandelic acid in the hepatic outflow than inflow indicate that this metabolic end product is produced largely from the norepinephrine and glycol metabolites removed by the liver. Renal elimination of vanillylmandelic acid (18-20 nmol/min), produced mainly by the liver (17 nmol/min), and of 3-methoxy-4-hydroxyphenylglycol sulfate (7-9 nmol/min), produced largely by mesenteric organs (7 nmol/min), compromised 86-91% of the total renal elimination of norepinephrine metabolites. The results show that mesenteric organs produce about one-half of the norepinephrine formed in the body. The liver removes substantial amounts of circulating norepinephrine and its glycol metabolites and converts these compounds to vanillylmandelic acid, which is then eliminated from the body by the kidneys. The sulfate conjugates are also metabolic end products eliminated by the kidneys. However, these metabolites are produced by extrahepatic tissues, in particular, mesenteric organs, which represent a significant source of sulfate-conjugated norepinephrine and 3,4 dihydroxyphenylglycol, and the main source of sulfate-conjugated 3-methoxy-4 hydroxyphenylglycol. PMID- 8627313 TI - The rate of valine incorporation into proteins with correction for valine recycling, measured in two brain tumor models and the cortex. AB - The tissue dilution factor (lambda) for the incorporation of valine into proteins in the rat cortex and in two different tumors, AA ascites and C6 glioma, was determined from measurements of specific activities in the tissue acid-soluble and aminoacyl-tRNA pools and in the plasma. A constant plasma specific activity was achieved by a constant infusion rate of [3H] valine. The data showed that the lambda for valine was the same in the cortex as in the tumors, and the recycling was approximately 36%. There was no difference in the lambda calculated on the basis of the specific activities in the tissue acid-soluble or aminoacyl-tRNA pools. The average dilution factor was found to be 0.64+/-0.05. The rate of valine incorporation into proteins was on average 3.2+/-0.4 and 4.9+/-0.4 nmol/g/min in the cortex for the groups of rats used in the AA ascites and C6 glioma experiments, respectively. In the AA ascites tumor the rate was approximately 41 and 29 nmol/g/min 4 and 7 days after tumor implantation, respectively, whereas in the C6 glioma the rate was approximately 41 and 72 nmol/g/min 6 and 13 days after inoculation, respectively. The tumors had, in comparison with the cortex, a significantly greater volume of distribution of valine. The amounts of valyl-tRNA were significantly greater in the tumors as compared with the normal cortex, with the exception of the glioma 6 days after implantation where the concentration was the same as in the cortex. PMID- 8627314 TI - alpha-Linolenic acid dietary deficiency alters age-related changes of dopaminergic and serotoninergic neurotransmission in the rat frontal cortex. AB - The effects of alpha-linolenic acid diet deficiency on rat dopaminergic and serotoninergic neurotransmission systems were investigated in the frontal cortex, striatum, and cerebellum of male rats 2,6,12, and 24 months of age. The diet deficiency induced severe decrease in the 22:6n-3 fatty acid levels in all regions and a compensatory increase in n-6 fatty acid levels. A recovery in the levels of 22:6n-3 was observed in deficient rats between 2 and 12 months of age; however, this recovery was lower in frontal cortex than in striatum and cerebellum. In the striatum and cerebellum, dopaminergic and serotoninergic receptor densities and endogenous dopamine and serotonin levels were affected by aging regardless of the diet. In contrast, a 40-75% lower level of endogenous dopamine in the frontal cortex occurred in deficient rats according to age. The deficiency also induced an 18-46% increase in serotonin 5-HT2 receptor density in the frontal cortex during aging, without variation in endogenous serotonin level, and a 10% reduction in density of dopaminergic D2 receptors. Monoamine oxidase-A and -B activities showed specific age-related variations but regardless of the diet. Our results suggest that a chronically alpha-linolenic-deficient diet specifically affects the monoaminergic systems in the frontal cortex. PMID- 8627315 TI - Presynaptic serotonergic markers in community-acquired cases of Alzheimer's disease: correlations with depression and neuroleptic medication. AB - Presynaptic serotonergic markers, serotonin uptake sites, and concentrations of serotonin and 5-hydroxyindoleacetic acid were studied in the frontal and temporal cortex of 20 community-acquired cases of Alzheimer's disease and 16 controls matched for age, sex, postmortem delay, and storage. Clinical assessments, including behavioural symptoms, of the Alzheimer patients were made at 4-month intervals during life. There was significant reduction in the number of serotonin uptake sites in Alzheimer cases in temporal but not frontal cortex. There was no significant alteration in the concentrations of serotonin or 5 hydroxyindoleacetic acid in either region. Alzheimer patients who had persistent depressive symptoms during life had significantly fewer serotonin uptake sites in both cortical areas compared with Alzheimer patients without these symptoms. In addition, Alzheimer patients who were receiving chronic neuroleptic medication had significantly lower concentrations of serotonin in frontal cortex and 5 hydroxyindoleacetic acid in temporal cortex than those patients not receiving such treatment. These data suggest previous studies that reported uniform serotonergic dysfunction may have been subject to unintentional selection of behaviourally disturbed Alzheimer cases or those receiving chronic neuroleptic medication. This study also provides a basis for the treatment of behaviourally disturbed Alzheimer patients with serotonomimetics. PMID- 8627316 TI - Polyamine modification increases the permeability of proteins at the blood-nerve and blood-brain barriers. AB - The permeability of the blood-nerve barrier (BNB) and the blood-brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient-surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with naturally occurring polyamines-putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD. In a similar manner, polyamine-modified insulin showed a 1.7-2.0-fold increase in PS of the BNB and BBB compared with the high values of native insulin. Polyamine-modified albumin showed a remarkable 54 165-fold increase in PS of the BNB and BBB compared with native albumin, whereas PUT-IgG resulted in an even higher increase in the PS that ranged from 111- to 349-fold for nerve and different brain regions compared with native IgG. Polyamine modification of proteins, therefore, can dramatically increase the permeability at the BNB and BBB of a variety of proteins with widely differing M(r) and function. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT>SPD>SPM). Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is suggested that the polyamine transporter may be responsible for the transport of these polyamine-modified proteins. Systemic administration of polyamine-modified peptides and proteins might prove to be an efficient approach to deliver therapeutic agents into the CNS and PNS for the treatment of a variety of neurological diseases. PMID- 8627317 TI - CCK-4-induced calcium mobilization in T cells is enhanced in panic disorder. AB - We investigated the effects of brain cholecystokinin (CCK) receptors on the intracellular calcium concentration and protein kinase C in human T cells. CCK-4 produced a transient increase in calcium in the absence of extracellular calcium. CCK-B agonists stimulated calcium mobilization in a dose-dependent manner in T cells. CCK-B antagonists suppressed CCK-4-induced calcium mobilization more potently than CCK-A antagonist. The recovery of desensitization of the CCK-4 induced response was delayed by phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A. The responsiveness to CCK-4 was also reduced by phorbol 12,13-dibutyrate (PDBu), and this effect of PDBu was abolished completely by preincubation with staurosporine. CCK-4-induced calcium mobilization was too small to attribute the desensitization to the protein kinase C transduction pathway. T cells from patients with untreated panic disorder exhibited significantly higher cholecystokinin-4-induced calcium mobilization than those from healthy controls or patients with treated panic disorder. These results suggest that cholecystokinin-B receptor function in T cells of patients with panic disorder is enhanced. Cholecystokinin-4-induced calcium mobilization in T cells may be state dependent and useful as a biological marker of panic disorder. PMID- 8627318 TI - Threshold effects and control of oxidative phosphorylation in nonsynaptic rat brain mitochondria. AB - The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV ), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by approximately 72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by approximately 70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed. PMID- 8627319 TI - Effect of hypothyroidism on the subcellular distribution of Ca2+/calmodulin stimulated protein kinase II in chicken brain during posthatch development. AB - In developing chicken brain Ca2+/calmodulin-stimulated protein kinase II (CaMPK II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T3) at 42 days. The concentrations of alpha- and beta-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca2+/calmodulin- or ZN2+-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca2+/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes. PMID- 8627320 TI - Expression of the beta-galactoside alpha 1,2-fucosyltransferase gene suppresses axonal outgrowth of neuro2a neuroblastoma cells. AB - The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the beta-galactoside alpha 1,2-fucosyltransferase (alpha 1,2-FT) gene. We recently cloned two types of rabbit alpha 1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene alpha 1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene alpha 1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and alpha-L-fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H type alpha1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension. PMID- 8627321 TI - Increased production of matrix metalloproteinases in enriched astrocyte and mixed hippocampal cultures treated with beta-amyloid peptides. AB - Growing evidence supports the notion of a functional relationship between the presence of the beta-amyloid (A beta) peptide and the production of inflammatory mediators in and around neuritic plaques of Alzheimer's disease. Tissue remodeling enzymes that are critical in peripheral inflammatory responses are the matrix metalloproteinases (MMPs), enzymes produced by neurons and glia. Thus, it was of interest to determine whether A beta may alter the expression of MMPs in glial and neuronal cultures. It was demonstrated that A beta (1-40) is a potent stimulator of MMP-9 and MMP-2 activity in addition to inducing the expression of a lower molecular weight, unidentified gelatinase activity in mixed hippocampal and astrocyte cultures. Shorter fragments of A beta were less effective in stimulating the production of these enzymes. The lower molecular weight activity was observed only in response to A beta, and not after treatment with various cytokines. In addition, both cultures express MMP-3 (stromelysin-1) in response to A beta peptides. These results suggest that MMPs may play a role in the development or progression of neuritic plaques, i.e., abnormal neurite outgrowth. PMID- 8627322 TI - Isolation and characterization of two fatty acid binding proteins from mouse brain. AB - Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP. PMID- 8627323 TI - Modulation of serine proteinases and metalloproteinases during morphogenic glial endothelial interactions. AB - The regulation of microvessel formation and the expression of CNS-specific endothelial properties are attributed to perivascular astroglia. Specific proteolytic pathways mediate processes such as tissue remodeling, differentiation, invasion, and metastasis. We used a co-culture system in which C6 glial cells induce CNS microvascular endothelial cells to form capillary-like structures to examine the role of plasminogen activators and collagenases in CNS microvessel morphogenesis. Fibrin zymography revealed the presence of high molecular weight urokinase-type plasminogen activator (uPA), low-molecular weight uPA, and uPA/inhibitor complexes within endothelial cultures and cocultures. Gelatin zymography revealed the presence of 92-, 72-, and 62-kDa type IV collagenases within endothelial cultures and cocultures. uPA activity was confirmed by incubating the extracts with amiloride, an inhibitor of uPA. Collagenase activity was confirmed by incubating the gels with EDTA, an inhibitor of metalloproteinases. Quantitative densitometry showed a six- to eightfold decrease in coculture uPA during capillary-like structure formation. Substantially less change in type IV 72-kDa procollagenase activity was seen in cocultures during capillary-like structure formation, but active type IV 62-kDa collagenase activity was significantly increased during capillary-like structure formation. These findings establish that uPA and activated type IV collagenase activity specifically regulates morphogenic endothelial responses to glial interactions and suggest mechanisms by which microvessels respond within the CNS. PMID- 8627324 TI - Glutamine transport in mouse cerebral astrocytes. AB - We measured initial influx and exchange of [14C]glutamine in primary astrocyte cultures in the presence and absence of Na+. Kinetic analysis of transport in Na+ -free solution indicated two saturable Na+ -independent components, one of which was identifiable functionally as system L1 transport. In the presence of Na+, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na+ dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by alpha-(methylamino) isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na+ -dependent uptake, preferential inhibition of an Li+ -intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and alpha-(methylamino) isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na+, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 microM glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li+ in place of Na+. PMID- 8627325 TI - Adenosine deaminase interacts with A1 adenosine receptors in pig brain cortical membranes. AB - Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high-affinity site of the receptor, which corresponds to the receptor-G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins. PMID- 8627326 TI - Ex vivo measurement of brain tissue nitrite and nitrate accurately reflects nitric oxide synthase activity in vivo. AB - The ex vivo tissue concentration of nitrite and nitrate (NOx) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N omega-nitro-L-arginine (L-NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NOx by up to 75% but had no effect on plasma NOx. Selective inhibition of central NOS by intracerebroventricular administration of L-NA methyl ester produced similar decreases in levels of whole-brain NOx. A residual concentration of NOx of 10-15 microM remained in all brain regions even after complete inhibition of brain NOS. Brain NOx content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NOx was found to reflect the in vivo efficacy of several different types of NOS inhibitor: L-NA, N omega monomethyl-l-arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate L-arginine increased brain NOx concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NOx is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo. PMID- 8627327 TI - Alcohol inhibits the depolarization-induced stimulation of oxidative phosphorylation in synaptosomes. AB - The effects of alcohol and Ca2+ transport inhibitors on depolarization-induced stimulation of oxidative phosphorylation and free-Ca2+ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K+ or veratridine and by the Ca2+ ionophore ionomycin. The stimulation by K+, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca2+. Depolarization-stimulated respiration was inhibited by verapamil, Cd2+, and ruthenium red but not by diltiazem. Synaptosomal Ca2+ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca2+. Ethanol, at pharmacological concentrations (50-200 mM), inhibited the Ca2+ dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage-gated Ca2+ channels, which inhibited the elevation of synaptosomal free Ca2+, and, in part, from the stimulation of the mitochondrial Ca2+/Na+ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca2+. The inhibition by ethanol of the excitation induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity. PMID- 8627328 TI - Differential cellular phosphorylation of neurofilament heavy side-arms by glycogen synthase kinase-3 and cyclin-dependent kinase-5. AB - To investigate the cellular mechanisms regulating neurofilament-heavy subunit (NF H) side-arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)alpha, GSK-3 beta, and cyclin-dependent kinase-5 (cdk-5), to phosphorylate NF-H in transfected cells. We analysed NF-H phosphorylation by using a panel of phosphorylation-dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF-H on sodium dodecyl sulphate-polyacrylamide gel electrophoresis because this is known to be affected by side-arm phosphorylation. Our results demonstrate that whereas GSK-3 alpha, GSK-3 beta, and cdk-5 will all phosphorylate NF-H, they generate different antibody reactivity profiles. GSK-3 alpha and GSK-3 beta induce a partial retardation of a proportion of the transfected NF-H, but only cdk-5 alters the rate of electrophoretic migration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 phosphorylate different residues or sets of residues within NF-H sidearm in cells. We further show that cdk-5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk-5. PMID- 8627329 TI - O-linked oligosaccharide on the 75-kDa neurotrophin receptor. AB - Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O-glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of disialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)(1-2-) Gal beta 1-3GalNAc. Modification of the O linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling. PMID- 8627330 TI - Increasing age alters transbilayer fluidity and cholesterol asymmetry in synaptic plasma membranes of mice. AB - Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3-4-, 14-15-, and 24-25-month old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins. PMID- 8627332 TI - Time-dependent increase in protein phosphorylation following one-trial enhancement in Hermissenda. AB - One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32 PO4 into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins, detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one trial conditioning. PMID- 8627331 TI - On the origin of extracellular glutamate levels monitored in the basal ganglia of the rat by in vivo microdialysis. AB - Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and gamma-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were 5 and approximately 5 and approximately 0.4 microM, respectively. Lactate and pyruvate concentrations were approximately 200 and approximately 10 microM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K+ depolarization, (b) Na+ channel blockade, (c) removal of extracellular Ca2+, and (d) depletion of presynaptic vesicles by local administration of alpha-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K+ depolarization and were increased by perfusing with tetrodotoxin or with Ca2+ free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K+-depolarizing conditions by alpha-latrotoxin. Because the effect of K+ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or L-trans-pyrrolidine-2,4 dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K+ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements. PMID- 8627333 TI - In situ analysis of Na, K-ATPase alpha1- and alpha3-isoform mRNAs in aging rat hippocampus. AB - Age-related changes in the expression of Na, K-ATPase alpha1- and alpha3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) alpha1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas alpha3 content shows the opposite ratio and (b) alpha3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1-3. In the 24-month-old rats as compared with the young animals, the alpha1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in the perikaryal layers. The alpha3 signal is reduced about threefold (p<0.0001, ANOVA, n=6) in perikaryal layers, is almost completely absent over interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase a isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age related alterations in hippocampal function, before cell loss. PMID- 8627334 TI - The differential response of protein kinase A to cyclic AMP in discrete brain areas correlates with the abundance of regulatory subunit II. AB - We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIalpha, RIIalpha, and RIIbeta) and of 150-kDa RIIbeta-anchor proteins for cyclic AMP (cAMP)-dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIbeta paralleled the presence of specific RII-anchoring proteins, high levels of membrane-bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C-PKA). Conversely, in brain areas showing low RIIbeta levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII-anchoring proteins, low levels of membrane-bound holoenzyme PKA, and high levels of cytosolic dissociated C-PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP-induced dissociation, without, however, a consistent translocation of C PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C-PKA and phosphorylation of cAMP-responsive element binding protein. Taken together, these data suggest that RIIbeta (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP-induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits. PMID- 8627335 TI - Embryonic expression of vasoactive intestinal peptide (VIP) and VIP receptor genes. AB - Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development. PMID- 8627336 TI - Overexpression of MARCKS, but not protein kinase C-alpha, increases phorbol ester stimulated synthesis of phosphatidylcholine in human SK-N-MC neuroblastoma cells. AB - To investigate the regulation of phorbol ester-stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine-rich protein kinase C substrate (MARCKS) and the alpha-isoform of protein kinase C (PKC-alpha) were overexpressed in a human neuroblastoma (SK-N-MC) cell line that does not increase PtdCho synthesis in response to 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl-3H] choline was stimulated 1.88-2.34-fold in the presence of 100-200 nM TPA. In clones overexpressing PKC-alpha (30-40-fold increased level of protein) or in mock-transfected vector controls, TPA had much less of a stimulatory effect (1.04-1.43 fold) on PtdCho synthesis. TPA caused translocation of PKC-alpha and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK-N-MC cells, MARCKS is required for TPA-stimulated synthesis of PtdCho and PKC-alpha alone is insufficient for supporting enhanced synthesis. PMID- 8627337 TI - Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in CATH.a cells. AB - We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element-binding protein (CREB) is down-regulated in CATH.a cells (a neural derived cell line) by activation of the cAMP pathway. We now demonstrate that this down-regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin-induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run-on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed. PMID- 8627338 TI - Molecular analysis of the presenilin 1 (S182) gene in "sporadic" cases of Alzheimer's disease: identification and characterisation of unusual splice variants. AB - Mutations of the presenilin 1 (PS-1) gene at the Alzheimer's disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in "sporadic" disease, we performed a molecular analysis of PS-1 transcripts extracted from brain tissues of a series of histologically confirmed cases of "sporadic" AD (n=10) and also from histologically "normal" (non-Alzheimer) age-matched brain controls (n=5). No sequence changes in the PS-1 coding sequence were detected after analysis by reverse transcription-PCR. This suggests that the frequency of mutations in the PS-1 (S182) coding region in "sporadic" Alzheimer's disease in very low. However, we demonstrated that the PS-1 gene is highly variably spliced. One splice variant involves the 5' untranslated region of the PS-1 gene only and hence encodes for normal PS-1. Six further splice variants involve coding regions of the PS-1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS-1 gene. One of these variants, resulting in the loss of transmembrane domain TM-VII, was found only in an AD patient. PMID- 8627339 TI - Glial inclusions in CNS degenerative diseases. PMID- 8627340 TI - In situ hybridization analysis for expression of myogenic regulatory factors in regenerating muscle of mdx mouse. AB - Precise temporal and spatial coordination of expression of the myogenic regulatory factors (MRF) plays a critical role in the development of skeletal muscle. Whether this pattern is recapitulated postnatally during regeneration of mature muscle after injury is not known. The aim of this study was to determine the cellular distribution of mRNA expression of the various myogenic regulatory factors (MyoD, Myogenin, Myf-5 and Myf-6) during regeneration in mature skeletal muscle. We used the mdx mouse, an animal model for Duchenne muscular dystrophy, which undergoes necrosis and vigorous regeneration of muscle fibers, as a natural model to study muscle fibers at various stages of maturity ranging from satellite cells to mature muscle fibers. In situ hybridization analysis revealed that MyoD expression was the most widespread and was expressed in satellite cells and small regenerating muscle fibers surrounding necrotic fibers. Myogenin, Myf5, and Myf6 were expressed weakly in some immature fibers and none of the MRF's could be detected in mature muscle. These findings, taken together with previous studies, suggests that the pattern of expression of the various MRF's in regenerating skeletal muscle differs from that in developing muscle in embryos and that MyoD may play a critical role in the initiation and progression of events which lead to regeneration in mature skeletal muscle. PMID- 8627341 TI - Expression of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) mRNA in adult human astrocytes: comparison with adult microglia and fetal astrocytes. AB - Astrocytes and microglia are cell populations which are implicated as being capable of regulating and effecting immune responses within the central nervous system (CNS). These functions are postulated to be mediated at least in part by production of soluble protein molecules termed cytokines. In this study, we utilized dissociated cultures of glial cells prepared from adult and fetal CNS tissue and a combined in situ hybridization-immunocytochemical technique in order to compare expression of TNF alpha and IL-6 mRNA between adult and fetal astrocytes and between adult astrocytes and microglia. Our results, using digoxygenin-labeled riboprobes, indicate that in contrast to fetal astrocytes only rare adult astrocytes express TNF alpha and IL-6 transcripts under our serum supplemented basal culture conditions. Activation with LPS and IFN gamma increased the proportion of adult astrocytes expressing detectable TNF alpha and IL-6 mRNA signals; however, the proportion was significantly less than for microglia contained in the same cultures. These results suggest that microglia rather than astrocytes are more likely to be sources of these cytokines within the adult human CNS. Further studies of cytokine expression by glial cells will need to consider both the age and species of the glial cells used. PMID- 8627342 TI - Expression of growth factors and growth factor receptors in capillary hemangioblastoma. AB - To elucidate the mechanisms underlying the regulation of growth and differentiation of capillary hemangioblastoma we studied the expression of selected growth factors and growth factor receptors by immunocytochemistry. As stromal cells of capillary hemangioblastoma express high levels of vascular endothelial growth factor (VEGF) and placental growth factor (P1GF) mRNA, we studied the distribution of the corresponding VEGF and P1GF proteins. We also studied the expression of epidermal growth factor receptor (EGFR) and platelet derived growth factor receptors (PDGFR) because their ligands have been reported to promote angiogenesis. The stromal cells expressed abundant EGFR and, in addition, some stromal cells expressed PDGFR-alpha but not PDGFR-beta. In contrast, the endothelial cells co-expressed PDGFR-alpha and PDGFR-beta. VEGF and P1GF were expressed by scattered stromal cells; however, more intense staining was observed in the endothelial cells of the intratumoral blood vessels, possibly indicating the secreted growth factors bound to their target receptors. We conclude that capillary hemangioblastomas express a variety of growth factor receptors and ligands, potentially involved in both autocrine and paracrine loops. The uniformly high EGFR expression is unique among brain tumors and may be associated with the typical morphology of capillary hemangioblastoma. The expression of highly angiogenic growth factors and their receptors may contribute to the rich vascularity of this enigmatic tumor. PMID- 8627343 TI - Differential expression of versican isoforms in brain tumors. AB - Versican is a member of the family of large aggregating chondroitin sulfate proteoglycans which are one of the major constituents of brain extracellular matrix (ECM). We examined the expression of versican splice variants at mRNA and protein levels in normal human brain and in gliomas, medulloblastomas, schwannomas, neurofibromas, and meningiomas. RT-PCR revealed transcripts for V0 and V1 in all tissues. V2 mRNA was restricted to gliomas and normal brain, while V3 mRNA was detected in all tissues except for medulloblastomas. Immunohistochemistry using antibodies to the glycosaminoglycan (GAG)-alpha attachment domain of versican (present in V0 and V2) revealed decreased staining of most glioma ECMs compared to normal neuropil, while some abnormal tumor vessels, but not normal cerebral vessels, were GAG-alpha-positive. Expression of the GAG-beta attachment domain (present in V0 and V1) was faint in normal neuropil and cerebral vessels, but increased in tumor vessels and was absent in most glioma ECMs. Both GAG-alpha and GAG-beta were expressed in connective tissue of all nonglial tumors. Our data suggest that V2 is the major versican isoform of normal neuropil and glioma ECM. Furthermore, increased expression in tumor vessels and decreased expression in glioma ECM of the anti-adhesive versican may be related to marked local invasivity and rarity of extracranial metastasis of gliomas. PMID- 8627344 TI - Neurofibrillary tangles in progressive supranuclear palsy contain the same tau epitopes identified in Alzheimer's disease PHFtau. AB - Neurofibrillary tangle (NFT)-rich brain samples from patients with progressive supranuclear palsy (PSP) or Alzheimer's disease (AD) were probed with a large panel of anti-tau antibodies to compare the species of tau present in PSP and AD NFTs by immunohistochemistry and Western blot methods. These antibodies have been shown to recognize phosphate-independent or -dependent epitopes that extend from the amino to the carboxy terminal domains of normal brain tau and the abnormal tau in the paired helical filaments (PHFs) of AD NFTs (PHFtau). The immunohistochemical studies showed that all of the tau epitopes detected in brainstem PSP NFTs also were found in hippocampal AD NFTs and vice versa. While Western blots demonstrated 2 PHFtau-like immunobands in PSP brainstem, a triplet of PHFtau proteins were seen in the AD and PSP hippocampus. Despite differences in the distribution, ultrastructure and immunoblot profile of NFTs in PSP and AD, the same constellation of tau epitopes is present in the abnormal tau proteins in PSP and AD NFTs. Thus, the generation of abnormal tau proteins in PSP (PSPtau) and AD (PHFtau) may have similar adverse biological consequences in both diseases. PMID- 8627345 TI - Production and characterization of two ependymoma xenografts. AB - Childhood ependymomas exhibit epidemiologic, anatomic, histologic, and biologic features and distinguish them from other gliomas. Because of their propensity to grow in functionally sensitive regions of the brain, adequate tumor sampling for basic and therapeutic research is limited. We have established xenografts in both subcutaneous and intracranial nude mouse systems (D528 EP-X, D612 EP-X) from the ependymomas of two nonrelated children. Median subcutaneous growth rates (reported in days to grow from 200 mm3 to 1000 mm3) are 82 days for D528 EP-X (n = 10) and 50 days for D612 EP-X (n = 10). D528 EP-X grows intracranially with a median postimplantation survival of 85 days (n = 10); D612 EP produces a median postimplantation survival of 72.5 days (n = 10). Both xenografts grow as well formed masses with no evidence of infiltration into either brain or subcutaneous tissues. While perivascular pseudopalisading is found in both xenografts, true ependymal rosette formation is absent. Ultrastructurally, neither xenograft exhibits cilia, but both produce abundant intermediate filaments. By light microscopy, the neoplastic cells are immunoreactive for the intermediate filaments glial fibrillary acidic protein, vimentin, and nestin. Karyotypically D528 EP exhibits 46,XX,del(6)(q22q26)/46,XX while D612 EP exhibits 50,XX, +X,t(1;8)(p11;q11),t(1;8)(p11;q11), +1,-4, der(5)t(4;5)(q12;q35), +der(5)t(4;5)(q12;q35),-6, +9, +9,-16, +der(17)t(6;17)(p11;p11), +mar. Restriction fragment length polymorphism studies comparing the primary brain tumor tissue from each patient against multiple passages of the resulting xenografts confirm the origin of both xenografts. These xenografts represent models on which future studies into the oncogenesis, progression and therapy of ependymomas can be performed. PMID- 8627346 TI - Myofibrillar myopathy with abnormal foci of desmin positivity. I. Light and electron microscopy analysis of 10 cases. AB - A number of myopathies whose common denominator is abnormal foci of desmin positivity have been described under the rubrics of spheroid body myopathy, cytoplasmic body myopathy, Mallory body myopathy, myopathy with granulofilamentous inclusions, desmin storage myopathy, and intermediate filament myopathy. In this study we reevaluate the light microscopic and ultrastructural features of the myopathy with abnormal foci of desmin positivity. In 10 cases of the disease, ultrastructural analysis reveals 2 major types of lesions: (a) foci of myofibrillar destruction and (b) hyaline structures that appear as spheroidal bodies on electron microscopy. The foci of myofibrillar destruction consist of fiber areas containing disrupted myofilaments, Z-disk-derived bodies, dappled dense structures of Z-disk origin, and streaming Z-disks that are sometimes adjacent to lakes of dense material. The spheroid bodies are composed of compacted and degraded myofibrillar elements. Membrane-bound vacuoles harboring degenerating membranous organelles are a less frequent and probably secondary abnormality. None of the lesions in muscle comprise 8 to 10 nm intermediate filaments. The findings imply that spheroid body myopathy, cytoplasmic body myopathy, Mallory body myopathy, and myopathy with granulofilamentous inclusions are consequences of a single or closely related pathologic processes. Because the common denominator appears to be focal dissolution of the myofibrils followed by accumulation of the products of the degradative process, we propose the term myofibrillar myopathy to cover the observed spectrum of pathologic changes. PMID- 8627347 TI - Myofibrillar myopathy with abnormal foci of desmin positivity. II. Immunocytochemical analysis reveals accumulation of multiple other proteins. AB - The two major types of lesions in myofibrillar myopathy consist of hyaline spheroidal structures composed of compacted myofibrillar residues, and nonhyaline lesions that comprise foci of myofibrillar destruction. We employed immunocytochemical analysis to further characterize these abnormalities. The nonhyaline lesions are depleted of actin, alpha-actinin, myosin, and, less consistently, of titin and nebulin. Thus, each major component of the myofibrils is lost or decreased. These lesions also react strongly for both NCAM and desmin. By contrast, the hyaline structures are highly enriched in actin, are immunoreactive for fast and slow myosin, and show increased expression of titin, nebulin, and alpha-actinin. They fail to react for NCAM and react variably for desmin. Both types of lesion react, but with differing intensities, for gelsolin, dystrophin, beta-amyloid precursor protein (beta APP) epitopes amino-terminal to the alpha-secretase site, alpha 1-antichymotrypsin, and ubiquitin, and both can be congophilic. The increased expressions of desmin, dystrophin and gelsolin in muscle are also confirmed by immunoblot studies. The results, in harmony with the ultrastructural findings described in the companion paper, suggest that myofibrillar myopathy is conditioned by abnormal activation of a degradative process that primarily affects the myofibrils. A structural abnormality of desmin alone may not be sufficient to disrupt the myofibrillar architecture, but abnormal activation of a phosphorylating process could account for dissolution of the myofibrils. The cause and significance of the ectopic overexpression of desmin, dystrophin, NCAM, and beta APP components, and the chemical basis of the congophilia remain unknown. PMID- 8627348 TI - Role of complement in the aetiology of Pick's disease? AB - Complement in the postmortem brains of 15 cases of Pick's disease has been widely analyzed immunohistochemically and, in 2 cases, by immunoelectron microscopy. Astrocytes and the Pick bodies and cytoplasm of ballooned neurons were immunoreactive with antibodies to classical pathway components C1, C1q, C4, C2 and C3 and the terminal complex components C5, C6 and C8. In almost all cases, no immunostaining was obtained with antibodies against C9 and neoepitopes in the membrane attack complex (MAC), the complement complex responsible for cytotoxicity. However, unequivocal staining with antibodies to two soluble complement regulatory proteins, S-protein and clusterin, and to the membrane complement inhibitor CD59 was found, although three other membrane inhibitors, CR1(CD35), DAF (CD55), and MCP (CD46), were not detected. The complement immunoreactivity of astrocytes and neurons could be the result of complement biosynthesis or attack. Complement attack will be restricted by the expressed regulatory proteins. However, neurons may be the victims of attack since they show pathological change. The internalization of complement-attacked membrane, perhaps involving the genesis of Pick bodies and ballooning, may explain the intracellular immunolocalization of complement in damaged neurons. Immunoglobulins, as a possible source of complement activation, were observed in only two cases, leaving unresolved the trigger for complement activation in the other cases. PMID- 8627349 TI - Reinduction of hyponatremia improves survival in rats with myelinolysis-related neurologic symptoms. AB - Brain myelinolysis occurs after excessive correction (delta SNa > 20 mEq/1/24 hours) of chronic hyponatremia. However, we showed recently that the mechanisms leading to brain myelinolysis remain reversible. Indeed, reinduction of the hyponatremia by water administration despite 12 hours of sustained excessive correction could prevent the development of demyelination in rats still asymptomatic at that time. Whether this therapeutic maneuver could be also beneficial to rats with preexisting myelinolysis-related neurologic symptoms is unknown. Therefore we evaluated here the effect of reinduction of the hyponatremia on the survival and on brain damage in rats presenting obvious neurologic symptoms after excessive correction of hyponatremia. After 3 days of severe hyponatremia induced by 2.5 D-glucose in water and continuous infusion of AVP, rats were submitted to a large correction (delta SNa approximately 30 mEq/l) by 2 i.p. injections of hypertonic saline given over 24 hours. In group I (n = 15) the rats developing neurologic symptoms during the first 24 hours of correction received one i.p. injection of distilled water which rapidly decreased the natremia to a final correction gradient <20 mEq/l/24 hour. In group II (n = 13, controls) the symptomatic rats were left permanently overcorrected. In group I, after water administration, the neurological manifestations were generally attenuated or disappeared. Seven of the 15 rats (47%) in this group survived up to day 10 with a mean survival time of 7.5 +/- 2 days, an outcome clearly improved as compared to group II (controls): only 1 of the 13 rats (7%, p < 0.03) was still alive on day 10 and the mean survival time was 3.3 +/- 2 days (p < 0.001) in this group II. The duration of the symptoms also influences the prognosis. In group I, in 9 rats the water administration was performed 4 hours after symptoms onset. These rats had a better outcome than the 6 rats with more sustained (8-10 hours) neurologic symptoms before water loading. Brain analysis in the 7 surviving rats of group I demonstrated demyelinating lesions in only 2 of them, suggesting the reversibility of the process even when neurologic manifestation developed. In conclusion, after exposure to an excessive correction of chronic hyponatremia, even when rats have developed myelinolysis-related neurologic symptoms, hypotonic fluids administration could improve survival and could prevent the subsequent development of brain myelinolysis. PMID- 8627350 TI - Listeria monocytogenes meningoencephalitis: murine models vs. the naturally occurring disease in ruminants. PMID- 8627351 TI - Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. AB - trkB encodes a receptor tyrosine kinase activated by three neurotrophins--brain derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5. In vivo, three isoforms of the receptor are generated by differential splicing- gp145trkB or the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145trkB, trkB.T1, and trkB.T2 by expressing the receptors in a Xenopus oocyte microinjection assay. We found that oocytes expressing gp145trkB, but not trkB.T1 or trkB.T2, were capable of eliciting 45Ca efflux responses (a phospholipase C-gamma-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145trkB, they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145trkB had similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145trkB appears to be an essential step in activation of the BDNF signaling cascade. PMID- 8627352 TI - cGMP-dependent protein kinase in dorsal root ganglion: relationship with nitric oxide synthase and nociceptive neurons. AB - Nitric oxide and cGMP influence plasticity of nociceptive processing in spinal cord. However, effectors for cGMP have not been identified in sensory pathways. We now demonstrate that cGMP-dependent protein kinase I (cGKl) occurs in the DRGs at levels comparable to that in cerebellum, the richest source of cGKl in the body. Immunohistochemical studies reveal that cGKl is concentrated in a subpopulation of small- and medium-diameter DRG neurons that partially overlap with substance P and calcitonin gene-related polypeptide containing cells. During development, cGKl expression throughout the embryo is essentially restricted to sensory neurons and to the spinal floor and roof plates. Neuronal nitric oxide synthase (nNOS) is coexpressed with cGKl in sensory neurons during embryonic development and after peripheral nerve axotomy. The primary target for cGKl in cerebellum, G-substrate, is not present in developing, mature, or regenerating sensory neurons, indicating that other proteins serve as effectors for cGKl in sensory processing. These data establish sensory neurons as a primary locus for cGMP actions during development and suggest a role for cGKl in plasticity of nociception. PMID- 8627353 TI - Study of receptor-mediated neurotoxins released by HIV-1-infected mononuclear phagocytes found in human brain. AB - Although there is growing evidence that neurotoxic molecules produced by HIV-1 infected mononuclear phagocytes damage neurons, the precise mechanisms of neuronal attack remain uncertain. One class of cytotoxin involves neuronal injury mediated via the NMDA receptor. We examined blood monocytes and brain mononuclear cells isolated at autopsy from HIV-1-infected individuals for the ability to release NMDA-like neuron-killing factors. We found that a neurotoxic amine, NTox, was produced by blood monocytes and by brain mononuclear phagocytes infected with retrovirus. In vivo injections of minute quantities of NTox produced selective damage to hippocampal pyramidal neurons. NTox can be extracted directly from brain tissues infected with HIV-1 and showed structural features similar to wasp and spider venoms. In contrast to NTox, HIV-1 infection did not increase the release of the NMDA excitotoxin quinolinic acid (QUIN) from mononuclear cells. Although we found modest elevations of QUIN in the CSF of HIV-1-infected individuals, the increases were likely attributable to entry through damaged blood-brain barrier. Taken together, our data pinpoint NTox, rather than QUIN, as a major NMDA receptor-directed toxin associated with neuro-AIDS. PMID- 8627354 TI - Invertebrate synapsins: a single gene codes for several isoforms in Drosophila. AB - Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed. PMID- 8627355 TI - Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. AB - The molecular mechanisms underlying the establishment of a postsynaptic receptor mosaic on CNS neurons are poorly understood. One protein thought to be involved is gephyrin, a peripheral membrane protein that binds to the inhibitory glycine receptor and functions in clustering this receptor at synapses in cultured rat spinal cord neurons. We investigated the possible association of gephyrin with synapses in cultured rat hippocampal neurons, where glutamate and GABA but not glycine are the principal transmitters. Gephyrin immunoreactivity was detected in axons as well as dendrites, changing from a predominantly axonal to a more dendritic distribution with time in culture. Gephyrin staining was not distributed uniformly, but always took the form of clusters. Small clusters of gephyrin (0.2 microns 2), present throughout development, were distributed widely and not restricted to synaptic sites. Larger clusters of gephyrin (0.4-10.0 microns 2, sometimes composed of groups of small clusters), which developed in older cells, were localized to a subset of contacts between axons and dendrites. These large clusters were not present at glutamatergic synapses (marked by immunostaining for GluR1), but were closely associated with GABAergic synapses (marked by immunostaining for GABA and glutamic acid decarboxylase). These results, together with previous findings, suggest that gephyrin may function to anchor GABA and glycine receptors, but not glutamate receptors, at postsynaptic sites on central neurons. They also raise the possibility that gephyrin has additional functions, independent of its role at synapses. PMID- 8627356 TI - Selective, activity-dependent uptake of histamine into an arthropod photoreceptor. AB - The synapses made by many arthropod photoreceptors are disinhibitory and use histamine as their transmitter. Because decreases and not increases in the cleft concentration of transmitter constitute the important event at these synapses, a transporter to clear the cleft of histamine would seem particularly crucial to signal transfer. We report here that 3H-histamine is taken up selectively into barnacle photoreceptors by a Na+-dependent mechanism, presumably a transporter. Using light microscopic autoradiography, we observe heavy label over axons and presynaptic terminals of these neurons when they are stimulated during uptake. The radioactivity taken up was identified as 3H-histamine by thin layer chromatography; no metabolites were detected, even after 5 hr. Radiolabeled 5 hydroxytryptamine and GABA are not taken up by the photoreceptor. 3H-histamine uptake into photoreceptors is decreased markedly by an excess of unlabeled histamine and by chlorpromazine and phenoxybenzamine. Unexpectedly for uptake dependent on the NA+ gradient, photoreceptor terminals label more intensely in the light (when depolarized) than in the dark (when hyperpolarized). Glia label more strongly than photoreceptors in dark-incubated preparations. The presence of presynaptic uptake strengthens the evidence that histamine is the neurotransmitter of arthropod photoreceptors and provides a mechanism by which this synapse could recycle transmitter, control its steady-state cleft concentration, and clear it from the cleft in response to decreases in its release from the photoreceptors. PMID- 8627357 TI - A macromolecular synthesis-dependent late phase of long-term potentiation requiring cAMP in the medial perforant pathway of rat hippocampal slices. AB - Memory storage consists of a short-term phase that is independent of new protein synthesis and a long-term phase that requires the synthesis of new proteins and RNA. A cellular representation of these two phases has been demonstrated recently for long-term potentiation (LTP) in both the Schaffer collateral and the mossy fibers of the hippocampus, a structure widely thought to contribute to memory consolidation. By contrast, much less information is available about the medial perforant pathway (MPP), one of the major inputs to the hippocampus. We found that both a short-lasting and a long-lasting potentiation (L-LTP) can be induced in the MPP of rat hippocampal slices by applying repeated tetanization in reduced levels of magnesium. This potentiation was dependent on the activation of NMDA receptors. The early, transient phase of LTP in the MPP did not require either protein or RNA synthesis, and it was independent of protein kinase A activation. By contrast, L-LTP required the synthesis of proteins and RNA, and was selectively blocked by inhibitors of cAMP-dependent protein kinase (PKA). Forskolin, an adenylate cyclase activator, also induced a L-LTP that was attenuated by inhibition of transcription. Our results demonstrate that, like LTP in the Schaffer collateral and mossy fiber pathways, MPP LTP also consists of a late phase that is dependent on protein and RNA synthesis and PKA activity. Thus, cAMP-mediated transcription appears to be a common mechanism for the late form of LTP in all three pathways within the hippocampus. PMID- 8627359 TI - Frequency and dendritic distribution of autapses established by layer 5 pyramidal neurons in the developing rat neocortex: comparison with synaptic innervation of adjacent neurons of the same class. AB - Synaptic contacts formed by the axon of a neuron on its own dendrites are known as autapses. Autaptic contacts occur frequently in cultured neurons and have been considered to be aberrant structures. We examined the regular occurrence, dendritic distribution, and fine structure of autapses established on layer 5 pyramidal neurons in the developing rat neocortex. Whole-cell recordings were made from single neurons and synaptically coupled pairs of pyramidal cells, which were filled with biocytin, morphologically reconstructed, and quantitatively analyzed. Autapses were found in most neurons (in 80% of all cells analyzed; n = 41). On average, 2.3 +/- 0.9 autapses per neuron were found, located primarily on basal dendrites (64%; 50-70 microns from the soma), to a lesser extent on apical oblique dendrites (31%; 130-200 microns from the soma), and rarely on the main apical dendrite (5% 480-540 microns from the soma). About three times more synaptic than autaptic contacts (ratio 2.4:1) were formed by a single adjacent synaptically coupled neuron of the same type. The dendritic locations of these synapses were remarkably similar to those of autapses. Electron microscopic examination of serial ultrathin sections confirmed the formation of autapses and synapses, respectively, and showed that both types of contacts were located either on dendritic spines or shafts. The similarities between autapses and synapses suggest that autaptic and synaptic circuits are governed by some common principles of synapse formation. PMID- 8627360 TI - The ganglionic eminence may be an intermediate target for corticofugal and thalamocortical axons. AB - In the nervous system of many species, growing axons associate transiently with cellular groupings along their path. Whether this mechanism applies to the development of corticothalamic and thalamocortical projections is unknown. Using carbocyanine dyes, we studied the early growth of both corticofugal and thalamocortical fibers in hamster embryos. At embryonic day 11.5 (E11.5), corticofugal fibers invade the lateral ganglionic eminence (LGE), and thalamocortical fibers invade the medial ganglionic eminence (MGE). At this age, both sets of fibers are not yet in contact with each other. At the same time, neurons in each subdivision of the GE grow toward the cortex and thalamus. During the next 24 hr, corticofugal and thalamocortical fibers remain within the confines of the GE, where they course at different radial levels and bear large and complex growth cones. In the LGE, corticofugal fibers are often found in close association with cells that are likely to be neuronal. Starting on E13.5, both early projections from the GE decrease, and corticothalamic and thalamocortical fibers invade their definitive target regions. To test whether the GE specifically orients the growth and trajectories of cortical fibers even in the absence of the reciprocal thalamic projection, we cocultured explants of cortex and GE from either hamster or mouse embryos. These experiments showed that the GE, but not other tested brain regions, is able specifically to orient the growth of cortical axons. We therefore suggest that the GE may be an intermediate target in the pathfinding of axons between the cortex and the thalamus. PMID- 8627358 TI - Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells. AB - Although remyelination of demyelinated CNS axons is known to occur after transplantation of exogenous glial cells, previous studies have not determined whether cell transplantation can restore the conduction properties of demyelinated axons in the adult CNS. To examine this issue, the dorsal columns of the adult rat spinal cord were demyelinated by x-irradiation and intraspinal injections of ethidium bromide. Cell suspensions of cultured astrocytes and Schwann cells derived from neonatal rats transfected with the (beta galactosidase) reporter gene were injected into the glial-free lesion site. After 3-4 weeks nearly all of the demyelinated axons were remyelinated by the transplanted Schwann cells. The dorsal columns were removed and maintained in an in vitro recording chamber; conduction properties were studied using field potential and intra-axonal recording techniques. The demyelinated axons exhibited conduction slowing and block, and a reduction in their ability to follow high frequency stimulation. Axons remyelinated by transplantation of cultured Schwann cells exhibited restoration of conduction through the lesion, with reestablishment of normal conduction velocity. The axons remyelinated after transplantation showed enhanced impulse recovery to paired-pulse stimulation and greater frequency-following capability as compared with both demyelinated and control axons. These results demonstrate the functional repair of demyelinated axons in the adult CNS by transplantation of cultured myelin-forming cells from the peripheral nervous system in combination with astrocytes. PMID- 8627361 TI - Microtubule stability decreases axon elongation but not axoplasm production. AB - Microtubules are a primary cytoskeletal constituent of axons and growth cones. In addition to serving as a scaffolding for axon assembly, they also provide a means of transport of organelles that are essential for outgrowth and maintenance of synaptic function. Pharmacological manipulations that disrupt net assembly of microtubules also interfere with growth cone advance and axon extension. Less is known after the effects of disrupting microtubule dynamics without affecting net assembly. To investigate this, we studied the effects of low doses of nocodazole on axon extension and microtubule organization in rat superior cervical ganglion neurons. We report that 165-330 nM nocodazole significantly reduces axon extension rate and increases axon diameter without affecting the rate of production of axoplasm or microtubule polymer, and without decreasing the average length or number of microtubules. Two observations suggested that microtubule dynamics were depressed by this dose of nocodazole. First, this treatment eliminated the highly divergent lengths and positions of microtubules characteristic of normal growth cones, inducing an array in which each microtubule terminated at roughly the same position in the proximal regions of the growth cone. Second, there was a decrease in the proportion of microtubule length containing mostly tyrosinated (newly assembled) alpha-tubulin and an increase in the proportion of microtubule length containing mostly acetylated (older, more stable) alpha-tubulin. Together, these data suggest that a decrease in dynamic instability of microtubules is sufficient to disrupt axon extension but does not interfere with axoplasm production. PMID- 8627362 TI - Influences of the thalamus on the survival of subplate and cortical plate cells in cultured embryonic mouse brain. AB - The afferent and efferent connections of the cerebral neocortex develop simultaneously toward the end of embryogenesis. At this stage, the neocortex comprises two main cell-dense layers: the thicker and more superficial cortical plate (future layers 2-6) and the thinner underlying subplate. Many early thalamocortical projections temporarily innervate the subplate before leaving it to locate their ultimate targets in the overlying cortical plate. The subplate then disappears. In this study, we performed in vitro experiments on late embryonic murine brain to test whether the thalamus can influence the survival of cortical plate and subplate cells at this stage. In isolated organotypic cortical explants from embryonic day 19 mice, most of the cells that had formed the subplate died. Coculture with a thalamic explant prevented this loss; coculture with additional cortical or cerebellar explants did not. By contrast, many cells in or destined for the cortical plate survived even in isolated cortical explants; coculture with a thalamic explant did not alter the numbers of these cells that survived. Our results suggest that the thalamus provides trophic support for subplate cells but not for late embryonic cortical plate cells. In vivo, a loss of thalamic-derived trophic support for the subplate late in embryogenesis, consequent on the movement of thalamocortical axons into the cortical plate, may contribute to subplate death. PMID- 8627363 TI - Synaptic modulation by neurotrophic factors: differential and synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor. AB - Extracellular application of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) to developing neuromuscular junctions in Xenopus nerve muscle cultures resulted in an increase in the frequency of spontaneous synaptic currents (SSCs) and in the amplitude of nerve-evoked synaptic currents. Analyses of the amplitude and time course of the SSCs suggest that these effects are attributable to elevation of presynaptic transmitter release. The actions of these two factors on the transmitter secretion process, however, are distinctly different. Fura-2 Ca2+ imaging showed that an increase in presynaptic cytosolic Ca2+ ([Ca2+]i) accompanied the synaptic potentiation by BDNF, whereas no change in [Ca2+]i was observed during synaptic potentiation by CNTF. Removing external Ca2+ also abolished the potentiating effect of BDNF but did not influence the CNTF effect. Moreover, the two factors exerted different effects on the short term synaptic plasticity. Paired-pulse facilitation normally found at these synapses was reduced by BDNF but unaffected by CNTF; CNTF, but not BDNF, reduced the extent of synaptic depression during high-frequency tetanic stimulation. Finally, the potentiation effect of BDNF and CNTF on spontaneous transmitter release was additive when both factors were applied together to the synapse at saturating concentrations (100 ng/ml) and was highly synergistic when low doses (1 and 10 ng/ml) of both factors were used. These results suggest that because of their differential effects on the secretory machinery, BDNF and CNTF may act cooperatively in modulating the development and functioning of synapses. PMID- 8627364 TI - Transgenic expression of embryonic MAP2 in adult mouse brain: implications for neuronal polarization. AB - The major neuronal microtubule-associated protein MAP2 is selectively localized in dendrites, where its expression is under strong developmental regulation. To learn more about its potential effects on neuronal morphogenesis and its sorting within the neuronal cytoplasm, we have raised transgenic mice that express high levels of the embryonic form, MAP2c, in the adult brain. One transgenic line expressed higher levels of MAP2c than endogenous adult MAP2. This had no detectable effect on either the arrangement or morphology of neurons, suggesting that although MAP2c is necessary for neuronal morphogenesis it is not involved in its regulation. Like endogenous adult MAP2, transgenic MAP2c was present in dendrites but not axons, indicating that the signal responsible for its cytoplasmic sorting is contained within the 1.5 kb of its coding sequence. In situ hybridization with specific probes showed that transgenic MAP2c mRNA was limited to cell bodies. Thus, the dendritic localization of MAP2c protein cannot be the result of previous transport of its mRNA but must depend on a signal associated with the protein itself. Furthermore, because the amino acid sequence of MAP2c is present in all forms of MAP2, this signal is also contained within adult high-M(r) MAP2 protein. This raises the possibility that, rather than the conventional scheme of mRNA sorting preceding protein localization, the transport of adult MAP2 mRNA into dendrites could depend on it being part of a translation complex in which the targeting signal is on the nascent protein. PMID- 8627365 TI - Experience-dependent plasticity of binocular responses in the primary visual cortex of the mouse. AB - An activity-dependent form of synaptic plasticity underlies the fine tuning of connections in the developing primary visual cortex of mammals such as the cat and monkey. Studies of the effects of manipulations of visual experience during a critical period have demonstrated that a correlation-based competitive process governs this plasticity. The cellular mechanisms underlying this competition, however, are poorly understood. Transgenic and gene-targeting technologies have led to the development of a new category of reagents that have the potential to help answer questions of cellular mechanism, provided that the questions can be studied in a mouse model. The current study attempts to characterize a developmental plasticity in the mouse primary visual cortex and to demonstrate its relevance to that found in higher mammals. We found that 4 d of monocular lid suture at postnatal day 28 (P28) induced a maximal loss of responsiveness of cortical neurons to the deprived eye. These ocular dominance shifts occurred during a well-defined critical period, between P19 and P32. Furthermore, binocular deprivation during this critical period did not decrease visual cortical responses, and alternating monocular deprivation resulted in a decrease in the number of binocularly responsive neurons. Finally, a laminar analysis demonstrated plasticity of both geniculocortical and intracortical connections. These results demonstrate that an activity-dependent, competitive form of synaptic plasticity that obeys correlation-based rules operates in the developing primary visual cortex of the mouse. PMID- 8627366 TI - Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K+ current development. AB - A developmental increase in density of delayed rectifier potassium current (IKv) in embryonic Xenopus spinal neurons shortens action potential durations and limits calcium influx governing neuronal differentiation. Although previous work demonstrates that maturation of IKv depends on general mRNA synthesis, it is not known whether increases in K+ channel gene transcripts direct maturation of the current. Accordingly, the developmental appearance of specific Kv potassium channel genes was determined using single-cell reverse transcription-PCR techniques after whole-cell recording of IKv during the period of its development. Detection of a coexpressed housekeeping gene along with the potassium channel gene controlled for successful aspiration of cellular mRNA and allowed scoring of cells in which Kv gene transcripts were not detected. Diverse types of Xenopus spinal neurons exhibit homogeneous development of IKv both in vivo and in culture. In contrast, transcripts of two genes encoding delayed rectifier current, Kv1.1 (Shaker) and Kv2.2 (Shab), are expressed heterogeneously during the period in which the current develops. Kv1.1 mRNA achieves maximal appearance in approximately 30% of cells, while IKv is immature; Kv2.2 mRNA appears later in approximately 60% of mature neurons. Kv1.1 and 2.2 are thus candidates for generation of IKv, and spinal neurons are a heterogeneous population with respect to potassium channel gene expression. Moreover, correlation of gene expression with current properties shows that neurons lacking Kv2.2 have a characteristic voltage dependence of activation of IKv. PMID- 8627367 TI - BEN as a presumptive target recognition molecule during the development of the olivocerebellar system. AB - It has been shown previously that in the chick embryo the cell adhesion molecule BEN/SC1/DM-GRASP is expressed by neurons in the inferior olive (IO) and by their terminal axonal arbors in the cerebellar cortex, the climbing fibers (Porquie et al., 1992b). Here, new information on the expression of BEN during the formation of the olivocerebellar projection adds the important notion that BEN is also expressed by the cerebellar targets of inferior olivary axons, Purkinje cells (PCs) and deep nuclear neurons. This expression is transient, starting at E7-E8 and vanishing shortly after hatching. More importantly, BEN expression is restricted to precise subsets of IO neurons and PCs. In the cerebellar cortex, BEN-immunoreactive (BEN-IR) structures are not found randomly but are distributed according to a reproducible pattern of parasagittal stripes. A maximum of four distinct sagittal stripes is found in each lobule, along the whole rostrocaudal extent of the cerebellum. Moreover, BEN-expressing stripes belong to two classes; one contains BEN-IR climbing fibers terminating on BEN-IR PCs and the other, more frequent class is solely composed of BEN-IR climbing fibers. Organotypic cultures of isolated cerebella have shown that the expression of BEN in the IO and in the cerebellum arise independently, probably because of an intrinsic developmental program. Thus, the cell adhesion molecule BEN meets all criteria for a recognition molecule involved in the formation of the olivocerebellar projection. PMID- 8627368 TI - Contingent vulnerability of entorhinal parvalbumin-containing neurons in Alzheimer's disease. AB - Calcium-binding proteins containing local circuit neurons are distributed ubiquitously in the human cerebral cortex where they colocalize with a subpopulation of cells that contain GABA. Several reports using a variety of pathological models, including Alzheimer's disease (AD), have suggested that cells containing calcium-binding proteins are resistant to pathological insults. In this report, we test the hypothesis that AD pathology can differentially affect parvalbumin-containing cells depending on their location in the entorhinal cortex and the state of projection neurons with which they are associated. Using cases with different quantities of AD pathology, we determined the density of immunostaining for parvalbumin in the entorhinal cortex, and we correlated this with the concomitant pathological lesions in the various layers of this cortex. Our results show a clear decrease in parvalbumin immunostaining in some parts of the entorhinal cortex when AD neuropathological markers are present. As the density of pathological markers in the entorhinal cortex becomes greater and more widespread, there is a decrease of parvalbumin immunostaining in additional layers, although in all cases, some cells persist. Parvalbumin-containing neurons are clearly vulnerable in AD, but not because of neurofibrillary tangle formation. Instead, they are rendered vulnerable only after substantial loss of projection neurons; only then do they, too, become part of the lesion. PMID- 8627369 TI - A novel entorhinal projection to the rat dentate gyrus: direct innervation of proximal dendrites and cell bodies of granule cells and GABAergic neurons. AB - Entorhinal fibers to the fascia dentata originating from layer II stellate neurons are known to terminate exclusively in the outer two thirds of the molecular layer, where they innervate distal dendritic portions of dentate neurons. Using anterograde tracing with Phaseolus vulgaris leucoagglutinin, we unraveled a previously unknown entorhinal projection that directly innervates proximal dendritic portions and somata of granule cells and GABAergic neurons. This projection originates from neurons located in entorhinal layers IV-VI of the medial entorhinal area. These fibers enter the outer two thirds of the molecular layer, traverse the inner molecular layer (IML) and granule cell layer, where they form numerous boutons, and finally arborize subjacent to the granule cells. Correlated light and electron microscopy revealed that the boutons formed by these fibers establish asymmetric synapses on dendrites in the IML, on spines and somata of granule cells, and on spineless dendrites subjacent to the granule cell layer. Postembedding immunogold staining indicates that this entorhino-dentate projection is not GABAergic and that it also terminates on GABAergic inhibitory neurons. These data demonstrate that not all entorhino-dentate fibers display a similar high laminar specificity for the outer molecular layer (OML). Although fibers from the superficial layers of the entorhinal cortex terminate exclusively in the OML, entorhinal fibers arising from deeper layers are not confined to laminar boundaries. Finally, the possibility that these supposedly excitatory entorhinal afferents may monosynaptically activate proximal dendrites and somata of dentate neurons needs to be incorporated into contemporary concepts of the hippocampal network. PMID- 8627370 TI - Projection cells and interneurons of the lateral and basolateral amygdala: distinct firing patterns and differential relation to theta and delta rhythms in conscious cats. AB - To study relations between the basolateral (BL) amygdaloid complex and major electroencephalogram (EEG) rhythms of the entorhinal cortex (delta and theta), neurons of the lateral and BL nuclei were recorded in conscious cats. An essential task to this end was to obtain criteria allowing the identification of projection cells and interneurons. BL projection cells, identified by their antidromic response to parahippocampal stimuli, generated stereotyped high frequency bursts (2-4 spikes at 140-250 Hz), which repeated at low rates. Projection cells of the lateral nucleus were virtually silent, but their presence was disclosed by cortical-evoked responses. In both nuclei, the firing rates and/or responsiveness of projection cells increased from waking to slow-wave sleep (S). In contrast with projection cells, presumed interneurons discharged at high rates (approximately 10-15 Hz) and displayed various discharge patterns ranging from tonic to phasic. The bipartite classification of BL neurons on the basis of their discharge patterns and synaptic responses was supported by the differential relation existing between EEG rhythms and the activity of the two cell types. Indeed, fast-firing and bursting cells of the BL nucleus tended to fire on opposite phases of the delta oscillation of S and entorhinal theta oscillation of paradoxical sleep. The unusual state-related changes in activity displayed by lateral and BL neurons point to functional similarities between the amygdala and hippocampus. This idea is supported by the presence of coherent theta oscillations in the amygdala-hippocampal circuit that might favor the emergence of recurring time windows when synaptic interactions will be facilitated in this limbic network. PMID- 8627371 TI - Efficient coding of natural scenes in the lateral geniculate nucleus: experimental test of a computational theory. AB - A recent computational theory suggests that visual processing in the retina and the lateral geniculate nucleus (LGN) serves to recode information into an efficient form (Atick and Redlich, 1990). Information theoretic analysis showed that the representation of visual information at the level of the photoreceptors is inefficient, primarily attributable to a high degree of spatial and temporal correlation in natural scenes. It was predicted, therefore, that the retina and the LGN should recode this signal into a decorrelated form or, equivalently, into a signal with a "white" spatial and temporal power spectrum. In the present study, we tested directly the prediction that visual processing at the level of the LGN temporarily whitens the natural visual input. We recorded the responses of individual neurons in the LGN of the cat to natural, time-varying images (movies) and, as a control, to white-noise stimuli. Although there is substantial temporal correlation in natural inputs (Dong and Atick, 1995b), we found that the power spectra of LGN responses were essentially white. Between 3 and 15 Hz, the power of the responses had an average variation of only +/-10.3%. Thus, the signals that the LGN relays to visual cortex are temporarily decorrelated. Furthermore, the responses of X-cells to natural inputs can be well predicted from their responses to white-noise inputs. We therefore conclude that whitening of natural inputs can be explained largely by the linear filtering properties (Enroth-Cugell and Robson, 1966). Our results suggest that the early visual pathway is well adapted for efficient coding of information in the natural visual environment, in agreement with the prediction of the computational theory. PMID- 8627373 TI - Low-frequency stimulation cancels the high-frequency-induced long-lasting effects in the rat medial vestibular nuclei. AB - In rat brainstem slices, we investigated the effects of low-frequency stimulation (LFS) of the primary vestibular afferents on the amplitude of the field potentials evoked in the medial vestibular nuclei (MVN). LFS induced long-term effects, the sign of which depended on whether the vestibular neurons were previously conditioned by HFS. In unconditioned slices, LFS evoked modifications of the responses that were similar to those observed after HFS but had a smaller extension. In fact, LFS caused long-lasting potentiation of the N1 wave in the MVN ventral portion (Vp) and long-lasting depression of the N2 wave in the MVN dorsal portion (Dp), whereas it provoked small and variable effects on the N1 wave. By contrast, when the synaptic transmission was already conditioned, LFS influenced the synaptic responses oppositely, reducing or annulling the HFS long term effects. This phenomenon was specifically induced by LFS, because HFS was not able to cause it. The involvement of NMDA receptors in mediating the LFS long term effects was supported by the fact that AP-5 prevented their induction. In addition, the annulment of HFS long-term effects by LFS was also demonstrated by the shift in the latency of the evoked unitary potentials after LFS. In conclusion, we suggest that the reduction of the previously induced conditioning could represent a cancellation mechanism, useful to quickly adapt the vestibular system to continuous different needs and to avoid saturation. PMID- 8627372 TI - AMPA receptor subunits underlying terminals of fine-caliber primary afferent fibers. AB - Postembedding immunogold electron microscopy was used to determine the relation of primary afferent terminals in superficial laminae of the spinal dorsal horn with AMPA receptor subunits. Immunogold particles coding for GluR1 and GluR2/3 were concentrated at synaptic sites, between 30 nm outside and 40 nm inside the postsynaptic membrane. Immunopositive synapses displayed round vesicles and asymmetric specializations, characteristic of terminals releasing excitatory neurotransmitters; symmetric synapses, characteristic of terminals releasing inhibitory amino acids, were immunonegative. In superficial laminae, large terminals of two main types at the center of a synaptic glomerulus originate from primary afferents: C1 terminals are mainly endings of unmyelinated afferent fibers; C2 terminals are mainly endings of thinly myelinated afferent fibers. Terminals of both types were presynaptic to AMPA subunits, but in different proportions: C1 terminals were related more to GluR1 than to GluR2/3, whereas the reverse was true for C2 terminals. These results suggest that functional properties of peripheral afferents to the spinal cord may be specified by the density and combination of receptor subunits in the postsynaptic membrane, and raise the possibility that calcium-permeable AMPA channels may play a special role in the mediation of sensory input by unmyelinated fibers. PMID- 8627374 TI - Blue-cone horizontal cells in the retinae of horses and other equidae. AB - The morphology of horizontal cells chiefly of the horse, but also of asses, mules, and a zebra, has been examined by Lucifer yellow injections into lightly fixed retinae and by immunocytochemistry. In common with other mammals, equids have a B-type horizontal cell, i.e., a cell with dendrites synapsing with cones and possessing a single axon synapsing with rods. Most mammalian retinae have a further type of horizontal cell, the A-type, also synapsing with cones but without an axon. The second type of horizontal cell in equids also has no axon; otherwise, it is most unusual. Compared with other mammalian A-type cells, it has a vary large dendritic field, both absolutely and relative to the dendritic fields of B-type cells. The dendrites are fine and sparsely branching. Their most striking feature is that they bear a low density of irregularly spaced synaptic terminal aggregates, suggesting their cone contacts are selective. Immunolabelling of S (blue)-cones in horse retina showed that they comprise, depending on retinal location, 10-25% of the cone population. For a single horse A-type cell, it is shown that 44 of its 45 terminal aggregates are congruent with the pedicles of S-cones. Immunostaining with a calbindin antibody demonstrated that each type of horizontal cell forms an independent regular mosaic. The density ratio of B- to A-type cells varied between 5 and 10. This is the first demonstration in a mammalian retina of a horizontal cell type with a direct input exclusively from S-cones. PMID- 8627375 TI - Interneurons containing calretinin are specialized to control other interneurons in the rat hippocampus. AB - Spine-free calretinin-immunoreactive (CR-IR) interneurons form a subpopulation of GABAergic cells in the rat hippocampus. A characteristic feature of these cells- located in all areas and layers--is the frequent dendro-dendritic and axo dendritic contacts they form with each other. In this study we examined in detail the connectivity of these neurons by reconstructing their dendritic and axonal arbor and by identifying their postsynaptic targets. Radially running dendrites of CR-IR cells, located in different layers, intermingled into long braids. An average cell was in contact with dendrites of three to seven other CR-IR cells. Reconstruction of the dendritic trees from six consecutive sections demonstrated that at least 15 cells may participate in a dendro-dendritically connected cluster. Electron microscopical examination revealed that regularly spaced zonula adherentia connect the touching dendrites. The postsynaptic targets of CR-IR neurons have been examined using postembedding immunogold staining for GABA. CR containing GABA-immunoreactive axons of local origin formed multiple symmetrical synaptic contacts (two to five) exclusively on GABAergic dendrites (CR-negative as well as CR-positive). Two to 10 CR-IR axons may converge onto a single CR-IR neuron, often from cells belonging to the same dendro-dendritically connected cluster. Using double immunocytochemistry, CR-IR cells were shown to heavily innervate calbindin D28k-containing interneurons and VIP-containing basket cells but avoided the parvalbumin-containing basket and axo-axonic cells. The unique connectivity of CR-IR cells may enable them to play a crucial role in the generation of synchronous, rhythmic hippocampal activity by controlling other interneurons terminating on different dendritic and somatic compartments of principal cells. PMID- 8627376 TI - Morphological correlates of bilateral synchrony in the rat cerebellar cortex. AB - Simultaneous recordings of the left and right crus IIA of the cerebellar cortex in the rat have demonstrated that Purkinje cells of both sides can be activated synchronously by their climbing fibers. Because climbing fibers arise exclusively from the contralateral inferior olive (IO), this physiological finding seems to contradict the anatomy. To define the structural basis responsible for the bilateral synchrony, we examined the possibilities that bilateral common afferent inputs to the IO and interolivary connections form the underlying mechanisms. The bilaterality of the major afferents of the olivary regions that project to crus IIA was studied using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. We found that the excitatory and inhibitory projections from the spinal trigeminal nucleus and dorsolateral hump of the interposed cerebellar nucleus to the transition area between the principal olive and dorsal accessory olive were bilateral. A second possible mechanism for bilateral synchrony, which is the possibility that axons of olivary neurons provide collaterals to the contralateral side, was investigated using biotinylated dextran amine as an anterograde tracer. Labeled axons were traced and reconstructed from the principal olive and dorsal and medial accessory olive up to the entrance of the contralateral restiform body. None of these axons gave rise to collaterals. The possibility that neurons in the left and right IO are electronically coupled via dendrodendritic connections was investigated by examining the midline region of the IO. The neuropil of the left and right IO is continuous in the dorsomedial cell column. Examination of Golgi impregnations of this subdivision demonstrated that (1) many dendrites cross from one side to the other, (2) neurons close to the midline give rise to dendrites that extend into both olives, and (3) dendrites of neurons in the dorsomedial cell column frequently traverse into adjacent olivary subdivisions such as the medial accessory olive and the transition area between the principal olive and dorsal accessory olive. Sections immunostained for dendritic lamellar bodies or GABAergic terminals showed the same pattern: the neuropils of the dorsomedial cell columns on both sides form a continuum with each other as well as with the neuropil of other adjacent olivary subdivisions. Ultrastructural examination of the dorsomedial cell column demonstrated that the midline area includes many complex glomeruli that contain dendritic spines linked by gap junctions. To verify whether the complex spike synchrony observed between left and right crus IIA could indeed be mediated in part through coupled neurons in the dorsomedial cell column, we recorded simultaneously from crus IIA areas and from left and right vermal lobule IX, which receives climbing fibers from the dorsomedial cell column. In these experiments we demonstrated that the climbing fibers of all four areas, i.e., the left and right crus IIA as well as the left and right lobule IX, can fire synchronously. The present results indicate that synchronous climbing fiber activation of the left and right crus IIA in the rat can be explained by (1) bilateral inputs to the transition areas between the principal olive and dorsal accessory olive and (2) dendrodendritic electrotonic coupling between neurons of the left and right dorsomedial cell column and between neurons of the dorsomedial cell column and adjacent olivary subdivisions. PMID- 8627377 TI - Molecular indices of neuronal and glial plasticity in the hippocampal formation in a rodent model of age-induced spatial learning impairment. AB - Spatial learning ability was quantitated in young and aged Long-Evans rats, and molecular markers were assessed in the striatum and hippocampal formation using immunocytochemical, immunoblotting, and in situ hybridization histochemical procedures. The mRNA for beta-amyloid precursor protein (beta APP), most likely the transcript encoding the 695-amino acid form of this protein, was elevated in pyramidal and granule cells in the hippocampus of aged rats exhibiting poorer spatial learning. In immunoblots of hippocampal protein extracts, however, the level of beta APP-like immunoreactivity was depressed in the more impaired subjects. Similarly, the level in hippocampus of the mRNA for manganese-dependent superoxide dismutase (Mn-SOD), a marker of oxidative stress, was positively correlated with the degree of behavioral impairment, but immunoblotting revealed that Mn-SOD protein was depressed in the aged hippocampus compared with young. The mRNAs for the neuronal form of nitric oxide synthase and for the astrocyte marker glial fibrillary acidic protein (GFAP) were elevated in the hippocampus in correlation with the extent of learning impairment. In the striatum, the levels of mRNA and protein for several candidate genes, including GFAP, were elevated in parallel with the learning index, but these were age effects. Several hippocampal proteins were unchanged (GFAP) or depressed (beta APP and Mn-SOD) in level, despite elevations in corresponding mRNAs. In the aged cohort, hippocampal GFAP mRNA, Mn-SOD mRNA, and beta APP emerged as predictors of behavioral impairment, suggesting the involvement of these hippocampal systems in age-related cognitive impairment. PMID- 8627378 TI - Implementation of action sequences by a neostriatal site: a lesion mapping study of grooming syntax. AB - The neostriatum and its connections control the sequential organization of action ("action syntax") as well as simpler aspects of movement. This study focused on sequential organization of rodent grooming. Grooming syntax provides an opportunity to study how neural systems coordinate natural patterns of serial order. The most stereotyped of these grooming patterns, a "syntactic chain," has a particularly stereotyped order that recurs thousands of times more often than could occur by chance. The purpose of the present study was to identify the crucial site within the striatopallidal system where lesions disrupt the syntax or serial order of syntactic grooming chains without disrupting constituent movements. Small excitotoxin lesions were made using quinolinic acid at bilateral sites within the dorsolateral, dorsomedial, ventrolateral, or ventromedial neostriatum, or in the ventral pallidum or globus pallidus of rats. An objective technique for mapping functional lesions was used to quantify cell death and to map precisely those lesions that disrupted grooming syntax. Our results identified a single site within the anterior dorsolateral neostriatum, slightly more than a cubic millimeter in size (1.3 x 1.0 x 1.0 mm), as crucial to grooming syntax. Damage to this site did not disrupt the ability to emit grooming actions. By contrast, damage to sites in the ventral pallidum and globus pallidus impaired grooming actions but left the sequential organization of grooming syntax intact. Neural circuits within this crucial "action syntax site" seem to implement sequential patterns of behavior as a specific function. PMID- 8627379 TI - Phasic firing of single neurons in the rat nucleus accumbens correlated with the timing of intravenous cocaine self-administration. AB - To examine potential neural mechanisms involved in cocaine self-administration, the activity of single neurons in the nucleus accumbens of rats was recorded during intravenous cocaine self-administration. Lever pressing was reinforced according to a fixed-ratio 1 schedule. On a time base comparable to the interinfusion interval, half the neurons exhibited phasic firing patterns time locked to the cocaine reinforced level press. For almost all neurons, this pattern consisted of a change in firing rate postpress, typically a decrease, followed by a reversal of that change. The postpress change was closely related to the lever press. Typically, it began within the first 0.2 min postpress and culminated within the first 1.0 min postpress. For a small portion of responsive neurons, the reversal of the postpress change was punctate and occurred within 1 3 min of either the last lever press or the next lever press so that firing was stable during much of the interinfusion interval. For the majority of neurons, the reversal was progressive; it began within 2 min after the previous level press, and it was not complete until the last 0.1-2.0 min before the next lever press. The duration of this progressive reversal, but not of the postpress change, was positively correlated with the interinfusion interval. Thus, in addition to exhibiting changes in firing related to the occurrence of self infusion, the majority of neurons also exhibited progressive changes in firing related to the spacing of infusions. In a structure that has been shown to be necessary for cocaine self-administration, such a firing pattern is a likely neurophysiological component of the mechanism that transduces declining drug levels into increased drug-related appetitive behavior. It is, thus, a neural mechanism that may contribute to compulsive drug-maintained drug taking. PMID- 8627380 TI - Ethanol self-administration restores withdrawal-associated deficiencies in accumbal dopamine and 5-hydroxytryptamine release in dependent rats. AB - Basal forebrain dopamine (DA) and 5-HT neurotransmission has been implicated in the mediation of the acute reinforcing actions of ethanol. Neuroadaptation theories predict that compensatory changes in neurochemical systems that are activated by alcohol acutely may underlie symptoms of withdrawal after chronic administration. To test this hypothesis, the release of DA and 5-HT was monitored by microdialysis in the nucleus accumbens of dependent male Wistar rats at the end of a 3-5 week ethanol (8.7% w/v) liquid diet regimen, during 8 hr of withdrawal, and during renewed availability of ethanol involving (1) the opportunity to operantly self-administer ethanol (10% w/v) for 60 min, followed by (2) unlimited access to the ethanol-liquid diet. Results were compared to control groups pair-fed with ethanol-free liquid diet and trained to self administer either ethanol or water. In nondependent rats, operant ethanol self administration increased both DA and 5-HT release in the NAC. Withdrawal from the chronic ethanol diet produced a progressive suppression in the release of these transmitters over the 8 hr withdrawal period. Self-administration of ethanol reinstated and maintained DA release at prewithdrawal levels but failed to completely restore 5-HT efflux. 5-HT levels recovered rapidly, however, within 1 hr of reexposure to ethanol liquid diet. These findings suggest that deficits in accumbal monoamine release may contribute to the negative affective consequences ethanol withdrawal and, thereby, motivate ethanol-seeking behavior in dependent subjects. PMID- 8627381 TI - Altered habituation of an identified escape circuit in Drosophila memory mutants. AB - Genetic approaches in Drosophila have advanced our understanding of the molecular mechanisms of different forms of learning, including habituation, but relevant neural components have not been explored. We show that a well defined neural circuit that underlies an escape response can be habituated, providing for the first time excellent opportunities for studying physiological parameters of learning in a functional circuit in the fly. Compared with other forms of conditioning, relatively little is known of the physiological mechanisms of habituation. The giant fiber pathway mediates a jump-and-flight escape response to visual stimuli. The jump may also be triggered electrically at multiple sites in the tethered fly. This response shows parameters of habituation, including frequency-dependent decline in responsiveness, spontaneous recovery, and dishabituation by a novel stimulus, attributable to plasticity in the brain. Mutations of rutabaga that diminish cAMP synthesis reduced the rate of habituation, whereas dunce mutations that increase cAMP levels led to a detectable but moderate increase in habituation rates. Surprisingly, habituation was extremely rapid in dunce rutabaga double mutants. This corresponds to the extreme defects seen in double mutants in other learning tasks, and demonstrates that defects of the rutabaga and dunce products interact synergistically in ways that could not have been predicted on the basis of simple counterbalancing biochemical effects. Although habituation is localized to afferents to the giant fiber, cAMP mutations also affected performance of thoracic portions of the pathway on a millisecond time scale that did not account for behavioral plasticity. More significantly, spontaneous recovery and dishabituation were not as clearly affected as habituation in mutants, indicating that these processes may not overlap entirely in terms of cAMP-regulating mechanisms. The analysis of habituation of the giant fiber response in available learning and memory mutants could be a crucial step toward realizing the promise of memory mutations to elucidate mechanisms in neural circuits that underlie behavioral plasticity. PMID- 8627382 TI - The echidna Tachyglossus aculeatus combines REM and non-REM aspects in a single sleep state: implications for the evolution of sleep. AB - Placental and marsupial mammals exist in three states of consciousness: waking, non-REM sleep, and REM sleep. We now report that the echidna Tachyglossus aculeatus, a representative of the earliest branch of mammalian evolution (the monotremes), does not have the pattern of neuronal activity of either of the sleep states seen in nonmonotreme mammals. Echidna sleep was characterized by increased brainstem unit discharge variability, as in REM sleep. However, the discharge rate decreased and the EEG was synchronized, as in non-REM sleep. Our results suggest that REM and non-REM sleep evolved as a differentiation of a single, phylogenetically older sleep state. We hypothesize that the physiological changes that occur during postnatal sleep development parallel certain aspects of the changes that have occurred during the evolution of sleep-waking states in mammals. PMID- 8627384 TI - Mesencephalic substrate of reward: axonal connections. AB - The behavioral version of the collision technique was used to study the existence of axonal linkage between reward-relevant sites in the ventral tegmental area (VTA) and posterior mesencephalon (PM) in six rats trained to self-administer trains of electrical brain stimulation. The combined use of fixed and moveable stimulation electrodes allowed us to carry out collision tests at a total of 46 different combinations of VTA-PM sites, and collision-like effects were observed at 24 of these. Stimulation of the VTA and the most caudal PM sites generally resulted in collision curves that were characterized by a single increase in paired-pulse effectiveness (E-values), whereas recovery in those collision curves obtained from stimulation of the VTA and more rostral PM sites was generally slower, and often characterized by a double rise. Despite little variability in interelectrode distances (1.0-3.8 mm), collision intervals varied widely, ranging from 1.5 to 7.3 msec. Recovery from refractoriness (initial 25%) was also estimated and ranged from 0.7 to 1.0 msec, resulting in conduction-time estimates of 0.7-6.3 msec. The lack of correspondence between interelectrode distances and conduction times suggests the presence of axonal branching. Results of this study constitute the first behavioral evidence of a reward-relevant axonal link between the VTA and the PM. In addition, the finding that in one animal the anterior electrode was located within the posterior portion of the lateral hypothalamus (LH) suggests that the reward-relevant axonal bundle linking the LH and VTA may extend as far back as the caudal regions of the PM. PMID- 8627383 TI - The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat. AB - Although much evidence suggests that the brain dopamine transporter (DAT) is susceptible to dopaminergic regulation, only limited information is available for the vesicular monoamine transporter (VMAT2). In the present investigation, we used a chronic, unlimited-access, cocaine self-administration paradigm to determine whether brain levels of VMAT2, as estimated using [3H]dihydrotetrabenazine (DTBZ) binding, are altered by chronic exposure to a dopamine uptake blocker. Previously, we showed that striatal and nucleus accumbens DAT levels, as estimated by [3H]WIN 35,428 and [3H]GBR 12,935 binding, are altered markedly using this animal model (Wilson et al., 1994). However, in sequential sections from the same animals, [3H]DTBZ binding was normal throughout the entire rostrocaudal extent of the basal ganglia (including striatum and nucleus accumbens), cerebral cortex, and diencephalon, as well as in midbrain and brainstem monoamine cell body regions, both on the last day of cocaine access and after 3 weeks of drug withdrawal. These data provide additional evidence that VMAT2, unlike DAT, is resistant to dopaminergic regulation. PMID- 8627386 TI - Chronic psychosocial stress causes apical dendritic atrophy of hippocampal CA3 pyramidal neurons in subordinate tree shrews. AB - We have shown previously that repeated laboratory restraint stress or daily corticosterone administration affects the structure of CA3 hippocampal neurons in rats. In the present study, we investigated the effect of repeated daily psychosocial stress on the structure of hippocampal CA3 pyramidal neurons in male tree shrews (Tupaia belangeri). Male tree shrews develop social hierarchies in which subordinates show characteristic changes in physiological and behavioral parameters when confronted with a dominant. In the present experiments, subordinate animals lost body weight soon after starting the daily social conflict, and urinary excretion of cortisol was elevated throughout the experiment as compared with the control period. Golgi-impregnated brain tissue from subordinates exposed to 28 d (1 hr/d) of social confrontations was compared with that from control nonstressed animals. The apical dendrites of the CA3 pyramidal cells from subordinates had a decreased number of branch points and total dendritic length as compared with controls. No differences were observed in apical dendritic spine density or in the basal dendritic tree morphology. The stress-induced CA3 apical dendritic atrophy in subordinates was prevented by administering daily oral doses of the antiepileptic drug phenytoin (Dilantin, Sigma, St. Louis, MO) (200 mg/kg), which interferes with excitatory amino acid (EAA) action. These results suggest that the naturalistic stressor psychosocial stress induces specific structural changes in hippocampal neurons of subordinate male tree shrews. These changes, like those in the rat after glucocorticoid treatment or restraint stress, probably are mediated by activation of the hypothalamo-pituitary-adrenal-axis acting in concert with endogenous EAAs from mossy fiber input. PMID- 8627385 TI - Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression. AB - In contrast to its inhibitory role in mature neurons, GABA can exert excitatory actions in developing neurons, including mediation of increases in cytosolic Ca2+. Modulation of this excitatory activity has not been studied previously. We used Ca2+ digital imaging with Fura-2 to test the hypothesis that neuropeptide Y (NPY) would depress GABA-mediated Ca2+ rises in neurons cultured from the developing suprachiasmatic nucleus (SCN). SCN neurons were chosen as a model system for this study because SCN neurons are primarily GABAergic, they express high levels of NPY and GABA receptors, and functionally, NPY causes profound phase-shifts in SCN-generated circadian rhythms. Vigorous GABA-mediated Ca2+ activity was found in young SCN neurons that were maintained in vitro for 4-14 d. NPY showed a dose-dependent rapid depression of the amplitude of Ca2+ rises generated by GABA released from presynaptic SCN axons. NPY exerted a long-term depression of cytosolic CA2+ in the majority of neurons tested, which lasted more than 1 hr after NPY washout. The magnitude of the NPY depression was dose dependent. NPY did not affect Ca2+ levels when GABAA receptor activity was blocked by bicuculline; however, when bicuculline and NPY were withdrawn from the perfusion solution, the subsequent CA2+ rise was either significantly reduced or completely absent, suggesting that the NPY receptor was activated in the absence of elevated intracellular Ca2+ and GABAA receptor activity, and that the latent effect of NPY was revealed only after depolarizing GABA stimulation was renewed. Pretreating neurons with pertussis toxin greatly reduced the ability of NPY to depress GABAergic Ca2+ rises, suggesting that the NPY modulation of the GABA activity was based largely on a mechanism involving pertussis toxin-sensitive Gi/Go proteins. NPY receptor stimulation depressed (< 30%) postsynaptic Ca2+ rises evoked by GABA (20 microM) application in the presence of tetrodotoxin (TTX). The effects of NPY were mimicked by the NPY Y1 receptor agonist [Pro34,Leu31] NPY and the Y2 receptor agonist NPY 13-36 and by peptide YY (PYY). Together, our data suggest that the Y1 and Y2 type NPY receptors act both presynaptically and postsynaptically to depress GABA-mediated Ca2+ rises. If related mechanisms exist in peptide modulation of inhibitory GABA activity in mature neurons, this could underlie long-term changes in the behavior of neurons of the SCN necessary for phase-shifting the circadian clock by NPY, NPY also modulated GABA responses in neuroendocrine neurons from the hypothalamic arcuate nucleus. NPY thus can play an important role in evoking long-term depression of GABA-mediated Ca2+ activity in these developing neurons, allowing NPY-secreting cells to modulate the effects of GABA on neurite outgrowth, gene expression, and physiological stimulation. This is the first example of such a cellular memory: that is, long-term Ca2+ depression based on modulation of depolarizing GABA activity. PMID- 8627387 TI - Selective effects of nerve growth factor on spatial recent memory as assessed by a delayed nonmatching-to-position task in the water maze. AB - Nerve growth factor (NGF) ameliorates age-related deficits in certain types of memory in rats. Although the effects of NGF on reference memory are well documented, the influence of NGF on recent memory is less well understood. The issue of recent memory is of primary importance in the design of therapies for cognitive disorders, because this type of memory is impaired in elderly humans and is severely affected early in the course of Alzheimer's disease (AD). The present study was designed to evaluate the efforts of NGF on recent memory in a task that used escape from water as the motivating stimulus and used the same design as forced-choice recognition tasks given to humans. Fischer-344 rats, 4 months old (4MO) or 23 months old (23MO), were pretested in a new spatial recent memory task designed for the Morris water maze, a delayed nonmatching-to-position task, and infused intraventricularly with recombinant human NGF or vehicle. After 2 weeks of NGF infusion, no substantial changes in behavior were observed in either age group. However, NGF treatment extended over 4 weeks improved considerably the choice accuracy of 23MO rats to a level similar to the performance of 4MO rats. These results, together with our previous work (Markowska et al., 1994), indicate that the effects of NGF on spatial recent memory are more intense than on spatial reference memory. NGF suppressed the body weight gain in 4MO rats but did not affect 23MO rats. In 23MO rats, NGF mildly counteracted age-related deficits in inhibitory avoidance, but did not have an effect in young rats. PMID- 8627388 TI - How wide is the gap in defining quality care? Comparison of patient and nurse perceptions of important aspects of patient care. AB - OBJECTIVES: The authors determine the importance that patients, nurses, and nurse managers place on aspects of care and measure nurses' care values based on their perceptions of their patients and nurse manager care values and their desire to meet these care expectations. BACKGROUND: The literature has documented gaps in how nurses and patients define quality and value specific care aspects, but little is known about the situation in the current continuous quality improvement and patient-centered care environment, which emphasizes a customer focus. Misunderstanding patients' values and expectations may impede service improvement. Information about any existing gaps could help managers begin to devise patient satisfaction improvement strategies. METHOD: Two thousand fifty one medical-surgical patients, 1264 staff members, and 97 nurse managers from 17 randomly selected hospitals participated in study activities related to selected aspects of patient care. Trained interviewers surveyed patients by telephone within 26 days of discharge using a pretested instrument. Staff members and managers completed a coordinated written tool. Descriptive and correlational statistics were used in individual and unit-level analyses. RESULTS: Staff members perceive correctly that patients value differently various aspects of care but do not agree with their managers on patients' value of aspects of care. Unit staff members' and managers' beliefs regarding patients' care values did not match those of their patients (-14 to 0.11 and -0.01 to 0.06 zero order correlations, respectively). CONCLUSIONS: A unit's errors in defining patients' values may be self-reinforcing. Strategies to reorient personnel, including adoption of those suggested by the diffusion of innovation literature, may help bridge the gap and change practice. PMID- 8627389 TI - Occupational stress and organizational commitment in nurse administrators. AB - OBJECTIVE: The authors explore levels of occupational stress (burnout) and organizational commitment (intent to stay), their inter-relationships, and their relationships to personal and organizational factors in nurse administrators. BACKGROUND: Although the concepts of burnout and turnover have been studied and reported for staff nurses, relatively few such studies exist in relation to nurse administrators. METHODS: Consequently, the Maslach Burnout Inventory, the Organizational Commitment Questionnaire, and a personal data sheet were completed by respondents to a mailed survey representing 65.6% of the membership of the state's nurse executive organization and 58.2% of the state's hospitals (N = 78). RESULTS: Scores indicate that half the respondents experience low levels of burnout whereas a third have high levels of burnout. Commitment scores were high for most nurse executives and were correlated inversely with burnout scale scores (depersonalization, personal accomplishment, emotional exhaustion), with the amount or phase of burnout, and with non-nursing management experience. Those with few opportunities to meet with colleagues scored higher on emotional exhaustion and lower on personal accomplishment scales. Intent to stay (high commitment scores) was related positively to perceived organizational support and to religiosity. CONCLUSIONS: Findings are consistent with those reported in other studies; however, the administrative role may require unique approaches to avoid or correct organizational stress. The role of social support, the determination of organizational/individual fit, and the adaptation of traditional organizational development techniques to the administrative role are discussed and proposed as areas for further study. PMID- 8627390 TI - Registered nurse empowerment. Model testing and implications for nurse administrators. AB - OBJECTIVE: The author developed and tested a model formulated to determine what combination of organizational, leadership, and personal characteristics maximize the prediction of registered nurse empowerment in an acute care hospital. BACKGROUND: Recent innovations in patient care delivery and nursing governance are more successful with an empowered nursing staff. However, the contexts for empowerment and management practices that foster empowerment are poorly understood. Previous empowerment studies suggest that personal characteristics such as age, experience, and education are related to empowerment. Additionally, specific leadership practices and organizational cultures have been shown to foster empowerment. METHODS: Registered nurses (N = 113) who were employed in an academic health center participated in the study. Participants completed measures of organizational culture, connective leadership, empowerment, and a demographic questionnaire designed to elicit the personal characteristics included in the model. RESULTS: Regression analysis identified constructive organizational culture and connective leadership as positive predictors of empowerment, whereas defensive culture was a negative predictor. The three variables combined to explain 45% of the variance in empowerment (P < 0.001). Personal characteristics were not significant predictors, but education and position level were significantly related to connective leadership (P < 0.01). CONCLUSIONS: The Registered Nurse Empowerment Model can serve as a framework for identifying and targeting areas requiring organizational change. The quantitative measures for organizational culture, connective leadership, and empowerment provide practical assessment and evaluation tools for data-based change. Education programs for nurses at all levels should address the behaviors and strategies that promote constructive cultures and connective leadership, minimize defensive cultures, and thus, foster empowerment of nursing staff. PMID- 8627391 TI - The interconnectedness of nurses' lives. Implications for nursing management. AB - OBJECTIVE: This study extends knowledge of how nurses' careers interact with their personal lives and professional development. BACKGROUND: Extant research suggests that nurses' career patterns are unstable or unplanned. However, these images may be a reflection of the models that are applied. Such models are overwhelmingly normative and do not reflect the actual life experience of nurses. METHODS: A series of interviews explored how nurses make changes in their lives. Questions focused on the stimulus for change and the resources used, problems associated with changes, and the effects of changes on the nurses themselves and others. RESULTS: The results show that nurses' careers, professional development, and personal lives are interconnected. CONCLUSIONS: The concept of interconnectedness reflects the confluence of events or people and their contemplation and integration by the nurses into their lives and relationships. The results contradict normative admonitions about career planning and have implications for the development of theories on nursing careers and for nurse managers. PMID- 8627392 TI - The Practice Environment Project. A process for outcome evaluation. AB - OBJECTIVE: Evaluating the practice environment is essential to determine if the practice environment is conducive to the delivery of quality patient care. The Practice Environment Project (PEP) was created to provide a framework for current and ongoing evaluation of the practice environment. BACKGROUND: Major changes in the delivery of patient care services have focused attention on the evaluation of the effectiveness of these changes. The extent and type of change may vary throughout an institution. The identification of core variables to assess the effectiveness of these changes sets the framework for ongoing evaluation of new unit-based models for delivery of services. METHODS: In part 1, quantitative data were collected from nursing staff members (job satisfaction, collaboration with physicians, autonomy), physicians (quality of nursing care, collaboration with nurses), and patients (satisfaction with nursing care). In part 2, focus groups were held with nursing personnel to discuss factors that affected the provision of services. Unit-based action plans were developed to manage barriers to the delivery of services. RESULTS: Patients and physicians reported a high degree of satisfaction with patient care. Physicians reported a higher level of collaboration with nurses than that reported by nurses. Nurses reported a high degree of autonomy in practice, however, in other areas of job satisfaction (development and recognition), they suggested areas for improvement. CONCLUSIONS: The PEP created a mechanism to evaluate the current state-of-the-practice environment by identifying core elements for evaluation of work redesign. It also provided a framework for managing barriers that disrupted the delivery of patient care services. PMID- 8627393 TI - Nursing administration. Its time has come. PMID- 8627394 TI - The effect of a professional practice model on staff nurse perception of work groups and nurse leaders. AB - OBJECTIVE: The objective of the study was to measure the effect of an enhanced professional practice model on perception of work groups and nurse leaders. BACKGROUND: Previous studies of staff nurse perception of work environment and nurse leaders were used to guide development of the professional practice model and the selection of variables. Implementation of the model was expected to result in more favorable perceptions of the work group and a desire for facilitative leadership style. METHODS: A quasi-experimental design was used to compare change over time in five-hospitals--an urban medical center, two community hospitals, and two rural hospitals. FINDINGS: Introduction of the model resulted in more favorable perceptions about the work group and a desire for a more facilitative nurse leader. CONCLUSION: Change in perception of work group and leader rather than job satisfaction may be an early indication of favorable outcome of planned change. Changes in nurse satisfaction may be a late outcome, which may be the reason findings from previous studies are mixed. PMID- 8627395 TI - Integrity and misconduct in research. PMID- 8627396 TI - The nature of nursing administration research. Knowledge building or fire stomping? PMID- 8627397 TI - Breastfeeding. PMID- 8627398 TI - Transforming prenatal care: reflections on the past and present with implications for the future. AB - The current model for delivery of prenatal care was developed more than 100 years ago. Evidence suggests that this model is no longer appropriate for meeting national health objectives or for meeting the needs of a diverse population of pregnant women. This article provides a historical overview of prenatal care; describes the current system for care delivery and problems associated with it; and suggests strategies for transforming care into an effective, comprehensive model. PMID- 8627399 TI - A nurse's guide to hormone replacement therapy. AB - Menopause is a natural event most women experience as they enter their 5th decade. As human life expectancy has lengthened, health issues concerning women in mid-life have become a major focus in holistic, preventive health care, which is heavily influenced by nurses. Controversy continues about the risks and benefits of hormone replacement therapy for women during their years of perimenopause and postmenopause. Evidence is compiling, however, that indicates the benefits of exogenous hormones may outweigh these concerns. As nurses, we must have a current knowledge of hormone replacement therapy to counsel our patients effectively. This article presents information to assist the nurse in meeting this goal. PMID- 8627400 TI - Nursing management of the infant with a congenital malignancy. AB - Care of the newborn diagnosed with a congenital malignancy is a challenge for the neonatal intensive-care unit nurse. Malignancies found in infants differ from those found in older children. Nursing care of the neonate suspected or diagnosed with congenital malignancy includes standard practices and problem identification as well as interventions unique to the patient with cancer. This article reviews the incidence, diagnosis, treatment, and nursing management of neoplasms diagnosed in neonates. PMID- 8627401 TI - Predictors of weight gain at 6 and 18 months after childbirth: a pilot study. AB - OBJECTIVE: To test the contributions of life-style and stress to postpartum weight gain after controlling for sociodemographic and reproductive influences. DESIGN: Longitudinal mail survey with retrospective data on gestational weight gain and prospective data on postpartum weight gain. SETTING: Multicounty community in the midwestern United States. PARTICIPANTS: After deleting from the sample women who became pregnant again, had confounding medical conditions, or had missing weight data, the sample consisted of 88 predominantly white mothers at 6 months after childbirth and 75 predominantly white mothers at 18 months after childbirth. MAIN OUTCOME MEASURES: Weight gain at 6 and 18 months after childbirth. RESULTS: Maternal race and gestational weight gain accounted for significant amounts of variance in 6-month and 18-month postpartum weight gain. Neither life-style nor perceived stress contributed significantly to predicting postpartum weight gains. Gestational weight gain was the most important predictor of postpartum weight gain. CONCLUSIONS: Given the contribution of gestational weight gain to postpartum weight gain, further study is needed of high gestational weight gain. PMID- 8627402 TI - Pregnancy outcomes among active and sedentary primiparous women. AB - OBJECTIVE: To describe the relationship between exercise during the last trimester of pregnancy and physiologic outcomes of mothers and newborns to determine whether differences exist between active and sedentary exercise patterns on these variables. DESIGN: Nonexperimental, descriptive. SETTING: Physicians' offices, Lamaze classes, and hospitals. PARTICIPANTS: One hundred one primiparous women (48 sedentary and 53 active), 20-30 years of age, with no medical complications, whose length of gestation was at least 32 weeks. OUTCOME MEASURES: Weight gain, weeks of gestation, length of labor, common discomforts of pregnancy, occurrence of cesarean section, birth weight, and Apgar scores. RESULTS: Brisk walking was the preferred physical activity among 47% of the participants. Regular, active exercise had no significant effect on maternal weight gain or neonate birth weight. Twenty-five percent of the women in the active exercise group reported five or fewer discomforts compared with 6% of the women in the sedentary group (X2 = 7.45, p < 0.01). For the discomforts of swelling, leg cramps, fatigue, and shortness of breath, 44% of the women in the active group reported having only one or two of them, compared with 21% of the women in the sedentary group (X2 = 6.15, p = 0.01). CONCLUSION: Women who engaged in active exercise during the last trimester of pregnancy had fewer of the common discomforts associated with pregnancy. PMID- 8627403 TI - Loneliness and social support in infertile couples. AB - OBJECTIVE: To determine differences between infertile wives' and husbands' levels of loneliness and perception of social support and to determine if there is a relationship between loneliness and social support. DESIGN: Comparative descriptive. SETTING: University infertility clinic and RESOLVE, an infertility support group. PARTICIPANTS: Convenience sample of 62 couples diagnosed as experiencing either primary or secondary infertility. OUTCOME MEASURES: Loneliness was measured using the Revised UCLA Loneliness Scale; social support was measured using the Interpersonal Relationship Inventory with subscales of Social Support, Reciprocity, and Conflict. RESULTS: Wives were significantly more lonely than husbands (t = 2.053, p = 0.04). There was no significant group difference on the social support total score or three subscale scores. Loneliness was inversely related to social support and reciprocity both for wives (r = 0.62, p = 0.001, and r = -0.50, p = 0.002, respectively) and husbands (r = -0.74, p = 0.001, and r = -0.56, p = 0.001, respectively); loneliness correlated with conflict for wives only (r = 0.48, p = 0.007). CONCLUSIONS: Although wives and husbands differed in loneliness, they were similar in perceived social support. Greater social support and reciprocity lessened feelings of loneliness for both groups. Wives who perceived increased conflict in their social relationships were more lonely. PMID- 8627404 TI - Adolescent condom use, the health belief model, and the prevention of sexually transmitted disease. AB - OBJECTIVE: To review factors associated with adolescent condom use for the prevention of sexually transmitted diseases (STDs). DATA SOURCES: Thirty-six references on adolescent development, STDs, STD risk factors, and factors influencing condom use among heterosexual adolescents. STUDY SELECTION: Twenty two research articles addressing issues influencing condom use by heterosexual adolescents. DATA SYNTHESIS: This review identifies unique risk factors related to adolescents and their risk for STDs, barriers to and facilitators of condom use, and suggestions for health care providers to increase condom use among adolescents. CONCLUSIONS: Although research on condom use among adolescents has inherent difficulties, current findings, along with reported high rates of STDs in this population, indicate infrequent and inconsistent condom use. Many factors contribute to the motivation for condom use and should be assessed individually. Using the Health Belief Model as a theoretical framework, health care providers can guide the adolescent to make realistic risk assessments and identify positive ways of incorporating condoms into their sexual lives. Further research then must be conducted to test the effectiveness of this approach. PMID- 8627405 TI - Cultural aspects of pain in childbearing women. AB - As the American population increases in ethnic diversity, nurses must prepare to care for women from various cultures. The American Nurses Association has stated that a knowledge of cultural diversity is vital at all levels of nursing. Culture is known to affect the patient's perception of pain and the nurse's inference of pain in the patient. Pain is expected in childbirth, and nurses should learn how cultures influences individual women in their expression of pain. Because each women is unique, nurses must combine information about culture with clinical assessment of the patient to provide culturally sensitive care. PMID- 8627406 TI - The discomforts of pregnancy. AB - During pregnancy, women may experience one or more of a wide variety of discomforts. Every pregnancy is different; discomforts felt during one pregnancy may not appear in another. Most discomforts experienced during pregnancy are thought to be the result of abundant hormonal changes. As pregnancy progresses, other discomforts are attributed to physical changes associated with the enlarging uterus. Selected discomforts of pregnancy are presented with suggestions for clinical management. Nurses can do something to decrease discomforts associated with pregnancy. PMID- 8627407 TI - The pain and discomfort of labor and birth. AB - One unique aspect of childbirth is the association of this physiologic process with pain and discomfort. However, the experience of pain during labor is not a simple reflection of the physiologic processes of parturition. Instead, labor pain is the result of a complex and subjective interaction of multiple physiologic and psychological factors on a woman's individual interpretation of labor stimuli. An understanding of labor pain in a multidimensional framework provides the basis for a woman-centered approach to labor pain management that includes a broad range of pharmacologic and nonpharmacologic intervention strategies. PMID- 8627408 TI - Drive-through deliveries. PMID- 8627409 TI - Spatiotemporal adaptation model for retinal ganglion cells. AB - An adaptation model for the level of the ganglion cell in the retina is presented. The model assumes separate adaptation mechanisms for each of the receptive field (RF) regions, i.e., before edge detection. According to the model, the decay in the response time course of each RF region reflects its adaptation process. A mathematical description of adaptation that includes its temporal properties is developed through the change in the semisaturation constant theta in the Naka-Rushton equation. The model and its simulations show a good agreement with a wide variety of physiological studies. PMID- 8627410 TI - Luminosity thresholds: effects of test chromaticity and ambient illumination. AB - Color constancy is often modeled on the assumption that color appearance in natural scenes is a function of the visual system's estimates of surface reflectance. Some stimuli, however, do not look like illuminated surfaces. Instead, they appear to be self-luminous. We hypothesized that the appearance of luminosity occurs when the visual system estimates a reflectance spectrum that is outside the gamut of physically realizable surfaces. To test this idea, we measured luminosity thresholds as a function of stimulus chromaticity and illuminant spectral power distribution. Observers adjusted the luminance of a test patch until it just appeared self-luminous. The test patch was spot illuminated by a computer-controlled projection colorimeter viewed in an experimental room lit diffusely by computer-controlled theater lamps. Luminosity thresholds were determined for a number of test patch chromaticities under five experimental illuminants. The luminosity thresholds define a surface in color space. The shape of this surface depends on the illuminant. We were able to describe much of the luminosity threshold variation with a simple model whose parameters define an equivalent illuminant. In the context of our model, the equivalent illuminant may be interpreted as the illuminant perceived by the observer. As part of our model calculations we generalized the classic notion of optimal stimuli by incorporating linear-model constraints. Given the equivalent illuminant, the model predicts that a patch will appear self-luminous when it is not consistent with any physically realizable surface seen under that illuminant. In addition, we show that much of the variation of the equivalent illuminant with the physical illuminant can be modeled with a simple linearity principle. The fact that our model provides a good account of our data extends the physics-based approach to judgements of self-luminosity. This in turn might be taken as support for the notion that the visual system has internalized the physics of reflectance. PMID- 8627411 TI - Current-source density analysis of the electroretinogram of the frog: methodological issues and origin of components. AB - The technique of current-source density (CSD) analysis for extracellular potentials is reviewed, along with some methodological features that are important for performing CSD analysis of the electroretinogram. In addition, three formulas for computing CSD's are examined on model circuits of resistors and current generators. Finally, CSD results from frog retina that bear on the origins of the b, d, and M waves, along with slow PIII, are presented. It is concluded that the b and d waves are generated primarily and directly by bipolar cells, whereas the M wave and the slow PIII are generated by Muller (glial) cells through the K+ spatial buffer mechanism. PMID- 8627412 TI - Inner retinal contributions to the primate photopic fast flicker electroretinogram. AB - The primate electroretinogram (ERG) recorded at the cornea in response to fast flickering light is thought to reflect primarily the cone photoreceptor potential. We investigated the origin of the photopic 33-Hz corneal flicker ERG to square-wave and photostrobe flashes by recording in the monkey before and after blocking postsynaptic responses with intravitreal injections of 2-amino-4 phosphonobutyric acid and/or cis-2,3-piperidiendicarboxylic acid or sodium aspartate. Blocking postsynaptic ON or OFF responses produced effects on the timing and the waveform of the 33-Hz flicker ERG similar to changes in the b and the d waves in the corneal single-flash ERG. When all the ERG waves of postsynaptic origin in the flash ERG were abolished the flicker response was greatly suppressed, suggesting the postsynaptic cells producing the b and the d waves make major contributions to the photopic fast flicker ERG. PMID- 8627413 TI - Photoreceptor function in infants and children with a history of mild retinopathy of prematurity. AB - Five infants and children with a history of mild retinopathy of prematurity (ROP) were tested for postulated alterations in rod photoreceptor function. The photoreceptor responses were derived from the electroretinographic alpha waves. Postreceptoral components, the beta wave and the oscillatory potentials, were also examined. The saturated amplitude and sensitivity of the rod photoreceptor responses were low, except for the sensitivity in one patient. The beta-wave sensitivity was low, but saturated amplitudes were within the 95% prediction interval for normal. The amplitudes of the oscillatory-potential responses were also attenuated. The results indicate that retinal dysfunction may be present in patients with a history of mild ROP long after the ROP has completely resolved. Additionally, the data suggest that the photoreceptors are the primary site of retinal dysfunction in mild ROP. PMID- 8627414 TI - Rod photoreceptor transduction is affected in central retinal vein occlusion associated with iris neovascularization. AB - Patients with central retinal vein occlusion who later develop iris neovascularization show changes in the amplitude, the timing, and the sensitivity of the electroretinogram beta wave. We determined the extent to which rod photoreceptor dysfunction contributes to these changes by recording single-flash electroretinograms from both eyes of 52 patients with unilateral central retinal vein occlusion and fitting the leading edges of alpha waves with a model of rod phototransduction. Eyes with central retinal vein occlusion showed reductions in photoreceptor gain but no changes in photoreceptor amplitude when compared with the fellow eyes. The reductions were larger in eyes that developed iris neovascularization and were predictive of this complication. Photoreceptor gain reductions accounted for only part of the beta-wave timing delays and sensitive loss; the remainder is attributed to functional loss in the inner nuclear layer. PMID- 8627415 TI - Rod phototransduction in transgenic mice expressing a mutant opsin gene. AB - Rod-mediated electroretinograms (ERG's) were recorded from transgenic mice expressing a mouse opsin gene with three point mutations (V20G, P23H, and P27L; termed VPP mice) and from normal littermates. The leading edge of the alpha wave was analyzed in relation to a computational model of rod phototransduction [J. Physiol. 499, 719 (1992)], in which values for the maximum response (RmP3), transduction gain (S), and transduction delay (td) are derived from alpha-wave data. VPP mice exhibited an age-related decrease in RmP3. This decrease was consistent with reductions in the number of rod photoreceptors and in the length of rod outer segments observed in previous histological studies of the VPP retina. Values of S determined for the VPP mice were within the normal range, consistent with a normal amplification of the visual signal in VPP rods. At high stimulus intensities, both normal and VPP mice exhibited a decrease in S, which may reflect depletion of a phototransduction substrate at these stimulus levels. We examined the recovery of the alpha wave after a bright conditioning flash by measuring the rod alpha-wave response to a probe flash presented at varying times after the conditioning stimulus. In both normal and VPP mice a fourfold (0.6-log unit) increase in conditioning stimulus intensity increased both T50%, the period required for half-maximal recovery, and tau, the exponential time constant describing recovery. However, the increases in T50% and tau were significantly greater in VPP mice, indicating an abnormally slow recovery of the flash response in VPP rods. PMID- 8627416 TI - Recovery kinetics of human rod phototransduction inferred from the two-branched alpha-wave saturation function. AB - Electroretinographic data obtained from human subjects show that bright test flashes of increasing intensity induce progressively longer periods of apparent saturation of the rod-mediated electroretinogram (ERG) alpha wave. A prominent feature of the saturation function [the function that relates the saturation period T with the natural logarithm of flash intensity (ln I(f)] is its two branched character. At relatively low flash intensities (I(f) below approximately 4 x 10(4) scotopic troland second), T increases approximately in proportion to ln I(f) with a slope [delta T/delta (ln I(f)] of approximately 0.3 s. At higher flash intensities, a different linear relation prevails, in which [deltaT/delta(ln I(f) is approximately 2.3 s [Invest. Ophthalmol. Vis. Sci. 36, 1603 (1995)]. Based on a model for photocurrent recovery in isolated single rods [Vis. Neurosci. 8, 9 (1992)], it was suggested that the upper-branch slope of approximately 2.3 s represents tau R*, the lifetime of photoactivated rhodopsin (R*). Here we show that a modified version of this model provides an explanation for the lower branch of the alpha-wave saturation function. In this model, tau E* is the exponential lifetime of an activated species (E*) within the transducin or guanosine 3', 5'-cyclic monophosphate (cGMP) phosphodiesterase stages of rod phototransduction; the generation of E* by a single R* occurs within temporally defined, elemental domains of disk membrane; and Ex, the immediate product of E* deactivation, is converted only slowly (time constant tau Ex) to E, the form susceptible to reactivation by R*. The model predicts that the decay of flash activated cGMP phosphodiesterase (PDE*) is largely independent of the deactivation kinetics of R* at early postflash times (i.e., at times preceding or comparable with the lifetime tau E*) and that the lower-branch slope (approximately 0.3s) of the a-wave saturation function represent tau E*. The predicted early-stage independence of PDE* decay and R* deactivation furthermore suggests a basis for the relative constancy of the single-photon response observed in studies of isolated rods. Numerical evaluation of the model yields a value of approximately 6.7s for the time constant tau Ex. PMID- 8627417 TI - Effects of background light on the human dark-adapted electroretinogram and psychophysical threshold. AB - We compared the effects of background light on the sensitivities of two components of the human electroretinogram, the cornea-negative scotopic threshold response (STR) and the cornea-positive PII (beta wave), as well as on the psychophysical sensitivity in a ganzfeld. The background illuminance necessary to reduce the STR (an inner retinal signal) measurably was approximately five times greater than that needed to raise the psychophysical threshold. A background illuminance at least 1 log unit greater still was needed to reduce PII (a signal reflecting activity of bipolar cells). These findings suggest (1) that the weakest backgrounds that reduce retinal sensitivity have their effect at a site that is proximal to the bipolar cells, a site that involves amacrine or ganglion cells, and (2) that very weak backgrounds have their effect on visual sensitivity at a site more proximal than the scotopic threshold response generator and perhaps more central than the retina. PMID- 8627418 TI - Photoreceptor and bipolar cell contributions to the cat electroretinogram: a kinetic model for the early part of the flash response. AB - The time course of the initial negative wave of the flash electroretinogram of the dark-adapted cat has been found to be critically dependent of contributions from cells of the inner retina, not only for very low-intensity flashes for which the negative scotopic threshold response is dominant but also when the stimulus is sufficiently intense for the rods themselves to contribute directly to the electroretinogram. However, if the inner-retinal responses are blocked pharmacologically or are suppressed by a steady adapting background, the initial negative wave of the remaining electroretinogram (the alpha wave) can be explained as the sum of photoreceptor and bipolar-cell components that can be modeled as described by Lamb and Pugh [J. Physiol. (London) 449, 717 (1992)] and Robson and Frishman [Vis. Neurosci. 12, 837 (1995)], respectively. PMID- 8627420 TI - Imaging localized retinal dysfunction with the multifocal electroretinogram. AB - Conventional electroretinographic techniques do not permit efficient mapping of retinal responsiveness for the detection of small dysfunctional areas. This study explores the application of a new technique that makes such mapping possible. It utilizes a multifocal electroretinogram technique based on binary m sequences that simultaneously tests a large number of small retinal areas by multiplexing their responses onto a single signal derived from the human cornea. The focal responses are subsequently extracted for the derivation of high-resolution maps that characterize retinal responsiveness. The required recording times are short enough to make such testing feasible in the clinic. In this study we demonstrate the high sensitivity of the technique by mapping a small area that has been partially bleached by a strobe flash in a normal retina and by mapping dysfunctional areas in three patients with different, well-documented retinal pathologies. The results suggest that the multifocal electroretinogram has the potential to become a valuable clinical tool. PMID- 8627419 TI - Beta wave of the scotopic (rod) electroretinogram as a measure of the activity of human on-bipolar cells. AB - The beta wave of the human electroretinogram (ERG) is widely believed to reflect the activation of on-bipolar cells. However, the shape of the beta wave is also influenced by the activity of other cell types. To assess how the activity of on bipolar cells is reflected in the human ERG, rod ERG's were recorded in the dark and on the steady fields. Derived P2 responses were obtained by computer subtraction of the receptor contribution to the ERG. The light-adapted derived P2 was shown to have properties similar to those predicted from previous studies of on-bipolar activity. This was also true of the dark-adapted derived P2 if a small (less than 10%) contribution from a negative potential was taken into consideration. The derived P2, and under certain conditions the beta wave, can be used to study rod on-bipolar activity. PMID- 8627421 TI - Electroretinogram flicker photometry and its applications. AB - The electroretinogram (ERG) has been a traditional tool for the measurement and the analysis of spectral sensitivity. With the appropriate choices of stimulus and measurement conditions, the ERG permits a noninvasive examination of photopigment complement and provides the means for studying the combination of spectral signals at various locations throughout the retina. There are a number of practical problems associated with making spectral measurements with the ERG. One approach to minimizing these problems is to exploit the advantages of a flicker-photometric procedure. We summarize a method used to conduct ERG flicker photometry and illustrate a range of problems to which this technique can be successfully applied. PMID- 8627422 TI - Analysis of retinal light adaptation with the flicker electroretinogram. AB - To study the retinal light adaptation we measured and analyzed the flicker electroretinogram response to stimuli that varied in temporal frequency, retinal illuminance, and modulation depth. The responses measured at 100% modulation showed the classic adaptation pattern, being independent of mean retinal illuminance at low temporal frequencies, consistent with Weber adaptation, and increasing in proportion to mean retinal illuminance at high temporal frequencies, consistent with linearity. At 25% modulation, however, high frequency linearity was not found. The response amplitude consistently showed a minimum at 40-48 Hz. When modulation was systematically varied, response amplitudes measured at 16 and 22 Hz showed Weber adaptation at all modulations and response phase was relatively constant with modulation, whereas response amplitudes at 40 and 48 Hz showed adaptation at low modulations but linearity at high modulations and response phase varied with modulation. We conclude that retinal gain controls also operate at high temporal frequencies. PMID- 8627423 TI - Outer-retina locus of increased flicker sensitivity of the peripheral retina. AB - We tested alternative hypotheses concerning the locus of enhanced flicker sensitivity observed in response to stimuli presented to the peripheral retina. The first hypothesis attributes increased temporal frequency sensitivity to ganglion cell and higher-order neural processing, whereas the second hypothesis states that the locus of these temporal effects is at the cone photoreceptors. To test these alternative hypotheses we measured retinal electrophysiological and psychophysical temporal modulation thresholds. We found that sensitivity for temporal frequencies < 30 Hz did not vary as a function of retinal location for either the focal electroretinogram (ERG) or the psychophysical measure. However, for both measures, sensitivity for temporal frequencies > or = 30 Hz was greater in the peripheral retina than in the central retina. In addition, critical flicker frequency for the central retina was linear as a function of retinal illuminance for both the psychophysical and the electrophysiological measures. For the peripheral retina the slopes of critical flicker frequency versus log illuminance functions were steeper than the central slopes for both threshold measures. Eccentrically measured focal ERG and psychophysical critical flicker frequency values showed a relative saturation, deviating from the linear slope above 3.5 log Td. The findings of similar focal ERG and psychophysical temporal sensitivity changes with eccentricity support an outer retinal origin of this phenomenon. PMID- 8627424 TI - Response of the retina at low temporal frequencies. AB - We investigated the low-frequency temporal response of the retina by measuring the corneal electroretinogram elicited by flickering lights. A sum of two temporal sine-wave modulations was used to generate difference frequencies between a 36-Hz standard stimulus and a series of low-frequency stimuli. The response of the retina at the difference frequency did not change as the low frequency component of the stimulus was varied from 0.5 to 4 Hz. We also replicated an earlier study, stimulating the retina with a sum of two sine waves that were varied in average frequency but keeping the difference frequency constant. These data showed no change in the amplitude of the difference frequency as the average stimulus frequency was varied from 8 to almost 40 Hz. Taken together, the two sets of data support the notion that the in vivo early retinal response is low pass and extends without attenuation to frequencies greater than 30 Hz, in contrast to the sensitivity of the visual system measured by psychophysical techniques. PMID- 8627425 TI - Raising couch potatoes (little spuds and how they grew--and grew) PMID- 8627426 TI - An ounce of prevention. PMID- 8627427 TI - Prone sleeping in healthy infants and victims of sudden infant death syndrome. PMID- 8627428 TI - Expanding spectrum of mitochondrial disorders. PMID- 8627429 TI - Etiology of nutritional rickets: geographic variations. PMID- 8627430 TI - Recommendations for the postnatal use of indomethacin: an analysis of four separate treatment strategies. PMID- 8627431 TI - Increased incidence of non-insulin-dependent diabetes mellitus among adolescents. AB - OBJECTIVE: To determine whether a rise in the diagnosis of non-insulin- dependent diabetes mellitus (NIDDM) has accompanied the rise in obesity in the pediatric population, as it has among adults. STUDY DESIGN: Medical records of 1027 consecutive patients from birth to age 19 years with a diagnosis of diabetes from 1982 to 1995 at a regional, university-affiliated pediatric diabetes referral center were reviewed and classified according to criteria of the National Diabetes Data Group. RESULTS: The number of patients with a diagnosis of NIDDM rose from approximately 4% of new diagnoses of diabetes in patients from birth to age 19 years before 1992, to 16% in 1994. Among patients 10 to 19 years of age, NIDDM accounted for 33% of diagnoses of diabetes in 1994. The incidence of adolescent NIDDM in Greater Cincinnati increased tenfold, from 0.7/100,000 per year in 1982 to 7.2/100,000 per year in 1994. The mean (+/- SD) age and body mass index at presentation were 13.8 +/- 1.9 years and 37.7 +/- 9.6 kg/m2, respectively. The overall female/male ratio was 1.7:1, and female patients were seen 1 year earlier than male patients (p < 0.01). Male subjects had a higher body mass index than female subjects (p < 0.05). A first-degree relative with NIDDM was identified for 65% of patients. At presentation, 21% of the patients had had a diagnosis of at least one other condition associated with obesity. CONCLUSION: There is an increasing incidence of NIDDM among adolescents in Greater Cincinnati, accompanying the national rise in adolescent obesity. Obesity and strong family histories of NIDDM are important risk factors. Because NIDDM leads to long-term morbidity, the prevention of obesity as well as early identification of overt disease, is critical. PMID- 8627432 TI - Face-straight-down and face-near-straight-down positions in healthy, prone sleeping infants. AB - OBJECTIVE: To determine the frequency and physiologic consequences of the face straight-down (FSD) position, a postulated mechanism for the sudden infant death syndrome in prone-sleeping infants. STUDY DESIGN: A survey of 151 infants, aged 1 to 7 months, in Montreal showed that 33% slept prone. Ten healthy prone-sleeping infants were studied in their homes at age 10 to 22 weeks. Infrared video and cardiorespiratory recordings were made on 3 consecutive nights in the prone (nights 1 and 3) and lateral (night 2) positions. RESULTS: Infants maintained the prone position during 17 of 19 studies, but only 4 of 9 infants maintained the lateral position. The FSD position was observed 27 times in 17 prone nights: median frequency, 0.6 times per night (interquartile range, 0 to 4), and median total duration, 3.3 minutes (0.8% of total sleep time). A related position, the face-near-straight-down (FNSD) position, occurred more often, 5.3 (1 to 10) time per prone night, for 22.4 minutes (5.8% of total sleep time). Most periods in the FSD and FNSD position had no physiologic consequences; however, 14% of FSD and 3% of FNSD episodes were associated with airway obstruction as indicated by snoring, paradoxical respiratory movements, apnea, and/or increased partial pressure of transcutaneous carbon dioxide. Spontaneous arousal and head turning terminated the FSD and FNSD episodes. CONCLUSION: The FSD and FNSD positions occur commonly in healthy prone-sleeping infants, and these positions can cause airway obstruction. We speculate that those infants with sudden infant death syndrome found in the FSD or FNSD position either have a congenital or an acquired defect in the arousal-head turning response or have encountered insurmountable environmental factors that prevent effective head turning. PMID- 8627433 TI - Prone sleep position and the sudden infant death syndrome in King County, Washington: a case-control study. AB - OBJECTIVE: To determine whether the prone sleep position was associated with an increased risk of the sudden infant death syndrome (SIDS). STUDY DESIGN: Population-based case-control study. PARTICIPANTS: Case subjects were infants who died of SIDS in King County, Washington. Control subjects were randomly selected infants born in King County. Up to four control subjects were matched on date of birth to each case subject. METHODS: During the study period, November 1992 through October 1994, sleep-position data were collected on infants who died of SIDS by the King Count Medical Examiner's Office during their investigation of the deaths. Parents of infants chosen as control subjects were contacted by telephone, and sleep position information was obtained. Infants who usually slept on their abdomen were classified as sleeping prone; those who usually slept on the side or back were categorized as sleeping nonprone. The adjusted odds ratio for prone sleep position as a risk factor for SIDS was calculated with conditional logistic regression after control for race, birth weight, maternal age, maternal marital status, household income, and maternal cigarette smoking during pregnancy. RESULTS: Sleep position data were collected on 47 infants with SIDS (77% of eligible infants) and 142 matched control subjects; 57.4% of infants who died of SIDS usually slept prone versus 24.6% of control subjects (p < 0.00001). The unadjusted odds ratio for prone sleep position as a risk factor for SIDS was 4.69 (95% confidence interval: 2.17, 10.17). After control for potentially confounding variables, the adjusted odds ratio for prone sleep position was 3.12 (95% confidence interval: 1.08, 9.03). CONCLUSION: Prone sleep position was significantly associated with an increased risk of SIDS among a group of American infants. PMID- 8627434 TI - Prophylactic indomethacin therapy in the first twenty-four hours of life for the prevention of patent ductus arteriosus in preterm infants treated prophylactically with surfactant in the delivery room. AB - OBJECTIVE: To determine whether a course of low-dose indomethacin therapy, when initiated within 24 hours of birth, would decrease ductal shunting in premature infants who received prophylactic surfactant in the delivery room. DESIGN: Ninety infants, with birth weights of 600 to 1250 gm, were entered into a prospective, randomized, controlled trial to receive either indomethacin, 0.1 mg/kg per dose, or placebo less than 24 hours and again every 24 hours for six doses. Echocardiography was performed on day 1 before treatment and on day 7, 24 hours after treatment. A hemodynamically significant patent ductus arteriosus (PDA) was confirmed with an out-of-study echocardiogram, and the nonresponders were treated with standard indomethacin or ligation. RESULTS: Forty-three infants received indomethacin (birth weight, 915 +/- 209 gm; gestational age, 26.4 +/- 1.6 weeks; 25 boys), and 47 received placebo (birth weight, 879 +/- 202 gm; gestational age, 26.4 +/- 1.8 weeks; 22 boys) (P = not significant). Of 90 infants, 77 (86%) had a PDA by echocardiogram on the first day of life before study treatment; 84% of these PDAs were moderate or large in size in the indomethacin-treated group compared with 93% in the placebo group. Nine of forty indomethacin-treated infants (21%) were study-dose nonresponders compared with 22 (47%) of 47 placebo treated infants (p < 0.018). There were no significant differences between both groups in any of the long-term outcome variables, including intraventricular hemorrhage, duration of oxygen therapy, endotracheal intubation, duration of stay in neonatal intensive care unit, time to regain birth weight or reach full caloric intake, incidence of bronchopulmonary dysplasia, and survival. No significant differences were noted in the incidence of oliguria, elevated plasma creatinine concentration, thrombocytopenia, pulmonary hemorrhage, or necrotizing enterocolitis. CONCLUSION: The prophylactic use of low doses of indomethacin, when initiated in the first 24 hours of life in low birth weight infants who receive prophylactic surfactant in the delivery room, decreases the incidence of left-to-right shunting at the level of the ductus arteriosus. PMID- 8627435 TI - Cardiorespiratory function before and after corrective surgery in pectus excavatum. AB - OBJECTIVE: To determine whether pectus excavatum (PE) results in cardiopulmonary abnormalities, and whether surgical repair results in improvement. METHODS: We performed pulmonary function testing and incremental exercise testing in 36 adolescents with PE (aged 16 +/- 3 (SD) years) and 10 age-matched, healthy control subjects. Fifteen PE subjects were reexamined postoperatively, as were six control subjects. RESULTS: Preoperatively, PE subjects had a significantly lower forced vital capacity than control subjects had (81% +/- 14% vs 98% +/- 9% of the predicted value; p < 0.001). Chest computed tomography ratios of internal transverse to antero-posterior diameters correlated inversely with total lung capacity (r = 0.56; p < 0.01). Fifty-eight percent of PE subjects had subjective complaints of exercise limitation. PE subjects exercised at a workload similar to that of control subjects. Maximal heart rate and O2 pulse did not differ between the two groups. Respiratory measurements during exercise were similar between the two groups. Respiratory measurements during exercise were similar between the two groups. Postoperatively there was no change in forced vital capacity (as a percentage of the predicted value). The PE subjects exercised for a slightly longer period and had a slightly higher O2 pulse, whereas control subjects showed no change. CONCLUSION: Some subjects with PE have mild restrictive lung disease, which is not affected by surgical repair. Postoperatively they have a slight increase in exercise tolerance and O2 pulse, which suggests improved cardiac function during exercise. However, the clinical implications of this modest improvement are unclear. PMID- 8627436 TI - Use of minors as bone marrow donors: current attitude and management. A survey of 56 pediatric transplantation centers. AB - OBJECTIVE: To determine the current attitude about the use of minors as bone marrow donors in pediatric bone marrow transplantation (BMT) centers in North America. STUDY DESIGN: A questionnaire was mailed to 70 North American BMT centers. The questionnaire asked for opinions on a number of ethical and clinical issues pertaining to the use of minors as marrow donors. A case history was included and respondents were asked to check all appropriate answers listed in the survey. RESULTS: Fifty-six (80%) of 70 centers responded. There was general consensus on many issues. Pediatricians endorse the validity of parental consent, even in potentially controversial situations. Most are prepared to extract marrow from young (about 6 months of age) infants and are willing to use the same donor more than once. There is general approval of performing BMT with experimental protocols, and the projected outcome of BMT does not affect the decision to use a minor as a marrow donor. There is less consensus regarding the optimal management of minors donating a large volume of bone marrow. CONCLUSION: This survey shows a fairly consistent attitude among pediatric BMT centers about the use of minors as marrow donors. The actual management of such donors was not evaluated in detail and requires further study. PMID- 8627437 TI - Immunogenicity of heptavalent pneumococcal conjugate vaccine in infants. AB - OBJECTIVE: To evaluate the safety, immunogenicity, and immunologic memory in young infants of a seven-valent (6B, 14, 19F, 23F, 18C, 4, 9V) pneumococcal vaccine conjugated to the outer membrane protein complex of Neisseria meningitidis. VACCINEES: Healthy 2-month-old infants 12- to 15-month-old control infants were recruited from participating private practices. METHODS: Infants (n = 25) were vaccinated at 2, 4, and 6 months of age with the conjugated pneumococcal vaccine, followed by a single dose of licensed pneumococcal polysaccharide vaccine (n = 20) at 12 to 15 months of age. Thirteen infants who had not received the investigational pneumococcal conjugate vaccine served as control subjects and were given a single dose of the licensed pneumococcal polysaccharide vaccine at 12 to 15 months of age. RESULTS: The investigational pneumococcal conjugate vaccine was well tolerated by infants. The vaccine was highly immunogenic in young infants, with significant increases in antibody to all seven serotypes after either two or three injections. At 12 to 15 months of age, infants who had been primed with the investigational pneumococcal conjugate vaccine had a brisk immunologic response to the booster injection of the licensed pneumococcal polysaccharide vaccine. Control infants, who received a single primary injection of the licensed pneumococcal polysaccharide vaccine, had negligible immunologic responses to four of the seven serotypes and low responses to the other three types. CONCLUSION: The investigational seven-valent pneumococcal conjugate vaccine administered to young infants was well tolerated and highly immunogenic and provided immunologic memory to an injection of the licensed pneumococcal polysaccharide vaccine. PMID- 8627438 TI - Increased risk of reported pertussis and hospitalization associated with pertussis in low birth weight children. AB - OBJECTIVES: To determine whether low birth weight (LBW) children are at greater risk of reported pertussis and complications of pertussis in the first 2 years of life than are normal birth weight (NBW) children. STUDY DESIGN: We performed a secondary analysis of three data sets containing statewide information among Wisconsin residents for children born between January 1, 1981, and December 31, 1990. We identified all reported cases of pertussis among children younger than 2 years of age and linked this information with birth certificate data and hospitalization data to determine the relative risk of reported pertussis in LBW compared with NBW children. We also compared the frequency of reported complications of pertussis in LBW and NBW children. RESULTS: We analyzed reports of 549 pertussis cases; 49 cases occurred in LBW children. The LBW children were significantly more likely to have reported pertussis than were NBW children (relative risk 1.86; 95% confidence interval 1.33, 2.38). The rates of pneumonia and seizures did not differ among LBW and NBW children; however, LBW children with reported pertussis were significantly more likely to be hospitalized than were NBW children (relative risk 1.40; 95% confidence interval 1.11, 1.69). CONCLUSION: In addition to timely vaccination of all infants, efforts are needed to determine additional ways to reduce the risk of pertussis among LBW infants and children. PMID- 8627439 TI - Topical ointment therapy benefits premature infants. AB - OBJECTIVE: Premature infants have an ineffective epidermal barrier. The aim of this study was to investigate the cutaneous and systemic effects of preservative free topical ointment therapy in premature infants. STUDY DESIGN: We conducted a prospective, randomized study of 60 infants less than 33 weeks' estimated gestational age. The treated infants received therapy for 2 weeks with twice daily preservative-free topical ointment therapy while the control group received no topical treatment or as-needed therapy with a water-in-oil emollient. Data collection included transepidermal water loss (TEWL) measurement, skin condition evaluations, fungal and quantitative bacterial skin cultures, analysis of fluid requirements, patterns of weight low or gain, and the incidence of blood and cerebrospinal fluid cultures positive for microorganisms. RESULTS: We found that topical ointment therapy significantly decreased TEWL during the first 6 hours after the initial application. TEWL was decreased by 67% (p = 0.0001) when measured 30 minutes after application and 34% (p = 0.001) when measured 4 to 6 hours after application. We also observed significantly superior skin condition scores in the treated group on study days 7 and 14 (p = 0.001) and 0.0004, respectively). Quantitative bacterial cultures revealed significantly less colonization of the axilla on day 2, 3, or 4 and on day 14 (p = 0.008 and 0.04, respectively). The incidence of positive findings in blood and/or cerebrospinal fluid cultures was 3.3% in the treated group of infants versus 26.7% in the control group (p = 0.02). There was no statistical difference in the fluid requirements or patterns of weight gain or loss during the 2 weeks of the study. CONCLUSIONS: Preservative-free topical ointment therapy decreased TEWL for 6 hours after application, decreased the severity of dermatitis, and decreased bacterial colonization of axillary skin. Infants treated with ointment had fewer blood and cerebrospinal fluid cultures positive for microorganisms. These data support the use of topical ointment therapy in very premature infants during the first weeks after birth. PMID- 8627440 TI - Neutrophil and cytokine activation with neonatal extracorporeal membrane oxygenation. AB - OBJECTIVE: To determine whether extracorporeal membrane oxygenation (ECMO), like cardiopulmonary bypass, produces systemic inflammatory responses that could potentiate organ injury in infants with respiratory failure. STUDY DESIGN: We evaluated the effects of neonatal ECMO on neutrophil surface adherence proteins, elastase release, and cytokine levels in blood samples from 15 patients before and during ECMO, and from banked blood and ECMO circuit blood before cannulation. Neutrophil elastase, tumor necrosis factor alpha, and interleukin types 1 beta, 6, and 8 were measured. Chest radiographs were evaluated by a radiologist using a lung injury score in blinded fashion. RESULTS: Primed ECMO circuit blood, in comparison with patient pre-ECMO blood, demonstrated marked up-regulation of CD11b (mean fluorescence intensity 1660 +/- 109 vs 361 +/- 81; p < 0.001 (mean +/ SEM)), shedding of L-selectin (mean fluorescence intensity 10 +/- 2 vs 89 +/- 38; p < 0.01), and elevated elastase levels (349 +/- 76 vs 154 ng/ml +/- 38; p < 0.001), consistent with neutrophil activation. During ECMO, neutrophil CD11b levels increased but L-selectin was not significantly shed. Concentrations of circulating neutrophil elastase increase significantly during ECMO. Corrected circulating quantities of interleukin-8 also rose significantly, but the responses of tumor necrosis factor alpha and interleukin-1 beta were minimal. Radiographic lung injury scores worsened with the initiation of ECMO (median score: 6 before ECMO vs 11 in first hour of ECMO; p = 0.012), in conjunction with indicators of neutrophil activation. CONCLUSION: Neonates with respiratory failure have activation of the inflammatory cascade. ECMO incites additional neutrophil and cytokine activation in association with early pulmonary deterioration. Routine leukodepletion of blood for circuit priming to remove activated neutrophils may be beneficial. PMID- 8627441 TI - Depletion of mitochondrial DNA in the liver of a patient with lactic acidemia and hypoketotic hypoglycemia. AB - An infant with feeding difficulties, hypotonia, lactic acidemia, and severe hypoketotic hypoglycemia died at the age of 7 months of liver disease. Electron microscopy revealed abnormal mitochondria. Biochemical studies of mitochondrial enzymes in liver showed a decreased activity of complexes I, III, and IV. Mitochondrial DNA (mtDNA) content was reduced in liver 7% of the mean value in control subjects) and in muscle (50%). In kidney, brain, and heart, the mtDNA content was normal. The liver-specific mtDNA depletion syndrome in this patient manifested itself with features of both a respiratory chain defect and a mitochondrial fatty acid oxidation defect. Syndromes involving depletion of mtDNA can be diagnosed only when the activity of the respiratory chain enzymes and the content of mtDNA are investigated in the most affected tissues. PMID- 8627442 TI - Depletion of mitochondrial deoxyribonucleic acid in a family with fatal neonatal liver disease. AB - We describe a family in which three children of consanguineous parents died of hepatic failure before the age of 3 months. The first child had clinical symptoms of liver disease with hypoglycemia that were evident at birth. The second child was healthy and has normal development. The third child had severe liver dysfunction noted a few days after birth. Liver failure also developed in the fourth child soon after birth. Recently a mitochondrial disorder was considered as a possible cause. Deficiency of respiratory chain enzymes that contain polypeptides encoded by mitochondrial DNA (mtDNA) and depletion of mtDNA were found in the liver of the fourth child, but mitochondrial abnormalities were absent in muscle of the third child. The similarities in clinical presentation suggest that liver-specific depletion of mtDNA was the cause of the hepatic failure in all three children. We conclude that liver dysfunction with onset in the perinatal period can be caused by depletion of mtDNA. PMID- 8627443 TI - Oxidative phosphorylation defect associated with primary adrenal insufficiency. AB - An 18-month-old girl with an oxidative phosphorylation defect had neonatal onset of chronic lactic acidosis, lipid storage myopathy, bilateral cataracts, and primary adrenal insufficiency. Chronic lactic acidosis responded to treatment with dichloroacetate. Sequential muscle biopsies demonstrated resolution of the lipid storage myopathy associated with the return to normal muscle free carnitine levels. This case demonstrates a new clinical phenotype associated with a defect in oxidative phosphorylation and the need to consider mitochondrial disorders in the differential diagnosis of primary adrenal insufficiency in childhood. PMID- 8627444 TI - Etiology of rickets in Nigerian children. AB - We studied 26 Nigerian children with active rickets (13 boys, 13 girls), aged 1 to 5 years, and compared results of biochemical studies with those of healthy control subjects. The plasma 1,25-dihydroxyvitamin D level was elevated (568 +/- 317 pmol/L) and the 25-hydroxyvitamin D level was (36 +/- 28 mol/L) in the children with rickets compared with the control subjects (369 +/- 134 nmol/L and 69 +/- 22 nmol/L, respectively). The results suggest that rickets in Nigeria is largely the result of calcium deficiency and that vitamin D deficiency and possibly end organ resistance may be contributory factors. PMID- 8627445 TI - Conjugated bilirubin in neonates with glucose-6-phosphate dehydrogenase deficiency. AB - We used a system capable of measuring conjugated bilirubin and its monoconjugated and diconjugated fractions in serum to assess bilirubin conjugation in 29 glucose 6-phosphate dehydrogenase (G6PD)-deficient, term, male newborn infants and 35 control subjects; all had serum bilirubin levels > or = 256 mumol/L (15 mg/dI). The median value for diconjugated bilirubin was lower in the G6PD-deficient neonates than in control subjects (0.06 (range 0.00 to 1.84) vs 0.21 (range 0.00 to 1.02) mumol/L, p = 0.006). Diglucuronide was undetectable in 11 (38.9%) of the G6PD-deficient infants versus 3 (8.6%) of the control subjects (p = 0.015). These findings imply a partial defect of bilirubin conjugation not previously demonstrated in G6PD-deficient newborn infants. PMID- 8627446 TI - Accelerated nodulosis during methotrexate therapy for juvenile rheumatoid arthritis. AB - We describe two patients with rheumatoid factor-positive, polyarticular-onset juvenile rheumatoid arthritis in whom accelerated nodulosis developed during methotrexate therapy. Although they had only a few nodules at diagnosis, the nodules increased in number and size 3 to 4 months after the start of methotrexate therapy in both patients. The nodules regressed after withdrawal of methotrexate therapy in one patient and were arrested with the addition of hydroxychloroquine in the other. Physicians treating patients with methotrexate for juvenile rheumatoid arthritis must be aware of this extraarticular side effect. PMID- 8627447 TI - Gastrointestinal hemorrhage complicating aspirin therapy in acute Kawasaki disease. AB - Although gastrointestinal hemorrhage is a well-recognized complication of aspirin therapy, this side effect has not been previously reported in patients with Kawasaki disease (KD). We describe two children with typical KD who had massive gastrointestinal bleeding that required hospitalization and transfusion. Physicians caring for patients with KD should instruct parents on the signs and symptoms of aspirin toxicity. Fortunately, gastrointestinal hemorrhage appears to be a rare complication of salicylate therapy in patients with Kawasaki disease. PMID- 8627448 TI - Spinal extradural hematoma in an infant with hemophilia A: an unusual presentation of a rare complication. AB - Epidural hematomas are a rare complication of hemophilia. This article documents the first case of an infant who initially had irritability alone without neurologic symptoms. The infant's disease was diagnosed and treated early and the child had a good neurologic outcome. PMID- 8627449 TI - Neonatal purpura fulminans in association with factor V R506Q mutation. AB - We report a case of neonatal purpura fulminans associated with activated protein C resistance. Analysis of DNA demonstrated heterozygosity for the factor V R506Q mutation. The neonate, at 8 hours of age, had progressive purpuric skin lesions and later had evidence of microvascular, hemorrhagic thrombosis in the brain. The baby was treated with fresh frozen plasma infusions and had complete resolution of the skin lesions and no apparent long-term complications. We suggest that activated protein C resistance testing be included in the initial evaluation of neonatal purpura fulminans. PMID- 8627450 TI - Screening infants for hearing impairment. PMID- 8627451 TI - Hattie E. Alexander (1901-1968). PMID- 8627452 TI - Reopening of the ductus arteriosus after closure with indomethacin: importance of sustained effective indomethacin serum concentrations. PMID- 8627454 TI - Molecular cause of Hirschsprung disease. PMID- 8627453 TI - Differences in immunoglobulin preparations and outcome of Kawasaki disease. PMID- 8627455 TI - Cardiomyopathy related to acquired immunodeficiency syndrome in children. PMID- 8627456 TI - Mild CF phenotype associated with T3381 missense mutation. PMID- 8627457 TI - Enteropathy and IgG subclass deficiency. PMID- 8627458 TI - Hypotonic dehydration in cystic fibrosis: mild or severe disease phenotype? PMID- 8627459 TI - Exercise-induced anaphylaxis related to specific foods. PMID- 8627460 TI - Utility of the National Cooperative Growth Study database for safety reporting. AB - The National Cooperative Growth Study (NCGS) maintains the largest database in the world, collecting information on patients with growth disorders treated with growth hormone (GH). More than 24,000 children have been monitored during its first decade (1985 through 1995). The database provides unique opportunities to learn about effectiveness of GH therapy in a real-world context. Its size also makes it possible to investigate rare adverse reactions, which is not reasonable in a randomized controlled trial (RCT). The frequency of adverse experiences reported in an observational study like the NCGS is lower than in an RCT, because the investigators in an observational study typically do not report events that they believe are clearly unrelated to the study drug. Nevertheless the NCGS has greatly facilitated collecting adverse-event data; approximately 75% of all adverse-event reports for GH received by the manufacturer are through NCGS data collection forms. The NCGS has thus amassed a repository of GH safety data that is unparalleled. Furthermore, in contrast to an RCT, the NCGS database reflects real-world experience with long-term GH therapy in North America. Although the advantages of an observational study such as the NCGS must be recognized, such a study does differ from an RCT in important ways. For example, because the NCGS protocol allows customized patient treatment and individualization of GH therapy, it may be difficult to use the database to address questions (e. g., estimation of dose-response relationships, true incidence of adverse events) that require RCT designs. PMID- 8627461 TI - National Cooperative Growth Study: Ten Years of Guidance in Growth. Proceedings of a meeting. New York, New York, October 12-15, 1995. PMID- 8627462 TI - Response to growth hormone in children with chondrodysplasia. AB - Theoretic concerns exist that children with chondrodysplasia will not grow in response to growth hormone (GH) therapy because of an inability of the abnormal growth cartilage to respond. Experience to date, however, suggests that there is an increase in growth velocity, especially during the first year of treatment, which may be beneficial. Growth has increased during the early phases of GH therapy in both patients with achondroplasia and patients with hypochondroplasia. Fourteen patients with achondroplasia in the National Cooperative Growth Study have been treated with an average dose of GH of 0.317 mg/kg per week for an average of 2.6 years and have gained an average of 0.7 SD in height. Twenty patients with hypochondroplasia in the National Cooperative Growth Study have been treated with an average dose of GH of 0.317 mg/kg per week for an average of 2.6 years and have gained an average of 0.7 SD in height. These data suggest that the abnormal growth cartilage in patients with chondrodysplasia responds to GH therapy. The effect on final height cannot be predicted with the currently available data. PMID- 8627463 TI - Growth hormone treatment in Noonan syndrome: the National Cooperative Growth Study experience. AB - We evaluated the response to growth hormone (GH) therapy in 150 children (97 boys) with Noonan syndrome (NS) by analyzing growth data from children with NS who were enrolled in the National Cooperative Growth Study and compared those data with National Cooperative Growth Study growth data from children with idiopathic growth hormone deficiency (IGHD) and Turner syndrome (TS). Children with NS were significantly shorter than those with IGHD and TS. The annualized growth rates for years 1, 2, 3, and 4 of therapy in patients with NS who were naive to previous GH therapy were significantly greater than baseline. Their growth rates for years 1, 2, 3, and 4 were intermediate between those in children with IGHD and TS and were significantly different from both. A significant improvement occurred in height SD scores for those 42 children with NS who have been monitored for at least 4 years of GH therapy. Three of six boys with NS for whom adult height data were available exceeded their pretreatment predicted heights. PMID- 8627464 TI - Linear growth in response to growth hormone treatment in children with short stature associated with intrauterine growth retardation: the National Cooperative Growth Study experience. AB - Short stature commonly follows intrauterine growth retardation (IUGR). Most patients are not growth hormone (GH)-deficient, but GH therapy has been used in IUGR. Early studies found a heterogeneous increase in initial growth rate that could not be maintained. Results of more recent studies with higher doses are more encouraging but do not establish whether final height is increased. Data from a large number of patients in the National Cooperative Growth Study were reviewed to evaluate the response to GH treatment in patients with IUGR associated short stature. Two hundred seventy such patients were identified and were categorized as those with unclassified IUGR and those with Russell-Silver syndrome/primordial short stature (RSS/PSS). Patients were treated with standard doses of recombinant human GH (approximately 0.3 mg/kg per week) and were assessed periodically for up to 4 years. The height SD score at baseline in patients with unclassified IUGR was -3.49 +/- 1.16, and their relative height improved with each year of therapy. Patients who completed 4 years of treatment reached a height SD score of -1.32 +/- 0.79. Results were similar in patients with RSS/PSS; their baseline height SD score was -3.83 +/- 1.05 and improved to 2.10 +/- 0.99 by year 4. Despite these encouraging results, no change occurred in predicted adult heights. Furthermore the number of patients who remained in treatment for 4 years decreased substantially, thus limiting the interpretation of the data. These data suggest that a beneficial response to GH occurs in some patients with IUGR-associated short stature and that little difference exists in the responses in patients with RSS/PSS compared with those in patients with unclassified IUGR. PMID- 8627465 TI - Growth hormone treatment of children with myelomeningocele. AB - From the National Cooperative Growth Study database 106 patients (53 boys) with myelomeningocele who were treated with recombinant human growth hormone (GH) at 56 centers were identified. Eighty-one patients (41 boys) were prepubertal at enrollment. The mean pretreatment growth rate (GR) in these prepubertal patients was 4.5 +/- 3.7 cm/yr, and the mean height SD score was -4.0 +/- 1.2. The maximal stimulated GH level was less than 10 micrograms/L in 71% of these patients and less than 7 micrograms/L in 49%. The mean chronologic age was 6.5 +/- 2.9 years, and the mean height age was 3.7 +/- 1.7 years. After GH treatment the year 1 GR in those who remained prepubertal was 8.5 +/- 3.3 cm/yr, a significant increase over baseline (p < 0.01). This increase was sustained through year 4 and remained significant through year 3 (p < 0.01). The height SD score showed sustained significant improvement through year 4, to -2.2 +/- 1.4 (p < 0.001). The GR and SD score for stature improve with GH treatment in children with myelomeningocele. PMID- 8627466 TI - Physiology of growth hormone secretion during sleep. AB - The temporal relation between the first few hours of sleep and the secretion of growth hormone (GH), which is present in normal persons of both sexes from early childhood until late adulthood, is reviewed. In adults the most reproducible pulse of GH secretion occurs shortly after the onset of sleep in association with the first phase of slow-wave sleep (SWS) (stages III and IV). In men approximately 70% of the GH pulses during sleep coincide with SWS, and the amount of GH secreted during these pulses correlates with the concurrent amount of SWS. Sleep-related secretion of GH appears to be primarily dependent on the release of growth hormone-releasing-hormone. Rodent and human studies have shown that growth hormone-releasing hormone injections decrease wakefulness and increase SWS. During the fourth decade of life (ages 30 to 40 years) the total amount of GH secreted over a 24-hour span decreases by two- to threefold. Similarly, the amount of SWS decreases dramatically over the same narrow age range. Because the sleep-onset GH pulse is often the major secretory output in adults, age-related decrements in sleep-related GH secretion likely play a major role in the hyposomatotropism of senescence. PMID- 8627467 TI - Overview of the National Cooperative Growth Study substudy of serial growth hormone measurements. AB - For the National Cooperative Growth Study II substudy, data on spontaneous growth hormone (GH) secretion were collected from 5106 children with short stature. Of these, 2123 with complete 12-hour samples were subsequently enrolled in the NCGS. Compared with NCGS enrollees who were not in the NCGS II substudy, these children were significantly older (11.3 +/- 3.3 years vs 9.9 +/- 4.2 years), had a higher maximum reported GH level (13.3 +/- 10.5 micrograms/L vs 9.2 +/- 8.7 micrograms/L), and were more likely to be male (71% vs 62%) and pubertal (27.3% vs 21.9%) (p<0.001) for all). Height deficit, bone age delay, and pretreatment growth rates were similar. Children who were classified as having GH deficiency on the basis of their response to standard pharmacologic tests had lower spontaneous GH secretion than those who were classified as having idiopathic short stature, but considerable overlap was seen between the two groups on all indexes of spontaneous GH secretion. This finding suggests that the investigators were using serial sampling studies in examining children with short stature who were not growing well but had "normal" GH responses to standard pharmacologic testing. PMID- 8627468 TI - Brain tumor recurrence in children treated with growth hormone: the National Cooperative Growth Study experience. AB - As of October 1993 the National Cooperative Growth Study included 1262 children with brain tumor who were treated with growth hormone. The type of brain tumor was specified in 947 (75%) of these children. The most common types were glioma, medulloblastoma, and craniopharyngioma, accounting for 91.3% of all those for which type was specified. Brain tumor recurred in 83 (6.6%) of the 1262 children over a total of 6115 patient-years at risk. The frequencies of tumor recurrence in children with low-grade glioma (18.1%), medulloblastoma (7.2%), and craniopharyngioma (6.4%) are lower than those in published reports of tumor recurrence in the general pediatric population with the same types of tumors. The analysis cannot conclusively show that no increased risk of tumor recurrence exists, however, because of the potential incompleteness of data reporting in the National Cooperative Growth Study. Nevertheless the findings are reassuring that children with the more common types of brain tumor who are treated with growth hormone do not seem to be at excessive risk for tumor recurrence. PMID- 8627470 TI - Growth hormone measurements versus auxology in treatment decisions: the Australian experience. AB - Growth hormone (GH) therapy is regulated in Australia by an expert national government committee. A national database (OZGROW) enables regular audits and rational guideline revisions. In 1988 the guidelines were revised to allow eligibility on auxologic criteria alone because of difficulties in diagnosing GH deficiency (GHD) and GH responses in non-GH-deficient children. Initial entry criteria were height less than the 3rd percentile and growth velocity less than the 25th percentile of bone age. Growth hormone testing was continued in most children. More than 3100 children have been treated since 1988 (35 percent with GHD, 12.5% with Turner syndrome, and 52% with other non-GHDs). Five-year responses (change in height SD scores) were best in the group with complete GHD (+2) (which received the lowest dose of GH) and similar in other groups, including those with partial GHD (+1.5). The increase in final height is 4 to 6 cm in subjects with Turner syndrome. This data is not yet available for subjects with other non-GHDs. In 1994 the guidelines were revised to restrict use of GH therapy to subjects with height less than the 1st percentile, and cessation of GH therapy was brought forward to bone age 13.5 years for girls and 15 years for boys. Subjects with maturational delay were excluded because of the finding that in the presence of significant bone age delay height prognosis was good. New patient accruals have decreased since 1992, from 100/yr to less than 50/yr. Expenditures have also fallen, from $31 to $16 in 1994-1995, because of reduced patient numbers and GH pricing. Australian use of GH is 68.7% that of the United States and 42.2% that of Sweden and is in the midrange internationally. In conclusion, an auxology-based GH program coupled with a comprehensive national database enables rational and economic use of GH in short children. PMID- 8627469 TI - National Cooperative Growth Study substudy. II: Do growth hormone levels from serial sampling add important diagnostic information? AB - The National Cooperative Growth Study includes growth data on more the 24,000 children in the United States and Canada who have been treated with growth hormone (GH). To determine whether dysregulation of GH release causes growth failure in children, we initiated the National Cooperative Growth Study substudy II to evaluate the diagnostic utility of serially sampled GH levels and to determine whether those patterns were responsible for the low growth rates in certain subsets of short children and whether children in any of the diagnostic categories would respond to GH therapy. A total of 3744 subjects whose mean height standardized for their chronological age was -2.8 SD and whose pretreatment growth rate was 4.2 cm/yr had complete 12-hour data sets-- serial samples obtained in a 12-hour overnight period. Pulsatile characteristics of GH release were assessed with the cluster algorithm. There was a virtually complete overlap of the GH pulsatile characteristics between control subjects and short children, but the insulin-like growth factor I (IGF-I) levels were markedly lower in the short children, suggesting impairment in the GH-IGF-I axis. THe growth response to administered GH showed only very weak correlations with the various cluster-derived parameters. Our results indicate that one must look beyond the release of GH to find an explanation for the short statures and low IGF-I levels in the subsets of children with idiopathic short stature. PMID- 8627471 TI - Assays for insulin-like growth factors and their binding proteins: practicalities and pitfalls. AB - Insulin-like growth factors (IGFs) are growth hormone-dependent anabolic peptides that circulate in biologic fluids complexed to a family of IGF binding proteins. Measurement of the serum concentrations of IGF peptides and IGF binding proteins has proved to be an effective means of evaluating functional growth hormone status and makes it possible to establish a diagnosis of IGF deficiency. PMID- 8627472 TI - Growth hormone deficiency in adults: characteristics and response to growth hormone replacement. AB - Despite adequate adrenal, gonadal, and thyroid hormone replacement, many adults with hypopituitarism have a recognizable syndrome of weakness and diminished sense of well-being, accompanied by alterations in metabolism and body composition, as well as increased mortality. Short-term treatment with human growth hormone improves many of these abnormalities, but a clear improvement in functional status has yet to be demonstrated. Until such an effect is shown, the use of growth hormone replacement in adults with hypopituitarism remains investigational. PMID- 8627473 TI - Physician and clinic charges for diagnosing growth hormone deficiency. AB - Physician and clinic charges for diagnosing growth hormone deficiency (GHD) in children are not generally known, whereas the charges for purchasing growth hormone (GH) are known. We recently surveyed the charges submitted to third-party payers for diagnosing GHD in five pediatric endocrine clinics throughout the United States: the Albert Einstein College of Medicine, Baylor College of Medicine, Health Science Schools of the State University of New York at Buffalo, Oregon Health Sciences University, and the University of Chicago. The financial data analyzed included charges for physician services and for GH testing. Different approaches to the medical examination of children with suspected GHD at these clinics prevented any comparison of physician or GH testing charges. However, the charges for diagnosing GHD could be determined for each pediatric endocrine clinic if the methods of examination were not considered. Contractual adjustments, net revenues, costs, and net margins were not surveyed. Subjective comments from the study sites suggest significantly reduced reimbursement amounts. We conclude that the total charges for diagnosing GHD submitted to third party payers at these institutions averaged $1719. PMID- 8627474 TI - Safety of human growth hormone therapy: current topics. AB - Current data on patients treated with human growth hormone (GH) were analyzed for the following safety topics. New leukemia. Thirteen of 46 new cases of leukemia were in non-Japanese patients without risk factors for leukemia (compared with at least 13 new cases expected). A possible increased occurrence of leukemia with GH treatment appears to be limited to patients with risk factors. Nonleukemic extracranial neoplasms. The number of cases reported (10) does not differ significantly from the number expected. Acute pancreatitis. In five of the seven cases reported risk factors (renal failure, valproic acid use, insulin-dependent diabetes mellitus) were present. The available data do not indicate a clear cause and-effect relation between GH therapy and pancreatitis. Prepubertal gynecomastia. Of 15 possible cases, two were pubertal, eight resolved or improved with continued GH therapy, and two resolved with the cessation of GH therapy. An effect of GH treatment on prepubertal gynecomastia remains unknown. Scoliosis. Scoliosis is reported in fewer than 1 percent of the patients in the National Cooperative Growth Study (general-population prevalence, 1.5% to 3%). Curvature progression can occur during growth acceleration, and a causal association with GH treatment is not substantiated. Pigmented nevi. Nevi growth may be increased with GH treatment. Biopsies have detected no neoplasia or premalignant nevi transformations. PMID- 8627475 TI - Vitamin B12 changes in Nippostrongylus brasiliensis in its free-living and parasitic habitats with biochemical implications. AB - Bacteria in rat feces cultures that had synthesized vitamin B12 were ingested by the free-living stages of Nippostrongylus brasiliensis and the vitamin was concentrated and stored in the third-stage infective filariform larvae. As assayed with Ochromonas malhamensis, the vitamin B12 content of a single filariform larva as well as the concentration expressed as microgram B12 per g filariform larvae reached extraordinarily high levels, the latter being the highest yet recorded for a metazoan organism. The stored B12 content of the filariforms surviving in fecal culture for as long as 104 days remained constant, whereas the B12 concentration rose due to gradual loss of larval body weight. This storage strategy ensured that a high level of the vitamin would be immediately available to the rapidly growing and differentiating worms following infection of the rat. The changing patterns of B12 content and concentration during the parasitic cycle were followed quantitatively and correlated with B12 turnover, increase in worm weight with growth, and incorporation of B12 into the eggs. The possible sources of B12 and its metabolic functions in nematodes are discussed. PMID- 8627476 TI - Viability of infective larvae of Haemonchus contortus, Ostertagia ostertagi, and Trichostrongylus colubriformis following exsheathment by various techniques. AB - Various techniques were examined to determine optimum conditions for exsheathing infective larvae of 3 important ruminant parasites (Haemonchus contortus, Ostertagia ostertagi, and Trichostrongylus colubriformis). In repeated experiments, aliquots of 10(5)-10(6) infective larvae, 1-2 mo old, of each parasite were incubated in each of 4 exsheathing media (distilled water, Earle's balanced salt solution + carbon dioxide, nematode washing buffer + carbon dioxide, or sodium hypochlorite) for 1 or 18 hr. In each case, the percentage of larvae exsheathed and infectivity for jirds was determined. Results of these studies indicate that no single exsheathing technique of those studied is optimum for every parasite. In addition, caution must be used in drawing conclusions from in vitro studies using exsheathed larvae because techniques that routinely provide high percentages of exsheathment also appear to reduce viability. PMID- 8627477 TI - The heat shock response and major heat shock proteins of Tritrichomonas mobilensis and Tritrichomonas augusta. AB - The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes. PMID- 8627478 TI - Proteolytic enzyme activity and Plasmodium falciparum sporogonic development in three species of Anopheles mosquitoes. AB - If proteolytic enzymes affect the innate vector competence of Anopheles mosquitoes for Plasmodium infections, then mechanistic effects should be most pronounced at the zygote to ookinete developmental transition. Anopheles freeborni, Anopheles gambiae, and Anopheles albimanus exhibit excellent, good, and poor susceptibility to P. falciparum, respectively. Aminopeptidase and trypsin activity were determined relative to the kinetics of P. falciparum ookinete development in these 3 Anopheles species. Ookinetes in A. freeborni, A. gambiae, and A. albimanus were first observed at 18 hr postinfection. For separate infection experiments, peak parasite densities were observed at either 18, 24, or 30 hr for A. albimanus, at 24 or 30 hr for A. freeborni, and at either 24, 30, or 36 hr for A. gambiae. Although the 3 species supported ookinete development equally, they had significantly different oocyst infection rates. Similar patterns of aminopeptidase activity were observed for the most susceptible species, A. freeborni, and the least susceptible species, A. albimanus. Anopheles gambiae had the lowest levels of aminopeptidase. Anopheles freeborni also had higher levels of trypsin activity than either A. albimanus or A. gambiae. Irrespective of differences in innate vector competence, the 3 species showed peak levels of aminopeptidase and trypsin that were coincident with peak ookinete densities. Thus, the close correspondence between the kinetics of ookinetes and enzymes associated with bloodmeal digestion indicates that proteolytic enzymes alone do not limit the early stages of sporogonic development in vector species of Anopheles. PMID- 8627479 TI - Characterization of extracellular proteinases of Tritrichomonas foetus. AB - Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis. PMID- 8627480 TI - Pathobiology associated with the spiruroid nematodes Camallanus oxycephalus and Spinitectus carolini in the intestine of green sunfish, Lepomis cyanellus. AB - The histopathology induced by Camallanus oxycephalus and Spinitectus carolini in the intestine of green sunfish Lepomis cyanellus was examined. At what is assumed to be more recent attachment sites, penetration of C. oxycephalus was restricted to the mucosal layer, causing complete destruction of the columnar epithelium. At what appeared to be older attachment sites, the parasite penetrated deep into the intestinal wall, even to the circular muscle layer. At these sites of attachment, there were flask-shaped ulcers in the mucosal and submucosal layers. Surrounding the ulcers, there was granulomatous tissue, with extensive fibrosis at the surface; the fibrosis probably reduced the loss of body fluids and blood cells from the intestinal wall. The number of goblet cells in the posterior end of the intestine increased significantly, from 92.6 cells/mm in uninfected fish to 168.4 cells/mm in fish infected with more than 5 C. oxycephalus. Adult S. carolini also causes severe damage to the intestinal wall of green sunfish. Parasite penetration was not limited to the mucosal layer. Mature worms were observed invading both the muscular and serosal layers. Penetration by the parasite was usually associated with the local loss of columnar epithelium and infiltration by lymphocytes and granulocytic leukocytes. Fibrosis in the mucosal layer was commonly observed. There was goblet cell hyperplasia in the anterior portion of the intestine, with numbers of goblet cells increasing from 64.4 cells/mm in uninfected fish to 95.0 cells/mm in heavily infected fish. PMID- 8627481 TI - Compromised microcirculation in acute murine Trypanosoma cruzi infection. AB - Microvascular compromise has been 1 of many factors implicated in the etiology of the cardiomyopathy associated with Chagas' disease. To further assess the effect of Trypanosoma cruzi infection on the microcirculation, we examined the cremaster microvascular model in CD-1 male mice infected with the Brazil strain at 20-25 days postinfection. There was a significant decrease in red cell velocity (Vrbc) in first and third-order arterioles and venules, which was reversed by verapamil treatment. Video recordings revealed a marked inflammatory response that was confirmed by transmission electron microscopy. A marked inflammatory response was not seen in verapamil-treated infected mice. Segmental vasospasm and dilatation was evident in the microvascular bed of infected mice. This was not seen in control or verapamil-treated mice. This model provides a readily accessible method to observe directly the effects of T. cruzi infection on the microcirculatory flow in vivo. In addition, it confirms and extends our previous observations regarding T. cruzi-associated microvascular spasm and underscores a role for verapamil, a calcium-channel blocker, in the amelioration of the Chagas' disease. PMID- 8627482 TI - Pseudohexabothrium taeniurae n. sp. (Monogenea: Hexabothriidae) from the gills of Taeniura lymma (Dasyatididae) from the Great Barrier Reef, Australia. AB - Pseudohexabothrium taeniurae n. sp. is proposed for a hexabothriid monogenean found on the gills of Taeniura lymma (Forsskal) off Heron Island, Great Barrier Reef, Queensland, Australia. Pseudohexabothrium taeniurae is distinguished from Pseudohexabothrium rajae Brinkmann, 1952, the type and only species of the genus, by the morphology of the sucker sclerites and eggs, and by the size of the hamuli, pharynx, and the "false" oral sucker. The generic diagnosis of Pseudohexabothrium is revised. A phylogenetic analysis places Pseudohexabothrium as the sister group to the rest of the family except Hexabothrium. PMID- 8627483 TI - Myxobolus cognati n. sp. (Myxosporea) from the opercular integument of Cottus cognatus (Cottidae) in Lake Michigan. AB - A myxosporean producing aggregations of white pseudocysts in skin covering the posterior margin of the operculum and perioral tissue of Cottus cognatus in Lake Michigan is described as Myxobolus cognati n. sp. (Myxosporea). Histological sections revealed that the parasite develops in vascularized regions of the dermis and with development protrudes beyond the surrounding skin surface. Fixed spores are oval in front view, measure 12.0-14.0 microns long and 9.5-10.5 microns wide, and contain 8-11 filament coils wound tightly and perpendicular to the longitudinal axis of the capsule. Myxobolus cognati resembles Myxobolus rhinichthidis Fantham, Porter and Richardson, 1939 from subepithelial tissue of the operculum of the cyprinid Rhinichthys cataractae. Spores in the 2 species are the same size and shape. However, in contrast to those of M. cognati, spores of M. rhinichthidis have filament coils wound loosely at 45 degrees to the axis of the polar capsule and have no sutural ridge folds. PMID- 8627484 TI - Burhinotaenia colombiana n. sp. (Cestoda, Cyclophyllidea) from the double-striped stone curlew Burhinus bistriatus (Aves, Charadriiformes) in Colombia. AB - Burhinotaenia colombiana n. sp. (Dilepididae) is described from the small intestine of the double-striped stone curlew Burhinus bistriatus (Burhinidae) captured at Carimagua, Colombia. The new species is distinguished from the most similar Burhinotaenia delachauxi (Baer, 1925), a parasite of the Old World Burhinus spp., by the longer cirrus-sac (375-590, avg. 514 microns vs. 322-393, avg. 354 microns) and longer rostellar hooks (412-451, avg. 440 microns vs. 358 367, avg. 364 microns). The validity of the genus Burhinotaenia Spasskii and Spasskaya, 1965 and its generic diagnosis as proposed by Bona (1994) are confirmed. This is the first record of a species of Burhinotaenia in the New World. PMID- 8627485 TI - The synlophe and other structural characteristics of Sarwaria bubalis (Nematoda: Trichostrongyloidea) from cattle in Guyana. AB - The synlophe (longitudinal, surface cuticular ridges) of Sarwaria bubalis is described for the first time. It is a tapering lateral synlophe of about 40 ridges. The synlophe of S. bubalis is similar to that of Ostertagia ostertagi but markedly different from that of species of Spiculopteragia and Mazamastrongylus. New information is provided also on the structure of the esophagus and perivulval pores. The esophageal valve is more than twice as long as wide. The bilateral perivulval pores were located 192-267 microns posterior to the vulva and dorsal to the lateral lines. The new information will be useful in a study of the generic level systematics of the Ostertaginae. Sarwaria bubalis appears to be well established in tropical South America where it infects its normal host, the Asian water buffalo Bubalus bubalis, as well as domestic cattle Bos taurus and mixed breed cattle B. taurus x Bos indicus. PMID- 8627486 TI - The effects of ivermectin on the hydatid cyst of Echinococcus granulosus after direct injection at laparotomy. AB - The effect of ivermectin on the hydatid cysts of Echinococcus granulosus was studied in vivo. Jirds were infected with secondary hydatid cysts of E. granulosus by intraperitoneal passage. One to one and a half years postinfection a laparotomy was performed on each of 20 jirds and the hydatid cysts located. Ivermectin or the vehicle (1% dimethyl sulfoxide in sterile physiological saline) was injected into a single hydatid cyst in each animal (10 with ivermectin; 10 with vehicle as control). The jirds were killed 44-58 days postsurgery and necropsied. The integrity of each injected cyst was assessed and tissue samples were taken for microscopic examination. Treated cysts were discolored, collapsed, and had no viable protoscolices. The germinal layer of treated hydatid cysts was necrotic; the laminated layer was intact. The effects were restricted to the ivermectin-treated cysts. Treated cysts were not infective to naive jirds by intraperitoneal passage. PMID- 8627487 TI - Effect of diethyldithiocarbamate on Cryptosporidium parvum infection in immunosuppressed rats. AB - Diethyldithiocarbamate was the only immune modulator of 7 evaluated to show activity against Cryptosporidium parvum in immunosuppressed rats. The model was then used to assess the drug's activity further. When administered prophylactically, oral doses > or = 75 mg/kg/day significantly (P < or = 0.05) reduced the severity of ileal infection and doses > or = 300 mg/kg/day significantly (P < or = 0.05) inhibited infection of the biliary tract. When administered to rats with established infection, the drug significantly (P < or = 0.05) reduced the parasite burden in the ileum but was ineffective against biliary tract infection. The data suggest that diethyldithiocarbamate is effective for treating cryptosporidiosis of the small intestine but is probably ineffective against chronic cryptosporidiosis involving the large intestine or biliary tract. PMID- 8627488 TI - Ultrastructural morphology of intermediate forms and forms suggestive of conjugation in the life cycle of Pneumocystis carinii. AB - The intrapulmonary life cycle of Pneumocystis carinii has been proposed to include asexual reproduction of haploid trophic forms and sexual reproduction to form cysts or ascospores containing 8 intracystic bodies or spores. This study presents the ultrastructural morphology of trophic forms and developing ascospores. Nuclear-associated organelles (NAO) were observed in these various forms and in a binucleated trophozoite. The unique configuration of the nuclei and NAO in this binucleated trophic form were similar to ultrastructural features observed in conjugation of Saccharomyces cerevisiae. NAOs in developing ascospores were present in pairs and were associated with multiple nuclei. In more mature ascospores, NAO were associated with individual spores. The intermediate forms described in this study support an asexual and sexual reproductive process in P. carinii. PMID- 8627489 TI - Gastrointestinal helminths of Arctic foxes (Alopex lagopus) from different bioclimatological regions in Greenland. AB - Nine species of gastrointestinal helminths were recovered from 254 arctic foxes (Alopex lagopus) from 8 different localities in Greenland. Prevalences of infection with the helminth species differed from area to area: Toxascaris leonina (39-68%), Strongyloides stercoralis (0-14%), Mesocestoides lineatus (0 58%), Diphyllobothrium dendriticum (0-15%), Taenia ovis krabbei (0-70%), Cryptocotyle sp. (0-3%), Plagiorchis elegans (0-6%), and Polymorphus sp. (0-3%). Additionally, a Taenia species, which appears to be different from T. ovis krabbei, had a prevalence of 24% but only on the east coast of Greenland. In general, the composition of the helminth fauna of arctic foxes in Greenland showed distinct differences geographically. Thus, the diversity of helminth species in foxes caught in the northern districts of Greenland seems lower than in the southern districts; only nematode species with direct life cycles were represented equally in all parts of the country. The diversity of the surrounding fauna, and thereby the food items available for the foxes, seems to determine the spectrum of helminth species. Helminths requiring rodents as intermediate hosts were absent on the west coast, even in the areas in northwest Greenland in close proximity to the Canadian Archipelago. Foxes from air bases, which are known to feed intensively on garbage, harbored similar numbers of species compared to foxes from settlements in the same regions. The number of T. leonina in animals less than 1 yr of age was significantly higher than in older individuals. For M. lineatus, the prevalence of infection was found to increase and worm burdens to decrease with increasing host age. Infections with 2 or more helminth species were most frequent in older animals (47% in foxes > or = 4 yr old and 33% in foxes < 4 yr old). PMID- 8627491 TI - Nocturnal questing by adult blacklegged ticks, Ixodes scapularis (Acari: Ixodidae). AB - Quantitative tick drag samples were taken at various times during the day and night from February through April 1994 on St. Catherines Island or on Sapelo Island, Georgia. For each month, there was no statistical difference between the numbers of adult blacklegged ticks, Ixodes scapularis, collected during any hour of daylight or darkness on St. Catherines Island, Adult I. scapularis also quested during both day and night on Sapelo Island, but on this island significantly more ticks were collected in 1 nocturnal sample during March. Nocturnal questing may partially explain why hosts that are principally nocturnal or that are active during both day and night are often heavily parasitized by adult I. scapularis. This observation could be epidemiologically important with respect to tickborne zoonoses such as Lyme disease and babesiosis. PMID- 8627490 TI - A Sarcocystis neurona-like organism associated with encephalitis in a striped skunk (Mephitis mephitis). AB - A Sarcocystis neurona-like organism was associated with granulomatous encephalitis in an ataxic male juvenile striped skunk (Mephitis mephitis) from Cape Cod, Massachusetts. Various stages of schizonts and merozoites of S. neurona were seen within some of the granulomata. PMID- 8627492 TI - Carmine-propionic acid stain for elucidation of fine cellular structure in nematodes. AB - Carmine-propionic acid staining was found to enhance the elucidation of cellular membranes in the esophagus and genital tract and the structure of sperm in strongylate nematodes. This regressive staining technique may also have general utility for differentiation of cellular membranes and nuclear structure in other taxa of parasitic helminths. PMID- 8627493 TI - Immunopurification and measurement of IgE in serum samples from bancroftian filariasis patients. AB - Patent infection with Wuchereria bancrofti is associated with increased levels of filaria-specific IgG4 and IgE. In vitro quantification of filaria-specific IgE is hampered by its small proportion in serum relative to other isotypes and by potential competition with IgG4 for the same epitopes on parasite antigens. To determine if IgG4 or other isotypes inhibit the detection of parasite-specific IgE, total IgE was affinity purified prior to filaria-specific IgE enzyme-linked immunosorbent assay. Briefly, anti-human IgE mouse monoclonal antibody 6H10 was coupled to Affigel, and 50 microliters of patient serum was incubated on microcolumns for 16 hr. Total IgE was eluted with 25 mM triethylamine (pH 11.2) and levels of total and filaria-specific IgE and total IgG4 were assessed in the filtrates and eluates. Sera from 14 patients with W. bancrofti microfilaremia (Mf+) and 17 amicrofilaremic patients with chronic pathology (CP) were assayed. Filtrates and eluates were devoid of IgE and IgG4, respectively. The average yield of total IgE in the eluates was 70% (SEM = 6.5; range 21-100%) of that measured in the serum. Antifilarial IgE levels in column eluates were significantly higher in serum samples from CP patients than Mf+ patients. Antibody inhibition of IgE was assessed by comparing the levels of anti-filarial IgE detected in eluates and serum. Evidence for antibody-mediated inhibition of IgE detection was obtained with 2/2 samples from Indian tropical pulmonary eosinophilia patients, but only 2/14 and 4/17 Mf+ and CP patients, respectively. PMID- 8627494 TI - Characteristics of naturally acquired Plasmodium relictum capistranoae infections in naive Hawaiian crows (Corvus hawaiiensis) in Hawaii. AB - Indigenous to Hawaii, the Hawaiian crow (Corvus hawaiiensis) is the world's most severely endangered species with only 3 reproductively active pairs remaining in the wild. Seven captive-reared, avian malaria-naive C. hawaiiensis were exposed in an outdoor aviary and hematologically and serologically monitored for 9 wk. Three birds showed Plasmodium relictum capistranoae parasitemia (6.35%, 2.15%, and 0.60%). All birds were seroconverted for malaria on week 7 as determined by enzyme-linked immunosorbent assay (ELISA). Malaria IgG levels of exposed parasitemic birds did not differ from those of exposed nonparasitemic C. hawaiiensis and were not significantly correlated with the level of parasitemia. Four of 9 hematological parameters, e.g., white blood cell count (WBC), relative and absolute lymphocytosis, and total solids (TS), showed significant increases related to ELISA-determined malarial infection. The sensitivity, specificity, and the positive predictive values of these 4 parameters for malarial infections in C. hawaiiensis were higher than 66%, with the WBC and TS sensitivity reaching 100%. The reference range of 9 hematological parameters was established based on uninfected, clinically healthy C. hawaiiensis. Seven birds were successfully treated and released, increasing the total wild C. hawaiiensis world population by approximately 50%. PMID- 8627495 TI - Disseminated toxoplasmosis in a captive porcupine (Coendou mexicanus) from Costa Rica. AB - An adult porcupine (Coendou mexicanus) from the National Zoo, San Jose, Costa Rica, died because of toxoplasmosis involving the heart, lungs, liver, and kidneys. Toxoplasma gondii was found in lesions and the diagnosis was confirmed by immunohistochemical staining with T. gondii-specific polyclonal rabbit antibodies. This is a new host record for T. gondii. PMID- 8627496 TI - Effect of ivermectin on Caenorhabditis elegans larvae previously exposed to alcoholic immobilization. AB - First-stage larvae of Caenorhabditis elegans were immersed in 0.15% 1-phenoxy-2 propanol to induce temporary paralysis, including the suppression of pharyngeal pumping. Subsequent addition of ivermectin (to give 50 micrograms/ml) induced coiling and prolonged immobilization of such larvae, as also of control larvae (previously immersed only in water). The results suggest that ingestion of drug by means of pharyngeal pumping is not a prerequisite for the uptake of ivermectin at levels sufficient for antinematodal action. PMID- 8627497 TI - Ascaris lumbricoides aggregation in relation to child growth status, delayed cutaneous hypersensitivity, and plant anthelmintic use in Madagascar. AB - The relationships between Ascaris lumbricoides worm burden, growth status, general delayed cutaneous hypersensitivity (DCH) response, and plant anthelmintic use were investigated in a 12-mo prospective survey of 663 children, 4-10 yr old, living in the Ranomafana rainforest, Madagascar. Initial fecal examinations revealed prevalences of 93% for A. lumbricoides, 55% for Trichuris trichuria, and 27% for hookworm. After anthelmintic treatment and a 12-mo reinfection period, 428 of the children participated in worm expulsion studies using pyrantel pamoate, revealing an overdispersed A. lumbricoides worm population, mean = 19.2 (SD = 20.4). Malnutrition was common with 72% of the children growth stunted, 61% underweight, and 6% wasted. The children were also skin tested to recall antigens for DCH, with 94% reacting. The DCH immune response was significantly decreased in the malnourished children; however, DCH was not reduced in relation to increasing worm intensity. Growth status, growth velocity, and triceps skinfold did not vary significantly in relation to A. lumbricoides worm burden. Traditional plant anthelmintic treatment was effective in significantly reducing worm intensity. This study indicates that, in human communities where the children are predominately stunted, A. lumbricoides does not aggregate in the most malnourished or immunodepressed children. PMID- 8627498 TI - Competition between two steinernematid nematode species for an insect host at different soil depths. AB - We studied interactions between 2 entomopathogenic nematode species, Steinernema carpocapsae, an ambusher forager, and Steinernema glaseri, a cruiser forager, when they were provided wax moth larvae as hosts at 0, 2, or 10 cm soil depth. Populations of infective juvenile nematodes in soil were monitored at 30-day intervals over 120 days using wax moth larvae as baits. After application of S. carpocapsae, S. glaseri, or the combination of both species, hosts were added at 30-day intervals. With hosts at 0 cm depth, each nematode species was negatively affected by the presence of the other species at the 30- and 60-day samples. At 90 and 120 days, S. carpocapsae numbers in the combined treatment were as high as in the single species treatment, whereas only few S. glaseri were recovered. With hosts at 2 or 10 cm depth, the presence of S. glaseri had a strong negative effect on S. carpocapsae, but S. glaseri was not affected by the presence of S. carpocapsae. In another experiment, S. carpocapsae dominated over S. glaseri in hosts located at 0 cm depth as measured by penetration efficiency into hosts and progeny production. In contrast, S. glaseri dominated at 2 cm depth. At 2 cm depth, S. carpocapsae penetrated into hosts too slowly to compete successfully with S. glaseri. Steinernema carpocapsae is superior to S. glaseri when competing for a host on the soil surface; however, below the surface S. glaseri is superior to S. carpocapsae. PMID- 8627499 TI - Prevalence of Toxoplasma gondii antibodies in hunter-killed white-tailed deer (Odocoileus virginianus) in four regions of Minnesota. AB - Sera from 1,367 white-tailed deer (Odocoileus virginianus) from 4 geographic regions in Minnesota collected during 4 hunting seasons (1990-1993) were tested for antibodies to Toxoplasma gondii using the modified direct agglutination test incorporating mercaptoethanol. Sera from 30% of the deer had antibody titers > or = 25; 8.6% were positive at a titer of 25, 11% at a titer of 50, and 10% at a titer > or = 500. There was a significant increase in seropositivity with age (P < 0.0001). Adult deer were twice as likely to be positive as yearlings; yearlings were 2.5 times as likely to be positive as fawns. There was no difference in prevalence by sex when adjusted for age (P = 0.316), nor was there age-sex interaction. Only males showed a slight increase in titer with age (P = 0.049). There were no significant differences in prevalence among the regions of northeast pine/aspen forest, southwest tall-grass prairie, southeast mixed hardwood forest, and aspen/oak suburban park land. There were no statistically significant differences by year of collection. The prevalence of T. gondii antibodies in white-tailed deer remains high and deer hunters and consumers should ensure that venison is well-cooked or frozen prior to consumption. PMID- 8627500 TI - Influence of temperature and host species on the development of Cryptocaryon irritans. AB - The course of infection of the parasitic ciliate Cryptocaryon irritans was followed on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C. The parasite was originally isolated from locally caught Acanthopagrus australis. At 20 C trophonts stayed on the fish longer, tomonts took longer to excyst, and the resulting theronts were larger than at 25 C. On L. calcarifer at 20 C, trophonts grew slowly at first but eventually became significantly larger (mean tomont diameter 466 x 400 microns) than at 25 C (mean diameter 373 x 320 microns). On M. novemaculeata, trophonts never grew as large as on L. calcarifer and at 20 C they grew poorly. The number of theronts produced per tomont was directly related to the size of the tomont but was not influenced by incubation temperature. The tomont incubation period was not related to the diameter of the tomont but was significantly influenced by the host origin of the tomont. Theront size was also significantly affected by the host origin of the tomont but not the diameter of the tomont. These results show that C. irritans exhibits variability in morphometrics on different hosts and under different temperature conditions. This variability needs to be taken into account if utilizing morphometric data for separating strains of C. irritans. PMID- 8627501 TI - Ehrlichia-like 16S rDNA sequence from wild white-tailed deer (Odocoileus virginianus). AB - The reservoir hosts of Ehrlichia chaffeensis, etiologic agent of human ehrlichiosis are unknown. Initially, white-tailed deer (WTD) were serologically implicated as possible reservoirs of E. chaffeensis. Subsequent studies showed that WTD were susceptible to infection with E. chaffeensis and that deer-to-deer transmission by a tick vector, Amblyomma americanum, is possible under experimental conditions. To determine if wild WTD were infected with E. chaffeensis, whole blood was collected from 10 deer from Oklahoma and Georgia. All 10 deer had antibodies reactive to E. chaffeensis. Whereas E. chaffeensis was not isolated, restriction enzyme mapping and sequencing of the 16S rDNA gene revealed that a unique Ehrlichia-like agent was present. All 10 deer appeared to be infected with the same agent. We suspect that A. americanum is the vector of this new agent based upon the previously published temporal association between the appearance of E. chaffeensis seropositive WTD and A. americanum. However, the taxonomic and antigenic relationships, geographic distribution, epidemiology, and zoonotic potential of this agent are yet to be determined. PMID- 8627502 TI - Host genetics and resistance to acute Trypanosoma cruzi infection in mice: profiles and compartmentalization of IL-2-, -4-, -5-, -10-, and IFN-gamma producing cells. AB - Survival of acute Trypanosoma cruzi infection by mice is influenced by genes inside and outside the major histocompatibility complex (MHC) and genes associated with resistance must be expressed in both the MHC and the genetic background or the host will die within a few weeks of infection. Both the levels and the kinetics of cytokine production have also been implicated as important factors for resistance. Antigen-stimulated spleen cells from mice that express the resistant H-2q MHC haplotype produced significantly more interferon (IFN) gamma than did cells from mice that share the susceptible H-2k haplotype. But, spleen cells from susceptible and resistant mice produce similar levels of IFN gamma when stimulated with concanavalin A. The kinetics of interleukin (IL)- 10 production by ConA (ConA)-stimulated spleen cell were inversely correlated with IFN-gamma levels throughout the course of acute infection in all mouse strains. Levels of IL-2 produced by ConA-stimulated spleen cells were also initially high (day 0) then decreased as acute infection progressed. Conversely, IL-4 production by ConA-stimulated spleen cells increased during infection, and mice that express the susceptible C3H background produced significantly more IL-4 than those that share the resistant B10 background. IL-2 production by lymph node cells from mice that express the susceptible C3H genetic background also declined during infection, while lymph node cells from B10 background mice showed a moderate increase in IL-2 secretion. These data suggest that both the levels and the kinetics of cytokine production may be genetically regulated and that cytokine responses are compartmentalized in the T. cruzi-infected host. PMID- 8627503 TI - Immunologic cross-reactivity among various strains of Sarcoptes scabiei. AB - Varieties of Sarcoptes scabiei from different hosts are highly host specific but they are morphologically indistinguishable. The purpose of this study was to investigate the immunologic cross-reactivity among several varieties of scabies mites using serum from a human scabies patient and from several other species of infested hosts. Homologous and heterologous crossed-immunoelectrophoretic (CIE) analysis of extracts prepared from var. canis (dog) and var. suis (pig) mites yielded very similar antigen profiles. Serum from a human patient infested with var. hominis had circulating IgE that bound to antigens present in extracts prepared from each animal mite variety. Antigen homology was further confirmed by fused peaks on tandem CIE. Additionally, sodium dodecyl sulfate polyacrylamide gel electrophoresis/immunoblot analysis showed that the 2 extracts contained proteins that bound antibody in serum from a var. suis-infested pig, a var. canis infested dog, var. canis-infested rabbits, and a var. hominis-infested human. The results of this study clearly indicate that different varieties of scabies mites, though host specific, introduce some immunologically cross-reactive molecules into the host. However, each serum from the 4 scabies-infested hosts also contained antibody that was specific for proteins in extract from only 1 variety of mite. These data indicated that each variety of scabies introduced some unique molecules into the host, each strain produced some similar molecules, or both, but different hosts responded immunologically to different sets of these. PMID- 8627504 TI - Taenia saginata oncosphere excretory/secretory peptidases. AB - To identify oncosphere excretory/secretory peptidases, Taenia saginata adult worms were collected from 3 patients. Eggs were hatched and activated in vitro and oncospheres cultured in vitro. The culture medium from the oncospheres was assayed with peptide substrates coupled to 7-amino-4-trifluoromethyl coumarin (AFC), and free AFC was detected fluorometrically. The endopeptidase substrates Z Phe-Arg-AFC and Z-Arg-AFC as well as the aminopeptidase substrate Arg-AFC were hydrolyzed when incubated with spent media from the oncospheres compared to control culture medium removed prior to incubation. Hydrolysis of Z-Phe-Arg-AFC was inhibited 78% by preincubation of the medium with the serine proteinase inhibitor Phenylmethylsulfonyl fluoride. Endopeptidase activity was partially enhanced in the presence of exogenous thiols and partially inhibited with the cysteine proteinase inhibitor E-64, suggesting the presence of both serine and cysteine endopeptidases. No significant inhibition was noted with pepstatin or phenanthroline. The peptidase activities detected with Z-Phe-Arg-AFC and Arg-AFC were separated by gel-filtration fast protein liquid chromatography and eluted at volumes corresponding to molecular weights of 18 and 30 kDa, respectively. These data demonstrate that T. saginata oncospheres produce excretory/secretory peptidases, including serine and cysteine endopeptidases and an aminopeptidase. These enzymes may play a role in invasion of the intestinal mucosa of the intermediate host. PMID- 8627505 TI - The occurrence of antibodies to Naegleria species in wild mammals. AB - Individuals from 13 wild mammalian species collected in southwestern Tennessee were serologically positive for anti-Naegleria spp. antibodies (ANA). This is the first report of ANA in wild mammals. Interspecific differences in the occurrence of ANA and titers indicate that wild mammals have differing degrees of contact with Naegleria spp. based on ecological or behavioral adaptations, and possibly the innate ability of a species to produce ANA. Intraspecifically, it appears that if a mammal is exposed to Naegleria spp. in the environment, titers of ANA will not significantly differ, regardless of age or sex. Adults of many species had significantly higher occurrences of ANA. Populations of juvenile wild mammals are probably at higher risk than adults to naeglerial infection in the environment, not because of lower titers, but because their chance of having ANA is less than adults. Differences in the occurrence of ANA in wild mammals may also reflect how humans are exposed to Naegleria spp. in the environment. PMID- 8627506 TI - IL-2-stimulated splenocytes reduce infections by Leishmania donovani in vivo. AB - The traditional treatment of infections produced by the obligate intramacrophage protozoan Leishmania donovani involves the use of antimonial drugs. Because these drugs may have toxic side effects (and are sometimes ineffective), the potential efficacy of alternative therapy with lymphokine-stimulated leucocytes was assessed. Macrophage-depleted C57BL/6 splenocytes from mice inoculated 2 wk earlier with L. donovani were stimulated in vitro with the interleukin-2 containing supernatant of MLA 144 cells and transferred intravenously into syngeneic infected mice. Compared to infected mice that had received unstimulated normal or infected spleen cells, animals treated with lymphokine-stimulated splenocytes had significantly reduced parasite loads. Efficacy was further enhanced significantly by supplementary intraperitoneal injections of the MLA 144 supernatant; the effector function of the stimulated splenocytes was dose dependent. The rescue of animals infected by L. donovani from parasite-induced down-regulation of immunity could be an important part of a strategy for the effective treatment of kala azar; lymphokine-stimulated cells are potential candidate agents to restore curative immune responses of experimental visceral leishmaniasis. PMID- 8627507 TI - Characterization of a chicken monoclonal antibody that recognizes the apical complex of Eimeria acervulina sporozoites and partially inhibits sporozoite invasion of CD8+ T lymphocytes in vitro. AB - A stable chicken hybridoma secreting a monoclonal antibody (mAb) that detects the apical complex of Eimeria acervulina sporozoites has been developed by fusing a thymidine kinase (TK)-deficient chicken myeloma with spleen cells from chickens immunized with sporozoite antigen. The mAb, designated as 6D-12-G10, recognized the apical complex of a sporozoite of 20-21 kDa molecular mass on western blots. Immunoelectron microscopic examination revealed that mAb 6D-12-G10 stained the conoid antigen. Furthermore, mAb 6D-12-G10 inhibited the invasion of sporozoites into CD8+ T cells in vitro. These results suggest that the conoid may play an important role in the recognition and invasion of host cells by Eimeria sporozoites. PMID- 8627508 TI - Detection and quantification of Ornithodoros-specific anti-tick antibody by competitive inhibition ELISA. AB - The objective of this study was to develop a highly specific enzyme-linked immunosorbent assay (ELISA) for the serological detection of anti-Ornithodoros tick antibodies in animals. Affinity-purified rabbit anti-Ornithodoros IgG antibodies were employed in indirect competitive inhibition ELISA assays designed to measure the anti-Ornithodoros antibody titers in other animal species using the domestic goat (Capra hircus) as a large animal model. Repeated infestation of goats with Ornithodoros coriaceus was found to elicit the formation of antibodies capable of inhibiting the binding of the Ornithodoros-specific rabbit IgG. Western blot analysis of goat and rabbit anti-tick antisera demonstrated both animal species to respond immunologically to a set of 9 major protein bands in O. coriaceus salivary gland extracts. The results of these experiments demonstrate that a history of animal exposure to O. coriaceus may be detected serologically by competitive inhibition ELISA. PMID- 8627509 TI - Behavioral basis of second intermediate host specificity among four species of Haematoloechus (Digenea: Haematoloechidae). AB - Cercarial behavior patterns were examined in 4 species of frog lung flukes (Haematoloechus spp.). Cercariae of Haematoloechus complexus, Haematoloechus medioplexus, Haematoloechus longiplexus, and Haematoloechus varioplexus were exposed to 3 species of experimental arthropods and an inanimate control. The number of cercariae attached to an experimental host at 5 min postexposure was recorded. Haematoloechus longiplexus and H. complexus cercariae attached to experimental hosts at higher rates than cercariae of H. medioplexus and H. varioplexus. Cercariae of H. longiplexus attached to experimental hosts in approximately the same numbers as H. complexus, but H. longiplexus penetrated only damselfly naiads, and only at the base of the zygopteran caudal gills. Cercariae of H. complexus, a second intermediate host generalist, were able to penetrate and enter several arthropod species at the intersegmental membranes. Haematoloechus medioplexus and H. varioplexus are restricted to development in dragonfly naiads and cercariae rarely attached to and never penetrated experimental hosts. These behavioral patterns dictate the range of hosts suitable for metacercarial development of H. complexus, H. longiplexus, H. medioplexus, and H. varioplexus. The evolution of disparate patterns of behavior among the cercariae of these 4 congeners has directly affected subsequent patterns of transmission to the definitive host. PMID- 8627510 TI - Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450s. AB - To assess the contribution of cytochrome P450 (P450) to the microsomal ethanol oxidation system (MEOS) in humans, we examined ethanol oxidization activity in human hepatic microsomes and multiple forms of human hepatic P450s expressed in B lymphoblastoid cells. Acetaldehyde produced by the MEOS was converted into a fluorescent derivate with cyclohexane-1,3-dione and analyzed by high-performance liquid chromatography using a fluorescence detector. The ethanol oxidation activity of seven forms of human P450s was investigated. P450s 2E1 and 1A2 formed acetaldehyde at high rates. In immunoinhibition studies, anti-P450 2E1 antibody inhibited the ethanol oxidation activity of human hepatic microsomes by 54%, and anti-P450 1A2 antibody inhibited ethanol oxidation activity by 21%. 7,8 Benzoflavone, an inhibitor of P450 1A forms, also inhibited this activity by 38%. The correlation of ethanol oxidation activity with the levels of immunoreactive P450 2E1 in individual human microsomes was highly significant (r = 0.91, P < .001), and the correlation of ethanol oxidation activity with the levels of P450 1A2 was also highly significant (r = 0.87, P < .001). The Km values of P450s 2E1 and 1A2 for ethanol oxidation were 16.5 and 23.6 mM, respectively. These results indicated that P450 2E1 was a major contributor to the MEOS in humans; however, P450 1A2 was considered to play an important role in the MEOS. PMID- 8627511 TI - Characterization of CYP2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes. AB - Nicotine is primarily metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine in human liver, which is a major metabolite of nicotine in humans. We studied the formation of trans-3' hydroxycotinine from cotinine in human liver microsomes. trans-3'-Hydroxycotinine formation demonstrated single enzyme Michaelis-Menten kinetics (Km, 234.5 +/- 26.8 MicroM; Vmax, 37.2 +/- 2.4 pmol/min/mg protein). Significant correlation (r = .967, P < .001) between cotinine 3'-hydroxylase activities at low (50 microM) and high (1 microM) cotinine concentrations in 20 human liver microsomes suggested the contribution of a single enzyme to cotinine 3'-hydroxylation. The cotinine 3'-hydroxylase activity correlated significantly with immunoreactive cytochrome P450 (CYP)2A6 contents (r = .756, P < .01) and coumarin 7-hydroxylase activity (r = .887, P < .001). The cotinine 3'-hydroxylase activity was inhibited by coumarin, alpha-naphthoflavone, chlorzoxazone and anti-rat CYP2A1 antibodies. Microsomes of B-lymphoblastoid cells expressing human CYP2A6 exhibited cotinine 3'-hydroxylase activity. The Km value of the expressed CYP2A6 (264.7 microM) was almost identical to that of human liver microsomes. In conclusion, cotinine 3' hydroxylation appears to be catalyzed solely by CYP2A6 in humans. Cotinine is a candidate for a new substrate for CYP2A6 in humans. PMID- 8627512 TI - Mapping the genes for haloperidol-induced catalepsy. AB - The strain means for haloperidol-induced catalepsy were determined in the 26 strain BXD recombinant inbred series. The ED50 values ranged from 0.55 mg/kg (strain 30) to 7.9 mg/kg (strain 2). Heritability for the catalepsy response was 0.78 and the number of effective loci was estimated to be four. The strain means were correlated with the strain distribution patterns for 1300 marker loci of known chromosomal location and polymorphic between the C57Bl/6J and DBA/2J strains. Six quantitative trait loci (QTLs) were identified at P < .01. Two of the six QTLs were confirmed in a sample of B6XD2 F2 animals (n = 144), phenotyped for haloperidol response and genotyped for microsatellites closely linked to the QTLs. The confirmed QTL on chromosome 4 is near the b locus. The confirmed QTL on chromosome 9 is closely linked to Drd2, the D2 dopamine receptor gene. One hundred of the F2 individuals were phenotyped for D2 dopamine receptor binding using the ligand [125I] epidepride as the ligand. Consistent with previous results, the nonresponsive F2 individuals showed modestly higher receptor binding in all brain regions examined: the nucleus accumbens core, the nucleus accumbens shell, the lateral caudate putamen, the dorsomedial caudate putamen, the substantia nigra zona compacta and the ventral tegmental area. The DBA/2J allele of the chromosome 9 QTL was associated with higher receptor binding in all brain areas except the ventral tegmental area. Overall, the data illustrate that either near or part of Drd2 is a QTL which has significant effects on both haloperidol response and D2 dopamine receptor binding. However, the data also illustrate that most of the genetic variance in either haloperidol response or D2 dopamine receptor binding is not associated with Drd2. PMID- 8627513 TI - Down-regulation of metallothionein expression in human and murine hepatocellular tumors: association with the tumor-necrotizing and antineoplastic effects of cadmium in mice. AB - Previously, we found that oral cadmium (Cd) treatment either prevented or substantially reduced N-nitrosodiethylamine (NDEA)-induced tumor formation in B6C3F1 mouse liver or lung regardless of exposure interval and even when the Cd was given well after tumors were formed. Because Cd salts are powerful emetics, oral exposure would probably be impractical in humans. Thus, we studied suppression of NDEA-initiated tumors in male B6C3F1 mice by a single i.v. dose of Cd. NDEA (776 mumol/kg i.p.) was given at time 0 followed by CdCl2 (16 mumol/kg i.v.) 40 weeks later. This dose of Cd had no effect on body weights through the conclusion of the study at 52 weeks. The NDEA-induced increase in hepatic tumor incidence (19 tumor-bearing mice/22 mice at risk, 86%) over control (5/24, 21%) was remarkably reduced by Cd treatment (13/27, 48%, P < or = .05). Multiplicity and size of liver tumors induced by NDEA (2.18 tumors/liver; 31.6 mm3 mean volume) were also substantially reduced by the Cd exposure (0.96 tumors/liver; 17.1 mm3 mean volume). NDEA-induced lung tumor incidence (22/22, 100%) and multiplicity (5.09 tumors/lung) were modestly, but significantly, reduced by Cd treatment (21/27, 78%; 3.89 tumors/lung). Clear evidence of tumor-specific cytotoxicity was observed as Cd treatment induced a necrotizing effect that was localized only within the hepatic tumors. Metallothionein (MT), an inducible metal-binding protein associated with tolerance to many metal including Cd, was not detected immunohistochemically in mouse liver tumors, even those undergoing Cd-induced necrosis, whereas the surrounding normal liver cells expressed high levels of MT after Cd exposure. Likewise, in human hepatocellular carcinomas MT was only poorly or erratically expressed relative to normal tissue. These results indicate that a single, nontoxic dose of Cd dramatically reduces liver tumor burden through tumor cell-specific necrosis due to a down-regulation of MT expression in hepatic tumors of murine origin and furthermore indicate that a similar down-regulation of MT occurs in human hepatocellular carcinomas. PMID- 8627514 TI - Vasopressin mediates the pressor effects of endothelin in the subfornical organ of the rat. AB - The potent vasoconstrictor peptide endothelin may affect central cardiovascular function in areas with incomplete blood-brain barrier such as the subfornical organ (SFO). In these studies, we determine whether microinjection of endothelin 1 (ET-1) into the SFO increases blood pressure (BP) in a dose-related manner and investigate the potential involvement of sympathetic and vasopressinergic mechanisms. In urethane-anesthetized Sprague-Dawley rats, BP was recorded intra arterially, and ET-1 (0.125-6.0 pmol/60 nl) was microinjected stereotaxically into the SFO. Whereas vehicle (60 nl) did not change mean BP or HR, ET-1 evoked a dose-related pressor and bradycardic effect. The maximal changes were noted at the 1-pmol dose. No significant hemodynamic effects were observed with ET-1 microinjection in areas immediately surrounding the SFO or into the SFO of rats pretreated with a specific endothelin antagonist. In animals instrumented for recording of renal sympathetic nerve activity (RSNA), the administration of ET-1 (1 pmol/60 nl) evoked pressor (14 +/- 5 mm Hg) and bradycardic (-41 +/- 12 bpm) effects with a decrease in RSNA (16% +/- 3%). The effects on HR and RSNA seem to be mediated by baroreflex changes because in sino-aortic denervated rats, ET-1 pressor effects occur without inhibition of HR or RSNA. We documented the involvement of vasopressin in ET-1 actions by using vasopressin antagonists that inhibited the effects evoked by ET-1 administration. In addition, increases in vasopressin plasma levels were demonstrated at the time of the maximal effect of this peptide. These results indicate that ET-1 acting in the SFO increases BP by a vasopressinergic mechanism. PMID- 8627515 TI - Properties of Ca++ release induced by puff adder lectin, a novel lectin from the snake Bitis arietans, in sarcoplasmic reticulum. AB - Puff adder lectin (PAL), a novel lectin venom purified from Bitis arietans, induced Ca++ release from the heavy fraction (HSR) but not from the light fraction of skeletal muscle sarcoplasmic reticulum with EC50 congruent to 10 microM. The potency of PAL was approximately 200-fold higher than that of caffeine. The bell-shaped profile of Ca++ dependence for PAL was almost the same as that for myotoxin a (MYTX), a peptide Ca++ releaser, but was different from that for caffeine. Typical blockers of Ca++ release channels, such as Mg+2, procaine, ruthenium red and ryanodine, markedly reduced PAL-induced Ca++ release from HSR. Interestingly, PAL inhibited 125I-MYTX binding to HSR with IC50 approximately equal to 20 microM. Scatchard analysis revealed that the mode of inhibition by PAL was noncompetitive, which suggests that PAL binds to a different site from that of MYTX. PAL did not affect 3H-ryanodine binding to HSR. These results suggest that PAL binds to a different site from that of MYTX to cause Ca++ release from HSR with novel properties. PMID- 8627516 TI - Biodistribution of the nitroimidazole EF5 (2-[2-nitro-1H-imidazol-1-yl]-N (2,2,3,3,3-pentafluoropropyl) acetamide) in mice bearing subcutaneous EMT6 tumors. AB - The characteristic reduction and binding of nitroimidazoles to cellular macromolecules in the absence of oxygen allows their use for detection and characterization of hypoxia. The biodistribution of a new nitroimidazole, EF5 (2 [2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide), in mice bearing EMT6 tumors is described. Detection methods based on radioactivity and monoclonal antibody techniques are compared for liver and tumor. All nonexcretory tissues demonstrated similar levels of radioactivity at 0.5 hr postinjection of drug, demonstrating equivalent access of EF5 to all tissues. At 24 hr, when unbound drug has been cleared, the tissues with the highest binding are the liver, esophagus, bladder and tumor. Typically, liver tissue contains the highest level of radioactivity at this time. Examination of tumor and liver tissue by use of fluorescence microscopy and Cy3-bound monoclonal antibodies specific for EF5 adducts showed that the patterns of binding in tumor are considerably more heterogeneous than those of liver. Histograms of fluorescence intensity, with use of these antibodies, demonstrate average and maximal binding higher in tumors than in the liver. This divergence from the radioactivity data was determined to be unrelated to sampling error, differential antibody access or staining efficiency of liver vs. tumor tissue. A possible cause is the scavenging of radioactive drug metabolites by liver. The data presented herein suggest that EF5 is useful as a hypoxia detector and that monoclonal antibody detection methods can give detailed information on the distribution of EF5 binding. This technology may allow an accurate estimation of the oxygenation and/or nitroreductase levels in both tumor and normal tissues. PMID- 8627517 TI - Molecular mechanism for alkyl sulfide-modulated carbon tetrachloride-induced hepatotoxicity: the role of cytochrome P450 2E1, P450 2B and glutathione S transferase expression. AB - The modulation of CCl4-induced hepatotoxicity in response to alkyl sulfides and alkyl ethers including allyl disulfide (ADS), allyl sulfide (AS), allyl ether (AE), propyl disulfide (PDS), propyl sulfide (PS), propyl ether (PE) and butyl sulfide (BS) was studied. Whereas pretreatment of rats with either ADS or AS (50 mg/kg, 7 days) blocked a CCl4-induced increase in plasma alanine aminotransferase (ALT) activity by 91 and 56%, respectively, AE, PDS, PS, PE or BS treatment enhanced CCl4-induced ALT activity by 52, 55, 238, 25 or 86%, respectively. Histochemical examinations supported the results of plasma ALT activity. Injection of GdCl3 to PS-pretreated rats failed to block the potentiated ALT increase, whereas GdCl3 completely prevented vitamin A-enhanced elevation of ALT activity. AS treatment completely blocked PS-potentiated CCl4 intoxication. Concomitant treatment of animals with both PS and vitamin A followed by a CCl4 insult resulted in super-potentiation of CCl4-induced hepatotoxicity, suggesting that the mechanism of PS-enhanced hepatotoxicity differs from that caused by vitamin A. Pyridine or phenobarbital potentiation of CCl4-induced increases in ALT activity implys that cytochrome P450 2E1 (P450 2E1) and P450 2B expression may be associated with the increased toxicity. P450 2E1 expression appeared to be associated with the alkyl sulfide-modulated hepatotoxicity, as evidenced by both immunoblot analyses and metabolic activity. P450 2B immunoblot analysis revealed that either AS or PS substantially induced hepatic P450 2B1/2 levels. Thus, PS enhanced CCL4 hepatotoxicity may be related in part with P450 2B induction. ADS, AS or PS treatment caused increases in the glutathione S-transferase (GST) conjugating activity toward 1-chloro-2,4-dinitro-benzene. ADS, AS or PS induced Ya and Yb1 subunits by 2- to 3-fold. ADS or AS treatment also significantly elevated the levels of Yc subunits. PS failed to induce Yc expression, although this agent effectively increased Yb2 expression. Northern blot analyses revealed that ADS and AS concomitantly stimulated GST Ya, Yb1 and Yc2 gene expression, whereas PS increased the levels of Ya, Yb1, and Yb2 mRNA, but not Yc2 mRNA levels. The expression of GST subunit Yc2 in response to these compounds might be associated with hepatoprotective effects. These results demonstrate that ADS and AS have distinct capability of blocking CCl4-induced hepatotoxicity, whereas certain saturated alkyl sulfides rather potentiate CCl4-induced hepatotoxicity and that the underlying mechanism is associated with P450 2E1 and P450 2B expression, and possibly with certain GST expression. PMID- 8627518 TI - Enhanced uptake of rsCD4 across the rodent and primate blood-brain barrier after conjugation to anti-transferrin receptor antibodies. AB - The delivery to the brain of nonlipophilic therapeutic compounds, especially proteins, is severely hindered by the presence of the blood-brain barrier, which is formed by the tightly apposed brain capillary endothelial cells. However, brain endothelial cells do possess specific receptor-mediated transport mechanisms so that substances required by the brain can cross the blood-brain barrier. By use of monoclonal antibodies that bind to the transferrin receptor present on the luminal surface of brain capillary endothelial cells, we have taken advantage of the transport system responsible for the delivery of iron to the brain to deliver recombinant human soluble CD4 (rsCD4), a potential anti-HIV therapeutic, across the blood-brain barrier. Anti-transferrin receptor antibody rsCD4 conjugates were synthesized with a disulfide linkage and characterized in vitro. Experiments that use immunohistochemistry to localize these conjugates after intravenous administration into the tail vein of rats have shown that both the carrier antibody and the protein "passenger" accumulate in brain capillaries. The carrier-mediated delivery of radiolabeled protein across the blood-brain barrier in vivo was also examined in both rodents and primates. With use of the technique of capillary depletion in rats, the amount of rsCD4 in the capillary fraction of the brain, which reaches a maximal value within 1 hr postinjection, was shown to decrease with time, whereas the amount in the brain parenchyma increased, which suggests that the protein was delivered across the blood-brain barrier. In primates rsCD4 levels in the brain were increased 5-fold when the protein was administrated intravenously in the form of an anti-transferrin receptor antibody-rsCD4 conjugate. PMID- 8627519 TI - Functional classification of antidepressants based on antagonism of swim stress induced fos-like immunoreactivity. AB - Autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake and immunocytochemical assessment of Fos-like immunoreactivity (Fos-LI) were used to assess swim stress-induced changes in metabolic activity in brain and to define the effect of chronic treatment with antidepressants from different pharmacological classes. Saline-treated rats processed in the forced swim test exhibited marked increases in Fos-LI in limbic cortical regions, lateral septum, medial amygdala and paraventricular nucleus of the hypothalamus (PVN). Uptake of 2-DG was increased by swim stress in some of the same brain regions where Fos-LI was induced, with the notable exception of a lack of a change in the PVN. Rats received injections for 3 wk with imipramine, desipramine, fluoxetine, nisoxetine, tranylcypromine or mianserin before being processed in the forced swim test. Chronic treatment with imipramine and desipramine alone induced Fos-LI in the central nucleus of the amygdala and the dorsolateral bed nucleus of the stria terminalis. After tranylcypromine treatment, Fos-LI was induced in many brain regions including limbic cortex, amygdala and paraventricular nucleus of the hypothalamus. None of the other antidepressants induced Fos-LI in any brain region examined. Chronic administration of imipramine, desipramine and nisoxetine antagonized the swim induced expression of Fos-LI in the PVN and in limbic cortical regions, including the medial prefrontal ventrolateral orbital and cingulate cortices. Chronic treatment with fluoxetine, tranylcypromine and mianserin did not alter swim stress-induced Fos-LI in any brain region. Thus, only antidepressant drugs that affect norepinephrine uptake (i.e., imipramine, desipramine and nisoxetine) antagonized swim stress-induced Fos-LI. In contrast to the action of chronic imipramine on Fos-LI induced by swim, chronic administration of imipramine did not antagonize the stress-induced changes in 2 DG uptake in limbic cortical regions. Acute administration of propranolol, which blocks beta-adrenergic receptors, reduced the number of cells staining for Fos-LI in limbic cortical regions, resembling the effects produced by chronic imipramine, desipramine and nisoxetine. In the PVN, neither propranolol nor prazosin (an alpha 1 antagonist) blocked the swim-induced Fos-LI, suggesting that swim-induced Fos-LI in the PVN is not under control of beta- or alpha 1 adrenergic receptors. These latter results imply that adaptation of noradrenergic receptors by chronic imipramine may not be related to the antagonism of stress induced Fos-LI. The clear functional differences of the various antidepressant agents on swim stress-induced Fos-LI after chronic administration provide a functional classification of antidepressant drug action not previously identified. PMID- 8627520 TI - Evaluation of immune parameters and lymphocyte production of prolactin immunoreactive proteins after chronic administration of cocaine to pregnant rats. AB - This study examined the effect of chronic cocaine exposure on selected immune parameters in pregnant rats. Cocaine hydrochloride, 60 mg/kg, was administered by i.p. injection as a divided daily dose on gestation days 8 to 19. This cocaine treatment regimen did not result in any change in maternal body weight, spleen and thymus body weight ratios or lymphocyte recovery from these organs. Cocaine treatment had no effect on the plasma levels of prolactin, growth hormone and insulin-like growth factor-1; hormones with immunoregulatory potential. In contrast, the plasma immunoglobulin G concentration in cocaine-treated animals was 48% higher (P < .05) than in control animals. Spleen lymphocytes and thymocytes were isolated and evaluated for their proliferative responses in vitro to a panel of T and B cell mitogens. Lymphocytes from cocaine-treated animals showed no significant differences in proliferative responses to concanavalin A (conA), phytohemagglutinin, pokeweed mitogen, interleukin-2 or lipopolysaccharide. The ability of conA-stimulated spleen lymphocytes to synthesize and secrete prolactin-immunoreactive proteins was further assessed by Western immunoblotting. We found that conA-stimulated spleen lymphocytes from cocaine-treated animals showed significantly decreased levels of intracellular and secreted 44,000-mw prolactin-immunoreactive proteins. In contrast, conA stimulated spleen lymphocytes from control and cocaine-treated groups secreted equivalent amounts of the cytokine interleukin-2. In conclusion, chronic administration of cocaine to female rats during pregnancy significantly altered serum immunoglobulin G levels and lymphocyte production of prolactin immunoreactive proteins in the absence of changes in lymphocyte proliferation in response to mitogens. PMID- 8627521 TI - Different effects of three bisphosphonates on nitric oxide production by RAW 264 macrophage-like cells in vitro. AB - The macrophage-suppressive properties of three bisphosphonates were evaluated by studying their effect on nitric oxide (NO) production by activated RAW 264 macrophage-like cells. The cells were activated with 10 micrograms/ml of lipopolysaccharide, and NO was determined as nitrite in the cell culture supernatant. The effect of the drugs on inducible NO synthase was determined by Western blot analysis. As free drugs, clodronate and pamidronate inhibited NO secretion in a dose-dependent manner, whereas alendronate had no effect. Liposome encapsulation enhanced the effect of clodronate by a factor of 7, but the potency of pamidronate weakened slightly when encapsulated in liposomes. The inducible NO synthase expression inside the cells was also decreased by liposomal clodronate. In contrast to pamidronate, clodronate could affect the NO secretion when given to the cells simultaneously with lipopolysaccharide, and the inhibitory action was still seen when the drug was added 2 h after lipopolysaccharide induction. The viability of the cells was not affected by free or liposomal clodronate, whereas pamidronate showed considerable cytotoxicity. This study shows the different actions of these three bisphosphonates on NO production by macrophages and suggests that liposomal clodronate is the most promising bisphosphonate as an anti-inflammatory agent, whereas aminobisphosphonates do not possess anti inflammatory properties. PMID- 8627522 TI - The 5-hydroxytryptamine2B receptor and 5-HT receptor signal transduction in mesenteric arteries from deoxycorticosterone acetate-salt hypertensive rats. AB - One of the most profound increases in vascular responsiveness in hypertension has been observed for serotonin (5-hydroxytryptamine, 5-HT). This study investigates the hypothesis that the increase in vascular responsiveness to 5-HT is the result of altered 5-HT receptor signal transduction. Mesenteric arteries were dissected from deoxycorticosterone- (DOCA) salt hypertensive and sham-normotensive rats for use in isolated tissue experiments. Agonist contractile potencies indicated that a 5-HT2 receptor mediates contraction to 5-HT in both sham and DOCA-salt arteries. In arteries from sham rats, ketanserin (5-HT2A/5-HT2C selective), LY53857 (5-HT2 selective) and spiperone (5-HT2A/5-HT2C selective) shifted contraction to 5-HT (pKB = 8.58, 8.35 and 9.52, respectively) indicating that a 5 HT2A receptor mediates contraction in arteries from normotensive rats. By contrast, ketanserin and spiperone did not shift contraction to 5-HT in DOCA-salt mesenteric arteries (pKB > 6.52, > 7.52, respectively). LY53857 did shift the response to 5-HT in DOCA-salt mesenteric arteries (pKB = 7.85). Thus, contraction in arteries from DOCA-salt rats is predominantly mediated by 5-HT2B receptors. Unlike the 5-HT receptor in the sham mesenteric artery and aorta (5-HT2A receptor), the 5-HT receptor in DOCA-salt mesenteric arteries and stomach fundus (5-HT2B receptor) were relatively insensitive to phenoxybenzamine (10-300 nM). These data suggest that the 5-HT2B receptor is insensitive to phenoxybenzamine, is increased in number or, alternatively, has increased G protein coupling. DOCA salt mesenteric arteries were more sensitive to contraction by the direct G protein stimulator AIF4- (-log EC50 [M]: DOCA-salt = 2.82 +/- 0.04; sham = 2.55 +/- 0.03, P < .05). PCR analyses indicated an increase in mRNA for the 5-HT2B receptor in mesenteric arteries of DOCA-salt hypertensive arteries, supporting an increase in receptor number. Taken together these studies demonstrate significant changes in 5-HT receptor signal transduction in DOCA-salt hypertension, both at the level of the receptor and G protein and may provide one reason why ketanserin has proved to be a relatively ineffective antihypertensive agent in some forms of hypertension. PMID- 8627523 TI - Cocaine inhibits sympathetic neural activity by acting in the central nervous system and at the sympathetic ganglion. AB - The effects of cocaine on spontaneous pre- and postganglionic sympathetic nerve activity (SNA), mean blood pressure and heart rate were assessed in anesthetized cats. I.v. administration of 2 and 4 mg/kg decreased preganglionic SNA by -24 +/- 7% (n = 4) and -63 +/- 15% (n = 5), respectively. The 4 mg/kg dose produced a decrease in mean blood pressure (-49 +/- 6 mm Hg) with no change in heart rate. Similar sympathoinhibition was obtained in animals with denervated cardiovascular reflexes. Cocaine methiodide (4 mg/kg), a quaternary derivative of cocaine with limited central nervous system access, reduced mean blood pressure (-47 +/- 9 mm Hg) but did not inhibit preganglionic SNA. Cocaine (4 mg/kg) also reduced sympathetic discharge to the adrenal medulla as evidenced by a -62 +/- 6% (n = 5) decrease in splanchnic nerve firing. Cocaine, 2 and 4 mg/kg i.v., decreased postganglionic cardiac SNA by -54 +/- 5% (n = 7) and -60 +/- 13% (n = 4), respectively. The 2 mg/kg dose depressed postganglionic SNA to a greater extent that it did preganglionic SNA. Furthermore, cocaine methiodide (2 mg/kg) reduced postganglionic SNA by -85 +/- 15%. The depressant effect of cocaine (2 mg/kg) on postganglionic cardiac SNA was attenuated after treatment with phentolamine (5 mg/kg i.v.). Lidocaine administered in doses equimolar to those of cocaine did not significantly affect sympathetic discharge. These results indicate that cocaine acts in the central nervous system and at the stellate ganglion to inhibit sympathetic discharge and that these sympathoinhibitory effects are unrelated to cocaine's local anesthetic action. PMID- 8627524 TI - Involvement of the ventral tegmental area in locomotion elicited from the nucleus accumbens or ventral pallidum. AB - This study was designed to evaluate the role of the circuit containing the nucleus accumbens, ventral pallidum (VP) and ventral tegmental area (VTA) in the motor stimulation produced by the microinjection of dopamine, alpha-amino-3 hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or [D-Ala2, MePhe4,Gly ol5]enkephalin (DAMGO) into VP or the shell and core compartments of the nucleus accumbens. Initial dose-response curves revealed that dopamine was approximately equipotent at producing motor activity after microinjection into the core and shell, AMPA was more effective in the core, whereas DAMGO was more potent in the shell. A role for the VTA in the motor responses elicited by dopamine, AMPA or DAMGO microinjection into the shell, core or VP was evaluated by microinjecting the tau-aminobutyric acidB agonist baclofen into the VTA to inhibit neuronal activity. Baclofen treatment abolished the motor responses elicited by AMPA from the shell, core and VP. The motor effect of DAMGO in the VP was abolished by baclofen, whereas the response in the shell was attenuated. The motor response to dopamine was unaltered by baclofen, regardless of the injection site. These data indicate that there exist differences between the core and shell of the nucleus accumbens in the capacity of neurotransmitter analogs to elicit motor activity, and that although AMPA-induced motor activity is dependent upon neurotransmission in the VTA after microinjection into the core, shell and VP, DAMGO-induced locomotion only requires such tone after microinjection into the VP and shell. PMID- 8627525 TI - Characterization of the cloned HEL cell thromboxane A2 receptor: evidence that the affinity state can be altered by G alpha 13 and G alpha q. AB - Thromboxane A2 (TXA2) induces activation of platelets and vascular smooth muscle contraction via cell surface receptors. A platelet type TXA2 receptor from the megakaryocyte-like HEL cell was cloned with a deduced amino acid sequenced identical to that previously reported for the human placental TXA2 receptor. Transient expression of the HEL cell TXA2 receptor cDNA and radioligand binding studies with the agonist 125I-BOP showed a single class of binding sites with an affinity comparable to a low affinity platelet TXA2 receptor. Using a series of 13-azapinane TXA2 analogs, which discriminate between TXA2 receptor subtypes in platelets and vascular smooth muscle, we found that the cloned HEL cell TXA2 receptor is characteristic of a platelet type TXA2 receptor and that its binding characteristics are different from those of vascular smooth muscle cells. The affinity of the HEL cell TXA2 receptor for 125I-BOP was significantly (P < .05) increased upon co-transfection with G alpha 13 alone, or with G alpha q alone and with G alpha 13 and G alpha 12 together (n = 4-6). GTP gamma S significantly (P < .05) decreased the affinity of the receptor for 125I-BOP in COS-7 cell membranes coexpressing HEL-TXR and G alpha 13 to a value comparable to HEL-TXA2 receptor alone. We conclude that 1) the cloned HEL cell TXA2 receptor has pharmacological characteristics of a low affinity platelet type receptor and 2) that the affinity state of this receptor may be influenced by interaction with G alpha 13 and G alpha q. PMID- 8627526 TI - Glial cell line-derived neurotrophic factor sustains axotomized basal forebrain cholinergic neurons in vivo: dose-response comparison to nerve growth factor and brain-derived neurotrophic factor. AB - Glial cell line-derived neurotrophic factor (GDNF) was infused continuously for 2 weeks into the ventricles of male Wistar rats that had received a unilateral knife transection of the fimbria/fornix. In vehicle-treated, control animals, there was a 70% loss of choline acetyltransferase (ChAT)-positive and a 60% loss of p75-positive neurons in the septum/diagonal band ipsilateral to the axotomy as identified by immunohistochemistry, with no loss in ChAT biochemical activity. GDNF treatment at 10 micrograms/day completely prevented the loss of p75-positive neurons, significantly reduced the loss of ChAT-positive neurons to 40% of normal, and stimulated ChAT biochemical activity to 40% more than normal in an axotomy-dependent manner. GDNF is 1 order of magnitude less potent than NGF but, unlike NGF, had little or no effect on normal, uninjured neurons. GDNF was 1 order of magnitude more potent than BDNF, and BDNF had no effect on ChAT biochemical activity. GDNF and NGF inhibited weight gain, whereas BDNF induced significant weight loss and death at the dosage of 100 micrograms/day. PMID- 8627528 TI - Determinants of the response of neuroblastoma cells to DNA damage: the roles of pre-treatment cell morphology and chemical nature of the damage. AB - Chemotherapeutic agent-induced DNA cleavage gives rise to apoptosis in a subpopulation of SK-N-SH human neuroblastoma cells; the remaining cells undergo Schwann cell-like differentiation. Like other neural crest and primitive neurectodermal tumor-derived cell lines, SK-N-SH cultures contain cells of neural (N-type) and epithelial (substrate-adherent, or S-type) phenotypes. Using isolated N-type and S-type cells from neuroblastoma, medulloblastoma, melanoma and glioma cell lines, we demonstrate that the determinants of the response to DNA cleavage are intrinsic properties of the cell. Furthermore, using a series of analogues of enediyne deoxyribonucleic acid (DNA) cleaving agents, we show that the molecular target of these agents is likely to be the same in N- and S-type cells, implying that the difference in response characteristics is a function of different distal pathways that are triggered by DNA cleavage. We demonstrate that the concentration of the DNA damaging agent used, and not the specific characteristics of the damage it produces, is the trigger for production of the cellular response. Response type does not correlate with previously published values for expression of the apoptosis modulators Bcl-2, Bcl-XL, wildtype p53, or, in medulloblastoma lines, p75. PMID- 8627527 TI - Procollagen gene expression is down-regulated by taurine and niacin at the transcriptional level in the bleomycin hamster model of lung fibrosis. AB - Taurine (T) and niacin (N) have previously been found to block the accumulation of collagen in the bleomycin (BL) model of interstitial pulmonary fibrosis. The present study was designed to evaluate whether the mechanism for the antifibrotic effect of combined treatment with taurine and niacin involves the down-regulation of BL-induced overexpression of procollagen I and III messenger ribonucleic acid (mRNA) levels in lungs. Hamsters were intratracheally instilled with three consecutive doses of saline or BL at weekly intervals (2.5, 2.0, 1.5 units/5 ml/kg). Four groups of animals were fed a diet throughout the experiment containing either 2.5% taurine and 2.5% niacin or the same diet without the drugs. The four groups were saline-instilled with the control diet (SA + CD), saline-instilled with TN in the diet (SA + TN), BL-instilled with the control diet (BL + CD), and BL-instilled with the TN diet (BL + TN). Steady state transcript levels in total RNA prepared from lungs of all four groups were determined at 0, 3, 7, 14 and 21 days after the last BL instillation by slot blot and Northern blot analyses. Results indicate that procollagen I mRNA levels are elevated compared with saline control by 2.5-, 2.4- and 2.0-fold at 7, 14, and 21 days after the last dose of BL instillation, respectively. Dietary treatment with taurine and niacin decreased the steady state level of BL-induced increases of procollagen I mRNA from day 0 through 21. We observed a similar pattern of procollagen III inhibition by taurine and niacin from day 3 through day 21. Transcription of procollagen I and III genes was readily detected in nuclei prepared from BL-treated lung samples at 14 days after treatment. In contrast, transcription of procollagen I and III genes was barely detectable in nuclei prepared at the same time point from BL + TN treated lungs. Our results suggest that procollagen I and III gene expression in BL-induced lung fibrosis in hamsters is transcriptionally down-regulated by combined treatment with taurine and niacin. PMID- 8627529 TI - Quantitative assessment of blood-brain barrier damage during microdialysis. AB - In view of the increasing use of microdialysis for monitoring drug uptake into the brain, the consequences of tissue/blood-brain barrier (BBB) damage that occurs on microdialysis probe insertion on the extent and rate of solute uptake need to be more carefully examined. In this study, both microdialysis and a classic method were used to compare the apparent brain uptake of two polar permeants, [3H]sucrose and [14C]urea. The blood-to-brain transfer constants of these compounds differ significantly, with the value of urea exceeding that of sucrose by a factor of approximately 20 when compared by the classic one-point per-animal method. The BBB selectivity to these nonmetabolized permeants as assessed by microdialysis provides a sensitive measure of the integrity of the BBB to polar nonelectrolytes within the molecular size range of most drugs. The following evidence for blood-brain barrier damage during microdialysis sampling was obtained: (1) the loss of [3H]sucrose from the extracellular fluid in brain on termination of an intravenous infusion is biphasic, with the initial phase evident immediately on termination by the infusion, suggesting that a fraction of the microdialysis probe resides in a region in rapid equilibrium with plasma; (2) complete loss of selectivity in the rate constants for CNS entry of sucrose vs. urea and (3) there were substantially higher area under the concentration vs. time curve AUCECF/AUCplasma ratios for both sucrose and urea generated by microdialysis than the corresponding ratios (AUCCSF/AUCplasma or AUCbrain/AUCplasma) obtained by classic methods. These results suggest that the BBB to small molecule transport likely remains compromised for some time after microdialysis probe insertion. PMID- 8627530 TI - Evidence for selective involvement of dopamine D1 receptors of the ventral tegmental area in the behavioral sensitization induced by intra-ventral tegmental area injections of D-amphetamine. AB - The induction of behavioral sensitization to D-amphetamine (AMPH) is known to result at least in part from an action of the drug in the ventral tegmental area (VTA), which contains the dopamine (DA) A10 cell bodies. To specify the cellular mechanisms through which AMPH acts in the VTA and leads to behavioral sensitization after repeated intra-VTA injections, the involvement of VTA DA D1 and D2 and serotonin2 receptors in this phenomenon was investigated in independent experiments. The results reported here confirm that repeated intra VTA AMPH injections (four injections of 5 micrograms/0.5 microliter every other day) induce behavioral sensitization, as revealed by the potentiation of the locomotor response to peripheral challenges with AMPH (0.5 mg/kg) 4 or 15 days after treatment. This behavioral sensitization induced by intra-VTA administration of AMPH cross-reacts with morphine (1 microgram/0.5 microliter) administered into the VTA 7 days after treatment. We demonstrated that the D1 receptor antagonist (R)-(+)-8-chloro-2,3,4, 5-tetrahydro-3-methyl-5-phenyl-1H-3 benzazepin-7-ol (0, 0.01, 0.1 and 1 microgram/ 0.5 microliter) coadministered in the VTA with AMPH dose-dependently prevents the behavioral sensitization to peripheral AMPH challenges (at either 4 or 15 days after treatment) as well as the cross-sensitization with intra-VTA morphine. Neither DA D2 nor serotonin2 receptor blockade, using sulpiride (10 micrograms/0.5 microliters) and ketanserin (1 micrograms/0.5 microliters), respectively, had any effect on the induction of behavioral sensitization. In conclusion, these results demonstrate the selective involvement of VTA DA D1 receptors in the process by which AMPH acts in the VTA to induce behavioral sensitization. PMID- 8627531 TI - Transdermal modification of platelet function: an aspirin patch system results in marked suppression of platelet cyclooxygenase. AB - Aspirin inhibits platelet cyclooxygenase and prevents thromboxane A2 (TXA2) production. Although it is an effective antithrombotic, even at a low doses aspirin may induce gastrointestinal toxicity. We examined the feasibility of delivering aspirin transdermally using two patch systems, one without (type A) and one with (type B) the permeation enhancer limonene. Daily application of two type A patches that had a total surface area of 100 cm2 and contained 84 mg/patch resulted in 85% +/- 6% reduction in serum TXB2 in six male subjects by day 14. Suppression of serum TXB2 was less marked in females (32% +/- 16%). Analysis of the residual drug in the patch showed that each patch delivered 18 +/- 3 mg on day 1 and 17 +/- 4 mg on day 14, with no difference between males and females. Daily application of a single patch B that had a surface area of 50 cm2 and contained 120 mg aspirin resulted in 60% +/- 11% suppression of serum TXB2 by day 14 in nine male subjects and 84% +/- 9% suppression by day 21. Analysis of the applied patches showed that patch B delivered 33 +/- 3 mg of aspirin daily. Plasma aspirin and salicylate were determined by gas chromatography, mass spectrometry. No aspirin was detected, whereas plasma salicylate was 157 +/- 38 ng/ml and 133 +/- 20 ng/ml by day 14 with patch A and patch B, respectively. Analysis of aspirin applied by patch to the skin in three subjects showed marked hydrolysis to the inactive product, salicylic acid. Aspirin can be delivered transdermally by patch in a dose that suppresses platelet cyclooxygenase. The delivery rate is low reflecting hydrolysis of the drug in the skin. Delivery is improved by the permeation enhancer limonene. This novel route of delivery may be applicable to other antithrombotics and may limit the risk of gastrointestinal toxicity. PMID- 8627532 TI - Protective effects of dilazep and its novel derivative, K-7259, on mechanical and metabolic derangements induced by hydrogen peroxide in the isolated perfused rat heart. AB - The effect of dilazep, a potentiator of the adenosine-mediated effects, on the hydrogen peroxide (H2O2)-induced mechanical and metabolic derangements was studied in the isolated rat heart, and compared with that of K-7259, a dilazep derivative having less potentiating action on the adenosine-mediated effects. The heart was perfused aerobically by Langendorff's technique at a constant flow and driven electrically. H2O2 (600 microM) decreased the left ventricular developed pressure and increased the left ventricular end diastolic pressure in the heart (i.e, mechanical dysfunction), decreased the tissue ATP level and increased the tissue AMP level (i.e., metabolic change) and increased the tissue level of malondialdehyde (i.e., lipid peroxidation). These mechanical and metabolic alterations induced by H2O2 were attenuated by dilazep (1 microM), and the effect of dilazep was not modified by 8-(p-sulfophenyl)-theophylline (20 microM), a nonselective adenosine receptor antagonist. K-7259 (1 microM) also attenuated the H2O2-induced mechanical and metabolic derangements. Nevertheless, neither dilazep nor K-7259 modified the tissue malondialdehyde level, which was increased by H2O2, and the mechanical function and energy metabolism of the normal (H2O2 untreated) heart. These results suggest that both dilazep and K-7259 attenuate mechanical and metabolic derangements induced by H2O2. The protective action of dilazep and K-7259 on the H2O2-induced derangements is not due to potentiation of adenosine-mediated effects, reduction of lipid peroxidation or preservation of energy. PMID- 8627533 TI - The angiotensin receptor antagonist 2-ethoxy-1-[[2'-(1H- tetrazol-5-yl) biphenyl 4-yl]methyl]-1H-benzimidazole-7-carboxylic acid (CV11974) attenuates the tubuloglomerular feedback response during NO synthase blockade in rats. AB - Nitric oxide (NO) produced in the juxtaglomerular apparatus may regulate the tubuloglomerular feedback (TGF) response. The inhibition of intrinsic NO results in significant renal hemodynamic changes, a phenomenon similar to that observed after angiotensin II (A-II) administration. We measured stop-flow pressure (Psf) during loop perfusion with artificial tubular fluid in Sprague-Dawley rats to establish whether alterations in TGF responsiveness during NO inhibition depend on the action of endogenous A-II. The NO synthase blocker N omega-nitro-L arginine-methyl-ester (L-NAME: 10 mg/kg i.v.) significant increased TGF responsiveness, defined as the change in Psf on increasing loop flow from 0 to 40 nl/min compared with control (delta Psf: -21.3 +/- 2.6 vs. -9.7 +/- 0.6 mm Hg, P < .001). After concomitant treatment with the nonpeptide A-II type 1 receptor antagonist 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-1H benzimidazole-7-carboxic acid (CV11974: 1 mg/kg i.v.) and L-NAME, the TGF response was attenuated significantly (delta Psf: -7.6 +/- 1.9 mm Hg, P < .001). On the other hand, Psf in the absence of loop perfusion was increased similarly by L-NAME treatment in the presence (53.7 +/- 2.2 mm Hg) or absence of CV11974 (Psf 50.7 +/- 3.2 mm Hg). These results suggest that augmentation of the TGF response by endogenous NO inhibition depends, at least in part, on the intrinsic A-II activity. PMID- 8627534 TI - The atypical neuroleptic profile of the glycine/N-methyl-D-aspartate receptor antagonist, L-701,324, in rodents. AB - The present study has examined the glycine/N-methyl-D-aspartate antagonist, L-701 324 [7-chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2 (H)quinolone] in rodent behavioral tests commonly used to predict antipsychotic potential and side effect liability in humans. Pretreatment with L-701,324 dose-dependently antagonized amphetamine induced hyperactivity in the mouse (ED50 = 1.12 +/- 0.45 mg/kg p.o.), an effect which was similar to that of the classical neuroleptic, haloperidol, and the atypical neuroleptic, clozapine. In addition, p.o. administration of L-701,324 (2.5 or 5 mg/kg) attenuated the hyperactivity response induced by amphetamine infusion into the rat nucleus accumbens. In contrast to haloperidol, however, stereotyped sniffing and licking/biting, induced by either the systemic administration of apomorphine or infusion of amphetamine into the striatum, was not altered in rats pretreated with L-701,324 (30 or 100 mg/kg p.o.). Furthermore, L-701,324 failed to impair spontaneous locomotor activity or induce catalepsy in the mouse at doses > or = 100 mg/kg. Although a significant reduction in spontaneous activity was observed in rats pretreated with L-701,324, the minimum effective dose (10 mg/kg p.o.) was 2-fold greater than that which abolished amphetamine-induced hyperactivity in this species. Thus, L-701,324 selectively blocks behaviors associated with the activation of the mesolimbic dopamine system suggesting that glycine/N-methyl-D-aspartate receptor antagonists may offer a novel approach to the treatment of schizophrenia in humans. PMID- 8627536 TI - Selective mechanism-based inactivation of rat CYP2D by 4-allyloxymethamphetamine. AB - The high selectivity of amphetamine and its derivatives for CYP2D-mediated oxidations suggested the use of the phenylisopropylamine skeleton as a template for a selective inhibitor of this important enzyme. Accordingly, 4 allyloxymethamphetamine-amine (ALLMA) was synthesized and its ability to selectively inactivate CYP2D was investigated both in in vitro and in vivo experiments. Incubation studies with rat liver microsomes demonstrated that this compound suppressed the CYP2D-mediated methylenedioxymethamphetamine (MDMA) demethylation in time- and dose-dependent manner and that the inhibition required the presence of NADPH. The development of irreversible inhibition was associated with oxidation at position 4 of the aromatic ring, the common site of CYP2D mediated oxidation of this group of compounds. In in vivo studies doses of ALLMA (1-10 mg/kg) were administered to adult male Sprague-Dawley rats and liver microsomes were obtained 3 hr later. Methamphetamine p-hydroxylation and low Km MDMA demethylation activities, both mediated by CYP2D, were reduced by more than 80% after a dose of 10 mg/kg. Cytochrome P-450 reactions attributed to P-450s other than CYP2D, such as aniline p-hydroxylation, the high Km system of MDMA demethylation and the N-demethylation of methamphetamine, benzphetamine, aminopyrine and erythromycin, all appeared to be minimally affected. The importance of aromatic ring oxidation in the metabolism is such that inhibition of CYP2D would be expected to cause a significant change in the pharmacokinetics of these compounds. The kinetics of MDMA metabolic activity in microsomes from ALLMA-pretreated rats were comparable to those from female Dark-Agouti rats, an animal model for CYP2D1 deficiency. PMID- 8627535 TI - In vivo characterization of a specific cannabinoid receptor antagonist (SR141716A): inhibition of delta 9-tetrahydrocannabinol-induced responses and apparent agonist activity. AB - SR141716A has been described as a cannabinoid receptor antagonist. This study was conducted to determine whether SR141716A was capable of antagonizing the pharmacological effects of the prototypical cannabinoid agonist delta 9-THC. The AD50 (+/- 95% confidence limits) obtained after a 10 min i.v. pretreatment with SR141716A in measures of hypoactivity, hypothermia, and antinociception were: 0.12 (0.02-0.66), 0.087 (0.037-0.201), and 0.16 (0.03-1.01) mg/kg, respectively. The effect of SR141716A lasted up to 1 hr (antinociception, 10 mg/kg), 4 hr (locomotion, 1 and 3 mg/kg), or 24 hr (hypothermia, 3 mg/kg). Further evaluation revealed an AD50 value of 2.7 mg/kg (1.7-4.4) in the PPQ-stretch procedure. Additionally, the ED50 (+/- S.E.) of morphine in the tail-flick antinociception procedure was increased by SR141716A (30 mg/kg, i.v.) from 3.2 (+/- 0.3) to 5.3 (+/- 0.6) mg/kg. Finally, SR141716A produced direct effects on locomotor activity at doses greater than 3 mg/kg. Locomotion was stimulated to more than 200% of control (20 mg/kg), with an ED50 value of 4.7 (+/- 1.5) mg/kg. The ED50 value represents stimulation to levels approximately 150% of control. It is not clear whether this pharmacological activity represents an uncharacterized action of SR141716A, or an index of tonic activity of an endogenous cannabinergic system. SR141716A will be useful in establishing the biochemical events responsible for the in vivo effects of exogenous cannabinoids, as well as in establishing the existence of a putative endogenous cannabinergic system. PMID- 8627537 TI - Identification of genetic markers for initial sensitivity and rapid tolerance to ethanol-induced ataxia using quantitative trait locus analysis in BXD recombinant inbred mice. AB - Rapid tolerance to rotarod ataxia has previously been demonstrated in mice after sequential ethanol injections. Here we tested DBA/2J and C57BL/6J mice for initial ethanol sensitivity; DBA/2J mice were more sensitive (0.40 +/- 0.17 mg/g brain) than C57BL/6J mice (1.44 +/- 0.12 mg/g). We then monitored the development of tolerance by quantifying blood ethanol concentrations at the recovery from ataxia over five sequential injections; tolerance reached a plateau in about 5 hr. DBA/2J mice became very tolerant (final ethanol threshold 3.47 +/- 0.16 mg/ml, an increase of 3.07 mg/ml, or 8.7-fold above base line); B6 became slightly tolerant (final ethanol threshold 2.62 +/- 12 mg/ml, and increase of 1.18, or 1.8-fold above base line). Therefore, by the end of the treatment regimen, the rank order of sensitivity of the two strains had reversed. We then tested 25 recombinant inbred strains from among strains representing a cross between C57BL/6J and DBA/2J inbred strains, followed by a quantitative trait locus analysis with a database of 1522 markers to identify provisional loci. This procedure identified 19 markers on 11 chromosomes for initial sensitivity, 18 markers on 9 chromosomes for tolerance (delta) and 21 markers on 11 chromosomes for tolerance (fold-increase). Of these, 17 markers were in common, which suggests that initial sensitivity and tolerance share substantial genetic codetermination. Major candidate loci will be confirmed by genotyping B6D2F2 offspring that have been tested for initial sensitivity and tolerance. PMID- 8627538 TI - Evaluation of potential genetic associations between ethanol tolerance and sensitization in BXD/Ty recombinant inbred mice. AB - Ethanol (EtOH) has both locomotor stimulant and locomotor ataxic effects. Repeated EtOH treatment can result in the development of behavioral sensitization (increased sensitivity) similar to that seen with the classical stimulant drugs amphetamine and cocaine. However, it has been suggested for EtOH that sensitization may be a by-product of the development of tolerance to the sedative/ataxic effects of EtOH. It is also possible that the converse is true: that tolerance develops as the result of sensitization development. We examined this notion by measuring EtOH sensitization and tolerance in the BXD/Ty recombinant inbred strains. Changes in locomotor activation and grid test ataxia were used as the measures of sensitization and tolerance, respectively. If a genetic relationship exists between sensitization and tolerance, then those strains most susceptible to sensitization should also develop the most robust tolerance. Genetic correlations did not support the presence of this relationship. In addition, the use of the BXD/Ty recombinant inbred strains enabled us to perform gene mapping by quantitative trait locus analysis for activity and ataxia measures. We found that 28% to 79% of the genetic variation in the various activity and ataxia responses could be explained by the identified quantitative trait loci associations. However, when associations of gene markers with behavioral phenotypes were compared, we obtained no strong evidence for common genes determining magnitude of sensitization and tolerance. Thus the results of this study do not support the hypothesis that sensitization results from development of tolerance to the sedative/ataxic effects of EtOH or, conversely, that tolerance is a by-product of sensitization. PMID- 8627539 TI - Common genetic determinants of the ataxic and hypothermic effects of ethanol in BXD/Ty recombinant inbred mice: genetic correlations and quantitative trait loci. AB - Sensitivity and tolerance to ethanol-induced ataxia and hypothermia are determined in part by genetic factors; some genes that affect one of these traits may affect others as well. To test this general hypothesis, we examined hypothermia and two tests of ataxia in the C57BL/6J and DBA/2J inbred mouse stains and in 18 to 25 of their recombinant inbred strains. Genetic correlations among strain mean responses revealed strong positive associations of genetic origin between sensitivity and tolerance for each of the three responses. Furthermore, tolerance to grid test ataxia and tolerance to hypothermia were positively associated. Sensitivity scores across the three responses were uncorrelated. The second method employed to assess genetic correlation was to examine the pattern of genetic locations of quantitative trait loci (QTLs) provisionally identified using genetic mapping procedures. This method identified 3 to 14 QTLs associated with each trait. Within each response, a number of these associations were in common for measures of sensitivity and tolerance; this suggests the existence of several specific genes that exert pleiotropic effects on sensitivity and tolerance. In a result consistent with the analyses of genetic correlations, there was modest evidence for QTLs associated across measures. Some QTLs associated with multiple traits mapped to chromosomal regions where candidate genes (e.g., genes for neurotransmitter receptors) have been mapped. In summary, the analyses presented suggest modest commonality of genetic influence on tolerance to some measures of ataxia and hypothermia, and they strongly support previous data indicating that sensitivity and tolerance to specific effects of ethanol share common genetic determinants. PMID- 8627540 TI - Evidence for sympathetic and adrenal involvement in the immunomodulatory effects of acute morphine treatment in rats. AB - The present study examined the involvement of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis in the immunomodulatory effects of acute morphine treatment in rats. Chlorisondamine, a ganglionic blocker, was used to assess the involvement of sympathetic and sympathoadrenal activity. Adrenalectomized rats were used to assess the involvement of the adrenal cortex, which is regulated primarily by hypothalamic-pituitary-adrenal axis activity, and the adrenal medulla, which is regulated primarily by sympathetic activity. The results showed that both chlorisondamine and adrenalectomy antagonize morphine's suppressive effects on the proliferative response of splenic lymphocytes to concanavalin A (Con A), lipopolysaccharide or ionomycin/phorbol myristate acetate. Chlorisondamine, but not adrenalectomy, antagonizes morphine's suppressive effects on phytohemagglutinin (PHA)-stimulated proliferation of splenic lymphocytes and interferon-gamma production by stimulated splenocytes. Adrenalectomy, but not chlorisondamine, blocks morphine's suppressive effects on the proliferative response of blood lymphocytes to Con A or PHA. Neither chlorisondamine nor adrenalectomy alters morphine's suppressive effect on splenic natural killer cell cytotoxicity. Collectively, these results suggest that sympathoadrenal activity is involved in the suppressive effects of acute morphine treatment on the proliferative response of splenic T and B cells to Con A, lipopolysaccharide or ionomycin/phorbol myristate acetate. Morphine's suppressive effects on the proliferative response of splenic T cells to PHA and the production of interferon-gamma by stimulated splenocytes also involve sympathetic activity, but not sympathoadrenal activity. The results suggest further that morphine's suppressive effects on the proliferative response of blood T cells to Con A or PHA do not involve sympathetic activity, but rather adrenocortical activity. Neither sympathetic nor adrenocortical activity appears to be involved in morphine's suppressive effect on splenic natural killer cell cytotoxicity. PMID- 8627541 TI - Neurotoxin-induced lesions to central serotonergic, noradrenergic and dopaminergic systems modify caffeine-induced antinociception in the formalin test and locomotor stimulation in rats. AB - Our study examined the effects of neurotoxin-induced lesions to central 5 hydroxytryptamine (5-HT), noradrenaline (NA) and dopamine (DA) systems on antinociception by caffeine in the formalin test and on locomotor stimulation by caffeine. Locomotor activity was determined simultaneously with responses to formalin, although in some studies, scores were derived in the absence of formalin. Rats were tested 10 to 12 days after toxin administration, and amine levels in brain regions and the spinal cord determined at the end of the experiment. Intracerebroventricular 5,7-dihydroxytryptamine (+/- desipramine) (which depleted central 5-HT systems) inhibited antinociception by caffeine but had no effect on locomotor stimulation. Intracerebroventricular 6-hydroxydopamine (which depleted NA in brain and spinal cord and DA in brain) augmented both antinociception and locomotor stimulation by caffeine. Both effects were due to NA depletion as they were mimicked by microinjection of 6-hydroxydopamine into the locus ceruleus that selectively depleted NA, not by intracerebroventricular 6 hydroxydopamine with desipramine that selectively depleted DA. In the absence of formalin, locus ceruleus lesions no longer had a significant effect on locomotor stimulation. Phentolamine, an alpha-adrenergic antagonist, inhibited caffeine induced locomotor stimulation in the absence of formalin. These neurotoxin induced lesions reveal an involvement of central 5-HT and NA (but not DA) systems in antinociception, and an involvement of central NA (but not 5-HT or DA) systems in locomotor stimulation. There appears to be an interrelationship between noxious stimulation and the expression of locomotor activity. PMID- 8627542 TI - Electrical stimulation at traditional acupuncture sites in periphery produces brain opioid-receptor-mediated antinociception in rats. AB - Previous studies in rats measuring latency to tail flick with radiant heat have shown that the antinociceptive effect induced by electrical stimulation of different frequencies at traditional acupuncture sites is mediated via different opioid receptors in the spinal cord. The present study was designed to observe (1) whether electrical stimulation at such sites could produce antinociceptive effects in the cold water tail-flick (CWT) test; (2) whether the antinociceptive effects could be blocked by s.c. injection of the opioid receptor antagonist naloxone and (3) whether i.c.v. injection of selective antagonists for mu (cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, CTAP), delta (naltrindole) or kappa (nor binaltorphimine) opioid receptors would block the antinociceptive effect produced by electrical stimulation. Sprague-Dawley rats were stimulated at frequencies of 2, 30 or 100 Hz with acupuncture needles inserted into acupoints Zusanli and Sanyinjiao in the hind leg for 30 min. Antinociception was assayed in the CWT. The results showed that (1) a significant, frequency-related increase in threshold in the CWT was observed in all electrical stimulation groups as compared with the placebo group and the antinociceptive effect lasted about 30 min poststimulation; (2) naloxone (s.c.) antagonized the antinociceptive effect induced by 2 Hz, 30 Hz or 100 Hz electrical stimulation and (3) either CTAP or naltrindole (i.c.v.) almost completely blocked the antinociceptive effect induced by 2 Hz or 30 Hz electrical stimulation, but was less effective in blocking antinociception induced by 100 Hz electrical stimulation; nor-binaltorphimine (i.c.v.) greatly reduced antinociception induced by 30 Hz or 100 Hz electrical stimulation, but not by 2 Hz electrical stimulation. These results indicate that the antinociception induced by 2 Hz electrical stimulation is mediated by both mu and delta opioid receptors; the antinociception induced by 100 Hz electrical stimulation is mediated primarily by the kappa receptor; and the antinociception induced by 30 Hz electrical stimulation is mediated by all three opioid receptor types. Thus, the antinociceptive effect induced by peripheral electrical stimulation, as measured by the CWT, involves opioid receptors in the rat brain. PMID- 8627543 TI - 4-(2'-Methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-iodobenzamido]ethyl] piperazine and 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p- fluorobenzamido]ethyl]piperazine, two new antagonists at pre- and postsynaptic serotonin-1A receptors. AB - p-MPPI [4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-iodobenzamido] ethylpiperazine] and p-MPPF [4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p- fluorobenzamido]ethyl]piperazine] competitively antagonized 5-HT1A receptor activation in the rat, as measured by hypothermia, forepaw treading and 5-HT turnover; they exhibited to partial agonist activity on any of these measures. When given i.p., p-MPPI and p-MPPF dose-dependently antagonized the hypothermia induced by 8-hydroxy-2- (di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/kg), with approximate ID50 of 5 and 3 mg/kg, respectively. The inhibitory effect caused by a fixed dose of p-MPPI (6 mg/kg) or p-MPPF (3 mg/kg) was surmounted by higher doses of 8-OH-DPAT. p-MPPI and p-MPPF also attenuated the hypothermia induced by gepirone. Forepaw treading caused by 8-OH-DPAT (2 mg/kg) in reserpine-pretreated rats (1 mg/kg, s.c., 18-24 hr before the experiment) was dose-dependently antagonized by p-MPPI and p-MPPF with approximate ID50 of 3 and 0.7 mg/kg, respectively. p-MPPI also antagonized forepaw treading induced by 5-methoxy-N,N, dimethyltryptamine (5-MeODMT) (5 mg/kg) and this antagonism was competitively overcome by higher doses of 5-methoxy-N,N,-dimethyltryptamine. p-MPPI and p-MPPF were able to antagonize the 8-OH-DPAT-(0.5 mg/kg) induced reduction in the 5 HIAA/5-HT ratio, a measure of 5-HT turnover in the hippocampus or striatum. No hypothermia or reciprocal forepaw treading was produced by either drug when given at doses as high as 10 mg/kg. Neither p-MPPI nor p-MPPF (10 mg/kg) given alone significantly altered the ratio of 5-HIAA/5-HT in the hippocampus or striatum. Also, binding of [3H]p-MPPF to hippocampal membranes was unaltered by the addition of 100 microM guanylyl-imidodiphosphate to the incubation medium. In conclusion, p-MPPI and p-MPPF behave, in vivo, as competitive antagonists of both postsynaptic 5-HT1A receptors and somatodendritic 5-HT1A autoreceptors. PMID- 8627544 TI - Differential efficacy of the class III agent MK-499 against programmed stimulation-induced and ischemic-induced ventricular arrhythmias in a canine model of previous myocardial infarction. AB - Class III activity has been proposed as a potential mechanism for the treatment of reentrant arrhythmias. The purpose of the present study was to assess the concordance in antiarrhythmic efficacy of MK-499, a selective blocker of IKr, the rapidly activating component of cardiac delayed rectifier K+ current, against programmed ventricular stimulation (PVS)-induced ventricular tachycardias and thrombotically induced lethal ischemic arrhythmias, and to characterize the electrophysiologic determinants of antiarrhythmic efficacy in the canine model of previous myocardial infarction. Single i.v. doses of 1.0, 3.0 and 10.0 micrograms/kg MK-499 were administered to anesthetized dogs with anterior myocardial infarctions. Protection (suppression + stabilization/slowing) vs. PVS induced ventricular tachycardias occurred in 5/11 (45%) preparations at 1.0 microgram/kg, in 9/12 (75%) preparations at 3.0 micrograms/kg and in 10/11 (91%) preparations at 10.0 micrograms/kg i.v. MK-499. The incidences of lethal ventricular arrhythmias developing in response to thrombotically induced posterolateral myocardial ischemia were 34/40 (85%) in vehicle controls, 7/11 (64%) at 1.0 microgram/kg, 6/12 (50%, P < .05) at 3.0 micrograms/kg and 4/11 (36%, P < .01) at 10.0 micrograms/kg i.v. MK-499. Low-dose i.v. MK-499 prolonged ECG QT interval and increased noninfarct zone and infarct zone ventricular refractoriness. However, there was a poor concordance (56%) between response to PVS with MK-499 and response to thrombotically induced acute myocardial ischemia. Furthermore, different trends of association between site and magnitude of Class III effect and antiarrhythmic efficacy were observed for PVS- vs. ischemia induced arrhythmias. Hence, although low-dose i.v. MK-499 provided significant protection against both electrically and ischemically triggered arrhythmias in the setting of previous myocardial infarction, protection against PVS-induced ventricular tachycardias was not highly predictive of protection against lethal ischemic arrhythmias in this preparation. PMID- 8627545 TI - Identification and characterization of a high-affinity leukotriene B4 receptor on guinea pig T lymphocytes and its regulation by lipoxin A4. AB - A single class of high affinity leukotriene B4 (LTB4) receptors has been identified on the surface of guinea pig peritoneal exudate T lymphocytes. The Kd of these receptors is 1.6 nM, with a Bmax of 25.2 fmol/10(7) cells (1500 sites/cell). Receptor binding activity can be blocked by specific LTB4 receptor antagonists, but not by a specific LTD4 receptor antagonist, lipoxins A4 or B4 (LXA4, LXB4) or K252a, a protein kinase C inhibitor. Pretreatment of T lymphocytes with phorbol myristyl acetate or LXA4, reduced LTB4 receptor density in a concentration-dependent manner, although similar concentrations of LXB4 had no effect. LTB4 receptor down-modulation by LXA4 was reversed by K252a. 4 alpha Phorbol 12,13-didecanoate, an inactive structural analogue of phorbol myristyl acetate, did not activate protein kinase C or decrease LTB4 receptor density. These results suggest that LTB4 receptor density on T cells may by ultimately down-regulated by a protein kinase C-dependent mechanism and are consistent with a physiological role of LXA4 in the modulation of inflammatory process. PMID- 8627546 TI - 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is N-demethylated by cytochromes P450 2D6, 1A2 and 3A4--implications for susceptibility to Parkinson's disease. AB - 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a Parkinson-like syndrome through biotransformation by monoamine oxidase B to the neurotoxic metabolite 1-methyl-4-phenylpyridine. Neuroprotection may be provided by parallel N-demethylation and N-oxidation pathways mediated by the microsomal cytochrome P450 and flavin monooxygenase systems, respectively. The aims of this study were to characterise the N-demethylation of MPTP by human liver microsomes over a wide range of concentrations, and to identify the cytochrome P450 enzymes involved in this reaction. The kinetics of the N-demethylation of MPTP (1 microM - 3 mM) by microsomes from the liver of an extensive metabolizer with respect to cytochrome P4502D6 (CYP2D6) activity were biphasic (apparent Km1 and Km2 values = 48 and 2882 microM). The high affinity activity was abolished in the presence of quinidine (1 microM) and was absent in microsomes from a genotypically poor metabolizer with respect to CYP2D6. Yeast microsomes containing heterologously expressed CYP2D6 N-demethylated MPTP (Km = 39 microM), and there was a high correlation between the quinidine-inhibitable N-demethylation of MPTP (50 microM) (0.7-91%, mean 44%, of total activity) and the alpha-hydroxylation of metoprolol in microsomes from 11 human livers (rs = 0.92; P < .001). At 50 microM MPTP, N demethylase activity in human liver microsomes was also inhibited by furafylline (10 microM) and ketoconazole (2 microM) (mean inhibition 39 and 13%, respectively; n = 11 livers). Yeast microsomes containing heterologously expressed human CYP1A2 N-demethylated MPTP with a Km of 2246 microM. These findings indicate that CYP2D6, CYP1A2 and, to a lesser extent CYP3A4, may have a role in protecting against Parkinson's disease induced by MPTP and other potential environmental neurotoxins. The data provide some biochemical support for the proposition that genotypically poor metabolizers with respect to CYP2D6 are overrepresented in some populations of Parkinson's patients, and that smokers (induced CYP1A2?) are underrepresented. PMID- 8627547 TI - The effects of administering quinacrine during ultraprofound hypothermia on warm ischemic kidney cortex tissue. AB - Recent advances have led to increased use of ultraprofound hypothermia for cardiopulmonary bypass, organ preservation and trauma patients and have introduced the possibility of targeted pharmacologic intervention during the hypothermic period. In this study, rabbit renal cortex slices were used to examine the effect of administering quinacrine (100 microM) during hypothermia induced after a warm ischemic injury (60 min at 37 degrees C) on recovery of biochemical function during 3.5 hr of simulated warm reperfusion. In ischemic tissue slices, ATP content was reduced to near zero and only recovered about 50% by the end of reperfusion. Hypothermic storage of ischemic slices for 18 hr restored slice ATP content to about 80% of control levels but was followed by a decline during reperfusion to levels similar to ischemic slices. Administering quinacrine (100 microM) during 18 hr of hypothermic storage of ischemic slices resulted in a significant and sustained increase in slice ATP content during warm reperfusion. Slices stored at hypothermia only 3 hr with quinacrine had reduced swelling during reperfusion even though total ATP content was unaffected. Administering quinacrine (100 microM) only during reperfusion after ischemia or hypothermia did not affect tissue ATP content. This study showed that drug administration during hypothermic storage has potential therapeutic benefits for resuscitating tissues after warm ischemia and is more effective than the same drug given only during reperfusion. Tissue pretreatment was not required to obtain improved function in this study which suggests that future adaptations of these principles may have practical applications for specific clinical conditions where ischemic and reperfusion injury are significant factors. PMID- 8627548 TI - Pharmacological characterization of tachykinin NK2 receptors on isolated human urinary bladder, prostatic urethra and prostate. AB - The contractile effect of two highly potent, selective and peptidase-resistant neurokinin (NK) 1 and NK2 receptor agonists, namely delta-Aminovaleryl-[L-Pro9, N MeLeu10]substance P-(7-11) (GR 73632) and [Lys3, Gly8-R-gamma-lactam-Leu9]NKA-(3 10) (GR 64349), respectively, was investigated on smooth muscle strips dissected from specimens of human detrusor, prostatic urethra and prostate. Furthermore, the potencies of two peptidic NK2 receptor antagonists, GR 87389 L 659,837, in antagonizing GR 64349-induced contractions were compared in these three tissues. In human detrusor muscle the rank order of agonist potency was: [beta Ala8 (NKA (4-10)] > GR 64349 >> NKA-(4-10) >> SP = GR 73632 >> SP-methylester. The NK2 receptor antagonist, GR 87389, antagonized GR 64349-induced contractions in a competitive manner, whereas L 659,837 was a noncompetitive antagonist. In the prostatic urethra the rank order of agonist potency was GR 64349 > NKA-(4-10) > SP > GR 73632, whereas in the prostate it was: GR 64349 >> [beta Ala8 (NKA-(4 10)] > NKA-(4-10) > SP; GR 73632 was ineffective up to 30 microM. In the prostatic urethra and in the prostate GR 87389 was a noncompetitive antagonist with a potency similar to that exhibited in the detrusor. On the contrary, L 659,837 appeared to be a competitive antagonist in the prostate and in the prostatic urethra, having approximately the similar potency in these two tissues. The selective NK3 agonist senktide was ineffective up to 30 microM in all three tissues. These results are discussed in the view of the proposed NK2 receptor subtypes and considering possible therapeutic implications in the treatment of urinary bladder disorders. PMID- 8627549 TI - Halothane constricts bovine pulmonary arteries by release of intracellular calcium. AB - In the canine lung, when compared with the conscious state, halothane causes vasoconstriction that is independent of blood flow. However, traditionally inhalational anesthetics have been shown to attenuate hypoxic pulmonary vasoconstriction and have therefore been considered pulmonary vasodilators. We have shown, in isolated bovine pulmonary artery, that halothane produces a transient contractile response. A variety of smooth muscle cellular mechanisms could be responsible for the vasoconstriction produced by halothane. The purpose of this study was to test the hypothesis that the halothane-induced contraction was caused by the release of sarcoplasmic reticular Ca++. Isometric tension was measured in isolated rings of bovine pulmonary artery with intact endothelium. Three protocols were followed. Rings were exposed to cyclopiazonic acid or ryanodine (modulators of sarcoplasmic reticular Ca++) (protocol 1), caffeine (protocol 2) verapamil or nicardipine (protocol 3). Halothane-induced contraction was measured before, during and after exposure to drug. In nominally Ca(++)-free buffer cyclopiazonic acid and ryanodine attenuated the halothane-induced contraction. Similar responses were seen with cyclopiazonic acid and ryanodine treatment when caffeine was substituted for halothane. The calcium channel blockers nicardipine and verapamil did not significantly alter the halothane induced contraction. Our data in bovine pulmonary artery segments are consistent with halothane effects seen in vascular smooth muscle from several other tissues and species. The results of our experiments support the conclusion that the release of intracellular Ca++ from sarcoplasmic reticular stores is responsible for the halothane-induced vasoconstriction that has been observed in this tissue. PMID- 8627550 TI - The 21-aminosteroid tirilazad mesylate protects against liver injury via membrane stabilization not inhibition of lipid peroxidation. AB - Whether tirilazad mesylate (U-74006F) protects against liver injury by inhibition of lipid peroxidation or by cell membrane stabilization was investigated. In male Fischer rats subjected to 20 min of hepatic ischemia followed by reperfusion and injection of 0.5 mg/kg Salmonella enteritidis endotoxin, developed of liver injury was accompanied by lipid peroxidation, as indicated by 81 to 184% increases in hepatic 8-, 9-, 11-, and 12-hydroxyeicosatetraenoic acid content and a 85% increase of plasma F2-isoprostane concentrations at 4 h of reperfusion. Treatment with U-74006F (two bolus doses of 3 mg/kg each; the first dose was injected i.v. 30 min before ischemia and the second dose, at the time of reflow) reduced hepatic injury by 60% but had no significant effect on either parameter of lipid peroxidation. In contrast, U-74006F treatment attenuated liver injury and lipid peroxidation at 24 h reperfusion. Pretreatment with U-74006F in vivo had no effect on lipid peroxidation and liver injury in vitro during perfusion with tert-butylhydroperoxide. However, U-74006F protected hepatocytes significantly against membrane damage induced by cell swelling due to perfusion with hypotonic medium or ischemia-reperfusion. These data support the conclusion that U-74006F enhances the resistance of liver cell membranes to injury by its membrane-stabilizing effect and not by directly scavenging free radicals in vivo. However, the cytoprotective effect of U-74006F can under certain circumstances inhibit recruitment and activation of inflammatory cells, which will then reduce the oxidant stress and lipid peroxidation in the liver. PMID- 8627551 TI - Alterations in calcium antagonist receptors and calcium content in senescent brain and attenuation by nimodipine and nicardipine. AB - Characteristics of L- and N-type calcium (Ca++) channel antagonist receptors in brains of senescence-accelerated prone mouse (SAMP8) showing age-related deterioration of learning and memory were examined by using (+)-[3H]PN 200-110 and [125I]omega-conotoxin GVIA as radioligands. There was a tendency toward consistent decrease in Bmax for both radioligands in seven brain regions of SAMP8 compared with the control mouse. The reduction in (+)-[3H]Pn 200-110 binding sites was statistically significant in the hippocampus, midbrain and pons/medulla oblongata, and that in [125I]omega-conotoxin binding sites was significant in the cerebral cortex, corpus striatum and pons/medulla oblongata. On the other hand, there was a marked elevation in Ca++ content in the brain of SAMP8. Chronic p.o. administration (0.3, 1 and 3 mg/kg/day for 3 weeks) of nimodipine and nicardipine to SAMP8 caused a significant increase in the Bmax values of (+)-[3H]PN 200-110 binding in the cerebral cortex and hippocampus. This may reflect up-regulation of brain Ca++ channel antagonist receptors as a result of the prolonged blockade by nimodipine and nicardipine. On the other hand, similar administration of amlodipine and nilvadipine failed to produce an enhancement of Bmax values of (+) [3H]PN 200-110 binding, whereas both drugs at high doses evoked a significant increase in the apparent dissociation constant. Furthermore, the brain Ca++ content in SAMP8 was markedly reduced by chronic p.o. administration of Ca++ channel antagonists, and the decrease was equivalently observed for all of four 1,4-dihydropyridine antagonists in spite of the difference in the effect on brain receptors. In conclusion, the present study suggests that there is an altered Ca++ homeostasis in the SAMP8 brain that is effectively attenuated by chronic administration of nimodipine and nicardipine. Hence SAMP8 may be a suitable animal model for evaluating the therapeutic effects of Ca++ channel antagonists on neurological disorders associated with the aging brain. PMID- 8627552 TI - Pharmacological evaluation of novel Alzheimer's disease therapeutics: acetylcholinesterase inhibitors related to galanthamine. AB - Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl) carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease. PMID- 8627553 TI - Discriminative effects of CGS 15943, a competitive adenosine receptor antagonist, in monkeys: comparison to methylxanthines. AB - Caffeine and related methylxanthines are competitive antagonists at A1- and A2 adenosine receptors, but have other actions at the cellular level that contribute to their effects on behavior. As an approach toward determining the role of adenosine receptors in the behavioural effects of drugs, four squirrel monkeys were trained to discriminate between injections of CGS 15943 (1.0 mg/kg i.m.), a nonxanthine adenosine receptor antagonist that does not inhibit phosphodiesterase, and its vehicle. All monkeys generalized dose-dependently and completely to six of seven methylxanthines: 3-isobutyl-1-methylxanthine (0.1-1.75 mg/kg), theophylline (0.03-3.0 mg/kg), paraxanthine (0.3-30 mg/kg), 8 cyclopentyltheophylline (0.3-30 mg/kg), theobromine (0.3-30 mg/kg) and caffeine (1.0-30 mg/kg). Three of four monkeys did not generalize to 8-p-sulfophenyl theophylline (1.0-30 mg/kg), which does not cross the blood-brain barrier. When the training dose of CGS 15943 was administered concurrently with adenosine receptor agonists, its effects were blocked dose-dependently and completely by CGS 21680 (A2 selective), only partially by cyclohexyladenosine (A1 selective), but were not blocked by 5'-N-ethylcarboxamidoadenosine (nonselective). CGS 21680 did not block responding on the CGS 15943-appropriate lever occasioned by 30 mg/kg of caffeine or 3.0 mg/kg of theophylline. These results suggest that stimulus control of behavior by CGS 15943 derives, in part, from blockade of A2 adenosine receptors located in the central nervous system. However, the potency order of methylxanthines as CGS 15943-like discriminative stimuli did not correlate with their relative affinities at either A2- or A1-adenosine receptors or their potencies for other known effects at the cellular level. Therefore, a novel mechanism of action might account for the CGS 15943-like discriminative effects of some or all of these drugs. PMID- 8627554 TI - Non-neuronal release of ATP and inositol 1,4,5-trisphosphate accumulation evoked by P2- and M-receptor stimulation in guinea pig ileal segments. AB - Intracellular signal transduction involved in non-neuronal ATP release evoked by alpha, beta-methylene ATP and bethanechol was evaluated in guinea pig ileal longitudinal muscle segments. alpha, beta-methylene ATP (100 microM) and bethanechol (10 microM) evoked ATP released that reached a peak about 3 min after administration. The evoked release of ATP was markedly inhibited by neomycin and spermine, inhibitors of phospholipase C, but not by treatment with pertussis toxin. In addition, the release of ATP was almost completely suppressed by 1 mM Li+, an inhibitor of inositol monophosphatase. These inhibitors, however, did not affect the contractions of the tissue evoked by these agonists. Forskolin and phorbol 12-myristate 13-acetate, activators of adenylate cyclase and protein kinase C, respectively, failed to enhance the evoked release. The accumulation of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] in the muscle segments were enhanced about 2 min after the administration of alpha, beta-methylene ATP. In the presence of 1 mM Li+, however, the enhancement of Ins(1,4,5)P3 accumulation by the P2 agonist was no longer elicited. These findings suggest that the release of ATP by receptor stimulation may result mainly from the activation of phospholipase C, which is coupled to a pertussis toxin-insensitive G-protein and subsequent accumulation of Ins(1,4,5)P3 in the smooth muscles. However, the discrepancy between the inhibitory effects of Li+ on the release of ATP and the accumulation of Ins(1,4,5)P3 to be clarified in future studies. PMID- 8627555 TI - Frequency modulation of acetylcholine-induced oscillations in Ca++ and Ca(++) activated Cl- current by cAMP in tracheal smooth muscle. AB - The effects of adenosine 3':5'-cyclic monophosphate (cAMP) on acetylcholine (ACh) induced oscillations in intracellular calcium concentration ([Ca++]i) and Ca(++) activated Cl- current (ClCa current) were determined in isolated tracheal smooth muscle cells. Whole-cell current was measured in individual smooth muscle cells with patch clamp methodology. At a holding potential of -80 mV, ACh (0.1 microM) elicits base line-separated oscillations in ClCa current which correlate with oscillations in [Ca++]i. The addition of the beta adrenoceptor agonist isoproterenol (ISO) (10 nM to 1 microM) in the continued presence of ACh caused a concentration-dependent decrease in the frequency of the oscillations in ClCa current with significant reductions in oscillation frequency of 21.4 and 81.5% in the presence of 0.01 and 0.1 microM ISO, respectively (P < .05). This effect was mimicked by both forskolin (FSK) (3 microM) and 3-isobutyl-1-methylxanthine (IBMX) (30 microM). ISO and forskolin also inhibited ACh-induced oscillations in [Ca++]i measured by confocal fluorescence microscopy in non-voltage-clamped cells loaded with the Ca(++)-sensitive dye, fluo3. The inhibition of ACh-induced oscillations in ClCa current by ISO was partially reversed by increasing extracellular Ca++. These data are consistent with previous observations that the frequency of ACh-induced oscillations in [Ca++]i and ClCa current is dependent on the concentration of extracellular Ca++ and the influx of Ca++ through a verapamil-sensitive pathway. Moreover, these results lend support to the hypothesis that beta adrenoceptors inhibit the ACh-induced increase in [Ca++]i through a cAMP-dependent mechanism that inhibits Ca++ influx and in independent of changes in membrane potential. PMID- 8627556 TI - Effect of ethanol on the excitability of the inferior olive in decerebrate ferret. AB - Climbing fibers, which originate in the inferior olive and project to Purkinje cells and Golgi cells in the cerebral cortex, were activated at low (0.4-Hz) and high (4-Hz) frequencies by periorbital stimulation in decerebrate ferrets. Climbing fiber responses were recorded as field potentials from the c3 zone of the cerebellar surface. When periorbital stimulation was applied at high frequency, the climbing fiber responses became strongly depressed within a few seconds. It has previously been shown that this high frequency depression (HFD) of climbing fiber responses is due to a cerebellar inhibition of the inferior olive, probably via the nucleo-olivary pathway. Acute administration of ethanol had small and variable effects on the amplitude of climbing fiber responses evoked by low-frequency stimulation. In contrast, medium concentrations (0.44 2.90 g/l) of ethanol led to a marked reduction of the HFD. Low ( < 0.44 g/l) systemic concentrations had no measurable effects on the HFD, whereas high concentrations ( > 2.90 g/l) caused either an increased HFD or a nonseptic reduction in olivary excitability. Because HFD has previously been shown to involve cerebello-olivary inhibition, the possibility of an interaction between ethanol and GABA-ergic responses in the interposito-olivary pathway is discussed. PMID- 8627557 TI - (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502) as a novel substrate for cytochrome P450 2A6 in human liver microsomes. AB - (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) was oxidized by human liver microsomes to produce the S-oxide as a sole metabolite. Indirect evidence suggested that the S-oxidation was catalyzed by cytochrome P450 (CYP). Eadie-Hofstee plots showed biphasic pattern, suggesting that at least two enzymes were involved in the S-oxidation in human liver microsomes. Kinetic parameters of the S-oxidase with high-affinity showed Km and Vmax values of 20.9 +/- 4.4 microM and 0.111 +/- 0.051 nmol/min/mg microsomal protein, respectively. The S-oxidase activity was inhibited by coumarin and anti-CYP2A antibody. Among the contents of forms of CYP 20 samples of human liver microsomes, the content of CYP2A6 correlated with S-oxidase activity measured with 50 microM SM-12502 (r = .808, P < .0005). A close correlation (r = .908, P < .0001) was observed between activities of SM-12502 S-oxidase and coumarin 7-hydroxylase. Microsomes from genetically engineered human B-lymphoblastoid cells expressing CYP2A6 metabolized SM-12502 to the S-oxide efficiently. The results indicate that CYP2A6 isozyme is a major form of CYP responsible for the S-oxidation of SM-12502 in human liver microsomes. Thus, SM-12502 will be a useful tool in further research to analyze a human genetic polymorphism of CYP2A6. PMID- 8627558 TI - Physiological release of striatal acetylcholine in vivo: modulation by D1 and D2 dopamine receptor subtypes. AB - Our experiments assessed the modulation of striatal acetylcholine (ACh) output by dopamine (DA) receptor subtypes under physiological conditions using in vivo microdialysis in awake rats. The degree to which the dopaminergic modulation of striatal cholinergic neurons might vary as a function of local extracellular ACh level also was examined by application of varying concentrations of the acetylcholinesterase (AChE), inhibitor neostigmine (NEO) in the microdialysis perfusate. Under physiological conditions (O NEO), the amount of ACh in the dialysates was 25.1 +/- 2.2 fmol/20-microliters sample (n = 20) whereas values of 67.9 +/- 3.5 (n = 35) and 527.7 +/- 56.1 (n = 13) fmol/20-microliters sample were obtained when the applied NEO concentration was 10 and 100 nM, respectively. In the absence of NEO, a low dose of the indirect DA agonist amphetamine (AMPH; 2 mg/kg i.p.) failed to affect striatal ACh output; a higher AMPH dose (10 mg/kg i.p.) significantly decreased the amount of ACh in dialysates. Under physiological conditions, the direct D2-selective agonist quinpirole (3 mg/kg i.p.) decreased extracellular ACh in striatum to nondetectable levels and the direct D1-selective agonist SKF-38393 (10 mg/kg i.p.) produced a significant increase in this measure. Analysis of the changes in striatal ACh output produced by administration of these DA compounds in the absence vs. presence of local NEO revealed that 10 nM NEO did not qualitatively alter the pharmacological responsivity of this system as compared to the physiological condition. However, in the presence of 100 nM NEO, 2 mg/kg AMPH elicited a significant increase in striatal ACh output. At the 100 nM NEO concentration it also was observed that the amplitude of the quinpirole-induced inhibition of ACh efflux did not increase further in proportion to basal ACh levels whereas the amplitude of the increase in ACh output produced by SKF-38393 was linearly related to basal ACh levels across all NEO concentrations. Under conditions where cholinergic pharmacological responsivity was minimally affected (10 nM NEO), the D2 receptor antagonist haloperidol (1 mg/kg i.p.) increased striatal ACh output by 50% and the D1 receptor antagonist SCH-23390 (0.5 mg/kg i.p.) decreased this variable by 41%. Under these conditions, the inhibitory action of quinpirole on ACh output could be reversed by subsequent administration of AMPH (5 mg/kg i.p.) and this effect of AMPH could then be blocked by administration of SCH-23390. Thus, under physiological or low NEO (10 nM) conditions a prevalent D2-mediated inhibition as well as an opposing D1-mediated excitation of striatal ACh output can be demonstrated. At a higher NEO concentration (100 nM), regulation of the striatal ACh system by DA receptor subtypes is differentially affected such that the D2 mediated inhibitory influence no longer predominates over the D1-mediated excitatory drive. Caution should be exercised when interpreting ACh efflux data obtained using microdialysis under conditions of AChE inhibition. PMID- 8627559 TI - Opioid antagonists and antisera to endogenous opioids increase the nociceptive response to formalin: demonstration of an opioid kappa and delta inhibitory tone. AB - The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta funaltrexamine. Additionally, antisera to [Leu5]enkephalin and dynorphin also resulted in a significant increase in formalin-induced flinching, whereas antisera to [Met5]enkephalin had no effect. On the basis of significant increases in formalin-induced flinching produced by 1) receptor-selective doses of delta and kappa, but not mu, opioid antagonists and 2) antisera to [Leu5]enkephalin and dynorphin A, but not [Met5]enkephalin, these data suggest the presence of an opioid inhibitory tone which acts to limit the intensity of the pain signal. This tone appears to be mediated via activation of delta and kappa receptors, possibly by a [Leu5]enkephalin- and dynorphin-like substance, respectively. PMID- 8627560 TI - Depletion and refilling of acetylcholine- and caffeine-sensitive Ca++ stores in tracheal myocytes. AB - We have previously shown that acetylcholine (ACh) induces oscillations in Ca++ and Ca++-activated Cl- currents (Clca) in tracheal myocytes. These oscillations are initiated by Ca++ release from inositol 1,4,5-trisphosphate-sensitive Ca++ stores and maintained by Ca++ influx, in part, through voltage-operated Ca++ channels. In the current study whole-cell Clca was measured in isolated tracheal smooth muscle cells as an index of changes in intracellular Ca++ concentration. We demonstrate that ACh-sensitive Ca++ stores and caffeine-sensitive Ca++ stores and caffeine-sensitive Ca++ stores are functionally linked but are refilled through distinct pathways. Two pathways responsible for replenishing ACh sensitive Ca++ stores were identified. Ca++ influx through verapamil-sensitive voltage-operated Ca++ channels and Ca++ uptake through cyclopiazonic acid sensitive Ca++ pumps accounted for 80% of the response. The other 20% of the response was both cyclopiazonic acid- and verapamil-insensitive. In contrast, the refilling of caffeine-sensitive Ca++ stores was not inhibited by 10 microM cyclopiazonic acid or 10 microM verapamil, but was dependent on extracellular Ca++ concentration. However, 0.2 microM thapsigargin, another more potent Ca++ pump inhibitor, completely and irreversibly eliminated ACh-induced transient Clca, whereas it reduced caffeine-induced Clca by 57%. The differences in refilling mechanisms and the functional overlap of ACh- and caffeine-sensitive Ca++ pools suggest that multiple interactive Ca++ stores play an important role in the generation of Ca++ signals in airway smooth muscle cells. PMID- 8627561 TI - Acetylcholine-induced Ca++-dependent chloride current oscillations are mediated by inositol 1,4,5-trisphosphate in tracheal myocytes. AB - We demonstrated previously that acetylcholine (ACh) induces Ca++ oscillations in tracheal myocytes. These oscillations, as measured with fluo3-loaded cells and confocal microfluorimetry, correlated with Ca++-dependent Cl- current (Clca) oscillations measured by whole-cell voltage-clamp recording. In the current study, we investigated the role of inositol 1,4,5-trisphosphate (IP3) in ACh induced oscillations in Ca++ and membrane currents. Both an IP3 receptor monoclonal antibody (5 micrograms/ml) and an IP3 receptor antagonist, heparin (5 mg/ml), directly introduced into the cells via the patch pipette, reduced or abolished oscillations in Clca induced by ACh. In addition, IP3 (1-100 microM) applied intracellularly, elicited concentration-dependent Clca oscillations that resembled those induced by ACh. Increasing external Ca++ concentration enhanced IP3-induced Clca oscillations, whereas verapamil (10 microM), a voltage-operated Ca++ channel blocker, attenuated IP3-induced Clca oscillations as well as both control and IP3-enhanced spontaneous transient outward currents. However, neither 5 microgram/ml IP3 receptor monoclonal antibody nor 5 mg/ml heparin altered the caffeine-induced transient Clca. Caffeine (10 mM) reversibly eliminated IP3 induced Clca oscillations as well as IP3-enhanced spontaneous transient outward currents, which indicates that caffeine releases Ca++ via a mechanism independent of the IP3 receptor. The findings are consistent with the hypothesis that ACh induced Ca++ oscillations can arise and be sustained via IP3-induced Ca++ release pathways. PMID- 8627562 TI - Identification of the human P450 enzymes involved in lansoprazole metabolism. AB - The aim of this study was to identify which human P450 enzymes are involved in the metabolism of lansoprazole. In the presence of NADPH and oxygen, human liver microsomes converted lansoprazole to lansoprazole sulfide, lansoprazole sulfone and 5-hydroxylansoprazole. Formation of lansoprazole sulfide occurred nonenzymatically. The formation of lansoprazole sulfone appeared to be catalyzed by a single, low-affinity enzyme (apparent Km approximately 100 microM). In contrast, lansoprazole 5-hydroxylation appeared to be catalyzed by two kinetically distinct enzymes (apparent Km approximately 100 microM and approximately 15 microM). When human liver microsomes (n = 16) were incubated with 100 microM lansoprazole, both the 5-hydroxylation and sulfoxidation of lansoprazole appeared to be catalyzed by CYP3A4/5 (based on correlation analyses). Antibodies against rat CYP3A enzymes inhibited the rate of both 5 hydroxylation (approximately 55%) and sulfoxidation (approximately 70%) and cDNA expressed CYP3A4 catalyzed both the 5-hydroxylation and sulfoxidation of lansoprazole (apparent Km approximately 100 microM). However, at the pharmacologically relevant substrate concentration of 1 microM, lansoprazole sulfoxidation was still highly correlated with CYP3A4/5 activity (r2 = .905), but lansoprazole 5-hydroxylation appeared to be catalyzed by CYP2C19 (r2 = .875) rather than CYP3A4/5 (r2 = .113). Antibodies and chemical inhibitors of CYP2C enzymes preferentially inhibited the 5-hydroxylation of lansoprazole, whereas lansoprazole sulfoxidation was preferentially inhibited by antibodies and chemical inhibitors of CYP3A4/5. The cDNA expressed enzymes CYP2C8, CYP2C9 and CYP2C19 catalyzed varying rates of lansoprazole 5-hydroxylation at a substrate concentration of 50 microM, but only CYPC19 catalyzed this reaction at 1 microM. These results suggest that at pharmacologically relevant concentrations, the 5 hydroxylation of lansoprazole is primarily catalyzed by CYP2C19, whereas the sulfoxidation of lansoprazole is primarily catalyzed by CYP3A4/5. It is possible that individuals lacking CYP2C19 will be poor metabolizers of lansoprazole. PMID- 8627563 TI - Lowered norepinephrine turnover as a sign of impaired ganglionic transmission after preganglionic lesioning by acetylcholinesterase antibodies. AB - Monoclonal antibodies to acetylcholinesterase are known to destroy preganglionic sympathetic terminals in rats. To investigate resulting changes in sympathetic tone, turnover of norepinephrine (NE) was examined in five adrenergically innervated tissues: submaxillary salivary gland, heart, spleen, vas deferens and kidney. At time zero, 50 mu Ci of [3H]NE was injected into the tail vein; turnover rates were determined from the loss of radioactive NE between 2 and 24 hr later. Experiments with ganglionic blocking agents showed that most NE turnover was related to impulse traffic. Combined treatment with atropine (4 mg/kg/day) and chlorisondamine (20 mg/kg/day) reduced the apparent turnover rate constant by two thirds or more in all organs except vas deferens. NE turnover was likewise slowed after treatment with acetylcholinesterase antibodies (1.6 mg i.v., 5 days earlier): apparent rate constants fell 50% or more in submaxillary gland, heart and kidney. The reduced NE turnover in these end organs suggested that preganglionic immunologic lesions blocked synaptic transmission in the respective sympathetic ganglia. Sustained turnover in the spleen, however, suggested that certain pathways through the celiac ganglion resisted immunologic lesion or recovered quickly. Hence, there may be structural or functional differences among the sympathetic ganglia, especially between pre- and paravertebral groups. PMID- 8627564 TI - Endogenous serotonin facilitates in vivo acetylcholine release in rat frontal cortex through 5-HT 1B receptors. AB - We characterized the role of endogenous serotonin (5-HT) in regulating in vivo acetylcholine (ACh) output in frontal cortex of freely moving rats using the microdialysis technique. Systemic (0.63, 1.25 and 2.5 mg/kg, i.p.) or local (20 and 40 microM, reverse dialysis) administration of the 5-HT releaser and uptake inhibitor, d-norfenfluramine, dose-dependently enhanced frontal cortex ACh output. The d-norfenfluramine-induced increase in cortical ACh release was tetrodotoxin sensitive and completely prevented by a 7-day chemical degeneration of the serotonergic afferents to the frontal cortex. Investigating the 5-HT receptors that might mediate the d-norfenfluramine cholinergic effect, we found that the 5-HT4 (GR 125487) and 5-HT2A/2C (ritanserin) receptor antagonists, at doses effective in other in vivo tests, did not prevent the increase in cortical ACh output induced by the maximal effective does of d-norfenfluramine. However, the 5-HT1A/1B receptor antagonists (-)-pindolol (8 mg/kg, s.c.) or (-)-propanolol (8.8 mg/kg, i.p.) antagonized the increasing effect of d-norfenfluramine although the selective 5-HT1A receptor antagonist WAY-100635 (1 and 2 mg/kg, s.c.) did not. In accordance with an involvement of the 5-HT1B receptor in the ACh facilitation induced by d-norfenfluramine is the finding that the selective 5 HT1B agonist, CP-93,129, given locally (2, 4 and 8 micrograms/side) does dependently raised cortical ACh release. In conclusion, the overall regulatory control exerted by endogenous 5-HT in vivo is to facilitate frontal cortex ACh release through 5-HT1B receptors located in the frontal cortex. The 5-HT1B receptors may act indirectly to facilitate ACh release probably by inhibiting cortical inhibitory inputs onto the cholinergic neurons. PMID- 8627565 TI - Transport characteristics of differently charged cephalosporin antibiotics in oocytes expressing the cloned intestinal peptide transporter PepT1 and in human intestinal Caco-2 cells. AB - To investigate whether multiple peptide transporters mediate absorption of beta lactams carrying different charges at physiological pH, we used the human intestinal cell line Caco-2 and Xenopus laevis oocytes expressing the cloned rabbit intestinal peptide transporter PepT1. Characteristics of transport of the anionic cefixime and the zwitterionic cefadroxil were assessed by 1) flux studies using radiolabeled compounds, by 2) measuring changes in pHin in cells and oocytes as a consequence of substrate-mediated proton influx and 3) by applying the two-electrode voltage clamp technique to assess the electrophysiological phenomena associated with beta-lactam transport in oocytes expressing PepT1. Both beta-lactams were rapidly taken up into Caco-2 cells and oocytes expressing PepT1 by a pH-dependent and saturable transport pathway. Mutual inhibition suggested that acidic and zwitterionic compounds may share a common transporter. Cefixime and cefadroxil caused a significant decline in intracellular pH as a consequence of proton coupled substrate influx. Uptake of cefixime and cefadroxil via PepT1 expressed in oocytes was electrogenic indicating that transport of both beta lactams is associated with movement of net positive charge. The more acidic pH required for rheogenic cefixime uptake in both cell systems, when compared to cefadroxil uptake in both cell systems, when compared to cefadroxil uptake, and the concomitant faster intracellular acidification indicates that cefixime most likely is taken up only in its nonionized form with an additional proton being cotransported. This is supported by the observation that cefixime uptake at different pH correlated significantly with the percentage of the nonionized species being present. From our studies we conclude that a single peptide transport system can mediate electrogenic uptake of the neutral form of beta lactam antibiotics into intestinal epithelial cells. PMID- 8627566 TI - In vitro and in vivo characterization of MDL 105,212A, a nonpeptide NK-1/NK-2 tachykinin receptor antagonist. AB - We have identified and characterized a novel, potent, nonselective tachykinin receptor antagonist, MDL 105,212A [(R)-1-[2-[3-(3,4- dichlorophenyl)-1-(3,4,5 trimethoxybenzoyl)-pyrrolidin-3-yl] -ethyl]- 4-phenylpiperidine-4-carboxamide, hydrochloride]. The compound binds with low nanomolar affinity and species specificity to human NK-1 and NK-2 receptors as well as to guinea pig NK-3 receptors. In vitro functional assays are consistent with potent competitive antagonism of substance P-(SP) or neurokinin A-(NKA) induced [3H]-inositol phosphate accumulation in NK-1 or NK-2 monoreceptor cell lines with pA2 values of 8.19 and 8.67, respectively. Its ability to inhibit SP, NKA and capsaicin mediated respiratory effects was examined in guinea pigs in vivo. MDL 105,212A attenuated SP-induced airway plasma protein extravasation (ED50 = 0.20 mg/kg, i.v.), NKA-induced respiratory collapse (ED50 = 5 mg/kg, i.v) and inhibited capsaicin-induced increases in pulmonary insufflation pressure (ED50 = 0.5 mg/kg, i.v.). Conscious guinea pigs responded to capsaicin aerosol exposure with dyspnea, coughs and gasps (significant respiratory events) and plasma protein extravasation. MDL 105,212A inhibited these responses in a dose-dependent manner after i.v. (ED50 = 5 mg/kg) or oral (ED50 = 50 mg/kg) administration. These data suggest that MDL 105,212A is a potent NK-1 and NK-2 receptor antagonist based on in vitro activity and its ability to inhibit SP and NKA mediated respiratory effects in vivo after exogenous administration or endogenous release and hence may be a useful therapeutic agent in neuroinflammatory disorders such as asthma in which a role for both tachykinins in the pathogenesis of the disease has been postulated. PMID- 8627567 TI - Differences in modulation of noradrenergic and serotonergic transmission by the alpha-2 adrenoceptor antagonists, mirtazapine, mianserin and idazoxan. AB - The effects of three compounds with alpha-2 adrenoceptor antagonistic properties, mirtazapine (Org 3770; Remeron), mianserin and idazoxan, were measured on hippocampal noradrenergic and serotonergic transmission in freely moving rats by using microdialysis. Dihydroxyphenylacetic acid (DOPAC) was measured as a correlate of noradrenergic presynaptic activity. Infusing 1 microM tetrodotoxin decreased extracellular serotonin (5-HT) and DOPAC by 65 and 40%, respectively. 5 Hydroxytryptophan (25 mg/kg s.c.) increased extracellular 5-HT by 500%, whereas 8 hydroxy-2-(di-n-propylamino)tetralin hydrobromide (0.5 mg/kg s.c.) decreased 5-HT release by 60%. Prazosin decreased 5-HT release to 60% of base-line in agreement with an alpha-1-mediated control of 5-HT transmission, whereas it increased DOPAC release with 80%. Both mirtazapine (2 and 5 mg/kg s.c.) and idazoxan (1 mg/kg s.c.) caused a rapid increase in DOPAC by up to 80%. Mianserin slowly increased DOPAC, reaching a maximal increase of 30 and 60% at 2 and 5 mg/kg s.c., respectively. Only mirtazapine caused a concurrent increase in 5-HT, reaching up to 80% above base-line within 60 min, whereas mianserin and idazoxan failed to change 5-HT levels significantly. Mirtazapine (5 mg/kg s.c.) only slightly affected DOPAC and homovanillic acid levels in the striatum, hardly affected 5-HT release, but clearly increased 5-hydroxyindole acetic acid. Thus, the antidepressants mirtazapine and mianserin markedly differ in their effects on noradrenergic and serotonergic transmission in vivo as measured with microdialysis in freely moving rats. These differences are explained by their different modulatory effects on noradrenergic transmission. PMID- 8627568 TI - Effect of the alpha-2 adrenoceptor antagonist mirtazapine on the 5 hydroxytryptamine system in the rat brain. AB - Mirtazapine ([(+/-)-MIR], Remeron, ORG 3770) is an alpha-2 adrenoceptor antagonist endowed with antidepressant activity in humans. The aim of the present study was to assess the effects of (+/-)-MIR and of its (-)enantiomer [(-)-MIR] on pre- and postsynaptic alpha-2 adrenoceptors and to characterize their putative modulation of 5-HT neurotransmission. (+/-)-MIR (25 micrograms/kg i.v.) enhanced the effectiveness of the electrical stimulation of the ascending 5-HT pathway by blocking both alpha-2 adrenergic auto- and heteroreceptors. (-)-MIR (10 micrograms/kg i.v.) enhanced the effectiveness of these stimulations due to a selective action of (-)-MIR on the alpha-2 heteroreceptors located on 5-HT terminals. Both (+/-)- and (-)-MIR (500 micrograms/kg i.v.) blocked the suppressant effect of microiontophoretically applied norepinephrine (NE) on the firing activity of CA3 dorsal hippocampus pyramidal neurons, indicating their antagonistic effects on postsynaptic alpha-2 adrenoceptors. (+/-)-MIR (10- 250 micrograms/kg i.v.) enhanced dose-dependently the firing activity of the 5-HT neurons in naive rats, but not in 6-hydroxydopamine-pretreated rats. (+/-)-MIR also significantly increased the firing activity of locus ceruleus NE neurons. In contrast, (-)-MIR (10-250 micrograms/kg i.v.) failed to change the firing rate of dorsal raphe 5-HT neurons. In conclusion, these results suggest that both (+/-) MIR and (-)-MIR are antagonists at postsynaptic alpha-2 adrenergic receptors, that (+/-)-MIR is an antagonist of somatodendritic as well as terminal alpha-2 adrenergic auto- and heteroreceptors, whereas (-)-MIR is a selective antagonist at alpha-2 adrenergic heteroreceptors. Furthermore, the inhibitory effect of (-) MIR on locus ceruleus NE neurons appears to be mediated via 5-HT neurons because it is abolished by a 5,7-dihydroxytryptamine pretreatment. PMID- 8627569 TI - Dual effect of clonidine on mesenteric artery adrenoceptors: agonistic (alpha-2) and antagonistic (alpha-1). AB - The effect of clonidine on the mesenteric vascular bed and the isolated mesenteric artery was examined in preparations in which tonus was induced by norepinephrine or endothelin. In preparations precontracted by norepinephrine, clonidine caused a relaxation which was not inhibited by the alpha-2 antagonists yohimbine and idazoxan or by the K+ channel blockers apamine, tetraethylammonium and glibenclamide. In preparations precontracted with endothelin, clonidine increased the depolarization and induced a contraction. Both these effects were inhibited by prazosin. In isolated mesenteric arteries, norepinephrine cause a significant depolarization that was inhibited by clonidine or prazosin. On the other hand, clonidine caused a hyperpolarization which was inhibited by idazoxan or yohimbine, but not by prazosin. This hyperpolarization was also abolished by apamine, tetraethylammonium and glibenclamide. It is concluded that clonidine acts on alpha-1 adrenoceptors as a partial agonist, causing relaxation of the mesenteric artery precontracted with norepinephrine or contraction of preparations precontracted with endothelin. Moreover, clonidine can open K+ channels and hyperpolarize the plasma membrane of mesenteric artery by acting on alpha-2 adrenoceptors. PMID- 8627570 TI - kappa-Opioid agonist, U50,488H, stimulates ovine fetal pituitary-adrenal function via hypothalamic arginine-vasopressin and corticotrophin-releasing factor. AB - The fetal hypothalamic-pituitary-adrenal axis is regulated by such factors as corticotrophin releasing factor, arginine vasopressin and the endogenous opioid peptides. The goal of this study was to determine whether activation of the kappa opioid system can modulate ovine fetal pituitary-adrenal function. The highly selective kappa-opioid agonist, U50,488H ?trans-(+/-)-3, 4-dichloro-N-methyl-[2 (1-pyrrolidinyl)-cyclohexy]benzeneacetamide ? (1 mg/kg, i.v.), was administered directly to the ovine fetus in utero and fetal plasma levels of immunoreactive adrenocorticotrophin (ir-ACTH) and cortisol (ir-cortisol) were measured via radioimmunoassay. U50,488H resulted in an immediate and highly significant (P = .00005) increase in ir-ACTH, with a concomitant, significant (P = .02) increase in ir-cortisol. The peak increase was 312.1 +/- 31.2 pg/ml and 17.9 +/- 5.4 ng/ml from predrug control values for ir-ACTH and ir-cortisol, respectively, at 60 min after administration of U50,488H. This stimulation was completely blocked by concurrent naloxone (12 mg/hr, i.v.) administration, indicating that U50,488H is acting at classical opioid receptors to elicit this effect. Pretreatment with antagonists of arginine vasopressin or corticotrophin releasing factor attenuated the U50,488H response and it was therefore concluded that U50,488H is most likely acting to modulate ir-ACTH and ir-cortisol levels through regulation of arginine vasopressin and corticotrophin releasing factor release via hypothalamic kappa opioid receptors. The results of this study should aid in the design of obstetrical analgesics that will not alter the fetal stress response. PMID- 8627571 TI - Pharmacologic characterization of CHIR 2279, an N-substituted glycine peptoid with high-affinity binding for alpha 1-adrenoceptors. AB - We characterize the in vitro and in vivo pharmacology of CHIR 2279, an N substituted glycine peptoid previously identified from a combinatorial library as a novel ligand to alpha 1-adrenoceptors. Competitive receptor-binding assays with [3H]prazosin showed that CHIR 2279 was similar to prazosin in binding to alpha 1A (rat submaxillary), alpha 1a, alpha 1b, and alpha 1 d (cDNA expressed in LTK- cells) with high and approximately equipotent affinity. Ki values for CHIR 2279 ranged from 0.7 to 3 nM, and were 10-fold weaker than with prazosin. Functional assays for postsynaptic alpha 1-adrenoceptors showed CHIR 2279 was approximately equipotent in antagonizing agonist-induced contractile responses with rat was deferens (alpha 1A), canine prostate (alpha 1A), rat spleen (alpha 1B) and rat aorta (alpha 1D). The pA2 for CHIR 2279 averaged 7.07 in these assays, indicating a 10- to 100-fold lower in vitro potency than prazosin. In dogs, CHIR 2279 antagonized the epinephrine-induced increase in intraurethal pressure (pseudo pA2, 6.86) and in rats antagonized the phenylephrine-induced increase in mean arterial blood pressure. In rats and guinea pigs, CHIR 2279 induced a dose dependent decrease in mean arterial blood pressure without eliciting the tachycardia commonly observed with other alpha 1-blockers. Pharmacokinetic/pharmacodynamic modeling showed the i.v. system clearance rate of CHIR 2279 was 60 and 104 ml/min/kg in rats and guinea pigs, respectively, and the in vivo potency for mean arterial blood pressure reduction was twice as great in guinea pigs (EC50, 520 ng/ml) than rats (EC50, 1170 ng/ml). PMID- 8627572 TI - Characterization of CP-122,721; a nonpeptide antagonist of the neurokinin NK1 receptor. AB - CP-122,721 [(+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzyl)amino-2 phenylpiperidine] interacts with high affinity (pIC50 = 9.8) at the human NK1 receptor expressed in IM-9 cells. In the presence of CP-122,721, there was a reduction in Bmax of [125I]BH-SP binding with no change in affinity suggesting that CP-122,721 does not interact with the NK1 receptor in competitive manner. In an in vitro functional assay. CP-122,721 blocked SP-induced excitation of locus ceruleus cells in guinea pig brain slices with a IC50 value of 7 nM. In vivo, CP 122,721 potently blocked plasma extravasation in guinea pig lung elicited by aerosolized capsaicin (1 mM) with an ID50 = 0.01 mg/kg, p.o. Orally administered CP-122,721 antagonized Sar9, Met (O2)11-SP-induced locomotor activity in guinea pigs with an ID50 = 0.2 mg/kg suggesting good entry into the central nervous system. In addition, consistent with insurmountable blockade observed in vitro, CP-122,721 (0.01, 0.03 0.3 mg/kg, p.o.) produced a rightward shift in the dose response curve for SP-induced hypotension in the awake dog that was accompanied by a decrease in the maximal response. Thus, in vitro and in vivo CP-122,721 appears to behave functionally as a non-competitive antagonist producing an insurmountable blockade of the actions of SP. PMID- 8627573 TI - Preclinical pharmacology of CB30900, a novel dipeptide inhibitor of thymidylate synthase, in mice. AB - CB30900 is a novel, potent thymidylate synthase inhibitor which can not be polyglutamated and may be active in cancers expressing low or defective folylpolyglutamate synthetase. Pharmacokinetics were studied in mouse tumors and tissues after bolus or infusion protocols. Elimination was triphasic after 100 mg kg-1 i.v. (T 1/2 alpha, 2.8 min; T 1/2 beta, 19.1 min and T 1/2 gamma, 4.1 hr). Peak concentrations were 716 microM; clearance, 1.19 ml g-1 hr-1; and area under the curve (AUC 0-2 hr), 131 microM hr. Biphasic elimination occurred after i.p. administration and was comparable to the i.v. route giving complete i.p. bioavailability. Kidney concentrations were similar to plasma (AUC 0-2 hr, 84.3 microM hr). CB30900 concentrations in the gut increased steadily with time (AUC 0 2 hr, 645 microM hr) and liver drug concentrations were 7-fold greater than plasma (AUC 0-2 hr, 847 microM hr). Peak tumor concentrations occurred at 30 min and were 27% of plasma concentrations, but tumor drug clearance was markedly slower than for plasma (T 1/2, 51 +/- 8.2 min, mean +/- S.E.). CB30900 was remarkably stable in vivo with 93% of an administered dose recovered unchanged after 48 hr. Plasma drug binding was concentration-dependent, ranging from 93.3 to 76% over 1 to 500 microM. During 24 hr infusion (50 mg kg-1 s.c.), steady state plasma concentrations were 3 microM, giving an AUC 0-24 hr of 71 microM hr. Kidney drug levels were similar to plasma but liver concentrations were elevated 7-fold. By contrast, tumor drug concentrations were about 0.5 microM (AUC 0-24 hr, 14.6 microM hr). However, these low plasma drug concentrations are growth inhibitory in vitro (24-hr exposure). PMID- 8627574 TI - Effect of tacrine on in vivo release of dopamine and its metabolites in the striatum of freely moving rats. AB - The effects of tacrine (THA) on extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were investigated in the striatum of freely moving rats, using a microdialysis technique in which tacrine was administered locally via the microdialysis membrane. THA in concentrations of 10(-8) to 10(-5) M, significantly elevated the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid, whereas a significantly increased content of extracellular DA was observed at higher concentrations of THA (10(-3) to 10(-2) M). Local administration of the muscarinic antagonist atropine (10(-6) M) or the nicotinic antagonist mecamylamine (10(-5) M) both prevented the effects of THA on DA and its metabolites. In vitro receptor binding studies showed that THA displaced the binding of muscarinic antagonists [3H]pirenzepine (IC50, 2.1 +/- 0.4 microM) and [3H]AFDX 384 (IC50, 3.4 +/- 0.2 microM) equally in striatal tissue, suggesting that THA binds with equal affinity to M1 and M2 muscarinic receptor subtypes. THA showed a 20-fold lower affinity to high-affinity nicotinic receptors compared with muscarinic receptors when determined by [3H]cytisine competition curves. The study indicates that THA enhances monoamine neurotransmission in the rat striatum, probably via an interaction with both muscarinic and nicotinic heteroreceptors. PMID- 8627575 TI - Pharmacokinetic properties of several novel oligonucleotide analogs in mice. AB - Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2' propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5' octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5' conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney. PMID- 8627576 TI - Sodium 2,3-dimercaptopropane-1-sulfonate challenge test for mercury in humans. III. Urinary mercury after exposure to mercurous chloride. AB - The sodium salt of 2,3-dimercaptopropane-1-sulfonic acid (Dimaval; DMPS) challenge test has been given previously to humans exposed to elemental mercury (vapor) or mercuric salts, but not mercurous salts. The test (300 mg p.o., after an 11-hr fast) was given to 11 factory workers who make a skin lotion that contains mercurous chloride, eight users of the skin lotion and nine controls. Urines were analyzed for total mercury by using cold vapor atomic absorption spectrophotometry. The mercury excreted for 6 hr before and 6 hr after DMPS treatment was 113 micrograms +/- 26 and 5037 micrograms +/- 682 S.E.M. for the skin lotion makers; 16.2 micrograms +/- 3.4 and 1410 micrograms +/- 346 S.E.M. for the skin lotions users; and 0.49 micrograms +/- 0.11 and 18.4 micrograms +/- 7.1 S.E.M. for the controls, respectively. The increases in urinary mercury resulting from the DMPS challenge test were 45-, 87- and 38-fold, respectively. The results demonstrate that, in humans exposed to mercurous chloride, DMPS increases the urinary excretion of mercury and that the DMPS/mercury challenge test is of value for a more realistic estimation of mobilizable mercury. An attempt to associate genotoxicity, as indicated by micronuclei content in buccal cells, with mercury exposure was inconclusive, perhaps because of the small number of subjects. PMID- 8627577 TI - Modulation of embryonic glutathione peroxidase activity and phenytoin teratogenicity by dietary deprivation of selenium in CD-1 mice. AB - Selenium (Se)-dependent and -independent glutathione (GSH) peroxidases detoxify H2O2 and lipid hydroperoxides, which may mediate the teratogenicity of phenytoin and related xenobiotics. To test this hypothesis, CD-1 mice were placed on Se deficient diets for 15, 25 or 40 days and bred so that the day of analysis corresponded to gestational day 11. In Se-replete control animals, embryonic peroxidase activities were only 5% of activities in maternal liver (P < .05). After 15 days of Se deprivation, maternal activities for H2O2 (reflecting Se dependent peroxidase) and cumene hydroperoxide (CmOOH) (reflecting both Se dependent and -independent peroxidases) were reduced to 20% (P < .05) and 35% of controls, respectively. At this time, the incidence of fetal cleft palates initiated by phenytoin (55 mg/kg intraperitoneally on gestational days 11, 12 and 13) was doubled, from 12% to 25% (P < .05). Selenite rescue (Na2SeO3, 350 micrograms/kg intraperitoneally on day 9) restored maternal and embryonic peroxidase activities and completely inhibited phenytoin-initiated postpartum lethality and fetal resorptions in animals that had been Se depleted for 15 days. After 40 days of Se deprivation, maternal and embryonic peroxidase/H2O2 activities were reduced to < 1% and 27% of Se-replete controls, respectively. In contrast, maternal peroxidase/CmOOH activity was increased to 70% of controls, reflecting induction of Se-independent peroxidase, compared with that with 15 days' depletion. Phenytoin-initiated cleft palates with 40 days' depletion appeared to be reduced (16%) compared with Se-replete controls (24%) (P < .07). In 40-day Se-depleted animals given selenite rescue, the 10% incidence of cleft palates was significantly lower than that in the 40-day Se-replete group (24%) but not the Se-depleted group (16%). This is the first demonstration of reduced Se-dependent GSH peroxidase activities in embryonic tissues with dietary Se deprivation. The results implicate reactive oxygen species and lipid hydroperoxides in the mechanism of phenytoin teratogenicity and suggest that GSH peroxidases are important embryoprotective enzymes. PMID- 8627578 TI - Neomycin selectively inhibits 5-hydroxytryptamine-induced contraction in the guinea pig trachea. AB - Neomycin (3 mM) inhibited maximal 5-HT-induced contraction by approximately 50% without inhibiting [3H]5-HT binding to 5-HT2A receptors. In contrast, neomycin had no effect on carbamylcholine- or histamine-induced contraction. Carbamylcholine (10 microM) and histamine (10 microM) both stimulated phosphatidylinositol (PI) hydrolysis but neomycin had no effect on the increase in PI hydrolysis. 5-HT (10 microM) did not stimulate PI hydrolysis in the absence or presence of neomycin, suggesting that neomycin inhibited 5-HT contraction in the guinea pig trachea independent of PI turnover. Although bradykinin stimulated phospholipase D (PLD) activity, 5-HT did not activate PLD, suggesting that the 5 HT2A receptor is not coupled to this enzyme in the guinea pig trachea. Neomycin (3 mM) and nitrendipine (1 microM) inhibited 5-HT-induced contraction to a similar extent, and neomycin did not further inhibit contraction in the presence of nitrendipine. These data indicate that neomycin inhibited 5-HT-induced contraction, like nitrendipine, via an effect on calcium influx through L-type calcium channels and did not affect intracellular calcium release. However, unlike nitrendipine which completely blocked KCl-induced contraction, neomycin only marginally reduced the maximal KCl-induced contraction. Taken together, these data suggest that neomycin may indirectly inhibit calcium influx through L type calcium channels in guinea pig tracheal smooth muscle. The mechanism by which neomycin inhibited calcium influx in the guinea pig trachea may provide insight into the novel signaling pathway of the 5-HT2A receptor in this tissue. PMID- 8627579 TI - Discriminative stimulus effects on enadoline in pigeons. AB - The discriminative stimulus effects of enadoline were characterized in pigeons responding under a fixed-ratio 20 schedule of food presentation and discriminating between intramuscular injections of the kappa opioid agonist enadoline and saline. Cumulative doses of enadoline dose-dependently increased drug-key responding with the training dose of enadoline (0.178 mg/kg) producing > or = 90% drug key responding (% DR). In time course studies, doses of enadoline larger than 0.32 mg/kg produced > or = 90% DR for more than 40 min. Naltrexone antagonized both the discriminative stimulus and the rate-decreasing effects of enadoline (pA2 = 6.79 and 6.73, respectively); in some pigeons, naltrexone produced an unsurmountable antagonism of the enadoline discriminative stimulus. Substitution tests using the kappa agonists U-50,488, spiradoline, U-69,593 and ethylketocyclazocine resulted in > or = 90% DR in most, but not all, pigeons; at larger doses, all compounds markedly decreased response rates. Up to rate decreasing doses, nalorphine, dynorphin A(1-13) amide (DYN), nalbuphine, butorphanol, morphine and ketamine failed to occasion > or = 90% DR; nalbuphine, nalorphine, butorphanol, but not DYN, antagonized the discriminative stimulus and the rate-decreasing effects of enadoline. This study established stimulus control with enadoline in pigeons and results from substitution studies in these pigeons support the view that the enadoline discriminative stimulus is mediated by kappa opioid receptors; these results further demonstrate that nalbuphine and butorphanol have kappa antagonist actions in pigeons. The negative results obtained with DYN are in contrast to previous demonstrations of kappa agonist effects for DYN and might provide support for the hypothesized importance of nonopioid systems in the effects of this peptide. PMID- 8627580 TI - Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100,907 as a potent 5-HT2A antagonist with a favorable CNS safety profile. AB - In preclinical studies, [R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4 fluorophenyl)ethyl]-4- piperidinemethanol] [formula: see text] (MDL 100,907), a putative atypical antipsychotic, was characterized in vitro as a potent and selective ligand for the serotonin2A (5-HT2A) receptor and was evaluated in vitro and in vivo as a potent 5-HT2A receptor antagonist. Furthermore, MDL 100,907's potential CNS safety profile and selectivity as a potential antipsychotic agent were evaluated and compared with benchmark compounds. MDL 100,907 demonstrated low nanomolar or subnanomolar binding in vitro at the 5-HT2A receptor and showed a > 100-fold separation from all other receptors measured. MDL 100,907 had subnanomolar potency as a 5-HT2A antagonist in vitro in reversing 5-HT-stimulated inositol phosphate accumulation in NIH 3T3 cells transfected with the rat 5-HT2A receptor. In vivo, MDL 100,907 potently inhibited 5-methoxy-N, N dimethyltryptamine-induced head twitches in mice or 5-hydroxytryptophan-induced head twitches in rats. In vivo functional tests in mice revealed a > 500-fold separation between doses that produced 5-HT2A antagonism and doses that produced alpha 1-adrenergic or striatal D2 antagonism. Using inhibition of D-amphetamine stimulated locomotion in mice as a measure of potential antipsychotic efficacy, MDL 100,907 showed a superior CNS safety index relative to the reference compounds, haloperidol, clozapine, risperidone, ritanserin, and amperozide, in each of five tests for side effect potential, including measures of ataxia, general depressant effects, alpha 1-adrenergic antagonism, striatal D2 receptor antagonism, and muscle relaxation. MDL 100,907 did not antagonize apomorphine induced stereotypes in rats, suggesting that it potentially lacks extrapyramidal side effect liability. MDL 100,907 showed selectivity as a potential antipsychotic in that it lacked consistent activity in selected rodent models of anticonvulsant, antidepressant, analgesic, or anxiolytic activity. In summary, these preclinical data indicate that MDL 100,907 is a potent and selective ligand at the 5-HT2A receptor. MDL 100,907's potent 5-HT2A antagonist activity might account for its activity in preclinical models of antipsychotic potential. Ongoing clinical evaluation with MDL 100,907 will test the hypothesis that 5-HT2A receptor antagonism is sufficient for antipsychotic activity in humans. PMID- 8627581 TI - Biotransformation of tirilazad in human: 1. Cytochrome P450 3A-mediated hydroxylation of tirilazad mesylate in human liver microsomes. AB - Tirilazad mesylate (Freedox), a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclusively via hepatic elimination. Characterization of three major microsomal metabolites of tirilazad by mass spectrometry indicated that hydroxylation had occurred in the pyrrolidine ring(s) and at the 6 beta-position of the steroid domain. A role for CYP3A4 in the formation of the three major metabolites (tirilazad hydroxylase activity) was established in human liver microsomal preparations: 1) Tirilazad hydroxylation was potently inhibited by troleandomycin and ketoconazole, specific inhibitors of CYP3A4. 2) The rates of tirilazad hydroxylation within a population of 14 human livers displayed a 9-fold interindividual variation and a significant correlation (r2 = .95) between tirilazad hydroxylation and testosterone 6 beta-hydroxylation. 3) Kinetic analysis of tirilazad hydroxylase activity in three human livers resulted in an apparent Km of 2.12, 1.68 and 1.66 microM, and Vmax = 0.85, 0.44 and 3.45 (nmol/mg protein/min) for HL14, HL17 and HL21, respectively. In addition, an apparent Km of 2.07 microM was established for tirilazad hydroxylation in a cDNA-expressed CYP3A4 microsomal system. Collectively, these data indicate that the metabolic clearance of tirilazad in humans is catalyzed primarily by CYP3A4 and provide an insight into factors (i.e., age, sex, drug drug interactions) that modulate the metabolic clearance of tirilazad in vivo. PMID- 8627582 TI - Biotransformation of tirilazad in human: 2. Effect of ketoconazole on tirilazad clearance and oral bioavailability. AB - The effect of ketoconazole, a CYP3A inhibitor, on the oral bioavailability of tirilazad mesylate was assessed in 12 healthy subjects, who received the following treatments in a crossover design: a) 10 mg/kg tirilazad mesylate solution orally on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily, b) 10 mg/kg tirilazad mesylate solution orally, c) 2 mg/kg i.v. tirilazad mesylate solution on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily and d) 2mg/kg i.v. tirilazad mesylate solution. Plasma concentrations of tirilazad mesylate and its active reduced metabolites (U-89678 and U-87999) were measured by high-performance liquid chromatography. Urinary ratios of 6 beta hydroxycortisol to cortisol (6 beta-OHC/C) were measured as an index of hepatic CYP3A activity. Ketoconazole increased mean tirilazad mesylate area under the curve (AUC) values by 67% and 309% for i.v. and oral administration, respectively. Mean AUC values for U-89678 were increased 472% and 720% by ketoconazole coadministration with i.v. and oral tirilazad, respectively, whereas increases of > 10-fold in mean U-87999 AUC values were observed. These differences were statistically significant. These results indicate that ketoconazole inhibits the metabolism of these three compounds, which suggests that all of the compounds are substrates for CYP3A. Urinary 6 beta-OHC/C ratios did not reflect this level of effect of ketoconazole on CYP3A; this probe may not be useful for assessing the effect of CYP3A inhibitors. The absolute bioavailability of oral tirilazad was 8.7 +/- 4.8%; ketoconazole increased the bioavailability to 20.9 +/- 6.5%. Ketoconazole increased tirilazad mesylate bioavailability by decreasing the first-pass liver and gut wall metabolism of tirilazad mesylate to similar degrees. PMID- 8627583 TI - Isosteric substitution of Asn5 in antagonists of oxytocin and vasopressin leads to highly selective and potent oxytocin and V1a receptor antagonists: new approaches for the design of potential tocolytics for preterm labor. AB - Substitution of Asn5 in oxytocin (OT) or vasopressin (VP) invariably leads to a dramatic loss of the biological activities of the peptides. Because of this observation, few structure-activity-relationship studies of OT and VP peptides have involved modifications in the 5 position. It is now recognized that peptide agonists and antagonists may use different structural and conformational features in their interactions with the receptors. Our prior studies showed that OT and VP antagonists, unlike the agonists, tolerate amino acid substitutions in the 5 position. This opens new approaches for the design of antagonists. We describe the effects of isosteric replacement of Asn5 by diaminopropionic acid (Dap) or diaminobutyric acid (Dab) in three OT and VP antagonists: (1) the V1a (vasopressor receptor) antagonist d(CH2)5[Tyr(Me)2]AVP; (2) the OT (uterine OT receptor) antagonist d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH29] OVT and (3) three selective OT antagonists, desGly-NH2,d(CH2)5[D-Tyr2, Thr4]OVT, desGly-NH2, d(CH2)5[D-Phe2, Thr4]OVT and desGly-NH2, d(CH2)5- [D-Trp2, Thr4]OVT. The Dap5 and Dab5 substitutions were tolerated remarkably well, with the less isosteric Dap5 substitution leading to a greater retention of anti-OT potency than the Dab5 substitution. Furthermore, the Dap5 and Dab5 and OT and VP antagonist analogues were surprisingly shown to be much more selective than their respective parent compounds. The Dab5 analogue of (1) was devoid of anti-OT activity. The three Dap5 analogues of (3) were devoid of anti-V1a activities. These appear to be the first single-receptor-type-selective OT and VP antagonists discovered to date. These findings could provide new leads for the development of single-receptor type-selective receptor probes for the localization and characterization of OT and VP receptors and potential selective tocolytics for the treatment of premature labor. PMID- 8627584 TI - Aspirin-mouthwash relieves pain of oral lesions. PMID- 8627585 TI - Medicinal plants of Seychelles. AB - Plants are known to contain pharmacologically active substances. Traditional medical practitioners have considerable knowledge of herbal medicines. The Seychelles have many unique plants which have not been studied in depth. The people of Seychelles originate from diverse ethnic backgrounds which has resulted in a culture with a strong tradition of use of herbal medicines and consultation with traditional healers. Some of the plants and their use in traditional healing are outlined. PMID- 8627586 TI - Climate change. PMID- 8627587 TI - Drugs and deprivation. PMID- 8627588 TI - Public health policy and the nursing profession. PMID- 8627589 TI - Coronary heart disease and public health policy. PMID- 8627590 TI - Characterisation of the effects of alcohol use and abuse on the brain. PMID- 8627591 TI - Occupational health services: supply, demand, & company size. PMID- 8627592 TI - Condensation and mould: the Canadian experience. AB - It has been estimated that up to 20% of the UK housing stock is significantly affected by dampness and associated with mould growth. The effects on the health of the occupants of affected homes are well documented. The recent imposition of VAT at 17.5% on domestic fuel is generally regarded as likely to worsen the problem. However, this deteriorating situation puts me in mind of a recent study tour to Canada where the problem of dampness in housing is being tackled very differently to the United Kingdom. Are there any lessons to be learnt? PMID- 8627593 TI - Smoking habit in female students of northwestern Greece: relation to other cardiovascular risk factors. AB - THE AIMS OF OUR STUDY WERE: (1) to detect the prevalence of cigarette smoking among female students in Northwestern Greece, and (2) to assess the relation of cigarette smoking with other harmful lifestyles (such as alcohol consumption, obesity, type of dietary fat), fasting lipids and blood pressure. A total of 590 female students in the last class of the local senior high schools was studied. Seventy-seven (13%) of the students were smokers. Interestingly, in this cohort of young female students clustering of the smoking habit with dyslipidemia and alcohol use was observed. However, smoking was associated with significantly lower levels of blood pressure, as well as with lower mean body weight and body mass index. In conclusion, a considerable though apparently decreasing number of Greek female students are smokers. The smoking habit is strongly associated with dyslipidemia and alcohol consumption in this population. PMID- 8627594 TI - Obesity among female university students in the United Arab Emirates. AB - The aim of the study was to determine the prevalence of obesity among female students from the United Arab Emirates University. A sample of 566 students was taken. Body weight and height were measured. The students were interviewed to collect data on age, home Emirate, obesity in childhood, obesity among parents, food intake between meals, the consumption of fast foods and physical activity. A student was considered overweight when the body weight was 110-120% of the reference weight of height and was considered obese when the body weight exceeds 120% of the reference value. The results show that 10.8% of the students were overweight and 30.6% were obese. While the prevalence of obesity increased with age, obesity among students was associated with obesity during childhood, the presence of obesity among one or both parents, food intake between meals and in particular fast foods. Limited physical exercise and long afternoon napping were important contributors to the development of obesity. PMID- 8627595 TI - Health risk behaviour of a medical student population: report on a pilot study. AB - A pilot study of a cross-sectional nature was carried out to observe and describe the health risk behaviour of a medical student population. The participants (242) were drawn from the students of the University Medical School of Szeged, Hungary. The students were aged 18-31 years (x = 23) and were randomly selected. The response rate was (73%). The project focussed on 4 harmful habits ranked in the following order of prevalence: excessive coffee drinking (35%), smoking (20.9%), regular alcohol use (6.8%) and illicit drug use (5.1%). The non-parametric (Chi square) test showed significant differences between the higher and lower physical activity groups in terms of psychological well-being (p < 0.05) and health behaviour changes (p < 0.005). Harmful habits, however, were reported more frequently by the higher physical activity group. Significant differences could be detected in terms of women's illicit drug use (p < 0.05). Using the Mann Whitney U-test, it was detected that those who performed more physical activities rated their health significantly higher (p < 0.001). This study will be pursued in an expanded study with a larger sample and concentrate especially on the relationship of physical activity behaviour to harmful habits. Follow-up methods are also planned to study the medical student population over time, which should yield some greater insight into these relationships. PMID- 8627596 TI - Design and synthesis of 2',3'-dideoxy-2',3'-didehydro-beta-L-cytidine (beta-L d4C) and 2',3'-dideoxy 2',3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), two exceptionally potent inhibitors of human hepatitis B virus (HBV) and potent inhibitors of human immunodeficiency virus (HIV) in vitro. PMID- 8627597 TI - 2(S)-((3,5-bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4- ((3-oxo-1,2,4-triazol 5-yl)methyl)morpholine (1): a potent, orally active, morpholine-based human neurokinin-1 receptor antagonist. PMID- 8627598 TI - Methotrexate resistance of mouse dihydrofolate reductase: effect of substitution of phenylalanine-31 by serine or tryptophan. AB - Steady state and preliminary pre steady state studies of the mouse DHFR indicate that the wild-type enzyme used for our mutagenic studies follows a significantly different in vitro kinetic pathway than previously reported. In particular, turnover does not appear to be governed by H4F release from the E.NADPH complex. The discrepancies in catalysis and binding behavior of the mouse DHFRs may be due to the isomeric nature of the DHFRs studied. The enhanced ability of the two mutations at position 31 to confer resistance to MTX, as expected, decreased the affinity of the enzyme for the inhibitor. A correlation between the increased size of the side chain at position 31 and decreased inhibitor affinity was observed. This findings is consistent with previous mutagenesis studies of mouse DHFR but is at odds with conclusions drawn from an analysis of the role of the position in inhibitor binding to human DHFR. It is generally agreed that a highly efficient enzyme is desired for most cellular metabolic functions; however, because substitution of position 31 with tryptophan impairs catalytic efficiency, it appears that there exists a high physiological tolerance for significantly impaired DHFR. Indeed, mice who have received transplants of bone marrow expressing the Trp-31 mutant or the severely impaired Arg-22 mutant are capable of surviving lethal doses of MTX. Nevertheless, the consequences in vivo of a reduction in the observed in vitro catalytic effectiveness of DHFR remain to be determined. Additional mutagenic studies attempting to select catalytically silent mutations that reduce inhibitor binding may further enhance the therapeutic potential of drug-resistant DHFR genes for improved folate antagonist mediated antitumor activity. PMID- 8627599 TI - Development of a high specific activity sulfur-35-labeled sulfonamide radioligand that allowed the identification of a new growth hormone secretagogue receptor. PMID- 8627600 TI - Nucleoside conjugates. 15. Synthesis and biological activity of anti-HIV nucleoside conjugates of ether and thioether phospholipids. AB - A series of the anti-HIV nucleoside conjugates of either (1-O-alkyl) and thioether (1-S-alkyl) lipids linked by a pyrophosphate diester bond has been synthesized as micelle-forming prodrugs of the nucleosides to improve their therapeutic efficiency. These include AZT 5'-diphosphate-rac-1-S-octadecyl-2-O palmitoyl-1-thioglycerol (1), 3'-azido-2',3'-dideoxyuridine 5'-diphosphate-rac-1 S-octadecyl-2-O-palmitoyl-1-thioglycerol (2) 2',3'-dideoxycytidine 5'-diphosphate rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (3), and AZT 5'-diphosphate-rac-1 O-tetradecyl-2-O-palmitoylglycerol (4). The conjugates form micelles by sonication (mean diameters ranging 6.8-55.5 nm). Conjugate 1 protected 80% of HIV infected CEM cells as low as 0.58 microM and lost the protection at 180 microM due to prevailing cytotoxicity, while the conjugate started to show the cytotoxicity at 100 microM. Pharmacokinetics studies showed a significant increase of half-life values (t1/2) of AZT and AZddU2 (respective t1/2 = 5.69 and 6.5 h) after administration of conjugates 1 and 2, while those after administration of AZT and AZddU were 0.28 and 0.89 h, respectively. The fractions of the prodrugs 1 and 2 converted to the parent compounds AZT and AZddU were 36% and 55%, respectively. The results indicate that AZT and AZddU thioether lipid conjugates 1 and 2 warrant further investigation. PMID- 8627601 TI - Synthesis and biological activity of novel nonsteroidal progesterone receptor antagonists based on cyclocymopol monomethyl ether. AB - A novel class of nonsteroidal progesterone receptor antagonists has been synthesized and was shown to exhibit moderate binding affinity for hPR-A, the ability to inhibit the transcriptional activity of human progesterone receptor (hPR) in cell-based assays, and anti-progestational activity in a murine model. Cyclocymopol monomethyl ether, a component of the marine alga Cymopolia barbata was weakly active in random screening against PR. Investigations into the SAR surrounding the core of this natural product lead structure resulted in improved in vitro activity. In contrast to the cross-reactivity profiles observed with known steroidal antiprogestins, compounds of the general structural class described display a high degree of selectivity for the progesterone receptor and no functional activity on the glucocorticoid receptor. PMID- 8627602 TI - Longimicins A-D: novel bioactive acetogenins from Asimina longifolia (annonaceae) and structure-activity relationships of asimicin type of annonaceous acetogenins. AB - Bioactivity-directed fractionation of the ethanol extract of Asimina longifolia led to the isolation of four novel bioactive annonaceous acetogenins: longimicins A-D (1-4). Compounds 1-4 represent the asimicin type of acetogenins; however, the locations of the adjacent bis-tetrahydrofuran (THF) ring moieties are shifted along the aliphatic chains compared to the known compounds of this type. They are the first examples among this type of acetogenins with the placements of the ring systems altered. Compounds 1-4 showed bioactivities in several bioassays, but they are less active than their structural isomers. Study of their structure activity relationships (SAR) reveals that the position of the adjacent bis-THF ring moiety is essential for maximization of the bioactivities among these asimicin type annonaceous acetogenins. PMID- 8627603 TI - Mapping the melatonin receptor. 4. Comparison of the binding affinities of a series of substituted phenylalkyl amides. AB - A series of 2-, 3-, and 4-substituted phenylalkyl amides were prepared as potential melatonin analogs in order to investigate the nature of the binding site of the melatonin receptor in chicken brain. The length of the alkyl chain was systematically varied from n = 1 to 4, and methoxyl substituents were incorporated into the phenyl ring at the 2-, 3-, and 4-positions. The maximum binding affinity was found to occur when n = 3 and when the methoxyl substituent was in the 3-position, the direct analog of the carbon framework of melatonin in which the 1,2-atoms of the indole ring have been removed. Whereas there was only a relatively small decrease in binding affinity for the corresponding 2-methoxy derivatives, 4-methoxyl substitution led to a large decrease in binding affinity, suggesting that the binding sites for the side chain and methoxyl group could not now be occupied at the same time. As in the indole analogs of melatonin, replacement of the methyl group of the amide by a longer alkyl chain led to an increase in binding affinity for ethyl and propyl with a subsequent decrease in binding affinity for butyl chains. Thus N-propanoyl-3-(3 methoxyphenyl)propanamine (6f) has a binding affinity of 5.6 nM, a remarkably high affinity for so simple a compound. Substitution of halogen for 3-methoxyl in the propanamide series gave a series of compounds with lower, but still substantial, binding affinities, the 3-chloro derivative 7e showing the highest affinity, 113 nM. In the case of the 3-fluoro propanamides, a maximum in the binding affinity was not observed in the series synthesized, and these derivatives will merit further exploration. These results demonstrate the utility of simple, readily modified phenylalkylamines as a "framework" for studying the effect of changes in the nature and position of substituents on the melatonin receptor binding affinity. PMID- 8627604 TI - Non-peptide cholecystokinin-B/gastrin receptor antagonists based on bicyclic, heteroaromatic skeletons. AB - A series of potent and selective cholecystokinin-B/gastrin receptor antagonists based on the dibenzobicyclo[2.2.2]octane (BCO) skeleton which have recently been described were found to show species-dependent behavior when examined in rat and dog models. We now report the discovery of compounds in which the BCO skeleton has been replaced with bicyclic, heteroaromatic frameworks, such as a 5,6 disubstituted indole or benzimidazole. These new ligands maintain the affinity and selectivity profile of the previous compounds in vitro but show a much more consistent behavior pattern in vivo. Representative examples of this class of compound have been shown to inhibit pentagastrin-stimulated acid secretion when administered intravenously at doses of 0.1 mumol kg-1 or less. PMID- 8627605 TI - Opioid antagonist activity of naltrexone-derived bivalent ligands: importance of a properly oriented molecular scaffold to guide "address" recognition at kappa opioid receptors. AB - The presence of a molecular scaffold to orient a basic group is important for potent and selective kappa opioid antagonist selectivity. An attempt to determine how the geometry of the scaffold affects this selectivity has led to the synthesis of a bivalent ligand (5) whose linker constrains the N17' basic nitrogen (the "address") to a position that is 6.5 A from N17' in the kappa antagonist norBNI (1) when these molecules are superimposed. The fact that compound 5 was found to be a highly selective and potent mu-selective antagonist supports the idea that the position of N17' in 5 precludes effective ion pairing with the nonconserved residue Glu297 on outer loop 3 of the kappa opioid receptor. The high mu receptor binding affinity and in vitro pharmacological selectivity of 5 coupled with its presumed low central nervous system bioavailability suggest that it may be a useful antagonist for the investigation of peripheral mu opioid receptors. PMID- 8627606 TI - Tyrosine kinase inhibitors. 10. Isomeric 4-[(3-bromophenyl)amino]pyrido[d] pyrimidines are potent ATP binding site inhibitors of the tyrosine kinase function of the epidermal growth factor receptor. AB - Following the discovery of the very high inhibitory ability of the 4-[(3 bromophenyl)amino]-quinazolines against the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (e.g., 3, IC50 0.029 nM), four series of related pyrido[d]pyrimidines bearing electron-donating groups at the 6- or 7 positions have been synthesized and evaluated. The compounds were prepared by nucleophilic substitution of the corresponding 6- and 7-fluoro analogues. While members of all series showed potent inhibitory activity against isolated EGFR, there were important differences between the different isomeric pyrido[d]pyrimidines and the parent quinazolines. Overall, the [3,4-d] and [4,3 d] series were the most potent, followed by the [3,2-d] compounds, with the [2,3 d] analogues being least active. Whereas in the parent quinazoline series the addition of steric bulk to a 6- or 7-NH2 substituent (i.e., NHMe and NMe2 groups) dramatically decreased potency, no such trend was discernable in the [3,2-d] series. Furthermore, in the 7-substituted pyrido[4,3-d]- and 6-substituted pyrido[3,4-d]pyrimidine series, and to a limited extent in the 7-substituted pyrido[2,3-d] series, such substitution increased potency dramatically, to the extent that the 7-(methylamino)pyrido[4,3-d]pyrimidine (5f) (IC50 0.13 nM) and 6 (methylamino)pyrido[3,4-d]pyrimidine (7f) (IC50 0.008 nM) constitute important new leads. Selected compounds were evaluated for their ability to inhibit EGFR autophosphorylation in A431 cells, and a positive quantitative correlation was found between this activity and inhibitory activity against the isolated enzyme. PMID- 8627607 TI - Nonclassical 2,4-diamino-8-deazafolate analogues as inhibitors of dihydrofolate reductases from rat liver, Pneumocystis carinii, and Toxoplasma gondii. AB - The synthesis and biological activity of 42 6-substituted-2,4-diaminopyrido[3,2 d]pyrimidines (2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in improved yields compared to previous classical analogues using modifications of procedures reported previously by us. Specifically, the S phenyl-; mono-, di-, and trimethoxyphenyl-; and mono-, di-, and trichlorophenyl substituted analogues with H or CH3 at the N10 position and methyl and trifluoromethyl phenyl ketone analogues with H, CH3, and CH2C identical to CH at the N10 position were synthesized. The S10 and N10 alpha- and beta-naphthyl analogues along with the N10 CH3 analogues were also synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg); selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference enzyme. Against pcDHFR the IC50 values ranged from 0.038 x 10-6 M for 2,4-diamino-6-[(N-methyl-2' naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 x 10(-6) M for 2,4 diamino-6[(2',4'-dimethoxyanilino)methyl]pyrido[3,2-d]pyrim idi ne (15). N10 methylation in all instances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was 2,4-diamino-6 [(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine (5) (IC50 0.0084 x 10(-6) M) and the least potent was 2,4-diamino-6[(2'-naphthylamino)methyl]-pyrido[3,2 d]pyrimidine (37) (IC50 0.16 x 10-6 M). N10 methylation afforded an increase in potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2'-chloro-N methylanilino)-methyl]pyrido[3,2-d] pyrimidine (13), 2,4-diamino-6-[(3',4',5' trimethoxyanilino)methyl]pyrido[3,2-d]pyr pyrimidine (29), and 2,4-diamino-6 [(2',4',6'-trichloroanilino)methyl]pyrido[3,2-d] pyrimidine (32) which combined high potency at 10-8 M along with selectivities of 8.0, 5.0, and 12.4, respectively. The potency of these three analogues are comparable to the clinically used agent trimetrexate while their selectivities for tgDHFR are 17-43 fold better than trimetrexate. PMID- 8627609 TI - Selective inhibitors of monoamine oxidase. 3. Structure-activity relationship of tricyclics bearing imidazoline, oxadiazole, or tetrazole groups. AB - Inhibition of monoamine oxidase A (MAO A) is believed to cause antidepressant and possibly antianxiety effects. The previous paper had developed structure activity relationships (SAR) for in vitro MAO A inhibition by tricyclic N arylamides. It is shown in this paper that the same in vitro SAR can be carried over to tricyclics whose potentially toxic amide function is replaced by an appropriately substituted imidazoline, a 1,2,4- or 1,3,4-oxadiazole, or an alkylated tetrazole moiety. Dialysis of the inhibitor from the enzyme was used as a measure of reversibility which correlates with a low ability to cause a blood pressure rise with ingested tyramine ("cheese effect"). PMID- 8627608 TI - Novel terphenyls as selective cyclooxygenase-2 inhibitors and orally active anti inflammatory agents. AB - A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure-activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5 difluorobenzenesulfonamide series are 21a-c,k,l,n (COX-2, IC50 = 0.002-0.004 microM), in which all have in vitro COX-1/COX-2 selectivity > 1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg. PMID- 8627610 TI - Synthesis and structure-activity relationship of (lactamylvinyl)cephalosporins exhibiting activity against staphylococci, pneumococci, and enterococci. AB - The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone. PMID- 8627611 TI - Structure-based design of novel, urea-containing FKBP12 inhibitors. AB - The structure-based design and subsequent chemical synthesis of novel, urea containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506. PMID- 8627612 TI - Structure-activity relationships of the antimalarial agent artemisinin. 3. Total synthesis of (+)-13-carbaartemisinin and related tetra- and tricyclic structures. AB - Provided by total synthesis, endoperoxides 18, 20, and 22 underwent intramolecular oxymercuration-demercuration leading respectively to formation of an isomeric tetracycle, (1aS, 3S, 5aS, 6R, 8aS, 9R, 12S)-10-deoxo-13 carbaartemisinin (19), (+)-10-deoxo-13-carbaartemisinin (21), and (+)-13 carbaartemisinin (4). Structure assignment to 19 and 21 was based on single crystal X-ray crystallographic analysis. Tricyclic endoperoxide 20 was converted to methyl and benzyl ethers 23 and 24 and reduced to saturated analog 25 which was also converted to ethers 26 and 27. In vitro antimalarial screening of both tri- and tetracyclic analogs was conducted using the W-2 and D-6 clones of Plasmodium falciparum. Neither target 4 nor 21 displayed substantial antimalarial potency in vitro against P. falciparum, but the diastereomeric peroxide 19 possessed good antimalarial potency in vitro. Tricyclic analogs were uniformly impotent. Iron(II) bromide-promoted rearrangement of 21 gave, in 79% yield, the unique tetracyclic alcohol 35, while 19 provided ring-opened cyclohexanone 41 (39%) along with the tricyclic epoxide 42 (20%). Neither 41 nor 42 possessed in vitro antimalarial activity, suggesting that epoxide-like intermediates are not responsible for the mode of action of this subclass of antimalarials. Rearrangement of 10-deoxoartemisinin (43) with FeBr2 gave a major product (79%) not encountered in the rearrangement of artemisinin that resulted from unraveling of the tetracyclic system cyclohexanone 46. Minor amounts of 1,10 dideoxoartemisinin (49) (8%) were also produced in this reaction. PMID- 8627613 TI - 3,3-Dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones: a new class of anticonvulsant agents. AB - A series of 3,3-dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones (lactams) have been prepared and evaluated for their anticonvulsant activities. In the pentylenetetrazole mouse seizure model, 3,3-diethyl lactam 7c and 3-benzyl 3-ethyl lactam 7j are the most effective anticonvulsants (ED50 = 46 and 42 mg/kg, respectively) and have protective index (PI = TD50/ED50) values of 5.65 and 3.00, respectively. These protective index values compare favorably to those of the clinically used antiepileptic drugs ethosuximide (ED50 = 161 mg/kg), phenobarbital (ED50 = 22 mg/kg), and valproic acid (ED50 = 133 mg/kg), which have PI values of 2.35, 4.00, and 2.12, respectively. The benzyl compounds [3 substituents are Bn, H (7h); Bn, Me (7i); and Bn, Et (7j)] are also very effective anticonvulsants against seizures induced by maximal electroshock (ED50 = 41, 55, and 74 mg/kg, respectively) and have PI values of 3.51, 3.04, and 1.70, respectively. The corresponding PI values for phenobarbital and valproic acid are 1.37 and 5.18, respectively. As a class of anticonvulsants, the 3,3-disubstituted 2-pyrrolidinones have a broad spectrum of action and may be useful for the treatment of human epilepsies. PMID- 8627614 TI - Synthesis and anticonvulsant activities of N-Benzyl-2-acetamidopropionamide derivatives. AB - Studies have demonstrated that 2-substituted N-benzyl-2-acetamidoacetamides (2) are potent anticonvulsants. A recent investigation has led to the hypothesis that an important structural feature in 2 for maximal anticonvulsant activity is the placement of a small, substituted heteroatom moiety one atom from the C(2) site. This paper validates this hypothesis. Twelve derivatives of N-benzyl-2 acetamidopropionamide have been prepared in which six different heteroatom substituents (chloro, bromo, iodo, oxygen, nitrogen, and sulfur) were incorporated at the C(3) site. Highly potent activities were observed for the two oxygen-substituted derivatives, N-benzyl-2-acetamido-3-methoxypropionamide (18) and N-benzyl-2-acetamido-3-ethoxypropionamide (19). The ED50 values in mice following intraperitoneal (i.p.) dosing for the maximal electroshock-induced seizure test for 18 and 19 were 8.3 and 17.3 mg/kg, respectively. These values compared favorably to the ED50 value found for phenytoin (ED50 = 6.5 mg/kg). Comparable activities were observed for 18 and 19 upon oral (p.o.) administration to rats (18, ED50 = 3.9 mg/kg; 19, ED50 = 19 mg/kg; phenytoin, ED50 = 23 mg/kg). Evaluation of the individual stereoisomers for 18 demonstrated that the principal anticonvulsant activity resided in the (R)-stereoisomer. The ED50 value for (R) 18 was 4.5 mg/kg, and the ED50 for (S)-18 exceeded 100 mg/kg. This difference in activity for the two stereochemical isomers surpassed comparable values for other members within this class of compounds. The protective indices (PI = TD50/ED50) (where TD50 represents a neurotoxic dose impairing rotorod performance) for (R) 18 in mice (i.p.) and in rats (p.o.) were 6.0 and > 130, respectively. PMID- 8627615 TI - 2-(Hydroxyalkyl)estradiols: synthesis and biological evaluation. AB - Synthetic estrogens possessing hydroxyalkyl side chains at the C-2 position of the A-ring were designed in order to further elucidate the structural and electronic requirements of the estrogen receptor to A-ring modifications. Furthermore, these compounds were envisaged as being stable analogs of the estradiol metabolite 2-hydroxyestradiol. The homologous series of 2 (hydroxyalkyl)estradiols 1-3 has been prepared by chain extension of 2 formylestradiol 6, which, in turn, was prepared via ortholithiation of estradiol. The substituted estradiols 1-3 were assayed for their abilities to bind to the estrogen receptor in MCF-7 cells and induce estrogen-responsive gene expression. The estradiol homologs exhibited significantly weaker affinity than estradiol for the MCF-7 cell estrogen receptor, with relative binding affinities (estradiol = 100) ranging from 1.11 for 2-(hydroxymethyl)estradiol (1) to 0.073 for 2 (hydroxypropyl)estradiol (3). The relative activities for mRNA induction of the pS2 gene by the estradiol homologs closely parallel the relative binding affinities for the estrogen receptor in MCF-7 cells. 2-(Hydroxymethyl)-estradiol exhibited similar estrogen receptor affinity and pS2 gene induction to the catechol estrogen 2-hydroxyestradiol and may prove useful in examination of the further biological effects of 2-hydroxyestrogen homologs. PMID- 8627616 TI - Highly selective aldose reductase inhibitors. 1. 3-(Arylalkyl)-2,4,5 trioxoimidazolidine-1-acetic acids. AB - A series of 3-(arylalkyl)-2,4,5-trioxoimidazolidine-1-acetic acids (1) was prepared and tested for aldose reductase (AR) and aldehyde reductase (ALR) inhibitory activities. These compounds showed strong inhibitory activity against AR without significant inhibitory activity for ALR. The ratio of IC50(ALR)/IC50(AR) was > 1000 in some compounds. On the basis of pharmacological tests such as the recovery of reduced motor nerve conduction velocity and toxicological profile, 3-(3-nitrobenzyl)-2,4,5-trioxoimidazolidine-1-acetic acid (NZ-314) was selected as the candidate for clinical development. PMID- 8627617 TI - Synthesis and pharmacological properties of novel 8-substituted imidazobenzodiazepines: high-affinity, selective probes for alpha 5-containing GABAA receptors. AB - The synthesis and pharmacological properties of imidazobenzodiazepines with both high affinity and selectivity for alpha 5-containing GABAA receptors are described. Four of these compounds (5, 6, 8, and 9) inhibited [3H]flunitrazepam binding to recombinant alpha 5 beta 2 gamma 2 GABAA receptors with IC50 values between approximately 0.4 and 5 nM. These compounds were > or = 24-75-fold more selective for recombinant receptors containing alpha 5 subunits compared to other, "diazepam-sensitive" GABAA receptors containing either alpha 1, alpha 2, or alpha 3 subunits. Imidazobenzodiazepine 9 (used as the prototypical alpha 5 selective ligand) inhibited [3H]flunitrazepam binding to hippocampal membranes with high- and low-affinity components (IC50 0.6 +/- 0.2 and 85.6 +/- 13.1 nM, respectively), representing approximately 16% and approximately 84% of the receptor pool. Inhibition of [3H]flunitrazepam binding to cerebellar membranes with imidazobenzodiazepine 9 was best fitted to a single population of sites with an IC50 of 79.8 +/- 18.3 nM. These imidazobenzodiazepines behaved as GABA negative ligands in recombinant GABAA receptors expressed in Xenopus oocytes and were convulsant in mice after parenteral administration. The relative potencies of flumazenil and zolpidem in blocking convulsions induced by 9 and DMCM, respectively, indicated that occupation of alpha 5-containing GABAA receptors substantially contributed to the convulsant properties of acetylene analog 9. These 8-substituted imidazobenzodiazepines (5, 6, 8 and 9) should prove useful in examining the physiological roles of GABAA receptors bearing an alpha 5 subunit and may also lead to the development of novel, subtype selective therapeutic agents. PMID- 8627618 TI - Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro. AB - Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparations techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (-15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM). PMID- 8627619 TI - Measurement of soft X-ray absorption spectra and elemental analysis in local regions of mammalian cells using an electronic zooming tube. AB - Direct measurement of absorption spectra for minute areas (2 microns x 2 microns) in a dried mammalian cell was attempted using X-ray contact images of a whole cell. The wavelength region used ranged from 1.5 to 10nm covering the absorption edges of the major cellular elements. The measurements were achieved taking advantage of synchrotron radiation as a tuneable light source and an electronic zooming tube as an X-ray detector with a high spatial resolution. The spectra in every intracellular area exhibited marked absorption changes at the absorption edges of carbon, nitrogen and oxygen, while minor but significant changes for iron and calcium were observed, particularly in the cytoplasmic areas. These results reveal the different spatial distributions of the constituent elements in a cell. PMID- 8627620 TI - Identifying interacting regions in the beta subunit of Escherichia coli RNA polymerase. AB - Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves. As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues. We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E). With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit. Allele specific suppression is a genetic criterion for protein-protein interaction. Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation. Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide. We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme. PMID- 8627621 TI - The stability of nucleosomes at the replication fork. AB - Purified simian virus (SV40) minichromosomes were photoreacted with psoralen under various conditions that moderately destabilize nucleosomes. This assay allows indirect distinction between stable nucleosomes, partially unravelled nucleosomes and nucleosomes containing (or lacking) histone H1. In replicating molecules the passage of the replication machinery destabilizes the nucleosomal organization of the chromatin fiber over a distance of 650 to 1100 bp. In front of the fork, an average of two nucleosomes are destabilized presumably by the dissociation of histone H1 and the advancing replication machinery. On daughter strands, the first nucleosome is detected at a distance of about 260 nucleotides from the elongation point. This nucleosome is interpreted to contain no histone H1, while no stepwise association of (H3-H4)2 tetramers with H2A/H2B dimers on nascent DNA can be detected in vivo. The second nucleosome after the replication fork appears to contain histone H1. The prolonged nuclease sensitivity of newly replicated chromatin described in the literature therefore may not be due to a slow reassociation of histone H1. PMID- 8627622 TI - The rate of CpG mutation in Alu repetitive elements within the p53 tumor suppressor gene in the primate germline. AB - Cytosine to thymine transition mutations at the CpG dinucleotide are the most common point mutations in cancer and genetic disease. We calculated the in vivo rate of CpG mutation in the primate germline by deriving a primordial consensus sequence for an Alu repetitive element which inserted into intron 6 of the primate p53 gene 35 to 55 million years ago. Comparison of this primordial sequence to the Alu sequence in intron 6 of present-day primates was used to determine the nature and rate of mutations which occurred during evolution. We estimate the half-life of a CpG nucleotide to be 24 to 60 million years, and the rate constant for mutation at this dinucleotide to be 1.2 x 1O(-8) to 2.9 x 1O( 8) years(-1). These results were confirmed by the analysis of a second Alu sequence in intron 10 of the p53 gene. The in vivo mutation rate is at least 1250 fold slower than the in vitro chemical rate of 5-methylcytosine deamination in double-stranded DNA, showing that current estimates of CpG mutation repair have been significantly underestimated. Furthermore, the mutability of the CpG dinucleotide has led to the depletion of this dinucleotide from the vertebrate genome, and calculations in this study suggest that current levels of the CpG dinucleotide in the primate genome are very close to a steady state equilibrium in which the rate of CpG mutation is equal to the rate of CpG formation by random mutation. PMID- 8627623 TI - Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots. AB - Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5' TC sites in the eight base palindrome 5' CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in the primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis. PMID- 8627624 TI - Nicotinic acetylcholine receptors in the nematode Caenorhabditis elegans. AB - Two cDNAs (Ce21 and Ce13) were isolated from a Caenorhabditis elegans library screened with a probe encoding conserved domains of the avian alpha5 neuronal nicotinic acetylcholine receptor (nAChR). Alignments to all nAChR subunits in the EMBL/Swissprot data base demonstrate that the Ce21 protein most resembles the vertebrate alpha7 subunit, whereas Ce13 is closest to the ARD subunit of Drosophila. The corresponding genes were isolated and hybridized to YAC grids: Ce21 maps on chromosome V near the his-23 gene, and Ce13 on chromosome I very near or at unc-29. The structure of the Ce21 gene was compared with that of other vertebrate and invertebrate nAChR genes and found to share by far the largest number of conserved splice sites with the vertebrate alpha7 gene. Upon expression in the Xenopus oocyte system, the Ce21 subunit assembled into a functional homomeric nAChR, whose properties were compared with those of the chicken alpha7 receptor. The anthelmintic nicotinic agonist levamisole is unable to activate the Ce21 and alpha7 receptors, but efficiently antagonises their responses to ACh. Both receptors desensitise quickly upon agonist application, are more sensitive to nicotine than to acetylcholine, and are efficiently blocked by dihydro-beta erythroidine. Unlike the alpha7 receptor, however, the Ce21 receptor is relatively insensitive to methyllycaconitine and to alpha-bungarotoxin. The similarities in protein sequence, gene structure and physiological properties between alpha7 and Ce21 suggest a very ancient lineage for the alpha7 class of nAChR subunits. PMID- 8627625 TI - Mutations in motB suppressible by changes in stator or rotor components of the bacterial flagellar motor. AB - Five proteins (MotA, MotB, FliG, FliM and FliN) may be involved in energizing flagellar rotation in Escherichia coli. To study interactions between the Mot proteins, and between them and the three Fli proteins of the switch-motor complex, we have isolated extragenic suppressors of dominant and partially dominant motB missense mutations. Four of the 13 motB mutations yielded partially allele-specific suppressors. Of the suppressing mutations, 57 are in the motA gene, eight are in fliG, and one is in fliM; no suppressor was identified in fliN. The prevalence of suppressors in fliG suggests that FliG interacts rather directly with the Mot proteins. The behaviour of cells in tethering and swarm assays indicates that the motA suppressors are more efficient than the fliG or fliM suppressors. Some of the suppressing mutations themselves confer distinctive phenotypes in motB+ cells. We propose a model in which mutations affecting residues in or near the putative peptidoglucan-binding region of MotB misalign the stator relative to the rotor. We suggest that most of the suppressors restore motility by introducing compensatory realignments in MotA or FliG. PMID- 8627626 TI - Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein. AB - Prohead-like capsid shells containing the scaffolding and head proteins of bacteriophage T7 were isolated after both proteins were expressed from the cloned genes in the same cell. When the head-tail connector protein was also expressed, the isolated capsids contained neither connector nor scaffolding protein and resembled mature phage capsids rather than proheads. However, only a small fraction of the head protein was converted to stable capsid structures in either case. Purified scaffolding protein (expressed individually from the cloned gene) appeared to be a monomer in solution; purified head protein appeared to be a tetramer. The purified proteins reacted in the presence of polyethylene glycol or dextran to produce prohead-like capsid shells and also polycapsids consisting primarily of head protein, similar to the polycapsids observed after infection by T7 mutants lacking connector or core proteins. Neither capsids nor polycapsids were produced in the absence of scaffolding protein. Polycapsids were usually the predominant product even when scaffolding protein was in excess, and a small fraction of scaffolding protein catalyzed the conversion of an excess of head protein to polycapsids. Our results suggest that the first step in the natural pathway to prohead formation is the assembly of incomplete prohead shells, which are normally closed by insertion of a connector-core complex. In the absence of a functional connector-core complex, incomplete capsid shells apparently react further to form polycapsids or completely closed capsid shells. PMID- 8627627 TI - Purification and characterization of T7 head-tail connectors expressed from the cloned gene. AB - Cloned gene 8, which specifies the protein of the head-tail connector of bacteriophage T7, was expressed in Escherichia coli. Extracts prepared in a low salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography perhaps because of dissociation of monomer-connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA. PMID- 8627628 TI - Structure of a hexanucleotide RNA hairpin loop conserved in ribosomal RNAs. AB - The structure of 5'-pppGGAC(GUAAUA)GUCC has been deduced from NMR data. The six nucleotide hairpin loop is highly conserved in large subunit ribosomal RNA and confers unusual stability on RNA hairpins. Standard assignment methods, including through-bond strategies for backbone protons, were used to assign all 31P resonances and all but two of the non-exchangeable protons. The model calculated by restrained molecular dynamics shows that the loop adopts a specific structure stabilized by five non-canonical hydrogen bonds. An edge-to-edge G-A pair (hydrogen bonds G5 N3-A10 NH6, G5 H2-A1O N7 and A10 NH6-G5 O2') closes the hairpin loop. A trans 5' P-O bond at A7 reverses the direction of the backbone and is stabilized by two base-backbone hydrogen bonds (U6 NH3-U9 OP and U6 O2'-A8 N7). The only unstructured part of the loop is U9, which is completely unstacked and also the only phylogenetically variable position. The U6-A7-A8 "U-turn" reproduces hydrogen bonds and backbone torsion seen in the tRNA anticodon loop and TTpsiC loop, and in an internal loop of the hammerhead ribozyme. G-A is a common closing mismatch in ribosomal RNA hairpin loops. The U-turn and G-A pair found in this hexaloop structure may therefore be common structural units of RNA hairpin loops. PMID- 8627629 TI - H NMR study of the solution structure of Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus. AB - The conformation in water of antimicrobial protein 2 from Amaranthus caudatus (Ac AMP2) was determined using 1H NMR, DIANA and restrained molecular modeling. Ac AMP2 is a 30 amino acid residue, lectin-like protein that specifically binds to chitin, a polymer of beta-1,4-N-acetyl-D-glucosamine. After sequence specific resonance assignments, a total of 198 distance restraints were collected from 2D NOESY buildup spectra at 500 MHz at pH 2, supplemented by a 2D NOESY spectrum at 600 MHz. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of Ac-AMP2 is presented as a set of 26 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol in the AMBER force field, with a backbone r.m.s.d. for the well defined Glu3-Cys28 segment of 0.69(+/-0.12) angstroms. The main structural element is an antiparallel beta-sheet from Met13 to Lys23 including a betaI-turn over Gln17-Phel8 with a beta bulge at Gly19. In addition, a beta'I turn over Arg6-Gly7, a beta'III turn over Ser11-Gly12 and a helical turn from Gly24 to Cys28 are identified. This structure is very similar to the equivalent regions of the X-ray structure of wheat germ agglutinin and the NMR structure of hevein. PMID- 8627630 TI - Effects of experimentally achievable improvements in the quality of NMR distance constraints on the accuracy of calculated protein structures. AB - New methods for collecting cross-relaxation data from proteins and nucleic acids make it possible to improve the accuracy and precision of interproton distance measurements used as input for NMR solution structure determinations. It thus is of interest to determine whether such experimentally achievable improvements in input distance constraints have significant effects on the precision and accuracy of the resulting structures. To answer this question, we have turned to a computational procedure involving the use of data simulated from a known structure, in order to allow unambiguous assessments of accuracy. The approach to improved distances evaluated here is that afforded by magnetization exchange network editing (MENE); MENE pulse sequences break the network of cross relaxation interactions into regions that are manipulated so as to defeat certain spin-diffusion terms. A target structure was prepared from the X-ray structure of a small protein, turkey ovomucoid third domain (OMTKY3). A normal NOESY spectrum and two varieties of MENE spectra, BD-NOESY and CBD-NOESY, were simulated by means of complete relaxation matrix analysis. These results were used to create different input data sets with the same number of constraints (perfectly accurate distances derived from the target structure, more accurate distances derived from the MENE simulations, and less accurate distances derived from the NOESY simulation), and these, interpreted at different levels of precision, were used as input for solution structure calculations. The results showed that the use of more precise input data measurably improves the local precision and accuracy of calculated structures, but only if the more precise data include the actual target distance. Incorporation of the experimentally achievable, accurate distances with higher precision afforded by the MENE pulse sequences into the set of input distances was found to improve the accuracy of the resulting structures, particularly in terms of side-chain conformation. PMID- 8627631 TI - Structural diversity in a family of homologous proteins. AB - An interesting example of a structurally diverse group of sequentially homologous proteins is analyzed at the level of molecular interactions. In this family, the EF-hand calcium-binding proteins, there are examples of at least three distinct mutual positions of the N and C-terminal domains, despite significant sequence homology between all members of this family. Why does a particular protein choose one arrangement over another? To answer this question, detailed models of all proteins in their native structures as well as all alternative sequence/structure combinations are built by comparative modeling. By studying and comparing interactions stabilizing native structures and destabilizing alternative conformations, it is possible to gain insight into how such conformational diversity is achieved. It is shown that some mechanisms used to achieve it are: correlated mutations on the surface of two units and the presence of additional domains/chain fragments stabilizing desired topologies. The implications of these findings, both for structure predictions for other members of this family as well as the general problem of quaternary structure formation, are discussed. PMID- 8627632 TI - Energy functions that discriminate X-ray and near native folds from well constructed decoys. AB - This study generates ensembles of decoy or test structures for eight small proteins with a variety of different folds. Between 35,000 and 200,000 decoys were generated for each protein using our four-state off-lattice model together with a novel relaxation method. These give compact self-avoiding conformations each constrained to have native secondary structure. Ensembles of these decoy conformations were used to test the ability of several types of empirical contact, surface area and distance-dependent energy functions to distinguish between correct and incorrect conformations. These tests have shown that none of the functions is able to distinguish consistently either the X-ray conformation or the near-native conformations from others which are incorrect. Certain combinations of two of these energy functions were able, however, consistently to identify X-ray structures from amongst the decoy conformations. These same combinations are better also at identifying near-native conformations, consistently finding them with a hundred-fold higher frequency than chance. The fact that these combination energy functions perform better than generally accepted energy functions suggests their future use in folding simulations and perhaps threading predictions. PMID- 8627633 TI - Motions in hemoglobin studied by normal mode analysis and energy minimization: evidence for the existence of tertiary T-like, quaternary R-like intermediate structures. AB - The normal mode analysis of human hemoglobin showed the presence in the deoxy T state of one main preferential direction that brings the structure close to the R state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like. PMID- 8627634 TI - Effects of estrogen on screening mammography: another complexity. PMID- 8627635 TI - Psychological distress and disease course for women with breast cancer: one answer, many questions. PMID- 8627636 TI - Transplants for liver cancer show promise, not proof. PMID- 8627637 TI - NCI's Klausner on challenges, solutions. Interview by Hugh McIntosh. PMID- 8627638 TI - Hormone replacement grows; some experts worried. PMID- 8627639 TI - Treatment network focuses on pediatric cancers. PMID- 8627640 TI - Effect of estrogen replacement therapy on the specificity and sensitivity of screening mammography. AB - BACKGROUND: Previous studies have demonstrated that mammographic breast density increases following the initiation of estrogen replacement therapy (ERT). The effect, if any, that this increase in density has on the specificity (related to false-positive readings) and the sensitivity (related to false-negative readings) of screening mammography is unknown. PURPOSE: Using a retrospective cohort study design, we assessed the effects of ERT on the specificity and the sensitivity of screening mammography. METHODS: Participants (n = 8779) were postmenopausal women, aged 50 years or older, who were enrolled in a health maintenance organization located in western Washington state and who entered a breast cancer screening program between January 1988 and June 1993. Two-view mammography was performed as part of a comprehensive breast cancer screening visit. Menopausal status, as well as demographic and risk-factor information, was recorded via self administered questionnaires. Hormonal replacement therapy type and use were determined from questionnaire data and from an automated review of pharmacy records. Individuals diagnosed with breast cancer within 12 months of their first screening-program mammograms were identified through use of a regional cancer registry. Risk ratios (RRs) plus 95% confidence intervals (CIs) of false-positive as well as false-negative examinations among current and former ERT users (with never users as the reference group) were calculated. Reported P values are two sided. RESULTS: The specificity of mammographic screening was lower for current users of ERT than for never users or former users. Defining a positive mammographic reading as any non-normal reading (either suspicious for cancer or indeterminate), the adjusted RR (95% CI) of a false-positive reading for current users versus never users was 1.33 (1.15-1.54) (P < .001); for former users versus never users, the RR (95% CI) was 1.00 (0.87-1.15). The adjusted mammographic specificities (95% CIs) for never users, former users, and current users of ERT were 86% (84%-88%), 86% (84%-87%), and 82% (80%-84%), respectively. Defining a positive reading more rigorously (i.e., as suspicious for cancer only), the adjusted RRs (95% CIs) of false-positive readings for current users and former users (versus never users) were 1.71 (1.37-2.14) (P < .001) and 1.16 (0.93-1.45), respectively. Sensitivity was also lower in women currently receiving ERT. The unadjusted RR (95% CI) of a false-negative reading for current users versus never users was 5.23 (1.09-25.02) (P = .04); for former users versus never users, the RR (95% CI) was 1.06 (0.10-10.87). The unadjusted mammographic sensitivities (95% CI) for never users, former users, and current users of ERT were 94% (80%-99%), 94% (69%-99%), and 69% (38%-91%), respectively. CONCLUSIONS AND IMPLICATIONS: Current use of ERT is associated with lower specificity and lower sensitivity of screening mammography. Lower specificity could increase the cost of breast cancer screening, and lower sensitivity may decrease its effectiveness. PMID- 8627641 TI - Relative weight, weight change, height, and breast cancer risk in Asian-American women. AB - BACKGROUND: Breast cancer incidence rates have historically been four to seven times higher in the United States than in China or Japan, although the reasons remain elusive. When Chinese, Japanese, or Filipino women migrate to the United States, their breast cancer risk rises over several generations and reaches that for white women in the United States, indicating that modifiable exposures are involved. In a previous report on this case-control study of breast cancer in Asian-American women, designed to take advantage of their diversity in risk and lifestyle, we demonstrated a sixfold gradient in risk by migration history, comparable to the international differences in breast cancer incidence rates. PURPOSE: In this analysis, we have examined the roles of adult height, adiposity, and weight change in breast cancer etiology. METHODS: A population-based, case control study of breast cancer was conducted among women of Chinese, Japanese, and Filipino ethnicities, aged 20-55 years, living in San Francisco-Oakland (CA), Los Angeles (CA), and Oahu (HI) during the period from April 1, 1983, through June 30, 1987. We successfully interviewed 597 (70%) of 852 eligible case subjects and 966 (75%) of 1287 eligible control subjects from August 1985 through February 1989. Subjects were asked about current height, usual adult weight, and usual weight in each decade of life, excluding the most recent 3 years and any periods of pregnancy. RESULTS: Height, recent adiposity (weight in the current decade of life/height 1.5), and recent weight change (between the current and preceding decades of life) were strong predictors of breast cancer risk after adjustment was made for accepted breast cancer risk factors. Risk doubled (relative risk [RR] = 2.01; 95% confidence interval [CI] = 1.16-3.49) over the 7 inch (17.8-cm) range in height (two-sided P for trend = .003), with comparable effects in both premenopausal and postmenopausal women. Except for reduced risk in the heavy, younger women (weight/height 1.5 > 29 kg/m 1.5 and < 40 years old), risk was positively associated with usual adult adiposity. Trends in risk became more striking as adiposity in each succeeding decade of adult life was considered. Women in their 50s and in the top quintile for their age group had twice the breast cancer risk (RR = 2.13; 95% CI = 1.17-3.87) of women in the bottom quintile (two-sided P for trend = .004). Women in their 50s, above the median adiposity for their age group, and with a recent gain of more than 10 pounds had three times the risk (RR = 3.01; 95% CI = 1.45-6.25) of women below the median adiposity and with no recent weight change. Recent weight loss was consistently associated with reduced risk (RRs of approximately 0.7) relative to no recent weight change. CONCLUSIONS: Adult adiposity, weight change, and height are critical determinants of breast cancer risk. Increased adiposity and weight gain in the decade preceding diagnosis are especially influential, suggesting that excess weight may function as a late stage promoter. IMPLICATIONS: Weight maintenance and/or reduction as an adult, possibly accompanied by specific changes in diet and physical activity, may have a significant and rapid impact on breast cancer risk. PMID- 8627642 TI - Psychological symptoms and disease-free and overall survival in women with stage II breast cancer. Cancer and Leukemia Group B. AB - BACKGROUND: The possible link between psychological factors and length of cancer survival has generated a literature of contradictory findings. Associations usually have not been found when general psychological symptoms are assessed. Associations usually have been found for predictors related to expressive versus repressive emotional coping (e.g., depression, "fighting spirit," hostility, and type C personality); however, even these associations have been relatively small, when compared with those for medical factors. Yet few studies have adequately controlled for medical and treatment-related factors. PURPOSE: Within a Cancer and Leukemia Group B (CALGB) national clinical trial of four adjuvant therapy regimens for stage II breast cancer (CALGB 8082), this study prospectively examined the contribution of potential psychological predictors to length of disease-free and overall survival over a 15-year period. METHODS: Subjects were 280 women with stage II breast cancer, out of a total of 899, who were randomly assigned to receive CMFVP (cyclophosphamide-methotrexate-fluorouracil-vincristine prednisone) for two 6-week cycles or six 4-week cycles, then subsequently randomly assigned to receive or not to receive VATH (vinblastine-doxorubicin thiotepa-fluoxymesterone). Subjects were recruited during the period between October 1980 and August 1984, inclusive, and followed until January 1996. Prior to chemotherapy, psychological symptoms were assessed using the Symptom Check List-90-Revised (SCL-90-R). SCL-90-R scores were trichotomized into categories representing high, medium, and low distress. Basic base-line sociodemographic data (including age, ethnicity, education, and marital status) and medical data (including lymph node status, estrogen receptor status, menopausal status, and performance status) were collected. Subjects with psychosocial data differed from those without psychosocial data solely in their higher percentage of classification in the mild limitation category of the Zubrod (Eastern Cooperative Oncology Group) performance status rating (subjects with psychosocial data: 14%; subjects without psychosocial data: 8%). RESULTS: In stepwise Cox regression analyses that controlled for sociodemographic and medical variables, there was no significant predictive effect of the level of distress (as measured by the SCL-90 R trichotomized scores) on length of disease-free and overall survival of the study subjects. Risk ratios for low versus high distress were 1.01 (95% confidence interval [CI] = 0.62-1.66) for disease-free survival and 1.03 (95% CI = 0.58-1.82) for overall survival. CONCLUSIONS: This study failed to provide evidence that psychological factors contributed to length of disease-free or overall survival of women who received adjuvant chemotherapy (either CMFVP alone or CMFVP followed by VATH) for treatment of stage II breast cancer. IMPLICATIONS: In the context of far more potent medical factors, the contribution of psychological factors to disease-free and overall survival is likely to be relatively small. Future research should focus on specific theory-driven predictors rather than on general psychological symptoms. Moreover, it should be based on clinical studies using a controlled, prospective design, in which the effects of medical factors may be distinguished and psychological predictors are clear antecedents of survival outcomes. PMID- 8627644 TI - Epidemiology of brain lymphoma among people with or without acquired immunodeficiency syndrome. AIDS/Cancer Study Group. AB - BACKGROUND: In recent years, brain lymphoma incidence has dramatically increased, presumably because of elevated risk of brain lymphoma among persons with acquired immunodeficiency syndrome (AIDS). PURPOSE: The objective of this study was to estimate independent incidence and survival rates of brain lymphoma among persons with or without AIDS and to understand the epidemiologic features of this cancer. METHODS: We linked AIDS and cancer registry reports at nine state and local health departments and compared the demographics, histology, and survival of brain lymphoma cases among persons with or without AIDS. The data were limited to people under 70 years of age. We calculated the incidence of brain lymphoma among persons with AIDS and compared observed cases with those expected. The differences were statistically analyzed using the Poisson test. Epidemiologic features of brain lymphoma in persons with or without AIDS were compared using the chi-squared test, the Student's t test, and the chi-squared test for linear trend. The logrank test was used to compare survival rates estimated by the Kaplan-Meier technique. All P values were two-sided. RESULTS: We matched 50,989 AIDS registry reports to 859,398 cancer registry reports (data from 1981 to 1990) and found 431 people with both AIDS and brain lymphoma. Among people with AIDS, those developing brain lymphoma versus those without brain lymphoma were more likely to be white (70% versus 59%; P < .001) and had homosexuality as their only human immunodeficiency virus risk factor (75% versus 64%; P < .001). Of the 431 patients, 223 developed brain lymphomas during 47,465 person-years of observation after diagnosis of AIDS. The absolute incidence rate of brain lymphoma among persons with AIDS was 4.7/1000 person-years (95% confidence interval = 4.1 5.3/1000 person-years), 3600-fold higher than the base-line rate in the general population. From 1980 through 1989, overall counts of brain lymphoma increased ninefold. Most of this increase was derived from persons with AIDS, but a substantial increase also occurred among persons without AIDS (0.04/100,000 in 1982 to 0.28/100,000 in 1989) (chi-squared test for trend; P < .05). The median survival was shortest for persons with AIDS and brain lymphoma (2 months), was intermediate for persons with brain lymphoma without AIDS (5-7 months), and was longest for persons with AIDS without brain lymphoma (14 months) (P < .05 for all comparisons). CONCLUSIONS: This analysis distinguishes the separate epidemiologies of brain lymphoma incidence among persons with or without AIDS and shows brain lymphoma incidence among persons with AIDS to be several thousand fold higher than that in the general population. The study documents the overwhelming effect of AIDS-associated brain lymphoma on the overall rate in the general population and demonstrates a significantly rising trend, although of a lesser magnitude, among persons without AIDS. IMPLICATIONS: This study emphasizes a greater need to bring health care resources to this burgeoning epidemic. PMID- 8627643 TI - Phase III study of bolus versus infusion fluorouracil with or without cisplatin in advanced colorectal cancer. AB - BACKGROUND: Phase II studies of fluorouracil (5-FU) administered by protracted intravenous infusion have suggested an improved response rate and decreased toxicity profile when compared with 5-FU given by bolus injection in patients with metastatic colorectal cancer. Additional studies have suggested further enhancement of infusion 5-FU activity when it is combined with low-dose weekly cisplatin administration. PURPOSE: This phase III study in adults with metastatic colorectal cancer was planned as a comparison of objective response rates, toxicity, and survival in patients receiving bolus versus protracted-infusion 5 FU with or without cisplatin. METHODS: Four hundred ninety-seven previously untreated patients with advanced, measurable metastatic colorectal cancer were randomly assigned to receive treatment A (bolus 5-FU at 500 mg/m2 for 5 days followed in 2 weeks by weekly bolus 5-FU at 600 mg/m2), treatment B (bolus 5-FU at 500 mg/m2 for 5 days followed in 2 weeks by weekly bolus 5-FU at 600 mg/m2, plus weekly cisplatin at 20 mg/m2), treatment C (5-FU at 300 mg/m2 per day by continuous infusion), or treatment D (5-FU at 300 mg/m2 per day by continuous infusion plus weekly cisplatin at 20 mg/m2). All drugs were administered intravenously. Enrollment in the trial occurred from August 1987 through December 1990, and follow-up was through September 1995. The Kaplan-Meier method was used to estimate overall and disease-free survival, and Cox regression models were used to assess the effects of patient characteristics on survival. All P values resulted from two-sided tests. RESULTS: Objective tumor response was observed in 28 (18%) of 153 patients receiving treatment A, in 45 (28%) of 159 patients receiving treatment C (C versus A; P = .045), and in 47 (31%) of 153 patients receiving treatment D (D versus A; P = .016). Because of excessive toxicity, treatment B was discontinued after only 12 patients had begun treatment. Median time to disease progression was 5.1 months for patients in arm A compared with 6.2 and 6.5 months for patients in arms C and D, respectively (C versus A, P = .007; D versus A, P = .017). Patterns of toxic effects differed substantially among the treatment arms. Forty-five percent of the patients receiving bolus 5-FU alone (A) experienced grade 3-4 leukopenia, with two sepsis-related deaths. Hand foot syndrome and mucositis were the major treatment-limiting toxic effects for patients in the two treatment arms involving infusion. Despite the improvement in response rates and time to disease progression with infusion 5-FU with or without cisplatin (C and D, respectively) (P = .003), the overall survival for the three groups (A, C, and D) was similar (P = .307). This may have been due in part to a longer median survival time of 10.4 months for patients in arm A, compared with an anticipated survival of 7 months. CONCLUSION: 5-FU given as a continuous infusion produced a higher objective response rate, a modest prolongation in time to disease progression, and less life-threatening myelosuppression in patients than bolus 5-FU. Concomitant treatment with low-dose cisplatin caused added toxicity and complexity of treatment and did not provide a major clinical benefit. No statistically significant survival differences were observed among the three treatment groups. PMID- 8627646 TI - Report on the Sixth International Congress on Anti-Cancer Treatment. PMID- 8627645 TI - Reduced survival in patients with ductal pancreatic adenocarcinoma associated with CDKN2 mutation. PMID- 8627647 TI - Prognostic value of Bcl-2 and p53 expression in urinary tract transitional cell cancer. PMID- 8627648 TI - Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases. AB - Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy terminal regulatory tyrosine and the SH2 domain prevents association with middle T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src. PMID- 8627649 TI - ras oncogene-dependent activation of the P4 promoter of minute virus of mice through a proximal P4 element interacting with the Ets family of transcription factors. AB - The P4 promoter of parvovirus minute virus of mice (MVMp) directs transcription of the genes coding for nonstructural proteins. The activity of promoter P4 is regulated by several cis-acting DNA elements. Among these, a promoter-proximal GC box was shown to be essential for P4 activity (J.K. Ahn, B.J. Gavin, G. Kumar, and D.C. Ward, J. Virol. 63:5425-5439, 1989). In this study, a motif homologous to an Ets transcription factor-binding site (EBS), located immediately upstream from the GC box, was found to be required for the full activity of promoter P4 in the ras-transformed rat fibroblast cell line FREJ4. In normal parental FR3T3 cells, the transcriptional function of P4 EBS was insignificant but could be restored by transient cell transfection with the c-Ha-ras oncogene. P4 EBS may thus contribute to the stimulation of promoter P4 in ras-transformed cells. Electrophoretic mobility shift assays using crude extracts from FREJ4 cells revealed the binding of a member(s) of the Ets family of transcription factors to the P4 EBS, as well as the interaction of two members of the Sp1 family, Sp1 and Sp3, with the adjacent GC box. When produced in Drosophila melanogaster SL2 cells, Ets-1 and Sp1 proteins acted synergistically to transactivate promoter P4 through their respective cognate sites. PMID- 8627650 TI - Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1. AB - Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1. PMID- 8627651 TI - The receptor-binding domain of pseudorabies virus glycoprotein gC is composed of multiple discrete units that are functionally redundant. AB - Many herpesviruses attach to cells in a two-step process, using the glycoprotein gC family of homologs to bind the primary receptor, heparan sulfate (HS) proteoglycan, and glycoprotein gD homologs to bind an unknown secondary receptor. We have previously shown by deletion analysis that the amino-terminal one-third of gC from pseudorabies virus (PRV), a swine herpesvirus, includes at least the principal HS receptor-binding domain. This portion of PRV gC contains three discrete clusters of basic residues that exactly or nearly match proposed consensus sequences for heparin-binding domains (HBDs); four additional potential HBDs lie in the distal two-thirds of the glycoprotein. We now specifically implicate each of the three amino-terminal HBDs in virus attachment. Mutational analysis demonstrated that any one of the three HBDs could mediate efficient virus infectivity; HS-dependent PRV attachment to cells was eliminated only after all three amino-terminal HBDs were altered. Furthermore, the binding dysfunction was due to a disruption of the specific HBDs and not to total charge loss. Thus, unlike previously described viral receptor-binding domains, the PRV gC receptor binding domain is composed of multiple, discrete units that can function independently of one another. These units may function redundantly either to increase binding affinity or perhaps to effectively increase the virus's host range. PMID- 8627652 TI - Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism. AB - Murine cytomegalovirus (MCMV) gene products dispensable for growth in cell culture are likely to have important functions within the infected host, influencing tissue tropism, dissemination, or immunological responses against the virus. To identify such genes, our strategy was to delete large regions of the MCMV genome likely to contain genes nonessential for virus replication in NIH 3T3 cells. Mutant virus RV7 contained a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I. This virus grew comparably to wild-type (WT) virus in NIH 3T3 fibroblasts, primary embryo fibroblasts, and bone marrow macrophages. However, RV7 failed to replicate in target organs of immunocompetent BALB/c mice and severe combined immunodeficient mice, which are exquisitely sensitive to MCMV infection. This defect in vivo growth may be related to the observation that RV7 grew poorly in the peritoneal macrophage cell line IC-21, which is highly permissive for growth of WT MCMV. Two other mutant viruses with an insertion or smaller deletion in the region common to the RV7 deletion grew comparably to WT virus in the macrophage cell line and replicated in salivary gland tissue. The poor growth of RV7 in IC-21 cells was due to a block in immediate-early gene expression, as levels of RNA from immediate-early gene IE1 were reduced eightfold compared with levels for WT virus in macrophages infected with RV7. Consequently, levels of RNA from early and late genes were also reduced. The lower expression of IE1 in RV7-infected IC-21 macrophages was not due to defective entry of virus into the cells, as equal amounts of viral DNA were present in cells 3 h after infection with RV7 or WT MCMV. These studies demonstrate that deletion of sequences in HindIII-J and -I confer altered cell and tissue tropism. PMID- 8627653 TI - Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II. AB - Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression. PMID- 8627655 TI - Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS. AB - Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) were isolated from three patients with AIDS who underwent multiple sequential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol., 47:237-244, 1995). We previously demonstrated that the three strains were genetically unrelated and that each of them was present as a single viral population in vivo. Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the present paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associated with a slower replication of HCMV strains in cell cultures. Sequencing of the UL97 and UL54 genes, GCV anabolism assays, and marker transfer experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460 --> Ile; VR4955, Ala-594 --> Val; VR5120, Leu595 --> Ser), while single amino acid changes in domain II of the DNA polymerase (VR4760 and VR5120, Val-715 --> Met; VR4955, Thr-700 --> Ala) were responsible for both the PFA resistance and the slow-growth phenotype. Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resistance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and (S)-1-(3-hydroxy-2 phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the clinical isolates, since the recombinant strains showed a marked delay in immediate-early antigen plaque formation and a reduction of infectious virus yield compared with AD169, from which they were derived. These results may have some important implications for the successful isolation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations. PMID- 8627654 TI - Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes. AB - Human immunodeficiency virus type 1 (HIV-1) infection has been associated with an increase in the binding of the transcription factor NF-kappa B to its consensus sequence in the viral promoter. Using cultures of primary human fetal astrocytes, we show that exogenous HIV-1 Tat protein, which has been demonstrated to be released from infected cells, is associated with an increase in the binding of this transcription factor to an HIV-1 long terminal repeat kappa B sequence. This effect occurs rapidly and is independent of new protein synthesis. We also demonstrate that extracellular Tat protein is associated with an increase in protein kinase C activity. If Tat functions similarly in other cell types, such findings could relate to some of this protein's previously described physiological effects. These effects include Tat's ability to upregulate the synthesis of specific cytokines and to act as a growth factor. PMID- 8627656 TI - Properties of the adenovirus IVa2 gene product, an effector of late-phase dependent activation of the major late promoter. AB - The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450 4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus. PMID- 8627657 TI - Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity. AB - The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types. PMID- 8627658 TI - Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A. AB - Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another. PMID- 8627659 TI - The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates. AB - The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction. PMID- 8627660 TI - Characterization of two novel YY1 binding sites in the polyomavirus late promoter. AB - NF-D is a ubiquitous nuclear factor that has been shown to bind specifically to a DNA element in the polyomavirus regulatory region. In this report, we demonstrate that NF-D is either identical or very similar to a transcription factor that has been variously named YY1, delta, NF-E1, UCRBP, or CF1. Moreover, we show the presence in the polyomavirus genome of a second DNA motif, located 40 bp from the first, which binds YY1/NF-D with high affinity. Both sites lie downstream of the major late transcription initiation sites. By site-directed mutagenesis, we demonstrate that both elements contribute positively to the activity of the late promoter, probably by a cooperative mechanism. We also demonstrate that the requirement of the YY1/NF-D function for late promoter activity varies with the cell line. PMID- 8627661 TI - A preferred region for recombinational patch repair in the 5' untranslated region of primer binding site-impaired murine leukemia virus vectors. AB - Transduction of primer binding site-impaired Akv murine leukemia virus-based retroviral vectors from the murine packaging cell lines psi-2 and omega E was studied. The efficiency of transduction of the neo marker of all mutated constructs was found to decrease by 5 to 6 orders of magnitude compared with that of the wild-type vector. Thirty-two of 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern of nucleotide differences from the Akv-based vector, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombination partner RNA was found in virions from psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions at or around the glutamine tRNA primer binding site. We propose that the forced recombination of primer binding site mutants involves initial priming on endogenous viral sequences and requires template switching during minus-strand synthesis in the region between the neo gene and the mutated primer binding site to allow correct second-strand transfer in reverse transcription. The system thereby selects for a reverse transcriptase-mediated recombination event in the 5' untranslated region. A panel of sequence differences between the recombination partners in this region has allowed mapping of the site of recombination for each transduction event. Interestingly, the majority of the recombination events were clustered within a narrow, 33-nucleotide region though to be involved in genomic RNA dimerization. PMID- 8627662 TI - Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein. AB - We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein. PMID- 8627663 TI - Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink. AB - The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed. PMID- 8627664 TI - Attenuation stem-loop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects. AB - The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075 5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner. PMID- 8627665 TI - Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic. AB - Tat is an 86- to 104-amino-acid viral protein that activates human immunodeficiency virus type 1 expression, modifies several cellular functions, and causes neurotoxicity. Here, we determined the extent to which peptide fragments of human immunodeficiency virus type 1 BRU Tat1-86 produced neurotoxicity, increased levels of intracellular calcium ([Ca2+]i), and affected neuronal excitability. Tat31-61 but not Tat48-85 dose dependently increased cytotoxicity and levels of [Ca2+]i in cultured human fetal brain cells. Similarly, Tat31-61 but not Tat48-85 depolarized rat hippocampal CA1 neurons in slices of rat brain. The neurotoxicity and increases in [Ca2+]i could be significantly inhibited by non-N-methyl-D-aspartate excitatory amino acid receptor antagonists. Shorter 15-mer peptides which overlapped by 10 amino acids each and which represented the entire sequence of Tat1-86 failed to produce any measurable neurotoxicity. Although it remains to be determined if Tat acts directly on neurons and/or indirectly via glial cells, these findings do suggest that Tat neurotoxicity is conformationally dependent, that the active site resides within the first exon of Tat between residues 31 to 61, and that these effects are mediated at least in part by excitatory amino acid receptors. PMID- 8627666 TI - Identification and characterization of a new and distinct molecular subtype of human T-cell lymphotropic virus type 2. AB - Molecular studies have demonstrated the existence of at least two major subtypes of human T-cell lymphotropic virus type 2 (HTLV-2), designated HTLV-2a and HTLV 2b. To further investigate the heterogeneity of this family of viruses, we have characterized the HTLV-2 subtypes present in several urban areas in Brazil. DNAs from peripheral blood mononuclear cells of a large number of infected individuals, the majority of whom were intravenous drug abusers, were analyzed by using PCR with restriction fragment length polymorphism and nucleotide sequencing analysis. Restriction fragment length polymorphism analysis of the env region suggested that all individuals were infected with the HTLV-2a subtype, and this was confirmed by nucleotide sequence analysis. In contrast, nucleotide sequence analysis of the long terminal repeat demonstrated that although the viruses were more related to the HTLV-2a than to the HTLV-2b subtype, they clustered in a distinct phylogenetic group, suggesting that they may represent a new and distinct molecular subtype of HTLV-2. This conclusion was supported by nucleotide sequence analysis of the pX region, which demonstrated that the Tax proteins of the Brazilian viruses differed from that of prototype HTLV-2a isolates but were more similar to that of HTLV-2b in that they would be expected to have an additional 25 amino acids at the carboxy terminus. In transient expression assays, the extended Tax protein of the prototype HTLV-2a subtype. The studies suggest that the Brazilian viruses analyzed in this study, while being phylogenetically related to the prototypic HTLV-2a seen in North America, are phenotypically more related to HTLV-2b and can be justifiably classified as a new molecular subtype, which has been tentatively designated HTLV-2c. PMID- 8627667 TI - Comparing transcriptional activation and autostimulation by ZEBRA and ZEBRA/c-Fos chimeras. AB - The lytic cycle of Epstein-Barr virus (EBV) can be activated by transfection of the gene for ZEBRA, a viral basic-zipper (bZip) transcriptional activator. ZEBRA and cellular AP-1 bZip activators, such as c-Fos, have homologous DNA-binding domains, and their DNA-binding specificities overlap. Moreover, EBV latency can also be disrupted by phorbol esters, which act, in part, through AP-1 activators. It is not known whether ZEBRA and AP-1 factors play equivalent roles in the initial stages of reactivation. Here the contribution of ZEBRA's basic DNA recognition domain to disruption of latency was analyzed by comparing ZEBRA with chimeric mutants in which the DNA recognition domain of ZEBRA was replaced with the analogous domain of c-Fos. Chimeric ZEBRA/c-Fos proteins overexpressed in Escherichia coli bound DNA with the specificity of c-Fos; they bound a heptamer AP-1 site and an octamer TPA response element (TRE). ZEBRA bound the AP-1 site and an array of ZEBRA response elements (ZREs). In assays with reporter genes, both ZEBRA and ZEBRA/c-Fos chimeric mutants activated transcription from Zp, a promoter of the ZEBRA gene (BZLF1) that contains the TRE and multiple ZREs. However, despite their capacity to activate reporters bearing Zp, neither ZEBRA nor the c-Fos chimeras activated transcription from Zp in the context of the intact latent viral genome. In contrast, ZEBRA but not ZEBRA/c-Fos chimeras activated Rp, a second viral promoter that controls ZEBRA expression. Hence, transcriptional autostimulation by transfected ZEBRA occurred preferentially at Rp. Both ZEBRA and the ZEBRA/c-Fos chimeras activated transcription from reporters with multimerized AP-1 sites. However, in the context of the virus, only ZEBRA activated the promoters of two early lytic cycle genes, BMRF1 and BMLF1, that contain an AP-1 site. Thus, overexpression of an activator that recognized AP-1 and TRE sites was not sufficient to activate EBV early lytic cycle genes. PMID- 8627668 TI - Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice. AB - The functional roles of the human immunodeficiency virus type 1 (HIV-1) accessory genes (nef, vpr, vpu, and vif) are as yet unclear. Using the SCID-hu model system, we have examined the infectivity, replicative capacity, and pathogenicity of strains of the molecular clone HIV-1NL4-3 that contain deletion mutations in these individual accessory genes. We determined that deletion of these genes had differential effects on both infectivity and pathogenicity. Deletion of vpr had little or no effect on viral infectivity, replication, and pathogenicity; however, deletion of vpu or vif had a significant effect on infectivity and moderate effects on pathogenicity. nef-minus strains were the most attenuated in this system, demonstrating significantly lower levels of infectivity and pathogenicity. However, deletion of these individual genes attenuated but did not abrogate the pathogenic properties of HIV-1. Mutant viruses still retained the ability to induce thymocyte depletion to various degrees if implants were infected with higher doses of virus or observed for longer periods of time. The relative contributions of these genes to in vivo pathogenic potential should be taken into consideration when one is contemplating a live attenuated vaccine for HIV-1. PMID- 8627669 TI - Functional comparison of PML-type and archetype strains of JC virus. AB - Isolates of the human polyomavirus JC can be grouped as either PML-type or archetype strains primarily on the basis of divergence in their regulatory regions. Only PML-type viruses have so far been found to be associated with the human demyelinating disease progressive multifocal leukoencephalopathy. Here we have compared the functional properties of archetype and PML-type regulatory regions with regard to DNA replication and viral gene expression. No significant differences could be detected between archetype and PML-type regions in their ability to direct episomal DNA replication in the presence of JC virus T antigen. When viral gene expression was examined, early- and late-gene promoters from all PML-type strains exhibited a significantly higher activity in glial than in nonglial cells. Surprisingly, archetype strain promoters were also preferentially active in glial cells, although this effect was less pronounced than in PML-type strains. Furthermore, all promoters from archetype strains reacted to the presence of viral T antigen or the glial transcription factor Tst-1/Oct6 in a manner similar to the promoters of the PML-type viral strain Mad-1. Interestingly, T antigen and Tst-1/Oct6 were found to function in a species specific and cell-type-specific manner, respectively. We concluded from our experiments that the differences in the regulatory regions cannot account for the different biology of archetype and PML-type viral strains. PMID- 8627670 TI - Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo. AB - Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection. PMID- 8627671 TI - Inhibition of Nef- and phorbol ester-induced CD4 degradation by macrolide antibiotics. AB - Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS. The simian immunodeficiency virus (SIV) causes a similar syndrome in macaques. The product of the nef gene of SIV has been shown to be important for virus replication and disease progression in vivo. In vitro, both SIV and HIV Nef downregulate surface expression of CD4 and accelerate total CD4 turnover. The mechanism by which Nef downregulates CD4 has not been established. A current model suggests that Nef enhances cell surface CD4 endocytosis and degradation in lysosomes. However, this was recently challenged when CD4 was found to accumulate in early endosomes of cells expressing Nef. Because inhibition of Nef function might halt virus replication and disease progression, we tested two macrolide antibiotics for their ability to inhibit Nef function. Concanamycin B (ConB) and bafilomycin A1 (BFLA1) are specific inhibitors of acidification of cell endosomes and lysosomes and, unlike other inhibitors, do not affect transport. Although ConB (25 nM) and BFLA1 (100 nM) blocked phorbol myristate acetate- and Nef induced CD4 degradation in human monocyte U937 cells, CD4 surface expression was not recovered. Instead, CD4 accumulated in lysosomes. To determine if Nef is directly responsible for CD4 degradation or if they bind to each other in a manner similar to Vpu, transcripts of human CD4 and HIV-1 nef were cotranslated in vitro. Our results indicate that under our experimental conditions, Nef does not affect CD4 stability and does not associate with CD4 in this in vitro system. Our data suggest that (i) CD4 downregulation by Nef results in degradation of CD4 in lysosomes, (ii) inhibition of CD4 degradation by macrolide antibiotics does not restore surface expression, and (iii) the inhibition of CD4 expression by Nef appears to be indirect and is likely to involve cellular factors. PMID- 8627672 TI - Downstream regulatory elements increase acute and latent herpes simplex virus type 2 latency-associated transcript expression but do not influence recurrence phenotype or establishment of latency. AB - The role of putative promoter or activator sequences downstream of the herpes simplex virus type 2 latency-associated transcript (LAT) promoter and upstream of the LAT intron was investigated in vivo by constructing and evaluating mutant viruses with deletions in this region. The deletion of LAT promoter sequences upstream of the primary LAT transcript reduced levels of LAT expression during productive infections, compared with the LAT expression level of wild-type virus, and abolished LAT expression during latency. The deletion of the putative downstream regulatory elements reduced but did not eliminate LAT expression during productive and latent infections. The deletion of both regions almost completely eliminated acute LAT transcription, although additional acute LAT region transcription directed by sequences upstream of either region was detected by reverse transcriptase PCR. The deletion of the downstream elements did not influence the ability of the virus to reactivate from latently infected guinea pigs relative to the ability of the wild-type virus to reactivate; thus, decreased LAT expression did not affect the frequency of recurrence. The deletion of both regions did not affect the ability of the virus to establish latency. We conclude that downstream regulatory elements are necessary for maximal acute LAT expression but do not constitute an independent promoter during latency and do not play an obvious role in the establishment of our reactivation from latency. PMID- 8627673 TI - Sequence requirements for binding of Rep68 to the adeno-associated virus terminal repeats. AB - We have used reciprocal competition binding experiments with mutant substrates and chemical modification interference assays to precisely define the sequences within the adeno-associated virus (AAV) terminal repeat (TR) that are involved in site-specific binding to the AAV Rep protein. Mutagenesis experiments were done with a 43-bp oligonucleotide which contained the Rep binding element (RBE) within the A stem of the TR. Experiments in which two adjacent base pairs of the RBE were substituted simultaneously with nucleotides that produced transversions identified a 22-bp sequence (CAGTGAGCGAGCGAGCGCGCAG) in which substitutions measurably affected the binding affinity. Although the 22-bp RBE contains the GAGC motifs that have been found in all known Rep binding sites, our results suggest that the GAGC motifs alone are not the only sequences specifically recognized by Rep. The effects of substitutions within the 22-bp sequence were relatively symmetrical, with nucleotides at the periphery of the RBE having the least effect on binding affinity and those in the middle having the greatest effect. Dinucleotide mutations within 18 (GTGAGCGAGCGAGC) of the 22 bp were found to decrease the binding affinity by at least threefold. Dinucleotide mutations within a 10-bp core sequence (GCGAGCGAGC) were found to decrease binding affinity by more than 10-fold. Single-base substitutions within the 10-bp core sequence lowered the binding affinity by variable amounts (up to fivefold). The results of the mutagenesis analysis suggested that the A-stem RBE contains only a single Rep binding site rather than two or more independent sites. To confirm the results of the mutant analysis and to determine the relative contribution of each base to binding, chemical modification experiments using dimethyl sulfate and hydrazine were performed on both the linear A-stem sequence and the entire AAV TR in both the flip and flop hairpinned configurations. Interference assays on the linear A stem identified the 18-bp sequence described above as essential for binding. G, C, and T residues on both strands contributed to binding, and the interference pattern correlated well with the results of the mutagenesis experiments. Interference assays with complete hairpinned TR substrates also identified the 18 bp sequence as important for binding. However, the interference patterns on the two strands within the RBE and the relative contributions of the individual bases to binding were clearly different between the hairpinned substrates and the linear A-stem binding element. Interference assays also allowed us to search for residues within the small internal palindromes of the TR (B and C) that contribute to binding. The largest effect was seen by modification of two T residues within the sequence CTTTG. This sequence was present in the same position relative to the terminal resolution site (trs) in both the flip and flop orientations of the TR. In addition, the interference pattern suggested that the remaining bases within the CTTTG motif as well as other bases within the B and C palindromes make contacts with the Rep protein, albeit with lower affinities. Regardless of whether the TR was in the flip or flop orientation, most of the contact points were clustered in the small internal palindrome furthest away from the trs. We also determined the relative binding affinity of linear substrates containing a complete RBE with hairpinned substrates and found that linear substrates bound Rep less efficiently. Our results were consistent with our previous model that there are three distinct elements within the hairpinned AAV TR that contribute to binding affinity or to efficient nicking at the trs: the A stem RBE, the secondary structure element which consists of the B and C palindromes, and the trs. PMID- 8627674 TI - Inducible translational regulation of the NF-IL6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells. AB - Respiratory syncytial virus (RSV), the most common etiologic agent of epidemic pediatric respiratory disease, infects and replicates in the human airway epithelium, resulting in the induction of cellular gene products essential for immune and inflammatory responses. We describe the effect of RSV infection on nuclear factor-IL6 (NF-IL6) expression, a human basic domain-leucine zipper containing transcription factor that alone in combination with other inducible transcription factors regulates the expression of cytokine and adhesion molecule genes. RSV-infected human type II pulmonary alveolar epithelial cells (A549) synthesize a single 45.7-kDa isoform of NF-IL6 rapidly and in a time-dependent manner. NF-IL6 is first detectable after 3 h of infection and continues to accumulate until 48 h (until the cells lose viability). NF-IL6 production could not be induced by UV-inactivated virus, demonstrating the requirement of viral replication for NF-IL6 synthesis. Immunoprecipitation after [35S]methionine metabolic labeling was done to investigate the mechanism for NF-IL6 production. There was robust NF-IL6 protein synthesis within RSV-infected (24 h) cells. Protein synthesis occurred without detectable changes in the abundance or size of the single 1.8-kb NF-IL6 mRNA. RNase protection assay of transfected chloramphenicol acetyltransferase reporter genes driven by either wild-type or mutated NF-IL6 binding sites show a virus-induced increase in NF-IL6-dependent transcription. These studies have demonstrated a novel inducible mechanism for translational control of NF-IL6 synthesis and identify this transcription factor as a potential effector of the host response to RSV infection. PMID- 8627675 TI - Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides. AB - To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function. PMID- 8627676 TI - Transport and processing of the Rous sarcoma virus Gag protein in the endoplasmic reticulum. AB - The Gag proteins of replication-competent retroviruses direct budding at the plasma membrane and are cleaved by the viral protease (PR) just before or very soon after particle release. In contrast, defective retroviruses that bud into the endoplasmic reticulum (ER) have been found, and morphologically these appear to contain uncleaved Gag proteins. From this, it has been proposed that activation of PR may depend upon a host factor found only at the plasma membrane. However, if Gag proteins were cleaved by PR before the particle could pinch off the ER membrane, then the only particles that would remain visible are those that packaged smaller-than-normal amounts of PR, and these would have an immature morphology. To distinguish between these two hypotheses, we made use of the Rous sarcoma virus (RSV) Gag protein, the PR of RSV IS included on each Gag molecule. To target Gag to the ER, a signal peptide was installed at its amino terminus in place of the plasma membrane-binding domain. An intervening, hydrophobic, transmembrane anchor was included to keep Gag extended into the cytoplasm. We found that PR-mediated processing occurred, although the cleavage products were rapidly degraded. When the anchor was removed, allowing the entire protein to be inserted into the lumen of the ER, Gag processing occurred with a high level of efficiency, and the cleavage products were quite stable. Thus, PR activation does not require targeting of Gag molecules to the plasma membrane. Unexpectedly, molecules lacking the transmembrane anchor were rapidly secreted from the cell in a nonmembrane-enclosed form and in a manner that was very sensitive to brefeldin A and monensin. In contrast, the wild-type RSV and Moloney murine leukemia virus Gag proteins were completely insensitive to these inhibitors, suggesting that the normal mechanism of transport to the plasma membrane does not require interactions with the secretory pathway. PMID- 8627677 TI - Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro. AB - Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN. PMID- 8627678 TI - Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles. AB - The 5'- and 3'-terminal sequences of the genomic RNA from classical swine fever virus (CSFV) were determined, and the resulting information was used for construction of full-length CSFV cDNA clones. After transfection of in vitro transcribed RNA derived from a cDNA construct, infectious CSFV was recovered from porcine cells. To confirm the de novo generation of infectious CSFV from cloned DNA, a genetically tagged CSFV was constructed. In comparison with parental CSFV, the recombinant viruses were retarded in growth by about 1 order of magnitude. Introduction of a deletion by exchange of part of the full-length construct for corresponding cDNA fragments derived from the genomes of cytopathogenic CSFV defective interfering particles (DIs) (G. Meyers and H.-J. Thiel, J. Virol. 69:3683-3689. 1995) resulted in recovery of cytopathogenic DIs in the DI genomes is responsible for their cytopathogenicity. The established system will allow novel approaches to analysis of pestiviral molecular biology and in particular to elucidation of the molecular basis of attenuation and cytopathogenicity of these viruses. PMID- 8627679 TI - A chimeric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecule exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1. AB - We have previously shown that hairpin ribozymes targeting the human immunodeficiency virus (HIV) genome can effectively inhibit virus replication in a variety of primary and cultured hematopoietic cells. To further increase antiviral potency and minimize the chance of viral resistance, we have now cloned the stem-loop II sequences of the HIV type 1 Rev response element into ribozyme transcription cassettes. Fusion RNA molecules were shown to function both as RNA decoys and ribozymes. Stable Molt-4/8 cell lines expressing fusion RNA of stem loop II and a ribozyme directed at the HIV-type 1 U5 sequence (MSLMJT) or its disabled counterpart (MSLdMJT) were generated. The expression of fusion RNA was persistent for at least 6 months without apparent cytotoxicity. When virus inhibition was examined after the cocultivation of transduced cells with chronically infected Jurkat cells, much greater protection was observed in MSLMJT cells than in MSLdMJT or MMJT (expressing only the ribozyme) cells. Furthermore, to specifically compare the ribozyme activities in various transduced cells, we determined the quantitative levels of proviral DNA in the first round of virus replication (7 h after infection with HXB2). By competitive PCR, the proviral DNA levels in MSLMJT and MMJT cells were found to be reduced to 1/7 and 1/3, respectively, compared with those in MSLdMJT and MdMJT cells. These results suggest not only that the greater inhibition afforded by this fusion RNA was due to its function both as decoy and ribozyme but also that the ribozyme activity may be facilitated. PMID- 8627680 TI - Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions. AB - The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor. PMID- 8627681 TI - A common mechanism for the enhancement of mRNA 3' processing by U3 sequences in two distantly related lentiviruses. AB - The protein coding regions of all retroviral pre-mRNAs are flanked by a direct repeat of R-U5 sequences. In many retroviruses, the R-U5 repeat contains a complete core poly(A) site-composed of a highly conserved AAUAAA hexamer and a GU rich downstream element. A mechanism that allows for the bypass of the 5' core poly(A) site and the exclusive use of the 3' core poly(A) site must therefore exist. In human immunodeficiency virus type 1 (HIV-1), sequences within the U3 region appear to play a key role in poly(A) site selection. U3 sequences are required for efficient 3' processing at the HIV-1 poly(A) site both in vivo and in vitro. These sequences serve to promote the interaction of cleavage and polyadenylation specificity factor (CPSF) with the core poly(A) site. We have now demonstrated the presence of a functionally analogous 3' processing enhancer within the U3 region of a distantly related lentivirus, equine infectious anemia virus (EIAV). U3 sequences enhanced the processing of the EIAV core poly(A) site sevenfold in vitro. The U3 sequences also enhanced the stability of CPSF binding at the core poly(A) site. Optimal processing required the TAR RNA secondary structure that resides within the R region 28 nucleotides upstream of the AAUAAA hexamer. Disruption of TAR reduced processing, while compensatory changes that restored the RNA structure also restored processing to the wild-type level, suggesting a position dependence of the U3-encoded enhancer sequences. Finally, the reciprocal exchange of the EIAV and HIV U3 regions demonstrated the ability of each of these sequences to enhance both 3' processing and the binding of CPSF in the context of the heterologous core poly(A) site. The impact of U3 sequences upon the interaction of CPSF at the core poly(A) site may therefore represent a common strategy for retroviral poly(A) site selection. PMID- 8627682 TI - The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep. AB - Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors. PMID- 8627683 TI - Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity. AB - We recently reported purification, determination of the nucleotide sequence, and cloning of a 60-nucleotide RNA (I-RNA) from the yeast Saccharomyces cerevisiae which preferentially blocked cap-independent, internal ribosome entry site (IRES) mediated translation programmed by the poliovirus (PV) 5' untranslated region (UTR). The I-RNA appeared to inhibit IRES-mediated translation by virtue of its ability to bind a 52-kDa polypeptide which interacts with the 5' UTR of viral RNA. We demonstrate here that the HeLa 52-kDa I-RNA-binding protein is immunologically identical to human La autoantigen. Moreover, I-RNA-mediated purified La protein. By using I-RNAs with defined deletions, we have identified sequences of I-RNA required for inhibition of internal initiation of translation. Two smaller fragments of I-RNA (16 and 25 nucleotides) inhibited PV UTR-mediated translation from both monocistronic and bicistronic RNAs. When transfected into HeLa cells, these derivatives of I-RNA inhibited translation of PV RNA. A comparison of protein binding by active and inactive I-RNA mutants demonstrates that in addition to the La protein, three other polypeptides with apparent molecular masses of 80, 70, and 37 kDa may influence the translation-inhibitory activity of I-RNA. PMID- 8627684 TI - Interspecies transmission of macaque simian T-cell leukemia/lymphoma virus type 1 in baboons resulted in an outbreak of malignant lymphoma. AB - An outbreak of malignant lymphoma has been observed in one of the baboon (Papio hamadryas) stocks of Sukhumi Primate Center. More than 300 cases in this "high lymphoma stock" have been registered since 1967. Human T-cell lymphotropic virus type 1 (HTLV-1)-related virus was implicated as the etiologic agent of Sukhumi baboon lymphoma. The origin of this virus remained unclear. Two possibilities were originally considered: the origin could be baboon simian T-cell leukemia/lymphoma virus type 1 (STLV-1) or HTLV-1 (before the outbreak started, some Sukhumi baboons were inoculated with human leukemic material). The third possibility entered recently: interspecies transmission of rhesus macaque STLV-1 to baboons. It was prompted by the finding of very close similarity between STLV 1 991-1cc (the strain isolated from a non-Sukhumi baboon inoculated with material from a Sukhumi lymphomatous baboon) and rhesus STLV-1. To test this hypothesis, we investigated 37 Sukhumi STLV-1 isolates from baboons of high-lymphoma stock by PCR discriminating rhesus type and baboon type STLV-1 isolates. All of them were proved to be rhesus type STLV-1. In contrast, all six STLV-1 isolates from baboons belonging to other stocks or populations were of baboon type. The PCR results were fully confirmed by DNA sequence data. The partial env gene gene sequences of all four STLV-1 isolates from Sukhumi lymphomatous baboons were 97 to 100% similar to the sequence of known rhesus STLV-1 and only 85% homologous with the sequence of conventional baboon STLV-1. Thus, interspecies transmission of STLV-1 from rhesus macaques (or closely related species) to baboons occurred at Sukhumi Primate Center. Most probably this event initiated the outbreak of lymphoma in Sukhumi baboons. PMID- 8627685 TI - Glycoprotein B of herpes simplex virus type 1 oligomerizes through the intermolecular interaction of a 28-amino-acid domain. AB - Herpes simplex virus type 1 glycoprotein B (gB) is an envelope component that plays an essential role in virus infection. The biologically active form of gB is an oligomer that contributes to the process of viral envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studies, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711. In this study, in vitro-transcribed and -translated gB test molecules were used to characterize the more active essential membrane-proximal domain. A series of gB test polypeptides mutated in this downstream oligomerization domain were assayed for their abilities to form oligomers with a mutant gB capture polypeptide containing the analogous wild-type domain. Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the capture polypeptide. Analysis of the immune-precipitated products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 626 to 653 and 653 to 675, each of which was independently sufficient to form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers. Moreover, this stretch of 28 amino acids was shown to form oligomers when introduced into the carboxy-terminal region of gB monomers lacking this domain at the normal site, thus indicating that this domain was functionally independent of its natural location within the gB molecule. Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered in individual amino acids revealed that cysteines 9 and 10 located at positions 596 and 633, respectively, were not required for oligomer formation but contributed to dimer formation and/or stabilization. The results of this study suggest that oligomerization of gB monomers is induced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelope membrane. PMID- 8627687 TI - Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. AB - The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D BP interaction constitutes an important step in the AAV life cycle. PMID- 8627686 TI - Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. AB - Present knowledge of human immunodeficiency virus type 1 (HIV-1) envelope immunobiology has been derived almost exclusively from analyses of subtype B viruses, yet such viruses represent only a minority of strains currently spreading worldwide. To generate a more representative panel of genetically diverse envelope genes, we PCR amplified, cloned, and sequenced complete gp160 coding regions of 35 primary (peripheral blood mononuclear cell-propagated) HIV-1 isolates collected at major epicenters of the current AIDS pandemic. Analysis of their deduced amino acid sequences revealed several important differences from prototypic subtype B strains, including changes in the number and distribution of cysteine residues, substantial length differences in hypervariable regions, and premature truncations in the gp41 domain. Moreover, transiently expressed glycoprotein precursor molecules varied considerably in both size and carbohydrate content. Phylogenetic analyses of full-length env sequences indicated that the panel included members of all major sequence subtypes of HIV-1 group M (clades A to G), as well as an intersubtype recombinant (F/B) from an infected individual in Brazil. In addition, all subtype E and three subtype G viruses initially classified on the basis of partial env sequences were found to cluster in subtype A in the 3' half of their gp41 coding region, suggesting that they are also recombinant. The biological activity of PCR-derived env genes was examined in a single-round virus infectivity assay. This analysis identified 20 clones, including 1 from each subtype (or recombinant), which expressed fully functional envelope glycoproteins. One of these, derived from a patient with rapid CD4 cell decline, contained an amino acid substitution in a highly conserved endocytosis signal (Y721C), as mediated virus entry with very poor efficiency, although they did not contain sequence changes predicted to alter protein function. These results indicate that the env genes of primary HIV-1 isolates collected worldwide can vary considerably in their genetic, phylogenetic, and biological properties. The panel of env constructs described here should prove valuable for future structure-function studies of naturally occurring envelope glycoproteins as well as AIDS vaccine development efforts targeted against a broader spectrum of viruses. PMID- 8627689 TI - Efficient initiation and strand transfer of polypurine tract-primed plus-strand DNA prevent strand transfer of internally initiated plus-strand DNA. AB - A critical step in retroviral reverse transcription is the initiation of plus strand DNA synthesis at the polypurine tract (PPT) and strand transfer of the PPT primed strong-stop DNA to the 5' end of the viral DNA. An attachment site (att) immediately 3' to the PPT is essential for proper integration of proviral DNA into the host chromosome. Plus-strand DNA synthesis is discontinuous in many retroviruses, indicating that sequences upstream of the PPT are also used to initiate plus-strand DNA synthesis (internally initiated DNA). Strand transfer of internally initiated DNA would result in "dead" viral DNA that lacks the att site needed for integration. Strand transfer of the internally initiated DNA could occur if DNA synthesis failed to initiate at the PPT or if the PPT-primed DNA was displaced before strand transfer. We sought to determine the efficiency of DNA synthesis initiating at the PPT and the proportions of PPT-primed DNA and internally initiated DNAs that are utilized for strand transfer. We constructed spleen necrosis virus-based retroviral vectors containing an internal PPT and an att site 5' of the normal PPT and att site. After one replication cycle of the retroviral vectors, the structures of the resulting proviruses were determined by Southern blotting. The analysis suggested that the PPT is an efficient and rapid initiator of plus-strand DNA synthesis and that internally initiated DNAs are rarely utilized for strand transfer. We hypothesize that efficient synthesis and strand transfer of PPT-primed DNA evolved to prevent lethal strand transfers of internally initiated DNAs. PMID- 8627688 TI - Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit. AB - A collection of influenza virus PB2 mutant genes was prepared, including N terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG primed reactions but required a higher beta-globin mRNA concentration than wild type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity. PMID- 8627690 TI - Molecular genetic analysis of Epstein-Barr virus Cp promoter function. AB - The Cp promoter of Epstein-Barr virus (EBV) directs most transcription of the EBNA genes in lymphoblastoid cell lines. The functions of two control regions in the Cp promoter have been studied by construction of recombinant EBV strains containing specific mutations in these elements. Mutation of the RBP-Jk (CBF1) binding site reduced but did not completely abolish EBNA-2-dependent Cp activity in transient transfection assays. The same mutation in recombinant virus gave only a modest average reduction in Cp function, ranging from full activity to almost no activity in different isolates. Separate deletion of a 262-bp region containing glucocorticoid response elements had little effect in a transient assay but caused a fivefold increase in the steady-state level of Cp RNA in recombinant virus. The results indicate that other elements in addition to the intensively studied RBP-Jk site are important in determining Cp activity in the whole virus. Clonal EBV-infected cell lines expressed RNA from both the Cp and Wp promoters, but the level of Wp RNA did not simply compensate for changes in the level of Cp RNA. The levels of EBNA proteins varied much less than the levels of Cp and Wp RNA, suggesting other types of control in addition to initiation of transcription. A survey of RNAs derived from the internal repeat region of the virus indicated that gene expression from this region of EBV in lymphoblastoid cell lines is accounted for by the known transcripts. PMID- 8627691 TI - The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. AB - Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G 497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P 501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent. PMID- 8627692 TI - Disruption of prion rods generates 10-nm spherical particles having high alpha helical content and lacking scrapie infectivity. AB - An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity. PMID- 8627693 TI - The human immunodeficiency virus type 1 transmembrane gp41 protein is a calcium binding protein and interacts with the putative second-receptor molecules in a calcium-dependent manner. AB - Fusion is a crucial event in the life cycle of the human immunodeficiency virus (HIV); is is initiated by the high-affinity binding between gp120, the external surface glycoprotein of HIV-1, and the differentiation antigen CD4 and finally results in the insertion of the hydrophobic amino terminus of the gp41 envelope glycoprotein into the plasma membrane of the target cell. Recent results suggest that this process is dependent upon calcium ions, but the mechanism or the proteins involved are not understood. Computer-assisted sequence analysis revealed a putative calcium-binding site within the extracellular part of gp41 that was highly reminiscent of the calcium-binding EF-hand structure. To test this hypothesis, calcium-binding experiments were performed. Binding of a recombinant soluble form of the transmembrane protein (rsgp41) to its putative second-receptor molecules in equilibrium was dependent upon calcium. The affinity was not influenced by calcium, but the maximum binding was increased in a dose dependent manner. Radioactive calcium bound to rsgp41 covalently attached to Sepharose but not to bovine serum albumin. Binding was inhibited by the addition of nonradioactive calcium, indicating that binding was specific. Neither magnesium nor manganese inhibited the binding of labeled calcium to rsgp41. Binding was dependent on the oxidative state of the rsgp41 molecule, suggesting the functional importance of the correctly folded structure of the rsgp41 protein. In this report, we demonstrate that the HIV-1 transmembrane protein gp41 is a calcium-binding protein and interacts with the putative second-receptor molecules in a calcium-dependent manner. PMID- 8627694 TI - Theiler's virus persistence and demyelination in major histocompatibility complex class II-deficient mice. AB - Mice with targeted disruption of the A beta gene of major histocompatibility complex class II molecules (Abo) were used to investigate the role of class II gene products in resistance or susceptibility to virus-induced chronic demyelination in the central nervous system (CNS). Class-II-deficient mice from the resistant H-2b [H-2b(Abo)] and nonmutant H-2b backgrounds were infected with Theiler's murine encephalomyelitis virus intracerebrally and examined for CNS virus persistence, demyelination, and neurologic clinical signs. Virus titers measured by plaque assays showed that 8 of 10 normally resistant nonmutant H-2b mice had cleared the virus within 21 days, whereas the other 2 mice had low titers. In contrast, all class II-deficient Abo mice had high virus titers for up to 90 days after infection (4.30 log10 PFU per g of CNS tissue). Virus antigens and RNA were localized to the brains (cortex, hippocampus, thalamus, and brain stem) and spinal cords of Abo mice. Colocalization identified persistent Theiler's murine encephalomyelitis virus in oligodendrocytes and astrocytes but not in macrophages. There was demyelination in 11 of 23 and 6 of 9 Abo mice 45 and 90 days after virus infection, respectively, whereas no demyelination was observed in infected nonmutant H-2b mice. Demyelinating lesions in Abo mice showed virus-specific CD8+ T cells and macrophages but no CD4+ T cells. Spasticity and paralysis were observed in chronically infected Abo mice but not in the nonmutant H-2b mice. These findings demonstrate that class II gene products are required for virus clearance from the CNS but not for demyelination and neurologic disease. PMID- 8627695 TI - Identification of a herpesvirus Saimiri cis-acting DNA fragment that permits stable replication of episomes in transformed T cells. AB - Herpesvirus saimiri is a lymphotropic herpesvirus capable of immortalizing and transforming T cells both in vitro and in vivo. Immortalized and transformed T cells harbor several copies of the viral genome as a persisting genome. The mapping of the cis-acting genetic cis-acting segment (oriP) required for viral episomal maintenance is reported here. Viral DNA fragments that potentially contain oriP were cloned into a plasmid that contains the hygromycin resistance gene. After several round of subcloning followed by transfection, oriP was mapped to a 1.955-kb viral segment. This viral fragment permits stable plasmid replication without deletion or rearrangement as well as episomal maintenance without integration or recombination. The function of oriP depends on a trans acting factor(s) encoded by the viral genome. The 1.955-kb viral segment includes a dyad symmetry region located between two small nuclear RNA genes and is located upstream of the dihydrofolate reductase gene homolog. Therefore, this oriP contains novel elements distinct from those of other DNA viruses. PMID- 8627696 TI - Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes. AB - Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein. PMID- 8627697 TI - Characterization of nuclear structures in cells infected with herpes simplex virus type 1 in the absence of viral DNA replication. AB - Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites. Replication-defective viruses containing subtle mutations in the helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented. PMID- 8627699 TI - Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding. AB - We have mutated amino acids within the receptor-binding domain of Moloney murine leukemia virus envelope in order to identify residues involved in receptor binding. Analysis of mutations in the region of amino acids 81 to 88 indicates that this region is important for specific envelope-receptor interactions. None of the aspartate 84 (D-84) mutants studied bind measurably, although they are efficiently incorporated into particles. D-84 mutants have titers that correspond to the severity of the substitution. This observation suggests that D-84 may provide a direct receptor contact. Mutations in the other charged amino acids in this domain (R-83, E-86, and E-87) yield titers similar to those of wild-type envelope, but the affinity of the mutant envelope in the binding assay is decreased by nonconservative substitutions in parallel to the severity of the change. These other amino acids may either provide secondary receptor contacts or assist in maintaining a structure in the domain that favors efficient binding. We also studied other regions of high hydrophilicity. Our initial characterization indicates that amino acids 106 to 111 and 170 to 188 do not play a major role in receptor binding. Measurements of relative binding affinity and titer indicate that most mutations in the region of amino acids 120 to 131 did not significantly affect receptor binding. However, SU encoded by mutants H123V, R124L, and C131A as well as C81A could not be detected in particles and therefore did not bind measurably. Therefore, the region encompassed by amino acids 81 to 88 appears to be directly involved in receptor binding. PMID- 8627698 TI - Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures. AB - Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis. PMID- 8627700 TI - Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA. AB - The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors. PMID- 8627701 TI - Cyclic AMP-responsive element-dependent activation of Epstein-Barr virus zebra promoter by human herpesvirus 6. AB - We have recently shown that infection of Epstein-Barr virus (EBV) genome-positive B cells by human herpesvirus 6 (HHV-6) results in the expression of the immediate early EBV Zebra gene, followed by virus replication (L. Flamand, I. Stefanescu, D. V. Ablashi, and J. Menezes, J. Virol. 67:6768-6777, 1993). Here we show that HHV-6 upregulates Zebra gene transcription through a cyclic AMP-responsive element (CRE) located within the Zebra promoter (Zp). Using human B- or T-cell lines transfected with ZpCat reporter gene constructs, we demonstrate that a region designated the ZII domain of Zp is the target of HHV-6 transactivation. Mutation of the consensus AP-1/CRE site within ZII abolished the inducibility of Zp by HHV-6, whereas positioning of the ZII domain upstream of the beta-globin minimal promoter conferred responsiveness following HHV-6 infection. Binding of these factors to ZII was prevented by oligonucleotides containing CRE but not by AP-1 consensus sequences. Antibodies against CRE-binding (CREB) protein but not against c-Fos or c-Jun were able to supershift the DNA-protein complex, identifying the nature of the transcription factor which binds to ZII as a member of the CREB family of proteins. Finally, transfection of CREB protein and protein kinase A expression vectors were found to activate Zp in Jurkat cells, suggesting that phosphorylated form of CREB protein can play a determining role in the EBV reactivation process. PMID- 8627702 TI - Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site. AB - Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector. PMID- 8627703 TI - Competitive selection in vivo by a cell for one variant over another: implications for RNA virus quasispecies in vivo. AB - Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies noted that the wild-type virus contained a Phe at glycoprotein (GP) residue 260 which correlated with neuron tropism compared with LCMV variants containing a Leu at residue 260 which showed selected tropism for cells of the immune system (C.F. Evans, P. Borrow, J. C. de la Torre, and M. B. A. Oldstone J. Virol. 68:7367-7373, 1994; L. Villarete, T. Somasundaram, and R. Ahmed, J. Virol 68:7490-7496, 1994). Here we (i) evaluated the ability of the viral variants with either a Phe or Leu at GP residue 260 to replicate in vivo in the spleen, liver, or brain, (ii) analyzed the ability of these viruses to compete against each other for cell (neuron)-specific selection following a single viral inoculation of different ratios of both viruses, and (iii) utilized genetic reassortants of both viruses to test their ability to replicate in neurons in vivo. We found that viral variants containing either a Phe or Leu at GP residue 260 were equally capable of replicating in neurons, but when inoculated together, neurons selected for the viral population containing Phe at GP residue 260 over viruses containing a Leu at this position. This was in contrast to selection in the liver and spleen that favored viruses with Leu and not Phe at GP residue 260. Analysis of inoculations with viral reassortants indicated that genes encoded on the short RNA (the GP and nucleoprotein, not the L [polymerase] and Z proteins that are encoded by the large RNA) were associated with neurotropism. Since the nucleoprotein sequences of wild-type Armstrong and clone 13 are identical, it is likely that specific cytoplasmic factors of the neurons play a fundamental role in the selection of virus with Phe at GP residue 260. PMID- 8627704 TI - A novel murine retrovirus identified during testing for helper virus in human gene transfer trials. AB - An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses. To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients. This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses. The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors. All of the helper viruses were in a single interference group. We have now shown that treatment of the M. dunni marker rescue assay cells with 5-iodo-2' deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M. dunni endogenous virus (MDEV). Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium. MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect. PMID- 8627705 TI - Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene. AB - Open reading frame P (ORF P) maps in the viral DNA sequences transcribed during latency and is located antisense to the gamma 1 34.5 gene. Earlier studies have shown that the expression of ORF P is repressed by an infected cell protein no. 4 binding site straddling the transcription initiation site. We have made monospecific polyclonal antibodies to the protein and constructed a virus, designated ORF P++, in which the infected cell protein no. 4 binding site has been mutagenized, thereby allowing full expression of an unmodified ORF P gene from its natural promoter. We report the following findings. (i) The native protein forms multiple bands on denaturing polyacrylamide gels suggestive of extensive processing and aggregation of the protein; (ii) the protein accumulates in the nucleus in rod-shaped structures perpendicular to the axis of attachment of the infected cell to the solid matrix; (iii) the virus was highly attenuated on inoculation into mice by the intracerebral or ocular route, and virus was not recovered upon explantation of trigeminal ganglia; (iv) although protein synthesis was not prematurely shut off in the human neuroblastoma cell line SK-N SH, gamma 1 34.5 protein was not detected in immunoblasts. Analyses of electrophoretically separated denatured RNAs indicated that in cells infected with the ORF P++ virus, there was a large increase in the amount of ORF P RNA and a corresponding decrease in the amount of gamma 1 34.5 RNA. We conclude that either the overproduction of ORF P protein blocks the expression of some herpes simplex virus 1 genes or derepression of the transcription of ORF P has a negative effect on the transcription of the antisense gamma 1 34.5 RNA. PMID- 8627706 TI - Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en. AB - The development of viral resistance to the neuraminidase (NA) inhibitor, 4 guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug-resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor. PMID- 8627707 TI - Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence. AB - Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses. PMID- 8627708 TI - Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues. AB - The first full-length hexon protein DNA and deduced amino acid sequences of a subgenus D adenovirus (AV) were determined from candidate AV48 (85-0844). Comprehensive comparison of this sequence with hexon protein sequences from human subgenera A, B, C, D, F, bovine AV3, and mouse AV1 revealed seven discrete hypervariable regions (HVRs) among the 250 variable residues in loops 1 and 2. These regions differed in length between serotypes, from 2 to 38 residues, and contained > 00% of hexon serotype-specific residues among human serotypes. Alignment with the published crystal structure of AV2 established the location and structure of the type-specific regions. Five HVRs were shown to be part of linear loops on the exposed surfaces of the protein, analogous to the serotype specific loops or "puffs" in picornavirus capsid proteins. The HVRs were supported by a common framework of conserved residues, of which 68 to 75% were hydrophobic. Unique sequences were limited to the seven HVRs, so that one or more of these regions contain the type-specific neutralization epitopes. A neutralizing AV48 hexon-specific antiserum recognized linear peptides that corresponded to six HVRs by enzyme immunoassay. Affinity-purification removal of all peptide-reactive antibodies did not significantly decrease the neutralization titer. Eluted peptide-reactive antibodies did not neutralize. Human antisera that neutralized AV48 did not recognize linear peptides. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not. We conclude that the AV48 neutralization epitope(s) is complex and conformational. PMID- 8627709 TI - Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers. AB - Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus. PMID- 8627710 TI - A novel mechanism for persistence of human cytomegalovirus in macrophages. AB - Human cytomegalovirus (HCMV) infection of monocyte-derived macrophages (MDM) results in delayed and nonlytic productive viral growth. During late stages of replication, infectious virus remains cell associated in cytoplasmic vacuoles. In order to understand HCMV survival and persistence in MDM, we examined mechanisms involved in the formation and trafficking of HCMV-containing vacuoles in these cells. Utilizing double-label immunofluorescence with antibodies to viral and cellular proteins, HCMV-containing vacuoles were associated with the Golgi apparatus marker mannosidase II but not with markers to early endosomes (transferrin receptor and rab5) or late endosomes and early lysosomes (LAMP-1 and -2). In addition, as late-stage viral infection progressed in MDM, the cells displayed increasing abnormalities in the Golgi apparatus. Analysis of structural features of infected cells revealed the disruption of the microtubule network. These observations suggest a novel mechanism by which HCMV is vacuolized in MDM, avoiding degradation and release from the cell. PMID- 8627711 TI - Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. AB - Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1. PMID- 8627712 TI - Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene. AB - The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice. PMID- 8627713 TI - Murine leukemia virus envelope protein in transgenic-mouse serum blocks infection in vitro. AB - Transgenic mice bearing a murine retroviral envelope transgene (Fv4) have Fv4 gp70env (SU) in their serum in amounts sufficient to block infection by ecotropic virus in vitro. Fv4 Env in serum is derived largely but not exclusively from hematopoietic cells. Tail cells from Fv4 mice and cell lines transduced with the Fv4 env transgene synthesize both components of the envelope protein (gp70 SU and p15E TM) but secrete the gp70 moiety, in the absence of retroviral particles. Blocking of the ecotropic viral receptor by secreted gp70 SU may contribute to resistance to retroviral infection in these mice. PMID- 8627714 TI - RNA-protein interactions at the 3' end of the hepatitis A virus RNA. AB - The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross linking/label transfer experiments, we have analyzed sequences of the 3' nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3' NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis. PMID- 8627715 TI - The herpes simplex virus type 1 VP5 promoter contains a cis-acting element near the cap site which interacts with a cellular protein. AB - The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter. PMID- 8627716 TI - The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis. AB - We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection. PMID- 8627717 TI - The primary target cells of the high-risk cottontail rabbit papillomavirus colocalize with hair follicle stem cells. AB - Papillomaviruses are small DNA tumor viruses with a life cycle inseparably linked to the differentiation of the pluristratified epithelium. The infection of epithelial layers of the skin may remain latent or may result in the development of benign tumors. A certain number of distinct papillomavirus types, however, cause lesions which have a high risk of progression into carcinomas, and extensive efforts have been made to understand this process. comparatively little is known about the initial events during the establishment of a persistent infection and papilloma development. Although it is generally accepted that the growth of a papilloma requires the infection of cells in the basal layer of the epithelium, it remains unknown which cells perform this task. We have analyzed by in situ hybridization biopsy samples taken at various time points after infection of domestic rabbits with cottontail rabbit papillomavirus. The positive cells detected at a low frequency in biopsy samples taken after 11 days predominantly expressed high levels of E6 and E7 mRNA and were localized in the outer epithelial root sheath and in the bulbs of hair follicles. A clonal analysis of keratinocytes isolated from different subfragments of individual rabbit hair follicles demonstrated a clear colocalization of cottontail rabbit papillomavirus mRNA-positive cells with clonogenic cells in hair follicles. These data suggest that the cells competent to establish papillomatous growth represent a subpopulation of keratinocytes in hair follicles with properties expected of epithelial stem cells. PMID- 8627718 TI - Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus. AB - A region of the infectious bronchitis virus (IBV) genome between nucleotide positions 8693 and 10927 which encodes the predicted 3C-like proteinase (3CLP) domain and several potential cleavage sites has been clones into a T7 transcription vector. In vitro translation of synthetic transcripts generated from this plasmid was not accompanied by detectable processing activity of the nascent polypeptide unless the translation was carried out in the presence of microsomal membrane preparations. The processed products so obtained closely resembled in size those expected from cleavage at predicted glutamine-serine (Q/S) dipeptides and included a protein with a size of 35 kDa (p35) that corresponds to the predicted size of 3CLP. Efficient processing was dependent on the presence of membranes during translation; processing was found to occur when microsomes were added posttranslationally, but only after extended periods of incubation. C-terminal deletion analysis of the encoded polyprotein fragment revealed that cleavage activity was dependent on the presence of most but not all of the downstream and adjacent hydrophobic region MP2. Dysfunctional mutagenesis of the putative active-site cysteine residue of 3CLP to either serine or alanine resulted in polypeptides that were impaired for processing, while mutagenesis at the predicted Q/S release sites implicated them in the release of the p35 protein. Processed products of the wild-type protein were active in trans cleavage assays, which were used to demonstrate that the IBV 3CLP is sensitive to inhibition by both serine and cysteine protease class-specific inhibitors. These data reveal the identity of the IBV 3C-like proteinase, which exhibits characteristics in common with the 3C proteinases of picornaviruses. PMID- 8627720 TI - Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA. AB - Mechanisms by which the plus-sense RNA genomes of picornaviruses are replicated remain poorly defined, but existing models do not suggest a role for sequences encoding the capsid proteins. However, candidate RNA replicons (delta P1 beta gal and delta P1Luc), representing the sequence of human rhinovirus 14 virus (HRV-14) with reporter protein sequences (beta-galactosidase or luciferase, respectively) replacing most of the P1 capsid-coding region, failed to replicate in transfected H1-HeLa cells despite efficient primary cleavage of the polyprotein. To determine which P1 sequences might be required for RNA replication, HRV-14 mutants in which segments of the P1 region were removed to frame from the genome were constructed. Mutants with deletions involving the 5'proximal 1,489 nucleotides of the P1 region replicated efficiently, while those with deletions involving the 3' 1,079 nucleotides did not. Reintroduction of the 3' P1 sequence into the nonreplicating delta P1Luc construct resulted in a new candidate replicon, delta P1Luc/VP3, which replicated well and expressed luciferase efficiently. Capsid proteins provided in trans by helper virus failed to rescue the nonreplicating delta P1Luc genome but were able to package the larger-than-genome-length delta P1Luc/VP3 replicon. Thus, a 3'-distal P1 capsid-coding sequence has a previously unrecognized cis-active function related to replication of HRV-14 RNA. PMID- 8627719 TI - Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch. AB - The essential herpes simplex virus type 1 (HSV-1) immediate-early IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3' processing. We have investigated the role of IE63 in the regulation of viral mRNA 3' processing and of late gene expression. Our in vitro 3' processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3' processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3' processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were recognized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3' processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components on the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre mRNA 3' processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis. PMID- 8627721 TI - Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine. AB - Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14 bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge. PMID- 8627722 TI - Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase. AB - The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site directed mutations within the highly conserved CHAE sequence of the postulated C terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD. PMID- 8627723 TI - Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes. AB - Herpes simplex virus type 1 mutants with certain lesions in the ICP27 gene show a 5- to 10-fold reduction in viral DNA synthesis. To determine how ICP27 promotes amplification of viral DNA, we examined the synthesis, accumulation, and stability of the essential viral replication proteins and steady-state levels of the replication gene transcripts throughout the course of ICP27 mutant virus infections. These studies reveal that in the absence of ICP27, expression of the UL5, UL8, UL52, UL9, UL42, and UL30 genes is significantly reduced at the level of mRNA accumulation. In contrast to that of these beta genes, ICP8 expression is unaltered in mutant virus-infected cells, indicating that ICP27 selectively stimulates only a subset of herpes simplex virus beta genes. Analysis of multiple ICP27 mutant viruses indicates a quantitative correlation between the ability of these mutants to replicate viral DNA and the level of replication proteins produced by each mutant. Therefore, we conclude that the primary defect responsible for restricted viral DNA synthesis in cells infected with ICP27 mutants is insufficient expression of most of the essential replication genes. Of further interest, this analysis also provides new information about the structure of the UL52 gene transcripts. PMID- 8627724 TI - Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes. AB - The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation. PMID- 8627725 TI - The CREB, ATF-1, and ATF-2 transcription factors from bovine leukemia virus infected B lymphocytes activate viral expression. AB - Efficient transcription and replication of the bovine leukemia virus (BLV) genome require both the viral long terminal repeat (LTR) and the virus-coded transcriptional activator Tax, which functions through a 21-bp sequence (Tax responsive element [TxRE]) which is repeated three times within the LTR. Since Tax does not bind directly to DNA, host cell transcription factors play a central role in BLV expression. Electrophoretic mobility shift assays with nuclear extracts prepared with infected bovine B lymphocytes revealed five TxRE-specific complexes (C1, C2, C3, C4, and C5). Here, by using a UV-induced indirect labeling technique (UV cross-linking) in conjunction with mobility shift assays, eight major polypeptides of 31, 33, 42, 46, 51, 57, 87, and 119 kDa were identified within these five complexes. Immunoprecipitation experiments identified the 57- and 119-kDa proteins as cyclic AMP response element-binding (CREB) proteins, the 46- and 51-kDa proteins as activating transcription factor-1 (ATF-1), and the 87 kDa as protein ATF-2. All of these proteins (except the ATF-1 protein of 51 kDa) belong to the complex C1, which is the major complex identified in freshly isolated BLV-infected lymphocytes from cattle with persistent lymphocytosis. In transient-cotransfection experiments, these three transcription factors were able to activate LTR-directed gene expression in the presence of protein kinase A or Ca2+/calmodulin-dependent protein kinase IV. CREB protein, ATF-1, and ATF-2 thus appear to be the major transcription factors involved in the early stages of viral expression. PMID- 8627726 TI - Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells. AB - Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis. PMID- 8627727 TI - Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli. AB - The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies. PMID- 8627728 TI - A 371-nucleotide region between the herpes simplex virus type 1 (HSV-1) LAT promoter and the 2-kilobase LAT is not essential for efficient spontaneous reactivation of latent HSV-1. AB - The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. However, neither the mechanism by which LAT carries out this function nor the region of LAT responsible for this function in known. LAT is transcribed as an unstable 8.3-kb RNA that gives rise to a very stable 2-kb LAT RNA that is readily detected in latently infected sensory neurons. We show here that 371 of the 662 nucleotides located between the start of LAT transcription and the 5' end of the 2-kb LAT RNA do not appear to be essential for wild-type levels of spontaneous reactivation in the rabbit ocular model of HSV-1 latency. We deleted LAT nucleotides 76 to 447 from both copies of the LAT gene (one in each viral long repeat) to produce the mutant dLAT371. Rabbits were ocularly infected with dLAT371, and spontaneous reactivation was measured in comparison with the marker rescued virus dLAT371R. Both dLAT371 and dLAT371R had spontaneous reactivation rates of approximately 13 to 14%. This was consistent with the parental McKrae wild-type virus (11.7%; P = 0.49) and significantly higher than the LAT transcription-negative mutant dLAT2903 (2.4%; P < 0.0001). Southern analysis confirmed that the spontaneously reactivated dLAT371 virus retained the deletion in both copies of LAT. Therefore, it appeared that the function of LAT involved in efficient spontaneous reactivation mapped outside the 371-nucleotide region deleted from the LAT gene of dLAT371. PMID- 8627729 TI - The recombination rate is not increased when retroviral RNA is missing an encapsidation sequence. AB - Retroviruses, as a result of the presence of two identical RNA molecules in their virions, recombine at a high rate. When nonhomologous RNA is present in the dimer RNA molecules, nonhomologous recombination occurs, although the rate is only 0.1% of the rate of homologous recombination. We developed a protocol to study transduction of cellular proto-oncogenes in a single cycle of retrovirus replication. The psi sequences is a cis required sequence for packaging viral RNA into viral particles. To test if the rate of nonhomologous recombination increases about 1,000 times when the psi sequence is deleted, as reported by other, we modified vectors we used previously (J. Zhang and H. M. Temin, Science 259:234-238, 1993). Our results indicated that the recombination rate did not undergo the increase of about 1,000 times when the psi sequence of a chimeric RNA was deleted. PMID- 8627730 TI - Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus specific cytotoxic activity. AB - Distinct functional CD8+ T-cell populations have been observed during human immunodeficiency virus (HIV) infection. One of these functions is the inhibition of viral replication by a noncytotoxic mechanism, which was shown to be mediated by the CD8+CD28+ subpopulation. On the other hand, CD8+ T cells exert an HIV specific cytotoxic activity. The present study shows that CD8+CD28- lymphocytes display this HIV-specific cytotoxic activity, which is detectable immediately after the cells are purified from peripheral blood. The CD28- population is also able to proliferate and to retain its cytotoxic activity after in vitro restimulation with autologous blast cells. Finally, HIV-specific cytotoxic T cells can be obtained in vitro from the CD8+CD28+ population. PMID- 8627731 TI - Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences. AB - Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus. PMID- 8627732 TI - Glycoprotein Bb, the N-terminal subunit of bovine herpesvirus 1 gB, can bind to heparan sulfate on the surfaces of Madin-Darby bovine kidney cells. AB - The present study confirms our previous findings made by using heparin affinity chromatography that bovine herpesvirus 1 gB can bind to heparin-like structures. In order to locate the functional domain for heparin binding, we expressed the extracellular portion of gB (gBt) and the large subunit of gB (gBb) in Madin Darby bovine kidney (MDBK) cells under the control of the bovine heat shock protein 70A gene promoter. The recombinant gBt and gBb were both efficiently secreted from the transfected cells. They were shown to have structural and antigenic properties similar to those of authentic gB. Like authentic gB, both gBt and gBb were able to bind heparin-Sepharose as well as heparan sulfates on MDBK cells. Thus, we suggest that at least one heparin-binding domain is localized in gBb, the N-terminal portion of gB, which agrees with the presence of clusters of prolines and basic residues, thought to be essential for heparin binding. PMID- 8627733 TI - In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects. AB - We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity. PMID- 8627734 TI - Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids. AB - We have constructed defined human cytomegalovirus (CMV) mutants by cotransfecting overlapping cosmid clones spanning the 230-kbp genome. Using this strategy, we have introduced a 13-kbp region of DNA from a virulent strain of CMV into a defined position within the avirulent CMV(Towne) genome. Although more than 80% of the genome of these recombinant viruses was derived from Towne DNA, their plaque morphology more closely resembled that of Toledo. To date, CMV is the largest virus and requires the greatest number of cosmids to be regenerated via overlapping cosmid cotransfection. PMID- 8627735 TI - Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo. AB - The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95 110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95 110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV. PMID- 8627736 TI - Growth of influenza A virus in primary, differentiated epithelial cells derived from adenoids. AB - Epithelial cells of adenoid origin were grown in tissue culture to examine viral replication in cells that are the primary target of many human pathogens. These cells remained highly differentiated, with subpopulations of cells which retained active ciliary motility and others which demonstrated specialized secretory functions. The epithelial cells were permissive for growth of influenza A virus. Primary respiratory epithelial cells provide a model system for examining virulence, cell tropism, and receptors which replicate in the nasopharynx. PMID- 8627737 TI - Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU. AB - We previously reported a strategy to redirect the retroviral host range by expressing single-chain antibodies (S. J. Russell, R. E. Hawkins, and G. Winter, Nucleic Acids Res. 21:1081-1085, 1993) or ligands (F.-L. Cosset, F. J Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell, J. Virol. 69:6314 6322, 1995) at the N terminus of Moloney murine leukemia virus (MoMLV) surface proteins (SU). Although such chimeric envelopes were able to bind the new receptors, the transduction efficiency of retargeted viruses was generally low. We hypothesized that conformational rearrangements of envelope glycoproteins were not optimally triggered following binding, and to overcome these postbinding blocks, we have generated here a set of chimeric MoMLV-derived envelopes targeted to the Ram-1 phosphate transporter in which we have varied the spacing between the Ram-1-binding domain and the MoMLV SU. All of the recombinant envelopes were correctly expressed on virions, and all bound efficiently to Ram-1. However, the interdomain spacing greatly affected the efficiency of gene transfer by retroviral vectors that had bound to Ram-1 via their chimeric envelopes. Optimal interdomain spacing allowed a 100-fold-increased viral transduction via Ram-1 compared to our previous results. PMID- 8627738 TI - Mutations in the zinc-binding motif of the reovirus capsid protein delta 3 eliminate its ability to associate with capsid protein mu 1. AB - Reovirus capsid protein delta 3 binds both double-stranded RNA (dsRNA) and zinc. Previous studies have revealed that the amino-terminal zinc finger is not required for the ability of delta 3 to bind dsRNA. We expressed wild-type and mutant delta 3 molecules by in vitro transcription/translation to evaluate the importance of the zinc finger for other functions of delta 3. delta 3 molecules with mutations in the zinc finger did not form complexes with capsid protein mu 1 but bound dsRNA more efficiently than wild-type delta 3 did. In contrast, a dsRNA binding mutant was unimpaired in its ability to associate with mu 1. Studies with delta 3 fragments support these findings and indicate that sequences critical for delta 3's interaction with mu 1 lie in the amino terminus of the molecule. Our finding that mu 1 and dsRNA do not compete for identical binding sites on delta 3 has implications for its function as a translational regulator in infected cells. PMID- 8627739 TI - Differential processing of sindbis virus glycoprotein PE2 in cultured vertebrate and arthropod cells. AB - A step in the maturation of Sindbis virus glycoproteins is the cleavage of the precursor glycoprotein PE2 into E3 and E2 by furin or a furin-like host cell protease. The results presented here suggest that PE2 cleavage is an obligatory event for Sindbis virus maturation in C6/36 cells and demonstrate that certain mutants display a cell-specific PE2 cleavage phenotype. We previously have described Sindbis virus variants which fail to cleave PE2 because of incorporation of a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage site but are viable in BHK-21 cells by virtue of an additional mutation at E2 216 or E2 191 (TRSB-NE2G216 and TRSB-NE2T191, respectively) (H. W. Heidner, K. L. McKnight, N. L. Davis, and R. E. Johnston, J. Virol. 68:2683-2692, 1994). Other viable PE2 cleavage-defective mutants were constructed by substituting the parental residue at E2 position 1 (Arg), with Leu or Val (TRSB E2L1 and TRSB-E2V1, respectively) (H.W. Heidner and R. E. Johnston, J. Virol. 68:8064-8070, 1994). When grown in BHK-21 cells, all four of these viruses replicated normally and incorporated PE2 in place of E2 in released virions. However, growth of TRSB-NE2G216 and TRSB-NE2T191 was severely restricted in cultured arthropod cells (C6/36 cells). Analysis of infected C6/36 cells by flow cytometry demonstrated that the restricted growth of TRSB-NE2G216 and TRSB NE2T191 was not due to an impaired ability to initiate infection. In addition, TRSB-NE2G216 and TRSB-NE2T191 remained growth restricted in C6/36 cells following introduction of in vitro transcriptions by electroporation. In contrast, the PE2 cleavage defect of TRSB-E2L1 and TRSB-E2V1 was cell type specific. In C6/36 cells, the majority of PE2 was converted to E2, and these viruses replicated normally in C6/36 cells. These results demonstrated a consistent link between expression of a PE2 cleavage defect and restricted growth in C6/36 cells and suggest that cleavage of PE2 is required for maturation of Sindbis virus late in infection of C6/36 cells. PMID- 8627740 TI - What is the orientation of DNA polymerases on their templates? PMID- 8627741 TI - A novel herpes simplex virus 1 gene, UL43.5, maps antisense to the UL43 gene and encodes a protein which colocalizes in nuclear structures with capsid proteins. AB - An open reading frame mapping antisense to the UL43 gene of herpes simplex virus 1 encodes a protein with an apparent Mr of 38,000. The protein was detected in wild-type-infected cells with rabbit monospecific polyclonal antibody directed against a fusion protein containing all of the sequences encoded by the open reading frame. The antibody did not react with mutants from which the open reading frame was deleted. Expression of this gene, designated UL43.5, was grossly decreased or abolished in infected cells incubated in medium containing inhibitory concentrations of phosphonoacetic acid, suggesting that it is regulated as a gamma gene. UL43.5 is dispensable in cell culture. UL43.5 protein colocalized with the major capsid protein (infected cell protein 5) and the capsid scaffolding proteins (infected cell protein 35) in nuclear structures situated at the periphery of the nucleus. The predicted amino acid sequence indicates that the UL43.5 protein is a highly hydrophilic protein. The colocalization of UL43.5 protein with capsid proteins in discrete nuclear structures suggests that the former may be involved in assembly of viral particles in an accessory role in cells in culture. PMID- 8627742 TI - n-Butyrate, a cell cycle blocker, inhibits early amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of duck hepatocytes. AB - During chronic hepadnavirus infection, virus persistence depends on the regulation of the pool of covalently closed circular DNA (cccDNA), which is the template for transcription of viral RNA species. The development of in vitro infection of duck hepatocyte primary cultures by duck hepatitis B virus (DHBV) provides a unique opportunity to study the regulation of cccDNA synthesis. After DHBV in vitro infection, cccDNA is detected 1 day later and is amplified to a high copy number after 1 week in culture. We studied whether this amplification occurs during cell cycle progression of duckling hepatocytes. By using [3H]thymidine incorporation, we found that hepatocytes obtained from 3-week-old ducklings spontaneously entered the S phase of the cell cycle when cultured in serum-free medium without added growth factors. Bromodeoxyuridine labeling confirmed that cellular DNA synthesis took place in more than 50% of parenchymal cells. Cytofluorometry analysis revealed the presence of asynchronous populations and polyploidization processes. The addition of a cell cycle blocker, n-butyrate, completely inhibited [3H]thymidine incorporation and blocked duckling hepatocytes in the G1 phase of the cell cycle. Simultaneously, butyrate inhibited cccDNA amplification and allowed the establishment of DHBV infection, as demonstrated by the detection of a basal level of cccDNA in treated hepatocytes. Both effects were reversible since active cell DNA synthesis was restored and cccDNA accumulated after drug withdrawal. PMID- 8627743 TI - Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus. AB - A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory. This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies. The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced. The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element. A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified. Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection. We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus. The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation. PMID- 8627744 TI - Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus strand synthesis: role of the nsP2 protein. AB - We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion of the initial, short-lived minus-strand replicase complex (RCinitial) into mature, stable forms that are replicase and transcriptase complexes (RCstable), producing 49S genome or 26S mRNA. Base changes at nucleotide (nt) 2166 (G-->A, predicting a change of Glu-163-->Lys), at nt 2502 (G-->A, predicting a change of Val-275-->Ile), and at nt 2926 (C-->U, predicting a change of Leu-416-->Ser) in the nsP2 N domain were responsible for the phenotypes of ts14, ts16, and ts19 members of subgroup 11 (D.L. Sawicki and S.G. Sawicki, Virology 44:20-34, 1985) of the A complementation group of Sindbis virus RNA-negative mutants. Unlike subgroup I mutants, the RCstable formed at 30 degrees C transcribed 26S mRNA normally and did not synthesize minus strands in the absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsP2 and did not stop the proteolytic processing of the polyprotein containing the nonstructural proteins. The distinct phenotypes of subgroup I and 11 A complementation group mutants are evidence that the two domains of nsP2 are essential and functionally distinct. A detailed analysis of ts14 found that its nsPs were synthesized, processed, transported, and assembled at 40 degrees C into complexes with the properties of RCinitial and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of RCstable occurred after cleavage of the minus-strand replicase P123 or P23 polyprotein into mature nsP1, nsP2, nsP3, and nsP4, indicating that structures resembling RCstable, were formed at 40 degrees C. However, these RCstable or pre RCstable structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase the rate of plus-strand synthesis after shift to 40 degrees C appears to result from failure to convert RCinitial to RCstable. We conclude that RCstable is derived from RCinitial by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, we predict that other nsP2 N-domain mutants are blocked also in the conversion of RCinitial to RCstable. Thus, the N domain of nsP2 plays an essential role in a folding pathway of the nsPs responsible for formation of the initial minus-strand replicase and for its conversion into stable plus-strand RNA-synthesizing enzymes. PMID- 8627745 TI - The UCUAAAC promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering RNA. AB - The 65-nucleotide leader on the cloned bovine coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild type leader of the helper virus following DI RNA transfection into helper virus infected cells. A model of leader-primed transcription in which free leader supplied in trans by the helper virus interacts by way of its flanking 5'UCUAAAC3' sequence element with the 3'-proximal 3'AGAUUUG5' promoter on the DI RNA minus strand to prime RNA replication has been used to explain this phenomenon. To test this model, the UCUAAAC element which occurs only once in the BCV 5' untranslated region was either deleted or completely substituted in input DI RNA template, and evidence of leader conversion was sought. In both cases, leader conversion occurred rapidly, indicating that this element is not required on input RNA for the conversion event. Substitution mutations mapped the crossover region to a 24-nucleotide segment that begins within the UCUAAAC sequence and extends downstream. Although structure probing of the bovine coronavirus 5' untranslated region indicated that the UCUAAAC element is in the loop of a prominent stem and thus theoretically available for base pair-directed priming, no evidence of an unattached leader early in infection that might have served as a primer for transcription was found by RNase protection studies. These results together suggest that leader conversion on the DI RNA 5' terminus is not guided by the UCUAAAC element and might arise instead from a high-frequency, region-specific, homologous recombination event perhaps during minus-strand synthesis rather than by leader priming during plus-strand synthesis. PMID- 8627746 TI - Persistent activation of NF-kappa B/Rel by human T-cell leukemia virus type 1 tax involves degradation of I kappa B beta. AB - Activation of the eukaryotic NF-kappaB/Rel transcription factors by various cytokines and mitogens is a transient event, reflecting the fact that these inducers trigger the degradation and resynthesis of the dynamic NF-kappaB/Rel inhibitor IkappaBalpha. However, the tax gene product of the human T-cell leukemia virus type 1 (HTLV-1) is known to induce the persistent nuclear expression of various NF-kappaB/Rel factors, especially the c-Rel proto oncoprotein, although the underlying mechanism remains unclear. In the present study, we demonstrate that Tax induces the degradation Of IkappaBbeta, another NF kappaB/Rel cytoplasmic inhibitor that differs from IkappaBalpha in signal responses. Unlike that observed with IkappaBalpha, the degradation Of IkappaBbeta is not associated with its rapid resynthesis, apparently because of the failure of Tax to stimulate IkappaBbeta gene transcription. Thus, expression of Tax in Jurkat T cells leads to the gradual depletion of IkappaBbeta, which is correlated with the induction of c-Rel-containing kappaB binding complexes. Remarkably, in the three HTLV-1-infected T-cell lines investigated, little or no detectable amount of IkappaBbeta was found. We further demonstrate that Tax is able to override the cytoplasmic retention of c-Rel by 1kappaBbeta in transiently transfected cells. Together, these studies suggest that Tax-mediated inactivation Of IkappaBbeta may play a role in the persistent nuclear expression of c-Rel induced by HTLV-I infection. PMID- 8627747 TI - Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. AB - Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system. PMID- 8627748 TI - Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons. AB - The influenza virus nucleoprotein (NP), matrix protein (M1), and ribonucleoproteins (vRNPs) undergo regulated nuclear import and export during infection. Their trafficking was analyzed by using interspecies heterokaryons containing nuclei from infected and uninfected cells. Under normal conditions, it was demonstrated that the vRNPs which were assembled in the nucleus and transported to the cytosol were prevented from reimport into the nucleus. To be import competent, they must first assemble into virions and enter by the endosomal entry pathway. In influenza virus mutant ts51, in which M1 is defective, direct reimport took place but was inhibited by heterologous expression of wild-type M1. These data confirm M1's role as the inhibitor of premature nuclear import and as the main regulator of nuclear transport of vRNPs. In addition to this vRNP shuttling, M1 also shuttled between the nucleus and the cytoplasm in ts51-infected cells. When NP was expressed in the absence of virus infection, it was also found to be a shuttling protein. PMID- 8627749 TI - Identification of a region in the Sindbis virus nucleocapsid protein that is involved in specificity of RNA encapsidation. AB - The specific encapsidation of genomic RNA by an alphavirus requires recognition of the viral RNA by the nucleocapsid protein. In an effort to identify individual residues of the Sindbis virus nucleocapsid protein which are essential for this recognition event, a molecular genetic analysis of a domain of the protein previously suggested to be involved in RNA binding in vitro was undertaken. The experiments presented describe the generation of a panel of viruses which contain mutations in residues 97 through 111 of the nucleocapsid protein. All of the viruses generated were viable, and the results suggest that, individually, the residues mutated do not play a critical role in encapsidation. However, one mutant which had lost the ability to specifically encapsidate the genomic RNA was identified. This mutant virus, which contained a deletion of residues 97 to 106, encapsidated both the genomic RNA and the subgenomic mRNA of the virus. It is proposed that the encapsidation of this second species of RNA, which is not present in wild-type virions, is the result of the loss of a domain of the nucleocapsid protein required for specific recognition of the genomic RNA packaging signal. The results suggest that this region of the protein is important in dictating specificity in the encapsidation reaction in vivo. The isolation and preliminary characterization of two independent second-site revertants to this deletion mutant are also described. PMID- 8627750 TI - A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. AB - Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid. PMID- 8627751 TI - In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus. AB - The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines. PMID- 8627752 TI - Simian virus 40 large T antigen alters the phosphorylation state of the RB related proteins p130 and p107. AB - p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus ElA protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb 1) have suggested that p130 and p107 may be important targets for SV40 large TAg mediated transformation (J.B. Christensen and M.J. Imperiale, J. Virol. 65:3945 3948, 1995; J. Zalvide and J.A. DeCaprio, Mol. Cell. Biol. 15:5800-5810, 1995). In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb. PMID- 8627753 TI - Varicella-zoster virus (VZV) transcription during latency in human ganglia: detection of transcripts mapping to genes 21, 29, 62, and 63 in a cDNA library enriched for VZV RNA. AB - Information on the extent of virus DNA transcription and translation in infected tissue is crucial to an understanding of herpesvirus latency. To detect low abundance latent varicella-zoster virus (VZV) transcripts, poly(A)+ RNA extracted from latently infected human trigeminal ganglia was enriched for VZV transcripts by hybridization to biotinylated VZV DNA. After hybridization, the RNA-DNA hybrid was isolated by binding to avidin-coated beads and extensively washed, and the RNA was released by heat denaturation. A lambda-based cDNA library was then constructed from the enriched RNA. PCR and DNA sequencing of DNA extracted from the cDNA library revealed the presence of VZV genes 21, 29, 62, and 63, but not VZV genes 4, 10, 40, 51, and 61, in the enriched cDNA library. These findings confirm the detection of VZV gene 29 and 62 transcripts on Northern (RNA) blots prepared from latently infected human ganglia (J.L. Meier, R.P. Holman, K.D. Croen, J.E. Smialek, and S.E. Straus, Virology 193:193-200, 1993) and the presence of VZV gene 21 transcripts in a cDNA library from mRNA of latently infected ganglia (R.J. Cohrs, K. Srock, M.B. Barbour, G. Owens, R. Mahalingam, M.E. Devlin, M. Wellish and D.H. Gilden, J. Virol. 68:7900-7908,1994) and also reveal, for the first time, the presence of VZV gene 63 RNA in latently infected human ganglia. PMID- 8627754 TI - Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis. AB - We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis. PMID- 8627755 TI - Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells. AB - Endogenous reverse transcription (ERT) of retroviruses has long been considered a somewhat artificial process which only mimics reverse transcription occurring in target cells, as detergents or amphipathic peptides have classically been used to make the envelopes of retroviruses in these reaction systems permeable. Recently, several studies suggested that ERT of human immunodeficiency virus type 1 (HIV-1) might occur without detergent treatment. However, this phenomenon could be due to damage of the retroviral envelope during the process of virion purification or freezing and thawing. In this report, intravirion HIV-1 ERT, without detergent induced permeabilization, is demonstrated to occur in the natural microenvironments of HIV-1 virions and is not caused by artificial processes. Therefore, this stage of the viral life cycle was termed natural ERT (NERT). The efficiency of NERT in HIV-1 virions was markedly augmented by several physiological substances in the extracellular milieu, such as polyamines and deoxyribonucleoside triphosphates. In addition, HIV-1 virions in seminal plasma samples harbored dramatically higher levels of full-length or nearly full-length reverse transcripts than virions isolated from peripheral blood plasma samples of HIV-1-seropositive men. When HIV-1 virions were incubated with seminal plasma samples, infectivity in initially nondividing cells was also significantly enhanced. Thus, we suggest that HIV-1 virions are actively altered by the extracellular microenvironment and that NERT may play an important role in viral infection of nondividing cells. PMID- 8627757 TI - Early region 3 of adenovirus type 19 (subgroup D) encodes an HLA-binding protein distinct from that of subgroups B and C. AB - Early region 3 (E3) of human adenoviruses (Ads) codes for proteins that appear to control viral interactions with the host. For example, the most abundant E3 protein, E3/19K, inhibits the transport of newly synthesized class I major histocompatibility molecules to the cell surface, thereby interfering with antigen presentation. So far, the E3 regions of Ad subgroups A, B, C, and F have been characterized. We have cloned the E3A region of Ad type 19a (Ad19a), which belongs to the largest subgroup, D, and causes epidemic keratoconjunctivitis in humans. The sequence reveals five open reading frames (ORFs) with the potential to encode the Ad19 equivalent of pVIII, as well as proteins 12.2K, 16.2K, and 18.6K. The last ORF predicts a novel 49K protein which has no counterpart in other subgroups. Both the sequence and the overall organization of the E3 region from Ad19a shows a closer relationship to group B than to group C Ads. The 18.6K ORF represents the Ad19 homolog of the Ad2 E3/19K protein. By using 293 cells stably transfected with the Adl9a E3A region, we showed by immunoprecipitation, pulse-chase experiments, and fluorescence-activated cell sorter analysis that the Ad19 E3/19K protein binds to and prevents the transport of major histocompatibility complex molecules to the cell surface. The similar but distinct functional activity of the Ad19 E3/19K protein, combined with the new sequence which differs from those of subgroup B and C proteins, allows a more precise definition of amino acids essential for HLA binding. PMID- 8627756 TI - Anti-human immunodeficiency virus type 1 activity of an oligocationic compound mediated via gp120 V3 interactions. AB - An oligocationic peptide compound (ALX40-4C) was developed for consideration in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This compound was designed to mimic the basic domain of the HIV-1 transactivation protein, Tat, and will competitively inhibit Tat binding to its specific RNA hairpin target (TAR [transactivation region]), found at the 5' end of all HIV-1 transcripts. Blocking Tat-TAR interactions can abrogate HIV-1 replication. ALX40 4C was shown to inhibit replication of HIV-1NL4-3 in a range of cell types, including primary cells and transformed cell lines, by as much as 10(4)-fold. In some experiments, virus rescue was not possible even after removal of ALX40-4C from the cultures. Strain-dependent resistance has been demonstrated for all antiretroviral agents tested; therefore, we tested for variable sensitivity to ALX40-4C. The cloned primary strains, HIV-JR-CSF and HIV-JR-FL, were less sensitive to ALX40-4C inhibition. Unexpectedly, determinants for efficient ALX40 4C inhibition were mapped by using recombinant virus strains to the V3 region of gpl20 and were shown to act at early events in viral replication, which include viral entry. If entry and reverse transcription are bypassed by transfection, a more modest, virus strain-independent inhibition is shown; this inhibition is likely due to blocking of Tat-TAR interaction. Thus, the highly basic oligocationic Tat inhibitor ALX40-4C appears to interfere with initial virus target cell interactions which involve HIV-1 gp120 V3 determinants, most efficiently for T-cell line-adapted strains. PMID- 8627758 TI - Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro. AB - The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein. PMID- 8627759 TI - Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern. AB - Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) caused excessive disease in infants upon subsequent natural infection with RSV. Recent studies with BALB/c mice have suggested that T cells are important contributors to lung immunopathology during RSV infection. In this study, we investigated vaccine-induced enhanced disease by immunizing BALB/c mice with live RSV intranasally or with FI-RSV intramuscularly. The mice were challenged with RSV 6 weeks later, and the pulmonary inflammatory response was studied by analyzing cells obtained by bronchoalveolar lavage 4 and 8 days after challenge. FI-RSV immunized mice had an increased number of total cells, granulocytes, eosinophils, and CD4+ cells but a decreased number of CD8+ cells. The immunized mice also had a marked increase in the expression of mRNA for the Th2-type cytokines interleukin-5 (IL-5) and IL-13 as well as some increase in the expression of IL 10 (a Th2-type cytokine) mRNA and some decrease in the expression of IL-12 (a Th1 type cytokine) mRNA. The clear difference in the pulmonary inflammatory response to RSV between FI-RSV- and live-RSV-immunized mice suggests that this model can be used to evaluate the disease-enhancing potential of candidate RSV vaccines and better understand enhanced disease. PMID- 8627760 TI - Replication of hepatitis A viruses with chimeric 5' nontranslated regions. AB - The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK 4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow growth phenotype of HAV. PMID- 8627761 TI - Astrovirus ribosomal frameshifting in an infection-transfection transient expression system. AB - Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection-transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell free translation system. Coupling of transcription and translation in a cell-free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting. PMID- 8627762 TI - Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis. AB - The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein. PMID- 8627763 TI - High-dose ocular infection with a herpes simplex virus type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation. AB - We report here that in the rabbit ocular model of herpes simplex virus type 1 (HSV-1) latency, spontaneous reactivation of the HSV-1 ICP34.5 deletion mutant d34.5 increased significantly in response to increasing infectious doses. At the highest infectious dose of d34.5, the spontaneous reactivation rate was indistinguishable from that of wild-type virus (average spontaneous reactivation rates for d34.5, 0.3 to 1.4% at 2 x 10(5) PFU per eye, 3.4% at 2 x 10(6) PFU per eye, and 6.3 to 11.5% at 1 x 10(8) PFU per eye; average spontaneous reactivation rates for marker-rescued virus, 7.7 to 19.6% at 2 x 10(5) PFU per eye). The percentage of latency-associated transcript (LAT) RNA-positive neurons in sections from trigeminal ganglia (TG) of rabbits latently infected with d34.5 demonstrated a similar dose-response effect as estimated by in situ hybridization (0.05% LAT RNA-positive neurons at 2 x 10(5) PFU per eye and 0.1% LAT RNA positive neurons at 1 x 10(8) PFU per eye; P = 0.002). In contrast, even at the highest infectious dose (1 x 10(8) PFU per eye), d34.5 was less virulent (23 of 23 survivors) than the normal infectious dose (2 x 10(5) PFU per eye) of marker rescued virus (14 of 27 survivors; P < 0.0001). In addition, at 1 x 10(8) PFU per eye, d34.5 produced virtually no corneal disease, compared with the production of severe corneal disease by 2 x 10(5) PFU of marker-rescued virus per eye (P < 0.0001). Thus, at increasing infectious doses of d34.5, both spontaneous reactivation and the percentage of neurons expressing LAT appeared to increase, without a corresponding increase in virulence. These results strongly suggest that (i) the phenotypes of neurovirulence and spontaneous reactivation are separable, (ii) the phenotypes of corneal disease and spontaneous reactivation are separable, and (iii) the decreased rate of spontaneous reactivation previously reported for d34.5 (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995) is at least partially due to a reduced rate of establishing latency. PMID- 8627764 TI - Frequent perinatal transmission of feline immunodeficiency virus by chronically infected cats. AB - Vertical transmission of feline immunodeficiency virus (FIV) was studied in cats infected with either of two FIV clinical isolates (FIV-B-2542 or FIV-AB-2771) prior to breeding and conception. Queens infected 4 to 30 months (mean = 14 months) prior to conception transmitted FIV to 59 of 83 (71%) kittens; 50.6% were virus positive on the day of birth. To examine potential routes of FIV transmission from mother to offspring, kittens were delivered via either vaginal or cesarean birth and nursed by either their virus-infected natural mothers or uninfected surrogate mothers. Comparison of FIV infection rates at birth with those at 6 months of age in kittens delivered by cesarean and surrogate raised demonstrated that late in utero transmission occurred in approximately 20% of kittens. Comparison of kittens nursed by FIV mothers with those by uninfected surrogate mothers demonstrated a 13.5% increase in infection rate of kittens exposed to milk-borne virus. Isolation of virus from 40% of maternal vaginal wash samples and the slightly greater infection rate in vaginally versus cesarean delivered surrogate-nursed kittens suggested that intrapartum transmission may occur. In addition, we found that low maternal CD4 count (<200 cells per microl), longer duration of maternal infection (>15 months), and maternal symptoms of clinical immunodeficiency correlated with increased rates of mother-to-kitten FIV transmission, paralleling observations in human immunodeficiency virus-infected women. We conclude that FIV infection provides a model in which to explore aspects of human immunodeficiency virus vertical transmission and intervention difficult to address in human patients. PMID- 8627765 TI - Antiviral cytotoxic T-cell memory by vaccination with recombinant Listeria monocytogenes. AB - Listeria monocytogenes is a facultative intracellular bacterium that is able to escape phagocytic vesicles and replicate in the cytoplasm of infected cells. As with viral vectors, this intracytoplasmic life cycle provides a means for introducing foreign proteins into the major histocompatibility complex class I pathway of antigen presentation. Using recombinant L. monocytogenes (rLM) strains expressing the full-length nucleoprotein (NP) or a single cytotoxic T-lymphocyte (CTL) epitope from lymphocytic choriomeningitis virus (LCMV), we analyzed antiviral CTL responses induced by rLM vaccination. After vaccination, rLM was cleared from the host within 7 days while inducing an LCMV-specific ex vivo CD8+ effector CTL response. Virus-specific CTL memory was maintained for 6 months postvaccination, as demonstrated by vigorous secondary CTL responses after in vitro stimulation. A single immunization with rLM that expressed either the full length NP gene or the CTL epitope alone resulted in LCMV NP-specific CTL precursor frequencies of approximately 1/10(4) CD8+ T cells. A second rLM vaccination resulted in enhanced virus-specific CTL activity and in vitro proliferation. rLM-vaccinated mice were protected against chronic viral infection by an accelerated virus-specific memory CTL response. These mice cleared infectious virus as well as viral antigen, suggesting that sterilizing immunity was achieved. In contrast to mice that received wild-type LM, rLM-vaccinated mice were protected from virally induced immunosuppression and splenic atrophy associated with chronic LCMV infection. The ability to elicit long-term cell mediated immunity is fundamental in designing vaccines against intracellular pathogens, and these results demonstrate the efficacy of recombinant LM vaccination for inducing protective antiviral CTL memory. PMID- 8627767 TI - Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro. AB - Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences. PMID- 8627768 TI - Permanent occupancy of the human immunodeficiency virus type 1 enhancer by NF kappa B is needed for persistent viral replication in monocytes. AB - This work aimed to ascertain the role of kappaB-responsive elements of the human immunodeficiency virus type 1 (HIV-1) enhancer not only in early initiation but also in long-term maintenance of proviral transcription in cells of the monocytic lineage. For this purpose, we used three main approaches. The first was to abruptly terminate tumor necrosis factor-induced NF-kappaB binding to the enhancer sequences in U1 monocytic cells, using a short pulse of exogenous tumor necrosis factor. This resulted in concomitant decrease in nuclear NF-kappaB DNA binding activity and endogenous long terminal repeat transcriptional activity. The second was to suppress the permanent NF-kappaB translocation induced by HIV-1 replication itself in chronically infected U937 cells, using a specific proteasome inhibitor (Z-LLL-H). As early as 2 h after addition of the inhibitor to the culture medium, there was an inhibition of both constitutive activation of NF-kappaB and HIV-1 genome expression. The third approach was to monitor the replication competence in U937 cells of an infectious HIV-1 provirus carrying point mutations in the kappaB-responsive elements of both long terminal repeats. Compared with its wild-type counterpart, this mutated provirus showed a profoundly decreased, Z-LLL-H-insensitive transcriptional and replicative activity in U937 monocytes. Together, our results indicate that occupancy of the viral enhancer by NF-kappaB (p50/p65) heterodimers is required for ongoing transcription of integrated HIV provirus in monocytes, even in cells chronically infected and permanently producing functional HIV Tat protein. Thus, the ability of HIV-1 replication to activate NF-kappaB is crucial to the intense self perpetuated viral transcription observed in cells of the monocytic lineage. PMID- 8627766 TI - Cyclin-dependent kinases phosphorylate the adenovirus E1A protein, enhancing its ability to bind pRb and disrupt pRb-E2F complexes. AB - The adenovirus E1A protein of 243 amino acids has been shown to affect a variety of cellular functions, most notably the immortalization of primary cells and the promotion of quiescent cells into S phase. The activity of E1A is derived, in part, from its association with various cellular proteins, many of which play important roles in regulating cell cycle progression. E1A is known to have multiple sites of phosphorylation. It has been suggested that cell cycle dependent phosphorylation may also control some of E1A's functions. We find now that immune complexes of cyclin-dependent kinases such as cdk4, cdk2, and cdc2 are all capable of phosphorylating E1A in vitro. Additionally, the sites on E1A phosphorylated by these kinases in vitro are similar to the E1A sites phosphorylated in vivo. We have also found that a phosphorylated E1A is far more efficient than an unphosphorylated E1A in associating with pRB and in disrupting E2F/DP-pRB complexes as well. On the basis of our findings and the differences in timing and expression levels of the various cyclins regulating cdks, we suggest that E1A functions at different control points in the cell cycle and that phosphorylation controls, to some extent, its biological functions. PMID- 8627769 TI - The complete DNA sequence and genomic organization of the avian adenovirus CELO. AB - The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions. PMID- 8627770 TI - Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies. AB - Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1 specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV 2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections. PMID- 8627771 TI - Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single chain antibody fragment-envelope fusion proteins. AB - As an approach to cell targeting by retroviruses, the lack of which constitutes one major limitation of retroviral vector technology, we engineered the Moloney murine leukemia virus ecotropic envelope glycoprotein. When inserted between amino acids 6 and 7 of the latter, a single-chain antibody fragment (ScFv) specific for human major histocompatibility complex class I molecules was shown to be able to redefine the tropism of ecotropic Moloney murine leukemia virus derived retroviral particles by allowing infection of major histocompatibility complex class I-positive human cells. At variance with other recently described experimental systems, the type of modification adopted here allowed targeted infection in the absence of coexpressed wild-type env-encoded protein molecules. Interestingly, the chimeric ScFv-env protein also retained the ability to recognize the ecotropic receptor and allowed infection of murine cells, albeit at a reduced efficiency. PMID- 8627772 TI - The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. AB - We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/psi element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/psi region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis. PMID- 8627773 TI - Intracellular posttranslational modifications of S1133 avian reovirus proteins. AB - Avian reovirus S1133 specifies at least 10 primary translation products, eight of which are present in the viral particle and two of which are nonstructural proteins. In the work presented here, we studied the covalent modifications undergone by these translation products in the infected cell. The structural polypeptide mu2 was shown to be intracellularly modified by both myristoylation and proteolysis. The site-specific cleavage of mu2 yielded a large carboxy terminal fragment and a myristoylated approximately 5,500-Mr peptide corresponding to the amino terminus. Both mu2 and its cleavage products were found to be structural components of the reovirion. Most avian reovirus proteins were found to be glycosylated and to have a blocking group at the amino terminus. In contrast to the mammalian reovirus system, none of the avian reovirus polypeptides was found to incorporate phosphorus during infection. Our results add to current understanding of the similarities and differences between avian and mammalian reoviruses. PMID- 8627774 TI - Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41. AB - Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537 10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence. PMID- 8627775 TI - Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome. AB - Two border disease virus (BDV) pairs each consisting of cytopathogenic (cp) and non-cp viruses have been analyzed at the molecular level. Within the NS2-3 (p125) encoding region of both cp viruses, insertions of cellular sequences were identified which were absent in the corresponding non-cp isolates. A comparative sequence analysis revealed that within each pair the cp and non-cp viruses are almost identical. This strongly suggests that the cp BDV isolates developed from the non-cp viruses by RNA recombination between the viral genome and cellular sequences. Nonstructural protein NS3 (p80) was demonstrated after infection with both cp BDV strains. In addition, fusion proteins composed of cellular and viral sequences were identified. In contrast, expression of NS3 and the fusion proteins was not found after infection with the respective non-cp counterparts. PMID- 8627776 TI - Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation. AB - The expression of the rice tungro bacilliform virus open reading frame I was studied in transiently transfected protoplasts. Expression occurs despite the presence of a long leader sequence and the absence of a proper ATG initiation codon. Translation is initiated at an ATT codon. The efficiency of initiation in rice protoplasts depends strongly on the mechanism by which ribosomes reach this codon. From the effects of scanning-inhibiting structures inserted into different leader regions, it can be deduced that this mechanism is related to the ribosome shunt described for cauliflower mosaic virus 35S RNA. The process delivers initiation-competent ribosomes to the region downstream of the leader and is so precise that only the second of two potential start codons only 12 nucleotides apart is recognized. The ATT codon that is used when it is present downstream of the leader is hardly recognized as a start codon by ribosomes that reach it by scanning. PMID- 8627777 TI - Molecularly cloned feline immunodeficiency virus NCSU1 JSY3 induces immunodeficiency in specific-pathogen-free cats. AB - A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1. PMID- 8627778 TI - Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication. AB - Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine. We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene. RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally. Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h. The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus. Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication. Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses. PMID- 8627779 TI - In vivo effects of a recombinant vaccinia virus expressing a mouse mammary tumor virus superantigen. AB - Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg reactive T cells. Stimulated T cells become anergic and are deleted from the T cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response. PMID- 8627780 TI - Bovine herpesvirus 1 U(s) open reading frame 4 encodes a glycoproteoglycan. AB - Sequence analysis of the short unique (Us) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the Us open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the Us ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the Us ORF3 and Us ORF4, whereas the 1.8-kb RNA constitutes the monocistronic Us ORF4 mRNA. To identify the predicted BHV-I gG, recombinant vaccinia virus expressing the Us ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61- and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the Us ORF4 deletion mutant BHV-l/gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N- and O-linked carbohydrates, and that the high-molecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan C (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-I gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino glycosylation, that has not yet been described for a herpesvirus-encoded protein. PMID- 8627781 TI - Modulation of cytokine expression by CD4+ T cells during coxsackievirus B3 infections of BALB/c mice initiated by cells expressing the gamma delta + T-cell receptor. AB - Two variants of coxsackievirus B3 have been used to investigate the pathogenesis of myocarditis in BALB/c mice. H3 virus induces moderate myocarditis and H310A1 virus induces minimal myocarditis, although both viruses infect and replicate in the heart. Cells expressing the gamma delta+ T-cell receptor composed 5 to 13% of the lymphocytes infiltrating the hearts of H3 virus-infected mice and belonged to either the CD4- CD8+ gamma delta+- or CD4- CD8- gamma delta+-cell population. Giving 5,000 gamma delta+ cells isolated from the hearts of H3 virus-infected mice to H310A1 virus-infected recipients restored myocarditis susceptibility in the recipient animals and shifted the pattern of cytokine production in the virus immune CD4+-cell population from being predominantly interleukin-4 producing to being predominantly gamma interferon producing in the H310A1 virus-infected mice. Apoptosis was evident in the infiltrating lymphocyte population in the myocardia of H3 virus-infected mice by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay and in splenic lymphocytes by DNA fragmentation in agarose gel electrophoresis and was confined to the CD4+ population. No apoptosis was observed in H310A1 virus-infected mice, but apoptosis was induced subsequent to gamma delta +-T-cell transfer. These results are consistent with the hypothesis that gamma delta+ T cells may help modulate cytokine responses during virus infections in vivo and that apoptosis might be involved in this modulation. PMID- 8627782 TI - Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques. AB - Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact. PMID- 8627783 TI - Regulation of avian leukosis virus long terminal repeat-enhanced transcription by C/EBP-Rel interactions. AB - The avian leukosis and sarcoma virus long terminal repeat (LTR) enhancers feature directly repeated CCAAT/enhancer element sequences which are also found in many viral and cellular gene enhancers. While most members of the CCAAT/enhancer element-binding protein (C/EBP) transcription factor family exhibit tissue restricted expression, there may be ubiquitously expressed C/EBP-like factors that regulate widespread CCAAT/enhancer element-driven transcription. An avian C/EBP-related factor designated Al/EBP was previ- ously shown to bind CCAAT/enhancer elements within the avian leukosis virus (ALV) and Rous sarcoma virus (RSV) LTR enhancers in a pattern identical to that of a B-cell LTR-binding factor (W. J. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). An Al/EBP specific antiserum recognizes a 40-kDa LTR CCAAT/enhancer element-binding protein purified from avian B lymphoma cells. A1/EBP is widely expressed at the mRNA and protein levels, suggesting that this protein could be important not only in regulating widespread expression of the AIN and RSV retroviruses but also in controlling the expression of other viral and cellular gene enhancers that possess CCAAT/enhancer motifs. We also found that an NF-KB/Rel-related protein is a component of the LTR CCAAT/enhancer element binding complex through its interaction with A1/EBP. At least one of the NF-kappaB family members, p65 (RelA), is capable of activating LTR CCAAT/enhancer element-driven transcription. These findings suggest a role for Rel-related factors in the regulation of AIN or RSV LTR-driven transcription via an interaction with Al/EBP. PMID- 8627784 TI - Tyrosine kinase-dependent release of an adenovirus preterminal protein complex from the nuclear matrix. AB - Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) through the interaction of Ad preterminal protein (pTP). The NM is a highly salt-resistant fibrillar network which is known to anchor transcription, mRNA splicing, and DNA replication complexes. Incubation of rATP with NM to which pTP was bound caused the release of pTP as a pTP-NM complex with a size of 220 to 230 kDa; incubation with 5' adenylylimidodiphosphate (rAMP-PNP) showed no significant release, indicating that rATP hydrolysis was required. With NM extracts, it was shown that a pTP-NM complex which was capable of binding Ad origin DNA could be reconstituted in vitro. A number of high-molecular-weight NM proteins ranging in size from 120 to 200 kDa were identified on Far Western blots for their ability to bind pTP. rATP-dependent release of pTP from the NM was inhibited in a dose-dependent fashion by the addition of tyrosine kinase inhibitors, such as quercetin, methyl-2,5-dihydroxycinnamate, or genistein. NM mediated phosphorylation of a poly(Glu, Tyr) substrate was also significantly abrogated by the addition of these compounds. rATP-dependent release of Ad DNA termini bound to the NM via pTP was also blocked by the addition of these inhibitors. These results indicate that a tyrosine kinase mechanism controls the release of pTP from its binding sites on the NM. These data support the concept that phosphorylation may play a key role in the modulation of pTP binding sites on the NM. PMID- 8627785 TI - The amino-terminal domains of Epstein-Barr virus nuclear proteins 3A, 3B, and 3C interact with RBPJ(kappa). AB - The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. We now show that EBNA3A and EBNA3B expressed in non-EBV infected Burkitt tumor lymphoblasts are similar to EBNA3C in binding to glutathione S-transferase-RBPJkappa in vitro and in coimmunoprecipitating from cell lysates with antibody to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis. PMID- 8627786 TI - Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. AB - Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotype-specific antibody-secreting cells (ASC were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirus-inoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease. PMID- 8627787 TI - Brucella abortus conjugated with a peptide derived from the V3 loop of human immunodeficiency virus (HIV) type 1 induces HIV-specific cytotoxic T-cell responses in normal and in CD4+ cell-depleted BALB/c mice. AB - We have previously shown that immunization of mice with human immunodeficiency virus (HIV)-derived proteins or peptides conjugated to inactivated Brucella abortus induces the secretion of virus-neutralizing antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype. In addition, B. abortus activates human CD4+ and CD8+ cells to secrete gamma interferon. Since these are both characteristics of a Th1-type immune response, which is associated with the development of cell-mediated immunity, it was important to determine if B. abortus conjugates would also act as a carrier to induce a cytotoxic T-lymphocyte (CTL) response. To test this hypothesis, we conjugated an 18-amino-acid peptide from the V3 loop of the MN strain of HIV-1 gp120 that contains both B- and cytotoxic T-cell epitopes to B. abortus (B. abortus-MN 18-mer). A 10-amino-acid fragment of this peptide has been shown to be the minimal CTL determinant presented by murine H-2Dd. It was found that two in vivo immunizations with 10(8) organisms of B. abortus-MN 18-mer followed by in vitro stimulation with peptide induced a virus-specific CTL response. Conjugation to B. abortus was required for in vivo priming, since there was no induction of memory CTLs when B. abortus was only mixed with peptide. Targets pulsed with peptide as well as those infected with a vaccinia virus encoding HIV gp160 were killed, demonstrating recognition of naturally processed envelope. Also, major histocompatibility complex incompatible L cells which were infected with vaccinia viruses that encoded H 2Dd, but not H-2Kd, and pulsed with peptide were lysed. This demonstrated the appropriate major histocompatibility complex class I restriction. Treatment of the mice with anti-L3T4 prior to immunization caused a severe depletion of CD4+ lymphocytes, yet it did not decrease the CTL priming. Thus, inactivated B. abortus can induce non-CD4+ cells to produce the cytokines required for CTL induction. We conclude that B. abortus stimulates a cellular as well as a humoral immune response, even in the relative absence of CD4+ helper cells. It may be a particularly useful vaccine carrier in HIV-1-infected individuals or others with impaired CD4+ T-cell function. PMID- 8627788 TI - A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity. AB - A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line. PMID- 8627789 TI - Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. AB - To explore the mechanism of sexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared HIV-1 gp120 sequences in longitudinal samples from five acute seroconvertors with those from their corresponding sexual partners (transmitters). We used a quantitative homoduplex tracking assay to compare the overall genetic composition of HIV-1 quasispecies in each transmission pair and to track the transmitted viruses during the acute and asymptomatic stages of HIV 1 infection. In the chronically infected transmitters, HIV-1 variants in genital secretions differed from those in blood and variants in cells differed from those in cell-free plasma, indicating remarkable sequence heterogeneity in these subjects as well as compartmentalization of the virus in different bodily sites. Conversely, two of five seroconvertors had only a few related variants and three of five harbored only one viral population, indicating that in these subjects the transmitted viruses were typically homogeneous. Transmitted viruses were evident in the donor's seminal plasma (one of five cases) and even more so in their seminal cells (three of five cases), suggesting that both cell-associated and cell-free viruses can be transmitted. In every pair studied, the transmitted variant(s) represents only a minor population in the semen of the corresponding transmitter, thereby providing evidence that HIV-1 selection indeed occurs during sexual transmission. PMID- 8627790 TI - Identification of amino acids involved in recognition by dengue virus NS3 specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones. AB - The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3 specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells. PMID- 8627791 TI - Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappaB and Sp1 binding elements. AB - Ten mutants of the simian immunodeficiency virus (SIV) SIVmac239 bearing deletions (delta) or substitutions (subst) in the NF-kappaB and/or Sp1 binding elements were created, and the replicative capacities of the mutants were analyzed. All mutants, including one extensively mutagenized strain entirely missing the NF-kappaB and four Spl binding elements, replicated with wild-type kinetics and to a wild-type level in peripheral blood mononuclear cell cultures in 50 to 100% of the experiments. One group of mutants replicated very similarly to SIVmac239 in kinetics and yield in CEMxl74 cells (2xNFKappaB > or = SlVmac239 approximately deltaNFkappaB approximately deltaSpl234 approximately substNFkappaB approximately substSpl2 approximately substSp23), while a second group replicated with delayed or slightly delayed kinetics in CEMxl74 cells (SIVmac239 > substSp34 > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSp1 > substSpl234). Reversions or additional mutations were not detected in the U3 and R regions of proviral DNA from CEMxl74 cells infected with the SIVmac239 mutants. Similar results were obtained when mutants of SIVmacMER (a macrophage-competent derivative of SIVmac239) were tested in peripheral blood mononuclear cell and CEMx174 cultures. However, the growth of most mutated viruses was suppressed in primary rhesus monkey alveolar macrophages (SIVmacMER approximately 2xNFkappaB approximately substNFkappaB > deltaNFkappaB > deltaNFkappaBdeltaSpl234 approximately deltaNFkappaBdeltaSpl > deltaSpl234 approximately substSpl2 > substSp23 approximately substSp34 approximately substSpl234 > or = SIVmac239). Thus, changes in the Sp1 binding sites had the most dramatic effects on SIVmac replication in primary macrophage cultures. Analysis of long terminal repeat driven secreted alkaline phosphatase activity in transient assays showed that, unlike human immunodeficiency virus type 1, the SIV long terminal repeat possesses an enhancer region just upstream of the NF-kappaB element which maintains significant levels of basal transcription in the absence of NF-kappaB and Sp1 sites. This region is responsive to transactivation by Tat. In addition, the SIV TATA box was shown to be stronger than that of human immunodeficiency virus type 1. Therefore, the surprisingly high replicative capacity of NF-kappaB and Sp1 binding site mutants of SIVmac is due to unique features or the enhancer/promoter region. PMID- 8627792 TI - Intratype variation in 12 human papillomavirus types: a worldwide perspective. AB - In this study, we have examined intratype human papillomavirus (HPV) sequence variation in a worldwide collection of cervical specimens. Twelve different HPV types including HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM9/PAP238A (recently designated HPV-73), and a novel partial genomic HPV sequence designated MM4/Wl3B were analyzed in this study. Cervical specimens were collected as part of epidemiological investigations conducted in New Mexico and an international study of invasive cervical cancer (IBSCC). Specimens from several countries including Argentina, Brazil, Bolivia, Benin, Cuba, Colombia, Chile, Germany, Mali, Panama, Paraguay, Spain, Algeria, Uganda, Guinea, Tanzania, Indonesia, Philippines, Thailand, and the United States were evaluated. Specimen DNAs were subjected to amplification with the MY09/11 L1 consensus PCR system. The PCR products were cloned, and an approximately 410-bp region in the L1 open reading frame was sequenced from 146 specimens (approximately 60,000 bp). Within a single HPV type, nucleotide diversity varied between 0.2 and 2.9% (i.e., between any pair of variants) and the majority of nucleotide changes were synonymous (amino acid conserving). These data provide information pertinent to HPV diagnostic probe development and are potentially relevant to future rational vaccine strategies. Similarly, amino acid diversity varied between 0 and 5.1%. Some of these amino acid changes may represent markers of intertype evolutionary relationships. Presuming that HPVs have evolved under the same constraints as their corresponding hosts, the limited genetic diversity observed for all HPVs studied to date may reflect an evolutionary bottleneck occurring in both virus and host populations. PMID- 8627793 TI - Quantitation of herpes simplex virus type 1 DNA and latency-associated transcripts in rabbit trigeminal ganglia demonstrates a stable reservoir of viral nucleic acids during latency. AB - In this investigation we determined the dynamics of herpes simplex virus type 1 (HSV-1) DNA and latency-associated transcripts (LAT) in the latently infected rabbit trigeminal ganglion. Rabbit eyes were infected with either the McKrae strain or the l7Syn+ strain of HSV-1. Rabbits were sacrificed between 5 and 360 days after infection and their trigeminal ganglia were analyzed for the number of HSV DNA genomes and the number of neuronal cells expressing LAT. There was no statistically significant change in the number of HSV genomes or the number of neuronal cells expressing LAT in these ganglia between 20 and 360 days after infection. For both strains, the amount of HSV DNA averaged 16.8 genomes per 100 cells, and 9.2% of the neurons expressed LAT. There were 17 to 34 HSV genomes per LAT-expressing neuronal cell. The number of LAT-expressing neurons did not change over the 360 days. Spontaneous reactivation (HSV-1 recovery in tear film) and recurrence (HSV-1-specific epithelial lesions) occurred during the period of this study; however, these events did not alter the quantity of HSV-1 DNA or the number of LAT-expressing cells. These results suggest that after the latent infection is established, the viral DNA in the ganglia does not replicate to any measurable extent over long periods of latency, since no significant change in the number of HSV genomes occurs. The results also suggest that only a very small number of latently infected neuronal cells are needed to produce infectious HSV-1 during reactivation. PMID- 8627794 TI - Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins. AB - A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells. PMID- 8627795 TI - Adenovirus-mediated expression of ribozymes in mice. AB - Ribozymes are a new pharmaceutical class of reagents that offer potential in treating a number of different medical disorders, including infectious diseases and cancer. As a first step towards using ribozymes for the treatment of liver disorders such as viral hepatitis, adenovirus vectors that contain a ribozyme expression cassette under the control of different promoters directed against human growth hormone (hGH) were constructed and infused into transgenic mice that produce hGH from the gastrointestinal tract and liver. Adenovirus-mediated transfer of expressed ribozymes resulted in up to a 96% reduction of hepatic hGH mRNA over a period of several weeks in the transgenic mouse model. Furthermore, the concentration of ribozyme RNA correlated with the degree of hGH mRNA reduction. This study clearly demonstrates that ribozymes can function during the period of expression in an intact organ after somatic gene transfer. PMID- 8627796 TI - Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome. AB - Ovine pulmonary carcinoma (OPC) is a contagious lung cancer of sheep that is presumed to be caused by an exogenous retrovirus of sheep, jaagsiekte sheep retrovirus (JSRV). The sheep genome carries 15 to 20 copies of endogenous sheep retrovirus (ESRV) loci that hybridize to JSRV DNA probes. In order to clarity the etiologic roles of ESRV and an exogenous JSRV-like retrovirus (exJSRV) in OPC, we assessed sequence differences between ESRV and JSRV. Molecular characterization of six ESRV loci revealed restriction sites specific for JSRV. Nucleotide sequences of ESRVs from sheep of different breeds were similar to those of JSRV in structural genes but divergent in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for use in the PCR. Of 13 tumor DNAs tested by PCR with these exogenous-virus U3 primers, 8 produced DNA fragments that hybridized with the JSRV gag probe, but neither lung DNAs from healthy sheep nor DNAs from nontumor tissues of diseased sheep produced similar DNA fragments. exJSRV PCR products from tumor DNAs of sheep with OPC from three continents had restriction profiles similar to each other but different from those of ESRVs upon digestion with EcoRI, HindIII, NdeI, KpnI, and ScaI. These exjSRVs could be classified into two genotypes according to U3 sequences and restriction profiles. U3 sequences of exJSRV proviruses in tumors strongly resembled those of JSRV but differed from those of ESRVs, suggesting that exJSRVs, rather than ESRVs, are primarily associated with oncogenesis in OPC. PMID- 8627797 TI - Branched structures in the intracellular DNA of herpes simplex virus type 1. AB - Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions. PMID- 8627798 TI - PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms. AB - Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms. PMID- 8627799 TI - Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques. AB - By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1. PMID- 8627800 TI - An env gene derived from a primary human immunodeficiency virus type 1 isolate confers high in vivo replicative capacity to a chimeric simian/human immunodeficiency virus in rhesus monkeys. AB - To explore the roles played by specific human immunodeficiency virus type 1 (HIV 1) genes in determining the in vivo replicative capacity of AIDS viruses, we have examined the replication kinetics and virus-specific immune responses in rhesus monkeys following infection with two chimeric simian/human immunodeficiency viruses (SHIVs). These viruses were composed of simian immunodeficiency virus SIVmac239 expressing HIV-1 env and the associated auxiliary HIV-1 genes tat, vpu, and rep. Virus replication was assessed during primary infection of rhesus monkeys by measuring plasma SIVmac p27 levels and by quantifying virus replication in lymph nodes using in situ hybridization. SHIV-HXBc2, which expresses the HIV-1 env of a T-cell-tropic, laboratory-adapted strain of HIV-1 (HXBc2), replicated well in rhesus monkey peripheral blood leukocytes (PBL) in vitro but replicated only to low levels when inoculated in rhesus monkeys. In contrast, SHIV-89.6 was constructed with the HIV-1 env gene of a T-cell- and macrophage-tropic clone of a patient isolate of HIV-1 (89.6). This virus replicated to a lower level in monkey PBL in vitro but replicated to a higher degree in monkeys during primary infection. Moreover, monkeys infected with SHIV 89.6 developed an inversion in the PBL CD4/CD8 ratio coincident with the clearance of primary viremia. The differences in the in vivo consequences of infection by these two SHIVs could not be explained by differences in the immune responses elicited by these viruses, since infected animals had comparable type specific neutralizing antibody titers, proliferative responses to recombinant HIV 1 gp120, and virus-specific cytolytic effector T-cell responses. With the demonstration that a chimeric SHIV can replicate to high levels during primary infection in rhesus monkeys, this model can now be used to define genetic determinants of HIV-1 pathogenicity. PMID- 8627801 TI - The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice. AB - The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host. PMID- 8627802 TI - Developmental analysis of the cytomegalovirus enhancer in transgenic animals. AB - The major immediate-early promoter (MIEP) of human, cytomegalovirus (HCMV) constitutes a primary genetic switch for viral activation. In this study, regulation of the enhancer-containing segment (nucleotides -670 to +54) of the HCMV MIEP attached to the 1acZ reporter gene was examined in the developing embryos of transgenic mice to identify temporal and tissue-specific expression. We find that the transgene reporter is first detected as a dorsal stripe of expression in the neural folds of embryos at day 8.5 postcoitum (p.c.). A broad expression pattern is exhibited in embryos at day 9.5 p.c. This pattern becomes more restricted by day 10.5 p.c. as organogenesis progresses. By day 14.5 p.c., prominent expression is observed in a subpopulation of central nervous system cells and spinal ganglia, endothelial cells, muscle, skin, thyroid, parathyroid, kidney, lung, liver, and gut cells, and the pancreas and submandibular and pituitary glands. This distribution pattern is discussed in relation to human congenital HCMV infection. These results suggest that the transcriptional activity of the HCMV MIEP may determine in part, the ability of the virus to specifically target developing fetal tissues in utero. PMID- 8627803 TI - Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. AB - The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols. PMID- 8627804 TI - In vivo model of adeno-associated virus vector persistence and rescue. AB - Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206 212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue. PMID- 8627805 TI - Subcellular localization of Aleutian mink disease parvovirus proteins and DNA during permissive infection of Crandell feline kidney cells. AB - Confocal microscopy allowed us to localize viral nonstructural (NS) and capsid (VP) proteins and DNA simultaneously in cells permissively infected with Aleutian mink disease parvovirus (ADV). Early after infection, NS proteins colocalized with viral DNA to form intranuclear inclusions, whereas VP proteins formed hollow intranuclear shells around the inclusions. Later, nuclei had irregular outlines and were virtually free of ADV products. In these cells, inclusions of viral DNA with or without associated NS protein were embedded in cytoplasmic VP protein. These findings implied that ADV replication within an infected cell is regulated spatially as well as temporally. PMID- 8627806 TI - A novel form of hepatitis delta antigen. AB - Hepatitis delta virus (HDV) is known to express a protein termed the small delta antigen, a structural protein which is also essential for genome replication. During replication, posttranscriptional RNA editing specifically modifies some of the HDV RNA, leading to the production of an elongated form of the delta antigen, the large form, which is essential for virus assembly. The present study showed that yet another form of HDV protein is expressed during genome replication. This novel form is not produced in all infected cells, but it arises during replication in transfected cells and in infected woodchucks, and as was previously reported, patients infected with HDV do make antibodies directed against it. These findings are an indicator of the complexity of gene expression during HDV infection and replication. PMID- 8627807 TI - Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein. AB - Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H 2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus. PMID- 8627808 TI - SFA-1, a novel cellular gene induced by human T-cell leukemia virus type 1, is a member of the transmembrane 4 superfamily. AB - A novel cellular gene termed SFA-1 was isolated by differential hybridization of a cDNA library, using probes obtained from an adult T-cell leukemia cell line in comparison with probes obtained from normal CD4+ T cells and the MOLT-4 cell line. The mRNA of the SFA-1 gene is approximately 1.6 kb in size and encodes a protein of 253 amino acids, containing four putative transmembrane domains, a number of cysteine residues, and one potential N-glycosylation site in a major hydrophilic region between the third and fourth transmembrane domains. Expression of the SFA-1 gene was either absent or present at a low level in lymphoid cells but was up-regulated after transformation by human T-cell leukemia virus type 1 and transactivated by Tax. SFA-1 was broadly expressed in many human cell types and conserved in different species. Computer-aided comparison showed that SFA-1 had significant sequence homology and common structural features with members of the transmembrane 4 superfamily. SFA-1 antigen was detected as a 29-kDa membrane protein by immunoblotting, using anti-SFA-1 monoclonal antibody. PMID- 8627809 TI - Cytokine production in the immune response to murine gammaherpesvirus 68. AB - Cytokine profiles were determined following intranasal infection of C57BL/6J mice with murine gammaherpesvirus 68 (MHV-68). Spleen, mediastinal, and cervical lymph node cells from infected mice produced high levels of interleukin 6 (IL-6) and gamma interferon (IFN-gamma) and lower levels of IL-2 and IL-10 following in vitro restimulation. Little or no IL-4 or IL-5 was detected. Cytokine production was generally maximal at 10 days after infection, correlating with viral clearance from the lung, although significant levels were seen as early as 3 days after administration of the virus. In vitro infection of naive splenocytes induced B-cell- dependent secretion of IL-6 and IL-10, whereas IFN-gamma and IL-2 were produced only by cells that had been primed in vivo. Depletion of B lymphocytes from primed splenocyte populations did not, however, abrogate IL-6 and IL-10 production. Highly purified immune T cells made IL-6, IL-10. and IFN gamma following in vitro restimulation with MHV-68. Thus, IL-6 and IL-10 are components of both the acquired and the innate host response. These cytokines have potential roles in the establishment and maintenance of persistent infection. PMID- 8627810 TI - Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. AB - Transfection of the E6 and E7 genes of the high-risk human papillomaviruses (HPVs) into primary genital keratinocytes generates colonies of proliferating cells which are resistant to calcium- and serum-induced terminal differentiation. To genetically map the HPV gene(s) responsible for this cellular phenotype, we cloned cDNAs for full-length E6, full-length E7, four truncated E6 splice variants (E6I to E6IV), and a series of E6 C-terminal truncation mutants into a simian virus 40 expression vector. The E6 proteins were tagged with the AU1 epitope at the C terminus to verify their expression in COS cells by immunoprecipitation and immunofluorescence. Transfection of the full-length E6 protein, either wild type or epitope tagged, induced calcium- and serum-resistant keratinocyte colonies, but the truncated E6 variants and full-length E7 protein did not. E6-induced colonies, while altered in response to serum and calcium, could not be established into cell lines without the combined presence of the E7 protein, further exemplifying the independent roles of E6 and E7 in cell differentiation and cell proliferation. The E6 C-terminal deletion mutants defined two distinct functional domains between amino acids 120 and 151. Amino acids 120 to 151 contained an apparent bipartite nuclear localization signal, whereas amino acids 132 to 141 were required for the induction of resistance to calcium- and serum-induced differentiation and for immortalization of human keratinocytes in conjunction with E7. PMID- 8627811 TI - Repression of the NF1 gene by Tax may expain the development of neurofibromas in human T-lymphotropic virus type 1 transgenic mice. AB - In familial neurofibromatosis type 1 (NF1), individuals with a germ line transmitted NF1 mutation develop multiple neurofibromas. To explain the observation that transgenic mice expressing the human T-lymphotropic virus type 1 (HTLV-1) tax gene under the control of the viral regulatory element also develop multiple neurofibromas, we demonstrate that the Tax trans-regulator can functionally repress NF1 gene expression through a cis-acting element located immediately upstream of its transcriptional start site, thereby allowing the development of benign neurofibromas without the need for direct mutations in NF1. We propose that such a mechanism would suffice to epigenetically alter NF1 gene expression. The fact that transgenic animals have localized rather than diffuse neurofibroma formation, however, suggests that additional genetic or epigenetic events may be required for neurofibroma formation. PMID- 8627812 TI - Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals. AB - Epstein-Barr virus infects resting B cells in vitro and activates them to continuously proliferating lymphoblasts. Activation is essential for the virus to convert its linear genome to the covalently closed circular episomal form in which it persists in proliferating cells. However, in vivo, Epstein-Barr virus persists in resting B cells. We found that in these cells also the virus is present as an episome, suggesting that the cells must, at some time, have been activated and then returned to a resting state. This is the first direct demonstration, for any herpesvirus, of this form of the viral genome in normal persistently infected tissue. Since no linear viral DNA was detected, we estimate that fewer than 1 in 40 cells replicates the virus in the peripheral blood of healthy donors. PMID- 8627813 TI - Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition. AB - Intracellular immunization to target the human immunodeficiency virus type 1 (HIV 1) regulatory protein Rev has been explored as a genetic therapy for AIDS. Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-1 replication have been previously reported by our laboratories. To further understand the molecular mechanisms and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D1O), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety. These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to after the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS. PMID- 8627814 TI - Assessment of the serological relatedness of genital human papillomaviruses by hemagglutination inhibition. AB - To assess the potential for cross-protection among genital human papillomavirus (HPV) types in virus-like particle (VLP)-based vaccinations, inhibition of HPV VLP-mediated hemagglutination by rabbit antisera raised against HPV type 6b (HPV 6b), HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 was analyzed. Only highly homologous types (HPV-6b and HPV-11, and HPV-18 and HPV-45) exhibited detectable serological cross-reaction for the class of antibodies that inhibit virion-to cell surface binding. However, analysis of neutralizing monoclonal antibodies to several animal and human papillomaviruses indicated that over half of these antibodies do not prevent cell surface binding, but these latter antibodies do not appear to be more cross-reactive in enzyme-linked immunosorbent assays than those that mediate inhibition of hemagglutination. The data strongly suggest that while there may be limited cross-protection between highly (>85% L1 amino acid identity) homologous types, protection by HPV VLP-based vaccines will be predominantly type specific. PMID- 8627815 TI - Human endogenous retrovirus K10 encodes a functional integrase. AB - We cloned a human endogenous retrovirus K1O DNA fragment encoding integrase and expressed it as a fusion protein with Escherichia coli maltose-binding protein. Integrase activities were measured in vitro by using a double-stranded oligonucleotide as a substrate mimicking viral long terminal repeats (LTR). The fusion protein was highly active for both terminal cleavage and strand transfer in the presence of Mn2+ on the K1O LTR substrate. It was also active on both Rous sarcoma virus and human immunodeficiency virus type 1 LTR substrates, whereas Rous sarcoma virus and human immunodeficiency virus type 1 integrases were active only on their corresponding LTR substrates. The results strongly suggest that K1O encodes a functional integrase with relaxed substrate specificity. PMID- 8627816 TI - Structure of the 3' terminus of the hepatitis C virus genome. AB - Hepatitis C virus (HCV), a positive-strand RNA virus, has been considered to have a poly(U) stretch at the 3' terminus of the genome. We previously found a novel 98-nucleotide sequence downstream from the poly(U) stretch on the HCV genome by primer extension analysis of the 5' end of the antigenomic-strand RNA in infected liver (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215: 744-749, 1995). Here, we show that the novel sequence is a highly conserved 3' tail of the HCV genome. We repeated primer extension analyses with four HCV-infected liver samples and found the 98-nucleotide sequence in all the samples. Furthermore, experiments in which RNA oligonucleotide was ligated to the 3' end of the HCV genome existing in infectious serum revealed nearly identical 3' termini with no extra sequence downstream from the 98-nucleotide sequence, suggesting that this sequence is the tail of the HCV genome. This tail sequence was highly conserved among individuals and even between the two most genetically distant HCV types, II/1b and III/2a. Computer modeling predicted that the tail sequence can form a conserved stem-and-loop structure. These results suggest that the novel 3' tail is a common structure of the HCV genome that plays an important role in initiation of genomic replication. PMID- 8627817 TI - Analysis of Rous sarcoma virus Gag protein by mass spectrometry indicates trimming by host exopeptidase. AB - We have used electrospray ionization-mass spectrometry to investigate Gag protein structure and processing in Rous sarcoma virus, the prototype of the avian sarcoma and leukemia viruses. Molecular masses determined for the mature virion proteins MA, CA, NC, and PR agree closely with those predicted by currently accepted models for their structures. However, the data for p10 imply that only about 10% of the product has the predicted mass while the remainder is missing the C-terminal methionine residue. Molecular masses also were obtained for products generated by PR cleavage in vitro of a Gag precursor polyprotein expressed in Escherichia coli. The data confirm the predicted Gag cleavage sites for PR. Thus, carboxypeptidase activity appears to be responsible for generating the des-Met form of p10. The same activity may account for the small amount of the mature des-Met CA, as previously reported. Analysis of cleavage products generated in vitro also serves to define the PR processing site separating the p2a and p2b peptides, Asn-164-Cys-165. In conjunction with published characterizations of these two peptides processed from the segment of Gag between MA and p10, these data suggest trimming of p2b by an aminopeptidase. Finally, the molecular masses determined for the MA-related species p19f, p23, and p35 now accurately define the structures of these proteins. PMID- 8627818 TI - Hybrid proteins between Pseudomonas aeruginosa exotoxin A and poliovirus 2Apro cleave p220 in HeLa cells. AB - Cleavage of p220, a component of the initiation factor eIF-4F, has been correlated with the inhibition of host translation during poliovirus infection. To obtain p220 cleavage in the absence of any other poliovirus gene products, hybrid proteins containing Pseudomonas aeruginosa exotoxin A and poliovirus protease 2Apro have been constructed. The addition of the hybrid molecules to cultured cells did not lead to substantial p220 cleavage. However, the simultaneous presence of the hybrid toxin with replicationally inactive chicken adenovirus particles results in efficient cleavage of p220 in the intact cells. Under these conditions, cellular translation continues unabated for several hours, arguing against a direct requirement for intact p220 in each round of the initiation of translation of cellular mRNAs. PMID- 8627819 TI - Characterization of long-term cultures of hepatitis C virus. AB - The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted. PMID- 8627820 TI - Reviews of chromosome studies in urological tumors. III. Cytogenetics and genes in testicular tumors. AB - PURPOSE: We reviewed available cytogenetic and molecular findings in testicular germ cell tumors, and their possible application to clinical, pathological and basic parameters. MATERIALS AND METHODS: Findings in the literature on testicular germ cell tumors as well as those from our laboratory were summarized, including a listing of the cytogenetic findings on testicular germ cell tumors to date with some illustrations. RESULTS: Testicular germ cell tumors are characterized in most cases by the presence of an i(12p) isochromosome. In tumors without such an abnormal chromosome studies using fluorescence in situ hybridization and molecular approaches have demonstrated either masking of the i(12p) or the presence of extra 12p sequences in the karyotype. Although testicular germ cell tumors are often associated with chromosome changes in addition to the i(12p), no other specifically recurrent structural chromosome changes have been found. Based on the cytogenetic and molecular findings in testicular germ cell tumors, a hypothetical scheme for the genetic events leading to these tumors is presented. CONCLUSIONS: The genetic events leading to genesis of testicular germ cell tumors in men appear to be related to aneuploidization followed by the formation of an i(12p) isochromosome, the latter characterizing the preponderant number of testicular germ cell tumors. The exact role of the i(12p) isochromosome in testicular germ cell tumor pathogenesis remains to be determined, as is true of the genes involved in or affected by these tumors. Based on presently available information, a hypothetical pathogenetic and oncogenetic model for the development of testicular germ cell tumors is presented. PMID- 8627821 TI - Preoperative reverse transcriptase polymerase chain reaction for prostate specific antigen predicts treatment failure following radical prostatectomy. AB - PURPOSE: We previously demonstrated than an enhanced reverse transcriptase polymerase chain reaction assay for prostate specific antigen (PSA) can predict final pathological stage in radical prostatectomy patients. The potential role of the assay in predicting serum PSA recurrence after radical prostatectomy was explored. MATERIALS AND METHODS: We evaluated 100 radical prostatectomy candidates by reverse transcriptase polymerase chain reaction preoperatively, and status was compared to serum PSA, Gleason score and final pathological results. Potential surgical failure was defined as tumor at the surgical margin or extending into the seminal vesicle. Patients were monitored postoperatively by serum PSA every 4 months. Kaplan-Meier analysis was used to evaluate the correlation between reverse transcriptase polymerase chain reaction and disease recurrence, defined as a PSA of 0.2 ng/ml. or greater. RESULTS: Enhanced reverse transcriptase polymerase chain reaction for PSA had a stronger correlation with potential surgical failure than preoperative serum PSA or Gleason score (relative risks 15.2, 5.9 and 3.2, respectively). The correlation between these modalities and PSA recurrence was evaluated during a mean followup of 13.6 months (range 5 to 26). Of 36 patients with positive reverse transcriptase polymerase chain reactions 9 had failure by PSA compared to 3 of 64 (4.7%) with negative polymerase chain reactions (p<0.0286). The relative risk for failure by reverse transcriptase polymerase chain reaction was 3.6. Gleason score and serum PSA had higher correlations with postoperative PSA elevations (relative risk 13.2 and 7.6, respectively). A Cox regression analysis model demonstrated that reverse transcriptase polymerase chain reaction for PSA can be used in conjunction with Gleason score and provides statistically significant risk information. CONCLUSIONS: Enhanced reverse transcriptase polymerase chain reaction for PSA is a statistically significant predictor of potential failure by pathological analysis and of disease recurrence by PSA. Longer followup data are required to define further the role of the assay in the management of patients with prostate cancer. PMID- 8627822 TI - Urolithiasis and cystic fibrosis. AB - PURPOSE: We determined whether cystic fibrosis patients are at increased risk for the development of urolithiasis. MATERIALS AND METHODS: Telephone interviews were conducted with 201 cystic fibrosis patients older than 15 years to identify those with a history of urolithiasis. Histories were confirmed by chart review, and only patients with urolithiasis documented by excretory urography, renal ultrasound or spontaneous passage of a stone were included. RESULTS: A total of 11 patients (5.5%) had documented urolithiasis, with 2 or more episodes in 7. Mean age at initial episode was 27.0 years (range 19 to 33). CONCLUSIONS: Cystic fibrosis patients may be at increased risk for urolithiasis compared to age specific prevalence rates for stone disease in the general population. PMID- 8627823 TI - New stone formation: a comparison of extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy. AB - PURPOSE: There is theoretical concern that stone recurrence rates may be higher following extracorporeal shock wave lithotripsy (ESWL) compared to other techniques because of residual stone debris. MATERIALS AND METHODS: We documented all new stone formations in 298 consecutive patients who initially achieved a stone-free status following ESWL for renal calculi less that 2 cm in largest dimension, and compared the findings to those of 62 patients treated with percutaneous nephrolithotomy without ultrasonic fragmentation. Stone-free status was assessed by a centrally reviewed plain abdominal film and renal tomograms at 3 months. A plain abdominal film was repeated at 12 and 24 months to detect recurrence. RESULTS: New stones formed in 22.2% of patients after ESWL and 4.2% after percutaneous nephrolithotomy at 1 year (p = 0.004), and in 34.8% versus 22.6%, respectively, at 2 years (p =0.190). Furthermore, more new stones recurred in the lower and mid calices compared to baseline location in the ESWL group (chi square <0.0001), which was not observed in the percutaneous nephrolithotomy group. CONCLUSIONS: Our data support a trend toward higher stone recurrence rates in ESWL treated patients, which may be due to microscopic sand particles migrating to dependent calices and acting as a nidus for new stone formation. PMID- 8627824 TI - Modified ureteral stripping as an alternative to open surgical ureterectomy. AB - PURPOSE: Using a compression technique instead of the classical invagination technique in cases of total nephroureterectomy improves traction during transurethral stripping of the ureteral stump. MATERIALS AND METHODS: Eight patients underwent our modified compression technique. After nephrectomy a kinked 5F ureteral catheter is attached to the ureteral stump with a double ligation. Traction on the ureteral catheter is used to achieve ureteral compression. RESULTS: There were no intraoperative difficulties except for 1 dislodged ureteral catheter. No complications were associated with ureteral detachment or resection. CONCLUSION: Our technique offers an alternative to the classic invagination technique by increasing transurethral traction on the ureteral stump, while substantially decreasing the risk of ureteral catheter dislodgment. PMID- 8627825 TI - The use of transureteroureterostomy in the management of complex ureteral problems. AB - PURPOSE: Transureteroureterostomy has primarily been performed for benign disease in children with reflux or undergoing undiversion, and in adults with lower ureteral injury. We report the use of transureteroureterostomy for other than these traditional indications, including malignant disease. MATERIALS AND METHODS: Transureteroureterostomy was performed in 6 patients at the time of tumor resection to bypass large ureteral defects and in 4 as a secondary operation, usually associated with a ureteral leak after previous surgery. RESULTS: Complications related to transureteroureterostomy included 1 ureteral stricture and 1 ureteral leak. Good renal function was maintained with a mean followup of 77.9 months. CONCLUSIONS: Our experience suggests that transureteroureterostomy can be useful for urinary diversion when a segment of lower ureter is involved with malignant disease. PMID- 8627826 TI - Percutaneous transrenal electro-incision of ureterointestinal anastomotic strictures: long-term results and comparison of fluoroscopic and endoscopic guidance. AB - PURPOSE: We determined the long-term outcome of a new technique for incising ureterointestinal anastomotic strictures using a transrenal percutaneously inserted papillotome. Procedures using fluoroscopic and endoscopic guidance were compared. MATERIALS AND METHODS: Of 33 stenoses incised in 30 patients 15 were in ileal conduit diversions and 15 were in enterocystoplasties. The papillotome was inserted percutaneously over a guide wire into the stenosis, and then deflected and gently withdrawn under fluoroscopic (11 cases) or endoscopic (22) guidance using a flexible pediatric gastroscope or a lateral duodenoscope inserted retrograde into the ileal loop or neobladder. Air filling provided an excellent view of the stenotic area. Operative time did not exceed 45 minutes. The only major complication was damage to a right internal iliac artery. RESULTS: Followup data were available for 31 stenoses, with 27 followed for longer than 12 months after stent removal. Of the stenoses 22 are completely patent (actuarial long term patency rate 71%), 3 showed partial improvement and 6 recurred requiring further treatment. CONCLUSIONS: Combined endoscopic and fluoroscopic guidance is preferable to fluoroscopy alone. The technique is simple if the endoscope is inserted retrograde. Long-term results are satisfactory and we believe that incision should be the initial approach to strictures of uro-digestive anastomoses. PMID- 8627827 TI - Complications of the afferent antireflux valve mechanism in the Kock ileal reservoir. AB - PURPOSE: Since 1982 the Kock ileal reservoir has been the primary form of urinary diversion in patients requiring lower urinary tract reconstruction at our institution. The intussuscepted afferent nipple valve of the Kock ileal reservoir is designed to prevent reflux and protect the upper urinary tract. Problems associated specifically with the afferent antireflux valve have been few. We defined and characterized all complications associated with the Kock pouch antireflux nipple valve. MATERIALS AND METHODS: From November 1984 through July 1992, 802 patients underwent construction of a continent Kock ileal reservoir. All complications associated with the afferent antireflux valve in this group and their management were identified. RESULTS: Overall, 79 of 802 patients (9.8%) had a total of 84 complications of the afferent antireflux valve (10.4%), including formation of stones on staples securing the afferent nipple valve in 42 cases (5.2%), stenosis of the afferent valve in 35 (4.3%) and prolapse of the valve in 7 (0.9%). A total of 81 patients required surgical intervention to correct the afferent valve complication: 56 (7.0%) were treated endoscopically and 25 (3.1%) required open surgical revision. CONCLUSIONS: We report an overall complication rate of 10.4% associated with the afferent antireflux nipple valve in the Kock ileal reservoir. Most complications can be treated endoscopically without difficulty on an outpatient basis with the use of local sedation. With these results, and only a 3% incidence of open surgical correction of all afferent nipple problems, we encourage the continued use of the intussuscepted afferent nipple valve whenever continent urinary diversion is performed. PMID- 8627828 TI - The ileal ureter neobladder is associated with a high success and a low complication rate. AB - PURPOSE: Several different methods to construct a bladder substitute after cystectomy have been described. We evaluated our experience with the Studer ileal ureter neobladder during the last 5 years. MATERIALS AND METHODS: We reviewed retrospectively the results in 32 patients who underwent construction of a slightly modified ileal neobladder from that originally described. Mean followup was 25 months (range 6 to 68). RESULTS: Patients experienced few complications and only 1 required reoperation. Daytime and nighttime continence rates were 94 and 74%, respectively. One patient sustained a ureteral stricture resulting in hydronephrosis (1 of 64 renal units). CONCLUSIONS: The results reveal the ileal neobladder to be an easily constructed pouch with a low complication rate, and a high success rate in regard to continence and the establishment of adequate capacity at low pressure. Within the study period upper tract preservation was excellent. However, a 10 to 15-year followup is indicated to confirm our initial results. PMID- 8627829 TI - Reconstruction and diversion. PMID- 8627830 TI - Cyclosporine in severe interstitial cystitis. AB - PURPOSE: Cyclosporine is a widely used immunosuppressive drug in organ transplantation and recently it has been used in several autoimmune disorders with good results. Because interstitial cystitis may have an autoimmune etiology, we wished to determine whether cyclosporine has any effect on symptoms in patients with severe interstitial cystitis. MATERIALS AND METHODS: A total of 11 patients, who fulfilled the criteria for interstitial cystitis according to an international accrual form, received cyclosporine for 3 to 6 months at an initial dose of 2.5 to 5 mg./kg. daily and a maintenance dose of 1.5 to 3 mg./kg. daily. Blood pressure, serum creatinine and cyclosporine concentrations were monitored regularly. The patients completed frequency-volume charts at 2-week intervals. RESULTS: The frequency-volume charts showed favorable effects. Micturition frequency decreased (p<0.01), and mean and maximum voided volumes increased significantly (p<0.001 and p<0.01, respectively). Bladder pain decreased or disappeared in 10 patients, allowing for storage of large urine volumes. Serum creatinine did not change with the dosages used. Mild hypertension occurred in 2 patients and resolved after the cyclosporine dose was lowered. After cessation of treatment symptoms recurred in the majority of patients. CONCLUSIONS: The findings revive the concept of interstitial cystitis as an autoimmune disease. PMID- 8627831 TI - Dynamic gadolinium-enhanced magnetic resonance imaging in staging of superficial bladder cancer. AB - PURPOSE: We evaluated the usefulness of dynamic enhanced magnetic resonance imaging (MRI) in the staging of superficial tumors following a bolus administration of gadopentetate dimeglumine. MATERIALS AND METHODS: In 48 patients with proved bladder tummors the results of preoperative plain spin echo T1 (repetition time/echo time 500/20 msec.) and T2 (repetition time/echo time 2,000/40 to 100 msec.)-weighted MRI, dynamic gadolinium-enhanced MRI (repetition time/echo time 200/15 msec.) and late gadolinium-enhanced MRI (repetition time/echo time 500/20 msec.) were compared and correlated with the histopathological findings. RESULTS: Unenhanced spin echo T1 and T2-weighted MRI sequences were able to stage correctly 14 (56%) and 17 (68%) of 25 superficial bladder cancers, respectively. Muscular infiltration (stages pT2 and pT3a) was correctly depicted in 3 (27%) and 6 (54%) of 11 cases respectively, with over staging being the most frequent error. On the basis of the dynamic gadolinium enhanced T1-weighted MRI appearance, superficial involvement of the bladder wall was correctly assessed in 21 of 25 cases (84%) and muscular infiltration (stages pT2 to pT3a) in 7 of 11 (63%). Delayed enhanced T1-weighted sequences showed a low accuracy rate in staging superficial tumors (44%). The overall accuracy of T1 and T2-weighted, dynamic T1-weighted and delayed T1-weighted MRI in staging bladder cancer was 58, 71, 81 and 56% respectively. CONCLUSIONS: The use of gadolinium improved the accuracy of dynamic enhanced MRI in staging superficial bladder cancer. On the contrary, delayed enhanced MRI was not useful for staging superficial bladder cancer. The degree of bladder distension was a determinant factor in staging superficial tumors. PMID- 8627832 TI - Impotence and incontinence after immediate realignment of posterior urethral trauma: result of injury or management? AB - PURPOSE: We examined further whether the injury or method of management is responsible for impotence and incontinence after immediate realignment of prostato-membranous urethral disruptions. MATERIALS AND METHODS: A total of 20 patients with complete urethral disruptions treated with immediate realignment (group 1) was compared to 12 with partial or complete injuries treated with retrograde catheterization alone (group 2). Followup status was obtained by patient questionnaire or telephone interview. RESULTS: Of the patients 83% in group 1 and 80% in group 2 are continent, and 76% and 70%, respectively, regained erections suitable for sexual intercourse. CONCLUSIONS: The results suggest that impotence and incontinence in this setting are the result of the injury and not of attempts at immediate surgical management. PMID- 8627834 TI - Outcome analysis of goal directed therapy for impotence. AB - PURPOSE: We assessed patient preference, satisfaction and overall outcome of goal directed management of erectile dysfunction. MATERIALS AND METHODS: The results of goal directed therapy of impotence were assessed by an independent telephone survey of 377 consecutive men who had not received prior therapy and who were followed for a minimum of 2 years. RESULTS: Patients preferred medical to surgical therapies despite significantly higher satisfaction rates achieved with surgery. Average number of treatment modalities chosen by each patient was 2 (range 0 to 5). Ultimately, only 40% of the patients achieved a long-term satisfactory result with goal directed therapy. The remainder were not satisfied with the last treatment but chose no further therapy, were lost to followup or refused therapy from the outset. CONCLUSIONS: Our results clearly demonstrate a patient preference for the least invasive forms of therapy. Patients avoid significantly more effective but also more invasive treatment options despite unsatisfactory results with less invasive methods. Future research efforts should be concentrated on the development of new medical therapies to enhance overall patient satisfaction. PMID- 8627833 TI - Improvement of sexual function in testosterone deficient men treated for 1 year with a permeation enhanced testosterone transdermal system. AB - PURPOSE: The effects of androgen replacement via a nonscrotal permeation enhanced testosterone transdermal system on the sexual function of men with hypogonadism were assessed. MATERIALS AND METHODS: An open label, multicenter study of testosterone supplementation and withdrawal was conducted with sexual function assessed by the Watts and Davidson questionnaires and RigiScan monitoring. RESULTS: When comparing results obtained during use of the testosterone transdermal system (with normalized testosterone levels) and during the androgen withdrawal period, nocturnal erections occurred more frequently with longer duration and greater rigidity, and patient assessments of sexual desire and weekly number of erections were higher. CONCLUSIONS: Sexual function improved significantly in men with hypogonadism treated with the testosterone transdermal system. PMID- 8627835 TI - Report of a multicenter clinical evaluation of the Dura-II penile prosthesis. AB - PURPOSE: Information was collected during the first 2 years of an ongoing, prospective, multi-institutional evaluation of the Dura-II penile prosthesis. Evaluation included data on clinical outcomes and a patient satisfaction survey, and will continue for 5 years after implantation for each patient. MATERIALS AND METHODS: To date 196 patients have been evaluated. Surgical data, adverse events and information from satisfaction surveys are reported. RESULTS: At a followup of 13.4 +/- 8.4 months postoperatively, adverse events occurred in 8.2% of the patients, resulting in reoperation in 5.1%. There have been no mechanical failures. Overall satisfaction rates were 85% at 3 months, 83% at 1 year and 91% at 2 years after implantation, and levels of sexual functioning were correspondingly high. A majority of patients assigned high scores to rigidity, concealability, ability to have intercourse and erection size, although satisfaction with erection size ranked lower than the other measures. CONCLUSIONS: The Dura-II penile prosthesis provides a satisfactory result and has an acceptable level of complications associated with its use. Further followup will assess temporal changes in satisfaction rates and sexual functioning for these patients. PMID- 8627836 TI - Complications of penile lengthening and augmentation seen at 1 referral center. AB - PURPOSE: Complications of the recent cosmetic technique of penile lengthening and girth enhancement are reviewed. MATERIALS AND METHODS: During a 16-month period 12 men presented with complications of penile augmentation performed elsewhere. All 12 patients had undergone release of the suspensory ligament and 10 had received autologous fat injection. RESULTS: The chief complaint was poor cosmetic appearance (irregular residual fat nodules in 7 men, skin deformity and scarring in 4 and scrotalization in 4). Reoperation was necessary in 6 patients, wound complications occurred in 6 and sexual dysfunction was reported by 4. Only 1 patient reported a subjective increase in penile length. CONCLUSIONS: Although a verifiable complication rate may never be available, the morbidity of elective penile lengthening and girth enhancement is noteworthy. These cosmetic techniques should be regarded as experimental. PMID- 8627837 TI - Long-term results with Hydroflex and Dynaflex penile prostheses: device survival comparison to multicomponent inflatables. AB - PURPOSE: We compared our experience with self-contained inflatable and multicomponent prostheses during similar intervals. MATERIALS AND METHODS: We followed 295 patients with self-contained prostheses and 1,026 with multicomponent inflatable devices for up to 8 years. Revision rates for mechanical failure, infection or patient dissatisfaction were statistically analyzed. RESULTS: Multicomponent inflatable prostheses were superior with regard to mechanical reliability and patient satisfaction, with no differences in rate of infection. Self-contained prostheses were best replaced with the AMS 700CX device, thus avoiding aneurysm. CONCLUSIONS: Self-contained inflatable prostheses may be of limited value for the treatment of impotence. The AMS 700CX is the device of choice for replacement of explanted self-contained prostheses. PMID- 8627838 TI - Impotence--improving patient treatment. PMID- 8627839 TI - Outcomes of initial surveillance of invasive squamous cell carcinoma of the penis and negative nodes. AB - PURPOSE: We determined factors predictive of inguinal nodal relapse in patients with stages T1 to 3NOMO squamous cell penile cancer treated initially with surveillance of inguinal nodes. MATERIALS AND METHODS: Between 1980 and 1994, in 42 patients with stages T1 to 3NOMO squamous cell penile cancer of 60 with invasive disease seen at our center the inguinal nodes were surveyed after definitive treatment of the primary tumor. Clinical inguinal nodal recurrences were treated with inguinal lymphadenectomy. RESULTS: A total of 26 patients (62%) had inguinal nodal recurrences during followup, with 50% occurring within 1.4 years and 75% within 2.8 years of resection of the primary tumor. The only factor predicting nodal relapse was grade of the primary tumor at initial treatment. Patients with grade 1 tumors had a 45% long-term actuarial relapse-free survival rate. All other groups had a 100% actuarial nodal relapse rate. Of the patients 10% had metastatic disease without nodal recurrence. CONCLUSIONS: Invasive penile cancer may be associated with inguinal lymph node and hematogenous metastasis. A strong case for prophylactic bilateral inguinal lymphadenectomy can be made in patients with primary tumors other than grade 1, since surveillance of these patients will not spare them eventual lymphadenectomy and may potentially compromise survival by delaying surgery. Patients with grade 1 tumors may be offered either careful surveillance or prophylactic bilateral inguinal lymphadenectomy depending on the clinical circumstances and patient preference. PMID- 8627840 TI - Seminal plasma of spinal cord injured men inhibits sperm motility of normal men. AB - PURPOSE: Seminal plasma was investigated as a contributor to the poor sperm motility of spinal cord injured men. MATERIALS AND METHODS: Seminal plasma of spinal cord injured men was mixed with sperm of normal men and vice versa. Sperm motility was analyzed at 5 and 60 minutes after mixing. RESULTS: At 5 (but not 60) minutes seminal plasma from spinal cord injured men inhibited motility of sperm from normal men. Concomitantly, seminal plasma from normal men improved motility of sperm from spinal cord injured men. CONCLUSIONS: Seminal plasma of spinal cord injured men contributes to poor sperm motility. PMID- 8627841 TI - Effect of varicocelectomy on sperm parameters and pregnancy rate in patients with subclinical varicocele: a randomized prospective controlled study. AB - PURPOSE: We assessed whether subclinical varicocele ligation improves fertility and/or semen parameters. MATERIALS AND METHODS: A total of 85 patients with a subclinical varicocele diagnosed by scrotal thermography presented with infertility. Patients were randomly assigned to groups 1 (high ligation of the internal spermatic vein) and 2 (followed without any treatment). At least 3 semen samples were obtained at study entry and 1 year later in both groups. RESULTS: The pregnancy rate in group 1 was 6.7% compared to 10% in group 2, and the difference was not statistically significant. Group 1 had significantly higher levels of sperm density and total motile sperm count at 1 year. There were no significant differences between groups 1 and 2 regarding change in seminal volume, sperm motility and abnormal sperm morphology. CONCLUSIONS: Subclinical varicocelectomy has some effect on spermatogenesis but no beneficial effect on pregnancy rate. PMID- 8627842 TI - Normal human ejaculatory duct anatomy: a study of cadaveric and surgical specimens. AB - PURPOSE: W examined the anatomy of the ejaculatory ducts in normal men and correlated findings with theories of ejaculatory duct obstruction. MATERIALS AND METHODS: Gross and microscopic anatomical studies were performed on cadaveric and operative specimens derived from radical prostatectomy. RESULTS: Histologically, the ejaculatory ducts are a continuation of the seminal vesicles. However, the thick muscle wall of the seminal vesicle is not present within the ejaculatory duct. Normal ejaculatory duct luminal and wall dimensions are remarkably uniform among men. A luminal diameter of greater than 2.3 mm. defines a dilated system statistically. CONCLUSIONS: The largely collagenous ejaculatory ducts may serve as simple semen conduits instead of muscular tubes with spasmodic, sphincteric or peristaltic properties. The anatomical findings presented suggest several possible mechanisms for the prevention of urinary reflux into the ejaculatory ducts. PMID- 8627843 TI - Male infertility redefined--back to the basics. PMID- 8627845 TI - Clinical diagnosis of bladder outlet obstruction in patients with benign prostatic enlargement and lower urinary tract symptoms: development and urodynamic validation of a clinical prostate score for the objective diagnosis of bladder outlet obstruction. AB - PURPOSE: We attempted to improve the method of objective clinical evaluation of patients with benign prostatic enlargement and lower urinary tract symptoms. MATERIALS AND METHODS: We compared the results of free uroflowmetry and transrectal ultrasound prostate size determination with those of pressure-flow analysis of bladder outlet obstruction in 871 consecutive elderly men. RESULTS: Maximal flow, prostate size, and post-void residual and voided volumes were correlated with bladder outlet obstruction to derive a clinical prostate score. CONCLUSIONS: Clinical prostate score shows a superior correlation with bladder outlet obstruction than isolated objective parameters or symptom scores. PMID- 8627844 TI - Urogenital anomalies in men with congenital absence of the vas deferens. AB - PURPOSE: We evaluated urogenital anomalies in men with congenital absence of the vas deferens. METHODS AND MATERIALS: A retrospective review was done of 104 subfertile men with congenital absence of the vas deferens (84 bilaterally and 20 unilaterally). RESULTS: Of men with unilateral or bilateral congenital absence of the vas deferens 26 and 11%, respectively, had renal agenesis. Of men with unilateral congenital absence of the vas deferens and infertility 80% had genitourinary anamalies affecting the contralateral testis. No man with congenital absence of the vas deferens and renal anomalies had cystic fibrosis transmembrane-conductance regulator (CFTR) gene mutations detected. CONCLUSIONS: Other urogenital anomalies are common for men with congenital absence of the vas deferens. CFTR gene mutations frequently contribute to maldevelopment of the vas deferens but vasal agenesis can occur without any evidence of CFTR defects. CFTR abnormalities are rarely detected in men with congenital absence of the vas deferens and renal anomalies. PMID- 8627846 TI - Pitfalls in interpreting prostate specific antigen velocity. AB - PURPOSE: The concept of prostate specific antigen (PSA) velocity as an improved marker for prostate cancer detection is intriguing. However, before this concept is applied to individual patients several confounding parameters must be addressed. We determined the variability of serum PSA levels in men without prostate cancer. MATERIALS AND METHODS: We reviewed data from a prostate cancer screening program, and determined inter-assay and individual variability of the serum PSA values for a 2-year followup period in 265 men clinically free of prostate cancer. RESULTS: Our average inter-assay coefficient of variation was 7.5%. Therefore, we considered only PSA changes exceeding +/- 15% as significant. Fluctuations in serum PSA occurred in 78% of the men during the observation period, and 12.5% had at least a single PSA increase exceeding 0.75 ng/ml. per year. Fluctuations were noted throughout the entire range of serum PSA levels but became progressively larger with an increasing mean PSA. CONCLUSIONS: The inter assay variability must be considered when interpreting PSA velocity. Individual fluctuations in serum PSA dictate an observation period of at least 2 years before PSA velocity is considered abnormal. PMID- 8627847 TI - The periurethral glands do not significantly influence the serum prostate specific antigen concentration. AB - PURPOSE: The periurethral glands are known to produce prostate specific antigen (PSA). With ultra-sensitive assays now routinely available, it is necessary to determine if the periurethral glands significantly influence serum PSA concentration after radical prostatectomy. MATERIALS AND METHODS: Serum PSA levels of 46 men, 51 to 89 years old (median age 67) who underwent radical cystoprostatectomy and total urethrectomy, were compared with those of 92 men 46 to 91 years old (median age 67) who underwent radical cystoprostatectomy only. All men had transitional cell carcinoma of the bladder without gross or microscopic evidence of prostate cancer and all underwent ileal conduit diversion. Serum was obtained at least 1 year postoperatively. Each specimen was analyzed using the Tosoh, Immulite, and Yu and Diamandis ultra-sensitive PSA assays with analytical detection limits of 0.02 ng./ml., 0.004 ng./ ml. and 0.002 ng./ml., respectively. RESULTS: Median PSA for the radical cystoprostatectomy with urethrectomy group was 0.00 ng./ml. (range 0.00 to 0.14) for each of the 3 assays. For the radical cystoprostatectomy only group the median Tosoh and Immulite PSA assay levels were 0.01 ng./ml. (range 0.00 to 0.22), and median Yu and Diamandis PSA assay level was 0.00 ng./ml. (range 0.00 to 0.31). CONCLUSIONS: The greatest difference in median PSA levels that could be found between men with and without periurethral glands when using 3 different ultra-sensitive assays was 0.01 ng./ml., indicating that the periurethral glands do not have a clinically significant effect on serum PSA concentration after radical prostatectomy. Thus, a serum PSA level above the residual cancer detection limit following radical prostatectomy, even if obtained with a ultra-sensitive assay, reflects either malignant or benign residual prostatic tissue, rather than the presence of periurethral glands. PMID- 8627848 TI - Prostate cancer--our patients' questions. PMID- 8627849 TI - Cryosurgical ablation of the prostate for localized adenocarcinoma: a preliminary experience. AB - PURPOSE: Cryosurgical ablation of the prostate has recently become recognized as a therapeutic option in the treatment of localized adenocarcinoma of the prostate. To assess the efficacy of cryoablation in this disease process several centers have instituted treatment protocols. MATERIALS AND METHODS: Our overall series includes 117 ultrasound guided percutaneous transperineal cryoablations performed on 104 patients with localized adenocarcinoma of the prostate. Followup consisted of digital rectal examinations and measurement of prostate specific antigen levels at 3-month intervals after cryosurgery. Additionally, prostate biopsies were obtained 3 to 6 months postoperatively. RESULTS: Of 63 patients who underwent initial cryosurgery and followup biopsy 47 (75%) had negative findings. Of the 16 patients with positive biopsies 10 consented to undergo a second cryosurgical ablation, and 7 of these patients subsequently had negative followup biopsies. Therefore, our disease-free rate at 3 months after 1 or 2 cryosurgical procedures was 95%. A total of 46 protocol patients in our series completed 12 months of evaluation and 40 (87%) had no evidence of disease. This same cohort showed only minimal disease progression, with disease-free rates of 96, 93, 87 and 87% at 3, 6, 9 and 12 months, respectively. Major complications were infrequent. CONCLUSIONS: At 1-year followup our clinical experience shows cryoablation of the prostate to be an effective therapy in select cases of prostatic adenocarcinoma. Long-term efficacy is still in question but, based on current disease-free rates, this therepeutic modality merits continued clinical investigation. PMID- 8627850 TI - Black race is an adverse prognostic factor for prostate cancer recurrence following radical prostatectomy in an equal access health care setting. AB - PURPOSE: We determined if black men with clinically localized adenocarcinoma of the prostate have the same recurrence-free outcome following radical prostatectomy, and whether they have similar preoperative, operative and pathological characteristics as white men in an equal access health care environment. MATERIALS AND METHODS: We studied consecutive single hospital case series of 366 white and 107 black patients who underwent radical prostatectomy between 1975 and February 29, 1995. Evaluation included comprehensive retrospective chart review, prospective data collection and proactive followup. Univariate and multivariate statistical analyses were done of preoperative, operative, pathological and recurrence data by race. RESULTS: Although the incidences of hypertension and diabetes, pretreatment prostate specific antigen (PSA) and serum creatinine measurements, elevated PSA as an indication for biopsy and clinical stage were greater in black men, the operative variables of blood loss, operative time and performance of a nerve sparing procedure were not different. The incidence of margin positivity was greater in black patients but pathological stage, Gleason score and seminal vesicle or nodal involvement were not different. Black race was an adverse prognostic factor for recurrence following radical prostatectomy after multivariate adjustment for pretreatment PSA and acid phosphatase, organ confinement status and tumor grade. CONCLUSIONS: The poorer recurrence-free outcome for black patients even after multivariate adjustment suggests a potentially more aggressive variant of prostate cancer in this population, the etiology of which is unknown. Race should be a stratification factor in clinical trials, especially those including radical prostatectomy and using recurrence-free outcome as an end point. PMID- 8627851 TI - Cellular proliferative fraction of metastatic lymph nodes predicts survival in stage D1 (TxN+M0) prostate cancer. AB - PURPOSE: We assessed the ability of tumor cellular proliferative fraction to predict long-term survival among patients with lymphatic metastases from prostate cancer. MATERIALS AND METHODS: We studied 50 patients with stage D1 (TxN+M0) prostate cancer who underwent pelvic lymphadenectomy and 125iodine seed implantation between 1970 and 1978. We used the MIB-1 monoclonal antibody to Ki67 to stain sections of the lymphatic metastases in these patients. The Ki67 proliferative fraction was defined as the fraction of positively stained malignant nuclei. We also used flow cytometry to determine the deoxyribonucleic acid content of the lymphatic metastases. RESULTS: Median followup was 6.1 years. Patients whose metastases had a Ki67 proliferative fraction of less than 0.1 had significantly longer survival compared to those with a proliferative fraction of greater than 0.1 (8.7 years versus 4.4 years, respectively, p = 0.005, log rank test). The Ki67 proliferative fraction and ploidy were not independent variables. Patients whose metastases were diploid had a significantly longer survival than those with aneuploid metastases (8.8 years versus 4.4 years, respectively, p = 0.01, log rank test). Multivariate analysis showed that ploidy had a slightly stronger effect on survival than did the Ki67 proliferative fraction. CONCLUSIONS: Cellular proliferative fraction of lymphatic metastases is useful to predict survival in patients with stage D1 prostatic carcinoma. Proliferative fraction may be useful as a marker of progression among patients with other stages of disease. PMID- 8627852 TI - Prostate cancer--adjusting the tiller. PMID- 8627854 TI - p53 immunohistochemical and genetic alterations are associated at high incidence with post-irradiated locally persistent prostate carcinoma. AB - PURPOSE: Several reports have shown that cells with p53 mutations display increased resistance to ionizing radiation, a treatment often used clinically for localized prostate carcinoma. MATERIALS AND METHODS: Totals of 18 post-irradiated locally recurrent prostatic carcinoma specimens and 25 (no radiation) stage D1 node-positive (TxN+MO) primary prostatic carcinoma specimens were tested for p53 immunoreactivity by immunohistochemistry. Of the 18 post-radiation locally recurrent prostatic carcinomas 10 were further analyzed by single strand conformational polymorphism to assess the validity of using this immunohistochemistry approach in irradiated tissue for detecting p53 alterations. Specimens showing p53 alterations by single strand conformational polymorphism were subjected to nucleotide sequence analysis or tested for loss of heterozygosity at a locus within the p53 gene. RESULTS: Of the 25 stage TxN+MO prostatic carcinomas without radiation 5 (20%) were immunoreactive (consistent with the reported incidence of positive immunoreactivity in clinical/surgical stage TxN+MO primary prostatic carcinomas). In contrast, 13 of 18 post-radiation locally recurrent prostatic carcinoma specimens (72%) were immunoreactive. Multivariate logistic regression analysis showed no dependence of p53 immunoreactivity to grade, stage or androgen status in the post-radiation locally recurrent prostatic carcinoma group, while 8 of 10 hormone naive prostatic carcinoma specimens (80%) were immunoreactive. The temporal relationship between p53 alterations and radiotherapy was assessed. Pre-irradiation prostatic carcinomas available from 5 patients with immunoreactive post-radiation locally recurrent disease were analyzed and all were immunoreactive. CONCLUSIONS: p53 Alteration in localized prostatic carcinoma is uncommon. Our study confirms others in that even aggressive locally advanced nonirradiated primaries (stage TxN+MO) contain only 20% incidence of p53 alterations. However, our study demonstrates that p53 alterations are found in the preponderant majority of post radiation locally recurrent prostatic carcinoma specimens. Limited evaluation of pretreatment prostatic carcinoma biopsies uniformly documented the presence of p53 alterations before ionizing radiation, thereby demonstrating that p53 alteration was already present and was not radiation-induced or only correlated with late stage disease. This finding suggests a potential for p53 immunoreactivity to be used as a pretreatment marker that might predict local treatment failure with ionizing radiation. Large scale prospective trials would appear warranted to evaluate conclusively the potential prognostic applicability of p53 pre-screening before enrollment in definitive radiotherapy. PMID- 8627853 TI - Influence of patient age and co-morbidity on outcome of a collaborative care pathway after radical prostatectomy and cystoprostatectomy. AB - PURPOSE: We determined whether standardized care patterns developed with a collaborative care methodology can be applied successfully across all patient groups with favorable effects on cost and quality. MATERIALS AND METHODS: We retrospectively analyzed financial and clinical outcomes in 109 radical retropubic prostatectomy and 47 radical cystectomy cases. Patients older than 70 years and/or with an American Society of Anethesiology status of 3 or greater were compared to younger, healthier patients undergoing these procedures. RESULTS: Standardized care patterns resulted in favorable financial and clinical outcomes in high and low risk patient groups. The only apparent difference was an increased need for rehospitalization after discharge for patients undergoing radical prostatectomy with a high American Society of Anesthesiology status. CONCLUSIONS: Standardized care patterns developed with a collaborative care methodology provide a high quality, cost-efficient approach to medical care. This methodology is applicable to all patient groups and is highly compatible with current medical practice. PMID- 8627855 TI - Ureteral replacement with reconfigured colon substitute. AB - PURPOSE: Upper ureteral defects are often too extensive to repair by direct anastomosis or with use of a bladder flap. Ureteral substitution may be the only remaining alternative to restoring urinary drainage from the kidney to the bladder. This effect is usually achieved by interposing a segment of small bowel between the proximal collecting system and bladder. If ileum is not available other substitution alternatives must be sought. MATERIALS AND METHODS: We report a new technique in which a tube constructed from a small piece of ascending colon was used to replace a large ureteral defect. RESULTS: Excellent short-term results were achieved by replacing the strictured ureteral segment with a reconfigured colon segment as shown by symptomatic and radiographic improvement. CONCLUSIONS: A reconfigured colon segment can be used for ureteral reconstruction in the patient with limited alternatives. This tube has several advantages for ureteral reconstruction over ileal segments. PMID- 8627856 TI - Technique of harvesting buccal mucosa for urethral reconstruction. AB - PURPOSE: Buccal mucosa has been used increasingly by urologists for urethral substitution in complex hypospadias repair. We have found buccal mucosa to be useful in reconstruction of bulbar urethral strictures, and describe a simple and reliable technique for harvest. MATERIALS AND METHODS: In 11 patients with refractory bulbar urethral strictures a nontubularized onlay patch of buccal mucosa was used for urethral reconstruction. All procedures were done with a 2 team approach in which 1 team (usually an oral surgeon and urologist) harvested the graft from the mouth, while the perineal team simultaneously exposed and calibrated the stricture. RESULTS: The length of buccal mucosa used ranged from 3.5 to 17 cm. (average 6.4). All patients achieved excellent results. No oral complications were noted, even in patients in whom multiple buccal mucosal grafts were obtained. CONCLUSIONS: With the technique reported, buccal mucosa is a reliable, easily obtained tissue for patch graft urethroplasty. Our 2-team approach decreased operative time considerably. PMID- 8627858 TI - Transitional cell carcinoma of the labia minor: evidence of contact implantation of bladder cancer. PMID- 8627857 TI - Occlusion therapy for an intractable transplant-ureteral fistula using fibrin glue. PMID- 8627859 TI - Metastatic umbilical transitional cell carcinoma from a bladder diverticulum. PMID- 8627860 TI - Prostate carcinoma metastatic to the penis: magnetic resonance imaging and brachytherapy. PMID- 8627861 TI - Metachronous presentation of bilateral Leydig cell tumors. PMID- 8627862 TI - RE: Combined use of bowel and the artificial urinary sphincter in reconstruction of the lower urinary tract: infectious complications. PMID- 8627863 TI - RE: The chance for fertility in adolescent boys after corrective surgery for varicocele. PMID- 8627864 TI - RE: Prostate specific antigen after gonadal androgen withdrawal and deferred flutamide treatment. PMID- 8627865 TI - RE: Prostate specific antigen after gonadal androgen withdrawal and deferred flutamide treatment. PMID- 8627866 TI - Renal cell carcinoma in children: the Detroit experience. AB - PURPOSE: We analyzed the presentation, treatment and survival of 6 children with renal cell carcinoma. MATERIALS AND METHODS: We retrospectively reviewed the pathological and hospital records of 6 children diagnosed with renal cell carcinoma at Children's Hospital of Michigan (5) and Henry Ford Hospital (1) from 1980 to 1995. RESULTS: The most common presenting complaints were flank pain (50%) and a palpable abdominal mass (50%), while gross hematuria was present in only 1 patient (16%). No patient had the classic triad of flank pain, hematuria and palpable mass. Only 1 patient had localized disease (stage II), while 5 of the 6 presented with stage III or IV disease. While followup is limited, all patients with stage III disease are without evidence of recurrence at a mean 38.5 months and 1 of 2 with stage IV disease is without evidence of disease at 22 months. CONCLUSIONS: Although renal cell carcinoma in childhood often presents at an advanced stage, the prognosis for those with isolated regional lymph node involvement appears to be encouraging. PMID- 8627867 TI - Video cystometric recording of dilating reflux in infancy. AB - PURPOSE: Bladder hypercontractility has previously been suggested to occur in male infants with gross bilateral reflux, and the causal relationship between hypercontractility and reflux has been questioned. MATERIALS AND METHODS: In this video urodynamic study of dilating reflux the association between reflux and bladder pressure was evaluated in 16 infants. RESULTS: In our 11 male patients hypercontractility was noted often, usually with reflux that occurred simultaneously with an increase in detrusor pressure. In the 5 female patients no hypercontractility was noted and reflux occurred without an increase in detrusor pressure. CONCLUSIONS: Our results further confirm the association between bladder dysfunction and dilating reflux in infants but with different patterns in male and female infants. PMID- 8627868 TI - Endoscopic treatment of vesicoureteral reflux with collagen: preliminary report and cost analysis. AB - PURPOSE: We determined therapeutic and operative cost efficacy of endoscopic collagen injection for vesicoureteral reflux. MATERIALS AND METHODS: We performed a multicenter study of 10 male and 23 female sequential patients (45 renal units) with vesicoureteral reflux and a surgical indication. Patients were skin tested with collagen and when negative, they underwent endoscopic injection. Renal ultrasound/cystography was done 3 months after treatment. Cost analysis was performed at 1 institution. RESULTS: Reflux was grade I in 2 renal units, II in 18, III in 15 and IV in 10. The majority of cases were single systems with primary reflux. We performed 1 treatment in 32 renal units, 2 in 6, 3 in 6 and 4 in 1. Cure was evident in 31 of 38 renal units (81.6%) and 21 of 27 patients (78%) 3 months after the last injection. Patient morbidity was minimal. Operative cost per renal unit was $1,599.68 for collagen injection and $9,144.47 for reimplantation. CONCLUSIONS: Endoscopic injection of collagen is effective treatment for vesicoureteral reflux. Furthermore, it causes minimal morbidity and may effect cost savings in health care management. PMID- 8627869 TI - Extravesical ureteral reimplantation: results in 128 patients. AB - PURPOSE: We evaluated the results of extravesical ureteral reimplantation in children. MATERIALS AND METHODS: We reviewed the records of 128 children (174 ureters) who underwent ureteral reimplantation via extravesical techniques. Primary vesicoureteral reflux was the most common diagnosis (73 patients). RESULTS: The extravesical technique produced a successful result in all patients (no reflux or obstruction). The 2 complications included postoperative urosepsis and transient urinary retention. CONCLUSIONS: Extravesical ureteral reimplantation is a reliable procedure with predictable results comparable to those of more traditional intravesical techniques. PMID- 8627870 TI - Enterocystoplasty: the star modification. AB - PURPOSE: A modification of sagittal cystoplasty is described that maximally reconfigures the native neuropathic bladder, as required in enterocystoplasty. MATERIALS AND METHODS: The star modification incorporates lateral cystotomies with anteroposterior cystotomy, as in the widely used sagittal clamshell technique. RESULTS: Enterocystoplasty was performed in 27 patients with various bowel segments using this technique and none has had complications attributable to cystoplasty after a mean followup of 2.5 years. CONCLUSIONS: Star reconfiguration defunctionalizes any potential noncompliant or hyperreflexic tendencies inherent to the neuropathic bladder before augmentation. In addition, the modification provides a technical advantage by increasing the linear length of the edge available for the anastomosis of bowel to bladder. PMID- 8627871 TI - Experience with ileal augmentation cystoplasty using a short pfannenstiel incision. AB - PURPOSE: Our study was done to evaluate the efficacy of performing ileal augmentation cystoplasty through a short Pfannenstiel incision. MATERIALS AND METHODS: From December 1986 to June 1988, 7 patients with neurovesical dysfunction secondary to myelodysplasia underwent ileal augmentation cystoplasty via a lower abdominal incision. From July 1989 to July 1993, 12 similar patients underwent ileal augmentation cystoplasty via a short Pfannenstiel incision. RESULTS: Operative time was slightly shorter and mean recovery time, as measured by the return of gastrointestinal function and hospital stay, was shorter in the Pfannenstiel incision group. CONCLUSIONS: In select patients a short Pfannenstiel incision is the incision of choice for ileal augmentation cystoplasty. PMID- 8627872 TI - Subpubic fistula: a urethral duplication. PMID- 8627873 TI - The long-term outcome of posterior urethral valves treated with primary valve ablation and observation. AB - PURPOSE: We believe that primary valve ablation with observation is the preferred management for posterior urethral valves. However, debate continues as to the role of high diversion. We examined the long-term outcome of a large series of patients treated with primary valve ablation, and compared it to the outcome of high diversion and vesicostomy. MATERIALS AND METHODS: We reviewed the records of 100 patients treated with primary valve ablation (74%), vesicostomy (13%) or high diversion (9%) before 1985. Median followup was 11.2 years. RESULTS: Overall 13% of our patients had end stage renal disease by age 15 years. Three patients initially treated with valve ablation and 3 initially treated with vesicostomy later underwent high diversion but none benefited from the secondary procedure. Four patients initially treated with valve ablation subsequently underwent vesicostomy but only 1 benefited. Bladder storage capacity was well preserved. Diurnal urinary continence developed in 46% of patients at age 10 years and only 1 remained incontinent after age 20 years. One patient with diversion who awaits transplantation had a small contracted bladder. Recent urodynamic studies in 10 cases of delayed urinary continence have not shown decreased bladder compliance or capacity. Kaplan-Meier analysis of outcomes of the different treatments indicated no statistical difference in patient age at end stage renal disease development. However, comparing the number of surgical procedures in the different treatment groups revealed a significant increase in the amount of surgery in infants with diversion. Our results were equivalent to those of the best published series, many of which strongly advocate high diversion. CONCLUSIONS: By avoiding diversion in most cases bladder function is preserved and the need for bladder augmentation is decreased. PMID- 8627874 TI - Modified Barcat balanic groove technique for hypospadias repair: experience with 295 cases. AB - PURPOSE: We reviewed the results of a large series of hypospadias repairs (311) using the modified Barcat balanic groove technique. MATERIALS AND METHODS: All patients who underwent modified Barcat repair with closure of the divergent corpus spongiosum were included in our study and were seen or contacted for postoperative followup. RESULTS: Of 295 patients available for followup 94% had excellent cosmetic results. The fistula rate after primary repair was 5% for a glanular or coronal meatus and 18% for a penile meatus, and meatal stenosis was rare. After secondary repair complications were common in patients with a penile meatus. CONCLUSIONS: The Barcat technique allows anatomically superior glans reconstruction in distal hypospadias repair. Fistula rates can be reduced by tissue coverage of the neourethra. PMID- 8627875 TI - Hemangiomatous penile horn. PMID- 8627876 TI - Biodistribution of protoporphyrin IX in rat urinary bladder after intravesical instillation of 5-aminolevulinic acid. AB - PURPOSE: Photodynamic therapy (PDT) has considerable potential for the treatment of superficial bladder neoplasia. Problems of detrusor muscle scarring and prolonged cutaneous photosensitivity with current photosensitizers may be reduced by using the new photosensitizer precursor, 5-aminolevulinic acid (ALA). We studied the fluorescence distribution of protoporphyrin IX (PpIX, the active derivative of ALA) in rat urinary bladder after intravesical administration of ALA as the first step to undertaking PDT using locally administered ALA. MATERIALS AND METHODS: Solutions of varying concentrations of ALA and pH were given intravesically to Wistar rats. The bladder was removed 1 to 24 hours later for measurement of the tissue levels of PpIX by fluorescence microscopy in the layers of the bladder wall. RESULTS: The stability of the ALA solution steadily decreased as the pH was increased from 2.1 to 7. The best value was pH 5.5, at which the stability of the solution and the urothelial tolerance were both acceptable. The maximum PpIX fluorescence intensity ratio between urothelium and the underlying muscle layer was 11 to 1. This was seen 5 hours after instillation of 1% ALA solution at pH 5.5. A 10% ALA solution gave higher levels of PpIX in the urothelium, but less selectivity between layers. CONCLUSIONS: Under appropriate conditions, intravesical instillation of ALA can be used to sensitize the bladder urothelium with a ratio between urothelial and muscle concentrations of PpIX of up to 11:1. PMID- 8627877 TI - Photodynamic therapy on rat urinary bladder with intravesical instillation of 5 aminolevulinic acid: light diffusion and histological changes. AB - PURPOSE: Photodynamic therapy (PDT) has the potential to treat extensive premalignant lesions and microinvasive tumors in the bladder, but its use has been hampered by the risk of detrusor muscle damage and prolonged skin photosensitivity. We have shown that the rat urothelium can be sensitized by selectively using a 10% solution of 5-aminolevulinic acid (ALA) at pH 5.5 administered intravesically. This paper evaluates the photodynamic effects on sensitized bladders. MATERIALS AND METHODS: The bladders ofs Wistar rats were instilled with ALA solutions of different concentrations at pH 5.5 and subsequently treated with laser light at 630 nm. Bladders were harvested 1 to 7 days after PDT for histological assessment. RESULTS: Under optimum conditions (10% intralipid diffusion medium, light dose 50J) uniform urothelial necrosis was seen after 1 to 2 days; it healed in 7 days without damage to the underlying muscle layer although some increase in collagen was seen in the lamina propria. Overtreatment or poor light distribution resulted in muscle necrosis and scarring. CONCLUSIONS: Selective urothelial necrosis is possible with PDT using intravesical ALA. There is now sufficient data for pilot clinical trials to start photodynamic therapy for management of superficial bladder cancer or carcinoma in situ. PMID- 8627878 TI - Relationship of p53 and bcl-2 to prognosis in muscle-invasive transitional cell carcinoma of the bladder. AB - PURPOSE: We examined the presence of the p53 and Bcl-2 oncoproteins, as detected by immunohistochemistry, in muscle-invasive bladder cancer and correlated this with survival. MATERIALS AND METHODS: Formalin-fixed cystectomy specimens from 41 consecutive patients with mean follow-up of 52 months were used. Five patients were either lost to follow-up or died of other diseases and were not included in the survival evaluation. RESULTS: Eighteen of 36 patients died of metastatic transitional cell carcinoma. p53 immunostaining was found in 61% of patients. In 21 of 23 this staining was homogeneous, with more than 75% of cancer cells staining using a DO-1/DO-7 antibody cocktail. p53 staining was not correlated with stage (p>0.25) or grade (p<0.10) in these invasive cancer specimens. Contrary to recent studies p53 immunostaining was not correlated with disease specific survival. Bcl-2 immunostaining was found in 28% of patients and was not correlated with grade (p>0.25) or disease-specific survival. No combination of p53 and Bcl-2 staining gave added predictive information. CONCLUSIONS: Cytoplasmic Bcl-2 is found in a small percentage of these cancers and does not correlate with prognosis. Further, p53 molecular overexpression is detected in the majority of muscle-invasive bladder tumors as a field defect. However, in patients undergoing cystectomy, it does not correlate with prognosis. PMID- 8627879 TI - Pharmacological characterization of the isolated canine prostate. AB - PURPOSE: The goal of the present study was to characterize the responses of the isolated normal canine prostate to various contracting and relaxing stimuli to determine which pharmacological agents may have utility against the dynamic component of benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Isometric force development was measured in isolated strips of prostate tissue. RESULTS: The alpha-adrenergic agonists were the most efficacious stimulants tested (phenylephrine EC50=2.1 microM.). Endothelin-1, acting primarily via ETA receptors, was more potent (EC50=27nM.) but less efficacious. Histamine (EC50=14.7 microM.), serotonin (EC50=0.12 microM.), carbachol (EC50=5.9 microM.) and KC1 (EC50=48.8 mM.) were also less efficacious than phenylephrine. Nifedipine was a potent (IC50=28 nM.) and efficacious (74% inhibition) inhibitor of phenylephrine-induced force. Potassium channel activator drugs were also efficacious relaxants, producing approximately 80% inhibition of force; rank order of potency was P1075 > cromakalim > diazoxide. Sodium nitroprusside was a weak relaxant, producing only approximately 40% relaxation at a concentration of 100 micronM. Both isoproterenol and forskolin were effective relaxants (75 to 90% relaxation). CONCLUSIONS: We conclude that potassium channel activators, adenylate cyclase stimulators, or endothelin antagonists may have utility against the dynamic component of outflow obstruction secondary to BPH. PMID- 8627880 TI - Phenylacetate is an inhibitor of prostatic growth and development in organ culture. AB - PURPOSE: Benign prostatic hyperplasia (BPH) is the most common proliferative disease affecting men, and the obstructive uropathy it causes results in serious morbidity and financial cost. Phenylacetate (PA) is a small molecule that is a product of phenylalanine metabolism and is normally present in the mammalian circulation at very low levels. It has long been safely used in humans to treat the hyperammonemia resulting from urea synthesis disorders and liver failure. It has recently been investigated as an anticancer agent because it decreased growth and increased differentiation of a variety of human neoplasms, including prostate cancer in which a phase I trial has recently been completed. MATERIALS AND METHODS: Because of PA's growth-inhibitory effects on a variety of cell lines and the idea that BPH is due to a reawakening of embryonic-like inductive activity in prostatic stromal cells, which then induce development of epithelial nodules, we examined the effect of PA on serum-free organ cultures of developing rat prostates. RESULTS: We found that PA markedly decreased rat prostatic growth and ductal morphogenesis at concentrations that have previously been well tolerated in patients. In ventral prostates grown for 7 days in organ culture, histodifferentiation was inhibited as measured by a marked decrease in ductal lumen formation and ductal branching morphogenesis. This inhibition of differentiation was confirmed by using cytokeratin antibodies specific for basal and luminal cells. Synthesis of DNA was also significantly decreased per organ with PA. The growth inhibitory effects of PA were reversible, and the mechanism did not appear to be due to glutamine or glycine deprivation, or androgen receptor inhibition. CONCLUSIONS: In common with earlier studies, we found that PA inhibits prostatic growth; however, in our organ culture system, differentiation was also largely inhibited. These studies indicate that there may be a role for PA in treating BPH or in elucidating the mechanism by which it occurs since BPH apparently involves the neoformation of ductal-acinar tissue in aged men via mechanisms fundamentally similar if not identical to those in fetal prostatic development. PMID- 8627881 TI - Argon laser induced autofluorescence may distinguish between normal and tumor human urothelial cells: a microspectrofluorimetric study. AB - PURPOSE: To assess the ability of argon laser-induced autofluorescence spectroscopy (LIAFS) to discriminate normal from tumor human urothelial cells. MATERIALS AND METHODS: Emission spectra of single living cells excited at 488 nm. have been studied with confocal microspectrofluorimeter. RESULTS: Cellular autofluorescence appeared as a broad band with a maximum in the same "green" spectral range, 550 to 560 nm., probably corresponding to oxidized flavoprotein emission. However, the maximum autofluorescence intensity of normal urothelial cells was much higher, 10 times (p<0.0001) that of any of the tumor cell types tested. CONCLUSIONS: These results, suggesting a significantly reduced oxidized flavoprotein concentration in tumor urothelial cells, should prompt us to evaluate argon LIAFS as a potential tool to detect occult urothelial severe dysplasia and carcinoma in situ. PMID- 8627882 TI - Antitumor effects of angiogenesis inhibitor 0-(chloroacetyl-carbamoyl) fumagillol (TNP-470) against murine renal cell carcinoma. AB - PURPOSE: The effect of 0-(chloroacetyl-carbamoyl) fumagillol (TNP-470) on tumor growth and metastasis is investigated. MATERIALS AND METHODS: BALB/c mice were inoculated with Renca murine tumor and graded doses of TNP-470 were given subcutaneously every other day beginning on day 1 and ending on day 9. Tumor angiogenesis was measured quantitatively by a colorimetric assay. RESULTS: TNP 470 inhibited tumor growth and prolonged the life span of Renca-bearing mice in a dose-dependent manner. Body weight-loss was not observed in the mice given less than 30 mg./kg./day. When the treatment was delayed on day 6, TNP-470 did not inhibit tumor growth, pointing to the importance of the timing of drug administration in relation to disease stage. Tumor angiogenesis was inhibited 33 to 62% of the control level by TNP-470. Furthermore, TNP-470 reduced pulmonary and hepatic metastatic foci of intravenously inoculated Renca and of the tumor inoculated in the spleen. CONCLUSION: These data suggest that TNP-470 may be effective as a treatment of renal cell carcinoma, especially when micrometastases are involved. PMID- 8627883 TI - Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells. AB - PURPOSE: Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necrosis factor are now being considered for clinical use. In this study, we used 2 renal cancer cell lines. SK-RC-42 and SK-RC-49, and determined the cytotoxic effects of tumor necrosis factor (TNF) and the possible mechanism of TNF resistance. MATERIALS AND METHODS: Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used. RESULTS: Cytotoxic assays with SK-RC-42 demonstrated that TNF at 50 ng./ml. for 24 hours resulted in 19% cytotoxicity of the cells. Similar assay with SK-RC-49 only demonstrated less than 4% cytotoxicity. Based on these results, we defined our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resistant cells. To determine whether protein kinase C (PKC), which is involved in the signal transduction pathway of a cell, could regulate endogenous basal TNF mRNA levels, comparative PCR analyses for TNF expression were used. The PCR results demonstrated that the TNF-resistant cell, SK-RC-49, had a higher basal expression of TNF mRNA than the TNF-sensitive cell SK-RC-42. With PMA, a PKC activator, for various time points, both cell lines demonstrated an induction of endogenous TNF mRNA. To further confirm our findings that PKC may regulate endogenous TNF expression, a PKC inhibitor, staurosporine, was used. When the cells were treated with staurosporine prior to PMA stimulation, no increase in TNF mRNA expression was seen. To determine whether PKC is involved in providing resistance against TNF, we incubated the SK-RC-42 and SK-RC-49 cells with staurosporine at 100 nM. and TNF at 50 ng./ml. for 24 hours. After factoring out the cytotoxicity of staurosporine, the TNF-mediated cytotoxicity increased to 39.3% and 28.7% for the SK-RC-42 and SK-RC-49 cells, respectively. Furthermore, by using a nuclease digestion assay, we demonstrated that TNF activated a Ca++/Mg++ dependent endonuclease responsible for programmed cell death or apoptosis. CONCLUSIONS: Our data suggest that protein kinase C may play a role in protecting renal cancer cells from undergoing cytokine-mediated apoptosis. PMID- 8627884 TI - Prevalence and clinical significance of HER/2neu, p53 and Rb expression in primary superficial bladder cancer. AB - PURPOSE: The usefulness of the expression of HER2/neu oncoprotein and of p53 and Rb suppressor gene product as predictors of tumor recurrence was evaluated by using a bank of prospectively collected primary papillary bladder tumors treated initially only by transurethral resection. MATERIALS AND METHODS: The expression of HER-2/neu oncoprotein and p53 and Rb suppressor genes was evaluated by immunohistochemistry in 256, 265 and 74 specimens of primary Ta/T1 superficial bladder tumors obtained from patients enrolled in a prospective study from 1990 to 1992. Survival analysis was used to evaluate the association between time to first recurrence and expression of each marker, the follow-up period extending to March 1994. Immunostaining for p53 and HER-2/neu was performed on paraffin embedded tissue and, for Rb, on frozen tissue only. RESULTS: Her-2/neu was expressed in 21 (8.2%) cases and p53 in 39 cases (14.7%); Rb expression was altered in 12 (16.2%). In this study, p53 expression was only related to earlier recurrence in a univariate analysis. Multivariate analysis, however, failed to recognize p53 expression as an independent predictor of recurrence. CONCLUSIONS: Our study demonstrates the low prevalence of HER2/neu, p53 and altered Rb expression in superficial TCC at initial resection. Only p53 expression was significantly associated with an earlier first tumor recurrence, but this association was not independent of other prognostic factors. PMID- 8627885 TI - Forskolin: a promising new adjunct to intracavernous pharmacotherapy. AB - PURPOSE: To evaluate the utility of forskolin as a potentially novel intracavernous therapy. MATERIALS AND METHODS: Forskolin- and prostaglandin E1 (PGE1)-induced intracorporal pressure changes were evaluated in vivo by cavernosometry performed on 2 male mongrel dogs, while systemic pressure changes were simultaneously monitored. Forskolin- and PGE1-induced intracellular cAMP accumulation was measured in vitro on homogeneous explant cultures of canine corporal smooth muscle cells. RESULTS: Forskolin and PGE1 elicited concentration dependent increases in cAMP accumulation in cultured canine corporal smooth muscle cells. Forskolin and PGE1 also elicited concentration-dependent increases in both the magnitude and duration of intracorporal pressure, up to a maximum of 80 to 90% of mean arterial pressure. Furthermore, the presence of threshold concentrations of forskolin was shown to significantly augment the activity of PGE1 both in vitro (increased cAMP) and in vivo (increased pressure). Moreover, there were no detectable systemic effects following the intracorporal injection of forskolin or a mixture of forskolin and PGE1. CONCLUSIONS: These observations suggest that the use of forskolin, alone or in combination with other drugs that increase intracellular cAMP levels, might represent an attractive opportunity for improved and more rational development of next generation intracavernous pharmacotherapeutic agents. PMID- 8627886 TI - Study of etiologic relationship of arterial atherosclerosis to corporal veno occlusive dysfunction in the rabbit. AB - PURPOSE: The aim of this study was to explore the possible etiologic relationship of hypercholesterolemia and atherosclerosis to corporal veno-occlusive dysfunction. MATERIALS AND METHODS: In the New Zealand White rabbit, the competence of the corporal veno-occlusive mechanism was examined at various intervals after exposure to control diet, high cholesterol diet, or aortoiliac atherosclerosis. RESULTS: Initially, all animals showed normal erectile function and corporal veno-occlusion. After 8 weeks and 16 weeks, the control animals preserved normal erection and corporal veno-occlusion, while most of the hypercholesterolemic and atherosclerotic animals developed corporal veno occlusive dysfunction. The incidence of corporal veno-occlusive dysfunction in the hypercholesterolemia and atherosclerotic animals increased with time. CONCLUSIONS: This study suggests that a close relationship exists between prolonged atherosclerotic occlusion of major penile arteries and the development of corporal veno-occlusive dysfunction. Ischemia-induced corporal veno-occlusive dysfunction may be the result of alterations in corporal smooth muscle relaxation or changes in the structure and fibroelastic properties of erectile tissue. PMID- 8627887 TI - Decreased urine glycosaminoglycan excretion in cats with interstitial cystitis. AB - PURPOSE: The urine protein and glycosaminoglycan (GAG) excretion of normal cats was compared with that of cats suffering from interstitial cystitis (IC), which is reported to decrease urine GAG excretion in humans. MATERIALS AND METHODS: Total urine GAG concentration was measured in random and 24-hour urine specimens by spectrophotometry by 1,9-dimethylmethylene blue (DMB) chloride. RESULTS: In both random and 24-hour urine samples, GAG and protein concentrations and GAG:creatinine and protein:creatinine ratios all were significantly lower in cats with IC. Total GAG and protein excretion also were significantly lower in 24-hour urine samples from cats with IC. CONCLUSIONS: GAG excretion was decreased in cats with IC, as it is in humans. It is unclear whether this is due to changes in synthesis, metabolism, or bladder permeability. PMID- 8627888 TI - Transfemoral endovascular repair of abdominal aortic aneurysm: results of the North American EVT phase 1 trial. EVT Investigators. AB - PURPOSE: This report describes the results of a phase 1 trial of endovascular repair of abdominal aortic aneurysm, conducted under FDA protocol in 13 U.S. medical centers from February 1993 to December 1994. METHODS: Forty-six patients 54 to 84 years of age underwent endovascular repair of abdominal aortic aneurysm (diameter, 3.8 to 7.1 cm). Fifteen were treated with the original device (EGS-I), and 31 with a revised over-the-wire system (EGS-II). All patients were periodically observed with contrast-enhanced computed tomographic scan, color flow duplex scan, and plain abdominal films to evaluate the stability of prosthetic location and to detect any vascular communication with or entry of blood into the aneurysm sac or change in aneurysm size. RESULTS: Thirty-nine implants (85%) were successful; average operating time was 194 minutes. Seven attempts were unsuccessful and were converted to open repair without complication (EGS-I, 5 of 15; EGS-II, 2 of 31). Conversions were caused by iliac stenosis in four patients, subintimal deployment in one, proximal displacement in one, and short distal neck in one. No patients died within 30 days of surgery. Complications included myocardial infarction in one patient, iliofemoral arterial injury in eight, wound infection in seven, required transfusion in eight, transient unexplained fever in nine, and minor emboli with foot petechiae in two. There were no amputations, major emboli, or episodes of mesenteric ischemia. Contrast enhancement outside the graft but within the aneurysm sac was detected initially in 17 grafts (44%), of which nine (53%) resolved spontaneously. Of eight persistent leaks into the aneurysm sac, one was controlled with transluminal balloon angioplasty and one required surgical explanation because of aneurysm enlargement. Six patients continued to have contrast enhancement, but had no evidence of aneurysm enlargement from 6 to 27 months after surgery. Hospital stay averaged 3.8 days (range, 1 to 13 days). Follow-up extends to 27 months, with one non-device related death of respiratory failure at 6 months. Metallic attachment system fracture, a device-related malfunction, was identified in nine implants (23%), which led to one removal; the remaining eight functioned normally with no untoward sequelae. The program was suspended while the defect was corrected. Preparations are complete for the phase 2 portion of the trial. CONCLUSIONS: Endovascular repair of abdominal aortic aneurysm appears to be safe and efficacious. Long-term results and late consequences of attachment system fracture have yet to be determined. The long-term results of perigraft leak into the aneurysm sac are unknown but worrisome in view of adverse outcomes reported by other investigators. PMID- 8627889 TI - Mid-term and long-term results with directional atherectomy of vein graft stenoses. AB - PURPOSE: The purpose of this study was to evaluate the outcomes of our 6-year experience with directional atherectomy used for treatment of stenoses in infrainguinal vein grafts. METHODS: From March 1988 to April 1994, 52 directional atherectomy procedures were undertaken in 42 patients to treat 67 stenoses in 44 vein grafts. Follow-up consisted of periodic physical examinations and graft surveillance; ankle/brachial indexes, pulse volume recordings, and color-flow duplex ultrasonography. Follow-up angiography (n = 18) was performed for recurrent symptoms, reproducible drop in ankle/brachial index of greater than 0.15, a twofold to threefold focal increase in peak systolic velocity, or incidentally during evaluation of the opposite leg. RESULTS: Forty-nine of 52 (94%) procedures were technically successful. In two the residual diameter stenosis was greater than 30%, and in one atherectomy could not be performed. Complications were minor in six (11%) and major in three (6%): two acute graft occlusions and one delayed pseudoaneurysm at the atherectomy site. There were no deaths at 30 days. With a mean follow-up of 21 +/- 18 months, 36 of 44 grafts (82%) remained patent without restenosis; 6 others were patent but considered "failed"--5 (11%) with restenosis, 1 with a pseudoaneurysm; and 2 grafts (5%) occluded. Clinically 33 of 44 extremities (75%) were asymptomatic during follow up. Claudication improved in five, recurred in three, and was unchanged in one. There was one below-knee amputation. Life-table analysis including all 52 procedures reveals cumulative primary atherectomy patency rates for the 44 grafts of 82%, 78%, and 78%, respectively, at 1, 2, and 3 years after atherectomy, and 86%, 83%, and 83% for the 67 individual stenoses treated. CONCLUSIONS: Directional atherectomy of vein graft stenoses has high technical and clinical success rates, acceptably low morbidity rates, and offers better sustained patency rates than balloon angioplasty. Its long-term patency rate seems to approach that of surgical vein patch angioplasty. PMID- 8627891 TI - Long-term follow-up of patients with early atherosclerosis. AB - PURPOSE: Patients with premature peripheral vascular disease may respond differently than their older counterparts. To determine the impact of early onset of atherosclerosis on outcome, we decided to compare a group of these patients with a group of patients with typical onset of atherosclerosis with regard to early complications, indications for intervention, site of disease at initial presentation (aortoiliac, infrainguinal, or cerebrovascular), and long-term outcomes (secondary revascularization, amputation, and death). METHOD: All patients younger than 50 years old requiring operative intervention between 1987 and 1992 were retrospectively compared with a group of patients greater than 60 years old, randomly selected from patients who underwent operation during the same time period. Patients were evaluated and compared for indications, risk factors, and early and late outcomes. RESULTS: Patients with early onset atherosclerosis at the aortoiliac or infrainguinal level had a higher late amputation rate (17% versus 3.9%, p = 0.02) and poorer overall outcome than their older cohorts. Patients with cerebrovascular disease in both cohorts had similarly good prognoses. CONCLUSION: Aortoiliac or infrainguinal disease diagnosed in patients less than 50 years of age portends a poorer outcome than does similar disease in an older patient population. PMID- 8627890 TI - Progression and regression of myointimal hyperplasia in experimental vein grafts depends on platelet-derived growth factor and basic fibroblastic growth factor production. AB - PURPOSE: The factors that lead to myointimal hyperplasia (MH) in arterial vein grafts (AVGs) are unknown. Platelet-derived growth factor (PDGF) and basic fibroblastic growth factor (bFGF) are two powerful mitogens for smooth muscle cells that have been implicated in the genesis of MH. The aim of this study was to analyze the correlation between progression and regression of MH and production of PDGF and bFGF in experimental vein grafts. MATERIALS: In 64 inbred Lewis rats, a 1-cm segment of inferior vena cava was inserted at the level of the abdominal aorta. The segments of inferior vena cava were obtained from syngenic rats. In 48 rats, the AVG was explanted 3 days (n = 8), 7 days (n = 8), 4 weeks (n = 24), and 12 weeks (n = 8) after surgery. In 16 rats the vein graft was explanted after being in the arterial system for 4 weeks and was reimplanted as a venous-venous bypass in syngenic Lewis rats. Reimplanted vein grafts (RVGs) were explanted 2 weeks (n = 8) and 8 weeks (n = 8) later. Grafts were analyzed by light and electron microscopy, morphometry, and histochemistry, and were put in organ culture to assess PDGF and bFGF production and mitogenic activity. RESULTS: We observed MH formation in AVGs and MH regression in RVGs (p < 0.001).PDGF and bFGF production correlated with the degree of MH (p < 0.01). Histochemistry showed PDGF and bFGF in the area of MH in AVG, which disappeared in RVG. Conditioned media from AVG had greater mitogenic activity than RVG or control veins. CONCLUSION: MH formation and regression in experimental vein grafts correlate with PDGF and bFGF production. PMID- 8627893 TI - Extracranial internal carotid artery aneurysms: results of a surgical series with long-term follow-up. AB - PURPOSE: The purpose of this study was to analyze mode of presentation, surgical treatment, and early and long-term results of a series of extracranial internal carotid artery aneurysms (EICAA). METHODS: A retrospective analysis was performed on all cases treated for EICAA in a single institution from March 1974 to March 1995. Patient follow-up was obtained by a surveillance protocol, with duplex scanning performed 3 months after surgery and yearly thereafter. RESULTS: Twenty four EICAA in 20 patients were treated over a 21-year period. The cause was fibromuscular dysplasia in 12 cases (50%), nonspecific "atherosclerosis" in nine (37.5%), previous carotid artery surgery in two (8.3%), and trauma in one case (4.1%). Neurologic symptoms were present in a total of nine cases (37.5%) and were hemispheric in seven (29.1%) and nonhemispheric in two (8.3%). Operative techniques were performed with patients receiving general anesthetic and included aneurysm excision with internal carotid artery reanastomosis (8 cases [33.3%]) or reimplantation onto the external carotid artery (1 case [4.1%]); interposition graft (10 cases [41.6%]), 7 veins, 3 polytetrafluoroethylene) or simple aneurysmectomy and closure of the wall defect either with (3 cases [12.5%]) or without (2 cases [8.3%]) a patch. Elective surgery was performed in 22 cases, with a 0% mortality rate and 4.5% stroke rate. Emergency operations were performed in two cases of ruptured aneurysms (one spontaneous and one iatrogenic); one patient (50%) died. Cranial nerve morbidity occurred in five cases (20.8%). Mean follow-up was 96.7 +/- 88.15 months (range 4 to 240 months) and included 2 of 7 (28%) complications in saphenous vein grafts, 1 (4.1%) late transient ischemic attack, and a recurrent aneurysm after 19 years. CONCLUSIONS: Symptoms and potential complications caused by EICAA suggest a broad surgical indication. EICAA can be treated safely because of the good early and long-term results. PMID- 8627892 TI - The natural history of the external carotid artery after carotid endarterectomy: implications for management. AB - PURPOSE: Most surgeons perform some type of endarterectomy of the external carotid artery (ECA) routinely during standard carotid endarterectomy (CEA). This approach has been shown to result in a small percentage of ECA occlusions, the clinical significance of which remains poorly understood. We have modified our approach to the management of the ECA during standard CEA by averting any attempt at external CEA. To evaluate the natural history of the untreated ECA after CEA, we reviewed the preoperative, postoperative, and follow-up duplex scans obtained from 232 CEAs over the past 4 years. METHODS: Preoperative and postoperative carotid artery duplex examinations with specific evaluation of the extent of ECA stenosis were available for review on 114 CEAs performed between January 1991 and July 1994. All CEAs were performed for internal carotid artery stenosis greater than 75% as determined by duplex scanning, which was confirmed by either contrast arteriography or magnetic resonance angiography. RESULTS: Seventy-three (64.0%) procedures were performed for symptomatic lesions, whereas 41 (36.0%) were performed for asymptomatic stenosis. There were no perioperative strokes or transient ischemic attacks in this group, and there was one postoperative death (0.9%). Short- and intermediate-term follow-up demonstrated insignificant changes in ECA diameter after operation, with no cases of ECA occlusion and only five cases progressing to greater than 75% on the 1-year follow up duplex examination. CONCLUSION: We conclude from these data that averting external CEA during standard CEA does not result in significant progression of ECA stenosis or occlusion. PMID- 8627894 TI - Hypertension-enhanced monocyte adhesion in experimental atherosclerosis. AB - PURPOSE: Hypertension is a known clinical risk factor for atherosclerosis. In experimental atherosclerosis, monocyte adhesion to the endothelial surface is enhanced and is considered to be an important early stage in plaque formation. We tested the hypothesis that hypertension enhances monocyte adhesion in experimental atherosclerosis. METHODS: Twenty-two New Zealand White rabbits were fed an atherogenic diet for 3 weeks to induce plaque formation. Aortic coarctation was created in eight rabbits by wrapping a Dacron band around the midportion of the descending thoracic aorta (stenosis group), whereas six rabbits underwent banding without aortic constriction (no stenosis group). Eight rabbits served as nonoperated controls. Monocyte binding to the aortic endothelial surface was counted with epifluorescent microscopy on standard aortic segments proximal and distal to the band. Immunohistochemistry was performed for the following antibodies: VCAM-1, RAM11, CD11b, and factor VIII. RESULTS: Mean blood pressure was 89 +/- 3 mm Hg in the aorta proximal to the stenosis, compared with 64 +/- 4 mm Hg in the no stenosis group and 74 +/- 3 mm Hg in the control group (p < 0.01). The mean aortic blood pressure gradient across the stenosis was 16 +/ 2 mm Hg in the stenosis group, whereas the aortic blood pressure gradient was 0.2 +/- 0.6 mm Hg in the no stenosis group and -0.3 +/- 0.4 mm Hg in the control group (p < 0.001). Monocyte adhesion to the aortic endothelial surface proximal to the stenosis was increased twofold compared with adhesion to the aorta distal to the stenosis and compared with the proximal aorta in the control group (p < 0.02). The proximal-to-distal aortic ratio of monocyte binding was enhanced in the stenosis group (2.2) compared with the no stenosis (0.76) and control (0.83) groups (p < 0.01). The intima area of the aorta proximal to the stenosis was significantly increased compared with the proximal aortas in the no stenosis and control groups (p < 0.01). RAM11, CD11b, and endothelial VCAM-1 expression were enhanced in the hypertensive region proximal to the stenosis. CONCLUSIONS: In the hypertensive region in the aorta proximal to the stenosis, monocyte adhesion and endothelial VCAM-1 expression were increased, with intimal thickening and accumulation of macrophages. These findings suggest that hypertension may promote atherosclerotic plaque formation by enhancing monocyte adhesion. PMID- 8627895 TI - Duplex-derived valve closure times fail to correlate with reflux flow volumes in patients with chronic venous insufficiency. AB - The best way to quantitate venous reflux is still a matter of debate. Duplex derived valve closure time (VCTs) have been used recently because they can be measured easily. We examined the relationships between VCT and duplex-obtained quantitation of venous volume and between VCT and air plethysmography (APG). Sixty-nine legs in 45 patients with varying clinical degrees of chronic venous insufficiency were studied by duplex scan and APG. VCTs were compared with duplex derived flow calculations and with APG-derived venous filling index and residual volume fraction. The patient's mean age was 47.5 +/- 13.9 years; the mean duration of their symptoms was 13 +/- 4 years. Twenty percent had a history of deep venous thrombosis, and 29% had undergone venous surgery. No correlation was found between VCT and flow volume or between VCT and flow at peak reflux at any of the anatomic locations studied: saphenofemoral junction, greater saphenous vein, lesser saphenous vein, superficial femoral vein, profunda femoris vein, and popliteal vein. Likewise, no correlation was found between total VCT and APG derived venous filling index or between total flow volumes and APG-derived residual volume fraction. Total VCT and total flow volumes did, however, have a moderate correlation (r = 0.65; p = 0.0003). Duplex-derived VCTs, although extremely useful in determining the presence of reflux, do not correlate with the magnitude of reflux, and should not be used to quantitate the degree of reflux. PMID- 8627896 TI - Valvular function of peripheral veins after hyperemic dilation. AB - PURPOSE: We wanted to answer the question of whether physiological dilation of normal extremity veins can induce temporary valvular leakage and reflux. METHODS: Directional flow was recorded in 22 forearm and popliteal veins by Doppler duplex scanning after distal compression. Reflux was assessed by valve closure time and calculation of a "reflux index," the ratio of backward to forward flow areas. RESULTS: Hyperemia and enhanced flow did not increase but lowered reflux. During control conditions the mean reflux ratio (backward/forward flow area) of 21 veins was 0.058 and decrease to 0.028 (p < 0.05). Reflux was slightly longer in patients in the erect position in the popliteal vein, compared to forearm veins (with the patients in the sitting position). Significantly increased reflux occurred during hyperemia in only one deep forearm vein (valve closure time 0.92 seconds). CONCLUSIONS: Most veins of the upper (forearm) and lower extremity (popliteal vein) were competent even after a maneuver that induced venodilation and an increase in blood flow (exercise hyperemia or postocclusion reactive hyperemia). Veins with an inherent valvular weakness can be identified by a hyperemia test with duplex flow analysis. PMID- 8627897 TI - Laboratory assays and duplex scanning outcomes after symptomatic deep vein thrombosis: preliminary results. AB - PURPOSE: The purpose of this article was to assess a number of hematologic and fibrinolytic assays at the time of diagnosis of deep vein thrombosis (DVT) and at several intervals over a period of 6 months afterward and to correlate these results with the results of serial duplex scanning. METHODS: Thirty-five patients (average age 61, range 18 to 82) with acute symptomatic DVT confirmed by duplex scanning were included. On diagnosis, blood was drawn, and plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI), D-dimer (DD), and tissue factor pathway inhibitor (TFPI) were determined. Duplex scanning and all laboratory assays were repeated 1 week, 1 month, 3 months, and 6 months thereafter. RESULTS: The rate of DVT complete resolution 6 months after diagnosis was 57%. Whereas plasma levels of PAI were similar throughout the 6 month follow-up period, t-PA increased significantly 1 week after diagnosis and decreased thereafter. Both DD and TFPI levels decreased significantly after diagnosis compared with presentation values. Comparing these assay levels between patients with complete resolution versus partial or no resolution, PAI levels were significantly higher during the first week in patients with poor outcome. Plasma levels of t-PA were higher in cases with good outcome, and DD levels were higher in patients with poor outcome. TFPI levels were similar in both outcome groups. CONCLUSIONS: Patients with complete DVT resolution on duplex scanning at 6 months had significantly lower levels of PAI on presentation and after 1 week than did those with incomplete lysis. Although differences were not significant, t-PA levels were higher and DD lower in patients with good outcome. Our results suggest that certain plasma fibrinolytic assays might correlate with the outcome of DVT, as assessed by duplex ultrasonography. PMID- 8627898 TI - The changing face of carotid endarterectomy. AB - PURPOSE: The economic milieu and improvements in care have altered the diagnostic and therapeutic algorithm of the patient with carotid stenosis. This study analyzes the efficacy and safety of these changes. METHODS: The records of patients who underwent 320 consecutive carotid endarterectomies performed by three surgeons at our institution from 1990 to 1994 were reviewed retrospectively. Use of diagnostic angiography, use of carotid duplex ultrasound, length of hospital stay, postanesthesia recovery observation, intensive care unit (ICU) observation, complications, and hospital charges were analyzed. RESULTS: The average length of hospital stay decreased from 6.18 days to 2.00 days (p < or = 0.001). The day of discharge decreased from 3.10 days to 1.24 days after surgery (p < or = 0.01). By 1993, 68% were discharged by the first day after surgery, increasing to 73% by 1994. From 1990 to 1992, average postoperative ICU observation time fluctuated between 18 and 25 hours; this time decreased to 12.2 hours by 1994. In 1993, only 12.5% of patients were admitted to the ICU, down from 94.8% in 1990; by 1994, only 7.3% were admitted to the ICU (p < or = 0.001). Postanesthesia recovery observation time decreased from 3.77 hours to 1.63 hours during this time (p < or = 0.04). With regard to preoperative diagnosis, angiography was performed in 93.1% of patients in 1990; by 1994, only 32.8% underwent this procedure (p < or = 0.0001). Average hospital charges decreased significantly (1990, $14,378; 1994, $10,436) with these modifications in patient care (p < or = 0.001). The complication rate reflected no significant changes over the course of the study. There were six incidences of cerebrovascular accident (6/320, 1.9%), including one death. There were four incidences of transient ischemic attack (4/320, 1.3%), with no significant differences noted from year to year. CONCLUSIONS: This study confirms the changing nature of carotid endarterectomy and documents that these changes have not adversely affected the safety of the operation. PMID- 8627899 TI - Duplex velocity characteristics of aortoiliac stenoses. AB - PURPOSE: It is now possible to grade aortoiliac stenoses in broad categories based on peak systolic velocity (PSV) changes. The goal of this study was to see whether additional simple Doppler parameters would improve the grading of aortoiliac obstructive disease. METHODS: Duplex parameters were compared in 112 aortoiliac segments with four categories of arteriographic diameter reduction and four categories of common femoral artery pressure measurements. These parameters were PSV, PSV ratio, PSV difference, end diastolic velocity (EDV), the presence or absence of reverse flow, and the presence or absence of spectral broadening. RESULTS: The discriminative value of the PSV ratio was better than that of either the absolute PSV value or the PSV difference. A PSV ratio < 1.5 combined with reverse flow and a clear systolic window in the Doppler spectrum predicted a diameter reduction < 20% (sensitivity, 100%; specificity, 58%; positive predictive value [PPV], 89%; negative predictive value [NPV], 100%; accuracy, 90%). For the detection of > or = 50% aortoiliac stenoses, a PSV ratio > or = 2.8 provided 86% sensitivity and 84% specificity (PPV, 84%; NPV, 85%, accuracy, 85%). An absolute PSV value of 200 cm/sec has a high sensitivity (95%) but a low specificity (55%) in identifying > or = 50% stenoses (PPV, 68%; NPV, 91%; accuracy 75%). An EDV > 0 cm/sec at the stenosis indicates a femorobrachial pressure index < 0.90 with 51% sensitivity and 89% specificity. An EDV > or = 40 cm/sec indicates a femorobrachial index < 0.80 with 50% sensitivity and 89% specificity, and indicates > or = 75% arteriographic stenoses with 70% sensitivity and 90% specificity (PPV, 64%; NPV, 92%; accuracy, 86%). A stenosis > or = 75% was also identified by a PSV ratio of 5.0 with 65% sensitivity and 91% specificity (PPV, 65%; NPV, 91%; accuracy, 86%). CONCLUSIONS: The PSV ratio is the most important parameter to grade aortoiliac stenoses into < 20%, 20% to 49%, 50% to 74%, and 75% to 99% categories, but additional parameters such as absolute PSV value, EDV, and the presence or absence of reverse flow and spectral broadening in the Doppler spectrum are helpful in gradation. PMID- 8627900 TI - Is completion arteriography mandatory after reversed-vein bypass grafting? AB - Many surgeons advocate uniform performance of operative completion arteriography after leg bypass surgery to ensure technical success and to optimize short- and intermediate-term graft patency. To determine the impact of this practice on the outcome of reversed-vein bypass surgery and associated patient charges, we reviewed our series of consecutive nonemergent leg bypass procedures. Ninety three infrainguinal bypass procedures were performed in 80 patients (76 men and 4 women) from September 1991 to August 1994. The patients' average age was 67 years (range, 30 to 92 years). Follow-up (mean, 113.1 months; range, 1 to 36 months) was available on 91 grafts (97%). Indications for surgery included limb salvage in 75 cases, claudication in 12 cases, and popliteal aneurysm exclusion in 6 cases. All patients survived surgery. Primary graft patency rates as determined by life-table analysis were 87%, 81%, 78%, and 78% at 6 months and at 1, 2, and 3 years, respectively. Limb-salvage rates were 95%, 91%, 87% and 87% at the same intervals. Bypass procedures were divided into two groups. The 25 grafts in group 1 were evaluated with inspection, continuous-wave Doppler insonation, and routine completion arteriography. The 68 grafts in group 2 were evaluated by inspection and insonation alone. Fourteen grafts occluded after surgery (average, 5 months; range, 1 to 12 months), five in group 1 and nine in group 2. The likelihood of graft occlusion was similar in both groups (p = 0.42). The optimal method of confirming technical adequacy after bypass surgery in the clinically satisfactory graft remains uncertain. Charges for completion arteriography at our institution average $700, including 15 minutes of additional operative time. In our experience, these charges do not appear to be justified by improved short- or intermediate-term graft patency rates in reversed-vein grafts when completion arteriography is performed. PMID- 8627901 TI - Carotid artery stenosis in peripheral vascular disease. AB - PURPOSE: The goal of the study was to assess the prevalence and severity of symptomatic and asymptomatic carotid artery disease in patients with peripheral vascular disease (PVD). METHODS: Consecutive patients with clinically and Doppler scanning-proven PVD (category 1 or greater) underwent prospective screening for the presence of carotid atherosclerosis with color-coded duplex ultrasonography. Preexisting risk factors were recorded with a standard questionnaire and included sex, age, diabetes mellitus, history of smoking, hypertension, prior stroke/transient ischemic attacks, and coronary artery disease. RESULTS: Three hundred seventy-three consecutive patients were studied over 2 years. The mean age of the patients was 70 +/- 10 years; there were 223 (60%) men and 150 (40%) women; 71% of the patients had a history of smoking, 47% had coronary artery disease, 43% had hypertension, and 21% had diabetes mellitus. Two hundred eleven (57%) patients had 30% or greater carotid artery stenosis detected by carotid artery duplex scanning. Sixty-seven (32%) of these had symptoms of ischemic cerebral events, of whom 22 had potentially operable carotid artery stenoses (70% to 99%), whereas 72 of the 144 symptom-free patients had 60% to 99% stenosis. An additional 34 patients would be eligible candidates for the ongoing carotid endarterectomy trials (North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial). Although all the risk factors were associated significantly with PVD and carotid artery disease (p < 0.002), male sex and prior stroke/transient ischemic attack were the strongest predictors. CONCLUSIONS: Routine carotid ultrasound screening of 373 consecutive patients with category I or greater PVD revealed that 22 patients with symptoms and 72 symptom-free patients were potential surgical candidates, representing 25% of the study cohort. An additional 34 patients were potential candidates for enrollment into the North American Symptomatic Carotid Endarterectomy Trial and European Carotid Surgery Trial. PMID- 8627902 TI - Inhibition of vein graft intimal and medial thickening by periadventitial application of a sulfated carbohydrate polymer. AB - PURPOSE: The purpose of this study was to determine whether the wall thickening observed in vein grafts after they were placed into the arterial circulation could be inhibited by periadventitial delivery of an insoluble sulfated polymer of beta-cyclodextrin (P-CDS) capable of tightly binding heparin binding growth factors. METHODS: Thirty-four New Zealand white rabbits underwent implantation of reversed autologous jugular vein interposition grafts in the common carotid artery and were randomized to receive either 20 mg P-CDS (n = 18) topically around the graft or no additional therapy (n = 16). Before being killed at 28 days, animals were given bromodeoxyuridine to assess smooth muscle cell proliferation. Histomorphometric analyses were performed after perfusion fixation. RESULTS: Compared to controls, treatment with P-CDS was associated with reduced mean intimal thickness (24 +/- 3 vs 38 +/- 4 microns; mean SEM, p < 0.01) and intimal area (0.25 +/- 0.03 vs 0.54 +/- 0.09 mm2; p < 0.01). There was also significantly less medial thickness in the P-CDS group (45 +/- 3 vs 63 +/-3, p < 0.001). There was no significant difference in intimal or medial smooth muscle cell proliferation between P-CDS-treated and control vein grafts at 28 days. The polymer persisted in the adventitia with a mild foreign body reaction. CONCLUSION: Periadventitial placement of P-CDS, a novel, insoluble, sulfated carbohydrate polymer, inhibits intimal and medial thickening of vein bypass grafts in this model of vein grafting. The persistence of P-CDS in vivo for prolonged periods, and the ease of topical application of P-CDS during vascular bypasses may have important implications for its future use in vascular surgery. PMID- 8627903 TI - Thrombolysis with prourokinase versus urokinase: an in vitro comparison. AB - PURPOSE: Despite advantages demonstrated in vitro, no single thrombolytic agent has been clearly shown to be superior to another in the clinical setting. Prourokinase has recently received attention as a new thrombolytic agent with higher fibrin specificity. The thrombolytic activity of prourokinase, however, remains ill defined. The purpose of this study was to evaluate thrombolysis with prourokinase in comparison to urokinase in vitro. METHODS: We used an in vitro parallel channel perfusion model that simulates catheter-directed thrombolysis in the peripheral arterial system. Radiolabeled thrombi were subjected to 90 minutes of endhole catheter-directed infusion with either prourokinase 5000 IU/ml, urokinase 5000 IU/ml; or 5% dextrose in water at 4 ml/hr. RESULTS: Prourokinase and urokinase were found to be equivalent with respect to thrombolytic effect. Percent lysis was maximal at 90 minutes in both the urokinase and prourokinase groups. Prourokinase and urokinase were found to be equally effective in restoring flow through thrombosed graft segments. CONCLUSION: Prourokinase appears to offer little benefit over urokinase with respect to thrombolytic activity in an in vitro model that closely resembles the clinical setting. If prourokinase is to be accepted as an alternative to urokinase, advantages must relate to differences in fibrin specificity. PMID- 8627904 TI - Effect of carotid artery geometry on the magnitude and distribution of wall shear stress gradients. AB - PURPOSE: Recent information indicates that large, sustained wall shear stress gradients are a dominant hemodynamic parameter associated with the location and severity of atherosclerosis and myointimal hyperplasia. This study computes the spatial values of wall shear stresses and their gradients for three carotid artery bifurcation geometries. METHODS: A computational fluid dynamics program was used to solve the transient two-dimensional partial differential equations that describe fluid flow. Blood was treated as both a Newtonian and a non Newtonian incompressible fluid. Solutions for the velocities, wall shear stresses, and wall shear-stress gradients were obtained for three carotid bifurcation geometries: a normal carotid bifurcation (similar to a primarily reconstructed carotid endarterectomy), a patch-reconstructed carotid endarterectomy, and a gradually tapered, low-angle carotid bifurcation (no carotid bulb). RESULTS: Computed velocity profiles closely match published experimental ones. Disturbed flow velocities are largest in the bulb segment of the normal carotid bifurcation. Peak and minimum wall shear stresses and peak shear stress gradients occurred in the lateral internal carotid artery wall. These were binodal in the normal or primarily reconstructed carotid artery, localized at the distal end of the patch-reconstructed carotid bifurcation, and minimal in the smooth, tapered carotid bifurcation. Wall shear stresses and their gradients were slightly higher for non-Newtonian than Newtonian fluids in the normal carotid artery but were similar in the other two geometric configurations. CONCLUSION: These results indicate that flow disturbances in general and wall shear stress gradients in particular are markedly reduced in carotid artery bifurcations that are smooth and gradually tapered and do not have a bulb. Abrupt geometric wall changes such as those occurring in the normal carotid bulb and at the distal end of a patch-reconstruction after carotid endarterectomy are harbingers of disturbed flow and high wall shear stress gradients. These results suggest that carotid endarterectomy reconstruction geometry characterized by a gradually tapered internal carotid artery may minimize the hemodynamically induced component of early myointimal hyperplasia and thrombosis and late atherosclerotic restenosis. PMID- 8627905 TI - Efficacy of balloon angioplasty of the superficial femoral artery and popliteal artery in the relief of leg ischemia. AB - PURPOSE: To assess the efficacy of balloon angioplasty in the superficial femoral artery (SFA) and popliteal artery for relief of lower-limb claudication and critical ischemia. METHODS: All patients who underwent SFA or popliteal balloon angioplasty at the Royal Adelaide Hospital between January 1989 and September 1994 were reviewed. Risk factors, indications, angiographic variables, and complications were assessed. Outcome was expressed in life-table form as patency, limb survival, and patient survival rates. RESULTS: One hundred seventy-six patients (96 men, 80 women) who underwent 200 balloon angioplasty procedures were monitored for a mean of 25 months. Seventy-four percent of procedures were for claudication relief and 26% for critical ischemia. The cumulative patency rate at 24 months for all cases was 46%. The limb salvage rate was 95%, and the patient survival rate was 91% at 24 months. CONCLUSION: Percutaneous transluminal angioplasty of the SFA and popliteal arteries is commonly used to treat claudication and critical ischemia but is associated with a high initial failure rate and poor patency at 24 months. Balloon angioplasty is not recommended to treat claudication. PMID- 8627906 TI - Albumin-coated vascular prostheses: A five-year follow-up. AB - In June 1989, we set out to implant 200 albumin-coated aortic bifurcation grafts and to track the patients for a period of 5 years to determine whether coating the prosthesis with albumin affected the patency or the incidence of complication. Two hundred and one prostheses were implanted between June 1989 and July 1991. The primary and secondary patency at 5 years was 95% and 98%, respectively. No relation between gender and patency or between the state of the runoff and patency was found, but there was a statistically significant relation between age and patency (p = 0.00). Graft infection was recorded in three patients (1.5%). There were no instances of bleeding through the graft at the time of implantation. The mean intra- and postoperative blood requirement was 2 units. There have been no incidences of false aneurysm in the groin. We conclude that there are no disadvantages of coating the prosthesis with albumin, and a trial of coated versus uncoated prostheses would be impractical. PMID- 8627907 TI - Iliac artery stenoses after percutaneous transluminal angioplasty: follow-up with duplex ultrasonography. AB - PURPOSE: To assess iliac artery stenosis before and up to 1 year after percutaneous transluminal angioplasty (PTA) with duplex ultrasound (DUS) to determine the incidence of residual and recurrent stenoses and correlate these findings to clinical outcome. PATIENTS AND METHODS: Sixty-one patients with 70 iliac artery segments treated with PTA were examined. The peak systolic velocity (PSV) ratio (PSV ratio = PSV in stenosis divided by PSV proximal or distal to stenosis) was determined by DUS before PTA and 1 day, 3 months and 1 year after PTA. Three categories of results were identified by using PSV ratios at the site of the treated stenosis 1 day and 1 year after PTA (good result, residual stenosis, and recurrent stenosis). The DUS-determined anatomic result was correlated with the clinical outcome at 1 year. Clinical outcome was classified according to Society for Vascular Surgery/International Society for Cardiovascular Surgery (SVS/ISCVS) criteria. RESULTS: Good results with DUS (PSV ratio 1 day and 1 year after PTA > or = to 2.5) were found in 45 of 70 segments (64.3%), residual stenoses (PSV ratio > or .5 1 day after PTA) in 15 of 70 segments (21.4%), and recurrent stenosis (PSV ratio 1 day after PTA < 2.5 and 1 year after PTA > or = 2.5) in 10 of 70 segments (14.3%). PSV ratios of residual stenoses decreased significantly between 1 day and 1 year after PTA because some residual stenoses improved hemodynamically in time. Clinical results were significantly better in patients with a good result compared with other patients. However, the clinical outcome of patients with residual stenoses was not significantly different from the patients with good DUS results. CONCLUSION: Some residual stenoses improved sonographically after PTA. Clinical results at 1 year are highly variable within different groups. Clinical outcome of patients with residual stenoses did not differ from patients with good DUS results, whereas clinical outcome in patients with recurrent stenoses was worse than in the other groups. PMID- 8627908 TI - Differential modulation of vascular endothelial and smooth muscle cell function by photodynamic therapy of extracellular matrix: novel insights into radical mediated prevention of intimal hyperplasia. AB - PURPOSE: Photodynamic therapy (PDT) has been demonstrated to inhibit experimental intimal hyperplasia and to lead to expedient reendothelialization but negligible repopulation of the vessel media. The mechanism that underlies the differential ingrowth of cells into PDT-treated vessel segments is not understood. Because the extracellular matrix (ECM) is known to modulate specific cell functions, this study was designed to determine whether PDT of isolated ECM affects the function of endothelial cells (ECs) and smooth muscle cells (SMCs). METHODS: PDT of bovine aortic EC-ECM was performed with chloroaluminum sulfonated phthalocyanine and 675 nm laser light. Control specimens included untreated ECM, ECM-free plates, and ECM exposed to either light or photosensitizer only. Cell function was characterized by attachment, proliferation, and migration of ECs or SMCs that were plated onto identically treated matrixes. RESULTS: SMC attachment (86% +/- 0.4% vs 95% +/- 0.4%), proliferation (46% +/- 0.5% vs 100% +/- 1.4%), and migration (40% +/- 1.0% vs 100% +/- 0.9%) were significantly inhibited after PDT of ECM when compared with untreated ECM (all p < 0.001). In contrast, PDT of ECM significantly enhanced EC proliferation (129% +/- 6.2% vs 100% +/- 6.2%; p < 0.03) and migration (118% +/- 2% vs 100% +/- 0.8; p < 0.01), but did not affect attachment. CONCLUSIONS: This report establishes PDT-induced changes in the ECM with a result of inhibition of SMCs and stimulation of EC functions. It provides insight into how PDT-treated arteries can develop favorable EC repopulation without SMC-derived intimal hyperplasia. These findings may help provide a better understanding of the interactions between cells and their immediate environment in vascular remodeling. PMID- 8627909 TI - Pelvic radiation therapy as a risk factor for ischemic colitis complicating abdominal aortic reconstruction. AB - Ischemic colitis is an infrequent but potentially devastating complication of abdominal aortic reconstruction. Identification of patients with predisposing risk factors for the development of ischemic colitis can guide intraoperative measures to preserve or restore colonic blood flow during aortic surgery. Previous radiation therapy for pelvic malignancy may be one such predisposing risk factor. Two cases are presented in which ischemic colitis complicated abdominal aortic reconstruction in the setting of previous pelvic irradiation. In the months after radiation therapy for prostate cancer, one patient underwent infrarenal abdominal aortic aneurysm repair. Ischemic infarction of the sigmoid colon developed acutely after surgery and required emergent sigmoid colectomy. The second patient underwent reconstruction of an infrarenal abdominal aortic aneurysm after having had radiation therapy for a bladder tumor. Despite an initial satisfactory result, the patient's abdominal pain and diarrhea progressively worsened and he eventually required sigmoid colectomy for severe ischemic colitis. In both of these patients, the inferior mesenteric arteries were patent and had not been reimplanted. The association of pelvic radiation therapy with ischemic colitis after aortic reconstruction should focus attention to the operative details for maintaining the colonic circulation in these patients. Reimplantation of the inferior mesenteric artery in particular may prevent both the acute and the insidious variants of this complication in patients who undergo aortic surgery and decrease the incidence of this complication in patients with a history of radiation therapy to the pelvis. PMID- 8627910 TI - Aneurysms of the pancreaticoduodenal artery. AB - True aneurysms of the pancreaticoduodenal artery are a rare form of visceral artery aneurysm. Two cases are presented and options for treatment are reviewed. PMID- 8627911 TI - Primary Clostridium septicum aortitis: a rare cause of necrotizing suprarenal aortic infection. A case report and review of the literature. AB - A 74-year old woman sought medical attention for general symptoms of nausea, vomiting, and back pain. A computed tomographic scan showed gas in the wall of the descending thoracic and suprarenal aortas. Emergency thoracoabdominal exploration revealed a necrotizing infection of the thoracic aorta extending to the origin of the celiac axis. After surgery Clostridium septicum was identified in tissue culture. Surgical management consisted of in-situ graft replacement of the thoracoabdominal aorta. Three months later, a pseudoaneurysm developed at the distal anastomosis. The patient refused further surgery and died 3 days later. The cause of death was presumed to be a ruptured mycotic aneurysm as a result of recurrent C. septicum infection. The relationship of C. septicum with occult gastrointestinal and hematologic malignancy has been documented. This patient represents the 10th reported case of C. septicum arteritis. Including the nine previous case reports of C. septicum arteritis, the mortality rate is 70%. When evaluating a patient with a mycotic aneurysm or aortitis, C. septicum should be considered. If it is found, a search should be carried out for an associated gastrointestinal or hematologic malignancy. Surgical repair should include extraanatomic revascularization and wide debridement of the infected field. Consideration should be given to lifelong antimicrobial therapy for this potentially fatal infection. PMID- 8627912 TI - Cocaine-induced multiple vascular occlusions: a case report. PMID- 8627913 TI - Chronic intestinal ischemia caused by intravenous cocaine use: report of two cases and review of the literature. AB - Chronic mesenteric ischemia caused by thrombosis of large visceral arteries due to cocaine abuse are reported in two young women. Both cases were managed successfully with visceral revascularization. PMID- 8627914 TI - Simplified, controlled limb reperfusion and simultaneous revascularization for acute aortic occlusion. AB - A 62-year old patient was admitted with a 24-hour history of ischemia in both legs caused by acute distal aortic occlusion and had had a total loss of sensitivity and motor function for 8 hours. Preoperative serum creatine phosphokinase level was 10,900 IU/ml. During aortofemoral reconstruction, both limbs were reperfused with a potassium-free, blood-cardioplegia-like perfusate. Fasciotomies were not necessary. After operation, maximal serum creatine phosphokinase levels remained below 10,000 IU/ml. Limb sensitivity and motor function were normal. Even for prolonged acute aortoiliac occlusion, a simplified controlled limb-reperfusion may preserve skeletal muscle and nerve function and prevent local and systemic complications caused by reperfusion damage. PMID- 8627915 TI - Regarding "The significance of microemboli detection by means of transcranial Doppler ultrasonography monitoring in carotid endarterectomy". PMID- 8627916 TI - Endothelial coverage of endovascular Dacron grafts in dogs. PMID- 8627917 TI - Intraluminal thrombus predicts rupture of an abdominal aortic aneurysm. PMID- 8627918 TI - Effects of embedded tungsten-bismuth-tin shot and steel shot on mallards (Anas platyrhynchos). AB - We assessed the potential for embedded steel and tungsten-bismuth-tin (TBT) shot to adversely affect health of mallards (Anas platyrhynchos). Ducks were implanted with three number four steel (n = 19) or TBT (n = 20) shot pellets in their pectoral muscles. None of seven hematology parameters measured differed in response to treatment (P > or = 0.17). At necropsy 1, 2, 4, and 8 wk posttreatment, we observed only localized tissue reactions to embedded steel or TBT shot. Reactions differed grossly: after wk 1, embedded steel shot were enveloped in 0.5 to 2 mm grayish capsules, whereas TBT shot were surrounded by thinner (< 0.5 mm), translucent capsules. Corrosion of steel shot was apparent. Microscopic lesions associated with steel shot were characterized by moderate to severe histiocytic and lymphocytic inflammation and considerable particle deposition, whereas histiocytic inflammation was mild and particle deposition minimal in TBT lesions. Overall scores of inflammation at steel shot implant sites were greater (P < or = 0.043) than at TBT sites during wk 1 and 8. Inflammation at steel implant sites was relatively consistent over the 8-wk period, but decreased (P = 0.0017) at TBT sites by wk 8. Weights of steel shot recovered from muscle tissue declined logarithmically (R2 = 0.978, P = 0.0014) over 8 wk, but TBT shot weights remained unchanged (P = 0.255). Embedded TBT shot, as compared to steel, resisted corrosion and induced comparatively mild inflammatory responses in mallard muscle tissue. However, inflammatory reactions to both embedded steel and TBT shot were localized and had no detectable systemic effects on mallard health under experimental conditions. PMID- 8627919 TI - Osteoporotic skull lesions in moose at Isle Royale National Park. AB - Osteoporotic lesions were evaluated in the skulls of moose (Alces alces) collected in Isle Royale National Park, Michigan (USA), from 1958 to 1994. Circular lesions which penetrated the outer table of the skull were distributed over the frontal and nasal bones asymmetrically in both sexes. About 32% of skulls recovered had some porotic lesions with slightly greater prevalence among males. PMID- 8627920 TI - Rehabilitation of native reptiles and amphibians in DuPage County, Illinois. AB - Between 1980 and 1994, 586 individuals of 20 native reptile and amphibian species were evaluated at a wildlife rehabilitation facility in DuPage County, Illinois (USA). On examination, 48% of reptiles and 52% of amphibians were injured, and 2% of each class were diseased; remaining animals were uninjured. The most frequent causes of injury to reptiles were automobile collisions (55%), ingestion of fishing tackle (19%), and domestic animal attacks (8%). Automobile collisions (38%), lawn and garden accidents (33%), and pet collection (19%) were the most frequent causes of injury to amphibians. We released 354 reptiles and 54 amphibians into appropriate habitat available in local forest preserves, representing case release prevalences of 84% and 68% for each class, respectively. PMID- 8627921 TI - Mortality of passerines adjacent to a North Carolina corn field treated with granular carbofuran. AB - Red-winged blackbirds (Agelaius phoeniceus) were collected during an epizootic in southeastern North Carolina (USA). Activity of brain cholinesterase (ChE) was inhibited by 14 to 48% in three of five specimens, and returned to normal levels after incubation. Gastrointestinal tracts were analyzed for 30 anti-ChE agents. Carbofuran, the only compound detected, was present in all specimens at levels from 5.44 to 72.7 micrograms/g wet weight. Application of granular carbofuran in an adjacent corn field, results of necropsy examinations, and chemical analyses are consistent with a diagnosis of carbofuran poisoning in these specimens. PMID- 8627922 TI - In vitro sensitivity of macropodid herpesvirus 2 to selected anti-herpetic compounds. AB - We tested the in vitro sensitivity of Macropodid Herpesvirus 2 to eight commonly used anti-herpetic compounds using plaque reduction tests, March and April, 1995. The virus was most susceptible to inhibition by (E)-5-(2'-bromovinyl)-2' deoxyuridine and adenine 9-beta-D-arabino-furanoside. Both compounds have been used for anti-herpetic therapy in humans and may prove useful in the treatment of macropodoids in captivity. PMID- 8627923 TI - Leptospira interrogans exposure in free-ranging elk in Washington. AB - Exposure to one or more serovars of Leptospira interrogans was observed in five of six sampled elk (Cervus elaphus roosevelti) killed in November 1993, from an isolated herd in southwest Washington, USA (46 degrees 45'N, 123 degrees 6'W). In April 1994, exposure to L. interrogans serovars was documented in nine of 11 captured cow elk from the same herd. Leptospires were not isolated from any of the exposed elk, and 10 of the 11 cows were pregnant. The high seroprevalence is evidence that exposure is widespread in the herd. Local productivity of elk was high, however, and the surrounding topography was not conducive for transmission to other elk populations. PMID- 8627924 TI - Coccidial infection in mouflon, Ovis musimon, in central Spain. AB - From February to September 1993, ten adult female mouflons (Ovis musimon) and their five offspring from central Spain were examined weekly for coccidial infection. All adult mouflons had Eimeria spp. infections with mean (+/- SD) intensity of 1,869 (+/- 1,264) oocysts per gram of feces the day of capture, increasing progressively during the first two months in captivity and later returning to the initial values (1,869 +/- 1,547). The mean (+/- SD) oocyst shedding in young animals was 16,800 (+/- 966) oocysts per gram at 1 mo and 18,796 (+/- 1,220) at 1.5 mo of age and more than 40,000 (40,250 to 52,000) at 3 mo of age; this high intensity was associated with a transient diarrhea. The species involved, in order of frequency, were E. bakuensis (syn. Eimeria ovina), E. ovinoidalis, E. crandallis, E. caprovina, E. parva, E. faurei, E. granulosa and E. intricata, and one more not previously described and recorded as Eimeria sp.. The predominant species for both age groups was E. bakuensis. PMID- 8627925 TI - An unusual coccidian parasite causing pneumonia in a northern cardinal (Cardinalis cardinalis). AB - In June 1993 an unusual coccidian parasite was identified in lung tissue from a northern cardinal (Cardinalis cardinalis), collected near Tucson, Arizona (USA), which died in respiratory distress. Histologically, there was evidence of severe, generalized interstitial pneumonia, associated with the parasite. Both asexual and sexual stages were seen. Schizonts, gamonts, and sporulated oocysts were seen in lung tissue. The parasite most closely resembled coccidia of the genus Lankesterella. This is the first report of such a coccidian parasite in the alveolar tissue of a cardinal. PMID- 8627926 TI - Avian tick paralysis caused by Ixodes brunneus in the southeastern United States. AB - Between 1988 and 1994, 16 definitive and 26 presumptive cases of tick paralysis were diagnosed in 10 species of birds from five southeastern states in the USA. All birds had engorged adult female Ixodes brunneus ticks on the head region and were partially paralyzed or dead. Cases occurred in the winter and early spring months, and most birds were passerines found in private yards or near feeders. All stages of I. brunneus feed exclusively on birds, and this species previously has been associated with avian tick paralysis. Little is known concerning the life cycle of this ixodid tick and its impact on wild bird populations. PMID- 8627927 TI - Prevalence of hepatic helminths and associated pathology in impala (Aepyceros melampus) in Swaziland. AB - The livers of impala (Aepyceros melampus) collected in the Mlawula-Mbuluzi Simunye Nature Reserve and Protected Area complex in northeastern Swaziland from October 1985 to September 1986 were infected with three species of helminths: a nematode (Cooperioides hepaticae), a cestode (Stilesia hepatica), and a trematode (Fasciola gigantica). Cooperioides hepaticae caused a severe cholangitis. Lambs were infected at 1.5 to 2 mo of age, and the prevalence increased to 100% by 5 mo of age. The prevalence was 82% in yearlings and 44% in adults, with the highest prevalence and intensity of infection at the end of the dry season. Stilesia hepatica was found primarily in adults. Heavy infections caused a thickening and distension of the main bile ducts, but no changes were evident in the liver parenchyma. Fasciola gigantica, found only in one adult male, caused a thickening and distension of main bile duct. There was no apparent association between the helminth infections and body condition. PMID- 8627928 TI - Variation in hematological and serum biochemical values of the mountain brushtail possum, Trichosurus caninus Ogilby (Marsupialia: Phalangeridae). AB - Hematological and serum biochemical values were determined in a wild population of the mountain brushtail possum (Trichosurus caninus) at Cambarville, central Victoria, southeastern Australia. Animals were sampled during two-week trapping periods in June, September, and December 1992, and April 1993. Values for hemoglobin, red cell count and hematocrit were significantly higher in males than females. Total protein and mean corpuscular volume (MCV) were significantly higher in female T. caninus. Significant seasonal variations were detected for total bilirubin, alkaline phosphatase, total protein, albumin, urea, absolute eosinophils, MCV, sodium, potassium, and phosphate. PMID- 8627929 TI - Field immobilization of Ethiopian wolves (Canis simensis). AB - Telazol (tiletamine hydrochloride and zolazepam hydrochloride combination) and a combination of ketamine hydrochloride and acepromazine were used to immobilize wild Ethiopian wolves (Canis simensis) in Ethiopia from 1988 to 1992. Telazol doses of 2.1 to 6.5 mg/kg resulted in a mean (+/- SD) induction time of 2.3 +/- 0.9 min and a mean (+/- SD) immobilization time of 82.2 +/- 28.6 min. Induction time did not differ by dose, wolf weight, or age, but was significantly longer for females. Immobilization time differed with dose, but not by wolf weight, age, or sex. Total recovery times ranged from 50 to 158 min. There were no apparent side effects on immobilized animals. Wolves immobilized using a combination of ketamine hydrochloride and acetylpromazine had longer induction time (3.0 +/- 0.8 min) and recovery time (114.7 +/- 29.2 min). Telazol is an effective and safe agent for immobilizing Ethiopian wolves and is preferred to ketamine/acetylpromazine. PMID- 8627931 TI - Consumption of deoxynivalenol-contaminated wheat by mallard ducks under experimental conditions. AB - Captive mallards (Anas platyrhynchos) were fed wheat containing 5.8 ppm deoxynivalenol (DON, vomitoxin) from an outbreak of Fusarium graminearium head blight that occurred on grain crops in Manitoba, Canada, during 1993. There was no evidence of taste aversion to this grain during a 10-day palatability trial. No significant differences were detected in serum protein, calcium, glucose, creatinine kinase, aspartate aminotransferase or uric acid levels, blood packed cell volume, or body or organ weight, between ducks fed contaminated wheat and those fed uncontaminated wheat during a 14-day feeding trial. No gross or microscopic lesions were detected in birds fed contaminated wheat for 14 days. Based on these results, ducks will consume grain containing moderate levels of DON and short-term exposure to this grain will not result in obvious adverse effects. PMID- 8627930 TI - Immobilization of muskrats (Ondatra zibethicus) with ketamine and xylazine. AB - The effectiveness of ketamine and xylazine as an immobilizing combination for muskrats (Ondatra zibethicus) was evaluated. Eleven muskrats were intramuscularly injected using a high (n = 7) or low (n = 4) dosage of a 20:1 mixture of ketamine (12 or 20 mg) and xylazine (0.6 or 1.0 mg) in Carlton County, Minnesota (USA) from 1 to 4 May 1995. Mean (+/- SD) induction times for muskrats receiving a high dosage (6.5 +/- 2.6 min) or low dosage (7.0 +/- 1.6 min) was similar (P = 0.71). In contrast, muskrats receiving a low dosage recovered sooner (37.0 +/- 15.1 min) than muskrats receiving a high dosage (62.2 +/- 15.6 min) (P = 0.04). There was a positive linear relationship (r2 = 0.75, P = 0.02) between the amount (mg/kg) of ketamine-xylazine injected and recovery time but not between the amount injected and induction time (r2 = 0.49, P = 0.18). Heart rate, respiratory rate, and body temperature were similar (P = 0.20 to 0.62) between high and low dose groups. No mortality occurred nor were short-term adverse effects observed in recaptured individuals. I conclude that a 20:1 mixture of ketamine-xylazine is a safe and effective immobilization agent for muskrats when conducting non-surgical field procedures. Immobilizing muskrats with 15 mg/kg ketamine and 0.75 mg/kg xylazine should provide about 10 min of handling time before arousal and allow full recovery in < 60 min. PMID- 8627933 TI - The possible importance of wintering yards in the transmission of Parelaphostrongylus tenuis to white-tailed deer and moose. AB - Terrestrial gastropods were collected, 15 June to 25 November 1994, from beneath cardboard sheets on deer range in northeastern Minnesota (USA) and examined individually for larvae of Parelaphostrongylus tenuis, the meningeal worm of white-tailed deer (Odocoileus virginianus). Overall, 10 (0.08%) of 12,096 snails and slugs were infected with a mean (+/- SD) of 3.2 +/- 2.5 P. tenuis larvae. The prevalence of infection in gastropods was greater in a traditional deer wintering yard (seven of 4,401, 0.16%), where deer aggregated for almost 5 months at a density of 50/km2, than on summer range (three of 7,695, 0.04%) where they occurred at 4/km2. Despite relatively low densities of infected gastropods, their ingestion purely by chance remains a tenable explanation for the high prevalence of P. tenuis infection observed in white-tailed deer. PMID- 8627932 TI - Efficacy of ivermectin against nematodes infecting field populations of snowshoe hares (Lepus americanus) in Yukon, Canada. AB - From July 1990 to February 1991, nematode numbers in free-ranging snowshoe hares (Lepus americanus) at Kluane Lake, southwestern Yukon, Canada, were manipulated by subcutaneous injection (0.4 mg/kg) of ivermectin. Three field experiments were conducted to determine the degree of helminth loss associated with a single administration of ivermectin; the length of time that ivermectin was effective in reducing worm numbers; and the effect of repeated ivermectin administration in reducing worm numbers. Numbers of the nematodes, Protostrongylus boughtoni and Nematodirus triangularis were reduced by approximately 80% 2 wk after treatment with a single dose of ivermectin, and were still significantly lower than controls at 4 wk. However, beyond 2 wk, ivermectin did not affect the rate of acquisition of new worms of either species. All treated groups contained one or more hares in which numbers of P. boughtoni and N. triangularis were not reduced. In addition, ivermectin had no effect on numbers of Trichuris leporis or Passalurus sp. Overall, ivermectin was not as effective against the nematodes of free-ranging hares as has been reported for nematodes of domestic and laboratory animals. PMID- 8627935 TI - Helminth and arthropod parasites of experimentally introduced whooping cranes in Florida. AB - Nine species of nematodes, unidentified larval nematodes, three species of trematodes, two species of acanthocephalans and a single species of chewing louse were collected from 1993 to 1995 from 25 introduced whooping cranes (Grus americana) in Florida (USA). In spite of a quarantine procedure involving anthelmintic therapy, three helminth parasites may have been introduced from captive populations. Other parasites acquired were similar to those found in a local congener, the Florida sandhill crane (Grus canadensis pratensis), or only occurred infrequently. PMID- 8627936 TI - Seroprevalence of Psoroptes sp. mites in free-ranging elk (Cervus elaphus) as determined by kinetic ELISA. AB - Western blots and a kinetic enzyme-linked immunosorbent assay (ELISA) were used to characterize and quantify the prevalence of antibodies to Psoroptes sp. mites in elk (Cervus elaphus) from nine herds in North America. Sera from infested (n = 18) and non-infested (n = 22) elk were used to optimize test methodology and to define cut-off values for negative, suspect, and positive samples. Among 357 samples, 35 (9.8%) of the animals were seropositive, 259 (73%) were negative, and 63 (18%) were suspect. Six of nine herds (67%) contained positive animals and two additional herds (22%) had suspect animals. Sex was not associated with prevalence of antibodies, but adults greater than 2 yr old were approximately five times more likely (95% confidence interval = 2.6-15.4) to be seropositive than calves. Based on these results, we propose that exposure to Psoroptes sp. mites may be widespread in free-ranging elk of North America. PMID- 8627937 TI - Sarcoptic mange in wild ruminants in zoological gardens in Israel. AB - Sarcoptic mange (Sarcoptes scabiei) occurred among wild ruminant species in five zoological gardens in Israel, from 1984 to 1994. Infestation of five ruminants by S. scabiei is reported for the first time: mountain gazelles (Gazella gazella), Nubian ibexes (Capra ibex nubiana), a barbary sheep (Ammotragus lervia), elands (Taurotragus oryx), and an Arabian oryx (Oryx leucoryx). All animals in the herds were administered ivermectin orally at a dose of 200 micrograms/kg body weight daily for 3 consecutive days. This was repeated three times at 2-wk intervals. The disease was eradicated in four small zoos, whereas in the biggest zoo, only control was achieved. Mortality among animals < 4 mo and > 8-yr-old animals composed 65% of mortality among all age classes. PMID- 8627934 TI - Natural infection by gastrointestinal and bronchopulmonary nematodes in mouflons (Ovis musimon) and their response to netobimin treatment. AB - Gastrointestinal and bronchopulmonary nematode infections and the efficacy of netobimin (Hapasil) were analyzed by way of fecal examination in 10 female mouflons (Ovis musimon), in central Spain, February 1993. Before treatment all 10 mouflons had Trichostrongylus axei, Teladorsagia circumcincta and Marshallagia spp.; sic had Nematodirus spp., two had Trichuris sp., one had Capillaria sp., seven had bronchopulmonary Dictyocaulus filaria and 10 mouflons had protostrongylid lungworms (Muellerius capillaris, Protostrongylus rufescens, Cystocaulus ocreatus or Neostrongylus linearis). Netobimin (7.5 mg/kg) was 100% effective against T. axei, T. circumcincta, Marshallagia spp., and D. filaria infections whereas one animal continued eliminating Nematodirus spp. eggs. The drug also was effective against Capillaria spp. but not against Trichuris spp. or protostrongylid infections. PMID- 8627938 TI - Susceptibility of elk (Cervus elaphus) to experimental infection with Anaplasma marginale and A. ovis. AB - Anaplasma ovis was experimentally transmitted from domestic sheep to elk (Cervus elaphus) and back to splenectomized sheep. No rickettsemias were detected but serum from three of seven experimentally inoculated elk developed Anaplasma spp. reactive antibody as measured by indirect immunofluorescence (IIF) or by the rapid card agglutination and complement fixation assays. Three elk were experimentally infected with A. marginale. The rickettsiae were detected in blood of these elk and caused disease in a splenectomized domestic bovine calf after subinoculation of blood from the elk. All three elk had positive titers with IIF. No clinical signs of illness were noted in any elk inoculated with either Anaplasma species. PMID- 8627939 TI - Pregnancy detection in bighorn sheep (Ovis canadensis) using a fecal-based enzyme immunoassay. AB - We developed and validated an enzyme immunoassay for immunoreactive pregnanediol 3-glucuronide (iPdG) in feces to monitor reproductive status in desert and Rocky Mountain bighorn sheep (Ovis canadensis). Fecal iPdG concentrations were strongly correlated (r = 0.71) with serum progesterone concentrations in paired fecal and blood samples collected from 34 free-ranging desert bighorn sheep. In bimonthly fecal samples collected from 12 captive ewes, fecal iPdG profiles were similar between desert and Rocky Mountain bighorn sheep and we selected a pregnancy detection cutoff value of iPdG > or = 1.8 ng/mg feces. Fecal iPdG concentrations always exceeded this cutoff value when samples were collected from about day 60 of pregnancy to a few days before parturition, but values < 1.8 ng/mg (false negatives) were common for samples collected during the first 60 days of gestation. Although we tested a small number of known pregnant and non-pregnant ewes, the accuracy of the assay was 100% when two samples, collected 2 wk apart, were evaluated for any given ewe. Based on these data, this direct enzyme immunoassay for fecal progesterone metabolites has promise as a diagnostic tool to monitor hormone excretion and pregnancy in a free-ranging ungulate species. PMID- 8627940 TI - Serum chemistry of bowhead whales (Balaena mysticetus). AB - Sera of 19 male and female bowhead whales (Balaena mysticetus) collected near Barrow, Alaska (USA) between 30 August and 13 October 1992 were evaluated for 18 serum chemistry values. Male bowhead whales had significantly greater creatinine and sodium concentrations, and significantly lower glucose concentrations than females. Pregnant females had greater triglyceride levels than non-pregnant females. The mean concentrations of creatinine, blood urea nitrogen, alkaline phosphatase, total bilirubin, total protein, sodium, potassium, chloride, phosphorus, and calcium were similar to those previously reported from bowhead whales. High aspartate aminotransferase and creatinine kinase levels were attributed to muscle damage associated with harpooning. PMID- 8627941 TI - Rabies and mortality in Ethiopian wolves (Canis simensis). AB - Between October 1991 and February 1992, 41 of 53 known adult and subadult Ethiopian wolves (Canis simensis) in five adjacent packs in the Bale Mountains National Park, Ethiopia, died or disappeared. Brain smears from two carcasses were positive for rabies by the immunofluorescence test, and rabies virus was isolated from the brains by mouse inoculation. Based on monoclonal antibody tests on the mouse brains, we identified the virus as a minor variant of the serotype 1 rabies viruses found in domestic dogs and wild canids of Africa. Sera from two of 15 Ethiopian wolves had rabies virus neutralizing antibody. PMID- 8627942 TI - Ockelbo virus (Togaviridae: Alphavirus) neutralizing antibodies in experimentally infected Swedish birds. AB - The ability of native Swedish birds to produce and maintain production of Ockelbo virus neutralizing (Nt) antibodies were evaluated experimentally between 6 June 1990 and 27 July 1991. After experimental infection of 57 birds of the orders Anseriformes and Passeriformes with Ockelbo virus, these birds were examined for Ockelbo virus Nt antibodies at 5 days, and at 1, 3, 6, 9, and 12 mo after inoculation. One month after inoculation, Nt antibodies were more prevalent in birds with a detectable viremia (100%, n = 22) than in non-viremic birds (65%, n = 26). The Nt antibody prevalence varied over time among taxa; detectable antibodies occurred earlier after inoculation and for a longer time in Anseriformes than in Passeriformes. By 5 days after inoculation, antibodies could be detected in 22 (71%) of 31 Anseriformes but in none of 16 Passeriformes. However, at 1 mo the antibody prevalences at 1 mo were similar: 84% among the Anseriformes and 73% among the Passeriformes; at 3 mo the prevalences were 50% in Anseriformes and 15% in Passeriformes. Forty-two percent of the Anseriformes had detectable antibodies even 12 mo after inoculation. PMID- 8627943 TI - Toxicologic evaluation of a high-selenium hay diet in captive pronghorn antelope (Antilocapra americana). AB - Five captive-raised pronghorn antelope (Antilocapra americana) were fed an alfalfa-grass hay diet containing 15 ppm total dietary selenium (Se) for 164 days. Four additional captive-raised pronghorns fed a similar diet containing approximately 0.3 ppm total dietary Se served as controls. None of the pronghorns had clinical signs attributable to the high Se hay. Plasma Se increased more rapidly than blood Se concentrations, from baseline concentrations (< 0.15 g/ml) to > 0.40 g/ml within the first 50 days on the high selenium diet, but thereafter declined to approximately 0.30 microgram/ml. Mean primary antibody response to hen egg albumin was less in pronghorn on Se hay. No significant gross or histological lesions attributable to selenosis were found, nor was there any evidence of dystrophic hoof growth. The greatest Se tissue concentrations were found in liver and kidney (5.67 to 10.4 micrograms/g and 2.36 to 3.14 micrograms/g, respectively) from experimental animals; liver and kidney from the controls contained considerably less (< or = 0.52 microgram/g and < or = 0.61 microgram/g, respectively). Exposure of pronghorns for more than 5 mo to a diet containing 15 ppm Se caused significant increases in plasma, liver and kidney Se concentrations, in the absence of clinical disease or pathologic lesions due to selenosis. Based on these results, we propose that pronghorns are less susceptible to selenosis than previously reported and that diagnostic criteria for the disease should be modified. PMID- 8627945 TI - New studies say viral burden tops CD4 as a marker of HIV disease progression. PMID- 8627944 TI - Experimental studies on Brucella abortus in moose (Alces alces). AB - Four moose (Alces alces) were inoculated conjunctivally with B. abortus biovar 1 to determine their susceptibility to brucellosis, and to describe the serology, bacteriology, hematology, clinical chemistry, and pathology associated with infection. All moose became infected. Two moose were killed at day 70 post exposure, one (83F) died acutely at day 85, and one was killed at day 166. None of the moose had clinical signs, except for 83F immediately before death. Infected moose were readily detected serologically by the buffered antigen plate test, Brewer card test, standard tube agglutination test, and complement fixation test as used for brucellosis in cattle. With the exception of samples from 83F taken 24 hours before death, clinical chemistry, and hematology results were stable for all moose, and similar to normal values seen in cattle. Lesions seen in all moose were indicative of endotoxemia, and moose 83F died of acute endotoxic shock. Brucella abortus biovar 1 was isolated from several tissues in all moose, most notably from lymph nodes where counts often exceeded 4 x 10(4) colony forming units per g of tissue. Thus infection with B. abortus will kill moose, and progression of the disease is likely rapid under field conditions. Moose appear to be a dead-end host for brucellosis. PMID- 8627946 TI - US military medical presence increases in Bosnia. PMID- 8627947 TI - Trials reveal no benefit, possible harm of beta carotene and vitamin A for lung cancer prevention. PMID- 8627948 TI - FDA gives calcium channel blockers clean bill of health but warns of short-acting nifedipine hazards. PMID- 8627949 TI - From the Health Care Financing Administration. PMID- 8627950 TI - From the Centers for Disease Control and Prevention. Carbon monoxide poisonings associated with snow-obstructed vehicle exhaust systems--Philadelphia and New York City, January 1996. PMID- 8627951 TI - From the Centers for Disease Control and Prevention. Hepatitis A among persons with hemophilia who received clotting factor concentrate--United States, September-December 1995. PMID- 8627952 TI - From the Centers for Disease Control and Prevention. Trends in cancer screening- United States, 1987 and 1992. PMID- 8627953 TI - A piece of my mind. Moments of love. PMID- 8627954 TI - Policies on medical decisions concerning the end of life in Dutch health care institutions. AB - OBJECTIVE: To describe the prevalence and some features of policies on medical decisions concerning the end of life (MDELs) in Dutch hospitals, nursing homes, and institutions for the mentally disabled. DESIGN: A cross-sectional descriptive postal survey of 558 Dutch health care institutions. SETTING: All Dutch hospitals, nursing homes, and general institutions for the mentally disabled. PARTICIPANTS: Directors of patients care of the institutions. MAIN OUTCOME MEASURES: Respondents' reports on the existence of policies and guidelines on the following MDELs: euthanasia/assisted suicide (EAS), life-terminating acts without explicit request of the patient, refusal of treatment by patient, withholding or withdrawing treatment, symptom and pain control, and do-not-resuscitate (DNR) decisions. RESULTS: Of 558 health care institution managers, 86% responded. Most of the hospitals (69.2%) and nursing homes (73.9%) but only 16.3% of the institutions for the disabled had a written EAS policy. Nursing homes with a ban on EAS often had religious affiliations. In 37% of nursing homes, 15% of hospitals, and 15% of institutions for the disabled, the management had written policies on terminating life without request. Sixty percent of the hospitals, 35% of the nursing homes, and 17% of the institutions for the disabled had guidelines for one or more of four other distinct MDELs. Forty-five percent, 20%, and 8% of hospitals, nursing homes, and institutions of mentally disabled, respectively, had guidelines on DNR decisions. The management of 89% of the hospitals and 94% of the nursing homes communicated their policies on EAS to physicians and nurses in their institutions without being asked. Far fewer of these hospitals (3.9%) and nursing homes (30.5%) made their policies on EAS known to patients without being asked. CONCLUSIONS: This study indicates that an important step toward policy development on EAS has been made by Dutch hospitals and nursing homes. Particularly with respect to policies on such decisions as withholding or withdrawing treatment, symptom and pain control, and DNR orders, an unexplored field is open to management for policy development in the Netherlands. PMID- 8627955 TI - Occupational exposure to environmental tobacco smoke. PMID- 8627956 TI - Occupational exposure to environmental tobacco smoke. PMID- 8627957 TI - Polyneuropathy after mechanical ventilation. PMID- 8627958 TI - Reconsideration of AMA opinion on anencephalic neonates as organ donors. PMID- 8627959 TI - Hepatitis B vaccine for medical students. PMID- 8627960 TI - Superficial femoral vein thrombosis: a potentially confusing term. PMID- 8627961 TI - Superficial femoral vein thrombosis: a potentially confusing term. PMID- 8627962 TI - Superficial femoral vein thrombosis: a potentially confusing term. PMID- 8627963 TI - Timing of drug administration to prevent drug interactions. PMID- 8627964 TI - Risperidone and neuroleptic malignant syndrome. PMID- 8627965 TI - Vegetable, fruit, and cereal fiber intake and risk of coronary heart disease among men. AB - OBJECTIVE: To examine prospectively the relationship between dietary fiber and risk of coronary heart disease. DESIGN: Cohort study. SETTING: In 1986, a total of 43,757 US male health professionals 40 to 75 years of age and free from diagnosed cardiovascular disease and diabetes completed a detailed 131-item dietary questionnaire used to measure usual intake of total dietary fiber and specific food sources of fiber. MAIN OUTCOME MEASURE: Fatal and nonfatal myocardial infarction (MI). RESULTS: During 6 years of follow-up, we documented 734 cases of MI (229 were fatal coronary heart disease). The age-adjusted relative risk (RR) for total MI was 0.59 (95% confidence interval [CI], 0.46 to 0.76) among men in the highest quintile of total dietary fiber intake (median, 28.9 g/d) compared with men in the lowest quartile (median, 12.4 g/d). The inverse association was strongest for fatal coronary disease (RR, 0.45; 95% CI, 0.28 to 0.72). After controlling for smoking, physical activity and other known nondietary cardiovascular risk factors, dietary saturated fat, vitamin E, total energy intake, and alcohol intake, the RRs were only modestly attenuated. A 10-g increase in total dietary fiber corresponded to an RR for total MI of 0.81 (95% CI, 0.70 to 0.93). Within the three main food contributors to total fiber intake (vegetable, fruit, and cereal), cereal fiber was most strongly associated with a reduced risk of total MI (RR, 0.71; 95% CI, 0.55 to 0.91 for each 10-g increase in cereal fiber per day). CONCLUSIONS: Our results suggest an inverse association between fiber intake and MI. These results support current national dietary guidelines to increase dietary fiber intake and suggest that fiber, independent of fat intake, is an important dietary component for the prevention of coronary disease. PMID- 8627966 TI - Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. AB - OBJECTIVE: To determine whether isolates of Mycobacterium tuberculosis from New York and elsewhere that are resistant to four or more primary antimicrobial agents and responsible for widespread disease in the 1990s represent a newly emerged clone or a heterogeneous array of unrelated organisms. SETTING: New York City area and selected locations in the United States. PATIENTS: M tuberculosis isolates from 1953 patients in New York and multidrug-resistant isolates from six patients from other US communities. DESIGN: Convenience sample of all M tuberculosis strains (M tuberculosis isolates resistant to rifampin, streptomycin, isoniazid, and ethambutol, and sometimes ethionamide, kanamycin, capreomycin, or ciprofloxacin) submitted to the Public Health Research Institute Tuberculosis Center since 1991 and samples submitted to the Centers for Disease Control and Prevention from throughout the United States. The samples submitted were representative of the New York City strains of M tuberculosis. MAIN OUTCOME MEASURE: Characterization of resistant M tuberculosis strains studied by IS6110 and polymorphic GC-rich repetitive sequence (PGRS) hybridization patterns, multiplex polymerase chain reaction (PCR) analysis, and automated DNA sequencing of genes containing mutations associated with resistance to rifampin (rpoB), isoniazid (katG and inhA locus), and streptomycin (strA and rrs). RESULTS: Multidrug-resistant M tuberculosis isolates were recovered from 253 New York City patients and had the same or closely allied IS6110 and PGRS patterns, multiplex PCR type, and gene mutations associated with resistance to rifampin, isoniazid, and streptomycin. Isolates with these same molecular characteristics were recovered from patients in Florida and Nevada, health care workers in Atlanta, Ga, and Miami, Fla, and an individual who recently moved from New York City to Denver, Colo, and caused disease or skin test conversion in at least 12 people in a nursing home environment. CONCLUSIONS: The results document the molecular origin and spread of progeny of a closely related family of multidrug-resistant M tuberculosis strains that have recently shared a common ancestor and undergone clonal expansion. The multidrug-resistant phenotype in these organisms arose by sequential acquisition of resistance-conferring mutations in several genes, most likely as a consequence of antibiotic selection of randomly occurring mutants in concert with inadequately treated infections. Dissemination of these difficult-to treat bacteria throughout New York City and to at least four additional US cities has adverse implications for tuberculosis control in the 21st century. PMID- 8627967 TI - Coffee consumption and coronary heart disease in women. A ten-year follow-up. AB - OBJECTIVE--To assess the relationship between coffee consumption and risk of coronary heart disease (CHD) among women. DESIGN--Prospective cohort study with coffee consumption measured in 1980, 1984, and 1986, and follow-up through 1990. SETTING--Female registered nurses in the United States. PARTICIPANTS--A total of 85,747 US women 34 to 59 years of age in 1980 and without history of CHD, stroke, or cancer. MAIN OUTCOME MEASURE--Ten-year incidence of CHD (defined as nonfatal myocardial infarction or fatal CHD). RESULTS--During 10 years of follow-up we documented 712 cases of CHD. After adjustment for age, smoking, and other CHD risk factors, we found no evidence for any positive association between coffee consumption and risk of subsequent CHD. For women drinking six or more cups of caffeine-containing coffee per day in 1980, the relative risk was 0.95 (95% confidence interval, 0.73 to 1.26) compared with women who did not consume this beverage. Similarly, there was no association when the first 4 years of follow-up were excluded, when nonfatal and fatal CHD end points were examined separately, or when we updated coffee consumption in 1984 or 1986 and examined only CHD during the next 2-year interval. Further, there was no association with caffeine intake from all sources combined or with decaffeinated coffee consumption. CONCLUSIONS--These data indicate that coffee as consumed by US women is not an important cause of CHD. PMID- 8627968 TI - Emerging bacterial zoonotic and vector-borne diseases. Ecological and epidemiological factors. AB - Among the etiologic agents of emerging infectious diseases are several bacterial organisms that naturally reside in animal and arthropod hosts. The most compelling emerging bacterial zoonotic and vector-borne diseases in the United States are Lyme disease; a Southern erythema migrans-like illness; human monocytic ehrlichiosis; human granulocytic ehrlichiosis; a novel cat flea associated typhus group rickettsiosis; bartonelloses of immunocompetent and immunocompromised persons, particularly with AIDS; and sylvatic plague. Some of these antimicrobial-treatable infections are life threatening. During the acute stage of illness when antimicrobial agents are most effective, the flulike clinical signs and symptoms and available laboratory tests frequently do not point to a particular diagnosis. Epidemiological factors determined by the ecology of the bacteria are often the most useful diagnostic clues. The recognition of these evolving problems emphasizes the need for development of better laboratory diagnostic methods, for surveillance for and tracking of disease, and for continued research into factors contributing to transmission of the organisms. The continual appearance of previously unidentified bacterial infections requires prospective national strategies for timely recognition of the syndrome, identification of the agent, establishment of criteria and methods for diagnosis, optimization of the treatment regimen, and determination of successful approaches to prevention and control. PMID- 8627969 TI - The spectrum of septic encephalopathy. Definitions, etiologies, and mortalities. AB - OBJECTIVE: To determine whether the severity of septic encephalopathy is correlated with gram-negative bacteremia and mortality and whether there exists a single or combination of metabolic derangements(s) that cause septic encephalopathy. DESIGN AND SETTING: Prospective case series in an academic medical center. PATIENTS: Fifty patients selected according to clinical and laboratory criteria for severe sepsis. The criteria included temperature, heart rate, respiratory rate, and hypotension and/or signs of systemic hypoperfusion. MAIN OUTCOME MEASURES: A single or combination of metabolic and laboratory derangements and organ failures, three different methods to grade the severity of septic encephalopathy, Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, gram-negative bacteremia and infection, and mortality. RESULTS: Encephalopathy was associated with an increase in mortality when graded by the Glasgow Coma Score; a score of 15 had 16% mortality, 13 to 14 had 20%, 9 to 12 had 50%, and 3 to 8 had 63% mortality (P < .05). Bacteremia was associated with encephalopathy; 13% of septic patients without encephalopathy vs 59% of patients with encephalopathy had bacteremia (P < .001) when graded by altered mental status. Septic encephalopathic patients had elevated serum urea nitrogen and bilirubin levels, increased APACHE II scores, and a higher incidence of renal failure. CONCLUSIONS: The severity of septic encephalopathy correlated with mortality, bacteremia, and renal and hepatic dysfunction. The Glasgow Coma Score is a useful tool for characterizing septic encephalopathy. Considerable variations can be found according to different criteria used to classify septic encephalopathy. PMID- 8627970 TI - Good care of the dying patient. Council on Scientific Affairs, American Medical Association. PMID- 8627971 TI - A 47-year-old man with chronic depression. PMID- 8627972 TI - A 72-year-old man with localized prostate cancer, 1 year later. PMID- 8627973 TI - High fiber intake. Indicator of a healthy lifestyle. PMID- 8627974 TI - Preventing multidrug-resistant tuberculosis. PMID- 8627975 TI - Increased protection of the ischemic myocardium by decreased aortic pressure. AB - The elevation of aortic pressure increases myocardial oxygen consumption and increases the blood supply to the myocardium. It is not obvious whether elevated systemic arterial pressure would enhance or diminish myocardial protection during acute myocardial ischemia. This report reviews previously published data on the effects of aortic pressure elevation on the coronary pressure-flow relationship, and on the ischemic myocardium in the dog. The shift of the maximally-dilated coronary pressure-flow line to the right on the pressure-flow diagram is an indication of the deterioration of the oxygen supply/demand ratio caused by aortic pressure elevation. The only maneuver recognized for protecting the ischemic myocardium is early reperfusion. In patients with de novo acute myocardial ischemia, we propose that myocardial protection can be enhanced by reduction of systemic blood pressure until coronary reperfusion therapy is initiated. PMID- 8627977 TI - Comparison of long-term efficacy of medical treatment versus coronary artery bypass grafting (CABG) in multivessel coronary artery disease. AB - The long-term results of medical therapy and coronary artery bypass grafting (CABG) were compared in patients with multivessel disease. All patients were confirmed to have > or = 75% luminal narrowing of major coronary arteries by coronary arteriogram. When multivessel disease was stratified into double- and triple- vessel disease, the outcomes varied. In triple-vessel disease, the outcome with CABG was good, but the outcome was unfavorable in those employing medical therapy, particularly in patients with decreased left ventricular (LV) function. In patients with double-vessel disease with good LV function, the long term results with medical therapy were just as favorable as those with CABG. However, double-vessel disease complicated by reduced LV function (ejection faction < or = 40%) had a clearly less favorable outcome when treated with medical therapy than with CABG. Thus, it is important for patients with multivessel disease to undergo revascularization if indicated, to improve their prognosis. On the other hand, the incidence of cardiac events arising from vein graft occlusions tended to increase in CABG patients after 5 years or more following surgery. PMID- 8627976 TI - Coronary dilating effects of intracoronary nicorandil. Comparison with isosorbide dinitrate. AB - Although nicorandil, N-(2-hydroxyethyl) nicotinamide dinitrate, is a nitrate ester, its cardiovascular action differs from that of nitrate compounds in several aspects. In this quantitative angiographic study, the acute coronary dilating effect of intracoronary nicorandil (0.25, 0.50, 1.0 mg) was compared with that of isosorbide dinitrate (ISDN; 1.0 mg) in 46 patients with or without ischemic heart disease (IHD). Dose-dependent right coronary dilating action was observed by intracoronary administration of nicorandil without any adverse effects. The same degree of right coronary dilation was achieved by the intracoronary application of equivalent doses of ISDN. We conclude that intracoronary administration of nicorandil is beneficial for the supportive treatment of IHD during coronary artery investigation and intervention without the risk of severe systemic hypotension. PMID- 8627978 TI - Evaluation of repeated balloon inflation in angioplasty as a clinical model of ischemic preconditioning. AB - Reevaluation of repeated balloon angioplasty as a model of ischemic preconditioning and of the role of collateral circulation and high-frequency electrocardiograms in repeated inflations was carried out. There have been few studies using angioplasty as a model of ischemic preconditioning of the collateral circulation during repeated inflations or of the use of high-frequency electrocardiograms during angioplasty. Twenty patients underwent 3 repeated balloon inflations, each with a duration of more than 138 seconds. During inflation, ipsilateral and contralateral coronary angiography and signal-averaged electrocardiography were performed. At the 1st inflation, the ST segment gradually increased as the inflation time elapsed. During the 2nd and 3rd inflations, in which ST elevation was nearly equal, the ST segment gradually increased, but it was not as prominent as that at the 1st inflation; thus, the concept that ischemia is gradually ameliorated (adaptation to ischemia) was not documented. Comparison of the maximal ST elevation and ipsilateral and contralateral circulation at each inflation revealed that the ST segment became elevated and the contralateral collateral circulation increased significantly at the 1st inflation compared with those in the control. ST elevation, however decreased significantly and collateral circulation remained unchanged at the 2nd inflation; thus, collateral circulation did not cause the ST elevation decrease. The total root mean square voltage detected by signal averaging decreased significantly only at the 1st inflation. Balloon angioplasty is not always suitable as a model of ischemic preconditioning and collateral circulation is not the cause of ischemic preconditioning. PMID- 8627979 TI - The effects of accumulated experience on radiofrequency ablation of accessory pathways. AB - Increasing experience in radiofrequency ablation for accessory pathways appears to reduce the procedure time, radiation time and radiofrequency pulse number, and results in a higher success rate. However, the effect of a learning curve on this procedure from the perspective of location and conduction direction of accessory pathways has not been reported before. The purpose of this study was to determine the effect of accumulated experience on the outcomes of radiofrequency ablation for accessory pathways and on the duration of the procedure parameters by analyzing the results of a dedicated ablation team. The first 512 patients with a single accessory pathway treated in this laboratory were included for analysis of the procedure parameters with respect to locations and conduction directions of accessory pathways. The results showed that the average procedure time, radiation time, and radiofrequency pulse number differed significantly among the different subgroups (left free wall, right free wall, posteroseptal and anteromidseptal location; manifest or concealed conduction). All subgroups except the anteromidseptal pathways showed a significant improvement of the procedure parameters with increased ablation experience. Although the initial rate of improvement was similar among the different subgroups, the rate of improvement in left free wall pathways nearly reached a plateau after 120 ablation procedures. Thus it was concluded that a certain number of ablation procedures was necessary before achievement of a high success rate with shorter procedure and radiation times and a lower radiofrequency pulse number. PMID- 8627981 TI - Palliative treatment for tetralogy of Fallot with percutaneous balloon dilatation of right ventricular outflow tract. AB - Percutaneous balloon pulmonary dilatation of the right ventricular tract was performed on 16 children with tetralogy of Fallot for palliative purposes after routine cardiac catheterization. Immediate improvement in aortic saturation from 73.4 +/- 6.8 to 84.0 +/- 4.8% (mean +/- SD = 10.6 +/- 2.7%, p < 0.001) and clinical symptoms were achieved in all 16 cases. The pressures in the right ventricle, pulmonary artery, left ventricle and aorta showed no remarkable changes after percutaneous balloon dilatation. The diameters of the proximal end and at the first branching of the right and left pulmonary arteries, pulmonary arterial index and the diameter of the descending aorta at the diaphragm increased significantly after balloon dilatation (p value 0.0004-0.006). One child suffered from repeated cyanotic spells in spite of the immediate improvement of aortic saturation. She received a left side Blalock-Taussig shunt 2 months after the balloon dilatation. None of the children had a significant complication. Eight had follow-up cardiac catheterization one year later and demonstrated much improvement in the diameters of the proximal end and at the first branching of the right and left pulmonary arteries, pulmonary arterial index and the diameter of the descending aorta at the diaphragm (p value 0.005 0.04). All 8 patients had their cardiac lesions successfully corrected. Percutaneous balloon dilatation is an alternative palliative therapy for children with tetralogy of Fallot. PMID- 8627980 TI - Notched T wave as evidence of autonomic nervous lability in Duchenne progressive muscular dystrophy. AB - We investigated the significance of notched T waves on the ECG in 30 patients with Duchenne progressive muscular dystrophy (DMD) and 50 age-matched controls using noninvasive cardiovascular examinations and measurement of urinary catecholamines. Notched T waves were more frequently observed in patients with DMD than in control subjects (46.7% vs. 20.0%, p < 0.05). Moreover, their frequency was age-independent in DMD, whereas they decreased with age in controls. Patients with notched T waves showed significantly increased heart rate, prolonged QTc and augmented excretion of urinary adrenaline compared with patients without them. There were no significant differences in casual BP or incidences of characteristic UCG abnormalities, such as mitral valve prolapse, and ECG abnormalities, such as tall R waves in the right precordial leads, between DMD patients with and without notched T waves. These findings suggest that notched T waves are associated with accelerated sympathetic nervous activity rather than progressive cardiac involvement in DMD. PMID- 8627982 TI - The first heart sound in atrial septal defect with reference to atrioventricular valve motion and hemodynamics. AB - To clarify the characteristics of the first heart sound in atrial septal defect (ASD) and its pathophysiological basis, 17 patients with ASD associated with incomplete right bundle branch block (IRBBB) and 7 with isolated IRBBB were studied using phonoechocardiography and Doppler echocardiography. Fifteen of the 17 ASD patients also were studied following surgical closure of the defect. Indices were compared among the preoperative ASD, postoperative ASD, and IRBBB groups including: P-Q intervals, loudness of the mitral and tricuspid component of the first heart sound (IM, IT), end-diastolic closing excursions of the mitral and tricuspid valves (Mx, Tx), and mitral and tricuspid inflow velocities during early diastole and atrial contraction. There were no significant differences in the P-Q intervals among these 3 groups. IM was attenuated, and IT was accentuated in ASD compared with IRBBB. Postoperatively, IM was augmented in all but 1 patient and IT was attenuated in all patients. Mx was significantly smaller, and Tx was significantly larger in ASD than in IRBBB. Postoperatively, Mx was significantly increased and Tx was significantly reduced; the maximal mitral inflow velocity during atrial contraction was increased while the maximal tricuspid inflow velocity was significantly reduced. Thus, the first heart sound in ASD is characterized by an attenuated mitral component and an accentuated tricuspid component. Hemodynamic alterations and consequent changes in closing energies of the atrioventricular valves probably account for these features. PMID- 8627983 TI - Transient prolongation of ventricular action potential duration after metabolic inhibition. AB - Transient prolongation of the action potential duration was observed in canine ventricular muscle during the reoxygenation period following metabolic inhibition. We investigated the effects of verapamil, lanthanum (La3+), and hexamethyleneamiloride (HMA) on the recovery time course of the action potential and its rebound prolongation. The time course of the intracellular resistivity was estimated from the conduction velocity and electrograms. The action potentials of canine left ventricular trabeculae were recorded by the conventional microelectrode technique. After a control tracing was obtained, the preparation was perfused with a hypoxic, acidic solution for 20 min and then reoxygenated with regular Tyrode's solution. After reoxygenation, action potential prolongation exceeding the control value by 21.0 +/- 7.3% was observed depending on the degree of metabolic inhibition. Verapamil depressed the rebound prolongation when it was added before the start of metabolic inhibition, but not when added after reoxygenation was started. La3+ and HMA depressed the rebound phenomenon. Intracellular resistivity was increased during metabolic inhibition, but showed no significant changes during the period of action potential prolongation. It was concluded that the rebound action potential prolongation was related to the accumulation of intracellular Ca2+ during metabolic inhibition. Other ions, such as Na+ and H+ may also contribute to the phenomenon by modulating outward currents. PMID- 8627985 TI - Effect of a calcium antagonist on renal hemodynamics in salt-loaded spontaneously hypertensive rats. AB - In the present study, we investigated the change in renal hemodynamics induced by a calcium antagonist in young (6 week-old) spontaneously hypertensive rats (SHR), a salt-sensitive hypertensive model. In acute experiments, SHR were fed either a 0.66% or 8.0% NaCl diet for 4 weeks. In acute experiments, manidipine, a calcium antagonist, was administered in a bolus dose of 10 microg/kg. In chronic experiments, SHR were fed a 0.66% NaCl, 0.66% NaCl plus 0.05% manidipine, 8.0% NaCl or 8.0% NaCl plus 0.05% manidipine diet for 4 weeks. Mean arterial pressure (MAP), glomerular filtration rate (GFR), and renal blood flow (RBF) were measured. Salt loading increased MAP in young SHR. Acute administration of manidipine decreased MAP more in salt-loaded SHR compared to non-salt-loaded SHR (-43.3 +/- 3.1 vs. -18.6 +/- 2.1 mmHg: p < 0.01). Moreover, chronic administration of manidipine attenuated the rise in MAP in salt-loaded SHR (155 +/- 3 mmHg vs. 196 +/- 5 mmHg; p < 0.01) and less so in non-salt-loaded SHR (150 +/-2 mmHg vs. 160 +/- 3 mmHg; p < 0.01). Salt loading elevated renal vascular resistance (RVR) but changed neither RBF nor GFR. The acute- and chronic administration of manidipine increased RBF (Acute; +0.77 +/- 0.22 ml/min/g kidney: p < 0.05, Chronic; 4.32 +/- 0.29 vs. 5.50 +/- 0.90 ml/min/g kidney: p < 0.01) in non-salt-loaded SHR, which was greater in salt-loaded SHR (Acute; +2.19 +/- 0.52 ml/min/g kidney: p 0.05 vs. non-salt-loaded SHR, Chronic; 4.29 +/- 0.53 vs. 6.09 +/- 1.41 ml/min/g kidney: p < 0.01) Manidipine also decreased RVR (Acute; -10.2 +/- 2.2 mmHg/ml/min/g kidney: p < 0.01, Chronic; 35.3 +/- 1.6 vs. 27.3 +/- 4.1 mmHg/ml/min/g kidney: p < 0.01) in non-salt-loaded SHR, which was greater in salt-loaded SHR (Acute; -21.1 +/- 3.1 mmHg/ml/min/g kidney: p < 0.01 vs. non-salt-loaded SHR, Chronic; 44.9 +/- 2.6 vs. 27.6 +/- 4.1 mmHg/ml/min/g kidney: p < 0.01). GFR did not change significantly following manidipine. It is suggested that the antihypertensive effect of the calcium antagonist, manidipine, was greater in salt-loaded SHR and was accompanied by profound amelioration of the abnormal renal hemodynamics. PMID- 8627984 TI - Plasma atrial natriuretic peptide levels in rabbits with alloxan monohydrate induced diabetes mellitus. AB - The effect of alloxan monohydrate-induced diabetes on the resting plasma atrial natriuretic peptide (ANP) level was investigated in 22 male New Zealand white rabbits. Alloxan monohydrate (100 mg/kg) dissolved in saline at a concentration of 50 mg/ml was administered by a single intravenous injection 3 months before the experimental analysis. The diabetic state was examined 72 h later by quantitative determination of blood glucose levels of >350 mg/dl. Beginning on day 3, 14 animals (Group 1) received a daily subcutaneous injection of 1 U insulin having moderate hyperglycemia (blood glucose concentration [BGC] between 300 and 400 mg/dl). Eight animals (Group 2; normoglycemic controls) received 3.2 U of insulin daily to maintain the BGC below 100 mg/dl. Eight healthy rabbits were included in the study as controls (Group 3). Blood samples for ANP analysis were obtained three months after administration of alloxan monohydrate. The plasma ANP levels in moderately diabetic rabbits (328 +/- 43 pg/ml) were significantly higher than those in normoglycemic (98.5 +/- 20 pg/ml) and healthy (76.6 +/- 18 pg/ml) controls (p < 0.001 for both). In addition, we found a significant correlation between plasma levels of glucose and levels of ANP (r = 0.665, p < 0.001). Our data indicate that further experiments need to be performed to investigate what is responsible for the elevation of plasma ANP levels in diabetic rabbits. PMID- 8627986 TI - Ultrafast computed tomography in the diagnosis and evaluation of anomalous origin of the right coronary artery. AB - Anomalous origin of the right coronary artery may lead to myocardial ischemia despite the absence of atherosclerosis. We report the case of a 52-year-old man who was admitted to our hospital with exertional chest discomfort and palpitations. An anomalous origin of the right coronary artery was demonstrated by coronary angiography. There was no evidence of atherosclerosis in either the left or right coronary arteries. However, detailed information regarding the proximal portion of the anomalous artery was not acquired by coronary angiography. In this patient, ultrafast computed tomography (UFT) revealed an acute angle takeoff of the anomalous right coronary artery from the aorta. Furthermore, the proximal portion of the right coronary artery traversed the aorta and pulmonary trunk. This case illustrates that UFT is useful for detecting an anomalous origin of the coronary arteries and evaluating the mechanism of myocardial ischemia in patients with anomalous origin of the coronary arteries. PMID- 8627987 TI - Isolated persistent fifth aortic arch with right-sided aortic arch. AB - A persistent fifth aortic arch was recognized in a 4-year-old boy by echocardiography, magnetic resonance imaging and angiocardiography. The case was unique in at least two respects: right-sided aortic arch, and systemic-to pulmonary connection without intracardiac lesion. This rare type of anomaly was correctly diagnosed and successfully repaired by patch closure of the arch. This is the first report of this combination of lesions in the Orient. PMID- 8627988 TI - Corrected transposition of the great arteries: dynamic three-dimensional echocardiography and volumetry. A new diagnostic tool in intensive care management. AB - Today, an increasing incidence of severe complications of cardiac malformations in adult patients must be expected since the life expectancy of such individuals is prolonged due to improved management. A 32-year-old woman with corrected transposition of the great arteries (CTGA) was admitted to the intensive care unit because of cardiac decompensation. Information provided by multiplane two dimensional transesophageal echocardiography was not sufficient for an unequivocal explanation of the cardiac decompensation. In CTGA uncommon complications may cause pulmonary edema. Using dynamic three-dimensional echocardiography to reveal function, volume parameters and the morphology of both ventricles and to exclude additional complications may be considered an adequate diagnostic tool for responding to this challenge. PMID- 8627989 TI - A surgically-treated case of left atrial myxoma complicating coronary artery fistula. AB - A surgically-treated case of left atrial myxoma complicating congenital coronary fistula is reported. A review of the literature indicates that this complication has not been reported previously. PMID- 8627990 TI - Left atrial myxoma associated with severe congestive heart failure, pulmonary hypertension, and multiple organ insufficiency. AB - We have experienced a case of left atrial (LA) myxoma with rapid progression of congestive heart failure and ensuing multiple organ insufficiency. After the tumor excision, the hemodynamic derangement was totally corrected and the patient dramatically recovered from kidney, liver, and lung insufficiency. Specifically, pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP), and central venous pressure (CVP) significantly decreased in the intensive care unit. Administration of prostaglandin E1 (PGE1) was effective in treating residual pulmonary hypertension. PMID- 8627991 TI - Endorectal ultrasonography. PMID- 8627992 TI - Hepatic cryosurgery precision: evaluation of ultrasonography, thermometry, and impedancemetry in a pig model. AB - One of the main problems of the use of liver cryosurgery is to be sure that a defined hepatic volume has been completely destroyed. We undertook an experimental pig study to determine histopathological evolution of cryolesions, to evaluate the value of intraoperative sonography, thermometry, and impedancemetry to monitor necrosis and to evaluate clinical and biological repercussions of hepatic cryosurgery. Forty-eight cryolesions were obtained by freezing each liver lobe of 12 experimental pigs during a 5-min contact with a flat cryoprobe cooled with liquid nitrogen. Cryolesions and the surrounding liver were monitored during cryosurgery by six thermocouple electrodes, five impedance electrodes, and intraoperative sonography. Animals were sacrificed immediately, 6 hr and between day 1 and day 32 after the procedure. Cryolesions were excised, and a full size pathological study was carried out. No morbidity or mortality was observed. At the end of the freezing time, cryolesions were hemispheric in shape, and their radius measured by sonography was 17.7 +/- 1.2 mm (mean +/- SD). Microscopic study showed sequential tissue alterations with edema, ischemic necrosis, tissue slough, and granulation. Cryolesions were sharply delineated from the normal liver tissue. The radius of necrosis at days 2 and 3 was 17 +/- 0.3 mm (mean +/- SD). It showed good correlation with the cryolesion size measured by intraoperative sonography. The temperature threshold to obtain complete normal liver necrosis was -15 degrees C. We found impedancemetry too difficult to use and not precise enough to monitor cryonecrosis. We conclude that intraoperative sonography and thermometry are useful means to monitor the extent of cryonecrosis during liver cryosurgery. PMID- 8627993 TI - Adenocarcinoma of the uterine cervix: a study of 37 cases. AB - In a study of 37 patients diagnosed with cervical adenocarcinoma between 1961 and 1994, clinical and pathologic findings were evaluated. Of the 37 patients, 27 (73%) had a pure adenocarcinoma, five (13.5%) had a collision tumor and five (13.5%) had an adenosquamous carcinoma. Twenty-six patients (70.3%) were diagnosed in Stage I, and 11 (29.7%) patients in Stage II, III, and IV. Two patients (5.4%) were treated with simple hysterectomy alone, nine (24.3%) with simple hysterectomy followed by radiotherapy, eight (21.6%) with radical hysterectomy alone, five (13.5%) with radical hysterectomy followed by radiotherapy, nine (24.3%) with radiotherapy alone, one (2.7%) with radiotherapy followed by simple hysterectomy, and three (8.1%) received no treatment. The actuarial 5-year survival rate was 69%. It is suggested that for patients with small early-stage disease, radical hysterectomy should be primary treatment and postoperative adjuvant radiotherapy would be advocated if high-risk features are histologically demonstrated. For all other patients, radiotherapy should be primary treatment. PMID- 8627994 TI - Potentiation of antitumor activity of mitomycin C by estradiol: studies of human breast carcinoma xenografts serially transplanted into nude mice. AB - The effect of experimental cancer chemotherapy with mitomycin C (MMC) was studied using three estrogen-receptor (ER)-positive (MCF-7, R-27, and Br-10) and one ER negative (MX-1) human breast carcinoma xenograft serially transplanted into nude mice, and the effect of estradiol (E2) priming on the antitumor activity of MMC was investigated. Intramuscular injection of E2 at 1 mg/kg changed the ER state and increased the growth fraction detected by flow cytometry, although the growth rate of ER-positive tumors was not effective by E2 priming. MMC suppressed the growth of the four xenografts in a dose-dependent manner. When 1 mg/kg E2 was administered 1 h before MMC treatment, which was given intraperitoneally at a dose of 3 mg/kg, the antitumor activity of MMC was increased in comparison with MMC alone in ER-positive strains, although the effect of MMC on MX-1 was not changed by E2-priming. Priming with E2 at this dose increases the growth fractions of ER-positive breast carcinoma cells, which are sensitive to MMC, resulting in increased antitumor activity of MMC. This E2-primed MMC chemotherapy may be of value in the treatment of ER-positive human breast cancer. PMID- 8627995 TI - Clinical manifestations in patients with hereditary nonpolyposis colorectal cancer. AB - The clinical manifestations of 1,042 Japanese patients with nonpolyposis colorectal cancer who underwent a resection between 1972 and 1992 at the National Kyushu Cancer Center were examined. Hereditary nonpolyposis colorectal cancer (HNPCC) was found in 39 (3.7%) patients. Some characteristic findings in HNPCC cases included early age of onset, a preponderance of right colon cancers, an increased frequency of colorectal cancers, and a favorable survival. Metachronous (postoperative) colorectal cancers developed significantly more often in cases with HNPCC than in those without (12.8% vs. 1.8%, P = 0.0001). Metachronous (postoperative) extracolonic cancers tended to develop more often in cases with HNPCC than in those without (10.2% vs. 3.5%, P = 0.053). In cases with HNPCC, the mean interval between the initial surgery and the diagnosis of the second cancer was 61 months (range; 12-153 months). These findings thus indicate the importance of routine and long-term follow-up to identify any second lesions, especially in patients with HNPCC. PMID- 8627996 TI - Recurrent esophageal carcinoma after esophagectomy with three-field lymph node dissection. AB - To evaluate the effect of the extended lymphadenectomy for thoracic esophageal carcinoma, the pattern of recurrence in the 50 patients with pT3 tumors who underwent esophagectomy with cervical, mediastinal, and abdominal lymph node dissection (3-F) (group A) was compared with that of 100 patients at pT3 who underwent esophagectomy without upper mediastinal and cervical lymphadenectomy (2 F) (group B). The cumulative 5-year survival rate for 115 patients who underwent 3-F was 50.9%. Cumulative 5-year survival rates for patients in groups A and B were 36.8% and 22.0%, respectively. The survival curve for group A was significantly better than group B (P = 0.02332). Lymphatic recurrence was noted less frequently in group A (8/23) than in group B (31/49) (chi 2 = 5.1149), whereas the rate of hematogenous recurrence was similar. Extension of the field of lymph node dissection reduced the lymph node recurrence in patients with thoracic esophageal carcinoma, which may have positively affected patient survival. PMID- 8627997 TI - Pre-, peri-, and postoperative chemotherapy for breast cancer: is one better than the other? AB - The purpose of the present study was to determine the relative efficacy of pre-, peri-, and postoperative chemotherapy in the prevention of breast cancer relapse and prolongation of host survival. The studies were performed using an experimental mouse breast cancer model. TA3Ha mouse mammary adenocarcinoma was transplanted into the mammary fat pad of syngeneic mice to obtain tumors in their natural organ. The tumors were surgically excised with a "curative" intent. A single treatment with 10 mg/kg doxorubicin was given intravenously pre-, peri-, or postoperatively. Among 74 mice whose tumors were resected but no doxorubicin was given, local recurrence, axillary metastasis, and lung metastasis were seen in 43%, 37%, and 16% of the mice, respectively. Seventeen (23%) mice had no evidence of disease. Doxorubicin given 4 days preoperatively reduced the rate of growth of primary tumor. Local recurrence was reduced in these mice by 30% and metastasis to the axillary lymph nodes and lung was completely prevented. Disease free survival was increased to 70% (P < 0.01). Similar beneficial effects were obtained when chemotherapy was administered 2 days prior to surgery. The peri operative chemotherapy group showed 8% (2/26) local recurrence, 4% axillary metastasis, and 0% lung metastasis. Proportion of mice without any evidence of disease increased to 92% (P < 0.00001). Chemotherapy given 4 days postoperatively resulted in 63% (10/16) local recurrence, 38% axillary metastasis, and 6.3% lung metastasis. Only 38% of the mice were disease-free. Thus in the model studied, perioperative chemotherapy offers the best chance for reduced recurrence and for improved disease-free survival. PMID- 8627998 TI - Long-term outcome after pneumonectomy for nonsmall cell lung cancer. AB - Long-term survivors (5 or more years) of pneumonectomy for nonsmall cell lung cancer are at risk for late death from cancer recurrence, second primary malignancies, and cardiopulmonary insufficiency related to the adverse physiological effects of pneumonectomy. A retrospective study of pneumonectomy patients was done to quantify the risks of late death from these causes. Of 246 patients treated for nonsmall cell lung cancer by pneumonectomy, medical records of 49 who survived 5 or more years were reviewed. Follow-up for the 49 long-term survivors ranged from 60 to 240 months, with a mean of 113 months. Twenty-five (51%) of the long-term survivors were alive at the time of the study. Twenty-four (49%) had died. Causes of death included late lung cancer recurrence (6 patients), second primary malignancies (7 patients), cardiopulmonary insufficiency (4 patients), and miscellaneous causes unrelated to cancer and its treatment (7 patients). Long-term survival after pneumonectomy for nonsmall cell lung cancer occurs in 20% of patients. Late lung cancer recurrence and second primary malignancies are important causes of death in these patients. Late cardiopulmonary insufficiency related to adverse physiological consequences of pneumonectomy is uncommon. Long-term follow-up is recommended after pneumonectomy for nonsmall cell lung cancer. PMID- 8627999 TI - Postradiotherapy hypothyroidism: radiation dose response and chemotherapeutic radiosensitization at less than 40 Gy. AB - To analyze our experience with iatrogenic hypothyroidism, we prospectively followed 84 patients, seen from 1984 to 1990, who had been diagnosed with either Hodgkin's disease (HD) or head and neck (H&N) carcinoma and subsequently treated with radiotherapy. Within these two diagnostic groups were subgroups whose treatment differed as to dose of therapeutic irradiation received or adjunctive use of chemotherapy. Approximately 50% of all patients and of each subgroup developed either clinical or subclinical hypothyroidism during follow-up. However, among the HD patients who received irradiation plus chemotherapy, a dose response relationship below a threshold limit of dose received, probably 40 Gy, was observed. PMID- 8628000 TI - Glutathione and phospholipid depletion of liver tumors after arterial ischemia. AB - Breakdown of membrane phospholipids is a causative event leading to irreversible cell injury after ischemia and reperfusion insults, which might be one mechanism leading to liver tumor cell death after repeated arterial ischemia as well. After 2 hr of hepatic dearterialization followed by 30 min of reperfusion tumor phospholipid was measured chromatographically, glutathione (GSH) analyzed by determining nonprotein sulfhydryl and activity of glutathione-S-transferase (GST) determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. A transient, arterial ischemia for 2 hr induced a substantial decrease of phosphatidylserine (PS) and phosphatidylinosital (PI) compared with sham treatment (P < 0.01). Although phosphatidylcholine (PC) and phosphatidylethanolamine (PE) did not significantly decline after a single arterial ischemia for 2 hr, they dropped dramatically following repeated arterial ischemia for 2 hr during 5 days (P < 0.01 and P < 0.05 respectively). GSH was depleted in tumors after both a single (P < 0.01) and repeated arterial ischemia (P < 0.05) and GST was inactivated as well (P < 0.001). By contrast, neither liver phospholipid nor liver GSH or GST was significantly changed. Tumor growth was significantly retarded in rats subjected to repeated arterial ischemia compared with sham treatment (P < 0.01). Repeated arterial ischemia facilitated degradation of tumor membrane phospholipids and induced depletion of GSH and inactivation of GST without affecting the normal liver. Thus, ischemia/reperfusion induced depletion of membrane phospholipids and of GSH might represent two mechanisms by which repeated arterial ischemia led to tumor growth delay. PMID- 8628002 TI - Poor prognosis of lower quadrant breast carcinoma. Nishi Nippon Study Group on Adjuvant Chemo-endocrine Therapy for Breast Cancer. AB - Although some lymphatic plexuses exist in lower quadrants of the breast, there have been no investigations of whether or not carcinoma located in this region is a prognostic factor for breast cancer. Of 914 patients with carcinoma of the breast who underwent curative resection following chemo-endocrine therapy between 1982 and 1985, 149 patients had disease of the lower quadrants. The recurrence free survival rate was lower in patients with the lower quadrants carcinoma than in those with carcinoma of other breast regions. Multivariate analysis showed that a lower quadrant tumor location was a significant prognostic factor for recurrence, especially soft tissue and visceral recurrence. The worse prognosis of patients with lower quadrant carcinoma of the breast suggests the possible existence of residual or occult tumor cells after surgical resection. PMID- 8628001 TI - Comparison between the in vitro intrinsic radiation sensitivity of human soft tissue sarcoma and breast cancer cell lines. AB - The purpose of this study is to evaluate the radiation sensitivity of human soft tissue sarcoma cell lines in vitro and to compare with that of human breast carcinoma and glioblastoma cell lines. The intrinsic radiation sensitivity parameters of seven human soft tissue sarcomas and eight breast carcinoma cell lines were investigated in vitro by clonogenic assays for single-dose irradiation under aerobic conditions on cells in exponential phase of growth. The results for sarcoma cell lines showed that the mean surviving fraction at 2 Gy (SF2) was 0.39 (SD +/- 0.09) with a range of 0.24 to 0.53, and the average mean inactivation dose (MID) was 1.92 (SD +/- 0.35) range from 1.36 Gy to 2.49 Gy. These values were not different from that of breast cell lines examined concurrently and using the same experimental methods (mean SF2 0.38, SD +/- 0.09; MID 1.9 Gy, SD +/- 0.37). However radiobiological parameters of nine karyotyped human malignant glioma cell lines determined earlier in this laboratory were significantly higher (mean SF2 0.50 +/- 0.14; mean MID 2.61 +/- 0.60). In conclusion, the data presented here do not support the view that cells of sarcomas show unusual radiation resistance. To the extent that the in vitro determined cellular radiation sensitivity reflects the tumor response in vivo, the success rate for radiation applied against sarcoma and breast carcinoma of comparable size could be similar. PMID- 8628003 TI - Palladium-103: a new radioactive source in the treatment of unresectable carcinoma of the pancreas: a phase I-II study. AB - Palladium-103 (Pd-103) is introduced in brachytherapy procedures because of its favorable physical properties, including its low energy, rapid dose fall-off, short half-life, and total cumulative dose delivery at a higher dose rate than iodine-125 (I-125) isotope. Intraoperative brachytherapy using I-125 pellets was reported to provide significant palliation and meaningful prolongation of life in highly selected patients with unresectable carcinoma of the pancreas. After considering some of the advantages of Pd-103 over I-125, we designed a phase I-II clinical trial to assess the feasibility of intraoperative Pd-103 in unresectable carcinoma of the pancreas to study the related morbidity when combined with chemotherapy and external beam radiation, and to evaluate the impact on palliation and local control rates. Between December 1989 and December 1993, 15 patients with biopsy-proven unresectable adenocarcinoma of the pancreas were treated with interstitial Pd-103 implants during laparotomy. In 13 patients the lesion was located in the head of the pancreas, in one patient in the uncinate process, and in one patient in the body of the pancreas. The stage distribution was as follows: T1 = 2; T2 = 6, and T3 = 7. In addition, all patients underwent biliary and gastric bypass. The mean number of Pd-103 pellets was 45; the mean total activity to obtain a matched peripheral dose (MPD) of 11,000 cGy was 68.9 mCi. The mean tumor volume encompassing the MPD was 16.5 cc. All patients received postoperative external beam radiation (4,500 cGy over 4 1/2 weeks) and chemotherapy (5-fluorouracil and mitomycin C). This combined treatment, consisting of intraoperative brachytherapy using Pd-103 and postoperative external beam radiation with chemotherapy, was well tolerated in all patients. These were no treatment-related mortalities, and no serious complications, such as bleeding or fistula formation. Pain relief was obtained within 3-6 weeks in 10 out of 12 patients presenting with pain. Survival ranged from 6 to 24 months (median 10 months). The study suggests that Pd-103 can be considered an alternative to I-125 for interstitial brachytherapy for unresectable carcinoma of the pancreas. Symptom relief appeared to occur faster and complications are significantly less. However, this study did not show any improvement in the median survival rate over I-125 due to the advanced stage cancer in the majority of patients in the study. PMID- 8628004 TI - Rapid resolution of necrolytic migratory erythema after glucagonoma resection. AB - A 55-year-old man presented with an 11-year history of necrolytic migratory erythema and glossitis. After the patient's serum glucagon was demonstrated to be elevated, computed tomography scan revealed a mass involving the head of the pancreas. The patient underwent a Whipple-type pancreatico-duodenectomy and his rash resolved completely 6 days after tumor resection. He received no adjuvant treatment. A discussion of the varying theories regarding the pathogenesis and treatment of glucagon-associated necrolytic migratory erythema is presented. PMID- 8628005 TI - Axillary dissection: preservation of the pectoralis minor. PMID- 8628006 TI - Completion thyroidectomy via the "retrograde access". PMID- 8628007 TI - bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells. AB - Parental and multidrug resistant HL60 leukemia cell lines were used to study coupling of expression of apoptotic/cytostatic (bcl-2, bax, bclxL, p21/Waf1, and c-myc) genes during differentiation. The multidrug resistant HL60 cell line, HL60/ADR, was less sensitive than parental cells to cytostatic activity of low (0.4-2 ng/ml) doses of PMA. However, during treatment with standard differentiating doses of PMA (10 ng/ml), no difference between the two cell lines in cytostasis and differentiation was found. Downregulation of c-myc and upregulation of p21/Waf1 proteins showed the same time-course in both cell lines. The bcl-2 mRNA was rapidly downregulated while bax and bclxL gene expression was not altered in both differentiating HL60 and HL60/ADR cells. Significant downregulation of bcl-2 protein occurred only in parental HL60 cells. In HL60/ADR, despite rapid cessation of bcl-2 protein synthesis, almost no change in steady-state bcl-2 protein level was found. The lack of bcl-2 protein downregulation was a result of the prolonged half-life of this protein in HL60/ADR cells. Thus, although downregulation of bcl-2 mRNA is coupled to differentiation, actual loss of bcl-2 protein is not required for accomplishment of the differentiation program. PMID- 8628008 TI - A day in the life of the Bcl-2 protein: does the turnover rate of Bcl-2 serve as a biological clock for cellular lifespan regulation? PMID- 8628009 TI - Infrequent microsatellite instability during the evolution of myelodysplastic syndrome to acute myelocytic leukemia. AB - Microsatellites are highly polymorphic, short-tandem repeat sequences dispersed throughout the genome. Instability of these repeat sequences at multiple gentic loci may result from mismatch repair errors and occur in hereditary nonpolyposis colorectal cancer and several other sporadic cancers, including chronic myelocytic leukemia as it progresses to blastic crisis. We investigated whether genetic instability occurred as myelodysplasia progressed to acute myelocytic leukemia. To this end, we studied microsatellite instability in 20 patients with myelodysplastic syndrome (MDS). These included five patients with refractory anemia (RA), three with refractory anemia with ringed sideroblast (RARS), nine with refractory anemia with excess blasts (RAEB) and three with chronic myelomonocytic leukemia (CMML). All of these patients transformed to acute myelocytic leukemia (AML) of various subtypes: three patients with M1, 11 with M2 and six patients with M4 (according to FAB classification). The DNA from both the MDS and AML phases of their disease was analyzed at 16 loci, and only four microsatellite instabilities were found in the 240 paired samples (1.6%) analyzed. These results indicate that mismatch repair errors such as microsatellite instability are not important in the evolution of MDS to AML. PMID- 8628010 TI - Enhanced topoisomerase II-induced DNA breaks and free radical production by a new anthracycline with potent antileukemic activity. AB - In a previous study we reported that a new anthracycline derivative (moflomycin) exhibited a higher antileukemic activity compared to other anthracyclines, such as daunorubicin and doxorubicin. To explain the superior antileukemic effect of moflomycin and to disclose a possible structure-activity relationship, we investigated the three main mechanisms by which anthracyclines are though to exert their antitumor effect: DNA binding, free radical production and topoisomerase II inhibition. The DNA interaction was assessed both by DNA binding and DNA unwinding assays, free radical generation was studied by electron spin resonance, and topoisomerase II interaction by analysis of the stimulation of enzyme-induced DNA breaks. The results showed a higher free radical production and a greater stimulation of topoisomerase II-mediated DNA cleavage by moflomycin than doxorubicin, associated with a lower DNA affinity. The different biochemical characteristics of moflomycin, particularly its interaction with topoisomerase II, are related to the structural modifications performed on the chromophore. These properties, associated with a higher stability of the molecule induced by the presence of an iodine atom on the sugar moiety, are probably responsible for the higher antileukemic activity of this compound. PMID- 8628011 TI - Modulation of metabolism and cytotoxicity of cytosine arabinoside with N (phosphon)-acetyl-L-aspartate in human leukemic blast cells and cell lines. AB - Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non hematological cells. PMID- 8628013 TI - Enhanced ex vivo drug sensitivity testing of chronic lymphocytic leukaemia using refined DiSC assay methodology. AB - Ex vivo drug sensitivity testing is of considerable benefit in aiding the choice of optimum chemotherapy for leukaemia patients, especially when several therapeutic options exist, e.g. for relapsed chronic lymphocytic leukaemia (CLL). We have used the Differential Staining Cytotoxicity (DiSC) assay to assess drug sensitivity in CLL for over a decade and here present many methodological improvements, including depositing multiple samples per microscope slide and performing a rapid LC90 evaluation. Using these improvements, 412/450 specimens were successfully tested. Failures were mainly due to extended specimen transit time. All 38 drugs tested exhibited dose-dependent cell kill and broad ranges of resultant LC90S were observed. Comparison of 2- and 4-day incubations underscored a requirement for 4-day incubation with pentostatin and steroids. The rapid, simple and streamlined DiSC assay presented here can aid choice of optimum therapy, identify novel anticancer agents and be used to study drug resistance. PMID- 8628012 TI - Interleukin-2-mediated growth of leukemic cells in lymph nodes of patients with adult T-cell leukemia/lymphoma. AB - We investigated the effect of interleukin-2 (IL-2) on tumor growth of primary adult T-cell leukemia/lymphoma (ATL) cells in biopsied lymph node cells obtained from 14 patients (seven [corrected] with acute-type disease, one with chronic type disease and six [corrected] with lymphoma-type disease). Biological activity of IL-2 in culture supernatants of the cells was detected in six out of 12 cases. The IL-2 mRNA in the lymph node cells was detected in four out of nine patients by northern blotting. However, it was detected in all nine patients examined by reverse polymerase chain reaction (PCR) method. Lymph node cells from 12 out of 14 patients showed a high or moderate proliferative response to IL-2; the remaining two patients showed a slight response. These results suggest that malignant growth of primary tumor cells in lymph nodes may be associated with the IL-2-IL-2 receptor system in patients with ATL more frequently than had been previously thought. PMID- 8628014 TI - Comparison of the effects of 2-chlorodeoxyadenosine and melphalan myeloma cell lines. AB - We have studied the effects of 2-chlorodeoxyadenosine (2-CdA) and melphalan on DNA synthesis and cell proliferation of five myeloma cell lines and one primary culture of highly purified myeloma cells. The DNA synthesis was estimated by thymidine incorporation and cell death was estimated by a coluorimetric assay sensitive to mitochondrial activity. The concentrations of 2-CdA giving 50% inhibition of DNA synthesis (ID50) in four cell lines were 8, 100, 500 and 2500 nM, whereas the corresponding concentrations of melphalan were 600, 600, 1000 and 7500 nM, respectively. In one cell line, the ID50 for melphalan was 400 nM, whereas 2-CdA apparently was without inhibitory effect and the ID50 was not reached. The ID50 for 2-CdA in the primary culture was 250 nM. When compared on a molar basis, 2-CdA had a more potent inhibitory effect than melphalan on four out of five myeloma cell lines. This is in contrast to clinical experiments which have shown lack of effect of 2-CdA in myeloma patients. Our study shows that 2 CdA has a marked heterogeneous effect on myeloma cell lines and opens up the possibility that a similar variation in sensitivity may also exist among myeloma cell clones in vivo. PMID- 8628016 TI - ASTA-Z 7557 impairs human natural killer (NK) cell activity. AB - Mafosfamide (ASTA-Z 7557) is a chemotherapeutic agent currently used for purging human bone marrow cells prior to autologous bone marrow transplantation. Adoptive cell-mediated immunotherapy has been shown to have a positive effect on the control of minimal residual disease and reinduction of remission post-bone marrow transplantation. Large granular lymphocytes and natural killer (NK) cells are believed to play a role in this effect. In the present work, we assessed the effect of ASTA-Z on the cytotoxic and proliferative capabilities of large granular lymphocytes (LGLs). The ASTA-Z significantly inhibited peripheral blood and bone marrow-derived LGL proliferation and cytotoxic capabilities, while this effect was less pronounced post-IL-2 treatment. We conclude that ASTA-Z purging may significantly impair the NK cell-mediated graft versus leukemia effect and therefore should be preceded by IL-2 therapy. PMID- 8628015 TI - Down regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation induced by 1,25 dihydroxyvitamin D3. AB - The NM23 gene, involved in the negative regulation of metastatic progression, has been found to be highly homologous to developmentally regulated genes such as the awd gene in Drosophila melanogaster and the Gip17 gene in Dyctiostelium discoideum. To ascertain whether the NM23 genes are involved in the differentiation processes of human cell lines, the NM23.H1 and NM23.H2 expression level has been determined during the monocyte-macrophage differentiation of HL-60 and U-937 cell lines induced by vitamin D3. In both lines, vitamin D3 produced induction of differentiative markers, inhibition of cell proliferation and a decrease of the NM23.H1, NM23.H2 and c-myc genes, behaving both as a differentiative and an antiproliferative agent. The fact that the c-myc transcriptional factor PuF is identical to the NM23.H2 gene and that NM23 protein could be a transcriptional factor suggests that the regulatory action exerted by vitamin D3 on c-myc transcription is mediated by NM23.H2. PMID- 8628017 TI - Cytogenetics and P-glycoprotein (PGP) are independent predictors of treatment outcome in acute myeloid leukemia (AML). AB - Clinical and biological features have recognized prognostic significance in acute myeloid leukemia (AML). To evaluate the interaction of these variables and weighted effect on treatment outcome, prognostic variables from 96 previously untreated patients were analyzed for association with expression of the MDR1 gene product P-glycoprotein (Pgp), and effect on response to induction chemotherapy, progression-free survival and overall survival. Multivariate relationships were analyzed using six prognostic variables, including age, cytogenetic pattern, gender, CD34+ surface phenotype, AML type (de novo versus secondary) and Pgp. Univariate comparisons indicate that Pgp (P = 0.0001), cytogenetic pattern (P = 0.0004) and a Cd34+ phenotype (P = 0.0005) are predictive of primary treatment failure, whereas Pgp (P = 0.0001) had the greatest predictive value in multivariate analysis. Only cytogenetic pattern retained prognostic significance (P = 0.0143) for response to induction therapy after adjustment for Pgp. Although all variable except gender were associated with Pgp, specimens harboring the favorable karyotypic abnormalities t(15;17), t(8;21) and inv(16) exclusively lacked Pgp expression. In a multivariate model, both Pgp and cytogenetic pattern predicted response and overall survival, whereas secondary AML and cytogenetic pattern influenced remission duration. These findings indicate that cytogenetic has prognostic relevance that is independent of Pgp, and implies the presence of undefined biological mechanisms affecting chemotherapy resistance. PMID- 8628018 TI - Cancers in relatives of children with non-Hodgkin's lymphoma. AB - We undertook a family study of children treated at the Institute Gustave-Roussy in France to investigate a familial aggregation of cancer in the families of children with non-Hodgkin's lymphoma (NHL). We obtained family dat for 284 children with NHL. Using the Standardized Incidence Ratio, we compared the observed and expected number of families with at least one proband relative affected by cancer at a young age (before 46 years). We found a small but non significant excess of all tumors in first-degree relatives (SIR = 1.3, 95% CI = 0.7-2.3) explained by a small but non-significant excess of hematological malignancies (SIR = 1.5, 95% CI = 0.2-5.5), particularly Hodgkin's disease and leukemia, and of osteosarcoma (SIR = 7.5, 95% CI = 0.1-41.4). This is probably a lower bound of the SIR, because the expected number of families was estimated from cancer incidence in France between 1978 and 1982, whereas most cancers occurred before this period. Other tumors were not in excess in first-degree relatives. PMID- 8628019 TI - Spontaneous granulocyte-macrophage colony growth by peripheral blood mononuclear cells in myeloproliferative disorders. AB - In the present study, the ability of peripheral blood (PB) progenitor cells to form granulocyte-macrophage (GM) colonies spontaneously in methylcellulose was investigated in healthy controls and patients with myeloproliferative disorders (MPDs). Spontaneous colony formation was observed in only one of the 18 control cases (6%), but in 22 of the 29 MPD patients (76%). The incidence of spontaneous GM colonies correlated both with the number of blast cells and the amount of c kit positive cells present in the initial sample. Spontaneous GM colony growth in PB mononuclear cells isolated from patients with MPDs seems to be a frequent phenomenon in contrast to the healthy controls and may present a marker of malignancy. PMID- 8628020 TI - Analysis of the novel cyclin-dependent kinase 4 and 6 inhibitor gene p18 in lymphoma and leukemia cell lines. AB - The genes for the CDK4/6-inhibitors p16INK4A/MTS1 and p15INK4B/MTS2 are frequently deleted in hematological malignancies. A new member of this family of CDK4/6 inhibitors is p18. In order to assess p18 growth-suppressor gene alterations in hematological neoplasms, we investigated 31 lymphoma and leukemia cell lines by PCR for both exons of this gene. No homozygous deletions were observed. Investigation of a new intragenic restriction fragment length polymorphism revealed no differences in allele distribution between the tumor cell lines and healthy volunteers. Our results suggest that homozygous deletion of the p18 gene does not play a major role in leukemogenesis or lymphomagenesis. PMID- 8628021 TI - Association of antineutrophil cytoplasmic antibodies with resistance to treatment of left-sided ulcerative colitis: results of a pilot study. AB - OBJECTIVE: To determine the frequency of antineutrophil cytoplasmic antibodies with perinuclear staining in patients with treatment-resistant left-sided ulcerative colitis. METHODS: We studied four groups: treatment-resistant left sided ulcerative colitis, treatment-responsive left-sided ulcerative colitis, ulcerative colitis historical controls, and healthy control subjects. Antineutrophil cytoplasmic antibodies were detected by enzyme-linked immunosorbent assay, and positive results were confirmed by demonstration of a perinuclear staining pattern by indirect immunofluorescence assay. RESULTS: The frequency of perinuclear antineutrophil cytoplasmic antibodies was significantly greater in treatment-resistant left-sided ulcerative colitis (90%) than in treatment-responsive left-sided ulcerative colitis (62%) (P = 0.03) or in ulcerative colitis historical controls (60%). CONCLUSION: The increased frequency of perinuclear antineutrophil cytoplasmic antibodies in treatment-resistant left sided ulcerative colitis suggests a possible association between these antibodies and relative resistance to medical therapy in patients with ulcerative colitis. PMID- 8628022 TI - A program to increase breast and cervical cancer screening for Cambodian women in a midwestern community. AB - OBJECTIVE: To determine local screening rates for breast and cervical cancer screening among Cambodian women older than 50 years of age who had used the health-care system, to compare these rates with those for non-Cambodian women, to identify barriers to screening among Cambodians, and to implement community screening. MATERIAL AND METHODS: From review of medical records, cancer screening rates for 1 year among Cambodians (N = 57) were compared with rates for a matched non-Cambodian sample (N = 57). Southeast Asian focus groups identified barriers to screening as a basis for intervention. The intervention included community informational programs in the Cambodian language, group screening appointments, provision of transportation, use of female physicians and interpreters, and an informal clinic setting. Cancer screening rates were compared before and after the intervention. RESULTS: Initial screening rates for Cambodians were significantly lower than for the non-Cambodians. Expressed barriers included lack of knowledge about cancer, shyness at physical examination, lack of transportation, fear of a large, technical medical center, and individual appointments. After the intervention, community screening rates were almost 5 times higher than at baseline. CONCLUSION: The intervention was successful in overcoming organizational, economic, and cultural barriers to cancer screening among older Cambodian women in this setting. PMID- 8628023 TI - Peak exercise blood pressure stratified by age and gender in apparently healthy subjects. AB - OBJECTIVE: To determine the peak blood pressure responses during symptom-limited exercise in a large sample of apparently healthy subjects, including both men and women over a wide range of ages. DESIGN: We retrospectively studied the blood pressure response during maximal treadmill exercise testing with use of the Bruce protocol in apparently healthy subjects. MATERIAL AND METHODS: Peak exercise blood pressures in 7,863 male and 2,406 female apparently healthy subjects who underwent a screening treadmill exercise test with the Bruce protocol between 1988 and 1992 were analyzed by age and gender. RESULTS: In this large referral population of apparently healthy subjects, peak exercise systolic and diastolic blood pressures and delta systolic blood pressure (rest to peak exercise) were higher in men than in women and were positively associated with age. In men, the 90th percentile of systolic blood pressure increased from 210 mm Hg for the age decade 20 to 29 years to 234 mm Hg for ages 70 to 79 years; the corresponding increase among women was from 180 mm Hg to 220 mm Hg. Delta diastolic blood pressure also increased with advancing age. The difference in peak and delta systolic blood pressures between men and women seemed to decrease after age 40 to 49 years. Exercise hypotension, defined as peak exercise systolic pressure less than rest systolic pressure, occurred in 0.23% of men and 1.45% of women and was not significantly related to age. CONCLUSION: Overall, peak exercise systolic and diastolic, as well as delta systolic, blood pressures were higher in men than in women and increased with advancing age. The reported data will enable clinicians to interpret more accurately the significance of peak exercise blood pressure response in a subject of a specific age and gender and will allow investigators to define exercise hypertension in statistical terms stratified by age and gender. PMID- 8628024 TI - Diagnosis and management of primary spinal epidural non-Hodgkin's lymphoma. AB - OBJECTIVE: To describe the diagnosis, management, and outcome in 10 patients with histologically confirmed primary spinal epidural non-Hodgkin's lymphoma. MATERIAL AND METHODS: We review the findings in a cohort of seven men and three women in whom this tumor was diagnosed between January 1979 and January 1993 and discuss the prognostic differences between primary and secondary spinal lymphomas. RESULTS: All patients (median age at diagnosis, 70 years) underwent a decompressive laminectomy, subtotal tumor resection, and spinal irradiation (median dose, 3,800 cGy). Nine of 10 tumors were of B-cell origin. Six patients are alive and well. In four patients, recurrent disease developed from 15 to 62 months after the original diagnosis; of these, one has died. The median duration of survival of all patients was 42 months; of those living more than 24 months after diagnosis, the median duration of survival was 80 months. CONCLUSION: A rapidly progressive spinal cord or cauda equina syndrome with neuroimaging findings consistent with an extradural compressive lesion should alert caregivers to the possibility of spinal epidural lymphoma. Although the prognosis for patients with secondary spinal epidural non-Hodgkin's lymphoma is often poor, primary spinal epidural non-Hodgkin's lymphoma can be associated with a favorable outcome if diagnosed and treated early. PMID- 8628025 TI - Hepatitis C genotypes: current trends and future implications. AB - OBJECTIVE: To review the geographic distribution and current understanding of hepatitis C virus (HCV) genotypes in regard to liver disease activity and response to treatment. MATERIAL AND METHODS: We review the relevant medical literature and discuss our recent findings relative to chronic HCV infection and the importance of HCV genotypes. RESULTS: HCV genotypes 1a and 1b are the most commonly found genotypes in patients with chronic HCV in the United States. Infection with HCV genotype 1b may be associated with more severe liver disease and may have a higher risk for the development of hepatocellular carcinoma. HCV genotype 2b seemed to be the most sensitive and HCV genotype 1b was the least sensitive to interferon therapy. CONCLUSION: The identification of the infectious HCV genotype may be beneficial in clinical settings and may assist in the selection of patients who would benefit from interferon treatment. PMID- 8628026 TI - Primary-care approach to the diagnosis and management of attention-deficit hyperactivity disorder. AB - Attention-deficit hyperactivity disorder (ADHD), a relatively common problem, affects 3 to 5% of school-age children. Physicians are often reluctant to treat children with ADHD, possibly because of the lack of a practical approach to the assessment. The medical model, with some modifications, is well suited for this purpose. Comprehensive treatment, including both medication and nonmedical intervention, should be coordinated by the primary-care provider. Logistic considerations are another barrier to effective treatment of ADHD. The physician must have a practical plan to provide high-quality care to children with ADHD. Primary-care physicians, who have detailed information about children and their families, are in an ideal position to treat patients with ADHD. PMID- 8628027 TI - Managing cancer pain: basic principles and invasive treatments. AB - Severe pain associated with cancer continues to be a substantial problem for patients, physicians, and the health-care system. During the past 2 decades, major advances have occurred in the understanding of the pathogenesis of pain. Likewise, considerable advances have occurred in the conceptualization of and clinical approach to cancer pain. This article briefly summarizes the basic principles of the treatment of cancer pain and in particular describes the World Health Organization 3-step "analgesic ladder" for the treatment of cancer pain. In addition, several invasive approaches for managing various refractory cancer pain syndromes are discussed. PMID- 8628028 TI - Hepatic adenomatosis. AB - This case report describes a 41-year-old woman with hepatic adenomatosis (multiple hepatic adenomas) associated with acute hemorrhage. She had no history of oral contraceptive use or corticosteroid therapy. Ultrasonography showed multiple masses in the right lobe of the liver. The clinical associations and potential complications of hepatocellular adenomas are discussed, and the histologic characteristics are provided. Their typical appearance on computed tomography, ultrasonography, magnetic resonance imaging, and nuclear scintigraphy is described. The differential diagnosis of hepatic adenomatosis and multiple solid liver masses is discussed. PMID- 8628029 TI - Roentgenographically occult small-cell lung cancer: case report and review of the literature. AB - This report describes a rare case of roentgenographically occult small-cell lung cancer in a 73-year-old man with hemoptysis. Fiberoptic bronchoscopy disclosed a 5-mm dome-shaped lesion; a biopsy established the diagnosis of small-cell lung cancer. The patient received a combination of chemotherapy and radiotherapy. More than 10 years later, he is still alive without recurrent disease. A review of the literature of roentgenographically occult small-cell lung cancer revealed the following: (1) a history of heavy smoking was common; (2) double primary bronchogenic carcinoma was noted; (3) hemoptysis or bloody sputum was an initial common symptom; (4) the sensitivity of sputum cytologic analysis was relatively low; (5) the tumor, which was shiny, smooth, and covered with bronchial epithelium, was often located at the bifurcation; and (6) lymph node metastatic involvement occurred. PMID- 8628030 TI - 68-year-old woman with dyspnea and chest pain. PMID- 8628031 TI - 22-year-old man with abdominal pain and rash. PMID- 8628032 TI - Psychogenic nonepileptic seizures. AB - Psychogenic nonepileptic seizures (NES) are commonly encountered in clinical practice, and they may pose difficult diagnostic problems. For appropriate evaluation and treatment of NES, a multidisciplinary team approach is needed; typically, a neurologist with expertise in epilepsy, a psychologist or psychiatrist, and a support staff should be involved. Psychogenic NES have no single initial clinical manifestation, and various etiologic factors may contribute to their development. Of importance, psychogenic NES are "real" seizures that may be as disabling as epileptic seizures. Most often, they occur on a subconscious level, and the patient may have no control over their occurrence. Precipitation or termination of a habitual seizure during video electroencephalographic monitoring has often been used to distinguish NES from epileptic seizures, but the results can sometimes be misleading. Numerous additional diagnostic techniques can be used to assist in making the diagnosis. Treatment is based on the type of psychiatric disorder present. Favorable prognostic factors include being female and having an independent lifestyle, normal electroencephalographic findings, higher intelligence, and no prior psychotherapy. PMID- 8628033 TI - Epileptiform electroencephalographic patterns. AB - Electroencephalography (EEG) is the most useful test for assessment of patients with epilepsy. It can help establish the diagnosis of epilepsy and determine the type of seizure disorder and its site of origin. Epileptiform abnormalities in the EEG tracing may be focal or generalized. The main types of focal epileptiform discharges arise from the temporal, frontal, occipital, centroparietal, centrotemporal, and midline regions of the brain. Generalized epileptiform discharges consist of the 3-Hz spike-and-wave, slow spike-and-wave, atypical spike-and-wave, paroxysmal fast activity, and hypsarrhythmic patterns. Status epilepticus is manifested by continuous epileptiform discharges or recurrent seizure activity without interim recovery, which can occur in a generalized or focal manner. Benign epileptiform variants unassociated with seizures can also be present in the EEG. Included in this category are the "14 & 6" positive bursts, small sharp spikes, wicket waves, 6-Hz spike-and-wave discharges, and rhythmic temporal theta activity. The EEG findings should be interpreted in the context of the overall clinical picture. PMID- 8628034 TI - Changes in forgoing life-sustaining treatments in the United States: concern for the future. PMID- 8628035 TI - pANCA and classification resistance in ulcerative colitis. PMID- 8628036 TI - Preventive health care in a multicultural society: are we culturally competent? PMID- 8628037 TI - Romanian physician contributes to the control of diabetes. PMID- 8628038 TI - Reflex sympathetic dystrophy. PMID- 8628039 TI - Reflex sympathetic dystrophy. PMID- 8628040 TI - Chiropractic and medical costs of low back care. AB - This study compares health insurance payments and patient utilization patterns for episodes of care for common lumbar and low back conditions treated by chiropractic and medical providers. Using 2 years of insurance claims data, this study examines 6,183 patients who had episodes with medical or chiropractic first contact providers. Multiple regression analysis, to control for differences in patient, clinical, and insurance characteristics, indicates that total insurance payments were substantially greater for episodes with a medical first-contact provider. Most of the cost differences were because of higher inpatient payments for such cases. Analysis of recurrent episodes indicates that chiropractic providers retain more patients for subsequent episodes and that patient exposure to a different provider type during early episodes significantly affects retention rates for later episodes. Patients choosing chiropractic and medical care were comparable on measures of severity and in lapse time between episodes. The lower costs for episodes in which chiropractors serve as initial contact providers along with the favorable satisfaction and quality indicators for patients suggest that chiropractic deserves careful consideration in gatekeeper strategies adopted by employers and third-party payers to control health care spending. More research is needed, especially in developing alternative measures of health status and outcomes. PMID- 8628041 TI - Financial pressure and competition. Changes in hospital efficiency and cost shifting behavior. AB - Using data from the American Hospital Association and the Medicare program, the authors analyzed the effects of financial pressure and market competition on changes in several measures of performance of 1,435 acute care hospitals between 1987 and 1989. Over the observation period, the least profitable hospitals constrained their growth in total expenses to half that for the most profitable hospitals (13.3% versus 27.6%) by limiting the growth of their staffs and their total assets. These changes were associated with a reduction in inefficiency of 1.8% (11.2%) compared with a very slight increase in inefficiency for the highest profit group. Similarly, hospitals in highly competitive markets controlled expenses relative to those in the least competitive areas. However, they also experienced slower revenue growth than did less competitive hospitals so that, in relative terms, their profit rates fell. The authors found no evidence to suggest that financial pressures created by either low profits or market competition resulted in hospitals engaging in cost-shifting. The authors conclude that health care reforms or market forces that put financial pressures on hospitals can result in cost-containment and improved efficiency without significant cost shifting. PMID- 8628042 TI - A 12-Item Short-Form Health Survey: construction of scales and preliminary tests of reliability and validity. AB - Regression methods were used to select and score 12 items from the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) to reproduce the Physical Component Summary and Mental Component Summary scales in the general US population (n=2,333). The resulting 12-item short-form (SF-12) achieved multiple R squares of 0.911 and 0.918 in predictions of the SF-36 Physical Component Summary and SF-36 Mental Component Summary scores, respectively. Scoring algorithms from the general population used to score 12-item versions of the two components (Physical Components Summary and Mental Component Summary) achieved R squares of 0.905 with the SF-36 Physical Component Summary and 0.938 with SF-36 Mental Component Summary when cross-validated in the Medical Outcomes Study. Test retest (2-week)correlations of 0.89 and 0.76 were observed for the 12-item Physical Component Summary and the 12-item Mental Component Summary, respectively, in the general US population (n=232). Twenty cross-sectional and longitudinal tests of empirical validity previously published for the 36-item short-form scales and summary measures were replicated for the 12-item Physical Component Summary and the 12-item Mental Component Summary, including comparisons between patient groups known to differ or to change in terms of the presence and seriousness of physical and mental conditions, acute symptoms, age and aging, self-reported 1-year changes in health, and recovery for depression. In 14 validity tests involving physical criteria, relative validity estimates for the 12-item Physical Component Summary ranged from 0.43 to 0.93 (median=0.67) in comparison with the best 36-item short-form scale. Relative validity estimates for the 12-item Mental Component Summary in 6 tests involving mental criteria ranged from 0.60 to 107 (median=0.97) in relation to the best 36-item short-form scale. Average scores for the 2 summary measures, and those for most scales in the 8-scale profile based on the 12-item short-form, closely mirrored those for the 36-item short-form, although standard errors were nearly always larger for the 12-item short-form. PMID- 8628043 TI - Responsiveness of a single-item indicator versus a multi-item scale: assessment of emotional well-being in an international adjuvant breast cancer trial. AB - A single-item linear analogue self-assessment scale for mood was compared with a 28-item adjective checklist for emotional well-being. To confirm its concurrent validity and responsiveness to treatment and recurrence in patients with breast cancer, emotional well-being was assessed every 3 months for 2 years and at 1 and 6 months after recurrence in 1,169 patients who were premenopausal and 960 patients who were postmenopausal. These patients were enrolled in two International Breast Cancer Study Group randomized clinical trials in operable breast cancer conducted from 1986 to 1993. To assess concurrent validity, Pearson's correlation between the linear analogue self-assessment scale and the adjective checklist were calculated for each time-point within each treatment group and for the two assessments after recurrence. Responsiveness to treatment and recurrence were analyzed using paired t tests and the squared ratio of these t tests, an estimate of relative efficiency. Concurrent validity of the mood linear analogue self-assessment was consistently confirmed across four language groups. Both measures were responsive; out of 24 changes over time, 19 were in the expected direction for the linear analogue self-assessment scale (p < or = 0.05 for 9 of 19) and 17 for the adjective checklist (p < or = 0.05 for 10 of 17). The linear analogue self-assessment scale was less but significantly efficient for detection of treatment effects, with relative efficiency estimates ranging from 0.16 to 2.45 and a median of 0.66 among the comparisons with relatively stable estimates (/t/ > or = 1.0) and more efficient for recurrence than the adjective checklist. The mood linear analogue self-assessment scale is a valid indicator of emotional well-being in patients with breast cancer in large multicenter, multicultural trials in which comprehensive scales are less feasible. This investigation supports the clinical relevance of linear analogue self-assessment scales as indicators of components of quality of life in cancer clinical trials. PMID- 8628044 TI - Medicaid health maintenance organizations. Can they reduce program spending? AB - A randomized trial to evaluate the Florida site of the Program for Prepaid Managed Care showed that the plan, a staff model health maintenance organization, was successful in attracting Medicaid enrollees. The evaluation established that the health maintenance organization was able to limit members' utilization. The savings were in the form of lower likelihood of using care. The amount of services received, once care was initiated, was the same in both fee-for service Medicaid and health maintenance organizations. The authors detected no differences in inpatient use or costs. Additionally, they found evidence that the plan attracted sicker than average enrollees, so this reduced utilization translates into Medicaid program savings. PMID- 8628045 TI - A guide for planning community-oriented health care: the health sector resource allocation model. AB - The objective of this study was to demonstrate the value of a planning model for the design and evaluation of community health services. The health status of Washtenaw County, Michigan was modeled. Data were obtained from the Michigan Department of Public Health, Medstat Systems, and the medical literature for 32 diseases or conditions, representing approximately 85% of causes of death and 56% of medical payments (excluding medication costs). An expanded life-table approach was used for 16 age-and sex-matched cohorts exposed to a disease attack rate, access-to-care rate, case fatality rate, morbidity, and costs. Rates can be modified to reflect changes due to treatment, secular trends, or prevention programs. Two alternative delivery methods were considered to show the potential impact of reducing cardiovascular deaths (worksite initiative), or increasing utilization of services (lay health promotion) on county health status and costs over time. Deaths, bed days, and annual medical payments were the main outcome measurements. Cardiovascular and cancer conditions are and will be the primary causes of death in this population. The most important causes of bed days are musculoskeletal conditions, chronic obstructive pulmonary disease, accidents, strokes, and depression. The major health-care payments are for angina pectoris and/or other cardiac conditions, musculoskeletal conditions, accidents, prenatal care, and/or childbirth, and depression. The two alternative scenarios illustrate how reductions in mortality are not necessarily equated with similar improvements in morbidity or costs. This model presents an overview of the current and projected health status of a community. With such a planning tool, a community can better understand the impact of potential prevention or intervention programs, and help design its health-care system within the constraints of available resources. PMID- 8628046 TI - Initiation of medical malpractice suits: a conceptualization and test. PMID- 8628047 TI - [4000 physicians answered a health status questionnaire. Physicians' mental health is worse compared to general population]. PMID- 8628048 TI - [A report on epidemiologic research. Research committees should increase the economic support]. PMID- 8628049 TI - [Salaries of American physicians are decreasing for the first time]. PMID- 8628050 TI - [French citizens irritated by reform plans. Street-protest of physicians against impaired working conditions]. PMID- 8628051 TI - [SFAM (Swedish Society of General Practice) wants to develop specialists' examination in general practice. Create a system of quality and life-long learning]. PMID- 8628052 TI - [Care agreements in Orebro County]. PMID- 8628053 TI - [Patients' position is weakened again!]. PMID- 8628054 TI - [Quality development with a national registry of diabetes]. PMID- 8628055 TI - [Facing death requires maturity and courage]. PMID- 8628056 TI - [The health insurance agency should pay more for the certificates]. PMID- 8628057 TI - [There should be "standards"!]. PMID- 8628058 TI - [Can tamoxifen relieve fibromyalgia?]. PMID- 8628059 TI - [Child murders in Bagdad]. PMID- 8628060 TI - [A flexible fiber endoscope is useful in complicated anesthetic interventions]. PMID- 8628061 TI - [How to get rid of a doctor. A noble story]. PMID- 8628062 TI - [Extended guaranteed care. What are we going to do?]. PMID- 8628063 TI - [More effective screening prevents cervical cancer]. PMID- 8628064 TI - [Gene therapy. Expectations and realization]. PMID- 8628065 TI - [A vaccine against diarrhea. Soon a reality in the fight against viral gastroenteritis in both developed and developing countries]. PMID- 8628067 TI - [Transplantation of retinal cells in man]. PMID- 8628066 TI - [A case report. Thrombocytopenia and granulomatous hepatitis caused by quinine]. PMID- 8628068 TI - [Extend screening for cervical cancer! Improved diagnosis with papillomavirus test and colposcopy]. PMID- 8628070 TI - [Drug insurance is an underutilized resource. Physicians should help more people to report]. PMID- 8628069 TI - [Alternative drugs against Chlamydia urethritis. Tetracycline offers the most cost-effective cure]. PMID- 8628071 TI - [Mutual exchange at a study visit to Lithuania]. PMID- 8628072 TI - [Performing surgery on patients in their 80's may be beneficial. Age alone is not a risk factor in heart surgery]. PMID- 8628073 TI - [A woman behind the Noonan syndrome. She described a hereditary disease in children with congenital heart defects]. PMID- 8628074 TI - [Conditions of the good death--an important debate in our time]. PMID- 8628075 TI - [Enteral nutrition at home. Every third child without any complications]. PMID- 8628076 TI - Evolution of research otolaryngology--head and neck surgery over the past century. PMID- 8628077 TI - Lorente de No's "Anatomy of the eighth nerve. I. The central projection of the nerve endings of the internal ear; III. General plan of structure of the primary cochlear nuclei." (Laryngoscope. 1933;43:1-38 & 327-350). PMID- 8628078 TI - A tribute to Max Goldstein, MD, founder and editor of The Laryngoscope. PMID- 8628079 TI - Laser versus cold instruments for microlaryngoscopic surgery. AB - Controversy has arisen concerning the merits of the CO2 laser in microlaryngoscopic surgery because of the potentially harmful effects that the injudicious application of the laser could have on voice production. In an effort to develop a logical approach to instrument selection, the author examined the use of both cold instruments and the CO2 laser in the treatment of various benign and malignant lesions. A retrospective review of 307 microlaryngeal procedures performed over a 3-year period revealed that 263 (86%) were glottal and 44 (14%) were supraglottal. Of the 263 glottal procedures, 203 (77%) employed cold instruments alone and 60 (23%) used both cold instruments and the CO2 laser. The laser was used to assist in all 44 supraglottal procedures. Therefore, 203 (66%) of 307 procedures were done with cold instruments alone, and 104 (34%) of 307 procedures were performed using the CO2 laser with cold instruments. Lesions were stratified on the basis of pathology and size, as well as surgical approach. A primary phonomicrosurgical principle in glottal surgery is to maximally preserve the vocal fold's layered microstructure (laminae propria and epithelium). Precise tangential dissection was necessary for achieving this goal. For limited lesions, this dissection was best accomplished with cold instruments alone. The CO2 laser facilitated hemostatic surgical dissection for all supraglottal lesions and for selected larger glottal lesions in which bleeding would obscure visualization of the microanatomy of the musculomembranous vocal fold. PMID- 8628080 TI - Enhanced magnetic resonance imaging and subtraction techniques in postoperative evaluation of craniofacial resection for sinonasal malignancy. AB - The use of magnetic resonance imaging (MRI) enhanced with gadolinium. and diethylenetriamine pentaacetic acid (DTPA) in the preoperative evaluation of sinonasal malignancy is well established and has an accuracy of over 98%. Since 1979, 208 patients have undergone craniofacial resection at London's Institute of Laryngology & Otology, primarily for sinonasal neoplasia affecting the anterior skull base. Thirty-five "high-risk" patients had MRI at follow-up. The MRI scans were assessed before the patients were examined under anesthesia. MRI was assessed before examination under anesthesia and the results compared with histologic findings demonstrating a reasonable degree of positive correlation but one which is inferior to that found preoperatively (80%). An extension of this technique using the subtraction of T1-weighted MRI with Gd-DTPA highlights areas of increased vascularity, which significantly improves the ability to predict recurrence at the skull base and above the surgical repair in the anterior cranial fossa (94%). PMID- 8628081 TI - Management of carcinoma of the supraglottic larynx: evolution, current concepts, and future trends. AB - The treatment of cancer of the supraglottic larynx has undergone an evolution. Better understanding of the anatomy and biology of cancer in this anatomic site has enabled surgeons to devise effective oncologic strategies while making every effort to preserve the function of the larynx. Certain recent concepts and changing trends have emerged in the treatment of cancer of the supraglottic larynx, including the treatment of the neck, significance of extracapsular spread of tumor in cervical lymph nodes, and conservation laser surgery. In 1985, Snyderman et al. reported the prognostic significance of extracapsular spread in patients with cancer of the supraglottic larynx. In 1990, Lutz et al. reported the results of our experience with the treatment of 202 patients. The review verified the significant risk of bilateral neck disease in these patients, even with adjuvant radiation therapy. Accordingly, since 1990 all patients having cancer of the supraglottic larynx have been treated in the Department of Otolaryngology at the University of Pittsburgh with bilateral neck dissections. The use of adjuvant radiation therapy has been based on the presence of extracapsular spread. This study documents the oncologic effectiveness of this treatment and confirms the efficacy of bilateral neck dissections in an attempt to control neck disease and the prognostic significance of extracapsular spread. We review the evolution of the treatment of cancer of the supraglottic larynx, present our results, and consider innovative surgical approaches. PMID- 8628082 TI - Echo-planar magnetic resonance imaging of deglutitive vocal fold closure: normal and pathologic patterns of displacement. AB - Abnormalities of vocal fold closure during deglutition predispose to aspiration due to impairment of airway protection. Conventional assessment of deglutitive vocal fold motion with laryngoscopy does not permit visualization through a complete adduction-abduction cycle. We determined spatiotemporal patterns of deglutitive vocal fold adduction through echo-planar magnetic resonance imaging in 15 normal volunteers and 6 patients with vocal fold paralysis. In normal volunteers, deglutitive vocal fold adduction was synchronized with laryngeal elevation, with complete vocal fold closure at the apex. Patients with unilateral vocal fold paralysis demonstrated reduced elevation and medial movement of the involved vocal fold. At maximal laryngeal elevation the uninvolved vocal fold attained a position superior to the paralyzed fold, resulting in level differences and an interglottic gap. Patients with bilateral vocal fold paralysis demonstrated reduced elevation and medial movement of both vocal folds. These findings indicate that normal and abnormal patterns of vocal fold displacement can be distinguished noninvasively through the use of echo-planar imaging. PMID- 8628083 TI - Longitudinal evaluation of vocal function after thyroplasty type I in the treatment of unilateral vocal paralysis. AB - This study investigated longitudinal changes of vocal efficiency and stability after primary thyroplasty type 1. Fifty-three patients with unilateral vocal-fold paralysis underwent vocal-function evaluation preoperatively and at periodic intervals of 1, 3, and 6 months postoperatively. Vocal-function assessment included videostrobolaryngoscopic examination, acoustical and aerodynamic analysis, and perceptual judgment of voice characteristics. Parameters that included glottic-gap size, maximum phonation time, glottic-flow rate, jitter, harmonic/noise ratio, breathiness, hoarseness, loudness, and phrasing showed significant improvement after thyroplasty and remained stable as early as 1 month postoperatively, with only slight fluctuations over a 6-month period. Postoperative voice outcome was not affected by age, sex, duration of vocal symptoms, cause of paralysis, or preoperative pulmonary function. PMID- 8628084 TI - Epithelial cell proliferation in nasal polyps could be up-regulated by platelet derived growth factor. AB - The modifications of epithelial differentiation and proliferation observed in nasal polyps (NP) could be related to local secretion of growth factors, among which platelet-derived growth factor (PDGF) could play a key role. We therefore prospectively studied, by immunohistochemistry, proliferating cell nuclear antigen (PCNA, an S-phase cell marker), PDGF, and CD-68 (activated macrophages marker) expression in NP and inferior turbinate mucosa (NM) in 11 patients. Our data show that PCNA and PDGF expression are increased in NP epithelium, while CD 68 expression is increased in NP epithelium and lamina propria when compared to NM. Increased local PDGF secretion by numerous activated macrophages could therefore be involved in epithelial cell proliferation up-regulation in NP. PDGF could also be involved in the pathogenesis of NP via its connective tissue remodeling actions. PMID- 8628085 TI - Three-dimensional computed imaging using a personal computer for nasal surgery. AB - Three-dimensional (3-D) computed imaging was applied before surgery in 16 patients with nasal or paranasal disease. The images obtained by computed tomography (CT) were scanned by a personal computer. The contours of the organs were outlined on the scanned images, and the images were then reconstructed using 3-D imaging software. The resulting 3-D images were evaluated and compared with actual surgical images. The 3-D images were found to be useful for surgical procedures, because they facilitated recognition of the topologic relation between structure and lesion. Although this method requires a personal computer and an image scanner, it is cheaper than, and in some respects even superior to, a 3-D CT system. PMID- 8628086 TI - Comparison of clinical evaluation and computed tomographic diagnostic accuracy for tumors of the larynx and hypopharynx. AB - Ninety-five patients with laryngeal and hypopharyngeal cancer were examined and staged preoperatively by clinical evaluation (CE) and computed tomography (CT). The CE and CT staging were compared to each other and to the pathologic (PT) staging of the tumors. The CT staging showed high accuracy in staging transglottic (88%), supraglottic (68%), and oropharyngeal tumors invading the larynx (68%) when compared to the PT findings. The CT staging was less effective in evaluating glottic tumors (46%), both overstaging (12%) and understaging (20%) cases. Combined CE-CT evaluation showed higher accuracy in staging all tumors (84%) compared to CE alone (52%) or CT alone (68%). The findings suggest that combined CE-CT should be used to evaluate laryngeal and hypopharyngeal tumors. PMID- 8628087 TI - An application of telemedicine technology for otorhinolaryngology diagnosis. AB - Applications of interactive video communications for providing remote telemedicine consultations are under study. Most of these applications use "off the shelf" medical peripheral devices that are not specifically designed for telemedicine examinations. This study reports a field trial of the telemedicine instrument pack (TIP), a small self-contained unit under development by the National Aeronautics and Space Administration for use in spaceflight. Patients with otorhinolaryngology problems were selected from a primary care practice and examined with the TIP by a technician under the direction of a remote consultant. The equipment proved adequate for examination of the posterior nose, hypopharynx, and larynx. Examinations of the ear and oropharynx with the TIP were not optimal, suggesting a need for further modifications of this prototype unit. PMID- 8628088 TI - Human nasal epithelium adsorbs complement C3-related fragments and expresses cell membrane complement regulatory proteins. AB - Recent evidence suggests that complement is activated in human nasal airways in inflammatory states. Activated complement protects the nasal mucosa against microorganisms, but also has the potential to lyse the host's normal cells. Complement-mediated cell lysis depends on adsorption of complement to the cell membrane and on uninterrupted activation of the complement cascade upon the same cell membrane. In the present study, the authors investigated first whether key complement components, C3-related fragments, are adsorbed to nasal epithelial cell membrane. Second, we investigated whether nasal epithelium expresses cell membrane complement regulatory proteins that are known as interruptors of complement activation. Studies were done using fresh nasal mucosa obtained at turbinectomies from allergic rhinitis and vasomotor rhinitis patients. In addition, in order to establish an in vitro model, studies were also done using primary cell cultures of nasal epithelium. We have found that complement C3 related fragments are present on cell membranes of fresh nasal epithelium and that C3-related fragments are adsorbed to the epithelial cell membrane in nasal mucosa tissue segments and in cell cultures that were incubated with autologous serum. Adsorption of C3-related fragments to the cell membrane of cultured nasal epithelial cells was found by flow cytometry analysis to be concentration dependent. In addition, we found that nasal epithelium in fresh tissue and in cell culture express three cell membrane complement regulatory proteins: membrane cofactor protein (MCP, CD46), decay-accelerating factor (DAF, CD55), and CD59. Our findings in fresh nasal epithelium suggest that complement activation may occur upon the nasal epithelial cell membrane during inflammation in vivo and that nasal epithelium might regulate this complement activation. Our in vitro cell culture model will allow further investigations of complement activation and regulation upon the human nasal epithelial cell membrane. PMID- 8628089 TI - Effect on the nasal mucosa of long-term treatment with oxymetazoline, benzalkonium chloride, and placebo nasal sprays. AB - A parallel, randomized, double-blind study was performed in 30 healthy subjects to investigate the effects on the nasal mucosa of a 1-month treatment with nasal sprays. Ten subjects received oxymetazoline nasal spray; 10 subjects used a nasal spray containing the preservative benzalkonium chloride, and the others were treated with a placebo nasal spray. The three variables that were studied --nasal mucosal swelling, symptom scores, and nasal reactivity-- were estimated by histamine challenge before and after 28 days of treatment. Rhinostereometry was used to measure nasal mucosal swelling and nasal reactivity. After 28 days of use, benzalkonium chloride spray alone induced an increase in nasal mucosal swelling. At the end of the month, the score for nasal stuffiness was significantly higher for the group treated with oxymetazoline than for those treated with benzalkonium chloride. Oxymetazoline nasal spray induced a pronounced increase in nasal reactivity, which was significantly greater than that induced in the placebo group. Long-term use of placebo and benzalkonium chloride nasal sprays also caused an increase in nasal reactivity, but not to the same extent as with the nasal sprays containing oxymetazoline. The authors concluded that long-term use of oxymetazoline induces a sensation of nasal stuffiness, which may be due to unconscious exaggeration of the degree of nasal stuffiness, induced nasal hyperreactivity, or a combination of both. These factors are probably the main reasons for the prolonged use of nasal decongestive sprays and the development of rhinitis medicamentosa. Benzalkonium chloride induces mucosal swelling, which explains why the presence of this preservative in a decongestant spray aggravates rhinitis medicamentosa. PMID- 8628090 TI - Preventive management of cerebrospinal fluid leakage in translabyrinthine surgery. AB - A simple technique can be used to prevent cerebrospinal fluid (CSF) leakage through the nose and the wound in translabyrinthine surgery. This method, evolved from Glasscock's technique of packing the eustachian tube, adds only 5 to 10 minutes to the procedure. The procedure was modeled in the temporal bones of 20 human cadavers, and the findings are described. With this technique, no nasal CSF leaks occurred in a consecutive series of 25 patients who underwent acoustic tumor surgery. Factors thought to be necessary for the success of the technique are discussed. PMID- 8628092 TI - Regeneration of the damaged endolymphatic sac epithelium. AB - The regeneration of endolymphatic sac (ES) epithelium after epithelial damage was examined using 5-bromo-2'-deoxyuridine (BrdUrd). Guinea pigs and anti-BrdUrd, antikeratin, and anti-basement membrane (BM) component antibodies were used. Animals were sacrificed at 3, 7, and 14 days following an immune injury of the ES. The animals were sacrificed after an administration of BrdUrd at the designated times. Decalcified temporal bones were examined immunohistochemically. At day 3, epithelial loss without subepithelial BM was seen in the region of marked cell infiltration. BrdUrd-positive cells could be detected in the epithelium at the edge of the wound. At day 7, cell infiltration decreased, and the epithelial lining with BM was repaired. These results reveal that the epithelium of the ES can regenerate within 7 days after an immune injury. PMID- 8628091 TI - Effects of oral and systemic immunization on nasopharyngeal clearance of nontypeable Haemophilus influenzae in BALB/c mice. AB - BALB/c mice were immunized orally or subcutaneously with formalin-killed nontypeable Haemophilus influenzae (NTHi). Salivary immunoglobulin A (IgA) antibody titers against NTHi were significantly increased by oral immunization, but not by subcutaneous immunization. Both immunization procedures remarkably increased the levels of serum antibody activities of both IgA and immunoglobulin G. Live NTHi were then inoculated into the naso-pharynx, and the clearance of the pathogen from the nasopharynx was observed. Significantly fewer bacteria were present in the nasopharynx of the orally immunized mice than in the control mice. However, there was no significant difference between the subcutaneously immunized mice and the control mice. The results indicate that oral immunization can enhance the ability of mice to clear NTHi from the nasopharynx. PMID- 8628093 TI - A histopathologic review of temporal bone exostoses and osteomata. AB - It is widely accepted that external auditory canal exostoses and osteomata are separate clinical entities that differ in their gross appearance. Disagreement still exists as to whether external auditory canal exostoses and osteomata should be considered similar or separate histopathologic entities. A chart review was performed of all patients who had external auditory canal exostoses or temporal bone osteomata excised from January 1991 to November 1994 at St. Vincent's Hospital, Sydney. A histologic examination was undertaken with a blind study method of the patients whose pathological specimens were suitable for assessment. Eight exostoses and five osteomata were available for examination. The results of this study demonstrate that exostoses and osteomata of the external auditory canal cannot be reliably differentiated by routine histopathological examination. PMID- 8628094 TI - Brain-derived neurotrophic factor and peripheral nerve regeneration: a functional evaluation. AB - The potential benefit of brain-derived neurotrophic factor (BDNF) on motor-nerve regeneration after transection injury in 24 adult rats was evaluated after entubulation repair. Gait analysis for ankle angle and tension transduction device (TTD) strain-gauge measurements yielded functional evaluation of regeneration. The BDNF (15 mg/mL) or phosphate buffered saline (control) was injected into the silicone elastomer (Silastic) channel. Gait analysis performed 0, 2, 4, 6, 10, and 12 weeks after injury demonstrated a significant difference between uninjured and injured legs of 23 and 43 degrees, respectively (P<.001, analysis of variance). The TTD evaluation 13 weeks after injury demonstrated a significant decrease in force development of injured compared to uninjured legs, 148 and 58 g, respectively (P<.001). No functional benefits were demonstrated between BDNF-treated versus control-treated animals in either model for a single exposure to BDNF. PMID- 8628095 TI - Predictive value of facial nerve electrophysiologic stimulation thresholds in cerebellopontine-angle surgery. AB - The predictive value of intraoperative stimulation thresholds for facial nerve function, using a constant-current system, was examined in 49 patients undergoing resection of cerebellopontine-angle tumors. Immediately after surgery, 75% of the 0.1-mA threshold group, 42% of the 0.2-mA group, and 18% of the 0.3-mA or greater group had good (grade I or II) facial nerve function. One year after surgery, 90% of the 0.1-mA group, 58% of the 0.2-mA group, and 41% of the 0.3-mA or greater group had grade I or II function. A statistically significant breakpoint of 0.2 mA was found to predict good postoperative facial function. Delayed facial paralysis occurred in 22% of patients, but the prognosis for these patients was favorable. Both current stimulation threshold and duration are necessary for a meaningful comparison of data between investigators. PMID- 8628096 TI - Indications and variations of transcochlear exposure of the ventral brainstem. AB - The transcochlear exposures represent a spectrum of three approaches (transotic to transcochlear to transpetrous) that provide progressively wider lateral skull base exposure. The approaches all combine the translabyrinthine approach with removal of the cochlea. The facial nerve remains in situ in the transotic approach. The facial nerve is mobilized posteriorly in the transcochlear approach, and the transpetrous exposure adds resection of the petrous apex with carotid artery mobilization. This report focuses on the indications for each of the variations of transcochlear exposure. These lateral skull base exposures can be individualized to the needs of each patient. PMID- 8628097 TI - Influence of chemical allograft preservation procedures on the human immunodeficiency virus. AB - Since chemically preserved allogenic transplants have an established place in reconstructive procedures, the possibility of transferring the human immunodeficiency virus (HIV) with these transplants has been intensively discussed. In this study the authors obtained brain and spleen samples from six HIV-infected cadavers and preserved them with Merthiolate, Cialit, and formaldehyde. After preservation, the tissues were examined for proviral HIV-1 DNA (gag, pol, env) using the polymerase chain reaction. Proviral sequences were clearly demonstrated after the preservation procedure. The results of this study indicate that HIV remains in tissues that have been treated with Merthiolate, formaldehyde, or Cialit. Further investigations are necessary to determine if the virus is in an inactivated or activated form. It can be concluded that, because of the possible transmission of HIV by chemically preserved homografts, serologic screening of donors should be mandatory. PMID- 8628098 TI - The communicating vein and its use in the radial forearm free flap. AB - Thrombosis is the most frequent cause of failure in microvascular free-tissue transfer. The large communicating vein of the cubital fossa connects the deep and superficial venous drainage of the radial forearm free flap (RFFF). This vein allows the surgeon to simultaneously drain both systems by means of the large veins of the cubital fossa. We prospectively collected data on the venous anatomy of the cubital fossa in 40 consecutive RFFFs over a 3-year period. We then retrospectively reviewed available data from the 14 cases preceding the cases in the prospective series. At least 78% of our patients had a communicating vein that facilitated dual venous drainage; 87% of our RFFFs were drained by both the superficial and deep venous systems, and 90% of our RFFFs had two or more venous anastomoses. We had no RFFF failures in our series of 54 flaps. We present our venous anatomy findings in this series of forearms as well as the venous anastomoses of our 54 patients. The surgi- cal-flap harvest, including the communicating vein and its use, may provide an advantage in the dependability and quality of venous outflow. PMID- 8628100 TI - Expanded polytetrafluoroethylene augmentation of the lower face. AB - Most options for rejuvenation of the lower face use soft-tissue fillers that augment the appropriate sites. Each of these options has associated risks and benefits. The U.S. Food and Drug Administration recently approved the use of expanded polytetrafluoroethylene (E-PTFE) as a soft-tissue filler in the face. From January 1991 through December 1993, the authors used E-PTFE soft-tissue patches for lower facial augmentation in 41 patients at 115 implant sites. Postsurgical follow-up has ranged from 2.5 to 4.5 years; during this time, complications have occurred in 4 patients. One implant had to be removed because of a seroma (1 patient), 4 implants required further secondary augmentation (2 patients), and 1 implant required revision because of malposition (1 patient). There have been no cases of implant infection, extrusion, long-term inflammation, or capsule formation. In this article, the authors review the technical aspects of E-PTFE use and discuss issues relating to the long-term efficacy of this new option for soft-tissue augmentation. The technique is also compared with other options for rejuvenation of the lower face. PMID- 8628099 TI - Triosite implants and fibrin glue in the treatment of atrophic rhinitis: technique and results. AB - Nine patients with primary or secondary atrophic rhinitis were treated by narrowing of the nasal fossae using a new surgical technique (derived from the Eryes procedure) in which a Triosite and fibrin glue mixture is implanted via the labial vestibule route. The results were good or excellent in seven patients. No rejections occurred. Osseocoalescence, as evaluated by computed axial tomography at 6 months, was good. Inspiratory intrasnasal pain in patients with postsurgical atrophic rhinitis improved following the operation. The surgical technique, which is quick and easy to perform, avoids the discomfort of nostril closure or the implantation of grafts from other parts of the body. Complicated flap procedures are also avoided. PMID- 8628101 TI - Preservation of spinal accessory nerve in modified neck dissections. PMID- 8628103 TI - Directory of otolaryngological societies. PMID- 8628102 TI - Sphenopalatine artery ligation: an alternative to internal maxillary artery ligation for intractable posterior epistaxis. PMID- 8628104 TI - ETA receptor-mediated role of endothelin in the kidney of DOCA-salt hypertensive rats. AB - Renal effects of FR139317, an endothelin ETA receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; -13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; -12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; -26%) accompanied by a potent renal vasodilation (RVR; -33%). FR139317 significantly increased renal blood flow only in the DOCA-salt rats. In contrast, FR139317 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-1) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-1 is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-1 and ETA receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-1 production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension. PMID- 8628105 TI - Nitric oxide mediates hyperphagia of obese Zucker rats: relation to specific changes in the microstructure of feeding behavior. AB - The presence of a nitric oxide synthetase (NOS) was demonstrated in the rat brain. It has been demonstrated recently that NOS-inhibitors reduce food intake in mammals and this suggest that nitric oxide (NO) might be a physiological mediator involved in the mechanisms controlling feeding behavior. Actually, there is no information about the acute central and peripheral effects of NOS inhibitors on feeding behavior in obese and lean Zucker rats. That is why we investigated the acute dose-dependent activity of NG-Nitro-Arginine-Methyl-Ester (L-NAME) on food intake and feeding behavior in these rats. When given peripherally in the obese rats, L-NAME produced a dose-dependent decrease in food intake (p<0.001). The calculated MED and the ED 50 were 0.50 mg/kg IP and 3.46 mg/kg IP, respectively. These effects could not be reproduced in the lean Zucker rats whatever the dose used (p=0.59). The anorectic properties of L-NAME were very well translated into the microstructure of the feeding behavior. Time spent to eat (p<0.001), meal duration (p<0.01) and meal number (p<0.01) were reduced in the obese rats. Interestingly, L-NAME produced the same effects in the lean rats, but meal size increased in a compensatory manner. Central administration of L NAME reproduced the same effects in the obese rats, but lean rats still remained insensitive. Central aminergic and/or peptidergic defects associated with the expression of hyperphagia might explain the differences observed between these lean and the obese animals. These results indicate a role of nitric oxide in the expression of hyperphagia and show that it might be a physiological mediator involved in the mechanisms controlling feeding behavior. PMID- 8628106 TI - Beta- and alpha-adrenergic mechanisms are involved in regulating type II thyroxine 5'-deiodinase in rat thymus. AB - The role of adrenergic receptors in regulation of rat thymus type II thyroxine 5' deiodinase (5'-D) activity was investigated. Our results show that norepinephrine, an alpha- and beta-adrenergic agonist elicited an increase in thymus 5'-D activity. Isoproterenol, beta-adrenergic agonist, also increased the enzyme activity, being less effective than norepinephrine. Moreover, alpha adrenergic agonists, i.e., methoxamine, an alpha1-agonist, and clonidine, an alpha2-agonist, did not increase 5'-D activity. The effect of isoproterenol was potentiated by methoxamine, but the potentiating effect was observed only at doses of isoproterenol which induce submaximal activation of the enzyme. Administration of propranolol, beta-adrenergic blocker, and prazosin, an alpha1 adrenergic blocker, inhibited significantly the activation of the enzyme induced by norepinephrine. However, yohimbine, an alpha2-adrenergic blocker, had small effect. These results show, in hypothyroid rats, a clear regulation by adrenergic mechanisms of 5'-D activity in the thymus, where alpha- and beta-adrenergic receptors might be involved. PMID- 8628107 TI - The membrane-associated 40 KD fatty acid binding protein (Berk's protein), a putative fatty acid transporter is present in human skeletal muscle. AB - Muscle tissue (1.1 +/- 0.1 grams) was obtained from seven healthy individuals (3 males, 4 females) using an open incision approach before and after ingestion of either 75 grams of dextrose (N=5) or water (N=2). Purified sarcolemmal membranes from the muscle were prepared using a sucrose step gradient. A polyclonal antibody raised against the purified (99%) rat hepatocyte 40 KD membrane fatty acid binding protein (mFABP-L) was used to probe for this putative transporter in the muscle membranes using Western blot. A single band at the 40 KD MW band was identified which reacted antigenically with the protein purified from rat livers. These response of Berk's protein 60-75 minutes after dextrose ingestion (or water) was erratic and no specific trend could be identified. Our data demonstrate that the 40 KD mFABP-L originally isolated from rat liver is also present in human skeletal muscle membrane. This protein may be involved in transport of fatty acids across the membrane of skeletal muscle, however its physiological role in human fatty acid metabolism remains to be established. PMID- 8628108 TI - Lysophosphatidic acid sensitizes mechanical stress-induced Ca2+ mobilization in cultured human lung epithelial cells. AB - We conformed that lysophosphatidic acid (LPA), which is known to be released from activated platelets, sensitizes response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stimulation in cultured epithelial cells (REPF-LC-AI cells) from human lung carcinoma. [Ca2+]i was transiently increased by spritzing of bath solution onto cells as mechanical stimulation in the presence of LPA with concentration-dependent manner (10-100 nM). The transient increase induced by the mechanical stimulation in the presence of LPA was inhibited by 10 microM Ga3+ or removing extracellular Ca2+, but not by 10 microM nicaridipine, suggesting that LPA sensitizes mechanical stimulation-induced Ca2+ influx through stretch activated ion channels. Phosphatidic acid (1 microM), but not lysophosphatidycholine (10 microM), histamine (100 nM), bradykinin (10 nM), nor ionomycin (100 nM), caused the same effect as that of LPA. This effect was observed in confluent cells, but not in subconfluent cells. These results show that LPA sensitizes mechanoreceptor-linked response in human lung epithelial cells, suggesting a possibility that LPA affects lung function, in particular, during pathological state. PMID- 8628109 TI - Changes in methionine-enkephalin levels in specific rat brain regions following repeated treatment with selective dopaminergic agonists and antagonists. AB - The present study was conducted to investigate the effects of repeated treatment with selective dopaminergic agents on the level of methionine-enkephalin (Met Enk) in rat brain cortex (CTX), hypothalamus, (HYPO), hippocampus (HIPP) and midbrain (MID). Male Sprague-Dawley rats were kept under controlled conditions for at least one week. After adaptation period, rats were randomly assigned into nine groups (7-9 rats per group) for intraperitoneal treatment with dopaminergic agents. Group 1 served as control, while, groups 2, 3 and 4 were treated with either SKF-81297, SCH 23390 or their combination, respectively. Groups 5, 6 and 7 received either LY 171555, (-)-sulpiride or their combination, whereas groups 8 and 9 were treated with nomifensine or selegiline, respectively. One hour after the last injection, rats were sacrificed, brains were removed and dissected into different regions, then extracted and their Met-Enk levels determined by radioimmunoassay (RIA). Administration of SKF-81297 or SCH 23390 significantly elevated Met-Enk levels in all brain regions examined, while their combination elevated Met-Enk levels in HYPO and HIPP only. On the other hand, treatment with LY 171555 or (-)-sulpiride, but not their combination, markedly increased Met-ENK levels in all brain regions investigated, whereas, treatment with nomifensine increased Met-Enk levels in all brain regions investigated, whereas, treatment with selegiline significantly elevated Met-Enk in HYPO, HIPP and MID but not in CTX. These findings clearly indicate that dopaminergic agonists and antagonists alter Met-Enk levels in specific rat brain regions. PMID- 8628110 TI - Effects of tacrine (THA) on spatial reference memory and cholinergic enzymes in specific rat brain regions. AB - Cognitive function of rats treated with saline (control), THA (8 mg/kg, i.p.), scopolamine (5 mg/kg, i.p.), or a combination of THA (8 mg/kg) and scopolamine (5 mg/kg) was tested in the Morris water maze. The latency to find the platform in the water maze was used to evaluate performance. THA did not significantly alter the latency period as compared to control rats. Scopolamine resulted in a highly significant (p<0.01) increase in latency period (183% increase) as compared to saline treated controls. However, when THA was concurrently administered with scopolamine, it was able to completely reverse the performance decrement induced by scopolamine. Immediately following spatial reference memory testing, animals were sacrificed by decapitation one hour post injection. Brains were immediately removed and the cortex, hippocampus, hypothalamus, and pituitary were dissected and their choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were determined spectrophotometrically. THA administration resulted in a significant increase in ChAT activity in the cortex (23% increase). However, when THA was concurrently administered with scopolamine, a significant increase in ChAT activity was observed in cortex (77% increase), hippocampus (32% increase), hypothalamus (97% increase), and pituitary (92.5% increase). THA administration resulted in a significant decrease in AChE activity (p<0.001) in cortex (62% decrease), hippocampus (78% decrease), and hypothalamus (90% decrease). When tacrine was administered with scopolamine, a significant increase was found in the cortex (197% increase) and the hippocampus (207% increase). In conclusion, the increase in ChAT activity produced by tacrine may in part explain its ability to reverse the scopolamine induced decrease in spatial reference memory and may play a role in its beneficial effect in improving cognitive ability. PMID- 8628111 TI - Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats. AB - Endothelin-1 (ET-1), the most powerful agent to cause constriction of the hepatic vasculature, is synthesized in the liver by sinusoidal endothelial cells. Circulating ET-1 levels have been shown to increase in liver cirrhosis. As liver could be a major source of increased plasma ET-1 as well as a target for its pathologic actions, this study was designed to determine hepatic ET-1 and ET receptor(s) in experimental liver cirrhosis. Cirrhosis was induced in rats by intraperitoneal administration of carbon tetrachloride for 8 weeks. Hepatic ET-1 was measured by radioimmunoassay and ET receptors were determined by radioligand competition binding procedure. A four fold increase in ET-1 concentration accompanied by a 65% increase in ET-receptor density was observed in the cirrhotic liver. There was no change in the ET receptor affinity. The capacity of the liver to metabolize ET-1 was reduced significantly in cirrhosis. Interestingly, transforming growth factor-beta, hepatic levels of which increase in cirrhosis, stimulated ET-1 synthesis in cultured Ito cells. It has been shown that ET-1 is a potent constrictor of Ito cells that proliferate and transform into highly contractile myofibroblasts in liver cirrhosis. Thus, interactions between ET-1 and Ito cells may have significant implications in the pathogenesis and complications of liver cirrhosis. PMID- 8628112 TI - Role of alpha1-adrenoceptors in the reduction of external carotid blood flow induced by buspirone and ipsapirone in the dog. AB - The effects of the 5-HT1A receptor agonist with anxiolytic properties, buspirone and ipsapirone, in the external carotid bed of anaesthetized dogs were analyzed. Since these agonists produce several vascular effects via activation of both 5-HT receptors and alpha1-adrenoceptors, their effects were compared with those elicited by the 5-HT agonist, quipazine, and the alpha1-adrenoceptor agonist, methoxamine. 1-Min intracarotid (i.c.) infusions of buspirone (300 microgram/min), ipsapirone (40 microgram/min), quipazine (300 microgram/min) and methoxamine (15 microgram/min) produced consistent decreases in external carotid blood flow (ECBF); since these changes in blood flow were not accompanied by modifications in systemic blood pressure, the agonists produced parallel increases in external carotid resistance. After interruption of the sympathetic tone by bilateral cervical vagosympathectomy, the vasoconstrictor responses to all the agonists remained unaffected. The intravenous (i.v.) administration of the nonselective 5-HT1-like receptor antagonist, methiothepin (1-100 microgram/kg), potently and dose-dependently antagonized buspirone-, ipsapirone- and quipazine-induced vasoconstriction; methiothepin similarly antagonized the vasoconstrictor responses to methoxamine. Interestingly, the alpha1-adrenoceptor antagonist, prazosin (1-100 microgram/kg, i.v.), also antagonized the vasoconstrictor responses to buspirone, ipsapirone and methoxamine in a dose dependent manner. Finally, buspirone (300 microgram/min, i.c.) and ipsapirone (40 microgram/min, i.c.) did not modify the responses to noradrenaline (10 microgram/min, i.c.) or tyramine (100 microgram/min, i.c.). It is concluded that canine external carotid vasoconstriction induced by buspirone and ipsapirone is mainly mediated by activation of alpha1-adrenoceptors located in vascular smooth muscle. These data further highlight the ability of the above anxiolytics to produce significant vascular effects under in vivo conditions. PMID- 8628113 TI - Opioids inhibit dopamine secretion from PC12 rat pheochromocytoma cells in a naloxone-reversible manner. AB - Opioids inhibit the release of catecholamines in the nervous system. Normal adrenal chromaffin cells produce delta opioids and they respond to them by suppressing the release of their catecholamines. Chromaffin cell tumors, the pheochromocytomas, produce mainly kappa opioids. The aim of this work was: (a) to test if pheochromocytomas retain the response of normal chromaffin cell catecholamines to delta opioids and to naloxone (a general opioid antagonist), and (b) to test if kappa opioids exert any specific effect on catecholamine release from these tumors. Since we have previously shown that, in common with human pheochromocytomas, the PC12 rat pheochromocytoma cells express the prodynorphin gene and secret its kappa opioid products, we used these cells to examine the effect of several opioid agonists and of naloxone on basal, nicotine , and KCl-induced dopamine release. Dopamine is the main PC12 catecholamine. We have found that the specific kappa opioid agonist U-69593 inhibited the release of dopamine in a dose-dependent manner (IC50=0.5 x 10(-8)M). Under basal conditions the mean concentration of dopamine in the culture media was 11.25 +/- 0.57 ng/mg of total cellular protein (n=13). A 30 min exposure to U-69593 at 10( 6) M suppressed basal dopamine release to 58 +/- 2% (n=7) of controls. A 12 hr pre-incubation with U-69593 caused the same degree of suppression. The effect of the synthetic kappa opioid agonist dynorphin A was indistinguishable from that of U-69593. DADLE (a mu and delta synthetic opioid agonist) was significantly less effective in suppressing dopamine release (IC50=10(-7)M). The concentration of dopamine following exposure to 10-6 M of DADLE for 30 min was 74 +/- 5% of the controls (n=4). The mu opioid agonist DAGO was ineffective. The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved. PMID- 8628114 TI - Effect of galanin on growth hormone (GH) response to thyrotropin releasing hormone of rat pituitary GH-secreting adenomatous cells (GH1) in culture. AB - The growth hormone (GH) releasing effect of thyrotropin-releasing hormone (TRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, alone or in combination was investigated in cultured rat pituitary tumor cells (GH1). TRH stimulated GH secretion in GH1 cells (maximal stimulation at the dose of 0.1 microM). Galanin alone had a significant GH inhibitory effect in GH1 cells at all the doses used. When the two peptides were administered in combination, no significant changes as compared to baseline levels were observed. The results of this study indicate that galanin has potent direct inhibitory effects on baseline and TRH-stimulated GH release from rat tumor cells. PMID- 8628115 TI - Nitric oxide synthase induction and relaxation in lipopolysaccharide-treated gastric fundus muscle of rats. AB - To investigate whether L-arginine/nitric oxide (NO) pathway activated after treatment with lipopolysaccharide (LPS) could relax the gastric fundus smooth muscle, we made functional examinations and measured NO synthase activity by the conversion of radiolabelled L-arginine to L-citrulline in rat gastric fundus strips treated with LPS in vitro. L-arginine caused a relaxation of the mucosa free gastric fundus strips which had been treated with LPS for 6 h in vitro and then contracted by PGF2alpha beforehand. This relaxation was partially reversed by N(G)-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor). Ca(2+)-independent NO synthase activity was induced after LPS-treatment. Co-incubation with LPS and cycloheximide for 6 h inhibited the relaxation to L-arginine and the induction of NO synthase. On the other hand, Ca(2+)-dependent NO synthase activity was decreased after LPS treatment. These results strongly suggest that Ca(2+)-independent NO synthase is induced by endotoxin in the gastric fundus muscle, resulting in inhibition of the contractile response. PMID- 8628116 TI - Knowledge, power and experience: variation in physicians' perceptions of breast cancer risk factors. AB - Recent theory in anthropology has increasingly been concerned with issues of power. Anthropology also has a long history of interest in variation in cultural knowledge, which, we argue, benefits from attention to power relations. To show this, we examine perceptions of breast cancer risk factors among physicians. Although physicians share a general cultural model of breast cancer risk factors, variation exists, especially between university-based physicians and community based physicians. The nature of the work performed in these two settings influences the acquisition of various sources of information and frames what is considered valid information. Similar to Foucault's argument, we find that physicians working in a university setting are more disciplined in discussing their perceptions of breast cancer risk factors, compared to community-based physicians, who move away from the centers of knowledge and power (universities). PMID- 8628117 TI - "Sympathy for my body": breast cancer and mammography at two Atlanta clinics. AB - This article examines beliefs about breast cancer and mammograms among low-income urban black women. Our research indicates that women associate breast cancer with domestic violence, believing that bruises resulting from physical abuse which is not reported or given medical attention can later turn into cancer. Some women fear that in "mashing" the breast, mammograms cause "knots" or bruises that can become cancerous. Mothers and daughters were found to have extensive knowledge of, and sense of responsibility for, each other's health. This bond can be used to encourage use of cancer screening procedures. While women assert that one's health is ultimately in God's hands, their faith appears to motivate health seeking behavior rather than promote a fatalistic or passive orientation. PMID- 8628118 TI - Patterns of sexual risk-taking among heterosexual men. AB - Heterosexual male STD (sexually transmitted disease) clinic patients (N=146) were classified by cluster analysis into four groups based on their self-reported frequency of participation in eleven sexual behaviors that varied in risk for contracting an STD or HIV. Significant differences among the four groups were found in their participation in some sexual practices that were not used in the clustering, and men in the four groups differed significantly by ethnicity, education, type of relationship, and motivations for engaging in sex. Results of this study have implications for the design of effective AIDS prevention programs to encourage safer-sex among heterosexual men. Given the differences in men's sexual practices, safer-sex messages need to be tailored to reach subgroups of STD patients with similar risk-taking patterns. PMID- 8628119 TI - Messages of distinction: the HIV/AIDS media campaign in Thailand. AB - In predominantly rural Thailand, television is a primary source of HIV/AIDS knowledge. Since 1990, HIV/AIDS warning messages have been aired regularly and repeatedly on television as part of the national strategy to minimize transmission of HIV. The education and prevention messages chosen do more than suggest measures to avoid infection. Within a logic of risk, these messages also define characteristics of people who are signified as threatening agents of infection. In Thailand, prostitutes and drug users are portrayed as the feared Other. Because commercial sex is so widespread, the demarcation of prostitutes as a high risk group signals a diffuse threat not easily subject to conceptual distancing. It is the pervasive and often fear-based associations born of the media material that, in large part, establish the basis for emergent practice when thoughts or actions are triggered by consideration of HIV/AIDS. PMID- 8628120 TI - Recasting the "ethno" in "epidemiology". AB - Collaboration between ethnography and epidemiology has a long and noble history, longer and nobler than most people realize. This article presents the argument that the growing interest in ethnography is, in fact, a way to reestablish nineteenth century epidemiology's concern with host and environment. Ethnography features meaning and context in ways that epidemiology used to, and features them in epistemological as well as methodological ways. PMID- 8628121 TI - Comparison of the profiles of seedborne fungi and the occurrence of aflatoxins in mould-damaged beans and soybeans. AB - The average percentage of fungal infection in twenty bean and soybean lots was determined by visual inspection (24.5 and 16.8%, respectively), and by laboratory isolation (58.5 and 33.7%), respectively. The mycoflora were more diverse in bean than soybean. Bean was more susceptible to fungal infection than soybean under similar environmental and cultural conditions. The most frequently isolated fungal genera for bean and soybean were Aspergillus, Penicillium, Rhizopus, Eurotium and Curvularia. Aflatoxins were not detected in the non-infected beans and soybeans, but aflatoxins (B1, B2, G1 and G2) were detected in the infected beans but not in the infected soybeans. The absence of aflatoxin in the fungal infected soybeans is discussed. PMID- 8628122 TI - Effect of a detergent Triton X-100 on growth and alpha-glucosidase production by the thermophilic fungus Malbranchea sulfurea. AB - A detergent Triton X-100 was found to affect maltose-induced synthesis of extracellular, mycelial and intracellular alpha-glucosidase in the thermophilic fungus Malbranchea sulfurea. The extracellular fraction of the total alpha glucosidase yield was found to be 90.7% and 40.4% in the presence and absence of the detergent, respectively. Data suggest that supplementation of the detergent in the medium resulted in the partial solubilization of the cell-bound alpha glucosidase and caused its release in the growth medium. PMID- 8628123 TI - Presence of sterile hyphae in moulds: relationship with inhibitory activity. AB - The relationship involving the presence of sterile hyphae in moulds and their inhibitory activity on 34 micro-organisms was investigated. From the results it was evident that antimicrobial activity varied when morphological changes were observed in the cultures. PMID- 8628124 TI - In vitro activity of imipenem and six other beta-lactam antibiotics against aminoglycoside resistant gram-negative bacilli. AB - The in vitro activity of imipenem and six other beta-lactams (ampicillin, cefamandole, cefoxitin, ceftriaxone, ceftazidime, cefotaxime) against twenty Gram negative bacilli resistant to clinically important aminoglycosides was studied. The bacterial strains showed high resistance (R) to gentamicin, tobramycin (R 100%), netilmicin (R 75%) and to the beta-lactams ampicillin (R 100%), cephamandole (R 90%) and cefoxitin (R 75%). The strains were susceptible to isepamicin (R 10%) and imipenem (R 10%) as well as susceptible to at least third generation cephalosporin. Three Pseudomonas aeruginosa strains were resistant to the majority of antibiotics, except for imipenem. PMID- 8628125 TI - Electron microscopic demonstration of transcription of ram sperm chromatin. AB - Transcription of ram sperm chromatin was examined by two electron microscopic techniques, namely spreading and thin sections. Labelling by Streptavidin-gold particles permits identification of transcription complexes, that have previously incorporated biotinylated uridine triphosphate. This supports previous electron microscopic data for randomly distributed transcription complexes and the presence of polymerase II molecules, documented by means of specific antibody using immunoelectron microscopy. Labelling of transcripts with biotin permits exact visualization of the transcription process, as well as identification of regions, where transcription occurs. PMID- 8628126 TI - Growth and photosynthetic activities of acclimated cyanobacteria in sewage effluents. AB - Various types of cyanobacteria were isolated from sewage of Kuwait. The growth and photosynthesis of these acclimated cyanobacteria on sterilized sewage effluents were studied. Primary treated effluent was found to support the growth of the cyanobacteria more than treated effluent. This indicated the possible tolerance of such cyanobacteria to more organic pollution in comparison with unacclimated cyanobacteria. The results explain in part the possible hazards of dumping sewage effluents into the sea or the possible uses of such cyanobacteria in mass culture as an animal food. PMID- 8628127 TI - Hepatitis C: questions to be answered. Questions remain about virus transmission, the natural history of infection and the role of interferon in management. PMID- 8628128 TI - That's the limit: alcohol consumption and health. Meta-analyses validate the NHMRC recommendations on responsible drinking. PMID- 8628129 TI - Oily fish and asthma--a fishy story? Further studies are required before claims can be made of a beneficial effect of oily fish consumption on asthma. PMID- 8628130 TI - Consumption of oily fish and childhood asthma risk. AB - OBJECTIVE: To investigate the association between diet and airway disease in children in the light of epidemiological studies suggesting that consumption of fish more than once a week reduces the risk of developing airway hyperresponsiveness (AHR). DESIGN: Diet was assessed by a detailed food frequency questionnaire and airway disease by respiratory symptoms or airway responsiveness to exercise. METHODS: A questionnaire, containing questions about the frequency of eating more than 200 foods, was sent to the parents of 574 children in whom we had measured recent wheeze (by questionnaire), AHR (by exercise) and atopy (by skin prick tests) six months before this study. We defined current asthma as the presence of both recent wheeze and AHR. RESULTS: Response rate to the questionnaire was 81.5% (n=468.) After adjusting for confounders such as sex, ethnicity, country of birth, atopy, respiratory infection in the first two years of life and a parental history of asthma or smoking, children who ate fresh, oily fish (>2% fat) had a significantly reduced risk of current asthma (odds ratio, 0.26; 95% confidence interval, 0.09-0.72; P<0.01). No other food groups or nutrients were significantly associated with either an increased or reduced risk of current asthma. CONCLUSION: These data suggest that consumption of oily fish may protect against asthma in childhood. PMID- 8628131 TI - Meta-analysis of alcohol and all-cause mortality: a validation of NHMRC recommendations. AB - OBJECTIVE: To compare the National Health and Medical Research Council (NHMRC) recommendations on responsible, hazardous and harmful alcohol intake with their effects on all-cause mortality in men and women and on the occurrence of 10 specific neoplastic, cardiovascular and alimentary diseases. DESIGN: Meta analyses of relative risks of mortality in relation to usual level of alcohol intake pooled from 16 cohort studies (mostly of adults over 35 years), and alcohol and selected conditions from a further 132 epidemiological studies. Results reported by authors were assigned to sex-specific exposure categories defined by the NHMRC based on median alcohol intakes. Pooled estimates of relative risk were calculated using precision-based weighting. SETTING: The assessment was part of comprehensive meta-analysis of epidemiological research undertaken for the National Drug Strategy. RESULTS: Relative risk of all-cause mortality in male drinkers compared with abstainers fell to 0.84 at 1.0-1.9 standard drinks per day, returned to 1.01 by 3.0-3.9 drinks, and increased to 1.37 at six or more drinks. In female drinkers the lowest relative risk (of 0.88) was at 0-0.9 drinks per day, and by 2.0-2.9 drinks the risk exceeded that in abstainers by 1.13; at six drinks the relative risk was 1.58. Based on NHMRC categories, the relative risks of mortality were 0.93 (0.93-0.94) in responsible drinkers, 1.24 (1.22-1.27) in hazardous drinkers and 1.37 (1.35-1.49) in harmful drinkers. Risk of cancers of the oropharynx, oesophagus, liver, larynx and female breast and of cirrhosis of the liver increased with increasing alcohol intake level. CONCLUSIONS: A pattern of usual alcohol intake consistent with the NHMRC recommendations will confer a mortality risk similar to or less than that observed in abstainers. The biologically effective dose of alcohol on mortality in women is approximately two standard drinks per day less than in men. Our validation is most reliable for drinkers aged 35 years or older. PMID- 8628132 TI - Acute rheumatic fever and rheumatic heart disease in the top end of Australia's Northern Territory. AB - OBJECTIVE: To describe the epidemiological and clinical features of acute rheumatic fever and rheumatic heart disease in the Top End of the Northern Territory. METHODS: A retrospective review (in some instances as far back as the 1960s) of all cases of known or suspected acute rheumatic fever or rheumatic heart diseases, with prospective validation of clinical status where necessary. Cases were ascertained from hospital and community medical clinic records and medical staff; and from records and health staff of 10 rural communities. RESULTS: Three hundred and eighty-six revised Jones criteria-confirmed episodes of acute rheumatic fever were documented in 249 individuals (99% Aboriginal). The annual incidence of confirmed acute rheumatic fever between 1989 and 1993 was 254 per 100,000 Aboriginal people aged 5 to 14 years. A more accurate estimate of 651 per 100,000 came from 10 rural communities with more complete information. As of 1995, there were 286 people living with established rheumatic heart disease (95% Aboriginal). The point prevalence of rheumatic heart disease among Aboriginal people was 9.6 per 1000, with a rate of 24 per 1000 in one large rural community. Sydenham's chorea was common, and associated with later rheumatic heart disease in 49% of cases. There was a preponderance of females with acute rheumatic fever, rheumatic heart disease and chorea. CONCLUSIONS: In Aboriginal people in rural northern Australia the incidence of acute rheumatic fever is higher than that reported anywhere in the world, and the prevalence of rheumatic heart disease is among the highest in the world. While continuing attention must be paid to alleviating the causes of these diseases of poverty, immediate action is needed to improve diagnosis of acute rheumatic fever, adherence to secondary benzathine penicillin prophylaxis regimens, and follow-up of rheumatic heart disease cases. PMID- 8628133 TI - Chronic hepatitis C and interferon alfa therapy: predictors of long term response. AB - OBJECTIVE: To identify independent patient, disease and viral characteristics that predict a sustained biochemical or viral response to interferon alfa therapy in patients with chronic hepatitis C. DESIGN: Comparison of interferon responders and non-responders by univariate and multivariate analysis. SETTING: The hepatitis clinic of the Alfred Hospital, Melbourne (a tertiary referral hospital), between July 1989 and June 1994. SUBJECTS: All patients with chronic hepatitis C who were treated with interferon alfa (IFN-alpha; 3 million IU, three times a week or more) for at least 12 weeks. OUTCOME MEASURES: Patient demographic and epidemiologic characteristics, pretreatment serum alanine aminotransferase (ALT) and 2-gamma-glutamyl transpeptidase (GGT) levels, histological grading of hepatic steatosis, necroinflammatory activity and fibrosis, serum hepatitis C virus (HCV) RNA titres and genotype and post treatment serum ALT levels and presence of HCV RNA. RESULTS: Of 58 patients, 13 (22%) had a sustained (six months or longer) biochemical response to IFN-alpha therapy, including 12 (21%) with a sustained viral response. Univariate analysis showed that young patients with a normal serum GGT level, grade 0-1 steatosis and fibrosis, low viral titre and infection with genotypes 3a and 2a were more likely to have a sustained response. Infection with genotypes other than 1a and 1b was the only independent variable associated with both a sustained biochemical and viral response. After adjusting for genotype, a hepatic fibrosis grade of 0-1 was also independently associated with viral response. This logistic regression model accurately predicted the virological response in 80% of cases. CONCLUSION: In Australian patients with chronic hepatitis C, a sustained viral response to IFN alpha therapy is most likely in those infected with a genotype other than 1a or 1b and with minimal hepatic fibrosis. PMID- 8628134 TI - When is hypospadias not hypospadias? AB - Hypospadias is a deformity of urethral and penile development in the male. Some cases are secondary to more serious disorders of sexual differentiation. Simple clinical rules can sort the common morphological defect of hypospadias from the rarer but more dangerous diagnosis of intersex. PMID- 8628135 TI - Caring for a vulnerable population: Who will take responsibility for those getting a raw deal from the health care system? PMID- 8628136 TI - Managing HIV. Part 3: Mechanisms of disease. 3.1 Structure and function of HIV. AB - The discovery of HIV in 1983 is a landmark of medical science in the 20th century. HIV is a fragile but stealthy virus that thrives within the cells of the immune system itself, subverting the body's defences against disease. Knowing the structure and life cycle of the virus is the key to understanding how it is transmitted, how it causes disease and how best to prevent or control infection. PMID- 8628137 TI - Managing HIV. Part 3. Mechanisms of disease. 3.2 How HIV produces immune deficiency. AB - Initially, the immune system controls HIV infection, but over time the virus depletes the population of CD4+ T cells and disturbs the homoeostasis and function of other cells in the immune system. Extensive destruction of the immune system eventually leaves the body defenceless. PMID- 8628138 TI - Managing HIV. Part 3: Mechanisms of disease. 3.3 How HIV leads to opportunistic infections. AB - HIV-related immune deficiency differs from that due to other causes in its severity and inexorably progressive nature. It leads to multiple opportunistic infections in patients with advanced HIV infection, mostly with common endogenous and environmental organisms that usually pose little threat to human health. PMID- 8628139 TI - Great expectations: the coroner's report on the South Australian haemolytic uraemic syndrome outbreak. PMID- 8628140 TI - Is there a medical litigation crisis? Individual viewpoints on the perceived medical litigation crisis. PMID- 8628141 TI - Automated screening of pap smears: a statement of caution. PMID- 8628142 TI - Hepatitis B surface antigenaemia in a child after vaccination. PMID- 8628143 TI - Screening--sensitivity, specificity and hepatitis C. PMID- 8628144 TI - Excessive wax in the ears. PMID- 8628145 TI - Streptococcus pneumoniae susceptible to benzylpenicillin but with reduced susceptibility to both cefotaxime and ceftriaxone. PMID- 8628146 TI - The decline in hospital autopsy rates. PMID- 8628147 TI - Blood-injury-injection fears in medical practice. PMID- 8628148 TI - Benefits of a best friend. PMID- 8628149 TI - Massive blood transfusion in a tertiary referral hospital. PMID- 8628150 TI - Rural practice: is the correct message getting through? PMID- 8628151 TI - Difficulties in admitting patients to metropolitan public hospitals: the view of medical practitioners. PMID- 8628152 TI - Duodenal papillary adenoma in a Jehovah's Witness. PMID- 8628153 TI - Does rheumatoid arthritis exist in the indigenous Australian Aboriginal population? PMID- 8628154 TI - Are cycling, physical exercise and traffic fumes compatible? PMID- 8628155 TI - Passive smoking: what are the limits to liberty? PMID- 8628156 TI - Cervical cancer screening in Australia: let's keep it in perspective. PMID- 8628157 TI - Is cryotherapy treating or infecting? PMID- 8628158 TI - Information for all by the year 2000. PMID- 8628159 TI - Passive smoking and respiratory function in very low birthweight children. AB - AIM: To determine if an adverse relationship exists between passive smoking and respiratory function in very low birthweight (VLBW) children at 11 years of age. SETTING: The Royal Women's Hospital, Melbourne. PATIENTS: 154 consecutive surviving children of less than 1501 g birthweight born during the 18 months from 1 October 1980. METHODS: Respiratory function of 120 of the 154 children (77.9%) at 11 years of age was measured. Exposure to passive smoking was established by history; no children were known to be actively smoking. The relationships between various respiratory function variables and the estimated number of cigarettes smoked by household members per day were analysed by linear regression. RESULTS: Most respiratory function variables reflecting airflow were significantly diminished with increasing exposure to passive smoking. In addition, variables indicative of air-trapping rose significantly with increasing exposure to passive smoking. CONCLUSION: Passive smoking is associated with adverse respiratory function in surviving VLBW children 11 years of age. Continued exposure to passive smoking, or active smoking, beyond 11 years may lead to further deterioration in respiratory function in these children. PMID- 8628160 TI - Cancer diagnosis after a report of negative cervical cytology. AB - OBJECTIVES: 1. To determine the annual rate of interval squamous cancer of the cervix after a negative Papanicolaou smear report. 2. To evaluate the proportion of women with cervical cancer who received negative cervical smear reports during the three years before the cancer diagnosis. DESIGN AND SETTING: Objective 1. A prospective study of the incidence of squamous cervical cancer from 1990 to 1993 among women who received negative cervical smear results in Victoria in 1990. Objective 2. A retrospective audit of preceding cervical smear results from 1990 to 1993 in women diagnosed with cervical cancer in Victoria in 1993. RESULTS: The average interval cervical rate was 2.54 squamous cancers per 100000 women per year (95% confidence interval, 1.75-3.67) during the first three years after a negative smear report. The interval cancer rate did not vary by age group nor by the endocervical status of the negative smear report. Of the 233 cases of cervical cancer diagnosed during 1993, 56 women (24%) had negative cervical cytology reported during the preceding three years. The frequency of preceding negative cervical cytology was greater for non-squamous cancer (22 women [33%] from 66 cases) than for squamous cancer (34 women [20%] from 167 cases. CONCLUSION: The rate of interval cancer diagnosis is very low compared with expected rates in the absence of screening, indicating the effectiveness of the cervical screening program in Victoria. PMID- 8628161 TI - Hospitalisations for rotavirus gastroenteritis among children under five years of age in New South Wales. AB - OBJECTIVE: To estimate the number of children under five years of age hospitalised for rotavirus gastroenteritis in New South Wales. DESIGN: Retrospective survey with comparison of patterns of hospital admissions for acute gastroenteritis in children under five with laboratory reports of rotavirus infection. SETTING: New South Wales, January 1991-December 1993. OUTCOME MEASURES: Laboratory reports of rotavirus infection to the Eastern Sydney Laboratory Surveillance Program (a voluntary laboratory reporting scheme) and hospital admissions of children under five with principal diagnosis classified under international classification of diseases, 9th revision, clinical modification (ICD-9-CM) codes 008.6, 008.8, 009.0-009.3 and 558.9 from NSW Inpatient Statistics Collection. RESULTS: Rotavirus infections were reported throughout each year (mean, 57 reports per month), with incidence peaks in August or September. Admissions for gastroenteritis showed the same seasonal pattern (correlation coefficient, 0.93). About 3700 children under five were admitted for rotavirus gastroenteritis annually in NSW at an estimated annual cost of 4.6 million dollars. Annual rates were highest for children aged 12-23 months (1800 per 100000 population in age group), intermediate for those aged 0-11 and 24-35 months (810 and 1000 per 100000 in age group, respectively) and lowest for those aged 36-47 and 48-59 months (450 and 190 per 100000 population, respectively, in age group). CONCLUSIONS: Rotavirus is a major cause of morbidity among young children in NSW. Routine infant vaccination against rotavirus could reduce this morbidity and the resulting health costs. PMID- 8628163 TI - Clinical exercise stress testing. Safety and performance guidelines. The Cardiac Society of Australia and New Zealand. AB - Clinical exercise testing has wide application in medicine, including the assessment of functional capacity, ventilatory function, gas exchange, muscle function, and endocrine and metabolic function, and as a test for claudication in peripheral vascular disease. The major use of exercise testing, however, is as a stress test in patients with known or suspected coronary artery disease. This article outlines the minimum safety and performance guidelines for exercise stress testing with electrocardiography, although many of the safety guidelines are common to other types of exercise tests, particularly exercise stress scintigraphy and echocardiography. PMID- 8628162 TI - Topical anaesthesia for minor lacerations: MAC versus TAC. AB - OBJECTIVE: To determine whether a solution of bupivacaine Marcain [Astra]), adrenaline and cocaine (MAC) is as safe and effective as tetracaine, adrenaline and cocaine (TAC) as topical anaesthesia for wound suturing. DESIGN: Double blind, randomised, prospective trial. SETTING: Emergency departments of two tertiary referral hospitals (one specialising in paediatric care) in Adelaide, South Australia, between February 1992 and April 1994. PARTICIPANTS: 181 patients, aged six or older, with simple dermal lacerations less than 5mm deep, not involving mucous membranes or areas with end-arterial blood supply. INTERVENTIONS: Patients received a weight-adjusted dose of either MAC or TAC. OUTCOME MEASURES: Needle-prick testing of wound for pain before suturing, pain ratings by patients and physicians during suturing, signs and symptoms of cocaine toxicity, wound complications and patient preference for topical anaesthesia. RESULTS: Topical anaesthesia was adequate for suturing in 73% of patients (83% or those with head wounds and 56% of those with extremity wounds). MAC and TAC did not differ significantly in efficacy overall or by wound location. Pain ratings from patients treated with MAC and TAC were comparable, as was patients acceptance of topical anesthesia (77%, MAC; 81%, TAC) and the incidence of adverse effects (4% infection rate overall). CONCLUSIONS: Topical anesthesia is a safe and effective means of anaesthetising selected lacerations for suturing. As we found no significant differences in either the efficacy or safety of the two solutions, we believe that MAC can be substituted for the less readily available TAC whenever expedient. PMID- 8628164 TI - Screening for prostate cancer: the case against. PMID- 8628165 TI - Managing HIV. Part 4: Primary therapy. 4.1 Antiretroviral therapies for HIV. AB - Zidovudine (AZT) is not the only drug with some effect on HIV. New agents and better-directed use of established agents can delay progression of the disease and prevent the onset of symptoms for some time. PMID- 8628166 TI - Managing HIV. Part 4: Primary therapy. 4.2 Immune-based therapy for HIV infection. AB - Immune-based therapy is in its infancy and to date no therapies are licensed in Australia. A number of the reagents are reaching phase III clinical trials and may be available within two years. Patient and physician interest is high because antiretroviral agents have not as yet had a significant impact on survival. PMID- 8628167 TI - Managing HIV. Part 4: Primary therapy. 4.3 The laboratory in managing HIV infection. AB - The CD4 cell count is a useful guide to the clinical stages of HIV infection, although it is only an indirect measure of viral activity. More direct measures of viral load will eventually become part of clinical practice. PMID- 8628168 TI - Sociopathy: forever forensic? AB - We suggest that the criteria for antisocial personality disorder, or "sociopathy", in the Diagnostic and statistical manual of mental disorders (4th edition) are deficient in that they define "failed sociopathy", and fail to include an equally valid group of individuals who, although seen by society as successful, display core features of the antisocial personality disorder. PMID- 8628169 TI - Reducing preventable deaths and containing costs: the expanding role of intensive care medicine. PMID- 8628170 TI - The evolution of the intensivist: from health care provider to economic rationalist and ethicist. AB - In contrast to many open intensive care units (ICUs) in the United State, where "parent" units (sometimes with few intensive care skills) admit and manage their own patients, Australia has closed units in which the intensivist has primary control of patient care while the patient remains in the ICU. This difference is important because in Australia, by virtue of having control of the ICU, the intensivist can be made responsible not only for patients care, but for resource allocation and financial constraint. Australian intensivists are called upon daily to make difficult decisions about who will be admitted, prematurely discharged or transferred from ICUs, and when treatment will be restricted or withdrawn. PMID- 8628171 TI - The Quality in Australian Health Care Study. PMID- 8628172 TI - The Quality in Australian Health Care Study. PMID- 8628173 TI - The Quality in Australian Health Care Study. PMID- 8628174 TI - The Quality in Australian Health Care Study. PMID- 8628175 TI - The euthanasia controversy. Decision-making in extreme circumstances. PMID- 8628176 TI - Reducing the incidence and mortality from cervical cancer. PMID- 8628177 TI - Prostate cancer: enthusiasm for screening. PMID- 8628178 TI - Prostate cancer: don't be too casual. PMID- 8628179 TI - Hazards of transrectal biopsy of the prostate. PMID- 8628180 TI - The centenary of x-rays. PMID- 8628181 TI - Acute reactions to drugs of abuse. PMID- 8628182 TI - Chlorzoxazone hepatotoxicity. PMID- 8628183 TI - [Social gerontology]. PMID- 8628184 TI - [Pathohistologic changes in aortocoronary venous grafts]. AB - This paper analyses histological changes of venous grafts in 52 obduction cases who had undergone aortocoronary bypass grafting and in whom death occurred after more than 30 days from surgery. The group consisted of 46 males and 6 females. The mean age at the moment of surgery was 51.5, while on the average the patients lived 4.83 years after the surgery. 144 venous grafts were implanted, that is 2.77 per patient. In 49 (34.03%) venous grafts fibrosis intima was established. As in 43 (29.86%) grafts it did not cause occlusion of the lumen greater than 25%, these kinds of grafts were marked as unchanged, normal. In 43 (29.00%) grafts atherosclerosis was established, in 14 (9.72%) fibrointimal proliferation, and in 38 (26.39) lumen obliteration. A year after the surgery the most important pathohistologic change was fibrointimal proliferation, and two years after the surgery and on-atherosclerosis. The percentage of certain pathohistologic types of graft changes, no matter how long patients survived after the surgery was the same in both patients with implants of the coronary artery and those with anterior interventricular branch of the left coronary artery. Grafts implanted into branches of the circumflex artery were statistically significantly more often obliterated. The locality of anastomosis of the venous graft and artery was not occluded in 67.31% of all obduction cases, whereas it was occluded in 32.69% of cases or 32 (22.22%) implanted grafts. Occlusions were caused by atherosclerotic change of the anastomosis in 84.37% of grafts, and by simultaneous occurrence of atherosclerotic change and thromb in 15.63% of grafts. PMID- 8628185 TI - [Herpes simplex virus and ovarian malignancies]. AB - Immunologic methods were used to examine 35 sera and the same number of malignant tissue samples taken from female patients with verified ovarian malignomas in order to establish presence of herpes simplex virus. In 23 preoperative sera presence of herpes simplex virus antibodies was established. In 23 postoperatively taken sera there was an evident decrease of antibody titres. In both samples antibodies were IgG class. In 25 samples of malignant tissues presence of herpes simplex virus antigen was established by method of indirect imunnofluorescency. In 9 samples antigen was of the type 1. In 6 cases it was herpes simplex virus of type 2. In order to isolate the complete infective virion, acellular filtrates taken from malignant tissues were used, inoculated with Vero cell cultures, as well as experimental intracerebrally inoculated mice. Cytopathogenic effect on cell cultures was typical but poor and it disappeared in the following passages. In inoculated mice symptoms were also typical, but incomplete. In the examined brains dyed by indirect imunnofluorescency method only focal fluorescence being different from diffuse which characterizes the complete infective virus, was established. PMID- 8628186 TI - [Difficulties in the treatment of depressive disorders]. AB - All troubles and difficulties of depressive patients are, from first atypical and mild symptoms, presented to full development of a depressive disease. Particularly, there are difficulties to make a difference between neurotic and functional depression, owing to atypical and masked forms of this disorder, as well as lack of real criteria for a selective clinical use of antidepressive agents. PMID- 8628187 TI - [Etiopathogenic, diagnostic and therapeutic aspects of stress fractures]. AB - Stress fractures occur with strenuous activity and represent a unique and relatively rare traumatic entity. Their diagnosis is difficult and therapy accompanied with specific problems. The purpose of the presented study is to explain basic characteristics of stress fractures and to approximate possibility of diagnosis and treatment better. Out of 26 fractures, 22 (84.62%) were nondisplaced and managed by bed rest, non-weight bearing or plaster of Paris immobilization for eight to ten weeks. On the other hand, four primarily displaced fractures (15.38%) were successfully treated with rigid internal fixation. Four conservatively managed patients (18.18%), two with fractures of the tibia and two with fractures of the femoral neck, attained a secondary angulation and pseudoarthrosis of the fracture site and, for these reasons, recorded injuries demanded a compensatory surgery management. It is suggested that in case of suspectability of stress fractures it is beneficial to use, parallel to native radiographic study, the bone scan imaging techniques which in the earlier phase of the disease establishes the diagnosis. Treatment of the stress fractures should be, as a rule, conservative. Moreover, "fatigue" fractures of the tibia and femoral neck ask for more continuous observations and a serious access. If non weightbearing regiment and immobilization do not decrease the difficulties; and fracture patterns progress, or if fracture becomes displaced because of delayed diagnosis, open reduction and rigid internal fixation should be done without delay. PMID- 8628188 TI - [Leptospirosis at the Clinic for Infectious Diseases in Novi Sad 1984-1993]. AB - We examined the clinical picture and course of leptospiral infections in 50 patients treated at the Clinic during the period 1984-1993. Most patients were 11 20 years of age (9 years the youngest and 77 years the oldest, mean age being 25.16). The disease more often occurred in male patients, 44 (88%). There were 30 (60%) patients with gastrointestinal symptoms, while 21 (42%) had liver and kidney infections. Meningeal syndrome was established in 25 (50%) and serous meningitis in 13 (26%). Leptospiral infections represented average and serious infections with complications in 8 (16%) patients, successful treatment in 47 (94%) patients while 3 (6%) patients died due to hematorenal insufficiency. PMID- 8628189 TI - [The effect of cilazapril and prazosin on insulin sensitivity in patients with essential hypertension]. AB - Two groups of drugs are actual for improving effects of insulin in vivo: alpha-1 adrenergic blockers and angiotensin-converting-enzyme-inhibitors. The objective of this study was to examine short-term effects of cilazapril and prazosin therapy on insulin sensitivity assessed by a 15' test of insulin tolerance of obese hypertensive persons. In groups of 8 patients insulin tolerance was tested prior to and 7 days after therapy by cilazapril dosed to 2.5 mg a day and prazosin 3 mean 1-2 mg a day. In this way satisfactory control of arterial tension was achieved. No significant changes of coefficient which enable assessment of insulin effects in vivo were established. However, in the group treated with cilazapril there were 6 out of 8 patients in whom coefficient increase was registered, so that among groups there was a significant difference in coefficient change in regard to therapy with better cilazapril effects. PMID- 8628190 TI - [Personal experience in the treatment of maxillary sinus diseases treated with functional surgery--nasoantrostomy]. AB - Authors analyzed results of functional surgery-low nasal antrostomy in treatment of certain maxillary sinus disease in 76 patients, 7-51 years of age. Advantages of this method were staded in relation to classical radical surgery of maxillary sinus from the aspect of simple procedures, preservation of surrounding region's integrity and absence of postoperative complications. The importance of the irrigation tube that is Foley catheter in the postoperative sinus lavage in children was pointed out. In our opinion this method is getting important due to increased use of endoscopic procedures concerning maxillary sinuses being easier because of previously performed nasal antrostomy. In order to achieve success, precise diagnose is necessary as well as right indications which results in 90% of cured patients. PMID- 8628191 TI - [Psychological study of rape victims]. AB - This paper covers a study of rape victims, according to the records of Clinic for Gynekology and Obstetrics in Novi Sad, in terms of the late psychological and psychiatric consequences. Several methods were used: special questionnaire, Minnesota Multiphasic Personality Inventory for estimating personality and selfdescriptive scale of depression, Beck Depression Inventory. Results emphasize that the group with the highest rape risk consists of female persons aged from 22 to 30 years, single, without children and with lower education level. Psychological and psychiatric and analysis shows pathological personality of victims in most cases. All victims tested, expressed intensive or mild degree of depression. Analysis of offense situation points to significant degree of victimization. PMID- 8628192 TI - [Hemorrhagic fever with renal syndrome in Vojvodina]. AB - This paper presents 4 serologically confirmed cases of hemorrhagic fever with kidney syndrome. The objective of this study was to point to occurrence of unexpected hemorrhagic fever with kidney syndrome, to different clinical pictures and differential-diagnostic difficulties as well as to therapeutic procedures of serious cases. The disease had a sudden onset with general signs of infection in all our patients. 3 out of 4 patients had had a dominant symptomatology in regard to gastrointestinal tract, whereas signs of acute renal insufficiency appeared later. In 3 patients there was a complete recovery without consequences, while one female patient died on the twelfth day of the disease. PMID- 8628193 TI - [A completely organized extracerebral hematoma--case report]. AB - Extracerebral hematomas, mostly caused by trauma, can be epidural or subdural according to their localization. According to their clinical picture they can be acute, subacute or chronic. Chronic epidural hematomas are extremely rare cases. Extracerebral hematomas are hemolyzed with time and fibrosis happens around them and, if not operated, they may appear finally as dural scars. Diagnostic and evolution of these hematomas can be presented by CT of the brain. They can be differential diagnostic problem in diagnosis of tumor of cranial bone, dura mater or brain. Definitive diagnosis is accomplished by pathoanatomic and pathohistologic examination. Treatment of these hematomas is mostly operative. This paper deals with a patient who suffered of completely organized dural scar that is very rare. PMID- 8628194 TI - [Occlusive hydrocephalus as a complication of von Recklinghausen's neurofibromatosis--case report]. AB - Phacomatoses are hereditary disease caused by germinative matrix disorder. Apart from known proliferative and tumor processes on peripheral nerves and their roots which make up a familiar picture of this disease to all neurologist, other tissue and organ malformations of octo and mesodermal origin may occur. This is a case report of a girl with neurofibromatosis type I after Riccardi with occlusive hydrocephalus complication. We pointed to a great number of neurofibromatosis complications, their prompt detection and treatment. PMID- 8628195 TI - [Factors which have an effect on infections in orthopedic surgery]. AB - In orthopedic reconstructive surgery infection is a fatal complication which compromises not only the functional result and worsens the starting, initial state, but sometimes, endangers the patient's life, especially when it is an elder person. In the literature there are data which state that in orthopedic reconstructive surgery there are certain risk factors, but there are no data about the following: which factors affect the infection incidence and how. It gets even a greater problem because various types of described infections are not well defined as characteristics of certain infections (superficial or deep) and they are not described in the same way by certain authors. That is why the goal of this paper was to enlighten these problems and to clear up the connection among risk factors and certain infections. That is how risk factors may be divided into factors typical for 1. infected tissue, 2. patient, 3. applied intervention, 4. direct postoperative period and 5. presence of a foreign body. In the conclusion it may be pointed out that the decrease in percentage of infections in orthopedic surgery cannot be achieved if all mechanisms and factors which affect it are not known. It is the characteristic of bone tissue that if it is once infected it cannot be healed spontaneously. However, there is no guarantee that there will be no relapse after an adequate therapy. Treatment of postoperative infections remains an open and one of the leading problems of the orthopedic septic surgery. First signs of infection should be promptly reacted to, because every deepening of infection and surgery prolongation prolongs the treatment to months and years, often without results. PMID- 8628196 TI - [90 years since the first congress of Serbian physicians]. AB - The first congress of the south Slav physicians was held in Belgrade in 1904 and was entitled the First Congress of the Serbian Physicians and Naturalists under the presidency of Dr. Jovan Danic, the president of the Medical Society of Serbia. The Congress was attended by 433 active participants, and the work was organized in sessions: medico-pharmaceutical, physico-chemical and mathematical, biological and abiological, veterino-agronomical. Papers were printed in extenso in the proceedings and were published in the native language of the lectureres (Serbian, Croatian, Bohemian, Slovenian and Bulgarian). Out of these papers 56 were presented in the medico-pharmaceutical session. The Congress was appraised very successful by the attenders and its honorary president, Prof. Dr. Jaroslav Hlava from Prague who concluded at the end of the Congress ".. the First Serbian Congress has prepared material for the future faculty of medicine". PMID- 8628198 TI - [Traumatological risk in ambulatory cardiological rehabilitation]. AB - BACKGROUND: In Israel in 1956 Gottheiner introduced outpatient rehabilitation programs in patients who had survived a myocardial infarction. In Germany one decade later these WHO phase III activities were established as well. At present any patient with cardiovascular disease is included unless suffering from acute illnesses or presenting with symptoms at rest. Gymnastics, stretching and the "historic" volleyball are completed by jogging, soccer, basketball and anaerobic exercise. Thus, a notable trauma rate would be expected especially in the elderly and those who are on anticoagulation. METHODS: A retrospective analysis evaluated questionnaires of 903 patients in 116 outpatient coronary care groups covering approximately equal to 270,000 patient exercise hours. RESULTS: 101 of 123 injuries (approximately equal to 1/2 200 patient exercise hours) occurred during games the majority of which having been distortions (53.7%), hematomas, or bruises (15.4%). Severe traumas included bone fractures (11.4%) and ruptured muscles, tendons, or ligaments (12.2%). One patient lost the sight of one eye due to a broken spectacle frame. Patients who had felt exercise as too exhausting (p < 0.001) or complained of cardiovascular symptoms (p < 0.01) showed a significantly elevated trauma rate. CONCLUSIONS: Thus, in outpatient cardiac rehabilitation the trauma rate is within the range of healthy sports men. To prevent injuries sports should be limited to the patient's level of activity. Improved skills in techniques and strategies as well as unbreakable glasses are recommended. PMID- 8628199 TI - [Female patient with kidney transplantation with acute pancreatitis]. PMID- 8628200 TI - [Therapy recommendations for the use of intravenously applicable immunoglobulins]. PMID- 8628197 TI - [Leiomyosarcoma of the stomach--clinical aspects, morphology and therapy]. AB - BACKGROUND: Due to the rarity of gastric leiomyosarcomas, only little is known on the results of diagnostics and therapy of these tumors. According to literature, the problem of suitable criteria in determining malignancy of these neoplasms is unsettled. PATIENTS AND METHOD: 21 patients (10 male, 11 female; aged 13 to 74 [53 +/- 13,9] years) with leiomyosarcomas of the stomach were treated from 1978 to 1993. In a retrospective study, the diagnostic and therapeutic results and the pathologic features of these patients are compared with the results of literature. RESULTS: Diagnosis was rarely confined preoperatively. Potentially curative resection was possible in 13/21 cases. Five year survival was 44% overall (R0-resections: 60%, R1/2-resections 22%) with a wide spread of individual cases. Pathologic examination of the tumors showed only half of the tumors classifiable according to the system of McGrath. Some well differentiated tumors revealed unclear malignancy criteria. Analysis of prognostic factors showed no statistically significant influence of surgical radicality but a significant influence of tumor size, mitotic activity and nuclear grading on survival and recurrence. CONCLUSIONS: Because of their rare incidence, the treatment of gastric leiomyosarcomas remains non-standardized. Surgical radical removal of the tumor seems to promise the largest benefit. The treatment of patients with residual or recurrent tumors remains individual, no adjuvant radio- or chemotherapeutical treatment can be generally recommended. PMID- 8628202 TI - [Long-term ambulatory blood pressure determination--I: Current status in the diagnosis and management of hypertension]. PMID- 8628201 TI - [Update nephrology--II. Causes of chronic kidney insufficiency and possibilities of affecting its progression]. PMID- 8628203 TI - [Pathogenesis and neuroprotective therapy trial in chronic degenerative diseases. Status of antioxidants]. PMID- 8628204 TI - [Torsade de pointes during quinidine and amiodarone therapy]. PMID- 8628205 TI - [Successful antibiotic therapy with ciprofloxacin in pleural empyema caused by Salmonella enteritidis]. PMID- 8628206 TI - Lyme disease--United States, 1995. AB - Lyme disease (LD) is caused by the tickborne spirochete Borrelia burgdorferi sensu lato. Surveillance for LD was initiated by CDC in 1982 and, during 1990, the Council of State and Territorial Epidemiologists designated LD as a nationally notifiable disease. For surveillance purposes, LD is defined as the presence of an erythema migrans rash > or = 5 cm in diameter or laboratory confirmation of infection with objective evidence of musculoskeletal, neurologic, or cardiovascular disease. This report summarizes cases of LD reported by state health departments to CDC during 1995 and indicates that the number of reported cases declined slightly from 1994. PMID- 8628207 TI - Update: National Breast and Cervical Cancer Early Detection Program--July 1991 September 1995. AB - During the 1990s, breast or cervical cancer will be diagnosed in an estimated 2 million women in the United States, and 500,000 will die as a result of these diseases. Screening mammography followed by timely and appropriate treatment can reduce breast cancer mortality by 30% for women aged 50-69 years, and routine use of the Papanicolaou (Pap) test followed by timely and appropriate treatment can prevent nearly all deaths from cervical cancer. The Breast and Cervical Cancer Mortality Prevention Act of 1990 established a nationwide, comprehensive public health program for increasing access to breast and cervical cancer screening services for underserved women. This report summarizes the impact of this initiative, CDC's National Breast and Cervical Cancer Early Detection Program (NBCCEDP), during July 1991-September 1995. PMID- 8628208 TI - Factors associated with prevalent self-reported arthritis and other rheumatic conditions--United States, 1989-1991. AB - Arthritis and other rheumatic conditions are among the most prevalent diseases in the United States, particularly for women and some racial/ethnic groups. In 1992, arthritis was the leading cause of disability and was associated with total direct and indirect costs of $64.8 billion; projections indicate that by 2020, arthritis will affect 59.4 million (18.2%) persons in the United States. Previous reports have documented marked differences in the prevalence rates of arthritis by age, sex, race, ethnicity, education, and body mass index (BMI). To examine the relative importance of these factors, CDC used data from the 1989-1991 National Health Interview Survey (NHIS) and a multivariate model to estimate the independent effect of each factor on self-reported arthritis. This report summarizes the results of that analysis, which indicate that a higher risk for arthritis is associated with older age, overweight, or obesity and that a lower risk is associated with being Asian/Pacific Islander or Hispanic or with having a higher education level. PMID- 8628209 TI - Outbreaks of postoperative bacterial endophthalmitis caused by intrinsically contaminated ophthalmic solutions--Thailand, 1992, and Canada, 1993. AB - Endophthalmitis is the syndrome of inflammation or infection of the ocular cavity and its adjacent structures and can result in severe sequelae, such as visual loss. Although most postoperative endophthalmitis following intraocular surgery is caused by infection with normal skin flora, cases associated with the intraoperative use of contaminated eye solutions or materials have been reported. This report summarizes outbreaks of postoperative bacterial (Pseudomonas aeruginosa or Bacillus spp.) endophthalmitis in Thailand and Canada; the outbreaks were associated with the intraoperative use of intrinsically contaminated basal salt solution (BSS) and hyaluronic acid. PMID- 8628210 TI - Availability of parenteral quinidine gluconate for treatment of severe or complicated malaria. PMID- 8628211 TI - Abortion surveillance--United States, 1992. AB - PROBLEM/CONDITION: From 1980 through 1992, the number of legal induced abortions reported to the CDC remained stable, varying each year by < or = 5%. REPORTING PERIOD COVERED: This report summarizes and reviews information reported to CDC regarding legal induced abortions obtained in the United States during 1992. This report also includes recently reported abortion-related deaths for 1988-1991 and an update on abortion-related deaths for 1985-1987. DESCRIPTION OF SYSTEM: For each year since 1969, CDC has compiled abortion data received from 52 reporting areas (i.e., the 50 states, the District of Columbia, and New York City). RESULTS: In 1992, 1,359,145 abortions were reported--a 2.1% decrease from 1991. The abortion ratio was 335 legal induced abortions per 1,000 live births, and the abortion rate was 23 per 1,000 women 15-44 years of age. Women who were undergoing an abortion were more likely to be young, white, and unmarried; most had had no previous live births and were obtaining an abortion for the first time. More than half (51%) of all abortions were performed at or before the 8th week of gestation, and 87% were before the 13th week. Approximately 14% of abortions were performed at < or = 6 weeks of gestation, 15% were performed at 7 weeks of gestation, and 22% at 8 weeks of gestation. Younger women (i.e., women < or = 19 years of age) were more likely to obtain abortions later in pregnancy than were older women. Sixteen deaths in 1988, 12 deaths in 1989, and five deaths in 1990 were associated with legal induced abortion. The case-fatality rates for 1988, 1989, and 1990, respectively, were 1.2, 0.9, and 0.3 abortion-related deaths per 100,000 legal induced abortions. INTERPRETATION: Since 1980, the number and rate of abortions have remained relatively stable, with only small year-to-year fluctuations of < or = 5%. However, since 1987, the abortion-to-live birth ratio has declined; in 1992, the abortion ratio was the lowest recorded since 1977. More pregnant women have been opting to carry their pregnancies to term rather than choosing to have an abortion. As in previous years, deaths associated with legal induced abortions occurred rarely (i.e., one or fewer deaths per 100,000 legal induced abortions). ACTIONS TAKEN: The number and characteristics of women who obtain abortions in the United States should continue to be monitored so that efforts to prevent unintended pregnancy can be assessed and the preventable causes of morbidity and mortality associated with abortions can be identified and reduced. PMID- 8628212 TI - Organization of the histone H3 genes in soybean, barley and wheat. AB - Several variants of the replacement histone H3 genes from soybean, barley and wheat have been cloned and sequenced. Analysis of segregating populations in barley and soybean, as well as analysis of clones isolated from a soybean genomic library, suggested that these genes are dispersed throughout the genome. Several genes contains introns located in similar positions, but of different lengths and sequence. Comparison of mRNA levels in different tissues revealed that the intron containing and intronless genes have different expression patterns. The distribution of the introns in the histone H3 genes across several plant species suggests that some of the introns might have been lost during the evolution of the gene family. Sequence divergence among introns and gene-flanking sequences in cloned gene variants allowed us to use them as specific probes for localizing individual gene copies and analyzing the genomic distribution of these variants across a range of genotypes. PMID- 8628213 TI - Identification of Tnr3, a suppressor-mutator/enhancer-like transposable element from rice. AB - We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion.Aa 3-bp sequence at the insertion site within p SINE1 appeared duplicated. The insertion sequence, 1539 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5' CACTA---3'; these repeats are similar to those found in members of the En/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to the En/Spm family and is thus named Tnr3 (transposable element in rice no. 3). In fact, Tnr carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of the En/Spm family. The subterminal repeat sequences in Tnr3 are, however, of two kinds, although they share homology with each other. Tnr3 and its relatives were present in multiple copies in rice. considering the length of Tnr3, it cannot represent an autonomous type element, but is a non autonomous element probably derived by deletion from an autonomous transposon. PMID- 8628214 TI - Distinct cis-acting elements direct the germination and sugar responses of the cucumber malate synthase gene. AB - The malate synthase gene (ms) promoter in cucumber (Cucumis sativus L.) was investigated with the aim of distinguishing DNA sequences mediating regulation of gene expression by sugar, and expression following seed germination. Promoter deletions were constructed and their ability to direct expression of the beta glucuronidase (gus) reporter gene was investigated in transgenic Nicotiana plumbaginifolia. Gene expression was assayed in germinating seeds and developing seedlings (the germination response) and in seedlings transferred from light into darkness with and without sucrose (the sugar response). As progressively more of the promoter was deleted from the 5' end, first the sugar response and then the germination response was lost. Thus, distinct regions of the promoter are required for carbohydrate control and for regulation of gene expression in response to germination. Sequence comparisons of the ms promoter with that of the isocitrate lyase gene (icl) of cucumber have previously identified four IMH(ICL MS-Homology) sequences. One such sequence, IMH2, is shown here to be implicated in the sugar response of the ms gene. The 17 bp sequences which when deleted from the ms gene results in loss of the germination response, contains a 14 bp sequence which is similar to a sequence in the icl promoter, which we refer to as IMH5. Furthermore, this sequence has similarity with amdI9-like sequences in filamentous fungi, which confer facB-mediated acetate inducibility on several genes, including those encoding ICL and MS. PMID- 8628215 TI - Regulation of SNM1, an inducible Saccharomyces cerevisiae gene required for repair of DNA cross-links. AB - The interstrand cross-link repair gene SNM1 of Saccharomyces cerevisiae was examined for regulation in response to DNA-damaging agents. Induction of SNM1 lacZ fusions was detected in response to nitrogen mustard, cis-platinum (II) diamine dichloride, UV light, and 8-methoxypsoralen + UVA, but not after heat shock treatment or incubation with 2-dimethylaminoethylchloride, methylmethane sulfonate or 4-nitroquinoline-N-oxide. The promoter of SNM1 contains a 15 bp motif, which shows homology to the DRE2 box of the RAD2 promoter. Similar motifs have been found in promoter regions of other damage-inducible DNA repair genes. Deletion of this motif results in loss of inducibility of SNM1. Also, a putative negative upstream regulation sequence was found to be responsible for repression of constitutive transcription of SNM1. Surprisingly, no inducibility of SNM1 was found after treatment with DNA-damaging agents in strains without an intact DUN1 gene, while regulation seems unchanged in sad1 mutants. PMID- 8628216 TI - Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28. AB - A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28. PMID- 8628217 TI - A retrotransposon-like sequence linked to the S-locus of Nicotiana alata is expressed in styles in response to touch. AB - We have identified a family of repetitive sequences in the genome of Nicotiana alata named Tna1 (Transposon of N. alata). The first element we characterised was a genomic clone for the N. alata s6-ribonuclease (S6-RNase), a gene required for self-incompatibility in this species. The DNA sequence of this element resembles the integrase domain of retrotransposons of the gypsy class and is most similar to a retrotransposon from Lilium henryi. A transcript present in N.alata styles (self-incompatibility genotype S6S6) hybridized to Tna1 and accumulated in the style following either pollination or touching. This transcript was cloned from a cDNA library and was encoded by second, partial Tna1 elements. Neither the transcribed sequence nor the original Tna1 element contain an open reading frame or is likely to be able to transpose. The second element was mapped using a population of N.alata plants segregating for alleles of the self-incompatibility locus and is closely linked to the S6-allele. The Tna1 element is present in a number of Nicotiana species and appears to have been active at least twice during the evolution of this genus. PMID- 8628218 TI - Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen. AB - The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed. PMID- 8628219 TI - Molecular analysis of the scrA and scrB genes from Klebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme II Scr of the phosphotransferase system and a sucrose-6-phosphate invertase. AB - The Klebsiella pneumoniae genes scrA and scrB are indispensable for sucrose (Scr) utilisation. Gene scrA codes for an Enzyme IIScr (IIScr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), while scrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscr AB DNA fragment has been cloned from K. pneumoniae and expressed in Escherichia coli. Its nucleotide sequence was determined and the coding regions for scrA (1371 bp) and scrB (1401 bp) were identified by genetic complementation, enzyme activity test and radiolabelling of the gene products. In addition, the nucleotide sequence of the scrB gene from conjugative plasmid pUR400 isolated from Salmonella typhimurium was also determined and errors in the previously published sequence of the scrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven IIScr proteins, a hypothetical model of the secondary structure of IIScr is proposed. PMID- 8628220 TI - Differential expression of genes in the vir regulon of Streptococcus pyogenes is controlled by transcription termination. AB - Streptococcal C5a peptidase (SCP), encoded by scpA in Streptococcus pyogenes, is a surface molecule which is able to cleave and inactivate the chemotactic factor C5a. The scpA gene is part of the vir regulon and subject to positive regulation by the Mga protein. It is down-regulated compared to another Mga-activated gene, emm. A chloramphenicol acetyltransferase (CAT) reporter gene was used to measure scpA promoter activity. Previous work had shown that when a large portion of the scpA promoter region was deleted, expression of CAT increased relative to the wild-type. This deleted region was found to contain an inverted repeat. In this study we show that the inverted repeat in the leader mRNA is the site of transcription termination, which down-regulates expression of scpA. This is a novel mechanism for regulation of gene expression in S. pyogenes. A specific deletion of the inverted repeat in the scpA promoter-CAT reporter construct was made using inverse PCR. Expression was measured from single-copy chromosomal integrants. When the inverted repeat was deleted, expression increased. Furthermore, Northern hybridization confirmed the existence of a truncated transcript, consistent with a transcription termination mechanism. PMID- 8628221 TI - chs-4, a class IV chitin synthase gene from Neurospora crassa. AB - In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase. PMID- 8628222 TI - Codon adjustment to maximise heterologous gene expression in Streptomyces lividans can lead to decreased mRNA stability and protein yield. AB - The impact of the codon bias of the mouse tumour necrosis factor alpha (mTNF) gene cloned in Streptomyces lividans on the efficiency of expression and secretion was analysed. Minor codons occurring in the mTNF gene were therefore adapted to the codon bias of Streptomyces by site-directed mutagenesis. No improvement in mTNF yield could be detected. The stability of the transcript derived from the construct was shown to be more important for determining the final level of mTNF production. A strong correlation was observed between the yield of secreted biologically active mTNF and the amount of mTNF mRNA present in the cells. PMID- 8628223 TI - A T7 promoter-specific, inducible protein expression system for Bacillus subtilis. AB - The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported. The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction. The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation of E. coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha amylase of Thermactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation. PMID- 8628225 TI - The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron. AB - The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequence is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5' and 3' splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3' splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3' splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene. PMID- 8628224 TI - Binding specificity and tissue-specific expression pattern of the Arabidopsis bZIP transcription factor TGA2. AB - The binding specificity and tissue-specific expression pattern of TGA2 (AHBP-1b), an Arabidopsis bZIP transcription factor have been determined. Filter-binding and gel-shift assays showed that TGA2 has high affinity for C-boxes (ATGACGTCAT). In this respect TGA2 is similar to other members of the Arabidopsis TGA family (such as TGA1, TGA3 and OBF4) and to tobacco TGA1a. Genomic Southern blot analysis confirmed that TGA2 is a member of the gene family. Northern blot analysis showed that the gene is expressed at similar levels in root, stem, leaf and flower t at somewhat lower levels in siliques. TGA3 was also found to be expressed at the same level throughout the plant, whereas genes encoding TGA1 and OBF4 have relatively high RNA expression levels in root. The differential expression of these genes suggests that they have distinct functions. PMID- 8628226 TI - Cloning and molecular analysis of the bifunctional dihydrofolate reductase thymidylate synthase gene in the ciliated protozoan Paramecium tetraurelia. AB - We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan, Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a approximately 500 kb macronuclear chromosome and is transcribed as an mRNA of approximately 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of approximately 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, the P. tetraurelia gene is most similar to that from Toxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences. PMID- 8628227 TI - The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains. AB - Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described. We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. The isfA mutation has now been demonstrated also to suppress mutator activity in E. coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability. PMID- 8628228 TI - Molecular analysis of the Arabidopsis pattern formation of gene GNOM: gene structure and intragenic complementation. AB - The GNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plant Arabidopsis thaliana. Mutations in the GNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo. We have isolated the GNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles. Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeast YEC2 gene, which is not essential for cell viability. Four fully complementing gnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non complementing alleles have splice-site lesions. Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through. PMID- 8628229 TI - Molecular analysis of the phosphate-specific transport (pst) operon of Pseudomonas aeruginosa. AB - The organization of the phosphate-specific transport (pst) operon in Pseudomonas aeruginosa has been determined. The gene order of the pst operon is pstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the known Escherichia coli pst operon is the lack of the pstS gene encoding a periplasmic phosphate (Pi)-binding protein. Even though the three pst genes were absolutely required for P(i)-specific transport, expression of the pst operon at high levels did not increase P(i) uptake in P. aeruginosa. DNA sequences for the pstB and phoU genes have been determined previously. The newly identified pstC and pstA genes encode possible integral membrane proteins of 677 amino acids (M(r) 73,844) and 513 amino acids (M(r) 56,394) respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in their E. coli counterparts. A chromosomal deletion of the entire pst operon rendered P. aeruginosa unable to repress P(i) taxis under conditions of P(i) excess. The phoU and pstB genes are essential for repressing P(i) taxis. However, mutants lacking either PstC or PstA alone were able to repress P(i) taxis under conditions of P(i) excess. PMID- 8628230 TI - Characterization of the replicon of plasmid pSW500 of Erwinia stewartii. AB - A 1.6-kb DNA region required for the replication of pSW500 from Erwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression of repA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained in Escherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability in E. coli. Curing of pSW500 from E. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism. PMID- 8628231 TI - Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli. AB - To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20. PMID- 8628232 TI - Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans. AB - The extracellular proteases of Aspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designated xprE, located on chromosome VI. The xprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations, xprF1, xprF2 and xprG1, which suppress xprE1, were characterised. Both xprF and xprG map to chromosome VII but the two genes are unlinked. The xprF1, xprF2 and xprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that the xprE1 and xprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA. PMID- 8628233 TI - Isolation and molecular characterisation of the gene encoding eburicol 14 alpha demethylase (cYP51) from Penicillium italicum. AB - The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14 alpha-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of Pl italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14 alpha-demethylase. PMID- 8628235 TI - Dissection of the Bacillus subtilis spoOE binding site for the global regulator AbrB reveals smaller recognition elements. AB - AbrB is a global transcriptional regulator of many genes that are expressed as Bacillus subtilis exits from active growth into stationary phase and sporulation. Previous results have suggested that binding of abrB at some promoters involves multiple sites of recognition and is a cooperative process. It is shown here that the binding site at spoOE can be subdivided into 5' and 3' halves, each capable of directing AbrB binding. In addition, the central portion of the intact site can promote AbrB binding. Examination of various heterologous and homologous tandem combinations of the half-sites confirms that the native site is a complex array of overlapping suboptimal sites, the precise arrangement of which is required for optimal AbrB binding. Other data suggest that binding of multiple AbrB units is needed for stable complex formation. A binding mechanism involving numerous steps of intermediate affinity is envisioned. PMID- 8628234 TI - Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis. AB - The sigE and sigK genes, encoding the sporulation-specific sigma factors sigma 35 and sigma 28 of Bacillus thuringiensis, were each disrupted by inserting a gene conferring resistance to kanamycin into their coding sequences. The B. thuringiensis SigE- and sigK- mutant strains were blocked at different sporulation stages and were unable to sporulate. The SigE-strain was blocked at stage II of sporulation, whereas the SigK- strain was blocked at stage IV. The expression of a cryIAa'-'lacZ transcriptional fusion was analysed in these genetic backgrounds and it was found that both sigma factors are involved in the in vivo transcription of this gene. However, the SigK- strain harbouring the cryIAa gene produced amounts of toxin similar to those produced by the B. thuringiensis Spo+ strain. The toxins accumulated in the mother cell compartment to form a crystal inclusion which remained encapsulated within the cell wall. Thus, transcription from the sigma E-dependent promoter alone (Bt I promoter) is sufficient to support high levels of toxin production in B. thuringiensis. PMID- 8628236 TI - Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm. AB - B- and C-hordein gene transcription is severely reduced in the endosperm of the regulatory barley mutant lys3a, and this is correlated with persistent hypermethylation of the promoters. In contrast, D-hordein is expressed at normal levels in the mutant. To confirm the connection between methylation and transcriptional activity, a genomic D-hordein clone was isolated and sequenced. The nucleotide composition of the promoter region revealed a CpG island and methylation analysis, using bisulphite treatment of genomic DNA, confirmed that the D-hordein promoter is unmethylated in endosperm and leaf tissue. Immunocytochemical studies localized D-hordein to the reticular component of protein bodies in both the wild-type Bomi and lys3a. Transient expression of GUS reporter gene constructs in barley endosperm, following transfection by particle bombardment revealed the D-hordein promotors. Comparison of transient expression in Bomi and lys3a endosperm demonstrated that the activities of the unmethylated D-hordein and the Hor1-14 C-hordein promoters were equivalent, while the activities in the mutant of the Horl-17 C-hordein and the Hor2-4 B-hordein promoters were reduced two- and tenfold, respectively. Methylation of plasmids in vitro prior to expression severely inhibited B- and D-hordein promoter activities. Based on these observations two categories of promoters for endosperm specific expression of storage proteins are recognized and a model involving methylation and modulation of chromatin structure in the regulation by the Lys3 gene is presented. PMID- 8628237 TI - Suppression of the Bgl+ phenotype of a delta hns strain of Escherichia coli by a Bacillus subtilis antiterminator binding site. AB - Bacillus subtilis, like Escherichia coli, possesses several sets of genes involved in the utilization of beta-glucosides. In E. coli, all these genes are cryptic, including the genes forming the bgl operon, thus leading to a Bgl- phenotype. We screened for B. subtilis chromosomal DNA fragments capable of reverting the Bgl+ phenotype associated with an E. coli hns mutant to the Bgl- wild-type phenotype. One B. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from the bgl gene. Deletion studies as well as subcloning experiments allowed us to prove that the putative B. subtilis of the E. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein of E. coli bgl operon by our putative B. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs. PMID- 8628238 TI - Transcriptional analysis of the mtA idiomorph of Neurospora crassa identifies two genes in addition to mtA-1. AB - In Neurospora crassa, mating and heterokaryon formation between opposite mating types is controlled by a single locus with two alternate forms termed mt A and mt a. Previously, an open reading frame (mt A-1) that confers mating identity and heterokaryon incompatibility was characterized in the 5.3 kb mt A idiomorph. In this study, we describe the structural and transcriptional characterization of two additional genes in the mt A idiomorph, Mt A-2 and mt A-3. The 373 amino acid mt A-2 ORF has 23% identity to the SMR1 ORF of Podospora anserina. DNA sequence analysis of a mutation affecting ascospore to 129 amino acids. The 324 amino acids mt A-3 ORF has an HMG domain and shows 22% amino acid identity to SMR2 of P. anserina. Transcripts from mt A-2 and mt A-3 are constitutively expressed during both vegetative and sexual reproduction. The presence of upstream ORFs in the mt A-2 and mt A-3 transcripts suggests the possibility of post transcriptional regulation of the expression mt A-2 and mt A-3 polypeptides. PMID- 8628239 TI - The str gene cluster for the biosynthesis of 5'-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway specific regulation by StrR. AB - Two divergently oriented operons, strXU and strVW, located within the gene cluster for 5'-hydroxystreptomycin (5'-OH-Sm) biosynthesis in Streptomyces glaucescens strain GAL.0 (ETH 22794), were analysed by DNA sequencing and transcription/regulation studies. Three genes, strU and strVW, are conserved in a similar arrangement but in a different location within the str/sts gene cluster of the Sm-producing strain S. griseus N2-3-11. The four putative products resemble NDP-4-ketohexose 3,5-epimerases (StrX, M(r) 20.2 kDa), NAD(P)-dependent oxidoreductases (StrU, 45.6 kDa), and ABC-transporters (StrV, 61.8 kDa; StrW, 63.4 kDa). These genes are apparently involved in the biosynthesis of 5'-OH-Sm because the promoters of both operons are activated in trans by the activator StrR of S. griseus N2-3-11, when cloned in S. lividans 66 TK23. A sequence motif resembling the consensus sequence GTTCGActG(N)11CagTcGAAc for binding of StrR was identified within the intergenic region of strX and strV. Specific binding of StrR to this site was demonstrated by gel retardation assays using purified His*Tag-StrR. PMID- 8628241 TI - Degradation of the Bacillus subtilis xynA transcript is accelerated in response to stress. AB - A popular method for the investigation of transcriptional regulation of gene expression is direct measurement of mRNA levels. As an internal control the level of a transcript from a constitutively expressed gene is often determined. To measure the induction rate of stress-responsive genes from Bacillus subtilis the transcript of the constitutively expressed xynA gene was used as a control. But the results presented in this communication prove that the degradation rate of the xynA transcript rises considerably in response to different kinds of stress. This response to stress is not dependent on protein biosynthesis. PMID- 8628240 TI - Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells. AB - The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes. PMID- 8628242 TI - Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox gene OSH1 lead to altered morphology of leaves in transgenic tobacco. AB - Transgenic tobacco plants were generated carrying a rice homeobox gene, OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicates arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotrophic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly, OHS1 expression was undetectable in mature leaves with the aberrant morphological features. Thus, OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate that PR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopic OSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. The OSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines. PMID- 8628243 TI - The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. AB - A chimeric gene composed of the coding sequence of the ble gene from Streptoalloteichus hindustanus fused to the 5' and 3' untranslated regions of the Chlamydomonas reinhardtii nuclear gene RBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome of C. reinhardtii by co transformation with the ARG7 marker yields Arg+ transformants of which approximately 80% possess the ble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against the ble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of the ble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome of C. reinhardtii and provides a useful dominant marker for nuclear transformation. PMID- 8628244 TI - Identification of the pheromone response element in Ustilago maydis. AB - The a mating type locus of Ustilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. Binding of pheromone to its cognate receptor includes mating competence in haploid cells and stimulates filamentous growth of the dikaryon. We have analyzed transcription of genes located in the a locus and demonstrate that all genes are induced by pheromone. Transcriptional stimulation is mediated by a 9 bp DNA element (ACAAAGGGA) that occurs in multiple copies in both alleles of the a locus. By fusing multimers containing this 9 bp sequence to the pheromone gene promoter and to a heterologous promoter we demonstrate that this sequence acts as a pheromone response element. In addition, we show that expression of the b genes, which regulate pathogenic development of the dikaryon, is also stimulated by pheromone. Pheromone-inducible genes can be divided into three classes depending on whether their expression is reduced, maintained, or increased after cell fusion. These differences may suggest some regulatory cross-talk between the a and b loci. PMID- 8628245 TI - Molecular and genetic characterization of SLC1, a putative Saccharomyces cerevisiae homolog of the metazoan cytoplasmic dynein light chain 1. AB - Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster. PMID- 8628246 TI - Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis. AB - In the biosynthesis of lipochitin oligosaccharides (LCOs) the Rhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence of nodA in the nodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which the nodA, nodB and nodC genes are separately present on different plasmids. Efficient nodulation was only obtained if nodC was present on a low-copy-number vector. Our results confirm the notion that nodA of Rhizobium leguminosarum biovar viciae is essential for nodulation on Vicia. Surprisingly, replacement of R. l. by viciae nodA by that of Bradyrhizobium sp. ANU289 results in a nodulation-minus phenotype on Vicia. Further analysis revealed that the Bradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of the R. l. by, viciae nodFE dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion that nodA is a common nod gene should be revised. PMID- 8628247 TI - A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination. AB - Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized one ms locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that the ms14 locus is located in a approximately 300 kb region. We have identified a YAC clone with an insert size of approximately 610 kb that contains the ms14-linked markers, reflects the organization of the physical map and therefore most probably contains the ms14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from approximately 105-140 kb/cM to less than 24kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination. PMID- 8628248 TI - An asexual fungus has the potential for sexual development. AB - The availability of cloned genes that control sexual reproduction (mating type genes) in high fungi has allowed us to consider the causes of failure to mate in asexual fungi. We report here that the asexual fungus Bipolaris sacchari has a homolog of the MAT-2 gene of its sexual ascomycete relative Cochliobolus heterostrophus. The B. sacchari MAT-2 sequence is highly similar to that of C. heterostrophus MAT-2 and, in fact, functions in transgenic C. heterostrophus. Thus, the asexual nature of B. sacchari is not due to absence or mutation of MAT. When either of the C. heterostrophus MAT genes was transformed into B. sacchari, the recipient could neither self nor cross with other B. sacchari strains, in contrast to transgenic C. heterostrophus strains which can do both. Persistent asexuality of B. sacchari, in spite of the presence of complementary functional MAT genes, suggests that this fungus lacks genes other than MAT which are essential for mating. Notably, the transgenic B. sacchari strains were sometimes able to initiate, but not complete, sexual development in interspecific pairings with C. heterostrophus. Transcript analysis showed that the B. sacchari MAT-2 gene is expressed in transgenic C. heterostrophus and that the C. heterostrophus MAT genes are expressed in transgenic B. sacchari. No transcript of the native B. sacchari MAT-2 gene was detected under any growth condition tested. PMID- 8628249 TI - Interaction of FLC and late-flowering mutations in Arabidopsis thaliana. AB - The phenotype caused by mutations that affect the timing of flowering in Arabidopsis thaliana has been most extensively analyzed in the Landsberg erecta (Ler) genetic background. In Ler, the late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed by the Ler allele of FLC. In this study, the interactions of nine mutations conferring late flowering with the FLC allele of the Columbia ecotype (FLC-Col), which does not suppress late flowering, were examined. The effect on flowering time of combining six of the mutations with FLC-Col was additive; plants containing FLC-Col with fd, gi, fwa, fha, ft, and fe flowered slightly later than plants containing these mutations with the Ler allele of FLC. In contrast, a synergistic effect was observed between FLC-Col and three mutations; fca, fpa, and fve plants became extremely late flowering when combined with FLC-Col. Maximum delay in flowering for the majority of the mutant strains required FLC-Col in a homozygous state, although for fpa and fe a single copy of FLC-Col allowed maximum lateness. In addition, the fd and fe mutations became more dominant in the presence of FLC-Col. PMID- 8628250 TI - Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration. AB - To allow the regulated expression of cloned genes in Candida albicans, a plasmid was constructed using the inducible promoter of the C. Albicans MAL2 gene. To demonstrate that the MAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of the C. albicans URA3 gene. This plasmid was introduced into a Ura- strain of C. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of the BamHI-linearized plasmid in the presence of BamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomal BamHI sites. All transformants examined were inducible for URA3 expression, which was determined by growth analysis and by measuring the level of URA3 gene product activity. The URA+ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes in C. albicans. PMID- 8628251 TI - Structure and expression of three genes encoding ACC oxidase homologs from melon (Cucumis melo L.). AB - The enzyme ACC oxidase catalyses the last step of ethylene biosynthesis in plants, converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. We have previously described the isolation and characterization of a cDNA clone (pMEL1) encoding an ACC oxidase homolog from melon (Cucumis melo L.). Here we report the isolation and characterization of three genomic clones, corresponding to three putative members of the ACC oxidase gene family in melon. All are transcriptionally active. The sequences of these genes have been determined. One genomic clone (CM-ACO1), corresponding to the cDNA previously isolated, presents a coding region interrupted by three introns. Its transcription initiation site has been defined with RNA from ripe fruit and ethylene-treated leaves. The other two genes (CM-ACO2, CM-ACO3) have only two introns, at positions identical to their counterparts in CM-ACO1. The degree of DNA homology in the coding regions of CM-ACO2 and CM-ACO3 relative to CM-ACO1 is 59% and 75%, respectively. CM-ACO2 and CM-ACO3 are 59% homologous in their coding regions. These three genes have close homology to PH-ACO3, a member of the ACC oxidase multigene family of petunia. The predicted amino acid sequences of CM-ACO1 and CM-ACO3 are 77% to 81% identical to those encoded by the tomato and petunia genes, while the deduced amino acid sequence of CM-ACO2 shows only 42% to 45% homology. RT-PCR analysis using gene-specific primers shows that the three genes are differentially expressed during development, ethylene treatment and wounding. CM-ACO1 is induced in ripe fruit and in response to wounding and to ethylene treatment in leaves. CM ACO2 is detectable at low level in etiolated hypocotyls. CM-ACO3 is expressed in flowers and is not induced by any of the stimuli tested. PMID- 8628252 TI - Vestigial gene expression in Drosophila melanogaster is modulated by the dTMP pool. AB - The vestigial (vg) gene of Drosophila melanogaster encodes a nuclear protein which plays a key role in wing formation but is also involved in other developmental processes. We have previously shown that depletion of the dTMP pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in vgBG/+ flies and also in individuals heterozygous for a deficiency of the vg locus (vgB/+). Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. In this paper, we show that: (1) depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcripts; (2) insertion of the retrotransposon 412 in the vgBG mutant, which is resistant to aminopterin, leads to the formation of a truncated transcript that is prematurely terminated in the long terminal repeat of this transposable element; and (3) aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on levels of vg transcripts; the resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript but, rather, is related to the expression of a modified Vg protein encoded by a truncated transcript. These results are compatible with a role for vestigial in modulating cell proliferation. PMID- 8628253 TI - Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis. AB - One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3' ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD. PMID- 8628254 TI - Stepwise transformation of rat embryo fibroblasts: c-Jun, JunB, or JunD can cooperate with Ras for focus formation, but a c-Jun-containing heterodimer is required for immortalization. AB - Among the Jun family of transcription factors, only c-Jun displays full transforming potential in cooperation with activated c-Ha-Ras in primary rat embryo fibroblasts. c-Jun in combination with Ras can both induce foci of transformed cells from rat embryo fibroblast monolayers and promote the establishment of these foci as tumoral cell lines. JunB can also cooperate with Ras to induce foci but is unable to promote immortalization. We report here that JunD, in cooperation with Ras, induces foci with an efficiency similar to that of JunB. Artificial Jun/eb1 derivatives from each of the three Jun proteins were also analyzed. These constructs carry a heterologous homodimerization domain from the viral EB1 transcription factor and are thought to form only homodimers in the cell. We show here that these Jun/eb1 chimeras are potent transactivators of AP1 sites and that they can cooperate with c-Ha-Ras to induce foci. However, among all the Ras-Jun and Ras-Jun/eb1 combinations tested, only foci from Ras-c-Jun can be efficiently expanded and maintained as long-term growing cultures. Therefore, we suggest that a heterodimer containing c-Jun might be required for in vitro establishment of these primary mammalian cells. PMID- 8628255 TI - The E2F transcription factor activates a replication-dependent human H2A gene in early S phase of the cell cycle. AB - Histone gene expression is restricted to the S phase of the cell cycle. Control is mediated by a complex network of sequence-specific DNA-binding factors and protein-protein interactions in response to cell cycle progression. To further investigate the regulatory functions that are associated at the transcriptional level, we analyzed the regulation of a replication-dependent human H2A.1-H2B.2 gene pair. We found that transcription factor E2F binds specifically to an E2F recognition motif in the H2A.1 promoter region. Activation of the H2A.1 promoter by E2F-1 was shown by use of luciferase reporter constructs of the intergenic promoter region. Overexpression of the human retinoblastoma suppressor gene product RB suppressed E2F-1 mediated transcriptional activation, indicating an E2F-dependent regulation of promoter activity during the G1-to-S-phase transition. Furthermore, the activity of the H2A.1 promoter was also downregulated by overexpression of the RB-related p107, a protein that has been detected in S-phase-specific protein complexes of cyclin A, E2F, and cdk2. In synchronized HeLa cells, expression of luciferase activity was induced at the beginning of DNA synthesis and was dependent on the presence of an E2F-binding site in the H2A.1 promoter. Together with the finding that E2F-binding motifs are highly conserved in H2A promoters of other species, our results suggest that E2F plays an important role in the coordinate regulation of S-phase-specific histone gene expression. PMID- 8628256 TI - A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures. AB - Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure. PMID- 8628257 TI - Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor. AB - Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed. PMID- 8628259 TI - Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation. AB - Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations. PMID- 8628258 TI - Yeast SNF1 protein kinase interacts with SIP4, a C6 zinc cluster transcriptional activator: a new role for SNF1 in the glucose response. AB - The SNF1 protein kinase has been widely conserved in plants and mammals. In Saccharomyces cerevisiae, SNF1 is essential for expression of glucose-repressed genes in response to glucose deprivation. Previous studies supported a role for SNF1 in relieving transcriptional repression. Here, we report evidence that SNF1 modulates function of a transcriptional activator, SIP4, which was identified in a two-hybrid screen for interaction with SNF1. The N terminus of the predicted 96 kDa SIP4 protein is homologous to the DNA-binding domain of the GAL4 family of transcriptional activators, with a C6 zinc cluster adjacent to a coiled-coil motif The C terminus contains a leucine zipper motif and an acidic region. When bound to DNA, a LexA-SIP4 fusion activates transcription of a reporter gene. Transcriptional activation by SIP4 is regulated by glucose and depends on the SNF1 protein kinase. Moreover, SIP4 is differentially phosphorylated in response to glucose availability, and phosphorylation requires SNF1. These findings suggest that the SNF1 kinase interacts with a transcriptional activator to modulate its activity and provide the first direct evidence for a role of SNF1 in activating transcription in response to glucose limitation. PMID- 8628260 TI - Role of the liver-enriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor V111 gene. AB - Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence ( 44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region. PMID- 8628261 TI - Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation. AB - The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB 2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID- 8628262 TI - The Oct-1 POU-specific domain can stimulate small nuclear RNA gene transcription by stabilizing the basal transcription complex SNAPc. AB - The RNA polymerase II and III human small nuclear RNA promoters have a common basal element, the proximal sequence element, which binds the TATA box-binding protein-containing complex SNAPc. They also contain an enhancer characterized by a highly conserved octamer sequence, which constitutes a binding site for the broadly expressed POU domain transcription factor Oct-1. The POU domain is a bipartite DNA-binding domain consisting of a POU-homeo (POUH) domain and a POU specific (POUs) domain joined by a flexible linker. Here, we show that the Oct-1 POU domain but not the related Pit-1 POU domain can facilitate the binding of SNAPc to the proximal sequence element, and activate transcription. The effect is probably mediated by protein-protein contacts, and 1 of 30 amino acid differences between the Oct-1 and Pit-1 POUs domains is the key determinant for the differential interaction with SNAPc and the ability to activate transcription. These results show that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain. In this case, subtle changes between activator DNA-binding domains, as subtle as a single amino acid difference, can profoundly affect interaction with the basal transcription machinery. PMID- 8628263 TI - Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster. AB - We report the molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eucaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24. PMID- 8628264 TI - Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation. AB - We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. PMID- 8628265 TI - ATP-dependent release of glucocorticoid receptors from the nuclear matrix. AB - Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome. PMID- 8628266 TI - Induction of circles of heterogeneous sizes in carcinogen-treated cells: two dimensional gel analysis of circular DNA molecules. AB - Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N' nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed. PMID- 8628267 TI - Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1. AB - Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE. PMID- 8628268 TI - Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex. AB - The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability. PMID- 8628269 TI - Relationship between nuclease-hypersensitive sites and meiotic recombination hot spot activity at the HIS4 locus of Saccharomyces cerevisiae. AB - Meiotic double-strand DNA breaks (DSBs), the lesions that initiate meiotic recombination at the HIS4 recombination hot spot, occur in a region upstream of the coding sequence associated with multiple DNase I-hypersensitive sites. Mutations in transcription factors that lead to loss of the DSBs result in the loss of some but not all DNase I-hypersensitive sites in the upstream region. A meiosis-specific change in chromatin structure is detected in strains with the wild-type hot spot but not in strains with alterations that elevate or reduce hot spot activity. The position and intensity of micrococcal nuclease-hypersensitive sites correlate poorly with the sites of DSB formation. PMID- 8628270 TI - Three functional classes of transcriptional activation domain. AB - We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH. PMID- 8628271 TI - Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX). AB - This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes. PMID- 8628272 TI - Characterization of the cooperative function of inhibitory sequences in Ets-1. AB - DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications. PMID- 8628273 TI - Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction. AB - Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction. PMID- 8628275 TI - The Saccharomyces cerevisiae IMP2 gene encodes a transcriptional activator that mediates protection against DNA damage caused by bleomycin and other oxidants. AB - Bleomycin belongs to a class of antitumor drugs that damage cellular DNA through the production of free radicals. The molecular basis by which eukaryotic cells provide resistance to the lethal effects of bleomycin is not clear. Using the yeast Saccharomyces cerevisiae as a model with which to study the effect of bleomycin damage on cellular DNA, we isolated several mutants that display hypersensitivity to bleomycin. A DNA clone containing the IMP2 gene that complemented the most sensitive bleomycin mutant was identified. A role for IMP2 in defense against the toxic effects of bleomycin has not been previously reported. imp2 null mutants were constructed and were found to be 15-fold more sensitive to bleomycin than wild-type strains. The imp2 null mutants were also hypersensitive to several oxidants but displayed parental resistance to UV light and methyl methane sulfonate. Exposure of mutants to either bleomycin or hydrogen peroxide resulted in the accumulation of strand breaks in the chromosomal DNA, which remained even after 6 h postchallenge, but not in the wild type. These results suggest that the oxidant hypersensitivity of the imp2 mutant results from a defect in the repair of oxidative DNA lesions. Molecular analysis of IMP2 indicates that it encodes a transcriptional activator that can activate a reporter gene via an acidic domain located at the N terminus. Imp2 lacks a DNA binding motif, but it possesses a C-terminal leucine-rich repeat. With these data taken together, we propose that Imp2 prevents oxidative damage by regulating the expression of genes that are directly required to repair DNA damage. PMID- 8628274 TI - Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB. AB - In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells. PMID- 8628276 TI - Adenovirus E1A proteins inhibit activation of transcription by p53. AB - p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53. PMID- 8628277 TI - Fos-Jun dimerization promotes interaction of the basic region with TFIIE-34 and TFIIF. AB - The regulation of RNA polymerase II-mediated transcription involves both direct and indirect interactions among regulatory proteins and the general transcription factors (GTFs) that assemble at TATA-containing promoters. Here we show that the oncogenic transcription factors Fos and Jun make direct physical contacts with three proteins of the basal transcription apparatus, TFIIE-34 (TFIIE-beta), TFIIF 30 (RAP30), and TFIIF-74 (RAP74). The interactions among the activator proteins and these three GTFs were not detected with other transcription factors, including some bZIP protein family members. Both coimmunoprecipitation and protein blotting experiments demonstrated that the interactions were strongly favored by dimerization of Fos and Jun and that they involved the basic region and basic region-proximal domain of both proteins. Mutations within the DNA binding domains of Fos and Jun abolished binding to GTFs, although the presence of DNA was not required for the association. Surprisingly, only a single basic region in the context of a protein dimer was sufficient for the interaction. Squelching of AP-1-dependent transcription in vitro by an excess of Fos-Jun dimers was relieved by the addition of TFIIE, indicating that it is a direct functional target of Fos and Jun. These results suggest that dimerization induces a conformational alteration in the basic region of Fos and Jun that promotes an association with TFIIE-34 and TFIIF, thus contributing to transcription initiation. PMID- 8628279 TI - Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. AB - The Wnt-1 proto-oncogene induces the accumulation of beta-catenin and plakoglobin, two related proteins that associate with and functionally modulate the cadherin cell adhesion proteins. Here we have investigated the effects of Wnt 1 expression on the tumor suppressor protein APC, which also associates with catenins. Expression of Wnt-1 in two different cell lines greatly increased the stability of APC-catenin complexes. The steady-state levels of both catenins and APC were elevated by Wnt-1, and the half-lives of both beta-catenin and plakoglobin associated with APC were also markedly increased. The stabilization of catenins by Wnt-1 was primarily the result of a selective increase in the amount of uncomplexed, monomeric beta-catenin and plakoglobin, detected both by affinity precipitation and size-exclusion chromatography of cell extracts. Exogenous expression of beta-catenin was possible in cells already responding to Wnt-1 but not in the parental cells, suggesting that Wnt-1 inhibits an essential regulatory mechanism for beta-catenin turnover. APC has the capacity to oppose this Wnt-1 effect in experiments in which overexpression of the central region of APC significantly reduced the size of the monomeric pool of beta-catenin induced by Wnt-1. Thus, the Wnt-1 signal transduction pathway leads to the accumulation of monomeric catenins and stabilization of catenin complex formation with both APC and cadherins. PMID- 8628278 TI - A role for a bent DNA structure in E2F-mediated transcription activation. AB - We examined the role of promoter architecture, as well as that of the DNA-bending capacity of the E2F transcription factor family, in the activation of transcription. DNA phasing analysis revealed that a consensus E2F site in the E2F1 promoter possesses an inherent bend with a net magnitude of 40 +/-2 degrees and with an orientation toward the major groove relative to the center of the E2F site. The inherent DNA bend is reversed upon binding of E2F, generating a net bend with a magnitude of 25 +/- 3 degrees oriented toward the minor groove relative to the center of the E2F site. We also found that three members of the E2F family, in conjunction with the DP1 protein, bend the DNA toward the minor groove, suggesting that DNA bending is a characteristic of the entire E2F family. The Rb-E2F complex, on the other hand, does not reverse the intrinsic DNA bend. Analysis of a series of E2F1 deletion mutants defined E2F1 sequences which are not required for DNA binding but are necessary for the DNA-bending capacity of E2F. An internal region of E2F1, previously termed the marked box, which is highly homologous among E2F family members, was particularly important in DNA bending. We also found that a bent DNA structure can be a contributory component in the activation of the E2F1 promoter but is not critical in the repression of that promoter in quiescent cells. This finding suggests that E2F exhibits characteristics typical of modular transcription factors, with independent DNA binding and transcriptional activation functions, but also has features of architectural factors that alter DNA structure. PMID- 8628281 TI - The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. AB - The aromatic hydrocarbon receptor (AhR) has been defined and characterized according to its ability to mediate biological responses to exogenous ligands, such as the synthetic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD). The natural ligand(s) for AhR is unknown, and we know relatively little about AhR function in the absence of TCDD. Here, we have exploited the availability of AhR-defective (AhR-D) mouse hepatoma (Hepa 1c1c7) cells to analyze AhR's effects under conditions in which TCDD is not present. Our results reveal that AhR-D cells exhibit a different morphology, decreased albumin synthesis, and a prolonged doubling time compared with wild-type cells. Introduction of AhR cDNA into AhR-D cells by stable transfection alters these characteristics such that the cells resemble wild-type cells. Conversely, introduction of antisense AhR cDNA into wild-type cells changes their phenotype such that they resemble AhR-D cells. Fluorescence microscopy reveals that AhR-D cells do not exhibit an increased rate of death. Flow cytometric and biochemical analyses imply that the slowed growth rate of AhR-D cells reflects prolongation of G1. Our findings reveal a potential link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. These effects of AhR occur in the absence of TCDD. We speculate that they represent responses to an endogenous AhR ligand in Hepa 1c1c7 cells. PMID- 8628280 TI - The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes. AB - Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein alpha2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of alpha2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in alpha2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity. PMID- 8628282 TI - The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma. AB - RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins. PMID- 8628283 TI - Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. AB - In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5' AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1. PMID- 8628284 TI - Genetic characterization of transactivation of the human T-cell leukemia virus type 1 promoter: Binding of Tax to Tax-responsive element 1 is mediated by the cyclic AMP-responsive members of the CREB/ATF family of transcription factors. AB - To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax. PMID- 8628286 TI - Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain. AB - We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action. PMID- 8628287 TI - Pre-mRNA topology is important for 3'-end formation in Saccharomyces cerevisiae and mammals. AB - Various signal motifs that are required for efficient pre-mRNA 3'-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3'-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3'- and 5'-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3'-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3'-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3'-end formation is discussed. PMID- 8628285 TI - A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene. AB - Binding of many cytokines to their cognate receptors immediately activates Jak tyrosine kinases and their substrates, STAT (signal transducers and activators of transcription) DNA-binding proteins. The DNA binding targets of STATs are sequence elements related to the archetypal gamma interferon activation site, GAS. However, association of interleukin 1 (IL-1) with Jak-STAT signaling has remained unresolved. We now report an element termed LILRE (lipopolysaccharide [LPS] and IL-1-responsive element) in the human prointerleukin 1beta gene (IL1B) which can be immediately induced by either lipopolysaccharide (LPS) or IL-1 protein to bind a tyrosine-phosphorylated protein. This LPS- and IL-1-induced factor (LIL factor) is recognized by an antibody raised against the N terminus of Stat1, but not by those specific for either the C terminus of Stat1 or any other GAS-binding STAT. Phosphotyrosine (P-Tyr) specifically inhibits formation of the LIL factor-DNA complex, suggesting the importance of P-Tyr for the DNA-binding activity, as has been found for all STAT dimers. Analysis of DNA-binding specificity demonstrates that the LIL factor possesses a novel GAS-like binding activity that contrasts with those of other STATs in a requirement for a G residue at position 8 (TTCCTGAGA). Further investigation has revealed that IL-6, but neither IL-4 nor gamma interferon, activates the LIL factor. Thus, the existence of such a STAT-like factor (LIL-Stat) relates the LPS and IL-1 signaling pathway to other cytokine receptor signaling pathways via the activation of STATs. Moreover, the unique DNA-binding specificity and antigenicity of this factor suggest that LPS, IL-1, and IL-6 may use a common signaling pathway. PMID- 8628288 TI - Human chromosome 3 mediates growth arrest and suppression of apoptosis in microcell hybrids. AB - Chemotherapeutic treatment of tumor cells leads either to tumor cell death (usually by apoptosis) or to the formation of drug-resistant subpopulations. Known mechanisms of cancer cell drug resistance include gene amplification and increased expression of drug transporters. On the other hand, normal cells survive many forms of chemotherapy with minimal damage probably because of their capacity for growth arrest and stringent control of apoptosis. Microcell hybrids between B78 (murine melanoma) and HSF5 (normal human fibroblasts) were analyzed to identify a new human chromosomal region involved in the promotion of drug induced growth arrest and suppression of apoptosis. In these hybrids, the presence of human chromosome 3 was strongly associated with suppression of apoptosis via G1 and G2 growth arrest during exposure to the antimetabolite N phosphonoacetyl-L-aspartate (PALA), suggesting that a gene(s) on chromosome 3 serves an antiproliferative role in a drug-responsive growth arrest pathway. PMID- 8628289 TI - Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. AB - The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions. PMID- 8628290 TI - A palindromic regulatory site within vertebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA-binding domain for high-affinity interaction. AB - GATA-1, a transcription factor essential for the development of the erythroid lineage, contains two adjacent highly conserved zinc finger motifs. The carboxy terminal finger is necessary and sufficient for specific binding to the consensus GATA recognition sequence: mutant proteins containing only the amino-terminal finger do not bind. Here we identify a DNA sequence (GATApal) for which the GATA 1 amino-terminal finger makes a critical contribution to the strength of binding. The site occurs in the GATA-1 gene promoters of chickens, mice, and humans but occurs very infrequently in other vertebrate genes known to be regulated by GATA proteins. GATApal is a palindromic site composed of one complete [(A/T)GATA(A/G)] and one partial (GAT) canonical motif. Deletion of the partial motif changes the site to a normal GATA site and also reduces by as much as eightfold the activity of the GATA-1 promoter in an erythroid precursor cell. We propose that GATApal is important for positive regulation of GATA-1 expression in erythroid cells. PMID- 8628291 TI - A glycine-rich region in NF-kappaB p105 functions as a processing signal for the generation of the p50 subunit. AB - Transcription factor NF-kappaB is generally considered to be a heterodimer with two subunits, p50 and p65. The p50 subunit has been suggested to be generated from its precursor, p105, via the ubiquitin-proteasome pathway. During processing, the C-terminal portion of p105 is rapidly degraded whereas the N terminal portion (p50) is left intact. We report here that a 23-amino-acid, glycine-rich region (GRR) in p105 functions as a processing signal for the generation of p50. A GRR-dependent endoproteolytic cleavage downstream of the GRR releases p50 from p105, and this cleavage does not require any specific downstream sequences. p50 can be generated from chimeric precursor p105N-GRR IkappaBalpha, while the C-terminal portion (IkappaBalpha) can also be recovered, suggesting that p105 processing includes two steps: a GRR-dependent endoproteolytic cleavage and the subsequent degradation of the C-terminal portion. We have also demonstrated that the GRR can direct a similar processing event when it is inserted into a protein unrelated to the NF-kappaB family and that it is therefore an independent signal for processing. PMID- 8628292 TI - Characterization of the roles of SH2 domain-containing proteins in T-lymphocyte activation by using dominant negative SH2 domains. AB - Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling. PMID- 8628293 TI - Transcription factor PU.1 mediates induction of c-fms in vascular smooth muscle cells: a mechanism for phenotypic change to phagocytic cells. AB - The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms. PMID- 8628294 TI - A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP. AB - The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription. The activation property of the C terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP. PMID- 8628295 TI - Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. AB - The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B cell-mediated immune responses is discussed. PMID- 8628296 TI - Evidence for posttranscriptional regulation of C/EBPalpha and C/EBPbeta isoform expression during the lipopolysaccharide-mediated acute-phase response. AB - The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42 kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors. PMID- 8628298 TI - Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica. AB - In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM 1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a mechanism is not known to operate in any naturally occurring prokaryotic or eukaryotic plasmid. PMID- 8628297 TI - SUI1/p16 is required for the activity of eukaryotic translation initiation factor 3 in Saccharomyces cerevisiae. AB - A genetic reversion analysis at the HIS4 locus in Saccharomyces cerevisiae has identified SUI1 as a component of the translation initiation complex which plays an important role in ribosomal recognition of the initiator codon. SUI1 is an essential protein of 12.3 kDa that is required in vivo for the initiation of protein synthesis. Here we present evidence that SUI1 is identical to the smallest subunit, p16, of eukaryotic translation initiation factor 3 (eIF-3) in S. cerevisiae. SUI1 and eIF3-p16 comigrate upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and cross-react with anti-SUI1 and anti-eIF3 antisera. Anti-SUI1 antisera immunoprecipitate all of the subunits of eIF3, whereas antisera against the eIF3 complex and the individual PRT1 and GCD10 subunits of eIF3 immunoprecipitate SUI1. Finally, the N-terminal amino acid sequence of a truncated form of eIF3-p16 matches the sequence of SUI1. eIF3 isolated from a sui1(ts) strain at 37 degrees C lacks SUI1 and fails to exhibit eIF3 activity in the in vitro assay for methionyl-puromycin synthesis. A free form of SUI1 separate from the eIF3 complex is found in S. cerevisiae but lacks activity in the in vitro assay. The results, together with prior genetic experiments, indicate that SUI1 is essential for eIF3 activity and functions as part of eIF3 and in concert with eIF2 to promote eIF2-GTP-Met-tRNAi ternary complex recognition of the initiator codon. PMID- 8628299 TI - Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing. AB - We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors. PMID- 8628300 TI - Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression. AB - COUP-TF, an orphan member of the nuclear receptor superfamily, has been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development. Since heterodimierization is a common theme within the nuclear receptor superfamily and has been demonstrated to modulate transcriptional properties of heterodimeric partners via allosteric interactions, we have devised a strategy to examine the silencing function of COUP-TF in a heterodimeric context. We find that the intrinsic active repression function of COUP-TF is not affected by heterodimerization. Moreover, COUP-TF can transrepress the ligand-dependent activation of its heterodimeric partners without its own DNA binding site. Using receptor deletion mutants in transfection assays, we show that the region necessary for COUP-TF silencing function is not sufficient for its transrepression activity. Furthermore, our studies indicate that in addition to its active repression function, COUP-TF can repress several different types of activator-dependent transactivation. However, this active repression function of COUP-TF may be differentially regulated by some other activator(s). These studies provide new insights into the molecular mechanism(s) of COUP-TF-mediated repression. PMID- 8628301 TI - IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice. AB - The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin. PMID- 8628303 TI - Rad23 is required for transcription-coupled repair and efficient overrall repair in Saccharomyces cerevisiae. AB - The repair of UV-induced photoproducts (cyclobutane pyrimidine dimers) in a well characterized minichromosome, genomic DNA, and a transcribed genomic gene (RPB2) of a rad23delta mutant of Saccharomyces care was examined. Isogenic wild-type cells show a strong bias for the repair of the transcribed strands in both the plasmid and genomic genes and efficient overall repair of both DNAs (>80% of the dimers were removed in 6 h). However, the rad23delta mutant shows (i) no strand bias for repair in these genes and decreased repair of both strands, (ii) partial repair of genomic DNA (approximately 45% in 6 h), and (iii) very poor repair of the plasmid overall approximately 15% in 6 h). These features, coupled with the decreased UV survival of rad23delta cells, indicate that Rad23 is required for both transcription-coupled repair and efficient overall repair in S. cerevisiae. PMID- 8628304 TI - A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins. AB - In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting. PMID- 8628302 TI - Heterogeneous nuclear ribonucleoprotein K is a transcription factor. AB - The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor. PMID- 8628305 TI - Complex alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms. AB - Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern. PMID- 8628306 TI - Identification of an inducible regulator of c-myb expression during T-cell activation. AB - Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation. PMID- 8628308 TI - Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts. AB - Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent. PMID- 8628307 TI - Helix-loop-helix proteins LYL1 and E2a form heterodimeric complexes with distinctive DNA-binding properties in hematolymphoid cells. AB - LYL1 is a basic helix-loop-helix (HLH) protein that was originally discovered because of its translocation into the beta T-cell receptor locus in an acute lymphoblastic leukemia. LYL1 is expressed in many hematolymphoid cells, with the notable exceptions of thymocytes and T cells. Using the yeast two-hybrid system to screen a cDNA library constructed from B cells, we identified the E-box binding proteins E12 and E47 as potential lymphoid dimerization partners for LYL1. The interaction of LYL1 with E2a proteins was further characterized in vitro and shown to require the HLH motifs of both proteins. Immunoprecipitation analyses showed that in T-ALL and other cell lines, endogenous LYL1 exists in a complex with E2a proteins. A preferred DNA-binding sequence, 5'-AACAGATG(T/g)T 3', for the LYL1-E2a heterodimer was determined by PCR-assisted site selection. Endogenous protein complexes containing both LYL1 and E2a bound this sequence in various LYL1-expressing cell lines and could distinguish between the LYL1 consensus and muE2 sites. These data demonstrate that E2a proteins serve as dimerization partners for the basic HLH protein LYL1 to form complexes with distinctive DNA-binding properties and support the hypothesis that the leukemic properties of the LYL1 and TAL subfamily of HLH proteins could be mediated by recognition of a common set of target genes as heterodimeric complexes with class I HLH proteins. PMID- 8628309 TI - Common core sequences are found in skeletal muscle slow- and fast-fiber-type specific regulatory elements. AB - The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb 0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5 kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles, indicating that these regulatory elements are sufficient to confer fiber-type-specific transcription. Alignment of rat TnIs and quail TnIf regulatory sequences indicates that there is a conserved spatial organization of core elements, namely, an E box, a CCAC box, a MEF-2-like sequence, and a previously uncharacterized motif. The core elements were shown to bind their cognate factors by electrophoretic mobility shift assays, and their mutation demonstrated that the TnIs CCAC and E boxes are necessary for transgene expression. Our results suggest that the interaction of closely related transcriptional protein-DNA complexes is utilized to specify fiber type diversity. PMID- 8628310 TI - Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1. AB - The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early growth-response transcription factors and dominant-negative helix-loop-helix proteins. PMID- 8628311 TI - Multiple roles of the novel protein tyrosine phosphatase PTP3 during Dictyostelium growth and development. AB - PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic protein tyrosine phosphatase domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow-growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys to Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of various sizes. Antiphosphotyrosine Western blot (immunoblot) analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with nonnutrient buffer led to reciprocal changes in the pattern of several phosphotyrosine proteins, including a protein of approximately 130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested that p130 is a potential substrate of PTP3. A transient posttranslational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data obtained strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2. PMID- 8628312 TI - mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. AB - The mdm-2 gene encodes a 90-kDa polypeptide that binds specifically to the p53 tumor suppressor protein. This physical interaction results in the inhibition of the transcriptional functions of p53 (J. Chen, J. Lin, and A. J. Levine, Mol. Med. 1:142-152, 1995, and J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). Experiments are described that demonstrate the ability of mdm-2 to abrogate both the p53-mediated cell cycle arrest and the apoptosis functions. In addition, the results presented here suggest that mdm-2 binding to p53 and the resultant inhibition of p53 transcription functions are critical for reversing p53-mediated cell cycle arrest. The N-terminal half or domain of the mdm-2 protein is sufficient to regulate these biological activities of p53, consistent with the possibility that the highly conserved central acidic region and the C-terminal putative zinc fingers of mdm-2 may encode other functions. PMID- 8628313 TI - Spi-1/PU.1 transgenic mice develop multistep erythroleukemias. AB - Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages. PMID- 8628314 TI - Identification of SLF1 as a new copper homeostasis gene involved in copper sulfide mineralization in Saccharomyces cerevisiae. AB - In Saccharomyces cerevisiae, at least 12 genes are important for cells to propagate in medium containing elevated concentrations of copper salts (J. Welch, S. Fogel, C. Buchman, and M. Karin, EMBO J. 8:255-260, 1989). Complementation studies were carried out on a copper-sensitive mutation (cup14) from this group. A new yeast gene, designated SLF1, was identified as a multicopy suppressor of the cup14 mutation. Slf1 is important for the physiological process of copper sulfide (CuS) mineralization on the surface of cells cultured in medium containing copper salts. CuS mineralization causes the cells to turn brown. Disruption of SLF1, which is located close to the telomere region of chromosome IV, leads to limited copper sensitivity, and the resulting cells lack the normal brownish coloration when grown in CuSO4-containing medium. Overproduction of Slf1 in wild-type cells confers superresistance to CuSO4 and enhances the coloration of cells cultured in the presence of CuSO4. Upon addition of KCN to Cu-grown cells, the brownish coloration was bleached instantly, and copper ions were solubilized. These data are consistent with Slf1-dependent accumulation of CuS complexes on the cell surface. Disruption of SFL1 also results in loss of the ability of yeast cells to deplete Cu but not Cd ions from the growth medium, whereas overexpression enhances Ca depletion ability and the resulting deposition of CuS particles. It is proposed that Slfl participates in a copper homeostasis pathway, distinct from the Cup1 detoxification system, that leads to sulfide generation and CuS biomineralization on the cell surface. This process may coordinate with the Cup1 pathway at different copper concentrations to prevent copper-induced toxicity. PMID- 8628315 TI - Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin. AB - Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. To identify functionally important regions in the cytoplasmic domain of the TPO receptor, mpl, we introduced wild-type mpl and deletion mutants of murine mpl into the granulocyte-macrophage colony-stimulating factor (GM-CSF)- or erythropoietin (EPO)-dependent human cell line UT7. TPO induced differentiation of UT7-Wtmpl cells, not parental UT7 cells, along the megakaryocytic lineage, as evidenced by decreased proliferation, changes in cell morphology, and increased surface expression and mRNA levels of megakaryocytic markers CD41, CD61, and CD42b. When UT7-mpl cells were cultured long-term in EPO instead of GM-CSF, the TPO effect was dominant over that of EPO. Moreover, the differentiation induced by TPO was more pronounced for cells shifted from EPO to TPO than for cells shifted from GM-CSF to TPO, as shown by the appearance of polyploid cells. Mutational analysis of the cytoplasmic domain of mpl showed that proliferation and maturation functions of mpl can be uncoupled. Two functional regions were identified: (i) the first 69 amino acids comprising the cytokine receptor motifs, box I and box 2, which are necessary for both TPO-induced mitogenesis and maturation; and (ii) amino acids 71 to 94, which are dispensable for proliferation but required for differentiation. Surprisingly, however, EPO could complement this latter domain for TPO-induced differentiation, suggesting a close relationship between EPO and TPO signaling. PMID- 8628317 TI - Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles. AB - Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras. PMID- 8628316 TI - Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae. AB - Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing. PMID- 8628318 TI - Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p. AB - The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids. PMID- 8628319 TI - The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. AB - The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells. PMID- 8628320 TI - Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1. AB - The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11 In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci. PMID- 8628321 TI - The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1. AB - We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1. PMID- 8628323 TI - Modulatory effects of polycyclic aromatic hydrocarbons on the mutagenicity of 1 nitropyrene: a structure-activity relationship study. AB - Benzo[a]pyrene (B[a]P) is able to inhibit the mutagenicity of 1-nitropyrene (1 NP) through the reduction of nitroreductase activity and formation of adducts with DNA. The relationships between the chemical structure of 9 polycyclic aromatic hydrocarbons (PAHs) and antagonistic effects on the 1-NP-induced mutation were evaluated by the binary mixtures of 1-NP and PAHs with Salmonella typhimurium TA98 in the absence of S9 mix. Remarkably different antagonistic effects of 9 PAHs on the mutagenicity of 1-NP were observed. Among the tested PAHs, coronene demonstrates the most antagonistic potential followed by benzo[g,h,i]perylene (B[g,h,i]P), benzo[e]pyrene (B[e]P), dibenzo[a,h]pyrene (DB[a,h]P), benzo[a]pyrene (B[a]P) and pyrene. Naphthalene, anthracene, and chrysene had only minor inhibitory activity on the 1-NP mutagenicity. The modifying effects of PAHs on the nitroreductase activity of TA98 strains in the presence of 1-NP were further examined from the production of 1-AP. The statistical analytical data showed that the inhibitory effect of PAHs on the mutagenicity of 1-NP significantly correlated with their effects on the nitroreductase activity (r = -0.69, p < 0.05). In addition, the formation of 1-NP DNA adducts of the binary mixtures of 1-NP and PAH was determined by the 32P postlabeling method. The results indicated that the modulatory effects of PAHs on the formation of 1-NP-DNA adducts were correlated well with their antagonistic activity (r = -0.91, P < 0.01). From the above results, the relationships between the chemical structure of PAHs and the antagonistic effects on the 1-NP mutagenicity were revealed by the surface area and electronic parameters of PAHs. The planar molecular area of PAHs was more convincingly correlated with the antagonistic effect on the mutagenicity of 1-NP (r = -0.81, p < 0.01) than that with the difference in energy, delta E, between EHOMO and ELUMO (r = 0.69, p < 0.05). According to the above, two possible mechanisms are involved in the interactive effect of the binary mixtures: (1) a higher binding affinity with nitroreductase for PAHs having a large planar surface area; and (2) a high energy of interaction between 1-NP and PAHs with a low delta E might decrease the nitroreductive capability. PMID- 8628322 TI - Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract. AB - Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4' hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis. PMID- 8628324 TI - Genotoxicity of the Ganges water at Narora (U.P.), India. AB - Water samples were collected from the river Ganga at Narora (U.P.). High performance liquid chromatography analysis of water samples by the liquid extraction procedure indicated the presence of several pesticides such as DDT, alpha-BHC, aldrin, endrin and dieldrin at concentrations of 1.36, 1.38, 0.95, 0.61 and 0.41 ppb, respectively. The organophosphorus pesticides such as dimethoate and methyl parathion also appear to be present at concentrations of 0.20 and 0.41 ppb, respectively. The XAD water concentrates and liquid-liquid extracted water samples were assayed for mutagenic potential by the Ames Salmonella/microsome test. The test samples exhibited a significant degree of mutagenicity with TA102, TA100 and TA98 strains both in the presence and absence of DNA repair defective mutants, recA, lexA and polA of E. coli was observed as compared to their wild-type counterpart in the presence of XAD water concentrates. PMID- 8628325 TI - Induction of hypoploidy and cell cycle delay by acrylamide in somatic and germinal cells of male mice. AB - Monomeric acrylamide was tested for its potential to induce aneuploidy in spermatocytes and bone marrow cells of mice. For this purpose, chromosomes from metaphase spreads were counted semi-automatically. In both test systems, cell proliferation was monitored, determining the meiotic index of spermatocytes and the average generation time of bone marrow cells after BrdU incorporation, respectively. No indications could be seen for different sensitivity of somatic and germinal cells towards acrylamide. With a dose of 120 mg/kg, the chemical caused cell cycle delay in both germ line and somatic cells. There was diverging response with respect to the balance of hypo- and hyperploidy. While the percentage of chromosome loss was significantly elevated in both test systems, acrylamide treatment did not increase the frequency of hyperploid cells. Interpreting these results on the basis of conventional test protocols, acrylamide should not be considered as an aneugen. The conservative approach, however, may be inadequate for the detection of aneugenic mechanisms different from non-disjunction. PMID- 8628326 TI - Shark-cartilage containing preparation protects cells against hydrogen peroxide induced damage and mutagenesis. AB - Natural products from flora and fauna are frequently used as nutritional supplements and medicaments. Two short-term assays were carried out and negative results were obtained for shark-cartilage containing preparation. The tests employed were the Salmonella/mammalian microsome assay using tester strains TA97, TA98, TA100, TA102 and TA1535 with or without S9 mix and the SOS-Chromotest with Escherichia coli strain PQ37. Evidence for shark-cartilage containing preparation functioning as an antimutagen was detected. Using bacterial survival assays with Escherichia coli fpg (BH20) and xthA (BW9091), we investigated the putative role of shark-cartilage containing preparation in protecting cells against lesions induced by hydrogen peroxide in normal and low iron level conditions. Our data suggest that shark-cartilage containing preparation can play a scavenger role for reactive oxygen species and protect against DNA lesions in both conditions. PMID- 8628327 TI - Assessment of DNA adducts and the frequency of 6-thioguanine resistant T lymphocytes in F344 rats fed 2,4-toluenediamine or implanted with a toluenediisocyanate-containing polyester polyurethane foam. AB - Toluenediamines have been of toxicological concern because of their industrial use as intermediates in polyurethane synthesis and because of the potential of their release from degradation of the Microthane polyesterurethane covering of some breast implants. In this study, we have assessed the extent of DNA damage in rats treated with a carcinogenic toluenediamine isomer, 2,4-toluenediamine (2,4 TDA), under conditions that result in tumor induction, and in rats implanted with Microthane polyesterurethane foam. Time and dose-dependent formation of adducts was observed in DNA from the liver and mammary gland of rats fed 10, 40, 80 and 180 ppm 2,4-TDA for up to 6 weeks. In assays conducted 1 to 32 weeks after the start of treatment, no adducts were detected in the DNA of T-lymphocytes isolated from the spleens of animals fed 40 or 180 ppm 2,4-TDA, nor was there an increase in mutations at the hprt locus in these lymphocytes. In rats fed 40 or 180 ppm, 2,4-TDA for 6 weeks, adducts were detectable in DNA isolated from liver and mammary gland for 26 to 43 weeks after termination of the treatment. No DNA damage, as assessed by both DNA adduct measurement and induction of T-lymphocyte hprt mutations, was observed in rats up to 42 weeks after receiving subcutaneous implants of polyesterurethane foam (67 or 267 mg/kg). Although 2,4-TDA is clearly capable of damaging DNA, the results of this study are consistent with the conclusion that Microthane foam-containing implants present a minimal risk of genotoxicity through release and subsequent metabolic activation of 2,4-TDA. The study also indicates that DNA adduct formation and mutation induction in lymphocytes are inadequate biomonitors for measuring exposure to toluenediamines. PMID- 8628328 TI - Inhibition of covalent DNA binding and mutagenicity of benzo[a]pyrene by isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl) carbamoyl]acetate (YH439), a novel hepatoprotective agent. AB - Isopropyl-2-(1,3-dithietane-2-ylidene)-2[N-(4-methyl-2-thiazol+ ++-2-yl) carbamoyl]acetate (YH439) was synthesized as a hepatoprotective drug for the treatment of chronic hepatitis and liver cirrhosis. In the present investigation, we have tested YH439 for its chemoprotective activity against the carcinogen benzo[a]pyrene. The drug exhibited dose-dependent protection against bacterial mutagenesis induced by benzo[a]pyrene its covalent binding to DNA in vitro mediated by rat hepatic postmitochondrial supernatant enriched with NADPH. The direct mutagenicity of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, the ultimate electrophilic and carcinogenic metabolite of benzo[a]pyrene, was also ameliorated by YH439 in a dose-dependent manner. The results of this study suggest that YH439 has a potential as a chemopreventive agent. PMID- 8628329 TI - In vivo effect of gamma ray pretreatment on sister-chromatid exchange induction by mitomycin C in murine bone marrow cells. AB - The effect of gamma ray pretreatment on sister-chromatid exchange induction by mitomycin C (MMC) was determined in murine bone marrow cells in vivo. A 30% reduction in the expected SCE frequency was observed assuming an additive effect. These results support the prediction of the replicative model for SCE formation with regard to the interactions between mutagens, and confirm previous results focused on the adaptive response. PMID- 8628330 TI - Micronucleus induction by chromium and selenium, and suppression by metallothionein inducer. AB - The clastogenic effects of chromium compounds (CrCl3 and K2CrO4) and selenium compounds (H2SeO3 and Na2SeO4) in mouse bone marrow cells have been investigated. K2CrO4 induced significant, dose-related increases in micronuclei. H2SeO3 also showed a significant micronucleus induction at the highest dose. CrCl3 and Na2SeO4 were negative for micronuclei induction in mice. The suppressive effect of Bi(NO3)3, a metallothionein inducer, on the micronucleus induction by K2CrO4 and H2SeO3 has been also investigated. Pretreatment with Bi(NO3)3 suppressed the micronucleus induction by K2CrO4 and H2SeO3. In addition, the incidence of micronucleus induction seemed to be reduced by double dosing with K2CrO4 and H2SeO3, compared to single dosing. It is generally recognized that a number of metal compounds induce metallothionein synthesis. These results suggest that the mechanism of suppression of micronucleus induction by K2CrO4 and H2SeO3 involve possible participation of metallothionein in mouse bone marrow. PMID- 8628331 TI - Co-administration of ethanol transiently inhibits urethane genotoxicity as detected by a kinetic study of micronuclei induction in mice. AB - Urethane (ethyl carbamate) is a genotoxic carcinogen that requires metabolic activation. Ethanol is known to inhibit urethane metabolism and genotoxicity. Since ethanol is eliminated rapidly in animals, the persistence of ethanol inhibition was studied in a mouse bone marrow and a peripheral blood micronucleus assays. In the bone marrow assay, male CD-1 mice were injected intraperitoneally (i.p.) with water (vehicle), urethane (1000 mg/kg), ethanol (2500 mg/kg) or urethane and ethanol (1000 and 2500 mg/kg, respectively) in single injections. Polychromatic erythrocytes (PCE) from bone marrow were obtained at 24 and 48 h after injection and scored for micronuclei. Urethane induced an increase of micronucleated PCE (MN PCE) frequency from 0.19% in the control to 8.63% at 24 h, followed by a decrease to 6.98% at 48 h. When urethane was co-administered with ethanol, the MN PCE frequency was suppressed to 0.49% at 24 h, but markedly increased to 7.35% at 48 h. This delay of MN PCE occurrence indicated that ethanol inhibition was transient. To pinpoint the duration of this delay, a peripheral blood micronucleus assay was conducted to monitor the kinetics of MN PCE induction. In this assay, male CD-1 mice were injected i.p. with water, ethanol, urethane, or urethane and ethanol as described above. Peripheral blood was scored for MN PCE at 8-h intervals for 4 days. Two additional dose groups injected with urethane or urethane and ethanol were also scored for MN PCE at 8 h intervals, but each blood sampling time was staggered 4 h later from the first four dose groups. The combined data provided MN PCE frequencies at 4-h intervals from 24 to 100 h after injection. Urethane alone induced a peak MN PCE frequency of 11.6% at 52 h. Urethane and ethanol induced a peak MN PCE frequency of 11.2% at 64 h, a delay of 12 h. Thus, ethanol delays but does not diminish urethane genotoxicity. PMID- 8628332 TI - Micronucleus test in erythrocytes of Barbus plebejus (Teleostei, Pisces) from two natural environments: a bioassay for the in situ detection of mutagens in freshwater. AB - Erythrocyte micronucleus frequencies in wild fish from two riverine environments and in fish reproduced and reared under controlled conditions (control group) were compared, with the aim to evaluate the suitability of the MN test for the in situ detection of mutagens in freshwaters. Fish were caught in different months in two rivers of central Italy which have different pollution levels. As indicator species, the barbel (Barbus plebejus) was chosen because of its ecological significance. Blood samplings were performed on wild fish immediately after capture and repeated at different time intervals on the same individuals, which were maintained in controlled conditions after capture. A total of 10,000 erythrocytes per specimen were scored. No significant differences in micronucleus frequencies were observed between the control group and fish from the unpolluted river (Mignone). A significantly higher frequency of micronuclei was observed in fish caught in the polluted river (Tiber), in comparison to both the controls and the Mignone river fish. No significant seasonal differences were observed. Barbels examined 50 and 100 days after capture presented a remarkable decrease in micronucleus frequency in comparison with the frequency observed in barbels at capture. The micronucleus test in fish erythrocytes was shown to be a sensitive bioassay for detecting mutagenic pollution in fresh water environments. PMID- 8628333 TI - The evaluation of clastogenic potential of trichloroacetic acid (TCA) in chick in vivo test system. AB - The objective of the present study was to evaluate the genotoxic potential of trichloroacetic acid (TCA) in chick bone marrow chromosomes, and the experiment was designed to study the dose, route, time and acute vs. sub-acute (fractionated) yield effects of the chemical. TCA induced chromosomal aberrations in a dose, route and time response manner. The results revealed the genotoxic property of TCA in the tested system. PMID- 8628334 TI - Sister chromatid exchanges in the peripheral lymphocytes of newborns with Down syndrome after in vitro exposure to blue or green light. AB - This study determines whether irradiation by blue or green light has an adverse effect on the DNA of Down syndrome (DS) cells by examining the sister chromatid exchange (SCE) frequency of peripheral lymphocytes obtained from five neonates with DS and five karyotypically normal neonates (control). Lymphocytes in G0 of the cell cycle were irradiated with blue or green fluorescent light for 1, 2, 4 or 6 h, and then cultured using a conventional method. Our results revealed that the induction of SCEs per cell in both groups increased in a dose-dependent manner, although more SCEs were respectively induced by the blue light. In addition, after 6 h of blue light irradiation, the net-induced SCEs in the DS groups were higher than those in the control groups. PMID- 8628335 TI - Effect of an enteric-coated fish-oil preparation on relapses in Crohn's disease. AB - BACKGROUND: Patients with Crohn's disease may have periods of remission, interrupted by relapses. Because fish oil has antiinflammatory actions, it could reduce the frequency of relapses, but it is often poorly tolerated because of its unpleasant taste and gastrointestinal side effects. METHODS: We performed a one year, double-blind, placebo-controlled study to investigate the effects of a new fish-oil preparation in the maintenance of remission in 78 patients with Crohn's disease who had a high risk of relapse. The patients received either nine fish oil capsules containing a total of 2.7 g of n-3 fatty acids or nine placebo capsules daily. A special coating protected the capsules against gastric acidity for at least 30 minutes. RESULTS: Among the 39 patients in the fish-oil group, 11 (28 percent) had relapses, 4 dropped out because of diarrhea, and 1 withdrew for other reasons. In contrast, among the 39 patients in the placebo group, 27 (69 percent) had relapses, 1 dropped out because of diarrhea, and 1 withdrew for other reasons (difference in relapse rate, 41 percentage points; 95 percent confidence interval, 21 to 61; P < 0.001). After one year, 23 patients (59 percent) in the fish-oil group remained in remission, as compared with 10 (26 percent) in the placebo group (P = 0.003). Logistic-regression analysis indicated that only fish oil and not sex, age, previous surgery, duration of disease, or smoking status affected the likelihood of relapse (odds ratio for the placebo group as compared with the fish-oil group, 4.2; 95 percent confidence interval, 1.6 to 10.7). CONCLUSIONS: In patients with Crohn's disease in remission, a novel enteric-coated fish-oil preparation is effective in reducing the rate of relapse. PMID- 8628336 TI - Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. Hepatoma Prevention Study Group. AB - BACKGROUND: In patients with hepatocellular carcinoma (hepatoma), the rate of recurrent and second primary hepatomas is high despite surgical resection and percutaneous ethanol-injection therapy. We developed an acyclic retinoid, polyprenoic acid, that inhibits hepatocarcinogenesis in the laboratory and induces differentiation and apoptosis in cell lines derived from human hepatoma. In a randomized, controlled study, we tested whether the compound reduced the incidence of recurrent and second primary hepatomas after curative treatment. METHODS: We prospectively studied 89 patients who were free of disease after surgical resection of a primary hepatoma or the percutaneous injection of ethanol. We randomly assigned the patients to receive either polyprenoic acid (600 mg daily) or placebo for 12 months. We studied the remnant liver by ultrasonography every three months after randomization. The primary end point of the study was the appearance of a histologically confirmed recurrent or new hepatoma. RESULTS: Treatment with polyprenoic acid significantly reduced the incidence of recurrent or new hepatomas. After a median follow-up of 38 months, 12 patients in the polyprenoic acid group (27 percent) had recurrent or new hepatomas as compared with 22 patients in the placebo group (49 percent, P = 0.04). The most striking difference was in the groups that had second primary hepatomas--7 in the group receiving polyprenoic acid as compared with 20 in the placebo group (P = 0.04 by the log-rank test). Cox proportional-hazards analysis demonstrated that as an independent factor, polyprenoic acid reduced the occurrence of second primary hepatomas (adjusted relative risk, 0.31; 95 percent confidence interval, 0.12 to 0.78). CONCLUSIONS: Oral polyprenoic acid prevents second primary hepatomas after surgical resection of the original tumor or the percutaneous injection of ethanol. PMID- 8628337 TI - Presynaptic dopaminergic deficits in Lesch-Nyhan disease. AB - BACKGROUND: Lesch-Nyhan disease is a rare, devastating, X-linked recessive disorder of purine synthesis. Patients present with hyperuricemia, choreoathetosis, dystonia, and aggressive and self-injurious behavior. Although the genetic and biochemical abnormalities have been identified, the causes of the neuropsychiatric syndrome remain unclear. METHODS: We used positron-emission tomography to measure presynaptic accumulation of fluorodopa F 18 tracer in the dopaminergic regions of the brains of 12 patients with Lesch-Nyhan disease (age, 10 to 20 years) and 15 healthy controls (age, 12 to 23). The results were expressed as ratios of specific to nonspecific radioactive counts. A low ratio indicates decreased dopa decarboxylase activity and dopamine storage. RESULTS: The fluorodopa F 18 ratio was significantly lower in the putamen (31 percent of control values), caudate nucleus (39 percent), frontal cortex (44 percent), and ventral tegmental complex (substantia nigra and ventral tegmentum; 57 percent) in the patients with Lesch-Nyhan disease than in the controls. Uptake of the tracer was abnormally low even in the youngest patients tested, and there was no overlap in the values between patients and controls. CONCLUSIONS: Patients with Lesch Nyhan disease have abnormally few dopaminergic nerve terminals and cell bodies. The abnormality involves all dopaminergic pathways and is not restricted to the basal ganglia. These dopaminergic deficits are pervasive and appear to be developmental in origin, which suggests that they contribute to the characteristic neuropsychiatric manifestations of the disease. PMID- 8628338 TI - Brief report: rifampin-resistant tuberculosis in a patient receiving rifabutin prophylaxis. PMID- 8628339 TI - Images in clinical medicine. Periodic breathing in an infant with hydrocephalus. PMID- 8628340 TI - Cardiopulmonary resuscitation on television. Miracles and misinformation. AB - BACKGROUND: Responsible, shared decision making on the part of physicians and patients about the potential use of cardiopulmonary resuscitation (CPR) requires patients who are educated about the procedure's risks and benefits. Television is an important source of information about CPR for patients. We analyzed how three popular television programs depict CPR. METHODS: We watched all the episodes of the television programs ER and Chicago Hope during the 1994-1995 viewing season and 50 consecutive episodes of Rescue 911 broadcast over a three-month period in 1995. We identified all occurrences of CPR in each episode and recorded the causes of cardiac arrest, the identifiable demographic characteristics of the patients, the underlying illnesses, and the outcomes. RESULTS: There were 60 occurrences of CPR in the 97 television episodes--31 on ER, 11 on Chicago Hope, and 18 on Rescue 911. In the majority of cases, cardiac arrest was caused by trauma; only 28 percent were due to primary cardiac causes. Sixty-five percent of the cardiac arrests occurred in children, teenagers, or young adults. Seventy five percent of the patients survived the immediate arrest, and 67 percent appeared to have survived to hospital discharge. CONCLUSIONS: The survival rates in our study are significantly higher than the most optimistic survival rates in the medical literature, and the portrayal of CPR on television may lead the viewing public to have an unrealistic impression of CPR and its chances for success. Physicians discussing the use of CPR with patients and families should be aware of the images of CPR depicted on television and the misperceptions these images may foster. PMID- 8628341 TI - New antiepileptic drugs. PMID- 8628342 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 18-1996. A newborn boy with multiple hemorrhagic vesicles, lymphadenopathy, and respiratory distress. PMID- 8628343 TI - Keeping Crohn's disease quiet. PMID- 8628345 TI - New approaches to understanding Lesch-Nyhan disease. PMID- 8628344 TI - Synthetic retinoids for the secondary prevention of hepatocellular carcinoma. PMID- 8628346 TI - Cardiopulmonary resuscitation on television. Exaggerations and accusations. PMID- 8628347 TI - Dietary fat and the risk of breast cancer. PMID- 8628349 TI - Improving the fecal occult-blood test. PMID- 8628348 TI - Dietary fat and the risk of breast cancer. PMID- 8628350 TI - Allelic loss of chromosome 1p in neuroblastoma. PMID- 8628351 TI - Coronary-artery bypass surgery with internal-thoracic-artery grafts. PMID- 8628352 TI - Coronary-artery bypass surgery with internal-thoracic-artery grafts. PMID- 8628353 TI - Deficiency of adhalin in a patient with muscular dystrophy and cardiomyopathy. PMID- 8628354 TI - Parkinsonism and Parkinson's disease. PMID- 8628355 TI - Metformin-associated mortality in U.S. studies. PMID- 8628356 TI - Obstetrical factors and the transmission of human immunodeficiency virus type 1 from mother to child. The Women and Infants Transmission Study. AB - BACKGROUND: A substantial proportion of perinatally acquired infections with the human immunodeficiency virus type 1 (HIV-1) occur at or near delivery, which suggests that obstetrical factors may have an important influence on transmission. We evaluated the relation of such factors and other variables to the perinatal transmission of HIV-1. METHODS: The Women and Infants Transmission Study is a prospective, observational study of HIV-1-infected women who were enrolled during pregnancy and followed with their infants for three years after delivery. We studied obstetrical, clinical, immunologic, and virologic data on 525 women who delivered live singleton infants whose HIV-1-infection status was known as of August 31, 1994. RESULTS: Among mothers with membranes that ruptured more than four hours before delivery, the rate of transmission of HIV-1 to the infants was 25 percent, as compared with 14 percent among mothers with membranes that ruptured four hours or less before delivery. In a multivariate analysis, the presence of ruptured membranes for more than four hours nearly doubled the risk of transmission (odds ratio, 1.82; 95 percent confidence interval, 1.10 to 3.00; P = 0.02), regardless of the mode of delivery. The other maternal factors independently associated with transmission were illicit-drug use during pregnancy (odds ratio, 1.90; 95 percent confidence interval, 1.14 to 3.16; P = 0.01), low antenatal CD4+ lymphocyte count (<29 percent of total lymphocytes) (odds ratio, 2.82; 1.67 to 4.76; P<0.001), and birth weight <2500 g (odds ratio, 1.86; 1.03 to 3.34; P = 0.04). CONCLUSIONS: The risk of transmission of HIV-1 from mother to infant increases when the fetal membranes rupture more than four hours before delivery. PMID- 8628357 TI - Nadolol plus isosorbide mononitrate compared with sclerotherapy for the prevention of variceal rebleeding. AB - BACKGROUND: Patients who have bleeding from esophageal varices are at high risk for rebleeding and death. We compared the efficacy and safety of endoscopic sclerotherapy with the efficacy and safety of nadolol plus isosorbide mononitrate for the prevention of variceal rebleeding. METHODS: Eighty-six hospitalized patients with cirrhosis and bleeding from esophageal varices diagnosed by endoscopy were randomly assigned to treatment with repeated sclerotherapy (43 patients) or nadolol plus isosorbide-5-mononitrate (43 patients). The primary outcomes were rebleeding, death, and complications. The hepatic venous pressure gradient was measured at base line and after three months. RESULTS: Base-line data were similar in the two groups, and the median follow-up was 18 months in both. Eleven patients in the medication group and 23 in the sclerotherapy group had rebleeding. The actuarial probability of remaining free of rebleeding was higher in the medication group for all episodes related to portal hypertension (P = 0.001) and variceal rebleeding (P = 0.002). Four patients in the medication group and nine in the sclerotherapy group died (P = 0.07 for the difference in the actuarial probability of survival). Seven patients in the medication group and 16 in the sclerotherapy group had treatment-related complications (P = 0.03). Thirty-one patients in the medication group underwent two hemodynamic studies; 1 of the 13 patients with more than a 20 percent decrease in the hepatic venous pressure gradient had rebleeding, as compared with 8 of the 18 with smaller decreases in the pressure gradient (P = 0.04) for the actuarial probability of rebleeding at two years). CONCLUSIONS: As compared with sclerotherapy, nadolol plus isosorbide mononitrate significantly decreased the risk of rebleeding from esophageal varices. PMID- 8628358 TI - Treatment of hereditary angioedema with a vapor-heated C1 inhibitor concentrate. AB - BACKGROUND: Hereditary angioedema results from a congenital deficiency of functional C1 inhibitor and is characterized by episodic bouts of edema, which may be life-threatening when they involve the larynx. We evaluated the effectiveness of a C1 inhibitor concentrate in the prevention and treatment of attacks of hereditary angioedema. The concentrate was vapor-heated to inactivate hepatitis and human immunodeficiency viruses. METHODS: We conducted two double blind, placebo-controlled studies. The first was a crossover study consisting of two 17-day trials in which prophylactic infusions of either C1 inhibitor (25 plasma units per kilogram of body weight) or placebo were given intravenously every third day to six patients with hereditary angioedema. The second study was conducted in patients with acute attacks of hereditary angioedema and assessed the length of time to a clinical response after infusions of either 25 plasma units of C1 inhibitor per kilogram (55 infusions in 11 patients) or placebo (49 infusions in 11 patients). RESULTS: The infusions of C1 inhibitor concentrate resulted in close to normal functional levels of C1 inhibitor and C4. As compared with placebo, prophylactic infusions of C1 inhibitor resulted in significantly lower daily symptom scores for the severity of edema of the extremities (P<0.01), larynx (P<0.05), abdomen (P<0.05), and genitourinary tract (P<0.05). Likewise, during the treatment study the time from the start of an infusion to the beginning of improvement in symptoms was shorter for the C1 inhibitor infusions than the placebo infusions (55 vs. 563 minutes, P<0.001). There was no evidence of toxicity. CONCLUSIONS: Infusions of a vapor-heated C1 inhibitor concentrate are a safe and effective means of both preventing attacks of hereditary angioedema and treating acute attacks. PMID- 8628359 TI - The influence of the wider use of surfactant therapy on neonatal mortality among blacks and whites. AB - BACKGROUND: Surfactant therapy reduces morbidity and mortality among premature infants with the respiratory distress syndrome (RDS). Fetal pulmonary surfactant matures more slowly in white than in black fetuses, and therefore RDS is more prevalent among whites than among blacks. We reasoned that the increased use of surfactant after its approval by the Food and Drug Administration (FDA) in 1990 might have reduced neonatal mortality more among whites than among blacks. METHODS: We merged vital-statistics information for all 1563 infants with very low birth weights (500 to 1500 g) born from 1987 through 1989 or in 1991 and 1992 to residents of St. Louis with clinical data from the four neonatal intensive care units in the St. Louis area; we then compared neonatal mortality during two periods, one before and one after the FDA's approval of surfactant for clinical use (1987 through 1989 and 1991 through 1992). RESULTS: The use of surfactant increased by a factor of 10 between 1987 through 1989 and 1991 through 1992. The neonatal mortality rate among all very-low-birth-weight infants decreased 17 percent, from 220.3 deaths per 1000 very-low-birth-weight babies born alive (in 1987 through 1989) to 183.9 per 1000 (in 1991 through 1992; P = 0.07). This decrease was due to a 41 percent reduction in the mortality rate among white newborns with very low birth weights (from 261.5 per 1000 to 155.5 per 1000; P = 0.003). In contrast, among black infants, the mortality rate for very-low-birth weight infants did not change significantly (195.6 per 1000 and 196.8 per 1000). The relative risk of death among black newborns with very low birth weights as compared with white newborns with similar weights was 0.7 from 1987 through 1989 and 1.3 from 1991 through 1992 (P = 0.02). The differences in mortality were not explained by differences in access to surfactant therapy, by differences in mortality between black and white infants who received surfactant, or by differences in the use of antenatal corticosteroid therapy. CONCLUSIONS: After surfactant therapy for RDS became generally available, neonatal mortality improved more for white than for black infants with very low birth weights. PMID- 8628360 TI - Images in clinical medicine. Angioedema of the intestine. PMID- 8628361 TI - Hyperbaric-oxygen therapy. PMID- 8628362 TI - Angiotensin receptors and their antagonists. PMID- 8628363 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 19-1996. Multisystem failure in a 33-year-old man after bone marrow transplantation. PMID- 8628364 TI - Reducing mother-to-infant transmission of HIV -- the door remains open. PMID- 8628365 TI - Hereditary angioedema. PMID- 8628366 TI - Euthanasia in Australia. PMID- 8628367 TI - Intensity of hemodialysis and response to erythropoietin. PMID- 8628368 TI - Intensity of hemodialysis and response to erythropoietin. PMID- 8628369 TI - Intensity of hemodialysis and response to erythropoietin. PMID- 8628370 TI - Intensity of hemodialysis and response to erythropoietin. PMID- 8628371 TI - Intensity of hemodialysis and response to erythropoietin. PMID- 8628372 TI - Viral load and response to treatment of HIV. PMID- 8628373 TI - Viral load and response to treatment of HIV. PMID- 8628374 TI - Viral load and response to treatment of HIV. PMID- 8628375 TI - Sudden cardiac death triggered by an earthquake. PMID- 8628376 TI - Smoking and impaired endothelium-dependent dilatation. PMID- 8628378 TI - The quandary over graduates of foreign medical schools in the United States. PMID- 8628377 TI - Resolution of refractory AIDS-related mucosal candidiasis after initiation of didanosine plus saquinavir. PMID- 8628379 TI - Health secretary backs AIDS office's budget authority. PMID- 8628380 TI - Cancer link to power cables 'exaggerated', say critics. PMID- 8628381 TI - Ministry 'knew high risk' of AIDS transmission. PMID- 8628382 TI - Nuclear watchdog tightens incident reporting rules. PMID- 8628384 TI - Commercial interests delay publication. PMID- 8628383 TI - 'Leak' rumours fuel debate on gene patent. PMID- 8628385 TI - French citations forge ahead but Europe still loses ground. PMID- 8628387 TI - Baboon cells fail to combat AIDS. PMID- 8628386 TI - University renames faculty after row over eugenics advocate. PMID- 8628388 TI - Organ transplants. PMID- 8628390 TI - Doctors watch the forecasts. PMID- 8628389 TI - Why Japan ought to legalize the pill. PMID- 8628391 TI - Digging up the roots of life. PMID- 8628392 TI - Yeast genes and human disease. PMID- 8628393 TI - A newly defined interleukin-1? PMID- 8628394 TI - From DNA sequence to biological function. AB - Genome sequencing is leading to the discovery of new genes at a rate 50-100 times greater than that achieved by classical genetics, but the biological function of almost half of these genes is completely unknown. In order fully to exploit genome sequence data, a systematic approach to the discovery of gene function is required. Possible strategies are discussed here in the context of functional analysis in the yeast Saccharomyces cerevisiae, a model eukaryote whose genome sequence will soon be completed. PMID- 8628395 TI - Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. AB - Disruption of the mouse dopamine transporter gene results in spontaneous hyperlocomotion despite major adaptive changes, such as decreases in neurotransmitter and receptor levels. In homozygote mice, dopamine persists at least 100 times longer in the extracellular space, explaining the biochemical basis of the hyperdopaminergic phenotype and demonstrating the critical role of the transporter in regulating neurotransmission. The dopamine transporter is an obligatory target of cocaine and amphetamine, as these psychostimulants have no effect on locomotor activity or dopamine release and uptake in mice lacking the transporter. PMID- 8628396 TI - Characterization of low-dimensional dynamics in the crayfish caudal photoreceptor. AB - Attempts to detect and characterize chaos in biological systems are of considerable interest, especially in medical science, where successful demonstrations may lead to new diagnostic tools and therapies. Unfortunately, conventional methods for identifying chaos often yield equivocal results when applied to biological data, which are usually heavily contaminated with noise. For such applications, a new technique based on the detection of unstable periodic orbits holds promise. Infinite sets of unstable periodic orbits underlie chaos in dissipative systems; accordingly, the new method searches a time series only for rare events characteristic of these unstable orbits, rather than analysing the structure of the series as a whole. Here we demonstrate the efficacy of the method when applied to the dynamics of the crayfish caudal photoreceptor (subject to stimuli representative of the animal's natural habitat). Our findings confirm the existence of low-dimensional dynamics in the system, and strongly suggest the existence of deterministic chaos. More importantly, these results demonstrate the power of methods based on the detection of unstable periodic orbits for identifying low-dimensional dynamics- and, in particular, chaos--in biological systems. PMID- 8628397 TI - Abnormal splicing of the leptin receptor in diabetic mice. AB - Mutations in the mouse diabetes (db) gene result in obesity and diabetes in a syndrome resembling morbid human obesity. Previous data suggest that the db gene encodes the receptor for the obese (ob) gene product, leptin. A leptin receptor was recently cloned from choroid plexus and shown to map to the same 6-cM interval on mouse chromosome 4 as db. This receptor maps to the same 300-kilobase interval as db, and has at least six alternatively spliced forms. One of these splice variants is expressed at a high level in the hypothalamus, and is abnormally spliced in C57BL/Ks db/db mice. The mutant protein is missing the cytoplasmic region, and is likely to be defective in signal transduction. This suggests that the weight-reducing effects of leptin may be mediated by signal transduction through a leptin receptor in the hypothalamus. PMID- 8628399 TI - Neural correlates of category-specific knowledge. AB - An intriguing and puzzling consequence of damage to the human brain is selective loss of knowledge about a specific category of objects. One patient may be unable to identify or name living things, whereas another may have selective difficulty identifying man-made objects. To investigate the neural correlates of this remarkable dissociation, we used positron emission tomography to map regions of the normal brain that are associated with naming animals and tools. We found that naming pictures of animals and tools was associated with bilateral activation of the ventral temporal lobes and Broca's area. In addition, naming animals selectively activated the left medial occipital lobe--a region involved in the earliest stages of visual processing. In contrast, naming tools selectively activated a left premotor area also activated by imagined hand movements, and an area in the left middle temporal gyrus also activated by the generation of action words. Thus the brain regions active during object identification are dependent, in part, on the intrinsic properties of the object presented. PMID- 8628398 TI - Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. AB - Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment, 20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines, suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells, and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias, attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker, AG-490, selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death, with no deleterious effect on normal haematopoiesis. PMID- 8628400 TI - Creation of a biologically active interleukin-5 monomer. AB - Interleukin-5 (IL-5) specifically induces the differentiation of eosinophils, which are important in host defence and the pathogenesis of allergies and asthma. Structurally, IL-5 is a unique member of the short-chain helical-bundle subfamily of cytokines whose canonical motif contains four helices (A-D) arranged in an up up-down-down topology. In contrast to other subfamily members, which fold unimolecularly into a single helical bundle, IL-5 forms a pair of helical bundles by the interdigitation of two identical monomers that contribute a D helix to the other's A-C helices. We predicted that the lack of bioactivity by an IL-5 monomer was due to a short loop between helices C and D which physically prevents unimolecular folding of helix D into a functionally obligate structural motif. Here we report that, by lengthening this loop, we have engineered an insertional mutant of IL-5 that was expressed as a monomer with biological activity similar to that of native IL-5. These studies demonstrate that all of the structural features necessary for IL-5 to function are contained within a single helical bundle. PMID- 8628401 TI - Identification of the gal4 suppressor Sug1 as a subunit of the yeast 26S proteasome. AB - The SUG1 gene of Saccharomyces cerevisiae encodes a putative ATPase. Mutations in SUG1 were isolated as suppressors of a mutation in the transcriptional activation domain of GAL4. Sug1 was recently proposed to be a subunit of the RNA polymerase II holoenzyme and to mediate the association of transcriptional activators with holoenzyme. We show here that Sug1 is not a subunit of the holoenzyme, at least in its purified form, but of the 26S proteasome, a large complex of relative molecular-mass 2,000K that catalyses the ATP-dependent degradation of ubiquitin protein conjugates. Sug1 co-purifies with the proteasome in both conventional and nickel-chelate affinity chromatography. Our observations account for the reduced ubiquitin-dependent proteolysis in sug1 mutants and suggest that the effects of sug1 mutations on transcription are indirect results of defective proteolysis. PMID- 8628402 TI - [The non-healing ulcer; think of squamous cell carcinoma of the skin]. PMID- 8628403 TI - [Socioeconomic differences in survival of cancer patients]. PMID- 8628404 TI - [Inhibition of saliva secretion caused by antidepressive drugs; a risk for oral health]. PMID- 8628405 TI - [Adenoviruses as causative agents in gastroenteritis]. PMID- 8628406 TI - [Clinical thinking and judgment in clinical practice. A patient with diarrhea]. PMID- 8628407 TI - [Consensus inflammatory intestinal diseases in children: ulcerative colitis and Crohn disease. Work Group Academic Medical Center, Amsterdam]. AB - Members of different societies, including Paediatrics, Gastroenterology, Clinical Chemistry, Radiology and Pathology, involved in the treatment of children with inflammatory bowel disease (IBD) reached consensus during 12 meetings about definition, clinical and diagnostic work-up as well as initial treatment options in paediatric IBD. On the basis of history and physical examination a distinction is made between colitis-like or Crohn-like appearance. Further laboratory investigations are required in the presence of malnutrition, extraintestinal manifestations or growth retardation. Pathology plays an important part in the final diagnosis. Special attention is given to pubertal staging and height measurements as routine aspects of the treatment of children with IBD. PMID- 8628409 TI - [A Chinese patient with pernicious anemia; a medical experience from the Indonesian period]. AB - On May 19th 1952 a 64-year-old Chinese man was admitted to a hospital at Yogyakarta (Indonesia) on account of a sawing noise in both ears and some soreness of the tongue. He had macrocytic anemia (haemoglobin: 3.7 mmol/l) and the tongue showed some smooth patches. A presumptive diagnosis of pernicious anaemia was confirmed by gastric analysis which revealed a histamine fast achlorhydria. On treatment with vitamin B12 the noise in the ears rapidly disappeared and there was a characteristic rise in reticulocytes and haemoglobin content. After 3 years the patient died of inoperable gastric carcinoma. There probably was a hereditary component as in a 54-year-old cousin, who also suffered (and died) from gastric carcinoma, gastric analysis showed a histamine fast achlorhydria. The patient is the first case of pernicious anaemia described in a Chinese resident of Indonesia. A survey of the literature revealed that until now pernicious anaemia has been recorded in 31 Chinese patients, in chronological order from the following countries: U.S (1945), Indonesia (1954), Singapore (1967), Hong-Kong (1969) and China (1990). In the autochthonous Chinese population no case has yet been reported. PMID- 8628408 TI - [Selection of patients for kidney transplantation; how to deal with scarcities in clinical practice]. AB - OBJECTIVE: To investigate what criteria are applied in the actual practice of patient selection for renal transplantation and how practice relates to guidelines developed in medical ethics and health law. SETTING: Two centres for renal transplantation and nine dialysis centres. DESIGN: Descriptive. METHODS: Data were collected by observation and open interviews with 33 physicians and other health professionals involved, and analysed using a computer programme for qualitative data. RESULTS: Formally, scarcity of donor organs did not affect the referral and indication of patients for renal transplantation. However, according to some respondents, fewer people were entered on the waiting list because of scarcity. This concerned mainly patients less likely to benefit from a transplantation in terms of life expectancy or enhancement of quality of life. CONCLUSION: There was some 'covert selection', in that scarcity implicitly or unintentionally was a factor in the decision whether or not to place patients on the waiting list for renal transplantation. The absence of consensus on acceptable selection criteria and the emphasis on medical criteria in the social debate on selection criteria may have contributed to this covert selection. PMID- 8628410 TI - [Stimulation Program Health Research. VI. Evaluation of the program section 'Interrelationship somatic and psychological disorders']. PMID- 8628411 TI - [Alexandria Diabetes Days]. PMID- 8628412 TI - [Sluder's method of adenotonsillectomy; with or without endotracheal intubation?]. PMID- 8628413 TI - [Fecal analysis for occult blood obsolete]. PMID- 8628414 TI - [Fecal analysis for occult blood obsolete]. PMID- 8628415 TI - [Fecal analysis for occult blood obsolete]. PMID- 8628416 TI - [Gout: current concept of etiology, diagnosis and therapy]. PMID- 8628417 TI - [Fractures and blood vessels]. PMID- 8628418 TI - [Recovery processes in interrupted peripheral nerves]. PMID- 8628420 TI - [Farmacotherapeutisch Kompas 1995]. PMID- 8628419 TI - [Therapeutic (im)possibilities in Raynaud's phenomenon]. PMID- 8628421 TI - [Improvement in the internists' outpatient report using a time control system and systematic feedback by the general practitioner]. AB - OBJECTIVE: To establish if the letters from internists contain the information wanted by the GP and if the quality of these letters can be improved by a time control system and by systematic feedback from the GP to the internist. DESIGN: Questionnaire study. SETTING: Outpatient clinic for General Internal Diseases, University Hospital Nijmegen. METHOD: The 284 GPs in the region were polled about their requirements for information in terms of speed and contents. Patient files were examined to find out to what extent the outpatient department met these requirements. The outpatient clinic physicians were informed of the findings. Subsequently a control system was introduced; every letter was accompanied by a postage-paid evaluation card inviting the GPs to comment. The findings obtained were reported back to the outpatient department physicians. RESULTS: GPs appreciated a preliminary report after the first outpatient visit (response rate: 70%). In the final report, they particularly wanted information on diagnosis, treatment, management to be conducted and information given to the patient. The control system accelerated final reporting. Of the evaluation cards 75% were returned; the explanation concerning diagnosis and treatment was experienced as sufficient, while mention of the information given to the patient was not always found satisfactory. CONCLUSION: The internists' letters did not optimally meet the GPs' need of information. A 'time control system' accelerated the final reporting on newly referred patients. A systematic feedback of the GPs' opinions on the quality of the letters improved the alignment of the information provided with the GPs' requirements. PMID- 8628422 TI - [Mediocre quality of discharge reports concerning psychiatric patients]. AB - OBJECTIVE: Determination of the quality of discharge letters concerning psychiatric patients. DESIGN: Descriptive. SETTING: 29 community mental health clinics in The Netherlands. METHOD: 420 hospital discharge letters were examined according to 13 quality criteria (reply time, legal status of the patient, reason for hospitalization, heteroanamnesis, source of income, alcohol use, drug use, physical findings, approach, structural diagnosis, DSM-III-R diagnosis, number of words, signature by psychiatrist) derived from a Dutch guideline for discharge letters. RESULTS: The letters did not reach the aftercare attendant in time. The length of the letters was usually within 1050 words. Some formal criteria, like classification according to the DSM-III-R and signature were mostly met (in 67.1% and 90.9% of the letters). Important other information (for instance about a heteroanamnesis (24.8%), the source of income (23.1%), the use of alcohol (24%) or drugs (14.7%) and how to cope with the patient (8%)) were not sufficiently mentioned. CONCLUSION: The quality of hospital discharge letters from community mental health clinics in The Netherlands was insufficient. Adequate exchange of information is essential to guarantee good care for psychiatric patients. Improvement of the quality of hospital discharge letters is therefore highly advisable. PMID- 8628423 TI - [Psychiatric admissions in Amsterdam according to ethnic background and diagnosis]. AB - OBJECTIVE: To determine whether patients from ethnic groups in Amsterdam are admitted to psychiatric institutes more often than Dutch natives. DESIGN: Descriptive. SETTING: Amsterdam, The Netherlands METHOD: Admission incidences were calculated from the Amsterdam registry of admissions to psychiatric institutions, 1992-1993, and the country of birth was determined. Only first generation migrants were studied. RESULTS: Patients from most ethnic groups were not admitted more frequently than Dutch natives. Only people from non industrialized countries were more often admitted. Schizophrenia was diagnosed significantly more often in Surinam men (a factor 2) than in Dutch natives, among Turkish men the diagnosis was less frequent. CONCLUSION: Earlier findings of two to-five-fold increased incidence of schizophrenia in first-generation migrants from Surinam, The Netherlands Antilles and Morocco, were in Amsterdam only partly confirmed. PMID- 8628424 TI - [3 children with velocardiofacial (Shprintzen) syndrome]. AB - In three children, girls of 3, 8, and 12 years old, who attended or had attended public health care facilities, velocardiofacial syndrome was diagnosed. The most important symptoms are cleft palate, cardiac anomalies, characteristic facies (almond-shaped eyes, wide nose, small ears) and learning problems. The syndrome has an autosomal dominant inheritance pattern with a very variable expression. Using fluorescence in situ hybridisation, microdeletions of the long arm of chromosome 22 have been identified in 70% of the patients (22qII). Velocardiofacial syndrome should be considered in any child with cleft palate, velopharyngeal insufficiency and/or hypernasal speech, and notably in children with nasal regurgitation of food in the first year of life, poor growth, developmental problems, facial dysmorphism and/or conotruncal cardiac anomalies. PMID- 8628426 TI - [What was Doctors without Borders doing in Albania?]. PMID- 8628425 TI - [Possible potentiation of phenprocoumon by clarithromycin and roxithromycin]. AB - Two patients, a women of 70 and a man of 75 years old, who were using phenprocoumon chronically, and were monitored by a regional thrombosis service, received a macrolide antibiotic, clarithromycin and roxithromycin respectively, for an airway infection. Both patients developed a serious increase in hypocoagulability, requiring administration of phytomenadione and temporary decreases in phenprocoumon dose. There were no bleeding complications. After the antibiotics were discontinued, the original dosage of phenprocoumon was needed again. It is suggested that in these patients the macrolide antibiotics may have potentiated the effect of phenprocoumon, perhaps as a result of inhibition of phenprocoumon transformation by liver enzymes. In patients receiving chronic treatment with phenprocoumon, coagulation parameters should be regularly checked if they are given a macrolide antibiotic such as clarithromycin, roxithromycin or erythromycin. PMID- 8628427 TI - [Does flattening of the skull have lasting sequelae for brain development?]. PMID- 8628428 TI - [What to do in a 60-year-old HIV-positive woman with a cerebral arteriovenous malformation? Decision analysis]. PMID- 8628429 TI - [2 years further on: a poignant WAO (Law for Work Disability Insurance)]. PMID- 8628430 TI - [Psychiatric drugs as a risk factor in fatal heat exhaustion]. PMID- 8628431 TI - [Compliance with consensus 'Diagnosis pulmonary embolism' in practice]. PMID- 8628432 TI - [Hereditary diseases are family diseases]. PMID- 8628433 TI - [Favorable effects of hormone substitution therapy in the menopause on risk factors for ischemic heart disease]. PMID- 8628434 TI - [The effect of menopause on risk factors for ischemic diseases]. PMID- 8628436 TI - [Preconception screening for carrier state in cystic fibrosis; testing against Health Council's criteria for genetic screening]. AB - OBJECTIVE: To determine if preconceptional screening for carriership of cystic fibrosis (CF) meets the requirements formulated by the Committee for Screening for Hereditary and Congenital Disorders of the Health Council. DESIGN: Theoretical evaluation. METHOD: Preconceptional screening for CF carriership by means of direct mutation analysis in a blood sample, cheek smear or mouth rinse sample of both partners of pairs desiring children was tested against the criteria of the Health Council. RESULTS: Preconceptional screening for CF carriership proves to meet most of the requirements for genetic screening. Current problems are the inadequate sensitivity of the screening (72% for pairs, 85% for individuals), the consequences of this for the meaning of the test results and reaching of the target group. Much attention is required for provision of information at all stages of the screening procedure. The desirability, feasibility and psychosocial effects of this type of screening have not yet been investigated sufficiently in the Netherlands. PMID- 8628435 TI - [Primary hyperaldosteronism as a cause of hypertension; diagnosis]. PMID- 8628437 TI - [Intravascular treatment of inoperable cerebral aneurysms using Guglielmi's spirals; initial results in The Netherlands]. AB - OBJECTIVE: Evaluation of endovascular treatment of inoperable cerebral aneurysms using electrolytically detachable platinum coils (Gugliemi Detachable Coils, GDC). DESIGN: Retrospective. SETTING: St. Elizabeth Hospital Tilburg, the Netherlands. METHOD: Fifteen aneurysms in 13 patients were treated using GDC; 14 of these aneurysms were inoperable and in three aneurysms surgical clipping had failed. RESULTS: Twelve of the 15 treated aneurysms were completely occluded. In another two, occlusion was 90% and in one, 70%. One patient with an inoperable basilar bifurcation aneurysm died of progressive thrombosis of both posterior cerebral arteries. One patient with an inoperable aneurysm of the anterior communicating artery developed an infarction of the A. centralis longa (recurrent artery of Heubner). CONCLUSION: GDC treatment of inoperable cerebral aneurysms is currently the only available option with a reasonable chance of success and acceptable risks. PMID- 8628438 TI - [Quality improvement project 'laboratory diagnosis by family physicians' leads to considerable decrease in number of laboratory tests]. AB - OBJECTIVE: Quality improvement of laboratory diagnostics by general practitioners (GPs) through introduction of a simplified, problem-oriented application form, supported by information and feedback. DESIGN: Prospective descriptive study. SETTING: Laboratory of Clinical Chemistry and Haematology of St. Maartens Gasthuis, Venlo, the Netherlands and the GPs in the region. METHODS: The effects of the intervention were measured by counting the analyses requested by all GPs in the region in 1992, 1993 and 1994. Furthermore requests by every GP for fifteen selected analyses in the first 6 months of 1992, 1993 and 1994 were counted and reported together with the anonymous data of all colleagues. RESULTS: After the intervention a 23% reduction of the total number of analyses request by GPs was noticed. Blood sampling increased by 8% and the mean number of laboratory test requests per patient decreased from 5.9 to 4.2. The largest request reductions were noticed for analyses not listed on the application form. Measurements in the first 6 months of 1992, 1993 and 1994 showed continuation of the trend and a fadeaway of 'redundant' analyses. CONCLUSION: The introduction of a simplified problem-oriented application form for GPs supported by feedback caused a marked decrease of the number of (redundant) laboratory requests. PMID- 8628440 TI - [Serum screening in pregnant women for Down syndrome and open neural tube defects; testing of Health Council's criteria for genetic screening]. PMID- 8628439 TI - [Pulmonary infection with Coccidioides immitis]. AB - A man aged 42 who was referred to the outpatient department for Pulmonary Diseases because of a cough of two weeks' standing and fever up to 40 degrees C, had recently spent 10 days in the desert (San Joaquin Valley, California). Physical examination revealed no distinct abnormalities. Radiologically, an infiltrative lesion in the left upper lobe of the lung was established, as well as swollen lymph nodes near the pulmonary hilus. Blood testing revealed leukocytosis and eosinophilia. Possibilities considered were tuberculosis and, because the patient had been in California, coccidioidomycosis. The Mantoux test and several Ziehl-Neelsen preparations were negative. However, the immunodiffusion test for Coccidioides immitis antibodies was positive. Also, C. immitis was cultured from the sputum. The diagnosis read ?pulmonary coccidioidomycosis with eosinophilia'. Because of aggravation of the pulmonary lesions and eosinophilia, treatment with ketoconazole was instituted. This was the first time in the Netherlands that a human infection with the fungus C. immitis could be confirmed serologically as well. PMID- 8628441 TI - [Serum screening in pregnant women for Down syndrome and open neural tube defects; testing of Health Council's criteria for genetic screening]. PMID- 8628442 TI - [Treatment of asthma; are there still questions?]. PMID- 8628443 TI - Is selective reporting of well-designed clinical research unethical as well as unscientific? PMID- 8628444 TI - [Ras-oncogene detection in feces of patients with colorectal tumors]. PMID- 8628445 TI - [Startle disease: growing rigid with fear]. PMID- 8628447 TI - Commodity trading and PC. PMID- 8628446 TI - [Opportunistic lung infections in patients with chronic obstructive lung disease; a side effect of inhalation steroids?]. PMID- 8628448 TI - Serotonin syndrome and fluvoxamine: a case study. AB - OBJECTIVE: To report a serotonin syndrome reaction in a patient taking fluvoxamine to replace an earlier SSRI agent. CASE REPORT: A female patient with Obsessive Compulsive Disorder on paroxetine after resurgence in her obsessive ruminations was started on fluvoxamine 50 mg daily. One week later she became suicidal and was hospitalized. The fluvoxamine was increased to 50 mg morning and 100 mg bedtime and the paroxetine was discontinued. Over the next few days she began to have trouble with her concentration. A low grade fever set in after she experienced auditory hallucinations. Fluvoxamine was discontinued and she had an uneventful recovery after twenty-four hours. DISCUSSION: Fluvoxamine is a recently approved serotonin selective reuptake inhibitor (SSRI) with few side effect profiles. It is effective in the treatment of Depressive Disorder and Obsessive Compulsive Disorder and is used to potentiate or replace other anti-OCD drugs including already available serotonin specific reuptake inhibitors (SSRI). We wish to draw attention to the potential for serotonin syndrome in patients on fluvoxamine who may have previously been on other SSRIs. PMID- 8628449 TI - Timing of breast cancer surgery during the luteal menstrual phase may improve prognosis. AB - A meta-analysis has been performed of available retrospective reports concerning the 5-15 year disease-free survival of 5,353 premenopausal breast cancer patients operated on either during the follicular or luteal phases of the menstrual cycle. Patients with surgery performed during the luteal phase (d 14-23+) had an overall mean 5% benefit compared to those operated on the follicular phase determined by date of onset of their last menstrual period p = 0.02 by Wilcoxon 2-tailed test. When nodal invasion was reported, node-negative patients had a 5 +/- 2% SEM benefit. Patients with positive nodes had a 34 +/- 3% SEM increase in survival (p = .05), including both estrogen and progesterone-receptor negative as well as positive neoplasms. In 3 of 4 reports from major cancer treatment centers, each containing 249-1175 cases, risk of recurrent cancer and/or death increased 5 to 6 fold after 10 years for women receiving surgery during d 7-14 of their cycle, compared to those resected during d 21-36. Improvement in prognosis was greatest for patients with the highest risk of recurrence due to node-invasive disease and receptor dysfunction. Several cell-mediated immunologic factors inimical to metastasis are maximal in the luteal phase of the menstrual cycle, including natural killer cell activity. A new drug which augments natural killer cell activity may extend any beneficial survival results to post-menopausal breast cancer patients in the future. We conclude that accurate menstrual histories should be included in the medical record from now on for all premenopausal women receiving any surgical procedure upon the breast, preferably using an objective method of determining the date of last ovulation. Prospective randomized clinical trials are necessary to determine the full extent of survival benefits of late luteal surgical timing. PMID- 8628450 TI - Chopart's fracture dislocation: a case report and review of the literature. AB - The prompt identification of peri-talar dislocation with immediate reduction and early range of motion will generally result in a good outcome. Higher energy injury with greater soft tissue compromise and associated fractures worsen the prognosis. Salvage procedure for post-traumatic degenerative changes require arthrodesis for relief of pain. The deformity is usually apparent on physical examination, but occasionally is masked by marked early swelling. A high index of suspicion should be maintained with high or low energy injury to the foot and ankle with normal ankle radiographs. This particular case is unusual in that the patient was able to function as a farmer even with the fracture dislocation. This again is probably explained by his mild peripheral neuropathy. This case, however, does demonstrate the difficulty in making the diagnosis. PMID- 8628451 TI - A focused review of fetal deaths in Nebraska in 1992. PMID- 8628452 TI - The relationship of multiple system atrophy to sporadic olivopontocerebellar atrophy and other forms of idiopathic late-onset cerebellar atrophy. PMID- 8628453 TI - How familial is familial tremor? The genetic epidemiology of essential tremor. AB - Essential tremor (ET) is commonly assumed to be partly genetic. This is due to a seemingly excess aggregation within certain families. There are many families containing more than one member with ET, and some large kindreds with multiple affected individuals. Despite this, the proportion of ET cases with affected family members is not known. Estimates vary from 17 to 100%. Additionally, none of the published studies employed control subjects. Therefore, it is unknown to what extent ET aggregates within families beyond that expected by chance, and the importance of genetic susceptibility in the etiology of ET at the population level has not been established. Additionally, whether or not some clinical subtypes of ET are more likely than others to be influenced by genetic susceptibility is unclear. Some studies suggest that early-onset ET may be familial, although increased awareness and earlier recognition of symptoms is an issue. Although many data support the view that ET susceptibility is inherited in an autosomal dominant manner, most studies are kindreds or service-based studies that might favor selection of families with apparent autosomal dominant modes of inheritance. One community-based study suggested an autosomal dominant mode of inheritance, but this has not been confirmed in studies of more heterogeneous populations. Further understanding of the extent of familial aggregation, extent of genetic heterogeneity, and mode of inheritance is essential for clinical counseling and for further research aimed at localizing and identifying susceptibility genes. PMID- 8628455 TI - Blepharospasm: report of a workshop. PMID- 8628454 TI - Chronic inflammatory demyelinating polyradiculoneuropathy: unusual clinical features and therapeutic responses. AB - We present three patients with atypical chronic inflammatory demyelinating polyradiculoneuropathy and discuss the management of patients who appear treatment resistant or present with unusual manifestations. The clinical features of the patients included massive nerve root hypertrophy causing myelopathy and movement-provoked paresthesia, pupillary dysfunction, visual loss due to increased intracranial pressure, and focal brachial plexus involvement. Each patient ultimately required prolonged courses of immune modulating therapy before benefit was attained, illustrating the importance of intensive and prolonged treatment combined with objective assessment of response to therapy. PMID- 8628456 TI - Survival and proliferation of nonneural tissues, with obstruction of cerebral ventricles, in a parkinsonian patient treated with fetal allografts. AB - BACKGROUND: Since 1985, treatment of idiopathic Parkinson's disease (PD) by surgical transfer of adult or fetal chromaffin tissue or of fetal central neural tissue to the brains of afflicted patients has been attempted, with variable clinical results. Neuropathologic studies of the status of these transplants are few and show wide variation in degree of graft survival. METHODS: We report the case of a 52-year-old man, who, 23 months earlier, received both intrastriatal implantation and intraventricular infusion of tissues derived from fetuses of 15 to 16 weeks and 5 to 6 weeks gestational age. Clinical improvement, as measured by increased amounts of "on" time with reduced levodopa requirements, seemed to occur over the subsequent months. He died suddenly at home after a several-hours interval of progressive lethargy and breathing difficulties. RESULTS: At autopsy, the diagnosis of PD was confirmed. Intrastriatal graft sites were identified, but contained no viable neurons; astrogliosis, focal microgliosis, and mixed inflammatory response, suggesting allograft rejection, were present. Surprisingly, the intraventricular tissues survived and showed ectodermal and mesenchymal, but no neural, differentiation, as well as cellular response; the left lateral and fourth ventricles were filled completely by this proliferated tissue. CONCLUSIONS: By intraventricular infusion, tissues from early-gestation sources can engraft successfully, grow, and survive for at least 23 months in the brain of a PD patient. However, contamination by, or differentiation into, nonneural tissues can occur, can lead to proliferation of tissues within ventricular spaces, and may result in ventricular obstruction. Grafts, whether intraventricular or intraparenchymal, are capable of inciting host responses, which in turn may limit their long-term survival. Finally, post-transplant clinical improvement in symptoms of PD may be unrelated to survival of engrafted neurons. PMID- 8628457 TI - Coexistence of migraine and idiopathic intracranial hypertension without papilledema. AB - Eighty-five patients with refractory transformed migraine type of chronic daily headache (CDH) had spinal tap as a part of diagnostic work-up. Twelve had increased intracranial pressure without papilledema, transient visual obscurations, or visual field defects. The headache profile of these 12 patients was not different from that of transformed migraine type of CDH. Acute headache exacerbations responded to specific antimigraine agents such as ergotamine, dihydroergotamine (DHE), and sumatriptan, whereas prophylactic antimigraine medications were only partially helpful. Addition of agents such as acetazolamide and furosemide, after the diagnosis of increased intracranial pressure, resulted in better control of symptoms. These observations suggest a link between migraine and idiopathic intracranial hypertension that needs further research. In refractory CDH with migrainous features, a spinal tap to exclude coexistent idiopathic intracranial hypertension without papilledema may be indicated. PMID- 8628458 TI - Frontal lobe injuries, violence, and aggression: a report of the Vietnam Head Injury Study. AB - Knowledge stored in the human prefrontal cortex may exert control over more primitive behavioral reactions to environmental provocation. Therefore, following frontal lobe lesions, patients are more likely to use physical intimidation or verbal threats in potential or actual confrontational situations. To test this hypothesis, we examined the relationship between frontal lobe lesions and the presence of aggressive and violent behavior. Fifty-seven normal controls and 279 veterans, matched for age, education, and time in Vietnam, who had suffered penetrating head injuries during their service in Vietnam, were studied. Family observations and self-reports were collected using scales and questionnaires that assessed a range of aggressive and violent attitudes and behavior. Two Aggression/Violence Scale scores, based on observer ratings, were constructed. The results indicated that patients with frontal ventromedial lesions consistently demonstrated Aggression/Violence Scale scores significantly higher than controls and patients with lesions in other brain areas. Higher Aggression/Violence Scale scores were generally associated with verbal confrontations rather than physical assaults, which were less frequently reported. The presence of aggressive and violent behaviors was not associated with the total size of the lesion nor whether the patient had seizures, but was associated with a disruption of family activities. These findings support the hypothesis that ventromedial frontal lobe lesions increase the risk of aggressive and violent behavior. PMID- 8628459 TI - Neural basis of confabulation. AB - We present a case of acute alcohol-induced Korsakoff amnesia. A severe amnestic confabulatory syndrome characterized the early clinical status. The initial neuropsychological tests demonstrated severe learning deficits plus impaired performance on many, but not all, tests of frontal lobe function. Single-photon emission CT (SPECT) at this stage showed hypoperfusion in the orbital and medical frontal regions and the medial diencephalic area. Four months later, the patient's amnesia remained but there was no confabulation. Repeat neuropsychological tests confirmed an ongoing severe amnesia, but performance on the frontal lobe tests now was normal. Repeat SPECT showed a return to normal perfusion in the frontal brain areas but little improvement in the medial diencephalic region. These findings along with data from the clinical literature suggest that confabulation results from dysfunction of orbital and a medial frontal cortex. PMID- 8628460 TI - A double-blind placebo-controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis. ALS CNTF Treatment Study Group. AB - Ciliary neurotrophic factor (CNTF) is a neuroactive cytokine found in Schwann cells, which appears to be released in response to nerve injury. The ALS CNTF Treatment Study (ACTS) clinical trial was a phase II-III randomized, placebo controlled, double-blind study designed to evaluate the safety, tolerability, and efficacy of subcutaneous administration of recombinantly produced human CNTF (rHCNTF) in slowing disease progression in 730 patients with amyotrophic lateral sclerosis (ALS). Patients were randomized to receive 30 micrograms/kg or 15 micrograms/kg rHCNTF or placebo subcutaneously three times a week for 9 months. The primary endpoint of the study, the slope of decline of isometric muscle strength in treated versus placebo patients, showed no statistically significant difference between rHCNTF and placebo-treated patients, and was complicated by an initial statistically significant decrease in strength early in rHCNTF-treated patients. Mortality was similar in all three study arms. There were no statistically significant treatment effects among the secondary measures. Side effects of rHCNTF included anorexia, weight loss, and cough and were sufficient to limit dosing in many patients. PMID- 8628461 TI - Pontine lesions in idiopathic narcolepsy. AB - Three patients had longstanding (37 to 50 years), highly disabling narcolepsy, poorly controlled by treatment. The clinical histories were typical, consisting of sleepiness, cataplexy, sleep paralysis, hypnagogic hallucinations, disturbed nocturnal sleep, and HLA-DR2 tissue typing. Polygraphic findings confirmed the diagnosis. Neurologic examination, spinal fluid, and evoked potentials were normal. On MRI scanning, all three patients showed overlapping bilateral and symmetric brainstem T2 hyperintensities circumscribed to the ventrolateral aspect of the midrostral pons. The nature of the lesions remains uncertain but their location corresponded to the pontine oral reticular formation, where the neuronal network generating REM sleep is located. This is the first report of MR signal abnormalities in patients with idiopathic narcolepsy and suggests a causal relationship between the disease and the central pontine lesions. PMID- 8628462 TI - Self-reported automobile accidents involving patients with obstructive sleep apnea. AB - We developed a routine survey instrument, which included data on self-reported motor vehicle accidents (MVA), among 253 patients who attended the UCLA Sleep Disorders Center to evaluate whether patients with diagnosed sleep apnea syndrome (SAS) have a higher ratio of MVAs. We used unconditional multiple logistic regression to determine the odds ratio between MVA and SAS, adjusted for the following covariates: age, sex, work shift, daytime nap, alcohol and coffee intake, and history of neurologic diseases. Thirty-one percent of patients with SAS compared with 15% of patients without SAS reported at least one MVA (p < 0.01). The adjusted odds ratio found through logistic multiple regression analysis was 2.99 (p < 0.01). The results demonstrate that patients with SAS had a higher ratio of self-reported MVA than did those without SAS and that the characteristics of falling asleep at inappropriate times and driving past destinations were two good indicators of probability of having an MVA. Nevertheless, sleep apnea is a very treatable condition; once treated the risk factors diminish considerably. PMID- 8628463 TI - Cataplexy and monoamine oxidase deficiency in Norrie disease. AB - Norrie disease (ND) is an X-linked recessive disorder causing ocular atrophy, mental retardation, deafness, and dysmorphic features. Virtually absent monoamine oxidase (MAO) type-A and -B activity has been found in some boys with chromosome deletions. We report the coexistence of cataplexy and abnormal REM sleep organization with ND. Three related boys, referred for treatment of medically refractory atonic spells and apneas, underwent extended EEG-video polysomnographic monitoring. They demonstrated attacks of cataplexy and inappropriate periods of REM sleep during which they were unarousable. One boy also had generalized tonic-clonic seizures. Previous testing revealed that all three have complete ND gene deletions. In all subjects, platelet MAO-B activity was absent, serum serotonin levels were markedly increased, and plasma catecholamine levels were normal. Data from the canine narcolepsy syndrome model implicate abnormal catecholaminergic and cholinergic activities in the pathogenesis of cataplexy. Our findings suggest that abnormal MAO activity or an imbalance between serotonin and other neurotransmitter levels may be involved in the pathogenesis of human cataplexy. PMID- 8628464 TI - Does monoamine oxidase type B play a role in dopaminergic nerve cell death in Parkinson's disease? AB - Evidence supports the role of hyperoxidation phenomena in the mechanism of nerve cell death in Parkinson's disease (PD). The oxidative degradation of dopamine, catalyzed by monoamine oxidase type B (MAO-B), produces free radicals and thus could be implicated in the degenerative process. For this reason, we investigated by immunohistochemistry the distribution of MAO-B-containing cells in the midbrain of five patients with PD and five matched control subjects. MAO-B-like immunoreactivity was detected in glial cells, fibers, and neurons. Although most of the MAO-B-positive neurons probably belonged to the raphe dorsalis, we demonstrated by double-labeling immunohistochemistry that some of them were also dopaminergic. MAO-B-positive dopaminergic neurons were present in all dopaminergic groups of the control midbrain. Within the substantia nigra pars compacta, most dopaminergic neurons were located in the dorsal part of the structure. MAO-B-positive dopaminergic neurons were still detected in PD midbrains. Compared with control subjects, the loss of dopaminergic neurons containing MAO-B (-45%) was no higher than that of MAO-B-negative dopaminergic neurons (-59%). The density of MAO-B-positive glial cells varied in the control midbrains: high in the least affected dopaminergic group (the central gray substance) and low in the most affected region (the substantia nigra pars compacta). The density of MAO-B-positive glial cells within dopaminergic cell subgroups in control midbrains were negatively correlated (r = -0.94; p < 0.02) to the estimated neuronal loss in PD. We conclude that the presence of MAO-B in dopamine-containing neurons does not contribute to vulnerability in PD. Moreover, its presence in some glial cells might have a protective effect against oxidative stress induced by dopamine metabolism. PMID- 8628465 TI - Case-control study of idiopathic Parkinson's disease and dietary vitamin E intake. AB - A nested case-control study of 84 incident cases of patients with idiopathic Parkinson's disease (PD) detected by June 30, 1994 and 336 age-matched control subjects, compared previously-documented intake of total dietary vitamin E and of selected vitamin E-containing foods. All study subjects had been followed for 27 to 30 years after diet recording in the 8,006-man Honolulu Heart Study cohort. We determined PD outcomes by periodic cohort re-examination and neurologic testing, private physician reports, examination of O'ahu neurologists' office records, and continual death certificate and hospital discharge diagnosis surveillance. Data on vitamin E intake, obtained from three dietary data sets at the time of cohort enrollment (1965 to 1968), included a food-frequency questionnaire and a 24-hour photograph-assisted dietary recall administered by trained dietitians. Although absence of PD was significantly associated with prior consumption of legumes (adjusted OR = 0.27, 95% CI 0.09 to 0.78), a dietary variable preselected for high vitamin E content, neither food categories nor quartiles nor continuous variables of vitamin E consumption were significantly associated with PD occurrence. Though consistent with prior reports of PD protection afforded by legumes, and with speculation on the possible benefits of dietary or supplemental vitamin E in preventing PD, these preliminary data do not conclusively document a beneficial effect of dietary vitamin E on PD occurrence. PMID- 8628466 TI - Possible environmental, occupational, and other etiologic factors for Parkinson's disease: a case-control study in Germany. AB - In a case-control study, we investigated the possible etiologic relevance to Parkinson's disease (PD) of rural factors such as farming activity, pesticide exposures, well-water drinking, and animal contacts; toxicologic exposures such as wood preservatives, heavy metals, and solvents; general anesthesia; head trauma; and differences in the intrauterine environment. We recruited 380 patients in nine German clinics, 379 neighborhood control subjects, and 376 regional control subjects in the largest case-control study investigating such factors and collected data in structured personal interviews using conditional logistic regression to control for educational status and cigarette smoking. The latter was strongly inversely associated with PD. There were significantly elevated odds ratios (OR) for pesticide use, in particular, for organochlorines and alkylated phosphates, but no association was present between PD and other rural factors. A significantly elevated OR was present for exposure to wood preservatives. Subjective assessment by the probands indicated that exposure to some heavy metals, solvents, exhaust fumes, and carbon monoxide was significantly more frequent among patients than control subjects, but this was not confirmed by a parallel assessment of job histories according to a job exposure matrix. Patients had undergone general anesthesia and suffered severe head trauma more often than control subjects, but a dose-response gradient was not present. Patients reported a significantly larger number of amalgam-filled teeth before their illness than control subjects. The frequency of premature births and birth order did not differ between patients and control subjects. Patients reported significantly more relatives affected with PD than control subjects. These results support a role for environmental and genetic factors in the etiology of PD. PMID- 8628467 TI - Limited usefulness of electroconvulsive therapy in progressive supranuclear palsy. AB - OBJECTIVE: To perform a pilot study of the efficacy of electroconvulsive therapy (ECT) in improving motor function in progressive supranuclear palsy (PSP). BACKGROUND: Few effective treatments are available for PSP. Tricyclic antidepressants and idazoxan (which increases central norepinephrine) have shown benefits in small clinical trials, and dopaminergic therapy has been reported, anecdotally, to be beneficial. ECT exerts effects on all of these transmitter systems, possibly by inducing increased receptor sensitivity. We postulated that by sensitizing dopaminergic and noradrenergic systems, ECT might improve motor symptoms of PSP. METHODS: Five patients with clinically diagnosed PSP were evaluated before and after nine ECT treatments using the Unified Parkinson's Disease Rating Scale (UPDRS) and an apomorphine challenge to assess dopaminergic responsiveness. RESULTS: No permanent side effects were seen. Transient side effects included confusion in all patients, worsening of speech and swallowing in two, and dystonic posturing of the foot in one. One patient experienced a dramatic response (going from a completely wheelchair-bound state to independent ambulation), two were mildly improved, and two were unchanged. CONCLUSIONS: Although ECT may ameliorate motor symptoms in PSP, the long hospitalization and the significant treatment-induced confusion limit the usefulness of this technique. PMID- 8628468 TI - Risk factors for Creutzfeldt-Jakob disease: a reanalysis of case-control studies. AB - To review the evidence for risk factors of Creutzfeldt-Jakob disease (CJD), we pooled and reanalyzed the raw data of three case-control studies. The pooled data set comprised 178 patients and 333 control subjects. The strength of association between CJD and putative risk factors was assessed by computing the odds ratio as estimate of the relative risk. The risk of CJD was statistically significantly increased for subjects with a family history of CJD (odds ratio = 19.1; 95% CI 1.1 to 348.0). Further, there was a significant association between the risk of CJD and a history of psychotic disease (odds ratio = 9.9; 95% CI 1.1 to 86.1). Although not significantly increased, there was an elevated risk of CJD for subjects with a family history of dementia, a history of poliomyelitis, subjects employed as health professionals, and subjects ever exposed to cows and sheep. No association could be shown with organ meat consumption, including brain. The negative results of this reanalysis reassures the absence of a common risk factor in all CJD patients. However, the ongoing epidemiologic surveillance of CJD in several European countries may provide more evidence to exclude any environmental exposure early in childhood. PMID- 8628469 TI - Intracranial hemorrhage associated with cocaine abuse: a prospective autopsy study. AB - OBJECTIVES: To determine the incidence of cocaine abuse in cases of fatal intracranial hemorrhage and to examine potential pathophysiologic mechanisms. DESIGN: Prospective clinical, autopsy, and toxicologic evaluation of all cases of fatal non-traumatic intracranial hemorrhage examined during 1 year (April 11, 1989 to April 10, 1990) at the Connecticut Office of the Chief Medical Examiner. Autopsy examination included exhaustive histologic evaluation of cerebral vessels and parenchyma for vasculitis and other vasculopathies. RESULTS: Ten of 17 (59%) of all non-traumatic intracranial hemorrhages were associated with a positive toxicology for cocaine. Seven (70%) of these were parenchymal hemorrhages, and the remaining three (30%) were subarachnoid hemorrhages (ruptured berry aneurysms). No vasculitis or other vasculopathy was identified. CONCLUSIONS: These findings implicate cocaine use as a significant risk factor for fatal brain hemorrhage and may explain, in part, the increased incidence of hemorrhagic stroke in some drug-using cohorts. The lack of specific pathologic findings suggests that cocaine-associated intracranial hemorrhages are a consequence of the pharmacodynamic effect of cocaine and not a cocaine-induced vasculopathy. PMID- 8628470 TI - Extensive brain calcifications, leukodystrophy, and formation of parenchymal cysts: a new progressive disorder due to diffuse cerebral microangiopathy. AB - A new cerebral disorder, described in three unrelated children, has recognizable clinical, radiologic, and neuropathologic findings. The onset occurs from early infancy to adolescence with slowing of cognitive performance, rare convulsive seizures, and a mixture of extrapyramidal, cerebellar, and pyramidal signs. CT shows progressive calcifications in the basal and cerebellar gray nuclei and the central white matter. MRI reveals diffuse abnormal signals of the white matter on T2-weighted sequences. A special feature is the development of parenchymal cysts in the cerebellum and the supratentorial compartment, leading to compressive complications and surgical considerations. Neuropathologic examination of surgically removed pericystic samples reveals angiomatous-like rearrangements of the microvessels, together with degenerative secondary changes of other cellular elements. Both the anatomic findings and the course of the disease suggest a constitutional, diffuse cerebral microangiopathy resulting in microcystic, then macrocystic, parenchymal degeneration. PMID- 8628471 TI - Stroke recurrence in patients with patent foramen ovale: the Lausanne Study. Lausanne Stroke with Paradoxal Embolism Study Group. AB - Patent foramen ovale (PFO) is more common in patients with stroke than in matched controls, but the stroke mechanism and late prognosis are not well known. We studied features, coexisting causes, and recurrences of stroke in 140 consecutive patients (mean age 44 +/- 14 years) with stroke and PFO admitted to a population based primary-care center. We selected the patients from 340 patients (41%) aged < or = 60 years with acute stroke. The initial event was brain infarction in 118 patients (84%) and TIA in 22 (16%). Intracranial embolic occlusions were present on angiography or transcranial Doppler in most patients admitted within 12 hours of onset, whereas a venous source was clinically apparent in only six patients (5.5%). Pulmonary embolism, Valsalva maneuver at onset, and coagulation abnormalities were rare, but one-fourth of the patients had an interatrial septum aneurysm (ISA) that coexisted with PFO. An alternative cause of stroke was present in only 22 patients (16%), usually cardiac (atrial fibrillation, severe mitral valve prolapse, akinetic left ventricular segment). During a mean follow up of 3 years, the stroke or death rate was 2.4% per year, but only eight patients had a recurrent infarct (1.9% per year). This low rate of recurrence contrasted with the severity of initial stroke, which left disabling sequelae in one-half the patients. Multivariate analysis showed that interatrial communication, a history of recent migraine, posterior cerebral artery territory infarct, and a coexisting cause of stroke were associated with recurrence, whereas ISA and treatment type (coagulant or antiaggregant therapy, surgical closure of PFO) were not. However, given the low number of events, these findings must be taken with caution. In conclusion, our study shows that stroke associated with PFO with or without ISA is not commonly due to a coexisting cause of stroke. It is usually embolic, although a definite source cannot often be demonstrated. The presenting stroke is often severe, but recurrence is uncommon. The demonstration of factors associated with a higher risk of recurrence in subgroups of patients is critical for the long-term management of these patients. PMID- 8628472 TI - Botulinum toxin type A in the treatment of upper extremity spasticity: a randomized, double-blind, placebo-controlled trial. AB - Spasticity is a disorder of excess muscle tone associated with CNS disease. We hypothesized that botulinum toxin, a neuromuscular blocking agent, would reduce tone in spastic muscles after stroke. This randomized, double-blind, placebo controlled, multicenter clinical trial evaluated the safety and efficacy of botulinum toxin type A (BTXA) in the treatment of chronic upper limb spasticity after stroke. Thirty-nine patients received IM injections of a total dose of either 75, 150, or 300 units of BTXA or placebo into the biceps, flexor carpi radialis, and flexor carpi ulnaris muscles. At baseline, patients demonstrated a mean wrist flexor tone of 2.9 and elbow flexor tone of 2.6 on the Ashworth Scale (0 to 4). Treatment with the 300-unit BTXA dose resulted in a statistically and clinically significant mean decrease in wrist flexor tone of 1.2 (p = 0.028), 1.1 (p = 0.044), and 1.2 (p = 0.026) points and elbow flexor tone of 1.2 (p = 0.024), 1.2 (p = 0.028), and 1.1 (p = 0.199) at weeks 2, 4, and 6 postinjection. In the placebo group, tone reduction at the wrist was 0.3, 0.2, and 0.0 and at the elbow was 0.3, 0.3, and 0.6 at weeks 2, 4, and 6 postinjection. BTXA groups reported significant improvement on the physician and patient Global Assessment of Response to Treatment at weeks 4 and 6 postinjection. There were no serious adverse effects. In this 3-month study, BTXA safely reduced upper extremity muscle tone in patients with chronic spasticity after stroke. PMID- 8628473 TI - Linkage and mutation analysis of Charcot-Marie-Tooth neuropathy type 2 families with chromosomes 1p35-p36 and Xq13. AB - A locus for autosomal dominant Charcot-Marie-Tooth disease type 2 (CMT2A) was assigned by linkage analysis to chromosome 1p35-p36. We examined 11 unrelated CMT2 families for linkage to CMT2A using short tandem repeat (STR) polymorphisms. Only one family showed suggestive evidence for linkage to 1p35-p36. Further, because of an overlap in electrophysiologic data between CMT2 and CMTX female patients, we screened 6 of 11 CMT2 families compatible with dominant X-linkage for mutations in the connexin 32 (Cx32) gene at Xq13. There was a Cx32 mutation in one family, whereas another family showed suggestive evidence for Xq13 linkage upon analysis with STR polymorphisms. Our results suggest that the CMT2A locus is a minor locus for CMT2, additional linkage studies are needed to localize other CMT2 loci, and Cx32 mutations may be the underlying genetic defect in some CMT2 families. PMID- 8628474 TI - Autosomal recessive hereditary motor and sensory neuropathy with focally folded myelin sheaths: clinical, electrophysiologic, and genetic aspects of a large family. AB - We describe 10 patients from a large family with early onset motor and sensory neuropathy. Six were still living at the time of the study. In all cases, early motor milestones had been achieved. Mean age at onset of symptoms was 34 months; these included progressive distal and proximal muscle weakness of lower limbs. Pes equinovarus developed in all patients during childhood. Slight facial weakness was present in four patients, and one of them also had bilateral facial synkinesia. Intellectual function was normal in all cases. There was no evidence of thickened peripheral nerves. All three adult patients (mean age, 27 years) were seriously handicapped and wheelchair-bound. Death occurred in the fourth to fifth decade of life and the duration of the illness varied from 27 to 39 years. Motor nerve conduction velocities ranged from 15 to 17 m/sec in the upper limbs of the youngest patients, and were undetectable in the adult patients. Sensitive action potentials were almost always absent. In all patients, auditory evoked potentials showed abnormally delayed interpeak I-III latencies. The most prominent pathologic finding was a highly unusual myelin abnormality consisting of irregular redundant loops and folding of the myelin sheath. The genealogic study gave strong evidence of autosomal-recessive inheritance. The molecular analysis failed to demonstrate either duplication in the chromosome 17p11.2-12, point mutations in the four exons of the PMP-22 (17p11.2) and the six exons of the Po (1q21-q25) genes, or linkage to chromosome 8q13-21.1. PMID- 8628475 TI - Intranuclear inclusions in oculopharyngeal muscular dystrophy among Bukhara Jews. AB - We studied, by electron microscopy, muscle biopsies from seven patients with autosomal dominant oculopharyngeal muscular dystrophy (OPMD) belonging to the recently described Bukhara-Jewish cluster. Typical tubulofilamentous intranuclear inclusions (INI) of 8.5 nm outer diameter were present in all cases. The INI were observed in 4.5 +/- 1.8% of the nuclei in five patients. In the other two, they occurred in 9.5 +/- 0.5% of the nuclei and often occupied a larger nuclear area. These two patients, offspring of intermarriage between affected cousins, had an unusually severe form of OPMD beginning in their early 30s, suggesting homozygote state. Our results confirm that INI are pathognomonic for OPMD and suggest that their frequency may be quantitatively related to the number of abnormal DNA copies. PMID- 8628476 TI - Multiple mitochondrial DNA deletions associated with autosomal recessive ophthalmoplegia and severe cardiomyopathy. AB - Six patients in two unrelated families from the eastern Arabian peninsula presented with childhood-onset progressive external ophthalmoplegia (PEO), mild facial and proximal limb weakness, and severe cardiomyopathy requiring cardiac transplantation. Muscle biopsies showed ragged-red and cytochrome c oxidase negative fibers. The activities of several complexes in the electron-transport chain were decreased and Southern blot analysis showed multiple mtDNA deletions. The apparent autosomal-recessive inheritance and the association with cardiomyopathy distinguish this syndrome from autosomal-dominant PEO with multiple mtDNA deletions. The combination of autosomal-recessive PEO, cardiomyopathy, and multiple mtDNA deletions appears to be another disease due to a defect of communication between the nuclear and mitochondrial genomes. PMID- 8628477 TI - A MERRF/PEO overlap syndrome associated with the mitochondrial DNA 3243 mutation. AB - We describe a two-generation family with combined clinical features of myoclonic epilepsy, progressive external ophthalmoplegia (PEO), proximal myopathy, pigmentary retinopathy, progressive deafness, basal ganglia calcification, and ragged-red fibers in a muscle biopsy specimen. One family member died unexpectedly at age 22 years. The molecular tests revealed an A-to-G transition at nucleotide position 3243 of the mitochondrial tRNA(Leu(UUR)) gene. No one in this family had stroke-like episodes. Although the propositus (a 28-year-old woman) had a significant number of white hairs, the percentage of mutant mtDNA in white-hair roots was not different from that in the colored-hair roots. Our findings suggest that the 3243 mutation can be associated with mixed clinical features of myoclonic epilepsy with ragged-red fibers (MERRF) and PEO and that a preferential increase in the levels of the mutant mtDNA is not related to graying of hair, and hence to the hypothesized production of premature aging of cells. PMID- 8628478 TI - Late-onset muscle weakness in partial phosphofructokinase deficiency: a unique myopathy with vacuoles, abnormal mitochondria, and absence of the common exon 5/intron 5 junction point mutation. AB - Three patients (ages 51, 59, and 79) from two generations of an Ashkenazi Jewish family had partial (33% activity) phosphofructokinase (PFK) deficiency that presented with fixed muscle weakness after the age of 50 years. MR spectroscopy revealed accumulation of phosphomonoesters during exercise. Muscle biopsy showed a vacuolar myopathy with increased autophagic activity and several ragged-red and cytochrome c oxidase-negative fibers. The older patient, age 79 at biopsy, had several necrotic fibers. Electron microscopy revealed subsarcolemmal and intermyofibrillar glycogen accumulation and proliferation of mitochondria with paracrystalline inclusions, probably related to reduced availability of energy due to impaired glycolysis. The common point mutation of exon 5/intron 5 junction seen in Jewish Ashkenazi patients with PFK deficiency was excluded. We conclude that late-onset fixed muscle weakness occurs in partial PFK deficiency and it may represent the end result of continuing episodes of muscle fiber destruction. Partial enzyme deficiency in two successive generations suggests a unique molecular mechanism. PMID- 8628479 TI - Respiratory-chain enzyme activities in isolated mitochondria of lymphocytes from untreated Parkinson's disease patients. Grupo-Centro de Trastornos del Movimiento. AB - We studied respiratory-chain enzyme activities in lymphocyte mitochondria from 36 untreated Parkinson's disease (PD) patients and in 30 age- and sex-matched healthy controls. The respiratory-chain enzyme activities did not differ significantly between patients and controls. Moreover, no patient showed respiratory-chain enzyme levels below normal range. Values for activities of complexes in the PD group did not correlate with age at onset, duration, scores of the Unified Parkinson's Disease Rating scales, or Hoehn and Yahr staging. These results suggest that the presence of defects of respiratory-chain complexes could depend on methodologic aspects, and that determinations of respiratory chain enzymes in cell homogenates are not generally appropriate for evaluating abnormal mitochondrial dysfunction, especially when the amount of the specific enzyme is relatively low, as is the case of blood cells. In addition, the method of measuring complex I activity is critical for evaluating the results. In conclusion, our finding of normal mitochondrial function in lymphocyte mitochondria suggests that this tissue cannot be used to develop a diagnostic test for PD. PMID- 8628480 TI - A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene. AB - We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases. PMID- 8628481 TI - Prenatal diagnosis of Duchenne muscular dystrophy using a single fetal nucleated erythrocyte in maternal blood. AB - We developed a method that allows prenatal diagnosis of Duchenne muscular dystrophy using a single nucleated erythrocyte (NRBC) isolated from maternal blood. Maternal blood was obtained at 8 to 20 weeks of gestation. NRBCs were separated with Percoll using a discontinuous density gradient method and then collected by micromanipulator under microscopic observation. The entire genome of a single cell was amplified by primer extension preamplification (PEP). Sex was determined from a small aliquot of the PEP reaction. After an NRBC was determined to be male and confirmed to be of fetal origin, dystrophin exons 4, 8, 12, 45, 48, 50, and 51 were determined from the same PEP reaction. This diagnostic method using maternal blood is safer than amniocentesis or cordocentesis and can be applied to other X-linked diseases. PMID- 8628482 TI - Disruption of muscle basal lamina in congenital muscular dystrophy with merosin deficiency. AB - We studied three new cases of congenital muscular dystrophy (CMD) with homogeneous clinical and laboratory features, represented by congenital muscle hypotonia and weakness, early contractures, elevated serum CK, and dystrophic pattern at muscle biopsy, without clinical impairment of CNS. Merosin, the laminin isoform that contains the alpha 2 heavy chain, was absent in muscle fibers of all the patients by immunohistochemistry and by immunoblot. By electron microscopy, we found a severe disruption of muscle fiber basal lamina, but not of blood vessel basal lamina, which contains the laminin alpha 1 heavy chain isoform. This disruption may play a key role in the degeneration of muscle fibers and in the abnormal proliferation of connective tissue seen in CMD. PMID- 8628483 TI - Central motor loop oscillations in parkinsonian resting tremor revealed by magnetoencephalography. AB - A variety of clinical and experimental findings suggest that parkinsonian resting tremor results from the involuntary activation of a central mechanism normally used for the production of rapid voluntary alternating movements. However, such central motor loop oscillations have never been directly demonstrated in parkinsonian patients. Using magnetoencephalography, we recorded synchronized and tremor-related neuromagnetic activity over wide areas of the frontal and parietal cortex. The spatial and temporal organization of this activity was studied in seven patients suffering from early-stage idiopathic Parkinson's disease (PD). Single equivalent current dipole (ECD) analysis and fully three-dimensional distributed source solutions (magnetic field tomography, MFT) were used in this analysis. ECD and MFT solutions were superimposed on high-resolution MRI. The findings indicate that 3 to 6 Hz tremor in PD is accompanied by rhythmic subsequent electrical activation at the diencephalic level and in lateral premotor, somatomotor, and somatosensory cortex. Tremor-evoked magnetic activity can be attributed to source generators that were previously described for voluntary movements. The interference of such slow central motor loop oscillations with voluntary motor activity may therefore constitute a pathophysiologic link between tremor and bradykinesia in PD. PMID- 8628484 TI - Abnormal cortical motor excitability in dystonia. AB - To assess the excitability of the motor system, we studied 11 patients with task specific dystonia and 11 age-matched normal subjects. The dominant side was affected in nine of the patients. We delivered transcranial magnetic stimuli at different stimulus intensities and with different levels of muscle facilitation to the side contralateral to the side of electromyographic recording, and recorded motor evoked potentials (MEPs) from the flexor carpi radialis muscles bilaterally. The threshold intensity for eliciting MEPs at rest did not differ between patients and normal subjects. We compared the affected side in patients with the dominant side in normal subjects. With facilitation, the percentage of the area of the MEP to the M wave (MEP area%) was similar in both groups at low stimulus intensities, but with increasing stimulus intensity the increase in the MEP area% was greater in patients than in normal subjects (ANOVA, p < 0.001). The increase in MEP area% was similar in both groups with increasing facilitation levels. The duration of the silent period was similar in patients and normal subjects. We conclude that cortical motor excitability is increased in dystonia. PMID- 8628485 TI - Characterization of postexercise facilitation and depression of motor evoked potentials to transcranial magnetic stimulation. AB - We studied the effects of exercise on motor evoked potentials (MEPs) to transcranial magnetic stimulation (TMS) and transcranial electrical stimulation (TES). Subjects performed 30-second periods of isometric exercise of the extensor carpi radialis until fatigue, which was defined as the inability to maintain half maximum force. The amplitude of MEPs to TMS recorded from the resting muscle after each exercise period was on average more than twice the pre-exercise value (postexercise MEP facilitation). After fatigue occurred, the MEP amplitudes were approximately 60% of the pre-exercise value (postexercise MEP depression). There was a gradual recovery of the depressed MEPs to pre-exercise values over several minutes of rest. Postexercise MEP facilitation was constant when exercise intensity ranged from 10 to 50% of maximum voluntary contraction and it decayed to baseline over several minutes after the end of exercise. There was no postexercise MEP facilitation to TES. We hypothesize that both postexercise MEP facilitation and MEP depression are due to intracortical mechanisms. PMID- 8628486 TI - Dose-response curve of human extensor digitorum brevis muscle function to intramuscularly injected botulinum toxin type A. AB - To develop a human model for measuring the effect of intramuscularly injected botulinum toxin, we injected both extensor digitorum brevis (EDB) muscles in 13 healthy volunteers with seven varying doses of botulinum toxin type A. We measured, pre- and postinjection, EDB M-wave amplitude, area, and mean rectified voltage (MRV) (obtained during maximal voluntary muscle activation). There was a logarithmic-appearing dose-response relationship between increasing doses of botulinum toxin and decline in EDB M-wave amplitude, area, and MRV. The decline was incrementally less at higher doses of toxin and appeared to level off at a maximal effect of 85 to 90% decrement from baseline (85 to 90% "paralysis") at 15 to 20 units. The peak toxin effect was present on day 6 postinjection. Measurement of EDB M-wave amplitude, area, and MRV is a simple objective method for quantifying the onset and degree of human muscle "paralysis" following botulinum-toxin injection. PMID- 8628487 TI - Activity-dependent conduction in single motor units. AB - Vertebrate sensory and motor axons vary in their responses to submaximal stimuli as a function of time since prior activation. When two equal but submaximal stimuli are presented in pairs, the response to the second stimulus may be greater or less than the response to the first stimulus, depending on the interstimulus interval (ISI). We studied both the supernormal period (ISI between 6 and 25 msec) and the subnormal period (ISI between 25 and 100 msec) under conditions where only single motor axons were stimulated. Twenty single motor units from eight normal subjects were studied. The behavior of single units was very similar to that observed in compound motor action potentials, with the supernormal period lasting approximately 20 msec, followed by a subnormal period lasting at least 80 msec. Surprisingly, a supernormal period could be evoked by a stimulus that did not produce a response in the motor unit being studied; however, the presence of subnormality was dependent on an action potential being generated in response to the first stimulus. Based on these results, we conclude that the supernormal period does not require the opening of voltage-dependent ion channels, in contrast to the later occurring subnormal period. PMID- 8628488 TI - Calcium channel blockers and transmitter release at the normal human neuromuscular junction. AB - Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals. PMID- 8628489 TI - Cortical projections to spinal motoneurons: changes with aging and amyotrophic lateral sclerosis. AB - Peristimulus time histograms (PSTHs) of discharging single motor units, recorded from the extensor digitorum communis (EDC) during randomly applied cortical magnetic stimulation, were obtained in 42 normal subjects aged 24 to 83 years and 42 patients with amyotrophic lateral sclerosis (ALS) aged 37 to 84 years. Normal subjects had an early period of increased firing probability occurring at about 20 msec poststimulus, reflecting an underlying compound excitatory postsynaptic potential (EPSP) induced by fast-conducting, descending volleys of the corticomotoneuronal core facilitating the single spinal motoneuron. There was an age-dependent, linear decline in the amplitude of the EPSP (r = 0.673). We estimated that by age 50 years about 35% of corticomotoneurons are lost or nonfunctioning in normal controls. Compared with age-matched controls, the EPSP in most patients with ALS was reduced, and it was unmeasurable in six. We postulate this reflects a loss of corticomotoneurons. Seven (16.7%) patients phenotypically the same as the others had EPSPs that were larger than age predicted values. This may reflect glutamate-induced excitotoxicity in a subset of ALS. In a single patient with chronic spinal muscular atrophy the EPSP was normal. PMID- 8628490 TI - Intensity dependence of auditory evoked potentials is pronounced in migraine: an indication of cortical potentiation and low serotonergic neurotransmission? AB - Migraine is associated with stimulus hypersensitivity, increased evoked cortical responses, and abnormal 5-HT levels in peripheral blood. We studied cortical auditory evoked potentials (AEPs) between attacks in 35 patients suffering from migraine without aura (MO, n = 25) or with aura (MA, n = 10) and in 25 healthy volunteers. Binaural tones were delivered at 40, 50, 60, and 70 dB sensation level (SL) in a pseudorandomized order. The intensity dependence of the auditory N1-P2 component was significantly greater in MO (p = 0.003) and MA (p = 0.02) patients than in healthy controls, resulting in a much steeper amplitude/stimulus intensity function slope. When three sequential blocks of 40 averaged responses were analyzed at the 40- and 70-dB SL intensities, N1-P2 amplitude decreased in second and third blocks at both intensities in controls, but increased in migraineurs, a difference that was significant in both blocks for the 70-dB SL stimulus. The strong interictal dependence of AEPs on stimulus intensity may thus be due to potentiation (instead of habituation) of the response during repetition of the high-intensity stimulation. In concordance with previous studies of visual evoked potentials, these results confirm that migraine is characterized between attacks by an abnormality of cortical information processing, which might be a consequence of low 5-HT transmission and favor cortical energy demands. PMID- 8628491 TI - Frequency of MBP and MBP peptide-reactive T cells in the HPRT mutant T-cell population of MS patients. AB - Somatic mutation as an index of in vivo T-cell amplification is a powerful tool to analyze the specificity and size of the autoreactive T-cell repertoire. Using this strategy, we determined the precursor frequency of T cells reactive to myelin basic protein (MBP) and overlapping MBP peptides spanning regions p84-168 in patients with MS and controls in the HPRT mutant T-cell population. Among 19 MS patients, nine had estimatable frequencies to MBP or MBP peptides, p93-112, p124-142 and p143-168 in the HPRT mutant T-cell population. Only one of the 10 controls showed measurable frequency to MBP in the HPRT mutant T-cell population. These studies suggest that increased frequency of T cells reactive to MBP and MBP peptides might indicate putative disease-related epitopes in MS. PMID- 8628492 TI - Clinical and radiologic correlates of a novel T lymphocyte gamma-interferon activated Ca2+ influx in patients with relapsing-remitting multiple sclerosis. AB - T lymphocytes are the main cellular mediators in MS pathogenesis, and their activity is modulated by a complex cytokine network in which gamma-interferon (gamma-IFN) is considered essential. We have recently identified a new transplasmalemma Ca2+ influx activated by gamma-IFN in T lymphocytes (mainly CD4+) from patients with MS that makes T cells more susceptible to proliferation. To define the possible role of this Ca2+ influx as a marker of disease activity, we correlated its appearance with clinical and MRI findings in a cross-sectional study of 67 patients with relapsing-remitting MS (RR-MS). We also conducted a short-term longitudinal evaluation (every 15 days over a 5- to 7-month period) in three of the RR-MS patients. Sixty-five percent of all clinically active RR-MS patients showed the gamma-IFN-activated Ca2+ influx. However, positivity was higher in the first week (78%) after the onset of a clinical exacerbation than the second (57%) and third (44%) weeks. The influx was also detected in 45% of clinically stable RR-MS patients, 30% of RR-MS patients with a "benign" course of the disease, 14% of the other active autoimmune or neurologic disease patients, and 9% of healthy subjects (RR-MS versus control subjects, p < 0.001). Brain-MRI gadolinium-enhancing lesions were more frequently found in influx-positive (72%) than in influx-negative (47%) patients (p < 0.005). In the longitudinal study, we recorded five intracellular Ca2+ ([Ca2+]i) elevations and three clinical attacks (one per patient). A peak increase of [Ca2+]i due to the gamma-IFN-activated Ca2+ influx always preceded the clinical attacks from 4 to 45 days and coincided to MRI evidence of inflammation. [Ca2+]i had returned to baseline levels by the time of the onset of two clinical attacks. This finding may account for the lack of detection of the gamma-IFN-activated Ca2+ influx in some RR-MS patients during the first week after clinical onset. The strong association between the influx and clinical and MRI evidence of disease activity supports its role in the early phases of cellular immune activation leading to demyelination in MS. The detection of [Ca2+]i elevations due to the gamma-IFN-activated Ca2+ influx may represent a valuable prognostic marker of disease activity and may be useful to monitor immunologic studies of MS patients in future clinical trials. PMID- 8628493 TI - Topography of interictal glucose hypometabolism in unilateral mesiotemporal epilepsy. AB - We mapped the regional cerebral glucose metabolism (rCMRGlu) in 20 patients suffering from medically refractory focal epilepsy of either left or right mesiotemporal origin (mTLE) during resting wakefulness. After temporal lobectomy, histology demonstrated hippocampal sclerosis in 18 patients. Pixel-by-pixel comparisons with healthy control subjects showed significant (p < 0.001) depressions of the mean rCMRGlu ipsilateral to the epileptic focus in the mesiotemporal region, including the hippocampus and the parahippocampal gyrus and middle temporal gyrus. Additional remote rCMRGlu depressions occurred bilaterally in the fronto-orbital cortex and ipsilaterally in the posterior insula and the thalamus. Patients with left-sided mTLE had additional rCMRGlu depressions in the left inferior frontal gyrus (Broca's region) and superior temporal gyrus at the parietotemporal junction, whereas corresponding rCMRGlu depressions were not present in patients with right mTLE. Neuropsychological testing showed impaired verbal fluency, verbal intelligence, and verbal memory in the left mTLE patients. Correlations of the specific mean rCMRGlu depressions and the neuropsychological deficits suggest that impaired language functions in patients with left mTLE could result from functional changes beyond the temporal lobe. PMID- 8628494 TI - Levodopa-induced changes in synaptic dopamine in patients with Parkinson's disease as measured by [11C]raclopride displacement and PET. AB - Changes in striatal binding of [11C]raclopride, a dopamine D2 receptor antagonist, induced by acute levodopa administration, were evaluated with PET in 10 patients with idiopathic Parkinson's disease (PD). The patients were scanned on two occasions: drug-free and 15 minutes after a 5-minute intravenous infusion of 3 mg/kg levodopa. Levodopa administration produced reductions in striatal [11C]raclopride uptake index with a rostrocaudal gradient. The most pronounced reduction was found in the posterior putamen (to 82% of baseline), followed by the anterior putamen (to 88% of baseline) and the caudate nucleus (to 94% of baseline). The magnitude of [11C]raclopride uptake index reduction correlated with drug-free disability. Moreover, in four hemiparkinsonian patients, a reduction in [11C]raclopride uptake index was measured in the putamen contralateral to the parkinsonian symptoms. The present results demonstrate a positive correlation between striatal dopaminergic nerve-terminal deficiency and the capacity for levodopa to increase synaptic dopamine and displace [11C]raclopride binding, which corresponds to an accelerated amine turnover in dopamine-depleted striatal tissue. We therefore suggest that dopaminergic degeneration in PD is paralleled by a progressive acceleration of amine turnover. This mechanistic consequence of nigrostriatal degeneration, the selective restoration of synaptic dopaminergic neurotransmission in denervated striatal subregions, may explain the effectiveness of levodopa in producing symptomatic benefits in early PD. However, we also suggest that in the vastly denervated striatum, as in advanced PD, an excessive acceleration of amine turnover results in swings in levodopa-induced synaptic dopamine levels that are far beyond normal. This phenomenon most likely plays a key role in the pathogenesis underlying the development of motor-response complications in PD. PMID- 8628495 TI - Synthetic peptide derived from the Bordetella pertussis bacterium reduces infarct volume after transient middle cerebral artery occlusion in the rat. AB - We explored the therapeutic potential of a peptide (F20) derived from the filamentous hemagglutinin of Bordetella pertussis in a model of ischemic cell injury after transient (2 hours) middle cerebral artery (MCA) occlusion in the rat. Animals were divided into two groups-(1) F20 peptide group: rats (n = 11) were subjected to 2 hours of transient MCA occlusion, and F20 peptide was administered intravenously (50 nmol) at 0 hours of reperfusion and intraperitoneally (150 nmol/dose) at 2, 4, 6, 8, 22, and 30 hours of reperfusion; (2) control group: rats (n = 10) were administered peptide F23 (a scrambled version of peptide F20) with the same experimental protocol as the F20 peptide group. Forty-six hours after reperfusion, animals were sacrificed, and brain tissue was stained with triphenyltetrazolium chloride for evaluation of tissue damage. To measure neutrophil numbers in ischemic tissue, myeloperoxidase (MPO) immunostaining was performed on a coronal cerebral section in each animal. There was a significant reduction of ischemic infarct volume (36%, p < 0.05) in the F20 group of animals compared with the F23 group. The area of the ischemic lesion was highly correlated with the numbers of the immunoreactive MPO cells (r = 0.78, p < 0.001). The data demonstrate that the F20 peptide significantly reduces infarct volume and intraparenchymal neutrophil numbers after transient MCA occlusion. PMID- 8628496 TI - Relationship of the Tufts Quantitative Neuromuscular Exam (TQNE) and the Sickness Impact Profile (SIP) in measuring progression of ALS. SSNJV/CNTF ALS Study Group. AB - The Tufts Quantitative Neuromuscular Exam (TQNE) is a standardized tool for measuring muscle strength and pulmonary function in patients with amyotrophic lateral sclerosis (ALS). We describe the relationship of TQNE scores to functional disability and health-related quality of life as measured by the Sickness Impact Profile (SIP) in 524 ALS patients. There was a significant relationship (p < 0.0001) between TQNE and SIP scores, both in cross section and over time. TQNE scores strongly relate to ALS patients' quality of life and ability to perform activities of daily living. PMID- 8628497 TI - Elevated serum levels of manganese superoxide dismutase in polymyositis and dermatomyositis. AB - We studied serum concentrations of manganese superoxide dismutase (Mn SOD) and copper-zinc superoxide dismutase (Cu/Zn SOD) in 22 patients with polymyositis and dermatomyositis (PM/DM), 87 patients with four types of muscular dystrophy, 20 patients with amyotrophic lateral sclerosis, and 15 patients with collagen vascular diseases (CVD). Serum levels of Mn SOD were increased only in the patients with PM/DM and CVD, and the elevation was more prominent in those with PM/DM. Levels of Cu/Zn SOD were slightly elevated in some patients with PM/DM and Duchenne muscular dystrophy. In patients with PM/DM, the change in Mn SOD levels corresponded to disease activity as closely as or more closely than those of creatine kinase. The results indicate that serum Mn SOD may be a useful clinical marker for PM/DM. PMID- 8628498 TI - Necrotizing myopathy with pipestem capillaries and minimal cellular infiltration: a case associated with cutaneous signs of dermatomyositis. AB - In 1991, Emslie-Smith and Engel described a distinct form of idiopathic inflammatory myopathy they called "necrotizing myopathy with pipestem capillaries, microvascular deposition of the complement membrane attack complex (MAC) and minimal cellular infiltration." We describe a patient with exercise dependent painful myopathy related to a necrotizing myopathy with pipestem capillaries in whom mild cutaneous signs of dermatomyositis were detected 7 years after onset and who subsequently developed multiple cerebral infarcts. PMID- 8628499 TI - Cure of a solitary brainstem abscess with antibiotic therapy: case report. AB - A solitary brainstem abscess is uncommon. The use of antibiotics and surgical aspiration or excision of a brainstem abscess has resulted in survivors. Survival after treatment of a brainstem abscess with antibiotics alone has been reported rarely, and we present the eighth study case. The patient made an excellent recovery after 12 weeks of antibiotics, with 8 weeks completed as an outpatient. Medical management of a solitary brainstem abscess in an immunocompetent patient is feasible and may result in a complete cure with antibiotics only. Completion of IV antibiotics as an outpatient is viable and cost-effective in selected patients. PMID- 8628500 TI - Inflammatory trigeminal sensory neuropathy mimicking trigeminal neurinoma. AB - Idiopathic trigeminal sensory neuropathy is a benign disorder. We report two patients with transient MRI abnormalities, suggesting transient inflammation of the trigeminal nerve caused temporary facial sensory symptoms. PMID- 8628501 TI - Felbamate-associated fatal acute hepatic necrosis. AB - Thirty-six cases of hepatic toxicity associated with felbamate therapy have been collected by the Food and Drug Administration. Five patients died. We describe a case of massive acute hepatic necrosis and death within 40 days of initiation of felbamate therapy for a generalized tonic-clonic seizure disorder. We describe the clinical and histopathologic features. PMID- 8628503 TI - Tactile spatial acuity at the human fingertip and lip: bilateral symmetry and interdigit variability. AB - We used the spatial threshold for discrimination of grating orientation to assess tactile spatial acuity at the lower lip and each fingertip, bilaterally, in seven normal human subjects. We confirmed previous findings that: (1) this test is highly reliable, and (2) acuity at the lip is substantially higher than at the fingertips. In addition, we showed that: (1) acuity does not differ significantly between right and left sides, and (2) among fingertips, acuity is lowest at the fifth digit and comparable on the other digits. PMID- 8628502 TI - Human brain GABA levels rise after initiation of vigabatrin therapy but fail to rise further with increasing dose. AB - Using 1H spectroscopy, we measured occipital lobe GABA levels serially in 18 patients enrolled in an ongoing open label trial of vigabatrin. Brain GABA levels were elevated twofold in patients taking vigabatrin (3 to 4 g/d) compared with nonepileptic subjects. Serial measurements suggested that brain GABA rose in proportion to vigabatrin dose up to 3 g/d. Doubling the dose from 3 to 6 g/d failed to increase brain GABA further. Serial measurements on three patients taking 6 g/d showed a gradual decrease in brain GABA in two patients over 1 to 2 years of treatment. These observations suggest that GABA synthesis may decrease at high GABA levels. PMID- 8628504 TI - Gowers' memory. PMID- 8628505 TI - Consensus statement on the definition of orthostatic hypotension, pure autonomic failure, and multiple system atrophy. The Consensus Committee of the American Autonomic Society and the American Academy of Neurology. PMID- 8628506 TI - Report of the AAN Task Force on access to health care: the effect of no personal health insurance on health care for people with neurologic disorders. Task Force on Access to Health Care of the American Academy of Neurology. AB - Access to medical care is limited for people with no health insurance. In the United States, an estimated 31 to 41 million people under age 65 have no health insurance. Among the uninsured, an estimated 340,000 new cases of neurologic disorders occur annually. The Task Force on Access to Health Care of the Academy analyzed data from four nationwide health surveys to describe the national population of people with neurologic disorders (PWND) by insurance status and to examine access to care, utilization of services, and expenses for health care of PWND. Health insurance status significantly affected access to and utilization of health care services. Compared with insured PWND, the uninsured less often had a usual source of medical care, saw a particular doctor, or visited a neurologist. The uninsured had fewer doctor's office visits and fewer hospital admissions than privately insured PWND. In the doctor's office they got fewer tests, fewer referrals for therapies, but more medications. In the hospital they received more diagnostic and therapeutic procedures overall, but those with cerebrovascular disease received fewer angiograms and endarterectomies. National health care reform may improve access to care for PWND if they are equitably included in the new systems. However, neurologists should assertively advocate for the needs of PWND to have adequate insurance and appropriate access to neurologic consultations, neurologic tests, and treatments. PMID- 8628507 TI - Ethics education in neurology residency programs: results of a survey. PMID- 8628508 TI - Apolipoprotein E allele in centenarians. PMID- 8628509 TI - A simple and efficient method for apolipoprotein E genotype determination. PMID- 8628510 TI - Misdiagnosis revealed by genetic linkage analysis in a family with Wilson disease. PMID- 8628511 TI - Anti-Yo-associated paraneoplastic cerebellar degeneration in a man with adenocarcinoma of unknown origin. PMID- 8628512 TI - Post-concussion syndrome. PMID- 8628513 TI - Mild traumatic brain injury. PMID- 8628514 TI - AAMI. PMID- 8628515 TI - The human primary motor cortex. PMID- 8628516 TI - Motor neglect: what do we mean? PMID- 8628517 TI - Lewy body disease in a patient with REM sleep disorder. PMID- 8628518 TI - Polyneuropathy in the mtDNA base pair 3243 point mutation. PMID- 8628520 TI - Hormone therapy. PMID- 8628519 TI - Polyneuropathy in the mtDNA base pain 3243 point mutation. PMID- 8628521 TI - Cough headache and the competency of jugular venous valves. PMID- 8628522 TI - Epilepsy in porphyria. PMID- 8628523 TI - Rhabdomyolysis and the acquired immunodeficiency syndrome. PMID- 8628524 TI - Prevention of chronic obstructive pulmonary disease: a challenge for the health professions. PMID- 8628525 TI - Implementing tuberculosis control guidelines in a hospital environment. AB - AIM: This review assessed the effectiveness and results of a programme implementing the 1992 Tuberculosis Control Guidelines to hospital staff at Green Lane and National Women's hospitals. METHODS: All staff tuberculosis surveillance and patient contact tracing contacts carried out by the occupational health unit and the resultant outcomes were assessed. RESULTS: A higher risk of Mantoux conversion does exist in hospital staff. These conversions occur in nonhigh risk occupational groups as well as the more recognized at risk groups. CONCLUSIONS: The 1992 guidelines proved impracticable to administer effectively and were resource intensive. Hospital staff form a distinct, well educated occupational group who are able to make their own risk assessments about the lifetime risks of tuberculosis infection. The British Thoracic Society Guidelines for screening healthcare workers make a practical surveillance programme. Tuberculosis remains a risk for healthcare workers and the emphasis of any staff programme should broaden from surveillance aimed at identifying primary infection having occurred to include improved environmental measures, work practices and the use of personal protection to reduce the work related exposure to the mycobacterium. PMID- 8628526 TI - Management of the wheezy infant. The Respiratory Committee of the Paediatric Society of New Zealand. PMID- 8628527 TI - Did Cook's sailors have Tapanui 'flu? --chronic fatigue syndrome on the Resolution. AB - The 1982 publication of the Resolution journal of Johann George Reinhold Forster provided justification for his recognition as a scientist, and gave a remarkable insight into his character. It also included an account of an illness suffered by many of the sloop's crew, including Forster, after a period ashore at Queen Charlotte Sound. The symptoms of the illness were remarkably similar to those now clustered as the chronic fatigue syndrome. PMID- 8628528 TI - Sexual contacts between doctors and patients. PMID- 8628529 TI - "Unavailable" and death certificates. PMID- 8628530 TI - A dilemma for clinicians. PMID- 8628531 TI - The incidence of fetal alcohol syndrome in New Zealand. PMID- 8628532 TI - The use of short acting calcium channel blockers. PMID- 8628533 TI - Cot death and cot mattresses. PMID- 8628534 TI - Education may help reduce Yersinia sepsis from blood transfusion. PMID- 8628535 TI - New Zealand melanoma research group. PMID- 8628536 TI - Trends in antihypertensive prescribing. AB - AIM: To identify trends in the prescribing of antihypertensive medications and measure the changes in government and patient expenditure resulting from any identified change. METHODS: The computerised records of 16 069 patients from six Otago practices from 1991-3 were examined. Those patients prescribed any antihypertensive medication in all 3 years were selected for investigation. The antihypertensives prescribed were assigned to one of seven classes. The cohort was then divided into two groups; those remaining on the same class of medication for the three years and those changing medication class at any stage. Reasons for any change were identified. The direct costs of the prescribing decisions taken were evaluated. RESULTS: 914 patients were prescribed antihypertensive in all 3 years. Of these 579 (63.3%) remained on the same class of medication, while 335 (36.7%) changed class. A clinical reason was identified for medication change class. A clinical reason was identified for medication change in 98% of cases available for examination. There was no significant shift in expenditure for those remaining on the same medication, while costs for those remaining on the same medication, while costs increased by 20.6% for those changing. CONCLUSION: For this cohort increased expenditure on antihypertensive was driven by those changing medication. Although these changes were prompted by clinical reasons, better health outcomes for patients cannot be assumed due to lack of objective indicators. PMID- 8628538 TI - Acute lymphoblastic leukaemia: the Wellington experience 1980-92. AB - AIMS: The study was designed to compare two populations of children with acute lymphoblastic leukaemia treated in the Wellington regional oncology unit over two six year periods, 1980-6 and 1986-92 when two different treatment protocols were used. METHODS: Fifty-eight cases were identified from the children's cancer registry and the relevant data collected from the hospital records. Overall survival and disease free survival in the two populations were compared. RESULTS: There was a significant difference for both outcomes between the two study populations, those treated prior to August 1986 having an actuarial survival of 53% and a disease free survival of 47% compared with 93% and 88% in those children treated after August 1986 (p=0.01 and 0.001 respectively). This was not explained by differences in sex, age, area of residence at diagnosis or disease risk. CONCLUSIONS: Increased intensity and duration of the induction and consolidation phase of treatment has significantly improved outcome for standard and high risk disease in children who have received the intensive phases of this treatment at a regional paediatric oncology centre. Outcome is not compromised if children receive the less intensive maintenance therapy close to their homes at the district or base hospital and close links are maintained with the regional unit. PMID- 8628537 TI - Schistosomiasis: a review of cases in Wellington 1993-4. AB - AIM: To review those cases of Schistosoma haematobium presenting in Wellington during 1993 and 1994. METHOD: All patients receiving praziquantel during 1993 and 1994 were traced through local pharmacy records. Their clinical records were reviewed and they were contacted by phone to ensure a complete set of information was obtained. This included timing of possible exposure to the parasite, symptoms, investigations and response to treatment. RESULTS: Nine patients were identified who had been treated with praziquantel during this period. The records of six patients were available for review, all of whom had recently travelled to Africa and had swum in water contaminated with the parasite. Four had presented to medical practitioners with irritative voiding symptoms and scant intermittent haematuria. One presented with haematospermia and one after becoming aware that a friend had contracted schistosomiasis. All were treated with a single dose of praziquantel. CONCLUSIONS: This paper has demonstrated an increased number of cases of Schistosoma haematobium being identified in Wellington. The prevalence of this condition is low, but has been seen to increase recently and relates to the increasing numbers of New Zealanders touring Africa. Patients present with relatively mild, nonspecific symptoms which require a high index of suspicion to ensure that appropriate investigations are ordered. The currently available treatment is efficacious and relatively well tolerated. PMID- 8628539 TI - Encapsulated filtering blebs. PMID- 8628540 TI - Baltimore Vision Screening Project. AB - PURPOSE: This study estimates the prevalence of common visual disorders (amblyopia, strabismus, refractive errors) in a group of inner-city school children. In addition, the study addresses the issue of access to care for vision screening programs, specifically for children with recognized difficulties in obtaining routine medical care. METHODS: School children from an inner-city elementary school were enrolled into a prospective vision-screening program combining the identification arm (screening) and diagnostic/treatment arm (ophthalmic examination). The screening consisted of Snellen E optotypes presented at a 10-foot test distance. Each child failing the vision screening was examined by an ophthalmologist at the school using standard protocol. This allowed the authors to examine all children identified through the vision screening program. RESULTS: Six-hundred eighty children were screened during the 1993 to 1994 school year. Eleven percent (76) failed the vision screening and were examined, 68 of whom failed the ophthalmic examination. The estimated prevalence of visual morbidity was as follows: amblyopia, 3.9%; strabismus, 3.1%, and refractive errors, 8.2%. CONCLUSION: Amblyopia, strasbismus, and refractive errors were found in relatively high frequencies for this population sample of inner city children. These findings underscore the necessity of comprehensive vision-screening programs that integrate follow-up care. Children with limited access to specialized eye care must be provided with a mechanism for obtaining these services. PMID- 8628541 TI - Visual-field deficits associated with proton beam irradiation for parapapillary choroidal melanoma. AB - PURPOSE: A large series of patients treated with proton irradiation for parapapillary choroidal melanoma were reviewed retrospectively to determine the frequency of radiation papillopathy and visual-field loss after treatment. METHODS: Among 249 patients with proton irradiation for parapapillary choroidal melanoma, the authors identified 59 patients who had visual-field testing performed before treatment and at least 18 months after treatment. The visual fields, color fundus photographs, and charts were reviewed to determine the prevalence of radiation papillopathy and visual-field loss after treatment. RESULTS: Nineteen of the 59 patients reviewed (31%) received a clinical diagnosis of radiation papillopathy. Progressive visual-field loss, defined as enlargement of absolute scotoma of greater than [corrected] or equal to 30 degrees as compared with the pretreatment visual field, was noted in 67% of patients with radiation papillopathy and 73% of patients without papillopathy. In both groups, visual-field loss correlated with the area of the retina predicted to be exposed to irradiation in the majority of patients. CONCLUSIONS: Progressive visual-field loss is common after proton irradiation for parapapillary choroidal melanoma. However, the scotoma usually correlates with the area of the retina exposed to irradiation. The development of radiation papillopathy does not appear to be associated with additional visual-field defects in most cases. PMID- 8628542 TI - Field of dreamers and dreamed-up fields: functional and fake perimetry. AB - PURPOSE: Hysterical and malingering patients can manifest visual field defects on perimetry (visual field testing), including defects suggestive of true visual pathway pathology. It has been shown that control subjects can easily imitate some pathologic defects with automated, computed perimetry. The authors sought to determine whether subjects could imitate the same pathologic defect with manual and automated perimetry. METHOD: Six subjects posed as patients with neurologic problems. They had manual perimetry with both an experienced and inexperienced technician followed by automated perimetry. They were later interviewed about the methods of the technicians and the difficulty of the exercise. RESULTS: Four of six subjects easily imitated the assigned defects with both technicians on manual perimetry and with automated perimetry. These included quadrantic, altitudinal, hemianopic, and enlarged blind-spot defects. Two subjects who were assigned cecocentral and paracentral scotomas instead produced enlarged blind spots by manual perimetry and defects suggestive of chiasmal pathology by automated perimetry. Paradoxically, some subjects found that experienced technicians were easier to fool than inexperienced technicians because of the systematic way in which experienced technicians defined defects. CONCLUSIONS: With minimal coaching, some subjects can imitate visual fields with enlarged blind spots, quadrantic, hemianopic, and altitudinal defects with ease and reproducibility by both automated and manual perimetry. Cecocentral and paracentral scotomas are harder to imitate but may be mistaken as representing chiasmal pathology. Paradoxically, experienced technicians may not be better at detecting hysterical or malingering individuals. PMID- 8628543 TI - A six-month, randomized, double-masked study comparing latanoprost with timolol in open-angle glaucoma and ocular hypertension. The Latanoprost Study Group. AB - PURPOSE: To compare the intraocular pressure (IOP)-reducing effect and side effects of 0.005% latanoprost administered once daily with 0.5% timolol administered twice daily in patients with open-angle glaucoma or ocular hypertension. METHODS: This was a randomized, double-masked study with two parallel groups and a treatment period of 6 months. The primary objective of the study is to compare the IOP-reducing effect of lantanoprost with that of timolol at the end of the 6-month treatment period. A total of 294 patients were included: 149 were in the latanoprost group and 145 were in timolol group. Latanoprost was administered in the evening. RESULTS: Diurnal IOP (9:00 am, 1:00 pm, 5:00 pm) was reduced from 25.2 to 16.7 mmHg (33.7%) with lantanoprost and from 25.4 to 17.1 mmHg (32.7%) with timolol as determined at the end of the 6 month treatment period. No upward drift in IOP occurred with either drug during the treatment period. Latanoprost caused a somewhat more conjunctival hyperemia than timolol and more corneal punctuate epithelial erosions. However, both drugs were generally well tolerated. The most significant side effect of latanoprost was increased pigmentation of the iris which was observed in 15 patients (10.1%). Timolol caused more systemic side effects than latanoprost. CONCLUSIONS: Latanoprost 0.005% administered once daily in the evening reduced IOP at least as well as timolol 0.5% administered twice daily. Latanoprost was generally well tolerated systemically and in the eye. However, the drug has an unusual side effect of increasing the pigmentation of the iris, particularly in individuals with green-brown or blue-brown eyes. PMID- 8628544 TI - Comparison of latanoprost and timolol in patients with ocular hypertension and glaucoma: a six-month masked, multicenter trial in the United States. The United States Latanoprost Study Group. AB - PURPOSE: Latanoprost, a new prostaglandin analogue, was compared with timolol for ocular hypotensive efficacy and side effects. METHODS: In a multicenter, randomized, double-masked, parallel group study, 268 patients with ocular hypertension or early primary open-angle glaucoma received either 0.005% latanoprost once daily or 0.5% timolol twice daily for 6 months. All except ten patients from each group successfully completed the study. RESULTS: Intraocular pressure (IOP) was significantly (P<0.001) reduced and maintained by both medications without evidence of a long-term drift over 6 months. Comparing 6 month with baseline diurnal IOP values, the IOP reduction (mean +/- standard deviation) achieved with latanoprost (-6.7 +/- 3.4 mmHg) was significantly (P<0.001) greater than that produced with timolol (4.9 +/- 2.9 mmHg). Four patients treated with timolol and none treated with latanoprost were withdrawn from the study because of inadequate IOP control. Pulse rate was significantly reduced with timolol, but not with latanoprost. Slightly more conjunctival hyperemia appeared in latanoprost-treated compared with timolol-treated eyes. Fewer subjective side effects occurred in latanoprost-treated eyes. Both eyes of a patient with a characteristic, concentric iris heterochromia (darker centrally) at baseline showed a definite, photographically documented increase in pigmentation during latanoprost treatment, making the irides uniformly darker. Three additional patients treated with latanoprost were suspects for this color change. Otherwise, no significant difference between treatment groups occurred visual acuity, slit-lamp examination, blood pressure, and laboratory values. CONCLUSION: Latanoprost has the potential for becoming a new first-line treatment for glaucoma. PMID- 8628545 TI - Ocular injuries in battered women. AB - PURPOSE: The purpose of this article is to examine the occurrence of battered women seen by ophthalmologists in an eye emergency department, and to make ophthalmologists aware that these women are not a rarity but often go unrecognized. METHODS: This retrospective study reviews a large urban eye center's emergency room charts over a 6-month period for documentation of injuries to women as a result of domestic violence. Seventy-nine charts were selected as part of this study. In 18 of these charts, battered women definitely were identified, and in 61 charts abuse was suspected but the abuser was not specifically identified. RESULTS: The patients ranged in age from 15 to 90 years. The abuser was most commonly a boyfriend using a fist as the means of inflicting the injury. Injuries ranged from lacerations and contusions to more serious injuries, including three hyphemas and three ruptured globes. Six patients were hospitalized because of their injuries. Substance abuse was documented in 5 of 79 patients. CONCLUSIONS: Many of these female victims of intentional violence in their homes go unrecognized or uncharted by physicians. Ophthalmologists see many of these women because of the high frequency of head and neck injuries in these cases, and should therefore maintain a high index of suspicion and be prepared to act appropriately with recognition and documentation of the injury and provision of social service referrals. PMID- 8628546 TI - Hypertropia and the posterior blowout fracture: Mechanism and management. AB - OBJECTIVE: To better understand the pathophysiology and proper management of a subgroup of patients with orbital blowout fracture which manifests by a vertical diplopia and hypertropia on the affected side. PATIENTS AND METHODS: This report is based on a series of ten consecutive patients with orbital floor blowout fractures who had diplopia and hypertropia on the affected side. All patients were followed through at least 13 days of conservative care. Computed tomography demonstrated a characteristic depressed fracture of the posterior orbital floor extending to the posterior wall of the maxillary sinus in all patients. In many patients, the inferior rectus looped inferiorly and then rose to contact the globe at a steep angle. Diplopia did not spontaneously resolve in any patient. At surgery, the orbital contents were elevated to the posterior extent of the fracture, and the floor defects were bridge. Patients were followed for resolution of diplopia. RESULTS: Eight patients had resolution of the hypertropia and diplopia within 2 months of surgery, and two patients had residual diplopia in extreme downgaze but were significantly improved. CONCLUSIONS: When hypertropia and vertical diplopia are noted after orbital trauma, a posterior blowout fracture should be suspected. In these patients, infraduction may be diminished due to changes in the effective origin and insertion of the inferior rectus muscle. The diagnosis of a posterior blowout fracture should be supported by characteristic findings on computed tomography. If the motility abnormality persists for 10 to 14 days, posterior orbital exploration and fracture repair should be undertaken. PMID- 8628547 TI - Primary ductal adenocarcinoma of the lacrimal gland. AB - PURPOSE: To their knowledge, the authors report the first recognized case of ductal adenocarcinoma of the lacrimal gland (histologic equivalent of salivary duct carcinoma). Primary adenocarcinoma of the lacrimal gland is rare and has been described generically. In contrast, primary adenocarcinomas of the major and minor salivary glands are much more common and have been classified into histopathologic subtypes that have different clinical characteristics and outcomes. METHODS: A 68-year-old man presented with a 6-month history of a painless mass in the right upper outer eyelid. The authors describe the clinical, radiologic, and histopathologic features of this case and review the lacrimal gland literature. RESULTS: A modified en bloc orbitectomy was performed, and postoperative radiotherapy was administered. The patient was alive and well without evidence of tumor recurrence 10 months after surgery. CONCLUSION: The World Health Organization classification of salivary adenocarcinomas (1991) provides a framework for further insight into the presentation and biologic behavior of the less common lacrimal carcinomas. PMID- 8628548 TI - Repeatability and reproducibility of optic disc measurement with the Zeiss 4 mirror contact lens. AB - BACKGROUND: A simple clinical quantitative method of optic disc diameter measurement using a 4-mirror gonioscope contact lens has been described recently. METHODS: Intraobserver and interobserver variations are assessed on 77 eyes of 41 patients. Of these eyes, 63 had refractive errors within 3 diopters of emmetropia. The disc was measured by projecting a slit beam of known height onto the disc. RESULTS: Intraobserver variation: the coefficient of variation was 3.04% for observer 1 (experienced) and 4.76% for observer 2 (novice). The mean of the three measurements by the two observers was not significantly different (P=0.2318). For observer 1, the mean variance of the eyes with higher refractive errors did not differ significantly from the eyes with lower refractive errors. Interobserver variation: the coefficient of variation was 4.52%. The measurements for the novice were slightly larger the smallest discs and slightly smaller for the largest discs (regression analysis P=0.0209). CONCLUSION: Repeatability and reproducibility for this method are comparable with other methods of measurement. Until experienced with the technique, the authors recommend that the mean of three readings is used. PMID- 8628549 TI - The differential diagnosis and classification of eyelid retraction. AB - BACKGROUND: Classification schemes are useful in the formulation of differential diagnoses. Thoughtful commentary has been devoted to the classification of blepharoptosis, but the causes of eyelid retraction have received less attention in published reports. Although eyelid retraction most frequently is associated with Graves ophthalmopathy, numerous other entities may cause the sign. This study was undertaken to provide a more comprehensive differential diagnosis and classification of eyelid retraction. METHODS: A series of patients with eyelid retraction was studied, and pertinent published reports were reviewed. RESULTS: Forty-four patients with different causes for eyelid retraction are described. Normal thyroid function and regulation were confirmed in all patients in whom Graves ophthalmopathy could not be excluded by clinical, biochemical, or historical criteria. CONCLUSION: Based on a series of patients and reported cases, a differential diagnosis for eyelid retraction is proposed using a classification system compromising three categories (neurogenic, myogenic, and mechanistic). PMID- 8628550 TI - Oculopalatal myoclonus after the one-and-a-half syndrome with facial nerve palsy. AB - PURPOSE: The one-and-a-half syndrome is an eye movement disorder characterized by a unilateral gaze palsy and an ipsilateral internuclear ophthalmoplegia. The authors describe a previously unrecognized association between the one-and-a-half syndrome and oculopalatal myoclonus (OPM). METHODS: Five clinical cases are presented, with pertinent physical findings and radiologic studies. RESULTS: A previously unrecognized association of the one-and-a-half syndrome with subsequent development of OPM appears to exist. Involvement of the facial nerve in patients with the one-and-a-half syndrome may be a predictor of the subsequent development of OPM. CONCLUSION: Patients with the one-and-a-half syndrome and facial nerve palsy should be followed closely for possible future development of OPM. PMID- 8628551 TI - Standardizing the measurement of visual acuity for clinical research studies: Guidelines from the Eye Care Technology Forum. PMID- 8628552 TI - Standardizing the measurement of intraocular pressure for clinical research. Guidelines from the Eye Care Technology Forum. PMID- 8628553 TI - Standardizing the measurement of visual fields for clinical research: Guidelines from the Eye Care Technology Forum. PMID- 8628554 TI - AMD research. PMID- 8628555 TI - The role of smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious keratitis. AB - PURPOSE: To examine the role of routine smears, cultures, and antibiotic sensitivity testing in the treatment of suspected infectious keratitis. METHODS: A retrospective chart and laboratory data review was performed for 81 consecutive patients seen in the Los Angeles County/University of Southern California Department of Ophthalmology between June 1991 and December 1993 with a primary diagnosis of community-acquired infectious keratitis. No patients were treated with antibiotics before evaluation in the author's department, and all underwent corneal scrapings for gram-stain and bacterial, fungal, and mycobacterium cultures. Ulcers were classified as moderate or severe. All initially were treated as inpatients with a regimen including fortified cefazolin and a fortified aminoglycoside. RESULTS: Of 81 patients, 74 ulcers were either culture- negative (n=18) or grew bacteria (n=56). Fungal infection was suggested in seven patients. Of the nonfungal ulcers, 33 were classified as moderate, and 41 as severe; all moderate ulcers improved without requiring a modification in antibiotic treatment, whereas 3 severe ulcers required a change in treatment. CONCLUSION: Most community-acquired bacterial ulcers resolve with broad spectrum empiric therapy. Alternatives to universal culture and sensitivity testing that might be considered include selectively performing cultures for more severe or suspected non-bacterial ulcers or routinely obtaining cultures in all cases, but pursuing identification and sensitivity studies only when those data are required for therapy modification. PMID- 8628557 TI - Improving clinical evaluations of new eye care technologies. PMID- 8628556 TI - Limbal transplantation. AB - BACKGROUND: Limbal transplantation is a surgical technique of ocular surface epithelial transplantation advocated for a variety of ocular surface disorders with presumed stem-cell deficiency. Limbal transplantation was performed in 18 patients with ocular surface disease, which included aniridia keratopathy, chronic contact lens-associated epitheliopathy, chemical injury, Stevens-Johnson syndrome, and corneal intraepithelial dysplasia. METHODS: Limbal allograft transplantation was performed in nine eyes with the use of heterologous limbal tissue from cadaveric donor eyes or live relatives, whereas nine eyes underwent conventional limbal autograft transplantation. RESULTS: Limbal allograft transplantation resulted in restoration of a stable ocular surface in seven of nine cases, with early visual rehabilitation and significant reduction in symptoms. At a mean follow-up period of 14.7 months, one patient was noted to have failure of the inferior graft related to postoperative microbial keratitis, whereas one patient had acute rejection episode after early cessation of oral cyclosporine. The mean follow-up period for limbal autografts was 27.1 months. Limbal autograft failure occurred in two patients with limbal autograft transplantation for chronic contact lens-associated epitheliopathy. One contact lens wearer had epithelial dysplasia in the fellow eye at the previous donor site. Subclinical involvement of the fellow eye is suggested as a reason for graft failure and donor eye complications in these eyes. CONCLUSIONS: Although it requires a longer follow-up period, limbal allograft transplantation is a viable procedure for bilateral ocular surface disease and for presumed bilateral disease (viz bilateral exposure to injurious agents) and may be a preferred alternative to limbal autograft transplantation for such patients. The role of immunosuppression is being evaluated. PMID- 8628558 TI - Corneal anesthetic abuse and Candida keratitis. AB - PURPOSE: Topical corneal anesthetic abuse is a self-inflicted injury, causing profound corneal morbidity. Superimposed infection is an important complicating factor. The authors report four patients with confirmed topical anesthetic abuse of the cornea, in whom Candida keratitis developed. METHODS: A retrospective review of the medical records of four patients with confirmed topical corneal anesthetic abuse and fungal keratitis. RESULTS: A 21-year-old woman, two 28-year old women, and a 35-year-old man were included in the study. All these patients sustained a corneal injury, prompting the chronic use of topical anesthetics (0.5% proparacaine hydrochloride in 3 patients, and 0.5% tetracaine hydrochloride and 0.4% benoxinate hydrochloride in the other). Corneal findings included epithelial defects in all patients, focal infiltrate in one patient, and ring shaped stromal infiltrate in three patients. Topical anesthetic was discontinued, all patients initially were treated empirically with antibacterial agents, and three patients received topical corticosteroids. Subsequent corneal cultures grew Candida spp, Candida albicans specifically in three patients, and local and systemic antifungal therapy was started. Corneas in two patients re epithelialized; a conjunctival flap was performed on another patient with a descemetocele; and the remaining patient was lost to follow-up, although repeat fungal cultures yielded no growth. CONCLUSIONS: Corneal superinfection with Candida may occur during topical anesthetic abuse. Therapy includes discontinuation of the anesthetic and institution of antifungal therapy. PMID- 8628559 TI - Clinical and surgical factors influencing corneal graft survival, visual acuity, and astigmatism. Corneal Transplant Follow-up Study Collaborators. AB - PURPOSE: To quantify clinical and operative factors that influence corneal graft outcome. METHODS: A multifactorial analysis was done on 2242 corneal grafts registered by the United Kingdom Transplant Service from July 1987 to June 1991. RESULTS: There was an increased risk of graft failure in patients with preoperative diffuse and other noncentral stromal edema, less-common eye diseases, small trephine size, difference in donor and recipient sizes greater than 0.25 mm, and use of mixed continuous and interrupted sutures. Visual acuity 3 months after surgery was poorer in patients who had glaucoma and low visual acuity preoperatively, small trephine size, and combined vitreous surgery. Use of interrupted sutures resulted in higher astigmatism at 3 months. CONCLUSIONS: After allowing for the effects of recipient factors, surgical factors significantly affected corneal graft outcome. No factors that showed significant benefits for graft survival also adversely affected visual performance. Details of medical history, clinical condition, and surgical method failed to predict more than a small proportion of observed variability in visual performance of functioning grafts. PMID- 8628560 TI - Preliminary results of photorefractive keratectomy in active-duty United States Navy personnel. AB - PURPOSE: To evaluate the safety, efficacy, and quality of vision after photorefractive keratectomy (PRK) in active-duty military personnel. METHODS: Photorefractive keratectomy (6.0-mm ablation zone) was performed on 30 navy/marine personnel(-2.00 to -5.50 diopters [D]; mean, -3.35 D). Glare disability was assessed with a patient questionnaire and measurements of intraocular light scatter and near contrast acuity with glare. RESULTS: At 1 year, all 30 patients had 20/20 or better uncorrected visual acuity with no loss of best-corrected vision. By cycloplegic refraction, 53% (16/30) of patients were within +/- 0.50 D of emmetropia and 87% (26/30) were within +/- 1.00 D. The refraction (mean +/- standard deviation) was +0.45 +/- 0.56 D (range, -1.00 to 1.63 D). Four patients (13%) had an overcorrection of more than 1 D. Glare testing in the early (1 month) postoperative period demonstrated increased intraocular light scatter (P<0.01) and reduced contrast acuity (with and without glare, (P<0.01). These glare measurements statistically returned to preoperative levels by 3 months (undilated) and 12 months (dilated) postoperatively. Two patients reported moderate to severe visual symptoms (glare, halo, night vision) worsened by PRK. One patient had a decrease in the quality of night vision severe enough to decline treatment in the fellow eye. Intraocular light scatter was increased significantly (>2S D) in this patient after the procedure. CONCLUSIONS: Photorefractive keratectomy reduced myopia and improved the uncorrected vision acuity of all patients in this study. Refinement of the ablation algorithm is needed to decrease the incidence of hyperopia. Glare disability appears to be a transient event after PRK. However, a prolonged reduction in the quality of vision at night was observed in one patient and requires further study. PMID- 8628561 TI - Effects of diclofenac eye drops on corneal epithelial structure and function after small-incision cataract surgery. AB - PURPOSE: To investigate prospectively the effect of diclofenac sodium (DfNa) eye drops on corneal epithelial structure and function. METHODS: Seventeen patients with bilateral age-related cataract undergoing phacoemulsification combined with intraocular lens implantation were studied prospectively. After the surgery on both eyes, one eye each of these patients was assigned randomly to receive 0.1% DfNa eye drops three time daily (DfNa group), and the other eye served as control (control group). Vital stainings, tear function test, corneal sensitivity measurement, specular microscopy for corneal epithelium and endothelium, pachymetry, anterior fluorometry to measure epithelial barrier function, and laser flare-cell-metry were performed before and after surgery. RESULTS: There were no statistically significant differences between the DfNa and control groups in any measurement examined except for laser cell-flare-metry, in which the DfNa group demonstrated a significantly lower flare value at day 7 postoperatively (compare with the preoperative level of 73% +/- 41% in the DfNa group, and 130% +/- 98% in the control group, P=0.035). Epithelial cells in the DfNa group showed slight elongation and increased permeability postoperatively; however, there were no statistically significant differences with the control group. CONCLUSIONS: Administration of DfNa eye drops did not cause significant abnormalities in the corneal epithelial structure or function of patients who had undergone small incision cataract surgery. Their safety for use in procedures such as excimer laser photorefractive keratectomy remains to be established. PMID- 8628562 TI - Characteristics of exudative age-related macular degeneration determined in vivo with confocal and indirect infrared imaging. AB - PURPOSE: To evaluate the current and future interventions in age-related macular degeneration (AMD), it is essential to delineate the early clinical features associated with later visual loss. The authors describe the retinal pigment epithelium (RPE)/Bruch membrane region in ten patients with advance exudative AMD using current angiographic techniques and a noninvasive method: infrared (IR) imaging with the scanning laser ophthalmoscope. METHODS: Ten patients with exudative AMD, evidence by choroidal neovascularization (CNV), fibrovascular scar formation, pigment epithelial detachment, or serous subretinal fluid,were examined using IR imaging, fluorescein angiography, indocyanine green angiography, and stereoscopic viewing of fundus slides. The authors determined the number and size of drusen and subretinal deposits and the topographic character of the RPE/Bruch membrane area and of CNV. RESULTS: In all patients, IR imaging yielded the greatest number of drusen and subretinal deposits. Sheets of subretinal material, but few lesions consistent with soft drusen, were seen. Infrared imaging provided topographic information of evolving CNV. Choroidal neovascularization appeared as a complex with a dark central core, an enveloping reflective structure which created a halo-like appearance in the plane of focus, and outer retinal/subretinal striae. CONCLUSIONS: Infrared imaging provides a noninvasive, in vivo method to image early changes in the RPE/Bruch membrane. It offers advantages over current imaging techniques by minimizing light scatter through cloudy media and enhancing the ability to image through small pupils, retinal hyperpigmentation, blood, heavy exudation, or subretinal fluid. It provides additional information regarding early CNV, and the character of drusen and subretinal deposits. PMID- 8628563 TI - Central serous chorioretinopathy in women. AB - BACKGROUND: Central serous chorioretinopathy is a disorder that typically affects young and middle-aged men. Although extensive information is available pertaining to the clinical features of central serous chorioretinopathy in men, little is known about this condition in women. MATERIALS AND METHODS: The authors reviewed the medical records and photographic files of women who received a diagnosis of central serous chorioretinopathy. The women were divided into three groups for data analysis: idiopathic, exogenous corticosteroid use, and pregnancy. RESULTS: Fifty-one women with active central serous chorioretinopathy were evaluated. These findings in women with idiopathic serous chorioretinopathy were similar to those described in men, with the exception that women tend to be older at the time of onset. Central serous chorioretinopathy in women taking exogenous corticosteroids more likely was characterized by bilateral involvement and subretinal fibrin. Central serous chorioretinopathy in pregnant women typically developed in the third trimester and resolved spontaneously within 1-2 months after delivery. CONCLUSION: Idiopathic central serous chorioretinopathy is similar in women and men, with the exception that women tend to be more older at the time of onset. The finding of exogenous corticosteroid use in a significant number of women in our study provides further support that cortisol may play a role in the development of central serous chorioretinopathy. The mechanism by which cortisol influences the development of central serous chorioretinopathy is unclear. PMID- 8628564 TI - Role of rheologic factors in patients with acute central retinal vein occlusion. AB - PURPOSE: To assess the rheologic findings in acute central retinal vein occlusion (CRVO) with respect to associated risk factors, the clinical appearance of ischemic or nonischemic CRVO, and to elucidate the etiology of possible changes. METHODS: The authors enrolled 173 patients with acute CRVO (ischemic, 33%; nonischemic, 67%) in this prospective study. One hundred seventy-three patients who were matched for age, sex, and cardiovascular risk factors served as control subjects. All patients underwent testing to determine hematocrit values, plasma viscosity (PV), erythrocyte aggregation (SEA), and erythrocyte rigidity (SER). RESULTS: Hemocrit values and PV were increased significantly (P<0.01) in patients with ischemic and nonischemic CRVO compared with control subjects but did not differ significantly between the two groups. No significant differences were found in SEA and SER values between the clinical subsets of patients with CRVO and when the patients were compared with matched control subjects. Analysis revealed that hemocrit and PV values were (P<0.001) increased significantly independent of associated cardiovascular risk factors. CONCLUSION: These results suggest that increased hemocrit and PV values may be contributing factors in the pathogenesis of CRVO. PMID- 8628565 TI - Retinopathy and optic neuropathy in bone marrow transplantation for breast cancer. AB - PURPOSE: To characterize the ocular toxicity of a bone marrow transplant regimen does not include total body or focal head irradiation. METHODS: Nine patients with advanced breast cancer were referred for visual symptoms after high-dose chemotherapy with cisplatin, cyclophosphamide, and carmustine and autologous bone marrow transplantation without total body irradiation or local head irradiation. RESULTS: Symptoms consistent with optic neuropathy and retinopathy developed in five patients. Retinopathy alone developed in three patients and optic neuropathy alone developed in one. Retinal abnormalities included cotton-wool spots, intraretinal hemorrhages, and macular exudate. Optic nerve findings included disk swelling and subsequent pallor. Symptoms and signs associated with retinopathy were generally reversible, whereas those associated with optic neuropathy often were permanent. Retinopathy and/or optic neuropathy developed in all of the patients from 1 to 5 months after bone marrow transplantation. Resolution or stabilization of findings was observed 2-4 months after presentation. Two patients with optic neuropathy showed progression of field and acuity loss after 4 months. When compared with control subjects, the exposure of patients to cyclophosphamide and carmustine was no different. However, cisplatin exposure was 1.2-fold higher in patients with ocular toxicity compared with control subjects. CONCLUSION: Optic neuropathy and retinopathy are presumed to arise from the administration of a high-dose chemotherapy regimen. As techniques in supportive care improve, long-term adverse effects of these therapies now are becoming apparent. PMID- 8628567 TI - Pentastomid parasites of the family Sebekidae Fain, 1961 in west African dwarf crocodiles Osteolaemus tetraspis Cope, 1851 from the Congo, with a description of Alofia parva n. sp. AB - The lungs and viscera of 23 dwarf crocodiles (Osteolaemus tetraspis) obtained from markets in Brazzaville, were examined for pentastomid infection. Twenty-one animals were infected and harboured a total of 82 pentastomids, all belonging to the family Sebekidae and representing at least two genera and three species. Sebekia okavangoensis Riley & Huchzermeyer, was present in the body cavity and lungs; the other two species were restricted to the lungs. Alofia parva n.sp. (14 female female; 10 male male) was unusual in that the fulcrum supporting the anterior hooks carried a cowl-like extension. The remaining species, provisionally allocated to the genus Sebekia, could not be classified to the generic level with certainty. PMID- 8628568 TI - In vivo effects of a novel calcium antagonist (R56865) against induced epoxyscillirosidin and tulp poisoning in sheep. AB - Two anaesthetized sheep were intoxicated with epoxyscillirosidin, the main cardio active bufadienolide, extracted from Homeria pallida (Natal yellow tulp). The epoxyscillirosidin was injected intravenously as a bolus of 50 micrograms/kg, followed 30 min later by a continuous infusion in a normal saline drip (0.9% NaCl) at 25 micrograms/kg/h. In addition, another two conscious sheep were poisoned by intraruminal dosing of 1,25 g/kg of dried H. pallida plant material. Electrocardiograms, heart and respiratory rates and venous-acid-base levels were recorded prior to and at approximately 30-60 min intervals during the course of the experiment. Additional recordings were made when animals showed signs of intoxication. R56865 (Janssen Pharmaceutica, Pty Ltd), a novel Ca++ antagonist, was administered at the first distinct signs of cardiac disturbances in the sheep given epoxyscillirosidin and after development of tachycardia and dyspnoea in those that received plant material. Activated charcoal was drenched at 3 g/kg to both sheep that received H. pallida about 1 h after the initial administration of R56865. All H. pallida sheep and one of the epoxyscillirosidin sheep survived. The signs of intoxication with H. pallida, namely groaning and tachypnoea, abated within minutes of treatment with R56865, but returned c. 30 min later in both animals. The treatment apparently had little effect on heart rate and EKG changes. One of the epoxyscillirosidin sheep was treated while exhibiting paroxysmal ventricular tachycardia. Although a transient improvement in conduction disturbance was recorded, the animal died soon afterwards. The results of this study indicate that the in vivo response of R56865 against induced bufadienolide cardiac disturbance in sheep is not as evident as that observed with R56865 against similar cardiac disturbance in vitro. The potential use of R56865 together with activated charcoal is discussed. PMID- 8628566 TI - Acquired unilateral night blindness associated with a negative electroretinogram waveform. AB - PURPOSE: The authors performed clinical, electrophysiologic, psychophysical, and immunologic studies in a patient who presented with an acquired night blindness in one eye to better define the clinical and functional changes in this rare disorder. METHODS: In addition to an ophthalmologic examination, the patient underwent the measurement of electroretinogram responses, dark-adapted thresholds using a Tubingen perimeter (Oculus, Tubingen, Germany), color vision assessment, kinetic visual-field testing using a Goldman perimeter, and immunologic testing to determine if the serum contained autoantibodies to retinal bipolar cells. RESULTS: Fundus examination showed no clinically apparent abnormality in either eye. The patient showed a selective reduction in the b-wave amplitude of the rod electroretinogram and an abnormality of the cone electroretinogram ON response in the affected left eye, whereas the rod and cone electroretinograms of the right eye were normal. Rod thresholds in the affected eye were elevated markedly, whereas rod thresholds in the right eye were normal centrally and slightly elevated in the far periphery. Immunologic testing did not show circulating autoantibodies to retinal cells. CONCLUSIONS: The patient examined in this study showed phenotypic similarities to patients with congenital stationary night blindness and to patients with an acquired form of night blindness associated with cutaneous melanoma (MAR syndrome). The electroretinogram findings from the patient are consistent with an acquired defect in signal transmission from photoreceptors to ON-type bipolar cells. However, the etiology of this unique form of unilateral night blindness remains obscure. PMID- 8628569 TI - The use of preserved colostrum for rearing replacement dairy calves: calf performance, economics and on-farm practicability in Kenya. AB - A total of 133 observations on mean daily mass gains from 19 calves reared on either whole milk (control) or preserved colostrum (treatment) were estimated. The control group had a total of 104 observations computed, while the treatment group had a total of 29 observations. There was no significant difference in the overall mean daily mass gains between the treatment and control groups which were 0.2257 and 0.3607 kg, respectively (P < 0.1). Partial budgeting analysis estimated that with an annual calf crop of 80 calves, the use of preserved colostrum would result in a direct saving of an estimated US$1,800 per year for the farm. PMID- 8628570 TI - Parasites of domestic and wild animals in South Africa. XXXIV. Arthropod parasites of nyalas in north-eastern KwaZulu-Natal. AB - Seventy-three nyalas (Tragelaphus angasii) in the Umfolozi, Mkuzi and Ndumu Game Reserves in northeastern KwaZulu-Natal were examined for arthropod parasites during 1983 and 1984. In addition, six animals were examined during 1994. Ten ixodid tick species, two louse species and a louse fly species were recovered. The nyalas were good hosts of all stages of development of Boophilus decoloratus, Rhipicephalus appendiculatus and Rhipicephalus muehlensi and the immature stages of Amblyomma hebraeum and Rhipicephalus maculatus. Adult male animals harboured more adult ticks, biting lice and louse flies than did adult females. B. decoloratus was generally most abundant from October to May. The larvae of R. appendiculatus peaked from April to October, nymphs from July to October and adults, on adult male nyalas, from February to May. Larvae of R. maculatus were most abundant from May to July and nymphs from June to October. The immature stages of A. hebraeum and all stages of R. muehlensi were present throughout the year. PMID- 8628571 TI - Causes of calf mortality in Kabete area of Kenya. AB - A total of 345 calf carcases of mainly dairy breeds from the farms around Kabete area were examined at the post-mortem facility in the Department of Veterinary Pathology and Microbiology, University of Nairobi, over a 10-year period (1980 1989). About 46.8% of the total deaths took place within the first 2 months, 31.8% of them in the first month and 13.3% in the first 2 weeks. In 23 cases (6.7%) no specific cause of death was determined. The major causes of death were diseases of the alimentary tract (31.3%)--mainly gastroenteritis (76/108) due to colibacillosis, salmonellosis, coccidiosis and helminthiasis, and bloat (20/108). The other major causes of death were diseases of the respiratory tract (16.8%)- mainly pneumonia (42/58), and tick-borne diseases (13.3%)--mainly east coast fever (ECF) (37/46). The alimentary and respiratory diseases were most common in the 1-30 d age group. The other causes of death occurred in the following frequencies: musculoskeletal system (7.0%), septicaemia (6.7%), malnutrition (6.1%), cardiovascular system (3.7%), nervous system (3.2%), liver (2.6%) and poisoning (2.6%). PMID- 8628572 TI - Characterization of mucus glycoproteins in the intestinal mucosa of the African elephant (Loxodonta africana) following lectin histochemistry. AB - The glycoproteins of the small intestines, caecum and colon of three adult elephants and one recently weaned elephant calf were examined by means of lectin histochemistry. Tissue sections were histochemically stained with peroxidase labelled concanavalin A (Con A), asparagus-pea (TPA), peanut (PNA) and wheat-germ (WGA) lectins. Con A and TPA showed no binding activity in the intestinal tract of the adult elephants or the duodenum and ileum of the elephant calf, but did show a small amount of binding activity in the caecum and colon of the calf. WGA bound very intensely throughout the intestinal tracts of the adults and of the calf--especially with the goblet cells located in the crypts of Lieberkuhn and the glands of Brunner--decreasing in intensity towards the luminal surface of the intestinal tract. PNA stained the glands of Brunner of the duodenum faintly and the goblet cells of the ileum moderately, with no staining of the caecum and faint staining of the colon. These results show the distribution of Con A-, WGA-, PNA- and TPA-binding sites, and the changes that take place in the type of glycoprotein secreted after a change in the diet of the animal. PMID- 8628573 TI - Sperm-storage tubules in the vagina of the ostrich (Struthio camelus). AB - Sperm-storage tubules have been described in a number of species of birds. The presence of these tubules in the Rhea has been mentioned, but no description of these structures in ratites is available. The purpose of this study was to determine the presence and morphology of sperm-storage tubules in the vagina of the ostrich. The study was performed with the use of conventional light- and electron-microscopic techniques. Sperm-storage tubules were located in a 200-mm wide band of the vagina adjacent to the utero-vaginal junction. The tubules were mostly branched and slightly coiled and lined by columnar epithelial cells. The cells contained a basal nucleus and displayed extensive apical junctional complexes. TEM revealed sperm in all the tubules examined. PMID- 8628574 TI - The role of Hyalomma ticks in foot infestations and temporary lameness of sheep in a semi-arid region of South Africa. AB - An outbreak of lameness amongst Merino lambs, associated with the presence of Hyalomma ticks, was investigated on a farm in the south-western Free State, South Africa. The purpose was to follow the progress of the condition and to determine the extent of involvement of the two Hyalomma species which occur in the region. The flock of experimental sheep (n = 460) ranged free in natural veld under extensive farming conditions. During September and October 1993, adult ewes and lambs in this flock were examined at weekly intervals to determine tick identity, abundance and attachment-site preferences on lame and unaffected animals. Lameness occurred only among lambs, of which 68 were affected during the 8-week period. Hyalomma ticks tended to aggregate and mean numbers of ticks/aggregation were significantly higher on lame lambs (mean = 11.3) than on either the unaffected lambs (mean = 6.9) or the ewes (mean = 7.1). Most tick aggregations (72.4%) on the lame lambs occurred on the lower legs and feet, 34 out of 55 of these on the fetlocks or interdigital clefts. Hyalomma truncatum dominated (> 97%) on all animals examined. Only 15.8% of the Hyalomma marginatum rufipes recovered from the lame animals were attached to the legs. At two other localities at which H. marginatum rufipes was more abundant, or even dominant, few ticks attached to the lower legs or feet. Those that did were mostly H. truncatum (> 90%). Both H. marginatum rufipes and H. truncatum may attach to the same ventral and anogenital body regions, but H. truncatum has a tendency to attach also to the feet and lower legs. Although attachment of one or a few ticks in the axillar region or upper legs may cause lameness in sheep, the attachment of ticks to the interdigital clefts and fetlocks almost always causes lameness. The latter condition is more likely to occur in regions where H. truncatum dominates. PMID- 8628575 TI - The prevalence of neosporosis in aborted bovine foetuses submitted to the Allerton Regional Veterinary Laboratory. AB - Combined histological examination and avidin-biotin immunohistochemical staining of formalin-fixed brain and myocardium from aborted bovine foetuses presented to the Allerton Regional Veterinary Laboratory were utilized for the diagnosis of bovine neosporosis. Two out of 144 cases were diagnosed positive for neosporosis, indicating a low prevalence of infection and confirming Neospora caninum to be a cause of sporadic abortion in the population surveyed. PMID- 8628576 TI - Attachment preferences of Hyalomma truncatum and Hyalomma marginatum rufipes ticks (Acari: Ixodidae) on two sheep breeds. AB - Hyalomma ticks were collected from Merino and black-headed Dorper sheep and their attachment preferences determined. The preferred site of attachment for Hyalomma marginatum rufipes on both Dorper and Merino sheep was the anogenital and inguinal areas (75-76%). On Dorper sheep, H. truncatum attached predominantly to the anogenital and inguinal areas (67.7%). On Merino sheep, most adults of this species attached to the feet (26%). Almost equal percentages also attached to the anogenital/inguinal and brisket areas (21.6 and 22.9%, respectively). These differences should be considered when pour-on acaricides are applied to different breeds of sheep. PMID- 8628577 TI - An improved technique for the cryopreservation of Gaigeria pachyscelis (Sandveld hookworm). AB - A technique for the cryopreservation of third-stage larvae of Gaigeria pachyscelis is described. It consists of incubating sheathed infective larvae at 37 degrees C in 40% (v/v) ethylene glycol for 7 min, followed by 2 min at 0 degree C in 82.8% (v/v) cryoprotectant mixture, prior to transferring the larvae to liquid nitrogen. The survival rate obtained with this technique is consistently high: 69.1% as assessed by motility. PMID- 8628578 TI - Radiotherapy for bone pain. PMID- 8628579 TI - The central nucleus of the amygdala contributes to the production of morphine antinociception in the formalin test. AB - The rat paw formalin test is a model of prolonged pain due to mild tissue injury. There is some evidence suggesting that morphine does not produce antinociception in the formalin test via the brain-stem and spinal cord circuitry normally associated with antinociception. Furthermore, morphine appears to require an intact forebrain in order to function as an analgesic for formalin pain. In the 2 experiments reported here, we investigated the possibility that the central nucleus of the amygdala (Ce) contributes to the production of morphine antinociception (MA) in the formalin test. Nociception in this test occurs in 2 phases, with the 1st phase occurring 0-5 min after formalin injection and the 2nd phase beginning 10-15 min after injection and continuing for approximately 1 h. In Exp. 1, bilateral neurotoxic lesions of the Ce, but not lesions of the adjacent basolateral nucleus (BL), reliably attenuated MA (7 mg/kg morphine sulfate) during the 2nd phase of the formalin test without affecting baseline nociception. These results were obtained regardless of whether the rating scale method or flinch-frequency method of nociceptive scoring was used. During the 1st phase, Ce lesions reliably attenuated MA as measured by the flinch-frequency method, but not as measured by the rating scale method. In Exp. 2, Ce lesions also reliably attenuated the antinociception produced by 12 mg/kg morphine sulfate during the 2nd phase of the formalin test. Antinociception appeared to be almost completely re-instated, however, if the dose of morphine was raised to 20 mg/kg. The results indicate that neurons originating from the Ce contribute to the production of MA during the 2nd phase, and possibly the 1st phase, of the formalin test, especially at relatively lower doses of morphine. This suggests that in addition to coordinating conditioned antinociceptive responses, the amygdala may be a component of endogenous antinociceptive circuitry. These and other issues are discussed with reference to the spino-ponto-amygdaloid nociceptive pathway, and the proposed role of the amygdala in the mediation of defense reactions. PMID- 8628580 TI - Modification of rhythmical jaw movements by noxious pressure applied to the periosteum of the zygoma in decerebrate rabbits. AB - The purpose of this study was to describe and quantify changes in the movement and EMG patterns caused by tonic noxious pressure on the periosteum of the zygoma during electrically induced rhythmic jaw movements in the decerebrate rabbit. Eight New Zealand rabbits were anesthetized with urethane. EMG electrodes were placed in the masseter and digastric muscles and a light was attached to the mandibular symphysis to track jaw movements. The animals were decerebrated and the anesthetic was discontinued. Rhythmic jaw movements were evoked by electrically stimulating the corticobulbar tracts (1-msec rectangular pulses, 50 Hz), in the absence and presence of tonic noxious pressure applied bilaterally to the zygomatic periosteum (range: 400-1500 kPa). The overall response to tonic noxious pressure was a statistically significant decrease in the frequency and amplitude of the rhythmic jaw movements and in the mandibular velocity during opening and closing. The slowing of the frequency was associated with a significant increase in the duration between muscle bursts. In those animals in which the jaw closing muscles were most active, there was a significant decrease in the area of the masseter muscle bursts during jaw closure. The changes in motor activity in response to the application of tonic noxious pressure are similar to those that have been reported for patients experiencing musculoskeletal pain, suggesting that pain modifies motor programs at the segmental level. Our data support the pain-adaptation model. PMID- 8628581 TI - Effects of intravenous ketamine, alfentanil, or placebo on pain, pinprick hyperalgesia, and allodynia produced by intradermal capsaicin in human subjects. AB - The importance of N-methyl-D-aspartate (NMDA) receptor-mediated sensitization of central nervous system (CNS) neurons is well established in animal models of acute and chronic pain. A human model of central sensitization would be useful in screening new NMDA antagonists and establishing dose regimens for clinical trials in patients with pain related to sensitization of CNS neurons. We used this model to examine the effects of intravenous infusions of two centrally acting analgesics, the NMDA receptor antagonist ketamine and the morphine-like opioid agonist alfentanil. Twelve normal subjects completed a 3-session, randomized, double-blind, crossover study. From 25 to 60 min after capsaicin injection, subjects were given intravenous infusions of ketamine (mean dose: 32 mg), alfentanil (mean dose: 3075 micrograms), or saline placebo. Both drugs significantly reduced ongoing pain and pinprick-evoked hyperalgesia during the infusion. The reduction in allodynia evoked by light stroking was statistically significant only for alfentanil. Mean reduction +/- SEM relative to placebo were for ongoing pain: ketamine, 36 +/- 9%; alfentanil, 51 +/- 5%; area of pinprick hyperalgesia: ketamine, 34 +/- 7%; alfentanil, 35 +/- 7%; and area of mechanical allodynia: ketamine, 52 +/- 20%; alfentanil, 70 +/- 12%. Because the drugs were given systemically and produced side effects in all subjects, we cannot specify the site or sites of action nor conclusively rule out a non-specific 'active placebo' response as the cause for reduction of symptoms. Arguing against an 'active placebo' response, however, was the lack of analgesic effect of intravenous midazolam (mean dose; 3.4 mg, titrated to produce side effects of similar magnitude to ketamine and alfentanil) given at 145 min after capsaicin in 9 subjects who had received saline from 25 to 60 min. The results of this study suggest that neural systems sensitive to NMDA receptor antagonists and opioids participate in capsaicin-evoked pain phenomena, and support the feasibility of pharmacological studies using the intradermal capsaicin model. PMID- 8628582 TI - Effects of intrathecal local anesthetics on spinal excitability and on the development of autotomy. AB - The effect of 500 micrograms intrathecal (i.t.) lidocaine and tocainide injected prior to unilateral sciatic nerve section on the development of autotomy behavior was examined in rats. Neither drug inhibited autotomy compared with saline controls. The effect of lidocaine and tocainide on the flexor reflex in decerebrate, spinalized rats was also examined. Both drugs at 500 micrograms i.t. totally blocked the flexor reflex and hyperexcitability following acute nerve section, with a longer duration of action with tocainide. Recovery from the drugs was followed by a prolonged period of reflex hyperexcitability, even without axotomy. Lower dose lidocaine (100 micrograms) briefly blocked the reflex and recovery was followed by reflex hyperexcitability. Very low dose i.t. lidocaine (1 and 10 micrograms) did not block the flexor reflex, but only induced a prolonged increase in reflex magnitude. The possible mechanisms underlying these observations are discussed. The results suggest that i.t. local anesthetic pretreatment alone may not be beneficial for the prevention of the development of postoperative or neuropathic pain. PMID- 8628583 TI - Analgesic effects induced by TENS and electroacupuncture with different types of stimulating electrodes on deep tissues in human subjects. AB - Effects of conditioning peripheral nerve stimulation with different types of stimulating electrodes on pain thresholds in various deep tissues were measured in human subjects. Cone-shaped metal (phi 13 mm), rubber (phi 13 mm), and large soft surface electrodes (50 x 150 mm) were used for transcutaneous electrical nerve stimulation (TENS), and insulated and non-insulated acupuncture needles (diameter: 240 microns) were used for electroacupuncture (EA). Two pairs of electrodes were placed around the point of deep pain measurement. Symmetrical positive and negative square pulses (0.1 msec at 100 Hz) of just below the pain tolerance intensity were used for both TENS and EA. Deep pain thresholds were measured at the center of the thigh with a pulse algometer and insulated needle electrodes. Pain thresholds of deep tissues were in the order periosteum < fascia < skin (including subcutaneous tissues) < muscle. TENS with surface electrodes significantly increased pain thresholds of skin and fascia but not those of muscle or periosteum. The shape, material and size of the surface electrodes hardly affected the degree of analgesic effect, except in the fascia by large soft electrodes. In contrast, EA with non-insulated needles induced a greater increase in pain threshold in skin, fascia and muscle, although statistically significant results were obtained in only the first two tissues. EA with insulated needle electrodes was the only technique with which we obtained a significant increase in pain threshold in muscle and periosteum. These results suggest that the choice of electrode and stimulus parameters is important for the production of sufficient analgesic effects in different somatic tissues and that insulated needle electrodes are useful for pain relief in deeper tissues such as muscle and periosteum. PMID- 8628584 TI - Relaxation and imagery and cognitive-behavioral training reduce pain during cancer treatment: a controlled clinical trial. AB - Few controlled clinical trials of psychological interventions for cancer pain relief exist in spite of frequent support for their importance as adjuncts to medical treatment. This study compared oral mucositis pain levels in 4 groups of cancer patients receiving bone marrow transplants (BMT): (1) treatment as usual control, (2) therapist support, (3) relaxation and imagery training, and (4) training in a package of cognitive-behavioral coping skills which included relaxation and imagery. A total of 94 patients completed the study which involved two training sessions prior to treatment and twice a week 'booster' sessions during the first 5 weeks of treatment. Results confirmed our hypothesis that patients who received either relaxation and imagery alone or patients who received the package of cognitive-behavioral coping skills would report less pain than patients in the other 2 groups. The hypothesis that the cognitive-behavioral skills package would have an additive effect beyond relaxation and imagery alone was not confirmed. Average visual analogue scale (VAS) report of pain within the therapist support group was not significantly lower than the control group (P = 0.103) nor significantly higher than the training groups. Patient reports of relative helpfulness of the interventions for managing pain and nausea matched the results of VAS reports. From these results, we conclude that relaxation and imagery training reduces cancer treatment-related pain; adding cognitive behavioral skills to the relaxation with imagery does not, on average, further improve pain relief. PMID- 8628585 TI - Effect of EMG biofeedback compared to applied relaxation training with chronic, upper extremity cumulative trauma disorders. AB - This study examined the relative effectiveness of EMG biofeedback, applied relaxation training and a combined procedure in the management of chronic, upper extremity cumulative trauma disorder. Forty-eight patients with a history of about 5-6 years of upper extremity pain were randomly assigned to 1 of 4 treatment conditions, namely applied relaxation training, EMG biofeedback, a combined approach or a wait-list control. Treatments were conducted on an individual basis, twice per week for 4 weeks. Patients in all 3 treatment conditions showed significant short-term reductions in pain and psychopathology in comparison to the wait-list group who showed minimal change. Six-month follow up data were obtained for patients in the treatment conditions, but not the wait list group. There was some evidence of relapse on measures of depression, anxiety and pain beliefs for treated patients during the 6-month follow-up period, although measures remained significantly below pre-treatment levels for most outcome indices. Self-monitored pain continued to decrease for the treatment groups through follow-up. Contrary to predictions, however, the strongest short term treatment benefits were shown by patients receiving applied relaxation training on measures of pain, distress, interference in daily living, depression and anxiety. By 6-month follow-up, differences between treatment groups were no longer evident. PMID- 8628586 TI - Humor as a cognitive technique for increasing pain tolerance. AB - Substantial research has demonstrated that cognitive psychological techniques including distraction can increase pain tolerance. In recent years, there also have been claims that humor and laughter possess unique characteristics for coping with pain and stress. Theoretically, explanations include the release of endorphins, the lowering of tension, as well as the distraction that results from humor. The question is whether humor is more effective than simple distraction. For this purpose humor was contrasted with a repulsive stimulus and a neutral stimulus controlled for interest level, that would also have distraction capabilities but not the unique aspects of humor. Pain tolerance was tested using cold pressor stimulation. Four groups (20 subjects in each) were tested. Three groups were shown a film: (1) a humorous film, (2) a repulsive film, (3) a neutral film. Group 4 was not shown any film. Results indicated that both the humor and repulsive groups showed a significant increase in pain tolerance as compared to the other groups. The repulsive group yielded the largest increase in pain tolerance although not different from the humor group. Except for sex differences, pain ratings did not show any group effects. Discussion focused on the type of distraction that would be meaningful for increasing pain tolerance and on the place of humor in pain control. PMID- 8628587 TI - Influence of eye orientation on pain as a function of anxiety. AB - The effect of eye orientation on pain was investigated as a function of the stimulus intensity and the subjects' anxiety. Right-handed female subjects received sequences of electric shocks gradually increasing in intensity. Unpleasantness threshold, pain threshold, tolerance threshold and pain range were obtained when eyes were directed towards a target LED situated in the direction of, or away from, the source of stimulation, in the ipsi- or contralateral hemispace. Directing the eyes ipsilaterally to the stimulated hand yielded a lower unpleasantness threshold when the right hand was stimulated. No significant effect was observed in high trait anxiety subjects. These results are interpreted in terms of hemispatial attention bias. Moreover, an ipsilateral eye orientation increased pain range but this effect reversed in the highest state anxiety subjects. These latter observations could reflect the interacting influences of both spatial attention and state anxiety in selecting the processing mode of painful stimuli. PMID- 8628588 TI - Differences in family functioning between patients with chronic headache and patients with chronic low back pain. AB - The family climate in 36 families, comprising 154 individuals, was investigated. The objective of this study was to compare families where the mother suffered from chronic headache to families with pain-free mothers and to those where the mother suffered from chronic low back pain. The Family Environment Scale (FES) was used to evaluate the family climate in these 3 groups as perceived by the members of the family. The results in sufficiently standardized groups show a significantly reduced intra-family openness (P < 0.0001) in families where the mother suffered from chronic headache. Both pain groups were less active in their leisure time than the pain-free families. Based on the findings of the present study, the impact of the psychosocial environment as a novel normative value for chronic pain syndromes is discussed in relation to the need for further research and treatment modalities. PMID- 8628589 TI - Central representation of chronic ongoing neuropathic pain studied by positron emission tomography. AB - This study was undertaken to explore whether the neural substrates demonstrated in brain imaging studies on experimentally induced pain are involved in the perception of chronic neuropathic pain. We investigated the cerebral representation of chronic lateralised ongoing pain in patients with painful mononeuropathy (PMN, i.e., pain in the distribution of a nerve, neuralgia) with positron emission tomography (PET), using regional cerebral blood flow (rCBF) as an index for neuronal activity. Eight patients (29-53 years) with PMN in the lower extremity (4 in the right, 4 in the left) were recruited. Paired comparisons of rCBF were made between the patient's habitual pain (HP) state and the pain alleviated (PA) state following a successful regional nerve block (RNB) with lidocaine. The ongoing neuropathic pain resulted in activation of bilateral anterior insula, posterior parietal, lateral inferior prefrontal, and posterior cingulate cortices as well as the posterior sector of the right anterior cingulate cortex (ACC), Brodmann area (BA) 24, regardless of the side of PMN. In addition, a reduction in rCBF was noted in the contralateral posterior thalamus. No significant change of rCBF was detected in the somatosensory areas, i.e., SI and SII. The cerebral activation pattern, while addressing the differences between the HP and PA states, emphasises the affective-motivational dimension in chronic ongoing neuropathic pain. The striking preferential activation of the right ACC (BA 24), regardless of the side of the PMN, not only confirms that the ACC participates in the sensorial/affectional aspect of the pain experience but also suggests a possible right hemispheric lateralisation of the ACC for affective processing in chronic ongoing neuropathic pain. Our data suggests that the brain employs different central mechanisms for chronic neuropathic pain and experimentally induced acute pain, respectively. PMID- 8628590 TI - Audible and ultrasonic vocalization elicited by single electrical nociceptive stimuli to the tail in the rat. AB - We describe audible and ultrasonic vocalization elicited in rats by a short electrical pulse applied to the tail. Three types of vocal emissions were recorded: (1) 'peep', characterized by a repartition of energy over a wide range (0-50 kHz) of frequencies without any clear structure; (2) 'chatters', characterized by an audible (frequencies in hearing range of humans) fundamental frequency (2.47 +/- 0.03 kHz) and harmonics; and (3) 'ultrasonic emissions', characterized by a succession of slightly modulated pulses with frequencies in the 20-35 kHz range. Peeps and chatters were never recorded before the application of the stimuli. Several different vocalization patterns were described in terms of these types of responses. Just after the stimulation, all the animals emitted a 1st peep, which was generally (61%) followed by a 2nd one. They appeared with reproducible latencies, durations and envelopes. The envelopes of the audible (peeps and chatters) responses were intensity-dependent. Experimental data (moving the stimulation site, lidocaine injection) indicated that the 1st and 2nd peeps were triggered by two different groups of peripheral fibres with mean conduction velocities of 7.3 +/- 0.8 and 0.7 +/- 0.1 m/sec, respectively. This suggested an involvement of A delta and C fibres. Morphine showed a naloxone-reversible and dose-dependent antinociceptive effect by decreasing the 1st and 2nd peep envelopes. It is concluded that a short stimulus applied to the tail triggers a complex behavioural repertoire. It is proposed that this model will be a useful tool for physiological and pharmacological studies of nociception. PMID- 8628591 TI - The development of opioid tolerance in the formalin test in the rat. AB - The formalin test produces persistent pain in animals and is believed to provide a better model of the pain experienced by humans than do tests that measure reflex nociceptive thresholds. The present study evaluated whether tolerance to morphine develops in the formalin test in the rat. Morphine (75 mg) or vehicle pellets were implanted subcutaneously for 5 consecutive days. On the 6th day, a subcutaneous (s.c.) dose of either morphine (10 mg/kg) or saline was given 30 min prior to the injection of 50 microliters of 5% formalin into the left hindpaw of the rats. In vehicle-pelleted rats administered saline, formalin evoked characteristic pain behavior consisting of licking, biting and flinching of the affected hindpaw. The pain behavior in morphine-tolerant rats given morphine did not differ significantly from the saline control; i.e., tolerance to the analgesic effect of morphine was demonstrable in the formalin test. These results do not agree with previous published reports. Rather, these results suggest that the mechanisms involved in morphine analgesia in the formalin test and in reflex nociceptive tests are similar and subject to the same problem of tolerance with chronic opioid administration. PMID- 8628592 TI - The influence of colonic temperature changes in anaesthetised rats on tail skin temperatures and repeated testing of tail-flick latencies. AB - Tail-flick (TF) response latencies were measured in pentobarbitone-anaesthetized rats and variations with time, body and tail temperatures and 5 tail stimuli positions analysed with a mixed model analysis of variance. Variation with time was not significant. Highly significant differences (P < 0.001) were found between tail-flick latencies (TFLs) for tail temperatures and stimulus position. The most proximal tail position showed significantly different relationships for TFL with time and body temperature from other positions. The method described allows multiple TFLs to be measured in 1 animal with the potential of reducing the total number of animals in an experiment. Tail stimuli positions from proximal to distal sites showed a variation in response from 4.3 sec (95% CI: 4.2, 4.4) to 6.7 sec (95% CI: 6.6, 6.9). Rat tail stimulus position should therefore be standardised to allow reproducible measures of TFL and body temperature maintained within normal limits. TFLs were found to be abnormal at body temperatures above 39 degrees C. PMID- 8628593 TI - Subcutaneous fentanyl and sufentanil infusion substitution for morphine intolerance in cancer pain management. AB - Eleven patients with cancer pain in a palliative care and chronic pain service required cessation of morphine due to unacceptable opioid side effects. In this retrospective study fentanyl was evaluated as a second-line subcutaneously infused opioid. Starting doses ranged from 100 to 1000 micrograms/24 h, and the duration of fentanyl infusion was 3-70 days. The clinically derived mean relative potency of fentanyl to morphine infusions was 68:1 (SD +/- 23; range: 15-100), and we now recommend cautious dose conversion at an approximate equivalence of 150-200 micrograms fentanyl for 10 mg morphine in non-opioid naive chronic cancer pain patients. All patients demonstrated an improvement in the adverse effect(s) for which the change in opioid was undertaken. Adequate pain relief was achieved in all but 1 patient with mixed nociceptive and neuropathic pelvic pain for whom an epidural infusion of a local anaesthetic/opioid mixture was required. Fentanyl was changed to the more potent synthetic opioid sufentanil in 2 patients for whom the fentanyl dose necessitated too large a volume for the portable syringe driver in use. The clinically derived sufentanil to fentanyl relative potencies were 24:1 and 16:1, respectively. This achieved good analgesia and maintained the favourable side-effect profile seen with fentanyl. Subcutaneous infusion appears to be a safe and viable route of fentanyl delivery, and provided effective analgesia with a low incidence of adverse effects in this small selected group of patients who were intolerant of subcutaneous morphine. We suggest a trial of subcutaneous fentanyl for selected patients who have intractable adverse effects on morphine, and it is now the second-line infusable opioid in our service. Further prospective evaluation of the role of these two synthetic mu opioid agonists in palliative care practice is warranted, as part of an evolving picture of variation in opioid side-effect profile seen with different drugs within the class. PMID- 8628594 TI - Massive opioid resistance in an infant with a localized metastasis to the midbrain periaqueductal gray. AB - We report the case of a 4-month-old infant with terminal malignancy who had systemic metastases and a localized metastasis to the dorsal midbrain periaqueductal gray (PAG). Extraordinary doses of opioids (dose equivalent of 2680 mg morphine sulfate/h, i.v.) were required to achieve adequate analgesia. The behavior of the infant, interpreted as being representative of a response to pain, may have been an aversive reaction due to the location of the lesion in the dorsal PAG. We propose that the lesion in the PAG impaired the responsiveness of this infant to the effect of opioids. This report is to alert clinicians to the possible role of the PAG in impaired opioid responsiveness in patients with terminal malignancy, as well as the possibility that pain-like signs (e.g., tachycardia, tachypnea, vocalization, facial grimacing) may indicate an aversive reaction rather than pain in non-verbal patients. PMID- 8628595 TI - Pertussis deaths: report of 23 cases in the United States, 1992 and 1993. AB - OBJECTIVE: To characterize pertussis deaths and to identify possible risk factors and prevention strategies. METHODS: A retrospective review of all deaths attributed to pertussis with disease onset during 1992 and 1993 reported to the Centers for Disease Control and Prevention. Hospital discharge summaries and autopsy reports were reviewed, and additional clinical information was provided by physicians involved in the care of the children. RESULTS: During 1992 and 1993, 23 deaths attributed to pertussis were reported to the Centers for Disease Control and Prevention. Cultures for Bordetella pertussis were positive in 18 (90%) of the 20 cases in which it was performed. Twenty (87%) of the 23 children who died were young than 1 year of age, and 18 (78%) of the children had received no doses orf pertussis vaccine. Among 20 children for whom gestational ages were known, 12 (60%) were born at36 weeks' gestation or earlier; in contrast, 10.7% of live births in the United States in 1992 were at 36 weeks' gestation or earlier. The median age of mothers whose children had fatal pertussis was 20 (range, 14 to 37) years in the 15 cases in which ages were known, compared with the national median age of 26.3 years in 1992. Pneumonia was a complication in all but 1 (96%) of the cases. Seizures occurred in 4 cases (17%), and acute encephalopathy occurred in 3 cases (13%). CONCLUSIONS: Pertussis continues to cause serious illness and death in the United States, particularly among infants who are not vaccinated. Preterm delivery and young maternal age may place infants at increased risk of death because of pertussis. Under the current pertussis vaccination schedule, three fourths of the infants who died were too young to have received three doses of pertussis vaccine, the minimum number of doses considered necessary for adequate protection against clinical pertussis. Additional strategies, to prevent deaths caused by pertussis in young infants, such as starting infant vaccination at an earlier age and booster doses to adolescents and adults, need to be evaluated. PMID- 8628596 TI - An evaluation of measles revaccination among school-entry-aged children. AB - BACKGROUND: A two dose measles vaccination schedule is recommended routinely for all school-entry-aged children. We evaluated this recommendation by determining both measles antibody seroprevalence and the response to revaccination in seronegative children in this age group. METHODS: Children 4 to 6 years of age who had received a single dose of measles vaccine between the ages of 15 to 17 months were tested for measles antibody by using enzyme-linked immunosorbent assay (ELISA) microneutralization technique. Seronegative children were revaccinated and again tested for measles antibody (immunoglobulin M [IgM] and neutralizing). RESULTS: Of 679 children tested, 37 (5.4%) were seronegative. Seronegativity was not significantly associated with age, sex, race, age at initial vaccination, time since vaccination, or maternal year of birth. However, children mothers with a college degree were 12 times more likely to be seronegative than children of mothers who never attended college (P < .01). Of the 37 seronegative children, 36 seroconverted after revaccination--33 producing IgM measles antibody, suggestive of a primary immune response. The cost per seroconversion would have been an estimated $415 if all 679 children had been revaccinated. CONCLUSIONS: Revaccination reduces the pool of children who are susceptible to measles. Although the cost per seroconversion is high, a two-dose schedule should reduce the substantial costs of controlling measles out breaks by reducing the number of outbreaks. PMID- 8628597 TI - Treatment of familial hypercholesterolemia in children and adolescents: effect of lovastatin. Canadian Lovastatin in Children Study Group. AB - OBJECTIVE: Familial hypercholesterolemia (FH), an inherited autosomal dominant disorder of lipoprotein metabolism, is associated with premature atherosclerosis. The recommended pediatric therapy consists of dietary intervention and, when necessary, treatment with bile acid-binding resins. However, compliance has been poor in many children. Therefore, our objectives were to determine the efficacy, safety, and tolerance of the short-term use of lovastatin, a 3-hydroxy 3 methylglutaryl coenzyme A reductase inhibitor, in the control of severe FH in a male pediatric population and to evaluate the dose-response relationship. METHODS: Sixty-nine male patients with FH 12.9 +/- 2.4 years of age (mean +/- SD) participated in this multicenter, randomized, double-blind trial. After a 4-week placebo period, the patients were allocated to four treatment groups (lovastatin 10, 20, 30, 40 mg/d) for 8 weeks. Plasma lipid and apolipoprotein (Apo) concentrations were measured every 2 weeks. Clinical and laboratory evidence of adverse events was monitored periodically throughout the study. RESULTS: All lovastatin doses reduced total cholesterol (-17% to -29%), low density lipoprotein cholesterol (-21% to -36%), and ApoB (-19% to -28%) concentrations. A dose-response relationship was seen, and between-group comparisons showed that results were significantly improved up to a dose of 30 mg/d. We observed a 7% increase in high-density lipoprotein cholesterol and a 4% increase in ApoA1 concentrations. The medication was well tolerated by all patients. No serious clinical adverse experience was reported. Lovastatin increased aspartate aminotransferase concentrations, but there was no evidence of a dose-response relationship, and no value exceeded two times the upper limit of normal. No significant change in alanine aminotransferase was observed. Three patients had marked (more than three times the upper limit of normal) asymptomatic elevations in their creatine kinase values, which returned spontaneously to normal, and no action was required regarding the drug. PMID- 8628598 TI - Pediatric care for children whose parents are gay or lesbian. AB - BACKGROUND: A growing number of children have at least one parent who is gay or lesbian. There is no evidence that these children experience any particular difficulties as a result of their parents' sexual orientation. Considerable evidence suggests that the provision of health care may not address the special needs and concerns of gay men and lesbians adequately. No research has been done regarding the pediatric care of children whose parents are gay or lesbian. It is likely that there are predictable challenges and development transitions for these children and parents for which pediatricians and other health care providers might be helpful advisers. OBJECTIVE: This exploratory project sought to describe the experiences that lesbian and gay parents and their children have had with the pediatric health care system. We were interested in describing experiences that had been especially affirming and others that had been troublesome and in gathering suggestions regarding changes in the structure and process of care. METHODOLOGY: Two hundred fifty-five parents completed an open ended questionnaire. Responses were coded and tabulated. RESULTS: Most parents described considerable success in obtaining pediatric care that was affirming, supportive, and satisfactory. On the other hand, many parents noted deficiencies in pediatric offices, clinics, emergency departments, and hospitals, many of which could be corrected easily. CONCLUSIONS: We have summarized the accumulated advice to pediatric health care providers and have described some of the developmental transitions that are potentially appropriate opportunities for pediatric intervention. PMID- 8628599 TI - Birth weight-specific mortality for extremely low birth weight infants vanishes by four days of life: epidemiology and ethics in the neonatal intensive care unit. AB - BACKGROUND: The persistent differences between those who question the appropriateness of aggressive resuscitative measures for many extremely low birth weight (ELBW) infants (birth weight < 1001 g) and those who generally initiate such treatment has been a source of ongoing tension for physicians, parents, judges, and policymakers. We believe that much of this tension may be a result of the way the issue is framed. We began this study with the intuition that although many ELBW infants die, most succumb quickly. Were this true, discussions that considered only survival rates might miss the point. A more relevant statistic might be the degree to which interventions prolong dying, extend suffering, or use resources for infants who will eventually die. METHODS: We determined the survival and nonsurvival for 429 ELBW infants admitted to our neonatal intensive care unit (NICU) for 3 years. We noted particularly the relationship between birth weight, illness severity (fraction of inspired oxygen [Fio2] requirement for infants requiring mechanical ventilation), and the time course of mortality for nonsurvivors. We next calculated a resource utilization index (NICU bed days occupied by survivors and nonsurvivors) for each patient and for the population as a whole. Finally, we determined how NICU resources were distributed among infants who eventually died and those who survived. RESULTS: Of the 429 ELBW infants alive on day of life (DOL) 1,202 (47%) survived to be discharged. on DOL 1, both birth weight and illness severity independently predicted likelihood of survival. Approximately 80% of ELBW deaths occurred in the first 3 days of life-- consequently, once an infant had survived to DOL 4, the likelihood of survival was dramatically enhanced (81% for the 249 patients alive on DOL 4). In addition, although survival for DOL 4 infants continued to depend on illness severity, survival no longer depended on birth weight. These observations on DOL 4 were confirmed in the subpopulation of 212 infants whose birth weight was < 750 g. Overall, although 53% of ELBW babies admitted died, only approximately 13% of all NICU bed-days (a proxy for resource allocation) were devoted to infants who did not survive. This figure did not vary as a function of birth weight. CONCLUSION: Generally, when we talk of survival rates to parents, ethics committees, or policy makers, we base our predictions largely on birth weight. The data presented here suggest that predictions should be corrected by including DOL and that, when this is done, the prognostic value of birth weight rapidly diminishes. In addition, birth weight-specific mortality and day of death for nonsurvivors correlated inversely; that is more of the smaller infants died, but the doomed ones died more quickly. Consequently, medical resources allocated to nonsurvivors remained low, and independent of birth weight. This formulation lends weight both to the reasonableness of physicians in offering NICU care to ELBW infants, with unlikely prospects for survival, and of parents and surrogate decision-makers in requesting/ assenting to it. PMID- 8628600 TI - Antenatal phenobarbital therapy and neonatal outcome. I: Effect on intracranial hemorrhage. AB - OBJECTIVE: To evaluate the effect of antenatal phenobarbital (PB) therapy on neonatal intracranial hemorrhage (ICH) in preterm infants. DESIGN: Prospective, randomized, controlled trial. SETTING: Single institution study. SUBJECTS AND INTERVENTIONS: Women in preterm labor ( < 35 weeks' gestation) were assigned to control and treatment groups. The treatment group received 10 mg/kg (maximum, 1000 mg) PB intravenously, followed by 100 mg orally daily, until delivery. Neonates did not receive PB after birth. Head sonograms were performed on days 3, 7, and 14 and at discharge. Hemorrhage was classified as mild, moderate, or severe by a single reader. OUTCOME MEASURES: Incidence of neonatal ICH in all infants, infants weighing less than 1250 g, and infants of multiple gestations. RESULTS: The study population comprised 110 women, 60 in the control group and 50 in the PB group. Neonates in the control group (n = 74, including 10 pairs of twins and 2 sets of triplets) were comparable to those in the treatment group (n = 62, including 7 pairs of twins, 1 set of triplets, and 1 set of quadruplets) regarding birth weight, gestational age, and other clinical risk factors for ICH. There was a trend for the incidence of any grade of hemorrhage to be lower in the PB group (22% [14 of 62]) compared with the control group (35% [26 of 74]). Moderate and severe hemorrhages were significantly lower in the PB group (1.6% [1 of 62]) compared with the control group (9.4% [7 of 74]). Among infants weighing less than 1250 g, overall ICH was lower in the PB group (23% [6 of 26]) compared with the control group (51% [18 of 35]). Among multiple-gestation infants, overall ICH was 4.7% (1 of 21) in the PB group, compared with 31% (8 of 26) in the control group. CONCLUSIONS: Antenatal PB therapy results in a significant decrease in moderate and severe ICH in infants born at less than 35 weeks' gestation. Antenatal PB therapy also resulted in a decrease in the incidence of all grades of ICH in infants weighing less than 1250 g and infants born of multiple gestations. PMID- 8628601 TI - Antenatal phenobarbital therapy and neonatal outcome. II: Neurodevelopmental outcome at 36 months. AB - OBJECTIVE: To evaluate the effect of antenatal phenobarbital (PB) therapy on neurodevelopmental outcome at 36 months. DESIGN: Prospective, randomized, controlled trial. SETTING: Single-institution study. SUBJECT AND INTERVENTIONS: Children born to women who participated in the study evaluating the effect of antenatal phenobarbital (PB) on neonatal intracranial hemorrhage were prospectively followed to 3 years of age. OUTCOME MEASURES: Physical growth, neurologic examinations, and developmental testing (McCarthy Scales of Children's Abilities). Comparisons between groups were made on all children and those born to multiple gestations. RESULTS: Forty-one children born to women who received 10 mg/kg PB before delivery and 55 children in the control group were evaluated. Three children, all in the control group, had growth parameters (height, weight, and head circumference) below the fifth percentile. The McCarthy General Cognitive Index (standard, 100 +/- 16) was 93 +/- 20 in the PB group and 85 +/- 18 in the control group. The subscores tended to be higher in the PB group than in the control group, with higher quantitative scores in the PB group (44 +/- 11 vs 39 +/- 8). Neurologic deficits were noted in 2 of 41 in the PB group and in 6 of 55 in the control group. CONCLUSIONS: Infants born to women who received antenatal PB therapy had similar neurodevelopmental outcomes as infants born to women who did not receive PB. No adverse effects of PB exposure were detected. PMID- 8628602 TI - Measles antibody in vaccinated human immunodeficiency virus type 1-infected children. AB - OBJECTIVES: The goals of this study were to evaluate the proportion of previously vaccinated human immunodeficiency virus (HIV) type 1-infected children with detectable postvaccination measles antibody; to assess risk factors for vaccine failure; and to evaluate the response to reimmunization. METHODS: A total of 81 perinatally HIV-infected children receiving medical care in the Bronx, New York who had previously received measles vaccine were enrolled. The Centers for Disease Control and Prevention (CDC) HIV class, lymphocyte subsets, and measles antibody were determined upon enrollment. Additional data abstracted from medical records included dates and number of prior measles vaccinations and CDC HIV class at the time of vaccination. Measles antibody was determined by microneutralization enzyme-linked immunosorbent assay (ELISA). RESULTS: The median age at time of study was 42 months (range, 9 to 168 months). Overall, 58 (72%) subjects had detectable measles antibody (microneutralization ELISA titer > 1:5). Children studied within 1 year of vaccination were more likely to have detectable measles antibody than children evaluated more than 1 year after vaccination (83% vs 52%, P < .01). The proportion of children with detectable measles antibody was higher among children with no or moderate immunosuppression compared to those with severe immunosuppression when immune status was based on CD4%. Children vaccinated at 6 to 11 months of age appeared to have a higher proportion of detectable measles antibody than those who received a first measles vaccination after age 1. Only 1 (14%) of 7 previously vaccinated children who were seronegative or had very low titers experienced a four-fold rise in measles antibody when reimmunized. CONCLUSION: These results support current recommendations to vaccinate HIV-infected children against measles. The proportion of children with detectable measles antibody among vaccinated HIV infected children is considerably lower than in vaccinated healthy children. HIV infected children may respond better to measles vaccine when it is administered before the first birthday. From our limited data it appears that reimmunization of previously vaccinated HIV-infected children with moderate to severe immunosuppression does not result in an antibody recall response. PMID- 8628603 TI - Persistent pulmonary hypertension of the newborn and smoking and aspirin and nonsteroidal antiinflammatory drug consumption during pregnancy. AB - OBJECTIVE: Prenatal causation of persistent pulmonary hypertension of the newborn (PPHB) is suggested by a specific pattern of pulmonary vascular remodeling observed immediately after birth in some infants with fatal PPHN. The goal of this study was to determine whether PPHN is associated with fetal exposure to: (1) tobacco and marijuana smoking (ie, contributors to fetal hypoxemia), (2) consumption of aspirin and other nonsteroidal antiinflammatory drugs (ie, inhibitors of prostaglandin synthesis), and (3) cocaine use (ie, a contributor to vasospasm). DESIGN: Case-control interview study. SETTING: Two Harvard-affiliated newborn intensive care units. PARTICIPANTS: Mothers of case infants who had PPHN or who met criteria for the referent group. INTERVENTIONS: During July 1985 through April 1989, we interviewed mothers of 103 infants with PPHN and 298 control infants. Because of potential selection bias that might result from recruiting only inborn control infants even though two-thirds of cases were outborn, separate analyses compared the 103 total and 35 inborn infants with PPHN with the 298 inborn control infants. Multivariate analyses were used to adjust for potential confounding factors, including maternal education and Medicaid health insurance (ie, two markers of socioeconomic status), other antenatal factors found to be associated with PPHN (ie, maternal urinary tract infection and diabetes mellitus), and the infant's sex. MAIN OUTCOME MEASURES: Self reported use or consumption of tobacco, marijuana, cocaine, aspirin, and other nonsteroidal antiinflammatory drugs during pregnancy. RESULTS: The adjusted odds ratios (and 95% confidence intervals) for maternal pregnancy exposures to the factors of principal interest among the total study population were: aspirin, 4.9 (1.6-15.3); and nonsteroidal antiinflammatory drugs, 6.2 (1.8-21.8); for the inborn group they were aspirin, 9.6 (2.4-39.0); and nonsteroidal antiinflammatory drugs, 17.5 (4.3-71.6). Although the association between tobacco smoking during pregnancy and PPHN was elevated in univariate analyses, with odds ratios (and 95% confidence intervals) of 2.0 (1.2-3.4) and 1.3 (0.6-3.3) for total and inborn populations, respectively, the relationship was not significant after adjustment for all other factors in the final logistic regression model. Acknowledged illicit drug use was too infrequent (3.2%) to evaluate. CONCLUSION: Maternal consumption of nonsteroidal antiinflammatory drugs and aspirin during pregnancy or the reasons these drugs were ingested seem to contribute to an increased risk of PPHN. PMID- 8628604 TI - Epidural hemorrhage: is it abuse? AB - OBJECTIVE: To determine whether children presenting with epidural hemorrhage (EDH) are as likely to have been abused as are children presenting with subdural hemorrhage (SDH). DESIGN: Retrospective chart review. SETTING: Level I regional trauma center and a regional children's hospital. PATIENTS: All children at both institutions 3 years old or younger with a diagnosis of EDH or SDH identified by a search of the computerized trauma registry and hospital medical records from 1985 through 1991. MEASUREMENT AND RESULTS: Complete records were found for 93 of 94 eligible subjects. The diagnosis of accidental or inflicted injury was ascertained from the patient's hospital medical record or the records of Child Protective Services. Of all subjects (n = 93), 52% (48/93) were male and the median age was 15 months. Abuse was diagnosed in 47% (28/59) of children with SDH and 6% (2/34) of those with EDH. Other significant injuries were found in 47% of children with SDH and 18% of children with EDH. There was no statistically significant difference between the two groups with respect to the likelihood of identifying a skull fracture, the need for surgical evacuation of the hemorrhage, or mortality. CONCLUSIONS: Our data are consistent with current biomechanical concepts of intracranial injury. EDHs results from brief linear contact forces that commonly occur in unintentional falls. SDHs are caused by global high-energy rotational acceleration/deceleration forces that are commonly generated in episodes of abuse. Compared with SDH, EDH rarely results from abuse. PMID- 8628605 TI - Changing hospital practices to increase the duration of breastfeeding. AB - OBJECTIVE: To change the breastfeeding policy in a university teaching hospital in accord with the Ten Steps to Successful Breastfeeding of the United Nations Children's Fund and World Health Organization and to assess the impact of hospital practices on the duration of breastfeeding. METHODS: One hundred ninety two and 392 postpartum women in a maternity ward were interviewed in 1990 and 1993, respectively, regarding how they were feeding their infants and feeding practices in the hospital. Between these two periods, the hospital infant-feeding policy was reviewed and revised in accord with the Ten Steps. Two hundred seventy of the mothers interviewed in 1993 were interviewed again when their infants were 4 months old regarding the duration of full and partial breastfeeding. RESULTS: By 1993, more newborns were put to the breast in the first hour of life (63.2% vs 24.8%); fewer breastfed infants were fed foods other than breast milk (27.9% vs 46.7%); and more mothers received breastfeeding guidance from hospital staff (81.9% vs 61.3%). The duration of breastfeeding in 1993 was longer for women who did not receive formula in the hospital, who were not given discharge packs containing formula and/or coupons, and who roomed-in more than 60% of the time. These associations persisted after controlling for confounding. CONCLUSION: Infant-feeding policies and practices are amenable to change, and policies such as the Baby-Friendly Hospital Initiative may contribute to an increase in the duration of breastfeeding. PMID- 8628606 TI - Do parents and professionals agree on the developmental status of high-risk infants? AB - OBJECTIVES: To examine the degree of agreement between parental reporting of the development of high-risk infants and professional assessment by a multidisciplinary team. METHODS: The developmental status of 196 infants discharged from neonatal intensive care units (NICUs) was assessed by their parents using the Infant Monitoring Questionnaire (IMQ) at 4, 8, or 12 months' corrected age. On the same day, a clinical assessment was done by a multidisciplinary team consisting of a developmental pediatrician, physical therapist, and psychologist. The kappa statistic was used to measure agreement between the assessments. Logistic regression was used to investigate factors that might influence agreement. RESULTS: Both the IMQ and the multidisciplinary team classified infants as developing normally ("normal"), being at risk for abnormal development ("suspect"), or developing abnormally ("abnormal"). Although the same proportion of children fell into the three categories by both assessments, parents and the multidisciplinary team showed poor agreement with respect to the classification of individual infants (kappa = 0.276). No infant or family characteristic was found to have an influence on agreement. CONCLUSIONS: For a group of high-risk infants discharged from NICUs, the agreement between parental assessment of developmental status using the IMQ and the professional assessment by a multidisciplinary team is poor in the first year of life. We do not recommend the use of this questionnaire as a substitute for clinical assessment of biologically at-risk infants discharged from NICUs. However, it may be useful for those groups of infants for whom no other information is available or as an adjunct to clinical assessment when infants are not behaving typically because of an unfamiliar setting or concurrent illness. PMID- 8628607 TI - Advance care planning for children with special health care needs: a survey of parental attitudes. AB - OBJECTIVES: This study explored parental attitudes about their interactions with their children's providers when decision making involved critical life situations. We evaluated parents' attitudes regarding the following questions: What was the parents' understanding of their children's health care issues, and what was the parental perception of the professionals' understanding of their children and of themselves? Who should be the principal decision makers for the children? What was the parents' knowledge about advance directives? Did parents want to participate in a process of advance planning to assist with critical life decision making for their children? METHODS: We surveyed all parents attending a conference sponsored by the Massachusetts Department of Public Health for parents of children with special needs. The questionnaire was provided to all parents attending the conference. An announcement was made at the conference requesting parental participation. The 76 respondents constitute a convenience sample of parents of children with special needs sufficient for this preliminary stage of investigation. RESULTS: Of 177 parents attending the conference, 76 (43%) completed the questionnaire. Eighty-eight percent of the participants strongly agreed that they understood their children's conditions. Twenty-one percent stated that they had sufficient understanding of their children's future medical needs, and 21% thought that they had a sufficient understanding of their children's developmental potential. Ninety-nine percent of parents strongly agreed that physicians should share information with parents no matter how serious or potentially upsetting. Ninety-four percent of those parents who thought that their children's physicians understood their own needs also thought that the physicians understood their children's needs. In contrast, only half (55%) of those parents who thought the physicians did not understand their needs thought the physicians understood their children's needs. Ninety-two percent of parents who thought that the physicians understood their needs agreed that the physicians would make the best decisions in crises versus 60% of those who did not think the physicians understood their needs. Seventy-four percent stated that they would consider written guidelines for their children that dealt with critical life situations. All parents who thought their children's conditions were not understood wanted written guidelines. Of those parents who had thought their children would not survive (15 parents), 94% wanted written guidelines. All seven parents who had been told their children would not survive wanted written guidelines. CONCLUSIONS: Parents in this study were generally satisfied with care being provided to their children. Nevertheless, the results clearly suggest goals that could lead to improved capacity for parents and providers to make critical life decisions for and with children. First, physicians must understand the needs of parents to be able to make decisions that would be in the children's best interests. Second, parents should participate fully in critical life decisions for their children and should use written guidelines to assist with the process of these critical life decisions. Our findings strongly support the development of a longitudinal process, initiated early after the onset or discovery of illness and maintained longitudinally throughout the course of a child's illness, to help parents and providers work together in this vital area of health care to children. PMID- 8628608 TI - Growth and physical outcome of children conceived by in vitro fertilization. AB - OBJECTIVE: To determine the growth and physical outcome at 2 years of age for children born after assisted reproductive techniques in the state of Victoria. DESIGN: Using a case-matched control study between January 1991 and July 1993, 314 children (196 singletons, 47 sets of twins, 8 sets of triplets) conceived after in vitro fertilization (IVF) and related techniques at the Monash IVF and Royal Women's Hospital Reproductive Biology Unit and 150 control children (113 singletons, 17 sets of twins, 1 set of triplets) randomly selected from the general population using the Victorian Perinatal Data Collection Unit records were enrolled to be examined for minor dysmorphic and major organ abnormalities. Singleton and twin cases were matched for plurality and gestation and date of birth. Triplets were not matched. RESULTS: IVF status was not a significant independent factor for physical outcomes, including malformation rates, nor for days of hospitalization postdischarge and operations. There was no significant interaction between IVF status and mean percentiles for weight and head circumference. The IVF group had a greater mean length percentile. Twins in both groups had significantly poorer physical outcomes than singletons on some measures. CONCLUSION: This study did not demonstrate an independent IVF effect on the growth and physical outcome of children at 2 years of age when matched for plurality and gestation. The poor outcomes where noted were related to the effects of multiple births. These findings must be viewed in context of the response rates and therefore representativeness of the data. The need for longitudinal studies is demonstrated. PMID- 8628609 TI - Postdischarge utilization of medical services by high-risk infants: experience in a large managed care organization. AB - BACKGROUND: Infants discharged from intensive care nurseries are a high-risk infant (HRI) population known to have increased utilization of medical services. Most studies tracking HRIs have been based on data obtained from individual chart review or direct patient contact. Given the high cost of such studies, it is desirable to develop less costly methods to track such infants. OBJECTIVES: Our goals were: (1) to identify an HRI cohort at two neonatal intensive care units; (2) to identify a control group of infants not meeting HRI criteria; and (3) to measure outpatient and inpatient utilization in both controls using computerized files in a managed care organization. METHODS: Using California Children's Services criteria as our starting point, we established an HRI definition. From a 1-year birth cohort of 7579 infants at two facilities, we identified 250 infants meeting the HRI definition at two neonatal intensive care units during 1990. We then matched the HRIs with a cohort of 896 randomly selected control newborns (those not meeting the HRI definition). Using organizational computer files and state of California death certificate tapes, we followed these infants until February 28, 1992. We measured the number of hospitalizations, total number of hospital days, and total number of outpatient visits and expressed these outcomes as rates per person-year. We also measured postdischarge mortality. RESULTS: The rate of hospitalization in the HRI group was 6.07 times (95% confidence interval [CI], 4.74-7.77) that in the control group. The utilization of hospital days by the HRI population (hospital days per 1000 person-months) was 13.24 times higher (95% CI, 11.00-16.04). The outpatient visit rate was 1.40 times higher (95% CI, 1.36-1.45) in the HRI population. CONCLUSION: Our findings in a large managed care organization corroborate previous studies showing that hospitalization rates are significantly higher among HRIs. In our study population, outpatient utilization was significantly higher as well. Our study also demonstrates the feasibility of using computerized files to study outcomes in selected pediatric populations. These methods can be used for epidemiologic studies, interventional trials, and planning for resource allocation. PMID- 8628610 TI - Effects of polychlorinated biphenyl/dioxin exposure and feeding type on infants' mental and psychomotor development. AB - OBJECTIVE: To evaluate the effects of in utero and lactational exposure to polychlorinated biphenyls (PCBs) and dioxins on the mental and psychomotor development of infants. DESIGN: Prenatal PCB exposure was estimated from the levels in maternal plasma during the last month of pregnancy. Postnatal PCB and dioxin exposure of breastfed infants was calculated from levels in human milk samples and the duration of breastfeeding. Infants were examined at 3, 7, and 18 months of age with the Bayley Scales of Infant Development. SETTING: General community. PARTICIPANTS: Voluntary sample of 207 mother-infant pairs. One hundred five infants were breastfed and 102 were bottle-fed. INTERVENTIONS: None. RESULTS: Higher in utero exposure to PCBs was associated with lower psychomotor scores at 3 months of age: a doubling of the PCB load resulted in a decrease of 3 points. Breastfed infants scored significantly higher on the psychomotor score at 7 months of age, compared with formula-fed infants. However, when corrected for confounders, the psychomotor score of the 66% highest-exposed breastfed infants ( > 756 pg total PCB-dioxin toxic equivalent) was negatively influenced by this postnatal exposure to PCBs and dioxins, and was comparable to the psychomotor score of the formula-fed infants. Breastfed infants also scored higher on the mental scale at 7 months of age in a dose-dependent way. There was no significant influence of the perinatal PCB and dioxin exposure on the mental outcome at 3 and 7 months of age. At 18 months of age neither the mental nor the psychomotor score was related to perinatal PCB or dioxin exposure, nor to the duration of breastfeeding. CONCLUSIONS: Prenatal PCB exposure has a small negative effect on the psychomotor score at 3 months of age. PCB and dioxin exposure through breastfeeding has an adverse effect on the psychomotor outcome at 7 months of age. The mental outcome at 7 months of age is positively influenced by breastfeeding per se; the perinatal exposure to PCBs and dioxins does not influence this outcome. At 18 months of age the development is affected neither by PCB and dioxin exposure nor by feeding type. PMID- 8628611 TI - Henna: a potential cause of oxidative hemolysis and neonatal hyperbilirubinemia. AB - OBJECTIVE: To evaluate the in vitro oxidation potential of lawsone (2-hydroxy-1,4 naphthoquinone). Lawsone is a chemical present in henna, the crushed leaves of which are used worldwide as a cosmetic agent to stain the hair, skin, and nails. METHODOLOGY: Venous blood from glucose-6-phosphate dehydrogenase (G6PD)-normal and G6PD A- subjects were incubated with various amounts of lawsone for 2 hours at 37 degrees C. Reduced glutathione and methemoglobin (MHb) levels were measured before and after incubation. RESULTS: Final molar concentrations of lawsone in normal blood of 1.4, 2.8, 5.7, and 8.6 x 10-3 mol/L increased MHb percentages from 0.5% to 2.2%, 8.3%, 9.5% and 12.5%, respectively. In a C6PD A- blood, MHb percentages were 19.8%, 32.2%, 44.9%, and 53.9%. At a lawsone concentration of 2.8 x 10-3 mol/L, blood from 15 healthy adults formed MHb percentages of 7.4% +/- 3.3% (+/- 1 SD); in blood from 4 G6PD A- adults, percentages were 44.5%, 40.6%, 41.3%, and 42.8%. Simultaneous measurements of reduced glutathione revealed preincubation values of greater than 40 mg/100 mL of red cells in blood of healthy and G6PD A- subjects. Postincubation values were greater than 40 in blood of healthy subjects and less than 40 in blood of G6PD A- subjects. CONCLUSIONS: These in vitro observations indicate that lawsone is an agent capable of causing oxidative hemolysis. In regions of the world where there is a high incidence of G6PD deficiency and unexplained hyperbilirubinemia, oxidative hemolysis secondary to the cutaneous application of henna could be the initiating event. PMID- 8628612 TI - Natural history of human immunodeficiency virus disease in perinatally infected children: an analysis from the Pediatric Spectrum of Disease Project. AB - OBJECTIVE: To describe the progression of human immunodeficiency virus (HIV) disease through clinical stages from birth to death among a large number of perinatally infected children. METHODS: The Pediatric Spectrum of Disease (PSD) project, coordinated by the Centers for Disease Control and Prevention (CDC), has conducted active surveillance for HIV disease since 1988 in seven geographic regions. PSD data are collected from medical and social service records every 6 months through practitioners at each participating hospital clinic. We analyzed data from perinatally HIV-infected children born between 1982 and 1993. The natural history of HIV disease was divided into five progressive stages using the clinical categories in the CDC 1994 pediatric HIV classification system: stage N, no signs or symptoms; stage A, mild signs or symptoms; stage B, moderate signs or symptoms; stage C, severe signs or symptoms; and stage D, death. A five-stage Markov model was fitted to the PSD data. To compare the estimates from the PSD project with the published estimates, we also fitted an alternative Markov model using acquired immunodeficiency syndrome (AIDS; 1987 case definition) in place of stage C and also calculated standard Kaplan-Meier estimates. RESULTS: A total of 2148 perinatally HIV-infected children were included in the analysis. The estimated mean times spent in each stage were: N, 10 months; A, 4 months; B, 65 months; and C, 34 months. We estimated that a child born with HIV infection has a 50% (95% confidence interval [CI], 40%-60%) chance of severe signs or symptoms developing by 5 years of age and a 75% (95% CI, 68%-82%) chance of surviving to 5 years of age. For a child in stage B, there is a 60% (95% CI, 49%-71%) chance of severe signs or symptoms developing within the next 5 years and a 65% (95% CI, 56%-73%) chance of surviving 5 more years. The estimated mean time from birth to stage C was 6.6 (95% CI, 5.7-7.5) years, and the estimated mean survival time from birth was 9.4 (95% CI, 8.1-10.7) years. From the alternative Markov model, the estimated mean time from birth to AIDS was 4.8 (95% CI, 4.5-5.2) years. CONCLUSION: Markov modeling using the revised pediatric classification system allowed us to describe the natural history of HIV disease in children before diagnosis of AIDS. On average, children progress to moderate symptoms in the second year of life and then remain moderately symptomatic for more than half of their expected lives, underscoring their need for clinical care before the onset of AIDS. The results from the Markov model are useful in family counseling, health care planning, and clinical trial designs. PMID- 8628613 TI - Methylene blue-induced phototoxicity: an unrecognized complication. AB - OBJECTIVE: To describe photosensitization after prenatal exposure to a toxic amount of methylene blue and to alert pediatricians that, in a review of the literature, photosensitization (which this dye is capable of) has not been reported as a complication of prenatal exposure. DESIGN AND PATIENTS: A descriptive report of physical findings and significant laboratory tests in a very low birth weight preterm infant with prenatal exposure to methylene blue and a comparison of this reported case with previously described patients' complications and treatment. SETTING: Neonatal intensive care unit. INTERVENTION: Monitoring of laboratory tests to assess for methylene blue toxicity: two exchange transfusions for methemoglobinemia, hemolytic anemia, and hyperbilirubinemia; phototherapy for hyperbilirubinemia; and pathologic examination of skin bullae. RESULTS: Within hours of exposure to phototherapy, redness developed on all exposed areas of the patient's skin (which was initially deep blue), followed by bullae and desquamation of about 35% of the total skin surface area. The desquamation of erythematous areas continued even after discontinuation of phototherapy. Complete re-epithelialization was attained by 3 weeks of age. In addition to this newly observed complication, the patient had other previously described toxic effects. CONCLUSION: We have reported a previously unrecognized complication associated with high prenatal exposure to methylene blue and treatment with phototherapy. Methylene blue phototoxicity may be related to the high prenatal dose of the dye relative to patient's small size and young gestational age. PMID- 8628614 TI - Nebulized budesonide is as effective as nebulized adrenaline in moderately severe croup. AB - OBJECTIVE: Nebulized budesonide and nebulized adrenaline have been shown to be effective in the treatment of moderately severe croup. However, there has been no direct comparison of these therapies. We undertook a multicenter, randomized, double-blind, parallel group study in 66 hospitalized children with viral or spasmodic croup. METHODS: Children 0.5 to 6 years of age were assessed using a validated croup symptom score (stridor, 0 through 4; cough, 0 through 3; retractions, 0 through 3; dyspnea, 0 through 3; and color, 0 through 4) at 0.5, 1, 1.5, 2, 12, and 24 hours after nebulization. Patients received either budesonide (2 mg/4 mL) or L-adrenaline (4 mg/4 mL) via nebulization. The primary outcome measure was change in the total croup symptom score. RESULTS: Thirty-five children received budesonide and 31 received adrenaline. There was no significant difference in baseline features, including croup score (mean [95% confidence interval]: budesonide, 7.1 [6.7-7.5]; adrenaline, 7.7 [7.3-8.1]). All patients had significant improvement from baseline, and there was not significant difference between the two treatments, as measured by change in croup scores, change in oxygen saturation, duration of hospitalization, number of subsequent treatments with systemic steroids or adrenaline, and adverse events. No child required intubation. CONCLUSION: This study does not show any difference in efficacy and safety between nebulized budesonide and nebulized adrenaline in the treatment of acute upper airway obstruction in patients with moderately severe croup. PMID- 8628615 TI - Percutaneous endoscopic gastrostomy in children: influence on gastroesophageal reflux. AB - OBJECTIVE: Few data exist in the literature about the relationship between percutaneous endoscopic gastrostomy (PEG) and gastroesophageal reflux (GER) in children, and the data that do exist are contradictory. The aim of the present study was to evaluate the effect of PEG on GER. METHODS: Twenty children underwent PEG for enteral nutrition. They were 55 +/- 55 months old and weighed 13 +/- 10 kg. A pH study was performed before and after PEG without treatment when GER status was unknown (n = 10) or under treatment (n = 10) if previous GER was demonstrated. In these cases, the pH study was performed under the same treatment before and after PEG. RESULTS: Six pH studies had abnormal results before PEG. After PEG, the GER of these 6 children significantly improved after the treatment was intensified (n = 50 or spontaneously normalized (n = 1). Results of 13 pH studies that were previously normal remained normal. Only one child with a normal reflux index before PEG had GER after it. For the 20 children, the mean reflux index did not change significantly after PEG (5.5% vs 5.6%). CONCLUSION: Contrary to surgical gastrostomy, PEG does not worsen GER. Therefore, GER is not a contraindication to PEG. PMID- 8628616 TI - Prenatal pediatrics: traditional specialty definitions no longer apply. PMID- 8628617 TI - The new edition of the Guide to Clinical Preventive Services. PMID- 8628619 TI - Recurrent thrombocytopenic purpura after repeated measles-mumps-rubella vaccination. PMID- 8628618 TI - The critical role of provider practices in undervaccination. PMID- 8628621 TI - Acute renal failure secondary to pyelonephritis. PMID- 8628620 TI - Gram-negative bacteremia in four patients with Klippel-Trenaunay-Weber syndrome. PMID- 8628622 TI - Avian mite dermatitis. PMID- 8628624 TI - Promotion of healthy weight-control practices in young athletes. American Academy of Pediatrics. Committee on Sports Medicine and Fitness. PMID- 8628623 TI - The adolescent's right to confidential care when considering abortion. American Academy of Pediatrics. Committee on Adolescence. AB - In this statement, the American Academy of Pediatrics (AAP) reaffirms its position that the rights of adolescents to confidential care when considering abortion should be protected. The AAP supports the recommendations presented in the report on mandatory parental consent to abortion by the Council on Ethical and Judicial Affairs of the American Medical Association. Adolescents should be strongly encouraged to involve their parents and other trusted adults in decisions regarding pregnancy termination, and the majority of them voluntarily do so. Legislation mandating parental involvement does not achieve the intended benefit of promoting family communication, but it does increase the risk of harm to the adolescent by delaying access to appropriate medical care. The statement presents a summary of pertinent current information related to the benefits and risks of legislation requiring mandatory parental involvement in an adolescent's decision to obtain an abortion. The AAP acknowledges and respects the diversity of beliefs about abortion and affirms the value of voluntary parental involvement in decision making by adolescents. PMID- 8628625 TI - School Transportation Safety. American Academy of Pediatrics. Committee on School Health and Committee on Injury and Poison Prevention. AB - The following policy statement is a revision of the American Academy of Pediatrics' 1985 statement entitled "School Bus Safety." It provides updated information regarding relevant federal regulations and outlines recommendations that can enhance community systems for addressing school bus safety education, awareness, and practices. Pediatricians can assist in this process by sharing these recommendations at both the community and state levels. PMID- 8628626 TI - Safe transportation of premature and low birth weight infants. American Academy of Pediatrics. Committee on Injury and Poison Prevention and Committee on Fetus and Newborn. AB - Special considerations are essential to ensure the safe transportation of premature and low birth weight infants. Both physical and physiologic issues must be considered in the proper positioning of these infants. This statement discusses current recommendations based on the latest research and provides guidelines for physicians who counsel parents of very small infants on the choice of the best car safety seats for their infants. PMID- 8628627 TI - Selecting and using the most appropriate car safety seats for growing children: guidelines for counseling parents. American Academy of Pediatrics. Committee on Injury and Poison Prevention. AB - Despite the existence of laws in all 50 states requiring the use of car safety seats or child restraint devices for young children, many children continue to be killed each year while riding in motor vehicles. Pediatricians and other health care professionals need to provide up-to-date, appropriate information for parents regarding their car safety seat choices and use. This policy statement discusses current recommendations based on the latest research and the factors parents should consider before selecting and using a car seat. PMID- 8628628 TI - Efforts to reduce the toll of injuries in childhood require expanded research. American Academy of Pediatrics. Committee on Injury and Poison Prevention. AB - Efforts to control injury, like those to control disease, are based on epidemiologic evidence that most occurrences are both predictable and preventable. Effective injury control programs depend heavily on evidence generated as a result of a strong research agenda. This policy statement outlines the magnitude of the injury toll and the prevention efforts that have expanded during the past decade. Even with this expansion, the level of federal commitment to research aimed at reducing the toll of injury does not match the magnitude of the injury problem. Much research remains to be done, and the AAP recommends a multilevel and multifaceted approach to prevention and treatment, based on injury morbidity and mortality data. As injury control methods of proven efficacy continue to be developed, pediatricians can apply them to their patients, and thereby reduce the toll of childhood injury. PMID- 8628629 TI - Practice parameter: the neurodiagnostic evaluation of the child with a first simple febrile seizure. American Academy of Pediatrics. Provisional Committee on Quality Improvement, Subcommittee on Febrile Seizures. AB - The American Academy of Pediatrics and its Provisional Committee on Quality Improvement, in collaboration with experts from the Section on Neurology, general pediatricians, consultants in the fields of neurology and epilepsy, and research methodologists, developed this practice parameter. This parameter provides recommendations for the neurodiagnostic evaluation of a child with a first simple febrile seizure. These recommendations derive from both a thorough review of the literature and expert consensus. Interventions of direct interest include lumbar puncture, electroencephalography, blood studies, and neuroimaging. The methods and results of the literature review and data analyses can be found in the technical report that is available from the Publications Department of the American Academy of Pediatrics. This parameter is designed to assist pediatricians by providing an analytic framework for the evaluation and treatment of this condition. It is not intended to replace clinical judgement or establish a protocol for all patients with this condition. It rarely will be the only appropriate approach to the problem. PMID- 8628631 TI - Homeopathic treatment of childhood diarrhea. PMID- 8628630 TI - Homeopathic treatment of childhood diarrhea. PMID- 8628632 TI - Homeopathic treatment of childhood diarrhea. PMID- 8628633 TI - Homeopathic medicine. PMID- 8628634 TI - Reappraisal of lytic cocktail/demerol, phenergan, and thorazine (DPT) for the sedation of children. PMID- 8628635 TI - Cerebral palsy in very low birth weight infants, pre-eclampsia and magnesium sulphate. PMID- 8628636 TI - Acute isoniazid neurotoxicity in an urban hospital. PMID- 8628637 TI - Isoniazid neurotoxicity. PMID- 8628638 TI - Rapid streptococcal tests. PMID- 8628639 TI - [Declaration or legislation on patients' rights?]. PMID- 8628640 TI - [Patients' rights--a Scandinavian perspective]. PMID- 8628641 TI - [More aggressive leadership by physicians is needed in future health care. Interview by Eva Oldinger]. PMID- 8628642 TI - [Diagnosis and treatment of conjunctivitis]. AB - Conjunctivitis is a frequent reason for consulting a doctor. Most often treatment consists of antibiotics, even though sensitive bacteria are only rarely demonstrated. In our part of the world conjunctivitis is self-limiting, and it may be worth considering the effect of treatment, if any. With this in mind, diagnosis and differential diagnoses are reviewed and with regard to treatment distinctions are made between neonatal conjunctivitis (gonococcus, chlamydia, virus), conjunctivitis in children (symptoms of upper respiratory tract infections), and conjunctivitis in adults and the elderly (dry eyes, epiphora in ectropion). PMID- 8628643 TI - [Health service's responsibility for sexually abused victims]. AB - The abuse of women is a global problem, a women's health problem, and an egalitarian issue. In Sweden alone, 15,000 cases of the abuse of women, where victim and perpetrator were acquainted, were reported in 1994. In 1995, the National Women's Centre was inaugurated at the University Hospital, Uppsala, for the care of woman exposed to assault, abuse and sexual violence. Both care, research and training of personnel are carried out at the centre. Every abused woman coming to the hospital is now taken care of by specially trained personnel. The centre works in close cooperation with the police and the voluntary crisis centre, where battered women can receive support and help--for instance with temporary accommodation. PMID- 8628644 TI - [Glucose toxicity--pros and cons. Useful adaptation and common cause of insulin resistance in diabetic patients]. AB - Insulin resistance is directly proportional to the degree of glycaemic control in patients with insulin-dependent or non-insulin-dependent diabetes mellitus (IDDM and NIDDM) in whom hyperglycaemia induces insulin resistance within 24 hours. Glucose toxicity (i.e., glucose-induced insulin resistance) explains why apparently unrelated therapeutic measures to improve glycaemic control (e.g., weight reduction, and sulphonylurea, metformin or insulin administration) all also increase insulin sensitivity. The improvement is manifest when insulin stimulated glucose uptake is measured at similar glucose and insulin concentrations in IDDM and NIDDM patients. In daily life, however, both IDDM and NIDDM patients utilise as much glucose in insulin-sensitive tissues as do non diabetic individuals since, due to the mass action effect of glucose, glucose uptake increases in response to hyperglycaemia. Recent studies have shown that the hexosamine pathway may act as a cellular glucose monitor maintaining steady state glucose flux under both normo- and hyper-glycemic conditions. Thus, glucose toxicity may be seen as a mechanism protecting insulin-sensitive tissues from excessive glucose uptake and diabetic complications. PMID- 8628646 TI - [Medical ethics and assistance in suicide]. PMID- 8628645 TI - [European practicing physicians want to improve education in general practice]. PMID- 8628648 TI - Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence. AB - The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17 specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo. PMID- 8628647 TI - Fate of direct and inverted repeats in the RNA hypermutagenesis reaction. AB - RNA hypermutagenesis results from cDNA synthesis in the presence of highly biased dNTP precursor concentrations and preferentially exploits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Such reaction conditions slow down DNA synthesis, which might be conducive to strand transfer and deletion. This has been investigated. A 6 bp inverted repeat nested between 10 bp repeats was efficiently deleted at dCTP concentrations typically used. Inter- or intramolecular strand transfer between 10 bp repeated sequences separated by runs of templated G residues occurred, but at lower concentrations. If RNA hypermutagenesis of a sequence containing direct and inverted repeats is unavoidable, avian myeloblastosis virus (AMV) reverse transcriptase could be used, as strand transfer occurs with much diminished dCTP substrate dependence. PMID- 8628649 TI - Characterization of the AB (AF-1) region in the muscle-specific retinoid X receptor-gamma: evidence that the AF-1 region functions in a cell-specific manner. AB - The retinoid X receptors alpha, beta and gamma (RXRs) share a highly conserved 'C' region or DNA binding domain (DBD). The conserved 'DE' region or ligand binding domain (LBD) of the RXRs is functionally complex, mediating dimerization and a ligand-dependent activation function (AF-2). The AB or N-terminal region of the RXRs is poorly conserved and encodes a ligand-independent activation function (AF-1). RXR gamma mRNA is preferentially expressed in skeletal and cardiac muscle, however, cell-specific steroid receptor-mediated trans-activation is a poorly understood phenomenon. We utilized the GAL4 hybrid assay system and have demonstrated that RXR gamma contains two functional domains in the AB and DE regions that activate transcription in a ligand-independent and -dependent manner respectively. The functions of the AB (AF-1) and DE (AF-2) domains were regulated by cAMP-dependent protein kinases, furthermore, the function of AF-2 in the LBD was activated by 8-Br-cAMP, independent of 9-cis-retinoic acid treatment. Deletion analysis demonstrated that the AF-1 of RXR gamma, is located between amino acids 1 and 103 and contained multiple motifs that were targets of cAMP dependent protein kinases. Transfection analyses in non-muscle and myogenic cells clearly demonstrated that: (i) the AF-1 of RXR gamma functions in a muscle specific manner and is required for optimal ligand-dependent trans-activation from an RXRE; (ii) RXR gamma trans-activates more efficiently in a myogenic background. PMID- 8628650 TI - A promotor directing alpha-amanitin-sensitive transcription of GARP, the major surface antigen of insect stage Trypanosoma congolense. AB - The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein), which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is alpha-amanitin sensitive. Whilst a transcriptional gap 5' of the first GARP gene in the cloned region of the 4.4 locus could not be detected, such a gap was present in the 5' flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and, using reporter gene constructs containing a putative GARP promoter region in transient transfection studies, we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens, GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5' RT PCR and demonstrated microheterogeneity in GARP polyadenylation by 3' RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals, although similar, are also species-specific. PMID- 8628651 TI - Distamycin-induced inhibition of formation of a nucleoprotein complex between the terminase small subunit G1P and the non-encapsidated end (pacL site) of Bacillus subtilis bacteriophage SPP1. AB - The small subunit of the Bacillus subtilis bacteriophage SPP1 terminase (G1P) forms a sequence-specific nucleoprotein complex with the SPP1 non-encapsidated end (pacL site) during initiation of DNA encapsidation. Gel mobility shift assay was used to study the G1P-pacL interaction. Distamycin, a minor groove binder that induces local distortion of the DNA, inhibits G1P-pacL complex formation. The competition of G1P with distamycin for DNA binding at the pacL site is independent of the order of addition of the reactants. Other minor groove binders, such as spermine or Hoechst 33258, which do not distort DNA, failed to compete with G1P for pacL DNA binding. Cationic metals, which generate a repertoire of DNA structures different from that caused by the minor groove binders, can partially reverse the distamycin-induced inhibition of G1P binding to pacL DNA. The major groove binder methyl green, which does not distort sequence-directed bending of pacL DNA, competes with G1P for binding at the pacL site. Our data suggest that the natural sequence-directed bend that exists within the pacL site is the architectural element that facilitates assembly of a nucleoprotein complex and hence initiation of DNA encapsidation by bacteriophage SPP1. PMID- 8628652 TI - Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two dimensional gel electrophoresis. AB - We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID- 8628653 TI - Investigation of the intracellular stability and formation of a triple helix formed with a short purine oligonucleotide targeted to the murine c-pim-1 proto oncogene promotor. AB - In our previous work we have shown that the oligonucleotide 5'-GGGGAGGGGGAGG-3' gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro[Svinarchuk, F., Bertrand, J.-R. and Malvy, C.(1994)Nucleic Acids Res., 22, 3742-3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3' terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells. PMID- 8628654 TI - Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development. AB - The cDNA clones encoding ARE(Na,K-ATPase alpha1 subunit gene regulatory element) binding protein AREC3 were isolated from myoblast C2C12 cells and mouse skeletal muscle cDNA library. At least four alternatively spliced forms of AREC3 cDNA were identified. Sequence analysis indicates that AREC3 has an extensive homology with the Drosophila sine oculis gene product required for development of the entire visual system [Cheyette et al.(1994) Neuron 12, 977-996]. The homologous region including a homeodomain is required for specific DNA binding to ARE. A transactivation domain was identified in the C-terminal part of the AREC3 by reporter gene assays using GAL4-AREC3 fusion protein constructs. Immunohistochemistry revealed that AREC3 localized to the nucleus and cytoplasm of myoblast C2C12 cells, and the production of AREC3 is augmented during muscle differentiation. Western blot analysis indicated that the 115 kDa form of AREC3 protein is increased in the cytoplasmic extract, and the 67kDa form is increased both in nuclear and cytoplasmic extracts of C2C12 cells during muscle differentiation. PMID- 8628655 TI - Backbone and benzoyl mustard carrying moiety modifies DNA interactions of distamycin analogues. AB - Alkylating distamycin derivative FCE-24517 (l) is the prototype of a novel class of alkylating agents. In the present study we have investigated the effect of further chemical modifications introduced in the alkylating distamycin-derived molecule with the aim of improving their ability to bind DNA. The new compound, MEN 10710 (II), has a four pyrrolecarboxamide backbone linked at its N-terminus and through a butanamido residue to a 4-[bis(chloroethyl)amino]phenyl moiety. We have demonstrated that the presence of the flexible trimethylene chain confers to the novel distamycin derivative a peculiar mode of interaction with DNA as compared with I or melphalan. In fact, interstrand cross-links are detected in DNA samples treated even with low concentrations of II (being 200-fold more efficient than melphalan) but not with I. Similar results were obtained with a related compound of II containing a three pyrrole ring backbone. Compound II induces a conformational change in the DNA structure as deduced from the inhibition of T4 DNA ligase activity. In alkylation experiments, unlike melphalan, both I and II induce DNA breaks at bases closely located to AT-rich tracts, however II was more potent than I in producing greater amount of covalent adducts. These data suggest that the new compound shows a different and peculiar mechanism of interaction with DNA. PMID- 8628656 TI - Cleaning the GenBank Arabidopsis thaliana data set. AB - Data driven computational biology relies on the large quantities of genomic data stored in international sequence data banks. However, the possibilities are drastically impaired if the stored data is unreliable. During a project aiming to predict splice sites in the dicot Arabidopsis thaliana, we extracted a data set from the A.thaliana entries in GenBank. A number of simple 'sanity' checks, based on the nature of the data, revealed an alarmingly high error rate. More than 15% of the most important entries extracted did contain erroneous information. In addition, a number of entries had directly conflicting assignments of exons and introns, not stemming from alternative splicing. In a few cases the errors are due to mere typographical misprints, which may be corrected by comparison to the original papers, but errors caused by wrong assignments of splice sites from experimental data are the most common. It is proposed that the level of error correction should be increased and that gene structure sanity checks should be incorporated--also at the submitter level--to avoid or reduce the problem in the future. A non-redundant and error corrected subset of the data for A.thaliana is made available through anonymous FTP. PMID- 8628657 TI - Visualization and analysis of unfolded nucleosomes associated with transcribing chromatin. AB - We have characterized the structure of transcriptionally active nucleosome subunits using electron spectroscopic imaging. Individual nucleosomes were analyzed in terms of total mass, DNA and protein content, while the ensemble of images of active nucleosomes was used to calculate a three-dimensional reconstruction. Transcriptionally active nucleosomes were separated from inactive nucleosomes by mercury-affinity chromatography thus making it possible to compare their structures. The chromatographic results combined with electron spectroscopic imaging confirm that active nucleosomes unfold to form extended U shaped particles. Phosphorus mapping indicated that the nucleosomal DNA also underwent a conformational change consistent with particle unfolding. The three dimensional structure of the Hg-affinity purified nucleosomes determined using quaternion-assisted angular reconstitution methods unites and resolves the different electron microscopic views of the particle and is concordant with a sulphydryl-exposing disruption of the H3-H4 tetramer. PMID- 8628658 TI - Unwinding of the third strand of a DNA triple helix, a novel activity of the SV40 large T-antigen helicase. AB - We present experiments indicating that the SV40 large T-antigen (T-ag) helicase is capable of unwinding the third strand of DNA triple helices. Intermolecular d(TC)(20)d(GA)(20)d(TC)(20) triplexes were generated by annealing, at pH 5.5, a linearized double-stranded plasmid containing a d(TC)(27).d(GA)27 tract with a (32)P-labeled oligonucleotide consisting of a d(TC)(20) tract flanked by a sequence of 15 nt at the 3'-end. The triplexes remained stable at pH 7.2, as determined by agarose gel electrophoresis and dimethyl sulfate footprinting. Incubation with the T-ag helicase caused unwinding of the d(TC)(20) tract and consequent release of the oligonucleotide, while the plasmid molecules remained double-stranded. ATP was required for this reaction and could not be replaced by the non-hydrolyzable ATP analog AMP-PNP. T-ag did not unwind similar triplexes formed with oligonucleotides containing a d(TC)(20) tract and a 5' flanking sequence or no flanking sequence. These data indicate that unwinding of DNA triplexes by the T-ag helicase must be preceded by binding of the helicase to a single-stranded 3' flanking sequence, then the enzyme migrates in a 3'--> 5' direction, using energy provided by ATP hydrolysis, and causes release of the third strand. Unwinding of DNA triplexes by helicases may be required for processes such as DNA replication, transcription, recombination and repair. PMID- 8628659 TI - DNA adducts of antitumor trans-[PtCl2 (E-imino ether)2]. AB - It has been shown recently that some analogues of clinically ineffective trans diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans [PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy. PMID- 8628661 TI - DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein. AB - Strand displacement amplification (9SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection. We have combined SDA with fluorescence polarization detection. A 5'-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single- to double strand conversion is monitored through an increase in fluorescence polarization. Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence. During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity. We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis. PMID- 8628662 TI - An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution. AB - The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3' endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering. PMID- 8628660 TI - CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells. AB - Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter. PMID- 8628663 TI - Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides. AB - We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides. PMID- 8628664 TI - The C-terminal region of Drosophila heat shock factor (HSF) contains a constitutively functional transactivation domain. AB - The heat shock transcription factor (HSF) is constitutively expressed in Drosophila cells as an inactive monomer. Upon heat shock HSF undergoes trimerization and acquires high affinity DNA binding ability leading to specific interaction with its cognate elements in heat shock promoters. Here we show that the transactivation function of HSF is conferred by the extreme C-terminal region of the protein. Deletion analysis of HSF fragments fused to the GAL4 DNA-binding domain demonstrates that transactivation is dependent on HSF residues 610-691. This domain is located beyond the C-terminal heptad repeat (leucine zipper 4) whose presence or integrity is dispensable for transactivation. The transactivation domain is functional in the absence of heat shock and can be replaced by the extreme C-terminal region of human HSF1. The Drosophila and human HSF transactivation domains are both rich in hydrophobic and acidic residues and may be structurally conserved, despite limited sequence identity. PMID- 8628665 TI - Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR. AB - The microreaction volumes of PCR chips (a microfabricated silicon chip bonded to a piece of flat glass to form a PCR reaction chamber) create a relatively high surface to volume ratio that increases the significance of the surface chemistry in the polymerase chain reaction (PCR). We investigated several surface passivations in an attempt to identify 'PCR friendly' surfaces and used those surfaces to obtain amplifications comparable with those obtained in conventional PCR amplification systems using polyethylene tubes. Surface passivations by a silanization procedure followed by a coating of a selected protein or polynucleotide and the deposition of a nitride or oxide layer onto the silicon surface were investigated. Native silicon was found to be an inhibitor of PCR and amplification in an untreated PCR chip (i.e. native slicon) had a high failure rate. A silicon nitride (Si(3)N(4) reaction surface also resulted in consistent inhibition of PCR. Passivating the PCR chip using a silanizing agent followed by a polymer treatment resulted in good amplification. However, amplification yields were inconsistent and were not always comparable with PCR in a conventional tube. An oxidized silicon (SiO(2) surface gave consistent amplifications comparable with reactions performed in a conventional PCR tube. PMID- 8628666 TI - Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips. AB - We examined PCR in silicon dioxide-coated silicon-glass chips (12 microl in volume with a surface to volume ratio of approximately 17.5 mm(2)/microl) using two PCR reagent systems: (i) the conventional reagent system using Taq DNA polymerase; (ii) the hot-start reagent system based on a mixture of TaqStart antibody and Taq DNA polymerase. Quantitative results obtained from capillary electrophoresis for the expected amplification products showed that amplification in microchips was reproducible (between batch coefficient of variation 7.71%) and provided excellent yields. We also used the chip for PCR directly from isolated intact human lymphocytes. The amplification results were comparable with those obtained using extracted human genomic DNA. This investigation is fundamental to the integration of sample preparation, polynucleotide amplification and amplicate detection on a microchip. PMID- 8628668 TI - RNA fingerprinting by molecular indexing. PMID- 8628667 TI - Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p. AB - DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences. PMID- 8628669 TI - Repair of products of oxidative DNA base damage in human cells. AB - Oxidative DNA damage is the most frequent type of damage encountered by aerobic cells and may play an important role in biological processes such as mutagenesis, carcinogenesis and aging in humans. Oxidative damage generates a myriad of modifications in DNA. We investigated the cellular repair of DNA base damage products in DNA of cultured human lymphoblast cells, which were exposed to oxidative stress by H2O2. This DNA-damaging agent is known to cause base modifications in genomic DNA of mammalian cells [Dizdaroglu, M., Nackerdien, Z., Chao, B.-C., Gajewski, E. and Rao, G. (1991) Arch. Biochem. Biophys. 285, 388 390]. Following treatment with H2O2, the culture medium was freed from H2O2 and cells were incubated for time periods ranging from 10 min to 6 h. DNA was isolated from control cells, hydrogen peroxide-treated cells and cells incubated after H2O2 exposure. DNA samples were analyzed by gas chromatography/isotope dilution mass spectrometry. Eleven modified bases were identified and quantified. The results showed a significant formation of these DNA base products upon H2O2 treatment of cells. Subsequent incubation of cells caused a time-dependent excision of these products from cellular DNA. The cell viability did not change significantly by various treatments. There were distinct differences between the kinetics of excision of individual products. The observed excisions were attributed to DNA repair in cells. The rate of repair of purine lesions was slower than that of pyrimidine lesions. Most of the identified products are known to possess various premutagenic properties. The results of this work may contribute to the understanding of the cellular repair of oxidative DNA damage in human and other mammalian cells. PMID- 8628670 TI - Palingol: a declarative programming language to describe nucleic acids' secondary structures and to scan sequence database. AB - At the DNA/RNA level, biological signals are defined by a combination of spatial structures and sequence motifs. Until now, few attempts had been made in writing general purpose search programs that take into account both sequence and structure criteria. Indeed, the most successful structure scanning programs are usually dedicated to particular structures and are written using general purpose programming languages through a complex and time consuming process where the biological problem of defining the structure and the computer engineering problem of looking for it are intimately intertwined. In this paper, we describe a general representation of structures, suitable for database scanning, together with a programming language, Palingol, designed to manipulate it. Palingol has specific data types, corresponding to structural elements-basically helices-that can be arranged in any way to form a complex structure. As a consequence of the declarative approach used in Palingol, the user should only focus on 'what to search for' while the language engine takes care of 'how to look for it'. Therefore, it becomes simpler to write a scanning program and the structural constraints that define the required structure are more clearly identified. PMID- 8628671 TI - Regulation of Cre recombinase activity by the synthetic steroid RU 486. AB - To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting. PMID- 8628672 TI - DNA-protein interactions at the telomeric repeats of Schizosaccharomyces pombe. AB - Gel retardation assays using a probe containing the repeat region of a Schizosaccharomyces pombe chromosomal telomere identified four specific DNA- protein complexes in S. pombe total protein extracts (I, I', IIa and IIb). The proteins responsible for these complexes bound to the telomeric repeat region irrespective of whether or not the repeats were in close proximity to the end of a DNA molecule, and none of them bound strongly to single-stranded DNA. The protein responsible for complex I (TeRF I) was separated from the activity responsible for complexes IIa and IIb (TeRF II) using heparin-Sepharose chromatography. Both factors were efficiently cross-competed by an oligonucleotide containing the 18 bp sequence 5'-GGTTACAGGTTACAGGTT-3', which corresponds to two complete telomeric repeat units. Mutation of the T residues at positions 4 and 11 in the oligonucleotide dramatically reduced binding by TeRF II, but had no affect on binding by TeRF I. The protein responsible for complex I' did not bind strongly to either the wild-type or mutant oligonucleotide. PMID- 8628673 TI - Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication. AB - We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro. PMID- 8628674 TI - Afut1, a retrotransposon-like element from Aspergillus fumigatus. AB - A repeated DNA sequence used for epidemiological studies of the human opportunistic pathogen Aspergillus fumigatus has been characterized. It is a retroelement of 6914 bp in length, bounded by long terminal repeats of 282 bp, with sequence and features characteristic of retroviruses and retrotransposons. A 5 bp duplication site was found at its borders. This element, designated Afut1, encodes amino acid sequences homologous to the reverse transcriptase, RNase H and endonuclease encoded by the pol genes of retroelements. Comparison of the peptidic sequences with other putative polypeptides of fungal LTR retrotransposons showed that Afut1 is a member of the gypsy group. This is the first report of a transposable element in A.fumigatus. Afut1 is a defective element: the putative coding domains contain multiple stop codons due exclusively to transitions from C:G to T:A. PMID- 8628675 TI - Multiplex messenger assay: simultaneous, quantitative measurement of expression of many genes in the context of T cell activation. AB - The hybridization signature approach, using colony filters and labeled complex probes, can provide high throughput measurement of gene activity. We describe here the implementation of this method to follow the expression levels of 47 genes in resting and activated T cells, as well as in epithelial cells. Using 4 fold spotting of colonies, imaging plate detection and various correction and normalization procedures, the technique is sensitive enough to quantify expression levels for sequences present at 0.005% abundance in the probe. Comparison with Northern blotting shows good consistency between the two methods. Upon activation of a T cell clone by an anti-CD3 antibody variations ranging from 2- to 20-fold are measured, some of which had not been reported previously. This 'multiplex messenger assay' method, performed using available commercial apparatus, can be used in many cases where simultaneous assessment of mRNA levels for many genes is of interest. PMID- 8628676 TI - Genomic position effects lead to an inefficient reorganization of nucleosomes in the 5'-regulatory region of the chicken lysozyme locus in transgenic mice. AB - The chicken lysozyme locus is gradually activated during macrophage development exhibiting a specific chromatin structure with each differentiation state. Its small size and the extensive characterization of its cis-regulatory elements allows us to study even subtle changes in chromatin structure of the entire gene locus during transcriptional activation. Tissue-specific and position independent expression of the lysozyme locus in transgenic mice requires the cooperation of all cis-regulatory elements. In order to elucidate further the molecular basis of locus activation, we have determined nucleosome positions within the complete 5' regulatory region of the chicken lysozyme locus in chicken myeloid cell lines and transgenic mice. Each cis-regulatory element develops its unique nucleosomal structure and each one remodels chromatin differently. The nucleosomal organization of the endogenous gene in chicken cell lines and the transgene in the mouse turned out to be identical, enabling us to study the influence of cis regulatory deletions on the development of an active chromatin structure in transgenic mice. Transgenes with a deletion of an important cis-regulatory element show an impediment in nucleosome reorganization as compared with the complete lysozyme locus. We demonstrate that multicopy transgene-clusters in position dependently expressing mouse lines exhibit a heterogeneous chromatin organization. PMID- 8628677 TI - Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding. AB - The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1. PMID- 8628678 TI - The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism. AB - For the first time, the interaction between a series of small molecules and the TAR RNA of HIV-1 has been investigated by electric linear dichroism (ELD). The compounds tested include the DNA intercalating drugs proflavine and ethidium bromide and an amsacrine-4-carboxamide DNA-threading intercalator as well as the AT-specific DNA minor groove binders netropsin, Hoechst 33258, berenil and DAPI. In all cases except for netropsin, negative reduced dichroism signals were measured in the drug absorption band. In agreement with previous studies, the results indicate that both classical and threading intercalation can occur with the TAR RNA. The ELD data show that the mode of binding of the drugs Hoechst 33258, berenil and DAPI to the TAR RNA is similar to their binding mode in GC rich regions of DNA and likely involves intercalation into the A-form TAR RNA helix. The wide and shallow minor groove of the TAR RNA is apparently not accessible to DNA minor groove binding drugs such as netropsin. The ELD technique appears uniquely valuable as a means of investigating the interaction of drugs with the TAR RNA. PMID- 8628679 TI - Age-dependent silencing of globin transgenes in the mouse. AB - Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation. PMID- 8628681 TI - The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma. AB - We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases. PMID- 8628680 TI - Structure of recombinant rat UBF by electron image analysis and homology modelling. AB - We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments. PMID- 8628682 TI - 2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver. AB - The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals. PMID- 8628683 TI - The catalytic core of RNase P. AB - A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA. PMID- 8628684 TI - Screening of differentially amplified cDNA products from RNA arbitrarily primed PCR fingerprints using single strand conformation polymorphism (SSCP) gels. AB - Arbitrarily primed PCR fingerprinting of RNA and differential display resolved on an acrylamide gel has been extensively used to detect differentially expressed RNAs. However, after a differentially amplified product is detected the next steps are labor-intensive: a small portion of the fingerprinting gel that contains the differentially amplified product is cut out, reamplified and the correct product is determined, typically by cloning and sequencing what is often a mixture of products of similar size. Here we use a native acrylamide gel to separate DNAs in the reamplified mixture based on single-stranded conformation polymorphisms. Reamplifications are performed for the region carrying the differentially amplified product and a corresponding region from an adjacent lane where the product is less prominent or not visible. Denaturation of the reamplified DNA followed by side-by-side comparison on an SSCP gel allows the classification of reamplified material into (i) those that can be directly cloned because the differentially amplified product is relatively pure, (ii) those that need to be reamplified from the SSCP gel before cloning and (iii) those that are too complex for further study. This screen should save considerable effort now wasted on directly cloning unsuitable products from RNA fingerprinting experiments. An example is presented of cloning a gene differentially expressed in Trypanosoma brucei life cycle. PMID- 8628685 TI - Oligonucleotide N3'-->P5' phosphoramidates as antisense agents. AB - Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3' oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents. PMID- 8628686 TI - SAGA: sequence alignment by genetic algorithm. AB - We describe a new approach to multiple sequence alignment using genetic algorithms and an associated software package called SAGA. The method involves evolving a population of alignments in a quasi evolutionary manner and gradually improving the fitness of the population as measured by an objective function which measures multiple alignment quality. SAGA uses an automatic scheduling scheme to control the usage of 22 different operators for combining alignments or mutating them between generations. When used to optimise the well known sums of pairs objective function, SAGA performs better than some of the widely used alternative packages. This is seen with respect to the ability to achieve an optimal solution and with regard to the accuracy of alignment by comparison with reference alignments based on sequences of known tertiary structure. The general attraction of the approach is the ability to optimise any objective function that one can invent. PMID- 8628687 TI - An element in the endogenous IgH locus stimulates gene targeting in hybridoma cells. AB - Gene targeting of the immunoglobulin (Ig) heavy chain locus is the basis of improved methods of investigating gene expression and of antibody engineering. The VH-Cmu intron is a convenient region for mediating homologous recombination events which result in production of Ig bearing an altered heavy chain. Also, this segment includes several elements which are important for gene expression, replication and isotype switching: in some cases it will be advantageous to alter these processes by modifying this intron. Considering that multiple targeting steps might be needed to accomplish all the requisite changes, it is important to know whether any of the anticipated modifications also alter the recombinogenicity of the IgH locus. To test this possibility we have measured the frequency at which a mutation in the Cmu3 exon of the endogenous mu gene is corrected by homologous recombination with a transfected segment of Cmu DNA. Comparison of recombination frequencies in several engineered hybridomas indicates that deletion of a 7.1 kb segment from the VH-Cmu intron depresses recombination by approximately 10-fold. PMID- 8628690 TI - Identification of members of several homeobox genes in a planarian using a ligation-mediated polymerase chain reaction technique. AB - I have used a novel single-sided specific polymerase chain reaction (PCR) strategy inspired by ligation-mediated PCR to clone fragments of divergent homeobox genes from a flatworm, the planarian Polycelis nigra. Eight homeobox containing fragments were amplified, belonging to the Hox, msh, NK-1 and NK-2 classes. Together with the results obtained from several genomes of platyhelminths, my screening shows the presence of the same array of homeodomain developmental regulators in planarians, traditionally regarded as primitive metazoans in terms of body plan, as in coelomate organisms. However, the presence of a Ubx/abd-A homolog may indicate that platyhelminths are more closely related to protostomes than to deuterostomes and supports the idea that flatworms have inherited an elaborate HOX cluster (seven or eight genes) from their ancestor. Likely homologs of the fly genes tinman, bagpipe and S59 suggest that the mesoderm might be patterned by the same genes in all bilaterally symmetrical animals. Finally, a msh-like gene, a family known to be involved in inductive mechanisms in vertebrates, has been found. These results support the hypothesis that the tremendous diversity of metazoan body plans is specified by a largely conserved array of homeobox-containing developmental genes. PMID- 8628689 TI - DNA repair deficiencies associated with mutations in genes encoding subunits of transcription initiation factor TFIIH in yeast. AB - Several proteins, including Rad3 and Rad25(Ssl2), are essential for nucleotide excision repair (NER) and function in the RNA polymerase II transcription initiation complex TFIIH. Mutations in genes encoding two other subunits of TFIIH, TFB1 and SSL1, result in UV sensitivity and have been shown to take part in NER in an in vitro system. However, a deficiency in global NER does not exclude the possibility that such repair-deficient mutants can perform transcription-coupled repair (TCR), as shown for xeroderma pigmentosum group C. To date, temperature-sensitive C-terminal truncations of Tfbl are the only TFIIH mutations that result in intermediate UV sensitivity, which might indicate a deficiency in either the global NER or TCR pathways. We have directly analyzed both TCR and global NER in these mutants. We found that ssl1, rad3 and tfb1 mutants, like rad25(ssl2-xp) mutants, are deficient in both the global NER and TCR pathways. Our results support the view that the mutations in any one of the genes encoding subunits of TFIIH result in deficiencies in both global and TCR pathways of NER. We suggest that when subunits of TFIIH are in limiting amounts, TCR may preclude global NER. PMID- 8628688 TI - Experimentally determined weight matrix definitions of the initiator and TBP binding site elements of promoters. AB - The basal elements of class II promoters are: (i) a-30 region, recognized by TATA binding protein (TBP); (ii) an initiator (Inr) surrounding the start site for transcription; (iii) frequently a downstream (+10 to +35) element. To determine the sequences that specify an Inr, we performed a saturation mutagenesis of the Inr of the SV40 major late promoter (SV40-MLP). The transcriptional activity of each mutant was determined both in vivo and in vitro. An excellent correlation between transcriptional activity and closeness of fit to the optimal Inr sequence, 5'-CAG/TT-3', was found to exist both in vivo and in vitro. Employing a neural network technique we generated from these data a weight matrix definition of an Inr that can be used to predict the activity of a given sequence as an Inr. Using saturation mutagenesis data of TBP binding sites we likewise generated a weight matrix definition of the -30 region element. We conclude the following: (i) Inrs are defined by the nucleotides immediately surrounding the transcriptional start site; (ii) most, if not all, Inrs are recognized by the same general transcription factor(s). We propose that the mechanism of transcription initiation is fundamentally conserved, with the formation of pre initiation complexes involving the concurrent binding of general transcription factors to the -30, Inr and, possibly, downstream elements of class II promoters. PMID- 8628691 TI - The structure of d(TpA), the major photoproduct of thymidylyl-(3'5') deoxyadenosine. AB - Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929 7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA. PMID- 8628692 TI - Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5') deoxyadenosine, in Escherichia coli under SOS conditions. AB - The biological activity of TA*, the major photoproduct of thymidylyl-(3',5') deoxyadenosine, has remained speculative since it was identified a decade ago. To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil containing (+)-strand of the phage. The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand. TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic. Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants. The most abundant mutation was a 3'A-->T substitution (31/46, 56%). The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*. PMID- 8628695 TI - A rapid and efficient method for concentration of small volumes of retroviral supernatant. PMID- 8628694 TI - Reduced extension temperatures required for PCR amplification of extremely A+T rich DNA. PMID- 8628693 TI - An NMR study of [d(CGCGAATTCGCG)]2 containing an interstrand cross-link derived from a distamycin-pyrrole conjugate. AB - Minor groove binding compounds related to distamycin A bind DNA with high sequence selectivity, recognizing sites which contain various combinations of A.T and G.C base pairs. These molecules have the potential to deliver cross-linking agents to the minor groove of a target DNA sequence. We have studied the covalent DNA-DNA cross-linked complex of 2,3- bis(hydroxymethyl)pyrrole-distamycin and [d(CGCGAATTCGCG)]2. The alkylating pyrrole design is based on the pharmacophore of mitomycin C and is similar in substructure to another important class of natural products, the oxidatively activated pyrrolizidine alkaloids. Ligand-DNA NOEs confirm that the tri(pyrrole-carboxamide) unit of the ligand is bound in the minor groove of the central A+T tract. Unexpectedly, it is shifted by 1 bp with respect to the distamycin A binding site on this DNA sequence. The cross-link bridges the 2-amino position of two guanine residues, G4 and G22. The C3.G22 and G4.C21 base pairs exhibit Watson-Crick base pairing, with some local distortion, as evidenced by unusual intensities observed for DNA-DNA NOE cross-peaks. The model is compared with a related structure of a cross-linked mitomycin C:DNA complex. PMID- 8628696 TI - Taq DNA polymerase blockage at pyrimidine dimers. PMID- 8628697 TI - Ready to use agarose encapsulated PCR reagents. PMID- 8628698 TI - Improved method for selecting RNA-binding activities in vivo. AB - RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo. We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein. PMID- 8628700 TI - A rapid method of DNA isolation using laundry detergent. PMID- 8628699 TI - High resolution restriction mapping of YACs using chromosome fragmentation. PMID- 8628702 TI - Hospital provides hospice are through palliative care service. PMID- 8628701 TI - Sleep disturbances in hospitalized patients with cancer. AB - PURPOSE/OBJECTIVES: To describe the relationship between nocturnal disturbances and sleep (length and quality) in hospitalized patients with cancer. DESIGN: Descriptive, correlational. SETTING: A regional oncology center located in a large teaching hospital. SAMPLE: Fifty hospitalized patients with cancer (58% female, x age = 48 years), all with solid tumors. METHODS: The investigator observed and recorded the number and characteristics of nocturnal disturbances using the Sleep Environment Observation Tool and the Taxonomy of Patient Participation. Patients completed the Verran and Snyder-Halpern Sleep Scale, which measures perceived sleep length and quality. MAIN RESEARCH VARIABLES: Number and timing of nocturnal disturbances, patient participation in care, sleep length, and sleep quality. FINDINGS: Number and total duration of nocturnal disturbances as well as level of patient participation in nocturnal care were negatively correlated with sleep quality. Sleep length was not correlated with any other variable. CONCLUSIONS: Nocturnal disturbances had a negative effect on sleep quality. IMPLICATIONS FOR NURSING PRACTICE: The number and duration of disturbances and the level of patient participation in care should be minimized during nighttime hours. PMID- 8628703 TI - Different approaches to transfusion practices used in hospice care. PMID- 8628704 TI - A patient's self-determination is sometimes difficult to accept. PMID- 8628705 TI - Possible diversion of pain medication occurs in home hospice setting. PMID- 8628706 TI - Did family attempt to hasten patient's death? PMID- 8628707 TI - Palliative management of dehydration in patients receiving hospice care at home. PMID- 8628708 TI - Reader encourages educating teenagers about the dangers of tobacco use. PMID- 8628709 TI - Hepatic cryosurgery for metastatic colorectal carcinoma. AB - PURPOSE/OBJECTIVES: To review an innovative, potentially curative surgical approach for the treatment of select patients with colorectal cancer metastatic to the liver. DATA SOURCES: Published articles and reported and unreported research results. DATA SYNTHESIS: The liver is the primary site of recurrence in 60%-80% of patients who develop recurrent disease following resection of the colorectal primary cancer. Less than 25% of patients with liver metastases are candidates for hepatic resection because of the location or number of liver metastases. Hepatic cryosurgery provides a viable treatment option for some patients with unresectable metastatic colorectal carcinoma confined to the liver, including patients with bilobar and multiple lesions. CONCLUSIONS: Because colorectal cancer is the second most common cause of cancer mortality in the United States, interventions that improve survival rates are an important focus of care. With knowledge of patient selection criteria and implications of hepatic cryosurgery, informed oncology nurses are resources for patients contemplating this therapy. IMPLICATIONS FOR NURSING PRACTICE: Preoperative evaluation and postoperative nursing care focus on careful assessment, education, and interventions aimed at preventing and detecting complications unique to hepatic cryosurgery. Knowledge of hepatic cryosurgery as a treatment option for colorectal liver metastases, patient selection criteria, and related implications allows oncology nurses to serve as resources for patients and families considering this therapy. PMID- 8628710 TI - "I hope I don't have cancer": colposcopy and minority women. AB - PURPOSE: To determine what women scheduled for colposcopy knew about the procedure and to understand their concerns about the test and its implications. DESIGN: Descriptive, exploratory, qualitative. SAMPLE AND SETTING: All women scheduled for colposcopy in a three-month period (n = 29) in an inner-city academic center in the northeast United States. The sample consisted of African American (66%), Hispanic (31%), and white (3%) women ranging in age from 17-59 years (x = 39 years). METHODS: Women were interviewed on the telephone or in person and were asked seven open-ended questions about why they were referred and what they expected would happen at the appointment. Interviews were content analyzed. FINDINGS: Four themes common to the women's responses emerged from the interview data: fear about their health, apprehension about the colposcopy, uncertainty about the meaning of the Pap test, and pervasive lack of knowledge. CONCLUSIONS: These results suggest that women in this study scheduled for colposcopy had little factual information about the test or its implications, were anxious about the appointment, and wanted more complete information. IMPLICATIONS FOR NURSING PRACTICE: Nurses cannot assume that women understand the reasons for their referral for colposcopy or the implications of the test. These study results imply that lengthened appointment time might be required to provide appropriate education and help alleviate women's anxiety. Future nursing research should examine the effect of anticipatory counselling and education for this group of women. PMID- 8628711 TI - Reproductive issues for men with cancer: technology and nursing management. AB - PURPOSE/OBJECTIVES: To review the current medical technologies available to protect the reproductive potential of adult males undergoing sterilizing cancer treatments; to describe the attributes and limitations of these technologies and how oncology nurses can access them for the patient; and to discuss psychosocial elements, including the legal considerations of oncology nurses who counsel patients. DATA SOURCES: Quantitative data from personal clinical records; personal clinical experience; published articles, abstracts, and books identified by bibliographic data base searches; and consultation with lawyers. DATA SYNTHESIS: Cancer treatment can have severe and adverse long-term iatrogenic effects on male fertility. Medical technologies that protect male reproduction potential from sterilizing procedures have progressed from unreliable to clinically practical over a period of 20 years. The present clinical means for preserving the potential reproductive capacity of men at risk is cryopreservation of sperm before treatment begins, followed by assisted reproductive technology when pregnancy is desired. Medical, legal, and religious issues relevant to counseling are involved. CONCLUSIONS: Current reproductive technology provides realistic hope for future procreation by men facing sterilizing cancer treatment. IMPLICATIONS FOR NURSING PRACTICE: Nursing intervention primarily involves providing patient counseling and arranging patient access to cryopreservation facilities. Oncology nurses can assist men making these types of reproductive decisions by assessing their medical and psychological need for information and by counseling them in regard to human sexuality, the fertility risk of oncologic therapy, the availability of reproductive interventions, and the social ramifications of using stored semen. PMID- 8628712 TI - Diarrhea associated with nasogastric feedings. AB - PURPOSE/OBJECTIVES: To determine the difference in the incidence of diarrhea among subjects given one of three formulas with varying fiber concentrations administered by nasogastric (NG) tube, variables affecting incidence of diarrhea, discomforts other than diarrhea associated with NG tube feedings, and effects of changing from continuous to interval feedings on incidence of diarrhea and discomforts. DESIGN: Prospective, double-blind, randomized study. SETTING: Midwestern tertiary care center otolaryngology nursing unit. SAMPLE: Eighty randomized subjects who were 18 years or older, English-speaking, and undergoing head and neck cancer surgery that required an NG tube postoperatively and who had no gastrointestinal (GI) illness within two weeks prior to surgery. METHODS: Subjects received continuous administration of formula containing no fiber, 7 gms/L of fiber, or 14 gms/L of fiber until they reached the caloric intake goal and then were advanced to interval feedings. Patients' medical records provided past medical history and information on medication administration. A bedside flow sheet was used for documenting incidence of diarrhea and other GI discomforts. MAIN RESEARCH VARIABLES: Amount of fiber in the formula administered, patient's genders and prior food aversions, and antibiotics' effect on diarrhea and other GI discomforts. FINDINGS: Multiple logistic regression showed significant odds ratios (ORs) for developing diarrhea in female subjects (OR = 7.96), subjects who had prior food aversions (OR = 2.67), and subjects receiving broad spectrum antibiotics (OR = 3.22). Diarrhea was four times more likely to occur in males who received fiber-free formula. Of all subjects, 70% experienced GI discomforts with continuous feedings, and 50% experienced discomforts when advanced to interval feedings. CONCLUSIONS: Fiber formulas reduced the incidence of diarrhea in male subjects but not in female subjects. Antibiotics' effect on diarrhea paralleled the findings of other studies. IMPLICATIONS FOR NURSING PRACTICE: Use formulas with fiber for males. Liquid stools do not require interruption of tube feeding; GI discomforts warrant interruption. Interval feeding schedules require monitoring similar to continuous feeding schedules. PMID- 8628713 TI - Self-blame and adjustment to breast cancer. AB - PURPOSE: To examine the relationship between self-blame and illness adjustment in women with breast cancer. DESIGN: Descriptive, correlational design. SETTING: Outpatient facilities of two metropolitan medical centers. SAMPLE: 234 women diagnosed with stage I or II breast cancer. METHODS: Structured and semistructured interviews using the Psychosocial Adjustment to Illness Scale (PAIS), the Global Adjustment to Illness Scale (GAIS), an attributions and blame interview, and several questions about control over the cause and course of the cancer. MAIN RESEARCH VARIABLES: Self-blame, controllability, and adjustment to illness. FINDINGS: 39% of the sample reported some self-blame. Analysis of variance indicated that subjects with high levels of self-blame had poorer scores on the PAIS and GAIS. CONCLUSIONS: The findings indicate the need for further study of the self-blame/adjustment relationship. Researchers must clarify the characteristics that compromise adjustment in patients with breast cancer. IMPLICATIONS FOR NURSING PRACTICE: The findings support the importance of assessing self-blame and providing information to discourage its use by patients with breast cancer. PMID- 8628714 TI - Determining crucial correlates of breast self-examination in older women with low incomes. AB - PURPOSE: To investigate the frequency and proficiency of breast self-examination (BSE) in older women with low incomes and to identify predictors of BSE to facilitate more effective clinical encounters and improved educational programs. DESIGN: Correlational, nonexperimental survey. SETTING: Urban tertiary-care center. SAMPLE: 204 women (ages 40-86) self-referred or recruited from the community for free breast cancer screening. Criteria for inclusion were age 40 or older, a household income of less than two-and-one-half times the poverty level, and uninsured or underinsured status. METHODS: The Mamon and Zapka Breast Self Examination Survey was modified for use with this population. Telephone surveys were conducted during the period between the initial contact and screening. MAIN RESEARCH VARIABLES: 102 independent variables were correlated with frequency and proficiency. Part correlations were calculated to determine unique contributions. Variables with significant part correlations were entered into regression equations. FINDINGS: The mean frequency and proficiency scores were 2.9 out of 4 and 3.4 out of 19, respectively. Four variables explained 40% of the variance in frequency, and eight explained 66% of the variance in proficiency. Frequency and proficiency were significantly related. CONCLUSIONS: 10 variables were found to predict BSE behavior in this population: confidence, confidence level, awareness of mammography, provider influence, desire for reconstruction, knowledge of parity risk factor, education, having been taught the correct time of month to perform BSE, adequate time devoted to BSE, and exposure to BSE messages from a clinician. IMPLICATIONS FOR NURSING PRACTICE: The study identifies variables to include in clinical encounters and educational programs designed to promote BSE. Of importance is considering the characteristics and specific needs of the recipients of educational and interventional programs. PMID- 8628715 TI - Wrong answers. PMID- 8628716 TI - Identifying patient symptoms after radiotherapy using a nurse-managed telephone interview. AB - PURPOSE/OBJECTIVES: To identify patients' symptoms following completion of radiotherapy for common cancers by a nurse-managed telephone interview. DESIGN: Quality assurance project. SETTING: Radiation therapy department in a community hospital in a large midwestern city. SAMPLE: One hundred eleven patients treated by radiotherapy for primary cancer of the prostate, head/neck, lung, and breast. METHODS: Two time points of data collection: nurses completed an end-of-treatment symptom evaluation within the last five days of treatment and conducted telephone interviews 14-21 days post-therapy. Interview questions were based on each individual's end-of-treatment symptoms and common site-specific side effects. MAIN RESEARCH VARIABLES: Symptoms at end of treatment and 14-21 days after therapy completion, nursing assessments and interventions, and length of telephone interview. FINDINGS: At the end of treatment, 104 (94%) patients were experiencing symptoms. Nurses contacted 106 (95%) patients by telephone 14-21 days after therapy completion and assessed symptoms in 84 (79%) patients. Nineteen (18%) patients reported the development of a new symptom. Nurses independently managed 95% of the calls. CONCLUSIONS: The majority of patients experienced symptoms in the immediate post-therapy period. Telephone follow-up interviews served as a mechanism for evaluating short-term morbidity and provided the opportunity for nurses to intervene with many patients. IMPLICATIONS FOR NURSING PRACTICE: A nurse-managed telephone follow-up program can be used as a component of a quality improvement process in radiation centers to assess patients' post-treatment symptoms and provide education and support. PMID- 8628717 TI - Changes in gene expression of growth factors and their receptors during castration-induced involution and androgen-induced regrowth of rat prostates. AB - To find candidates for the mediator of the growth-promoting action of androgen in rat prostates, the changes in the steady-state levels of mRNAs coding for several growth factors and their receptors were examined by Northern blot analysis during castration-induced involution, and subsequent regrowth induced by androgen in the ventral and dorsolateral lobes. The changes in the growth factor systems and a typical secretory protein in the ventral lobe were similar to, but more prominent than, those in the dorsolateral lobe, showing the higher androgen dependency of the ventral lobe. Among the growth factors and their receptors investigated, only epidermal growth factor (EGF) showed apparent positive androgen dependency: EGF mRNA content in the ventral lobe decreased to about 30% of the normal level within 24 hr after castration, and increased, attaining about 200-300% of the normal level 3-5 days after androgen administration to castrated rats. mRNAs coding for all other factors examined, i.e., transforming growth factor-alpha (TGF-alpha), EGF receptor, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1, TGF-beta1, TGF-beta type II receptor, hepatocyte growth factor (HGF), and c-MET/HGF receptor, increased after castration in greater or lesser degree, and after a brief pause or a decrease some of them increased again attaining a second peak 3-5 days after androgen replacement. The second increase was evident in TGF-alpha, EGF receptor, KGF, and c-MET mRNAs. These results indicate the possibility that multiple growth factor receptor systems participate in the androgen-dependent regrowth of castrated rat prostates. PMID- 8628718 TI - Expression of CD44 isoforms in human prostate tumor cell lines. AB - We have examined the expression of the transmembrane glycoproteins CD44 in four human prostate tumor cell lines. Expression was examined at the protein level by flow cytometric analysis and Western blot, and at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR). All four cell lines (DU145, LNCaP, PC3, and ND1) expressed the standard CD44 isoform (CD44s) at the mRNA level and all cell lines except LNCaP expressed CD44s at the protein level. All four cell lines contained one or more isoforms containing the v6 region (exon 10) at the mRNA level, which has been associated with metastatic potential. However, a subpopulation of LNCaP and ND1 cells showed protein expression of v6. In addition, soluble CD44 isoforms were identified in cultured supernatants from all cell lines except LNCaP. These results show that CD44 isoforms are expressed on human prostate tumor cell lines, including the expression of variant isoforms containing the v6 region, and provide a rationale for the further study of this cellular adhesion molecule in prostate cancer. In addition, preliminary results indicate altered expression of CD44 in human prostatic adenocarcinomas examined immunohistochemically. PMID- 8628719 TI - Low incidence of androgen receptor gene mutations in human prostatic tumors using single strand conformation polymorphism analysis. AB - It is possible that structural changes of the androgen receptor (AR) contribute to the insensitivity of prostatic carcinomas to endocrine therapy. We have isolated DNA from 58 human prostate tumor specimens (31 carcinomas pretreatment, 13 carcinomas after relapse to hormonal therapy, and 14 benign prostatic hyperplasia), three established human prostate carcinoma cell lines and two transplantable human prostatic carcinoma xenografts. Twelve pairs of oligonucleotide primers were used to amplify the majority of the coding region of the AR gene and the products screened for mutations using single-strand conformation polymorphism (SSCP) techniques. In one tumor sample a cytosine to guanine transition in exon F which leads to substitution of glutamic acid for the wild type glutamine at position 798 of the ligand binding domain was detected. The same mutation was also found in the patient's genomic DNA and as been described in a patient with partial androgen insensitivity syndrome. Intronic mutations were detected in two of the benign prostatic hyperplasia samples, and a silent mutation at nucleotide 995 was found to be present in eight poorly differentiated carcinomas, one BPH specimen, as well as in the cell line DU145 (18% of the samples studied). In agreement with most of the literature, these studies indicate that AR mutations are rare both prior to therapy and even in androgen relapsed tumors. PMID- 8628720 TI - Inhibitory effects of the nucleoside analogue gemcitabine on prostatic carcinoma cells. AB - Gemcitabine (2',2'difluoro-2'deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen-sensitive tumor cell line LNCaP and the androgen insensitive cell lines PC-3 and DU-145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co-administration of 10-100 microM of its natural analogue deoxycytidine. In view of a future clinical application of this anti-tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic-macrophage progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic-macrophagic progenitor cells required a concentration of 9 nM. Co administration of deoxycytidine to gemcitabine-treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma. PMID- 8628721 TI - Control of LNCaP proliferation and differentiation: actions and interactions of androgens, 1alpha,25-dihydroxycholecalciferol, all-trans retinoic acid, 9-cis retinoic acid, and phenylacetate. AB - There is increasing evidence that growth and differentiation of prostatic carcinoma cells may be modulated not only by androgens and growth factors but also by vitamin D, retinoids, and phenylacetate (PA). The latter agonists may have a role in the prevention and therapy of prostate cancer but their exact therapeutic potential remains unclear. Since both retinoids and vitamin D act via nuclear receptors, the same way androgens do, we studied the interactions of these compounds with androgen-induced proliferation and differentiation using LNCaP cells as a model of androgen-responsive tumor cells. PA was included because of its suspected different mode of action [H3]-thymidine incorporation was used as a measure of proliferative activity, secretion of prostate-specific antigen (PSA) as a measure of differentiated function. The present data show that 1alpha,25-dihydroxycholecalciferol (VD3), all-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and PA stimulated LNCaP cell-differentiated function in the presence or absence of androgens. The effects on cell growth were more complicated. In the absence of androgens growth stimulatory effects were observed for the retinoids and under some conditions for VD3. These effects were limited, however, and tended to be more pronounced at low cell densities. In the presence of androgens nearly exclusively growth inhibitory effects were observed. On a molar basis VD3 was the most effective antiproliferative agonist (ED50 = 10(-9) M). It completely neutralized the stimulatory effects of androgens. Growth inhibition was not due to a decrease in the concentration of androgen receptor: whereas atRA, 9cRA, and PA did not alter androgen receptor levels, VD3 provoked a twofold increase. Neither in the presence nor in the absence of androgens did we observe any cooperativity in the growth stimulatory or inhibitory effects of VD3, atRA, or 9cRA. To test whether treatment with any of the studied agonists resulted in a phenotypic reversion and sustained growth arrest, LNCaP cells were pretreated with VD3, atRA, 9cRA, or PA for 6-12 days and reseeded at equal densities as untreated cells. In all cases tested [3H]-thymidine incorporation was restored within 6 days suggesting that none of these compounds caused irreversible growth inhibition. PMID- 8628723 TI - The case for early endocrine treatment of advanced or metastatic prostate cancer. PMID- 8628722 TI - Transurethral prostatectomy: mortality and morbidity. AB - In 1989 two large-scale multicenter studies on the mortality and morbidity of transurethral resection of the prostate were published [Mebust et al.: J Urol 141:243-247, 1989; Roos et al.: N Engl J Med 320:1120-1124, 1989]. These studies caused us to perform a retrospective study on a total of 1,211 consecutive patients who underwent transurethral resection of the prostate at our department between January 1988 and July 1991. The mortality rate in the 1,211 patients subjected to transurethral resection of the prostate was 0.00%; none of the patients died of intraoperative or postoperative complications. Intraoperative complications were observed in 8.9% of the patients, while the rate of early postoperative complications was 15.8%. Of the 1,211 patients operated on, 775 were followed for at least 1 year postoperatively. Late complications were noted in 11.2% of the patients. Repeat resection had to be performed in 0.9% of patients within 1 year, and in 2.5% within 3 years after surgery. A comparison of the studies by Mebust et al. and Roos et al. yielded similar rates of intraoperative and postoperative complications, whereas our mortality rate and repeat transurethral resection rate were significantly lower. PMID- 8628724 TI - Carcinoma of prostate: case against immediate hormonal therapy. PMID- 8628725 TI - Neutron scattering redux? PMID- 8628726 TI - Helical fold prediction for the cyclin box. AB - The smooth progression of the eukaryotic cell cycle relies on the periodic activation of members of a family of cell cycle kinases by regulatory proteins called cyclins. Outside of the cell cycle, cyclin homologs play important roles in regulating the assembly of transcription complexes; distant structural relatives of the conserved cyclin core or "box" can also function as general transcription factors (like TFIIB) or survive embedded in the chain of the tumor suppressor, retinoblastoma protein. The present work attempts the prediction of the canonical secondary, supersecondary, and tertiary fold of the minimal cyclin box domain using a combination of techniques that make use of the evolutionary information captured in a multiple alignment of homolog sequences. A tandem set of closely packed, helical modules are predicted to form the cyclin box domain. PMID- 8628727 TI - Molecular dynamics study of glucocorticoid receptor-DNA binding. AB - Molecular dynamics simulations have been conducted to investigate the binding of the glucocorticoid receptor (GR) dimer to DNA. For this purpose simulations of the complex formed by a DNA segment and a dimer of GR-DNA binding domains (GR DBD) have been carried out, employing an available X-ray structure. A second set of simulations was based on this structure as well, except that the DNA segment was altered to the consensus glucocorticoid response element (GRE). Simulations of a single GR-DBD and of the uncomplexed GRE served as controls. For the simulations, each system was encapsulated in an ellipsoid of water. Protein-DNA interactions, dimer interactions, and DNA structural parameters were analyzed for each system and compared. The consensus GRE is found to yield more favorable and symmetric interactions between the GR-DBDs and the GRE, explaining the ability of the GR dimer to recognize this DNA segment. Further analysis focused on deformations of the DNA that are induced by the binding of GR. The deformations observed involve a 35 degree bend of the DNA, an unwinding, and a displacement of the helical axis. These deformations are consistent with a mechanism for transcriptional regulation that involves a change of nucleosome packing upon GR binding. Significant protein-protein and protein-DNA interactions, both direct and water mediated, develop due to the deformations of the GRE and are indicative of an increased recognition achieved through DNA deformation. The interactions include direct interactions between the GRE and glycine-458 and serine-459, side groups which differentiate GR from other members of the nuclear hormone receptor family. PMID- 8628728 TI - Crystallization of glycosylated and nonglycosylated phytohemagglutinin-L. AB - In the seeds of legume plants a class of sugar-binding proteins can be found, generally called legume lectins. In this paper we present the crystallization of phytohemagglutinin-L (PHA-L), a glycosylated lectin from the seeds of the common bean (Phaseolus vulgaris). Single PHA-L crystals were grown by vapor diffusion, using PEG as precipitant. The protein crystallizes in the monoclinic space group C2, and diffracts to a resolution of 2.7 angstroms. The unit cell parameters are a=106.3 angstroms, 121.2 angstroms, c=90.8 angstroms, and beta=93.7 degrees. The asymmetric unit probably contains one PHA-L tetramer. Crystals of a recombinant nonglycosylated form of PHA-L, grown under identical conditions, and crystals of the native PHA-L, grown in the presence of isopropanol, did not survive the mounting process. PMID- 8628729 TI - Crystallization of ADP-ribosyl cyclase from Aplysia californica. AB - ADP-ribosyl cyclase synthesizes the secondary messenger cyclic ADP-ribose from NAD+. Diffraction quality crystals of the enzyme from ovotestes of Aplysia californica have been obtained. Crystallographic analysis of this enzyme will yield insight into the mode of binding of the novel cyclic nucleotide and the mechanism by which NAD+ is cyclized. PMID- 8628730 TI - Crystallization and preliminary x-ray analysis of volvatoxin A2 from Volvariella volvacea. AB - Volvatoxin A2, an ion channel disturbed cardiotoxic and hemolytic protein from the edible mushroom, Volvarilla volvacea, has been crystallized by the vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in sodium acetate buffer pH 4.6. The best crystals belong to the monoclinic space group C2 with unit cell dimensions a = 155.25 angstroms, b = 58.06 angstroms, c = 116.92 angstroms, and beta = 119.5 degrees. These crystals diffract to at least 2.2 angstroms and there are four molecules of molecular weight 24 kDa per asymmetric unit with a solvent content of 48%. PMID- 8628731 TI - Secondary structure prediction and unrefined tertiary structure prediction for cyclin A, B, and D. AB - We present heuristic-based predictions of the secondary and tertiary structures of cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a beta-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure. PMID- 8628732 TI - Identification of a pattern in protein structure based on energetic and statistical considerations. AB - We carry out a statistical analysis of the nonbonded interactions in 10 high resolution nonhomologous protein structures, using original algorithms. We observe a tendency of nonbonded interactions which contribute significantly (i.e., with an energy lower than the average value, referred to as "strong") to protein stability, to be concentrated in clusters of residues that are strongly sequence correlated. We characterize this sequence correlation and subsequently define a "system" as the pattern that describes these clusters. In order to study the distribution of the systems in the proteins we build a matrix for each protein and for each term of the empirical potential function used to compute the nonbonded interactions; each ij element is the number of common residues between the systems i and j. The analysis of the matrices shows the presence of compact blocks that define units in the protein structure which concentrate strong and weak interactions inside the unit itself and display relative independence with respect to the rest of the protein. Comparing the blocks defined by the three nonbonded energy components (electrostatic, hydrogen bonds, and van der Waals interactions) we observe a one-to-one correspondence between the blocks of different energy components with an average overlap of 90% of the residues forming each block. PMID- 8628733 TI - Predicting human immunodeficiency virus protease cleavage sites in proteins by a discriminant function method. AB - Based on the sequence-coupled (Markov chain) model and vector-projection principle, a discriminant function method is proposed to predict sites in protein substrates that should be susceptible to cleavage by the HIV-1 protease. The discriminant function is defined by delta = phi+ - phi-, where phi+ and phi- are the cleavable and noncleavable attributes for a given peptide, and they can be derived from two complementary sets of peptides, S+ and S-, known to be cleavable and noncleavable, respectively, by the enzyme. The rate of correct prediction by the method for the 62 cleavable peptides and 239 noncleavable peptides in the training set are 100 and 96.7%, respectively. Application of the method to the 55 sequences which are outside the training set and known to be cleaved by the HIV-1 protease accurately predicted 100% of the peptides as substrates of the enzyme. The method also predicted all but one of the sites hydrolyzed by the protease in native HIV-1 and HIV-2 reverse transcriptases, where the HIV-1 protease discriminates between nearly identical sequences in a very subtle fashion. Finally, the algorithm predicts correctly all of the HIV-1 protease processing sites in the native gag and gag/pol HIV-1 polyproteins, and all of the cleavage sites identified in denatured protease and reverse transcriptase. The new predictive algorithm provides a novel route toward understanding the specificity of this important therapeutic target. PMID- 8628734 TI - Crystal structure of the Trypanosoma cruzi trypanothione reductase.mepacrine complex. AB - The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 angstoms resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser 109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp 21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease. PMID- 8628735 TI - High pressure effects on protein structure and function. AB - Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity. PMID- 8628736 TI - Structure, energetics, and dynamics of lipid-protein interactions: A molecular dynamics study of the gramicidin A channel in a DMPC bilayer. AB - The microscopic details of lipid-protein interactions are examined using molecular dynamics simulations of the gramicidin A channel embedded in a fully hydrated dimyristoyl phosphatidylcholine (DMPC) bilayer. A novel construction protocol was used to assemble the initial configurations of the membrane protein complex for the simulations. Three hundred systems were constructed with different initial lipid placement and conformations. Seven systems were simulated with molecular dynamics. One system was simulated for a total of 600 psec, four were simulated for 300 psec, and two for 100 psec. Analysis of the resulting trajectories shows that the bulk solvent-membrane interface region is much broader than traditionally pictured in simplified continuum theories: its width is almost 15 angstroms. In addition, lipid-protein interactions are far more varied, both structurally and energetically, than is usually assumed: the total interaction energy between the gramicidin A and the individual lipids varies from 0 to -50 kcal/mol. The deuterium quadrupolar splittings of the lipid acyl chains calculated from the trajectories are in good agreement with experimental data. The lipid chains in direct contact with the GA are ordered but the effect is not uniform due to the irregular surface of the protein. Energy decompositions shows that the most energetically favorable interactions between lipid and protein involve nearly equal contributions from van der Waals and electrostatic interactions. The tryptophans, located near the bulk-membrane interface, appear to be particularly important in mediating both hydrogen bonding interactions with the lipid glycerol backbone and water and also in forming favorable van der Waals contacts with the hydrocarbon chains. In contrast, the interactions of the leucine residues with the lipids, also located near the interface, are dominated by van der Waals interactions with the hydrocarbon lipid chains. PMID- 8628737 TI - Synthesis and hypolipidemic evaluation of beta-alkylaminopropiophenone and beta alkylaminopropio-2'-naphthone derivatives in rodents. AB - A series of beta-alkylamino-(4'-alkyl)-propiophenone or beta-alkylamino(7' methyl)-propio-2'-naphthone derivatives were prepared and found to have hypolipidemic activity by lowering both serum cholesterol and triglyceride levels in rodents. The electron donating substituent at the para position of the phenyl ring seems to decrease the hypolipidemic activity when compared to non substituted or electron withdrawing group substituted analogs as investigated previously in this laboratory. In comparison with lovastatin or clofibrate, most of these analogs showed similar or higher activity in lowering both serum cholesterol or triglyceride levels. beta-Pyrrolidino-(4'-methyl)-propiophenone (1) demonstrated the best activity after 16 d, i.p. administration in mice at 8 mg/kg/d. Further detailed studies in rats indicated that beta-pyrrolidino-(4' methyl)-propiophenone also showed decreased serum cholesterol and triglyceride levels with increased HDL-cholesterol and triglyceride levels after 14 d. In hyperlipidemic mice and rats, this compound was observed to be effective in lowering serum lipid levels as well as tissue lipid levels. The activities of hepatic acetyl CoA synthetase, phosphatidylate phosphohydrolase, and hepatic lipoprotein lipase were moderately inhibited by beta-pyrrolidino-(4'-methyl) propiophenone. PMID- 8628738 TI - Synthesis and anticonvulsant activity of some amino acid derivatives. Part 2: Derivatives of Gly, Ala, Leu, Pro, Trp, Phe(4 Cl), Ala(alpha-Me). AB - Fourteen amides of N-substituted natural and anatural amino acids have been designed and synthesized as potential anticonvulsants. They were evaluated in the maximal electroshock seizure (MES) test, the subcutaneous Metrazol seizure threshold (sc Met) test and the rotorod neurotoxicity (Tox) test. According to the classification of the Anticonvulsant Screening Project (ASP) of the Antiepileptic Drug Development Program (ADDP) eight of synthesized compounds received class I, two class II and four class III. One of the compounds classified in class I (18) was tested quantitatively after i.p. administration in mice. It showed MES ED50 = 67.41 mg/kg and protective index (PI) = 4.5. Conformational models of the synthesized compounds were compared to one another, as well as to models of some standard compounds. PMID- 8628739 TI - Rapid HPLC assay for verapamil and its metabolites: use for application to in vitro studies. AB - An improved method using isocratic reversed phase HPLC is presented for the extraction and rapid determination of verapamil and its main metabolites in microsomal preparations and cell culture media. Possibilities for using the method to estimate cytochrome P450 enzymes in microsomal test systems and hepatocyte cultures are described. The studies show that primary hepatocyte cultures are suitable for studying the metabolism and interactions of pharmaceuticals in vitro and could be superior to microsomal systems in many cases. PMID- 8628740 TI - Studies on dressings for mucosa of the oral cavity. Part 3: Effect of preparation technology on the physical and chemical properties of stomatological xerogel dressings. AB - Adhesion of xerogel dressings prepared on Eudragit (E), methylcellulose (MC) and glycerol compositions, remaining within the range of 625-650 g, after addition polyvinylpyrolidone (PVP) amounts to 450-1250 g. Dissolution time from xerogel dressings without PVP additive both in water and in an artificial gastric juice amounts to 3 h. Addition of PVP results in reduction of the elution rate. In dependence upon the PVP/E/MC ratio elution time amounts to 3-3.5 h. Pharmaceutical availability of the Kunitz protease inhibitor in particular groups of then dressings depends upon concentration of hydrophilizing agent. The semi liberation times amounts to 3.65-17.50 h. PMID- 8628741 TI - Effect of local anesthetic [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid on the activity of purified sarcoplasmic reticulum (Ca-Mg)ATPase. AB - Local anesthetic [2-(alkyloxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid modulate the activity of the purified sarcoplasmic reticulum (Ca-Mg)ATPase. The phase of insensitivity or slight stimulation of the activity at lower anesthetics concentrations is followed by the inhibition phase at higher concentrations. The potency to inhibit the activity at high concentrations is maximal for the homologue with the hexyloxy substituent and decreases for shorter and longer substituents. The inhibition of activity can be partially reversed by addition of n-decane. The inhibition could be caused by the binding of anesthetics to binding sites at the protein--lipid bilayer interface. The changed thickness of the hydrophobic part of this interface might be responsible for the n-decane reversible inhibition and its dependence on the alkyloxy substituent chain length, while the changed structure of the polar part of this interface could be the cause of the n-decane irreversible inhibiton. PMID- 8628742 TI - A new cholestane derivative of Abutilon bidentatum Hochst. and its bioactivity. PMID- 8628743 TI - Psoralen photobiology: recent advances. AB - Clinical efficacy may arise from psoralen-induced photodamage by the induction of a panoply of biomolecules. In cellular studies with UV or photoactivatable agents, the activation of NF-kappa B, probably the most widely studied transcription factor, occurs after its release from an inhibiting factor, I kappa B. The activation of transcription factors has also been correlated with the UV absorption spectrum of DNA (35). Furthermore, it was shown that the photoadducts do not need to be processed or repaired because transcription factor induction is observed in repair-deficient cells. In these latter cells lower UV doses are required to induce these transcription factors. As a result of transcription factor induction, treated cells may have a greater number of class I molecules on their surface and an altered cytokine profile. It is possible (and likely) that these effects occur in different cells to different extents. The important message is that activational events may occur that could alter the cell's (or cells') ability to regulate a disease process. Many studies have shown that the time frame for this type of damage-induced event may range from seconds to minutes (36). Clearly 8-MOP/UVA-induced events such as those described above could be initiated as the result of a single photochemotherapy session. PMID- 8628744 TI - Cytolytic response to HIV in patients with HIV disease treated with extracorporeal photochemotherapy: preliminary study. AB - Extracorporeal photochemotherapy (photopheresis), an immunomodulatory therapy that targets circulating T helper lymphocytes, has been applied to the management of human immunodeficiency virus (HIV) disease. Any therapy that exerts its actions on CD4+ T cells has the potential of exacerbating HIV infection. Therefore, it was necessary to observe immune function during treatment. Because cytotoxic T lymphocytes (CTL) and natural-killer cells are thought to play an important role in the response against HIV infection, we examined the effect of photopheresis on HIV cytolytic activity. The study group consisted of seven patients with late-stage HIV disease who had not received any previous treatment for HIV infection. Patients were treated exclusively with photopheresis on two consecutive days each month for 14-32 months (average, 25 months). Peripheral lymphocytes, collected at various points during treatment, were used as effectors in a 51Cr release assay. Epstein-Barr virus (EBV)-transformed autologous B cell lines transfected with recombinant vaccinia vectors that expressed the HIV env (gp120, gp41) and gag (p24) proteins were used as target cells. All seven patients demonstrated relatively constant levels of cytolysis (>10% above controls) during treatment in the context of stable CD4+ T cell counts and a stable clinical status. These results suggest that extracorporeal photochemotherapy did not impair the cytolytic response to HIV infection and may have enhanced it in some patients. PMID- 8628745 TI - Inactivation of Trypanosoma cruzi trypomastigote forms in blood components with a Psoralen and Ultraviolet A light. AB - Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4' aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 micrograms/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi, the trypomastigote, was achieved at a UVA (320-400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cm2 the trypomastigote motility continues for at least 16 h-post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of 3H-AMT indicated that at the lethal UVA influence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi, the causative agent of Chagas disease. PMID- 8628747 TI - Kinetic analysis of apoptosis induction in human cell lines by UVA and 8-MOP. AB - Whereas previous studies have indicated that DNA damage as a result of 8 methoxypsoralen (8-MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8-MOP and UVA necessary to induce apoptosis in human T-lymphocytic and monocytic cell lines. In order to asses apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8-MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm2 UVA and 12.5 ng/mL of 8-MOP. A striking observation was that UVA alone at doses > or = 1.0 J/cm2, but not 8-MOP alone (0-300 ng/mL), induced significant apoptosis in the Sup-T1 cells induced by UVA alone was not as great as that of 8 MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8-MOP (ng/mL) times the dose of UVA (J/cm2) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA-induced DNA damage to apoptosis induction. moreover, it will provide a means by which early events in the apoptotic pathway can be dissected. PMID- 8628746 TI - PUVA treatment selectively induces a cell cycle block and subsequent apoptosis in human T-lymphocytes. AB - Psoralen plus UVA (320-400 nm radiation; PUVA) is a highly effective therapy for cutaneous diseases caused by skin infiltration with normal or neoplastic T lymphocytes. In comparing the effects of pharmacologically relevant, low-dose PUVA treatment on growth of human keratinocytes, peripheral blood leukocytes (PBMC), and T-lymphocyte cell lines, we determined that PBMC or T-lymphocytes were > 50-fold more sensitive to cytotoxic effects of PUVA, while antiproliferative effects were produced by similar PUVA levels in all cell types. Low doses of PUVA (10 ng/mL 8-methoxypsoralen and 1-2 J/cm2) were highly cytotoxic for phytohemagglutinin-activated normal lymphocytes or transformed T lymphocytes as assessed by two viability assays and by flow cytofluorometry. Altered lymphocyte morphology, nuclear fragmentation, TUNEL+ nuclei or nuclear fragments, and the appearance of a sub-G1 DNA peak indicated that cell death occurred by apoptosis, beginning about 1 day after PUVA treatment and continuing for several days thereafter. From assessment of cell cycle progression in mimosine-synchronized cells, PUVA treatment markedly slowed cell cycle progression, eventually producing cell cycle arrest and apoptotic entry. We propose that the probable basis for disease remissions (psoriasis, cutaneous T cell lymphoma) produced by PUVA treatment is through selective cytotoxic effects on clonal T-lymphocyte populations that are concentrated in diseased skin. PMID- 8628748 TI - The lack of efficacy of 4,6,6'-trimethylangelicin to induce immune suppression in an animal model for photopheresis: a comparison with 8-MOP. AB - Photopheresis is an extracorporeal form of photochemotherapy with 8 methoxypsoralen (8-MOP) and UVA (PUVA). Patients ingest 8-MOP and then a psoralen rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell-mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8-MOP. Upon UVA exposure 8-MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross-links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross-links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4'-trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8-MOP under conditions relevant to the in vivo situation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppression in vivo, even after extensive irradiation. In addition a dose dependency of 8-MOP/UVA versus the induced immune suppression was carried out. It was shown that there is a log dose/response correlation of r=0.9205. PMID- 8628749 TI - Photoimmunology of lupus erythematosus and dermatomyositis: a speculative review. AB - David Norris has proposed a four step model for the pathogenesis of LESSD (1): (1) exposure to UV light induces the release of proinflammaotry epidermal and dermal mediators such as IL-1 and TNF-alpha; (2) these mediators induce changes in epidermal and dermal cells including the induction of adhesion molecules and promotion of the translocation of normally intracellular autoantigen such as Ro/SS-A to the surface of epidermal cells; (3) autoantibody from the circulation binds to autoantigens such as Ro/SS-A that have been translocated to the surface of epidermal keratinocytes and (4) keratinocyte cytotoxicity ensues as the results of lymphoid cells that have been recruited from the circulation recognizing and responding to the Fc domains of autoantibody molecules bound to autoantigen expressed on the surface of keratinocytes (i.e. antibody-dependent cell-mediated cytotoxicity). Although this remains among the most attractive of hypotheses for the explanation of Ro/SS-A antibody-associated forms of LESSD such as SCLE and neonatal LE, it does not address the pathogenesis of other forms of LESSD such as DLE, which are not associated with high-level Ro/SS-A antibody production or other known autoantibody specificities (low-level Ro/SS-A autoantibody production has been noted recently in DLE patients (155)). In addition, this hypothesis implies that the fundamental abnormality in SCLE and neonatal LE is the production of high levels of Ro/SS-A autoantibody; however, equally high levels of Ro/SS-A antibodies having similar molecular specificities are frequently encountered in other conditions such as Sjogren's syndrome in which LESSD is seen only infrequently. Also, a nude mouse model of anti-Ro/SS-A autoantibody in deposition grafted human skin has been developed; however, no inflammation or epidermal injury occurs in these animals (83). Most work has indicated that the action spectrum for Ro/SS-A autoantigen modulation in human epidermal keratinocytes is limited to the UVB spectrum; however, recent studies have suggested that UVA is involved in the elicitation of certain forms of photosensitive cutaneous LE such as SCLE. The hypothesis that CD4+ T cells that are specific for autoantigens in the skin whose expression is altered by UVR exposure might play a role in the expression of LESSD needs to be explored further. Because LE is thought to be a polygenic autoimmune disease, it is possible that polymorphism of genes that govern the skin's response to UVR might be involved in the pathogenesis of photosensitive LESSD. Candidate genes would include: The Ro/SS-A autoantigenic polypeptides and h-YRNA; cytokines, cytokine receptors and adhesion molecules induced on epidermal keratinocytes and dermal endothelial cells by UVR; molecules involved in DNA repair; components of pathways leading to the generation and quenching of oxygen free radicals and components of the UVR-induced apoptosis cascade. Unfortunately, so little is known about DM photosensitivity that it is difficult to even speculate about pathogenetic mechanisms that might be involved other than to extrapolate from the observations and currents of thought relating to photosensitive cutaneous LE. A truly limiting aspect of our understanding in this area has been the absence of working models of the patterns of inflammation seen in LESSD and cutaneous DM. Until such models become available, considering the limitations of human studies, alone it is highly likely that reviews of this subject will continue to be based on much in speculation as observation. PMID- 8628750 TI - Physiological activity of retinoids in natural and artificial visual pigments. PMID- 8628751 TI - Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells. AB - Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments. PMID- 8628752 TI - Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin in single melanoma cells. AB - Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo-PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge-coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (10(3)-4 x 10(3) point pixels in a single cell). The net result was the intensity I (x, y, gamma), for each pixel of the image (x,y), at any wave-length (gamma). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5 aminolevulinic acid (ALA). The fluorescence image of ALA-treated cells revealed endo-PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one subcellular site to another. Photoirradiation of the cells induced point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal-lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo-products could be visualized at sites of formation in subcellular compartments. PMID- 8628753 TI - Photooxidation of troglitazone, a new antidiabetic drug. AB - Troglitazone (CS-045) is a new oral antidiabetic drug reported to be effective in insulin-resistant diabetes and to show antihypertensive effects. Photooxidation of troglitazone gave the quinone and quinone epoxide as the major final stable products. An intermediate observed by NMR spectroscopy was shown to be the hydroperoxydienone, which is moderately stable at room temperature. The rate constant of singlet oxygen quenching by troglitazone is 2.14 x 10(8) M(-1) s(-1) and the reaction rate constant in acetone-d6 is 8.64 X 10(6) M(-1) s(-1). Only the chroman ring of troglitazone reacts with and quenches singlet oxygen significantly, and its reactivity and products are analogous to those of alpha tocopherol. The reactivity of CS-45 toward singlet oxygen is much larger than that of the related compounds lacking the chroman ring. PMID- 8628754 TI - Erythemal ultraviolet insolation in New Zealand at solar zenith angles of 30 and 45.. AB - Solar UV radiometers with spectral responsivities that are close to the erythemal/carcinogenic action spectrum of skin have been installed at several centers of population in New Zealand, including Auckland, 37 degrees S, Wellington, 41 degrees S and Christchurch, 43.5 degrees S. The data set covers the period from the time the radiometry program commenced in 1988/1989 to the end of the southern summer, March 1995. The radiometers were recalibrated annually and the data were corrected for changes in the absolute responsivity of the radiometers. Erythemally effective UV irradiances at solar zenith angles of 30 degrees and 45 degrees were then extracted from the data set. No monotonic trend in these data is apparent, although there are statistically significant differences in mean irradiances from one year to the next. An example of this is the decrease observed in all sites following the Mt. Pinatubo eruption in June 1991. The maximum erythemally effective insolations at solar zenith angles of 30 degrees and 45 degrees were consistently lower in Christchurch than in the other two New Zealand sites. This could arise from higher levels of atmospheric turbidity and/or tropospheric ozone at this location. Also, a seasonal increase in erythemally effective UV insolation from spring to autumn was observed each year in all three New Zealand sites. PMID- 8628755 TI - Overexpression of Medaka (Oryzias latipes) photolyase gene in Medaka cultured cells and early embryos. AB - To study the role and the regulation of the photolyase gene in the Medaka (small teleost), we constructed a eukaryotic expression plasmid of the Medaka photolyase gene and introduced it into Medaka cells in vivo and in vivo. The expression plasmid contains a cytomegalovirus enhancer and a thymidine kinase promoter to overexpress the photolyase gene of the Medaka. First, we transfected this construct into cultured Medaka cells and established several lines of transfectant. Every transfectant showed enhanced ability of pyrimidine dimer repair in the presence of fluorescent light. In the transfectant that showed the most enhanced ability of photorepair, the augmented transcription of photolyase gene was observed compared with that of progenitor OL32 cells. In this transfectant, we also observed an enhanced rate of UV survival with 20 min of fluorescent light treatment after irradiation with a 400 J/m2 UV sunlamp. Next, the expression construct was microinjected into the embryos of the Medaka at the one cell stage. Compared with the nontreated counterparts, the overexpression of a photolyase gene was detected in the microinjected embryos, but we failed to detect a significant increase in photo-reactivability of death at the midblastula stage. PMID- 8628756 TI - Quantitation of the reactive oxygen species generated by the UVA irradiation of ascorbic acid-glycated lens proteins. AB - The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol. 62, 454-463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/cm2 of absorbed UVA light (gamma > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 microM as determined by the superoxide dismutase (SOD)-dependent increase in hydrogen peroxide formation and of 52 microM by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 microM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system. Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N-dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H2O2 were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen. PMID- 8628757 TI - Liposome-mediated delivery of photosensitizers: localization of zinc (II) phthalocyanine within implanted tumors after intravenous administration. AB - CGP55847, liposomal zinc(II)-phthalocyanine (Zn-Pc), was administered by the intravenous route to Swiss mice bearing intramuscularly implanted Ehrlich carcinomas or to C57/BL6 mice bearing subcutaneously implanted B16 melanomas. Tumors were removed 3 h or 24 h after dosing the intratumoral distribution determined by fluorescence microscopy. Localization of the photosensitizer occurred more rapidly in the Ehrlich carcinoma than in the B16 melanoma; this difference in photosensitizer uptake may be related to a higher degree of vascularization of the carcinoma. The photosensitizer was found in association with blood vessels at 3 h but not 24 h after dosing and appeared to have a greater affinity for areas of tissue necrosis within the tumor compared to viable tumor tissue. Little or no Zn-Pc was detected in the muscle tissue invaded by the Ehrlich carcinoma and was associated with the membranes and the cytosol, but not the nucleus, of cells in both tumors. PMID- 8628758 TI - The effects of ex vivo handling procedures on the near-infrared Raman spectra of normal mammalian tissues. AB - Recent studies in the literature have investigated the feasibility of tissue diagnostics based on Raman spectroscopy. The majority of these compare the ex vivo spectra of normal and diseased tissue. Due to the time lapse between tissue excision and spectroscopic examination, samples must be frozen or otherwise preserved to maintain their native biochemical states. In order to establish optimum procedures for ex vivo Raman spectroscopy of tissue, the effects of tissue drying, formalin fixing, snap freezing, tissue freezing in optimal cutting temperature (OCT) medium and extended post-thaw durations were studied to determine if any of these handling procedures introduced spectral artifacts. Experiments on representative tissues indicated that tissue heating due to the excitation light did not change the spectra significantly. With minor exceptions, OCT and formalin did not contaminate tissue spectra, so that samples stored for histological examination could also be studied with Raman spectroscopy. Tissue dehydration caused disruption of the protein vibrational modes, which caused spectral artifacts. It is concluded that ex vivo tissue samples should be frozen in OCT. Prior to spectral analysis, the tissue should then be acclimatized at room temperature in phosphate-buffered saline (PBS) and immersed in PBS during spectroscopic examination. PMID- 8628759 TI - Effects of ultraviolet-B exposure on the resistance to Listeria monocytogenes in the rat. AB - A rat infection model using the bacterial pathogen Listeria monocytogenes was employed to analyze the immunosuppressive activity of UVB radiation. Rats were exposed to suberythemal doses of UVB radiation for 5 or 7 consecutive days, using Kromayer or FS40 lamps respectively. Subsequently, the rats were infected subcutaneously or intravenously with Listeria. Exposure to UVB resulted in an increased number of bacteria in the spleen 4 days after infection. Listeria specific lymphocyte proliferation assays as well as delayed-type hypersensitivity reactions demonstrated that T cell-mediated immunity to Listeria was impaired by UVB as measured 4 and 8 days after infection. In addition, UVB exposure decreased phagocytotic activity of peripheral blood macrophages. This study demonstrated that suberythemal doses of UVB radiation caused a delay in the clearance of Listeria bacteria from the spleen of the rats and that this was probably caused by impaired nonspecific phagocytosis of Listeria by macrophages in addition to an impaired activity of Listeria-specific T cells. PMID- 8628760 TI - The photochemistry of the retinoids as studied by steady-state and pulsed methods. AB - The retina and retinal pigment epithelium contain a number of retinoids in a metabolic pathway that eventually forms the visual pigments. This study investigates the photochemistry of those retinoids that may contribute to light induced damage to the retina. These include retinal (RAL), retinol (ROL), retinylpalmitate (ROLpal) and the protonated Schiff-base of retinal (RALsb). Their photochemistry was followed by both EPR spin-trapping techniques and the direct detection of singlet oxygen via its luminescence at 1270 nm. Irradiation (> 300 nm) of RAL, ROL in methanol (MeOH) or RALpal in dimethylformamide, produces free radicals from both solvents. Illumination of RALsb in MeOH containing NADH with light above 400 nm (and even above 455 nm) generates the superoxide radical. We also determined that the quantum yields for singlet oxygen sensitization by RAL, ROL or RALpal in MeOH are 0.05, 0.03 and < 0.01, respectively. These values are at least 75% less than those previously found using chemical methods. These observations indicate that a major photochemical process for these retinoids may be an electron (or hydrogen) process that will lead to radical products, and that the singlet oxygen mechanism is of relatively minor importance in protic solvents. These results may explain the action spectra obtained from light-induced damage to the retina. PMID- 8628761 TI - Blue-light receptor requirement for gravitropism, autochemotropism and ethylene response in Phycomyces. AB - Light, gravity and ethylene represent for plants and fungi important environment cues for spatial orientation and growth regulation. Coordination of the frequently conflicting stimuli requires signal-integration sites, which, however, remain largely unidentified. The genetic and physiological basis for signal integration was investigated with a set of phototropism mutants (genotype mad) of the UV- and blue-light-sensitive fungus Phycomyces blakesleeanus, which responds also to gravity, ethylene and nearby obstacles (autochemotropism or avoidance response). Both, class 1 and class 2 mutants display a reduced sensitivity to visible light. Class 1 mutants with defects in genes madA, B, C, I have preserved their sensitivity to gravity and ethylene, whereas class 2 mutants with defects in genes madD,E,F,G,J have lost it. We found that the phototropic sensitivity of class 1 mutants is affected roughly to the same extent in far UV and blue light. In contrast, the sensitivity loss of class 2 mutants is restricted mainly to the near-UV and the blue-light region, whereas the sensitivity to far UV is only mildly affected. This behavior of the class 2 mutants indicates that different photoreceptors mediate phototropism in far-UV and in near-UV/ blue light. The photogravitropic action spectra for two class 2 mutants with defects in genes madF and madJ display distortions between 342 and 530 nm and a bathochromic shift relative to the action spectrum of the wild type. These features indicate that the madF and madJ mutants are affected at the level of the blue-light photoreceptor system. As an implication we infer that an intact near-UV/blue light photoreceptor system is required even in darkness for negative gravitropism, the ethylene response and autochemotropism. In Phycomyces, signal integration occurs, at least in part, at the level of the near-UV/blue-light photoreceptor system. PMID- 8628762 TI - Studies in cranial suture biology: part II. Role of the dura in cranial suture fusion. AB - The biology underlying normal and premature cranial suture fusion remains unknown. The purpose of this study was to investigate the role of the dura mater in cranial suture fusion. In the Sprague Dawley rat model, the posterior frontal cranial suture fuses between 10 and 20 days of postnatal life. The effect of separating the posterior frontal cranial suture from its underlying dura mater with an intervening silastic sheet was studied. Sixty rat pups, age 8 days, were divided into four groups of 15. Group A served as unoperated controls. Group B, the experimental group, underwent craniotomy, dural elevation, and insertion of a silicone sheet between the posterior frontal cranial suture and the underlying dura. Two operative sham groups were included. Group C underwent craniotomy and dural deflection only. Group D underwent craniotomy alone without dural deflection. The rats were sacrificed at 15, 22, and 30 days of age. The results showed that the unoperated animals (group A) demonstrated normal initiation of suture fusion at 15 days and complete fusion by 22 days. Group B animals, with silicone sheet barriers placed, showed persistent patency of sutures at 22 days. Initiation of suture fusion was delayed until 30 days. Sham group C, animals with craniotomy and dural deflection, showed that initiation of fusion was delayed until 22 days with complete fusion by 30 days of age. Sham group D, craniotomy alone, had the same normal temporal sequence of suture fusion as the unoperated control group A. These data indicate that normal cranial suture fusion is delayed when the suture-dural interaction is interrupted by a surgically place barrier or by simple dural deflection. Furthermore, interaction between the dura and the overlying suture appears to direct suture fusion. PMID- 8628763 TI - The concept of the sagittal orbital-globe relationship in craniofacial surgery. AB - Euophthalmos, the normal relationship of the orbital rims to the eyes, is critical to planning and to surgical correction of craniofacial deformities. The four most easily localized anthropometric (soft tissue) landmarks for the sagittal orbital-globe relationship are orbitale superius (os), orbitale inferius (oi), orbitale laterale (ol), and nasion (n), all referenced to apex corneae (acor). The normal adult values for os, oi, ol, and n were extracted from the literature. Age-specific anthropometric landmarks were computed from age-specific Bolton cephalometric templates. A vernier caliper was used to measure preoperatively the surface orbital landmarks in patients with various syndromic and nonsyndromic craniosynostotic disorders. Preoperative measurements were compared with the derived normative data to determine the necessary sagittal orbital translocation for frontal advancement (n=19) and frontal-midfacial advancement (n=2). Postoperative orbital anthropometry documented the degree of normalization of the sagittal orbital-globe relationship. The problems with current instrumentation for orbital anthropometry are discussed. PMID- 8628764 TI - Reliability of the fibular osteocutaneous flap for mandibular reconstruction: anatomical and surgical confirmation. AB - There is ongoing controversy regarding the reliability of the skin island associated with the fibular osteocutaneous flap for mandibular reconstruction. Anatomical dissections and a clinical series of mandibular reconstructions using the fibular osteocutaneous flap have demonstrated unequivocally that a skin flap can be reliably harvested with the fibula based purely on the septal perforators, without needing to incorporate portions of the soleus or flexor hallucis longus muscles or to perform any intramuscular dissection or anastomosis of the muscle perforators. However, the skin island should be designed more distally over the distal third of the lower leg at the junction of the middle and distal thirds of the fibula. A fibular osteocutaneous flap was designed over the distal third of the fibula in 60 fresh cadavers, and each flap was completely isolated on the septum and all muscle perforators were ligated before dye injection. A major perforator through the soleus muscle or flexor hallucis muscle was identified in 41 of 60 dissections (67 percent) and discrete septal perforators were identified under loupe magnification in 45 dissections (75 percent). All 60 flaps demonstrated 100 percent reliable perfusion of the skin island after injection of the proximal peroneal artery with methylene blue or red latex. This anatomical study was corroborated with 100 percent survival of 34 fibular osteocutaneous flaps for mandibular reconstruction with the skin island designed over the distal third of the lower leg and based only on septal perforators without incorporating the soleus or flexor hallucis muscles. Reliability of this fibular osteocutaneous flap for mandibular reconstruction is attributed to (1) design of the skin island more distally over the distal third of the lower leg, (2) preoperative precision Doppler mapping of the perforators, and (3) design of the closing wedge osteotomies of the fibula to protect the septocutaneous perforators transversing through the posterior periosteum of the fibula. PMID- 8628765 TI - Postoperative function after implant insertion in vascularized bone grafts in maxilla and mandible. AB - Between 1988 and 1992, 80 Branemark-type implants were inserted in 18 patients during reconstruction of the mandible or maxilla with vascularized iliac crest or scapula grafts with or without additional soft tissue pedicles. In these procedures, nine vascularized bone grafts were combined with a primary insertion of 32 implants and a secondary insertion of 48 implants. Twelve patients are currently wearing the implant-borne dentures. From 32 implants inserted primarily, eight could not be used for prosthodontic rehabilitation because three were lost with a graft, three were left as sleepers, and two demonstrated a lack of osseointegration. None of the implants inserted secondarily in grafts were lost. Primary implant insertion should be performed only in close cooperation with the prosthodontist and in selected cases, for example, in free-end reconstruction of the mandible with a straight graft and where a limited number of implants is needed. Although restoration of masticatory function in patients with head and neck cancer can be achieved, compared with a healthy control group, functional impairments remain. Patients subjectively favor the nonreconstructed side of the mandible or maxilla for chewing. These findings can be correlated with a postoperative follow-up investigation using a miniature force transducer and the T-scan system. PMID- 8628766 TI - The fate of teeth transfixed by osteosynthesis screws. AB - Rigid internal fixation with plates and screws for osteosynthesis of facial fractures and osteotomies in the cranio-maxillofacial skeleton is often undertaken in situations in which the exact position of the underlying tooth roots cannot be determined. Therefore, a screw may be inadvertently placed into a root. There is scant data in the literature addressing the consequences of tooth impingement. This 5-year retrospective study documents the long-term outcome of teeth transfixed by osteosynthesis screws in a series of 387 consecutive facial fractures at a Level I trauma center. The incidence of root impingement per screw was 0.47 percent (13 transfixed teeth per 2340+ screws). Mandibular teeth were more "at risk" than maxillary teeth by a ratio of 10:3. No transfixed teeth became infected or required extraction in this series. In conclusion, inadvertent tooth root impingement by osteosynthesis screws appears to have minimal adverse consequences. PMID- 8628767 TI - Hemimandibular hyperplasia. AB - True hemimandibular hyperplasia is an uncommon maxillofacial deformity. Patients with this affliction present clinically with varying degrees of asymmetry characterized by an increase in ramus height, a rotated facial appearance with kinking at the mandibular symphysis, and prominence of the lower border of the mandible. In the advanced form, maxillary and mandibular alveolar bone overgrowth result in a compensatory canting of the occlusal plane and a significant functional malocclusion requiring bimaxillary surgical correction. Nine patients with the above condition were treated with various surgical methods over a 9-year period. The clinical results were satisfying and the long-term results showed good postoperative stability. The pathology of hemimandibular hyperplasia is described and the preexisting nomenclature further defined. PMID- 8628768 TI - Oral commissure burns in children. AB - Electrical burns of the mouth are relatively common in young children. Early intervention to prevent complications remains controversial. A chart review was conducted of 24 patients with oral commissure burns who were treated at the University of Iowa from 1975 to 1988. All of these patients were treated conservatively without splinting or early surgery. Only one patient underwent oral splinting before receiving care at the University of Iowa Hospitals and Clinics. While under our care, no patients suffered significant hemorrhage at eschar separation. Commissuroplasty and/or reconstructive lip surgery were performed at various times after the burn injury was healed and the functional or aesthetic impairment was established. Long-term follow-up was from 5 to 17 years, allowing for longitudinal evaluation of the postburn scars and their influence on facial growth. Our review revealed that (1) younger children with more severe burns have a less favorable outcome; (2) no hemorrhage was observed immediately after the burn or at eschar separation; and (3) conservative surgical treatment after scar maturation- and in some cases following steroid injections- resulted in a successful functional and esthetic outcome. PMID- 8628769 TI - Free flaps based on the anterior interosseous artery. AB - Free flaps based upon the anterior interosseous artery were defined anatomically by 15 dissections. The anterior interosseous artery runs distally for the entire length of the anterior interosseous nerve on the palmar surface of the interosseous membrane. A dorsal branch of the anterior interosseous artery penetrates the interosseous membrane about 5 cm proximal to the distal radioulnar joint and gives off a tributary radially. This tributary ascends the ulnar aspect of the radius to provide blood to the radius and the dorsal wrist skin. Based on these anatomical findings, four types of free flaps were possible: fasciocutaneous, osteocutaneous, neurovascular flaps, and one muscle flap. All seven dorsal wrist flaps successfully covered the defects. Three of four nerve flaps demonstrated patency of the arterial pedicle with digital Allen's test and good sensory recovery of the reconstructed area. One reconstruction of Blauth type II hypoplastic thumb using an ipsilateral pronator quadratus free-muscle flap provided a functionally and cosmetically satisfactory result. PMID- 8628770 TI - In vitro measurement of silicone bleed from breast implants. AB - A method to measure gel bleed from intact silicone gel-filled breast implants was developed. This nondestructive technique permits accurate and reproducible serial measurements of silicone bleed from smooth wall breast implants (n=10) under simulated physiologic conditions in vitro. Gel bleed rates from new low bleed gel filled implants and intact explants (unbarriered, low bleed, double lumen) were determined. These results demonstrate the reliability of this method to quantify silicone gel bleed and may permit a meaningful comparison of bleed rates from implants in the future. PMID- 8628771 TI - The subcutaneous pedicle tensor fascia lata flap. AB - The presence of a consistent subcutaneous vascular plexus allows carrying of a distal skin island safely on the iliotibial tract. A distal skin island can be designed on the lateral thigh and can be raised on a subcutaneous pedicle that is proximally supplied by the lateral femoral circumflex artery. This technique preserves the lateral thigh skin and employs subcutaneous tunneling to overcome the traditional drawbacks of the conventional extended tensor fascia lata flap. The subcutaneous pedicle tensor fascia lata flap's sensate potential, thin skin, durable fascia, extensive reach, and 360 degree arc of rotation make it an appealing donor site for coverage of lower midsection and pelvic defects or for penile reconstruction. PMID- 8628772 TI - Neovaginal reconstruction after exenteration using an omental flap and split thickness skin graft. AB - The earliest efforts at neovaginal reconstruction used split-thickness skin grafts when bladder and rectum remained in place. In patients undergoing total pelvic exenteration, the pelvic organs are not available to accept the skin graft. By modifying the omental flap normally used to close off the pelvic inlet after total pelvic exenteration with or without lower coloproctostomy, a cylinder can be created that provides anterior, posterior, and lateral walls for the neovagina. When this omental cylinder is lined with a split-thickness skin graft and secured in the postoperative period using a soft vaginal form, a satisfactory neovagina can be created. This article presents the authors' experience with 20 patients who underwent radical pelvic exenteration for gynecological malignancy and neovaginal reconstruction using an omental cylinder flap lined with a split thickness skin graft. In this series, all flaps and skin grafts have remained soft and completely viable with no pelvic infections, perineal fistulae, or hernias, and they offer the potential for sexual function in approximately 80 percent of patients. Average reconstruction operating time is less than 2 hours. In the properly selected patient, this method provides distinct advantages over reconstruction with myocutaneous flaps, which may be too bulky, too difficult to pass into the pelvis, and require additional donor-site incision with prolonged operative time. Myocutaneous flaps may have greater potential for partial or complete tissue loss. Neovaginal reconstruction using an omental cylinder flap lined with a split-thickness skin graft compares favorably with previously described methods by providing support for the pelvic floor with primary healing while restoring the potential for sexual function with minimal overall morbidity. PMID- 8628773 TI - Prostacyclin production at the human microvascular anastomosis: its effect on initial platelet deposition. AB - The human microvascular anastomosis represents a localized environment with strongly thrombotic tendencies. In previous studies, an increase in initial platelet deposition at a human ex vivo anastomosis was measured. It is postulated that this increase in anastomotic platelet deposition was due to a reduction in anastomotic prostacyclin production as a consequence of local endothelial cell injury or loss. Instead, in this study, an increase in anastomotic prostacyclin production over unsutured controls (control 1093 +/- 222 pg/ml of 6-keto prostaglandin F (PGF) 1-alpha, n=21; anastomosis 2494 +/- 414, n=21, mean +/- 1 SEM, p=0.005) is demonstrated. Anastomotic prostacyclin production was augmented by addition of arachidonic acid (0.1 mM) (39,000 +/- 11,300 pg/ml of 6-keto PGF 1 alpha, n=7, p<0.001) and suppressed by the preincubation of vessel segments with aspirin in a dose-dependent fashion (1mM) (83+/-22 pg/ml of 6-keto PGF 1-alpha, n=21, p<0.001); aspirin (0.1 mM) (312 +/- 56 pg/ml of 6-keto PGF 1-alpha, n=7, p<0.001). In further studies using a perfusion apparatus of human blood pumped through human placental artery segments, suppression of prostacyclin production did not augment initial platelet deposition (control anastomosis 4.9 +/- 2.2 x10(6) platelets per cm2, aspirin treatment 6.0 +/- 2.8 x 10(6) platelets per cm2, n=5, mean +/- 1 SEM, p>0.05). Suppression of platelet function with aspirin (0.1 mM) also did not decrease initial platelet deposition onto the anastomosis (5.8 +/- 2.8 x 10(6) platelets per cm2, n=r, p>0.05). In this model system, initial platelet deposition at the anastomosis may not be dependent upon cyclooxygenase pathways. PMID- 8628774 TI - Microvascular anastomoses. A comparative study of fibrinogen adhesive and interrupted suture techniques. AB - A modified sleeve technique was developed for making microsurgical anastomoses using a commercially produced fibrinogen adhesive called Tisseel. A controlled study was then carried out to compare the new fibrinogen adhesive anastomoses with conventional suture anastomoses in a bilateral groin flap model using 50 consecutive rabbits. Statistical analysis of the results indicated that flap survival rate and vascular patency rate were comparable for the two techniques. The fibrinogen adhesive anastomoses took less time to complete and, subjectively, were less difficult technically. The suture anastomoses were more versatile. Histologic studies revealed that the adhesive did not flow through the sleeve into the lumen, and that, although there was a brief inflammatory response associated with healing, this inflammation was very localized and did not involve the inner layers of the vessel wall or lumen. It was concluded that the new technique was a useful addition to techniques already available. PMID- 8628775 TI - Advantages of autologous fascia versus synthetic patch abdominal reconstruction in experimental animal defects. AB - Although prosthetic patches (i.e., expanded polytetrafluoroethylene) are commonly used to repair abdominal fascial defects, autologous tissue is preferred in the presence of wound contamination. This study was undertaken to discover (1) whether fascial grafts are revascularized and incorporated as living tissue, and (2) whether fascial grafts are more resistant to bacterial contamination than prosthetic patches. In the first experiment, 18 New Zealand White rabbits underwent full-thickness resection of the central abdominal wall preserving only panniculus carnosus and skin. Six control animals had only skin repaired, and all developed large ventral hernias. Twelve animals had the defect repaired with thoracodorsal fascia patches. At 3- and 6-week intervals, no hernias were present and all patches were incorporated with minimal contraction. Fluorescein angiography verified revascularization from the surrounding abdominal wall. Next, 36 rabbits underwent similar resection followed by repair with either autologous fascia (n=18) or expanded polytetrafluoroethylene (n=17). Six rabbits of each repair group were inoculated with 10(4) Staphylococcus aureus and twelve rabbits with each repair were inoculated with 10(9) S. aureus. All rabbits receiving 10(4) S. aureus were infection-free survivors. Seven of the twelve expanded polytetrafluoroethylene-repaired animals receiving 10(9) S. aureus developed necrotizing wound infections and died. Only 2 of 12 rabbits with autologous fascia repairs died from wound sepsis and 1 died of diarrhea with a healed wound. Differences in wound infection rates achieved statistical significance, whereas survival differences approached significance (Fisher's exact test), suggesting that revascularized fascial grafts may be more resistant to bacterial contamination than expanded polytetrafluoroethylene patches at this concentration (10(9) S. aureus). PMID- 8628777 TI - Academic plastic surgery: perspectives in a changing health care environment. PMID- 8628776 TI - Assessing the role of presuturing on wound closure. AB - The efficacy of presuturing was tested by quantitating the tissue gains for standardized wounds in a white swine piglet (9 to 11-kg) model. Also measured were the changes in wound closure tension for the wounds. Measurements were carried out at 1, 4, 17, 24, 48, and 72 hours in a total of 20 piglets. Presuturing was carried out on one of the flanks and the opposite flank served as the control. Presuturing achieved a modest tissue gain, amounting to 67 mm at 4 hours and a maximum of 1.4 cm at 48 and 72 hours, for a 4-cm defect. The encouraging tissue gain was unfortunately not paralleled by a decrease in the tension required to approximate the wound edges. At 4 hours, the tension to close the wound was 1.2 N (only 120 g) less than the control wound. This initial small reduction in wound-closing tension was diminished thereafter, and at 72 hours the wounds were stiffer and harder to close than the control. This appeared to be due to tissue edema and was confirmed by an increase in measurable tissue water with increasing time. In this model, presuturing produces a minimal tissue advancement and even less reduction in wound-closing tension. It is concluded, from this work and from previously published work, that undermining will prove generally to be a more useful technique in closing broad defects. PMID- 8628779 TI - An accurate technique for fixation in endoscopic brow lift. PMID- 8628778 TI - Idiosyncratic allergic reaction to textured saline implants. AB - In the literature, multiple conditions, including hematomas, self-limited cutaneous eruptions, and generalized systemic complaints, have been attributed to breast implants. We report the first case of idiosyncratic allergic reaction to the textured surface of a mammary prosthesis. The reaction was documented by patch testing of the textured surface compared with smooth-surface silicone controls. Symptoms resolved with removal of the implants and have not recurred after insertion of smooth-walled implants. Whereas the physiology of this condition remains unclear at this time, it is important to recognize the possibility of a delayed hypersensitivity reaction when considering reconstruction with a textured breast implant. PMID- 8628780 TI - Alar rim excision: a method of thinning bulky nostrils. AB - A thick nostril is a disturbing deformity and one for which surgeons and patients may be reluctant to employ the traditional "piece of pie" skin excisions commonly described. As an alternative in selected circumstances, a method of internally thinning the nostril rim using an alar base groove incision known as alar rim excision is presented. An alar base resection can accompany this, or the incision can be used solely for access when reducing a bulky nostril, while avoiding additional scarring or the risk of alar rim elevation. PMID- 8628781 TI - Subcutaneous approach for elevation of the malar fat pad through a prehairline incision. AB - In summary, vertical suspension of the malar fat pad has been a safe procedure that results in the creation of a more youthful cheek and a lessening of the prominence of the nasolabial folds. No complications were encountered in this series with the suture suspension of the malar fat pad. PMID- 8628783 TI - Reconstruction of the lower lip and chin with local flaps. AB - We present a single patient with reconstruction of a rectangular defect of 40% of the lower lip and chin. The method used constitutes a modification of the Karapandzic method, with the addition of an advancement of the lateral cheek and remaining chin. The technique restores form, sensation, and function- the principles of a successful reconstruction. PMID- 8628782 TI - A technique for correcting witch's chin deformity. AB - A new technique for soft-tissue correction of the retraced submental fold in the Witch's Chin deformity is described. An illustrative case report demonstrating our technique, results, and the pathogenesis of this condition is presented. An anatomical analysis of the underlying etiology of the condition and tailored treatment strategies are outlined. PMID- 8628784 TI - Adipofascial axial pattern cross-finger flap. PMID- 8628785 TI - Scar formation: the spectral nature of fetal and adult wound repair. PMID- 8628786 TI - Solving the mystery of the scalpel blades: what do the numbers mean? PMID- 8628787 TI - Maxillofacial injuries. PMID- 8628788 TI - Resection of the corrugator supercilii muscles through blefaroplasty incision. PMID- 8628789 TI - Repair of the incomplete earlobe cleft. PMID- 8628790 TI - Curettage: an alternative to capsulectomy after breast implant removal. PMID- 8628791 TI - Necrobiotic palisading granulomas associated with face cream injections. PMID- 8628792 TI - Microsurgical replantation of the avulsed scalp: report of 20 cases. AB - Our 4-year experience with 20 patients who had suffered avulsion of 75 percent or more of the scalp is reviewed. All patients underwent replantation using microsurgical technique with 100 percent survival in 16, partial survival in 3, and failure in only 1 case. The emergency management and indications for replantation are demonstrated. The roles of sufficient preoperative preparation, generous debridement of damaged vessels, interpositional vein grafts, and the shortening of operative time in contributing to this success are emphasized. We developed a new surgical procedure called simultaneous vein grafts on donor and recipient sites in an effort to use less time in the anastomosis of interpositional vein grafts. Furthermore, we anastomosed the extra artery of the scalp to the vein on the recipient head when no suitable vein could be found. Intraoperative repair of the scalp sensory nerve and no postoperative use of any vasodilator or anticoagulant are discussed. PMID- 8628793 TI - Transcutaneous PO2 of the scalp in male pattern baldness: a new piece to the puzzle. AB - Our study was designed to measure the transcutaneous PO2 of the scalp to determine if there was a relative microvascular insufficiency and associated tissue hypoxia in areas of hair loss in male pattern baldness. A controlled prospective study was performed at Butterworth Hospital, Grand Rapids, Michigan. Eighteen nonsmoking male volunteers aged 18 years and older were studied. Nine men had male pattern baldness (Juri degree II or III), and nine were controls (no male pattern baldness). Scalp temperature and transcutaneous PO2 were obtained at frontal and temporal sites in each subject. Peripheral circulation was assessed from postocclusive transcutaneous PO2 recovery time by means of maximum initial slope measurements. Statistical significance was assessed at p < 0.05. There was no significant difference in scalp temperature between male pattern baldness subjects and controls. Temporal scalp blood flow was significantly higher than frontal scalp blood flow in male pattern baldness subjects; however, there was no significant difference in controls. Transcutaneous PO2 was significantly lower in bald frontal scalp (32.2 +/- 2.0 mmHg) than in hair-bearing temporal scalp (51.8 +/- 4.4 mmHg) in men with male pattern baldness. In controls, there was no significant difference in transcutaneous PO2 of frontal scalp (53.9 +/- 3.5 mmHg) and temporal scalp (61.4 +/- 2.7 mmHg). Transcutaneous PO2 also was significantly lower in the frontal scalp of male pattern baldness subjects (32.2 +/- 2.0 mmHg) than in either frontal or temporal scalp of controls (53.9 +/- 3.5 mmHg and 61.4 +/- 2.7 mmHg, respectively). There is a relative microvascular insufficiency to regions of the scalp that lose hair in male pattern baldness. We have identified a previously unreported tissue hypoxia in bald scalp compared with hair-bearing scalp. PMID- 8628794 TI - Monobloc and facial bipartition osteotomies for reconstruction of craniofacial malformations: a study of extradural dead space and morbidity. AB - This study evaluated the presence of extradural dead space following a monobloc or facial bipartition osteotomy and examined its natural history and relationship to postoperative infection and the presence of a ventriculoperitoneal shunt at the time of osteotomy in a consecutive series of patients with craniofacial dysostosis, frontonasal dysplasia, midline cranio-orbital clefts, and orbital hypertelorism. Only patients followed for at least 1 year were included in the study (range 1.3 to 5.5 years). The 23 patients studied were divided into three groups: 10 patients (mean age 9 years) underwent a monobloc osteotomy with advancement, 7 (mean age 8 years) a facial bipartition osteotomy with advancement, and 6 (mean age 7 years) a facial bipartition osteotomy without advancement. Standard craniofacial computed tomographic (CT) scans were obtained for each patient early after surgery (within 2 weeks in 13 patients and at 6 to 8 weeks in 10 patients) and again 1 year after surgery in every case. The extradural dead space was measured from a reproducible axial CT scan slice for each patient at each postoperative interval. An initial dead space was documented in the retrofrontal region of the anterior cranial fossa when the reconstruction incorporated forward projection of the osteotomy parts. This space was found to be obliterated by the expanded brain by 6 to 8 weeks in the patients examined by CT scan slice for each patient at each postoperative in all patients. Perioperative complications also were documented. The presence of a ventriculoperitoneal shunt at the time of osteotomy (7 of 23 patients) did not increase the risk of complications or alter the pattern of dead space closure after operation. Two patients developed infectious complications that were managed without long-term consequences. PMID- 8628795 TI - The effect of insulin-like growth factor 1 on craniofacial bone healing. AB - Human insulin-like growth factor 1, a known regulator of bone formation, was investigated for its possible effect on membranous bone formation in a rat model. Full-thickness bone defects (10 x 10 x 1 mm) were created in the rat calvarium, and insulin-like growth factor 1 was administered by an osmotic minipump directly into the defect enclosed by the periosteum and dura mater. The dose of insulin like growth factor 1 was 100 micrograms every 2 weeks. The defects were studied radiographically, macroscopically, and microscopically at 3, 6, 9, and 12 weeks. The group treated with insulin-like growth factor 1 showed qualitative and quantitative differences when compared with the control group. The amount of new bone formation in the group treated with insulin-like growth factor 1 was significantly larger than that of the control group. In the insulin-like growth factor 1 group, the location of new bone formation occurred in the center and at the margin of the bone defect. In the control group, bone was formed only around the margin of the bone defect. This study suggests that insulin-like growth factor 1 improved membranous bone healing in vivo and that insulin-like growth factor 1 makes mesenchymal precursor cells of bone differentiate directly into bone-forming cells. PMID- 8628796 TI - Surgical correction of submucous cleft palate with Furlow palatoplasty. AB - Many surgeons have favored using the pharyngeal flap as the primary treatment for the velopharyngeal insufficiency associated with submucous cleft palate. However, the increasing number of reports of sleep apnea and airway compromise as a result pharyngeal flap surgery support the need to eliminate any unnecessary pharyngeal flap surgery. From 1988 to 1993, 35 Chinese submucous cleft palate patients with velopharyngeal insufficiency received surgery. A Furlow palatoplasty was used in 30 patients (3 to 26 years old). The follow-up duration was 9 months to 5 1/2 years. These patients were selected after a thorough study for velopharyngeal insufficiency including intraoral examination, perceptual speech assessment, videonasopharyngoscopy, and/or multiview videofluoroscopy. The criteria for selection included age, intraoral finding of an obviously anterior inserted levator palatine muscle, size of velopharyngeal gap, pattern of velopharyngeal closure, degree of lateral pharyngeal wall movement, and response to biofeedback speech therapy. In general, younger patients with circular or sagittal pattern closure, a velopharyngeal gap less than 5 mm, or good response to biofeedback speech therapy were considered to be the best candidates for a Furlow palatoplasty. The 5 patients who did not fulfill these criteria and whose velopharyngeal function failed to improve on preoperative biofeedback therapy were treated by pharyngeal flap operation. Twenty-nine patients (96.7 percent) achieved competent velopharyngeal function after the Furlow palatoplasty. The procedure corrected the velopharyngeal insufficiency in 3 patients older than 20 years with a velopharyngeal gap of less than 2 mm. The only patient with an unsatisfactory result was a 26-year old woman who had very prominent action of the musculus uvulae before the surgery. The results show that a Furlow palatoplasty can satisfactorily correct velopharyngeal insufficiency in carefully selected submucous cleft palate patients and thus avoid the serious complications of pharyngeal flap surgery. PMID- 8628797 TI - A comparative study of miniplates used in the treatment of mandibular fractures. AB - The purposes of this study were to investigate the differences in mechanical properties of major miniplating systems used for noncompression miniplate osteosynthesis of mandibular fractures and to determine whether these properties influence treatment outcome. The study was conducted in two parts. First, six of the major miniplate systems currently used at the Royal Adelaide Hospital were subjected to bending tests at the University of Adelaide Engineering Department to quantify the relative stiffness of each plate. Second, a prospective sample of patients presenting with mandibular fractures was analyzed. These patients were treated with a variety of the miniplating systems. The results of treatment as a whole were compared to identify any direct benefit consequent on the miniplate selected. While significant differences in stiffness were identified between the plating systems, no significant differences in treatment outcome were identified, between the noncompression plates employed. Since no observable benefits have been identified by choice of miniplate, selection should be based on surgical preference, biocompatibility, CT compatibility and unit cost. Because of the variations in materials, design, properties, CT compatibility, and unit cost, it is important not to regard all miniplates as equal and interchangeable. PMID- 8628798 TI - The multipoint contact plate in fracture treatment of the atrophied mandible: animal study and clinical application. AB - Rapid consolidation of osseous fractures requires stable immobilization of the fragments. In the atrophied mandible, the nutrient supply should be ensured by protecting the periosseous soft tissue, i.e., the periosteum. In the conventional placement of osteosynthesis plates, however, the subimplant nutrition is restricted because of the high surface pressure between the osteosynthesis plate and bone. In order to improve the subimplant nutrient supply, we designed a new osteosynthesis plate with a "knobbed" underside enabling multipoint contact. Histomorphologic comparative studies were conducted on 16 Gottingen minipigs with an average weight of 47.5 +/- 8 kg. In each minipig, one side of the mandible was fractured and one side was left intact. One side of each mandible was plated with a six-hole dynamic multipoint contact plate and the other side with a conventional plate. The plates were applied, either epiosseous or epiperiosteal, with bicortical screws under defined torque. The results demonstrate the advantages of multipoint contact plates over the conventional ones and create the clinical basis for a "bio-logical" plate design that allows callus to grow between the knobs and therefore provide adequate nutritional and vascular supply. The plate's knobbed profile also promotes extraosseous venous drainage without impeding intraosseous fluid flow. This plate is the logical choice for rapid fracture consolidation in the high-grade atrophied mandible. PMID- 8628799 TI - Cost and outcome of osteocutaneous free-tissue transfer versus pedicled soft tissue reconstruction for composite mandibular defects. AB - Thirty-nine patients underwent reconstruction of composite mandibular defects following resection for squamous cell carcinoma. Thirty-four underwent immediate reconstruction, while 5 were reconstructed secondarily. Twenty-one received soft tissue reconstruction only with a pectoralis major myocutaneous flap, 14 underwent osteocutaneous free-tissue transfer, and 4 received a reconstruction plate with free-tissue transfer for soft-tissue coverage. The mandibular defects in the pectoralis major myocutaneous flap group tended to be posterolateral, while free-tissue transfer defects were more severe, usually involving the anterior mandible. Length of surgery and duration of intensive care unit care were significantly longer for free-tissue transfer patients, while flap complications were more common in the pectoralis major myocutaneous flap patients. Facial appearance scores were higher for the free-tissue transfer group by both patient and physician assessment. Social function, speech, and oral function did not differ significantly. Patients reconstructed secondarily with free-tissue transfer reported significant improvement in appearance, oral continence, and social function, with little change in speech intelligibility, deglutition, or diet tolerance. The cost of the main hospitalization was significantly higher in the free-tissue transfer group than in the pectoralis major myocutaneous flap group, although when the costs of subsequent hospitalizations are included, the difference in total cost narrows. Despite more adverse defects, free-tissue transfer provided more predictable aesthetic results and expeditious return to normal social function than did pectoralis major myocutaneous flap reconstruction. The fiscal impact of these complex reconstructions is, however, significant. Cost-containment issues are presented and recommendations are made. PMID- 8628800 TI - Bleed of and biologic response to triglyceride filler used in radiolucent breast implants. AB - Radiolucent breast implants filled with triglyceride oil have recently entered limited clinical trials. To investigate the questions of oil bleed and the fate of triglycerides that might escape from ruptured breast implants, experiments reported here used peanut oil labeled with radioisotopes so that it could be traced in the urine, feces, and organs of two groups of rabbits. In one experiment, 18 rabbits were implanted with peanut oil-filled implants labeled with tritium to determine whether triglycerides diffuse across silicone elastomer shells. In another experiment, 19 rabbits were injected with 14C-labeled peanut oil to study what might happen to the oil if an implant ruptures. At the end of the follow-up period, we measured radioisotope levels in tissue samples taken from the periprosthetic capsule or injection site of each rabbit, as well as from major organs and the subcutaneous fat on the dorsum opposite the experimental site. One experiment revealed that triglycerides do bleed across the implant shells. Tritium levels were highest in the implant capsule, the omentum, the aorta, and the subcutaneous fat on the nonexperimental side. In the experiment simulating implant rupture, 14C levels were above the background radiation count at the injection site and in the same tissue sites as in the bleed experiment. Both in vivo radiolabeling studies indicate that triglycerides freed from implants by means of bleed or rupture would be absorbed, metabolized, and either excreted or redistributed to the body's normal fat storage sites if they are not needed for energy. In a third in vitro experiment, triglyceride oil specimens were inoculated with various microorganisms associated with wound infections: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and diphtheroids. The data demonstrate that neutral triglycerides used as a breast implant filler do not support growth of common infection producing bacteria and suggest that triglycerides may have bactericidal properties. PMID- 8628801 TI - Z-plasty template: an innovation in Z-plasty fashioning. AB - Although Z-plasty is one of the basic skills in the armamentarium of a plastic surgeon, one is rarely found using a protractor to measure the exact angles. Two types of simple gadgets are described here to fashion Z-plasties with desired (fixed or variable) angles useful in various types of Z-plasties. These gadgets are made from transparent plastic sheets. They can be sterilized by formalin vapor or ethylene oxide gas. Since they are pliable, they can be adapted to the contours on the body. These are found to be particularly useful in cases where asymmetrical Z-plasties are needed. PMID- 8628802 TI - Textured or smooth implants for submuscular breast augmentation: a controlled study. AB - Capsular contracture consistently has been the most frequently noted complication of submuscular and subglandular breast augmentation. The etiology of this complication is still unknown, although silicone bleed, hematoma, infection, foreign bodies, and surgical trauma have been implicated. In this prospective, double-blind study, 61 women undergoing submuscular breast augmentation were randomized between Dow Corning textured and smooth-walled silicone gel implants. Any consequent capsular contracture was assessed by an independent plastic surgeon and also by the patients themselves. Objective evaluation was made by applanation tonometry. It was found that depending on doctors, patients, and objective method used, 3 to 9 percent grade III and IV encapsulation followed submuscular augmentation with textured implants and 10 to 20 percent with smooth walled implants after 1 year. The differences were significant according to both patient assessment and applanation tonometry but not according to the physicians' evaluations. There was no correlation of capsular contracture with the age of the patient, duration of the operation, or degree of blood loss. There was a small but inconclusive difference in capsular contracture rate that favored the placement of textured rather than smooth implants in the submuscular pocket. PMID- 8628803 TI - Silicon tissue assays: a comparison of nonaugmented cadaveric and augmented patient levels. AB - Tissue silicon assays were performed on 10 nonaugmented cadavers and 25 augmented women to confirm our previous cadaveric data and to establish silicon levels at local and distant sites in augmented women undergoing explant and further reconstruction. All assays were performed by inductively coupled plasma atomic emissions spectroscopy (ICP-AES). Cadaveric tissues were sampled from six sites: liver, spleen, breast, nipple, axilla (soft tissue and nodes), and subcutaneous tissue (abdominal). Augmented women undergoing explant surgery had a portion of their implant capsule, breast tissue, and for those undergoing autogenous reconstruction, a portion of the autogenous donor site or distant tissue sampled. Twenty-four women had silicone implants; one had saline. Results revealed baseline silicon levels in all 10 non-augmented cadavers. These results were equivalent to our previous data, confirming the validity of these baseline data. Silicon tissue levels in the 25 augmented women revealed elevated levels within the implant capsule and surrounding breast tissue. However, silicon levels obtained from distant sites in augmented women were equivalent to the baseline nonaugmented cadaveric levels. There was no correlation between intact or ruptured implants and symptoms of collagen-vascular disease. In conclusion, study validates our original data of baseline silicon levels in nonaugmented cadavers. Contrary to what some may perceive as silicone floating throughout the body, we have found elevated levels around the implant only, and levels at distant tissue sites were equivalent to the baseline cadaveric data. PMID- 8628804 TI - Strength of silicone breast implants. AB - Rupture and leakage are recognized problems associated with silicone breast implants. Data are scarce about the durability of the silicone shell, and the life span of this device is unknown. The purpose of this study was to investigate the strength of silicone breast implants. Thirty implant shells were subjected to mechanical testing. Twenty-nine of the shells were tested after explanation, and one unused implant served as a control to validate the testing method. Implantation time varied from 4 months to 20 years, and all shells were tested, regardless of condition. Fourteen implant shells were intact, eight were leaking, and seven were ruptured. All ruptured implants had been in place for 10 years or longer. The breaking force of all excised shell specimens ranged from 2.6 to 22.4 N (0.6 to 5.0 lb.). Specimens from the control "high performance" shell required 15.5 to 25.6 N (3.5 to 5.8 lb) of force to fail. The weakest group was from thin shelled implants between 10 and 16 years of age. More than half these specimens failed with less than 1 lb of force. The average breaking force of ruptured shell material was less than that of intact shells. A comparison of strength data in this study with manufacturers' data suggests that breaking force is dependent on implant type, shell thickness, and implantation time. PMID- 8628805 TI - Labial masses following vaginoplasty in male transsexuals: the differential diagnosis. AB - Following vaginoplasty and vulvoplasty for male-to-female transsexualism, a mass may evolve in one of the major labia in a minority of patients. From April of 1989 to April of 1994, we treated seven patients with such masses. The case reports presented illustrate some of the differential diagnoses of this long-term complication. More often than not, the swelling was caused by infection. The funiculus or even testicular rest in itself also may represent the mass. Intralabial urethral fistula appears to be the second cause of swelling, either with or without inflammation. As an exceptional cause we observed a swelling representing a cyst of prostatic origin. In all, the mass could be excised completely. PMID- 8628806 TI - The plastic surgery teaching conference: a horse and buggy educational vehicle for supersonic times. PMID- 8628807 TI - Complications, untoward events, and reality. PMID- 8628808 TI - Scrotal elephantiasis associated with hidradenitis suppurativa. AB - Hidradenitis suppurativa is a chronic relapsing infection of the apocrine sweat glands. Its association with penoscrotal lymphedema is not well recognized. A case of massive scrotal elephantiasis associated with chronic hidradenitis of the perineum and scrotum is described. A wide resection of the scrotal mass and perineum was performed, with reconstruction of the perineum and penis carried out using local skin flaps and split-thickness skin grafts. This one-stage treatment yielded an excellent cosmetic and functional outcome. PMID- 8628809 TI - Gigantic epidermoid cyst of the skull. AB - One case of an unusually giant epidermoid cyst of the skull is described. The neoformation had been present for 40 years, becoming extremely large in the last 20 years, in a 65-year-old woman. Difficulty in wearing glasses was the sole complaint of the patient, who otherwise concealed the mass by wearing a wig. Preoperative examinations (x-rays, CT scan, MRI) were important to better evaluate the nature of the cyst and to make a differential diagnosis. Dissection of the mass from the surrounding tissues was extremely easy because of its capsule. Sixteen months of follow-up have been pleasant, with no evidence of recurrence. PMID- 8628810 TI - SMAS autografts for the nasal dorsum. AB - Many patients of rhytidectomy age have had previous rhinoplasty or nasal injury and have dorsal irregularities or saddle-nose deformities. SMAS tissue ordinarily discarded at rhytidectomy may be used for nasal dorsal augmentation and padding. Six patients undergoing rhytidectomy who also had prior nasal surgery or deformity were selected. Two of the patients had saddle deformities, and four had palpable and visible bony or cartilaginous irregularities. In all patients, substantial improvement was noted. In the saddle deformities, significant long term results were achieved, with graft longevity as great as 24 months. The technique should be considered in rhytidectomy patients with concomitant nasal dorsal deformities. PMID- 8628811 TI - A simple atraumatic technique to hold tendon stumps during flexor or extensor tendon tenorrhaphy. PMID- 8628812 TI - History of the American Association of Plastic Surgeons, 1921-1996. PMID- 8628813 TI - A member's experience with the state medical board. PMID- 8628814 TI - Blowout fractures of the orbit. PMID- 8628815 TI - The pulsed-dye laser for infantile hemangiomas. PMID- 8628816 TI - The patient with a potential to bleed. PMID- 8628817 TI - Prefabrication of a secondary TRAM flap. PMID- 8628818 TI - Transient axillary-upper inner arm subcutaneous fibrous banding following transaxillary subpectoral endoscopic breast augmentation. PMID- 8628819 TI - Idiopathic osteomyelitis of the zygoma. PMID- 8628820 TI - Inferior pedicle technique for reduction mammaplasty after a Strombeck reduction. PMID- 8628821 TI - Burn of a reconstructed breast. PMID- 8628822 TI - Autotransfusion and reduction mammaplasty. PMID- 8628823 TI - The real cause of silicone autoimmune disease. PMID- 8628824 TI - The bubble cast: to monitor and protect flaps. PMID- 8628825 TI - Fixation for forehead endoscopic lifting: a simple, easy, no-cost procedure. PMID- 8628826 TI - Carpal tunnel release using minimally invasive technique. PMID- 8628827 TI - Mammography before abdominoplasty. PMID- 8628828 TI - Silicon capsule assays with low-bleed silicone gel implants. PMID- 8628829 TI - 200-GM reduction of breast should be enough for insurance coverage of reduction mammaplasty. PMID- 8628831 TI - Smoke is senseless. PMID- 8628830 TI - Techniques to minimize blood loss and simplify postoperative care of buttock skin graft donor sites. PMID- 8628832 TI - Current infection control policies must be challenged. PMID- 8628833 TI - Moisture susceptibility of resin-modified glass-ionomer materials. PMID- 8628834 TI - A new computer-assisted method for fabrication of crowns and fixed partial dentures. AB - The availability of high-technology systems that use computer-aided design and computer-aided machining is on the increase. One such system is the Procera system, which is currently providing cost-effective, high-quality dental restorative services to dental laboratories and to dentists. A reduction in cost to the dentist, and ultimately to the patient, is a major advantage of the Procera system. Cost benefits combined with its continued success in producing crowns and fixed partial dentures that meet professional standards of care should enhance the acceptance of this new technology. PMID- 8628835 TI - Direct pulp capping with a dentinal adhesive resin system: a pilot study. AB - The clinical and histological responses of exposed pulpal tissue after direct capping appear to be more closely related to bacterial infiltration than to direct material toxicity. Dentinal bonding agents have been suggested as a replacement for calcium hydroxide for pulpal protection. In this pilot study, direct pulp capping with a glutaraldehyde-containing dentinal adhesive was performed on eight permanent premolars and molars. The teeth were immediately restored with a resin composite restoration. The clinical technique included the use of a calcium hydroxide paste to cover the exposure site during completion of the preparation. Enamel and dentin were etched with phosphoric acid. All teeth remained vital and without symptoms during the initial observation period of 2 to 6 months. PMID- 8628836 TI - Effectiveness of three types of sterilization on the contents of sharps containers. AB - The purpose of this study was to test the effect of treatment in a gravity steam autoclave, high-vacuum steam autoclave, or an unsaturated chemical vapor sterilizer on endospores present on strips or placed inside of dental anesthetic cartridges held within sharps containers. Strips with 1.7 X 10(5) Bacillus stearothermophilus endospores were used; the cartridges were soiled with an equal number of spores or spores mixed with blood. If sterilization was not accomplished after the initial period, additional exposure time was added. Neither the presence of blood or anesthetic solution nor the position of the container affected the efficiency of sterilization. Soiled cartridges were much more difficult than strips to sterilize. Intact cartridges could not be sterilized by two runs in a gravity steam autoclave or an unsaturated chemical vapor sterilizer or one run in a high-vacuum steam autoclave. Sterilization occurred after two runs in the gravity steam autoclave and unsaturated chemical vapor sterilizer only when one end of the cartridge was removed prior to processing. Results indicated that unopened spore-soiled cartridges are not readily sterilized by commonplace office sterilizers, even after extended exposure. PMID- 8628837 TI - Complications after mandibular third molar extraction. AB - The records of 1,797 patients were retrospectively examined to analyze the possible relationships between postoperative complications following mandibular third molar extraction and parameters such as age, sex, indication for surgery, position of the molar, surgical experience, surgical technique, and postoperative care. Older patients tended to suffer more often from complications. Surgery performed while there were signs of pericoronal inflammation also resulted in more complications. There was no statistically significant difference in the mean complication rate arising from surgery performed by staff members and the rate when surgery was performed by residents. There seems to be no reason for patients to return routinely for removal of resorbable sutures or other postoperative care because this practice does not result in a decrease in postoperative symptoms. PMID- 8628838 TI - Bond strength and clinical evaluation of a new dentinal bonding agent to amalgam and resin composite. AB - Amalgambond Plus with HPA (high-performance additive) was evaluated for its ability to bond a resin composite and an amalgam to deep primary dentin. Tensile bond strengths for resin and amalgam were 6.40 +/- 2.17 MPa and 2.95 +/- 0.92 MPa, respectively. There was a statistically significant difference between the groups. The modes of failure of the specimens were 100% adhesive for amalgam and 76% adhesive-cohesive and 24% adhesive for resin composite. The clinical performance of amalgam and resin composite mesio-occlusodistal restorations bonded with Amalgambond Plus was also evaluated over 15 months. Periapical radiographs were taken to support the clinical examination. The following parameters were assessed: marginal adaptation, secondary caries, postoperative hypersensitivity, and retention. Both types of restoration exhibited excellent marginal adaptation and retention and no secondary caries or hypersensitivity. PMID- 8628839 TI - Papillon-Lefevre syndrome--successful treatment with a combination of retinoid and concurrent systematic periodontal therapy: case reports. AB - Papillon-Lefevre syndrome is a rare autosomal-recessive congenital differentiation disorder; the external signs are hyperkeratosis of the palms and soles. Intraorally, the most salient manifestations are dystrophic periodontal problems that affect both the primary and permanent dentitions and frequently lead to premature tooth loss. Two children were treated with acitretin 0.5 mg/kg of body weight per day from November 1992 to November 1993, and another child since October 1993. Concurrently, the children received professional oral hygiene care (scaling, root planing, and curettage). The combination of retinoid therapy and periodontal treatment improved the dermatologic and periodontal conditions. PMID- 8628840 TI - Articulatory speech performance in patients with salivary gland dysfunction: a pilot study. AB - Difficulty with speech is a common complaint of patients with xerostomia resulting from salivary gland dysfunction. The purpose of this pilot study was to assess and compare speech tasks in three patient groups with salivary gland dysfunction arising from different etiologies (primary Sjogren's syndrome, secondary Sjogren's syndrome with systemic lupus erythematosus, and irradiation therapy for head and neck cancer) and a matched control group. Diadochokinetic speech tasks were quantified clinically and videofluoroscopically. The results indicated that significantly fewer speech tasks were completed, with or without water, by the groups with salivary gland dysfunction than by the control group. Videofluoroscopy provided excellent quantitative analysis, yielding results similar to those of the clinical measurement. In a subjective self-assessment, subjects with salivary gland dysfunction reported more problems with speech than did control subjects. PMID- 8628841 TI - Porosity in resin composite core restorations: the effect of manipulative techniques. AB - Forty acrylic resin duplicates of an endodontically treated and extracted human molar were restored with two resin composite materials for core restorations. Two different manipulation techniques, bulk insertion and syringe application, were used. In a simulated clinical setup, the core buildups produced by the different manipulation techniques were investigated for voids. In 20 duplicates, a post was placed to simulate a metal post and resin composite core buildup. The syringe technique produced significantly fewer incorporated voids for both resin composite core materials than did the bulk insertion technique. It was not possible to demonstrate a relationship between the presence of voids and the presence of a metal post. PMID- 8628842 TI - Marginal sealing ability of three cervical restorative systems. AB - The purpose of this in vitro study was to evaluate the enamel and dentinal marginal sealing ability of three different cervical restorative systems. Class V preparations were made at the cementoenamel junction of 36 freshly extracted premolars. The teeth were randomly divided into three groups of 12 and restored with either a polyacid-modified resin composite ("compomer"), resin composite, or resin-modified glass-ionomer cement. The restored teeth were stored in saline at 37 degrees C for 1 week, thermally stressed for 500 cycles, and subjected to dye penetration testing. The results showed that there was no significant difference in dentinal margin sealing ability among the three materials evaluated. When the margins were in enamel, the resin composite restorations had significantly less leakage than did compomer or resin-modified glass-ionomer cement restorations. The marginal sealing ability of all three materials was significantly poorer in dentin than in enamel. PMID- 8628843 TI - Managed care: the reality must be controlled. PMID- 8628844 TI - Pneumocystic infections: the radiologist's perspective. PMID- 8628845 TI - Can government legislation improve screening mammography use? PMID- 8628846 TI - Problems in the detection and characterization of small renal masses. PMID- 8628847 TI - Generally speaking. PMID- 8628848 TI - Consensus quest: reshaping the future of radiology. RSNA Hartman Lecture/Eugene P. Pendergrass New Horizons Lecture. AB - We are practicing medicine in an era of economic revolution and find ourselves re examining virtually all of our beliefs, practices, rituals, habits, and ethics. This revolution is not driven by the government but--in large part--by economic factors that work to lower costs by restricting access to the health care system. Re-thinking all that we do and formulating responses in this new era should be viewed in a positive light; opportunities abound for radiologists to revive enthusiasm, stimulate creativity, deepen their involvement, and intensify their commitment. Are we capable of identifying what is valuable in our specialty and of safeguarding its stature? Can we develop a vision of what our future should be? Can we convince the majority of radiologists that change is inevitable and that a reshaping of our profession is critical? Can we develop a consensus about what we value most? Can we continue to entice bright individuals into our field? Can we identify our sundry missions and use them as guideposts? Can we create effective strategies for dealing with discontinuous change? Our rich heritage of innovation and our skills as consummate managers of medical information suggest that we can. The challenges will be great but the rewards are worth our best efforts. PMID- 8628849 TI - Capitated contracting in radiology: negotiating techniques, financial calculations, and utilization management. AB - The authors describe how to develop and manage a capitated outpatient radiology contract. Aspects of a capitated fee structure and the need to apportion fees between a hospital and its radiology group are discussed. During negotiations with a managed care organization (MCO), certain information and commitments should be obtained. Utilization data should also be obtained from the MCO, but if they are not available (or are thought to be unreliable), certain norms can be used. Once a utilization projection is arrived at, reimbursement calculations can be made and compared with assumed reimbursements under a known fee schedule, such as that of Medicare. This procedure allows one to estimate whether a capitated proposal is financially feasible. Once a contractual agreement is instituted, the radiology group must then track and manage utilization. An understanding of these principles should enable radiologists to deal effectively with both practice and fiscal concerns presented by managed care. PMID- 8628850 TI - Effect of a legislative mandate on mammography use and coding practices in Maryland. AB - PURPOSE: To analyze the effect of the 1991 Maryland legislative mandate of screening mammography benefits. MATERIALS AND METHODS: Claims submitted between January 1991 and December 1993 for outpatient mammograms obtained in women covered under Blue Cross Blue Shield of Maryland insurance indemnity contracts were analyzed for the distribution of services and charges. RESULTS: For 184,723 women, 285,241 claims were submitted by 851 Maryland providers. Claims for "mammography bilateral," which were considered "diagnostic," represented 67%; 24% were submitted for "screening mammography bilateral," and 9% were submitted for "mammography unilateral." Mammography claims increased only 25% during the 3 years, despite an estimated fivefold increase in the number of women with the screening mammography benefit. Mammography coding shifted from bilateral to screening. CONCLUSION: The number of mammograms obtained increased only modestly after the mandate, but claims coded for mammography bilateral declined dramatically. Removal of financial barriers appears to be insufficient to increase appropriate use of screening mammography. PMID- 8628851 TI - Normal mammograms and the practice of obtaining previous mammograms: usefulness and costs. AB - PURPOSE: To determine the usefulness and cost of acquiring and comparing mammograms previously obtained at a different facility with normal mammograms. MATERIALS AND METHODS: Comparison mammographic reports obtained within 52 months in 1,297 consecutive women (aged 33-82 years; mean age, 53 years) were retrospectively reviewed. Mammograms in which a normal interpretation was altered after comparison were reviewed. The clinical importance and the cost of obtaining previous mammograms were determined. RESULTS: Initially, interpretation was normal in 756 (58%) comparison mammograms and abnormal in 541 (42%). Of the 756 normal mammograms, 197 (26%) were not compared with previous mammograms. Of the remaining 559, 551 (98%) had no change. In eight of the 559 (1%), the original interpretation was altered. Seven of these eight mammograms were available for review: In one, a developing density was detected; in three, differences were attributable to technique; and in three, radiologists had different interpretations. No malignancies were detected. The average labor and postage cost was $21.49. CONCLUSION: Considering the cost and low diagnostic yield, obtaining previous mammograms is of limited usefulness. PMID- 8628852 TI - Stereotaxic core needle biopsy of breast microcalcifications: correlation of target accuracy and diagnosis with lesion size. AB - PURPOSE: To determine if lesion size or number of calcifications affects the ability to obtain microcalcifications or a specific histologic diagnosis at stereotaxic core needle biopsy (SCNB). MATERIALS AND METHODS: Mammographic findings and histopathologic reports of 138 lesions in 124 patients (aged 30-87 years; mean age, 56.2 years) who underwent SCNB of calcifications were reviewed. Calcifications in the specimen and attainment of a specific diagnosis were correlated with lesion size and number of calcifications. RESULTS: Calcifications were obtained in 118 cases (86%). A specific diagnosis was reported in 72 cases (52%). Differences in retrieval of calcifications or ability to establish a specific diagnosis with decreasing lesion size or decreasing number of calcifications were not statistically significant. Attainment of a specific diagnosis was significantly related to retrieval of calcifications (P<.005). CONCLUSION: SCNB was successful in obtaining calcifications in a high percentage of cases regardless of lesion size or number of calcifications. When calcifications were retrieved, a specific diagnosis was attained in most cases (72 of 118). PMID- 8628853 TI - Malignant and benign clustered microcalcifications: automated feature analysis and classification. AB - PURPOSE: To develop a method for differentiating malignant from benign clustered microcalcifications in which image features are both extracted and analyzed by a computer. MATERIALS AND METHODS: One hundred mammograms from 53 patients who had undergone biopsy for suspicious clustered microcalcifications were analyzed by a computer. Eight computer-extracted features of clustered microcalcifications were merged by an artificial neural network. Human input was limited to initial identification of the microcalcifications. RESULTS: Computer analysis allowed identification of 100% of the patients with breast cancer and 82% of the patients with benign conditions. The accuracy of computer analysis was statistically significantly better than that of five radiologists (P = .03). CONCLUSION: Quantitative features can be extracted and analyzed by a computer to distinguish malignant from benign clustered microcalcifications. This technique may help radiologists reduce the number of false-positive biopsy findings. PMID- 8628855 TI - Pulmonary tuberculosis: comparison of CT findings in HIV-seropositive and HIV seronegative patients. AB - PURPOSE: To determine the differences in the computed tomographic (CT) appearance of pulmonary tuberculosis (TB) between patients with and patients without human immunodeficiency virus (HIV) infection. MATERIALS AND METHODS: CT scans and chest radiographs of 42 HIV-seropositive and 42 HIV-seronegative patients with pulmonary TB were reviewed. CD4 T-lymphocyte counts, measured in 40 seropositive patients, were at least 200 cells per microliter in 10 patients and were less that 200 cells per microliter in 30. RESULTS: Seropositive patients had a higher prevalence of lymphadenopathy at chest radiography (P< .05). The seropositive patients had a lower prevalence of consolidation (P< .05), cavitation (P< .01), and postprimary pattern (P< .05) at CT. HIV-seropositive patients had a higher frequency of miliary (P< .01) and extrapulmonary disease (P< .001). Similar features of pulmonary TB were observed in seropositive patients with mild and severe immunosuppression. CONCLUSION: HIV-seropositive patients had a lower prevalence of localized parenchymal disease and a higher prevalence of disseminated disease at CT. PMID- 8628854 TI - Microbubble contrast agent for color Doppler US: effect on breast masses. Work in progress. AB - PURPOSE: To evaluate the effects of a new microbubble contrast agent for ultrasound (US) on breast masses. MATERIALS AND METHODS: Thirty-four patients underwent color Doppler US before and after intravenous injection of a "contrast agent" containing microbubbles. The authors subjectively evaluated the increase in intensity of the Doppler signals, the changes in the vascular patterns, and the timing of the transit of the microbubble bolus. The diagnostic confidence was assessed before and after administration of contrast material. RESULTS: After contrast material injection, there was greater and longer signal enhancement in the cancers than in the benign lesions. The cancers displayed characteristic vascular morphologic features, with more additional vessels visualized in relation to the lesion and a greater increase in vascular tortuousity. Shunts between vessels were demonstrated in all cancers but were not seen in any benign lesion. The diagnostic confidence increased with use of the contrast agent. The appearance at contrast-enhanced US led to a change in the US diagnosis in four patients. This increased both sensitivity and specificity to 100%. CONCLUSION: Injection of a microbubble agent enabled accurate differentiation of benign masses from carcinomas. PMID- 8628856 TI - Extracorporeal membrane oxygenation in adults: radiographic findings and correlation of lung opacity with patient mortality. AB - PURPOSE: To describe the radiographic appearance of extracorporeal membrane oxygenation (ECMO) in adults and to correlate lung opacity with physiologic parameters and mortality. MATERIALS AND METHODS: Chest radiographs of 50 adults treated with ECMO were reviewed; pre-ECMO radiographs were available in 35 patients. Lung opacity was assigned a score of 0-4. Complications of ECMO seen at chest radiography were recorded. RESULTS: The lung opacity scores of the first post-ECMO radiographs were higher than those of the pre-ECMO radiographs in 17 of 35 patients (P = .0005). Maximum opacity score was significantly lower for patients who survived compared with those who died (P = .001). Twelve of 14 patients (86%) with a maximum opacity score of 4 died, compared with eight of 29 patients (28%) with a maximum score of 3. Sixteen of 26 patients (61%) with evidence of baro-trauma died, compared with six of 24 patients (25%) without pneumothorax (P = .02). Four patients developed hemothorax. CONCLUSION: Lung opacity increases immediately after initiation of ECMO. Increased opacity corresponds to decreased pulmonary function, and severe opacity correlates strongly with mortality. PMID- 8628857 TI - Neural networks in ventilation-perfusion imaging. AB - PURPOSE: To optimize the performance of artificial neural networks in the prediction of pulmonary embolism from ventilation-perfusion (V-P) scans. MATERIALS AND METHODS: Neural networks were constructed with a set of V-P scan criteria that included sharpness and completeness of perfusion defects and involved quantification of abnormalities by using a continuous numeric scale. Several network parameters were systematically varied. Networks were trained with 150 cases and tested with 30 different cases. Findings were compared with those of pulmonary angiography. RESULTS: Networks capable of performing as well as experienced nuclear medicine physicians could be constructed with few V-P scan features. A brief training period was optimal (50-100 iterations). Further training diminished network performance. CONCLUSION: Effective neural networks can be constructed by using a limited number of unconventional V-P scan features. Several parameters can be adjusted to optimize performance. PMID- 8628858 TI - Neural networks in ventilation-perfusion imaging. Part II. Effects of interpretive variability. AB - PURPOSE: To evaluate the usefulness of a neural network developed by one physician and used by another. MATERIALS AND METHODS: Intra- and interobserver variability were analyzed in image categorization of ventilation-perfusion (V-P) scans. This information was used to estimate network performance when it was used by a physician who did not train the network. RESULTS: Network training was optimized by using input parameters that demonstrated both individually high correlations with pulmonary embolism and good reproducibility in multiple interpretations. CONCLUSION: Potential variability exists in the performance of a network when it is supplied with input data by different physicians. The clinical usefulness of a network depends heavily on the similarity of interpretive styles between the network trainer and the user. PMID- 8628859 TI - Transthoracic needle biopsy with a coaxially placed 20-gauge automated cutting needle: results in 122 patients. AB - PURPOSE: To determine the utility of coaxial transthoracic needle biopsy (TNB) with use of a 20-gauge automated cutting biopsy needle in the diagnosis of thoracic lesions. MATERIALS AND METHODS: A retrospective review was performed in 122 patients. Computed tomography was used to guide coaxial TNB, which was performed with aspirating (n = 87) and automated cutting (n = 99) needles. The sensitivities for malignant and benign lesions were determined, with a comparison of the relative yields from the two techniques. RESULTS: The overall diagnostic yield for coaxial TNB was 88%. For malignancy the sensitivity was 95%, whereas a specific benign diagnosis was obtained in 91%. Although no difference was found for fine-needle aspiration versus core biopsy of malignant lesions (92% vs 86%), a statistically significant difference was found for benign lesions (44% vs 100%, P<.05). Pneumothorax occurred in 54%. CONCLUSION: Coaxial TNB performed with an automated cutting needle helps provide a diagnosis in the majority of patients with focal chest disease and is particularly useful in the diagnosis of benign lesions. PMID- 8628860 TI - Pulmonary embolism: interobserver agreement in the interpretation of conventional angiographic and DSA images in patients with nondiagnostic lung scan results. AB - PURPOSE: To assess interobserver agreement in the interpretation of digital subtraction angiographic (DSA) images and conventional angiograms in patients with nondiagnostic lung scans. MATERIALS AND METHODS: One hundred thirty of 397 consecutive patients (65 men, 65 women; mean age, 57 years; age range, 18-92 years) in this prospective cohort study underwent angiography. Conventional, selective series were obtained between 1991 and 1992 in 45 patients, and intraarterial DSA series were obtained between 1992 and 1994 in 85 patients. All angiograms were read immediately by the attending radiologist, independently by two radiologists after 6 months, and later by means of consensus of the two radiologists. RESULTS: Results of the two methods were comparable. Interobserver disagreement occurred in 20%-36% (n = 9-16) of patients with conventional images and 4%-11% (n = 3-9) of patients with DSA images. Initial diagnoses were changed after the images were reviewed by means of consensus in 20% and 12% of patients with conventional and DSA series, respectively. In 70% of these patients, initial findings were false-positive; in 25%, inconclusive; and in 5%, false-negative. CONCLUSION: Interobserver agreement is better with selective, intraarterial DSA than with conventional techniques. PMID- 8628861 TI - Three-dimensional gadolinium-enhanced MR angiography for aortoiliac inflow assessment plus renal artery screening in a single breath hold. AB - PURPOSE: To develop a magnetic resonance (MR) angiography protocol, with use of breath-hold techniques, for simultaneous aortoiliac inflow assessment and renal artery screening in patients with lower extremity ischemia or aortic aneurysm. MATERIALS AND METHODS: Breath-hold three dimensional gadolinium-enhanced MR angiography was performed in 50 patients (conventional arteriography in 47 was the standard of reference). After multiple strategies were tested in the first 18 patients, a final protocol was formulated and tested in the subsequent 32 patients. RESULTS: The final protocol comprised a single-slab (28 3-mm-thick partitions) coronal acquisition (repetition time, 7 msec; echo time, 2.8 msec; flip angle, 60 degrees) during a single breath hold, enhanced with 30 mL gadoteridol. In the final 32 patients, sensitivity and specificity, respectively, for obstructive lesions were 100% and 100% for the aorta, 100% and 98% for common iliac arteries, 100% and 89% for external iliac arteries, 100% and 89% for main renal arteries, and 100% and 62% for accessory renal arteries. CONCLUSION: This breath-hold protocol improves the accuracy of aortoiliac inflow assessment, but low resolution limits evaluation of small renal arteries. PMID- 8628862 TI - Central intraluminal saturation stripe on MR angiograms of curved vessels: simulation, phantom, and clinical analysis. AB - PURPOSE: To investigate the appearance of reduced signal intensity in the center of blood vessels on magnetic resonance (MR) angiograms that can mimic intraluminal thrombus. MATERIALS AND METHODS: Simulations and phantom studies were performed to analyze MR angiogram appearance distal to a pronounced curve. RESULTS: Saturation effects substantially lower the signal strength in the center of the vessel relative to that at the vessel periphery. These effects appeared even though the flow was well ordered and laminar. In curved geometries, secondary flow patterns produced counter-rotating vortices, which moved the fastest-moving particles to the outside of the curve and folded the slow-moving particles to the center of the vessel. CONCLUSION: Imaging parameter choices that reduce saturation, such as acquisition of a two-dimensional section transverse to the vessel and through the questionable region, effectively eliminate the central hypointensity effect in vivo. PMID- 8628863 TI - Evaluation of coagulation tests as predictors of angiographic bleeding complications. AB - PURPOSE: To determine whether prothrombin time (PT), partial thromboplastin time (PTT), and platelet count are useful predictors of postangiographic hematoma. MATERIALS AND METHODS: The authors prospectively studied 1,000 consecutive patients who underwent femoral arterial puncture for a diagnostic or therapeutic vascular procedure. Demographic and procedural variables were recorded, including patient age and sex, history of medications and bleeding, procedure type and length, catheter size, and experience level of radiologist applying compression for hemostasis. RESULTS: Abnormal results of coagulation tests were not correlated with an increased occurrence of hemorrhagic complications, but bleeding complications did occur more often in patients with thrombocytopenia. Hematomas occurred in 8.1% (10 of 123) of patients with any abnormal coagulation test results and 9.7% (85 of 877) of patients with normal test results. A platelet count of less than 100 X 10(9)/L was correlated with a higher occurrence of hematoma (P = .002). CONCLUSION: Abnormal PT and PTTs do not correlate with an increased risk of postangiographic hematoma, but a low platelet count is associated with more bleeding complications. PMID- 8628864 TI - Coronary flow reserve: noninvasive measurement in humans with breath-hold velocity-encoded cine MR imaging. AB - PURPOSE: To measure coronary vasodilator reserve with breath-hold velocity encoded cine magnetic resonance (MR) imaging. MATERIALS AND METHODS: Eight healthy adult volunteers underwent 1.5-T MR imaging. Velocity-encoded cine images were acquired at seven to 13 temporal phases in 25 seconds, with k-space segmentation and view-sharing reconstruction (+/- 1 m/sec velocity-encoding value) (repetition time msec/echo time msec = 16/9). Flow velocity in the left anterior descending (LAD) artery was measured twice before and twice after administration of dipyridamole (0.56 mg per kilogram of body weight). RESULTS: Peak diastolic coronary flow velocity in the LAD artery was 14.8 cm/sec +/- 1.9 (mean +/- standard deviation) in the baseline state. It increased significantly (P< .01) to 46.3 cm/sec +/- 10.2 after dipyridamole administration, with an average coronary reserve of 3.14 +/- 0.59. Interstudy and interobserver reproducibilities for measurement of peak diastolic velocity were, respectively, 9.5% +/- 1.6 and 7.0% +/- .2.5 in the baseline state and 6.8% +/- 2.2 and 3.4% +/ 1.5 after dipyridamole administration. CONCLUSION: Breath-hold velocity-encoded cine MR imaging provided reproducible assessment of coronary flow reserve in humans. PMID- 8628865 TI - Tc-99m sestamibi cardiac SPECT imaging during coronary artery occlusion in humans: comparison with dipyridamole stress studies. AB - PURPOSE: To compare the magnitude of change in regional myocardial perfusion during dipyridamole stress with that during coronary occlusion. MATERIALS AND METHODS: The authors prospectively studied 14 men with more than 50% diameter stenosis in at least one major coronary artery. Same-day rest and dipyridamole technetium-99m sestamibi single photon emission computed tomography (SPECT) was performed 24 hours before coronary angioplasty. During angioplasty, while the vessel was occluded, 15 mCi(555 MBq) of tracer was injected, and SPECT studies were obtained 60 minutes later. Extent of regional perfusion abnormalities was estimated. RESULTS: Regional perfusion defect was greater during stress and occlusion than during rest (20%, P = .001 and 14%, P = .009, respectively). SPECT defect during coronary occlusion was similar to that obtained during pharmacologic stress with dipyridamole (53% vs 47%, P = .02). CONCLUSION: Tc-99m sestamibi SPECT with dipyridamole stress is a good predictor of the extent of perfusion abnormalities that occur during coronary occlusion and may facilitate estimation of the total myocardium in jeopardy from a stenotic lesion. PMID- 8628866 TI - Endomyocardial fibrosis: electron-beam CT features. AB - PURPOSE: To present morphologic and functional electron-beam computed tomographic (CT) features of endomyocardial fibrosis. MATERIALS AND METHODS: Twelve patients with histologic evidence of endomyocardial fibrosis and/or definitive echocardiographic and angiocardiographic findings were examined with electron beam CT. RESULTS: Endomyocardial fibrosis was depicted as linear calcifications and/or a thin tissue band of low attenuation within the endomyocardium. In right ventricular involvement, CT depicted obliteration of the apex associated with extension along the free wall and/or papillary muscles of the tricuspid valve. In left ventricular involvement, a large amount of endocardial tissue led to severe obliteration within the inflow tract and/or of the apex. At ventricular volume analysis, restrictive patterns of the disease were determined. CONCLUSION: Electron-beam CT allows direct visualization of endomyocardial fibrosis and the resulting volumetric changes and enables distinction of the disease from constrictive pericarditis. PMID- 8628867 TI - Colorectal obstruction: treatment with metallic stents. AB - PURPOSE: To evaluate the usefulness of self-expandable metallic stents in the treatment of acute colonic obstruction secondary to colorectal neoplasm. MATERIALS AND METHODS: Stents were placed in 12 patients with clinical and radiologic signs of acute colonic obstruction. After symptom improvement, patients underwent radiologic staging. Single-stage surgery was performed in patients without disseminated disease. RESULTS: Stent placement was successful in all patients. Signs and symptoms of intestinal obstruction resolved within 24 hours of stent placement in 10 patients (83%). In two patients with massive bowel dilation, improvement was evident 2 and 4 days after stent placement. Ten patients underwent elective single-stage surgery with partial colonic resection and creation of a primary end-to-end anastomosis without major complications. In two patients with disseminated neoplastic disease, stent placement was considered the primary palliative treatment. CONCLUSION: Metallic stent placement to relieve acute colonic obstruction secondary to colorectal carcinoma is a safe therapeutic alternative, allowing single-stage surgery in suitable cases. In patients who are not surgical candidates it is an adequate palliative option. PMID- 8628868 TI - Temporary use of a Bird's Nest filter during iliocaval thrombolysis. AB - A jugular Bird's Nest filter (Cook, Bloomington, Ind) was partially deployed in the suprarenal cava for prophylaxis to prevent pulmonary embolism in a young woman with phlegmasia cerulea dolens. It was effective in capturing large emboli during thrombolysis of a loose iliocaval thrombus. It was safely removed 6 1/2 hours later, after lysis of most retained filter clots. PMID- 8628869 TI - Percutaneous transhepatic liver biopsy with tract embolization. AB - PURPOSE: To determine the success and safety of percutaneous transhepatic liver biopsy with tract embolization (PBTE) in patients at risk for standard transhepatic biopsy. MATERIALS AND METHODS: Eighty biopsies were performed in 76 patients with diffuse liver disease: 57 biopsies in patients with coagulopathy (11 of whom also had ascites), 16 in patients with mild coagulopathy and ascites, and seven in patients with ascites only. Biopsy was performed with a cutting needle placed through a 10-cm vascular sheath. Gelatin sponge was the embolization agent. RESULTS: All 80 biopsy specimens (100%) were adequate for histopathologic diagnosis. Six complications (8%) resulted from the procedure: one bleeding complication due to incomplete tract embolization, two bowel injuries, one hemobilia, one intercostal artery injury, and one posterior liver capsule perforation with bleeding. All complications occurred in patients with coagulopathy and with the operator's first or second PBTE. CONCLUSION: PBTE produces excellent diagnostic specimens. The high complication rate empirically appears to be related to the degree of coagulopathy and operator experience. PMID- 8628870 TI - Intestinal malrotation in children: tutorial on radiographic diagnosis in difficult cases. AB - PURPOSE: To analyze difficult diagnostic cases of malrotation to identify features crucial to accurate diagnosis. MATERIALS AND METHODS: The authors reviewed the radiographs and records of 81 symptomatic children who underwent surgery with a preoperative diagnosis of malrotation. Eleven had subtle rotational abnormalities (potential false-negative examinations), and 12 had false-positive upper gastrointestinal examinations. RESULTS: Subtle signs of malrotation included unusual redundancy of the duodenum to the right of the spine and location of the duodenojejunal junction (DJJ) medial to the left pedicle. Nevertheless, two children with variations of malrotation had normal upper gastrointestinal examinations. False-positive diagnoses resulted from failure to recognize normal variants: jejunum in the right upper quadrant as the sole finding, DJJ over the left pedicle on the anteroposterior view, "duodenum inversum," and "duodenum mobile." Three children had bowel distention that displaced the DJJ. CONCLUSION: Diagnosis of difficult cases of malrotation may depend on recognition of anatomic subtleties. False-positive diagnoses may be avoided by appreciation of normal duodenal variants. PMID- 8628871 TI - Osteopenia in children: CT assessment. AB - PURPOSE: To assess the value of computed tomographic (CT) measurements of cortical bone in children with osteopenia. MATERIALS AND METHODS: The area and density of cortical bone in the midshaft of the femur were measured with CT in 37 children with osteopenia. Twenty had osteoporosis in one leg, nine had osteogenesis imperfecta (IO), and eight had vitamin D-resistant rickets. Comparisons were made between the CT measurements of the normal and abnormal extremities and between patients with OI or rickets and a group of 17 healthy, matched children. RESULTS: Sex, age, height, and weight did not influence cortical bone density; values were similar for the 17 control subjects. Children with osteoporosis and IO had reduced bone area but normal bone density. Compared with control subjects, patients with rickets had similar bone area but reduced bone density (869 mg/cm3 K2HPO4 +/- 79 [standard deviation] vs 1,132 mg/cm3 K2HPO4 +/- 41). CONCLUSION: CT measurements of area and density of cortical bone aided the differentiation of the various disorders that cause osteopenia in children. PMID- 8628872 TI - Small (< or = 3-cm) renal masses: detection with CT versus US and pathologic correlation. AB - PURPOSE: To determine the sensitivities of computed tomography (CT) and ultrasound (US) for detection and characterization of surgically verified small renal lesions. MATERIALS AND METHODS: Twenty-one patients with von Hippel-Lindau disease or hereditary papillary renal cancer underwent CT and US before partial nephrectomy or enucleation; 205 renal masses were removed (92% were <3 cm). Detection rates and accuracy of CT and US in the characterization of renal morphology were correlated with lesion size. RESULTS: CT and US detection rates for lesions of 0-5 mm were respectively 47% and 0%; 5-10 mm, 60% and 21%; 10-15 mm, 75% and 28%; 15-20 mm, 100% and 58%; 20-25 mm, 100% and 79%; and 25-30 mm, 100% and 100%. Among the lesions 10-35 mm, 80% and 82% were correctly characterized with CT and US, respectively. CONCLUSION: A substantial proportion of lesions under 1 cm were not detected with either modality. Neither CT nor US was superior in the characterization of lesions 3 cm or less. CT and particularly US screening studies in patients with von Hippel-Lindau disease should be interpreted cautiously because missed or mischaracterized small renal lesions are a frequent problem in these patients. PMID- 8628873 TI - Angiomyolipoma and renal cell carcinoma: US differentiation. AB - PURPOSE: To analyze whether shadowing and other ultrasound (US) features were helpful for distinguishing angiomyolipoma (AML) from renal cell carcinoma (RCC). MATERIALS AND METHODS: US images were reviewed of 49 patients with RCC and 35 patients with AML. Each tumor was evaluated for size, location, echogenicity, homogeneity, shadowing, hypoechoic rim, and intratumoral cysts. When available, computed tomographic (CT) scans of AMLs were analyzed for the amount of fat and soft tissue in each lesion. RESULTS: AMLs tended to be smaller and more frequently echogenic than RCCs, but statistically significant overlap occurred. Shadowing was seen in 12 (33%) AMLs but was not seen in RCCs. Hypoechoic rims and intratumoral cysts were seen only in RCCs (numbers were too small to perform further statistical analysis). CONCLUSION: In hyperechoic renal masses, the presence of shadowing, a hypoechoic rim, and intratumoral cysts are important findings that may help distinguish AML from RCC. PMID- 8628874 TI - Three-dimensional H-1 MR spectroscopic imaging of the in situ human prostate with high (0.24-0.7-cm3) spatial resolution. AB - PURPOSE: To evaluate if three-dimensional hydrogen-1 magnetic resonance spectroscopic imaging (3D MRSI) when combined with a clinical MR imaging examination could discriminate prostatic adenocarcinoma from normal prostatic zonal anatomy and benign prostatic hyperplasia (BPH) on the basis of observable metabolite levels. MATERIALS AND METHODS: Combined phased-array, endorectal MR imaging and 3D MRSI was performed in nine young healthy volunteers, five patients with BPH, and 85 patients with prostate cancer and BPH. Volume MR imaging and 3D MRSI data were analytically corrected for the reception profile of the endorectal and pelvic phased-array coils, aligned with the MR imaging data, and compared with postoperative pathologic histology findings. RESULTS: Statistically significant variations in metabolite levels with prostatic zonal anatomy, age, and pathologic condition were detected with a 3D MRSI examination added to a clinical MR imaging examination. Significantly higher choline levels and significantly lower citrate levels were observed in regions of cancer compared with BPH and normal peripheral zone tissues. The ratio (choline + creatine/citrate) in regions of cancer (2.1 +/- 1.3 [standard deviation]) had no overlap with normal peripheral zone values and minimal overlap with BPH values (0.61 +/- 0.21). An estimate of the spatial extent of prostate cancer was determined by generating metabolite images in which this metabolite ratio significantly exceeded normal peripheral zone values in multiple contiguous sections. CONCLUSION: These results suggest that a 3D MRSI examination added to a clinical MR imaging examination may help define the presence and spatial extent of prostate cancer. PMID- 8628875 TI - Prostate carcinoma: MR imaging findings after cryosurgery. AB - PURPOSE: To determine the morphologic changes in the prostate gland after cryosurgery and to assess the value of magnetic resonance (MR) imaging in detecting residual tumor. MATERIALS AND METHODS: Twenty-seven patients with prostate carcinoma underwent endorectal MR imaging after cryosurgery. Eighteen patients also underwent MR imaging before cryosurgery. MR findings were correlated with posttreatment clinical data, prostate-specific antigen level, and biopsy results. RESULTS: Mean prostate volume had decreased by 52% in patients examined 8 weeks or more after cryosurgery. Zonal differentiation was lost in 22 of 27 patients (81%). Areas of intraprostatic necrosis were identified in 14 of 27 patients (52%). A thick capsule enveloped the gland and neurovascular bundles in 24 of 27 patients (89%). Osteonecrosis of the symphysis pubis was seen in six of 27 patients (22%). Positive and negative predictive values for MR assessment of recurrent tumor were 44% and 73%, respectively. CONCLUSION: Cryosurgery induced changes in the prostate gland preclude reliable detection of residual prostate carcinoma at MR imaging. PMID- 8628876 TI - Mammary carcinoma model: correlation of macromolecular contrast-enhanced MR imaging characterizations of tumor microvasculature and histologic capillary density. AB - PURPOSE: To determine the relationship between capillary density, a recognized surrogate of tumor angiogenesis, and magnetic resonance (MR) imaging-derived estimates of plasma volume (PV) and microvascular permeability in two mammary carcinoma models. MATERIALS AND METHODS: Dynamic spin-echo imaging was performed by using albumin-(gadolinium-diethylenetriaminepentaacetic acid)34, a prototype blood-pool contrast medium, in 14 rats with a subcutaneously implanted slow- or fast-growing subtype of R3230 mammary carcinoma. Data were fitted to an established two-compartment kinetic model to estimate PV and permeability. RESULTS: MR imaging-derived tumor PVs and permeabilities increased exponentially with increasing capillary density. MR imaging-derived microvascular characteristics correlated strongly with histologic capillary density, with an r2 of .85. CONCLUSION: Contrast medium-enhanced MR imaging may prove useful in estimating angiogenic activity in carcinomas. MR imaging may be superior to histologic assay because it is noninvasive, can be used to "sample" the entire tumor, and reflects both anatomic and physiologic characteristics. PMID- 8628877 TI - Regional metastasis in head and neck squamous cell carcinoma: revised value of US with US-guided FNAB. AB - PURPOSE: To verify the acclaimed accuracy of ultrasound (US) combined with US guided fine-needle aspiration biopsy (FNAB) in the detection of lymph node metastasis in the neck and to evaluate the interobserver variability. MATERIALS AND METHODS: In a prospective, multicenter study of 185 patients with head and neck squamous cell carcinoma, US (n=238 neck sides) with US-guided FNAB (n=178 neck sides) was used for evaluation of the lymph node status of the neck. Findings were correlated with those of histopathologic examination in 238 neck sides. RESULTS: US with US-guided FNAB had a sensitivity of 77% and a specificity of 100%. Nineteen of 178 aspirations were nondiagnostic. There were no significant differences between the four participating hospitals or the individual sonologists (P>.05). CONCLUSION: Sensitivity of US with US-guided FNAB was slightly lower compared with previous reports. Specificity was similar to previous reports. Interobserver variability appeared to be low. The validity of US with US-guided FNAB is high and warrants widespread use of the procedure for evaluation of the neck. PMID- 8628878 TI - Performance of neuroradiologic examinations by nonradiologists. AB - PURPOSE: To determine the level of participation by nonradiologists in performing neuroradiologic examinations. MATERIALS AND METHODS: Medicare part B claims data from fiscal year 1992 were analyzed for CPT (current procedural terminology) codes related to computed tomography (CT) and magnetic resonance (MR) imaging of the brain, head and neck, and spine, as well as myelography, angiography, and diskography. Data were tabulated by place of service (hospital-based vs freestanding imaging centers) and by medical specialty. RESULTS: Among 363,224 Medicare claims for CT and MR imaging of the brain, head and neck, and spine, 91% of the examinations were performed in hospitals and 9% in offices or freestanding centers; 98% of studies were interpreted by a radiologist. The largest share of radiology billing by nonradiologists was from office-based or freestanding imaging centers (9%), versus 2% at hospital-based facilities. CONCLUSION: Radiologists perform the vast majority of neuroradiologic examinations. Most neuroradiologic examinations performed by nonradiologists are from neurologists at freestanding/office-based imaging centers. PMID- 8628879 TI - Gliomatosis cerebri. AB - PURPOSE: To characterize gliomatosis cerebri on computed tomographic (CT) and magnetic resonance (MR) images. MATERIALS AND METHODS: MR and CT studies of 22 patients with cerebral gliomatosis were reviewed retrospectively. Tumor was confirmed with autopsy (n=5) or biopsy. Distribution and extent of disease were assessed, and disease progression was followed. RESULTS: Tumor involved at least two lobes of the brain in all patients, with extension to the corpus callosum in 12, basal ganglia and thalamus in 17, brain stem in three, and cerebellum in two patients. Widespread invasion with hyperintensity was noted on proton-density- and T2-weighted MR images. At CT, areas of hypo- or isoattenuation were noted, and no contrast enhancement occurred. Extent of tumor was greater on MR images than on concurrent CT scans in all patients. The MR findings closely correlated with the autopsy findings. CONCLUSION: Gliomatosis cerebri is best detected with MR imaging. The pattern is infiltrative with enlargement of cerebral structures. PMID- 8628880 TI - Alzheimer disease: improved visual interpretation of PET images by using three dimensional stereotaxic surface projections. AB - PURPOSE: To compare the diagnostic usefulness of three-dimensional (3D) stereotaxic surface projection (SSP) with that of standard transaxial display in brain positron emission tomography (PET) in Alzheimer disease (AD). MATERIALS AND METHODS: Standard transaxial section display and 3D-SSP PET image sets obtained after administration of 2-deoxy-2-[fluorine-18]fluoro-D-glucose in 39 patients with probable AD (aged 53-82 years; 15 men, 24 women) and 40 subjects without AD (aged 21-78 years; 14 men, 26 women) were randomly interpreted. Receiver operating characteristic (ROC) analysis was performed. RESULTS: Diagnostic performance was superior with 3D SSP (Az[section]=0.94,Az[3D SSP]=0.99[Az=area under the ROC curve];P=.043). With 3D SSP, diagnosis of AD was equally good in beginners and experts. The sensitivity and specificity in questionable or mild dementia were 94% and 99% with 3D SSP and 79% and 88% with standard transaxial display. CONCLUSION: Accuracy of detecting AD was improved in PET with 3D SSP. PMID- 8628881 TI - Enlargement of the third intercondylar tubercle of Parsons as a sign of osteoarthritis of the knee: a paleopathologic and radiographic study. AB - PURPOSE: To correlate presence and size of the third intercondylar tubercle of Parsons (TITP) with occurrence and severity of osteoarthritis of the knee. MATERIALS AND METHODS: In 100 paleopathologic tibial specimens and on 160 clinical knee radiographs, size of the TITP was correlated with osteoarthritis of medical and lateral femorotibial joints (FTJ) and with prominence of tibial spines. RESULTS: TITP size correlated significantly with osteoarthritis of the tibial plateau in the paleopathologic specimens (P<.0001). TITP size correlated significantly with osteoarthritis of medial FTJ and tibial spines on radiographs (P<.0001). Of 85 patients without osteoarthritis of the medial FTJ, 49 (58%) had no TITP, 33 (39%) had small TITPs, and three (4%) had medium or large TITPs. TITPs were found in 15 (88%) of 17 patients with pronounced osteoarthritis. CONCLUSION: Frequency and prominence of TITPs are increased in patients with osteoarthritis of the medial FTJ and prominent tibial spines. Prominent TITP is a secondary sign of osteoarthritis. At histologic examination, increasing size of TITPs represents osteophytosis or enthesopathy of the TITP. PMID- 8628882 TI - Does altered biomechanics cause marrow edema? AB - PURPOSE: To determine if altered weight bearing causes the appearance of marrow edema on magnetic resonance (MR) images. MATERIALS AND METHODS: Twelve volunteers underwent MR imaging with a short inversion time inversion-recovery (STIR) sequence at 1.5 T. The hips, knees, ankles, and feet were evaluated before and 2 weeks after altered weight bearing achieved with overpronation of one foot. Three volunteers underwent imaging a third time, 2 weeks after overpronation was stopped. Two observers assessed the images for evidence of marrow edema. RESULTS: Changes were seen on images in 11 volunteers; the overpronated side only was affected in 10. Most changes occurred in the foot followed by the tibia and the femur. Most changes were a diffuse increase in marrow edema. In two volunteers, the changes resembled those of stress fractures. CONCLUSION: Altered weight bearing should be added to the list of causes of increased medullary signal intensity (ie, marrow edema) on MR images. PMID- 8628883 TI - Articular cartilage volume in the knee: semiautomated determination from three dimensional reformations of MR images. AB - PURPOSE: To determine the accuracy of semiautomated quantification of articular cartilage volume from three-dimensional (3D) reformations of magnetic resonance (MR) images. MATERIALS AND METHODS: Sagittal, fat-suppressed, 3D, spoiled gradient-recalled-echo MR imaging of two bovine and two human cadaver knees was performed. Articular cartilage volume was calculated from 3D reformations of the MR images by using a semiautomated program written at the authors' institution. Calculated volumes were compared with directly measured volumes of the surgically removed articular cartilage. RESULTS: The percentage of error of the MR imaging determined volumes was 6.53% +/- 4.75 (mean +/- standard deviation). A strong correlation between the two sets of observations was shown (r=.997). Linear regression showed the calculated volumes to be highly accurate (slope=1.002, P>.25). Repeated reformations yielded volumes that were reproducible (mean absolute error, 0.013 mL +/- 0.019) and not significantly different from the measured volume (P>.10). CONCLUSION: Semiautomated quantification of knee articular cartilage from MR images yields highly accurate cartilage volumes. PMID- 8628885 TI - Extrahepatic bile duct carcinoma: US characteristics and accuracy in demonstration of tumors. AB - PURPOSE: To assess the accuracy of ultrasonography (US) in the demonstration of extrahepatic biliary tree cholangiocarcinoma and to analyze US features of the different morphologic types of this tumor. MATERIALS AND METHODS: US findings were reviewed retrospectively in 49 patients with pathologically proved cholangiocarcinoma. Findings from percutaneous transhepatic cholangiography (n = 47) and endoscopic retrograde cholangiopancreatography (ERCP) (n = 2) were compared with those from US. RESULTS: US demonstrated the tumor in 47 (96%) of the 49 patients; a mass was demonstrated in 44 patients, and a focal or diffuse thickening of the bile duct wall was seen in three patients. CONCLUSION: The accuracy of US in depicting cholangiocarcinoma was attributable to the skill of the radiologists who performed the study and evaluated the findings and to the high frequency of tumor at the hilar level, where the liver and gallbladder allowed a clear acoustic window that facilitated detection of tumors. PMID- 8628884 TI - Three-dimensional spiral CT cholangiography in patients with suspected obstructive biliary disease: comparison with endoscopic retrograde cholangiography. AB - PURPOSE: To evaluate the diagnostic potential of spiral computed tomography (CT) performed after the administration of cholangiographic contrast material (spiral CT cholangiography) in patients with suspected obstructive biliary disease. MATERIALS AND METHODS: After infusion of meglumine iodoxamate, 29 patients underwent upper abdominal spiral CT with subsequent three-dimensional rendering of the biliary tract. In 27 patients, the presence, site, and extent of biliary obstruction were compared with that at endoscopic retrograde cholangiography (ERC). RESULTS: Spiral CT cholangiography correctly depicted biliary obstruction in 14 of 27 patients, with no false-positive or false-negative cases. In one patient, the precise length of a common bile duct stenosis could not be assessed with spiral CT cholangiography. ERC demonstrated intrahepatic ductal stenoses more clearly in two patients. In two patients with hilar cholangiocarcinomas, spiral CT cholangiography depicted undrained areas not seen with ERC. CONCLUSION: Spiral CT cholangiography allows accurate assessment of the biliary system in patients with suspected obstructive biliary disease. PMID- 8628886 TI - Cystic fibrosis: MR assessment of pancreatic damage. AB - PURPOSE: To evaluate magnetic resonance (MR) imaging for assessment of pancreatic damage in cystic fibrosis. MATERIALS AND METHODS: Twenty-seven patients with cystic fibrosis and 12 control subjects underwent T1-weighted imaging for visual assessment for pancreatic hyperintensity and mixed spin-echo-inversion-recovery imaging for quantitative measurement of T1. Pancreatic insufficiency, pulmonary status, and genotype were recorded. Statistical correlation was conducted. RESULTS: Four patterns of pancreatic involvement were noted: diffuse hyperintensity with a lobular pattern, diffuse homogeneous hyperintensity without residual lobular pattern, hyperintensity with focal areas of sparing, and no structural or signal intensity changes. Statistically significant reduction of the T1 was noted in 21 patients compared with that of controls. Statistically significant correlation between T1 shortening and pulmonary clinical-radiologic compromise and pancreatic insufficiency was found. CONCLUSION: MR imaging may offer further indication of pancreatic and, indirectly, pulmonary damage during the clinical course of cystic fibrosis. A pattern of fibrofatty infiltration of the pancreas, of undetermined clinical significance, is reported. PMID- 8628888 TI - Calcification in focal nodular hyperplasia: a new problem for differentiation from fibrolamellar hepatocellular carcinoma. AB - PURPOSE: To describe calcification in focal nodular hyperplasia (FNH) of the liver, which poses a new problem for the differentiation of FNH from fibrolamellar hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Ultrasound, computed tomography, and magnetic resonance imaging findings of 357 FNH lesions diagnosed in the past 5 years in 295 patients (274 female, 21 male; aged 14-72 years) were retrospectively reviewed with emphasis on intralesional calcification. RESULTS: Calcifications were seen in five FNH lesions (1.4%) as small, solitary spots located centrally or peripherally within the lesions. Morphologic features of these calcifications were similar to those of calcifications in two of six fibrolamellar HCCs in the same period. CONCLUSION: Calcification in FNH is a rare and atypical finding that poses further difficulty for differentiation from fibrolamellar HCC. PMID- 8628887 TI - Superparamagnetic iron oxide--enhanced versus gadolinium-enhanced MR imaging for differential diagnosis of focal liver lesions. AB - PURPOSE: To assess AMI-25- versus gadolinium-enhanced magnetic resonance (MR) imaging in the differential diagnosis of liver tumors. MATERIALS AND METHODS: Twenty-nine patients with liver tumors underwent unenhanced, AMI-25-enhanced (15 micromol/kg), and gadolinium-enhanced(0.1 mmol/kg) imaging within 2 weeks. RESULTS: A significant (P< .05) difference in percentage signal intensity loss (PSIL) was seen in benign tumors on AMI-25-enhanced proton-density-weighted images (nine focal nodular hyperplasia [FNH], 41%; one adenoma, 32.4%) versus malignant tumors. Gadolinium-enhanced T1-weighted gradient-echo images showed strong enhancement in benign lesions (seven FNH, 147.5%; one adenoma, 91.3%) and moderate enhancement in malignant tumors (eight hepatocellular carcinomas, 116.2%, 11 metastases, 39.7%). Receiver operating characteristic analysis revealed a threshold PSIL of 10% on AMI-25-enhanced images as the most essential criteria to distinguish benign from malignant lesions (sensitivity, 88%; specificity. 89%). Interobserver analysis for two observers revealed specificity of 93% for AMI-25-enhanced imaging versus 81.5% for gadolinium-enhanced MR imaging. CONCLUSION: AMI-25 decreased the SI of benign tumors and helped differentiate benign from malignant tumors. PMID- 8628889 TI - Contrast-enhanced MR imaging assessment of tumor capillary permeability: effect of irradiation on delivery of chemotherapy. AB - PURPOSE: To assess the effect of x rays on tumor capillary permeability with macromolecular contrast medium (MMCM)-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: One of paired R3230 mammary adenocarcinomas implanted in the flanks of 48 Fischer rats was treated with a single 5- or 15-Gy dose of x rays. After 1 or 3 days, MR imaging in 30 rats was performed unenhanced and enhanced with albumin-(gadolinium diethylenetriaminepentaacetic acid)30 (0.02 mmol gadolinium per kilogram body weight). Signal intensity enhancement was analyzed to estimate fractional blood volume and permeability-surface area product (PS). In 18 irradiated rats, 2.5 mg/kg cisplatin was injected intravenously, and fractional tumor platinum concentrations were measured spectrometrically. RESULTS: MR imaging showed highest capillary permeability 1 day after 15 Gy (PS 110% above control [P<.001]); PS was elevated 57% 1 day after 5 Gy and 63% 3 days after 15 Gy (P<.05). The largest mean tumor platinum fraction occurred 1 day after 15 Gy: 48% versus 20% (control)(P<.05). CONCLUSION: Transient irradiation-induced increase in tumor capillary permeability to cisplatin can be quantified with MMCM-enhanced MR imaging. PMID- 8628890 TI - Improved peripheral MR angiography with temperature regulation in healthy patients. AB - Peripheral magnetic resonance (MR) angiography was performed in eight volunteers, with a temperature-controlled water blanket. After warming of the right foot, unobserved vessels became conspicuous in all patients: Average increase in signal intensity was nearly twofold in the major artery and vein depicted. A similar improvement was observed in an examination in one patient with diabetes. Peripheral MR angiography performed after warming provided additional diagnostic information. PMID- 8628891 TI - Automatic motion correction for breast MR imaging. AB - In 29 gadolinium-enhanced breast magnetic resonance (MR) examinations, breast motion prevented accurate and efficient image processing. To compensate for global rotations and translations, an automatic motion correction method with a ratio-variance minimization algorithm was used to align images at multiple time points through an iterative process. This method reduced breast motion and improved the accuracy and efficiency of lesion detection. PMID- 8628892 TI - Reference lines for oblique axial MR imaging of the brain. AB - Reference lines that correspond to the three reference lines used at computed tomography were determined on 50 normal midsagittal magnetic resonance brain images. Lines were drawn from the mammillary body to the posterior tentorial attachment to the sinus (orbitomeatal), from the center of the pituitary gland to the posterior tentorial attachment to the sinus (Reid baseline), from the mammillary body to the posterior edge of the fourth ventricle (supraorbitomeatal). PMID- 8628893 TI - Names for gadobenate dimeglumine. PMID- 8628894 TI - Thrombosed hemodialysis grafts. PMID- 8628895 TI - Plantar compartments of the foot: MR appearance in cadavers and diabetic patients. PMID- 8628896 TI - Magnetic resonance. PMID- 8628897 TI - Thoracic radiology. PMID- 8628898 TI - Cardiac radiology. PMID- 8628899 TI - Cardiovascular/interventional radiology. PMID- 8628900 TI - Neuroradiology. PMID- 8628901 TI - Oncology/radiation therapy. PMID- 8628902 TI - Medical physics. PMID- 8628903 TI - Computer applications and digital imaging. PMID- 8628904 TI - Technical exhibits. PMID- 8628905 TI - Nursing and minority research revisited. PMID- 8628906 TI - Development of a functional capacity model for the elderly. AB - This study is based on the belief that the amount of nursing care each elderly client requires is determined more by his/or her functional capacity than by diagnosis of medical problems. A functional capacity theoretical model was developed, based on an ecological framework. Environment and personal competence (sensory competence) were hypothesized as predictor constructs, depression as a mediator, and functional capacity as an outcome variable in this model. The model was tested for fit with data from 209 female outpatients of two eye clinics in Seoul, Korea. The results indicated that the hypothesized model fit with the data. Environmental subconcepts showed an indirect effect on functional capacity through depression. This mediating effect of depression was not shown between sensory competence and functional capacity. PMID- 8628907 TI - Explication of the construct of shared care and the prevention of pressure ulcers in home health care. AB - The purpose of this investigation was to render a more complete understanding of subjective perceptions of pressure ulcers from the perspective of family dyads, and to study the effect of these subjective experiences on preventive behaviors and pressure ulcer outcomes. A naturalistic inquiry, combined with objective measures, was used. Twenty-one dyads participated in four in-depth interviews to explore how they mentally represented and responded to the risk of pressure ulcers. Through the process of concept development, a lay representation of pressure ulcers was developed. This process produced a new concept, identified as "shared care," that explained how the dyad's interaction influenced preventive behavior. Shared care consists of three elements: communication of symptoms, decisions about how to respond to symptoms, and appraisals of reciprocity. Two contrasting patterns of care were identified: shared and directed/discrepant. In the shared care group, 10 patients were at risk for pressure ulcers but only 4 developed ulcers. In this discrepant care group, 3 patients were at risk and 2 developed pressure ulcers. Shared care was a pattern of interaction used successfully by family members to prevent pressure ulcers in patients at risk. PMID- 8628908 TI - Pathways of influence on fifth and eighth graders' reports about having had sexual intercourse. AB - The purposes of this study were to explore the differences between fifth and eighth graders' self-reports of having initiated sexual intercourse and the factors related to their early sexual debuts. The urban sample consisted of African American and white fifth (n = 59) and eighth (n = 169) graders. Among fifth graders 46% and among eighth graders 55% reported they had initiated sexual intercourse. Sexual intercourse was more common than other risk behaviors such as cigarette smoking and drinking. The multivariate analyses indicated that the influences on the early initiation of sexual intercourse were complex. Specifically, gender, use of alcohol, costs (i.e., perceptions of negative consequences), and personal norms (i.e., beliefs about when to initiate sexual intercourse) had no direct influence on having initiated sexual intercourse, but indirectly affected the initiation of sexual intercourse through the frequency of other intimate behaviors. The findings are discussed in terms of gender-specific socialization into sex roles and the need to design risk reduction interventions that incorporate messages from multiple social sources. PMID- 8628909 TI - Concerns about analgesics among patients and family caregivers in a hospice setting. AB - Patients receiving curative treatment for cancer have concerns about reporting pain and using analgesics. These concerns are associated with underutilization of analgesics. To extend knowledge about such concerns to the context of palliative care, the concerns of hospice patients and family caregivers were compared. Within 5 days of admission to hospice, 35 patients with cancer and their caregivers each completed a measure of eight concerns such as fear of addiction, worry about tolerance, and worry about side effects. There was no correlation between caregiver and patient concerns and means for the two groups were similar, indicating that within a given dyad either the patient or the caregiver may have greater concerns. The findings highlight the need for patient and caregiver education about reporting pain and using analgesics. PMID- 8628910 TI - Relationships among sleep dimensions and factors that impair sleep after cardiac surgery. AB - Factors associated with the attempted length, disturbance, effectiveness, and nap supplementation of sleep were analyzed in 97 patients recovering from cardiac surgery a few days before hospital discharge. Patients rated sleep for the prior night and factors that impaired their sleep after transfer from the critical care unit. The group averaged little sleep, with moderate disturbance and effectiveness and low nap supplementation. The disturbance, effectiveness, and attempted length of sleep were associated with an inability to perform their usual routine before sleep, inability to get comfortable, pain, noises, procedural care, and an unfamiliar bed. Patients encounter difficulties with sleep, even near discharge from the hospital. Interventions should be tested to mitigate specific factors that affect selected dimensions of sleep. PMID- 8628911 TI - Mechanisms related to psychological well-being in older women with chronic illnesses: age and disease comparisons. AB - The influence of type of illness (breast cancer versus osteoarthritis) and age on physical health and functioning, self-system interpretive mechanisms, and women's psychological well-being were examined. Self-system interpretive mechanisms are considered mediators of the effects of physical health on psychological well being and include social comparisons, social integration, and illness perceptions. Young-old (aged 60-74) and old-old (75 +) women, diagnosed with osteoarthritis (N = 102) or breast cancer (N = 86), who volunteered for the study were interviewed using structured self-report scales. Young-old and old-old women did not differ in terms of health status, interpretive mechanisms, or on three of five indices of psychological well-being. On the other hand, women with arthritis, regardless of age, reported more functional health problems, more symptoms, and perceived their illness as more severe, more chronic, and less controllable than women with breast cancer. Although women with arthritis and breast cancer differed significantly in terms of physical health, they did not differ on multiple measures of psychological well-being. For both groups, women who made more positive social comparisons and who had more extensive social networks had higher levels of psychological well-being, regardless of physical health problems. PMID- 8628912 TI - Using multiple indicators to test the dimensionality of concepts in the health promotion model. AB - Data from 197 randomly selected male adults were analyzed in order to examine the dimensionality of two of the concepts in the Health Promotion Model-perceived control of health and definition of health. The model, tested with LISREL, examines the impact of age on exercise with the key concepts modeled as intervening variables. The addition of multiple indicators resulted in a model that failed to meet the data constraints, thereby calling into question the putative dimensionality of perceived control of health and definition of health. The proportionality constraints within multiple-indicator structural equation models seem to provide a more stringent test of whether several indicators measure the same concept than the test provided by the internal consistency constraints implicit in factor analysis. PMID- 8628913 TI - Focus on quantitative methods. Determining cut-off scores. AB - The major methods for determining cut-off scores for certification and licensing examinations, and other criterion-referenced tests that result in pass/fail decisions, are described. Two major categories of methods are available: examination-centered and examinee-centered. Particular emphasis is given to the widely used Angoff (1971) method, and its common modifications. Some of the outstanding procedural and statistical issues are briefly discussed, including the need for more attention given to determining cut-off scores for measures used in clinical and research settings. PMID- 8628914 TI - Natural and endogenous DNA adducts as detected by 32P-postlabeling. PMID- 8628915 TI - DNA damage: chemistry, repair, and mutagenic potential. AB - This paper is an overview on the chemical and biochemical effects of three different human carcinogens on DNA in vitro in terms of adducts, their effect on replication, in vitro and in vivo, and their repair by enzymes from human cells. The carcinogenic compounds chosen for specific discussion are N-nitroso alkylating agents: the vinyl chloride metabolite, chloroacetaldehyde (CAA), and the benzene metabolite, p-benzoquinone (p-BQ). PMID- 8628916 TI - Endogenous production of DNA adducts. PMID- 8628917 TI - Significance of DNA adducts at low dose: shortening the time to spontaneous tumor occurrence. AB - Formation and repair of adducts from DNA-reactive carcinogens as well as induction of genetic changes is expected to be proportional to dose as long as the rates of the enzymatic reactions are proportional to the substrate concentration and as long as the kinetics of cell division are not affected. Deviations from linearity are expected in situations of saturated or induced kinetics and whenever toxicity stimulates regenerative processes and accelerates the fixation and accumulation of mutations resulting from primary DNA lesions. Background DNA damage is postulated to drive the process of carcinogenesis resulting in what is called "spontaneous" tumor formation. Exposure to a low dose of a DNA-reactive carcinogen can result in a dose-dependent acceleration of the spontaneous process but it cannot induce cancer "out of the blue." The shortening of the time to occurrence of a tumor is estimated for different situations using the multistage model. For a tumor which has a high background rate and a large number of steps, life shortening is small, while at a low background incidence rate and a small number of stages, life shortening can be larger. For the majority of tumors, therefore, the approach presented should be able to relieve from the idea that a low dose of a carcinogen could induce cancer early in the life of an individual who without exposure would have lived a much longer, tumor free life. PMID- 8628919 TI - Dose-time response in mouse skin tumor induction by 7, 12 dimethylbenz[a]anthracene and 12-O-tetradecanoyl-phorbol-13-acetate. AB - The question was addressed whether the dose-response relationship derived from a carcinogenicity study can be used for mechanistic interpretation and to what extent the shape of the curve is dependent on the duration of the bioassay and the time of analysis. The mouse skin tumor model was used. It allows recording of the time of tumor appearance without interim sacrifice. Groups of 16 female NMRI mice were treated twice weekly by dermal administration with combinations of (a) 2.5 nmol 12-O-tetradecanoyl-phorbol-13-acetate (TPA) plus 0, 0.3, 1, 3, or 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) or with (b) 2.5 nmol DMBA plus 0, 0.1, 0.3, 1, 3, or 10 nmol TPA. The appearance of the first papilloma was recorded for each animal and the cumulative incidence data were analyzed in two ways: (i) With the usual dose-prevalence representation at a fixed time point, the dose response for TPA was sigmoidal, while it was linear-superlinear for DMBA. This was observed at all time points, indicating that the dose-response information may be used for a distinction between DNA-reactive and tumor-promoting mechanisms of action. (ii) When time-to-tumor and loss of tumor-free lifetime was analyzed as a function of dose, there was again a marked difference between DMBA and TPA. The tumor-free lifetime increased with each step of dose reduction but the slope was about four times larger for TPA compared with that for DMBA. Further reduction of the TPA dose could result in a situation in which the natural life span sets a limit to an observable effect. Under the conditions of this bioassay for mouse skin papilloma induction by a combination treatment with DMBA plus TPA, the findings support the idea of a no-effect low-dose threshold for the tumor promoting agent. PMID- 8628918 TI - Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. AB - In 1987, an evaluation of the nitrate drinking water standard was performed with a primary focus on the effects of nitrate on methemoglobinemia and reproductive/developmental effects (Fan et al. (1987). Regul. Toxicol. Pharmacol. 7, 135-148). The present review presents an updated overview and evaluation of the available information on the same health effects of nitrate and nitrite with an emphasis on data not included in the previous review, which should be used as a compendium to this report. Recent epidemiologic data have suggested an association between developmental effects in offspring and the maternal ingestion of nitrate from drinking water, but a definite conclusion on the cause and effect relationship cannot be drawn. Animal experimental data have shown reproductive toxicity associated with high exposure levels to nitrate or nitrite, which are not likely to be encountered in drinking water. No teratogenic effects were observed in rats, mice, rabbits, and hamsters tested. Several cases of methemoglobinemia have been reported in infants in the United States using water containing nitrate at levels higher than the current maximum contaminant level (MCL) of 45 ppm (mg/liter) nitrate (NO3) or 10 ppm nitrate-nitrogen (nitrate-N), but none at or lower than the MCL. The uncertainties in the data base are discussed, noting that no uncertainty factor was applied in deriving the MCL in order to account for the uncertainties that exist in the data base. PMID- 8628920 TI - A proposed design for conducting target animal safety studies for developing new veterinary pharmaceuticals. AB - Target animal safety studies are required for the regulatory approval of any new veterinary pharmaceutical registered almost anywhere in the world. Both the United States and Europe have extensive guidelines for conducting these studies. Due to new veterinary drug labeling requirements in the United States, changes in the overall design of target animal safety studies will be required for the United States. The guideline changes proposed in this paper are more evolutionary than revolutionary. While many new ideas are suggested, a number of existing practices that seem to work well were retained. Several new abbreviated drug development pathways are also proposed, an idea missing from current guidelines. This proposal, and others like it, should serve as a basis for representatives from the U.S. Food and Drug Administration, the European Commission on Veterinary Medical Products, the veterinary pharmaceutical industry, and practicing veterinarians to develop internationally harmonized target animal safety study guidelines that are clear in their intent, scientifically sound, and practical. PMID- 8628921 TI - Variable responses of species and strains to white mineral oils and paraffin waxes. AB - Recent dietary studies on mineral hydrocarbon (MHC) white oils and waxes have shown inflammatory effects in Fischer 344 (F-344) rats, but not in other rat strains or dogs. Histopathologic effects seen in F-344 rats include mesenteric lymph node histiocytosis, liver granulomas, and inflammation of the mitral valve (only seen with paraffin waxes). Human ingestion of MHC can result in noninflammatory lipogranulomas (oil droplets) in tissues which are regarded as clinically unimportant. It is speculated that inherent interspecies differences in pharmacokinetics and/or immune function may contribute to the differential response to MHC seen in F-344 rats. The F-344 rat retains greater amounts of MHC in target tissues compared to other rat strains and dogs and appears to be more sensitive immunologically to MHC than other species, including humans. This strain may be predisposed to these effects as indicated by a high background incidence of inflammatory granulomatous lesions in control female F-344 rats. Because of its apparently unique sensitivity, relevance of effects seen in F-344 rat to human health is questionable and requires further investigation. PMID- 8628922 TI - Establishing the timing of implantation in the Harlan Porcellus Dutch and New Zealand White rabbit and the Han Wistar rat. AB - The purpose of this study was to establish the timing of the onset of implantation in both the Harlan Porcellus Dutch and New Zealand White rabbit and the Han Wistar rat. Implantation was initiated on Day 5 (rat) and 7 (rabbit) and established by Day 7 and 8 of gestation in the rat and rabbit, respectively. Recent guidelines on toxicity testing during embryo-fetal development studies require that maternal exposure to pharmaceutical compounds does not occur until after implantation has taken place. In order to ensure that this is the case, female Harlan Porcellus Dutch and New Zealand White rabbits and Han Wistar rats were sacrificed on different days of gestation, over the expected periods of implantation. The presence of preimplantation blastocysts in the uterus was investigated, and evidence of established implantation sites was assessed. PMID- 8628923 TI - A critical assessment of the relationship between silicone breast implants and connective tissue diseases. AB - Concerns regarding the possible role of breast implants (particularly silicone breast implants) in the development of connective tissue diseases were raised by case reports of connective tissue diseases in women with breast implants. Case reports, however, are not appropriate for causation assessment. Within the past few years, epidemiologic studies have begun to appear. Based on a comprehensive literature search, 15 epidemiologic studies on breast implants and connective tissue diseases, which satisfied certain basic epidemiologic requirements, were included in the critical assessment. These studies utilized either the case control or the cohort study design. Although each individual study was relatively small, and the statistical power to detect a modest risk increase in specific categories of connective tissue diseases was limited, the results of these studies, however, were strikingly consistent, particularly those reported in case control studies. To increase statistical power and to take the consistency of results into consideration, meta-analyses were used to summarize results from individual studies quantitatively. Based on data from case-control studies, meta analyses of rheumatoid arthritis, systemic sclerosis (scleroderma), and systemic lupus erythematosus were performed. These case-control studies represented a combined database of approximately 4000 cases of connective tissue diseases, and the power was sufficient to detect a relatively small increase in risk. Based on the meta-analyses, the relative risks (95% confidence intervals) were 0.85 (0.48 1.51) for rheumatoid arthritis, 0.82 (0. 50-1.35) for systemic sclerosis, and 0.33 (0.06-2.03) for systemic lupus erythematosus, indicating that there was no increased risk of connective tissue diseases associated with breast implants. The findings derived from the meta-analyses of case-control studies were supported by results from cohort or prospective studies. It was concluded that epidemiologic data did not provide any evidence for a causal relationship between silicone breast implants and connective tissue diseases. PMID- 8628924 TI - [Acting and thinking in medicine]. PMID- 8628925 TI - [The reduction of ferromagnetic artifacts by using a fast-spin-echo sequence in the postoperative assessment of degenerative diseases of the cervical spine]. AB - This study was aimed at investigating if fast spin echo (FSE) pulse sequences can increase the diagnostic capabilities of MRI in the patients with degenerative disease of the postoperative cervical spine, considering their lower magnetic susceptibility than that of conventional spin echo (SE) sequences. The patient population consisted of 15 patients submitted to diskectomy after Cloward and Caspar. MR images were acquired with a 1.5-T unit (Signa, General Electric) with T2-weighted FSE versus conventional T1- and T2-weighted SE images. Thanks to fewer metallic artifacts, FSE images of the postoperative cervical spine yielded more pieces of information than conventional SE sequences in demonstrating the spine and its content. The best results were obtained in the patients with canal stenosis (8 patients) and myelomalacia (2 patients). In addition, the myelographic effect, another major semiologic feature of the FSE technique, provided further diagnostic information in these patients. Moreover, shorter examination times resulted in better patients tolerance, especially in those with recent surgery. Finally, reducing motion, blood flow or CSF flow artifacts definitely improved FSE image quality. PMID- 8628926 TI - [Meniscal deformities associated with fractures of the tibial proximal extremity. Considerations in 7 cases]. AB - Lateral tibial plateau fractures are a fairly frequent event in emergency clinical practice. In these fractures, when bone depression exceeds 5 mm, surgery is indicated. On the rule, conventional plain films combined with tomography can answer diagnostic questions about bone trauma. CT and MRI permit to study associated meniscocapsular injuries for better therapeutical management. Since February, 1991, we have examined 24 patients with tibial plateau fractures with conventional radiography and CT. CT was performed using thin sections, within 0 to 48 hours of the traumatic event. In our series, 7 patients had a lateral meniscal trauma associated with a fracture of the homologous tibial plateau; in all of these 7 women, surgery confirmed complete meniscal avulsion. In these cases, CT showed the following signs of meniscocapsular disinsertion: marked diastasis between capsular structure, popliteal tendon and meniscal profile; associated hypodense hemorrhagic fluid in the popliteal recess; inhomogeneous densitometry of the popliteal tendon resulting from hemorrhagic infarction. Furthermore, CT showed a characteristic and constant morphological alteration of the lateral meniscus with fibrocartilage deformation, that is with a wider or more narrow pattern relative to its normal "C"-like shape. We conclude that this morphological alteration of meniscal fibrocartilage, when associated with a tibial fracture, is a diagnostic CT sign of complete meniscal avulsion. This finding can be a useful integration to other CT signs of this meniscal injury, towards better and more complete therapeutical management. PMID- 8628927 TI - [Short stature due to a partial idiopathic deficiency of the growth hormone. The role of the TW2 method in assessing treatment with r-hGH]. AB - The Tanner-Whitehouse 2 (TW2) method to assess skeletal maturation (reviewed by Nicoletti for the Italian population) was used to study, from January, 1991, to December, 1994, a series of 26 Italian patients. The patients, 18 men (69.3%) and 8 women (30.7%), came from inhomogeneous family stocks and were all affected with short stature due to partial idiopathic GH deficiency; they were treated with replacement therapy with the biosynthetic recombinant human growth hormone (r hGH). Each patient underwent 3 wrist and left hand radiographs, the first one made on the basis of medical and endocrinologic assessment. The patients were reexamined after one and two years of treatment with the analysis of stature growth and of the skeletal maturation of hand bones. At one year, the average chronological age of our patients was 12.42 year (range: 9.4-15.2 years), their average bone age was 11.13 years (range: 6.5-14 years) and their average height was 137.81 cm (range: 117-155.5 cm). The patients were then retrospectively examined on the basis of bone maturation and final height, at the end of two years' therapy. At the end of treatment, height was above the third percentile in all patients and therefore within the expected personal target on the basis of genetic stature. The TW2 indexes of bone maturation, after one year of treatment, had increased by 44.84% (range: 27-77%) of the total maturation increase at two years. Moreover, after one year of treatment, average stature increase was 55.81% (range: 42-72%) of the total stature increase at two years. After two years of treatment, TW2 indexes showed an average 55.16% increase in bone maturation (range: 23-73%) of total maturation and average stature increase was 44.19% (range: 28-56%) of the total stature increase. Our results confirmed that skeletal growth and bone maturation are two distinct processes. Particularly, we noted that, while after one year of r-hGH therapy skeletal growth (especially in the long bones) prevails over bone maturation, after two years maturation prevails. In conclusion, our experience confirms the TW2 method as a simple and highly informative method which can be used in any radiologic center. PMID- 8628928 TI - [The procedures and results of a quality control program in mammography carried out on a regional basis]. AB - In 1990 the region of Tuscany instituted a quality control program for mammography, in order to check the working conditions of mammography centers in the region, to investigate the feasibility of applying some working standards in line with European recommendations, and to promote the training of personnel. The program involves the following: a test of newly acquired mammographic equipment to establish that it meets the required standards; an annual test of generators, X-ray beams, grid, AEC, screen-film system, film processing, dose and image quality; daily test (phantom radiography and film sensitometry) performed by the personnel of each mammographic Unit; and a sustained advisory service for the technical problems occurring between checks. Thirty-nine of 57 centers participated on a voluntary basis, and 135 checks were performed annually. The results of the program show a performance below the recommended standard in relation to AEC, film sensitometry and the adequacy of lights in dark rooms in 40%, 50% and 63%, respectively, of the centers. Entrance exposure was over 1500 mR, with a maximum value of 3450 mR, in 6 cases in the first round. 5 cases in second, and only 1 case in the third. In general, the results are slightly improving, but mammography optimization is still a long way off. It is necessary to carry out suitable programs for quality control in mammography on a regional basis and to establish precise guidelines for individual mammographic Units to follow in order to meet the European standards. PMID- 8628929 TI - [The correlation between morphology, electrolytic content and risk factors in breast cysts]. AB - Many studies on the biochemical composition of the liquid aspirated from breast cysts have identified three types of cysts: type I (apocrine) cysts, with a high concentration of K+ and low levels of Na+ and C1-; type II (transudate) cysts, with an electrolytic content similar to that of plasma and high Na+ levels and, finally, type III cysts, with intermediate characteristics. The literature data appear to indicate that the women with type I cysts are at higher risk for breast cancer. The authors report the results of a study carried out on 143 women from October, 1991, through October, 1994, in the Radiology Department of the University of Bologna, to investigate the correlations between some risk factors for breast cancer, the characteristics of cyst fluid and the morphology of the cysts after pneumocystography. Of 186 cysts, 104 (55.9%) were type I, 49 (26.4%) were type II, and 33 (17.7%) were type III. Among the risk factors we considered, only the premenopausal state (41 to 45 years of age) exhibited a statistically significant correlation with the presence of type I cysts. The morphological study of the cysts after pneumocystography showed a surprisingly high correlation between the honeycomb pattern and type I cysts. The constant correlation between cyst morphology and electrolytic content may allow the easy identification of the subgroups of patients eligible for a closer follow-up. PMID- 8628930 TI - [The role of ductal galactography in the differential diagnosis of breast carcinoma]. AB - The authors report on a series of 1009 consecutive patients submitted to clinical examination, mammography, cytology and galactography for the presence of nipple discharge. Galactography was performed mainly in the presence of hematic nipple discharge. Surgical biopsy was performed in 392 cases, and 52 cancers (31 invasive and 21 intraductal lesions) were diagnosed, which were suspected at palpation, mammography, cytology and galactography in 17, 17, 18 and 31 cases, respectively. Forty of 52 cancers were suspected on the basis of combined exam findings, while 8 cancers (7 intraductal and 1 invasive lesions) were submitted to biopsy on the basis of a galactographic diagnosis of multiple benign papilloma, an (invasive) lesion with a diagnosis of single papilloma, and 3 (invasive) lesions because of persistent hematic discharge. Cancer was suspected with only one exam in 14 cases: 3 at cytology, 2 at mammography, 1 at clinical examination and 8 at galactography. Hematic discharge alone is not a sufficient reason to indicate surgery, because its positive predictive value for cancer is low (< or = 10%), while cytology is of limited help because of its poor sensitivity (34.6%). In contrast, galactography (59.6% sensitivity) gives a greater contribution to differential diagnosis and is always indicated in the presence of hematic discharge. Galactographic evidence suspicious for cancer (67.3% positive predictive value) or for multiple papilloma (9.7% positive predictive value) is sufficient to advise open biopsy. The surgical removal of single papillomas is of questionable benefit, since single papillomas are benign lesions, with no clear evidence of progression to cancer. In our series, only one cancer was misdiagnosed as a single papilloma at galactography (0.5% positive predictive value). PMID- 8628931 TI - [Magnetic resonance myelography with a fast-spin-echo sequence]. AB - A major application of Magnetic Resonance Imaging (MRI) consists in the "myelographic effect", characterized by high signal intensity of the CSF, which permits to visualize spinal subarachnoid spaces and nerve root sheaths. Such new ultrafast pulse sequences as fast spin echo (FSE) further enhance CSF high signal intensity. In addition, suppressing background with the fat suppression technique and with maximum intensity projection (MIP), a particular algorithm already used in MR angiography, FSE sequences yield 3D myelography-like images. Our study was aimed at assessing the clinical role and the reliability of this technique. Our preliminary experience is based on 21 patients with pain in the lower back and legs and on 5 healthy volunteers; all the myelography-like images were obtained with a 1.5-T MR system (Signa, General Electric). The parameters for FSE myelography-like images included: TR = 6000, TE = 200, ETL = 32, thickness = 3 mm, matrix = 256 x 224, Nex = 4, FOV = 20 cm, fat presaturation, no phase wrap, acquisition time = 2 min 58 s. MR myelography sequences were acquired after a standard FSE T1/T2-weighted exam. MR-myelography results confirmed the diagnosis of disk herniation made on conventional T1/T2-weighted images, especially in thecal sac compression and/or amputation of nerve root sheaths. MR myelography appeared to be especially useful in migrated disk herniation or in abnormal origin of nerve roots. Moreover, it allowed full and panoramic visualization of the subarachnoid spaces and excellent definition of the thecal sac, nerve roots and nerve root sheaths. In one patient only movement artifacts affected image quality. To conclude, even though it fails to yield any functional information, MR myelography appears to be an easy, rapid and noninvasive support to conventional MRI of the lumbar spine, to replace conventional myelography. PMID- 8628932 TI - [Cerebral SPECT with iodine-123 IBZM in patients with extrapyramidal system disorders: the evaluation of its sensitivity in therapy with dopaminergic drugs]. AB - The functional integrity of striatal post-synaptic dopamine D2 receptors is requested for an effective pharmacologic treatment in patients with extrapyramidal movement disorders. Iodine-123 IBZM Single Photon Emission Computed Tomography (SPECT) is a noninvasive radionuclide technique for the morpho-functional imaging of post-synaptic dopamine D2 receptors. In this study, the results of iodine-123 IBZM SPECT and those of apomorphine tests were compared in 32 patients with extrapyramidal movement disorders--22 patients with idiopathic Parkinson's disease (IPD) and 10 with Parkinson's plus syndrome (PPS). Iodine-123 IBZM uptake was measured as the ratio between striatum and frontal cortex activities. Twenty of 22 IPD patients (91%) responded to apomorphine administration, while in 8 of 10 PPS patients (80%) the apomorphine test was negative. Iodine-123 IBZM uptake was significantly higher (p < 0.01) in IPD patients (1.39 +/- 0.114) than in PPS patients (1.27 +/- 0.078). Similarly, iodine-123 IBZM uptake was significantly higher (p < 0.01) in the patients with positive than in those with negative apomorphine test (1.38 +/- 0.113 vs. 1.26 +/ 0.078). In conclusion, the results of this study demonstrate that iodine-123 IBZM SPECT is a radionuclide technique capable of characterizing the patients with extrapyramidal movement disorders and of selecting the subjects who may respond to pharmacological dopamine treatment. PMID- 8628933 TI - [Magnetic resonance and computed tomography compared in the staging of rhinosinusal neoplasms. A cost-effectiveness evaluation]. AB - This study was aimed at comparing CT and MRI in the preoperative staging of sinonasal malignancies and at developing the most cost-effective diagnostic strategy to determinate resectability and choice of surgery and to assess surgical demolition extent. Forty-nine patients with sinonasal tumors were examined during a 10 years' period, with CT (20 patients), MRI (13 patients) and combined CT and MRI (16 patients); 42/49 patients underwent surgical resection (16 patients with craniofacial resection and 6 with orbital exenteration). Cost effectiveness of the two techniques was assessed comparing five protocols using CT and MRI either alone or combined. In 40/98 orbits the tumor was in contact with a bone wall. MRI was more accurate than CT in assessing orbital invasion (100% sensitivity, 94% specificity, 86% positive predictive value and 100% negative predictive value, vs. 50%, 93%, 75% and 82%, respectively, for CT). MRI showed the tumor in contact with the skull base in 24/49 patients and CT in 23/49 patients, with no statistically significant difference between the two techniques. Dural infiltration, however, was better demonstrated with MRI in 3 patients with minimal bone erosion of the nasal vault. In 5 patients CT and MRI showed pterygopalatine fossa invasion (4 true positives and 1 false positive for both techniques). Since the final decision about orbital exenteration needs accurate surgical mapping, CT appears to yield all the pieces of information necessary for surgical planning. The most cost-effective protocol seems to be a CT examination for all patients. MRI is needed only for better prognostic assessment in the patients with minimal bone erosion of the floor of anterior cranial fossa. In fact, dural invasion, which is a markedly negative factor for survival, may be missed by CT. PMID- 8628934 TI - [The prototype of an expert system for the diagnosis of pseudotumorous lesions and tumors of the jaws: ADAPT-M. Archiviazione e Diagnosi Automatica di Pseudotumori e Tumori delle ossa Mascellari]. AB - Many different tumors and pseudotumors may affect the jaw bones. The number and the rarity of most of these lesions, however, make both classification and differential diagnosis quite difficult. Computer software for statistical calculations and logical-deductive reasoning on vast amounts of data can improve diagnostic skills. These applications are defined as computer-assisted decision making, medical decision support, or expert systems. This study was aimed at developing a prototype probabilistic expert system, based upon knowledge from an "ad hoc"computerized data base, as an aid in the radiologic diagnosis of jaw tumors and pseudotumors. This program has been called ADAPT-M. The study considered 92 patients with benign space-occupying and fully documented lesions of the jaw bones. For each case, a list of parameters concerning different radiologic exams was considered. From all these pieces of information a data base was built, to calculate both the prevalence of each type of lesion and the frequency of many variables in the single conditions. For each kind of lesion 44 variables were considered. ADAPT-M used a formula based on Bayes' theorem to calculate the "a posteriori" probability of a diagnosis in the presence of a symptom. Overall diagnostic probability rate was high when the highest score hypothesis was matched with pathologic findings (80%) and even higher (96.1%) when the two most probable diagnoses were considered together. As expected, ADAPT M had higher sensitivity when used with lesions with typical semiology. This results in an unquestionable limitation, especially in the patients in whom a predictive diagnosis would be most desirable. The creation of a larger data base of known cases and software development may help increase the diagnostic accuracy of the ADAPT-M system. PMID- 8628936 TI - [The echographic anatomy of the larynx and the perilaryngeal structures]. AB - The normal laryngeal and perilaryngeal structures (neither nodal nor vascular) of 120 healthy and informed volunteers and of 46 patients with extralaryngeal neck conditions were studied with ultrasonography (US). The study was performed with a high-frequency linear probe on the transverse and sagittal planes; the patients were examined supine, with neck hyperextension, during quiet breathing, in inspiratory and expiratory breath-holding, during Valsalva maneuver or phonation. Axial scans clearly depict, in cranio-caudal direction, the base of the tongue, the hyoid and adjacent muscular structures. At the laryngeal vestibule epiglottis is always clearly demonstrated as a thin hypoechoic curvilinear rim and the pre epiglottic space as a fat-filled and markedly echogenic structure. Pyriform sinuses are more difficult to study with US and should therefore be always distended by air during Valsalva because no suitable US contrast agent is available for their exploration yet. The ossification of thyroid laminae prevents or hinders the exploration of endolaryngeal structures because of consequent posterior acoustic shadow. In case of partial ossification or cartilaginous thyroid laminae, paraglottic spaces are easily demonstrated with US, as well as the false cords and, partially, arytenoids and posterior laryngeal wall muscles. The muscular structure of the true cords, their mobility in the M-mode and, in half of the cases, the thin vocal plicae joining anteriorly in the anterior commissure, are easily depicted with US. To conclude, US permits to demonstrate normal laryngeal anatomy by a quick and harmless examination in several breathing and phonation phases, which is an essential premise to the correct interpretation of abnormal US findings. PMID- 8628935 TI - [Color Doppler echography in the study of obstruction-related lung collapse]. AB - The typical US pattern of obstructive atelectasis consists in a triangular hypoechoic area with anechoic bands inside related to fluid-filled bronchial structures--the US fluid bronchogram sign. According to some authors, this US sign within a chest mass indicates pulmonary parenchyma disease. Furthermore, it suggests the diagnosis of lung collapse. Sixty-one patients with obstructive atelectasis confirmed with conventional radiography, conventional and computed tomography, and bronchoscopy were submitted to B-mode and color-Doppler US to assess the importance of the US fluid bronchogram sign in obstructive pulmonary atelectasis. In this condition, B-mode US showed tubular anechoic bands in 59/61 patients. Power Doppler venous sampling showed a Doppler spectrum with marked phase oscillations. Arterial sampling showed a Doppler spectrum with high distal impedance-with poor or totally absent diastolic component. To conclude, in the atelectasis area, B-mode US showed in 96% of patients some anechoic bands with no apparent pulsatility. Color-Doppler showed color flow in 100% of cases, which confirmed the vascular nature of the masses. Thus, the US fluid bronchogram, which is frequently described in the literature, was never observed in our series. Power Doppler spectral flow analysis can be useful in the diagnosis of obstructive atelectasis because it depicts the hemodynamics of atelectasis parenchyma. Indeed, the arterial spectrum with high distal resistance is consistent with the effects of hypoxia on intra-atelectatic blood vessels. Further research is necessary to assess the role of color-Doppler US in the hemodynamic study of intra-atelectatic vessels. However, our preliminary results open new perspectives for the acquisition of physiopathologic data on abnormal blood flow in obstructive atelectasis. PMID- 8628937 TI - [The assessment of the activity of idiopathic pulmonary fibrosis by high resolution computed tomography]. AB - The authors report the results of the study performed with high resolution CT (HRCT) in a group of 29 patients affected with idiopathic pulmonary fibrosis (IPF). Each patient underwent HRCT at the beginning of the study and after one year. A complete clinico-functional assessment was available in 20 cases and functional CT correlation was made in these patients; 15/20 subjects underwent immunosuppressive therapy with corticosteroids and cyclophosphamide. Disease severity was assessed with chest radiography and HRCT. On the basis of CT findings the patients were classified into three groups, according to Wells classification: predominant ground-glass pattern, mixed pattern and predominant reticular disease with honeycombing. Furthermore, a visual score was assigned to total disease extent and a different score to ground-glass and reticular opacities. Our data confirm the poor accuracy of chest radiography in assessing disease type and overall severity, versus the outstanding diagnostic accuracy of HRCT. We also found a high incidence of mediastinal adenopathies (37.9% of the patients) and signs of pulmonary arterial hypertension (62%), together with low extent of pulmonary emphysema (65.5% of the patients; mean extent: 5.4%). Ground glass attenuation is an early sign of IPF and might suggest alveolitis activity. In our series, however, the patients with grade I disease were rare (6.8%), but likely to benefit from therapy. Different from Wells, we found no significant difference in the evolution of the patients with grade II versus grade III disease. Wells grading was useful in early disease assessment, but the visual score of total disease extent and the score of ground-glass and reticular opacities were much more useful in the follow-up because they can assess disease progression. Furthermore, in the few patients with predominant alveolitis, who improve after therapy, the relative prevalence of the reticular pattern might allocate the patient in a higher Wells group with a "paradoxical" worsening, if the visual score of the extent of the primary lesion is not used. PMID- 8628938 TI - [Computed tomography in the study of gastrointestinal perforation]. AB - Plain radiography is the method of choice in suspected perforating pneumoperitoneum. Nevertheless, especially when air collections are small, the technique must be very accurate, with patient mobilization and long examination times, which may be unfeasible in acute abdomen patients. To overcome these limitations, such cross-sectional imaging methods as US and especially CT are increasingly used. Our series consisted of 38 patients with gastrointestinal tract perforation examined 1990-94; thirty-one of them had surgical confirmation. CT had high sensitivity, demonstrating the presence of free intraperitoneal gas in more patients than conventional radiography (92% vs. 74%). Pneumoperitoneum was depicted between liver surface and anterior abdominal wall in 30 cases, in the subhepatic region in 17, posterior to the abdominal wall at paraumbilical level in 14, between the mesenteric folds in 8, in the pelvis in 7 and in other locations in 11. Extraluminal fluid collections were apparent in 79% of cases and contrast agent collections in 73%. The three most common findings were: intraperitoneal gas, fluid effusion and extraluminal contrast agent leaks (61.5%), gas and effusion (29%) and gas only (16%). The origin of the perforation was demonstrated in 82% of cases and its cause in 37%. CT was particularly useful in the diagnosis of clinically occult, of early and of confined perforations. Small gas bubbles, mild fluid effusion or minimum contrast agent leaks near perforation site are valuable signs. In selected cases CT can play an integrative role, thus improving the diagnostic accuracy of plain films. PMID- 8628939 TI - [Anal endosonography: the study technic and the correlations between the normal and echographic anatomy]. AB - The authors report on 16 healthy volunteers examined with anal endosonography. The US examinations were performed with a dedicated Bruel and Kjaer 1846 unit equipped with a 7-MHz probe (type 1850) with 360 degrees circular mechanic movement. A rigid plastic cone filled of degassed water is mounted on the probe to ensure the symmetrical representation of the anal canal. A condom with gel on both the internal and the external surfaces is put on the cone. The peculiar anatomy of the single layers which form the anal canal determines their different US patterns. Endosonography was performed on three levels, i.e., deep, intermediate and superficial planes. In the deep plane (at the anorectal junction) anal endosonography demonstrates: epithelial-subepithelial complex (intermediate echogenicity); internal sphincter muscle (low echogenicity), made of smooth muscular fibers; longitudinal muscle (intermediate echogenicity), made of smooth and striated muscular fibers; puborectal muscle (high echogenicity), made of striated muscular fibers, and perineal body, which has a longitudinal shape in men and a transverse shape in women. In the intermediate plane anal endosonography demonstrates: epithelial-subepithelial complex, internal sphincter muscle, longitudinal muscle, and external sphincter muscle (high echogenicity), made of striated muscular fibers. In the superficial plane (distal anal canal extremity), anal endosonography demonstrates: epithelial-subepithelial complex and external sphincter muscle. Internal sphincter muscle thickness was measured and the results follow: while the average thickness was 2.1 mm, we had 1.8 mm in 12 patients under 44 and 2.6 mm in 4 patients over 44 years old. This finding is in agreement with literature data. External sphincter muscle thickness did not vary significantly in the two groups. PMID- 8628941 TI - [Magnetic resonance pyelography: optimization of the technic and the preliminary results]. AB - Magnetic resonance pyelography (MRP) is a new noninvasive method which demonstrates dilated urinary tracts with no need of contrast agent injection. This study was aimed at technique optimization, using new fast sequences with high intrinsic contrast, to demonstrate the urinary tract in obstructive uropathy patients. Twelve consecutive patients and 4 healthy volunteers were included in this prospective study; all the exams were performed with a high gradient power 0.5-T unit using T2- weighted turbo SE sequences, acquired three-dimensionally on the coronal plane. Obstructive uropathy was caused in 9 patients by neoplastic lesions, in 2 by postoperative strictures and in 1 by inflammatory tissue. In all patients MRP depicted the dilated urinary tract optimally, with good morphological detailing and the accurate assessment of both level and cause of obstruction. In the healthy volunteers, the absence of dilatation did not permit the complete visualization of the urinary tract. To conclude, MRP is a new technique which permits high-quality imaging of the urinary tract. Further studies are needed to assess its actual potentials and clinical role. PMID- 8628940 TI - [The integrated diagnosis of hepatic focal nodular hyperplasia: echography, color Doppler, computed tomography and magnetic resonance compared]. AB - The findings were reviewed relative to twelve patients with focal nodular hyperplasia selected from a series of 130 patients with hepatic focal lesions examined with color-Doppler US, dynamic CT and MRI. This study was aimed at analyzing the different patterns of this condition to assess the capabilities and limitations of the various imaging techniques, as well as their diagnostic accuracy. Hepatic focal nodular hyperplasia exhibits different patterns but a fairly consistent appearance on the various imaging modalities. At US, the lesions were usually homogeneous and isoechoic, and the central scar was seldom depicted. Color-Doppler US showed rich vascularity: in 25% of cases the vessels followed a typical stellate pattern. Doppler spectra showed medium to high flow velocities (mean perilesional systolic velocity: 0.71 m/s, 0.34 KHz; mean intralesional systolic velocity: 0.33 m/s, 1.6 KHz). Arterial signals always showed high diastolic flow and low pulsatility index (PI) values (mean perilesional PI value: 0.70; mean intralesional PI value: 0.69). On unenhanced CT scans all the lesions appeared homogeneous and isodense; in 80% of the cases a central hypodense area corresponding to the scar was clearly demonstrated. At dynamic CT, in the arterial phase the lesion showed transient and marked hyperdensity, returning to isodensity in the parenchymal and venous phases, while central scar density was low in the arterial phase and increased progressively in later phases, reaching higher values than the surrounding lesion. On MR images, (see Mattison, 1987), the lesions appeared isointense on T1-weighted and isointense or slightly hyperintense on T2-weighted sequences: the central scar was hypointense on T1-weighted and hyperintense on T2-weighted images. Postcontrast MR images showed similar patterns to those of dynamic CT. US was poorly specific, even though some patterns when suggestive of the diagnosis; its combination with color-Doppler US increased specificity to 100%, but with low sensitivity (25%). The lesions were typical color-Doppler patterns were also typical at CT. Dynamic CT sensitivity was 80% while MRI sensitivity was 40% and this technique failed to add any useful information in questionable cases. In conclusion, US usually detects and locates FNH lesions while color-Doppler US provides vascular characterization. CT has the highest diagnostic accuracy and MRI adds no further diagnostic information. PMID- 8628942 TI - [An analysis of insurance claims of civil responsibility in radiology. The first Italian data]. AB - The purpose of this study is to provide information to Italian radiologists regarding claims and patient injuries in medical malpractice claims. The assurance claims of Italian radiologists over a two-year period (1994-95) were anonymously analyzed, based on pertinent data provided by the Insurance Company of the Italian Society of Medical Radiology. The incidence risk-rate of claims was 9.1 per thousand person/year. Alleged malpractice accounted for more than 85% of the claims. In nearly one-half of the cases (44.4%), the plaintiff's argument was based on a <>. The most common misdiagnosis was failure to diagnose fracture or dislocation. The second most common claim category (40.7%) were complications, most commonly occurring during interventional radiology and contrast media injection. The third claim category (14.8%) was patient's accidents occurring during the diagnostic procedure. The most frequent types of injuries experienced by patients were death (6 cases), loss of chance for survival and permanent disability. Claims were more frequent in public health services, and mostly related to emergency examinations and interventional procedures. Misdiagnoses almost exclusively involved public health radiologists. Private health care, on the other hand, had a higher rate of fatal injuries, mostly related to technical complications. PMID- 8628944 TI - [The biological significance and clinical prospects of apoptosis within the parameters of radiotherapy response]. PMID- 8628943 TI - [3-dimensional echography. Its applications to the urogenital system]. PMID- 8628945 TI - [An experimental in-vitro evaluation of membrane antigen expression and of interleukin production by monocytes exposed to magnetic resonance]. AB - The interactions of magnetic fields and radiofrequency with biological systems have been described by several authors. However, no definitive conclusions have been drawn yet as to the safety of the magnetic fields and radiofrequencies used in clinical examinations. The immune system is one of the most complex biological systems, in which a network of intracellular signals regulates the immune response. Interleukins are released by an activation process which involves, at least in part, intra- and/or extracellular calcium mobilization. The latter step can be influenced by the in vitro effect of this type of nonionizing radiations produced by an MR system on peripheral blood mononuclear cells. Our results show that the 2-hour exposure to magnetic fields (0.5 T) and radiofrequency caused an increase in the spontaneous release of IL2, IL4, IL10, TNF alpha and INF gamma, while the amount of the same cytokines induced by PHA stimulation was decreased. No differences were observed in the spontaneous or PHA-induced release of IL6 by exposure to magnetic fields (MRI). Furthermore, the expression of the CD 11b molecule was increased at the same time. These results may be useful for us to understand the interactions between magnetic fields and radiofrequencies and the immune system. PMID- 8628946 TI - [The radiocesium level in children from Byelarus]. AB - In 1994, the Institute of Applied Physics of Milan carried out a study to measure cesium 137 levels in some boys and girls from Belarus, mainly the Gomel district, which is still a risk area for radioactive contamination. In the summer, nearly 700 boys and girls (age range: 8-14 years) were accommodated by some Italian families in Piedmont and Lombardy. Our research was just meant to collect some pieces of information and dealt with whole body counter measurements (70 children) and with the analysis of radiocesium levels in urine samples (50 children). Our Institute collaborated with the Service of Radiation Protection in Ispra and with the ENEL Service of Radiation Protection in Trino Vercellese. In all, we examined 70 children, divided in groups of 8-10 children each. The results of our measurements show that radiocesium levels ranged from about ten Bq to some thousands Bq. In some children who exhibited high radiocesium levels, its distribution in the body was also studied and shown to be uniform. Finally, the comparison between body and urine Cesium levels allowed us to calculate the biological half-life of soluble cesium. PMID- 8628947 TI - [Tarsal metastases from breast carcinoma: a case report and review of the literature]. PMID- 8628948 TI - [Magnetic resonance in delayed postanoxic encephalopathy following acute carbon monoxide poisoning. A case report]. PMID- 8628949 TI - [Sellar metastases simulating the Tolosa-Hunt syndrome]. PMID- 8628950 TI - [The magnetic resonance assessment of craniofacial fibrous dysplasia. The completion of the study with gadolinium administration and magnetic resonance angiography]. PMID- 8628951 TI - [Thyroid amyloidosis diagnosed by echo-guided needle aspiration with a fine needle]. PMID- 8628952 TI - [Petit's inferior lumbar hernia. A case report]. PMID- 8628953 TI - [A rare pediatric tumor of the pancreas: pancreatoblastoma]. PMID- 8628954 TI - [Cystadenoma of the seminal vesicles. A case report]. PMID- 8628955 TI - [Contiguity spondylodiscitis following percutaneous femoral artery catheterization. A case report]. PMID- 8628956 TI - [What is your diagnosis?]. PMID- 8628958 TI - [Intra-disciplinary therapy strategy in esophageal carcinoma from a surgical viewpoint]. AB - Extensive long segment resection for carcinoma of the esophagus including also consequent mediastinal and celiac lymph node dissection is able to achieve satisfactory radicality only in early tumor stages while there is little influence on long term prognosis for advanced tumor stages. Only better risk analyses and improved surgical outcome with reduced operative mortality has improved the overall outcome. In locally limited primary tumors and even more in locally advanced stages generalisation or dissemination of tumor disease is to be expected. Therefore it is generally agreed upon that only multimodal therapy including systemic chemotherapy and local radiotherapy is able to improve therapeutic results in this disease with otherwise very poor prognosis. We report our own experience on 200 esophageal resections including modification of our strategy and protocol presently in use for tumor stages IIB to IV. PMID- 8628959 TI - [Preoperative radiochemotherapy in esophageal carcinoma]. AB - Patients with esophageal cancer still have on unfavourable prognosis. After standard treatment, only 10% will survive five years. During the last years a lot of study groups intended to improve patient's prognosis by the use of multimodality treatment including chemotherapy. The concept of preoperative chemotherapy seems to be most promising. In the case of stage I and II cancer (potentially resectable tumors) multimodal therapy has not proved superior to surgery alone, so far. The results of well done phase III trials have to be waited for. Prognosis of patients with locally advanced tumors has been significantly improved by preoperative radiochemotherapy compared to surgery alone. In general, patients with localized esophageal cancer should be treated in experienced cancer centers within prospective trials. PMID- 8628957 TI - [Intraoperative radiotherapy in gastrointestinal tumors]. AB - Intraoperative radiotherapy has proved its worth for curative and palliative treatment of gastrointestinal tumors. IORT indications comprise cancer of the stomach, of the pancreas and colorectal carcinomas. Most authors are cautious to apply it to carcinoma of the esophagus, of the small intestine and of the hepatobiliary system. IORT is applied as adjuvant treatment alone or in combination with postoperative external-beam photon irradiation +/- chemotherapy. There is evidence of an improved local control rate. Pain can be relieved in symptomatic patients by an IORT dose of > or = 20 Gy. Until now, however, there is no evidence of a prolonged relapse-free and overall survival. PMID- 8628960 TI - [Percutaneous and intraluminal radiotherapy and radiochemotherapy in esophageal carcinoma]. AB - Next to standard external beam radiation therapy, combined treatment schedules of percutaneous and endoluminal radiotherapy as well as simultaneous radiochemotherapy became important over the past ten years, especially for primarily inoperable, advanced carcinomas of the esophagus. Analyzing representative treatment protocols, the following conclusions are evident: the combination of external high-voltage therapy and endoluminal brachytherapy using high-dose afterloading techniques leads to intensified biologically effective tumor doses with increasing tumor control. The simultaneous application for radio and chemotherapy with 5-fluorouracil, cisplatinum or mitomycin results in a longer median survival compared to irradiation alone, and it is comparable to results in historical controls with radical esophagectomy. Up to now, no reduction of distant metastases was seen after simultaneous radiochemotherapy regimen alone. There is some evidence, that intensified chemotherapy before or after radiochemotherapy might result in improved survival rates and decreased distant metastases. PMID- 8628961 TI - [Benign tumors and pseudotumor of the maxilla in children]. AB - The underlying causes in series of benign tumours and pseudo-tumours and cysts of the maxillary bone observed in children over a ten-year period in the Saint Vincent-de-Paul Hospital in Paris are reported. Besides the frequently encountered odontogenic cysts, most cases involved aneurysmal cysts and benign odontogenic tumours, as well as rare tumours including a hydatic cyst. Several lesions were discovered on a panoramic X ray performed for orthodontal survey. PMID- 8628962 TI - [Venous hemangioma of the temporal muscle. Review of the literature: apropos of a case]. AB - A 41-year-old woman progressively developed a painful tumefaction in the left temporal fossa. The scan showed a isodense intramuscular structure weakly enhanced by contrast medium. Surgical exeresis was performed via the coronal access. Histology of the surgical specimen reported the diagnosis of venous muscular haemangioma. The diagnosis and management of these rare tumours is discussed. PMID- 8628963 TI - [Schwannoma of the parotid region. Apropos of a case report]. AB - Schwannomas are rarely located in the parotid regions. We report a case in a 6 year old child and recall the epidemiology of this type of tumours together with the clinical presentations and therapeutic approaches. PMID- 8628964 TI - [Metastasizing pleomorphic adenomas. Clinical and cytometric analysis]. AB - Metastasizing pleomorphic adenoma is a rare lesion, histologically benign but with a malignant clinical behaviour. Two cases are reported with a flow cytometric study. Recurrent adenoma and its metastasis were aneuploid in one case. PMID- 8628965 TI - [Primary tuberculosis of the oral cavity]. AB - Primary tuberculosis in the oral cavity is a rare entity. Usually, the microorganisms need a disruption of the oral mucosa to become pathogenic. In this article the authors describe a clinical case of primary oral tuberculosis, on a female of 52 years-old who suffered an exodontia 20 days before. The bacteria identificated was Mycobacterium tuberculosis hominis. The microbiologic identification is essential to assure the efficacy of the treatment. PMID- 8628966 TI - [Cervico-facial non-Hodgkin's lymphomas. Apropos of a case of orbital localization]. AB - The orbital localization of the non-Hodgkin's lymphoma is not the more common site of presentation. The most classical sites in the head and neck are the cervical lymph nodes and Waldeyer's ring. The treatment is depending on the Staging and includes radiation therapy, chemotherapy and surgery or a combination of these modalities. We describe here an orbital localization of intermediate grade. The clinical and pathological features are discussed, and the literature is summarized. PMID- 8628967 TI - [Cephalometric study of changes in the upper airways after orthognathic surgery. Significance in the management of obstructive sleep apnea syndromes. Apropos of 43 cases]. AB - The aim of this work was to study long-term airways space changes after maxillomandibular osteotomy in order to assess benefit of this surgery in case of obstructive sleep apnea syndrome. Preoperative and postoperative cephalometric airway spaces evaluation of 43 patients (who did not have obstructive sleep apnea) was done. Analysis revealed no charge in velopharyngeal space, lingual pharyngeal augmentation in case of mandibular advancement. Hyoid bone position was not modified by osteotomy. Signification and interest of this finding for patients with obstructive sleep apnea are discussed through literature data. PMID- 8628968 TI - [Permanent constriction of the jaws in children: 3 cases with extra-articular etiology]. AB - Three cases of permanent constriction of the jaw are reported. One was a sequela after a noma, the second occurred in a child with arthrogryposis and the third was an inborn malformation. Each case raised specific problems for diagnosis and treatment. Mechanicotherapy is essential postoperatively and requires a specific electric apparatus to mobilize the joint. PMID- 8628969 TI - [Paradental cyst]. AB - The paradental cyst is an odontogenic inflammatory cyst (WHO classification, 1992). It has been rarely described in the french literature and includes: The inflammatory paradental cyst which occurs on the buccal or distal aspects of an erupted third mandibular molar or of a canine (first description by Main in 1970); the mandibular infected buccal cyst which occurs on the buccal surface of the mandibular second or first molar in children around 6-8 years of age with an associated history of inflammatory signs (Stoneman and North, 1993). The histological features are the same as those of the radicular cyst. Prognosis is excellent after enucleation. This paper present an additional case report of a paradental cyst located on a second mandibular molar. PMID- 8628970 TI - Serum level of IL-6 in patients with primary and secondary Sjogren's syndrome. AB - The level of interleukin-6 in the serum of 17 patients suffering from the primary SJogren's syndrome and of 16 with secondary Sjogren's syndrome were determined by means of ELISA. The mean value of IL-6 in primary and secondary Sjogren's syndrome was 2.41 pg/ml (1.45-3.37 pg/ml) and 3.01 pg/ml (1.63-4.39 pg/ml). The normal serum level of IL-6 was below 3.0 pg/ml. The serum level of IL-6 was not elevated in Sjogren's syndrome patient, there was no significant difference in the serum level of IL-6 between both groups of the patients. PMID- 8628971 TI - Neural transplantation of the rat midgestation trophoblast. AB - The trophoblast may serve as an example of tissue that is endowed with invasive properties under physiological conditions. Invasiveness of the trophoblast is enabled by the secretion of various proteases capable of degrading extracellular matrix components. Trophoblast invasive behaviour is strictly controlled by anti invasive factors produced by uterine decidual cells. To assess invasive abilities of trophoblast cells we have transplanted E9 and E14 rat trophoblast (i.e. the trophoblast obtained on embryonic day 9 and 14) into the brain of adult rats. The brain parenchyma as an immunologically privileged site is suitable for acceptance of grafts of different tissues. Moreover, the trophoblast placed into the CNS lacks inhibitory influence of anti-invasive factors that normally regulate trophoblast invasivity in the course of intrauterine gestation. To visualize migration of grafted cells the nuclei of the trophoblast were labelled with bromodeoxyuridine prior to neural grafting. The transplant of E9 and E14 trophoblast cells obtained a blood nourishment from host vessels. A proper vascularization is necessary for a further transplant growth. The transplant contained labyrinthine trophoblast cells and giant cells that are typical for the rat placenta. Vital trophoblast cells were found in all grafts whose age did not exceed a lifespan of normal rat trophoblast cells, i.e. 21-22 days. In the centre of the graft, no blood vessels were observed. Interstitial spaces of neighbouring trophoblast cells were filled with the host blood and morphology of these spaces mimicked lacunae of the placental trophoblast. E9 and E14 rat trophoblast continued to differentiate after transplantation into the CNS of adult rats. Histological structure of the grafts were compared with microscopical morphology of the normal rat placenta. E9 and E14 trophoblast is considerably differentiated and it does not invade a neighbouring tissue. Trophoblast cells located at the graft periphery may migrate on free surfaces but they do not invade through the host parenchyma. Migration occurs at a limited distance from the transplant and the cells remain in a close contact with other trophoblast cells in the graft via their cytoplasmatic processes. The ability to lyse host blood vessels and form vascular lacunae is well preserved in E9 and E14 trophoblast after grafting into the CNS. This ability is necessary for a proper transport of nutrients from the host blood stream to fetal tissue that normally occurs in the placenta. PMID- 8628972 TI - Histochemical localization of succinate dehydrogenase in intracerebral mouse submandibular gland transplants. AB - Histochemical localization of succinate dehydrogenase in developing intracerebral auto- and homotransplants of the mouse submandibular gland was investigated in the course of a 5-month period after transplantation. Eight weeks after grafting, the enzyme pattern in the parenchyma of homotransplants was comparable with the histochemical picture of a fully mature submandibular gland in situ. At this time, numerous acini showed a weak activity, very numerous striated ducts a strong activity, and less frequent developing convoluted granular tubules slightly weaker activity, than in the striated ducts. Beginning histochemical differentiation of convoluted granular tubules was noted only in homotransplants, located in the brain of male recipients. On the other hand, only a weak activity in the cytoplasm of non-differentiated submandibular gland of duct-like structures of some autotransplants was seen. Homotransplants of non differentiated submandibular gland of newborn donors were found to be a more suitable transplantation object capable of postnatal development of gland parenchyma than autografts of fully differentiated gland of adult animals that did not enter the cytodifferentiation stage. PMID- 8628973 TI - Changes of some biochemical and hematological parameters following administration of daunorubicin in rabbits. AB - The effects of the repeated i.v. administration of daunorubicin (50 mg/m2, once weekly, max. 9 weeks) were investigated in rabbits in vivo to analyze biochemical and hematological changes. Noninvasive polygraph records were used to evaluate the function of the heart. The administration of daunorubicin induced changes especially in levels of protein (decrease in total protein and albumin) and of some ions (decrease in calcium, magnesium and phosphorus) as well as in hematological parameters (decrease in erythrocytes, leukocytes and thrombocytes). the results obtained correlate with data on mechanisms of daunorubicin toxicity. PMID- 8628974 TI - Convulsive properties of N-(3,5-dimethoxy-4-propoxyphenyl-ethyl)-aziridine and their influencing by diazepam and triazolam. AB - Convulsive activity of N-(3,5-dimethoxy-4-propoxy-phenyl-ethyl)-aziridine (FAZ 4), a newly synthesized aziridine compound was studied in rats. There is a lack of tonic component of major paroxysm in comparison with the classical convulsive agent pentylenetetrazol. This effect of FAZ-4 is probably due to the forebrain mechanism without the midbrain involvement. Both anticonvulsants tested suppressed seizures in a different manner, however triazolam exerted stronger anticonvulsive activity than the same dose of diazepam did. PMID- 8628975 TI - Motion onset VEPs improve the diagnostics of multiple sclerosis and optic neuritis. AB - In addition to standard pattern-reversal VEPs, the motion on-set VEPs were examined in 50 patients with acute unilateral retrobulbar neuritis (RN) and in 187 patients with possible or definite multiple sclerosis (MS). In MS patients (without sign or history of RN), the results of both types of VEPs correlated only partially. 26.2% of them displayed changes only in the motion-onset VEPs having the pattern-reversal VEPs completely normal. That is why we suppose that the magnocellular system (tested by motion-onset VEPs) can be affected by demyelination separately. In 28 patients with "pure" RN (without any other sign indicating demyelination disease) the always abnormal pattern-reversal VEPs were accompanied by delayed motion-onset VEPs in only 28.6% of patients. In contrast, much higher rate--68.2%--of delayed motion-onset VEPs was found in the 22 RN patients simultaneously suspected of MS. These results indicate that RN affects predominantly the parvocellular visual system (tested by reversal VEPs). Distinct latency changes of the motion-onset VEPs in RN patients seem to signal a linkage between RN and demyelination. PMID- 8628976 TI - Bleeding to adrenal gland in adults. AB - At the Department of Urology in Hradec Kralove five patients were treated in 1991 to 1993 with bleeding to the adrenal glands. We present our reports of these patients and stress the difficulty of preoperative diagnosis. PMID- 8628977 TI - Selective cyclooxygenase inhibition: promise for future NSAID therapy? PMID- 8628978 TI - Clinical implications of selective cyclooxygenase-2 inhibition. AB - Inducible cyclooxygenase-2 (COX-2), but not constitutive COX-1, can be upregulated in rheumatoid synovial tissue by interleukin-1 beta and phorbol esters and is inhibited by dexamethasone. This supports the role of COX-2 in acute inflammation in arthritis. Selective inhibition of COX-2 by non-steroidal anti-inflammatory drugs (NSAIDs) has been proposed as an approach to reduce their associated side effects while maintaining efficacy. The improved safety profile of selective COX-2 inhibitors will allow more widespread and sustained use than is currently possible with standard NSAIDs. In rheumatoid arthritis they may be used as effective symptomatic relief, in combination with disease modifying therapy at an early stage of disease. In osteoarthritis, and, more particularly, soft tissue rheumatism, pain contributes to the development of chronic disease, therefore the main benefit of selective COX-2 inhibition will be to provide safe, effective pain relief to maintain mobility and reduce disability. PMID- 8628979 TI - Review of clinical trials and benefit/risk ratio of meloxicam. AB - Meloxicam is a new once daily non-steroidal anti-inflammatory drug (NSAID). Double-blind trials in over 5000 patients with osteoarthritis and rheumatoid arthritis have shown that meloxicam 7.5 mg and 15 mg are significantly more effective than placebo and comparable in efficacy to standard NSAIDs such as naproxen 750-1000 mg, piroxicam 20 mg and diclofenac 100 mg slow release. In a global safety analysis, both meloxicam doses produced significantly fewer gastrointestinal (GI) side effects than the comparators (p < 0/05). Severe GI side effects, discontinuations due to GI side effects and less serious events such as dyspepsia and abdominal pain were also significantly less frequent with meloxicam. Perforations, ulcerations and bleedings occurred in 0.1%, 0.2%, 1.2%, 0.6% and 2.1% of meloxicam 7.5 mg, 15 mg, piroxicam, diclofenac and naproxen patients respectively (p < 0.05 for piroxicam and naproxen compared with meloxicam). This improved safety profile is likely to be due to meloxicam's selective inhibition of COX-2 relative to COX-1. PMID- 8628980 TI - Toward an understanding of NSAID-related adverse events: the contribution of longitudinal data. AB - The ARAMIS (Arthritis, Rheumatism and Ageing Medical Information System) databanks have been used to objectify and quantify drug toxicity. The relative risk of a gastrointestinal (GI)-provoked hospitalization was more than five times greater in patients taking non-steroidal anti-inflammatory drugs (NSAIDs) than in non-NSAID-treated patients, with an excess hospitalization rate of 1.3% per annum. Additionally, there was an excess GI-related death rate of around 3% in rheumatoid arthritis (RA) patients compared with the normal population. Age, previous NSAID-related GI events, prednisone use, higher doses and greater disability predicted high-risk patients. A toxicity index showed clear differences between NSAIDs, with aspirin, salsalate and ibuprofen emerging as the least toxic, and meclofenamate and indomethacin as the most toxic. Disease modifying anti-rheumatic drugs (DMARDs) were, surprisingly, found to have similar toxicity scores to the NSAIDs. This supports the contemporary practice of employing DMARDs earlier and more aggressively in the course of RA. PMID- 8628981 TI - Mechanism of action of anti-inflammatory drugs. AB - Cyclooxygenase (COX) is the pivotal enzyme in prostaglandin biosynthesis. It exists in two isoforms, constitutive COX-1 (responsible for physiological functions) and inducible COX-2 (involved in inflammation). Inhibition of COX explains both the therapeutic effects (inhibition of COX-2) and side effects (inhibition of COX-1) of non-steroidal anti-inflammatory drugs (NSAIDs). A NSAID which selectively inhibits COX-2 is likely to retain maximal anti-inflammatory efficacy combined with less toxicity. The activity of a number of NSAIDs has been investigated in several test systems, showing that most of those marketed have higher activities against COX-1 or are equipotent against both isoforms. Adverse event data of marketed NSAIDs show a relationship between a poor safety profile and more potent inhibition of COX-1 relative to COX-2. There are several new non steroidal COX-2 inhibitors in development. The most clinically advanced is meloxicam, which consistently demonstrates higher activity against COX-2 than COX 1 in several test systems. PMID- 8628982 TI - Biological determinism. PMID- 8628984 TI - Gene therapy: false expectations? PMID- 8628983 TI - Biological determinism. PMID- 8628985 TI - ARC affair troubles French scientists. PMID- 8628986 TI - NIH clinical center gets a boost. PMID- 8628987 TI - Results on new AIDS drugs bring cautious optimism. PMID- 8628988 TI - Science journals go wired. PMID- 8628989 TI - Electronic preprints point the way to 'author empowerment'. PMID- 8628990 TI - Indeterminate organization of the visual system. PMID- 8628991 TI - On hierarchies: response to Hilgetag et al. PMID- 8628992 TI - Intercalation, DNA kinking, and the control of transcription. AB - Biological processes involved in the control and regulation of transcription are dependent on protein-induced distortions in DNA structure that enhance the recruitment of proteins to their specific DNA targets. This function is often accomplished by accessory factors that bind sequence specifically and locally bend or kink the DNA. The recent determination of the three-dimensional structures of several protein-DNA complexes, involving proteins that perform such architectural tasks, brings to light a common theme of side chain intercalation as a mechanism capable of driving the deformation of the DNA helix. The protein scaffolds orienting the intercalating side chain (or side chains) are structurally diverse, presently comprising four distinct topologies that can accomplish the same task. The intercalating side chain (or side chains), however, is exclusively hydrophobic. Intercalation can either kink or bend the DNA, unstacking one or more adjacent base pairs and locally unwinding the DNA over as much as a full turn of helix. Despite these distortions, the return to B-DNA helical parameters generally occurs within the adjacent half-turns of DNA. PMID- 8628993 TI - DNA: an extensible molecule. AB - The force-displacement response of a single duplex DNA molecule was measured. The force saturates at a plateau around 70 piconewtons, which ends when the DNA has been stretched about 1.7 times its contour length. This behavior reveals a highly cooperative transition to a state here termed S-DNA. Addition of an intercalator suppresses this transition. Molecular modeling of the process also yields a force plateau and suggests a structure for the extended form. These results may shed light on biological processes involving DNA extension and open the route for mechanical studies on individual molecules in a previously unexplored range. PMID- 8628994 TI - Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. AB - Single molecules of double-stranded DNA (dsDNA) were stretched with force measuring laser tweezers. Under a longitudinal stress of approximately 65 piconewtons (pN), dsDNA molecules in aqueous buffer undergo a highly cooperative transition into a stable form with 5.8 angstroms rise per base pair, that is, 70% longer than B form dsDNA. When the stress was relaxed below 65 pN, the molecules rapidly and reversibly contracted to their normal contour lengths. This transition was affected by changes in the ionic strength of the medium and the water activity or by cross-linking of the two strands of dsDNA. Individual molecules of single-stranded DNA were also stretched giving a persistence length of 7.5 angstroms and a stretch modulus of 800 pN. The overstretched form may play a significant role in the energetics of DNA recombination. PMID- 8628995 TI - Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. AB - When mammalian cells were treated with triplex-forming oligonucleotides of sufficient binding affinity, mutations were specifically induced in a simian virus 40 vector contained within the cells. Triplex-induced mutagenesis was not detected in xeroderma pigmentosum group A cells nor in Cockayne's syndrome group B cells, indicating a requirement for excision repair and for transcription coupled repair, respectively, in the process. Triplex formation was also found to stimulate DNA repair synthesis in human cell extracts, in a pattern correlating with the inhibition of transcription in such extracts. These findings may have implications for therapeutic applications of triplex DNA and raise the possibility that naturally occurring triple helices are a source of genetic instability. PMID- 8628996 TI - Cell killing by the Drosophila gene reaper. AB - The reaper gene (rpr) is important for the activation of apoptosis in Drosophila. To investigate whether rpr expression is sufficient to induce apoptosis, transgenic flies were generated that express rpr complementary DNA or the rpr open reading frame in cells that normally live. Transcription of rpr from a heat inducible promoter rapidly caused wide-spread ectopic apoptosis and organismal death. Ectopic overexpression of rpr in the developing retina resulted in eye ablation. The occurrence of cell death was highly sensitive to the dosage of the transgene. Because cell death induced by the protein encoded by rpr (RPR) could be blocked by the baculovirus p35 protein, RPR appears to activate a death program mediated by a ced-3/ICE (interleukin-1 converting enzyme)-like protease. PMID- 8628997 TI - Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER. AB - Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1. PMID- 8628998 TI - Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. AB - The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS. PMID- 8628999 TI - PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells. AB - Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics. PMID- 8629000 TI - Cold-induced expression of delta 9-desaturase in carp by transcriptional and posttranslational mechanisms. AB - Poikilothermic animals respond to chronic cold by increasing phosphoglyceride unsaturation to restore the fluidity of cold-rigidified membranes. Despite the importance of this compensatory response, the enzymes involved have not been clearly identified, and the mechanisms that control their activity are unknown. In carp liver, cold induces an 8- to 10-fold increase in specific activity of the microsomal stearoyl coenzyme A desaturase. Cold-induced up-regulation of gene transcription resulted in a 10-fold increase in desaturase transcript amounts after 48 to 60 hours. However, this increase was preceded by the activation of latent desaturase, probably by a posttranslational mechanism. These two mechanisms may act sequentially to match desaturase expression to the demands imposed by a progressive decrease in temperature. PMID- 8629003 TI - Whistleblower protection. PMID- 8629001 TI - Identification of a committed precursor for the mast cell lineage. AB - Mast cells originate from hematopoietic stem cells, but the mast cell-committed precursor has not been identified. In the study presented here, a cell population in murine fetal blood that fulfills the criteria of progenitor mastocytes was identified. It is defined by the phenotype Thy-1loc-Kithi, contains cytoplasmic granules, and expresses RNAs encoding mast cell-associated proteases but lacks expression of the high-affinity immunoglobulin E receptor. Thy-1loc-Kithi cells generated functionally competent mast cells at high frequencies in vitro but lacked developmental potential for other hematopoietic lineages. When transferred intraperitoneally, this population reconstituted the peritoneal mast cell compartment of genetically mast cell-deficient W/Wv mice to wild-type levels. PMID- 8629004 TI - Academy's about-face on forensic DNA. PMID- 8629002 TI - Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. AB - Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting. PMID- 8629005 TI - SIV transmission. Monkey study prompts high-level public health response. PMID- 8629006 TI - Likely HIV cofactor found. PMID- 8629007 TI - Just how old is that DNA, anyway? PMID- 8629008 TI - California social climbers: low water prompts high status. PMID- 8629009 TI - Revised Galileo data leave Jupiter mysteriously dry. PMID- 8629010 TI - Impact of DNA replication errors put to the test. PMID- 8629011 TI - Transcription factor IIA: a structure with multiple functions. PMID- 8629012 TI - Getting down to the core of homologous recombination. PMID- 8629013 TI - Dengue hemorrhagic fever. PMID- 8629014 TI - Crystal structure of the yeast TFIIA/TBP/DNA complex. AB - The crystal structure of the yeast TFIIA/TBP/TATA promoter complex was solved to 3 angstrom resolution by double-edge multiple wavelength anomalous diffraction from two different species of anomalous scattering elements in the same crystal. The large and small subunits of TFIIA associate intimately to form both domains of a two-domain folding pattern. TFIIA binds as a heterodimer to the side of the TBP/TATA complex opposite to the side that binds TFIIB and does not alter the TBP/DNA interaction. The six-stranded beta-sandwich domain interacts with the amino-terminal end of TBP through a stereospecific parallel beta-strand interface and with the backbone of the TATA box and the 5'-flanking B-DNA segment. The four helix-bundle domain projects away from the TBP/TATA complex, thereby presenting a substantial surface for further protein-protein interactions. PMID- 8629015 TI - Galileo probe: in situ observations of Jupiter's atmosphere. AB - The Galileo probe performed the first in situ measurements of the atmosphere of Jupiter on 7 December 1995. The probe returned data until it reached a depth corresponding to an atmospheric pressure of approximately 24 bars. This report presents a brief overview of the origins and purpose of the mission. Science objectives, entry parameters and mission events, and results are described. The remaining reports address in more detail the individual experiments summarized here. PMID- 8629016 TI - The Galileo probe mass spectrometer: composition of Jupiter's atmosphere. AB - The composition of the jovian atmosphere from 0.5 to 21 bars along the descent trajectory was determined by a quadrupole mass spectrometer on the Galileo probe. The mixing ratio of He (helium) to H2 (hydrogen), 0.156, is close to the solar ratio. The abundances of methane, water, argon, neon, and hydrogen sulfide were measured; krypton and xenon were detected. As measured in the jovian atmosphere, the amount of carbon is 2.9 times the solar abundance relative to H2, the amount of sulfur is greater than the solar abundance, and the amount of oxygen is much less than the solar abundance. The neon abundance compared with that of hydrogen is about an order of magnitude less than the solar abundance. Isotopic ratios of carbon and the noble gases are consistent with solar values. The measured ratio of deuterium to hydrogen (D/H) of (5 +/- 2) x 10(-5) indicates that this ratio is greater in solar-system hydrogen than in local interstellar hydrogen, and the 3He/4He ratio of (1.1 +/- 0.2) x 10(-4) provides a new value for protosolar (solar nebula) helium isotopes. Together, the D/H and 3He/4He ratios are consistent with conversion in the sun of protosolar deuterium to present-day 3He. PMID- 8629017 TI - The helium mass fraction in Jupiter's atmosphere. AB - On 7 December 1995, the NASA Galileo probe provided in situ measurements of the helium abundance in the atmosphere of Jupiter. A Jamin interferometer measured the refractive index of the jovian atmosphere in the pressure region from 2 to 14 bars. These measurements indicate that the atmospheric helium mole fraction is 0.136 +/- 0.004. The corresponding helium mass fraction is slightly below the presolar value, which suggests that separation of helium from hydrogen in Jupiter's interior is only in its early stages. PMID- 8629018 TI - Solar and thermal radiation in Jupiter's atmosphere: initial results of the Galileo probe net flux radiometer. AB - The Galileo probe net flux radiometer measured radiation within Jupiter's atmosphere over the 125-kilometer altitude range between pressures of 0.44 bar and 14 bars. Evidence for the expected ammonia cloud was seen in solar and thermal channels down to 0.5 to 0.6 bar. Between 0.6 and 10 bars large thermal fluxes imply very low gaseous opacities and provide no evidence for a deep water cloud. Near 8 bars the water vapor abundance appears to be about 10 percent of what would be expected for a solar abundance of oxygen. Below 8 bars, measurements suggest an increasing water abundance with depth or a deep cloud layer. Ammonia appears to follow a significantly subsaturated profile above 3 bars. Unexpectedly high absorption of sunlight was found at wavelengths greater than 600 nanometers. PMID- 8629019 TI - Results of the Galileo probe nephelometer experiment. AB - The nephelometer experiment carried on the Galileo probe was designed to measure the jovian cloud structure and its microphysical characteristics from entry down to atmospheric pressure levels greater than 10 bars. Before this mission there was no direct evidence for the existence of the clouds below the uppermost cloud layer, and only theoretical models derived from remote sensing observations were available for describing such clouds. Only one significant cloud structure with a base at about 1.55 bars was found along the probe descent trajectory below an ambient pressure of about 0.4 bar, although many indications of small densities of particle concentrations were noted during much of the descent. PMID- 8629020 TI - Amino acid racemization and the preservation of ancient DNA. AB - The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber. PMID- 8629021 TI - Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA. AB - The molecular structure of the Escherichia coli RecA protein in the absence of DNA revealed two disordered or mobile loops that were proposed to be DNA binding sites. A short peptide spanning one of these loops was shown to carry out the key reaction mediated by the whole RecA protein: pairing (targeting) of a single stranded DNA to its homologous site on a duplex DNA. In the course of the reaction the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a beta structure. These events probably recapitulate the underlying molecular pathway or mechanism used by homologous recombination proteins. PMID- 8629022 TI - HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein coupled receptor. AB - A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated "fusin," is a putative G protein-coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4 expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV 1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target cells. Fusin messenger RNA levels correlated with HIV-1 permissiveness in diverse human cell types. Fusin acted preferentially for T cell line-tropic isolates, in comparison to its activity with macrophagetropic HIV-1 isolates. PMID- 8629023 TI - Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. AB - Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen independence occurs in the mid-to late G1 phase of the cell cycle and is called the restriction point. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulated the cyclin-dependent kinase (CDK) inhibitor p27Kip1. This was correlated with inactivation of essential G1 cyclin CDK complexes and with cell cycle arrest in G1. The ability of specific mitogens to allow transit through the restriction point paralleled their ability to down regulate p27, and antisense inhibition of p27 expression prevented cell cycle arrest in response to mitogen depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic signals to the cell cycle at the restriction point. PMID- 8629024 TI - A U1/U4/U5 snRNP complex induced by a 2'-O-methyl-oligoribonucleotide complementary to U5 snRNA. AB - Nuclear messenger RNA splicing involves multiple interactions between the five spliceosomal small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, and U6 and numerous spliceosomal proteins. Here it is shown that binding of a 2' O-methyl-oligoribonucleotide complementary to U5 small nuclear RNA (snRNA) nucleotides 68 to 88 (BU5Ae) disrupts the initial U4/U5/U6 tri-snRNP complex, enhances the U2/U6 interaction, and induces a Ul/U4/U5 snRNP complex. The Ul/U4/U5 snRNP complex interacts specifically with an RNA oligonucleotide containing the 5' splice site sequence and may therefore represent a transitional stage in the displacement of U1 from the 5' splice site by U5 snRNP. PMID- 8629025 TI - Genetically engineered resistance to dengue-2 virus transmission in mosquitoes. AB - The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus. PMID- 8629026 TI - Two genetically separable steps in the differentiation of thymic epithelium. AB - The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation. PMID- 8629028 TI - Current areas of treatment. PMID- 8629027 TI - An enhanced immune response in mice lacking the transcription factor NFAT1. AB - Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response. PMID- 8629029 TI - Role of paclitaxel in the treatment of breast cancer: the American Cooperative Group Experience. AB - Phase I and II trials to evaluate paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) as a single agent and combined with other drugs have generated considerable enthusiasm for this interesting new agent. Despite these trials, or because of them, many important questions remain regarding the use of paclitaxel. Ongoing Cooperative Oncology Group trials investigating the use of paclitaxel in breast cancer include CLB 9342, examining the question of paclitaxel dose intensity; the National Surgical Adjuvant Breast Project trial B 26, investigating the question of duration of infusion; E1193, testing the relative activity and synergy of paclitaxel and doxorubicin in a metastatic setting; and CLB 9344, focusing on the adjuvant use of paclitaxel. The cooperative groups are currently investigating novel paclitaxel-based combination therapy in phase II trials. PMID- 8629030 TI - Paclitaxel and doxorubicin, a highly active combination in the treatment of metastatic breast cancer. AB - The activity of single-agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) has been documented in untreated and previously treated metastatic breast cancer, including both patients with anthracycline-resistant disease and those with extensive pretreatment. Such activity has prompted investigations of the optimal doses and schedules of paclitaxel/doxorubicin combinations. With one exception, paclitaxel has been administered as either a 24- or a 3-hour infusion, while the administration times for doxorubicin vary from bolus injection to 72 hour infusion. Results of these completed phase I and II trials are reviewed. Also reported are two European trials that have achieved promising results. In Milan, a phase I/II trial has shown a preliminary response rate exceeding 90% in 32 chemotherapy-naive patients treated with an alternating schedule of paclitaxel given over 3 hours and intravenous bolus doxorubicin. At doses of paclitaxel 200 mg/m2 and doxorubicin 60 mg/m2, the dose-limiting toxicities were neutropenia, oral mucositis, myalgias, and peripheral neuropathy. Congestive heart failure occurred in six patients. A phase I/II study of a 30-minute doxorubicin infusion preceding a 3-hour paclitaxel infusion every 3 weeks in minimally pretreated patients also is reported. Of 29 patients evaluable for response, 17 have achieved partial responses and seven complete responses, for an overall response rate of 83% (95% confidence interval, 79% to 99%). Toxicities observed were grades 3 to 4 neutropenia and moderate paresthesias, nausea/vomiting, alopecia, myalgia, and mucositis. Cardiotoxicity also occurred, as 15 patients had a significant decrease in left ventricular ejection fraction measured by isotope cardiography. Six of these developed congestive heart failure. This effect has been observed only in studies using short infusions of both drugs, and it is now being investigated whether lowering the peak doxorubicin concentration will preclude it. PMID- 8629031 TI - A Phase I/II study of paclitaxel and doxorubicin in the treatment of advanced breast cancer. AB - We designed a clinical study in which fixed doses of doxorubicin were infused by intravenous bolus 16 hours before escalating doses of paclitaxel (Taxol; Bristol Myers Squibb Company, princeton, NJ) for the treatment of patients with advanced breast cancer, an interval selected to allow systemic clearance of doxorubicin before administration of paclitaxel in an outpatient setting. Courses of fixed dose doxorubicin 50 mg/m2 by intravenous bolus and paclitaxel (doses escalated from 130 mg/m2 to 250 mg/m2 via dose escalation of 30 mg/m2) were repeated every 21 days, to a maximum of eight cycles. Maximum tolerated dose was reached if two or more of six patients at a given dose level were affected by the following events: absolute neutrophil count less than 500/microliter for > or = 7 days, absolute neutrophil count less than 100/microliter for > or = 3 days, insufficient hematopoietic recovery with absolute neutrophil count less than 1,500/microliter on day 21, febrile neutropenia, grade 4 thrombocytopenia, any World Health Organization grade 3 nonhematologic toxicity for more than 7 days. There were 19 patients enrolled; the patients received a total of 128 treatment courses. Grade 4 neutropenia was the main side effect, occurring in 20% of courses but generally not associated with clinical events. No relevant clinical cardiac toxicity or alteration of left ventricular ejection fraction was observed. Other toxicities included complete alopecia, mild peripheral neuropathy, and mild myalgia. There was a reduction of one dose level for moderate myalgia in one patient (190 mg/m2 level). Complete alopecia was always present. Maximum tolerated dose was not reached at paclitaxel 250 mg/m2. Ultimately, the introduction of this combination in the adjuvant setting is warranted. PMID- 8629032 TI - Phase I study of paclitaxel and epirubicin in patients with metastatic breast cancer: a preliminary report on safety. AB - Attempting to develop a new active, convenient regimen, we initiated a phase I study of paclitaxel (Taxol; Bristol-Myers squibb Company, Princeton, NJ) combined with epirubicin (Farmitalia Carlo Erba, Milan, Italy) in patients with metastatic breast cancer. In addition to standard eligibility criteria, patients with chemotherapy-naive metastasis and at least one measurable lesion had to have left ventricular ejection fractions of at least 50%; the metastatic relapse had to have occurred more than 6 months after adjuvant treatment. Anthracycline pretreated patients could not have received cumulative doses of more than 300 mg/m2 doxorubicin, 450 mg/m2 epirubicin, or 70 mg/m2 mitoxantrone. An intravenous bolus dose of epirubicin was followed by a 3-hour paclitaxel infusion, with courses repeated every 3 weeks. To date, seven dose levels have been investigated and 31 patients have been treated, 19 of whom had already received anthracyclines. Grades 3 and 4 neutropenia occurred in 37% and 19% of 123 courses, respectively, with five episodes of febrile neutropenia. Grade 2 or 3 neurotoxicity has been observed in 42% of patients and cardiac toxicity in four patients (13%), all of whom had already received anthracyclines. One patient experienced transient myocardial ischemia, one had an asymptomatic decrease in ejection fraction, and two patients had clinical heart failure that required treatment. Dose-limiting toxicity was reached at dose level 5 (paclitaxel 200 mg/m2 plus epirubicin 60 mg/m2), with two of three patients experiencing febrile neutropenia. Reducing the epirubicin dose to 50 mg/m2, however, allowed the paclitaxel dose to be escalated to 250 mg/m2. At this dose level, only one of six patients experienced febrile neutropenia. At a preliminary response evaluation (dose levels 1 to 6), 11 patients (44%) had partial responses, 12 patients (48%) had stable disease, and disease progressed in two patients. We conclude that the combination paclitaxel/epirubicin is safe for patients with metastatic breast cancer and, at this early evaluation, shows promising antitumor activity. Additional patients will be treated at dose level 5 to confirm whether dose limiting toxicity occurs at this step. Indeed, we took into consideration that dose-limiting toxicity observed at this particular dose level in two of three patients might be due to hazard, since paclitaxel dose escalation up to 250 mg/m2 was further possible in association with 50 mg/m2 epirubicin. PMID- 8629033 TI - Activity and safety of epirubicin plus paclitaxel in advanced breast cancer. AB - We performed a dose-escalation study to evaluate the maximum tolerated dose (MTD) of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) plus a fixed dose of epirubicin. Epirubicin was administered as a 90 mg/m2 bolus immediately followed by a 3-hour infusion of paclitaxel starting at 135 mg/m2 and escalating by 20mg/m2 for each triplet of patients as long as no dose-limiting toxicity had occurred; courses were repeated every 3 weeks. The MTD was defined as that at which any of the following toxicities occurred in at least two of six patients: absolute neutrophil count less than 500/microliter for more that 7 days or less than 100/microliter for more than 3 days; any episode of febrile neutropenia requiring intravenous antibiotics and hospitalization; grade 4 thrombocytopenia requiring platelet transfusion; failure to recover absolute neutrophil count to > or = 1,500/microliter and/or platelets to > or = 100,000/microliter by day 28; and any grade > or = 3 nonhematologic toxicity. Two MTDs were defined: the first without granulocyte colony-stimulating factor (MTD 1) and the second with granulocyte colony-stimulating factor given either to accelerate recovery of grade 4 neutropenia lasting more than 72 hours or immediately in case of febrile neutropenia (MTD 2); granulocyte colony-stimulating factor was never used prophylactically. To date, 22 patients have been entered into the study; the median patient age was 55 years (age range, 30 to 66 years). Nineteen (86%) patients had received adjuvant chemotherapy that included anthracyclines in 12 cases (55%). The viscera were the dominant sites of disease in 55% of patients. Median baseline ventricular ejection fraction was 58% (range, 53% to 67%). Short lasting grade 4 neutropenia occurred in 61% of courses; however, only four episodes of febrile neutropenia were recorded. Grade 4 thrombocytopenia was reported in 8% and grade 3 anemia in 3% of courses; four patients experienced peripheral neuropathy (three patients grade 1, one patient grade 2); complete alopecia was universal. The cardiac effects of the combination were surprisingly low: median ejection fraction at study entry was 58%, and after a cumulative dose 1,080 mg/m2 it was 56%. Three complete responses and 12 partial responses have been documented for an overall response rate of 83.3% (95% confidence interval, 58% to 96%). In conclusion, neutropenia is the most frequent toxicity of this novel combination. However, the MTD has not yet been reached. The combination of epirubicin plus paclitaxel is highly active, and no signs of cumulative myocardiopathy have been observed. PMID- 8629034 TI - Interim Analysis of a Phase II study of epirubicin and paclitaxel as first-line therapy in patients with metastatic breast cancer. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), the first taxane to be used routinely in clinical practice, has aroused considerable interest for its high single-agent activity in breast cancer and its novel mechanism of action. The 4' epimer of doxorubicin, epirubicin is an agent with high activity against breast cancer but a lower rate of toxic side effects, especially cardiotoxic events, than its parent compound. Although the paclitaxel/doxorubicin combination has yielded response rates between 63% and 94% in phase I/II studies, some severe cardiotoxic events were reported. The rationale for our study was to evaluate the paclitaxel/epirubicin combination, focusing mainly on cardiotoxicity. In all, 57 patients with metastatic breast cancer entered the study, 28% of whom had primary metastatic breast cancer with large tumors at the primary site. Half of the patients had received adjuvant chemotherapy. Study medication consisted of 60 mg/m2 epirubicin given intravenously over 1 hour, followed by paclitaxel 175 mg/m2 administered as a 3-hour intravenous infusion after premedication with steroids, antihistamines, and H2 antagonists. The main toxicity was neutropenia (World Health Organization toxicity index grade 3/4, 72%). Other hematologic side effects were rare and no febrile neutropenia was reported. Peripheral neuropathy, arthralgia, and myalgia were mild (only World Health Organization grade 1 and 2). All patients had alopecia. The paclitaxel dose was escalated to 200 mg/m2 in eight patients, four of whom received a further escalation to 225 mg/m2. Severe neutropenia necessitated dose reductions in eight patients. No cardiac adverse events were reported. Of 41 patients evaluable for response, seven had complete remissions and 21 had partial remissions (68%). An additional 12 patients (29%) had stable disease. The combination of paclitaxel 175 mg/m2 and epirubicin 60 mg/m2 can be administered safely to patients with metastatic breast cancer. Although response was not the primary end point of this trial, the response data are nonetheless encouraging and suggest that further evaluation of the role of this combination in the first line treatment of metastatic breast cancer is warranted. PMID- 8629035 TI - Paclitaxel couplets with cyclophosphamide or cisplatin in metastatic breast cancer. AB - Determining active combinations containing paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) to treat metastatic breast cancer has been the focus of recent clinical development. Paclitaxel combined with either cyclophosphamide or cisplatin has several potential advantages: cisplatin and cyclophosphamide are active single agents against previously untreated metastatic breast cancer, colony-stimulating factors can modulate overlapping toxicities like myelosuppression, and no mechanisms of cross-resistance between paclitaxel and these agents are yet known. Major questions include the optimal schedule of administration and the sequence dependence of toxicities with these combinations. Paclitaxel schedules with cisplatin include either two dose levels using the 24 hour infusion or a novel biweekly 3-hour infusion. The sequence in the three available studies was paclitaxel followed by cisplatin. Hematologic toxicities were dose limiting with the biweekly and low-dose 24-hour paclitaxel/cisplatin combinations; with granulocyte colony-stimulating factor, neurotoxicity became a prominent cumulative toxicity of the high-dose paclitaxel/cisplatin combination. Response rates in the first-line treatment of metastatic breast cancer ranged from 49% to 85%. In the three completed studies with cyclophosphamide, paclitaxel has been administered over either 72, 24, or 3 hours. Paclitaxel followed by cyclophosphamide had greater hematologic toxicity than the opposite schedule or concurrent administration. Pharmacokinetic factors do not seem to account for this sequence-dependent toxicity. As expected, dose-limiting toxicity in all studies has been hematologic. However, granulocyte colony-stimulating factor has ameliorated myelosuppression and allowed considerable dose escalation of cyclophosphamide. This combination has demonstrated activity in previously treated patients with metastatic breast cancer, including the anthracycline refractory subpopulation that will be reviewed. PMID- 8629036 TI - Single-agent paclitaxel for the treatment of breast cancer: an overview. AB - Initial trials using a 24-hour intravenous infusion of 250 mg/m2 paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in the treatment of breast cancer yielded objective regression in 56% to 62% of patients with no or only one prior chemotherapy regimen. Tolerance to single-agent paclitaxel seemed acceptable, but after multiple cycles, peripheral neuropathy developed in a significant fraction of patients. Lower doses and, more recently, the 3-hour infusion schedule still produced objective responses, albeit lower, in the range of 20% to 35%. It became apparent that toxicity was dose and schedule dependent, and likely there was a dose-response correlation. A 96-hour infusion schedule yielded a maximum tolerated dose of 140 mg/m2, and no hypersensitivity reactions despite omission of the standard triple-drug premedication. More recently, a 1 hour infusion schedule (plus standard triple-drug premedication) was well tolerated, with activity in both lung and breast cancer similar to that observed after a 3-hour infusion treatment. Paclitaxel retained therapeutic activity, even among patients with anthracycline-refractory breast cancer, in clinical trials using the 3-, 24-, and 96-hour infusion of paclitaxel. Current ongoing trials will explore the range of paclitaxel activity of various doses and by alternative schedules, both as second-line therapy and in the adjuvant and neoadjuvant setting. PMID- 8629037 TI - Preclinical and clinical study results of the combination of paclitaxel and 5 fluorouracil/folinic acid in the treatment of metastatic breast cancer. AB - Results of phase II studies have demonstrated high efficacy and low toxicity for a weekly schedule of high-dose 5-fluorouracil/folinic acid (5-FU/FA) when given to intensively pretreated patients with metastatic breast cancer. In a phase I/II study of outpatients, we have added paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) to this regimen in an attempt to improve the results. Patients were treated weekly for 6 weeks (days 1, 8, 15, 22, 29, and 36) with high-dose 5-FU 2.0 g/m2 by 24-hour infusion with FA 500 mg/m2 given as a 2-hour infusion prior to 5-FU. Paclitaxel 175 mg/m2 by 3-hour infusion was administered on days 1 and 22 prior to 5-FU/FA. Each cycle comprised 6 weeks followed by 2 weeks rest; the number of cycles depended on response and toxicity. To date, 40 patients have been entered into this trial during phase II. Pretreatment included adjuvant chemotherapy among 12 patients, prior chemotherapy for metastasis in nine patients, and both adjuvant therapy and treatment for metastasis in 19 patients. Of 24 anthracycline-pretreated patients, 20 had anthracycline-resistant disease. The observed toxicities were of mild to moderate intensity, especially with regard to myelosuppression. Thirty-four patients have been evaluable for response. Response rates included 3% complete response (one of 34 patients) and 50% partial response (17 of 34 patients) for an overall response rate of 53% (95% confidence interval, 37% to 69%). Additionally, 41% (14 of 34 patients) had stable disease and 6% had progressive disease (two of 34 patients). Among 20 anthracycline-resistant patients, 55% responded (11 of 20 patients). The median number of treatment cycles per patient was three (range, one to five); time to maximum response, 2 months (range, 1 to 5 months); and remission duration, 9 months (range, 2 to 17 months). Median time to progression was 10 months (range, 3 to 17 months). In conclusion, the combination of paclitaxel with weekly high dose 5-FU/FA is well tolerated as second-line treatment of metastatic breast cancer, with high activity, even in patients with anthracycline-resistant disease. The regimen can be administered safely on an outpatient basis. PMID- 8629038 TI - Paclitaxel and 5-fluorouracil in metastatic breast cancer: the US experience. AB - Paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) is an effective drug in the treatment of metastatic breast cancer (MBC). In the salvage setting, 5-fluorouracil (5-FU) and folinic acid have proved to be effective against MBC as well. Recent preclinical data suggest that paclitaxel plus 5-FU has additive cytotoxicity. Given these observations, we initiated a phase II trial in which 38 women with MBC have been treated with a combination of all three drugs. All patients are currently evaluable for toxicity and 34 are evaluable for response. All women had histologically proven and assessable disease. Patients with prior exposure to paclitaxel were ineligible. Patient characteristics include a median age of 51 years (age range, 31 to 73 years) and a median performance status of 1 (range, 0 to 2). Thirty-three patients have received prior chemotherapy, of whom 23 had adjuvant chemotherapy only. Fifty-eight percent of the patients (22 of 38) had received prior doxorubicin or mitoxantrone; four patients had only hormonal therapy. Four patients had bone-only disease, and three patients had lymphangitic spread or cytologically positive pleural effusion as the only evaluable disease. Treatment consisted of paclitaxel 175 mg/m2 over 3 hours (day 1 only), followed by folinic acid 300 mg over 1 hour, followed by 5-FU 350 mg/m2 on days 1 to 3. Patients received standard paclitaxel premedications. To date, 175 cycles have been administered (median cycle length, 29 days; median number of cycles per patient, five). Toxicities included grade 3/4 infections in nine cycles (5%), grade 3/4 mucositis in three cycles, grade 3/4 nausea/vomiting in three cycles, grade 1 paresthesias in 12 patients (32%), alopecia 100%, and 17 cycles (10%) associated with dose reduction. Based on Cancer and Leukemia Group B toxicity criteria, arthralgia/myalgias were modest and graded mild (32 cycles), moderate (nine cycles), or severe (two cycles). There were two major hypersensitivity reactions, prompting removal of those patients from further protocol treatment. Four patients are unassessable for response due to hypersensitivity reactions (two) and unevaluable disease (two). Among the 34 patients evaluable for response, there were three complete responses, 18 partial responses, one minor response, nine stable disease, and three progressive disease (response rate, 62%). Responses were seen in patients who had received prior doxorubicin or mitoxantrone (11 of 22 patients) and in anthracycline/naive patients (10 of 16 patients). Responses were observed in all metastatic sites: soft tissue, viscera, and bone. Paclitaxel/5-FU/folinic acid appears to be an effective and well tolerated outpatient regimen for women with MBC, even after failure of anthracycline-containing therapy. PMID- 8629039 TI - Paclitaxel-containing combination chemotherapy for metastatic breast cancer. AB - After demonstration of the marked antitumor activity against metastatic breast cancer of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), other clinical trials explored the possibility of combining this new active agent with other cytotoxic drugs with proven efficacy against breast carcinoma. Paclitaxel plus doxorubicin, thought to be the most effective single agents against breast cancer, yielded remission rates ranging from 60% to 80%, including some complete remissions. Schedule-dependent toxic interactions were observed when paclitaxel preceded the administration of doxorubicin. Paclitaxel by 3-hour infusion plus doxorubicin by bolus proved to be a highly tolerable regimen, with overall remission rates in excess of 90% and complete remission rates approaching 50%. A paclitaxel plus cisplatin combination has been studied at numerous schedules and doses with variable activity and tolerability, although one group in Vancouver, Canada, reported an 85% overall response rate with the combination administered on a 14-day schedule in previously treated patients, most of whom had received doxorubicin. Paclitaxel also has been combined with cyclophosphamide, mitoxantrone, edatrexate, 5-fluorouracil, and other agents for the treatment of breast cancer. Of interest are recent reports on paclitaxel and vinorelbine, showing this combination to be clearly active, with good tolerability and rapid recovery after myelosuppression. Trials of this combination are ongoing with granulocyte colony-stimulating factor support, on an every-14-day schedule. The doxorubicin/paclitaxel doublet remains the most promising in terms of activity, although other combinations with a high degree of activity and good tolerance are being sought. PMID- 8629040 TI - Sequential adjuvant therapy: the Memorial Sloan-Kettering Cancer Center experience. AB - Adjuvant chemotherapy has a real but modest impact on the disease-free and overall survival of patients with breast cancer. Recent attempts to improve its effectiveness have focused on dose intensity and new agents. Sequential therapy maximized dose intensity while limiting overlapping toxicity. Sequential therapy using doxorubicin followed by cyclophosphamide/methotrexate/5-fluorouracil (CMF) has been found superior in patients with high-risk resectable breast cancer. The novel chemotherapy agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) is now known to be highly active in advanced breast cancer and appears to be clinically non-cross-resistant with doxorubicin. Therefore, this drug is being studied as a component of the next generation of adjuvant chemotherapy regimens. The most appropriate way to incorporate paclitaxel has not yet been defined, but its concurrent administration with other agents has, in some cases, been troublesome. Based on the demonstrated advantage of the sequential plan for doxorubicin and CMF, we conducted a series of pilot trials testing sequential high-dose therapy. Initially, we studied multiple cycles of doxorubicin followed by cyclophosphamide; we later added paclitaxel to this regimen. These phase II studies demonstrate the feasibility of sequential therapy with doxorubicin, paclitaxel, and cyclophosphamide, and early disease-free survival results are promising. Cooperative group projects are under way or planned to further define the activity of these regimens. PMID- 8629041 TI - Future developments for paclitaxel in the treatment of breast cancer. AB - Several clinical studies worldwide have demonstrated clearly the remarkable activity of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in treating metastatic breast cancer. Future directions should focus on two strategic points: the optimal combination of paclitaxel with other cytotoxic drugs and the most appropriate time to administer a paclitaxel-containing combination. The Milan experience using doxorubicin plus paclitaxel indicated an objective response rate of 95% (complete remissions, 41%) among chemotherapy naive patients with disseminated breast cancer. This very high response rate was recently confirmed in a study involving 12 women with primary inoperable disease (stage IIIB). To avoid cardiac effects, only four treatment cycles were used; the excellent tumor shrinkage was followed by mastectomy. Since no signs of myocardial toxicity were detected, we are currently testing the same drug schedule in women with smaller breast tumors (eg, 2.5 to 5.0 cm largest diameter) in an attempt to increase the frequency of pathologic complete remission following breast-conserving surgery. Thus, future research programs should exploit the efficacy of paclitaxel plus doxorubicin (or epirubicin) in the early stages of breast cancer. In turn, such studies should enable research physicians to build up more effective strategies with preoperative (neoadjuvant) and postoperative (adjuvant) breast cancer treatments. PMID- 8629042 TI - Substance abuse and the domestic assault of women. AB - Social workers often meet with practice situations, sometimes unknowingly, where domestic assault coincides with substance abuse by the batterer or victim. Knowledge for practice in this complex area is both minimal and remote. This article examines current research on the involvement of substance abuse in woman abuse and discusses issues of assessment and intervention. PMID- 8629043 TI - Suicide risk among people living with AIDS. AB - Using data from Louisiana vital statistics records, the authors found an increased suicide risk for people with AIDS. Between 1987 and 1991 the suicide rate of people with AIDS (175 per 10,000) was 134.6 times that of the general population (1.3 per 10,000). The suicide rate for people with AIDS in the metropolitan New Orleans area (126 per 10,000) was half that of the state's nonmetropolitan areas (249 per 10,000). This article explores the implications of this astounding suicide rate for social work practice. PMID- 8629044 TI - Substance-abusing women: false stereotypes and real needs. AB - Substance-abusing women are a diverse group, but some of them are among the most disadvantaged individuals in the United States. This article reviews and interprets some recent literature on substance abuse problems and treatments among women. Contrary to popular stereotypes, alcohol and drug abuse among women occurs at similar rates among poor and nonpoor people and among white people and people of color. Major risk factors include childhood sexual or physical abuse, adult victimization by domestic violence, and a spouse or partner who abuses substances. Standard treatment programs are based on male processes of recovery, and there are enormous problems of access to treatment for women with minor children. Although most studies have found that white women and women of color use substances during pregnancy at similar rates, women of color are disproportionately tested for drug use and receive more child protective services interventions. The findings discussed in this article indicate that prevention of substance abuse-related problems among women requires more than just education. PMID- 8629045 TI - HIV in health care workers: managing fear through a telephone information line. PMID- 8629046 TI - The model illusion in health care. PMID- 8629047 TI - In vivo study of the response of Plasmodium falciparum to standard mefloquine/sulfadoxine/pyrimethamine (MSP) treatment among gem miners returning from Cambodia. AB - An in vivo study of the response of P. falciparum to the combination drug, MSP, was conducted among gem miners who contracted malaria from Cambodia in 1991-1992. High level resistance (RII, RIII responses) was observed in 22.5% of the 40 cases attending Mae Sot malaria clinic, west Thailand border, and in 28.1% of the 96 cases attending Bo Rai malaria clinic, east Thailand border. The observations on in vitro studies conducted prior to the MSP treatment and after recrudescence, together with the findings on adequate mefloquine blood levels strongly indicated the serious deterioration of mefloquine efficacy. The first line treatment for the malaria control program needs to be revised and the use of qinghaosu derivatives considered. Intensive measures to combat spreading of the highly resistant strains to other parts of the country should be taken into account. PMID- 8629048 TI - Behaviors in self-prevention of malaria among mobile population in east Thailand. AB - The study investigated preventive behaviors among mobile villagers in Chanthaburi and Trat Provinces where the slide positive rates (SPR) of malaria in 1987 were 10.3 and 11.1%, respectively. The study was conducted in four villages namely Wang Thong, Khao Thong in Chanthaburi, and Manow and Khow in Trat by using a cross-sectional survey. Household and in-depth interviews with the movers were done together with field observations. The sizes of the villages were 52, 180, 184 and 209 households and the movement rates were 23.1, 12.2, 37.5 and 28.7%, respectively. The population involved in short term migration was 729 villagers living in 153 households or 23.7% of the total. Malaria prevalence rates in the four villages were 7.5%, 5.9%, 7.3% and 2.2%, respectively. The results indicate that the mobile population had a moderate 40% rating score of knowledge about malaria. Apparently, the rating score of preventive behaviors of the disease appeared to be as small as 20%. After six months of field investigations, 1,083 thick blood smears collected from the mobile population, there were 657 episodes (60.7%) of malaria that could be categorized with age specific prevalence of 60.3% in 19-29 years, 80.8% in 30-39 years, 54.8% in 40-49 years, 38.6% in 50-59 years and 35.7% in 60 years and over. Villagers aged between 30-39 years with frequent movement into the forest had the highest impact of malaria risk. Moderate levels of knowledge and attitude scores did not appear to enable the people to protect themselves against malaria. PMID- 8629049 TI - Humoral immune responses to the Plasmodium falciparum antigen Pf155 RESA in adults with differential clinical conditions from an Indian zone where malaria is endemic. AB - Ring infected erythrocyte surface antigen of Plasmodium falciparum (Pf155/RESA) has been considered as a vaccine candidate. However, the relative immunogenicity of this antigen has not been studied in Indian populations. Pf155/RESA was investigated for its immunogenicity by studying humoral immune responses against Pf155/RESA and Pf155/RESA derived peptides (P1, P2 representing immunodominant epitopes from the 3' and P3 from the 5' repeat regions) by erythrocyte membrane immunofluorescence (EMIF) assay and by enzyme linked immunosorbent assay (ELISA) in P. falciparum primed donors living in hyperendemic malarious areas (Orissa State, India) where P. falciparum infections are highly prevalent. Subjects of different clinical status namely acute (A), clinically immune (CI) and acute with history of repeated P. falciparum infections (R) were included in this study. All the donors were seropositive against the crude antigen. There was considerable variation in the responses among the donors. While humoral responses in the plasmas against the P2 peptide (EENV)4 were significantly higher in magnitude and in frequency in the CI donors than in the A donors, no positive response was seen in the R donors. The responses to the peptides P1 (EENVEHDA)2 and P3 (DDEHVEEPTVA)2 were poor both in the A and in the CI groups. Whereas, most of the R donors were seropositive against the P3. The present results indicate that Pf155/RESA contains B cell epitopes which were recognized differently by the immune system of individuals living in malaria-hyperendemic areas of India who have been primed by natural infection. Our studies also suggest that in order to investigate the possible functional role of a given antigen, study of immune responses against the antigen in donors of different clinical status may be useful. PMID- 8629050 TI - Susceptibility of Plasmodium falciparum to chloroquine in tea garden tribes of Assam, India. AB - P. falciparum was the predominant parasite ( > 80%) species in malaria ridden tea estates of Assam. A simplified 3 day in vivo test to determine chloroquine sensitivity in tea garden tribes revealed that the majority of the cases (85%) were S/RI, 7% were RI, and 3% were RII and 5% were RIII, respectively. Early case detection and treatment were deemed necessary to reduce morbidity and mortality due to P. falciparum in these tea estates. PMID- 8629051 TI - Establishment and characterization of cell lines from liver fluke-associated cholangiocarcinoma induced in a hamster model. AB - Cholangiocarcinoma (CCA) is a relatively rare tumor that occurs primarily in tropical countries and particularly in those with a high incidence of liver fluke infection. A hamster model for a liver fluke-associated CCA has been described previously. In the present study, hamster cholangiocarcinoma cell lines were established and characterized in order to obtain information regarding diagnostically useful tumor marker which could shed light for a future investigation for human cholangiocarcinoma. Two related cell lines, one from the original intrahepatic bile duct tumor and one from an allotransplanted tumor, were established. The established cell lines were found to have population doubling times of 31 and 26 hours respectively, and were maintained in Ham's F12 medium supplemented with 10% fetal bovine serum for over 80 passages. The cell monolayers were subjected to scanning and transmission electron microscopic study and found to have ultrastructural characteristics, including cytoplasmic lumens, consistent with those of adenocarcinoma cells of epithelial origin. An immunoperoxidase study using monoclonal antibodies (MAbs) specific for tumor antigens showed the cytoplasm and membrane of both cell lines to be positive. These antigens were also secreted in soluble form into the culture medium, judging from polyacrylamide gel electrophoresis in the presence of SDS and from immunoblot analyses. Different lines of evidence presented suggested that a 200 kDa glycoprotein produced and secreted by the tumor cell lines could be considered a cholangiocarcinoma-associated marker which has diagnostic potential. PMID- 8629052 TI - alpha 1-antitrypsin phenotype PiMZ, a risk factor for liver cirrhosis but not for liver cancers in Thailand. AB - The risk of developing liver cirrhosis, hepatoma (HCC) and bile duct carcinoma (BDC) have been associated with homozygous alpha 1-antitrypsin (AT) deficiency especially linked to the Z allele. While the association between liver cancers and AT deficiency remains debatable, the risk of adult AT deficiency carriers to develop liver cirrhosis has not been assessed quantitatively. Liver cancers and liver diseases with subsequent liver cirrhosis are highly prevalent in tropical countries such as Thailand and heterozygous AT phenotypes are rather common in this country as well. The aim of this study was to assess the risk of developing liver cirrhosis, HCC and BDC by means of case-control studies with Thai patients and controls in connection with AT deficiency. For hepatitis, HCC and BDC to association with AT deficiency was detected. Carriers of PiMZ phenotype in Thailand have a high risk to develop liver cirrhosis (odds. ratio of 10.8, 95% confidence interval = 1.3-88.1). Patients with predisposing diseases should be screened for Pi phenotypes so that rigorous measures to combat the occurrence of liver cirrhosis can be implemented. PMID- 8629053 TI - Prophylactic treatment for hemophilia A patients: a pilot study. AB - Prophylactic treatment with factor VIII concentrate was given to six hemophilia A boys whose factor VIII:C ranged from 1% to 3.5% at Ramathibodi Hospital. The age ranged from 11 to 16 years with the median age of 12 years old. Each patient received factor VIII concentrate twice a week in the dosage of 8-10 unit per kg for one year. During the prophylactic period, bleeding episodes seldom occurred. They did not need hospitalization. The absence from school was reduced. They became muscular from regular daily exercise. They could join the activity at school and lived a near normal life. The patients and family were very happy since they did not have to worry about bleeding. No adverse effect was found. The only constraint was the cost. It cost 180,000 baht (US$ 7,200) per year or 15,000 baht (US$ 600) per month for a 25 kg hemophiliac boy. PMID- 8629054 TI - Detection of Mycobacterium tuberculosis in sputum, pleural and bronchoalveolar lavage fluid using DNA amplification of the MPB 64 protein coding gene and IS6110 insertion element. AB - Two gene sequences specific for Mycobacterium tuberculosis were evaluated for the diagnosis of pulmonary tuberculous (PTB) in pleural fluid (PF), bronchoalveolar lavage fluid (BAL) and sputum (Sp). The 240 bp sequence (nts 460-700) coding for the MPB 64 protein coding gene and the 123 bp IS6110 insertion element present in multiple copies in the mycobacterial genome were amplified using the polymerase chain reaction. Fifty-nine clinical specimens were studied. The diagnosis of PTB was confirmed by positive M. tuberculosis cultures in 14 specimens, and by the presence of characteristic histological features of granuloma and Langerhan's giant cells on pleural biopsy in 3 PF specimens through cultures for M. tuberculosis were negative. The remaining 42 specimens were obtained from patient's with non-tuberculosis pulmonary infections or malignancy, and these served as negative controls. Our results showed that the IS6110 insertion element and MPB 64 gene sequence were detected in all 14 culture positive PTB cases, although detection of the latter sequence required both DNA amplification and oligonucleotide hybridization. There was however one false positive specimen with the MPB 64 detection protocol. More importantly, both the MPB 64 sequence and IS6110 insertion element protocols were unable to detect M. tuberculosis DNA in the 3 PF samples diagnosed by histological characteristics on pleural biopsy and culture negative. We conclude that DNA amplification for M. tuberculosis-specific sequences is a useful method for rapid diagnosis of PTB in culture positive specimens. However, the false negative results with TB culture negative cases of tuberculosis pleurisy, limits its usefulness for the diagnosis of tuberculous pleurisy. PMID- 8629055 TI - PCR to detect Mycobacterium tuberculosis DNA in sputum samples from treated leprosy patients with putative tuberculosis. AB - In this study of leprosy patients with putative tuberculosis, the polymerase chain reaction (PCR), one of the most reliable and sensitive molecular diagnostic methods, was carried out for the specific detection of Mycobacterium tuberculosis DNA. Sputum samples from 43 patients at Baba Baghi Leprosarium in Iran were tested. The DNA extraction method was based on the lysing and nuclease inactivating properties of guanidinium thiocyanate (GuSCN) together with the nucleic acid binding properties of diatoms or silica particles. Primers for a 123 base pair (bp) fragment of the repetitive DNA sequence of M. tuberculosis were used for the PCR assay. The results of PCR were compared with direct microscopy and culture. In total, 14% of the patients in this study were found to be PCR positive for M. tuberculosis. No positive results were found by direct microscopy for acid fast bacilli (AFB) and culture. It was thought probable that the positive PCR results indicated the tuberculosis (TB) in such treated leprosy patients. PMID- 8629056 TI - Comparison of multiplex PCR and culture for detection of Legionellae in cooling tower water samples. AB - We compared multiplex polymerase chain reaction (PCR) and culture for detecting the presence of Legionella pneumophila and Legionella spp in cooling tower water samples. Multiplex PCR was performed after phenol extraction of DNA from the samples. The set of primers for the PCR assay involved the 5S rRNA (Legionella spp) and the mip (macrophage infectivity potentiator gene, specific for L. pneumophila) genes as target sequences for amplification. Both the sensitivity and the specificity of the PCR assay were 100% when the 5S rRNA gene was used as target sequence. Isolation of Legionellae from the samples was observed only with the PCR-positive samples. We propose that PCR be used as a screening test before attempting to culture Legionellae from cooling tower water samples. PMID- 8629057 TI - Evaluation of WHO monoclonal antibody kit for diagnosis of acute respiratory viral infections. AB - In 1990 and 1991, six laboratories located in the WHO Western Pacific Region (WPR) and South East Asian Region (SEAR) were selected, based on their experience in the immunofluorescence antibody technique (IFAT), to participate in the evaluation of a WHO monoclonal antibody (Mab) kit to detect respiratory syncytial (RS) virus, influenza A virus, influenza B virus, parainfluenza virus and adenovirus. Despite differences in the initial standardization procedures, the WHO monoclonal antibodies were found to be of high quality, sensitivity and specificity when tested on clinical specimens. The constant supply of affordable high quality reagents from WHO would enable their use in clinical virological laboratories in the developing countries as well as promote the utilization of IFAT as an adjunct to cell culture isolation in the diagnosis of acute respiratory viral infections. PMID- 8629058 TI - Immunogenicity and safety of an inactivated hepatitis A vaccine amongst Singaporeans. AB - The immunogenicity and reactogenicity of an inactivated hepatitis A virus (HAV) vaccine was studied in healthy Singaporean adult volunteers. One hundred and forty healthy volunteers with normal alanine (ALT) and aspartate (AST) transaminases and no previous exposure to HAV, received three 1 ml doses (720 ELISA units) of an inactivated HAV vaccine (Smithkline Beechams Biologicals) following a 0, 1, 6 months vaccination schedule. All subjects were asked to record and grade the severity of any reactions for three consecutive days after each dose. Serum ALT and AST as well as anti-HAV were measured at 0, 1, 2, 6 and 7 months after the first vaccine dose. Anti-HAV seroconversion occurred when levels rose above 40 mIU/ml. Eighty-five percent of vaccinees seroconverted after the first innoculation and 99% after the second injection. All vaccinees seroconverted after the third dose. Geometric mean anti-HAV titers (GMTs) were, respectively, 119, 391, 4406 mIU/ml one month after each of the three doses. The most common side effect was transient pain and tenderness at the vaccination site. No elevation of ALT or AST levels were noted during the study period. The inactivated hepatitis A vaccine used in this study is safe and highly immunogenic in the local adult population. Two doses one month apart appeared to give adequate protection. PMID- 8629059 TI - Japanese encephalitis virus is an important cause of encephalitis among children in Penang. AB - This study was carried out to determine if Japanese encephalitis virus is an important causative agent of viral encephalitis among pediatric admissions in Penang, Malaysia. 195 children with CNS symptoms and 482 children with non specific febrile illness admitted into the Pediatric Ward of Penang Hospital during a 16 month period were entered into the study. The presence in serum of cerebrospinal fluid (csf) of Japanese encephalitis virus (JEV) specific IgM was determined by an IgM capture ELISA and cytomegalovirus (CMV) specific IgM was determined using a commercially available kit (Behringwerke AG). It was determined that 5 of 13 children with a discharge diagnosis of viral encephalitis had JEV specific IgM in csf, indicating that 38.5% of the viral encephalitis cases was due to JEV. One of the non-JEV cases was found to have mumps virus specific IgM in csf, while no etiology was determined for the other cases. It was also determined that 4 of the 195 (2.1%) cases with CNS symptoms had IgM to CMV, suggesting CMV may be an agent of encephalopathy in children in Penang. Other viruses found to be associated with CNS symptoms in children admitted into our study were measles and herpes simplex virus. A viral etiology was confirmed for 13 or the 195 cases (6.7%). We also screened 482 non-specific febrile cases for IgM to JEV and to dengue viruses and found that 2 (0.4%) had IgM specific for JEV and 9 (1.9%) had IgM specific for dengue virus. PMID- 8629060 TI - Socio-economic status and micro-environmental factors in relation to the risk of Japanese encephalitis: a case-control study. AB - In a population-based case-control study in Southern Henan Province, central China, children suffering from Japanese encephalitis (JE) were compared with neighborhood controls matched by age and sex in terms of several social and environmental variables. Factors found by crude analysis to be associated with an increased risk of JE included lower family income, lower parental education, living in houses near the periphery of villages and poor quality of houses. When adjustment was made for confounding variables, only the association of house location within village remained of borderline significance (OR = 0.51, 95% CI = 0.15 approximately 1.03). It is suggested that the beneficial effect of higher family income and parental education was partly due to the fact that those parents might be more conscious about having their children vaccinated in the situation where there was a shortage of JE vaccine in the study area. PMID- 8629061 TI - Protective and risk factors for acute respiratory infections in hospitalized urban Malaysian children: a case control study. AB - We performed a case control study to examine protective and risk factors for acute respiratory infections (ARI) in hospitalized children in Kuala Lumpur. Consecutive children between the ages of one month and five years hospitalized for pneumonia (n = 143), acute bronchiolitis (n = 92), acute laryngotracheobronchitis (n = 32) and empyema (n = 4) were included as cases and were compared with 322 children hospitalized during the same 24 hour period for non-respiratory causes. Potential risk and protective factors were initially analysed by univariate analysis. Logistic regression analysis confirmed that several home environmental factors were significantly associated with ARI. The presence of a coughing sibling (OR = 3.76, 95%CI 2.09, 6.77), a household with more than five members (OR = 1.52, 95%CI 1.03, 2.19) and sleeping with three other persons (OR = 1.45, 95%CI 1.00, 2.08) were independent risk factors. Significant host factors were history of allergy (OR = 2.50, 95%CI 1.74, 3.61) and ethnicity (Malay race) (OR = 2.07 95%CI, 1.27, 3.37). Breast feeding for at least one month was confirmed as an independent protective factor (OR = 0.58, 95%CI 0.38, 0.86). However, the study was not able to demonstrate that domestic air pollution had an adverse effect. This study provides further evidence that home environmental factors, particularly those associated with crowding, may predispose to ARI and that breast feeding is an important protective factor. PMID- 8629062 TI - Childhood malnutrition: an analysis of the effects of nutritional advice. AB - An analysis of malnutrition was done in 65 infants and preschool children (18 boys and 47 girls) who were under the third percentile of weight for age. Fifty seven percent of cases has hematocrits of less than 36%, 7% had hematocrits of less than 30% and two had iron deficiency anemia which improved after iron supplement. Eosinophils of more than 400 cells/mm3 were found in 35% of cases. Eleven percent had eosinophils of more than 1,000 cells/mm3. Parasites were found on stool examination in 12.5% of cases. Bone development was retarded in 39% of 23 cases. In 7 cases with bone development delayed more than 6 months, thyroid function and trace elements were analysed and found to be within normal limits. In 5 cases with delayed bone development and height less than 5 cm/year, growth hormones showed normal levels. Proper nutritional advice resulted in improvement in body weight and height in 57% of cases, tricept skin fold in 73%, bicept skin fold in 60%, arm muscle area in 50% and arm fat area in 29% of cases. Improvement was not associated with family income or education of the people who cared for the patients. PMID- 8629063 TI - Smokeless tobacco use among rural Kadazan women in Sabah, Malaysia. AB - A survey was conducted to document and bring attention to the use of smokeless tobacco among rural Kadazan women in Sabah, East Malaysia. Of the 472 women interviewed, 59.5% had used tobacco among the ingredients that they habitually chewed. Women with low education were more likely to be chewers. The chewing habit was usually acquired during the teenage years and the practice was perceived mainly as a cultural norm. 73.3% of these smokeless tobacco users were unaware of any adverse health effect of this type of tobacco use as compared to 53.9% of the non-tobacco users. The high prevalence of smokeless tobacco use is easily maintained as tobacco is cheap, locally produced and its use is socially accepted. The low level educational status of the women compounds the problem and intervention programs to curb this form of tobacco use is warranted. PMID- 8629064 TI - An evaluation of the future role of non-governmental organizations currently engaged in leprosy control in India. AB - The mass implementation of short term multi-drug therapy in India, has led to dramatic falls in the prevalence of leprosy. This paper addresses the future role of non-governmental organizations currently involved in leprosy control. This evaluation is based on current trends in leprosy control, projected health needs in the future and the necessity to maximize health care outputs in the face of limited resources. PMID- 8629065 TI - A preliminary study of filariasis related acute adenolymphangitis with special reference to precipitating factors and treatment modalities. AB - Episodic adenolymphangitis (ADL) is one of the important clinical manifestations of lymphatic filariasis. Recurrent ADLs contribute to the progress of the disease and also have important socioeconomic implications since they cause significant loss of man days. The present study was conducted in order to identify the precipitating factors responsible for ADL attacks and also to examine the different modalities of treatment. Sixty-five individuals with filariasis related ADL attacks, who are residents of Alleppey district (endemic for Brugia malayi) were studied. All efforts were taken to identify the precipitating factors for ADLs in these individuals. They were hospitalized for a period of five days or more. All of them received symptomatic antipyretic/antiinflammatory therapy and topical antibiotic/antifungal treatment of the affected limbs. They were then randomly allocated to one of the following four regimens: group I - symptomatic alone; group II - symptomatic plus antibiotics; group III - symptomatic followed by diethylcarbamazine citrate (DEC) and group IV - symptomatic plus antibiotic followed by DEC. Patients in groups III and IV received DEC every three months up to one year. There was a significant relationship between the number of ADL attacks and the grade of edema. Presence of focus of infection in the affected limb could be identified in 28 of the 65 patients. In the majority of patients (48) response to treatment was rapid (resolution in less than five days). Neither antibiotics nor DEC (given at intervals of three months) appeared to alter the frequency of ADL attacks. On the otherhand simple hygienic measures combined with good foot care and local antibiotic/antifungal cream application (where required), were effective in reducing the number of ADL attacks. PMID- 8629066 TI - Experimental studies on early treatment of schistosomal infection with artemether. AB - An early treatment with artemether given in appropriate regimens was tested in mice, rabbits and dogs for prevention purposes. Artemether was administered intragastrically (ig) to the hosts on day 7 after infection with Schistosoma japonicum cercariae at a single dose, and the same dose of artemether was repeated every 1 or 2 weeks for 2-4 times. As a result, most of the female worms were killed before their oviposition with female worm reduction rates of 90-100%, resulting in protection of the host from damage induced by schistosome eggs. When rabbits were treated ig with artemether 10 mg kg-1 on day 7 after infection, followed by repeated dosing every week for 4 times, some parameters related to acute schistosomiasis, such as temperature, eosinophil count and eggs in the feces were negative, and low specific antigen and antibody levels in serum were seen. Further study showed that the appropriate regimens of Artemether were also effective in early treatment of reinfection with cercariae. When rabbits infected with 48-52 cercariae once every other day for 5 times were treated ig with artemether 15 mg kg-1, followed by repeated dosing every 1 or 2 week for 2- 3 times, the female worm reduction rates were 92.1-98.4%. Histopathological examination of the livers showed that the above-mentioned early treatment with Artemether exhibited a promising protective effect on dogs and rabbits. The major features included normal appearance of the liver resembling those of uninfected dogs and rabbits; few or no dispersed miliary egg tubercles appeared on the surface of the liver; the structure of the hepatic lobules was normal with normal arrangement of the liver bundles; few or no eggs appeared in the portal vein area and there was apparent diminution of total egg granuloma, comprising inflammatory, fibrous or scarred egg granuloma. On the basis of above-mentioned results, early treatment with Artemether could be recommended for field trial for controlling acute schistosomiasis, reducing infection rate and intensity of infection. PMID- 8629067 TI - Possible formation of new brood capsule by the previously formed brood capsule in Echinococcus multilocularis metacestodes. AB - In Echinococcus multilocularis metacestodes obtained from the peritoneal cavity of an experimentally infected jird, cellular accumulations were found not only on the inner surfaces of germinal layers but also on the outer surfaces of brood capsules. These cellular accumulations are believed to represent at incipient form of the brood capsule. It has been thought that brood capsules are produced by the germinal layer, but we speculate that the brood capsule itself, as well as the germinal layer, may have the potential to produce new brood capsules. PMID- 8629068 TI - Ascaris and Trichuris do not contribute to growth retardation in primary school children. AB - To access the effectiveness of the treatment of soil-transmitted helminthiasis (STH) on the growth of primary school children, 353 children were block stratified to receive either mebendazole plus pyrantel oxantel pamoate every three months or a placebo. The children were followed for two years with 89% completing the trial. Follow-up stools indicated that the treatment was efficacious for ascariasis and trichuriasis. There was virtually no hookworm infection. The children were malnourished as measured by the number below -2 SD of height and weight standards. There was no difference in height or weight between the treatment and control groups by sex initially or at the end of two years of follow-up. The treatment of Ascaris and Trichuris had no effect on growth parameters. The effect of STH on growth may be mediated through hookworm infections. PMID- 8629069 TI - Use of culture-filtrated antigen in an ELISA and a dot immunoassay for the diagnosis of melioidosis. AB - An enzyme-linked immunosorbent assay (ELISA) and a dot immunoassay with culture filtrated antigen were developed for detection of Burkholderia pseudomallei specific antibodies in melioidosis patients. Sixty-eight sera of bacteriologically confirmed melioidosis patients, 45 sera of other bacterial infected patients and 80 sera of healthy blood donors from endemic area were investigated. The samples were subjected to those assays im comparison with indirect hemagglutination (IHA). The sensitivity, specificity, positive and negative predictive values in this dot immunoassay were 94.1%, 99.2%, 98.5% and 96.9%, respectively, with cut-off dilution at 1:4,000, whereas those in ELISA were 92.6%, 96.8%, 94.0% and 96.0%, respectively, with cut-off value of OD = 0.47 at 490 nm. Meanwhile, those in IHA were 64.7%, 93.6%, 84.6%, 83.0% respectively, with a cut-off value of > or = 1:80. The results in this study demonstrated that the dot immunoassay was more reliable and rapid than ELISA as the serological test for diagnosis of melioidosis. PMID- 8629070 TI - Cryptosporidiosis in HIV infected patients in Thailand. AB - Diarrhea, mostly chronic diarrhea and weight loss are common in patients with AIDS. Cryptosporidium had been identified as responsible for chronic, debilitating secretory diarrhea in HIV infected patients. We performed a retrospective study of the prevalence, clinical features and laboratory findings of cryptosporidiosis in HIV infected patients (adults and children), in the period of 6 years from January 1988 to December 1993 at Bamrasnaradura Hospital in Nonthaburi, Thailand. In this study, Cryptosporidium was found in 22 (8.8%) by detection in stool specimens of 250 HIV infected patients with diarrhea and was found throughout the year. The prevalence rates of cryptosporidiosis in this study among children and adults were 19% and 7.9%, respectively. The common features were chronic diarrhea (84.6%), mostly watery diarrhea and weight loss/malnutrition (100%). A few fecal leukocytes were found in 15.4%. PMID- 8629071 TI - Chlamydia pneumoniae infection in Thai children: serological diagnosis. AB - Recent Chlamydia pneumoniae infections were investigated in children with respiratory tract infections and in normal children. Four groups of sera were tested for C. pneumoniae antibody IgG and IgM serum fraction by the method of MIF test. A total of 7 cases of recent infection were detected, 3 of 116 with pneumonia, 3 of 123 with other respiratory tract infections, 1 of 263 normal school children and none in sera from cord blood. The cases with recent C. pneumoniae infection were as young as 24 days and 2 months old. PMID- 8629072 TI - Widespread emergence of Vibrio cholerae 0139 in India. AB - The National Institute of Communicable Diseases (NICD) has been monitoring the incidence of laboratory confirmed cases of cholera in Delhi in collaboration with Infectious Diseases Hospital (IDH) since 1965. Cholera and cholera-like cases from all hospitals in Delhi are admitted in IDH and the rectal swabs of all such cases are processed for isolation of Vibrio cholerae at NICD laboratory. Since April 1993, there has been isolation of Vibrio cholerae serotype 0139, in increasing numbers (831 out of 2,830, 29.2%) The isolates have been characterized and enterotoxin studies carried out. As a referral laboratory NICD has also confirmed the causative role of Vibrio cholerae 0139 in diarrhea outbreaks from various parts of the country. The implications of establishment of this newer serotype of Vibrio cholerae, as a potential epidemic strain are discussed. PMID- 8629073 TI - Shigellosis in Thai children: experience from a rural hospital 1985-1993. AB - Six hundred and ninety-four cases of shigellosis in Nakhon Nayok Hospital from January 1985 to December 1993 were studied to determine epidemiologic and microbiological features. Forty-five percent of cases were children under the age of 14 years. The majority of cases were in children under the age of four. The organism was found throughout the year, with peak incidence in June and July. The most common type isolated was Shigella flexneri, about 74.43%. Only 0.32% of organisms were Shigella dysenteriae. Shigella isolates showed a high rate of resistance to ampicillin and co-trimoxazole, in 1993 only 16.67% and 22.22% were sensitive respectively to these 2 drugs, but 100% were still sensitive to nalidixic acid. Fewer cases of shigellosis were isolated in recent years possible due to widespread use of quinolones in the treatment of acute infective diarrhea in adults. PMID- 8629074 TI - Development and preliminary evaluation of an IgM dot-immunobinding assay (IgM DIA) for rapid serodiagnosis of scrub typhus infection. AB - An IgM dot-immunobinding assay (IgM-DIA) was developed for the diagnosis of scrub typhus infection. The whole cell antigens of Karp, Kato and Gilliam strains of Rickettsia tsutsugamushi were immobilized onto nitrocellulose paper and reacted with patients sera. The presence of IgM R. tsutsugamushi specific antibody in the patient sera could be detected by the observation of a visible brown dot on the nitrocellulose paper. The IgM-DIA has a sensitivity of 90.4% and specificity of 81.4% as compared to the indirect immunoperoxidase test. The IgM-DIA is rapid, simple, cost-effective, does not require microscope or incubator. It is recommended as a rapid screening test for the diagnosis of scrub typhus infection in the field or rural area within the hyperendemic region. PMID- 8629075 TI - The impact of permethrin impregnated bednets on the malaria vector Anopheles maculatus (Diptera: Culicidae) in aboriginal villages of Pos Betau Pahang, Malaysia. AB - The effect of permethrin impregnated bednets on Anopheles maculatus Theobald was studied in four villages in Pos Betau, Pahang, Malaysia from August 1990 to July 1992. Collections of mosquitos were carried out indoors and outdoors from 1900 to 0700 hours. All mosquitos were dissected for sporozoites and parity. In May 1991 two villages received bednets impregnated with permethrin at 0.5 g/m2 and two villages received placebo bednets. There was a significant difference in the sporozoite and parous rates between the treated and control villages after the distribution of bednets (p < 0.05). There was no significant difference in the bites/man/night of An. maculatus between the pre and post treatment periods in the control villages. However there was a significant difference in bites/man/night between pre and post treatment in the treated villages (p < 0.001). PMID- 8629076 TI - A report of Anopheles (Diptera: Culicidae) attracted to cow bait in a malaria endemic village in Peninsular Malaysia near the Thailand border. AB - Twelve species of Anopheles were collected by using cow-baited net trap in a malarial endemic village in northern Peninsular Malaysia. Anopheles maculatus which is the main malaria vector with its densities were related to drought. An. aconitus, An. kochi and An. philippinensis were less susceptible to P. falciparum and P. vivax infection, and are not considered important in falciparum or vivax malaria transmission. Biting activities of An. kochi and An. vagus were primarily active after dusk and steadily declined after midnight. An. maculatus and An. aconitus showed biting activities throughout the night but An. maculatus showed two peaks of biting activity in the first half of the night. PMID- 8629077 TI - An epidemic of tetrodotoxin poisoning following ingestion of the horseshoe crab Carcinoscorpius rotundicauda. AB - At certain seasons of the year in Thailand, the horseshoe crab Carcinoscorpius rotundicauda may be toxic to human and fatal poisoning occasionally occur. Tetrodotoxin (TTX) and its derivatives were major toxins in the toxic eggs of the horseshoe crab. An epidemic of poisoning by eating toxic eggs of the horseshoe crab affected 71 persons in Chon Buri which located in the eastern coast of Thailand. Patients generally presented with neurologic symptoms such as paresthesia, vertigo, weakness, respiratory paralysis, altered consciousness with unreactive dilated pupils in addition to gastrointestinal symptoms such as nausea and vomiting. Nineteen patients required artificial ventilation and there were two deaths. This is the first large outbreak of tetrodotoxin poisoning recognized in Thailand. PMID- 8629078 TI - Effect of minor engineering intervention in the control of breeding of Phlebotomus papatasi (Scopoli) sandflies. PMID- 8629079 TI - Lambdacyhalothrin impregnated bednets control malaria in Sabah, Malaysia. PMID- 8629080 TI - Cyclospora infection in an HIV infected patient with ultrastructural study. PMID- 8629081 TI - Intestinal microsporidiosis: first reported case in Thailand. PMID- 8629082 TI - Treatment of cryptococcal meningitis with triple combination of amphotericin B, flucytosine and itraconazole. PMID- 8629084 TI - Asia-Pacific Conference on Medical Genetics. Bangkok, Thailand, July 27-29, 1994. PMID- 8629083 TI - Prenatal diagnosis and screening of common genetic diseases in Hong Kong. PMID- 8629085 TI - A clinical approach to inborn errors of metabolism. AB - Asking physicians to diagnose and treat all varieties of inborn errors or metabolism, without access to an extensive and experienced laboratory, is rather like asking a surgeon to perform his job without a scalpel. Nonetheless, astute clinicians armed with knowledge of the clinical manifestations of these disorders and using some simple algorithms can achieve considerable success although it should be said that tentative and unconfirmed diagnoses can be dangerous both for the patient and for use in genetic counseling. For these reasons it seems critical that areas of the world, such as Southeast Asia, where many of the countries have sophisticated medical systems, should develop regional services which can provide the contemporary procedures critical for accurate diagnosis of inborn errors of metabolism. In either case, the financial costs are substantial (Table 12). PMID- 8629086 TI - IBEM in Thailand. AB - The incidence of IBEM in Thailand is yet unknown, however, by estimation it is generally accepted to be 1 in 5,000. From a recent survey in 7 medical schools from different parts of the country and a largest pediatric hospital in Bangkok, we found numerous cases of IBEM nationwide. These are amino acids disorders, carbohydrate disorders, urea cycle defects, peroxisomal, lysosomal storage disorders and many others. Since Thais are quite homogeneous in their genetic make-up; it is, therefore, very likely that IBEM is much more prevalent than we realized. With the exception of thalassemias, IBEM is very common in Thailand and other countries in the Asia-Pacific region. It is a real burden, indeed, since the consequences of IBEM are very serious, eg permanent damage to the CNS causing mental retardation, epilepsy, blindness, deafness and shortened life-span of individuals. Newborn screening for IBEM has been initiated at Siriraj and Ramathibodi Hospital Medical Schools of Mahidol University in 1993. There is yet no national screening program, although a pilot program was launched in the North, Northeast and the South by the Ministry of Health in 1991. Main problems we are facing include: only a handful of clinicians and scientists with expertise in IBEM; no well-equipped laboratory facilities; lack of funding and well organized plan. PMID- 8629087 TI - Genetic problems and genetic services in Malaysia: a country report. PMID- 8629088 TI - Red cell enzymopathies as a model of inborn errors of metabolism. AB - The molecular abnormalities of erythroenzymopathies associated with hereditary hemolytic anemia have been determined using molecular techniques. Pyruvate kinase (PK) deficiency is the most common and well-characterized enzyme deficiency involving the glycolytic pathway and causing hereditary hemolytic anemia. We have identified six distinct missense mutations and a form of splicing mutation in 11 unrelated families with homozygous PK deficiency. Mutations located near the substrate binding site may change the conformation of the active site, resulting in a drastic loss of activity and severe clinical symptoms. Up to now, including these genetic defects, 21 missense, 1 nonsense and 2 splicing mutations, 2 insertions, and 3 deletions have been determined. G6PD deficiency is the most common metabolic disorder, and is associated with chronic and drug- or infection induced hemolytic anemia. To date, sixty different mutations have now been identified. Except for three kinds of variants with small gene deletions or three nucleotide substitutions, all of those were found to be produced by one or two nucleotide substitutions. Molecular studies disclosed that all the class 1 variants associated with chronic hemolysis have the mutations surrounding either the substrate or the NADP binding site. Among rare enzymopathies, missense mutations have been determined in glucosephosphate isomerase deficiency, aldolase deficiency, triosephosphate isomerase (TPI) deficiency, phosphoglycerate kinase deficiency, and adenylate kinase deficiency. Compound heterozygous cases with missense mutation/nonsense mutation and missense mutation/decreased mRNA have been reported in TPI deficiency and diphosphoglyceromutase deficiency, respectively. In phosphofructokinase (PFK) deficiency, three kinds of 5'-splice junction mutations resulting in abnormally spliced PFK-M mRNA were identified. An exception is a hemolytic anemia due to increased adenosine deaminase activity. The basic abnormality appears to result from overproduction of structurally normal enzyme. PMID- 8629089 TI - HPLC analysis of amino acids in inborn errors of metabolism. AB - Analysis of amino acids in blood or urine is a valuable diagnostic tool in cases of suspected metabolic disorders. The presence of a characteristic pattern of elevated amino acids is very useful in the diagnosis of these rare disorders. The detection of an apparently normal pattern of amino acids is also helpful to the clinician since it will eliminate many inborn errors of metabolism from the list of potential disorders. Methodologies for amino acid analysis in physiological fluids range from the very simple thin layer chromatography to automated low pressure or high pressure chromatography. Low pressure chromatography using a Beckman analyzer or similar instrument is the most common methodology for physiological amino acid analysis. Chromatography (HPLC) systems for amino acid analysis of proteins are available that can be modified for use with physiological samples. Waters makes a system called Picotag(TM) and Applied Biosystems makes an automated analyzer. These HPLC systems have some advantages over LPLC systems, including lower equipment cost, less caustic buffer systems and improved separation of certain amino acids. PMID- 8629090 TI - Newborn mass screening and molecular genetics of phenylketonuria in east Asia. AB - Phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH), and is performed with newborn mass screening. PKU causes irreversible mental retardation that can be prevented by a strict low-phenylalanine diet. More than 100 different mutations have been identified world wide and it has been revealed that PKU is a highly heterogeneous disorder. Here, we describe the progress of the molecular genetics of PKU in East Asia. Approximately 60% of all PKU alleles in East Asians have been characterized with 10 PKU mutations. Two major PKU mutations, R413P and IVS4nt-1, may have originated in different populations, spreading in prehistoric times through the Asian continent due to the founder effect, genetic drift, and bottleneck effect. We found different mutations in Caucasians and East Asians, thus PKU mutations have occurred after ethnic divergence between Caucasians and East Asians. Furthermore, PKU genotype and in vitro PAH activity in expression analysis correlates to the clinical and biochemical phenotypes in East Asians. The molecular defects at the PAH gene regulate the in vivo PAH activities and clinical manifestations. PMID- 8629091 TI - High performance liquid chromatography (HPLC) method for confirming thin layer chromatography (TLC) findings in inborn errors of metabolism children in Malaysia. AB - High performance liquid chromatography (HPLC) with phenylisothiocyanate (PITC) is recently used for confirming the diagnosis of inborn errors of metabolism (IEM) especially amino acid disorders in Malaysian children. The method of HPLC used is a precolumn derivatization of amino acids with phenylisothiocyanate and is separated by reversed phase chromatography using 3.9 x 300 mm free amino acid columns and is detected by a UV/Vis detector. The samples are obtained from cases suspected of inborn errors of metabolism, especially of amino acid disorders, which are detected clinically by pediatricians. Initially, samples from patients suspected of inborn errors of metabolism, either urine or serum, are run on one dimensional thin layer chromatography and supplementary chemical tests to detect the abnormal bands and associated abnormalities respectively. Positive samples are further run on HPLC to determine the specific amino acids abnormality. An examples of a case of maple syrup urine disease is discussed, based on the thin layer chromatography findings and HPLC findings. PMID- 8629092 TI - Inborn errors of metabolic diseases in Malaysia: a preliminary report of maple syrup urine diseases for 1993. AB - The Malaysian level of health care has greatly improved so that many of the infectious diseases are now under control. However, perinatal death or death due to unknown childhood diseases remains high (10.3%) being second on the list of causes of death amongst Malaysians. Could inborn metabolic diseases be the main cause of death among these children? Recently, with our success in the development of confirmatory techniques for amino acid disorders using high performance liquid chromatography (HPLC), we have examined 404 samples received from all over the country in 1993. Each specimen with abnormal findings from screening tests by one-dimensional thin layer chromatography was confirmed using HPLC. 41% had generalized aminoacidurias and 4.2% had maple syrup urine disease (MSUD). Patients were aged between 11 days to 6 years. Most of them were Malay males and presented with a history suggestive of MSUD. With this preliminary finding, further studies will be carried out in order to have an investigation and management protocol for the diseases and more importantly to formulate a strategy of screening for the country. PMID- 8629093 TI - Glucose-6-phosphate dehydrogenase deficiency in the newborn: its prevalence and relation to neonatal jaundice. AB - Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in Thailand. This condition can cause acute hemolysis during oxidative stress and also severe hyperbilirubinemia in the newborn in some populations. Our aim was to study the prevalence of G6PD deficiency in relation to neonatal jaundice. We performed quantitative red blood cell (RBC) G6PD assay in the cord blood of 505 male subjects. Observation of jaundice and determination of bilirubin level as well as work up for other causes of jaundice were made in the G6PD deficiency group compared to a G6PD normal group. Questionnaires were also sent for further follow up to both groups. The results of the study were as follows: Sixty-one of 505 male (12.08%) had RBC G6PD deficiency (Group I). The rest (444 cases) had normal G6PD (Group II). In Group I, 49.15% developed neonatal jaundice, of which 28.82% were physiologic and 20.33% were pathologic jaundice. In group II, 23.68% developed jaundice; 16.51% were physiologic and 7.17% were pathologic jaundice, respectively. Onset of jaundice, date of peak bilirubin and peak bilirubin level in Group I and Group II were not statistically different. ABO incompatibility was associated with Group I in 17.24% and with Group II in 9.09%. Hospitalization day in Groups I and II were not statistically different. Other associated diseases were found in both groups, ie infection, congenital malformation, respiratory distress syndrome, but there was no significant difference in terms of jaundice. Phototherapy was required in 18.64% and 10.28% in Group I and II with a duration of 3.91 +/- 1.24 and 3.21 +/- 1.75 days, respectively. One case in Group I who was also premature received one exchange blood transfusion due to severe sepsis but he did not survive. One case in Group II who had polycythemia was successfully treated by partial exchange transfusion with plasma. PMID- 8629094 TI - Growth response in Turner syndrome with recombinant human growth hormone therapy. AB - Ten Turner syndrome girls whose mean age was 10.9 +/- 2.7 years were treated with recombinant human growth hormone (rhGH), dose 0.6 U/kg/week. Five of them had classical 45, XO karyotype. The mean height velocity increased from 2.8 +/- 1.3 cm/year before treatment to 6.1 +/- 2.06 cm/year after treatment for a period of 1.4 years. The response of treatment correlated well with pretreatment height velocity (<3 cm/year) but not with karyotype. However, the response has been decreasing and an increased dose after the first year of treatment is recommended. PMID- 8629095 TI - Evolution and the origins of man: clues from complete sequences of hominoid mitochondrial DNA. AB - Dating the origins of Homo sapiens sapiens is a central problem in human population genetics and anthropology. Do we descend from a single recent ancestral population in Africa, or from multiple ancestral populations in various regions of the world which one million years ago simultaneously began developing into H.s.sapiens? The high substitution rate of mitochondrial DNA (mtDNA) makes this molecule suitable for genealogical and chronological research on closely related hominoid species. We have analyzed the complete mtDNA sequences of three humans (African, European and Japanese) and two African apes (common chimpanzee and gorilla) in an attempt to estimate more accurately the substitution rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated at an approximately constant rate. Under the assumption, supported by the fossil record, that the orangutan and African apes diverged 13 million years ago, we have previously obtained 4.7 million years as the divergence time between humans and chimpanzees. Using this date, we calibrated the substitution rates at synonymous sites and in the displacement-loop region as 4.03 and 7.25 x 10(-8)/site/year, respectively. Based on these rates together with the observation that the African sequence presented here is most diverged from all other human sequences, we inferred the age of the last common ancestor of the human mtDNAs as 140,000 +/- 18,000 years. The result strongly supports the recent African origin of modern humans, H.s. sapiens. PMID- 8629096 TI - Update in molecular genetics: mitochondrial energy transduction disorders. PMID- 8629097 TI - Genetics in the Philippines. PMID- 8629098 TI - Mitochondrial genome analysis in Kearns-Sayre syndrome. AB - We analysed the mitochondrial genome of one patient with chronic and progressive bilateral ophthalmoplegia. This patient also had abnormal EKG showing cardiac conduction defects and pigmentary retinopathy, suggestive of the Kearns-Sayre syndrome. On muscle biopsy, with Gomori trichrome stain, the fibers showed an increase in red-staining material in the intermyofibrillary network and the subsarcolemmal region. On electron microscopy, aggregations of abnormal mitochondria were demonstrated, confirming the diagnosis of mitochondrial myopathy. Analysis of mitochondrial DNA (mtDNA) from the patient and her mother showed no deleted mtDNA. PMID- 8629099 TI - Duchenne muscular dystrophy. AB - Duchenne muscular dystrophy (DMD) is a common inherited disease with a worldwide incidence of 1 in 3,500 male births. Recent molecular study on the DMD gene identified a 14-kb mRNA encoded by 79 exons distributed over 2.5 million bp of the X-chromosome. The protein named dystrophin contains 3,685 amino acids. Most of the genetic events (mutations) that inactivate the dystrophin gene have been shown to be deletions, with over 65% of patients exhibiting the loss of one or more of the exons at the genomic DNA level. The mechanism of the inactivation of the dystrophin gene in one third of patients with DMD/BMD is unknown. PMID- 8629100 TI - Detection of DMD gene deletions in Thai children patients. AB - For the dystrophin gene, it has been shown that about 65% of DMD/BMD patients have detectable deletions. The majority of deletions are clustered in exons 45-53 and at the 5' terminus. We studied 14 X-linked muscular dystrophy (DMD) Thai child patients for detection of gene deletions by amplification of nine exons plus the promoter of the dystrophin gene in two multiplex polymerase chain reactions that included hot spot region (exons 45-53 and 5' terminus). There were 8 DMD patients who had incomplete gene deletion and most of the deletions were around exon 49. PCR-base assays will allow deletion detection from dry blood spot samples and prenatal diagnosis. PMID- 8629101 TI - Usefulness of dinucleotide polymorphism markers in genetic analysis of Duchenne's muscular dystrophy cases in Singapore. AB - Dinucleotide polymorphisms are short tandem repeat sequences that can be used as probes for haplotype analysis in Duchenne's muscular dystrophy (DMD). There are approximately a total of 50,000 to 100,000 such loci in the human genome, and they are highly informative due to the variability of allele lengths at these loci. Primers can be designed to amplify across such repeats located in the dystrophin gene to provide diagnostic information when RFLP analysis is uninformative. We report the usefulness of three such loci for analysis of DMD families in Singapore. The STR50 marker consists of (CA)n repeats located in intron 50 of the dystrophin gene while DYS1 marker is located upstream to the transcriptional start site for the brain dystrophin promoter and BSTRH marker is identified in the 3' untranslated region of the gene. End-labeled PCR products were resolved on 6% denaturing polyacrylamide sequencing gel. Alleles were identified by comparison with sequencing markers. PCR product typically ranged between 174 bp to 255 bp with five to six alleles observed. The heterozygosity rates estimated from 50 X chromosomes of unrelated individuals were 76.0% (BSTRH), 86.6% (DYS1) and 93.3% (STR50). In 38 DMD families studied, the results obtained show that these markers were highly informative and reveal Mendelian mode of inheritance. They were useful for linkage analysis, identification of deletion mutations, confirmation of paternity and mapping of gene recombination. PMID- 8629102 TI - Uses of dinucleotide/trinucleotide repeats in the diagnosis of genetic diseases. PMID- 8629103 TI - Human T-lymphotropic retrovirus type-1 (HTLV-1) and its distribution in Asian populations. AB - Distribution of human T-lymphotropic retrovirus type1 (HTLV-1) among Asian populations is reviewed from an anthropological point of view. The incidence of HTLV-1 infection among New Mongoloid and Indo-Aryan populations was quite low. There were no close phylogenetic relations among HTLV-1 endemic populations. HTLV 1 has been retained among peoples who have been isolated and/or depended on a primitive mode of living. PMID- 8629104 TI - Medical genetics in Singapore. AB - This paper is a brief review of the scope of research and clinical work in human genetics in Singapore. Clinical genetics and karyotyping were established in the early sixties. G6PD deficiency was discovered then as the commonest cause of kernicterus in the newborn. Screening of all newborns was instituted. The measures taken have been very successful and kernicterus is virtually unknown since the early 1970s. Numerous G6PD variants have been discovered and characterized. During the 1980s the emphasis shifted to molecular genetics. Work on the molecular genetics of alpha- and beta-thalassemias, Duchenne muscular dystrophy, hemophilia and retinoblastoma have been established, and good progress on diseases such as neurofibromatosis, leukemias, and lymphoid malignancies. The diagnosis of tuberculosis by DNA amplification (PCR) has been successfully implemented. Numerous papers have been published on the molecular genetics of coronary artery disease, as well as in population genetics. PMID- 8629105 TI - Multistep carcinogenesis in colorectal cancers. AB - Recent advances in molecular genetics have revealed that multiple genetic alterations including activation of oncogenes and inactivation of tumor suppressor genes are required for tumor development and progression. Tumorigenesis of colorectal cancer, in which most cancers are considered to arise from preceding benign adenomas, has been well documented at the molecular level. Familial adenomatous polyposis (FAP), which is characterized by the development of hundreds to thousands of adenomatous polyps in the colon and rectum, one or more of which can progress to cancer if left without surgical treatment, is a good model for elucidation of genetic alterations involved in colorectal tumorigenesis. The adenomatous polyposis coli (APC) gene responsible for FAP was isolated in 1991, and germinal and somatic mutations of the APC gene have been identified. Moreover, activation of K-ras oncogene and inactivation of several tumor suppressor genes such as MCC, p53, and DCC are supposed to play important roles at specific stages of colorectal tumorigenesis. More recently, two genes, MSH2 and MLH1, responsible for hereditary non-polyposis colorectal cancer (HNPCC) have been identified. Thus the molecular mechanism of colorectal tumorigenesis now seems to be more complicated than has been supposed. PMID- 8629106 TI - Detection of BCR/ABL fusion gene in CML: a preliminary report. AB - The BCR/ABL fusion gene in 31 patients with chronic myeloid leukemia (CML) was detected by RT/PCR. In 18 cases of Ph' positive patients, 13 had BCR 3/ABL II rearrangement, 1 had BCR 2/ABL II rearrangement and 4 had both rearrangements. One case with complex translocation: 46,XY,t(9;9;22), had BCR 3/ABL II rearrangement. In 8 cases of Ph' negative patients, 4 had BCR 3/ABL II rearrangement, 3 had both rearrangements while 1 had no BCR/ABL rearrangement. Interestingly, in 4 patients who had no cytogenetic result, we could observe BCR 3/ABL II rearrangement in 3 cases and both rearrangements in 1 case. The results suggest that this procedure is sensitive and independent of the presence or absence of an identifiable Ph' chromosome. PMID- 8629107 TI - DNA polymorphisms for carrier detection of hemophilia in Thailand. AB - The assessment of carrier state based on the pedigree and laboratory testing in 55 females from 34 Thai hemophilia families (24 affected by hemophilia A, 10 by hemophilia B) was studied. The laboratory testing included phenotypic analysis (FVIII:C/vWF: Ag ratio, FIX:C) and two types of DNA polymorphisms, restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR) in/and close to the factor VIII genes (Bcl I, Xba I RFLP, St 14 VNTR) and factor IX genes (Mse I, Dde I RFLP). Fifteen out of seventeen (88%) obligate hemophilia A carriers and one out of five (20%) obligate hemophilia B carriers were diagnosed by phenotypic analysis. All hemophilia A carriers were informative for at least one polymorphism (Bcl I, Xba I or St 14) while 42% of hemophilia B carriers were informative for Mse I RFLP only. DNA polymorphism analysis has advantage over phenotypic analysis since it generally gives an absolute diagnosis when informative. Most DNA polymorphism analyses are performed by PCR technique which is a simple, inexpensive and quick procedure. However, it is limited by non informativeness and high incidence of new mutations. PMID- 8629108 TI - Molecular examination of GH gene deletion in familial growth hormone deficiency. AB - The human growth hormone gene (GH gene) from nine members of a family with familial growth hormone deficiency was examined. The patients were diagnosed as having growth hormone deficiency clinically and by response to hormonal treatment. PCR amplification was carried out using total DNA extracted from leukocytes. The flanking regions of the GH gene which are highly homologous were amplified by one pair of primers. PCR products of 1900 bp and 1919 bp were obtained. By using the combination of restriction enzymes BgII, HaeII and SmaI to digest these PCR products, the various sizes of GH gene deletion can be detected. None of the possible deletions was found in these patients and their relatives by either PCR or Southern blot analysis. PMID- 8629109 TI - Molecular analysis of the human fetal-to-adult globin switching. AB - Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by continued expression of the gamma-globin gene in adult life. Analysis of a Japanese HPFH family had revealed that the C-T transition at position -114 within the distal CCAAT box of the gamma-globin gene associated with the HPFH allele. In the vicinity of the distal CCAAT box, other two mutations (-117 C-T, 13 bp del) had been identified in individuals with a HPFH phenotype. Functional analysis of these mutant promoters in erythroid cell lines suggested that the distal CCAAT box works positively in the fetus but negatively in the adult on the expression of the gamma-globin gene. Further study on transgenic mice showed that the -114 mutation was responsible for the elevated expression of the gamma-globin gene in the adult. To elucidate the molecular mechanism underlying the persistent expression of the gamma-globin genes associated with the HPFH mutations, interaction of the mutant promoters with nuclear factors was analyzed. Relevance of the nuclear factor, NFE3, to the gamma-globin regulation was suggested by the affected binding of NFE3 to the altered distal CCAAT boxes with HPFH mutations ( 117, -114, 13 bp del). PMID- 8629110 TI - Molecular pathology of beta-thalassemia in Indonesia. PMID- 8629111 TI - The clinical severity of beta-thalassemia mutations in West Malaysia. AB - Beta-thalassemia in West Malaysia is caused by 14 molecular defects with differing clinical severity. In Chinese patients from West Malaysia, the main beta-thalassemia mutations seen were (a) a 4 base pair-TCTT deletion in codon 41 42 [frameshift mutation (FSC 41-42)]; (b) a C to T substitution at the second intervening sequence (IVS2-654); (c) an A to G substitution in the TATA box [-28 (A to G)], and (d) an A to T substitution in codon 17[17 A to T]. In the Malays, the main mutations seen were (a) a G to C in nucleotide 5 at the intervening sequence I [IVS1-5 (G to C)]; (b) G to T substitution in nucleotide I at the intervening sequence I [IVS1-1 (G to T)]; (c) a A to T substitution in codon 17 (17 A to T); (d) removal of C from codon 35 [codon 35 (-C)], and (e) a 4 base pairs-TCTT deletion in codon 41-42 [frameshift mutation (FSC 41-42)]. A scoring system (Tha1 CS) has been formulated to predict clinical severity. It is the type of beta-thalassemia mutation present that decides on the clinical phenotype. The most severe beta-thalassemia mutation is assigned a score of 4. A score of 8 indicates severe thalassemia. PMID- 8629112 TI - The spectrum of beta-thalassemia mutations in southern Thailand. AB - Beta-thalassemia mutations in 282 alleles of 253 unrelated individuals originating from various provinces in the south of Thailand were characterized by dot blot hybridization, specific PCR-amplification and direct DNA sequencing. It was possible to characterize the mutations in 274 (97.2%) of alleles studied. Twelve different point mutations and two different large deletions of the beta globin gene were identified. Seven common mutations, namely 4 bp deletion at codons 41/42. IVS1 position 5 (G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 position 1 (G-T), position -28 (A-G) and 3.5 kb deletion, accounted for about 91.5%. The mutations at mRNA cap site + 1 (A-C) and IVS1 position 1 (G-A), previously undescribed in Thailand, were found in 1 and 2 individuals, respectively. A novel mutation of 105 bp deletion at the 5' end of beta-globin gene was detected in a family originating from this area. The knowledge from this study should be useful for planning of genetic counseling and prenatal diagnosis programs for patients with beta-thalassemia in the south of Thailand. PMID- 8629113 TI - Molecular mechanisms of thalassemia in southeast Asia. AB - Hemoglobinopathies are the most common genetic disorders in Southeast Asia. alpha Thalassemia is most often due to a alpha-globin gene deletion. Hb Constant Spring (CS) occurs from the mutation at the termination codon of the alpha-globin gene resulting in an elongated polypeptide; alpha(CS)-globin mRNA is also unstable and only small amounts of Hb CS are produced. Thus Hb CS has an alpha-thalassemia 2 like effect. beta-Thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions or deletions or insertions of one to four nucleotides. Hemoglobin E occurs from a Glu --> Lys substitution at position 26 of the beta-globin chain. The abnormal gene also results in reduced amounts of beta E-mRNA and hence of beta E-globin chains. Therefore, Hb E has a mild beta + thalassemia phenotype. Homozygous beta-thalassemia and beta-thalassemia/Hb E are the major beta-thalassemic syndromes in Southeast Asia. In spite of seemingly identical genotypes, severity of beta-thalassemia/Hb E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta thalassemia. A number of genetic factors have been shown to determine the differences in severity of anemia in beta-thalassemia/Hb E, including co inheritance of alpha-thalassemia determinants and co-inheritance of other determinants which elevate Hb F expression. A correlation between the extent of beta E-globin mRNA cryptic splicing and the severity of anemia in beta(zero) thalassemia/Hb E patients has been observed. Complete characterization of mutations causing hemoglobinopathies will help to bolster the establishment of prenatal diagnosis of these genetic disorders in the region. PMID- 8629114 TI - Role of alternatively spliced beta E-globin mRNA on clinical severity of beta thalassemia/hemoglobin E disease. AB - In spite of seemingly identical genotypes, severity of beta thalassemia/hemoglobin (Hb) E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. Since mutation in codon 26 of the beta E-globin gene can lead to an alternative splicing, Hb E acts like a mild beta(+)-thalassemia. Variation in the amount of beta E-globin mRNA may also govern the difference in severity of anemia in beta thalassemia/Hb E patients who otherwise have the same genetic determinants. We have determined the percentage of the alternatively spliced beta E-globin mRNA by the RT-PCR technique in 14 patients and found that the amount of abnormal spliced beta E-globin mRNA in those patients with severe symptoms ranged between 2.9 to 6.1%, whereas those with milder symptoms had the values which ranged between 1.6 to 2.6%. The extent of beta E-globin mRNA cryptic splicing was better associated with clinical severity of the patients than did the patterns of the Xmn I polymorphism at position -158 of the G gamma-globin gene or levels of Hb F. PMID- 8629115 TI - Expression of hemoglobin E in newborn. AB - Hemoglobin E(HbE) is an abnormal hemoglobin resulted from a point mutation in codon 26 (GAG-AAG) of beta-globin gene. The mutation causes an amino acid substitution (Glu-Lys) and abnormal splicing in exon 1 that reduce the amount of beta E chain synthesis. While in adult, the HbE can easily be diagnosed, its level in newborn is usually underrepresent. In this study, we examined a relationship between genotype of HbE and the amount of HbE in cord blood. 145 Cord blood specimens were analysed by starch gel electrophoresis and the amounts of HbE were determined by microcolumn chromatography. The zygosity of beta E globin gene was determined by the polymerase chain reaction. The levels of HbE were 3.17 +/- 1.79% for 59 heterozygotes, 8.55 +/- 2.52% for 3 homozygotes and 0.48 +/- 0.22% in 83 normal newborns. This result provides useful data for a neonatal screening program and implies that expression of HbE in newborn dependent on a copy number of beta E globin gene in each individual. PMID- 8629116 TI - Molecular basis of alpha (0)-thalassemia in northeast of Thailand. AB - The molecular basis of alpha(0) thalassemia were studied in 95 patients who attended at Srinagarind Hospital, Khon Kaen University during September 1993 January 1994. From these 95 patients, hemoglobin electrophoresis indicated that there were 14 cases with A2AH, 21 cases with A2ABart'sH, 6 cases with ConSpA2AH, 31 cases with ConSpA2ABart'sH, 6 cases with EABart's, 3 case with EFBart's, 4 cases with ConSpEABart's, 5 cases with Portland Bart's and 5 cases with A2A. White blood cell lysate was prepared from peripheral blood leukocytes of these patients and was subjected to the polymerase chain reactions for detection of the SEA type alpha thalassemia gene deletion. Altogether 100 chromosomes with the SEA deletion were detected from all patients examined, the result indicated that this type of alpha(0) thalassemia gene deletion is the most common among these Thai population. These data will be useful for a carrier screening and a prenatal diagnosis program of homozygous alpha (0) thalassemia which is a lethal condition in northeast of Thailand. PMID- 8629117 TI - Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family. AB - Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/ alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E. PMID- 8629119 TI - Medical genetics in Thailand. AB - The development of medical genetics as a discipline in Thailand is relatively recent. It originated through interest in hematological disorders which occur in high frequency, namely the thalassemias and hemoglobinopathies, together with glucose-6-phosphate dehydrogenase deficiency. Although a complete registry of genetic diseases in Thailand has not yet been established, considerable data has accumulated through special interest groups. PMID- 8629118 TI - Alterations and pathology of thalassemic red cells: comparison between alpha- and beta-thalassemia. AB - Two main types of thalassemia have been categorized according to defective production of the globin gene ie alpha-thalassemia and beta-thalassemia. We report different red cell abnormalities between these two types. The study included 139 thalassemic patients including 91 patients with hemoglobin (Hb) H disease (52 cases with the classical genotype and 39 cases with Hb Constant Spring) and 48 were beta-thalassemia/Hb E disease. The deformability index of thalassemic red cells measured by laser diffractometer was significantly lower than that of normal red cells. Increased susceptibility of the thalassemic red cells to monocyte phagocytosis was markedly noted. Few sialic acid molecules were scattered on red cell surface of thalassemic red cells. Reticulocytes with delayed maturation stage were also observed in thalassemia indicating enhanced release from the bone marrow. The alpha-thalassemic red cells had relatively better deformability, increased susceptibility to phagocytosis, reduced sialic acid content and greater degree irregular distribution of sialic acid on red cell surface as compared to beta-thalassemic red cells. The alpha-type with hemoglobin Constant Spring (Hb CS) had increased percentage of reticulocyte and young reticulocyte (high fluorescent intensity) as compared to beta-thalassemic red cells. The different abnormalities between alpha- and beta-thalassemic red cells may lead to different mechanism of red cell destruction and different severity of the disease. PMID- 8629120 TI - Prevention of thalassemia. PMID- 8629121 TI - Phitsanulok population: the highest incidence of hemoglobin E in the northern provinces of Thailand and PND counseling. AB - Phitsanulok is a province situated in the southern part of northern Thailand. Studies of hemoglobinopathies of 2,806 individuals during the period 1988-1990 showed an overall incidence of hemoglobinopathies of 38.89%, with HbE as high as 25% which is the highest incidence of HbE in the North of Thailand. Buddhachinaraj Hospital conducted a maternal screening study on 1,015 pregnant women in 1991 as part of a prevention/control program and found that 22.56% (229 women) had hemoglobinopathies. Of those, 102 (44.54%) individuals (83.33% HbE heterozygotes and 8.82% HbE homozygotes) with their spouses participated in a prenatal diagnosis (PND) counseling program; 100% of the females and 96% of the male were willing to accept PND; 71% of the females and 75.6% of the males had no moral objection in PND. PMID- 8629122 TI - Prevalence of hemoglobin E, alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok. AB - Thalassemia hemoglobinopathies and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency are prevalent in Thailand. We studied the prevalence of these disorders from 1,000 cord bloods collected during 14 months period, using EDTA as anticoagulant. Red blood cell G-6-PD quantitative assay was performed in all male subjects. Nine hundred and eighty five specimens were available for hemoglobin (Hb) typing by starch gel electrophoresis. Further evaluation by cellulose acetate electrophoresis and follow up were made in the cases who had Hb E and/or high level of Hb Bart's. It was found that out of 505 males, 61 cases (12.08%) had G-6-PD deficiency. Among 985 cases studied for Hb typing, 61.92% revealed normal Hb type AF while Hb E was present in 18.68% and Hb Bart's designated alpha thalassemias were present in 25.18% respectively. Of these 985 cases, 18.78% had low Hb Bart's level ie detectable to 8.2% consistent with alpha-thal2, Hb Constant Spring (CS) or alpha-thal1 trait. Ten cases (1.02%) had high levels of Hb Bart's ranging from 16.1-35% without or with Hb CS and E, and further follow up revealed homozygous Hb CS, Hb A-E-Bart's, Hb H and Hb H with Hb CS disease. The other 53 cases (5.38%) had low level of Hb Bart's with Hb E consistent with alpha-thalassemia trait with Hb E trait. There were 127 cases (12.89%) who had only Hb E trait and 3 cases (0.3%) who had Hb F and E without Hb A initially. PMID- 8629123 TI - Clinical phenotypes and genotypes diagnosis of thalassemia in children. AB - Thalassemia is a relatively common hemolytic anemia in Southeast Asia. Alpha and beta thalassemia, hemoglobin (Hb) E, and Hb Constant Spring (CS) are prevalent in Thailand. Different gene combinations lead to over 60 thalassemic syndromes. One hundred and forty-nine thalassemia families were retrospectively studied. They were 4 homozygous beta-thalassemia (beta-thal/ beta-thal), 79 beta-thal/Hb E, 22 Hb H disease, 32 Hb with Hb CS, and 6 AE Bart's disease. The first clinical manifestation and hematologic data including hemoglobin electrophoresis were analysed. Most homozygous beta-thalassemia and beta-thal/Hb E presented with anemia (100% vs 81%), hepatomegaly (40% vs 21%), and splenomegaly (20% vs 27%). In Hb H disease and Hb H with Hb CS, the clinical findings were anemia (74% vs 79%), hepatomegaly (9% vs 8%), splenomegaly (9% vs 13%), jaundice (24% vs 13%), and fever (18% vs 25%). The 317 hematologic data and hemoglobin types of the patients, their parents and relative were also analyzed. These findings can be used as reference values for childhood thalassemia and heterozygous states. PMID- 8629125 TI - Prenatal diagnosis for beta-thalassemia syndromes using HRP-labeled oligonucleotide probes at Siriraj Hospital. AB - Characterization of the molecular defect of beta-thalassemia in Thais has enabled us to establish prenatal diagnosis for homozygous beta-thalassemia and beta thalassemia/Hb E. The nature of the beta-thalassemia mutation of each high risk couple or of the previous affected child was firstly identified after counseling. Detection of beta-thalassemia mutations was performed by dot-blot hybridization of the amplified DNA with a set of HRP-labeled ASO-probes specific for the common mutations. If the mutation could be characterized, prenatal diagnosis (PND) would be performed by using DNA extracted either from the chorionic villi (CVS) or amniotic fluid fibroblast in the first trimester of pregnancy or from fetal blood in the second trimester. DNA analysis was carried out in 23 couples at risk of having homozygous beta-thalassemia and 88 couples at risk for beta-thalassemia/Hb E. However, PND was performed by this technique in 22 pregnancies from 21 couples at risk of having homozygous beta-thalassemia children and 86 pregnancies from 71 couples at risk for beta-thalassemia/Hb E; 9 couples underwent more than one prenatal diagnosis. The results showed that, although there are more than 20 beta thalassemia mutations in the Thai population, PND by DNA analysis could be carried out in more than 95% of the risk couples by using beta(E) and 10 different HRP-labeled ASO probes. This technique was simple, economic and avoided the use of radioactive isotope. PMID- 8629124 TI - A simple non radioactive method for detecting beta-thalassemia/hbe disease: application to prenatal diagnosis. AB - We have developed allele specific polymerase chain reaction (ASPCR) that allows rapid screening of the beta E-globin and common beta-thalassemia genes in Thailand. These non-radioactive methods are based on the amplification by the polymerase chain reaction of the beta E and beta-thalassemia specific DNA fragments using specific primers. With this approach, both heterozygote and homozygote for the disease could readily be identified on agarose gel electrophoresis of the amplified DNA. We have applied the method for a prenatal diagnosis of beta-thalassemia/HbE disease in a Thai family at the second trimester of pregnancy. The result obtained was comparable to that of conventional dot blot hybridization using radioactive probes. The simplicity, accuracy and non isotopic of the approach make it a highly promising method for a carrier screening and a prenatal diagnosis of this common disorder. PMID- 8629126 TI - Prenatal diagnosis of Hb Bart's hydrops fetalis by PCR technique: Pramongkutklao experience. AB - Hb Bart's hydrops fetalis is very common in Southeast Asia, especially in Thailand. As the mother of such an infant may suffer from toxemia of pregnancy, ante- or post-partum hemorrhage as well as the psychological burden for carrying a nonviable fetus to term, so prenatal diagnosis is indicated and the family should be given the choice of early termination of the pregnancy. Seven high risk pregnancies with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia 1) were studied. Amniocentesis was done at 16-33 weeks of gestation. DNA analysis was performed by polymerase chain reaction (PCR) using 2 techniques, 1) three nucleotide primers and 2) four nucleotide primers. After either therapeutic abortion or birth, heart blood or cord blood was drawn to confirm the diagnosis by Hb electrophoresis and DNA analysis. Of 7 high risk fetuses, 3 were recognized as Hb Bart's hydrops fetalis, 2 showed the alpha-thal 1 trait, 1 showed alpha thal 2 trait and 1 was a normal fetus. The technique was entirely suitable for prenatal diagnosis of Hb Bart's hydrops fetalis. This technique was a rapid, simple non-radioactive method, less expensive and available in most PCR laboratories. PMID- 8629127 TI - Study of hematopoietic progenitor cells in thalassemia after bone marrow transplantation. AB - The hematopoietic committed progenitor cells (BFU-E and CFU-GM) in blood and bone marrow were studied in thalassemic patients before and after bone marrow transplantation. Eighteen transplants were performed in 17 patients with thalassemia. Five were homozygous beta-thalassemia and 12 were beta thalassemia/hemoglobin E disease. The age ranged from 1.2-14 years (median = 3.4 years). The conditioning regimen comprised busulfan 3.5-4 mg/kg/day for 4 days and cyclophosphamide 50 mg/kg/day for 4 days. Cyclosporin in combination with methotrexate were administered post transplant for GVHD prophylaxis. Before transplantation, BFU-E and CFU-GM in the blood of the patients were significantly higher compared with normal (p < 0.05) but were normal in the bone marrow. Only the CFU-GM in the bone marrow of the successful cases after transplantation recovered to the normal level at the first month through the 12th month whereas the BFU-E were low. Both CFU-GM and BFU-E in the blood were lower than normal after follow up to the 12th month. Inspite of the low number of progenitor cells, there was hematological recovery in the blood of the patients. It may be due to the capacity of the hematopoiesis react to stress which could be amplified the differentiation compartment or the shortened-transit time through the stem cell compartment. PMID- 8629128 TI - DNA fingerprinting and forensic medicine. AB - In forensic medicine, DNA fingerprinting for identification is becoming a necessary procedure. A method to radiolabel M13 DNA probe by primer extension using a specific oligonucleotide primer was developed. The method specifically labeled the two 15 bp repeats in M13 DNA which hybridize to target DNA giving rise to DNA fingerprinting patterns. The M13 probe labeled by this method has proven useful for individual identification, paternity testing and monitoring reconstitution in bone marrow transplantation. The genetic locus D1S80 and D17S30 containing a variable number of tandem repeats (VNTR) have also been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels. Analysis of this VNTR locus was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permited good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another. PMID- 8629129 TI - The burden of genetic disorders in India. PMID- 8629130 TI - HLA haplotype frequencies in Thais. PMID- 8629131 TI - Study of genotoxic effects of antidiarrheal medicinal herbs on human cells in vitro. AB - The use of medicinal herbs has been a common practice in Asia but their genotoxic properties are little known. In the present study, genotoxic effects of three antidiarrheal herbs, guava leaf, mangosteen peel and pomegranate peel, were examined using established human cell lines, Raji and P3HR-1. Cells were treated with boiled-water extract of the herbs at various concentrations for 24 and 48 hours in vitro. Cell growth and viability were dose dependently reduced. No apparent chromosomal aberrations were induced by the treatment. Administration of pomegranate extract induced apoptotic DNA fragmentation. This genotoxicity test system is simple and convenient for the primary screening. PMID- 8629132 TI - The Malaysian experience in the typing of genetic variants of alpha-1 antitrypsin. AB - It is known that alpha1-antitrypsin deficiency is associated with emphysema in adults and liver cirrhosis in neonates. The phenotypes PiZZ and PiSZ are considered to be high risk groups. alpha1-antitrypsin deficiency is one of the most common lethal congenital disorders in Europe and the USA, occurring in approximately 1 in 2,000 caucasians of North European descent. Studies in Malaysia have found that the phenotypes PiZ and PiS are present in our population. Out of 950 samples analyzed, it was found that 10 samples were shown to be apparently Z homozygous phenotype. The phenotype is determined by high resolution isoelectrofocusing on an ultra-thin polyacrylamide gel embedded with narrow range Pi phamarlyte. The isoelectrofocused bands are confirmed by immunofixation and the plasma alpha1-antitrypsin levels determined by electroimmunoassay. The abnormal phenotypes are further confirmed by polymerase chain reaction using allele specific oligonucleotides. PMID- 8629133 TI - Prenatal sex determination in Thai pregnant subjects by nested-PCR amplification of fetal Y chromosome-specific sequence. AB - Detection of a Y-specific sequence in either amniotic cells or the maternal circulation has clinical importance for prenatal sex diagnosis, especially in mothers with severe X-linked disorders. Deoxyribonucleic acid sequences of human ZFY (zinc-finger-Y) gene, a Y-chromosome-specific gene and candidate for the testis-determining factor, were amplified by in vitro nested PCR technique, in amniotic cells or peripheral blood of Thai pregnant subjects carrying male fetuses. This technique permits detection of ZFY gene DNA sequences in as few as a single male cell among 100,000 female cells. The subjects in this study were a group of Thai pregnant women who had been given prenatal fetal sex diagnosis by the cytogenetic method. The results herein demonstrated the usefulness of PCR detection of Y-specific sequences in amniotic cells. The ZFY gene detection method for prenatal sex prediction by nested PCR has been developed experimentally. However, further investigation is necessary to increase reliability of clinical application. With properly designed guidelines, the established nested-PCR technique may be an alternative for the determination of the fetal sex for those pregnancies at risk of X-linked genetic disorders. PMID- 8629135 TI - Epstein-Barr virus DNA in nasopharyngeal carcinoma in Thai patients at Ramathibodi Hospital, Bangkok. AB - Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in Thailand. The association of Epstein-Barr virus (EBV) and NPC, especially undifferentiated type, has been documented. There is, however, conflicting evidence with regard to the squamous cell type. We have therefore investigated EBV-DNA in all the three types of NPC to assess the association of EBV and NPC in Thai patients. EBV-DNA was detected in formalin-fixed paraffin-embedded tissues from the patients of Ramathibodi Hospital, Bangkok using polymerase chain reaction. A primer pair that amplified EBV nuclear antigen gene was used in the reactions and the amplification products were hybridized with a specific EBV probe. EBV-DNA was identified in 24 of 28 of tissue samples from patients with undifferentiated NPC, 25 of 40 samples from patients with squamous cell NPC and 3 of 4 samples with nonkeratinizing NPC. None of 12 nasopharyngeal tissue samples without NPC contained detectable EBV-DNA. Our results indicated a strong association of EBV with undifferentiated as well as non-keratinizing NPC. EBV-DNA was demonstrated in most cases of squamous cell NPC but the association of EBV in this type of carcinoma was not as frequent as in the other two types of NPC. PMID- 8629134 TI - PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria. AB - Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites. PMID- 8629136 TI - Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections. AB - Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results. PMID- 8629137 TI - Analysis of RNA polymerase gene mutation in three isolates of rifampicin resistant Mycobacterium tuberculosis. AB - Drug resistance in tuberculosis (TB) has become a major public health threat, particularly when the disease cannot be 100% controlled by BCG vaccination. In Thailand, resistance to rifampicin, a major component of multidrug regimens of treatment, is the common cause of tuberculosis recurrence. The mechanism of rifampicin resistance involves alterations of the RNA polymerase subunit beta (rpo B) gene. Mutations in rpo B gene were often found to cluster within a region of 23 amino acids starting from amino acid residue 511 to residue 533. Direct PCR sequencing was utilized to compare base changes in rpo B gene in three rifampicin resistant phenotypes of M. tuberculosis isolated from Thai patients. The sequences showed one base substitution at codon 531 resulting in an amino acid change from serine (TCG) to leucine (TTG) in a multidrug resistant isolate compared to that of a sensitive isolate, whereas a point mutation at codon 516 causing a change from aspartic acid (GAC) to tyrosine (TAC) was detected in a multidrug resistant isolate from a HIV positive patient. In an isolate resistant only to rifampicin a double mutation at codon 531 changing serine (TCG) to phenylalanine (TTT) was found. No mutations were observed in the same region in streptomycin, ethambutol or isoniazid resistant isolates. This finding reports two new types of mutation (GAC to TAC at codon 516 and TCG to TTT at codon 531) and confirms a direct correlation between rpo B gene alteration and rifampicin resistant phenotype in M. tuberculosis. PMID- 8629138 TI - Clinical and molecular cytogenetics and gene mapping: principles and techniques. AB - This article reviews the history of human cytogenetics with respect to technical advances from chromosome banding to molecular cytogenetics. Technologies such as in situ hybridization, chromosome painting, comparative genomic hybridization and interphase cytogenetics and their applications are discussed. The assignments of genes to chromosome regions by somatic cell genetics is illustrated by molecular analyses of somatic cell hybrid panels. The generation of complete physical maps of human chromosomes, by radiation hybrid mapping of sequence-tagged sites and establishment of chromosome-specific yeast artificial chromosome (YAC) banks and clone overlaps (contigs), is exemplified by studies of chromosome 18. The last section outlines the recent and future advances in clinical cytogenetics made possible by progress in molecular genetics. PMID- 8629139 TI - Congenital generalized lipodystrophy, a case report. AB - Generalized lipodystrophy is a rare condition which can be divided into congenital and acquired types, based on the age at presentation and pattern of inheritance. The congenital type of generalized lipodystrophy or Lawrence-Seip syndrome presents in first two years of life and is inherited in an autosomal recessive pattern. The diagnosis is made on the basis of loss of body fat, muscular hypertrophy, acanthosis nigricans, hirsutism, hepatomegaly with fatty liver, hyperlipidemia and hyperglycemia with insulin resistance. A 2 1/2-year-old Thai girl with the clinical features of Lawrence-Seip syndrome is reported. Abnormal platelet function was detected in this girl. PMID- 8629141 TI - Problems of genetic diseases and their services in Indonesia: a country report. PMID- 8629140 TI - Zellweger syndrome: first reported case in Thailand and literature review. AB - We describe an infant boy with facial dysmorphism, profound hypotonia, psychomotor retardation, seizure and hepatomegaly. Biochemical study revealed elevation of very long chain fatty acids and pipecolic acid, consistent with peroxisomal disorder. He died at the age of 4 months. Electron microscopic study demonstrated decreased amounts of peroxisomes in liver and kidneys. The clinical characteristic, accompanied the biochemical and microscopic findings led to the diagnosis of Zellweger syndrome. The recognition of this syndrome is important since it is a fatal disease. The pattern of inheritance is autosomal recessive, hence genetic counseling is necessary. We emphasize that peroxisomal disorder should be included in the differential diagnosis in patients with infantile hypotonia. This patient is the first reported case of Zellweger syndrome in Thailand. PMID- 8629142 TI - Clinical uses of computerized chromosome databases. PMID- 8629143 TI - Lysosomal storage disorders in Thailand: the Siriraj experience. AB - Lysosomal storage disorders are a heterogeneous group of biochemical genetic disorders; currently 40-50 are known. The clinical phenotype is determined by the tissue distribution of the storage material and degree of enzyme deficiency. The genetic transmission is mostly autosomal recessive. Lysosomal storage disorders can be divided into three groups according to the major organ system pathology: (1) Primary involvement of the central nervous system without significant somatic or skeletal pathology. Disorders of grey matter, eg gangliosidosis and disorders of white matter eg the leucodystrophy are the most common; (2) Primary involvement of the reticuloendothelial system with or without associated neuropathology, eg Niemann-Pick disease and Gaucher disease; (3) Multisystem involvement in which skeletal manifestations are prominent features. The mucopolysaccharidosis and mucolipidoses are the two major forms with this clinical phenotype. Lysosomal storage disorders identified at Siriraj Hospital are neuronal ceroid lipofuscinosis, GMI gangliosidosis, mucolipidosis II, Maroteaux-Lamy, sialidosis, Sly syndrome, Hunter syndrome, Morquio syndrome, Gaucher disease, Niemann-Pick, Sandhoff disease, Pompe's disease and many more. Most patients came from the provinces where consanguinity is common. Confirmation usually is done by enzyme assays using skin fibroblast culture or leucocytes. Genetic counseling is extremely important and prenatal diagnosis is recommended to high-risk couple. PMID- 8629144 TI - Genetic skeletal dysplasia in Thailand: the Siriraj experience. AB - Genetic skeletal dysplasias are a heterogeneous group of genetic disorders associated with abnormalities in the skeletal system frequently presenting with disproportionate short stature. There are over 100 distinct skeletal dysplasias which have been classified primarily on the basis of the clinical or radiographic characteristics. We have identified many genetic skeletal dysplasia disorders at Department of Pediatrics, Siriraj Hospital, Bangkok, Thailand. We have cases of achondroplasia, hypochondroplasia, pseudoachondroplasia, atelosteogenesis, pyknodysostosis, spondyloepiphyseal dysplasia (SED) congenita, spondylometaepiphyseal dysplasia (SMED), osteogenesis imperfecta type I, II and III, Ellis-van Creveld syndrome, cleidocranial dysostosis, thanatophoric dysplasia, rhizomelic chondrodysplasia punctata, trichorhinophalangeal syndrome, mucopolysaccharidosis I, II, IV and VI, mucolipidosis II, osteopetrosis, camptomelic dysplasia, metaphyseal dysplasia with spine involvement (Kozlowski type), Langer-Gideon syndrome and hypophosphatemic rickets. We have established a Genetic Skeletal Dysplasia Clinic at Siriraj Hospital since 1992, and see referrals from around the country. Genetic counseling is provided, including prenatal diagnosis and a multidisciplinary approach. PMID- 8629145 TI - The centromere: kinetochore complex. AB - At the meta-anaphase transition the centromeres in a genome separate in non random sequential manner. This sequential separation depends upon the timing of replication of DNA located in the pericentric and centromeric region. Cells in long term cultures as well as some newborn humans carry dicentric chromosomes. The inactive centromeres in these dicentric chromosomes do not show any sequence of separation. Whether or not a dicentric chromosome would segregate equationally depends upon if only one centromere binds to microtubules or both are functional. In man and other higher apes, a 171 base pair long DNA repeat (the alphoid sequence) is present on all centromeres. In mouse, the minor satellite fraction is said to constitute the centromere. These two DNAs also carry a 17 bp long sequence, the CENP-B 'box' to which the CENP-B antigen is bound. Other species eg, rat, pig, fish, Chinese hamster-exhibit still different sequences at the centromere and do not carry the CENP-B 'box' even though the antigen is ubiquitously present in all species. It is not clear why so many diverse sequences constitute the centromere when all centromeres look alike and perform the same function. I propose that the primary constriction owes its property not necessarily to its DNA composition but to some stereophysical property, eg the curvature and that the region is held together till late metaphase-anaphase due to a specific proteinaceous factor. The mammalian centromeres bind a complex of several proteins dubbed as CENtromere Proteins (CENP's). This complex, however, is not what constitutes the trilamellar kinetochore structure as see under the electron microscope.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 8629146 TI - Fragile X syndrome and other dynamic mutation diseases. PMID- 8629147 TI - FISH as a tool for genome mapping and positional cloning. PMID- 8629148 TI - Prenatal diagnosis and cytogenetics. PMID- 8629149 TI - Medical genetics in Japan. AB - In Japan genetic diseases are getting more popular in medicine, because of increased awareness of the role of genetic determinants of diseases. Care for patients with inherited disease is one of the current big problems. In this review, programs developed to support Duchenne muscular dystrophy patients are described as an example of medical services available for genetic diseases in Japan. PMID- 8629150 TI - Identification of chromosome 21 materials using the whole chromosome 21 specific library. AB - The chromosome in situ suppression hybridization or chromosome painting technic was applied to confirm and eliminate the markers involving chromosome 21 segments using a chromosome 21 DNA library. The library ATCCLL21SNO2 was amplified, directly biotinylated using the polymerase chain reaction. The results demonstrated a translocation of chromosome 21 material on chromosome 2 and X and eliminate the origin of the marker. Thus, the technique provides an important tool to complement the conventional G-banding technic. PMID- 8629151 TI - Chromosome analysis of unfertilized oocytes after in vitro fertilization program: a preliminary report. AB - To assess the contribution of chromosome anomalies to high failure of in vitro fertilization (IVF), a total of sixty three eggs from 37 women participating in the IVF program were cytogenetically investigated. The mean age of the oocytes donors was 36.8 years. Chromosome karyotype was obtained in 30 of unfertilized oocytes: 16 oocytes (53.3%) had a normal haploid chromosome complement; 5 (16.7%) were hypohaploid; 3 (10.0%) were hyperhaploid; 3 (10.0%) were diploid, 3 (10.0%) were polyploid. It may explain for early pregnancy loss, low pregnancy rate after in vitro fertilization. PMID- 8629152 TI - [Conservative surgical treatment of kidney tumors: indications, techniques, results]. AB - At the Urological Department in Hradec Kralove the authors operated in 1989-1993 287 patients with renal tumours, in 45 of them a conservative procedure was used (15.6%). The group comprises 29 men and 16 women, mean age 56.5 years. The contralateral kidney was normal in 29 patients (64%), in 5 patients nephrectomy was performed on account of carcinoma and in 9 patients (20%) the second kidney was hypofunctional (hydronephrosis, cysts). In two patients a conservative procedure was used on account of malignity of the blood. In 33 patients the tumour was enucleated (73.3%), in 12 the upper or lower pole of the kidney was resected. In 6 patients the tumour was in the first clinical stage, but in four already in T 3a (imperative indication). Twenty-seven tumours were evaluated by the histologist as medium differentiated, 2 of 35 as poorly differentiated. Thirty-five tumours were carcinomas (77.7%), 8 were adenomas, one was angiomyolipoma and in one instance the scar after PEC was removed which was not reliably diagnosed before surgery. The preoperative examination comprised also immunological examination which, however, was not made in two patients. The immunologist evaluated the criteria as favourable in 21 patients (60%), as risky in 12, incl. 6 were immunotherapy was administered. One patient developed a urinous fistula which healed on a stent. During the investigation period no local relapse was observed. PMID- 8629153 TI - [Immunomodulation in Pseudomonas urinary infection]. AB - The urological department is as a rule of department with the highest incidence of nosocomial infections. They are often caused by Pseudomonas aeruginosa. In 1992 the authors started to use prophylactic immunization with antipseudomonas vaccine PSAEVA in 58 patients indicated for surgery on account of an prostate adenoma. During and after operation the incidence of Pseudomonas infection in immunized and control patients was compared. In the immunized group the incidence after operation was 1.72%, in the control group 12%. The drop of Pseudomonas infections in operated patients immunized with the anti-pseudomonas vaccine as compared with non-immunized ones was by 10.28% lower which is statistically significant--p.0.05. PMID- 8629154 TI - [Surgical correction of hypospadias using a vascularized onlay flap]. AB - The principle of the onlay technique is to create a neourethra from the preserved urethral plate to which the vascularized flap of the prepuce is applied. Thus 51 patients were operated 10 months to 27 years old, half of them were under 5 years. By this method hypospadias was treated from coronary to perineal ones. In 16 patients chordectomy was performed, in 6 plications of the cavernous bodies because of their disproportion of the ventral and dorsal side. In three instances it was necessary to transsection the urethral plate and supplement the onlay flap by tubulization of the neourethra. The method was also used in secondary strictures of the urethra after urethroplasty and in occlusions of a fistula. Complications of the neourethra were recorded in 4 (7.8%) patients. Another four patients had a partial necrosis of the skin cover. The onlay method is becoming universal for resolving the proximal variant of hypospadias, it is associated with a considerably smaller number of complications than tubulized plastic operations and makes possible a definite one-stage solution already at an early age. PMID- 8629155 TI - [Is ureteral dilatation necessary in ureterorenoscopy?]. AB - A retrospective analysis of 136 patients, who were submitted rigid ureteroscopy for stone removal, was performed. Patients were divided into two groups. First group consisted of 63 patients with routine balloon dilation of intramural part of ureter before ureteroscopy. 73 patients of the second group were submitted ureteroscopy without previous ureter dilation. The total success rate was achieved in the first and second group of the patients in 90.5% and 91.8%. The success rate achieved in removing of the stone of upper, middle and lower part of the ureter was in the first group 81.8%, 88.9% and 94.1% and in the second group 78.6%, 95%, 94.9%, respectively. None of the patients have demonstrated vesico renal reflux. With respect to our similar success rate in both of the groups they don't consider routine dilation of ureter as necessary before ureteroscopy. PMID- 8629156 TI - [Ultrasonographic examination in the follow-up of patients after kidney transplantation]. AB - The authors followed-up in 1988-1994 a total of 134 patients after renal transplantation by ultrasonographic examinations in the B mode, Doppler frequency analysis and coloured Doppler mapping. They summarize the basic sonographic pictures during a normal development after transplantation and in case of surgical and non-surgical complications. They evaluate sonography as the basic examination method which is not quite specific for different complications but makes it possible to detect them in the initial stage and to indicate in time further examinations and treatment. PMID- 8629157 TI - [Subjective symptoms before and after prostate surgery. The International Symptom Scoring System, I-PSS]. AB - In this thesis are evaluated the questionnaires of the I-PSS retrieved from 415 patients (TVPE 104, TURP 272, TUIP + PRP 25 and TUIP 14) operated on the BPH at the department of Urology, FN Motol, during 1988-1993. The average value of S in the whole group of patients was 18.9 in the pre-surgery period and 6.9 after surgery. Before the surgery, the prominent difficulties were recorded in 50.8% of patients (S = 20-35) and after surgery this number fell down to 7%. Without any major difficulties (S = 0-7) were 66.3% of patients after the surgery. The average value of L before and after surgery was 4.2 and 1.6 respectively. The quality of life after the surgery was perceived by the patients more convenient than should correspond to the symptom scoring value. The best results (both the average S and L) were recorded in patients after TVPE (S 5.1 and L 1.1), followed by TURP (S 7.4 and L 1.7), than TUIP + PRP (S 8.4 and L 2.1) and only TUIP alone closed scale (S 8.9 and L 2.4). The differences of postsurgery S and L are statistically significant (p < 0.05). The comparison between the subjective better performance and the quality of life, when taken from TVPE resp. TURP point of view, does not prove to be the statistical difference. The comparison between S changes after the standard operation (TVPE and TURP) and minor prostatic surgery (TUIP + PRP and TUIP) also does not occur as statistically significant. Only the quality of life (L changes) is recorded by the patients as significantly worse after the minor prostatic surgery (p < 0.05). The pre-surgery worst perceived symptoms are: weak stream, nycturia and polakisuria (questions Nr. 5.7 and 2). After the surgery, nycturia is the leading worst symptom. PMID- 8629158 TI - [Results of surgical treatment of benign prostatic hyperplasia]. AB - The authors have evaluated the results of the BPH surgery in the computer study on 669 patients (out of them 181 after TVPE and 488 after TURP) operated on at the department of Urology, FN Motol during 1988-1992. The average age of the patients was 70.6 years. The occult cancer was found in 6.7%. The incidence of bladder stones rose with the age (in the 8th decade at the quarter of patients). The average weight of the resected tissue was 63.7 gms in TVPE and 16.7 gms in TURP and it rose with the age. The average surgery time 47.1 mins at TVPE and 50.8 mins at TURP did not change statistically. The average demand on the blood transfusion was higher at TVPE (440.1 mls) than at TURP (95.5 mls) and rose with the resected tissue. The death rate was 0.5% among all the patients included (TVPE 0, TURP 0.6%). All three patients after TURP died from myocardial infarction. In this thesis is evaluated the number of early and late complications (incl. the transient incontinence), which have occurred in five consecutive years. The total morbidity was 27.2% and the further surgery was necessary in 2.8% after TVPE and 5.7% after TURP respectively. The pre-surgery performance status was aggravated in 79.4% of patients. In spinal anesthesia was operated on in 84.2% (subarachnoidal 56.5% and epidural 27.7%) and in general anesthesia in only 15.8% of patients. The pre-surgery urinary infection was found in 38.7% and after operation in 29.3%. Out of the latter, 30% were nosocomials. This was in accordance with the higher rate of late complications after both types of operations. PMID- 8629159 TI - [Sexual consequences of benign prostatic hyperplasia]. AB - In this thesis are evaluated the sexual questionnaires retrieved from 412 patients (TVPE 101, TURP 272, TUIP + PRP 25, TUIP 14), who had been operated on during 1988-1993 for the BPH at the department of Urology, FN Motol. Before the surgery, 69.7% of patients were sexually active yet (28.6% regularly, 41% irregularly). Without sexual intercourse were 29.9% in the time of surgery and 2 patients (0.5% did not respond the questions. The sexual activity was rapidly declining in the 7th decade, in the 8th decade still 41% of the patient argued sexual activity prior to surgery. The libido remained unchanged in 62.9%, worsened in 24.3% and improved in 10.7%. The changes in libido was neither parallel to the aging nor the type of prostatic surgery. After the surgery, the intercourse was admitted only by 49.3% of patients. Without any intercourse remained 50%. The difference compared to the pre-surgery responds is statistically proved (p < 0.01). The erectile dysfunction seemed to be the main cause of sexual intercourse decline. The impotence margin after TVPE or TURP is not statistically significant. On the other hand, the reawakening of the sexual activity at the patients, who had already not experienced intercourse preoperatively, was recorded only in 6 cases (1.5%). The unchanged ejaculation after surgery argued 13.1% of patients, the weaker ejaculation was recorded in 21.6% of patients and ejaculation was completely absent in 62.4% (retrograde ejaculation). Between TVPE and TURP was no significant margin found. The ejaculation damage after minimal prostatic surgery (TUIP + PRP and TUIP alone) is significantly minor (p < 0.01) than after the standard prostatectomy (TVPE and TURP). PMID- 8629160 TI - [Intubation in reconstructive surgery of posterior urethral strictures]. AB - The authors operated in recent years (1989-1993) three patients with iatrogenic strictures of the posterior urethra which developed after transvesical prostatectomy. In one instance a stricture was involved which was difficult to intubate and in two instances a stricture impossible to intubate which was treated temporarily by epicystostomy. With regard to the site of the stricture which affected partly the prostatic and membranous urethra the authors selected the method of intubation plastic surgery in the modification of Michalowski Modelski, nowadays rarely mentioned in the literature. During a period of one to two years after operation the patients were able to urinate without difficulties, the urethra was patent for intubation, in one patient mild stress incontinence persists. The authors consider intubation plastic surgery, making use of the intact urethra as a suitable alternative method of treatment of short strictures in the prostatic and membranous part of the urethra. It is a relatively simple one-stage operation with satisfactory functional results. PMID- 8629161 TI - [Prevention of urinary infections as a complication in endoscopic surgery]. AB - With regard to the changing resistance of microorganisms it is important to develop and test steadily new chemotherapeutic agents. The authors used the preparation CIPROBAY of Bayer Co. (ciprofloxacin) prophylactically in 60 patients of a group before instrumental endoscopic examination of before endoscopic operation. Evaluation of the results of Ciprobay administration revealed a high antibacterial and very good tolerance and simple administration of the preparation. PMID- 8629162 TI - [Implantation of inflatable penile prostheses as an effective alternative surgical treatment for erectile disorders]. AB - The authors present an account of their own experience with results and complications of inflatable penile prostheses implanted in a group of 30 patients with organic erectile dysfunction. They discuss possible complications of this operation as well as the place of penile prostheses in the treatment of erectile dysfunction. PMID- 8629163 TI - [An unusual case of plastic penile induration]. AB - The presence of calcified plaques is one of the few unequivocal indications for surgery of Peyronie's disease. The mean size of plaques described in the literature is 1.5-2 cm whereby it may vary from 1 mm all along the penis. The authors treated successfully an unusually large calcified plaque 7 x 0.8 cm with excellent postoperative results--restoration of the patient's full sexual activity which was not possible before operation. PMID- 8629164 TI - [Treatment of iatrogenic renal vascular lesions using selective renal embolization]. AB - Two patients--one man (62 years) and one woman (69 years) with a iatrogenic renal arterial lesion were treated by percutaneous embolization. Both patients had undergone percutaneous nephrolitholapaxy. Both developed massive haematuria after surgery. Because haematuria could not be controlled by conservative means, angiography was indicated and subsequent superselective embolization of the bleeding artery. In both patients Gelfoam particles were used. The operation was without complications in both patients and produced a prompt effect. Within 4 months after operation no relapse developed. The authors did not observe an adverse effect associated with embolization. PMID- 8629165 TI - [Diagnosis and treatment of benign prostatic hyperplasia in the North Bohemia Region in 1994]. AB - The author investigated equipment, diagnostic and therapeutic procedures in benign hyperplasia of the prostate in urological in-patient departments in the North Bohemian region in 1994. The assembled data from nine health institutions revealed relatively good equipment with instruments for the diagnosis of BHP. The diagnostic procedures were consistent with recommendations of WHO. For surgical treatment transurethral prostatectomy was used in 62.8%, open prostatectomy in 32.1% and transurethral incision of the prostate in 5.1%. As regards non-surgical treatment, in three departments thermotherapy was used and in one laser therapy. PMID- 8629166 TI - [Traumatic dissection of the duodenal wall in a child]. AB - The authors present the case of a 6-year-old girl admitted to the department three days after a blunt abdominal injury. On surgical revision a dissecting haematoma of the wall of retroperitoneal duodenal sections was revealed. After its treatment and nutritive puncture jejunostomy the subsequent postoperative course was free from complications. This is an infrequent GIT injury in childhood which can be treated also by conservative methods. PMID- 8629167 TI - [Contusion of the lungs in childhood]. AB - The authors evaluated in a retrospective study a group of 45 patients with contusion of the lungs (age 1 to 15 years) who were treated at the Faculty Hospital in Motol in 1989-1993. In 38 children contusion of the lungs was part of multiple injuries with signs of failure of vital functions. These children were admitted after the injury to the in-patient department of the Anaesthesiological and Resuscitation Clinic. Seven children without alterations of vital functions were treated at the Department of Paediatric Surgery. The diagnosis was based on the case-history, clinical examination, X-ray examination and laboratory signs of lung injuries. An isolated pneumothorax was found in 17.8% of the injured children, haemothorax in 8.9% and in 24.5% pneumothorax and haemothorax. Severe craniocerebral injuries were diagnosed in 71% of patients, 37.8% children had concomitant injuries of the abdominal organs and retroperitoneum and 42% had fractures of the long bones. 46.6% of the children had rib fractures. The condition of 35 injured children called for endotracheal intubation (on average for 17 days) and artificial ventilation (on average for 14.4 days). 98% of the injured children were given antibiotics, in 84% steroids were administered, in 18 of the injured children (40%) drainage of the thoracic cavity was essential. Six patients (13.3%) drainage of the thoracic cavity was essential. Six patients (13.3%) died--the cause of death were severe injuries of the CNS. PMID- 8629168 TI - [The first 100 laparoscopic procedures in children]. AB - Laparoscopic operations (LO) of adults are performed as a routine in many departments, while LO in children are only at their onset. During September 1994 to May 1995 at the Clinic of Paediatric Surgery and Traumatology in Brno the first 100 LO and two thoracoscopic operations were performed. The group is formed by children aged 3 to 17 years. Their mean age was 10.6 years. The number comprised 14 acute operations, the remainder were planned operations. There was no perioperative fatality. Complications included one abscess of the Douglas bag, once the necessity of minilaparotomy on account of minor haemorrhage after resection of Meckel's diverticulum by means of endoGIA and once because the size of the tumour. Early conversion was necessary in two patients. PMID- 8629169 TI - [Effectiveness of dilatation of distal urethral stenosis in girls]. AB - Dilatation of distal urethral stenoses is one of the relatively frequent minor operations in child urology. The authors present a group of 830 female patients age 2-16 years treated by dilatation of the urethra in 1988-1994. According to the predominating finding they divide the cases into three groups--vesicoureteral reflux (VUR), infection of the urinary pathways (IUP), enuresis--and in these they evaluate the effectiveness of the operation, which was greatest in the group of patients with IUP (37%). Finally they discuss both possibilities of treatment of stenoses--dilatation and urethrotomy. PMID- 8629170 TI - [Current results of endoscopic therapy of esophageal varices in children]. AB - The authors present their experience with the endoscopic treatment of oesophageal varices in children. During 1991-1994 they treated 21 children where they performed a total of 156 sclerotizations. The mean age of the patients was 10.08 years. In 17 instances the prehepatic type of portal hypertension was involved, in four the intrahepatic type. In 13 children the varicosities were eradicated. A relapse was recorded in three cases. From the total number of sclerotizations the authors recorded complications 14 times. They emphasize the success of the method used: a minimal number of complications; a reduction of the number of pretentious devascularization operations in haemorrhage from oesophageal varices. Also the number of sclerotizations proper decreased, as many patients suffer no longer from haemorrhage and they do not have varices which require sclerotizations. PMID- 8629171 TI - [Treatment of congenital diaphragmatic hernia using ECMO]. AB - The authors present a case dealing with the treatment of a congenital diaphragmatic hernia. The procedure comprises surgery, extracorporeal membrane oxygenation--ECMO, classical and non-conventional pulmonary ventilation and pharmacological treatment. By using these methods it is possible to treat successfully a certain group of neonates with a congenital diaphragmatic hernia. PMID- 8629172 TI - [Teamwork in saving the life and lower extremities of a child]. AB - The authors describe the case of a successfully cured severe multiple injury in a seven-year-old boy, who after falling from his bicycle under a lorry suffered a brain injury, injury of the small intestine and of the neurovascular bundle in the retroperitoneal and inguinal area. The author emphasizes that in multiple injuries the life can be saved and best possible function recovered only by a correct algorithm of different surgical procedures in therapeutic care focused first on saving the patient's life and subsequently on restoration of all important functions and application of the maximum of contemporary therapeutic possibilities. The authors describe the life saving revision of the abdominal cavity and arrest of massive haemorrhage, the urgent reconstruction of blood vessels of the left inguinal area made within six hours after the accident and the delayed reconstruction of the femoral nerve made within four months after the injury. The resultant condition is evaluated after a six-year period. The general condition of the injured boy is normal, the functional condition of the left lower extremity can be evaluated clinically and on the basis of EMG as excellent. PMID- 8629173 TI - [Disorders of intestinal rotation and fixation in older children]. AB - The authors discuss five older children where malrotation of the gut was diagnosed. The youngest child was three years old, the oldest 16 years. In three children the malrotation was diagnosed from clinical symptoms and examination by imaging methods, in two it was detected by chance during operation. In the two children where the impaired rotation of the gut was detected on operation, non rotation was involved. In the remaining three children twice a left side paraduodenal hernia was diagnosed and once an external stenosis of the duodenum by Ladd's bands. PMID- 8629174 TI - [Malformation of the atrioventricular valve in atrioventricular septal defects and methods of plastic reconstruction in children]. AB - The authors studied the surgical anatomy of atrioventricular valves (AV) in defects of the atrioventricular septum (AVSD) in a group of 73 preparations of the heart. Evidence was provided of a continual spectrum of changes of the entire valvular apparatus from a common AV orifice (71.2%) to two-separate orifices (28.8%). In the defect with a common orifice the authors differentiated six grades of overlapping of the left-sided component of the anterior common cusp with the right ventricle. In 12.3% hearts the authors revealed stenosis of the left AV orifice, which was most frequently caused by a thickened valve with reduced mobility. In 19.2% hearts the authors found a potentially stenotic configuration of the left AV orifice. From the surgical aspect it was possible to divide AVSD into the complete, transitional or partial type. The results of the morphological investigation were used during correction of 121 patients with AVSD. Of those 54 suffered from the complete form, 10 from the transitional and 57 from the partial form. During correction the authors closed the defect by using one or two patches and they performed an aimed plastic operation of the AV valves. The plastic repair involved closure of the cleft of the septal cusp of the left AV orifice, commissuroplasty, suture of abnormal commissures and straightening of irregular margins of the valve. In eight patients the plastic operation could not be performed because of a potentially stenotic configuration. The early mortality rate was 7.5% in the partial and transitional form and 18.5% in the complete form. The results were adversely affected by an unfavourable anatomy of the left AV orifice with severe regurgitation. From the great variability of the anatomy of AV valves and the authors' clinical experience ensues the necessity of an exact surgical solution of AVSD with an aimed and individually modified reconstruction of AV valves. PMID- 8629175 TI - [Colo(ileo)rectoplasty in the treatment of Hirschsprung's disease and congenital neural malformations of the distal intestines]. AB - The objective of the presented paper is to define the characteristics of colo(ileo)rectoanastomosis for the treatment of Hirschsprung's disease (H. d.) and other congenital malformations in the innervation of the distal gut (CMDI). During 1979-1994 at the Clinic of Paediatric Surgery of the Second Medical Faculty in Prague-Motol 137 patients (100 boys and 37 girls), aged 5 months to 18 years with H. d. and CMDI were operated. In 124 patients Kasai's colorectoplasty was used, in 40 of them supplemented by partial sphincteromyectomy of the internal sphincter of the anus (SPME) and Swenson's transanal colorectal anastomosis. In 10 patients with total aganglionosis of the colon (TCA) in three instances ileorectoplasty and Martin's anastomosis was used, in seven instances only ileorectoplasty. In three patients the authors used Soave's endorectal pull through. 85 patients (62.1%) had no postoperative complications. Early infection was recorded in 12 patients (8.6%), dehiscence of the surgical wound in seven patients (5.1%). Dehiscence of the colo(ileo)rectorectal anastomosis occurred in 13 patients (9.5%), stricture in 10 patients (7.2%). Postoperative obstruction of the gut was recorded in 7 (5.1%) patients, postoperative enterocolitis in three patients (2.2%). There were no deaths. Regular opening of the bowels after 1-2 day intervals was achieved in 110 patients (84.1%). Patients after surgery of TCA have on average 2-5 stools per day. Sixteen patients developed chronic constipation and subileous conditions. Incontinence of faeces was found in two patients with Down's disease. Colo(ileo)rectoplasty in H. d. and other CMDI should meet the following conditions: maximal resection of the affected portion- creation of a satisfactory anastomosis with a minimal occurrence of strictures and dehiscences--preservation of satisfactory continence--elimination of anorectal sphincteroachalasia. Modified Kasai's rectoplasty or Swenson's procedure meet these conditions. In case of TCA a 10-15 cm ileorectocolic anastomosis is quite sufficient. For reoperations of strictures and inflammatory complications after previous colo(ileo)rectoplasty Soave's technique is probably the best choice. PMID- 8629176 TI - [Prospective comparison of laparoscopic surgery of inguinal hernia using pre peritoneal surgical mesh implantation with the classic open approach]. AB - The authors focused their attention on a comparison of laparoscopic hernioplasty and the classical operation of inguinal hernia in a prospective study. The study comprised fifty consecutive patients operated at the First Surgical Clinic in Olomouc between November 1994 and April 1995. The study compares surgical and postoperative complications, postoperative pain and the hospitalization period. Every patient was followed up for one month after operation. The results indicate that laparoscopic hernioplasty is an equally valuable method as classical surgery of inguinal hernia. According to the authors the main advantage of laparoscopic plastic surgery is a better postoperative course--i.e. less pain, a shorter hospitalization period and more rapid return to full activity. The disadvantage is the intraabdominal approach and all possible complications ensuing from it. PMID- 8629177 TI - [Tension-free surgery of femoral hernias using the Lichtenstein method]. AB - Based on 12 operations, the author report on the advantages of "plug tension free" plastic operations. The femoral canal is along its entire course sealed by a cylinder of rolled up polyester net. This operation reduces the postoperative discomfort and danger of the development of a relapse. PMID- 8629179 TI - [Successful acute aortomesenteric bypass as a result of favorable circumstances]. AB - The author presents an account of a case-history involving ischaemia of the entire small intestine, an acute vascular episode, caused by occlusion of the cranial mesenteric artery. The successful aortomesenteric bypass is presented as an example of the coincidence of favourable circumstances in the solution of the case. PMID- 8629178 TI - [Lymphatic mapping and biopsy of sentinel nodes--a new approach in lymphadenectomy. Review]. PMID- 8629180 TI - [Obstructive ileus--a complication of laparoscopic cholecystectomy]. AB - A 45-year old patient admitted with confirmed calculous pancreatitis underwent the laparoscopic cholecystectomy at the 14th day of the hospitalization. 4 days after the operation he had to be reoperated due to small-bowel-obstruction caused by the herniation of the loop through the supraumbilical port. The small bowel was reduced and plasty of the operative wound was done. Postoperative course was uncomplicated and the patient was discharged from the hospital 6 days after the operation. PMID- 8629181 TI - [The beginnings of the Czech Surgical Society]. AB - The authors present a brief of the position of Czech Surgery in medicine during the last century. They mention the conditions for association of the Czech doctors in the second half of the 19th century as a manifestation of national revival efforts. In 1860 the Czech Medical Society was founded and in 1862 the first issue of the Czech Medical Journal was published. The climax of Czech desires of revival was the separation of Czech and German clinics in 1883. The development of some medical disciplines, incl. surgical disciplines, made the surgeons abandon the Czech Medical Society and after the establishment of the committee of the Czechoslovak Republic they founded in 1920 the committee of the Czechoslovak Society for Surgery and Gynaecology. In April 1921 the first congress of the Czechoslovak Surgical and Gynaecological Society was held. At this occasion the first issue of the Surgical and Gynaecological Review (Rozhledy v chirurgii a gynaekologii) was published. Both events laid the foundations of the contemporary Czech Society of Surgeons. PMID- 8629182 TI - [Advantages and disadvantages of cervical esophageal anastomosis using a stapler]. AB - The authors evaluate the advantages and shortcomings of cervical anastomosis of the oesophagus by means of stapler according to the following criteria: 1. difficulty of implementation, 2. immediate results--fistulae, 3. long-term results--stenoses, 4. costs. The authors operated, using a stapler, 26 patients who developed in four instances /15.4%/ a fistula. By comparison with a retrospective group of 21 patients with eight fistule /38%/ where the anastomosis was sutured manually, they found that by using a stapler the number of complications declined. A disadvantage is the high price of staplers. PMID- 8629183 TI - Endoscopic transthoracoscopic sympathectomy--the Durban experience. AB - Advances in optics, illumination and video-technology together with refinements in operative technique have made endoscopic transthoracoscopic sympathectomy (ETS) the method of choice for upper thoracic sympathectomy. Palmar hyperhidrosis is by far the main indication for ETS. The procedure is technically easy and well tolerated by patients, and complications are few and minor. PMID- 8629184 TI - Acute traumatic rupture of the thoracic aorta. A comparison of techniques. AB - Twenty-eight patients were treated for acute blunt thoracic aortic rupture at Groote Schuur Hospital between January 1984 and March 1994. Aortic arch ruptures occurred in 2 patients and were successfully repaired by means of hypothermic circulatory arrest. Descending aortic ruptures were repaired more safely by insertion of an interposition graft as opposed to direct suture reapproximation, and with the aid of partial heparinless bypass as opposed to simple aortic cross clamping. PMID- 8629185 TI - Volvulus of the sigmoid colon at Baragwanath Hospital. AB - Fifty-six patients treated for sigmoid volvulus over a period of 5 years are reviewed. The purpose of this study is to evaluate our management policy and to compare it with other studies, particularly from other parts of Africa. The patients were evaluated by retrospective allocation into one of three treatment modalities. Patients with clinical evidence of ischaemia underwent a Hartmann's procedure. There was a 33% mortality rate in this group. In those patients who had sigmoidoscopic reduction, a second-stage sigmoidectomy and primary anastomosis were performed. The results in this group were excellent. In the group of patients with failed sigmoidoscopic resection the majority underwent a Hartmann's procedure with a mortality rate of 15%. PMID- 8629186 TI - Primary gastric tuberculosis. A report of 3 cases. AB - Gastric tuberculosis is an uncommon clinical entity. Great difficulty is encountered in making the pre-operative diagnosis. Other chronic granulomatous conditions (Crohn's disease, sarcoidosis and isolated granulomatous gastritis) should always be considered. Surgery, with postoperative antituberculosis treatment, remains the main modality for accurate diagnosis and treatment. PMID- 8629187 TI - Surgical management of worm volvulus. AB - A retrospective study of 50 consecutive patients with small-bowel volvulus complicating intestinal ascariasis is reported. The commonest presenting features were abdominal distension (44 patients) and abdominal pain (38 patients). Thirty three patients presented with vomiting, of whom 8 vomited worms. Twenty-five patients presented with peritonitis. In 33 patients there was evidence of worm infestation on radiographs while volvulus was diagnosed radiographically in 16 patients. Thirty-two patients required emergency surgery, while 16 were observed for a mean of 2.6 days before surgery. Thirty-nine patients had gangrenous bowel that required resection. The overall mortality rate was 14.5% and all deaths were from the gangrenous group. The results show that volvulus complicating ascariasis still carries a high mortality and morbidity rate. We stress the importance of early detection and early operative intervention. PMID- 8629188 TI - Metastatic carcinoma in the neck from an occult primary lesion. AB - Over an 11-year period, from January 1980 to December 1990, 23 patients with metastatic carcinoma of the neck were admitted to Addington Hospital with an unknown primary lesion. More than 50% of patients presented with disease in the N3 nodal stage. Sixteen patients had squamous cell carcinoma, 6 undifferentiated carcinoma and 1 adenocarcinoma. The diagnoses were made by means of fine-needle aspiration cytology and histological assessment of the neck nodes. Twenty-one patients were treated with radiotherapy and 2 with surgery. The primary tumour became apparent in only 4 (17%) patients, 7, 8, 9, and 12 months respectively after initial presentation. The sites were the base of tongue in 2, the pyriform fossa in 1 and the scalp in the remaining patient. The overall 3-year and 5-year survival rates were 26% and 17% respectively. PMID- 8629189 TI - The relief of acute pain. PMID- 8629190 TI - Massive haemoperitoneum due to spontaneous rupture of ovarian cyst in children. A report of 2 cases. AB - Two girls with haemoperitoneum and haemorrhagic shock caused by spontaneous ovarian cyst rupture are described. The first case could not be diagnosed pre operatively, and cyst excision and repair of the ovary were carried out at emergency laparotomy. The second case was diagnosed by ultrasound, and conservative treatment was successful. PMID- 8629191 TI - Chondroid syringoma of the abdominal wall. A case report and review of the literature. AB - A case of an unusually large chondroid syringoma of the abdominal wall is described. Chondroid syringomas are rare neoplasms believed to be of sweat gland origin. They are often slow-growing and benign. Local recurrence and distal spread characterise the malignant form. PMID- 8629192 TI - The influence of surgical training on the practice of surgery. Are changes necessary? AB - Changes in health care financing are having a significant effect on surgical practice. These changes require adjustments in what surgical trainees must learn as well as where and how they must learn. To ensure educational quality, surgical educators must have a clear definition of their educational mission. PMID- 8629193 TI - The management of hernia. Considerations in cost effectiveness. AB - Approximately 700,000 herniorrhaphies are performed annually in the United States for primary, recurrent, and bilateral inguinal hernias. This article describes the components of cost regarding the approach and management of groin hernias. The trends toward outpatient procedures, regional anesthetic agents, and early return to work are analyzed. The common types of repair are compared with reference to recurrence and complication rates. The advances and results of laparoscopic hernia are reviewed. In summary, a cost-effective approach for the management of inguinal hernias is presented that could reduce the yearly cost of hernia repair by hundreds of millions of dollars. PMID- 8629194 TI - "Routine" preoperative studies. Which studies in which patients? AB - The utility of mass screening of preoperative patients has never been demonstrated for the majority of tests. Although screening patients to uncover occult disease appears logical, in reality it has resulted in excessive expenditure of our health care dollars with limited benefit. More than $30 billion is spent annually on preoperative examinations, 60% of which are unnecessary. In addition, iatrogenic injury has resulted from the further evaluation and treatment of false-positive results. A selective utilization of routine examinations can accurately supplement the clinician's evaluation, providing the patient with a complete preoperative assessment. The benefits of selective testing must be balanced against the possible omission of warranted examinations, highlighting the need for a more reliable system for test ordering. PMID- 8629195 TI - Managing gallbladder disease in a cost-effective manner. AB - The advent of laparoscopic cholecystectomy has resulted in an increase in total health care costs for gallstone disease despite a reduced unit cost for the procedure. The total number of cholecystectomies performed annually has increased, reflecting an alteration in referral patterns and threshold for surgery in these patients. Cost-effective management of gallstones is possible by adopting strategies to limit length of stay, with discharge possible on the day of surgery for many patients. Selective cholangiography, laparoscopic common bile duct exploration, and judicious postoperative use of ERCP also have a role in cost-effective gallstone treatment. Institutions must analyze all costs and patterns of use to make informed decisions about reusable and disposable instrumentation. PMID- 8629196 TI - Technology and surgery. Dilemma of the gimmick, true advances, and cost effectiveness. AB - The key to evaluating a procedure in regard to a true advance versus a gimmick is to determine its value. This can be done only by physicians cognizant of a disease process. The value is determined by assessing a procedure's utilization, outcomes, and costs. Utilization allows early treatment and avoids neglected disease. Therefore, the appropriateness of the utilization can be determined only by an outcome study. An outcome study is another term for quality assessment. Outcomes deal with morbidity, mortality, and also the long- and short-term effects of the procedure on the disease. Overall, an increase of quality in a global perspective decreases the costs of the procedure to the health care community. Costs must remain secondary to outcomes. An attempt to decrease costs directly is a maneuver that, when applied by nonmedical individuals, will most likely decrease quality. When the quality can be maintained (as assessed only by a practitioner), then a decrease in global costs will increase value. The concept of increasing value by increasing quality without an attempt to decrease costs is a very important principle that the health care system must learn in our ever challenging medical environment. Is a new procedure a gimmick or a true advance? The decision is made jointly by the stakeholders in our health care system--the patient, provider, payer, employer, and industry. If the procedure does not receive negative votes, then its adoption is almost assured. Comparing two procedures through these perspectives ultimately allows us to determine the potential for new procedures. A procedure not adopted through this method could be called a gimmick. PMID- 8629197 TI - Quality assurance and medical outcomes in the era of cost containment. AB - Market forces are driving health care organizations to "prove" quality while diminishing costs. Payers for health care, led by large employers and insurance companies, are demanding clinical, financial, and satisfaction outcomes from providers. To meet the challenge, traditional quality assurance based on inspection and rooting out "bad apples" is rapidly being replaced by the industrial engineering principles of continuous quality improvement. A philosophical shift is occurring from a focus on episodes of care delivered by physicians to the delivery of processes of care by teams of health care personnel. We are seeing a shift in emphasis from a fascination with intensive care delivered to sick patients to cost-effective preventive services delivered to populations of well patients. The locus of care delivery is moving from inpatient hospitals to ambulatory clinics and home care. The need for this information is leading to innovation in computer systems and health care organizations. New partnerships are emerging between physicians, nurses, and hospitals. Traditional oversight bodies including the JCAHO and the HCFA sponsored PROs are restructuring to meet these new demands. New organizations such as the National Committee on Quality Assurance and state governmental agencies are being established to fill the perceived void. Individual surgeons have begun to receive performance data on their individual and group practices. Professional societies have collaborated in the development of clinical guidelines and outcomes data bases. This massive reorganization will take several more years to play out. With careful development it has the potential to dramatically improve patient care through the efficient application of new scientific knowledge and the sustained flow of information back to physicians and patients. PMID- 8629198 TI - How to increase efficiency in the operating room. AB - The key to increasing operating room efficiency is increasing productivity. Standardizing and streamlining of internal procedures reduce bottlenecks, and computers speed the flow of information so that continuous improvement of the system becomes possible. Patterns and themes can be discovered only when one sits back and listens and watches, shifting the focus from fixing problems to discovering patterns and the structures underlying them. Rethinking the system and evaluating all aspects of the care delivery cycle, abandoning the "sacred cows" of operating room practice, and creating a vision for health care in the future are essential to survival in the managed care environment. PMID- 8629199 TI - Cost effectiveness in the intensive care unit. AB - Effective policies to reduce true costs will require integrated information systems and demand behavioral changes from providers. A congenial environment must be created among medical educators, providers, vendors, and consumers if cost reduction is to be accomplished without compromising quality or access to critical care services. Physicians should do everything they believe may be of benefit for their patients, but we have an obligation to educate the public about the limitations of our art and the fact that "doing everything" is not always best for the patient or the grieving family. A significant method of controlling ICU costs is closely monitoring which patients are admitted and when they are discharged. Laboratory tests represent a source of cost reduction, and physicians must learn to order specific tests and not simply a battery of tests which includes the actual test desired. Limits should be placed on the tests that are ordered in terms of number and frequency. Improved efficiency of the utilization of resources should improve the care of our patients. The largest budget item of any or most critical care units is nursing; it is paramount that this essential and invaluable resource be utilized in a cost-effective manner. Diminishing unnecessary activity will both decrease complications and have salutary effects. Having more time to be with patients and their families will decrease our sense of failure and fulfill the important goal of caring. Physicians and nurses can return to thinking, assessing, and decision making instead of frenetically ordering, reacting, and intervening, which, we believe, accurately describes informational overload created by undue emphasis on high technology. In this way, we can respond to Fuch's exhortation that "physicians consider the possibility of contributing more by doing less." In responding, however, we must never forget that the societal, not merely the economic impact of medical care, is our principal consideration. We must first contribute more by achieving a greater understanding of the medical care process. Only then can we know how to do less at the bedside. We can and must distinguish between costly and high-quality care- they are not necessarily synonymous. PMID- 8629200 TI - Cost-effective preoperative evaluation, operative treatment, and postoperative follow-up in the breast cancer patient. AB - Although medical criteria based on retrospective and prospective studies form the basic foundation for the strategies enunciated in this article, efficiency in management and cost effectiveness follow because of the simplification that has been found to be appropriate by analyzing reports over the past decade and using common sense. In addition, the realization of the effectiveness of mammographic screening programs should encourage all surgeons to promote and practice yearly mammographic screening in appropriately aged women. At present, this means women between the ages of 50 and 75, but there is increasing evidence that women between the ages of 40 and 49 may benefit. A compromise would suggest women over 45 years of age as appropriate, recognizing the controversy now raging over the effectiveness of screening in women 40 to 49 years old. Wide appreciation of the major gains in breast cancer detection by use of mammographic screening programs needs to be emphasized. We have predicted that within a decade the median maximum diameter of all invasive breast cancer in the United States will be only 1 cm if mammographic screening programs are widespread. If this occurs, there would be extraordinary gains in reduced patient morbidity and cost savings and undoubtedly changes will occur in the previously unyielding age-adjusted mortality rate. The goal of entirely outpatient management in the vast majority of breast cancer patients can be achieved by such mammographic screening as well as by the adaptation of induction chemotherapy and breast conservation in the majority of advanced primary breast cancer patients that still appear. PMID- 8629202 TI - Cost effectiveness in trauma care. AB - The above discussion brings together a vast body of data that together proclaim with fervent clarity: Traumatic injuries are expensive. The expense is paid in productive lives lost, in permanent disability, in pain and suffering, and in health care resources consumed. As local and regional trauma systems struggle for development and survival, competition for the health care dollar casts in the additional necessity of providing the service of trauma care with maximum efficiency. Despite the variety of cost-efficiency measures described above, a majority of trauma centers continue to operate "in the red." Such cannot continue indefinitely. Fiscal responsibility dictates that health care institutions must balance budgets in order to maintain operations. Four primary strategies for cost containment appear from the above discussion: 1. Improve reimbursement rates from trauma patients. 2. Increase outside funding from government sources. 3. Improve cost efficiency of diagnostic and therapeutic procedures used in trauma patient management. 4. Increase efforts aimed at primary prevention of intentional and unintentional injuries. In the final analysis, most authors agree that the last strategy offers the best hope. As stated in their article, "The Economic Impact of Injuries," Harlan and colleagues conclude that "the most effective medical and cost reduction strategy would be prevention." The same article goes on to detail how greater funding for research into optimal prevention modalities could reap societal and economic benefits far beyond the value of the initial outlay. Yet such research funding continues to be inadequate. For every dollar spent on medical care of cancer patients, nine cents is directed to research. For every dollar spent on trauma care, less than a penny is spent on research. Until the public recognizes the terrible toll trauma extracts in lives, livelihood, and money wasted and until it realizes the pre-eminent importance of prevention, care of the trauma patient cannot truly achieve cost efficiency. PMID- 8629201 TI - Screening of average-risk individuals for colorectal cancer and postoperative evaluation of patients with colorectal cancer. AB - Screening of the average-risk individual for disease creates a different relationship between practitioner and subject than the normal doctor-patient relationship. Screened individuals are asymptomatic, have a whole range of motivations from denial to neurosis, and, for the most part, derive no benefit from the screen, as they are destined never to contract the disease. Rewards to the few must clearly outweigh the risks to the many. The screening strategy must be economical when applied to a large population, safe, and effective in achieving the ultimate goal, which, in the case of colorectal cancer, is reduction in the mortality rate. Prediagnostic screening of asymptomatic adults over the age of 45 years for primary disease has been shown to fulfill all of these criteria. Postoperative screening of asymptomatic individuals after curative resection of colorectal cancer for recurrence has been shown to fulfill none of them. PMID- 8629203 TI - Malignant obstructive jaundice. Evaluation and management. AB - A large number of laboratory tests, radiologic studies, and endoscopic techniques are available for the evaluation of the jaundiced patient. Similarly, the therapeutic options have increased with the development and improvement of endoscopic, percutaneous, and laparoscopic procedures, and the morbidity and mortality rates associated with open surgery have decreased. The challenge is to select, on an individual basis, the most efficient and cost-effective evaluation as well as the management with the lowest morbidity and mortality rates and the best short- and long-term goals. PMID- 8629204 TI - Cost-effective evaluation and management of the acute abdomen. AB - 1. Managed care gate-keeper financial responsibility must be balanced with specialist diagnostic and therapeutic responsibility to maximize cost effectiveness and quality of medical service. 2. Cost in health care is closely tied to length of stay and operating room time; extremes of patient age are associated with increased costs. 3. Through years of training and subsequent experience, surgeons are best qualified to direct work-up of patients with an acute abdomen, especially when the work-up requires costly imaging (e.g., CT scan, ultrasonography, angiography). 4. Cost is increased when the surgeon is not involved early in cases of acute abdomen. 5. Surgeons must be willing to enter the evaluation phase of the patient with an acute abdomen patient early and follow during the observation period to best reduce unnecessary laboratory work or tests. 6. Health care teams willing to implement pathways and then document the effect such pathways exert will be most successful in assessing new technologies' cost effectiveness. PMID- 8629205 TI - A cost-effective approach to the patient with peptic ulcer bleeding. AB - The average hospital cost to manage patients hospitalized at Virginia Mason Hospital who bleed from a peptic ulcer is approximately $5000 per patient in our series of 30 patients. Because there are 150,000 admissions per year in the United States for peptic ulcer bleeding, the total hospital cost can be estimated to be $750 million. The actual cost may be higher because our 30 patients had minimal complications and were discharged on average in less than 4 days. The majority of hospital cost is incurred by the intensive care unit or the hospital nursing floor. There is a close to linear relation between the length of stay and the total hospital cost. Upper gastrointestinal endoscopy is a major advance in the treatment of peptic ulcer bleeding. It can provide significant cost savings by identifying some patients with bleeding peptic ulcers who have clean bases on endoscopy who are then eligible for prompt discharge from the hospital. In addition, endoscopic thermal therapy (with multipolar electrocautery or heater probe) and injection therapy cost less than $50 in incremental cost and can reduce further bleeding by 43%, reduce the need for urgent surgery by 63%, and reduce the mortality rate by 60%. Some patients still require urgent surgical intervention, which is substantially more costly than endoscopic hemostasis but is highly effective. Preliminary studies show promise in predicting further bleeding, with clinical scoring systems such as the Baylor Bleeding Score and with the use of Doppler ultrasonography. Better prediction of further bleeding should guide the choice of durable hemostasis early in the hospitalization. Additional studies should clarify the role of NSAID avoidance and H. pylori eradication in the long-term prevention of recurrent peptic ulcer bleeding. PMID- 8629206 TI - Is outpatient laparoscopic cholecystectomy wise? AB - The authors report a prospective analysis of their experience with 506 consecutive laparoscopic cholecystectomies to examine the appropriateness of outpatient or same-day laparoscopic cholecystectomy. Thirty-eight patients experienced at least one postoperative complication. The complication was clinically evident or suspected in only 4 of these 38 patients within 8 h following surgery. Thirty-nine percent and 76% of complications were clinically detected at 24 and 48 h, respectively. Nausea and vomiting occurred among 32% of all patients on the day of operation and extended into the 1st postoperative day in 10%. Compared to predicted values, forced vital capacity was 61 +/- 5% 1 h postoperatively in 32 patients studied. At 6 and 24 h postoperatively, forced vital capacity was 63 +/- 7% and 66% respectively. Postoperative analgesic medication requirement was determined in 220 patients who were provided with a patient-controlled intravenous morphine analgesia machine with no basal rate. Consumption of morphine was highly variable but substantial on the day of operation: 17 +/- 16 mg. Most complications of laparoscopic cholecystectomy, including life-threatening complications, are not apparent by 8 h postoperatively and may not be apparent at 24 h. The potential for delay in the diagnosis and treatment of complications, variable but substantial analgesic requirements, impaired postoperative ventilation, and postoperative gastrointestinal dysfunction argue for the need to use great caution in selecting patients for outpatient laparoscopic cholecystectomy. Criteria are proposed to identify patients who are safest for outpatient laparoscopic cholecystectomy. PMID- 8629207 TI - Laparoscopic ultrasonography as compared with static or dynamic cholangiography at laparoscopic cholecystectomy. A prospective multicenter trial. AB - We compared laparoscopic ultrasonography (LICU) with static (S) or dynamic (D) cholangiography (IOC) for assessment of duct anatomy an calculi in 209 patients. LICU visualized ducts in 88% compared with 93% for IOC (P = 0.046). Nineteen patients (9%) had stones: 17 were found by LICU (89%) and 10 (53%) by IOC (P = 0.032). Time to perform LICU (7 +/- 3 min) was less than IOC (13 +/- 6 min) (P < 0.0001). Time to perform SIOC (12 +/- 5 min) and DIOC (14 +/- 6 min) did not differ (P = 0.48), nor did these tests differ in accuracy. LICU provided useful anatomical information but IOC better defined anatomic anomalies. LICU required less time but was less reliable at defining anatomy and complete duct visualization. LICU was more sensitive for stones. SIOC and DIOC did not differ objectively. LICU and IOC are complementary. PMID- 8629208 TI - Bile leakage following laparoscopic cholecystectomy. AB - Laparoscopic cholecystectomy (LC) is now the treatment of choice for gallstones, but there has been concern that bile leakage with LC is more frequent than after open cholecystectomy (OC). We have analyzed our experience of this complication with regard to both its incidence and management. From a consecutive series of 500 LC, in which both operative cholangiography and drainage of the gallbladder bed were routine, bile leakage was identified in ten patients (2%). There was no bile duct injury. Nine of the ten patients presented with bile in the drain within 24 h of operation and one patient presented 1 week after operation with a subphrenic collection. Of the ten patients, five settled spontaneously. Of the five remaining patients, two needed laparotomy--one for a subphrenic collection not responding to percutaneous drainage and one for biliary peritonitis. One patient was treated by relaparoscopy and suture of a duct of Luschka and one patient had successful percutaneous drainage of an infected collection; the fifth patient who presented with a late subphrenic collection of bile was shown at endoscopic retrograde cholangiopancreatography (ERCP) to have a cystic duct stump leak and was treated with an endoscopic stent. Bile leakage is seen more frequently after LC than OC for reasons that are currently unclear. We believe that the use of routine gallbladder bed drainage is justified for this reason alone. The majority of bile leaks settle either spontaneously or with minimally invasive intervention. PMID- 8629209 TI - Laparoscopic splenectomy in children. Preliminary results and comparison with the open technique. AB - To determine the safety and efficacy of laparoscopic splenectomy (LS) in children, a retrospective review of our preliminary experience using LS was compared to results in patients who previously underwent open splenectomy (OS). From July 1993 to January 1995, we performed eight LS procedures in six children with hereditary spherocytosis (HS) and two with immune thrombocytopenic purpura (ITP). Laparoscopic cholecystectomy was simultaneously done in one case with HS. There were 4 males and 4 females who ranged in age from 5 to 15 years--an average age of 8.8 years. Two cases in the early series required a counterincision because of bleeding. Eleven patients who previously underwent OS in our department were used to compare demographics, operative courses, and surgical outcomes. The ages, genders, diseases, body weights, and spleen weights were comparable between LS group and OS groups. The operative time for the LS group was statistically longer than for the OS group (226 +/- 24 min vs 101 +/- 8 min, P < 0.001). The estimated blood loss in the LS group was similar to that of the OS group (100 +/- 39 ml vs 73 +/- 11 ml. P = 0.97). There were no peri- or postoperative complications in two groups. The postoperative hospital stay of LS group was statistically shorter than that of the OS (6.8 +/- 0.6 days vs 10.4 +/- .05 days, P < 0.0001). LS provided better cosmesis and minimized trauma in children over OS. LS appears to be a safe and effective procedure in children, and is useful in the management of pediatric patients with HS or ITP. PMID- 8629210 TI - Physiological outcome following laparoscopic highly selective vagotomy. A controlled study in a pig model. AB - Open highly selective vagotomy (HSV) has withstood the rigors of objective evaluation to become the optimal surgical treatment for chronic duodenal ulcer refractory to medical therapy in many centers. Laparoscopic HSV has not been subjected to the same scrutiny before entering clinical practice. A controlled animal study was conducted to demonstrate the physiological validity of laparoscopic HSV. Experimental groups underwent laparoscopic and open highly selective vagotomies, and control groups underwent a sham laparoscopic gastric mobilization or no operative procedure. Gastric acid output was measured by an aspiration technique, liquid gastric emptying was calculated by the double sampling technique of George, and gastroesophageal reflux was assessed by 8-h ambulatory pH monitoring. Laparoscopic HSV was as effective as its open counterpart in reducing basal acid output, and laparoscopic HSV did not interfere with liquid gastric emptying in contrast to open HSV. Neither open nor laparoscopic HSV was observed to precipitate gastroesophageal reflux. These data suggested that the continued use of laparoscopic HSV in clinical practice is appropriate. PMID- 8629211 TI - Laparoscopic hepatic resection for hepatocellular carcinoma. AB - Despite recent progress in diagnostics for hepatocellular carcinoma, the rate of resectability remains low, mainly because of the advancement of the underlying liver disease. We report a case of a 54-year-old man with a hepatocellular carcinoma and poor liver function that was treated successfully with a laparoscopic hepatic resection. Laparoscopic hepatic resection is considered to be feasible with the aid of an ultrasonic dissector and a microwave coagulator; however, close attention should be paid to the development of air embolism and hepatic vein injury. PMID- 8629212 TI - Video-assisted thoracoscopic resection of type I cystic adenomatoid malformation in a 3-month old girl. AB - A 3-month Chinese girl with type I cystic adenomatoid malformation involving both lobes of her left lung was successfully operated upon using the video-assisted thoracoscopic surgical (VATS) approach. The conventional endoscopic stapling device was used for resection without complications. PMID- 8629213 TI - Laparoscopic resection of a large right adrenal gland cyst. AB - A case of a benign cyst of the right adrenal gland resected laparoscopically is presented. The approach was through the right subcostal space mobilizing the right lobe of the liver and the right colonic flexure. The procedure was of 75 min duration and was uneventful. The patient was discharged the 3rd postoperative day free of postoperative pain. The advantages and disadvantages of this new modality for the treatment of adrenal gland cysts are discussed. PMID- 8629214 TI - Peritoneal seeding of gallbladder cancer after laparoscopic cholecystectomy. AB - We report a case of peritoneal seeding of an unsuspected adenocarcinoma of the gallbladder following laparoscopic cholecystectomy despite the use of a retrieval bag. The metastasis developed at the umbilical trocar site, which was also used to extract the resected gallbladder. There was no evidence foe a leak of the retrieval bag. Most likely malignant cells became desquamated during the operation, implanting themselves in the tissue during the removal of the bag. Taking into consideration previous reports and the dismal prognosis of the disease, we discuss the management in the case of an incidental carcinoma. PMID- 8629215 TI - Laparoscopic bipolar strip-tease appendicectomy. A new endosurgical technique. AB - Since its conception, several techniques have been described for laparoscopic appendicectomy. We describe a technique which utilizes a 5-mm bipolar forceps designed to coagulate and cut tissues at the same time; 50 pediatric patients underwent laparoscopic appendicectomy for acute appendicitis using this "stripping and teasing" technique. No bleeding complications occurred. There were only two postoperative abscesses in the series. We believe that the bipolar laparoscopic striptease appendicectomy technique as developed by the senior author is safe, quick, and effective, even for severe appendicitis. PMID- 8629216 TI - The use of a large, transparent cannula, with a beveled tip, for safe laparoscopic management of hydatid cysts of liver. AB - Aspiration of hydatid cysts and instillation of scolicidal fluid as part of the surgical treatment should be done very carefully to prevent any spillage that might cause anaphylaxis and dissemination of the disease. We describe herein a laparoscopic technique for safe aspiration of hydatid cysts of liver by using an assembled large, transparent trocar sleeve with a beveled tip. The method described allows laparoscopic access even to hydatid cysts that are located under the diaphragm. PMID- 8629217 TI - Laparoscopic vs inguinal hernia repairs. Outcomes and costs. PMID- 8629218 TI - Open vs laparoscopic hernia repair. Analysis of costs, charges, and outcomes. PMID- 8629219 TI - Laparoscopic colon resection for cancer. PMID- 8629220 TI - Laparoscopic vs open colon surgery. Costs and outcome. PMID- 8629221 TI - Examination of outcome and cost data of open and laparoscopic antireflux operations at Virginia Mason Medical Center in Seattle. PMID- 8629222 TI - Results of Nissen fundoplication. A cost analysis. PMID- 8629223 TI - Postoperative complications of anti-reflux surgery. Outcomes and costs. PMID- 8629224 TI - Radiosurgery patterns of practice. AB - We distributed a questionnaire on radiosurgery patterns of practice to members of the International Stereotactic Radiosurgery Society (ISRS). Responses were obtained from physicians at 52 facilities, who had treated more than 13,000 patients. Most respondents were found to work within a multidisciplinary team, and averaged 17.3 specialist-hours devoted per patient on the day of radiosurgery. These results will enable radiosurgeons to determine if their practice differs from the norm and to adjust their practice standards, if appropriate. PMID- 8629225 TI - Gamma knife radiosurgery for cerebral arteriovenous malformations: an autopsy report focusing on irradiation-induced changes observed in nidus-unrelated arteries. AB - BACKGROUND: In radiosurgical treatment for an arteriovenous malformation (AVM), the effects of irradiation on the intranidal and perinidal angioarchitectures have seldom been analyzed histologically. An autopsy case is reported, studying an AVM treated by gamma knife radiosurgery. Postmortem studies following AVM unrelated death were performed after a 2-year angiography had demonstrated complete nidus obliteration. Irradiation-induced changes were also observed in surrounding nidus-unrelated arteries and the choroid plexus, both of which were within the irradiation target. METHODS: Microscopic studies were performed using a coronal section of the brain including the center of the AVM, on which the percent isodose volume gradient, corrected with a magnification rate, was superimposed. RESULTS: This study disclosed that intimal hypertrophy can occur in a normal, AVM-unrelated pial artery due to irradiation of 10 Gy or more and that more remarkable intimal hypertrophy with fragmentation of the elastic laminae, or even complete occlusion, can occur in these arteries with 25 Gy. Similarly, irradiation-induced degeneration was present in the choroid plexus, which had been exposed to doses varying from 10 Gy to 25 Gy. CONCLUSIONS: A normal surrounding blood vessel may also be affected by high-dose, single-fraction irradiation though the abnormal vessels have been reported to be more susceptible. PMID- 8629226 TI - Holographic imaging of human brain preparations--a step toward virtual medicine. AB - BACKGROUND: Holography is the only technique that can record the full tri dimensional quality of an object, and allow the observer to easily see this as an image that is truly 3-D. The pulsed laser can capture even a moving object's three dimensional form as a hologram because of its short wavelength. METHODS: Using a modification of the Klinger processing sequence, formalin-fixed human brains were subjected to periods of freezing and thawing. This assisted the visual demarcation between white and gray matter during neuroanatomical dissection. Holographic methodology with the Lumonic HLS2 pulsed ruby laser was employed to create three dimensional images from brain preparations. RESULTS: Master holograms of the human brain preparations demonstrating the various pathways such as vision, cortico-spinal, etc. are made using the ruby laser at wavelength 694.3 nanometers. From this master an infinite number of holograms can be copied. CONCLUSION: By providing immediate three dimensional information, holograms uniquely facilitate the spontaneous understanding of human neuroanatomical relationships which cannot be as efficiently learned with photographs or diagrams. This points the way toward future educational and biomedical applications of this emerging technology. PMID- 8629227 TI - Primary intraventricular hemorrhage in adults: clinical features, risk factors, and outcome. AB - BACKGROUND AND PURPOSE: Nontraumatic primary intraventricular hemorrhage in adults is uncommon. The purpose of this study was to identify clinical features, risk factors, and outcome of primary intraventricular hemorrhage in adults. METHODS: We identified computed tomography scans for nontraumatic primary intraventricular hemorrhage performed between 1982 and 1993 at our institutions and reviewed medical records to determine clinical features, risk factors, and outcomes of these patients. RESULTS: Of 14 cases studied, the onset of symptoms was acute in all and the most common symptom was headache (78%), followed by nausea/vomiting (71%), and mental status change (71%). Seizures as well as focal neurologic deficits were uncommon. The associated risk factors were hypertension in 7 cases, aneurysm or arteriovenous malformation in 5, coagulopathy in 1, and fibromuscular dysplasia in 1 case. Five patients died acutely, and all of the survivors returned to functional independence. The patients with lethargy, pupillary and extraocular movement abnormalities, quadriparesis, hydrocephalus, and aneurysmal intraventricular hemorrhage had worse outcomes. CONCLUSION: Patients with nontraumatic primary intraventricular hemorrhage present with sudden onset of diffuse neurologic symptoms. The mortality in early phase is high (36%) and the prognosis for survivors is good. Factors correlating with the outcome are discussed. PMID- 8629228 TI - Management of intracranial hemorrhage associated with anticoagulant therapy. AB - BACKGROUND: Intracranial hemorrhage may be a particularly devastating complication of anticoagulant therapy. Very few accounts have reported data on the duration of anticoagulant discontinuation following intracranial hemorrhage or the intensity of anticoagulation during treatment for it, although we must adequately manage such a complication. METHODS: We analyzed the management of warfarin-related intracranial hemorrhages in 27 patients with cardiac diseases. We evaluated the degree of anticoagulation using the thrombotest. Anticoagulants were stopped as soon as the diagnosis of intracranial hemorrhage was established by computed tomographic scan. RESULTS: Mechanical valve prosthesis patients, who required intensive long-term anticoagulant therapy, constituted the majority of our series (74.1%). Intraoperative hemostasis was brought under control despite low thrombotest values (13%-68%) at the time of surgery except for the acute subdural hematoma (SDH) patients with cerebral contusion. Early resumption of anticoagulant therapy (within 3 days) did not cause intracranial rebleeding in any operative patient. All the chronic SDH patients and some of the subcortical hematoma patients had a good outcome. All three patients with acute SDH and contusion, however, had a fatal outcome because of intracranial rebleeding within a short period of time or ineffective intraoperative hemostasis. CONCLUSIONS: The patients with anticoagulant-related intracranial hemorrhage may undergo surgery with thrombotest values approximately between 20% and 60%, and anticoagulants can be resumed after an interval of 3 days. Aggressive surgery should particularly be performed in patients with anticoagulation-related chronic SDH or subcortical hemorrhage, as in the cases of anticoagulant-unrelated intracranial hemorrhage. PMID- 8629229 TI - Clinical findings in patients with recurrent intracerebral hemorrhage. AB - BACKGROUND: The use of computerized tomography has led to the detection of second intracerebral hemorrhage (ICH) in some patients. There have, however, only been a few clinical studies of second ICH. SUBJECTS AND METHODS: Thirty patients with a second ICH were analyzed according to clinical criteria. These patients comprised 5.9% of all patients admitted to Chugoku Rosai Hospital for ICH between 1984 and 1992. RESULTS: The mean interval between the first and second ICH was 27.7 months (range 1-144). The incidence of second hemorrhage was especially high within the first year after the initial ICH. Twelve patients bled bilaterally into the basal ganglia or thalamus during either the first or second attack. Most of these patients had poor outcomes and prognoses. The nine patients with good prognoses included patients with high activity of daily living (ADL) prior to the second attack, and those with neurologic grade 1 following the second attack. All of these patients were managed with conservative therapy. The 10 patients who underwent surgery had poor prognoses. CONCLUSIONS: The incidence of second ICHs is greater than that of initial ICHs. Many patients who experience a second ICH will have a poor prognosis, possibly worse than expected. Surgical therapy should not be recommended in such patients, since our data suggest that they will not do well even after surgery. PMID- 8629230 TI - The anatomical basis for the cerebellar infarcts. AB - BACKGROUND: Very little can be found in the literature concerning the variation of the irrigation area of the cerebellar arteries, as well as the characteristics of anastomoses among these vessels. The anatomical features may determine certain features of cerebellar infarcts. Consequently, we examined the irrigation area of and the anastomoses among the cerebellar arteries. METHODS: The anatomical features of the posterior inferior cerebellar artery (PICA), the anterior inferior cerebellar artery (AICA), and the superior cerebellar artery (SCA) were studied in 26 cerebella injected with india ink, while their irrigation areas and anastomoses were examined in 8 of these cerebella. RESULTS: The PICA, which most often (82%) arose from the vertebral artery, was found most commonly (81.3%) to supply the largest part of the occipital surface of the cerebellar hemisphere, the caudal or caudomedial part of the tentorial surface, and the inferior vermis. The AICA, which usually (92%) arose from the basilar artery, commonly (68.8%) supplied most of the petrosal surface of the hemisphere and the flocculus. The SCA, which divided into the medial and the lateral trunks, always irrigated most of the tentorial surface of the cerebellum, the superior vermis, and the dentate nucleus. The PICA, AICA, and SCA were always interconnected by anastomoses, which ranged from 40 microns to 420 microns in diameter. CONCLUSIONS: Cerebellar infarcts were documented by computed tomography (CT) or magnetic resonance imaging (MRI) examinations in 10 patients. The infarcts were located in the PICA territory (60%) or the SCA region (40%). The authors compared the obtained anatomic data to the features of the cerebellar infarcts in these patients. PMID- 8629232 TI - Expanded cavum septi pellucidi and cavum vergae associated with behavioral symptoms relieved by a stereotactic procedure: case report. AB - CASE REPORT: A 6-year-old boy had symptoms such as unstable gait, behavioral symptoms, and irregular appetite, evacuation, and sleep. Giant cavum septi pellucidi and cavum vergae of 3 x 3 x 5 cm were revealed by computed tomography (CT) and magnetic resonance imaging (MRI). After stereotactic cyst-peritoneal shunting, regression of the cavities was revealed by CT and MRI, in association with marked improvement in the above-described symptoms. In the corpus callosum particularly, which had been preoperatively revealed on sagittal MRI sections to be extended circumferentially as a result of compression by a cyst; the compression was relieved postoperatively, and compression of the pericallosal brain tissue was also relieved. CONCLUSIONS: Compression of the brain tissue around the cyst (limbic system, etc.) was considered the most important cause of the behavioral symptoms in this patient. PMID- 8629231 TI - Investigation of chemoresistance-related genes mRNA expression for selecting anticancer agents in successful adjuvant chemotherapy for a case of recurrent glioblastoma. AB - BACKGROUND: Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy. The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance. Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs. METHODS: We present a 45-year old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy. In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor. RESULTS: Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5 pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance. On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low. Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable. Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression. For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis. Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16. CONCLUSIONS: The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy. PMID- 8629233 TI - "Preplacement" of the back of the halo vest in patients undergoing cervical traction for cervical spine injuries: a technical note. AB - BACKGROUND: In patients with unstable cervical spine injuries initially managed with in-line traction, there is some risk of loss of cervical alignment and of new neurologic deficit when the halo vest is applied. This report outlines a simple technique which facilitates halo application in these patients. METHODS: The back of the halo vest is "prepositioned" before traction is instituted. Once alignment is optimized, halo vest application is easily completed without lifting the patient or discontinuing cervical traction. RESULTS: The authors have used this technique in four patients without complication. CONCLUSIONS: In patients with unstable cervical spine injuries who require an initial period of in-line traction, halo vest application is made easier and safer by "prepositioning" the back of the vest. PMID- 8629234 TI - Complication of epidural fat graft in lumbar spine disc surgery: case report. AB - A left-sided L5-S1 lumbar disc herniation operation was performed on a 36-year old woman in 1987. After a 6-year period, the same patient came back to the clinic with the problem of severe sciatic pain on the same side as before. Upon diagnosis, it was seen that the left S1 radix was compressed with a piece of free autofat graft in the foramen, which was used in the first operation. As far as the writers are informed, this is the first report about a later complication of epidural autofat graft in lumbar spine disc surgery. PMID- 8629235 TI - Invasive aspergilloma of the frontal base causing internal carotid artery occlusion. AB - BACKGROUND: Aspergilloma of the nasal cavity or paranasal sinuses rarely extends through the skull base into the intracranial space. PATIENT: The patient was a 79 year-old man in a nonimmunosuppressive state who had an invasive aspergilloma of the base of the frontal lobe. CLINICAL COURSE: The patient, whose initial symptom was a visual disturbance, eventually developed an occlusion of the right internal carotid artery and died. The diagnosis was established by a transsphenoidal biopsy. CONCLUSIONS: A rare case of invasive aspergilloma of the frontal base is described with emphasis on the findings of low-intensity mass on the T2-weighted magnetic resonance imaging (MRI). The prognosis in this disease is very poor. Early diagnosis and surgical treatment, combined with postoperative antifungal drug therapy, would have improved the outcome. PMID- 8629237 TI - Informed consent. PMID- 8629236 TI - Parasellar Aspergillus granuloma extending from the sphenoid sinus: report of two cases. AB - BACKGROUND: Sphenoid sinus aspergillosis is a rare disease known to show an aggressive course with high mortality. Early diagnosis, though difficult, is required to prevent lethal fungal meningoencephalitis. CASE REPORT: We describe two cases of parasellar Aspergillus granuloma extending from the sphenoid sinus clinically indistinguishable from intracranial neoplasms. In the first patient, the fungus colony was visualized by computed tomography (CT) and magnetic resonance imaging (MRI) as a calcified concretion and total removal was curative. In the second patient, partial removal and subsequent antifungal therapy had minimal effect. CONCLUSIONS: The prognosis of the patients with this disease depends on prompt surgical treatment before intradural invasion occurs, and CT and MRI are useful diagnostic maneuvers for detecting calcified Aspergillus colonies. PMID- 8629239 TI - Trigeminal neuralgia. PMID- 8629238 TI - Withdrawal of glucocorticoid therapy in neurosurgical patients. PMID- 8629240 TI - Giant aneurysms of the anterior circle of Willis: management outcome of open microsurgical treatment. AB - BACKGROUND: There is no uniform agreement to date regarding the optimal management of giant aneurysms (GAs) of the anterior circle of Willis. Endovascular therapeutic techniques have yielded unacceptable rates of aneurysm growth and recanalization (endosaccular) or high rates of complications (distal parent vessel occlusion). Despite size, frequent thrombosis and calcification (incollapsibility), and splaying of parent vessels, these aneurysms are readily amenable to direct surgical exposure and control of parent vessels intracranially. Published series have not considered these lesions separately and have often reflected a mixture of management strategies for these and other GAs. METHODS: Thirty-eight consecutive patients with symptomatic GAs of the anterior circle of Willis were managed by the senior author over a 7-year period. Twenty six of these patients (68%) presented with subarachnoid hemorrhage (SAH). Temporary occlusion was performed under a protocol to enhance brain protection. Direct clip reconstruction or trapping was used in all instances, with intraoperative angiographic control. Revascularization procedures and suture vascular reconstructions were not used in any case. RESULTS: All patients were considered for direct microsurgical treatment. One patient refused surgery, and two patients were deemed a prohibitive medical risk. Thirty-five patients were treated surgically with complete obliteration of the aneurysm in 34 cases (97%), and patency of all parent arteries in 30 cases (86%). Overall mortality was 6% in the surgical cohort, with good or excellent clinical outcome in 71%. Mortality and poor outcome occurred exclusively in the setting of recent hemorrhage. CONCLUSIONS: The results are compared to the natural history of these lesions and to outcome (safety and effectiveness) of currently available endovascular techniques. This experience supports direct microsurgical intervention as the primary therapeutic modality for these lesions. PMID- 8629242 TI - Early surgery improves the cure of aneurysm-induced oculomotor palsy. AB - BACKGROUND: Aneurysm of the internal carotid-posterior communicating artery (ICA PCoA) is the most frequent cause of sudden unilateral oculomotor palsy. Timely surgery for the aneurysm is the most important factor for third nerve recovery. METHODS: We scrutinized the world literature with nearly one thousand cases of isolated unilateral oculomotor palsy caused by intracranial aneurysms and treated with surgery. Only those reports (one-third of all) in which the time interval between onset of oculomotor palsy and surgery could be determined were included. We treated 1314 patients with cerebral aneurysms (183 = 14% with ICA-PCoA aneurysms) from our catchment area in Eastern Finland during years 1977-1992. Twenty-eight patients having oculomotor palsy caused by ICA-PCoA aneurysm had surgery as soon as the diagnosis was made. RESULTS: Eight of 9 patients operated within three days (0-3) and 4 of 6 patients operated on within 4 to 6 days the onset of oculomotor palsy had complete recovery of their third nerve function, in contrast to only 4 of 13 patients operated on later. Especially those operated on more than four weeks later had a dismal outcome: only 1 of 6 had complete recovery. CONCLUSIONS: We recommend immediate admission and acute or early surgery for aneurysm-induced third nerve palsy, preferably within 3 days, to avoid functionally and cosmetically invalidizing disability. PMID- 8629241 TI - Evaluation of outcome after intracranial aneurysm surgery: the neuropsychiatric approach. AB - BACKGROUND: With the reduction of mortality and gross neurologic morbidity of patients undergoing intracranial aneurysm surgery, the interest in outcome is shifting towards more subtle aspects such as cognitive deficits and psychosocial adjustment. METHODS: We discuss two different ways of measuring outcome in a sample of 20 patients who had intracranial aneurysm surgery. Patients were evaluated at discharge using the Karnofsky Scale and the Glasgow Outcome Scale. Six months after discharge we conducted a neuropsychiatric evaluation including cognitive, behavioral, and mood status assessment. RESULTS: Although 13 of out patients had a "good recovery, " 18 had some neuropsychiatric impairment. Comparing patients with "good recovery" with the remainder, patients with poorer outcomes tended to have a left pterional approach, a poorer "drive", and language disorders (p < 0.05). There was no correlation between out cognitive, mood, and behavioral assessment and the results of the Karnofsky and Glasgow Outcome Scales ( p > 0.05). CONCLUSIONS: we conclude that neuropsychiatric deficits are common after intracranial aneurysm surgery and that for our study the Karnofsky Scale and Glasgow Outcome Scale were not sensitive enough to detect residual impairment. therefore, it is important to develop brief tests and scales able to identify these problems and to complement the standard clinical neurological examination. PMID- 8629243 TI - False localization of rupture by computed tomography in bilateral internal carotid artery aneurysms. AB - BACKGROUND: Focal clot on computed tomographic (CT) scan is one of the most reliable clues in determining the site of rupture in patients with multiple intracranial aneurysms. However, unusual presentation of clot may cause false localization of the ruptured aneurysm in bilateral aneurysms, particularly when they are located near the midline. METHODS: We describe two cases of bilateral internal carotid artery (ICA) aneurysms presenting contralateral distribution of clot on CT scan. RESULTS: Misjudgment of rupture site by the CT scan resulted in clipping of the wrong aneurysm in one patient. Disastrous rebleeding occurred before the second operation for the ruptured one. In the other patient, the rupture site was assumed correctly despite contralateral situation of clot on CT, and both aneurysms were treated in one operative session. CONCLUSIONS: Adhesion and obliteration of the subarachnoid cisterns from previous hemorrhage will deviate the direction of hemorrhage and present contralateral clot on CT scan in bilateral aneurysms located near the midline as ICA aneurysms. Medial direction of rupture may obscure accurate localization of rupture side in such aneurysms. PMID- 8629244 TI - Cisternal talc injection in dog can induce delayed and prolonged arterial constriction resembling cerebral vasospasm morphologically and pharmacologically. AB - BACKGROUND: The possible role of inflammation in the pathogenesis of cerebral vasospasm has been noted in recent studies. In order to examine the role of inflammation, we examined the vasocontractile activity of talc, which is known to cause severe inflammation, using a canine cisternal talc injection model. METHODS: Under general anesthesia, a sterile talc powder suspended in saline was injected into the cisterna magna of the dog. Serial vertebral angiography and postmortem histologic changes of the harvested basilar artery were examined. The morphologic and pharmacologic features of talc-induced vessel spasm were compared with the usual autologous blood-induced artery spasm. RESULTS: Cisternal injection of sterile talc powder caused no early spasm, but induced definite basilar arterial constriction 2 days after injection. This vascular constriction was observed to continue up to 7 days after injection. Ultrastructural study of the constricted vessel revealed several morphologic changes, such as corrugation of the elastic lamina, subintimal proliferation, migration of smooth muscle cells, detachment of endothelial cells, etc.; findings that are compatible with the changes observed in vasospasm. Pharmacologic study showed a moderate decrease in the maximal contraction to KCl and UTP. Endothelium-dependent relaxation was markedly disturbed, while endothelium-independent relaxation was preserved. These pharmacologic properties were also similar to those reported in vasospasm. CONCLUSIONS: Our present study indicates that the several changes of vascular properties, which had been considered to be specific to cerebral vasospasm, can be regarded as a nonspecific biologic defense reaction against the foreign body. The analysis of the common pathway from talc and autologous blood to vasospasm may lead to the pathogenesis of cerebral vasospasm. PMID- 8629245 TI - Sugita aneurysm clip sterilizing tray. PMID- 8629246 TI - Near-infrared monitoring of cerebral oxygenation state during carotid endarterectomy. AB - BACKGROUND: Recent studies have indicated that near-infrared spectroscopy (NIRS) could continuously and noninvasively observe the changes in cerebral oxygenation state during hypoxia and ischemia, using their optical properties. Its validity and usefulness during cerebrovascular surgery, however, still remain to be clarified. METHODS: Using NIRS, we continuously monitored the changes in the concentration of oxyhemoglobin, deoxyhemoglobin, and total hemoglobin ([oxy-HB], [deoxy-Hb], and [total Hb], respectively) and redox state of cytochrome oxidase (cyt ox) during carotid endarterectomy for 22 patients, and we compared the NIRS responses with those of intraoperative somatosensory evoked potentials (SEP) and regional cerebral blood flow (rCBF). RESULTS: In 9 of 22 patients, cross-clamping of the carotid artery caused a continuous decrease [oxy-Hb] and [total Hb], and an increase in [deoxy-Hb]. Cyt ox was partially reduced during the clamping. These NIRS responses demonstrated the occurrence of severe hypoxia in the ipsilateral cerebral tissue. These patients showed a marked decrease in the N20 amplitude of SEP and rCBF. In contrast, the other 13 patients did not show a significant decrease in the cerebral oxygenation state, which showed no remarkable changes in either SEP or in rCBF. CONCLUSIONS: NIRS could successfully jude the cerebral oxygenation state noninvasively during carotid surgery and was more sensitive to ischemic crisis than other indirect methods. PMID- 8629247 TI - Evaluating cerebral concussion. AB - Years ago we developed a rat model that was consistently comatose for 1-2 seconds following 50 G acceleration of the head. Motor normalcy returned in about 10 seconds, and normal memory in an hour. There were immediate electroencephalogram and transmitter changes. All these returned to normal within an hour following 1 or 20 such concussions. We could find no light or electron microscopic changes. Halothane anesthesia precludes measurement of coma and introduces distortion of metabolite findings. To date our findings indicated concussion at this level is completely reversible. PMID- 8629249 TI - Cerebellopontine angle glioneuronal hamartoma. AB - Roughly 90% of cerebellopontine angle tumors are acoustic neuromas. Other prevalent lesions include meningiomas and epidermoid tumors; additional lesions are rare. We describe a patient with a neuroglial hamartomatous mass of the internal auditory canal who was thought to have a schwannoma preoperatively. In this case, the hamartomatous tissue containing neuroectodermal elements may have become separated from the developing neuraxis during neural migration. Hamartomas should be considered when masses are discovered originating from eighth nerve branches other than the superior vestibular nerve and when magnetic resonance signal characteristics vary from the T1 enhancement typically seen with schwannomas and meningiomas. PMID- 8629248 TI - The first primary brain tumor operation in America. AB - On February 25, 1886, in San Francisco, California the first documented resection of a primary brain tumor in the United States was performed by Drs. Hirschfelder and Morse. This operation followed fourteen months after the first recognized resection of a primary brain tumor in history was performed by Mr. Rickman Godless in London, England, November 25, 1884. Hirschfelder and Morse's operation is presented, and the contemporary medical technology that made this operation possible is summarized. The unique circumstances that explain the occurrence of this milestone in the history of American neurosurgery are documented. PMID- 8629250 TI - Preoperative MRI diagnosis of Lhermitte-Duclos disease: case report with associated enlarged vessel and syrinx. AB - BACKGROUND: The most common primary cerebellar tumor is hemangioblastoma, a lesion which is associated with magnetic resonance imaging (MR)-detectable vascularity in over 60%. Lhermitte-Duclos disease is an uncommon cause of a cerebellar mass that is not typically vascular. METHODS: Computed tomography (CT), MRI with and without contrast, and magnetic resonance venography was performed in a patient with a cerebellar mass. RESULTS: The cerebellar mass was noted to have a prominent vessel, as well as an associated syrinx. In spite of MRI-detectable vascularity, the striped appearance of the lesion was felt to be typical of Lhermitte-Duclos disease. At surgery, the mass was resected and the diagnosis of Lhermitte-Duclos disease was confirmed. CONCLUSIONS: The diagnosis of Lhermitte-Duclos disease should be made when MRI shows a parallel linear "tiger-striped" lesion of the cerebellum. The presence of an enlarged vessel and/or syrinx should not deter one from making the preoperative diagnosis. PMID- 8629251 TI - Unilateral tongue atrophy due to an enlarged emissary vein in the hypoglossal canal. AB - A 16-year-old girl presented to our clinic with right-sided tongue atrophy and fasciculations of 1-year duration. Enlargement of the outer opening of the hypoglossal canal was reveal by conventional and computed tomography of the skull. Magnetic resonance imaging disclosed an enlarged venous system extending from the jugular vein to the internal jugular vein on the right, with low signal density suggestive of a flow void. A right-sided occipital craniotomy was performed. When the hypoglossal canal was opened, an enlarged emissary vein compressing the hypoglossal nerve was identified. This is the first reported case of unilateral tongue atrophy and an enlarged hypoglossal canal due to an enlarged emissary vein. PMID- 8629253 TI - Is how you look important? PMID- 8629254 TI - Thou shall not bear false witness. PMID- 8629255 TI - Retirement: what to do? PMID- 8629252 TI - Tirilazad does not protect rat brain from brachytherapy-induced injury. AB - BACKGROUND: Acute and chronic brain injury are common sequelae of high-dose focused radiation, as in radiosurgery and brachytherapy. Development of protectors of radiation injury, which would work in brain but not in tumor, would help enhance the therapeutic ratio of focused-radiation therapy. METHODS: Radiation protection by a clinically available 21-aminosteroid, Tirilazad, was studied in a rat brain brachytherapy model, both in tumor and non-tumor bearing animals. For the tumor model, 9L Glioma/SF line cells were implanted stereotactically into the right frontal lobe of F-344 rats and grew to a sphere of 5.0-mm diameter after 12 days. Animals received a standard brachytherapy dose of 80 Gy to a 5.5-mm radius volume administered by a high-activity removable iodine-125 seed. Radiation damage was evaluated 24 hours after removal of the seeds in all animals and again at 3 months in non-tumor-bearing animals, by T1 weighted gadolinium-enhanced and T2-weighted magnetic resonance imaging (MRI) on a 1.5-T unit. Treated animals received Tirilazad 5 mg/kg intravenously 15 minutes prior to implant, 1 hour after implant, every 6 hours for the duration of the implant, and for 24 hours after removal of the seed. Control animals were administered vehicle only. RESULTS: In both non-tumor-bearing and tumor-bearing rats, no difference in the volume of lesions on enhanced T1 or T2 MRI was seen between the Tirilazad-treated and control groups. In the non-tumor-bearing rats, volume of both T1 enhanced and T2 MRI lesions was significantly reduced at 3 months compared to the values at 24 hours. CONCLUSIONS: In the present model, Tirilazad failed to reduce the volume of radiation brain injury from brachytherapy as seen on MRI, studied acutely in tumor-bearing and non-tumor bearing animals and also at 3 months in non-tumor-bearing rats. PMID- 8629256 TI - The physician's role in curing social ills. PMID- 8629257 TI - Medicare and HMOs. PMID- 8629258 TI - Neutral changes and modifications of the genetic code. PMID- 8629259 TI - [Significance, diagnosis and prognosis in the cytogenetic analysis in acute leukemias and myelodysplastic syndromes]. AB - Cytogenetic analysis of leukemic cells has been shown to be a mandatory part of the diagnosis of malignant hemopathies. Recurring abnormalities may be divided into those exclusively found in myeloid disorders, those associated with lymphoid diseases and those detected in both types of hemopathy. Several of the common defects are characteristic of specific FAB types or subtypes and associated with clinico-pathologic manifestations. Cytogenetic abnormalities have served to identify relatively homogenous subsets of malignant hemopathies. In view of the significant progress realized in the treatment of malignant hemopathies, the assessment of prognostic factors is particularly important for therapeutic decision making. The chromosome status at diagnosis has proven to be a major prognostic indicator for survival and outcome in individual patients. This article will focus on the diagnostic and prognostic significance of the karyotype in de novo acute leukemia and primary myelodysplastic syndromes. PMID- 8629260 TI - [Current status of therapy and prognosis in acute adult leukemia]. AB - In the last two decades complete remission [CR] rates for adults with acute leukemia has increased to 60 to 80%. In acute myelogenous leukemia [AML], this is due to the application of intensive induction therapies, comprising an anthracycline and a cytarabine. In acute lymphoblastic leukemia [ALL], the introduction of intensive early consolidation and CNS prophylaxis played an additional important role. These myeloablative treatments became feasible because of considerable improvements in the management of infectious and bleeding complications. The standard induction for AML further on remains the '3 + 7' schedule [daunorubicin 45 to 60 mg/m2/day x 3, I.V. and Ara-C 100 to 200 mg/m2/day x 7, 24-h infusion]. In ALL, prednisone, vincristine, L-asparaginase and an anthracyeline are the backbone of the induction therapy. Unfortunately, there has been less improvement for overall long-term survival, which is about 15 to 20% in AML and 20 to 35% in ALL beyond 5 years. More intensive post-remission regimens which include high-dose Ara-C in AML and intermediate-dose methotrexate and cyclophosphamide in ALL seem to improve these results to some extent. Allogeneic bone marrow transplantation has the most powerful anti-leukemic potential; however, because of the high peritransplant mortality [20 to 25%], its use in first CR tends to be restricted to patients with adverse prognostic features predicting early relapse, while good-risk patients are transplanted at the first signs of relapse or in second CR. In both AML and ALL, the optimal form of post-remission treatment needs to be defined. PMID- 8629261 TI - [Value of the blood picture and flow cytometry immunotyping in the early diagnosis of low-grade lymphoma]. AB - Low-grade malignant non-Hodgkin lymphoma [NHL] and chronic lymphocytic leukemia [CLL] as its special form are slowly progressing malignancies which may present with lymphadenopathy, splenomegaly or, more rarely, hepatomegaly. The diagnosis is made by bone marrow or lymph node histology, while laboratory tests are relatively unspecific and may only hint towards the diagnosis. In contrast to high-grade malignant lymphoma, low-grade malignant NHL is often associated with the appearance of malignant lymphoma cells in peripheral blood. These malignant lymphocytes may be differentiated microscopically from normal lymphocytes, so that the diagnosis of NHL may be suspected not only because of clinical symptoms or lymphocytosis, which may present late in the natural history of the disease, but also on morphological grounds. Three types of low-grade malignant NHL cells may be recognized in peripheral blood: A mature appearing lymphocytic type with only slight alterations of the nucleus, a lymphoplasmocytic type, and a lymphocytic type with prominent alterations of the nucleus. The appearance of smudge cells and a monotony in lymphocyte morphology may serve as further diagnostic aids. Once the diagnosis has been suspected on morphological grounds, it may be verified in the case of B-cell lymphomas by flow cytometry. The clonality of T-cell lymphoproliferative disorders in addition has to be proven by demonstrating a clonal rearrangement of the T-cell receptor in Southern blots. An early diagnosis of low-grade malignant NHL may not only provide new insights into the natural history of monoclonal cytopathies but may also be of importance in the clinical management of patients. PMID- 8629262 TI - [Low-grade malignant non-Hodgkin lymphomas--current status and trends in therapy]. AB - Low-grade Non-Hodgkin Lymphomas [NHL] comprise a heterogenous group of disorders both in terms of their cellular and histological composition as in terms of their clinical course. The currently mostly applied classification systems, the Working Formulation and the Kiel Classification as well as the recently proposed Revised European American Lymphoma Classification, discriminate between low-, intermediate and high-grade subtypes. Low-grade NHL are characterized by a low to moderate proliferative activity and a long clinical course with median survival times ranging for approximately three years for centrocytic [CC] or mantle cell lymphomas [MCL] to five to eight years for centroblastic centrocytic [CB-CC] or follicular lymphomas [FL]. Recent cytogenetic and molecular biologic analyses indicate that these differences may result from distinct genetic abnormalities such as the translocation t [14; 18] which is frequently observed in FL-NHL and is associated with a bcl 2 overexpression, or the deregulation of the PRAD 1 in MCL-NHL induced by the translocation t [11; 14]. Therapy of low-grade lymphomas depends mainly on the extend of the disease. In the early stages I and II, at which approximately 15-20% of low-grade NHL are diagnosed, an extended field or a total nodal radiotherapy may be applied with curative intention. The treatment of patients with more advanced stages III and IV is controversial. The currently available information justifies a conservative approach by observing the natural course of the disease until therapeutic intervention is required due to the occurrence of B-symptoms, hematopoietic insufficient or lymphoma progression. Intensive chemotherapy seems not to translate into an improved disease-free or overall survival and can, therefore, not be recommended for first-line treatment. The most recent data of the German Low-Grade Lymphoma Study Group indicate, however, that after successful initial cytoreductive therapy maintenance treatment with interferon-alpha prolongs the disease-free interval and possibly also overall survival. New perspectives have arisen from the introduction of novel cytostatic agents such as purine analogues and the development of immunotoxins and antibody conjugated radioisotopes. Most promising at the present time appears the application of myelo-ablative radiochemotherapy followed by autologous peripheral blood stem cell transplantation which may provide a curative approach even for advanced stages of the disease. PMID- 8629263 TI - [High-grade non-Hodgkin lymphoma: diagnosis and therapy]. AB - High grade non-Hodgkin's lymphomas comprise a group of heterogenous disorders of the lymphatic system with aggressive clinical behaviour. Extranodal disease manifestations are common, especially in immunocompromised patients [e.g. AIDS]. High-grade NHL are chemo- and radiosensitive. Anthracyclin-containing chemotherapy regimens have led to a significant improvement in prognosis. The CHOP-regime, consisting of doxorubicin, cyclophosphamide, oncovin and prednisolon, is the standard therapy that should be given outside of clinical trials. Age, bad performance status, elevated serum-LDH, presence of more than one extranodal manifestation and disease stage III or IV have been defined as clinically relevant prognostic factors. Current treatment strategies include dose intensification by interval shortening and dose escalation as well as high-dose chemotherapy, followed by autologous stem cell rescue. Patients receiving these experimental therapies should be treated within multicenter clinical trials. PMID- 8629264 TI - [Hodgkin disease--pathogenesis and therapy]. AB - Hodgkin's lymphoma exerts characteristics of a true hematopoietic neoplasia as well as of an atypic immune response. In affected lymphatic tissue a minority of malignant Hodgkin-Reed Sternberg [H-RS] cells is surrounded by a majority of reactive, nonmalignant cells. Clinical features and biological studies suggest a pronounced, but inefficient, T-cell response against a yet undefined [viralS/B] target antigen expressed on H-RS cells. The role of the Epstein-Barr virus [EBV] which can be detected in the H-RS cells in about 50% of cases remains to be defined. By micromanipulation of single H-RS cells from frozen sections and subsequent enzymatic amplification of nucleic acids [single cell PCR] for the first time a monoclonal B-cell origin of H-RS cells could be demonstrated in several cases of Hodgkin's disease. Therapeutic strategies in Hodgkin's disease, in contrast to non-Hodgkin's lymphoma, are not based on the histological subtype, but rather on precise determination of the stage of disease. Treatment of choice in limited disease stages without risk factors is radiation therapy. Intermediate stages are treated with combined chemo- and radiation therapy, advanced stages with intensive chemotherapy [subsequent radiation only on bulk tumors]. Thus, about 85% of all patients suffering from Hodgkin's disease, can be cured definitely. High-dose chemotherapy followed by autologous stem cell transplantation in relapsed Hodgkin's lymphoma is currently proofed in clinical studies. PMID- 8629265 TI - [Multiple myeloma]. AB - Multiple myeloma is a low-grade non-Hodgkin lymphoma. Treatment is palliative, and an individually adapted strategy for therapy is needed for each patient. A review about diagnostic methods for staging, prognostic factors and different aspects of therapy is presented. PMID- 8629267 TI - [Malignant hematologic diseases]. PMID- 8629266 TI - [Indications for bone marrow and peripheral stem cell transplantation in malignant hematological diseases]. AB - Transplantation of hematopoietic precursor cells is an established therapy today in the treatment of hematological malignancies. Cells from different sources [bone marrow, peripheral blood, cord blood] and from different donor types [autologous, syngeneic or allogeneic] are used for transplantation. The aim of autologous transplantation is to apply intensive high-dose chemo-radiotherapy and to shorten the duration of aplasia. Allogeneic cells, in addition, are free of potentially contaminating precursor cells and provide a graft-versus-leukemia effect. For all patients, transplantation should be considered at diagnosis as an integral part of treatment strategy and, depending on risk factors, be performed early in the course of disease. Preferred time for patients with high-risk acute leukemias is first complete remission, second complete remission for standard or low-risk acute leukemias. For chronic myeloid leukemia, allogeneic transplantation should be performed within one year of diagnosis, preferably still in first chronic phase. Autologous transplantation can be considered in a protocol setting. For patients with myelodysplastic syndromes of the FAB subtype refractory anemia or refractory anemia with sideroblasts, allogeneic transplantation is the treatment of choice as initial therapy. For patients with refractory anemia and excess of blasts with or without transformation, remission induction should be attempted before transplantation. Autologous transplantation is the preferred treatment strategy for patients with Hodgkin's and non-Hodgkin's lymphoma, for high-risk patients in first complete remission, for other patients in chemotherapy-sensitive first relapse. For patients with myeloma, transplantation should be considered after first line therapy. Age is the main individual patient's risk factor, transplant-related mortality immediately increases in parallel to increasing age. Autologous transplants are limited to patients below 60 to 65 years, allogeneic HLA-identical sibling transplants to patients below 50 to 55 years, and unrelated transplants to patients below 40 to 45 years. Prerequisites for transplant are availability of a donor, access to a transplant bed, informed consent of patient and donor, as well as financial guarantee. Indications for the different hematological malignancies and the major risk factors are discussed. PMID- 8629268 TI - [Chronic myeloid leukemia]. AB - The chronic myelogenous leukemia [CML] is a clonal disease of hematopoietic stem cells with unknown etiology. The incidence is around 2/100,000/year, the median age at diagnosis about 47 years. The course of CML is characterized by a chronic phase with few symptoms and good therapeutic response of about 4 to 5 years duration and by transition to a prognostically unfavourable blast phase of about 3 months duration. Therapy of choice, at present, is early allogenous bone marrow transplantation [BMT], which is curative in 40 to 80% of transplanted cases. In patients below 55 years, a donor search should be started at the earliest possible time after diagnosis. Drug therapy of choice are interferon alpha [IFN] and hydroxyurea, which are both superior to busulfan with regard to duration of chronic phase and survival. Complete cytogenetic remissions are observed in 5 to 9% of IFN-treated patients in randomized studies, but virtually all remain positive for bcr/abl by PCR. Whether and in how far IFN is superior to hydroxyurea appears, at least in part, to depend on the treatment intensity with hydroxyurea and on patients characteristics. In analyzing median survival times, the risk profiles of the patients have to be considered. In the future, intensive chemotherapy with or without autografting might play an important role in the therapy of chronic-phase CML. Forthcoming trials have to consider both, conventional and new experimental treatment modalities. An example is the treatment strategy of the ongoing randomized study of the German CML Study Group which compares allogenous BMT with the best available drug therapy and, in addition, analyses the influence of intensified drug therapy on survival. PMID- 8629269 TI - [Morphological, immunological and cytogenetic diagnosis in acute leukemias]. AB - Current diagnosis of acute leukemia includes traditional morphology and cytochemistry supplemented with immunophenotypic, cytogenetic and molecular biologic analyses. This multiparameter approach has revealed the biological heterogeneity of acute leukemias and has enabled the identification of leukemic syndromes with distinct clinical and biological features. Morphology and cytochemistry are of particular importance for the classification of acute myeloid leukemia, except for certain subtypes such as minimally differentiated acute myeloid leukemia [AML-M0] or acute megakaryoblastic leukemia [AML-M7], requiring additional immunophenotypic or ultrastructural analyses. In acute lymphoblastic leukemia [ALL], immunophenotyping is essential for the diagnosis and lineage assignment [B- and T-lineage ALL] of leukemic blasts. Furthermore, it allows the characterization of the maturation stage and certain subtypes, i.e. ALL with coexpression of myeloid antigens [My+ ALL]. Cytogenetic and molecular analyses of leukemic cells have contributed important informations to the understanding of pathogenetic mechanisms in leukemogenesis and have led to the definition of prognostic risk groups and the development of subtype-specific or risk-adapted therapy strategies. PMID- 8629270 TI - [Myeloproliferative syndromes]. AB - Myelodysplastic syndromes [MDS] are clonal disorders of hematopoietic stem cells leading to a deregulation of proliferation and differentiation of the bone marrow cells. Clinically the patients present with symptoms and signs of anemia, thrombocytopenia, and neutropenia. About a third of the patients will develop acute myeloid leukemia. Supportive care is the mainstay of therapy in these mostly elderly patients. G-CSF should only be given in cases of neutropenia and infection, but not prophylactically. Selected patients with severe or transfusion dependent anemia will respond to treatment with erythropoietin. In advanced MDS aggressive chemotherapy should be considered, while in patients below 50 years of age and an HLA-identical sibling donor allogeneic bone marrow transplantation is the treatment of choice. PMID- 8629271 TI - [Study of the elimination of residues and local tissue injury following intramuscular injection of a solution of the combination trimethoprim/sulfatroxazole in pigs]. AB - Two different studies are described. The first study deals with the elimination of residues of trimethoprim (TMP), sulfatroxazole (STX) and its main metabolite N4-acetyl-sulfatroxazole (N4-acetyl-STX) in pigs. Thirty -six pigs were treated with trimethoprim/sulfatroxazole IM in the nec k at a dosage of 16 mg/kg body weight for five days. Groups of four pig s were slaughtered at different time intervals. The study showed that concentration of STX, N4-acetyl-STX and TMP in edible tissues and at the injection sites were below 0.1 ppm on day nine after the last injection. S TX was eliminated the slowest, and STX can therefore be selected as a marker for residues of the trimethoprim/sulfatroxazole formulation in the tissues. The second study deals with irritation aspects of this trimethoprim /sulfatroxazole formulation. Four pigs of 32-35 kg were treated IM w with trimethoprim/sulfatroxazole and benzylpenicillin sodium. Each pig received the same injection volume, namely four trimethoprim/sulfatroxazole injections (16 mg/kg body weight per injection site), two in the back and two in the neck muscle, and two benzylpenicillin sodium injections (20,000 I.U./kg body weight per injection site), in the back muscle. All pigs were slaughtered 14 days after treatment and the extent of the irritation was compared. There were no differences between trimethoprim/sulfatroxazole and benzylpenicillin sodium with regard to irritation at the injection site in the back muscle. The irritation in the neck site was statistically less prominent than that in the back muscle and was considered not to affect the quality of the meat. PMID- 8629272 TI - [Prion disease in humans and animals]. PMID- 8629273 TI - [An eye on the customer]. PMID- 8629274 TI - [Epidural anesthesia]. PMID- 8629275 TI - [Premedication in dogs with increased anesthesia risk]. PMID- 8629276 TI - Corticosteroid treatment and X-ray irradiation in adult rats induce the reexpression of fetal markers in cortical thymic epithelial cells. AB - The phenotype of cortical thymic epithelial cells (TEC) following hydrocortisone treatment and sublethal X-ray irradiation in adult rats was studied by immunohistochemistry. It was found that during thymic regeneration (2-16 days) a TEC subset, predominantly in the outer cortex, transiently expressed cytokeratin (CK) 19 and an antigen defined by PT13D11 monoclonal antibody (mAb). These markers are characteristic for fetal, but not adult cortical TEC. To examine whether regenerating thymocytes may influence the phenotype of cortical TEC we cultivated a rat cortical TEC line (R-TNC 1.1) with thymocytes isolated from the thymuses at day 7 after hydrocortisone treatment. The R-TNC 1.1 TEC line, although established from adult rat thymus, constitutively expresses PT13D11, but not CK19. The appearance of CK19 in the R-TNC 1.1 cells was not inducible neither by coculture of this line with thymocytes, nor by the influence of IL-1, IL-2, IL 6, TNF-alfa and IFN-gamma. These results demonstrated the phenotypic plasticity of cortical TEC in adult rats. PMID- 8629277 TI - Microbial parasites versus developing T cells: an evolutionary 'arms race' with implications for the timing of thymic involution and HIV pathogenesis. AB - The thymus attempts to ensure that T cells which emerge from it are able to discriminate self from nonself. As such, it is a potential 'backdoor' through which microbial parasites can enter, manipulate the host into perceiving them as 'self', and thereby avoid immune surveillance. It is proposed that the host has evolved to overcome this parasitic strategy by rapidly producing large numbers of long-lived T cells very early in life (closing the backdoor), before the developing individual has significant contact with infectious organisms, and while still under the protection of its mother's intact immune system. Hence the capacity of the thymus to function efficiently early in the lifespan would have been strongly favored by natural selection. It is well established in evolutionary biology that strong selection favoring enhanced early function easily accommodates, through pleiotropy, the accumulation of later occurring negative effects, and it is through this process that thymic involution and subsequent immune system senescence may have evolved. Once a large pool of competent T cells has been produced, even those microbes capable of contaminating the thymus usually can be eliminated, or at least contained. However, microbes that both destroy peripheral T cells (particularly peripheral T cells that are activated against them), and contaminate the thymus (leading to deletion of potential replacements of the destroyed peripheral cells), may be able to eventually overcome the immune system, thus producing disease after a long period of apparent latency. Human immunodeficiency virus, which is initially well controlled by the immune system, may become unleashed via this process. PMID- 8629278 TI - Thymic stromal cells eliminate T cells stimulated with antigen plus stromal Ia molecules through their cross-talk involving the production of interferon-gamma and nitric oxide. AB - We previously established a thymic stromal cell clone capable of inducing differentiation of immature thymocytes and described a clonal elimination model in which T cell clones are killed on the monolayer of this stromal clone by stimulation of their T cell receptors (TCR) with antigen plus stromal Ia molecules. This study investigated molecular mechanisms underlying this phenomenon. Antigenic stimulation on thymic stromal cells produced large amounts of interferon-gamma (IFN-gamma) and small amounts of tumor necrosis factor-alpha (TNF-alpha). Addition of anti-IFN-gamma monoclonal antibody (mAb) to these cultures largely prevented death of TCR-stimulated T cells. T cell death was also induced when cultures were treated with recombinant IFN-gamma (rIFN-gamma) or rTNF-alpha instead of the relevant antigen, showing that these lymphokines are involved in the process of T cell death. It was further demonstrated that these lymphokines, especially IFN-gamma, induced the expression of mRNA for the inducible type of nitric oxide (NO) synthase in thymic stromal cells and that enhanced levels of NO were produced by stromal cells cultures with T cells plus antigen or stimulated with rIFN-gamma or rTNF-alpha. NO was found to be critically responsible for inducing T cell death on the stromal cell monolayer following stimulation of T cells with antigen or of stromal cells with rIFN-gamma or rTNF-alpha, because T cells death was completely prevented by addition of NG monomethyl-L-arginine (L-NMMA), which is capable of inhibiting NO production. These results indicate that elimination of TCR-stimulated T cells on thymic stromal monolayers with the capacity to support thymocyte differentiation is induced by the cross-talk between IFN-gamma/TNF-alpha-producing T cells and stromal cells capable of producing NO in response to these lymphokines. PMID- 8629279 TI - Isolation, cultivation and phenotypic characterization of rat thymic dendritic cells. AB - Rat thymic dendritic cells (TDC) were isolated from thymic cell suspension by Nycodenz gradients of different densities and osmolarities. After cultivation of these cells for 3 days in the conditioned medium (TE-R 2.5 + HT supernatant) prepared by cocultivation of a medullary thymic epithelial cell line (TE-R 2.5) and hydrocortisone resistant thymocytes, the purity (75-85%) and survival of TDC significantly increased. The supernatant contained moderate activities of IL-1 and IL-6, low levels of TNF-alpha and IL-2 and factor(s) that strongly stimulated the proliferation of a mouse macrophage cell line RAW 264.7. TDC survival in culture was significantly increased by GM-CSF and decreased by IL-6 and IFN gamma. The phenotype of TDC was studied by flow cytometry using a panel of monoclonal antibodies (mAb) to rat cell surface markers. It was found that almost all freshly isolated TDC expressed major histocompatibility complex (MHC) class I and class II molecules as well as CD45. Most TDC were LFA-1 (CD11a)+, CD18+, ICAM 1 (CD54)+ and CD53 (OX- 44)+, but only certain subsets expressed CD11b and thymocyte markers Thy1, CD2, CD4 and CD8. Upregulation in the expression of almost all the markers was observed after cultivation of TDC. In addition, cultivated, but not freshly isolated TDC expressed CD25 (IL-2R alpha) and CD45RC (OX-22) molecules. Cultivated TDC had strong accessory function in autologous thymocyte proliferation. PMID- 8629280 TI - [Hearing impairment in quietude and noise; 2 different forms of expression of presbyacusis]. AB - Many elderly persons have problems in understanding speech in the presence of voice babble, music, etc. Fitting a hearing aid often does not improve their speech intelligibility. For a better insight, we should distinguish between two different modes of presbyacusis. The first mode expresses itself in attenuation of all incoming sounds, which can be compensated successfully by the hearing aid's electronic amplification. The second mode has to do with a reduced capacity of the ear to separate simultaneous sounds. As the hearing aid amplifies both the wanted and the unwanted sounds, its effect is in this case nihil. Extensive investigations have indicated the extent to which each of the two modes is actually present in the elderly. The data have confirmed that the amplification of the hearing aid cannot solve the hearing problems of the majority of the elderly. They need primarily a hearing aid that would help to separate simultaneous sounds. At the moment, the most promising solution of this difficult technical challenge is improving the directional sensitivity. PMID- 8629281 TI - [Experienced quality of life of somatic nursing home patients: a review of measuring instruments]. AB - Research into the quality of life of somatic patients in nursing home is scarce in the Netherlands. In this article, 33 quality of life instruments for somatic nursing home patients are reviewed with respect to content, psychometric characteristics and resident-friendliness. A combination of a health status instrument and a life satisfaction instrument seems most suitable for the assessment of somatic patients in nursing homes: Eight instruments, including six health status instruments (McMaster Health Index Questionnaire, MOS SF-36, Nottingham Health Profile, Sickness Impact Profile, Quality of Well-being scale and COOP/WONCA charts) and two life satisfaction instruments (Philadelphia Geriatric Center Morale Scale and Life Satisfaction Index Z) are qualified and should be further investigated. PMID- 8629282 TI - [Item series of the cognitive screening test compared to those of the mini-mental status examination]. AB - The items of the ?mini-mental state examination' (MMSE) and a Dutch dementia screening instrument, the ?cognitive screening test' (CST), as well as the ?geriatric mental status schedule' (GMS) and the ?Dutch adult reading test' (DART), were administered to 4051 elderly people aged 65 to 84 years. This study was part of the Amsterdam Study of the Elderly (AMSTEL-project), which is a population survey of cognitive decline and dementia. Based on the item-pool, CST and MMSE scores were calculated. Both tests were comparable as far as their validity as dementia screeners is concerned (dementia criterion was GMS Organic syndrome, cut-point 2/3). The abbreviated version of the CST (CST-14) has a somewhat lower validity. The reliabilities of the unabbreviated CST (CST-20) and the MMSE are also comparable. The influence of age, education, depression, and premorbid intelligence (DART-IQ) was most notable in the MMSE. Thus, the CST-20 item set has slightly better psychometric properties than the MMSE. A figure is presented by which CST scores can be transformed into MMSE scores. PMID- 8629283 TI - [Health care for the aged; not only an economic problem]. PMID- 8629284 TI - [Personal meaning in the elderly: sources of meaning, welfare, coping and attitude to death]. AB - There are many different ways to experience one's life and aging as meaningful. The present study looks for different patterns of personal meaning in the elderly. Respondents were 376 older adults (221 women and 155 men), their mean age was 65.9 years. They completed several standardized questionnaires including measures of sources of meaning (SOMP), ultimate meaning (PMI), life satisfaction (LSIA), depression (SRDS), anxiety (STAI), coping orientations (COAP), and death attitudes (DAP-R). A cluster analysis performed on the sources of meaning revealed three groups each with a different pattern of meaning. The smallest group I (n=23) found most meaning in values that indicate self-preoccupation. Self-transcendent sources of meaning were on the top of the hierarchy of values of the second group II (n=123). The third and largest group III (n=230) valued self-transcendent sources of meaning as well as sources of meaning that indicate self-realisation. The three clusters also differed with regard to ultimate meaning (group I having the highest and group III the lowest score), well-being (group I reporting more depressive feelings and more feelings of anxiety than group II and group III), coping with aging (group III reporting most instrumental coping), and death attitude (group II having the most positive attitude). PMID- 8629285 TI - Cytomegalovirus prophylaxis in solid organ transplant recipients. PMID- 8629286 TI - Permanent skin replacement using chimeric epithelial cultured sheets comprising xenogeneic and syngeneic keratinocytes. AB - The present study was undertaken to evaluate the possibility of permanent skin replacement using chimeric xenogeneic-syngeneic graftable sheets previously obtained in vitro. Newborn (<3 days old) BALB/c and human keratinocytes were isolated and cocultured in different ratios as follows: 50% BALB/c to 50% human and 25% BALB/c to 75% human keratinocytes. Four to 5 days after culture and prior to their grafting, all chimeric sheets contained both cell types in ratios similar to those used to seed the initial chimeric cultures. Fourteen and 30 days after chimeric sheet grafting onto BALB/c mice dorsum, the newly generated cutaneous tissue showed a histologically well-organized epidermis presenting basal and suprabasal cell layers. Cutaneous cells in these structures secreted laminin and type IV collagen in blood vessels, and at ground level of the dermoepidermal junction there were signs of physiologically active skin. Cell phenotyping revealed the presence of only syngeneic keratinocytes, whereas xenogeneic cells were passively eliminated without a total rejection of the chimeric implant. This selective and passive elimination of xenogeneic keratinocytes went through cellular and humoral immunity activation. Data suggest that this chimeric culture method can be used for cutaneous therapies such as large congenital nevi, skin ulcers, and extensively burned skin. Indeed, for large third-degree wounded skin treatment, this culture method may shorten the time (4-5 weeks) needed for cell growth and graftable sheet production. Moreover, since the ultimate aim in allogeneic and xenogeneic transplantation is to achieve an immunological acceptance and tolerance to these foreign tissues, the chimeric culture approach may provide ways to lighten tolerance phenomena on cutaneous tissue. PMID- 8629287 TI - New synthetic sulfated oligosaccharides prolong survival of cardiac xenografts by inhibiting release of heparan sulfate from endothelial cells. AB - Binding of recipient natural antibodies to the endothelium of the graft, complement activation, endothelial cell activation, and microvascular thrombosis are major events in the hyperacute rejection of organ xenografts. The aim of this study was to investigate the effects of two new synthetic sulfated oligosaccharides (A and B) on the survival of discordant cardiac xenografts in the guinea pig-to-rat model. In untreated recipients, hyperacute rejection occurred in 5 min (median; range, 4-6 min) and immunohistological analysis of all the grafts revealed deposition of IgM and C3 along the endothelium. Administration of oligosaccharides A and B prior to revascularization prolonged the survival of xenografts in a dose-dependent manner, up to 113 min (median; range, 42-145 min) and 86 min (median; range, 35-108 min), respectively, when doses of 20 mg/kg were used. There were no bleeding complications. Histological examination of the rejected grafts showed a picture of hyperacute rejection, with no difference in IgM and C3 deposition as compared with the untreated animals. In cell culture experiments, the release of heparan sulfate from guinea pig cardiac endothelial cells induced by rat serum was inhibited by both saccharides in a dose-dependent manner. The results indicate that these new synthetic sulfated oligosaccharides are effective for prolongation of discordant xenograft survival, possibly by interfering with endothelial cell activation. Such substances may be of value in other xenotransplant combinations. PMID- 8629288 TI - Assessment of hyperacute rejection in a rat-to-primate cardiac xenograft model. AB - We studied a rat-to-cynomolgous monkey model for xenotransplantation of vascularized organs and found that a rat heart was rejected in 5.5 +/- 1.4 min (n = 10). This hyperacute rejection (HAR) was consistent with kinetic experiments in vitro that showed damage to rat endothelial cells (ECs) after 3 min of incubation with primate serum. Histopathology and ultrastructural analysis of rejected hearts showed marked EC damage and early adherence of platelets and polymorphonuclear leukocytes to the endothelium. Immunohistochemical analysis revealed deposition along endothelial surfaces of IgG, IgM, and complement (C) components of the classical but not the alternative pathway, suggesting that, as in the pig-to-primate model, HAR is mediated by the binding of recipient xenogeneic natural antibodies and C activation. The effect of C depletion on xenograft survival was evaluated in two recipients that were treated with cobra venom factor (CVF). CVF caused complete C inactivation, demonstrated by lack of serum hemolytic activity and C-dependent EC cytotoxicity at engraftment and until the animals died. The rat cardiac transplants survived for at least 9 hr and 77 hr. Histology showed massive interstitial hemorrhage, edema, and cellular infiltration with scanty fibrin deposits. These results in CVF-treated recipient monkeys indicate that C activation mediates the development of HAR in this rat-to primate model. We suggest that the model may be of interest as an alternative to the more expensive and time-consuming pig-to-primate model for testing the efficacy of transgenic modification of donor organs to prolong xenograft survival and for studying mechanisms of discordant xenograft rejection. PMID- 8629289 TI - Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice. AB - Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen were in minority and mainly located in the peripheral parts of the ICC xenograft. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 double-positive cells were found to be larger and more granular than the CD4 single-positive cells. No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (approximately 15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive beta 2 microglobulin-deficient mutant, C4.4-25-, lymphoma cell line EL-4 served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but, readily rejected the NK cell-sensitive lymphoma cell line. Taken together, ICC xenograft rejection in mice seems to be T cell dependent, as evidenced in the nude mice model, while the main effector cell appears to be a macrophage with a unique phenotype. PMID- 8629290 TI - CTLA4Ig alone or in combination with low-dose cyclosporine fails to reverse acute rejection of renal allograft in the rat. PMID- 8629291 TI - Delayed endocrine pancreas graft function after simultaneous pancreas-kidney transplantation. Incidence, risk factors, and impact on long-term outcome. AB - BACKGROUND: The incidence of delayed endocrine pancreas graft function and its impact on long-term outcome after simultaneous pancreas-kidney transplantation are unknown. METHODS: We studied 54 technically successful adult type I insulin dependent diabetic recipients of cadaver, whole organ, bladder-drained simultaneous pancreas-kidney transplants (mean age, 37.6 years; 65% male, 35% female; 9% pancreas retransplants; 63% on chronic pretransplant dialysis; mean duration of diabetes, 25.1 years). Insulin was administered during the first 2 weeks after transplantation, as needed, to keep blood glucose < 150 mg/dl. Delayed endocrine pancreas graft function was defined as total, cumulative insulin requirement of > 30 U between day 5 and day 10, and/or > 15 U between day 11 and 15. Quadruple immunosuppression was used for all recipients. RESULTS: The incidence of delayed endocrine pancreas graft function was 69%. By univariate analysis, delayed endocrine graft function was associated with pretransplant recipient weight > 80 kg (P = 0.04), donor age > 45 years (P = 0.02), and cardiocerebrovascular (P = 0.06) and nontraumatic causes of donor death (P = 0.02). The incidence of acute pancreas rejection episodes was similar for recipients without and with delayed endocrine pancreas graft function. Pancreas graft survival at 1 and 3 years was 94% and 82% without versus 76% and 59% with delayed endocrine graft function (P = 0.03). CONCLUSIONS: Increased pancreas graft failure after delayed endocrine function was a consequence of insufficient functional reserve (e.g., older donors) rather than increased immunogenicity. Pretransplant reduction of recipient weight and careful donor selection are therefore crucial in order to decrease the incidence of delayed endocrine pancreas graft function and its negative impact on long-term outcome. PMID- 8629292 TI - Delayed graft function in the absence of rejection has no long-term impact. A study of cadaver kidney recipients with good graft function at 1 year after transplantation. AB - We previously reported that delayed graft function (DGF) in the absence of biopsy proven acute rejection (Rej) had no effect on outcome of primary cadaver kidney transplantation (TX). By contrast, DGF in combination with Rej strongly predicted poor long-term graft survival. We asked whether this poor long-term outcome was due to early graft loss associated with DGF, or to an ongoing process leading to late graft loss. To answer this question, we studied a subset of 298 cadaver kidney transplant recipients who had not suffered early graft loss and had a serum creatinine level < or = 2.0 mg/dl at 1 year after TX. The incidence of DGF (defined by dialysis during the first week after TX) in this subset was 19%. DGF was associated with cold ischemia time >24 hr (P = 0.0003) and Rej (P = 0.06). For grafts with versus without DGF, the incidence of late acute Rej (>1 year after TX) was similar. Actuarial graft survival was similar for Rej-free recipients with versus without DGF (P = 0.9) and was worse for those with Rej and no DGF (P < 0.02). Importantly, however, in our recipients who all had a serum creatinine level < or = 2.0 mg/dl at 1 year after TX, the worst long-term outcome was noted in the subgroup with both DGF and Rej (P < 0.0001). By multivariate analysis, DGF was also only a risk factor in combination with Rej (P = 0.002, relative risk = 3.7), while a 0-antigen HLA mismatch had no impact. Patient survival decreased for recipients with both DGF and Rej by univariate (P = 0.009) and multivariate (P = 0.02, relative risk = 2.9) analyses. We conclude that DGF without Rej has no impact on long-term survival. However, our data for recipients with both DGF and Rej suggest that a chronic ongoing process leads to late graft failure. Further research is necessary to identify the exact pathophysiology of this process, which appears to be, at least in part, HLA antigen independent. PMID- 8629293 TI - Interstitial rejection, vascular rejection, and diffuse thrombosis of renal allografts. Predisposing factors, histology, immunohistochemistry, and relation to outcome. AB - Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis. PMID- 8629294 TI - How best to use tacrolimus (FK506) for treatment of steroid- and OKT3-resistant rejection after renal transplantation. AB - Nineteen patients with biopsy-confirmed ongoing acute rejection of renal allografts were converted from standard immunosuppression to FK506. Eight grafts showed vascular rejection and 11 had cellular rejection on biopsy. All patients had already received intravenous high-dose steroid treatment. Ten patients also had additional OKT3 rescue therapy. Initial FK506 doses were 0.13 +/- 0.06 mg/kg/day; the FK506 whole blood trough level after 3 days of treatment was 9.3 +/- 4.5 ng/ml. After conversion to FK506 all but four patients also received azathioprine, 1.5-2 mg/kg/day, and all patients received oral prednisolone. Concomitant with initiation of FK506, an anti-infective prophylaxis was prescribed, consisting of ganciclovir and trimethoprim/sulfamethoxazole. Sixteen out of 19 of the grafts (84%) were rescued successfully, including two grafts of patients already on hemodialysis at the time of conversion. Graft function of the responders improved from an average serum creatinine level of 364 +/- 109 mumol/L to 154 +/- 49 mumol/L. Of the patients receiving high-dose steroids alone prior to FK506 initiation, 8/9 responded to FK506 treatment, compared with 8/10 of those who had also received OKT3. During the mean follow-up of 35 weeks after conversion, no clinically apparent cytomegalovirus infection and no pneumonia were seen. Treatment with FK506 may successfully suppress ongoing acute rejection, even if antilymphocyte preparations have failed. FK506 can be used at a lower dose than so far recommended without impairing the antirejection potential. An additional anti-infective prophylaxis seems effective in preventing severe complications in the first months after rejection therapy. PMID- 8629295 TI - Identification of high- and low-risk second kidney grafts. AB - The purpose of this study was to identify recipients who are at low or high risk of early cadaveric regraft failure by segregating results of the flow cytometric crossmatch (FCXM) test with previous graft survival time (PGST). Early immunologic kidney regraft failure was analyzed in 103 multicenter recipients by cross-stratifying FCXM negative/positive status with < or =3- and >3-month PGST. T cell and B cell cytotoxicity crossmatches were negative. All were tested retrospectively in the T cell FCXM and 60 of the 103 were also tested in the B cell FCXM. A positive T and B cell FCXM was defined as a mean channel shift of > or = 9 (256 channel log scale) or > or = 40 (1024 channel log scale) for pretransplant crossmatch serum above negative control serum. Recipients received triple immunosuppression therapy and limited-use antilymphocyte induction therapy. Early cadaveric regraft losses were biopsied. Comparably good rates of second kidney graft survival at 3 years were found among three ow risk subsets: 78% for 18 FCXM-positive patients with PGST >3 months, 78% for 49 FCXM-negative patients with PGST >3 months, and 84% for 19 FCXM-negative patients with PGST < or =3 months. in contrast, 53% 3-month and 44% 3-year regraft survival rates occurred in 17 high-risk FCXM-positive recipients with a PGST < or =3 months. The odds ratio for increased relative risk of early second graft loss was 4.5 (confidence interval: 1.32-1.67) for the high-risk versus low-risk subsets (P = 0.009). Within the high-risk subset, 56% (5 of 9) of those who were FCXM T negative B positive experienced early regraft loss. A positive B cell FCXM has an adverse clinical impact only for high-risk regraft recipients. Pretransplant panel reactive antibody levels, pregnancy, number of blood transfusions between grafts, repeat donor HLA mismatches, and regraft recipient HLA mismatches did not correlate with early regraft loss. We conclude that kidney regraft survival rates in low-risk recipients (PGST >3 months/FCXM negative or positive [T and/or B cell] and PGST < or = 3 months/FCXM negative) approach primary graft survival rates and justify retransplantation, but the rate in high-risk regraft candidates (PGST < or =3 months/FCXM positive T and/or B cell) suggests that retransplantation should be performed only with a negative FCXM. PMID- 8629296 TI - Hepatic artery thrombosis after liver transplantation in children under 5 years of age. AB - The incidence of hepatic artery thrombosis (HAT) following orthotopic liver transplantation in children varies from 4% to 26% and represents a significant cause of graft loss. The purpose of this study was to analyze the risk factors for HAT following liver transplantation in children less than 5 years old. Seventy-three transplants were performed in 62 children under 5 years of age, including 16 for acute hepatic failure, 46 for chronic liver disease, and 11 retransplants. Twenty-four whole liver grafts (WLG) and 49 reduced size grafts (3 right lobes, 16 left lobes, and 30 left lateral segments) were transplanted. The recipient common hepatic artery was used to provide arterial inflow in 22 transplants and an infrarenal iliac conduit in 51 transplants. The overall incidence of HAT was 8 out of 73 transplants (11%). The cold ischemia time (14.3 +/- 3.03 hr) in this group was significantly longer than the cold ischemia time for those without HAT (11.7 +/- 3.94 hr) (P = 0.049). The incidence of HAT for whole and reduced grafts was 25% (6/24) and 4% (2/49), respectively (P = 0.01). HAT occurred in 6 of 22 grafts (27.3%) revascularized from the recipient common hepatic artery, compared with 2 of 51 grafts (3.9%) using an infrarenal arterial conduit (P = 0.008). The combination of recipient hepatic arterial inflow to a WLG resulted in HAT in 50% (6/12), whereas there were no cases of HAT with an iliac conduit to a WLG (P = 0.01). Of the eight patients with HAT, five are alive (median follow-up, 20 months; range, 7-27 months). Five patients were retransplanted, three within the first 2 weeks and two at 4 and 5 months for abnormal liver function in association with clinical and histological features of chronic rejection. Prolonged cold ischemia time and use of a whole graft with recipient hepatic arterial inflow are risk factors for developing HAT. The use of reduced size grafts and infrarenal iliac arterial conduits are associated with a low incidence of HAT. PMID- 8629297 TI - Improved outcome of orthotopic liver transplantation for chronic hepatitis B cirrhosis with aggressive passive immunization. AB - Passive immunization with hepatitis B surface antibody (anti-HBs) is important to prevent hepatitis B virus (HBV) recurrence after orthotopic liver transplantation for chronic HBV cirrhosis. Hepatitis B immune globulin (HBIG) dosing regimens have been poorly defined, utilize numerous routes of administration, and result in a high rate of HBV relapse and mortality. Twenty-five of 27 (93%) patients transplanted (four retransplants) for chronic HBV cirrhosis show no evidence of recurrent HBV (range, 2-55 months). Anti-HBs titers necessary to minimize the risk of hepatitis B surface antigen detectability were >500 IU/L for days 0 to 7, >250 IU/L for days 8 to 90, and >100 IU/L thereafter. Pretransplant HBV E antigen (HBeAG)-positive patients required more HBIG to achieve these goals than HBeAG negative individuals. The elimination of anti-HBs changed continually for the initial 3 posttransplant months. The anti-HBs half-life increased from 0.7 days to 14.1 days. Anti-HBs elimination was significantly different in HBeAG+ and HBeAG- patients for the first week, but was subsequently indistinguishable after week 1. After 3 months, the half-life was statistically less for HBeAG+ patients, but the difference did not influence the clinical treatment regimens. Quantitative hepatitis B DNA levels did not predict the amount of HBIG required. HBV recurrence after orthotopic liver transplantation can be reduced by aggressive passive immunization. Pharmacokinetic analysis of anti-Hbs elimination can improve immunoglobulin therapy and prevent recurrence of clinical hepatitis. PMID- 8629298 TI - Tacrolimus for primary treatment of steroid-resistant hepatic allograft rejection. AB - Twelve patients who experienced steroid-resistant rejection after primary liver transplantation while receiving cyclosporine-based therapy were converted to tacrolimus without receiving OKT3 or additional steroids. The indications for conversion were ongoing biopsy-confirmed rejection. All patients had received one course of high-dose intravenous steroids, which failed to reverse the rejection episode. No other antirejection therapy was given. Tacrolimus was initiated to reverse rejection and for maintenance therapy. The tacrolimus target level was 15 20 ng/ml (whole blood, IMX). All 12 patients had rapid reversal of the rejection episode and did not experience recurrent rejection (mean follow-up: 8.2 +/- 1.2 months). The mean bilirubin level dropped from 6.1 mg/dl at the initiation of tacrolimus therapy to 4.4 mg/dl by day 7 of therapy, 2.5 mg/dl by day 14, and 1.5 mg/dl by day 21 (P < 0.003). Serum glutamic pyruvic transaminase demonstrated a similar response. The serum creatinine level was unchanged at 1.5 mg/dl. No major adverse reactions were noted in this group of patients. Patient and graft survival rates were 100%. Four of the eight patients with a follow-up of >4 months are no longer receiving steroid therapy. Tacrolimus is effective as the primary therapy for the treatment of steroid-resistant rejection and provides a rapid and sustained biochemical response. Patients with mild to moderate rejection may be safely converted from cyclosporine to tacrolimus without an additional steroid bolus or OKT3 therapy. Early "preemptive" conversion to tacrolimus prior to the use of additional steroids or OKT3 may decrease overall rejection therapy requirements. This approach has promise for improved graft survival and fewer infectious and immunologic complications. PMID- 8629299 TI - Early graft loss secondary to massive hemorrhagic necrosis following orthotopic liver transplantation. Evidence for cytokine-mediated univisceral Shwartzman reaction. AB - Massive hemorrhagic necrosis (MHN) of the liver following orthotopic liver transplantation (OLT) occurs infrequently during an otherwise uneventful recovery 1 week after OLT. It is characterized by fever and sudden deterioration of allograft function leading to failure in the absence of vascular thrombosis. The etiology is unknown, although it is usually preceded by some degree of allograft rejection. Between 6 and 8 days after OLT, four patients (out of 150) became febrile, hypotensive, and experienced a rapid rise in transaminases within 48 hr. Two patients had evidence of mild rejection; the other two had moderate to severe acute cellular rejection. All patients were ABO identical, crossmatch negative. Bolus steroids were given followed by OKT3 in the two patients with severe rejection. Although sepsis was suspected, antibiotic therapy did not ameliorate the clinical course. Each patient progressed to MHN with severe centrilobular necrosis and variable portal infiltrate. High levels of interferon-gamma and tumor necrosis factor-alpha occurred prior to the rise in transaminases in each MHN patient (155 +/- 39 pg/ml and 414 +/- 201 pg/ml, respectively) compared with levels in OLT patients with severe rejection (14 +/- 4 pg/ml and 26 +/- 5 pg/ml, respectively, P < 0.05). These data support the concept of a cytokine-mediated inflammatory response leading to a univisceral Shwartzman reaction in the transplanted liver. Early recognition of this syndrome and retransplantation are critical for survival. PMID- 8629300 TI - An analysis of late deaths after liver transplantation. AB - Late deaths (after more than 1 year) after liver transplantation were analyzed in a series of 464 consecutive patients who received liver grafts between 1982 and 1993. Recipients who survived the first posttransplant year (n = 365) had actuarial 5- and 10-year survival rates of 92% and 84%, respectively. Thirty-five patients died between 1.1 and 7.6 years after transplantation (mean, 3.2 +/- 1.9 years). The most common causes of death were related to immunosuppression (40%), namely, chronic rejection, opportunistic infection, and lymphoma. The second most common causes of death were related to the primary disease for which liver transplantation was performed (34.3%), mainly recurrence of hepatobiliary malignancy and hepatitis B. Eight patients (22.9%) died of unrelated and unpredicted causes, most commonly of cardiovascular disease. Although the survival of liver recipients who live beyond the first posttransplant year is excellent, control of rejection and the consequences of chronic immunosuppression are continual threats. Modification of immunosuppression may help in decreasing the mortality of long-term survivors. In addition, better selection of recipients and effective adjuvant therapies (antiviral and antineoplastic) are needed in patients in whom the primary liver disease is notorious for recurrence. PMID- 8629301 TI - Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism. AB - Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts. PMID- 8629302 TI - Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted human hearts. AB - Allograft inflammatory factor-1 (AIF-1), a cytokine-responsive macrophage molecule, was originally identified and cloned from rat cardiac allografts with chronic cardiac rejection. We performed the present study to determine whether AIF-1 was also involved in the inflammatory response associated with human cardiac transplant rejection. AIF-1 gene transcripts were identified by the reverse-transcriptase polymerase chain reaction in endomyocardial biopsy specimens from human heart transplants and in macrophage cell lines. In the 441 base pair coding region of the human and rat cDNAs, the nucleotide sequences were 86% identical and the deduced amino acid sequences were 90% identical. Consistent with our studies in the rat. AIF-1 was selectively expressed in human macrophage like cell lines, and immunostaining in human heart allografts localized the AIF-1 gene product to a subset of CD68+ macrophages in the interstitial and perivascular spaces. The parallels between rat and human AIF-1 expression suggest that AIF-1 may have a common effect on the function of activated macrophages in cardiac allografts. PMID- 8629303 TI - Comparison of T cell responses in patients with a long-term surviving renal allograft versus a long-term surviving liver allograft. It's a different world. AB - The aim of the present study was to analyze whether acquired transplantation tolerance had developed in patients with a long-term surviving renal or liver allograft. Analysis of antidonor cytotoxic T cell precursor frequencies was performed in 31 renal allograft recipients and 9 liver allograft recipients with good graft function 2 years after transplantation. The results demonstrated that, before transplantation, normal antidonor T cell responses were generated in both groups of patients. Two years after transplantation, donor-specific CTL nonresponsiveness had developed in a minority of the renal transplant recipients. In contrast, 8 out of 9 liver transplant recipients showed donor-specific mixed lymphocyte culture and CTL nonresponsiveness. These findings indicate that development of donor-specific T cell nonresponsiveness is not a common event after kidney transplantation, whereas liver transplantation seems to induce, at least in vitro, a state of donor-specific T cell nonresponsiveness. PMID- 8629304 TI - Impact of N-acetylcysteine on the hepatic microcirculation after orthotopic liver transplantation. AB - Recent observations showed an improvement of hepatic macro- and microhemodynamics as well as survival rates after warm ischemia of the liver following treatment with N-acetylcysteine (NAC). In this study we assessed the influence of NAC on the hepatic microcirculation after orthotopic liver transplantation (OLT) using intravital fluorescence microscopy. OLT with simultaneous arterialization was performed in 16 male Lewis rats following cold storage in University of Wisconsin solution for 24 hr. Within the experimental group (n = 8) donors received NAC (400 mg/kg) 25 min before hepatectomy. In addition, high-dose treatment of recipients with NAC (400 mg/kg) was started with reperfusion. Control animals (n = 8) received an equivalent amount of Ringer's solution. Intravital fluorescence microscopy was performed 30-90 min after reperfusion assessing acinar and sinusoidal perfusion, leukocyte-endothelium interaction, and phagocytic activity. Treatment with NAC reduced the number of nonperfused sinusoid from 52.4 +/- 0.8% to 15.7 +/- 0.5% (p = 0.0001) (mean +/- SEM). Furthermore, we achieved a significant reduction of leukocytes adhering to sinusoidal endothelium (per mm2 liver surface) from 351.9 +/- 13.0 in controls to 83.6 +/- 4.2 in the experimental group (P = 0.0001). In postsinusoidal venules, treatment with NAC decreased the number of sticking leukocytes (per mm2 endothelium) from 1098.5 +/- 59.6 to 425.9 +/- 37.7 (P = 0.0001). Moreover, bile flow was significantly increased after therapy with NAC (4.3 +/- 1.2 vs. 2.2 +/- 0.7 ml/90 min x 100g liver) (P < 0.05). Phagocytic activity was not influenced by application of NAC. We conclude that high-dose therapy with NAC in OLT attenuates manifestations of microvascular perfusion failure early after reperfusion and should be considered as a means to reduce reperfusion injury. PMID- 8629305 TI - The liver neither protects the kidney from rejection nor improves kidney graft survival after combined liver and kidney transplantation from the same donor. AB - It has been proposed that the liver protects a simultaneously transplanted kidney from acute rejection. Using the United Network for Organ Sharing database, we compared the kidney allograft data from 248 combined liver and kidney transplants (LKT) with a control group comprising 206 contralateral kidney alone transplants (KAT) from the same donor. The LKT and KAT groups were identical with respect to most baseline parameters, save a greater degree of HLA matching in the KAT group. The overall 3-year graft survival rate was higher in the KAT group compared with the LKT group (80% vs 68%, P < 0.01). When these data were censored to remove death as a cause of graft loss and to minimize the matching effect, the 3-year survival rates were not statistically different (78% for KAT and 81% for LKT, P = NS). We conclude that the liver neither protects the kidney from rejection nor improves kidney allograft function or survival after LKT. PMID- 8629306 TI - Prophylactic immunosuppression with anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 versus OKT3 in liver allografting. A two-year follow-up study. AB - A prospective trial was conducted in 129 recipients of primary liver transplantation, to compare induction immunosuppression using triple drug therapy (cyclosporine, steroids, and azathioprine; group 1, n = 42), versus triple drug therapy with a 10-day course of OKT3 (group 2, n = 44) or of the anti-interleukin 2 receptor monoclonal antibody LO-Tact-1 (group 3, n = 43). Two-year actual patient survival rates were 64%, 79%, and 93% in groups 1, 2, and 3, respectively (1 vs. 2, NS; I vs. III, P = 0.003; 2 vs. 3, NS). Up to 2 years after transplantation, 18%, 44%, and 53% of the grafts in groups 1, 2, and 3, respectively, had not experienced steroid-resistant acute rejection (1 vs. 2, P = 0.002; 1 vs. 3, P = 0.007; 2 vs. 3, NS). The overall incidence of chronic rejection was 4%. OKT3 therapy, but not LO-Tact-1, significantly increased the incidence of cytomegalovirus infections (P = 0.019). In conclusion, immunoprophylaxis with LO-Tact-1 seemed to provide a liver graft acceptance rate at least as satisfactory as that with OKT3, without an increase in the incidence of infections. PMID- 8629307 TI - Rapid resolution of chylous ascites after liver transplantation using somatostatin analog and total parenteral nutrition. AB - Chylous ascites is the accumulation of chylomicronrich lymphatic fluid within the peritoneal cavity. It is a rare complication of retroperitoneal surgery, and may occur spontaneously in 0.5% of patients with cirrhosis. Its management is controversial, and despite a variety of treatment options with limited efficacy, the course is usually indolent. In this article, we report a case of rapid resolution of chylous ascites after liver transplantation following 10 days of treatment using somatostatin analog (Octreotide, 100 micrograms sc. t.i.d.) and total parenteral nutrition (TPN). A 55-year-old man underwent liver transplantation for hepatitis C cirrhosis, and developed an infected chylous fistula on the 10th day. Treatment by fasting, TPN, and somatostatin analog resulted in a rapid falloff in fistula output, with complete resolution of ascites within 2 days. This is the first report, to our knowledge, of somatostatin analog and TPN used in combination for rapid and successful closure of a chylous fistula. PMID- 8629308 TI - Recurrent hepatic allograft injury in erythropoietic protoporphyria. PMID- 8629309 TI - Humoral graft-versus-host disease after pancreas transplantation with an ABO compatible and Rh-nonidentical donor. Case report and a rationale for preoperative screening. AB - Severe hemolysis and graft ischemia complicating solitary pancreas transplantation with an ABO-compatible, Rh-negative, anti-D-positive donor to Rh positive recipient is described in this article. A brief review of the literature is presented. A rationale for preoperative screening for red cell antibodies during solid organ transplantation in this special setting is discussed. PMID- 8629310 TI - Glyoxalase II and glutathione levels in rat liver mitochondria during cold storage in Euro-Collins and University of Wisconsin solutions. AB - Glutathione and glyoxalase II levels were evaluated in cytosolic and mitochondrial compartments of rat liver after 7 and 24 hr of cold storage in University of Wisconsin (UW) and Euro-Collins solutions. 1-4 A similar time dependent depletion of cytosolic glutathione up to about 60% of control values was observed in both Euro-Collins and UW solutions. Cytosolic glyoxalase II showed activity oscillations in livers stored in Euro-Collins but not in UW. Mitochondrial glutathione and glyoxalase II were severely depleted soon after 7 hr of cold storage in Euro-Collins, whereas the same parameters did not change in liver stored in UW after 24 hr. UW is confirmed to be the most suitable solution for liver cold storage and we conclude that mitochondrial glutathione and glyoxalase II can be important parameters in assessing mitochondrial and cell function. PMID- 8629311 TI - A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation. AB - A newly developed, reliable, DNA-based method for typing for alleles of the HLA-C locus has been applied in the context of unrelated, volunteer donors for bone marrow transplantation. Some donors matched for HLA-A, -B, -DR, and -DQ have been found to generate in vitro high frequencies of CTL reactive with the recipient's cells. Here we demonstrate that there is a highly significant correlation of the frequencies of CTL precursors and incompatibility at the HLA-C locus. These data indicate that HLA-C locus incompatibility should be avoided in unrelated donor bone marrow transplantation. PMID- 8629312 TI - Complement: a cascade with neuro, immune, and endocrine functions. PMID- 8629313 TI - Metabolism of azathioprine. PMID- 8629314 TI - Interleukin-10 and Epstein-Barr virus-induced posttransplant lymphoproliferative disorder. PMID- 8629315 TI - Distal lower limb bone pain after renal transplantation. PMID- 8629316 TI - [Inorganic anions with a potential goitrogenic effect in drinking water supply for humans and animals]. AB - The levels of inorganic anions (NO3-, NO2-, SO(2-)4, F- and HPO(2-)4 with the following average values (mg/l) were determined isotachophoretically in 82 samples of potable water for humans and animals: 39.7 +/- 53.1 for nitrates, min. 0.0 - max. 363.3; 0.206 +/- 0.954 for nitrites, min. 0.0 - max. 7.82; 37.5 +/- 32.4 for chlorides, min. 0.0 - max. 137.9; 74.2 +/- 74.1 for sulfates, min. 4.2 - max. 369.2; 0.208 +/- 0.138 for fluorides, min. 0.032 - max 0.605 and 0.350 +/- 1.197 for phosphates, min. 0.0 - max. 17.4 (Tabs. I-IV). Within the set of samples examined, 22.0 of samples exceeded the limit value of nitrites; the respective values of nitrites, chlorides, sulfates and phosphates were 13.4, 6.1, 2.4 and 1.2. None of the samples exceeded the limit value of fluorides. 34.1% of samples complied with the value recommended by the standard for nitrites. Nitrate concentrations in drinking water for cows were compared with urine iodine content in some cows in the total of 398 urine samples from 14 iodine content in urine was recorded at some localities. Correlation analysis proved this relationship to be statistically highly significant (P < 0.01), Tab. VIII. The values of nitrate and iodine contents at DYJ locality are expressly different (Tab. VII). PMID- 8629317 TI - [The effect of prolonged starvation on changes in the activity of selected adaptive enzymes in rat liver]. AB - Male rats of Wistar SPF stain (Velaz Prague) were used to investigate the influence of prolonged starvation on changes in the activity of selected adaptive enzymes in the liver and corticosterone in serum. Analyses were carried out on days 1,2,3,5 and 7 of starvation. The activity of tyrosine aminotransferase significantly increased in the period between days 2 and 5 of starvation, after which a decrease to the level of satiated animals was observed in the terminal period. Activities of tryptophane-2-3-dioxygenase and alanine aminotransferase increased in two phases reaching maximum values on days 2 and 7 of starvation. The activity of aspartate aminotransferase showed a progressive significant increase in dependence on the length of starvation. A more than threefold increase in corticosterone concentration was observed in the serum of starved animals in comparison with satiated rats. PMID- 8629318 TI - [Q-fever--occurrence and significance of this disease in the Czech Republic and Slovak Republic]. AB - The paper summarizes the most important knowledge of Q fever and its causative agent Coxiella burnetti since the first descriptions of nosologic unit and causative agent until now. With respect to disease occurrence, distribution and importance the attention is focused on the territory of the former Czechoslovakia, i.e. currently on the territories of the Czech Republic and Slovak Republic. Taking into account normal occurrence of C. burnetii in cows which is however shown in latent form only, taking into account that no cases of human clinical infections related to these latent infections in cattle have been reported at present either in the territory of the Czch Republic or of the Slovak Republic, and considering that the results of experimental infections in sheep by the autochthonous strain of C. burnetii did not reveal its virulence, it can be stated that the strains of C. burnetti currently circulating on cattle farms are little virulent or even avirulent. This decrease in virulence can be ascribed to repeated subcultures of C. burnetti strains in cattle populations excluding ticks from this circulation. Q fever prevention should currently include measures to avoid in the territories of the Czech and Slovak Republics uncontrolled imports of infected animals and/or contaminated raw materials from animal or plant sources to introduce foreign fully virulent strains that can cause clinical infections both in humans and in domestic animals. PMID- 8629319 TI - Suspected adverse reactions, 1994. PMID- 8629320 TI - Improved performance of a large pig complex after sequential nursery depopulation. AB - An attempt was made to compare the productivity and financial benefits of nursery depopulation on five large pig farms which were all part of one complex. Each farm had been experiencing poor post weaning performance for 12 months, and had previously been infected with porcine reproductive and respiratory syndrome virus (PRRSV). A plan to depopulate each nursery sequentially was established, and the pigs were moved to fattening facilities on one of the farms (farm 3) where space was available. Over a four week period, the nurseries of farms 1, 2, 4 and 5 were emptied, cleaned and disinfected, and any changes in nursery performance, mortality and the seroprevalence of antibodies to PRRSV were then assessed for one year. The financial benefit to the entire farm complex was analysed by using partial budget methods. During the year a net benefit of $1,708,431 was assessed to the farm complex owing to the increased numbers of marketable pigs and the reduced antibiotic costs. There were highly significant improvements in nursery growth rate and decreases in mortality on farms 1, 2, 4 and 5, and antibodies to PRRSV were detected on farms 3 and 4 but not on farms 1, 2 and 5. The inability to empty the farm 3 fattening facility, which housed the pigs from the other sites, may have led to the maintenance of its PRRSV positive status and could have served as the source of virus for farm 4. PMID- 8629321 TI - Application of real-time ultrasonography for the detection of tarsal vein thrombosis in cattle. AB - The clinical and ultrasonographic features of the thrombosis of three tarsal veins in a six-year-old dairy cow are described. Thrombosis and metastatic abscessation developed on the left tarsus six days after the amputation of the lateral claw of the left hindlimb. Originally, the cow suffered from a severe purulent arthritis of the distal interphalangeal joint and a retroarticular abscess, caused by interdigital necrobacillosis. By means of real-time ultrasonography, using a 7.5 MHz transducer, a marked subcutaneous oedema, a subcutaneous abscess, and a thrombosis of the ramus cranialis and ramus caudalis of the vena saphena lateralis reaching the confluence into the vena saphena lateralis and a thrombosis of the ramus caudalis of the vena saphena medialis could be identified. The thrombosed veins were not compressible, were oval and had an increased diameter of up to 9 x 12 mm. Intraluminal masses were visualised as hypoechoic structures and the veins distal to the thrombosis were distended up to 10 x 13 mm. The differential diagnosis and pathogenesis of the thrombosis and the abscessation are discussed, and the clinical course and the sonographic observations of the thrombosis during a six week period are described. PMID- 8629323 TI - Incidence of bovine hypomagnesaemia in Northern Ireland and methods of magnesium supplementation. PMID- 8629322 TI - Actinobacillus suis septicaemia in two foals. AB - A 24-hour-old Hackney ony filly developed signs of weakness, depression and a poor suck reflex, with harsh lung sounds over both fields, and a 48-hour-old Arabian colt from a normal birth which had sucked vigorously developed loose stools and became depressed, weak and anorectic. Both foals had serum IgG concentrations greater than 800 mg/dl, but each had a severe neutropenia with a left shift, and blood cultures from both of them yielded Actinobacillus suis. The A suis isolates had different antimicrobial susceptibility patterns and, in the case of the Arabian, the isolate was resistant to commonly used broad spectrum antimicrobial agents. PMID- 8629324 TI - Enteritis associated with adenovirus-like particles in a Spanish kid. PMID- 8629325 TI - Rabbit viral haemorrhagic disease in Northern Ireland. PMID- 8629326 TI - TB testing. PMID- 8629328 TI - Animal transport proposals 'less than satisfactory'. PMID- 8629327 TI - Wildlife diseases. PMID- 8629329 TI - Feline tuberculosis: a literature review and discussion of 19 cases caused by an unusual mycobacterial variant. AB - The literature relating to feline mycobacterial disease is reviewed and 19 cats with tuberculosis caused by a previously unknown strain of mycobacterium are discussed. The bacteria were found to have characteristics between those of Mycobacterium tuberculosis and M bovis. The paper considers the clinical signs, epidemiology and diagnosis of the cases, and discusses the possible origins of the organism, treatment regimens and zoonotic potential. PMID- 8629330 TI - Comparison of urethral pressure profilometry and contrast radiography in the diagnosis of incompetence of the urethral sphincter mechanism in bitches. AB - Three diagnostic indicators of urethral sphincter mechanism incompetence were compared in 25 continent and 25 incontinent anaesthetised bitches: the resting urethral pressure profile, the stressed urethral pressure profile and the radiographic position of the bladder neck. Logistic regression indicated that the best predictor of continence status was the stressed urethral pressure profile as assessed by the percentage of negative peaks extending below the resting intravesical pressure; it classified 43 of the 50 dogs correctly. The radiographic position of the bladder neck was a better predictor of continence than either the measurement of functional profile length or the maximum urethral closure pressure from the resting urethral pressure profile, whether alone or in combination. By combining the percentage of negative peaks on the stressed profile with the position of the bladder neck, 46 of the 50 dogs were classified correctly. Cut-off values for the percentage of negative peaks on the stressed urethral pressure profile, and for the radiographic position of the bladder neck are suggested for use in evaluating incontinent bitches in clinical practice. PMID- 8629331 TI - Health and welfare of rehabilitated juvenile hedgehogs (Erinaceus europaeus) before and after release into the wild. AB - Thirteen wild caught juvenile hedgehogs were treated and overwintered in a rehabilitation centre and 12 were released into the wild and monitored by radiotelemetry. Clinical examinations were carried out before they were released and twice afterwards, and any hedgehogs found dead were examined post mortem. The health of the animals was generally good but dental disease, obesity and minor injuries were common. One hedgehog died before it was released and had cardiovascular and respiratory lesions, and one was euthanased 28 days after its release and had verminous enteritis and parasitic bronchitis. Three hedgehogs were killed by badgers, two were killed by road traffic and four were known to have survived when the study ended. PMID- 8629332 TI - Granular cell tumour in the central nervous system of a ferret (Mustela putorius furo). PMID- 8629333 TI - Multigeneric resistance to oxfendazole by nematodes in cattle. PMID- 8629334 TI - Serological evidence for the presence of A/equine-1 influenza virus in unvaccinated horses in Croatia. PMID- 8629335 TI - Longevity and diet. PMID- 8629336 TI - Intestinal obstruction with hemp bedding. PMID- 8629337 TI - Causes of death in cattle. PMID- 8629338 TI - Mystery hum. PMID- 8629339 TI - [Diagnosis of nodal mastopathy: present-day views]. AB - The concept of nodal mastopathy is accumulative. Both fibrotic segments and fibroadenomas, cysts, lipomas and other formations with atypical manifestations may serve as a substrate for forming a node. In addition to the clinical method, a diagnostic set should involve mammography that detects the true nature of a formation in 75-91% of cases, traditional sonography providing additional information in cysts in 88% of cases, Doppler sonography suspecting enhanced proliferative processes by the higher blood flow velocity and defining indications for surgical treatment, as well as new puncture bioptic procedures yielding better information materials by 25-30%. The comprehensive assessment of non-specific manifestations of nodal mastopathies from the data obtained by diagnostic techniques enables a more exact preoperative diagnosis to be made. PMID- 8629340 TI - [Functional disorders of the digestive tract in children with vertebrogenic diseases]. AB - The paper provides the results of comprehensive clinical and X-ray studies of the digestive tract by using ultrasonography, fiber gastroscopy, heat imaging. The X ray symptomatology of its dysfunctions was examined in 89 children with vertebrogenic diseases (osteochondrosis, scoliosis, developmental anomalies, sequelae of birth injury to the neck, spine, vertebral arteries). PMID- 8629342 TI - [Possibilities of computerized tomography in the diagnosis of recurrences of cancer of the large intestine]. AB - To study the potentialities of CT in the diagnosis of recurrent cancer of the large bowel, 325 patients underwent various surgical interventions for colorectal carcinoma were examined. The surgery-CT study interval ranged from 2,5 months to 11 years. It was found that CT might detect various intra- and extraintestinal relapses and generalize a tumorous process. With this, the efficiency of CT is higher than that of other diagnostic tools in the detection of extraintestinal relapses and generalizing the tumorous process. To timely reveal a relapse the CT interval should exceed 4-5 months. PMID- 8629341 TI - [Computerized tomography diagnosis of splenic and hepatic lesions in malignant non-Hodgkin's lymphomas]. AB - Seventy eight patients with varying malignancy non-Hodgkin's lymphomas were examined. Signs of extranodal damage of the spleen and liver were studied. Spleno and hepatomegaly were signs of the commonest diffuse visceral involvements. In terms of the malignancy (low- or high-grade), splenomegaly was seen in 71.4 and 82.5% of patients and hepatomegaly was observed in 43.9 and 28.6%, respectively. In addition, irregular visceral outlines, tuberosity, changed density of the parenchyma and its homogeneity. A high correlation was found between the enlargement of the viscera and their decreased parenchymal density. Focal visceral involvements are less difficult to be diagnosed than diffuse ones, but they are much rarer. Computerized tomographic findings indicate the malignancy of a process. PMID- 8629343 TI - [Possibilities of polyposture methods in x-ray diagnosis of renal and ureteral calculi and associated complications]. AB - A comprehensive radiation examination of 47 apparently healthy patients has ascertained that one of the angles formed by the renal lateral axis and the body's frontal plane (BFP) is 35-45 degrees and medially open, the second made by the long axis of the kidney and BFT is 28-32 degrees and caudally open. The densitometric findings from the X-ray films of 17 patients with ureteral calculi (UC) significantly suggest that a contrast medium rapidly moves to the dilated lower urinary tract due to changes in the relative position of the renal cavities against BFP. Twelve patients underwent separate studies of diuresis, glomerular filtration and tubular reabsorption in the supine position and in the position of the lower body dropped at an angle of 45 degrees (for 20-min periods). Excretory urography followed by measurements of the mean ureteral diameter was performed in 36 patients with dilated ureters and in 31 control subjects. There was a close correlation between the size of ureteral lumen and diuretic values and there was an inverse relationship to the magnitude of tubular reabsorption. PMID- 8629344 TI - [Caval urography in children with large and giant omphalocele]. AB - The paper presents the results of using a caval urographic procedure in 37 children with large and giant omphalocele prior to the radical operation displacement of organs from hernial protrusion into the abdominal cavity. High percentages (54 and 96%) of changes in the vena cava and urinary tract, respectively, make the author recommend this procedure for all children with this congenital anomaly. The findings help surgeons choose treatment policy and avoid undesirable complications. PMID- 8629345 TI - [A case of tracheomegaly with diffuse constriction of peripheral bronchi]. PMID- 8629346 TI - [Complex x-ray diagnosis in multiple heart defects]. PMID- 8629347 TI - [Methods of determination and ways of lowering radiation loads in x-ray angiography room personnel]. PMID- 8629348 TI - [Computerized tomography in sequelae of brain contusions]. AB - Follow-up clinical and computed tomographic (CT) studies were performed in 140 patients with focal brain contusions at the acute stage of brain injury (BI). A total of 133 victims were followed up and the time course of CT changes were examined in the intervening and late BI periods. Despite the favourable natural history of acute BI, mild, moderate, and severe posttraumatic changes were shown to appear as cicatricial-adhesive and atrophic processes, intracerebral cysts, porencephaly, which result in posttraumatic epilepsy, hydrocephalus, etc. The magnitude of diffuse changes rather than focal changes in the area of the prior medullary lesion was found to play the leading role in the victims' disability. PMID- 8629349 TI - [Contrast media for computerized and magnetic resonance tomography. Basic principles]. AB - The paper gives a review of the chemical structure, pharmacology and clinical use of currently available contrast media for CT and MRT examinations. PMID- 8629350 TI - [History of the development of radiographic services in the Republic of Mari-El]. PMID- 8629351 TI - [Interventional radiology in postoperative recurrent goiter]. AB - Roentgeno-endovascular functional thyroidectomy was used to suppress the pathological activity of the thyroid in postoperative recurrent goiter. The method consists in vascular isolation of hyperplastic stump of the thyroid by catheterization of the left and right thyroid arteries, followed by their material occlusion. For embolization, a wide spectrum of nonlysed synthetic, organic, and inorganic materials were used. The results of roentgeno-endovascular functional thyroidectomy in 14 patients with postoperative recurrent goiter are analyzed. The advantages of roentgeno-endovascular occlusion of the thyroid arteries before subtotal thyroidectomy are emphasized. A stabile clinical and hormonal remission and reduction of the thyroid in size to stage 1 were observed during three years following roentgeno-endovascular intervention. PMID- 8629352 TI - [Short-term hospitalization for patient education--effect on metabolic compensation in type I diabetics]. AB - The objective of the work was to evaluate the effect of short-term hospitalization on metabolic compensation in type 1 diabetics with an intensified insulin regime who are admitted to hospital with the main purpose to participate in an intense educational and therapeutic programme. Twenty patients were examined, mean age 26.3 +/- 6.5 years, mean duration of diabetes 13.2 +/- 8.0 years, who participated in a 5-day educational therapeutic programme which takes place in the authors' department. The level of metabolic compensation was evaluated at the onset and at the end of the hospitalization by means of fructosamine (F), mean blood sugar level, Michaelis index, MAGE (mean amplitude glycaemic excursion), the M value, MDD. During hospitalization in the whole evaluated group an average 6% drop of F was recorded. In the sub-group of patients with a baseline F > 2.6 mmol/l a statistically significant decline occurred from 2.91 +/- 0.14 mmol/l to 2.44 +/- 0.31 mmol/l (p < 0.05). The changes of different indexes calculated from the whole group did not reach statistical significance. In the sub-group with a poorer compensation (F > 2.6 mmol/l), however, the mean blood sugar level declined from 13.48 +/- 2.45 mmol/l to 9.17 +/- 2.05 mmol/l (p < 0.05) and the M value improved from 141.4 +/- 56.6 to 60.5 +/- 40.7 (p < 0.05). The MAGE index deterioration significantly during hospitalization only in the sub-group with a baseline F < 2.6 mmol/l from 2.70 +/ 1.11 to 3.65 +/- 1.28 (p < 0.05). The results indicate that in patients with type 1 diabetes mellitus short-term hospitalization with an educational therapeutic programme need not be associated with a deteriorated metabolic compensation. In those whose compensation is unsatisfactory marked improvement is recorded. PMID- 8629353 TI - [Colchicine in the treatment of liver cirrhosis. 12 years' experience]. AB - The authors evaluate more than 12 years of their own experience with the administration of colchicine to patients with cirrhosis of the liver. Colchicine did not produce any apparent undesirable effects. The authors emphasize the absence of knowledge of prognostic parameters (except the stage of the disease and its activity) which would make it possible to evaluate in advance the effect of many years' treatment. They deal separately with a patient who entered their investigation in a very advanced stage of the disease and survived after he had recovered full compensation and did not depend on diuretic treatment. PMID- 8629354 TI - [Free radial activity in patients with pneumoconioses]. AB - The authors examined indicators of the effect of free radicals (MDA, SOD, GSHPx, selenium) in 20 patients with notified simple silicosis or miner's pneumoconiosis, in 11 patients with complicated silicosis or miner's pneumoconiosis and in 10 patients exposed to fibrogenic dust without an X-ray finding of pneumoconiosis. No statistically significant differences between individual groups were found. Subsequent investigations should be focused on subjects with incipient pneumoconiosis who are not yet entitled to damages. PMID- 8629355 TI - [Crouzon's syndrome, arthritis and crural ulcers]. AB - The author describes a special inflammatory rheumatic disease in a 48-year-old female patient with Crouzon's syndrome which most probably could be included among seronegative spondarthritis. The author discusses the possible ratio of inborn disease in the atypical course of the rheumatic disease. PMID- 8629356 TI - [Diabetic dyslipidemia and its treatment]. AB - A review on the lipid metabolism in IDDM and NIDDM. The paper deals with quantitative and qualitative lipid changes and their relationship with macrovascular diseases. Part of the paper is devoted also to practical problems of dietetic and medicamentous treatment of lipid disorders in diabetes and different hypolipidaemic agents are discussed in detail. PMID- 8629357 TI - [Diurnal changes in blood pressure, albuminuria and urinary excretion of retinol binding protein in type I diabetics]. AB - We studied 24-h ambulatory systolic and diastolic blood pressure (SBP, DBP), 16-h daytime and 8-h nighttime urinary excretion of albumin (UAE) and retinol-binding protein (URBP) in 20 type 1 diabetic patients (group 1) with normoalbuminuria (UAE < 20 micrograms/min) and 20 type 1 diabetic patients (group 2) with microalbuminuria and low proteinuria (UAE 20-500 micrograms/min). The groups were comparable in age, diabetes duration and actual glycaemic control. Daytime and nighttime SBP and DBP were higher in group 2 compared to group 1 (p < 0.01). Nighttime decrease in SBP and DBP correlated with nighttime decrease in UAE in group 2 (p < 0.05, p < 0.001). There was no correlation between BP and actual glycemic control in either group. Daytime UAE was found in group 2 by 20% higher than nighttime UAE. We found higher daytime and nighttime URBP in group 2 compared to group 1 (p < 0.05). We conclude, that microalbuminuric and low proteinuric patients had elevated BP and nighttime decrease in BP correlated with nighttime decrease in UAE but not with actual glycemic control. Increased URBP in these patients suggests impaired proximal renal tubular function in early stages of diabetic nephropathy. PMID- 8629358 TI - [Insulin resistance and pathogenesis of non-insulin-dependent diabetes mellitus (I)]. AB - The authors present a review of current knowledge of insulin mechanism on cellular level and of the defects leading to diminished cellular insulin sensitivity and to development of insulin resistance (IR). Attention is focused primarily on IR in subjects with non-insulin dependent diabetes mellitus (NIDDM) with main stress on the postreceptor level of insulin effect which includes glucose transport, storage (glycogen-synthesis) and glycolysis. The defects of these mechanisms (namely the decrease in glycogen-synthase activity) are considered the main causes of IR in patients with NIDDM. Beside the above mentioned factors the authors discuss some of the less well-known mechanisms that can be found in the background of IR in patients with NIDDM (tissue blood flow, capillary density, transport of insulin across the endothelial barrier, glucose toxicity, role of intracellular calcium and others. PMID- 8629359 TI - [Use of beta-blockers in the treatment of chronic heart failure]. AB - Recently in the world literature with increasing frequency reports are found on clinical studies investigating the use of beta-blockers in the treatment of chronic heart failure. The authors present a review of the problem comprising pathophysiological mechanisms at a receptor level up to investigations of the effect of beta-blockers on haemodynamics, the functional state of the left ventricle and survival. Although this promising and frequently controversial treatment is not accepted universally so far, partial data in the mentioned investigations indicate that when a more accurate dosage pattern is established and indications are specified in more detail, the use of beta-blockers is one way how to approach - in addition to the use of diuretics, cardiotonics and angiotensin converting enzyme inhibitors - a comprehensive therapeutic control of chronic heart failure. PMID- 8629360 TI - [Non-invasive prognostic parameters in chronic heart failure]. AB - The relationship between baseline clinical, laboratory and auxiliary indicators on the one-year mortality was investigated in 125 patients with chronic heart failure caused by ischaemic heart disease or cardiomyopathy associated with dilatation. During the baseline examination all patients had cardiac symptoms functional class NYHA II-IV- and their ejection fraction assessed by echocardiography was < 40% and/or their cardiothoracic index was > 50%. Within twelve months after the baseline examination 19 (15.2%) patients died. Signs of pulmonary congestion and the cardiothoracic index were the most significant prognostic indicator of the one-year mortality (p < 0.001). As to other indicators, the following were statistically significant: sodium level, urea level, the duration of the ergometric test and the patients' body weight. Statistical significance was not recorded in echocardiographic indicators and the NYHA classification. These data, in particular the newly introduced four-grade classification of pulmonary congestion, make it possible to assess a more accurate prognosis of high risk patients with chronic heart failure. PMID- 8629361 TI - [Chronic renal insufficiency. Present views on pathogenesis, clinical aspects and treatment]. AB - The authors present contemporary views on the pathogenesis, clinical picture and treatment of chronic renal failure. A comprehensive approach to the pathogenesis of renal failure revealed new therapeutic methods such as treatment with erythropoietin, vitamin D etc. The authors emphasize substantial changes in the quality and range of the clinical picture which developed due to the prolonged survival of patients with dialyzation treatment. New problems developed such as e.g. dialyzation amyloidosis, aluminium intoxication, endocrine changes, increased occurrence of malignities etc. These must be foreseen, diagnosed and treated. While during the first half of this century uraemia was a fatal disease, at the end of the nineties patients survive due to the dialyzation and transplantation programme. PMID- 8629362 TI - [Standard care of the diabetic foot]. PMID- 8629363 TI - [Kubat: Lactic acidosis--a rare or common disease?]. PMID- 8629364 TI - [Levels of lactic acid in type II diabetics treated with buformin]. AB - The objective of the work was to assess the blood lactate levels in type 2 diabetics selected at random, treated for prolonged periods with buformin. 77% of the investigated group of diabetics (N = 70) had elevated blood lactate levels and 16% of them had hyperlactataemia (more than 5.0 mmol/l). In 33% patients with an originally elevated lactataemia after 12 weeks of discontinued buformin treatment the lactic acid blood levels reached normal values. The lactate levels declined significantly (by more than 25% of the original value) after 6 weeks without buformin treatment in another 45% of the diabetic patients. In 18% of the investigated patients, who had high lactate levels even after discontinuation of treatment, in two-thirds neither impaired function of the kidneys and liver nor cardiac disease or alcoholism were detected. PMID- 8629365 TI - Morphological analysis of saphenous vein used for aortocoronary graft. AB - Histological, histochemical, immunohistochemical and ultrastructural analyses of 120 saphenous veins (of 120 patients) prepared for aortocoronary graft, revealed in 57 cases lesser or larger morphological changes, such as endothelial damage, hyperplasia of t. intima, or t. media, and fibrosis of t. adventitia that could be the result of postoperative complications (thrombosis of the graft lumen, or its occlusion due to progressive hyperplasia of t. intima). The analyses have proved significantly increased number of smooth muscle cells and fibroblasts in t. intima of the damaged veins in relation to t. media, contrary to the findings in normal veins, and it was suggested that these smooth muscle cells in the damaged veins migrated from t. media to t. intima causing hyperplasia, as was confirmed by histochemical, immunohistochemical and ultrastructural findings. Since in 57 of 120 analyzed veins morphological changes were found which developed before the implantation of vein and which could compromise the vein function as the aortocoronary graft and cause severe postoperative complications, forming of vein banks is recommended from the chosen donors to avoid vein implantation with the already existent morphological changes, that could be proven by their immediate histological examination. PMID- 8629366 TI - The correlation of clinical and laboratory parameters of the acute phase with the evolution of poststreptococcal glomerulonephritis in adults. AB - Prospective clinical study included the group of 44 patients who had the acute phase of poststreptococcal glomerulonephritis (PSGN) between the age of 18 and 22 during their military service. After the acute phase the patients were followed up to 5 years in order to investigate the influence of some clinical and laboratory parameters in the acute phase of PSGN on the evolution of this disease. The evolution of the disease, estimated on the basis of pathological findings in urine (proteinuria and erhytrocyturia), was found in 61% of patients. It was shown that severity of the clinical picture and the degree of proteinuria in the acute phase of PSGN were in direct correlation with the evolution of disease. On the other hand, the elevated concentrations of circulatory immune complexes (CIC) and cryoglobulin in the acute phase did not significantly affect the evolution of the disease although in some degree it contributed to the severity of pathological findings in the urine. PMID- 8629367 TI - [The effect of drivers' health status on the occurrence of traffic accidents]. AB - The analysis of the influence of the drivers' health condition on the occurrence of traffic accidents was presented. The tested group consisted of 930 drivers with diseases or conditions that demanded the constant health control and monitoring. Control group was composed of 320 completely healthy drivers. Both groups were approximately similar in relation to the other factors that could influence the occurrence of traffic accidents. The behavior of these drivers was followed up in the four-years period. The results suggested that drivers with certain diseases and conditions, (psychological-psychiatric conditions and diseases, arterial hypertension, diabetes mellitus, hearing and sight impairments, varices cruris, stomach and duodenal ulcers) significantly more often cause the traffic accidents, compared to healthy drivers. The need for constant and rigorous health selection for drivers is imposed in order to alleviate the epidemic of traffic traumatism. PMID- 8629368 TI - [Aspiration bronchopneumonia as a complication of acute poisoning with psychotropic drugs]. AB - In the five year period, retrospectively and prospectively, the frequency, clinical, radiographic and bacteriological characteristics of the aspiration bronchopneumonia (ABPN) were followed in the acute poisoning by psychotropic drugs (PD). In 1769 patients with acute poisoning by PD, ABPN was determined in 44 (2.49%) patients, and most frequently in groups with polymedicamentous (5.99%) and neuroleptic (5.17%) poisoning, and rarely in the group poisoned by anxiolytics (0.77%). Severest poisonings by PD were complicated by ABPN in 16.84% of cases. High conformity of clinical and radiographic finding of bronchopneumonia was achieved. The diagnosis of bronchopneumonia was established on the day of admission at the Clinic in 81.9% of cases on the other day in 13.6% and on the third day in 4.5% of patients. Bacteriological examination of sputum revealed the pathogens in 63.6% of patients, but in no patients the anaerobic bacteria were isolated. The treatment was very complex, and beside the detoxification measures, it was necessary to remove the aspirated contents from the respiratory tract (usually during endotracheal intubation or therapeutic bronchoscopy) and immediately apply antibiotics. The examinees were hospitalized twice longer than the patients whose course of poisoning by PD was not complicated by ABPN. The poisoning ended lethally in two (4.5%) patients. In no cases the ABPN was the immediate cause of death, but it significantly contributed to the lethal outcome. PMID- 8629369 TI - [Cytologic characteristics of cerebrospinal fluid in patients with serous meningitis caused by enteroviruses, mumps virus or Koch's bacillus]. AB - The qualitative and quantitative analysis of the cytogram of cerebrospinal fluid (CSF) was performed in the acute phase of enteroviral meningitis (ENTERO MGT. 31 patients), mumps meningitis (MUMPS MGT, 25 patients) and tubercular meningoencephalitis (TBC ME, 15 patients), to observe the basic characteristics and answer the question whether the cytological response of the central nervous system (CNS) is ethyologically determined. The CSF sediment was obtained in Saykov chamber and was dyed according to Pappenheim. In the group of ENTERO MGT the significant representation of neutrophils with the presence of basophile was observed. The main characteristic of MUMPS MGT was mononuclear response with various morphological forms of lymphocyte cells and neglectable presence of neutrophils. TBC ME was characterized by the neutrophilia in the first 12 days of illness; till the end of the fourth week there was an equal representation of neutrophils and mononuclear cells, followed by a significant lymphocytosis. The cells of the monocytemacrophage system and the plasma-cells were constantly present. It could be concluded that differences in cytological response of CNS between ENTERO and MUMPS group were mainly quantitative and dynamic, probably caused by the various pathogenesic mechanisms of inflammation. The cytological picture of CSF during TBC ME in relation to other two groups had certain qualitative, quantitative and dynamic characteristics which could indicate the diagnosis of the specific meningoencephalitis, making this analysis very useful in the early diagnostics of this illness. PMID- 8629370 TI - [Personal experience with ex tempore diagnosis of neurosurgical biopsies using the smear method]. AB - The results obtained by the use of the smear method were analyzed in ex tempore diagnostics of 240 neurosurgical biopsies, done in the period from 1991 to 1993. Thirty three different pathologic lesions were found that had their special morphology on smears, indicating the high specificity of this method. In 226 cases (94.17%) the diagnosis was established correctly, but in 14 cases (5.83%) it was not: in three cases the diagnosis was falsely negative and in 11 falsely positive. The main causes of the diagnostic errors were analysed and they included the subjective factors and uncommonness of the analyzed tissue samples. The accuracy of 94.17% enabled sufficiently reliable use of this method in ex tempore diagnostics of neurosurgical biopsies. PMID- 8629371 TI - [Epidemiologic characteristics of malignant tumors of the digestive tract in Serbia 1969-1990]. AB - The epidemiological situation of the chosen malignant tumors of digestive tract for the period 1969 - 1990, was analyzed in Serbia without autonomous provinces, based on the data of mortality rate. During this observed 22-year period the average standardized mortality rate (%000) was lowest for the oesophageal cancer (men - 2.2; women - 0.6) and gall bladder cancer (1:1.8) and highest for the stomach cancer (14.3:7.1). Men in relation to women died more of oesophageal cancer (3.7 times), stomach (2 times), colon (1.2 times), rectum (1.4 times) and pancreas (1.7 times). In both male and female population the highest average specific mortality rates were observed for persons over the age of 75 for all observed malignancies of digestive system. The standardized mortality rates for both sexes for the period 1969-1990 demonstrated the trend of increase for the cancers of oesophagus, colon, rectum, gall bladder, and pancreas, and the decrease for the stomach cancer. PMID- 8629372 TI - [Carboplatin and radical radiotherapy in the treatment of locally advanced urinary bladder carcinoma]. AB - Our prospective nonrandomized study included 46 patients with invasive urinary bladder carcinoma of the clinical T3a and b stages. The patients were treated by radical radiotherapy (65 Gy, conventional fractioning) and by Carboplatin (150 mg per week during radiotherapy; total 900 mg). Of 35 patients for whom the response was evaluated, the complete regression of tumor was reached in 85.71% and partial in 5.7% of patients. Hematological toxicity of I and II grade was seen in 36/46 of patients. The average follow-up time was 9 months (interval 4-15), overall one year survival rate was 85%, and disease-free survival was 87%. The results have shown that simultaneous application of radical radiotherapy and Carboplatine promises the improvement in the treatment of the invasive urinary bladder carcinoma, but the short follow-up time does not allow the definitive conclusions. PMID- 8629373 TI - [The significance of tumor suppressor genes and viral oncoproteins in the onset of malignant cell transformation]. PMID- 8629374 TI - [Multiple sclerosis--a new approach to pathophysiology, diagnosis and therapy]. PMID- 8629375 TI - [Modern therapy of scleroderma]. PMID- 8629376 TI - [Modern technologic development and progress in diagnostic radiology (II)]. PMID- 8629377 TI - [Symptomatic congenital anomalies of the internal carotid arteries. 2 case reports]. PMID- 8629378 TI - [Iatrogenic injuries of the rectum]. PMID- 8629379 TI - [A severe form of leptospirosis]. PMID- 8629380 TI - The role of communication in surgical wound infection surveillance. PMID- 8629381 TI - Technique and early results of videoscopic lumbar sympathectomy. AB - The authors describe their technique of videoscopic (VS) lumbar sympathectomy (LSE), compared to the open LSE. From 1992 to 1994, 21 open and 19 VS LSE were performed. The indication was reflex sympathetic dystrophy in 17 and arterial insufficiency in 23 patients. In the open LSE the mean duration of anaesthesia was 80 min (55-115) and of surgery 37 min (25-65). The length of the chain removed varied from 1 to 3 ganglia (6-7 cm). Complications were noted in 5 patients: 1 pneumonia, 2 superficial wound problems and 2 cases of postsympathectomy neuralgia. Hospital stay of patients with RSD varied from 2 to 5 days. Of the 19 attempts to perform a VS LSE 4 had to be converted to the open technique. The duration of anesthesia was 150 min (90-280) and of surgery 92 min (45-240). Lengths of chain removed varied from 1 to several ganglia (6-7 cm). A pneumoperitoneum was present in 10 procedures, but a Veress needle was placed in only 4 of these. Complications were present in 9 patients: 1 important subcutaneous emphysema, 1 severe costal pain, 2 neuralgia, 1 temporary psoas dysfunction, 1 haemorrhage from a lumbar vein with conversion to the open technique and 3 minor superficial wound problems. The hospital stay ranged from 2 to 5 days. This study suggests that the VS LSE has no benefit over the open technique as far as the operative and early results are concerned. Whether this technique avoids some of the late disadvantages of a lumbotomy remains to be seen. PMID- 8629382 TI - Kinking of the internal carotid artery: clinical significance and surgical management. AB - The authors report on 62 surgical corrections for kinking of the internal carotid artery during a 13-year period (1980-1993). This represents 2.8% of all carotid operative procedures (n = 2188) in the same period. It always concerned a significant (< 60 degrees) angulation of a redundant internal carotid artery, that in all but 3 cases was associated with atherosclerotic involvement of the carotid bifurcation. The indication to surgery included transient hemispheric or ocular ischaemia in 25.5% of cases, a regressive neurologic deficit in 8%, a minor stroke in 3%, a stroke in evolution in 11%, and non-lateralized cerebral ischaemia in 21%. In 19 patients (31%) it concerned an asymptomatic high degree stenosis. The surgical technique consisted in carotid transposition reimplantation after eversion endarterectomy in 37 cases, in posterior transverse plication with patch angioplasty in 20 cases, and in segmental excision with venous interposition graft in 5 cases. There was one postoperative death. The morbidity include one ipsilateral non-fatal stroke and 3 transient ischaemic attacks. A complete long-term follow-up (mean duration 3.4 years) is available for 57 patients. The late incidence of stroke is 1.5% per year. The 5-year survival attains 67%. These long-term results are comparable to the outcome of standard endarterectomy in the same institution. The authors discuss the indication, techniques, and outcome of surgical correction of kinked internal carotid artery. They recommend a shortening procedure, often associated with endarterectomy for severely kinked vessels (angulation 60 degrees or less), symptomatic or not. PMID- 8629383 TI - Role of thoracoscopy (VATS) in pleural and pulmonary pathology. AB - Although thoracoscopy is not a new procedure, there was a real revival after the introduction of laparoscopy. VATS (video-assisted thoracic surgery) has potential advantages of reduced pain due to smaller incisions and a shorter hospital stay. Main disadvantages are expensive equipment and a probably less adequate operation, especially for therapy of malignant disorders where long-term results are not known. VATS is very useful for investigation of pleural effusion and malignancy. Spontaneous pneumothorax is a very good indication for thoracoscopic treatment, as well as lung biopsy and diagnostic resection of lung metastases. Therapeutic metastasectomy, however, should not be performed by VATS. Regarding bronchogenic carcinoma, VATS is indicated for staging of lung cancer, facilitating sampling of mediastinal and hilar lymph nodes, investigation of pleural effusion, possible pleural dissemination and suspicion of intrapulmonary metastases. Wedge excision of solitary pulmonary nodules by VATS readily reveals the exact diagnosis, but its role in therapy of lung cancer is very limited at the present time. PMID- 8629384 TI - Contralateral inguinal hernial development and ipsilateral recurrence following unilateral hernial repair in infants and children. AB - The aim of the present study was to evaluate the frequency of contralateral hernial development and ipsilateral recurrence following surgery for unilateral inguinal hernia in infants and children. During the period 1961-1970, 161 infants and children (143 males and 18 females; 0-12 years old) underwent surgery for unilateral inguinal hernia. The patients were followed for 20-29 years (until 1990). 16/143 males (11.2%), and 1/18 females developed a contralateral inguinal hernia; the frequency among patients less than seven years old was significantly higher than the frequency among older children (16/118 > 1/43; p < 0.05). No significant difference was observed between patients primarily operated for rightsided and leftsided hernias. Four/143 boys (2.7%) experienced testicular maldescens. Recurrence after herniotomy was seen in 9/143 males (6.3%), and in 1/18 females. The low frequency of contralateral hernial development, as the risk of surgical complications, indicate that routine contralateral inguinal exploration may not be recommended while operating upon unilateral symptomatic inguinal hernias. Our recurrence rate of 6.2% may mirror the conditions in a general surgical department, where several surgeons and residents operate upon a limited number of paediatric patients. PMID- 8629385 TI - Surgical wound infection surveillance: results from the Belgian hospital network. AB - Since October 1992, the Belgian National Programme for the Surveillance of Hospital Infections has been implemented successfully in more than two-thirds of all Belgian acute-care institutions. Practitioners from hospitals participating in the surgical wound infection surveillance describe a selection of surgical procedures, practices connected (antimicrobial prophylaxis), and record the eventual infection, enabling the calculation of wound infection rates. The network allows comparisons of each hospital results with the national picture. From October 1992 to June 1993, 16,799 procedures were recorded by 51 hospitals; the crude incidence rate of infection was 1.47 per 100 operations. However, this figure may be an underestimation of the reality because of potentially missed post-discharge infections. Risk factors significantly associated with infection include length of preoperative stay, emergency, duration of surgery, wound contamination class, and American Society of Anesthesiologists (ASA) score. The National Nosocomial Infections Surveillance system (NNIS) risk index, a combination of the three latter shows a good correlation for predicting infection. Increased length of stay attributable to the infection was computed at 8.9 days. Micro-organisms isolated reveal a staphylococcal predominance. Antibiotic prophylaxis prescription present satisfactory quality performances regarding duration and time of initiation but rational prescribing about the indications is still of concern. PMID- 8629386 TI - Single and double stapled anastomoses in rectal cancer surgery; a retrospective study on the safety of the technique and its indication. AB - Low anterior resection is commonly believed the main indication to double stapled (DS) technique, because placing the purse-string suture on the distal rectum is difficult or impossible. This study was designed to figure out the safety of the DS technique and to better define its role in rectal cancer surgery. The data of 34 patients that had a DS anastomosis were retrospectively compared to those of 43 that had a single-stapled (SS) anastomosis after anterior resection. Three deaths after SS (7%) and one after DS procedures (3%) were recorded (p = 0.62). Rates of clinical leaks were 12% (four cases) in the DS group and 14% (six cases) in the SS group (p = 0.41). The mean distance of the rectal tumour from the anal verge was significantly lower for DS (mean = 7.7 cm) respect to SS (mean = 12.7 cm) anastomoses (p < 0.0001) and the blood consumption at surgery was significantly greater in patients that had DS (mean = 375 ml) compared to SS anastomoses (mean = 180 ml) (p = 0.028). Thus, the DS technique was mostly used in patients at high risk for leakage. The study shows that DS technique is a safe and reliable method to perform colorectal anastomosis after anterior resection for cancer. For cancers located in the upper rectum the routine adoption of the DS increases the cost of surgery and does not offer advantages over the SS technique with the exception of making feasible end-to-end mechanical anastomoses involving bowel segments having different diameters. PMID- 8629387 TI - Initial experience with the use of preoperative transarterial chemoembolization in the treatment of liver metastasis. AB - We retrospectively evaluated the influence of preoperative Transarterial Chemoembolization (TAE) on technique and complications, tumour histology, and disease-free survival after surgery for hepatic metastasis. In a 2-year period, a total of 23 patients were treated. In a first group of 14 patients, preoperative TAE was performed; in a second group of 9 patients only surgical resection was done. Extensive tumour necrosis was seen in the majority of patients treated with TAE; in tumours with an important fibrotic component, embolization was less effective. No significant effect was seen on operating time, transfusion requirement or perioperative complication rate. In the group of patients who underwent TAE, survival rate was 93% after a mean follow-up period of 15.5 months (SD: 12.5); recurrence was seen in only 8% of the survivors. In the second group, however, mortality was 33% after a median follow-up of 17.5 months (SD: 10), and recurrence was present in 66.7% of the survivors. These results indicate that preoperative TAE reduces the recurrence rate in the first postoperative year. Thereby survival may be improved in patients with resectable metastatic liver cancer. PMID- 8629388 TI - Gallbladder volvulus: a case report. Could the ultrasound be the key of the early diagnosis? AB - Gallbladder volvulus is a rare disease which may affect the elderly. It mimics clinically an acute cholecystitis, but the ultrasound signs may provide an early diagnosis: a very large, anteriorly floating gallbladder, with very thickened, multi layered wall, could orientate the physician to this rare disease. We present one case of gallbladder volvulus with its radiological signs. The symptoms, the etiology and the radiological examinations are discussed. PMID- 8629389 TI - The role of lactulose in the prevention of bacterial translocation in surgical trauma. AB - Surgical trauma (ST) is one of the causative factor of bacterial translocation. In this study we investigated the prevention of bacterial translocation with lactulose in a surgical trauma model. The study was designed in 3 experimental groups consisting of 15 rats in each. Group 1 was sham operated controls, group 2 was ST + physiologic saline treated and group 3 was ST + lactulose treated animals. Lactulose and physiologic saline were given by oro-gastric intubation in a dose of 2 ml of 33.5% solution/d and 2 ml/d respectively starting 3 days prior to surgery. Bacterial translocation was investigated 48 hours after the operations. In sham operated controls only 1 bacterial translocation to the mesenteric lymph nodes (MLN) was observed. In the ST + physiologic saline treated group bacterial translocation to the MLN and portal venous blood (PVB) were significantly increased compared with both sham operated controls and the ST + lactulose treated group (p < 0.001). In rats with lactulose treatment the results of caecal bacterial counts showed a significant decrease in the number of gram negative aerobes and facultative anaerobe bacteria (p < 0.01) and a significant increase in the number of lactobacilli (p < 0.001) compared to the sham operated controls. Measurement of the mucosal height showed a significant increase at the terminal ileum and the caecum compared with the sham operated controls and the surgical trauma+physiologic saline treated group (p < 0.001). We conclude that oral lactulose treatment 3 days prior to the surgical trauma, reduced the incidence of bacterial translocation to the MLN and PVB. PMID- 8629390 TI - Fluorescence in situ hybridization studies on breast tumor samples for distinguishing between different subsets of breast cancer. AB - OBJECTIVES: To evaluate fluorescence in situ hybridization for distinguishing between malignant and benign breast tumors and determining genetic subgroups of breast cancers. STUDY DESIGN: Touch preparations from 94 surgically removed breast tumors (17 benign and 77 malignant) were hybridized with a DNA probe specific for centromeric DNA sequences of chromosome 1. Twenty samples were additionally hybridized with a chromosome 9-specific probe. RESULTS: We investigated the heterogeneity of the cell populations on the basis of the number of signals per nucleus. All benign tumors showed two signals per nucleus. In contrast, carcinomas revealed a broad spectrum of hybridization patterns. Some showed almost exclusively two signals per nucleus, and others exceeded four signals. CONCLUSION: The hybridization patterns of individual tumors can be used for defining different subsets of breast cancer. The results may have prognostic impact, leading to "molecular-cytogenetic grading" of breast cancer. PMID- 8629391 TI - Staining for dipeptidyl aminopeptidase IV activity in nodular thyroid diseases. AB - OBJECTIVE: To determine the effectiveness of staining for dipeptidyl aminopeptidase IV (DAP IV) activity in thyroid nodular diseases. STUDY DESIGN: Imprint smears were made in 76 cases of papillary carcinoma, 10 of follicular carcinoma, 32 of follicular adenoma and 48 of adenomatous goiter after surgery, and staining for DAP IV activity was performed in all cases. RESULTS: All papillary carcinomas stained positively, and most adenomatous goiter cases were negative. Follicular carcinoma and follicular adenoma cases exhibited various degrees of positivity, though the former tended to be stained more than the latter. Of the total 166 cases of thyroid nodules, 53 showed negative staining; of those 53, all except 1 were benign. Follicular carcinoma cases that had vascular invasion tended to show a high DAP IV staining pattern, but no statistically significant difference between it and follicular carcinoma that did not show vascular invasion was found. CONCLUSION: DAP IV activity staining might reveal malignant potential and could have some value in the preoperative diagnosis between thyroid nodular diseases. The DAP IV method is a reliable indicator of benign disease if negative results are obtained. PMID- 8629392 TI - Fluorescence in situ hybridization for detecting erbB-2 amplification in breast tumor fine needle aspiration biopsies. AB - OBJECTIVE: To evaluate the feasibility of erbB-2 amplification analysis of fine needle aspiration (FNA) biopsies. STUDY DESIGN: FNA smears and dissociated nuclei from 58 breast cancer samples were examined by dual-labeling fluorescence in situ hybridization (FISH) with probes for centromere 17 and the erbB-2 gene. The results were compared with the outcome of erbB-2 immunohistochemistry. RESULTS: Tumors were categorized according to the erbB-2/centromere 17 signal ratio. There were 23 tumors with high-level amplification, four cases with a low-level erbB-2 gain and 27 tumors with normal erbB-2 content. Four tumors showed an erbB-2 deletion, all in patients < or = 42 years of age. ErbB-2 amplification was strongly associated with positive erbB-2 immunostaining (P < .0001). Comparison of FISH analysis on dissociated cells and on FNA biopsies showed high correspondence (P < .0001). CONCLUSION: FISH allows reliable detection of erbB-2 gene amplification on FNA biopsies. PMID- 8629393 TI - Modified technique for fine needle aspiration biopsy that eliminates needle manipulation. AB - OBJECTIVE: To evaluate a modified technique of fine needle aspiration biopsy that eliminates needle manipulation after aspiration. STUDY DESIGN: Fine needle aspiration biopsy of 112 palpable lesions was performed using both the conventional and modified techniques. Cellularity, cell preservation and diagnostic yield of the new procedure were compared to those of the conventional technique. RESULTS: Cytologic smears prepared from each method were comparable in quality, quantity and diagnostic yield. CONCLUSION: The modified approach eliminates the hazard of needle manipulation without diminishing the diagnostic efficacy of fine needle aspiration biopsy. PMID- 8629394 TI - Expression of type VI collagen in psammoma bodies: immunofluorescence studies on two fresh human meningiomas. AB - OBJECTIVE: To demonstrate that psammoma bodies in human meningiomas contain type VI collagen and laminin. STUDY DESIGN: Two fresh human meningiomas were obtained from surgery and the frozen sections examined for type VI collagen and laminin with specific antibodies. RESULTS: Type VI collagen was found on the surface of the psammoma bodies. In addition, it was detected in the interstitial tissues surrounding the tumor cells and on the tumor cells themselves. Laminin was detected mainly in the walls of capillaries and large blood vessels. In contrast, in some psammoma bodies, laminin was detectable internally. Laminin was not found in the interstitial tissues or tumor cells. CONCLUSION: This is the first report to describe the involvement of type VI collagen in psammoma bodies and whorl formations in meningiomas. This specific type of collagen may serve an anchoring function in psammoma body development. PMID- 8629395 TI - Percutaneous and intraoperative aspiration biopsy cytology of pancreatic neuroendocrine tumors: cytomorphologic study with an immunocytochemical contribution. AB - OBJECTIVE: To evaluate the efficacy and accuracy of fine needle aspiration biopsy (FNAB) in the diagnosis and recognition of pancreatic neuroendocrine tumors (NETs). STUDY DESIGN: The study group consisted of six cases of pancreatic tumors correctly diagnosed or strongly suggested to be NET based on fine needle aspiration biopsy (FNAB) cytology and was retrospectively reviewed. Also reviewed and examined were the immunocytochemical (ICC) stains applied to five of the aspirates and electron microscopic (EM) study done on one case. RESULTS: Five cases were collected by computed tomography (CT)-guided aspiration, and one case was obtained intraoperatively by the surgeon. All six cases showed characteristic cytomorphologic features sufficient for their recognition and separation from pancreatic adenocarcinoma and other lesions. The ICC staining results and EM study were very helpful in confirming the cases' neuroendocrine cell origin. Histologic confirmation was available for four cases. CONCLUSION: Intraoperative and CT-guided FNAB of the pancreas is a valuable method in the recognition of NET of the pancreas, particularly when coupled with ICC studies and appropriate clinical and radiologic settings. PMID- 8629396 TI - Peritoneal fluid cytology in advanced mucinous cystadenocarcinoma of the pancreas. AB - OBJECTIVE: To assess the role of peritoneal cytology in the management of mucinous cystadenocarcinoma. STUDY DESIGN: A review of the peritoneal fluid cytologic findings in eight selected cases of pancreatic mucinous cystadenocarcinomas. RESULTS: Four of eight cases (50%) were positive for malignant cells. All four patients with positive peritoneal cytology had unresectable tumors and extensive intraabdominal metastases. Of the four patients with negative cytology, three had unresectable, locally invasive tumors without metastases, and one had a resectable tumor confined to the pancreas. CONCLUSION: Peritoneal cytology is frequently positive in unresectable mucinous cystadenocarcinomas with intraabdominal metastases, but cytology may often be negative in unresectable cases. Therefore, negative cytologic findings in these cases do not reliably ensure resectability, while positive findings clearly correlate with the presence of intraabdominal metastases. A larger study of unselected patients, including more with resectable tumors, will be required to define the full utility of this technique in preoperative assessment. PMID- 8629397 TI - Fine needle aspiration biopsy in patients with diffusely enlarged spleens. AB - OBJECTIVE: To evaluate the diagnostic value and potential risk of fine needle aspiration biopsy (FNAB) of the spleen in patients with diffuse splenomegaly due to an undetermined cause. STUDY DESIGN: Retrospective evaluation of the medical records and cytologic material from 58 patients on whom FNAB was performed between 1967 and 1993. RESULTS: Sixty-five FNABs were performed on 58 patients. Lymphoproliferative diseases were found in six; in four additional cases, metastatic adenocarcinoma, sarcoidosis, Candida albicans and enterococcal infection were demonstrated. The remaining 55 biopsies either showed normal splenic tissue or were nondiagnostic. No complications were recorded. CONCLUSION: Splenic FNAB may be useful in some patients with diffuse splenomegaly. The diagnostic yield is rather low. The procedure, however, is simple, safe and well tolerated. PMID- 8629398 TI - Estimation of growth fraction in fine needle aspirates from non-Hodgkin's lymphoma using anti-proliferating cell nuclear antigen: correlation with the Kiel classification. AB - OBJECTIVE: This study was undertaken to assess whether fine needle aspirates from non-Hodgkin's lymphoma (NHL) could be used for growth fraction analysis with proliferating cell nuclear antigen (PCNA) staining and if there was a relationship between the growth fraction and cytomorphologic classification according to the Kiel classification. STUDY DESIGN: The study group consisted of 40 patients with NHL diagnosed by fine needle aspiration (FNA) cytology. The cytologic classification of the lymphomas was made by two cytopathologists on May Grunwald-Giemsa-stained slides using the Kiel classification. There were 27 cases of low and 13 of high grade lymphoma. The estimation of the growth fraction was made by PCNA immunoreactivity. The PCNA index was quantitated in smears by counting an average of 1,000 cells, and the count was correlated with the cytomorphologic classification. RESULTS: There was a strong correlation between the PCNA index and lymphoma grading. High grade lymphomas exhibited a mean PCNA positivity of 74.0%, which was significantly higher (P < .001) than that of low grade lymphomas (17.6%). CONCLUSION: Our study showed that PCNA evaluation is suitable for smears obtained by FNA on NHL, correlates with increasing grades of lymphoma according to the Kiel classification and may offer a method of monitoring treatment of lymphoma. PMID- 8629399 TI - Relationship between cytomegalovirus cells and survival in acquired immunodeficiency syndrome patients. AB - OBJECTIVE: To determine whether cytopathic changes due to cytomegalovirus (CMV) in human immunodeficiency virus-infected patients are prognostic. STUDY DESIGN: Three-month mortality was compared in three groups: 36 patients with positive CMV cytology, 38 with negative cytology but culture positive, and 40 with no evidence of CMV. Bronchoalveolar lavage, Papanicolaou-stained cytocentrifuge smears were quantitated using an average of two slides per case. Additionally, coinfection with Pneumocystis carinii and Cryptococcus neoformans was evaluated. RESULTS: There was a statistically significant increase in mortality at three months in CMV cytology-positive patients versus those with no evidence of CMV. Ten patients had quantitative CMV counts of less than 2, with a median survival of 3.0 months (range, 0.3-13.0); seven patients had counts of 2 or 3, with a median survival of 5.5 months (0.4-13.5); and 11 patients had CMV counts greater than 3, with a median survival of 7.2 months (0.3-14.0). There was no significant difference between the groups. Coinfection was P Carinii (12) or C neoformans (2) showed no difference from patients without coinfection (chi2 = 0.81). CONCLUSION: The presence of CMV cytopathic changes is associated with poorer survival, but an increased number of CMV-infected cells is not related to higher mortality. PMID- 8629400 TI - Cystic brain lesions: cytologic examination and carcinoembryonic antigen assay in fine needle aspirates. AB - OBJECTIVE: To compare the sensitivities of cytologic diagnosis by fine needle aspiration (FNA) with carcinoembryonic antigen (CEA) content of cystic brain tumors and to determine if CEA assay enhances the sensitivity of cytologic diagnosis and suggests a probable primary in carcinoma with an unknown primary source. STUDY DESIGN: Thirteen consecutive aspirates were studied. Cytologic diagnosis was confirmed by histologic examination and follow-up. Fluid varied from 3 to 50 mL and from clear to bloody to mucinous. Cytologic examination was performed on Papanicolaou-stained smears. CEA was measured by enzyme immunoassay, with a cutoff of 5 ng/mL. RESULTS: Benign: 3 (subdural hematoma, 2; arachnoid cyst, 1). Malignant: 10 (medulloblastoma, 1; glioblastoma multiforme, 4; metastatic adenocarcinoma with an unknown primary, 1; of breast, 1; oat cell, 3). Sensitivities: cytology, 50%; CEA, 50%; combined, 60%; specificity, 100%. High CEA (> 880 ng/mL) differentiated papillary adenocarcinoma of lung/gastrointestinal tract from renal and thyroid carcinoma. Mean CEA: oat cell, 216.7 ng/mL; adenocarcinoma, 0.5, > 880 ng/mL; glioblastoma and medulloblastoma, 1.6 ng/mL; benign, 0.6 ng/mL. CONCLUSION: High CEA content enhanced the sensitivity of cytologic diagnosis of cystic metastases and favored a primary carcinoma of the lung or gastrointestinal tract. PMID- 8629401 TI - Value of image-guided needle aspiration cytology in the assessment of thoracolumbar and sacrococcygeal masses. AB - OBJECTIVE: To investigate tumors and nontumorous processes in the thoracolumbar and sacrococcygeal region by the noninvasive method of needle aspiration cytology (NAC) and whether such preoperative assessment is useful in deciding between conservative and radical management. STUDY DESIGN: NAC was performed under computed tomographic guidance on 22 patients suspected of having a thoracolumbar sacrococcygeal mass. Cytologic examination was performed on site after staining smears with the Papanicolaou method. In addition, air-dried smears, fixed smears, filter preparations from needle washings and cell blocks were studied. The NAC diagnosis in all cases was supported by examining cell blocks, which illustrated the reliability of histologic architecture; further support was obtained with a tissue biopsy in eight cases. Additionally, immunoperoxidase and/or histochemical studies were done in cases with a NAC diagnosis of chordoma and some cases of metastatic carcinoma. RESULTS: Five cases were diagnosed as inflammatory/degenerative lesions, and in one case NAC diagnosis of neurofibroma was made. In 14 cases a variety of metastatic tumors were diagnosed, while in two cases a NAC diagnosis of chordoma was made. Immunohistochemical staining results were useful in supporting the cytohistologic diagnosis. CONCLUSION: NAC, as the first line of investigation, is not only useful in the diagnosis of space occupying lesions of the thoracolumbar-sacrococcygeal region but can also save the patient from a radical surgical procedure to obtain tissue for diagnosis. Also, histologic architecture from cell blocks can be correlated with cytology, and such material can be used for histochemical and immunomarker studies. PMID- 8629402 TI - The unicum and cytobrush plus spatula for cervical cytologic sampling: a comparison. AB - OBJECTIVE: To compare the efficacy of the Unicum, a new ectocervical-endocervical nylon brush, with that of the Cytobrush plus wooden spatula in the collection of cervical-endocervical material. STUDY DESIGN: Thirty-eight patients who underwent routine gynecologic examinations agreed to have a smear taken with the wooden spatula plus Cytobrush and the Unicum. RESULTS: The two methods were equally effective in collecting endocervical-ectocervical smears. CONCLUSION: With our method, sampling is done with only one device; the ectocervix and endocervix are sampled simultaneously, and adequate sampling of the transformation zone must be achieved. PMID- 8629403 TI - Observer variation in the assessment of adequacy and neoplasia in cervical cytology. AB - OBJECTIVE: To measure variation in reporting within and between cytotechnologists in a routine cytology laboratory and to assess if attending training courses and discussing many cases alter agreement levels. STUDY DESIGN: The study involved nine cytotechnologists each screening 100 cervical smears and recording the results. The same 100 smears were given a different identity number and the process repeated about six months later. Between the two occasions, two participants attended a training course, and two others discussed many other cases with the aim of reaching agreement on diagnostic criteria. RESULTS: The number of smears judged by the participants on either occasion as adequate varied between 87 and 100, and as adequate and negative, between 36 and 65. Percentage intraobserver agreement on the first round diagnosis of a negative smear varied between 88.5 and 100, and of an abnormal smear, between 58.5 and 91.2. CONCLUSION: Attending the same training course and in-depth discussion of other cervical smears appear to have had the effect of increasing some aspects of interobserver agreement between the two pairs of individuals involved. PMID- 8629404 TI - Cytology in the follow-up of cervical cancer. AB - OBJECTIVE: To determine the value of cytology in the follow-up of cervical cancer. STUDY DESIGN: The study group consisted of 230 patients with invasive cervical carcinoma who were followed for one to seven years. Forty-four patients developed recurrences or metastases. During this period, cytologic investigations involved 795 exfoliative smears from the cervix or vaginal vault, 10 fine needle aspirates and 5 fluids. RESULTS: Thirty-three patients had positive or inconclusive cervical or vault smears that were histologically proven to be recurrences, and the other 11 patients had clinically obvious recurrences that were not smeared. Cytology first alerted the clinicians to recurrence in eight patients. Of 25 cervical or vault smears reported as malignant, 24 (96%) were histologically confirmed, and 1 showed radiation change on biopsy. In all 22 cases of smears reported as inconclusive, a biopsy followed, and in 9 (41%) of these, recurrence was demonstrated histologically. Inability to distinguish radiation change from recurrent malignancy was the chief cause of inconclusive smears. Five fluids and seven fine needle aspirates were diagnosed as malignant, saving patients an invasive diagnostic procedure. CONCLUSION: Cytology is a useful, cost-effective, noninvasive and accurate investigation in the follow-up of cervical cancer. PMID- 8629405 TI - Early cervical neoplasia confirmed by conization: diagnostic accuracy of cytology, colposcopy and punch biopsy. AB - OBJECTIVE: To investigate the accuracy rates of cytology, colposcopy and punch biopsy in early cervical neoplasia confirmed by conization. STUDY DESIGN: During the 10 years from 1984 to 1993, cold knife conization was performed on 151 patients with early cervical neoplasia proven by punch biopsy at our department. The accuracy rates of cytology, colposcopy and punch biopsy were investigated. RESULTS: The accuracy rates of cytology, colposcopy and punch biopsy were 52% (78 of 151), 66% (100 of 151) and 66% (100 of 151), respectively. CONCLUSION: These results suggest that a composite diagnosis with cytology, colposcopy and punch biopsy is necessary for a correct evaluation. Early cervical neoplasms are frequently seen in young women, and conservative procedures, such as conization, cryosurgery and laser vaporization, are the treatments of choice in order to preserve reproductive function. We recommend conization as the best conservative procedure, with preservation of reproductive function. PMID- 8629406 TI - Apocrine carcinoma vs. apocrine metaplasia with atypia of the breast. Use of aspiration biopsy cytology. AB - OBJECTIVE: To solve the problem of diagnosing apocrine carcinoma (APCA) through distinguishing it from benign apocrine metaplasia with atypia (APMA). STUDY DESIGN: The study group consisted of five histologically confirmed cases of uncommon infiltrating apocrine carcinoma and a case of noninfiltrating apocrine carcinoma of the breast by aspiration biopsy cytology. The control group consisted of 103 cases of benign apocrine metaplasia with no atypia (APMN), 4 cases of APMA and 34 cases of common-type adenocarcinoma that were encountered in 662 breast aspirations from 1988 to 1992 at Hekinan Municipal Hospital. RESULTS: In APCA the average age of patients (65 +/- 17.7 SD)(mean +/- SD) was more than 20 years older than APMA, and APCA generally measured > or = 2 cm or more in diameter as compared to < or = 2 cm in APMA lesions. In APCA the apocrine cells had high cellularity, with the cells occur singly and with syncytia in the background. Numerous degenerated apocrine cells and characteristic cell detritus were found. APCA was also characterized by apocrine cells with more marked nuclear abnormalities, including hyperchromasia and irregular nuclear shape, frequently with irregular nucleoli; more nuclei measure > or = 12 micron in diameter than in APMA. These findings, however, were present only to a mild degree in APMA, if at all. APMA may coexist with APCA. CONCLUSION: If APMA is diagnosed, an open biopsy should be performed to distinguish it from APCA. PMID- 8629407 TI - Fine needle aspiration biopsy of extramedullary leukemia. AB - OBJECTIVE: To study the cytologic features and role of fine needle aspiration biopsy (FNAB) in the diagnosis of extramedullary leukemia. STUDY DESIGN: Forty one cases of extramedullary leukemia diagnosed by FNAB were analyzed along with their detailed clinical and hematologic features. RESULTS: Common sites of leukemic involvement were lymph nodes (34), skin (4), orbit (1), eyelid (1) and breast (1). The most common variety of leukemia was chronic myeloid in the chronic phase (17). Twenty-six patients were referred to the cytology clinic for FNAB as the initial screening test. In the majority of fresh cases, leukemia was not the first possibility considered, and FNAB played an important diagnostic role. No gross discrepancy was noted in any of the cases. CONCLUSION: FNAB is helpful in the diagnosis of extramedullary involvement by leukemia because of the good morphologic detail of blasts and other granulocytic cells. However, for more accurate subclassification of a hematologic disorder, other hematologic investigations are mandatory. PMID- 8629408 TI - Fine needle aspiration biopsy of hepatic lymphomas: cytomorphology and ancillary studies. AB - OBJECTIVE: The utility of fine needle aspiration biopsy (FNAB) for the diagnosis of epithelial hepatic neoplasms is now widely recognized. The liver may also play host to malignant nonepithelial stromal and lymphoreticular neoplasms, both metastatic and rarely primary. The cytomorphology of these hepatic tumors in aspiration smears is much less well known. In the current study, we examined FNAB material from hepatic lymphomas, including ancillary studies. STUDY DESIGN: We collected 16 cases of lymphoma involving the liver and diagnosed by FNAB. The patients included seven women and nine men, with ages ranging from 34 to 84 years. Nine patients did not have a diagnosis of lymphoma prior to FNAB. In all cases, aspiration smears were stained by both Diff-Quik and Papanicolaou stain. RESULTS: Dispersed, monomorphic lymphoid cells were usually numerous, whereas benign hepatocytes were scanty. In addition to the lymphoid appearances of individual cells, helpful clues included the presence of lymphoglandular bodies and the absence of true intercellular cohesion. There were 6 large cell, 3 immunoblastic, 2 small cell, 1 small cell cleaved, 2 mixed cell and 2 unclassified lymphomas in our FNAB series. Immunocytochemical studies were performed on 9 aspirated specimens, flow cytometry on 4 and gene rearrangement on 1. All these ancillary studies supported the cytomorphologic diagnosis of lymphoma, with approximately 90% classified as of B-cell lineage. All seven subsequent histologic examinations were confirmatory. CONCLUSION: Other entities that must be considered in the FNAB differential diagnosis of hepatic lesions are nonlymphoreticular small cell neoplasms, inflammatory processes and sinusoidal hematopoietic cells. FNAB with ancillary studies can successfully establish the diagnosis of involvement of the liver by lymphoma. PMID- 8629409 TI - Kala-azar: liver fine needle aspiration findings in 23 cases presenting with a fever of unknown origin. AB - OBJECTIVE: To study the role of liver fine needle aspiration (FNA) in the diagnosis of kala-azar with an atypical presentation. STUDY DESIGN: The study group consisted of 23 patients (aged 18-37). All were admitted to Shiraz University Hospitals for the investigation of fever of unknown origin. The immunofluorescent antibody titer for kala-azar was positive (> 1:256 dilution). However, routine abdominal sonography revealed multiple small, hypoechoic lesions in the liver, more suggestive of metastatic tumor or miliary tuberculosis. The lesions were aspirated for cytologic diagnosis. RESULTS: The smears revealed many atypical hepatocytes, groups of epithelioid histiocytes and, in 15 patients, macrophages containing few to many Leishman bodies, allowing a diagnosis of kala azar. The liver needle biopsy specimens confirmed the cytologic diagnosis of kala azar and liver cell atypia. The patients were given glucantime therapy, and 21 recovered; 1 patient was lost to follow-up, and 1 died during treatment. CONCLUSION: Liver FNA is a useful procedure for the diagnosis of kala-azar, particularly in cases with atypical clinical presentations. PMID- 8629410 TI - Filter immunocytology: methodologic evaluation of a new assay for the diagnosis of urothelial cancer. AB - OBJECTIVE: Several investigators have demonstrated the high sensitivity of immunocytology in the diagnosis of transitional cell carcinoma (TCC). A new technique, designated "filter immunocytology" (FLIC), simplifies the technique of quantitative immunocytology, considerable decreases assay time and increases the percentage of assessable specimens. STUDY DESIGN: Voided urine samples were obtained from 89 patients without evidence of TCC and from 91 patients with histologically proven TCC. The cells were transferred onto a polycarbonate membrane. Immunostaining was performed using monoclonal antibody. Due ABC 3, directed against a differentiation antigen on urothelial cells. Specimens containing > 35% positive urothelial cells were regarded as abnormal. RESULTS: Of 153 specimens 180 (85%) were assessable. The investigation of 76 specimens from control patients and 77 from patients with TCC yielded a specificity of 86% and a sensitivity of 75%, respectively. Sensitivity did not correlate with tumor grade. Despite high interobserver and intrapatient variations regarding the amount of antigen-positive cells, a concordant attribution to either "normal" or "abnormal" was made in > 95% of cases. Intraobserver variation was small and did not influence the test result. CONCLUSION: These results suggest that FLIC assay may be a valuable adjunct to conventional cytology. A careful prospective investigation appears to be worthwhile to further define the indications for this technique. PMID- 8629411 TI - Diagnostic efficacy of endometrial cytology with the Abradul cell sampler supplemented by laser scanning confocal microscopy. AB - OBJECTIVE: To study the diagnostic efficacy of endometrial cytology with the Abradul cell sampler and the effect of supplementing it, in cases with overly thick smears, with laser scanning confocal microscopy. STUDY DESIGN: Sampling was performed in 1,684 women. All patients underwent subsequent histology. In eight cases with overly thick smears the original smears were restained to allow confocal microscopy. RESULTS: Efficacy was high, with 1,593 good samples. Thirty one endometrial carcinomas were signed out as cytologically positive and 10 as atypia. In addition, 10 squamous cell carcinomas of the cervix and 1 ovarian carcinoma were diagnosed with this method. Only one case (endocervical adenocarcinoma) was not detected in the sample. In the eight cases in which confocal microscopy was performed, the images were high quality, allowing a correct diagnosis. In addition, the transition from hyperplasia to carcinoma could be visualized in the cytologic smear. CONCLUSION: Endometrial cytology in an outpatient setting is highly effective, and confocal microscopy in selected cases is not only helpful but also highly instructive. PMID- 8629412 TI - Chondrosarcoma diagnosed by fine needle aspiration cytology. AB - OBJECTIVE: To define and discuss the cytologic findings in 6 conventional chondrosarcomas (CS), 1 chondroblastic osteosarcoma, 1 extraskeletal myxoid CS and 20 chondroma cases. STUDY DESIGN: Study of fine needle aspiration cytology (FNAC) of cases air dried and May-Grunwald-Giemsa stained. RESULTS: Tumor cells were embedded in a pink, amorphous, chondroid matrix in conventional CS. They were larger than chondroma cells and had vacuoles and small, pink, chondroid substance-like granules. Chondroblastic osteosarcoma was diagnosed as high grade CS by FNAC, but extremely pleomorphic chondrocytes, osteoid matrix, osteoblastic cells, and clinical and radiologic findings should have produced the proper diagnosis. Extraskeletal myxoid CS had a pinkish, granular, myxoid background. The tumor cells had granulated, dark blue cytoplasm and oval-round, lobulated, slightly indented, hyperchromatic nuclei. CONCLUSION: FNAC is efficient for the diagnosis of CS and its variants as long as it is evaluated with radiologic and clinical findings by a pathologist familiar with bone pathology and cytology. PMID- 8629413 TI - Adenoid cystic (cylindromatous) carcinoma associated with squamous cell carcinoma of the cervix uteri: cytologic presentation of a case with histologic and ultrastructural correlations. AB - BACKGROUND: Adenoid cystic (cylindromatous) carcinoma is a rare tumor. It accounts for approximately < 1-3% of primary adenocarcinomas of the cervix uteri. Its origin is debatable. It has a higher incidence in postmenopausal women but can develop in patients under 40. An association of adenoid cystic carcinoma with squamous cell carcinoma has been reported. CASE: A case of invasive adenoid cystic (cylindromatous) carcinoma associated with an in situ squamous cell carcinoma of the cervix detected on Papanicolaou smears is reported. Ultrastructural studies provided findings that confirmed that the hyaline material corresponded to the remnants of the lamina densa of the basement membrane of malignant cells. CONCLUSION: The concurrence of these two tumors supports the hypothesis that adenoid cystic carcinoma of the cervix may develop from multipotent reserve cells. PMID- 8629414 TI - Psammoma bodies and cells from in situ fallopian tube carcinoma in endometrial smears: a case report. AB - BACKGROUND: Primary in situ fallopian tube carcinomas are rarely diagnosed, and malignant cells from this lesion have not been previously described as occurring in endometrial smears. CASE: A 57-year-old, postmenopausal woman had an endometrial smear that revealed adenocarcinoma cells associated with psammoma bodies. She also had smooth muscle cells in the smear compatible with uterine leiomyomas. Examination of the hysterectomy specimen disclosed a primary in situ carcinoma of the left fallopian tube and uterine leiomyomatosis. CONCLUSION: Endometrial cytology plays an important role in the diagnosis of pathologic processes in the uterus. It may also contribute useful information on extrauterine diseases. PMID- 8629415 TI - Microfilariae in cytologic smears: a report of six cases. AB - BACKGROUND: Filariasis is a major public health problem in tropical countries, including India. Despite this high incidence, it is unusual to find microfilariae in fine needle aspiration cytology. (FNAC) smears and body fluids. Six cases of filariasis diagnosed by FNAC are reported. CASES: In case 1, a thyroid aspirate from a 23-year-old female showed a microfilaria with colloid and thyroid follicular cells. In case 2, pericardial fluid from a 50-year-old male with breathlessness and chest pain showed clusters of malignant cells and many microfilariae. In case 3, a bronchial aspirate from a 55-year-old male with fever and chest pain showed aggregates of acute inflammatory cells and a microfilaria. Cases 4 and 5 were lymph node aspirates from a 29-year-old male and 3-year-old female. Both smears showed microfilariae in a background of reactive lymphoid cells. In case 6, FNAC from a 21-year-old female with a cystic swelling in the breast showed clusters of microfilariae, lymphocytes and granular debris. CONCLUSION: In three of the six cases microfilariae were the cause of symptoms, whereas in the other three cases, microfilariae were associated with other diseases, including malignancy. Thus, careful screening of FNAC smears might be helpful in detecting microfilariae, even in asymptomatic patients. PMID- 8629416 TI - Choroidal cells in ovarian cyst fluid: a case report. AB - BACKGROUND: The ovary is conspicuous for the wide diversity of histologic tumor types that it contains. Unusual or rare tumors may present diagnostic difficulty on cytologic examination. CASE: A 36-year-old woman presented with an 80-mm cystic lesion in the left ovary. Fine needle aspiration provided 30 ml of transparent, light yellow fluid. On cytologic examination, numerous clusters and rosettes of benign cells, resembling choroid plexus cells, were seen. Surgical resection confirmed the presence of an ependymal cyst adjacent to a mature cystic teratoma (dermoid cyst). CONCLUSION: We report this unique case in order to alert cytologists unfamiliar with cerebrospinal fluid cytopathology to the rare entity of ovarian ependymal cysts. PMID- 8629417 TI - Pneumocystis carinii thyroiditis diagnosis by fine needle aspiration cytology: a case report. AB - BACKGROUND: Extrapulmonary infection or dissemination of Pneumocystis carinii (PC) is rare, but under certain conditions the parasite can spread via the bloodstream or lymphatic vessels. Systemic pneumocystosis most often involves the lymph nodes, stomach, spleen, liver, skin, pancreas, choroid and eye. Isolated lesions containing PC have also been identified in the thyroid. CASE: A 41-year old homosexual male infected with the human immunodeficiency virus (HIV) developed a PC infection in the thyroid gland. The patient had had thrush and anal herpes since being diagnosed as HIV positive in 1984. In 1992 the patient developed a mass in the area of the right lobe of the thyroid gland. Smears from fine needle aspiration cytology of the thyroid mass revealed epithelioid cells. However, a cell block revealed numerous PC organisms on Gomori methenamine-silver stain; that finding was confirmed by an excisional biopsy. The patient had not previously been diagnosed with PC pneumonia. CONCLUSION: Our case of thyroid involvement with PC expands the clinical spectrum of extrapulmonary pneumocystosis in patients with the acquired immunodeficiency syndrome. We believe that in these patients the incidence of Pneumocystis thyroiditis will continue to rise and be reported. PMID- 8629418 TI - Papillary Hurthle cell tumor of thyroid: report of a case with a cytomorphologic approach to diagnosis. AB - BACKGROUND: Hurthle cells are founded often in fine needle aspirates of the thyroid from nonneoplastic and neoplastic lesions. Papillary Hurthle cell tumors of the thyroid are composed of oxyphilic cells with a prominent papillary component. Papillary Hurthle cell carcinoma is a relatively uncommon type of thyroid carcinoma. CASE: A 42-year-old man had two asymptomatic nodules (2.5 and 3.5 cm) in the left lobe and one (2.5 cm) in the right lobe of the thyroid. Fine needle aspiration biopsy of the tumor in the left lobe showed large cells in monolayers and papillary clusters with moderate to abundant eosinophilic, granular cytoplasm with sharp borders, a granular nuclear enlargement with pleomorphism and single nucleoli. Histologic examination showed capsular invasion and infiltration of vessels. CONCLUSION: The cytologic appearance of papillary Hurthle cell carcinoma is not specific but can be suspected on fine needle aspiration. PMID- 8629419 TI - Fine needle aspiration cytology of an ectopic parathyroid adenoma: a case report. AB - BACKGROUND: Surgical exploration in the region of the neck identifies a majority of parathyroid adenomas; however, in individuals in whom surgical exploration has failed, fine needle aspiration (FNA) biopsy can be useful. Ectopic parathyroid adenomas can be rapidly diagnosed by the cytologic appearance of aspirate smears with the identification of stromal and intracellular lipid on oil red O-stained smears. CASE: A 59-year-old male presented with hyperparathyroidism; surgical exploration of the neck in the region of the thyroid gland failed to identify any parathyroid abnormality. Subsequent imaging with (99m)technetium and (201)thallium showed a mass in the left supraclavicular region that, on FNA biopsy, was confirmed as an ectopic adenoma. Removal of the adenoma resulted in normal serum calcium levels. CONCLUSION: FNA can be successfully utilized in the localization of ectopic parathyroid tissue. PMID- 8629420 TI - Dermoid cyst of the floor of the mouth diagnosed by fine needle aspiration cytology: a case report. AB - BACKGROUND: Dermoid cyst of the floor of the mouth is a rare, benign lesion requiring surgical intervention. To our knowledge, the preoperative diagnosis of this entity by fine needle aspiration (FNA) cytology has not been previously reported in the cytologic literature. CASE: The patient presented with a massive swelling of the floor of the mouth that displaced the tongue superiorly. A computed tomographic (CT) scan demonstrated a cystic lesion thought to be consistent with a lymphangioma. FNA was performed without complications and demonstrated numerous anucleated and some nucleated squames consistent with a diagnosis of dermoid cyst. Establishment of the correct diagnosis by FNA enabled the surgeon to proceed with the appropriate operation; that outcome might not have been possible had the erroneous CT diagnosis of lymphangioma been trusted. CONCLUSION: A preoperative diagnosis of dermoid cyst of the floor of the mouth was established by FNA. In contrast to reports in the older literature, the FNA procedure caused no complications. In patients presenting with lesions of the floor of the mouth, FNA should be the diagnostic procedure of first choice. PMID- 8629421 TI - Fine needle aspiration cytology of appendiceal mucinous cystadenoma: a case report. AB - BACKGROUND: Fine needle aspiration (FNA) of the appendiceal mucocele is generally avoided because of fear that puncture of a distended viscus will lead to rupture or seeding of neoplastic cells and result in localized or diffuse pseudomyxoma peritonei. Cytologic reports regarding FNA of appendiceal mucoceles are virtually nonexistent. CASE: A 55-year-old male with a preoperative diagnosis of an infectious process with interloop abscess formation underwent computed tomography guided aspiration of the mass. FNA showed clusters of intestinal type epithelium with mild nuclear crowding, hyperchromasia and atypia in a mucinous background. CONCLUSION: The differential diagnosis of a neoplastic appendiceal cystic tumor based on the FNA findings could be difficult and should be made only in conjunction with clinical findings. FNA of an appendiceal mucocele may be safe when a small-gauge needle and retroperitoneal approach are utilized. PMID- 8629422 TI - Aspiration cytodiagnosis of dermal analogue tumor, a rare subtype of salivary gland monomorphic adenoma: a case report. AB - BACKGROUND: Fine needle aspiration (FNA) cytology can be used to diagnose a rare subtype of monomorphic adenoma, dermal analogue tumor. CASE: A 70-year-old male presented with a mass in the parotid gland. An FNA aspirate was obtained using multiple passes in the mass. Filter preparations from aspirated material were made and stained by the Papanicolaou method, while cell blocks from aspirate and excised tissue were stained by hematoxylin-eosin after processing. The cytohistologic features in the cellular sample were characterized by aggregates of uniform epithelial cells with regular nuclei; fine chromatin; a few, regular nucleoli; scanty, ill-defined cytoplasm; no nuclear pleomorphism or mitotic activity; and cell groups bordered by thick, basement membrane-like material. CONCLUSION: Dermal analogue tumor of the salivary gland mimics dermal cylindroma and can recur. This was true in the case presented since the tumor recurred after 11 months. An analogy between adenomas of salivary glands and those of skin adnexa is not surprising since both arise in germinal epithelium of ectodermal origin. Also, the biologic behavior of these adenomas is similar, with a close histogenetic relationship. The findings suggest that the cytomorphologic expression of dermal analogue tumor is distinctive and that diagnosis of this neoplasm by FNA cytology is possible. PMID- 8629423 TI - Breast fibroadenoma with atypical features: a case report. AB - BACKGROUND: Although the cytologic features of fibroadenoma are well established, some of the smears from these tumors cause serious diagnostic problems. CASE: In the cytologic smears from a deeply located breast nodule in a 53-year-old female we found, besides the typical features of fibroadenoma, atypical cells suggesting carcinoma, with intranuclear indentations, nuclear molding, mitotic activity and intracytoplasmic vacuoles. Histologic examination confirmed the diagnosis of fibroadenoma, but marked ductal cell hyperplasia was also revealed. CONCLUSION: Some disturbing atypical changes may occur in fibroadenoma smears. PMID- 8629424 TI - Primary squamous cell carcinoma of the liver: a case report. AB - BACKGROUND: Bile cytology from percutaneous transhepatic cholangio-drainage (PTCD) is a useful procedure in case of obstructive jaundice. CASE: Repeated cytology via PTCD from a cystically dilated duct in a 56-year-old female with a long clinical history permitted a diagnosis of squamous cell carcinoma (SCC). The smears showed excessive benign-appearing squamous cells with or without a nucleus and a few keratinized- and nonkeratinized-type SCC cells. No adenocarcinoma derived cells were identified. No focus of SCC was detected clinically. The resected hepatic specimen disclosed a keratinized-type SCC simultaneous with benign metaplastic squamous epithelium. CONCLUSION: This case of cytology of primary SCC of the liver seems to be the first reported. Even when showing a great number of benign-appearing squamous cells, the possibility of malignancy should not be ruled out. Repeating the cytology from PTCD is effective for a diagnosis. PMID- 8629425 TI - Hemangioblastoma in the cerebellar vermis: a case report. AB - BACKGROUND: The cytologic characteristics of hemangioblastoma have not been well described, although hemangioblastoma is a common primary neoplasm in the cerebellar vermis, and its histologic feature is well known. CASE: Hemangioblastoma was found in the cerebellar vermis of a 47-year-old male. Stamp specimens, made from the cut surface of a solid, yellow-tan tumor, showed many foamy cells considered to be stromal cells and capillaries in a hemorrhagic background. The stromal cells had round or oval nuclei with relatively fine chromatin and lacelike or granular, often vacuolated cytoplasm slightly stained light green. The stromal cells tended to gather around capillaries. CONCLUSION: Distinguishing stromal cells from astrocytes or histiocytes was difficult in this case. Good knowledge of the cytologic characteristics, along with the clinical findings, seems necessary for an accurate diagnosis. PMID- 8629426 TI - Cerebral cysticercosis mimicking malignant glioma: a case report. AB - BACKGROUND: Cysticercosis is the most common parasitic infection of the central nervous system in the United States. CASE: A case of cerebral cysticercosis radiographically and pathologically mimicked a high grade glioma. A symptomatic solitary cerebral hemispheric lesion in a child, unknown at the time to have been born in Korea and adopted by U.S. residents, was initially evaluated by stereotactically guided needle biopsy and diagnosed as having malignant glioma in accord with the radiographic impression. The subsequently excised mass revealed cysticercus with an exuberant granulomatous inflammation and gliosis surrounding a fibrous-walled cyst. CONCLUSION: In the setting of a solitary parenchymal cyst, the radiographic differential diagnosis of neurocysticercosis often includes a primary neoplasm. However, to our knowledge, this is the first report of cysticercosis's also simulating a brain neoplasm pathologically. PMID- 8629427 TI - Multiple myeloma with monoclonal surface immunoglobulin expression: a case report. AB - BACKGROUND: Plasma cells from patients with multiple myeloma have been found to express the immunophenotype of normal plasma cells without surface immunoglobulin expression. CASE: A case occurred of multiple myeloma with monoclonal surface immunoglobulin expression, defined by morphology and flow cytometric immunophenotyping of a fine needle aspiration biopsy of an osteolytic rib lesion and a bone marrow aspirate as well as urine and serum protein electrophoresis with immunofixation. CONCLUSION: The clinical significance of monoclonal surface immunoglobulin expression in rare cases of multiple myeloma is uncertain, and other parameters with clinical significance (CD10 positivity, multiple myeloid antigen expression) will continue to be more useful until additional cases accrue. PMID- 8629428 TI - Atypical cytomorphologic appearance of Cryptococcus neoformans: a report of five cases. AB - BACKGROUND: Cryptococcus neoformans is not generally recognized as producing pseudohyphae. Although atypical morphologic forms have been described in the microbiology literature, we believe this is the first complete cytologic report describing this uncommon and unusual cytologic appearance of cryptococcal infection. CASES: In five cases of cryptococcal infection, C neoformans formed chains of budding yeasts, pseudohyphae and germ tube-like structures. The atypical forms of C neoformans were seen in cerebrospinal fluid, imprints and in histopathologic sections from multiple organs from two human immunodeficiency virus (HIV)-positive patients; in pleural fluid from a patient with non-Hodgkin's lymphoma; in crush smears from a stereotactic biopsy of the brain; and in a fine needle aspirate of a lung nodule in two patients with no known risk factors for HIV infection. CONCLUSION: Recognition of atypical cytomorphologic variants of C neoformans is important since there are potential diagnostic pitfalls for confusing these atypical-appearing organisms with a Candida-type species or fungal contaminants. Special stains for capsular material and culture can be helpful in making a correct diagnosis. PMID- 8629429 TI - Rapid, simple method of extracting DNA from archival Papanicolaou-stained cervical smears. PMID- 8629430 TI - Cytodiagnosis of dermal cylindroma. PMID- 8629431 TI - Cholesterol-rich thyroglossal cyst: report of a case diagnosed by fine needle aspiration. PMID- 8629432 TI - Neuroblastoma arising in a mature ovarian teratoma. PMID- 8629433 TI - Endometriosis of the bladder: cytologic findings and differentiation from transitional cell carcinoma. PMID- 8629434 TI - Detection of metastatic medulloblastoma in a fine needle breast aspirate. PMID- 8629436 TI - Lingual thyroid diagnosed by fine needle aspiration cytology. PMID- 8629435 TI - Cytologic findings in a case of malignant eccrine poroma. PMID- 8629437 TI - Unique association of tuberculous enteritis and Giardia lamblia. PMID- 8629438 TI - Expression of the latent membrane protein of Epstein-Barr virus in Hodgkin's disease. Age and subtype distribution in Polish patients. AB - We examined immunohistochemically 135 cases of HD (4, lymphocytic predominance (LP); 34, mixed cellularity (MC); 90, nodular sclerosis (NS) and 7, lymphocytic depletion (LD) for the presence of EBV latent membrane protein (LMP). Ten patients were younger than 14 years, 55 were young adults (15-34 years), 45 were 35-49-years old and 26 were older than 50 years. The average LMP-immunopositivity was 33% of all HD cases. The highest proportion of LMP-immunopositivity was found in the MC subtype (61.8%), followed by the NS (NS 1-25%, NS 2-26%) and the LD subtype (14%), but none in the LP subtype of HD. The differences between LMP immunopositivity in the MC and NS subtypes were statistically significant in children and young adults (p < 0,01). The LMP-positivity was mainly associated with the MC subtype which occurred predominantly in children, while the lowest proportion of LMP-positive cases was found in the young adult group. This report supports a notion that there are regularities in age and subtype distribution of EBV in HD. PMID- 8629439 TI - Influence of hormone replacement therapy (17 beta-OH estradiol and medroxyprogesterone acetate) on plasma concentration of prothrombin fragment F1+2 and thrombin-antithrombin III complex in postmenopausal woman. AB - The influence of hormone replacement therapy (HRT) on coagulation system has not been completely explained yet. We measured plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III complex before the treatment and after 6 and 12 months of HRT in 116 postmenopausal women HRT considered of transdermal estradiol and oral medroxyprogesterone acetate. We observed no statistically significant differences in plasma levels of the above coagulation markers before and during the treatment. PMID- 8629440 TI - [Comparison of anti-human and anti-porcine factor VIII antibody titers in patients with hemophilia]. AB - The development of antibodies to factor VIII (circulating anticoagulant, factor VIII inhibitor) is a serious complication following substitution therapy in patients with hemophilia A. These antibodies are rarely specific of human factor VIII, tending to cross-react to a variable extent with factor VIII from other species. Their neutralizing activity is usually weaker against porcine than human factor VIII. In this study, anti-human and anti-porcine factor VIII antibodies (Ab) were measured in 41 patients with severe hemophilia A. The anti-human Ab titers ranged from 0.5 to 1200 BU/ml, and the anti-porcine Ab titers ranged from 0 to 170 BU/ml. The median cross-reactivity was 28% (mean 28.9 +/- 17.2%, range 0 75%). Of the 24 patients with anti-human Ab titers > 5 BU/ml, 11 (46%) had a low anti-porcine Ab level (< or = 5 BU/ml). These patients could be treated effectively with porcine factor VIII concentrate (Hyate: C). PMID- 8629441 TI - [The diagnostic significance of blast immunophenotype assay in patients with acute leukemia]. AB - A detailed analysis of immunophenotypes of 120 adult newly diagnosed patients with acute leukaemias was performed. Using the immunopheno-typing, it was possible to defined 96,7% of leukemia cases. The proportion of leukemia subtypes was: AML in 62,5%, ALL in 32,5%, acute biphenotypic leukaemia 1,7% and acute undifferentiated leukaemia (AUL) in 3,3%. The diagnosis initially made according FAB criterias in 12,5% cases after the immunophenotyping was verified. Above analyses showed the existence of the atypical blasts phenotypes in 31%: co expression of CD 19, CD2 and CD7 markers in AML, co-expression of CD 33 marker in ALL, co-expression of CD 19 marker in T cell ALL and biphenotypic (mixed-lineage) leukaemia. PMID- 8629442 TI - Leukemic cells growth fraction in the central nervous system in blastic phase of chronic myelogenous leukemia. AB - Clinical and neuropathological investigations were carried out in 6 patients, deceased due to blastic phase of chronic myelogenous leukemia (BPCML). Growth fraction of leukemic cells in peripheral blood, cerebrospinal fluid and in the central nervous system (CNS) was studied, using mitotic index and immunohistochemical staining technique with the monoclonal antibody antiproliferating cell nuclear antigen (anti-PCNA). The results suggest that in BPCML the proliferative activity of leukemic cells is low both in peripheral blood, cerebrospinal fluid, cerebral leukostasis and within the leptomeningeal and intracerebral infiltrates, even in cases with a very high white blood cells count. It can confirm the opinion that in BPCML, accumulation rather than proliferation of leukemic cells plays an important role in the development of the CNS leukemia. PMID- 8629443 TI - [Bone marrow changes in patients with hairy cell leukemia after 2 chlorodeoxyadenosine treatment]. AB - Bone marrow abnormalities were assessed in 6 patients with hairy cell leukemia after 2-chlorodeoxyadenosine treatment. In spite of clinical and haematological remission in all patients, hairy cells and fibrosis were found within the marrow. However, the hairy cells number, the thickness of argentophilic fibres and the extent of fibrosis. It was stressed that in addition to histological analysis with hematoxylin-eosin also other stainings for fibrosis should be applied in the bone marrow evaluation, followed by immunohistochemical and molecular methods which allow to exclude minimal residual disease after leukemia treatment. PMID- 8629444 TI - [Treatment of multiple myeloma--present status and perspectives]. AB - Multiple myeloma (MM) remains incurable. Despite many chemotherapy programs for large numbers of patients, there has been little improvement in outcome during the past 25 years. For many years, intermittent courses of melphalan and prednisone have represented the standard chemotherapy for newly diagnosed symptomatic MM. Many other drug combinations have been assessed, including regimens using multiple alkylating agents, and programs with vincristine, or an anthracycline, and have failed to show any superiority to melphalan-prednisone. Interferon alpha (IFN alpha) inhibits plasma cell growth and has induced responses in approximately 15% of previously untreated patients. This cytokine may have a role when used in those patients who have reached a good "plateau phase" with low tumor burden at the end of a chemotherapeutic program or after a transplantation procedure. The results of myeloablative therapy with allogenic or autologous marrow transplantation are promising and suggest possibility of a cure in some patients. Important problem in the management of MM patients is the treatment of complications, especially bone destruction, hypercalcemia, anemia and infections. Experimental modalities, especially immunotherapy, hold promise for use in humans and may also provide further insights into the pathogenesis of MM. PMID- 8629445 TI - [Techniques of positive isolation of CD34(+) cells. Personal experience with the immunomagnetic method]. AB - In this article the contemporary methods of positive stem cells isolation have been reviewed, with special regard to their application in the hematological transplantology, and the results of our own studies on isolation with the immunomagnetic method have been presented. PMID- 8629446 TI - [The frequency of cytomegalovirus infection (CMV) in patients immunocompromised patients with malignant and nonmalignant blood disorders]. AB - Studies on CMV infection were carried out in a group of 110 patients, aged from 15 to 78 (average 38), treated in the Department of Haematology. During the course of observation most of the patients were examined repeatedly. Diagnosis was based on serology (CFT, ELISA) and virus isolation from the clinical material. Primary infection (seroconversion) was confirmed in 4 (21%) out of 19 seronegative patients. Seropositive patients comprised 83 per cent. Active CMV infection was determined in 20 (22%) of those patients. PMID- 8629447 TI - [Transthyretin and albumin in cerebrospinal fluid in patients with acute leukemias or lymphomas of high grade malignancy]. AB - Transthyretin and albumin in lumbar cerebrospinal fluid (CSF) and in serum were repeatedly assessed in 40 patients with acute leukemias or high grade non Hodgkin's lymphomas. The patients were divided into 3 groups. Group I-5 individuals with clinical manifestations of leukemic or lymphomatous meningosis; Group II-33 cases with no clinical data of central nervous system involvement by neoplasm (leukemia/lymphoma); Group III-2 patients in whom extramedullary solid leukemic or lymphomatous infiltrations were diagnosed. It was revealed that blood brain-barrier dysfunction due to neoplastic infiltration of leptomeninges went with an increase of albumin blood-CSF barrier dependent- and with decrease of blood-CSF barrier-independent transthyretin concentrations. In patients with extramedullary tumors an evident increase of albumin and total-CSF transthyretin and no blood-CSF barrier-independent transthyretin concentrations below the tumor location were observed. It can betray a complete block of the vertebral canal subarachnoid space below the neoplastic compression of the spinal cord. Cytostatics application, either general or intrathecal, did not influence the CSF albumin and transthyretin levels. PMID- 8629448 TI - Acute myelofibrosis in children: report on two cases. AB - In our report, myelofibrosis in children is discussed and two cases of acutely developing myelofibrosis in association with acute megakaryoblastic leukaemia (M7) are presented. In the first case (girl, 34 months), it was acute myelofibrosis of hypocellular marrow. Diagnosis of M7 was confirmed by positive reaction of blasts from peripheral blood with CD42 and CD61 monoclonal antibodies. In the other child (girl, 5 years old), Ph1(+) chronic myeloid leukaemia diagnosed 22 months earlier transformed to M7. Similar to the first case, no marrow aspirate could be obtained and the diagnosis of M7 was made by the bone marrow histology that showed the presence of grossly fibrosed, hypercellular marrow with a large number of dysplastic, maturing megakaryocytes. Neither of the children had Down's syndrome. Although according to FAB classification both cases represent the same haematological entity, their clinical and histopathological presentations are very different. PMID- 8629449 TI - [Unclassified chronic myeloproliferative Ph(-); i(17q); +8 syndrome with mixed myelo-megakaryoblastic crisis--case report]. AB - We present a case of a 17-year old patient with extreme hepatosplenomegaly, hyperthrombocytosis, hyperleucocytosis and the presence of myelo- and megakaryoblasts in the peripheral blood film. Numerous complications that occurred in the course of the disease made cytostatic treatment difficult. Since Ph chromosome and hybrid gene bcr/abl were absent, the diagnosis of unclassified chronic myeloproliferative syndrome in the phase of blast crisis was established. Immunophenotyping confirmed a mixed myelo- megakaryoblastic character of the crisis. In the differential diagnosis other myeloproliferative syndromes were taken into account including i(17q) syndrome. The patient died after a 13-month observation due to neoplasm progression and sepsis. PMID- 8629450 TI - [RHnull syndrome--diagnostic and therapeutic problems]. AB - A very rare Rhnull phenotype as a cause of haemolytic anaemia in a female patients was for the first time detected in Poland. The beneficial influence of splenectomy was demonstrated. It was proved that a single transfusion of Rh negative red cell concentrate, because of the lack of Rhnull blood, did not cause any acute adverse reactions. However, a transient, subclinical delayed haemolytic reaction due to the stimulation of anti-c and anti-e was observed. PMID- 8629451 TI - Avascular necrosis of the proximal femur in developmental dislocation of the hip. Incidence, risk factors, sequelae and MR imaging for diagnosis and prognosis. AB - Avascular necrosis of the proximal femur still remains the major complication of the treatment for developmental dislocation of the hip. In a three part study I reviewed this problem. Part I analyzed incidence, causes, and risk factors of avascular necrosis. In 105 children with 113 hips who developed avascular necrosis out of 636 consecutive patients with 823 hips treated nonoperatively for developmental dislocation of the hip in the years 1972-1976 the risk factors of avascular necrosis were determined. A method of treatment in most cases was Frejka pillow. Conventional radiographs obtained in AP views during the course of treatment and follow-up were analyzed. Avascular necrosis was found in 14 percent of the hips, classified as mild (49%), moderate (14%), and severe (37%). The differences between mild and severe cases were significant as regards age at the onset of treatment (p 0.006); with higher average age in mild forms, and degree of dislocation (p 0.01) with higher values in severe forms. The older the child was at the onset of treatment, the greater the risk of necrosis, notably if treatment was begun after 6 months of age. However, the incidence of the more severe cases was higher in the group up to 6 month of age. In general, avascular necrosis was more likely to occur in cases with high degree of initial dislocation and the differences between groups with low and high degree of dislocation were significant. In the group with highest initial dislocation the number of both mild and severe forms was high. Part II evaluated the growth and remodeling of the hip joint with avascular necrosis after nonoperative treatment of developmental dislocation on the basis of conventional radiography. An attempt was also made to determine the correlation between the severity of necrosis as seen in conventional radiography and the clinical and radiographic appearance of the hip after completion of growth. Finally the prognostic value of conventional radiography in prediction of deformities of the proximal femur due to necrosis was estimated. 68 patients with 98 involved hips treated exclusively nonoperatively for developmental dislocation of the hip in whom avascular necrosis developed were selected for the study. The average age at the time when the final radiograph was made was 25 (18-36) years and the average follow-up period was 23 (18-35) years. 16 patients (27 hips) were examined twice after completion of growth with the time interval of 10 years, the second examination being at an average age of 30 (26-36) years. In this group also progress of signs and symptoms of degenerative changes in clinical and radiographic examination was noted. To achieve sufficient data necessary to establish indications for further operative treatment in 2 patients also CT examinations with three-dimensional surface reconstruction were performed after physeal closure. Physical examinations were performed in all patients after completion of growth. Radiographs made before the onset of treatment for developmental dislocation of the hip, during treatment, at the child's age of 4-6 years, all obtained until the cessation of growth, and at final assessment, were studied. By physical evaluation 77 hips were rated as excellent or good, being pain free or with only occasional mild pain after walking long distances, with a good range of hip motion and negative Trendelenburg sign. The reasons for 21 fair or poor clinical end-results were pain, mostly with activity, and limp due to pain and abductor weakness. By radiographic evaluation in this group there were 50 hips rated as excellent or good, and 48 hips rated as fair or poor. In 29 hips excellent or good clinical findings at final review contrasted with fair or poor radiographic scores. In no case fair or poor clinical end-result coexisted with excellent radiographic ones. In the group examined twice after completion of growth with the time interval of 10 years no difference in clinical score was found in PMID- 8629452 TI - Basic fibroblast growth factor for stimulation of bone formation in osteoinductive or conductive implants. AB - Basic Fibroblast Growth Factor (bFGF) is one of the endogenous factors found in bone matrix. bFGF is a mitogen for many cell types, including osteoblasts and chondrocytes. It can stimulate angiogenesis and osteoblast gene expression. The purpose of this study was to investigate whether exogenous bFGF can stimulate the formation of bone in bone grafts and in a bone graft substitute. In a model using demineralized bone matrix implants for bone induction, a dose of 15 ng bFGF per implant increased the number of chondrocytes and the amount of bone, whereas 1900 ng greatly inhibited cartilage and bone formation. These results are consistent with previous studies with this model, showing that a lower dose of bFGF increased bone calcium content and a higher dose reduced it. Thus, exogenous bFGF can stimulate proliferation during early phases of bone induction. A new device, the bone conduction chamber, was developed for the application of bFGF to bone conductive materials. This model made it possible to demonstrate a difference between the conductive properties of bone grafts and porous hydroxyapatite. bFGF increased bone ingrowth into bone graft inside the chamber and showed a biphasic dose-response curve, so that 8-200 ng per implant (0.4-10 ng/mm3) increased bone ingrowth, but higher or lower doses had no effect. The same doses had the same effects in porous hydroxyapatite. In both bone grafts and porous hydroxyapatite, the highest dose still caused an increase in ingrowth of fibrous tissue. The effect on bone ingrowth was first detected after 6 weeks, regardless if administration of bFGF started at implantation or 2 weeks later, using an implanted minipump. Hyaluronate gel was effective as a slow-release carrier for bFGF. In conclusion, bFGF stimulates bone formation in bone implants, depending on dose and method for administration. PMID- 8629453 TI - Effect of corticotropin-releasing factor on the pituitary-ovary axis in human luteal phase. AB - In order to test the possible negative effect of corticotropin-releasing factor (CRF) on the pituitary-gonadal axis, two gonadotropin-releasing hormone (GnRH) tests, on two consecutive days, were performed in five normally cycling women in the midluteal phase of the menstrual cycle. The first GnRH test was performed after 2 h during which time basal blood samples were collected every 10 min. On the second day, the GnRH test was repeated after a 2-h CRF infusion which was continued throughout the GnRH test. Our results demonstrate that while CRF increases cortisol levels it affects neither the basal nor the GnRH-stimulated gonadotropin levels. Similarly, both basal and luteinizing hormone (LH) stimulated progesterone concentrations were not modified by the CRF infusion thereby also excluding a possible direct effect of the peptide on corpus luteum steroidogenesis. PMID- 8629454 TI - Bone mineral content in girls with precocious puberty treated with gonadotropin releasing hormone analog. AB - In order to evaluate the effects of gonadotropin-releasing hormone (GnRH) analogs on calcium metabolism, we studied 12 girls with central precocious puberty (CPP) who were treated with the GnRH agonist D-Trp6-GnRH every 28 days. The patients' mean age +/- SD was 5.9 +/- 2.1 years. The patients were studied before commencement and after 6 and 12 months of treatment. We also studied 12 age matched healthy girls who served as controls. Bone mineral content was measured by dual-photon densitometry with 125I, in the distal third of the left radius. We evaluated the serum levels of calcium, phosphate, magnesium, parathyroid hormone, calcitonin, 25-hydroxy-vitamin D and the 24-h urinary excretion of calcium, phosphate and magnesium. All of these parameters were found to be normal before and during the treatment in both groups. At the beginning of the study, the patients with CPP had significantly higher bone mineral content than controls (0.51 +/- 0.12 g/cm2 vs. 0.39 +/- 0.09, p < 0.001); after 6 months contents were 0.42 +/- 0.11 vs. 0.41 +/- 0.05, p < 0.01; and after 12 months 0.44 +/- 0.11 vs. 0.44 +/- 0.05, NS, for treatment and control groups, respectively. This difference remained after 6 months of treatment, while after 12 months no significant difference between patients and controls was found. Our study shows that girls with CPP have an increased bone mineral content and that GnRH analogs modify bone density with a consequent reduction, it seems, that is not related to any of the calcium parameters studied. PMID- 8629455 TI - A study of premature ovarian failure in Turkish women. AB - The parameters that could be responsible for or could be the end results of the premature ovarian failure were evaluated in 100 patients and compared with the same parameters of a control group consisting of 30 ovulatory healthy women with regular menstrual cycles. The incidence of premature ovarian failure was 6.6%. The mean age of the premature ovarian failure group was 39.2 +/- 4.1 years (range 30-58) whereas this was 45.6 +/- 3.1 years (range 39-52) for the control group. The mean menopausal age was 4.0 +/- 4.3 years (range 1-30). The prevalence of being a widow, divorcee or single was high in the premature ovarian failure group (p < 0.01). Of the hormonal and metabolic parameters, thyroid-stimulating hormone (TSH) and high-density lipoprotein (HDL) levels showed significant differences between the two groups (p < 0.01 and p < 0.05, respectively) and of the parameters reflecting bone metabolism, alkaline phosphatase levels showed a significant difference (p < 0.05). The bone mineral densitometric measurement values were lower in the premature ovarian failure group (p < 0.05). Because premature ovarian failure is not a rare disorder and because the patients have an early estrogen deficiency we concluded that, whatever the etiologic factor, hormone replacement therapy needs to be given as early as possible. PMID- 8629456 TI - Administration of unmodified progesterone by nasal spray in fertile women. AB - A 11.20-mg dose of progesterone was administered by nasal spray to five healthy fertile women in the follicular phase of the menstrual cycle. Serial blood samples were collected and Cmax (the maximum progesterone concentration reached), Tmax (the time at which Cmax was reached) and the area under the curve (AUC), with the time limits of 0 and 720 min, were calculated. Serum progesterone levels were assayed by means of a non-extraction [125I]radioimmunoassay. The mean Cmax was 4.50 +/- 2.31 ng/ml at a Tmax of 30 min; levels returned to baseline after 720 min. The mean AUC value was 1180.50 +/- 613.90 ng.h/ml. The progesterone administered by nasal spray in fertile women was effective in reaching physiological progesterone levels. Even if a nasal first-pass metabolic effect is taken into account, this route allows progesterone to avoid liver first-pass metabolism and its metabolic consequences. PMID- 8629457 TI - A severe case of ovarian hyperstimulation syndrome: 65 liters of ascites aspirated in an on-going IVF-ET twin pregnancy. AB - Ovarian hyperstimulation syndrome (OHSS) is the most serious life-threatening iatrogenic complication of ovulation induction. Presented here is an unusual case where 62 l of intraperitoneal and 3.2 l of intrapleural fluid were aspirated in order to stabilize a severe life-threatening case of OHSS resulting from an on going IVF-ET twin pregnancy. PMID- 8629458 TI - The influence of the dose of ethinylestradiol in oral contraceptives on follicle growth. AB - This prospective, randomized comparative clinical study involving 416 women investigated follicle development over a period of 12 oral contraceptive treatment cycles. Women were allocated to two groups, one group (n = 207) received a preparation containing 30 micrograms ethinylestradiol and 75 micrograms gestodene daily, and the other group (n = 209) received 20 micrograms ethinylestradiol and 150 micrograms desogestrel, daily. Follicular development was monitored by transvaginal ultrasonography of the ovaries, during days 18-21 in the pretreatment cycle and in treatment cycles 1, 3, 6, 9 and 12. Follicular development was found to be twice as frequent in the group receiving 20 micrograms ethinylestradiol/desogestrel as in the group receiving 30 micrograms ethinylestradiol/gestodene. For all cycles, follicles of 10-30 mm were found in 18% of women in the desogestrel group, compared with 9.7% in the gestodene group, whilst follicles with a diameter of >30 mm were present in 5% of the desogestrel group compared with 1.9% of the gestodene group. The difference between the treatment groups with respect to follicle diameters of 10-30 mm and >30 mm was statistically significant (p < 0.05 and p < 0.001, respectively). No ruptured follicles were observed in either group throughout the study, suggesting that there was no escape ovulation, however, there was one pregnancy in the desogestrel group that could not be explained either by drug interactions or missed pills. It can be concluded that the ethinylestradiol dose in an oral contraceptive has a significant effect on follicular ovarian activity, and that reducing the dose to 20 micrograms is associated with a significant increase in follicle size. PMID- 8629459 TI - Concentrations of various arachidonic acid metabolites in menstrual fluid are associated with menstrual pain and are influenced by hormonal contraceptives. AB - In a pilot study we investigated the association between concentrations of various eicosanoids in menstrual blood with pain and oral contraceptive use. Menstrual fluid was collected on tampons by 12 women who did not use an oral contraceptive but suffered from slight primary dysmenorrhea and by three pain free women who used an oral contraceptive. Eicosanoids (cyclooxygenase products: 6-ketoprostaglandin F1 alpha, thromboxane B2, prostaglandin E2, prostaglandin F2 alpha, 13,14-dihydro-15-ketoprostaglandin F2 alpha, 12-hydroxy-heptadecatrienoic acid; lipoxygenase products: 5-, 12-, 15-hydroxy-eicosatetraenoic acid (HETE), leukotriene B4, leukotriene C4, leukotriene D4, leukotriene E4) and female sex steroids (17 beta-estradiol and progesterone) were analyzed by the combined use of high-performance liquid chromatography and radioimmunoassay. 12-HETE was the main arachidonic acid metabolite. An increased metabolism of arachidonic acid was associated with pain, especially when synthesis of 12-HETE was elevated. Oral contraceptive use decreased the synthesis of prostaglandins as well as leukotrienes. The concordant changes of cyclooxygenase and lipoxygenase products in dysmenorrhea or in oral contraceptive use may be explained by an increased or decreased phospholipid metabolism, respectively. PMID- 8629460 TI - Recurrent spontaneous ovarian hyperstimulation syndrome associated with polycystic ovary syndrome. AB - Ovarian hyperstimulation syndrome (OHSS) is the most serious potentially life threatening iatrogenic complication of ovulation induction. Presented here is the first reported case of recurrent severe OHSS which developed spontaneously in a women with polycystic ovary syndrome, diagnosed early in her second pregnancy, and necessitated intensive fluid and colloid therapy. PMID- 8629461 TI - Expression of sex hormone-binding globulin mRNA in uterine leiomyoma, myometrium and endometrium of human subjects. AB - This study was designed to evaluate the effect of sex hormone-binding globulin (SHBG) on the estrogen-dependent growth of human uterine leiomyoma. Levels of SHBG mRNA were analyzed using competitive reverse transcription-polymerase chain reaction-Southern blot analysis (RT-PCR-SBA). In normal uterine endometria, the levels of SHBG mRNA in the early/mid and late proliferative phases of the menstrual cycle were significantly lower than in the secretory phase (p < 0.01). In uterine myometria and leiomyomas, SHBG mRNA level showed no significant differences during the menstrual cycle, while the ratio of leiomyoma SHBG level to the corresponding myometrium SHBG level was >1 in 21 out of 23 cases (91%). Our results suggest that steroidal regulation of intracellular SHBG synthesis in the myometrium and the leiomyoma might differ from that in the endometrium, and that the mechanism of SHBG expression in leiomyoma might, in the process of tumorigenesis, be altered from that of corresponding normal myometrium, contributing to SHBG overexpression and the abundant estrogen supply. PMID- 8629462 TI - Spontaneous episodic release of adenohypophyseal hormones in hypothalamic amenorrhea. PMID- 8629464 TI - Multiple modes of periodic breathing during sleep. PMID- 8629463 TI - Breathing patterns under enflurane, halothane and propofol sedation in humans. PMID- 8629465 TI - Volume history response of airway resistance. PMID- 8629466 TI - Non-stationarity of breath-by-breath ventilation and approaches to modelling the phenomenon. PMID- 8629467 TI - Effect of repetitive testing on breathlessness. PMID- 8629468 TI - Exercise hyperpnea. Chairman's introduction. PMID- 8629469 TI - Respiratory compensation, as evidenced by a declining arterial and end-tidal PCO2, is attenuated during fast ramp exercise functions. PMID- 8629470 TI - Acute ventilatory response to ramp exercise while breathing hypoxic, normoxic, or hyperoxic air. PMID- 8629472 TI - Is the pattern of breathing at rest chaotic? A test of the Lyapunov exponent. PMID- 8629471 TI - Ventilatory responses during ramp exercise in hyperoxia. PMID- 8629473 TI - Respiratory compensation for the metabolic acidosis of severe exercise as a modulator of muscular capillary O2-unloading. PMID- 8629474 TI - Effects of base line changes in work rate on cardiorespiratory dynamics in incremental and decremental ramp exercise. PMID- 8629475 TI - Simulation of asymmetrical O2 uptake kinetics during incremental and decremental ramp exercise. PMID- 8629476 TI - Core temperature thresholds for ventilation during exercise. Temperature and ventilation. PMID- 8629477 TI - Modelling the effect of taper on performance, maximal oxygen uptake, and the anaerobic threshold in endurance triathletes. AB - The purpose of this study was to determine the nature of taper required to optimize performance in Ironman triathletes. Eleven triathletes (26 +/- 4 yrs, 77.0 +/- 6.5 kg) took part in 3 months of training interspersed with two taper periods, one of 10 days (Taper 1) and another six weeks later for 13 days (Taper 2). Reducing training volume by 50% in an exponential fashion (tau < or = 5 days) in one group of triathletes during Taper 1 resulted in a 46 second (4%) improvement in their 5 km criterion run time and a 23 W (5%) increase in maximal ramp power output above the same measurement at the beginning of taper. A 30% step reduction in training volume in the second group did not result in any significant improvement in physical performance on the same measures. Training volume was reduced exponentially from the end of training in both a high volume group (tau > or = 8 days) and a low volume group (tau < or = 4 days) during Taper 2. Criterion run time improved significantly by 74 seconds (6%) and 28 seconds (2%) in the high and low volume groups respectively, while maximal ramp power increased significantly by 34 W (8%) only in the low volume taper group. Maximal oxygen uptake increased progressively from 62.9 +/- 5.8 ml.kg-1.min-1 two weeks prior to taper, to a significantly higher level 68.9 +/- 4.2 ml.kg-1.min-1 during the final week of Taper 2 (p < or = 0.5). The anaerobic threshold determined by a non-invasive method was also observed to increase from 70.9% to 74.9% of a subject's maximal oxygen uptake during Taper 2. These results demonstrate that proper placement of training volume during taper is a key factor in optimizing performance for a specific competition and a high volume of training in the immediate days preceding an event may be detrimental to physical performance. PMID- 8629478 TI - Is the slow component of exercise VO2 a respiratory adaptation to anaerobiosis? PMID- 8629479 TI - Effects of age on VO2 kinetics during calf and cycling exercise. PMID- 8629480 TI - VO2 on-transient kinetics with a centrally acting calcium channel blocker. PMID- 8629481 TI - Dynamics of the pulmonary O2 uptake to blood flow ratio (VO2/Q) during and following constant-load exercise. PMID- 8629482 TI - Control of intermittent ventilation in lower vertebrates. A computer dynamic model. PMID- 8629483 TI - Exercise ventilation and K+ in patients with COPD. Positive and negative work. PMID- 8629484 TI - Phase-coupling of arterial blood gas oscillations and ventilatory kinetics during exercise in humans. Phase coupling and the exercise hyperpnoea. PMID- 8629485 TI - Optimization of respiratory pattern during exercise. PMID- 8629486 TI - The influence of chemical and mechanical feedback on ventilatory pattern in a model of the central respiratory pattern generator. PMID- 8629487 TI - Breathing in exercising quadrupeds. There ain't no such thing as a free breath! PMID- 8629488 TI - Dynamic chemoreceptiveness studied in man during moderate exercise breath by breath. PMID- 8629489 TI - CO2 retention during exercise. A role for the carotid chemoreceptors? PMID- 8629490 TI - Hypoxic exercise does not elicit long-term modulation of the normoxic exercise ventilatory response in Goats. PMID- 8629491 TI - Respiratory responses to hypoxia peripheral and central effects. Chairman's introductory communication. PMID- 8629492 TI - Hypoxic ventilatory depression may be due to central chemoreceptor cell hyperpolarization. AB - By re-examining the results of various studies of HVD, of the localization of medullary CO2 chemosensory cells, and of their acid secretion, an hypothesis has been developed suggesting that the neurones which detect increased CO2 or CSF acid respond to decreased transmembrane H+ gradient, i.e. a greater fall in ECF than in ICF pH. Hypoxic lactic acid generated within these cells depresses activity, which can be restored by an appropriate rise of Paco2, disclosing both normal peripheral chemoreceptor hypoxic sensitivity and normal medullary integrative response. PMID- 8629493 TI - Central hypoxic chemoreceptors in the ventrolateral medulla and caudal hypothalamus. PMID- 8629494 TI - Ventilatory responses to isocapnic hypoxia in the eighth decade. PMID- 8629496 TI - A comparison between the effects of 8 hours of isocapnic hypoxia and 8 hours of poikilocapnic hypoxia on respiratory control in humans. PMID- 8629495 TI - Hypoxic ventilatory response near normocapnia. PMID- 8629498 TI - Changes in blood flow in the middle cerebral artery in response to acute isocapnic hypoxia in humans. PMID- 8629497 TI - Individual differences in ventilatory and HR responses to progressive hypoxia following 100% O2 exposure in humans. PMID- 8629499 TI - Role of acetylcholine as an essential neurotransmitter in central respiratory drive. PMID- 8629500 TI - Middle cerebral artery blood flow velocity studied during quiet breathing, reflex hypercapnic breathing and volitionally copied eucapnic breathing in man. Voluntary control of breathing. PMID- 8629501 TI - The effects of hypoxia and hyperoxia on the 1/F nature of breath-by-breath ventilatory variability. PMID- 8629502 TI - Neurobiology of breathing control. Where to look and what to look for. PMID- 8629503 TI - Cholinergic dimensions to carotid body chemotransduction. PMID- 8629504 TI - Gases as chemical messengers in the carotid body. Role of nitric oxide and carbon monoxide in chemoreception. PMID- 8629505 TI - Interactive ventilatory effects of carotid body hypoxia and hypocapnia in the unanesthetized dog. PMID- 8629506 TI - The postnatal potentiation of chemoreceptor sensitivity to O2 and CO2 in the in vitro rat carotid body is blunted by chronic hypoxaemia. Development of chemosensitivity. PMID- 8629507 TI - The excitation of carotid body chemoreceptors of the cat by potassium and noradrenaline. PMID- 8629508 TI - Effects of GABA receptor antagonists on the raphe magnus-induced inhibition of bulbar and spinal respiratory neural activities in the cat. PMID- 8629509 TI - Activation of limbic structures during CO2-stimulated breathing in awake man. PMID- 8629510 TI - Improvements to the PRBS method for measuring ventilatory response to CO2. PMID- 8629511 TI - Intralaryngeal CO2 reduces the inspiratory drive in cats by sensory feedback from the larynx. PMID- 8629512 TI - Investigation of central CO2-sensitivity around eucapnia in awake humans using a brief hypoxic stimulus. PMID- 8629513 TI - Central-peripheral ventilatory chemoreflex interaction in humans. PMID- 8629514 TI - Subcellular control of oxygen transport. PMID- 8629515 TI - Muscle perfusion and control of breathing. Is there a neural link? PMID- 8629516 TI - Retrotrapezoid nucleus (RTN) metabotropic glutamate receptors and long-term stimulation of ventilatory output. RTN glutamate receptors and breathing. PMID- 8629517 TI - Expression of c-fos in the brain stem of rats during hypercapnia. PMID- 8629518 TI - Two distinct descending inputs to the cricothyroid motoneuron in the medulla originating from the amygdala and the lateral hypothalamic area. AB - 1. The retrograde labelling study revealed that there are at least two independent descending pathways from the limbic system to the ventral medulla, i.e., the hypothalamo-medullary pathway and the amygdalo-medullary pathway. 2. The stimulation in the lateral hypothalamic area produced parasympathetic excitation and vocalization response: the recruited motor unit of CT muscle occurred in the late expiratory phase. By contrast, the stimulation at the medial part of the amygdala evoked sympathetic excitation and expiratory braking: the recruited motor unit of CT muscle occurred in the early expiratory phase or in the post-in-spiratory phase. 3. The present physiological study provided further important information on dual innervation of the cricothyroid muscle of the larynx: one motor unit with inspiratory firing is generated in the medulla, whereas the other motor unit with expiratory firing is evoked by a descending input from the limbic system. PMID- 8629519 TI - Trigeminal motor nucleus and pontile respiratory regulation. PMID- 8629520 TI - Axon branching of medullary expiratory neurons in the sacral spinal cord of the cat. PMID- 8629521 TI - Vagal cooling and the origin of pulmonary reflexes in cats. PMID- 8629522 TI - New computational models of the respiratory oscillator in mammals. AB - Understanding how respiratory oscillations are generated and controlled in the mammalian brainstem requires synthesis of neural function at cellular, synaptic, and network levels. Modeling the respiratory network has entered a new era where simulations incorporating complex cellular properties and network interactions exhibited by respiratory neurons are possible. Given the complexity and unknowns of the real system, there are obvious limitations of the modeling in its present form. Nevertheless, if we are to produce neurobiologically satisfying, mechanistic explanations that synthesize cellular and network phenomena, the modeling approach outlined here should have distinct advantages. The approach allows a close interaction between experimental and computational studies, and will enable simulations with increasing neurobiological realism as additional experimental data becomes available. The modeling summarized here represents the initial stages of establishing a computational framework for storing information about neuron and network properties, and for exploring complex dynamical properties in ways not possible experimentally. PMID- 8629523 TI - Apneic snout immersion in trained pigs elicits a "diving response". PMID- 8629524 TI - Interaction between expiratory time and inspiration in conscious humans. PMID- 8629525 TI - Control of the respiratory cycle in conscious humans. PMID- 8629526 TI - Intellectual work using a video game inhibits post hyperventilation hyperpnoea following voluntary hyperventilation while it stimulates breathing at rest. PMID- 8629527 TI - Introduction to session on the pathophysiology of breathing control and breathing: awake and asleep. PMID- 8629528 TI - Possible genomic mechanism involved in control systems responses to hypoxia. PMID- 8629529 TI - Asynchronous thoracoabdominal movements in chronic airflow obstruction (CAO). Active expiration during spontaneous breathing in sleep and wakefulness. PMID- 8629530 TI - Consumer campaign is directed at women with HIV or AIDS. PMID- 8629531 TI - Counseling for healthy lifestyles: exercise prescription. PMID- 8629532 TI - Carotid artery stenosis: to screen or not to screen. PMID- 8629533 TI - The death-with-dignity dilemma. PMID- 8629534 TI - The death-with-dignity dilemma. PMID- 8629535 TI - Successful technique for treating ingrown toenails. PMID- 8629536 TI - Diagnostic imaging in the evaluation of dysphagia. AB - Evaluation of dysphagia is a challenge commonly encountered by family physicians. Dysphagia may be classified as either the oropharngeal type or the esophageal type and may have a variety of etiologies. Possible causes of oropharyngeal dysphagia include Zenker's diverticulum, pharyngeal carcinoma, pharyngeal webs and strictures, lateral pharyngeal pouches and neuromuscular diseases. Esophageal dysphagia can be caused by esophageal carcinoma, esophageal stricture and webs, achalasia, diffuse esophageal spasm and scleroderma, caustic esophagitis and infectious esophagitis. Studies using different textures of barium allow evaluation of the swallowing mechanism. Static images are obtained to evaluate the integrity of the mucosa. PMID- 8629537 TI - Prevention of stroke caused by carotid bifurcation stenosis. AB - Prevention of stroke caused by carotid bifurcation stenosis can be achieved by accurate identification and evaluation of patients at risk. A consensus report from the National Institute of Neurologic Disorders and Stroke has standardized diagnostic criteria and symptoms related to this disease. Recent prospective, randomized trials have identified effective treatment for both asymptomatic and symptomatic carotid stenosis. The risk factors for carotid stenosis are similar to those for atherosclerosis--hypertension, diabetes, cigarette smoking and hyperlipidemia. A carotid bruit is the most common clinical finding, although its positive predictive value is only about 60 to 70 percent. Recent clinical trials have identified patient groups that benefit from surgical and medical therapy, depending on the degree of carotid stenosis and the presence or absence of symptoms. Symptomatic patients with carotid stenosis greater than 70 percent benefit from surgical therapy. Asymptomatic patients who have carotid stenosis greater than 60 percent and are good surgical candidates should be referred for surgical consultation. PMID- 8629538 TI - Management of ocular emergencies and urgent eye problems. AB - Evaluation of the patient with an acute eye problem begins with documentation of the level of vision in each eye, except in the case of a splash injury. In such cases, immediate copious irrigation is of critical importance. Subconjunctival hemorrhage is common and, typically, completely benign. Herpes simplex infection is painful and can lead to extensive damage. Herpes zoster infection is usually accompanied by skin lesions and can be effectively treated with oral acyclovir or famcyclovir. In patients with Bell's palsy, the eye must be carefully protected to prevent secondary injury. Corneal abrasions heal rapidly when antibiotics and patch protection are provided. Acute infections of the eyelids and conjunctivae usually respond well to topical antibiotics and warm compresses. Traumatic injuries require careful evaluation and, frequently, referral to an ophthalmologist. PMID- 8629539 TI - Prescribing exercise for health: a simple framework for primary care. AB - Physicians need to become committed to promoting the preventive benefits of regular physical activity. Practical strategies can help physicians efficiently and effectively incorporate exercise counseling into their practices. These strategies include regularly asking about leisure activity and linking the agenda of an office visit to the benefits of exercise. The physician and the patient should work together to formulate a mutually acceptable plan for the patient to adopt and maintain a healthful exercise lifestyle. In the same way that prescriptions are written for medications, a prescription can be given for an exercise plan suited to the individual patient. PMID- 8629540 TI - Angiotensin-II receptor antagonists: a new class of antihypertensive agents. AB - Pharmacologic agents that attenuate the influence of the renin-angiotensin aldosterone system are known to reduce systemic arterial blood pressure through vasodilatory action and enhanced renal clearance of sodium and water. Angiotensin converting enzyme inhibitors are known to antagonize the renin-angiotensin aldosterone system through their ability to inhibit conversion of angiotensin I to angiotensin II. A new class of antihypertensive agents, angiotensin-II receptor antagonists, has recently been developed. These agents specifically block the receptor for angiotensin II, thereby limiting angiotensin II-mediated vasoconstriction and reducing aldosterone secretion. These effects result in a reduction in systemic arterial blood pressure through reduced vascular tone and enhanced sodium and water clearance. Clinical trials have demonstrated the efficacy of these agents in reducing blood pressure. These new antihypertensive agents also have uricosuric actions and are well tolerated, with a low incidence of cough and angioedema, side effects that are seen with angiotensin-converting enzyme inhibitors. Clinical trials are underway to see if these drugs will be useful in the treatment of diseases other than hypertension, such as congestive heart failure and chronic renal disease. PMID- 8629541 TI - Prevention and care of diabetic foot ulcers. AB - The foot is frequently overlooked in the management of diabetic patients. Failure to control diabetic foot ulcers at an early stage can lead to life-threatening infection or amputation. Preventive care should emphasize patient education, glycemic control, careful daily foot hygiene and appropriate footwear. Early management of a diabetic foot ulcer should include culture-directed antibiotic therapy when there is evidence of infection, moist dressings and adjustment of footwear or casting to avoid pressure on the wound site. All patients with foot ulcers should be evaluated for evidence of foot ischemia. Surgical intervention to debride infected tissue and bone or to revascularize ischemic tissue can aid in ulcer healing. Serious infection or severe ischemia, unfortunately, often necessitates amputation. PMID- 8629542 TI - Preventive dentistry and the family physician. AB - Family physicians care for infants and toddlers during a critical period for dental health, and before most children have seen a dentist. The risk of dental disease can be reduced in children by encouraging appropriate diet and hygiene, avoidance of harmful bottle habits, optimal fluoride intake and application of dental sealants to permanent posterior teeth. Fluoride effectively reduces dental caries, but combined intake from water, beverages, foods, dentifrices and inappropriate supplementation has led to an increase in dental fluorosis. New guidelines for fluoride use recommend that fluoride supplementation be delayed until the child is six months of age. Physicians can provide parents with valuable counsel related to teething concerns, thumb-sucking and prevention of dental trauma and disease. PMID- 8629543 TI - Evaluating the child with chronic diarrhea. AB - The occurrence of chronic diarrhea in infants younger than three months suggests disaccharidase deficiency, cow's milk or soy protein intolerance, cystic fibrosis or an immunodeficiency state, while chronic diarrhea in children three to 18 years of age suggests celiac disease, late-onset primary lactose deficiency and inflammatory bowel disease. Gastrointestinal infection is the most common cause of chronic diarrhea in children of all ages. Diarrhea that develops after the introduction of cow's milk, cereals and fruits suggests an enzyme deficiency or protein intolerance. Watery, explosive stools suggest sugar intolerance, and foul smelling, greasy, bulky stools suggest fat malabsorption. Marked weight loss suggests malabsorption, inflammatory bowel disease, hyperthyroidism or malignancy. The presence of neutrophils or red blood cells in the stool indicates bacterial gastroenteritis or inflammatory bowel disease, while the presence of eosinophils suggests protein intolerance or parasitic infestation. A toddler who is thriving and cheerful despite having diarrhea may have chronic nonspecific diarrhea of childhood. PMID- 8629544 TI - Varicella vaccine: rationale and indications for use. AB - The recently released varicella vaccine is effective and cost-effective. Routine immunization with one dose of varicella vaccine is now indicated for all children 12 months to 18 months of age. Catch-up immunization for children 18 months to 12 years of age is indicated if the child does not have a history of chickenpox. For susceptible persons over 12 years of age, two doses given four to eight weeks apart are recommended. Because the varicella vaccine contains live virus, proper evaluation for contraindications is necessary. The varicella vaccine should be administered subcutaneously and is sensitive to heat. PMID- 8629545 TI - Current management of breast cancer. AB - Breast cancer accounts for more than 25 percent of all new cases of cancer in women. Family physicians have an important role in the management of patients who are diagnosed with breast cancer. The clinical staging system is a two-phase postsurgical process that assesses relevant prognostic factors and directs selection of adjuvant therapy after surgery. In stage I or II breast cancer, a patient's chances of disease recurrence and death depend on the number of cancer positive regional lymph nodes at the time of diagnosis. Survival rates in patients with metastatic disease vary with the location and type of metastases. Whether surgery, chemotherapy or radiation therapy is chosen, patients and their families need continuous advice and support, especially when the cancer is advanced. PMID- 8629546 TI - A practical approach to the patient with back pain. AB - Management of back pain is most likely to be effective when treatment is based on a specific diagnosis, when patients are followed proactively to recovery and when psychosocial factors receive appropriate attention. Selected non-habit-forming drugs are useful, especially when taken on a regular basis rather than on an "as needed" basis. Rest is less useful in the management of back pain than previously believed and may be counterproductive. Surgery has value only in carefully selected patients, primarily those with well-documented sciatic nerve injury that has not responded to a course of conservative treatment. PMID- 8629547 TI - Cardiovascular medicine. Recommended core educational guidelines for family practice residents. American Academy of Family Physicians. PMID- 8629548 TI - ACOG releases practice guidelines on vaginal delivery after previous cesarean birth. PMID- 8629549 TI - AAFP recommends seven action steps for family physicians in the battle against tobacco. PMID- 8629550 TI - Lamivudine as adjunct therapy in HIV. PMID- 8629551 TI - Saquinavir for treatment of patients with HIV. PMID- 8629552 TI - Gynecologic care. PMID- 8629553 TI - The minimally abnormal Pap smear. A conservative approach. PMID- 8629554 TI - ASCUS and LSIL Pap smear. Results: triage considerations. PMID- 8629555 TI - Nifedipine-induced gingival hyperplasia. PMID- 8629556 TI - Traditional Jewish circumcision technique of Bris. PMID- 8629557 TI - Group A streptococcal infection and polysplenia syndrome. PMID- 8629558 TI - White coat hypertension: measures to improve diagnosis. PMID- 8629559 TI - Management of BPH: educating patients about PSA. PMID- 8629560 TI - Elevated cholesterol levels and healthy lifestyles in childhood. PMID- 8629561 TI - STDs and the American Social Health Association. PMID- 8629562 TI - Pelvic inflammatory disease: a contemporary approach. AB - Pelvic inflammatory disease (PID) results from an ascending polymicrobial infection of unclear pathogenesis. One in 10 women in the United States has PID during her reproductive years. Medical costs of managing the disease and its sequelae are over $5 billion per year. One out of four women with PID has serious sequelae, including infertility, ectopic pregnancy or chronic pelvic pain. Patients with PID present with a spectrum of clinical symptoms and signs, none of which conclusively makes the diagnosis. The diagnosis of PID relies on a high index of suspicion, coupled with empiric therapeutic intervention and careful follow-up. Revised guidelines from the Centers for Disease Control and Prevention include recommendations for the use of broad-spectrum antibiotics, which are initiated before culture results are obtained. Indications for hospitalization are more liberal now than under the previous CDC recommendations. Prompt recognition, patient compliance with recommended therapy and treatment of the sexual partner decrease the risk of sequelae. Family physicians can make significant contributions to the health care of women through skillful management of PID. PMID- 8629563 TI - The minimally abnormal Papanicolaou smear. AB - The Bethesda system has helped to standardize the nomenclature for cervical cytology. Previously used cytologic classification systems failed to define modern histopathologic concepts. The Bethesda system also has dramatically increased the number of Papanicolaou smears classified as minimally abnormal. While high-grade squamous intraepithelial lesions (cervical intraepithelial neoplasia 2 and 3) clearly require colposcopic evaluation, the minimally abnormal Pap smear may not always require colposcopic assessment. Common minimally abnormal cytologic categories include atypical squamous cells of undetermined significance, low-grade intraepithelial lesions and atypical glandular cells. New guidelines propose a conservative and expectant approach for these mild abnormalities. Understanding the meaning of these Pap smear reports and instituting appropriate treatments will improve patients care, reduce clinician anxiety and help ensure the best care for patients. PMID- 8629564 TI - Differentiating normal and abnormal findings of the vulva. AB - Proper evaluation of the vulva required background in general dermatology and an understanding of the special nature of many vulvar conditions. A firm understanding of normal vulvar findings is necessary to guard against overdiagnosis and unnecessary treatment. In the past few years, the most common overdiagnosis has been that of micropapillae of the inner labia minora, or acetowhite changes of the vulva, secondary to human papillomavirus (HPV) infection. Differentiating normal variants and minor nonspecific findings from well-developed disease is not always easy. Vulvar biopsy should be performed if a diagnosis cannot be made confidently by visual inspection alone. Vulvar intraepithelial neoplasia is a precancerous lesion of squamous origin associated with HPV infection. It usually occurs in younger patients. Vulvar intraepithelial neoplasia in older women is associated with chronic inflammation, especially in areas of hyperplasia and atrophy. The older patient with vulvar intraepithelial neoplasia has a higher risk of developing invasive vulvar cancer. PMID- 8629565 TI - Evaluation of amenorrhea. AB - Pregnancy is the most common cause of amenorrhea and must be ruled out before proceeding with diagnostic evaluation. A careful history and physical examination may reveal evidence of androgen excess, estrogen deficiency or other endocrinopathies. Serum prolactin and thyroid-stimulating hormone (TSH) levels should be checked in all women who are not pregnant. Galactorrhea by history or on examination and/or an elevated prolactin level should be investigated with an imaging study to rule out a pituitary adenoma. If serum prolactin and TSH levels are normal, a progesterone challenge test should be performed to determine outflow tract patency and estrogen status. In women with hypoestrogenic amenorrhea, indicated by a negative challenge test and a competent outflow tract, serum gonadotropin, follicle-stimulating hormone and luteinizing hormone levels may be measured to determine whether amenorrhea represents ovarian failure or pituitary or hypothalamic dysfunction. Hypothalamic amenorrhea is common in women with a history of weight loss, stress or vigorous exercise. Amenorrheic women with adequate estrogen levels should receive cyclic progesterone. Hormonal therapy and calcium supplementation in hypoestrogenic amenorrhea. PMID- 8629566 TI - Acute otitis media: making an accurate diagnosis. AB - The diagnosis of acute suppurative otitis media is not as easy and straightforward as it may seem. Many of the signs and symptoms in children with acute otitis media are also observed in children without it. Furthermore, several of the "classic" findings of acute otitis media, such as fever and earache, are often absent, even in cases confirmed by myringotomy. An otoscope with a fresh bulb and a good power source, as well as a view of the tympanic membrane that is not obstructed by cerumen, are essential to making the diagnosis of acute otitis media. A bulging, cloudy, immobile tympanic membrane is highly associated with otitis media. Erythema of the eardrum alone, however, is often the result of viral infection, crying or attempts to remove cerumen and should not be the sole basis for the diagnosis of acute otitis media. To avoid the common problem of overdiagnosing acute otitis media, the clinician should consider the predictive values of the various symptoms and physical examination findings associated with ear infections. PMID- 8629567 TI - Halitosis. AB - Although halitosis is generally thought of as a social handicap related to poor oral hygiene or disease of the oral cavity, it may also indicate a serious systemic illness that requires diagnosis and treatment. Halitosis can be the result of pathologic or nonpathologic factors, both local systemic. A complete physical examination, with laboratory and radiographic studies as indicated by the history and physical examination, will generally identify a treatable cause for halitosis. Treatment is directed at the underlying cause. Simple measures, including careful oral hygiene, may be employed to lessen breath odor. PMID- 8629568 TI - The challenge of irritable bowel syndrome. AB - Irritable bowel syndrome is a common disorder with symptom complexes that can include diarrhea, constipation, pain and bloating. After other disorders have been excluded, treatment should be directed at the predominant symptoms and gauged to their severity. A careful balance between a thoughtful investigation and the expense and dangers of overtesting must always be considered. Most patients continue to have persistent symptoms several years after the original diagnosis. PMID- 8629569 TI - Claudication: diagnosis and treatment. AB - Claudication is exercise-induced lower extremity pain that is caused by ischemia and relieved by rest. This underreported condition affects at least 10 percent of persons over 70 years of age and 2 percent of those 37 to 69 years of age. Claudication is usually caused by atherosclerotic narrowing of the arteries that supply blood to the lower extremities. The diagnosis may be suspected based on the history and the physical examination, and it is confirmed by Doppler segmental pressures and an ankle/brachial index. Initial treatment includes vigorous risk factor modification and an exercise program. Further treatment includes pentoxifylline and, occasionally, endovascular or bypass procedures. PMID- 8629570 TI - An approach to diagnostic imaging of suspected pulmonary embolism. AB - Risk factors for pulmonary embolism include immobilization, trauma and surgery, particularly for hip fracture. Patients may present with acute respiratory symptoms, including tachypnea, tachycardia and rales. Chest radiographs and clinical and laboratory findings alone cannot provide a firm diagnosis. A completely normal chest radiograph may be seen in up to 40 percent of patients with pulmonary embolism, and as many as 30 percent of persons with pulmonary embolism and no prior cardiopulmonary disease will have a PaO2 greater than 80 mm Hg. The ventilation/perfusion (V/Q) lung scan is central to guiding clinical decisions. V/Q scans interpreted as either normal, near normal or high probability are reasonably diagnostic. A low probability V/Q scan can exclude the diagnosis of pulmonary embolism only if the patient has a clinically low probability of pulmonary embolism. Intermediate V/Q scans are not diagnostic and call for further evaluation. Compression ultrasonography is sensitive in detecting symptomatic deep venous thrombosis in the thigh. When clinical suspicion remains high and noninvasive imaging studies are uncertain, pulmonary angiography is likely to be diagnostic. PMID- 8629571 TI - Abdominal trauma in the pregnant patient. AB - Physicians who care for pregnant women must be prepared to evaluate and treat abdominal trauma during pregnancy. The evaluation and management of even minimal abdominal trauma in pregnancy is often problematic, and the use of laboratory tests, fetal monitoring, radiography and ultrasonography depends on the extent of the trauma and the viability of the fetus. Intervention ranges from reassurance to cardiopulmonary resuscitation and surgery. Common laboratory studies include a complete blood count, coagulation panel, urinalysis and blood type and screen. Ultrasonography is useful for evaluation of fetal weight and status of amniotic fluid, but tocodynamometry is more sensitive for diagnosis of placental abruption. Patients with minimal trauma and no bleeding, uterine contractions or abdominal pain can be safely discharged after four to six hours of monitoring, but patients with any of these findings should be admitted to the hospital for overnight observation with continuous fetal heart rate monitoring. PMID- 8629572 TI - Treatment options for early Parkinson's disease. AB - Proper treatment of tremor in any clinical setting depends on correct diagnosis. Essential, or familial, tremor is symptomatic with movement and involves the limbs, head and voice. Parkinson's disease tremor occurs at rest, involves the jaw and limbs and is associated with bradykinesia, rigidity and falling. Parkinson's disease is treated with a number of medications, but levodopa, a dopamine precursor, is considered most effective. Other therapies in the early stages of Parkinson's disease may include neuroprotective agents, dopamine agonists, dopamine reuptake inhibitors, anticholinergics and/or amantadine. Polypharmacy is often necessary to minimize or avoid drug side effects. PMID- 8629573 TI - A rational approach to the diagnosis of arthritis. AB - The evaluation of patients with joint pain begins during the medical history and the physical examination, when the differential diagnosis can often be narrowed to a few likely possibilities. Many patients with joint pain have mild, self limited symptoms that are best managed conservatively. Exceptions are patients with acute monoarthritis with effusion, in whom septic arthritis must often be excluded by diagnostic joint aspiration. Patients with evidence of active joint inflammation also require prompt evaluation. Laboratory tests for rheumatologic problems lack the sensitivity and specificity to allow their use as screening tools. Laboratory tests are most valuable when used selectively in patients with a clinically significant likelihood of disease. A single test used to rule in or rule out the most likely joint disease is more helpful than a panel of tests used for every patient with joint pain. Testing should aid in determining prognosis or planning treatment. PMID- 8629574 TI - Behavioral factors in the management of back pain. AB - Most persons with acute back problems recover promptly. Comprehensive evaluation of those who have not significantly improved within two weeks will often reveal significant behavioral factors that may be impeding recovery. These factors may include interpersonal, economic or occupational stress; psychologic disorders, including anxiety, depression or somatization, and counterproductive beliefs about back disorders. Effective management strategies include building a constructive doctor-patient relationship, addressing life stress issues, keeping patients physically active and prescribing psychotropic and/or analgesic medication when appropriate. PMID- 8629575 TI - Minimizing prescribing errors in infants and children. AB - Family physicians frequently prescribe medications for children. The high volume of pediatric prescribing, particularly of antibiotics, means that even a low rate of prescription error would result in adverse consequences for a substantial number of children. Errors may be attributed to factors associated with drug names, packaging or formulation, particularly when a liquid preparation is marketed in several concentrations, as well as to human error in selecting and prescribing medications. A systematic approach that minimizes distractions during the prescribing process, provides a safety-checking mechanism by office staff and encourages the consistent use of standard techniques for calculating pediatric dosages is recommended. In the interest of clarity, prescriptions should be written legibly, abbreviations should be avoided, the indication for the medication should be specified and the times of day and number of times each day the medication is to be taken should be clearly stated. PMID- 8629576 TI - Sports and recreational medicine. AAFP core educational guidelines. PMID- 8629577 TI - American Academy of Family Physicians releases a procedural skills paper on EGD by family physicians. PMID- 8629578 TI - CDC issues recommendations for screening for tuberculosis in high-risk populations. PMID- 8629579 TI - Ganciclovir for cytomegalovirus infection. PMID- 8629580 TI - AAP and ACOG statement on HIV screening. PMID- 8629581 TI - Analysis of microvascular integrity, contractile reserve, and myocardial viability after acute myocardial infarction by dobutamine echocardiography and myocardial contrast echocardiography. AB - The purpose of this study was to evaluate, in postinfarction dysfunctioning myocardium, the relative potential of myocardial contrast and low-dose dobutamine echocardiography in detecting myocardial viability, and the relation between microvascular integrity, contractile reserve, and functional recovery at follow up. Twenty-four patients with recent myocardial infarction were studied before hospital discharge with low-dose dobutamine and myocardial contrast echocardiography. In the dysfunctioning infarct area, wall motion score index was calculated at baseline, during low-dose dobutamine, and at 3-month follow-up. Revascularization of the infarct-related artery was performed if clinically indicated. Eighteen patients (group A) had myocardial enhancement of the dysfunctioning infarct area at myocardial contrast echocardiography of >50%, whereas the remaining patients (group B) had an increase of < or = 50%. Wall motion score index was similar at baseline in groups A and B (2.6 +/- 0.4 and 2.8 +/- 0.2; p = NS), but it improved during low-dose dobutamine and at follow-up only in group A (1.9 +/- 0.9 and 1.9 +/- 0.7, respectively; p <0.001 vs baseline). In group B, wall motion score index was 2.7 +/- 0.4 with low-dose dobutamine and 2.8 +/- 0.2 at follow-up (p = NS vs rest). In identifying viable myocardial segments, myocardial contrast echo had 100% sensitivity and 46% specificity, whereas low-dose dobutamine echo had 71% sensitivity and 88% specificity. Thus, microvascular integrity after acute myocardial infarction is a fundamental prerequisite for ensuring myocardial contractile reserve and regional functional recovery. Myocardial contrast and low-dose dobutamine echocardiography have different, but complementary, diagnostic characteristics in detecting myocardial viability. PMID- 8629582 TI - Acute and long-term effects of thrombolysis after anterior wall acute myocardial infarction with serial assessment of infarct expansion and late ventricular remodeling. AB - This study investigates the impact of thrombolysis on infarct expansion and subsequent left ventricular (LV) remodeling in patients with anterior wall acute myocardial infarction (AMI). We evaluated 51 consecutive patients (24 treated with thrombolysis) with anterior wall AMI by 2-dimensional echocardiography in the following sequence: days 1, 2, 3, and 7, after 3 and 6 weeks, and after 3, 6, and 12 months. LV end-diastolic and end-systolic volume indexes were determined from apical 2- and 4-chamber views using Simpson's biplane formula. Infarct and total LV perimeters were determined in the same views and their ratio expressed as infarct percentage. Infarct expansion was defined as: (1) an increase in infarct percentage and total perimeter >5% on days 2 to 3 in either of the views, or (2) initial infarct percentage >50% with an increase in total perimeter >5% on days 2 to 3. Coronary angiography was performed in 43 patients before discharge, and patency of the infarct-related artery was assessed using Thrombolysis in Myocardial Infarction trial criteria. Infarct expansion was detected in 23 patients. Infarct perimeter steadily decreased in patients with versus without thrombolysis and in patients with patent versus occluded infarct-related arteries. Furthermore, by logistic regression, thrombolysis (p = 0.007) and potency of the infarct-related artery (p = 0.02) were strong negative predictors of expansion, whereas initial infarct perimeter (p = 0.009) was directly associated with subsequent expansion. End-systolic volume index was higher in patients with expansion from day 1 (p = 0.003) through the end of the study (p = 0.021), and end-diastolic volume index was higher in these patients from day 2 (p = 0.012) through 12 months (p = 0.015). Thus thrombolysis, initial infarct size, and infarct-related artery patency are major predictors of infarct expansion after anterior wall AMI. PMID- 8629583 TI - Stent implantation in acute myocardial infarction. AB - Among 138 patients treated with coronary angioplasty during acute myocardial infarction (AMI), 35 (25%) had stent implantation. Mean age was 56 years and 83% were men. Mean onset of chest pain was 6.0 +/- 5.3 hours, and previous thrombolytic therapy was given to 10 patients (29%). Infarct location was anterior in 19 (54%), inferior in 14 (40%), and lateral in 2 patients (6%). Thrombolysis in Myocardial Infarction trial flows 0,1, and 2 were seen in 24 (69%), 6 (17%), and 5 patients (14%), respectively. The culprit vessel was the left anterior descending artery in 18 (51%), right coronary artery in 14 (40%), left circumflex in 2 (6%), and left main coronary artery in 1 patient (3%). Mean vessel diameter was 3.3 +/- 0.3 mm. Indications were: primary in 5 (14%), suboptimal result in 8 (23%), nonocclusive dissection in 14 (40%), and occlusive dissection in 8 patients (23%). Angiographic thrombus after initial angioplasty was present in 12 patients (34%). A total of 46 stents were implanted; mean balloon diameter and pressure were 3.4 +/- 0.4 mm and 15.5 +/- 2.2 atm, respectively. Residual diameter stenosis was 4 +/- 7%. There were 2 deaths; sudden 1, and after elective coronary artery bypass grafting in the other; 2 patients (6%) had groin hematomas. Mean hospitalization was 9.9 +/- 5.0 days. Repeat angiography revealed no stent occlusion. With initial intravenous heparin for 3 to 7 days, all patients received aspirin and ticlopidine for 1 month. Thus, AMI is not a contraindication for stent implantation. The benefits of stenting are a high success rte, low residual diameter stenosis, and low incidence of in hospital recurrent ischemia. Reduction in restenosis rate in this setting is likely but remains to be determined. PMID- 8629584 TI - Usefulness of on-line three-dimensional reconstruction of intracoronary ultrasound for guidance of stent deployment. AB - The additional information provided by automated on-line 3-dimensional (3-D) reconstruction of intracoronary ultrasound (ICUS) was assessed in 42 patients (62 stents) who underwent stent deployment after achieving an optimal quantitative angiographic result. In 10 of 42 patients, 3-D ICUS was also performed before stenting. ICUS images of stents and adjacent reference segments were acquired by using a motorized pullback at a constant speed (1 mm/s) and immediately processed in the catheterization laboratory. Optimal stent expansion was detected by 3-D ICUS in case of complete apposition of stent struts to the vessel wall. Furthermore, an attempt was made to maximize the intrastent lumen area to match lumen area of the reference segment and to cover with stents all the segments with residual significant lesions (plaque burden >50%). Three-dimensional automated reconstruction of ICUS was successful in 8 of 10 patients (80%) before, and in 36 of 42 patients (86%) after stent deployment. In all 8 patients who underwent successful 3-D ICUS assessment before stent implantation, the selection of stent length was facilitated by accurately measuring the lesion length. After stenting, 3-D ICUS modified the management strategy in 21 of 36 patients (58%), triggering additional high-pressure dilatations in 13 patients (36%) and additional stent deployment in 8 (22%). In conclusion, on-line 3-D ICUS facilitates stent selection and strongly modifies the revascularization strategy by accurately detecting stent underexpansion and presence of uncovered lesions. PMID- 8629585 TI - Effect of spinal cord stimulation on myocardial blood flow assessed by positron emission tomography in patients with refractory angina pectoris. AB - Spinal cord stimulation in angina pectoris increases exercise capacity and reduces both anginal attacks and ischemic electrocardiographic signs. This suggests an anti-ischemic action, perhaps through changes in myocardial blood flow. In 9 patients, regional myocardial blood flow was studied with positron emission tomography before and after 6 weeks of spinal cord stimulation, both at rest and during a dipyridamole stress test. Frequency of anginal attacks and consumption of short-acting nitrates were assessed by patient diaries. Exercise duration and time to angina were measured with treadmill exercise tests. After 6 weeks of stimulation, both frequency of daily anginal attacks and nitrogen consumption decreased (3.7 +/- 1.7 vs 1.4 +/- 1.0 [p <0.01] and 2.8 +/- 2.2 vs 1.1 +/- 1.2 tablets [p = 0.01], respectively); exercise duration and time to angina increased (358 +/- 165 vs 493 +/- 225 seconds [p <0.01] and 215 +/- 115 vs 349 +/- 213 seconds [p = 0.02], respectively); and ST-segment depression during dipyridamole stress testing was reduced (0.17 [0 to 0.5] mV vs 0.09 [0 to 0.2] mV, p = 0.04) (all data mean +/- SD). Total resting blood flow remained unchanged (115 +/- 29 vs 127 +/- 31 ml/min/100 g, p = 0.31), but flow reserve decreased (146 +/- 43% vs 122 +/- 39%, p = 0.04). The coefficient of variation of flow, representing flow heterogeneity, decreased after treatment, both at rest (20.1 +/ 3.8% vs 17.4 +/- 2.6%, p = 0.04) and after dipyridamole stress (26.2 +/- 4.4% vs 22.9 +/- 5.5%, p = 0.02). Thus, spinal cord stimulation is clinically effective due to homogenization of myocardial blood flow. Since flow reserve decreases despite clinical improvement, the dipyridamole effect may be blunted by spinal cord stimulation. PMID- 8629586 TI - Ultrastructural characteristics of human atherectomy tissue from coronary and lower extremity arterial stenoses. AB - In animal studies, smooth muscle cell phenotype conversion has been suggested to be an essential prerequisite for subsequent migratory and proliferative events leading to (neo)intima formation. To determine ultrastructural characteristics of individual smooth muscle cells and to relate them to specific lesion types and intimal cell density, we compared atherectomy samples from 17 restenotic and 32 primary coronary and peripheral lesions using transmission electron microscopy and histology. Ultrastructural analysis of cell-rich tissue, predominantly of restenotic origin, revealed smooth muscle cells full of synthetic organelles. Moreover, these cells were frequently found to be surrounded by loose extracellular matrix and partially fragmented basement membrane components. In contrast, plaques exhibiting low cell density, as exclusively seen with primary lesions, displayed an extensive buildup of extracellular matrix containing sparse numbers of microfilament-rich smooth muscle cells. The central finding of our study is a morphometrically quantitated, twofold greater (p <0.001) volume fraction of synthetic organelles (VS) within smooth muscle cells in restenotic versus primary plaques, indicating a more dedifferentiated cellular phenotype as a typical feature of restenotic lesions. Equally enhanced VS values were seen for restenotic coronary and peripheral plaques. No VS decrease was observed during time after angioplasty (2.2 to 30 months) regardless of previous revascularization procedures (balloon angioplasty or atherectomy). Despite intra- and interlesional variability, VS and intimal cell density were strongly correlated (r = 0.74; p <0.001). This correlation was observed more often with clinical restenoses and, importantly, in a portion (10% to 15%) of primary lesions. Data from restenotic lesions indicate that a dedifferentiated smooth muscle cell phenotype, pericellular matrix disintegration, and intimal hypercellularity are long-lasting biologic responses to previous smooth muscle cell injury. Similar tissue characteristics expressed in several primary lesions suggest that comparable pathogenic mechanisms are related to the progression and/or acuity of chronic lesions. PMID- 8629587 TI - Comparison of the effects on quality of life and of the efficacy and tolerability of lovastatin versus pravastatin. The Quality of Life Multicenter Group. AB - This multicenter study compared the effects of lovastatin (40 mg) and pravastatin (40 mg) on quality of life. Men, aged 20 to 65 years old, with primary hypercholesterolemia (types IIa and IIb) were eligible for enrollment if baseline low-density lipoprotein cholesterol met the first National Cholesterol Education Program adult treatment guidelines. Eligible patients were enrolled into a 6-week diet baseline period, followed by a 6-week diet-plus-placebo period. Patients whose low-density lipoprotein cholesterol still met National Cholesterol Education Program guidelines for drug therapy were randomized into the double blind, active-treatment period to receive either lovastatin 40 mg or pravastatin 40 mg/day for 12 weeks. Clinic visits were scheduled every 4 weeks and included complete serum lipid profiles, monitoring of adverse experiences, and patient completion of health-related quality-of-life questionnaires. The primary end point of the study was the change in quality of life, as measured by the Nottingham Health Profile (part 1) after 12 weeks of treatment. Secondary end points included responses to a general health question from the National Health and Nutrition Examination Survey, sexual function questions from the Medical Outcomes Study, and stress/life event questions from the National Institutes of Health Post-Coronary Artery Bypass Grafting Study. No significant differences between the 2 groups were observed in tolerability, health-related quality-of life measures, or changes in lipid profiles. PMID- 8629588 TI - Influence of digitalis on left ventricular functional response to exercise in congestive heart failure. AB - This was a double-blind, placebo-controlled, crossover study designed to determine the influence of digitalis treatment on left ventricular (LV) response to physical exercise in patients with congestive heart failure (CHF). In 10 patients with CHF (ejection fraction 29 +/- 2%), LV function was assessed during upright bicycle exercise using an ambulatory radionuclide detector for continuous noninvasive monitoring of cardiac function. Exercise was performed during control conditions and after a 3-week treatment with digoxin (0.25 mg/day orally) or placebo. Ten normal volunteers matched for sex and age constituted the control group. In normals, exercise ejection fraction and end-diastolic volume increased (both p <0.001), while end-systolic volume decreased progressively (p <0.001). In control conditions, patients with CHF had a sharp increase in heart rate during exercise, while ejection fraction did not change; both end-diastolic and end systolic volumes increased significantly (both p <0.001) during exercise. During digoxin treatment, heart rate response to exercise recorded in patients with CHF was comparable to that recorded in normal subjects. In addition, a significant increase in ejection fraction during exercise was detected (P <0.001), and the increase in end-systolic volume was significantly smaller than that observed in control conditions (p <0.05). When patients received placebo, the responses of LV function to exercise were comparable to those observed in control conditions. These findings demonstrate that digitalis has a favorable influence on LV functional adaptation to exercise in CHF. PMID- 8629589 TI - Combined alpha-beta blockade (doxazosin plus metoprolol) compared with beta blockade alone in chronic congestive heart failure. AB - There has been growing evidence for the benefits of beta blockers, but alpha blockers have not shown sustained benefits in chronic congestive heart failure (CHF). Thirty patients with moderate to severe CHF (New York Heart Association class II to IV) were sequentially assigned to receive metoprolol 6.25 mg with the alpha-1 antagonist doxazosin 4 mg/day or metoprolol alone. The dose of metoprolol was gradually increased to a target dose of 50 mg orally twice daily. Hemodynamic measurements were obtained before drug therapy, 2 hours after the first dose of combined alpha-beta therapy or metoprolol alone, and after 3 months of continuous treatment. Nuclear ejection fraction, plasma norepinephrine, and submaximal and maximal exercise capacity were also measured before and after chronic therapy. With initial combined drug administration, mean arterial pressure, left ventricular filling pressure, and systemic vascular resistance decreased significantly compared with results after metoprolol alone. However, after 3 months of continuous therapy, both treatment groups showed similar and significant reductions in systemic vascular resistance and heart rate, with significant increases in cardiac index, stroke volume index, stroke work index, ejection fraction, and exercise capacity. Furthermore, the next dose of chronic combined medication no longer showed vasodilating effects. Chronic therapy with fixed-dose doxazosin and increasing doses of metoprolol produced identical effects as those seen in patients receiving metoprolol alone. PMID- 8629590 TI - Delayed improvement in skeletal muscle metabolism and exercise capacity in patients with mitral stenosis following immediate hemodynamic amelioration by percutaneous transvenous mitral commissurotomy. AB - The abrupt improvement in hemodynamics after successful percutaneous transvenous mitral commissurotomy (PTMC) does not immediately enhance exercise capacity. Improved exercise capacity several months after PTMC has been reported. We hypothesized that the delayed improvement in exercise capacity is due partly to the slow improvement in the metabolism of skeletal muscle. This study examined the short- and long-term effects of PTMC on exercise capacity and skeletal muscle metabolism in patients with mitral stenosis. Treadmill exercise testing with respiratory gas analysis was performed in 11 patients with symptomatic mitral stenosis before and 3, 30, and 90 days after successful PTMC. On the same schedule, forearm metabolism of high-energy phosphates was measured by magnetic resonance spectroscopy during and after handgrip exercise. Ten healthy volunteers were examined. PTMC resulted in an immediate symptomatic improvement. However, exercise capacity and skeletal muscle metabolism remained unchanged 3 days after PTMC. At 30 days after PTMC, there were significant improvements in peak oxygen consumption (p <0.05), intracellular pH at end-exercise (p <0.05), and time constant for phosphocreatine recovery (mean +/- SD 88.9 +/- 11.3 vs 106.3 +/- 11.7 seconds, p <0.01) compared with these baseline values. These improvements remained even at 90 days after PTMC. Exercise capacity improved with some time delay after immediate hemodynamic amelioration by PTMC. Long-term improvement in exercise capacity depends partly on the slowly progressing improvement in skeletal muscle metabolism after long-standing mitral stenosis. PMID- 8629591 TI - Effects of dual-chamber pacing in hypertrophic cardiomyopathy on left ventricular outflow tract obstruction and on diastolic function. AB - Hypertrophic cardiomyopathy (HC) is characterized by impaired diastolic function and, in about 1/4 of patients, left ventricular (LV) outflow tract obstruction. Atrioventricular (AV) pacing diminishes LV outflow tract gradient in HC, but impairs diastolic function in the experimental animal and in different categories of patients. To investigate the effects of AV pacing on hemodynamics and LV function in obstructive HC, 16 patients with HC were studied by cardiac catheterization and simultaneous radionuclide angiography during atrial and AV pacing. The resting LV outflow tract gradient decreased with AV pacing from 60 +/ 34 to 38 +/- 37 mm Hg (mean +/- SD; p <0.001). Regional ejection fraction decreased significantly at the septal level from 0.81 +/- 0.21% to 0.69 +/- 0.27% (p <0.01). Pulmonary artery wedge pressure increased from 10 +/- 5 to 15 +/- 6 mm Hg (p <0.001). AV pacing induced asynchrony (i.e., the coefficient of variation of the time to end-systole increased from 7 +/- 4% to 14 +/- 10% (p <0.01). The time constant of isovolumetric relaxation (t) increased from 58 +/- 24 to 74 +/- 33 ms (p <0.02), and peak filling rate decreased from 491 +/- 221 to 416 +/- 184 ml/s (p <0.05). Thus, AV pacing greatly diminishes resting obstruction through a reduction in septal ejection fraction (i.e., an increase in LV outflow tract width in systole), but impairs active diastolic function and increases filling pressures. These latter effects are potentially detrimental in patients with HC in whom diastolic dysfunction is present. PMID- 8629592 TI - Associated cardiac anomalies in isolated and syndromic patients with tetralogy of Fallot. AB - To detect in children with tetralogy of Fallot (ToF) the prevalence of associated cardiac anomalies in syndromic and isolated cases, the additional cardiac defects of 150 consecutive patients with ToF (102 isolated and 48 syndromic cases) were evaluated by review of echocardiographic, angiocardiographic, and surgical reports. Syndromic patients were classified into groups with branchial arch defects, Down syndrome, and other genetic conditions. ToF is significantly associated with additional cardiac malformations in patients with branchial arch (11 of 21, p <0.01) and Down (10 of 20, p <0.0001) syndromes. The subarterial ventricular septal defect with deficiency of the infundibular septum (4 of 21, p <0.01) and the right aortic arch (6 of 21, p <0.05) were prevalent in patients with branchial arch syndromes, whereas atrioventricular canal (10 of 20, p <0.0001) was associated with ToF in patients with Down syndrome. Peculiar anatomic cardiac patterns are present in children with ToF and may alert the cardiologist to look at additional cardiac anomalies. Moreover, the presence of some associated cardiac anomalies may suggest careful clinical evaluation for genetic syndromes. PMID- 8629593 TI - Left ventricular filling pattern in uncomplicated obesity. AB - To determine if uncomplicated obesity is associated with systolic dysfunction or impairment of left ventricular (LV) filling, 40 normotensive, white, asymptomatic, obese subjects (16 men and 24 women, mean +/- SD age 35 +/- 13 years; body mass index 36 +/- 6 kg/m2) and 40 normotensive, normal-weight, white volunteers matched for age and sex distribution, were studied by Doppler echocardiography. Endocardial and midwall shortening did not show differences between groups (obese = 33 +/- 4% and 17 +/- 2%; normal weight = 33 +/- 3% and 18 +/- 2%, respectively). LV mass index was higher in obese than in normal-weight subjects (p <0.0001). Obese persons had prolonged isovolumic relaxation time (p <0.0001), lower transmitral peak early diastolic filling wave (E) velocity (p <0.02), higher E velocity deceleration time (p <0.002) and lower E/atrial diastolic filling wave (A) flow velocity ratio (p <0.01) than did normal-weight subjects, even after controlling for age and blood pressure. Between-group differences in E and E velocity deceleration time disappeared when controlling for LV mass index, whereas prolonged isovolumic relaxation time in obesity was independent of LV mass, chamber dimension, and end-systolic stress. LV filling variables were not statistically related to endocardial or midwall shortening, both as absolute value or as a percentage of that predicted from wall stress. We conclude that uncomplicated obesity is associated with primary impairment of LV isovolumic relaxation; abnormalities of early passive filling flow in obesity are associated with increased LV mass. PMID- 8629594 TI - Dissociation between wall thickening of normal myocardium and cyclic variation of backscatter during inotropic stimulation. AB - The purpose of this study was to determine the relation between increased myocardial wall thickening during inotropic stimulation and quantitative acoustic properties of normal myocardium in humans. We first validated a new 2-dimensional ultrasonic backscatter imaging approach for measurement of cyclic variation in the parasternal long-axis view against conventional M-mode integrated backscatter technique in 41 patients and controls (group A). We then performed 2-dimensional ultrasonic integrated backscatter imaging in 18 patients (group B) with normal segmental function at baseline to determine the magnitude of the cyclic variation of the septum and the posterior wall before and during infusion of dobutamine (10 and 20 microgram/kg/min). Group A patients showed a close correlation of the cyclic variation obtained by the new 2-dimensional ultrasonic integrated backscatter imaging approach and the conventional M-mode technique. Group B patients had mean values of cyclic variation that remained unchanged in the septum (4.4 +/- 1.4, 4.3 +/- 1.7, and 4.8 +/- 1.6 dB at baseline and at each dobutamine stage, respectively, p = NS) and in the posterior wall (6.4 +/- 1.7, 6.4 +/- 1.8 and 6.1 +/- 1.9 dB, respectively, p = NS) despite progressive dobutamine-induced increases in percent wall thickening (septum: 38 +/- 10% to 57 +/- 17% and 68 +/- 19%, respectively, and posterior wall 42 +/- 13% to 72 +/- 20% and 77 +/- 18%, respectively; both p <0.001 vs baseline for both walls). Thus, physical properties of normal myocardium remain unchanged during inotropic stimulation. PMID- 8629595 TI - Can serial tilt testing be used to evaluate therapy in neurally mediated syncope? AB - The role of serial head-up tilt testing for the evaluation of therapeutic efficacy of neurally mediated syncope is reviewed. The evidence available suggests that guiding therapy based on serial head-up tilt response may not be appropriate, and large placebo-controlled trials should be conducted to address this issue. PMID- 8629596 TI - Five-year experience with implantable defibrillators in children. AB - Cardioverter-defibrillators were implanted in children aged 4 to 16 years over a 5-year period with no mortality and eventual clinically appropriate shocks in 6 of 11 patients. Both transvenous and epicardial implantable cardioverter defibrillators were safe and effective in children resuscitated from sudden death or at high risk for sudden death. PMID- 8629597 TI - Effect of biphasic endocardial countershock on pacing thresholds in humans. AB - The use of non-thoracotomy endocardial implantable defibrillators with pacing capabilities has increased substantially over the past 2 years. This report demonstrates that the pacing threshold increases in some patients after endocardial defibrillation, and substantiates the practice of using maximal pacing output after endocardial defibrillation. PMID- 8629599 TI - The PAPERCUFF, a new disposable blood pressure cuff. AB - We demonstrated the accuracy of a new disposable BP cuff (CAS PAPERCUFF) compared with 2 widely used cuffs. The PAPERCUFF should limit the possibility of spread of infection and reduce costs. PMID- 8629598 TI - Effects of aspirin on status of thrombin generation in atrial fibrillation. AB - Aspirin 330/mg/day suppressed platelet function in patients with atrial fibrillation, although it did not affect the increased coagulation activity in these patients. Findings support the premise that anticoagulant therapy is more appropriate than aspirin for the prevention of systemic embolism in patients with atrial fibrillation. PMID- 8629600 TI - Inhaled nitric oxide to test the vasodilator capacity of the pulmonary vascular bed in children with long-standing pulmonary hypertension and congenital heart disease. AB - Nitric oxide-induced vasodilator capacity greatly varies among children with pulmonary hypertension and elevated vascular resistance. The decline of this selective response seems to parallel the progression of established vascular disease and thus may be helpful for the selection of patients for operation. PMID- 8629601 TI - Echocardiographic diagnosis of single coronary artery in double-outlet right ventricle. AB - We report the echocardiographic diagnosis of the combination single coronary artery and double-outlet right ventricle, initially recognizing this coronary artery variation by imaging in the subcostal coronal axis. This combination occurs in approximately 11% of patients with double-outlet right ventricle and raises important issues regarding coronary artery transfer in an arterial switch repair. PMID- 8629603 TI - Survival into adulthood of patients with unoperated single ventricle. AB - Adult patients with double-inlet left ventricle and perfectly balanced circulation may survive into the sixth decade with good functional capacity and preserved ventricular function. This should be considered before such patients are referred for a Fontan repair. PMID- 8629602 TI - Intraoperative transesophageal echocardiography using high-resolution biplane 7.5 MHz probes with continuous-wave Doppler capability in infants and children with tetralogy of Fallot. AB - This study reports the role of biplane transesophageal echocardiography in monitoring surgical repairs of tetralogy of Fallot. In our patients, 3 repairs were revised based on transesophageal echocardiography and continuous-wave Doppler results, and intraoperative management was altered in 2 others. PMID- 8629604 TI - Effect of vitamin E on the anticoagulant response to warfarin. AB - To clarify whether vitamin E enhances the pharmacologic effect of warfarin, we completed a double-blind clinical trial in which 21 subjects taking chronic warfarin therapy were randomized to receive either vitamin E or placebo. None of the subjects who received vitamin E had a significant change in the international normalized ratio, and thus it appears that vitamin E can safely be given to patients who require chronic warfarin therapy. PMID- 8629605 TI - Association of systemic to pulmonary collateral arteries with tetralogy of Fallot and absent pulmonary valve syndrome. AB - Two patients with tetralogy of Fallot and absent pulmonary valve syndrome with significant systemic to pulmonary collateral arteries are described. Search for these rarely reported vessels should be included in the workup of these complex patients. PMID- 8629606 TI - Cigarette smoking as a predictor of death from prostate cancer in 348,874 men screened for the Multiple Risk Factor Intervention Trial. AB - The association of cigarette smoking and mortality from prostate cancer was evaluated in 348,874 black and white men who were screened as part of the Multiple Risk Factor Intervention Trial (MRFIT). Current smoking status was assessed, serum cholesterol was measured, and demographics were recorded at screening; however, no information was collected on history of smoking, prostate screening, or diet. The vital status of each member of this cohort was ascertained through 1990. Death certificates were obtained from state health departments and coded by a trained nosologist. A total of 826 deaths due to prostate cancer occurred over an average of 16 years follow-up. The proportional hazards model was used to study the joint association of age, race, income, cigarette smoking, serum cholesterol level, and use of medication for diabetes mellitus on risk of death from prostate cancer. Statistically significant associations were observed with age (p < 0.01), cigarette smoking status (relative risk (RR) = 1.31, p < 0.01), black race (RR = 2.70, p < 0.01), and serum cholesterol (RR = 1.02 for 10 mg/dl higher cholesterol level, p < 0.05). Similar results were obtained when deaths that occurred during the first 5 years were excluded. Among cigarette smokers, there was some evidence of a dose response relation (p = 0.20). The relative risk for those who reported that they smoked 1-25 cigarettes per day compared with nonsmokers was 1.21 (p = 0.04); the relative risk for those who reported smoking > or = 26 cigarettes per day compared with nonsmokers was 1.45 (p = 0.0003). These findings add to the limited evidence that cigarette smoking may be a risk factor for prostate cancer. PMID- 8629607 TI - Determinants of mortality from cystic fibrosis in Canada, 1970-1989. AB - The frequency, prevalence, and mortality patterns of cystic fibrosis were analyzed in 3,795 patients documented in the Canadian Patient Data Registry in 1970-1989. Cystic fibrosis frequency in the 1970-1979 birth cohort was virtually identical to the commonly quoted 1 in 2,500. In 1985-1989, median survival age was 36.7 years for males and 27.8 years for females, compared with 26.6 and 19.7, respectively, in 1970-1974. However, there were significant regional differences when Canada was divided into the four regions, East, Quebec, Ontario, and West. In Quebec, patients were younger at diagnosis and until recently had greater mortality than patients in other regions, which suggests more severe disease; dramatically improved survival in the 1980s coincided with a change from a restricted fat diet to a high fat diet. Improved survival in Ontario in the 1970s accompanied this change in dietary therapy, which may also account for good survival throughout the study period in the East. The West showed gradually improving survival, similar to that reported in other parts of the world. Proportional hazards analysis showed pulmonary function to be the best predictor of survival. Poorer survival in females was associated with poorer weight, but the interrelation of declining pulmonary function, weight maintenance, sex, and mortality requires further study. The effect of pulmonary colonization with Pseudomonas aeruginosa was confounded with degree of pulmonary dysfunction, but colonization with Burkholderia cepacia (previously Pseudomonas cepacia) was associated with increased mortality at all levels of pulmonary function. PMID- 8629608 TI - Prevalence of diabetes mellitus in New Caledonia: ethnic and urban-rural differences. CALDIA Study Group. CALedonia DIAbetes Mellitus Study. AB - The prevalence of diabetes mellitus was assessed in the multiracial population of New Caledonia in the South Pacific with the use of a two-step procedure. The first step included 9,390 subjects aged 30-59 years who were visited at home for screening with glucose strips. All subjects who had a fasting capillary blood glucose value > or = 110 mg/dl were invited to come to the health center for a more detailed examination. In non-fasting subjects, a capillary blood glucose value of 140 mg/dl was chosen as the cut-off point for further examination. A 2 hour glucose tolerance test was performed according to World Health Organization (WHO) recommendations on 424 positive screenees as well as in a sample of 517 negative screenees. In all, 219 previously known diabetic subjects were found; among subjects classified as positive at the first step, 277 were detected as new cases or diabetes. After correction to take into account false negative subjects, the age-adjusted prevalence rate of diabetes was estimated to be 8.9% overall; Polynesians (15.3%) were found to be more at risk than Melanesians or Europeans (8.4% in two other groups). A higher prevalence of diabetes was found in Melanesians who lived in the urban area compared with Melanesians who lived in villages. This finding confirms the deleterious effect of western life in this population subject to rapid modernization. PMID- 8629609 TI - Relations of changes in coronary disease rates and changes in risk factor levels: methodological issues and a practical example. AB - One of the main hypotheses of the World Health Organization (WHO) MONICA Project is that trends in the major coronary disease risk factors are related to trends in rates of fatal and non-fatal coronary disease events. The units of study are populations rather than individuals. The WHO MONICA Project involves continuous monitoring of all coronary disease events in the populations over a 10-year period and periodic risk factor surveys in random samples of the same populations. Estimation of associations between average annual changes in mortality and risk factor levels is illustrated with the use of data from a subset of MONICA centers. Crude estimates of regression coefficients are compared with estimates obtained by weighting for standard errors in both the outcome and explanatory variables. The results show that the strength of association may be either underestimated or overestimated if these errors are not taken into account. PMID- 8629610 TI - No long-term excess mortality after measles infection: a community study from Senegal. AB - Because measles immunization has been found in all studies to reduce mortality with more than the share of deaths attributed to acute measles, the authors examined mortality after measles infection in a study in a rural area of Senegal that included 6,924 unimmunized children, whom 1,118 developed measles. Age adjusted post-measles mortality was similar to the mortality of unvaccinated, uninfected children (mortality ratio (MR) = 1.04, 95% confidence interval (CI) 0.80-1.35). When controlling for source of infection, mortality rate was significantly different for children who contracted measles from a person outside the home (index cases vs. unvaccinated, uninfected MR = 0.27, 95% CI 0.09-0.85) and for children infected at home (secondary cases vs. unvaccinated, uninfected MR = 1.10, 95% CI 0.80-1.51). Hence, secondary cases had markedly higher long term mortality than did index cases (MR = 4.13, 95% CI 1.26-13.58). These estimates were essentially unchanged when the effects of season, period, separation from mother, size of community, and size of compound were investigated using a multivariate Cox regression model. The authors conclude that measles infection was not associated with increased mortality after the acute phase of infection and that index cases had lower mortality than uninfected, unvaccinated children. The reduction in mortality after measles immunization can therefore not be explained by the prevention of post-measles mortality. PMID- 8629611 TI - Impact of institution size, staffing patterns, and infection control practices on communicable disease outbreaks in New York State nursing homes. AB - Institutional risk factors associated with the occurrence of nosocomial respiratory or gastrointestinal disease outbreaks in 1992 were examined in a case cohort study of New York State nursing homes conducted in 1993. Facility size, staffing patterns, and employee sick leave policies were the principal effects found in an unconditional logistic regression model. The risk of having respiratory or gastrointestinal disease outbreaks was greater in larger nursing homes (adjusted risk ratio (RR) = 1.71 for each 100-bed increase in size, 95% confidence interval (CI) 1.20-2.42), for nursing homes with a single nursing unit (adjusted RR = 3.93, 95% CI 0.98-15.71), or those with multiple nursing units with shared staff (adjusted RR = 2.51, 95% CI 1.07-5.89). The risk was less for nursing homes with paid employee sick leave policies (adjusted RR = 0.38, 95% CI 0.15-0.99). Other potential risk factors examined in this study, such as the ratio of beds per unit, type of sponsorship, daily review of laboratory test results, and the proportion of private beds and patient-to-staff ratio, were not significantly associated with the risk of disease outbreaks. The results of this study have direct implications for control of nosocomial disease outbreaks in nursing homes. PMID- 8629612 TI - Sex differences in work-related injury rates among electric utility workers. AB - Few epidemiologic studies have evaluated sex differences in work-related injury rates. In this study, the authors examined injury trends by type of injury, severity of injury, and how the injury occurred among a cohort of 9,582 female and 26,898 male electric utility workers employed during 1980-1992 by the Southern California Edison Company. Sex-specific unadjusted injury rates were higher throughout the period for male workers. However, after adjustment for occupation, job experience, and age, elevated rate ratios indicate that female workers have higher injury rates. Mantel-Haenszel summary rate ratios and 95% confidence intervals were 1.49 (1.43-1.54) for all types of injuries, 1.27 (1.16 1.39) for head and neck injuries, 1.48 (1.38-1.58) for upper extremity injuries, 1.11 (1.01-1.21) for back injuries, and 2.11 (1.97-2.25) for lower extremity injuries. The rate ratios were slightly higher for more severe injuries, which suggests that potential reporting bias was not a likely explanation for these findings. The authors conclude that differences between male and female workers in training, physical capacity, task assignments, and other factors could explain these injury trends. PMID- 8629613 TI - Adjusting survival curves for confounders: a review and a new method. AB - When reporting results from survival analysis, investigators often present crude Kaplan-Meier survival curves and adjusted relative hazards from the Cox proportional hazards model. Occasionally, the investigators will also provide a graphical representation of adjusted survival curves based on regression estimates and the average covariate values in the study groups. In this paper, the authors review the limitations of this approach and examine alternative approaches to obtaining adjusted survival curves that have been proposed. Furthermore, a new method to obtain multivariate adjusted survival curves is described. This method is based on direct adjustment of the observed conditional probability of survival at the time of each event. When an unexposed group is used as a standard for adjusting an exposed group, the survival curve in the exposed group is adjusted to the covariate distribution among the unexposed at the time of the event. This method has the advantage over the average covariate method of allowing for the possibility that the adjusted survival curves differ in shape. The method can handle multiple fixed or time-dependent categorical covariates as well as left truncated data, and it allows for estimation of confidence intervals. The authors have written a macro in SAS language that produces the adjusted survival estimates and graphs. This macro is available on request and can be downloaded through the World Wide Web. PMID- 8629614 TI - Mismeasurement and the resonance of strong confounders: uncorrelated errors. AB - Greenland first documented (Am J Epidemiol 1980; 112:564-9) that error in the measurement of a confounder could resonate--that it could bias estimates of other study variables, and that the bias could persist even with statistical adjustment for the confounder as measured. An important question is raised by this finding: can such bias be more than trivial within the bounds of realistic data configurations? The authors examine several situations involving dichotomous and continuous data in which a confounder and a null variable are measured with error, and they assess the extent of resultant bias in estimates of the effect of the null variable. They show that, with continuous variables, measurement error amounting to 40% of observed variance in the confounder could cause the observed impact of the null study variable to appear to alter risk by as much as 30%. Similarly, they show, with dichotomous independent variables, that 15% measurement error in the form of misclassification could lead the null study variable to appear to alter risk by as much as 50%. Such bias would result only from strong confounding. Measurement error would obscure the evidence that strong confounding is a likely problem. These results support the need for every epidemiologic inquiry to include evaluations of measurement error in each variable considered. PMID- 8629615 TI - Prior to use of estrogen replacement therapy, are users healthier than nonusers? AB - Observational studies have demonstrated that women who have used postmenopausal estrogen replacement therapy (ERT) are at reduced risk of coronary heart disease. The authors examined whether premenopausal women who subsequently elected to use ERT during menopause had a better cardiovascular risk factor profile prior to use than did nonusers. A total of 541 premenopausal women had their cardiovascular risk factors and psychosocial characteristics evaluated at study entry. After approximately 8 years, 355 women had become postmenopausal, and 157 women reported ERT use during the follow-up period (mean = 93.4 months). The authors compared the premenopausal characteristics of users with those of nonusers. Relative to nonusers, ERT users were better educated (63 vs. 81% with at least some college), and prior to the use of ERT had higher levels of high density lipoprotein (HDL) cholesterol (1.49 vs. 1.59 mmol/liter), HDL2 (0.50 vs. 0.57 mmol/liter), HDL3 (0.98 vs. 1.02 mmol/liter), leisure physical activity (5, 122 vs. 7,158 Kjoules), and alcohol intake (7.5 vs. 9.7 g/day), and lower levels of apolipoprotein B (0.97 vs. 0.90g/liter), systolic blood pressure (112.1 vs. 107.1 mmHg) and diastolic blood pressure (73.8 vs. 71.4 mmHg), weight (68.5 vs. 64.2 kg), and fasting insulin (9.10 vs. 7.66 microU/liter). Prior to use of ERT, in comparison with nonusers, subsequent users reported on standardized questionnaires that they often exhibited Type A behavior, more aware of their feelings, motives, and symptoms, and had more symptoms of stress. Women who elect to use ERT have a better cardiovascular risk factor profile prior to the use of ERT than do women who subsequently do not use this treatment during the menopause, which supports the hypothesis that part of the apparent benefit associated with the use of ERT is due to preexisting characteristics of women who use ERT. This study underscores the widely recognized importance of randomized clinical trials to estimate the direct benefit of postmenopausal ERT for protecting women from cardiovascular disease. PMID- 8629616 TI - Invited commentary: can selection bias explain the cardiovascular benefits of estrogen replacement therapy? PMID- 8629617 TI - Prospective study of relative weight and risk of breast cancer: the Breast Cancer Detection Demonstration Project follow-up study, 1979 to 1987-1989. AB - Despite extensive research on obesity and breast cancer in recent decades, inconsistencies in the literature exist. The authors examined prospectively the relation between adult relative weight (weight (kg)/height (m)1.5) and breast cancer risk in a cohort of 54,896 women aged 31-89 years who had previously participated in the Breast Cancer Detection Demonstration Project. During a mean follow-up period of 7 years, 226 of the premenopausal women and 1,198 of the postmenopausal women developed breast cancer. Analysis was performed using Cox proportional hazards regression methods with age as the underlying time variable and adjusted for the effects of potential confounders. Among postmenopausal women, the risk of breast cancer increased with increasing relative weight (p < 0.05 for trend); relative risk for the highest compared with the lowest quintile for relative weight was 1.3 (95% confidence interval (CI) 1.1-1.6). This association was modified by age at diagnosis, with relative risks of 1.1 (95% CI 0.8-1.4), 1.2 (95% CI 0.8-1.7), and 1.8 (95% CI 1.3-2.5), respectively, for women aged < 60, 60-64, and > or = 65 years. The higher risk of breast cancer among the older and overweight women was largely confined to women whose weights were measured during the postmenopausal but not the premenopausal period. This risk pattern was observed among the naturally menopausal women, but was also apparent in the smaller group of women with bilateral oophorectomy or hysterectomy with one ovary retained. Among premenopausal women, adult relative weight was not associated with breast cancer risk. These findings suggest that the inconsistencies in the literature on obesity and breast cancer may be due in part to the differing age distributions of the populations studied. The authors conclude that prevention of obesity throughout adulthood, particularly after menopause, may help reduce breast cancer among older women. PMID- 8629618 TI - Gravid health status, medication use, and risk of neuroblastoma. AB - The epidemiology of neuroblastoma suggests that prenatal exposures may be important etiologic factors in this disease. The authors describe the role of maternal health status and prenatal medication usage and risk of neuroblastoma. This retrospective study was based on completed interviews with 183 histologically confirmed neuroblastoma cases aged 0-14 years diagnosed among residents of New York State (excluding New York City) between 1976 and 1987. Controls were matched to cases on year of birth and race and were drawn from the New York State live birth certificate registry. Interviews were satisfactorily completed with 85% of the cases and 87% of controls. Significantly elevated odds ratios were noted for vaginal infections during pregnancy (odds ratio (OR) = 2.2, 95% confidence interval (CI) 1.2-4.0), medical treatments for vaginal infection during pregnancy (OR = 2.4, 95% CI 1.2-4.9), and any reported use of sex hormones during pregnancy (OR = 3.0, 95% CI 1.3-6.9). Point estimates for any hormone use suggested elevated risk among male offspring (OR = 4.4, 95% CI 1.5-13.3). Among the individual exposures comprising any hormone use, only hormone use related to infertility was observed to be significant (OR = 10.4, 95% CI 1.2-89.9). A protective effect was noted for self-reported vitamin use (OR = 0.28, 95% CI 0.03 0.69). Although it is not possible to presume a specific role for prenatal hormone exposure as initiator or promoter, these findings lend support to an association between prenatal hormone exposure and risk of neuroblastoma. PMID- 8629619 TI - Treatment of hepatic failure--1996: current concepts and progress toward liver dialysis. AB - Liver failure, especially in its acute form, is a medical emergency that quickly leads to failure of multiple other organs. Many of these end-organ failures can be supported temporarily by drugs or medical devices, but the support is invariably short-lived if liver function is not restored. In most instances, liver function can only be restored by transplantation, although patients with acute disease have the potential to recover by regeneration ("spontaneous recovery"). Unfortunately, spontaneous recovery from acute liver failure is uncommon, so the two most important aspects of patient management are highly skilled intensive care and early recognition of patients in need of liver transplantation. Even under these circumstances, the mortality of liver failure remains high because we have no easy way of replacing liver function on demand and donor organs are becoming increasingly difficult to obtain in time. The development of techniques for liver assist offer the possibility that patients with liver failure will become a simple management problem, analogous to the options available in the treatment of acute and chronic renal failure. PMID- 8629620 TI - Serologic study of immunoglobulin A-fibronectin aggregates in immunoglobulin A nephropathy. AB - The immunoglobulin A (IgA)-fibronectin aggregates, detected by enzyme-linked immunosorbent assay using either antifibronectin or collagen I as binding protein, were previously found to be raised in the circulation of patients with IgA nephropathy (IgAN). It has been suggested that IgA-fibronectin aggregates are involved in the pathogenesis and that the plasma IgA-fibronectin level may even be of diagnostic value in IgAN. Nevertheless, a recent report has questioned the specificity of these assays as plasma IgA may interact with immobilized IgG and these assays detect not only IgA-fibronectin, but also total plasma IgA. These doubts render the interpretation of raised IgA-fibronectin aggregates in IgAN impossible. We isolated total IgA, in plasma by jacalin-agarose. Monomeric and polymeric IgA1 were distinctly separated by fast protein liquid chromatography. When the fast protein liquid chromatography fractions were analyzed for IgA fibronectin using the antifibronectin capture assay, increased optical density values were predominantly observed in polymeric IgA but not in monomeric IgA. Similar findings were found when the fast protein liquid chromatography fractions were studied using a novel gelatin-anti-IgA assay that avoided nonspecific interaction between plasma IgA and immobilized IgG used as the capture antibody in antifibronectin capture assay. Using our gelatin-anti-IgA assay, we failed to demonstrate a diagnostic increase in IgA-fibronectin aggregates in polymeric IgA from patients with IgAN compared with controls. Our finding of circulating IgA fibronectin aggregates in patients with IgAN comparable to those of healthy controls did not support the notion that these aggregates may have a pathogenetic role or diagnostic value in IgAN. PMID- 8629621 TI - Incidence of biopsy-proven primary glomerulonephritis in an Italian province. AB - Between January 1, 1970, and December 31, 1994, 1,926 cases of biopsy-proven primary glomerulonephritis (PGN) were diagnosed in an adult population (> 15 years of age) in a northwestern region of Italy with approximately 3.5 million inhabitants. The principal long-term changes were an increase in the absolute number of biopsies per year, an increase in the mean age of patients undergoing biopsy (from 29.3 +/- 12.2 years to 47.0 +/- 17.8 years), an increase in the percentage of patients older than 65 years (from 1.7% to 20.4%), and an increase in the percentage of isolated urinary abnormalities as an indication for biopsy (from 3.5% to 29.6%). In the total biopsy material, immunoglobulin A glomerulonephritis (IgA-GN) is the most frequent type (26%), followed by membranous glomerulonephritis (MGN; 20%). An incidence study was begun in 1990; this survey was restricted to the population of the province of Torino (approximately 2 million inhabitants) as only this area completely refers to the nephrologic centers that entered patients into this study. The overall incidence of PGN is 4.68 new cases/yr/10(5) population with a predominance of males (> 2:1); IgA-GN is the most common type (1.47/yr/10(5) population [34.5%]) in the overall population. In the elderly, cases of PGN are twice as high as in adults (8.19/yr/10(5) population v 4.02/yr/10(5) population in the 65 to 74 year and 45 to 54 year age groups, respectively); MGN mainly accounts for this high incidence (3.4/yr/10(5) population), while the nephrotic syndrome is the most common indication for biopsy (53.8%). A comparison with the incidence in the same area in the early 1970s is evaluable only for PGN, which was mainly registered in the age groups for which an unrestricted biopsy policy was already in place (15 to 35 years). In contrast with a misleading increase of all types of PGN, which is in reality due to the extension of the biopsy policy to older and asymptomatic patients, membranoproliferative glomerulonephritis type I shows a countercurrent decrease from 0.43 to 0.13/yr/10(5) population. Evidence of a simultaneous decrease in severe cardiac valvulopathy, due to rheumatic fever, is also provided. We feel that before epidemiologic conclusions can be reached, a clear understanding of one's own biopsy policy is essential. An apparent change in the PGN rate in our region over the last 25 years mainly depends on modifications in our biopsy policy, most probably coupled with a change in the threshold of detection of symptoms in the general population. At present, according to our experience, IgA-GN is the most common type of PGN in the total bioptic material, as demonstrated in other European countries, while the elderly show a peculiar pattern with a higher PGN incidence, mainly represented by MGN and heralded by the nephrotic syndrome. We also confirm that membranoproliferative glomerulonephritis type I is indeed decreasing in parallel with changes in the microbiologic environment. PMID- 8629622 TI - Monocyte chemoattractant protein-1 levels in patients with glomerular disease. AB - Chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 have been implicated in recruiting leukocytes to the glomerulus during immune renal injury. To detect the expression of MCP-1 in human glomerular disease, we measured urinary MCP-1 levels in patients with a variety of glomerulopathies. These data demonstrated that MCP-1 was present in the urine of patients with glomerular disease and that patients with inflammatory glomerulopathies had higher levels of urinary MCP-1. Urinary MCP-1 correlated with the extent of proteinuria (r = 0.71, P < 0.0001), but not with serum MCP-1 levels (r = 0.14, P > 0.3). The MCP-1 present in urine was biologically active, increasing monocyte migration in a microchemotaxis assay. This activity was attenuated by the addition of anti-MCP-1 antibody to the samples. Enumerating glomerular macrophages demonstrated that glomerular inflammation was correlated with urinary MCP-1 levels (r = 0.53, P < 0.02). Individuals with light microscopic evidence of severe glomerular injury (ie, crescents, necrosis, endocapillary proliferation) had significantly higher levels of MCP-1 in their urine than patients with less severe glomerular changes (1,962 +/- 612 pg MCP-1/mg creatinine v 314 +/- 45 pg MCP-1/mg creatinine; P < 0.002). Taken together, these results suggest that urinary MCP-1 reflects the extent to which MCP-1 that is produced in the glomerulus participates in the glomerular inflammatory response. PMID- 8629623 TI - The racial prevalence of glomerular lesions in nephrotic adults. AB - We retrospectively evaluated the prevalence of primary glomerular lesions in adults who had a renal biopsy for nephrotic proteinuria. From July 1975 to May 1994, 340 patients undergoing renal biopsies evaluated at Rush-Presbyterian-St Lukes Medical Center had the primary glomerular lesions of minimal-change disease, focal segmental glomerular sclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephropathy, immunoglobulin A nephropathy, and immunotactoid glomerulopathy. The patients had a mean age of 43 +/- 17 years, 57% were male, and 50% were white, 36% were black, 7% were of other race, and 7% were of unknown race. The distribution of lesions for black patients was minimal-change disease 14%, FSGS 57%, MGN 24%, membranoproliferative glomerulonephritis 2%, immunoglobulin A 2%, and immunotactoid glomerulopathy 1%; for white patients, the distribution of lesions was minimal-change disease 20%, FSGS 23%, MGN 36%, membranoproliferative glomerulonephropathy 6%, immunoglobulin A 8%, and immunotactoid glomerulopathy 6%. The prevalence of FSGS was significantly greater (P < 0.0001) and that for MGN, immunoglobulin A, and immunotactoid glomerulopathy was significantly less (P < 0.05) for black patients compared with white patients. In a logistic regression analysis, race remained the only significant predictor of FSGS (P = 0.0001), with black patients four times as likely to have FSGS as white patients. The distribution of lesions among white patients was similar based on gender, age, and biopsy period. For black patients the distribution was also similar based on gender and age, but over 20 years the incidence of FSGS increased from 39% (1975 to 1984) to 64% (1985 to 1994) (P < 0.01). Thus, significant racial differences in the distribution of primary glomerular lesions exists. This has important prognostic and therapeutic implications for nephrotic adults. PMID- 8629625 TI - Subxiphoid pericardial window for pericardial effusion in end-stage renal disease. AB - Fifty-seven patients with end-stage renal disease who were on maintenance dialysis underwent pericardial fluid drainage surgically between January 1980 and December 1991. All patients had echocardiographically proven pericardial effusion of more than 300 to 500 mL. Seven patients had pericardiectomy by left thoracotomy under general anesthesia in the first 2 years. Subsequently, 50 patients underwent a subxiphoid pericardial window by a left subcostal incision. A pericardial drainage tube was inserted at surgery and removed after 4 to 5 days. All but five patients undergoing subxiphoid pericardial window surgery received local anesthesia. The xiphoid process was not resected during surgery and steroids were not instilled in the pericardial cavity. There were minimal complications, no surgery-related deaths, and no recurrence of fluid in patients after pericardial window surgery. With our present experience, we advise a subxiphoid pericardial window with pericardial drainage under local anesthesia for all end-stage renal disease patients on dialysis who have a symptomatic or large pericardial effusion of more than 300 to 500 mL. Steroid instillation is not necessary for the prevention of recurrence of effusion. PMID- 8629624 TI - Effects of dietary protein restriction on the progression of advanced renal disease in the Modification of Diet in Renal Disease Study. AB - Patients with advanced renal disease randomized to the very low-protein diet group in the Modification of Diet in Renal Disease (MDRD) Study had a marginally (P = 0.066) slower mean glomerular filtration rate (GFR) decline compared with patients randomized to the low-protein diet group. The objective of these secondary analyses was to determine the relationship between achieved, in addition to prescribed, dietary protein intake and the progression of advanced renal disease. A randomized controlled trial was conducted in patients with chronic renal diseases of diverse etiology. The average follow-up was 2.2 years. Fifteen university hospital outpatient nephrology practices participated in the study, which comprised 255 patients aged 18 to 70 years with a baseline GFR 13 to 24 mL/min/1.73 m2 who participated in MDRD Study B. Patients with diabetes requiring insulin were excluded. The patients were given a low-protein (0.58 g/kg/d) or very low-protein (0.28 g/kg/d) diet supplemented with keto acids-amino acids (0.28 g/kg/d). Outcomes were measured by comparisons of protein intake from food or from food and supplement between randomized groups, and correlations of protein intake with rate of decline in GFR and time to renal failure or death. Comparison of the randomized groups showed that total protein intake from food and supplement was lower (P < 0.001) among patients randomized to the very low protein diet (0.66 g/kg/d) compared with protein intake from food only in patients randomized to the low-protein diet (0.73 g/kg/d). In correlational analyses, we combined patients assigned to both diets and controlled for baseline factors associated with a faster progression of renal disease. A 0.2 g/kg/d lower achieved total protein intake (including food and supplement) was associated with a 1.15 mL/min/yr slower mean decline in GFR (P = 0.011), equivalent to 29% of the mean GFR decline. After adjusting for achieved total protein intake, no independent effect of prescription of the keto acid-amino acid supplement to slow the GFR decline could be detected. If the GFR decline is extrapolated until renal failure, a patient with a 29% reduction in the rate of GFR decline would experience a 41% prolongation in the time to renal failure. Additional analyses confirmed a longer time to renal failure in patients with lower total protein intake. In conclusion, these secondary analyses of the MDRD Study suggest that a lower protein intake, but not the keto acid-amino acid supplement, retards the progression of advanced renal disease. In patients with GFR less than 25 mL/min/1.73 m2, we suggest a prescribed dietary protein intake of 0.6 g/kg/d. PMID- 8629626 TI - Ankle-arm blood pressure index as a predictor of mortality in hemodialysis patients. AB - The ankle-arm blood pressure index (AAI, ratio of ankle to arm systolic blood pressure), a simple, noninvasive, and inexpensive screening test, has recently been found to be highly predictive of subsequent mortality in several populations. The purpose of this study was to evaluate the relationship of the AAI to cardiovascular and all-cause mortality in hemodialysis patients. A cohort of 132 patients was followed for 1 year. The primary outcome measures were cardiovascular and all-cause mortality. An AAI of <0.9 was associated with a relative risk (RR) of cardiovascular mortality of 7.5, (95% CI, 2.3 to 24.8). Other predictive variables included diabetes mellitus RR 3.0, (95% CI, 1.2 to 7.3), and a history of any vascular disease RR 2.6 (95% CI, 1.0 to 7.0). An AAI of <0.9 was also predictive of all-cause mortality, RR 2.4 (95% CI, 1.2 to 4.7). Other predictive variables for all-cause mortality included older age, RR 1.4 per 10 years (95% CI, 1.0 to 2.1), decreased serum albumin RR 0.9 per 0.1 mg/dL (95% CI, 0.8 to 1.0), and diabetes mellitus RR 2.0 (95% CI, 1.0 to 3.7). Multivariate analysis showed an AAI of <0.9 and diabetes mellitus to be the only independent predictors of cardiovascular mortality, and an AAI of <0.9, older age, and a decreased serum albumin were independent predictors of all-cause mortality. In conclusion, we have found an AAI of <0.9 to be a powerful, independent predictor of mortality in hemodialysis patients. PMID- 8629627 TI - Urea concentration gradients during conventional hemodialysis. AB - To elucidate the intradialytic urea concentration gradients, we examined 26 hemodialysis patients wearing a double-lumen central venous catheter during their first or second fistula-punctured dialysis session. In 17 patients (group A), after 60 and 240 minutes of treatment with a mean blood flow of 196.4 +/- 9.9 mL/min, blood urea nitrogen (BUN) was measured in blood samples taken simultaneously from the central venous catheter, a vein in the arm opposite the access site, and the arterial and venous lines of the dialyzer. In 16 patients (group B), after 60 minutes of treatment with a mean blood flow rate of 197.5 +/- 12.3 mL/min, BUN was measured in blood samples taken from the dialyzer arterial line and then, after decreasing the blood flow to 50 to 60 mL/min for 1 minute, in samples taken from a vein in the arm opposite the access site, the central venous catheter, and the dialyzer arterial line. In group A, the mean BUN values in the dialyzer arterial line at 60 and 240 minutes were found to be 3.7% +/- 3.7% and 3.5% +/- 3.4% higher than the corresponding values in the central veins, respectively (P = NS between 60 and 240 minutes). In group B, after 1 minute of low blood flow, this difference was 1.5% +/- 2.4% (P = 0.06 compared with group A). The peripheral veins in group A patients at 60 and 240 minutes had 9.7% +/- 5.2% and 10.9% +/- 5.3% higher BUN values, respectively, compared with the central veins. This difference in group B patients after 1 minute of low blood flow was 6.8% +/- 4.2%. Urea access recirculation rate in group A, calculated by the classical three-samples method, was found to be 7.6% +/- 5.0% at 60 minutes and 9.9% +/- 5.8% at 240 minutes (P = NS). In group B, BUN values in the dialyzer arterial line after 1 minute of low blood flow increased significantly by 3.4% +/ 4.5% (P < 0.01). Our study shows that during conventional hemodialysis with a blood flow rate of 200 mL/min, urea concentration in the central veins is lower than in the dialyzer arterial line. This gradient after 1 minute of low-flow dialysis had a tendency to decrease. At the same time, however, the urea concentration gradient between the peripheral and central veins remained high, indicating that during conventional hemodialysis, intercompartmental disequilibrium plays a significant role in the arteriovenous gradient. PMID- 8629628 TI - Vitamin B6 and hemodialysis: the impact of high-flux/high-efficiency dialysis and review of the literature. AB - High-flux/high-efficiency (HF/HE) dialysis is associated with improved clearance for larger molecules, which include a wide variety of middle molecules and water soluble vitamins. Our study attempted to measure in vivo clearances of serum pyridoxal-5'-phosphate (PLP), the active metabolite of vitamin B6, on standard cuprophan versus cellulose triacetate HF/HE dialysis for patients maintained on 10 mg daily pyridoxine supplements. A longitudinal evaluation of PLP after 3 months on HF/HE dialysis was performed simultaneously. The average in vivo PLP clearance for six patients on standard hemodialysis increased by more than 50%, from 86 +/- 61.7 mL/min using a cuprophan membrane to 173 +/- 90.2 mL/min using a cellulose triacetate dialyzer, at average blood flows of 375 mL/min (P < 0.05). Levels of PLP decreased from a baseline of 50 +/- 13.8 ng/mL to 24 +/- 9.7 ng/mL (P < 0.05) after 3 months of HF/HE treatments; the levels returned to 45 +/- 6.4 ng/mL on resumption of standard dialysis treatments. Although not achieving statistical significance, the average hematocrit increased from 31.2% +/- 1.66% to 32.7% +/- 1.24% while on HF/HE dialysis without an increase in erythropoietin requirements. We conclude that HF/HE dialysis treatments can have a dramatic impact on vitamin B6 homeostasis. Further investigation to evaluate the effects of different membranes and reprocessing should be performed on more heterogeneous patient populations in whom compliance problems with diet and vitamin supplementation may exist. The increased clearance of vitamin B6 may have significantly more detrimental effects in these settings. PMID- 8629629 TI - Clinical and economic effects of mupirocin calcium on preventing Staphylococcus aureus infection in hemodialysis patients: a decision analysis. AB - This study was performed to determine the clinical and economic consequences of alternative strategies of preventing Staphylococcus aureus infection in chronic hemodialysis patients by use of intranasal mupirocin calcium to clear nasal carriage of S aureus. Decision analysis evaluated clinical outcomes and cost effectiveness of three likely management strategies to address S aureus nasal carriage and prevent subsequent infection in chronic ambulatory hemodialysis patients: (1) screen for S aureus nasal carriage every 3 months and treat those with a positive test result with mupirocin calcium; (2) treat all patients weekly with mupirocin calcium; or (3) no prevention strategy, treat infection only. Rates of nasal carriage of S aureus, S aureus infection rates, proportion of infections attributable to nasal carriage, efficacy of mupirocin, natural history of infection, and patient management strategies were derived from the published literature and supplemented by a panel of experts. Actual payments for medical services were obtained from Medicare parts A and B. Incremental cost effectiveness was calculated from the perspective of Medicare and subjected to sensitivity analyses. Assuming that 75% of S aureus infections are attributable to nasal carriage in hemodialysis patients, eliminating nasal carriage of S aureus with mupirocin calcium (with or without screening) markedly reduces the number of infections (45% to 55%) and also reduces health care expenditures relative to treating infections when they occur. Annual savings to Medicare are $784,000 to $1,117,000 per 1,000 hemodialysis patients, depending on the prevention strategy. Preventing S aureus infection by eradicating nasal carriage in chronic hemodialysis patients reduces morbidity while simultaneously reducing medical care costs. The decision to eliminate nasal carriage on a regular basis or use a screening test to guide antibiotic therapy is dependent on the tradeoff between improved short-term clinical and cost benefits and the potential for bacterial resistance that may arise from widespread use of mupirocin calcium. PMID- 8629630 TI - A randomized trial of Staphylococcus aureus prophylaxis in peritoneal dialysis patients: mupirocin calcium ointment 2% applied to the exit site versus cyclic oral rifampin. AB - The objective of this study was to compare prophylaxis for Staphylococcus aureus infections in peritoneal dialysis patients using 600 mg cyclic oral rifampin for 5 days every 3 months versus mupirocin calcium ointment 2% applied daily to the exit site. The study design was a prospective randomized trial, controlling for S aureus nasal carriage. Eighty-two continuous ambulatory and continuous cyclic peritoneal dialysis patients (54% male, 71 % white, 34% insulin-dependent, mean prestudy time on peritoneal dialysis 1.2 years) were randomly assigned to cyclic rifampin (n = 41 patients) or daily exit site mupirocin prophylaxis (n = 41 patients). Mean follow-up was 1 year. S aureus catheter infection rates were 0.13/yr with mupirocin and 0.15/yr with rifampin (P = NS). Both rates were significantly lower than the center's historical rate (the period between 1983 and 1992) of 0.46/yr prior to the study (P < 0.001). S aureus peritonitis rates were 0.04/yr with mupirocin and 0.02/yr with rifampin (P = NS), both significantly lower than the center's historical rate of 0.16/yr (P < 0.02). Catheter loss due to S aureus infections was 0.02/yr with mupirocin and 0/yr with rifampin (P = NS), both significantly lower than the center's historical rate of 0.12/yr (P < 0.001). There were no side effects in patients using mupirocin, but 12% were unable to continue rifampin due to side effects. We conclude that mupirocin ointment at the exit site and cyclic oral rifampin are equally effective in reducing S aureus catheter infections. In addition, rifampin or mupirocin significantly reduced S aureus peritonitis and catheter loss due to S aureus infections. Mupirocin at the exit site provides an excellent alternative prophylaxis for S aureus infections, particularly in patients who cannot tolerate oral rifampin therapy. PMID- 8629631 TI - Divergent effects of calcium channel and angiotensin converting enzyme blockade on glomerulotubular function in cyclosporine-treated renal allograft recipients. AB - The effects of amlodipine and perindopril on renal hemodynamics and tubular function in cyclosporine-treated hypertensive renal allograft recipients were evaluated in a randomized, double-blind crossover fashion. Ten patients were studied after a 2-week placebo run-in and, after 8 weeks of active treatment, allowing a 2-week placebo washout between treatments. At the end of each period, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured as 51Cr-EDTA and 123I-hippuran clearance, respectively, and tubular function evaluated by the lithium clearance technique was determined. Both drugs maintained GFR and ERPF and lowered mean arterial pressure (MAP, mm Hg) to a similar extent (time x treatment, P = 0.466): amlodipine from 126.9 +/- 2.5 to 115.9 +/- 2.2; perindopril from 126.9 +/- 2.5 to 117.9 +/- 3.9 (time effect of all treatments together, P = 0.003). Accordingly, renal vascular resistance (RVR, mm Hg/mL/min/1.73 m2) was equally reduced (time x treatment, P = 0.955): amlodipine from 0.36 +/- 0.03 to 0.30 +/- 0.02; perindopril from 0.36 +/- 0.03 to 0.32 +/- 0.01 (time effect all treatments together, P = 0.043). Sodium clearance and fractional excretion of sodium were not affected by either drug. Output of fluid from the proximal tubules measured as clearance of lithium (CLi, mL/min) and uric acid (CUr, mL/min) was higher after amlodipine than after perindopril (CLi 19.1 +/- 2.1 v 16.5 +/- 1.7, P =0.036 and CUr 7.0 +/- 0.6 v 5.9 +/- 0.4, P = 0.007). As a consequence, after amlodipine, distal absolute reabsorption of sodium was higher (DARNa 2.57 +/- 0.28 v 2.19 +/- 0.22 mEq/min, P = 0.027) and serum uric acid was lower (5.9 +/- 0.3 v 6.7 +/- 0.4 mg/dL, P = 0.001) in comparison with perindopril. In cyclosporine-treated renal allograft hypertensives, amlodipine and perindopril lower blood pressure equally and reduce RVR to the same extent. Overall sodium excretion is not affected by either agent. Urate clearance is higher and serum uric acid lower on amlodipine as compared with perindopril. PMID- 8629632 TI - Early recovery of the actin cytoskeleton during renal ischemic injury in vivo. AB - The actin cytoskeleton of proximal tubule cells is important for both the maintenance of membrane domains and attachment to neighboring cells and underlying substrata. Adenosine triphosphate (ATP) depletion during ischemic injury causes early alterations in the actin cytoskeleton, resulting in loss of membrane domains and cellular attachment. We examined the actin cytoskeleton during recovery from ischemic injury. As shown previously in cell culture studies, ATP depletion to 14% of control values from in vivo ischemia resulted in decreases in G-actin consistent with net polymerization of the cytoskeleton. After 20 minutes of recovery restored ATP levels to 24% of control values, percent G-actin increased back to control values, yet cytoplasmic actin polymerized with little evidence of apical recovery. After 120 minutes of recovery, ATP levels had increased to 48% of control values with little qualitative or quantitative change in actin polymerization from 20 minutes of recovery. When ATP levels recovered to 65% of control values at 360 minutes after ischemia, movement of F-actin back toward the apical surface was observed. These data, along with prior data using maleic acid, suggest that thresholds of cellular ATP may cause differing effects on distinct cellular actin pools. We conclude that actin cytoskeletal recovery occurs very early and may be necessary for reestablishment of polarity essential for normal reabsorptive functions. PMID- 8629633 TI - Nephrotic syndrome and renal insufficiency associated with lithium therapy. AB - Although the therapeutic administration of lithium in the psychiatric setting has been associated with various renal side effects, only a few reported cases have suggested a possible link to glomerular disease. We report two patients who, while taking lithium, developed heavy proteinuria caused by minimal change disease and acute renal insufficiency attributable to acute tubular necrosis. These clinical findings resolved on discontinuation of the drug, suggesting a role for lithium in their development. We also postulate that lithium, with its unique properties as a modulator of the phosphoinositol pathway, may play a key role in the pathogenesis of minimal change disease. PMID- 8629634 TI - Renal infarction and thrombosis of the infrarenal aorta in a 35-year-old man with primary antiphospholipid syndrome. AB - Renal manifestations of the "primary" antiphospholipid syndrome are rare. We report the case of an athletic 35-year-old man with an unremarkable medical history who suddenly developed hypertension and a renal infarction. Laboratory and radiological investigations showed a complete thrombosis of the infrarenal aorta, extensive collateral circulation arising from the superior mesenteric artery, and the primary antiphospholipid syndrome. Eight cases of renal infarction have previously been reported in the primary antiphospholipid syndrome. To our knowledge, this represents the first case of an infrarenal aortic thrombosis attributable to this syndrome. PMID- 8629635 TI - Right atrium thrombosis in patients on hemodialysis. AB - Vascular access has been the Achilles heel of hemodialysis for many years, and placement of temporary subclavian and internal jugular vein catheters has been a daily practice for the nephrologist. Now, concern about central vein stenosis, well described with the use of subclavian catheters in end-stage renal disease (ESRD), has prompted the use of internal jugular vein permanent catheters to avoid this complication, so as not to hinder future arteriovenous grafts. Permanent catheter access is not without its own special problems, and we describe here two patients that developed thrombosis of the right atrium while receiving hemodialysis through a permanent internal jugular catheter. PMID- 8629636 TI - Sclerosing peritonitis occurring in a hemodialysis patient. AB - The pathogenesis of sclerosing peritonitis is poorly understood. In patients with end-stage renal failure, it has been described in association with continuous ambulatory peritoneal dialysis (CAPD), although it has not been described in patients treated exclusively by hemodialysis. We present a case of sclerosing peritonitis occurring in a 47-year-old man who was treated solely by hemodialysis. Additionally, the patient was diagnosed as having "nephrogenic ascites" 5 years before developing sclerosing peritonitis, and we discuss a possible link between these two conditions. PMID- 8629637 TI - Fatal Aeromonas hydrophila bacteremia in a hemodialysis patient treated with deferoxamine. AB - A 49-year-old woman undergoing long-term hemodialysis and treated with deferoxamine (DFO) 1.5 g twice weekly for aluminum bone disease developed fever and bilateral calf pain caused by myonecrosis with gas gangrene. She had a rapidly fatal outcome. The cultures of blood and aspirates from both calf muscles demonstrated Aeromonas hydrophila. No obvious entry point could be traced. The in vitro growth of the patient's strain was found to be stimulated by the deferoxamine-iron complex in an iron-deprived medium. It is suggested that high dose DFO therapy in this patient was responsible for promoting a bacterial infection by this microorganism. PMID- 8629638 TI - Biocompatibility of peritoneal dialysis solutions. PMID- 8629639 TI - Nutritional management of the continuous ambulatory peritoneal dialysis patient. PMID- 8629640 TI - Techniques for modelling adequacy in patients on peritoneal dialysis. PMID- 8629641 TI - Management of catheter-related infections. PMID- 8629642 TI - Documentation of the hemodialysis prescription: a current evaluation. PMID- 8629643 TI - Diagnosis of iron deficiency in dialysis patients. PMID- 8629644 TI - ABIM looks toward the future. PMID- 8629645 TI - Choosing resuscitation late in life: problems with the paradigm. PMID- 8629646 TI - Choices of seriously ill patients about cardiopulmonary resuscitation: correlates and outcomes. SUPPORT Investigators. Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments. AB - PURPOSE: For patients hospitalized with serious illnesses, we identified factors associated with a stated preference to forgo cardiopulmonary resuscitation (CPR), examined physician-patient communication about these issues, and determined the relationship of patients' preferences to intensity of care and survival. PATIENTS AND METHODS: The study was a cross-sectional evaluation of patient preferences. The setting was five geographically diverse academic acute-care medical centers participating in the SUPPORT (Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments) project. Study participants were hospitalized patients > or = 18 years of age with 1 of 9 serious illnesses who were interviewed between days 3 and 6 after qualifying for the study. Using standardized interviews, patients provided information on demographics, preferences for CPR and other treatments, quality of life, functional status, perceptions of prognosis, and whether the patient had discussed CPR preferences with his or her physician. Data abstracted from the medical record included physiologic measures, therapeutic intensity, whether CPR was provided, and whether there was a do-not-resuscitate order. RESULTS: Of 1,995 eligible patients, 84% were interviewed (mean age 62 years, 58% men, inhospital mortality 7%, 6-month mortality 33%). Of the respondents, 28% did not want CPR. Factors associated independently with not wanting CPR included: hospital site; diagnosis; being older; being more functionally impaired; and patient perception of a worse prognosis. Only 29% of patients had discussed their preferences with their physician; 48% of those who did not want CPR reported such discussions. After adjusting for illness severity and factors associated with CPR preferences, patients not wanting CPR had lower intensity of care; similar inhospital mortality; and higher mortality at 2 and 6 months following study entry. CONCLUSIONS: The diagnosis, patients' perception of the prognosis, and hospital site were significantly associated with patients' resuscitation preferences after adjusting for patient demographics, severity of illness, and functional status. The rate of discussing CPR was low even for patients who did not want CPR. Patient preferences not to receive CPR were associated with a small decrease in intensity of care but no difference in hospital survival. PMID- 8629647 TI - Evaluation and outcomes of patients with palpitations. AB - PURPOSE: To determine: (1) the etiologies of palpitations, (2) the usefulness of diagnostic tests in determining the etiologies of palpitations, and (3) the outcomes of patients with palpitations. PATIENTS AND METHODS: One hundred and ninety consecutive patients presenting with a complaint of palpitations at a university medical center were enrolled in this prospective cohort study. Patients underwent a structured clinical interview and psychiatric screening. The charts were abstracted for results of the physical exam and tests ordered by the primary physician. Assignment of an etiology of palpitations was based on strict adherence to predetermined criteria and achieved by consensus of the two physician investigators. One-year follow-up was obtained in 96% of the patients. RESULTS: An etiology of palpitations was determined in 84% of the patients. The etiology of palpitations was cardiac in 43%, psychiatric in 31%, miscellaneous in 10%, and unknown in 16%. Forty percent of the etiologies could be determined with the history and physical examination, an electrocardiogram, and/or laboratory data. The 1-year mortality rate was 1.6% (95% confidence interval [CI] 0% to 3.4%) and the 1-year stroke rate was 1.1% (95% CI 0% to 2.6%). Within the first year, 75% of the patients experienced recurrent palpitations. At 1-year follow up, 89% reported that their health was the same or improved compared to that at enrollment, 19% reported that their work performance was impaired, 12% reported that workdays were missed, and 33% reported accomplishing less than usual work at home. CONCLUSIONS: The etiology of palpitations can often be diagnosed with a simple initial evaluation. Psychiatric illness accounts for the etiology in nearly one third of all patients. The short-term prognosis of patients with palpitations is excellent with low rates of death and stroke at 1 year, but there is a high rate of recurrence of symptoms and a moderate impact on productivity. PMID- 8629648 TI - Intensity of murmurs correlates with severity of valvular regurgitation. AB - PURPOSE: To evaluate the relationship between the intensity of murmurs and severity of mitral and aortic regurgitation. PATIENTS AND METHODS: Consecutive patients with chronic isolated aortic (n = 40) or mitral (n = 170) regurgitation undergoing echocardiographic quantitation of regurgitation between 1990 and 1991 were studied. Regurgitant volume and fraction were measured using two simultaneous methods (quantitative Doppler echocardiography and quantitative two dimensional echocardiography); the intensity of the regurgitant murmur (grade 0 to 6) was noted by physicians unaware of the study. RESULTS: Correlations between murmur intensity and regurgitant volume and fraction were good in aortic regurgitation (r = .60 and r = .67, respectively; P < 0.001) and mitral regurgitation (r = .64 and r = .67, respectively; P < 0.001) but weaker (r = .47 and r = .45, respectively) in the subset of mitral regurgitation of ischemic or functional cause. Murmur intensity grades > or = 3 for aortic regurgitation and > or = 4 for mitral regurgitation predicted severe regurgitation (regurgitant fraction > or = 40%) in 71% and 91% of patients, respectively. Murmur grades < or = 1 for aortic regurgitation and < or = 2 for mitral regurgitation predicted "not severe" regurgitation in 100% and 88% of patients, respectively. Murmur grades 2 for aortic regurgitation and 3 for mitral regurgitation were not correlated to degree of regurgitation. The severity of regurgitation was the most powerful determinant of intensity of murmur. CONCLUSIONS: Murmur intensity correlates well with the degree of chronic organic aortic and mitral regurgitation, and can be used as a predictor of regurgitation severity and as a simple guideline for diagnostic testing in these patients. PMID- 8629649 TI - Glycemic control in diabetes mellitus: have changes in therapy made a difference? AB - PURPOSE: New methods of measuring and controlling glycemia in diabetes mellitus have been developed and implemented in the past 10 years. We examined whether glycemia, as measured by glycosylated hemoglobin, changed in outpatient insulin dependent diabetes mellitus (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM) populations between 1985 and 1993 and whether contemporaneous changes in therapy could account for observed changes in glycemia. PATIENTS AND METHODS: Outpatients were selected based on having glycated hemoglobin (HbA1c) measured in the Massachusetts General Hospital laboratory during March 1985 (IDDM n = 94 and NIDDM n = 137) or during March 1993 (IDDM n = 89 and NIDDM n = 118). Chart reviews established demographic and clinical characteristics, including frequency of blood glucose self-monitoring, insulin injections, office visits, and HbA1c measurements during the year prior to the HbA1c result. RESULTS: Mean HbA1c level was significantly lower in the 1993 IDDM cohort compared with the 1985 cohort (8.77% +/- 1.7% versus 9.47% +/- 2.1%, P = 0.014). In the NIDDM cohorts, the difference in mean HbA1c did not achieve statistical significance (8.35% +/- 1.6% in 1993 versus 8.75% +/- 2.1% in 1985, P = 0.09); however, when adjusted for differences in NIDDM duration, HbA1c in the 1993 cohort was significantly lower than that in the 1985 cohort. The largest decrease in HbA1c in NIDDM was in patients treated with insulin (9.53% +/- 2.0% versus 8.54% +/- 1.5% in 1985 and 1993, respectively, P = 0.004). Multiple linear regression analyses demonstrated that increased frequency of self-monitoring and of insulin injections were associated with lower HbA1c in IDDM. CONCLUSIONS: The level of average glycemia has decreased in IDDM patients over the past 8 years, attributable, at least in part, to an increased frequency of monitoring and of insulin injections. Glycemia decreased in NIDDM, especially in the subset of patients treated with insulin. This temporal shift in glycemic control should have a salutary effect on the development of long-term microvascular and neurologic complications. PMID- 8629650 TI - Thoracic endometriosis syndrome: new observations from an analysis of 110 cases. AB - PURPOSE: To determine the demographics, clinical presentations, pathological findings, and the effectiveness of treatment in 110 patients with thoracic endometriosis syndrome (TES). METHODS: Retrospective analysis based on data published in the English medical literature. RESULTS: The mean age at presentation of TES was 35 +/- 0.6 years (+/- standard error of the mean) with a range from 15 to 54. The trends of age-specific incidence for pelvic endometriosis and TES were similar. The peak incidence for pelvic endometriosis occurred between 24 and 29 years, whereas the peak incidence for TES was between 30 and 34 years. Pneumothorax was the most common presentation, occurring in 80 of 110 (73%), followed by hemothorax in 15 (14%), hemoptysis in 8 (7%), and lung nodules in 7 (6%). The right hemithorax was involved in more than 90% of all manifestations except for nodules. Hemothorax was more often associated with presence of pleural and pelvic endometriosis compared with other manifestations (P < 0.003, P < 0.02). Compared with hormonal treatment, surgical pleurodesis resulted in low recurrence rate for pneumothorax or hemothorax among patients treated with danazol or oral contraceptives. CONCLUSIONS: There is a significant association between the presence of pelvic endometriosis and TES, with the latter occurring approximately 5 years later. Pneumothorax is the most common manifestation. The most plausible explanation for pathogenesis involves peritoneal-pleural movement of endometrial tissue through diaphragmatic defects and microembolization through pelvic veins. Diagnosis is established on clinical grounds in most cases. Surgical pleural abrasion is superior to hormonal treatment in the long-term management of pneumothorax. Earlier diagnosis and effective therapy of TES can decrease the morbidity of this disease in women during their reproductive period. PMID- 8629651 TI - The use of respiratory-tract cultures in the diagnosis of invasive pulmonary aspergillosis. AB - PURPOSE: To define the role of lower-respiratory-tract cultures in the diagnosis of invasive pulmonary aspergillosis (IPA) in immunocompromised hosts. METHODS: Immunocompromised patients with a positive, nonbiopsy, lower-respiratory-tract culture for Aspergillus species were classified as having definite, probable, indeterminate, or no IPA. Culture data, positive predictive values (PPVs), correlation with clinical and radiographic findings, and the relationship between the number of specimens submitted and the likelihood of recovering Aspergillus were assessed. RESULTS: Definite or probable IPA was diagnosed in 72% of episodes from patients with hematologic malignancy, granulocytopenia, or bone-marrow transplant; in 58% of those with solid-organ transplant or using corticosteroids; and in 14% of those with human immunodeficiency virus infection. The PPV of cultures ranged from 14% in the latter group to 72% in the first group (bone marrow-transplantation subgroup, 82%). Fungal cultures were more often positive than were routine cultures (P < 0.001). Clinical and radiographic findings suggestive of IPA were present more frequently in infected than uninfected patients (59% versus 24%, P < 0.025); and 73% versus 6%, (P < 0.0001, respectively). Infected patients with > or = 1 positive node had more cultures submitted than a control group of patients with no positive cultures (5.8 +/- 4.7 versus 2.1 +/- 2.2 cultures, P < 0.001). CONCLUSION: Recovery of Aspergillus species from high-risk patients is associated with invasive infection. Clinical and radiographic correlations help to separate true- from false-positive cultures. At least 3 sputum specimens should be submitted for fungal culture whenever fungal infection is suspected. PMID- 8629652 TI - Diagnostic features of tuberculous peritonitis in the absence and presence of chronic liver disease: a case control study. AB - PURPOSE: To determine diagnostic features of tuberculous peritonitis (TBP) in the absence and presence of chronic liver disease. PATIENTS AND METHODS: Thirty-four patients with TBP (13 without [Group I] and 21 with chronic liver disease [Group II] and 26 controls with cirrhosis and uninfected ascites (Group III) were studied. RESULTS: The clinical features in Groups I and II were similar and all patients had elevated ascitic fluid total mononuclear cell count. In Groups I, II, and III, respectively, ascitic fluid protein was > 25 g/L in 100% (13/13), 70% (14/20), and 0% (0/26); serum-ascites albumin gradient (SAAG) was > 11 g/L in 0% (0/13), 52% (11/21), and 96% (25/26), (0% [0/13], 71% [15/21], and 96% [25/26] after correction for serum globulin); and ascitic fluid lactate dehydrogenase (LDH) level was > 90 U/L in 100% (12/12), 84% (16/19), and 0% (0/20), respectively. In Groups I and II combined, ascitic fluid acid-fast stain was negative in all but Mycobacterium tuberculosis culture was positive in 45% (10/22); peritoneal nodules occurred in 94% (31/33), granulomas in 93% (28/30), and positive peritoneal M tuberculosis culture in 63% (10/16). CONCLUSIONS: In patients with suspected TBP, ascitic fluid protein of > 25 g/L, SAAG of < 11 g/L and LDH of > 90 U/L have high sensitivity for the disease. With coexistent chronic liver disease, a lower protein level and higher SAAG are usually not helpful but LDH > 90 U/L is a useful parameter for screening. Diagnosis is best confirmed by laparoscopy with peritoneal biopsy and M tuberculosis culture. PMID- 8629653 TI - Tuberculin skin test reactivity, anergy, and HIV infection in hospitalized patients. Longcope Firm of the Osler Medical Housestaff. AB - PURPOSE: Detection of latent tuberculosis infection is an important step in the control of tuberculosis because high-risk persons may be given preventive therapy. The value of tuberculin skin testing in individuals with human immunodeficiency virus (HIV) infection, however, is limited by anergy. We evaluated the prevalence of tuberculin skin test reactivity, anergy, and HIV infection in a group of hospitalized patients in an area where both tuberculosis and HIV infection are prevalent. PATIENTS AND METHODS: Three hundred fifty-one patients consecutively admitted to a medical service of a large urban teaching hospital were enrolled in the study. All those with no documented history of a positive tuberculin skin test were evaluated on admission with purified protein derivative (PPD) by the Mantoux test, and with anergy testing using a multiple puncture device. HIV testing was offered to all patients who did not have a known history of HIV infection, and was performed when informed consent was obtained. RESULTS: Forty-one patients (12%) had a documented history of a positive PPD. Of the remaining 310 patients, 62 (20%) had a PPD response of > or = 10 mm induration. Fifty-two (15%) of the 351 patients were HIV positive. None of the HIV-infected patients was PPD positive. Anergy was found in 63% of the HIV infected patients and 28% of the HIV-seronegative patients. Independent risk factors for a positive PPD included age > 55, male sex, and hypertension. HIV infection, current steroid use, and a history of cancer were associated with a negative PPD. Independent risk factors for anergy included HIV infection, current corticosteroid use, renal failure pneumonia, and a history of cancer. Of the 62 new PPD-positive patients, 30 (48%) were candidates for chemoprophylaxis. Additionally, 30 (63%) of 48 HIV-seropositive patients who were completed testing were anergic and might be candidates for chemoprophylaxis. Almost all of the patients eligible for chemoprophylactic therapy would have been detected if only patients at increased risk for developing tuberculosis were screened. CONCLUSIONS: Tuberculosis infection, HIV infection, and anergy were common in patients admitted to this medical service. Interpretation of PPD reactivity was confounded by a high prevalence of anergy, particularly in HIV-infected patients. A large proportion of patients tested were candidates for chemoprophylaxis. Routine tuberculin skin testing with anergy testing for high-risk patients on admission to the hospital is useful for identifying patients who might benefit from prophylaxis to help control the spread of tuberculosis. PMID- 8629654 TI - Long-term survival of patients with giant cell arteritis in the American College of Rheumatology giant cell arteritis classification criteria cohort. AB - OBJECTIVE: To characterize survivorship among patients with giant cell arteritis in a well-defined, multicenter cohort. PATIENTS AND METHODS: Follow-up was obtained for 205 (95.8%) of the 214 patients enrolled in the 1990 American College of Rheumatology vasculitis classification study. Standardized mortality ratios (SMR) were calculated comparing mortality data from this group of patients with giant cell arteritis versus the general population. RESULTS: There were 49 deaths (33 women and 16 men among the 205 patients available for follow-up. Survivorship was virtually identical to that of the general population (SMR = 1.034 +/- 0.121), and was similar for women (SMR = 1.022 +/- 0.149) and men (SMR = 1.078 +/- 0.206) (SMR = 1 indicates that expected and observed survival are identical). CONCLUSION: The life expectancy of patients with giant cell arteritis is the same as that of the general population. PMID- 8629655 TI - Drug therapy for hypercholesterolemia in patients with cardiovascular disease: factors limiting achievement of lipid goals. AB - PURPOSE: To determine the extent that goal lipid levels derived from the National Cholesterol Education Program (NCEP) are achievable in clinical practice, and to identify factors associated with the achievement of these goals. PATIENTS AND METHODS: We conducted a retrospective cohort study consisting of a consecutive sample of 244 patients with either coronary artery disease or peripheral vascular disease treated for hypercholesterolemia at a large Veterans Affairs medical center. Primary outcomes, recorded prospectively, were lipid levels and lipoprotein cholesterol response, and tolerance and compliance to drug therapy. Goal lipid levels were defined as low-density lipoprotein cholesterol (LDL-C) < or = 130 mg/dL and triglyceride (TG) < or = 200 mg/dL. RESULTS: Lipid-lowering drug therapy reduced LDL-C from 25% to 42% below baseline in patients with hypercholesterolemia varying from mild (130 to 160 mg/dL) to severe ( > 220 mg/dL), respectively. Approximately 75% of patients with LDL-C < or = 160 mg/dL ultimately achieved their lipid goal with drug therapy; however, less than half of patients with baseline LDL-C > 160 mg/dL achieved target lipid values. Multivariate analysis indicated that lower baseline LDL-C and triglycerides, use of combinations of drug therapy rather than monotherapy, and patient adherence to treatment predicted the achievement of goal lipid levels. CONCLUSIONS: Successful implementation of NCEP guidelines, frequently requires combination drug therapies, and is limited by poor patient tolerance and acceptance of niacin and the sequestrants. PMID- 8629656 TI - Diagonal earlobe creases and prognosis in patients with suspected coronary artery disease. AB - PURPOSE: To determine whether high-risk patients with unilateral, bilateral, or no earlobe creases (ELC) have different prognoses for common sequelae of coronary heart disease. PATIENTS AND METHODS: Two hundred sixty-four consecutive patients from a university-based coronary care unit or catheterization laboratory were blindly followed up for 10 years, using questionnaires, medical records, and death certificates. The primary outcome measure was time to cardiac event; namely, coronary artery bypass graft (CABG), myocardial infarction (MI), or cardiac death. Analyses included log-rank tests and Cox proportional hazards regression modelling. RESULTS: The number of creased ears was significantly associated, in a graded fashion, with 10-year cardiac event free survival: 43.5% +/- 5.7%, 33.0% +/- 6.7%, or 17.5% +/- 4.6% (mean +/- standard error for 0, 1 or 2 ELC, respectively; P = 0.003). After adjustment for 10 known cardiac risk factors, including age and left ventricular ejection fraction, the relative risk for a cardiac event for a unilateral ELC, relative to 0 ELC, was 1.33 (95% confidence interval [CI] 1.10 to 1.61, P = 0.02), and for bilateral ELC, it was 1.77 (95% CI 1.21 to 2.59, P = 0.003). CONCLUSIONS: Ear lobe creases are associated, in a graded fashion, with higher rates of cardiac events in patients admitted to hospital with suspected coronary disease. In such patients, ELC may help to identify those at higher risk for sequelae for coronary disease. PMID- 8629657 TI - Perceived benefits in a behavioral-medicine insomnia program: a clinical report. AB - PURPOSE: This clinical replication series assessed the perceived outcome of individuals with chronic insomnia who spontaneously sought treatment at a hospital behavioral-medicine insomnia program. PATIENTS AND METHODS: Chronic insomnia patients who were treated with a group multifactor behavioral intervention completed posttreatment (n = 102) and 6-month follow-up (n = 70) questionnaires that assessed improvement. RESULTS: All patients reported improved sleep at posttreatment, with the majority (58%, 59) reporting significant improvement. Of sleep medication users, 91% (62/68) either eliminated or reduced medication use. At 6-month follow-up, 90% (63/70) of respondents rated improvement in sleep as either maintained or enhanced. CONCLUSION: These results suggest that patients spontaneously seeking treatment for insomnia, including sleep medication users and those with psychological comorbidity, derive significant benefit from a group multifactor behavioral intervention. Several factors, including maintenance of therapeutic gains at long-term follow-up, the average pretreatment duration of insomnia, previous unsuccessful treatment with psychotherapy and pharmacotherapy, and previous research, argue against nonspecific effects playing a significant role in these results. PMID- 8629658 TI - Subclinical thyroid disease. AB - Widespread use of improved measurements for serum thyroid hormones and thyroid stimulating hormone (TSH) has resulted in characterization of the syndromes of subclinical hypothyroidism, characterized by normal free T4 estimate and raised serum TSH, and subclinical hyperthyroidism, in which patients have normal serum free T4 estimate and decreased serum TSH. Therapy for these two disorders in generally recommended but must be individualized according to the patient's general medical condition. Additional studies are needed to assess the benefits of treatment. This report reviews subclinical hypothyroidism and subclinical hyperthyroidism, describing their causes, diagnostic criteria, complications, and indications for treatment. A brief review of testing for thyroid function is presented, and each of the subclinical disorders is compared with the classic syndromes of hypothyroidism and hyperthyroidism. PMID- 8629659 TI - Balancing service and education: improving internal medicine residencies in the managed care era. AB - BACKGROUND: Internal medicine training programs must adapt to health care systems faced with balancing the competitive priorities of patient-care responsibilities and educational needs. OBJECTIVE: To evaluated the effects of a major organizational change on the inpatient service of an internal medicine residency program in a vertically integrated health system. METHODS: We changed the structure of our program from a system in which the hospitalized patients' primary physicians were responsible for daily inpatient management, while teaching was assigned to a defined teaching rounder, to a method in which the rounding attending was responsible for both teaching and patient care. Measurements before and after the change in the rounding system included: the McGill University clinical tutor evaluations, time-motion observations of house staff, patient satisfaction surveys, average length of stay data, and physician focus groups to assess physician satisfaction. RESULTS: The rounding attendings consistently received excellent to superior ratings by the house staff both before and after the implemented change. Compared to time-motion observations performed before the change, observations recorded after the change suggested that a greater percent of house staff time was spent on educational activities. The responses of patient satisfaction surveys indicated that the perception of quality of care remained high after the system change. Lastly, the average length of stay for patients on the general internal medicine and subspecialty services was reduced from 7.6 days before the change to 6.6 days after the change, a difference of 0.92 day (95% confidence interval 1.3 to 0.6, P < 0.001). CONCLUSIONS: Through organizational restructuring, it is possible to improve the quality of patient care and improving the efficiency of patient-care management. PMID- 8629660 TI - Current views on obesity. AB - Although the disorders associated with obesity have been extensively studied, little attention has been paid to the fact that obesity is itself a chronic disease. This misunderstanding of the nature of obesity has contributed to the stigmatization of obese persons and to the use of inappropriate or inadequate treatment regimens. Although the etiology of obesity is still unclear, genetic, metabolic, and social factors are all believed to play a role in its development and progression. Behavioral therapy, exercise, very-low-calorie diets, drug therapy, and surgery affect the treatment of obesity of differing levels of severity. The regaining of weight following treatments other than surgery is very frequent, in part because periods of weight loss are rarely followed by maintenance programs. An increasing awareness of the chronic, multifactorial nature of obesity will ideally lead to the development of new long-term treatment programs that are safe and effective. Such programs are urgently needed in light of new data that show that the prevalence of obesity is increasing in the United States, as much as 30% in the last decade. PMID- 8629661 TI - Spinal epidural hematoma associated with warfarin therapy. PMID- 8629662 TI - Nephrotoxicity associated with olsalazine. PMID- 8629663 TI - Therapeutic options in patients with hormone-refractory prostate cancer. PMID- 8629664 TI - Interchangeability of levothyroxine preparations. PMID- 8629665 TI - Interchangeability of levothyroxine preparations. PMID- 8629666 TI - Interchangeability of levothyroxine preparations. PMID- 8629667 TI - Interchangeability of levothyroxine preparations. PMID- 8629668 TI - The potential impact of graduate medical education policy initiatives on internal medicine residency programs--response from a survey of program directors. PMID- 8629669 TI - The quality of consent. PMID- 8629670 TI - The impact of disease severity on the informed consent process in clinical research. AB - PURPOSE: To assess the efficacy of informed consent in subjects differing in disease severity, ranging from those with immediately life-threatening disease to healthy volunteers. SUBJECTS AND METHODS: A total of 127 subjects, enrolled in four types of clinical research protocols, were tested. Subjects completed questionnaires before entry into the protocol, within 24 hours of signing the primary protocol's consent document, and 4 to 6 weeks after entry. RESULTS: Healthy volunteers retained the most information about risks and side effects, and severely ill Phase I subjects retained the least (P <0.0001). Phase I and II subjects had the best long-term retention of information about procedures, whereas Phase III subjects and healthy volunteers retained the least (P <0.001). Information about the scientific purpose and confidentiality of data were retained best by symptom-free, Phase III subjects (P <0.05). Phase I subjects entered the study primarily for treatment purposes, and the consent document was rated less useful by subjects with more advanced disease (P <0.05). CONCLUSIONS: Subjects with differing disease processes and illness severities focused on and retained different aspects of experimental protocols for dissimilar reasons. During the informed consent process, research staff should inquire of potential subjects' personal goals for participating in experimental protocols and develop means for ensuring subjects' understanding of the inherent risks and alternative interventions available. PMID- 8629671 TI - Low-molecular-weight heparins and unfractionated heparin in the treatment of patients with acute venous thromboembolism: results of a meta-analysis. AB - PURPOSE: To obtain reliable estimates of the relative efficacy and safety of low molecular-weight heparins (LMWHs) and unfractionated heparin (UFH) in the treatment of patients with venous thromboembolism. METHODS: A literature search of randomized trials evaluating LMWH and UFH for the period from 1980 to 1994 was conducted to obtain data for a meta-analysis. Studies were classified as level 1 if they were double-blind or if there was blinded assessment of outcome measures, and level 2 if they did not provide assurance of blinded outcome assessment. RESULTS: In level 1 studies, the relative risk (RR) of recurrent venous thromboembolism during the first 15 days and over the entire period of anticoagulant therapy was 0.24 (95% confidence intervals [CI] 0.06 to 0.80, P = 0.02) and 0.39 (95% CI 0.30 to 0.80, P = 0.006), respectively, in favor of LMWH treatment. The RR for major bleeding was 0.42 (95% CI 0.2 to 0.9, P = 0.01), in favor of LMWH. In level 2 studies, no significant differences in the rates of recurrent venous thromboembolism or major bleeding were observed. Pooling level 1 and level 2 studies, the RR for overall mortality and mortality in cancer patients was 0.51 (95% CI 0.2 to 0.9, P = 0.01), and 0.33 (95% CI 0.1 to 0.8, P = 0.01), respectively in favor of LMWH. CONCLUSIONS: LMWH are likely to be more effective than UFH in preventing recurrent venous thromboembolism, to produce less major bleeding, and to be associated with a lower mortality rate, particularly in the subgroup of patients with cancer. PMID- 8629672 TI - Variation in the management of deep vein thrombosis: implications for the potential impact of a critical pathway. AB - PURPOSE: To evaluate the potential impact of a practice guideline in the form of a critical pathway on variation and quality of care in patients with deep vein thrombosis (DVT). METHODS: Goals were identified for key steps and processes that were believed to be important for meeting a length-of-stay (LOS) goal of 5.5 days, and for improving quality of care for patients with DVT. Data collected via chart review were used to determine the percentage o patients with uncomplicated DVT admitted in the year after October 1, 1992, whose management would have met these goals. RESULTS: Only 11 (12%) of 92 eligible patients with a primary discharge diagnosis of DVT met the LOS goal. In 30%, the activated partial thromboplastin time (aPTT) was >60 seconds within a target of 12 hours after admission. The goals for the initiation of warfarin (within 12 hours after aPTT >60 seconds) and the achievement of a therapeutic international normalized ratio (INR) level (within 120 hours) were met in 51% and 58% of patients, respectively. The target duration of intravenous heparin therapy was achieved in 78% of patients. Only 18% of patients, however, were discharged within 12 hours after 96 hours of heparin therapy had been given and a therapeutic INR had been achieved. CONCLUSIONS: These data demonstrate considerable variation in management of uncomplicated DVT at a single hospital, suggesting that a critical pathway could have impact on both LOS and quality of care. PMID- 8629673 TI - A critical pathway to treat proximal lower-extremity deep vein thrombosis. AB - To address variation in treatment of deep vein thrombosis (DVT) while maximizing the efficiency and quality of care, our institution developed a critical pathway guideline. This paper presents this critical pathway and the clinical rationale underlying its recommendations. The DVT pathway synthesizes recommendations for all aspects of patient care, including laboratory evaluation at admission, dosing and management of heparin therapy, timing of warfarin initiation, elements of patient education, discharge planning, and anticipated duration of heparinization and hospitalization. Differences among interpretations of the medical literature, patient populations, physician skills, test availability, and other variables make it unlikely that all elements of this pathway would best meet the needs of another institution. Nevertheless, the critical pathway format and the specific contents of this pathway may serve as a useful benchmark for others involved in creating clinical guidelines for this patient population. PMID- 8629674 TI - Treatment of 100 patients with primary amyloidosis: a randomized trial of melphalan, prednisone, and colchicine versus colchicine only. AB - PURPOSE: A clinical trial designed to test whether treatment with melphalan, prednisone, and colchicine (MPC) is superior to colchicine (C) alone was performed in patients with primary amyloidosis (AL), a nonmalignant plasma cell dyscrasia. PATIENTS AND METHODS: Patients were randomized to MPC or C with stratification according to sex, time from diagnosis to study entry (ie, less than 3 months or 3 to 12 months), and dominant organ system involvement (ie, cardiac, renal, neurologic, or others). Data were gathered monthly from patients, quarterly from physicians, and annually in the Clinical Research Center. One hundred consecutive patients with AL amyloidosis admitted between 1987 and 1992 who met eligibility requirements were treated and followed for a minimum of 18 months. Fifty patients (group A) received daily oral colchicine and 50 patients (group B) received cycles of oral melphalan and prednisone every 6 weeks for 1 year as well as colchicine. RESULTS: The principal outcome measure was median survival, which was compared in the two treatment groups and in the subgroups. The overall survival of all patients from study entry was 8.4 months. Comparing group A (C) to group B (MPC), the survival was 6.7 months versus 12.2 months (P = 0.087). Both treatment groups had poor survival for patients in the cardiac subgroup, longest survival in the renal group, and significant differences favoring MPC treatment only in patients whose major system manifestations were neurologic (P = 0.037) or other (P = 0.007). Multivariate analysis showed a strongly significant treatment effect (P = 0.003) and improved survival associated with not having cardiac or gastrointestinal involvement. CONCLUSIONS: MPC was advantageous for patients whose major manifestations of amyloid disease were other than cardiac or renal. Better survival regardless of treatment was noted in patients for whom a satisfactory supportive treatment such as transplant or dialysis exists for their organ failure. PMID- 8629675 TI - Allogeneic bone marrow transplantation for poor-prognosis lymphoma: response, toxicity and survival depend on disease histology. AB - PURPOSE: To evaluate outcomes and identify prognostic factors in allogeneic bone marrow transplantation in patients with end-stage lymphoma. PATIENTS AND METHODS: Data were retrospectively analyzed of 64 patients (42 men and 22 women) 18 to 48 years of age with recurrent or refractory lymphoma who underwent allogeneic bone marrow transplantation from matched sibling donors (or in 1 case from a one antigen-mismatched relative) between May 1981 and July 1994. RESULTS: Twelve patients survived free of disease. They were 8 of 15 with low-grade lymphoma (disease-free survival at 2 years 59% +/- 13%); 3 of 25 with lymphoblastic lymphoma (disease-free survival 17% +/- 8%); and 1 of 10 with diffuse small non cleaved cell lymphoma (disease-free (10% +/- 9%). Survival and disease-free survival of patients with low-grade lymphoma were significantly superior compared to any other subgroup of patients (P <0.01). Only 2 patients with low-grade lymphoma had disease progression (9% +/- 9% actuarial risk at 2 years) as opposed to 5 of 15 with intermediate-grade lymphoma (39% +/- 14%), 9 of 25 with lymphoblastic lymphoma (28% +/- 9%), and 8 of 10 (80% +/- 13%) with diffuse small non-cleaved lymphoma. The actuarial risk for disease progression was significantly lower for patients with low-grade lymphoma than for any other histologic subgroup (P <0.02). It was significantly higher for those with diffuse small non-cleaved cell lymphoma than for other histologic subgroups (P < or = 0.003). CONCLUSIONS: Allogeneic bone marrow transplantation is an effective procedure in patients with refractory low-grade lymphoma. It results in long-term remissions and should be considered in younger patients with recurrent disease who have a matched sibling donor. The late recurrence in 1 patient indicates the necessity of continued follow-up. A small fraction of patients with end-stage intermediate- and high-grade lymphoma can obtain prolonged disease-free survival, but recurrence and regimen-related toxicity remain major problems. The results could be improved by the development of conditioning regimens with less toxicity and by the use of bone marrow transplantation earlier in the course of the disease. PMID- 8629676 TI - Long-term intraperitoneal deferoxamine for hemochromatosis. AB - Intraperitoneal deferoxamine is a well established treatment for aluminum accumulation syndrome in patients with end-stage renal disease receiving peritoneal dialysis, but the use of intraperitoneal deferoxamine has not been described outside of the setting of chronic renal failure. We present here a case of secondary hemochromatosis, complicated by cirrhosis and cardiomyopathy, in which a chronic peritoneal dialysis catheter was used both to treat ascites and to deliver parenteral deferoxamine for iron overload. Daily urinary iron excretion was similar to that achieved when using standard routes of deferoxamine administration. Over a 2-year period, reversal of both the biochemical indicators and the clinical manifestations of iron overload was accomplished. PMID- 8629677 TI - Upper gastrointestinal hemorrhage clinical--guideline determining the optimal hospital length of stay. AB - PURPOSE: Physicians lack objective outcome data to define the medically appropriate length of stay (LOS) for patients hospitalized with acute upper gastrointestinal hemorrhage (UGIH), resulting in wide variations in resource utilization and quality of care. A clinical practice guideline with the ability to assign relative risk for adverse events is proposed. METHODS: A comprehensive scoring system was derived from the literature by using four variables; hemodynamics, time from bleeding, comorbidity, and esophagoduodenoscopy findings. The discriminatory ability, potential safety, and impact on resource utilization of the clinical practice guideline was measured in a retrospective, observational study. RESULTS: Seventy percent of UGIH patients (349 of 500) achieved low-risk status according to the guideline, and, were therefore potentially suitable for early discharge from the hospital. If low-risk patients were discharged based upon the guideline, mean (+/- SD) hospital LOS would have been decreased from 4.8 +/- 2.4 days to 2.7 +/- 1.4 days; mean reduction was 2.1 bed-days per patient (95% CI 1.8 to 2.3, P <0.001). LOS would have increased in 4% of cases. Adopting the guideline's recommendation of early discharge would have resulted in a 0.6% (95% CI 0.07 to 2.1) complication rate, with no worsening of quality of care, as judged by implicit review. CONCLUSIONS: The proposed clinical practice guideline may safely reduce hospital LOS for selected low-risk patients with acute UGIH. Moreover, it also may reduce premature discharge of high-risk patients prone to life-threatening events. PMID- 8629678 TI - Isoetharine versus albuterol for acute asthma: greater immediate effect, but more side effects. AB - PURPOSE: To compare the magnitudes of the immediate effects of the nebulized beta agonists isoetharine and albuterol in the treatment of acute severe asthma. PATIENTS AND METHODS: Fifty-one adults presenting with severe asthma exacerbations (forced expiratory volumes in the first second of exhalation [FEV1] <40% of predicted) to the emergency department were randomized (double-blind) to receive hourly inhaled nebulization treatment with either isoetharine (5 mg) or albuterol (2.5 mg). The FEV1 was measured immediately before and after each nebulized treatment. Any side effects were recorded. RESULTS: Immediately after the first nebulized treatment, the isoetharine group improved its mean FEV1 (+/ SEM) by a significantly greater amount than did the albuterol group: 60% +/- 11% versus 39% +/- 5%, respectively (P <0.05). One hour later the mean FEV1 were equivalent. This pattern repeated itself after the second hourly treatment. The two groups did not differ in any outcome parameters (FEV1 at discharge, number of nebulized treatments required, the number of inpatient admissions, number of clinical relapses after discharge). More patients treated with isoetharine had side effects (36% versus 4% for albuterol, P <0.01), 1 of whom required discontinuation from the study. CONCLUSIONS: Both medications were equally effective in alleviating bronchospasm. The immediate effect of isoetharine was significantly greater, but equalized that of albuterol within an hour after treatment. There were more side effects with isoetharine. PMID- 8629679 TI - Long-term outcome of mothers of children with complete congenital heart block. AB - OBJECTIVES: To determine the health of mothers of offspring with complete congenital heart block (CHB) both at the time of delivery of the affected child and in the long-term, and the percentage of mothers and children with CHB who had anti-SSA/Ro and/or SSB/La antibodies. PATIENTS AND METHODS: Sixty-four mothers of 64 children with CHB (seen between 1964 and 1993) were identified through the Cardiology database of The Hospital for Sick Children, Toronto, Canada. Medical information from these of children with CHB was evaluated. Data were obtained from the mothers by mailed questionnaire, telephone interview, and/or from the attending physicians. The presence of anti-Ro antibodies and anti-La antibodies were evaluated by ELISA assay. RESULTS: The mean age at the time of delivery of the first child with CHB was 28 +/- 6 years. At the time of delivery 42 (66%) mothers were healthy, 2 (3%) had systemic lupus erythematosus (SLE), 2 (3%) had linear scleroderma, 2 (3%) had rheumatoid arthritis; 3 (5%) had a history of rheumatic fever (but were otherwise well), 1 (2%) had Sjogren's syndrome (SS), and 12 (19%) had an undifferentiated autoimmune syndrome (UAS) (arthralgia, myalgia, photosensitivity, skin vasculitis, Raynaud's phenomenon). The mean time to follow-up from deliver to study was 121 +/- 88 months. The mean maternal age at study was 38 +/- 9 years. Three of 12 mothers who initially had a UAS progressed to SLE (average follow-up time of 80 months, median 96), and 2 developed SS (with average follow-up time 140 months, median 132) and 1 went into remission. The mean follow-up time for the other mothers who did not develop an autoimmune disease was 150 +/- 102 months. Thirty-six of the 42 initially healthy mothers remained well. One mother developed SLE; 1 developed hyperthyroidism; 1 developed anky-losing spondylitis; and 3 developed an UAS. The mean follow-up time of the 36 mothers who remained healthy was similar (123 +/- 97 months) to the 6 initial healthy mothers who developed an autoimmune disease (121 +/- 36 months). Anti-Ro and/or anti-La antibodies were positive in 32 of 53 (60%) mothers tested. Fourteen of the 53 mothers were symptomatic at the time of delivery and 39 were asymptomatic. Anti-Ro and/or anti-La antibodies were positive in 12 of 13 mothers tested at the time of delivery. CONCLUSIONS: The long-term maternal outcome in our cohort was very good as most of the initially healthy mothers remained well at follow-up. Twenty-five percent of the mothers with a UAS and only 2% of the initially healthy mothers developed SLE. The development of an autoimmune disease in an asymptomatic mother identified by the birth of a child with CHB was less common in our study than in previous studies. However, close follow-up of mothers with UAS is warranted. PMID- 8629680 TI - Long-term, nightly benzodiazepine treatment of injurious parasomnias and other disorders of disrupted nocturnal sleep in 170 adults. AB - PURPOSE: To assess the efficacy, dose stability, safety, and abuse potential of long-term, nightly benzodiazepine treatment of chronic disorders of disrupted nocturnal sleep. PATIENTS AND METHODS: During a 12-year period, one author evaluated and treated 170 adult referrals for > or = 6 months with nightly benzodiazepine therapy for longstanding, sleep-disruptive disorders: injurious sleepwalking and sleep terrors (69); rapid eye movement sleep behavior disorder (52); chronic, severe insomnia (25); and restless legs syndrome/periodic limb movement disorder (24). RESULTS: Complete/substantial control of the sleep disorders was achieved by 146 patients (86%); 8% had adverse effects requiring medication changes; 2% had relapses of alcohol or chemical abuse requiring hospitalization; another 2% at times misused their medications. A total of 136 patients received clonazepam nightly for a mean 3.5 (+/- 2.4) years, with no significant difference in inital versus final mean dose: 0.77 mg (+/- 0.46) versus 1.10 mg (+/- 0.96). Similar results were obtained with chronic alprazolam treatment and with other benzodiazepine treatments. CONCLUSION: Long-term, nightly benzodiazepine treatment of injurious parasomnias and other disorders of disrupted nocturnal sleep resulted in sustained efficacy in most cases, with low risk of dosage tolerance, adverse effects, or abuse. Data from this study on the treatment of chronic, severe insomnia (a small subset of all insomnia) are not generalizable to the typical insomnia patient. PMID- 8629681 TI - Preserving the patient referral process in the managed care environment. AB - Referrals are a central component of the American health care system, defining the relationship among generalists, patients, and specialists. The dynamics of the referral process as they existed in a fee-for-service medical environment will evolve under managed care, but retain the basic "Try-out" approach of the generalist and "Rule-out" approach of the specialist. A managed care, contract based health care system alters some of the assumptions on which the referral relationship has been structured. A four-step approach to assuring quality interactions among patient, generalist, and specialist within the managed care environment is described, including: (1) engage; (2) anticipate; (3) feedback; and (4) reassess. When the referral process is structured as suggested, it can be evaluated for quality and efficacy. Armed with mutual respect and understanding, the forces that polarized specialist and generalist care in the 1980s can be redirected to enhancing patient care in the 1990s. PMID- 8629682 TI - The present role of radiofrequency catheter ablation in the management of cardiac arrhythmias. AB - Radiofrequency catheter ablation can be used to treat a variety of cardiac arrhythmias. Yet while the spectrum of arrhythmias amenable to catheter ablation continues to expand, the protocol for whether or not catheter ablation should be recommended for the management of an individual patient has not been firmly established. This article summarizes the experience of one medical center with radiofrequency catheter ablation. The varieties of arrhythmias that may be effectively treated with catheter ablation, the probability of success, and the attendant risks of these procedures in the series are presented. In addition, the future applications of catheter ablation to cardiac arrhythmias are discussed. PMID- 8629683 TI - Hypertension and myocardial and cerebral infarctions. PMID- 8629684 TI - Quality of life: who can make the judgment? PMID- 8629686 TI - Mycobacterium bovis vertebral osteomyelitis as a complication of intravesical administration of Bacille Calmette-Guerin. PMID- 8629685 TI - Prospective trial of paromomycin for cryptosporidiosis in AIDS. PMID- 8629687 TI - Hyperglycemia during physical stress. PMID- 8629688 TI - Sputum testing for TB: getting good specimens. PMID- 8629689 TI - Metformin precaution. PMID- 8629690 TI - Perpetuating oppression? PMID- 8629691 TI - Dangers of new drugs. Does your facility or team have a system for incorporating new agents? PMID- 8629692 TI - Getting physicians to change their practices. PMID- 8629693 TI - When patients refuse pain medications. PMID- 8629694 TI - The overlooked epidemic: HIV in older adults. PMID- 8629695 TI - Clinical snapshot: permanent pacemaker. PMID- 8629696 TI - Re-entry: when a chemically dependent colleague returns to work. PMID- 8629697 TI - No code. PMID- 8629698 TI - Managing vascular leg ulcers. Part 2: treatment. PMID- 8629699 TI - Emergency! Bleeding gastric ulcer. PMID- 8629700 TI - Missed DNR order. PMID- 8629701 TI - Not too late to sue. PMID- 8629702 TI - RN seen victim of secondhand smoke. PMID- 8629703 TI - New accrediting regs pose issues for schools, NLN. PMID- 8629704 TI - On the perversion of public policy. PMID- 8629705 TI - Eras, paradigms, and the future of epidemiology. PMID- 8629706 TI - The promise of risk-based allocation trials in assessing new treatments. PMID- 8629707 TI - Toward a holistic approach to public health surveillance. PMID- 8629708 TI - From disease surveillance to the surveillance of risk factors. PMID- 8629709 TI - Uncertainties in the estimation of HIV prevalence and incidence in the United States. PMID- 8629710 TI - On the Holm, Simes, and Hochberg multiple test procedures. PMID- 8629711 TI - Emerging objectives and methods in epidemiology. PMID- 8629712 TI - Surveillance in environmental public health: issues, systems, and sources. AB - This article describes environmental public health surveillance and proposes a framework to enhance its practice in the United States. Special issues for surveillance in environmental public health are examined, and examples of existing systems useful for environmental public health practice are provided. Current and projected surveillance needs, as well as potential sources of data, are examined. The proposed framework for conducting environmental public health surveillance involves data from three points in the process by which an agent in the environment produces an adverse outcome in a host: hazards, exposures, and outcomes. Environmental health practitioners should build on efforts in other fields (e.g., infectious diseases and occupational health) to establish priorities in the surveillance of health conditions associated with exposure to environmental toxicants. For specific surveillance programs, existing data systems, as well as data gaps, should be identified. Coordinated surveillance systems can facilitate public health efforts to prevent and control disease, injury, and disability related to the interaction between people and their environment. PMID- 8629713 TI - Comment: Toward a coordinated system for the surveillance of environmental health hazards. AB - The rapid increases in the numbers and quantities of chemicals released into the environment have been accompanied by a lack of adequate prerelease testing for adverse health outcomes. Environmental health surveillance is used both to track changes in exposures that are known to have adverse health effects and to identify previously unrecognized hazards. Surveillance data can directly aid in the design of interventions to reduce the level of hazardous agents in the environment or the opportunities for human contact with them. Components of an ideal environmental health surveillance system are discussed. For well-recognized hazards, databases related to exposure alone are adequate. However, for uncovering previously unrecognized associations, linkage between exposure and outcome databases that are collected or aggregated at the same geographic scale and for regions of relatively homogeneous exposures are needed. PMID- 8629715 TI - Viral infections in short-term injection drug users: the prevalence of the hepatitis C, hepatitis B, human immunodeficiency, and human T-lymphotropic viruses. AB - OBJECTIVES: The purpose of this study was to estimate the prevalence and correlates of four blood-borne viral infections among illicit drug injectors with up to 6 years of injecting experience. METHODS: We analyzed data from 716 volunteers recruited in 1988 and 1989. Test results for hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus, type 1 (HIV), and human T lymphotropic virus types I and II (HTLV) were examined across six sequential cohorts defined by duration of drug injection. RESULTS: Overall, seroprevalence of HCV, HBV, HIV, and HTLV was 76.9%, 65.7%, 20.5% and 1.8%, respectively, and 64.7%, 49.8%, 13.9%, and 0.5%, respectively, among those who had injected for 1 year or less. Among the newest initiates, HCV and HBV were associated with injecting variables, and HIV was associated with sexual variables. CONCLUSIONS: The high rates of HCV, HBV, and HIV infections among short-term injectors emphasizes the need to target both parenteral and sexual risk reduction interventions early. Renewed efforts at primary prevention of substance abuse are indicated. PMID- 8629714 TI - The estimated prevalence and incidence of HIV in 96 large US metropolitan areas. AB - OBJECTIVES: This study sought to estimate the size and direction of the human immunodeficiency virus (HIV) epidemic in US metropolitan statistical areas (MSAs) with populations greater than 500,000. METHODS: A "components model" from review of more than 350 documents, several large datasets, and information from 220 public health personnel was used. Data review focused on injection drug users, men who have sex with men, and high-risk heterosexual men and women. RESULTS: In the 96 MSAs, there are, broadly, an estimated 1.5 million injection drug users, 1.7 million gay and bisexual men, and 2.1 million at-risk heterosexuals, and, among them, an estimated 565,000 prevalent and 38,000 incident HIV infections. This implies about 700,000 prevalent and 41,000 new HIV infections yearly in the United States. Roughly half of all estimated new infections are occurring among injection drug users, most of them in northeastern cities, Miami, and San Juan. Gay and bisexual men still represent most prevalent HIV infections, although incidence--except in young and minority gay men--is much lower now than it was a decade ago. Relatively high prevalences of HIV in at-risk heterosexual persons in several cities indicate the potential for an increase in transmission among them. CONCLUSIONS: This review and synthesis outline the comparative epidemiology of HIV in major US cities and identify populations for interventions. PMID- 8629716 TI - The risk of suicide among wounded Vietnam veterans. AB - OBJECTIVES: This study was undertaken to determine whether an association exists between combat trauma and risk of postservice suicide among Vietnam veterans. METHODS: Risk of suicide for 34,534 veterans who were wounded in Vietnam was evaluated for severity of wound and number of times wounded. RESULTS: There was a trend of increasing risk of suicide with increased occurrence of combat trauma, the highest relative risk (1.82, 95% confidence interval [CI] = 1.12, 2.96) being observed for those veterans who were wounded more than once and hospitalized for a wound. In comparison with the US male general population, veterans hospitalized because of a combat wound or wounded more than once had a significantly increased risk of suicide (standardized mortality ratios [SMRs] of 1.22 [95% CI = 1.00, 1.46] and 1.58 [95% CI = 1.06, 2.26], respectively). Those wounded more than once and hospitalized had the highest increased risk of suicide (SMR = 1.73, 95% CI = 1.10, 2.60). CONCLUSIONS: This study suggests that, among wounded Vietnam veterans, there is an increased risk for suicide associated with increased occurrence of combat trauma. PMID- 8629717 TI - Choosing a future for epidemiology: I. Eras and paradigms. AB - To inform choices about the future of epidemiology, the present condition of epidemiology is examined, in terms of its evolution through three eras, each demarcated by its own paradigm: (1) the era of sanitary statistics with its paradigm, miasma; (2) the era of infectious disease epidemiology with its paradigm, the germ theory; and (3) the era of chronic disease epidemiology with its paradigm, the black box. The historical context in which these eras arose is briefly described. In each era, the public health was at the center of the concerns of the founders and early protagonists of the prevailing paradigm. Around this intellectual development we weave a further theme. We argue that in the present era, the public health has become less central a concern. At the same time, in epidemiology today the dominant black box paradigm is of declining utility and is likely soon to be superseded. PMID- 8629718 TI - Choosing a future for epidemiology: II. From black box to Chinese boxes and eco epidemiology. AB - Part I of this paper traced the evolution of modern epidemiology in terms of three eras, each with its dominant paradigm, culminating in the present era of chronic disease epidemiology with its paradigm, the black box. This paper sees the close of the present era and foresees a new era of eco-epidemiology in which the deployment of a different paradigm will be crucial. Here a paradigm is advocated for the emergent era. Encompassing many levels of organization- molecular and societal as well as individual--this paradigm, termed Chinese boxes, aims to integrate more than a single level in design, analysis, and interpretation. Such a paradigm could sustain and refine a public health-oriented epidemiology. But preventing a decline of creative epidemiology in this new era will require more than a cogent scientific paradigm. Attention will have to be paid to the social processes that foster a cohesive and humane discipline. PMID- 8629719 TI - Traditional epidemiology, modern epidemiology, and public health. AB - There have been significant developments in epidemiologic methodology during the past century, including changes in basic concepts, methods of data analysis, and methods of exposure measurement. However, the rise of modern epidemiology has been a mixed blessing, and the new paradigm has major shortcomings, both in public health and in scientific terms. The changes in the paradigm have not been neutral but have rather helped change--and have reflected changes in--the way in which epidemiologists think about health and disease. The key issue has been the shift in the level of analysis from the population to the individual. Epidemiology has largely ceased to function as part of a multidisciplinary approach to understanding the causation of disease in populations and has become a set of generic methods for measuring associations of exposure and disease in individuals. This reductionist approach focuses on the individual, blames the victim, and produces interventions that can be harmful. We seem to be using more and more advanced technology to study more and more trivial issues, while the major causes of disease are ignored. Epidemiology must reintegrate itself into public health and must rediscover the population perspective. PMID- 8629720 TI - Genetic screening for reproductive planning: methodological and conceptual issues in policy analysis. AB - OBJECTIVES: This paper explores several critical assumptions and methodological issues arising in cost-effectiveness analyses of genetic screening strategies in the reproductive setting. METHODS: Seven issues that arose in the development of a decision analysis of alternative strategies for cystic fibrosis carrier screening are discussed. Each of these issues required a choice in technique. RESULTS: The presentations of these analyses frequently mask underlying assumptions and methodological choices. Often there is no best choice. In the case of genetic screening in the reproductive setting, these underlying issues often touch on deeply felt human values. CONCLUSIONS: Space limitations for published papers often preclude explaining such choices in detail; yet these decisions determine the way the results should be interpreted. Those who develop these analyses need to make sure that the implications of important assumptions are understood by the clinicians who will use them. At the same time, clinicians need to enhance their understanding of what these models truly mean and how they address underlying clinical, ethical, and economic issues. PMID- 8629721 TI - Clinical and prophylactic trials with assured new treatment for those at greater risk: I. A design proposal. AB - OBJECTIVES: The accepted sine qua non for estimating the difference in efficacy between a new and a standard treatment is a randomized controlled clinical trial. Yet in some situations it is either practically or ethically impossible to conduct such a trial. For example, patients who are desperately ill may decline to participate when they learn they may not receive the new treatment, especially when that treatment is readily available outside the experimental protocol. Likewise, in a prophylactic trial of a promising vaccine, recruitment of persons at greater risk may falter or fail. Our objective is to demonstrate that a rigorous comparison of treatments may still be attainable. METHODS: The features of a controlled clinical or prophylactic trial are reviewed from the perspectives of Food and Drug Administration regulations, ethical considerations, and practical problems. RESULTS: An explicit risk-based allocation method of design and analysis is proposed, one guaranteeing that all subjects at greater risk will receive the new treatment. CONCLUSIONS: Under certain conditions, a risk-based allocation trial can furnish consistent estimates of both standard and experimental treatment effects for those at greater risk while avoiding certain difficulties caused by randomized treatment allocation. PMID- 8629722 TI - Clinical and prophylactic trials with assured new treatment for those at greater risk: II. Examples. AB - OBJECTIVES: The preceding article proposed an assured treatment design that would address certain difficulties in recruiting persons who are at greater risk into randomized clinical trials. The purpose of this article is to illustrate the statistical validity of the design in a practical setting. METHODS: Three actual randomized clinical trials were considered as case studies; in each, the data that would have been obtained under assured allocation were identified. Then, with only these data, together with a reasonable choice of model describing the response of subjects under standard treatment as a function of initial severity, the treatment effect was estimated for the subjects at greater risk. The estimates were compared with conventional estimates for the sicker patients randomized in the original trials. RESULTS: In each case, the estimates produced in the assured treatment trial were close to those observed in the randomized trial. CONCLUSIONS: Risk-based allocation trials deserve serious consideration when randomized clinical trials are difficult or impossible to execute. The proposed designs and analyses would allow physicians to offer persons at greater risk assurance that they would receive the new treatment, while researchers would retain the ability to draw valid statistical conclusions about treatment efficacy. PMID- 8629723 TI - Differences in the misreporting of chronic conditions, by level of education: the effect on inequalities in prevalence rates. AB - OBJECTIVES: Many studies of socio-economic inequalities in the prevalence of chronic conditions rely on self-reports. For chronic nonspecific lung disease, heart disease, and diabetes mellitus, we studied the effects of misreporting on variations in prevalence rates by respondents' level of education. METHODS: In 1991, a health interview survey was conducted in the southeastern Netherlands with 2867 respondents. Respondents' answers were compared with validated diagnostic questionnaires in the same survey and the diagnoses given by the respondents' general practitioners. RESULTS: Misreporting of chronic lung disease, heart disease, and diabetes may be extensive. Depending on the condition and the reference data used, the confirmation fractions ranged between .61 and .96 and the detection fractions between .13 and .93. Misreporting varied by level of education, and although various patterns were observed, the dominant pattern was that of more underreporting among less educated persons. The effects on prevalence rates were to underestimate differences by level of education to a sometimes considerable degree. CONCLUSIONS: Misreporting of chronic conditions differs by respondents' level of education. Health interview survey data underestimate socioeconomic inequalities in the prevalence of chronic conditions. PMID- 8629724 TI - The expanded racial and ethnic codes in the Medicare data files: their completeness of coverage and accuracy. AB - OBJECTIVES: This paper evaluates the new race/ethnicity codes for Asian Americans, Hispanics, and Native Americans that have recently been added to the Medicare enrollment database. METHODS: The race/ethnicity code revisions made by the Health Care Financing Administration are described and evaluated by (1) comparing the numbers of persons identified as Asian Americans, Hispanics, and Native Americans with corresponding population census projections and (2) determining whether Medicare enrollees born in Asian and Hispanic countries are assigned Asian and Hispanic codes. RESULTS: Among persons 65 years of age and older, approximately 24% of Hispanics, 17% of Native Americans, and 56% of Asian Americans are identifiable by the new codes. From 18% to 29% of enrollees 65 years old or older born in Mexico, Puerto Rico, and Cuba are coded as Hispanic, and from 14% to 73% of enrollees born in nine Asian countries are classified as Asian American. Classification is not random but is related to timing of migration and to country of origin. CONCLUSIONS: Researchers should resist the temptation to base analyses on the revised Health Care Financing Administration race/ethnicity codes, since coverage is incomplete and biased. PMID- 8629725 TI - Tracking community sentinel events: breast cancer mortality and neighborhood risk for advanced-stage tumors in Denver. AB - OBJECTIVES: The incidence of related sentinel events--breast cancer mortality and neighborhood-specific morbidity for advanced stage at diagnosis--were calculated for women likely to use a community health center in Denver, Colo. METHODS: For the center's service area, neighborhoods (n = 37) were defined by program use. Mortality rates and proportional hazards regression models were estimated for 4189 breast cancer cases recorded between 1979 and 1990. Neighborhood-specific standard morbidity ratios of advanced-stage tumors were based on age-specific rates applied to the entire community. RESULTS: Service area residents were more likely to present with advanced tumors (odds ratio [OR] = 1.4; 95% [CI] = 1.2, 1.5). After adjustment, advanced-stage disease and socioeconomic-demographic status, but not race-ethnicity, contributed significantly to survival. Two neighborhoods (6.5% of the population at risk) with standard morbidity ratios of 2.1 (95% CI = 1.3, 3.4) and 1.7 (95% CI = 1.2, 2.5) accounted for 42% of the excess cases of advanced-stage tumors between 1986 and 1990. CONCLUSIONS: Neighborhood variation in advanced-stage cancer can serve as the basis for efforts to improve access to breast cancer screening. PMID- 8629727 TI - Adjusting for multiple testing when reporting research results: the Bonferroni vs Holm methods. AB - Public health researchers are sometimes required to make adjustments for multiple testing in reporting their results, which reduces the apparent significance of effects and thus reduces statistical power. The Bonferroni procedure is the most widely recommended way of doing this, but another procedure, that of Holm, is uniformly better. Researchers may have neglected Holm's procedure because it has been framed in terms of hypothesis test rejection rather than in terms of P values. An adjustment to P values based on Holm's method is presented in order to promote the method's use in public health research. PMID- 8629726 TI - Trends in US urban black infant mortality, by degree of residential segregation. AB - Trends in Black infant mortality rates from 1982 through 1991 in large US metropolitan statistical areas were examined. In some least-segregated areas, the total Black infant mortality rate reached a low of 13 per 1000 live births in 1985; it increased sharply after 1985 in the West but not in the South. The explanation for these trends is unknown, but variation in regional trends in Black postneonatal infant mortality rates suggested that social and medical-care differences among Blacks should be examined. A high Black infant mortality rate for a group of most-segregated metropolitan statistical areas persisted and contributed to the rising Black-White ratio of rates. PMID- 8629728 TI - Tuberculosis surveillance in the United States: case definitions used by state health departments. AB - Health departments in all 53 reporting areas in the United States were asked to submit the case definition they used for tuberculosis surveillance. Sixteen areas used the 1990 case definition; two areas sent 1977 guidelines; and 34 areas sent other definitions. Case reports sent to the Centers for Disease Control and Prevention (CDC) in 1992 were analyzed; 4% of cases did not meet the 1990 definition. Tuberculosis case reporting criteria are not uniformly applied in the United States. CDC, in collaboration with state and local health officials, is evaluating the current definition and will implement uniform national criteria for tuberculosis surveillance. PMID- 8629729 TI - The surveillance of birth defects: the usefulness of the revised US standard birth certificate. AB - To assess the sensitivity and positive predictive value of birth defects reported on the 1989 revision of the US Standard Birth Certificate, a population of 76,862 Atlanta-area births during 1989 and 1990 was used as the basis for comparing 771 birth certificates that reported birth defects with 2428 live-born infant records in a birth defects registry that uses multiple sources of case ascertainment. Only 14% of birth defects in the registry records were reported on birth certificates. After the analysis was restricted to defects recognizable at birth, the sensitivity and positive predictive value of the birth certificates were 28% and 77%, respectively. Birth certificates underestimate birth defect rates and should be used cautiously for birth defect surveillance and epidemiological studies. PMID- 8629730 TI - Reporting vaccine-associated paralytic poliomyelitis: concordance between the CDC and the National Vaccine Injury Compensation Program. AB - This paper compares cases of paralytic poliomyelitis reported to the systems operated by the National Vaccine Injury Compensation Program and the Centers for Disease Control and Prevention (CDC) for reporting of adverse events associated with vaccination. Of the 118 cases of vaccine-associated paralytic poliomyelitis determined by either system, 18 were reported initially only to the compensation program, 50 only to the CDC, and 50 to both. The annual incidence of vaccine associated paralytic poliomyelitis determined from data from both systems varied from 6 to 13 cases (mean = 9.1) a year, with an increase of 1.4 cases a year when initial reports only to the compensation program are included. Thus, the compensation program provides important supplemental incidence data. PMID- 8629731 TI - Cuban epidemic neuropathy, 1991 to 1994: history repeats itself a century after the "amblyopia of the blockade". AB - The 1991 to 1994 epidemic of neuropathy in Cuba has been one of the more devastating in recent history, affecting more than 50,000 people throughout the entire country with clinical manifestations of optic and peripheral neuropathy. Although the causes are not entirely clear, it seems that a combination of acute nutritional deficiency and the toxic effects of tobacco and possibly other unidentified toxic substances is involved. The epidemic coincided with the acute worsening of the economic situation on the island following political changes in Eastern European countries and a tightening of the US economic embargo. This paper reviews reports of a strikingly similar epidemic known as the "Amblyopia of the Blockade," which occurred in Cuba almost a century ago when the island was undergoing a US naval blockade during the Cuban-Spanish-American war. It discusses the parallelism with the recent epidemic as well as the implications of this historical evidence to clarify further the ultimate causes of these epidemics. PMID- 8629732 TI - Intensive follow-up of control subjects: is it necessary? PMID- 8629733 TI - Who are these "nonsmokers"? PMID- 8629734 TI - Capture-recapture estimates of cancer incidence, adjusting for geographic effects: an alternative perspective. PMID- 8629735 TI - The Department of Energy's Comprehensive Epidemiologic Data Resource: a public use database on radiation exposure. PMID- 8629736 TI - A graphical method for pooling epidemiological studies. PMID- 8629737 TI - Monitoring vendor compliance with tobacco sales laws: payment vs no payment approaches. PMID- 8629738 TI - Firearm injuries among Virginia juvenile drug traffickers, 1992 through 1994. PMID- 8629739 TI - [The role of rh-erythropoietin in the framework of methods for excluding allogenic blood. Symposium, Mannheim, 13-14 November 1992]. PMID- 8629740 TI - Respiratory effects of a balanced anesthetic technique--revisited fifteen years later. AB - Five hundred and fifty patients underwent general anesthesia with fentanyl, diazepam, and methohexital. Forty-seven (8.5%) developed signs of hypoventilation or airway obstruction. Arterial blood gas analysis revealed mild hypoxemia in three of the 47 cases and mild hypercarbia in six. Airway obstruction was more predictive of abnormal blood gas values than was hypoventilation. PMID- 8629741 TI - Pulse oximetry signals local anesthetic-induced methemoglobinemia. AB - An otherwise healthy patient with a fractured mandible was scheduled to undergo an open reduction under general anesthesia. Just before transport to the operating room, bimaxillary arch bars were placed under local anesthesia with 4% prilocaine and 1:200,000 epinephrine. Although induction of anesthesia and nasoendotracheal intubation were uneventful, pulse oximetry values fell to 89% despite adequate ventilation and an inspired oxygen concentration of 50%. Inquiry by the anesthesiologist and arterial blood gas measurements revealed that methemoglobinemia had developed in response to the large amount 576 mg) of prilocaine administered. A total of 150 mg of methylene blue administered in two doses corrected the problem. The oral surgeon, having recently switched to prilocaine because of a manufacturer's recall of lidocaine, was unaware of the potential of prilocaine to cause this disorder. PMID- 8629742 TI - Asystole and bradycardia during maxillofacial surgery. AB - A Chinese female undergoing maxillary osteotomy developed asystole when the maxillary tuberosity was cut. Surgery was stopped. After about 10 sec and before instituting cardiac massage, sinus rhythm and bradycardia ensued. Atropine was administered intravenously, resulting in an increase in heart rate. No further episodes of asystole or bradycardia were encountered. PMID- 8629743 TI - A case of sinus arrest caused by opening the mouth under general anesthesia. AB - We report a case in which transient sinus arrest was observed under general anesthesia. This was associated with opening the mouth and was thought to be caused by a trigeminovagal reflex. The reflex was interrupted by blocking bilaterally the third division of trigeminal nerve. PMID- 8629745 TI - Use of peak expiratory flow rate in emergency department evaluation of acute exacerbation of chronic obstructive pulmonary disease. AB - STUDY OBJECTIVE: The purpose of the study was to compare peak expiratory flow rate (PEFR) against 1-second forced expiratory volume (FEV1) as a measure of airway obstruction in patients with acute exacerbation of chronic obstructive pulmonary disease (COPD). METHODS: The participants were patients older than 50 years of age who presented with an acute exacerbation of COPD to the emergency department of a large, urban medical center. Pulmonary function was measured with a computerized Fleisch pneumotachygraphic spirometer before, during, and after treatment. PEFR and FEV1 were compared both as absolute values and after conversion to percent of predicted normal values (PPVs). RESULTS: Five hundred fifty-six paired sets of measurements of FEV1 and PEFR were obtained from 199 patients and compared. There was good correlation between PEFR and FEV1, both in terms of absolute value (r=84; P<.001) and in terms of PPV (r=81; P<.001). Despite good correlation, further analysis revealed that there was not uniformly good agreement between the PPVs for FEV1 and PEFR. Although the mean difference between the PPVs obtained from the two measurements was only 4.3%, the 95% limits of agreement ranged widely, from -15% to 24%. CONCLUSION: Although PEFR can be used as an alternative measure of airway obstruction in instances in which FEV1 is not available, there may be clinically significant discrepancies between the two tests. Measurement of the FEV1 is preferable because it allows comparison with baseline studies and previously published guidelines. PMID- 8629744 TI - Effective postoperative pain prevention through administration of bupivacaine and diclofenac. AB - The efficacies of bupivacaine and lidocaine together with a preoperatively administered single-dose oral combination of normal- and sustained-release preparations of diclofenac in preventing postoperative pain after third molar removal were compared in a double-blind crossover study. Bilaterally impacted lower third molars were removed in two sessions. Each patient was given one type of local anesthetic on one session and the other in the second. Pain was recorded using a visual analog scale. When the diclofenac combination (150 mg) was given before the operation, postoperative analgesia was better with bupivacaine plus diclofenac than with lidocaine plus diclofenac. Twenty-five out of 40 patients preferred bupivacaine to lidocaine for local anesthesia. It is possible to achieve effective postoperative pain prevention by combining bupivacaine and preoperative normal- and sustained-release preparations of diclofenac. PMID- 8629746 TI - Effects of a novel hemoglobin-based oxygen carrier on percent oxygen saturation as determined with arterial blood gas analysis and pulse oximetry. AB - STUDY OBJECTIVE: Hemoglobin-based oxygen carrier 201 (HBOC-201) is a polymerized hemoglobin of bovine origin being developed for use in hemorrhage during surgery or trauma. Pulse oximetry is commonly used in clinical practice to assess percent saturation of hemoglobin (Spo2). The ability to measure Spo2 in the presence of HBOC-201 will be important for the use of this compound in patient care. METHODS: We carried out a randomized, single-blind, placebo-controlled study at the Upjohn Research Clinics in Kalamazoo, Michigan, with normal, healthy adult men and women as subjects. The members of four groups of adult subjects (N=24) each received 45 g of HBOC-201 (nine each, men and women) or a control solution (Ringer's lactate) (three each, men and women). Each subject underwent phlebotomy (about 15% of estimated blood volume) followed by 3:1 hemodilution with Ringer's lactate and then either HBOC-201 or control solution. An indwelling arterial catheter in the radial artery was used for serial arterial blood gas sampling. Arterial blood gas measurements were made with a cooximeter (Instrumentation Laboratories). Fingertip pulse oximetry was used (Criticare 504-US; Criticare, Incorporated). Paired pulse oximetry and arterial blood gas sampling were made serially (at approximately hourly intervals) over 24 hours. RESULTS: The mean +/- SEM difference for Spo2 for arterial blood gas analysis compared with the pulse oximetry reading in the presence of HBOC-201 was 1.1% +/- 0.75%; in controls it was .1% +/- 0.64% (P<.001) for each) over the 24 hours after dosing. This relationship was constant despite increased concentrations of plasma hemoglobin (between 1 and 2g/dl [10 to 20 g/L]) in the HBOC-201 groups. CONCLUSION: Accurate determinations of Spo2 can be made with pulse oximetry in subjects given HBOC-201 over the normal range of Spo2. PMID- 8629747 TI - Randomized, double-blind, placebo-controlled trial of intravenous ketamine in acute asthma. AB - STUDY OBJECTIVE: To evaluate the efficacy of IV ketamine in the management of acute, severe asthma. METHODS: This prospective, randomized, double-blind, placebo-controlled clinical trial at an urban teaching hospital emergency department involved 53 consecutive patients aged 18 to 65 with a clinical diagnosis of acute asthmatic exacerbation and a peak expiratory flow of less than 40% of the predicted value after three albuterol nebulizer treatments. All patients received oxygen, continuous nebulized albuterol, and methylprednisolone sodium succinate (Solu-Medrol). Patients then received either ketamine hydrochloride in a bolus of .2 mg/kg followed by IV infusion of .5 mg/kg per hour for 3 hours or a placebo bolus and infusion for 3 hours. Because of the occurrence of dysphoric reactions, the bolus dose was lowered to .1 mg/kg after the first 9 patients; the infusion dose was kept the same. RESULTS: The first nine patients were eliminated from analysis. Repeated ANOVA testing on the remaining 44 patients determined significant improvements over time within each treatment group in peak flow (F=3.637, P=.004). Borg score (F=22.959, P=.001), respiratory rate (F=8.11, P=.0001). and 1-second forced expiratory volume (F=9.076, P=.001). However, no difference could be detected over time between treatment groups (power, 80%). Patients receiving ketamine gave the treatment a rating of 4.3 on a scale of 1 to 5, whereas those receiving placebo scored their treatment 3.7 (P=.0285). The hospital admission rate was not different between treatment groups (P=.1088). CONCLUSION: IV ketamine at a dose low enough to avoid dysphoric reactions demonstrated no increased bronchodilatory effect compared with standard therapy in treating exacerbations of asthma in the ED. Although there was a slight increase in satisfaction in the ketamine group, no clinical benefit in terms of hospital admission rate was noted. PMID- 8629748 TI - Appropriateness of hospitalization of patients with community-acquired pneumonia. AB - STUDY OBJECTIVE: To investigate the association between the appropriateness of hospitalization and the course of hospitalization in patients with community acquired pneumonia (CAP). METHODS: We carried out a prospective study of 346 adult patients hospitalized with community-acquired pneumonia (CAP). Appropriateness of hospitalization was assessed with a modified appropriateness evaluation protocol (AEP) based on vital signs and laboratory tests. Hospitalizations that ended in death, lasted more than 4 days, or involved resuscitation, incubation, monitoring, or supplemental oxygen therapy were considered complicated. RESULTS: According the AEP protocol, hospitalization was not appropriate for 210 of the 346 patients (61%). However, AEP proved to be an insensitive tool for the identification of patients with complicated hospital courses. Half of the 346 patients had complicated courses, including 82 of the 210 patients with inappropriate hospitalization (39%), according to the AEP. Four independent factors, age greater than 50 years, female sex, no antibiotic treatment before hospitalization, and more than 4 days of illness before admission predicted a complicated course in patients with inappropriate hospitalization as determined with the AEP criteria. CONCLUSION: It is important to avoid the unnecessary hospitalization of patients with CAP. However, this should not be achieved at the expense of unjustified discharge from the emergency department. In the decision to hospitalize, additional prognostic factors, such as those presented here, should be taken into consideration to improve the admission process. This is particularly relevant for cases in which the AEP is invalid and indications for hospitalization are not clear cut. In these patients, a simpler and more precise scoring system should be developed. PMID- 8629749 TI - Signal analysis of the human electrocardiogram during ventricular fibrillation: frequency and amplitude parameters as predictors of successful countershock. AB - STUDY OBJECTIVE: To determine whether there is information in the human ventricular fibrillation (VF) ECG signal that is predictive of successful countershock. METHODS: We carried out a retrospective analysis of ECG signals recorded during out-of-hospital treatment of adult patients in VF. Four parameters--centroid frequency (FC), peak power frequency (FP), average segment amplitude (SA), and average wave amplitude (WA)--were extracted from the recorded ECG signal immediately before each countershock and compared with countershock outcome. RESULTS: The outcome of each countershock (total, 128 countershocks) administered to 55 patients in VF was determined from available emergency medical services data sheets and time-domain ECG signal and voice recordings. The original 4-second time-domain ECG segment immediately before the countershock was used to extract SA and WA. The 4-second ECG segment immediately before each countershock was transformed into the frequency domain by means of Fourier analysis, and the parameters FC and FP were extracted from the result. These parameters were compared with countershock outcome by means of Kolmogrov-Smirnov analysis. Sensitivity and specificity of these parameters, as well as receiver operating characteristic curves, were constructed. FC was statistically higher for successful countershocks (FC, 5.48 +/- .67 Hz) than for successful countershocks (FC, 4.85 +/- 1.16 Hz; P=.012). We found no statistical difference for FP (P=.066), SA (P=.549), and WA (P =.337). FP and FC, when used in combination and in certain ranges (3.5 Hz < or = FP < or = 7.75 Hz and 3.86 Hz < or = FC < or = 6.12 Hz) had a sensitivity of 100% and a specificity of 47.1% in predicting successful countershock. The probabilities of predicting countershock outcome for FC, FP, SA, and WA were .72, .70, .52, and .53, respectively. CONCLUSION: FC and FP are predictive of countershock outcome for patients in VF and hold the potential to guide therapy during cardiac arrest. PMID- 8629750 TI - Feasibility study of the use of bilevel positive airway pressure for respiratory support in the emergency department. AB - STUDY OBJECTIVE: To determine the feasibility of bilevel positive airway pressure (BiPAP) support for acute respiratory distress (ARD) in the emergency department. METHODS: A convenience sample of patients in ARD as a result of any nontraumatic cause was recruited for a prospective, noncontrolled clinical trial in the ED of an urban tertiary care teaching hospital. Hemodynamically unstable patients and those requiring immediate endotracheal intubation were excluded. After an initial arterial blood gas (AGB) analysis was obtained, the patient was placed on BiPAP (Bi-PAP S/T noninvasive ventilator; Respironics, Incorporated) by nose mask or face mask to provide noninvasive pressure support at 5 cm H20. Settings were titrated to patient tolerance and satisfactory pulse oximetry. After at least 30 minutes on a stable setting, arterial blood gases were remeasured. The cause of respiratory distress, vital signs, assessment of need for intubation, arterial blood gas results, and patient disposition were recorded. Success of noninvasive support was defined as the presence of (1) improvement in ABG parameters, (2) clinical improvement and decrease in evident dyspnea, and (3) avoidance of endotracheal intubation and mechanical ventilation. RESULTS: Fifty patients were studied. Causes of ARD included acute congestive heart failure (CHF; n=16), exacerbation of chronic obstructive pulmonary disease (COPD; n=9), mixed COPD/CHF (n=3), pneumonia (n=10), status asthmaticus (n=6), and other causes of acute respiratory failure (eg, stroke, overdose; n=6). Noninvasive management was successful in 43 patients (86%), with patients in all etiologic categories being equally likely to respond favorably to therapy. All patients were admitted to the hospital, but 52.5% of those who ordinarily would have required ICU beds were admitted to lower (and less costly) levels of care. Three patients were eventually intubated, all after admission to the ICU on BiPAP. Two patients did not tolerate BiPAP, and two others were considered ED treatment failures but were not intubated because of advance directives. CONCLUSION: As has been reported from other critical care settings, use of BiPAP is feasible and has potential utility in the management of ARD in the ED. PMID- 8629751 TI - Test of the acute cardiac ischemia time-insensitive predictive instrument (ACI TIPI) for prehospital use. AB - STUDY OBJECTIVES: To test diagnostic performance for acute cardiac ischemia (ACI) in a manually calculated and in a computerized, ECG-calculated ACI time insensitive predictive instrument (ACI-TIPI) in prehospital chest pain patients. METHODS: We carried out prospective inclusion and data acquisition with retrospective analysis. Over a 6-month period, 439 adult emergency medical services patients with chest pain underwent prehospital electrocardiography. Because of incomplete data, 77 cases were excluded, leaving a study sample of 362 patients. Excluded patients did not differ significantly with respect to age, sex, final diagnosis, or history of myocardial infarction, heart surgery, diabetes, or stroke. ACI-TIPI probabilities of ACI were computed on the basis of the prehospital ECGs as interpreted retrospectively and independently by two study investigators blinded to patient outcome, with a specially programmed electrocardiograph, and with a computer algorithm further modified by logistic regression analysis. RESULTS: Diagnostic performance on the basis of receiver operating characteristic (ROC) curve areas of the ACI-TIPI was scored, by the two physician readers, .73 and .74; and by ECG, .75. Patients with low ACI-TIPI probability (0% to 9%) had no acute myocardial infarctions, a 2.3% incidence of angina, and no prehospital life-threatening events. CONCLUSION: ACI-TIPI probabilities of ACI as generated by a specially programmed electrocardiograph are comparable to those based on physician ECG interpretations and may be useful in the prehospital evaluation of chest pain. PMID- 8629752 TI - Geriatric use of emergency medical services. AB - STUDY OBJECTIVE: To quantify use by geriatric patients of emergency medical services (EMS) compared with that by young adult patients. METHODS: We conducted a retrospective, consecutive case series over a 6-month period in a suburban, all paramedic municipal EMS system serving 76,500 residents, of whom approximately 15% are 65 years of age or older and 33% are between 25 and 45 years old. Patient age, the sole entry criterion, was used to distinguish two groups: the young adult group, defined as patients 25 to 45 years old; and the geriatric group, defined as patients 65 years or older. RESULTS: Of the 2,712 patients whose cases were reviewed during the study period, 1,734 (65%) met the entry criterion. The geriatric group (n=1,043) accounted for 39% of the total call volume, compared with the young adult group (n=690), which accounted for 25% of total call volume. Patients in the young adult group were 7.3 times more likely to have been in a motor vehicle accident, whereas the GP group was 2.6 times more likely to have cardiorespiratory complaints, 1.8 times more likely to have fallen, and 1.7 times more likely to have minor medical problems requiring transportation and more frequently required advanced life support (ALS) care (54% versus 33%) (P<.001 for all comparisons). Scene times for geriatric patients were found to be longer than those for young adults (ALS, P<.001; basic life support [BLS], P<.05). However, costs billed to the patient were greater for young adults for all care rendered (BLS, P<.001; ALS, P<.05). CONCLUSION: Use by geriatric patients of EMS differed significantly from that by young adults. Geriatric patients used EMS more frequently and required more ALS care than did young adults. Although geriatric patients required longer scene times for EMS care, young adults incurred greater charges for service. These findings, although perhaps system specific, speak to the need for ongoing analysis of EMS health care delivery to better serve a population increasing in age. PMID- 8629753 TI - Injuries and small-wheel skates. AB - STUDY OBJECTIVE: To determine the types of injuries sustained during the use of in-line skates and to compare them with injuries sustained during the use of roller skates and skateboards, which have similar riding mechanics; and to assess the protection afforded by wrist, elbow, and knee guards. METHODS: The study population was a consecutive series of injured patients who presented to the emergency department of a Level 1 trauma center between May 1992 and October 1993. RESULTS: Of the 137 patients with skating injuries evaluated in the ED during the study period, 63 (46%) were in-line skaters, 36 (26%) were roller skaters, and 38 (28%) were skateboarders. Minor injuries (sprains, bruises, lacerations) were more common than fractures, and there was no statistical difference in the types of injury between skate groups (P=NS). The most common serious injury was fracture of the distal arm, which occurred in each of the three skater groups (43%, n=59). Of these patients 37% (n=21) required open or closed orthopedic reduction. More fractures of the distal forearm or elbow occurred among skaters who had not been wearing wrist guards (P=.013; risk ratio, 2.07; 95% confidence interval, 1.37 to 3.13). Only 25% of skaters used any protective equipment. In-line skaters owned and used protective equipment more often than did roller-skaters or skateboarders. Most injuries occurred while the patient was travelling in the street or on the sidewalk. Injuries occurred more commonly because the skater was going too fast (35%), because the skater struck an object in the pavement (20%), or because the skater was unable to brake (19%) than because of equipment failure (2%) or interference from motor vehicles (3%). CONCLUSION: Injuries sustained by in-line skaters were similar to those sustained by roller skaters and skateboarders. The risk of wrist or elbow fracture is greater when wrist guards are not worn. PMID- 8629754 TI - Safe at home? Domestic violence and other homicides among women in New Mexico. AB - STUDY OBJECTIVE: To define the contribution of domestic violence (DV) to homicides in women in New Mexico and to examine differences in ethnicity, mechanism, previous documented injuries, incidence of sexual assault, and use of alcohol or illicit drugs between DV- and non-DV-related homicides. METHODS: We carried out a retrospective analysis of reports of the state office of the medical investigator (OMI) reports from all female homicides from 1990 to 1993 in New Mexico. A homicide was defined as being related to DV if the perpetrator was a current or former male intimate partner. The chi-squared and Mann-Whitney tests were used to analyze data. RESULTS: The OMI investigated 134 homicides in women for an overall fatality rate of 4.3 per 100,000. A male intimate partner was the perpetrator in 62 cases (46%). The rate of DV homicide among American Indians (4.9 per 100,000) was significantly higher than that among Hispanics (1.7) and non-Hispanic whites (1.8)(RR=2.8; 95% confidence interval (CI), 1.5 to 5.1). Firearms were almost two times as likely to be used in DV homicides as in non-DV homicides (RR=1.8; 95% CI, 1.2 to 2.6). Evidence of old injuries was found more often in DV homicide cases (35.5%) than in non-DV cases (83%) (RR=4.3; 95% CI, 1.8 to 9.8). The presence of alcohol or other drugs was higher among non-DV homicide victims (69%) than DV homicide victims (54.3%) (P=.03). CONCLUSION: American Indian women are at particularly high risk of homicide, including DV homicide. Firearms were overrepresented in DV homicides, suggesting that removing firearms from the homes of previous DV perpetrators would be a useful public health strategy. Alcohol or illicit drugs were found in approximately two thirds of New Mexico women who were victims of homicide. The high prevalence of history of previous injuries among DV homicide victims indicates that early identification of DV victims in the emergency department and other health care settings is an important point of intervention. PMID- 8629755 TI - Effect of integration of injury control information into a high school physics course. AB - STUDY OBJECTIVE: To investigate the effects on knowledge, attitudes, and self reported behaviors of a 1-week course of injury control and crash safety information integrated within a high school physics curriculum. METHODS: Students in an intervention high school (n=129) were compared with students in a control high school (n=74) enrolled in a comparable physics curriculum. A standardized survey was administered before instruction (time T1), and at 2 weeks (T2) and 6 months (T3) after instruction was completed. The behaviors measured were self reported use of seat belts, speeding, drinking and driving, and intention to use seat belts in the future. RESULTS: At T2, students in the intervention group reported attitudes that were less favorable toward risk-taking in regard to speeding and seat belt use than those of the control group. At T3, there was still a difference in attitudes toward speeding but not toward seat belt use. The intervention significantly altered the knowledge level of the course participants, and these changes persisted to T3. The strongest and most persistent change was that students in the intervention group reported increasing their use of seat belts when riding as a passenger. (Seat belt use as a driver was high for both groups.) The intervention group showed a significant increase in their 1-year intentions to use seat belts both as a driver and as a passenger. CONCLUSION: This study demonstrated that driver safety education can be successfully integrated into a mainstream high school science curriculum. Future studies measuring the effects of this curriculum on observed behaviors are needed. PMID- 8629756 TI - Health maintenance organizations: managed care or mismanaged care? PMID- 8629757 TI - The budget crisis and emergency medicine: a view from Los Angeles. PMID- 8629758 TI - TennCare in the emergency department: the first 18 months. PMID- 8629759 TI - Health care reform and the safety net. PMID- 8629760 TI - Correlation versus agreement: methods of measurement in medicine. PMID- 8629761 TI - Ethical dilemmas in emergency medical services: the perspective of the emergency medical technician. PMID- 8629762 TI - Asthma research: future directions for emergency medicine. The NHLBI Asthma Working Group. PMID- 8629763 TI - Emergency, disaster, and defense medicine: the Swedish model. PMID- 8629764 TI - Unusual cause of abdominal pain: pelvic vein thrombosis in a nonpregnant woman. AB - We present a case of isolated pelvic vein thrombosis in which clinical diagnosis was difficult and the results of routine diagnostic tests were normal. We eventually used ventilation-perfusion scanning and abdominal computed tomography to make the correct diagnosis. PMID- 8629765 TI - Five days of whole-bowel irrigation in a case of pediatric iron ingestion. AB - The maximum duration and volume of polyethylene glycol electrolyte solution (PEG ELS) that can be safely administered during whole-bowel irrigation of the poisoned patient are poorly defined. We present a case of a 33-month-old boy who ingested at least 160 mg/kg elemental iron and received 44.3 L of PEG-ELS (2,953 ml/kg) over 5 days because of the persistence of iron tablets in teh gastrointestinal tract. The child remained clinically well after initiation of PEG-ELS therapy, and further significant iron absorption did not appear to occur. The rectal effluent cleared within 2 days of the start of PEG-ELS therapy despite the persistence of iron in the gastrointestinal tract as shown on radiography. No adverse effects resulted from teh large volume or duration of the PEG-ELS therapy. This is the greatest reported volume of PEG-ELS to be used for whole bowel irrigation in the treatment of a toxic ingestion. PMID- 8629766 TI - Sternoclavicular septic arthritis in a patient with end-stage liver disease. AB - Sternoclavicular septic arthritis is an uncommon clinical entity that is often misdiagnosed on initial presentation. It has generally been described in IV heroin users and immunocompromised hosts. We report the case of a 43-year-old woman with endstage liver disease who presented with a fever, a painful sternoclavicular joint, and gastrointestinal bleeding. The clinical presentation, diagnosis, and treatment of sternoclavicular septic arthritis are reviewed. PMID- 8629767 TI - Wayne and his bipap babushka. PMID- 8629768 TI - Velocity and air gun injuries. PMID- 8629769 TI - "ALS" and "BLS": outdated terminology? PMID- 8629770 TI - Chemical decontamination. PMID- 8629771 TI - Using metered-dose inhalers. PMID- 8629772 TI - Efficacy of widow spider antivenin. PMID- 8629773 TI - Proceedings of the Second Chicago Symposium on Advances in CPR Research and guidelines for laboratory research: foreword. PMID- 8629774 TI - Resuscitation medicine research: quo vadis. AB - Laboratory research should have clinical relevance. Topics should be selected according to need, gaps in knowledge, and opportunities; the investigator's background, expertise, interests, and ambitions; scientific, clinical, and socioeconomic importance; and feasibility of successful performance and conclusion. The current explosion of knowledge and sophistication of methods will require research by multidisciplinary teams. Systematic goal-oriented studies should be conducted in environments that encourage serendipitous discoveries. Mechanism- and outcome-oriented research, in laboratories and on patients, is needed. In cardiac arrest research, hearts and brains "too good to die" offer many challenges. In trauma research, particular challenges include protection preservation during uncontrolled hemorrhagic shock, suspended animation for delayed resuscitation in exsanguination, and prevention of brain swelling after traumatic brain injury. Emergency physicians have the unique opportunity to initiate clinical resuscitation research in unexplored territory: the prehospital arena. PMID- 8629775 TI - Newer methods of improving blood flow during CPR. PMID- 8629776 TI - Aortic-based therapy for cardiac arrest. AB - After the failure of electrical countershock, the successful treatment of cardiac arrest is a function of raising aortic pressure so as to improve vital organ perfusion. Pharmacologic pressor agents have until recently been the most direct means of increasing aortic pressure. We have now begun to reevaluate direct aortic techniques including occlusion, infusion, counter-pulsation, and combinations of these. Clinical studies have demonstrated that the aorta can be accessed quickly and reliably even under emergency conditions. Initial laboratory studies indicate that some nonpharmacologic aortic therapies hold promise as adjuncts to external chest compression, or even as stand-alone therapies. Considerable research will be needed to identify the most effective approach before clinical trials can be considered. PMID- 8629777 TI - Reassessing the need for ventilation during CPR. AB - In the United States debate continues about the necessity of ventilation during CPR because of fear of contracting infectious diseases. Three questions will be considered in this article. First, is ventilation necessary for the treatment of cardiac arrest? Second, is mouth-to-mouth ventilation any better than no ventilation at all? Third, are other techniques of ventilation as effective or more effective than mouth-to-mouth ventilation during basic life support CPR? Although research is still inconclusive with regard to the need for ventilation during CPR, recent findings have clarified the effect of ventilation during low blood flow states and how ventilation influences resuscitation. Ventilation affects oxygenation, carbon dioxide elimination, and pH during times of low rates of blood flow. Ventilation may be unnecessary during the first few minutes of CPR. Under conditions of prolonged, untreated cardiac arrest, ventilation during CPR affects return of spontaneous circulation. Isolated hypoxemia and hypercarbia independently have adverse effects on survival of cardiac arrest. Because ventilation with exhaled gas contains as much as 4% CO2 and less oxygen than air, it may have adverse effects during CPR. Spontaneous gasping may provide sufficient ventilation during CPR. Chest compression alone provides some pulmonary ventilation and gas exchange. Active chest compression-decompression may improve gas exchange better than does standard chest compression. Other forms of manual ventilation may also have a role in CPR. PMID- 8629778 TI - The mystery of bradyasystole during cardiac arrest. PMID- 8629779 TI - Global brain ischemia and reperfusion. AB - Brain damage accompanying cardiac arrest and resuscitation is frequent and devastating. Neurons in the hippocampus CA1 and CA4 zones and cortical layers III and V are selectively vulnerable to death after injury by ischemia and reperfusion. Ultrastructural evidence indicates that most of the structural damage is associated with reperfusion, during which the vulnerable neurons develop disaggregation of polyribosomes, peroxidative damage to unsaturated fatty acids in the plasma membrane, and prominent alterations in the structure of the Golgi apparatus that is responsible for membrane assembly. Reperfusion is also associated with vulnerable neurons with prominent production of messenger RNAs for stress proteins and for the proteins of the activator protein-1 complex, but these vulnerable neurons fail to efficiently translate these messages into the proteins. The inhibition of protein synthesis during reperfusion involves alteration of translation initiation factors, specifically serine phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (elF-2 alpha). Growth factors--in particular, insulin--have the potential to reverse phosphorylation of elF-2 alpha, promote effective translation of the mRNA transcripts generated in response to ischemia and reperfusion, enhance neuronal defenses against radicals, and stimulate lipid synthesis and membrane repair. There is now substantial evidence that the insulin-class growth factors have neuron-sparing effects against damage by radicals and ischemia and reperfusion. This new knowledge may provide a fundamental basis for a rational approach to "cerebral resuscitation" that will allow substantial amelioration of the often dismal neurologic outcome now associated with resuscitation from cardiac arrest. PMID- 8629780 TI - Esophageal detector device versus detection of end-tidal carbon dioxide level in emergency intubation. AB - STUDY OBJECTIVES: To confirm the ability of the esophageal detector device (EDD) to indicate positioning of endotracheal tubes (ETTs) in patients intubated under emergency conditions and to compare the performance of the EDD with that of end tidal carbon dioxide (ETCO2). METHODS: This single-subject study comprising a prospective case series was conducted in the emergency department of an urban university hospital. All adult patients were intubated either in the ED or by paramedics in the field. ETT position was initially evaluated by means of auscultation, then EDD, and, finally, spectrographic qualitative ETCO2 monitoring in each patient. Discrepancies between the EDD and ETCO2 results were resolved by means of direct laryngoscopy. RESULTS: In 100 intubated patients, both the EDD and ETCO2 monitoring detected the single esophageal intubation that occurred. Of the remaining 99 tracheal intubations, the EDD correctly indicated tracheal placement in 98 (sensitivity, 99%) and was indeterminate in 1 case because of blockage of the ETT by secretions resulting from pulmonary edema. By comparison, ETCO2 monitoring correctly indicated tracheal placement in 86 cases (sensitivity, 87%) and was incorrect in 13 cases (P < .01). ETCO2 monitoring failed in 2 patients with pulmonary edema and in 11 patients with cardiac arrest. Among the 37 patients in the cardiac arrest group, the EDD correctly indicated ETT placement in 37 patients (sensitivity, 100%). In contrast, ETCO2 monitoring correctly indicated ETT placement in 26 patients (sensitivity, 70%; P < .01). CONCLUSION: The EDD reliably confirms tracheal intubation in the emergency patient population. The EDD is more accurate than ETCO2 monitoring in the overall emergency patient population because of its greater accuracy in cardiac arrest patients. [Bozeman WP, Hexter D, Liang HK, Kelen GD: Esophageal detector device versus detection of end-tidal carbon dioxide level in emergency intubation. PMID- 8629781 TI - Using tomography to diagnose occult ankle fractures. AB - STUDY OBJECTIVES: To determine the incidence of occult ankle fractures and to determine whether an ankle effusion identified radiographically correlated with the presence of an occult fracture. METHODS: Attending physicians in the emergency department of a university hospital prospectively identified patients with acute ankle injuries that produced ecchymosis or edema over two or more major ligaments of the ankle. Plain radiographs of these patients demonstrated ankle effusions without fractures. A consecutive sample of patients with acute ankle injuries was selected. Thirty-six of 1,151 patients were eligible to enter the study; 2 of these patients subsequently were excluded from the data analysis. All eligible patients underwent plain ankle tomography at 3-mm intervals in the coronal and sagittal planes. The types and frequencies of occult ankle fractures identified on plain film tomography were noted. The distention of the ankle joint capsule of enrolled patients was measured to determine if the degree of effusion correlated with the presence of an occult fracture. RESULTS: The minimum incidence of occult fractures in the general population with ankle injuries was 1%. Twelve of 34 patients (35%) with acute, severe injuries had occult fractures. Depending on the size, an effusion was correlated with the presence of an occult fracture in 35% to 85% of the study population. CONCLUSION: Plain film tomography should be used when evaluating ankle injuries if both edema or ecchymosis over two or more stabilizing ligaments and radiographic evidence of an effusion are present simultaneously. PMID- 8629782 TI - ACEP chest pain policy: emergency physician awareness. AB - STUDY OBJECTIVE: To assess emergency physician knowledge of and effect on clinical practice of the ACEP "Clinical Policy for Management of Adult Patients Presenting With a Chief Complaint of Chest Pain, With No History of Trauma." METHODS: We conducted a written survey of attending physicians by mail and in person at emergency medicine meetings. Residents were surveyed at their academic departments. The survey recipients were emergency physicians and residents from university, community teaching, urban, and rural hospitals. RESULTS: Of the 338 surveyed physicians, 163 (48%; 95% confidence interval [CI], 43% to 54%) said that they were aware of the policy. Fifty-four percent of ACEP members said they were aware of the policy, compared with 24% of nonmembers. Of the physicians who said they were aware of the policy, 63% did not know that the policy contains rules. Seventy-one percent of the physicians who were aware of the policy incorrectly believed the policy requires treatment with thrombolytic therapy in certain cases of myocardial infarction. Physicians said they learned about the policy by reading it (42%), by word of mouth (16%), or in department/quality assurance meetings (13%). Twelve percent of physicians believe the policy has changed their clinical practice. CONCLUSION: Fewer than half the emergency physicians we surveyed were aware of the policy. Of the physicians who said they had been aware of the policy, most did not know important specifics of the policy. PMID- 8629783 TI - Effect of selective aortic arch perfusion on median frequency and peak amplitude of ventricular fibrillation in a canine model. AB - STUDY OBJECTIVE: To determine whether the computer-derived measures of median frequency or peak amplitude of ventricular fibrillation (VF), obtained by fast Fourier transform of the VF waveform, change during selective aortic arch perfusion in a canine model of cardiac arrest. METHODS: Eight mongrel dogs (including 4 control animals) were sedated, intubated, catheterized, and instrumented to record the electrocardiogram (digitally at 100 Hz, filtered with a finite impulse response filter at 2 Hz), right atrial pressure, and aortic pressure during resuscitation in a model of VF-induced cardiac arrest. After 10 minutes of VF-induced arrest, cardiopulmonary resuscitation (CPR) with a mechanical chest compression device was initiated. Beginning 2 minutes later, the 4 study animals received, every 2 minutes, 45 seconds of selective aortic arch perfusion (SAAP) with autologous blood infusions under high pressure. Defibrillation was attempted after 3 minutes of CPR and every minute thereafter. Both study and control groups received standard-dose epinephrine (.01 mg/kg) every 3 minutes by means of an intraaortic catheter. The median frequency, peak amplitude, and coronary perfusion pressure (CPP) during the 5-second period just before defibrillation were obtained with the use of computer algorithms. RESULTS: All SAAP animals and 1 control animal were resuscitated. Baseline measures of median frequency (8.4 +/- 1.5 versus 6.6 +/- 1.0 Hz) and peak amplitude (.18 +/- .05 versus .36 +/- .13 mV) were not different between the SAAP and control groups, respectively, at the start of CRP. SAAP infusion resulted in significant increases in the SAAP group compared with the control group: median frequency, 9.6 +/- .4 versus 7.3 +/- 1.4 Hz; peak amplitude, .74 +/- .21 versus .39 +/- .15 mV; and CPP, 40.5 +/- 7.1 versus 18.0 +/- 15.0 mm Hg, respectively. Median frequency correlated with CPP (r2 = .67). Peak amplitude did not correlate with CPP (r2 = .06). CONCLUSION: Median frequency and peak amplitude increase with SAAP during cardiac arrest in a canine model. This method of resuscitation was reliable in allowing restoration of a stable perfusing rhythm after defibrillation. Changes in measures of peak amplitude and median frequency may reflect interventions that enhance the likelihood of successful defibrillation and may thereby offer a noninvasive means of monitoring interventions during cardiac arrest. PMID- 8629784 TI - Efficacy of several modes of continuous-flow insufflation for resuscitation of a canine model of acute respiratory arrest. AB - STUDY OBJECTIVE: To test the efficacy of several modes of continuous-flow insufflation on the maintenance of physiologic parameters in a model of respiratory arrest, and the effect of these modes on neurologic outcome. METHODS: Anesthetized dogs were slowly infused with tetrodotoxin over 75 minutes to the point of respiratory arrest. We used two different modes of continuous-flow insufflation: endobronchial insufflation (EI) of air 3 cm distal to the carina (.25 or 1.0 L.kg-1.min-1); and tracheal insufflation of oxygen (TRIO) 1 cm proximal to the carina (.08 or .2 L.kg-1.min-1). RESULTS: EI at either flow rate provided ventilation sufficient to allow the dogs to recover effective spontaneous breathing and be removed from ventilation after 4 hours. By this time, almost all cardiovascular variables and blood gas values were normal. TRIO at .2 L.kg-1.min-1 also resulted in successful recovery, although Pa02, as well as systemic and pulmonary arterial pressures and vascular resistances, remained increased at the end of the 4-hour period. TRIO at the low flow rate, however, resulted in deterioration of blood gas values and systemic arterial pressure; dogs required conventional mechanical ventilation after 45 minutes of low-flow TRIO. CONCLUSION: EI can be used to maintain oxygenation in acute respiratory arrest when conventional techniques are not feasible; TRIO at .2 L.kg-1.min-1 is also effective. PMID- 8629785 TI - Factors associated with the higher traumatic death rate among rural children. AB - STUDY OBJECTIVE: To examine medical and demographic factors associated with traumatic deaths among children in Kentucky. METHODS: This was a retrospective review and multiple regression analysis of all deaths in children younger than 18 years reported to the Kentucky Office of Vital Statistics from 1988 to 1992. RESULTS: All 1,024 pediatric trauma deaths that occurred from 1988 to 1992 were analyzed. Death rates were calculated for each type of trauma for each county in the state. Motor vehicle accidents accounted for most of the pediatric deaths, but this finding was markedly age dependent. Death rates were higher in rural Kentucky for all forms of trauma and were highest in the Appalachian region. Multiple Poisson regression analysis identified variables associated with the traumatic pediatric death rates. Rural setting was associated with higher traumatic death rates, whereas the availability of a hospital with 24-hour emergency services in the county and the presence of advanced life support prehospital care were associated with lower death rates. Children in Appalachia were at an increased risk compared with other Kentucky children, even when we controlled for the rural nature of Appalachia. CONCLUSION: Demographic and medical system factors are associated with traumatic death rates in Kentucky children. Access to care and advanced prehospital support were both significantly associated with lower pediatric death rates. Increased access to quality care and training of prehospital providers in advanced life support should be priorities in the planning of trauma systems for this state. PMID- 8629787 TI - Relationship of timeliness of paramedic advanced life support interventions to outcome in out-of-hospital cardiac arrest treated by first responders with defibrillators. AB - STUDY OBJECTIVE: We sought to determine whether the interval between the arrival of first responder/defibrillators and paramedic advanced life support (ALS) interventions is associated with outcome. METHODS: We carried out a prospective observational study of adults in out-of-hospital cardiac arrest treated by both first responders and paramedics in an urban emergency medical services system between July 15, 1992, and May 27, 1993 (N = 544). RESULTS: The gap between first responder and medic arrival was short (3.2 minutes); medics arrived before first responder shock in 22% of ventricular fibrillation (VF) cases. Just 10% of patients has a pulse when medics arrived, but the presence of pulse on medic arrival was a powerful predictor of hospital discharge (odds ratio [OR], 20.5; sensitivity, 39%; specificity, 98%; positive predictive value, 55%; negative predictive value, 97%) or a Cerebral Performance Category score on discharge of 1 or 2 (OR, 2.9). No response or individual ALS treatment interval was related to outcome, including the interval from first-responder to medic arrival. ALS interventions by medics were associated with poorer outcomes; even the need for nothing more than additional defibrillation by medics decreased the survival rate of VF patients threefold. By contrast, bystander CPR improved survival more than fourfold and early defibrillation of VF by first responders more than ninefold. Ninety-one percent of all patients discharged from the hospital who received only minimal ALS other than intubation had good neurologic outcome and longer survival after discharge. Half the total survivors of VF arrest (and 59% of all arrest survivors) were resuscitated by medics with aggressive ALS measures, but 80% had very poor neurologic outcomes and 50% died within a year of hospital discharge. Even the need for only additional defibrillation by medics worsened neurologic outcome by a factor of 2.8. CONCLUSION: Faster response by medics, or any individual ALS intervention other than first-responder defibrillation, demonstrated no benefit in this urban population with short intervals between responder arrivals. Aggressive ALS increased the number of survivors but also decreased their neurologic quality. The benefit of rapid ALS backup to first responder/defibrillators needs further study in other systems. System performance cannot be judged without knowledge of neurologic outcome. PMID- 8629786 TI - Adolescent health care in a pediatric emergency department. AB - STUDY OBJECTIVE: We examined the use of the pediatric emergency department of an urban children's hospital by adolescents. This study included visits by all adolescents aged 13 to 18 years in a 1-week period from each season during 1992. RESULTS: The ED saw 426 adolescents, representing approximately 15% of the total ED visits. Fifty percent of these patients were male. More than half of the patients were black; a few were members of other minority groups. Eighteen percent had emergency, 60% urgent, and 21% nonurgent conditions. Forty-three percent of the patients came to the ED between 3 PM and 11 PM. Injuries accounted for 47% of male visits and 42% of female visits. Approximately half of the injuries resulted from violent events. Twenty-seven percent of the visits were for exacerbation of a chronic illness such as asthma or diabetes. The most common reason for adolescent female visits was gynecologic problems. Injury was the most common reason for adolescent male visits. Only 27% of the adolescents lived in a two-parent home. In 16% of the cases, the adolescents were treated without consent. Nineteen percent of the adolescents were uninsured, and nearly 50% were publicly insured. CONCLUSION: Issues of violence, consent, and insurance present problems for many adolescents in the pediatric ED. Injuries, particularly those related to violent events, are cause for many adolescent visits. ED staff members should develop plans to care for the complex psychosocial and medical problems of adolescents. PMID- 8629788 TI - Field termination of unsuccessful out-of-hospital cardiac arrest resuscitation: acceptance by family members. AB - STUDY OBJECTIVE: To determine whether family members accept field termination of unsuccessful out-of-hospital cardiac arrest resuscitation. METHODS: We carried out a prospective cohort study, using a structured interview, in an urban, municipal, advanced life support emergency medical services (EMS) system. The interview subjects were family members present at the scene in a consecutive series of unsuccessful out-of-hospital resuscitation attempts. RESULTS: During the 4-month study period, 140 out-of-hospital cardiac arrests occurred. Follow-up with a family member was performed in 42 of the 53 cardiac arrests that met the inclusion criteria (79%). When resuscitation was terminated in the field (n = 25), 24 family members (96%) reported satisfaction with the decision. When resuscitation was terminated in the emergency department (n = 17), 14 family members (82%) reported satisfaction with the decision to transport the victim to the hospital. However, responses indicated that 13 of the family members (76%) might have accepted termination of resuscitation in the home. In all cases, relatives reported satisfaction with the paramedics' care and with the manner in which they were informed of the victims' deaths. CONCLUSION: Family members accept termination of unsuccessful out-of-hospital cardiac arrest resuscitation in the field. PMID- 8629789 TI - Emergency medicine clinical guidelines: we can make them, but will we use them? PMID- 8629790 TI - Evaluation of pancreatic injury after blunt abdominal trauma. AB - Pancreatic injury after blunt abdominal trauma can be subtle and delayed in its onset. We present a 24-year-old man who became jaundiced 16 days after he experienced blunt trauma. He was found to have an isolated pancreatic contusion. We discuss the initial evaluation for blunt pancreatic injury, the differential diagnosis of jaundice resulting from the trauma, and, finally, three methods of treating pancreatic contusions that cause jaundice. PMID- 8629791 TI - Bloody neonatal diaper. AB - As part of the ongoing effort to minimize health care expenses, mothers and newborns are being discharged after shorter hospital stays. Problems that previously would have been noticed in the hospital nursery are now being seen in the emergency department. We report the case of a 1-day-old infant who was brought to our ED with grossly bloody stool. An Apt test was performed to determine whether the blood was of infant or maternal origin. After determining that the blood was the infant's, we transferred the child to a pediatric specialty center, where a diagnosis of necrotizing enterocolitis was made. PMID- 8629792 TI - A flexible solution for emergency intubation difficulties. AB - The inability to correctly position the patient may cause difficulty during oral endotracheal intubation. Examples of such circumstances include cases of suspected cervical spine injury and cases of restricted access to the patient in the prehospital environment. The Eschmann tracheal tube introducer, more commonly called the "gum elastic bougie", is a valuable aid to oral intubation. The case reported herein, of a successful bougie-assisted oral intubation in the prehospital setting, highlights the usefulness of the technique. Physicians considering the use of the gum elastic bougie for intubation difficulties after rapid sequence induction should seek specific training in the use of the instrument. PMID- 8629793 TI - Sabbatical. PMID- 8629794 TI - Activated charcoal and salicylate. PMID- 8629795 TI - Organ transplantation from victims of carbon monoxide poisoning. PMID- 8629796 TI - Coronary artery spasm during anaphylaxis. PMID- 8629797 TI - Adult thermal epiglottitis. PMID- 8629798 TI - Is one needle better than two? Should anybody care? PMID- 8629799 TI - Emergency medicine is not a Ford Pinto. PMID- 8629800 TI - "Do not attempt resuscitation" directives in the out-of-hospital setting. American College of Emergency Physicians. PMID- 8629801 TI - Expert witness guidelines for the specialty of emergency medicine. American College of Emergency Physicians. PMID- 8629802 TI - Providing telephone advice from the emergency department. American College of Emergency Physicians. PMID- 8629803 TI - Physician credentialing and delineation of clinical privileges in emergency medicine. American College of Emergency Physicians. PMID- 8629804 TI - Poison information and treatment systems. American College of Emergency Physicians. PMID- 8629805 TI - [Human familial autosomal reciprocal translocations]. AB - Reciprocal translocations are one of the most frequently observed structural chromosome abnormalities. They are defined by a segment exchange between two non homologous chromosomes. A great number of different translocations exist since any chromosome can be involved in the translocation and the position of the breakpoint can vary. Though generally silent these translocations can be expressed in the form of reproduction failure or, more seriously, as offspring showing mental retardation/malformation syndromes. Since the risk of malformation varies from one translocation to the next, genetic counselling and prenatal diagnosis strategies should be adopted to suit the particular malformation risks of each individual translocation. This is currently not the case. Different prediction methods (for the most probable mode of unbalance at birth, the risk of unbalance at term) are presented. A computer system, called Reci-Conseil brings these different functionalities together to create a new aid for genetic counselling. The data base on which it is founded (approx 2000 families) offers interesting perspectives for genomic mapping of partial trisomies and monosomies. PMID- 8629806 TI - [Genetic variation in the apolipoprotein B gene]. AB - It is well known that coronary heart disease (CHD) is multifactorial, with environmental and inherited risk factors both playing a role. Apolipoprotein B (apo B) is of major importance in lipoprotein metabolism and might play a central role in atherogenesis. The apo B gene is the obvious candidate gene to study the relations between lipid concentrations and CHD. Some rare mutations in the apo B gene affect plasma cholesterol levels, leading to either familial hypobetalipoproteinemia or familial defective apolipoprotein B100. Other frequent polymorphisms have little biological effect but, because of their high frequency, might contribute to the development of CHD in a given population. Many apo B gene polymorphisms are associated with variations in plasma lipid concentrations, including the response of plasma lipids to dietary intervention, and peripheral and coronary atherosclerosis. Age, body mass index and gender affect the degree and nature of the association between apo B genetic markers and normal lipid and lipoprotein levels. However, negative and contradictory results have also been reported. One likely explanation is differences between studies, including populations of different geographic origin, arbitrary definition of cases and controls and multiple criteria for CHD. Future work on the effect of the apo B locus on hyperlipidaemia and atherosclerosis must involve large numbers of patients belonging to carefully defined populations. Prospective studies using a combination of genetic markers in well-defined populations should lead to firm conclusions on the role of apo B in atherogenesis and coronary heart disease. PMID- 8629807 TI - Diagnostic usefulness of the polymorphism of the GT dinucleotide and the polythymidine tract in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. AB - 15-20% of the CF mutation are expected to be rare and escape detection by systems designed to screen for common mutations. The highly polymorphic simple repeats would be particularly useful for genetic diagnosis in CF families where the mutations have not been identified. In this study, we used denaturing gradient gel electrophoresis with psoralen-modified oligonucleotide primers to study the GTnTm polymorphism previously identified at the intron 8 - exon 9 junction of the CFTR gene. Twelve characteristic patterns were identified. The most frequent genotype in CF alleles was GT10T9 and in non-CF alleles GT11T7. In this study, the heterozygous incidence is 70% in unrelated CF carriers. This polymorphism is full informative in 45% and half-informative in 50%. We conclude, that this polymorphism, easy to study by a relatively simple, rapid and cheap procedure, would be particularly useful in genetic counselling for CF and prenatal diagnosis in CF families in which mutations have not been yet identified. PMID- 8629808 TI - Geographic distribution of 119 alleles of the alpha and beta globin genes detected in 432 French Caucasian carriers of haemoglobin variant. AB - The French population is the result of mixing of different peoples including the Celts, Saxons, Germans, Italians and Hispanics. Between 1981 and 1993 patients were selected during investigations in France for haematological disorders associated or otherwise with the presence of a haemoglobin (Hb) variant. Further carriers of abnormal Hb were identified by HPLC measurement of glycated Hb in diabetics and by neonatal screening. Four-hundred and thirty-two subjects were found to be heterozygous for one of the 119 different alpha and the beta gene alleles encountered. These variants were characterised by a combination of 6 electrophoretic methods and in some cases by protein structure determinations. Some mutants reflected the population movements in and into France. A few mutants are frequently described in the French Caucasian population: Hb Lepore Boston, Hb D Punjab, whereas others appear to be anthropological markers. Hb Winnipeg has only been found in the West of France (Normandy); Hb J Baltimore is mainly found in French subjects of Spanish origin. Several cases of sporadic and previously undescribed mutations of Hb were identified. The last immigration waves from Africa and Asia appear to have contributed to the evolution of the pattern of haemoglobinopathies in the French population (Hb S, Hb O Arab or Hb C). PMID- 8629809 TI - Recurrent regular trisomy-21 in two Bedouin families. Parental mosaicism versus genetic predisposition. AB - Two young, unrelated multiplex families with Bedouin ancestors, each confirmed to have 3 sibs with recurrent regular trisomy-21 are reported. Low grade mosaicism for trisomy-21 (3/350 cells) was confirmed in the mother in one of the families but not in the other. However, two of the relatives (first degree) were confirmed to have Down syndrome. The recurrence risk for trisomy-21 based on livebirth and prenatal diagnosis data were estimated at 1 to 2 percent for couples 35 years old or younger at the time of conception. However, such risk estimates are not available for recurrence of simple trisomy-21 to a particular couple (<35 years). Clustering of trisomy-21, trisomy-18 has been reported among Bedouins. The possibility of cryptic parental mosaicism as well as <> should be considered in genetic counselling of families with sibships of regular trisomy-21 or other aneuploidy. PMID- 8629810 TI - Polymorphic variations in peripherin-RDS gene in the Spanish population. AB - The authors report a study of polymorphisms in the peripherin-RDS gene in 21 Spanish families affected with Autosomal Dominant Retinitis Pigmentosa and 56 unrelated normal individuals. We found 3 variants in first exon and nine variants in the third exon in an SSCP analysis, all corresponding to different previously described polymorphisms. PMID- 8629811 TI - Terminal deletion in chromosome region 8p23.1-8pter in a child with features of velo-cardio-facial syndrome. AB - A child with an atrial septal defect, hypernasal speech with an immobile soft palate, learning difficulties and behavioral problems is reported. The clinical diagnosis in this child was velo-cardio-facial syndrome (VCFS), but cytogenetic analysis showed the presence of a small terminal deletion in 8p23.1-8pter. This observation shows that deletions in 8p23 may lead to features similar to VCFS. This suggests that in patients with VCFS but without a deletion in 22q11, attention should be focussed on 8p. PMID- 8629812 TI - The proteinase 3 (PRTN3) gene is localized on 19p13.3 and is distal to the E2A gene. AB - The gene encoding the proteinase 3. (PRTN3, myeloblastin) was localized to chromosomal band 19p13.3 by radioactive and fluorescent in situ hybridization (FISH) to normal metaphase chromosomes. This localization was shown to be telomeric to the E2A gene by performing FISH to metaphases with balanced translocation t(1;19)((q23;p13) characteristic for a pre-B subtype of acute leukemia. In the unbalanced form of this translocation, a monoallelic loss of the PRTN3 locus was observed with FISH. PMID- 8629813 TI - Herpes simplex type 1:lacZ recombinant viruses. I. Characterization and application to defining the mechanisms of action of known antiherpes agents. AB - Recombinant viruses with the lacZ gene placed under the control of the HSV-1 ICP4, TK and gD regulatory regions were constructed by recombination into the TK locus of HSV-1. Difficulty in isolating ICP4 and gD recombinant viruses with high level, regulated expression of beta-galactosidase was overcome by the use of HSV 1 translational initiation sequences of these genes in place of vector-derived sequences. beta-Galactosidase expression displayed the kinetics particular to each viral class. The maximal expression of beta-galactosidase from the recombinant viruses within a 22-h period (m.o.i. 5) (relative to the ICP4 virus) was gD(3) > gC(2) > ICP4(1) > TK(0.5). The ICP4 virus produces easily quantifiable levels of beta-galactosidase activity for multiplicities of infection from 5 x 10(-4) through 5 over 48 h postinfection. At multiplicities of infection between 2 and 5, ICP4-driven activity was measurable within 2 h postinfection from a monolayer of 3 x 10(4) Vero cells in microtiter wells. Mechanisms of inhibition of several antivirals were probed by using the regulated expression of beta-galactosidase from the ICP4 virus as a marker for viral growth. An experimental antiviral (E3925, IC50 1 microgram/ml) and a neutralizing gD MAb (DUP55306, IC50 0.6 microgram/ml) acted prior to immediate early synthesis, consistent with inhibition of viral entry or uncoating. IFN-gamma inhibited expression of immediate-early synthesis, while having no effect on viral entry. IC50 values for E3925 obtained using either the ICP4 or gD viruses at m.o.i. 0.005, were in good agreement with those obtained by standard plaque assays, but were determined in only 1 day, using a microtiter plate format. Thus, these reporter viruses are useful tools for defining the mechanisms of action of antiherpes agents, while quantitatively reproducing the results for IC50 determinations from standard plaque assays within 24 h in a microtiter plate format. PMID- 8629814 TI - Herpes simplex type1:lacZ recombinant viruses. II. Microtiter plate-based colorimetric assays for the discovery of new antiherpes agents and the points at which such agents disrupt the viral replication cycle. AB - A panel of microtiter plate-based colorimetric assays for monitoring HSV-1 growth has been made. The panel consists of 4 different HSV-1 (strain KOS) lacZ recombinant viruses which express beta-galactosidase under the control of different HSV-1 promoters derived from each class of herpes simplex gene expression: immediate-early (ICP4), early (TK), delayed early (gD) and late (gC). Inhibitors of HSV-1 growth were evaluated using differential effects on each of the reporter viruses as a measure of which points in the viral replication cycle an inhibitor was acting. Aphidicolin (DNA synthesis inhibitor) was studied as a model compound. At an m.o.i. of 0.05, at 24 h postinfection (h p.i.), aphidicolin inhibited 80% of viral growth at 1 micrograms/ml, as determined by a reduction in ICP4-driven activity within the second cycle of infection. At m.o.i. 5, within the first infectious cycle, aphidicolin had no effect on the signals from either the ICP4 or TK viruses at 3 micrograms/ml, while largely suppressing gD and fully inhibiting gC-driven signals at 2 micrograms/ml. This profile is consistent with the behavior expected of a DNA synthesis inhibitor. Five inhibitors of unknown mechanism were evaluated. Two compounds inhibited ICP4-driven activity within the first infectious cycle and were classified as potential inhibitors of viral entry, uncoating or IE gene expression (XF884, BT318). One compound inhibited gD and gC-driven activity without inhibiting signal from the ICP4 and TK viruses, and was classified as a potential DNA synthesis inhibitor (DS810). Two compounds (S5193, ER622) had effects on gD- and gC-driven activity which were somewhat different from aphidicolin and DS810, but which could be interpreted as inhibition of viral assembly and/or egress. The potency of XF884 varied with the time postinfection at which it was added to cells (IC50 3.7 to > 10 micrograms/ml) while the effects of BT318 were independent of time of addition (IC50 11.4 micrograms/ml). These results suggest XF884 inhibits viral entry while BT318 is acting after viral entry, possibly as a direct inhibitor of ICP4 gene expression. Together, these results suggest the panel of recombinant herpes viruses has utility in aiding in the identification of the points in the herpes life cycle at which antiherpes drug candidates, of unknown mechanisms, are acting. PMID- 8629815 TI - Antisense oligonucleotides are effective inhibitors of hepatitis B virus replication in vitro. AB - Antisense oligonucleotides are currently being used in numerous laboratories as potential anticancer and antiviral agents. The unique replication cycle of hepatitis B virus (HBV) contains several different steps which are potentially amenable to modulation by these molecules. We have examined the ability of 56 different single-stranded, oligodeoxyribonucleotides (14-23 nucleotides in length), which target several HBV-specific functions, to inhibit HBV replication in the human hepatoblastoma cell line, 2.2.15. None of the oligonucleotides examined were toxic at concentrations up to 500 microM. Oligonucleotides directed against the HBV surface antigen (HBsAg) gene (S gene), the preS1 open reading frame, and the HBV core antigen (HBcAg) gene (C gene) were effective at depressing virus production, while molecules targeting the HBV e antigen (HBeAg) open reading frame and the HBV polymerase (POL) gene were ineffective. Oligonucleotides directed against the HBV encapsidation signal/structure (epsilon) comprised some of the most effective antiviral molecules against HBV. None of 5 oligonucleotides complementary (i.e., 'sense' orientation) to the antisense oligonucleotides targeting HBsAg, HBcAg, HBeAg, preS1 and POL had any measurable effect on HBV production. The relative effectiveness of oligonucleotides targeting the S and C genes on HBV replication was highly correlated with an effect on HBsAg or HBcAg levels, respectively. None of the antisense oligonucleotides examined affected either the levels or the sizes of HBV-specific RNA transcripts. Since antisense oligonucleotides can exert biologic effects on HBV in 2.2.15 cell cultures in a sequence-specific manner which are consistent with predicted modes of action, such molecules may have practical applications in the therapy of chronic HBV infection. PMID- 8629816 TI - Antiviral activity of 9-[[(ethoxyhydroxyphosphinyl)-methoxy]methoxy] guanine against cytomegalovirus and herpes simplex virus. AB - An isosteric analog of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG), 9 [[(ethoxyhydroxyphosphinyl)methoxy]methoxy]guanine (SKI 1008), was evaluated for its in vitro antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), murine cytomegalovirus (MCMV), and human cytomegalovirus (HCMV), and its in vivo antiviral efficacy against MCMV in mice. The in vitro anti-HSV activity of SKI 1008 was much lower than that of acyclovir, even though SKI 1008 showed similar antiviral activity against thymidine kinase positive (TK+) and thymidine kinase negative (TK-) strains. Like ganciclovir and PMEG, SKI 1008 selectively inhibited plaque formation of MCMV; the 50% effective concentration (EC50) and the 50% cytotoxic concentration (CC50) of SKI 1008, ganciclovir, and PMEG being 0.51 and 600, 1.65 and 461, and 0.06 and 12.1 micrograms/ml, respectively. The in vitro EC50 value of SKI 1008 against HCMV was comparable to that of ganciclovir (0.24 vs 0.16 microgram/ml) and was 12-fold higher than that of PMEG in a plaque reduction assay, but the therapeutic indices (the ratios of CC50 to EC50) of SKI 1008 and ganciclovir were higher than that of PMEG. The in vivo antiviral efficacy of SKI 1008 in MCMV-infected normal BALB/c and severe combined immunodeficiency (SCID) mice was lower than that of ganciclovir in terms of mortality and mean survival time. PMID- 8629818 TI - In vivo drug screening applications of HIV-infected cells cultivated within hollow fibers in two physiologic compartments of mice. AB - Previous studies demonstrated that human cell lines can be cultivated in hollow fibers in the subcutaneous and intraperitoneal compartments of mice. We have extended the range of cell lines to include cells infected with the human immunodeficiency virus (HIV). Furthermore, these HIV-infected cells have been shown to replicate in the hollow fibers located in both physiologic compartments (intraperitoneal and subcutaneous) of SCID mice. Treatment of the host mice with antiviral agents can suppress virus replication in these hollow fiber cultures. The potential use of this system for early in vivo screening of anti-HIV compounds is discussed. PMID- 8629817 TI - Immunomodulating and antiviral activities of the imidazoquinoline S-28463. AB - Recently, a new class of immunomodulating agents, represented by the molecules imiquimod and R-842, has demonstrated potent antiviral and antitumor activities in animal models. In this study, another representative of this class, S-28463 (4 amino-2-ethoxymethyl-alpha,alpha-dimethyl-1H-imidazo[4,5-c]quinoline- 1- ethanol) was evaluated for its immunomodulating and antiviral activities. S-28463 induced IFN and other cytokines in vivo in mice, rats, monkeys and in vitro in human peripheral blood mononuclear cell cultures. S-28463 showed potent antiviral activity against herpes simplex virus-challenged guinea pigs when given subcutaneously, dermally, or intravaginally 24 h before infection. Antiviral activity in guinea pigs correlated with the induction of serum 2',5' oligoadenylate synthetase activity. Thus, S-28463, like the other imidazoquinolines, demonstrates potent antiviral and immunomodulating effects in a number of models. PMID- 8629819 TI - Compression therapy in painful piezogenic pedal papules. PMID- 8629820 TI - Research and dermatologic excellence. AB - Nearly everyone, even within our small specialty, has a slightly different idea, or bias, as to what constitutes research. We hear of clinical research and academic research and pharmaceutical research and descriptive research and cutting-edge research, but the distinctions often blur and are unimportant. What is important is that research of all kinds increases knowledge and adds information that improves patients' well-being. Perhaps we can agree with a general definition: Research is inquiry. In its simplest form, it may be nothing more than a literature review pertinent to an observation or interest. More often we think of systematic research (a progression starting with questions and ideas) moving forward to a hypothesis, then planning approaches and methods for experiments, all directed toward characterizing normal features and disease mechanisms to prevent and treat illness. PMID- 8629822 TI - Bullous pemphigoid and epidermolysis bullosa acquisita. Differentiation by fluorescence overlay antigen mapping. AB - BACKGROUND AND DESIGN: From previous studies, we concluded that the fluorescence overlay antigen mapping (FOAM) technique could be of value to the differential diagnosis of the acquired subepidermal bullous skin disorders, bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). In these diseases, ultrastructural identification of the site of skin-bound IgG deposits at the epidermal basement membrane zone (EBMZ) may be essential to the correct diagnosis. Since ultrastructural studies are more expensive, time-consuming, and less widely available than immunofluorescence, we addressed the question of whether the FOAM technique can reliably identify the site of IgG deposits at the EBMZ, and distinguish BP from EBA. For this purpose, the technique was applied to perilesional skin from seven patients with BP and six with EBA, using computer aided imaging of red-stained type VII collagen and green-stained IgG, according to previous findings. RESULTS: Digitized multicolor FOAM images of perilesional skin from patients with BP showed nonoverlap band patterns of green-stained lamina lucida IgG deposits (ultrastructurally proven) and red-stained type VII collagen. By contrast, FOAM images of EBA skin typically showed overlap patterns of green-stained sublamina densa IgG deposits and red-stained type VII collagen. These findings were observed also in skin tissue stored in Michel's transport medium or stored frozen for 15 years. CONCLUSIONS: The computer-aided FOAM technique may have great potential in distinguishing between IgG deposits above (BP) and just below (EBA) the lamina densa of the EBMZ in skin tissue. The technique is not as simple as saline-split skin methodology but offers more flexibility, and it certainly is quicker and less expensive than electron microscopy. Furthermore, the use of digitized fluorescence images offers improved possibilities for evaluating the various "linear" patterns of immune reactant deposition at the EBMZ in subepidermal bullous autoimmune skin diseases. PMID- 8629821 TI - Generalized atrophic benign epidermolysis bullosa. Either 180-kd bullous pemphigoid antigen or laminin-5 deficiency. AB - BACKGROUND: Generalized atrophic benign epidermolysis bullosa (GABEB) is a form of nonlethal junctional epidermolysis bullosa, clinically characterized by generalized blistering after birth, atrophic healing, and incomplete universal atrophic alopecia with onset in childhood. Recently, we discovered a deficiency of the 180-kd bullous pemphigoid antigen (BP180) and a reduced amount of BP180 messenger RNA in three patients with GABEB. It is not yet clear, however, whether GABEB is invariably caused by BP180 deficiency. RESULTS: We examined 18 patients with nonlethal junctional epidermolysis bullosa from unrelated families; nine of these individuals presented with the clinical characteristics of GABEB. Specimens of clinically normal skin obtained from the patients were stained by immunofluorescence with monoclonal antibodies to BP180 and laminin-5. The BP180 epitopes were not expressed in eight patients, all of whom were sharing the typical clinical features of GABEB. In one of the nine patients with GABEB, the BP180 level was sufficient, but the laminin-5 level was reduced. Among the nine patients with junctional epidermolysis bullosa without atrophic alopecia, laminin 5 level was not expressed in one patient, while in the other patients both antigens were normally expressed. CONCLUSIONS: Not all patients with GABEB are deficient in BP180, since some individuals with GABEB only exhibit reduction of the laminin-5 expression. The BP180 deficiency in the skin invariably seems to result in GABEB. Immunofluorescence analysis using monoclonal antibodies against BP180 (and laminin-5) may allow early subtyping, which is of prognostic significance, in children born with junctional epidermolysis bullosa. PMID- 8629823 TI - Complications of cutaneous surgery in patients who are taking warfarin, aspirin, or nonsteroidal anti-inflammatory drugs. AB - BACKGROUND AND DESIGN: No controlled studies exist with regard to the risks of continuing therapy with warfarin sodium or platelet inhibitors or the benefits of briefly discontinuing therapy with these agents in patients who are undergoing cutaneous surgical procedures. Our objective was to determine the frequency of complications of cutaneous surgery in patients who were receiving warfarin or platelet inhibitors and to evaluate whether preoperative discontinuation reduces complications. A retrospective, controlled study was performed of complications of excisional and Mohs micrographic surgery in 653 patients who were being treated with warfarin or platelet inhibitors or with their medications being briefly withheld. RESULTS: Severe complications of cutaneous surgery in patients who are taking warfarin or platelet inhibitors are uncommon, occur in 1.6% of cases, and are not significantly increased compared with complications in control subjects. Furthermore, there was no statistically significant reduction in the rates of severe complications in patients who had their medications preoperatively held. CONCLUSION: Cutaneous surgery in patients who receive warfarin or platelet inhibitors is associated with a low risk of severe complications, not significantly reduced by brief preoperative discontinuation. PMID- 8629824 TI - Longitudinal melanonychia in children. A study of eight cases. AB - BACKGROUND AND DESIGN: Longitudinal melanonychia is rare in white children and represents a difficult clinical challenge. Because of the fear of malignant melanoma, a surgical excision is usually performed, sometimes with definitive aesthetic and functional consequences. Eight children with longitudinal melanonychia who were between the ages of 2 and 14 years underwent follow-up. Surgical excision was performed in five cases. For three children, a wait-and-see policy was adopted, because their longitudinal melanonychia had been unchanged for years at the time of their examination. RESULTS: Melanoma was never observed in our cases (follow-up, 5.5 years). Histologic examinations performed in five cases showed junctional nevi of the nail matrix, often with dysplasia as commonly seen in juvenile nevi. Two children had postoperative nail dystrophy. CONCLUSION: In white children, longitudinal melanonychia rarely disappears. Since an ungual melanocytic band can appear at the age when other nevi appear, surgical excision should not be undertaken on different grounds than for other congenital or acquired nevi in children. PMID- 8629826 TI - Psoriatic onycho-pachydermo-periostitis. A variant of psoriatic distal interphalangeal arthritis? AB - BACKGROUND: Joint and nail involvement in psoriasis is relatively common. In contrast, bony involvement of the terminal phalanx under a psoriatic nail is rare. We report on this psoriatic onycho-pachydermo-periostitis and suggest pathophysiologic mechanisms. OBSERVATIONS: Two new cases are reported and the data are compared with the data from eight similar cases reported in the literature. All subjects presented with similar changes--onychopathy, soft-tissue thickening, and radiologic features consisting of bone erosions and a periosteal reaction of the terminal phalanx. Of the 10 patients, two had no history of psoriasis before a diagnosis of psoriatic onycho-pachydermoperiostitis was made. The inflammation is likely transmitted from the psoriatic nail to the adjacent underlying bone by the same mechanism as in enthesopathies. CONCLUSION: Psoriatic onycho-pachydermo-periostitis should be recognized as a specific entity in the spectrum of psoriatic disease. PMID- 8629825 TI - Large congenital melanocytic nevi and the risk for the development of malignant melanoma. A prospective study. AB - BACKGROUND AND DESIGN: Patients with large congenital melanocytic nevi have been described to have an increased risk for the development of malignant melanoma (MM). Ninety-two patients with large congenital melanocytic nevi were followed up prospectively for the development of MM. Matched individuals from the general population served as control subjects. RESULTS: Ninety-two patients (median age, 3 years) were followed up prospectively for an average of 5.4 years. In three patients (3%), MM developed in extracutaneous sites. The cumulative 5-year life table risk for the development of MM was calculated to be 4.5% (95% confidence interval, 0% to 9.3%). In individuals in the general US population, matched for age, sex, and length of follow-up to the 92 study patients, 0.013 would be expected to develop MM. The standardized morbidity ratio (adjusted relative risk) was calculated to be 239, which was highly significant (P < .001). CONCLUSIONS: Patients with large congenital melanocytic nevi are at a significantly increased risk for the development of MM and should be kept under continuous surveillance for the development of cutaneous as well as noncutaneous primary MM. PMID- 8629827 TI - Methotrexate osteopathy in long-term, low-dose methotrexate treatment for psoriasis and rheumatoid arthritis. AB - BACKGROUND: In dermatology and rheumatology, methotrexate is frequently prescribed in low dosages per week; in oncology, high dosages per week are prescribed. Methotrexate osteopathy was first reported in children with leukemia treated with high doses of methotrexate. In animal studies, low doses of methotrexate proved to have an adverse effect on bone metabolism, especially on osteoblast activity. OBSERVATIONS: Methotrexate osteopathy is a relatively unknown complication of low-dose methotrexate treatment. We describe three patients treated with low-dose oral methotrexate in whom signs and symptoms were present that were similar to those found in children treated with high doses of methotrexate. All three patients had a triad of severe pain localized in the distal tibiae, osteoporosis, and compression fractures of the distal tibia, which could be identified with radiographs, technetium Tc 99m scanning, and magnetic resonance imaging. CONCLUSIONS: Methotrexate osteopathy can occur in patients treated with low doses of methotrexate, even over a short period of time. As pain is localized in the distal tibia, it is easily misdiagnosed as psoriatic arthritis of the ankle, but the diagnosis can be correctly made by careful investigation and use of imaging techniques. The only therapy is withdrawal of methotrexate. It is important that more physicians become aware of this side effect of methotrexate therapy, which can occur along with arthritic symptoms. PMID- 8629828 TI - Neonatal Malassezia furfur pustulosis. AB - BACKGROUND: Papulopustular eruptions of the face in neonates are frequently referred to as neonatal acne or sebaceous miliaria. Our findings suggest that there is an association between this type of eruption and Malassezia furfur infection. OBSERVATIONS: Direct examination of pustule smears showed M furfur yeasts in eight of 13 cases involving neonates with erythema and papulopustules of the face, neck, and scalp (mean age at onset, 22 days [range, 7 to 30 days]). The pustules were predominantly neutrophilic. Treatment with 2% ketoconazole cream applied topically twice daily was effective in 1 week. CONCLUSION: Malassezia furfur is frequently associated with a common nonfollicular pustulosis of the newborn, probably improperly termed neonatal acne. PMID- 8629829 TI - Chronic lupoid leishmaniasis. Evaluation by polymerase chain reaction. AB - BACKGROUND: The cutaneous lesions in chronic lupoid leishmaniasis resemble those of lupus vulgaris, both clinically and histologically. The differential diagnosis is difficult and may depend on the detection of a few Leishmania amastigotes in the histologic sections, the growth of the promastigotes in cultures, or the identification of amastigotes by other techniques. Polymerase chain reaction was used to detect Leishmania amastigote DNA in tissue samples obtained from 65 patients with chronic lupoid leismaniasis, and the results were confirmed by Southern blot analysis. OBSERVATIONS: The histologic findings of a predominantly epithelioid cell granuloma surrounded by lymphocytic infiltrate in chronic lupoid leishmaniasis are very similar to those observed in lupus vulgaris. Extensive histologic examination of the sections in this series revealed occasional macrophages containing a few amastigotes in only 12 cases. Cultures in NNN medium yielded Leishmania promastigotes in 20 cases. Polymerase chain reaction studies using a Leishmania-specific primer identified Leishmania DNA in 30 of 63 cases, and those using a Mycobacterium tuberculosis primer were found to be negative for mycobacteria in 47 cases tested, including 11 cases with a positive tuberculin skin reaction. CONCLUSIONS: The histologic findings in chronic lupoid leishmaniasis resemble those of lupus vulgaris. Polymerase chain reaction studies were useful in identifying amastigotes in 30 (47.6%) of 63 cases. This study confirms the presence of DNA molecules of Leishmania amastigotes in samples of formalin-fixed, paraffin-embedded granulomatous tissue obtained from patients with chronic lupoid leishmaniasis. PMID- 8629830 TI - The adjuvant therapy of pemphigus. An update. AB - Systemic corticosteroids remain the mainstay of therapy for pemphigus. Their use has transformed what was almost invariably a fatal illness into one whose mortality is now below 10%. Unfortunately, the high doses and prolonged administration of corticosteroids that are often needed to control the disease result in numerous side effects, many of which are serious. Most patients who die of pemphigus at present die of complications of therapy. This has led to a continued search for alternative treatments that might reduce the need for steroids. Those treatments now in use are usually given in combination with steroids and, for that reason, are referred to as adjuvant therapies. This article summarizes progress in the adjuvant therapy of pemphigus since the last review of this subject 10 years ago. PMID- 8629832 TI - Advances in the diagnosis of subepidermal bullous diseases. PMID- 8629831 TI - Psoriatic arthritis. New types, new treatments. AB - Arthritis represents the most common complication of psoriasis, affecting a substantial proportion of patients. Various clinical manifestations are recognized with different prognoses. The clinical spectrum of psoriatic arthritis has been recently extended by newly described entities such as pustulosis palmoplantaris with osteoarthritis sterno-clavicularis and psoriatic onycho pachydermo-periostitis. Besides methotrexate, cyclosporine has emerged as the drug of choice for the treatment of psoriatic arthritis, since it influences both cutaneous and arthritic manifestations of the disease. New immunosuppressive drugs, such as tacrolimus (FK 506), offer therapeutic promise. Future progress will depend on new insights into the pathogenesis of psoriatic arthritis. PMID- 8629833 TI - Atrophic outpouchings of abdominal skin. Nodular cutaneous amyloidosis. PMID- 8629834 TI - Nail dystrophy in a 35-year-old man. Subungual enchondroma. PMID- 8629835 TI - A yellow plaque with keratotic papules on the abdomen. Perforating calcific elastosis (periumbilical perforating pseudoxanthoma elasticum [PXE], localized acquired cutaneous PXE). PMID- 8629836 TI - Acquired clubbing of the great toenail. Digital mucoid cyst (pseudocyst). PMID- 8629837 TI - Failure of topical polymyxin B to improve mild plaque psoriasis. PMID- 8629838 TI - Severe facial edema following root canal treatment. PMID- 8629839 TI - Tinea corporis resembling dermatophyte colonies on Sabouraud's agar in a patient with the human immunodeficiency virus. PMID- 8629840 TI - Pseudallescheria boydii in an immunocompromised host. Successful treatment with debridement and itraconazole. PMID- 8629841 TI - Rx: Capitation... a bitter pill for the near future. 2400 American dermatologists in the year 2000? PMID- 8629842 TI - Pulsed carbon dioxide laser resurfacing of photo-aged facial skin. AB - BACKGROUND AND DESIGN: public demand for procedures to rejuvenate photoaged skin have stimulated the use of high-energy short-pulsed carbon dioxide lasers as a precise and predictable treatment modality. The purpose of this study was to determine the degree of clinical improvement achieved in treating perioral and periorbital wrinkles with a high-energy, microsecond-domain pulsed CO2 laser. Photodamaged skin in the perioral (n=73) and periorbital (n=38) regions was treated with multiple passes of confluent single pulses of CO2 laser energy (10 600 nm, 3-mm collimated beam, <1- millisecond pulse, 450 mJ per pulse, 2 to 5 W), with the tissue being cleansed and debrided with normal saline between passes. A nine-point clinical scoring system was devised for evaluation of the degree of wrinkling and photodamage present. Preoperative and postoperative photographs were independently scored by four ?blinded? reviewers. The patients were observed postoperatively for 1 to 12 months for the course of healing, and adverse events were recorded. RESULTS: All three classes (mild, moderate, and severe) of photoaging of the skin responded equally well, showing an average wrinkling score reduction of 2.25 for the periorbital region and 2.34 for the perioral region, the most superficial wrinkles and photodamage being eliminated and the more severe being markedly improved. An unexpected finding was tightening of loose and folded skin. Side effects included transient erythemia and postinflammatory hyperpigmentation, and one instance of an isolated hypertrophic scar. CONCLUSIONS: Resurfacing of photoaged skin by means of a high-energy, microsecond domain pulsed CO2 laser with a specific clinical treatment protocol results in predictable improvement in perioral and periorbital wrinkling and photodamage with minimal risks. Heat-induced collagen shrinkage appears to contribute to these results by tightening loose skin and folds. PMID- 8629843 TI - Skin reflectance-guided laser selections for treatment of decorative tattoos. AB - BACKGROUND AND DESIGN: A fundamental idea of laser treatment of tattoos is that the wavelength must be well absorbed by the tattoo color. In this study, absorption by different tattoo colors was therefore measured in vivo by skin reflectance to establish optimal laser wavelengths for different tattoo colors. Spectral reflectance by tattooed skin and by normal, uninvolved skin was measured in 10-nm steps in a wavelength range from 300 to 800 nm on eight human volunteers with a total of 13 tattoos, which consisted of 14 different tattoo colors. Wavelength ranges for maximal absorption were established. RESULTS: We found that tattoos absorbed maximally in the following ranges: red tattoos, from 505 to 560 nm (green spectrum); green tattoos, from 630 to 730 nm (red spectrum); and a blue green tattoo, in two ranges from 400 to 450 nm and from 505 to 560 nm (blue purple and green spectrums, respectively). Yellow tattoos absorbed maximally from 450 to 510 nm (blue-green spectrum), purple tattoos-absorbed maximally from 550 to 640 nm (green-yellow-orange-red spectrum), blue tattoos absorbed maximally from 620 to 730 nm (red spectrum), and orange tattoos absorbed maximally from 500 to 525 nm (green spectrum). Black and gray absorbed broadly in the visible spectrum, but these colors were most effective from 600 to 800 nm. Optimal and suboptimal laser wavelengths are proposed. CONCLUSIONS: We recommend that wavelength ranges should be established for maximal tattoo absorption before laser treatment of decorative tattoos to select the most optimal laser wavelength present. PMID- 8629844 TI - Motivation for tattoo removal. AB - BACKGROUND AND DESIGN: Motivational issues surrounding tattoo removal are important to understand because tattooing is flourishing, thus creating many requests for tattoo removal. A descriptive study and a 67-item survey were used to examine characteristics of tattooed patients seeking laser therapy for tattoo removal. The setting was the Laser Dermatology Center, Massachusetts General Hospital in Boston, a large metropolitan ambulatory clinic. Patients queried were 64 tattooed males and 41 tattooed females between the ages of 17 and 62 years with a wide variety of vocational and professional occupations. We assessed risk factors surrounding tattooing decisions and experiences that might later influence their motivation to have the tattoos removed by laser therapy. RESULTS: Motivation, treatment, and cost, in terms of money, pain, and risk of disfigurement all entered into the decision making to have the tattoos removed. Strong elements of purchase and possession risks were documented as well as an improved sense of self and maturity. The patient's maturation was in contrast to the notion of a waiting room filled with ill-behaved stereotypical tattooed individuals. Most participants impulsively obtained their tattoos for internal expectations of self-identity at an early age and were still internally motivated to dissociate from the past and improve self-identity. CONCLUSIONS: Poor decision making and subsequent personal regret seem to be frequent motivations for tattoo removal, thus viable methods and accessibility to tattoo removal programs are important. In addition, educational programs for adolescents about tattooing to reduce risks and promote dissuasion should be implemented. PMID- 8629845 TI - Systemic tacrolimus (FK 506) is effective for the treatment of psoriasis in a double-blind, placebo-controlled study. The European FK 506 Multicentre Psoriasis Study Group. AB - BACKGROUND AND DESIGN: Fifty patients with severe recalcitrant plaque-type psoriasis were randomized to receive treatment with either oral tacrolimus (FK 506) (n=27) or placebo (n=23) for 9 weeks. The two treatment groups were comparable with respect to baseline demographic data. The initial dose was 0.05 mg/kg per day and, in cases of insufficient efficacy, could be increased to 0.10 and 0.15 mg/kg per day at the end of weeks 3 and 6, respectively. Treatment efficacy was based on the percentage reduction in the Psoriasis Area and Severity Index compared with baseline data. Patients were defined as responding to therapy if the percentage change in the Psoriasis Area and Severity Index from baseline after 3, 6, and 9 weeks was 20% or greater, 45% or greater, and 70% or greater, respectively. Safety was assessed on the basis of all adverse events reported. RESULTS: At the end of week 9, tacrolimus+-treated patients had a significantly greater reduction in the Psoriasis Area and Severity Index than did placebo treated patients (tacrolimus, -83; placebo, -47; P<.02). Similar numbers of patients in both groups responded to therapy at the end of week 3, but at the end of weeks 6 and 9, more tacrolimus-treated patients responded to therapy (week 6 ,12 tacrolimus- and six placebo-treated patients; week 9, 12 tacrolimus- and three placebo-treated patients). Diarrhea, paresthesia, and insomnia were the most frequently reported causally related adverse events in the tacrolimus treated group. All of the reported adverse events were mild or moderate in severity, and all resolved without a change in study medication. CONCLUSION: Compared with placebo, tacrolimus is efficacious in the treatment of recalcitrant plaque-type psoriasis. PMID- 8629846 TI - Histologic evaluation of preauricular and postauricular human skin after high energy, short-pulse carbon dioxide laser. AB - BACKGROUND: Despite its growing use in dermatalogic surgery, the effects of high energy, short-pulse carbon dioxide laser on human skin have not been well documented. OBJECTIVES: To study the histologic effects of this high-energy, short-pulse CO2 laser on human skin and to compare these changes with the effects of standard chemexfoliation procedures. OBSERVATIONS: Twenty-four hours after laser administration, there was extensive epidermal necrosis and coagulative change in the superficial papillary dermis. With increasing doses of laser energy, there was a statistically significant increase in the depth of dermal wounding (P<.001 for days 1 and 3, F-test). Reepithelialization occurred in most specimens by day 3. By day 90, most specimens showed a subepidermal dermal repair zone consisting of compact new collagen fibers overlying collagen with evidence of solar elastosis. CONCLUSION: This high-energy, short-pulse CO2 laser produces morphologic changes similar to those seen with medium-depth chemical peels. This laser can ablate skin precisely and bloodlessly with little interference in the wound healing process, suggesting that it may serve as an alternative treatment for photoaged skin. PMID- 8629847 TI - Partial spontaneous regression of Bowen's disease. AB - BACKGROUND: Although the lesions of Bowen's disease often show conspiciously irregular configurations, there have been no studies performed to elucidate what these clinical features imply. Of 90 patients with Bowen's disease, 34 (group 1) had such irregular lesions with patches of normal-appearing interlesional skin. Their clinical backgrounds were compared with those of 56 patients without irregular lesions (group 2). Specimens of lesional, interlesional, and perilesional skin from 17 patients in group 1 were examined to evaluate the histopathologic evidences of regression. OBSERVATIONS: The lesions of group 1 patients were more commonly located on the trunk, larger in size, and of longer duration than those of group 2 patients. The specimens of lesional skin from the 17 group 1 patients revealed amyloid deposition (12 patients), melanophages (three patients), increased vascularity (15 patients), and inflammatory cell infiltrates (17 patients). The interlesional skin also exhibited amyloid deposition (12 patients), melanophages (six patients), increased vascularity (15 patients), dermal fibrosis (16 patients), inflammatory cell infiltrates (15 patients), and epidermal atrophy (eight patients). The perilesional skin showed no amyloid depositions, melanophages, or increased vascularity. CONCLUSION: These histopathologic findings in normal-appearing interlesional skin may represent partial spontaneous regression of Bowen's disease. PMID- 8629848 TI - Prevention and early detection strategies for melanoma and skin cancer. Current status. PMID- 8629849 TI - Cutaneous malignant melanoma and sun exposure. Recent developments in epidemiology. AB - BACKGROUND: The rapidly increasing incidence of cutaneous malignant melanoma in white populations world-wide stresses the need for identification of risk factors for the disease. The most important risk factor seems to be sun exposure, but its relationship to melanoma appears to be complex. OBSERVATIONS: Epidemiological studies that examine the association of sun exposure with melanoma are reviewed. The results of these studies concern the anatomic distribution of the disease and its incidence with regard to latitude of residence, sun sensitivity, different patterns of sun exposure, and sun exposure in childhood. CONCLUSIONS: Sun sensitive individuals, children, and individuals with an intermittant pattern of sun exposure seem to be very vulnerable to sunlight, as far as melanoma formation is concerned, and should be thoroughly protected from sun exposure. PMID- 8629850 TI - Laser skin resurfacing. PMID- 8629851 TI - Violaceous rash of dorsal fingers in a woman. Diagnosis: chilblain lupus erythematosus (perniosis). PMID- 8629852 TI - Nodular lesions on fingertips. Diagnosis: cutaneous sarcoidosis. PMID- 8629853 TI - Asymptomatic blue nevus-like macule. Diagnosis: localized argyria. PMID- 8629854 TI - Chronic finger dermatitis after trauma. Diagnosis: posttraumatic eczema with allergic contact dermatitis to neomycin, bacitracin, and topical corticosteroids. PMID- 8629855 TI - Mycosis fungoides palmaris et plantaris. PMID- 8629856 TI - Pulsed carbon dioxide laser, trichloroacetic acid, Baker-Gordon phenol, and dermabrasion: a comparative clinical and histologic study of cutaneous resurfacing in a porcine model. PMID- 8629857 TI - Coexisting malignancies in patients with malignant melanoma. PMID- 8629858 TI - Chlorinated hydrocarbons in human bone marrow of healthy individuals and leukemia patients. AB - In this pilot study, concentrations of chlorinated hydrocarbons in bone marrow were determined by capillary-column gas chromatography. Bone marrow was obtained from a total of 29 healthy adults and from patients with leukemia or lymphoma. The chlorinated hydrocarbons in adults that occurred in the highest concentrations were dichlorodiphenyltrichloroethane and its derivatives (mean = 129 mg/g DNA). Hexachlorobenzene, the hexachlorohexane isomers, and dieldrin were also found, but in reduced concentrations (i.e., mean concentrations: 49.5 mg/g DNA, 14.9 mg/g DNA, and 5.9 mg/g DNA, respectively). Patients who had leukemia and lymphoma did not have significantly increased concentrations of chlorinated hydrocarbons. PMID- 8629859 TI - Reduced IL-6 levels among individuals in Hudson County, New Jersey, an area contaminated with chromium. AB - Hudson County, New Jersey, was a major center for the processing of chromium ore. After processing, some of the ore residue that contained low concentrations of chromium became distributed in population centers throughout the county. There now exists concern in the county about possible health effects from chromium exposure. Our previous research suggested that immune-function assays would make useful biomarkers for chromate exposure in humans. Blood samples were drawn from 46 individuals who lived and/or worked in Hudson County and from 47 controls. Only one of the immune-associated assays performed on these samples showed any statistically significant differences between the Hudson County and control groups. The mean level of IL-6 produced by pokeweed mitogen-stimulated mononuclear cells isolated from the Hudson County group was 64% of the control value--a highly significant decrease (p<.001). There was also a significant correlation between the proliferative responses of the mononuclear cells to pokeweed mitogen and the levels of IL-6 produced by these cells. No differences were detected in the IL-6 responses that resulted from age, gender, or smoking status. The reliability of the IL-6 assay was found to be 90%. To our knowledge, there have been no reports, until now, that describe reduced production of any cytokine in individuals who are exposed to chromate. PMID- 8629860 TI - Tumor necrosis factor alpha gene expression in human monocytic THP-1 cells exposed to beryllium. AB - Chronic beryllium disease, which results from occupational exposure to particulate beryllium, is characterized by the development of lung granulomas and progressive pulmonary fibrosis. Increased production of proinflammatory cytokines (e.g., tumor necrosis factor alpha and interleukin-1 beta) by pulmonary alveolar macrophages occurs in many chronic fibrotic lung diseases and is thought to contribute to the disease process. The purpose of the present study was to investigate cytokine production by human monocytic cells exposed to beryllium in vitro. The results indicated that such cells respond to beryllium ions in the presence of fluoride by accumulation of messenger ribonucleic acid for both tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that inhaled beryllium may directly stimulate the production of these cytokines by alveolar macrophages in vitro. PMID- 8629862 TI - Home dampness and childhood respiratory symptoms in a subtropical climate. AB - The association between measures of home dampness and symptoms of respiratory illness was evaluated in 1 340 8- to 12-y-old children in the Taipei area. The following were reported to occur in the homes: self-dampness (i.e., home considered damp by residents), 36.8% of the homes; "classified" dampness (i.e., presence of mold, water damage, or flooding), 72.3%; visible mold, 38.3%; stuffy odor, 33.9%; water damage, 47.8%; and flooding, 15.1%. Moreover, the prevalence of all respiratory symptoms was consistently higher in homes for which the occurrence of molds or dampness was reported. The adjusted odds ratios ranged from 1.37 (95% confidence interval: 1.03-1.83) for allergic rhinitis to 5.74 (95% confidence interval: 2.20-14.95) for cough. In addition, the observed high prevalence of home dampness/mold indicated that dampness in the home was very common in the subtropical region studied, and home dampness was a strong predictor of respiratory symptoms. PMID- 8629861 TI - Effects of smog on absenteeism in forestry workers. AB - Absenteeism among 161 Dutch forestry workers was investigated in a cohort study. The rate of taking sick leave was related to the concentrations of sulfate and ozone in ambient air, the air temperature, and the relative humidity. The incidence of absenteeism was treated as a Poisson process, with the size of population at risk as the offset factor and with the environmental monitoring data as hazard factors. There appeared to be some overdispersion; therefore, in a second analysis the incidence of absenteeism was also treated as a negative binomial outcome. With the exception of a separate overdispersion parameter in the negative binomial approach, both methods yielded approximately the same results. Although no significant association was found between absence rate and the measured ambient-air-quality data, there appeared to be a time lag of 3 d between a peak in temperature and 1 d in absenteeism. PMID- 8629863 TI - A method for assessing small airways independent of inspiratory capacity. AB - Reduced forced vital capacity may confound assessment of small-airway function. In 17 healthy and 16 asthmatic volunteers, we validated a method for measuring mean expiratory flow during the middle half of the forced vital capacity, mean expiratory flow during the third quarter of the forced vital capacity, instantaneous forced expiratory flow at 50% of forced vital capacity , and instantaneous expiratory flow at 75% of forced vital capacity. These measurements were conducted at the same absolute lung volume (isovolume) when forced vital capacity was reduced voluntarily to 100%, 85%, and 75% of maximum, and the variances, expressed as the coefficients of variations, were compared. Absolute lung volumes above residual volume were determined with two reference spirograms: 100% and 75% forced vital capacity. In normals, means of flow rates at the same absolute lung volume did not differ with the three forced vital capacities, regardless of whether the 100% or 75% forced vital capacity served as the reference spirogram. Reduced forced vital capacity among asthmatics was associated with modest increases in isovolume flow rates, an effect that may underestimate airway narrowing. Intrasubject variability was least among volume averaged flow rates (e.g., mean expiratory flow during the middle half of the forced vital capacity). Volume-adjusted flow rates can be used to assess small airways narrowing when forced vital capacity is reduced, and volume-averaged rates provide the least variability. PMID- 8629864 TI - Chlorpyrifos (Dursban)-associated birth defects: report of four cases. AB - Extensive and unusual patterns of birth defects noted in four children included defects of the brain, eyes, ears, palate, teeth, heart, feet, nipples, and genitalia. Brain defects were present in the ventricles, corpus callosum, choroid plexus, and septum pellucidum, and genital defects included the testes (undescended), microphallus, and labia (fused). All children had growth retardation, and three had hypotonia and profound mental retardation. The children were exposed in utero to chlorpyrifos (Dursban). Published literature and unpublished documents by the U.S. Environmental Protection Agency contain reports that identify similarities in defects found in test animals and in children exposed to Dursban. A pattern of defects found in the four children in this study may represent a heretofore unrecognized syndrome that should be considered when Dursban-exposed women have children with birth defects. PMID- 8629865 TI - Levels of lead in blood and bone of women giving birth in a Boston hospital. AB - Blood lead levels declined among Americans aged 1-74 y between the years 1976 and 1991 (National Health and Examination Survey [NHANES III]). In 1990, umbilical cord blood lead levels were surveyed among 223 women who gave birth in a Boston hospital obstetrical service. In a survey conducted 10 y earlier, women had a mean umbilical cord blood lead level of 6.56 micrograms/dl (standard deviation = 3.19). In another subgroup of 41 women who were 1-4 wk postpartum, bone lead levels were surveyed with a K-x-ray fluorescence instrument and by analysis of venous blood lead levels. In 1990, umbilical cord blood lead levels were very low (i.e., mean and median of 1.19 [standard deviation = 1.32] and 1.0 micrograms/dl, respectively. In the subgroup of postpartum women, lead levels in cortical bone (i.e., tibia) and trabecular bone (i.e., patella) were also low (i.e., tibia mean and median of 4.5 [standard deviation = 4.0] and 4 micrograms/g bone mineral, respectively); patella mean and median of 5.8 [standard deviation = 4.5] and 5 micrograms/g, respectively). Maternal age was the only factor associated (i.e., borderline [p=.076]) with bone lead. Umbilical cord blood lead levels among women served by this Boston hospital from 1980 to 1990 declined dramatically, thus paralleling the National Health and Examination Survey. Bone lead levels were also low, but lead remains a reproductive hazard to women in select high-risk groups. PMID- 8629866 TI - Blood lead levels in Toronto children and abatement of lead-contaminated soil and house dust. AB - South Riverdale in Toronto, Canada, underwent a lead-abatement program. In 1988, lead-contaminated soil was replaced at 970 properties, and in 1989, professional housecleaning for lead removal was conducted in 717 households. The effect of "abatement" on blood lead levels in young children was investigated. Data were analyzed from 12 cross-sectional blood-screening surveys that were conducted during an 8-y period in South Riverdale and in two comparison areas. Responses regarding behavioral, household, lifestyle, neighborhood, and environmental factors, all of which were gleaned from associated questionnaires, were also analyzed. Response rates varied between 32% and 75%. During the years between 1984 and 1992, blood lead decreased in all study areas. There appeared to be a minimal blood lead level of 2-3 micrograms/dl for urban Ontario children who were less than 6 y of age. The significant difference between South Riverdale and the control areas disappeared by 1992. Although abatement activity in South Riverdale was associated with an accelerated decline in blood lead levels, it was difficult to distinguish this from effects of decreased Toronto air lead levels or decreased smelter emissions. Within South Riverdale, abatement appeared to be associated with a slower decline in blood lead levels over time, likely the result of selection bias, soil mixing, or recontamination from the smelter. No difference was observed between the separate effects of housecleaning or soil replacement. The findings could neither strongly support nor refute beneficial effects of abatement. PMID- 8629867 TI - Results of a lead decontamination program. AB - An epidemiological survey was conducted in August 1991 to evaluate the impact of a public health program. The objective of the program was to decrease the level of exposure to lead of children who lived within 200 m of a lead- reclamation plant. In 1989, these children had a geometric mean blood lead level of 9.2 micrograms/dl (0.44 mu mol/l). Children who were 6 mo to 10 y of age (N = 101) participated in the survey. Demographic and behavioral characteristics of the children and their parents were ascertained by interviewers. Blood samples were taken by venipuncture. Geometric mean blood levels were calculated by age in 1991 and by residence in 1989. In 1991, the geometric mean blood lead level had decreased to 5.0 micrograms/dl (0.24 mu mol/l) in children. There was no difference in mean blood lead levels with respect to age or residence. From 1989 to 1991, a significant decrease in the proportion of children who engaged in hand to-mouth activities was also observed. The lead-poisoning prevention program reached its main objective stated above. The success of this program was attributed to the coordinated actions of public agencies at both the provincial and local levels. PMID- 8629868 TI - Lead exposure in the city of Arar, Saudi Arabia. AB - In follow-up to a case of lead encephalopathy, high prevalences of lead exposure (23%) and iron deficiency (60%) were found in children who lived in Arar, Saudi Arabia. Environmental factors had minor effects on the blood lead concentrations of these children. We concluded that traditional cosmetics and remedies were the major sources of lead exposure in this Arar population. PMID- 8629869 TI - Environmental lead and renal effects in children. AB - The effect of lead on five renal-effect parameters was studied in 151 children (i.e., 3-6-y-olds) who resided at different distances from a lead smelter in Baia Mare, Romania. A relationship was found between concentration of lead in blood (mean +/- standard deviation: 342 +/- 224 microgram/l) and the activity of N acetyl-beta-D-glucosaminidase in urine, as demonstrated by a 14% increase of N acetyl-beta-D-glucosaminidase per 100 micrograms/l blood lead that was indicative of renal tubular damage. No relationship was found between blood lead level and the renal-effect parameters albumin, alpha-1-microglobulin, retinol binding protein, or alanine aminopeptidase in urine. Cadmium in blood was not elevated. It is well known that N-acetyl-beta-D-glucosaminidase is a sensitive parameter for renal effects, resulting from lead exposure in adults and from diabetes and nephrotoxic medicines in children. This study is the first to demonstrate an effect of environmental lead exposure on renal integrity in children. PMID- 8629871 TI - Renal Parenchymal Hypertension: current concepts of pathogenesis and management. AB - Renal parenchymal disease is a common but often unrecognized cause of hypertension. Chronic renal disease and systemic hypertension may coexist in two distinct settings. First, essential hypertension is an important cause of chronic renal disease. Second, renal parenchymal disease is a well-established cause of secondary hypertension. Renal parenchymal disease is the most common cause of secondary hypertension, accounting for 2.5% to 5.0% of all cases of systemic hypertension. Secondary hypertension may also accelerate the decline in renal function if inadequately controlled. Therefore, hypertension is both a cause and a consequence of renal disease, and it may be difficult to distinguish them clinically. PMID- 8629870 TI - Neuropsychiatric and somatic characteristics of young adults with and without self-reported chemical odor intolerance and chemical sensitivity. AB - The psychological, neuropsychiatric, and somatic characteristics of young adults who have different degrees of cacosmia (i.e., feeling "ill" from the odor of xenobiotic chemicals) and who have self-described "chemical sensitivity" were examined. A total of 800 college students completed the following: a self-rating scale for frequency of odor intolerance for 10 common substances, Simon Environmental Illness Symptom Survey, the SCL-90-R, Barsky Amplification Scale, Pearlin-Schooler Mastery Scale, Cheek-Buss and Kagan Shyness scales, Marlowe Crowne Social Desirability Scale, and a health-symptom and physician-diagnosed checklist. Two pairs of groups were compared: (1) subjects in the top 16% (i.e., cacosmics) and bottom 15% (noncacosmics) of the sample with respect to odor intolerance scale scores; and (2) subjects from the entire sample who did (28%) or did not (72%) consider themselves to be "especially sensitive to certain chemicals.? Cacosmics and the chemically sensitive subjects scored significantly higher on measures of psychological distress and amplification of somatic symptoms, but there was little evidence of lifestyle change, as assessed by the Simon Survey. Compared with their respective comparison groups, cacosmic and chemically sensitive groups had significantly higher incidences of illnesses associated with chemicals, alcohol intake, opiate drug use, and caffeine use, even after controlling for the psychological measures and histories of atopic allergy. Subjects with and without neuropsychiatric symptoms were differentiated with respect to chemical odor intolerance, but subjects with and without atopic allergies and possible autoimmune diseases were differentiated with respect to chemical sensitivity. Females were more cacosmic than males. Cacosmia is defined by a population subset, with or without occupational xenobiotic exposures or disability, that has distress and symptom amplification and neuropsychiatric and somatic symptoms, none of which are explained fully by psychological measures. Prospective clinical studies are possible with such individuals. The data are also consistent with a time-dependent sensitization model for illness from low level chemical exposures. PMID- 8629872 TI - Long-term follow-up of patients with undiagnosed fever of unknown origin. AB - BACKGROUND: A casual diagnosis cannot be established in 10% to 25% of the patients who are studied for fever of unknown origin (FUO). The long-term clinical outcome of these patients cannot be inferred from the literature. This study describes the results of a 5-year follow-up of 61 patients studied for FUO and discharged from the hospital with no causal diagnosis being established. METHODS: Patients meeting the classic criteria for FUO who were studied in the 1980s and discharged from the hospital without a casual diagnosis were followed up for at least 5 years or until death. Follow-up was performed by review of the patients' medical records or by consulting the treating physician and occasionally the patients themselves. The final diagnosis, clinical course (resolution of the fever and required treatments), and morality rate were studied. RESULTS: Of a cohort of 199 patients with FUO, 61 individuals (30%) were discharged from the hospital without a final diagnosis being established. A definite diagnosis could be established in 12 cases, mostly (eight of 12) within 2 months after discharge. Thirty-one individuals became symptom free during hospitalization or shortly following discharge. Eighteen patients had persisting or recurring fever for several months or even years after discharge, but 10 of them were considered to be finally cured. Four patients were treated with corticosteroids and six patients required intermittent therapy with nonsteroidal anti-inflammatory agents. Six patients died, but the cause of death was considered to be related to the disease that caused FUO in only two cases. CONCLUSION: No single disease, particularly not tuberculosis, was found to be a cause of undiagnosed FUO. Most cases resolved spontaneously, and corticosteroids were seldom required. Most symptomatic patients could be treated with nonsteroidal anti-inflammatory drugs. The mortality rate in patients with undiagnosed FUO who were followed up for 5 years or more was only 3.2%. PMID- 8629873 TI - The incidence of hypertension in insulin-dependent diabetes. AB - BACKGROUND: There are few epidemiologic data describing the long-term incidence of hypertension in people with diabetes. METHODS: In a population-based study performed in southern Wisconsin, 765 individuals diagnosed as having diabetes when they were younger than 30 years and taking insulin participated in baseline, 4-year, and 10-year examinations. Blood pressure was measured by standardized protocols,and hypertension was defined as a mean systolic blood pressure of 160 mm Hg or more ( > or = 140 mm Hg in those younger than 25 years) and/or mean diastolic blood pressure of 95 mm Hg or more ( > or = 90 mm Hg in those younger than 25 years) and/or history of hypertension with the use of antihypertensive medication. RESULTS: The prevalence of hypertension at baseline was 17.3%. The 10 year incidence of hypertension was 25.9%. The incidence of hypertension was greater with older age, longer duration of diabetes, higher glycosylated hemoglobin level, proteinuria, more severe retinopathy, and male gender. After other risk factors were controlled for, the 10-year incidence of hypertension was significantly related to higher glycosylated hemoglobin level (odds ratio, 1.23 per percentage increase; 95% confidence interval, 1.13 to 1.34) presence of gross proteinuria (odds ratio, 3.64; 95% confidence interval, 2.26 to 5.85), longer duration of diabetes (odds ratio, 1.03 per year of diabetes; 95% confidence interval, 1.01 to 1.04), and being male (odds ratio, 1.93; 95% confidence interval, 1.34 to 2.77). CONCLUSION: These data suggest that control of hyperglycemia and prevention of gross proteinuria may lead to a reduction in the long-term incidence of hypertension. PMID- 8629874 TI - Smoking and mortality among older women: the study of osteoporotic fractures. AB - BACKGROUND: Relatively few studies have been focused on the effect of smoking among older individuals. The goal of this study is to investigate the relationship between smoking status and cause-and age-specific mortality among elderly women. METHODS: Women aged 65 years and older and living in four geographical areas (Baltimore, Md, Minneapolis, Minn, Pittsburgh, Pa, and Portland, Ore) were recruited from various population-based listings for participation in the multicenter Study of Osteoporotic Fractures between September 1986 and October 1988 (N=9704). During a mean follow-up of 4.9 years (<99% complete), 751 deaths occurred. The date and cause of death were ascertained, and the relationship between mortality and current and past smoking status was analyzed using Cox proportional hazards modeling techniques. RESULTS: Compared with nonsmokers, women smokers aged 65 to 74 years have a more than twofold increase in mortality attributable to increases in both cardiovascular and cancer mortality; death from smoking-related cancers increased eight- to 10 fold. Women 75 years and older who smoke have a small overall increased relative risk (RR) of mortality (RR=1.4; 95% confidence interval [CI], 0.9 to 2.3), but more than five-fold increased risk of dying from a smoking-related cancer (RR=5.2; 95% CI, 1.6 to 16.8). All-cause and cardiovascular death rates approach those of nonsmokers within 10 years after a woman quits smoking; morality from smoking-related cancers remains elevated for at least 23 years. CONCLUSIONS: The harmful effects of continuing to smoke are apparent even among women aged 75 years and older. PMID- 8629875 TI - Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen study. AB - BACKGROUND: Epidemiological studies suggested that consumption of fruit and vegetables may protect against stroke. The hypothesis that dietary antioxidant vitamins and flavonoids account for this observation is investigated in a prospective study. METHODS: A cohort of 552 men aged 50 to 69 years was examined in 1970 and followed up for 15 years. Mean nutrient and food intake was calculated from cross-check dietary histories taken in 1960, 1965, and 1970. The association between antioxidants, selected foods, and stroke incidence was assessed by Cox proportional hazards regression analysis. Adjustment was made for confounding by age, systolic blood pressure, serum cholesterol, cigarette smoking, energy intake, and consumption of fish and alcohol. RESULTS: Forty-two cases of first fatal or nonfatal stroke were documented. Dietary flavonoids (mainly quercetin) were inversely associated with stroke incidence after adjustment for potential confounders, including antioxidant vitamins. The relative risk (RR) of the highest vs the lowest quartile of flavonoid intake ( > or = 28.6 mg/d vs <18.3 mg/d) was 0.27 (95% confidence interval [CI], 0.11 to 0.70). A lower stroke risk was also observed for the highest quartile of beta carotene intake (RR, 0.54; 95% CI, 0.22 to 1.33). The intake of vitamin C and vitamin E was not associated with stroke risk. Black tea contributed about 70% to flavonoid intake. The RR for a daily consumption of 4.7 cups or more of tea vs less than 2.6 cups of tea was 0.31 (95% CI, 0.12 to 0.84). CONCLUSION: The habitual intake of flavonoids and their major source (tea) may protect against stroke. PMID- 8629876 TI - Functional outcomes of acute medical illness and hospitalization in older persons. AB - BACKGROUND: Short-stay hospitalization in older patients is frequently associated with a loss of function, which can lead to a need for postdischarge assistance and longer-term institutionalization. Because little is known about this adverse outcome of hospitalization, this study was conducted to (1) determine the discharge and 3-month postdischarge functional outcomes for a large cohort of older persons hospitalized for medical illness, (2) determine the extent to which patients were able to recover to preadmission levels of functioning after hospital discharge, and (3) identify the patient factors associated with an increased risk of developing disability associated with acute illness and hospitalization. METHODS: A total of 1279 community-dwelling patients, aged 70 years and older, hospitalized for acute medical illness were enrolled in this multicenter, prospective cohort study. Functional measurements obtained at discharge (Activities of Daily Living) and at 3 months after discharge (Activities of Daily Living and Instrumental Activities of Daily Living) were compared with a preadmission baseline level of functioning to document loss and recovery of functioning. RESULTS: At discharge, 59% of the study population reported no change, 10% improved, and 31% declined in Activities of Daily Living when compared with the preadmission baseline. At the 3-month follow-up, 51% of the original study population, for whom postdischarge data were available (n=1206), were found to have died (11%) or to report new Activities of Daily Living and/or Instrumental Activities of Daily Living disabilities (40%) when compared with the preadmission baseline. Among survivors, 19% reported a new Activities of Daily Living and 40% reported a new Instrumental Activities of Daily Living disability at follow-up. The 3-month outcomes were the result of the loss of function during the index hospitalization, the failure of many patients to recover after discharge, and the development of new postdischarge disabilities. Patients at greatest risk of adverse functional outcomes at follow up were older, had preadmission Instrumental Activities of Daily Living disabilities and lower mental status scores on admission, and had been rehospitalized. CONCLUSION: This study documents a high incidence of functional decline after hospitalization for acute medial illness. Although there are several potential explanations for these findings, this study suggests a need to reexamine current inpatient and postdischarge practices that might influence the functioning of older patients. PMID- 8629877 TI - Cholesterol crystal embolization in the Netherlands. AB - OBJECTIVE: To clarify the incidence and clinical features of cholesterol crystal embolization (CCE). METHODS: Analysis of the relevant data of 842 diagnosed cases of CCE filed in the Dutch National Pathology Information System from 1973 through 1994. RESULTS: No report of CCE was recorded from 1973 through 1979. Since then, its incidence rose from 0.9 case per million population in 1980 to 6.0 cases per million population in 1985, but stabilized thereafter. Among autopsy reports, the relative percentage of CCE was similar over the years, with 0.35% in 1982 and 0.30% in 1994 (mean 0.31% range, 0.20% to 0.42%). Nine patients in whom CCE was found in their renal transplant were excluded from the study. Thus, among a total of 833 elderly (mean age, 72.1 years), predominantly male (73.9%) patients, 1066 CCE sites were found in 323 biopsy reports, 264 resection reports, and 287 autopsy reports. CONCLUSION: In the Dutch population, CCE is reported steadily, with an average frequency of 6.2 cases per million population per year since 1985. It occurs predominantly in elderly men with a history of atherosclerotic disease and hypertension. Symptoms may be absent, go unrecognized, or mimic other disease processes. It can also be a coincidental finding. The primary CCE site is the kidney, followed by the skin and gastrointestinal tract. PMID- 8629878 TI - Fecal hemoglobin excretion in elderly patients with atrial fibrillation: combined aspirin and low-dose warfarin vs conventional warfarin therapy. AB - BACKGROUND: Antithrombotic prophylaxis using combined aspirin and low-dose warfarin is under evaluation in several clinical trials. However, therapy may result in increased gastrointestinal blood loss and clinical bleeding vs conventional single-agent antithrombotic therapy. METHODS: To assess differences in gastrointestinal blood loss, we measured quantitative fecal hemoglobin equivalents (HemoQuant, Mayo Medical Laboratory, Rochester, Minn) in 117 patients, mean age 71 years, 1 month after initiation of assigned therapy in the Stroke Prevention in Atrial Fibrillation III Study. Sixty-three of these patients who had characteristics for high risk of stroke were randomly assigned to conventional adjusted-dose warfarin therapy (international normalized ratio, 2.0 to 3.0) or low-dose combined therapy (warfarin [international normalization ratio,<1.5] plus 325 mg/d of enteric-coated aspirin). The remaining 54 patients with low risk of stroke received 325 mg/d of enteric-coated aspirin. RESULTS: Among the 63 at high risk of stroke, abnormal values (>2mg of hemoglobin per gram of stool) were detected in 11% and values greater than 4 mg of hemoglobin per gram of stool were found in 8%. Mean ( +/- SD) values were more for those randomly assigned to receive combined therapy (1.7 +/- 3.3 mg of hemoglobin per gram of stool vs adjusted-dose warfarin therapy, 1.0 +/- 1.9 mg/g; P=.003). The 54 nonrandomized patients with low risk of stroke receiving aspirin alone had a mean (+/- SD) HemoQuant value of 0.8 +/- 0.7mg of hemoglobin per gram of stool 1 month after entry in the study. CONCLUSIONS: Abnormal levels of fecal hemoglobin excretion were common in elderly patients with high risk of atrial fibrillation 1 month after randomization to prophylactic antithrombotic therapy. Combined warfarin and aspirin therapy was associated with greater fecal hemoglobin excretion than standard warfarin therapy, suggesting the potential for increased gastrointestinal hemorrhage. PMID- 8629879 TI - The importance of quality of primary studies in producing unbiased systematic reviews. AB - BACKGROUND: Traditional and largely qualitative reviews of evidence are now giving way to much more structured systematic overviews that use a quantitative method to calculate the overall effect of treatment. The latter approach is dependent on the quality of primary studies, which may introduce bias if they are of poor methodologic quality. OBJECTIVE: To test the hypothesis that the inclusion of poor-quality trials in meta-analyses would bias the conclusions and produce incorrect estimates of treatment effect. METHODS: An overview of randomized trials of antiestrogen therapy in subfertile men with oligospermia was performed to test the hypothesis. Data sources included online searching of MEDLINE and Science Citation Index databases between 1966 and 1994, scanning the bibliography of known primary studies and review articles, and contacting experts in the field. After independent, blind assessment, nine of 149 originally identified studies met the inclusion criteria and were selected. We assessed study quality independently. Outcome data from each study were pooled and statistically summarized. RESULTS: There was a marginal improvement in pregnancy rate with antiestrogen treatment (odds ratio, 1.6; 95% confidence interval, 0.9 to 2.6). Sensitivity analyses on the basis of methodologic quality demonstrated that poor-quality studies produced a positive effect with treatment, whereas no benefit was observed with high-quality studies. CONCLUSION: The results of a meta analysis are influenced by the quality of the primary studies included. Methodologically, poor studies tend to exaggerate the overall estimate of treatment effect and may lead to incorrect inferences. PMID- 8629880 TI - Sacral insufficiency fractures: an easily overlooked cause of back pain in elderly women. AB - Sacral insufficiency fractures (SIF) that usually present as nonspecific pelvic or low back pain are often overlooked in the elderly. In a retrospective study conducted in a department of internal medicine, 16 patients with SIF were identified during a 6-year period. All patients were elderly women (mean age of 81 years) who presented with low back or pelvic pain. Radicular pain in the lower limb was common. Ten patients were bedridden. All 16 patients were osteopenic. Plain radiographs of the pelvis were nondiagnostic in 11 patients. Radionuclide bone scan showed a typical H-shaped pattern of increased uptake in 11 patients, and computed tomographic scan confirmed SIF (9/9 patients). With bed rest and analgesics, outcome was favorable in all patients. This study confirms the nonspecific presentation of SIF and the need to maintain a high index of clinical suspicion to make a prompt diagnosis and avoid unnecessary and sometimes invasive procedures. PMID- 8629881 TI - Erythromycin-induced clozapine toxic reaction. AB - Clozapine, used in the treatment of patients with schizophrenia resistant to other neuroleptic medication, is metabolized by the hepatic microsomal system to demethyl-clozapine and clozapine-N-oxide. Changes in clozapine serum concentrations have been documented after initiation of therapy with medications known to induce or inhibit liver microsomal enzymes. These interactions are of clinical importance when diminished efficacy or increased toxic effects of clozapine therapy occur. A 34-year-old schizophrenic man had increased clozapine serum concentrations, leukocytosis, and adverse effects as a result of concomitant erythromycin therapy given for suspected lower respiratory tract infection. Symptoms included somnolence, difficulty in coordination and ambulation, slurred speech, disorientation, and incontinence. The symptoms resolved after treatment with clozapine and erythromycin were discontinued, and treatment with clozapine was gradually resumed. PMID- 8629882 TI - Severe hyponatremia during therapy with fluoxetine. AB - We described two elderly patients with severe hyponatremia that was probably caused by inappropriate secretion of antidiuretic hormone. The hyponatremia arose during fluoxetine (Prozac) antidepressant therapy. PMID- 8629883 TI - Benefits of isoniazid preventive therapy. PMID- 8629884 TI - Morning report: not just a matter of attitude. PMID- 8629885 TI - Isoniazid preventive therapy in areas of high isoniazid resistance. PMID- 8629886 TI - Systematic changes in cerebral glucose metabolic rate after successful behavior modification treatment of obsessive-compulsive disorder. AB - BACKGROUND: We sought to determine in a new patient sample whether symptomatic improvement in obsessive-compulsive disorder treated with behavior modification is accompanied by significant changes in glucose metabolic rates in the caudate nucleus, measured with positron emission tomography, as seen in a previous study. Second, by combining samples from this and the previous study, we also examined whether there were pathologic correlational relationships among brain activity in the orbital cortex, caudate nucleus, and thalamus that obtained before behavioral treatment of obsessive-compulsive disorder, but that decreased significantly with symptom improvement. METHODS: Nine patients with obsessive-compulsive disorder were studied with positron emission tomography before and after 10 weeks of structured exposure and response prevention behavioral and cognitive treatment. Results were analyzed both alone and combined with those from nine similar subjects from the previous study. RESULTS: Behavior therapy responders had significant (P < .05) bilateral decreases in caudate glucose metabolic rates that were greater than those seen in poor responders to treatment. Before treatment, there were significant correlations of brain activity between the orbital gyri and the head of the caudate nucleus and the orbital gyri and the thalamus on the right. These correlations decreased significantly after effective treatment. CONCLUSIONS: These results replicate and extend previous findings of changes in caudate nucleus function with behavior therapy for obsessive-compulsive disorder. A prefrontal cortico-striato-thalamic brain system is implicated in mediation of symptoms of obsessive-compulsive disorder. PMID- 8629887 TI - Clinical and biochemical effects of catecholamine depletion on antidepressant induced remission of depression. AB - BACKGROUND: Most hypotheses of the therapeutic mechanism of action of antidepressant drugs have focused on the role of the monoamines. We examined the effect of catecholamine depletion on antidepressant-induced remission. METHOD: The tyrosine hydroxylase inhibitor alpha-methylparatyrosine and the antihistamine diphenhydramine hydrochloride were administered, during separate test sessions, to depressed patients in remission maintained with either norepinephrine reuptake inhibitors (desipramine [n = 7] or mazindol [n = 2]) or serotonin reuptake inhibitors (fluoxetine hydrochloride [n = 9] or sertraline hydrochloride [n = 1]). Because of considerable sedation associated with alpha-methylparatyrosine testing, diphenhydramine was used as an active control rather than an inactive placebo. The effects of alpha-methylparatyrosine and diphenhydramine on depression, anxiety, and plasma catecholamine metabolites were assessed. RESULTS: alpha-Methylparatyrosine produced similar significant decreases in plasma 3 methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid levels in the treatment groups. alpha-Methylparatyrosine produced a robust increase in depressive symptoms on the Hamilton Depression Rating Scale, including depressed mood, decreased concentration, anhedonia, loss of interest, and feelings of worthlessness, helplessness, and hopelessness, in the desipramine-mazindol but not in the fluoxetine-sertraline group. Diphenhydramine had no effects on mood in either treatment group. CONCLUSIONS: The therapeutic effects of norepinephrine reuptake inhibitors, but not serotonin reuptake inhibitors, are reversed by catecholamine depletion. Considered with previous reports that serotonin depletion produces depressive relapses in patients in remission maintained with serotonin reuptake inhibitors, but not norepinephrine reuptake inhibitors, these findings suggest that antidepressants may not work via a single monoamine-related mechanism. PMID- 8629888 TI - A hospital-based twin register of the heritability of DSM-IV unipolar depression. AB - OBJECTIVES: To estimate the contribution of genes and shared family environment to the liability to DSM-IV major depression and to examine the influence of certain proband characteristics on twin concordance. METHODS: We studied 177 probands with major depressive disorder ascertained via the Maudsley Hospital Twin Register (London, England) and their same-sex co-twins. Diagnostic assessments were carried out blind to zygosity and information on the other member of the twin pair. Probandwise concordances were used to compute correlations in liability, and model fitting was performed using maximum likelihood procedures. RESULTS: The probandwise concordance was 46% in monozygotic (n = 68) and 20% in dizygotic (n = 109) twins, a statistically highly significant difference. There was no evidence of a sex difference in heritability or of shared environmental effects. Depending on the assumed population risks for DSM-IV, major depression estimates of heritability were between 48% and 75%. A duration of longest episode of less than 13 months, multiple episodes, and an endogenous rather than neurotic pattern of symptoms as established by the International Classification of Diseases, Ninth Revision, in the proband were associated with a trend toward a higher monozygotic-dizygotic concordance ratio. Using log-linear analysis, only the association between duration of episodes and monozygotic-dizygotic concordance ratio was significant. CONCLUSIONS: Liability to DSM-IV major depression has a substantial heritable component, and there is no evidence of an effect of shared family environment. Some proband characteristics, especially shorter duration of episodes, may be associated with a larger degree of genetic determination. PMID- 8629889 TI - Increased number of vasopressin- and oxytocin-expressing neurons in the paraventricular nucleus of the hypothalamus in depression. AB - BACKGROUND: Cerebrospinal fluid levels of arginine vasopressin (AVP) and oxytocin (OXT) have been found to change in mood disorders. In the present study, the numbers of AVP-immunoreactive (IR) and OXT-IR neurons were determined in the paraventricular nucleus (PVN) of the human hypothalamus. METHODS: Postmortem brain tissue was fixed in formalin, embedded in paraffin, and stained for AVP and OXT using immunocytochemical techniques. The number of IR neurons in the PVN was estimated by morphometry in eight depressed patients ranging in age from 21 to 85 years and eight age-matched controls ranging in age from 23 to 88 years. RESULTS: The numbers of AVP-IR and OXT-IR neurons in the PVN of patients with mood disorder were increased by 56% and 23%, respectively. No differences were found in AVP-IR or OXT-IR cell numbers between three patients with major depression and three patients with bipolar depression. The numbers of AVP-IR and OXT-IR neurons in two patients with depression not otherwise specified were within the same range as in the six other patients with a mood disorder. CONCLUSIONS: The AVP and OXT neurons were activated in the PVN in patients with major depression or bipolar disorder. This activation may be associated with activation of the hypothalamic-pituitary-adrenal axis in these patients, since both AVP and OXT are known to potentiate the effects of corticotropin-releasing hormone. Because of their central effects, activation of AVP and OXT neurons may also be related to symptoms of major depression or bipolar disorder. PMID- 8629890 TI - Electroencephalographic sleep profiles during depression. Effects of episode duration and other clinical and psychosocial factors in older adults. AB - BACKGROUND: Limited evidence suggests that polysomnographic alterations may be more prominent early in a depressive episode. Whether the effects of episode duration extend beyond middle age and appear in late-life depression as well has important implications for treatment decisions and for understanding depressive illness across the life span. Furthermore, the impact of episode duration on sleep has not been examined in the context of other factors related to clinical history and psychosocial status. METHODS: Eighty-three persons aged 60 years or older with recurrent depression were studied: 34 had been depressed for 2 to 16 weeks and 49 for longer periods. An age- and gender-matched group of 48 persons with no history of major depression served as controls. Initial univariate analyses examined duration effects on electroencephalographic (EEG) sleep measurements. Multivariate analyses considered the combined effects of episode duration, clinical variables, and psychosocial variables on EEG sleep profile. RESULTS: Episode duration was strongly associated with sleep continuity, architecture, and rapid eye movement: subjects who were earlier in their depressive episodes had their sleep impaired more than those later in their episodes, who, in turn, were more impaired than controls. Moreover, clinical characteristics of subjects' depressive illness, demographic variables, and psychosocial stressors and supports had unique effects on the EEG sleep profile. CONCLUSION: Episode duration appears to be a potent factor to consider when evaluating sleep during depression. The additional contribution of clinical and psychosocial characteristics to the prediction of the EEG sleep profile demonstrates the importance of incorporating these variables into models of the psychobiologic characteristics of depression. The results are relevant to the timing and focus of therapeutic interventions. PMID- 8629891 TI - Agoraphobia, simple phobia, and social phobia in the National Comorbidity Survey. AB - BACKGROUND: Data are presented on the general population prevalences, correlates, comorbidities, and impairments associated with DSM-III-R phobias. METHODS: Analysis is based on the National Comorbidity Survey. Phobias were assessed with a revised version of the Composite International Diagnostic Interview. RESULTS: Lifetime (and 30-day) prevalence estimates are 6.7% (and 2.3%) for agoraphobia, 11.3% (and 5.5%) for simple phobia, and 13.3% (and 4.5%) for social phobia. Increasing lifetime prevalences are found in recent cohorts. Earlier median ages at illness onset are found for simple (15 years of age) and social (16 years of age) phobias than for agoraphobia (29 years of age). Phobias are highly comorbid. Most comorbid simple and social phobias are temporally primary, while most comorbid agoraphobia is temporally secondary. Comorbid phobias are generally more severe than pure phobias. Despite evidence of role impairment in phobia, only a minority of individuals with phobia ever seek professional treatment. CONCLUSIONS: Phobias are common, increasingly prevalent, often associated with serious role impairment, and usually go untreated. Focused research is needed to investigate barriers to help seeking. PMID- 8629892 TI - Public-speaking fears in a community sample. Prevalence, impact on functioning, and diagnostic classification. AB - BACKGROUND: Recent epidemiologic studies have revealed that social phobia is more prevalent than has been previously believed. An unresolved issue is the extent to which public-speaking fears constitute a recognizable form of social phobia in a community sample and, moreover, to what extent these fears are associated with functional morbidity. METHODS: To examine the prevalence and impact of public speaking fears and their relationship to social phobia in a community sample, we conducted a randomized telephone survey of 499 residents of Winnipeg, Manitoba, a medium-sized midwestern metropolitan area. RESULTS: One third of the respondents reported that they had excessive anxiety when they spoke to a large audience. The onset of fears was early (ie, 50%, 75%, and 90% by the ages of 13, 17, and 20 years, respectively). Anxious cognitions about public speaking included the following fears: doing or saying something embarrassing (64%), one's mind going blank (74%), being unable to continue talking (63%), saying foolish things or not making sense (59%), and trembling, shaking, or showing other signs of anxiety (80%). In total, 10% (n = 49) of the respondents reported that public-speaking anxiety had resulted in a marked interference with their work (2%), social life (1%), or education (4%), or had caused them marked distress (8%). Twenty-three persons (5%) had public-speaking anxiety in isolation (ie, without evidence of additional kinds of social fears). CONCLUSIONS: These data support the inclusion of severe forms of public-speaking fears within the social phobia construct and, furthermore, suggest that public-speaking anxiety may have a detrimental impact on the lives of many individuals in the community. PMID- 8629893 TI - Depressed mood and the incidence of Alzheimer's disease in the elderly living in the community. AB - BACKGROUND: It remains unclear whether depression increases the risk for dementia in the elderly. We evaluated the relationship between depressed mood at baseline and the incidence of dementia, particularly Alzheimer's disease, in the elderly living in the community. METHODS: A total of 1070 elderly individuals, aged 60 years or older, were identified as part of a registry for dementia in the Washington Heights community of North Manhattan, NY. In a prospective, longitudinal design with follow-up for 1 to 5 years, annual physician evaluation and neuropsychological testing were used to assess levels of cognitive impairment and to diagnose dementia. Depressive symptoms were evaluated with the 17-item Hamilton Rating Scale for Depression. Based on clinical considerations and a validity study, a positive score for the depressed mood item was used in statistical analyses. To confirm the results, the total Hamilton Rating Scale for Depression score was also evaluated as the "depression" variable. RESULTS: Of the 1070 subjects, 218 met criteria for dementia at baseline evaluation. In the 852 subjects without dementia, depressed mood was more common in individuals with greater cognitive impairment. In a follow-up study of 478 of these subjects without dementia (mean +/- SD, 2.54 +/- 1.12 years of follow-up), the effect of baseline depressed mood on the end-point diagnosis of dementia (93% had possible or probable Alzheimer's disease) was evaluated in a Cox proportional hazards model. Depressed mood at baseline was associated with an increased risk of incident dementia (relative risk, 2.94; 95% confidence interval, 1.76 to 4.91; P < .001). This effect remained after adjustment for age, gender, education, language of assessment, Blessed Memory Information and Concentration test scores, and Blessed Functional Activity Scale scores (relative risk, 2.05; 95% confidence interval, 1.16 to 3.62; P < .02). Similar results were obtained when the total Hamilton Rating Scale for Depression score was used as the depression variable, with the use of the same covariates (relative risk, 1.07 per point interval; 95% confidence interval, 1.02 to 1.11; P < .01). CONCLUSIONS: Depressed mood moderately increased the risk of developing dementia, primarily Alzheimer's disease. Whether depressed mood is a very early manifestation of Alzheimer's disease, or increases susceptibility through another mechanism, remains to be determined. PMID- 8629894 TI - Abnormal electroencephalographic sleep profiles in major depression: association with response to cognitive behavior therapy. AB - BACKGROUND: To test the hypothesis that depressed patients with selected neurobiologic disturbances are less responsive to psychotherapy, we examined responses to cognitive behavior therapy in relation to electroencephalographic sleep profiles. METHODS: Under a prospective, case-control design, 90 outpatients with probable or definite endogenous major depression (Schedule for Affective Disorders and Schizophrenia and Research Diagnostic Criteria) were stratified into abnormal and normal sleep subgroups (on the basis of an empirically validated electroencephalographic sleep profile) and more severe and less severe depression subgroups (on the basis of pretreatment Hamilton scores). Response to 16 weeks of treatment was analyzed for both intention-to-treat and completers (n = 82) samples. Outcomes during a 36-month prospective follow-up were assessed with survival analyses. RESULTS: Abnormal sleep profiles and higher pretreatment depression severity were independently associated with poorer outcomes on several analyses. The association between sleep abnormality and cognitive behavior therapy response was not significant in the completers analyses, however, largely because of differential attrition. During follow-up, pretreatment depression severity was predictive of relapse and a lower recovery rate, whereas sleep abnormality was predictive of a lower recovery rate and a higher risk of recurrence. CONCLUSIONS: Depressed patients characterized by higher severity and/or an abnormal electroencephalographic sleep profile were relatively less responsive to cognitive behavior therapy. These associations are hypothesized to result from a constellation of neurophysiologic disturbances that interfere with the acquisition, application, and implementation of the skills emphasized in cognitive behavior therapy. PMID- 8629895 TI - Asbestos contamination in paraffin tissue blocks. PMID- 8629896 TI - Surgical pathology specimen identification and accessioning: A College of American Pathologists Q-Probes Study of 1 004 115 cases from 417 institutions. AB - OBJECTIVE: To examine and suggest improvements for deficiencies occurring in the specimen identification and accessioning process in the surgical pathology laboratory. DESIGN: Using the College of American Pathologists' and the Joint Commission for Accreditation of Healthcare Organizations' requirements as the standard, each laboratory was asked to prospectively document deficiencies in specimen identification and accessioning for 4 months, or until a maximum of 4000 cases or 400 deficiencies were accrued. PARTICIPANTS: Four hundred seventeen laboratories in the College of American Pathologists' voluntary quality improvement program, Q-Probes, participated in this study. RESULTS: Identification and accessioning deficiencies were found in 60 042 (6%) out of a total 1 004 115 cases accessioned (median deficiency rate of 3.4%). Errors related to specimen identification accounted for 9.6% of these deficiencies, discrepant or missing information items were present in 77%, and 3.6% involved specimen handling. The most common deficiency was "no clinical history or diagnosis present on the requisition slip," which represented 40% of all deficiencies. Deficiencies were most often detected by the person assigned to accessioning duties or by histology personnel. In 66% of cases, no action was taken to remedy the deficiency, but this varied dramatically according to the specific type of deficiency. An action was taken to remedy deficiencies in 69% of cases involving specimen identification errors, in 58% of specimen handling errors, and in 27% of cases with discrepant or missing information. Peer group stratifiers were associated with a lower deficiency rate. Laboratories with lower numbers (<15 000) of accessioned cases and laboratories with a formal written plan for the detection of errors in accessioning and specimen identification reported lower rates of deficiencies. Factors that correlated with a higher rate of deficiencies included submitting the specimen container and requisition slip in a unique secondary container (P<.005) and labeling the specimen container with only a patient's name or unique patient identification number (as opposed to both identifiers). CONCLUSIONS: The majority of deficiencies occurring in surgical pathology specimen identification and accessioning are related to missing or inaccurate clinical information. Deficiencies are detected in multiple locations, including areas not typically thought of as quality check points, such as transcription. A variable amount of effort occurs to rectify deficiencies; this effort is largely dependent on the type of deficiency involved. Finally, laboratories with a formal error detection plan had fewer deficiencies. PMID- 8629897 TI - Intralaboratory timeliness of surgical pathology reports. Results of two College of American Pathologists Q-Probes studies of biopsies and complex specimens. AB - OBJECTIVE: To develop multi-institutional reference databases for intralaboratory timeliness of surgical pathology routine biopsies and complex specimens from the time of specimen accessioning to report completion, and to examine the influence of laboratory characteristics and practices on turnaround time (TAT). DESIGN: Participants in the Q-Probes quality improvement program of the College of American Pathologists took part in two separate studies, the first conducted in 1992 and 1993 and the second in 1993 and 1994. Each participant tracked the number of days from specimen accessioning to report completion for 30 routine biopsies and 30 complex specimens in each study. Based on this intralaboratory time interval, performance was compared with the College of American Pathologists' laboratory accreditation standard of 2 working days. PARTICIPANTS: Five hundred twenty-five surgical pathology laboratories responded to the study of routine biopsies, and 489 laboratories responded to the study of complex specimens. Participants were mainly located in the United States, but there were respondents from Canada, Australia, New Zealand, and Hong Kong as well. RESULTS: In the first study, evaluation of 15 725 biopsy cases showed that the cumulative aggregate percentage of routine biopsy cases processed from the time of specimen accessioning to report completion was 79% by 1 working day, 95% by 2 working days, and 98% by 3 working days. Individual participant's data revealed that all reports were completed by the second working day in 90% of the laboratories and by the third working day in 95% of laboratories. Factors that significantly contributed to increased report TAT included larger institutional size, a greater number of surgical pathologists, greater annual surgical pathology volume processed, technical processing resulting in delayed slide availability, pathology practices that integrated residency training, and reduced staffing levels of histotechnologists/technicians and transcriptionists. Shorter TATs were achieved in those institutions that had previously established a TAT goal for routine biopsy specimens. In the second study of 14 298 aggregate complex specimen cases, 68% required routine processing and 32% required special handling. Overall, 56% of all complex specimen reports were processed and completed in 1 working day, 81% in 2 working days, 91% in 3 working days, and 95% in 4 working days. On average, the percentage of cases processed and reports signed out in 2 working days or less was 80% for all complex specimen cases, 90% for routine cases, and 60% for special-handling cases. The mean of all participants' median TATs was 1.5 days (range 0-5 days) for complex specimens, 1.3 days (range 0-5 days) for cases requiring routine handling, and 2.6 days (range 0-13.5 days) for cases requiring special handling. Several factors were associated with increased report TAT: institutional occupied bedsize greater than 450, routine responsibility for gross dissection assigned to residents only, earliest availability of slides after 12 pm, resident involvement in sign-out, interposing a day between availability of slides and final slide sign-out for resident education purposes, and a greater number of surgical pathologists. CONCLUSIONS: We have documented that for the majority of routine cases, the College of American Pathologists Laboratory Accreditation Program's TAT standard of report completion time within 2 working days for the intralaboratory component of TAT is a reasonable goal. This standard was successfully met by participants in 95% of routine biopsy cases and 91% of routine complex specimens. Special handling procedures for complex specimens contributed, on average, an additional delay of 1.3 days. To our knowledge these are the first systematic studies to describe timeliness from the time of specimen accessioning to report completion for surgical pathology specimens, and they may serve as reference databases for benchm PMID- 8629898 TI - Effect of chorioamnionitis on the levels of serum proteins in the cord blood of premature infants. AB - OBJECTIVE: To determine the relationships among complements, other serum proteins (including acute-phase reactant), and the cytokine interleukin-6. DESIGN: Prospective observational study. SETTING: A perinatal center, children's hospital, and research institute in Osaka, Japan. PATIENTS: Two hundred fifteen newborn infants ranging from 17 to 42 weeks in gestational age. MAIN OUTCOME MEASURES: We measured the serum levels of several proteins and complements in the cord blood of neonates with and without chorioamnionitis by immunological assays. RESULTS: The levels of C3d, haptoglobin, interleukin-6, IgM, C-reactive protein, and IgA were not influenced by gestational age, and levels of C5, C1q, C2, albumin, C9, and IgG were not influenced by chorioamnionitis status. The levels of C3, C4, CH50, factor B, and orosomucoid were influenced by both factors. CONCLUSIONS: Our investigation of the mutual relation of the data suggests that the increase of cytokine interleukin-6 affects each other component. We suggest that, compared with the serum levels of proteins in neonates with chorioamnionitis, levels in neonates without chorioamnionitis are more similar to those in the normal fetus. PMID- 8629899 TI - Enhancing autopsy performance and reporting. A system for a 5-day completion time. AB - OBJECTIVE: To develop a system for enhancing the performance and reporting of autopsies in an effective and clinically useful manner. DESIGN: Twelve steps were defined as essential for the completion of the autopsy. Each step of the process was evaluated for usefulness and effectiveness. SETTING: Autopsies performed in a university hospital from 1992 through 1994. PARTICIPANTS: Pathology residents and staff, clinicians, and clinical team house staff. INTERVENTION: Participants followed the 12-step process, with emphasis on involving the clinical team in the interview, prosection, and final rounds. The final rounds conference was designated a working conference, where the perfused-fixed brain was cut, histologic sections of the case were submitted, and the provisional diagnosis was written with the clinicians. A next-day microscopic slide review session was scheduled to "sign out" the case. Establishing a philosophy of status equal to all other department functions facilitated implementation. MAIN OUTCOME MEASURE: All autopsies performed for a period of 3 years (2 retrospective and 1 prospective) were included. RESULTS: The autopsy completion time was reduced from a mean of 57 days (range 7 to 174) in 1992 to 4.8 days (range 1 to 16) in 1994. CONCLUSION: The autopsy completion time was reduced, increasing its usefulness for teaching and quality assurance. Relationships with the clinical staff were enhanced with consultation-style final reports. Enthusiasm for, and satisfaction with, the new process was expressed by clinicians, pathology staff, residents, and technical support staff. PMID- 8629900 TI - Stage IE non-Hodgkin's lymphoma involving the dura: A clinicopathologic study of five cases. AB - OBJECTIVE AND DESIGN: Non-Hodgkin's lymphomas rarely present as a localized mass involving the dura. In this report we describe the clinical, histologic, and immunohistochemical features of five cases of stage IE non-Hodgkin's lymphoma involving the dura. PATIENTS: Four women and one man, 36 to 67 years of age (median 50.6 years). RESULTS: Myelography and magnetic resonance imaging scans revealed discrete expansile masses involving the dura of the cervical, thoracic, and lumbar regions of the spinal cord and the frontal lobe of the brain. Histologically, the tumors were classified in the Working Formulation as small lymphocytic (2), diffuse large cell (2), and large cell immunoblastic (1) (anaplastic large cell lymphoma). Four tumors were of B-cell lineage and the anaplastic large-cell lymphoma was of T-cell lineage. The two small lymphocytic neoplasms had immunoglobulin heavy-chain gene rearrangements as shown by either Southern blot hybridization or the polymerase chain reaction. Four patients underwent decompression laminectomy; three received spinal radiation; two received chemotherapy (one intrathecal, one systemic) for lymphocytosis of the cerebrospinal fluid. The dural mass overlying the frontal lobe was excised and focally irradiated. Clinical follow-up was available for all patients. Four patients were alive 12 to 40 months after diagnosis and showed no evidence of recurrent or disseminated disease. The patient with anaplastic large-cell lymphoma died 10 days after laminectomy, secondary to pulmonary thromboemboli. CONCLUSIONS: We conclude that non-Hodgkin's lymphomas of varied histologic types and of either B- or T-cell lineage may rarely present as a stage IE dural mass. These lesions appear to have a good initial response to treatment; however, longer clinical follow-up is necessary to assess the incidence of relapse and final outcome. PMID- 8629902 TI - Immunohistochemical observation of foci of muscle actin-positive tumor cells in meningiomas. AB - OBJECTIVE: It has been suggested that muscle actin is expressed in some meningiomas. The purpose of this study was to examine this hypothesis in a large number of meningiomas and to examine possible explanations for this expression. DESIGN: Sixty-five meningiomas were examined using immunohistochemistry for muscle actin. Meningiomas with foci of muscle actin-positive cells (n = 6) were further examined using monoclonal antibodies for desmin, epithelial membrane antigen, cytokeratin, and von Willebrand factor. These specimens were also stained for reticulin. SETTING: All tissues were obtained from surgical biopsy specimens at a large academic medical center. MAIN OUTCOME MEASURES: Meningioma cells were considered to express muscle actin if the expression was shown to localize specifically in meningothelial cells and whorls that were also positive for epithelial membrane antigen. RESULTS: Seventeen of 65 meningiomas were shown to contain muscle actin-positive cells. Scattered cells were positive in 11 cases, and in the remaining six cases we observed foci of muscle actin-positive cells, which had a propensity to form cellular whorls. These foci expressed epithelial membrane antigen, but stains for desmin, cytokeratin, von Willebrand factor, and reticulin were negative. CONCLUSIONS: Our findings suggest that meningioma cells elaborate muscle actin without showing differentiation into specific cell types. PMID- 8629901 TI - Renovasculopathies of hypertension in Mexico City. AB - OBJECTIVE: To examine the severities of renovasculopathies at autopsy in Mexico City, comparing birth cohorts, cause of death categories, and urban versus rural residence. DESIGN: Autopsies conducted in 1954, 1974, and 1991 were chosen from archived materials to represent men and women aged 15 to 84 years. Hematoxylin- and eosin-stained sections of kidney were retrieved and evaluated morphometrically. SETTING: Department of Pathology, Hospital General, Mexico City. PATIENTS: Patients of the Hospital General evaluated at autopsy. MAIN OUTCOME MEASURES: Renovasculopathies of hypertension include the defining features of benign nephrosclerosis, arterial intimal fibroplasia and arteriolar hyalinization. Fibroplasia is measured as average intimal thickness of renocortical arteries, expressed as percent of outer diameter. Hyalinization is measured by counting the number of affected arterioles in a unit area of cortical section. RESULTS: This study confirms a former study that shows much lower levels of renovasculopathy in Mexico City than in blacks and whites of New Orleans. The cohort born in 1894 showed slightly more involvement than those of 1914, 1934, 1954, and 1971, which did not differ from each other. No differences were found between urban and rural residence nor among the categories of tuberculosis, cirrhosis, cancer, and other conditions unrelated to atherosclerosis. CONCLUSIONS: The remarkable constancy of the renovasculopathies in most comparisons includes little change throughout the 20th century, keeping stable the substantial Mexico-US differences over much of this time frame. PMID- 8629903 TI - Altered expression of alpha-smooth muscle isoactin in Hirschsprung's disease. AB - OBJECTIVE: To analyze the expression of smooth muscle actin (SMA) contractile proteins in the intestinal muscle of patients with Hirschsprung's disease. DESIGN: A total of 56 colonic whole-wall specimens were tested with immunohistochemistry and monoclonal antibodies directed against, respectively, alpha and pan (alpha and gamma) SMA. PATIENTS: The study included both aganglionic and dilated normoganglionic colonic samples from 29 children who had undergone definitive surgery for Hirschsprung's disease. Morphologically normal (n = 6) and dilated colons (n = 5) from 11 patients suffering from conditions unrelated to Hirschsprung's disease served as controls. RESULTS: The alphaSMA immunostaining showed a pattern identical to normal controls in all the aganglionic areas and in 70% of the normoganglionic segments. In contrast, a lack of alphaSMA expression confined to the circular muscle cells was observed in 30% of the normoganglionic specimens and in the five dilated control specimens. The same areas showed a faintly reduced immunoreactivity with the antibody to pan SMA, consistent with the absence of alpha-isoactin, but implying the presence of gamma-isoactin. In the Hirschsprung's disease cases, the absence of alphaSMA expression at the time of surgery was associated with the occurrence of long-term postoperative motility problems (p<.01). CONCLUSION: These data suggest that alphaSMA expression defect in Hirschsprung's disease may be acquired and related to chronic colonic obstruction and subsequent dilatation. The observed changes correlate with disturbances of peristalsis and could be used to assess the risk of intestinal dysmotility frequently reported after surgical removal of the aganglionic bowel. PMID- 8629904 TI - Monoclonal B-cell population mimicking lymphoma in a patient with multiple sclerosis. AB - BACKGROUND: Diagnosis of intraparenchymal brain lesions has usually required invasive diagnostic procedures, because too few cells are shed into cerebrospinal fluid to permit cytologic diagnosis. Polymerase chain reaction technology makes it possible to identify cell populations that are present at a much lower frequency than traditional techniques. CASE REPORT: A young woman presented with multiple brain lesions raised the question of primary central nervous system lymphoma. Polymerase chain reaction analysis of cerebrospinal fluid showed evidence of a monoclonal B-cell population heightening suspicion of lymphoma. Brain biopsy showed acute demyelination most consistent with multiple sclerosis. CONCLUSION: Although T-cell restriction has been demonstrated in multiple sclerosis lesions, the finding of a monoclonal B-cell population was unexpected and to our knowledge has not been previously reported. This case emphasizes that monoclonality is not always indicative of a neoplastic process, particularly in the central nervous system. PMID- 8629906 TI - Tacrolimus-related posttransplant lymphoproliferative disorder presenting as autoimmune hemolytic anemia. AB - Tacrolimus (formerly known as FK506) is a macrolide immunosuppressant that has been used to prevent rejection of solid organ allografts. Acute hemolytic anemia is one of the side effects associated with tacrolimus therapy, and two mechanisms have been described to account for acute hemolytic anemia in patients receiving tacrolimus: drug-induced hemolysis and alloimmune hemolysis resulting from donor lymphocytes derived from the allograft (passenger lymphocyte syndrome). We report a case of a liver transplant recipient who developed fatal autoimmune hemolytic anemia while under treatment with tacrolimus for allograft rejection, and in whom postmortem examination revealed a clinically unsuspected posttransplant lymphoproliferative disorder. This case implicates autoimmune hemolytic anemia as a novel mechanism of acute hemolysis in patients treated with tacrolimus and further suggests that acute hemolytic anemia in this group of patients may herald an occult lymphoproliferative disorder. PMID- 8629905 TI - Extramedullary plasmacytoma mimicking primary colonic carcinoma in a patient with Crohn's disease. Case report and literature review. AB - We present the case of a 45-year-old man who underwent segmental resection for what was considered clinically to be a primary colonic carcinoma. Histopathologic evaluation revealed the tumor to be a malignant plasmacytoma of the colon. Additionally, the entire segment of distal ileum and colon was involved by an inflammatory process with the features of Crohn's disease, accompanied by focal glandular dyplasia of the colonic mucosa. This case reemphasizes that plasmacytoma of the colon can closely mimic carcinoma of the colon in its presentation. The association between plasmacytoma of the colon and Crohn's disease has not, to our knowledge, been described previously. PMID- 8629907 TI - Hodgkin's disease primarily involving Waldeyer's ring. Case report and review of the literature. AB - We report a rare case of mixed cellularity Hodgkin's disease primarily involving Waldeyer's ring, which was confirmed by immunohistochemistry and gene rearrangement studies. Our literature search revealed only 14 cases of this entity; six of these represented lymphocyte-predominant Hodgkin's disease not confirmed by immunohistochemistry and, thus, were possibly not truly Hodgkin's disease. Our case demonstrates the diagnostic difficulties in this rare entity and we recommend appropriate immunoperoxidase staining in atypical lymphoid proliferations involving otolaryngic mucosa-associated lymphoid tissue. PMID- 8629908 TI - Primary Epstein-Barr virus-related non-Hodgkin's lymphoma of the pleural cavity following long-standing tuberculous empyema. AB - Primary non-Hodgkin's lymphomas of the pleural cavity have been described mostly in Japan. We report a case of high-grade non-Hodgkin's lymphoma (immunoblastic type) of the pleural cavity occurring in a nonimmunocompromised patient 55 years after an artificial pneumothorax was performed for the treatment of pulmonary tuberculosis. Immunohistochemical study revealed a B phenotype (CD20), and an in situ hybridization detected small nuclear RNAs encoded by Epstein-Barr virus in most lymphomatous cells. A link between primary pleural lymphoma and the local long-standing chronic inflammation, inducing a clonal transformation of Epstein Barr virus-infected immortalized B lymphocytes, is suspected. PMID- 8629909 TI - Liposarcoma of the base of tongue and tonsillar fossa: A possibly underdiagnosed neoplasm. AB - Liposarcomas of the head and neck are exceedingly rare, and fewer than 90 cases have been reported in the literature. Liposarcoma of the oral cavity is an even less common entity, and to our knowledge only nine cases have been reported to date. We report the clinical and pathologic findings of a case of well differentiated liposarcoma of the base of tongue and tonsillar fossa. The patient is a 76-year-old white man with a long-standing history of a mass in the oral cavity and hypopharynx. The mass had been resected several times over the span of 23 years, and diagnoses of lipoma, neurofibroma, mesenchymoma, and angiofibrolipoma have been rendered on different occasions. At the last admission, a polypoid mass of the left tonsillar fossa and base of tongue was resected. The tumor was multinodular and measured 2.5 cm in greatest diameter. Histologically the tumor was ill-defined with infiltrating borders and was composed predominantly of mature adipose tissue with occasional lipoblasts. A small proportion of the tumor consisted of clusters of spindle cells and pleomorphic lipoblasts. Mitotic activity was not seen. The pleomorphic cells were positive for S100 protein and negative for muscle-specific markers. Ultrastructural analysis confirmed the nature of the lipoblasts. Our case depicts the typical natural history and histologic features of liposarcoma of the oral cavity. This tumor is usually well differentiated and has a high recurrence rate and almost no tendency for metastasis. Based on our case and review of the literature, it appears that well-differentiated liposarcoma of the oral cavity can occasionally be underdiagnosed because of the low mitotic activity and long latent period between the original diagnosis and first recurrence. PMID- 8629910 TI - Malignant granular cell tumor. Report of a case with DNA ploidy analysis. AB - Malignant granular cell tumor is a rare neoplasm that has been reported to occur at various sites. Although there are no reliable histologic criteria to differentiate the benign from the malignant form of granular cell tumor, in many reported cases cellular pleomorphism and the presence of mitotic activity suggested an aggressive nature. We report a case of malignant granular cell tumor that involved the subcutaneous tissue in the sacrococcygeal area. The tumor showed cellular atypia on histologic sections, but mitotic figures were rare. The tumor recurred and produced widespread metastasis, resulting in the death of the patient. DNA flow cytometry demonstrated DNA diploidy, suggesting that such analysis may not be beneficial in predicting the behavior of these tumors. PMID- 8629911 TI - Peritoneal silicosis. AB - A 73-year-old man with a clinical diagnosis of pulmonary silicosis (long-standing exposure to silica, pulmonary infiltrates, and flu-like symptoms) presented to the emergency room with fever, acute biliary colic, and cholelithiasis. The patient had a 2-year status postchemotherapy with complete remission of hepatic and splenic malignant lymphoma. At laparotomy we found studding of the undersurface of the diaphragm with multiple small dark nodules. Owing to the patient's history of previously treated abdominal malignant lymphoma, the lesions were grossly interpreted as abdominal lymphomatosis. The microscopic appearance of the lesions suggested silicotic nodules, which were confirmed by digital scanning electron microscopy and roentgenographic microanalysis performed on formalin-fixed, paraffin-embedded tissue. This is an unusual extrapulmonary pattern of peritoneal seeding in silicosis. PMID- 8629912 TI - Luteinized thecoma with sclerosing peritonitis. AB - Clement and colleagues recently described a group of six patients who had spindle cell proliferations of the ovary resembling luteinized thecomas. A fatal case of extensive sclerosing peritonitis associated with an identical ovarian proliferation is reported. In addition, several other cases in the literature that almost certainly represent the same disease entity have been identified. To our knowledge, this report increases the number of cases to 13. The extensive peritoneal disease along with the high mitotic activity of the ovarian tumor resulted in the diagnosis of peritoneal dissemination of an ovarian cancer in some cases, including the one currently described. Awareness of this newly described condition, which may be more common than previously thought, may help prevent further misdiagnosis. PMID- 8629913 TI - Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study. AB - A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor. PMID- 8629914 TI - Concurrent pulmonary arterial dissection and saccular aneurysm associated with primary pulmonary hypertension. AB - We report concurrent pulmonary arterial dissection and saccular aneurysm resulting from primary pulmonary hypertension in a 26-year-old man. The pulmonary trunk was dissected 9 cm along its entire circumference, 3 cm above the pulmonary valvular cusps. In addition, a saccular aneurysm 3.5 cm in diameter had formed at the right pulmonary hilus. Histopathology revealed marked medical degeneration and fragmentation of the elastic laminae in the former lesion and a true aneurysm with attenuation and fragmentation of elastic laminae in the latter. The peripheral vasculature in the lungs showed evidence of increased pulmonary arterial pressure, including plexiform and angiomatoid lesions. We present this unique case and discuss the pathomorphogenesis of these lesions in conjunction with primary pulmonary hypertension. PMID- 8629915 TI - Physiological parameters associated with the performance of a distracting task and genital self-stimulation in women with complete spinal cord injuries. AB - OBJECTIVE: To compare the physiological sexual responses of women with complete spinal cord injuries (SCI) with those of able-bodied women. DESIGN: Controlled laboratory-based analysis of responses to a distracting task coupled with manual genital stimulation versus masturbation. SETTING: The sexual physiology laboratory at our free-standing rehabilitation hospital. PARTICIPANTS: A volunteer sample of 10 women with complete SCI along with 10 able-bodied women, matched for age and educational status. INTERVENTIONS: A 78-minute protocol using 6-minute baselines alternating with 12-minute testing conditions. DEPENDENT VARIABLES: Vaginal pulse amplitude, subjective arousal, heart rate, respiratory rate, and blood pressure. RESULTS: Subjective sexual arousal increased in able bodied women with manual genital stimulation in conjunction with performance of a distracting task and was further augmented by removal of the distracting task and continuing with masturbation. In contrast, complete SCI subjects did not demonstrate increased subjective arousal with performance of the distracting task in conjunction with manual genital stimulation; however, they did evidence significant increases in arousal when the distracting task was eliminated. CONCLUSIONS: Genital responses tended to parallel subjective responses in able bodied women; however, women with SCI revealed nonsignificant changes in genital responses throughout the treatment protocol. It is hypothesized that the genital and subjective responses of able-bodied subjects reflect the additive components of reflex and then psychogenic sexual arousal. Furthermore, the lack of responsivity in the SCI subjects was thought to be related to compromised upper extremity function in the majority of our subjects. Further work is necessary to validate these hypotheses. PMID- 8629916 TI - Reliability and validity of measures obtained from stroke patients using the Balance Master. AB - OBJECTIVE: To determine the test-retest reliability and validity of data obtained using the Balance Master (BM), a computerized balance assessment and training tool. DESIGN: Data were collected on three occasions, 1 week apart. Intraclass correlation coefficients (ICCs) were calculated if significant (p < .05) between subject variance was found using a univariate analysis of variance for repeated measures. Concurrent validity of the BM data was determined using the Berg Balance Scale and gait velocity as criterion standards. PARTICIPANTS: Twenty ambulatory hemiparetic subjects who had no history of lower extremity orthopedic problems, no neurological deficits apart from stroke, and had not trained using the BM. MAIN OUTCOME MEASURES: BM data relating to static and dynamic balance performance, gait velocity, and the total score from the Berg Balance Scale. RESULTS: ICCs indicated that only the BM test requiring subjects to shift their center of gravity to randomly highlighted targets (positioned in a circle representing 75% of the individual's limits of stability) was reliable, both in terms of movement path (ICC = .84) and movement time (ICC = .88). Concurrent validity of the BM data was established for the dynamic measures of balance only, which correlated with both the Berg Balance Scale and gait velocity outcomes (r > or = .48, p < .05). CONCLUSIONS: These findings suggest that in stroke patients the test-retest reliability of data obtained using the BM is greatest for complex tests of balance and that dynamic rather than static balance measures are valid indicators of functional balance performance. PMID- 8629917 TI - Intermodal agreement of follow-up telephone functional assessment using the Functional Independence Measure in patients with stroke. AB - OBJECTIVE: To examine the intermodal agreement of Functional Independence Measure (FIM) ratings when obtained by two commonly used approaches: telephone interview and in-person assessment of functional performance. DESIGN: A random sample of 40 persons with hemiparesis was tested by two registered nurses trained in FIM definitions and telephone interview techniques. The two assessments occurred within 5 days of each other. The raters were blind to previous assessments. The administration of assessments was alternated to minimize bias and order effects. SETTING: All subjects were assessed at home, between 3 and 10 months after discharge from rehabilitation. PATIENTS: The criteria for inclusion were: (1) diagnosis of cerebral vascular accident (CVA); (2) completion of a minimum of 2 weeks in an acute rehabilitation program; (3) currently living at home; (4) living within a 30-mile radius of the hospital; and (5) cognitive and verbal skills adequate to complete a telephone interview. From a population of 103 patients, 40 subjects were randomly selected, 18 women and 22 men ranging in age from 37 to 90 years. MAIN OUTCOME MEASURES: The intermodal agreement between FIM ratings obtained by telephone interview and in-person assessment was examined using the intraclass correlation (ICC). FIM item scores were analyzed for agreement using the Kappa coefficient. The stability of the responses was determined by computing the coefficient of variation and plotting the data to visually examine the relationship between the two methods of administration. RESULTS: Data analysis revealed that there was no statistically significant difference (p > .05) between the two methods of administration for total FIM score. The total FIM ICC was .97. ICC values for FIM subscales ranged from .85 to .98, except for social cognition. Kappa scores for noncognitive items ranged from .49 (bowel movement) to .93 (grooming). The coefficient of variation computed to examine cognitive and communication items with reduced variability indicated good stability across all items. CONCLUSION: The results indicated good intermodal agreement for follow-up telephone assessment using the Functional Independence Measure. The findings were limited to persons with effective communication skills. PMID- 8629918 TI - The performance of a limited set of items from the Functional Independence Measure for use in acute trauma care and rehabilitation. AB - OBJECTIVE: To evaluate the performance of a limited set of three items from the Functional Independence Measure--eating, walking, and expression--(henceforth referred to as the Set) for use in acute trauma care and rehabilitation. DESIGN: Retrospective comparison of the Set-scores (3-item total) to the Functional Independence Measure (FIM) scores in 83 consecutive patients with a primary diagnosis related to trauma (90% of those eligible) over a 2-year period, on an acute rehabilitation unit in a tertiary care university hospital. METHODS: The age distributions of gender and diagnostic categories were examined. The sensitivity and specificity of the Set-score in detecting changes in the FIM were evaluated across a range of cut-off values. Correlation between FIM scores and Set-scores at admission, at discharge, and for interval change were assessed using the intraclass correlation coefficient. Sources of the Set's weaknesses were explored. RESULTS: Age distribution reflects the predisposition of older women to hip fractures and males to the other trauma. No degree of change in the Set-score provided a combination of sensitivity and specificity satisfactory for detecting potentially important changes in the FIM. Intraclass Correlation Coefficients (ICC) for Set-scores and FIM scores on admission and discharge were both .12. The ICC for the interval change data was .11. (95% confidence limits:- .11 to .34). CONCLUSION: This Set is not a useful measure of functional disability in a heterogeneous population of trauma patients. PMID- 8629919 TI - The SIP68: a measure of health-related functional status in rehabilitation medicine. AB - OBJECTIVE: To demonstrate the usefulness of the SIP68, a recently developed short version of the sickness impact profile (SIP), for measuring health-related functional status in rehabilitation medicine. DESIGN: Survey, oral interviews. SETTING: Patient's homes. PATIENTS: 315 persons (out of 423 that could be reached) with a spinal cord injury whose mean average age was 39.4 years and who were living in the community at the time of the interview. MAIN OUTCOME MEASURES: Internal consistency is tested by computing Cronbach's alpha. Construct validity is tested by principal components analysis and computing Cattell's similarity index. Criterion validity is tested by comparing SIP68 results with the level of the spinal cord lesion and with results of specific measures of disability (Barthel Index) and life satisfaction (Life Satisfaction Questionnaire), and with vocational status. RESULTS: SIP68 scores and subscale scores indicate that our spinal cord injured group falls well within the scope of this instrument. Internal consistency figures are good and the proposed six-dimensional structure is confirmed. Criterion validity figures are also satisfactory. Barthel Index scores show high agreement with the scores of the subscale "somatic autonomy," moderate agreement with the other physically related and socially related subscales, and low agreement with the mentally related subscales of the SIP68. LSQ scores show low agreement with the physically related subscales and moderate agreement with the mentally and socially related subscales. Figures of vocational status show strongest agreement with the socially oriented subscales. CONCLUSION: The SIP68 is recommended as a useful generic outcome measure for research in rehabilitation medicine. PMID- 8629920 TI - A cane reduces loss of balance in patients with peripheral neuropathy: results from a challenging unipedal balance test. AB - OBJECTIVE: To test the hypothesis that use of a cane in the nondominant hand during challenging balance tasks would significantly decrease loss of balance in patients with peripheral neuropathy while transferring from bipedal to unipedal stance on an unsteady surface. DESIGN: Nonrandomized control study. SETTING: Tertiary-care institution. PARTICIPANTS: Eight consecutive patients with peripheral neuropathy (PN) and eight age- and gender-matched controls (C) with a mean (SD) age of 65 (8.2) years. METHODS: Subjects were asked to transfer their weight onto their right foot, despite a rapid +/- 2 degrees or +/- 4 degrees frontal plane tilt of the support surface at 70% of weight transfer, and balance unipedally for at least 3 seconds. The efficacy of their weight transfer was evaluated over 112 consecutive randomized and blocked trials by calculating loss of balance as failure rates (%FR) with and without visual feedback, and with and without use of a cane in the nondominant (left) hand. Results were analyzed using a 2 x 2 x 2 x 2 x 2 repeated-measures analysis of variance (rm-ANOVA) and post hoc t tests. RESULTS: The rm-ANOVA showed that the FR of the PN subjects (47.6% [18.1%]) was significantly higher than C (29.2% [15.2%], p = .036). Removing visual feedback, simulating the dark of night, increased the FR fourfold (p = .000). Use of a cane in the contralateral nondominant hand significantly reduced the FR (p = .000), particularly in the PN group (cane x disease interaction: p = .055). Post hoc t tests showed that with or without visual feedback, the cane reduced the FR of the PN group fourfold and enabled them to perform more reliably than matched controls not using a cane (p = .011). An inversion perturbation resulted in a higher FR than an eversion perturbation (p = .007). The PN group employed larger mean peak cane forces (21.9% BW) than C (13.6% BW) in restoring their balance (p = .000). CONCLUSION: Use of a cane by PN patients significantly reduced their risk of losing balance on unstable surfaces, especially under low light conditions. PMID- 8629922 TI - Prospective study on the use of bolus intrathecal baclofen for spastic hypertonia due to acquired brain injury. AB - OBJECTIVE: To determine if the intrathecal delivery of baclofen will decrease spastic hypertonia caused by brain injury. PATIENTS: Eleven patients more than 1 year after their brain injury with disabling lower extremity spastic hypertonia. SETTING: University tertiary care outpatient rehabilitation clinic. DESIGN: Patients were a consecutive sample randomized in a double blind, placebo controlled crossover study. INTERVENTIONS: Bolus intrathecal injection of either normal saline or 50 micrograms baclofen. MAIN OUTCOME MEASURES: Data for Ashworth rigidity scores, spasm scores, and deep tendon reflex scores were collected for both the upper extremities (UE) and lower extremities (LE). Changes over time were assessed via Friedman's test. Differences between the placebo and active drug at any given time were assessed via Wilcoxon signed-rank. RESULTS: Four hours after injection with the active drug (maximum effect) the average LE Ashworth score decreased from 4.2 +/- 0.8 (SD) to 2.2 +/- 0.6 (p = .0033), spasm score from 3.1 +/- 1.0 to 1.0 +/- 0.7 (p = .0032), and reflex score from 3.3 +/- 0.5 to 1.0 +/- 1.3 (p = .0033). The average UE Ashworth score decreased from 3.3 +/- 1.3 to 1.9 +/- 0.8 (p = .0033), spasm score from 1.8 +/- 1.3 to 0.6 +/- 1.0 (p = .007), and reflex score from 2.7 +/- 0.5 to 1.7 +/- 0.6 (p = .0111). No trend was observed over time with placebo administration. There were significant reductions in the average for LE Ashworth (p < .0001), spasm (p < .0001), and reflex (p < .0001) scores and for UE Ashworth (p < .0001) and spasm (p < .0004) scores observed over 4 hours (maximum effect) with active drug administration. No significant differences were noted between the active drug and placebo groups before administration in LE and UE Ashworth, spasm, or reflex scores. There were significant differences between the active drug and placebo groups at 4 hours after administration for LE and UE Ashworth, spasm, and reflex scores (p < or = .0272). CONCLUSION: Intrathecal injection of baclofen is capable of reducing the spastic hypertonia associated with brain injury. PMID- 8629921 TI - Diabetic sensory neuropathy effect on ankle joint movement perception. AB - OBJECTIVE: To determine if diabetic subjects with lower extremity cutaneous sensory neuropathy also have a loss of ankle joint movement perception. The strength of association between measurements of ankle joint movement perception and measures of cutaneous sensory function was also investigated. DESIGN: Diabetic subjects with and without sensory neuropathy and individuals without diabetes participated in this study. SETTING: All subjects were community-living individuals. PARTICIPANTS: Fifty-one subjects, ages 40 to 68. Seventeen of the 34 subjects with diabetes had significant distal sensory neuropathy as determined by cutaneous perception of mechanical vibration. All individuals without diabetes were volunteers from the community. Most subjects with diabetes were recruited through direct referral from their physicians. INTERVENTIONS: Ankle joint movement perception threshold (JMPT) was assessed using a device designed for this study. Cutaneous sensory function under both halluces was measured for vibration perception using a vibrometer and for touch-pressure perception using Semmes-Weinstein monofilaments. MAIN OUTCOME MEASURES: Ankle JMPTs (degrees) were compared to measurements of cutaneous vibration perception (volts) and touch pressure perception (monofilaments force ratings). RESULTS: Diabetic subjects with cutaneous sensory neuropathy demonstrated a significant loss of ankle movement perception (p < .01). Correlation between JMPT and cutaneous sensory tests ranged from Spearman's rank r = .43 to .67. CONCLUSIONS: Although individuals with cutaneous sensory loss secondary to diabetic neuropathy also demonstrated loss of movement perception at the ankle, the relatively low explained variance between the two types of assessment (18% to 45%) indicates that the severity of ankle joint movement perception deficits cannot be directly implied from cutaneous sensory tests. PMID- 8629923 TI - Generator sources for the early and late ulnar hypothenar premotor potentials: short segment electrophysiologic studies and cadaveric dissection. AB - OBJECTIVE: Determine the generator sources for the ulnar hypothenar premotor potentials (PMPs). DESIGN: Observational. SETTING: EMG laboratory. SUBJECTS: Ten asymptomatic adult volunteers, three cadaver hands. MAIN OUTCOME MEASURE: Far field versus near-field characteristics of recorded PMPs as determined by bipolar and referential recording electrode montages. A possible anatomic basis for any observed differences between ulnar PMPs and previously studied median PMPs were explored through cadaveric dissection. RESULTS: An early PMP (E-PMP) had a latency that varied with changes in the position of G1 only. A late PMP (L-PMP was seen only when G1 and G2 were on different volumes (palm vs fifth digit, or second digit vs fifth digit); its latency did not vary significantly with changes in the position of G1 and G2. CONCLUSIONS: (1) E-PMP is a near-field potential generated by the ulnar nerve passing near the G1 electrode. (2) L-PMP represents a far-field potential generated by the ulnar digital nerves as they traverse from the hand volume containing G1 to the finger volume containing G2. (3) Greater L PMP-to-CMAP separation in the median than in the ulnar nerve was explained by cadaveric dissection, which revealed that the motor branch (responsible for the trailing CMAP) is longer in the median nerve than in the ulnar nerve relative to each nerve's corresponding digital sensory branch (responsible for the preceding L-PMP). (4) The PMP that is typically recorded with G1 at the hypothenar motor point and G2 on the fifth digit most likely represents E-PMP. (5) Any proposed diagnostic use of the ulnar PMPs must take into consideration these generator sources. PMID- 8629924 TI - Hand dominance effect on median and ulnar sensory evoked amplitude and latency in asymptomatic workers. AB - OBJECTIVE: To examine the relative effect of hand dominance on the median and ulnar sensory evoked responses and grip strength in active workers. DESIGN: A cross-sectional or survey design. SETTING: Workers from 4 different sites underwent on-site testing of the median and ulnar sensory nerves in both hands (antidromic stimulation, 14 cm), and testing of bilateral grip strength. PATIENTS: 224 workers, asymptomatic of hand, wrist, or finger pain, numbness, or tingling: 87% were right handed. MAIN OUTCOME MEASURES: Amplitude and latency of the median and ulnar sensory response and the grip strength of each hand. RESULTS: The amplitude of the median sensory response in the dominant hand was significantly lower in right handed individuals (34.6 microm V versus 38.8 microm V) but not in left handed workers (34.2 microm V versus 34.3 microm V). A similar relationship held for the ulnar evoked responses and the grip strength. Median and ulnar sensory latencies in right handed individuals did not differ side to side, whereas left handed workers had a slightly shorter latency on the left. CONCLUSIONS: The left hand of a right handed worker may not be exposed to as much trauma, resulting in relative protection of the nerves in the hand and a higher amplitude of the left median and ulnar evoked response. Left handed individuals may be more likely to use both hands equally and thus expose the nerves in each hand to an equal amount of trauma, resulting in their sensory amplitudes being equivalent (and also equal to the dominant hand of a right handed individual). PMID- 8629925 TI - Precision of surface measurements for below-knee residua. AB - OBJECTIVE: To determine the absolute and relative precision of geometric measurements made of below knee (BK) residua and their BK plaster positive casts using calipers, electromagnetic digitizer, optical surface scanner (OSS), and spiral x-ray computed tomography (SXCT). DESIGN: The experimental measurement protocol for a single measurement session was as follows: Dot markers were placed on the residuum, and volume and distances were measured using water displacement and calipers; residuum was measured using electromagnetic digitizer; residuum was scanned using three-dimensional (3D) OSS; a negative plaster cast of subject's residuum was made; the residuum was scanned using SXCT scanner. These steps were repeated at a second measurement session. Plaster positive casts were constructed and subsequently measured using the same protocol. PARTICIPANTS: Thirteen adult below-knee amputee volunteers (subjects) participated in the study, and nine subjects returned for a second measurement session. The study group consisted of 9 men and 4 women; 10 Caucasians and 3 African Americans. RESULTS: Distance measurements for all measurement devices were repeatable within 1% in vivo and within 0.5% on plaster casts; and volumes were within 1% in vivo and within 0.1% on plaster casts. Distance measurements for each device were precise within 3% in vivo and within 1% on plaster casts; and volumes were within 5% in vivo and within 6% on plaster casts when compared with caliper and water displacement measures. CONCLUSION: These measurement systems were found to be substantially equivalent in terms of repeatability and precision for measurement of lower extremity residua. PMID- 8629926 TI - The stretch reflex response in the normal and spastic ankle: effect of ankle position. AB - OBJECTIVE: The influence of stretch of the gastrocnemiussoleus muscle on the stretch reflex activity was studied, by varying the ankle angle in steps from 10 degrees of plantarflexion (PF) to 5 degrees of dorsiflexion (DF). DESIGN: Nonrandomized control trial. SETTING: Department of Rehabilitation Medicine of a university medical center. PATIENTS: Sixteen subjects with and 16 subjects without spasticity. MAIN OUTCOME MEASURES: The passive elastic stiffness and active reflex response, expressed by the total and elastic path lengths, were determined at each ankle angle as a sinusoidal displacement of 5 degrees was applied to the joint at frequencies from 3 to 12 Hz. RESULTS: The elastic stiffness showed no difference between the spastic and normal subjects for all ankle angles (p > .05). The elastic stiffness increased linearly similarly in both groups when the ankle was dorsiflexed. The reflex response was significantly greater in the spastic group for all positions (p < or = .01). The total and elastic path lengths showed a linear increase in both groups when the ankle angle was varied from PF to DF. The spastic group, however, had a significantly faster increase (p < or = .005). Between-group comparison showed a significant quadratic trend in the elastic path length for the spastic group (p < or = .05), with a maximum at 2.5 degrees of DF. CONCLUSIONS: This study showed that the stretch reflex activity varies with the ankle position. This must be considered when performing spasticity tests subsequent to an intervention that has changed the available range of motion and when comparing subjects measured at different ankle positions. PMID- 8629927 TI - Laryngotracheal stenosis after intubation or tracheostomy in patients with neurological disease. AB - OBJECTIVE: This retrospective study evaluated the incidence of airway complications in neurological patients following translaryngeal intubation, tracheostomy, or both. DESIGN: The medical records of 315 consecutive patients (200 with traumatic brain injuries, 31 traumatic tetraplegics, and 84 with other neurological disorders) were reviewed. The type of artificial airway, duration of intubation, and use of nocturnal ventilation were recorded. Eighty-six percent of the patients underwent some combination of tracheal tomograms, flow-volume loop analysis, and fiberoptic tracheolaryngoscopy. Stenosis was classified as severe if it required surgery, if it required maintaining the tracheostomy, or was lethal. It was classified as benign if it was successfully treated by medical or local means. RESULTS: Fifty-five percent of the patients were intubated translaryngeally only (mean = 17 days). Three percent underwent tracheostomy only, and 42% underwent tracheostomy after intubation for a mean of 13 days. The overall incidence of airway stenosis was 20%, 1/4 of which was severe. Fifteen percent of these patients died as a result of tracheal complications. The incidence of stenosis was higher following tracheostomy than following intubation only (29% vs 13%, p < .01). The incidence of severe stenosis in intubated-only patients was low (1%) compared with that following tracheostomy (10%, p < .01). No significant relationship was found between the length of intubation or the timing of tracheostomy. CONCLUSION: Fewer complications are associated with transtracheal intubation than with tracheostomy. The data suggest that longer periods of intubation be used when attempting ventilator weaning before restoring to tracheostomy if weaning fails. PMID- 8629928 TI - Statistical methods in rehabilitation literature: a survey of recent publications. AB - OBJECTIVE: To document the use of statistical methods in the recent rehabilitation research literature. DESIGN: Descriptive survey study. METHODS: All 1,039 articles published between January 1990 and December 1993 in the American Journal of Physical Medicine and Rehabilitation and the Archives of Physical Medicine and Rehabilitation were reviewed for the use of statistical methods. RESULTS: There were 682 (66%) research articles in the sample that included systematic data collection and analysis. The most frequently encountered analytic tests and techniques included: (1) analysis of variance, (2) t tests, (3) correlation analysis, (4) contingency table analysis, (5) regression, and (6) nonparametric tests. Results were compared with results of a review of the 1982 rehabilitation literature by Wainapel and Kayne. Our study showed an increased sophistication in statistical methodology as well as a more intense use of such methods. In addition, there was a large number of relatively obscure and poorly documented tests encountered in the more recent literature. There was also a lack of adherence to a standardized format for describing statistical methods. CONCLUSIONS: The following recommendations are made: (1) Training curricula for rehabilitation professionals should provide instruction in the most commonly encountered statistical methods and should be revised as needed to reflect changes in statistical method usage. (2) When less common statistical tests are applied, the responsibility of the authors to fully describe and justify their methods should be recognized. (3) Critical assessment of the literature is facilitated when statistical methods are reported in a standardized format. PMID- 8629929 TI - Pregnancy in spinal cord injured women. AB - The management of pregnancy, labor, and delivery in women with spinal cord injury (SCI) has received increased attention by investigators and clinicians in recent years. Physicians who care for women with SCI need to become familiar with the general principles of care during pregnancy. This article presents a summary of publications identified by a MEDLINE search of the topics spinal cord injury, paraplegia, tetraplegia, and pregnancy, and by reviewing the reference lists of these articles. The current medical literature suggests excellent overall maternal and neonatal outcome. Certain medical complications such as urinary tract infections and autonomic hyperreflexia are predictable and can be managed successfully. Obstetrical management is changed little by SCI but needs to account for the risk of unattended delivery in the patient with a high level of lesion. PMID- 8629930 TI - Botulinum toxin for spasticity and athetosis in children with cerebral palsy. AB - Botulinum toxin A has been used therapeutically in humans for a variety of conditions since 1980. Over the past few years, it has been used more frequently for spasticity management. We describe the use of botulinum toxin for spasticity or athetosis management in three children with cerebral palsy. Two of these children had severe spasticity or athetosis that was unresponsive to other forms of treatment. The injection of botulinum toxin decreased pain and improved ease of care in these two children. Another child with left hemiparetic cerebral palsy underwent injection of botulinum toxin into upper and lower limb muscles to improve function. In this patient, the injections were combined with other forms of treatment. In this patient spasticity was decreased and function was increased temporarily. The use of botulinum toxin in general is discussed and related to these three cases. PMID- 8629931 TI - Acute gastric dilatation in Duchenne muscular dystrophy: a case report and review of the literature. AB - Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder of childhood. Its clinical characteristics that derive from skeletal muscle involvement have been well described. Less well known is that visceral smooth muscle is affected in DMD. We report a case of a 19-year-old man with DMD who presented with severe nonradiating epigastric pain. He was initially sent home from the emergency department with a diagnosis of costochondritis. Acute gastric dilation was not considered in the differential diagnosis despite supportive history, physical examination findings, and radiographs. The case illustrates the lack of familiarity by clinicians of the gastrointestinal manifestations of DMD, including gastric dilatation and intestinal pseudoobstruction. Following a case discussion, the literature relevant to acute gastric atony is reviewed. PMID- 8629932 TI - Gait analysis of transfemoral amputee patients using prostheses with two different knee joints. AB - OBJECTIVE: To evaluate the gait of transfemoral amputee patients using a prosthesis with a 4-bar linkage knee joint with either a mechanical swing phase control (Otto Bock 3R20) or a pneumatic swing phase control (Tehlin knee). DESIGN: Randomized cross-over trial. SETTING: Rehabilitation Department of a university hospital in The Netherlands. PATIENTS: Twenty-eight subjects with unilateral transfemoral amputation for reasons other than chronic vascular disease; ages between 16 and 65 years and familiar with the use of the Otto Bock 3R20 knee. SELECTION PROCEDURE: a consecutive sample. INTERVENTION: The Otto Bock 3R20 was used by the subjects before they entered the study. The patients changed to the Tehlin knee at random either after 1 or 2 assessments with the Otto Bock 3R20. MAIN OUTCOME MEASURES: Temporal and kinematic variables in gait analysis (speed recordings were taken before and after the equipment for the measurements of the other parameters was placed on the patients). Subjective scores for comfortable and fast ambulation were obtained by means of 2 questionnaires. Questionnaire A consisted of multiple choice questions (maximum score: 5) and questions using an 8-point rating scale (maximum score: 7). In questionnaire B, the patient was asked to compare the present prosthesis with the previous one. RESULTS: Fast walking speed in gait analysis was higher with the Tehlin knee than with the 3R20 (without equipment 95% confidence interval (CI) .02-.09 m/sec, with equipment CI .04-.11), while comfortable walking speed was not higher with the Tehlin knee (without equipment CI -.20-.20 m/sec, with equipment CI .00-.05). Symmetry of walking as regards swing phase duration was closer to 100% with the Tehlin knee than with the 3R20 (comf. walking CI 4% to 19%, fast walking CI 7% to 17%). Knee joint range of motion during swing phase was smaller with the Tehlin knee than with the 3R20 (comf. walking CI 1.8 degrees-8.7 degrees, fast walking CI 2.0 degrees-9.5 degrees). Knee flexion duration during swing phase was shorter for the Tehlin knee than for the 3R20 at fast walking speed (CI 6-46msec), while knee flexion duration from and to 10 degrees flexion was shorter for the Tehlin knee at comfortable speed (CI 18-67msec) and fast speed (CI 20-64msec). In questionnaire A the amputees reported the Tehlin knee to be better for fast walking (part A1 CI .01-.52, part A2 CI .13-.98) and in questionnaire B for both comfortable and fast walking. PMID- 8629933 TI - Cost-effectiveness in stroke rehab. PMID- 8629934 TI - Cost-effectiveness in stroke rehab. PMID- 8629935 TI - Sinemet in locked-in syndrome. PMID- 8629936 TI - Ipsilateral pushing in stroke. PMID- 8629937 TI - Species-specific and interspecies relatedness of NSP1 sequences in human, porcine, bovine, feline, and equine rotavirus strains. AB - We have sequenced gene 5 encoding NSP1 for three human, two porcine, two bovine, one feline, and five equine rotavirus strains, and compared the nucleotide and deduced amino acid sequences with the published sequences for other various strains. Subgroup I human strains L26, 69M, and DS-1 were found to have a similar NSP1 sequence despite their different G serotypes, VP4 genotypes, and RNA patterns. The NSP1 sequence of the human strain K8 showed a high degree of homology to those of porcine strains OSU and YM. A high degree of homology was found among three equine strains (H2, FI-14, and FI23), but they differed from the other equine strains L338 and H1. The strain H1 was similar to the porcine strains. The feline strain Cat2 showed a high homology to bovine strains UK, RF, and A44. Thus, species-specific and interspecies relatedness of NSP1 sequences among human, porcine, bovine, feline and equine rotaviruses was found. Overall genomic relatedness of strains L26 and YM to various human and animal strains was also examined by RNA-RNA hybridization assay. The present and previous hybridization results showed that there is a good correlation in most strains between overall genomic property (or genogroup) and NSP1 sequence homology. PMID- 8629938 TI - Topography and immunogenicity of bluetongue virus VP7 epitopes. AB - The core of bluetongue virus (BTV) consists of ten dsRNA viral genome segments and five proteins, including two major (VP7 and VP3) and three minor (VP1, VP4 and VP6) components. The major core protein VP7 is believed to be an important structural constituent because it interacts, not only with the underlying core protein VP3, but also with two outer capsid proteins (VP2 and VP5). In this communication we summarise data on the mapping of at least six different epitopes of VP7 distributed along the molecule. Two of the six epitopes have not been mapped previously. The accessibility of these epitopes in intact virions and core particles was analysed using immunoelectron microscopy. The epitope located near the N-terminus of VP7 was accessible at the surface of intact virions and core particles. Epitopes in other parts of the VP7 molecule were detected weakly in core particles but not in intact virions. These results support the proposal that VP7 molecules are orientated with their N-terminus accessible on the surface of either the particle or at least one of the three different channels observed by cryoelectron microscopy in the outer capsid layer. Analysis of the immune response to BTV-infected or -immunised sheep and rabbits to three selected epitopes, which are located in different regions of the VP7 molecule, demonstrated that all of them were recognised by the animals tested. These results provided further molecular evidence suggesting that VP7 is indeed a major immunogenic antigen ideal for BTV antibody detection. PMID- 8629939 TI - Molecular evidence that the whitefly-transmitted sweetpotato mild mottle virus belongs to a distinct genus of the Potyviridae. AB - Complementary DNA representing 2 108 nucleotides at the 3' end of the genomic RNA of the whitefly-transmitted sweetpotato mild mottle virus (SPMMV) was cloned after PCR. Sequence analysis revealed an open reading frame of 1 797 nucleotides which codes for a protein of 599 amino acids, followed by a 3' non-coding region of 311 nucleotides. Alignment of the deduced amino acid sequence with corresponding sequences of other members of the Potyviridae demonstrated that part of the presumptive RNA-dependent RNA polymerase and the coat protein coding regions of SPMMV are found at the 3' end of its genome, in that order. Alignment of the amino acid sequence of the core of SPMMV coat protein with those of selected members of the Potyviridae showed limited identity, thus demonstrating- with phylogenetic analysis--that SPMMV belongs to a distinct genus of the family Potyviridae. PMID- 8629940 TI - Strain-dependent differences in the human cytomegalovirus replication origin. AB - The nucleotide sequence of the human cytomegalovirus replication origin of strain Towne (an AatII-SacI fragment corresponding to nt 90372-94637 of strain AD169) was determined and compared with AD169. Two differences were found in the nucleotide sequence level. One was the alteration of structural organization (a major difference): a 189-bp region of AD169 (nt 93337-93525) was directly repeated three times in Towne. The other was a change in the nucleotide residue level including substitution, insertion, or deletion (a minor difference). The divergent residues were predominantly localized within the nt 92591-92855 region of AD169. A replication assay revealed that replication ability remained after deletion of the 189-bp repeat but disappeared after either a 1.5-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. The 1.5- and 0.9-kb regions were relatively conserved. These results indicate that at least two regions essential for replication ability lie outside of both the relatively variable region and the 189-bp repeat and suggest that these essential regions support replication even with a spatial separation of either one (AD169) or three repeats (Towne) of the 189-bp region. PMID- 8629941 TI - Improved diagnosis of cowpea aphid-borne mosaic virus in Africa: significance for cowpea seed-indexing, breeding programs and potyvirus taxonomy. AB - Large-scale surveys in Africa for blackeye cowpea mosaic (B1CMV) and cowpea aphid borne mosaic (CABMV) showed that several CABMV isolates from Southern Africa were either not or poorly recognized by monoclonal antibodies prepared to isolates collected in West Africa. Selection of three new monoclonal antibodies prepared against the Maputo (Mozambique) isolate of CABMV, and their incorporation into a revised panel of monoclonal antibodies, resulted in the assignment of four of these new CABMV isolates to existing serotypes (II, IV, and V) and three others to a new serotype (VI). The South African isolate of passiflora mosaic virus was shown to be related to CABMV isolates in serotype IV. It is proposed that CABMV isolates be assembled into a distinct species in the legume-infecting, aphid transmissible potyviruses. PMID- 8629942 TI - Detection of proviral human T-cell lymphotrophic virus type I DNA in mouthwash samples of HAM/TSP patients and HTLV-I carriers. AB - Human T-cell lymphotrophic virus type I (HTLV-I), is a member of the oncogenic retroviruses family endemic in several parts of the world and also recently identified in the Jewish Mashhadi population who immigrated from Iran to Israel. The virus is the causative agent of adult T-cell leukemia (ATL) and a chronic myelopathy known both as tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM). The known modes of HTLV-I transmission are by sexual intercourse, from mother to child in breast milk, via blood transfusion, and by sharing of needles by parenteral drug users. In the present study we examined the presence of HTLV-I provirus genomic DNA by nested polymerase chain reaction (PCR) and by DNA hybridization in mouthwash samples obtained from 13 Mashhadi-born Iranian Jews with spastic paraparesis associated with HTLV-I, 4 Mashhadi-born Iranian Jews asymptomatic carriers for HTLV-I and 21 healthy controls. Proviral HTLV-I DNA was detected by mouthwash PCR in 12 of 17 HTLV-I infected subjects (71%) but in none of 21 controls. Proviral DNA was also detected in mouthwash samples using HTLV-I probe by dot blot hybridization assay. The presence of HTLV I proviral DNA in whole saliva may suggest a possible transmission of the virus via saliva and explain the increased rate of infection in elderly Mashhadi-Jewish population. PMID- 8629943 TI - Identification of a unique VP4 serotype that is shared by a human rotavirus (69M strain) and an equine rotavirus (H-2 strain). AB - A cDNA clone representing the VP4-encoding gene of human rotavirus strain 69M(VP7 serotype 8) was constructed and inserted into a baculovirus expression vector. Baculovirus recombinants that expressed the 69 M VP4 protein in Spodoptera frugiperda (Sf9) cells were screened by immunofluorescence with hyperimmune antiserum to the 69M strain and purified by terminal dilution. The expressed VP4 was detected by Coomassie blue staining of PAGE-separated proteins. The antigenic relationships between the VP4 of the 69M strain and those of various human and other animal rotavirus strains representing ten established VP4 serotypes were examined by plaque reduction neutralization. Hyperimmune antiserum produced in guinea pigs following immunization with a lysate of Sf9 cells infected with a 69M gene 4-baculovirus recombinant neutralized the infectivity of the homologous human rotavirus 69M strain as well as heterologous equine rotavirus H-2 strain to a high titer. The anti-69M VP4 hyperimmune antiserum did not neutralize significantly other rotavirus strains of human, simian, porcine, bovine, or murine origin. It thus appears that the human rotavirus 69M strain has a distinct VP4 serotype (designated as P serotype 4) which is closely related antigenically to equine rotavirus H-2 VP4. PMID- 8629944 TI - Acetylcholine activates latent pseudorabies virus in pigs. AB - Pseudorabies virus (PrV) was isolated from the nasal swabs and the cultured trigeminal ganglia of latently infected pigs after they were treated with acetylcholine (ACH). These results indicate that ACH activates latent infections of PrV. PMID- 8629945 TI - Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell-cell fusion. AB - Monoclonal antibodies specific for equine herpesvirus 1 (EHV-1) glycoproteins (gB, gD, gp2 and a cleaved translation product of gene 71) were tested for ability to inhibit cell-cell fusion as measured by syncytium formation in EHV-1 infected cell cultures. Syncytium formation was inhibited by a complement dependent neutralising antibody (7B10) which recognised the large subunit of EHV 1 gB. This indicated that EHV-1 gB, in common with gB homologues of herpes simplex virus and other herpesviruses, plays a role in the cell-cell fusion process. PMID- 8629947 TI - Molecular cloning and nucleotide sequencing of the 3'-terminal region of a South African isolate of ryegrass mosaic virus RNA and in vitro expression of the coat protein gene. AB - The 2094 nucleotides at the 3'-terminus of a South African isolate of ryegrass mosaic virus (RGMV) was cloned and sequenced. Two putative polyprotein cleavage sites were found: Q/L and E/A, both of which are novel in the Potyviridae. The RGMV-SA cDNA was cloned into an expression vector, pUEX, and a fusion protein of 185 kDa was obtained which reacted strongly to anti-RGMV-SA antiserum. Alignment of the predicted amino acid sequence of RGMV-SA with those of other Potyviridae members showed limited identity, indicating that RGMV-SA is a definite and distinct virus. PMID- 8629946 TI - Sequence analysis of the 5' ends of tomato spotted wilt virus N mRNAs. AB - Messenger RNAs transcribed from the tomato spotted wilt virus (TSWV) RNA genome have characteristic extra non-templated heterogeneous sequences at their 5' ends which may be the result of a cap-snatching event involving cellular mRNAs. In order to investigate the genetic origin of these extra sequences and to gain more insight in the process of cap-snatching as performed by TSWV, nucleocapsid protein (N) mRNAs derived from the TSWV S RNA were cloned and sequenced. Twenty clones were obtained which contained 5'-proximal sequences of non-viral origin, ranging in length from 12 to 21 nucleotides. None of the sequences analyzed were identical and no base preference at the endonucleolytic site was observed. PMID- 8629948 TI - Nucleotide sequence of the capsid protein gene of papaya leaf-distortion mosaic potyvirus. AB - The DNA complementary to the 3'-terminal 1 404 nucleotides [excluding the poly(A) tail] of papaya leaf-distortion mosaic potyvirus (PLDMV) RNA was cloned and sequenced. The sequence starts within a long open reading frame (ORF) of 1 195 nucleotides and is followed by a 3' non-coding region of 209 nucleotides. Capsid protein (CP) is encoded at the 3' terminus of the ORF. The CP contains 293 residues and has a Mr of 33 277. The CP of PLDMV exhibits 49 to 59% sequence similarity at the amino acid level to the CPs of papaya ringspot potyvirus (PRSV) and other potyviruses. This result is consistent with the absence of a serological relationship between PLDMV and PRSV or other potyviruses. The results support the assignment of PLDMV as a distinct member of the genus Potyvirus. PMID- 8629949 TI - Genetic complementation between replication-defective mutants of HIV-1 and SIVagm. AB - To investigate the functional complementation of essential genes for virus growth between HIV-1 and SIVagm derived from African green monkeys, we co-transfected replication-defective molecular clones containing mutations in gag, pol, env, tat or rev, and monitored transient complementation by reverse transcriptase assay (RT), cytopathic effect (CPE) and immunofluorescence assay (IFA). The following results were obtained: 1) No complementation was observed in combinations of the gag and pol mutants. 2) The rev mutant of HIV-1 was minimally complemented by other SIVagm mutants, although the rev mutant of SIVagm was significantly complemented by other HIV-1 mutants. 3) Among all combinations tested, the env mutant of HIV-1 was the most effectively complemented by SIVagm mutants. 4) CPE was mostly absent in combinations of the env mutant of SIVagm and the gag, pol, or tat mutants of HIV-1, although there were significant positive results in RT and IFA assays. These findings provided basic information about the functional compatibility of pathogenic HIV-1 and nonpathogenic SIVagm which will be useful for generating chimeras of these two viruses. PMID- 8629950 TI - Identification and analysis of the simian varicella virus thymidine kinase gene. AB - The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes. PMID- 8629951 TI - Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD, and gE. AB - We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV 1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope. PMID- 8629952 TI - In vivo administration of serum thymic factor (FTS) prevents EMC-D virus-induced diabetes and myocarditis in BALB/cAJcl mice. AB - The effect of serum thymic factor (FTS) on the D-variant of encephalomyocarditis (EMC-D) virus-induced diabetes and myocarditis in BALB/cAJcl mice was investigated. Mice pretreated with 50 or 10 micrograms of FTS were infected with 10 or 10(3) PFU of EMC-D virus. In the mice inoculated with 10 PFU of virus, 40% developed diabetes on post-infection day (PID) 14, whereas those treated with FTS (50 micrograms/administration) on day 2 and 1 before infection did not develop diabetes. FTS (10 micrograms)-pretreated mice developed diabetes. In histological observation, FTS non-treated mice which developed diabetes showed severe necrosis and inflammation of mononuclear cells in the islets of Langerhans and myocardia on 19 PID. Mice pretreated with 50 micrograms of FTS, however, manifested mild islet degeneration without any myocardial inflammation. Furthermore, in FTS non treated mice, immunohistological staining showed a loss of insulin granules. This loss was markedly reversed and insulin granules remained largely intact in FTS pretreated mice. Viral titers in pancreas of FTS-pretreated mice approximated well to those of non-treated mice on PID 4, 7 and 19. In mice inoculated with higher titer of EMC-D virus (10(3) PFU), however, 50 micrograms of FTS pretreatment did not change the course of these acute pathological developments (diabetes and myocarditis observed from PID 4). PMID- 8629953 TI - Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence, genome organization and homology to other tospoviruses. AB - The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22-34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82-86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To. PMID- 8629954 TI - Genetic variation in populations of kennedya yellow mosaic tymovirus. AB - Kennedya yellow mosaic tymovirus (KYMV) occurs along the eastern Australian seaboard in the perennial legumes Desmodium triflorum and D. scorpiurus in the north, and Kennedya rubicunda in the south. The genetic variation of more than 100 isolates of KYMV, most of them from the north, has been studied using an RNA hybrid mismatch polymorphism (RHMP) method. The method clearly separated the isolates into two groups; all the northern Desmodium isolates formed one group and all the Kennedya isolates from the south another. These sub-populations were themselves variable and the Desmodium population alone was more variable than that of the related turnip yellow mosaic tymovirus in the Kosciusko alpine area. PMID- 8629956 TI - The stability of facial osteotomies. 3. Chin advancement. AB - Surgical repositioning of the chin can be used to improve function and aesthetics. The most commonly employed technique involves a sectioning of the lower border of the anterior mandible, thus allowing a sliding movement of the chin. It is generally accepted that the maintenance of a soft tissue attachment provides a clinically stable skeletal and predictable soft tissue change with minimal postoperative osseous resorption. Part 3 of this series of papers addresses the issues of treatment planning, surgical technique and clinical indications for undertaking advancement genioplasty. These, combined with the surgeon's artistic sense of facial harmony, are pre-conditions necessary for successful chin surgery. PMID- 8629955 TI - Major maxillofacial infections. An evaluation of 107 cases. AB - A review of 107 cases of acute maxillofacial infections managed at the Royal Melbourne Hospital was undertaken, and details of the presentations, demography, management, and outcomes of these patients are presented. The results indicated that many of the patients had sought treatment from dentists in general practice, and that a significant proportion had received sub-optimal management prior to referral. Thus a review of the principles of management and guidelines for the referral of patients with maxillofacial infections is also presented. PMID- 8629957 TI - The provision of orthodontic services by general dental practitioners. 1. Methods and descriptive results. AB - Information regarding orthodontic service provision by general dental practitioners in Australia is limited. The aim of this survey was to determine the amount and variety of orthodontic services provided by general dental practitioners in the Melbourne Statistical Division, Victoria, Australia. A random sample of 307 dentists drawn from the Victorian Dentists Register was surveyed by mailed questionnaire: 218 (71%) replied. Data were collected using a fortnight log. During this time 59 per cent of the dentists saw at least one orthodontic patient; one dentist saw 66 orthodontic patients. Removable orthodontic appliances were used by 35 per cent of the dentists and fixed orthodontic appliances by 18 per cent. Twenty-six per cent provided comprehensive orthodontic treatment, 22 per cent aligned incisors, and 21 per cent corrected anterior crossbites. The general dental practitioners surveyed provided a wide range of preventive and interceptive orthodontic services to generally a small percentage of their patients. PMID- 8629958 TI - Temporomandibular disorders. 1. Clinical evaluation. AB - The treatment of temporomandibular disorders continues to provide dental practitioners with a difficult challenge that has yet to overcome numerous major obstacles. In this, the first of a series of three articles, an overview of the current understanding of the diagnosis of temporomandibular disorders will be presented. The subsequent two articles will present an overview of the management strategies that have appeared in the literature in recent years. PMID- 8629959 TI - Methodological aspects of a computer-assisted telephone interview survey of oral health. AB - Despite the reported benefits of computer-assisted telephone interview (CATI) methods, experiences from their use in Australian oral health surveys have not been described. This report aimed to present methodological aspects of a CATI survey conducted in the five mainland states. A response rate of 66 per cent was obtained, yielding 4050 completed interviews. Analysis revealed generally small levels of non-response bias: persons who avoided or delayed dental treatment because of cost and non-health card holders were harder to contact, while non English speakers and persons aged 20-29 years were less likely to participate. A total of 1770 person hours of interview time was spent on the survey: 64.5 per cent of that time was spent on the telephone with an average of 10 minutes 17 seconds per call (13 minutes 37 seconds per completed call). Only seven questions had missing data for more than 1 per cent of respondents. Comprehension of questions and cooperation with the interview was rated by interviewers as 'good' or 'very good' for more than 90 per cent of respondents. The CATI method was highly efficient and yielded good quality data for the survey. PMID- 8629960 TI - Caries experience in rural Victorian adolescents. AB - An oral health status survey was conducted on 818 adolescents aged 12 to 16 years in a non-fluoridated area in rural Victoria. The prevalence and severity of dental caries experience was found to increase with age. The occlusal surface was the predominant surface affected by dental caries for all age groups. Filled surfaces represented the largest component of the DMFS index and were responsible for the increasing dental caries experience with age. Fewer fissure sealants were present with increasing age and second molars were sealed less often than the first molars. Although no difference was evident in the total caries experience between those with and without access to public dental services, a statistical difference was found to exist in unmet need between these two groups. Untreated caries levels were significantly higher in health card holders. The provision of preventive and dental treatment services to this group should be a priority in the future. PMID- 8629961 TI - Torus palatinus and torus mandibularis: a review of the literature. AB - The torus has been mentioned in the literature for about 180 years. However, little has been revealed about it until the last two decades when great advances were made in the field of genetics. Its occurrence in various ethnic groups ranges from 9 to 66 per cent. Even between similar ethnic groups living in different environments, different figures have been reported. It has been statistically proven that differences do occur between various ethnic groups and the sexes. In current thinking, the occurrence of tori is considered to be an interplay of genetic and environmental factors. The quasi-continuous genetic or threshold model seems to hold the answers to their formation. This theory proposes that the environmental factors responsible must first reach a threshold level before the genetic factors can express themselves in the individual. Hence, both genetic and environmental factors determine liability, making the system multifactorial. PMID- 8629962 TI - Dimensional changes of relined denture bases with heat-cured, microwave activated, autopolymerizing, and visible light-cured resins. A laboratory study. AB - The dimensional changes of relined denture bases were evaluated in a laboratory study. Heat-cured resin, two autopolymerizing resins, microwave-activated resin and a new visible light-cured resin were used as relining materials in this study. The gaps between the metal die and the relined denture bases were measured immediately after the relining procedure and subsequently on the first day, first week, fourth week, and eight week. The relined denture bases were stored in distilled water at 37 degrees C between measurements. It was shown that dimensional changes in relined denture bases were influenced by the relining materials and procedures, and that heat-cured resin proved to have the smallest dimensional change as well as the greatest dimensional stability in comparison with the other relining materials and procedures. PMID- 8629963 TI - Serum alkaline phosphatase, calcium, and phosphate levels following clinical use of natural coral. Case reports. AB - Measurement of alkaline phosphatase enzymatic activity is the most commonly used serum marker to assess bone formation. In this present study serum alkaline phosphatase, calcium, and phosphate were measured in 12 patients where natural coral was implanted in surgical bony defects. Blood samples were obtained preoperatively which served as control, and at 1, 7, 15, and 30 days. No statistically significant increase in serum alkaline phosphatase, calcium, and phosphate was observed. PMID- 8629964 TI - Dental air turbine handpiece performance testing. AB - Air turbine handpieces are expected to continue to be widely used as the main means of carrying out dental cutting work and scope exists for further design improvements. An understanding of the theoretical principles governing the performance of these devices seems essential for the systematic development of better handpiece designs and methods of specification. Furthermore, for experimental work on cutting behaviour with air turbine equipment, this knowledge is required for appropriate characterization of the performance of the particular handpiece used with respect to actual rates of energy disposition. The literature relating to air turbine handpiece performance is critically reviewed to assess currently available methods of measuring important variables such as speed, torque, and power. In this, consideration is given to the current state of knowledge of the influence on these variables of air pressure, flow and turbine design features. It is apparent that, although various measurement methods have been described and data for individual handpieces published, no attempt has yet been made to explore the functional relationships that exist between the variables. It is concluded that there is a need to identify the factors influencing turbine performance, to develop measurement systems which would provide adequate accuracy and precision and then to investigate the functional relationships between these relevant variables. PMID- 8629965 TI - The amalgam issue still simmers. PMID- 8629966 TI - Methamphetamine and childhood and adolescent caries. PMID- 8629967 TI - Protocol for single-tooth implants in general dental practice. PMID- 8629968 TI - Single-tooth implants and the soft tissue relationship. PMID- 8629969 TI - Endodontic cellulitis 'flare-up': clinical implications. PMID- 8629970 TI - What should the non-colorectal surgeon do when faced with a patient with acute fulminating colitis? PMID- 8629971 TI - The risk of an unplanned return to the operating room in Australian hospitals. AB - BACKGROUND: The unplanned return of the patient to the operating room (OR) after a previous procedure has implications concerning the quality of surgery, but little has been written on this subject. METHODS: The relationship of bed-size and hospital type (private or public) was studied using data on this clinical indicator submitted to the Australian Council on Healthcare Standards Care Evaluation Program (ACHS CEP) by hospitals presenting voluntarily for accreditation in 1993. RESULTS: The mean rate of an unplanned return to OR was 0.6% (95% confidence interval 0.5-0.7). After adjusting for potential confounders in a logistic model, the risk of unplanned return to OR did not significantly differ by type of hospital (private or public), and location (rural, metropolitan). The risk of unplanned return to OR was higher in large compared with small hospitals. CONCLUSIONS: The finding of the risk of the event being greater in large compared with small hospitals is likely to be a reflection of casemix. An interval review of results (for any facility) is obviously necessary. With some operations a higher incidence of return to the OR may indicate vigilance in peri-operative management. PMID- 8629972 TI - Laparoscopic cholecystectomy: the experience of community hospital. AB - BACKGROUND: The practice of laparoscopic cholecystectomy in a community hospital is presented. The morbidity of the procedure is analysed and recommendations for improvement are made. Laparoscopic cholecystectomy was introduced into this 200 bed community hospital in October 1990. All five general surgeons accredited to the hospital agreed to participate in a quality assurance programme to determine the incidence of complications and to make recommendations for improvement. METHODS: The records of all 534 patients having laparoscopic cholecystectomy between October 1990 and September 1993 were reviewed, and all complications recorded. RESULTS: Of the 534 cases reviewed in the study 470 were considered uncomplicated and 64 patients experienced a total of 85 postoperative complications. The death of one patient was caused by a pulmonary embolus and another patient experienced a myocardial infarction. Twenty patients has postoperative atelectasis or pneumonia and urinary infection or retention occurred in seven. Complications of laparoscopic cholecystectomy requiring a conversion to open cholecystectomy occurred in eight patients, biliary complications occurred in 18 and 11 required re-operation. CONCLUSIONS: Three areas of concern were identified. They were the incidence of major biliary injury (0.37% of all cases) and its management, the role of cholangiography, and the incidence and prophylaxis of deep venous thrombosis and pulmonary embolism. Recommendations for improvement in these areas were made. PMID- 8629973 TI - Operative cholangiography in the laparoscopic era: a retrospective review of the quality and interpretation of this investigation. AB - BACKGROUND: Operative cholangiograms during the year of introduction of laparoscopic cholecystectomy were reviewed to examine their quality and interpretation. METHODS: 149 operative cholangiogram films (34 open and 115 laparoscopic) were reviewed retrospectively by a panel and scored for their ability to demonstrate biliary anatomy and detect bile duct stones. RESULTS: Cholangiography performed by the cystic duct was of similar quality, whether performed laparoscopically or open. Cholangiography via the cystic duct (conventional films) produces superior results for both anatomical delineation and detection of choledocholithiasis (80% adequate) than cholangiography performed by direct gallbladder puncture (29-35% adequate). CONCLUSION: There is no clear evidence to suggest that an intra-operative specialist radiological review of cholangiograms performed by the cystic duct would improve the detection of bile duct stones. PMID- 8629975 TI - Unexplained septicaemia and pneumoperitoneum in Vietnamese boat people: a self inflicted condition? AB - BACKGROUND: Ten Vietnamese boat people with septicaemic shock or pneumoperitoneum were admitted between April and July 1993. METHODS: In order to investigate the cause of unexplained septicaemic shock and spontaneous pneumoperitoneum in Vietnamese boat people, an audit was carried out. Attention was paid to the mode of presentation, abnormal physical signs, presence of injection marks, radiological signs, subsequent progress outcome. RESULTS: The clinical and radiological findings were all in favour of self-inflicted conditions. A retrospective review from the hospital record revealed that the emergency admission rate and disappearance rate of the Vietnamese boat people was higher than those of other patients (P<0.001). CONCLUSION: It is concluded that self inflicted conditions are a serious problem among the Vietnamese boat people, who may use a hospital admission to avoid the detention centre. PMID- 8629974 TI - Nodular fasciitis and related pseudosarcomatous lesions of soft tissues. AB - BACKGROUND: Nodular fasciitis is characterized by a proliferation of spindle cells which may be misdiagnosed as sarcomas due to the rich cellularity, mitotic activity and variant morphologic patterns. METHODS: This report includes 42 cases of nodular fasciitis and related conditions of skin and soft tissue from the pathology files of the Royal Brisbane Hospital, Queensland, Australia. There were 33 cases of nodular fasciitis, three cases of ossifying fasciitis, three cases of proliferative fasciitis, two cases of proliferative myositis and one case of intravascular fasciitis. Two-thirds of cases were referred from outside Royal Brisbane Hospital. RESULTS: Nodular fasciitis and ossifying occurred most commonly in young adults with 16 patients (44%) between the ages of 20 and 29. However, the other variants of nodular fasciitis including proliferative myositis and intravascular fasciitis occurred in older people (six patients being older than 49 years) and more commonly occurring in men (n=5). A painless rapidly growing mass was most common. The lesions of nodular fasciitis were most often located in the upper extremity with the forearm most commonly affected. CONCLUSIONS: Nodular fasciitis and related conditions are benign. All patients are alive and well 3 months to 13 years (mean 7.2 years) after simple local resection. PMID- 8629976 TI - The surgical treatment of brachial plexus injuries. AB - BACKGROUND: Brachial plexus injuries cause a devastating loss of function in the arm. The aim of this study was to review the results of surgical treatment of patients with brachial plexus injuries. METHODS: Forty-seven patients were reviewed. Five patients did not undergo surgery. Forty-two patients had exploration and 38 had primary surgery with neurolysis, nerve graft or neurotization or a combination. Four patients had other reconstructive surgery primarily. Secondary reconstructive surgery consisted of joint fusions and tendon transfers to enhance or replace the primary surgery. RESULTS: Nerve grafting achieved 62% Medical Research Council (MRC) grade M3 or better. Intercostal neurotization (ICN) of the musculocutaneous nerve (MCN) for elbow flexion achieved M3 or better in 69% of patients. CONCLUSIONS: Primary nerve reconstruction, combined with joint fusions and tendon transfers, provides a worthwhile return of function to many patients. We advise early exploration (i.e. within 2 weeks when possible) for patients with complete C5-T1 lesions or C5,6,7 lesions in conjunction with high energy injuries. In order to obtain optimal results patients with brachial plexus injuries should be referred to appropriate units as early as possible. PMID- 8629977 TI - Single-layer continuous anastomosis in gastrointestinal surgery: a prospective audit. AB - BACKGROUND: Single-layer intestinal anastomoses have been constructed conventionally using an interrupted suture technique. It is however, increasingly popular to perform such anastomosis using a continuous suture. METHODS: One hundred and eighty consecutive patients with 254 continuous single-layer anastomoses performed over a 4 year period were included in the study. Sixty-one patients underwent oesophagectomy, oesophageal bypass or gastrectomy, 32 underwent biliary bypass, hepatic, biliary or pancreatic resection and 88 had colorectal operations. The median age was 67 years. RESULTS: There were 254 anastomoses of which four leaked (1.6%). Fifteen patients (8.3%) died in hospital. CONCLUSION: These results show that the single-layer continuous suture technique is safe in gastrointestinal anastomoses. PMID- 8629978 TI - The effect of duodenal distension upon antro-pyloric motility and liquid gastric emptying in pigs. AB - BACKGROUND: This study has investigated first the role of the antrum and pylorus in the retardation of gastric emptying during distension of the duodenum, and second whether ascending duodenal intramural nerves contribute to control of both antro-pyloric motility and liquid gastric emptying in response to distension of the duodenum. METHODS: Studies were performed on 18 pigs. In six the duodenum was transected 1-2 cm distal to the pylorus, to interrupt intramural nerves, in six the pylorus was excised and a further six pigs without any transection or resection acted as controls. Motility of the antrum, pylorus and duodenum was recorded by a sleeve/side hole manometric catheter. Gastric emptying was measured by drainage of the duodenum through a cannula. RESULTS: In control animals distension of the duodenum inhibited antro-pyloric pressure waves (APPW), from 1.52 waves/min at minimum distension to 0.25/min at maximum distension (P=0.0007), stimulated isolated pyloric pressure waves (IPPW), from 0.56/min to 1.80/min (P=0.034) and slowed emptying of a 1000 mL load of 5% dextrose over 30 min from 788 mL to 251 mL (P=0.0001). Duodenum transected animals did not show the duodenal distension-induced stimulations of IPPW (maximum distension: 0.93/min), but both the distension-induced inhibition of APPW (maximum distension: 0.85/min) and slowing of emptying (maximum distension: 52 mL emptied) were unaltered. Similarly in pylorus-excised animals, duodenal distension inhibited APPW (maximum distension: 0.47/min) and slowed liquid emptying (maximum distension: 267 mL), effects which did not differ from control animals. Retardation of gastric emptying by duodenal distension may be due in part to inhibition of antral contractions. CONCLUSIONS: Under the conditions of this experiment, increased pyloric resistance to flow does not play a major role in the slowing of emptying by duodenal distension, but the stimulation of the pylorus by duodenal distension depends on duodenal intramural neural pathways. Duodenal distension-induced feedback control of emptying is mediated primarily via pathways other than ascending intraduodenal nerves. PMID- 8629979 TI - Euthanasia. PMID- 8629980 TI - Laparoscopic endoluminal gastric surgery. AB - Laparoscopic endogastric surgery is a novel surgical approach that introduces the viewing laparoscope and instruments into the gastric lumen. The excellent visualization, improved instrument handling and versatility afforded by this technique enable local resection of mucosal lesions that are beyond the scope of peroral endoscopy. The authors describe this particular approach for the excision of gastric villous adenoma and early gastric cancer. PMID- 8629981 TI - Is the mid-inguinal point an accurate landmark for the common femoral artery in vascular patients? AB - BACKGROUND: An accurate guide to the common femoral artery (CFA) is important in vascular patients as the CFA pulse may not me palpable. At least four different descriptions of the surface anatomy of the CFA are given in standard anatomic texts. This study was undertaken to determine the accuracy of the mid-inguinal point as a guide to the CFA in patients undergoing femoral arteriography. METHODS: The surface anatomy of the CFA was determined using true anteroposterior arteriograms of 30 patients [20 men, 10 women, age (mean +/- s.d.) 67 +/- 6.8 years, range 56-82]. RESULTS: The distance between the anterior superior iliac spine (ASIS) and the symphysis pubis was measured with callipers and defined as the inguinal distance [(mean +/- s.d.) 16.03 +/- 1.06 cm, range 14.00-18.50]. The midpoint of this line defined the mid-inguinal point. Similarly, the distance was measured from the symphysis pubis to the midpoint of the CFA where it crossed the inguinal line on the arteriogram, and was defined as the femoral distance [(mean +/- s.d.) 7.99 +/- 0.51 cm, range 6.80-9.00]. The location of the CFA coincided with the mid-inguinal point in only six (10%) of the 60 measurements. The difference between the CFA and mid-inguinal point varied from 1.25 to 1.50 cm either side of the mid-inguinal point, and in six (10%), the difference was 1 cm more, and therefore of clinical significance. CONCLUSIONS: On the basis of these findings we conclude that the relationship between the mid-inguinal point, found using bony landmarks, is an appropriate guide to the CFA as it can be expected to lie within 1.5 cm either side of the mid-inguinal point. The natural variability observed could account for the different anatomical descriptions of the surface anatomy of the CFA. No exact definition may be possible. PMID- 8629982 TI - Recent developments in upper gastrointestinal surgery. AB - Important changes have occurred in the field of upper gastrointestinal surgery in the past few years. The change with the greatest impact has been the introduction of laparoscopic/thoracoscopic surgery in the treatment of the functional upper gastrointestinal disorders. However, new therapies in such diverse fields as bleeding oesophageal and peptic ulcer disease have greatly lessened the role of the surgeon in the elective setting. Malignant disease of the upper gastrointestinal tract has shown a marked swing away from cancer of the stomach towards cancer in the region of the gastro-oesophageal junction. There remains no consensus on the place of radical surgery in the treatment of these conditions. PMID- 8629983 TI - Anal pathology in patients with Crohn's disease. AB - BACKGROUND: A distinctive feature of patients suffering from Crohn's disease is a predisposition to develop a variety of anal complications. The aetiology of such conditions is unclear, and the reported incidence of anal involvement in Crohn's disease varies party due to the various criteria used for classification. This study aims to review the management of patients with symptomatic anal pathology associated with Crohn's disease at St Vincent's Hospital, Melbourne. METHODS: A database of 306 patients with Crohn's disease referred to the department between January 1978 and October 1994 was reviewed to identify those patients with symptomatic anal disease. The anal pathology was recorded and classified. Demographic data and the clinical and surgery history of the patient were recorded. RESULTS: Of the 306 patients with Crohn's disease, 129 (42.4%) were identified as having symptomatic anal pathology. Patients were likely to present with anal symptoms after they had been diagnosed as having intestinal Crohn's disease (46.1%). The commonest presentations were perianal abscess (29.5%), anal fissure (27.6%), and low anal fistula (26.7%). A minority of patients presented with high/complex anal fistulae (3.8%), or recto-vaginal fistulae (5.2%). Five per cent of patients had Crohn's disease localized to the anal area. The pattern of intestinal disease in the remaining patients was small bowel 21.1%. small bowel and colon 31.9%, and colon 43.0%. A total of 244 local anal and surgical procedures were performed on these patients; the commonest of these were drainage of an abscess (38.5%), examination under anaesthetic (29.1%), and laying open of a low anal fistula (22.5%). Following surgical treatment, the recurrence rate for perianal abscesses was 13%, and for low anal fistulae 6%. CONCLUSIONS: The majority of patients with Crohn's disease who develop anal pathology have an excellent prognosis. A minority of patients develop complex anal complex anal fistulae and these remain a therapeutic challenge. PMID- 8629984 TI - Controlled duodenostomy for difficult duodenal stump. PMID- 8629985 TI - Controlled duodenostomy for difficult duodenal stump. PMID- 8629986 TI - Closure of a benign broncho-oesophageal fistula by endoscopic injection of bovine collagen, cyanoacrylate glue and gelfoam. AB - Endoscopic application of adhesives and occlusives has been used to treat both benign and malignant tracheo-oesophageal fistulae. Successful management of a large benign broncho-oesophageal fistula by endoscopic reduction of the opening with injected bovine collagen and occlusion of the lumen with gelfoam and cyanoacrylate plugs reported. PMID- 8629987 TI - An unusual cause of stress fracture of the first rib. AB - An unusual case of stress fracture of the first rib that occurred in a young male as a result of the constant loading due to carrying a heavy schoolbag is reported. This case demonstrates the difficulty in diagnosis and highlights the need for awareness of this condition, thus avoiding invasive investigation. PMID- 8629988 TI - Skin migration following periurethral polytetrafluorethylene injection for urinary incontinence. AB - We present a patient who was treated unsuccessfully for urinary incontinence with the injection of particulate polytetrafluoroethylene (Polytef). Following removal of the resultant periurethral mass she developed fever, malaise, polyatrhopathy, a raised erythrocyte sedimentation rate, and she had a skin rash which contained refractile foreign material surrounded foreign body giant cells. PMID- 8629989 TI - Elastic properties of single titin molecules made visible through fluorescent F actin binding. AB - Titin (also known as connection) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several indirect observations have implicated titin as playing a fundamental role in the generation of passive force of muscle, driven by titin's elastic properties. A direct observation of the mechanical properties of titin, however, has not been demonstrated. Here we have used the recently shown strong actin-binding property of titin to indirectly visualize and manipulate single molecules of titin. Titin molecules were immobilized on a microscope coverslip by attaching them to anti titin antibody. The titin molecules were detected by attaching fluorescent actin filaments to them. The titin molecules were subsequently stretched by manipulating the free end of the attached actin filaments with a glass microneedle. Titin is shown here to possess a high degree of torsional and longitudinal flexibility. The molecule can be repetitively stretched at least fourfold, followed by recoil. Titin's unloaded elastic recoil proceeded in two stages: an initial rapid process (15 ms time constant) was followed by a slower one (400 ms time constant). The force necessary to fully extend titin--estimated by observing the breakage of the titin-bound actin filaments--may reach above approximately 100 pN (longitudinal tensile strength of actin). Attachment of fluorescent actin filaments to titin provides a useful tool to further probe titin's molecular properties. PMID- 8629990 TI - Plant calreticulin is specifically and efficiently phosphorylated by protein kinase CK2. AB - Calreticulin isolated from spinach leaves has been specifically phosphorylated in vitro by protein kinase CK2 while animal calreticulin from rabbit liver is not a substrate of this kinase under the same conditions. Phosphoserine is the only phosphoamino acid detected. High affinity binding (Km = 4.4 microM) and a nearly stoichiometric incorporation of phosphate was determined. Partially purified spinach calreticulin is phosphorylated at the same site(s) by a copurifying protein kinase sharing biochemical properties very similar if not identical to those of mammalian CK2. Other plant calreticulins isolated from Liriodendron tulipifera appear to be also phosphorylated by CK2. PMID- 8629991 TI - Isolation and characterization of Caenorhabditis elegans extracellular matrix. AB - The extracellular matrix (ECM) plays an important structural and functional role in multicellular organisms. Because of the similarities between C. elegans and vertebrate development, this nematode could serve as a simplified model to study the biology of the ECM. In this study, a method for extracting mammalian ECM was adapted for the extraction of C. elegans ECM. ECM components from C. elegans were found to be homologous to mammalian ECM by immunoblotting. It was also demonstrated that antibodies generated against C. elegans ECM stained basement membrane-like structures in C. elegans eggs, larvae, and adults. PMID- 8629992 TI - Activated lymphocytes induce promoter activity of the TCA3 gene in mast cells following cell-to-cell contact. AB - Aggregates of mast cells and lymphocytes have been found in inflamed tissues suggesting that lymphocytes may have the ability to activate mast cells through cell-to-cell contact. To examine this hypothesis, murine mast cells were transfected with a T cell activation gene-3 (TCA3)-chloramphenicol acetyl transferase (CAT) construct, and these cells co-cultured with murine EL-4 (T), CH12.LX (B), WEHI-3 (myelomonocytic) or 3T3(fibroblast) cell lines. Co-culture of activated EL-4 or CH12.LX cells, but not WEHI-3 or 3T3 cells, with transfected mast cells induced a 5 to 7 fold increase in CAT expression which was dependent on the lymphocyte to mast cell ratio. Supernatants from activated EL-4 or CH12.LX cells did not induce CAT expression in transfected mast cells. These data demonstrate that activated lymphocytes have the ability to induce the promoter of the TCA3 gene in mast cells through a mechanism requiring cell-to-cell contact, and suggest the possibility that activated lymphocytes may effect other biologic processes in mast cells as well through such heterotypic activation. PMID- 8629993 TI - In vivo phosphorylation of type II myosin in Saccharomyces cerevisiae. AB - Phosphorylation of the myosin heavy chain has been shown to be a key regulatory mechanism of several non-muscle myosins. In this study we present evidence demonstrating that the yeast type II myosin heavy chain is phosphorylated in vivo. Phosphorylation of serine residues was confirmed by direct metabolic labeling with [32P] and by indirect immunostaining of phosphoserine with a specific monoclonal antibody. Loss of immunoreactivity in a targeted deletion of the 26 amino acid carboxyl terminal segment of the type II myosin heavy chain suggests that the phosphorylation occurs at one or more serine residues located between residues 1903 and 1928. PMID- 8629994 TI - Triiodothyronine increases glucose transporter isotype 4 mRNA expression, glucose transport, and glycogen synthesis in adult rat cardiomyocytes in long-term culture. AB - Effects of T3 treatment (day 2-day 12) on the expression of the insulin-regulated GLUT4, on 2-deoxyglucose uptake, and on glycogen synthesis were studied in ARC after 12 days of culture in T3-depleted 20% fetal calf serum. GLUT4 mRNA expression was low in controls, but increased in a dose-dependent manner by T3 treatment up to 2.8-fold at 100 nM. In parallel, 100 nM T3 increased basal 2 deoxyglucose uptake 1.95-fold and insulin-stimulated uptake 1.75-fold. In addition, T3 enhanced basal and insulin-stimulated [14C] glucose incorporation into glycogen 1.86- and 1.5-fold, respectively. Hence, ARC may meet part of their increased energy requirements in response to T3 by enhancing GLUT4 expression. PMID- 8629995 TI - Brain-specific interaction of a 91-kDa membrane-bound protein with the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor. AB - The cytoplasmic tail of the 300 kDa mannose 6-phosphate receptor (MPR 300-CT) is thought to play an important role in sorting and targeting of lysosomal enzymes and the insulin-like growth factor II along the biosynthetic and endocytic pathway. In this study a brain specific 91 kDa protein and a 35 kDa protein salt washed from membranes (referred as TIP 91-M and TIP 35-M) were found to interact with the cytoplasmic receptor tail as assayed by cross-linkage with recombinant [32P] labeled MPR 300-CT. Subcellular fractionation revealed a distinct pattern of distribution of TIP 35-M and TIP 91-M in microsomal and synaptosomal fractions. Furthermore, the formation of cross-link complexes with membrane proteins appeared to be developmentally and regionally regulated in the brain and inhibited upon ATP hydrolysis. The data suggest the requirement of specific protein interactions for MPR 300 functions in neuronal cells. PMID- 8629996 TI - Increase of mitochondrial PLA2-released fatty acids is an early event in tumor necrosis factor alpha-treated WEHI-164 cells. AB - We have previously reported that TNF induced changes in mitochondrial enzymes, one of which, succinate-dehydrogenase, is specifically activated in various TNF sensitive cell lines. In an attempt to further characterize the mechanism of trans-membrane signalling at the mitochondrial level, we have oriented our investigation to the study of phospholipase A2 activity localized in this organelle isolated from TNF-treated WEHI-164 cells. Under physiological conditions, this enzyme has a very low basal activity near the resting state, while under TNF treatment its activity is dramatically increased. This event is induced by TNF concentrations which also cause cytolysis; however, the activity of this enzyme is increased several hours before maximum cytotoxicity occurs. The activation of the mitochondrial phospholipase A2 is not arachidonoyl or fatty acid selective, as is the case for the cytosolic species. Phospholipase A2 and succinate-dehydrogenase display higher activities simultaneously under TNF treatment. This change in activity is linked to morphologic modifications. Mitochondria in particular display an orthodox state characterized by a large and clear matrix space and few crests. PMID- 8629997 TI - Role of protein phosphatase 2A in the regulation of mitogen-activated protein kinase activity in ventricular cardiomyocytes. AB - Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 +/- 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 +/- 65% of control levels at 5 min, and 316 +/- 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 +/- 17% of control values at 5 min and 125 +/- 7% at 15 min). Okadaic acid (1 microM ) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity. PMID- 8629998 TI - Inhibition of human immunodeficiency virus type 1 long terminal repeat-driven transcription by an in vivo metabolite of oltipraz: implications for antiretroviral therapy. AB - Metabolite III (MIII, 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-alpha]pyrazine), a major in vivo metabolite of oltipraz (OLT, 5-pyrazinyl-4-methyl-1,2-dithiole-3 thione), appears to disrupt human immunodeficiency virus type 1 (HIV-1) replication at a point distal to integration of the viral genome into host DNA. We report that MIII (but not OLT) is a nontoxic inhibitor of long terminal repeat (LTR)-driven expression of beta-galactosidase in phorbol-12-myristate-13-acetate (PMA)-stimulated and unstimulated 293.27.2 cells (ED50 = 14 +/- 1 and 41 +/- 4 microM, respectively). Electrophoretic mobility-shift assays (EMSA) reveal that MIII does not significantly reduce the PMA-induced DNA binding activities of NF kappa B or AP-1. Although the mechanism by which MIII inhibits LTR-driven transcription remains unclear, the antiviral synergism of OLT and MIII in vitro are likely due their independent activities. Whether this translates into antiviral synergy in vivo is being examined by comparing OLT and MIII pharmacokinetics to the pharmacodynamic effects of orally-administered OLT in patients with p24 antigenemia. PMID- 8629999 TI - A mechanism of regulation of hepatic Fru 2,6-P2 concentration upon refeeding: involvement of xylulose 5-P and cyclic-AMP. AB - In order to determine the mechanism for delayed increase in Fructose 2,6-P2 in livers of refed rats, the time course of changes in various metabolites upon refeeding NIH or high sucrose diet was investigated. Kinetics of increase in Fructose 2,6-P2 and Xylulose 5-P were similar but different from hexose 6-P or glycogen in the livers of 48 h starved rats refed with NIH diet. The increase in the Fructose 2,6-P2 level was a result of a combination of changes in Fructose 6 P,2-kinase and Fructose 2,6-bisphosphatase activity ratios, indicating dephosphorylation of the bifunctional enzyme and decreased cAMP. A similar correlation between Fructose 2,6-P2 and Xylulose 5-P and dephosphorylation was observed with refeeding high sucrose diet and also with 16 h starved rats. These kinetic results are consistent with the idea that a specific protein phosphatase 2A, activated by Xylulose 5-P, dephosphorylates Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase and also decreased protein kinase A activity, resulting in increased hepatic Fructose 2,6-P2. PMID- 8630000 TI - Lens-specific expression of a chicken beta A3/A1-crystallin promoter fragment in transgenic mice. AB - Beta A3/A1-crystallin is one of the major refractive proteins of the chicken eye lens. Previously we showed that a fragment from -382 to +22 bp of the beta A3/A1 crystallin gene functions as a promoter in transfected lens cells. Here we show by use of the bacterial chloramphenicol acetyltransferase reporter gene that the 143/+22 fragment is sufficient for lens-specific promoter activity in transgenic mice. DNase I footprinting shows that lens nuclear proteins protect several regions within the minimal promoter fragment. PMID- 8630001 TI - Retinoic acid-stimulated liver stellate cells suppress the production of albumin from parenchymal cells via TGF-beta. AB - We studied a cell-cell interaction via transforming growth factor-beta (TGF-beta ) between liver stellate cells (SCs) and parenchymal cells (PCs) using co cultures of rat primary SCs and PCs. Both TGF-beta added exogenously to the culture medium, and TGF-beta produced endogenously from SCs after stimulation with retinoic acid (RA), suppressed the production and secretion of albumin from PCs. This effect occurred at the translational level, but not at the transcriptional level; TGF-beta, as well as SC culture medium conditioned by RA, did not affect the albumin mRNA levels, but decreased the biosynthesis of [35 S]methionine-labeled albumin without altering its post-translational degradation rate. These results suggest that TGF-beta generated from SCs facilitates the development of liver cirrhosis not only by inducing the production of fibrotic components from SCs, but also by impairing the function of the surrounding PCs. PMID- 8630002 TI - The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP 1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors. AB - Jun and Fos (AP-1) transcription factors were recently proposed to mediate induction of the mouse heme oxygenase-1 gene by different agents including heme and cadmium. In this report we show that the AP-1 binding sequence, TGAGTCA, is necessary but insufficient for gene activation in response to heme or cadmium. The minimal heme response element was identified as an extended AP-1 binding site, (T/C)GCTGAGTCA. In addition to the AP-1 heptad, this element also contains an interdigitated antioxidant response element, GCnnnGTCA. Specific antioxidant response elements from the NAD(P)H:quinone oxidoreductase-1 and the glutathione S transferase Ya subunit genes were in fact responsive to heme but not all sequences that conform to the consensus antioxidant response element were activated by this agent. The heme response element resembles the consensus binding sites for the product of the maf oncogene and for the transcription factor NF-E2. The potential role of these and related transcription factors and the implication of the composite heme response element in heme oxygenase-1 gene regulation are discussed. PMID- 8630003 TI - Bradykinin increases intracellular free Ca2+ concentration and promotes insulin secretion in the clonal beta-cell line, HIT-T15. AB - We have examined the effects of bradykinin (BK) on both the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in the hamster beta-cell line, HIT-T15 cells. BK evoked a rise in [Ca2+]i in a dose-dependent manner. This response was suppressed by neomycin, suggesting that BK mobilizes Ca2+ from intracellular store via promotion of the phosphatidyl inositol turnover. Furthermore, BK also evoked insulin secretion. Both the BK-evoked rise in [Ca2+]i and insulin secretion were suppressed by the BK2 receptor antagonist, but not by the BK1 receptor antagonist. These results indicate that BK increases [Ca2+]i via BK2 receptor, thereby promoting insulin secretion in HIT-T15 cells. PMID- 8630004 TI - Cell proliferation-dependent expression of two isoforms of the nucleolar phosphoprotein p130. AB - A highly phosphorylated human nucleolar protein p130 (130-kDa) was found to be expressed in synchrony with cell-growth activation. It was not detectable in the resting lymphocytes, but its expression was increased rapidly after mitogenic stimulation. During the terminal differentiation-coupled growth-arrest of HL60 cells, the mRNA and protein of p130 reduced significantly within 24 h after the induction. In addition to the previously identified form (now referred to as p130 alpha ), a novel isoform p 130 beta was found. It contains an insert of ten amino acids located within a region corresponding to the fourth proline-rich basic domain of p130 alpha. Both isoforms were coexpressed in cell lines of different origins, with the beta-transcript exhibiting much less compared to the alpha transcript. Furthermore, both alpha- and beta-transcripts diminished when cells returned to the quiescent stage. cDNA transfection experiments demonstrated that the two p130 isoforms existed stably in the nucleoli and showed the same nucleolar distribution pattern. It implies that the ten-amino-acid-insert does not alter significantly the interactions of p130 with other nucleolar components in interphase cells. PMID- 8630005 TI - Cloning and tissue distribution of the human P2Y1 receptor. AB - Sets of degenerate oligonucleotide primers synthesized on the basis of the best conserved regions of the chick brain P2Y/P2Y1 and the murine neuroblastoma P2U/P2Y2 receptors were used in polymerase chain reaction experiments on human genomic DNA. An amplified fragment of 712 base pairs was then used as a probe to screen a human genomic DNA library. Several clones were isolated and sequencing revealed an intronless 1122 base pair open reading frame. The corresponding amino acid sequence revealed 83% identity with the chick brain P2Y1 receptor and 34% with the murine neuroblastoma P2Y2 receptor. In COS-7 cells transfected with the coding sequence inserted into the pcDNA3 expression vector, 2-methylthioATP and ATP produced a strong stimulation of inositol phosphates, a typical response of a P2Y1 receptor. Northern blot analysis detected a 6.7 kilobase messenger RNA in most human tissues, the strongest signals being observed in prostate and ovary. PMID- 8630006 TI - Potentiation of sodium butyrate-induced apoptosis by vanadate in human promyelocytic leukemia cell line HL-60. AB - Vanadate (10 microM ), a potent inhibitor of tyrosine phosphatase, added simultaneously potentiated the sodium butyrate (BuONa)-induced growth inhibition. Furthermore, at 1 mM BuONa alone, after 96 h of incubation, about 20 +/- 5% of cells exhibited the morphological characteristic of apoptosis, as established by nuclear changes (condensed and fragmented nuclei) and decrease in cell size. After treatment of cells with 1 mM BuONa in the presence of 10 microM vanadate, apoptotic cells became more abundant; 90 +/- 3% of cells presented morphological characteristics of apoptosis after 96 h of incubation. Flow cytometric measurement of DNA content demonstrated the accumulation of cells in G1 phase after 72 h of incubation with 1 mM BuONa alone. In the presence of vanadate (10 microM ), accumulation of cells in G1 phase appeared after shorter times of incubation (48 h) with BuONa. A substantial increase in the proportion of cells with degraded DNA characteristic of apoptosis was observed after 48- to 72-h incubation with BuONa in the presence of vanadate. BuONa-induced apoptosis was accompanied by the increase of tyrosine phosphorylation of cellular proteins pp37 and pp97. Our results raised the possibility that regulation of tyrosine phosphorylation of pp37 and pp97 is an important event that heralds the BuONa induced apoptosis. PMID- 8630007 TI - Suppression of translation frameshift by upstream termination codon. AB - Retroviruses employ translation frameshift for gene expression. Translation frameshift takes place at the slippery heptanucleotide sequence on mRNA. A downstream stem-loop structure (and a pseudoknot formed by the stem-loop) enhances the level of ribosomal frameshifting. Here, we describe that an upstream termination codon suppresses the ribosomal frameshifting, leading to reduction in the expression of downstream genes. PMID- 8630008 TI - Rapid protein identification using N-terminal "sequence tag" and amino acid analysis. AB - Proteins can be identified by amino acid analysis and database matching, but it is often desirable to increase the confidence in identity through the use of other techniques. Here we describe a rapid protein identification method that uses Edman degradation to create a 3 or 4 amino acid N-terminal "sequence tag," following which proteins are subjected to amino acid analysis protein identification procedures. Edman degradation methods have been modified to take only 23 min per cycle, and rapid amino acid analysis techniques are used. The Edman degradation and amino acid analysis is done on a single PVDF membrane-bound protein sample. A computer database matching program is also presented which uses both amino acid composition and "sequence tag" data for protein identification. This method represents the most inexpensive, accurate, and rapid means of protein identification, which is ideal for the screening of proteomes separated by 2-D gel electrophoresis. The creation of N-terminal Edman degradation "sequence-tags" prior to peptide mass fingerprinting of samples should also be useful. PMID- 8630009 TI - Promoter variation in the liver glucokinase is a risk factor for non-insulin dependent diabetes mellitus. AB - We have previously reported a common variation in the liver promoter of the human glucokinase, which is regulated by insulin, in the patients with non-insulin dependent diabetes mellitus (NIDDM). The variation occurred within a 10-bp region completely conserved between human and rat. Its basic motif was almost identical to the insulin regulatory element of the phosphoenolpyruvate carboxykinase gene. In vitro transfection experiment showed that the G-to-A variation causes a 58% reduction in the promoter activity. After oral glucose challenge, the homozygous A/A subjects had the highest stimulated insulin levels at 60 and 90 minutes and the highest insulin area under the curve as compared to the subjects with other genotypes, which suggested the homozygous A/A subjects were more insulin resistant. As insulin resistance is a risk factor of NIDDM, we concluded that this promoter variation is a risk factor for NIDDM. PMID- 8630011 TI - Monoclonal antibodies to the EGF receptor act as betacellulin antagonists. AB - Betacellulin (BTC), a recently discovered member of the EGF family that is produced by certain tumour cells, is thought to be involved in autocrine growth by activating the EGF receptor (EGFR). We have investigated whether monoclonal antibodies (mAbs) to the EGFR which act as EGF, TGF alpha and HB-EGF antagonists could also be effective as BTC antagonists by blocking its binding to the EGFR. We report that the binding of 125I-BTC to a range of tumour cells expressing the EGFR was inhibited by rat mAbs that bound to three distinct epitopes on the extracellular domain of the EGFR. We show that one of these mAbs (ICR62) also prevents activation of the EGFR by betacellulin as evidenced by reversal of the effects on the growth of human fibroblasts and the head and neck carcinoma cell line HN5 and by inhibition of receptor phosphorylation in HN5 cells. We conclude that mAbs such as ICR62 have potential for use as therapeutic agents by blocking betacellulin-induced growth of tumours which over-express the EGFR. PMID- 8630010 TI - Intestinal expression of apolipoprotein A-IV and C-III is coordinately regulated by dietary lipid in newborn swine. AB - The purpose of this study was to determine the effects of dietary fatty acids on intestinal apolipoprotein (apo) A-IV and C-III expression in the newborn piglet. Two-day-old female piglets received one of three isocaloric formulas containing medium-chain triglycerides (TG) (MCT), intermediate-chain saturated TG (ICST), or long-chain polyunsaturated TG (LCPUT) by continuous duodenal infusion for 24 hrs. Control groups received a low-TG elemental formula infusion (LTG group) and a high-TG long-chain unsaturated lipid emulsion infusion (HTG group). Jejunal synthesis of both apo A-IV and C-III was increased by all infusions compared to the LTG group. Apo A-IV and C-III mRNA levels paralleled synthesis levels except for apo C-III with the MCT diet, where regulation appeared to occur at the translational level. Among all groups, apo A-IV synthesis and mRNA abundance were linearly correlated with that of apo C-III. PMID- 8630012 TI - Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond. AB - A site-directed mutagenesis in AprP, an alkaline protease isolated from Pseudomonas sp. KFCC 10818 was carried out in order to obtain increased thermostability. Sites for cysteine substitutions to form disulfide bond within AprP were chosen by comparing the sequences with aqualysin I, an alkaline thermostable serine protease whose disulfide bonds seems to be important for its thermostability. Gly199 and Phe236 residues were each replaced with cysteine by site-directed mutagenesis. The G199C/F236C mutant enzyme appeared to form a disulfide bond spontaneously during its expression. It also showed improved kinetic parameters for the hydrolysis of a synthetic peptide substrate at pH 8.5 and 10.5 compared to those of the wild-type enzyme. The half-life of the G199C/F236C mutant was found to be 2 to 4.8 times longer than that of wild-type under various experimental conditions, except when tested under reducing condition, where no significant differences in the half-life of the two types were observed. Therefore, it is concluded that the introduction of the disulfide bond enhanced the thermostability and the catalytic efficiency of the enzyme AprP. PMID- 8630013 TI - Cysteine-699, a possible palmitoylation site of the thyrotropin receptor, is not crucial for cAMP or phosphoinositide signaling but is necessary for full surface expression. AB - We investigated the role of the cysteine residues in the cytoplasmic tail of the thyrotropin receptor (TSHR) in TSH binding and signal transduction by individually mutating two cysteine residues to serine. Neither mutant exhibited changes in basal, TSH- or Graves' IgG-stimulated cAMP or inositol phosphate responses. However, mutation of Cys-699 significantly decreased TSH binding Bmax without significantly changing binding affinity, amount or sizes of TSHR forms on Western blots. These findings suggest that Cys-699, which is a potential site for lipid modification and whose equivalent in other receptors was shown to be palmitoylated, is important for efficiency of proper membrane insertion and/or stability of the receptor. PMID- 8630014 TI - Identification of promoter in the 5'-flanking region of the E. coli thioredoxin linked thiol peroxidase gene: evidence for the existence of oxygen-related transcriptional regulatory protein. AB - E. coli thiol peroxidase (Tpx) linked to the thioredoxin as an in vivo thiol regenerating system acts as an antioxidant enzyme removing peroxides and H2O2. In order to elucidate the mechanism regulating tpx gene expression in E. coli in response to oxygen stress, we made 5' progressive deletions of upstream region from tpx gene, and fused to lacZ gene. LacZ activity was increased 6-fold by oxygen stress and inverted repeat sequence located between -47 and -33 nt was proven to be essential for the oxygen response of tpx promoter. Primer extension experiment and analysis of upstream sequence revealed transcription start point, 10, and -35 regions, which are in good agreements with the consensus sequences recognized by E sigma 70. Northern hybridization showed that expression of tpx gene is regulated at the transcriptional level. DNA binding assays using inverted repeat sequence including -35 region provides preliminary evidence that expression of tpx requires additional transcriptional factor in response to oxygen stress. PMID- 8630015 TI - Functional analysis of the preproendothelin-1 gene promoter in pulmonary epithelial cells and monocytes. AB - At least two human preproendothelin-1 mRNAs are produced by a single gene through the use of different promoters, and the mechanisms controlling the production of these alternative transcripts might be cell-specific. In human lung, endothelin-1 is produced by vascular endothelial cells, bronchial epithelial cells, and pulmonary monocytes/macrophages. Given the important role of endothelin-1 in the pathogenesis of some pulmonary diseases, we carried out a functional analysis of the preproendothelin-1 gene promoter(s) in bronchial epithelial cells and lung monocytes. Two distinct preproendothelin-1 mRNAs were expressed in the two cell populations. Using deletion mutants and enhancer trap transfection experiments, we also identified different regions of the preproendothelin-1 gene necessary for endothelin-1 expression in bronchial epithelial cells and pulmonary monocytes. PMID- 8630017 TI - The effects of ruthenium red, an inhibitor of calcium-induced calcium release, on phasic myometrial contractions. AB - Ruthenium red inhibits calcium-induced calcium release from the ryanodine sensitive intracellular calcium stores; this study sought to evaluate the effects of ruthenium red on agonist-stimulated phasic myometrial contractions. Ruthenium red was found to significantly inhibit in vitro isometric contractions stimulated in response to oxytocin, prostaglandin F2 alpha, aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium-induced calcium release from ryanodine-sensitive calcium stores is an important event during agonist-stimulated phasic myometrial contractions. PMID- 8630016 TI - Anti-TNF treatment reverts increased muscle ubiquitin gene expression in tumour bearing rats. AB - Implantation of the ascitic tumour Yoshida AH-130 hepatoma (a cachectic tumour) resulted in important increases in muscle ubiquitin gene expression. Administration of daily injections of 25 mg/kg b.w. polyclonal goat anti-murine TNF IgG preparation to tumour-bearing rats abolished the increase in muscle ubiquitin gene expression observed in the control (non-anti-TNF-treated) tumour bearing rats. It is concluded that TNF can have an important role in the activation of the ubiquitin-dependent proteolytic system during tumour growth. PMID- 8630018 TI - DNA polymerases from a parasitic protozoa Leishmania donovani UR6: evidence of presence of a novel kind of DNA polymerase. AB - DNA polymerases of Leishmania donovani have been isolated and purified. The cell extract has been chromatographed on a phosphocellulose column that separated into three peaks. The activity peak 1 was further purified to homogeneity. The DNA polymerase is a 64 KDa polypeptide, resistant to N-ethylmaleimide and aphidicolin. It requires MnCl2 and a high concentration of KCl (0.5 M) for maximal activity. It has both 3' to 5' and 5' to 3' exonuclease activities that reside in the same polypeptide. PMID- 8630019 TI - Nitric oxide inhibits migration of cultured endothelial cells. AB - Endothelial cell migration is an important event in both physiological and pathophysiological processes. Although nitric oxide (NO) plays a critical role in regulating vascular functions, it is not known whether NO modulates migration of endothelial cells. We show here that chemically-derived NO inhibited the serum induced migration of cultured human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. The sensitivity of inhibition by S-nitroso-N acetylpenicillamine (SNAP, a NO donor) was 2.36 +/- 1.032 x 10(-4) M (n = 4). This effect was attributed to NO since (1) other NO donor (e.g., sodium nitroprusside) also exhibited antimigratory effect, (2) pre-incubated SNAP (72 h) had no effect, (3) hemoglobin, a NO scavenger, eliminated the effect; while (4) superoxide dismutase, a NO protector, enhanced the antimigratory effect. Furthermore, 8-bromo-cGMP also inhibited the serum-induced migration of HUVEC. These data appear to support the notion that NO may serve as an important signaling molecule for neovascularization. PMID- 8630020 TI - Rab3A delayed catecholamine secretion from bovine adrenal chromaffin cells. AB - To study the role of Rab3A in regulated secretion, recombinant Rab3A protein plus various guanine nucleotides were perfused into bovine adrenal chromaffin cells via a patch pipette, and depolarization-evoked catecholamine secretion from individual cells was measured by amperometry. Rab3A plus GTP, GDP, and GTP gamma S all delayed the occurrence of exocytosis while Rab3A, GTP, or GDP alone had no effect. The delay of evoked-secretion is specific for Rab3A plus guanine nucleotides, since treatments with GTP plus BSA or GDP beta S plus Rab3A had no effect on the appearance of secretion events. Rab3A plus GTP increased the frequency of the exocytosis of smaller packets of catecholamine. These results are consistent with the notion that an excess of Rab3A in the cytoplasm depletes the regulatory proteins responsible for regulating the interconversion between GTP- and GDP-bound forms of Rab3A and that Rab3A is involved in the final steps of regulated exocytosis. PMID- 8630021 TI - Fine mapping of the human endothelin-converting enzyme gene by fluorescent in situ hybridization and radiation hybrids. AB - Based on the sequence of the human endothelin-1-converting enzyme (hECE-1) cDNA, we cloned a 1982 bp cDNA fragment and we amplified by PCR a 3' fragment located on the last exon (exon 19). Human metaphase chromosomes were studied by Fluorescent in Situ Hybridization (FISH) using the 1982 digoxigenated hECE-1 fragment as a probe and chromosome 1-specific probes. Twin spot signals on each of the two homologous chromosomes 1 were found by FISH on band p36. The results of monochromosomal hybrids confirmed that hECE-1 is on chromosome 1. Radiation hybrid mapping localized the hECE-1 gene 3.15 cR far from D1S2436 (WI-3177), at about 25 cM from the telomere of the short arm, possibly at the border between 1p36.3 and 1p36.2. PMID- 8630022 TI - Epsilon PKC acts like a marker of progressive malignancy in rat liver, but fails to enhance tumorigenesis in rat hepatoma cells in culture. AB - We have analyzed the expression pattern of epsilon protein kinase C (PKC) in normal liver tissue, in hyperplastic liver nodules and in hepatocellular carcinomas generated in the rat with the Solt-Farber protocol. A progressive increase in PKC epsilon expression was observed in nodules and carcinomas compared to normal liver tissue, suggesting that the expression level of this PKC isoenzyme could be associated with increased malignancy. To test this hypothesis, the well differentiated, poorly tumorigenic MH1C1 rat hepatoma cell line was stably transfected with a full length epsilon PKC cDNA. No increase in growth rate, saturation density, soft agar growth or in vivo tumorigenicity was observed in transfected cells, compared to parental or mock-transfected cells. These results indicate that epsilon PKC does not seem to participate in signaling pathways involved in neoplastic transformation or malignant progression in our liver cell model. The fact that epsilon PKC overexpression is tumorigenic in several other cell types suggests that this effect might be strictly cell- and tissue-specific. PMID- 8630023 TI - Relative contribution of the selectins in the neutrophil recruitment caused by the chemokine cytokine-induced neutrophil chemoattractant (CINC). AB - Rat CINC induced a dose- and time-dependent accumulation of neurophils into murine air-pouches, a response which was inhibited by two selective H1 antagonists, mepyramine and triprolidine (approximately 60%). As pretreatment with fucoidin abolished CINC effect, the relative time-related contribution of selectins on this process was then investigated by using specific monoclonal antibodies (mAb). Anti-CD62L mAb gave a similar degree of inhibition of CINC induced cell accumulation both at the 2h and 4h time-point (approximately 75%). Anti-CD62P mAb, but not the anti-CD62E mAb, inhibited PMN accumulation at 2h (65%), but only co-administration of these two mAbs inhibited the cell response to CINC at the 4h time-point (90%). Thus endogenous histamine, CD62L, CD62P, and CD62E, though to a different degree, are required for PMN extravasation observed in response to CINC administration. PMID- 8630024 TI - Coexpression of flt-1, flt-4 and KDR in freshly isolated and cultured human endothelial cells. AB - Vascular endothelial cell growth factors (VEGF) are key modulators of endothelial cell growth and function. The class III receptor tyrosine kinases KDR and Flt-1 are high affinity receptors for VEGF, while Flt-4 is a receptor for the recently identified VEGF-C. We have examined the expression of flt-1, flt-4 and KDR in human microvascular and large vessel endothelial cells and in a variety of other cell types in vitro. Endothelial cells proliferated and exhibited increased procoagulant activity in response to VEGF. Flt-1, flt-4 and KDR were detected in both freshly isolated endothelial cells, and in sparse and confluent endothelial cell cultures by RT-PCR. Attempts to modulate receptor expression by culturing cells at reduced oxygen tensions (2%) did not induce consistent changes in flt-1, flt-4 or KDR expression. Incubation with tumor-conditioned medium or co-culture of endothelial cells with a range of breast and small cell lung carcinoma cell lines did not reproducibly alter receptor mRNA expression. However, flt-1, flt-4 and KDR transcript levels were enhanced following treatment with tetradecanoylphorbol acetate. PMID- 8630025 TI - Stimulation by C-type natriuretic peptide of the differentiation of clonal osteoblastic MC3T3-E1 cells. AB - We examined the effects of C-type natriuretic peptide (CNP) and B-type natriuretic peptide receptor (NPR-B) system, which stimulates the intracellular production of cGMP, on osteoblastic differentiation using clonal murine calvarial MC3T3-E1 cells. CNP-like immunoreactivity was detected in the conditioned medium and in lysates of MC3T3-E1 cells. Exposure of cells to CNP caused an increase in the intracellular production of cGMP and the increase was dose-dependent, while ANP had no effect. These results imply that CNP regulates osteoblastic metabolism via NPR-B in an autocrine manner. Northern blot analysis revealed that treatment of MC3T3-E1 cells with CNP increased the steady-state levels of mRNAs for type-I collagen, cellular alkaline phosphatase (ALPase), and osteocalcin, which are well known as markers of osteoblastic differentiation. Our observations suggest the possibility that CNP functions as a local regulator of osteoblastic differentiation, acting via a cGMP-mediated pathway. PMID- 8630026 TI - Apoptosis in vascular smooth muscle cells: role of cell shrinkage. AB - Cell volume decrease is known to be one of the earliest steps of apoptosis in immune system cells. In this study, we compared the kinetics of apoptosis and cell volume adjustment in cultured vascular smooth muscle cells (VSMC) from the aorta of normotensive Brown-Norway (BN.1x) as well as spontaneously hypertensive (SHR) rats and in Mardin-Darby canine kidney (MDCK) cells. The transfer of VSMC to serum-deprived medium led to a transient cell volume decrease and to increased apoptosis. Both the cell volume decrease and apoptosis displayed faster kinetics in SHR than in BN.1x VSMC. Increased tonicity of serum-deprived medium by the addition of 200 mM mannitol augmented apoptosis in VSMC by 2.5- to 3-fold. In contrast to VSMC, neither apoptosis nor the cell volume of MDCK cells was affected by serum deprivation. Apoptosis in MDCK cells was also insensitive to tonicity of serum-deprived medium. There results demonstrate an initial volume decrease in VSMC undergoing apoptosis and suggest that this phenomenon is involved in triggering the apoptotic process. PMID- 8630027 TI - Arachidonic acid potentiates ACh receptor currents by protein kinase C activation but not by receptor phosphorylation. AB - The effects of arachidonic acid on ACh-gated channel currents were examined using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Arachidonic acid decreased ACh-evoked currents during treatment, to a greater extent in Ca(2+) free extracellular solution. The currents were enhanced for more than 30 min after washing, reaching 150 and 170% in Ca(2+)-containing and -free extracellular solutions, respectively. The current enhancement was inhibited by the selective protein kinase C (PKC) inhibitor, GF109203X, whereas the current depression was not affected. Furthermore, arachidonic acid-evoked current depression was blocked in mutant ACh receptors with PKC phosphorylation site deletions on the alpha and delta subunits, but the long-lasting potentiation effect remained. These results indicate that arachidonic acid may decrease ACh receptor currents by a direct binding to PKC phosphorylation sites of the ACh receptors and may potentiate the currents via a novel pathway related to arachidonic acid-regulated PKC activation, but not via PKC phosphorylation of the ACh receptor itself. PMID- 8630028 TI - Specific in vitro binding of p53 to the promoter region of the human mismatch repair gene hMSH2. AB - MSH2 is one of the genes involved in DNA-mismatch repair. Mutations in the coding region of the human gene (hMSH2) have been shown to be directly involved in microsatellite instability in hereditary nonpolyposis colorectal tumors. Examination of the promoter region of hMHS2 revealed a site with homology to the p53 consensus binding sequence. Using gel mobility shift experiments we were able to show that purified p53 has at least in vitro the potential to specifically bind the hMSH2-p53 motif. This binding activity was even stronger than the binding activity measured with the p53-consensus site. These data identify the hMSH2 gene as a possible novel p53-regulated target gene and indicate a direct involvement of p53 in repair mechanisms via DNA binding of a mismatch repair gene. PMID- 8630030 TI - Modulation of in vitro kidney cell growth by hepatic transfer RNAs. AB - The quiescent and proliferating status of the cell appears characterized by two different tRNA populations. The tRNA species changed both as relative percentage of total tRNA and in absolute concentration. The changes seem causally related to the proliferation status of the cell as tRNAs from proliferating hepatocytes were able to stimulate in vitro kidney cell growth. Moreover, tRNAs from quiescent hepatocytes inhibited kidney cell growth. It was concluded that the composition of tRNA population can modulate the proliferative behaviour of the cell. PMID- 8630029 TI - In vivo detection of mouse liver nitric oxide generation by spin trapping electron paramagnetic resonance spectroscopy. AB - Nitric oxide, a paramagnetic molecule synthesized in biological systems, plays an important role in many pathophysiological processes. In vivo electron paramagnetic resonance spectroscopy/imaging could be a useful tool to study, in situ and in real time, nitric oxide generation. In this study the intracellular production of nitric oxide in tissues of living septic-shock mouse was detected by the spin trapping technique in combination with electron paramagnetic resonance spectroscopy. A lipophilic spin trap agent was used and nitric oxide formation was determined by the intensity of its iron-mononitrosyl complex. Among all examined tissues at 20 degrees C, the highest signal intensity of the trapped nitric oxide was found in the liver homogenates (n = 5). The amount of complex found in the kidneys was about 40% of that found in the liver. In the brain and lung, around 10% was found. This study reports, for the first time, the in vivo detection of nitric oxide generation in the upper abdomen of septic-shock mice (n = 3). Within 1 h after the trap injection, the signal was stable, indicating that the formation had reached a steady state. PMID- 8630031 TI - Molecular cloning of the precursors of pigment dispersing hormone in crustaceans. AB - The cDNAs encoding the precursors of a chromactive crustacean hormone, Pigment Dispersing Hormone (PDH) of the shrimp Penaeus vannamei, were studied by PCR and molecular cloning. Three different cDNAs were isolated and sequenced. The PDH precursor consists of a putative 22- or 23-amino acid signal peptide, a 34-amino acid PDH-Precursor Related Peptide (PPRP) of unknown function, and the 18-amino acid mature PDH. The deduced mature PDH amino acid sequences are identical except the change of a Leucine by an Isoleucine in one variant and are very similar to those of other species. The signal peptides appear highly variable. The variability between the PPRP sequences is low between the different species, suggesting that this peptide may have a physiological role. PMID- 8630032 TI - Molecular cloning and characterization of a novel stromal cell-derived cDNA encoding a protein that facilitates gene activation of recombination activating gene (RAG)-1 in human lymphoid progenitors. AB - The activation and expression of recombination activation genes (RAGs) in lymphoid progenitors are regulated by signals from surface molecules of stromal cells and/or cytokines. Using a mRNA differential display method, we isolated a novel stromal cell-derived cDNA clone, C2.3, whose transcripts were intensively expressed in RAG-1-inducible stromal cell line, but rarely expressed in RAG-1-non inducible mutant cell line (PA6). The cDNA sequence had no homology to the known genes. The sequence revealed an open reading frame that encodes a 221 amino acid protein with 4 potential transmembrane domains, suggesting a possible role of C2.3 product as a membrane receptor. Introduction of C2.3 cDNA into PA6 mutant line restored the ability to activate RAG-1 gene in lymphoid progenitors, indicating that a C2.3 product may be involved in the induction of RAG-1 gene activation. PMID- 8630033 TI - Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro. AB - Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations. PMID- 8630034 TI - Novel polypeptides induced by the insecticide lindane (gamma hexachlorocyclohexane) are required for its biodegradation by a Sphingomonas paucimobilis strain. AB - When exposed to the potent insecticide gamma-hexachlorocyclohexane or lindane, a Sphingomonas paucimobilis strain rapidly synthesized 7 novel polypeptides and concomitantly gained the ability to degrade lindane. Synthesis of these proteins was switched-off subsequent to the disappearance of lindane from the medium. Treatments which induced the synthesis of identical proteins also conferred on cells the ability to degrade lindane. In contrast, cells blocked in protein synthesis could not be induced to degrade lindane. The close correspondence observed between expression of lindane-induced proteins and gamma hexachlorocyclohexane catabolism strongly implicates these novel proteins in lindane biodegradation. PMID- 8630035 TI - Evidence for a direct inhibitory effect of extracellular H+ on store depletion activated Ca2+ entry in vascular endothelial cells. AB - Modulation of store depletion-activated Ca2+ entry by acidosis was investigated in ECV304 endothelial cells. Lowering extracellular pH from 7.4 to 6.9 markedly suppressed Ca2+ entry elicited by direct depletion of Ca2+ stores with thapsigargin (100 nM), but did not significantly affect leak Ca2+ entry. Acidosis diminished thapsigargin-induced Ca2+ entry by 53.7 +/- 7.8% at 2.5 mM extracellular Ca2+. A similar degree of inhibition was observed in cells depolarized by high extracellular K+ (100 mM). Reduction of extracellular pH from 7.4 to 6.9 was associated with a decrease in intracellular pH from 7.23 +/- 0.01 to 7.01 +/- 0.03. Propionate (20 mM) caused a reduction of intracellular pH to 6.97 +/- 0.02, but failed to suppress store depletion-activated Ca2+ entry at 2.5 mM extracellular Ca2+ significantly. Our results suggest that an increase in extracellular proton concentration inhibits store depletion-activated Ca2+ entry through a direct, membrane potential-independent effect on the plasmalemmal Ca2+ channel. PMID- 8630036 TI - Molecular cloning and characterization of human bone morphogenic protein (BMP)-5 gene promoter. AB - The bone morphogenic proteins (BMPs) constitute a novel subfamily of the transforming growth factor type beta (TGF-beta) supergene family and play a critical role in modulating mesenchymal differentiation and inducing the processes of cartilage and bone formation. In this study we isolated the 5' flanking region of the human BMP-5 gene. Nucleotide sequencing, primer extension, DNase I foot printing and functional analysis by transient expression showed that the cloned 1.5 kb promoter region contains two transcription start sites, a canonical TATA box (-17 approximately -12) and a number of consensus recognition sequences including GATA-1 (-582 approximately -576) and engrailed (-549 approximately -541). PMID- 8630037 TI - The mechanism for mechanochemical energy transduction in actin-myosin interaction revealed by in vitro motility assay with ATP analogues. AB - We investigated in vitro motility of F-actin on heavy meromyosin (HMM) and nucleotide triphosphatase activity of acto-HMM by using ATP analogues of various nucleotide triphosphates (NTPs) and enzymatically cleaved actins. The sliding velocity did not correlate with the actin activated HMM-NTPase activity, but correlated strongly with the reciprocal of NTPase activity of HMM itself, i.e., the cycle time of HMM NTPase. This indicated that with ATP the complex of myosin with the product, M.ADP.Pi, at the long lived intermediate state of the rate limiting step would play a key role for efficient mechanochemical energy transduction during actin-myosin interaction. PMID- 8630038 TI - Activation of phosphatidylinositol 3-kinase by prolactin in Nb2 cells. AB - In the present studies, using anti-phosphotyrosine (PY20) and PI3-kinase (p85) antibodies, we have shown that PRL causes activation of phosphatidyl inositol 3 kinase (PI3-kinase) in vitro in a dose- and time-dependent manner in Nb2 cells. PRL activated PI3-kinase was completely inhibited by LY294002 (1 microgram/ml). Stimulation of the cells with PRL also increased tyrosine phosphorylation of the 85-kDa regulatory subunit. Moreover, in vitro kinase assay followed by SDS-PAGE protein separation demonstrated the phosphorylation of several other proteins besides the p85. However, no direct association between p85 and JAK2 tyrosine kinase was observed. These results indicate, for the first time, the involvement of PI3-kinase in PRL-stimulated Nb2 cell growth. PMID- 8630039 TI - Interleukin 3 activates not only JAK2 and STAT5, but also Tyk2, STAT1, and STAT3. AB - It has been described that interleukin 3 (IL3) activates JAK2, which in turn stimulates STAT5 activation. We found, however, that IL3 induces tyrosine phosphorylation of Tyk2 as well as JAK2 in IL3-dependent mouse cell lines, FDC-P2 and Ba/F3. Furthermore, we found that IL3 induces activation of not only STAT5 but also STAT1 and STAT3. Taken together with other observations, these results indicate that IL3, erythropoietin and thrombopoietin share a common JAK-STAT signaling pathway. PMID- 8630040 TI - Vesicular monoamine transporter in microvesicles from bovine posterior pituitaries is immunologically similar to but distinct from the chromaffin granule counterpart in its sensitivities to 1-methyl-4-phenylpyridinium and histamine. AB - The microvesicles (MVs) in bovine posterior pituitaries contain the reserpine sensitive vesicular monoamine transporter (Moriyama et al. (1995) J. Biol. Chem. 270, 11424-11429). An antibody against the N-terminal region of the monoamine transporter from bovine chromaffin granules recognized a polypeptide in the MVs with a similar molecular mass to the chromaffin granule counterpart. 1-Methyl-4 phenylpyridinium inhibited the norepinephrine uptake by the MVs and chromaffin vesicles, the concentrations required for 50% inhibition being 8 and 150 microM, respectively. Histamine also showed similar effect. These results indicated that the monoamine transporter in MVs is immunologically similar to, but distinguishable pharmacologically, from the chromaffin granule counterpart, and suggested the polymorphism of the transporter in bovine tissues. PMID- 8630042 TI - Organization of the human prostacyclin synthase gene. AB - We determined the exon/intron organization of the human prostacyclin synthase gene. The gene, which spans approximately 70 kilobases, is composed of 10 exons. PMID- 8630041 TI - Androgen-dependent expression of fibroblast growth factor-1 in submaxillary gland of mouse. AB - We have purified a 16,000 dalton protein that stimulates growth of human umbilical cord vein-derived endothelial cells (HUV-EC) from mouse submaxillary glands by using heparin-Sepharose affinity and C4 reverse phase chromatography. The purified molecule was identified as an FGF-1 on the basis of its biological activities, its affinity for heparin and its N-terminal amino-acid sequence. The concentrations of FGF-1 in the submaxillary gland of male or testosterone-treated female mice were about 12 times those of untreated females or castrated males. The 2.3 and 4.1 kb FGF-1 mRNAs were expressed in the glands of male mice older than 4 weeks but not in the glands of female mice. These results suggest that FGF 1 may have important functions for growth, differentiation and development of mouse submaxillary glands, and it may act as an endocrine hormone. PMID- 8630043 TI - Expression of functional melanocortin 1 receptors in insect cells. AB - We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4,D Phe7]alpha-MSH. Both saturation and competition analysis, using alpha-, beta-, and gamma 1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells. PMID- 8630044 TI - Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme. AB - A systematic study of tethering various groups on 6-phenyl ring of 4-hydroxy-6 phenyl-3-[(2-isopropylphenyl)thio]pyran-2-one was performed to increase the binding affinity with HIV protease. This tethering approach was aimed to fill S3 pocket of the enzyme. Thus, tethering hydrophilic groups resulted in more potent inhibitors. Similarly, various aromatic hydrophobic rings as well as heterocyclic rings were explored as tethering substituents to alter the physical properties as well as to enhance the binding affinity with HIV protease. Inhibitor 24, 4 hydroxy-3-[(2-isopropylphenyl)thio]-6-[4-(3-pyridinylmethoxy+ ++ ) phenyl]-2H pyran-2-one, was evaluated as a prototypic lead structure to study various physical as well as pharmacological properties of this class of HIV protease inhibitors. PMID- 8630045 TI - Kinetic analysis of inositol trisphosphate binding to pure inositol trisphosphate receptors using scintillation proximity assay. AB - Inositol 1,4,5-triphosphate (InsP3) receptors are regulated by many intracellular signals including proteins and small messengers. By linking purified cerebellar InsP3 receptors to scintillation proximity assay beads, binding of radioligands can be measured without separation of bound from free ligand. InsP3 receptors assayed by scintillation proximity assay bound heparin with high affinity and stereoselectively bound InsP3 with similar affinity to cerebellar membranes. By rapidly freezing scintillation proximity assay reactions and then counting the frozen samples, both fast and slow components of [3H] InsP3 association and dissociation were identified. Our novel freeze-quench method in combination with conventional stopped-quench equipment and scintillation proximity assay allows the rapid kinetics of the interactions of pure receptors with their ligands to be resolved. PMID- 8630046 TI - Involvement of an endogenous sialidase in skeletal muscle cell differentiation. AB - We have previously observed that the cytosolic sialidase gene is highly expressed in rat skeletal muscle and that it contains an enhancer/promoter region which is transcriptionally active in rat L6 myogenic cells. Here we present evidence for the involvement of cytosolic sialidase in myoblast differentiation. During L6 myoblast differentiation induced by serum depletion, cytosolic sialidase is increased in activity as well as in terms of mRNA level. Sialidase activity is essentially lacking in untreated myoblasts but appears concomitantly with myotube formation after induction of differentiation. The mRNA becomes able to be detected after 3 days at the time which myogenin mRNA reaches a maximum level. Myotube formation can, in fact, be blocked by the addition of an antisense oligodeoxyribonucleotide complementary to the first 8 codons of the cytosolic sialidase. PMID- 8630047 TI - Fluctuation analysis of myosin-coated bead movement along actin bundles of Nitella. AB - Movements of myosin-coated magnetic beads on the actin bundles of Nitella axilliformis were studied using fluctuation analysis. Regulated force applied by magnetic field could hold magnetic beads stationary against their driving force. Thus beads selected by the same magnetic force had almost the same number of active motor proteins. When the magnetic field was switched off, the beads began sliding again and reached a steady sliding velocity. The fluctuations of bead movements were estimated from the deviations of displacement and those of velocity. The results indicate that (1) the mean step-size was 6.8 nm, and (2) the equivalent stiffness of motor proteins increased during the steady sliding compared with the stationary state. PMID- 8630048 TI - Polymorphonuclear leukocytes present laminin peptides in endocytic compartments. AB - Rat and Human neutrophils presented cytoplasmic vacuoles immunoreactive for laminin at the electron microscopy level. Colocalization of the anti-laminin labeling with albumin-gold complexes and alkaline phosphatase activity in rat PMN suggest an endocytic nature for this compartment. Immunoblot analysis of human normal peripheral blood neutrophils revealed the presence of two laminin peptides around 100 kDa. PMID- 8630049 TI - Identification of a transcriptional silencer in the protein-coding region of the mouse major inducible Hsp70 gene. AB - The mouse Hsp70.3 gene encodes the major inducible HSP70 protein and is located in close proximity to the Hsc70t gene, a testis-specific variant of the Hsp70 gene family. The two genes are arranged head-to-head, transcribed in divergent directions, and separated by approximately 600 base pairs. During transient expression analysis of the promoter for Hsc70t in mouse L cells, we have found that the Hsp70.3 coding region between -1844 and -800 relative to the transcription start site of Hsc70t possesses a silencer activity. The negative regulatory region containing this sequence also inhibited the expression from the heterologous simian virus 40 early promoter independently of orientation and position. It is possible that this silencer may not only be involved in testis specific expression of Hsc70t but also function in down-regulation of Hsp70.3 induction. PMID- 8630050 TI - Role of c-jun in the inhibition of erythropoietin receptor-mediated apoptosis. AB - Human bone marrow cells express both a truncated and full-length form of the erythropoietin receptor (EpoR-T and EpoR-F, respectively). Transfection experiments using the murine interleukin (IL)-3-dependent cell line, Ba/F3, revealed that the cells coexpressing EpoR-F and EpoR-T (Ba/F3-FT) were more likely to undergo programmed cell death (apoptosis) than cells expressing EpoR-F (Ba/F3-FF), even in the presence of erythropoietin (Epo). When Ba/F3-FF cells were stimulated with Epo or IL-3, rapid induction of c-myc, c-fos, c-jun and junB genes was observed. A similar effect was also seen in IL-3 stimulated Ba/F3-Ft cells. However, in Ba/F3-FT cells expression of the c-jun gene was not induced by Epo stimulation. Administration of Epo could prevent apoptosis induced by IL-3 deprivation in Ba/F3-FT cells expressing ectopic c-Jun protein. These results indicate that induction of c-Jun through the Epo signaling pathway has an important role in the inhibition of apoptosis. PMID- 8630051 TI - Effect of hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes in rats. AB - The effect of human hepatocyte growth factor on the proliferation of intrasplenically transplanted hepatocytes was investigated. A human hepatocyte growth factor (360 micrograms/kg BW) was administered intravenously every 24 hr for 5 days following hepatocyte transplantation in rats. A significant increase in hepatocyte proliferation was observed morphologically and immunohistochemically (p < 0.05). The bromodexyuridine labeling index was significantly greater in human hepatocyte growth factor treated rats than in the control rats (p < 0.05). Hepatocyte growth factor may promote the proliferation of intrasplenically transplanted hepatocytes. PMID- 8630052 TI - A novel homozygous mutation of the myelin Po gene producing Dejerine-Sottas disease (hereditary motor and sensory neuropathy type III). AB - We have previously reported that heterozygosity for myelin Po gene mutations were associated with Charcot-Marie-Tooth disease type 1B (CMT1B) or Dejerine-Sottas disease. We investigated the Po gene in a family with clinical Dejerine-Sottas disease and found two children were homozygous for a deletion of Phe 64. The parents were heterozygous first cousins with subclinical CMT1B and slow nerve conduction velocities. These results suggest that the effect of homozygous Phe 64 deletion on impairment of myelination is dosage-dependent. Clinical phenotype and/or myelin impairment may be determined both by the type of mutation and by the dosage of mutated gene. PMID- 8630053 TI - Effects of chronic ethacrynic acid exposure on glutathione conjugation and MRP expression in human colon tumor cells. AB - Chronic exposure to ethacrynic acid of a subcloned HT29 human colon cancer cell line produces a 3- to 4-fold increase in the level of resistance to this agent. The resistant cells (HT6-8) have an enhanced capacity to metabolize the parent drug and efflux it from the cell. This is reflected in a 5-fold enhanced decompositioning rate constant for ethacrynic acid in HT6-8 (3.47 x 10-3 min-1) versus wild type cells (1.58 x 10-2 min-1). We observed that the glutathione conjugate of ethacrynic acid is an effective competitive inhibitor for binding to the multidrug resistance-associated protein by [35S]azidophenacyl-glutathione, a photoaffinity analog of glutathione. In addition, the HT6-8 cells overexpressed multidrug resistance-associated transcript 2- to 3-fold. These results suggest that resistance to ethacrynic acid results from a concerted, coordinate increase in defense mechanisms which detoxify the drug and remove its conjugate via plasma membrane efflux. PMID- 8630054 TI - Genomic aberrations of human papillomavirus recovered from cervical cancers. AB - Human papillomaviruses (HPV) contribute to the development of malignancies of the uterine cervix and the viral E6 and E7 oncogenes are invariably retained and expressed in cervical cancer tissues. Minor, but not major, structural aberrations have been found quite frequently in viral DNA recovered from cervical cancer tissues. We examined the presence of the DNA sequence of HPV type 18 in 33 cervical cancer tissues by polymerase chain reaction. HPV type 18 DNA sequences was found in 24 of these 33 cervical cancer tissue specimens, and at least 21 of these 24 specimens did not appear to retain all the region and open reading frames examined. Twelve of these 24 tissues seemed to harbor only the E6 and/or E7 genes. These results can be construed to suggest that the absence of viral genes other than E6 and E7 is quite frequent in HPV recovered from cervical cancer tissues and that the E6 and E7 genes are important in the carcinogenesis of cervical carcinoma. It is possible that the E6 and/or E7 alone may be sufficient to maintain the transformed phenotype of cervical cancer. PMID- 8630055 TI - Functional properties of Drosophila dopamine D1-receptors are not altered by the size of the N-terminus. AB - Molecular cloning revealed the existence of at least five pharmacologically different dopamine receptors in vertebrates. Functionally, dopamine receptors either activate (D1-type), inhibit or do not interact with adenylate cyclase (D2 type). A recently cloned dopamine receptor from Drosophila melanogaster shares many structural and functional properties with vertebrate D1-type receptors but the pharmacological properties are very different. In contrast to most aminergic receptors, DmDop1 contains a long N-terminal extension. Here we describe a deletion-mutagenesis approach to study whether the N-terminus of DmDop1 participates in receptor-ligand interactions. All mutants gave rise to functional receptors after heterologous expression in HEK 293 cells. The pharmacological properties, however, remained unchanged. A comparison of DNA and deduced amino acid sequences revealed that some Drosophila strains express a truncated version of the DmDop1 receptor. PMID- 8630056 TI - The N-terminal sequences (5-20) of thymosin beta 4 binds to monomeric actin in an alpha-helical conformation. AB - The relationship between the conformation of a peptide in solution and its interaction capacity is generally unclear. Trifluoroethanol (TFE), which stabilizes alpha-helical conformations, can be used to induce definite folding in synthetic peptides. The N-terminal part of thymosin beta 4, including the 5-20 sequences, is implicated in binding to monomeric actin. The corresponding peptide was synthesized and its conformation studied by CD. The peptide is unstructured in solution, and becomes folded at medium TFE concentrations, below 30%. In contrast, TFE does not significantly modify the conformation of monomeric actin which conserves its intrinsic properties, such as gelsolin interaction and DNase I inactivation. We report here that the apparent affinity of the synthetic peptide to monomeric actin is increased by an order of magnitude in the presence of TFE, which implies that the peptide adopts a folded conformation needed for accurate interaction. PMID- 8630057 TI - CD63 associates with transmembrane 4 superfamily members, CD9 and CD81, and with beta 1 integrins in human melanoma. AB - CD63 belongs to the Transmembrane 4 superfamily (TM4SF) of membrane proteins whose functions are largely unknown. Previous results have suggested that CD63 may play an important role in the regulation of melanoma progression. To explore the role of CD63 in melanoma we have examined its association with other molecules by immunoprecipitation of CD63 from detergent induced lysates of melanoma cells. These results are the first to demonstrate an association between CD63 and two other TM4SF members, CD9 and CD81 in 2 human melanoma cell lines. We are also able to identify an association between CD9 and CD63 with beta 1 integrins in melanoma. The results suggest that CD63 is capable of forming multicomponent complexes with TM4SF members and beta 1 integrins on the surface of melanoma. These findings provide further insights into the function of CD63. PMID- 8630058 TI - Inhibition of protein prenylation down-regulates signalling by inflammatory mediators in human keratinocytes. AB - Several inflammatory mediators have been shown to activate phospholipase C in human keratinocytes via GTP-binding protein-coupled receptors. Since GTP-binding proteins are prenylated proteins, we have examined the role of prenylation in signal transduction in HaCaT keratinocytes. Indirect inhibition of prenylation with the HMG CoA reductase inhibitors fluvastatin or compactin decreased bradykinin-stimulated inositol 1,4,5-triphosphate generation. This effect was abolished by mevalonic acid but not by serum, indicating a requirement for a non sterol metabolite for signal generation. The BK response was also inhibited by zaragozic acids B and C, known inhibitors of prenyl protein transferases. These results suggest that protein prenylation may be a novel therapeutic target in dermatological conditions where an up-regulation of the inositol lipid pathway has been demonstrated. PMID- 8630059 TI - Lack of endothelin ETB receptor binding and function in the rat with a mutant ETB receptor gene. AB - Congenital aganglionosis rat is a mutant with an autosomal recessive gene (sl). Recent studies have revealed that the endothelin ETB receptor gene of sl/sl rat has a deletion of 301-bp region spanning exon 1 and intron 1 corresponding to the first and the second transmembrane domains of the receptor. In the present experiments, we examined the functions of ETB receptors in the sl/sl rats. In the membranes of cerebellum, heart, and lung of control (+/+ and sl/+) rats, ET-1 induced a monophasic, competitive displacement of [125I]ET-1 binding, whereas ET 3, IRL 1620, and BQ-123 showed biphasic displacement. In the membranes of sl/sl rats, in contrast, ET-1, BQ-123, ET-3, and IRL 1620 showed only monophasic displacement. Scatchard analysis revealed a single [125I]ET-3 binding site in the membrane of control heart but not in the sl/sl rat heart, and the specific binding sites for [125I]ET-1 in both control and sl/sl rat hearts. In the control rat aorta but not in the sl/sl rat aorta, ET-3 induced endothelium-dependent relaxation. These results suggest that sl/sl rats do not have functional ETB receptors. PMID- 8630060 TI - Cloning of SEZ-12 encoding seizure-related and membrane-bound adhesion protein. AB - SEZ-12 is one of the seizure-related cDNAs which was isolated by differential hybridization from primary cultured neurons from the mouse cerebral cortex with or without pentylenetetrazol (PTZ). SEZ-12 expression is transiently down regulated in the mouse brain by injection of PTZ. To characterize SEZ-12, isolation of full-length cDNA and nucleotide sequence analysis were performed. The deduced amino acid sequence of SEZ-12 revealed that it encodes membrane-bound C-type lectin and has a significant homology to that of human cDNA, DGCR2 and IDD, which were cloned from a balanced translocation breakpoint associated with the DiGeorge syndrome. The isolated cDNA was about 4 kb in length and the message was expressed ubiquitously in various organs with low-abundance. Previously, we also cloned a transmembrane protein which is probably involved in cell-cell interaction by the differential hybridization technique. These findings suggest that transmembrane signaling in neuronal cells may have an important role in PTZ induced seizure. PMID- 8630061 TI - Characterization of monoclonal antibodies to epitopes of human transcobalamin II. AB - Cellular uptake of cobalamin (Cbl) is mediated by transcobalamin II (TCII), a Cbl binding protein in the plasma. The TCII-Cbl complex binds to a cell surface receptor and is internalized by endocytosis. We have generated monoclonal antibodies (mAbs) to human TCII that can be distinguished into three functional types on the basis of interaction with three different regions of the protein. Type 1: Receptor blocking. This mAb binds holo-TCII and inhibits the cellular uptake of Cbl. Type 2: Cbl blocking. This mAb binds apo-TCII at or near the Cbl binding domain and inhibits the formation of holo-TCII. Type 3: Precipitating. This mAb binds both holo-TCII and apo-TCII but does not interfere with Cbl binding. Whereas type 1 and type 2 mAb, following incubation with TCII-[57Co]Cbl or apo-TCII, respectively, inhibit the uptake of radio-labeled Cbl by K562 cells, type 3 mAb has no such activity with either form of TCII. These properties of type 1 and type 2 mAb that inhibit the cellular uptake of Cbl, may serve to induce rapid Cbl deficiency and provide a model to study the effect of selective Cbl depletion on cell division and differentiation as well as on the pathways dependent on the two Cbl cofactors, methyl-Cbl and 5'-deoxyadenosyl-Cbl. PMID- 8630062 TI - Basal regulatory promoter elements of the hsp27 gene in human breast cancer cells. AB - The small human heat shock protein hsp27 has been shown to play important roles in diverse cellular processes such as actin polymerization, thermotolerance, growth, and chemotherapeutic drug resistance. Two breast cancer cell lines MCF-7 and MDA-MB-231 were used as a model to study the molecular mechanisms important for basal hsp27 promoter transcriptional activity. A genomic clone containing 1.1 kb of the hsp27 promoter was sequenced and the regulatory elements were characterized. The first 200 bp within this 5'-flanking region holds the majority of the transcriptional activity, according to transient transfection assays using a series of hsp27 promoter deletion fragments in luciferase reporter vectors. The basal activity of this fragment is largely confined to a G/C-rich region containing overlapping SP1 and AP2 transcription factor binding sites. PMID- 8630063 TI - SFA-2, a novel bZIP transcription factor induced by human T-cell leukemia virus type I, is highly expressed in mature lymphocytes. AB - A novel cellular gene, SFA-2, was isolated by differential hybridization of a cDNA library, using probes obtained from an adult T-cell leukemia cell line in comparison with normal CD4+ T cells and MOLT-4 cell line. The mRNA of the SFA-2 gene is approximately 0.9-kb in size and encodes a protein of 125 amino acids, containing a basic region-leucine zipper DNA-binding domain. The N-terminal region of SFA-2 is rich in serine and contains a consensus sequence for casein kinase II phosphorylation. The SFA-2 gene was strongly expressed in mature T and B lymphocytes, and was up-regulated after transformation by human T-cell leukemia virus type I. The SFA-2 did not homodimerize efficiently but formed heterodimer preferentially with c-Jun. The SFA-2/c-Jun heterodimer bound preferentially to the AP-1 and CRE sites. PMID- 8630064 TI - Absence of imprinting in U2AFBPL, a human homologue of the imprinted mouse gene U2afbp-rs. AB - The mouse gene U2 auxiliary factor binding protein related sequence (U2afbp-rs) has previously been shown to be genomically imprinted with monoallelic expression from the paternal allele. To determine if the human homologue is imprinted and contains conserved structural features which regulate imprinting, we isolated genomic clones from a human P1-derived artificial chromosome (PAC) library that map to human chromosome 5q22-31, a region syntenic to the proximal portion of mouse chromosome 11 where U2afbp-rs resides. A genomic subclone was isolated which contained an open reading frame with high homology to the mouse gene. This subclone also maintained the intronless character of the mouse gene. A KpnI polymorphism within the open reading frame of the gene was found to occur in 21% (8/38) of the alleles tested from human placental tissue samples. RT-PCR analysis of human placentas using the KpnI polymorphism to determine the parental origin of the alleles indicates biallelic expression of the human chromosome 5 U2AFBPL gene. PMID- 8630065 TI - Cloning and expression of human homolog HSMT3 to yeast SMT3 suppressor of MIF2 mutations in a centromere protein gene. AB - A human HSMT3 cDNA encoding a homolog of the yeast SMT3, a suppressor of MIF2 mutations in a centromere protein gene, was identified and sequenced. The sequence of 95 amino acids deduced from the human HSMT3 cDNA exhibited 51.1% identity and 69.6% similarity to the yeast Smt3p sequence. The HSMT3 transcripts of 1.35Kb were found to be abundantly expressed in various human tissues. PMID- 8630066 TI - Modulation of vitellogenin II gene by estradiol and progesterone in the Japanese quail. AB - Estrogen and progesterone receptors are reported to functionally cooperate in gene activation if their cognate binding sites are close to one another in the gene. Our studies show that the expression of the vitellogenin (VTG) gene is induced by estradiol alone or along with progesterone. Progesterone alone inhibits the expression completely. Methylation status of the VTG gene remains unaltered by steroid hormones. Gel mobility shift assay shows that qualitative and quantitative changes occur in the trans-acting factor(s) that bind to estradiol and progesterone responsive elements (ERE and PRE) after administration of these steroid hormones. We, therefore, conclude that the interaction of trans acting factors that bind to ERE and PRE play a role in the regulation of VTG gene expression. PMID- 8630067 TI - PLC gamma 1 Src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts. AB - Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways. PMID- 8630068 TI - Phenotype-linked amino acid alteration in leptin receptor cDNA from Zucker fatty (fa/fa) rat. AB - The mouse obese (ob) gene product (leptin), expressed specifically in adipose cells, regulates energy balance in mice. Both mouse diabetes (db) and rat fatty (fa) gene products are thought to play major roles in leptin signaling pathways in the hypothalamic area. Mutations of these genes in murines result in marked obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. Reported herein are the cloning and sequencing of one of spliced variant forms of rat leptin receptor (OB-R) cDNA with a short intracellular domain. In the Zucker (fa/fa) rat, no changes in either the gene structure or the expression levels were observed. However phenotype-linked nucleotide alteration exists in the cDNA from Zucker (fa/fa) rat, which results in an amino acid substitution. PMID- 8630069 TI - Intestinal preconditioning is mediated by a transient increase in nitric oxide. AB - The effect of ischemic preconditioning on the intestine, as well the implantation of nitric oxide and prostacyclin in this process has been evaluated. Thus, intestinal ischemia-reperfusion was induced in rats, and the protection conferred by previous preconditioning was evaluated. In addition, the effect of nitric oxide inhibition and the administration of nitric oxide were determined. Results show that the increases observed in LDH release after ischemia-reperfusion were prevented after preconditioning. Inhibition of nitric oxide synthesis abolished the protective effect of preconditioning, and nitric oxide administration replicated this effect. Also, an increased synthesis of nitric oxide has been detected after preconditioning. Increases in 6 keto PGF1 alpha were independent of nitric oxide. Altogether indicates that preconditioning is triggered by an initial increase in nitric oxide synthesis. PMID- 8630070 TI - 2,2,2-Trifluoroethanol induces helical conformation in an all beta-sheet protein. AB - The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue II (CTX II), from the Taiwan cobra (Naja naja atra) is studied. Using circular dichroism studies, it is found that higher concentrations of TFE induced a structural transition from beta-sheet to alpha helix, both in the native state (nCTX II) and in denatured but not disulfide reduced CTX II (dCTX II) samples. The beta-sheet to alpha-helix conversion is shown to be cooperative. However, in denatured and reduced CTX II (rCTX II), a TFE transforms a portion(s) of the protein backbone a random coil to an alpha helical conformation. Based on the solution structure of CTX II and the physical property of TFE, a possible mechanism for the observed backbone structural transitions induced by TFE is discussed. The results described in this paper question the significance of the structure of the "molten globule" intermediate(s) obtained in organic solvents such as TFE. PMID- 8630071 TI - A modular approach to HIV-1 proteinase inhibitor design. AB - HIV-1 proteinase represents a promising target for antiviral chemotherapy. We have designed, synthesized, and tested modular inhibitors combining an active site inhibitor tethered to a structure targeted to the dimerization domain of the enzyme. At pH 5 the parent active site inhibitor, the equimolar mixture of active site and dimerization inhibitors, and the best compound from our series of modular inhibitors show the same inhibition activity. At neutral pH, however, the combination of the dimerization and active-site inhibitors shows a synergistic effect. Moreover, the modular inhibitor has an IC50 value 5x lower than the parent active site inhibitor and 2x lower than the equimolar mixture of the two parent inhibitors. The Lineweaver-Burk plot for modular inhibitors corresponds to a pattern for mixed type inhibition. PMID- 8630072 TI - Role of hydrogen peroxide in the formation of DNA adducts in HL-60 cells treated with benzene metabolites. AB - We have investigated the influence of peroxides on DNA adduct formation in HL-60 cells treated with polyphenolic metabolites of benzene. Treatment of HL-60 cells with 50 microM hydroquinone (HQ), 500 microM catechol (CAT) or 200 microM 1,2,4 benzenetriol (BT) resulted in adduct levels of 0.27, 0.21 and 0.21 x 10(-7), respectively. Addition of 50-250 microM H2O2 or 250 microM cumene hydroperoxide to HL-60 cells increased DNA adduct formation 2.7-10-fold following treatment with HQ or CAT but had no effect on adduct formation by BT. Treatment of HL-60 cells with the combinations of HQ plus either BT or phorbol myristate acetate (PMA) potentiated DNA adduct formation by 2.5-4-fold. Significant elevations of cellular H2O2 levels occurred after treatment of HL-60 cells with either PMA, CAT or BT. These results indicate that cellular levels of H2O2 regulate the peroxidase dependent activation of benzene metabolites to form DNA adducts. PMID- 8630073 TI - Hematopoietic cell phosphatase (HCP) regulates p56LCK phosphorylation and ZAP-70 binding to T cell receptor zeta chain. AB - Ligation of the T cell receptor complex and CD4 leads to activation of the protein tyrosine kinases p56lck and p59fyn resulting in phosphorylation of TcR zeta chain and the recruitment of ZAP-70. In this study, we have reconstituted p56lck phosphorylation of TcR zeta and ZAP-70 recruitment in heterologous cells and examined the role of the tyrosine phosphatase HCP in regulating the process. Both p56lck and p59fyn induce significant phosphorylation of TcR zeta. However, under conditions of comparable p56lck and p59fyn expression, p56lck was found to induce three to four fold greater in vivo phosphorylation of TcR zeta. HCP dephosphorylated p56lck, ZAP-70 and the TcR zeta chain. Further, dephosphorylation of the different TcR zeta isoforms results in disruption of the interaction between TcR zeta and ZAP-70. These results indicate that HCP acts to negatively regulate signal transduction pathways in T cells. PMID- 8630074 TI - Generation of active immunotoxins containing recombinant restrictocin. AB - Restrictocin, a toxin produced by the fungus Aspergillus restrictus, is a potent inhibitor of eukaryotic protein synthesis. Recombinant restrictocin was made in Escherichia coli and purified to homogeneity in large amounts. The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally. Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage. The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture. Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage. Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy. PMID- 8630075 TI - Reduced growth capacity of hepatocytes from c-myc and c-myc/TGF-alpha transgenic mice in primary culture. AB - We have previously shown that coexpression of c-myc and TGF-alpha in the liver results in accelerated replicative senescence and promotes tumor development in young adult transgenic mice. Here we describe the characteristics of hepatocyte proliferation in primary cultures established from 10-week-old control, c-myc and c-myc/TGF-alpha transgenic mice. A variety of cellular and functional changes occurred in the transgenic livers at this age including enhanced polypoidization and impairment of hepatic functions. Control mouse hepatocytes demonstrated a high level of DNA synthesis in serum-free medium with a maximum at day three in culture at which time 70% of the cells were in S phase. In contrast, DNA synthesis peaked one day later and was reduced by 50% in the cultured c-myc and c myc/TGF-alpha hepatocytes. Also, higher frequency of apoptosis was observed in the transgenic hepatocytes. However, in hepatocytes isolated from c-myc/TGF-alpha mice, which show early appearance of preneoplastic lesions in vivo, the DNA synthesis continued for 6 days in culture in contrast to a sharp decrease in the labeling index of control and c-myc hepatocytes after 3-4 days in culture. The results suggest that proliferative features of the transgenic hepatocytes in vitro reflect the general properties of these cells in vivo and thus may provide a model for studies on senescence and transformation of hepatocytes. PMID- 8630076 TI - Identification of bovine invariant chain (Ii) gene by nucleotide sequencing. AB - Four overlapping cDNA clones encoding the bovine invariant chain (Ii) were isolated and characterized. The bovine Ii cDNA clone, NI3 with a 1,381-bp insert, encoded a translated product of 204 amino acids. The amino acid sequences deduced from this clone revealed that the bovine Ii gene is more closely related to human Ii gene than to genes for rodents, such as mouse and rat, but lacked 10 amino acids of the 3' end of the extracellular domain comparable to those in proteins from other species. Of interest is that the class-II-associated invariant chain peptide (CLIP) segment is present in the protein encoded by NI3, and this region exhibits a high degree of overall similarity to sequences encoded by human, mouse and rat, suggesting that bovine CLIP may have the biological function of CLIP binding to MHC class II molecules. Thus, it seems likely that this cDNA clone encodes a functional product which might perform an important function in MHC class II antigen presentation, as previously established in studies in mouse and man. PMID- 8630077 TI - Augmentation of apoptosis in bronchial exuded rat eosinophils by cyclosporin A. AB - The effect of cyclosporin A on apoptosis in eosinophils was examined to clarify the inhibitory mechanisms of cyclosporin A on allergen-induced eosinophilia in the airway. Eosinophils in bronchoalveolar lavage fluid from sensitized rats after inhaling an allergen were used. More than 50% of the eosinophils that died spontaneously by apoptosis within 24 hour incubation in RPMI 1640 medium contained 10% fetal calf serum. The addition of cyclosporin A or dexamethasone significantly enhanced the eosinophils apoptosis at concentrations of more than 0.1 microM. Apoptosis in eosinophils was considerably suppressed in the presence of culture supernatant of activated splenocytes as a source of various cytokines. Even in the presence of culture supernatant of activated splenocytes, cyclosporin A or dexamethasone facilitated apoptosis in eosinophils. These results suggest that apoptotic death of activated eosinophils is augmented with cyclosporin A and that accelerated apoptosis in eosinophils of the airway may account for the inhibitory effect of cyclosporin A on eosinophilia. PMID- 8630078 TI - Alzheimer's disease amyloid beta peptide 25-35 is localized in the membrane hydrocarbon core: x-ray diffraction analysis. AB - Alzheimer's disease (AD) neuropathology is characterized by neuritic plaques composed primarily of amyloid beta peptide (A beta). An elevation in A beta in the cerebral cortex has been implicated in the pathophysiology of AD but its mechanism of action is unknown. The addition of A beta protein to neuronal cell cultures produces changes in the activity of various membrane proteins, including ion channels and receptors, potentially as a result of intercalating into the membrane bilayer. In this study, the interactions of the A beta fragment 25-35 [A beta(25-35)] with liposomes were directly examined by small angle x-ray diffraction approaches. One-dimensional electron density profiles generated from the diffraction data demonstrated that the addition of A beta(25-35) produced a discrete increase in electron density 0-12 A from the center of the lipid bilayer. Under these conditions, the membrane concentration of A beta(25-35) was 860-fold higher than in the aqueous buffer. These findings indicate that this peptide is highly lipophilic and inserts into the membrane hydrocarbon core. Following the intercalation of A beta(25-35) to this location in the membrane, the protein fragment may interact with regulatory membrane proteins. PMID- 8630079 TI - Monoepoxide production from linoleic acid by cytochrome c in the presence of cardiolipin. AB - We found that cytochrome c (Cyt c) could oxidize cardiolipin (CL), and detected monoepoxides of linoleic acid (LA) in the fatty acids constituting the oxidized CL. We also found that in the presence of CL and Cyt c, free LA was oxidized and LA monoepoxides were produced. The aim of this study was to elucidate the mechanism of this lipid peroxidation. We concluded that ferric Cyt c produced some radical species from water-soluble oxygen in the presence of CL (CL-Cyt c system) and that radicals oxidized free LA or CL. The CL-Cyt c system may be another LA monoepoxide producing system in the neutrophil and may account for the lipid peroxidation observed in the ischemia-reperfusion-induced cardiac injury. PMID- 8630080 TI - Both reductive forms of 17 beta-hydroxysteroid dehydrogenase (types 1 and 3) are expressed during development in the mouse testis. AB - Androstenedione is reduced to form testosterone by 17-beta-hydroxysteroid dehydrogenase (17 beta HSD) and two different reductive isoforms of the enzyme have been identified (types 1 and 3). In this study, levels of mRNA encoding both reductive isoforms have been measured during fetal and post-natal development in the mouse. In fetal and neonatal testes mRNA encoding both type 1 and type 3 isoforms was present at relatively high levels reaching a peak at postnatal day 5. Thereafter, mRNA levels of both 17 beta HSD isoforms fell to low levels until day 30 when there was a marked increase in the levels of the type 3 isoform. The presence of the type 1 17 beta HSD enzyme in fetal testes may explain the virilization of the mesonephric (Wolffian) duct which occurs in pseudohermaphrodite individuals lacking the type 3 isoform. PMID- 8630082 TI - Regulation of ionotropic receptors by protein phosphorylation. AB - The regulation of synaptic signal transduction is of central importance to our understanding of normal and abnormal nervous system function. One mechanism by which signal transduction can be affected is the modification of cellular sensitivity by alterations of transmembrane receptor properties. For G-protein coupled receptors, protein phosphorylation is intimately involved in many stages of receptor regulation. This appears to be true for ionotropic receptors as well. Evidence of a role for protein kinase and protein phosphatase activity in the multi-staged ionotropic receptor regulation cascade is presented and a comparison to G-protein coupled receptor regulation is considered. PMID- 8630081 TI - Differential effects of overexpression of PKC alpha and PKC delta/epsilon on cellular E2F activity in late G1 phase. AB - Introduction of a reporter gene containing E2F binding sites linked to the luciferase gene permitted us to detect transient cellular E2F activity in late G1 phase rat 3Y1 fibroblasts. Overexpression of three major protein kinase C (PKC) isozymes expressed in 3Y1 cells caused differing effects on E2F activity depending on the isozymes overexpressed. Overexpression of PKC alpha inhibited E2F activity while the overexpression of PKC delta or PKC epsilon enhanced it, suggesting that these PKC isozymes play different roles in the regulation of E2F activity. Consistent with previous findings that the activation of PKC by TPA in late G1 phase results in the inhibition of DNA synthesis (Huang, C., and Ives, H.E., 1987, Nature 329, 849-850), the addition of TPA in late G1 phase specifically inhibited E2F activity. Overexpression of PKC isozymes resulted in an enhancement of the TPA-induced inhibition of E2F in late G1 phase. This enhancement was observed for all three PKC isozymes examined, suggesting that these PKC isozymes all are potent mediators of the TPA-induced inhibition of E2F activity in late G1 phase. PMID- 8630083 TI - P-glycoprotein induction in rat liver epithelial cells in response to acute 3 methylcholanthrene treatment. AB - Expression of P-glycoprotein (P-gp), a plasma membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in nonparenchymal rat liver epithelial (RLE) cells in response to acute exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). High levels of mdr mRNAs were evidenced by Northern blotting in two independent RLE cell lines after treatment by either 3-methylcholanthrene (MC) or benzo-(a)pyrene. MC-mediated mdr mRNA induction was demonstrated to be dose-dependent; it occurred through enhanced expression of the mdr 1 gene, as indicated by reverse transcriptase-polymerase chain reaction analysis using rat mdr gene-specific primers and paralleled an induction of a 140 kDa P-gp as demonstrated by Western blotting. In addition, MC-induced P-gp appeared to be fully functional because RLE cells exposed to MC displayed enhanced cellular efflux of rhodamine 123, a known P-gp substrate, compared to their untreated counterparts. Analysis of time-course induction revealed that mdr mRNA levels were maximally increased when RLE cells were treated for 48 to 96 hr and returned to low levels after the PAH was removed. In contrast to P-gp, both cytochrome P-450 1A1 and cytochrome P-450 1A2 were not detected after exposure to MC, thus indicating that these liver detoxification pathways are not coordinately regulated with P-gp in RLE cells. In addition, MC-mediated P-gp regulation was not associated with major cellular disturbances such as alteration of protein synthesis and, thereby, differed from the known mdr mRNA induction occurring in response to cycloheximide. Moreover, cotreatment with MC and cycloheximide led to a superinduction of mdr mRNAs, thus suggesting that the effects of the two xenobiotics were, at least partly, additive. In contrast to MC and benzo(a)pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(e)pyrene were unable to increase P-gp expression. These results indicate that some PAHs can act as potent inducers of P-gp in RLE cells and may be interpreted as an adaptive reaction of these cells in lowering cellular accumulation of toxic drugs, including carcinogens transported by P-gp and, therefore, conferring protection on these compounds. PMID- 8630084 TI - Characterization of peripheral benzodiazepine receptors in purified large mammal pancreatic islets. AB - In this work, we evaluated the biochemical properties of peripheral benzodiazepine receptors (PBRs) in the porcine endocrine pancreas and their role in insulin release. Binding of [3H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3 isoquinolinecarboxa mide ([3H]PK-11195), a specific ligand of PBRs, to islet membranes was saturable and Scatchard's analysis of saturation curve demonstrated the presence of a single population of binding sites, with a dissociation constant (Kd) value of 4.75 +/- 0.70 nM and a maximum amount of specifically bound ligand (Bmax) of 4505 +/- 502 fmol/mg of proteins. The pharmacological profile of PBRs was determined as the ability of PK-11195 and several benzodiazepine compounds to displace [3H]PK-11195 from these binding sites. The rank order of potency yielded the following affinity results: PK-11195 > 7-chloro 1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2 -on (Ro 5-4864) > diazepam > or = flunitrazepam >> flumazenil. Secretion studies demonstrated that PK-11195 (1 and 10 microM) and Ro 5-4864 (10 and 50 microM) significantly potentiated insulin secretion from freshly isolated porcine islets at 3.3 mM glucose. This potentiating effect was not observed at 16.7 mM glucose concentration nor by the addition of clonazepam. These results show the presence of PBRs in purified porcine pancreatic islets and suggest an implication of PBRs in the mechanisms of insulin release. PMID- 8630085 TI - Ebselen inhibition of apoptosis by reduction of peroxides. AB - We investigated the capacity of ebselen [2-phenyl-1,2-benzisoselenazol-3(2H) one], a glutathione peroxidase mimic, to protect cells from radiation-induced apoptosis. Incubating mouse thymocytes with 25 microM ebselen immediately after 60Co gamma-radiation exposure (5 Gy) inhibited morphological changes associated with apoptosis. Treatment of thymocytes with ebselen before, during, or after irradiation completely blocked internucleosomal DNA fragmentation, a biochemical marker for apoptosis. We measured peroxides formed in cells during and after irradiation, using the oxidation-sensitive fluorescent probe 2',7' dichlorofluorescin diacetate. By 2 min postirradiation, levels of peroxides in irradiated thymocytes were approximately 10-11 times greater than those in the same cells before irradiation, and levels continued to increase with time. We also measured membrane lipid peroxidation using cis-parinaric acid, a naturally fluorescent polyunsaturated fatty acid that readily incorporates into cell membranes. The oxidation of cis-parinaric acid also began soon after irradiation and increased with time. Peroxide generation and membrane lipid peroxidation preceded both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. Treatment of cells with ebselen reduced peroxide levels and appeared to protect thymocytes from radiation-induced apoptosis by scavenging peroxides generated during and after irradiation. The results suggest that peroxide generation and membrane lipid peroxidation may be important signaling events that trigger apoptosis in irradiated cells. PMID- 8630086 TI - Hydrolysis of P2-purinoceptor agonists by a purified ectonucleotidase from the bovine aorta, the ATP-diphosphohydrolase. AB - Pharmacologists are becoming more and more aware of the possibility that certain ATP analogues currently used to classify the P2-purinoceptors are dephosphorylated by ectonucleotidases. In this study, we provide evidence that in the vascular system, these purine analogues are hydrolysed by an ATP diphosphohydrolase (ATPDase). This enzyme is known as the major plasma membrane nucleotidase of endothelial and smooth muscle cells, and is believed to dephosphorylate extracellular triphospho- and diphosphonucleosides. Assays were conducted with a purified ATPDase from smooth muscle cells of bovine aorta. At a concentration of 250 microM, adenosine 5'-(alpha,beta-methylene) triphosphonate (alpha,beta-metATP), adenosine 5'-(beta,gamma-methylene) triphosphonate (beta,gamma-metATP), adenosine 5'-(alpha,beta-methylene) disphosphonate (alpha,beta-metADP), adenylyl 5'-(beta,gamma-imido) diphosphonate (beta,gamma imidoATP) and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) all resisted dephosphorylation, whereas 2-chloroadenosine triphosphate (2-chloroATP), 2 methylthioadenosine triphosphate (2-MeSATP) and 8-bromoadenosine triphosphate (8 bromo-ATP) were hydrolysed at 99, 63, and 20% of the rate of ATP hydrolysis, respectively. All the non-hydrolysable analogues tested, except alpha,beta metADP, competed with ATP and ADP for the ATPDase catalytic site, reducing their hydrolysis by 35-50%. Apparent Km values for ATP and ADP were estimated at 14.1 and 12.0 microM, respectively, whereas apparent Km and Ki values for the purine analogues ranged from 12 to 28 microM. These results strongly support the view that (1) the ATPDase is expected to reduce substantially the P2-response induced by ATP, ADP, and some hydrolysable agonists; and (2) by competing with the hydrolysis of endogenously released ATP and ADP, non-hydrolysable analogues could alter the amplitude or direction of the cellular response induced by these natural substrates. PMID- 8630087 TI - Structurally modified trimethine thiacarbocyanine dyes. Effect of N-alkyl substituents on antineoplastic behavior. AB - The effect of dye localization and dye distribution on the antineoplastic behavior of photosensitizers was investigated with a homologous series of trimethine thiacarbocyanine dyes in L1210 leukemia and A549 lung carcinoma cells. These dyes were synthesized with N-alkyl groups of different sizes (ethyl to octadecyl) to vary their lipophilic properties without compromising their photophysics. While dyes with smaller N-alkyl groups (ethyl to decyl) were already cytotoxic in the dark, longer chain cyanines exhibited antineoplastic activity only after exposure to light. Results from this study indicate that the switch from dark cytotoxicity to phototoxicity occurred when dyes, due to a decrease in cationic character with increasing size of alkyl substituents, were no longer able to cross the plasma membrane. Dark cytotoxicity decreased with increasing size of N-alkyl groups and was cell-line independent. On the other hand, photodynamic damage varied by several orders of magnitude depending on the cell line and the length of the alkyl substituents. The most effective photosensitizer was the dioctadecyl dye which achieved a 4- to 5-log reduction of leukemia cells, although it had very modest triplet and singlet oxygen quantum yields of 0.008 and 0.006, respectively. This study also showed that photobiological performance can be improved greatly by optimizing dye binding properties via structural modifications. PMID- 8630088 TI - Further characterization of rat brain flavin-containing monooxygenase. Metabolism of imipramine to its N-oxide. AB - Flavin-containing monooxygenase (FMO) activity was compared in rat liver and brain microsomes by estimating the actual amount of imipramine N-oxide relative to the corresponding activity, measured using substrate-stimulated rates of NADPH oxidation. The activities measured as NADPH oxidation rates were significantly higher than those estimated from the N-oxide formed. The brain FMO activity was detectable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The antibody to rabbit pulmonary FMO selectively inhibited imipramine N-oxidation. The antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect on imipramine N oxidation, indicating the noninvolvement of cytochrome P-450 in the above metabolic pathway. A flavin-containing monooxygenase was partially purified from the rat brain microsomes using sequential chromatography on n-octylamino Sepharose 4B, DEAE-Sephacel and 2',5'-ADP agarose. The purified FMO was resolved by SDS-PAGE into two bands (approximately 57 and 61 KDa, respectively) both of which cross-reacted with antibody to rabbit pulmonary FMO. The purified enzyme metabolized imipramine and the model substrate methimazole to their respective N oxide and S-oxides. PMID- 8630089 TI - Inhibition of squalene synthase of rat liver by novel 3' substituted quinuclidines. AB - Squalene synthase (SQS) is a key enzyme in the biosynthetic pathway for cholesterol and is a target for improved agents to lower plasma levels of low density lipoprotein (LDL). A series of novel 3' substituted quinuclidines have been discovered as inhibitors of the rat liver microsomal enzyme. In this study, we demonstrate the inhibitory effects in vitro and in vivo, of two examples of the series. When microsomes were preincubated with compounds, before addition of substrate, both 3-(biphenyl-4-yl)quinuclidine (BPQ) and 3-(biphenyl-4-yl)-3 hydroxyquinuclidine (BPQ-OH) were found to cause biphasic inhibition of the enzyme with apparent inhibition constants (K'i) for the sensitive phases of 12 nM and 15 nM, respectively. The K'i values for the insensitive phases were 1.8 microM and 2.9 microM, respectively. The two examples inhibited equally both steps of the SQS-catalysed reaction, as shown by parallel inhibition of 3H+ release and labelled squalene formation from [1-3H]farnesyl pyrophosphate (FPP). BPQ and BPQ-OH were shown to be inhibitors of hepatic sterol synthesis from mevalonate with ED50 values of 10.6 and 7.1 mg/kg, respectively, after acute oral administration to the rat. BPQ-OH was chosen for further study and, to determine its selectivity of effect on the mevalonate pathway in vivo, the effect of a dose of 70 mg/kg on the pattern of labelled mevalonate incorporation into the various lipid fractions of the rat liver was examined. As expected, the incorporation into squalene and sterol products was inhibited by about 70%. An appearance of label in fractions corresponding to farnesyl and geranylgeranylpyrophosphates, as well as the corresponding alcohols, was observed in treated but not control animals. In addition, the administration of compound resulted in the appearance of peaks of mevalonate-derived radioactivity in an acidic fraction believed to represent metabolites of farnesol. Such results are consistent with inhibition of the mevalonate pathway at, and not before, SQS. In contrast, there was a significant increase in the incorporation of labelled mevalonate into ubiquinone 10, and the synthesis of dolichols was apparently unchanged. The results suggest a specific effect of BPQ-OH on rat liver SQS. The compound is, therefore, an interesting lead for further investigation of this class of compounds. PMID- 8630090 TI - Inhibition of squalene synthase in vitro by 3-(biphenyl-4-yl)-quinuclidine. AB - Squalene synthase (SQS) catalyses a step following the final branch in the pathway of cholesterol biosynthesis. Inhibition of this enzyme, therefore, is an approach for the treatment of atherosclerosis with the potential for low side effects. We have characterised the inhibition of rat liver microsomal SQS by 3 (biphenyl-4-yl)quinuclidine (BPQ). BPQ follows slow binding kinetics in that the rate of accumulation of product decreases with time if the inhibitor is added when the assay is started. Preincubation of BPQ and SQS leads to a biphasic dose response where accumulation of product is linear with time only for the sensitive phase. When the farnesyl pyrophosphate (FPP) substrate is present at 19.6 microM, approximately 77% of the SQS activity is sensitive to the inhibitor (vOs) and the remainder is insensitive (vOi). The apparent inhibition constants (K'i values) are respectively K'is = 4.5 nM and K'ii = 1300 nM. Similar biphasic behaviour is exhibited by other inhibitors and in microsomes prepared from human and marmoset liver. As the concentration of FPP is reduced below 19.6 microM, there is a decrease in the relative contribution from vOi. Conversely, the value of K'is for BPQ remains constant when the FPP concentration is changed, showing noncompetitive kinetics with respect to this substrate. Possible causes of the observed kinetics are discussed. Inhibition by BPQ is said to follow tight binding kinetics because the value of K'is is similar to the concentration of inhibitor binding sites. Thus, to avoid an artefactual variation in potency when the enzyme concentration is varied, it is necessary to allow for the effects of depletion of free inhibitor. Furthermore, estimates of potency that average activity across the two phases are influenced by the relative contributions of each phase. These contributions differ according to the FPP concentration and the species used as the source of microsomes. Thus, it is necessary to separate the phases to compare measurements made in different experiments. Our observations indicate that careful experimental design and data analysis are required to characterise the kinetics of SQS inhibitors. PMID- 8630091 TI - Effects of isoquinoline derivatives structurally related to 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) on mitochondrial respiration. AB - Isoquinoline derivatives exert 1-methyl-4-phenylpyridinium (MPP+)-like activity as inhibitors of complex I and alpha-ketoglutarate dehydrogenase activity in rat brain mitochondrial fragments. We now examine the ability of 19 isoquinoline derivatives and MPP+ to accumulate and inhibit respiration in intact rat liver mitochondria, assessed using polarographic techniques. None of the compounds examined inhibited respiration supported by either succinate + rotenone or tetramethylparaphenylenediamine (TMPD) + ascorbate. However, with glutamate + malate as substrates, 15 isoquinoline derivatives and MPP+ inhibited state 3 and, to a lesser extent, state 4 respiration in a time-dependent manner. None of the isoquinoline derivatives were more potent than MPP+. 6,7-Dimethoxy-1-styryl-3,4 dihydroisoquinoline uncoupled mitochondrial respiration. Qualitative structure activity relationship studies revealed that isoquinolinium cations were more active than isoquinolines in inhibiting mitochondrial respiration; these, in turn, were more active than dihydroisoquinolines and 1,2,3,4 tetrahydroisoquinolines. Three-dimensional quantitative structure-activity relationship studies using Comparative Molecular Field Analysis showed that the inhibitory potency of isoquinoline derivatives was determined by steric, rather than electrostatic, properties of the compounds. A hypothetical binding site was identified that may be related to a rate-limiting transport process, rather than to enzyme inhibition. In conclusion, isoquinoline derivatives are less potent in inhibiting respiration in intact mitochondria than impairing complex I activity in mitochondrial fragments. This suggests that isoquinoline derivatives are not accumulated by mitochondria as avidly as MPP+. The activity of charged and neutral isoquinoline derivatives implicates both active and passive processes by which these compounds enter mitochondria, although the quaternary nitrogen moiety of the isoquinolinium cations favours mitochondrial accumulation and inhibition of respiration. These findings suggest that isoquinoline derivatives may exert mitochondrial toxicity in vivo similar to that of MPTP/MPP+. PMID- 8630092 TI - Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells. AB - We previously reported that a hydroquinone-type antioxidant, 2,5-di(tert-butyl) 1,4-hydroquinone (DTBHQ), increases intracellular free Ca2+ concentration ([Ca2+]i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five-hydroquinone-type and phenolic antioxidants (2,5-di(tert-amyl) 1,4-hydroquinone [DTAHQ], 2-tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di(tert butyl)-4-hydroxytoluene [BHT], 3,5-di(tert-butyl)-4-hydroxyanisole [DTBHA], and 3 tert-butyl-4-hydroxyanisole [MTBHA]) on [ca2+]i and degranulation (beta hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (> or = 3 microM) showed effects similar to those of DTBHQ (10 microM) on [Ca2+]i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 microM) and DTBHA (50 microM) caused [Ca2+]i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca2+]i and degranulation, even at 50 microM. The degree of Ca2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca2+]i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca2+-ATPase. PMID- 8630093 TI - Redox cycling activity of monoamine-serotonin binding protein conjugates. AB - It has been shown recently that the covalent binding of labelled dopamine and serotonin to serotonin binding proteins (SBP) from bovine frontal cortex is potently inhibited by their related neurotoxins. The present study reveals that the monoamine-SBP conjugates of serotonin, dopamine, and related toxins are able to catalyse redox cycling reactions. Using an improved method to detect quinoproteins in SDS-PAGE gels, we were also able to demonstrate that the redox cycling activity corresponded to two major protein components with molecular weights of 45 and 56 kDa. The covalent monoamine-SBP conjugates may be referred to as "artificial quinoproteins." PMID- 8630094 TI - Biochemical evaluations of the effects of loreclezole and propofol on the GABAA receptor in rat brain. AB - The effects of loreclezole on the function of the gamma-aminobutyric acid type A (GABAA) receptor complex in rat cerebral cortical membrane preparations were compared with those of propofol and diazepam. Loreclezole and propofol modulated [3H]muscimol binding and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to washed and unwashed membranes with potencies and efficacies greater than those of diazepam. Loreclezole and propofol enhanced [3H]flunitrazepam binding to washed membranes with efficacies lower than those of GABA and muscimol. Both loreclezole and propofol showed biphasic effects on [35S]TBPS binding to washed membranes: at low concentrations (5 to 10 microM), both drugs, with different efficacies, enhanced [35S]TBPS binding whereas, at higher concentrations (30 to 100 microM), they inhibited this biochemical parameter. In contrast, diazepam enhanced [35S]TBPS binding to washed membranes at all concentrations tested. The combination of loreclezole with GABA, at a concentration (0.3 microM) that only slightly increased [35S]TBPS binding to washed membranes, reversed the increase in binding elicited by loreclezole (5 to 10 microM) and significantly potentiated the inhibitory effect exerted by higher concentrations (30 to 100 microM) of this drug. Similar effects were observed with the combination of GABA and propofol. However, GABA had no effect on the enhancement of [35S]TBPS binding induced by diazepam. The ability of GABA to reverse and potentiate the effects of loreclezole and propofol on [35S]TBPS binding to washed membranes was shared by pentobarbital (200 microM) and alphaxalone (3 microM). These anesthetics showed greater efficacies in combination with pentobarbital (200 microM) and alphaxalone (3 microM). These anesthetics showed greater efficacies in combination with propofol than with loreclezole. These results suggest that, unlike diazepam, loreclezole and propofol may activate the receptor-associated Cl- channel in the absence of GABA. Furthermore, the difference in the pharmacological profiles of loreclezole and propofol may result from their different effectiveness in activating the receptor Cl- channel directly. PMID- 8630095 TI - Regulation of epidermal growth factor receptor activity by crotoxin, a snake venom phospholipase A2 toxin. A novel growth inhibitory mechanism. AB - Crotoxin (CT), a phospholipase A2 (PLA2) derived from the venom of Crotalus durissus terrificus, is a heterodimeric protein composed of subunit B with enzymatic activity and a binding regulatory subunit (A) without enzyme activity. Although the PLA2 activity of CT may be important in its anti-proliferative activity, its cytostatic mechanism is unknown. In this study, we examined the cytostatic effect of PLA2-associated CT activity on squamous carcinoma cells expressing distinct levels of epidermal growth factor receptor (EGFr). CT was most effective in suppressing growth on cells expressing high intrinsic levels of EGFr. Cardiotoxin, another membrane active toxin with no intrinsic PLA2 activity, had no differential anti-proliferative activity on cells expressing high EGFr levels, suggesting a correlation between EGFr expression and CT-directed anti proliferative activity. Both chemically modified CT (MCT) devoid of PLA2 activity and covalently cross-linked CT (CCT), which is functionally unable to utilize cellular membranes as PLA2 substrate, were also without growth inhibitory activity. No evidence for direct binding of CT to EGFr was found, although pretreatment with EGF was able to partially suppress the anti-proliferative activity of CT. Tyrosine phosphorylation of EGFr, however, was stimulated by CT in intact A431 cells. Tyrosine phosphorylation of EGFr was concentration dependently stimulated (3- to 8-fold) in cellular membranes of A431 cells treated in vitro with CT but not with anti-proliferatively inactive MCT or CCT. The data provide evidence for transmembrane receptors involved in growth signaling (namely EGFr) as cellular targets and potential effectors of PLA2-mediated anti proliferative activity of snake venom. PMID- 8630096 TI - Induction of differentiation and down-regulation of c-myb gene expression in ML-1 human myeloblastic leukemia cells by the clinically effective anti-leukemia agent meisoindigo. AB - Meisoindigo, a second generation derivative of indirubin, is an effective chemotherapeutic agent with very low toxicity used in the treatment of chronic myeloid leukemia. To determine the nature of this activity, the effect of a nontoxic concentration (0.72 micrograms/mL) of this compound on ML-1 human myeloblastic leukemic cells was examined. At such a concentration, differentiation induction was found to be the most pronounced drug effect. During the 3-day drug incubation period, the viable cell number remained essentially constant, with approximately 48% of the cells demonstrating a mature phenotype with increased acid phosphatase activity and nitroblue tetrazolium dye reduction. As observed with other DNA-specific agents, induction of ML-1 differentiation by meisoindigo was accompanied by the down-regulation of c-myb gene expression. These data suggest that induction of leukemic cell differentiation associated with decreased c-myb expression may be one of the mechanisms of the antitumor action of meisoindigo. PMID- 8630097 TI - Cell cycle-dependent inhibition of the proliferation of human neural tumor cell lines by iron chelators. AB - The current studies were designed to examine the conditions under which the ferric iron chelator desferrioxamine (DFO) arrested cell cycle progression and hence the proliferation of neural cell lines in vitro. DFO arrested proliferation at different stages of the cell cycle depending on the concentration and duration of drug exposure. Twenty-four-hour treatment with 160 microM DFO arrested glioma cells in G1, whereas 72-hr treatment with 10 microM DFO acted to slow the passage of glioma cells through the cell cycle, eventually accumulating in G2/M. Another iron chelator, ADR 529, also inhibited the proliferation of glioma cells by lengthening the period of the cycle and causing the cells to arrest in G2/M. The effects of 10 and 160 microM DFO were irreversible after 24 and 48 hr, respectively, and 10 microM DFO became cytotoxic after 3 days. These observations demonstrate that DFO has different effects on the proliferation of neural tumor cell lines depending on the concentration and time of exposure, which result in different sites of cell cycle arrest. These different in vitro actions of DFO may have ramifications for the successful application of iron chelator therapy in vivo. PMID- 8630098 TI - Kinetic analysis of the interaction of cidofovir diphosphate with human cytomegalovirus DNA polymerase. AB - Cidofovir [CDV,(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic cytosine nucleoside phosphonate analog with potent in vitro and in vivo activity against a broad spectrum of herpesviruses. CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is a competitive inhibitor of dCTP and an alternate substrate for human cytomegalovirus (HCMV) DNA polymerase. HCMV DNA polymerase used a synthetic DNA primer-template with a Km value of 90 +/- 8 nM and incorporated dCTP approximately 42 times more efficiently than CDVpp. HCMV DNA polymerase also utilized a synthetic DNA primer containing a single molecule of CDV at the 3'-terminus. The Km value for this DNA primer-template was 165 +/- 42 nM and incorporation of dCTP was approximately 17 times more efficient than that of CDVpp. The slower rate of incorporation of CDVpp was due mostly to the higher Km value of CDVpp toward the enzyme-primer-template complexes. These data demonstrate that incorporation of a single CDV into DNA by HCMV DNA polymerase does not lead to chain termination. PMID- 8630099 TI - Iron coordination by catechol derivative antioxidants. AB - Iron complexes of nitrocatechols with different substituent groups [1: -CH = CR2; 2: -CH2-CHR2; 3: -CH = CR'(R")] were synthesized and their effects on iron induced free radical reactions of biological importance investigated. Catechol and nitrocatechol derivatives effectively inhibited iron-induced lipid peroxide dependent lipid peroxidation. In the Fenton-like reaction, iron-catechol generated hydroxyl radicals more strongly than did iron citrate, and iron nitrocatechol derivative 2 generated a small amount of hydroxyl radicals. The iron complexes of derivatives 1 and 3 did not generate hydroxyl radicals. Iron catechol had the highest ratio of reduction to oxidation rate constants and the second was iron-nitrocatechol 2, suggesting that iron chelated by nitrocatechols 1 and 3 may be most difficult to reduce. To elucidate the structure and physical properties of the iron complexes, UV/vis absorption spectroscopic, ESR and 1H NMR studies were performed in aqueous and DMSO solutions. In aqueous solution at pH 7.4, iron complexes of the nitrocatechol derivatives were high-spin tris(nitrocatecholato)ferrate(III) with a characteristic ligand-to-metal charge transfer absorbance (pi -> d pi). The lambda max of iron-nitrocatechol derivative 2 was shorter than those of iron-nitrocatechol derivatives 1 and 3, suggesting that the reduction potential of iron-nitrocatechol 2 is higher than that of iron nitrocatechols 1 and 3. Nitrocatechol derivatives with a conjugation structure can sequester the chelated iron more effectively than catechol and the derivative without the conjugation against free radical generation by keeping the iron in the ferric state, probably because of the reduction potentials. PMID- 8630100 TI - In vivo induction of cytochrome P450 CYP3A expression in rat leukocytes using various inducers. AB - Although the induction of cytochromes P450 3A (CYP3A) is relatively well characterized in liver, its inducibility in an easily available tissue such as the peripheral leukocytes is not known. The purpose of this study was, therefore, to determine if CYP3A is inducible in vivo in peripheral leukocytes. Microsomes from rat leukocytes and liver were examined for CYP3A protein expression using Western blotting with a rabbit polyclonal antibody against rat CYP3A. Although CYP3A was not detected in control leukocytes, in vivo treatment with known CYP3A inducers (dexamethasone, clotrimazole, phenobarbital, pregnenolone-16 alpha carbonitrile) resulted in CYP3A leukocyte levels of 0.2-0.8 pmol/mg protein. This leukocyte induction was approximately 1000-fold lower than in induced liver. Interestingly, there was an apparent linear relationship between leukocyte and liver CYP3A contents (r2 = 0.748, n = 29). These results not only demonstrate for the first time that CYP3A is inducible in rat leukocytes after in vivo treatment with various CYP3A inducers, but also suggest that peripheral leukocytes could be used to assess induction in vivo. PMID- 8630101 TI - Enhancement of SPARC (osteonectin) synthesis in arthritic cartilage. Increased levels in synovial fluids from patients with rheumatoid arthritis and regulation by growth factors and cytokines in chondrocyte cultures. AB - OBJECTIVE: To investigate the roles of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) in arthritis, using cartilage and synovium specimens and synovial fluids (SF) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), and to examine the effects of cytokines, growth factors, and hormones on SPARC synthesis by chondrocytes in culture. METHODS: SPARC in cartilage and synovium was immunostained with monoclonal antibodies. SPARC synthesis by cultured chondrocytes was measured by Northern blot analysis, immunoblotting, and sandwich enzyme-linked immunosorbent assay. RESULTS: SPARC was identified in numerous chondrocytes in the superficial and middle zones and in regenerating chondrocytes of RA and OA joints, whereas such staining was absent in these zones of normal cartilage, except for weak signals from a few chondrocytes in the deep zone. In addition, SPARC synthesis was enhanced in synovial cells of RA and OA joints. The average SPARC level in SF was 10-fold higher in the RA than in the OA population. In rabbit articular chondrocyte cultures, administration of transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 increased SPARC levels at 24-48 hours, whereas interleukin-lbeta (IL-1 beta), IL-1 alpha, tumor necrosis factor alpha, lipopolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone decreased SPARC levels at 24-72 hours. TGF beta increased SPARC messenger RNA (mRNA) levels at 24 hours, whereas IL-1 beta caused a marked decrease in SPARC mRNA levels at 24 hours. Furthermore, IL-1 decreased the glycosylation of SPARC. CONCLUSION: These findings suggest that various growth factors and cytokines, including TGF beta 1 and IL-1 beta, regulate the production of SPARC by chondrocytes at pre- and posttranslational levels, and that SPARC synthesis is markedly enhanced in arthritic joints. PMID- 8630102 TI - TSG-6 expression in human articular chondrocytes. Possible implications in joint inflammation and cartilage degradation. AB - OBJECTIVE: The hyaluronan-binding protein TSG-6 (tumor necrosis factor-stimulated gene 6) forms a stable complex with the serine protease inhibitor, inter-alpha inhibitor, potentiates the inhibition of plasmin activity, and has antiinflammatory effects in vivo. This study examines the expression of TSG-6 in human articular chondrocytes and cartilage. METHODS: Human articular chondrocytes and cartilage explants were stimulated with cytokines, growth factors, and other agents. TSG-6 expression was analyzed by imaging-assisted Northern and Western blotting. RESULT: TSG-6 messenger RNA (mRNA) expression was upregulated by cytokines and growth factors, predominantly interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), platelet-derived growth factor AA (PDGF-AA), and transforming growth factor beta 1 (TGF beta 1). TSG-6 mRNA induction by TGF beta 1 was delayed as compared with IL-1beta. Treatment of the cells with the glucocorticoid dexamethasone neither induced TSG-6 mRNA nor did it affect IL-1 beta-induced transcript levels. TSG-6 mRNA induction may involve several signal transduction pathways. The strong transcriptional stimulation by phorbol myristate acetate suggests protein kinase C (PKC)-mediated signaling. In contrast, PKA- and Ca- dependent signals are only marginally involved as messengers leading to increased TSG-6 levels after IL-1beta and TNF alpha treatment. In chondrocyte and cartilage organ cultures, both free TSG-6 (35 kd) and the complex with inter-alpha-inhibitor (120 kd) were present and upregulated by IL-1 beta, TNF alpha, or TGF beta 1. CONCLUSION: Chondrocytes are a source of TSG-6 which may play a role in cartilage remodeling and joint inflammation. PMID- 8630104 TI - Fibronectin synthesis in superficial and deep layers of normal articular cartilage. AB - OBJECTIVE: To study the distribution and synthesis of fibronectin (FN) in superficial and deep layers of normal articular cartilage. METHODS: Superficial and deep bovine and human articular cartilage slices were used to extract and quantitate FN by radioimmunoassay. Chondrocytes were also isolated by collagenase digestion for FN extraction and culture. Superficial and deep cartilage explants were cultured with and without stimulation by cytokines. Quantitation of newly synthesized FN was carried out by incubation with 35S-methionine. FN was purified on gelatin-agarose columns and further characterized by polyacrylamide gel electrophoresis. FN messenger RNA (mRNA) was quantitated by Northern blot analysis. RESULTS: Freshly isolated bovine chondrocytes from deep cartilage contained 2.3 +/- 0.2 times more FN than was found in superficial cells (P < 0.025). Deep cartilage explants contained 1.2 times more FN than was found in superficial tissue. Explants obtained from deep cartilage synthesized 2.4 times more FN per cell than did superficial tissues (P < 0.01). FN synthesis as a fraction of total protein synthesis was significantly greater in deep explants (P < 0.01) compared with superficial tissues. Isolated deep chondrocytes in culture synthesized 1.89 +/- 0.33-fold more FN than did superficial cells (P < 0.05). Cytokine-stimulated superficial cartilage explants failed to respond in terms of FN synthesis. FN mRNA quantitation showed no significant differences between superficial and deep populations. CONCLUSION: Since FN plays a major role in cell adhesion to damaged cartilage surfaces, our results suggest that modulation of FN synthesis near the articular surface of cartilage may be one of the factors that impede pannus invasion following an inflammatory insult to the joint. PMID- 8630103 TI - Stimulation of TIMP-1 production by oncostatin M in human articular cartilage. AB - OBJECTIVE: To investigate the effects of the interleukin-6 (IL-6) family cytokines, such as IL-6, IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM), on the production of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) in human articular cartilage. METHODS: Effects of IL-6 family cytokines and growth factors on TIMP-1 production were studied in human articular chondrocytes, grown as monolayer and cartilage explant culture. TIMP-1 protein levels in the cultured medium were measured by sandwich enzyme immunoassay. TIMP 1 messenger RNA levels in the cultured chondrocytes were analyzed by Northern blotting. Western blot analysis was performed to evaluate the release of matrix metalloproteinases (MMPs) in the cultured medium. Cell proliferation of chondrocytes was determined by 3H-thymidine uptake. RESULTS: IL-6 family cytokines induced TIMP-1 expression in monolayer and explant culture, and the production of TIMP-1 was most stimulated by OSM. In contrast, OSM did not modulate the release of MMPs and cell proliferation. CONCLUSION: These results suggest that OSM may be characterized as one of the chondroprotective mediators in cartilage destruction. PMID- 8630105 TI - src-related tyrosine kinases regulate transcriptional activation of the interstitial collagenase gene, MMP-1, in interleukin-1-stimulated synovial fibroblasts. AB - OBJECTIVE: To characterize tyrosine kinases that contribute to the transcription of interstitial collagenase. METHODS: Four thousand six hundred fourteen basepairs of the rabbit collagenase promoter region were cloned and sequenced. Plasmids containing collagenase promoter fragments linked to the luciferase reporter gene were transiently transfected into primary rabbit synovial fibroblasts. Regulation of gene activation by inflammatory mediators and tyrosine kinase inhibitors was assessed. To identify specific tyrosine kinases that contribute to collagenase gene expression, v-src was transiently expressed in rabbit synovial fibroblasts along with collagenase promoter constructs, and basal and interleukin-(IL-l)-induced collagenase transcription was assayed. RESULT: An inhibitor of src-related tyrosine kinases, herbimycin A, inhibited increases of collagenase messenger RNA in IL-1- and phorbol myristate acetate-treated fibroblasts. Transcriptional activation of collagenase by IL-1 was also inhibited by herbimycin A. Expression of v-src in synovial fibroblasts enhanced basal and IL-1-inducible transcription. CONCLUSION: Activation of collagenase transcription by inflammatory mediators involves activation of an src-related tyrosine kinase. PMID- 8630106 TI - Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue. AB - OBJECTIVE: The transcription factor nuclear factor kappaB (NF-kappaB) has been implicated in the inflammatory response and is known to be activated by a process involving reactive oxygen intermediates. The purpose of the present study was to demonstrate the presence and distribution of activated NF-kappaB in synovium samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and from autopsy subjects with no known history of arthritis. METHODS: Immunohistochemical staining was performed using both polyclonal and monoclonal "activity-specific" antibodies to the Rel-A (p65) subunit of NF-kappaB (anti-Rel A nuclear location sequences). Histologic features of inflammation were also scored. RESULTS: Both antibodies demonstrated positive staining of synovial tissue, with a cellular distribution that was nuclear. The staining was associated with specific cell types within the tissue, in particular, type A synoviocytes and vascular endothelium. Notably, lymphoid aggregates were unstained. Using the monoclonal antibody, a further study was carried out to investigate the distribution of staining in tissues from patients with different disease activities and clinical diagnoses, as well as in normal control tissue obtained at autopsy. Patients with acute RA more commonly showed vessel staining (P = 0.05) and, conversely, showed less frequent staining of the synovial lining (P < 0.005) compared with OA patients. Synovial tissue from controls exhibited either no staining or only weak staining in the synovial lining. CONCLUSION: The activation of NF-kappaB in vascular endothelium and type A synovial lining cells is a feature of synovial tissue from both RA and OA patients. The distribution of this staining appears to be related to the clinical diagnosis. PMID- 8630107 TI - Cell-type specificity of anti-CD45 autoantibodies in systemic lupus erythematosus. AB - OBJECTIVE: To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation. METHODS: CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies. RESULTS: IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed. CONCLUSION: Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms. PMID- 8630108 TI - Defective antigen-presenting cell function in patients with systemic lupus erythematosus. AB - OBJECTIVE: Patients with systemic lupus erythematosus (SLE) who exhibit defective in vitro responses to recall antigens and normal responses to alloantigens have been shown to have an abnormality in antigen-presenting cell (APC) function. This study was undertaken to further characterize this defect in APC function in lupus patients. METHODS: Mononuclear cells (MNC) from the peripheral blood of patients with SLE and from normal individuals were cultured in the presence of either recall antigen tetanus toxoid (TT), anti-CD3 (OKT3) monoclonal antibody, or alloantigens, and proliferative or interleukin-2 responses were assessed. Cell surface expression of B7-1 was assessed by flow cytometry. RESULTS: MNC from all normal individuals and from 7 patients with SLE responded to both TT and alloantigen and were designated +/+. Twelve SLE patients did not respond to TT but did respond to alloantigen stimulation and were designated -/+. In both normal subjects and SLE patients, the ability to respond to OKT3 correlated strongly with the ability to respond to recall antigen. A defect in APC costimulatory function was suggested by data demonstrating that interferon-gamma induced expression of B7-1 was significantly reduced in SLE patients compared with controls. Neither controls nor SLE patients expressed detectable amounts of surface B7-1 molecule on resting APC. Defective recall and anti-CD3-stimulated responses could be enhanced in SLE patients in the presence of B7/BBl-transfected P815 murine mastocytoma cells underscoring an SLE-associated defect in costimulatory activity. However, nontransfected P815 cells were also able to enhance responses to OKT3 in -/+ patients; blocking experiments showed that this was mediated through an IgG Fc receptor-dependent mechanism. CONCLUSION: These data indicate that SLE-associated defects in APC function in vitro can be accounted for by abnormalities in APC surface membrane molecules such as B7, IgG Fc receptors, and possibly others. PMID- 8630109 TI - Polymyositis, arthritis, and uveitis in a macaque experimentally infected with human T lymphotropic virus type I. AB - OBJECTIVE: To establish a simian model of human T lymphotropic virus type I (HTLV I) infection and disease. METHODS: Irradiated HTLV-I-producing cells were used to infect two 2-year-old rhesus macaque monkeys (Macaca mulatta). One monkey was also simultaneously inoculated with a cell-free suspension of simian immunodeficiency virus (SIV). Evidence of infection was monitored by serial clinical examinations and by serologic, molecular, and virologic assays. RESULTS: Both HTLV-I-inoculated monkeys became persistently infected following inoculation. Clinical disease was observed in the singly inoculated monkey, which developed arthritis (with synovial fluid positive for HTLV-I by culture and polymerase chain reaction), anterior chamber uveitis, and steroid-responsive polymyositis confirmed by electrophysiologic studies. The dually inoculated animal remained clinically healthy, despite high levels of SIV and HTLV-I virus expression and loss of HTLV-I-specific antibodies. CONCLUSION: These results indicate the utility of a nonhuman primate model for studying HTLV-I disease pathogenesis and the dynamics of SIV-1/HTLV-I retroviral coinfection. PMID- 8630110 TI - Clinical images: Wegener's granulomatosis of the lungs. PMID- 8630111 TI - Reduction in long-term disability in patients with rheumatoid arthritis by disease-modifying antirheumatic drug-based treatment strategies. AB - OBJECTIVE: Therapeutic strategies for rheumatoid arthritis (RA) have been evolving from the traditional "pyramid" approach toward one based upon early and sustained use of disease-modifying antirheumatic drugs (DMARDs), in the hope of improving long-term health outcomes. However, few data to have been presented to document the effects of this approach. We sought to directly assess associations between consistent DMARD use and long-term functional outcomes. METHODS: We studied 2,888 RA patients who were followed up prospectively for up to 20 years (average 9 years) at 8 databank centers. The independent variable was the proportion of patient encounters that resulted in treatment with > or = 1 DMARD (hydroxychloroquine, sulfasalazine, auranofin, intramuscular gold, D penicillamine, methotrexate, and/or azathioprine). The dependent variable was each patient's last recorded Disability Index value from the Health Assessment Questionnaire (HAQ). RESULTS: Increased DMARD use was strongly associated with better long-term Disability Index values (P < 0.0001). The association was strengthened when restricted to more seriously affected (rheumatoid factor (RF) positive) patients. The magnitude of the effect, unadjusted, was a difference of 0.53 HAQ Disability units (scale 0-3) between 100% DMARD use and 0%. Correlation coefficients ranged up to 0.26. Effects were similar for all disease duration periods (0-4, 5-9, 10-14, 15-19, and 20+ years). "Control" correlations, with variables computed to represent the proportion of time in which patients were taking either nonsteroidal anti-inflammatory drugs or prednisone, failed to show positive associations. A multiple linear regression model, which controlled for age, disease duration, sex, RF positivity, proportion of visits under a prednisone regimen, and initial disability level, included the proportion of time in which patients were taking DMARDs (P < 0.0001), with a model R2 of 0.54. These results were obtained despite an adverse selection bias in which more severely affected individuals were given DMARDS more frequently, and despite absence of data on drug use early in the disease course of many patients. Thus, these results, which suggest up to a 30 percent reduction in long term disability with consistent DMARD use, are most likely conservative. CONCLUSION: An association between consistent DMARD use and improvement in long-term functional outcomes in RA is supported by these data. PMID- 8630112 TI - A pilot trial of oral type II collagen in the treatment of juvenile rheumatoid arthritis. AB - OBJECTIVE: To evaluate the efficacy of oral chicken type II collagen (CCII) in the treatment of juvenile rheumatoid arthritis (JRA). METHODS: Ten patients with active JRA were treated with CCII for 12 weeks. Efficacy parameters, which included swollen and tender joint count and score, grip strength, 50-foot walking time, duration of morning stiffness, and patient and physician global scores of disease severity, were assessed monthly. RESULTS: All patients completed the full course of therapy. Eight patients had reductions in both swollen and tender joint counts after 3 months of CCII. The mean changes from baseline in swollen and tender joint counts for the 8 responders at the end of the study were -61% and 54%, respectively. Mean values for other efficacy parameters also showed improvement from baseline. There were no adverse events that were considered to be treatment related. CONCLUSION: Oral CCII may be a safe and effective therapy for JRA, and its use in this disease warrants further investigation. PMID- 8630113 TI - Elevated levels of the soluble form of bone marrow stromal cell antigen 1 in the sera of patients with severe rheumatoid arthritis. AB - OBJECTIVE: Bone marrow stromal cell antigen 1 (BST-1) is a novel glycosyl phosphatidylinositol-anchored ectoenzyme, which is overexpressed on bone marrow stromal and synovial cell lines derived from patients with rheumatoid arthritis (RA). To investigate the pathophysiologic roles of BST-1 in RA, we established an enzyme-linked immunosorbent assay (ELISA) system to detect the soluble form of BST-1 (sBST-1) and examined levels of sBST-1 in the sera of RA patients. METHODS: Concentrations of sBST-1 in sera from healthy donors and from patients with RA, osteoarthritis, Sjogren's syndrome, and systemic lupus erythematosus were measured with the ELISA. RESULTS: In 7% of the RA patient samples (10 of 143), concentrations of serum sBST-1 were higher (approximately 30-50-fold) than in non RA samples. Serum sBST-1 concentrations showed no correlation with age, C reactive protein level, or rheumatoid factor level. All RA patients with high concentrations of serum sBST-1 had severe disease with involvement of several large joints. CONCLUSION: We believe the measurement of serum sBST-1 may have prognostic value, but further analysis is necessary to clarify the clinical significance of elevated sBST-1 in RA. PMID- 8630114 TI - Epstein-Barr virus clonality in lymphomas occurring in patients with rheumatoid arthritis. AB - OBJECTIVE: A causative role for Epstein-Barr virus (EBV) in the development of lymphoma in patients with rheumatoid arthritis (RA) has been proposed. We investigated the molecular features of EBV-positive diffuse large cell lymphomas in 2 patients with RA. METHODS: Southern blot analysis for immunoglobulin gene rearrangements, terminal repeat analysis for clonality of the EBV genome, and double-labeling of the lymphoma cells by in situ hybridization and immunoperoxidase staining were performed. RESULTS: In both cases, double-labeling studies localized the EBV genome to the malignant B cells. Both neoplasms contained clonal immunoglobulin gene rearrangements and clonal EBV genomes. CONCLUSION: Our data indicate that EBV infection was an early step in the development of these neoplasms. The findings further extend knowledge on the similarity of this subset of lymphomas to posttransplantation lymphomas and emphasize the role of immunosuppression in their genesis. PMID- 8630115 TI - Elevated nitric oxide production in rheumatoid arthritis. Detection using the fasting urinary nitrate:creatinine ratio. AB - OBJECTIVE: To develop a simple method for assessing endogenous nitric oxide (NO) production applicable to routine clinical practice in rheumatology. METHODS: NO production was assessed in patients with rheumatoid arthritis (RA) as serum nitrate levels and as the urinary nitrate:creatinine ratio in morning samples of urine following an overnight fast. The influence of dietary intake of nitrate on these measurements was investigated in healthy volunteers. The clinical value of the urinary nitrate:creatinine ratio was validated in patients with infectious gastroenteritis, in whom its production is known to be increased. RESULTS: Urinary nitrate:creatinine ratios were significantly elevated in patients with RA (average 3-fold elevation over controls; P < 0.005) or infectious gastroenteritis (average 10-fold elevation, P < 0.001). Serum nitrate was significantly elevated only in patients with infectious gastroenteritis (P < 0.001). Dietary intake of nitrate had no significant influence on the fasting morning urinary nitrate:creatinine ratio in the healthy volunteers, showing that this parameter is a useful indicator of endogenous NO production. PMID- 8630116 TI - Do antioxidant micronutrients protect against the development and progression of knee osteoarthritis? AB - OBJECTIVE: Cumulative damage to tissues, mediated by reactive oxygen species, has been implicated as a pathway that leads to many of the degenerative changes associated with aging. We hypothesized that increased intake of antioxidant micronutrients might be associated with decreased rates of osteoarthritis (OA) in the knees, a common age-related disorder. METHODS: Participants in the Framingham Osteoarthritis Cohort Study underwent knee evaluations by radiography at examinations 18 (1983-1985) and 22 (1992-1993). Usual dietary intake was assessed using the Food Frequency Questionnaire, administered at examination 20 (1988 1989). Knees without OA at baseline (Kellgren and Lawrence [K&L] grade < or = 1) were classified as having incident OA if they had a K&L grade > or = 2 at followup. Knees with OA at baseline were classified as having progressive OA if their score increased by > or = 1 at followup. Knees were also classified as having cartilage loss or osteophyte growth if their maximal joint space narrowing or osteophyte growth score increased by > or = 1 (range 0-3). The association of vitamin C, beta carotene, and vitamin E intake, ranked in sex-specific tertiles, with incidence and progression of OA was compared with that of a panel of nonantioxidant vitamins, Bl, B6, niacin, and folate, using logistic regression and generalized estimation equations to adjust for correlation between fellow knees. The lowest tertile for each dietary exposure was used as the referent category. Odds ratios (OR) were adjusted for age, sex, body mass index, weight change, knee injury, physical activity, energy intake, and health status. RESULTS: Six hundred forty participants received complete assessments. Incident and progressive OA occurred in 81 and 68 knees, respectively. We found no significant association of incident OA with any nutrient. A 3-fold reduction in risk of OA progression was found for both the middle tertile (adjusted OR = 0.3, 95% confidence interval [95% CI] 0.1-0.8) and highest tertile (adjusted OR = 0.3, 95% CI 0.1-0.6) of vitamin C intake. This related predominantly to a reduced risk of cartilage loss (adjusted OR = 0.3, 95% CI 0.1-0.8). Those with high vitamin C intake also had a reduced risk of developing knee pain (adjusted OR = 0.3, 95% CI 0.1-0.8). A reduction in risk of OA progression was seen for beta carotene (adjusted OR = 0.4, 95% CI 0.2-0.9) and vitamin E intake (adjusted OR = 0.7, 95% CI 0.3-1.6), but was less consistent. No significant associations were observed for the nonantioxidant nutrients. CONCLUSION: High intake of antioxidant micronutrients, especially vitamin C, may reduce the risk of cartilage loss and disease progression in people with OA. We found no effect of antioxidant nutrients on incident OA. These preliminary findings warrant confirmation. PMID- 8630117 TI - Hair dye use and the risk of developing systemic lupus erythematosus. AB - OBJECTIVE: To investigate the role of hair dye use in the etiology of systemic lupus erythematosus (SLE). METHODS: Participants included 106,391 women enrolled in the Nurses' Health Study, a prospective cohort study. The subjects were ages 30-55 years in 1976, and were free from SLE and any other connective tissue disease at the time of enrollment. In 1976, 1978, 1980, and 1982, subjects were classified as never-users or ever-users of permanent hair dye, based on self report. Incidence rates of SLE meeting American College of Rheumatology classification criteria were ascertained and confirmed by chart review. RESULTS: Compared with never-users of permanent hair dye, the age-adjusted relative risks (RR) for the development of SLE among ever-users (n = 85 cases) was 0.96 (95% confidence interval [95% CI] 0.63-1.47). Duration of hair dye use was not related to risk of SLE. Women with 15 or more years of use had no increased risk (RR = 0.92, 95% CI 0.46-1.83). There was no relationship between frequency of use or time since first use and risk of SLE. The results were similar when less stringent criteria for SLE were used. CONCLUSION: We found no evidence that permanent hair dye use, age at first use, frequency of use, or duration of use is associated with the development of SLE. PMID- 8630118 TI - Homozygous hereditary C1q deficiency and systemic lupus erythematosus. A new family and the molecular basis of C1q deficiency in three families. AB - OBJECTIVE: To describe a new kindred with Clq deficiency and to identify the molecular lesions responsible for complete functional C1q deficiency in this and 2 other previously described kindreds. METHODS: The A-, B-, and C-chain genes of C1q were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations. RESULT: Patient 1 had a homozygous G-to-A change at codon 6 of the C chain, causing an amino acid change from Gly to Arg. Patient 2 had a homozygous deletion of a C nucleotide at codon 43 of the C-chain, causing a frame shift, leading to a premature stop codon at codon 108. Patient 3 had a homozygous C-to-T mutation at amino acid position 41 of the C chain, resulting in a premature stop codon. CONCLUSION: In the homozygous state, the mutations are sufficient to cause complete deficiency of Clq. The mutation in patient 1 has been previously reported in a patient of different ethnic origin. A survey of a series of 158 DNA samples from patients with systemic lupus erythematosus showed no other examples of this mutant allele. PMID- 8630119 TI - Antiribosomal P antibodies in pediatric patients with systemic lupus erythematosus and psychosis. AB - OBJECTIVE: To study antibodies directed against ribosomal P proteins, a sensitive and specific marker of depression and psychosis in systemic lupus erythematosus (SLE), in pediatric patients with SLE. METHODS: One hundred six serum samples were obtained from 79 patients with SLE. Sixty age- and sex-matched control sera were obtained, and 12 samples were obtained from children with primary psychosis. Antibodies to recombinant ribosomal P (rRP) protein were detected using a standard enzyme-linked immunosorbent assay. RESULTS: All 12 children with non-SLE associated psychosis had normal levels of anti-rRP antibodies. Elevated levels of anti-rRP were found in 11 of 64 pediatric SLE patients without a history of psychosis (17%). In the group of 13 SLE patients with psychosis, 5 (38%) had increased anti-rRP antibody levels during the time of acute psychosis, and which significantly decreased during remission. In addition, most of the SLE patients without a history of psychosis had a good correlation between anti-rRP antibody levels and disease activity. The patients with psychosis had significantly less renal involvement than the patients without a history of psychosis. This apparent protection from renal disease was not related to the presence or absence of either antiribosomal P or anti-DNA antibodies. CONCLUSION: Elevated serum levels of antibodies to ribosomal P protein can distinguish SLE-associated psychosis from primary psychosis of childhood. In SLE, elevated antiribosomal P antibody levels were not specific for psychosis. Serial assays were useful for monitoring the disease activity. PMID- 8630120 TI - Thallium perfusion defects predict subsequent cardiac dysfunction in patients with systemic sclerosis. AB - OBJECTIVE: To determine the significance of thallium perfusion defects in patients with systemic sclerosis (SSc). METHODS: This is a followup study of a series of 48 SSc patients who underwent thallium perfusion scans in the early 1980s. Their cardiac history and survival information over the last 10 years were obtained as part of the Pittsburgh Databank's yearly evaluation. We determined the frequency of subsequent development of arrhythmias requiring treatment or of congestive heart failure through patient and physician information. RESULTS: Patients with larger thallium perfusion defects had a significantly increased risk of developing subsequent cardiac events or death. The size of the initial thallium defect was the best predictor of later adverse events compared with other disease-related features, in a logistic regression analysis. CONCLUSION: We conclude that patients with SSc who have significant thallium perfusion defects are at a significantly increased risk of developing subsequent cardiac disease or death. PMID- 8630121 TI - A prospective long-term study of fibromyalgia syndrome. AB - OBJECTIVE: To ascertain the long-term natural history of fibromyalgia syndrome (FMS). METHODS: Patients with a history of FMS, seen in an academic rheumatology referral practice, were originally surveyed soon after onset of symptoms, and then were reinterviewed 10 years later in a prospective followup cohort study. A validated telephone survey was administered that inquired into current symptoms, medical care and treatments used, and work disability. The results were compared with the prior surveys. RESULTS: Of the original 39 patients, there were 4 deaths. Of the remaining 35 patients, 29 (83%) were reinterviewed. Mean age at current survey was 55 years, and mean duration of symptoms was 15.8 years. All patients had persistence of some fibromyalgia symptoms, although almost half (48%) had not seen a doctor for them in the last year. Moderate to severe pain or stiffness was reported in 55% of patients; moderate to a lot of sleep difficulty was noted in 48%; and moderate to extreme fatigue was noted in 59%. These symptoms showed little change from earlier surveys. In 79% of patients, medications were still being taken to control FMS symptoms. Despite continuing symptoms, 66% of patients reported that FMS symptoms were a little or a lot better than when first diagnosed. Fifty-five percent of patients said they felt well or very well in terms of FMS symptoms, and only 7% felt they were doing poorly. With the exception of sleep trouble, which was persistent, baseline survey symptoms correlated poorly with symptoms at the 10-year followup. CONCLUSION: FMS symptoms last, on average, at least 15 years after illness onset. However, most patients experience some improvement in symptoms after FMS onset. PMID- 8630122 TI - Enhancement of anti-DNA topoisomerase I autoantibody response after lung cancer in patients with systemic sclerosis. A report of two cases. AB - Anti-DNA topoisomerase I (anti-topo I) antibody profiles were compared before and after lung cancer in 2 patients with systemic sclerosis (SSc). Both patients developed adenocarcinoma of the lung late in the course of SSc and died of the cancer. Anti-topo I antibody levels, determined by double immunodiffusion and enzyme-linked immunosorbent assay, increased markedly at the time of diagnosis of lung cancer. Furthermore, patients' sera obtained after lung cancer reacted with multiple epitopes on the entire topo I molecule, some of which had not previously been recognized. These results further support the concept that anti-topo I antibody production in SSc patients is due to an antigen-driven process. PMID- 8630123 TI - Coexistence of two antisynthetases in a patient with the antisynthetase syndrome. AB - We describe the immunologic findings in a patient with the antisynthetase syndrome characterized by prominent arthritis, lung fibrosis, and subclinical myositis. At disease onset and during the followup, this patient's serum showed 2 different subsets of antisynthetase autoantibodies: anti-Jo-1, which reacted with histidyl-transfer RNA (tRNA) synthetase by immunoblot and inhibited its enzymatic function; and anti-0J, which immunoprecipitated the multi-enzyme complex of synthetases, and reacted with lysyl-tRNA synthetase by immunoblot. This is the first report of anti-Jo-1 and another antisynthetase antibody being found together in the same patient. PMID- 8630124 TI - IgA multiple myeloma presenting as Henoch-Schonlein purpura/polyarteritis nodosa overlap syndrome. AB - We report the unusual case of a man with a 5-year history of relapsing Henoch Schonlein purpura (HSP) and macroscopic polyarteritis nodosa (PAN) as early manifestations of IgA kappa multiple myeloma. The glomeruli contained monoclonal IgA kappa deposits, without other immunoglobulins or lambda light chains. Glomerular deposits lacked the usual electron density but could be demonstrated by immunoelectron microscopy. Multisystem large vessel vasculitis (antineutrophil cytoplasmic antibody negative) produced aneurysms of renal and hepatic arteries, whereas small vessel vasculitis affected the skin and glomeruli, producing a symptom complex of HSP with dermal and glomerular deposits of IgA. We conclude that HSP/overlap syndrome may be the initial manifestation of IgA myeloma and should be considered within the spectrum of monoclonal IgA deposition disease. PMID- 8630125 TI - Intravenous immunoglobulin therapy: modification of the immunofluorescence pattern in the skin of six patients with systemic lupus erythematosus. PMID- 8630126 TI - Familial primary antiphospholipid antibody syndrome. PMID- 8630127 TI - Mannose-binding protein in Chinese patients with systemic lupus erythematosus. PMID- 8630128 TI - Chondrocyte metalloproteinase activation through integrin receptors and interleukin-1: comment on the article by Arner and Tortorella. PMID- 8630129 TI - Who has the rheumatology service they need?: comment on the article by Yelin et al. PMID- 8630130 TI - Sulfasalazine in psoriatic arthritis: a new or established indication? PMID- 8630131 TI - Are emergency departments really a "safety net" for the medically indigent? AB - This study was designed to quantify the willingness of emergency departments (EDs) and private care practitioners to see medically indigent patients. Three case scenarios were developed to represent severe, moderate, and mild problems that typically confront ED physicians. A female investigator made telephone calls using these scenarios, each time declaring herself to be medically indigent. All EDs received calls about all three scenarios, but only the least severe scenario was used for private practitioners. The timing and order of all calls were randomized. A control survey of the same population was subsequently performed in which the caller related that she had third-party insurance and had the minimal (rash) problem. The participants were all 54 nonmilitary EDs in Arizona and 69 randomly chosen private primary care practitioners in the same locales as the EDs. Calls to EDs were made during all time periods and days of the week; private practitioners were called only during their weekday office hours. The majority of all EDs were willing to see medically indigent patients, recommending that the caller come to the ED immediately 76% of the time. This response did not vary by geography or the facility's size, although ED personnel suggested initial home treatment more commonly at smaller hospitals (P = .02), and suggested coming to the ED more often on weekends (P < .02). Some EDs, however, clearly did not comply with their own telephone advice policies, and some ED personnel failed to give medically appropriate advice. In contrast to the EDs (P < .001), 62% of private practitioners' staffs stated they were not taking new patients or required at least $30 in advance. Private practitioners in the largest communities were significantly more reluctant to see the medically indigent than their peers in smaller communities (P < .05). For an insured caller, 55% of private practitioners would see the caller for < $30 and only 35% were not taking new patients or provided referral. In contrast to most private primary care practitioners, EDs are at least willing to serve as a triage point for the medically indigent and are often the primary-care "safety net" for the medically indigent. PMID- 8630132 TI - Does warming local anesthetic reduce the pain of subcutaneous injection? AB - The most frequent complaint noted with the use of lidocaine (or other amide local anesthetic) is stinging or burning pain associated with subcutaneous infiltration. The purpose of this study was to evaluate the efficacy of warming buffered lidocaine for reducing the pain of infiltration. Forty adult volunteers were entered into a randomized, crossover study conducted at a community teaching hospital. Blinded subjects underwent 1-mL subcutaneous injections of the study agent through 27-gauge needles over 30 seconds. Following a crossover protocol, "room temperature" buffered lidocaine (20 degrees C) was injected into one midvolar forearm and "body temperature" buffered lidocaine (37 degrees C) into the opposite arm. The order and the handedness of the two injections were randomized; an independent observer prepared the anesthetic solutions. Pain was assessed using a 100-mm visual analog pain scale and subjects' comparison of pain on injection. Twenty subjects (50%) reported that 20 degrees C buffered lidocaine was more painful and 17 (42.5%) reported that the 37 degrees C solution was more painful (sign test, P = .74). Similarly, a median pain score difference of 5.0 mm favoring 37 degrees C lidocaine was not statistically significant (sign rank test, P = .42). The order or the initial side of the injection did not influence the pain scores. The study had a power of 80% to detect a 10-mm difference between the two solutions at alpha = .05. These results suggest that warming buffered lidocaine to body temperature (37 degrees C) does not reduce the pain of subcutaneous infiltration. PMID- 8630133 TI - C-reactive protein in the diagnosis of acute appendicitis. PMID- 8630134 TI - Fingertip injuries: immediate reconstruction versus delayed healing. PMID- 8630135 TI - Racemic epinephrine in the treatment of laryngotracheitis: can relapse be prevented? PMID- 8630136 TI - Titratable acid/alkaline reserve is not predictive of esophageal perforation risk after caustic exposure. PMID- 8630137 TI - Axillary artery pseudoaneurysm and axillary nerve palsy: delayed sequelae of anterior shoulder dislocation. PMID- 8630138 TI - End-tidal carbon dioxide during preclinical CPR: correlation with primary outcome. PMID- 8630139 TI - ST segment elevations without myocardial infarction in a patient on clozapine. PMID- 8630140 TI - Snake stone for snake envenomization. PMID- 8630141 TI - Revisiting ancient times. PMID- 8630142 TI - The mentality of contraction. PMID- 8630143 TI - ED census and day of the week. PMID- 8630144 TI - Self-administered electroshock. PMID- 8630145 TI - A survey of the contract terms used in academic emergency medicine. PMID- 8630146 TI - Demand valve ventilation in a swine pneumothorax model. AB - Bag-valve-mask (BVM) and oxygen-powered demand valve (OPDV) are two available adjuncts for artificial ventilation. Use of OPVDs has been limited by concern for causing or worsening pneumothorax. This study examined the effect of OPDV and BVM ventilation in swine with pneumothorax. This was a randomized, crossover design. Five female swine (16 to 23 kg) were sedated (ketamine/xylazine), anesthetized (alpha chloralose), and intubated. Right chest injury was induced, through an incision, using a 14-gauge needle to produce disruption of underlying lung parenchyma. An 18F Foley catheter was inserted and sealed in the right pleural space at the cut-down site. For each trial, a pneumothorax was induced by instilling 300 mL of air. Each subject underwent 10-minute ventilation trials with OPDV and with BVM by the same investigator, blinded to instrumentation. Post trial pneumothorax volume, heart rate, blood pressure, end-tidal CO2 (ETCO2), and arterial blood gases were measured. There was no significant difference in pneumothorax volume after OPDV ventilation (310.8 mL) versus BVM ventilation (315.4 mL) (P = .821). There were no significant differences between heart rates, blood pressures, or arterial blood gases, although ETCO2 showed statistically significant decreases from baseline in both OPDV (33.0 to 28.6) and BVM (33.8 to 30.2) trials. However, multiple comparisons showed no significant differences between OPDV and BVM at any time points. In this model, OPDV and BVM ventilation did not differ in their effects on pneumothorax volume or hemodynamic variables. No animal showed signs of tension pneumothorax. PMID- 8630147 TI - Evaluation of pulse oximetry in sickle cell anemia patients presenting to the emergency department in acute vasoocclusive crisis. AB - This prospective study was done to evaluate the accuracy of pulse oximetry as an indicator of hypoxemia in sickle cell anemia patients presenting to the ED in acute vasoocclusive crisis. Thirteen patients (age older than 18 years) presenting in crisis, with pulse oximetry readings less than 95% had arterial blood gas (ABG) analysis performed. The mean pulse oximetry reading was 91.4% +/- 3.2% (SD); the mean ABG oxygen saturation (ABG O2 sat) was 95.3% +/- 1.4% (SD). The mean O2 sat difference (ABG O2 sat minus pulse oximetry) was 4.5% (P < .001 versus a control group of ED patients receiving ABG and pulse oximetry). Pulse oximetry correlated with ABG O2 sat (r = 0.889, P < .001) and O2 sat difference (r = -0.927, P < .001). No correlation existed between complete blood count, reticulocyte count, historical and physical examination findings, and O2 sat or O2 sat difference. By linear regression, ABG O2 sat = 0.44 (pulse oximetry) + 55.6. This study suggests that pulse oximetry underestimates oxygenation in sickle cell anemia patients presenting in painful crisis. PMID- 8630148 TI - Emergency department discharge instructions and patient literacy: a problem of disparity. AB - This two-part study was designed to determine the reading level necessary to understand commonly used emergency department (ED) discharge instructions and the functioning reading level of adult patients treated in an urban hospital ED. In the first phase, 10 preprinted patient discharge instructions were analyzed using the Flesch Reading Ease Score. The average reading level required to comprehend these instructions was 9.8, with a range from the 8th to the 14th grade. In the second phase of the study, a reading exam, based on the Stanford Diagnostic Reading Test, was administered to 82 ED patients prospectively. The mean reading level was 9.8, with a median level of 10; approximately 45% of the ED patients would be unable to understand the preprinted discharge instructions. ED discharge instructions are frequently written at a level beyond the comprehension of a significant portion of the population. PMID- 8630149 TI - Outcome of patients who refused out-of-hospital medical assistance. AB - Activation of the emergency medical services (EMS) system does not always result in transport of a patient to the hospital. This study assessed the outcomes of patients who refused medical assistance in the field, to determine if refusal of medical assistance (RMA) is associated with poor outcomes. Four high-volume suburban volunteer ambulance corps participated in the study. Consecutive patients who refused medical assistance were prospectively enrolled. Medical and identifying data were collected for each patient. Telephone follow-up was conducted to determine the patient's condition and if the patient sought further care after RMA. Primary endpoints were whether the patient sought further care, was admitted to a hospital, or died subsequent to RMA. Follow-up was successfully obtained for 199 of 321 patients enrolled (62%). Of these 199 patients, 95 (48%) sought further medical care within 1 week for the same complaint, with 13 being admitted to the hospital. Six of the 13 admitted patients had chief complaints of a cardiac or respiratory nature. One patient died during hospital admission. Even if none of the patients lost to follow-up had sought further care, a substantial number of patients who refuse out-of-hospital medical assistance seek further care. Some of these patients require hospital admission, especially those with cardiac or respiratory complaints. Efforts to minimize RMA should be especially focused on patients with such complaints. PMID- 8630150 TI - Effects of chemical pretreatment on posttraumatic cortical edema in the rat. AB - The purpose of this study was to evaluate the effects of mannitol (Man), dexamethasone (DM), dichloroacetic acid (DCA) and 1,3-butanediol (BD) in reduction of posttraumatic cortical edema following brain deformation injury to rats. Ten minutes prior to fluid percussion injury, each animal received one of four pretreatments or placebo: Man, 1 g/kg intravenously, DM 3.0 mg/kg intravenously, DCA 25 mg/kg intraperitoneally BD 0.5 mg/kg intraperitoneally (n = 12 per treatment group), or equivolume saline (n = 8 per corresponding trauma group). Six hours after trauma, cortical tissue was harvested. Using a benzene kerosene gradient column calibrated with potassium sulfate standards, the specific gravity (SpG) of cortical tissue from each group was measured and compared (ANOVA, P < .05). The measured cortical SpG from traumatized animals receiving Man (mean 1.037 +/- SEM .001), DCA (1.038 +/- .001), and BD (1.039 +/- .001) were equal to SpG from untraumitized cortex (1.041 +/- .001), and were significantly greater than SpG from traumatized cortex for animals receiving DM (1.035 +/- .001) or placebo (1.033 +/- .002). Pretreatment with DCA, Man, and BD appears to protect against development of posttraumatic cortical edema when measured 6 hours after blunt head trauma in the rat. Each of these chemical treatments appears effective in preventing or reducing posttraumatic cortical edema. PMID- 8630151 TI - Emergent use of echocardiography in a post-myocardial infarction patient with acute dyspnea. AB - Acute dyspnea in a post-myocardial infarction patient may prompt the physician to use further diagnostic testing to evaluate apparent worsening left ventricular function, ischemia, mitral valve dysfunction, chordae or valvular rupture, or a ventricular septal defect producing a left to right shunt. We present a case of a 62-year-old woman with an unrecognized myocardial infarction at home who presented to the emergency department (ED) acutely dyspneic and free of chest pain. Prompt evaluation in the ED with echocardiography and Doppler imaging proved time-efficient and allowed early lifesaving surgery to be performed. We propose that access to emergent echocardiography in select cases should be a standard service in every ED. PMID- 8630152 TI - In-vivo binding of lithium using the cation exchange resin sodium polystyrene sulfonate. AB - The purpose of this study was to determine if the exchange resin sodium polystyrene sulfonate increases the clearance of lithium in a healthy volunteer. A single healthy volunteer received 900 mg lithium for each of three study phases. A sodium polystyrene sulfonate dose of either 0, 20, or 40 grams was administered one half hour after each lithium dose to assess the effect on lithium clearance. Multiple blood samples were obtained for serum lithium concentrations over a 36- to 48-hour time period. Pharmacokinetic parameter estimates were calculated and compared. Sodium polystyrene sulfonate was found to increase the clearance of lithium in a single volunteer. Sodium polystyrene sulfonate may be useful in the treatment of lithium intoxication by increasing the clearance of lithium. PMID- 8630153 TI - Severe burn injury from recreational gasoline use. AB - This report describes the case of a 13-year-old boy who suffered severe burns with inhalation injury during the recreational use of gasoline. Gasoline sniffing is a popular form of solvent abuse for young children and adolescents in isolated, rural communities. In addition to the neurological and physiological complications of gasoline inhalation, gasoline sniffers face the significant threat of severe burn injury or death resulting from ignition of the gasoline vapor as well as from extensive physical contact with the constituents of liquid gasoline. PMID- 8630154 TI - Can prehospital personnel detect hypoxemia without the aid of pulse oximeters? AB - Although pulse oximeters have been proven accurate in the prehospital environment, they have not been proven to be necessary. This study was undertaken to determine if emergency medical services (EMS) providers can identify hypoxemia without pulse oximetry. An oximeter was placed at the ambulance entrance to the emergency department (ED), and EMS personnel obtained saturation levels on all patients on arrival. Hypoxemia was defined as a saturation level of 95% or less. The hypoxemia was classified as "recognized" if the patient received aggressive intervention and "unrecognized" if the patient did not. One hundred eighty patients were enrolled in the study; 30 had a saturation level of 95% or less. Twenty-seven (90%) of those patients had "unrecognized" hypoxemia. Twenty-three (85.2%) of the 27 patients with "unrecognized" hypoxemia did not complain of respiratory distress. Thus, there are patients whose hypoxemia is unrecognized by EMS providers, and this occurs most frequently in patients who do not complain of respiratory distress. PMID- 8630155 TI - Abdominal pain and hemoperitoneum in the gravid patient: a case report of placenta percreta. AB - A 24-year-old woman, G4P3 at 14 weeks gestation, presented to the ED with acute abdominal pain, hemoperitoneum, and fetal demise. Emergent laparotomy showed placenta percreta, requiring hysterotomy for delivery of the fetus and gestational sac followed by oversewing of the uterine defect. Although an uncommon occurrence, clinicians should consider placenta percreta in the gravid patient who presents with acute abdominal pain and shock. PMID- 8630156 TI - Use of the endotracheal tube as a pharyngeal airway. AB - An animal study was conducted to determine whether an endotracheal tube placed above the vocal cords in the pharynx can be used for ventilation. Four dogs undergoing general anesthesia were ventilated through an endotracheal tube placed in the oropharynx with the remainder of the airway occluded. Ventilation was performed for 3 of every 5 minutes during a total period of 25 minutes. Arterial PCO2 was compared in sequential samples alternating apnea and pharyngeal ventilation. Ventilation via the pharyngeal tube significantly reduced the arterial PCO2 from 48.8 mm Hg (SD 16) during apnea to 30.1 mm Hg (SD 10.9). Repeated measures ANOVAF = 8.2, P < .001. All PCO2 levels during ventilation were in or below the normal range of 34 to 46. Provided that the mouth and nose can be sealed, an endotracheal tube placed in the pharynx above the cords allows for adequate ventilation. PMID- 8630157 TI - Intravenous calcium chloride in the conversion of paroxysmal supraventricular tachycardia to normal sinus rhythm. AB - This brief report describes several cases of paroxysmal supraventricular tachycardia that converted promptly to normal sinus rhythm within 1 to 2 minutes of receiving intravenous calcium salts as pretreatment in anticipation of verapamil therapy. A review of calcium's hemodynamic and dromotropic effects suggests that this probably was due to electrophysiological effects rather than mere coincidence. Calcium raises blood pressure, which may reflexively increase cardiac parasympathetic tone, and also has a direct slowing effect on atrioventricular conduction. Adenosine remains the drug of choice in the treatment of paroxysmal supraventricular tachycardia. However, in addition to preventing hypotension when used as pretreatment to verapamil, intravenous calcium itself may terminate supraventricular tachycardia. PMID- 8630158 TI - Boerhaave's syndrome presenting with abdominal pain and right hydropneumothorax. AB - This case of Boerhaave's Syndrome had several unusual features including a delayed presentation and right-sided esophageal perforation. The patient's initial episode of hematemesis may have been caused by a small mucosal laceration in the area of the Barrett's lesion that later ruptured. On the other hand, if initially there was an esophageal rupture, it did not violate the parietal pleura or mediastinum. The overlying pleura remained intact until digested by gastric contents, thereby causing a right-sided hydropneumo thorax and a marked increase in symptoms, which promoted the patient to come to the ED. Because the patient initially appeared stable and had a history of emesis 4 days before presentation, and because an initial chest X-ray interpretation overlooked the right-sided apical pneumothorax, Boerhaave's Syndrome was not considered initially. Aspiration pneumonia, pancreatitis, alcoholic gastritis, or active peptide ulcer disease were in our initial differential. It was only after the repeat chest X ray, which more obviously showed the pneumothorax, and insertion of the chest tube that the correct diagnosis was made. Had the pneumothorax not been overlooked initially, the diagnosis may have been made earlier. It is apparent from this case and a review of the literature that Boerhaave's Syndrome is an uncommon clinical entity and has varying modes of presentation, making the diagnosis a difficult clinical challenge. Boerhaave's Syndrome should be considered in all ill-appearing patients presenting with the combination of gastrointestinal and respiratory complaints. The single most important test may be the upright chest X-ray. However, if it is normal, and there is a high index of suspicion, esophagograms and or chest CT may be required to demonstrate the lesion. Because survival is directly related to the time to diagnosis and treatment, a high clinical suspicion can decrease the substantial morbidity and mortality associated with Boerhaave's Syndrome. PMID- 8630159 TI - Oral Ambu-bag insufflation to remove unilateral nasal foreign bodies. AB - The nose is perhaps the most common site for the insertion of foreign bodies by children. Removal is essential. We report the successful use of the oral Ambu-bag insufflation technique to remove unilateral nasal foreign bodies in three children. Conscious sedation was not required. There were no complications. PMID- 8630160 TI - Initiation of mechanical ventilation in the emergency department. AB - Mechanical ventilation is frequently initiated by emergency physicians. Further, the physician on duty in the emergency department is frequently responsible for evaluating ventilated patients who decompensate in the intensive care unit when other physicians are not present in the hospital. A bewildering array of features on new mechanical ventilators has made their appropriate and effective use increasingly complex. Knowledge of the pathophysiology of acute respiratory failure and changes in lung physiology during positive pressure ventilation will aid the emergency physician in choosing an appropriate ventilator modality and initial settings to maximally benefit patients with respiratory insufficiency due to various causes. An appreciation of the adverse effects of mechanical ventilation and problems commonly encountered in patients on ventilators will prepare the emergency physician to rapidly assess and effectively manage the patient who deteriorates in this setting. PMID- 8630161 TI - Physicians' children are treated differently in the emergency department. AB - The objective was to determine whether children with a physician parent receive treatment different from that of children of nonphysician parents when they present to the emergency department (ED). The design was a retrospective cohort study. The setting was a university-affiliated children's hospital ED. All children with a physician parent seen in the ED during a 16-month period were identified. Children with a nonphysician parent were matched to children with a physician parent by date and time of visit. Length of stay in ED, performance of laboratory or radiological testing, evaluation by a consultant, and training level of the least experienced physician to evaluate the patient in the ED were measured. The authors identified 92 children with a physician parent and 181 children with nonphysician parents. Children of physician parents were similar to controls with regard to age, sex, nursing acuity level, length of stay, and whether a laboratory or radiographic procedure was performed. Children with a physician parent saw significantly fewer nonconsultant physicians while in the ED (P = .005). Compared with controls, the most junior member of the medical team seen by children of a physician parent was less likely to be a medical student (relative risk [RR] = 0.22) or a resident (RR = 0.71) and more likely to be an ED staff physician (RR = 1.52) or consultant (RR = 1.84). This trend was statistically significant (P = .002). The children of physician parents are more likely to see only an ED staff physician and/or consultant and less likely to see trainees than other children presenting to the pediatric ED. PMID- 8630162 TI - Mycotic aneurysm: a diagnostic challenge. PMID- 8630163 TI - Implications of current procompetitive approaches on EDs. AB - Health reform is now predicted to occur in the marketplace without much further governmental intervention. The major vehicles driving this fortuitous trend are the managed care plans, capitated payment, and the development of complex and fiscally awesome health networks organized by hospitals, physicians, and health insurance companies. In the context of this procompetitive environment, potential decreases are projected in current emergency department (ED) volumes, demand for emergency medicine (EM) physicians, and anticipated workload and remuneration of doctors working in EDs. Eventually, mandated universal insurance for 260 million Americans will create a slight increase in total ED visits; additional cost constraints will force the closure of a number of hospital EDs; and EM specialists will experience less ideal working conditions, requiring them to work more hours to maintain their current incomes. PMID- 8630164 TI - Cardiac arrest in cocaine users. PMID- 8630165 TI - EMS characteristics in an Asian Metropolis. AB - A prospective citywide cohort study was conducted from August 1, 1993, through May 31, 1994 to analyze the epidemiological characteristics of emergency medical services (EMS) in an Asian city. Of 5,459 studied cases, the leading 3 causes were trauma (49.7%), alcohol intoxication (8.6%), and altered mental status (AMS) (6.9%). Half of the studied cases needed no prehospital care and 16.4% needed advanced life support (ALS) care. Traffic accidents accounted for 68% of trauma cases. Of 897 cases requiring ALS care, the two most common causes were AMS and dead on arrival (DOA) (32.1% and 21.2% in medical group, 10.1% and 4.5% in trauma group, respectively). The response time, time on scene, and transportation time were 4.6, 4.3, and 9.4 minutes, respectively. This Oriental EMS system experienced very short prehospital times, many traffic accidents, and extremely few DOA cases. Because few patients required ALS care, an emergency medical technician-based EMS system would probably be able to handle the majority of prehospital patients. PMID- 8630167 TI - Measuring heart attack care performance: new indices and understanding. PMID- 8630166 TI - Outcomes in severely ill patients transported without prehospital ALS. AB - Because of the debate regarding the impact of advanced life support (ALS) care on the outcome of prehospital patients, we monitored the influence of lack of sophisticated prehospital treatment in cases of severe illness arriving by ambulance to the emergency department (ED). A prospective cohort study to examine and compare the outcome of trauma- and nontrauma-induced "ALS-eligible" cases in the setting of no prehospital care was carried out from August 1, 1993 through May 31, 1994. On arriving at the ED, patients meeting the criteria for ALS cases and sent by EMS public prehospital personnel were assessed for subjective and objective status and change in severity by triage nurses as well as being followed up for neurological status until discharged from the hospital. Chi Square method was used to compare the data between two groups and P < .05 was considered statistically significant. Of 667 studied ALS cases (155 trauma and 512 nontrauma), < 20% had their condition change subjectively and < 10% had their condition change objectively; 68% of medical patients and 60% of trauma cases were discharged from the hospital (neurologically intact). However, subgroup analysis showed that objective measures worsened in transit in nearly 18% of trauma victims, a rate nearly 3 times greater than that of medical cases. Moreover, neurological outcome was particularly poor in trauma cases. These results suggest that ALS care may be valuable for severely ill trauma victims. PMID- 8630168 TI - Carbon monoxide detectors and emergency physicians. PMID- 8630169 TI - Risperidone overdose. PMID- 8630170 TI - A bodybuilder with diuretic abuse presenting with symptomatic hypotension and hyperkalemia. PMID- 8630171 TI - Gas gangrene secondary to subcutaneous insulin injection. PMID- 8630173 TI - Inaccurate fit of implant superstructures. Part II: Efficacy of the Preci-disc system for the correction of errors. AB - The Preci-disc prosthetic system for dental implants is designed to compensate for casting inaccuracies and to reduce stresses generated on the superstructure and implant components. This laboratory study compared stresses induced on the implant superstructure with various sizes of vertical discrepancy of fit, with and without the application of the Preci-disc system. A series of test casts carrying four abutment replicas were fabricated incorporating various sizes of discrepancies (10 to 100 micrometers) relative to a master superstructure. The stress generated on the superstructure was quantified using photoelastic stress analysis. It was found that the Preci-disc system was effective in reducing stresses induced on the surface of the implant superstructure particularly with greater discrepancies of fit (60 to 100 micrometers). PMID- 8630172 TI - Persistent hypoglycemia and hyperinsulinemia: caution in using glucagon. PMID- 8630174 TI - The effect of die-spacing on crown retention. AB - Ten extracted molar teeth were prepared to a standardised complete crown preparation. Five stone dies were constructed for each tooth and coated with zero, two, four, six, and eight layers of a paint-on die-spacer. Crowns were fabricated on each die and the force required for removal from each tooth was measured prior to cementation. The crown elevation and force required for removal were measured after luting with zinc phosphate cement. The force required to remove the crowns before cementation decreased with increasing layers of die spacer. Following cementation, the mean crown elevation decreased from 547 micrometers (zero layers) to 38 micrometers (eight layers); while the mean removal force increased from 250 N (zero layers) to 375 N (eight layers). PMID- 8630175 TI - Effects of grinding, polishing, and overglazing on the flexure strength of a high leucite feldspathic porcelain. AB - The objective of this study was to determine how surface treatments and moisture affect the flexure strength of a high-leucite feldspathic porcelain. Uniaxial flexure strength was measured for porcelain beams whose surfaces were coarse ground, overglazed, or polished. Half of the specimens were stored in distilled water and tested while their surfaces were coated with distilled water. The other half were stored in a dry environment and tested immediately in dry air. The high leucite feldspathic porcelain was found to be sensitive to roughness and surface stresses, similar to leucite-free and low-leucite feldspathic porcelains. The experimental method used, however, was not sensitive enough to detect susceptibility to moisture. PMID- 8630177 TI - Changes in the rabbit temporomandibular joint associated with posterior displacement of the mandible. AB - The purpose of this investigation was to study the changes in temporomandibular joint tissues, in particular the disc and its attachments, associated with muscle powered continuous posterior displacement of the mandible for 33 days. After measurement of incisal relationships, food and water consumption, histologic and morphometric analysis, and radiologic data, it was determined that spatial relationships of joint tissues were altered. Mandibular retrusion leads to alteration in disc morphology and induces anterior positioning of the disc relative to the condylar head. Nutritional intake was disrupted by distal displacement crowns placed on the maxillary incisor teeth. PMID- 8630176 TI - Failure rates of restorations for single-tooth replacement. AB - Failure rates of traditional fixed partial dentures, resin-bonded prostheses, and implant restorations for the replacement of a single missing tooth are compared in this literature review. A lack of documented longevity studies involving conventional fixed partial dentures makes failure rates difficult to determine. Reported failures ranged from only 3% over 23 years to 20% over 3 years. The major causes of loss were caries and endodontically and periodontally related complications. Research concerning resin-bonded fixed partial dentures was more prevalent, but indicated that overall retention of these prostheses may be unpredictable. Failure rates were quite divergent, ranging from 10% over 11 years to 54% over 11 months. Debonding was the usual cause of failure. Clinical reports of single-tooth implant replacements have followed stricter protocols than the other two alternatives. Short-term studies indicated favorable success rates for these restorations, but these must be confirmed by long-term evaluations. Failures ranged from 9% over 3 years to 0% over 6.6 years. These comparisons should be considered in treatment planning a restoration for the replacement of a single missing tooth. PMID- 8630178 TI - Combination syndrome in relation to osseointegrated implant-supported overdentures: a survey. AB - Thirteen patients who had worn a maxillary conventional denture and mandibular osseointegrated implant-supported overdenture for at least 3 years were evaluated for subjective assessment of fit of the maxillary denture, occlusal integrity, and the status of the anterior maxillary residual ridge. The findings of this study support the view that this combination of prostheses can result in perceived loosening of the maxillary denture, loss of posterior occlusion, increased anterior occlusal pressure, and anterior maxillary bone loss, similar to the effects seen in Combination Syndrome. It is therefore important to ensure that where an implant-supported mandibular overdenture is planned for the edentulous patient, some form of stabilisation of the maxillary arch is also considered. PMID- 8630179 TI - Diagnosis and dental management of eating disorder patients. AB - Anorexia nervosa and bulimia nervosa are serious eating disorders that affect a significant number of young adults. Dentists play an important role in the early diagnosis and implementation of comprehensive treatment of patients with these disorders. Dental treatment modalities vary with the severity of the erosion of the dentition and the mental health status of the patient. The decision to proceed with definitive treatment is predicated upon the patient seeking psychotherapy and ceasing to vomit. A description of the early signs with various degrees of dental destruction, are presented. PMID- 8630180 TI - The evaluation of factors affecting the castability of metal ceramic alloy- investment combinations. AB - An evaluation of the castability of metal ceramic alloy-investment combinations using a mesh test and utilizing a factorial design concept has shown that different metal ceramic alloys (gold-based, palladium-based, and nickel-chromium based alloys) react differently, as far as castability is concerned, to various factors involved in the casting process. These factors were divided into two groups: procedural factors, that included sprue diameter, investing technique, and melting and casting method; and operator factors, which included alloy casting temperature, mould temperature, and heat-soak times. Each of these factors had two or three levels. PMID- 8630181 TI - Marginal fit of prefabricated crowns of the Ha-Ti implant system: an in vitro scanning electron microscopic study. AB - This study investigated the marginal fit of the prefabricated crowns for the Ha Ti implant system using scanning electron microscopy. The results demonstrated that the mean gap between the crown margin and the implant base was less than 3 micrometers following simulated ceramic firing and continuous loading in vitro. PMID- 8630182 TI - Clinical follow-up study of ceramic veneered titanium restorations--three-year results. AB - Eighty-four titanium restorations having 125 ceramic veneers were placed for 32 patients. One hundred sixteen (93%) of the veneers could be reexamined after 21 to 41 months. Two of the restorations required removal because of a partial veneer loss. A Kaplan-Meier survival analysis was performed, giving a survival probability of .85 for single crowns and .59 for fixed partial dentures after a time interval of 30 months, regarding the veneer cracking or chipping. There was a significantly higher survival probability for single crowns than for fixed partial dentures (P=.001, logrank test). It was concluded that ceramic veneered titanium restorations should be limited to single crowns. PMID- 8630183 TI - Techniques for making spacers in interstitial brachytherapy for tongue cancer. AB - The use of a spacer has been recommended in interstitial brachytherapy for tongue cancer to reduce the radiation dose to the mandible. This article describes the development of two types of acrylic resin spacers, a mouth guard-type spacer for dentate patients, and a replica denture-type spacer for edentulous patients. These spacers can be used without suture retention and are comfortable for patients. PMID- 8630184 TI - Sequence-specific binding of antitumour bisquaternary ammonium heterocycles to DNA and inhibition of polymerase activity in vitro. AB - Ten bisquaternary ammonium heterocycles (BQA) active against experimental tumours were investigated for possible sequence-selective binding to DNA. Footprinting analyses indicated that several bound preferentially to dAdT runs consisting of at least four base pairs. Shortening of one or two spacer groups between the aromatic rings of the ligands (by replacement of CONH with NH) emerged as a prerequisite for sequence-specific binding. Other relevant factors concerned the overall shape of the ligands and the relative position of their positive charges. Footprinting plots evaluated for the BQA compound SN 6132 on the 167mer EcoRI RsaI restriction fragment from plasmid pBR322 yielded the highest individual binding constant for the symmetrical base sequence AATTTAA, with approximate K(A) = 2.0 x 10(6)/M. Polymerase-catalysed syntheses of DNA and RNA in vitro were inhibited by all BQA derivatives, but the inhibition was much more pronounced with the sequence-specific binders SN 6999 and SN 6132 than with the non-specific ligand SN 6113. PMID- 8630185 TI - Pharmacokinetics and anti-tumour activity of B.4152, a reactive molecular combination of 5-fluorouracil and N-(2-chloroethyl)-N-nitrosourea, and its uracil analogue. AB - The original design of the previously described molecular combinations of 5 fluorouracil (5-FU) and a chloroethyl nitrosourea (CNU) moiety was on the basis that a single drug would be able to deliver two cytotoxic moieties to tumours following hydrolytic release in vivo of free 5-FU. Subsequently experiments have shown to date that the observed activity is due mainly to the alkylating effect of the CNU. This study investigates a molecular combination of 5-FU/CNU (B.4152) which is the most readily hydrolysed under acid conditions of all the 5-FU seco nucleosides so far prepared, and compares it with the unsubstituted uracil analogue B.4184. In vitro cytotoxicity studies against three murine colon tumour cell lines showed large differences in IC50 values between the two analogues, those for B.4184 being > 10-fold greater than those for B.4152. These differences could not be accounted for by drug stability as half-lives in tissue culture medium were similar. In vivo, B.4152 was also more active against the tumour lines and was marrow sparing at therapeutic doses. Pharmacokinetic studies demonstrated that improved activity was not due to release of free 5-FU, but differences in activity, toxicity and plasma pharmacokinetics between B.4152 and B.4184 are quite marked, indicating that the 5-FU moiety does have an important role to play. PMID- 8630186 TI - Synthesis and biological evaluation of a new series of phenylhydroquinone derivatives as inhibitors of EGF-R-associated PTK activity. AB - In order to design new potent inhibitors of the epidermal growth factor receptor (EGF-R) associated protein tyrosine kinase (PTK) activity as antitumor agents, several families of phenylhydroquinone derivatives were synthesized. Some of these compounds were shown to block PTK activity in vitro, but also efficiently to inhibit EGF-stimulated DNA synthesis in ER 22 cells (CCL 39 hamster fibroblasts transfected with EGF-R) with IC50 values in the range 1-10 microM. In some cases, a correlation between the two sets of data was observed, allowing structure-activity relationships to be established. However, inhibitors which had in vitro specificity with regard to other kinases were not specific in the cellular test. Similar effects on DNA synthesis were observed after stimulation by various activating agents, suggesting that our compounds may also act against other cellular targets involved in the EGF-dependent pathways leading to cell proliferation. PMID- 8630187 TI - Photolysis of an iodoplatinum(IV) diamine complex to cytotoxic species by visible light. AB - The feasibility of photolyzing the Pt(IV) complex trans,cis-[PtCl2I2(en)] to cytotoxic species by visible light was evaluated. The synthesis of trans, cis [PtCl2I2(en)] was achieved by the oxidation of [PtI2(en)] with PCl5 in tetrahydrofuran at room temperature for 30 min in the dark. The UV-Vis spectrum of trans, cis-[PtCl2I2(en)] in water showed a broad ligand-to-metal charge transfer (LMCT) band with lambda(max) = 396 nm (epsilon = 1191/M/cm). Although trans,cis-[PtCl2I2(en)] was relatively stable in water in the dark, irradiation at lambda(irr) = 410 nm brought about its rapid decomposition. A detailed analysis of the photodecomposition products was not carried out, but two lines of evidence suggest that I2 and [PtCl2(en)], a known antitumor agent, may be formed as a result of a reductive-elimination type reaction: (i) irradiation of trans, cis-[PtCl2I2(en)] in water at lambda(irr) = 410 nm led to the same spectral changes as when [PtCl2(en)] and I2 together were irradiated at the same wavelength; (ii) the photoinduced loss of trans,cis-[PtCl2I2(en)] was accompanied by the covalent binding of Pt to DNA at a rate comparable to that of [PtCl2(en)] at 37 degrees C, and the presence of 100 mM chloride suppressed this DNA platination. On the other hand, the combined photolysis products, formed when trans,cis-[PtCl2I2(en)] was irradiated in culture medium at lambda(irr) > 375 nm for 60 min, were less potent than [PtCl2(en)] at inhibiting the growth of two human cancer cell lines. Two limitations make the use of trans,cis-[PtCl2I2(en)] in the therapy of cancer impractical: (i) trans,cis- [PtCl2I2(en)] was relatively unstable in the presence of serum: however, [PtI2(en)] did not appear to be a product of the reaction; (ii) the LMCT band extends only weakly into the region of the electromagnetic spectrum (i.e. lambda > 600 nm) where maximal tissue penetration would be expected. In conclusion, these investigations demonstrate that iodo-Pt(IV) diamines can be photolyzed to cytotoxic species by visible light, but the aforementioned limitations must be overcome before this new class of Pt(IV) complexes can be used as antitumor agents. PMID- 8630188 TI - International Seminar-cum-School on Macromolecular Crystallographic Data at Calcutta, India. PMID- 8630189 TI - Photodamaging effects of tetraphenylporphycene in a human carcinoma cell line. AB - The photosensitizing effects of tetraphenylporphycene (TPPo) and light on HeLa cells, with emphasis on cell viability and the microtubular network, have been investigated. The survival of the cells incubated with TPPo was dependent on both drug concentration and light dose. The integrity of microtubules (MTs) was evaluated by immunofluorescence staining of alpha-tubulin. Interphasic and mitotic MTs were altered after 30 min of incubation with 5 microM TPPo followed by light irradiation. The degree of damage depended on light exposure time: 3 or 15 min corresponded to survival rates of approximately 50% or < 5%, respectively. Sublethal treatment led to the gradual accumulation of cells in metaphase, which caused an increase in the mitotic index (MI), with a maximum being found 6 h later. The number of cells in metaphase, as well as the MI, were within control values 24 h after sublethal photodynamic treatment. Lethal treatment provoked irreversible damage of interphasic and mitotic MTs. In addition, cell surface modifications such as bleb projections of the plasma membrane were also observed immediately after lethal treatment. It is concluded that both cellular structures, plasma membrane and MTs, constitute important targets for the phototoxic action of TPPo. PMID- 8630190 TI - Functional outcome from traumatic brain injury: unidimensional or multidimensional? AB - This study compared multidimensional and unidimensional measures of functional outcome from traumatic brain injury in a referred sample of 67 persons with moderate to severe traumatic brain injury who resided in the community or in a nonhospital facility. Four outcome indicators (independence in self-care, independence in travel, paid employment, and friendship) were correlated with the patients' ratings on two unidimensional outcome measures, the Disability Rating Scale (DRS) and Glasgow Outcome Scale (GOS), and on a multidimensional outcome measure, the Craig Handicap Assessment and Reporting Technique (CHART). The results showed that the CHART Occupation and Social Integration scales were associated with different outcome indicators, controlling for overall outcome as measured by the DRS and GOS. DRS and GOS ratings were associated more strongly with the self-care and travel indicators than with the employment or friendship indicators. The results support a multidimensional model of functional outcome from brain injury, suggest that the CHART may measure some outcome dimensions more specifically than do the DRS and GOS, and point to limitations of unidimensional outcome scales. PMID- 8630192 TI - The Uniform Data System for Medical Rehabilitation: report of first admissions for 1994. PMID- 8630191 TI - Effectiveness of an intensive outpatient rehabilitation program for postacute stroke patients. AB - The effectiveness of ongoing rehabilitation services for postacute stroke survivors is poorly documented. We designed a randomized control, single-blinded study to demonstrate the effectiveness of intensive outpatient therapy. The treatment intervention consisted of 1 hr each of physical and occupational therapy, four times per week, for 12 wk; therapy focused on neuromuscular facilitation and functional tasks. All subjects were screened before the therapies and after 3 mo and 9 mo. Forty-nine stroke survivors, who were at least l yr (mean, 2.9 yr) poststroke, were randomized with two treated patients to each control (no treatment supplied). All patients had received inpatient rehabilitation at the time of their acute stroke, but no patient had any ongoing therapy within the last 6 mo. The outcome measures included the Functional Independence Measure (FIM), Brunnstrom stages of motor recovery, timed mobility tasks, and the Jebson hand evaluation. We also evaluated the level of depression, self-esteem, and socialization. The treated patients demonstrated an improvement of 6.6 points over the 3 mo of therapy compared with only 1.5 points in the control group in the FIM motor score transformed using Rasch analysis. The change from time 0 to 3 mo was significant in the treated group but not in the controls. Treated patients maintained their gains at the 9-mo follow-up, and controls lost ground. The treated group improved in terms of socialization and self-esteem as evidenced by a lower Sickness Impact Profile, whereas the controls tended to get worse. There was a trend toward less depression, but this did not reach a P = 0.05 level of significance. This study demonstrates that significant functional gains can still be attained in the postacute stroke survivor, despite prior inpatient rehabilitation services. PMID- 8630193 TI - Pressure ulcers in SCI: does tension stimulate wound healing? AB - Pressure ulcers are an immense problem among older and disabled populations. Although there are many studies in the literature about the etiology, interface pressures, natural history, and epidemiology of skin breakdown, there is relatively little information about factors that stimulate the repair of body wall tissues after breakdown. Specifically, there is a paucity of information about the effects of mechanical stress on healing. This is a particularly important consideration for those areas, such as the perineal tissues, that bear large mechanical forces. The purpose of this paper is to discuss the research that addresses skin and tissue repair in response to tension. Two case studies that support the hypothesis that tension stimulates wound healing are presented from the Spinal Cord Injury Center at the Seattle VAMC. After weeks of nonhealing, each of these cases healed within 2 weeks of initiating a range of motion program that applied tension to the nonhealing portion of a myocutaneous flap. PMID- 8630194 TI - Management of conversion disorder. AB - Conversion disorder is a psychologically produced alteration or loss of physical functioning suggestive of a physical disorder. Conversion symptoms are often superimposed on organic disease and can be overlooked. Psychological techniques are central to the management of conversion symptoms. Principles of psychological management include the following: avoiding confrontation with the patient; avoiding reinforcement or trivializing the symptoms; reviewing results of tests and exams and creating an expectation of recovery; educating the patient by providing a benign explanatory model of symptoms; evaluating the patient's emotional adjustment and referring for psychotherapy when possible. Use of behavior therapy reinforcement and double-bind psychotherapy strategies may be helpful with more chronic or resistant symptoms, especially when there is a history of vague or excessive somatic complaints or significant secondary gain. Four case studies are presented to illustrate these principles. PMID- 8630195 TI - Analyzing the National Resident Match data. Are there too many physical medicine and rehabilitation training positions? A commentary. PMID- 8630196 TI - Qualitative inquiry. Expanding rehabilitation medicine's research repertoire: a commentary. PMID- 8630198 TI - The compleat physiatrist. PMID- 8630197 TI - Educational goals and objectives in physical medicine and rehabilitation for the medical school graduate. Association of Academic Physiatrists Undergraduate Education Committee Workgroup. PMID- 8630199 TI - Effect of mexiletine on spinal cord injury dysesthetic pain. AB - Severe pain occurs in 5-30% of the spinal cord-injured (SCI) population and is difficult to treat. Subarachnoid lidocaine has been used in selected patients with some success. Mexiletine, an analog of lidocaine that acts at Na+/K+ channels in the peripheral nerve, has been found effective in persons with diabetic dysesthetic neuropathy. The effect of mexiletine in the treatment of spinal cord dysesthetic pain was examined in this study. Fifteen patients were enrolled, and 11 patients completed the prospective, randomized, placebo controlled, double-blind, crossover design trial. Inclusion/exclusion criteria were carefully defined. A 1-wk washout period was followed by a 4-wk drug trial of either mexiletine (450 mg/day) or placebo. This was repeated for the second medication in the second arm of the study. Patients were followed weekly with McGill and visual analog pain scales. Baseline, midpoint, and endpoint Barthel function scores were recorded. The Wilcoxon's signed-rank test and paired t test were used for statistical analysis. Results showed no significant effect of mexiletine on SCI dysesthetic pain scales or Barthel index. In conclusion, in this trial, mexiletine did not appear to decrease spinal cord injury-related dysesthetic pain. PMID- 8630200 TI - Use of an obstacle course to assess balance and mobility in the elderly. A validation study. AB - An obstacle course, consisting of a series of 12 simulated functional tasks, has been developed to aid in the evaluation of rehabilitation interventions for elderly individuals with balance and mobility dysfunction. The validity of the obstacle course as a measure of balance and functional mobility was tested by comparing obstacle course performance scores of a sample of 237 community dwelling veterans, aged 67 to 93 yr, with clinical indicators of balance and mobility including age, history of falls, symptoms of balance dysfunction, medication use, orthostatic blood pressure changes, neurologic examination abnormalities, muscle strength, joint range of motion, self-reported activity level, and functional status. Significant correlations were found between obstacle course scores and all clinical indicators except blood pressure changes and age. Multiple regression analysis of selected variables showed that activity level and neurologic abnormalities best determined both qualitative and quantitative obstacle course performance. Non-fallers performed significantly better on the obstacle course than fallers. These results suggest that the obstacle course is valid and has potential as a useful tool in the evaluation of older persons with balance and mobility impairment. PMID- 8630201 TI - A new pressure ulcer risk assessment scale for individuals with spinal cord injury. AB - Each year, one-fourth of the 200,000 individuals with spinal cord injury in the United States develop pressure ulcers. No method currently exists, however, to accurately identify which of these individuals are at increased risk for development of pressure ulcers. We studied 219 spinal cord-injured patients, seen at a Veterans Affairs Medical Center, during a 6-yr period. Our goal was to develop a pressure ulcer risk assessment scale, specifically for persons with SCI. Each risk factor had to meet four criteria: (1) statistical association with pressure ulcer development; (2) biologically plausible mechanism; (3) literature support; (4) improved prediction. Among the 219 spinal cord-injured patients evaluated, 176 (80.4 percent) had a history of one or more pressure ulcers. Fifteen risk factors met the four criteria for inclusion into the risk assessment scale. They were as follows: restricted activity level, degree of immobility, complete spinal cord injury, urinary disease, impaired cognitive function, diabetes, cigarette smoking, residence in a nursing home or hospital, hypoalbuminemia, and anemia. Compared with the more general scales available, for quantifying the risk of pressure ulcer development, preliminary results suggest that this new scale is a significant improvement for the spinal cord-disabled. PMID- 8630202 TI - The otolaryngology lane on the information superhighway. PMID- 8630203 TI - The effect of intravenous dexamethasone in pediatric adenotonsillectomy. AB - OBJECTIVE: To determine whether the intravenous administration of dexamethasone sodium phosphate before tonsillectomy and adenoidectomy can reduce the morbidity from, and increase the safety of, this procedure. DESIGN: Prospective, randomized, double-blind, placebo-controlled clinical trial. SETTING: A university medical center, caring for both ambulatory and hospitalized children. PATIENTS: Eighty children aged 3 to 15 years undergoing tonsillectomy and adenoidectomy for either chronic tonsillitis or adenotonsillar hypertrophy (obstructive sleep apnea and/or upper airway obstruction). INTERVENTIONS: Forty one children received intravenous dexamethasone sodium phosphate (1 mg/kg up to 16 mg) and 39 received placebo before undergoing an electrocautery dissection tonsillectomy and adenoidectomy. MAIN OUTCOME MEASURES: Postoperative oral intake, pain, vomiting, temperature, and complications. RESULTS: Patients who received intravenous dexamethasone had significantly less trismus, vomiting, and elevations of temperature 6 hours after surgery and more oral intake (liquids and soft solids) at 24 hours. Three children, all of whom were in the placebo group, had emergency department visits for pain and dehydration. Each group had one child who had a secondary hemorrhage (no surgery needed), one child who had pneumonia, and one child who had night terrors. CONCLUSIONS: Treatment with intravenous dexamethasone before electrocautery tonsillectomy and adenoidectomy is safe, increases early postoperative oral intake, and decreases morbidity. PMID- 8630204 TI - Angiofibroma. Changes in staging and treatment. AB - OBJECTIVE: To identify specific preoperative tumor characteristics and potential surgical decisions that ultimately place a patient at a greater risk for tumor recurrence. DESIGN: The clinical presentation, management, and prognosis of 23 consecutive cases of juvenile nasopharyngeal angiofibroma were reviewed retrospectively from January 1, 1977, to June 30, 1993. A minimum follow-up of 12 months was necessary for study inclusion. SETTING: A single, tertiary care pediatric facility. INTERVENTIONS: All available preoperative imaging studies were reevaluated to ensure consistency in reporting. Preoperative computed tomography was performed in 21 patients, but only 18 scans were available for review. Preoperative angiography with embolization was performed in 21 of 23 patients. Surgical excision was the primary mode of treatment in 22 of 23 patients, and complete surgical excision was possible in 21 of 23 patients. MAIN OUTCOME MEASURES: The rate of recurrence was examined with respect to time of presentations, initial tumor stage, intraoperative blood loss, and surgical approach. RESULTS: When compared with patients without a recurrent tumor, there was no difference in age at presentation, primary symptom, or duration of symptoms before diagnosis. Preoperative tumor stage was found to be the primary factor affecting tumor recurrence. A recurrence rate of 21.7% (five of 23 patients) was identified after an average 6-year follow-up. A trend toward use of the midfacial degloving approach for surgical exposure was identified and was not associated with an increased risk of recurrence. All patients were ultimately cured of their tumor without the need for open craniotomy despite a 32% incidence of stage IIIA and IIIB tumors. No deaths were reported during the study. CONCLUSIONS: Juvenile nasopharyngeal angiofibromas are benign tumors occurring almost exclusively in adolescent males. Recent advances in radiographic imaging techniques allow for more accurate preoperative staging, especially in regard to skull base involvement. Recognition of the extent of the tumor before surgical extirpation reduces the risk of recurrence. PMID- 8630205 TI - The scala vestibuli for cochlear implantation. An anatomic study. AB - BACKGROUND: Traditionally, cochlear implantation has used the scala tympani (ST) for electrode insertion. When faced with ST ossification, the surgeon may elect to drill out the cochlea to accomplish partial electrode insertion. Theoretically, another option in this situation is to insert the electrode into the scala vestibuli (SV). OBJECTIVE: To determine whether or not the dimensions of the SV are sufficient to accommodate an electrode array so as to assess the feasibility of SV cochlear implantation. METHODS: The study of 20 normal human temporal bones, comparing the maximum diameter and surface area of the ST with those of the combined SV and scala media. RESULTS: The dimensions of the SV and scala media were comparable to those of the ST and appeared sufficient to accommodate a cochlear implant electrode array. CONCLUSION: It appears that the combination of SV and scala media is a viable alternative route for electrode insertion, at least on the basis of anatomic dimensions, in those cases in which the ST is obliterated. PMID- 8630206 TI - A contemporary analysis of acute mastoiditis. AB - BACKGROUND: Acute mastoiditis persists as a serious infection despite a dramatic decline in incidence coincident with the introduction of antibiotic therapy. OBJECTIVE: To assist the contemporary practitioner in the recognition and management of acute mastoiditis through the assessment of a large series of patients. DESIGN: Retrospective case series comprising 124 patients with acute mastoiditis. SETTING: Pediatric and adult otology referral center. MAIN OUTCOME MEASURES: Selected clinical parameters. Risk factors for necessity of surgical intervention and for increased length of hospitalization were analyzed by a stepwise logistic regression model. RESULTS: A history of antecedent acute otitis media was absent in 45% of patients. Pain (98%) was the most common presenting symptom. Physical signs included an abnormal-appearing tympanic membrane (88%), fever (83%), a narrowed external auditory canal (80%), and postauricular edema (76%). Streptococcus pneumoniae was the most commonly isolated organism. Mastoid surgery was required in 62% of the patients. An elevated white blood cell count (relative risk [RR], 7.4; P < .01), proptosis of the auricle (RR, 4.5; P = .03), and fever on admission (RR, 7.3; P = .05) were risk factors for surgical intervention. All 33 patients with complications (27%) proceeded to surgical intervention. The average length of hospital stay was 7.9 days. The strongest predictor for an increased length of hospital stay was whether the patient required surgery (RR, 3.7; P = .002). CONCLUSIONS: Acute mastoiditis remains a potentially serious otologic infection. Not all patients present with a classic history or physical examination. Therapeutic mastoidectomy is often required. PMID- 8630207 TI - Development of the Tinnitus Handicap Inventory. AB - OBJECTIVE: To develop a self-report tinnitus handicap measure that is brief, easy to administer and interpret, broad in scope, and psychometrically robust. DESIGN: A standardization study of a self-report tinnitus handicap measure was conducted to determine its internal consistency reliability and convergent and construct validity. SETTING: Audiology clinics in tertiary care centers in two sites. PARTICIPANTS: In the first investigation, 84 patients reporting tinnitus as their primary complaint or secondary to hearing loss completed the 45-item alpha version of the Tinnitus Handicap Inventory (THI). In the second investigation, 66 subjects also reporting tinnitus completed the 25-item beta version. OUTCOME MEASURES: Convergent validity was assessed using another measure of perceived tinnitus handicap (Tinnitus Handicap Questionnaire). Construct validity was assessed using the Beck Depression Inventory, Modified Somatic Perception Questionnaire, symptom rating scales (annoyance, sleep disruption, depression, and concentration), and perceived tinnitus pitch and loudness judgments. RESULTS: From the alpha version of the THI, we derived a 25-item beta version with the items grouped into functional, emotional, and catastrophic subscales. The total scale yielded excellent internal consistency reliability (Cronbach's alpha = .93). No significant age or gender effects were seen. Weak correlations were observed between the THI and the Beck Depression Inventory, Modified Somatic Perception Questionnaire, and pitch and loudness judgments. Significant correlations were found between the THI and the symptom rating scales. CONCLUSION: The THI is a self-report measure that can be used in a busy clinical practice to quantify the impact of tinnitus on daily living. PMID- 8630208 TI - Meta-analysis of the sensitivity and specificity of platform posturography. AB - OBJECTIVE: To compare the sensitivity and specificity of platform posturography with other vestibular tests for patients with peripheral vestibular deficits (PVD), Meniere's disease, benign paroxysmal positional vertigo (BPPV), and central nervous system-vestibular impairment (CNS). DATA SOURCES: A computed search was conducted using the Index Medicus database (1966-1994) and Current Contents Science Editions. STUDY SELECTION: Studies were selected for analysis if the article addressed the sensitivity and/or specificity of platform posturography, compared posturography with another objective test of vestibular function, identified the basis for abnormal test results, and reported the data with sufficient detail to calculate an effect size from a 2 x 2 contingency table. DATA EXTRACTION: A count of the normal and abnormal test results for posturography and the criterion standard were retrieved from each article, analyzed using a chi 2 statistic, and converted to an effect size. A positive effect size indicated that posturography identified abnormalities in patients who had normal tests on the criterion standard. DATA SYNTHESIS: Sensitivity and specificity of posturography were about 50%. The overall effect size was small (0.13) but positive. The diagnostic category had a significant influence on the predictive value of abnormal results (73% for Meniere's disease and BPPV, compared with 41% for PVD, and 44% for mixed CNS and PVD (F2,12 = 5.26, P = .02) and on the magnitude of the effect size (0.41 for mixed CNS and PVD compared with 0.22 for Meniere's disease and BPPV, and -0.10 for PVD (F2,12 = 13.95, P = .001). CONCLUSIONS: Platform posturography provides a measurable supplement to the standard vestibular examination. The enhancement was most notable when the target population included patients with CNS deficits. PMID- 8630210 TI - Central auditory dysfunction, cognitive dysfunction, and dementia in older people. AB - OBJECTIVES: To determine in older people the relation between auditory dysfunction and cognitive dysfunction, and if central auditory test abnormalities predict the onset of clinical dementia or cognitive decline. DESIGN: Prospective population-based cohort study. SETTING: Framingham Heart Study outpatient biennial examinations 18 and 21. PARTICIPANTS: Members of the Framingham Heart Study cohort with normal findings from cognitive screening tests at biennial examination 18. MEASUREMENTS: Peripheral audiometric thresholds and word recognition in quiet; Synthetic Sentence Identification with Ipsilateral Competing Message (SSI-ICM); Mini-Mental State Examination; and detailed neuropsychological testing of subjects with abnormal findings from the Mini Mental State Examination. Relative risk of dementia was determined using age adjusted Cox proportional hazards regression models. RESULTS: Hearing loss significantly lowered performance on the verbal parts of the Mini-Mental State Examination. The relative risk of subsequent clinical dementia or cognitive decline was 6 in subjects with very poor scores (< 50%) in one ear on the SSI-ICM (P = .02); the relative risk was 12.5 if the poor scores were present in both ears (P = .001). CONCLUSIONS: Central auditory dysfunction precedes senile dementia in a significant number of cases and may be an early marker for senile dementia. Hearing tests should be included in the evaluation of persons older than 60 years and in those suspected of having cognitive dysfunction. PMID- 8630209 TI - Expression of messenger RNA for keratinocyte growth factor in human cholesteatoma. AB - OBJECTIVE: To understand the interaction between cholesteatoma epithelium and subepithelial connective tissue as a paracrine regulation by keratinocyte growth factor. DESIGN: Preparation of a specific DNA probe from human fetal fibroblast and detection of localization of keratinocyte growth factor messenger RNA in subepithelial granulation tissue of middle-ear cholesteatoma by a nonradioactive in situ hybridization method. PARTICIPANTS: The cholesteatoma specimens were excised from 12 patients during surgery. Normal skin specimens collected from the external ear canal of six patients were used as controls. RESULTS: Signals specific for keratinocyte growth factor messenger RNA were not expressed in the normal skin of the external ear canal, but were observed in fibroblasts of subepithelial connective tissue of cholesteatoma specimens. Signals were observed only in specimens collected from patients whose subepithelial connective tissue was thick and proliferated and whose inflammation was strong. CONCLUSIONS: A paracrine regulation mechanism involving keratinocyte growth factor may exist for proliferation of epithelium of cholesteatoma. The subepithelial connective tissue of cholesteatoma may play an important role in the proliferation and development of the cholesteatoma, especially under inflammatory conditions. PMID- 8630211 TI - Transforming growth factor beta 1 improves wound healing and random flap survival in normal and irradiated rats. AB - OBJECTIVES: To evaluate the effect of chronic irradiation on wound healing and random flap survival (FV), and the effect of transforming growth factor beta 1 (TGF-beta 1) in this setting using an animal model. DESIGN: A randomized, controlled study with four groups of rats to study the effect of irradiation 4 months before surgical intervention. The effect of TGF-beta 1 on FV and wound healing also was evaluated in the irradiated and nonirradiated groups. SUBJECTS: Ninety-five rats were available for evaluation. Group 1 (n = 10) was the control; group 2 (n = 28) received TGF-beta 1; group 3 (n = 28) received radiation therapy; and group 4 (n = 29) received radiation therapy and TGF-beta 1. INTERVENTION: The irradiated groups received 15 Gy to their dorsal skin. Four months later all received McFarlane skin flaps. Groups 2 and 4 received topical TGF-beta 1, 4 micrograms, to the bed of the flap; groups 1 and 3 received saline. On postoperative day 7 all rats were evaluated for tensile strength and FV, and histologic staining with hematoxylin-eosin for collagen and TGF-beta 1 was done. The slides were evaluated in a "blinded" fashion. RESULTS: Irradiation decreased tensile strength and FV, but not to a notable degree. Transforming growth factor beta 1 improved tensile strength in the irradiated (P = .04, Student's t test) and nonirradiated groups (P = .05, Student's t test). Transforming growth factor beta 1 also improved FV in all groups, but significantly in the irradiation plus TGF-beta 1 group (P = .001, Student's t test). The TGF-beta 1 group had the most mature collagen present at the wound edge. No qualitative difference was seen in the immunohistochemical staining for the four groups. CONCLUSIONS: Transforming growth factor beta 1 improves wound healing and random FV in radiated and nonirradiated rat skin. Further study is needed to determine the radiation dose necessary to create an "impaired wound-healing model" in rats, and the optimum dose of TGF-beta 1 in this setting. PMID- 8630212 TI - Microcystic adnexal carcinoma. Case report and review of the literature. AB - Microcystic adnexal carcinoma is an aggressive, locally destructive cutaneous neoplasm with a high rate of recurrence. This tumor is often misdiagnosed clinically and histologically. It usually occurs in middle-aged to older adults. We describe a 44-year-old man with a large microcystic adnexal carcinoma that was present for more than 20 years. The tumor invaded the perichondrium, muscle, nerve, and blood vessel adventitia. A review of the literature suggests that these tumors are often histologically misdiagnosed because the biopsy specimens may be too small to reveal all the characteristic histologic features. The clinical presence of marked induration, a smooth surface, and, possibly, sensory changes should alert the clinician to the possibility of this neoplasm. The initial biopsy specimen must be large enough to demonstrate the identifying histologic features. Mohs surgery is currently the treatment of choice for microcystic adnexal carcinoma, as it often spreads far beyond clinically evident tumor. PMID- 8630213 TI - Microvascular replantation of a traumatically amputated ear. AB - We describe the replantation of a traumatically severed auricle using microvascular anastomosis to reestablish blood flow to the ear. Microvascular reattachment of the severed auricle occurred 10 hours after the trauma. Postoperatively, adjunctive measures, including anticoagulation and the use of medicinal leeches, were used to relieve venous congestion of the replanted auricle. The replanted auricle healed completely with 100% survival, resulting in an essentially normal-appearing external ear. In the management of a traumatically severed auricle, microvascular replantation should be considered as the intervention of first choice in selected cases. If the procedure is successful, the cosmetic results are excellent; if it is not successful, a number of other reconstructive techniques remain as options. PMID- 8630214 TI - Coccidioides immitis subperiosteal abscess of the temporal bone in a child. AB - A destructive granulomatous lesion of the temporal bone caused by Coccidioides immitis disseminated from a pulmonary lesion was found in a 4-year-old immunocompetent child. To our knowledge, it is the first case of coccidioidomycosis of the temporal bone reported in the world literature. The child presented with pain in her right ear and a 6-month history of intermittent fever, which partially responded to multiple courses of antibiotics. A tender erythematous postauricular swelling consistent with a subperiosteal abscess subsequently developed over 1 month. A mastoidectomy showed granulation tissue with pockets of purulence, and histologic evaluation of the specimen revealed spherules of C immitis, later verified by culture. The patient responded to intravenous amphotericin B therapy, without evidence of disease recurrence. Coccidioides immitis is endemic in regions of the Southwestern United States, with extremely infectious characteristics and relative resistance to medical therapy. Coccidioidomycosis should be considered in the differential diagnosis of a granulomatous lesion of the temporal bone. PMID- 8630215 TI - Pathologic quiz case 1. Temporomandibular pigmented villonodular synovitis. PMID- 8630216 TI - Pathologic quiz case 2. Bilateral herpes zoster oticus. PMID- 8630217 TI - Cochlear implantation in a patient after removal of an acoustic neuroma. PMID- 8630218 TI - Prevalence of IgE-mediated hypersensitivity in children with adenotonsillar disease. PMID- 8630219 TI - Cupulolithiasis and canalolithiasis are two photos of a story called 'the destiny of the otoconia'. PMID- 8630220 TI - Prior authorship. PMID- 8630222 TI - Terminal duct carcinoma. PMID- 8630221 TI - Cocaine and phenylephrine. PMID- 8630223 TI - Control of tumor progression by maintenance of apoptosis. AB - The ability to induce multiple apoptotic regressions of an androgen-dependent tumor cell population by repeated cycles of androgen withdrawal and replacement may be advantageous in therapeutic strategies aimed at delaying or preventing tumor progression. With greater insight into factors that either initiate or limit apoptosis, more efficient application of intermittent therapy might be achieved, especially if methods could be devised to increase the length or number of treatment cycles. Both calreticulin and clusterin represent proteins with a potential role in the regulation of apoptosis. Calreticulin may inhibit target gene transcription by interacting with steroid hormone receptors, thereby masking their DNA-binding sites and triggering the onset of the apoptotic process. Clusterin, on the other hand, is a membrane-stabilizing protein that appears to be involved in limiting the autophagic lysis of epithelial cells during apoptosis. Also, the increasing tendency for nuclear localization of clusterin after androgen withdrawal may preserve the nuclear environment, limiting the lethal effect of treatment. Thus, tumor progression, characterized by the loss of apoptotic potential, appears to be linked in part to the inappropriate activation of TRPM-2 gene, which accounts for the constitutive expression of clusterin. PMID- 8630224 TI - Growth factors as mediators of androgen action during male urogenital development. AB - Studies on the developing prostate and seminal vesicle suggests that androgens act via mesenchymal androgen receptors to elicit synthesis and secretion of keratinocyte growth factor and probably other paracrine factors that regulate epithelial growth and morphogenesis. Clearly, the overall regulation of prostatic epithelial growth and ductal branching morphogenesis is complex and multifactorial, involving the interaction of both positive and negative regulators, extracellular matrix, cell surface receptors for growth factors, and vascularization. Future progress in our understanding of normal and abnormal prostatic growth will certainly be dependent upon the utilization of appropriate, biologically relevant models to examine the respective roles of these complex developmental process. PMID- 8630225 TI - Future directions in prostate cancer treatment: an oncologist's perspective. AB - Prostate cancer has become one of the most important national health problems, as its incidence increases yearly. Conventionally, single-modality local therapy has been the standard of care for clinically localized stage disease and systemic therapy, in the form of primary or combined androgen deprivation, is reserved for advanced, usually metastatic, prostate cancer. Despite improvements in local therapy, a significant number of patients with clinically localized prostate cancer will suffer a systemic relapse after potentially curative local therapy and, despite 50 years of experience in hormone therapy, significant survival prolongation for patients with metastatic prostate cancer remains a challenge. This paper explores potential future systemic therapy strategies in the treatment of prostate cancer. Specifically, the role of adjuvant systemic therapy as part of a multimodality treatment approach for localized prostate cancer and alternative applications of hormone and chemohormone therapies for patients with metastatic disease are discussed. PMID- 8630226 TI - Neuroendocrine differentiation and hormone-refractory prostate cancer. AB - There is an intriguing link between differentiation of neuroendocrine cells and tumor progression in prostate cancer. Neuroendocrine differentiation appears to be associated with the androgen-independent state, for which there is currently no successful therapy. However, the role of the neuroendocrine cells is complex, both in the normal prostate and in the pathway toward malignancy. One important area of research is to investigate the hormones expressed by prostatic neuroendocrine cells and, in particular, to elucidate their significance to androgen independence. It is hoped that an understanding of the specific roles of hormones such as somatostatin, bombesin, and serotonin in prostate cancer may lead to improved therapeutic approaches. PMID- 8630227 TI - Metastasis suppressor genes for prostate cancer. AB - To examine the role of human chromosomes in the development of metastatic prostate cancer, we introduced a copy of human chromosomes into highly metastatic Dunning R-3327 rat prostatic cancer cells by microcell-mediated chromosome transfer. Each microcell hybrid clones containing human chromosomes 8, 10, 11, and 17, respectively, showed decreased ability to metastasize to the lung, without any loss of tumorigenicity. This finding demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in human chromosome 10, 11, and 17 studies. In the human chromosome 8 study, irradiated microcell mediated chromosome transfer was performed to enrich chromosomal arm deletions of human chromosome 8. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained metastasis suppressor gene(s) for prostate cancer. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes 8, 10, and 11 were located on 8p23-q12, 10q, 11p13-11.2, respectively. Further analyses are proposed to confirm the potentially useful advantage of this assay system to identify metastasis suppressor gene(s) for prostate cancer. PMID- 8630228 TI - DNA methylation, molecular genetic, and linkage studies in prostate cancer. AB - Molecular biologic studies have now identified a number of important genetic and epigenetic mechanisms that cause alterations in growth and differentiation genes in prostate cancer. In addition to DNA deletion and point mutation, DNA methylation represents a new paradigm for the inactivation of tumor suppressor or growth suppressor genes. The identification of new genes, including a prostate cancer susceptibility locus, may furnish further insight into the molecular characteristics of prostate cancer and permit the early identification of affected individuals. PMID- 8630229 TI - Genetic regulation of androgen action. AB - The androgen receptor (AR) belongs to the superfamily of nuclear receptors that employ complex genetic mechanisms to guide the development and physiological functions of different target tissues. Upon interaction with its cognate hormone, AR activates or represses gene transcription through association with specific DNA elements and/or proteins. This review summarize briefly our current view of androgen action, with a special emphasis on genetic factors that may modulate the response. PMID- 8630230 TI - Role of androgen in prostate growth and regression: stromal-epithelial interaction. AB - The prostate is a secretory gland in which secretions produced by its cells are transported through the ductal system and discharged into the urethra. Each prostatic ductal system can be traced from the opening in the urethra as a single tubular structure from which branches and sub-branches are formed in a manner like the branching pattern of a tree. Owing to the distance from the urethral orifice, regions of the prostatic ductal system can be classed into the proximal, intermediate, and distal regions. In the distal region, the tips of the ductal system (equivalent to the top of the tree), the epithelial cells are tall and columnar in shape, and active in cell division. Cells of the intermediate region (equivalent to the majority of the body of the tree) are also of tall and columnar type but are mitotically quiescent. Cells in this region are the only ones that have the ability to secrete. Cell death is not evident in these two regions. Cells in the proximal region, a region that is immediately adjacent to the urethra (equivalent to the tree trunk) are low cuboidal or flat in shape and are actively undergoing cell death. These observations indicate that cells in different regions of the prostatic ductal system are not the same, even though they are exposed to the same circulating level of androgen. The recognition of this regional heterogeneity in cell shape and activity in the ductal system has advanced our understanding of the basic biology of the prostate. For example, our understanding of the cellular mechanism of action of androgen in the prostate should be re-evaluated. In the past, the convention concept of androgen action has been a stimulatory one, and a depletion of this androgenic support leads to prostatic cell death. The recognition of a regional heterogeneity in cellular activity has created a situation in which all prostatic cells in the same prostatic ductal system are exposed to the same level of circulating androgen. However, these cells are not responding to the same amount of androgen in the same manner: some are multiplying while others are dying. These observations indicate that the effect of androgen vary according to the location of prostatic cells in the ductal system. A new concept of the role of androgen in prostatic growth, differentiation, and cell death is discussed. PMID- 8630231 TI - A glimpse at the future of some endocrine aspects of prostate cancer. AB - There is new scientific information on the relationship between androgenic hormones and prostate cancer. In spite of a plausible association between androgenic hormones and the development of prostate cancer, precise mechanisms are lacking. Racial variability in the incidence of prostate cancer may be related to hormonal influences but suggestive observations were noted in studies with a small sample size. Currently, a serum hormone profile will not identify groups or individuals at higher risk of prostate cancer. The earlier integration of hormonal therapy into treatment plans for prostate cancer will substantially alter the model. Early treatment may be vastly more expensive, and the long-term benefit is uncertain. The aggregate costs of hormonal treatment for prostate cancer to the health care system will require a re-evaluation of less expensive, traditional methods for androgen deprivation. PMID- 8630232 TI - Responses of LNCaP prostatic adenocarcinoma cell cultures to LY300502, a benzoquinolinone human type I 5alpha-reductase inhibitor. AB - We evaluate the metabolic inhibitory, antiproliferative, and antisecretory effects of LY300502, a benzoquinolinone human-specific type I-selective steroid 5alpha-reductase inhibitor in LNCaP human prostatic adenocarcinoma cell cultures. Reductive metabolism of [3H-T] in the LNCaP cells was inhibited in a concentration-dependent manner by LY300502 (IC50 approximately 5.77 nM). The proliferative responses of LNCaP cells to LY300502 were examined in the presence of 0.1 NM testosterone (T), a concentration that stimulates maximal LNCaP cell numbers 40% above control levels. LY300502 significantly anatagonized T-induced stimulation of LNCaP cellular proliferation at concentrations greater that 10 nM (P<0.05), and at 1,000 nMcompletely blocked the mitogenic effects of T on LNCaP cells. In the absence of androgen, LY300502 had no effect on LNCaP cellular proliferation. In the presence of 100 nM T, an androgen concentration that maximally stimulates in vitro PSA production, LY300502 significantly antagonized T-induced PSA secretion at a concentration equal to or greater than 30 nM (P<0.05). These studies provide the basis for additional investigations into the pathophysiologic significance of type I 5alpha-reductase to prostatic cancer and the potential utility of selective inhibitors as therapeutic agents. PMID- 8630233 TI - Benign prostatic hyperplasia: a review of its histogenesis and natural history. AB - Although the exact etiology of benign prostatic hyperplasia (BPH) is not well defined, it is thought to occur as a result of epithelial-stromal interactions in the appropriate hormonal milieu. Benign prostatic hyperplasia originates in the periurethral and transition zones of the prostate in a microscopic (histologically identifiable) state as early as the third decade of life. With advancing age and the presence of androgens, approximately 50% of microscopic BPH will develop into macroscopic (palpably enlarged prostate) BPH. However, clinically significant BPH, necessitating treatment, will develop in only 50% of men with an enlarged prostate gland. In the United States, the estimated risk of a 50-year old man undergoing therapeutic intervention in his lifetime is approximately 40%. If left untreated, a significant number of symptomatic patients will remain stable or improve without adverse sequelae. PMID- 8630234 TI - Cellular biology of prostatic growth factors. AB - The etiology of prostate cancer or of benign prostatic hyperplasia (BPH) is essentially not understood. It is becoming clear, however, that major determinants of the malignant or hyperplastic phenotype are various growth stimulatory or -inhibitory factors and their receptors, whose inappropriate expression or loss disrupts normal regulation of cell proliferation and differentiation. Cell culture has been a versatile tool for studying the expression and interaction of growth factors in prostatic cells. Immunohistochemistry and in situ hybridization have provided a view of growth factor expression coupled with histopathology. The eventual definition of autocrine, paracrine, and endocrine pathways of growth regulation in the human prostate will facilitate the design of new preventative, diagnostic, and therapeutic strategies. PMID- 8630235 TI - Androgen regulation of gene expression. AB - Androgen receptors are important transcription factors regulating the expression of a number of genes in androgen-responsive cells and may play a role in prostate cancer. This article describes transcriptional suppressors and other transcription factors which may play important roles in modulating the expression of androgen receptors. PMID- 8630237 TI - Role of dihydrotestosterone in androgen action. AB - Androgen action differs from that of most hormones in the testosterone, the major androgen secreted from the testes and the most abundant androgen in the circulation of men, is not the principal androgen within target cells. Indeed, abundant evidence indicates that most androgen actions are mediated by the 5alpha reduced metabolite dihydrotestosterone that is formed in target tissues. The conversion of testosterone to dihydrotestosterone is mediated by two isoenzymes; mutations in the steroid 5alpha-reductase 2 gene cause a rare autosomal-recessive form of male pseudohermaphroditism, and inhibition of this enzyme causes regression of the prostate gland. Dihydrotestosterone binds more tightly to the androgen receptor that does testosterone, but it is not clear whether this property is the sole explanation for its essential role in androgen action. Nor is it clear whether some androgenic effects may be mediated by circulating dihydrotestosterone acting as a hormone. PMID- 8630236 TI - 5alpha-reductase inhibitors/finasteride. AB - Benign prostatic hyperplasia (BPH) is a disease diagnosed by the presence of prostatic enlargement and lower-tract urinary obstruction. Finasteride (Proscar), is a potent and specific inhibitor of 5alpha-reductase, which inhibits the conversion of testosterone (T) to dihydrotestosterone (DHT) an important promoter of prostatic growth. It has provided a new therapeutic alternative for the treatment of BPH. The safety and efficacy of finasteride in the treatment of symptomatic BPH have been demonstrated by two multi-center placebo-controlled studies. After 12 months of treatment with 5 mg finasteride daily, prostate volume, DHT and prostate-specific antigen (PSA) levels were reduced and maximum urinary flow rates and symptom scores were improved. Finasteride was well tolerated. Upon completion of the controlled studies, patients were eligible to enter an open-label extension study in which all patients received 5 mg finasteride. Approximately half of the 543 patients randomized to the 5 mg finasteride group in the controlled studies have now been treated with 5 mg finasteride continuously for 3 years. The data provided by this group of patients on the long-term safety and efficacy of finasteride in the treatment of symptomatic BPH are reviewed. PMID- 8630238 TI - Provocative aspects of androgen genetics. AB - Androgens play a key role in prostate structure and function, leading to the hypothesis that effects of the hormone are an important component in the development of prostatic disease. Differences in serum testosterone levels and 5alpha-reductase activities between ethnic and racial groups have been implicated in the variable incidence of prostate cancer among certain populations. Androgen receptors transduce the steroid signal within cells, but attempts to correlate differences in receptor levels with prostatic disease have been unsuccessful. However, molecular studies of androgen receptor gene structure have recently provided new insights toward defining a genetic basis for the pathology associated with three diseases--spinal bulbar muscular atrophy, breast carcinoma, and prostate cancer--affecting middle-aged and older men. In summary, epidemiologic data on androgen biosynthesis, metabolism, and action of androgens and molecular genetic analysis of gene structure have led to a new understanding of the interrelationships between environmental and genetic factors that may impact on the incidence of certain pathologic conditions in men. PMID- 8630240 TI - Muscarinic modulation of sodium current by activation of protein kinase C in rat hippocampal neurons. AB - Phosphorylation of brain Na+ channels by protein kinase C (PKC) decreases peak Na+ current and slows macroscopic inactivation, but receptor-activated modulation of Na+ currents via the PKC pathway has not been demonstrated. We have examined modulation of Na+ channels by activation of muscarinic receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording. Application of the muscarinic agonist carbachol reduced peak Na+ current and slowed macroscopic inactivation at all potentials, without changing the voltage dependent properties of the channel. These effects were mediated by PKC, since they were eliminated when the specific PKC inhibitor (PKCI19-36) was included in the pipette solution and mimicked by the extracellular application of the PKC activator, OAG. Thus, activation of endogenous muscarinic receptors on hippocampal neurons strongly modulates Na+ channel activity by activation of PKC. Cholinergic input from basal forebrain neurons may have this effect in the hippocampus in vivo. PMID- 8630239 TI - A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. AB - We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K(ATP) channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell KATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K(ATP) channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K(ATP) channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors. PMID- 8630241 TI - ATP serves as a negative feedback inhibitor of voltage-gated Ca2+ channel currents in cultured bovine adrenal chromaffin cells. AB - Modulation of voltage-gated Ca(2+) channel current (I(Ca)) regulates secretion of catecholamines from adrenal chromaffin cells. Previous work demonstrated that I(Ca) can be augmented by phosphorylation to increase secretion or that inhibition of I(Ca) results in diminished catecholamine secretion. In the current manuscript, we show that stimulation of chromaffin cells results in the release of an "endogenous inhibitor" that suppresses I(Ca). The inhibition is due to the secretion of ATP, which is stored at high concentrations in secretory granules and is coreleased with catecholamines upon stimulation. The ATP exerts its actions through P(2 gamma) purinoceptors and inhibits both N- and P/Q-type Ca (2+) channels in a voltage-dependent manner but with different efficacies. Overall, we have identified and characterized a negative feedback pathway that may serve as an important regulatory mechanism for catecholamine secretion in chromaffin cells. PMID- 8630242 TI - Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. AB - We used serial cysteine mutagenesis to study the structure of the outer vestibule and selectivity region of the voltage-gated Na channel. The voltage dependence of Cd(2+) block enabled us to determine the locations within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances of the substituted cysteines were compared with the differential sensitivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, while the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage dependence for Cd(2+) block produced changes in monovalent cation selectivity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recognized as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrate the membrane to varying depths to form the channel pore. PMID- 8630243 TI - A transgenic rat model of Charcot-Marie-Tooth disease. AB - Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy in humans and has been associated with a partial duplication of chromosome 17 (CMT type 1A). We have generated a transgenic rat model of this disease and provide experimental evidence that CMT1A is caused by increased expression of the gene for peripheral myelin protein-22 (PMP22, gas-3). PMP22-transgenic rats develop gait abnormalities caused by a peripheral hypomyelination, Schwann cell hypertrophy (onion bulb formation), and muscle weakness. Reduced nerve conduction velocities closely resemble recordings in human patients with CMT1A. When bred to homozygosity, transgenic animals completely fail to elaborate myelin. We anticipate that the CMT rat model will facilitate the identification of a cellular disease mechanism and serve in the evaluation of potential treatment strategies. PMID- 8630244 TI - Drug addiction: the yin and yang of hedonic homeostasis. PMID- 8630245 TI - Under siege: The brain on opiates. PMID- 8630246 TI - Addiction to cocaine and amphetamine. PMID- 8630247 TI - Molecular and cellular aspects of nicotine abuse. PMID- 8630248 TI - Alcohol addiction: an enigma among us. PMID- 8630249 TI - Ion channels: too complex for rational drug design? PMID- 8630250 TI - Mechanisms of neuronal degeneration in Alzheimer's disease. PMID- 8630251 TI - A septum-derived chemorepulsive factor for migrating olfactory interneuron precursors. AB - During mammalian brain development, immature neurons often migrate considerable distances. A dramatic example is the rostral migration of olfactory interneuron precursors from near the septum to the olfactory bulb via a subventricular pathway. Heterotopic transplantations establish that this migration is unidirectional and that guidance cues operate over a considerable distance. The guidance cues for this translocation have not been identified, and the present studies provide evidence that a diffusible chemorepulsive factor, secreted by caudal septum but not by other tissue regions surrounding the pathway, may be involved. This activity is functionally distinct from that produced by factors that influence vertebrate axon outgrowth, such as netrin-1, netrin-2, and collapsin-1/semaphorin-III. The presence of this activity in the floor plate/ventral spinal cord as well as the septum suggests that it may influence other types of cell migration. PMID- 8630252 TI - Functional analysis of the weaver mutant GIRK2 K+ channel and rescue of weaver granule cells. AB - In the neurological mutant mouse weaver, granule cell precursors proliferate normally in the external germinal layer of the cerebellar cortex, but fail to differentiate. Granule neurons purified from weaver cerebella have greatly reduced G protein-activated inwardly rectifying K+ currents; instead, they display a constitutive Na+ conductance. Expression of the weaver GIRK2 channel in oocytes confirms that the mutation leads to constitutive activation, loss of monovalent cation selectivity, and increased sensitivity to three channel blockers. Pharmacological blockade of the Na+ influx in weaver granule cells restores their ability to differentiate normally. Thus, Na+ flux through the weaver GIRK2 channel underlies the failure of granule cell development in situ. PMID- 8630253 TI - Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK. AB - Nerve-induced clustering of the nicotinic acetylcholine receptor (AChR) requires rapsyn, a synaptic peripheral membrane protein, as well as protein-tyrosine kinase activity. Here, we show that rapsyn induces the clustering of the synapse specific receptor-tyrosine kinase MuSK in transfected QT-6 fibroblasts. Furthermore, rapsyn stimulates the autophosphorylation of MuSK, leading to a subsequent MuSK-dependent increase in cellular tyrosine phosphorylation. Moreover, rapsyn-activated MuSK specifically phosphorylated the AChR beta subunit, the same subunit that is tyrosine phosphorylated during innervation or agrin treatment of muscle. These results suggest rapsyn may mediate the synaptic localization of MuSK in muscle and that MuSK may play an important role in the agrin-induced clustering of the AChR. PMID- 8630254 TI - A local action of neurotrophin-3 prevents the death of proliferating sensory neuron precursor cells. AB - The role of neurotrophin-3 (NT-3) in early development of the dorsal root ganglion was investigated. Excessive cell death in the dorsal root ganglion of mice that carry a deleted NT-3 gene (NT-3-/- mice) preceded the period of programmed cell death, detected by the TUNEL method, and caused a reduction in the number of proliferating precursors without altering the proportion of proliferating cells to total number of neurons. Furthermore, the majority of proliferating cells detected by bromodeoxyuridine incorporation also stained with the TUNEL method. NT-3 mRNA was expressed locally in the embryonic, but not the postnatal dorsal root ganglion. Most cultured early embryonic NT-3-/- neurons died in the absence of exogenous NT-3 as did the wild-type neurons when cultured with NT-3 neutralizing antibodies, suggesting that NT-3 acts locally to prevent the death of proliferating sensory precursor cells during neurogenesis. Thus, NT 3 may inflict constraints on the number of proliferating precursor cells and thereby affect the number of neurons generated during development of the peripheral nervous system. PMID- 8630255 TI - Induction of CRE-mediated gene expression by stimuli that generate long-lasting LTP in area CA1 of the hippocampus. AB - Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, we made mice transgenic for a CRE regulated reporter construct. We focused on long-lasting long-term potentiation (L-LTP), because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus. PMID- 8630256 TI - Subunit stoichiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function. AB - Cyclic nucleotide-gated (CNG) ion channels are multimeric structures containing at least two subunits. However, the total number of subunits per functional channel is unknown. To determine the subunit stoichiometry of CNG ion channels, we have coexpressed the 30 pS conductance bovine retinal channel (RET) with an 85 pS conductance chimeric retinal channel containing the catfish olfactory channel P region (RO133). When RO133 and RET monomers are coexpressed, channels with four distinct intermediate conductances are observed. Dimer constructs reveal that two of these conductance levels arise from channels with the same subunit composition (2 RO133:2 RET) but distinct subunit order (like subunits adjacent to each other versus like subunits across from each other). Thus, the data demonstrate that cyclic nucleotide-gated ion channels are tetrameric like the related voltage gated potassium ion channels; the order of subunits affects the conductance of the channel; and the channel has 4-fold symmetry in which four asymmetric subunits assemble head to tail around a central axis. PMID- 8630257 TI - Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors. AB - Drosophila vision involves a G protein-coupled phospholipase C-mediated signaling pathway that leads to membrane depolarization through activation of Na+ and Ca2+ channels. InaD mutant flies have a M442K point mutation and display a slow recovery of the Ca2+ dependent current. We report that anti-INAD antibodies coimmunoprecipitate TRP, identified by its electrophoretic mobility, cross reactivity with anti-TRP antibody, and absence in a null allele trp mutant. This interaction is abolished by the InaD point mutation in vitro and in vivo. Interaction was localized to the 19 amino acid C-terminus of TRP by overlay assays, and to the PDZ domain of INAD, encompassing the point mutation. Given the impaired electrophysiology of the InaD mutant, this novel interaction suggests that INAD functions as a regulatory subunit of the TRP Ca2+ channel. PMID- 8630259 TI - Increased interleukin-10 messenger RNA expression in atopic allergy and asthma. AB - Interleukin-10 (IL-10) inhibits T-lymphocyte proliferation and production of cytokines. We have examined expression of IL-10 messenger RNA (mRNA) in atopic asthma and in allergen and tuberculin skin responses by in situ hybridization. The proportion of bronchoalveolar lavage (BAL) cells positive for IL-10 mRNA was increased in a group of 10 symptomatic asthmatics when compared with control subjects (17.5% versus 5.2% BAL cells positive; P < 0.001). In a separate group of six mild atopic asthmatics, there was an increased proportion of BAL cells positive for IL-10 mRNA 24 h after allergen inhalation challenge compared with diluent challenge BAL from the same subjects (24% versus 10%; P < 0.005). By simultaneous in situ hybridization and immunocytochemistry, IL-10 mRNA was localized to both CD3+ T cells and CD68+ alveolar macrophages in BAL, with a significantly more prominent T-cell signal in the symptomatic asthmatics compared with control subjects and after allergen challenge compared with diluent challenge of the mild asthmatic subjects. It has been suggested that IL-10 production is a late event after T-cell activation. To examine kinetics and specificity of IL-10 mRNA expression, skin biopsies were obtained from atopic, tuberculin-sensitive subjects at 1, 6, and 48 h after cutaneous injection of allergen or tuberculin. With both stimuli, there was an increase in IL-10 mRNA positive cells at 6 h when compared with control sites injected with appropriate diluent which were biopsied 24 h after injection (P < 0.01 for allergen and P < 0.02 for tuberculin). These findings are compatible with the hypothesis that IL 10 mRNA is expressed in both macrophages and T lymphocytes in the airway in asthma and that IL-10 mRNA expression is induced from T lymphocytes in response to allergen. This response may also occur in other types of cell-mediated inflammation. PMID- 8630260 TI - Regulation of gene transcription in respiratory epithelial cells. PMID- 8630258 TI - Mutations in a C. elegans Gqalpha gene disrupt movement, egg laying, and viability. AB - We find that C. elegans egl-30 encodes a heterotrimeric G protein a subunit more than 80% identical to mammalian Gqalpha family proteins, and which can function as a Gqalpha subunit in COS-7 cells. We have identified new egl-30 alleles in a selection for genes involved in the C. elegans acetylcholine response. Two egl-30 alleles specify premature termination of Gqalpha and are essentially lethal in homozygotes. Animals homozygous for six other egl-30 alleles are viable and fertile, but exhibit delayed egg laying and leave flattened tracks. Overexpression of the wild-type egl-30 gene produces the opposite behavior. Analysis of these mutants suggest that their phenotypes reflect defects in the muscle or neuromuscular junction. PMID- 8630261 TI - Characterization of the human surfactant protein D promoter: transcriptional regulation of SP-D gene expression by glucocorticoids. AB - We have previously described the characterization of genomic clones encoding the entire translated sequence of human pulmonary surfactant protein D (SP-D). We now describe the characterization of a genomic fragment (H5E7) that encodes the entirety of the first translated exon (Exon 2), Intron 1, a short transcribed untranslated sequence (Exon 1; 39 bp), and approximately 4 kb of sequence upstream from the transcription initiation site. The start site was identified by 5'-RACE-PCR cloning and primer extension. A putative TATA box (CATAAATA) was identified approximately 30 bp upstream of the start site. Complete sequencing of a HindIII/SacI fragment (HS-1674) encoding approximately 1.7 kb of sequence 5' to the TATA demonstrated multiple potential cis-regulatory elements including half site glucocorticoid response elements (GRE), a canonical AP-1 consensus, several AP-1 like sequences, E-box sequences, NF-IL-6 and PEA3 motifs, and putative interferon response elements. H441 lung adenocarcinoma cells, which express low levels of SP-D mRNA, and liver HepG2 cells, were transiently co-transfected with chloramphenicol acetyl transferase (CAT) reporter constructs containing up to 3,000 base pairs of upstream sequence, and with constructs encoding beta-gal. H441 cells transfected with constructs containing at least 161 bp of upstream sequence gave normalized levels of CAT activity greater than or equal to that obtained for parallel positive control transfections using pTK-CAT. Treatment of the cells for 48 h with 50 nM dexamethasone (Dex) gave a 2- to 5-fold increase in CAT activity. Interestingly, a 5'-deletion construct containing 161 bp of upstream sequence (pFS161-CAT) conferred both cell type-restricted and dexamethasone-responsive expression. These studies emphasize the potential complexity of SP-D gene regulation, and further support the hypothesis that the effects of glucocorticoids on SP-D production in vivo are regulated at the level of transcription. PMID- 8630262 TI - TGF-beta 1, but not TGF-beta 2 or TGF-beta 3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. AB - Although it is recognized that three isoforms of transforming growth factor-beta (TGF-beta) exist in mammals, their expression, distribution, and function in injury and repair are not well characterized. Using immunohistochemistry and antibodies to synthetic peptides of TGF-beta 1, TGF-beta 2, and TGF-beta 3, we determined the distribution of TGF-beta isoforms in lung sections with acute and chronic lesions of idiopathic pulmonary fibrosis (IPF), chronic asbestosis and hypersensitivity pneumonitis, as well as non-specific pneumonitis. In lung sections with advanced pulmonary fibrosis and honeycombing, irrespective of the diagnosis, TGF-beta 1 was prominently expressed in epithelial cells and macrophages and was found to be associated with the extracellular matrix. In lungs with early lesions of IPF and only inflammatory changes, TGF-beta 1 was present in alveolar macrophages but TGF-beta 1 was not present in epithelial cells. Small amounts of matrix-associated TGF-beta 1 were present subepithelially in areas of lung sections from patients with IPF with minimal inflammation and no fibrosis. In normal lungs with no evidence of inflammation or fibrosis TGF-beta 1 was not seen in alveolar macrophages, epithelial cells, or extracellularly. TGF beta 2 and TGF-beta 3 were expressed in alveolar macrophages, epithelial cells, and smooth muscle cells of vessels and bronchi of normal lungs and lungs with both inflammatory and fibrotic changes. Our findings suggest that while TGF-beta 2 and TGF-beta 3 are ubiquitously expressed in the lung, TGF-beta 1 is expressed in epithelial cells of fibrotic lungs where the presence of TGF-beta 1 is not disease-specific but an indication of the chronicity of the injury. PMID- 8630263 TI - The role of Na+/H+ exchange and growth factors in pulmonary artery smooth muscle cell proliferation. AB - Chronic hypoxia produces pulmonary hypertension, in part because of hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PA SMC). Platelet derived growth factor (PDGF) and epidermal growth factor (EGF) have been shown to stimulate SMC proliferation and may be involved in these vascular changes. Both factors cause a rise in intracellular pH (pHi) in systemic vascular SMC through stimulation of the Na+/H+ exchanger, an event that has been thought to be permissive, allowing cell proliferation in response to the growth factor. The present studies examined the possibility that the activation of Na+/H+ exchange is involved in the PA SMC mitogenic response to these growth factors. Na+/H+ exchange activity was assessed by monitoring pHi in cultured cells using the pH sensitive dye, 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). PDGF (60 ng/ml) exposure led to a marked activation of Na+/H+ exchange, evidenced by a rise in pHi (mean +/- SEM) of 0.20 +/- 0.03 pH units (n = 5, P < 0.05). EGF (60 ng/ml) exposure produced a rise in pHi of 0.27 +/- 0.03 pH units (n = 5, P < 0.05). Dimethyl amiloride (DMA, 50 microM), a competitive inhibitor of Na+/H+ exchange, blocked the pH response to PDGF and EGF. PA SMC showed a proliferative response when exposed to PDGF and EGF which was attenuated by 50 microM DMA (n = 6). Thus, activation of the Na+/H+ exchanger may be important in pulmonary cell signaling in response to growth factors as it has been found to be in systemic vessels. PMID- 8630264 TI - Mucin production by SPOC1 cells--an immortalized rat tracheal epithelial cell line. AB - An airway epithelial mucous goblet cell line would be useful towards understanding mechanisms underlying the common problem of respiratory mucus hypersecretion. SPOC1 is a novel rat tracheal epithelial (RTE) cell line that developed cytologic features suggestive of mucous goblet cells when grown in tracheal grafts in vivo (Am. J. Respir. Cell Mol. Biol. 1995; 12:385-395). Our aims were to determine whether SPOC1 cells were capable of mucin synthesis and to directly compare mucin production by SPOC1 cells and RTE cells. Towards this end, we validated the use of monoclonal antibody (mAb) RTE11 (Exp. Lung Res. 1992; 18:323-342) as an immunologic probe for rat airway secretory mucin. Our results strongly suggest that mAb RTE11 detects a carbohydrate antigen that is a sensitive and specific marker for rat tracheobronchial secretory mucin. SPOC1 cells in tracheal grafts in vivo contained granules with ultrastructural features similar to mucous granules in normal rat airway goblet cells and they were strongly stained by mAb RTE11. Retinoic acid (RA) and culture on porous supports are known to profoundly modify airway epithelial cell phenotype in vitro. Expression of several retinoid-responsive proteins was similar in cultured SPOC1 and primary RTE cells, but major differences in mucin production were noted. Primary RTE cells in vitro only made mucin when grown on porous supports in the presence of RA, whereas SPOC1 cells produced mucin when grown on plastic or glass surfaces and even in the absence of RA. Interestingly, RA enhanced mucin secretion by SPOC1 cells during the early plateau stage of culture but there were no differences due to RA late in the culture period. SPOC1 cells are capable of mucin production and will be a useful tool for studying select aspects of airway secretory cell differentiation and function. PMID- 8630265 TI - Alveolar macrophage uptake of the environmental particulate titanium dioxide: role of surfactant components. AB - Pulmonary surfactant components can modulate uptake of microorganisms and viruses by alveolar macrophages (AMs), but little is known about their role in the uptake and clearance of inert environmental particulates. We tested the hypotheses that surfactant components [e.g., surfactant protein A (SpA) and the artificial bovine surfactant Survanta] modulate phagocytosis of inert environmental particulates by acting as particle opsonins, or by direct activation of AMs. AM uptake of a model inert particulate [titanium dioxide (TiO2)] was measured using flow cytometry to quantitate increased right angle scatter caused by particle uptake (e.g., fold increase in right angle scatter versus control: 2.6 +/- 0.3; and 5.0 +/- 0.2 for AMs plus TiO2, 20 and 80 micrograms/ml TiO2, respectively). Opsonization of TiO2 with surfactant components resulted in a modest increase in AM uptake compared with that of unopsonized TiO2 [e.g., fold increase, uptake of TiO2 (50 micrograms/ml), opsonized with SpA, Survanta, and rat immunoglobulin G, respectively: 1.6 +/- 0.1; 1.3 +/- 0.01; 1.5 + 0.02, n = 3-4]. Uptake of inert latex beads was similarly enhanced after opsonizing with SpA and Survanta (beads per cell: unopsonized, 3.2 +/- 0.40; SpA, 5.0 +/- 0.55; Survanta, 6.0 +/- 0.12; n = 3-6). Pretreating AMs with surfactant components and measuring the subsequent uptake of unopsonized TiO2 resulted in approximately the same magnitude of enhancement. The data indicate that surfactant components can increase AM phagocytosis of environmental particulates in vitro, but only slightly relative to the already avid AM uptake of unopsonized particles. PMID- 8630266 TI - Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation. AB - Ceruloplasmin (CP) is an important extracellular antioxidant and free radical scavenger. Although CP is expressed mainly in the liver, recent studies have identified the lung as another major site of CP synthesis. The sites and cell types that are responsible for CP expression in baboon and mouse lung are described. CP mRNA is detected in primordial bronchial epithelium in baboon fetuses by 60 days of gestation. At 140 days of gestation and thereafter, CP mRNA is found in airway epithelium and in the ductal cells of the submucosal glands. In developing and mature mice, CP mRNA is present in epithelial cells throughout the airway. In endotoxin-treated mice, the amount of CP mRNA increases several fold in large airways but increases only moderately in small airways. This suggests that the high concentration of CP in the mucus lining of the upper airway, which serves to filter harmful substances, is particularly important during stressful conditions. Endotoxin treatment in mice also results in the induction of high levels of CP mRNA in a subset of alveolar wall cells. The data suggest that the airway epithelial cells are the major source of CP in the lung fluid and support ceruloplasmin's critical role in host defense against oxidative damage and infection in the lung. PMID- 8630267 TI - Effect of hypoxia on release of IL-1 and TNF by human alveolar macrophages. AB - Our previous work demonstrated that hypoxia decreases transcription of the human prostaglandin H synthase-2 (PGHS-2) gene during exposure to lipopolysaccharide (LPS), resulting in decreased prostaglandin E2 (PGE2) synthesis (J. Biol. Chem. 269:32979-32984, 1994). Because PGE2 is reported to inhibit interleukin 1 (IL-1) and tumor necrosis factor (TNF), it is likely that hypoxia, through changes in PGE2, will alter IL-1 and TNF release from the human alveolar macrophage. In addition, like PGHS-2, the TNF and IL-1 promoters contain oxidant-sensitive elements which might be altered by hypoxia. Therefore, we hypothesized that LPS induced release of TNF and IL-1 would be altered by hypoxia. To test this, human alveolar macrophages were cultured for 24 h with 0 to 1 microgram/ml LPS in a room-air incubator with 5% CO2 or a hypoxia incubator continuously perfused with 5% CO2/95% N2 (O2 < 0.05%). With room air, LPS increased IL-1 beta mRNA and increased IL-1 beta protein release into the culture medium in a dose-dependent manner. Hypoxia increased the LPS-stimulated release of IL-1 beta 30% above that of room-air controls. However, immunoblots showed that hypoxia caused no change in intracellular IL-1 beta compared with room-air controls. There was also no change in LPS-induced IL-1 beta message with hypoxia. The inhibitor of IL-1, IL 1RA, was apparently decreased by hypoxia, but this decrease was not statistically significant. TNF-alpha mRNA and release of protein also increased during LPS exposure in room air. Hypoxia markedly increased LPS-induced TNF-alpha message and release of TNF-alpha compared with LPS-exposed room-air controls. Consistent with our prior observations, hypoxia decreased LPS-induced PGHS-2 message and protein, and also the PGHS-2 product, PGE2. Because PGE2 is reported to inhibit the expression of IL-1 and TNF genes, we inhibited PGE2 synthesis with indomethacin during culture in room air; the result was an increase in the release of IL-1 and TNF. In additional studies, adding PGE2 inhibited TNF release from the hypoxia cells to values near those of room-air controls. In summary, hypoxia increases the release of the cytokines IL-1 beta and TNF-alpha. This increase may be due to decreased PGE2 synthesis during hypoxia. These results demonstrate that the response of the human alveolar macrophage to hypoxia is complex. Hypoxia increases the LPS-stimulated release of the inflammatory cytokines IL-1 and TNF, whereas synthesis of PGHS-2, which generates the anti inflammatory prostaglandin PGE2 is decreased. PMID- 8630268 TI - Postnatal age at onset of hyperoxic exposure influences developmentally regulated tropoelastin gene expression in the neonatal rat lung. AB - Upregulation of tropoelastin (TE) gene expression in rat lung interstitial fibroblasts normally occurs during alveolar septation. TE message increases at the end of the first week of life, peaks on days 9-11, and returns to barely detectable levels over the next 7-10 days. Our previous in situ hybridization studies indicated that exposure of pups to > 95% oxygen from 3 to 13 days of age interfered with the increased in TE gene expression in interstitial fibroblasts normally seen during septation. However, when the pups were returned to room air, lung fibroblast TE message levels increased, exceeding levels seen in control lungs during the exposure. In addition, TE message levels remained elevated for a week after levels in control lungs had returned to background. A possible interpretation of these results was that the developmentally regulated increase in TE messenger RNA (mRNA) was downregulated by the hyperoxic exposure but resumed when the pups were returned to a normoxic environment. We report herein the results of a subsequent study conducted to determine whether continued hyperoxic exposure beyond day 13 would further delay the peak in TE mRNA. Rat pups were exposed to 95% O2 from 5 to 17 days of age. TE and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) message levels in lung interstitial fibroblasts were assessed by in situ hybridization. As observed in pups exposed from 3 to 13 days, hyperoxic exposure from days 5 to 17 also extended the period during which TE mRNA levels were elevated. After exposure, TE message levels were 99%, 262%, and 223% of controls on days 19, 21, and 23 respectively. In addition, delaying the exposure 2 days until the pups were 5 days old resulted in an upregulation of TE message, relative to control values, during the hyperoxic exposure. In hyperoxic pups, values for TE message expression were 105%, 152%, 168%, and 144% of control pups on days 9, 11, 13, and 16 respectively. The influence on peak TE message expression of postnatal age at the time of exposure was further explored to verify the results of the 3-13 and 5-17 day exposures. When pups were exposed continuously from 2, 3, 4, 5, or 6 days until 11 days of age, the results of both in situ hybridization and Northern blot analysis confirmed our previous observations, demonstrating that the postnatal age at which hyperoxic exposure is initiated influences TE message expression in the developing lung. PMID- 8630269 TI - Differential enhancement of gamma-glutamyl transpeptidase and gamma glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. AB - Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of gamma-glutamyl transpeptidase (GGT) and gamma glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress. PMID- 8630270 TI - Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity. AB - Tert-butylhydroquinone (TBHQ) is a monofunctional Phase II enzyme inducer, which produces reactive oxygen species. Incubation with a sublethal concentration of TBHQ increased the activities of both gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS), although the mechanisms are different (Liu and colleagues, accompanying manuscript). In this study, we found that TBHQ increased intracellular glutathione (GSH) content in rat lung epithelial L2 cells. L2 cells pretreated with a nontoxic concentration of TBHQ (50 microM) acquired resistance to a subsequent challenge with a normally lethal concentration of TBHQ (200 microM). Pretreatment with L-buthionine S,R sulfoximine (BSO), an inhibitor of GCS, prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Similarly, pretreatment with acivicin, an inhibitor of GGT, also prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Nevertheless, blockage of GGT by acivicin could be bypassed using 2 oxothiazolidine-4-carboxylate (procysteine) to provide the cell with a source of cysteine. This allowed an increase in GSH and restored resistance in the TBHQ pretreated cells. The results suggest that elevation of GCS and GGT activities participated in acquired resistance to quinone toxicity. PMID- 8630271 TI - Plasma from hemorrhaged mice activates CREB and increases cytokine expression in lung mononuclear cells through a xanthine oxidase-dependent mechanism. AB - Hemorrhage rapidly increases plasma xanthine oxidase levels as well as the expression of proinflammatory and immunoregulatory cytokines in the lungs. To determine the role of circulating xanthine oxidase (XO), as well as other plasma factors, in affecting pulmonary cytokine expression, we conducted studies in which plasma from hemorrhaged mice was transferred into unhemorrhaged recipient mice. Administration of posthemorrhage plasma to recipient mice increased the levels of mRNA for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta 1 (TGF-beta 1) in lung mononuclear cells. No enhancement of mRNA levels for these cytokines was found in the lungs of mice given allopurinol-treated posthemorrhage plasma or fed a tungsten-enriched, XO-depleting diet prior to transfer of posthemorrhage plasma. Among the nuclear transcriptional regulatory factors examined, only the cyclic AMP response-element binding protein (CREB) was activated in nuclear extracts from lung mononuclear cells of mice that were given posthemorrhage plasma. No activation of nuclear factor-kappa B (NF-kappa B), nuclear factor interleukin 6 (NF-IL6), activating protein-1 (AP-1), or serum protein-1 (SP-1) was found. These results suggest that the mechanism for hemorrhage-induced increases in pulmonary cytokine expression is by activation of the enhancer CREB through a tissue XO dependent pathway initiated by plasma-borne mediators. PMID- 8630273 TI - Hypotonic intraperitoneal cisplatin chemotherapy for peritoneal carcinomatosis in mice. AB - The intraperitoneal (i.p.) administration of cisplatin (CDDP) is one of the most effective therapies for cancers that are confined to the abdominal cavity. However, the effect of fluid osmolarity on the therapeutic efficacy of i.p. administration of CDDP has not been well established. In the current study, hypotonic (154 mosmol 1-1), isotonic (308 mosmol 1-1) and hypertonic (616 mosmol 1-1) solutions of CDDP were prepared for an evaluation of their therapeutic efficacy in an experimental system. After i.p. administration, uptake of CDDP in vivo by tumor cells in hypotonic solution was significantly greater than in isotonic or hypertonic solution. The 50% lethal dose (LD50) value of CDDP in hypotonic solution (12.1 mg kg(-1)) was lower than that in isotonic solution (16.9 mg kg(-1)) and than that in hypertonic solution (28.6 mg kg(-1)). However, when a dose equal to one-half of the LD50 was administered in each solution to mice with i.p. tumours, survival of mice given the CDDP in hypotonic solution was significantly prolonged as compared with the survival of the other mice. These results demonstrate that the therapeutic efficacy of i.p. CDDP in mice is augmented when the drug is administered in hypotonic solution. PMID- 8630272 TI - Modulation of bryostatin 1 muscle toxicity by nifedipine: effects on muscle metabolism and oxygen supply. AB - Bryostatin 1, an anti-neoplastic agent and protein kinase C activator, has dose limiting toxicity manifesting as myalgia. Studies in vivo have suggested that this myalgia may be caused by impairment of oxidative metabolism as mitochondrial capacity, muscle reoxygenation and proton washout from muscle are reduced by bryostatin, possibly as a result of vasoconstriction. To investigate these mechanisms further, and to enable use of bryostatin for prolonged periods, the effect of a vasodilator on the established effects of bryostatin on calf metabolism was studied using 31P magnetic resonance spectroscopy and near infrared spectroscopy. Six patients with disseminated melanoma were examined on four occasions: before and 1 week after initiation of long-term nifedipine (10 mg twice daily) treatment and then 4 and 48 h after bryostatin infusion (25 micrograms m(-2)). Nifedipine impaired muscle oxidative metabolism but had no effect on proton efflux or muscle reoxygenation rate. In the presence of nifedipine, two of the effects of bryostatin, impaired reoxygenation rate and reduced proton efflux, were abolished, but the impaired mitochondrial activity remained. These results show that nifedipine counteracted the vasoconstrictive effect of bryostatin 1. However, because nifedipine itself had an unexpected effect on mitochondrial metabolism, it was not possible to assess whether nifedipine modified bryostatin's effect on this variable. There was no additive detrimental effect of bryostatin on mitochondrial metabolism and nifedipine did not reduce the clinical toxicity of bryostatin 1, which cannot therefore be due to vasoconstriction. PMID- 8630274 TI - Localisation of [131I]MIBG in nude mice bearing SK-N-SH human neuroblastoma xenografts: effect of specific activity. AB - The biodistribution of no-carrier-added (n.c.a.) meta-[131I]iodobenzylguanidine ([131I]MIBG) and that prepared by the standard isotopic exchange method were compared in athymic mice bearing SK-N-SH human neuroblastoma xenografts. No advantage in tumour uptake was observed for the n.c.a. preparation. BALB/c nu/nu mice exhibited lower uptake in highly innervated normal tissues (heart and adrenals) than normal BALB/c mice. In another experiment, the distribution of n.c.a. [131I]MIBG in the absence or presence (3-9 micrograms) of MIBG carrier was determined. At both 4 h and 24 h, the heart uptake was reduced by a factor of 1.5 even at a dose of 3 micrograms MIBG. Tumour uptake was not significantly altered by various amounts of unlabelled MIBG at either time point. PMID- 8630276 TI - Inhibition of angiogenesis and murine tumour growth by laminarin sulphate. AB - LAM S5 is a polysulphated derivative of the glucan laminarian that inhibits basic fibroblast growth factor (bFGF) binding and the bFGF-stimulated proliferation of fetal bovine heart endothelial (FBHE) cells. This report demonstrates that LAM S5 has anti-angiogenic activity, as shown by inhibition of tubule formation by endothelial cells cultured on Matrigel and inhibition of vascularisation of the chick chorioallantoic membrane. In addition, LAM S5 caused a tumour growth delay of the murine RIF-1 tumour of 2.6 days (P = 0.01). PMID- 8630275 TI - Effects of neocarzinostatin-chimeric Fab conjugates on the growth of human pancreatic carcinoma xenografts. AB - Neocarzinostatin (NCS) was bound covalently to human/mouse chimeric Fab fragments of MAb A7 (chA7Fab) directed against human pancreatic carcinoma. The anti-tumour effect of chA7Fab-NCS was tested in a nude mouse model on pancreatic carcinoma and compared with A7-NCS or NCS alone. The anti-tumour effect of chA7Fab-NCS increased in a dose-dependent manner and was significantly greater than either A7 NCS or NCS. Tumour growth was completely suppressed after the administration of chA7Fab-NCS. An enzyme-linked immunosorbent assay with rabbit anti-mouse immunoglobulin was performed to examine the antigenicity of chA7Fab. ChA7Fab had less reactivity with rabbit anti-mouse immunoglobulin than either whole antibody A7 or murine Fab fragments of A7. Thus, chA7Fab-NCS can inhibit human pancreatic cancer growth in an animal and may be useful for targeting chemotherapy to pancreatic cancer in humans. PMID- 8630277 TI - Novel anti-tumour activity of 2,3,5-trimethyl-6-(3-pyridylmethyl)-1,4- benzoquinone (CV-6504) against established murine adenocarcinomas (MAC). AB - 2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5 lipoxygenase and thromboxane A2 synthase and a scavenger of active oxygen species, has been shown to exhibit profound anti-tumour activity against three established murine adenocarcinomas (MACs) that are generally refractory to standard cytotoxic agents. For the cachexia-inducing MAC16 tumour, optimal anti tumour activity was seen at dose levels of 10 and 25 mg kg-1 day-1, together with a reversal of cachexia and a doubling of the time to sacrifice of the animals through cachexia from 8 days to 17 days. The remaining tumour fragments showed extensive necrosis in regions distal from the blood supply. Growth of the MAC13 tumour was also effectively suppressed at dose levels between 5 and 50 mg kg-1 day-1, resulting in a specific growth delay between 1.0 and 1.2. Growth of the MAC26 tumour was also inhibited a concentration-related manner, with doses of 25 50 mg kg-1 day-1 being optimal. Anti-tumour activity towards all three tumours at low dose levels of CV-6504 was effectively suppressed by concurrent administration of linoleic acid (1 g kg-1 day-1), suggesting that inhibition of linoleate metabolism was responsible for the anti-tumour effect. Tumour sensitivity may be correlated with increased DT-diaphorase that are required to metabolise CV-6504 to the active hydroquinone, which inhibits 5-lipoxygenase activity. PMID- 8630278 TI - Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance. AB - As a first step towards elucidating the potential role(s) of bcl-2 and bcl-2 related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-x1-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl 2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts. PMID- 8630280 TI - Effect of hormone depletion on cell survival in the EMR-86 rat mammary carcinoma. AB - Growth of the transplantable EMR-86 rat mammary carcinoma depends on elevated prolactin levels which are induced by oestrogenic stimulation of the pituitary. We investigated histological and cell kinetic changes during tumour regression after removal of implanted oestrogen pellets (EP), and we especially focused on the role of apoptosis. After EP removal, serum prolactin decreased to basal levels in 5 days, reaching its largest depletion during the first day. Similarly, S-phase cell fractions, assessed by bromodeoxyuridine (BrdUrd) incorporation, decreased to half the initial value during the first day and developed into a gradual decrease to basal levels thereafter. Within 10 days, tumour volumes were reduced to 20% without striking changes in tissue architecture. To quantify apoptosis, we applied a method that stains DNA breaks in tissue sections and subsequently measured the stained area by automated image cytometry. This procedure was necessary, as the subtle changes could not be detected by histological examination alone. One day after the rapid decline of the S-phase fraction, a 3-fold increase in apoptotic area was observed that remained for about 3 days and then gradually decreased. This correlated with the histologically observed reduction of tumour cells. In spite of the major cell loss, regression came to a halt after about 10 days. The surviving cell fraction is discussed within the context of a stem cell hypothesis, in which tumour cells with stem cell characteristics are less susceptible to hormone-induced apoptosis than their (non-stem) daughter cells. This notion has implications for the eradication of residual tumour cells, because a diminished susceptibility might also apply to apoptosis induced by radio- or chemotherapy. PMID- 8630279 TI - Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide. AB - Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide. PMID- 8630281 TI - Lack of mutation in tumour-suppressor gene p53 in gestational trophoblastic tumours. AB - The objectives of this study were to better our understanding of the carcinogenesis of gestational trophoblastic tumours and to investigate the possible presence of mutational alteration of the p53 tumour-suppressor gene in these tumours. Amplification-based direct DNA sequencing was performed on 14 hydatidiform moles, six invasive moles, eight choriocarcinomas and ten normal early placental tissues. No mutation in exons 5-8 was detected in any of these 38 tissue specimens. These results suggest that a mutation in p53 tumour suppressor either does not exist or is a very rare event in gestational trophoblastic tumours. The gestational trophoblastic tumours probably involve a tumour suppressor gene other than p53 gene or may follow a completely different pathway to their malignant phenotype. PMID- 8630282 TI - Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer. AB - The development of familial and sporadic breast cancer is based on genetic alterations of tumour-suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation. To investigate LOH of BRCA1 (17q21) and BRCA2 (13-q12-13) in sporadic breast cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for detection of microsatellite polymorphisms was applied. A total of 137 breast cancer and 15 benign breast specimens with matched normal tissue were examined. Fluorescent-labelled PCR products were analysed in an automated DNA sequencer (ALFTM Pharmacia). Losses at both loci were correlated with different histological types, age, tumour size, lymph node status, grading and steroid hormone receptor expression, [SHR: oestrogen receptor (ER), progesterone receptor (PgR)]. For BRCA1 (D17S855, THRA1, D17S579) losses could be detected in invasive ductal carcinoma (IDC; n = 108) in 32-38%, invasive lobular carcinoma (ILC; n = 19) in 21-42% depending on the marker applied, but not in benign breast tumours (n = 15). Losses of BRCA1 markers correlated with larger tumour size, higher grade, and PgR expression. For BRCA2 (D13S260, D13S267, D13S171) losses could be detected in 108 IDCs in 30-38%, in 19 ILCs in 17-39% depending on the marker applied, but not in benign breast tumours. Losses of BRCA2 markers correlated only with higher grade. Microsatellite analyses combined with detection of fluorescent-labelled PCR products by an automated laser DNA sequencer can be used for routine determination of LOH. In sporadic breast cancer, LOH of BRCA1 of BRCA2 does not add decisive prognostic value as stated for familial breast cancer. PMID- 8630283 TI - Pro-gastrin-releasing peptide (31-98) as a tumour marker of small-cell lung cancer: comparative evaluation with neuron-specific enolase. AB - We attempted to clarify whether serum levels of a carboxy-terminal fragment of ProGRP, ProGRP(31-98), could serve as a more accurate tumour marker in patients with SCLC than neuron-specific enolase (NSE). ProGRP(31-98) and NSE were measured retrospectively in 101 newly diagnosed untreated patients with SCLC, 111 with non small-cell lung cancer (NSCLC) and 114 patients with non-malignant lung diseases. ProGRP(31-98) and NSE levels were determined using a sandwich enzyme-linked immunosorbent assay. Sensitivity in SCLC patients was 72.3% for ProGRP(31-98) and 62.4% for NSE. Comparing the area under curve (AUC) of 'receiver operator characteristics' of ProGRP(31-98) with that of NSE, ProGRP(31-98) was the more powerful marker in the diagnosis of SCLC (P = 0.0001). Serum levels of ProGRP(31 98) were higher in the 40 patients with extensive disease than in the 61 patients with limited disease (P = 0.0082). ProGRP(31-98) was significantly higher in patients with pure small-cell carcinoma than in patients with mixed small cell/large-cell carcinoma (P = 0.02). In serial measurement in 16 patients responding to treatment, a high degree of correlation was noted between the decrease in serum ProGRP(31-98) levels and clinical response during the second week after treatment (P = 0.0045). These results indicate that the determination of serum ProGRP(31-98) levels plays an important role in the diagnosis and treatment of SCLC patients. PMID- 8630284 TI - Immunohistochemical expression of the c-kit proto-oncogene product in human malignant and non-malignant breast tissues. AB - The immunohistochemical expression of c-kit proto-oncogene product in 57 breast cancer tissues was studied using anti-c-kit proto-oncogene product antibody in comparison with 20 normal breast tissues and 58 benign breast tumours. In normal breast tissues, the c-kit proto-oncogene product was strongly expressed on cell membrane and/or cytoplasm of alveolar and ductal cells. The immunoreactive score (IRS) of c-kit proto-oncogene product in normal mammary epithelia was 6.22 +/- 2.11 (mean +/- s.d.). In benign breast diseases, the c-kit proto-oncogene product was detected heterogeneously with a reduced IRS (3.33 +/- 2.44). In breast cancer tissues, the expression of the immunoreactive c-kit proto-oncogene product was often deleted and the average IRS was significantly reduced compared to those of normal breast tissues or benign breast diseases tissues. Among benign diseases, the average IRS of intraductal papilloma was significantly reduced (1.34 +/- 1.70) and the staining intensity and pattern were found to be similar to those seen in breast cancer. The results in this study suggested that the c-kit proto oncogene product is correlated with the growth control or the differentiation of normal breast epithelium. Also, the loss of the expression of this protein may indicate the change of the signal transduction in relation to malignant transformation in human mammary epithelium. PMID- 8630285 TI - Identification of leukaemic cells in bone marrow and blood samples by a new cytofluorometric assay. AB - The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. We used this fact to detect neoplastic cells in samples freshly taken from leukaemia patients and kept frozen in liquid nitrogen until analysis. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to identify leukaemic cells in a surplus of normal ones. Our results demonstrate the benefits of this assay for leukaemia diagnosis. PMID- 8630286 TI - A predictive index of axillary nodal involvement in operable breast cancer. AB - We investigated the association between pathological characteristics of primary breast cancer and degree of axillary nodal involvement and obtained a predictive index of the latter from the former. In 2076 cases, 17 histological features, including primary tumour and local invasion variables, were recorded. The whole sample was randomly split in a training (75% of cases) and a test sample. Simple and multiple correspondence analysis were used to select the variables to enter in a multinomial logit model to build an index predictive of the degree of nodal involvement. The response variable was axillary nodal status coded in four classes (N0, N1-3, N4-9, N > or = 10). The predictive index was then evaluated by testing goodness-of-fit and classification accuracy. Covariates significantly associated with nodal status were tumour size (P < 0.0001), tumour type (P < 0.0001), type of border (P = 0.048), multicentricity (P = 0.003), invasion of lymphatic and blood vessels (P < 0.0001) and nipple invasion (P = 0.006). Goodness-of-fit was validated by high concordance between observed and expected number of cases in each decile of predicted probability in both training and test samples. Classification accuracy analysis showed that true node-positive cases were well recognised (84.5%), but there was no clear distinction among the classes of node-positive cases. However, 10 year survival analysis showed a superimposible prognostic behaviour between predicted and observed nodal classes. Moreover, misclassified node-negative patients (i.e. those who are predicted positive) showed an outcome closer to patients with 1-3 metastatic nodes than to node-negative ones. In conclusion, the index cannot completely substitute for axillary node information, but it is a predictor of prognosis as accurate as nodal involvement and identifies a subgroup of node-negative patients with unfavourable prognosis. PMID- 8630287 TI - Prediction of survival and recurrence by serum and cytosolic levels of CEA, CA125 and SCC antigens in resectable non-small-cell lung cancer. AB - Risk of death and risk of recurrence in 108 potentially curable non-small-cell lung cancer patients were analysed with respect of TNM stage, histological type and carcinoembryonic antigen (CEA), CA125 antigen and squamous cell carcinoma antigen (SCC) levels in serum and cytosol. CA125 and CEA levels were closely related to outcome figures. Multivariate analyses indicated that TNM stage and histological type had the best predictive power, but serum and cytosolic CA125 and serum CEA contained additional, independent prognostic information. Predictive information drawn from serum and cytosolic levels proved mutually complementary. We conclude that CA125 and CEA complement TNM classification and histological type for the purpose of quantifying risk of death or recurrence. PMID- 8630289 TI - A phase II study of continuous-infusion 5-fluorouracil with cisplatin and epirubicin in inoperable pancreatic cancer. AB - Carcinomas of the exocrine pancreas respond poorly to most chemotherapy regimens. Recently continuous infusional 5-fluorouracil (200 mg m-(2)day-1) with 3 weekly cisplatin (60 mg m-2) and epirubicin (50 mg m-2) (the ECF regimen) has proven to be an active regimen in gastric and breast cancer and consequently worthy of further study in pancreatic cancer. Thirty-five patients were treated with the ECF regimen as above, of whom 29 were evaluable for response and 32 were evaluable for toxicity. The mean age was 59 years (range 37-75). Sixteen patients had locally advanced disease at presentation and 19 had metastases. Objective tumour responses were documented in five (17.3%) patients who achieved a partial response; in 18 (62%) patients there were no change and six (20.7%) patients progressed on therapy. Patients with either stable disease or partial response had a significantly improved overall survival (median = 253 days) compared with patients who progressed (median = 170 days; P = 0.01). Grade 3/4 (WHO) toxicity (all cycles) included alopecia in 18 (56%) patients, nausea/vomiting in eight (25%) stomatitis in three (9%) and diarrhoea in seven (22%) patients, with rhinorrhoea and excessive lacrimation in one patient each. Neutropenic sepsis occurred in 13 cycles in ten patients, and there was one toxic death due to sepsis. There were eight other episodes of non-neutropenic sepsis requiring hospital admission. Fourteen patients (40%) experienced complications with their Hickman lines, including thrombotic episodes (six patients) or their line falling out (five patients). ECF can prolong survival in patients with locally advanced or metastatic pancreatic cancer who demonstrate a response or stabilisation of their disease. However, this is associated with considerable toxicity. PMID- 8630288 TI - Comparison between immunocytochemical and polymerase chain reaction techniques for detection of oestrogen receptor and transforming growth factor beta in breast cancer. AB - The utility of the polymerase chain reaction (PCR) as a technique for determining the expression of transforming growth factor beta (TGF-beta) and of the oestrogen receptor (ER) in clinical breast cancer tissue was examined. PCR analysis was compared with immunocytochemical assays for TGF-beta and for ER. Seventy confirmed breast carcinoma samples were analysed for ER using both techniques with a statistically highly significant concordance (P < 0.001) between the two methods. Nineteen samples were observed to be ER positive and 46 samples were found to be ER negative by both techniques. Forty-eight samples were analysed for TGF-beta using both PCR and immunocytochemistry. Of the 24 samples observed to be positive for TGF-beta by immunocytochemistry, all were found to be positive for TGF-beta mRNA (PCR). Similarly, the 24 samples observed to be TGF-beta negative by immunocytochemistry were also negative for TGF-beta mRNA, indicating 100% specificity and 100% sensitivity of the PCR technique. PCR is therefore considered a viable technique for analysis of both ER and TGF-beta in small samples such as fine-needle aspirates. PMID- 8630290 TI - Phase II study of a short course of weekly high-dose cisplatin combined with long term oral etoposide in metastatic colorectal cancer. AB - In a phase I study of weekly administered cisplatin combined with oral etoposide, we observed a partial response in 4 out of 11 patients with metastatic colorectal cancer. Subsequently, we performed a phase II study to investigate the activity of this combination as first-line treatment in this disease. Fourteen patients with metastatic colorectal cancer were enrolled in this study. Treatment consisted of cisplatin, administered in 3% sodium chloride, at a dose of 70 mg m 2 on days 1, 8 and 15 and days 29, 36 and 43 combined with oral etoposide 50 mg absolute dose daily on days 1-15 of both courses. Patients with stable disease or better continued treatment with etoposide 50 mg m-2 orally on days 1-21 every 28 days. A partial response was observed in two patients with liver metastases (14%; 95% confidence limits 2-42%) for 30 and 32 weeks. Five patients had stable disease. Toxicity consisted mainly of anaemia, leucocytopenia, nausea and vomiting. Tinnitus was reported by six patients. The activity of the combination cisplatin-oral etoposide in the schedule is only minimal in metastatic colorectal cancer. PMID- 8630291 TI - Bioavailability and pharmacokinetics of oral topotecan: a new topoisomerase I inhibitor. AB - The results of preclinical and clinical studies indicate enhanced antineoplastic activity of topotecan (SKF 104864-A) when administered as a chronic treatment. We determined the apparent bioavailability and pharmacokinetics of topotecan administered orally to 12 patients with solid tumours in a two-part crossover study. The oral dose of 1.5 mg m-2 was administered as a drinking solution of 200 ml on day 1. The i.v. dose of 1.5 mg m-2 was administered as a 30 min continuous infusion on day 2. The bioavailability was calculated as the ratio of the oral to i.v. area under the curve (AUC) calculated up to the last measured time point. The oral drinking solution was well tolerated. The bioavailability revealed moderate inter-patient variation and was 30% +/- 7.7% (range 21-45%). The time to maximum plasma concentration after oral administration (Tmax) was 0.78 h (median; range 0.33-2.5). Total i.v. plasma clearance of topotecan was 824 +/- 154 ml min 1 (range 535-1068 ml min(-1)). The AUC ratio of topotecan and the lactone ring opened hydrolysis product (hydroxy acid) was of the same order after oral (0.34 1.13) and i.v. (0.47-0.98) administration. The bioavailability of topotecan after oral administration illustrates significant systemic exposure to the drug which may enable chronic oral treatment. PMID- 8630292 TI - High-dose chemotherapy and autologous bone marrow transplant in relapsed Hodgkin's disease--a pragmatic prognostic index. AB - High-dose chemotherapy with autologous bone marrow transplantation is used in the treatment of relapsed or high-risk Hodgkin's disease. As prospective randomised studies have proved difficult to accrue to, current recommendations are based on the reports of large series of prospectively collected data. We have looked at the outcome of 89 patients treated in this way at a single institution and have developed an index to predict outcome. Of 89 patients, with a median age of 29 years (range 15-51 years), eight patients were in first complete remission/partial remission (CR/PR), 17 in second or later CR, 37 were responding relapses, 13 resistant relapses, 11 primary refractory and three untested relapses. Combinations of melphalan, BCNU and etoposide were given in all cases except in ten patients who received melphalan alone. The median follow-up was 43 months (range 6-77 months). A total of 24 patients were in CR at the time of autologous bone marrow transplantation (ABMT), 33 achieved CR with ABMT, 16 PR, to give a response rate to ABMT of 49/65 = 74% (95% CI 60-83%) with a CR rate of 51% (CI 36-62%). In a Cox's multivariate analysis the most important factors in predicting outcome after ABMT were response to treatment before entry, number of previous treatments and previous chemosensitivity. Using these factors we devised a prognostic index which reliably selects a group of patients (65%) with at least a 70% chance of being progression free from 1 year onwards. Patients who have never achieved a CR and have received three or more chemotherapy regimens do not benefit from high-dose chemotherapy as used in this study. PMID- 8630293 TI - Space-time clustering of childhood leukaemia in Greece: evidence supporting a viral aetiology. AB - The method introduced by Knox for evaluation of space-time clustering has been applied to 872 cases of childhood (0-14 year old) leukaemia diagnosed in Greece over the 10 year period 1980-89. Greek towns are characterised by substantial population mixing due to internal migration, whereas there is relative isolation in mountainous rural areas. Predetermined space (5 km) and time (1 year) limits were used on the basis of previous reports in order to define the clustering cell. There is highly significant evidence for clustering of childhood leukaemia in Greece as a whole, the observed number of pairs that are close in both spaces and time exceeding the expected number by 5.2% (P = 0.004). The excess is particularly evident for leukaemia cases in 0 to 4-year-old children, among whom the observed number of pairs that are close in both space and time exceeded the expected number by 9.4% (P = 0.004). There is no evidence of space-time clustering for leukaemia cases older than 5 years. The overall pattern is descriptively similar in urban and semiurban areas and is especially marked for acute lymphoblastic leukaemia at the childhood peak ages (2-4 years) with an excess of 19% (P = 0.0006). In the rural population there is evidence for clustering of cases belonging to older and broader age groups, a phenomenon compatible with a delay in the development of herd immunity against putative infectious aetiological agents. The findings of the present study provide support for the hypothesis that a substantial proportion of cases of childhood leukaemia may arise as a rare sequel to exposure to an agent or agents, most probably viral in nature. PMID- 8630294 TI - Dietary factors and the risk of endometrial cancer: a case--control study in Greece. AB - In a hospital-based case-control study of endometrial cancer undertaken in Athens (1992-94), 145 women residents of Greater Athens with confirmed cancer of the endometrium were compared with 298 control patients with orthopaedic diseases. Personal interviews were conducted in the hospital setting, and diet was assessed using a validated semiquantitative food frequency questionnaire. Nutrient intakes for individuals were calculated by multiplying the nutrient intake of a typical portion size for each specified food item by the frequency at which the food was consumed per month and summing these estimates for all food items. Data were modelled through logistic regression, controlling for demographic, reproductive and somatometric risk factors for endometrial cancer as well as for total energy intake. No macronutrient was significantly associated with endometrial cancer risk, but increasing intake of monounsaturated fat, mostly olive oil, by about one standard deviation was associated with a 26% risk reduction (odds ratio = 0.74; 95% confidence interval 0.54-1.3). Among micronutrients, only calcium intake was significantly inversely associated with endometrial cancer risk, whereas there was evidence against retinol and zinc imparting protection against the disease. With respect to food groups, there was weak and non-significant evidence that vegetables are protective, whereas consumption of pulses was positively associated with disease possibly because they contribute substantially in Greece to energy intake in excess of physical activity-dependent requirements. PMID- 8630295 TI - Oral contraceptives and cervical cancer--further findings from the Oxford Family Planning Association contraceptive study. AB - In 1983, we reported results from the Oxford Family Planning Association contraceptive study regarding the association between oral contraceptives (OCs) and cervical neoplasia, after a 10 year follow-up of a cohort of 17,000 women. Further findings from this study are reported here after an additional 12 years of follow-up. A nested case--control design was used in which cases were all women diagnosed under 45 years of age with invasive carcinoma (n = 33), carcinoma in situ (n = 121) or dysplasia (n = 159). Controls were randomly selected from among cohort members and matched to cases on exact year of birth and clinic attended at recruitment to study. Conditional logistic regression analysis was used to determine odds ratios (ORs) and 95% confidence intervals (CIs) associated with various aspects of OC use relative to never users adjusted for social class, smoking, age at first birth and ever use of diaphragm or condom. Ever users of OCs had a slightly elevated OR for all types of cervical neoplasia combined (OR = 1.40, 95% CI 1.00-1.96). Odds ratios were highest for invasive carcinoma (OR = 4.44, 95% CI 1.04-31.6), intermediate for carcinoma in situ (OR = 1.73, 95% CI 1.00-3.00) and lowest for dysplasia (OR = 1.07, 95% CI 0.69-1.66). The elevated risk associated with OC use appeared to be largely confined to current or recent (last use in the past 2 years) long-term users of OCs. Among current or recent users, ORs for all types of cervical neoplasia combined were 3.34 (95% CI 1.96 5.67) for 49-72 months of use, 1.69 (95% CI 0.97-2.95) for 73-96 months and 2.04 (95% CI 1.34-3.11) for 97 or more months. These results suggest a possible effect of OC use on later stages of cervical carcinogenesis, although residual confounding due to sexual factors or human papillomavirus (HPV) infection cannot be ruled out. PMID- 8630296 TI - Epidemiology of in situ and invasive breast cancer in women aged under 45. AB - The incidence of in situ breast cancer in the USA has increased rapidly in recent years, even among young women. A population-based case-control study of 1616 breast cancer cases aged under 45 in the USA was used to examine risk factors for in situ, local and regional/distant tumours. Almost 60% of in situ tumours were detected by routine mammograms compared with 18% of local tumours and 8% of regional/distant tumours. After adjustment for screening history and established risk factors, family history of breast cancer in a first-degree relative and African-American race were associated with an increased risk of all stages of breast cancer. The associations with nulliparity, a previous breast biopsy and body mass index were significantly stronger for in situ tumours than for local or regional/distant disease. Alcohol consumption was associated with an increasing trend in risk of regional/distant tumours but not of earlier stage tumours, indicating that alcohol may be involved in late-stage events. Analyses by histological type of in situ tumours suggested that both ductal and lobular carcinoma in situ were associated with most established breast cancer risk factors, and the magnitude of association tended to be greater for the ductal form. PMID- 8630298 TI - Biologic therapy of cancer. PMID- 8630297 TI - Importance of anatomical subsite in correlating risk factors in cancer of the oesophagus--report of a case--control study. AB - In Bangalore, cancer of the oesophagus is the third most common cancer in males and fourth most common in females with average annual age-adjusted incidence rates of 8.2 and 8.9 per 100,000 respectively. A case-control investigation of cancer of the oesophagus was conducted based on the Population-based cancer registry, Bangalore, India. Three hundred and forty-three cases of cancer of the oesophagus were age and sex matched with twice the number of controls from the same area, but with no evidence of cancer. Chewing with or without tobacco was a significant risk factor. In both sexes chewing was not a risk factor for cancer of the upper third of the oesophagus. Among males, non-tobacco chewing was a significant risk factor for the middle third but not for the other two segments and tobacco chewing was a significant risk factor for the lower third of the oesophagus, but not for the other two segments. Bidi smoking in males was a significant risk factor for all three segments being highest for the upper third, less for the middle third and still less for the lower third. The risk of oesophageal cancer associated with alcohol drinking was significant only for the middle third. PMID- 8630299 TI - Antenatal serology testing in pregnancy. PMID- 8630300 TI - Pseudomyxoma peritonei. PMID- 8630301 TI - Assessing the costs of assisted reproductive techniques. PMID- 8630302 TI - Birth under water--to breathe or not to breathe. PMID- 8630303 TI - Velamentous umbilical cord insertion may be suspected from maternal serum alpha fetoprotein and hCG. AB - OBJECTIVE: Patients with unexplained elevations in second trimester maternal serum alpha-fetoprotein or human chorionic gonadotrophin (hCG) concentrations are at increased risk as regards a variety of pregnancy complications and adverse perinatal outcomes. Evidence suggests that elevated alpha-fetoprotein and hCG concentrations may, in some case, be sensitive indicators of underlying placental pathology, either vascular or inflammatory in nature. The present study was carried out to compare these biochemical markers in pregnancies complicated by velamentous umbilical cord insertion (VCI) with normal pregnancies. DESIGN: An observational study. PARTICIPANTS: Maternal serum hCG and alpha-fetoprotein concentrations were measured in samples from 76 singleton pregnancies complicated by VCI and from 5200 chromosomally normal controls at 15 weeks of gestation. RESULTS: Maternal serum hCG concentrations were elevated (mean 1.47 multiples of median (MoM)) in affected pregnancies, whereas alpha-fetoprotein levels were lower (mean 0.88 MoM) in the subjects than in the controls. In relation to Down's syndrome risk assessment, the pattern of the two markers indicated high risk more often in VCI than in pregnancies with normal umbilical insertion. Accordingly, the increased false positive rate (26.3% compared with 6.6%) resulted in a higher rate of invasive techniques for fetal karyotyping in these pregnancies. CONCLUSIONS: In obstetric practice, elevated maternal serum hCG concentrations may in some cases be explained as being solely the result of abnormal insertion, which provides a link between unexplained hCG elevation and adverse pregnancy outcome. We suggest colour flow Doppler imaging of cord insertion in pregnancies followed because of unexplained hCG elevation or a false positive result in Down's syndrome screening. PMID- 8630304 TI - Children whose mothers had second trimester amniocentesis: follow up at school age. AB - OBJECTIVE: To explore potential long term consequences of second trimester amniocentesis. DESIGN: Children exposed to second trimester amniocentesis have been followed prospectively from birth to school age and compared with children whose mothers declined the test. SETTING: The Hospital for Sick Children, Toronto. SAMPLE: Eighty-six children exposed to amniocentesis and 44 children whose mothers declined the test. INTERVENTIONS: Second trimester amniocentesis for the late maternal age indication. MAIN OUTCOME MEASURES: Standardised tests of intelligence, academic achievement, speech articulation, visual-perceptual motor ability and fine motor coordination were administered. Parents reported on child behaviour using standardised measures of behaviour problems, social competence, hyperactivity and temperament. Physical growth was also measured, and parents reported on child health. RESULTS: Group differences were not observed. CONCLUSIONS: Second trimester amniocentesis does not appear to compromise child development, behaviour, growth or health. PMID- 8630305 TI - Non-invasive prenatal diagnosis by isolation of both trophoblasts and fetal nucleated red blood cells from the peripheral blood of pregnant women. AB - OBJECTIVE: To isolate fetal trophoblasts and nucleated red blood cells from the peripheral blood of pregnant women. DESIGN: Trophoblasts were isolated from whole blood of women in the first trimester of pregnancy by a specific monoclonal antibody, 340. Nucleated red blood cells were isolated by separating whole blood on a triple gradient, staining with ferromagnetic particles coated with an antitransferrin monoclonal antibody and separated on a mini magnetic activated cell sorting (MACS) column. Sorted cells were sexed using a nested polymerase chain reaction for a specific sequence on the Y chromosome and the sex was confirmed by karyotyping of chorionic villus samples. PARTICIPANTS: Patients between 10 and 14 weeks of pregnancy who were undergoing elective chorionic villus sampling for the detection of fetal aneuploidies. MAIN OUTCOME MEASURE: Fetal sex determined by polymerase chain reaction on fetal cells sorted from maternal blood. RESULTS: When both trophoblasts and nucleated red blood cells were sorted, fetal sex was correctly predicted in 12/13 cases (92%), which included correct diagnosis of five of six male pregnancies. More importantly the two techniques were complementary, with only one male pregnancy being diagnosed on both trophoblasts and nucleated red blood cells, two being detected only with trophoblasts and two on nucleated red blood cells alone. No false positives (male signal from a female pregnancy) were diagnosed with either trophoblasts or nucleated red blood cells even with the highly sensitive nested polymerase chain reaction technique, which is very prone to contamination. This study also shows that it is possible to isolate both trophoblasts and nucleated red blood cells from the same sample of maternal blood. CONCLUSION: Fetal cells can be isolated from maternal blood at around 10 weeks of pregnancy. PMID- 8630306 TI - The standard primipara as a basis for inter-unit comparisons of maternity care. AB - OBJECTIVE: To assess the suitability of the standard primipara (a subset of the obstetric population that has relatively low risk or intervention and of adverse outcome) for making inter-unit comparisons of indicators of the process and outcome of maternity care. DESIGN: Inter-unit comparison of 10 indicators of obstetric intervention and adverse outcome derived from routinely collected computerised data held on the St Mary's Maternity Information System. SETTING: Fifteen maternity units in the former North West Thames Region. PARTICIPANTS: 15,463 primiparae who were delivered in 1992. MAIN OUTCOME MEASURES: Proportion of primiparae within the standard definition; degree to which standard primiparae are associated with lower rates of intervention and adverse outcome, as compared to other primiparae. RESULTS: Within the database, 42.6% of all primiparae were found to be standard, with rates varying between units from 25.9% to 57.7%. As expected, the standard primiparous woman is at less risk of intervention or adverse outcome than other primiparae. All but one component variable of the standard definition is a significant risk factor for at least four of the 10 indicators. Statistically significant differences in indicator rates are seen between standard and nonstandard primiparae within units. Within the standard group, significant differences in rates of intervention and adverse outcome are seen between units. Units with relatively high levels of intervention within the higher risk nonstandard group also have relatively high levels of intervention within the standard group. CONCLUSIONS: Use of the standard primipara, rather than the whole obstetric population, as the basis for inter-unit comparisons of maternity care will control for the substantial difference in case mix seen in different units, thereby increasing the validity of those comparisons. The technique has the additional benefit of clarifying the relationship between everyday clinical decision making and a unit's performance in comparative indicator reports. The approach must be combined with a separate study of the other groups in the case mix, such as multiparae and high risk primiparae. Additional nonoverlapping groups, homogeneous in terms of risk factors, should be defined and used to extend the basis on which comparisons may be made. PMID- 8630307 TI - Very preterm birth--a regional study. Part 1: Maternal and obstetric factors. AB - OBJECTIVE: To ascertain the demographic, pregnancy and obstetric factors associated with the delivery of a liveborn very preterm infant ( < 33 weeks of gestation) and to investigate any differences in these factors between identifiable aetiological groups. DESIGN: Cohort analytical study. SETTING: King Edward Memorial Hospital for Women (KEMH), Western Australia. MAIN VARIABLES EXAMINED: Maternal demographic and obstetric history, primary complication associated with delivery, obstetric management and mode of delivery. RESULTS: Six hundred and eight women who were delivered of 693 liveborn very preterm infants in Western Australia between 1.1.90 and 31.12.91, representing 1.22% of all women who were delivered of a liveborn infant in those years. Singleton pregnancy occurred in 517 (85%) and 541 (89%) were delivered in KEMH. Mean maternal age was 28 years with an excess of mothers less than 20 years of age and older than 34 years compared with the statewide perinatal data. Pre-eclampsia (n = 128, 21.1%), preterm prelabour rupture of membranes (n = 148, 24.3%), idiopathic preterm labour (n = 195, 30.4%) and antepartum haemorrhage (n = 111, 18.3%) were associated with 94.1% of deliveries. These proportions varied with plurality and period of gestation. Demographic details, use of antenatal steroids, exposure to labour and caesarean section delivery differed between mothers depending on the primary complication. Overall 322 (53.0%) received antenatal steroids and 297 (48.8%) were delivered by caesarean section. Factors associated with decreased use of steroids were gestational age of less than 27 weeks (odds ratio (OR) 0.54; 95% CI 0.36-0.83), preterm prelabour rupture of the membranes (OR 0.48; 95% CI 0.29-0.78) and idiopathic preterm labour (OR 0.56; 95% CI 0.35-0.91). Factors associated with increased use of steroids were multiple pregnancy (OR 1.70; 95% CI 1.02-2.81) and pre-eclampsia (OR 1.87; 95% CI 1.09-3.19). CONCLUSIONS: These very preterm deliveries account for only a small proportion of all deliveries. There are differences in the mother's demographic history, obstetric management and delivery depending on the primary aetiological factor. PMID- 8630308 TI - Very preterm birth--a regional study. Part 2: The very preterm infant. AB - OBJECTIVE: To ascertain the growth characteristics, delivery room management and hospital mortality of very preterm liveborn infants (< 33 weeks of gestation) and to identify differences between infants associated with the aetiological factor related to their very preterm delivery. DESIGN: Cohort analytical study. SETTING: King Edward Memorial Hospital for Women, Western Australia. MAIN VARIABLES EXAMINED: Gestational age, birthweight, birthweight ratio, condition at birth and mortality. RESULTS: Six hundred and ninety-three liveborn very preterm infants were born to 608 mothers between 1.1.90 and 31.12.91. This was 1.37% of all liveborns in Western Australia. Three hundred and eighty-five (55.6%) were male. Growth characteristics (birthweight, birthweight ratio and proportion small for gestational age) differed between infants depending on the primary obstetric complication associated with the very preterm delivery. Overall 217 (31%) infants were small for gestational age, 34(4.9%) had a congenital anomaly, and 102 (14.7%) died. Corrected mortality, excluding major fatal congenital anomaly, was 86 (12.7%). The majority of infants died on the first day (n = 59 (57.8%)). The only factors associated with an increased or decreased mortality were decreasing gestation (adjusted odds ratio (AOR) 1.7, 95% CI 1.50-1.93), decreasing birthweight ratio (small for gestational age) (AOR 1.3, 95% CI 1.08-1.53), antepartum haemorrhage as primary complication (AOR 3.1, 95% CI 1.25-7.69) and any antenatal steroids (AOR 0.26, 95% CI 0.14-0.51). In comparison with other studies, survival in the extremely preterm group, defined as a gestational age of less than 28 weeks, is improving. CONCLUSIONS: Very preterm infants account for a large proportion of perinatal mortality. Further studies are required to explore the differences between infants on the basis of the primary obstetric complication and to ensure that increased survival is not associated with an increase in disabilities. PMID- 8630309 TI - Vaginal hysterectomy for the large uterus. AB - OBJECTIVE: To assess the feasibility and safety of performing vaginal hysterectomy on enlarged uteri the equivalent of 14 to 20 weeks of gestation in size. DESIGN: A prospective observational study. SETTING: The Royal Free Hospital, London. PARTICIPANTS: Fourteen consecutive women undergoing vaginal hysterectomy for uterine fibroids up to 20 weeks in size. INTERVENTIONS: Vaginal hysterectomy with or without bilateral salpingo-oophorectomy or oophorectomy. MAIN OUTCOME MEASURES: Uterine size and weight, techniques used to reduce uterine size, surgical outcome, operative time, estimated operative blood loss, intra- and post-operative complications, duration of hospitalisation. RESULTS: The mean uterine size was 16.3 weeks (range 14 to 20 weeks). All hysterectomies were completed successfully by the vaginal route. The uteri weighed 380 to 1100 g, with a mean of 638.7 g. Bisection combined with myomectomy and morcellation were used in most cases to obtain reduction in uterine size, whereas coring was only utilised in two cases. The mean operating time was 84.3 min with a range of 30 to 150 min. The only complications were transient haematuria (n = 6) and superficial vaginal grazes (n = 5). One of the women required a blood transfusion. The mean post-operative hospital stay was 3.7 days (range 2 to 9 days). CONCLUSION: Enlargement of the uterus to a size equivalent to 20 weeks of gestation should no longer be considered a contraindication to vaginal hysterectomy. Many more hysterectomies should be carried out vaginally without resorting to abdominal or laparoscopic surgery. PMID- 8630310 TI - A comparative study of the cosmetic appeal of abdominal incisions used for hysterectomy. AB - OBJECTIVE: To assess the cosmetic appeal of abdominal incisions used for hysterectomy. DESIGN: A comparative study. SETTING: St James's University Hospital, Leeds. PARTICIPANTS: One hundred women, including 50 consecutive women attending a gynaecology clinic for the first time and 50 hospital staff. RESULTS: Sixty-eight percent of women preferred a Pfannenstiel incision as the incision of first choice, while 31% chose the laparoscopic assisted vaginal hysterectomy incisions (LAVH). When women who had undergone previous abdominal surgery were compared with women with no previous surgery, there was a significant difference in their choice (80% Pfannenstiel, 18% LAVH compared with 54% Pfannenstiel, 45% LAVH). CONCLUSIONS: In gynaecological surgery there is already a cosmetically favoured incision. One cannot assume that if a gynaecologist uses a minimally invasive surgical technique at hysterectomy that this will be cosmetically acceptable to the woman. Gynaecologists should not use cosmetic appeal in counselling women for LAVH and should concentrate on the other proven benefits of minimally invasive surgery. PMID- 8630311 TI - The Burch colposuspension for women with and without detrusor overactivity. AB - OBJECTIVE: To compare the results of the Burch colposuspension in women with stress urinary incontinence and detrusor overactivity with those obtained in women with stress incontinence and stable bladders. Additionally, to analyse the value of two detrusor instability indexes and of standard cystometric parameters in predicting the surgical outcome. DESIGN: Retrospective cohort study. SETTING: Secondary referral centre Urodynamics Unit, San Gerardo Hospital, Monza. SAMPLE: Forty-four women with detrusor overactivity (21 with low compliance bladder and 23 with detrusor instability) and 44 with stable bladders. They were matched for age, parity, dystocia, menopause, body mass index, previous vaginal surgery, heavy work, urethral functional length and maximum urethral closure pressure. RESULTS: At two years follow up, the cure rate of stress incontinence was 95% in the control group (patients with stable bladders) and 75% in detrusor overactivity group (odds ratio 0.1, 95% confidence interval 0.01-0.9, P = 0.02). No significant differences were observed between cured and failed patients according to both detrusor instability indexes. Likewise, mean pre-operative standard cystometric values of cured and failed patients showed no differences. CONCLUSIONS: Although results were better in women with stable bladders, we believe that the success of the Burch colposuspension in cases with mixed incontinence should be considered satisfactory. No pre-operative cystometric parameter consistently predicting the surgical outcome on stress incontinence was identified. PMID- 8630313 TI - Impaired implantation after in vitro fertilisation treatment associated with hydrosalpinx. AB - OBJECTIVE: To study whether the presence of hydrosalpinx affected success after in vitro fertilisation (IVF) in women with inflammatory tubal damage. DESIGN: Retrospective, nonrandomised study. SETTING: University private IVF clinic with complete fertility services. PARTICIPANTS: Two study groups (79 women with hydrosalpinges and 198 women with inflammatory tubal damage but no hydrosalpinx) and a third group (22 sterilised, previously fertile women) for comparison. MAIN OUTCOME MEASURES: Clinical pregnancy and livebirth rates per embryo transfer cycle; implantation and live baby rates per individual embryo transferred. RESULTS: The hydrosalpinx group had a significantly lower implantation rate per embryo (8.2%) and chance of a live baby per embryo transferred (5.6%), compared with the nonhydrosalpinx group (14.9% and 11.2%, respectively). The hydrosalpinx group also had a lower clinical pregnancy rate (23%) and live birth rate per transfer cycle (17%) compared with the nonhydrosalpinx group (30% and 26%), but these differences were not significant. CONCLUSIONS: The presence of a hydrosalpinx halves the chance of an embryo implanting, probably due to physical or toxic chemical effects of fluid draining from the hydrosalpinx into the uterine cavity. Women with hydrosalpinges may benefit from distal salpingostomy or salpingectomy as a drainage procedure before in vitro fertilisation treatment, even though such surgery may not increase the chances of natural conception. PMID- 8630312 TI - The effect of oestrogen dose and progestogen type on haemostatic changes in women taking low dose oral contraceptives. AB - OBJECTIVE: To determine the effect of oestrogen dose and progestogen type on the coagulation and fibrinolytic systems of a group of normal healthy women taking three different oral contraceptive combinations. DESIGN: Plasma levels of factor VII, X, antithrombin III, protein C, fibrinogen, tissue plasminogen activator activity, plasminogen activator inhibitor I antigen and fibrin (D-dimer) degradation products were measured at pretreatment, 6, 14, 22 weeks of treatment and at 6 weeks post-treatment in a group of 67 women taking either 30 micrograms ethinyloestradiol/150 micrograms desogestrel (n = 21), 20 micrograms ethinyloestradiol/150 micrograms desogestrel (n = 24), 30 micrograms ethinyloestradiol/75 micrograms gestodene (n = 22). PARTICIPANTS: Sixty-seven healthy normal women, 18 to 34 years, smoking fewer than 15 cigarettes per day. The subjects were within 10% of their normal body weight and had no history of thromboembolic disease. SETTING: Coombe Women's Hospital and St James's Hospital, Dublin, Ireland. RESULTS: Factor VII and X levels were significantly raised on treatment with both the 30 micrograms ethinyloestradiol/desogestrel and gestodene combinations. Higher levels of factor VII activity were observed in the 30 micrograms ethinyloestradiol/desogestrel combination compared with the gestodene combination. Factor VII and X were not significantly affected by the 20 micrograms ethinyloestradiol combination. Increased plasminogen, fibrinogen and D dimer levels and decreased plasminogen activator inhibitor I antigen levels were observed during the treatment phases in all three groups. Antithrombin III and protein C activity did not change during treatment with any of the oral contraceptives studied. CONCLUSIONS: Low dose oral contraceptives cause an activation of the coagulation system which is balanced by an activation of the fibrinolytic system. Reducing the dose of ethinyloestradiol from 30 micrograms to 20 micrograms reduces the effect on factor VII and X. This effect can be modified by the progestogen. The lesser effect of the 20 micrograms combination may make this a safer option for some women than pills containing a higher dose of oestrogen. PMID- 8630315 TI - Short term blood pressure and metabolic effects of tibolone in postmenopausal women with non-insulin dependent diabetes. PMID- 8630314 TI - Diet in pregnancy and the offspring's blood pressure 40 years later. AB - OBJECTIVE: To determine how diet of the mother in pregnancy influences the blood pressure of the offspring in adult life. DESIGN: A follow up study of men and women born during 1948-1954 whose mothers had taken part in a survey of diet in late pregnancy. SETTING: Aberdeen, Scotland. POPULATION: Two hundred and fifty three men and women born in Aberdeen Maternity Hospital. MAIN OUTCOME MEASURE: Systolic and diastolic blood pressure. RESULTS: The relations between the diet of mothers and their offsprings' blood pressure were complex. When the mothers' intake of animal protein was less than 50 g daily, a higher carbohydrate intake was associated with a higher blood pressure in the offspring (a 100 g increase in carbohydrate being associated with a 3 mmHg increase in systolic pressure (P = 0.02)). At daily animal protein intakes above 50 g, lower carbohydrate intake was associated with higher blood pressure (a 100 g decrease in carbohydrate being associated with an 11 mmHg rise in systolic blood pressure (P = 0.004)). These increases in blood pressure were associated with decreased placental size. CONCLUSION: Mothers' intakes of animal protein and carbohydrate in late pregnancy may influence their offsprings' adult blood pressure. This may be mediated through effects on placental growth. PMID- 8630316 TI - Vasa praevia: second trimester diagnosis using colour flow imaging. PMID- 8630317 TI - Is cervical cytology screening of teenagers worthwhile? PMID- 8630318 TI - Intra-vesical foreign body: an unusual cause. PMID- 8630319 TI - Amnioinfusion: a question of benefits and risks. PMID- 8630320 TI - The chronically symptomatic vulva: aetiology and management. PMID- 8630321 TI - A comprehensive philosophy for nursing: the total approach. AB - The political, social, economic, and technological forces being experienced within health care today demand that we apply creative models and philosophies for the delivery of nursing services. This paper describes the application of a "Quality Management Model" and presents a "Philosophy of Nursing Quality" that incorporates the elements of quality, cost, caring and nursing in a holistic manner. Application of this Model and Philosophy could provide nurse managers the guidance required to survive the turbulent 90's. In combination, they maximize the potential for success for both the health care recipient and care provider. PMID- 8630322 TI - Implementation of unit-based continuous quality improvement approach to quality management: applying concepts to practice. AB - This article describes the implementation of unit-based continuous quality improvement approach to quality management and the resultant fostering of autonomy and decision-making among clinical nurses. Restructuring, education and evaluation activities during implementation are described. Nurses in the post implementation evaluation survey stated that quality of care was improved by unit based continuous quality improvement. Unit-based continuous quality improvement promotes a high level of clinical nurse involvement as evidenced by quality improvement initiatives and projects completed. Accompanying this involvement, is a system of continuous quality improvement which facilitates patient care improvement. PMID- 8630323 TI - Innovation adoption: introducing I.V. patient controlled analgesia to the nursing workplace. AB - Technology in the nursing workplace is inevitable but what is not certain is how well these technologies will be initially accepted by nurses. One technological innovation that has appeared in some Canadian hospitals is IV Patient Controlled Analgesia (IV PCA), an innovative technique for pain management, employing the use of a computer-driven pump. Few nursing studies have examined the adoption of technological innovations. This qualitative study examines the incorporation of IV PCA to the nursing workplace and job-related factors that influenced it. A five-phase integration process was identified along with factors impacting on this process. Incorporating IV PCA into nursing practice involved the nurses' judgements of the influence of this innovation on the nurses' comfort with care delivery; the patient/nurse relationship; managing the technology; and the relationship with other health care professionals and patients' family/visitors. Anxiety accompanied the integration process, peaking as IV PCA was introduced to patient care. Findings of this study suggest that actions and decisions initiated by nurse administrators and educators have the potential to influence the successful adoption of this innovation. PMID- 8630324 TI - How a better understanding of adult learning can help improve your practice as a nurse administrator. AB - Theorists tell us that we are smack in the middle of the information age. We cannot help but realize that knowledge is expanding exponentially. The challenge for us as nurses and as nurse leaders is to "keep up", and to do so in a manner which is both efficient and effective. The education of nurses (both basic and ongoing) has historically operated in the pedagogical mode. Nurse learners were expected to be ready and willing to "absorb" whatever the teacher gave out. They were not to demonstrate too much initiative, nor were they held responsible for what or how they learned. Their nursing experience (even when that experience was significant) counted for little. Furthermore, individual nurses have rarely been afforded the opportunity to examine how they learn. Nor have nurse leaders taken into consideration their protegees' preferred learning styles. As a profession, we are very slow to accept and utilize modern principles and practices of learning--principles and practices which we desperately need to cope with the challenges of the information explosion! In undertaking this literature search, I chose to focus on theories of adult and organizational learning, and selected a variety of theories which I believe are timely and relevant to this task. PMID- 8630325 TI - A model for professional nursing in organizations: the Rainbow. AB - This paper presents a model of professional nursing in organizations that provides cohesive direction to the development of patterns that shape how nursing is structured in an organization. Such cohesive direction can foster integration between the practice and the discipline components of nursing. The model can influence the structure of nursing on a macro level within the organization and on a micro level by influencing patterns of nursing care delivery. A set of values, assumptions, dimensions, constructs, principles and propositions have been identified and brought together in this model. As a framework, it can be used to shape the structures and processes that contribute to professional nursing excellence in practice settings. PMID- 8630326 TI - Administrative challenges in a collaborative baccalaureate program. AB - The collaboration between Vancouver General Hospital and the University of British Columbia in offering the four-year baccalaureate nursing program is unique, as it is the first of its kind in Canada. This paper describes the administrative challenges that occurred at VGH as a result of this collaboration. These challenges arose as a result of altered roles and responsibilities, as well as changes in organizational structure and function. Nurse administrators, who are involved in educational collaborations, may find that they are facing similar challenges. PMID- 8630327 TI - Uptake and metabolism of [3H]retinoic acid delivered to human foreskin keratinocytes either bound to serum albumin or added directly to the culture medium. AB - Retinoic acid (RA), a potent modulator of cell proliferation and differentiation is present in plasma bound to serum albumin. The biologic significance or source of plasma RA is not clear. Although most cellular RA is believed to be made in situ via the oxidation of retinol, plasma RA could potentially provide target cells with a source of preformed RA. To investigate RA uptake, we have used a model system of human foreskin keratinocytes (HKc) cultured in serum-free media to compare the uptake and metabolism of [3H]RA added directly to the culture medium in ethanol to that delivered bound to bovine serum albumin (BSA). [3H]RA added directly to the culture medium was rapidly taken up by HKc during the first 10 min of incubation (25-35% of the applied RA), no further accumulation occurred between 10 min and 90 min, and then cell-associated radioactivity rapidly decreased to about 3-5% of the applied dose by 12 h. In contrast, when [3H]RA was delivered to HKc bound to BSA, total cell-associated radioactivity reached about 2.5% of the applied dose by 5 min, increased to 3-5% of the applied radioactivity by 1 h, and no further accumulation or loss occurred over the next 23 h. The uptake by HKc of [3H]RA delivered bound to BSA or added directly to the culture medium was not influenced by pre-treatment of the cells for 72 h with unlabeled RA or by excess unlabeled RA added at the time of uptake. Analysis of the cells and media by high-performance liquid chromatography for RA metabolites found that [3H]RA added directly to the medium is rapidly converted by HKc to polar compounds that are subsequently excreted back into the medium. Also, RA added directly to the medium was susceptible to degradation in the absence of cells. In marked contrast, [3H]RA added to the media bound to BSA was much less susceptible to degradation in the absence of cells, and few [3H]RA metabolites were found in the media even after exposure to HKc for 24 h. The binding of RA to albumin clearly protects RA from conversion to polar metabolites, and also provides for a controlled delivery of RA from the aqueous extracellular environment to the cell surface. PMID- 8630328 TI - Expression of Rab3D in dispersed chief cells from guinea pig stomach. AB - Rab3 proteins are low molecular weight GTP-binding proteins that are expressed in neurons and other secretory cells. These proteins are localized to secretory vesicles and may play a role in regulated exocytosis. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have been identified. We examined the expression of Rab3 isoforms in dispersed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cytosolic and membrane fractions, but staining was more intense in membrane fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To determine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obtained by reverse transcription and subjected to PCR using degenerate primers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced amino acid sequence was identical to that of mouse Rab3D (amino acids 16-83). Moreover, Rab3D was the only isoform detected in chief cells by these methods. To identify rab3 transcripts, the guinea pig rab3D fragment obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in chief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These results indicate that Rab3D is expressed in gastric chief cells. PMID- 8630329 TI - Secretagogue-induced proteolysis of cAMP-dependent protein kinase in intact rat alveolar epithelial type II cells. AB - Stimulation of secretion from rat alveolar epithelial type II cells by the beta adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion. PMID- 8630330 TI - Modulation of queuine uptake and incorporation into tRNA by protein kinase C and protein phosphatase. AB - It has been suggested that the rate of queuine uptake into cultured human fibroblasts is controlled by phosphorylation levels within the cell. We show that the uptake of queuine is stimulated by activators of protein kinase C (PKC) and inhibitors of protein phosphatase; while inhibitors of PKC, and down-regulation of PKC by chronic exposure to phorbol esters inhibit the uptake of queuine into cultured human fibroblasts. Activators of cAMP- and cGMP-dependent kinases exert no effect on the uptake of queuine into fibroblast cell cultures. These studies suggest that PKC directly supports the activity of the queuine uptake mechanism, and that protein phosphatase activity in the cell acts to reverse this. Regardless of the modulation of uptake rate, the level of intracellular queuine base saturates in 6 h. However, there is still an effect on the incorporation rate of queuine into tRNA of fibroblast cultures even after 24 h. We now show that the incorporation of queuine into tRNA in cultured human fibroblasts by tRNA guanine ribosyltransferase (TGRase) is also stimulated by activators of PKC and inhibitors of protein phosphatase; while inhibitors of PKC decrease the activity of this enzyme. These studies suggest that PKC supports both the cellular transport of queuine and the activity of TGRase in cultured human fibroblasts, and that protein phosphatase activity in fibroblasts acts to reverse this phenomenon. A kinase-phosphatase control system, that is common to controlling both intracellular signal transduction and many enzyme systems, appears to be controlling the availability of the queuine substrate and the mechanism for its incorporation into tRNA. Since hypomodification of transfer RNA with queuine is commonly observed in undifferentiated, rapidly growing and neoplastically transformed cells, phosphorylation of the queuine modification system may be a critical regulatory mechanism for the modification of tRNA and subsequent control of cell growth and differentiation. PMID- 8630331 TI - Interactions between Ca2+- and cAMP-dependent stimulatory pathways in parietal cells. AB - Isolated rat parietal cells were used to investigate the role of intracellular Ca2+ in the action of cAMP-dependent secretagogues and cross talk between cAMP- and Ca2+ -dependent stimulatory pathways. Aminopyrine accumulation (an index of acid produced and trapped by the parietal cells), cytosolic free Ca2+, morphological transformation and cell viability were used to investigate parietal cell function and stimulation. The increase of cytosolic free Ca2+ promoted by gastrin, or carbachol, was abolished by the intracellular Ca2+ chelator 1,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 microM). Also, the morphological transformations induced by dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), gastrin, and Sp-adenosine-cyclic-3', 5' monophosphothioate (Sp-cAMPS) were completely abolished by BAPTA (10 microM). In aminopyrine accumulation the action of 1 mM DBcAMP was dose-dependently reduced by BAPTA. The Ca2+ ionophore A23187 alone, in the range of 1 pM to 1 microM, had no effect but it dose-dependently potentiated the action of 1 mM DBcAMP in aminopyrine accumulation. The inhibitory actions of BAPTA on DBcAMP- and histamine-stimulated aminopyrine accumulation were dose-dependently reversed by A23187. Histamine-stimulated protein kinase activity and viability parameters as cellular lactate dehydrogenase (LDH) and trypan blue exclusion were not changed by BAPTA. These results indicated that in isolated parietal cells: (1) the action of cAMP-dependent secretagogues in aminopyrine accumulation and morphological transformation are dependent on cytosolic free Ca2+; (2) Ca2+ -induced morphological transformation is essential for aminopyrine accumulation; (3) a threshold level of one second messenger is required for stimulation of aminopyrine accumulation by the other second messenger. PMID- 8630332 TI - Role of clusterin in cell adhesion during early phases of programmed cell death in P19 embryonic carcinoma cells. AB - This study explored the role of clusterin in mechanisms of cell adhesion and apoptosis in P19 embryonic carcinoma cells. We found that serum deprivation induced transient but dramatic elevation in cell adhesion strength to the culture substrate and eventually led to apoptotic cell death. The time course of cell adhesion increase overlapped temporally with the elevation of clusterin mRNA (peak 8 h after serum deprivation). The coincidental elevation of clusterin expression and cell adhesion strength preceded the schedule of apoptotic cell death. Clusterin antiserum partially antagonized cell adhesion, but did not modify the course of apoptosis. These data suggest that clusterin expression may partially control cell adhesion with no influence on apoptosis in P19 cells, under defined conditions. PMID- 8630333 TI - 5-Iodotubercidin and proglycosyn: a comparison of two glycogenic compounds in hepatocytes from fasted rats. AB - 5-Iodotubercidin (Itu) and proglycosyn (Pro) have similar glycogenic properties. To compare their mechanisms of action, we tested them in hepatocytes from fasted rats. We show that both compounds are similar in that they stimulated glycogen synthesis, increased the concentration of synthase a, decreased that of phosphorylase a and lowered the concentration of F-2,6-P2 in the presence of glucose, lactate-pyruvate and amino acids. However, when amino acids were absent, Pro was the better stimulator of glycogenesis than Itu and in combination they elevated glycogen and synthase a concentrations synergistically. Further they differ in that (1) Itu enhanced the levels of cyclic AMP whereas Pro did not; (2) Pro depressed glucose production from gluconeogenic substrates, whereas Itu stimulated this process; (3) the inhibition of F-2,6-P2 formation and glycolysis by Pro became much weaker than that by Itu when glucose concentrations were raised from 10 to 20 mM. Inhibition of glycolysis but not that of glycogen synthesis was partly due to a phosphorylated metabolite of Itu. The present study indicates that despite their similar glycogenic effects, Itu and Pro do not share a common mechanism of action. Further, the inhibition of glycolysis and F-2,6-P2 formation by Itu cannot be explained if it acts solely as a general inhibitor of protein kinases. PMID- 8630334 TI - Neurotrophin-3 increases the DNA-binding activities of several transcription factors in a mouse osteoblastic cell line. AB - In the mouse osteoblastic cell line MC3T3-E1, the signaling responses of several DNA-binding proteins induced by the treatment of neurotrophin-3 were examined using electrophoretic mobility shift assay. Neurotrophin-3 increased binding activities in nuclear extracts of MC3T3-E1 cells to TPA-responsive element (TRE), cyclic AMP-responsive element (CRE) and serum-responsive element (SRE), but not binding activity in the nuclear extracts to c-Myc binding DNA element. Competition experiments revealed that the binding activity to TRE in the nuclear extracts of neurotrophin-3-treated MC3T3-E1 cells was entirely inhibited by the both unlabeled TRE and CRE probes. On the other hand, the binding activity to CRE was abolished by the unlabeled CRE probe but not by the same amount of unlabeled TRE probe. Moreover, immunodepletion/supershift assay using antibodies directed to Fos, Jun and CREB proteins, showed that the binding activities to TRE and CRE in the nuclear extracts were derived in part from these proteins. PMID- 8630335 TI - The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line. AB - Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi. PMID- 8630336 TI - Early and late G2 arrest of cells undergoing radiation-induced apoptosis or micronucleation. AB - Conventional flow cytometric DNA measurements combined with the microscopic detection of cells in the late G2 phase of the cell cycle (characterized by the occurrence of paired kinetochores) enabled us to differentiate and to quantify early and late G2 cells 0-40 h after irradiation using a radioresistant (L929) and a radiosensitive (HL-60) cell line. This approach provided us with (1) a new kind of G2 arrest characteristic revealing changes in the G2 phase which can not be detected by flow cytometric DNA measurements, (2) cell line dependent differences in the radiation-induced transition through G2, accompanied by the occurrence of micronucleation and apoptosis, and (3) the characterization of apoptotic cells occurring probably during early G2 and bearing a rapidly reduced number of kinetochores in contrast to mitotic cells, suggesting processes different from those that operate in mitosis. PMID- 8630337 TI - Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium. AB - In the normal hamster cheek pouch epithelium, cell proliferation takes place with a pronounced circadian rhythm. We tested our previous hypothesis that all cells having S phase DNA content are actively synthesizing DNA and thus participating in the daily cohort of proliferating cells. We found no evidence of resting S phase cells in the normal epithelium. Using labelling with tritiated thymidine followed by fluorescence activated cell sorting according to DNA content and by autoradiography of the sorted nuclei, it was demonstrated that during the 24 h period almost all cells with mid S phase DNA content were active in DNA synthesis. PMID- 8630338 TI - Proliferative response of mouse spermatogonial stem cells after irradiation: a quantitative model analysis of experimental data. AB - The testes of CDF1 mice were irradiated with single doses of X-rays ranging from 2-16 Gy. The number of haploid cells in the testis at different times after irradiation (42-350 days) was determined by one-parameter flow cytometry both for irradiated animals and for age-matched controls. Based on literature data on the kinetics of the spermatogenesis in mice, a mathematical model of the (hierarchical) germ tissue was developed. Using this model, the processes of radiation-induced cell loss and subsequent recovery were simulated and free parameters of the model were estimated by fitting the model prediction to the experimental data. One of the aims of the study was to investigate the kinetic behaviour of spermatogonial stem cells and the corresponding control mechanisms. In order to fit the data, the model has to include the following features: (i) A preferential self-repopulation of spermatogonial stem cells following tissue injury. The model-estimated probability of a self-renewing division rises from 50% (the steady-state value) to 95% if the stem-cell population is reduced to 10% of its normal size. (ii) A relatively low, almost constant turnover rate of the stem-cell compartment. It is suggested by the analysis that less than 10% of the spermatogonial stem cells present in the testis divide per day, regardless of the degree of cellular depletion. (iii) A mechanism responsible for incomplete recovery. The observed incomplete recovery of spermatogenesis after single doses exceeding 10 Gy can be described quantitatively assuming that the stem cells are organized into discrete proliferative structures, the number of cells per structure being about 60. PMID- 8630340 TI - Computerizing your practice: ready to surf the Internet? PMID- 8630341 TI - Dinosaurs walk among us. PMID- 8630339 TI - Genesis and evolution of high-ploidy tumour cells evaluated by means of the proliferation markers p34(cdc2), cyclin B1, PCNA and 3[H]-thymidine. AB - Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34(cdc2), cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal- and in high-ploidy subpopulations, almost all cells were p34(cdc2)-positive. In the modal-ploidy subpopulation only 17.1% cells were cyclin B1-positive and 85.6% PCNA-positive; in contrast, in the high-ploidy subpopulation up to 91.8% cells were cyclin B1-positive and 97.3% cells were PCNA-positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34(cdc2), both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24-h incubation with 3[H]-thymidine followed by withdrawal of the isotope, high-ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modal-ploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal-ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis. PMID- 8630342 TI - MEDLINE goes on-line. PMID- 8630343 TI - Local application of adenosine induces an increase of capillary diameter in skeletal muscle of anesthetized rabbits. AB - The effects of locally applied adenosine (ADO) and/or femoral artery pressure reduction (induced by complete aorta occlusion) on capillary diameter were investigated in the tenuissimus muscle of anesthetized rabbits. Capillaries were visualized by means of intravital video microscopy. Diameters were measured using an image shearing device. During control femoral artery pressure (median: 83 mm Hg) and without ADO, median capillary diameter was 4.3 microns (range: 3.2-5.3 microns; 27 capillaries in 7 animals). Complete aorta occlusion (median femoral artery pressure: 18 mm Hg) resulted in a reduction of capillary diameter to 3.9 microns (2.7-4.7 microns; p < 0.0001). Subsequent reactive hyperemia resulted in an increase in diameter to 5.2 microns (3.7-6.0 microns; p < 0.0001). Locally applied ADO (10(-4) M) probably led to complete vasodilation of the arterioles, because their diameters did not further increase during reactive hyperemia after complete occlusion. ADO (10(-4) M) induced an increase of control capillary diameter to 5.5 microns (4.1-6.4 microns; median relative increase: 27%; p < 0.0001), resulting in a decrease of capillary resistance by 61%. In the presence of ADO, aorta occlusion resulted in a capillary diameter decrease to 4.7 microns (3.4-6.1 microns); p < 0.0001). Subsequent reactive hyperemia resulted in an increase to maximally 5.6 microns (4.3-6.4 microns; p < 0.0001). This diameter was approximately the same as the control diameter during ADO. During occlusion in the presence of ADO, capillary diameter was significantly larger (11%; p < 0.0001) than during control without ADO. The capillary diameter changes induced by the various interventions were mainly passive, i.e., proportional to capillary transmural pressure changes. However, capillary diameter was larger during aortal occlusion in the presence of ADO than during control femoral artery pressures without ADO, even though capillary pressure was probably higher in the latter case. It is proposed that the prolonged increase in transmural capillary pressure due to ADO may induce changes in capillary wall configuration, leading to larger diameters. PMID- 8630344 TI - Modulatory role of endothelium-derived relaxing factors on the response to vasoconstrictors and flow-pressure curve in the isolated perfused rat kidney. AB - The aim of this study was to compare the effects of the blockade of nitric oxide (NO) and of endothelium-derived hyperpolarizing factor (EDHF) on the response to vasoconstrictors (VC) and on the flow-pressure curve in the isolated perfused rat kidney. To this end, dose-response curves to phenylephrine and BaCl2 were studied in the renal vasculature under basal conditions and after the infusion of N(omega)-nitro-L-arginine methyl ester (L-NAME) and tetraethylammonium (TEA) in endothelium-intact and endothelium-denuded (CHAPS-treated) preparations. In another experiment, renal flow-pressure curves were obtained under basal conditions and after the infusion of L-NAME or TEA. The flow-pressure curve was obtained by increasing renal perfusion flow (RPF) from 2.5 to 15 ml/min/g kidney weight (KW) over the basal level (5 ml/min/g KW). L-NAME or TEA produced a leftward shift of the dose-response curves of both VC, and the simultaneous administration of L-NAME and TEA produced a greater shift to the left in the pressor response curves. CHAPS treatment produced a leftward shift of the dose response curves of both VC, and the dose-response curves were not significantly modified by the infusion of L-NAME or TEA. L-NAME markedly shifted the flow pressure curve upward, especially at higher levels of RPF. However, the administration of TEA, perfusate solution (Tyrode) or D-NAME did not significantly change the flow-pressure curve. These results suggest that NO is released in response to VC as well as to increased perfusion flow in the isolated renal vascular bed. However, EDHF release seems to be stimulated by VC but not by increased perfusion flow. PMID- 8630345 TI - Estrogen replacement increases beta-adrenoceptor-mediated relaxation of rat mesenteric arteries. AB - The purpose of the present study was to determine whether estrogen replacement in ovariectomized rats could modulate arterial diameter responses to beta adrenoceptor activation. Under relaxed conditions (0.1 mM papaverine) there were no differences in the lumen diameter of isolated, pressurized (50 mm Hg) mesenteric arteries from nontreated (191.7 +/- 13.8 microns; n = 19) versus those from estrogen-treated (190.1 +/- 11 microns; n = 14) ovariectomized Sprague Dawley rats. In arteries precontracted with noradrenaline (0.3-1 microM), isoprenaline (0.01-10 microM)-induced relaxation was significantly increased in arteries from ovariectomized estrogen-treated rats (52.4 +/- 2% of the maximal relaxation induced by 0.1 mM papaverine, vs. 33.3 +/- 6.5%; p < 0.01). The half maximal concentration value was 0.04 +/- 0.05 microM in estrogen-treated rats and 0.4 +/- 0.1 microM in nontreated rats (p < 0.01). This response was inhibited by propranolol (1 microM) in both groups to a comparable extent (61.5%), and was unaffected by endothelial removal. Forskolin (0.01-10 microM) induced similar concentration-dependent vasodilation in arteries of both groups of rats with no differences in sensitivity or maximal response. These results suggest that isoprenaline acts through beta-adrenoceptors present on vascular smooth muscle and that estrogen replacement enhances the relaxant responses induced by beta adrenoceptor activation by an endothelium-independent mechanism. PMID- 8630346 TI - Diadenosine polyphosphates increase cytosolic calcium and attenuate angiotensin II-induced changes of calcium in vascular smooth muscle cells. AB - The effects of diadenosine tetraphosphate (AP4A), diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the cytosolic free calcium concentration ([Ca2+]i) were evaluated in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent dye technique. A concentration-dependent increase of [Ca2+]i by AP4A, AP5A, and AP6A was observed in VSMC. Additions of 10 micromol/l AP4A, AP5A, and AP6A significantly increased [Ca2+]i in VSMC by 224 +/ 98 nmol/l (n = 6; p < 0.01, 205 +/- 27 nmol/l (n = 14; p < 0.01), and 269 +/- 98 nmol/l (n = 5; p < 0.05), respectively. Additions of AP4A, AP5A, and AP6A only 120 s prior to angiotensin II (Ang II) administration significantly attenuated the Ang-II-induced changes of [Ca2+]i in VSMC from 1,053 +/- 174 nmol/l to 283 +/ 42 nmol/l, 591 +/- 112 nmol/l, and 477 +/- 79 nmol/l, respectively (each p<0.01) as compared to the control). The AP6A-induced changes of [Ca2+]i were inhibited by the blockers of P2 purinoceptors, suramin and pyridoxal-phosphate-6-azophenyl 2',4'-disulphonic acid, but not by the inhibitor of P2y purinoceptors, reactive blue. Adenosine triphosphate (ATP) also increased [Ca2+]i in VSMC, whereas the purinoceptor P2x agonist, alpha,beta-methylene-ATP, had no effect on [Ca2+]i in VSMC. Therefore diadenosine polyphosphates may induce changes of [Ca2+]i by interacting with purinoceptors and may be involved in local regulation of vascular resistance evoked by the Ca(2+)-dependent contractile response of VSMC. PMID- 8630348 TI - Endothelial cell recoverage and intimal hyperplasia after endothelium removal with or without smooth muscle cell necrosis in the rabbit carotid artery. AB - Interventional-injury-induced intimal hyperplasia involves smooth muscle cell proliferation that may be limited by endothelial cell coverage. We hypothesized that endothelial cell coverage modulates intimal hyperplasia. Therefore rabbit carotid arteries were injured with (2-french Fogarty balloon) and without media necrosis (Prolene loop). After termination at 3, 7, 21, or 42 days, endothelial cell coverage was assessed with an antibody to CD31 and cross-sectional intimal hyperplasia area was measured morphometrically. At 21 and 42 days, maximal intimal hyperplasia thickness was measured at a site where endothelium was present and where endothelium was absent. Proliferating cells were identified with an antibody to the nuclear antigen Ki-67. From 3 to 42 days, endothelial cell coverage progressed from the caudal and cranial ends of the lesion towards the center and was slower in balloon- versus loop-injured arteries (p<0.001, Anova). At 21 and 42 days, intimal hyperplasia area was larger after balloon than after loop injury (21 days: 0.20 +/- 0.01 vs. 0.09 +/- 0.04 mm2, p<0.05; 42 days: 0.26 +/- 0.03 vs. 0.08 +/- 0.02 mm2, p<0.01). At 21 days, the intimal hyperplasia was maximal at the center of the lesion and diminished towards the edges in both balloon- and loop-injured arteries. Surprisingly, at 21 and 42 days, maximal intimal hyperplasia thickness was larger in CD31-positive compared to CD31 negative regions (104 +/- 8 vs. 72 +/- 12 micron, p<0.01, paired t test). At 3 and 7 days, more medial proliferation was found after balloon than after loop injury (3 days: 46.2 +/- 8.8 vs. 0.2 +/- 0.1%, p<0.01; 7 days: 18.5 +/- 6.4 vs. 1.0 +/- 0.4%, p<0.01). In the same period, abundant adventitial proliferation was found after balloon injury that was entirely absent after loop injury. We conclude, first, that endothelial cell recoverage proceeded at a lower rate over damaged than over normal media. This retarded endothelial cell recoverage may contribute to enhanced intimal hyperplasia. Second, at a late stage of vascular healing, when intimal hyperplasia had already been formed, reendothelialization seems to have been enhanced over areas with thick intimal hyperplasia. Third, dilation-induced medial damage was accompanied with massive adventitial cell proliferation. PMID- 8630347 TI - Long-term guanethidine sympathectomy suppresses flow-induced release of ATP and endothelin from endothelial cells isolated from adult rat aorta. AB - Chronic rather than acute changes in the autonomic innervation of the vasculature are a feature of ageing and several cardiovascular disorders. To investigate the long-term influence of perivascular innervation on the vascular endothelium, the release of vasoactive substances which have been localized in endothelial cells, namely ATP, endothelin, substance P and vasopressin, was monitored from cells isolated from adult rat thoracic aorta following neonatal guanethidine sympathectomy. The endothelial cells were initially perfused at 0.5 ml/min and exposed to two periods of increased flow at 3.0 ml/min. Cells isolated from control rats released significantly more ATP on both occasions of switching from the lower to higher flow rate and significantly more endothelin on the second exposure to the higher flow rate. In contrast, endothelial cells isolated from sympathectomised rats showed no increased release of either ATP or endothelin with increase flow, although the release of endothelin at the initial flow rate of 0.5 ml/min was higher than in the controls. Substance P and vasopressin levels in the perfusate were the same in controls and after sympathectomy. In summary, long-term sympathectomy suppresses increased flow-induced release of selected vasoactive substances from the endothelium, thus shear-stress-induced changes in local blood flow may be impaired when there are chronic disturbances in the autonomic innervation. PMID- 8630350 TI - Endothelial modulation of ouabain-induced contraction and sodium pump activity in aortas of normotensive Wistar-Kyoto and spontaneously hypertensive rats. AB - The influence of vascular endothelium on ouabain-induced contractions and sodium pump activity in aortic segments of Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) was analyzed. De-endothelialization increased and reduced ouabain-induced contractions in WKY and SHR segments, respectively. The effects of de-endothelialization were not reproduced by pretreatment of the segments with NG-nitro-L-arginine methyl ester, indomethacin, or 5, 8, 11, 14-eicosatetraenoic acid, acetyl salicylic acid, dazoxiben, phosphoramidon, BQ-123, or superoxide dismutase. Bioassay experiments suggest that ouabain releases a diffusible factor from endothelial cells that inhibits or facilitates digitalis-induced contractions in WKY and SHR segments, respectively. In a potassium-free solution, potassium-induced relaxation in segments of both strains was abolished by ouabain in de-endothelialized aortas and reduced in intact ones. Ouabain-sensitive 86Rb+ uptake was significantly reduced by de-endothelialization both in WKY and in SHR. These results suggest that the vascular endothelium of WKY and SHR aortas releases a diffusible factor that stimulates the sodium pump and/or protects it from ouabain blockade. Ouabain also releases a diffusible endothelium-derived factor in SHR aortas that facilitates ouabain-induced contractions. PMID- 8630349 TI - Thrombogenicity of the human arterial wall after interventional thermal injury. AB - Thermal injury has been shown to reduce platelet adhesion (PA) in vitro but not in vivo. The controversy may be based on the mode of thermal injury, the anticoagulation regimen, or species differences. Human and rabbit arteries were dilated by a radio-frequency (RF)-heated balloon (RF dilation) or by immersion in heated buffer. The artery segments were perfused in an annular perfusion chamber with blood anticoagulant by citrate or heparin (37 degrees C, 5 min, shear rate: 1,300 s-1). To determine PA to deep wall layers, 6-micron cross-sections of heated arteries were perfused in a rectangular perfusion chamber (37 degrees C, 5 min, 1,300 s-1). After RF dilation of human arteries at 55 and 90 degrees C, subendothelical PA (citrated blood) decreased from 28.9% at 37 degrees C to 6.8%, and increased to 39.6%, respectively (in both cases p<0.05). Heparin anticoagulation resulted in subendothelial fibrin deposition that was equal after 37 and 90 degrees C, and decreased after 55 degrees C. Heating of cross-sections of atherosclerotic coronary arteries to 55 and 90 degrees C, showed increased and decreased PA, respectively, to the intima and media. No effect was observed on the highly reactive adventitia and atherosclerotic plaque. We conclude that thermal balloon angioplasty at 90 degrees C reduces PA to the arterial subendothelium, but not to the adventitia or the atherosclerotic plaque. As thermal balloon angioplasty in patients will always produce a region with increased PA at 55 degrees C and as heparin anticoagulation permits fibrin deposition that is not affected by heat, it is unlikely that thermal balloon angioplasty alone will reduce thrombotic complications. PMID- 8630351 TI - Mechanical properties of porcine small coronary arteries in one-kidney, one-clip hypertension. AB - The purpose of this study was to analyse the mechanical properties of small coronary arteries from pigs with one-kidney, one-clip hypertension and normotensive control animals and to determine whether these could contribute to vascular structural changes observed in hypertension. Small coronary artery wall tension-internal circumference relationships were determined for coronary arteries mounted in a myograph and used to calculate incremental elastic modulus stress relationships for arteries from normotensive and hypertensive pigs. Wall tension-internal circumference relationships were shifted to the left in arteries from hypertensive pigs, but the stress-strain and incremental elastic modulus stress relationships were not influenced by the increased blood pressure load. These data indicate that small coronary arteries from hypertensive pigs are not less distensible than those from normotensive controls. Therefore the previously reported increased media thickness:lumen diameter ratios in coronary arteries in this model of hypertension are not a consequence of abnormal elastic properties and can be ascribed to a remodelling process. PMID- 8630352 TI - Multiple substrates for cGMP-dependent protein kinase from bovine aortic smooth muscle: purification of P132. AB - Elevation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) in vascular smooth-muscle cells produces relaxation, but mechanisms distal to PKG activation are not well understood. Few PKG substrates have been described in smooth muscle that may mediate the action of PKG, including P240, P132 and phospholamban. None of them is a specific PKG substrate, raising the question of whether any specific PKG substrates possibly exist in vascular smooth muscle that may play roles in relaxation. In this study PKG substrates were detected in aortic smooth muscle by adding purified exogenous PKG and [gamma-32P]-ATP. Very few PKG substrates were detectable in whole-tissue homogenates or detergent solubilized fractions, due to the high basal activity of other protein kinases and the large numbers of other phosphoproteins. Heat or acid treatment of such fractions, to remove any endogenous protein kinase activity and achieve partial protein purification, revealed many potential PKG substrates. Of the 3 substrates identified previously, P240 and P132 were partly heat-stable. Thirty-one new PKG substrates were found: 14 in the initial heat-stable extract and 9 in the heat- and acid-soluble extract, whereas the others were revealed only after chromatography. All of the heat-stable PKG substrates were bound and salt-eluted from a DEAE-cellulose column in 2 major peaks called pool I and II. After sequential application to Q-Sepharose and S-Sepharose columns, 7 PKG substrates were found in pool I, in particular a group of 4 substrates of 40, 33, 28 and 22 kD virtually coeluted through all 3 columns. The former 3 produced similar phosphopeptide maps, suggesting a relationship. All the new substrates from pool I were relatively specific for PKG because they were poorly phosphorylated with exogenous cAMP-dependent protein kinase and not with Ca2+/phospholipid-dependent protein kinase. Further chromatography of the proteins in pool II resulted in an extensive purification of P132 as well as a group of 4 PKG substrates of 33-30 kD. Phosphopeptide mapping of the 132-kD protein revealed a close homology to the 132-kD PKG substrate previously described in rat aortic smooth muscle. These data demonstrate the presence of multiple substrates for PKG in aortic smooth-muscle tissue. PMID- 8630353 TI - [New knowledge: universities on the threshold of the 21st century]. PMID- 8630354 TI - [Computerized tomography volumetry of the cerebrospinal fluid by semiautomatic contour recognition and gray value histogram analysis]. AB - PURPOSE: A program (VAC, Siemens) using self-made, anthropomorphous phantoms to measure semiautomatically the volume of the cerebral liquor in CT scans of the whole skull was tested. MATERIAL AND METHODS: Cerebral tissue was simulated by ellipsoid bodies made from Agar and NaI which were placed in a human skull. Volumes of the ventricular and subarachnoidal liquor could be defined arbitrarily. RESULTS: A correlation coefficient of r > = 0.9 using a slice thickness of 1-5 mm was found (thickness 8 mm: r = 0.75). The volume of the cerebral liquor was underestimated by 25-68%. Separate measurements of the ventricular and subarachnoidal liquor spaces showed a high accuracy of the measurements done in the ventricles (r = 0.997, y = 0.996 x -1). The volume of the subarachnoidal liquor was not detected completely due to partial volume effects which were seen especially in the basal and apical scans. CONCLUSION: The program VAC is useful for the semiautomatic measurement of the volume of the ventricles. The assessment of the subarachnoidal liquor is limited to semiquantitative results which may, however, be useful for follow-up studies. PMID- 8630355 TI - [MRI of the pancreas: value of standard versus fat-suppressed pulse sequence at 0.5 T]. AB - PURPOSE: To compare five pulse sequences in 60 patients with pancreatic disease by means of MRI at 0.5 T. METHODS: T1 weighted Spin-Echo sequences (T1W SE) before and after i.v. contrast medium administration, fat-suppressed T1W SE sequences after i.v. contrast medium administration, T2 weighted Turbo-Spin-Echo sequences (T2W TSE), and fat-suppressed T2W TSE were evaluated within an ROC study. RESULTS: The standard sequences (T1W SE before and after i.v. contrast medium administration, T2W TSE) were superior to the fat-suppressed T1W and T2W (T)SE sequences in delineating the anatomy and for correct diagnosis. The T1W sequence with and without i.v. contrast media detected all anatomic structures best. In most instances the correct diagnosis and coexistent findings in the diseased pancreas were obtained with the T2W sequence. CONCLUSION: Fat-suppressed sequences are less useful in the diagnosis of pancreatic disease by MRI at medium field strength compared to the standard sequences. PMID- 8630356 TI - [2-D-time-of-flight MR angiography of the peripheral blood vessels. Experimental and clinical studies on value of this method in arterial occlusive diseases]. AB - PURPOSE: A retrospective study was performed in 70 patients with peripheral vascular disease who underwent MR angiography of the lower extremity. MATERIAL AND METHODS: MR angiography was performed with a 2-D-TOF sequence including a travelling presaturation to suppress the venous signal. Postprocessing images were obtained with the MIP algorithm. The MRA results were compared with conventional or digital angiography in all patients. RESULTS: Only 19 stenoses out of 31 which showed a degree of stenosis between 30 and 99% could be visualised by MR angiography. 9 stenoses were correctly classified by MRA. In 9 cases the degree of stenosis was overestimated, in one case it was underestimated. All 56 occlusions were correctly detected. 12 severe stenoses diagnosed with conventional angiography were graded as occlusions with MR angiography. CONCLUSIONS: MR angiography cannot be accepted in preoperative staging of patients with peripheral vascular disease. The postoperative examination seems to be a practical noninvasive alternative. PMID- 8630357 TI - [3-D-TONE magnetic resonance angiography in the detection of intracranial aneurysms compared with digital subtraction angiography. A prospective study]. AB - PURPOSE: To evaluate the efficiency and accuracy of the optimized 3-D-MT-TONE magnetic resonance angiography (MRA) with that of intraarterial digital subtraction angiography (DSA) in the detection of intracranial aneurysms. MATERIAL AND METHODS: From March 1994 to March 1995 30 patients with 38 aneurysms were prospectively subjected to MRA as well as to DSA. Respiratory insufficient patients were excluded from MRA. RESULTS: The aneurysm size ranged from 3-50 mm with an average size of 9 mm. Correct diagnosis was done in 33 aneurysms (86.8%) in 26 of the patients. In 4 patients (13.3%) with 5 aneurysms, the blinded readers made an incorrect diagnosis due to degradation of image quality. One (2.6%) aneurysm was falsely described via MRA as well as via DSA. The sensitivity was 86.8%. In the control group of 30 patients three aneurysms were found, therefore the specificity was 90% and we could calculate a positive predictive value of 97.1%, a negative predictive value of 84.4% and a correct diagnosis of 90.1%. CONCLUSION: Our results show that MT-TONE-MRA is a useful non-invasive technique with improved vascular detail and vascular background for detection of intracranial aneurysm. Further technical improvements are necessary for better visualisation of pathological findings in the carotid siphon, where only 60% of the aneurysms could be clearly detected. PMID- 8630358 TI - [Initial experiences with dilatation of dialysis shunts with color-coded duplex ultrasonography monitoring]. AB - PURPOSE: Aim of the study was to evaluate the technical aspects of colour coded duplex sonography guided interventions of peripheral vessels. METHODS: During 15 months 39 stenoses of shunt veins in 24 patients were dilated guided by colour coded duplex sonography. RESULTS: 38 stenoses were dilated without complications. The blood flow volume was increased from 361.9 +/- 83.5 to 718.9 +/- 189.2 ml/min. In one case it was not possible to dilate the stenosis because of a vasospasm. CONCLUSION: PTA guided by colour coded duplex sonography is an alternative to DSA guided interventions of superficial vessels without x-ray exposure or contrast agent application. PMID- 8630359 TI - [Evaluation system in assessing radiological examinations of clinical significance for the patient]. AB - PURPOSE: The purpose of this study was to test a scoring system and its utility for the assessment of the clinical significance of radiological examinations. MATERIAL AND METHODS: The reports of all diagnostic studies obtained in patients with a clinical suspicion of pancreatitis, urinary tract obstruction, biliary or gallbladder diseases, ileus and gastrointestinal perforation were scored prospectively in 4 categories from 0 to 3. These categories were related to the ability to establish a definite diagnosis, to exclude suspected disease, to influence further diagnostic work-up and therapeutic decision-making. RESULTS: This scoring system showed significant differences of the efficacy of different imaging modalities in these 5 clinical settings. CONCLUSION: This scoring system was helpful to determine the clinical significance of radiological examinations. PMID- 8630361 TI - [Intravascular therapy of aortic aneurysms: initial clinical results]. AB - PURPOSE: A clinical study aiming at examining the implantation technique and the clinical results of a new vascular prosthesis in endovascular therapy of aneurysms of the aorta. MATERIAL AND METHODS: In 21 patients (20 men of 55 to 87 years of age, average age 70 years) with aneurysms of the infrarenal abdominal aorta (AAA n = 19) and the thoracic aorta (n = 2), a self-expanding nitinol stent with outer Dacron sheath (tubular prosthesis n = 4; bifurcation prosthesis n = 17) was implanted by means of an F-18 introductory set after surgical opening of the inguinal artery. RESULTS: By means of the 4 tubular prostheses and 13 of the 17 bifurcation prostheses the aneurysm was bypassed completely. In 4 cases residual perfusion through leaks (n = 3) or collateral blood supply (n = 1) was seen. In 6 patients 7 complications occurred which did not require to be operated on. No patient died in consequence of the surgical intervention or during the hospital stay. Follow-ups up to 8 months after surgery did not reveal any late complications to date. CONCLUSIONS: Endovascular implantation of vascular prostheses is a promising new method for interdisciplinary treatment of aneurysms of the aorta. PMID- 8630360 TI - [Endoluminal therapy of infrarenal abdominal aortic aneurysms: a new interventional technique]. AB - PURPOSE: With a series of 31 transluminal stent-graft procedures, we assessed the feasibility and clinical effectiveness of a new stent-graft for the treatment of infrarenal abdominal aortic aneurysms (AAAs). METHODS: We treated the patients (male; mean age 71 years) with straight or bifurcated nitinol stents covered with woven dacron graft material for infrarenal eccentric saccular AAA (n = 4) or AAA involving the bifurcation and the common iliac arteries (n = 27). The 18-F delivery system was advanced via a surgical arteriotomy and the stent-graft was placed under fluoroscopic control. Follow-up period ranged from 8 days to 9 months. RESULTS: The implantation of the stent-grafts was technically successful in 30/31 cases. Procedure-related complications were acute hepatic failure due to gastric bleeding in a patient with liver cirrhosis, graft occlusion due to emboli originating from the left atrium (n = 1), macro-embolisation (n = 3), local haematoma (n = 1) and av-fistula (n = 1) requiring surgery. A post-implantation syndrome with leukocytosis and elevated C-reactive protein was observed in all patients. CONCLUSIONS: Endoluminal repair of infrarenal AAA with use of dacron covered nitinol stent-grafts is feasible, safe and clinically effective. However, careful long-term evaluation is necessary. PMID- 8630362 TI - [Technique of MRT-guided core biopsy in the abdomen using an open low-field scanner: feasibility and initial clinical results]. AB - PURPOSE: Due to high soft-tissue contrast and multiplanar imaging capabilities MRI is an interesting modality to perform image-guided biopsies. We checked on the feasibility of MR-guided core biopsies of abdominal masses with an open low field scanner (0.2 Tesla; vertical field axis). METHODS: 9 patients underwent MR guided biopsies of abdominal target lesions (6 focal liver lesions, two adrenal masses, one pelvic mass). Different MR-compatible core biopsy instruments were used (needle diameters 14G-18G). MR scans during the procedures were obtained applying T1-weighted gradient echo sequences suitable for breath-hold imaging. RESULTS: In each case, needle guidance was reliable, so that the biopsy instrument could be positioned correctly. Multiplanar imaging capabilities enabled even angled approaches to upper abdominal masses to be realized safely. The combination of magnet design and table design offered suitable access to the patient. CONCLUSION: Using an open low-field scanner, MR-guided core biopsies of abdominal masses are practicable. PMID- 8630363 TI - [In vitro studies on pulsed laser angioplasty in liquid and gaseous medium]. AB - PURPOSE: In a vascular model the mechanisms of a pulsed dye laser working in a liquid and a gaseous environment was tested and documented by means of a high speed camera. METHODS: The pulse application (630 nm wavelength) on a target of calcified arterial plaque material was detected at intervals of 20 microseconds up to 800 microseconds total time. RESULTS: In water the laser beam created a cavitation bubble which expanded from 0 to 450 microseconds from a radius 0 to 3 mm and collapsed afterwards. The average expansion velocity of the bubble was 80 m/s in the first 20 microseconds and the resulting velocity of the tube model wall was 10 m/s. Compared with gaseous atmosphere there was no bubble and consecutively no wall stress. CONCLUSION: It is obvious that laser angioplasty causes wall stress and destruction of different vascular layers. This might be the initial stimulation for restenosis. In gaseous atmosphere laser angioplasty should be more protective. PMID- 8630364 TI - [Clinical relevance of mammography in men]. AB - PURPOSE: During a period of four years 104 mammograms were performed in 89 men. Mastectomies were carried out on 23 men (group 1). 66 patients (group 2) were followed up clinically and in 15 patients serial mammograms were obtained. MATERIAL AND METHOD: In group 1 there were 5 patients with bilateral gynaecomastia, 9 with unilateral gynaecomastia and two with pseudogynaecomastia and there were 7 patients with malignancies. In group 2 there were 46 patients with bilateral gynaecomastia and 10 patients with unilateral gynaecomastia. Pseudogynaecomastia was found in 25 patients. There were 7 malignancies, of which 5 had been suspected clinically and one had been diagnosed as gynaecomastia by mammography. Two carcinomas in situ were missed clinically and also by mammography. CONCLUSION: When malignancy is not suspected on clinical grounds the first examination should be sonography, mammography being reserved for cases of doubt. Where, however, malignancy is suspected, and for follow-up, mammography retains its primary position. PMID- 8630365 TI - [Secondary stabilization of aortic bifurcation Chuter endoprostheses by implantation of self-expanding metal stents]. AB - The use of conventional graft fabric for the bifurcated Chuter endovascular graft may result in a kink instability since crimping has been removed. Especially in elongated arteries that are frequently found with aortic aneurysms this may cause stenosis of the kinked graft and subsequent thrombosis. Bilateral stabilisation of the graft limbs along its total length proved sufficient to stabilise the graft thus preventing secondary thrombosis. PMID- 8630366 TI - [3-D visualization of thoracic tumors from MR images]. AB - AIM: In cooperation with the thoracic surgery department 3-D visualizations of tumors were generated to support the surgeons in preoperative planning. As opposed to 3-D reconstructions of CT data, those representations based on MRI images remain an exception. In this paper different methods of three dimensional visualization of lung tumors are presented and compared to each other. DESIGN: These methods are 1. contour based surface models, 2. threshold based surface models and 3. rendered scenes of segmented volume data with transparent, color coded display. RESULTS: Combinations of all three methods in one single image are possible as well with the software we developed. Furthermore, cut planes through the original data can be integrated into the 3-D scene. The segmentation of anatomical objects is performed either manually or automatically with various procedures. CONCLUSIONS: Adequate possibilities of manipulation and archiving allow a fast handling and processing even of large data sets. The calculated volumes of the anatomical objects can be used for quantitative studies. Arbitrary views of the three dimensional reconstructions can be generated within a few seconds. Even animation can easily be calculated and displayed with 30 frames/second. PMID- 8630367 TI - [Thoracic aortic aneurysm with acute aortopulmonary fistula]. PMID- 8630368 TI - [HIV infection with abdominal non-Hodgkin's lymphoma presenting as necrotizing pancreatitis]. PMID- 8630370 TI - [Radiological and clinical aspects of Poland syndrome]. PMID- 8630369 TI - [Inflammatory pseudotumor (plasma cell granuloma) of the lung in childhood: a case report]. PMID- 8630371 TI - [Treatment of radiogenic ureteral stenosis with a wall stent]. PMID- 8630372 TI - Biologic basis for interleukin-1 in disease. AB - To understand the role of the proinflammatory cytokine interleukin-1 (IL-1) in disease, investigators have studied how production of the different members of the IL-1 family is controlled, the various biologic activities of IL-1, the distinct and various functions of the IL-1 receptor (IL-1R) family, and the complexity of intracellular signaling. Mice deficient in IL-1Beta, IL-1Beta converting enzyme, and IL-1R type I have also been studied. Humans have been injected with IL-1 (either IL-1alpha or IL-1beta) for enhancing bone marrow recovery and for cancer treatment. The IL-1-specific receptor antagonist (IL-1Ra) has also been tested in clinical trials. The topics discussed in this review include production and activities of IL-1 and IL-1Ra molecules, the effects of IL 1 on gene expression, functions of cell-bound and soluble IL-1 receptors, the importance of the IL-1R accessory protein, newly discovered signal transduction pathways, naturally occurring cytokines limiting IL-1 production or activity, the effects of blocking cyclooxygenase and nitric oxide, and the outcomes of IL-1 and IL-1 Ra in human trials. Special attention is paid to IL-1beta converting enzyme and programmed cell death. The roles of IL-1 in hematopoiesis, leukemia, atherosclerosis, and growth of solid tumors are also discussed. This is a lengthy review, with 586 citations chosen to illustrate specific areas of interest rather than a compendium of references. At the end of each section, a short commentary summarizes what the author considers established or controversial topics linking the biology of IL-1 to mechanisms of disease. PMID- 8630373 TI - Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G-CSF-mediated activation of signaling complexes of the p21ras route. AB - Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G-CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G-CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R. PMID- 8630374 TI - Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets. AB - The involvement of platelets and the c-mpl receptor in the regulation of thrombopoietin (TPO) plasma concentrations and tissue mRNA levels was investigated in both normal mice and mice defective in c-mpl (c-mpl-/-). Although c-mpl-/- mice have fewer platelets and higher plasma TPO activity than normal mice, there was no increase in TPO mRNA levels as measured by an S1 nuclease protection assay. After the intravenous injection of 125I-TPO, specific uptake of radioactivity by the spleen and blood cells was present in the normal mice, but absent in the c-mpl-/- mice. Platelet-rich plasma (PRP) from normal mice was able to bind and internalize 125I-TPO, whereas PRP from c-mpl-/- mice lacked this ability. Analysis of 125I-TPO binding to normal PRP indicated that binding was specific and saturable, with an approximate affinity of 560 pmol/L and 220 receptors per platelet. PRP from normal mice was also able to degrade 125I-TPO into lower molecular weight fragments. After the intravenous injections, c-mpl-/- mice cleared a dose of 125I-TPO at a much slower rate than did normal mice. Injection of washed platelets from normal mice into c-mpl-/- mice resulted in a dramatic, but transient, decrease in plasma TPO levels. These data provide evidence that platelets regulate plasma TPO levels via binding to the c-mpl receptor on circulating platelets. PMID- 8630375 TI - Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. AB - Mice with a null mutation in the thrombopoietin (TPO) receptor c-Mpl were generated by gene targeting. c-mpl-deficient mice developed normally but were deficient in megakaryocytes and severely thrombocytopenic. The hematocrit and numbers of mature circulating leukocytes were normal in mpl-/- mice, as was the distribution of morphologically identifiable precursors in hematopoietic tissues. Bone marrow and spleen cells of adult mpl-/- mice lacked specific binding sites for TPO, were unresponsive to TPO in culture, and displayed a marked deficiency in progenitor cells with megakaryocytic potential. Significantly, total hematopoietic progenitor cell numbers were also reduced in mpl-/- mice including multipotential, blast cell, and committed progenitors of multiple lineages. The megakaryocyte deficiency was evident as early as 14 days of gestation in mpl deficient mice, although reductions in progenitor cell numbers arose only later in development. The data suggest that the critical function of c-Mpl signalling in megakaryocytopoiesis is in maintenance of mature megakaryocyte numbers through control of progenitor cell proliferation and maturation. Moreover, our results also imply an important role for TPO and c-Mpl in the production of primitive pluripotent progenitor cells as well as progenitor cells committed to nonmegakaryocytic lineages. PMID- 8630376 TI - Mutations in the Wilms' tumor gene WT1 in leukemias. AB - The tissue-specific Wilms' tumor gene WT1 is expressed in a range of acute leukemias and hematopoietic cell lines. Using single-strand conformational polymorphism analysis, we have found mutations in the WT1 gene in 4 of 36 acute leukemias. WT1 mutations are found in 15% of cases of acute myeloid leukemia, in which they are associated with a poor response to chemotherapy. The mutations comprise small insertions in exons 1 and 7 and a nonsense mutation in exon 9. All are predicted to produce a truncated WT1 protein with absence or disruption of the zinc finger region. These are the first mutations in the WT1 gene to be described in sporadic leukemia. PMID- 8630377 TI - Multiple tumor-suppressor gene 1 inactivation is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia. AB - No constant genetic alteration has yet been unravelled in T-cell acute lymphoblastic leukemia (T-ALL), and, to date, the most frequent alteration, the SIL-TAL1 deletion, is found in approximately 20% of cases. Recently, two genes have been identified, the multiple tumor-suppressor gene 1 (MTS1) and multiple tumor-suppressor gene 2 (MTS2), whose products inhibit cell cycle progression. A characterization of the MTS locus organization allowed to determine the incidence of MTS1 and MTS2 inactivation in T-ALL. MTS1 and MTS2 configurations were determined by Southern blotting using 8 probes in 59 patients with T-ALL (40 children and 19 adults). Biallelic MTS1 inactivation by deletions and/or rearrangements was observed in 45 cases (76%). Monoallelic alterations were found in 6 cases (10%). The second MTS1 allele was studied in the 4 cases with available material. A point mutation was found in 2 cases. The lack of MTS1 mRNA expression was observed by Northern blot analysis in a third case. A normal single-strand conformation polymorphism pattern of MTS1 exons 1alpha and 2 was found and MTS1 RNA was detected in the fourth case, but a rearrangement occurring 5' to MTS1 exon 1 alpha deleting MTS1 exon 1Beta was documented. One case presented a complex rearrangement. Germline configuration for MTS1 and MTS2 was found in only 7 cases. The localization of the 17 breakpoints occurring in the MTS locus were determined. Ten of them (59%) are clustered in a 6-kb region located 5 kb downstream to the newly identified MTS1 exon 1Beta. No rearrangement disrupting MTS2 was detected and more rearrangements spared MTS2 than MTS1 (P<.01). MTS1 but not MTS2 RNA was detected by Northern blotting in the human thymus. These data strongly suggest that MTS1 is the functional target of rearrangements in T-ALL. MTS1 inactivation, observed in at least 80% of T-ALL, is the most consistent genetic defect found in this disease to date. PMID- 8630378 TI - Long-term leukemia-initiating capacity of a CD34-subpopulation of acute myeloid leukemia. AB - Acute myeloid leukemia (AML) proliferation in vivo is maintained by a small fraction of progenitor cells. These cells have been assumed to express an immature phenotype and to produce most colony-forming units (CFU-AML). For one case of AML (French-American-British [FAB] M1, normal cytogenetics), we examined the capacity of the CD34+ (25% of unseparated AML cells) and CD34- fractions to initiate leukemia in severe combined immunodeficient (SCID) mice. In addition, the production of CFU-AML and nucleated cells (NC) of these subsets was investigated in long-term bone marrow culture (LTBMC). The frequencies of cobblestone area-forming cells (CAFC) were also estimated; early appearing cobblestone areas (CAs) are indicative of relatively mature progenitors and late CAs represent the progeny of primitive progenitors. In mice transplanted with CD34- (98% pure) or CD34+ (98% pure) grafts, similar AML cell growth was seen throughout an observation period of 106 days. The capacity to establish long-term growth from the CD34- cells was confirmed by renewed outgrowth after retransplantation. In vitro, the CD34- fraction contained both immature and mature CAFCs and produced high numbers of CFU-AML and NC in LTBMC. The CD34+ fraction produced only small numbers of CFU-AML, NC, and mature CAFCs. Therefore, the expression of CD34 and the content of CFU-AML were not associated with long term growth of AML. However, similar frequencies of primitive CAFCs were observed in both fractions. Thus, both CD34- and CD34+ subsets of this AML sample contained immature progenitors with the capacity to initiate long-term AML growth as characterized in vivo (in SCID mice) as well as in vitro (in CAFC assay), indicating asynchrony between functional and immunophenotypical maturation of AML progenitor cell compartments. PMID- 8630379 TI - Allogeneic cell therapy with donor peripheral blood cells and recombinant human interleukin-2 to treat leukemia relapse after allogeneic bone marrow transplantation. AB - Allogeneic bone marrow transplantation (BMT) is the only effective treatment for hematologic malignancies resistant to conventional chemotherapy. Until recently, no cure existed for patients who relapsed post-BMT. We present our long-term observations on remission induction, after relapse post-BMT, by allogeneic cell therapy (allo-CT) and the feasibility of remission induction in allo-CT-resistant patients by activation of antileukemia effector cells with recombinant human interleukin-2 (rhIL-2) in vitro and in vivo. The longest observation of successful allo-CT (event-free survival, greater than 8 years) was made in a patient with resistant pre-B lymphoblastic leukemia who received infusions with graded increments of donor (female) peripheral blood lymphocytes (PBL) as soon as bulky hematologic and extramedullary relapse was noticed early post-BMT. The patient is currently without evidence of residual host (male) cells as determined by polymerase chain reaction (PCR). Of 17 patients with acute and chronic leukemia in relapse after BMT, 10 were reinduced into complete remission. Four patients with cytogenetic relapse responded to allo-CT alone, while five of six patients with overt hematologic relapse responded only after additional activation of donor with rhIL-2. Allo-CT can, therefore, successfully reverse chemoradiotherapy-resistant relapse of both acute and chronic leukemia. Moreover, in patients resistant to donor lymphocyte infusion, remission can be accomplished by additionally activating donor PBL in vitro and/or in vivo with rhIL-2. Based on our observations, after BMT, allo-CT should be considered the treatment of choice for patients with hematologic malignancies resistant to conventional anticancer modalities. Allogeneic activated cell therapy (allo ACT) should be considered for patients with tumor cells resistant to allo-CT. Although allo-CT, followed if indicated by allo-ACT, can be effective for patients with overt hematologic relapse, reversal of persistent minimal residual disease or documented molecular/cytogenetic relapse early after BMT may also be considered as a possible indication for allo-CT. PMID- 8630380 TI - Prospective, randomized trial of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide chemotherapy in combination with the interleukin-3/granulocyte macrophage colony-stimulating factor (GM-CSF) fusion protein (PIXY321) versus GM CSF in patients with advanced breast cancer. AB - We conducted a prospective randomized trial to evaluate the ability of the interleukin-3/granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion protein, PIXY321, to ameliorate cumulative thrombocytopenia after multiple cycles of 5-fluorouracil, leucovorin, doxorubicin, cyclophosphamide (FLAC) chemotherapy compared with GM-CSF in patients with advanced breast cancer. Fifty-three patients were randomized to receive either PIXY321. 375 microg/m2 twice a day subcutaneously, or GM-CSF, 250 microg/m2 daily subcutaneously after FLAC chemotherapy. PIXY321 was less well tolerated than GM-CSF, with more patients developing chills and local skin reactions and more patients stopping PIXY321 due to intolerance. While no difference in the neutrophil nadirs was seen with the two cytokines, the duration of the absolute neutrophil count less than 1,000/muL for all cycles was significantly longer with PIXY321 than with GM-CSF. Fifty percent of patients treated with multiple cycles of FLAC chemotherapy on both study arms developed dose-limiting thrombocytopenia. No differences in platelet nadirs, duration of thrombocytopenia, or need for platelet transfusions were observed with PIXY321 versus GM-CSF. The average delivered doses of FLAC chemotherapy were somewhat higher in the GM-CSF study arm. PIXY321 was not superior to GM-CSF in ameliorating the cumulative thrombocytopenia observed with multiple cycles of FLAC chemotherapy and was less well tolerated. PMID- 8630381 TI - The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells. AB - Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting-gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation. PMID- 8630382 TI - Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb. AB - To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA 2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5' flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment. PMID- 8630383 TI - Immobilized anti-KIT monoclonal antibody induces ligand-independent dimerization and activation of Steel factor receptor: biologic similarity with membrane-bound form of Steel factor rather than its soluble form. AB - Interaction of a tyrosine kinase type receptor and its ligand induces receptor dimerization or -oligomerization followed by transphosphorylation and activation of its intrinsic kinase, which leads to a series of intracellular signals. We have previously reported that the membrane-bound form of Steel factor (SLF) induces more persistent tyrosine kinase activation and longer life span of c-kit encoded protein (KIT) than its soluble form (Miyazawa et al, Blood 85:641, 1995). In this study, we used YB5.B8 monoclonal antibody (MoAb) that recognizes the extracellular domain of KIT to investigate whether immobilized anti-KIT MoAb can substitute for SLF as a potent activator of KIT by cross-linking receptors and further compared its effect with each SLF isoform in a factor-dependent cell line M07e. YB5.B8 MoAb in a soluble state suppressed SLF-induced M07e cell proliferation in a dose-dependent manner. By contrast, once this antibody was immobilized on the goat-antimouse MoAb (GAM)-coated culture plates, it supported the growth of M07e cells in the absence of any growth factors, whereas culture the cells in GAM alone or YB5.B8 without GAM-coated plates resulted in rapid cell death within 24 hours. As with the natural ligand SLF, immobilized YB5.B8 MoAb synergized with granulocyte-macrophage colony-stimulating factor (GM-CSF) in inducing cell proliferation compared with either YB5.B8 MoAb or GM-CSF alone. Immunoblotting with antiphosphotyrosine MoAb showed that interaction of M07e cells with immobilized YB5.B8 induced tyrosine phosphorylation of a series of intracellular proteins including KIT (145 kD). In addition, cross-linking studies using a water-soluble cross linking reagent bis-sulfosuccinimidyl-suberate showed that immobilized YB5.B8 MoAb induced dimerization and activation of KIT. However, as with stimulation by the membrane-bound form of SLF, the kinetics of KIT activation with YB5.B8 MoAb was more prolonged compared with the cells treated with recombinant soluble SLF. Flow cytometry showed that, unlike the cells treated with soluble SLF, no downmodulation of cell-surface KIT expression was observed in M07e cells cultured with immobilzed YB5.B8 MoAb. These data suggest that immobilized antibodies against hematopoietic receptors may replace their ligand-stimulators; however, their activities may resemble the membrane-bound form rather than the soluble form of natural ligands. PMID- 8630384 TI - The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques. AB - The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states. PMID- 8630385 TI - Antibody ligation of CD9 modifies production of myeloid cells in long-term cultures. AB - The KMC8.8 monoclonal antibody was made by immunizing rats with the BMS2 stromal cell clone, and was selected for further study because its ability to inhibit production of myeloid cells in Dexter cultures but not that of lymphoid cells in Whitlock-Witte cultures. The influence on myeloid progenitors might have been indirect, since the antibody did not prevent responsiveness to colony-stimulating factors in semisolid agar cultures. Furthermore, there was no inhibition, and some augmentation, of cell production when the antibody was added to established Dexter cultures. A cDNA clone that encoded the KMC8.8-recognized molecule was isolated by expression cloning and found to be identical in sequence to a previously published murine CD9 homologue. The antibody and cDNA clone were used to establish that CD9 is expressed by stromal cells, megakaryocytes, platelets, myeloid cells, and subpopulations of mature lymphocytes in mice. Treatment with the KMC8.8/CD9 antibody slightly augmented adhesion between myeloid cells and stromal cells, consistent with previous reports that this member of the tetraspan family of proteins can transmit proadhesive signals to human platelets and lymphoid cells. CD9 might participate in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow. PMID- 8630386 TI - The role of stem cell factor (c-kit ligand) and inflammatory cytokines in pulmonary mast cell activation. AB - Mast cells play a critical role in allergic airway responses via IgE-specific activation and release of potent inflammatory mediators. In the present study, we have isolated and characterized primary mast cell lines derived from the upper airways of normal mice. The primary mast cell lines were grown and maintained by incubation with interleukin-3 (IL-3) and stem cell factor (SCF) and shown to be c kit (SCF receptor) positive by flow cytometry. Subsequently, we examined the proliferation of both airway and bone marrow derived mast cell lines in response to inflammatory and hematopoietic cytokines, including SCF, IL-1, IL-3, interferon-gamma, IL-4, and IL-10. The results from the pulmonary mast cell lines were compared with those from bone marrow derived mast cells. Pulmonary mast cell lines were capable of proliferating in response to IL-3, IL-4, IL-10, and SCF, whereas the combination of SCF with the other cytokines did not increase the response over SCF alone. In contrast, the bone marrow-derived mast cells proliferated strongest to SCF or IL-3, but only modestly to IL-4 and IL-10. Furthermore, the combination of SCF with IL-3, but not the other cytokines, exhibited an increase in bone marrow-derived mast cell proliferation. Cytokine specific stimulation of histamine release in the airway-derived and bone marrow derived mast cells showed parallel results. SCF was the only cytokine shown to induce substantial histamine release. However, when certain nonhistamine releasing cytokines were combined with SCF, a synergistic increase in histamine release was induced in upper airway, but not bone marrow-derived mast cells. The results of these studies suggest that cytokines differentially modulate induction of proliferation and degranulation of bone marrow and upper airway-derived mast cells and may further indicate a cytokine activational cascade in tissue mast cells. PMID- 8630387 TI - Association of the expression of an SR-cyclophilin with myeloid cell differentiation. AB - The role of a 150-kD SR-cyclophilin (NK-TR1) in monocyte differentiation was investigated. Using an antipeptide monoclonal antibody, we have detected NK-TR1 in human peripheral blood monocytes and HL-60 cells. Unstimulated monocytes showed a low intracellular level of NK-TR1 protein that increased over 3 days of lipopolysaccharide + interferon-gamma treatment, consistent with the kinetics of monocyte differentiation. Normal HL-60 cells also had a low level of NK-TR1 protein, and exposure to 1.25% dimethyl sulfoxide (DMSO) resulted in a marked transient increase in expression that returned to basal levels before the development of granulocyte differentiation-associated biochemical changes. Phorbol myristate acetate, a promoter of monocytic differentiation in HL-60 cells, also caused a significant increase in NK-TR1 over basal levels. Transfection of a vector expressing NK-TR1 antisense RNA into HL-60 cells suppressed DMSO-mediated growth arrest. In addition, the development of a more mature phenotype, as measured by expression of CD16, and the ability to reduce nitroblue tetrazoleum dye was inhibited in transfectants when compared with controls. These results are consistent with the hypothesis that the NK-TR1 gene product is required for the progression towards a mature differentiated phenotype. PMID- 8630388 TI - CD4 Expression by erythroid precursor cells in human bone marrow. AB - Flow cytometry was used to assess CD4 expression in 62 consecutive bone marrow specimens from patients with a variety of clinical conditions. Using a lysed whole-blood technique for labeling with monoclonal antibodies, two populations of CD4+ cells were identified within the lymphocyte/blast-cell fraction in 58 (94%) of these specimens. These consisted of (1) a population of T helper cells with high density expression of CD4 and (2) a second population of cells with low density expression of CD4, which ranged from 1% to 36% of the gated cells. This latter population was present regardless of age, sex, or clinical condition including 21 of 21 specimens (100%) categorized as unremarkable bone marrows both morphologically and by flow cytometry and in four of four patients (100%) with human immunodeficiency virus-type 1 (HIV-1) infection. Coexpression of the erythroid lineage marker, glycophorin A, with the majority of cells in this second population was demonstrated in all 11 randomly selected samples using two color flow cytometric analysis. These cells also expressed low levels of the myeloid markers, CD13 and CD33, but CD34 expression could not be demonstrated. These results provide evidence for expression of CD4 on cells of erythroid lineage in human marrow, and offer a potential mechanism for direct infection of erythroid precursor cells and deranged erythropoiesis in patients with HIV-1 infection. PMID- 8630389 TI - A hematopoietic organ-specific 49-kD nuclear antigen: predominance in immature normal and tumor granulocytes and detection in hematopoietic precursor cells. AB - A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a novel mouse monoclonal antibody (B92) raised against stromal cells. The protein was found in the immunizing cells using a sensitive method. However, its detection in the bone marrow by the B92 antibody seemed to stem from the abundance of p49 in immature cells of the myeloid lineage. Study of the bone marrow following in vivo irradiation or 5-fluorouracil (5-FU) treatment, in vitro culture with differentiation-inducing factors and long-term culture, and cell sorting all pointed in the same direction: the protein was found in early myeloid cells and in hematopoietic precursor cells. These results were in accordance with the specific presence of p49 in primary radiation-induced myeloid leukemia and its absence in spontaneous B lymphoma. Immunofluorescent staining using B92 antibody detected a nuclear antigen forming a dotted pattern in early myeloid cells and day 12 colony-forming units-spleen (CFU-S). Nuclear localization of p49 was further demonstrated by subcellular fractionation followed by Western blotting. We thus identified a nuclear protein that within the hematopoietic population is detected in hematopoietic precursor cells, predominates in early myeloid cells, and is reduced following differentiation. These properties imply that p49 might be involved in the regulation of hematopoietic cell growth or differentiation. PMID- 8630390 TI - Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C. AB - The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N-terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF-dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D' domain were found to be involved in the protection of factor VIII from inactivation by aPC. PMID- 8630391 TI - Initiation of the tissue factor pathway of coagulation in the presence of heparin: control by antithrombin III and tissue factor pathway inhibitor. AB - Activation of factor X by both the unactivated tissue factor:factor VII complex (TF:VII) and the activated tissue factor:factor VIIa complex (TF:VIIa) has been studied in the presence of tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and heparin. At near-plasma concentrations of TFPI, ATIII, and factor X, factor X activation that occurs in response to TF:VII is essentially abolished in the presence of heparin (0.5 micromol/L). This effect requires both inhibitors, acting on different targets: (1) ATIII, which in the presence of heparin blocks the activation of TF:VII, and (2) TFPI, which inhibits the TF:VIIa that is generated. In the absence of ATIII, TFPI alone with heparin reduces but does not abolish factor X activation. Conversely, in the absence of TFPI, ATIII + heparin reduces but does not abolish TF:VIIa generation and allows continuing activation of factor X. These results indicated that when the unactivated TF:VII complex is the initiating stimulus, heparin-dependent reduction in the rate and extent of factor X activation requires both ATIII and TFPI. In contrast, if TF:VIIa is used to initiate activation, only TFPI is involved in its regulation. PMID- 8630392 TI - Expression of cyclic ADP-ribose-synthetizing CD38 molecule on human platelet membrane. AB - CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation. PMID- 8630393 TI - Hemodynamic forces modulate the effects of cytokines on fibrinolytic activity of endothelial cells. AB - We investigated the effects of hemodynamic force on fibrinolytic activity of cultured human umbilical vein endothelial cells stimulated by cytokines, using a modified cone-plate viscometer in which well-controlled and -defined shear forces were generated. Treatment of the cells with interleukin (IL)-beta or tumor necrosis factor alpha (TNFalpha) under static conditions had no effect on tissue plasminogen activator (t-PA) secretion, while release of plasminogen activator inhibitor 1 (PAI-1) increased. When cells were exposed to increasing shear stress up to 24 dynes/cm2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNFalpha, the increase in production of t-PA and the t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells in similar experimental conditions using t-PA and PAI 1 cDNA probes revealed no t-PA mRNA in 3 microg total RNA from static endothelial cells under resting or cytokine-stimulated conditions, but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm2, and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine-stimulated, nonsheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines. These results indicate a role for hemodynamic forces in regulating fibrinolytic activity with or without cytokine stimulation. PMID- 8630395 TI - Identification of complement activation sites in human immunodeficiency virus type-1 glycoprotein gp120. AB - Recombinant glycoprotein 120 (rgp120) of human immunodeficiency virus type-1 (HIV 1) activates the human complement system in the absence of anti-gp120 antibodies. HIV-1 glycoprotein gp120 can dissociate from the viral envelope either spontaneously or after binding of HIV-1 to the CD4 molecule. As a consequence, gp120 can circulate in the patient's serum and attach to the surface of uninfected CD4+ T cells. Complement activation by cell-bound HIV-1 glycoprotein gp120 with subsequent opsonization may represent a mechanism for the elimination of uninfected CD4+ cells by the reticuloendothelial system, thereby enhancing the progression of HIV disease. In the current study, the complement proteins C4,C3,C5,C9, and properdin were found to bind to a synthetic peptide covering positions 233-251 of the gp120BRU sequence on incubation with normal human serum. Complement activation by the peptide was comparable with that induced by aggregated IgG, complete rgp120, and the previously described complement activating gp41-peptide 609-623. Activation occurred via the classical pathway and was abrogated in the presence of EDTA, Mg2+/EGTA, or C4-deficient human serum. Peptides partly overlapping the sequence 233-251 activated complement to a lesser extent. The complement-activating capacity of the gp120 sequence 233-251 was not restricted to the HIV-1BRU isolate, because a peptide from the corresponding sequence of the HIV-1MN strain was also capable of activating complement. An additional strong complement-activating site was identified in the gp120 sequence 321-360 of the HIV-1MN strain. These data indicate that distinct sites in gp120 are able to activate human serum complement via the classical pathway in the absence of anti-gp120 and independent of glycosylation. PMID- 8630394 TI - Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets. AB - von Willebrand factor (vWF) is a multimeric glycoprotein that forms an adhesive link following vascular injury between the vessel wall and its primary ligand on the platelet surface, glycoprotein Ib (GpIb). Type 2b von Willebrand disease (vWD) is a qualitative form of vWD resulting from enhanced binding of vWF to platelets. Molecular characterization of the vWF gene in patients with type 2b vWD has resulted in identification of a panel of mutations associated with this disorder, all clustered within the GpIb binding domain in exon 28 of the vWF gene. We have expressed six of the most common type 2b vWD mutations in recombinant vWF and show that each mutation produces a similar increase in vWF binding to platelets in the absence or presence of ristocetin. Furthermore, expression of more than one type 2b vWD mutation in the same molecule (cis) or in different molecules within the same multimer (trans) failed to produce an increase in vWF platelet binding compared with any of the individually expressed mutations. Taken together, these data support the hypothesis that the vWF GpIb binding domain can adopt either a discrete "on" or "off" conformation, with most type 2b vWD mutations resulting in vWF locked in the on conformation. This model may have relevance to other adhesive proteins containing type A domains. PMID- 8630397 TI - HLA-target antigens and T-cell receptor diversity of activated T cells invading the skin during acute graft-versus-host disease. AB - To study the repertoire and specificity of T lymphocytes infiltrating skin lesions during graft-versus-host disease (GVHD), we performed an exhaustive molecular and functional analysis of 146 T-cell clones derived from the skin of three patients undergoing an acute GVHD after allogeneic bone marrow transplantation (BMT) from HLA-mismatched related donors. Analysis of T-cell receptor (TCR) rearrangement and TCR chain junctional sequences demonstrated the presence of 11 distinct clones among the 64 derived from patient UPN1, six among the 58 derived from patient UPN2, and seven among the 24 derived from patient UPN3. Three of the 11 T-cell clones from patient UPN1, and all clones from patients UPN2 and UPN3 reacted with mismatched HLA alleles between the bone marrow donor and recipient. Moreover, both HLA class I (HLA-A2 and -B27) and class II (HLA DP101, DP401, DP1301, DQ8, and DR402) molecules were recognized during this early antihost response. Finally, both TCR alpha and beta chains turned out to be extremely diverse, even within populations of clones derived from the same patient and directed against the same HLA allele. Taken together, these results indicate that any HLA mismatch is potentially targeted during early GVHD, and that the T-cell response at the onset of GVHD is both oligoclonal and highly diversified. PMID- 8630396 TI - Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase. AB - In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates. PMID- 8630398 TI - Eosinophils express a functional receptor for interferon alpha: inhibitory role of interferon alpha on the release of mediators. AB - Recent reports describe the beneficial use of alpha interferon (IFNalpha) for the treatment of idiopathic hypereosinophilic syndrome (HES) unresponsive to conventional therapy. A clinical improvement associated with a rapid decrease of peripheral blood eosinophilia suggested possible direct effects of IFNalpha on eosinophils through the presence of IFNalpha receptors (IFNalphaR). Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytochemistry were used respectively to detect the presence and define the distribution of IFNalphaR on enriched eosinophil preparations purified from blood cells. IFNalphaR was found on eosinophils collected from patients with various eosinophilic disorders. In addition, IFNalpha inhibited the release of eosinophil granule proteins such as eosinophil cationic protein (ECP), neurotoxin (EDN, or interleukin-5 (IL-5). Moreover, antiparasite cytotoxicity was also strongly reduced in a dose-dependent manner by IFNalpha. These results provide the first evidence that human eosinophils express a functional receptor for IFNalpha and represent a potential basis for the beneficial effects of IFNalpha in patients with hypereosinophilic syndromes. PMID- 8630399 TI - Inhibition of CD4 cross-linking-induced lymphocytes apoptosis by vesnarinone as a novel immunomodulating agent: vesnarinone inhibits Fas expression and apoptosis by blocking cytokine secretion. AB - Evidence is accumulating that T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals show accelerated cell death through apoptosis. We have recently demonstrated that the cross-linking of CD4 molecules (CD4XL) results in death of normal peripheral T cells through apoptosis and imbalanced cytokine secretion (ie, induction of tumor necrosis factor-alpha [TNF-alpha] and interferon-gamma [IFN-gamma] in the absence of interleukin-2 [IL-2] or IL-4 secretion). These upregulated cytokines (TNF-alpha/IFN-gamma) largely contributed to upregulation of the apoptosis-inducing cell surface molecule, Fas (APO-1/CD95) and apoptosis induction. The present study investigated the effect of vesnarinone as a novel immunomodulating agent on CD4XL-induced T-cell apoptosis. The addition of vesnarinone to peripheral blood mononuclear cells (PBMC) significantly inhibited CD4XL-induced lymphocyte apoptosis. This apoptosis-inhibitory effect of vesnarinone was associated with the blocking of CD4XL-induced TNF-alpha IFN-gamma secretion and of Fas antigen upregulation. However, vesnarinone did not block effects of exogenously supplemented TNF-alpha/IFN-gamma on Fas induction. These data suggest that vesnarinone inhibits CD4XL-induced TNF-alpha/IFN-gamma secretion, thereby blocking subsequent Fas upregulation and apoptosis induction. Given the potent pathogenic role of imbalanced cytokine secretion observed in HIV infection, an agent such as vesnarinone may be of therapeutic value in slowing disease progression. PMID- 8630400 TI - The natural killer-related receptor for HLA-C expressed on T cells from CD3+ lymphoproliferative disease of granular lymphocytes displays either inhibitory or stimulatory function. AB - Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related "p58" receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T-cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb-triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2 cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart. PMID- 8630401 TI - Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells. AB - We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells. PMID- 8630402 TI - Decreased inducible expression of CD80 and CD86 in human monocytes after ultraviolet-B irradiation: its involvement in inactivation of allogenecity. AB - Ultraviolet-B (UV-B) irradiation of antigen presenting cells (APCs) modifies their allogenecity, resulting in inhibition of the proliferative response of T cells in mixed lymphocyte reaction (MLR). Costimulation by the CD28 ligand CD80 (B7/B7-1) and CD86 (B70/B7-2) plays an important role during T-cell proliferation by augmenting synthesis of interleukin-2 (IL-2) and other cytokines. In this study, we demonstrated induced expression of both CD80 and CD86 during allogeneic MLR, though human freshly isolated monocytes express CD86 constitutively with a much lower level of CD80. A monoclonal antibody (MoAb) against CD86, but not CD80, efficiently inhibited allogeneic T-cell proliferative responses stimulated with highly purified monocytes. UV-B exposure (0 to 1,000 J/m2) of monocytes inhibited the proliferation of T lymphocytes in MLR in a dose-dependent manner. Flow cytometric analysis showed that UV-B exposure of monocytes impaired the constitutive expression of CD54 (intercellular adhesion molecule-1) by 24 hours after irradiation, but the effect on CD86 was relatively less. The surface expression of CD80, CD86, CD54, and HLA-DR on monocytes was further augmented by interferon (IFN)-gamma; this cytokine-induced expression was dose-dependently reduced by UV-B irradiation. Similarly, the upregulation of these molecules following allogeneic MLR was downregulated by UV-B irradiation. UV-B irradiation of monocytes inhibited the expression of IL-2 mRNA in monocyte-stimulated allogeneic MLR. In contrast, the addition of anti-CD28 MoAb at the onset of MLR prevented, at least partially, the reduction of IL-2 mRNA. These results strongly suggest that the impairment of inducible expression of CD86 and CD80 may contribute to the reduced MLR response following exposure of monocytes of UV-B. PMID- 8630403 TI - Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow. AB - Natural killer (NK) cells develop from the nonadherent cell component of NK long term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK-LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5-fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500. PMID- 8630404 TI - Increased activity and sensitivity of mitochondrial respiratory enzymes to tumor necrosis factor alpha-mediated inhibition is associated with increased cytotoxicity in drug-resistant leukemic cell lines. AB - The drug-resistant leukemic cell lines, CEM/VLB100 and K/DAU600, are more sensitive to tumor necrosis factor alpha (TNFalpha)-mediated cytotoxicity compared with their parental cell lines, CCRF-CEM and K562 cl.6. Drug-resistant leukemic cell lines have more active mitochondrial function, which is associated with a greater susceptibility to TNFalpha-induced respiratory inhibition. TNFalpha blocked electron transfer at three sites, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Respiratory rate and electron transport chain enzyme activities were significantly inhibited in the drug-resistant, TNF-sensitive cell lines. Respiratory inhibition preceded cell death by at least 5 to 8 hours. The respiratory failure was not compensated for by appropriate up-regulation of the glycolytic pathway. Increasing mitochondrial respiratory rate and enzyme activities by long-term culture with 2 mmol/L adenosine 5'-diphosphate (ADP) and Pi sensitized both drug-sensitive and drug-resistant cells to TNFalpha-induced cytolysis. Intramitochondrial free radicals generated by paraquat only had a limited and delayed effect on respiratory inhibition and cytolysis in comparison with the effect of TNFalpha. We conclude that TNFalpha-induced cytotoxicity in leukemic cells is, at least in part, mediated by inhibition of mitochondrial respiration. Free radical generation by TNFalpha may not directly lead to the observed inhibition of the mitochondrial electron transport and other mechanisms must be involved. PMID- 8630405 TI - Target-induced death by apoptosis in human lymphokine-activated natural killer cells. AB - Activated human natural killer (NK) cells undergo rapid apoptotic cell death after ligand binding to the Fc receptor (CD16). We examined whether human NK cells die after engagement in cytolytic functions. Peripheral blood NK cells, with and without prior activation in vitro with interleukin-2 (IL-2), were tested for the occurrence of cell death after incubation with K562, the prototype NK sensitive target cell. A proportion (15.2%) of NK cells that were stimulated for 3 days with IL-2 and then incubated for 4 hours with K562 cells showed rapid cell death, but NK cells not stimulated with IL-2 did not. This cell death was found to involve nuclear condensation and fragmentation and DNA cleavage, all of which are characteristic of apoptosis. These data indicate that a proportion of activated human NK cells undergo apoptosis as they engage in target cell lysis. Target-induced NK cell death may represent an important mechanism for regulation of inflammatory processes involving NK cells. PMID- 8630406 TI - Transcript synthesis and surface expression of the interleukin-2 receptor (alpha , beta-, and gamma-chain) by normal and malignant myeloid cells. AB - Expression of the interleukin-2 receptor alpha-(IL-2Ralpha-), IL-2Rbeta-, and the recently identified IL-2Rgamma-chain was examined on a wide range of cells of myeloid origin including neutrophils, monocytes, normal bone marrow-derived myeloid progenitors enriched for CD34+ cells, bone marrow blasts obtained from acute myelogenous leukemia (AML) patients, and permanent myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction and surface membrane analysis using receptor chain-specific monoclonal antibodies and flow cytometry. Expression of the p75 IL-2Rbeta- and the p64 IL-2Rgamma-chain was a common finding in most of the myeloid cell samples investigated, whereas IL-2Ralpha chain was less frequently expressed. Although the high-affinity IL-2R form (ie, the alpha+, beta+, gamma+ IL-2R form) was detectable in a small minority of primary AML samples as well as the KG-1 cell line and IL-2 binding to these cells was sufficient to initiate signal transduction as evidenced by an increase in overall protein tyrosine phosphorylation and more specifically in tyrosine phosphorylation of the Janus kinase (JAK) 3, in none of these cell types did exposure to IL-2 affect cell growth kinetics. These results suggest that, in myeloid cells, the IL-2R may not stimulate mitogenic responses or that its components may be expressed in a combinational association with receptors for other cytokines and that IL-2Rgamma may play a regulatory role in normal and malignant myelopoiesis possibly independent from IL-2. Because recent studies by others have indicated that the IL-2Rgamma- chain may be shared by the IL-4R, the IL-7R, and most likely the IL-9R, expression of mRNA of these receptor types was also investigated in these cell samples. Surprisingly, in a substantial part of the myeloid lineage cells examined, an IL-2Rgamma+, IL-4R-, IL7R- configuration was noted that was, however, frequently associated with expression of IL-9R. Sharing of IL-9R/IL-2R components was furthermore suggested by inhibition of 125I IL-2 binding to primary AML cells with excess of unlabeled IL-9. PMID- 8630407 TI - Nodular lymphocyte predominance Hodgkin's disease: a monoclonal or polyclonal B cell disorder? AB - Nodular lymphocyte predominance Hodgkin's disease (NLPHD) is characterized by the presence of atypical putatively neoplastic cells (L & H cells) with a B-cell phenotype. A proportion of patients with NLPHD develop a simultaneous or subsequent large cell B lymphoma (LCL) that is thought to evolve directly from the L & H cells of NLPHD. However, the clonal nature of L & H cells remains controversial, and the relationship between NLPHD and complicating LCL has not been fully established. In an attempt to determine the clonality of L & H cells and to clarify the link between NLPHD and complicating LCL, we used polymerase chain reaction (PCR) to analyze 33 cases of NLPHD, including 15 cases with simultaneous or subsequent LCL, for clonal immunoglobulin (lg) heavy chain variable region (VH) gene rearrangements. PCR amplifications with consensus primers covering framework 2 or framework 3 to joining region were performed on paraffin-embedded tissue sections and, in 12 cases, on microdissection-enriched L & H cells. No clonal Ig rearrangements were detected. In eight of the 15 LCL, monoclonal IgVH regions were amplified, four of which were cloned and sequenced. Clone specific primers were designed based on the unique N region sequences. These allowed detection of LCL clones at a sensitivity up to 1,000 times greater than the consensus primers, as determined by dilution assays. However, no LCL clones were detected in the preceding NLPHD, including microdissection-enriched L & H cells. Our results suggest that populations of L & H cells do not carry monoclonal Ig rearrangements and provide no evidence for a clonal link between NLPHD and complicating LCL. PMID- 8630408 TI - Epstein-Barr virus (EBV)-associated lymphoproliferations in severe combined immunodeficient mice transplanted with Hodgkin's disease lymph nodes: implications of EBV-positive bystander B lymphocytes rather than EBV-infected Reed-Sternberg cells. AB - To establish an in vivo model for the study of Hodgkin's disease and Reed Sternberg (RS) cells, 25 lymph node tissue samples involved by Hodgkin's disease were grafted into severe combined immunodeficiency (SCID) mice. Ten Epstein-Barr virus (EBV)-associated tumors were obtained in SCID mice. EBV-positive tumors growing in SCID mice were correlated with the presence of EBV-positive nonneoplastic B cells in patient tumors (90% v 26.6%; P<.01) and was independent of the EBV status of RS cells. Our results suggested that EBV-positive tumors growing in SCID mice originated from normal EBV-positive small lymphocytes (bystander B lymphocytes). We also compared the characteristics of these tumors with those obtained after transplantation of 15 non-Hodgkin's lymphoma and four reactive lymph nodes. The latent period to observe a growing tumor in SCID mice was similar between the two groups (12.86 +/- 5.59 weeks for Hodgkin's disease v 13.6 +/- 5.36 weeks for non-Hodgkin's lymphoma and reactive lymph nodes). The relatively high number of EBV-positive small lymphocytes detected in Hodgkin's disease and T-cell lymphoma compared with B-cell lymphoma may account for the greater percentage of EBV-positive tumors obtained in SCID mice. Our results show that SCID mice do not provide the growth conditions that are required for in vivo growth of RS cells. We noted in some SCID tumors, the presence of binucleated and/or multinucleated giant cells resembling RS cells. However, the presence of such cells was not restricted to mice grafted with lymph nodes involved by Hodgkin's disease. We postulate that in previous reports, cells resembling RS cells were just binucleated EBV-positive lymphoma blastoid cells rather than actual RS cells. PMID- 8630409 TI - Activation of Hodgkin cells via the CD30 receptor induces autocrine secretion of interleukin-6 engaging the NF-kappabeta transcription factor. AB - The CD30 surface molecule is a recently identified member of the tumor necrosis factor/nerve growth factor receptor superfamily. Within the cytoplasmic signal transducing domain, CD30 shares no significant homology to other members of this family. Signaling events engaged via CD30 are still unknown. We here identify the NF-kappabeta transcription factor as a target of the CD30-induced signal pathway in Hodgkin's disease (HD) cells. Exposure of HD cells to CD30 ligand induces release of interleukin-6 (IL-6) that can be duplicated by cross-linking HD-cells to an agonistic anti-CD30 specific monoclonal antibody (alphaCD30), but not by cross-linking to an isotype-identical irrelevant monoclonal antibody. Cross linking of HD cells to alphaCD30 leads to enhanced accumulation of IL-6 mRNA in a time-dependent fashion resulting from transcriptional activation of the IL-6 promoter. Transient transfection assays using a series of deleted IL-6 promoter constructs linked to the human growth hormone gene as a reporter gene furthermore indicate that transcriptional activation of the IL-6 promoter requires the presence of an intact NF-kappabeta binding site. In addition, introduction of an NF-kappabeta binding site appeared to be sufficient to confer inducibility of an heterologous promoter on activation of CD30 in HD cells. Cross-linking of CD30 promotes rapid and transient binding activity of nuclear proteins to the NF kappabeta recognition site of the IL-6 promoter. Supershift experiments using a series of monoclonal antibodies recognizing distinct members of the NF-kappaBeta transcription factor family furthermore indicate that in CD30 cross-linked HD cells p50, p65/Rel-A, and Rel-B are present, whereas the c-rel protein is not. PMID- 8630410 TI - Activation-dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells. AB - Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML. PMID- 8630411 TI - The abnormal eosinophils are part of the leukemic cell population in acute myelomonocytic leukemia with abnormal eosinophils (AML M4Eo) and carry the pericentric inversion 16: a combination of May-Grunwald-Giemsa staining and fluorescence in situ hybridization. AB - The French-American-British subtype acute myelomonocytic leukemia with abnormal eosinophils (FAB AML M4Eo) with pericentric inversion of chromosome 16 is cytomorphologically defined by a myelomonoblastic blast population and abnormal eosinophils. Until now, it remained an open question whether these abnormal eosinophils are part of the malignant clone or an epiphenomenon. We analyzed five cases of AML M4Eo with inv(16) and combined May-Grunwald-Giemsa staining with fluorescence in situ hybridization using yeast artificial chromosome clone 854E2, which spans the inv(16) breakpoint on 16p. In the case of inv(16), three instead of the normal two hybridization signals can be observed both on metaphase spreads and in interphase cells. With this approach, we were able to show inversion 16 in abnormal eosinophils and, therefore, identified them as a part of the leukemic cell population. PMID- 8630412 TI - Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia. AB - The monoclonal antibody LRP56 recognizes a 110-kD major vault protein (lung resistance protein [LRP]) overexpressed in several P-glycoprotein-negative (Pgp ), multidrug resistant tumor cell lines. To determine the frequency of LRP overexpression, its prognostic significance, and its relation to Pgp, we analyzed bone marrow specimens from 87 consecutive patients with acute leukemia. Diagnoses included de novo acute myeloid leukemia (AML; 21 patients), leukemia arising from an antecedent hematologic disorder or prior cytotoxic therapy (secondary AML; 27 patients), AML in relapse (29 patients), and blast phase of chronic myeloid leukemia (CML-BP; 10 patients). A granular cytoplasmic staining pattern was detected by immunocytochemistry in 32 (37%) cases, including 7 (33%) de novo AML, 13 (48%) secondary AML, 11 (38%) relapsed AML, and 1 of 10 CML-BP. Among 66 evaluable patients with AML, LRP overexpression was associated with an inferior response to induction chemotherapy (P = .0017). Remissions were achieved in 35% of LRP+ patients as compared with 68% of LRP- patients. Although Pgp adversely affected response in univariate analysis (P = .0414), only LRP had independent prognostic significance when compared in a logistic regression model (P = .0046). Differences in remission duration (P = .075) and overall survival (P = .058) approached significance only for LRP. Sequential specimens from remitting patients receiving treatment with the Pgp modulator cyclosporin-A showed emergence of the LRP phenotype despite a decrease or loss of Pgp at the time of treatment failure (P =.0304). Significant associations were observed between LRP and age greater than 55 years (P = .017), Pgp (P = .040), and prior treatment with mitoxantrone (P = .020) but not with CD34. These findings indicate that overexpression of the novel transporter protein LRP is an important predictor of treatment outcome in AML. PMID- 8630413 TI - Frequent expression of the cell death-inducing gene Bax in Reed-Sternberg cells of Hodgkin's disease. AB - The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax positive tumors also expressed either Bcl-2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3' OH-DNA end-labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequently expressed in Hodgkin's disease, providing a potential explanation for the good chemoresponses generally obtained for patients with this neoplastic disorder. PMID- 8630414 TI - Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture. AB - A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3-) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2 supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation. PMID- 8630415 TI - Retroviral vector-mediated transfer of the tumor necrosis factor alpha gene into human cancer cells restores an apoptotic cell death program and induces a bystander-killing effect. AB - Tumor necrosis factor alpha (TNFalpha) may induce tumor cell death by apoptosis, the physiologic program of cell death usually lost during neoplastic progression. However, many tumor cells are resistant to its effect unless high doses are administered. By retroviral vector-mediated gene transfer, we have transduced the TNFalpha gene into the DNA of human tumor cells to investigate whether the indefinite neoplastic cell proliferation could be blocked and the lost physiologic program of cell death restored. Evidence is provided that high TNFalpha-producing clones generated from a human lymphoma T-cell line (ST4) can undergo apoptosis following transduction of the TNFalpha gene. Internucleosomal DNA cleavage was documented by May-Grunwald-Giemsa and by propidium iodide staining, as well as by gel electrophoresis. The induced apoptotic phenomenon is TNFalpha-mediated, since it can be reverted following incubation with anti TNFalpha monoclonal antibodies (MoAbs), and it occurs with cytokine levels released in the supernatant by the engineered cells much lower(>100 times) than those required to promote the same effect on parental ST4 cells following administration of exogenous recombinant TNFalpha. The process is associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53, genes usually involved in promoting apoptosis, persists. Mixed culture experiments performed coincubating TNFalpha-transduced and untransduced ST4 cells allowed documentation of a bystander-killing effect on the parental cells. This phenomenon still occurred at transduced to parental cell ratios as low as 1:20 and was blocked in the presence of an anti-TNFalpha MoAb. These findings indicated that TNFalpha may play a regulatory role in the proliferation of human tumor cells, and suggest potential new antitumor therapeutic strategies based on the direct delivery of the TNFalpha gene into cancer cells. PMID- 8630416 TI - Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5. AB - To determine the incidence of MLL rearrangement in acute myeloid leukemia (AML) French-American-British (FAB) type M1 and to evaluate optimal screening strategies for the characterization of such abnormalities, we analyzed specimens from 41 patients with AML by Southern blotting with two MLL genomic probes and compared the capacities of reverse transcription-polymerase chain reaction (RT PCR) and fluorescent in situ hybridization (FISH) to identify the types of rearrangement found in AML M1 with those observed in AML M5. MLL rearrangement was found in 6 of 29 (20%) AML M1 and 6 of 10 AML M5 cases. RT-PCR characterization of 11 cases showed four MLL self-fusions, four MLL-AF6, two MLL AF9, including a novel AF9 breakpoint, and one uncharacterized t(11:19). Only 5 of 10 MLL-rearranged cases tested demonstrated karyotypic 11q23 abnormalities. FISH analysis of nine cases with an MLL-specific yeast artificial chromosome (YAC) confirmed the cytogenetic abnormalities in two cases, clarified them in one, and did not detect six cases, including three MLL self-fusions, one case with a probable MLL-rearranged subclone not represented karyotypically, and twoMLL-AF6. A whole chromosome 11 paint detected one of these MLL-AF6, and an AF6 cosmid demonstrated that the other was probably due to insertion of a submicroscopic portion of chromosome 6, including part of AF6, into an apparently normal chromosome 11. We conclude that MLL rearrangements are common in adult AML M1, that MLL self-fusion and MLL-AF6 are the most frequent types of abnormalities, and that RT-PCR is preferable to 11q23 FISH analysis for their characterization. PMID- 8630417 TI - Clonal evolution in B-lineage acute lymphoblastic leukemia by contemporaneous VH VH gene replacements and VH-DJH gene rearrangements. AB - B-cell acute lymphoblastic leukemia (B-ALL), more frequently than any other B lineage neoplasm, exhibits oligoclonal Ig heavy chain (IgH) gene rearrangement in 15% to 43% of all cases studied. To study the molecular processes that promote multiple IgH rearrangements, a comprehensive sequence analysis of a B-ALL case was performed in which seven clonal IgH gene rearrangements were identified. The genetic profiles suggested that a single leukemic progenitor clone evolved into several subclones through dual processes of variable (VH) to preexisting diversity-joining (DJH) gene segment rearrangement and VH to VH gene replacement. Predominant IgH-V usage and the uniquely rearranged clonotype-specific VHDJH region gene sequences were identified using a novel DNA-based gene amplification strategy. Polymerase chain reaction (PCR) was directed by an IgH-J generic primer and a complement of family-specific IgH-V primers that defined the major B-cell IgH-V gene usage. Clonality of rearranged VHDJH bands was substantiated by high resolution denaturant gel electrophoretic analysis. Sequence patterns of the amplified VHDJH fragments segregated into two groups defined by common DJH sequences. Partial N region homology at the VHD junction as well as shared DJH sequences firmly established VH to VHDJH gene replacement as a mechanism generating clonal evolution in one group. In the second subset, oligoclonality was propagated by independent VH gene rearrangements to a common DJH precursor. The contributions of all clonal Ig-VHDJH repertoires for each group was approximately 50% and reflected a symmetric distribution of leukemic subclones generated by either process. Thus, oligoclonal rearrangements evolved by two independent, yet seemingly contemporaneous molecular genetic mechanisms. All seven clones displayed nonfunctional Ig-VHDJH recombinations. These observations may have relevance to the recombinatorial opportunities available during normal B cell maturation. PMID- 8630419 TI - Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control of the alpha-globin locus control region. AB - The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human beta-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position-independent expression in erythroid cells. The regulatory element known as the beta-globin locus control region (BetaLCR) can provide a linked Beta-globin gene with these properties. However, addition of BetaLCR sequences to a retrovirus carrying a beta-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human gamma-globin gene is placed under the control of the alphaLCR, the major regulatory element of the alpha-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10(6) colony-forming units (CFU)/mL. In addition, we show that the transduced gamma-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous Betamaj-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neor) gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human beta-globin gene disorders such as sickle cell disease and beta thalassemia. PMID- 8630418 TI - A critical role of VLA-4 in erythropoiesis in vivo. AB - Hematopoiesis requires specific interactions with the microenvironments, and VLA 4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo. PMID- 8630420 TI - Ferritin translation by interleukin-1and interleukin-6: the role of sequences upstream of the start codons of the heavy and light subunit genes. AB - Interleukin-1beta (IL-1beta) elevates H- and L-ferritin subunit synthesis in both human hepatoma cells (HepG2) and primary human umbilical vein endothelial cells. Ferritin induction is greater than the increase in total HepG2 protein synthesis in response to IL-1. IL-6 causes a moderate increase in L-subunit synthesis. The levels of the mRNAs for the ferritin H-subunits (H-mRNA) and light subunits (L mRNA) remain unchanged, indicating that expression of the iron storage protein, ferritin, is regulated by translational mechanisms during inflammation. We have found a translational enhancer region in the L-ferritin mRNA 5'UTR that confers two-fold baseline and twofold IL-1-dependent translational regulation to a CAT reporter message. The L-mRNA motif is related to a 61 nucleotide (nt) G+C-rich translational enhancer within 70 nt of the H-ferritin start codon. Sequences upstream of the start codons (SUS elements) in both H-mRNA and L-mRNAs confer IL 1beta but not IL-6-dependent translation to hybrid ferritin/CAT reporter mRNAs. The H- and L-ferritin mRNA SUS elements contain a motif similar to a consensus reported for the 5' leaders of other acute phase response mRNAs. Transfected hybrid H-mRNA SUS/CAT mRNAs with a three nucleotide deleted version of the H-mRNA SUS displays an eightfold reduced level of translation and no longer confer IL 1beta-dependent translation. PMID- 8630421 TI - Hereditary spherocytosis with spectrin deficiency due to an unstable truncated beta spectrin. AB - Red cell membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and direct quantitation by radioimmunoassay or cytofluorometry defines four distinct subsets of patients with hereditary spherocytosis: Patients with isolated spectrin deficiency, combined spectrin and ankyrin deficiency, band 3 deficiency, and protein 4.2 deficiency. In regard to the first group, only one mutation of beta spectrin has been reported in the literature. We describe a spectrin variant characterized by a truncated beta chain, and associated with hereditary spherocytosis and isolated spectrin deficiency. The clinical phenotype consists of a moderate hemolytic anemia with spherocytosis and frequent spiculation of the red cells. We present the biochemical characteristics of this mutant protein and show that it constitutes only 12% of the total spectrin on the membrane. We show that the truncation of the protein is the result of a single point mutation at position +1 (G-->A) of the donor consensus splice site of intron 17 leading to an aberrant beta spectrin transcriptional message lacking exons 16 and 17. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we provide evidence that the mutated gene is transcribed but its mRNA is less abundant than its normal counterpart in reticulocytes; we also show that the mutant protein is synthesized in decreased amounts in the cytoplasm of erythroid progenitor cells, and appears to be susceptible to proteolytic degradation. This mutant spectrin underscores the importance of the regulatory role played by the beta spectrin molecule in the assembly of alphabeta spectrin heterodimers on the membrane. PMID- 8630422 TI - Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria. AB - The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable. PMID- 8630423 TI - Dynamic changes in the locus control region of erythroid progenitor cells demonstrated by polymerase chain reaction. AB - The locus control region (LCR) far upstream of the human beta-like globin genes is defined by the preferential chromatin accessibility/DNase I hypersensitivity of four constituent DNA sites HS4, 3, 2, and 1. In an attempt to understand the mechanism of LCR function during early stages of erythropoiesis, a new polymerase chain reaction (PCR) method has been developed to examine the chromatin structure/DNase I hypersensitivity of the LCR in progenitor cells logistically available in limited cell numbers. In erythroid progenitors as well as in multipotent cells with erythroid potential, hypersensitive sites HS4, 3, 2, and 1 were present and the chromatin structure of the LCR was accessible. Moreover, the chromatin structure of the LCR underwent dynamic changes during erythropoiesis. In early erythroid progenitors, the HS2 site was more accessible than the HS3 site. In more mature erythroid progenitors, HS2 became less accessible than HS3 and the other sites. The results indicate that the transcriptional program of the globin genes is encoded, at least in part, in the chromatin accessibility of the LCR. This globin program was apparently initiated in multipotent cells and maintained in erythroid progenitors. Furthermore, the program could be modulated in response to cellular changes accompanying differentiation of the progenitor cells. PMID- 8630424 TI - Mutations in the erythropoietin receptor gene are not a common cause of Diamond Blackfan anemia. AB - Diamond-Blackfan anemia (DBA) is an inherited pure red blood cell aplasia that often requires lifelong transfusional support. The origin of the imperfect erythrogenesis is not known. The existence of more than one molecular basis for DBA is indicated by its different modes of inheritance and widely variable clinical phenotypes. Several erythroid growth factors have been thought to have a role in the pathogenesis of DBA. However, there is neither molecular nor clinical evidence for the involvement of stem cell factor or interleukin-3. The observation of elevated erythropoietin (EPO) concentrations and an impaired in vivo and in vitro response to pharmacologic doses of recombinant human EPO has suggested a defective EPO function in the pathogenesis of DBA. We have investigated the possible involvement of the EPO receptor (EPO-R) gene in 23 patients by screening its coding sequence for mutations using single-strand conformation polymorphism (SSCP). A Southern blot and hybridization with an EPO-R probe was also performed on DNA from seven patients. No causal mutations were identified. The absence of concordant segregation of the disease with the EPO-R gene in two informative families ruled out its role in their DBA children. These findings demonstrate that DBA is not commonly associated with EPO-R gene mutations. PMID- 8630425 TI - Phospholipase A2 levels in acute chest syndrome of sickle cell disease. AB - Acute chest syndrome (ACS) is associated with significant morbidity and is the leading cause of death in patients with sickle cell disease (SCD). Recent reports suggest that bone marrow fat embolism can be detected in many cases of severe ACS. Secretory phospholipase A2 (sPLA2) is an important inflammatory mediator and liberates free fatty acids, which are felt to be responsible for the acute lung injury of the fat embolism syndrome. We measured SPLA2 levels in 35 SCD patients during 20 admissions for ACS, 10 admissions for vaso-occlusive crisis, and during 12 clinic visits when patients were at the steady state. Eleven non-SCD patients with pneumonia were also evaluated. To determine if there was a relationship between sPLA2 and the severity of ACS we correlated SPLA2 levels with the clinical course of the patient. In comparison with normal controls (mean = 3.1 +/ 1.1 ng/mL), the non-SCD patients with pneumonia (mean = 68.6 +/- 82.9 ng/mL) and all three SCD patient groups had an elevation of SPLA2 (steady state mean = 10.0 +/- 8.4 ng/mL; vaso-occlusive crisis mean = 23.7 +/- 40.5 ng/mL; ACS mean = 336 +/- 209 ng/mL). In patients with ACS sPLA2 levels were 100-fold greater than normal control values, 35 times greater than values in SCD patients at baseline, and five times greater than non-SCD patients with pneumonia. The degree of SPLA2 elevation in ACS correlated with three different measures of clinical severity and, in patients followed sequentially, the rise in SPLA2 coincided with the onset of ACS. The dramatic elevation of SPLA2 in patients with ACS but not in patients with vaso-occlusive crisis or non-SCD patients with pneumonia and the correlation between levels of SPLA2 and clinical severity suggest a role for SPLA2 in the diagnosis and, perhaps, in the pathophysiology of patients with ACS. PMID- 8630426 TI - A gene transfer strategy for making bone marrow cells resistant to trimetrexate. AB - Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers. PMID- 8630427 TI - Comparison of genomic DNA and cDNA for detection of residual disease after treatment of chronic myeloid leukemia with allogeneic bone marrow transplantation. AB - To test whether patients in remission after allogeneic bone marrow transplantation (BMT) possess a pool of chronic myeloid leukemia (CML) cells that do not express BCR-ABL mRNA, we have compared the results and sensitivity of amplification of BCR-ABL from genomic DNA with conventional reverse transcription polymerase chain reaction (RT-PCR). Bubble PCR was used to amplify the genomic BCR-ABL translocation breakpoints from chronic-phase DNA of 10 patients with CML who subsequently underwent BMT. After cloning and sequencing of the amplification products, patient-specific ABL primers were synthesized and tested for both specificity and sensitivity in nested or heminested combinations with a variety of primers derived from the major breakpoint cluster region of the BCR gene. In all cases, combinations of primers were selected that enabled the detection of chronic-phase DNA from a specific patient at up to a 10(5)x dilution into DNA from a normal individual. Patterns of residual disease obtained by serial RT-PCR and DNA-PCR analyses of blood and bone marrow samples obtained after BMT were similar for most patients, including one treated for relapse by infusion of donor leukocytes. Of the 24 samples for direct comparison of RT-PCR and DNA-PCR, results were concordant in 19 (79%) cases. Five results were discordant. In two instances, RT-PCR was positive, while PCR from genomic DNA was negative; this discrepancy might have arisen due to the slightly greater sensitivity of RT-PCR compared with DNA-PCR. In three samples from three patients, two of whom had been transplanted in the accelerated phase, PCR from genomic DNA was positive while RT PCR was negative; this could mean that some CML cells in these samples had a reduced or absent capacity to express BCR-ABL mRNA post-transplant. Of these three patients, one subsequently relapsed; and two are in remission at 21 and 24 months after the discordant result. Thus, the finding of a single DNA-PCR- positive, RT-PCR-negative results does not necessarily predict relapse. Because the great majority of samples (79%) gave concordant results with the two assays, we believe that patients in remission do not generally harbor a substantial pool of CML cells that do not express BCR-ABL mRNA. PMID- 8630428 TI - The development of cellular immunity to Epstein-Barr virus after allogeneic bone marrow transplantation. AB - Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a potentially lethal complication during the first 6 months after allogeneic bone marrow transplantation (BMT). To determine whether deficiencies of EBV-specific cellular immunity contribute to EBV-LPD susceptibility and distinguish patients at risk, we performed limiting dilution analysis to quantify anti-EBV cytotoxic T lymphocyte precursor (CTLp) frequencies in 26 recipients of unmodified or T-cell depleted (TCD) grafts from EBV-seropositive donors. At 3 months post-BMT (n = 26), only five patients had EBV CTLp frequencies in the range of seropositive normal controls, irrespective of the type of transplant administered. By 6 months post-BMT, 9 of 13 patients tested had EBV CTLp frequencies within the normal range. The time period in which these patients had deficient cellular immunity to EBV corresponds to the period in which we have observed EBV-LPD in most prior patients. One patient with a low EBV CTLp frequency at 4 months post-BMT developed an EBV-LPD. Within 2 weeks of receiving an infusion of donor peripheral blood mononuclear cells (PBMC) providing less than 1,200 EBV-specific cytotoxic T cell precursors, populations of EBV-specific CTL in the circulation were restored to levels detected in normal seropositive adults. Concurrently, the patient achieved a regression of the EBV-LPD, which has been sustained without further therapy. These studies indicate that recipients of both unmodified and TCD marrow grafts have profound deficiencies of EBV-specific T cell-mediated immunity early posttransplant, and that the period of risk for EBV-LPD closely corresponds to this interval of severe deficiency. Treatment of one patient with EBV-LPD with marrow donor-derived PBMC induced a rapid expansion of EBV-specific cytotoxic T cell populations that occurred contemporaneously with the clinical regression of disease. PMID- 8630429 TI - The accurate definition of protein S deficiency may avoid the misestimation of the frequency of this defect. PMID- 8630430 TI - Novel CBFB-MYH11 fusion transcripts or reverse transcription-polymerase chain reaction artifacts? PMID- 8630431 TI - Reticuloendothelial origin and metastasis of Kaposi's sarcoma: historical update on the literature. PMID- 8630432 TI - Variation of fetal hemoglobin and F-cell number with the LCR-HS2 polymorphism in nonanemic individuals. PMID- 8630433 TI - Visualization of IgG-binding loci on the surface of sickle erythrocytes. PMID- 8630434 TI - A genetic view on the etiology of the inhibitor complication. PMID- 8630435 TI - Flow cytometry and monoclonal antibodies identify normal liver cell populations antigenically related to oval cells. AB - Oval cells, a non-parenchymal cell population induced to rapidly proliferate in animals treated with carcinogens, are thought to be related to the hypothesized liver stem cells. In normal liver there are poorly defined cells antigenically related to oval cells. These oval cell antigen positive (OCAP) cells present in normal animals are thought to include hepatocyte and bile duct cell precursors. To isolate them, we modified the existing protocols designed for oval cells and used it on normal neonatal rat livers. Using flow cytometry, the percentage of normal liver OCAP-cells varied with the monoclonal antibody (MoAb) to the different oval cell membrane markers used: 12% (MoAb 18.2), 23% (MoAb 270.38), 27% (MoAb 18.11), 31% (MoAb 18.13), and 37% (MoAb 374.3). Macrophages consisted 10% of the cells (MoAb MCA 275); hepatocytes were essentially absent ( < 1%, MoAb 236.4). Our results demonstrate that is possible to obtain significant numbers of normal cells antigenically related to oval cells and that using different MoAbs, different cell populations can be sorted for use in experimental studies testing liver progenitor cell hypothesis. PMID- 8630436 TI - Dihydronicotinamide adenine dinucleotide diaphorase in the central nervous system of the crested newt. AB - By using a histochemical procedure, the distribution of neurons containing dihydronicotinamide adenine dinucleotide diaphorase (NADPHd) was examined in the brain, retina and olfactory epithelium of the urodele amphibian Triturus carnifex. Positive nerve fibers and terminals were observed throughout the brain and cell bodies were seen within the telencephalon, optic tectum, brain stem, cerebellum and spinal cord. In the retina, NADPHd labeling was localized in the outer segment of photoreceptors and in some amacrine cells as well as in the outer and inner plexiform layers. In the olfactory epithelium, NADPHd labeling was found in the olfactory neurons. By comparing NADPHd distribution with nitric oxide synthase (NOS) immunoreactivity (using a polyclonal antiserum raised against mouse cerebellar NOS) it was found that NADPHd labeling and NOS immunoreaction patterns generally matched. The organization of NADPHd and NOS containing neurons in the central nervous system of the crested newt, which is simpler than in other vertebrates investigated, shows some peculiarities, such as the occurrence of NADPHD in the pinealocytes of the epiphysis, nucleus rubber of the brain stem and eminentia cerebellaris ventralis of the cerebellum. PMID- 8630437 TI - L-aspartate aminotransferase and L-asparaginase in rat lymph node: a histoenzymological and immunohistochemical study. AB - L-asparaginase and L-aspartate aminotransferase are both involved in the synthesis of L-aspartic acid. It has been observed that L-asparaginase is involved in the immunosuppressor morphine-dependent syndrome in lymphoid cells whereas L-aspartic acid blocks the development of this syndrome. The aim of the present study was to clarify the localization of L-AATase activity and L asparaginase in rat lymph nodes using histoenzymological and immunohistochemical methods, respectively. No positive reaction was demonstrated for L-AATase while L asparaginase shown to be present in lymphocytes and lymphoblastic cells. These observations lead us to suggest that L-asparaginase is the enzyme mainly responsible for the synthesis of the L-aspartic acid necessary for satisfying the living requirements of lymphoid cells. Therapeutically administered L asparaginase could exert its action intracellularly after crossing the cell membrane. PMID- 8630438 TI - Histochemical analysis of lizard testicular glycoconjugates during the annual spermatogenetic cycle. AB - The distribution of glycoconjugates in the testis of lizard Podarcis s. campestris De Betta was investigated by HRP-conjugated lectins during the annual spermatogenetic cycle. In addition, pretreatments of sections with neuraminidase and removal of alkali-labile O-linked sequences by beta-elimination allowed the structure of glycoconjugates to be further explored. Leydig cells displayed changes of lectin-binding sites during annual cycle, and during the abortive spermatogenesis period lacked N-linked sialylgalactosyl glycans. Sertoli cells stained by Con A, WGA, RCA120, BS I-B4, showed, except in July, O-linked sialylgalactosyl glycans. Spermatogonia bound Con A and WGA. Spermatocytes bound also BS I-B4, SBA, UEA I, and during spring spermatogenesis, revealed O-linked sialylgalactosyl glycans. The acrosomes of spermatids were also stained by RCA120 and PNA, whereas the heads of spermatozoa did not bind SBA and PNA. During the abortive spermatogenic period, the acrosomes showed O-linked sialylgalactosyl glycans and N-linked glycans terminating in beta-galactosyl residues. During the reproductive period, the acrosomes of spermatozoa expressed O- and N-linked sialylgalactosyl glycans and beta-galactosyl terminal residues on O- and N-linked glycans. This, in the testis of lizard, the two spermatogenesis periods show the emergence of different types of glycosylation. PMID- 8630439 TI - Immuno- and enzyme-cytochemical studies on the pancreatic D-cells in the chicken following vagotomy. AB - To study the appearance of polymorphic, dense multivesicular body-like structures (MBLS) occurring in the D-cells of pancreatic islets following abdominal vagotomy in the chicken, the D-cells were evaluated by means of quantitative electron microscopy, identified by immunogold labeling and characterized by a method of enzyme cytochemistry. Following vagotomy, secretory granules of the pancreatic D cells decreased significantly in number, whereas MBLS increased significantly in number. the mean area of the cell, nucleus and mitochondria displayed no significant changes. MBLS were rarely labeled for somatostatin when subjected to immunogold staining. MBLS arranging along the Golgi stacks may possibly be formed in the Golgi apparatus. consequently, it seems unlikely that MBLS are secondary lysosomes revealing crinophagy. Some MBLS showed acid phosphatase (AcPase) activity, although the direct fusion of primary lysosomes exhibiting AcPase activity with MBLS was seldom observed. As a result, it is possible that MBLS may be a kind of modified form of secretory granules and that they may be destroyed by lysosomes under the storing condition, judging from the AcPase reaction. PMID- 8630440 TI - Iron and copper deposition in chronic active hepatitis and liver cirrhosis; pathogenetic role in progressive liver cell damage. AB - Iron and copper deposition were examined in patients with chronic active viral hepatitis (CAH) and posthepatitic liver cirrhosis (LC) by Berlin blue, rhodanine, or Victoria blue staining and X-ray microanalysis. Considerable iron or copper deposition was demonstrated in the peripheral zones of hepatic lobules in both CAH (53% of specimens) and LC (63% of specimens). Frozen sections taken from the 2 CAH surgical sections with iron depositions were examined by photoncounting image analysis, and superoxide liberation from the metal granules were demonstrated. In areas of metal deposition, vacuolation of liver cell nuclei, accumulation of lipofuscin, and induction of metallothionein (69% of rhodanine- or Victoria blue-positive specimens) were often demonstrated, whereas induction of ferritin was found only in 14% of Berlin blue-positive specimens. The PCNA index was significantly lower in areas of metal deposition than in the adjacent areas without metal deposition, indicating lowered proliferative capability in the former. These results indicate that cell-mediated immune mechanisms causing the disturbance of bile secretion and heavy metal deposition in the peripheral zones of hepatic lobules may be involved in the progression of viral hepatitis from its acute phase to CAH and finally to LC phase, resulting in piecemeal necrosis. However, cholangitis could not be demonstrated in the present study. PMID- 8630441 TI - Morphometric and densitometric approach in hypertrophic cardiomyopathy (HCM). AB - The aim of this study was to apply an easy method for the quantitative evaluation of changes in myocardial fibrocell size (area, diameter, circular shape factor, nuclear area, DNA content), and in the fibrous-interstitial area in hypertrophic cardiomyopathy (HCM) by mean of a computerized image analysis. A Vidas image analyser was employed for the morphometric study. The following parameters were assessed: area, maximum and minimum diameters, circular shape factor of myocytes; percentage of fibrosis; DNA content (integrated optical density) nuclear area of myocytes. The morphometric results in HCM compared to the ones in normal hearts, indicate an increase in the myocyte area and the transverse diameters, especially in the septum and left ventricle, altogether illustrating the hypertrophic condition of the myocytes, and increased fibrotic area was found in the left ventricular wall and septum (22.6 +/- 2.1% and 15.9 +/- 2.9% respectively). Densitometric analysis showed a significant increase in all test samples then compared with controls. The increase in the two nuclear parameters (area and DNA) also suggests hyperplasia. It is concluded that the morphometric determination of the morphologic abnormalities occurring in HCM by a rapid and less laborious approach, is an extremely useful method to describe the characteristics of this pathologic condition. PMID- 8630442 TI - [Development of a new telemetry recording system for measuring of gastrointestinal contractile activity in unrestrained and conscious small animals]. AB - Myoelectrogram, strain gauge force transducer or manometry has been commonly used to record contractile activity of the gastrointestinal (GI) tract in small animals, but protecting the lead wires and tubes is troublesome when conducting experiments. To solve this problem, we have developed a new telementric recorder which can be implanted in the abdominal cavity of a small animal. The elemeter is a cylinder (phi 10 x 35 mm) with a strain gauge force transducer (4 x 3 mm) connected by fine lead wires. The telemeter includes a battery and amplification, transmission and power supply circuit to the transducer. The battery has a 1,500 hr life and is designed to be turned on and off from outside the body by means of a magnetic switch. The device weights 4 g and is waterproofed with silicon. Five male Wister rats weighing 300-400 g were used. Under general anesthesic, the force transducer was sutured onto the serosa in the gastric antrum, and the telemeter was fixed in the corner of the peritoneal cavity. During measurement, the rats were housed in individual cages under unrestrained conditions and the cage was placed on the receiver. Gastric motility could be continuously recorded for up to 60 days, although body movements sometimes affected the recordings slightly due to adhesion. There was no noticeable trouble related to the device implanted in the abdominal cavity. Gastric motility recorded with this telemeter was identical with that measured by other devices, and consisted of two different patterns, the fasted and fed patterns divided into two phases, as reported previously. In the fasted state, cyclic occurrence of intense contractions was observed, and regular phasic contractions were observed in the fed state. Bethanechol induced strong contractions and atropine inhibited contractile activity. The newly developed telemeter is a useful and reliable device to use in measuring GI motility in small animals. PMID- 8630444 TI - [Pressure changes in the renal pelvis of the dogs in response to a sudden rise in intravesical pressure, examined in the presence of resico ureteral reflux with artificial ureter and/or pelvis]. AB - This study is to determine which part or the upper urinary tract, ureter or pelvis causes more significant delay in the pressure wave-front propagation from the bladder to the pelvis when a reverse flow of the urine occurs. A sudden rise in the intravesical pressure of the dog was produced by stimulating the vesical branches of pelvic nerve with a train of electrical impulses with 10 Hz for 60 sec. In order to eliminate the physiological mechanism that presents a reverse flow of the urine at the uretero-vesical junction, a 3 cm vinyl tube was inserted into the ureteral orifice in all experiments except the cases with ureteral substitution (group II and III). Changes in intrapelvic pressure were measured from 7 dogs without substitution of either ureter or pelvis, and used as the control responses for comparison with those recorded from the dogs with artificial ureter and/or pelvis. Artificial ureter was made by a vinyl tube of 25 cm, whereas artificial pelvis was made by a 3 ml plastic chamber. Twenty-eight dogs were divide into 3 groups. Group I consisted of 7 dogs whose pelvises were replaced by artificial ones. Group II included 8 dogs whose ureters were substituted by artificial ones, and group III consisted of 6 dogs whose both ureter and pelvis were replaced by artificial ones. Results obtained were as follows; (1) There was no significant difference between the maximum intravesical pressure and the maximum intrapelvic pressure recorded from every dog in all groups. (2) The onset time of intrapelvic response recorded from the control dog showed a delay from that of intravesical response by 2.05 +/- 1.03 sec. (3) The onset time of intrapelvic response recorded from group I, II and III were 0.51 +/ 0.94 sec., 1.95 +/- 1.10 sec., 0.10 +/- 0.25 sec., respectively. (4) Statistically significant difference in the delay of response was observed only between the responses recorded from the control or group II and the responses recorded from group I or III. The above results suggested that the major site causing a significant delay in the pressure wave-front propagation from the bladder to the pelvis is the pelvic area including caryx, but not the ureteral region, when a reverse flow of the urine occurs. PMID- 8630443 TI - [Changes in intestinal motility after massive small bowel resection with a reversed jejunal segment]. AB - To evaluate the functioning and effectiveness of a 20-cm reversed jejunal segment after 75-80% massive small bowel resection (MSBR), and whether migrating polarity changes or not, we continuously measured the postoperative bowel motility (using bipolar electrodes and/or contractile strain gage force transducers) in interdigestive and postprandial conscious dogs in short- (2-5 weeks) and long term (6-10 months) after surgery. The fasting migrating myoelectric (or motor) complex (MMC) arising from the duodenum was often interrupted at the jejunum above the proximal anastomosis and did not migrate smoothly to the reversed segment or terminal ileum. In addition, brief small discordant contractions were frequent in the jejunum above the proximal anastomosis and the proximal part of the reversed segment. The duodenal MMCs predominantly propagated to the ileum through the inherent anatomic continuity of the bowel. These findings of the MMC propagation pattern are very similar in short- and in long-term after surgery. The duration of the postprandial period without duodenal MMC activity was markedly longer in short-term, but shorter in long-term (both were significantly longer than in controls). Marked dilatation of the jejunum and reversed jejunal segment was noted across the proximal anastomosis. These results suggest that the transit time and passage of intestinal contents can be delayed and stagnated for at least 10 months after MSBR with a 20-cm reversed jejunal segment. Although, reports on the polarity of peristalsis in the reversed segment in long-term followup have been contradictory in both experimental and clinical studies, this results support the conclusion that the reversed jejunal segment maintains its inherent propagative polarity and pattern over a long postoperative period. PMID- 8630445 TI - Urodynamic and clinical characteristics of small intestine reservoir directly connected to urethral sphincter. AB - Eight patients in whom a small intestine reservoir with direct anastomosis to the urethral sphincter had to be performed after radical cystectomy for invasive carcinoma of the urinary bladder, were included in the study. The function of small intestine reservoir was clinically and urodynamically monitored during 24 months postoperatively. All patients urinated per urethram, with abdominal strain or with urethral sphincter relaxation without abdominal strain. After 21 months, diurnal continence was maintained in 87.5%, and circadian continence in 75% of the patients. Urodynamic tests showed it to be a low-pressure reservoir, where the basal reservoir pressure never exceeded 30 cm H2O during filling. The values of maximal cystometric capacity showed that a certain time of reservoir maturation had to elapse after the reservoir construction. After 3-6 months, the mean maximal cystometric capacity was 250 ml, and after 9 months it was 520 ml. The proportion of continent patients was observed to rise with the increase in the small intestine reservoir capacity. We believe that three substantial issues are ensured by the described surgical technique, i.e. the antireflux mechanism, appropriate capacity and acceptable continence. PMID- 8630446 TI - The need and value of routine screening of all pregnant women for hepatitis B surface antigen. AB - A prospective study was performed to determine whether the risk factor for hepatitis B, proposed by Centers for Disease Control (CDC), USA, are reliable predictors for the hepatitis B surface antigen (HBsAg) carrier state in an obstetric population. In the period between January 1, 1991 and December 31, 1992, all pregnant women from geographically defined areas of the Istrian and Rijeka districts were routinely screened for hepatitis B surface antigen (HBsAg). Among 10,627 pregnant women, 107 (1%) HBsAg positive cases were registered. History risk factors recommended by CDC were recorded in 46 (43%) out of 107 HBsAg positive women. The other 61 subjects had no recognizable risk factors. The screening of pregnant women for HBsAg only on the basis of the CDC recommended history guidelines, would have left 57% of our HBsAg positive mothers undetected and therefore their children unvaccinated against hepatitis B infection. Our results confirmed the need and value of the new CDC recommendations about routine prenatal screening of all pregnant women for HBsAg until hepatitis B vaccine is included in the scheme of compulsory vaccination of all newborns. PMID- 8630447 TI - Aldosterone and blood pressure in obese subjects during short-term starvation. AB - Changes in plasma renin activity (PRA) and aldosterone concentration during 10 days of starvation were studied, together with the relationship among PRA, aldosterone, plasma volume, sodium balance and mean blood pressure (MBP). Study subjects were 16 obese individuals (13 females and 3 males; age ranged 18-50 years) with a body mass index greater than 30 kg/m2. Total fasting induced a significant decrease in body weight, MBP, plasma volume and urinary sodium concentration, and a rise in PRA and aldosterone concentration. Before starvation, there was a significant correlation between aldosterone concentration and MBP. This data support the hypothesis that aldosterone is one of the factors which elevate blood pressure in obese subjects. Much of the initial fall in blood pressure during starvation seemed to be due to an imbalance between the cessation of sodium intake and excessive natriuresis. PMID- 8630448 TI - Relationship of eight basic emotions, life needs satisfaction and Type A behavior with body mass index and some biochemical characteristics in healthy blue-collar workers. AB - Studies of the relationship of eight basic emotion dimensions according to Plutchik, life needs satisfaction and Type A behavior with plasma cholesterol, triglyceride and glucose concentrations, and body mass index (BMI) were performed in 137 healthy blue-collar workers. Plasma cholesterol correlated positively with emotion dimensions of destruction and deprivation (r = +0.18, +0.21; p < 0.05) and negatively with the components of love in EPI (reproduction and incorporation; r = -0.24, -0.25; p < 0.01) and life needs satisfaction (family relationships, relationship with friends; r = -0.20, -0.17; p < 0.05). Triglycerides correlated negatively with satisfaction of life needs concerning health, family and friend relationships (r = -0.20, -0.20, 0.25; p < 0.05). Plasma glucose correlated positively with emotions of destruction and orientation (r = +0.22, +0.20; p < 0.05), and negatively with incorporation and reproduction (r = -0.22, -0.24; p < 0.01). BMI correlated positively with plasma cholesterol, triglyceride and glucose concentrations (r = +0.37, +0.40, +0.24; p < 0.01), and emotion of deprivation (r = +0.21; p < 0.01). Results of the study suggested the successful approach to the prevention of coronary heart disease to be related with certain basic emotions, life needs satisfaction and BMI. PMID- 8630449 TI - A new approach to surgical treatment of diabetic retinopathy. AB - Results of surgical treatment performed in 48 patients with proliferative diabetic retinopathy are presented. The technique of excision and vitrectomy of the posterior cortex and neovascular membranes was used in a single act. The authors are inclined to believe that membranes can be more completely and with less bleeding removed using this technique than with the technique of circumcision and segmentation. The procedure was most frequently complicated by perioperative formation of posterior ruptures (13 patients) and peripheral ruptures (11 patients). All ruptures were treated by endolaser photocoagulation. Additional or panretinal photocoagulation was carried out when necessary. During the postoperative period, retina remained attached in 33 patients, whereas in 15 patients complications in terms of reproliferation and neovascular glaucoma occurred. PMID- 8630450 TI - Antibody-dependent cellular cytotoxicity, ingestion and digestion in Dermatophagoides pteronyssinus-sensitive asthmatic children. AB - Dermatophagoides pteronyssinus (DP) is, for unknown reasons, the commonest cause of asthma attacks in children suffering from reaginic bronchial asthma. The underlying immune disorder is also unclear. The authors analyzed phagocytosis (ingestion), digestion and antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood leukocytes in 20 asthmatic children hypersensitive to DP, aged 2 to 14 years. The tests were performed while the children were entirely asymptomatic and under no therapy. The aim was to determine the possible difference in comparison to healthy children and to assess the correlation of these results with the total serum IgE level, DP-specific IgE and duration of the disease. Ingestion in asthmatics did not differ significantly from that in controls, while digestion and ADCC were significantly (P < 0.01) lower in asthmatics. This phenomenon could contribute to their difficulties in the elimination of allergens, immune complexes and microbial, particularly viral antigens, making them more susceptible to allergic reaction and infections. No significant correlation to the total serum IgE level, DP-specific IgE and duration of the disease was found. PMID- 8630451 TI - A biomechanical aspect of the lumbal approach to the kidney. AB - Functional differences between surgically attacked musculature of the lumbal region and intact musculature of the contralateral side were assessed in 71 patients by the method of Multic-Labar, Moire topography, electromyography and X ray analysis of the thoraco-lumbar spine. The following conclusions were derived from the results obtained. In the intact musculature of the lumbal segment of the spine, sliding forces reached 200 kN in males an 170 kN in females aged >25 and <30 years. In younger and older age groups, the values were lower, assuming a form of the ascending and descending limb of a parabole, respectively. Upon healing, the lumbal musculature damaged by surgical resection showed decreased values of the pulling and sliding forces by 15 and 25 kN on an average, respectively, as definite values independent of age and sex. Kinematic alterations included myogenic geneses of a stationary form, with normal innervation, involving the whole damaged musculature. Summa summarum, results of analyses suggested the values of kinematic forces of pulling and sliding forces of the musculature damaged by surgical resection to be definitely significantly reduced, thus confirming the premise of the study. PMID- 8630452 TI - Cigarette smoking and sarcoidosis. AB - The aim of the study was to analyze the effect of cigarette smoking on the occurrence of sarcoidosis. Sixty patients were examined (17 smokers had 43 nonsmokers). A control group consisted of 60 healthy subjects (33 smokers and 27 nonsmokers). The study showed sarcoidosis to more frequently occur in nonsmokers. Apart from the confirmation that sarcoidosis is predominantly a disease of nonsmokers, our aim was to determine whether smoking has an effect on the extent, course and outcome of the disease. Thus, at the beginning of the study the following examinations were performed in patients with sarcoidosis: chest X-ray, functional respiratory tests, determination of arterial blood acid-base status, blood cells, elastase and C-reactive protein in serum, and the cellular component of the bronchoalveolar lavage. Chest X-ray, functional respiratory tests and acid base component of arterial blood were repeated after one year. In our patients, hilopulmonary sarcoidosis was most frequent (66.7%). No significant difference was found between smokers and nonsmokers according to radiographic extent of the disease. Analysis of the cellular component of the bronchoalveolar lavage showed significantly more macrophages and less lymphocytes in smokers. Analysis of serum indicators showed no differences between smokers and nonsmokers. Smokers and nonsmokers did not differ according to values of forced expiratory volume in the first second (FEV1). However significantly more nonsmokers had mild degrees of decreased FEV1 (75%-60%) at the beginning of the study, and significantly more smokers had a higher degree of respiratory pathway obstruction (60%-45% and less), both at the beginning and end of the study. At the beginning of the study, significantly higher values of residual volume (RV) and total lung capacity (TLC) were recorded in smokers. No statistically significant difference was found between smokers nad nonsmokers according to the extent of the disease, frequency of extrathoracic localization of disease and use of corticosteroids. Comparison of radiographic finding at the end of the study showed no significant difference between smokers and nonsmokers. There was no difference according to outcome, depending on the therapy used. Based on the results of this study the authors conclude that smoking plays a certain protective role in the occurrence of sarcoidosis, although smoking has no effect on the extent, course and outcome of the disease. PMID- 8630453 TI - Medullary carcinoma of the thyroid: histomorphological, histochemical and immunohistochemical analysis of twenty cases. AB - Histomorphological patterns of twenty primary medullary carcinomas of the thyroid were studied by light and polarized microscopy in relation to the content of calcitonin and thyroglobulin determined by the immunoperoxidase method: Out of 20 tumors, 10 showed classical, 7 glandular and 3 insular histomorphological pattern. In 19/20 cases, the cytoplasm of tumor cells contained various amounts of calcitonin, and the intensity of Immunoreaction was strong in 2/19, moderate in 7/19 and weak in 10/19 cases. Tumor stroma contained calcitonin in 7/20 cases. In 1/20 case, which did not show calcitonin immunoreactivity in the cell cytoplasm, the stroma contained a considerable amount of calcitonin. Thyroglobulin immunoreactivity was found in 4/20 tumors, 2 of them with classic and 2 with glandular histomorphological picture only in the cytoplasm of tumor cells. These tumors are considered medullary carcinomas with thyroglobulin immunoreactivity, since they do not fulfil the WHO criteria for "mixed medullary follicular carcinomas". PMID- 8630454 TI - Inotropic agents in the treatment of postoperative low cardiac output syndrome. AB - Two basic groups of inotropic drugs that are in the treatment of postcardiotomy low cardiac output syndrome (LCOS) are presented. The authors emphasize the advantages of phosphodiesterase inhibitors (PDE III) as compared to catecholamines. PMID- 8630455 TI - Conventional retinal surgery for rhegmatogenous retinal detachment with proliferative vitreoretinopathy. AB - During the last two years, 79 consecutive patients (i.e. eyes) with rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR), stages B and CP according to the classification of Machemer and associates, were treated. Conventional retinal surgery (scleral buckling) was used. Retina was successfully reattached in 54 (68.4%) eyes in the early postoperative period. Thirty-five (81.4%) out of 43 patients with PVR detachment stage B, and 19 (52.8%) out of 36 patients with PVR detachment stage CP achieved total retinal attachment. The difference was statistically significant (p = 0.006). In stage CP, larger areas of retinal involvement by fixed retinal folds are inversely related to the rate of anatomical success. A higher degree of preoperative PVR means less success of conventional retinal detachment surgery, so it is advised to apply a conventional surgical technique up to CP-6 stage of PVR, whereas in more advanced stages of PVR pars plana vitrectomy is needed. PMID- 8630456 TI - Continuous hemofiltration in patients with acute renal failure. AB - Many intensive care unit (ICU) patients with acute renal failure (ARF) have multiple organ failure (MOF) and cardiovascular instability. Continuous hemofiltration (CH) is a widely accepted technique for the treatment of such critically ill patients. The authors treated ten patients with ARF and MOF. Continuous arteriovenous hemofiltration (CAVH) and venovenous hemofiltration (CVVH) were performed in five patients each. The mean duration of treatment was 88.6 +/- 82.8 h and the mean ultrafiltrate rate 392 +/- 272 ml/min. Serum urea i creatinine were stable, with good control of fluid balance. Only two of our patients survived: one of them recovered renal function and the other required chronic hemodialysis (HD). CVVH offered better control of blood flow and ultrafiltration rate, longer hemofilter use and has the advantage of requiring only venous access. PMID- 8630457 TI - [Genetic methods of diagnosing tuberculosis]. PMID- 8630458 TI - [Serologic and chromatographic methods of diagnosing tuberculosis]. PMID- 8630459 TI - [Value of neuron specific enolase levels and squamous cell carcinoma antigen in bronchial lavage fluid in patients with lung cancer]. AB - The aim of the study was to assess a value of NSE and SCC Ag level determination in bronchial lavage fluid in patients with lung cancer. It was found out that NSE levels in bronchial lavage fluid were much lower than in serum and did not differentiate patients with small cell lung cancer from patients with non small cell lung cancer and nonmalignant lung diseases. Bronchial lavage SCC Ag levels were significantly higher than serum SCC Ag levels. Although determination of SCC Ag levels in bronchial lavage was also not helpful in differential diagnosis. PMID- 8630460 TI - [The value of determining neuron specific enolase for diagnosis of complete remission during treatment of small cell lung cancer]. AB - The aim of this study was to assess whether the lowest serum NSE obtained during treatment of small cell lung cancer patients can be helpful in the diagnosis of complete remission (CR). The material consisted of 68 patients with small cell lung cancer, treated in the Institute o Tuberculosis and Lung Diseases from 1.III.1993 to 15.II.1995. In the course of treatment CR was obtained in 13 patients, partial remission (PR) in 37 and no remission (NR) in 18. The distribution and median of the lowest NSE serum levels were the same in CR and RP patients. NSE serum levels remained above normal, that is above 12.5 ng/ml, in two CR patients and in 4 PR patients. 3 patients (2 with CR and I with PR) are still living for 26, 27 and 41 months in spite of NSE serum levels 14.3, 15.6 and 13.6 ng/ml respectively. In those patients in whom NR was obtained the lowest NSE level above 20 ng/l was connected with bad prognosis. We conclude that the estimation of the lowest NSE serum level in the course of treatment can not help to differentiate CR from PR. PMID- 8630461 TI - [Immediate and follow up results of surgical therapy for mesenchymal mediastinal tumors]. AB - 35 patients (11 men, 24 women, aged 5-73) with mesenchymal tumor of mediastinum were surgically treated in the period 1970-1993. This group stated 10,3% of all patients treated because of cysts and tumors of mediastinum in this time. In 17 cases tumors were benign and in 18 malignant. In 31 pts total resection and in 3 partial resection were done. 5 out of 15 pts with malignant tumor are alive. PMID- 8630463 TI - [Indications for prophylactic cranial irradiation in patients with small cell lung cancer]. AB - Brain metastases are one of the important causes of failure in the treatment of SCLC patients. Intracranial metastases are reported in 10% of the newly diagnosed SCLC pts. Further 20% brain metastases develop during therapy. Autopsy studies showed that 40%-60% of pts had evidence of intracranial spread at the time of death. The frequency of brain metastases increases with the duration of survival, reaching a probability of 80% after 2 years of observation. The high risk of brain metastases which developed in spite of chemotherapy was the reason of introducing PCI to the treatment. PCI reduces the risk of brain metastases to less than 10%, but unfortunately, does not prolong median survival. Probably this is due to the fact, that in the majority of cases the brain is one of many sites of metastases at the time of progression. In addition, in some pts brain metastases developed in spite of PCI and/or brain toxicity may develop after this treatment. Thus, up till now the role of PCI in the treatment of SCLC pts remains controversial. The indication for this treatment could improve if we have more exact information which pts have the greatest probability do develop metastases restricted to the brain only. This question was the main aim of our study. PMID- 8630462 TI - [Comparison of conventional chest radiography and computed tomography for evaluation of the degree of regression in neoplasms of patients treated for small cell lung cancer]. AB - The aim of the study was to assess how objective is the estimation of the chest lesions regression on RT picture and how extent chest CT can increase the precision of this assessment. The material consisted of 66 SCLC pts observed in the Institute of Tuberculosis and Lung Diseases in Warsaw between 1987 and 1994 in whom RT and CT were performed to estimate the degree of response to treatment. Chest RT were estimated by 4 specialists independently. In 28 cases (42%) opinion concerning the degree of tumor regression was the same. In 23 cases (35%) the opinions were discordant and in 15 cases (13%) (mainly after chest irradiation) all examiners agreed that degree of response is impossible to precise (it ranged between PR and CR). In the second part of this study the degree of cancer regression was assessed in the same patients by 2 specialists independently estimated CT pictures. Opinions were different only in 4 cases. The comparison between these two methods was done. CI examination allowed exact estimation of response degree in 20 out of 24 pts (83%) in whom this assessment was impossible using chest radiography. In 9 cases (21%) the assessment of chest CT changed the previous estimation made using chest radiography (in 5 cases from CR to PR and in 4 cases from PR to CR). In conclusion-estimation of tumor regression using chest RT is very subjective method and many errors are possible. Chest CT estimation is a much better method for estimation of tumor response especially in irradiated patients. PMID- 8630464 TI - [Initial prospective evaluation of cancer incidence in relation to metabolism of antipyrine and family history in patients with lung cancer]. AB - Our previous studies revealed faster antipyrine metabolism among lung cancer patients and their first degree relatives in comparison with subjects without cancer history in their families. After 8 years 39-70% previously investigated first degree relatives of lung cancer patients and 55-73% previously investigated healthy subjects without cancer in their families were examined. One lung cancer case was noticed in the group of relatives and this subjects had very fast antipyrine metabolism. Also 2 cases of non-smoking related cancer were observed in subjects with intermediate antipyrine metabolism. Only one case of non-smoking related cancer was noticed among controls and this subjects had rather slow antipyrine metabolism. PMID- 8630465 TI - [Mutation of gene p53 in lung cancer]. PMID- 8630466 TI - [Changes of smoking habits over 10 years among II year medical students]. AB - In 1983 and 1993 among second year medical students surveys were conducted with regard to their cigarette smoking habits. After 10 years we found decrease in smoking habit among medical students (in 1983 48% of students smoked cigarettes, in 1993 only 28% smoked cigarettes). In 1983 only 27% but in 1993 75% of the students who smoked had started smoking prior to their university studies. In 1983 69% of the students smoked fewer than 5 cigarettes a day. Today, the figure is 36%. The corresponding figures of students smoking more than 20 cigarettes a day are 4.5% and 5.6% respectively. The reasons for smoking--chiefly among women students--are stress and sociability. PMID- 8630467 TI - [Bradykinin levels in BAL fluid in patients with sarcoidosis]. AB - In 14 patients with sarcoidosis and in 9 healthy controls the bradykinin level was estimated in broncho-alveolar lavage fluid. The statistically significantly higher bradykinin concentrations was found in sarcoid patients than in controls. No relationship was found between level of bradykinin in BAL and lymphocytes % and no relationship was found between bradykinin level and pulmonary function parameters. PMID- 8630468 TI - [Treatment of patients with pulmonary aspergilloma with itraconazole]. AB - The study aimed to assess effectiveness of itraconazole in treating pulmonary aspergilloma. Treatment with this triazole derivative was employed in 11 patients with pulmonary aspergilloma. Nine patients underwent a complete 6 month treatment at 200-400 mg dose. The treatment was discontinued in lesions 2 patients who demonstrated progression of lesions. Haemoptysis withdrew in 5 patients, the same number of patients displayed negative fungus culture. Only in 1 patient radiological appearances improved and negative sputum culture as well as cessation of haemoptysis were observed. No significant adverse reactions were reported for any patients. Six months after completion of the treatment haemoptysis recurred in 2 patients, renewed growth of A. Fumigatus was quoted for 3 patients and intensified precipitating test results for 2 patients. Only 1 patient improved within all the required parameters. The results of the conducted study indicate that itraconazole by infiltrating lung cavity inhibits fungus growth on the surface of aspergilloma, which, in isolated cases can lead to aspergilloma necrosis. The results of itraconazole treatment are not sufficiently satisfactory to justify application of itraconazole in routine treatment of pulmonary aspergilloma. PMID- 8630469 TI - [Pleural needle biopsy in diagnosis of pleural effusion]. AB - Diagnostic reliability of percutaneous parietal pleural biopsy with use of the Abrams needle technique performed over a period 1989-1994 was assessed in 37 patients with pleural effusion of unknown etiology, aged 24-82 years. Adequate diagnostic specimens were obtained in 33 patients. Histopathological diagnosis established neoplasma (39,4%), tuberculosis (9.1%) and chronic nonspecific pleuritis (51,5%). No complications of the procedure were encountered. Pleural needle biopsy should be more widely applied as a basic method in the diagnosis of pleural effusions of unknown etiology. PMID- 8630470 TI - [Tuberculosis of the larynx in material from the ORL ward of the District General Hospital in Czestochowa in the years 1980-1992]. AB - 11 cases of tuberculosis of the larynx were presented in this paper. Provisional diagnosis was tumor of the larynx. Sex ratio (male to female patients) was 9:2. Age of patients ranged from 36 to 79. PMID- 8630472 TI - [Wegener's granulomatosis. Three cases with different clinical courses]. AB - Three cases of Wegener's granulomatosis are presented. In every case the different clinical picture was observed. In the first case dominated the pulmonary changes and fingers necrosis. In that case the excellent therapeutic effect of etoposide was observed. In the second case the most important clinical sings illustrated severe renal insufficiency. The third case demonstrated pericardial and skin changes. The significance of early diagnosis and early institution of proper therapy was discussed. PMID- 8630471 TI - [Diffuse alveolar hemorrhage in Wegener's granulomatosis]. AB - Two cases of Wegeners Granulomatosis were presented. In the both cases diffuse alveolar hemorrhage was found. PMID- 8630473 TI - [Gall bladder tuberculosis and stomach cancer]. AB - The case of a seventy year old woman with delayed diagnosis of stomach cancer due to misinterpretation of signs and symptoms is presented. A few months earlier a gall bladder tuberculosis was found in this patient. Abdominal symptoms which appeared afterwards were therefore interpreted as manifestation of tuberculosis until the thorough examination allowed diagnosing a gastric cancer. PMID- 8630474 TI - [Intravenous injection of metallic mercury--case report]. AB - A case of a 16 year old male who injected intravenously metallic mercury is presented. The patient was admitted to the hospital due to slightly elevated fever and hemoptysis. Both symptoms presented themselves 18 months following intravenous injection of metallic mercury. Radiological examination disclosed fine metallic interstitial shadowing in the lung parenchyma. Metallic shadows were also present in the subcutaneous region of the cubital fossa. CT scans and USG examination disclosed similar deposition of metal in the liver, kidneys, lymph nodes and heart. The mercury serum and urine levels exceeded the normal values by hundreds. Encountered diagnostic problems are presented, also prognosis basing on data from literature is discussed. PMID- 8630475 TI - [A case of tuberculous lymphadenitis--diagnostic difficulties]. AB - There is presented the case of tuberculous cervical lymphadenitis of 60 years old patient who was hospitalized in Clinic of. Parasitic Diseases and Neuroinfection in 1992 and diagnostic difficulties connected with it. PMID- 8630477 TI - Romanovs find closure in DNA. PMID- 8630476 TI - [Cyfra 21-1--new marker for lung cancer]. PMID- 8630478 TI - One FISH, two FISH, red FISH, blue FISH. PMID- 8630479 TI - Friedreich's in relief. PMID- 8630480 TI - Keeping an eye on eye development. PMID- 8630481 TI - Complexity in a monogenic disease. PMID- 8630482 TI - A manic depressive history. PMID- 8630483 TI - Genetic dissection of complex traits. PMID- 8630484 TI - Genetic dissection of complex traits. PMID- 8630485 TI - XNP mutation in a large family with Juberg-Marsidi syndrome. PMID- 8630486 TI - Reconstructing the ancient mariners of humans. PMID- 8630487 TI - The most beautiful people. PMID- 8630488 TI - Creation of genomic methylation patterns. AB - There are two biological properties of genomic methylation patterns that can be regarded as established. First, methylation of 5'-CpG-3' dinucleotides within promoters represses transcription, often to undetectable levels. Second, in most cases methylation patterns are subject to clonal inheritance. These properties suit methylation patterns for a number of biological roles, although none of the current hypotheses can be regarded as proved or disproved. One hypothesis suggests that the activity of parasitic sequence elements is repressed by selective methylation. Features of invasive sequences that might allow their identification and inactivation are discussed in terms of the genome defense hypothesis. Identification of the cues that direct de novo methylation may reveal the biological role (or roles) of genomic methylation patterns. PMID- 8630489 TI - Karyotyping human chromosomes by combinatorial multi-fluor FISH. AB - We have developed epifluorescence filter sets and computer software for the detection and discrimination of 27 different DNA probes hybridized simultaneously. For karyotype analysis, a pool of human chromosome painting probes, each labelled with a different fluor combination, was hybridized to metaphase chromosomes prepared from normal cells, clinical specimens, and neoplastic cell lines. Both simple and complex chromosomal rearrangements could be detected rapidly and unequivocally; many of the more complex chromosomal abnormalities could not be delineated by conventional cytogenetic banding techniques. Our data suggest that multiplex-fluorescence in situ hybridization (M FISH) could have wide clinical utility and complement standard cytogenetics, particularly for the characterization of complex karyotypes. PMID- 8630490 TI - Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. AB - Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off PMID- 8630491 TI - A novel X-linked gene, G4.5. is responsible for Barth syndrome. AB - Barth syndrome is a severe inherited disorder, often fatal in childhood, characterized by cardiac and skeletal myopathy, short stature and neutropenia. The disease has been mapped to a very gene-rich region in distal portion of Xq28. We now report the identification of unique mutations in one of the genes in this region, termed G4.5, expressed at high level in cardiac and skeletal muscle. Different mRNAs can be produced by alternative splicing of the primary G4.5 transcript, encoding novel proteins that differ at the N terminus and in the central region. The mutations introduce stop codons in the open reading frame interrupting translation of most of the putative proteins (which we term 'tafazzins'). Our results suggest that G4.5 is the genetic locus responsible for the Barth syndrome. PMID- 8630492 TI - Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. AB - Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development. PMID- 8630493 TI - An X-chromosome linked locus contributes to abnormal placental development in mouse interspecific hybrid. AB - Interspecific hybridization between closely related species is commonly associated with decreased fertility or viability of F1 hybrids. Thus, in mouse interspecific hybrids, several different hybrid sterility genes that impair gametogenesis of the male hybrids have been described. We describe a novel effect in hybrids between different mouse species that manifests itself in abnormal growth of the placenta. Opposite phenotypes, that is, placental hypotrophy versus hypertrophy, are observed in reciprocal crosses and backcrosses. The severity of the phenotype, which is mainly caused by abnormal development of the spongiotrophoblast, is influenced by the sex of the conceptus. In general, placental hypertrophy is associated with increased fetal growth. Hypotrophy of the placenta frequently leads to growth impairment or death of the fetus. One of the major genetic determinants of placental growth maps to the proximal part of the mouse X chromosome. PMID- 8630494 TI - Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. AB - Duplications of a chromosome Xp21 locus DSS (Dosage Sensitive Sex reversal) are associated with male to female sex reversal. An unusual member of the nuclear hormone receptor superfamily, DAX1, maps to the DSS critical region and is responsible for X-linked adrenal hypoplasia congenita. Here we describe the isolation of the mouse Dax1 gene and its pattern of expression during development. Expression was detected in the first stages of gonadal and adrenal differentiation and in the developing hypothalamus. Moreover, Dax1 expression is down-regulated coincident with overt differentiation in the testis, but persists in the developing ovary. Comparison of the predicted protein products of the human and mouse genes show that specific domains are evolving rapidly. Our results suggest a basis for adrenal insufficiency and hypogonadotropic hypogonadism in males affected by adrenal hypoplasia congenita and are consistent with a role for DAX1 in gonadal sex determination. PMID- 8630496 TI - Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. AB - In 1991, nine sets of skeletal remains were excavated from a mass grave near Yekaterinburg, Russia which were believed to include the Russian Tsar Nicholas II, the Tsarina Alexandra, and three of their daughters. Nuclear DNA testing of the remains verified such a family group, and mitochondrial DNA (mtDNA) sequences of the presumed Tsarina matched a known maternal relative, Prince Philip. mtDNA sequences from bone of the presumed Tsar matched two living maternal relatives except at a single position, where the bone sample had a mixture of matching (T) and mismatching (C) bases. Cloning experiments indicated that this mixture was due to heteroplasmy within the Tsar; nevertheless, the 'mismatch' fueled a lingering controversy concerning the authenticity of these remains. As a result, the official final report on the fate of the last Russian Royals has been postponed by Russian authorities pending additional, convincing DNA evidence. At the request of the Russian Federation government, we analysed the skeletal remains of the Tsar's brother Georgij Romanov in order to gain further insight into the occurrence and segregation of heteroplasmic mtDNA variants in the Tsar's maternal lineage. The mtDNA sequence of Georgij Romanov, matched that of the putative Tsar, and was heteroplasmic at the same position. This confirms heteroplasmy in the Tsar's lineage, and is powerful evidence supporting the identification of Tsar Nicholas II. The rapid intergenerational shift from heteroplasmy to homoplasmy, and the different heteroplasmic ratios in the brothers, is consistent with a 'bottleneck' mechanism of mtDNA segregation. PMID- 8630495 TI - A microdeletion in cytochrome c oxidase (COX) subunit III associated with COX deficiency and recurrent myoglobinuria. AB - We have identified a 15-bp microdeletion in a highly conserved region of the mitochondrially encoded gene for cytochrome c oxidase (COX) subunit III in a patient with severe isolated COX deficiency and recurrent myoglobinuria. The mutant mitochondrial DNA (mtDNA) comprised 92% of the mtDNA in muscle and 0.7% in leukocytes. Immunoblots and immunocytochemistry suggested a lack of assembly or instability of the complex. Microdissected muscle fibres revealed significantly higher portions of mutant mtDNA in COX-negative than in COX-positive fibres. This represents the first case of isolated COX deficiency to be defined at the molecular level. PMID- 8630497 TI - Pendred syndrome (goitre and sensorineural hearing loss) maps to chromosome 7 in the region containing the nonsyndromic deafness gene DFNB4. AB - Inherited causes account for about 50% of individuals presenting with childhood (prelingual) hearing loss, of which 70% are due to mutation in numerous single genes which impair auditory function alone (non-syndromic). The remainder are associated with other developmental anomalies termed syndromic deafness. Genes responsible for syndromic forms of hearing loss include the COL4A5 gene in Alport syndrome and the PAX3 and MITF genes in Waardenburg syndrome. Pendred syndrome is an autosomal recessive disorder associated with developmental abnormalities of the cochlea, sensorineural hearing loss and diffuse thyroid enlargement (goitre). Pendred syndrome is the most common syndromal form of deafness, yet the primary defect remains unknown. We have established a panel of 12 families with two or more affected individuals and used them to search for the location of the Pendred gene by linkage analysis. We excluded localization to four previously mapped nonsyndromic deafness loci but obtained conclusive evidence for linkage of the Pendred syndrome gene to microsatellite markers on chromosome 7q31 (D7S495 Zmax 7.32, Qmax = 0). This region contains a gene, DFNBL, for autosomal recessive non syndromic sensorineural hearing loss. Multipoint analysis indicates that DFNB4 and Pendred syndrome co-localize to the same 5.5 centiMorgan (cM) interval flanked by D7S501 and D7S523. These data raise the possibility that Pendred syndrome is either allelic with DFNB4 or may represent an inherited contiguous gene disorder, not clinically manifest in the heterozygote. PMID- 8630498 TI - Pendred syndrome maps to chromosome 7q21-34 and is caused by an intrinsic defect in thyroid iodine organification. AB - Exactly 100 years ago, in 1896, Pendred first described the association of congenital deafness with thyroid goitre (MM#274600). The incidence of Pendred syndrome is estimated at 7.5-10/100,000, and may be responsible for as much as 10% of hereditary deafness. The cause of the congenital deafness in Pendred syndrome is obscure, although a Mondini type malformation of the cochlea exists in some patients. The reason for the association between the thyroid and cochlear defects is similarly obscure, leading some investigators to suggest that the two recessive defects may be occurring together by chance in highly consanguineous families. An in vivo defect in thyroid iodine organification in Pendred syndrome patients has been reported. However, the molecular basis of this defect is unknown and the presence of an intrinsic thyroidal defect has not been conclusively demonstrated. We have adopted a genetic linkage study as a first step towards identifying the gene. The availability of an inbred Pendred syndrome kindred allowed us to utilize an efficient DNA pooling strategy to perform a genome-wide linkage search for the disease locus. In this way, we have mapped the disease locus to an approximately 9-cM interval between GATA23F5 and D7S687 on chromosome 7. In addition, we demonstrate an intrinsic thyroid iodine organification defect in a patient's thyroid cells as the cause of the thyroid dysfunction. PMID- 8630499 TI - A locus for bipolar affective disorder on chromosome 4p. AB - The main clinical feature of bipolar affective disorder is a change of mood to depression or elation. Unipolar disorder, also termed major depressive disorder, describes the occurrence of depression alone without episodes of elevated mood. Little is understood about the underlying causes of these common and severe illnesses which have estimated lifetime prevalences in the region of 0.8% for bipolar and 6% for unipolar disorder. Strong support for a genetic aetiology is found in the familial nature of the condition, the increased concordance of monozygotic over dizygotic twins and adoption studies showing increased rates of illness in children of affected parents. However, linkage studies have met with mixed success. An initial report of linkage on the short arm of chromosome 11 (ref. 4) was revised and remains unreplicated. Reports proposing cosegregation of genes found on the X chromosome with bipolar illness have not been supported by others. More recently bipolar disorder has been reported to be linked with markers on chromosomes 18, 21, 16 and a region on the X chromosome different from those previously suggested. We have carried out a linkage study in twelve bipolar families. In a single family a genome search employing 193 markers indicated linkage on chromosome 4p where the marker D4S394 generated a two-point lod score of 4.1 under a dominant model of inheritance. Three point analyses with neighbouring markers gave a maximum lod score of 4.8. Eleven other bipolar families were typed using D4S394 and in all families combined there was evidence of linkage with heterogeneity with a maximum two-point lod score of 4.1 (theta = 0, alpha = 0.35). PMID- 8630500 TI - A genome-wide search for chromosomal loci linked to bipolar affective disorder in the Old Order Amish. AB - The most characteristic features of bipolar affective disorder (manic-depressive illness) are episodes of mania (bipolar I, BPI) or hypomania (bipolar II, BPII) interspersed with periods of depression. Manic-depressive illness afflicts about one percent of the population, and if untreated, is associated with an approximately 20% risk of suicide. Twin, family and adoption studies provide compelling evidence for a partial genetic aetiology, but the mode(s) of inheritance has not been identified. Nonetheless, the majority of genetic linkage studies have assumed classical mendelian inheritance attributable to a single major gene. Although segregation analyses have yielded inconsistent results (with most studies rejecting a single locus inheritance model), the best single gene model is dominant inheritance if only BPI is considered. Reported linkages of bipolar affective disorder on chromosomes 11, 18, 21 and X have been difficult to substantiate, and additional studies are required for replication or exclusion of these regions. We now present the results of our genome-wide linkage analyses that provide evidence that regions on chromosomes 6, 13 and 15 harbour susceptibility loci for bipolar affective disorder, suggesting that bipolar affective disorder in the Old Order Amish is inherited as a complex trait. PMID- 8630501 TI - Genetic mapping using haplotype, association and linkage methods suggests a locus for severe bipolar disorder (BPI) at 18q22-q23. AB - Manic depressive illness, or bipolar disorder (BP), is characterized by episodes of elevated mood (mania) and depression. We designed a multistage study in the genetically isolated population of the Central Valley of Costa Rica to identify genes that promote susceptibility to severe BP (termed BPI), and screened the genome ot two Costa Rican BPI pedigrees (McInnes et al., submitted). We considered only individuals who fulfilled very stringent diagnostic criteria for BPI to be affected. The strongest evidence for a BPI locus was observed in 18q22 q23. We tested 16 additional markers in this region and seven yielded peak lod scores over 1.0. These suggestive lod scores were obtained over a far greater chromosomal length (about 40 cM) than in any other genome region. This localization is supported by marker haplotypes shared by 23 of 26 BPI affected individuals studied. Additionally, marker allele frequencies over portions of this region are significantly different in the patient sample from those of the general Costa Rican population. Finally, we performed an analysis which made use of both the evidence for linkage and for association in 18q23, and we observed significant lod scores for two markers in this region. PMID- 8630502 TI - Mutation of the endothelin-3 gene in the Waardenburg-Hirschsprung disease (Shah Waardenburg syndrome). AB - Hirschsprung disease (HSCR) and Waardenburg sundrome (WS) are congenital malformations regarded as neurocristopathies since both disorders involve neural crest-derived cells. The WS-HSCR association (Shah-Waardenburg syndrome) is a rare autosomal recessive condition that occasionally has been ascribed to mutations of the endothelin-receptor B (EDNRB) gene. WS-HSCR mimicks the megacolon and white coat-spotting observed in Ednrb mouse mutants. Since mouse mutants for the EDNRB ligand, endothelin-3 (EDN3), displayed a similar phenotype, the EDN3 gene was regarded as an alternative candidate gene in WS-HSCR. Here, we report a homozygous substitution/deletion mutation of the EDN3 gene in a WS-HSCR patient. EDN3 thus becomes the third known gene (after RET and EDNRB) predisposing to HSCR, supporting the view that the endothelin-signaling pathways play a major role in the development of neural crests. PMID- 8630503 TI - A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome). AB - Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients. PMID- 8630504 TI - Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. AB - Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease. PMID- 8630505 TI - Exclusion of SNRPN as a major determinant of Prader-Willi syndrome by a translocation breakpoint. AB - The predominant genetic defects in Prader-Willi syndrome (PWS) are 15q11-q13 deletions of paternal origin and maternal chromosome 15 uniparental disomy (UPD). In contrast, maternal deletions and paternal chromosome 15 UPD are associated with a different neurogenetic disorder, Angelman syndrome (AS). In both disorders, these mutations are associated with parent-of-origin specific methylation at several 15q11-q13 loci. The critical PWS region has been narrowed to a approximately 320-kb region between D15S63 and D15S174, encoding several imprinted transcripts, including PAR5, IPW, PAR1 (refs 7,8) and SNRPN, which has so far been considered a strong candidate for the PWS gene. A few PWS-associated microdeletions involving a putative imprinting centre (IC) proximal to SNRPN have also been observed. We have mapped the breakpoint of a balanced translocation (9;15)pat associated with most of the PWS features between SNRPN and IPWIPAR1. Methylation and expression studies indicate that the paternal SNRPN allele is unaffected by the translocation, while IPW and PAR1 are unexpressed. This focuses the attention on genes distal to the breakpoint as the main candidate for PWS genes, and is consistent with a cis action of the putative IC, and suggests that further studies of translocational disruption of the imprinted region may establish genotype-phenotype relationships in this presumptive contiguous gene syndrome. PMID- 8630506 TI - Genetic mapping of a pulmonary adenoma resistance (Par1) in mouse. AB - Lung cancer, a major cause of death in the Western world, has a poor prognosis. So far, therapeutic strategies have had only a limited effect. Lung cancer risk is strongly associated with cigarette smoking and lung cancer pedigrees are rare. However, a possible polygenic nature of inherited predisposition to this cancer has been envisaged. Mouse inbred strains with inherited predisposition and resistance to lung cancer provide an important tool for the dissection of the genetics of this complex disease. The A/J strain carries the pulmonary adenoma susceptibility 1 (Pas1) locus and develops many lung tumours. We have mapped the M. spretus-derived locus that strongly resists the lung tumorigenesis in Pas1/+ mice. This locus, pulmonary adenoma resistance 1 (Par1) maps to mouse chromosome 11, near the Rara locus, with a lod score of 5.3. In Pas1/+ mice Par1 accounts for 23% of the phenotypic variance and 10 fold reduction in total tumour volume. These results provide evidence for a major resistance locus affecting the expression of an inherited predisposition to lung cancer. PMID- 8630507 TI - Human choroideremia protein contains a FAD-binding domain. PMID- 8630508 TI - Endocrinology in France: present status and future. PMID- 8630509 TI - Forecasting the future of endocrinology in Europe. PMID- 8630510 TI - Recent developments in the understanding of the pathophysiology of osteopetrosis. AB - Osteopetrosis is a rare metabolic bone disease characterized by a generalized increase in skeletal mass. It is inherited in a number of mammalian species, including man, and results from a congenital defect in the development or function of the osteoclasts. The consequent impairment of bone resorption prevents formation of bone marrow cavities, causes delayed or absent tooth eruption and results often in abnormally shaped bone. The pathogenetic defect may be intrinsic either to the osteoclast lineage or to the mesenchymal cells that constitute the microenvironment supporting the development and activation of the osteoclasts. In the first example, the disease can be cured by transplantation of hemopoietic cells. In some cases, bone marrow transplantation has also been successful in curing human osteopetrosis. This, together with the variability in the age of onset and severity of clinical aspects, suggests that a multiplicity of genetic mutations may cause the human disease. In recent years the genetic effects of some osteopetrotic mutations have been identified. This new information has been essential for the understanding of osteoclast biology. Colony stimulating factor 1 (CSF-1), the growth factor for cells of the mononuclear phagocytic system, is also essential for the development of osteoclasts. In the osteopetrotic (op) mouse, no biologically active CSF-1 is synthesized due to a point mutation in the coding region of its gene. This leads to an almost complete lack of osteoclast development and to impaired bone resorption. Altered CSF-1 production seems also to be involved in the toothless (tl) rat osteopetrosis. Recently, the mutation responsible for the microphthalmic (mi) mouse osteopetrosis has been identified in the gene encoding a member of the basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein family of transcription factors. The mi gene product seems to play a role in the fusion process of osteoclast precursor cells. Finally, osteopetrosis has been the result of experimental gene disruption in mice. Targeted disruption of the c-src proto oncogene encoding a nonreceptor tyrosine kinase leads to a form of osteopetrosis where osteoclasts are present but inactive. This indicates that pp60c-src, localized primarily on ruffled border membranes and vacuoles of the osteoclasts, is important for osteoclastic function. Disruption of the c-fos proto-oncogene, a major component of the AP-1 transcription factor complex, leads to an osteopetrotic phenotype characterized by a complete absence of osteoclasts. The defect is intrinsic to hemopoietic precursors that are unable to progress beyond an early stage of osteoclast differentiation. In humans, deficiency of carbonic anhydrase II has been identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. A lack of expression of the vacuolar proton pump has been observed in osteoclasts of a patient with craniometaphyseal dysplasia. In conclusion, the disease, although rare, is of great pathophysiological relevance for our understanding of the processes that govern the development and function of osteoclasts. PMID- 8630511 TI - Von Hippel-Lindau tumor suppressor protein and transcription elongation: new insights into regulation of gene expression. PMID- 8630512 TI - Co-activators and co-repressors: mediators of gene activation by nuclear hormone receptors. PMID- 8630513 TI - Bone mineral density in immigrants from southern China to Denmark. A cross sectional study. AB - Immigration from Japan to USA has been shown to increase bone mineral density (BMD) and body fat in women. The effects of immigration between other geographical areas on bone mass and body composition are largely unknown, especially in men. In the present study, we measured bone mass and body composition by dual energy X-ray absorptiometry (Hologic QDR-2000) in 73 healthy premenopausal women (age 35 +/- 8 years) and 69 men (age 40 +/- 12 years) who had immigrated from southern China to Denmark 2 months to 36 years ago. The BMD measurements (Total BMD, trunk BMD and leg BMD) were related positively to years since immigration (YSI) (R2 = 0.10-0.16, p < 0.05) in premenopausal women, but not in men. Fat distribution was related mainly to age in both premenopausal women and men (R2 = 0.16-0.26, p < 0.05). For comparison, we included 51 white, Danish premenopausal women (age 36 +/- 6 years). Chinese premenopausal women with a YSI below or equal to 12 years (N = 38) had significantly lower total and regional BMD (trunk, legs, arms) (p < 0.05), while women with a YSI above 12 years (N = 35) had significantly lower BMD in the legs only (p < 0.05) when compared to the Danish premenopausal women. After correction for age weight and height, Chinese premenopausal women with a YSI below or equal to 12 years still had significantly lower BMD in all regions (4-7%, p < 0.05), whereas no differences in BMD were found between Chinese premenopausal women with a YSI above 12 years compared with Danish premenopausal women. In conclusion, Chinese premenopausal women who immigrated to Denmark more than 12 years ago have a similar BMD to that of Danish premenopausal women. In the group who immigrated less than 12 years ago, a significantly lower BMD was found. PMID- 8630514 TI - Body composition and energy expenditure in thyroidectomized patients during short term hypothyroidism and thyrotropin-suppressive thyroxine therapy. AB - Thyroid hormone levels are a major determinant of energy balance and are thought to modify body composition by their effects on metabolism of lipids, carbohydrate and protein. The present study evaluates changes of body composition and basal energy expenditure (BEE) in thyroidectomized patients studied during short-term profound hypothyroidism while off all thyroid hormone before diagnostic whole body (131)I-imaging and while on thyrotropin-suppressive thyroxine therapy. Basal energy expenditure was assessed by indirect calorimetry, and four-point body impedance analysis was used to estimate body composition. Patients were compared with healthy controls matched with respect to sex, age, height and weight. Compared to healthy controls the percentages of body water and body cell mass were significantly lower while the percentage of fat was significantly higher in patients during short-term hypothyroidism. Weight did not change significantly when patients were put on thyroxine treatment, but body fat (-0.95 +/- 2.25 kg, p < 0.01) decreased while body water (+0.94 +/- 1.31 kg, p < 0.01) and body cell mass (+0.9 +/- 2.5 kg, p < 0.05) increased. With thyroxine replacement, body composition was not significantly different between patients and controls. Compared to healthy controls, BEE was significantly lower in patients without thyroxine replacement (5265 +/- 766 kJ/24h vs 6362 +/- 992 kJ/24h; p < 0.001). With thyroxine treatment, BEE increased (6492 +/- 967 kJ/24h) but was not significantly different from the controls (p > 0.05). Neither body composition nor BEE was significantly different in a subgroup of thyroxine-treated patients with free triiodothyronine or thyroxine values above the normal range. In conclusion, both body composition and energy expenditure showed significant changes when patients were deprived of thyroid hormone. However, no evidence of excess metabolic effects of thyroid hormone during thyrotropin-suppressive thyroxine therapy was found. PMID- 8630515 TI - Secretory rhythm of vasopressin in healthy subjects with inversed sleep--wake cycle: evidence for the existence of an intrinsic regulation. AB - The objective of this paper was to find out if the higher night levels of vasopressin described in previous studies are a manifestation of a permanent and stable rhythm bound to the different periods of the day or if they are independent of them and due to other causes. Vasopressin secretion was studied in a group of seven healthy subjects with an inverted sleep--wake cycle (night workers who sleep and rest during the day). The study was performed during the last week of their working period after at least 3 weeks of continuous night shift. Plasma samples for vasopressin determination were taken every 4 h during a 24-h period while the subjects were performing their normal night work and with their usual sleeping habits during the day. Plasma osmolality, electrolytes and blood pressure were also assessed during the test. In contrast to previous studies where higher nocturnal values have been reported, we found significantly higher vasopressin levels during the day, giving as a whole a characteristic pattern with the highest vasopressin levels measured at 16.00 h followed by a progressive decrement that reached its nadir at 04.00 h. The total measured secretion of vasopressin was significantly higher during the day than during the night (p = 0.0313). No significant difference was found, on the other hand, between day samples, with the exception of samples taken at 16.00 h and at 12.00 h (p = 0.031). Plasma osmolality and electrolytes were within the normal range during the test and no statistical difference was observed at the various points. It was concluded that the secretion of vasopressin is higher during sleep and rest time and lower during the active part of the 24 h. The secretory pattern of vasopressin is not bound to the different periods of the day as such, nor to variations in plasma osmolality or electrolytes. It seems therefore reasonable to assume that the secretion of vasopressin has an intrinsic daily rhythm that is not related to known regulatory agents but is modulated by other unidentified factors. Several hypotheses are discussed. PMID- 8630516 TI - Low frequency of p53 mutations in human thyroid tumours; p53 and Ras mutation in two out of fifty-six thyroid tumours. AB - OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours. PMID- 8630517 TI - Plasma free insulin-like growth factor I concentrations in growth hormone deficiency in children and adolescents. AB - Serum levels of total insulin-like growth factor I (IGF-I) correlate with growth hormone (GH) secretory status and are a useful parameter in the diagnostic evaluation of GH deficiency. Serum total IGF-I levels represent the combined quantity of free or unbound IGF-I and IGF-I that is bound to specific IGF binding proteins. Free IGF-I (fIGF-I), which is postulated to be the bioactive fraction, accounts for only a small fraction of the total amount. We have recently developed a new immunoradiometric assay (IRMA) for plasma fIGF-I and have investigated fIGF-I in relation to GH status. The simple, non-extraction assay procedure involves the capture of unbound IGF-I by anti-IGF-I antibody coated to polystyrene beads and detection by a radiolabelled anti-IGF-I antibody directed to a separate epitope. Preliminary studies demonstrated that the fIGF-I IRMA does not measure IGF-I that is complexed to IGF-binding proteins and that the equilibrium between the free and bound fractions is not disturbed during the assay. Free IGF-I levels were compared to total IGF-I levels measured in the same IRMA after acid-ethanol extraction of the samples. Normal levels of fIGF-I from infancy through adulthood were found to have a close correlation with total IGF-I levels, with the lowest levels occurring in infancy and peak levels during puberty. Patients with complete GH deficiency had low levels of both fIGF-I and total IGF-I, with 94% and 100% of the levels below the 5ht percentile for age, respectively. On the other hand, approximately 90% of patients with normal IGF binding protein-3 levels among partial GH deficiency and normal short children had free and total IGF-I levels above the 5th percentile for age. These data indicate that the clinical utility of plasma fIGF-I measurements is similar to measurements of total IGF-I in the evaluation of childhood GH deficiency. PMID- 8630518 TI - Effect of growth hormone on follicular fluid androgen levels in patients treated with gonadotropins before in vitro fertilization. AB - Forty normally ovulating women aged 25-38 years from one private and two university in vitro fertilization (IVF) centres were used in this randomized, double-blind, parallel, placebo-controlled study to explore the effect of recombinant human growth hormone (GH) on follicular fluid (FF) levels of steroid hormones, particularly androgens. All the women had tubal factor infertility and were classified as poor responders with at least two previously performed and failed IVF treatments in which less than five oocytes had been retrieved following ovarian hyperstimulation. Growth hormone (GH 0.1 IU/kg body wt per day) or placebo was given as pretreatment during down-regulation with gonadotropin releasing hormone agonist and during stimulation with human menopausal gonadotropin (hMG) according to the randomized protocol. Follicular fluid concentrations of steroids were measured and changes related to the levels of insulin-like growth factor I (IGF-I) and IGF binding proteins 1 and 3 and to the mode of GH administration. Pretreatment with GH, i.e. administration of GH before hMG stimulation only, caused significantly elevated follicular fluid concentrations of estrone, testosterone and dehydroepiandrosterone (DHEA) and higher values for markers of aromatase activity (ratios between estrone and androstenedione and between estradiol-17 beta and androstenedione) than in the placebo group, as well as in the two groups receiving GH during hMG stimulation. The highest values for markers of steroid sulfatase activity (ratios between DHA and DHEA sulfate and between unconjugated and conjugated estrone) were found in the patients pretreated with GH. Positive correlations were found between follicular fluid IGF-I and IGF binding protein 3 on the one hand and androgens on the other. This study showed that the administration of adjuvant GH to women who were poor responders to gonadotropins alters the endocrine/paracrine ovarian response to gonadotropins. PMID- 8630519 TI - Proinsulin, C-peptide, and insulin in normal subjects during an 8-h hyperglycemic clamp. AB - Increased concentrations of proinsulin immunoreactive material (PIM) absolutely or relative to insulin is a characteristic finding in patients with non-insulin dependent diabetes mellitus (NIDDM). The aim of this study was to test if 8 h or mild hyperglycemia (7-9 mmol/l) in healthy subjects could induce a preferential secretion of PIM from B cells. Serum concentrations of insulin, C-peptide and PIM were measured every 10 min during the 8 h of continuous glucose infusion in nine normal-weight healthy subjects without diabetes among their first-degree relatives. After a gradual rise in B-cell peptides, a steady state was reached. From 4 to 8 h no further difference in insulin, C-peptide or PIM concentration was found. Fasting PIM/C-peptide and PIM/insulin ratios of 0.5% and 2.3% increased during the glucose clamp to levels of 1.4% and 7.6%, respectively. Neither testing the regression slope nor comparing individual time points showed any significant difference for the PIM/C-peptide ratio from 2 to 8 h and for the PIM/insulin ratio from 3 to 8 h. These results do not support the hypothesis that an increased glucose drive per se results in an altered B-cell function with increasing PIM/C-peptide ratio. At least 8 h of mild hyperglycemia in healthy subjects does not progressively alter B-cell function. PMID- 8630520 TI - Relationship between serum dehydroepiandrosterone sulfate, androstenedione, and sex hormones in men and women. AB - Previous reports of a correlation between serum dehydroepiandrosterone sulfate (DHEAS) and testosterone in both men and women have led to the suggestion that adrenal and gonadal secretion are related. In the present study, the correlation of DHEAS with testosterone and free testosterone (FT) in both normal men and women was tested. Androstenedione, estradiol, sex hormone binding globulin (SHBG), and insulin were also measured and their correlations determined. All correlations were controlled for age and body mass index. In the men in the study, DHEAS did not correlate with testosterone or FT but correlated strongly with androstenedione. In the women, DHEAS correlated strongly with testosterone, FT, and androstenedione; androstenedione in turn correlated strongly with testosterone and FT. DHEAS showed no correlations with estradiol, SHBG, or insulin in the men or women. The lack of a correlation between DHEAS and testosterone in normal men is consistent with the independent secretion of these hormones by the adrenal and testis, respectively. The finding of a strong DHEAS testosterone correlation in normal women may be explained by parallel adrenal secretion in response to trophic stimuli, i.e., without invoking an adrenal gonadal interaction. PMID- 8630521 TI - Effect of modified brain histamine contents on prolactin and thyrotropin secretion in male rats. AB - Effects of modified brain histamine contents on thyrotropin and prolactin secretion were studied in male rats. Under basal conditions the histamine content in the hypothalamus was approximately 8-10-fold higher than that in the striatum and the rest of the brain. L-histidine (1000 mg/kg, ip), a histamine precursor, and metoprine (20 mg/kg, ip), an inhibitor of histamine methyltransferase, elevated histamine content in the brain by 65% and 167%, respectively. When the treatments were given together an additive effect (119-250% increase) on brain histamine was observed. Metoprine significantly decreased serum prolactin levels, while L-histidine had no effect. This effect of metoprine was not modified by treatment with L-histidine. Thus, metoprine has an inhibitory effect on prolactin secretion that is not related to elevated brain histamine contents. The increased brain histamine content after L-histidine treatment had no effect on prolactin secretion. Basal levels of serum thyrotropin were decreased by both L-histidine and metoprine, L-histidine being more potent. In rats treated with alpha fluoromethylhistidine, an inhibitor of L-histidine decarboxylase, the cold induced (rats kept for 60 min at +4 degrees C) thyrotropin secretion was increased while the stress-induced prolactin secretion was decreased. In these rats, metoprine did not affect thyrotropin release but blunted the prolactin response. In conclusion, endogenous histamine inhibits thyrotropin secretion but does not affect prolactin release. Owing to its other effects, metoprine is not suitable as a tool to elevate endogenous histamine contents in the brain, at least when the regulation of anterior pituitary hormone release is being studied. PMID- 8630522 TI - Response of triiodothyronine-dependent enzyme activities to insulin-like growth factor I and growth hormone in cultured rat hepatocytes. AB - Triiodothyronine (T3) is involved in the regulation of the growth hormone-insulin like growth factor I (GH-IGF-I) axis. In this study we investigated the effect of GH and IGF-I on the metabolic response of T3 in target tissues by evaluating the activity of two T3-dependent liver enzymes: mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in rat hepatocytes in primary culture. Growth hormone (35 nmol/l) as well as IGF-I (0.5 mumol/l) reduced alpha-GPD and ME activities (p < 0.01) compared to the control group. Timecourse studies indicated that IGF-I (1.5 mumol/l) significantly decreased alpha-GPD and ME activities (P < 0.01) after 24 h, whereas the effect of GH (35 nmol/l) was recorded only after 36 h (p < 0.01). This delayed effect of GH compared to IGF-I suggested the possibility that the effect of GH could be mediated by IGF-I synthesis. To test this hypothesis, the effect of GH on the two enzyme activities was studied in the presence of anti-IGF-I antibodies. A gradual recovery of alpha-GPD and ME activities (p < 0.01) was observed in the presence of GH (35 nmol/l) plus increasing concentrations of anti-IGF-I antiserum. The maximal alpha-GPD and ME activities attained after the incubation of the liver cells with 1 mumol/l T3, a concentration high enough to fully saturate the nuclear T3 receptors for 24 h, were lowered significantly by 1.0 mumol/l IGF-I (p < 0.01). This finding suggests that the IGF-I effect might be independent of the saturation of the nuclear T3 receptors. In conclusion, in cultured rat hepatocytes, GH and IGF-I reduced the metabolic response of T3 evaluated by two liver T3-dependent enzyme activities. The effect of GH was mediated at least in part by IGF-I. PMID- 8630523 TI - Effects of 5, 5'-diphenylhydantoin on the thyroid status in rats. AB - Treatment of rats with phenytoin (DPH), an anti-epileptic drug, results in lower tissue thyroid hormone (TH) levels. To investigate if this is accompanied by tissue hypothyroidism, rats were treated for 3 weeks with DPH (50 mg/kg body wt in food). Thyroid hormone-dependent parameters were measured, and the results were compared to those of control rats and to those of athyreotic rats substituted with thyroxine + triiodothyronine (Tx + TH) to reach the same plasma TH levels as DPH-treated rats. These rats were mildly hypothyroid with regard to their TH and TSH levels and TH-dependent parameters. Both DPH and Tx + TH led to a decrease in plasma thyroxine (T4) and triiodothyronine (T3) (+/-70% of the control). The percentage free T4 was unchanged. Plasma thyrotropin (TSH) was increased only in the Tx + TH rats (sixfold). For DPH rats, pituitary hormone content was not different from the control; growth hormone was lower and TSH was higher in Tx + TH rats. In DPH and Tx + TH rats, an increase in hepatic T4 and T3 uridine-diphosphate glucuronyltransferase activity was found, likewise indicating a change in the metabolic pathway of TH. Hepatic iodothyronine deiodinase (ID) type I activity decreased in Tx + TH rats but did not alter in DPH rats. Hepatic alpha-glycerophosphate dehydrogenase (alpha-GPD) decreased in DPH and Tx + TH rats. Malic enzyme in liver was enhanced in DPH rats. In the brains of DPH rats the level of alpha-GPD activity was raised; in Tx + TH it was lowered. The ID type II activity in the brain was reduced in DPH rats, but ID type III did not change for either group. Total body oxygen consumption increased in DPH rats (13%); it decreased in Tx + TH rats (9%). Our results show that DPH causes changes comparable to mild hypothyroidism. The lack of or a diminished hypothyroid response can be explained as the attenuating agonistic effect of DPH, which is supported by O2 consumption, brain ID type II and alpha-GPD activities. The T4 content was reduced by 30% in thyroid digests; this, together with a reduced T4 secretion, can lead to serious hypothyroxinemia during prolonged DPH treatment. PMID- 8630524 TI - Restoration of human chorionic gonadotropin response in human myometrial smooth muscle cells by treatment with follicle-stimulating hormone (FSH): evidence for the presence of FSH receptors in human myometrium. AB - Human myometrial smooth muscle cells contain receptors for human chorionic gonadotropin (hCG)/luteinizing hormone (LH). Exogenous hCG and LH can cause a modest hyperplasia in myometrial smooth muscle cells in culture. This response is lost after about the third subculture of the cells. The present study investigated whether the loss of hCG response could be restored by co-culturing with human follicle stimulating hormone (FSH). The results showed that co culturing with FSH can indeed restore a modest mitogenic response of hCG. However, FSH alone was not mitogenic. The FSH restoration of hCG response can be blocked by antibodies to FSH or hCG but not by non-specific rabbit IgG. The FSH treatment resulted in an increase of steady state levels of hCG/LH receptor mRNA and protein in myometrial smooth muscle cells. Since the FSH actions could be receptor mediated, we investigated the presence of FSH receptor mRNA transcripts and protein in freshly dispersed myometrial smooth muscle cells. Northern blotting demonstrated that myometrial smooth muscle cells, just as rat ovary, a classical target of FSH action, contain multiple FSH receptor mRNA transcripts. Western immunoblotting demonstrated that myometrial smooth muscle cells also contain a 60 kDA FSH receptor protein just as rat ovary and human granulosa cells used as positive control tissues. The immunocytochemistry also demonstrated that myometrial smooth muscle cells, as rat ovary and human granulosa cells, contain FSH receptor immunostaining. In summary, it is novel that FSH could restore the mitogenic response of hCG in human myometrial smooth muscle cells and these cells contain FSH receptors. These findings may have functional implications for direct regulation of human myometrium not only by hCG/LH but also by FSH. PMID- 8630525 TI - Androgen-dependent sex differences in the hypothalamic serotoninergic system. AB - This study has attempted to fulfill our knowledge on the sex differences in the hypothalamic serotoninergic (5-HT) system in adult rats, and also to evaluate the role of neonatal androgens in the appearance of this sexual dimorphism. Such integrative characteristics of the 5-HT system as 5-HT content and specific uptake were estimated and compared in the anterior and middle hypothalami in intact adult females and males, as well as in neonatally castrated adult males. According to our data, the 5-HT content and [3H]5-HT specific uptake both in the anterior and middle hypothalami of intact females exceeded significantly those in intact males. Neonatal castration of males resulted in an increase of the 5-HT content and [3H]5-HT uptake in both hypothalamic regions up to their levels in intact females. Thus, the present study provides new information on the sex differences in the hypothalamic 5-HT system, which are apparently related to the neonatal masculinization of the hypothalamus. PMID- 8630526 TI - Progesterone together with estradiol promotes luteinizing hormone beta-subunit mRNA stability in rat pituitary cells cultured in vitro. AB - The present study examined the role of ovarian steroids, estradiol and/or progesterone in the regulation of luteinizing hormone beta-subunit (LH-beta) mRNA levels and LH release in the rat anterior pituitary cells cultured in vitro. When estradiol (10 nmol/l) and/or progesterone (100 nmol/l) were added to the cultures, neither estradiol or progesterone nor both together altered the basal LH-beta mRNA levels or LH release. Continuous exposure to gonadotropin-releasing hormone (GnRH, 0.2 nmol/l) for 24 h markedly induced LH-beta mRNA accumulation, and in this experimental condition, progesterone alone and progesterone + estradiol further augmented GnRH-induced LH-beta mRNA levels and LH release. Then we explored further the possibility that ovarian steroids are involved in modulating LH-beta mRNA stability in cultured rat pituitary cells where transcription was inhibited by actinomycin D. Anterior pituitary cells were preincubated with GnRH (0.2 nmol/l) for 16 h and, after removing GnRH from culture medium, the cells were incubated further in the presence of actinomycin D (5 mumol/l) for 24 h. The LH-beta mRNA levels gradually declined to about 30% of the control values (zero time point after GnRH removal) in a time-dependent manner. During this period, either progesterone alone or progesterone + estradiol clearly blocked the degradation of LH-beta mRNA species. These results indicate that ovarian steroids promote LH-beta mRNA stability, thereby contributing to the maintenance of GnRH-stimulated LH-beta mRNA levels. PMID- 8630527 TI - Possible contribution of dopaminergic receptors in the anteroventral third ventricular region to hyperosmolality-induced vasopressin secretion in conscious rats. AB - We have reported previously that regions encompassing the cerebral ventricle may contain dopamine receptors responsible for facilitatory roles in the osmotic release of vasopressin in conscious rats. In order to explore the location of these receptors, we injected (0.5 mul) the dopamine antagonist haloperidol (13.3 nmol) or dopamine (26.4 nmol) topically into the anteroventral third ventricular region or the paraventricular nucleus of rats, and their effects on the levels of plasma vasopressin and its controlling factors were examined in the presence or absence of an osmotic stimulus. The effects of haloperidol injections into the ventral tegmental area were also tested to study whether information associated with drinking behavior may affect the osmotic vasopressin secretion. Intravenous infusion (0.1 ml kg-1 body wt min-1) of hypertonic saline (2.5 mol/l) enhanced plasma vasopressin 15 and 30 min later, and this was accompanied by an augmentation of plasma osmolality, sodium and chloride, and by elevated or unaltered arterial pressure. The vasopressin response was abolished by haloperidol injection into the anteroventral third ventricular region 10 min before the beginning of the hypertonic saline infusion. The injection sites were confirmed histologically to have been in or near the organum vasculosum of the laminae terminalis and a ventral part of the median preoptic nucleus. Similarly, a partial but significant reduction of the vasopressin response was noted after bilateral injections of haloperidol into the ventral tegmental area, whereas bilateral haloperidol injections into the paraventricular nucleus had no appreciable effect. The responses of plasma osmolality, electrolytes and arterial pressure to the osmotic load were not affected significantly by haloperidol injections into the anteroventral third ventricular region, ventral tegmental area or the paraventricular nucleus. The iv infusion of isotonic saline (0.15 mol/l) did not change plasma vasopressin and the other variables significantly, and this was also the case when preceded by application of haloperidol into the anteroventral third ventricular region, ventral tegmental area or the paraventricular nucleus. Dopamine injection into the anteroventral third ventricular region increased plasma vasopressin 5 min later, without affecting plasma osmolality, electrolytes or arterial pressure. On the basis of these results, we concluded that dopamine receptors responsible for facilitatory roles in osmotically stimulated vasopressin secretion may exist in the anteroventral third ventricular region and ventral tegmental area. PMID- 8630528 TI - Interleukin-1 beta-induced nitric oxide production from isolated rat islets is modulated by D-glucose and 3-isobutyl-1-methyl xanthine. AB - Interleukin-1 beta has been proposed to cause selective beta-cell destruction via the induction of nitric oxide synthesis. The cytotoxic effect of interleukin-1 beta is modulated by the concentration of D-glucose in the medium. The aim of this study was to investigate if D-glucose-mediated modulation of interleukin-1 beta effects on insulin release from isolated rat islets was related to modulation of nitric oxide production. Further, we wished to investigate the effects of agents increasing the intracellular concentration of cAMP on interleukin-1 beta-induced nitrite production. We demonstrated that D-glucose potentiated interleukin-1 beta-induced nitrite production in rat islets without affecting the mRNA level of the inducible nitric oxide synthase. This effect was dissociated from interleukin-1 beta action on insulin release, since a relative protection against interleukin-1 beta effects on acute insulin release was found at high (28 mmol/l) concentrations of D-glucose, and blocking nitrite production by the L-arginine analog aminoguanidine, which selectively inhibits the cytokine inducible nitric oxide synthase, did not result in protection against the inhibitory action of interleukin-1 beta. Neither L-glucose nor the secretagogues L-leucine, tolbutamide and 3-isobutyl-1-methyl xanthine shared the potentiating effect of D-glucose. The phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine reduced interleukin-1 beta-induced nitrite production at 3.3 mmol/l D-glucose, an effect that could be reproduced by the cAMP analog dibutyryl cAMP. Addition of 3 isobutyl-1-methyl xanthine resulted in a threefold reduction in the mRNA level of interleukin-1 beta-induced inducible nitric oxide synthase. We conclude that interleukin-1 beta-induced islet nitric oxide synthesis is augmented by D glucose, but not by non-substrate secretagogues, and that secretagogues that elevate cAMP inhibit islet nitric oxide production. PMID- 8630529 TI - A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species. AB - Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well studied, medically-important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non-vector species, and two or more member species are often found sympatrically. Until the late 1950, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes. A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species-specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA-based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA-based identification techniques have been developed, including hybridization assays based on species-specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species-specific sequences. In this review we discuss the relative marks of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species diagnostic PCR assays. PMID- 8630530 TI - Molecular characterization of the Anopheles gambiae 2L telomeric region via an integrated transgene. AB - A Drosophila P-element derivative (pUChsneo) integrated into the telomeric region of the left arm of the second chromosome of Anopheles gambiae was used to clone the proximally flanking An. gambiae sequences. Molecular analyses revealed that the pUChsneo construct was partially duplicated and had integrated into a subterminal minisatellite. This satellite has a repeat unit of 820 bp and is located exclusively at the tip of 2L. No sequence similarity to subterminal minisatellites from other dipterans was detected, but some structural features such as tandem subrepeats are shared. The end of the chromosome was mapped with respect to restriction sites in pUChsneo at approximately generation 100 after the integration event. Considering inevitable terminal nucleotide loss due to incomplete DNA replication, we conclude that the chromosome end must have undergone a dramatic elongation process since it was mapped in generation 23. PMID- 8630531 TI - Comparison of preservation techniques for DNA extraction from hymenopterous insects. AB - Two species of parasitic wasp, Venturia canescens and Leptomastix dactylopii, were killed and preserved by various methods used for Hymenoptera and in mass collecting devices. Total genomic DNA was subsequently extracted and a 524 bp fragment of the mitochondrial 16S ribosomal RNA gene amplified by PCR. Results for these techniques were compared with that for fresh material and museum specimens. Material from -80 degrees C, 100% ethanol, air-drying in a desiccator, and critical-point dried from alcohol all yielded good results after short and long-term storage, as did specimens from ethylene glycol but not formalin (the latter two being commonly used in pitfall and flight intercept traps). Specimens killed in ethyl acetate vapour and air-dried yielded very degraded DNA which did not successfully PCR. The use of this killing agent is a likely reason for previous reports of inconsistent results obtained from museum specimens, and the now widespread use of critical-point drying of wasps and other insects from alcohol is advocated as a potential source of DNA from rare taxa. PMID- 8630532 TI - Aedes aegypti midgut early trypsin is post-transcriptionally regulated by blood feeding. AB - Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal. PMID- 8630533 TI - Primary structure of ribosomal proteins S3 and S7 from Manduca sexta. AB - We have isolated from Manduca sexta full-length cDNAs encoding proteins homologous to human ribosomal proteins S3 and S7. These are the first ribosomal protein sequences obtained from non-Dipteran insects. M. sexta ribosomal protein S3 has a molecular mass of 26,715 Da. Ribosomal protein S7 has a mass 21,870 Da. Both are basic proteins, with abundant Lys and Arg residues that may interact with ribosomal RNA in the ribosome. Southern blot hybridization suggests the presence of single genes for both ribosomal proteins in the M. sexta genome. Alignments with other S3 and S7 sequences available In the database indicate regions of the ribosomal proteins that have been the most highly conserved in evolution and may point to important functional regions in the proteins. Ribosomal protein S3 appears to be more highly conserved then ribosomal protein S7. This may be due to greater constraints on the structure of S3 because of its dual functions in translation as a ribosomal protein and in DNA repair in the nucleus. PMID- 8630534 TI - Overlapping Lsp-2 gene sequences target expression to both the larval and adult Drosophila fat body. AB - Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster encodes one of the major hexameric haemolymph proteins of third-instar larvae and a major component of adult serum. Regulated transcription of Lsp-2 results in high-level, ecdysone stimulated expression throughout the larval fat body and low-level, spatially restricted expression in the adult fat cells. To localize cis-acting regulatory sequences responsible for the stage- and tissue-specific activity at Lsp-2, the expression of Lsp-2-lacZ fusion genes was studied by P element-mediated germline transformation of Drosophila. A 230 base pair larval enhancer, which includes an ecdysone response element (EcRE), specifically targets gene activity to the larval fat body. Although the adult mode of Lsp-2 expression depends on the larval enhancer, additional negative regulatory elements dictate both tissue specificity and unique spatial restriction within the adult fat body. Implications of these findings for the identification of fat body-specific gene regulatory units in other insects are discussed. PMID- 8630536 TI - Isolation and characterization of three serine protease genes in the mosquito Anopheles gambiae. AB - Three genes encoding serine proteases (Sp6A, Sp6T and Sp8T) were isolated from the malaria mosquito An. gambiae. The proteins that are conceptually translated from these genes contain all amino acids that have been described for this class of proteolytic enzymes, namely the His, Asp and Ser residues at the active site, and the six cysteine residues that form the three disulphide bridges in invertebrate serine proteases. The genes are expressed at low levels and the transcripts were detected only by PCR. Analysis of the nucleotide sequences of the three genes and their pattern of expression indicate that none of the genes code for digestive enzymes, but rather that the proteins have features of the tethered type of serine proteases. PMID- 8630535 TI - Evolution of the mitochondrial DNA control region in the Anopheles gambiae complex. AB - We have sequenced the AT-rich control region of the mitochondrial DNA (mtDNA) of six species in the Afrotropical Anopheles gambiae complex and the closely related A. christyi. Contrary to expectations, the AT-rich region in this group is evolving rather slowly, more slowly than the third position of mtDNA protein coding genes. Despite being relatively conserved between species, we detected intraspecific and intra-individual (heteroplasmy) variation in this region. Phylogenetically, we found we could place the rare endemic A. bwambae as a sister taxon to A. melas, the same evolutionary position as indicated by chromosomal inversions. The outgroup, A. christyi, gave evidence of the root of the tree. In comparing the molecular trees with that deduced by chromosomal inversions, they are completely congruent with the exception of the placement of A. arabiensis. The anomalous position of this species can be explained by introgression with A. gambiae. From the phylogenetic position, we could infer mtDNA gene flow from A. gambiae to A. arabiensis. PMID- 8630537 TI - Precise limitation of concerted evolution to ORFs in mosquito Hsp82 genes. AB - Two Hsp82 genes were isolated from the malaria vector Anopheles albimanus in a single lambda phage clone. The two genes are in a head-to-head arrangement separated by approx. 0.9 kbp. Northern hybridizations and 5' RACE demonstrate that both genes are transcribed, have moderate levels of constitutive transcription, and are also heat-inducible with maximum transcript accumulation occurring after 40 degrees C heat shocks. Both genes have typical heat-shock promoters and conserved intron boundaries in the untranslated leaders. The open reading frames are 99.6% identical differing in only nine silent nucleotide positions in the 2166 bp ORFs. However, precisely outside the ORFs, the flanking DNA of the two genes shows no evidence of common derivation. The high degree of identity between the two ORFs appears to be a result of gene conversion occurring by a process similar to that previously suspected in the A. albimanus Hsp70 genes and several D. melanogaster genes arranged as palindromes. This process probably involves a stem-loop intermediate and is restricted in extent by flanking sequence divergence. These Hsp82 genes clearly demonstrate the extreme precision with which gene conversion can lead to protein-coding-region homogeneity yet allow flanking DNA divergence. PMID- 8630538 TI - Noninvasive positive-pressure ventilation in acute respiratory distress without prior chronic respiratory failure. AB - We evaluated the efficacy of noninvasive mechanical ventilation (NIMV) in alleviating distress and avoiding intubation in patients with de novo acute respiratory failure complicating primary medical disorders. Eleven consecutive patients with severe respiratory distress were entered. In all patients a decision to intubate on an urgent basis had been made, but NIMV could be initiated within minutes. The patients suffered from acute pulmonary edema (five), sepsis/ARDS (two), status asthmaticus (two), and severe pneumonia (two). Dyspnea score (max=10) was (+/- SD) 8.4 +.- 1.6, scale for accessory muscle use (max=5) was 4.2 +/- 0.7, and respiratory rate was 37.6 +/- 3.8 min -1. Pa CO2, pH, and base excess (BE) were 48 +/- 18 mm Hg, 7.27 +/- 0.13, and -5.5 +/- 7.4, respectively, with five patients showing severe metabolic acidosis (BE < - 10). NIMV was applied using proportional assist ventilation. There were three early failures. These included the two patients with sepsis/ARDS who did not tolerate the mask. One patient failed because Pa CO2 and pH deteriorated despite subjective improvement. The remaining eight patients demonstrated progressive improvement, and none required intubation. The duration of NIMV was 3 h to 2 d. We conclude that when NIMV is made available on a "few minutes" basis, selected patients with severe de novo respiratory distress/failure caused by reversible medical disorders, who would otherwise have been intubated, can be given substantial relief and be spared intubation. PMID- 8630539 TI - Sedation of critically ill patients during mechanical ventilation. A comparison of propofol and midazolam. AB - Propofol (P) and midazolam (M) are frequently given by continuous infusion for sedation in critically ill, mechanically ventilated patients. We compared these drugs with regard to: (1) time-to-awaken; (2) reproducibility of bedside assessments of level of sedation; (3) time-to-sedation; and (4) change in oxygen consumption (V O2) from awake to sedated state. Seventy-three patients were prospectively randomized to receive either P (n=37) or M (n=36). Wake-up times after stopping the drug were assessed by blinded and unblinded observers, by asking patients to perform simple tasks. Times to sedate were assessed by consensus agreement among nurses and investigators. Demographics and APACHE II scores were not different between P and M. The P group had a significantly narrower range of wake-up times with a higher likelihood of waking in less than 60 min. Blinded versus unblinded observations had excellent correlation. Average time to sedate and decrease in V O2 were not different. We conclude that in this patient population: (1) both P and M achieved optimal sedation in a large fraction of patients when administered by specified dosing protocols; (2) P had a faster, more reliable, wake-up time; (3) assessments of time-to-awaken were objective and reproducible; (4) time to sedation was not significantly different; (5) V O2 decreased similarly with both. PMID- 8630540 TI - Tracheal gas insufflation-pressure control versus volume control ventilation. A lung model study. AB - Tracheal gas insufflation (TGI) has been recommended as an adjunct to mechanical ventilation in the presence of elevated Pa CO2. Based on our initial clinical experience with continuous flow TGI and pressure control ventilation (PCV), we were concerned about elevation in peak airway pressure as TGI was applied. In a lung model, we evaluated the effects of continuous flow TGI during both PCV and volume control ventilation (VCV). A single compartment lung model was configured with an artificial trachea into which an 8-mm endotracheal tube was positioned. TGI was established with a 16-G catheter positioned 2 cm beyond the tip of the endotracheal tube. Ventilation was provided by a Puritan-Bennett 7200ae ventilator with PCV 20 cm H2O or VCV with a tidal volume (VTt) similar to that with PCV. A rate of 15 breaths/min and PEEP of 10 cm H2O were used throughout. Inspiratory times (TI) of 1.0, 1.5, 2.0, and 2.5 s were used with TGI of 0, 4, 8, and 12 L/min. Lung model compliance (ml/cm H2O) and resistance (cm H2O/L/s) combinations of 20/20, 20/5, and 50/20 were used. Auto-PEEP, VT, and peak alveolar and airway opening pressures increased as TGI and Ti increased, regardless of lung mechanics settings (p<0.01). All increases were greater with VCV than PCV (p<0.05). Continuous flow TGI with both PCV and VT-uncorrected VCV may result in marked increases in Vt and system pressures, especially at long TI. PMID- 8630541 TI - Effects of assisted ventilation on the work of breathing: volume-controlled versus pressure-controlled ventilation. AB - During assisted ventilation, the same tidal volume can be delivered in different ways, with the possibility for the physician to vary the ventilatory target (pressure or volume) and the peak flow setting. We compared the effects on the respiratory work rate of assisted ventilation, delivered either with a square wave flow pattern (assist control ventilation [ACV]) or with a decelerating flow pattern and a constant pressure (assisted pressure-control ventilation [APCV]). In the first part of the study where seven patients were studied, inspiratory time and tidal volume were similar in the two modes of ventilation. High and moderate levels of tidal volume (VT) were studied (12 ml/kg and 8 ml/kg, respectively). To obtain moderate VT, inspiratory time was kept constant and, therefore, mean inspiratory flow was reduced. At high VT, no difference between ACV and APCV was noted for breathing pattern, respiratory drive indexes, respiratory muscle work, or arterial blood gases. All patients exhibited respiratory alkalosis. At moderate VT, normal pH was achieved. In this situation significantly lower levels were observed during APCV than during ACV for the power of breathing (10 +/- 2 versus 19 +/- 5 J/min, p<0.05), transdiaphragmatic pressure swing (7 +/- 1 versus 11 +/- 2 cm H2O, p<0.05), and pressure-time index (252 +/- 43 versus 484 +/- 114 cm H2O.s, p<0.05), even though breathing pattern and gas exchange were similar. In the second part of the study where six additional patients were studied, tidal volume was kept constant at a moderate level (8 ml/kg), and we studied the effect of shortening inspiratory time and increasing mean inspiratory flow. At moderate VT and high inspiratory flow, no significant differences could be found between ACV and APCV, and although pressure-time index tended to be lower during APCV, absolute levels of effort were of small magnitude (56 +/- 55 versus 76 +/- 55 cm H2O.s). We conclude that at moderate VT and low flow rates only, inspiratory assistance delivered at a constant pressure reduces the respiratory work rate more effectively than assist control ventilation. PMID- 8630542 TI - O2 supply dependence of respiration in patients with mitral stenosis undergoing valvuloplasty. AB - Although systemic oxygen consumption (V O2) is independent of O2 delivery (Q O2) in normal subjects, studies have suggested that supply dependence of V O2 may occur in patients with chronic diseases associated with reduced Q O2. In this regard, we previously found that Q O2 and V O2 increased when cardiac output was improved after balloon valvuloplasty in patients with aortic stenosis. However, their increases in Q O2 were relatively small, and it was not known whether the increase in V O2 was caused by the increase in delivery or was merely a response to the transient hypotension induced by valvuloplasty. Because patients with mitral stenosis frequently exhibit greater improvements in cardiac output after valvuloplasty than do patients with aortic stenosis, the present study sought to determine (1) whether V O2 is increased after valvuloplasty in patients with mitral stenosis, and (2) whether the magnitude of the increase in V O2 correlates with the magnitude of the improvement in cardiac output and Q O2. Oxygen delivery, V O2, and hemodynamics were measured in 57 patients with mitral stenosis before and 20 to 30 min after undergoing balloon valvuloplasty. After valvuloplasty, Fick-derived oxygen delivery increased by 13.0% (95% confidence interval: 10.8 to 15.2%), whereas V O2 (expired gas) increased by 8.3% (95% confidence interval: 9.5 to 12.3%). A correlation between Fick-derived Q O2 and V O2 was found (p<0.005) with a slope of 0.66 (95% confidence interval: 0.07 to 1.24), but the O2 extraction ratio did not change (-1.0%; 95% confidence interval: -2.7 to 0.5%). A significant correlation between the change in Q O2 and the change in V O2 was also seen (p<0.02). These findings suggest that the increase in V O2 may have been a consequence of the increase in Q O2 rather than a response to the procedure itself. PMID- 8630543 TI - Flow cytometric analysis of lung lymphocytes in lung transplant recipients. AB - Lung transplantation is an accepted therapeutic modality in end-stage lung disease. Presently, histologic examination of tissue by transbronchial biopsy remains as the definitive diagnostic procedure for determining rejection. To begin addressing the usefulness of flow cytometric analysis of bronchoalveolar lavage fluid (BALF) in acute lung rejection, we prospectively studied the expression of markers on lymphocytes from BALF samples removed from 10 lung and heart-lung transplant recipients and compared their pattern with that of BALF lymphs obtained from normal volunteers (Norm) and nonrejecting heart transplant recipients (HT) who were receiving similar immunosuppressive regimens. Compared with both Norm and HT subjects, CD4+ lymphocytes in the BALF of lung transplant recipients was significantly reduced. A greater percentage of the CD4+ lymphocytes in nonrejecting lung transplant subjects also expressed the interleukin-2 receptor, but only during the early post-transplant period, suggesting possible reactivity to persistent donor cells. However, the CD8+ lymphocytes were increased only in lung transplant recipients undergoing acute lung rejection. We conclude that the immunologic milieu is indeed altered in the transplanted lung. Further studies in lung transplant recipients are required to evaluate the role of flow cytometry in the early detection of acute lung rejection. PMID- 8630544 TI - Heat-killed Corynebacterium parvum enhances endotoxin lung injury with increased TNF production in guinea pigs. AB - Corynebacterium parvum (CP) is known to increase susceptibility to endotoxin, which is associated with increased production of tumor necrosis factor (TNF). We investigated the effect of CP-priming on the pathogenesis of acute lung injury caused by intratracheal Escherichia coli endotoxin (lipopolysaccharide [LPS]). Guinea pigs were divided into four groups: (1) control (n=6), (2) CP-alone (n=6), (3) LPS-alone (n=6) and (4) CP + LPS (n=6). A CP dose of 4 mg/kg was injected intraperitoneally 7 d before the study. Animals were observed for 4 h after intratracheal administration of 0.02 mg/kg of LPS. The lung wet-to-dry weight ratio (W/D), [125I] albumin concentration ratio of lung tissue to plasma (T/P) and of bronchoalveolar lavage (BAL) fluid to plasma (B/P) and differential cell count in BAL fluid were examined. In the LPS-alone group, neither excess lung water nor increased albumin leakage was observed. The CP + LPS group showed increased lung water and albumin leakage as compared with the other three groups (p<0.05). We also observed increased cell counts in BAL fluid (p<0.05), in the CP + LPS group. The spleen weight was increased in guinea pigs pretreated with CP, indicating reticuloendothelial system (RES) activation. In the CP + LPS group, the TNF level was increased in both plasma and BAL fluid. We conclude that pretreatment with CP enhances LPS-induced acute lung injury in parallel with increasing TNF production, which suggests that the activation of mononuclear phagocytes contributes to increased susceptibility to intratracheal endotoxin in guinea pigs. PMID- 8630545 TI - Tobacco smoking induces expression of very-high-affinity nicotine binding sites on blood polymorphonuclear cells. AB - The targets of nicotine in the central nervous system (CNS) have been identified as nicotinic acetylcholine (ACh) receptors. Because of their localization in the brain, no data are available about their in vivo modifications. However, nonneuronal nicotine binding sites have been identified on peripheral blood cells. The present study was designed to evaluate the effects of tobacco smoking on granulocyte nicotine binding sites. Binding assays were performed on granulocyte membranes using (-)(3H)nicotine and were analyzed by the Scatchard method in nonsmokers (n=10), smokers (n=10), and ex-smokers (n=10). In nonsmokers, a single-affinity binding site was detected with Kd = 1.08 +/- 0.05 x 10 -9 M. In contrast, in smokers, two different classes of binding sites were identified: one with Kd1 = 2.80 +/- 0.81 x 10 -11 M and another with Kd2 = 3.92 +/- 0.58 x 10 -9 M, similar to the binding site in nonsmokers. Moreover, a twofold increase in nicotine binding-site density was observed in smokers. Ex smokers recovered a single class of binding sites similar to those observed in nonsmokers when they had stopped smoking for more than 1 yr, whereas recent ex smokers (1 to 8 mo) displayed a pattern similar to that in smokers, as demonstrated by the persistence of two classes of nicotine binding sites. Chronic nicotine exposure induces modifications of nicotine binding sites on granulocytes. It is noteworthy that the persistence of such alterations corresponds to the very high rate of relapse into smoking observed during the early stages of tobacco cessation. Consequently, monitoring of nicotine binding sites could constitute an interesting approach to understanding the pharmacologic mechanisms involved in tobacco dependence. PMID- 8630546 TI - Tolerance to phosgene is associated with a neutrophilic influx into the rat lung. AB - Exposures to 100% oxygen, ozone, nitrogen oxides, and phosgene increase both lung lavage protein concentrations and neutrophils. The inhibition of the neutrophil influx can diminish lavage protein concentrations after exposures to these oxidant gases. Similarly, this injury can be reduced by pre-exposure to either the same (tolerance) or a different (cross-tolerance) oxidant gas. We tested the hypothesis that diminished injury after the development of tolerance of phosgene (COCl2) is associated with a decreased incursion of neutrophils. Sixty-day-old rats (n=12/group) were exposed to varying concentrations of COCl2. Lung lavage (n = 6/group) 24 h after a first phosgene exposure demonstrated an increase in both protein concentrations and percentage neutrophils. The remaining animals (n = 6/group) were exposed to COCl2 2 ppm x 60 min 1 wk later. Lavage confirmed the development of tolerance with protein concentrations diminished after the second exposure in those rats that had inhaled higher doses of COCl2 during the first exposure. However, the neutrophilic influx was not diminished but rather was increased. The association of the neutrophil incursion with a protective effect was further established in studies employing colchicine and dextran. Colchicine decreased neutrophil influx occurring after the first exposure and subsequently diminished the development of tolerance after a second exposure. Intratracheal instillation of dextran produced a neutrophil incursion and subsequently decreased injury after a phosgene exposure. In investigations using both colchicine and dextran, neutrophil influx increased with the development of adaptation. Thus, lung injury after the development of tolerance to phosgene provides a unique animal model of a respiratory distress syndrome in which neutrophils are not associated with injury but rather with a protective effect. PMID- 8630547 TI - Immune dysregulation in the aging human lung. AB - Aging has been associated with diminished lung function and increased susceptibility to lung infection. To determine whether changes suggestive of immune dysregulation and inflammation appear in the lungs of clinically normal individuals as a function of advancing age, we performed bronchoalveolar lavage (BAL) on discontinuous age groups (20-36, 45-55, and 65-78 yr old) of clinically normal volunteer subjects. We measured immunoglobulin (IgG, IgA, IgM), albumin, interleukin-6 (IL-6), and interleukin-10 concentrations in BAL fluid. Bronchoalveolar cell profiles, cell surface antigen expression, and superoxide anion production were also measured. A significant increase in total cell concentration, neutrophils, and BAL immunoglobulin content was observed in the oldest age group compared with the youngest age group. Mean lymphocyte subset (CD4+/CD8+) ratios were significantly increased in blood (2.6 +/- 0.4 versus 1.6 +/- 0.1; p<0.03) and to a greater degree in BAL (4.8 +/- 1.0 versus 1.9 +/- 0.2; p<0.01) for the oldest versus youngest age groups. Similarly, BAL-derived cells displayed significantly increased phorbol myristate acetate-stimulated release of superoxide anion (8.8 +/- 1.3 versus 4.5 +/- 0.7 nmol/5 x 10 5 cells/h; p<0.01) for the oldest versus youngest subject group, and mean BAL IL-6 concentrations were significantly elevated in the oldest age group (0.86 +/- 0.13 ng/ml) compared with the youngest age group (0.53 +/- 0.03 ng/ml; p<0.01). Our observations suggest that altered inflammatory cell profiles and low-grade inflammation exist in the lower respiratory tracts of many asymptomatic, clinically normal volunteers of advanced age. PMID- 8630548 TI - Obstructive sleep apnea syndrome and circadian rhythms of hormones and cytokines. AB - Several cytokines exhibit a high degree of temporal regulation as well as somnogenic potency (e.g., interleukin-1 [IL-1], tumor necrosis factor-alpha [TNF alpha]). Seeking the underlying cause of obstructive sleep apnea syndrome (OSAS), we investigated whether circadian rhythms of cytokine release were altered in 10 patients with OSAS. Ten healthy volunteers served as the control population. Seven of the 10 OSAS patients were reexamined after 3 mo of therapy with nasal continuous positive airway pressure (nCPAP) mask ventilation. Circadian cytokine release (IL-1, IL-6, gamma-interferon [gamma-IFN], TNF-alpha) was investigated ex vivo by short-term culture of blood samples. The circadian rhythm of TNF-alpha release was significantly disturbed in OSAS patients: nocturnal physiologic peaks in this cytokine had almost disappeared and an additional daytime peak had developed. Circadian variations in IL-1, IL-6, and gamma-IFN, and in the immunoregulatory hormones melatonin and cortisol, did not differ from those in the controls. Because TNF-alpha is a known modulator of sleep, and nCPAP therapy did not normalize TNF rhythms, we assume that TNF-alpha could well play a pathophysiologic role in OSAS. Further studies should be directed at whether a physiopathologic and/or pathogenic link exists between TNF-alpha and OSAS. PMID- 8630549 TI - Effect of gas density on respiratory sounds. AB - The generation of normal lung sounds by turbulent air flow has been questioned because gas density appears to have only a minor effect. We studied whether gas density has a greater influence on lung sounds at higher frequencies than traditionally measured. Six healthy adult men breathed air followed by a mixture of 80% helium and 20% oxygen (He-O2) at a target flow of 1.5 L/s. Flow and sounds at the trachea and posterior right lower lobe were simultaneously acquired by computer. Fourier analysis was applied to sounds at target flow +/- 0.2 L/s. Average power spectra were computed for each recording site, respiratory phase, and respired gas. He-O2 reduced the power of inspiratory lung sounds below 300 Hz by only 1.7 +/- 1.5 dB whereas power between 300 and 600 Hz was reduced by 4.6 +/ 1.4 dB (p<0.05). Tracheal sound power was reduced less consistently but all subjects showed an upward frequency shift in power maxima on He-O2, similar to formant shifts observed in helium speech. Our findings suggest that flow turbulence is the major determinant of lung sounds at higher frequencies. Current instruments for auscultation and recording of respiratory sounds may have to be modified to optimize their response in this higher frequency range. PMID- 8630550 TI - Tracheal microvascular responses to beta-adrenergic stimulation in anesthetized rats. AB - Previous study of adrenergic control of the tracheal vasculature in rats (1) demonstrated that beta-adrenergic blockade heightened arteriolar and large venular contractile responses to norepinephrine, a nonselective alpha-adrenergic agonist. The present study was undertaken to confirm the presence of functional beta-adrenergic receptors and to determine which beta-adrenergic receptor subtypes mediate vasodilation in this tissue. Tracheal adventitial arterioles (12.0 to 47.0 micro m initial diameter, n=39) and venules (48.0 to 98.5 micrometers initial diameter, n=44) were observed through a video microscope, and vessel diameters were measured. Vessels were preconstricted with 10(-4) M phenylephrine (PHE), a selective alpha 1-adrenergic agonist, to achieve sufficient tone for measurement of dilation responses. When vessels were treated only with PHE, arterioles and venules constricted to 55.9% and 67.6% of their initial diameter, respectively, after 15 min of suffusion. When preconstricted vessels were treated with the nonselective beta-adrenergic agonist isoproterenol (10(-5) M), both arterioles and venules significantly dilated from 63.4% to 82.9% and from 71.5% to 81.3% of their initial diameters. At high concentration (10(-5) M), the putative beta 2-adrenergic agonist terbutaline also caused preconstricted arterioles and venules to significantly dilate from 70.8% to 79.8% and from 71.5% to 83.4% of their initial diameters. The selective beta 1-adrenergic antagonist atenolol (10(-6) M) did not affect terbutaline-induced dilation in preconstricted arterioles, but greatly attenuated dilation in preconstricted venules. From these data, we conclude that beta 2-adrenergic receptors are present in and mediate dilation of tracheal arterioles, and also, that the dilation in large tracheal venules is mediated in large part through beta 1-adrenergic receptors. PMID- 8630551 TI - A longitudinal evaluation of bronchial responsiveness to methacholine in children: role of baseline lung function, gender, and change in atopic status. AB - A longitudinal study was done to evaluate the determinants of bronchial responsiveness (BR) to methacholine in children and adolescents. A cohort of 892 7- to 11-yr-old schoolchildren was restudied after a 3.5-yr interval. The same protocol for methacholine challenge (up to 64.0 mg/ml) and skin prick testing was employed at both the baseline survey and follow-up. An overall decline in the level of BR was observed, the geometric mean slope (percent decline in FEV1 per mg/ml of methacholine) decreasing from 0.68 (95% CI=0.61 to 0.75) to 0.51 (95% CI=0.46 to 0.57) (p<0.001). At both surveys, the strongest determinants of slope were baseline pulmonary function level (FEF25-75) and degree of atopic status. After adjusting for log FEF25-75, no gender difference was found in the first survey, whereas girls had greater BR than boys at follow-up. Longitudinal changes in skin prick test reactivity were associated with the BR level. Subjects in whom an initially positive skin prick test became negative (5.3%) had an increased slope at baseline but a follow-up slope similar to that of never skin-reactors. Conversely, those whose skin prick test converted from negative to positive (13.0%) had a slope similar to that of never-reactors at baseline but became stronger responders to methacholine than never-reactors at follow-up. Finally, responsiveness was highest in the presence of persistently positive skin prick testing (13.5%). In conclusion, BR declines from childhood to adolescence, paralleling the increase in lung function during this period; the decline is less pronounced in females. Changes in atopic status are associated with modifications in the BR level. PMID- 8630552 TI - Stroke volume during exercise in cystic fibrosis. AB - Previous studies comparing cardiac output (Q) and stroke volume (SV) between cystic fibrosis (CF) patients and control subjects have shown conflicting results: some found lower SV in CF patients with severe airflow limitation, and others showed no difference between CF and control subjects. Methodologic problems could explain these discrepant findings. The aim of this study was to better characterize Q and SV with exercise in CF patients with mild as well as severe airflow obstruction. Subjects included 18 CF patients with FEV1 ranging from 28 to 80% of predicted without pulmonary hypertension, and 16 matched control subjects. Cardiac output was measured at three levels of upright cycle exercise using the indirect Fick (CO2) method with blood gas sampling. Q on exercise was similar among control and CF subjects. SV was lower in CF patients, particularly those with FEV1 < or = 55% predicted, than in control subjects. Stepwise regression of SV on height, percent ideal body weight, and FEV1 showed a significant effect of relative underweight on SV. Despite this, well-nourished patients with FEV1 56 to 80% of predicted also had lower SV. As these findings were consistent across the range of severity of lung disease and age, even in the absence of malnutrition, they imply that another mechanism accounts for SV limitation during exercise in CF. PMID- 8630553 TI - Evaluation of a pneumonia practice guideline in an interventional trial. AB - There are few available data to define the medically necessary duration of stay for patients hospitalized with pneumonia. Therefore, we investigated the safety and effectiveness of a practice guideline that provided information about switching patients from parenteral to oral antimicrobials and early hospital discharge. The study was a prospective controlled study with an alternate month design. The practice guideline was studied in 146 "low-risk" pneumonia patients hospitalized during a 22-month period. Medical care consistent with the practice guideline occurred in 64% and 76% of patients during control and intervention periods, respectively (p=0.15). There were no differences in patient outcomes in the control and intervention groups when measured 1 mo after hospital discharge, including hospital readmission rates, health-related quality of life, and patient satisfaction. Explicit and implicit review revealed that 98.6% (95% confidence interval [CI]: 95.1%, 99.8%) of low-risk patients would not have benefited from continued hospitalization after the fourth hospital day. The 30-d survival rate of the low-risk pneumonia patients was 99.3% (95% CI: 96.2%, 100%) and patient outcomes appeared to be favorable compared with previously published values. We conclude that duration of hospital stay was frequently consistent with the practice guideline in both study groups, and patient outcomes remained unchanged. The guideline will require additional testing before it can be recommended for use. PMID- 8630554 TI - Antibody to surfactant protein A increases sensitivity of pulmonary surfactant to inactivation by fibrinogen in vivo. AB - It has been suggested that surfactant protein-A (SP-A) protects surfactant activity from inhibitors such as fibrinogen. Substantial evidence indicates that inhibition of surfactant activity is often important in the pathogenesis of acute respiratory failure. Studies on surfactant function in the pulsating bubble surfactometer imply that SP-A helps to maintain low surface tension in the presence of inhibitors such as fibrinogen. We tested whether SP-A acts in this way in vivo. Rabbit pups, 29 d gestational age, were treated with a monoclonal antibody to rabbit SP-A (R5) followed by fibrinogen, or with control preparations (normal IgG and saline, respectively). Lung compliance was measured during ventilation throughout these experiments. Air-space volume and pulmonary edema were quantitated morphometrically. Animals receiving anti-SP-A antibody + fibrinogen showed substantial and significant impairment in lung compliance compared with control littermates receiving normal IgG and/or saline. Lungs from these animals showed decreased pulmonary air-space volume and increased alveolar edema. We conclude that SP-A protects pulmonary surfactant from inhibition by fibrinogen in vivo. This protective activity may be important in the pathogenesis of both adult and neonatal respiratory distress syndromes, and it may also be useful in devising therapies for these diseases. PMID- 8630555 TI - Ciliary disorientation alone as a cause of primary ciliary dyskinesia syndrome. AB - Ciliary disorientation has been proposed as a variant of primary ciliary dyskinesia (PCD); cilia have normal ultrastructure and normal or near normal ciliary beat frequency (CBF) but lack efficacy because their beat direction is disorientated. We have identified 11 patients, including two siblings, with the clinical features of PCD, who satisfy these criteria. A chest radiograph, pulmonary function tests, nasal mucociliary clearance (NMCC), CBF, ciliary ultrastructure, and orientation were assessed in each subject. One patient had biopsies taken from the nose and both main bronchi. Eight patients had a computed tomography scan (CT) of the thorax; the clinical features were compatible with PCD. Cilia ultrastructure was normal and NMCC was absent in all cases. Mean CBF was normal (11.6-14.9 Hz) in five cases and slow in six (range 8.4-9.7 Hz). Ciliary beat pattern was stiff in seven cases, six of which had slow CBF. The cilia were disorientated when measured by both the central pair (range, 21.8 degrees - 26.4 degrees) and basal feet (range, 20.6 degrees - 28.9 degrees) compared with 16 normal controls (range, 11.0 degrees - 15.5 degrees and 12.3 degrees - 17.6 degrees, respectively). Two siblings had the clinical features of PCD and ciliary disorientation alone on repeated biopsies taken 10 yr apart. Orientation of cilia from the nose and bronchus was similar. Two cases had unchanged ciliary disorientation after 3 mo of treatment with antibiotics and topical corticosteroids. We concluded that ciliary disorientation alone can lead to the clinical syndrome of PCD. PMID- 8630556 TI - A human respiratory-tissue organ culture incorporating an air interface. AB - The immersion of respiratory tissue in organ cultures is unphysiologic and may influence the interactions of the tissue with experimental agents. We have assessed an organ culture of human nasal turbinate tissue with an air interface by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), with and without replacement of culture medium. Without replacement of medium, ciliary beat frequency (CBF) was normal (11.3 +/- 0.5 Hz) at 5 d, but fell significantly (p<0.05) to 7.9 +/- 0.8 Hz at 10 d. The degree of ciliation decreased significantly (p<0.05) at 4 and 10 d. Nuclear heterochromatin in all cell types was significantly (p<0.05) reduced at 5 d. Significant (p<0.05) mitochondrial abnormalities occurred in ciliated cells at 5 d and in both ciliated and unciliated cells at 10 d. With daily replacement of medium, CBF fell significantly (p<0.05) from 11.6 +/- 0.2 Hz at Time 0 to 10.6 +/ 0.3 Hz after 20 d. The proportions of ciliated and nonciliated cells did not change after 20 d, but the proportion of mucus cells was higher at 20 d (26.3 +/- 5.4%) than at Time 0 (9.8 +/- 2.7%). No mitochondrial abnormalities, changes in nuclear heterochromatin levels, or reduction in cilial density on ciliated cells were present. The amount of damaged epithelium was less at 20d (7.2 +/- 3.8%) than at Time 0 (19.0 +/- 5.8%). This model more closely reproduces physiologic conditions in vitro than do models involving the immersion of respiratory tissue in media. Its long viability will permit studies of virus and bacterial infections, and of the effects of pharmacologic agents and environmental factors. PMID- 8630557 TI - CO2 and Nd:YAG laser-induced pulmonary parenchymal lung injury in a rabbit model. AB - Laser exposure of the pulmonary parenchyma during treatment of emphysema and other clinical indications causes acute lung injury. Animal investigations are needed to understand and control laser-induced lung injury. We hypothesized that lung injury is deeper from Nd:YAG laser exposures than CO2 exposures because of deeper penetration of Nd:YAG wavelength light. We compared the temporal evolution of histologic injury in rabbits resulting from continuous mode shallow CO2 and Nd:YAG laser pulmonary parenchymal exposures applied in rabbits. Forty-six New Zealand white (NZW) rabbits underwent treatment with CO2 laser (n=18), Nd:YAG laser (n=18), or sham thoracotomy control (n=10) to the visceral pleural surface using 1 min of exposure (5 watts, defocused to 70 W/cm2 power density for both lasers). Animals were killed at 0, 4, 7, 21, and 49 d after exposure. Lung injury, similar to that seen clinically in humans, developed in all laser-treated animals. Injury progressed from ischemia and vascular congestion, to edema and necrosis, followed by pleural and parenchymal fibrosis. The acute injury was qualitatively distinct and slightly deeper in CO2 than Nd:YAG-treated animals (p<0.02) despite the shallower depth of penetration of the CO2 laser. These findings may imply that higher absorption coefficient for CO2 laser energy results in greater focal temperatures and injury in the areas of direct exposure, and suggest that Nd:YAG laser exposure at these settings may cause shallower injury than CO2 lasers in humans undergoing clinical treatment. PMID- 8630558 TI - Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis. AB - The acute phase protein, C-reactive protein (CRP), can increase more than a thousandfold during acute inflammatory states, and it is known to modulate neutrophil-mediated inflammatory responses. We have previously shown that CRP inhibits chemotaxis of C5a-stimulated neutrophils in vitro and that rabbits with elevated CRP blood levels exhibit diminished pulmonary vascular permeability and neutrophil infiltration in a model of alveolitis. To study the effect of CRP on alveolitis induced by different chemoattractants, transgenic mice capable of expressing rabbit CRP in a dietary-inducible fashion were treated with inflammatory doses of the chemoattractants. Intratracheal installation of FMLP (8 x 10(-10) mol), LTB4 (2 x 10(-11) mol), or IL-8 (5 x 10(-12) mol) in normal CF1 mice resulted in significant (p<0.05) influx of neutrophils and protein into the alveolar space. Transgenic mice with elevated plasma levels of CRP showed significantly (p<0.05) diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and significant reduction in BALF protein compared with that in normal mice. Rabbit CRP (10 to 500 micrograms/ml) inhibited in vitro neutrophil chemotaxis in a concentration-dependent fashion when stimulated by the various chemoattractants examined. These data show that rabbit CRP can modify both in vivo and in vitro neutrophil responses to several classes of chemoattractants and that CRP has a significant protective effect in alveolitis by reducing neutrophil influx and protein leakage into the lung. PMID- 8630559 TI - Surfactant nebulization does not alter airflow obstruction and bronchial responsiveness to histamine in asthmatic children. AB - To test the hypothesis that surfactant nebulization improves pulmonary function and reduces bronchial responsiveness to histamine, we studied 12 children with asthma. We measured before and after inhalation of 100 mg nebulized natural derived bovine surfactant (Alveofact (registered)) and after nebulization of 0.9% NaCl the change in peak expiratory flow, vital VC, FEV1, and forced expiratory flows at 50 and 75% of the flow-volume curve. In addition, we performed a histamine inhalation challenge. We did not find any significant changes in these parameters after nebulization of surfactant. These findings indicate that the inhalation of nebulized surfactant does not alter airflow obstruction and does not alter bronchial responsiveness to histamine in asthmatic children with airflow obstruction. PMID- 8630560 TI - The pleural space and organ transplantation. AB - Pleural disease both before and after organ transplantation has important implications. Pleural effusions are common in candidates for heart, liver, and kidney transplantation. A thoracentesis is not mandatory in these patients, but it should be performed if clinical or radiologic features suggest that the effusion is not the result of organ failure. Posttransplant pleural infections and pleural PTLD relate to the level and duration of immunosuppression and are probably not organ-specific. Organ-specific pleural complications include pleural effusion from hepatic venoocclusive disease, spontaneous pneumothorax associated with obstructive airway disease from chronic GVHD after bone marrow transplantation, and early pleural effusion from urinothorax and late effusion from perirenal lymphocele years after kidney transplantation. The treatment of pleural disease in potential lung transplant candidates should minimize the extent of pleurodesis. Pleural effusions are expected sequelae after lung transplantation, and they may be harbingers of acute rejection. Interpleural communication, an expected finding after heart-lung transplantation or double lung transplantation with a "clamshell" incision, has therapeutic implications. PMID- 8630561 TI - Transmission of Mycobacterium tuberculosis to recipients of single lung transplants from the same donor. AB - Recipients of organ transplants are at increased risk for infection both because of immunosuppression and because of the transfer of microbes through the donor organs. We report two cases of M. tuberculosis disease in recipients of single lung transplants who shared a common donor. Both recipients developed pulmonary tuberculosis, one having fever and pulmonary infiltrates and the other having subclinical disease with M. tuberculosis organisms being recovered from bronchoalveolar lavage. Restriction fragment length polymorphism analysis on both isolates of M. tuberculosis revealed a common source. The donor of both lungs had a normal chest radiograph and no known prior history of M. tuberculosis infection of disease. These cases are the first report of two single lung recipients developing pulmonary tuberculosis from a common donor. PMID- 8630562 TI - Refractory hypoxemia during liver cirrhosis. Hepatopulmonary syndrome or "primary" pulmonary hypertension? AB - We report an uncommon mechanism of severe hypoxemia in two cirrhotic patients under long-term beta-blocker therapy. Our patients presented with profound hypoxemia refractory to oxygen therapy, normal lung radiography and pulmonary function tests, and evidence of right-to-left anatomic shunt. Although these features are highly suggestive of hepatopulmonary syndrome, pulmonary hypertension was present, and a right-to-left shunt through a patent foramen ovale was demonstrated by contrast-enhanced echocardiography. No cause of pulmonary hypertension other than portal hypertension was identified. Pulmonary hypertension and intracardiac right-to-left shunt eventually regressed after discontinuation of beta-blocker therapy. We conclude that "primary" pulmonary hypertension associated with portal hypertension may because of severe hypoxemia during liver cirrhosis. Differential diagnosis of hepatopulmonary syndrome relies upon contrast-enhanced echocardiography and may be of critical importance because of possible therapeutic implications. PMID- 8630563 TI - Effects of an inhaled steroid (budesonide) on skin collagen synthesis of asthma patients in vivo. AB - Skin atrophy has been observed after prolonged use of inhaled corticosteroids. We therefore studied the effect of inhaled budesonide and nedocromil in patients with asthma on concentrations of procollagen propeptides in suction blister fluid reflecting skin collagen synthesis in vivo. Both types I and III procollagen propeptide concentrations decreased significantly after 6 wk of either 1,600 micro g/day (n=10) or 400 micro g/day (n=9) of inhaled budesonide but not in control subjects using inhaled nedocromil 16 mg/day (n=9). The reduction in mean propeptide concentrations ranged from 39 to 63%; the effects of the two budesonide doses did not differ significantly. Thus, even a low dose of inhaled corticosteroid represses skin collagen synthesis within a relatively short period. PMID- 8630564 TI - Patient-triggered ventilation decreases the work of breathing in neonates. AB - During conventional intermittent mandatory ventilation (IMV) in neonates, asynchrony between mechanical and spontaneous breaths is frequent. We tested the hypothesis that patient-triggered ventilation (PTV) reduces the work of breathing (WOB) by providing synchronized assistance for each breath. Accordingly, six intubated preterm infants were studied at the median postnatal age of 34 days while they were being weaned from mechanical ventilation (MV). Patients were ventilated using the Draeger Babylog 8000 (software #3) and studied in four successive modes of MV with a constant level of positive end-expiratory pressure. They were randomly assigned to IMV, PTV with peak inspiratory pressure of either 10 cm H2O (PTV10) or 15 cm H2O (PTV15), and spontaneous ventilation with continuous positive airway pressure. PTV was achieved in the assist/control mode. During PTV, infants adapted their pattern of breathing in response to an increase in tidal volume (median 7.5 ml/kg in IMV versus 8.2 in PTV10 and 8.5 in PTV15, p<0.05) by decreasing their respiratory rate, thus maintaining minute ventilation (439 ml/min/kg in IMV versus 422 in PTV10 and 455 in PTV15, NS) and transcutaneous CO2. WOB fell significantly during PTV compared with its level during IMV (0.81 J/L in IMV versus 0.48 and 0.47 during PTV10 and PTV15, respectively, p<0.05). Power of breathing decreased in the same proportions. These results demonstrate that PTV mode allows reduction of the workload imposed on the respiratory muscles. PMID- 8630565 TI - Interindividual variation in pubertal growth patterns of ventilatory function, standing height, and weight. AB - We studied interindividual variation in pubertal growth patterns from peak growth velocities (PGV) and peak growth ages (PGA) of ventilatory function, standing height, and weight in a selection of 144 boys from a longitudinal survey of 404 pupils in a Dutch secondary school. Measurements were made at intervals of approximately 0.5 yr between 1978 and 1985. Between 9 and 14 measurements were available for each selected individual. Average age on enrollment was 12.7 years. Ventilatory function was characterized by FVC, FEV1, peak expiratory flow (PEF), and maximal expiratory flow at 50% of the FVC (MEF50), derived from maximum expiratory flow volume (MEFV) curves. PGVs and PGAs were derived from monotonically increased regression splines, fitted to the data of each individual and each variable separately. The 90% percentile ranges of PGA were approximately 4.5 yr in all variables. In almost all boys, the PGA of height occurred earlier than that of ventilatory function, but the magnitude of the time lag varied considerably. Median PGAs agreed well with peak growth ages derived from average growth velocity curves fitted on exactly the same data. However, median PGVs were 1.25 to 1.40 times higher than the corresponding estimates from the average curves. The latter finding implies that in almost all cases, individual development deviates considerably from development suggested by average growth profiles. No differences in PGA and PGV were found between subjects with a prepubertal history of respiratory symptoms and those without. The large interindividual variations in PGA and PGV, and in the time lag between growth of height and of ventilatory function, are not accounted for in cross-sectional reference equations. These equations are therefore not suitable to predict individual development during adolescence. PMID- 8630566 TI - CPAP with hypervolemia. PMID- 8630567 TI - Randomized prospective trial of noninvasive positive pressure ventilation in acute respiratory failure. PMID- 8630568 TI - Tumor necrosis factor and sleep apnea. PMID- 8630569 TI - Upper airway shape: Is it important in the pathogenesis of obstructive sleep apnea? AB - It is widely accepted that the size of the airway and the level of upper airway muscle activity contribute to the pathogenesis of obstructive sleep apnea (OSA). However, some individuals with normal airway size and apparently normal upper airway muscle activity nonetheless develop OSA. Recent data and analysis suggest that airway shape may also contribute to the development of OSA; patients with OSA tend to have elliptical airways, with the long axis of the ellipse oriented along the anteroposterior axis, and this orientation may adversely affect upper airway muscle function. By incorporating airway shape, airway size, and upper airway muscle activity into a unified hypothesis of the pathogenesis of OSA, it may be possible to reconcile discrepant anatomic and electromyographic findings in patients with OSA, to explain the difficulty of selecting appropriate surgical therapy for patients with OSA using currently available diagnostic tools, and to understand the often disappointing response to upper airway surgery in these patients. PMID- 8630570 TI - Intrasubject variability in airway inflammation in biopsies in mild to moderate stable asthma. AB - We have studied intrasubject variability with respect to counts of immunostained inflammatory cells in snap frozen endobronchial biopsies from a group of patients with clinically mild to moderate stable asthma. Fiberoptic bronchoscopy was used to obtain endobronchial biopsies from the upper and lower lobes in 12 volunteer subjects with asthma on two separate occasions 1 mo apart. During this period there was no significant change in asthma treatment, symptom scores, pulmonary function, or airway hyperresponsiveness. With the aid of immunohistochemistry and interactive image analysis, subepithelial counts for T lymphocytes, T-lymphocyte subsets, and eosinophils were made. There was wide intrasubject variability between anatomic site and with time for all the inflammatory cell counts. In each case the intrasubject variability with time was greater than lobar differences. Power calculations were made to establish the optimal sample size required for each marker. We conclude that even in stable asthma considerable biologic variability compounds sampling variability. Such sources of variability need to be borne in mind when calculating the likely power of intervention studies using biopsy data as end points. Power calculations suggest that approximately 15 subjects would be an optimal number with little advantage in increasing beyond this. PMID- 8630571 TI - Ozone-induced decrements in FEV1 and FVC do not correlate with measures of inflammation. AB - In order to test the hypothesis that changes in lung function induced by ozone (O3) are correlated with cellular and biochemical indices of respiratory tract injury/inflammation, we exposed 20 healthy subjects, on separate days, to O3 (0.2 ppm) and filtered air for 4 h during exercise. Symptom questionnaires were administered before and after exposure, and pulmonary function tests (FEV1, FVC, and SRaw) were performed before, during, and immediately after each exposure. Fiberoptic bronchoscopy, with isolated left main bronchus proximal airway lavage (PAL) and bronchoalveolar lavage (BAL; bronchial fraction, the first 10 ml of fluid recovered) of the right middle lobe, was performed 18 h after each exposure. The PAL, bronchial fraction, and BAL fluids were analyzed for the following end points: total and differential cell counts, and total protein, fibronectin, interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. The study population was divided into two groups, least-sensitive (n = 12; mean O3-induced change in FEV1 = -7.0%) and most sensitive (n = 8; mean O3-induced change in FEV1 = -36.0%). We found a significant O3 effect on SRaw (p<0.001) and lower respiratory symptoms (p<0.001) for all subjects combined, but no significant differences between the least- and most-sensitive groups. Ozone exposure increased significantly percent neutrophils in PAL (p=0.01); percent neutrophils, total protein, and IL-8 in bronchial fraction (p<0.001, p<0.001, and p<0.01, respectively); and percent neutrophils, total protein, fibronectin, and GM-CSF in BAL (p<0.001, p<0.001, p<0.01, p=0.05, respectively) for all subjects combined; there were no significant differences, however, between least- and most-sensitive groups. Our results indicate that levels of O3-induced symptoms and respiratory tract injury/inflammation were not correlated with the magnitude of decrements in FEV1 and FVC. PMID- 8630572 TI - Bronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. AB - Although bronchial hyperresponsiveness in asthma is associated with inflammation within the airways, it is not known whether the degree and type of inflammation influence the response to different stimuli and whether pathologic changes of airway structure influence the bronchoconstrictive responses. Therefore, number of inflammatory cells in the epithelium and the lamina propria and the basement membrane thickness were estimated from bronchial biopsies taken in 27 asthmatic subjects (range percent predicted FEV1: 75.6 to 132.1, range of daily PEF variability: 1.9% to 20%) and related to the degree of bronchial responsiveness to ultrasonically nebulized distilled water (UNDW) and methacholine (M). PD20UNDW (provocative dose) was measurable in 15 of 27 patients and ranged between 1.01 and 20.4 ml. PC20M (provocative concentration) ranged between 0.15 and 31.7 mg/ml. In the 15 responders to UNDW, total inflammatory cells (p<0.04) and eosinophils (p<0.015) within the epithelium were higher than in 12 nonresponders to UNDW (PD20 > 34.8 ml). There was no correlation between PD20UNDW and any cell counts whereas negative correlations were found between PC20M and both total inflammatory cells (rs = -0.57; p<0.005) and eosinophils (rs = -0.63; p< 0.0015) within the epithelium. The degree of thickening of subepithelial layer ranged between 7 and 16 micrometers+ (n=26). Thickness correlates both with total inflammatory cells (rs = 0.49; p<0.025) and eosinophils (rs = 0.61; p< 0.003) within the epithelium. Moreover, it was correlated with baseline FEV1 (rs = 0.57; p<0.003) and daily peak expiratory flow (PEF) variability (rs = 0.51; p<0.01). A weak but significant correlation was also found between subepithelial layer thickness and PC20M (rs = -0.42; p<0.04). The results of this study demonstrate that eosinophilic inflammation of bronchial epithelium plays a role in determining UNDW and M responsiveness in asthma. Moreover, they suggest that remodeling of the airways such as thickening of subepithelial layer correlates with indices of asthma severity and could contribute to the degree of M but not to UNDW responsiveness. PMID- 8630573 TI - Evidence that epithelium-derived relaxing factor released by bradykinin in the guinea pig trachea is nitric oxide. AB - Bradykinin, applied locally to the airways, is a weak bronchoconstrictor agent in guinea pigs in vivo and it may cause constriction or dilatation of guinea pig airways smooth muscle in vitro. We examined the motor effect of bradykinin perfused through the lumen of isolated guinea pig tracheal tubes with or without nitric oxide (NO) synthase inhibitors. In the presence of NG-nitro-D-arginine methyl ester (D-NAME) or NG-monomethyl-D-arginine (D-NMMA) intraluminal bradykinin caused a moderate concentration-dependent relaxation. In contrast, in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L arginine (L-NMMA) tracheas developed a sustained increase in tone, and bradykinin caused a marked, concentration-dependent contraction, both effects being reversible by pretreatment with L-arginine, but not with D-arginine. The ability of bradykinin to relax (in the presence of D-NAME) or contract (in the presence of L-NAME) guinea pig tracheal tubes was not affected by indomethacin. Bradykinin contracted epithelium-denuded tracheas in the presence of either L-NAME or D NAME. Both contraction and relaxation by bradykinin were blocked by the kinin B2 receptor antagonist, HOE 140. Baseline production of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in strips of guinea pig trachealis in vitro was markedly reduced by L-NAME, but not by D-NAME. Bradykinin increased baseline cyclic GMP concentration. These results indicate that bradykinin releases NO or a NO-related molecule, which, possibly by increasing cyclic GMP concentrations, mediates relaxation and opposes contraction induced by bradykinin itself, and further, that bradykinin releases NO from the tracheal epithelium. PMID- 8630574 TI - Fluticasone propionate does not influence bone metabolism in contrast to beclomethasone dipropionate. AB - Inhaled corticosteroids (ICS) in dosages above 1,000 micrograms/d may influence parameters of bone metabolism. Fluticasone propionate (FP) is a new ICS with a higher clinical potency than beclomethasone dipropionate (BDP) combined with negligible oral bioavailability. The aim of this study was to evaluate the effects of FP and BDP in clinically equipotent dosages on indices of bone metabolism and morning cortisol. FP 750 micrograms/d and BDP 1,500 micrograms/d were compared in a randomized, double-blind, crossover study consisting of two 6 wk treatment periods, each preceded by a 3-wk single-blind placebo period. Twenty one nonsmoking asthmatic completed the study. FP had the same effect on FEV1 and peak expiratory flow as the double dose of BDP. Both treatments did not change morning cortisol. BDP decreased both osteocalcin and procollagen type 1 carboxyterminal propeptide, indices of bone formation, significantly by 18.5 and 21.9%, respectively. In contrast, FP did not change any variable of bone formation. FP and BDP did not increase type 1 collagen carboxyterminal telopeptide and deoxypyridinoline crosslinks, both markers of bone resorption. In changes in parameters of bone metabolism indicate adverse effects on bone quality in the long term, FP may offer an advantage over BDP. PMID- 8630575 TI - Inhibition of exercise-induced bronchospasm by zileuton: a 5-lipoxygenase inhibitor. AB - Recent evidence suggests that leukotrienes may have a causative role in exercise induced asthma. Twenty-four subjects with exercise-induced asthma received either 600 mg zileuton, a 5-lipoxygenase inhibitor, or a placebo four times daily for 2 d prior to exercise challenge (a total of nine doses). The last dose was administered in the laboratory 2 h before the exercise challenge. There was no bronchodilation after nine doses of the 5-lipoxygenase inhibitor (p=0.95). The administration of zileuton inhibited bronchospasm after exercise challenge by 40.75% as compared with placebo. Five minutes after the completion of exercise, the zileuton group's FEV1 was 85.76% of the preexercise value, compared with 73.92% of the preexercise value in the placebo group (p<0.01). The maximum percent change in baseline FEV1 after zileuton was a 15.58% decrement from the preexercise level, as compared with a 28.1% decrease after placebo (p<0.001). Five minutes after exercise, the FVC after zileuton was 92.76% of the preexercise value, as compared with 86.26% after placebo (p<0.05). This is the first study in which a 5-lipoxygenase inhibitor has been shown to attenuate exercise-induced asthma. These results suggest that leukotrienes are important biochemical mediators in the development of exercise-induced bronchospasm, and that leukotriene inhibit may have a role in the treatment of this disorder. PMID- 8630576 TI - A neurokinin 1-receptor antagonist improves exercise-induced airway narrowing in asthmatic patients. AB - Recent reports suggest the involvement of vascular phenomena in exercise-induced asthma. Sensory neuropeptides, such as substance P (SP), which causes airway vascular dilatation and plasma leakage, have been demonstrated to play a role in hyperpnea-induced airway narrowing in animal studies. The purpose of this study was to investigate the importance of tachykinins in exercise-induced airway narrowing in patients with asthma using a selective neurokinin 1-receptor (NK1 receptor) antagonist, FK-888. In a double-blind, placebo-controlled, crossover trial, nine subjects with stable asthma were given FK-888 (2.5 mg) or placebo by inhalation 20 min before each exercise at a level previously demonstrated to cause a fall of at least 40% in specific airway conduction (SGaw). Inhalation of FK-888 had no significant effect on baseline SGaw. While the recovery from exercise-induced airway narrowing was significantly faster after treatment with FK-888 the area under the curve for SGaw during the 50 min after exercise was significantly reduced (p<0.05) and the time taken for the SGaw to recover to within 65% of baseline after exercise was also significantly shorter with FK-888 than the placebo (p<0.05). However, treatment with FK-888 did not significantly attenuate the maximal fall in SGaw. These results suggest that NK1-receptor mediated mechanisms are involved in the recovery phase of exercise-induced airway narrowing. The possible mechanisms of these phenomena are discussed. PMID- 8630577 TI - Vocal cord dysfunction masquerading as exercise-induced asthma. a physiologic cause for "choking" during athletic activities. AB - Exercise-induced bronchospasm is a common clinical problem that is particularly troubling for patients who engage in strenuous physical activity, such as athletes. When such individuals develop this condition, the associated airway narrowing can materially interfere with performance; however, events other than asthma can also produce these symptoms and their differentiation is essential for proper treatment. The present report describes seven elite athletes with psychogenic vocal cord dysfunction who presented with acute dyspnea during sporting competitions. Although the combination of exertion and wheezing suggested the diagnosis of exercise-induced asthma, the patients' histories were sufficiently unique so as to represent a recognizable syndrome. The patients underwent clinical physiologic evaluations including bronchoprovocations with isocapnic hyperventilation of frigid air, methacholine and/or exercise. Direct laryngoscopy was also performed in some subjects. The findings that differentiated these patients from asthmatics were a lack of consistency in the development of symptoms when exposed to identical stimuli, the onset of breathing difficulties during exercise, and poor therapeutic and prophylactic responses to anti-asthma medications. The clinical impression of a functional disorder was confirmed by bronchoprovocations that demonstrated the variable extrathoracic airway obstruction of vocal cord dysfunction. Patients with atypical exertional complaints require careful clinical and physiologic evaluation. The mere association of exercise and airway obstruction is not sufficient to establish the diagnosis of asthma. PMID- 8630578 TI - Increased bronchial responsiveness in workers sawing Scots pine. AB - The purpose of the present investigation was to study bronchial responsiveness and pulmonary function in Swedish sawmill workers, who are not exposed to plicatic acid, the sensitizer in red wood cedar asthma. Bronchial responsiveness, transfer factor, spirometry, and precipitating antibodies in serum against sawmill fungi were measured in 164 workers at five sawmills. The results from workers inside the sawing area (sawyers, n=59), in the trimming department (trimmers, n=66), and from other workers in the sawmill (sawyer-referents, n=39) were compared. Sawyers had higher bronchial responsiveness than referents. In 55% of the sawyers FEV1 decreased by 20% or more within the highest dose of methacholine compared with 31% of sawyer-referents and 41% of trimmers (p<0.01, sawyers/referents). Sawyers decreased 74% more in FEV1 per milligram of inhaled methacholine compared with referents (geometric means, p<0.01). The transfer test in never-smokers was 13% lower in sawyers than in trimmers (p<0.01) and 8% lower compared with sawyer-referents (nonsignificant p<0.1). Presence of precipitating antibodies was not associated with changes in pulmonary function. Some agents in the sawing area of sawmills appear to increase bronchial responsiveness and decrease diffusion capacity. PMID- 8630579 TI - Influence of inhaled steroids on recovery from occupational asthma after cessation of exposure: an 18-month double-blind crossover study. AB - Occupational asthma (OA) is a useful model for the study of asthma in humans. The possibility that inhaled corticosteroids, in addition to withdrawal from the workplace, could improve clinical and functional recovery from OA can be hypothesized. We assessed clinical, functional, and behavioral characteristics of 32 subjects (22 male, 10 female), in all but one of whom OA was confirmed by specific inhalation challenges induced by either high- (n=13) or low-molecular weight (n=19) agents within 3 mo after cessation of exposure. In this randomized, crossover, double-blind study, subjects (paired for baseline PC20 and duration of symptoms after exposure) received either placebo or 1,000 micrograms of inhaled beclomethasone daily for 1 yr, followed by the alternate medication for 6 mo. Various clinical, functional, and behavioral parameters were examined at each 3 mo visit. Significant improvement in clinical (nocturnal symptoms, cough), functional (morning and evening peak expiratory flow rates), and behavioral (quality of life) parameters were detected in the active-treatment period, although the magnitude of the improvement was relatively small. Side effects (oropharyngeal, reduced cortisol) were similar in the placebo and treatment groups. Distinguishing subjects who started with the active preparation from those who were given placebo first showed that most clinical and behavioral parameters improved in the former instance, whereas there was no significant difference in the latter. We conclude that inhaled corticosteroids induce a small but significant overall improvement of the asthmatic condition in subjects with occupational asthma caused by high- and low-molecular-weight agents after withdrawal from exposure. The beneficial effect is, however, more pronounced if inhaled steroids are given early after diagnosis. PMID- 8630580 TI - Nutritional status and mortality in chronic obstructive pulmonary disease. AB - The role of nutritional status in the prognosis of subjects with severe chronic obstructive pulmonary disease was studied in a cohort of Canadian men and women followed for 3 to 5 yr. A total of 348 subjects who were recruited for a study of negative pressure ventilation were evaluated for lung function and body weight, and a subset who entered hospital for the study (n=184) had baseline measures of diffusing capacity, maximal inspiratory and expiratory mouth pressure (PImax and PEmax), and blood gases. Predictors of survival were analyzed using Cox regression models. In the total cohort, low body mass index (BMI) and use of home oxygen were independently associated with reduced survival. In the hospitalized group, predictors of respiratory mortality were elevated PaCO2 and low BMI, PImax, and diffusing capacity. PaO2 (measured on oxygen therapy), FEV1, PEmax, age, smoking behavior, and gender were not associated with survival. The predictors of total mortality were similar, except that BMI was no longer significant. In conclusion, low body weight, a potentially modifiable factor, was associated with respiratory mortality, but whether it has a casual effect or is a marker of declining health can only be resolved through an intervention trial. PMID- 8630581 TI - Inhaled bronchodilators reduce dynamic hyperinflation during exercise in patients with chronic obstructive pulmonary disease. AB - Dynamic hyperinflation (DH) is a major pathophysiologic consequence of airflow limitation during exercise in patients with chronic obstructive pulmonary disease (COPD) and an important contributing factor to breathlessness. In this study we aimed to examine the effect of inhaled beta agonist therapy on DH during exercise in these patients and the relationship between changes in DH and breathlessness. In 13 COPD patients (mean age 65.1 +/- 2.0, FEV1 1.20 +/- 0.17, FEV1/FVC 40 +/- 3) we measured pulmonary function tests, exercise breathlessness by Borg score, and exercise flow volume and pressure volume loops on two separate days. Prior to testing, patients randomly received inhaled placebo or albuterol on the first test day and the alternative medication on the second test day. From measurements of exercise inspiratory capacity (IC), we calculated the end-expiratory and end inspiratory lung volumes (EELV, EILV). We used esophageal pressure recordings to measure peak inspiratory esophageal pressure (Pesins) during exercise and this was related to the maximal capacity for pressure generation taking into account lung volume and airflow changes (Pcapi). Bronchodilator caused significant increase in both FEV1 and FVC (+0.23 and +0.51, p<0.01). Comparisons of breathlessness, exercise volumes, and pressures were made at the highest equivalent work load. There was a significant reduction in the peak exercise EELV/TLC (80 +/- 0.02% to 76 +/- 0.02%, p<0.05) while the peak EILV/TLC decreased by 2% (97 +/- 1% to 95 +/- 1%, p<0.05). The peak Pesins/Pcapi decreased (0.79 +/- 0.10 to 0.57 +/- 0.05, p<0.05), and the Pcapi - Pesins increased (7.4 +/- 3 to 13.0 +/- 3 cm H2O, p<0.05). There was significant improvement in neuroventilatory coupling for volume change (Pesins/Pcapi/VT/TLC 5.45 +/- 0.5 to 3.25 +/- 1.0, p<0.05). There was a significant reduction in breathlessness as measured by Borg score (4.5 +/- 0.7 to 3.1 +/- 0.5, p<0.05) and there was a significant correlation between delta Borg and delta EILV/TLC (r=0.771, p<0.01) with a trend for Pesins/Pcapi/VT/TLC (r=0.544, p=0.067). There was also a significant correlation between delta EELV/TLC and delta Pesins/Pcapi/VT/TLC (r=0.772, p<0.01). The relationships between delta Borg, delta resting volumes, and flow rates were not significant. We conclude that in patients with COPD, inhaled bronchodilator reduces exercise DH and improves inspiratory pressure reserve and neuroventilatory coupling. Changes in DH and neuroventilatory coupling were the main determinants of reduced breathlessness. PMID- 8630582 TI - Peripheral muscle weakness contributes to exercise limitation in COPD. AB - Recently, it was suggested that fatigue of peripheral muscles could contribute to exercise limitation in patients with chronic obstructive pulmonary disease (COPD). In order to quantify the role of peripheral muscle force, we restudied potential determinants of exercise capacity (6-min walking distance [6 MWD] and maximal oxygen consumption [V02max]) in 41 consecutive COPD patients (FEV1, 43 +/ 19% of predicted, TLCO, 56 +/- 25% of predicted) admitted to our pulmonary rehabilitation program. VO2max (incremental cycle ergometer test), 6 MWD (best of three), lung function (FEV1, FVC, TLC, FRC), diffusing capacity (TLCO), isometric quadriceps force (QF), hand grip force (HF), and maximal inspiratory (PImax) and expiratory (PEmax) pressures were measured. Patients had a poor 6 MWD (372 +/- 136 m) and VO2max (1.35 +/- 0.60 L, 71%), reduced respiratory (PImax 65 +/- 27%) and peripheral muscle force (QF 74 +/- 27%, HF 82 +/- 23%). In single regression analysis, significant correlations (r) were found for VO2max and TLCO (0.68), FEV1 (0.64), QF (0.55), HF (0.53), and body weight (0.49). Walking distance was significantly correlated with QF (0.63), HF (0.61), PImax (0.49), and TLCO (0.38). In stepwise multiple regression analysis, the variables significantly contributing to 6 MWD were QF and Plmax. For VO2max, variables significantly contributing were TLCO, QF, and FEV1. We conclude that lung function and peripheral muscle force are important determinants of exercise capacity in COPD. PMID- 8630583 TI - Noninvasive determination of cardiac output in patients with severe airflow limitation. AB - The noninvasive measurement of cardiac output (Q) by the Indirect Fick CO2 rebreathing technique requires mixed venous P CO2 (P CO2) to be determined by the rebreathing maneuver, and Pa CO2 to be estimated from end-tidal P CO2 (PET CO2). Previous work has suggested that although P CO2 can be determined, Pa CO2 cannot be accurately estimated in patients with significant airflow limitation. Nineteen patients with cystic fibrosis who had severe airflow limitation (%FEV1, 29.3 +/- 7.12 SD) were studied during steady-state exercise at 50% of their measured maximal work capacity. Estimated Pa CO2 was slightly lower than Pa CO2 measured from blood samples obtained from an indwelling arterial catheter (measured: 45.2 +/- 4.92; estimate: 42.7 +/- 5.68 mm Hg). To calculate arterial blood content, the values derived from Pa CO2, pH, hemoglobin (Hb), and O2 saturation were compared with those derived from PET CO2 and O2 saturation, where (1) pH was assumed to be 7.40 and Hb was measured, and (2) pH was assumed to be 7.40 and Hb was assumed to be 15 g/dl (measured mean pH, 7.34; Hb, 14.4 g/dl). No difference in arterial CO2 content was seen between the three methods (measured: 47.53 +/- 5.17; estimate 1: 49.57 +/- 6.58; estimate 2: 49.12 +/- 6.61 ml/100 ml). As pH and Hb can also affect mixed venous CO2 content, the effect on Q was also assessed. Both estimates fit closely with measured Q (r2=0.77 and 0.76), with intercepts not different from zero and slopes not different from 1, and coefficients of variation of 13.5 and 14.6%. When viewed with regard to the confidence intervals for Q as a function of O2 consumption, Q was altered to a minor extent. We conclude that the use of PET CO2 to estimate Pa CO2 can give reasonable values for Q determined noninvasively in patients with severe airflow limitation. PMID- 8630584 TI - Alveolar neutrophil functions and cytokine levels in patients with the adult respiratory distress syndrome during nitric oxide inhalation. AB - It was recently demonstrated that nitric oxide (NO) inhalation improves arterial oxygenation in patients with the adult respiratory distress syndrome (ARDS). However, the potential adverse reaction of NO on inflammatory cells and mediators in the lung has not yet been investigated. In this study, we evaluated the impact of NO inhalation on lung polymorphonuclear neutrophil (PMN) activation and proinflammatory cytokine release, both of which are involved in the pathophysiology of ARDS. Two groups of patients with ARDS of similar etiologies were compared; one received NO (n=9) and the other did not (n=5). After 4 d of NO inhalation (18 ppm), PMN form bronchoalveolar lavage (BAL) showed a reduction in both spontaneous H2O2 production (p<0.05) and beta 2 integrin CD11b/CD18 expression (p<0.05). Moreover, the high levels of IL8 and IL-6 decreased in BAL fluid supernatants after NO inhalation (p<0.05). In the NO-untreated group of patients with ARDS, neither PMN activation nor levels of IL-8 and IL-6 in BAL fluid changed significantly on Day 4. These results suggest that NO inhalation might reduce lung inflammation in ARDS, as reflected by PMN activation status and IL-8/IL-6 release. PMID- 8630585 TI - Direct comparison of inhaled nitric oxide and aerosolized prostacyclin in acute respiratory distress syndrome. AB - Inhalation of NO and aerosolization of PGI2 have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with ARDS. We directly compared these two modes of transbronchial vasodilator therapy in 16 ARDS patients mechanically ventilated (mean lung injury score [1] 2.75 +/- 0.05). Patients were randomized to receive either first NO and then PGI2, or vice versa. Each drug was individually titrated to find the maximum improvement of arterial oxygenation. Gas exchange variables, including data from the multiple inert gas elimination technique (MIGET), and hemodynamics under application of NO/PGI2 were compared with pre- and post-challenge values. NO (17.8 +/- 2.7 ppm) increased Pa O2/FI O2 from 115 +/- 12 to 144 +/- 15 mm Hg (p<0.01) and reduced the shunt-flow from 33.1 +/- 3.6 to 26.6 +/- 4.5% (p<0.05). Aerosolized PGI2 (7.5 +/- 2.5 ng/kg min) augmented Pa O2/FI O2 from 114 +/- 12 to 135 +/- 12 mm Hg (p<0.01), and decreased shunt from 33.5 +/- 3.8 to 26.0 +/- 3.9% (p<0.05). In 10 patients, both NO and PGI2 caused an increase in Pa O2/FI O2 by at least 10 mm Hg. Two further patients displayed an improvement of arterial oxygenation in response to either NO or PGI2. NO decreased mean pulmonary artery pressure from 34.8 +/- 2.2 to 33.0 +/- 1.8 mm Hg, and PGI2 from 35.0 +/- 2.2 to 31.9 +/- 1.7 mm Hg (p<0.05). We conclude that individually titrated doses of inhaled NO and aerosolized PGI2 effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to well-ventilated regions with nearly identical efficacy profiles. PMID- 8630586 TI - Evaluation of a knowledge-based system providing ventilatory management and decision for extubation. AB - We evaluated whether a knowledge-based system (KBS) connected to a ventilator in pressure support mode could correctly predict the ability of patients to tolerate total withdrawal from ventilatory support. The KBS was designed to continuously adapt ventilatory assistance to the needs of the patient, to manage a strategy of gradually decreasing ventilatory assistance, and to indicate when the patient was able to breathe without assistance. Thirty-eight patients for whom weaning was being considered were evaluated using a conventional battery of parameters, including weaning criteria, tolerance of a T-piece trial, and outcome 48h after permanent withdrawal of ventilation. The results of this evaluation were compared with the suggestions made by the KBS at the end of a period of KBS-driven mechanical ventilation inserted in the conventional weaning procedure. The positive predictive value of the KBS was 89%, versus 77% for the conventional procedure and 81% for the rapid shallow breathing index alone. The KBS correctly predicted the course of five patients who tolerated a T-piece trial but required ventilation within 48 h. We conclude that our KBS ensured appropriate patient management during the weaning period and improved our ability to predict responses to weaning. PMID- 8630587 TI - Analysis of breath sounds in bronchial provocation tests. PMID- 8630588 TI - We reap what we sow. PMID- 8630589 TI - Lung sounds during allergen-induced asthmatic responses in patients with asthma. AB - We postulated that the distinct pathophysiologic mechanisms of airway narrowing during the early (EAR) and the late (LAR) asthmatic responses to inhaled allergens are reflected by the generation or transmission of lung sounds in asthma. Therefore, we measured FEV1 and recorded lung sounds in eight mildly asthmatic subjects before a standardized allergen challenge (PRE), during the EAR, during the recovery phase at 2 h (MID), during the LAR at 7 h, and after inhalation of a bronchodilator (POST). The recordings were made during flow- and volume-standardized quiet breathing, and during maximal forced breathing maneuvers. Airflow-dependent power spectra were analyzed for lung sound intensity (LSI), quartile power points (Q25, Q50, Q75), and extent of wheezing (W). These sound characteristics were compared among the various stages of the challenge in the presence (EAR, LAR) and absence (PRE, MID, POST) of acute airway obstruction using ANOVA. LSI, Q25 - Q75, and W were all elevated during airway obstruction. When matched for percent fall in FEV1, during the EAR and the LAR (mean +/- SD: 26.7 +/- 4.0, and 28.9 +/- 5.7, respectively; p = 0.385), the increase in Q25, and Q50 with airflow during quiet expiration, as well as the extent of wheezing, were higher during the LAR than during the EAR (p < or = 0.042 and p < or = 0.012, respectively). At similar levels of FEV1 (p > or = 0.156), LSI on expiration was higher at POST than at PRE or MID (p < or = 0.067), whereas Q25 (p < or = 0.047) and Q50 (p < or = 0.064) were lower at POST than at PRE. During forced expiration W was higher at MID and POST than at PRE (p < or = 0.014). We conclude that LSI, frequency content, and the extent of wheezing vary during the subsequent stages of allergen-induced bronchoconstriction in asthma despite matched values of FEV1. This suggests that airflow-standardized phonopneumography is a sensitive method for detecting differences in the pathophysiology of airway narrowing in asthma. PMID- 8630590 TI - Comparison of addition of salmeterol to inhaled steroids with doubling of the dose of inhaled steroids. AB - A study was done to compare the efficacy and safety of the coprescription of salmeterol 50 microgram twice daily or 100 microgram twice daily with beclomethasone dipropionate (BDP) 500 micrograms twice daily (SALM 50 and SALM 100) with BDP 1,000 microgram twice daily (BDP 1,000) in patients with asthma not controlled by BDP 500 microgram twice daily (or the equivalent). Following a run in period, 738 patients at 72 centers were randomized to treatment for 24 wk in a double-blind, parallel-group study during which they maintained a daily record of peak expiratory flow rates (PEFRs) and symptom scores. At about 40 of the centers, bronchial hyperresponsiveness (BHR) to histamine was measured during and at 3 and 14 d after stopping treatment. Both groups taking salmeterol showed an improvement of more than 45 L/min in their morning PEFR and 30 L/min in their evening PEFR, compared with respective improvements of 16 L/min and 6 L/min in the group taking BDP 1,000. Both the SALM 50 and SALM 100 groups had a significantly increased percentage of symptom-free and rescue-free days and nights compared with the BDP 1,000 group, and there was no difference between the two salmeterol groups. None of the treatments altered BHR. Exacerbation rates did not differ among the three groups. We conclude that in this selected group of symptomatic patients taking BDP 500 micrograms twice daily, the addition of salmeterol provides better improvement in lung function and symptom control, without altering BHR or increasing exacerbation rates, than does doubling the dose of BDP. PMID- 8630591 TI - Functional and binding characteristics of long-acting beta 2-agonists in lung and heart. AB - Salmeterol and formoterol, two new long-acting beta 2-agonists were equipotent (values of negative log molar concentration eliciting half-maximal effect [pD2] 9.2 +/- 0.03 and 8.9 +/- 0.03, respectively) in relaxing maximally contracted guinea pig tracheal spirals (histamine, 100 microM). Both agonists were 10 times more potent than L-isoproterenol and fenoterol and 100 times more potent than albuterol. L-Isoproterenol and fenoterol induced > 90% relaxation (percentage of maximal aminophylline relaxation). Formoterol and albuterol were equally efficacious. Formoterol was more efficacious (86 +/- 5%) than salmeterol (62 +/- 3%) or soterenol (59 +/- 3%). In minimally contracted tissues (10 microM histamine), agonist potencies increased 10-fold and relaxation was complete. In [125I]iodocyanopindolol-labeled bronchial membranes, formoterol and salmeterol induced high-affinity states of the beta 2-receptor (pKh 9.6 +/- 0.4 and 10.4 +/- 0.7, respectively), the former inducing a higher percentage (57 +/- 6 versus 28 +/- 4, p < 0.05). Only low-affinity binding (pKI) was observed when guanine nucleotide was present. pD2 values were similar to pKh values and relative efficacies significantly correlated with percentage of pKI sites. Formoterol and salmeterol were highly selective for the beta 2 versus beta 1-subtype (pKI values were 8.2 +/- 0.09 and 6.25 +/- 0.06 and 8.3 +/- 0.04 and 5.7 +/- 0.04, respectively). Albuterol (5.83 +/- 0.06 and 4.71 +/- 0.16) and fenoterol (6.33 +/ 0.07 and 5.67 +/- 0.05) were less selective. These results can explain the potencies and efficacies of salmeterol and formoterol in humans. PMID- 8630593 TI - Effect of hypertonic saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. AB - In patients with cystic fibrosis (CF), dehydration of airway secretions leads to a decrease in mucociliary clearance (MCC). We examined the acute effect of MCC of a single administration by aerosolization of hypertonic saline (7%) (HS), amiloride (0.3% in 0.12% NaCl) (AML) and a combination of AML and HS (AML + HS) in 12 patients with CF using a radioaerosol technique. Isotonic saline [0.9%] (IS) was used as a control solution. As both the AML and HS solutions induced cough in some patients, the last nine patients studied also underwent a cough clearance day. This was to eliminate the possible confounding effect of cough on MCC measurement. Patients ranged from 18 to 28 yr (mean +/- SD, 22 +/- 3) with an FEV1 of 27 to 112% predicted (61 +/- 30%). Following deposition of the radioaerosol, baseline clearance was assessed for 30 min. This was followed by a 30-min intervention period. Assessment of post-intervention clearance for a further 30 min was then performed. Comparison of the amount of radioaerosol cleared from the right lung was made at 60 min (%C60) and 90 min (%C90) using repeated measures ANOVA. The percent cleared at 60 and 90 min was significantly increased with HS (%C60 = 26.5%, %C90 = 29.4%) and the combination of AML + HS (%C60 = 23.1%, %C90 = 27.4%) compared with both IS (%C60 = 14.7%, %C90 = 17.5%) and COUGH (%C60 = 18.0%, %C90 = 19.5%), p < 0.01. Inhalation of hypertonic saline is a potentially useful treatment in patients with cystic fibrosis. PMID- 8630592 TI - The tachykinin NK2 receptor antagonist SR 48968 inhibits citric acid-induced airway hyperresponsiveness in guinea pigs. AB - Airway hyperresponsiveness is a main feature of asthma, and several lines of evidence suggest that tachykinins might be involved in the pathogenesis of airway hyperresponsiveness in rodents. We conducted a study designed to describe an original model of airway hyperresponsiveness induced by citric acid administered as aerosol to guinea pigs, and to investigate the effects of the nonpeptide neurokinin1 (NK1) and neurokinin2 (NK2)-receptor antagonists, SR 140333 and SR 48968, respectively, on the development of this airway hyperresponsiveness. Animals received thiorphan 1 mg/kg intraperitoneally and 30 min later were exposed to an aerosol of citric acid 0.4 M for 1 h. After 24 h, the animals were anesthetized and ventilated. Airway hyperresponsiveness was evidenced by significant shifts to the left of dose-response curves for intravenous acetylcholine (ACh) without a change in maximum responses to ACh. Exposure to citric acid induced an airway hyperresponsive that was abolished by chronic pretreatment with capsaicin (120 mg/kg, 5 d before citric acid exposure). SR 48968 1 mg/kg intraperitoneally, given once at 30 min before the citric acid exposure, inhibited airway hyperresponsiveness, whereas SR 140333 1 mg/kg or codeine 30 mg/kg given under similar conditions did not. The inhibition of airway hyperresponsiveness by SR 48968 did not result from functional antagonism, since SR 48968 did not affect ACh-induced bronchoconstriction, nor did it result from inhibition of tachykinin, which could have been released under the influence of ACh in hyperresponsive animals, since SR 48968 given after the exposure to aerosolized citric acid failed to inhibit airway hyperresponsiveness. In conclusion, these results show that inhaled citric acid can induce the development of an airway hyperresponsiveness in the guinea pig through a release of tachykinins, and also demonstrate that NK2-receptor stimulation plays a predominant role in the development of airway hyperresponsiveness. PMID- 8630594 TI - Elevated nitric oxide concentrations in isolated lower airway gas of asthmatic subjects. AB - Previous studies have raised the possibility that the measurement of nitric oxide (NO.) concentrations in expired air may represent a noninvasive measure of lower airway inflammation. To address the question of whether the elevated NO. recovered in mixed expired air from asthmatic subjects is a reflection of the pulmonary airway microenvironment or merely nasopharyngeal contamination, mixed expired NO. determinations were performed in five normal and five asthmatic subjects before and after orotracheal intubation (thereby isolating the lower airway gas from ambient air contamination or gas conditioned in the nasopharynx). The mixed expired NO. concentrations determined in patients with asthma were significantly elevated (p < 0.05 or less) above those of normal subjects in both the pre- and postintubation samples. After intubation, mixed expired NO. levels were 4.7 +/- 1.3 ppb and 13.2 +/- 2.0 ppb in normal and asthmatic individuals, respectively; the difference in these values was statistically significant (p < 0.01). Lower airway gas, sampled through the bronchoscope during a breathhold, was found to contain NO. concentrations of 7.0 +/- 1.2 ppb and 40.5 +/- 5.6 ppb at the tracheal carina of normal and asthmatic individuals, respectively. The asthmatic values were significantly (p < 0.01) elevated above those found in normal subjects. These findings indicate that the difference in mixed expired NO. of normal subjects and asthmatics reflects a difference in NO. concentration present in the lower airway. PMID- 8630595 TI - Does oxygen help dyspnea in patients with cancer? AB - Dyspnea in patients with advanced cancer is a common symptom that is difficult to treat. This study investigated whether oxygen helps to relieve rest dyspnea in patients with advanced cancer. In a single-blind controlled trial, oxygen and air were administered in random order to hospice patients reporting dyspnea at rest. Measurements of arterial oxygen saturation, lung function, and dyspnea (using a visual analogue scale [VAS] and Borg score) were made before and after each gas had been given for 15 min. Data from 38 patients were used: analysis of variance revealed that mean VAS levels during baseline conditions, breathing room air (59 mm), were significantly reduced after administration of either air (48 mm; p < 0.001) or oxygen (45 mm; p < 0.001); there was no significant difference for the mean VAS scores between oxygen and air administration. There was no statistically significant order of treatment effect. There was no difference in the response to oxygen or air in patients with a history of cardiopulmonary disease. The improvement in dyspnea with oxygen could not be predicted from a subject's initial level of hypoxia. Results suggested that benzodiazepines may potentiate the effect of oxygen. The overall conclusion is that oxygen and air can have a significant effect in reducing dyspnea at rest in patients with advanced cancer. PMID- 8630596 TI - Eosinophil and neutrophil activity in asthma in a one-year trial with inhaled budesonide. The impact of smoking. AB - The object of this investigation was to study the long-term effects of antiasthma treatment on blood markers of inflammation and lung function in adult asthmatic subjects. For this purpose 85 allergic and nonallergic asthmatic subjects were randomized into three groups, which were given high-dose (1,600 micrograms/d) inhaled budesonide, low-dose (400 micrograms/d) inhaled budesonide, and oral theophylline (600 mg/d), respectively, and were followed for 11 mo with testing of lung function and blood sampling for the assay in serum of eosinophil cationic protein (ECP), eosinophil protein x/eosinophil derived neurotoxin (EPX/EDN) as eosinophil markers, and myeloperoxidase (MPO) and lactoferrin (LF) as neutrophil markers. Lung functions (FEV1% predicted, and histamine PC20) and the eosinophil markers ECP and EPX/EDN were improved and reduced, respectively, by budesonide in a dose-dependent and temporally parallel fashion. Theophylline did not alter lung functions but reduced ECP and EPX/EDN after prolonged treatment. The treatment efficacy of budesonide was attributed solely to an effect on nonsmoking asthmatic subjects, since neither lung functions nor eosinophil markers changed in smokers even with high-dose budesonide. MPO but not LF was reduced after several months of treatment in all three groups, but only in nonsmokers. We conclude that ECP and EPX/EDN may be used to monitor antiinflammatory treatment in asthmatic patients, and that smoking asthmatic subjects are resistant to inhaled corticosteroids. PMID- 8630597 TI - Association of chronic mucus hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity. Copenhagen City Heart Study Group. AB - The aim of this study was to examine the association between chronic mucus hypersecretion, and FEV1 decline, and subsequent hospitalization from chronic obstructive pulmonary disease (COPD). We used data from The Copenhagen City Heart Study on 5,354 women and 4,081 men 30 to 79 yr of age with assessment of smoking habits, respiratory symptoms, and spirometry at two surveys 5 yr apart. Information on COPD hospitalization during 8 to 10 yr of subsequent follow-up was obtained from a nationwide register. Chronic mucus hypersecretion was significantly associated with FEV1 decline; the effect was most prominent among men, where chronic mucus hypersecretion at both surveys was associated with an excess FEV1 decline of 22.8 ml/yr (95% confidence interval, 8.2 to 37.4) compared with men without mucus hypersecretion, after adjusting for age, height, weight change, and smoking; in women, the excess decline was 12.6 ml/yr (0.7-24.6). Chronic mucus hypersecretion was associated with subsequent hospitalization due to COPD after adjusting for age and smoking; relative risk was 5.3 (2.9 to 9.6) among men and 5.1 (2.5 to 10.3) among women. After further adjusting for FEV1 at the second survey, the relative risk was reduced to 2.4 (1.3 to 4.5) for men and 2.6 (1.2 to 5.3) for women. Chronic mucus hypersecretion was significantly and consistently associated with both an excess FEV1 decline and an increased risk of subsequent hospitalization because of COPD. PMID- 8630599 TI - Lack of a relationship of elastin peptide level to emphysema assessed by CT scans. AB - Clinical reports suggest that peptide (EP) concentration may be used as a subclinical marker of pulmonary emphysema. This hypothesis was tested in a clinical study by comparing EP concentration in male emphysematous patients with the level in two control groups, and by exploring the relation of elastin peptide level to high-resolution computed tomography (HRCT) scan emphysema score among emphysematous patients. Serum EP level was determined among male emphysematous patients with at least 20% of emphysema (n = 27) and in two populations of male workers, drawn from epidemiologic studies (227 coal miners and 310 policemen). No difference in elastin peptide concentration was observed between emphysematous patients and control subjects (mean +/- SD = 2.39 +/- 1.18 micrograms/ml in patients versus 2.55 +/- 1.34 micrograms/ml in policemen and 2.59 +/- 1.20 micrograms/ml in coal miners). The correlation of elastin peptide concentration with percentage of pulmonary emphysema was negative and of borderline significance (r = -0.36; p = 0.06). These results cast doubts on the usefulness of elastin peptide level as a predictive marker of pulmonary emphysema. PMID- 8630598 TI - Lung mechanics and dyspnea after lung transplantation for chronic airflow obstruction. AB - Single lung transplantation (SLT) is widely used to treat chronic airflow obstruction (CAO). During exercise the native lung should increase end-expiratory lung volume (EELV) and result in a different respiratory sensation compared with double lung transplantation (DLT). Eight SLT recipients and 12 DLT recipients demonstrated a similar maximal work load and achieved VO2. VEmax/MVV was 67.2 +/- 4.0% in SLT recipients and 48.5 +/- 3.6% in DLT recipients (p = 0.003). All SLT recipients demonstrated an increase in EELV during exercise, which was seen in only three of 12 DLT recipients. The change in absolute EELV from rest to peak exercise was different between SLT recipients (+0.37 +/- 0.10 L) and DLT recipients (-0.10 +/- 0.06, p = 0.0002). Tidal flow volume loop analysis demonstrated encroachment of the expiratory limb in four of seven SLT patients but in only one of 12 DLT recipients. A lesser peak breathlessness in DLT recipients approached statistical significance (p = 0.051), although the relation of respiratory sensation versus VE or VO2% predicted did not differ between the two groups. EELV increases in SLT recipients at peak exercise, although overall aerobic response is preserved and respiratory sensation is similar. PMID- 8630600 TI - Pulmonary fibrosis deaths in the United States, 1979-1991. An analysis of multiple-cause mortality data. AB - We sought to describe pulmonary fibrosis mortality in the United States from 1979 through 1991 by analyzing death certificate reports compiled by the National Center for Health Statistics. Of the 26,866,600 people who died during the study period, 107,292 had a diagnosis of pulmonary fibrosis listed on their death certificates. Among men, age-adjusted mortality rates increased from 48.6 per 1,000,000 in 1979 to 50.9 per 1,000,000 in 1991 and, among women, these rates increased from 21.4 per 1,000,000 in 1979 to 27.2 per 1,000,000 in 1991. Among both men and women rates were higher in older age strata than in younger age strata. Age-adjusted mortality rates were consistently higher among whites and people of races than blacks. The frequency with which pulmonary fibrosis was listed as the underlying cause of death increased from 40% in 1979 to 56% in 1991. Age-adjusted mortality rates varied by state, with lowest rates in the Midwest and Northeast, and the highest rates in the West and Southeast. We conclude that the age-adjusted rate of pulmonary fibrosis among decedents in the United States increased, and pulmonary fibrosis was listed as the underlying cause of death with increasing frequency, over the study period. We cannot determine whether the differences we detected between regions, sexes, and races are related to characteristics of the disease or problems in death certification and coding. PMID- 8630601 TI - Respiratory health of California rice farmers. AB - Rice farmers are occupationally exposed to agents that may affect respiratory health, including inorganic dusts and smoke from burning of agricultural waste. To assess respiratory health of this occupational group, we conducted a cross sectional study, including a self-administered health and work questionnaire, spirometry, and chest radiography among 464 male California rice farmers. Mean age +/- SD was 48.3 +/- 15.2 yr; mean duration of rice farming was 25.7 +/- 14.3 yr. Prevalences for respiratory symptoms were: chronic bronchitis (6.3%), physician-diagnosed asthma (7.1%), and persistent wheeze (8.8%). Chronic cough was reported by 7.1% of respondents and was associated with reported hours per year burning rice stubble. Mean FEV1 and FVC were at expected values. FEV1 was inversely associated with years working in rice storage and use of heated rice dryers. Mean FEF25-75 was 93% of expected and was inversely associated with rice storage activities involving unheated rice driers. ILO profusion scores > or = 1/0 for small irregular opacities were seen in 18 (10.1%) of 178 chest radiographs. Study findings suggest increased asthma prevalence among California rice farmers. Radiologic findings consistent with dust or fiber exposure were increased compared with those of the general population, although no associations with specific farming activities were identified. PMID- 8630602 TI - Coal mine dust exposure and spirometry in experienced miners. AB - In a previous study of new miners from the National Study of Coal Workers' Pneumoconiosis (NSCWP), researchers examined changes in spirometry values associated with coal mine dust exposure (Br J Ind Med 1993; 50:929-937). An unusual pattern of dust-related effects was observed: initial sharp decrements in FVC and FEV1 were followed by partial recovery. In the current study, similar methods were used to analyze data from experienced miners. Each of 1,915 male subjects contributed data from two of the NSCWP field surveys: either Round 1 (1969-71) and Round 2 (1972-75) and Round 4 (1985-88). From the cross-sectional analysis at Round 1 or Round 2 (R1/R2), changes of +0.6 ml FVC and -0.5 ml FEV1 were associated with each mg/m3-yr of cumulative coal mine dust exposure, but were not statistically significant (p > 0.05). From the analysis of longitudinal change in spirometry from R1/R2 to Round 4 (R4), annual declines in FVC (-0.10 ml/yr per mg/m3-yr, p = 0.003) and FEV1 (-0.07 ml/yr per mg/m3-yr, p = 0.006) were associated with pre-R1/R2 exposure. Both the pattern and the magnitude of the exposure-response relationship were different for experienced versus new miners. Possible reasons for these contrasts include differences in cumulative exposure between the two groups and the healthy worker effect among experienced miners. PMID- 8630603 TI - Lung hyaluronan decreases during group B streptococcal pneumonia in neonatal piglets. AB - Neonatal Group B streptococcus (GBS) sepsis and pneumonia result in lung injury and remain a major cause of morbidity and mortality in the newborn. Increased lung hyaluronan (HA) content is an important component of the lung's early response to damage in diseases such as adult respiratory distress syndrome (ARDS), infant respiratory distress syndrome (IRDS), and bleomycin-induced fibrosis. It is known, however, that GBS virulence factors include specific secretory enzymes such as hyaluronidase, an enzyme which breaks down HA. We therefore hypothesized that in lobar GBS pneumonia, lung HA would be decreased compared with normal values, and that in lobar pneumonia with atelectasis, lung HA would be further decreased because of increased substrate availability. The right lower lobes (RLL) and left lower lobes (LLL) of anesthetized piglets 16 +/- 2 d old were each selectively inoculated with 1 x 10(9) colony-forming units (CFU) GBS via an endobronchial catheter (n = 7). The LLL was subsequently collapsed by endobronchial occlusion following 10 min of 100% O2. Control animals (n = 6) was anesthetized, instrumented, and ventilated without exposure to GBS. At 4 h, lungs were removed and HA extracted and assayed using a competitive inhibition assay. HA extracted from areas of lobar pneumonia was significantly decreased (27 +/- 6.6 micrograms/g wet lung, p < 0.005) when compared with control values of control piglets (51 +/- 19.6 micrograms/g wet lung). Atelectasis plus lobar pneumonia further decreased lung HA to 10 +/- 13.3 micrograms/g wet lung, p < 0.0001. We conclude that lobar GBS decreases lung HA and that this process is augmented by collapsed lung regions, and speculate that this departure from the usual early lung response to injury contributes to GBS invasion of lung parenchyma. PMID- 8630604 TI - Efficacy of dead-space washout in mechanically ventilated premature newborns. AB - The prosthetic dead space makes a significant contribution to the total dead space in low-birth-weight premature newborns receiving artificial ventilation in response to respiratory distress. Use of an endotracheal tube with capillaries molded into the tube wall enables washout of the dead space without insertion of a tracheal catheter. In 10 premature newborns (mean gestational age, 27.5 +/- 2.2 wk; mean weight, 890 +/- 260 g) receiving continuous positive-pressure ventilation (Paw = 12.7 +/- 1.8 cm H2O; FIO2 = 39 +/- 17%), tracheal gas insufflation (TGI) for CO2 washout was conducted using this technique. The flow of tracheal insufflation (0.5 L/min) was derived from the inspiratory line of the ventilator circuit and blown into the trachea. Intratracheal pressures showed little or no TGI-related modification ( < 1 cm H2O). A control system enabled TGI discontinuation in the event of a pressure rise. At constant ventilation pressure, PaCO2 decreased by 12.1 +/- 5.9 mm Hg (delta PaCO2 = -26 +/- 12%) under TGI, whereas PaO2 remained unchanged. While maintaining PaCO2 constant, peak inspiratory pressure (PIP) was decreased by 5.4 +/- 1.7 cm H2O (delta PIP = -22.0 +/- 8.3%). TGI showed immediate efficacy (PCO2 reduction of at least 5 mm Hg) in nine of the 10 newborns who then received chronic TGI (14 to 138 h). TGI appears to be an effective method, suitable for long-term clinical application, enabling a reduction in the aggressive nature of conventional ventilation. PMID- 8630605 TI - Sepsis depresses the metabolic oxygen reserve of the coronary circulation in mature sheep. AB - This study was undertaken to describe the metabolic O2 reserve of the coronary circulation in an awake sheep model of hyperdynamic sepsis. Forty-eight hours after sheep were randomized to either a SHAM group (n = 8) or a cecal ligation and perforation (CLP) group (n = 8), we measured hemodynamics, organ blood flows, and systemic and myocardial O2 metabolism variables at baseline and through four stages of progressive hypoxia. A significant elevation in arterial lactate levels occurred at a higher O2 delivery in the CLP group (527 +/- 55 ml/min/m2) than in the SHAM group (357 +/- 29 ml/min/m2, p < 0.05). The heart's metabolic O2 reserve (difference in circulatory determinants of O2 availability between baseline and where O2 uptake could not be sustained) was exhausted at an O2 content of 56.9 +/ 4.2 ml O2/L in SHAM sheep and 79.6 +/- 7.2 ml O2/L (p < 0.05) in CLP sheep. An increase in coronary blood flow was three times greater in SHAM than in CLP animals. Myocardial O2 extraction increased in hypoxia in SHAM sheep (0.78 +/- 0.03 to 0.88 +/- 0.02, p < 0.05), but not in CLP sheep (0.79 +/- 0.02 to 0.80 +/- 0.04). We conclude that the metabolic O2 reserve of the coronary circulation is depressed in this model of hyperdynamic sepsis as the ability to increase both coronary blood flows and myocardial O2 extraction was significantly limited. PMID- 8630607 TI - Home versus intensive care pressure support devices. Experimental and clinical comparison. AB - A bench study using an artificial lung model and a clinical study in patients were performed to evaluate six commercially available home pressure support devices. Six devices were tested in the in vitro study, including five designed for home use and one designed for use in intensive care units. Minimal positive end-expiratory pressure (PEEP) varied across home devices, from 0.5 cm H2O to 4.3 cm H2O. Work imposed during exhalation varied up to six-fold across devices. A substantial rebreathing volume has present for the three home devices with a common inspiratory and expiratory line. This rebreathing volume decreased with increasing PEEP level, as expected, but remained substantial at the widely used PEEP level of 5 cm H2O. Use of a non-rebreathing valve increased both the work imposed by the circuit during the exhalation phase and the time required to attain the relaxation equilibrium. Except for two home devices and a bilevel positive airway pressure (BiPAP) device equipped with a non-rebreathing valve, differences in inspiratory trigger sensitivities were small between home and intensive care devices. During pressure support, the total work performed by the machines did not differ by more than 15% between devices, whereas differences of more than 300% were observed in flow acceleration. Only one home device gave a flow acceleration similar to or better than that obtained with the intensive care device. In a randomized, crossover clinical study, we compared a home device to a device specially designed for intensive care use in seven intubated patients during weaning from mechanical ventilation. The main differences between the two devices were trigger sensitivity and initial flow acceleration. For the same level of pressure support, there were no significant differences in arterial PCO2, tidal volume, respiratory rate, or minute ventilation between these two devices. However, the esophageal pressure-time product was 30% higher with the home device (165 +/- 93 versus 119 +/- 80 cm H2O/min, p < 0.05). In conclusion, differences exist between devices in terms of occurrence of rebreathing, speed of attainment of stable pressure support level, and expiratory resistance. These differences characterizing the delivery of pressure support may have clinical impact on the inspiratory effort of patients. PMID- 8630606 TI - Immunoglobulin A levels in bronchial samples during mechanical ventilation and onset of nosocomial pneumonia in critically ill patients. AB - Local immunoglobulins play a key role in host defense against lung infection. We investigated the pattern of evolution of bronchial albumin, IgA, and IgG levels in ventilated ICU patients in relation to nosocomial pneumonia. Immunocompetent, critically ill patients underwent serial blood and bronchial protein determinations on Day 1 (intubation day), and on Days 3, 7, 10, and 14. The variations in proteins levels were compared with corresponding Day 1 values in the whole population, and between patients who developed lung infections (Group A) and the remaining population (Group B). Forty-four patients were included into the study. In the whole population, when compared with the baseline value, bronchial IgA/albumin ratio increased significantly (Day 3, +58%, p = 0.04); Day 14, +171%, p < 0.01), but serum IgA/albumin and serum and bronchial IgG/albumin ratios did not change significantly. In Group A, the increase in the IgA/albumin ratio was less than in Group B (Day 3, +15% versus +87%, p = 0.04; Day 14, +29% versus +210%, p < 0.01). No significant differences were observed between the two groups for bronchial and plasma albumin and IgG levels and for bronchial polymorphonuclear elastase levels. Bronchial IgA production was enhanced in ventilated patients. A reduction in this enhanced bronchial IgA production might account for the development of nosocomial pneumonia. PMID- 8630608 TI - Local Pseudomonas instillation induces contralateral lung injury and plasma cytokines. AB - We investigated whether local bacterial instillation leads to lung injury in noninstilled lung regions and examined local and systemic cytokine accumulation. Rats were challenged by intrabroncheal instillation of Pseudomonas aeruginosa, 10(7) colony-forming units (CFU) (HD group, n = 11), 4 x 10(6) CFU (LD group, n = 10), or saline (control group, n = 12). 99mTc-labeled macroaggregated albumin was added to the P. aeruginosa or saline solution for later documentation of the instilled area. At 4 h the right lung, including instilled segment, and the left lung were sampled. Lung injury was assessed by lung tissue to plasma 125I-labeled albumin (T/P) and lung wet-dry (W/D) ratios. We measured plasma and bronchoalveolar lavage fluid (BALF) levels of tumor necrosis factor (TNF) and cytokine-induced neutrophil chemoattractant (CINC). HD bacterial instillation induced neutrophil recruitment and TNF and CINC elevation in BALF (p < 0.05) associated with increased T/P (p < 0.005) and W/D (p < 0.05) ratios in both instilled and the noninstilled lungs as compared with the saline-instilled and noninstilled controls. LD bacterial instillation induced neutrophil recruitment and TNF and CINC elevation only in the instilled lung (p < 0.05), and not in the noninstilled lung, and did not increase the T/P or W/D ratio. Plasma levels of TNF and CINC were increased in the HD, but not the LD, group when compared with the saline controls (p < 0.05). These data indicate that, when the dose is high enough to cause an excess inflammatory response, local bacterial instillation leads to neutrophil sequestration, lung injury, and cytokine elevation in the noninstilled lung associated with systemic cytokine release. PMID- 8630609 TI - Clinical efficacy of the amplified Mycobacterium tuberculosis direct test for the diagnosis of pulmonary tuberculosis. AB - The amplified Mycobacterium tuberculosis direct test (MTD) is a rapid diagnostic test based on a nucleic acid amplification technique, which can be used directly on processed clinical specimens. We evaluated the clinical utility of the MTD for diagnosing pulmonary tuberculosis by comparing the sensitivity and specificity of the test with acid-fast smear, mycobacterial culture, and clinical evaluation. The study included 844 respiratory tract specimens from 421 patients, which were submitted to the microbiology laboratory of our urban teaching hospital over a 6 mo period. Compared with culture, MTD had a sensitivity of 93.6% and specificity of 97.8%. MTD was more sensitive in detecting pulmonary tuberculosis in patients with previously undiagnosed disease (74.7%) than in those with established disease receiving chemotherapy (29.2%), and in smear-positive (95.5%) than in smear-negative (70.0%) disease. There were two false positive MTD results in patients with nontuberculous mycobacteria, for a specificity in this population of 97.3%. We conclude that MTD, when used in conjunction with routine smear and culture, is a useful rapid diagnostic test for suspected pulmonary tuberculosis. PMID- 8630610 TI - High-dose tumor necrosis factor alpha produces an impairment of hamster diaphragm contractility. Attenuation with a prostaglandin inhibitor. AB - We have investigated the influence of the cytokine tumor necrosis factor alpha (TNF alpha), an important mediator of sepsis, on in vitro hamster diaphragm contractility. Costal diaphragm strips were excised and mounted on an experimental apparatus consisting of a force transducer and servomotor. Preparations were randomized to incubation in one of the following solutions: (1) indomethacin 10(-6) M (n = 5); (2) TNF alpha (0.1 ng/ml) (n = 5); (3) TNF alpha (500 ng/ml) (n = 5); and (4) TNF alpha (500 ng/ml) plus indomethacin (10(-6)) (n = 5). Baseline contractile parameters measured at optimal length included twitch and tetanic tension, half relaxation time, time to peak tension, force frequency response (10-80 Hz), and fatigability to response to repetitive stimulation. After 90-min incubation in one of the solutions, an identical stimulation protocol was repeated. Initial twitch and tetanus parameters were similar between groups. Maximal twitch tension and tetanic tension decreased significantly, as did tetanic stimulations at 10-80 Hz in the TNF group (500 ng/ml) (p < 0.05). Coincubation with indomethacin decreased but did not completely abolish changes in diaphragm function caused by the higher dose of TNF. There were no significant changes in twitch or tetanus parameters, or in response to repetitive stimulation after incubation in the lower dose TNF group (0.1 ng/ml). We conclude that TNF causes impairment of in vitro diaphragm contractility at high incubation concentrations of TNF and that this effect can be partially blocked by prostaglandin synthetase inhibition. No significant deleterious effect on in vitro contractility was detected at concentrations of TNF similar to serum levels in human sepsis. PMID- 8630611 TI - Nasal two-level positive-pressure ventilation in normal subjects. Effects of the glottis and ventilation. AB - The purpose of this study was to examine the behavior of the glottis during intermittent positive-pressure ventilation (nIPPV) using a two-level positive pressure ventilator and to compare the glottic adaptation to this ventilatory mode with the one observed using volumetric ventilators, recently reported by us. Six healthy subjects were studied during both wakefulness and sleep. Their glottis was continuously monitored through a fiberoptic bronchoscope. We measured breath by breath the widest inspiratory angle formed by the vocal cords at the anterior commissure, the corresponding tidal volume, and other indices. We used the controlled ventilatory mode. The expiratory pressure was kept at 4 cm H2O, and the inspiratory pressure was increased by steps from 10 to 15 to 20 cm H2O. Increases in inspiratory pressure did not always lead to increases in effective ventilation reaching the lungs. This was due to a significant narrowing of the glottis by adduction of the vocal cords in all subjects. Periodic breathing with or without apneas were common during wakefulness, but especially during sleep, representing 10.5 +/- 11% (SD) of total sleep time. We conclude that effective ventilation during nIPPV using a two-level positive-pressure ventilator in the controlled mode is less predictable and less stable than during nIPPV using volumetric ventilators. PMID- 8630612 TI - Effects of non-REM sleep on the response of respiratory output to varying inspiratory flow. AB - It has been shown in mechanically ventilated awake normal humans that increasing inspiratory flow rate (VI) exerts an excitatory effect on respiratory output. It is not known if this effect persists during sleep. To test this, seven normal adults were studied during wakefulness and non-rapid eye movement (non-REM) sleep. Subjects were connected through a nose mask to a volume-cycled ventilator in the assist/control mode, and VI was increased in steps (3 to 4 breaths each) from 30 to 70 L/min and then back to 30 L/min. VI pattern was square, and all breaths were subject-triggered. Forty-one trials during non-REM sleep and 10 during wakefulness were analyzed. Both during sleep and wakefulness minute ventilation increased and total breath duration (Ttot) decreased significantly in a graded and reversible manner as VI increased. These changes were complete in the first breath after VI transition. The response was significantly less during sleep than during wakefulness (p < 0.050; at 30 L/min Ttot, expressed as percent of that at 70 L/min, was 110.2 +/- 1.3% during sleep and 127.8 +/- 3.9% during wakefulness. During wakefulness, the rate of change in airway pressure before triggering the ventilator (dp/dt), an index of respiratory drive, increased significantly (p < 0.05) with increasing VI. During sleep dp/dt was not affected by VI changes. In four sleeping subjects the increase in VI was sustained for 1.5 to 2 min. There was no evidence for adaptation of the response; Ttot, averaged over the last three breaths, did not differ from that obtained with VI was sustained for only 3 to 4 breaths. We concluded that VI exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism, and the gain of this reflex is attenuated by sleep. PMID- 8630613 TI - Measurement of exhaled nitric oxide by three different techniques. AB - The purpose of the study was to compare exhaled nitric oxide (NO) determined by three techniques. Ninety-one subjects performed a slow vital capacity maneuver: (1) through the mouth directly into a NO chemiluminescence analyzer (peak oral NO), (2) through the mouth into a collection bag (mean oral NO), and (3) through the nose into a collection bag (mean nasal NO). Peak oral NO was higher in patients with asthma (n = 18, 174.2 +/- 27.0 ppb), but lower in smokers (n = 36, 39.6 +/- 4.8 ppb) compared with nonsmoking control subjects (n = 23, 105.5 +/- 8.4 ppb, p < 0.05 both comparisons). Mean oral NO levels were significantly lower than peak oral NO levels (p < 0.05), but still higher in patients with asthma in comparison with nonsmoking healthy control subjects and asymptomatic smokers (27.2 +/- 3.5 versus 14.5 +/- 1.1 and 7.3 +/- 0.7 ppb, respectively, p < 0.05). In contrast, there was no significant difference in mean nasal NO levels between the three groups. Peak oral NO and mean oral NO levels correlated (r = 0.772, p < 0.0001). Determination of exhaled oral NO levels is qualitatively independent of the technique used, but nasal exhalation may affect NO determination in conditions associated with airway inflammation. PMID- 8630614 TI - The inhalation device influences lung deposition and bronchodilating effect of terbutaline. AB - The development of new inhalation devices for asthma drugs raises the issue of the relationship between pulmonary deposition and therapeutic effect of inhaled drugs in patients with obstructive lung diseases. We thus conducted a randomized, double-blind and double-dummy, four-period crossover study in 13 patients with moderate asthma (mean age 36 yr; FEV1 59% of predicted), who inhaled 0.25 and 0.5 mg terbutaline sulphate on separate occasions either via a pressurized metered dose inhaler (pMDI) or Turbuhaler (TBH). Pulmonary deposition was 8.1 +/- 2.7% and 8.3 +/- 2.3%, respectively, of the nominal dose for pMDI and 19.0 +/- 7.3%, and 22.0 +/- 8.1% for TBH. The FEV1 increase after 0.25 mg terbutaline sulphate via TBH was significantly greater than after 0.25 mg via pMDI. No significant differences in FEV1 increase were observed between 0.25 mg via TBH, 0.5 mg via pMDI, or 0.5 mg via TBH. Other lung function variables showed similar dose- and device-related changes. We concluded that: (1) the dose of terbutaline sulphate deposited in the lungs is dependent on which inhalation system is used; (2) TBH delivers about twice the amount of drug to the lungs as the pMDI; and (3) the observed difference in deposition is reflected in the bronchodilating effect. PMID- 8630615 TI - Variability of fine particle deposition in healthy adults: effect of age and gender. AB - Recent epidemiologic studies suggest increased mortality among the elderly in association with particulate air pollution. We investigated the variability in fractional deposition (DF) of inhaled particles (2 microns mass median aerodynamic diameter [MMAD]) in 62 subjects with normal lung function, aged 18 to 80 yr. Each subject inhaled 2-microns monodisperse carnauba wax particles while following a breathing pattern previously determined by respiratory inductance plethysmography in that subject (i.e., particles inhaled) was determined by laser aerosol photometry and pneumotachometry at the mouth. DF (mean DF = 0.29 +/- 0.06 (ages 18 to 40 yr), 0.29 +/- 0.07 (ages 41 to 60 yr), and 0.26 +/- 0.06 (age over 60 yr) was independent of age. There was a tendency toward greater DF in female than in male subjects; DF = 0.30 +/- 0.07 (females) and 0.27 +/- 0.06 (males) (p = 0.06); however, because the males had 45% higher minute ventilations than the females, the deposition rate (Drate), or particles depositing per unit of time, was 30% greater in males than in females (p = 0.004). Multiple regression analysis showed that among all subjects, the variability in DF was best predicted by variability in the breathing period (T) associated with the pattern used to breathe the particles, and by the subject's specific airway resistance (sRAW). These results may prove useful in determining age- and gender-relative risks that may be associated with the inhalation of pollutant particles in ambient air. PMID- 8630616 TI - Bronchial elastic fibers in normal subjects and asthmatic patients. AB - Elastic fibers required to maintain bronchial patency during ventilation may be damaged in asthma as a result of repair following inflammation or stretching during exacerbations. Fifteen normal subjects and 40 asthmatics of variable severity were studied. Bronchial biopsies were obtained from a subsegmental bronchus using a flexible bronchoscope. The elastic fibers were examined using orceine-eosine sustaining and/or immunohistochemistry with two monoclonal antibodies against elastin or transmission electron microscopy (six asthmatics and four control subjects). Orceine-eosine staining revealed that most normal subjects had normal fibers throughout the submucosa whereas of the 21 asthmatics analyzed only three had a normal superficial elastin network. In five patients, elastin had virtually disappeared. In the remaining patients, fibers appeared fragmented. The deeper layer of elastic fibers was abnormal in 17 asthmatics, fibers being patchy, tangled, and thickened. The fragmentation of the superficial network of elastic fibers shown in asthmatics was confirmed by immunohistochemistry. Electron microscopy studies suggested that the elastinolytic process and fragmentation of elastic fibers occurred in asthmatics. Elastinolysis occurs in the airways of asthmatics possibly as a result of repair elicited by chronic inflammation. Mechanical stretch induced by breathing and edema may lead to the fragmentation of fibers in asthmatic airways. PMID- 8630617 TI - Phenotypic and molecular characteristics of nasal mucosal gamma delta T cells in allergic and infectious rhinitis. AB - T cells expressing the T-cell receptor (TCR) gamma delta home in on various epithelia and may play an important role in local immunity to foreign antigens. The nasal mucosa is a potential site for chronic inflammatory diseases, yet little is known about the characteristics of nasal mucosal gamma delta T cells. Using flow cytometry, immunohistochemistry, and RT-PCR, we elucidated the characteristics of nasal mucosal gamma delta T cells in patients with perennial allergic rhinitis (PAR), chronic infective rhinitis (CIR), and seasonal allergic rhinitis (SAR) and in normal subjects. The gamma delta T cells were significantly increased in the nasal mucosa of patients with PAR (PAR, 24.3 +/- 4.5%; CIR, 12.9+/- 2.7%, p < 0.01), unrelated to those in autologous peripheral blood (PAR, 5.6 +/- 0.8%; CIR, 9.6 +/- 2.8%), and they were preferentially distributed in the epithelial compartment (26.7 +/- 2.3%) rather than in the lamina propria (5.4 +/- 2.5%). Of the expanded population of nasal mucosal gamma delta T cells in patients with PAR, CD4+ and CD4-8- gamma delta T cells were selectively increased (p < 0.01). Although nasal intraepithelial gamma delta T cells from all groups of patients and normal subjects dominantly expressed the V gamma 1/V delta 1 genes, and a bias for V gamma 3 gene expression was noted in those of patients with PAR, a significantly larger fraction of nasal mucosal gamma delta T cells in patients with PAR expressed the V gamma 1/V delta 1 TCR (p, 0.01), whereas those of the peripheral blood expressed the V gamma 2/V delta 2 TCR. More than 60% of V gamma 1/V delta 1 TCR+ cells in patients with PAR, were CD45RO+ ("memory cells"), independent of those in their peripheral blood (p < 0.01). Furthermore, a substantial proportion of nasal mucosal gamma delta T cells in patients with PAR synthesized IL-4 and IL-5 but negligible amounts of IFN-gamma. These observations of an increase in the proportions and activation of distinct subsets of nasal mucosal gamma delta T cells and their Th2-type cytokine profile in patients with PAR, unrelated to those in autologous peripheral blood suggest an important role for the oligoclonally expanded expanded nasal mucosal gamma delta T cells in the pathogenesis of PAR. PMID- 8630618 TI - Eosinophils, neutrophils, and venular gaps in the airway mucosa at epithelial removal-restitution. AB - Shedding of epithelium, increased venular permeability, and traffic of activated eosinophils and neutrophils may characterize asthmatic airways. This in vivo study involving briefly anesthetized guinea pigs examines whether epithelial denudation itself affects airway venules and granulocytes. Using an oral probe, a de-epithelialized tracheal zone (0.8 x 30 mm) was produced without bleeding or damage to the basement membrane. After 10 min, 2, 8, and 48 h, the tracheal tissue was examined by scanning and transmission electron microscopy. Silver staining revealed endothelial cell borders. Histochemistry identified neutrophils and eosinophils. Confirming previous observations, epithelial restitution started promptly and occurred speedily under a plasma exudation-derived, leukocyte-rich gel. Ten minutes after de-epithelialization, venular gaps (silver dots) were recognized as plasma exudation sites and, separately, silver rings at endothelial cell borders indicated attachment and extravasation of leukocytes. Tissue neutrophils were increased from 10 min to 48 h. Normally occurring eosinophils decreased in numbers during re-epithelialization, partly due to migration into the airway lumen and local cell death. Clusters of extracellular eosinophil granules were increased from 10 min to 8 h. Gentle removal of airway epithelium thus produced venular gaps, infiltration of neutrophils, and migration, activation, and death of eosinophils. Epithelial shedding-restitution processes may cause part of the microvascular and leukocyte changes that occur in inflammatory airway diseases. PMID- 8630619 TI - GM-CSF, IL-8, IL-1R, TNF-alpha R, and HLA-DR in nasal epithelial cells in allergic rhinitis. AB - Epithelial cells potentially contribute to airways inflammation by antigen presentation and the production of proinflammatory cytokines. This study investigated the immunocytochemical localization of interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor (IL-1R Type I), tumor necrosis factor-alpha receptor (TNF-alpha R; 55kD), and human leukocyte antigen-DR (HLA-DR) on epithelial cells obtained by nasal brushing from 10 patients with allergic rhinitis in season and 15 healthy, nonallergic subjects. Six of the 15 nonallergic asymptomatic subjects had macroscopic evidence of nasal mucosal inflammation, and their brushings contained more than 10% neutrophils ("subclinical inflammation"). In normal control subjects, 8 +/- 7.5% of epithelial cells stained for HLA-DR, approximately one quarter stained for IL-8 and GM-CSF, and about one third stained positive for IL 1R and TNF-alpha R. The findings in subjects with allergic rhinitis in season and with subclinical neutrophilia were similar, and the numbers of cells staining for HLA-DR expression correlated with both neutrophil and lymphocyte content. These findings further support the conclusion that epithelial cells can contribute to inflammatory processes in the nasal mucosa. The findings emphasize the need to identify asymptomatic nasal mucosal inflammation in studies of the nasal mucosa. PMID- 8630620 TI - Affected sib-pair and mutation analyses of the high affinity IgE receptor beta chain locus in Italian families with atopic asthmatic children. AB - Previous studies reported linkage between maternally inherited atopy and the beta subunit of the high affinity IgE receptor (Fc epsilon RI-beta) located on chromosome 11q13. We have investigated 45 Italian families with atopic asthmatic children, for a total of 213 subjects, including 148 patients. Genotyping was carried out with two microsatellite DNA markers: one (Fc epsilon RI-beta CA) located inside the gene, and one (CI11-319 CA) closely linked to it. Affected sib pair analysis in families with several affected children indicated 128 pairs in which either both markers were informative. An excess of maternal allele sharing was observed, although not significant. The allele-specific DNA amplification test for the FcRI-beta Ile181Leu mutation, described previously in 17% of atopic English families by Shirakawa and coworkers, was negative in all our families, as well as in 42 Italian children with atopic asthma and without family histories of the disease. PMID- 8630621 TI - Muscle weakness in mechanically ventilated patients with severe asthma. AB - Patients who undergo mechanical ventilation for severe asthma are at risk of developing diffuse muscle weakness because of acute myopathy. The relative importance of corticosteroids and neuromuscular paralysis in causing the myopathy is controversial, and it is uncertain whether the chemical structure of the drug used to induce paralysis influences the risk of myopathy. Using a retrospective cohort study design, we evaluated 107 consecutive episodes of mechanical ventilation for severe asthma to assess (1) the incidence of clinically significant weakness in patients treated with corticosteroids alone versus corticosteroids with neuromuscular paralysis, (2) the influence of the duration of paralysis on the incidence of muscle weakness, and (3) the relative risk of weakness in patients paralyzed with the nonsteroidal drug atracurium versus an aminosteroid paralytic agent (pancuronium, vecuronium). The use of corticosteroids and a neuromuscular blocking agent was associated with a much higher incidence of muscle weakness as compared with the use of corticosteroids alone (20 of 69 versus O of 38, p < 0.001). The 20 weak patients were paralyzed significantly longer than the 49 patients who received a neuromuscular blocking agent without subsequent weakness (3.4 +/- 2.4 versus 0.6 +/- 0.7 d, p < 0.001). Eighteen of the 20 weak patients had been paralyzed for more than 24 h. The incidence of weakness was not reduced when paralysis was achieved with atracurium as opposed to an aminosteroid neuromuscular blocking agent. In conclusion, corticosteroid-treated patients with severe asthma who undergo prolonged neuromuscular paralysis are at significant risk for the development of muscle weakness, and the risk of weakness is not reduced by use of atracurium. PMID- 8630622 TI - Bronchoalveolar lavage in adult sickle cell patients with acute chest syndrome: value for diagnostic assessment of fat embolism. AB - Fat embolism of necrotic bone marrow could be a frequent cause of acute chest syndrome (ACS) in sickle cell syndromes (SC), as suggested by postmortem findings. To check this hypothesis in living patients, we evaluated the presence of fatty macrophages recovered by bronchoalveolar lavage (BAL) in ACS. We investigated 20 consecutive cases of ACS by BAL, and identification of alveolar cells containing fat droplets was performed using oil red O (ORO), a specific neutral fat stain. The specificity of the method was determined on control groups, including eight SC patients without acute chest syndrome and 15 non-SC patients. A cut-off of > 5% of alveolar macrophages containing fat droplets was determined from the control groups to assess the diagnosis of fat embolism. In 12 ACS episodes, BAL exhibited > 5% of fatty macrophages, ranging from 10% to 100% (median value 46.5%). In 11 cases, fat embolism was associated with proven (n = 8) or probable (n = 3) bone marrow infraction, which mostly predated ACS. Eight ACS episodes were associated with a low percentage (< or = 5%) of fatty alveolar macrophages and could be related to a cause other than fat embolism in six episodes, such as sepsis, in-situ thrombosis, or rib infarcts generating hypoventilation. This study supports the diagnostic yield of BAL for fat embolism, which can be incriminated in 60% of cases of ACS in this adult population. PMID- 8630623 TI - Peroxynitrite induces airway hyperresponsiveness in guinea pigs in vitro and in vivo. AB - Peroxynitrite (ONOO-) is a cytotoxic product of the rapid reaction between nitric oxide and superoxide that may initiate inflammation. Isolated perfused tracheas from guinea pigs were incubated from the mucosal side for 15 min with peroxynitrite (1 to 100 muM). Thereafter, concentration-response curves to histamine and methacholine were constructed on the preparations. Peroxynitrite (10 muM) caused a significant hyperresponsiveness; the maximal contractions in response to histamine and methacholine were enhanced by 30% and 40%, respectively. In the peroxynitrite-treated group, clear epithelial damage as well as eosinophil destruction were detected. Moreover, 3, 5, and 10 days after intratracheal instillation of peroxynitrite (100 nmol), a significant rise in pulmonary resistance to histamine of anesthetized animals was observed. It is suggested that the generation of peroxynitrite from nitric oxide superoxide radicals during inflammatory processes induces epithelial damage, mediator release, and hence airway hyperresponsiveness. These findings may have clinical implications, because airway inflammation, epithelial damage, and hyperresponsiveness are characteristic features in patients suffering from asthma. PMID- 8630624 TI - Time course of the protective effect of inhaled heparin on exercise-induced asthma. AB - We have previously shown that heparin attenuates allergic bronchoconstriction in sheep, inhibits anti-IgE mediated histamine release in isolated mast cells, and prevents the bronchoconstrictor response in subjects with exercise-induced asthma (EIA). The purpose of the present study was to determine the pharmacokinetics of anti-asthmatic activity of inhaled heparin in EIA and compare it with cromolyn sodium, a mast cell stabilizing agent with established efficacy in EIA. Nine subjects with a history of EIA were studied on 10 different experiment days. After obtaining baseline pulmonary functions on day 1, subjects performed a standardized exercise challenge to document the presence of EIA. While monitoring minute ventilation and heart rate, exercise challenge was performed on a treadmill with increasing workload, until 85% of predicted maximum heart rate was achieved. The subjects then continued the exercise at that workload for 10 min. EIA was assessed by measurements of specific airway conductance (SGaw) before and after exercise. On experiment days 2-10, the exercise challenge was performed after the subjects inhaled 4 ml of either heparin (20,000 u/ml), cromolyn (20 mg), or placebo solutions with increasing pretreatment intervals of 15 min, 1 h, 3 h, or 6 h in a single-blind, randomized fashion. Maximum decreases in SGaw (mean +/- SE) at 3 to 5 min after exercise on control (39 +/- 2.1%) and placebo (37 +/- 2.6%) days were reproducible. Heparin and cromolyn had no effect on baseline SGaw but attenuated the EIA in a time-dependent fashion. Heparin inhibited the bronchoconstrictor responses to exercise by 58%, 78%, and 67% (p < 0.05) when nebulized 15 min, 1 h and 3 h, respectively, before exercise; cromolyn attenuated the responses by 37%, 46%, and 41%, respectively, (p < 0.05). Although heparin offered greater protection than cromolyn (at 1 to 3 h), both agents were ineffective when administered 6 h before exercise. These data demonstrate that inhaled heparin prevents EIA for up to 3 h and is more effective than cromolyn. PMID- 8630626 TI - Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies. A consensus statement, American Thoracic Society, November 1995. PMID- 8630625 TI - Relationship of isoniazid resistance to human immunodeficiency virus infection in patients with tuberculosis. AB - To investigate the relationship between isoniazid resistance and HIV infection in patients with tuberculosis, we evaluated data in the Los Angeles County tuberculosis registry on 1,506 patients for whom drug susceptibility results were available. Among 235 HIV-infected patients, isoniazid resistance was less common than in 1,271 patients who were HIV-seronegative or who had not been tested for HIV, with an unadjusted odds ratio of 0.3. After adjustment for other factors that affect drug resistance (ethnicity, country of birth, prior diagnosis of tuberculosis, and cavitation), the frequency if isoniazid resistance remained lower than that in patients without HIV infection, with an odds ratio of 0.4 (95% confidence interval, 0.2 to 0.8; p = 0.02). We conclude that in Los Angeles, a setting where there is no ongoing outbreak of drug-resistant tuberculosis, isoniazid-resistant tuberculosis is not more common in HIV-infected patients. PMID- 8630627 TI - Inhaled beclomethasone dipropionate and growth in children with mild asthma. PMID- 8630629 TI - Introduction: mechanism of action of NSAIDs. PMID- 8630628 TI - Airway eosinophilia in chronic bronchitis during exacerbations. PMID- 8630630 TI - A review of the clinical pharmacokinetics of meloxicam. AB - Meloxicam is a new preferential cyclooxygenase-2 (COX-2) inhibitor currently for the treatment of osteoarthritis and rheumatoid arthritis. Its pharmacokinetic profile is characterized by a prolonged and almost complete absorption and the drug is > 99.5% bound to plasma proteins. Meloxicam is metabolized to four biologically inactive main metabolites, which are excreted in both urine and faeces. The elimination half-life (t1/2) of meloxicam is approximately 20 h. This is reflected in a total plasma clearance (CL) of 0.42-0.48 1/h. Steady-state plasma concentrations are achieved within 3-5 days. The pharmacokinetic parameters of meloxicam are linear over the dose range 7.5-30 mg and bioequivalence has been shown for a number of different formulations. No interactions were observed following the concomitant administration of food, cimetidine, antacid, aspirin, beta-acetyldigoxin, methotrexate, warfarin or furosemide. Neither hepatic insufficiency nor moderate renal dysfunction have any relevant effects on the pharmacokinetics of meloxicam and dosage adjustments in the elderly are not required. PMID- 8630631 TI - A double-blind, three-week study to compare the efficacy and safety of meloxicam 7.5 mg and meloxicam 15 mg in patients with rheumatoid arthritis. AB - A multicentre, double-blind, randomized study was conducted in patients with rheumatoid arthritis (RA) in order to compare the efficacy and safety of two different doses of meloxicam, a new preferential cyclooxygenase-2 (COX-2) inhibitor. Four hundred and twenty-three patients were randomized to receive once daily oral meloxicam 7.5 mg (n = 216) or meloxicam 15 mg (n = 207) for 3 weeks. The Ritchie joint index and pain in the morning were significantly improved versus baseline (P < 0.001) in both groups. There were no significant differences between the effects of each dose with respect to these measures nor with respect to final assessment of global efficacy by the patients. However, the 15 mg dose was associated with a significantly (P < 0.05) better effect on morning stiffness and grip strength. No differences between the doses were observed with regard to the other secondary efficacy parameter (pain at night, body weight and erythrocyte sedimentation rate). Both doses of meloxicam were well tolerated. There were no differences between the doses with respect to global tolerance as assessed by the patient and the patients, 'general condition'. In conclusion, meloxicam at a once-daily dose of either 7.5 or 15 mg is well tolerated and effective in the treatment of patients with RA. PMID- 8630632 TI - A six-month double-blind trial to compare the efficacy and safety of meloxicam 7.5 mg daily and naproxen 750 mg daily in patients with rheumatoid arthritis. AB - Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which preferentially inhibits cyclooxygenase-2 over cyclooxygenase-1. A double-blind parallel-group trial compared meloxicam 7.5 mg once daily (n = 199) with naproxen 750 mg (n = 180) in rheumatoid arthritis. There was no significant difference between the groups regarding the primary efficacy variables (global efficacy assessment by patient and investigator, number of painful/tender and swollen joints) and eight of the ten secondary efficacy endpoints. Only the swollen joint severity index and the number of discontinuations due to lack of efficacy favoured naproxen 750 mg significantly over meloxicam 7.5 mg. Meloxicam was better tolerated in the gastrointestinal (GI) tract, with fewer GI adverse events in the meloxicam-treated group (30.3%) than in the naproxen-treated group (44.7%), where two patients developed ulcers. No ulcers were seen in meloxicam patients. Significantly more patients discontinued due to GI adverse events in the naproxen group. Additionally, there was a significant decrease in haemoglobin and a significant increase in serum creatinine and urea in the naproxen group compared with the meloxicam group. In conclusion, meloxicam 7.5 mg once daily is a promising treatment in rheumatoid arthritis, with efficacy comparable to naproxen 750 mg. Meloxicam has the advantage of a significantly lower incidence of GI and renal side effects. PMID- 8630633 TI - A long-term study to evaluate the safety and efficacy of meloxicam therapy in patients with rheumatoid arthritis. AB - Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID), which has a higher activity against cyclooxygenase-2 (COX-2) than against cyclooxygenase-1 (COX-1), with potentially high anti-inflammatory and analgesic action. This study was designed to assess the long-term safety and efficacy of meloxicam 15 mg daily. Three hundred and fifty-seven patients (aged 19-84 yr, mean 56 yr) with rheumatoid arthritis (RA) received meloxicam 15 mg orally once daily, for up to 18 months. Sixty-six per cent of patients remained on therapy for 18 months. Mean global efficacy, assessed by each patient on a visual analogue scale (0 cm = excellent, 10 cm = useless), was 3.32 +/- 3.1 cm at the last study visit (all patients included) and 2.33 +/- 2.25 cm after 18 months. Health status, general condition, morning stiffness, grip strength of right hand, Ritchie joint index, pain in the morning and pain at night all improved significantly. Efficacy was maintained through the study. Only 11.4% of patients discontinued prematurely due to lack of efficacy. Mean global tolerance was good. Twenty-eight per cent of patients experienced gastrointestinal (GI) adverse events, 21% musculoskeletal system disorders, 18% skin disorders and 15% respiratory disorders. Only 13.7% of patients discontinued due to adverse events. Severe GI effects, such as perforation, ulcer and bleeding, occurred in only three patients (0.8%). Withdrawals due to GI adverse events occurred in 3.9% of patients. Meloxicam 15 mg once daily was effective and compared favourably with standard NSAIDs regarding tolerance when administered to patients with RA over an 18 month period. PMID- 8630634 TI - A double-blind study to compare the efficacy and safety of meloxicam 15 mg with piroxicam 20 mg in patients with osteoarthritis of the hip. AB - Meloxicam 15 mg once daily (n = 128) was compared with piroxicam 20 mg (n = 127) in this 6 week, double-blind, parallel-group, randomized, multicentre study in out-patients with symptomatic osteoarthritis (OA) of the hip. Assessments of pain, global efficacy and global tolerance were made on a 10 cm horizontal visual analogue scale; severity of OA was evaluated by Lequesne's index. Efficacy results showed significant improvement compared with baseline, with no significant difference between meloxicam 15 mg and piroxicam 20 mg. The type and frequency of adverse events were comparable for the two drugs. The most frequent events reported were gastrointestinal (GI) disorders, occurring in 21 and 23% of meloxicam and piroxicam patients respectively. The global tolerance assessment by patients at the end of treatment favoured meloxicam. In conclusion, meloxicam at a dose of 15 mg/day is comparable in efficacy and safety to piroxicam 20 mg. PMID- 8630635 TI - Meloxicam in osteoarthritis: a 6-month, double-blind comparison with diclofenac sodium. AB - A multicentre, double-blind, randomized study was conducted in patients with osteoarthritis (OA) of the hip or knee in order to compare the efficacy and safety of the new cyclooxygenase-2 (COX-2) inhibitor, meloxicam, with diclofenac sodium, a conventional treatment for this condition. Three hundred and thirty-six patients were treated with oral meloxicam 7.5 mg once daily or diclofenac 100 mg slow release once daily for 6 months. There were no significant differences between the treatment groups with respect to overall pain, pain on movement, global efficacy or quality of life scores at the end of treatment, all of which showed good levels of improvement. Sixty-six patients were withdrawn after the start of the double-blind phase due to adverse events (n = 21, meloxicam; n = 31, diclofenac) or to lack of efficacy (seven in each group). The median of dose paracetamol taken concomitantly was statistically significantly lower in the meloxicam group than in the diclofenac group (185 vs 245 mg/day; P = 0.0123) with a comparable proportion of patients taking concomitant paracetamol therapy in both groups. Both drugs were well tolerated, although severe adverse events, treatment withdrawal and clinically significant laboratory abnormalities were more common with diclofenac than with meloxicam. Thus, meloxicam 7.5 mg is a safe and effective treatment for OA of the hip and knee which demonstrates a trend towards an improved safety profile compared with diclofenac. PMID- 8630636 TI - Pharmacology of meloxicam, a new non-steroidal anti-inflammatory drug with an improved safety profile through preferential inhibition of COX-2. AB - This review focuses on the key pharmacological findings with a new NSAID, meloxicam. Unlike established NSAIDs, it preferentially inhibits inducible COX-2 in guinea-pigs peritoneal macrophages and human COX-2 in COS cells. Compared with other NSAIDs, meloxicam is the most potent inhibitor of prostaglandin biosynthesis in pleural and peritoneal exudate, but only a weak inhibitor in the gastric tract and kidney. Ulcerogenicity in the rat stomach is weak in relation to anti-inflammatory potency, resulting in a high therapeutic index. Meloxicam's high anti-inflammatory potency combined with good tolerability can be explained by its preferential inhibition of COX-2. In adjuvant arthritis rats, meloxicam inhibits not only paw swelling, but also bone and cartilage destruction and systemic signs of disease. It inhibits leucocyte migration, but has no effect on leucotriene B4 or C4. Meloxicam shows a long-lasting anti-inflammatory and analgesic effect on inflammatory pain and reduces pyrogen-induced fever, but has no central nervous system effects. The pharmacokinetic profile of meloxicam in the rat is similar to that in man. Metabolites are inactive. PMID- 8630637 TI - Local tissue tolerability of meloxicam, a new NSAID: indications for parenteral, dermal and mucosal administration. AB - Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which has potent anti-arthritic activity and a reduced potential to induce gastric irritation in animals. The present series of animal studies investigated the local and/or systemic tolerance of meloxicam formulations: intravenous, intramuscular and subcutaneous injections, eye-drops, gel and suppositories. The concentration and formulations were as intended for therapeutic use in man. An in vitro haemolysis test demonstrated that the parenteral formulation of meloxicam produced only minimal haemolysis. In comparison, NSAIDs such as piroxicam, ketoprofen and indomethacin showed comparable haemolysis only after dilution. Diclofenac and ibuprofen caused considerable haemolysis even when diluted. In all studies, the local tolerance of meloxicam was good and did not differ from placebo, even when administered daily for 4 weeks. Few abnormal histopathological findings indicative or organ toxicity were observed. There were only small, transient macroscopic changes at the site of administration, with no striking histopathological changes directly attributable to meloxicam. Intramuscular piroxicam and diclofenac, however, resulted in development of an extensive, solitary necrotic area. Other formulations tested were also very well tolerated. In conclusion, all meloxicam formulations tested exhibited excellent tissue tolerability. Therefore, meloxicam appears to be suitable for parenteral, dermal and mucosal administration. PMID- 8630639 TI - An open study to assess the safety and tolerability of meloxicam 15 mg in subjects with rheumatic disease and mild renal impairment. AB - Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which has shown potent anti-inflammatory properties but good gastrointestinal (GI) renal tolerability. The safety and tolerability profile of orally administered meloxicam 15 mg given once daily over a 28 day treatment period in renally impaired patients with rheumatic disease is presented here. A total of 25 patients (aged 43-78 yr, mean age 70 yr) with rheumatic disease and mild renal impairment were enrolled in this multicentre, open-label study, with 22 patients completing the 28 day treatment period. The median estimated creatinine clearance and N-acetyl-beta-glucosaminidase/creatinine ratios (a marker of renal tubular damage) recorded at day 14, day 28 or 4-7 days after meloxicam treatment was terminated, were not statistically significantly different from baseline values. There was no evidence of accumulation of meloxicam. Overall, meloxicam was well tolerated. The most common adverse events were GI complaints of abdominal pain and dyspepsia. No adverse events related to the urinary system, or increases in serum urea or potassium were recorded. The results suggest that meloxicam, 15 mg once daily, does not further compromise renal function or result in accumulation of meloxicam over this treatment period in patients with pre-existing mild renal impairment. PMID- 8630638 TI - Tolerability of multiple administration of intramuscular meloxicam: a comparison with intramuscular piroxicam in patients with rheumatoid arthritis or osteoarthritis. AB - This multicentre, randomized, open controlled study compared the local and overall tolerability of i.m. meloxicam with i.m. piroxicam in 211 patients with rheumatoid arthritis (RA) (n = 95) or osteoarthritis (OA) (n = 116). Of these, 210 patients were randomized (2:1) to receive meloxicam 15 mg (n = 144) or piroxicam 20 mg (n = 66) for 7 days. Local tolerability of meloxicam was significantly better than piroxicam with respect to occurrence of redness after the first injection (P = 0.03) and global assessment after the first and final injections (P < 0.05). No rise in creatinine phosphokinase levels (a marker of muscle fibre damage) was observed with meloxicam, in contrast to piroxicam (P = 0.0001). The overall tolerability of both treatments was good. Significant differences in favour of meloxicam were observed for global efficacy assessed by the patient in RA (P < 0.05) and for overall pain intensity in OA patients (P = 0.02). In conclusion, i.m. meloxicam is safe and effective for the treatment of acute rheumatic pain and shows some superiority over piroxicam. PMID- 8630640 TI - A 4-week, double-blind, parallel-group study to compare the gastrointestinal effects of meloxicam 7.5 mg, meloxicam 15 mg, piroxicam 20 mg and placebo by means of faecal blood loss, endoscopy and symptom evaluation in healthy volunteers. AB - Meloxicam is a new non-steroidal anti-inflammatory drug (NSAID) which preferentially inhibits cyclooxygenase-2 (COX-2) over cyclooxygenase-1 (COX-1). Gastrointestinal (GI) tolerability of meloxicam 7.5 and 15 mg vs piroxicam 20 mg was evaluated in a 4-week, double-blind, parallel group, placebo-controlled study in 51 healthy male volunteers, using a combination of oesphago-gastro-duodenal endoscopy, faecal blood loss measurement and symptom evaluation. Analysis of covariance found no significant difference in faecal blood loss between the groups. However, significantly higher bleeding was found with piroxicam 20 mg compared with placebo using a Student's t-test on the weighted means. Endoscopy score were significantly higher with piroxicam than with meloxicam 7.5 mg or placebo (P < 0.01). A significant difference from baseline was observed in the meloxicam 15 mg and piroxicam groups (P < 0.05), but not in the meloxicam 7.5 mg and placebo groups. Six piroxicam-treated volunteers were withdrawn following a poor endoscopic score, but no such withdrawals occurred in the meloxicam and placebo groups (P < 0.01). Meloxicam 7.5 mg caused less GI damage compared with piroxicam 20 when administered to healthy young volunteers for 28 days; a possible dose dependency effect in GI tolerability was also suggested for meloxicam 7.5 and 15 mg, in relation to endoscopic findings. PMID- 8630641 TI - Safety of meloxicam: a global analysis of clinical trials. AB - Meloxicam is a new preferential cyclooxygenase-2 (COX-2) inhibitor for the treatment of rheumatic disease. This paper presents a global safety analysis of data from meloxicam clinical studies, focusing on gastrointestinal (GI) adverse events. Meloxicam 7.5 and 15 mg (n = 893 and 3282) were compared with piroxicam 20 mg (n = 906), diclofenac 100 mg slow release (n = 324) and naproxen 750-1000 mg (n = 243). With respect to all GI adverse events, meloxicam 7.5 and 15 mg were significantly better than all comparators in a pooled analysis of double-blind studies in rheumatoid arthritis (RA) and osteoarthritis (OA). When examining non serious GI events, severe GI events, discontinuous due to GI events, dyspepsia, abdominal pain and upper GI events, both meloxicam doses were significantly better than comparator non-steroidal anti-inflammatory drugs (NSAIDs) in most cases. Where statistical significance was not demonstrated, there was generally a trend in favour of meloxicam. With respect to upper GI perforations, ulcerations and bleedings, the most serious of NSAID-associated side-effects, meloxicam was better tolerated than the comparators, reaching statistical significance for piroxicam and naproxen. Meloxicam's improved GI safety profile is likely to be due to its preferential inhibition of inducible COX-2 relative to constitutive COX-1. PMID- 8630643 TI - Psychological correlates of adolescent depression. AB - PURPOSE: To identify psychological correlates of adolescent depression. METHODS: The subjects (N = 156) represented a convenience sample of adolescents who were attending public high schools in four Midwestern rural communities. Instruments used were: The Center for Epidemiologic Studies Depression Scale of Children, Loneliness Inventory-Short Form, Rosenberg Self-Esteem Scale, Family Strengths Inventory, and Parent-Adolescent Communication Inventory. FINDINGS: Higher levels of depression were associated with loneliness (r = .646, p < or = .001) and low self-esteem (r = .596, p < or = .001). Depression was negatively related to family strengths (r = -.293, p < or = .001). Older adolescents were more depressed than younger adolescents (r = .332, p < or = .001). A stepwise regression identified three pertinent factors associated with depression: loneliness, self-esteems, and age. CONCLUSION: Implications for nursing practice include interventions that minimize the exacerbating factors associated with depression in the adolescent population. PMID- 8630642 TI - What maintains the myth of the wicked stepmother? AB - TOPIC: A critical analysis of the myth of the wicked stepmother, its origins and social purposes, as well as factors that influence the myth and contribute to its maintenance. PURPOSE: To explore the myth of the wicked stepmother as represented in literature for professionals, parents, and children regarding the dynamics of relationships between children and stop-parents. SOURCES: Published literature and clinical observations. CONCLUSIONS: A cyclical process can be identified by reviewing the influence of myth on relationships between stepchildren and stepmothers as well as the influence of stepfamily relationships on the maintenance of myth. Solutions for stepfamily relationships require broader possibilities than the dichotomous answers provided by myth. Answers to many of the questions raised concerning the complexities of stepfamily life require an increased focus on clinical research. PMID- 8630644 TI - Threads in the professional fabric. PMID- 8630645 TI - Toward the national plan for research in child and adolescent mental health disorders: midterm report card. PMID- 8630646 TI - An interview with Carol Bush, PhD, RN. Interview by Linda M. Finke. PMID- 8630647 TI - Pharmacologic treatment of children and adolescents with obsessive-compulsive disorder. PMID- 8630648 TI - Adolescents' perceived barriers to healthcare services. AB - PURPOSE: To explore adolescents' perceptions of information needed to feel both mentally and physically healthy and their perceptions of barriers that exist to obtaining health-care services related to these needs. METHODS: A convenience sample of 64 adolescents ages 11-18 years, living in a metropolitan southern community. Content analysis was used to analyze the written responses of the subjects to open-ended questions. FINDINGS: Adolescents wanted information about what activities were defined as healthy and unhealthy, including facts about exercise, nutrition, and expressing feelings. Perceived barriers included money, time, personal characteristics, parents, and unavailability of appropriate resources. CONCLUSIONS: Findings have implications for designing services to meet the mental health care and educational needs of adolescents. Involving adolescents in the planning of these services is an important step in promoting health and self-care competence. PMID- 8630649 TI - One-to-one midwifery. PMID- 8630650 TI - Preparation for caseload management. PMID- 8630651 TI - How to find out what women want. PMID- 8630652 TI - Maternal infections. Part 2: Cytomegalovirus. PMID- 8630653 TI - Supporting families with a very low birthweight baby. PMID- 8630654 TI - Chlorhexidine swabbing in labour. PMID- 8630655 TI - When is a nurse a midwife? PMID- 8630657 TI - Support in labour. PMID- 8630656 TI - Watch out for stormy weather ahead. PMID- 8630658 TI - Fibrinogen is a predictor of mortality in coronary heart disease patients. The Bezafibrate Infarction Prevention (BIP) Study Group. AB - Results of epidemiological studies have indicated that fibrinogen is an important primary cardiovascular risk factor. The role of fibrinogen as a predictor of mortality in coronary heart disease (CHD) patients is unclear. We investigated the association between fibrinogen and mortality in a large cohort of CHD patients screened for participation in a secondary prevention clinical trial. Of the total investigated, 3092 men who were not included in the trial and for whom vital status was known were followed up for a mean period of 3.2 years. In 54.4% of the 111 men who died, mortality was attributed to CHD. Mean baseline plasma fibrinogen levels were 29.4 mg/dL higher in patients who died than in the survivors. All-cause and CHD mortality rates increased with increasing fibrinogen levels. This relationship was also demonstrated within categories of the primary variables predicting mortality in these patients. The contribution of fibrinogen to CHD and all-cause mortality was assessed by multivariate analysis adjusting for age, CHD severity, and comorbidity. Risk of CHD and all-cause mortality for patients in the highest fibrinogen tertile were 1.67 and 1.75, respectively, relative to patients in the lowest tertile, and an increase of about 1 SD of plasma fibrinogen level (75 mg/dL) was found to increase risk of CHD and all cause mortality 29% and 31%, respectively. These results indicate clearly that fibrinogen level is associated with significantly increased mortality in CHD patients. Implementation of a standardized measuring method is required to allow assessment of risk in CHD patients on the basis of fibrinogen levels. PMID- 8630659 TI - Antithrombin III and fibrinogen as predictors of cardiac events in patients with angina pectoris. AB - Because measurements of hemostatic factors might aid the prediction of cardiovascular clinical events, we investigated the long-term prognostic importance of selected hemostatic factors in patients with angina pectoris. At recruitment, 209 patients underwent clinical assessment and coronary angiography, and a range of hemostatic factors were measured. During the follow-up period of 9 years, 58 patients (28%) suffered a cardiac event (acute myocardial infarction or death from cardiac causes). The risk of cardiac events was positively related to baseline measurements of fibrinogen (risk ratio per SD [RR] increase 1.29, 95% confidence interval [CI] 0.99 to 1.68, P=.06) and negatively related to antithrombin III activity measurements (RR 0.75, 95% CI 0.59 to 0.95, P=.02). No other hemostatic factor measured was significantly related to the risk of having a cardiac event. Worsening of angina in the few weeks before and ejection fraction evaluation at the initial angiography were both strongly related to the risk of cardiac events. However, the relationships of fibrinogen and antithrombin III measurements to risk remained almost unchanged after adjusting for worsening of angina and ejection fraction. Fibrinogen and antithrombin III may have an important etiologic role in the prognosis of patients with angina pectoris. PMID- 8630660 TI - Association of chronic stress with plasminogen activator inhibitor-1 in healthy middle-aged men. AB - The effect of chronic stress on tissue-type plasminogen activator (TPA) and plasminogen activator inhibitor-1 (PAI-1) antigens was studied in 69 healthy middle-aged men. Chronic stress, defined as feelings of fatigue, lack of energy, increased irritability, and demoralization, was positively associated with plasma concentrations of PAI-1 antigen but was unrelated to TPA. The association remained unaltered after controlling for age, smoking, alcohol consumption, and physical activity but became nonsignificant after further controlling for abdominal obesity, BMI, and serum insulin and triglyceride levels. This attenuated association implies that the relationship between vital exhaustion and PAI-1 may be secondary to the effects of the metabolic variables. Thus, the present study shows that long-term stress affects the fibrinolytic system and suggests that obesity and insulin and triglyceride concentrations, which are closely correlated with the fibrinolytic parameters, may mediate the association. These findings are consistent with the hypothesis that chronic stress causes increased synthesis of PAI-1, thus promoting the risk for atherothrombotic disease by decreasing the likelihood of spontaneous fibrinolysis and increasing the likelihood of fibrin deposition. PMID- 8630661 TI - Visceral fat accumulation and its relation to plasma hemostatic factors in healthy men. AB - The associations between abdominal visceral fat and the plasma hemostatic system were examined in 38-year-old healthy men (n=52) with a wide range of fatness and fat distribution. Plasma hemostatic factors and metabolic parameters, including glucose tolerance, were measured, and body fatness and adipose tissue distribution were assessed by using computed tomography. The men with more visceral fat (ie, higher than the median value [n=26]) had a less favorable metabolic profile than the men with less visceral fat (n=26). They also had significantly (P<.05) higher plasma fibrinogen, factor VIII clotting activity, tissue-type plasminogen activator antigen, and plasminogen activator inhibitor-1 (PAI-1) activity (19.2+/-2.4 versus 8.5+/-1.6 AU/mL, P<.001) and lower basal tissue-type plasminogen activator activity. After adjustment for plasma insulin, the men with larger abdominal visceral fat area still had significantly higher plasma PAI-1 activity, but no difference was found in any of the other hemostatic factors. In multiple linear regression analysis, abdominal visceral fat area was a positive predictor of plasma PAI-1 activity, but it failed to show any significant association with other hemostatic factors after controlling for plasma insulin. These results suggest the presence of relationships between abdominal visceral fat and several plasma hemostatic factors that are largely mediated by concomitant alterations in plasma insulin concentration. In addition, our results suggest that abdominal accumulation of visceral fat is an independent predictor of plasma PAI-1 activity. PMID- 8630662 TI - Effects of partially hydrogenated fish oil, partially hydrogenated soybean oil, and butter on hemostatic variables in men. AB - We have compared the effects of partially hydrogenated fish oil (PHFO diet), partially hydrogenated soybean oil (PHSO diet), and butterfat (butter diet) on fibrinolytic and coagulation variables in 31 young men. The three test margarines, which contributed 78% of total fat in the diets, contained 70% butterfat, PHSO, or PHFO, each with 30% of soybean oil. Fat provided approximately 35% of energy, and the content of trans-fatty acids was 0.9%, 8.5%, and 8.0% of energy in the butter diet, PHSO diet, and PHFO diet, respectively. All diets contained 420 mg cholesterol per 10 megajoules per day. All subjects consumed all three test diets for 3 weeks, in a random order (crossover design). The PHSO diet resulted in higher levels of plasminogen activator inhibitor type 1 antigen and plasminogen activator inhibitor type 1 activity than the two other test diets. Fibrinogen increased on the butter diet compared with the PHFO diet. No significant differences in the levels of factor VII, fibrinopeptide A, D dimer, tissue plasminogen activator or beta-thromboglobulin were observed between the three test diets. The PHFO and the PHSO diets have previously been shown to result in higher levels of Lp(a) compared with the butter diet. The present findings indicate that PHSO has unfavorable antifibrinolytic effects relative to PHFO and butter and that butter may be procoagulant relative to PHFO. More controlled studies are needed to assess definitely the impact of different hydrogenated fats on risk of coronary heart disease. PMID- 8630663 TI - Plasminogen activator inhibitor-1, tissue-type plasminogen activator, and fibrinogen: Effect of dieting with or without exercise in overweight postmenopausal women. AB - This study assessed the short- and long-term effects of an energy-restrictive diet with or without exercise on plasminogen activator inhibitor-1 antigen (PAI-1 Ag) and PAI-1 activity, tissue-type plasminogen activator antigen (TPA Ag), and fibrinogen serum levels. Healthy, overweight postmenopausal women (age, 53.8+/ 2.5 years; body mass index, 25 to 42 kg/m2; n=121) were randomly assigned to one of three groups: control, 4200-kJ/d diet, or 4200-kJ/d diet with combined aerobic and anaerobic exercise. PAI-1 activity and PAI-1 Ag, TPA Ag, and fibrinogen levels were measured at baseline, after a 12-week intervention, and after a further 6-month follow-up. PAI-1 Ag and activity and TPA Ag were positively correlated with serum triglyceride levels, the abdominal-to-total-body fat ratio (as assessed by total-body dual-energy x-ray absorptiometry), fasting blood glucose, and systolic BP and negatively with HDL cholesterol and sex hormone binding globulin. The diet led to profound decreases and normalization of PAI-1 activity (approximately 50%), PAI-1 Ag (approximately 30%) and TPA Ag (approximately (29%), but exercise conferred no additional effect. Fibrinogen did not change. At follow-up there were no longer any significant changes (P>.05). In conclusion, PAI-1 Ag and activity as well as TPA Ag seem to be part of the metabolic syndrome X. The diet made the blood less thrombogenic in the short term with no effect of the added exercise. PMID- 8630664 TI - The acute rise in plasma fibrinogen concentration with exercise is influenced by the G-453-A polymorphism of the beta-fibrinogen gene. AB - We have investigated the effects of chronic physical training and acute intensive exercise on plasma fibrinogen levels and the relationship of these responses to beta-fibrinogen G-453-A polymorphism genotype. One hundred fifty-six male British Army recruits were studied at the start of their 10-week basic training, which emphasizes physical fitness. Cohorts were restudied between 0.5 and 5 days after a major 2-day strenuous military exercise (ME) undertaken in their final week of training. Changes in fibrinogen concentration were adjusted for the effects of age, body mass index, and smoking history. Compared with baseline values, fibrinogen concentrations were significantly lower (11.9%, P=.04) at day 5 after ME, consistent with the beneficial effect of training. However, they were higher on days 1 through 3 after ME (suggesting an "acute-phase" response to strenuous exercise) and were maximal on days 1 and 2 (27.2%, P<.001 and 37.1%, P<.001 respectively). Fibrinogen genotype was available in 149 individuals. As expected from previous studies, men with one or more fibrinogen gene A-453 alleles had plasma fibrinogen concentration slightly but significantly higher at baseline (4.5%, P=.11). During the acute-phase response (days 2 and 3), however, the degree of rise was strongly related to the presence of the A allele, being 26.7+/ 5.4% (mean+/-SE), 36.5+/-11.0%, and 89.2+/-30.7 for the GG, GA, and AA genotypes, respectively (P=.01). These results confirm that chronic exercise training lowers plasma fibrinogen levels, that intensive exercise generates an acute-phase rise in levels, and that this acute response is strongly influenced by the G/A polymorphism of the beta-fibrinogen gene. PMID- 8630665 TI - Lys and fibrinogen binding of wild-type (Trp72) and mutant (Arg72) human apo(a) kringle IV-10 expressed in E coli and CHO cells. AB - In a previous study, we identified a lysine (Lys)-binding-defective form of human lipoprotein(a) and attributed this defect to the presence of a Trp72-->Arg mutation in apolipoprotein(a) [apo(a)] kringle IV-10. To document this relationship, we expressed both wild-type (wt) and mutant (mut) forms of kringle IV-10 in Escherichia coli (nonglycosylated form) and Chinese hamster ovary (CHO) cells (glycosylated form). The Arg72 mut was prepared by introducing the T-->A mutation in apo(a) kringle IV-10 amplified from human liver mRNA by the reverse transcriptase polymerase chain reaction technique. All expressed kringles were tested for their ability to bind Lys and plasmin-modified fibrinogen (PM fibrinogen). wt kringle IV-10 expressed in both E coli and CHO cells bound to Lys Sepharose with comparable affinity. In contrast, the Arg72 mut expressed in both systems exhibited no Lys-binding capacity. Moreover, the wt kringle IV-10 expressed in both systems bound to PM-fibrinogen and exhibited two binding components, one Lys mediated (inhibitable by epsilon-amino-n-caproic acid) and one Lys insensitive, occurring in about the same proportions. Only the latter type of binding was present in the Arg72 mut expressed in E coli. We conclude that kringle IV-10 of human apo(a) has Lys- and PM-fibrinogen-binding capacities that are independent of glycosylation and require the presence of Trp72, one of the seven amino acids that constitute the Lys-binding site of kringle IV-10. Our results also show that the binding of kringle IV-10 to PM- fibrinogen is more complex than that to Lys, in that the former requires an additional binding site or sites outside the Lys-binding site. PMID- 8630666 TI - Increased expression of genes for platelet-derived growth factor in circulating mononuclear cells of hypercholesterolemic patients. AB - Platelet-derived growth factor (PDGF) is implicated in the accumulation of smooth muscle cells in atherosclerotic lesions following monocyte migration through the vascular endothelium. We show here a 15- to 20-fold increase in expression of PDGF-A and -B genes (as measured by a quantitative reverse transcription polymerase chain reaction assay of mRNA concentration) in circulating monocytes of hypercholesterolemic and hyperlipidemic patients compared with normocholesterolemic individuals. Strong positive correlations between PDGF-A and -B mRNA concentrations indicate that the two genes are coordinately regulated in mononuclear cells in both normal and hypercholesterolemic individuals. PDGF gene expression in patients correlates with concentrations of plasma total cholesterol and low-density lipoprotein cholesterol, a proven risk factor for atherosclerosis. Activation of monocyte PDGF expression may be an important component of the atherosclerotic risk associated with raised cholesterol levels and may represent an essential step in the early stages of atherogenesis. However, the marked increases in PDGF mRNA levels in patients with modest hypercholesterolemia compared with normal subjects suggest that other factors are involved. The relationship of monocyte PDGF expression to other atherosclerotic risk factors and to the different stages of atherosclerosis needs to be carefully evaluated. PMID- 8630667 TI - Expression of the VLDL receptor in endothelial cells. AB - In this article we describe the cellular distribution of the very low density lipoprotein receptor (VLDLR), a transmembrane protein that is expressed at high concentrations in skeletal muscle, heart, adipose tissue, and brain but in only trace amounts in the liver. Indirect immunofluorescence localization studies were performed in murine and bovine tissues using a rabbit polyclonal anti-human VLDLR antibody. Immunoreactive VLDLR protein was detected in the endothelium of capillaries and small arterioles but not in veins or venules of bovine skeletal muscle, heart, ovary, and brain. In the liver, there was intense staining of the capillaries and arterioles that supply the capsule and hepatic vessels but no staining of the sinusoidal surfaces. We failed to detect any signal from nonendothelial cells in the liver or peripheral organs. The VLDLR was also expressed at high levels on the endothelial surface of bovine coronary arteries; in contrast, little or no staining was seen in aortic endothelium. Antibody staining of cultured bovine coronary artery endothelial cells demonstrated punctate cell-surface staining, as well as staining of large and small cytoplasmic vesicles. This tissue and cell pattern of expression suggests that the VLDLR plays a role in the transport of VLDL or another plasma constituent from the vascular compartment to adjacent tissues. PMID- 8630668 TI - Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. AB - The inhibition of experimental atherosclerosis by antioxidants and the presence of oxidized LDL (oxLDL) in atherosclerotic lesions indicate that oxLDL may play what is perhaps a primary role in atherogenesis. LDL promotes the growth of arterial smooth muscle cells (SMCs), and oxLDL has cytotoxic effects. Since excessive intimal growth alternating with necrosis is typical of atherosclerotic lesions, we wondered whether these extreme changes in the lesions could be related to the extreme effects of LDL and oxLDL on cells. We therefore examined the effects of increasing LDL oxidation on its capacity to induce cell growth or cell death and whether the latter could be due to apoptosis. Cells of the types present in the atherosclerotic artery used, ie, SMCs (human arterial), macrophages (human macrophage-like cell line THP-1), and human fibroblasts. Growth was evaluated by measuring the synthesis of DNA and culture size (MTT method) and apoptosis by using the in situ labeling of internucleosomally degraded DNA and, in the case of SMCs, the appearance of chromatin condensation. The oxidation of LDL was by UV or Fe ions. Shortly oxidized LDL had a markedly increased growth-promoting effect on all cell types. With prolonged exposure to UV, but not to Fe, LDL became increasingly cytotoxic, and this toxicity was paralleled by the appearance of apoptosis in all cell types. After prolonged UV treatment, low-molecular-weight material from the partially degraded LDL was responsible for the induction of apoptosis. The dual effect of oxLDL, ie, its strong growth-promoting effect or the induction of cell death by apoptosis, depending on the degree of change by oxidation, is compatible with the notion that oxLDL plays a role not only in atherogenesis but also more extensively in the development of the structure typical of the atherosclerotic lesion, with focal excessive growth alternating with necrosis. PMID- 8630669 TI - Evidence for a synergistic effect of calcium channel blockers with lipid-lowering therapy in retarding progression of coronary atherosclerosis in symptomatic patients with normal to moderately raised cholesterol levels. The REGRESS Study Group. AB - To date, lipid-lowering therapy appears to be the most effective medical intervention to retard progression of coronary atherosclerosis. In spite of promising experimental results, clinical trials completed so far have failed to demonstrate that calcium channel blockers (CCBs) alone influence the evolution of established coronary atherosclerosis. To assess whether the two therapies may have an additive or synergistic beneficial effect on human atherosclerosis, we reviewed in this regard the data of the angiographic Regression Growth Evaluation Statin Study (REGRESS) trial. REGRESS was designed to determine the effect of lipid-lowering therapy with pravastatin in symptomatic patients with normal to moderately raised cholesterol levels. Angiographically, with respect to the minimum obstruction diameter, in the pravastatin group, patients had on average 0.05 mm (95% confidence interval [CI]: 0.01-0.09) less progression if cotreated with CCBs compared with no CCB treatment, whereas in the placebo (no pravastatin) group, no effect of CCB treatment was observed (interaction test for differential effect of CCB treatment in patients with pravastatin compared with patients receiving placebo: P=.0016). With respect to the mean segment diameter, similar although not significant (P=.33) results were found. With respect to new lesion formation, in the pravastatin group, there were 50% (CI: 25-83) fewer patients with new angiographic lesions if cotreated with CCBs compared with no CCB cotreatment, whereas in the placebo (no pravastatin) group, no significant effect of CCB treatment was observed (interaction test: P=.0026). No beneficial effects of CCB treatment on clinical events were observed. Although the REGRESS trial was not designed to evaluate combination therapy, the results suggest strongly that addition of CCBs to 3-hydroxy-3-methyl-glutaryl-coenzyme reductase inhibitor therapy (pravastatin) acts synergistically in retarding the progression of established coronary atherosclerosis. PMID- 8630670 TI - Oral estrogen replacement therapy in postmenopausal women selectively raises levels and production rates of lipoprotein A-I and lowers hepatic lipase activity without lowering the fractional catabolic rate. AB - To characterize the apolipoprotein (apo) subfraction specificity of the increase in high density lipoprotein (HDL) levels that is induced by oral estrogen and to explore the metabolic mechanisms thereof, six healthy, postmenopausal women were studied during each of two 5-week periods of a low-fat diet with and without ethinyl estradiol 0.05 mg/d. With estrogen, HDL cholesterol levels increased 36% (mean+/-SD, 47+/-15 versus 63+/-18 mg/dL, basal versus estrogen periods, respectively; P=.004), plasma levels of apo A-I increased by 27% (147+/-24 versus 186+/-34 mg/dL; P=.003), and apo A-II levels increased 17% (39+/-5 versus 46+/-4 mg/dL; P=.0003). Apo A-I mass in particles that contained apo A-I but no apo A-II (ie, Lp A-I) increased 66% (43+/-13 versus 71+/-17 mg/dL; P=.002) while the sum of apo A-I and apo A-II mass in particles with both apo A-I and apo A-II (ie, Lp A-I/A-II) showed only a nonsignificant 14% increase (144+/-23 versus 163+/-29 mg/dL; P=.12). In radioiodinated-protein turnover studies the production rate (PR) of each species changed in proportion to the change in its plasma concentration: Lp A-I PR increased 76% (4.7+/-1.8 versus 8.2+/-2.5 mg x kg(-1) x day(-1); P=.001) while Lp A-I/A-II PR showed only a nonsignificant increase of 22% (14.1+/-1.8 versus 17.2+/-5.4 mg x kg(-1) x day(-1); P=.2). Hepatic lipase (HL) activity in postheparin plasma decreased 66% with estrogen (10.4+/-4.3 versus 3.5+/-0.8 micromol x mL(-1) x h(-1); P=.005) while lipoprotein lipase activity was unchanged (7.1+/-1.6 versus 7.6+/-1.6 micromol x mL(-1) x h(-1); P>.2). Despite the large decrease in HL activity, the fractional catabolic rate of Lp A-I did not decrease (0.241+/-0.048 versus 0.258+/-0.066 pool/d; P=.2), nor was that of Lp A-I/A-II lessened (0.221+/-0.030 versus 0.233+/-0.053 pool/d; P>.2). Thus, oral estrogen replacement therapy has a novel and potentially antiatherogenic effect: a large and selective increase in Lp A-I levels. The metabolic mechanism of the estrogen-induced increase in Lp A-I concentration appears to be the increase in its PR. Although the ability of estrogen to suppress HL activity has been confirmed, surprisingly this decrease was not accompanied by any decrease in the fractional catabolic rate of Lp A-I or Lp A I/A-II. This suggests that HL may not play a major role in regulating HDL protein catabolism during suppression of HL by estrogen. PMID- 8630671 TI - Increased cholesteryl ester transfer activity in plasma from analbuminemic patients. AB - Hypercholesterolemia associated with analbuminemia, an inherited disease manifesting low plasma albumin concentration, is characterized by enhanced LDL cholesterol levels and reduced HDL cholesterol levels. In addition, compared with normal counterparts, the esterified cholesterol:triglyceride ratio tends to be higher in analbuminemic apoB-containing lipoproteins and lower in analbuminemic HDL. The aim of the present study was to investigate the mechanism that may account for the association of a hypoalbuminemic state with alterations in the concentration and composition of plasma lipoprotein fractions. To this end, endogenous cholesterol esterification activity, phospholipid transfer activity, and cholesteryl ester transfer activity were measured in total plasma from three analbuminemic patients and five control subjects. Whereas endogenous cholesterol esterification and phospholipid transfer rates were not significantly affected in analbuminemia, the transfer of radiolabeled cholesteryl esters from HDL toward apoB-containing lipoproteins was constantly higher in analbuminemic plasmas than in normal control plasma (473.6+/-107.3% x h(-1) x mL(-1) versus 227.5+/-84.0% x h(-1) x mL(-1), respectively; P=.036). The rise in cholesteryl ester transfer protein (CETP) activity in analbuminemic plasma was due to a significant increase in the transfer of radiolabeled cholesteryl esters toward LDL but not toward the triglyceride-rich lipoproteins. The CETP mass was higher in analbuminemic patients than in control subjects, but the difference did not reach the significance level (5.18+/-0.82 mg/L versus 3.13+/-1.19 mg/L respectively; P=.07). Since abnormally elevated nonesterified fatty acid (NEFA) levels were shown to be associated with analbuminemic lipoproteins, mostly LDL, the direct role of lipoprotein-bound NEFA in enhancing CETP activity was suspected. In support of this view, supplementation of total plasmas with fatty acid-poor albumin was shown to reduce CETP activity to a significantly greater extent in analbuminemic plasmas than in normal control plasma. It is concluded that hyperlipidemia associated with the hypoalbuminemic state can relate, at least in part, to the combined effect of CETP and NEFA in promoting the transfer of cholesteryl esters from the antiatherogenic HDL toward the proatherogenic apoB containing lipoproteins. PMID- 8630672 TI - Evidence of impaired glomerular charge selectivity in nondiabetic subjects with microalbuminuria: relevance to cardiovascular disease. AB - Microalbuminuria is associated with excess cardiovascular morbidity and mortality in diabetic and nondiabetic subjects. Loss of glomerular charge selectivity may explain the development of microalbuminuria in diabetic subjects. The primary population in this cross-sectional study was 124 subjects aged 40 to 75 years without glucose intolerance and with a previous (3 years before the present study) urinary albumin excretion rate (UAE) in the normal (<20 microgram/min) or microalbuminuric (>20 microgram/min) range. The secondary population consisted of 39 offspring aged 15 to 40 years. The main outcome measures included UAE, urinary IgG/IgG4 selectivity index (SI), and the presence of ischemic heart disease as determined by questionnaire or ECG. Among the primary population, a significant inverse correlation was found between SI and UAE (r=-.40, P<.001). Reduction in SI could be demonstrated in subjects with UAE >10 microgram/min. In multiple regression analysis reduction in SI was found with increasing age, independent of UAE. In 20 subjects with clinical cardiovascular disease a reduction in SI was found (1.9+/-0.6 versus 2.6+/-1.3, P=.001) without concomitant elevation in UAE (P=.99). Offspring from parents with microalbuminuria had an SI comparable to offspring from parents with normal UAE (2.7+/-0.7 versus 3.3+/-1.6, P=.17). In nondiabetic subjects the development of microalbuminuria is associated with reduced SI, suggesting impairment of glomerular charge selectivity. SI may offer a more sensitive monitoring of abnormalities in glomerular permselectivity than does measurements of UAE, but the ability of SI to predict development of cardiovascular disease needs to be evaluated prospectively. PMID- 8630673 TI - Factor VII hyperactivity and endothelial cell damage are found in elderly hypertensives only when concomitant with microalbuminuria. AB - We studied the relationship among albuminuria, factor VII (FVII) hyperactivity, and endothelial cell damage in 6 elderly hypertensive subjects. The plasma levels of activated FVII (FVIIa), FVII coagulant activity, FVII antigen (FVIIag), von Willebrand factor (vWF), and thrombomodulin were measured to assess FVII hyperactivity and endothelial cell damage, and urinary albumin excretion rate (UAE) was calculated using 12-hour nighttime (7 pm to 7 am) urine collection (mean for 2 consecutive nights). We performed 24-hour ambulatory blood pressure monitoring in all 61 hypertensive patients and classified them into a white-coat hypertension group (n=12) and a sustained hypertension group (n=49). For the levels of FVII, vWF, and thrombomodulin, there were no differences between the white-coat hypertension group and normotensive control subjects (n=25). In the sustained hypertensive group, only the microalbuminuric subgroup (UAE, 15 to 300 microgram/min: n=30) showed significant elevation compared with the normotensive group for the level of FVIIa (mean [95% confidence interval]: 4.0 [3.6 to 4.4] versus 3.0 [2.6 to 3.3] ng/mL, P<.001), the FVIIa/FVIIag ratio (an indicator of activation of FVII zymogen to FVIIa) (1.33 [1.19 to 1.50] versus 1.04 [0.92 to 1.19], P<.01), the level of vWF (188 [165 to 214] % versus 144 [129 to 160] %, P<.01), and thrombomodulin (11.7 [10.3 to 13.3] versus 9.3 [8.5 to 10.3] ng/mL, P<.01). In contrast, none of these levels in the normoalbuminuric hypertensive group (UAE <15 microgram/min, n=19) differed from that in the normotensive control group. These results suggest that among elderly hypertensives, only those with microalbuminuria show enhancement of FVII activation and endothelial cell damage, while patients with white-coat hypertension and normoalbuminuric hypertensives do not show these accompanying abnormalities. Thus, increased levels of FVII activity and markers of endothelial cell damage might account for the higher risk of cardiovascular events in essential hypertension with microalbuminuria. PMID- 8630674 TI - Multiple risk intervention in high-risk hypertensive patients. A 3-year ultrasound study of intima-media thickness and plaques in the carotid artery. Risk Intervention Study (RIS) Group. AB - In spite of optimal blood pressure control, available data indicate that the risk of coronary heart disease remains high in many patients with hypertension. Multifactorial risk intervention programs have therefore been advocated. The aim of the present randomized study was to analyze whether a favorable change in risk factors caused by a comprehensive risk factor modification program (focused mainly on nonpharmacological intervention) might beneficially affect ultrasound assessed far-wall common carotid intima-media thickness or plaques in the carotid artery in high-risk hypertensive patients (n=81) in comparison with those undergoing usual care (n=83). A further aim was to analyze whether risk factors measured at baseline or follow-up were related to the change recorded in intima media thickness during follow-up. The results showed in the intervention group a favorable change in LDL cholesterol (-9%), in smoking habits (32% of smokers quit smoking), and in HbA1c (-17% in patients with diabetes mellitus) over the 3.5 year observation period. However, no difference between the two randomization groups could be observed for far-wall common carotid intima-media thickness or plaque status during follow-up. Of all tested potential risk factors, only fasting insulin at baseline (available in nondiabetic patients) was significantly related to the change in mean intima-media thickness during follow-up (r=.25, n=92, P<.01). The relationship (negative) between follow-up serum HDL and change in mean intima-media thickness during the preceding follow-up was of borderline significance. Patients with moderate to large plaques in the carotid artery region at baseline had a significantly larger increase in common carotid artery intima-media thickness during follow-up than patients with no or only small plaques. The results are disappointing and may indicate that either the change in risk factors occurred too late in life or a considerably larger change in concomitant risk factors than we observed is needed to favorably affect intima media thickness during an observation period of around 3 years in high-risk hypertensive patients. PMID- 8630675 TI - Race and gender differences in the association of Lp(a) with carotid artery wall thickness. The Atherosclerosis Risk in Communities (ARIC) Study. AB - The association of lipoprotein(a) [Lp(a)] with preclinical atherosclerotic disease is not well established in any race group, particularly African Americans. This report examined the association of Lp(a) with preclinical extracranial carotid atherosclerosis in middle-aged black and white participants in the Atherosclerosis Risk in Communities (ARIC) Study. Study participants (15 124: 2417 black women, 1522 black men, 5907 white women, and 5278 white men) who were 45 to 64 years old at baseline were examined during the period 1987 to 1989. Carotid intimal-medial far-wall thickness was determined by B-mode ultrasonography and expressed as the overall wall thickness mean at six sites to approximate atherosclerosis in the carotid system. Lp(a) was measured as its total protein component, Lp(a) protein, by a double-antibody ELISA for apolipoprotein(a) detection. Mean Lp(a) protein levels were higher in blacks than whites (169.1 and 147.0 microgram/mL in black women and black men, respectively, compared with 86.6 and 75.1 micrograms/mL in white women and white men). Mean carotid wall thickness (in millimeters) varied by race and gender: 0.798 in white men, 0.779 in black men, 0.718 in black women and 0.695 in white women. Multivariable-adjusted Lp(a) protein was independently associated with wall thickness (in millimeters) in white men and black men; among women, however, this association appeared to be stronger when smoking and diabetes were present. A 100 microgram/mL difference in Lp(a) protein was associated with 0.049- and 0.043-mm higher wall thickness values in black men and white men, respectively. Among white women who smoked, the difference in wall thickness was 0.051 mm compared with 0.032 mm for former/never smokers and 0.21 mm in black female diabetics compared with 0.031 mm in black female nondiabetics. These results suggest that Lp(a) is associated with preclinical carotid atherosclerosis in both blacks and whites, but that this association may be affected by the presence of other cardiovascular risk factors, particularly in women. PMID- 8630676 TI - Role of intimal hyperplasia and arterial remodeling after balloon angioplasty: an experimental study in the atherosclerotic rabbit model. AB - The arterial response to injury appears to be an important factor in the development or restenosis. Traditionally, intimal hyperplasia has been thought to be the primary mechanism responsible for restenosis. However, recent studies have found that arterial remodeling is a major determinant of lumen loss after balloon angioplasty. In this study, we evaluated the actual separate contributions of intimal hyperplasia and arterial remodeling to the restenotic process after balloon angioplasty in the atherosclerotic rabbit model. One month after induction of focal atherosclerotic lesions, femoral arteries were randomized to receive treatment with either two or six balloon inflations. One group of rabbits was euthanized immediately after angioplasty to evaluate the initial degree of injury with each dilation strategy ("acute group"), and the rest were euthanized 28 days after angioplasty ("chronic group"). Arteries that had been treated with six inflations had a higher injury score than those treated with two (4.0+/-3.0 versus 1.9+/-1.5, P<.05). In the chronic group, there was a significant increase in intimal area in the six inflation-treated arteries compared with the two inflation group (0.617+/-0.06 versus 0.432+/-0.05 mm2, P<.004). However, there was no significant difference in lumen cross-sectional area between groups. By multivariate analysis, the most important independent predictor of lumen area was the external elastic lamina (EEL) area, although the degree of intimal thickening was also a significant independent predictor. There was a strong, positive correlation between intimal area and EEL area: the larger the intimal area, the larger the EEL area (r=.703, P<.0001). The intimal area was similar in both restenotic and nonrestenotic lesions. In contrast, EEL area was significantly larger (due to remodeling) in nonrestenotic lesions. This study confirms previous findings that the degree of injury determines the degree of neointimal proliferation and supports recent findings that chronic arterial remodeling plays a major role in the final lumen area. Understanding and controlling the remodeling process rather than concentrating solely on intimal hyperplasia may yield better results after balloon angioplasty in the future. PMID- 8630677 TI - Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. AB - Thrombin increases endothelial permeability in a rapid and reversible way. This effect requires the catalytic activity of the enzyme and thrombin receptor engagement. Endothelial cell permeability is mostly regulated by intercellular junction organization. In the present study, we investigated whether opening of intercellular gaps after thrombin treatment could be related to changes in adherence-junction molecular organization. By immunofluorescence analysis, we found that thrombin stimulation of endothelial cells caused a marked alteration of the distribution of vascular endothelial (VE)-cadherin and of the associated catenins. These molecules, which are strictly localized at intercellular boundaries in confluent resting cells, were absent in the areas of intercellular retraction. Immunoprecipitation analysis indicated that thrombin disrupted the VE cadherin/catenin complex. This effect was reversible and correlated with the increase in endothelial permeability. The use of a protein kinase C inhibitor (calphostin C) blocked both thrombin-induced permeability and disassembly of adherence-junction components. We propose that thrombin's effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent. PMID- 8630678 TI - Cisplatin and continuous infusion of fluorouracil followed by radiation and weekly carboplatin in the treatment of locally advanced head and neck cancer: a Hellenic Cooperative Oncology Group study. AB - Induction chemotherapy followed by radiation has been extensively studied in an effort to improve local control and possibly overall survival of patients with locally advanced head and neck cancer. From June 1989 until May 1991, 39 patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) were treated with 3 cycles of induction chemotherapy, consisting of cisplatin (100 mg/m2 d 1) and fluorouracil (1000 mg/m2 d 2-6) followed by radiation potentiated by weekly administration of carboplatin (60 mg/m2). Surgery was performed in selected patients with residual disease after the combined modality approach. Four cycles of adjuvant chemotherapy with carboplatin (325 mg/m2) and bleomycin (15 u) were administered in those patients who demonstrated a partial response after locoregional treatment. There were 36 men and 3 women with a median age of 56 (range 39-74) years and Karnofsky performance status of 70 (range 60-100). The primary site of the tumor was nasopharynx (8), oropharynx (8), hypopharynx (3), oral cavity (4), larynx (13), paranasal sinus (2), and salivary glands (1). Thirty-two (82%) patients presented with stage IV disease. After the completion of induction chemotherapy, 14 (36%, 95% CI 21-53%) patients achieved a complete response (CR). This CR rate was increased to 56% (95% CI, 42-74%) after locoregional treatment. Main toxicities included nausea/vomiting (56%), leukopenia (40%), anemia (30%), thrombocytopenia (10%), stomatitis (28%), diarrhea (17%), and alopecia (12%). Median relapse-free survival was 18 (1-50) months, median time to progression was 13 (0.3-58.5) months, and median survival 19 (0.3-59) months. Induction chemotherapy with cisplatin and fluorouracil followed by radiation potentiated with carboplatin is feasible. However, this combined modality approach, as applied in the present study, does not appear to yield superior results than those reported with chemotherapy followed by radiation alone. PMID- 8630679 TI - Treatment of advanced gastric cancer with a modified regimen of etoposide/leucovorin/5-fluorouracil. AB - The efficacy and toxicity of a combination of etoposide 100 mg/m2/day iv on day 2 4, leucovorin 300 mg/m2/day iv, and 5-FU 500 mg/m2 day iv on day 1-5 every 4 weeks were assessed in 21 patients with advanced gastric cancer with measurable or evaluable diseases. Eight patients had an objective response, including 3 in CR. The overall response rate was 38.1% (95% CI 33.4-42.8%). Five of 8 patients who exhibited locally advanced and unresectable diseases had an objective response (2 CR, 3 PR). The response rate in patients with metastatic disease was 23.0% (95% CI 14.4-31.6%). The median progression-free interval and overall survival time were 7 and 10 months, respectively. The most frequent side effect was alopecia (Gr I/II 71.4%). No treatment-related death occurred. Modified ELF is a relatively effective and tolerable combination regimen for advanced gastric cancer and can be safely administered to elderly patients and patients with systemic diseases. PMID- 8630680 TI - Treatment of advanced malignancies with high-dose acetaminophen and N acetylcysteine rescue. AB - High-dose acetaminophen (HDAC) produces hepatocellular necrosis and cytotoxic changes in other tissues that express mixed-function-oxidase (MFO) activity. N acetylcysteine (NAC), administered within 8 hr of HDAC exposure, replenishes reduced glutathione and prevents these effects. Numerous cell culture and animal studies have demonstrated that NAC may differentially protect normal cells compared with malignant cells from the toxic effects of chemotherapeutic agents and radiation. It was therefore proposed that HDAC with NAC rescue may be effective in malignancies that express MFO activity. To test this hypothesis, a phase I trial of HDAC with NAC rescue was conducted on 19 patients with advanced cancer. HDAC was escalated from 6 to 20 g/m2 PO using a standard IV NAC rescue regimen. A total of 78 treatments were administered. Moderate fatigue, anorexia, and weight loss were the main toxicities observed. Transient grade 3 liver toxicity was noted following 1 treatment. Alopecia and renal and hematological toxicities were not observed. Responses after 4 courses administered weekly were as follows: response in at least 1 site-8 (partial 3, improved 3, mixed 2); stable disease-3; progressive disease-3; inevaluable-5. In conclusion, HDAC was tolerated with moderate fatigue, anorexia, and weight loss but few other effects using a standard IV NAC rescue regimen. A maximum tolerated dose was not reached at 20 g/m2. A 3/19 (15.8%) partial response rate was observed. PMID- 8630681 TI - Human monoclonal antibody against colon cancer. AB - The purpose of the study was to produce human monoclonal antibodies (hMcAb) against human colon cancer for use in radioimmunoimaging. Human-mouse heterohybridomas were developed by fusing SHM-D33 mouse-human hybrid heteromyeloma cells with human lymphocytes from colon cancer tumor-draining lymph nodes. The hybridomas capable of secreting human monoclonal antibodies were screened by using human colon cancer cell lines and pathological biopsies with ELISA and immunohistochemical methods. hMcAb clone H11 was selected for a large scale antibody production, which was purified from mouse ascites. Biodistribution study demonstrated that specific uptake of 125I-hMcAb H11 by human colon cancer xenografts was significantly higher than by normal tissues. Radioimmunoimaging of human colon cancer xenografts exhibited distinct tumor visualization during the period of 72-96 hr after intraperitoneal injection of 125I-hMcAb H11. The development of human monoclonal antibodies such as hMcAb H11 may be useful for radioimmunodetection and therapy of colon cancer. PMID- 8630682 TI - A phase I pilot study of BCNU plus thymidine in patients with refractory cancer. AB - Thymidine (dThd) has been shown to increase the activity of BCNU in mice, possibly due to its ability to inhibit poly(ADP-ribose)polymerase (PADPRP), an enzyme thought to be active in DNA repair. The present phase I study characterized the pharmacokinetics and toxicity of dThd combined with BCNU. Sixty patients with refractory malignancies were infused with escalating doses of dThd from 7.5g/m2/day to 105.5 g/m2/day for 48 hr, along with 100 mg/m2/day of BCNU for 2 doses. Further dose escalation of dThd was limited by large fluid volumes required; therefore, the BCNU dose was escalated to a maximum of 160 mg/m2/day for 2 days. Plasma dThd concentrations were determined using high-performance liquid chromatography. At doses above 37.5 g/m2/day, steady-state concentrations of dThd approached or exceeded 1 mM, a concentration that nearly completely abolished BCNU-induced PADPRP activity in preclinical studies. Myelosuppression was consistent with BCNU dose but was not apparently increased by the coadministration of dThd. One patient had a partial response to therapy. Both the lack of effect of increasing dThd doses on BCNU-induced myelosuppression and the low response rate suggest that the schedule of drug administration was not optimal to inhibit PADPRP, or that PADPRP may not be essential in repairing BCNU mediated DNA damage in humans. PMID- 8630683 TI - Clusters of lymphoma in ferrets. AB - Cluster outbreaks of lymphoma and leukemia have been associated with viral infections in many species including humans, cattle, and cats. This study describes epidemiological, clinical, and pathological features of cluster outbreaks of lymphoma in multiferret households and examines and compares the Aleutian disease virus (ADV) and feline leukemia virus (FeLV) status of cases, ferrets at risk, and controls. Three ferret groups with 21 cases of histologically diagnosed lymphoma (12.6% cumulative incidence) and their cohabitants (n = 35) were examined and compared with three control groups (n = 52) of cohabitating ferrets without lymphoma. A familial distribution was observed in one group but most cases were not consanguinous. Ferrets greater than 3 years of age developed chronic disease in two of the groups and 2-year-old adults had acute disease in the remaining group. Lymphocytosis, splenomegaly, and lymphadenopathy were prominent features. Histologically, predominantly small noncleaved cell and polymorphous lymphoid lesions were observed. All of the ferrets with lymphoma that were tested for ADV and FeLV using serology or PCR were negative. The rate of ADV antibody among cases or ferrets at risk was not significantly different from controls. None of the cluster ferrets were seropositive for FeLV p27 antigen using a monoclonal ELISA. Infection with a novel ferret virus is suspected, but an etiological agent has not yet been identified. PMID- 8630684 TI - Tumor necrosis factor as marker for monocyte function in chronic myeloid leukemia. AB - The intactness of monocyte function in chronic myeloid leukemia (CML) patients as assessed by their ability to secret tumor necrosis factor was evaluated. Monocytes from CML patients continued to respond to lipopolysaccharide (LPS) stimulation even during the refractory period, suggesting different pathways for stimulation by LPS and malignant processes. PMID- 8630685 TI - Hydroxyurea may increase the activity of fluorouracil plus folinic acid in advanced gastrointestinal cancer: phase II study. AB - In a phase II trial, 36 patients with advanced gastrointestinal cancer were treated with: folinic acid (FA) 500 mg/m2 in a 2-hr intravenous (IV) infusion, 5- fluorouracil (5-FU) 600 mg/m2 as an IV push injection 1 hr after FA, and hydroxyurea (HU) 35 mg/kg/day given p.o. in three administrations (every 8 hr) 6 hr after 5-FU. Cycles consisted of six weekly treatments for 6 weeks, followed by a 2-week rest period. Thirty-three patients were evaluable for response and 36 for toxicity; 73% had previous chemotherapy. The response rate was 30% (CR + PR), the median duration of response was 21 weeks (range 5-36), and time to failure was 17 weeks (range 3-51). The response in patients previously exposed to chemotherapy was 29% and 44% in chemotherapy-naive patients. The median survival for all entered patients was 28 weeks (range 6-54). The most common toxicity was gastrointestinal: diarrhea 22/36 (61%), mucositis 15/36 (42%), and nausea and vomiting 15/36 (42%); hematological toxicity was mild. We conclude that HU can potentiate the activity of 5-FU plus FA in advanced gastrointestinal cancer; in particular, HU can restore the activity of 5-FU in patients previously exposed to chemotherapy. PMID- 8630686 TI - High-dose ifosfamide by infusion with Mesna in advanced refractory sarcomas. AB - Twenty patients with advanced sarcomas entered a pilot study with ifosfamide (IF) and mercaptoethane sulfonate sodium (Mesna) as a second-line treatment for six planned cycles. All patients had received prior doxorubicin- and cyclophosphamide based chemotherapies. IF was administered at a dose of 3 g/m2 given as continuous intravenous infusion for 24 hr on day 1-5 with Mesna. In the absence of disease progression, chemotherapy was planned to be repeated every 4 weeks for six consecutive cycles. Following chemotherapy, only 2 patients (11%) achieved partial response with response durations of 6 and 9 months. There was no complete response. When considered for only high-grade tumors, the response rate reached up to 22%. Toxicity was reported for 48 cycles and the dose-limiting toxicities were myelosuppression (22%) and encephalopathy (17%). Chemotherapy protocol was changed after two or three courses in 16 patients with stable or progressive disease. IF/Mesna chemotherapy at this dose and schedule was not found to be very promising in refractory sarcomas as a second-line chemotherapy. PMID- 8630687 TI - Oligodendroglioma. PMID- 8630688 TI - Cell surface antigens in leukemias and lymphomas. PMID- 8630689 TI - Medical malpractice reform and defensive medicine. PMID- 8630690 TI - Combined modality treatment of locally advanced head and neck cancer. PMID- 8630691 TI - Breakthroughs and slow progress: tumor targeting with antibodies. PMID- 8630692 TI - In search of better prognostic indicators for patients with oligodendrogliomas. PMID- 8630693 TI - Ifosfamide in sarcomas: is it a schedule-dependent drug? PMID- 8630694 TI - Maximum life span predictions from the Gompertz mortality model. AB - This study examined maximum life span predictions obtained with the Gompertz mortality rate model, which assumes that there is a constant rate of acceleration in the age-related mortality of adult populations. The influence of population size N on the maximum life span (tmax) was shown to be small, because the numeric impact of N is reduced to ln[ln(N)]. In contrast, the Gompertz exponential mortality coefficient alpha has much more influence on the tmax, which varies as 1/alpha. Examination of select mammals and birds showed that tmax as reported for local populations agrees very well with that calculated from mortality rate coefficients for these local populations. However, the tmax as reported from the world literature, which is designated here as the "world record, " shows major discrepancies for some species from the predicted tmax based on the local population. We demonstrate that these discrepancies are not due to population size, but represent other factors that may include genotype, diet, and environmental dangers. Potential increases in human tmax will depend mostly on slowing the age-related acceleration of mortality. If the degree of mortality rate slowing achieved in rats by diet restriction is applied to humans, then the median human life expectancy would approach the present tmax of 120 years. PMID- 8630695 TI - Muscle torque in young and older untrained and endurance-trained men. AB - Plantar flexor torque was measured in 24 young (25 +/- 1.4 y) and older (62 +/- 2 y) untrained and endurance-trained men to test the hypothesis that age-associated declines in muscle function would be attenuated in older men who also endurance trained. Endurance-trained subjects averaged 7-9 h/wk of aerobic activity for 10 12 years. These subjects had not engaged in resistance training previously in the past 10 years. Plantar flexor torque was measured at velocities between 0 and 5.23 rads. s-1. In absolute terms, maximal isometric torque was 23% lower in older men compared to young men, regardless of their training status. On the other hand, relative measures of isometric strength (i.e., torque.muscle cross sectional area-1 and torque.muscle volume-1) were similar in young and older men but were higher in trained than in untrained men. Isokinetic torque.muscle cross sectional area-1 and torque.muscle volume-1 was greater at contraction velocities of 0.26-2.09 rads.s-1 for trained subjects. These data suggest that endurance training does not attenuate the age-associated loss of muscle mass or absolute strength. However, endurance training might reduce the extent of loss of relative strength because torque-muscle cross-sectional area-1 and torque.muscle volume-1 are greater in endurance-trained older men than in untrained older men. PMID- 8630696 TI - A 12-year follow-up study of ankle muscle function in older adults. AB - The purpose of this study was to examine changes in strength over time in a cohort of healthy elderly people who underwent assessments of ankle muscle function 12 years earlier. The isometric strength and contractile characteristics of the dorsiflexors and plantarflexors were studied in 11 male and 11 female subjects, ranging from 73-97 yrs (mean age 84 +/- 7.1 yrs). The same footplate apparatus was used as during the original testing. From 1982 to 1994, plantarflexor strength decreased 2.1% per year in females, and 2.5% per year in males (p < .01). The loss was relatively less in the dorsiflexor muscles; strength decreased 0.3% per year in females, and 0.8% per year in male (p > .05). There were no significant changes in evoked twitch torque in either muscle group, which may be due to the fact that passive tension of the connective tissue increased (p < .01) over the 12-year period. We conclude from this longitudinal assessment of ankle muscle function that the rate of loss of voluntary strength can vary considerably between antagonistic muscle groups. Factors influencing this variable loss warrant further investigation. PMID- 8630697 TI - Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. AB - The primary purpose of this study was to determine the impact of brief dietary restriction (DR; 5 or 20 days) on skeletal muscle glucose transport activity (GTA) of 24-month-old female Fischer 344 rats. Basal GTA of isolated epitrochlearis muscles was unaffected by DR. Insulin-stimulated GTA was significantly increased by DR only at 20 days (51%). We also assessed the influence of DR on energy sources (blood-borne and stored). An approximately 20% decline in glycemia occurred in each DR group, but plasma-free fatty acid and beta-hydroxybutyrate concentrations were unaffected. Plasma insulin was reduced by 50% after 20 days. Hepatic glycogen was rapidly mobilized (-69% at 5 days; 83% at 20 days). The depletions of visceral adipose stores was slower (no significant decline at 5 days; -30% at 20 days), but the eventual reduction accounts for a significant amount of energy. The results demonstrate that muscle from old rats can rapidly upregulate GTA in response to brief DR. The relative magnitude of this increase represents a substantial portion of the increases previously observed after prolonged DR. PMID- 8630698 TI - Growth hormone supplementation increases skeletal muscle mass of old male Fischer 344/brown Norway rats. AB - Growth hormone (GH) supplementation can increase the body weight of old rats, but the individual tissues affected were previously unidentified. Therefore, the masses of the heart, spleen, kidney, epididymal fat pads, and five skeletal muscles were assessed in male Fischer 344/Brown Norway rats (9, 20, 31, months) injected with recombinant human GH (0.7 mg/kg) or vehicle twice daily for 10 days. Muscle composition (fiber type, protein concentration, dry weight/wet weight ratio, citrate synthase activity) was also evaluated. Muscle mass was increased with GH treatment, and this increment was undiminished in old age. Fiber type, protein concentration, and dry weight/wet weight ratio were unaffected by GH. Citrate synthase activity declined in the plantaris and increased in the soleus with GH treatment. GH supplementation elevated heart and spleen mass, but not fat pad or kidney weight. The data demonstrate that the capacity for GH-induced hypertrophy of skeletal muscle, myocardium, and spleen is retained during old age. PMID- 8630699 TI - Prolactin receptor gene expression in specific hypothalamic nuclei increases with age. AB - Increasing prolactin levels or increasing responsiveness to prolactin may contribute to reproductive aging by influencing the secretory patterns of hypothalamic GnRH, pituitary gonadotropins, and/or ovarian steroids. Some studies have documented changes in the levels of prolactin in peripheral plasma. The goal of this study was to determine whether prolactin receptor mRNA levels in the brain change with aging, which may lead to increasing responsiveness to prolactin. Young (2-4 months) and middle-aged (9-11 months), demonstrating 3 consecutive estrous cycles, and old (16-19 months) and very old (20-21 months) rats, exhibiting repeated pseudopregnancies, were bilaterally ovariectomized. They were implanted with Silastic capsules containing estradiol-17 beta one week later, and killed 2 days after capsule implantation. Changes in prolactin receptor gene expression were assessed using in situ hybridization. The level of prolactin receptor mRNA in choroid plexus, periventricular area of the preoptic nucleus, and arcuate nucleus increased significantly by the time the animals were old. In the lateral ventromedial nucleus, prolactin receptor gene expression did not change significantly during aging, even in the oldest group of rats. These findings suggest that changes in the prolactin receptor gene may influence the ability of prolactin to exert effects and may allow older animals to be more responsive to prolactin than young rats. PMID- 8630701 TI - Gender-related differences in left ventricular filling dynamics in older subjects after endurance exercise training. AB - The present study was designed to determine if gender affects the adaptive response to endurance exercise training of left ventricular filling dynamics in older individuals. Recently, it was shown that gender influences the cardiovascular responses to endurance exercise training in older subjects. Older men improve left ventricular systolic performance and increase maximal cardiac output in response to endurance exercise training, whereas older women do not. Twelve men (65 +/- 1 years old; mean +/- SE) and 10 women (64 +/- 1) were studied before and after 9 months of endurance exercise training. Maximal O2 uptake was determined during treadmill exercise. Left ventricular filling dynamics and ejection fraction (EF) at rest and during supine exercise were assessed by Tc-99m radionuclide ventriculography. When expressed relative to body weight, maximal O2 uptake (VO2 max) was increased by 24% (27.3 +/- 1.5 to 34.0 +/- 1.5 ml/kg/min; p < .01) in men and 27% (21.9 +/- 1.0 to 27.8 +/- 1.0 ml/kg/min; p < .01) in women in response to endurance exercise training. In men, the time-to-peak filling rate (TPFR) decreased (-19.8 +/- 6.7 ms; p < .05) during exercise at a comparable heart rate in response to training. In contrast, the change in TPFR in women (+2.7 +/- 6.0 ms) was small and insignificant. Peak filling rate (PFR) at rest and during exercise was similar before and after training in men and women. The change in left ventricular systolic reserve at a comparable heart rate from pre to posttraining improved in men (delta EF 4 +/- 3%; p < .05), but not in women ( 2 +/- 3%). The results indicate that the adaptive response of left ventricular filling dynamics to endurance exercise training is influenced by gender in older subjects. Older men show improvement in left ventricular filling dynamics, whereas older women do not. PMID- 8630700 TI - Effects of benzodiazepine receptor inverse agonists and nicotine on behavioral vigilance in senescent rats. AB - Previous experiments demonstrated that, compared with 6-month-old rats, the performance of 20-month-old rats in a behavioral vigilance task was characterized by an impairment in their ability to detect visual signals, whereas their ability to discriminate between longer signals and nonsignal events was unaffected. The benzodiazepine receptor (BZR) agonist chlordiazepoxide potently and selectively interacted with the effects of age on the relative number of hits. However, negative modulators of GABAergic transmission (Zk 93 426, beta-CCtB, RU 33965) failed to attenuate the effects of age on behavioral vigilance. the present experiment tested the hypothesis that the performance of senescent animals (28 months) is further impaired and thus would allow the demonstration of beneficial effects of BZR inverse agonists or nicotine. However, administration of ZK 93 426 (0.39, 1.56, 6.25 mg/kg), Ru 33965 (0.1, 0.5 mg/kg), or nicotine (0.09, 0.287, 0.689 mg/kg) did not beneficially affect the performance of senescent animals; rather, detrimental effects were found. Considering the beneficial behavioral effects of these compounds in animals with experimentally induced impairments in cholinergic function, the present finding point to limitations of normal aging as a variable in animal experiments on BZR inverse agonist or nicotine-induced attenuation of cognitive impairments that result from cholinergic hypofunction. PMID- 8630702 TI - Event-related potential prolongation in Alzheimer's disease signifies frontal lobe impairment: evidence from SPECT imaging. AB - BACKGROUND: The N2 and P3 components of auditory event-related potentials (ERPs) and single-photon emission computed tomographic (SPECT) images are separate independent biological markers of cerebral function and are abnormal in Alzheimer's disease (AD). The relationship between ERP N2 and P3 latencies and regional cerebral perfusion abnormalities in AD is unknown. METHODS: ERP and SPECT data were obtained one week apart in 18 patients with "probable" AD of mild or moderate severity, and 12 healthy age-matched elderly controls. Average premotor frontal, anterior temporal, inferior parietal, and occipital cortical/cerebellar perfusion ratios were calculated from the SPECT data and correlations with ERP N2 and P3 latencies derived for AD and control groups separately. RESULTS: ERP N2 latency was correlated significantly with the average frontal perfusion ratio (r = -.59; p < .009), but correlations with average temporal, parietal, and occipital ratios were nonsignificant in the AD group. Similarly, ERP P3 latency was correlated significantly with the average frontal perfusion ratio (r = -.65; p < .004), but not with the other perfusion ratios in the AD group. In the control group, a partial correlation between the average frontal perfusion ratio and the ERP N2 latency was noted (r = -.52; p < .09), but no other ERP/SPECT correlations approached statistical significance. CONCLUSION: ERP N2 and P3 latency delay in AD is a function of differential frontal lobe hypoperfusion. PMID- 8630703 TI - Subjective sleep characteristics of 1,485 males and females aged 50-93: effects of sex and age, and factors related to self-evaluated quality of sleep. AB - BACKGROUND: This epidemiologic study cross-sectionally examined the effects of sex and age on subjective characteristics of sleep and the factors related to self-evaluated sleep quality in a Dutch noninstitutionalized elderly population. METHODS: 1,692 sleep questionnaires were mailed to all attenders of the general practice serving Krimpen aan de Lek, The Netherlands, aged 50 or over. Both target population and responders (1,485 subjects) were virtually representative of the Dutch population regarding sex and age (50 +) characteristics. RESULTS: Overall, females reported significantly poorer quality of sleep, longer sleep latencies, more nighttime awakenings, less frequent napping, and more frequent use of sedative-hypnotic drugs when compared to males. Additionally, there was a female predominance in the prevalence of disturbed sleep onset and sleep maintenance, whereas a male predominance was observed in the prevalence of excessive daytime sleepiness. Across subjects, a significant age-related increment was found for sleep latency time and time spent in bed. The number of nighttime awakenings increased significantly with age only in males. No significant correlations were found between health status and sex, age, or subjective sleep quality. The most frequently reported causes of disturbed sleep onset and sleep maintenance were worries and nocturia, respectively. Subjective quality of sleep was mostly associated with self-estimated sleep latency. CONCLUSIONS: Our findings extend those of previous epidemiologic studies reporting that sleep disorders are common in the general elderly population. Future studies should further elucidate the nature and extent of geriatric sleep disorders to satisfy the increasing need for its accurate diagnosis and treatment. PMID- 8630704 TI - Stepping over obstacles: dividing attention impairs performance of old more than young adults. AB - BACKGROUND: Tripping over an obstacle is a common cause of falls in the elderly. An earlier study of abilities to avoid stepping on suddenly appearing obstacles found that, although healthy old adults had a lower rate-of-success than young adults, the magnitude of that difference was not large. The present study inquired whether dividing attention during such a task would differentially affect young and old healthy adults. METHODS: Rates-of-success were observed in 16 young and 16 old healthy adults (mean ages 24 and 72 years) in avoiding stepping on a band of light that was suddenly projected across their gait path while they walked at their comfortable gait speed. This virtual obstacle was placed at predicted next-footfall locations to give 350 or 450 msec available response times before footfall. During most of the trials the subjects were asked, in addition to trying not to step on the obstacle, simultaneously to respond vocally as quickly as possible when red lights near the end of the walkway turned on. These attention-dividing reaction time tests were of two types: synchronized, when only red lights lit at intervals synchronized with the appearance of the obstacle, and unsynchronized, when green or yellow lights lit in addition to the red lights, with lighting intervals not synchronized with the appearance of the obstacle. RESULTS: When synchronized and unsynchronized reaction time tests were conducted concurrently with the obstacle avoidance tasks, mean rates-of-success in avoidance decreased significantly in both young and old adults. With available response times of 350 msec, mean success rates decreased from their no-division values in the young adults by 14.7% for synchronized reaction and by 19.9% for unsynchronized reaction, attention dividing tests. Corresponding mean decreases for the old adults were 32.0 and 35.7%. This age difference in the effects of dividing attention was significant. CONCLUSION: Both young and old adults had a significantly increased risk of obstacle contact while negotiating obstacles when their attention was divided, but dividing attention degraded obstacle avoidance abilities of the old significantly more than it did in the young. Diminished abilities to respond to physical hazards present in the environment when attention is directed elsewhere may partially account for high rates of falls among the elderly. PMID- 8630705 TI - Progressive versus catastrophic disability: a longitudinal view of the disablement process. AB - BACKGROUND: There is little epidemiologic data on the development of disability over time in older persons. This study uses prospective data from cohorts followed annually for 6 to 7 years to identify persons who developed severe disability and to characterize the time course of their disabling process and subsequent mortality. METHODS: Incidence rates of severe disability, defined as need for help in three or more activities of daily living (ADLs), were estimated for 6,640 persons who had not reported severe disability at baseline and at the first four annual follow-up visits. Among persons developing severe disability, those who reported no need for help in ADLs in previous interviews were defined as cases of catastrophic disability, and those who had previously reported some disability in ADLs were defined as cases of progressive disability. RESULTS: Overall, 212 subjects developed progressive and 227 developed catastrophic disability. The rates of progressive disability and catastrophic disability were 11.3 and 12.1 cases per 1,000 person-years, respectively. For both types of disability, incidence rates increased exponentially with age, but the increase was steeper for progressive disability. At ages 70-74, less than 25% of severe disability was progressive, while over age 85 progressive disability represented more than half of severe disability. Incidence rates of total and both types of severe disability were similar in men and women. Mortality after severe disability onset was extremely high. Survival was unrelated to age at disability onset and type of disability but was significantly longer in women than in men (median 3.44 vs 2.12 years; p < .0001). CONCLUSION: Tracking the development of disability provides new and important insights into the disability experience in older men and women that are potentially relevant in planning preventive, intervention, and long-term care strategies. PMID- 8630706 TI - Regulatory environment and psychotropic use in board-and-care facilities: results of a 10-state study. AB - BACKGROUND: Largely unsupervised administration of drugs and the potential for overuse of psychotropic agents in residential care facilities have emerged as major public policy concerns. In a large multistate study, we examined patterns of psychotropic prescription and use by facility licensure status and the extensiveness of state regulations. METHODS: Descriptive analyses were based on a sample of 2,949 residents from 493 board-and-care facilities in 10 states, drawn via a complex, multistage sampling design. States were purposively selected based on the stringency of their board-and-care regulatory system, and samples of facilities were drawn, stratified by licensure status and home size. Residents were randomly selected within the sampled facilities. Weighted analyses were performed with Software for Survey Data Analysis (SUDAAN), accounting for the complex sampling design. RESULTS: Approximately 43% of the residents were prescribed and 41% used at least one psychotropic agent, primarily on a routinely scheduled basis. Antipsychotics were prescribed to 22% and used by 21%; antidepressants were prescribed to 17% and taken by 16%; antimanic agents were prescribed to and used by 4%; and anxiolytics, sedatives, or hypnotics were prescribed to 17% and used by 14%. Among psychotropic users, over 50% had not had mental health services in the prior year; 25% had no psychiatric history. Among licensed facilities, prescription and use of psychotropics, particularly antipsychotics, was significantly higher among residents of homes located in states with limited regulatory systems. CONCLUSIONS: Results revealed high rates of psychotropic prescription and use, and not inconsequential levels of polypharmacy - within and across therapeutic classes - among board-and-care facility residents. Frequently, psychotropics were not used as an adjunct to alternative treatments, and were not associated with a psychiatric history. Extent of psychotropic use was related to the regulatory environment, suggesting that more extensive regulations and monitoring may reduce psychoactive drug use in board-and-care facilities, and more adequately ensure the appropriateness of such treatment. PMID- 8630707 TI - Sexuality and aging--usual and successful. AB - BACKGROUND: As aging research increasingly reflects an effort to dissociate true time-driven changes from those that can be improved, sexuality in later life remains largely unexplored. Several problems are evident. There is a lack of normative data, a lack of a conceptual framework relating to the biology, psychology, and sociology of sex, and an attitudinal resistance that obscures the entire topic. METHODS: We conducted a three-part instructional series on major topics involved with sexuality and aging. We surveyed our group of attendees (n 158, average age 68 for males, 65 for females) before and after the series. RESULTS: A remarkably robust sex life was evidenced by both the men and the women, even until advanced old age. Yet, a substantially decreased involvement was reported from 10 years earlier. Despite current activities, people of both sexes wished they were participating even more than they currently were. Impotency was identified as the major negative feature for the men; relationship problems were for the women. A questionnaire 6 months after the series reported improved sexual attitudes, but no change in sexual activities from the earlier survey. CONCLUSION: Sexuality is a major quality-of-life issue which persists into old age. Our study showed that the usual sexual practices reported by our group were not considered by them to be ideal. The intervention of this instructional series provided improved sexual attitudes but not performance. Additional studies are encouraged. PMID- 8630708 TI - Atrial natriuretic peptide levels in the elderly: differentiating normal aging changes from disease. AB - BACKGROUND: Atrial natriuretic peptide (ANP) levels increase with advancing age and in patients with cardiac dysfunction. Previous studies have failed to differentiate the elevated ANP levels of normal aging from those of cardiac disease. METHODS: To differentiate the increased ANP levels seen in normal aging from that of disease, fasting supine ANP was measured in healthy young (n = 24), healthy old (n = 90), and clinically stable but cardiovascularly diseased old (n = 269) residents of a life care facility. ANP levels were correlated with physical exam findings, blood chemistries, measures of physical and cognitive function, and medications. RESULTS: ANP levels were almost fourfold higher in the healthy elderly than in the young (11.4 +/- 1.1 (SEM) vs 3 +/- 0.3 pmol/L, p < .01), and two-and-one-half times higher in the cardiovascular-diseased elderly than the healthy elderly (29 +/- 1.9 vs 11.4 +/- 1.1 pmol/L, p < .01). An ANP value of 21 pmol/L has a sensitivity of 83% and specificity of 52% in distinguishing those elders classified as healthy from those classified as having chronic cardiovascular disease. ANP levels had positive univariate correlation with age (even from 70 to 102 years) and systolic blood pressure. ANP rose progressively with increasing numbers of markers of cardiovascular comorbidity. ANP was higher in subjects with jugular venous pressure > 10 cm, presence of a third heart sound, peripheral edema, artificial cardiac pacemaker, atrial arrhythmias, and in those taking digoxin, diuretics, or nitrates. On multivariate analysis independent predictors of ANP levels were, in descending order, nitrates, age, diuretics, and atrial arrhythmias. CONCLUSION: These data suggest that ANP levels greater than 21 pmol/L are associated with cardiovascular comorbidity in a clinically stable elderly cohort. PMID- 8630709 TI - Varicella vaccine. PMID- 8630710 TI - New concepts in the immunology of sickle cell disease. AB - OBJECTIVE: Our objective is to review the role of adhesion molecules, cytokines, and inflammation in the abnormal adherence of sickle red blood cells to vascular endothelia in the pathogenesis of vascular complication in patients with sickle cell anemia. DATA SOURCES: The MEDLINE database was used to review the hematologic, immunologic, and allergy literature in English with respect to the adhesion molecules involved in sickle hematopoiesis and vascular complications. STUDY SELECTION: Studies selected for review were those that identified the adhesion molecules involved in reticulocyte-endothelial adhesion and the influence that cytokines, infections, and atopy have upon the expression of these molecules. RESULTS: In sickle cell disease, a constant low level of inflammation caused by abnormal adhesion of sickle erythrocytes to endothelial cells in the microvasculature produces low-level tissue ischemia. Allergic and infectious inflammations are likely to lead to increased sickle erythrocyte trapping in the microvascular endothelia which progresses to vessel obstruction, end organ ischemic damage, and dysfunction. CONCLUSION: The identification of underlying immune defects that predispose patients to infections and inflammation needs to be emphasized. Anti-inflammatory medications, anti-adhesion molecule monoclonal antibodies, and adhesion molecule binding-site analogs may have a future in the treatment of the acute vascular complications of sickle cell disease. PMID- 8630711 TI - A 54-year-old woman with rhinitis and a right ethmoid sinus mass. PMID- 8630712 TI - Bela Schick Lecture, 1995. The three Ps: unabridged. PMID- 8630713 TI - Labiatae allergy: systemic reactions due to ingestion of oregano and thyme. AB - BACKGROUND: There are no cases described in the medical literature of systemic allergic reactions due to oregano (Origanum vulgare) or thyme (Thymus vulgaris). These herbs belong to the Lamiaceae (Labiatae) family which comprises other plants such as hyssop (Hyssopus officinalis), basil (Ocimum basilicum), marjoram (Origanum majorana), mint (Mentha piperita), sage (Salvia officinalis) and lavender (Lavandula officinalis). OBJECTIVE: We describe three systemic allergic reactions caused by oregano and thyme in the same patient. METHODS: Skin tests with inhalant allergens and plants of the Labiatae family were done. We used the prick by prick technique with dried commercial plants and prick tests with extracts prepared with the Frugoni method in our patient and in ten control patients. Total serum IgE was determined by Phadezym IgE PRIST (Pharmacia). Specific IgE was measured by two methods: CAP system (Pharmacia) and Phadezym RAST (Pharmacia Diagnostics, Uppsala, Sweden) with activated discs of the allergenic extracts that were prepared in our laboratory. RESULTS: Skin tests with inhalants were positive to grasses. Skin tests with plants of the Labiatae family were positive in all cases when the skin prick technique was used; tests were negative with basil and lavender, and positive with all the others when we used the prick by prick technique. We did not detect any positive skin tests nor specific IgE to plants of the Labiatae family in control patients. Total serum IgE was 406 U/mL. Specific IgE was detected to all herbs tested; higher levels were obtained with the CAP system. CONCLUSIONS: Plants belonging to the Labiatae family seem to show cross-sensitivity on the basis of clinical history and in vitro and in vivo test results. PMID- 8630714 TI - Possible involvement of an environmental agent in the development of acute eosinophilic pneumonia. AB - BACKGROUND: Although the pathogenesis of acute eosinophilic pneumonia remains largely unknown, it has been suggested that it may include a hypersensitivity phenomenon induced by inhaled environmental antigens. METHODS: To investigate this possibility, we studied the effect of environmental challenges in three patients with acute eosinophilic pneumonia. Symptoms and laboratory findings were evaluated before and after the challenge tests in the patient's homes and their places of work. RESULTS: After the provocation challenges to their homes, all three patients developed fever, cough, and fatigue and two of them presented with dyspnea. Inspiratory crackles became audible in all cases, and there was a decreased Pao2 level in two. Similar challenges at their workplaces were negative. After moving out of their homes, the patients engaged in their usual work but had no recurrent episodes. CONCLUSIONS: These results suggest that environmental factors in the home can be the cause of acute eosinophilic pneumonia. In order to elucidate the pathogenesis of the disease, it is important to further investigate environmental factors. PMID- 8630715 TI - Occupational asthma due to deer dander. AB - BACKGROUND: The literature on allergy to deer is limited. The allergenic component remains to be identified. METHODS: We report a case of occupational asthma and rhinitis caused by deer dander. A 44-year-old male farmer had raised three red deer on his farm for 2 years, prior to occurrence of asthmatic symptoms. Dander extract was prepared from the patient's deer. RESULTS: Skin prick test elicited positive reactions to dander extracts from goat, sheep, camel, and cow as well as to deer dander extract. Bronchoprovocation test with deer dander extract elicited an early asthmatic response. Serum-specific IgE antibody to the deer dander extract was detected by enzyme-linked immunosorbent assay (ELISA). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) and immunoblot analysis showed five IgE binding components (110, 72, 59, 45, and 21 kilodalton) within the deer dander extract. CONCLUSIONS: These results suggest that deer dander can induce occupational asthma through an IgE-mediated mechanism in a farmer raising deer. PMID- 8630716 TI - Fetal exposure to involuntary maternal smoking and childhood respiratory disease. AB - BACKGROUND: Several studies have now gone beyond the effects of smoking during pregnancy to examine the effects of involuntary maternal smoke exposure on fetal development. A link has also been indicated between postnatal environmental smoke exposure and long-term respiratory problems in infants. OBJECTIVE: Our study examines whether an association exists between maternal exposure to environmental tobacco smoke and childhood respiratory disorders. METHODS: A cross-sectional design was implemented utilizing a maternal questionnaire survey. The surveys were distributed throughout the county's health department and outlying clinic immunization sites. The county population is a diverse community of all racial and socioeconomic levels and includes a metropolitan population of approximately 300,000. The questionnaire comprised inquiries about the child's wheeze, acute respiratory illnesses, and maternal and paternal exposure to passive smoke and/or other environmental air pollutants. RESULTS: Chi-square and logistic regression analysis showed no statistically significant difference between passively smoke exposed-in-utero subjects and non-smoke-exposed in-utero subjects on the outcomes of allergy and wheeze. Asthma, however, did show a statistically significant association to passive smoke exposure (chi2 = 12.4, P = .05 and Log reg = 8.7, P = .03). CONCLUSION: Passive maternal exposure to tobacco smoke during pregnancy is associated with increased incidence of asthma and supports other research findings that children born of mothers who are passively exposed to smoke during pregnancy are at risk for patterns of negative developmental outcomes. PMID- 8630717 TI - Efficacy of acrivastine plus pseudoephedrine for symptomatic relief of seasonal allergic rhinitis due to mountain cedar. AB - BACKGROUND: Acrivastine is a second-generation H1-antagonist chemically related to triprolidine, but more polar and with less central nervous system penetration than triprolidine. OBJECTIVE: The efficacy of the antihistamine-decongestant combination product (Semprex-D capsules) containing acrivastine 8 mg plus pseudoephedrine HCl 60 mg was evaluated for the treatment of seasonal allergic rhinitis symptoms. METHODS: A total of 676 patients sensitive to mountain cedar pollen was enrolled into a 6-center, randomized, double-blind, placebo controlled, parallel, 4-group study designed to compare acrivastine + pseudoephedrine, acrivastine, pseudoephedrine, and placebo. Patients with demonstrable diary symptom scores at baseline took study medication (4 doses/day) and recorded symptom scores twice daily for 2 weeks. The effectiveness of the acrivastine + pseudoephedrine combination was examined relative to the individual components and placebo in terms of changes in diary symptom scores. RESULTS: Over the 2-week period, the combination of acrivastine plus pseudoephedrine was significantly more effective than (1) acrivastine, pseudoephedrine, and placebo (P < .001) for relief of all symptoms; (2) pseudoephedrine (P < .001) for relieving allergy symptoms, ie, running nose, sneezing, itchy nose/throat and tearing; and (3) acrivastine (P < .001) for reducing nasal congestion. Relative to placebo, small increases in adverse experience rates were observed with acrivastine + pseudoephedrine for dry mouth, insomnia, somnolence, and headache. CONCLUSION: These findings in a large clinical trial demonstrate (1) the efficacy of acrivastine and (2) that each component of the combination of acrivastine 8 mg plus pseudoephedrine HCl 60 mg contributes to the overall efficacy, thereby supporting the conclusion that the combination is rational, safe, and effective for the treatment of allergic rhinitis. PMID- 8630718 TI - Evaluation of cetirizine in patients with allergic rhinitis and perennial asthma. AB - BACKGROUND: Cetirizine, a highly specific H1 receptor antagonist with modest sedation and anticholinergic effects at the higher doses required for bronchodilation, may be useful for treating concomitant allergy and symptoms of asthma. OBJECTIVE: This study evaluated the effectiveness of cetirizine compared with placebo in patients with allergic rhinitis and concomitant symptomatic mild to moderate perennial asthma. METHODS: Twenty-eight patients (13 females and 15 males) aged 13 to 59 years having allergic rhinitis and mild to moderate perennial asthma were treated for 26 weeks with 20 mg cetirizine or placebo. Daily diary entries of symptoms, medications taken and peak expiratory flow rate measurements (morning and evening), biweekly pulmonary function tests, and monthly global evaluations by investigators were performed. RESULTS: Cetirizine treatment significantly reduced baseline severity of several symptoms of rhinitis (itchy nose, nasal congestion, and watery eyes), and asthma (chest tightness, wheezing, shortness of breath, and nocturnal asthma). Although not statistically significant, cetirizine also reduced other symptoms of rhinitis (sneezing, itchy eyes, postnasal drip, and runny nose) and asthma (cough and sputum production), reduced albuterol use, and increased mean peak expiratory flow rates compared with placebo. No differences between treatment groups were noted for patient satisfaction, patient and physician global assessments of asthma management, pulmonary function, and methacholine challenge tests. Of two cetirizine patients with adverse events, one event, moderate drowsiness, was considered to be related to study drug. CONCLUSIONS: Cetirizine reduced symptoms of rhinitis as well as symptoms of asthma in patients with allergic rhinitis and concomitant perennial asthma. PMID- 8630719 TI - Comparative study of cetirizine and terfenadine versus placebo in the symptomatic management of seasonal allergic rhinitis. AB - BACKGROUND: Cetirizine is a new antihistamine with greater selectivity for the histamine H1 receptor and a low rate of hepatic metabolism. Cetirizine once daily is effective in the symptomatic treatment of seasonal and perennial allergic rhinitis and chronic idiopathic urticaria. OBJECTIVE: The efficacy and safety of cetirizine 10 mg qd, terfenadine 60 mg bid, and placebo were compared in patients with seasonal allergic rhinitis. METHODS: A multicenter, prospective, double blind, randomized, parallel study was conducted for 2 weeks during the ragweed pollen season in patients with documented allergic rhinitis. Total symptom complex and total symptom complex plus nasal congestion scores, global efficacy, overall satisfaction, and adverse events were assessed at baseline and after 1 and 2 weeks of treatment. RESULTS: Of the 311 patients randomized to treatment, 283 completed the study. Cetirizine produced a marked improvement in symptoms scores compared with placebo after 1 week of therapy (P = .001). By the end of week 1, total symptom complex scores were improved by 37% with cetirizine compared with 29% for terfenadine, and 23% for placebo. An overall treatment effect was evident at week 1 (P = .0019), with marked differences between cetirizine and both placebo (P = .0004) and terfenadine (P = .0464) but not between terfenadine and placebo (P = .1215). A more marked treatment effect was evident during the first week of the study; this appeared to be related to spontaneous resolution of symptoms, since mean pollen counts derived for each patient declined significantly each week of the study. Therapy was generally well tolerated. Headache was the most common side effect in each group. Four patients on cetirizine, one on terfenadine, and two on placebo withdrew because of side effects. Somnolence was reported in 12 patients on cetirizine (P < .05), 2 on terfenadine, and 3 on placebo. CONCLUSION: Cetirizine produced a greater improvement in symptoms of seasonal allergic rhinitis than terfenadine or placebo. PMID- 8630720 TI - Fluticasone propionate improves quality of life in patients with asthma requiring oral corticosteroids. AB - BACKGROUND: Fluticasone propionate is a potent inhaled corticosteroid that is effective in improving pulmonary function and symptoms in patients with asthma. OBJECTIVE: To evaluate the effects of fluticasone propionate on quality of life in patients with severe asthma requiring oral corticosteroids. METHODS: A total of 96 patients with severe asthma participated in a randomized, double-blind, placebo-controlled, parallel-group, oral steroid-sparing study. Patients received fluticasone propionate aerosol, 750 or 1000 micrograms bid, or placebo for 16 weeks; 91 of these patients continued in a 1-year open-label study, in which everyone initially received fluticasone propionate, 1000 micrograms bid. At regular intervals, patients completed the Medical Outcomes Study Short Form-36 (SF-36), a general health status questionnaire measuring eight dimensions of quality of life, plus one question on change in health from the previous year. RESULTS: Compared with the US population, patients scored significantly lower at baseline for five of eight SF-36 dimensions (P < .01). After 16 weeks, patients receiving fluticasone propionate, 1000 micrograms, improved significantly (P < or = .02) in physical functioning, role-physical, general health, and change in health, compared with the placebo group. After 1 year of open-label treatment with fluticasone propionate, these improvements were maintained. SF-36 scores in the placebo group during the double-blind period either worsened or remained unchanged; however, when these patients were switched to fluticasone propionate during the open-label period, their SF-36 scores also improved. Forced expiratory volume in 1 second (FEV1) at the end of the double-blind period was positively correlated with mean quality of life scores on physical functioning, role physical, vitality, social functioning, and change-in-health status. CONCLUSION: Health-related quality of life improved in patients with severe asthma following 16 weeks of treatment with fluticasone propionate, 1000 micrograms bid. These improvements were maintained during subsequent fluticasone propionate treatment over a 1-year period. PMID- 8630721 TI - Extended-release albuterol in the treatment of 6- to 12-year-old asthmatic children. AB - BACKGROUND: Albuterol sulfate, in the syrup and tablet form for oral administration, has been an effective treatment for adults and children with bronchial asthma. Extended-release albuterol sulfate tablets (Proventil Repetabs, Schering Corp.) provide a convenient, twice-daily dosing regimen, but are indicated only for patients > or = 12 years of age. OBJECTIVE: This study was undertaken to determine whether patients 6 to 12 years of age could be effectively and safely treated with extended-release albuterol tablets. METHODS: This was a randomized, double-blind, placebo-controlled, parallel group study of 157 patients in five centers. Patients were randomized to 4 weeks' treatment with extended-release albuterol tablets, 4 mg twice daily (q 12h), increasing up to 12 mg q 12h, or placebo. Efficacy was evaluated based on pulmonary function tests (PFTs), physician and patient evaluations, and data collected from patients' diaries on PEFR, asthma symptoms, number of nighttime awakenings, and number of tablets taken. The primary efficacy parameter was area under the curve (AUC) for FEV1, evaluated for 8 to 12 hours post-dosing. Safety was evaluated based on vital signs, electrocardiograms, and adverse events. RESULTS: Mean AUCs for FEV1 were significantly greater in the albuterol group at days 1 and 8 (P < or = .03). The albuterol group showed consistently lower severity scores for asthma symptoms. Physicians' and patients' global evaluations favored the albuterol group over the placebo group. No serious, treatment-related adverse events were reported. There were no clinically meaningful changes from baseline in either treatment group for vital signs or electrocardiograms. CONCLUSIONS: Extended release albuterol tablets (4 mg), administered to children 6 to 12 years old in divided doses of up to 24 mg/day, improved pulmonary function and asthmatic symptoms and were well tolerated. PMID- 8630722 TI - Inhibition of leukotriene synthesis by azelastine. AB - BACKGROUND: Azelastine, oxatomide, and ketotifen are used for patients with allergic diseases. These drugs inhibit the release of chemical mediators including the leukotrienes; however, the mechanism involved is unclear. OBJECTIVE: To clarify the mechanism of inhibition, we investigated the effects of three drugs on the function of phospholipase A2, 5-lipoxygenase, leukotriene C4 synthase, and leukotriene A4 hydrolase, which are all catabolic enzymes involved in synthesizing leukotriene C4 and leukotriene B4 in rat basophilic leukemia (RBL)-1 cells. METHODS AND RESULTS: The production of leukotriene C4 and leukotriene B4 was measured by high performance liquid chromatography (HPLC). All three drugs inhibited the production of leukotriene C4 and leukotriene B4 when cells were stimulated with A23187. All three drugs also inhibited the A23187 stimulated release of 3H-arachidonic acid from membrane phospholipids. Azelastine inhibited the production of leukotriene C4, but not leukotriene B4, when either arachidonic acid or leukotriene A4 free acid was used as the substrate in our cell free system. Oxatomide and ketotifen did not inhibit the synthesis of either leukotriene C4 or leukotriene B4 in the same cell free study. CONCLUSION: Results indicated that oxatomide and ketotifen inhibit the production of leukotriene C4 and leukotriene B4 by inhibiting phospholipase A2 activity, whereas, azelastine inhibits the leukotriene C4 production by inhibiting phospholipase A2 and leukotriene C4 synthase. PMID- 8630723 TI - Diagnosis of allergy to latex. PMID- 8630724 TI - Terfenadine, astemizole, and ebastine produce QTc interval prolongation in an experimental model predictive of adverse clinical ECG effects. PMID- 8630725 TI - Asthma peak flow diary improves care. PMID- 8630726 TI - Safety questions for skin test devices. PMID- 8630727 TI - CD40 and its ligand in host defense. PMID- 8630728 TI - Polarity of T cell shape, motility, and sensitivity to antigen. AB - T cell activation requires contact with APCs. We used optical techniques to demonstrate T cell polarity on the basis of shape, motility, and localized sensitivity to antigen. An intracellular Ca2+ clamp showed that T cell shape and motility are extremely sensitive to changes in [Ca2+]i (Kd = 200 nM), with immobilization and rounding occurring via a calcineurin-independent pathway. Ca2+ dependent immobilization prolonged T cell contact with the antigen-presenting B cell; buffering the [Ca2+]i signal prevented the formation of stable cell pairs. Optical tweezers revealed spatial T cell sensitivity to antigen by controlling placement on the T cell surface of either B cells or alpha-CD3 MAb-coated beads. T cells were 4-fold more sensitive to contact made at the leading edge of the T cell compared with the tail. We conclude that motile T cells are polarized antigen sensors that respond physically to [Ca2+]i signals to stabilize their interaction with APCs. PMID- 8630729 TI - Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. AB - Programmed cell death is first observed at stage 11 of embryogenesis in Drosophila. The systematic removal of apoptotic cells is mediated by cells that are derived from the procephalic mesoderm and differentiate into macrophages. We describe a macrophage receptor for apoptotic cells. This receptor, croquemort (catcher of death), is a member of the CD36 superfamily. Croquemort-mediated phagocytosis represents the concept that phagocytosis evolved primarily as a cellular process for the removal of effete cells. Our findings support the idea that the primordial function of macrophages may have been in tissue modeling and that their adapted role is in host defense. PMID- 8630730 TI - A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. AB - Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess. PMID- 8630731 TI - TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene. AB - In vivo genomic footprinting (IVGF) was used to examine regulatory site occupancy during the activation of the murine inflammatory response gene MCP-1/JE by TNF. In response to TNF, both promoter distal and proximal regulatory regions became occupied in vivo. EMSA analysis showed that while some of the factors involved in expression, including NF-kappa B, were translocated to the nucleus following TNF treatment, others were already present and able to bind DNA in vitro. Protein kinase inhibitor studies showed that protein phosphorylation was required for TNF activation but not factor assembly. These studies provide evidence for a multistep model of TNF-mediated gene regulation involving chromatin accessibility, transcription factor complex assembly, and protein phosphorylation. PMID- 8630732 TI - IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses. AB - IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFN gamma following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFN gamma secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFN gamma production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12. PMID- 8630734 TI - TCR activation of ZAP70 is impaired in CD4+CD8+ thymocytes as a consequence of intrathymic interactions that diminish available p56lck. AB - The fate of developing CD4+CD8+ thymocytes is determined by signals transduced through surface TCR complexes. Here, we report that cross-linking of TCR on CD4+ CD8+ thymocytes fails to activate ZAP70 protein tyrosine kinase and fails to initiate downstream signaling events, unless the TCR are coaggregated with surface coreceptor molecules. TCR signaling in CD4+CD8+ thymocytes is impaired because the number of available p56lck molecules is diminished by intrathymic CD4 Ia interactions that initially activate p56lck molecules, which are subsequently degraded. As a consequence of intrathymic CD4-Ia interactions, TCR zeta chains are initially phosphorylated to recruit ZAP70 molecules, but the recruited ZAP70 molecules are not subsequently phosphorylated, resulting in TCR complexes that are stably associated with inactive ZAP70 molecules. Thus, intrathymic interactions that diminish p56lck regulate TCR signaling thresholds and affect TCR structure in developing CD4+CD8+ thymocytes. PMID- 8630733 TI - Defective B-1 cell development and impaired immunity against Angiostrongylus cantonensis in IL-5R alpha-deficient mice. AB - We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice. PMID- 8630735 TI - A point mutation in HLA-A*0201 results in failure to bind the TAP complex and to present virus-derived peptides to CTL. AB - Mutating the HLA-A*0201 heavy chain from threonine to lysine at position 134 (T134K) results in a molecule that presents exogenous peptide, but cannot present endogenously derived antigen. This is reflected in diminished cell surface expression and altered intracellular trafficking of T134K. The failure of T134K to present endogenous antigen can be overcome by using an ER targeting sequence, suggesting that the antigen presentation defect is restricted to TAP-dependent peptide loading. The ability of T134K to load peptide in a TAP-dependent manner is dramatically reduced compared with HLA-A*0201. By coimmunoprecipitation there is no detectable association of the T134K molecule with the TAP complex. Thus, T134K selectively affects TAP association and peptide loading, suggesting a requirement for the direct interaction of MHC class I heavy chain and the TAP complex for efficient presentation of endogenous antigen. PMID- 8630736 TI - Regulation of Btk function by a major autophosphorylation site within the SH3 domain. AB - Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development. Overexpression of Btk with a Src family kinase increases tyrosine phosphorylation and catalytic activity of Btk. This occurs by transphosphorylation at Y551 in the Btk catalytic domain and the enhancement of Btk autophosphorylation at a second site. A gain-of-function mutant called Btk* containing E41 to K change within the pleckstrin homology domain induces fibroblast transformation. Btk* enhances the transphosphorylation of Y551 by endogenous Src family tyrosine kinases and autophosphorylation at the second site. We mapped the major Btk autophosphorylation site to Y223 within the SH3 domain. Mutation of Y223 to F blocks Btk autophosphorylation and dramatically potentiates the transforming activity of Btk* in fibroblasts. The location of Y223 in a potential ligand binding pocket suggests that autophosphorylation regulates SH3-mediated signaling by Btk. PMID- 8630737 TI - AIDS: newer concepts in the immunopathogenic mechanisms of human immunodeficiency virus disease. PMID- 8630738 TI - America's medical research enterprise: a time for rededication. PMID- 8630740 TI - Acceptance of the George M. Kober Medal for 1994. PMID- 8630739 TI - Presentation of the George M. Kober Medal to David M. Kipnis. PMID- 8630741 TI - Combination antiretroviral therapy for the treatment of human immunodeficiency virus type-1 infection. PMID- 8630743 TI - T cell costimulation through the CD28 receptor. PMID- 8630742 TI - Mechanisms of action of immunosuppressive agents: cyclosporin A, FK506, and rapamycin. PMID- 8630744 TI - Proprotein convertases and the pathophysiology of human diseases: prospective considerations. PMID- 8630745 TI - Hypoglycemia-induced autonomic failure in insulin-dependent diabetes mellitus. AB - Iatrogenic hypoglycemia is a major problem for patients with IDDM. The principles of glucose counterregulation, the physiological mechanisms that normally prevent or correct hypoglycemia, are now known. In concert with decrements in insulin, increments in glucagon, and in the absence of the latter increments in epinephrine, stand high in the hierarchy of redundant glucose counterregulatory factors. In IDDM, iatrogenic hypoglycemia is the result of the interplay of absolute or relative insulin excess and compromised glucose counterregulation. Syndromes of compromised glucose counterregulation include hypoglycemia unawareness (loss of the warning, neurogenic symptoms of developing hypoglycemia), defective glucose counterregulation (the result of combined deficiencies of the glucagon and epinephrine responses to falling glucose levels), and elevated glycemic thresholds (lower glucose levels required) for autonomic activation and symptoms during effective intensive therapy. These have been conceptualized as examples of hypoglycemia-associated autonomic failure. Hypoglycemia unawareness, but not defective glucose counterregulation, is reversible during scrupulous avoidance of iatrogenic hypoglycemia. Clearly, we need to learn to replace insulin in a much more physiological fashion, or to prevent, correct, or compensate for compromised glucose counterregulation, or both, if we are to eliminate hypoglycemia from the lives of people with IDDM without compromising glycemia control. In the meantime we must practice hypoglycemia risk factor reduction with our patients, continue to seek better insight into the fundamental mechanisms of compromised glucose counterregulation, and develop practical preventive clinical strategies. PMID- 8630746 TI - Atrial natriuretic peptide increases creatinine clearance and reduces need for dialysis in patients with established acute renal failure. PMID- 8630747 TI - Molecular cloning of the renal diuretic-sensitive electroneutral sodium (potassium)-chloride cotransporters. PMID- 8630748 TI - AIDS-associated Kaposi's sarcoma: a new perspective of its pathogenesis and treatment. PMID- 8630750 TI - Simultaneous surgery for bilateral pediatric cataracts. AB - BACKGROUND AND OBJECTIVES: To perform a review of the literature comparing the safety of bilateral simultaneous lensectomies in children versus the risk of more than one general anesthesia within a short time frame, and to study the results of bilateral surgery in a small group of patients. PATIENTS AND METHODS: Bilateral simultaneous lensectomies were performed in 9 children (18 eyes), in whom increased anesthetic risks warranted this approach. RESULTS: There were no postoperative complications for at least 6 months. CONCLUSION: Data in the current literature on endophthalmitis after cataract surgery and on the risks of repetitive anesthesia are inadequate to weigh the risk of bilateral endophthalmitis against the reduced risk of one anesthesia versus two and the advantages of simultaneous early visual rehabilitation. Until such information becomes available, simultaneous removal of bilateral infantile cataracts should probably be reserved for selected cases where the anesthetic risk is higher than average. PMID- 8630749 TI - Human leukocyte antigen-derived peptides as novel immunosuppressives. AB - Synthetic peptides corresponding to linear sequences of HLA class I and class II molecules can potently inhibit T lymphocytes responses both in vitro and in vivo. The class I and class II peptides studied to date seem to function by different mechanisms. Nevertheless, several different peptides have been shown to potently induce T cell anergy. Opelz and Terasaki first demonstrated that blood transfusions improved graft survival in transplant patients (19). Data from several sources indicate that blood transfusions are immunosuppressive in: 1) increasing infections in trauma patients who have received transfusions (20), 2) increasing metastases and/or relapses in cancer patients who have been transfused (21), and 3) remissions of autoimmune diseases associated with pregnancy and/or transfusion (22). It is likely that the active constituent of blood transfusions is soluble HLA molecule (23,24). Liver transplants, which are profoundly immunosuppressive in themselves, produce large amounts of soluble HLA (25). Both B and T lymphocytes in culture secrete soluble HLA (26). We hypothesize that soluble HLA is a natural immunoregulatory molecule involved in dampening of the immune response, but, even if this is not the case, synthetic peptides corresponding to HLA sequences have profound effects on T lymphocytes and may prove to be effective for the induction of clinical tolerance in transplant patients. PMID- 8630751 TI - Penetration of topical 0.3% ciprofloxacin into human aqueous humor. AB - BACKGROUND AND OBJECTIVE: Investigation of the penetration of topical ciprofloxacin into aqueous humor. PATIENTS AND METHODS: Topical 0.3% ciprofloxacin drops were administered to 17 patients with cataracts before lens extraction surgery. The time interval between the first dose and sampling was 3 hours in 9 patients (group A) and 6 hours in 8 (group B). In both groups, aqueous samples were collected 30 minutes after the last ciprofloxacin application. Ciprofloxacin concentration was determined by high-performance liquid chromatography-fluorescence detection. RESULTS: Mean aqueous concentration was 0.83+/-0.48 microgram/ml in group A and 2.42+/-1.42 micrograms/ml in group B. CONCLUSION: Topical 0.3% ciprofloxacin penetrates into the aqueous in a significant concentration. PMID- 8630752 TI - Mechanized keratomicropigmentation: corneal tattooing with the blepharopigmentor. AB - BACKGROUND AND OBJECTIVE: A mechanized micropigmentor with a triple prong tip was used to tattoo two corneas for cosmetically unacceptable blemishes. PATIENTS AND METHODS: Two adult patients with cosmetically disfiguring corneal blemishes in sightless eyes requested cosmetic improvement. With a mechanized micropigmentor, appropriate pigment was directly inoculated through the epithelium. RESULTS: After 2 and 3 years of follow-up, respectively, there have been no complications and there has been minimal fading. CONCLUSION: This mechanical device facilitates many more penetrations to introduce pigment, with a more uniform depth than can be achieved by freehand methods. PMID- 8630753 TI - Photorefractive keratectomy for myopia. PMID- 8630754 TI - Surgical management of coexisting cataract and glaucoma. PMID- 8630755 TI - Contact versus noncontact diode laser transscleral cyclophotocoagulation in cadaver eyes. AB - BACKGROUND AND OBJECTIVE: The optimal placement and laser energy levels of a new contact probe with a variable focus used for transscleral diode cyclophotocoagulation were compared with the diode laser noncontact technique. MATERIALS AND METHODS: Fresh cadaver eyes were evaluated by a modified posterior Miyake view using videotape, light microscopy, and scanning electron microscopy. RESULTS: The noncontact diode technique at maximum energy (1.2 J) and contact diode applications at similar energy levels failed to produce obvious gross tissue ciliary body change and mild alterations by histology. Consistent gross, histologic, and scanning electron microscopic damage was noted at 3.0 J or more with the contact probe placed at the surgical limbus and defocused 0.5 to 1.5 mm posteriorly. CONCLUSIONS: This study indicates that the Multilase contact diode laser causes transscleral ciliary body destruction in a cadaver eye model and may have potential clinical usefulness. PMID- 8630756 TI - Transscleral flow of aqueous humor: an in vitro experiment. AB - BACKGROUND AND OBJECTIVE: The minimal scleral flap thickness to ensure transscleral flow following a trabeculectomy has never been determined. The present study was designed to determine, in vitro, the critical scleral flap thickness that allows transscleral flow. MATERIALS AND METHODS: The apparatus consisted of two horizontal glass chambers (A and B) connected to each other by a customized scleral disc holder. High-pressure chamber A (at 25 mm Hg) was filled with sodium pertechnetate (99mTc) labeled normal saline and low-pressure chamber B (at 5 mm Hg) with normal saline. Transscleral flow of labeled normal saline from high-pressure chamber A via varying thickness scleral discs to low-pressure chamber B was observed over 16 hours using a gamma camera. Computer analysis was performed on the obtained images. RESULTS: Transscleral flow of labeled saline was observed only with scleral discs 0.5 mm thick or less. CONCLUSIONS: If the findings hold true for the in vivo situation, aqueous humor may reach the subconjunctival space following trabeculectomy via the transscleral route, provided scleral flap thickness is less than 0.5 mm. In addition, normal uveoscleral aqueous outflow may occur across sclera less than 0.5 mm thick, e.g., posterior to extraocular muscle insertions. PMID- 8630758 TI - Inadvertent intraocular injection of depot corticosteroids. PMID- 8630757 TI - Successful visual recovery in delayed onset Bacillus cereus endophthalmitis. PMID- 8630759 TI - Transient myopia after pars plana vitrectomy. PMID- 8630761 TI - Prophylaxis of endophthalmitis. PMID- 8630760 TI - The ciliary ganglion. PMID- 8630762 TI - Long-term effect of cataract extraction on the function of an established filtering bleb. AB - BACKGROUND AND OBJECTIVE: For patients with advanced glaucoma, care must be taken in the management of cataract to prevent loss of long-term intraocular pressure (IOP) control. The authors evaluated the effect of extracapsular cataract extraction (ECCE) with posterior chamber intraocular lens (PC IOL) implantation via the inferotemporal limbal approach on the function of an established glaucoma filtering bleb. PATIENTS AND METHODS: Twenty-three eyes of 21 patients with a minimum of 48 months of follow-up (range 48 to 97 months) were retrospectively evaluated. IOP, bleb status, visual field, and visual acuity were compared before and after operation. RESULTS: Average IOP increased from 8.7 mm Hg preoperatively to 12.2 mm Hg at last follow-up. Six blebs (26.1%) decreased in size, 11 (47.8%) did not change, 4 (17.4%) increased in size, and 2 (8.7%) were lost and required refiltration. At last follow-up, 23 eyes had an IOP of less than 20 mm Hg and did not show progression of glaucoma damage by optic disc or visual field evaluation. Six eyes required resumption of medical therapy and 2 eyes required refiltration for adequate control. CONCLUSION: ECCE with PC IOL implantation via the inferotemporal limbal approach in the presence of an established filtering bleb is effective in the long-term management of the glaucomatous eye with cataract. PMID- 8630763 TI - The selective approach to successful stomal management at home. AB - Practical aspects of caring for a patient's tracheal stoma at home are reviewed, based on our experience with 1,000 head and neck cancer and/or laryngotracheal stenosis patients treated at a large, multidisciplinary medical center. Successful management depends on understanding the various types of stomas and relating basic principles of hygiene to the fundamental concepts of wound healing and inflammation. There is no standard formula. Each case requires individual attention and planning. PMID- 8630764 TI - Chronic suppurative otitis media without cholesteatoma management. AB - Chronic suppurative otitis media without cholesteatoma (CSOMWC) is a significant disease process within the pediatric population. The definition, epidemiology, pathogenesis, pathology, and microbiology of CSOMWC are described. Recommendations for the medical management of CSOMWC are supported by the clinical outcomes of three studies. PMID- 8630765 TI - The etiology and management of acute and late sequelae of radiation therapy in persons with head and neck cancers. AB - Radiation therapy is an important primary modality in the treatment of cancers of the head and neck. Persons with advanced cancers often receive combined-modality treatment with concomitant radiation and chemotherapy. The sequelae of radiation alone and that of combined therapies create an extremely vulnerable individual. The nurse caring for the person receiving cancer treatment must understand the effect of radiation on normal tissues of the head and neck region and the methods designed to ameliorate the sequelae of therapy. PMID- 8630766 TI - Practice guidelines: cerumen/foreign body. Society of Otorhinolaryngology and Head-Neck Nurses. PMID- 8630767 TI - Practice guidelines: lymphadenopathy of head & neck. Society of Otorhinolaryngology and Head-Neck Nurses. PMID- 8630769 TI - ORL data elements: the foundation of our specialty practice. PMID- 8630768 TI - Practice guideline: carotid artery rupture. Society of Otorhinolaryngology and Head-Neck Nurses. PMID- 8630770 TI - Becoming a more politically active nurse. AB - From the earliest days of nursing's history, the profession has been known for promotion of human rights, opposition to injustice and inequity, and advocacy on behalf of those who could not advocate for themselves. These accomplishments have been achieved through the efforts of politically active nurses within our ranks. This article establishes why nurses need to be involved politically, examines levels of political participation, considers barriers to such participation, and provides strategies for the nurse interested in becoming more politically involved. PMID- 8630771 TI - [The mono-oxygenase system as a target in the action of hypoxia in combination with hyperthermia]. PMID- 8630772 TI - [The activity of natural and antibody-dependent leukocyte cytotoxicity in lympholeukemia and multiple myeloma]. AB - A study was made of cytotoxic activity of lymphocytes and neutrophils in the peripheral blood of 16 patients with advanced stage Kahler's disease, 4 patients in terminal stage, 14 patients with advanced stage chronic lymphoid leukosis, and 6 patients in the terminal stage disease. It has been shown that the level of cytotoxic activity of leucocytes in tumor diseases on the immunity B-system is a criterion for the extent to which the tumor process has been disseminated. PMID- 8630773 TI - [The prognostic significance of factors predisposing to the occurrence of pyelonephritis]. AB - With the purpose of studying factors which might be pathogenetically interrelated with predisposition to pyelonephritis and used in clinical settings for diagnosis, the authors analyzed 275 records of pediatric patients having been examined in an in-patient facility for pyelonephritis. Clinical records of 106 children free from uronephrologic pathology were used as a comparison group. The examination was done by clinical, laboratory and biochemical, immunologic, functional and roentgenoradiologic techniques, as well as by making use of a medicogenetic approach. The analysis done allowed identification of both eliminable and uneliminable risk factors for pyelonephritis in the children, with the assessment-prognostic table having been provided for giving prognosis of risk of pyelonephritis development in children in each particular case, to be used in clinical settings. PMID- 8630774 TI - [The morphofunctional changes in the kidneys of patients with staghorn nephrolithiasis operated on using a block of the renal blood flow]. AB - A study was made of morphofunctional changes in the kidneys depending on duration of ischemia and use of the drug combination proposed by the contributors to the article, for pharmacological protection of the kidney. The examination of intraoperative renal biopsy specimens revealed the hystological structure of the kidney to be more intact, with its functional capacity tending to restore more readily than in control, in those patients in whom pharmacological protection had been embarked on. Constricting the renal artery and carrying on pharmacological protection of kidney we succeeded in effecting a complete elimination of the coral-like calculus fragments in favourable conditions on a "dry" organ, being able to manage with almost non-traumatic nephrotomy associated with minimum loss of blood, which fact resulted in the reduction of the lithogenesis recurrence rate by 17 %. PMID- 8630775 TI - [The validation of the use of prostatilen and testilin for treating male infertility]. AB - The paper substantiates the use in clinical setting of medicinal preparations obtaining from prostate and tests, in male infertility. Using male rats for an experimental model of infertility it was shown that prostatilene and testilin exert a stimulating effect on spermatogenesis and androgenic functions of the testis. Those agents appear to correct androgenic-estrogenic balance in the animal organism as a model of infertility. The experiment permitted a conclusion to be drawn to the effect that prostatilene and testilin are liable to be of clinical benefit when used to stimulate spermatogenesis in infertility and hypoandrogenization; besides, the rise in androgens may stimulate copulative function in patients with sexual problems. PMID- 8630776 TI - [The role of collateral cerebral blood flow in the course of atherosclerotic circulatory encephalopathy]. AB - The paper is concerned with a study into compensatory capabilities of cerebral hemodynamics in patients with atherosclerotic dyscirculatory encephalopathy in constricting lesions of major arteries of the head. A detailed analysis is provided of hemodynamic mechanisms of the natural collateral's work, viz., via anterior communicating artery and conditions for creation of suprablock anastomosis with the internal carotid artery being occluded. The paper also addresses the advantages and disadvantages of compensatory collaterals in the course of the medical condition in question. PMID- 8630777 TI - [The health status of children up to 14 years old living in an area of long-term post-accident exposure to low doses of ionizing radiation]. AB - An assessment is submitted of morbidity rates and physical development of children aged under 14, residing in the territories being monitored after the Chernobyl Power Plant accident. A high level of disharmony in physical development of the children examined was recordable, as was an excess in morbidity of both general and separate classes of disease entities among the pediatric population having been victims of the Chernobyl accident, as compared to that in relatively "clean" areas and in Ukraine as a whole. PMID- 8630778 TI - [The characteristics of the limbic-diencephalic disorders in patients with early forms of cerebral circulatory disturbances]. AB - The limbic and diencephalic disorders observed in patients with prestroke forms of cerebrovascular disturbances depend on clinical forms of cerebral circulation. Timely therapy is conducive to a more favourable course of cerebrovascular disturbances. PMID- 8630780 TI - [Clinico-biochemical comparisons in the acute period of craniocerebral trauma]. AB - The content of prostaglandin F2 alpha was evaluated in the venous blood of 114 young male patients with acute craniocerebral injury of light to moderate severity. Prostaglandin content was determined by a gas/liquid chromatography technique. In the investigations carried out, the blood level of prostaglandin F2 alpha was found to show a strong tendency to increase from the moment of injury and up to day 3 to 5 following which day it began to slowly decline. There seems to be an association between the above process and severity of injury. Mathematic analysis has shown the blood level of prostaglandin to correlate primarily with disorders in the vegetative subdivision of the nervous system. PMID- 8630779 TI - [Hemato-encephalic barrier function in strokes in patients with arterial hypertension under drug correction]. PMID- 8630781 TI - [The action of medichronal (glucose-glycine-formate) on the serotonin blood level in alcoholism patients]. AB - The effects of a novel antialcoholic medication medichronal on the serotonine concentration in blood of patients with second stage alcoholism was investigated. It has been discovered that remission in 75% of the patients was connected with its hyper- and hyposerotoninemic influence, depending on the initial lowered or increased serotonine content. Similarity of medichronal and unithiol effects was observed in the form of palyndromoinversion regulation of serotoninergic activity. In a portion of the patients the increased, compared to healthy persons, serotonine concentration before and after the treatment does not differ from the average values in the whole group. PMID- 8630782 TI - [The mechanism of the immunomodulating action of Beres Drops Plus]. AB - Beres Drops Plus were found to raise the number of E-RFCs, being formed by lymphocytes of peripheral blood of subjects presenting with low levels of T cells, and to increase the production of substances possessing thymosine-like activity (STLA) by lymphocytes and epithelial cells of the murine thymus gland. In vitro the drug preparation in question enhances endocrine function of thymus in normal animals and the organism's capacity to induce STLA synthesis following thymectomy. These findings are useful in devising rational schemes of rehabilitation of immune system with the drug preparation Beres Drops Plus in secondary immunodeficiency states developing in cases presenting with precancer conditions, malignant neoplasia, in those subjects having taken part in the elimination of the Chernobyl NPP accident aftermath, those residing on the territories controlled, as well as evolving on account of aging. PMID- 8630783 TI - [An evaluation of the reactivity of the dermal connective tissue in patients with eczema, diffuse neurodermatitis and the cutaneous form of psoriasis]. PMID- 8630784 TI - [The role of the sympathoadrenal system in the development of acute muscle fatigue]. AB - A substantial decrease in the sympathoadrenal system catecholamines was found to be the case in muscular fatigue (the animals were running on a treadmill to refusal), as evidenced by experimental studies on white rats. Sympatholytics bring about marked reduction in the catecholamine content in hypothalamus, cerebral hemispheres and the heart, which fact contributes to the development of muscular fatigue. That this is really the case is indicated by a substantial shortening of time during which the animals were running until they came to be at the end of their tether subsequent upon administration of raucedil (2.5 mg/kg), (from 2 h 58 min. to 12 min). Fatigue was found out to be manifest as a result of the sympathoadrenal system reserves depletion. PMID- 8630785 TI - [The clinico-functional status of patients with unstable stenocardia (based on Holter electrocardiographic monitoring data)]. AB - As many as 50 patients presenting with unstable angina were examined for clinicofunctional status, using bicycle ergometry [correction of veloergometry], transesophageal electrocardiostimulation and 24-hour Holter ECG monitoring during 1-yr follow-up. The results of exercise tests at enrollment into the study suggested a dramatic decrement in the patients' exercise tolerance, as to Holter, with the painless myocardial ischemia tending to be more common among them than the painful one. A follow-up study a year later showed the course of the illness to be most variable among those cases presenting with unstable angina. A correction of their management being warranted, the latter was then tailored to the individual on the basis of findings from a comprehensive evaluation of each of them at the original admission to a hospital. PMID- 8630786 TI - [The frequency of behavioral type A among young myocardial infarct patients and their personality characteristics]. AB - Prevalence was studied of subjects presenting with behavioral type A among patients with myocardial infraction (MI) of young age, personality traits of the cases and lipid content in each of the groups identified. In spite of certain methodological difficulties existing in diagnosis of behavioral type of a personality we did not find type A to be predominant among young MI cases. It was subjects with psychosthenic traits, lowered tolerance to stresses that tended to prevail among MI patients of young age. PMID- 8630787 TI - [The age-related characteristics of the clinical picture of duodenal ulcer in women]. AB - A total of 53 women with duodenal ulcer were examined. Two groups were formed: group I-the premenopausal period, group II-the menopause proper, the post menopausal period. The evaluation done showed the illness run a graver course in group II women. PMID- 8630788 TI - [The metabolic aspects of the sequelae of the accident at the Chernobyl Atomic Electric Power Station in the course of kidney diseases]. AB - As many as 159 patients with chronic pyelonephritis and 100 those with glomerulonephritis residing in ecologically favourable, and on contaminated territories for 6-8 years after Chernobyl accident, were examined for indices of lipid peroxidation, antioxidant system and energy exchange. The above renal disease were found to be accompanied by a rise in the activity of lipid peroxidation and accumulation of excess of their products in cellular membranes and urine. In the antioxidant system, the activity of NAD-H2-dependent factors and an inhibitor of free radical oxidation of superoxiddismutase tends to decrease. There occur changes in isoenzyme spectrum of lactate and malatedehydrogenases. The above shifts appear to be manifest in those persons having been exposed to low radiation doses. Antioxidants and stabilizers of the biological membranes were shown to be useful in correcting the above metabolic processes. PMID- 8630789 TI - [The dynamics of the glycocholic acid content of the bile in patients with chronic inactive hepatitis]. AB - The studies made showed the content of bile acids in the bile, in particular that of glycocholic one, to get moderately reduced in chronic inactive hepatitis, which fact is thought to bring about dyspeptic events showing up in the above conditions: changeable stools, periodic moderate abdominal distention, nausea and other manifestations. It has been shown that the hepatic cells functional capacity may be medically controlled. Combined use in a therapeutic complex of riboxin and ricavit resulted in normalization of the glycocholic acid content in the patients' bile. Riboxin and ricavit used separately in the therapeutic programme proved to be ineffective as a treatment option. PMID- 8630791 TI - [The rehabilitation of colostomy patients]. AB - The most rational way of managing colostomy patients was found to be alternate use of all the methods available for care of colostomy (control of its activity by avoiding misuse or favouring one of the above more than the others). Permanent colostomy is not contraindication to rational work. Just on the contrary, a habitual work distracts the patient's mind from thinking too much about his/her bodily defects. There is a need for a society of colostomy patients to be organized, who, with the help of the fund "Miloserdie" (Charity) could improve their rehabilitation. PMID- 8630790 TI - [Problems in the echographic assessment of the kinetic capacity of the walls of the gallbladder and its sphincter apparatus]. AB - The function of bile secretion is an intricately regulated process. Apart from hormonal control there exists conditioned-reflex regulation (vagus-sympathicus- n. phrenicus dex.). Such a many-sided influence appear to facilitate the development of discoordination of the gallbladder motor-evacuatory function. The present paper addresses advantages of echography in studying the functional status of the bile-secretion system. A technique of assessing the gallbladder kinetics in its interrelation with the sphincter apparatus is proposed, with their various combinations having been identified. A correct assessment of their interrelations, differentiated approach to therapy will, we believe, help in attempts to reduce the recurrence rates and the gravity of the course of the hepatobiliary system inflammatory diseases. PMID- 8630792 TI - [Plasmapheresis efficacy in the treatment of patients with chronic cholestatic hepatitis and primary biliary liver cirrhosis]. AB - Effectiveness of plasmapheresis in a combined treatment of 46 patients with chronic cholestatic hepatitis and 30 with primary biliary cirrhosis of the liver was studied. Gratifying therapeutic results from plasmapheresis were achieved in 62 patients (81.5%). Time-related clinical manifestations of the illness, biochemical and immunological indices in the early post-plasmapheresis period were different in the groups studied. The mechanisms of therapeutic effect of plasmapheresis in patients with chronic cholestatic diseases of the liver were found to be chiefly due to its immunocorrective action, elimination of toxic products, returning to normal of rheological parameters of blood. PMID- 8630793 TI - [The central hemodynamic indices of patients with ulcerative gastroduodenal hemorrhage]. AB - Changes in certain indices for central hemodynamics in the early period after emergency operation were evaluated in 79 patients with helcoid gastroduodenal hemorrhage, the patients' age ranging between 18 to 80 years. At admission all patients demonstrated lowered cardiac and stroke output, cardiac and stroke indices, with the total peripheral vascular resistance being slightly increased. Operation aggravates disturbances in hemodynamics caused by loss of blood. Under the effect of corrective therapy, from day 3 to 5, indices for central hemodynamics tend to slowly return to normal as do those for hepatic bloodflow. The impairment of the function of the circulatory system appear to be greater in cases presenting with deficiency of globular volume of excess of 50%, whose compensatory potential is under too severe a strain. At discharge from the hospital, the percentage of those cases presenting with hypodynamic type circulation amounted to 67.3%, normodynamic having been 32.7%. PMID- 8630794 TI - [The role of disorders in the pulmonary surfactant system in the pathogenesis of acute pneumonia]. AB - Findings from the electronic microscopy investigation into the surfactant system suggest that the trigger mechanism in the pathogenesis of the inflammatory diseases of the lungs may be disarrangements in the surfactant system on ultrastructural and micromolecular levels, that precede the clinical manifestations. As a result of profound, uncorrectable changes in the organism's surfactant system the inflammatory process tends to run a protracted course. PMID- 8630795 TI - [The treatment of chronic prostatitis complicated by sexual disorders with vibrotherapy]. AB - The authors used intrarectal local vibrotherapy in 151 patients with chronic prostatitis; of these, 72 had corticospinal impotence. The most numerous group of cases with chronic prostatitis complicated by impotence was constituted of those patients with an inflammatory process in prostate, whose parenchymas were badly injured by a pathologic process leading to different stages fibrosis. The authors regard impotence as a sequence of events in the course of a pathologic process in spinal cord according to the Law of Hysteresis, bearing in mind that the process of hysteresis is reversible. All patients derived apparent therapeutic benefit from vibratory message in a combined treatment of chronic prostatitis, having got well in the long run. Recovery of sex function was observed in 52%. In cases of fibrosis development in patients with impotence the only current hope for improvement in the condition is offered by stimulation with electrical current of the colliculus seminalis zone. PMID- 8630797 TI - [Anticyan--a new highly effective antidote in cyanide poisoning]. AB - Anticyan is a highly effective antidote of cyanic acid and cyanide-containing compounds. It was created at the Institute of Pharmacology and Toxicology of AMS of Ukraine. Anticyan differs from other existing cyanide antidotes in that it possesses a tready methemoglobin-formation activity, has no hypotensive effect and is capable of direct stimulation of cyanide-inhibited tissue respiration. PMID- 8630796 TI - [Bettolepsy as a variant of transient ischemic attacks]. AB - Bettolepsy (B) refers to episodes of fainting of abrupt onset in patients with nonspecific pulmonary diseases, occurring at the paroxysm of coughing. The authors conducted a comprehensive evaluation in 12 patients liable to B attacks, that incorporated study of the state of the vegetative nervous system, EEG, REG, ultrasonic dopplerography of major vessels of the brain, computerized tomography to the brain, echocardioscopy, roentgenography of the spinal column. The data obtained allow B episodes to be regarded as an acute disturbance in cerebral bloodflow in vascular beds of the vertebrobasilar system of transient ischemic attacks type, which fact leads to derangement in the ascending impacts of the reticular formation, and is a syncopal state. PMID- 8630798 TI - [A new sorbent for concentrating viruses from an aqueous medium]. AB - Sorption activity was studied of a naturally occurring dispersive mineral saponite towards certain representatives of the families of Picornaviridae and Reoviridae: poliomyelitis type II viruses, Coxsackie B 1 and B 6, monkey disease rotavirus. In increasing rank order in respect of specific sorption activity is naturally occurring bentonite--sodium form of bentonite--naturally occurring saponite. Possible mechanisms of sorption of viruses on the sorbent's surface, related to structural and chemical properties of the mineral are discussed. PMID- 8630799 TI - [Work capacity and the factors limiting it in patients with III- to IV-degree obesity]. AB - There is a marked decrement in physical performance of those patients with III to IV grade obesity as compared to apparently healthy volunteers going into training. Deficient reserve capabilities of the system for utilization of oxygen and oxygen transport appear to be those factors determining and limiting physical fitness of obese individuals. Exercise testing involving recording of indices for gas exchange in real time appears to be a valuable method of diagnosis of particular features of oxygen supply in patients with III to IV grade obesity, providing a very helpful information. PMID- 8630800 TI - [The Lvov school of clinical pathologists]. AB - The story of pathology in Lviv can be briefly summarized in two stages, the first one beginning with 1785 when the University of Lviv medical faculty was organized, the second stage dating back to 1896 when the Medical Institute of Lviv and the department of pathology were found. In 1896 the first autopsy was performed at the pathology department of the Medical Institute of Lviv. Beginning with 1901 the Institute has been conducting postmortal, histological and microbiological investigations; since 1923 they have been obtaining biopsies and operative material, in 1955 histochemistry division was organized, and in 1967 electronic microscopy was first applied to studies and has become accepted practice since then. The beginning of the pathology scientific school in Lviv dates back to the end of the 19th century. It is possible to make out four principal periods of activity at the second stage of the department's development, viz., those between 1896 and 1920, 1920 and 1941, 1945 and 1965, and from 1966 up to the present day. Each of these was approximately of 20 years' duration. During the first period, A. Obzhut and Z. Dmokhovs'kyi, eminent pathologists, showed themselves first-rate lenders, the main fields of their activity having been creation of text-books for students, organization of the department's macro-museum, study into inflammation. During the second period it was V. Novits'kyi who came to the fore having devoted himself to study of infections diseases, carcinogenesis, problems of goitre; and also known for publication of a three-volume text-book on pathologic anatomy. During the third period the department was headed by M.V. Voino-Iasenets'kyi and Ie.I. Pal'chevs'kyi who distinguished themselves in the study of infectious diseases and endemic pathology. At present, D. Zerbino is in charge of the department of pathology. His principal lines of scientific activity are those of environmental and cardiovascular pathology. PMID- 8630801 TI - [The "people's hospital" in Lvov (on the 90th anniversary of its establishment)]. PMID- 8630803 TI - [Medical support for Ukrainian national military units in the first half of the 20th century]. PMID- 8630802 TI - [The clinical and biochemical characteristics of the status of stomach cancer patients who were exposed to ionizing radiation as a result of the accident at the Chernobyl Atomic Electric Power Station]. AB - As many as 208 patients with carcinoma of the stomach were examined, with 98 of these, having been victims of Chernobyl accident. It has been established that in spite of the fact that the clinical picture was practically identical in both case groups those patients having been exposed to mixed radiation demonstrated more clearly manifest endogenous intoxication, impairement of erythrocytic membrane function, which fact calls correction of these changes in the preoperative period. PMID- 8630804 TI - [The scientific and public activities of physicians in ancient Ostrog]. PMID- 8630805 TI - [Immune and microcirculatory disorders and their correction in the participants in the cleanup of the aftermath of the accident at the Chernobyl Atomic Electric Power Station who have neurosomatic pathology]. AB - A study was made of the immune status as well of microhemodynamics in those 312 patients presenting with neurosomatic pathologies who had taken part in the elimination of aftereffects of Chernobyl NPP accident. The degree of alterations in immunity and microcirculation was found to be associated with the gravity of cerebrovascular and concurrent gastrointestinal pathologies. Use in a combined treatment of ultraviolet-radiated blood, laser irradiation of blood as well as administration of parmidine permit rapid elimination of the abnormalities revealed. An association was established between values for immunity and those for microcirculation, which fact pathogenetic interrelationship between these findings. PMID- 8630806 TI - [The systematics of mental disorders related to the sequelae of the accident at the Chernobyl Atomic Electric Power Station]. AB - A dynamic survey was undertaken of mental status of those subjects exposed to ionizing radiation after Chernobyl NPP breakdown (n = 476). The examinees exhibited delayed course (up to 8 years) of psychopathologic disorders with the onset consequent upon working in the alienation zone or evacuation. On instituting some measures to form the pathological mental changes in the subjects described a process or psychopathologic development was noted to occur in 74 (15.5%) and 402 (84.5%) respectively. The psychopathologic disorders detected were arranged in the following classes: psycho-organic, neurotic, chiefly posttraumatic stressor, and psychosomatic ones, the relevant figures being as follows: 14.5% (n = 68), 12.05% (n = 58) and 58.0% (n = 176). A scheme of mental disorders related to the Chernobyl NPP breakdown aftereffects was suggested, which is likely to be amended in the course of further study. A specialized Ukrainian Psychoneurologic Rehabilitation Centre is envisaged, designed to give help to the Ch NPP accident survivors. PMID- 8630808 TI - [The system of the integration of man with the environment]. AB - Systemic integrity of man consists not only in comprehensive evaluation from the medical-biological and social standpoints-nothing can be said against that-but also in systemic approach to the environment he lives and acts in, -and not from the point of view of its investigation, which is in itself indisputable, but mainly in a final goal-control of the quality of the external environment through influence on economic structures and their functioning. It is a matter of ecological-hygienic optimization of economic structure according to toxicometric criteria. In view of the aforestated, it is suggested that a uniform regional ecologic-and-hygienic rating (standardization) of environmental factors be developed. PMID- 8630807 TI - [The functional status of the hypophyseal-thyroid and hypophyseal-adrenal systems in breast cancer patients taking into account their exposure to the factors of the accident at the Chernobyl Atomic Electric Power Station]. AB - Breast cancer patients residing in those territories controllable because of Chernobyl APP accident demonstrated significant disturbances in functional state of hypophyseal-thyroid and pituitary-adrenal axes, with central regulatory mechanisms being affected to the greatest extent. The above disturbances may arouse augmentation of prolactin secretion, which fact tends to aggravate the morbus course worsening the prognosis as well as leading to a variety of disorder in the reproductive system. PMID- 8630809 TI - [Tolerance for physical loading and its determinative factors in persons who took part in the cleanup of the aftermath of the accident at the Chernobyl Atomic Electric Power Station]. PMID- 8630810 TI - [Nitric oxide in the mechanism of the action of cardiovascular agents]. PMID- 8630811 TI - [The role of lead and its compounds in the development of arterial hypertension]. PMID- 8630812 TI - [The current aspects of the treatment of patients with chronic lympholeukemia]. PMID- 8630813 TI - [The role of neurohumoral factors and hemodynamic disorders in the development of primary juvenile arterial hypertension]. PMID- 8630814 TI - [Pain in the upper extremity after a stroke]. AB - Pain in the upper extremity presenting itself after prior stroke occurs in about 84% patients with hemiplegia. It may result from central/peripheral nervous system pathology or both. The arising and developing pain as well as its degree and pronouncement are thought to be influenced by additional factors such as aphasia, abnormalities in the affected extremity sensibility, depression. Treatment of post-stroke patients with the upper extremity pain syndrome is a difficult problem. Therefore it is essential to determine and, as far as possible, to adequately manage the source of pain since pain is an additional, and sometimes, a dominating factor in the patients' sufferings. PMID- 8630815 TI - [The experimental and clinical aspects of eicosapentaenoic amd docosahexaenoic acids (a review of the literature and the author's own data)]. AB - The author analyses data from relevant literature as well as results of his own studies on mechanism of angioprotective action of higher polyunsaturated fatty acids, such as eicosapentaenic and docosahexaenic ones in particular. The most important effect of the diet saturated with the above acids is conditioned by their influence on the cellular membrane lipid composition, activity of membrane associated enzymes, prostaglandin system, and, above all, synthesis of prostacyclin and thromboxane. The ability of exogenous polyunsaturated fatty acids to get incorporated into membrane phospholipids of blood constituents and endothelial cells of blood vessels appears to influence the basic properties of plasmolemma and function of receptors. Angioprotective properties of eicosapentaenic and docosahexaenic acids having been confirmed experimentally and in clinical settings, the preparation of these acids was considered liable to be of help in prophylaxis and treatment of vascular affections, in particular, those of a diabetic nature. PMID- 8630817 TI - [The syndromal principle in the diagnosis and construction of a working classification of cardiac arrhythmias]. PMID- 8630816 TI - [The physical work capacity of patients with neurocirculatory dystonia and the early ventricular repolarization syndrome]. AB - A total of 35 patients with neurocirculatory dystonia (NCD) presenting with a syndrome of early repolarization of ventricles (SERV), who comprised the principal group, were studied for an effect of the syndrome under conditions of the bicycle ergometer [correction of veloergometer] exercise test on EKG as well as on the indices for physical performance. The results obtained were compared to those in 32 NCD cases without SERV signs (control group) and 20 essentially healthy subjects. NCD SERV cases, while in bicycle ergometry [correction of veloergometry], developed depression up to the isometric line of the elevated St segment, and in a portion of cases it was 0.5--1 mm under the isoline with subsequent restoration of EKG findings to the baseline level after the cessation of the test. SERV occurrence in NCD patients did not influence exercise tolerance, increments of heart rate, arterial blood pressure and indices for "double product" under bicycle ergometric [correction of veloergometric] testing. Pattern of changes in the above indices depended, for the most part, on the condition of the baseline vegetative tone. PMID- 8630818 TI - [An experimental study of the mechanism of the combined action on the body of ionizing radiation, pesticides, nitrates and lead and cadmium salts]. AB - The studies made showed that repeated combined action of chemical factors (pesticides of different chemical groups, Cd and Pb salts, nitrates) in the doses studied resulted in an increase of the lipid peroxidation (LPO) in tissues, accumulation of its end products in biosubstrates, and activation of catalase and peroxidase oxidoreductases in blood of test animals. Combined action of chemical factors and gamma radiation was found out to make for increasing in lipid peroxidation and involving of ceruloplasmin in the LPO inhibition. Depletion of the antioxidant system in repeated irradiations against the background of chemical loading was accompanied by accumulation of the LPO products. We conclude that in the pathogenesis of the revealed changes in associated effects of ionizing radiation, pesticides, nitrates, Plumbum and Cadmium salts, the LPO activation in the presence of the antioxidant system depletion is most important, in view of which fact the specific criteria for the studied factors combined effects on the organism appear to be the activity of the oxidation-reduction enzymes and the level of the-active products, with the chief preventive measures being directed towards increasing of radioresistance of the organism through correction of the antioxidant deficiency. PMID- 8630819 TI - [The relationship between the copper and zinc intake with food and the prevalence of ischemic heart disease and its risk factors]. AB - There has been studied the actual dietary intake of copper and zinc in the open male population aged 20-59 years old (n = 1556) consisting of residents of Kiev. An age-associated tendency has been disclosed towards lowering copper intake and increase in the diet content of zinc. A correlation has been established between low intake of copper, high content of zinc and prevalence of ischemic heart disease and of such risk factors for its onset as dyslipoproteinemia, arterial hypertension and excessive body mass. PMID- 8630820 TI - [The combined use of diprospan and laser irradiation of the joints in rheumatoid arthritis patients]. AB - An evaluation was done of effectiveness of diprospane (prolonged action glucocorticosteroid) in rheumatoid arthritis. There have been studied parameters characterizing the articular syndrome, laboratory values during the course of complex therapy that incorporated intraarthrous administration of diprospane and a course of laser irradiation of the joints against a background of intake of non steroid antiinflammatory preparations and basis therapy with methotrexate. Topical therapy incorporated into the complex of therapeutic measures applied in patients with rheumatoid arthritis was found to be associated with most stable and pronounced effect, especially in the arthrous form of the malady. Combined use of intraarthrous administration of diprospane and a course of laser therapy permits achieving a favourable effect after a single administration of this drug preparation. PMID- 8630821 TI - [The indices of central and peripheral hemodynamics and of autonomic nervous system function in subjects with arterial hypertension working under different manufacturing conditions]. AB - As many as 318 persons with arterial hypertension working at an industrial enterprise, engaged in agricultural production, and in the sphere of mental work, were examined. The control group consisted of 40 essentially healthy subjects from the above cohorts. The following subgroups were identified in the groups observed: borderline arterial hypertension, I and II stage hypertensive disease. The examination involved electrocardiography, tetrapolar rheography, echocardiography, rheoencephalography, phonotacho-oscillography, variation pulsography, orthostatic test. Several distinguishing features were noted of central and peripheral hemodynamics, state of the vegetative nervous system in different occupational groups, which fact allows adoption of optimal, pathogenetically substantiated therapy of arterial hypertension in the cohorts followed-up. PMID- 8630822 TI - [The effect of radon baths at the Khmel'nik health resort on the central hemodynamic indices and calcium-regulating hormones in patients with essential hypertension]. AB - A total of 132 patients with stage I, II, essential hypertension were examined. Indices for central hemodynamics were determined as were the levels of parathormone, calcitonin, and ionized calcium. Radon baths in eukinetic and hypokinetic types of circulation caused a significant reduction in mean hemodynamic pressure and resulted in improvement of patency of arterioles. In so far as hyperkinetic type of hemodynamics is concerned, there was a significant reduction in cardiac index and an increase in specific peripheral resistance. The level of ionized calcium got increased in all the patients, the same being true of the levels of parathormone and calcitonin in cases of eukinetic and hyperkinetic types of circulation. PMID- 8630823 TI - [The use of tardyferon in the combined treatment of patients with chronic atrophic gastritis and peptic ulcer complicated by hemorrhage]. AB - Tardipherone is capable of maximum absorption in the initial portion of small intestine, iron being in its retarding form, which fact ensures a prolonged action of the drug, mucoprotease being a part of tardipherone composition acting to guard gastric and duodenal mucosae against irritating effect of iron. Tardipherone was found to be efficacious in treating patients with ulcer disease complicated by bleeding. Under its effect the hemogram indices get normalized as does the ratio of aggressive to protective factors of gastric juice, which factors play a major part in the emergent relapse of the illness. The use of tardipherone was found to be justified in the treatment of patients with chronic atrophic gastritis, being associated with gratifying shifts in the clinical picture of the condition and improvement in the hemogram characteristics. PMID- 8630824 TI - [Complement-binding antibodies to alpha-amylase in the diagnosis of chronic pancreatitis]. AB - Results are reported of measurement of blood levels of compliment-fixing antibodies to alphaamilase in 20 patients with chronic pancreatitis, 20 patients with different affections of the alimentary canal and no involvement of pancreas, and 20 essentially healthy subjects. Measuring antibodies to alphaamilase we obtain a new diagnostic aid identifying chronic pancreatitis, that of high sensitivity (95%), specificity and prognostic value. PMID- 8630825 TI - [The ultrastructural characteristics of the duodenal mucosa in peptic ulcer patients undergoing treatment with EHF-range electromagnetic radiation]. AB - An effect was studied of electromagnetic radiation of the millimetric wave band (EHF EMR) on ultrastructural processes of the mucosa and healing of ulcer in 10 patients with duodenal ulcer. Biopsy specimens were taken from the base of the margin of the ulcer and in the area 1 cm from the margin of the ulcer defect before the treatment, after 5 and 10 sessions of EHF EMR to acupuncture points. EHF EMR was found to exert a stimulating effect on the processes of ulcer regeneration. Seen after session 5 are layers of proliferating poorly differentiated enterocytes, activation of immune and autoimmune processes, normalization of the hemomicrovessel endothelial cell structure, filling in the ulcer defect with a newly formed granulation tissue in which, among fibroblasts, macrophages, mast cells and lymphocytes, there prevail plasmic cells with an augmented secretion of immunoglobulins, increase in proliferation of epitheliocytes, with their differentiation slowing down, and a complete epithelialization of the ulcer following ten treatment procedures. PMID- 8630826 TI - [The role of and changes in the copper and zinc levels and the activity of their related metalloenzymes in the blood of patients with destructive pulmonary tuberculosis during chemotherapy]. AB - Patients in acute phase of destructive pulmonary tuberculosis experienced a considerable rise in the activities of ceruloplasmin and carboangidrase as well as in the content of copper and zinc. In so far as destructive pulmonary tuberculosis is concerned, the therapeutic results were far from being gratifying. Loss of copper and zinc arising in the organism rapidly lead to a depletion of the latter and deficiency of trace elements, which fact may, in its turn, disturb the work of the compensatory-defense mechanisms. This warrants correction of the above trace elements in the organism of a patient with destructive pulmonary tuberculosis. PMID- 8630827 TI - What to do with physicians who commit sexual abuse. PMID- 8630828 TI - What to do with physicians who commit sexual abuse. PMID- 8630829 TI - View of US litigation challenged. PMID- 8630830 TI - Prevalence of HIV in Quebec. PMID- 8630831 TI - Sex differences in physicians' activity level. PMID- 8630832 TI - Sex differences in physicians' activity level. PMID- 8630833 TI - Heroes of early radiology. PMID- 8630834 TI - Health care spending Alberta-bound? PMID- 8630835 TI - A sociobehavioural perspective on genetic testing and counselling for heritable breast, ovarian and colon cancer. AB - Testing for susceptibility to heritable breast, ovarian and colon cancer has unique psychosocial costs. Negative test results may not be sufficient to relieve anxiety, and positive results can cause sufficient distress to compromise patient compliance with surveillance and risk reduction measures. More needs to be learned about how sociocultural factors affect the understanding of risk, how decisions to undergo testing are made and how information about increased risk affects family dynamics. As the demand for testing and counselling grows, health care providers will be faced with new challenges and dilemmas. A better understanding of genetics by the public is needed to mitigate deterministic attitudes that can lead to the neglect of health promotion. Also of concern are the socioeconomic implications of being identified as having a high risk for heritable cancer and the dangers inherent in using genetics to explain sociological phenomena. Health care providers must take the lead in ensuring that developments in genetics are used to the benefit of all. PMID- 8630837 TI - Recommendations for ensuring early thrombolytic therapy for acute myocardial infarction. The Heart and Stroke Foundation of Canada, the Canadian Cardiovascular Society and the Canadian Association of Emergency Physicians for the Emergency Cardiac Care Coalition. AB - OBJECTIVE: To recommend practical steps to ensure early thrombolytic therapy and thereby reduce mortality and morbidity associated with acute myocardial infarction (AMI). OPTIONS: Various factors were considered that influence time to thrombolysis related to patients, independent practitioners and health care systems. OUTCOMES: Reduction in morbidity and mortality associated with AMI. EVIDENCE: Early initiation of thrombolytic therapy reduces morbidity and mortality associated with AMI. The ECC Coalition analysed the factors that might impede early implementation of thrombolytic therapy. VALUES: Published data were reviewed, and recommendations were based on consensus opinion of the Emergency Cardiac Care (ECC) Coalition. The ECC Coalition comprises 20 professional, nongovernment and government organizations and has a mandate to improve emergency cardiac care services through collaboration. BENEFITS, HARMS AND COSTS: Early thrombolytic therapy reduces morbidity and mortality associated with AMI. Implementation of the recommendations will result in reduced time to thrombolytic therapy, streamlining of current practices and enhanced cooperation among health care professionals to expedite care. Depending on existing practices, implementation may require protocol development, and public and professional education. Although costs are associated with educating the public and health care professionals, they are outweighed by the financial and social benefits of reduced morbidity and mortality. RECOMMENDATIONS: Early recognition of AMI symptoms by the public and health care professionals, early access to the emergency medical services system and early action by emergency care providers in administering thrombolytic therapy (within 30 minutes after the patient's arrival at the emergency department). VALIDATION: No similar consensus statements or practice guidelines for thrombolytic therapy in Canada are available for comparison. PMID- 8630839 TI - Adverse events temporally associated with meningococcal vaccines. AB - OBJECTIVE: To determine the incidence of severe adverse events temporally associated with meningococcal vaccines administered as part of a mass vaccination program. DESIGN: Retrospective descriptive study of events reported to a passive provincial surveillance system. SETTING: The province of Quebec. PARTICIPANTS: The 1,198,751 individuals aged 6 months to 20 years who were vaccinated against meningococcal disease between Dec. 27, 1992, and Mar. 31, 1993. OUTCOME MEASURES: Total numbers and rates of severe adverse events, including allergic reactions, anaphylactic reactions, neurological events (other than abnormal crying and screaming) and other serious or unusual events. RESULTS: A total of 118 reports of severe adverse events were selected from the surveillance system. The most frequent were allergic reactions (9.2 per 100,000 doses). Few anaphylactic or neurologic reactions were reported (0.1 and 0.5 per 100,000 doses respectively). There were no reports of sequelae or of encephalopathy, meningitis or encephalitis. CONCLUSION: Meningococcal vaccines seem to be associated with fewer adverse events than have previously been reported. Existing surveillance programs are useful for determining the incidence of adverse events temporally associated with vaccines. PMID- 8630836 TI - Periodic health examination, 1996 update: 1. Prenatal screening for and diagnosis of Down syndrome. Canadian Task Force on the Periodic Health Examination. AB - OBJECTIVE: To make recommendations to physicians providing prenatal care on (1) whether prenatal screening for and diagnosis of Down syndrome (DS) is advisable and (2) alternative screening and diagnosis manoeuvres. OPTIONS: "Triple-marker" screening of maternal serum levels of alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol; fetal ultrasonographic examination; amniocentesis; and chorionic villus sampling (CVS). OUTCOMES: Accuracy of detection of DS in fetuses, and risks to the mother, including psychologic distress, and to the fetus from the screening and diagnostic interventions. EVIDENCE: A MEDLINE search for relevant articles published from Jan. 1, 1966, to Mar. 31, 1994, with the use of MeSH terms "Down syndrome," "prenatal diagnosis," "screening," "prevention," "amniocentesis," "chorionic villus sampling," "ultrasonography," "anxiety," "depression" and "psychological stress" and a manual search of bibliographies, recent issues of key journals and Current Contents. VALUES: The evidence-based methods and values of the Canadian Task Force on the Periodic Health Examination were used. A high value was placed on providing pregnant women with the opportunity to determine whether they are carrying a fetus with DS and to make choices concerning the termination of the pregnancy. The economic issues involved are complex and were not considered. BENEFITS, HARMS AND COSTS: Triple-marker screening identifies an estimated 58% of fetuses with DS, but it has an estimated rate of true-positive results of 0.1% and of false-positive results of 3.7% (given a risk cut-off of one chance in 190 of DS). These rates vary with maternal age and the risk cut-off chosen. Women with a known risk of having a fetus with DS (e.g., those who have had a previous child with DS) may benefit from a reduction in anxiety after confirmation that their fetus does not have DS. Screening allows women at low risk of having a child with DS to detect fetuses with the syndrome, but may cause psychologic distress if there is a false-positive screening test result. Up to 20% of women with positive results of screening tests may decline to undergo a subsequent amniocentesis. Amniocentesis and CVS are very accurate in diagnosing DS in fetuses and have a very low rate of serious complications for the mother. Amniocentesis is associated with a 1.7% rate of fetal loss when it is performed after 16 weeks' gestation, whereas the rate among controls is 0.7% (for a difference of 1%, 95% confidence interval 0.3% to 1.5%). CVS entails a greater risk of fetal loss than amniocentesis (odds ratio 1.32, 95% confidence interval 1.11 to 1.57). There is little evidence from controlled trials of significant associations between amniocentesis or CVS and neonatal morbidity or malformations; however, samples have been too small to show differences in rare outcomes. Results from some case-control studies suggest that CVS increases the risk of transverse limb deficiency. Costs were not considered because they are beyond the scope of this review. RECOMMENDATIONS: There is fair evidence to offer triple-marker screening through a comprehensive program to pregnant women under 35 years of age (grade B recommendation). Women given detailed information about serum-marker screening show more satisfaction with the screening than those not given this information. There is fair evidence to offer amniocentesis or CVS to pregnant women 35 years of age and older and to women with a history of a fetus with DS or of a chromosome 21 anomaly (grade B recommendation). Information on the limitations and advantages of each procedure should be offered. Triple-marker screening may be offered as an alternative to CVS or amniocentesis to pregnant women over 35. VALIDATION: Recommendations concerning prenatal diagnosis are similar to those of the US Preventive Services Task Force, the Society of Obstetricians and Gynaecologists of Canada, the Canadian College of Medical Geneticists and the Cochrane Pregnancy and Childbirth Group. No previous specific recommendations concerning triple-maker screening exist. SPONSORS: These guidelines were developed and endorsed by the Canadian Task Force on the Periodic Health Examination, which is funded by Health Canada and the National Health Research and Development Program. PMID- 8630838 TI - Has laparoscopic cholecystectomy changed patterns of practice and patient outcome in Ontario? AB - OBJECTIVE: To examine the effect of the introduction of laparoscopic cholecystectomy (LC) on patterns of practice (number of cholecystectomy procedures, case-mix and length of hospital stay) and patient outcomes in Ontario. DESIGN: Cross-sectional population-based time trends using hospital discharge data. SETTING: All acute care hospitals in Ontario where cholecystectomy was provided. PATIENTS: All 119,821 Ontario residents who underwent cholecystectomy between 1989-90 and 1993-94. After exclusions (initial bile duct exploration, cancer, incidental cholecystectomy, or missing codes for age, sex or residence) 108,442 patients remained. OUTCOME MEASURES: Number of cholecystectomy procedures, proportion of patients with acute or chronic gallstone disease, length of hospital stay, and rates of death, readmission, and bile duct injury and other in-hospital complications after cholecystectomy by year. RESULTS: The number of cholecystectomy procedures increased by 30.4% between 1989-90 and 1993-94. The number of patients with chronic gallstone disease increased by 33.6%, and the number who underwent elective surgery increased by 48.3%. The proportion of procedures performed as LC increased from 1.0% in 1990-91 to 85.6% in 1993-94. Patients who received LC tended to be younger female patients with chronic gallstone disease with no coexisting conditions undergoing elective operations. The mean length of stay, adjusted for case-mix differences, was significantly lower in 1993-94 than in 1989-90 (2.6 days v. 7.5 days) (p < 0.05); the values for LC and open cholecystectomy in 1993 94 were 1.8 days and 7.3 days respectively. The decrease in the crude death rate over the study period (0.3% to 0.2%) was not significant (relative odds 1.10, 95% confidence interval [CI] 0.72 to 1.69). In 1993-94 the adjusted risk of readmission to hospital within 30 days was 1.38 (95% CI 1.19 to 1.58) as compared with 1989-90. Over the 5 years the rate of bile duct injuries tripled (0.3% in 1989-90 v. 0.9% in 1993-94). The adjusted risk of having at least one complication after cholecystectomy in 1993-94 was 1.90 (95% CI 1.75 to 2.07) as compared with 1989-90. CONCLUSIONS: LC has had a substantial effect on the number of cholecystectomy procedures performed, the type of patient having the gallbladder removed and the length of hospital stay. Death rates are unchanged, but the odds of readmission and in-hospital complications are both increased. Future research should be directed toward determining the reasons for the overall increase in rates, developing methods to reduce bile duct injuries and identifying other relevant outcomes, such as patient satisfaction with the procedure. PMID- 8630840 TI - Initiating thrombolytic therapy for acute myocardial infarction: whose job is it anyway? AB - Although thrombolytic therapy has clearly become the standard of care for acute myocardial infarction (AMI), its delivery in Canada continues to be extremely variable. Significant unnecessary delays in the initiation of this treatment still occur in many hospitals and constitute the most common avoidable cause of death in patients with AMI. The authors agree with the statement by representatives of the member organizations of the Emergency Cardiac Care Coalition (see pages 483 to 487 of this issue) that emergency service providers must get patients to hospital sooner and that all eligible patients should receive thrombolytic therapy within 30 minutes of their arrival at hospital. This objective requires that thrombolytic therapy be initiated by emergency physicians and be supported by well-established guidelines for its use. PMID- 8630841 TI - An artificial heart with the right connection. AB - Led by Dr. Tofy Mussivand at the University of Ottawa Heart Institute, a team of Canadian medical scientists, engineers, economists, physicians and medical devices manufacturers is working to bring a prototype artificial heart closer to clinical trials. Key to this effort is the development of an electrical connector that can function for a long period in the harsh environment of the human body. Such a connector will enable various components of the artificial heart, such as the internal battery, to be replaced independently. As well as being of obvious benefit to people with end-stage heart disease, a Canadian-made artificial heart has export potential that should help to redress the country's large trade deficit in medical devices. PMID- 8630842 TI - Future of Medicare system on the table as forums spark debate across country. AB - Society needs to reach some conclusions about cost-efficient primary care and how medicare dollars should be spent, panelists told a recent conference in Vancouver. The discussion covered a wide range of subjects, including the use of alternate providers and the principle of universality. PMID- 8630843 TI - Larger private-sector role in health care needed now, think tank warns. AB - The Fraser Institute, a conservative think tank based in Vancouver, has called for the establishment of a parallel private health care sector to relieve pressure on the public system, encourage the development of medical technology, stimulate the economy and create jobs. The proposal includes establishment of competition by separating the roles of purchaser and provider of health care, and the creation of a medical premium account for each Canadian that would be funded by government but apportioned by the patient. PMID- 8630844 TI - Ontario's accelerated war against Medicare misuse another sign of leaner health care times. AB - A special-investigations unit is helping the Ontario Health Insurance Plan (OHIP) curb the fraud and abuse that has been draining millions of health care dollars from the province. The government is taking a tougher line on foreigners who use friends' or relatives' OHIP cards, people who use misplaced, stolen or counterfeit cards, and on snowbirds who deliberately bend residency requirements as they try to hang on to medicare benefits. In 1994-95, Ontario spent $74 million on health care for Ontarians travelling or living abroad. PMID- 8630845 TI - Jehovah's Witnesses leading education drive as hospitals adjust to no blood requests. AB - Jehovah's Witness representatives have visited more than 10 Canadian medical schools and 200 hospitals in an attempt to educate future and practising physicians about nonblood medicine. The trend is becoming more popular since the advent of HIV, and there are now about 100 bloodless medicine and surgery centres around the world, including 52 in the US. However, a Jehovah's Witness spokesman says Canada is "conspicuously absent" from the list of countries that offer bloodless-medicine programs. PMID- 8630847 TI - Medical ethics and truth telling in the case of androgen insensitivity syndrome. AB - Should a physician always tell the truth to a patient? Is biomedical ethics too "politically correct" in certain situations? The second-place winner in the 1995 Logie Medical Ethics Essay Contest discusses whether telling the truth is the proper course for a physician dealing with certain patients. PMID- 8630846 TI - Newfoundland pathologist driving force as province finally adopts medical examiner's act. AB - Newfoundland's former chief forensic pathologist is pleased that the provincial government has adopted a new medical examiner's act that he helped develop. He hopes the new act will help clear up confusion about who does what when a body is found in the province. PMID- 8630849 TI - Pretrial preparations can improve a physician's value as an expert witness. AB - Many physicians appear in court as expert witnesses, but the quality of their testimony varies considerably, says a Nova Scotia judge. Pretrial preparation will improve the quality of a physician's testimony, reduce stress and save time, says Judge Timothy Daley, who provides some suggestions about how to prepare to be an expert witness and what to expect in the courtroom. PMID- 8630848 TI - Social isolation can be major factor if patients are unable to deal with stress, researcher says. AB - Many people can cope with demanding work environments, busy lives, personal losses and tragedies without becoming ill. Yet others become ill under the same circumstances, some as a result of only relatively small amounts of stress. A Montreal researcher says social isolation plays a major role in patients' inability to deal with stress. PMID- 8630851 TI - Addressing concerns about dental alloys. PMID- 8630850 TI - New laws mean MDs must keep up to date on living-will and related legislation. AB - Lawyer Karen Capen discusses the legal aspects of documents such as living wills, advance directives and "proxies for personal care." Most provinces are enacting legislation to govern such directives, and physicians should keep abreast of new legislation and regulations in their jurisdiction. PMID- 8630852 TI - IPS Classic. Science, art and nature: a case report. AB - The ceramo-metal restoration still forms the backbone of modern restorative dentistry, despite many new systems. This article discusses a unique ceramo-metal system, its advantages, and clinical and technical applications; teamwork between the dentist and the technician is emphasized. IPS Classic (Ivoclar Williams) is a ceramic system with several exclusive features. It encompasses Color Visions, a computer-generated shade system, and the IPS Impulse modifier system allows the ceramist unlimited creativity in color development. PMID- 8630853 TI - Provipont & Provilink. Maximizing aesthetics and function when fabricating provisional restorations. AB - Only a few decades ago, the use and fabrication of provisional restorations was still a neglected area of restorative dentistry. Recently, a new recognition has been given to the importance of provisional restoration--maintenance of space for the tooth being, restored, acceptable interim aesthetics, cover against bacterial infiltration, and the registration of vital information for the fabrication of final restorations. This two-part article presents the clinical procedures for the utilization of provisional restorations in the anterior area and in inlay/onlay preparations. PMID- 8630854 TI - Over a decade of success with Heliomolar. AB - In developing improved dental materials and application techniques, high standards and goals are set by the clinicians and the manufacturers. This article summarizes the prerequisites and desirable qualities required of composite resins for long-term success. A particular composite has demonstrated predictable and consistent long-term results and has established its place in quality dentistry. The applications for this composite resin are outlined in a clinical procedure. Its anterior/posterior universality is recognized. Several pre- and postoperative examples, in function for up to 12 years, are used to illustrate the results. PMID- 8630855 TI - IPS Empress. Restoration of a single anterior crown. AB - All-ceramic restorations have gained patient acceptance and are increasingly being used in aesthetic appearance-related restorations. This case report of a single anterior crown matching natural dentition utilizes some of the new special effect ceramic powders designed for use with the layering technique. These powders help to properly replicate incisal, mamelon, and gingival areas. The clinical procedure is described, using photographs of the case to illustrate the steps involved. PMID- 8630856 TI - An interview with Dr. Ronald Jackson, DDS, on esthetic inlays and onlays. Interview by Dr. Gary Severance. PMID- 8630857 TI - TPS Probe. True Pressure Sensitive periodontal probe. Probing the future. PMID- 8630858 TI - Understanding the distribution of cancer within the breast is important for optimizing breast-conserving treatment. PMID- 8630859 TI - The relation between an esophageal cancer and associated cancers in adjacent organs. AB - BACKGROUND: The relation between esophageal cancers and head and neck tumors was studied in order to improve the treatment results in patients with multiple cancers. METHODS: We reviewed the records of 3,375 patients with an indexed squamous cell carcinoma of the head and neck treated at our institution between 1960 and 1994; and 81 patients were found to have an associated esophageal carcinoma. Similarly, the records of 434 patients with an indexed esophageal cancer were reviewed; and 54 patients had cancers in other organs or in the residual esophagus. A total of 135 esophageal cancers with 154 synchronous or metachronous cancers were entered into the analysis. RESULTS: The risk of developing esophageal cancer was ten times higher in male patients with head and neck cancer than in female patients. Synchronous or metachronous esophageal cancer associated with head and neck cancer was most frequently seen with pharyngeal cancer (28/360 = 7.8%), followed by in the oral cavity (47/2148 = 2.2%). CONCLUSIONS: Better knowledge of the relation between an esophageal cancer and a head and neck cancer may lead to the early detection of subsequent small, potentially curable neoplasms sited in either the esophagus or the head and neck region. PMID- 8630860 TI - Carcinoid tumors in Denmark 1978-1989 and the risk of subsequent cancers. A population-based study. AB - BACKGROUND: Previous studies have suggested an excess cancer risk in patients with carcinoid tumors. This association was reexamined using truly population based data. METHODS: By means of data from the Danish Cancer Registry all carcinoid tumors diagnosed in Denmark between 1978 and 1989 were identified. All patients with primary carcinoid tumors were studied for the occurrence of subsequent cancers. The numbers of subsequent cancers observed in the follow-up period were compared with the expected numbers calculated from population rates. RESULTS: A total of 1029 patients with carcinoid tumors were identified (464 men and 565 women). The annual age-adjusted incidence rates (world standardized) for carcinoid tumors during 1978-1989 were stable at about 1.1 per 100,000 person years for both men and women. The patients were followed for the occurrence of subsequent cancers over a period comprising 2512 person-years. Thirty subsequent cancers were identified in 29 patients. The overall relative risk of subsequent cancers was 1.1 (95% CI, 0.8-1.6). Subsequent cancers of the thyroid were in excess (RR, 21.4; 95% CI, 2.4-77.1; n = 2), as were tumors of the brain and nervous system (RR, 5.4; 95% CI, 1.1-15.9; n = 3) and non-Hodgkin's lymphomas (RR, 5.8; 95% CI, 1.2-16.9; n = 3). CONCLUSIONS: Overall, this population-based study does not support previous studies of an excess cancer risk in patients with carcinoid tumors. Increased risks of cancers of the thyroid, tumors of the brain and nervous system, and non-Hodgkin's lymphomas were observed, but these findings were based on few cases. PMID- 8630861 TI - Docetaxel (Taxotere) associated scleroderma-like changes of the lower extremities. A report of three cases. AB - BACKGROUND: Docetaxel (Taxotere) is a microtubule-stabilizing agent that is potentially important in chemotherapy for a variety of malignancies. METHODS: A clinical study of the cutaneous reactions experienced by a group of patients receiving docetaxel chemotherapy was undertaken. Patients were examined before initiation of therapy, before and after each cycle of therapy, and were followed subsequent to the completion of docetaxel chemotherapy. RESULTS: Three patients developed diffuse lower extremity edema (3-18 kg) and subsequent scleroderma-like changes after receiving multiple cycles of docetaxel therapy. These patients had different underlying malignancies and dissimilar prior therapy. Rheumatoid factor, antinuclear antibodies, anticentromere, and topoisomerase antibodies were not present in any patient. The diffuse lower extremity edema did not resolve with diuretic therapy. Cutaneous biopsies in two patients revealed diffuse sclerosis. One patient had a normal lymphangiogram during the edematous phase. Discontinuation of docetaxel correlated with resolution of edema and softening of the skin. CONCLUSION: The etiology of the scleroderma-like skin changes is unclear but appears to be either a toxic effect of docetaxel or an effect of polysorbate 80 (Tween 80), the vehicle for docetaxel. PMID- 8630862 TI - Infusion site soft-tissue injury after paclitaxel administration. AB - BACKGROUND: Paclitaxel is a diterpenic plant product that has significant activity in several solid tumors, including epithelial ovarian cancer. After promising results in Phase I and II studies, its use has increased dramatically. With this increased use, isolated reports of local tissue reactions to paclitaxel have been described. The purpose of this study was to characterize further the presentation and clinical course of this toxic effect. METHODS: Nine hundred fifty-five courses of paclitaxel were administered to patients with gynecologic malignancies at M. D. Anderson Cancer Center during a 13-month period. Nineteen (2%) local infusion-site injuries in 17 patients were observed. RESULTS: The primary disease site was the ovary in 13 patients, the peritoneum in 2, and the endometrium in 2. Paclitaxel was administered as initial therapy in 6 patients and as salvage therapy in 11. Clinical evidence of infiltration was documented during infusion in 8 of 19 (42%) reported episodes. Immediate reactions consisting of mild discomfort, erythema, and edema were observed in six patients, three of whom had complete resolution of the lesion within 1 month. The remaining patients noted initial development of injury between 3 and 13 days after paclitaxel infusion. The typical injury was a discolored, raised, rounded, and indurated lesion that was moderately painful. Two patients (11%) had Grade 1 lesions, 13 (68%) had Grade 2 lesions, and four (21%) had Grade 3 lesions with central ulceration. Cellulitis requiring intravenous antibiotic therapy was associated with two lesions. At last follow-up, 13 injuries had persistent discoloration and induration; one patient had persistent ulceration present at the time of her death 6 months after presentation and one patient underwent excision of a persistent lesion at 12 months. CONCLUSIONS: Given the incidence of Grade 2 and Grade 3 local reactions, it appears that paclitaxel should be considered a vesicant. PMID- 8630863 TI - Minimally invasive surgery in children with cancer. AB - BACKGROUND: The safety and efficacy of minimally invasive oncologic procedures in children have not been well defined and only limited anecdotal experience has been published. METHODS: A retrospective review of all patients undergoing either a laparoscopic or thoracoscopic procedure at Childrens Cancer Group institutions between December 1, 1991, and October 1, 1993, was performed. RESULTS: Eighty five children underwent 88 minimally invasive surgical procedures as part of the evaluation or treatment for cancer at 15 participating centers. In 25 patients, laparoscopy was performed and 60 patients underwent 63 thoracoscopic operations. Tissue biopsies were taken in 67 cases and diagnostic material was obtained in 99% of the biopsies. Seven complications occurred, all within the thoracoscopic group. These included conversion of six operations to an open procedure. One patient developed atelectasis postoperatively. CONCLUSIONS: In pediatric patients with suspected cancer, laparoscopy was highly accurate with minimal morbidity; thoracoscopy was nearly as efficient with slightly higher morbidity. Both modalities are useful for assessment of resectability, for staging purposes, and for evaluation of recurrent or metastatic disease. PMID- 8630864 TI - European randomized study of screening for prostate cancer. Progress report of Antwerp and Rotterdam pilot studies. AB - BACKGROUND: The feasibility of screening and early detection of prostate cancer are controversial issues at this time. To conduct a randomized screening study with prostate cancer mortality as the major endpoint is one possible solution to the present controversy. METHODS: Eight pilot studies have been conducted in the Netherlands (Rotterdam) and Belgium (Antwerp) to evaluate the feasibility of a large scale European randomized study of screening for prostate cancer. Randomization and all other administrative steps necessary to conduct a large scale screening study were evaluated in the two centers. Participation rates were from 30%-42%. Essential adjustments in pilot protocols were made during the study and led to significant changes. RESULTS: Administrative procedures necessary to run a large scale randomized study were successfully established at the two centers. The experience can be used to establish similar procedures in other European countries. Follow-up in the no screening arm and information with relation to the major endpoint, prostate cancer mortality, are not yet available. In the screening arm, detection rates varied from 3.2% to 3.6%. Major changes in the application of the screening tests made during the course of the pilot studies were the use of random biopsies for prostate specific antigen (PSA) values between 4 and 10 ng/ml as well as the biopsy indication for all suspicious lesions in those men with a PSA below 4.0 ng/ml. One-third of all cancer cases were detected in this latter group. The application of the screening tests to men with low PSA values is still under evaluation. CONCLUSIONS: The pilot studies led to a common, agreed set of minimal requirements for participation in the European study. These features include randomization, PC mortality as major endpoint, age 55-70 years, biopsy policy, rescreening interval, treatment policies, and follow up. A randomized screening study seems to be feasible in Europe. PMID- 8630865 TI - Cancer at a crossroads. PMID- 8630866 TI - A clinical trial of continuous cisplatin-fluorouracil induction chemotherapy and supracricoid partial laryngectomy for glottic carcinoma classified as T2. PMID- 8630867 TI - Tumor angiogenesis in human lung adenocarcinoma. PMID- 8630868 TI - Two cases of extramedullary acute promyelocytic leukemia. PMID- 8630869 TI - Breast cancer diagnosis by lactate dehydrogenase isozymes in nipple discharge. PMID- 8630871 TI - Decline in incidence of medulloblastoma in children. PMID- 8630870 TI - High dose chemotherapy with ifosfamide, carboplatin, and etoposide combined with autologous bone marrow transplantation for the treatment of poor-prognosis germ cell tumors and metastatic trophoblastic disease in adults. PMID- 8630872 TI - Transferrin receptor expression in nonsmall cell lung cancer. Histopathologic and clinical correlates. AB - BACKGROUND: In the search for tumor-related antigens with survival-predictive value, previous studies have yielded varied conclusions regarding the expression of one such antigen, the transferrin receptor in lung cancer. The goal of this study was to define the frequency of expression of transferrin receptor in lung cancer specimens and gather preliminary data regarding the prognostic value of this tumor-related antigen. METHODS: Tissue immunoreactivity was studied with a murine monoclonal antibody to transferrin receptor in patients with nonsmall cell lung cancer who underwent surgical resection at the Medical Center Hospital of Vermont during the period from January, 1988, to May, 1991. RESULTS: The study group consisted of 32 patients (21 males and 11 females) with an average follow up length of 27 months (standard deviation of 16 months). There were 17 patients with adenocarcinoma, 14 with squamous cell carcinoma, and 1 with large cell carcinoma. At the end of data accumulation, a total of 16 deaths had been recorded (8 with squamous cell, 8 with adenocarcinoma). Normal lung tissue did not stain for transferrin receptor; however, 13 of 17 (76%) adenocarcinomas, 13 of 14 (93%) squamous cell carcinomas, and the 1 large cell carcinoma stained positively for transferrin receptor. Staining for transferrin receptor was graded according to pattern and intensity and categorized as absent-weak or strong. Survival analysis was performed to evaluate patient outcome based on a variety of clinical and experimentally determined characteristics. Groups based on N-status (N0 vs. N1 + N2, P = 0.08), stage (Stage 1 vs. Stage 2 + 3 P = 0.13), age (younger than 60 vs. 60 years or older, P = 0.09), and transferrin receptor staining (absent-weak vs. strong, P = 0.14) achieved nearly significant differences in survival. Further analysis of the differences in survival for groupings based on transferrin receptor staining found that these differences in survival reached significance for patients with larger tumors (T2 or T3, P = 0.02). CONCLUSIONS: Transferrin receptor is expressed in the majority of lung cancers and the presence of transferrin receptor in nonsmall cell lung cancers may be an indicator of poorer prognosis in certain groups of patients. PMID- 8630873 TI - Specific cutaneous infiltrates of B-cell chronic lymphocytic leukemia arising at the site of herpes zoster and herpes simplex scars. AB - BACKGROUND: Cutaneous lymphoid infiltrates at sites of herpes zoster scars in patients with B-cell chronic lymphocytic leukemia (B-CLL) often are diagnosed as benign lymphoid hyperplasia ("pseudolymphomas"). The histologic and immunophenotypic features of these lesions are not well characterized. Appearance of skin lesions in B-CLL patients is considered a poor prognostic sign. METHOD: Eight punch biopsies from five patients (three males, two females; mean age, 66.7 years) affected by B-CLL and presenting with lesions at sites of previous herpes simplex (upper lip, one patient) or herpes zoster (trunk, four patients; forehead, one patient) infections were included in the study. Histologic examination was performed on routine sections stained with hematoxylin and eosin and Giemsa. Immunohistologic stainings were performed with a standard three-step immunoperoxidase technique on formalin fixed, paraffin embedded tissue sections. RESULTS: Specific cutaneous infiltrates of B-CLL were diagnosed histopathologically and immunophenotypically in eight biopsies from all five patients. Clinically, patients presented with erythematous papules or plaques confined to the area of previous herpes virus eruptions. Histopathologic features in most cases were characterized by a variably dense perivascular and periadnexal infiltrate of small hyperchromatic lymphocytes throughout the entire dermis, reaching the subcutaneous fat. In one case, a dense, diffuse infiltrate involving the entire dermis was observed. A granulomatous reaction with presence of epithelioid and multinucleated giant cells was a prominent feature in four biopsies from three patients. Light areas containing large lymphoid cells with features of prolymphocytes and paraimmunoblasts (so-called "proliferation centers") could be observed only in the case characterized by a diffuse infiltrate. Immunohistology revealed an aberrant CD20+/CD43+ phenotype of neoplastic B cells, which is not found in normal B lymphocytes (CD20+/CD43-). Reactive T lymphocytes were present in all lesions and had a normal CD20 /CD43+/CD45Ro+ phenotype. At the time of this writing, four patients were alive without signs of skin disease after a mean follow-up of 58.5 months, and one patient died of B-CLL 24 months after the cutaneous eruption. CONCLUSIONS: Specific cutaneous infiltrates of B-CLL are not uncommon at sites of herpes virus scars. The diagnosis can be confirmed by histopathologic and immunophenotypic criteria. The prognosis is better than previously reported. PMID- 8630874 TI - Intraductal extension of primary invasive breast carcinoma treated by breast conservative surgery. Computer graphic three-dimensional reconstruction of the mammary duct-lobular systems. AB - BACKGROUND: Intraductal tumor extension is a characteristic feature of primary breast carcinoma, and is an important consideration in patients undergoing breast conservative surgery. However, there have been no reports of studies of intraductal extension within the mammary ductal tree. METHODS: Quadrantectomy specimens from 20 patients with primary invasive breast carcinoma were examined by subgross and stereomicroscopic technique to visualize intraductal tumor extension. Serial 2 mm-thick sections were subjected to two-dimensional (2-D) tumor mapping, measuring the distances and angles of extension, and to three dimensional (3-D) reconstruction of the mammary duct-lobular systems by means of computer graphics. RESULTS: Intraductal tumor extension was found in 16 of 20 specimens (80.0%), extending continuously from the primary invasive carcinoma through the mammary ductal tree. The distances and angles of extension were larger in tumors with microcalcifications, papillotubular invasive ductal carcinoma, 30% or more of intraductal component, and comedo-type intraductal tumor extension. The 3-D reconstructions demonstrated three types of extension; central (11 cases), peripheral (3 cases), and mixed (2 cases). Further, there were some ductal branches anastomosing with different mammary duct-lobular systems at various sites. In one specimen, intraductal tumor extended widely from the primary invasive carcinoma through a branch connecting adjacent mammary duct lobular systems. CONCLUSIONS: Three-dimensional reconstruction images of intraductal extension of invasive breast carcinomas are presented for the first time to the authors' knowledge. Examples of ductal anastomoses were observed, and should be considered as a risk factor for possible widespread intraductal extension through multiple mammary duct-lobular systems. PMID- 8630875 TI - Olfactory neuroblastoma. An immunohistochemical, ultrastructural, and flow cytometric study. AB - BACKGROUND: Olfactory neuroblastoma is an uncommon neuroectodermal tumor of the upper nasal cavity, microscopic features of which are not always homogeneous. No morphologic features have been found to correlate reliably with prognosis. METHODS: Twenty-six olfactory neuroblastomas occurring in 14 females and 12 males, ages 18-78 years, were studied by immunohistochemistry, electron microscopy, and DNA flow cytometry. Survival rates were statistically analyzed relative to several variables. RESULTS: Microscopically, 22 tumors formed a morphologic spectrum intermediate between paraganglioma (PG) and neuroblastoma (NB). Others included two ganglioneuroblastomas (GNB), one lesion exhibited biphasic (neuronal and epithelial) differentiation, and one tumor showed predominantly epithelial features. Immunoreactivity for neuronal and neuroendocrine markers included synaptophysin in 77%, neurofilament protein in 38%, class III beta-tubulin in 81%, and chromogranin A in 77%. In 88% of cases, elongated S-100 protein-positive cells surrounded tumor lobules. Cytokeratin and epithelial membrane antigen immunoreactivity were noted in six (23%) and two (8%) tumors, respectively. Aberrant p53 expression was detected in 16 tumors (62%). The Ki-67 labeling index (LI) varied from 0%-43.8% (mean, 7.4%). Ultrastructurally, 80-230 nm dense core granules were noted within perikarya and as in microtubule-containing processes in all of the 11 tumors studied by electromicroscopy. Lobules of seven tumors were surrounded by electron-dense sustentacular cells. Epithelial tumors exhibited obviously epithelial features in addition to neuronal differentiation. DNA flow cytometry demonstrated a high incidence of polyploidy and aneuploidy (78%) and a wide range of percent S phase fractions (1.5%-21.8%; mean, 9.0%). The study showed that longer survival rates are related significantly to (1) the occurrence of metastases which was linked to tumor subtype, (2) to a higher incidence of S-100 protein-positive cells, and (3) to a low (< 10%) Ki-67 labeling index. CONCLUSIONS: The present study indicates that (1) although typical olfactory neuroblastomas exhibit PG/NB differentiation, they more closely resemble PG, (2) occasional tumors show GNB and/or epithelial differentiation, and (3) survival rates may correlate with S-100 protein immunoreactivity and Ki-67 LI. Cancer 1995; 76:4-19. PMID- 8630876 TI - Factors related to enrollment in the breast cancer prevention trial at a comprehensive cancer center during the first year of recruitment. AB - BACKGROUND: Using an a priori theoretic model of behavior change, factors predicting enrollment in a randomized chemoprevention trial during the first year of recruitment were assessed prospectively. METHODS: Eligible participants were asked to complete a 90-item semistructured questionnaire after attendance at an informational meeting. Components of the Health Belief Model (including perceived susceptibility, perceived severity, perceived benefits and barriers, cues to action, and health motivation), health status, preventive health behaviors, and social influence were assessed in relation to enrollment. RESULTS: Overall, 331 women attended one of the meetings, and 73% completed a questionnaire; 45% enrolled on the trial and 55% did not. In bivariate analyses, all but one of the perceived barriers were associated negatively with enrollment; however, items assessing perceived susceptibility, perceived severity, and perceived benefits were not. Nonparticipants also were more likely to be over 49 years of age, to be currently or to have been on estrogen replacement therapy, and to have had hot flashes. In logistic regression analysis, not being able to take estrogen replacement therapy was the strongest predictor of nonparticipation (odds ratio [OR], 12.13, 95% confidence interval [CI], 3.63, 40.60). Other factors associated with nonparticipation were concern about side effects of tamoxifen (OR, 5.06; CI, 2.37, 10.80); the possibility of getting a placebo (OR, 7.75; CI, 1.51, 39.67); the costs associated with the trial (OR, 3.21; CI, 1.12, 9.24); and absence of concern that significant others would be reassured if the respondent was taking tamoxifen (OR, 2.58; CI, 1.04, 6.41). CONCLUSIONS: These findings support the view that recruitment efforts for chemoprevention trials should address barriers specific to their circumstances. In addition, increasing the support available from personal social networks may enhance recruitment to chemoprevention trials for breast cancer. PMID- 8630877 TI - p53 mutation and MDM2 amplification are rare even in human papillomavirus negative cervical carcinomas. AB - BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg- >Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors. PMID- 8630878 TI - Effects of external radiotherapy on uterine blood flow in patients with advanced cervical carcinoma assessed by color Doppler ultrasonography. AB - BACKGROUND: This study was designed to evaluate radiation-induced changes in tumor blood flow by color Doppler ultrasonography. METHODS: Color Doppler examination was performed on 14 patients with advanced cervical carcinoma treated with external radiotherapy. The total dose of radiation varied from 30 to 65 Gy and was given as 1.9 Gy daily fractions, 5 days/week. Tumor vascularity and blood flow impedance were measured by one pretreatment and five follow-up examinations. RESULTS: At the baseline examination, 11 of 14 patients had very low tumor blood flow impedance (< 0.70). Radiotherapy caused a significant decrease in tumor vascularity (P = 0.0001) and in presence of very low blood flow impedance. The decrease of tumor vascularity during the treatment was associated with better outcome, whereas persistence of excessive vascularity or of vessels with low blood flow impedance at the end of radiation was associated with modest therapeutic response. Eight of 10 patients with increased tumor vascularity at the end of radiation needed further treatment or died of disease. Only one of four patients with normal vasculature at the end of radiotherapy needed further treatment and all four were clinically disease free during the follow-up (mean, 13 months; range, 6-26 months). CONCLUSIONS: These results suggest that color Doppler ultrasonography may be useful in early assessment of therapeutic response during radiotherapy and in for planning individualized treatment schedules. PMID- 8630881 TI - High incidence of point mutation in K-ras codon 12 in carcinoma of the fallopian tube. AB - BACKGROUND: Adenocarcinoma of the fallopian tube is a rare tumor with a poor prognosis. Whether these carcinomas possess any genetic changes that contribute to their malignant behavior is unknown and to date few studies regarding the molecular pathogenesis of these tumors have been reported. In adenocarcinoma of the endometrium, mutations in the first exon of K-ras, although relatively infrequent, were observed to be an independent risk factor for poor clinical outcome. METHODS: Eight patients with adenocarcinoma of the fallopian tube were examined for mutations in the 12th codon of K-ras. DNA was obtained from single sections of paraffin embedded tumor tissue and the first exon of K-ras was amplified by the polymerase chain reaction. Point mutations were assayed using a nonradioactive restriction fragment length polymorphism technique. RESULTS: The eight patients in this study varied in clinical stage from I-IV and were all treated with surgery and chemotherapy. Six of eight of the patients died and one of the surviving patients had metastases in the vertebrae. K-ras point mutations were detected at codon 12 in seven of the eight tumors (87.5%). CONCLUSIONS: K ras mutations occurred with high frequency in this series of eight patients with fallopian tube carcinoma, suggesting that mutations of this protooncogene could play an important role in the molecular pathogenesis of this lesion. PMID- 8630879 TI - Mutation and allelic loss of the p53 gene in endometrial carcinoma. Incidence and outcome in 92 surgical patients. AB - BACKGROUND: Alterations of the p53 gene are involved in the development of diverse human malignancies, but their incidence and clinicopathologic features are still not well characterized for endometrial carcinoma. METHODS: To investigate the clinicopathologic significance of p53, mutations and loss of heterozygosity (LOH) in endometrial carcinoma in 92 patients with this disease were examined. RESULTS: Mutations of p53 were detected in 20 (22%) of the 92 patients with carcinoma, and LOH was detected in 23 (32%) of the 72 patients in whom heterozygosity of the gene was available. There was a significant correlation between the occurrence of mutation and LOH. Mutations and LOH were more frequent in patients with Grade 3 tumors than in those with Grades 1 and 2 tumors (P = 0.0498, P = 0.0051, respectively). Patients with LOH had a poorer postoperative survival than those without LOH (P = 0.0022, log-rank test), and patients with both LOH and mutation showed the worst prognosis (P < 0.0001, log rank test). Loss of heterozygosity of the p53 gene showed a significant relation to prognosis that was independent of tumor stage, histologic grade, and muscular invasion. CONCLUSIONS: Mutation and LOH of the p53 gene are prognostic indicators in patients with endometrial carcinoma, suggesting that alterations of p53 may play an important role in the development of this cancer. PMID- 8630880 TI - Saporin toxins directed to basic fibroblast growth factor receptors effectively target human ovarian teratocarcinoma in an animal model. AB - BACKGROUND: The antitumor activity of the chemical conjugate and recombinant forms of the mitotoxin basic fibroblast growth factor (bFGF) saporin (SAP) and the bFGF receptor-directed immunotoxin 11A8-SAP against human ovarian teratocarcinoma PA-1 was examined in athymic nude mice. Alternative administration schedules to prolong therapeutic efficacy were explored. METHODS: Intravenous dosing (0.01-125 micrograms/kg) of chemical conjugate and recombinant bFGF-SAP or 11A8-SAP beginning 5 days after subcutaneous implantation of PA-1 cells was administered by i) weekly injection for 4 weeks, ii) continuous infusion for one week, or iii) daily injection five times a week for 4 weeks. RESULTS: Weekly injections (31.25 micrograms/kg) of chemical conjugate bFGF-SAP or 11A8-SAP, the latter of which is 25% the molarity of the former, resulted in mean tumor volumes that were, respectively, 35% or 52% of controls (day 30) and 52% or 76% of controls (day 60). Chemical conjugate or recombinant bFGF-SAP administered weekly resulted in mean tumor volumes that were, respectively, 51% or 77% (0.5 microgram/kg) and 42% or 31% (50 micrograms/kg) of controls (day 30). A mean tumor volume of 35% of controls (day 30) and of 49% of controls (day 60) were observed in animals treated by constant infusion of chemical conjugate bFGF SAP (125 micrograms/kg, total dose). Alternatively, tumors of animals receiving daily injections (125 micrograms/kg, total dose) exhibited a mean volume of 21% of controls (day 30) and prolonged growth inhibition as demonstrated by a mean tumor volume of 22% of controls (day 60). CONCLUSIONS: These studies suggest a therapeutic potential for bFGF-receptor-directed saporin toxins in the treatment of ovarian teratocarcinoma and the importance of frequency of administration in achieving optimal tumor responses. PMID- 8630882 TI - Prognostic criteria in patients with stage D2 prostate cancer. Correlation with mean nuclear volume. AB - BACKGROUND: Histologic grading, especially the Gleason score (GS), is considered to be of value in determining the prognosis of patients with prostate cancer. However, subjective histologic grading is characterized by low reproducibility. Conversely, estimates of mean nuclear volume (MNV), developed by Gunderson and Jensen based on a stereologic technique, is a method with high reproducibility. Furthermore, it has been reported that MNV provides an accurate prognosis of bladder cancer. In this study MNV was compared with two histologic grading methods in determining the prognosis of Stage D2 prostate cancer. METHODS: A retrospective, prognostic study of 31 patients with Stage D2 prostate cancer treated with transurethral resection of the prostate or needle biopsy between January, 1983, and July, 1994, was performed. Unbiased estimates of MNV were compared with age at the time of diagnosis, histologic grading according to World Health Organization (WHO) classification, and GS on the prognostic value. RESULTS: Age at the time of diagnosis, WHO classification and GS had no value as prognostic criteria, and only MNV correlated significantly with prognosis of Stage D2 prostate cancer (P = 0.0017). CONCLUSION: Results of this study indicate that MNV is prognostically superior to morphologic grading of malignancy, such as GS and WHO classification, in Stage D2 prostate cancer. PMID- 8630884 TI - The natural history of ductal carcinoma in situ. Implications for clinical decision making. PMID- 8630883 TI - Prostate specific antigen levels and clinical response to low dose dexamethasone for hormone-refractory metastatic prostate carcinoma. AB - BACKGROUND: It has been suggested that suppression of adrenal androgens may provide benefit to patients with metastatic prostate cancer refractory to initial hormonal therapy (e.g., orchiectomy). METHODS: The records of 38 patients with metastatic prostate cancer that had progressed after orchiectomy who were placed subsequently on low dose dexamethasone (DXM) with no other concurrent therapy (36 patients received 0.75 mg twice daily and two received 0.75 mg three times daily) were reviewed. Symptomatic status, prostate specific antigen (PSA) measurements, and available radiographic assessments were recorded. Bone scans were reviewed by an independent, blinded evaluator. RESULTS: Symptomatic improvement was experienced by 24 patients (63%), 20 (83%) of whom also had decreases in PSA. Prostate specific antigen values decreased in 30 patients (79%) with decreases 50% or greater and 80% or greater in 23 (61%) and 13 (34%) patients, respectively. Of the 23 patients with PSA decreases 50% or greater, 8 (35%) had radiographic evidence of disease regression, 5 (22%) were stable, 7 (30%) had disease progression, and 3 (13%) did not have serial radiographic exams. Flutamide was discontinued shortly before DXM treatment for 2 of the 23 patients. CONCLUSIONS: Low dose DXM may produce important symptomatic improvement and decreased PSA levels in the majority of patients with hormone-refractory prostate cancer. In addition, a substantial percentage of those patients with decreases in PSA also will have radiographic evidence of disease regression. These results suggest the need for additional prospective controlled studies of DXM as a therapy for hormone-refractory prostate cancer. PMID- 8630885 TI - Aspirin and reduced risk of esophageal carcinoma. AB - BACKGROUND: Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) have been shown experimentally to inhibit chemically induced esophageal cancers. An epidemiologic study of more than 600,000 adults in the United States followed for 6 years found that aspirin use was associated with a reduced risk of death from esophageal cancer. METHODS: The relation of aspirin use and esophageal cancer was examined using data from the National Health and Nutrition Examination Survey (NHANES I) and the National Epidemiologic Follow-up Studies (NEFS). Of the 14,407 United States residents followed for 12-16 years, esophageal cancer developed in 15. Persons were classified as nonusers, occasional users, or regular users of aspirin based on their response to two questions at the baseline examination: whether they had taken aspirin in the past 30 days and whether they had used pain medications regularly during the prior 6 months. RESULTS: Occasional use was associated with a 90% decreased risk (95% confidence interval, 0.01-0.76) of developing esophageal cancer, and no person classified as a regular user developed the disease. Adjusting for cigarette smoking (ever vs. never) and alcohol intake (at least monthly vs. not) did not explain the finding. CONCLUSION: Aspirin use was associated with a 90% decreased risk of developing esophageal carcinoma. Further studies to determine whether aspirin is protective against both squamous cell esophageal carcinoma and adenocarcinoma of the esophagus are indicated. PMID- 8630887 TI - High dose folinic acid and 5-fluorouracil bolus and continuous infusion for patients with advanced colorectal cancer. AB - BACKGROUND: Palliative chemotherapy remains a challenge for oncologists. The combination of high dose folinic acid (HDFA) with 5-fluorouracil (5-FU) improves response rates, as do continuous infusions of 5-FU. These protocols have limiting toxicities such as diarrhea, stomatitis, and leukopenia. Another schedule of 5-FU and folinic acid has proven effective and is very well tolerated. The results of a similar regimen are reported. METHODS: Eighty-six eligible patients with evolutive advanced colorectal cancer were treated, after informed consent was obtained, with chemotherapy consisting of HDFA (200 mg/m2 in a 2-hour infusion, followed by 5-FU (400 mg/m2 IV bolus injection, then 600 mg/m2 in a 22-hour infusion) on days 1 and 2, every 15 days. Seven of 86 had received prior therapy for metastatic disease. RESULTS: Two complete and 31 partial responses were noted for an overall response rate of 38.3% (95% confidence interval, 0.25-0.45). Toxicity was low, as more than 60% of the patients had no or minor toxicity. Grade III or IV World Health Organization toxicities included stomatitis (7 patients), leukopenia (3 patients), diarrhea (2 patients), and cardiac toxicity (3 patients). The overall median survival was 10.3 months, and for those having a response, 17.1 months. CONCLUSIONS: High dose folinic acid combined with 5-FU bolus and continuous infusion for 2 days every 2 weeks is an effective regimen. Its toxicity appears low. Moreover, this chemotherapy is feasible on an outpatient basis. PMID- 8630886 TI - Incidence and distribution of distant metastases from newly diagnosed esophageal carcinoma. AB - BACKGROUND: It is important to diagnose distant metastases disease in patients with newly diagnosed esophageal carcinoma, so that unwarranted surgery and its attendant risks are avoided. The purpose of this study was to determine (1) the percentage of esophageal cancer patients with distant metastases (M1) at presentation, (2) the locations of these distant metastases, and (3) how the metastases were diagnosed. METHODS: All patients at the University of Michigan Medical Center with newly diagnosed esophageal cancer between 1982 and July, 1993, were identified. Records for these 838 patients were reviewed, and patients were classified as having M0 or M1 disease at presentation. For patients with M1 disease, the locations of distant metastases and the methods of diagnosis were recorded. RESULTS: One hundred forty-seven of 838 (18%) patients had M1 disease. In 110 of 147 (75%) patients, M1 disease was detected before surgery via imaging or physical examination, including 102 of 147 (69%) via chest or abdominal computed tomography (CT). In no case staged as M0 by abdominal and chest CT was M1 disease detected on bone scan or head CT. Distant metastases were most commonly diagnosed in abdominal lymph nodes (45%), followed by liver (35%), lung (20%), cervical/supraclavicular lymph nodes (18%), bone (9%), adrenal (5%), peritoneum (2%), brain (2%), and stomach, pancreas, pleura, skin/body wall, pericardium, and spleen (each 1%). CONCLUSION: A significant percentage of patients with esophageal cancer have M1 disease at presentation. Imaging of the chest and abdomen is an effective method of screening such patients for M1 disease before treatment. PMID- 8630888 TI - Monosomy of chromosome 18 detected by fluorescence in situ hybridization in colorectal tumors. AB - BACKGROUND: At least two different evolutional pathways of colorectal cancer, namely the adenoma-carcinoma sequence and de novo carcinogenesis, have been indicated. However, whether there is a difference between them in affected chromosomes and genes has not yet been elucidated. Chromosomal examination is expected to provide a clue to an answer to this question. In this study, the relation of aberrations in chromosome 18 to type of colorectal cancer was examined. METHODS: Numeric aberrations in chromosome 18 were investigated in 71 colorectal tumors by means of fluorescence in situ hybridization, using an alphoid satellite DNA probe specific for the pericentromeric region on chromosome 18. RESULTS: The loss of one chromosome 18 was found in 33% (6 of 18) of patients with early cancer, excluding those with cancer in an adenoma. The loss was frequent in early colorectal carcinomas without foci of adenoma (four of six patients, 67%), whereas monosomy 18 was not significant in those with foci of adenoma except for patients with a hereditary background. Specimens exhibiting monosomy 18 were macroscopically classified as flat lesions, but the converse was not true. No adenoma showed monosomy 18. It was encountered in 44% of nine cancers with invasion to the muscularis propria. However, no significant subpopulation of monosomy 18 was present in cancers penetrating through the serosa or adventitia in which polysomic populations were often identified alternatively. Furthermore, tetrasomy for this chromosome was exclusive in these advanced cancers. CONCLUSIONS: The loss of chromosome 18 is closely related to colorectal cancers without foci of adenoma. PMID- 8630889 TI - Diagnosis of hepatocellular carcinoma by concanavalin A affinity electrophoresis of serum alpha-fetoprotein. AB - BACKGROUND: Concanavalin A (Con A) affinity electrophoresis of serum alpha fetoprotein (AFP) can distinguish hepatocellular carcinoma (HCC) from other malignancies when the serum AFP concentration is elevated. However, Con A has not been able to distinguish HCC from benign chronic liver disease such as cirrhosis or chronic hepatitis. METHODS: The Con A affinity electrophoresis of serum AFP was analyzed in patients with a serum AFP concentration greater than 50 ng/mL by antibody affinity electrophoresis and Western blotting in an attempt to distinguish hepatocellular carcinoma from benign chronic liver disease. Before the assay, the serum AFP concentrations were adjusted between 100 ng/ml and 300 ng/ml by concentrating or diluting the samples. RESULTS: Of 180 patients with HCC, 44 (24%) had a single band and 91 (51%), 35 (19%), and 10 (6%) had 2, 3, and 4 bands, respectively. All 35 patients with chronic hepatitis had a single band. All but 1 of 72 patients with cirrhosis had a single band. Multiple AFP bands on Con A affinity electrophoresis appear to be diagnostic of HCC. This method has a sensitivity of 76%, a specificity of 99%, a positive predictive value of 99%, and a negative predictive value of 71% for detecting HCC. The number of AFP bands correlated with serum AFP concentration and tumor size in patients with HCC. CONCLUSIONS: This assay is useful for distinguishing HCC from benign chronic liver diseases. PMID- 8630890 TI - Predictors of survival in patients with carcinoma of the gallbladder. AB - BACKGROUND: The role of resectional surgery in patients with advanced stages of gallbladder carcinoma has not been fully defined. It is generally believed that the survival depends on the stage of the disease, rather than on the treatment option. METHODS: Seventeen selected risk factors were analyzed using univariate and multivariate analyses to predict survival in 87 patients with gallbladder carcinoma who had undergone some form of surgical treatment. Similarly, a subset of 55 patients with American Joint Committee on Cancer Stage IV disease also was analyzed separately. RESULTS: Palpable mass, tumor (T) status, local infiltration, lymph node involvement, distant metastasis, TNM stage, and the type of surgical treatment (laparotomy alone, bypass, or resection) were significant risk factors by univariate analysis. In addition to palpable mass and the type of surgical treatment, age was also a significant predictor of survival by multivariate analysis. Multivariate analysis of patients with Stage IV disease revealed the same three factors to be significant. In this subset of patients, the median survival after resectional surgery was 16.3 months; after biliary and/or gastric bypass, 4.8 months; and after laparotomy alone, 1.6 months. CONCLUSIONS: The type of surgical treatment significantly influenced survival. Resectional surgery was associated with better survival compared with biliary and/or gastric bypass or laparotomy alone for patients with all stages of the disease, including those with advanced carcinoma of the gallbladder. PMID- 8630891 TI - p53 protein accumulation in tumors of the ampulla of Vater. AB - BACKGROUND: Accurate preoperative diagnosis of tumors of the ampulla of Vater is difficult because ampullary biopsies have a high false-negative rate. Recently, it has been suggested that p53 mutations in tumors of the ampulla of Vater are associated with the transformation of adenomas and low grade carcinomas to high grade carcinomas. The purpose of this study was to determine the extent of p53 protein accumulation in tumors of the ampulla of Vater, and to determine whether p53 accumulation can be detected in false-negative biopsies. METHODS: Using a monoclonal anti-p53 antibody, sections of 4 normal ampullas, 5 adenomas, 17 carcinomas, and 9 initial biopsies of 9 of the tumors of the ampulla of Vater that had no morphologic evidence of carcinoma were immunostained. RESULTS: None of the 4 normal ampullas (0%), 2 of 5 adenomas (40%), and 16 of 17 carcinomas (94%) were positive for p53. This p53 positivity was present through all stages of ampullary carcinoma. Of the nine initial biopsies negative for carcinoma, seven were positive for p53 and, of these, six (86%) were found to be carcinomas upon resection. CONCLUSIONS: 1) The molecular events leading to p53 accumulation in tumors of the ampulla of Vater occur early in the neoplastic process. 2) Tumors of the ampulla of Vater with biopsies negative for malignancy but positive for p53 are very likely to be carcinomas. PMID- 8630892 TI - Aggressive psammomatoid ossifying fibromas of the sinonasal region: a clinicopathologic study of a distinct group of fibro-osseous lesions. AB - BACKGROUND: Psammomatoid ossifying fibromas represent a unique subset of fibro osseous lesions of the sinonasal tract. They have distinctive histomorphologic features and a tendency toward locally aggressive behavior, including invasion and destruction of adjacent anatomic structures. METHODS: Seven cases of psammomatoid ossifying fibromas of the sinonasal tract were identified in the files of the Otolaryngic Tumor Registry at the Armed Forces Institute of Pathology. Medical records, including the clinical history, location of the lesions, radiographs, treatment, and follow-up were reviewed in each case. Follow up information was available in all of the cases. RESULTS: Four of the patients were male and three were female. The patient's ages ranged from 5 to 54 years (median age, 33 years). Symptoms included facial swelling, nasal obstruction, pain, sinusitis, headache, and proptosis. Radiographic studies confirmed the presence of an osseous and/or soft tissue mass varying in appearance from well demarcated without invasion or erosion to invasive with bone erosion and intracranial extension. Sites of involvement included the nasal cavity and all paranasal sinuses, particularly the ethmoid and maxillary sinuses. Often, more than one sinus was involved and extension of disease included involvement of the orbit, nasopharynx, palate, and anterior cranial fossa. The histologic appearance was characterized by the presence of small mineralized (psammomatoid) bodies admixed with a cellular stroma with a variable amount of myxomatous material and scattered giant cells. Confusion with other osseous and soft tissue tumors may occur resulting in too limited or too aggressive management. En bloc surgical excision is the treatment of choice and may prove curative. Aggressive behavior with recurrence(s) or invasion into adjacent structures occurred. At the time of this writing, the patients are alive over follow-up periods ranging from 6 months to 7 years. CONCLUSIONS: Gnathic and midfacial fibro-osseous proliferations are a diverse group of lesions. A subset of these fibro-osseous lesions with predilection for the sinonasal tract were identified. These lesions are characterized by their distinctive histology, including psammomatoid ossicles and their locally aggressive growth. Complete surgical removal is the treatment of choice. PMID- 8630894 TI - Cartilaginous tumors: prognostic applications of cytophotometric DNA analysis. AB - BACKGROUND: Traditionally, selection of cancer therapy is based on the assessment of the prognosis of the individual patient. The specific type of tumor and the stage of disease have been the most reliable indicators of prognosis. METHODS: Image cytometry to determine DNA content was used in conjunction with clinicopathologic parameters and patient survival to investigate 16 cartilaginous tumors. Histopathologic characteristics, cytometric DNA ploidy status, 2c deviation index (2cDI), DNA malignancy grade (DNA-MG), and 5c-exceeding event (5cEE) were used to learn more about the determination of tumor prognosis. Prognosis was analyzed with a maximum follow-up of 148 months. RESULTS: DNA ploidy status, 2cDI, DNA-MG, and 5cEE are indicators of prognosis. After 148 months of follow-up, patients with aneuploid tumors had a significantly lower overall survival rate compared with those with diploid tumors (P < 0.05). Patients with DNA-MG less than 0.8 or 2cDI less than 1.5 had a significantly longer overall survival rate with respect to the group of patients with a DNA-MG greater than 0.8 or 2cDI greater than 1.5 (P < 0.001). A significant difference was noted in the overall survival rates between patients with tumors with 5cEE less than 3 and 5cEE 3 or greater (P < 0.001). CONCLUSION: Image cytophotometry DNA ploidy status, 2c deviations index, DNA malignancy grade, and 5c exceeding event were investigated and were found to be of prognostic value for patients with cartilaginous tumors. PMID- 8630893 TI - Lung cancer-related changing profile of B-cell epitopes recognized on mucosal antigens: the Der p1 model. AB - BACKGROUND: The natural immunoglobulin G (IgG) response towards antigens presented at the mucosal level in patients with lung cancer, the most frequent mucosal malignancy in humans was studied. Compared with healthy control subjects, patients with lung cancer display lower IgG antibody titers toward antigen p1 of the house dust mite, Dermatophagoides pteronyssinus (Der p1), chronically presented to the respiratory mucosa. The present study further characterizes this defect in terms of antibody specificity. METHODS: Antibody specificity was studied by comparing the IgG binding to native Der p1 (nDer p1) and its products of pepsin hydrolysis (dDer p1) in a solid phase enzyme-linked immunosorbent assay (ELISA) in 148 patients with lung carcinoma, 148 healthy control subjects, and 50 patients with chronic bronchitis. Antibody specificity was also evaluated before and after (5 +/- 1 weeks) surgical excision of the tumor in competition ELISA using streptavidin-biotin technology. RESULTS: Lung cancer sera had a higher degree of binding to dDer p1 compared with nDer p1, whereas control group sera bound similarly to the two forms of the antigen. Lung cancer sera and control group sera showed distinct capacities to prevent the binding of pooled IgG from each group to nDer p1. The inhibition capacity displayed by cancer sera is changed 5 weeks after cancer removal. CONCLUSION: These results, somewhat similar to those observed for bovine betalactoglobulin, document the recognition of a different set of epitopes on Der p1 and more generally on mucosal antigens by lung cancer IgG compared with the IgG from control patients. This distinct profile of epitope specificity changes partially soon after cancer removal, suggesting a tumor-dependent disturbance and opens the way to a new class of markers in lung cancer. Furthermore, this study documents a surprising but striking influence of the clinical status on the choice of B-cell immunodominant epitopes in mucosal responses. PMID- 8630895 TI - Cutaneous plexiform schwannoma associated with neurofibromatosis type 2. AB - BACKGROUND: Plexiform schwannoma (PS) is a rare benign tumor of the nerve sheath that can be located either in the deep soft tissues or in the dermis or subcutis. The tumor predominantly affects young adults and occurs most commonly as a slowly growing asymptomatic solitary nodule in the head and neck region, trunk, and upper extremities. METHODS: A cutaneous PS located in the preauricular region of a 19-year-old white female is reported. The patient exhibited six "cafe-au-lait" spots in the trunk and the extremities. Magnetic resonance imaging examination showed bilateral tumors in both acoustic nerves (considered schwannomas) and also masses in the right major sphenoidal wing, falx, and T2-T3 level of rachis and a solid and cystic tumor in the low medulla oblongata. Tumors of the preauricular region, medulla oblongata, spinal cord at level T2-T3, and major sphenoidal wing area were surgically removed. The tumors were studied by immunohistochemistry and diagnosed as PS, pilocytic astrocytoma, and meningiomas, respectively. RESULTS: Seventy-eight cases of PS have been reported in the literature: 8 (10.2%) have been associated with clinical schwannomatosis, 6 (7.7%) with multiple cutaneous schwannomas syndrome, and only 3 (3.8%) with neurofibromatosis type 1 (NF-1). CONCLUSIONS: In this report, to the authors' knowledge, for the first time PS is described associated with neurofibromatosis type 2. The tumor does not appear to have significant association with NF-1. Plexiform schwannoma should be recognized because it may be misdiagnosed as plexiform neurofibroma or other plexiform malignant tumors. Differentiation from plexiform neurofibroma is important, because the latter is virtually pathognomonic of neurofibromatosis type 1 and has a propensity for malignant transformation. PMID- 8630896 TI - Molecular and immunohistochemical p53 status in liposarcoma and malignant fibrous histiocytoma: identification of seven new mutations for soft tissue sarcomas. AB - BACKGROUND: p53 mutations are the most frequently observed tumor-related genetic changes. Mutational analysis concerns mostly carcinomas and is not comprehensive for soft tissue sarcomas. Among soft tissue sarcomas, malignant fibrous histiocytoma (MFH) and liposarcoma represent the most frequent tumor types. Most of the few identified mutations for soft tissue sarcomas are localized in the core domain of p53. A correlation between p53 positive immunoreactivity, missense mutations, and a poor prognosis is generally assumed. However, the character of p53 mutations and their functional importance for the clinical process is still unknown. METHODS: Sixty-two soft tissue sarcoma samples were investigated for the presence of p53 mutations and for p53 immunoreactivity. Exons 4-9 of the p53 gene were amplified from genomic DNA with the polymerase chain reaction. A prescreen for mutations was performed by nonradioactive single strand conformation polymorphism analysis; striking cases were sequenced directly. For an evaluation of the immunohistochemical status, five p53 antibodies were used. RESULTS: In 10 tumor samples 7 new p53 mutations and one polymorphism were identified. Mutations were detected for five liposarcomas (four patients) and four MFHs (three patients). Of the seven mutations, three were missense point mutations, three were deletions, and one was a complex conversion. All mutations but one were localized in the core domain of p53. Of 62 tumor samples, 56% (14 of 32 liposarcomas and 21 of 30 MFHs) were positive for p53 immunostaining. CONCLUSIONS: The mutations identified in the core domain affect codons that are structurally or functionally involved in DNA binding. A relation between p53 positive immunoreactivity and a poor prognosis, but not with an exclusively high tumor grade, is evident. p53 mutations in soft tissue sarcomas have a similar spectrum to those in carcinomas. PMID- 8630897 TI - Continued local recurrence of carcinoma 15-25 years after a diagnosis of low grade ductal carcinoma in situ of the breast treated only by biopsy. AB - BACKGROUND: The stratification of ductal carcinoma in situ (DCIS) of the human breast into prognostically relevant categories by size and histologic pattern is a current concern. Few studies have been able to follow women after the identification of any type of DCIS when they have had biopsy only. METHODS: This is an extension of a follow-up study of a group of 28 women with small, noncomedo ductal carcinomas in situ that were excised by biopsy only, published in 1982. All these women have now been successfully followed for an average of almost 30 years. RESULTS: The overall risk of development of invasive carcinoma for these women over almost 30 years is nine times that of the general population (95% confidence interval, 4.7-17). This is similar to the 11-fold elevation in relative risk that was determined after about 15 years of follow-up. All invasive carcinomas have developed in the same area in the same breast. There were two women in whom invasive carcinoma developed between 20 and 30 years after initial biopsy. One other woman had an extensive noncomedo DCIS that was identified 25 years after her initial biopsy, but had no evidence of invasive disease. CONCLUSIONS: The natural history of small, noncomedo DCIS can last over at least 2 decades, with invasive carcinoma developing at the same site in which DCIS was previously discovered in a significant percentage of women (broadly, between 25% 50%). This is quite different from the natural history of comedo DCIS or any type of DCIS treated purposefully by surgery alone. PMID- 8630898 TI - p53 protein detected by immunohistochemistry as a prognostic factor in patients with epithelial ovarian carcinoma. AB - BACKGROUND: The clinical significance of p53 suppressor gene nucleoprotein immunostaining in ovarian epithelial cancer has not been determined. METHODS: p53 protein expression was studied by immunohistochemistry from paraffin embedded tissue in a series of 136 patients with malignant ovarian epithelial tumors. The median follow-up time of the patients still alive was 10 years. RESULTS: Sixty (44%) carcinomas stained clearly positive for p53 protein. Positive staining for p53 protein was associated with the serous histologic type (P = 0.0006), a higher than the median S-phase fraction size determined by DNA flow cytometry (P = 0.02), and poor histologic grade of differentiation (P = 0.04), but not with the International Federation of Gynecology and Obstetrics (FIGO) stage, age at diagnosis, or DNA ploidy. Cancers with positive staining had only 17% 5-year and 9% 15-year survival rates compared with 42% 5-year and 36% 15-year survival rates corrected for intercurrent deaths among the rest of patients (P = 0.002). In a multivariate analysis, positive p53 staining was associated with poor survival (relative risk of death, 1.8, 95% confidence interval [CI], 1.2-2.9) together with less than radical surgery (nonradical vs. radical: RR, 5.5; 95% CI, 2.2 13.6), and advanced FIGO stage (RR, 1.4; 95% CI, 1.0-2.0). CONCLUSION: Although p53 protein immunostaining is associated with several other prognostic factors in epithelial ovarian cancer, it may also have independent prognostic value in this disease. PMID- 8630899 TI - Malignant mixed mullerian tumors of the ovary: experience with surgical cytoreduction and combination chemotherapy. AB - BACKGROUND: The role of surgical cytoreduction and combination chemotherapy for malignant mixed mullerian tumors (MMMT) of the ovary was evaluated. METHODS: A retrospective review of 27 women with ovarian MMMT treated from 1980 to 1990 was performed. RESULTS: The International Federation of Gynecology and Obstetrics stages for the 27 women were 1 Stage I, 3 Stage II, 17 Stage III, and 6 Stage IV. Only 10 of the 23 patients with Stage III or IV disease were cytoreduced optimally. With respect to postoperative therapy, 3 women received no treatment, 6 were treated with whole abdomen radiotherapy, 1 received melphalan, and 17 received chemotherapy incorporating a platinum agent (3), doxorubicin (4), or both (10). The significant prognostic factors were stage (P < 0.001) and, for women with Stage III or IV disease, the feasibility of cytoreductive surgery (P = 0.03). The four patients in Stages I or II remained disease free after an interval of at least 5 years. The median and 5-year survival rates for patients with Stages III or IV disease was 18 months and 8%, respectively. Patients in Stage III or IV for whom optimal cytoreduction was not possible had a 2-year survival of 14%, whereas optimal cytoreduction was associated with a 52% 2-year survival. Median survival for the 14 women with Stage III or IV ovarian MMMT treated with combination chemotherapy was 25 months and nine women achieved progression free intervals of greater than 18 months. CONCLUSIONS: Aggressive surgical cytoreduction followed by combination chemotherapy may result in improved progression free intervals for women with advanced ovarian MMMT. However, a major improvement in prognosis for this rare malignancy has not yet been achieved. PMID- 8630901 TI - Myositis associated with interleukin-2 therapy in a patient with metastatic renal cell carcinoma. AB - BACKGROUND: Interleukin-2 (IL-2) has been used successfully in the treatment of some patients with metastatic renal cell carcinoma and melanoma, with a partial response rate of 15%-20%. It produces a well documented spectrum of side effects. Autoimmune diseases have been associated with IL-2 immunotherapy and the development of autoimmune thyroiditis may correlate with antitumor clinical response. METHODS: A patient with metastatic renal cell carcinoma is described who developed a polymyositis-like myopathy after an autologous tumor vaccine and IL-2 therapy. RESULTS: The patient had a delayed response for 15 months after developing this previously unreported toxicity. CONCLUSIONS: To the authors' knowledge, this represents the first reported case of necrotizing myositis in association with IL-2 therapy. Subsequent continuous partial response of the advanced malignancy was observed for 15 months. In this case, IL-2 may have broken tolerance to both normal muscle cells and tumor cells. PMID- 8630900 TI - Evaluation of ovarian findings in asymptomatic postmenopausal women with color Doppler ultrasound. AB - BACKGROUND: To evaluate the prevalence and significance of abnormal ovarian findings in asymptomatic postmenopausal women, screening for ovarian cancer with color Doppler ultrasound was performed. METHODS: One thousand three hundred sixty four asymptomatic women aged 56-61 years (mean, 59 years) were examined by color Doppler sonography. Ninety-six percent of the examinations were transvaginal and 4% transabdominal. The criteria for abnormality were an ovarian volume 8 cm3 or greater, nonuniform echogenicity, and/or pulsatility index (PI) of the ovarian artery or tumor vessel, if present, 1.0 or less. Repeat sonograms were performed 1-3 months later on all patients with abnormal findings, and exploratory laparotomy was performed if a malignant tumor was suspected. RESULTS: Abnormal ovarian findings were detected in 160 women (12%). At the time of repeat sonogram there were 28 persisting abnormalities (2%). At that time, the ovary was regarded as normal if it still contained a small clear cyst with an unchanged greatest dimension of less than 20 mm. Three women had a low PI value but all had also abnormal ovarian sonographic morphology. Two ultrasound-guided cyst punctures were performed and three patients had surgery; one benign serous cyst, one benign serous cystadenoma, and one serous cystadenoma of borderline malignancy were detected. The remaining abnormal findings disappeared or remained unchanged during a minimum follow-up of 2 years. One case of Stage IA ovarian cancer has been reported 2 years after a negative screening and one abdominal carcinomatosis 2 1/2 years after a negative screening result. CONCLUSIONS: There is a high frequency of small ovarian cysts in asymptomatic postmenopausal women. A large percentage of these cysts regress spontaneously or remain unchanged. Transvaginal color Doppler ultrasound is an effective method for detecting these lesions. Color Doppler does not increase substantially the number of operations for benign reasons. However, as a primary screening modality, the conventional sonography seems to be quite sufficient. PMID- 8630902 TI - Blood flow and metabolism of central neurocytoma: a positron emission tomography study. AB - BACKGROUND: New World Health Organization classifications have categorized central neurocytomas as neuronal tumors. The differential diagnosis between central neurocytomas and other tumors is important for selection of the optimal therapy modality for the management of intraventricular tumors. To characterize the pathophysiology and proliferating activity of central neurocytoma accurately, cerebral blood flow and metabolism in five patients with central neurocytoma were studied using positron emission tomography (PET). METHODS: Tracers used for the present study included C15O2, C15O, 15O2, and 18F-fluorodeoxyglucose (FDG). Regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), oxygen extraction fraction (rOEF), cerebral metabolic rate of oxygen (rCMRO2), and cerebral metabolic rate of glucose (rCMRGl) were quantitatively analyzed in tumor lesions and the contralateral gray matter. Four patients with central neurocytoma underwent a complete PET study, including all circulatory and metabolic parameters; one patient was studied with 11C-methyl-L-methionine and FDG tracers. RESULTS: Tumor rCBF and rCBV were higher than comparable values in the contralateral gray matter in three of four patients. This high level of perfusion corresponds to angiographic findings that show intense tumor staining in tumors fed by perforated arteries. Tumor rOEF and rCMRO2 were significantly lower than corresponding values in the gray matter (rOEF, P < 0.01; rCMRO2, P < 0.05 by Student's t test). Tumor rCMRGl ranged from 2.68 to 6.26 mg/100 ml/minutes and did not exceed contralateral gray matter values in any of the five patients. Tumor rCMRGl was significantly lower (P < 0.02) than the gray matter rCMRGl. One tumor exhibited a relatively high value of rCMRGl (comparable to gray matter rCMRGl), and increased in size 4 months after partial resection. No other tumors appeared during postoperative follow-up periods that ranged from 4 to 135 months. CONCLUSIONS: Circulation and metabolism parameters measured by PET offer insight into the biologic characteristics of central neurocytoma. Tumor rCMRGl may be an indicator of the proliferating activity in central neurocytoma. PMID- 8630904 TI - Centromeric instability of chromosome 1 resulting in multibranched chromosomes, telomeric fusions, and "jumping translocations" of 1q in a human immunodeficiency virus-related non-Hodgkin's lymphoma. AB - BACKGROUND: Acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas are associated with the B-cell chromosomal translocation t(8;14)(q24; q32). The most common secondary chromosome aberrations in these patients involve 1q and are believed to be associated with tumor progression. A mechanism for the origin of these 1q aberrations has not been demonstrated. To their knowledge, the authors report the first human immunodeficiency virus (HIV)-positive patient to have centromeric decondensation and multibranched chromosome aberrations of chromosomes 1 and 16 resulting in telomeric associations and "jumping translocations" of 1q. METHODS: Tumor cells from peritoneal fluid of an HIV positive patient were cultured for 24, 48, and 72 hours and analyzed by both conventional G-banding and fluorescence in situ hybridization. RESULTS: G-band analysis showed a stemline with t(8;14)(q24;q32), but also showed the progression from centromeric decondensation to multibranched chromosome configurations of chromosomes 1 and 16. The interchange and duplications of chromosome arms resulted in the gain of extra copies of 1q material on a number of different chromosomes, but also the loss of 16q in at least one sideline and the formation of micronuclei. Fluorescence in situ hybridization analysis demonstrated that micronuclei predominantly involved chromosome 1 and, to a lesser extent, chromosome 16. CONCLUSIONS: The cytogenetic findings in this unique case suggest that immunodeficiency may be a factor involved in centromeric instability, multibranching, and the progression to the subsequent formation of telomeric fusions and multiple unbalanced translocations of 1q (jumping translocations). The striking similarity of the centromeric instability in this patient to those with ICF syndrome (variable immunodeficiency, centromeric heterochromatin instability, and facial anomalies) suggests hypomethylation as the etiologic mechanism for the chromosome instability. PMID- 8630903 TI - Evaluation of 96-hour infusion fluorouracil plus cisplatin in combination with alpha interferon for patients with advanced squamous cell carcinoma of the head and neck: a Southwest Oncology Group study. AB - BACKGROUND: Recurrent cancer of the head and neck after primary therapy is almost always fatal. The combination of 5-fluorouracil (5-FU) and cisplatin is considered the best available therapy but complete response rates remain too low to affect survival. This study was designed to evaluate the complete response rate and toxicity of 5-FU, cisplatin, and alpha-interferon (alpha-IFN) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). METHODS: Fifty eligible patients with recurrent or metastatic SCCHN and no prior chemotherapy (40 men, 10 women; age range, 26-77 years; median, 59 years; 82% white; 88% had prior surgery and 92% had prior radiation therapy) were treated every 21 days with 96-hour infusion of 5-FU 1000 mg/m2/day; cisplatin 100 mg/m2, day 1; and alpha-IFN 5 x 10(6) units/day, days 1-4. RESULTS: One hundred fifty-seven courses of chemotherapy were administered, with a median of three courses. Thirty-seven patients experienced Grade 3 or 4 toxicity. Of the 17 patients with Grade 4 toxicity; 12 had hematologic toxicity, 3 stomatitis, and 2 vomiting. Two additional patients died of myelosuppression-related sepsis. Of the 50 patients, 3 (6%) achieved a complete response, five (10%) had a partial response, 3 (6%) had unconfirmed response (1 complete and 2 partial), 10 (20%) had stable disease, 17 (34%) progressed, and 12 (24%) were considered nonresponders owing to early death (6) or inadequate assessment (6). The median survival was 5 months. CONCLUSION: The complete response rate of patients with recurrent or metastatic SCCHN treated with 5-FU, cisplatin, and alpha-IFN does not appear to be superior to that observed for 5-FU and cisplatin. Alpha interferon appears to augment hematologic and gastrointestinal toxicities associated with this combination. PMID- 8630905 TI - The investigation of Epstein-Barr viral sequences in 41 cases of Burkitt's lymphoma from Egypt: epidemiologic correlations. AB - BACKGROUND: Epstein-Barr virus (EBV) is associated with many human neoplasms, including Burkitt's lymphoma (BL). Endemic BL in central Africa is more often EBV associated than BL in the United States, where seroconversion for EBV occurs somewhat later than in Africa. Therefore, the EBV association rate in BL may correlate more with the socioeconomic status of the population studied, which influences the age of EBV seroconversion, than with such factors as malaria, which may relate to the overall higher incidence rate in endemic regions. METHODS: Forty-one patients with BL in Egypt, which differs both climatically and racially from central African countries (i.e., Kenya, Uganda) where BL is endemic, were analyzed. All biopsies were evaluated for EBV-encoded RNAs (EBER1) by RNA in situ hybridization, analyzed for p53 protein expression using the monoclonal antibody D07, and immunophenotyped using a panel of monoclonal antibodies that included L26 (CD20), Leu 22 (CD43), and A6 (CD45RO). Twelve cases were evaluable for EBV subtype by polymerase chain reaction with EBV-specific primers. RESULTS: The median age at diagnosis was 9 years (range, 2-22 years). The biopsy site was extranodal in 29 patients and nodal in 12 patients. All 41 cases were documented as B-cell neoplasms. A hybridization signal for EBER1 RNA was identified in greater than 95% of the neoplastic cells in 30 of 41 cases (73%), whereas no signal was observed in 11 cases (27%). Epstein-Barr virus subtype 1 was found in 10 patients, subtype 2 in two patients. Immunostaining for p53 was observed in greater than 5% of the neoplastic cells in 9 of 37 cases (24%). No significant correlation was observed between EBV positivity and sex, biopsy site, or p53 immunostaining. CONCLUSIONS: The prevalence of EBV in BL from Egypt is slightly lower than in BL in endemic regions, but significantly higher than in sporadic BL. Epstein-Barr virus positivity probably reflects the socioeconomic status of the patient population, and age at seroconversion. The prevalence of EBV subtype 1 suggests that immunodeficiency does not play a role in Egyptian Burkitt's lymphoma, in contrast to endemic Burkitt's lymphoma, in which holoendemic malaria is thought to contribute to immunodeficiency, a higher incidence rate, and the observed prevalence of subtype 2. PMID- 8630906 TI - Enhanced human lymphokine-activated killer cell function after brief exposure to granulocyte-macrophage-colony stimulating factor. AB - BACKGROUND: Lymphokine-activated killer (LAK) cell function can be generated in peripheral blood mononuclear cells (PBMC) after brief exposure of high dose interleukin-2 (IL-2) over the course of 1 or 2 days' culture in plain culture medium (IL-2-pulsed PBMC). The aim of the present study was to investigate the ability of granulocyte-macrophage-colony stimulating factor (GM-CSF) to augment LAK induction in low dose IL-2-pulsed PBMC derived from patients with cancer undergoing immunotherapy with IL-2. METHODS: Peripheral blood mononuclear cells were collected from patients with cancer receiving a 5-day cycle of local (intraperitoneal or intrapleural) infusions with IL-2. The cells were incubated with IL-2 in the presence or absence of GM-CSF for 1 hour and then tested as effectors against allogeneic tumor cells and LAK-sensitive cell lines. RESULTS: Granulocyte-macrophage-colony stimulating factor at doses between 10 and 100 ng/ml was synergized with low dose IL-2 (100 IU/ml) in the generation of LAK activity in PBMC. Lymphokine-activated killer cell-mediated cytotoxicity derived from PBMC cultures incubated with IL-2 and GM-CSF was significantly higher (up to three-fold) compared with that generated with IL-2 alone. The GM-CSF-induced enhanced LAK activity was maintained when tested at day 5. GM-CSF increased the percentages of IL-2 receptor (R) positive (+) and CD8+ cells in the IL-2-pulsed PBMC. In contrast to CD56+ cells, highly purified CD8+ cells isolated from PBMC pulsed with IL-2 and GM-CSF responded with increased LAK activity, thus representing the cell-type that mediates the augmenting effect of GM-CSF. Major histocompatibility complex (MHC) molecules or the CD3 surface antigens were not involved in the GM-CSF-mediated enhancement of LAK induction because anti-MHC class I and class II monoclonal antibodies (MoAb) or MoAb against the CD3 molecules remained without any effect in this system. The GM-CSF-mediated LAK enhancement was IL-2-dependent because MoAb against IL-2 receptor completely inhibited the generation of LAK activity. CONCLUSIONS: The use of GM-CSF for the enhancement of IL-2-induced LAK activity in 1 hour cultures may improve clinical results in cancer immunotherapy. In addition, implementation of this procedure could eliminate the high cost of cell culture which usually accompanies IL-2/LAK cell therapy as well as eliminate the known toxic side effects associated with this kind of therapy. PMID- 8630907 TI - Bulky centroblastic non-Hodgkin's lymphoma of the cranium vault mimicking brain involvement managed with chemotherapy: a case report. AB - BACKGROUND: Patients with central nervous system (CNS) involvement by high grade non-Hodgkin's lymphoma (NHL) have a poor prognosis. The roles of computed tomography, radiotherapy, and intrathecal and systemic chemotherapy still need to be defined. METHODS: A patient with bulky cranial lymphoma mimicking brain involvement is reported. A 62-year-old man was admitted with a huge scalp lump, headache, fatigue, and focal and generalized neurologic symptoms. Computed tomography showed an abnormal mass in the frontoparietal region involving the subcutaneous scalp, osteolytic destruction of the cranial vault, and a bulky mass that was interpreted to be intracranial. A systemic survey also revealed bulky retroperitoneal involvement and focal involvement of the spleen. Biopsy revealed a B-cell NHL of centroblastic type according to the Kiel classification. RESULTS: The patient was treated with a modified combination of cyclophosphamide plus mitoxantrone plus vincristine plus prednisone (CNOP) and intrathecal methotrexate. The patient responded with complete remission, including partial bone restoration of the cranium. At the time of this writing, his relapse free survival lasted 5 years. CONCLUSIONS: The initial interpretation of this case indicated that systemic chemotherapy with modified CNOP plus intrathecal methotrexate would be useful in the management of NHL with CNS involvement. The clinical outcome with rapid neurologic repair and also bone restoration of the cranial vault within 5 years suggests that the lymphoma probably never penetrated the dura and a successful treatment was achieved with combination chemotherapy only. PMID- 8630908 TI - Advance directives in the intensive care unit of a tertiary cancer center. AB - BACKGROUND: Advance directives are associated with considerable controversy. The goal of this study was to evaluate the outcomes of critically ill patients with cancer who were admitted to the intensive care unit and who previously had executed an advance directive. The problems associated with interpreting and honoring such documents in a tertiary cancer center also were reviewed. METHODS: A prospective observational study of patients with cancer with advance directives who were admitted to the intensive care unit of a major cancer hospital was undertaken. Twenty-six patients with directives were followed from the time of admission to the intensive care unit or, in the case of patients who presented their directives after admission, from the time of presentation of the directive until either discharge or death. RESULTS: Twenty four of the 26 patients were placed on mechanical ventilators. Eight patients died while on the ventilator, nine were terminally weaned, and seven were weaned and survived for at least 24 hours. Of these seven patients, six died before being discharged from the hospital and one was discharged home. Delay in presenting the advance directive, conflict between the dictates of the living will and the wishes of the person named in the durable power of attorney, and controversy among health-care providers as to when in the course of disease the spirit of the advance directive had been met were the most frequent problems encountered; a number of other concerns were also identified. CONCLUSIONS: Considerable controversy exists regarding advance directives, and such documents often leave room for confusion about patients' desires in particular clinical situations. Many of the problems identified in this study might be avoided and considerable cost savings achieved by the timely presentation of documents and by the evaluation of clinical goals on an ongoing basis. PMID- 8630909 TI - Effect of methionine-deprived total parenteral nutrition on tumor protein turnover in rats. AB - BACKGROUND: Previous studies have shown that a methionine-lacking diet inhibited tumor growth in rats. The aim of this study was to determine how methionine free total parenteral nutrition (MTPN) can result in the inhibition of tumor growth on tumor protein metabolism in rats. METHODS: On day 0, AH109A rat ascites hepatoma cells were implanted subcutaneously into male Donryu rats (n = 68, body weight, 200-225 gm). On day 10, a catheter for total parenteral nutrition (TPN) was placed and either MTPN or standard TPN solution was given for 5 days. On day 15, 1-14C-leucine was infused continuously to measure tumor protein synthesis. Tumor proteolysis was calculated from tumor regional blood flow, using the 85Sr microsphere injection method. RESULTS: 1) Tumor weight was reduced with MTPN. 2) MTPN did not affect tumor protein synthesis, probably because endogenous methionine production was increased with MTPN (87.3 +/- 13.5 mumole methionine/kg/hour vs. 218.6 +/- 29.5, P < 0.01); however, MTPN caused an increase of tumor proteolysis (2.68 +/- 0.53 mumole leucine/g/hour vs. 3.79 +/- 0.73, P < 0.05). CONCLUSION: The enhanced tumor protein breakdown contributed to the inhibition of tumor growth that was found with the rats given the methionine free diet. PMID- 8630910 TI - Opioid pharmacotherapy in the management of cancer pain: a survey of strategies used by pain physicians for the selection of analgesic drugs and routes of administration. AB - BACKGROUND: This survey documents the strategies used by pain control physicians in the selection of opioid drugs and routes of administration in the management of inpatients referred to a cancer pain service. METHODS: The following approaches were prospectively evaluated during the treatment of 100 consecutive inpatients: 1) the influence of the evaluation of the goals of care on decision making, 2) selection of opioid drugs, 3) indications for changing opioid drugs and the frequency with which this strategy is used, and 4) selection of route of administration. RESULTS: Eighty of the 100 patients underwent a total of 182 changes in drug, route, or both drug and route before discharge or death. The major reasons for change were to improve the convenience of treatment regimen in the setting of adequate pain relief (31.4%), diminish side effects in the setting of controlled pain (25.0%), reduce the invasiveness of therapy in the setting of controlled pain (19.3%), and simultaneously improve pain control and reduce opioid toxicity (17.7%). When opioid toxicity was the reason for change, physicians changed the opioid drug in 71% of cases and the route in 29%. When convenience or invasiveness were targeted, the physicians changed the route in 61% of cases and the opioid in 39%. Forty-four patients required one or more change in the opioid, and 20 required 2 or more changes (range, 2-6 changes). At the time of discharge (n = 82), morphine was more commonly selected than hydromorphone or fentanyl (39% vs. 23% vs. 17%) and the routes of administration were oral (57%), transdermal (18%), intravenous (18%), subcutaneous (5%), and intraspinal (4%). Therapeutic changes were associated with improvement in physician-recorded pain intensity and a lower prevalence of cognitive impairment, hallucinations, nausea and vomiting, and myoclonus among patients who were discharged from the hospital. CONCLUSIONS: These data illustrate the application of strategies for selections of opioid drugs and their route of administration that are recommended in current guidelines for the management of cancer pain. PMID- 8630911 TI - Breast cancer trials on trial: a case of conflicting ethical interests. PMID- 8630912 TI - Autologous bone marrow transplantation in children with advanced neuroblastoma. PMID- 8630913 TI - The timely publication of manuscripts. A status report from the Editor-in-Chief. PMID- 8630914 TI - Twenty-year review of a breast cancer screening project. Ninety-five percent survival of patients with nonpalpable cancers. AB - BACKGROUND: In 1973, the Breast Cancer Detection Demonstration Project at the Georgia Baptist Medical Center began screening supposedly asymptomatic women for breast cancer. The project has been reviewed and now 20 years later, the follow up of those women with detected cancer who were matched with a group of the original cohort with negative screens is reported. Early criticism of the project was its "lead-time bias," which, after 20 years, should pose no problem. METHODS: Approximately 9043 women were screened for 5 years. The remainder of the total of 10,000 women were screened at the Emory University Department of Radiology. The 128 patients who were found to have breast cancer were observed for over 20 years. A matched group of 1609, who had negative results when originally screened, were also observed for 20 years. RESULTS: After 20 years, approximately 86% of the 128 women diagnosed with breast cancer were still free of disease, including 88.3% of women with lesions of less than 1.1 cm and 95.1% of those with nonpalpable carcinomas. CONCLUSIONS: Over 95% of the 67 women with nonpalpable lesions were alive after 20 years of follow-up. Women not screened yearly tend to have larger lesions than those screened on a regular basis. Regular screening is currently the best way to control this disease. PMID- 8630915 TI - Higher frequency of gene amplification in breast cancer patients who received adjuvant chemotherapy. AB - BACKGROUND: Amplification of certain genes is involved in resistance to chemotherapy. The development of such amplification in patients by drug treatment has not yet been established. We have assessed the appearance of gene amplification in breast cancer patients with recurrent disease. One group of patients had previously received adjuvant chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracil [CMF]) after surgery. The second group had not. All of these patients had developed recurrent disease and were receiving first-line endocrine treatment (tamoxifen). METHODS: Tumor cells were obtained from the recurrent carcinoma using fine-needle biopsies. Gene copy number was determined for dihydrofolate reductase and thymidylate synthase with semiquantitative polymerase chain reaction. Dihydrofolate reductase is involved in resistance to methotrexate (M) and thymidylate synthetase in resistance to 5-fluoropyrimidines (F), two targets for the CMF regime. RESULTS: We found that amplification of the examined genes develops in higher frequency among patients who previously received chemotherapy (p = 0.002). CONCLUSIONS: These findings provide strong evidence for gene amplification induced by drug treatment. PMID- 8630916 TI - Infiltrating lobular carcinoma of the breast. Clinicopathologic analysis of 975 cases with reference to data on conservative therapy and metastatic patterns. AB - BACKGROUND: The clinicopathologic features of infiltrating lobular carcinoma (ILC), which represents 5% to 15% of all breast cancers, are still controversial. In particular, the high frequency of multicentric lesions has led to questioning of the effectiveness of conservative treatment for this type of cancer. By studying a large number of cases, we aimed to compare the clinicopathological features of ILC with those of nonlobular infiltrating carcinoma (NLIC) and to assess the advisability of conservative therapy in the management of ILC. METHODS: The population analyzed included 726 cases of ILC, 249 cases of mixed ILC/invasive ductal carcinoma (ILC/IDC), and 10,061 cases of NLIC. The age of patients, TNM status, estrogen- and progesterone-receptor status (ER, PR), and histologic grades of the 3 groups were compared. The follow-up was carried out on a subgroup of 5846 cases. RESULTS: At diagnosis, ILC tumors were found to be larger on average and were detected in patients older than those with NLIC, but the degree of lymph node involvement was lower in patients with ILC than in NLIC. In ILC, tumors are more frequently grade I and ER-positive than in NLIC. Multicentric lesions were not significantly more frequent in ILC than in NLIC. The overall survival, locoregional control, disease free interval, and metastatic spread rates were not different among the three groups neither by univariate nor multivariate analysis, but the pattern of metastatic dissemination was different. In 480 cases of ILC considered for conservation therapy, the local recurrence and overall survival rates were similar to those observed for IDC. CONCLUSIONS: Our analysis specifies the clinicopathological features of ILC and confirms that conservation therapy may be an appropriate treatment for this type of cancer. PMID- 8630917 TI - Persistence of human papillomavirus (HPV) infections preceding cervical carcinoma. AB - BACKGROUND: A persistent genital infection with an oncogene-type of human papillomavirus (HPV) is considered to be essential for the development of most cervical carcinomas. Therefore, HPV analysis has been proposed as a possible complementary cytological screening program. The authors have developed a technique to analyze archival Pap smears, which has enabled them to study the relation between persistent HPV infection and the development of cervical cancer. METHODS: Nested polymerase chain reaction was used to demonstrate the presence of HPV DNA, and sequencing of the obtained amplimer was performed to establish HPV type. The authors analyzed a series of 88 smears taken 1.5 to 7 years prior to the diagnosis of an HPV-containing cervical carcinoma (12 invasive adenocarcinomas, 18 invasive squamous carcinomas, and 58 squamous carcinoma in situ), which were compared with age-matched controls with no tumor development. RESULTS: HPV DNA was present in a majority of the smears preceding a cancer, with an odds ratio of around 15 for all tumor groups. Infections with a persisting HPV type were demonstrated in most samples from a series of eight patients, from who multiple smears were available, covering the 7-year period preceding the cancer diagnosis. CONCLUSIONS: Persisting infection can be demonstrated in exfoliated cells many years before cancer is diagnosed. The results are complementary to those obtained with cytology, that is, HPV is detected also in those at-risk patients whose Pap smears are morphologically normal. However, the results are still insufficient to justify a general recommendation to use HPV testing for health control purposes. PMID- 8630919 TI - Evidence for coupling of phosphotyrosine phosphatase to gonadotropin-releasing hormone receptor in ovarian carcinoma membrane. AB - BACKGROUND: Gonadotropin-releasing hormone (Gn-RH) receptor (Gn-RHR) has been demonstrated in epithelial ovarian carcinoma (Imai et al., Cancer 1994; 74:2555 61). To examine whether Gn-RHR mediates direct antiproliferative effects, we attempted to determine stimulatory regulation by Gn-RH of phosphotyrosine phosphatase (PTP) activity in plasma membranes isolated from ovarian carcinoma samples. METHODS: Surgically removed ovarian carcinomas were screened for Gn-RHR expression prior to plasma membrane isolation. The phosphotyrosine level was observed by: (1) immunoblotting of membrane extracts with antiphosphotyrosine antibodies, and (2) dephosphorylation from 32P-labeled membrane protein. Membrane PTP activity was determined using the synthetic substrate p-nitrophenyl in a spectrophotometric assay. RESULTS: A Gn-RH analog alone, or guanosine thiotriphosphate (GTP-gamma-S) alone, caused a remarkable loss of phosphotyrosine from a 35-kD protein of the membranes; incubation with a Gn-RH analog and GTP gamma-S produced a further dephosphorylation of this endogenous protein. The Gn RH analog buserelin stimulated the PTP activity of the membranes in a dose dependent manner (P < 0.01). GTP-gamma-S enhanced the stimulatory action of Gn-RH on PTP; GDP-gamma-S reversed the Gn-RH action. A similar stimulation of PTP was observed (P < 0.01) when carcinoma tissue slices were exposed to Gn-RH analog in vivo prior to assay in vitro. CONCLUSIONS: Activation of PTP by Gn-RH stimulated the loss of phosphotyrosine from endogenous proteins through GTP-binding protein within plasma membrane isolated from Gn-RHR-expressing ovarian carcinoma. The antimitogenic action of the hormone may occur by counteracting tyrosine phosphorylation to promote cell growth. PMID- 8630920 TI - Familial prostate cancer in Sweden. A nationwide register cohort study. AB - BACKGROUND: Although prostate carcinoma is not widely recognized as a familial cancer, familial aggregation of this disease has been shown in some retrospective case-control studies. To study familial prostate cancer in Sweden, a population based cohort study was performed, that attempted to avoid possible bias connected with some earlier studies of familial prostate cancer. METHODS: A nationwide register cohort study was conducted using an unselected study population. The study cohort of 5496 sons of Swedish men found to have prostate cancer between 1959 and 1963 was identified through parish offices. All prostate cancer patients reported between 1958 and 1990 in this cohort were identified through linkage to the Swedish Cancer Register. The expected number of prostate cancer patients was calculated using incidence rates obtained from the same register. RESULTS: A highly significant increased overall standardized incidence ratio (SIR) of 1.70 (95% confidence interval, 1.51-1.90) was obtained for prostate cancer in this cohort, with 302 observed cases compared with 178 expected prostate cancers. The SIR was 3.38 among patients aged 45-49 years at diagnosis, with the risk gradually decreasing to a SIR of 1.35 among patients older than 80 years (trend, P = 0.013). Among sons with a father whose prostate cancer was diagnosed at an early age (< 70 years), a significant trend (P = 0.01) for prostate cancer risk was observed due to early onset of the disease. CONCLUSIONS: This cohort study provides further evidence that a positive family history of prostate cancer is a risk factor for developing the disease in an unselected population. The increased risk was found for all ages, but was more pronounced in younger men. PMID- 8630921 TI - Paclitaxel-induced apoptosis in human gastric carcinoma cell lines. AB - BACKGROUND: Gastric cancer is one of the most common cancers in Asia. Chemotherapy and radiation therapy have had limited success. Recently, paclitaxel has been found to be effective against a variety of cancers, including lung, breast, ovary, melanoma, and prostate. Whether paclitaxel is effective in the treatment of gastric cancer is not known and is worthy of investigation. METHODS: Human gastric carcinoma cell lines NUGC-3 and SC-M1 were examined for response to paclitaxel treatment. Cancer cells were treated with paclitaxel (0.001, 0.01, 0.1, and 1 microM) for 1-3 days. Cell number was counted by hemocytometer and cell viability was determined by the trypan blue exclusion method. Cell cycle progression and expression of proliferating cell nuclear antigen (PCNA) were examined by flow cytometry. The percentage of apoptotic cells was determined after staining with hematoxylin and eosin. RESULTS: Paclitaxel was cytotoxic to the two human gastric carcinoma cell lines examined. The growth-inhibiting dose was 0.01 microM. Paclitaxel-treated gastric carcinoma cells were arrested mainly in G2/M phases before apoptosis. However, treatment with 0.01 microM of paclitaxel resulted in a decrease of cells at G0/G1 phases without an increase of cells at G2/M phase indicating that paclitaxel was also cytotoxic to gastric carcinoma cells at G0/G1 phases. In addition, the expression of PCNA was significantly increased in 0.1 and 1 microM paclitaxel-treated cells, suggesting that DNA repair was increased in these cells. CONCLUSIONS: Paclitaxel is effective in growth inhibition of gastric carcinoma cell lines in clinically attainable concentrations. Our results suggest that paclitaxel is a potential chemotherapeutic drug for gastric carcinoma. PMID- 8630918 TI - Survival of women with advanced ovarian cancer and complete pathologic response at second-look laparotomy. AB - BACKGROUND: The purpose of the study was to analyze the determinants of long term survival in women with advanced ovarian cancer and negative second-look laparotomy. METHODS: A series of 140 advanced (Stage III-IV) ovarian cancer patients (median age, 54 years; range, 22-74 years) with negative second-look laparotomy after primary surgery and chemotherapy is included in the analysis. At first diagnosis, all patients were treated with radical or debulking surgery. After primary surgery, the patients were treated with a chemotherapy regimen based on cisplatin or carboplatin alone or in combination with other drugs. All second-look laparotomies were performed 6-8 months after first surgery. RESULTS: The overall survival rates were 76% at 3 years, 66% at 5 years, and 51% at 8 years. The corresponding rates for disease free survival were 57, 50, and 43%, respectively. Survival rates were better for women with a residual tumor 1 cm or less after primary surgery. The 5-year probability of survival was 78% in this group, compared with 55% in women with a residual tumor more than 1 cm (log rank test, P < 0.05). Survival rates for women with tumor Grade 3 tended to be worse than Grades 1-2, but the difference was only of borderline statistical significance. No relationship emerged between survival and age, histotype, and presence of ascites at diagnosis. Women with a residual tumor 1 cm or less and positive lymph nodes had a 66% 5-year probability of survival, compared with 85% for women with a residual tumor 1 cm or less and negative lymph nodes. This difference was significant (log rank test, P = 0.05). The 5-year survival probabilities were 47 and 58%, respectively, in women with a residual tumor more than 1 cm and positive or negative lymph nodes. CONCLUSIONS: This analysis shows a favorable long term survival rate for women with advanced ovarian cancer and complete pathologic response after debulking surgery and postoperative chemotherapy. It further suggests that lymph nodal status is a prognostic factor for women with minimal residual tumor after surgery. PMID- 8630922 TI - In vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP, DU145, and PC3 models of the human prostate. AB - BACKGROUND: Proliferation of normal and tumoral prostate tissue is regulated by androgens and various growth factors. We characterized the in vitro proliferative influence of prolactin (PRL) in androgen-sensitive and androgen-insensitive human prostate cancers. METHODS: The biologic models employed included the androgen sensitive LNCaP and the androgen-insensitive DU145 and PC3 cell lines. PRL induced influences (0.1-10 mIU/ml of medium) on proliferation were assessed using the colorimetric methylthiotetrazole assay. Androgen sensitivity in the three cell lines was determined by assessing the proliferative influence of dihydrotestosterone (DHT) (0.1-10 nM). PRL-induced modifications in PC3 cell kinetics were assessed using Feulgen-stained nuclear image cytometry. RESULTS: Although DHT markedly stimulated LNCaP proliferation, it had no proliferative effect on the DU145 and PC3 cell lines. By contrast, PRL significantly modulated the proliferation of the DU145 and PC3 lines, but exerted weak, if any, effect on the proliferation of the LNCaP cell line. PRL increased the percentage of PC3 proliferating cells (i.e., cells in the S/G2 phases of the cell cycle) at low doses (0.1 mIU/mL) and decreased this percentage at high doses (10 mIU/ml). CONCLUSIONS: Proliferation of androgen-insensitive human prostate cell lines can be significantly modulated by prolactin. PMID- 8630923 TI - The results of a five-year early prostate cancer detection intervention. Investigators of the American Cancer Society National Prostate Cancer Detection Project. AB - BACKGROUND: The American Cancer Society-National Prostate Cancer Detection Project (ACS-NPCDP) is a multidisciplinary evaluation of early prostate cancer detection interventions. This report summarizes the experience of the investigators to date and describes the overall and relative performance of the different detection modalities studied in this project. METHODS: Two thousand nine hundred ninety-nine men aged 55 to 70 years at entry who were not already under evaluation for prostate cancer were recruited to participate in up to 5 annual examinations by prostate specific antigen (PSA), digital rectal examination (DRE), and transrectal ultrasound (TRUS). In the course of 5 years of intervention, ACS-NPCDP investigators have completed 9937 examinations, recommended 1215 biopsies, and detected 203 cancers. RESULTS: Loss to cohort follow-up was greatest in the first year. Overall, TRUS led to twice the number of recommendations for biopsy compared with DRE (8.9% versus 4.4%). Elevated PSA was observed in 13.0% of 9535 measurements performed. The overall cancer detection rate declined significantly during the five years of intervention. Detection was significantly associated with age and symptom status at entry. DRE had lower sensitivity compared with TRUS or PSA, particularly in later years of follow-up. The specificity of TRUS was lower than that for DRE. PSA was elevated in 69.2% of examinations that led to cancer detection, compared with only 10.9% when cancer was not found. PSA level, PSA density, and PSA change were all related to the presence of cancer. Less than 6% of the cancers detected in this study were clinically advanced at the time of diagnosis. CONCLUSIONS: These data quantify the yield of early cancer detection that may be expected when PSA, DRE, and TRUS are used in populations comparable to the men participating in the ACS NPCDP. Continued follow-up and further research is needed to assess whether men receiving early prostate cancer interventions benefit as a result. PMID- 8630925 TI - Prognostic value of ploidy and proliferation markers in renal cell carcinoma. AB - BACKGROUND: The prognosis for patients with renal cell carcinoma depends mainly on pathological stage and grade of the tumor at the time of surgery. Cellular proliferation may prove to be another measure for predicting biologic aggressiveness and, therefore, the prognosis. METHODS: The authors compared four different methods to assess proliferation in a series of 87 curatively resected (R0) renal cell carcinomas: flow cytometry analysis (FCM), silver-stained nucleolar organizer regions (AgNOR), and immunohistochemical assessment of the MIB-1 (Ki-67) antigen, and proliferating cell nuclear antigen (PCNA). The results obtained were compared with pathologic stage (according to the International Union Against Cancer [UICC]) and grade with disease-related survival rate; finally, we assessed whether the methods led to similar results. RESULTS: In each carcinoma examined, we could demonstrate MIB-1, PCNA, and AgNOR dots in varied proportions. Statistical correlations were seen between the tumor grade, the rate of nuclear positivity for MIB-1 and PCNA, and the number of AgNOR dots. Additionally, the MIB-1 index was significantly higher in more advanced tumor stages. A good correlation between MIB-1 and AgNOR as well as for PCNA was found. In univariate survival analysis, tumor stage and grade, MIB-1 and PCNA index, and mean AgNOR number were related significantly to patient survival. On multivariate Cox disease-related survival analysis, stage of disease and MIB-1 were significant independent prognostic factors. Flow cytometry was not related to prognosis nor to other examined parameters. CONCLUSIONS: These results indicated that MIB-1 immunostaining is an additional prognostic parameter for patient outcome. MIB-1 and PCNA immunostaining, as well as AgNOR, demonstrated good correlations among themselves. We failed to establish flow cytometry as a method to predict proliferative capacity or prognosis in renal cell carcinoma patients. PMID- 8630924 TI - Cisplatin-based chemotherapy in renal transplant recipients. A case report and a review of the literature. AB - BACKGROUND: Renal transplant recipients have a high incidence of cancer. The main side effect of cisplatin, nephrotoxicity, has special implications in renal transplant recipients. This is particularly true in view of the routine use of cyclosporine as an immunosuppresant. Nephrotoxicity is also one of the main side effects of cyclosporine. METHODS: We report a patient with a renal allograft who was receiving cyclosporine for immunosuppression and developed metastatic transitional cell carcinoma of the bladder and was treated with cisplatin-based chemotherapy. The literature regarding cisplatin-containing chemotherapy in patients with different cancers and a single transplanted kidney is reviewed. RESULTS: The patient received four cycles of methotrexate, vinblastine, doxorubicin, and cisplatin while on continuous cyclosporine therapy. His renal function remained stable. He responded to chemotherapy initially, but this response was short. Ten patients with renal transplants and cancer who were treated with cisplatin have been reported previously. Two were maintained on cyclosporine for immunosuppression throughout chemotherapy. No patient developed renal failure during or shortly after administration of cisplatin. Two of five patients treated for testicular cancer developed renal failure at 3 and 6 years after completion of chemotherapy. However, in both cases the cause of renal failure was attributed to chronic rejection of the transplanted kidney. CONCLUSION: Renal transplant recipients usually tolerate cisplatin-based chemotherapy well. It should be offered to patients with potentially curable cancer (e.g., germ cell tumor). This case and a review of the literature suggest that these patients retain baseline renal function even if cisplatin-based chemotherapy and cyclosporine are given simultaneously. PMID- 8630926 TI - Differentiated thyroid cancer. Impact of adjuvant external radiotherapy in patients with perithyroidal tumor infiltration (stage pT4). AB - BACKGROUND: The role of adjuvant external radiotherapy in the survival of patients with differentiated thyroid cancer (DTC) is controversial. To our knowledge, no attempt has been undertaken thus far to assess the impact of this therapy with respect to the papillary and follicular types of thyroid cancer as separate entities. METHODS: Between 1979 and 1992, 238 patients with differentiated papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) with Stage pT4 have been treated and followed in our clinic. One hundred sixty nine patients free of metastases at the final staging, which was performed after the second radioiodine therapy, were included in this study. The standard treatment comprised total thyroidectomy, ablative radioiodine therapy, and thyroid-stimulating hormone-suppressive therapy with levothyroxin. Ninety-nine patients free of disease after the final staging received additional external radiotherapy to the neck (with a dose of 50-60 Gy), whereas the remaining 70 patients were treated with the standard treatment protocol only. Distributions of age, sex, and follow-up time were comparable in both irradiated and nonirradiated groups. Multivariate analysis of the influence of age, sex, histologic subtype, and lymph node status as well as of external radiotherapy on the time to first locoregional and distant failure (LDF), and the time to locoregional recurrence (LR), was accomplished using Cox's proportional hazard model. RESULTS: In patients with DTC, external radiotherapy was a predictive factor for improvement of both LR (P = 0.004) and locoregional and distant failure (P = 0.0003). When the time to first locoregional and distant failure was calculated separately for patients with PTC and FTC, there was a significant difference in the PTC group in favor of irradiated patients (P = 0.0001), whereas there was no effect of external radiotherapy in the FTC group (P = 0.38). Further analyses disclosed that this effect was significantly present only in patients with PTC and lymph node involvement (P = 0.002), whereas those without lymph node involvement did not benefit from an additional adjuvant radiotherapy (P = 0.27). Because none of the patients younger than age 40 years died due to the disease nor had progressive disease during follow-up, we reassessed our results in patients older than age 40 years. The effect of external radiotherapy could be confirmed in this subgroup of patients (P = 0.0009) and in the subgroup of lymph node positive patients older than age 40 years with invasive PTC (P = 0.01). CONCLUSIONS: In addition to total thyroidectomy, treatment with radioiodine, and TSH-suppressive therapy with thyroid hormone, adjuvant external radiotherapy improves the recurrence-free survival in patients older than age 40 years with invasive PTC and lymph node involvement. PMID- 8630927 TI - A long-term assessment of adjuvant chemotherapy on outcome of patients with extracapsular spread of cervical metastases from squamous carcinoma of the head and neck. AB - BACKGROUND: Extracapsular spread (ECS) of cervical lymph node metastases of squamous cell carcinoma from head and neck sites portend poor prognosis. Therefore, a program of combined surgery, postoperative irradiation therapy, and adjuvant methotrexate and 5-fluorouracil (5-FU) was initiated in 1982 for such patients. METHOD: All patients operated on between June 1982 and December 1992 by the full-time faculty of the Department of Otolaryngology at the University of Pittsburgh School of Medicine were eligible and reported in this trial. All patients had negative surgical margins of excision of the primary carcinoma, and histologic evidence of cervical metastases with ECS. Postoperative irradiation included 50-60 cGy for 5 to 6 weeks followed by methotrexate and 5-FU administered on an outpatient basis on days 1 and 8 every 21 days. All patients were followed for 30 or more months for evidence of recurrent disease. RESULT: A total of 371 patients met eligibility criteria. Of this group, 53 (14%) were treated with surgery only, 187 (50%) received surgery and postoperative irradiation, and 131 (35%) received surgery, irradiation therapy, and chemotherapy. The primary site, extent of nodal involvement, and stage of the three patient groups were similar. However, performance status (Karnofsky) was best in the patients who received chemoradiation (average 90) when compared with those who received surgery and irradiation (average 80) or surgery only (average 70). Absolute disease free survival rate (30 months) was 9.5% in patients treated with surgery only, 34% in patients treated with surgery plus irradiation, and 53% in patients treated with surgery, irradiation, and chemotherapy. When adjusted for patients who died of intercurrent disease with less than 30 months follow-up, survival rates became 17%, 40%, and 58%, respectively. These differences are highly significant (P < 0.001). CONCLUSION: Results of this study suggest that postoperative chemoradiation may improve survival in patients with ECS of cervical metastases. Compliance with the chemoradiation was suboptimal and suggests that improved strategy must be developed. PMID- 8630928 TI - Epstein-Barr virus in Hodgkin's disease patients in Japan. AB - BACKGROUND: The association of Epstein-Barr virus (EBV) and Hodgkin's disease (HD) has been suggested by serologic, epidemiologic, and molecular biologic studies. The high level of EBV association with HD in the developing countries was discussed in relation to the high HD incidence in these areas. Japanese HD shows a distinct peak incidence in older adults. In contrast, Western HD shows a bimodal pattern, the first peak in young adulthood and a second peak in older patients. In the present study, the EBV association with HD in Japan was investigated, and the results were compared with those reported from industrialized and developing countries. METHODS: Fifty-seven patients with HD were studied for the presence or absence of the EBV genome by the polymerase chain reaction and in situ hybridization methods. Seven cases were excluded from the analysis for EBV because of poor preservation of nucleotides in the specimens. RESULTS: EBV genomes were detected in the Reed-Sternberg (R-S) cells of 32 of the 50 patients examined (64%). The EBV association was independently affected by histologic subtype (84% in mixed cellularity and 44% in others), sex (76% in males and 31% in females), and age (76% in patients aged 40 years and older and 38% in patients younger than 40 years of age; P < 0.01). High EBV association is found at the peak in older adults predominantly with mixed cellularity type. Previous studies revealed that the high EBV was associated with the older peak of the bimodal peaks in Western HD, and a unimodal peak in childhood in developing countries. The EBV subtype was predominantly type A, which is identical to the immunocompetent type of HD reported previously. CONCLUSIONS: The results of the current study, together with those reported previously, showed that the presence of the Epstein-Barr virus correlated with mixed cellularity type, age older than 40 years, and male sex. PMID- 8630929 TI - Gastric DNA content in postgastrectomy patients. Relationship to mucosal dysplasia. AB - BACKGROUND: Gastric mucosal cellular DNA content was assessed in patients who had undergone gastric surgery for peptic ulcer disease more than 20 years previously, with the aim of examining the relationship between abnormal DNA content and gastric mucosal dysplasia, as well as determining the effect of different types of surgery on DNA content. METHODS: Sixty-five subjects underwent upper gastrointestinal endoscopy. In each, six biopsies were taken from the stoma or antrum and graded for severity of dysplasia. Cellular DNA was quantified using a microprocessor-controlled image analysis system with a fast densitometer card on Feulgen-stained slides. DNA histograms were evaluated using the 2c deviation index (2cDI) for proliferative activity and the 4c exceeding rate (4cER) and the 5c exceeding rate (5cER) as indices of malignant potential. RESULTS: In subjects with Billroth II operations, all the above DNA criteria were higher than in Billroth I (P < 0.05), vagotomy and pyloroplasty (P < 0.001), and controls (P < 0.0001). DNA values increased as dysplasia progressed in severity (2cDI, Rs = 0.67; 4cER, Rs = 0.61; 5cER, Rs = 0.72; respectively, P < 0.0001). Among subjects with no dysplasia, more aneuploid cells were found in the Billroth II group, (p < 0.005) compared with the other types of operation. CONCLUSIONS: Cellular DNA content is abnormal at an early stage in dysplasia and may even predate it. Increasing values of abnormal DNA content are related to the severity of dysplasia. DNA analysis may be a useful additional tool in surveillance programs to select high-risk patients for screening. PMID- 8630930 TI - Soft tissue sarcoma of the head and neck in children and adolescents. AB - BACKGROUND: The experience of one institution in treating soft tissue sarcomas of the head and neck in a pediatric population is presented. METHODS: Case materials of 134 patients younger than 20 years who were referred to the University of Texas M. D. Anderson Cancer Center between 1970 and 1989 for treatment of sarcoma of the head and neck were retrospectively reviewed. Patients with rhabdomyosarcoma underwent multimodality treatment consisting of surgery, irradiation, and chemotherapy. Wide resection was the treatment used for patients with nonrhabdomyosarcomatous soft tissue sarcomas (NRSTS). Adjuvant chemotherapy and irradiation were used to treat high grade neoplasms and residual disease. The clinical response to therapy was measured in terms of the disease-specific survival rate. RESULTS: Seventy-nine of 134 patients presented with untreated or biopsy-proven disease. Fifty-six had rhabdomyosarcoma and 23 had NRSTS: At 2 and 5 years, the disease-specific survival rates for patients with rhabdomyosarcoma were 74% and 63%, respectively, and patients with NRSTS had 80% and 75% disease specific survival rates at 2 and 5 years, respectively. CONCLUSIONS: Rhabdomyosarcoma of the head and neck in children is effectively treated with multimodality therapy. Prognostic indicators for rhabdomyosarcoma include completeness of tumor resection and the development of recurrent disease. Aggressive surgical resection is the treatment of choice for patients with NRSTS: Prognostic indicators for NRSTS include completeness of tumor resection and the development of recurrent disease. PMID- 8630932 TI - Malignant breast masses detected only by ultrasound: a retrospective review. PMID- 8630931 TI - Childhood cancer in the United States. A geographical analysis of cases from the Pediatric Cooperative Clinical Trials groups. AB - BACKGROUND: After injuries, cancer is the leading cause of death in children younger than 15 years in the United States. Despite dramatic increases in 5-year survival rates, more than 100,000 person-years of life are lost to childhood cancer each year. The exact proportion of pediatric cancer patients who receive care at centers that utilize up-to-date therapeutic protocols [such as those affiliated with the Childrens Cancer Group (CCG) or the Pediatric Oncology Group (POG)] remains unknown. The purpose of this study was to estimate the proportion and geographic distribution of childhood cancer patients in the United States who are seen at participating centers of the CCG and the POG. METHODS: Patients included 21,026 incident pediatric cancer cases diagnosed from January 1, 1989 through December 31, 1991. Observed numbers of pediatric cancer cases were compared with expected numbers of cases calculated from incidence rates obtained from the Surveillance, Epidemiology, and End Results (SEER) Program and population counts obtained from the 1990 U.S. Census Bureau. RESULTS: Approximately 94% of children younger than 15 years diagnosed with cancer are seen at an institution that is a member of either POG or CCG. Comparisons of the observed-to-expected numbers of incident cases in the age groups (birth-4 years, 5-9 years, 10-14 years, and 15-19 years) demonstrated ascertainment rates of 100, 93, 84, and 21%, respectively. In some regions within the United States the observed number of cases was significantly less than that expected number, including areas of Idaho, Oklahoma, and Virginia. In addition, there were areas in which the observed number of childhood cancer cases was substantially greater than that expected, including areas in California and Florida. CONCLUSIONS: The membership of the cooperative clinical trials groups (the POG and CCG) ascertain and provide access to state-of-the-art treatment protocols for the vast majority of pediatric cancer patients in the country. For the younger age groups, the CCG and POG may represent a mechanism of case ascertainment that approximates a population-based series for the continental United States. Those areas that have large enough discrepancies in observed-to-expected numbers may warrant further investigation. PMID- 8630933 TI - Timing of weight gain and breast cancer risk. PMID- 8630934 TI - Progression in gastric carcinoma relative to the ratio of CD44 epithelial variant transcript to CD44 hematopoietic variant transcript. AB - BACKGROUND: It has been suggested that CD44 splice variants play a role in the progression of certain epithelial cancers and non-Hodgkin's lymphoma. In this study, we investigated the epithelial variant/hematopoietic variant (E/H) ratio (the amount of the CD44 epithelial variant transcript relative to the CD44 hematopoietic variant transcript) in human gastric carcinoma compared with normal gastric mucosa. METHODS: The ratio was determined for tumors and adjacent noncancerous mucosa from 30 gastric carcinoma patients using reverse transcription-polymerase chain reaction and Southern blotting. We also determined the tumor (T)E/H--noncancerous mucosa (N)E/H (the difference between E/H ratios of tumor tissue and adjacent noncancerous mucosa) and examined these measurements for correlations with the pathologic features of gastric carcinoma, as well as for their usefulness as an indicator of tumor progression. RESULTS: The E/H ratio in tumor tissue was significantly higher than in adjacent noncancerous mucosa (P < 0.01). The TE/H--NE/H in patients with lymph node metastases was 0.16 +/- 0.11, compared with 0.07 +/- 0.08 in cases without lymph node metastases (P < 0.05). Significant correlations also were observed between the TE/H--NE/H and the depth of invasion, blood vessel invasion, and lymphatic vessel invasion (P < 0.03, P < 0.03, and P < 0.01, respectively). CONCLUSIONS: Our results suggest that increases in the E/H ratio may be a useful indicator of progression in gastric carcinoma. PMID- 8630935 TI - Flat adenoma, flat carcinoma, and de novo carcinoma of the colon. PMID- 8630936 TI - Genetic counseling in a Navajo hereditary nonpolyposis colorectal cancer kindred. AB - BACKGROUND: Cross-cultural genetic counseling was provided to an extended Navajo Indian family in which the MLH1 gene mutation for hereditary nonpolyposis colorectal cancer (HNPCC) had been identified. The family had been observed by the authors since 1983 and over the years had been provided with intensive education regarding the natural history of HNPCC as well as recommendations for cancer surveillance and management that was responsive to this natural history. METHODS: Following identification of the MLH1 mutation, DNA from family members was evaluated by a reference laboratory (OncorMed, Gaithersburg, MD), where sequences were checked in both the forward and reverse directions against the published sequence for MLH1. The 4bp deletion beginning at the first nucleotide of codon 727 was easily visualized in the heterozygous condition in both affected and predispositional individuals. The family was reeducated as a group and then provided further education individually during genetic counseling sessions, at which time they were appraised of potential penalties, such as insurance and employer discrimination, and psychological sequelae that could result from knowledge of the MLH1 mutation. Strict confidentiality of this information was assured. RESULTS: DNA testing was performed on 51 family members. Twenty-three individuals were counseled, seven of whom were positive for MHL1. Reactions ranged from full acceptance of the genetic implications to traditional Navajo reasoning such as the family had been cursed. CONCLUSIONS: DNA-based genetic counseling requires comparison and empathy, coupled with intensive preeducation regarding potential penalties and advantages that might emanate from this knowledge. Special care must be given to patients' culture, beliefs, and traditions. PMID- 8630937 TI - Postoperative chemotherapy for colorectal cancer by combining 5-fluorouracil infusion and 1-hexylcarbamoyl-5-fluorouracil administration after curative resection. AB - BACKGROUND: Colorectal cancer is one of the major malignant diseases and, recently, its incidence appears to be increasing. Surgical resectability is an important prognostic determinant; however, recurrent tumors are commonly noted, even after apparently curative surgery. Because such metastatic disease cannot be cured, better adjuvant therapies are urgently called for. METHODS: We studied the effect of postoperative chemotherapy using 5-fluorouracil (5-FU) infusions and 1 hexylcarbamoyl-5-fluorouracil (HCFU) oral administration for curatively resected Stage II to IV colorectal cancer. This study was prospectively randomized and controlled and 251 (93.3%) of 269 patients were determined to be candidates for statistical assessment. The inductive regimen for Group A included a total of 6 5 FU intravenous injections, 10 mg/kg, on postoperative days 0, 1, 2, 7, 8, and 9. For maintenance therapy, Group A also received oral HCFU, 300 mg daily for 52 weeks beginning 2 weeks after surgery. The regimen for Group B included only 5-FU injections of Group A. RESULTS: There were no differences in the prognostic factors or doses of 5-FU between Groups A and B. In addition, no difference was observed in the toxicity rate between the two groups. Group A, with 5-FU infusions plus oral HCFU administration, produced a reduction in the recurrence rate and a prolongation of the survival time for patients with rectal cancer. In a retrospective analysis, this protocol was also effective for patients with Stage III to IV, wall invasion-positive, and lymph node metastasis-positive colorectal cancers. CONCLUSIONS: This study suggests that the combination of 5-FU infusions and the continuous oral administration of HCFU is a reasonable therapeutic approach for patients with surgically resected colorectal cancer and a high risk of recurrence. PMID- 8630938 TI - Histopathological studies of superficial-type early colorectal carcinoma. AB - BACKGROUND: Superficial-type early colorectal carcinoma (SCa) is presently not a rare finding and is very important in discussions regarding the development of large bowel cancers, although the histologic characteristics of SCa remain obscure. METHODS: Using 54 SCa lesions (34 intramucosal adenocarcinomas (SCa-m) and 20 adenocarcinomas with invasion to the submucosa (SCa-sm)), the largest dimension of the depressed region of the lesion and the greatest dimension of the entire lesion were measured by the computed image analyzer, and the expression of p53 and ras of SCa were examined immunohistochemically. RESULTS: The percent of depressed regions in SCa lesions measuring less than 5 mm in greatest extent was larger than in those measuring more than 6 mm, and the percent of depressed regions in SCa-sm with deeper carcinoma invasion was lower than that of SCa-sm with shallower invasion. There was a positive correlation between the depth of invasion and the maximum dimension of the carcinoma. The frequency of association with adenoma in all SCa-m was 21% and was 32% in SCa-m that were more than 6 mm in greatest extent, although all minute SCa-m lesions less than 5 mm were pure carcinomas without any adenomatous component. Positive expression of ras was noted in 41% of SCa-m and 36% of SCa-sm, respectively, while positive expression of p53 was noted in 63% of SCa-m and 88% of SCa-sm, respectively. CONCLUSIONS: These results suggested that 70% to 80% of SCa developed via a de novo carcinoma theory and showed the depression form in the initial histologic stage and thereafter in the flat-protrusion form, while 20% to 30% of SCa arose from the preexisting flat adenoma via the adenoma-carcinoma sequence theory. The results also suggested that p53 was related to the enlargement and deeper invasion of SCa, regardless of the sequence of development of colorectal cancer. PMID- 8630939 TI - Spindle cell hepatocellular carcinoma. A clinicopathologic and immunohistochemical analysis of 15 cases. AB - BACKGROUND: Spindle cell hepatocellular carcinoma (SpHCC) has rarely been reported, and its clinicopathologic characteristics, histogenesis, and prognosis after hepatic resection have yet to be clarified. METHODS: Fifteen cases of SpHCC, including 13 surgically resected patients and 2 autopsy cases, were studied clinicopathologically and immunohistochemically. RESULTS: In 13 resected patients, all except 1 were male and the serum alpha-fetoprotein (AFP) was positive in 6 (46%). Portal venous invasion and intrahepatic metastases were frequent. In an immunohistochemical analysis of 13 SpHCC tumors, cytokeratin CAM 5.2 and AFP were positive in 8 (62%) tumors and 3 (23%) tumors in both ordinary HCC and spindle cell components, respectively. The spindle cell components, but not ordinary HCC components, revealed a positive reaction for vimentin in 8 (62%) tumors, S-100 protein and HAM-56 in 3 (23%) tumors, HHF-35 in 2 (15%) tumors, and alpha-smooth muscle actin, desmin, and KP-1 in 1 (8%) tumor, respectively. p53 overexpression was found in two SpHCC tumors. The survival curve after hepatic resection in the 13 patients with SpHCC was significantly worse than that of the 371 patients with Stage II-IV ordinary HCC (P < 0.001). CONCLUSIONS: These results suggest that SpHCC represents a sarcomatous transformation of HCC. The spindle cell components revealed an immunohistochemical expression of several markers of mesenchymal cells. In addition, poor survival after hepatic resection was documented. PMID- 8630940 TI - DNA ploidy analysis of mucinous cystic tumors of the pancreas. Correlation of aneuploidy with malignancy and poor prognosis. AB - BACKGROUND: Benign and malignant pancreatic mucinous tumors differ in proliferative activity, production of tumor markers, and expression of growth factors. DNA ploidy is a useful index of aggressiveness and poor survival in ductal adenocarcinoma, but has not been studied in pancreatic cystic tumors. METHODS: The DNA ploidy status of Fuelgen-stained tissue sections of pancreatic mucinous cystic tumors was evaluated by image cytometry and related to clinical outcome obtained by case record review. RESULTS: Ploidy status correlated with malignancy and poor clinical outcome. All benign mucinous cystadenomas (n = 13) were diploid and cured by resection. Patients with diploid cystadenocarcinomas (n = 6) had an 83% survival rate following resection, while patients with aneuploid cystadenocarcinomas (n = 5) all died (four of disease and the fifth of another cause). The difference in survival between the diploid and aneuploid carcinomas was significant (P = 0.04). CONCLUSIONS: DNA ploidy status of pancreatic mucinous tumors by image analysis provides quantifiable information that may be predictive of clinical outcome. DNA aneuploidy appears to be a significant differential factor in pancreatic mucinous tumors and is associated with shortened survival in mucinous cystadenocarcinomas. PMID- 8630941 TI - Dose escalation study of carboplatin with fixed-dose etoposide plus granulocyte colony stimulating factor in patients with small cell lung carcinoma. A study of the Lung Cancer Study Group of West Japan. AB - BACKGROUND: We performed a Phase I-II trial to determine the maximum tolerated dose of carboplatin (CBDCA) with a fixed dose of VP-16 and granulocyte-colony stimulating factor (G-CSF) in small cell lung cancer (SCLC) patients. METHODS: Treatment consisted of a starting dose of CBDCA, 400 mg/m2 (i.v., Day 1); VP-16, 100 mg/m2 (i.v., Days 1-3), and G-CSF, 2 micrograms/kg (s.c., Days 4-17) every 4 weeks for four cycles. The dose of CBDCA was escalated in increments of 50 mg/m2 until Grade IV toxicity on the World Health Organization scale developed in two thirds or more of the patients. RESULTS: Seventy-five previously untreated patients with pathology confirmed SCLC were entered into the trial. Seventy-one patients were eligible and 70 patients were evaluated for response. Forty-five patients had limited disease (LD) and 26 had extensive disease (ED). The response rate of the 70 patients who could be evaluated was 81%, with 23% attaining a complete response (CR) and 58% attaining a partial response (PR). The response rate was 80% in LD patients (CR, 23%; PR, 57%) and 85% in ED patients (CR, 23%; PR, 62%). The major dose-limiting toxicity was thrombocytopenia. Nephrotoxicity, neurotoxicity, and ototoxicity were uncommon. The doses of CBDCA that resulted in unacceptable thrombocytopenia were 700 mg/m2 in patients younger than 70 years and 500 mg/m2 in patients older than 70 years. Overall median survival time (MST) was 9 months. MST of LD patients and ED patients were 11 months and 7 months, respectively. The dose-limiting toxicity of CBDCA with a fixed dose of VP-16 and using G-CSF as bone marrow rescue was age-related thrombocytopenia. The maximum tolerated dose of CBDCA was 650 mg/m2 if patients were younger than 70 years and 450 mg/m2 if they were 70 years or older. CONCLUSIONS: When we retrospectively compared our results with those using standard chemotherapy regimens, we saw no therapeutic benefit from increasing planned doses of CBDCA up to 700 mg/m2 in combination with G-CSF in patients with SCLC. PMID- 8630942 TI - Prognostic implication of proliferative markers MIB-1 and PC10 in esophageal squamous cell carcinoma. AB - BACKGROUND: Proliferative markers are related to tumor behavior. The commonly used markers are proliferating cell nuclear antigen (PCNA) and Ki-67. The aim of this study is to evaluate the usefulness of MIB-1 (for Ki-67) and PC10 (for PCNA) in the assessment of the clinicopathologic features and prognosis in patients with esophageal squamous cell carcinoma. METHODS: One hundred patients (88 males, 12 females; mean age, 63 years [range, 39 to 83 years]) with surgically resected esophageal squamous cell carcinoma (32 well differentiated, 51 moderately differentiated, and 17 poorly differentiated) were studied. The clinicopathologic features and survival data of these patients were noted. Representative tissue was collected from each tumor and immunohistochemical preparations for MIB-1 and PC10 were made. RESULTS: The percentages of cells that tested positive for PC10 and MIB-1 were much higher in tumor cells than in nonneoplastic cells. The pattern of expression of both markers varied with the differentiation of the tumor. The results observed with MIB-1 staining were better than those with PC10; because MIB-1 had less background staining, as well as stronger and more uniform positive signals compared with PC10. Thus, further investigation was performed on MIB-1-stained sections. The tumor cell MIB-1 scores ranged from 169 to 964 positive cells per 1000 cells (mean 598 +/- 211; median, 636). Although it was significantly associated with the differentiation of the tumor (P = 0.0001), the score had no significant relationship to the tumor size, location, or stage, or to the patients' age and sex. The prognosis depended on the size and stage of the lesion. In Stage III lesions (n = 83), patients with MIB-1 scores below 300 had longer actual survival rates than those with a score of 300 or above. However, the survival rates of patients in the latter group were better if the greatest dimension of the tumor diameter was 7.5 cm or less. CONCLUSIONS: Proliferative activity in esophageal squamous cell carcinoma, as defined by the MIB-1 immunohistochemical method, is significantly related to tumor differentiation. It is also potentially valuable as a prognostic marker in addition to its use in tumor staging and size. PMID- 8630943 TI - ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma. AB - BACKGROUND: It has been reported that the c-erbB-2 protooncogene is frequently amplified and overexpressed in many types of cancers, except sarcomas and hematological malignancies. METHODS: Expression of ErbB-2 in the tumors of 26 patients with conventional osteosarcoma was evaluated by immunoblotting. DNA from osteosarcoma tissues that expressed ErbB-2 were analyzed by Southern blot hybridization to examine gross rearrangement of the gene. The DNA was also surveyed for the presence of genetic mutation in the transmembrane domain of ErbB 2 by polymerase chain reaction-single-stranded DNA conformation polymorphism analysis. In addition, possible correlation of ErbB-2 expression with gender, age, histopathologic subtype, and response to chemotherapy was analyzed. Survival analysis was performed by the Kaplan-Meier test using the approximate chi-square statistic for the log-rank test. RESULTS: The ErbB-2 protein was detected in 11 of 26 osteosarcoma tissues (42%) by immunoblot analysis. Expression of ErbB-2 was confirmed by immunohistochemical studies using specific anti-ErbB-2 monoclonal antibody. However, neither amplification of the c-erbB-2 gene nor evidence of significant genetic mutation was found in these osteosarcomas. Expression of ErbB 2 examined by immunoblotting was most strongly correlated with early pulmonary metastases (P < 0.05). Among the entire group of 26 patients in this study, Kaplan-Meier life table survival of the patients with apparent ErbB-2 expression was significantly worse than that of the patients with little ErbB-2 expression (P < 0.01). CONCLUSIONS: In 42% of the osteosarcomas, the tumor cells expressed ErbB-2. Expression of ErbB-2 was strongly correlated with early pulmonary metastasis and poor survival rate for the patient. These data suggest that ErbB-2 plays a significant role in aggressive tumor growth and in the promotion of metastatic potential in osteosarcomas. ErbB-2 in the osteosarcoma tissues would be a useful prognostic marker for patients. PMID- 8630944 TI - Hodgkin's disease presenting as a solitary bone tumor. A report of four cases and review of the literature. AB - BACKGROUND: Hodgkin's disease (HD) rarely presents as a solitary bone tumor. Fewer than 20 such cases have been reported in the English literature; many of these were reported prior to the development of immunohistologic markers for HD and T- and B-cell lymphomas. In this report, we describe four cases of HD that presented as a localized solitary mass in bone; the diagnosis was confirmed by immunohistochemical studies in all cases. METHODS: The biopsy specimens of four cases identified in our files were studied by conventional histopathology and immunohistochemistry. Clinical data and follow-up information were obtained for all patients. RESULTS: Three cases presented as a localized, solitary mass in the ilium and one case in the vertebra (T12). Three patients were female and one male. The average age was 43 years. Three of the patients presented with lower back pain without constitutional symptoms. All had solitary osteoblastic lesions. All four cases were diagnostic problems, and the diagnosis was confirmed in each case only after finding lymph node involvement. Bone biopsies showed fibrosis and a mixed inflammatory infiltrate with rare atypical cells. All four patients were subsequently found to have nodal involvement by HD. The histology of the associated nodal disease was mixed cellularity in two cases and nodular sclerosis in two. On immunohistochemical staining, the neoplastic cells in all cases expressed CD15 and CD30 and lacked CD45 and other B- and T-cell antigens. Three patients who were treated for HD are alive and well, 1, 6, and 14 years later. CONCLUSIONS: Although rare, HD should be considered in the differential diagnosis of solitary bone lesions. Most patients who present with apparently solitary HD of bone prove to have nodal involvement. Long-term survival is possible with aggressive treatment. PMID- 8630945 TI - Evaluation of interleukin-2 administered by continuous infusion in patients with metastatic melanoma. AB - BACKGROUND: Interleukin-2 (IL-2) has been used widely in the treatment of advanced melanoma, most often using a high dose bolus schedule of administration. We have evaluated the antitumor activity and toxicity of IL-2 when administered by a continuous infusion schedule in patients with metastatic melanoma. METHODS: Thirty-three patients with metastatic melanoma were treated with IL-2 using the maximum tolerated dose level of 12 x 10(6) IU/m2 as a continuous infusion over 24 hours x 4d/week for 4 weeks every 6 weeks. All patients but one had previously received and failed chemotherapy and had evidence of progressive disease. They were required to have normal organ functions and a performance status of 0 to 1. RESULTS: We observed 1 complete response and 6 partial responses among 31 evaluable patients for a response rate of 22% (95%, confidence interval; 10% to 41%). The median response duration was 6 months, with a range of 4 to 18 months. The toxicity of IL-2 was severe but manageable on the general inpatient ward. One patient died of hepatic necrosis that was probably related to IL-2. Five patients required dose reduction of IL-2 due to toxicity in the form of hepatic or renal insufficiency, which was rapidly reversible. CONCLUSIONS: IL-2, used as a continuous infusion at a dose level of 12 x 10(6) IU/m2/day, 4 times every week for 4 weeks, has activity against metastatic melanoma similar to that reported with high dose IL-2 given in a bolus schedule. PMID- 8630946 TI - Younger women with breast carcinoma have a poorer prognosis than older women. AB - BACKGROUND: It is controversial whether breast cancer in young women is more aggressive than in older women. This study was initiated to determine age associated outcome of women with breast carcinoma. METHODS: Patients with breast carcinoma, who were identified in a statewide tumor registry, were divided into age groups based on 10-year intervals (ages 40 and younger, 41 to 50, 51 to 60, 61 to 70, 71 to 80, and older than 80 years). Age at diagnosis, American Joint Committee on Cancer classification, 5-year disease free (5DFS) and cancer specific (5CSS) survival estimates using Kaplan-Meier analysis were determined. RESULTS: Between 1985 and 1992, 3722 women were diagnosed with invasive breast carcinoma. Approximately 5.6% (210) of the women were 40 years old or younger. The youngest age group had the worst 5CSS of 69.7%, followed by the oldest age group (> 80, 5CSS = 71.45%). The age groups 41 to 50, 51 to 60, 61 to 70, and 71 to 80 years had 5CSS of 80.30%, 78.45%, 82.06%, and 84.27%, respectively. The oldest age group (> 80) had the worst 5DFS (39.88%) followed by the youngest age group (< or = 40, 5DFS = 60.79%). The age groups 41 to 50, 51 to 60, 61 to 70, and 71 to 80 years had 5DFSs of 73.22%, 66.87%, 71.53%, and 63.11%, respectively. Analyzed by stage, young (< or = 40 years) women had a worse 5CSS when compared with the other age groups, except for those with Stage I disease. CONCLUSIONS: Our results indicate that women 40 years of age and younger have a worse 5CSS than their older counterparts. This difference in survival is not solely a reflection of more advanced disease but may reflect differences in tumor biology. PMID- 8630947 TI - The expression of CD44 adhesion molecules on seminoma cells. A new marker for early detection of the tumor. PMID- 8630948 TI - Demonstration of monoclonal origin of human parotid gland pleomorphic adenoma. AB - BACKGROUND: Parotid gland pleomorphic adenoma is histologically comprised of epithelial and mesenchymal elements. It remains to be established whether this neoplasm arises from epithelial and mesenchymal elements, or solely from the epithelial element. METHODS: In an attempt to resolve this issue, we have conducted clonal analysis on five pleomorphic adenomas. The method for clonal analysis was based on the trinucleotide repeat polymorphism of the x-chromosome linked androgen receptor gene and on random inactivation of this gene by methylation. The epithelial and mesenchymal elements were obtained separately from the paraffin sections of the pleomorphic adenomas using a microdissection technique and then subjected to clonal analysis. RESULTS: Clonal analysis revealed that both epithelial and mesenchymal elements were monoclonal. In addition, the same allele of the androgen receptor gene was inactivated in both elements in every case. CONCLUSIONS: It is unlikely that the epithelial and mesenchymal elements of different origin happen to inactivate the same allele of the androgen receptor gene in all five tumors. Rather, it is more reasonable to consider that these two elements have a common single cell origin. PMID- 8630949 TI - Prevalence and prognostic significance of tumor-associated tissue eosinophilia in nasopharyngeal carcinoma. AB - BACKGROUND: Tumor-associated tissue eosinophilia (TATE) has been associated with an improved prognosis in a variety of neoplasms. METHODS: Diagnostic biopsy specimens from 96 patients with nasopharyngeal carcinoma (NPC) were reviewed for the presence of TATE by an observer blinded to the patients' clinical histories. Comparisons between patients with and without TATE with respect to the probabilities of local recurrence, distant metastasis, and survival were performed using the log rank test on Kaplan-Meier product-limit estimates and the Cox proportional hazards model. RESULTS: The prevalence of TATE in these patients was 32%, and was not significantly associated with local recurrence, distant metastasis, or survival. CONCLUSIONS: These results are discordant with those of studies in other tumor models, although comparison is hampered by varying definitions of TATE. The differing results may be due to variations in the degree of activation of the eosinophils present in TATE in different tumors. PMID- 8630951 TI - K-ras and p53 mutations in stage I gallbladder carcinoma with an anomalous junction of the pancreaticobiliary duct. AB - BACKGROUND: An anomalous junction of the pancreaticobiliary duct (AJPBD) was thought to be an important risk factor for gallbladder carcinoma in Japan. In this report, we compared K-ras and p53 mutations in Stage I gallbladder carcinomas (GC) of patients with AJPBD with those in patients without AJPBD: METHODS: We examined 6 GC of patients with AJPBD and 20 GC of patients without AJPBD: Immunohistochemistry was performed for p53 protein. K-ras and p53 mutations were examined using genomic DNA extracted from the cancer regions. The methods of polymerase chain reaction (PCR) single strand conformation polymorphism analysis were performed for mutations in exons 5-8 of p53. The methods of PCR restriction fragment length polymorphism were performed for mutation in codon 12 of K-ras. RESULTS: p53 positivity was 67% in GC of patients with AJPBD and 65% in GC of patients without AJPBD: p53 mutations were found in exons 7 and 8 in GC of patients with AJPBD and in exons 5, 6, and 7 in GC of patients without AJPBD: The incidence of K-ras mutation in GC of patients with AJPBD (50%) was greater than that in patients without AJPBD (6%) (P < 0.05). CONCLUSIONS: These results suggest that K-ras mutation may be important in the early stage of carcinogenesis of the gallbladder mucosa with AJPBD, and that p53 mutations may also contribute to the early stage of carcinogenesis of the gallbladder mucosa, regardless of AJPBD: PMID- 8630950 TI - Relationship between 5-fluorouracil (5-FU) dose intensity and therapeutic response in patients with advanced colorectal cancer receiving infusional therapy containing 5-FU. AB - BACKGROUND: A phase II prospective trial was carried out to study the concept of 5-fluorouracil (5-FU) dose-intensity in patients with advanced colorectal cancer. Forty patients were treated with 5-FU plus leucovorin (LV), with individually increasing doses of 5-FU. A 5-FU pharmacokinetic follow up was performed and a relationship was sought between its metabolism and its response to treatment, and between 5-FU's toxicity and patient survival. METHODS: 5-FU was administered weekly by 8 hour continuous infusion. The initial dose of 1000 mg/m2 was individually increased every 3 weeks by 250 mg/m2 steps, potentiated by 400 mg/m2 LV. 5-FU plasma concentrations were determined weekly by liquid chromatography. RESULTS: Eighteen overall objective responses and 22 minor responses, stabilizations, or progressions (NR) were observed. 5-FU plasma levels were significantly higher in cases of complete or partial response, whatever the dose. They reached about 2000 micrograms/l as early as the second dose level (1250 mg/m2). Only seven patients who experienced NR reached equivalent levels after the fourth step (1750 mg/m2). High 5-FU plasma levels were predictive of an objective response and better survival (difference not significant). The acute toxicity, whatever the type, was correlated with 5-FU levels > 3000 micrograms/l and not with the dose. CONCLUSIONS: This study shows the wide variability of 5-FU metabolism, whatever the dose, the clear relationship between 5-FU plasma levels, toxicity, and efficacy. This relationship points out the problem of the polymorphism of 5-FU metabolism, the usefulness of the therapeutic range determination and the usefulness of the individual 5-FU dose adaptation. PMID- 8630952 TI - Nuclear DNA content and pathology in radically treated pancreatic carcinoma. The prognostic significance of DNA ploidy, histology and nuclear grade. AB - BACKGROUND: Nuclear DNA content and pathology are considered to be prognostically relevant in several solid tumors, but controversial findings have emerged in pancreatic carcinoma (PC). Histopathology and DNA ploidy were each correlated with survival in radically treated PC to ascertain the hierarchy of their prognostic significance. METHODS: DNA ploidy was assessed by flow cytometry (FC) in neoplastic tissue samples from 60 patients with PC who were followed until death. Representative neoplastic areas were obtained by microdissection from archival paraffin embedded material (excluding any carcinoma with a coefficient of variation of the G0/G1 peak higher than 8%). Histologic data and FC patterns were related to prognostic behavior using univariate multivariate statistical analysis. RESULTS: Aneuploid cancers were detected in 39 of 60 patients. Univariate analysis showed that histologic grade, nuclear grade, and ploidy were significantly related to prognosis. On multivariate analysis, only histologic grade and DNA ploidy (diploid vs. aneuploid) were significant with significant interaction. CONCLUSIONS: The prognostic value of pathology and ploidy was demonstrated in patients treated radically for PC. As in other tumors characterized by a short survival, the clinical usefulness of any prognostic parameters is somewhat limited. However, the significant relationship between prognosis and DNA ploidy might be of interest in a cost-benefit analysis for selecting patients in whom an attempt at radical surgical treatment or adjunctive chemotherapy may be justified. PMID- 8630953 TI - Malignant mesenchymoma. AB - BACKGROUND: Malignant mesenchymomas are rare soft tissue tumors that contain two or more distinct histologic subtypes of sarcoma within the same tumor (exclusive of a fibrosarcomatous or hemangiopericytomatous component). They are generally considered high grade neoplasms and are associated with a poor prognosis, although experience with these tumors is limited. METHODS: We report 8 patients seen at our center over the last 22 years and describe the clinical course of a patient with a malignant mesenchymoma arising in the retroperitoneum whose experience typifies the aggressive behavior of this tumor. RESULTS: All eight patients had large, high grade tumors located in the retroperitoneum or thigh. Six of the 8 died of disease and 2 were alive with disease at a median of 30 months from diagnosis. CONCLUSIONS: Malignant mesenchymoma represents a particularly aggressive form of soft tissue sarcoma. Our experience with this disease highlights the need for more effective treatment strategies for these patients. PMID- 8630954 TI - Expression of fatty acid synthase (FAS) as a predictor of recurrence in stage I breast carcinoma patients. AB - BACKGROUND: Fatty acid synthase (FAS) is a molecule found in tumor cells from breast carcinomas of patients whose prognosis is very poor. Recently, this molecule has been identified as the key enzyme in fatty acid biosynthesis. This study was done to test the strength of FAS as a prognostic indicator for disease free survival (DFS) and overall survival (OS). METHODS: Clinical records, histologic features, immunohistochemical expression of cathepsin D and c-erbB-2, and estrogen and progesterone receptor status of 110 Stage I breast carcinoma patients were all associated with FAS by a chi-square test. The patterns of DFS and OS were estimated over a ten-year follow-up period using the Kaplan-Meier method. Univariate and multivariate analysis were evaluated using a log logistic regression model. Multivariate regression analysis was based on the Cox proportional hazard model. To detect FAS, cathepsin D and c-erbB-2 expression as well as estrogen and progesterone receptor status, we used the unlabeled immunoperoxidase technique on formalin fixed, paraffin embedded tissue. RESULTS: FAS was significantly associated with a higher risk of recurrence because it predicted both DFS (P = 0.0001) and OS (P = 0.003) when evaluated as a continuous variable and DFS (P = 0.0001) when evaluated with other prognostic markers. Peritumoral lymphatic vessel invasion was the other most significant independent predictor for DFS (P = 0.001) and OS (P = 0.003). CONCLUSIONS: FAS is a reliable prognostic marker to predict DFS and OS in patients with early breast cancer. PMID- 8630955 TI - A randomized trial of megestrol acetate with or without premarin in the treatment of potentially responsive metastatic breast cancer. A Study of the Eastern Cooperative Oncology Group (E2185). AB - BACKGROUND: Human breast cancer cells in vitro exhibit increased levels of progestin receptors (PgR) after brief exposure to physiologic concentrations of estrogens. Prior clinical studies have positively correlated the responsiveness of metastatic breast cancer to progestin therapy with the level of PgR in the tumor cells. METHODS: These observations were used as the scientific basis for a randomized clinical trial by the Eastern Cooperative Oncology Group (ECOG) to compare the effectiveness of megestrol acetate (MEG) alone in a daily dose of 160 mg with MEG alternated with premarin in a dose of 1.25 mg/day on the first 3 days of a 14 day cycle (PRE/MEG). From 1985 through 1989, 266 eligible and fully evaluable patients were randomized to 1 of the treatment arms and accrued to this trial. All patients were postmenopausal with biochemical estrogen cytosol protein receptor (ER) positive (> or = 10 fm/mg) tumors. The treatment groups were balanced with respect to performance status, number of involved organ systems, and PgR levels. RESULTS: Forty-five of 135 (33%) (95% confidence interval [CI], 25-42%) patients receiving MEG experienced a partial (PR) or complete (CR) response. Thirty-one of 131 (23%) (95% CI, 17-32%) patients receiving PRE/MEG achieved a PR or CR. Survival was not influenced by treatment selection. However, median time to progression was seven months for patients receiving MEG and four months for the group receiving PRE/MEG (P = 0.03). The treatment failure hazard rate was higher for patients with a short disease free interval after primary treatment of the breast cancer, poor performance status, non-white race, and visceral disease. Survival was negatively impacted by short disease free interval, administration of prior radiation therapy, prior treatment for metastatic disease, and hepatic involvement. CONCLUSIONS: Sequential treatment with premarin and megestrol acetate is not superior to treatment with megace alone in potentially hormone responsive patients with advanced breast cancer. PMID- 8630956 TI - Male breast carcinoma: an evaluation of prognostic factors contributing to a poorer outcome. AB - BACKGROUND: Although breast cancer in men is far less common than breast cancer in women, it is associated with a less favorable prognosis. Conventional histopathologic features and new prognostic markers were evaluated to explain the less favorable survival outcome. METHODS: Forty-six consecutive male breast carcinomas were studied for size, histologic and nuclear grade, histologic subtype, presence of carcinoma in situ, nipple involvement, lymphovascular invasion, hormone receptor status, c-erbB-2 protein overexpression, and p53 protein accumulation. These findings were correlated with survival. RESULTS: Of the 46 carcinomas, 4 were noninvasive and 42 were invasive. In the invasive carcinomas, the median patient age was 64 years, and the median tumor size was 2 cm. The predominant histologic patterns were invasive ductal (45%) and mixed invasive ductal and cribriform (28%). Most tumors were of low histologic and nuclear grades (histologic grades: I, 17%; II, 50%; III, 33%; nuclear grade: I, 12%; II, 44%; III, 44%). Of those surgically staged, 22 patients (60%) were lymph node positive and 15 patients (40%) were node negative. Stage at presentation was higher than in women (0, 10%; 1, 17%; 2, 50%; 3, 13%; 4, 10%). The estrogen and progesterone receptor status was positive in 76% and 83% of tumors, respectively. Lymphatic vessel invasion (63%) and nipple involvement (48%) were also more common than in women. True Paget's disease of the nipple was not seen; all cases with nipple ulceration were the result of direct tumor extension to the epidermis. Of the 17 tumors tested, 41% were c-erbB-2 positive and 29% were p53 positive. Survival analysis was limited by the relatively small cohort size. Five and 10-year adjusted overall survival rates for invasive tumors were 76 +/- 7% and 42 +/- 9%, respectively. Skin and nipple involvement (P = 0.03) and c-erbB-2 positivity (P = 0.03) were significant predictors of adverse survival. CONCLUSIONS: Male breast carcinoma presents in an advanced stage with less favorable survival, despite low histologic grade, high estrogen receptor content, and small size. Anatomic factors may have been responsible for the poor survival outcome (i.e., paucity of breast tissue and close tumor proximity to skin and nipple, facilitating dermal lymphatic spread and early regional and distant metastasis). PMID- 8630957 TI - bcl-2 expression in the spectrum of preinvasive breast lesions. AB - BACKGROUND: The bcl-2 gene encodes for a protein that blocks apoptosis. Although the bcl-2 protein has been identified in some invasive breast cancers, its expression in preinvasive breast lesions has not been well characterized. METHODS: We studied bcl-2 expression using immunohistochemistry in cases of normal breast tissue (n = 10), ductal hyperplasia (n = 18), atypical ductal hyperplasia (ADH) (n = 10), atypical lobular hyperplasia/lobular carcinoma in situ (lobular neoplasia, LN) (n = 10), and ductal carcinoma in situ (DCIS) (n = 42). We also related bcl-2 expression to p53 expression in these lesions because the p53 gene is altered in many invasive breast cancers and is also involved in the regulation of apoptosis. RESULTS: bcl-2 was consistently expressed in the epithelial cells in all normal breast tissue, ductal hyperplasia, ADH, and LN lesions. In contrast, bcl-2 expression was present in 76% of the DCIS cases and was related to the histologic grade of DCIS. Staining for bcl-2 was observed in 100% of the well differentiated DCIS cases, 90% of intermediately differentiated cases, and 33% of poorly differentiated cases (P < 0.001). No immunoreactivity for the p53 protein was seen in any of the cases of normal breast tissue, ductal hyperplasia, ADH, or LN lesions. However, 24% of the DCIS cases were p53 positive. The bcl-2+/p53- phenotype, as seen in all cases of normal breast tissue, ductal hyperplasia, ADH, and LN was also observed in 67% of the DCIS cases. In contrast, the remaining 33% of DCIS cases showed combinations of bcl-2 and p53 expression that differed from that of normal breast epithelium and the other pathologic lesions studied. Most lesions with altered bcl-2/p53 phenotypes, including all bcl-2-/p53+ cases, represented examples of poorly differentiated DCIS. CONCLUSIONS: The bcl-2 protein is consistently expressed in normal breast epithelium, ductal hyperplasia, ADH, and LN. bcl-2 expression is variable in DCIS. Among DCIS cases, bcl-2 is most common in well differentiated and intermediately differentiated lesions. Most DCIS lesions are bcl-2+ and p53-, similar to normal epithelium, benign proliferative lesions, and LN. However, a minority of DCIS lesions show combinations of bcl-2 and p53 expression that differ from normal breast epithelium and from the other pathologic lesions studied. It is possible that such lesions may represent a subset of DCIS in which the regulation of apoptosis is no longer under normal control mechanisms, resulting in enhanced tumor cell survival and tumor growth. PMID- 8630958 TI - Molecular genetic analysis of clear cell adenocarcinomas of the vagina and cervix associated and unassociated with diethylstilbestrol exposure in utero. AB - BACKGROUND: Prenatal exposure to the synthetic estrogen diethylstilbestrol (DES) is associated with the subsequent development of clear cell adenocarcinoma of the lower reproductive tract in young women, and data concerning the molecular genetic alterations involved in the etiology of this tumor type have not previously been reported. Such knowledge would be of potential value by providing insight into the molecular mechanisms of hormonal carcinogenesis in general, as well as by suggesting molecular markers for risk assessment in the estrogen exposed population. METHODS: A total of 24 samples of clear cell adenocarcinoma of the vagina or cervix, 16 associated with exposure in utero to DES and 8 with no history of DES exposure, were obtained as archival fixed and embedded tissue specimens. DNA was purified from these tissues and used to examine a number of biologically plausible molecular genetic endpoints for tumor specific alterations. RESULTS: No evidence was found for mutations in the K-ras or H-ras protooncogenes, the Wilms' tumor (WT1) tumor suppressor gene, or the estrogen receptor gene. Sporadic overexpression of the p53 tumor suppressor gene was detected in some tumor cell nuclei by immunohistochemistry, but in the absence of detectable p53 gene mutation. Genetic instability as manifested by somatic mutation of microsatellite repeats was widespread in these tumors, with evidence of microsatellite instability in all DES-associated tumors examined, and in 50% of those tumors not associated with DES exposure. CONCLUSIONS: These data are consistent with the hypothesis that the induction of genomic instability may be an important mechanism of DES-induced carcinogenesis. PMID- 8630959 TI - Prognostic significance of nuclear DNA content and proliferative activity in renal cell carcinomas. A clinicopathologic study of 58 patients using mitotic count, MIB-1 staining, and DNA cytophotometry. AB - BACKGROUND: For a variety of human malignancies, static DNA cytophotometry and immunostaining for the Ki-67 antigen using the antibody MIB-1 have provided significant prognostic information. METHODS: Surgical specimens of 58 renal cell carcinomas (RCCs) were investigated by conventional histology, DNA cytophotometry, and MIB-1 immunostaining. RESULTS: The MIB-1 indices and DNA data were found not only to be significantly correlated with various other morphologic parameters, but also to the clinical behavior of RCC. In the course of this study (median observation period: 31 months), 27% of patients died from RCC. None of these patients belonged to the group of 37 patients with RCCs exhibiting diploid or euploid DNA histograms. Lethal outcome occurred in only 16 of the 21 patients (76%) with noneuploid or aneuploid histogram tumors (P < 0.0001). According to their MIB-1 indices and upon choosing different cutoff levels, the 58 RCCs were categorized into 2 groups with either low or high proliferative activity. Using the median and the mean MIB-1 index as cutoffs, none of the patients with tumors showing low proliferative activity had died, whereas 16 of 29 patients (55%) or, respectively, 16 of 25 patients (64%) with tumors exhibiting high proliferative activity, had died from RCC (P < 0.0001). CONCLUSIONS: In addition to tumor grade and stage, both a high MIB-1 index and a noneuploid or aneuploid DNA histogram of a given RCC have the potential to identify tumor patients with an impaired prognosis. PMID- 8630960 TI - Prospective evaluation of a chamomile mouthwash for prevention of 5-FU-induced oral mucositis. AB - BACKGROUND: Stomatitis has been found to be a major dose-limiting toxicity from bolus 5-fluorouracil-based (5-FU) chemotherapy regimens, despite the use of oral cryotherapy. Pursuant to preliminary data which suggested that a chamomile mouthwash might ameliorate this toxicity, a prospective trial was developed to test chamomile in this situation. METHODS: A Phase III, double-blind, placebo controlled clinical trial was designed. Patients were entered into the study at the time of their first cycle of 5-FU-based chemotherapy. All patients received oral cryotherapy for 30 minutes with each dose of 5-FU. In addition, each patient was randomized to receive a chamomile or placebo mouthwash thrice daily for 14 days. Stomatitis scores were determined by health care providers and by patients themselves. RESULTS: There were 164 evaluable and well-stratified patients equally randomized to both treatment groups. There was no suggestion of any stomatitis difference between patients randomized to either protocol arm. There was also no suggestion of toxicity. Subset analysis did reveal unsuspected differential effects between males and females that could not be explained by reasons other than chance. CONCLUSION: The resultant data from this clinical trial did not support the prestudy hypothesis that chamomile could decrease 5-FU induced stomatitis. PMID- 8630961 TI - Cisplatin-based chemotherapy of primary extragonadal germ cell tumors. A single institution experience. AB - BACKGROUND: Extragonadal germ cell tumors account for only 2-5% of all germ cell neoplasms in adult males. Because these tumors are rare and, in part, biologically distinct from their testicular counterparts, their optimal management continues to be defined. METHODS: The medical records of 51 patients with extragonadal germ cell tumors were reviewed. All patients were treated with cisplatin-based chemotherapy at a single institution between 1981 and 1994. RESULTS: Thirty-five patients had nonseminomatous germ cell tumors and 16 had pure seminomas. Sixteen tumors arose in the mediastinum (12 nonseminomas, 4 seminomas), and 35 in the retroperitoneum (23 nonseminomas, 12 seminomas). Six of 12 patients (50%) with mediastinal nonseminomas survived with no evidence of disease (NED) at 33-137 months (median, 96 months); all had undergone surgery as part of their treatment. Fifteen of 23 patients (65%) with retroperitoneal nonseminomas are alive with NED at 2-145 months (median, 39 months). Fifteen of 16 patients (94%) with extragonadal seminomas survived with NED at 2-141 months (median, 66 months), and 1 patient died from late irradiation-related toxicity. Three patients with retroperitoneal nonseminomas developed a testicular seminoma at 35, 42, and 77 months, respectively; all are currently disease free. CONCLUSIONS: Mediastinal and retroperitoneal nonseminomas have distinct clinical features. As in other series, clinical outcome is somewhat inferior for mediastinal nonseminomas compared with retroperitoneal nonseminomas. Regardless of the site of presentation, the vast majority of patients with extragonadal seminomas can expect cure. It remains controversial, however, whether retroperitoneal germ cell tumors are metastatic from a primary testicular germ cell tumor. PMID- 8630962 TI - Epidermal growth factor receptor and c-erbB-2 oncoprotein expression in female genital tract carcinosarcomas (malignant mixed mullerian tumors). Clinicopathologic study of 82 cases. AB - BACKGROUND: Epidermal growth factor receptor (EGFR) and c-erbB-2 (also known as HER-2/neu) oncoprotein (p185erbB-2) are members of the subfamily of tyrosine kinase, transmembrane receptors often implicated in human carcinogenesis. We hypothesize that expression of EGFR and p185erbB-2 adds useful prognostic and histogenetic information regarding female genital tract carcinosarcomas (FGTCSs). METHODS: Paraffin sections from 82 FGTCS (61 endometrium, 14 ovary, 5 cervix, and 2 fallopian tube), 56% of which exhibited heterologous elements, were stained using anti-EGFR (clone 31G7, Triton Diagnostics, Alameda, CA) and anti-p185erbB-2 (clone CB11, Novocastra Labs, UK). RESULTS: EGFR reactivity was present in 11 (13.4%) FGTCSs (55% carcinomatous component [CC] only, 18% sarcomatous component [SC] only, and 27% in both). EGFR was associated with adenosquamous histology of the CC (P < 0.05) and heterologous rhabdomyosarcomatous differentiation in the SC (P < 0.05); no other histopathologic features were correlated. p185erbB-2 reactivity was present in 79 (87.8% strong [S], 78% membrane [M], and 8.5% weak) FGTCSs (1% CC only, 0% SC only, and 99% in both). p185erbB-2 did not correlate with histopathologic features or EGFR. Seventy-seven patients had clinical follow up for longer than 12 months. Approximately 49.3% and 72.3% of patients had recurrent disease by 12 and 80 months, respectively; all but 1 were dead from disease. 27% of patients were disease free after 15 to 307 months (median, 77 months; mean, 92 months). EGFR, but not p185erbB-2 expression predicted disease recurrence (P < 0.05). Recurrent disease was associated with Stage greater than I (P < 0.0001), vascular space invasion in resection specimens (P < 0.01), and deep myometrial invasion in hysterectomies (P < 0.05). EGFR was associated with Stage greater than I and did not help predict recurrence in good prognosis groups. CONCLUSIONS: p185erbB-2 overexpression in both CC and SC of FGTCS suggests a common carcinogenic mechanism for both components and supports the conversion histogenesis hypothesis implicating a dominant role for the CC with the SC arising as a metaplastic change from the CC. EGFR may be expressed in either component and indicates aggressive biologic behavior; however, its prognostic utility is limited by its low predictive value for recurrence (40.3%), inability to foretell recurrence in good prognosis groups, and dependence on stage. High frequency of overexpression and dismal prognosis make FGTCS patients good candidates for trials of therapeutic strategies involving the p185erbB-2 receptor manipulations. PMID- 8630963 TI - A comparison of polymerase chain reaction examination of cerebrospinal fluid and conventional cytology in the diagnosis of lymphomatous meningitis. AB - BACKGROUND: Inherent limitations of conventional cytology often result in a failure to diagnose lymphomatous meningitis in cerebrospinal fluid (CSF) specimens from patients who actually have the disease. The development of polymerase chain reaction (PCR) techniques for the diagnosis of lymphoma based on the detection of clonal rearrangements of the immunoglobulin or T-cell receptor genes offers an alternative, DNA-based test for the diagnosis of lymphoma in the CSF. METHODS: In this retrospective study, 31 CSF specimens from 21 patients were examined by a PCR technique that can detect clonal immunoglobulin gene rearrangements. Twenty-four of the specimens came from 14 patients who eventually had definitive histologic or cytologic diagnoses of B-cell lymphoma. The other seven patients had other neurologic diagnoses, including two patients with reactive lymphocytosis, three with glioblastoma, one with metastatic carcinoma, and one with multi-infarct dementia. The results of the PCR examinations were compared with cytologic evaluation of the same CSF specimens. RESULTS: Five of seven specimens from patients with central nervous system lymphoma that were suspicious for, but not diagnostic of, lymphoma by conventional cytology were positive by PCR. Of 13 specimens from patients with lymphoma that showed no cytologic evidence of malignancy, 5 were positive by PCR. Two of four specimens for which conventional cytology showed definitive evidence of lymphoma were positive by PCR. Two specimens from patients with a reactive lymphocytosis showed a polyclonal pattern by PCR. Specimens from patients with other neurologic diseases were negative by PCR even when cytologically malignant (glioblastoma) cells were present in the specimen. CONCLUSIONS: PCR examination of CSF is practical, complements conventional cytology, and sometimes provides the correct diagnosis when conventional cytology yields only ambiguous results. PMID- 8630964 TI - Mesothelioma, asbestos, and reported history of cancer in first-degree relatives. AB - BACKGROUND: Although malignant mesothelioma is known to be strongly related to asbestos exposure, its relationship to familial factors is unclear. METHODS: We compared reported histories of cancer in first-degree relatives, obtained from telephone interviews with the next-of-kin of 196 patients who had a pathologic diagnosis of mesothelioma, and with those from 511 decreased controls. RESULTS: Among men exposed to asbestos, we found a statistically significant twofold elevation in the risk of mesothelioma for patients reporting cancer in two or more first-degree relatives. We found no significant elevation in women or among the small number of men without asbestos exposure. The next-of-kin of three patients (but no controls) reported a possible mesothelioma in a first-degree relative; asbestos exposure could not be ruled out in those relatives. Associations of asbestos with pleural mesothelioma were stronger among men with a reported family history of cancer than men without, although no statistical evidence of an interaction was detected. CONCLUSIONS: These results provide suggestive, but limited, evidence that a family history of cancer may be a risk factor for mesothelioma, or may indicate an increased susceptibility to mesothelioma given asbestos exposure. PMID- 8630965 TI - Long term survivors of childhood brain stem gliomas treated with hyperfractionated radiotherapy. Clinical characteristics and treatment related toxicities. The Pediatric Oncology Group. AB - BACKGROUND: Over the past decade, the principal focus of research in pediatric brain stem gliomas has been on the use of hyperfractionated radiotherapy (HRT). The purpose of this study was to evaluate the clinical characteristics and treatment related toxicities of long term survivors of HRT treatment. METHODS: Of the 130 children with brain stem tumors treated with escalating doses of HRT on Pediatric Oncology Group (POG) #8495, there are only 9 long term survivors. Prospectively collected data, including flow sheets and all pretreatment and follow-up radiologic studies, were reviewed for these patients. Additional information was requested from the treating institutions with regard to sequelae of treatment. RESULTS: Clinical characteristics (including age, sex, duration of symptoms, and presenting signs) for the nine surviving patients were not different from the total population of patients treated on POG #8495. Pretreatment imaging, however, revealed that only four of the nine patients had typical diffuse intrinsic pontine lesions and, conversely, that at least three of the nine patients had lesions that would now be considered relatively favorable. Complete information regarding treatment related toxicity was available for eight patients, only one of whom is without sequelae. Seven have schooling difficulties, two have a seizure disorder, five have hearing loss, and two have required growth hormone replacement. Follow-up imaging findings were striking in four of the eight patients because of white matter changes consistent with leukoencephalopathy (two patients), diffuse microhemorrhages (one patient), and dystrophic calcification (one patient) in the radiation field. CONCLUSIONS: The high frequency of treatment related sequelae in long term survivors of HRT suggests a need for caution in the use of HRT, particularly in patients who have brain stem tumors with a more favorable prognosis. PMID- 8630966 TI - Ethnicity and cure rates of Texas children with acute lymphoid leukemia. AB - BACKGROUND: Ethnic differences in the survival of children treated for acute lymphoid leukemia (ALL) have been described in several locations. Children of African, Polynesian, Native American, and Mexican ancestry had a less favorable outcome than children of European ancestry when treated in a similar manner by the same physicians and nurses. METHODS: We reviewed the medical records of the 94 European-American (E-A) and 84 Mexican-American (M-A) Texas children registered and treated in national collaborative ALL therapy trials at the M. D. Anderson Cancer Center in Houston between 1974 and 1985 and followed through June 1994. Information was collected regarding age, sex, presenting clinical features, risk for relapse grouping, protocol assignment, event free survival, and the financial status of their families. Cure was defined as initial continuous complete remission for more than seven years and cessation of therapy for more than four years. Presenting characteristics of E-A and M-A children were compared, and then related to cure rates by univariate and multivariate analyses. Event free survival rates of E-A and M-A children were determined together and by sex. RESULTS: Comparing presenting features, financial status as identified by pay code was significantly less for M-A children. Other features were not significantly different. By univariate analysis, an age of 2 to 6 years, female sex, initial white blood cell count below 10,000/microL, low risk grouping, registration on the most recent protocol, and full pay status were significantly associated with higher cure rates. By multivariate analysis, male gender, high risk group, and registration on earlier protocols were found to be significantly associated with a low cure rate. Event free survival and cure rate were lower for M-A children, but the differences were not statistically significant. CONCLUSIONS: Further study of larger numbers of patients, including contemporary immunophenotypic and genotypic characterization of ALL, is needed for better definition of possible ethnic differences. Ethnicity and financial status should be included in the analysis of clinical trials of ALL therapy. PMID- 8630967 TI - Molecular analysis of the cyclin-dependent kinase inhibitor family: p16(CDKN2/MTS1/INK4A), p18(INK4C) and p27(Kip1) genes in neuroblastomas. AB - BACKGROUND: Chromosomal abnormalities involving band 1p32, especially deletions, are frequent in neuroblastomas, indicating that a tumor suppressor gene(s) is localized at this region. The p18 gene, one of the cyclin-dependent kinase inhibitor (CDKI) genes, maps to this chromosomal region. Complexes of cyclin and cyclin-dependent kinase (CDK) play important roles in the cell cycle. CDKIs inhibit the kinase activities of these complexes and block transitions of the cell cycle. Some of the CDKI genes may be tumor suppressor genes. For example, the CDKI genes p16 and p15 are frequently deleted in various malignancies and are thought to contribute to cellular transformations. METHODS: To elucidate the importance of CDKI genes, including the p18 as well as the p16 and p27 genes in tumorigenesis of neuroblastoma, 25 neuroblastomas were analyzed for deletions by Southern blot analysis and for point mutations by polymerase chain reaction single strand conformational polymorphism. RESULTS: No deletions, rearrangements, nor mutations were detected in these genes, however, polymorphisms reported previously were detected. CONCLUSIONS: Abnormalities, including deletions and point mutations of the p16, p18, and p27 genes, were not observed in this series of neuroblastomas. Other mechanisms to inactivate these genes, such as transcriptional or translational defects, must be analyzed. CDKI genes rarely contributed to tumorigenesis in neuroblastomas. PMID- 8630969 TI - The importance of correct stage grouping in oncology. Results of a nationwide study of oropharyngeal carcinoma in The Netherlands. PMID- 8630968 TI - Existential well-being is an important determinant of quality of life. Evidence from the McGill Quality of Life Questionnaire. AB - BACKGROUND: The McGill Quality of Life Questionnaire (MQOL) is being developed to correct what we perceive to be a flaw in existing quality of life instruments: neglect of the existential domain. METHODS: This study reports the first use of MQOL for people with cancer at all phases of the disease, including those with no evidence of disease after therapy. RESULTS: The data suggest that MQOL is comprised of an item measuring physical well-being and four subscales: physical symptoms, psychological symptoms, existential well-being, and support. MQOL is acceptable to oncology outpatients. Correlation of the MQOL total and subscale scores with a single item scale measuring overall quality of life and with the Spitzer Quality of Life Index suggests that MQOL has construct and concurrent validity. CONCLUSIONS: The hypothesis that the existential domain is important, especially to those patients with a life-threatening illness, is supported because multiple regression showed that the existential subscale is at least as important as any other subscale in predicting a single item scale measuring the overall quality of life and plays a greater role in determining the quality of life of patients with local or metastatic disease than in patients with no evidence of disease. PMID- 8630970 TI - Cellular schwannoma: a clinicopathologic, DNA flow cytometric, and proliferation marker study of 70 patients. PMID- 8630971 TI - Ethnic differences in risk and prognosis factors for breast cancer. PMID- 8630972 TI - Instability of chromosomes 1, 3, 16, and 17 in primary breast carcinomas inferred by fluorescence in situ hybridization. AB - Abnormalities of chromosomes 1, 3, 16, and 17 were examined in 203 metaphase cells from 12 cases of primary breast carcinoma using fluorescence in situ hybridization with chromosome painting probes. The most common structural abnormalities were chromosomal rearrangements, especially translocations, and chromosome 17 was most frequently involved in these types of changes. Chromosome 16 was preferentially involved in the losses and deletions, while chromosomes 1 and 17 were more involved in the gains, including amplifications, than other chromosomes. This approach has revealed a different profile of abnormalities from those normally shown by G-banding analysis. Some of these changes are likely to be novel and may be biologic or clinical importance in breast cancer. PMID- 8630973 TI - Cytogenetic and histologic correlation of peripheral nerve sheath tumors of soft tissue. AB - Cytogenetic analysis was performed on 11 peripheral nerve sheath tumors of soft tissue from 10 patients. They include 6 benign and 5 malignant schwannomas. Five cases which include two benign, one cellular and two malignant schwannomas had a known association with a nerve, but only one patient with malignant schwannoma has clinically documented neurofibromatosis type I. All the patients had a normal diploid constitutional karyotype. Two cases of cellular schwannoma were analyzed by routine cytogenetic analysis and fluorescence in situ hybridization (FISH). One tumor was karyotyped as 45, XX,-13,-22 +mar; and the other case had a 45,X, Y,t(1;17) (p12;q11.2) karyotype. In the latter, the breakpoint in 17q occurred below the centromere and is at or in the region of the Neurofibromatosis Type 1 (NF1) gene. Four benign tumors had a normal diploid karyotype. One hypodiploid malignant schwannoma with myxoid features demonstrated monosomy of chromosomes 17 and 22 by FISH analysis. The rest of the malignant schwannomas showed a wide range of numerical and structural aberrations, with frequent loss of 22q and gains of chromosomes 2 and 7. Loss of a sex chromosome was observed in cellular as well as malignant schwannomas. Regional karyotypic evolution was noted in one malignant schwannoma. Cytogenetic analysis may prove to be useful in identifying tumors, such as cellular schwannomas, which, because of their histologic features may be inadvertently categorized as malignant. Simultaneous involvement of NF1 and NF2 genes, which are located on chromosomes 17q and 22q, respectively, should be investigated at a molecular level in both benign and malignant tumors of peripheral nerves. PMID- 8630975 TI - Spontaneous genomic fragility and cell cycle progression in lymphocytes of patients with cervical carcinoma. AB - Thirty patients with invasive carcinoma of the cervix, along with 15 age-matched healthy females as controls, were studied to examine the frequency of spontaneous SCEs, chromosomal aberrations (CAs), and cell cycle progression in lymphocytes. The frequency of SCEs and TCAs was statistically significant in the patients over the control group. The correlation of SCEs and TCAs with the stage of cancer, age, and number of pregnancies was also made. The cell cycle of lymphocytes showed its prolongation in the patients, as is evident from the higher proportion of cells at M1 (metaphase of first cell division after 72 hours). PMID- 8630974 TI - Translocation (9;11;22)(p22;q23;q11). A new type of complex variant translocation of t(9;11)(p22;q23) with MLL rearrangement. AB - We describe a patient with acute monocytic leukemia (M5a, FAB classification) associated with a new type of variant translocation (9;11). Southern blot analysis showed the rearrangement of the MLL (ALL-1/HRX) gene at 11q23. Fluorescence in situ hybridization (FISH) with painting probes of chromosomes 9, 11, and 22 revealed the translocation as t(9;11;22) (p22;q23;q11). This is more evidence that the production of chimeric mRNA following the translocation of the LTG9 (MLLT3/AF9) gene at 9p22 to 11q is a critical event in this leukemia subtype. PMID- 8630977 TI - A novel reciprocal translocation (14;15)(q11;q24) in a congenital mesoblastic nephroma. AB - We describe a novel reciprocal translocation, t(14;15)(q11;q24), as the sole cytogenetic aberration in a congenital mesoblastic nephroma. The tumor was predominantly of "classic" morphology, but with small cellular islands of larger cells indicating early transformation to the cellular type. This is the first report of a reciprocal translocation as the sole anomaly in a congenital mesoblastic nephroma. PMID- 8630976 TI - Lack of microsatellite instability in giant cell tumor of bone. AB - Microsatellite instability was searched for at six different loci on chromosome arms 5q, 18q, 15q, 17p, 19q, and 11p in 22 patients (12 men and 10 women; average age of 31.8 years, range of 20-55 years) with giant cell tumor of bone (GCT). These loci were chosen because of their use in microsatellite instability studies in other tumors such as colorectal cancer (e.g., 5q, 18q, 17p) or because of the presence of chromosomal abnormalities such as telomeric associations commonly occurring at 19q and 11p termini (thus the reason for including the 19q and 11p termini microsatellites in our study of GCT). No microsatellite instability or loss of heterozygosity were detected when comparing normal and tumor cells from any of the GCT patients. Unlike several other tumors, our study indicates that microsatellite instability does not appear to play a role in the tumorigenesis of GCT although other abnormal cytogenetic, biochemical, and molecular genetics data do exist for this musculoskeletal tumor. PMID- 8630978 TI - Cytogenetic responses to G2 phase x-irradiation of cells from retinoblastoma patients. AB - Fibroblast cell lines from 20 retinoblastoma (RB) patients with the hereditary bilateral form of disease compared with 16 lines from normal donors had a significantly higher chromatid aberration frequency (CAF), and more displaced and nondisplaced breaks per 100 metaphase cells after x-irradiation during the G2 phase of the cell cycle. The mean CAF was 39 +/- 1.0, range 30-46, for cells from normal subjects, compared to a mean of 245.6, range 101-506, for cells from hereditary RB patients (p < 10(-6). Of fibroblast lines from eight patients with unilateral RB, four had a CAF comparable to that of lines from normal donors (< 60) and four had a high CAF (> 130), resembling that of hereditary forms; two of the latter four lines were from patients with familial or deletion 13 forms of RB. Furthermore, in two families, PHA-stimulated blood lymphocytes from RB patients, one bilateral and one unilateral, and from certain unaffected first degree relatives after G2 phase X-irradiation had a high CAF (> or = 110) compared to a CAF (> or = 53) of cells from three normal donors sampled at the same time. These results were shown not to be related to differences in cell cycle progression or initial extent of chromatid damage. The results suggest that the high frequency of chromatid aberrations in the cells from hereditary RB patients results from a genetic deficiency in DNA repair. PMID- 8630979 TI - Translocation (X;18) in primary synovial sarcoma of the lung. AB - Primary sarcomas of the lung are extremely rare. Among the most common to occur in this location are leiomyosarcoma, fibrosarcoma, and hemangiopericytoma. Many difficulties are encountered when establishing these sarcoma diagnoses, or one of another pathologic type, because of overlapping histologic features and morphologic similarities between primary and metastatic lesions. In this study, the diagnosis of a primary monophasic synovial sarcoma of the lung was aided by the observation of the X;18 translocation characteristic of this neoplasm. To the best of our knowledge, this is the first cytogenetic report of a primary pulmonary synovial sarcoma. PMID- 8630980 TI - Cytogenetic findings in four malignant mixed mesodermal tumors of the ovary. AB - Short-term cultures of four malignant mixed mesodermal tumors of the ovary were cytogenetically analyzed. The primary tumor was examined in three cases, whereas in one case the sample was obtained from a residual tumor mass after chemotherapy. The tumor sampled after cytostatic treatment had a relatively simple karyotype with numerical changes that included pentasomy 12 and an i(1)(q10) as the only structural abnormality. Karyotypic analysis of the three primary tumors revealed extensive structural as well as numerical aberrations, i.e., a picture similar to that seen in the few malignant mixed mesodermal tumors with karyotypic anomalies described previously. Rearrangements of chromosome 1, leading to loss of distal 1p, and homogeneously staining regions have so far been the most frequent cytogenetic changes in this tumor type. Malignant mixed mesodermal tumors of the ovary thus seem to be karyotypically identical to the more numerous mixed mesodermal tumors of uterine origin, and they do not differ substantially in this respect from pure ovarian carcinomas. PMID- 8630981 TI - Cytogenetic, cytomorphologic, and immunologic analysis in 55 children with acute lymphoblastic leukemia. AB - This paper presents the results of cytogenetic analysis in 55 children with acute lymphatic leukemia (ALL). Acquired chromosome aberrations were identified in 35 (63.6%) children. Difference in frequency of clonal aberrations depending on age of patients was observed. Thus, acquired aberrations were detected in all three children up to 6 months of age, in 57.8% in the group from 1-10 years, and in six (85.7%) of seven children older than 10 years of age at diagnosis. The analysis revealed chromosome aberrations specific for ALL. Hyperdiploidy >50 was identified in 20% of children, and the association with CALLA+ early pre-B and L2 ALL was observed. del(6) and t(1;19) were identified in 7.3% and 5.5% of children, respectively. No association with a specific morphology or specific immunophenotype for both structural aberrations has been established. The association of structural aberrations involving regions 14q11-12 and T-cell ALL, however, has been observed, as an aberration was identified in two (3.6%) children, i.e., in 25% of our T-cell leukemias. Interstitial deletion of the long arm of chromosome 13, a rare chromosomal aberration in ALL, was identified in addition to del(9)(q31) in a 17-month-old girl with constitutional trisomy of chromosome 21 and B-cell ALL-L2. Interesting is the finding of hyperdiploidy with 52 chromosomes and structural aberrations of chromosome 1 in a 1-month-old girl with morphologically unclassified CALLA+ pre-T acute leukemia. To our knowledge this is the first case of hyperdiploidy >50 in a neonatal leukemia. PMID- 8630982 TI - L-MYC allelotype in renal cell carcinoma. AB - The L-MYC restriction fragment-length polymorphism (RFLP) revealed by EcoR1 has been suggested to be of prognostic significance in lung, breast, and kidney cancer. The presence of the smaller allele, in either the homozygotic (S-S) or heterozygotic form (L-S), is felt to convey a worse prognosis than the homozygotic form for the larger allele (L-L). The significance of this relationship in lung cancer has been questioned recently. The objective of the present study was to test the prognostic significance of the L-MYC allelotype in a group of renal cell carcinoma (RCC) patients. Tumor and normal tissue were obtained from 59 patients who underwent radical nephrectomy for RCC between 1986 and 1990. EcoR1 restriction digests were performed on isolated DNA and hybridized with the L-MYC probe. Allelotypes were correlated with pathologic parameters and clinical outcome using the chi 2 test. The L-MYC alleolotype (L-L versus L-S and S-S) did not correlate with any pathologic parameter or likelihood of disease recurrence and does not offer any clinical utility in patients with RCC. PMID- 8630983 TI - The use of FISH with chromosome-specific repetitive DNA probes for the follow-up of leukemia patients. Correlations and discrepancies with bone marrow cytology. AB - The use of fluorescence in situ hybridization (FISH) for the purpose of repeated follow-up examination of bone marrow samples from 38 leukemia patients was investigated. On the basis of conventional cytogenetic analysis, patients with acute leukemia whose leukemic cells carried numerical chromosomal aberrations were selected and followed with repetitive DNA probes that specifically hybridize to one chromosome type. Repeated cytogenetic metaphase analyses would have been laborious and not sensitive or quantitative enough to follow declining numbers of aberrant cells. FISH, as an interphase cytogenetic technique, provides a rapid and simple alternative with high sensitivity. Although FISH data before and after chemotherapy were in agreement with bone marrow cytology in 30 of 38 patients, discrepancies were noticed in specific cases. These could be explained by the presence of cytogenetically distinct subclones that behave differently during treatment, the presence of differentiated leukemic cells, changes in the chromosomal constitution caused by clonal relapse, or the fact that a numerical aberration is found by conventional chromosome banding analysis while the target region to which the probe is directed is still present in the nucleus as a diploid set. PMID- 8630984 TI - Inversion (14)(q11q32) in a patient with childhood T-cell acute lymphoblastic leukemia. AB - We report a rare example of inversion (14)(q11q32) in childhood T-ALL and its further involvement in a subsequent translocation. We discuss the possible clinical significance of inv(14) in childhood T-ALL. PMID- 8630985 TI - Detection of genetic alterations in micrometastatic cells in bone marrow of cancer patients by fluorescence in situ hybridization. AB - Detection of micrometastatic tumor cells in bone marrow of cancer patients has been shown to be of prognostic significance. To further characterize these cells, we combined antibody labeling and fluorescence in situ hybridization (FISH). For detection of numerical changes of chromosome 17, nine patients with proven breast cancer whose bone marrow contained epithelial tumor cells were evaluated. Epithelial cells were stained by anticytokeratin antibody. Afterwards FISH was performed using an alpha-satellite probe specific for chromosome 17. In a second series bone marrow epithelial cells of eight patients with breast cancer and of six with prostatic cancer were evaluated for the amplification of HER-2/neu by using a gene-specific DNA probe. In the first series four patients had only single epithelial cells in their bone marrow. Only one single cell showed five hybridization signals, whereas all other single cells showed two or less. Five patients had clusters of epithelial cells in bone marrow with or without additional single cells. One hundred four cells had three or more hybridization signals and 103 of these polysomic cells were located in tumor cell clusters. In the second series we could detect HER-2/neu amplification in bone marrow epithelial tumor cells in two of eight patients with breast cancer but in none of the prostatic cancer patients. These results show that it is possible to detect numerical chromosomal changes and oncogene amplification in bone marrow micrometastatic epithelial cells of cancer patients by combining immunophenotyping and FISH. PMID- 8630986 TI - The relationship between age and karyotypic abnormalities in myelodysplastic syndromes. AB - We performed a retrospective analysis of 143 consecutive patients with myelodysplastic syndrome to study the possible relationship between patient age and cytogenetic findings in this disorder. There were 96 men and 47 women, with a mean age of 63 years. Eighty-six patients were 63 years old and above, and 57 patients were younger than 63 years of age. The distributions of the five FAB subtypes were comparable in both groups of patients, except for a higher percentage of refractory anemia with ringed sideroblasts in older patients. The incidences of cytogenetic abnormalities were similar in the two groups. However, the younger patients tended to have a higher frequency of involvement of chromosomes 5 or 7 than the elderly patients (p < 0.05). The implications of our findings in relation to the biology of myelodysplastic syndrome are discussed. PMID- 8630987 TI - Derivative (y)t(Y;1)(q12;q12),+9 in a patient with polycythemia vera during transition into myelodysplasia. AB - Cytogenetic analysis of bone marrow cells of a 63-year-old male Caucasian patient with polycythemia vera (PV) who developed anemia, thrombocytopenia, and increased granulocytic immaturity revealed a 47, X,der(Y) t(Y;1)(q12;q12),+9 karyotype. The breakpoint in chromosome 1 appeared to map to q12 and not to q21, as has been described in previous reports without FISH confirmation. In the 4 years before this transition the patient was polycythemic and, accordingly, treated with phlebotomy and three short courses of busulfan. The cytogenetic picture observed has been described before in seven patients: three with PV, three with myelodysplasia, and one with Fanconi anemia. In 5/7 cases, like in our patient, the abnormality was observed during transition of the disease into either myelodysplasia or AML. PMID- 8630988 TI - Dicentric (1;15) in myeloid disorders. AB - We report three cases of myeloid disorders with a dic(1;15)(p11;p11), resulting in trisomy of the long arm of chromosome 1. A review of the literature showed six cases, reported as t(1;15). We suggest that these cases have the same anomaly and should be reappraised as dic(1;15). PMID- 8630989 TI - Two additional ANLL cases with chromosome 3 rearrangements involving bands q21 and q26. PMID- 8630990 TI - Down syndrome, acute lymphoblastic leukemia, and t(8;14)(q11;q32) PMID- 8630991 TI - Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer. AB - The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET 1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A 127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced. PMID- 8630992 TI - Both cell proliferation and apoptosis increase with lesion grade in cervical neoplasia but do not correlate with human papillomavirus type. AB - Recent molecular studies suggest that the expression of high-risk but not low risk human papillomavirus (HPV) oncoproteins E6 and E7 can significantly alter normal cell cycle regulation. The alterations in cell cycle regulation may be reflected by changes in the balance between cell growth and cell loss through apoptosis in cell populations expressing E6 and/or E7. We evaluated the kinetic indices of cell proliferation and apoptosis in a histopathological spectrum of cervical neoplasia and compared low-versus high-risk HPV-associated lesions. The cell proliferation index, as determined by detection of the nuclear antigen Ki67, increased with increasing lesion grade. Apoptotic cells were identified with terminal deoxynucleotidyl transferase-labeling of the 3'-hydroxyl ends of DNA nucleosomes. No apoptosis was observed in normal epithelium, and only occasional apoptotic cells were seen in low-grade lesions. However, there was a low but measurable apoptotic index in the higher grade lesions, which increased with lesion grade. There was no significant difference in the proliferative and apoptotic indices in similar grade lesions when stratified into low-versus high risk HPV types. These findings suggest that apoptosis in HPV-infected lesions correlates with proliferative activity rather than HPV type. PMID- 8630993 TI - Retinoic acid metabolism and inhibition of cell proliferation: an unexpected liaison. AB - The rationale for the use of all-trans-retinoic acid (RA) as an anticancer agent is based on its ability to inhibit growth and promote differentiation of some neoplastic cells. However, RA is not effective in all conditions of cell culture, and in some cases, it may stimulate cell growth. We used a serum-free culture system to study the effect of RA on cell proliferation. Following 2 days of RA exposure, 9 of a total of 15 cell lines showed an inhibition of cell growth (RA sensitive), while 6 of 15 cell lines showed resistance to RA (RA-resistant cells). Metabolic studies and high-performance liquid chromatography analysis of the cell-associated and medium extracts from cells incubated with [3H]RA revealed that all nine RA-sensitive cells showed a very high activity to metabolize RA to polar metabolites found in the medium. In sharp contrast, RA-resistant cells retained about 60% of the original RA at 76 h. However, conditioned medium from the sensitive cells was without activity on the growth of sensitive and resistant cells. We conclude that a relationship exists between RA inhibition of cell growth and intracellular RA metabolism. These data may help design useful strategies in cancer therapy by retinoids and dispel the notion that RA itself is responsible for the inhibition of cell growth. PMID- 8630994 TI - Suppression of carcinogenesis in the intestines of min mice by the soybean derived Bowman-Birk inhibitor. AB - We have performed experiments to determine whether the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), has the ability to affect intestinal carcinogenesis in Min mice. Min mice have an autosomally dominantly inherited predisposition to multiple intestinal neoplasms and are known to have a very high spontaneous rate of tumor development in both the small intestine and colon. BBI was administered in the diet as BBI Concentrate (BBIC), the form of BBI which is currently being evaluated in human trials as a cancer chemopreventive agent. We observed that 0.5% dietary BBIC led to a 42-50% reduction in the number of tumors/mouse in the small intestine and colon and a 41% reduction of tumorigenesis in the colon when the data are analyzed in terms of the fraction of mice bearing tumors. Thus, tumor-development in both the small intestine and colon of Min mice can be significantly suppressed by BBIC, despite the fact that the animals carry a predisposing mutation that leads to a markedly increased intestinal tumor incidence and mortality rate. PMID- 8630995 TI - Immunoperoxidase detection of 8-hydroxydeoxyguanosine in aflatoxin B1-treated rat liver and human oral mucosal cells. AB - An immunoperoxidase method using a monoclonal antiserum that recognizes 8 hydroxydeoxyguanosine has been developed for detection and quantitation of oxidative damage in single cells. The method was initially applied to cultured cells treated with H2O2 or aflatoxin B1 and then to cryostat liver sections of rats treated with aflatoxin B1. To demonstrate that the method has sufficient sensitivity for detection of damage in human samples, oral mucosal cells from a total of 12 pairs of smokers and nonsmokers were analyzed. Mean staining intensity of oral cells of smokers was 1.6-fold higher than in nonsmokers. The immunoperoxidase method, requiring a small number of cells and eliminating the need for isolation of DNA, will be useful for evaluation of oxidative damage in a wide range of biological samples. PMID- 8630996 TI - A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. AB - The p53 protein is a multifunctional transcriptional regulator involved in cellular response to DNA damage and has been implicated as a putative determinant of sensitivity of tumor cells to cytotoxic agents. Since the p53 gene becomes inactivated in over one-half of advanced ovarian carcinoma, in this study we have examined the relationships between p53 gene alterations, p53 immunoreactivity, and response to cisplatin-based chemotherapy in ovarian cancer patients. All patients had advanced (FIGO stage III or IV) ovarian carcinoma and, with one exception, were untreated at the time of collection of tumor specimens. After initial debulking surgery, patients received high-dose cisplatin therapy. Tumor samples were analyzed for p53 gene mutations and for p53 protein accumulation, and the findings were correlated with tumor responsiveness. Of the 33 tumors examined, p53 gene mutations were found in 20 cases, including 15 missense mutations, 2 deletions, 2 nonsense mutations, and a base substitution at splice site. Twenty tumors showed positive immunostaining for p53. Only missense mutations were associated with positive immunostaining. In addition, p53 overexpression was detected in five tumors in the absence of mutations. Most (12 of 14) of the missense mutations associated with p53 protein stabilization were found refractory to therapy, as well as tumors overexpressing wild-type p53 (4 of 5). A significant correlation has been found between p53 accumulation, type of mutation (i.e., missense mutations), and pathological response to cisplatin-based therapy. In conclusion, the present results are consistent with a role of p53 as a determinant of chemosensitivity of ovarian carcinoma. PMID- 8630997 TI - Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. AB - Wild-type p53 is involved in several aspects of cell cycle control and suppression of transformation, inducing either apoptosis or G1 block in cell cycle progression. Using a recombinant adenovirus containing the wild-type p53 cDNA, the biological effects of the newly expressed wild-type p53 protein were examined in six human glioma cell lines. Three cell lines (U-251 MG, U-373 MG, and A-172) expressed endogenous mutant p53, and the other three (U-87 MG, EFC-2, and D54 MG) expressed wild-type p53. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis as assessed by morphological studies using nuclear staining, electron microscopy, and flow cytometric assays. In wild-type p53 cell lines, however, the overexpression of wild-type p53 did not result in apoptosis but inhibited cellular proliferation rather drastically and modified the neoplastic phenotype. Differential effects suggest two pathways for glioma oncogenesis and a possible therapeutic strategy. PMID- 8630998 TI - Expression of rTS correlates with altered growth regulation of thymidylate synthase. AB - The recently discovered rTS gene is convergent with and overlaps the thymidylate synthase gene in the antisense orientation. K562 B1A and KB 1BT are two methotrexate-resistant cell lines that have amplified genes for dihydrofolate reductase. K562 B1A cells have increased levels of rTS mRNA and protein compared with their parental K562 cells, whereas KB 1BT cells show unaltered rTS expression compared with their parental (KB) cells. Altered levels and loss of growth-regulation of in situ thymidylate synthase activity were found in methotrexate-resistant K562 B1A cells but not in K562, KB, and KB 1BT cells. These data point to a link between rTS and the regulation of TS activity. PMID- 8630999 TI - In vitro and animal studies of sodium thiosulfate as a potential chemoprotectant against carboplatin-induced ototoxicity. AB - When carboplatin (cis-diammine-1,1-cyclobutane-dicarboxylato-platinum) delivery to brain tumors is optimized with osmotic blood-brain barrier disruption (BBBD), high frequency hearing loss can result. Treatment with sodium thiosulfate (STS) blocked carboplatin cytotoxicity against the LX-1 human small cell lung carcinoma cell line in vitro. STS decreased carboplatin-induced ototoxicity in a guinea pig model, as determined by electrophysiological measurements and analysis of inner ear outer hair cell numbers. Protection was found when STS was administered up to 8 h subsequent to carboplatin but not 24 h after carboplatin. In a rat model of osmotic BBBD, STS was neurotoxic when given immediately after BBBD but not when given 60 min after BBBD, when the barrier is reestablished. Thus, delayed administration of STS may provide a mechanism to reduce the cochlear toxicity caused by BBBD-enhanced carboplatin delivery to the brain. PMID- 8631000 TI - Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam. AB - C57BL/6J-Min/+mice (n = 56), heterozygous for a nonsense mutation in the Apc gene, were randomized at weaning to seven groups, including groups treated with piroxicam at 0, 50, 100, and 200 ppm in the AIN93G diet. After only 6 weeks of treatment, intestinal adenomas and aberrant crypt foci were counted, and serum levels of piroxicam and thromboxane B2 were quantitated. Tumor multiplicity was decreased in a dose-dependent manner from 17.3 +/- 2.7 in the control to 2.1 +/- 1.1 (12%) in the high-dose piroxicam group (P < 0.001). Thromboxane B2 levels in plasma also decreased monotonically in parallel to the decrease in tumor multiplicity, consistent with the prostaglandin inhibitory effect of piroxicam. The Min mouse model demonstrates that the nonsteroidal anti-inflammatory drug piroxicam has strong biological and therapeutic effects, potentially useful for prevention of the early adenoma stage of tumor development. PMID- 8631002 TI - Localization of an ovarian cancer tumor suppressor gene to a 0.5-cM region between D22S284 and CYP2D, on chromosome 22q. AB - The detection of loss of heterozygosity, indicative of the presence of a tumor suppressor gene, has been reported to occur frequently on chromosome 22q in human ovarian cancer. In this study, 110 sporadic ovarian tumors were analyzed using 8 polymorphic loci to define a minimum region of loss. Fifty-eight (53%) tumors showed loss of heterozygosity, and of these 6 exhibited partial loss, enabling the identification of two candidate tumor suppressor gene loci. One region, of less than 0.5 cM, is flanked by D22S284 and CYP2D, and a second region lies distal to D22S276. Analysis of loss of heterozygosity with respect to grade and stage suggests that chromosome 22q loss of heterozygosity is of more relevance in tumor progression rather than initiation. PMID- 8631001 TI - Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma. AB - The efficacy of combination therapy including an oral gelatinase inhibitor (CT1746) and cytotoxic agent was analyzed using the murine Lewis lung carcinoma model. Primary tumors, pulmonary metastases, and sera from tumor-bearing animals had increased gelatinase B activity that was inhibited by CT1746 levels achievable in vivo. The combination of CT1746 and cyclophosphamide (CTX) was significantly more effective than either single agent in delaying local tumor growth (CT1746/CTX, 30.9 +/- 1.7 days; CT1746, 2.6 +/- 0.3 days; CTX, 19.5 +/- 1.1 days; P < .001) and reducing the number and size of pulmonary metastases [CT1746/CTX, 5 +/- 2 (15% metastases > 3 mm); CT1746, 15 +/- 4 (55% > 3 mm); CTX, 11 +/- 3 (63% > 3 mm); no treatment, 24 +/- 5 (62% > 3 mm); P < .001]. These data support the notion of combining matrix metalloproteinase inhibitors and cytotoxic agents to treat certain epithelial malignancies. PMID- 8631003 TI - Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B. AB - The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms. PMID- 8631004 TI - The t(6;16)(p21;q22) chromosome translocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene. AB - Very little is known about the molecular and genetic mechanisms involved in prostate cancer. Previous studies have shown frequent loss of heterozygosity (40%) at chromosomal regions 8p, 10q, and 16q, suggesting the presence of tumor suppressor genes in these regions. The LNCaP cell line, established from a metastatic lesion of human prostatic adenocarcinoma, carries a t(6;16)(p21;q22) translocation. To determine whether this translocation involved genes important in the process of malignant transformation, we cloned and sequenced the t(6;16) breakpoint of this cell line. Sequence analysis showed that the breakpoint is within the haptoglobin gene cluster on chromosome 16, and that, on chromosome 6, the break occurs within a novel gene, tpc, similar to the prokaryotic S10 ribosomal protein gene. The translocation results in the production of a fusion transcript, tpc/hpr. PMID- 8631005 TI - G1 delay in cells overexpressing prostaglandin endoperoxide synthase-2. AB - Colorectal cancer is the second leading cause of death from cancer in the United States. Continuous use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to reduce the risk of colorectal cancer in humans by 40 50%. Patients with familial adenomatous polyposis who take NSAIDs, such as sulindac, undergo a regression of intestinal adenomas. Rodents exposed to carcinogens that cause colon cancer have a 50-60% reduction in the size and number of colonic tumors when treated continuously with NSAIDs. One common target for these drugs is prostaglandin endoperoxide synthase, also referred to as cyclooxygenase (COX). We and others have shown recently that COX-2 levels are increased dramatically in 85-90% of human colorectal adenocarcinomas and in 40 50% of colonic adenomas. We prepared intestinal epithelial cells that express the COX-2 gene permanently and found that they have altered adhesion properties and resist undergoing apoptosis. We report here that these cells also have a 3-fold increase in the duration of G1, lower levels of cyclin D1 protein, and a marked decrease in retinoblastoma kinase activity associated with cyclin-dependent kinase 4. The delay in G1 transit may relate to the resistance of these cells to undergo programmed cell death, which could affect their tumorigenic potential. PMID- 8631006 TI - Frequent loss of heterozygosity in region of the KIP1 locus in non-small cell lung cancer: evidence for a new tumor suppressor gene on the short arm of chromosome 12. AB - To refine the chromosomal localization of a putative tumor suppressor gene, we analyzed the loss of heterozygosity (LOH) of chromosome 12 in 36 primary non small cell lung cancer (NSCLC) samples with matched normal DNA using 22 highly informative polymorphic markers. Twelve cases showed LOH at one or more loci on chromosome 12. LOH of chromosome arm 12p was more frequent in large cell carcinoma than squamous cell carcinoma, indicating molecular genetic heterozygosity within the major NSCLC subtypes. We identified the smallest commonly deleted region on chromosome 12p13. This region is flanked by D12S269 and D12S308, including the KIP1 gene. Mutational analysis of KIP1 using PCR single strand conformation polymorphism and Southern Blot analysis showed no homozygous deletions, rearrangements, or point mutations, suggesting that the altered gene in this region is not the KIP1 gene. These data suggest that a new tumor suppressor gene which is involved in tumorigenesis of NSCLC is in the region of KIP1. PMID- 8631007 TI - Refinement of two chromosome 11q regions of loss of heterozygosity in ovarian cancer. AB - Loss of heterozygosity on chromosome 11q23.3-qter is a frequent event in ovarian carcinoma, implying the existence of an important ovarian tumor suppressor gene(s) within the region. To refine a minimum region(s) of loss, 67 ovarian tumors were analyzed for loss of heterozygosity with eight microsatellite markers spanning 11q23.3-qter. Forty tumors (61%) demonstrated allelic losses. Twenty seven of these had allelic losses on only part of 11q23.3, which enabled the identification of two distinct regions likely to harbor ovarian tumor suppressor genes. The proximal region, flanked by markers D11S925 and D11S1336, is less than two megabases while the second more distal region, flanked by markers D11S912 and D11S439, is approximately eight megabases. The refinement of these candidate tumor suppressor gene loci will facilitate future loss of heterozygosity studies and enable the isolation of candidate genes from these regions. PMID- 8631008 TI - Fatty acid synthase (FAS): a target for cytotoxic antimetabolites in HL60 promyelocytic leukemia cells. AB - Many human cancers express elevated levels of fatty acid synthase (FAS), with correspondingly increased fatty acid synthesis and abnormal fatty acid utilization. Recent studies have shown that the FAS inhibitor, cerulenin, is selectively cytotoxic to cell lines derived from human malignancies, suggesting that those carcinoma cells are dependent upon endogenous fatty acid synthesis for growth. These data further suggest that the fatty acid synthesis pathway is a potential target for chemotherapy development. The present studies demonstrate that cerulenin cytotoxicity is mediated by fatty acid pathway inhibition. Proliferating HL60 promyelocytic leukemia cells express high levels of FAS mRNA and protein and synthesize fatty acid predominantly for membrane phospholipid. Following exposure to 12-O-tetradecanoylphorbol-13-acetate, the FAS expression in HL60 cells is abolished, fatty acid synthesis diminishes, and the cells become insensitive to cerulenin while acquiring a differentiated, macrophage-like phenotype. HL60 cells adapted to growth in serum- and fatty acid-free medium show a dose-dependent sensitivity to cerulenin, which is reversed by palmitate, the major product of FAS, indicating that cerulenin cytotoxicity is mediated through fatty acid starvation. Cells grown in the presence of exogenous fatty acid partially downmodulate FAS expression and increase mean cell volume (phospholipid mass/cell) but retain their sensitivity to cerulenin, which is reversed by 3-fold excess oleate supplementation. These results demonstrate that malignant cells can retain dependence on endogenous fatty acid synthesis and sensitivity to FAS inhibitors in the presence of physiological fatty acid levels and thus support the notion that FAS inhibitors may be useful in treating cancer in vivo. PMID- 8631009 TI - Serum deprivation, but not inhibition of growth per se, induces a hypermutable state in Chinese hamster G12 cells. AB - Spontaneous mutagenesis is thought to play a crucial role in spontaneous carcinogenesis. We recently described a new mathematical model for estimation of the spontaneous mutation rate (mutation/gene/generations) based on the assumption that mutations are fixed in the S-phase of the cell cycle. With this definition, the spontaneous mutation rate should be independent of the growth rate. In the present study, we tested this hypothesis, using cell line G12, a transgenic Chinese hamster V79 derivative, which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis. The growth rate was modulated by varying the serum concentration or the seeding density, or by addition of suramin, transforming growth factor beta, or dichlorobenzimidazole riboside to the medium. Significant increases in the spontaneous mutation rate occurred when cell proliferation was blocked by serum deprivation. Density-dependent inhibition of growth and inhibition of growth by suramin, transforming growth factor beta, or dichlorobenzimidazole riboside did not result in significant increases in spontaneous mutation rates. The level of oxidants in cells cultivated in the presence of low concentrations of serum was higher compared to control cells, suggesting that the increases in the spontaneous mutation rates under low serum conditions may be partly a result of oxidative stress due to a lack of serum antioxidants. This was shown to be the case, because spontaneous mutation rates were significantly reduced in serum-depleted cells when antioxidants were added to the medium. We suggest that during carcinogenesis, when tumors are in a prevascularized state, the spontaneous mutation rate may be elevated, and this process may contribute to the genetic instability of the tumor cells. PMID- 8631010 TI - Carcinogenesis in human skin grafted to SCID mice. AB - To directly examine a multistage carcinogenesis model and the role of UV light in human tissues, we grafted human skin onto mice with severe combined immunodeficiency disease. We found that the maximum dose of UV radiation in the B range (UVB; 280-320 nm) tolerated by these grafts was 500 J/m2 three times weekly. One hundred fifty-one grafted mice were then randomized and observed for a median of 9 months in five groups: no treatment, chemical initiation alone, UVB as a complete carcinogen, initiation plus UVB promotion, and initiation plus UVB and phorbol ester promotion. Actinic damage and squamous atypia were found in grafts of all groups receiving UV treatment; unequivocal human squamous carcinomas developed in two of these. Species origin was verified by human specific bisbenzimide staining and in situ hybridization for human-specific Alu segment. Overall basal proliferation, measured immunohistologically, was reduced in UV-treated grafts, but foci of hyperproliferation were seen in conjunction with the dedifferentiated expression of cytokeratins 1, 10 and 5, 8. Murine tumors also developed frequently, confirming the biological relevance of the carcinogenic strategies tested. These findings demonstrate that development of malignant human tumors can be experimentally accelerated in human tissue. PMID- 8631011 TI - Reexpression of thyroid peroxidase in a derivative of an undifferentiated thyroid carcinoma cell line by introduction of wild-type p53. AB - Loss of function of p53 is believed to result in transformation through impairment of its properties as a transcription factor, which interferes with the regulation of the cell cycle and under certain conditions, with programmed cell death. We report that stable transfection of clonal undifferentiated thyroid carcinoma cell lines harboring endogenous p53 mutations with a wild-type p53 expression vector only rarely yields transfectants expressing authentic wild-type p53. Among these, most exhibited an increase in doubling time and an impairment of colony formation in soft agar. Only one clonal wild-type p53-overexpressing derivative of the NPA papillary carcinoma cell line was obtained, and these cells were found to reexpress thyroid peroxidase (TPO). This clone also demonstrated reexpression of the paired box domain transcription factor Pax-8, which specifically activates transcription of TPO. Wild-type p53 did not directly stimulate transcriptional activity of a TPO promoter construct. Although the low frequency of authentic wild-type p53 stable transfectants limits the power of this analysis, these data suggest that in addition to its role in malignant transformation, p53 may be significant in the determination or maintenance of cell differentiation in thyroid neoplasms. PMID- 8631012 TI - Chemopreventive efficacy of arylalkyl isothiocyanates and N-acetylcysteine for lung tumorigenesis in Fischer rats. AB - The purpose of this study is to evaluate the efficacy of three promising sulfur containing compounds, 6-phenylhexyl isothiocyanate (PHITC), phenethyl isothiocyanate (PEITC), and N-acetylcysteine (NAC), as chemopreventive agents in a long-term bioassay for lung tumorigenesis in F344 rats. PEITC occurs as a constituent of certain cruciferous vegetables, PHITC is a synthetic homologue, and NAC is an endogenous substance. Male F344 rats were treated with the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by s.c. injection at a dose of 1.5 mg/kg body weight three times weekly for 20 weeks. This dose regimen induced a 67% tumor incidence in the lung, a major target organ of NNK. PHITC or PEITC administered in the diet for 22 weeks, a period covering from 1 week before to 1 week after the NNK treatment, exhibited significant inhibition of lung tumorigenesis induced by NNK. The lung tumor incidences in the NNK-treated groups, fed a diet containing 4 mmol/kg (876 ppm) or 2 mmol/kg (438 ppm) PHITC, were 24 and 19% and were 9 and 17% in groups fed PEITC at concentrations of 8 mmol/kg (1304 ppm) or 4 mmol/kg (652 ppm), respectively. In contrast to isothiocyanates, NAC given in the diet at 80 mmol/kg (13056 ppm) or 40 mmol/kg (6528 ppm) exerted no inhibitory effects on the NNK-induced lung tumorigenesis. At the dose studied, NNK did not induce liver and pancreatic tumors in the treated animals, but a significant increase of nasal cavity tumor incidence was observed in the NNK-treated group. However, none of the test compounds showed any effect on the tumorigenesis in this tissue. This study demonstrated that PHITC and PEITC were potent chemopreventive agents for the NNK induced lung tumorigenesis in F344 rats, whereas NAC was not active at all. These results support further evaluation of these compounds in chemoprevention studies. PMID- 8631013 TI - High levels of AP-2-binding activity in cell lines infected with human T-cell leukemia virus type I: possible enhancement of AP-2 binding by human T-cell leukemia virus type I tax. AB - Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Recently, the transcription factor AP-2 has been demonstrated to be capable of activating gene expression from HTLV-I long terminal repeat. To determine whether changes occur in the levels of AP-2-binding activity in HTLV-I-infected cell lines, we compared the levels of AP-2 binding of nuclear extracts obtained from HTLV-I-infected T-cell lines with those of nuclear extracts obtained from uninfected T-cell lines. High levels of AP-2-binding activity were observed in HTLV-I-infected cell lines (MT-2, HUT-102, and SLB-1) using the mobility shift assay. In contrast, in the uninfected cell lines (Jurkat, HUT-78, and MLA 144), AP-2-binding activity was obviously low compared with that in the HTLV-I-infected cell lines. HTLV-I transactivator protein tax activates expression of both viral and cellular genes. We have demonstrated further that introduction of the tax gene into Jurkat cells stimulated AP-2-binding activity. These results indicate that HTLV-I-infected T cells exhibit constitutive AP-2-binding activity, and that tax may increase the DNA binding activity of AP-2. PMID- 8631014 TI - Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine. AB - We tested the hypothesis that the level of the DNA repair protein O6-alkylguanine DNA alkyltransferase in brain tumors was correlated with resistance to carmustine (BCNU) chemotherapy. Alkyltransferase levels in individual cells in sections from 167 primary brain tumors treated with BCNU were quantitated with an immunofluorescence assay using monoclonal antibodies against human alkyltransferase. Patients with high levels of alkyltransferase had shorter time to treatment failure (P = 0.05) and death (P = 0.004) and a death rate 1.7 times greater than patients with low alkyltransferase levels. Furthermore, the size of the subpopulation of cells with high levels of alkyltransferase was correlated directly with drug resistance. For all tumors the variables most closely correlated with survival, in order of importance, were age, tumor grade, and alkyltransferase levels. For glioblastoma multiforme, survival was more strongly correlated with alkyltransferase levels than with age. These results should encourage prospective studies to evaluate alkyltransferase levels as a method, for identifying brain tumor patients with the best likelihood of response to BCNU chemotherapy. PMID- 8631015 TI - Synergism between cisplatin and topoisomerase I inhibitors, NB-506 and SN-38, in human small cell lung cancer cells. AB - Topoisomerase I-targeting anticancer agents such as 7-ethyl-10-[4-(1-piperidyl)-1 piperidyl]carbonyloxy-camptothecin (CPT-11) and 6-N-formylamino-12,13-dihydro 1,11-dihydroxy-13-(beta-D- glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4 c]carbazole-5,7(6H)-di one (NB-506) have been developed and show strong antitumor activity against various cancers. We examined the interaction of these drugs and cisplatin (CDDP), and biochemical mechanisms of synergism between them. Interaction of drugs in human small cell lung cancer cells, SBC-3, was analyzed using the isobologram method. Combinations of CDDP with NB-506, CPT-11, and an active metabolite of CPT-11, 7-ethyl-10-hydroxy-CPT (SN-38), showed synergistic effects. Formation of DNA interstrand cross-links (ICLs) on the cells was analyzed using an alkaline elution assay and increased ICLs were observed by simultaneous exposure to CDDP (1.5 microM) and NB-506 (10 nM) compared with that in response to CDDP alone. DNA repair after ICL formation induced by 3-h exposure to CDDP (1.5 microM) was reduced by NB-506 (10 nM) exposure. On the other hand, a higher concentration of CDDP (150 microM) enhanced the topoisomerase I inhibitory activity of NB-506 and SN-38 determined by relaxation of supercoiled Escherichia coli DNA. These biological interactions might result in synergistic interactions between CDDP and NB-506 or SN-38. Topoisomerase I inhibitors and CDDP may be a key regimen for cancer chemotherapy and merit further examination. PMID- 8631016 TI - Cytotoxic effects of gemcitabine-containing regimens against human non-small cell lung cancer cell lines which express different levels of p185neu. AB - A novel pyrimidine analogue, gemcitabine, has been found to inhibit DNA replication and repair. We speculated that gemcitabine in combination with DNA damaging agents might be more active against high- than low-p185neu expressing non-small cell lung cancer (NSCLC) cells because the high-p185neu expressors were proposed to posses a more effective DNA repair ability. We therefore compared the combination effects of gemcitabine plus cisplatin, gemcitabine plus etoposide, and cisplatin plus etoposide in a panel of 12 NSCLC cell lines. We also investigated the correlations between the level of p185neu and the cytotoxicities of each single agent and the three combinations. We found that as single agents the cytotoxicities of cisplatin and etoposide but not gemcitabine were significantly correlated with the level of p185neu. In contrast to the tight cross-resistance between cisplatin and etoposide, gemcitabine demonstrated little cross-resistance to either etoposide or cisplatin. Both gemcitabine-containing combinations demonstrated equivalent or more active cytotoxicities compared to cisplatin plus etoposide, with gemcitabine plus cisplatin showing a greater synergistic activity which was effect (dose) dependent. The effect of cisplatin plus etoposide was not p185neu related, whereas gemcitabine-containing regimens, especially gemcitabine plus cisplatin, had a greater cytotoxicity against the high- than the low-p185neu expressors. Our findings indicate that gemcitabine in combination with cisplatin is active against NSCLC cells in vitro. The gemcitabine-cisplatin interaction is more active than the etoposide-cisplatin interaction in cells with high-p185neu expression. PMID- 8631017 TI - Preclinical pharmacology of the natural product anticancer agent bryostatin 1, an activator of protein kinase C. AB - Bryostatin 1, a natural product anticancer agent isolated from a marine bryozoan, has been shown in tissue culture to activate protein kinase C. This agent has recently undergone Phase I testing in humans given either as a bolus i.v. injection or a continuous infusion. To understand how bryostatin 1 might be used best as an anticancer agent, a study of the pharmacokinetics, tissue distribution, metabolism, and elimination of bryostatin 1 in mice was undertaken, using [C26-3H]-labeled bryostatin 1. Following i.v. administration, the plasma disappearance curve for bryostatin 1 could be described by a two-compartment model, with half-lives of 1.05 and 22.97 h, respectively. In contrast, the plasma disappearance curve for bryostatin 1 administered i.p. was better described by a first order absorption one-compartment model, with an absorption half-life of 0.81 h and an elimination half-life of 28.76 h, respectively. The majority of radioactivity in plasma was associated with the intact drug for up to 24 h after dosing. In the first 12 h after i.v administration, urinary excretion represented the major pathway of elimination, with 23.0 +/- 1.9% (mean +/- SD) of the administered dose excreted. Within 72 h after i.v. administration, approximately equal amounts of radioactivity (40%) were excreted in feces compared to urine. Bryostatin 1 was widely distributed in many organs but concentrated in the lung, liver, gastrointestinal tract, and fatty tissue. The concentration in the gastrointestinal tract, along with the fecal excretion, suggests the possibility of enterohepatic circulation of this drug. In summary, this study demonstrates that bryostatin 1 is relatively stable in vivo, widely distributed but concentrated in some major tissues, and rapidly excreted first through urine and at later times through the feces. The data from this animal study should be useful in the design of future human trials with this anticancer drug. PMID- 8631018 TI - Preparation of a trivalent antigen-binding construct using polyoxime chemistry: improved biodistribution and potential for therapeutic application. AB - In an attempt to improve the pharmacokinetic behavior of an antitumor radioimmunoconjugate, we have prepared a trivalent antigen-binding construct formed from three Fab' fragments derived from the parent murine monoclonal antibody (MAb) 35 directed against the carcinoembryonic antigen. The construct was generated by a novel approach using polyoxime chemistry. This approach leads to a homogeneous construct, as judged by SDS-PAGE and by mass spectrometry, which was found to retain full immunoreactivity. A comparison of the monovalent, divalent, and trivalent F(ab')n materials in vitro revealed the expected trend of increasing association constant with increasing valency. The in vivo biodistribution of the 125I-labeled trivalent construct was studied in xenograft bearing nude mice. Absolute tumor accumulation seen with the trivalent construct (10.8% injected dose/g) was lower than that seen with the intact MAb35 (15.2% injected dose/g). This finding and the more rapid loss of activity from tumor are presumably the consequence of the quicker blood clearance of the trivalent material. However, the construct showed tumor:blood ratios up to 10-fold higher than those seen for the parent antibody, and ratios of tumor:normal tissue accumulation were generally greatly improved. These improvements were achieved despite only modest reduction in maximum tumor accumulation when compared to the parent MAb35, and this augurs well for an improved potential for this novel construct as an agent for radioimmunotherapy and radioimmunoscintigraphy. PMID- 8631019 TI - Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. AB - Paclitaxel at low concentrations (10 nM for 20 h) induces approximately 90% mitotic block at the metaphase/anaphase transition in HeLa cells, apparently by suppressing dynamics of spindle microtubules (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993). It is not known, however, whether inhibition of mitosis by such low paclitaxel concentrations results in cell death. In the present work, we found that after removal of paclitaxel (10 nM-1 microM), blocked cells did not resume proliferation. Instead, cells exited mitosis abnormally within 24 h. They did not progress through anaphase or cytokinesis but entered an interphase-like state (chromatin decondensed, and an interphase-like microtubule array and nuclear membranes reformed). Many cells (> or = 55%) contained multiple nuclei. Additional DNA synthesis and polyploidy did not occur. DNA degradation into nucleosome-sized fragments characteristic of apoptosis began during drug incubation and increased after drug removal. Cells died within 48-72 h. Incubation with paclitaxel (10 nM for 20 h) resulted in high intracellular drug accumulation (8.3 microM) and little efflux after paclitaxel removal; intracellular retention of paclitaxel may contribute to its efficacy. The results support the hypothesis that the most potent chemotherapeutic mechanism of paclitaxel is kinetic stabilization of spindle microtubule dynamics. PMID- 8631020 TI - Selective loss of human leukocyte antigen class I allele expression in advanced renal cell carcinoma. AB - The expression of human leukocyte antigen (HLA) class I alleles was analyzed in 65 renal cell carcinomas using one-dimensional isoelectric focusing. Normal organ tissue and peripheral blood lymphocytes were used as controls. The patients were serologically typed using the standard microcytotoxicity test. Forty-two patients were staged as pT1 or pT2, and 23 patients had advanced tumor stages (pT3/pT4). In all cases the HLA-A,B phenotypes were confirmed using one-dimensional isoelectric focusing. The expression of HLA expression was reduced in two tumors [1 x HLA-A1(pT2); 1 x HLA-A28 (pT2)]. In three carcinomas the expression of HLA A1 was lost. One tumor showed a combined loss of HLA-A2 and HLA-B38. These selective losses occurred in tumor stage pT3 (n = 1) or pT4 (n = 3; P = 0.013, Fisher's exact test). This leads to the conclusion that the loss of HLA expression is predominantly present in advanced tumor stages. PMID- 8631021 TI - Neutral endopeptidase: variable expression in human lung, inactivation in lung cancer, and modulation of peptide-induced calcium flux. AB - Neutral endopeptidase (NEP; CALLA, CD10, EC 3.4.24.11) is a cell surface endopeptidase that hydrolyses bioactive peptides, including the bombesin-like peptides, as well as other neuropeptides. Bombesin-like peptides and other neuropeptides are autocrine growth factors for both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Low expression of NEP has been reported in SCLC and NSCLC cell lines. NEP inhibition has been shown to increase proliferation in one cell line. To date, NEP expression has not been quantitatively evaluated in normal adult lung, SCLC or NSCLC tumors, paired uninvolved lung from the same patient, or in other pulmonary neoplasms such as mesotheliomas and carcinoids. We examined the expression of NEP in these tissues and human cell lines using immunohistochemistry, flow cytometry, enzyme activity, ELISA, Western blot, and reverse transcription (RT)-PCR. Uninvolved lung tissue from different individuals displayed considerable variation in NEP activity and protein. By immunohistochemistry, NEP expression was detectable in alveolar and airway epithelium, fibroblasts of normal lung, and in mesotheliomas, whereas it was undetectable in most SCLC, adenocarcinoma, squamous cell carcinoma, and carcinoid tumors of the lung. NEP activity and protein levels were lower in all SCLC and adenocarcinoma tumors when compared to adjacent uninvolved lung, often at levels consistent with expression derived from contaminating stroma. NEP expression and activity were reduced or undetectable in most SCLC and lung adenocarcinoma cell lines. NEP mRNA by RT-PCR was not expressed or was in low abundance in the majority of lung cancer cell lines. The majority of lung tumors did not express NEP by RT-PCR as compared with normal adjacent lung. In addition, recombinant NEP abolished, whereas an NEP inhibitor potentiated, the calcium flux generated by neuropeptides in some lung cancer cell lines, demonstrating potential physiological significance for low NEP expression. NEP, therefore, is a signal transduction and possibly a growth modulator for both SCLC and NSCLC, emphasizing the role of neuropeptides in the pathogenesis of the major histological forms of lung cancer. PMID- 8631022 TI - An abnormality in the p53 pathway following gamma-irradiation in many wild-type p53 human melanoma lines. AB - DNA-damaging agents such as ionizing radiation (IR) activate the tumor suppressor p53, and, in turn, p53 transactivates a number of downstream effector genes such as GADD45, CIP1/WAF1, and MDM2. The induction of these downstream genes following IR appears to be strictly dependent upon the presence of wild-type functional p53 known to evoke G1 arrest. In this study, we characterized 56 cell lines from 9 different tumor types with predetermined p53 genotype by measuring the induction of GADD45, CIP1/WAF1, and MDM2 relative mRNA levels after IR. A higher fraction of melanoma lines had wild-type (wt) p53 (5/8, or 63%) compared to the nonmelanoma lines (11/48, or 23%). Most wt p53 (nonmelanoma) cell lines (11/12, or 92%) showed clear induction of both GADD45 and CIP1/WAF1. On the other hand, many wt p53 melanoma lines (4/5, or 80%) showed normal induction of CIP1/WAF1, but little or no induction of GADD45. Despite this defect in GADD45 induction, we found that all wt p53 melanoma lines exhibited strong G1 arrest and increased levels of p53 protein after IR. The results demonstrated that radiation-induced G1 arrest could occur by the p53-CIP1/WAF1 pathway without appreciable induction of GADD45 in melanoma lines. Time course experiments demonstrated prolonged induced expression of CIP1/WAF1 mRNA transcripts in melanoma lines in which GADD45 induction was lacking, suggesting some sort of compensatory mechanism involving CIP1/WAF1, in cell lines with defective GADD45 induction. We could reproduce this compensatory effect in RKO colon carcinoma cells in which GADD45 expression was blocked by constitutive antisense vectors. These findings reveal that defective induction of GADD45 following IR is common in human melanoma cell lines. PMID- 8631024 TI - Karyotypic comparisons of multiple tumorous and macroscopically normal surrounding tissue samples from patients with breast cancer. AB - Many tumor tissues are made up of genetically different cell populations, and the study of the causes and consequences of this heterogeneity must play a central role in cancer research. We have studied breast cancer clonal heterogeneity by cytogenetic analysis of 4123 cells from 52 successfully short-term-cultured tumorous, metastatic, and macroscopically normal breast tissue samples from 6 women with this disease. All 7 carcinomas (one woman had bilateral disease) contained 1 to 9 karyotypically related as well as unrelated clones, unevenly distributed among the tumor quadrants. Two clonal chromosome abnormalities were recurrent: interstitial 3p deletions were found in 5 carcinomas, whereas del(1)(q42) was detected in another 2 tumors. Both successfully analyzed metastatic lesions (one axillary lymph node and one metastasis in the subcutis) contained only one of several clones present in the primary tumor, thus exemplifying a reduction in overall karyotypic complexity during carcinoma spreading. In the case with the cytogenetically abnormal lymph node, another karyotypically unrelated clone was found to invade locally in the surrounding breast; also, histological evidence of carcinoma infiltration was seen in these tissue samples. In none of the other cases were clonal karyotypic changes found in macroscopically normal, extratumorous breast tissue. We conclude that a large proportion of breast carcinomas are polyclonal with cytogenetically distinct cell subpopulations expanding within separate domains of the growing tumor. Karyotypically disparate neoplastic cells may have different capacities to display malignancy-specific features (e.g., to grow invasively and set up distant metastases). It is presumed that their synergetic action is required for the full blown carcinoma phenotype. PMID- 8631023 TI - Inactivation of the P16INK4/MTS1 gene by a chromosome translocation t(9;14)(p21 22;q11) in an acute lymphoblastic leukemia of B-cell type. AB - We have reported previously a preliminary study of a t(9;14)(p21-22; q11) in B cell acute lymphoblastic leukemia. This translocation had rearranged the TCRA/D locus on chromosome band 14q11 and the locus encoding the tumor suppressor gene P16INK4/MTS1 (P16) on band 9p21 (D. Duro et al., Oncogene, 11: 21-29, 1995). In the present report, the breakpoints were precisely localized on each chromosome partner. On the 14q- derivative, the sequence derived from chromosome 9 was interrupted at 1.0 kb upstream of the first exon of P16, close to a consensus recombination heptamer, CACTGTG. In addition, the chromosome 14 breakpoint was localized at the end of the TCRD2 (delta 2) segment, and 22 residues with unknown origin were present at the translocation junction. On the 9p+ derivative, chromosome 9 sequences were in continuity with those displaced onto chromosome 14, and the 14q11 breakpoint was located within TCRJA29 segment. These features are consistent with aberrant activity of the TCR gene recombinase complex. Although all three coding exons of P16 were displaced onto the chromosome 14q derivative, no P16 transcript was detected in the leukemic cells. Because the region spanning the P16 exon 1 was not inactivated by methylation and because the other P16 allele was deleted, the implication is that the chromosome breakpoint was likely to disrupt regulatory elements involved in the normal expression of the gene. As a whole, then, our results show that translocations affecting band 9p21 can participate to the inactivation of P16, thus justifying a systematic survey of translocations of the 9p21 band in acute lymphoblastic leukemia. PMID- 8631025 TI - Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. AB - In this report, we describe a novel cDNA isolated from a primary human breast adenocarcinoma and differentially expressed in several breast carcinoma cell lines. The protein encoded by this cDNA, which we have named mammaglobin, is homologous to a family of secreted proteins that includes rat prostatic steroid binding protein subunit C3, human Clara cell 10-kilodalton protein, and rabbit uteroglobin. Expression of the mammaglobin gene is restricted to the adult mammary gland. More significantly, in an analysis of 35 breast tumor biopsies, mammaglobin mRNA levels were increased at least 10-fold relative to normal breast tissue in 23% of cases. The breast-specific expression of this potentially secreted protein and its frequent overexpression in primary human breast tumors suggest that mammaglobin may be a novel marker for the management of breast cancer. PMID- 8631026 TI - Apoptotic death of human leukemic cells induced by vascular cells expressing nitric oxide synthase in response to gamma-interferon and tumor necrosis factor alpha. AB - The host defense against tumor cells is in part based on the production of nitric oxide (NO) by activated macrophages. However, cells of the blood vessels can also participate in antitumor defense responses. They produce NO either constitutively [endothelial cells (ECs)] or after stimulation by proinflammatory cytokines (ECs and vascular smooth muscle cells). We have used a tumor cell-vascular cell coculture system to evaluate whether vascular cells can mediate cytotoxic effects on tumor cells. Treatment with IFN-gamma and tumor necrosis factor-alpha induced death of human erythroleukemic K562 cells cocultured with rodent vascular smooth muscle cells or ECs. The synergistic antitumor activity of the two cytokines depended on de novo gene expression of the inducible isoform of NO synthase and on synthesis of reactive nitrogen intermediates (RNIs) in the vascular cells. K562 cells did not produce any appreciable levels of NO, but they were targeted by RNIs released from the cytokine-stimulated vascular cells, as demonstrated by electron paramagnetic resonance spectrometry, which showed formation of nonheme iron-nitrosyl complexes in the tumor cells. Assays for mitochondrial respiration demonstrated that the tumor cells suffered from a block of the complexes I and II of the mitochondrial respiratory chain. Further analysis of the cytotoxic mechanism by fluorescent microscopy, flow cytometry, and DNA electrophoresis revealed that K562 cells attacked by NO-producing vascular cells underwent apoptosis with plasma membrane blebbing, cell volume reduction, condensation of cytoplasm and chromatin, and fragmentation of genomic DNA at internucleosomal sites. In contrast, only a few vascular cells exhibited these apoptotic changes, suggesting that these cells resist the RNI attack. Inhibition of NO production in vascular cells by NG-monomethyl-L-arginine, an inhibitor of NO synthases, significantly reduced the death of the K562 cells. These observations suggest that vascular cells induce apoptotic death of tumor cells by producing RNIs in response to cytokine stimulation. PMID- 8631027 TI - Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials. AB - B16-F10 and B16-BL6 are B16 mouse melanoma sublines that preferentially metastasize to the lung following i.v. and s.c. injections, respectively. To study molecular mechanisms underlying the different metastatic behaviors exhibited by the B16 melanoma sublines, we performed differential hybridization of the genes transcribed in these cells and compared their expression levels. We isolated four genes that were highly expressed in B16-F10 cells but not in B16 BL6 cells: TI-225 (polyubiquitin), TI-229 (pyruvate kinase), TI-241 (LRF-1 homologue), and TI-227 (novel gene). Triosephosphate isomerase, 10 formyltetrahydrofolate dehydrogenase, tyrosinase-related protein 2, cytochrome c oxidase, ATP synthetase alpha subunit, RNA helicase, and ribosomal protein (L37, J1, acidic phosphoprotein), however, showed higher expression in B16-BL6 cells than in B16-F10 cells. Among these clones, transfection of TI-241 into the low metastatic clone F1 converted the parental cells from low- into high-metastatic cells. TI-241 may regulate the expression of various genes as a transcription factor in the complex process of metastasis. PMID- 8631028 TI - Gastrimmune raises antibodies that neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer. AB - The effect of gastrin neutralization was evaluated on the in vivo growth of the rat colon line, DHDK12, which expressed cholecystokinin B/gastrin receptors and secreted glycine-extended gastrin-17 (G17). Gastrin neutralization was achieved by administration of the immunogen, Gastrimmune, which is composed of the amino terminal portion of G17 linked to a diphtheria toxoid. A rat-specific version of Gastrimmune was used to preimmunize rats, with control animals receiving diphtheria toxoid only. The antibodies raised neutralized both carboxy-amidated and glycine-extended G17. The tumor was implanted into the muscle layer of the abdominal wall, and rats immunized with Gastrimmune had significantly reduced median cross-sectional tumor areas (70.2% reduction; P = 0.005) and weights (56.5% reduction; P = 0.0078)) when compared to control rats. Histological analysis revealed that the tumors had an enhanced degree of necrosis, with the area of viable tumor in the Gastrimmune-immunized rat reduced to 40.3% compared to 58.6% in the control rats (P = 0.003). Immunization with Gastrimmune raised antibodies that inhibited the growth of a rat colon tumor. This could have been mediated by neutralization of both serum G17 and cell-associated precursor gastrin molecules. PMID- 8631029 TI - Interleukin 1 alpha and gamma-interferon induction of nitric oxide production from murine tumor-derived endothelial cells. AB - The role of nitric oxide (NO) in vascular function, host tumoricidal activity, and antiinflammatory effects is well documented. A number of cytokines induce NO from a variety of cell types. We have demonstrated in murine models that interleukin 1 alpha (IL-1 alpha) induces acute hemorrhagic necrosis, microvascular injury, and enhanced clonogenic tumor cell kill. Effects on the vasculature are observed only in tumor and not in normal tissues. Using methods established previously in our laboratory, murine tumor-derived and normal endothelial cells were cultured with IL-1 alpha, IFN-gamma, or IL-1 alpha/IFN gamma at various doses with NO production quantitated through the measurement of nitrite by the Griess reaction. In tumor-derived endothelial cells, we demonstrated that neither cytokine alone was capable of inducing nitrite but that the combination of IL-1 alpha/IFN-gamma induced dose-dependent nitrite, with peak levels observed after 4 days incubation. When tumor-derived, normal yolk sac, mouse brain, or mouse aortic endothelial cells were treated with IL-1 alpha (100 units/ml)/IFN-gamma (10 units/ml), tumor-derived endothelial cells produced significantly more nitrite when compared to the normal endothelial cells. Nitrite production from IL-1 alpha/IFN-gamma was sensitive to the nitric oxide synthase inhibitors, NG-methyl-L-arginine or NG-nitro-L-arginine in a dose-dependent manner. In addition, dexamethasone significantly inhibited nitrite production from IL-1 alpha/IFN-gamma-treated, tumor-derived endothelial cells. These studies suggest that the antitumor activity of IL-1 alpha may be mediated through the production of NO from tumor-derived endothelial cells. PMID- 8631030 TI - Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. AB - Many tumor types have p53 and/or RB mutations, and it is unclear what role the mutations of these tumor suppressor genes have on the efficacy of chemotherapeutic agents. The effect of p53 and RB inactivation on sensitivity to chemotherapeutic drugs was examined using a model system in which p53 or RB was inactivated in normal human foreskin fibroblasts (HFFs) by acute expression of human papillomavirus (HPV) 16E6 or 16E7. Cytotoxicity assays showed that HFFs expressing HPV 16E6 were 6- to 9-fold more sensitive to the DNA crosslinkers cisplatin and carboplatin and 7.8- to 11.5-fold more sensitive to the tubulin polymerizing agent paclitaxel than were LXSN-expressing cells. Analysis of mouse embryonal fibroblasts lacking p53 (p53-/-) compared with mouse embryonal fibroblasts homozygous (p53+/+) and heterozygous (p53+/-) for wild-type p53 confirmed the role of p53 in the enhanced sensitivity to cisplatin. Treatment with the alkylating agents melphalan and nitrogen mustard resulted in 3.8- to 7.3 fold greater sensitivity in HPV 16E6- or 16E7-expressing cells compared with LXSN expressing cells. Enhanced sensitivity to cisplatin in cells lacking p53 function was explored by examination of its effects on cell cycle progression after exposure. When treated with cisplatin, HFFs expressing 16E6 showed delayed progression through S phase relative to HFFs expressing LXSN. The delay in S phase progression was coincident with the induction of p53 protein levels in LXSN containing HFFs, suggesting a role for p53 in DNA repair of cisplatin-induced damage. These results indicate that the inactivation of p53 in the absence of other genetic alterations leads to enhanced sensitivity to multiple chemotherapeutic agents rather than to increased resistance. PMID- 8631031 TI - erbB family receptor expression and growth regulation in a newly isolated human breast cancer cell line. AB - A new human breast cancer cell line (SUM-52PE), originating from a malignant pleural effusion specimen, that can be cultured under serum-free conditions has been isolated. Experiments were conducted to examine the relationship between expression of the erbB family of growth factor receptors and growth regulation in these cells. SUM-52PE cells are epidermal growth factor receptor negative but express single copy levels of erbB-2 protein. Southern blot analysis indicates that the erbB-2 gene is not amplified in these cells. The cells also express mRNA for both erbB-3 and erbB-4. Phosphotyrosine Western blot analysis of membrane protein obtained from SUM-52PE cells indicates the presence of a constitutively tyrosine phosphorylated M(r) 185,000 protein. Immunoprecipitation, using antibodies to erbB-2 or erbB-3, coupled to phosphotyrosine Western blot analysis indicates that both erbB-2 and erbB-3 are constitutively tyrosine phosphorylated in proliferating SUM-52PE cells. Conditioned medium obtained from SUM-52PE cells does not induce tyrosine phosphorylation of p185erbB-2 in a sensitive indicator cell line, suggesting that an erbB-2 activating factor is not secreted by these cells. However, neu differentiation factor/heregulin (NDF/HRG) mRNA is expressed by the cells, and Western blot analysis of SUM-52PE membrane protein revealed the presence of a M(r) 90,000 immunoreactive NDF/HRG protein. Thus, SUM-52PE cells synthesize a membrane bound form of NDF/HRG that may activate erbB-2 and erbB-3 via a juxtacrine mechanism. The addition of exogenous beta-2-NDF/HRG to the culture medium of SUM-52PE cells yields enhanced tyrosine phosphorylation of p185erbB-2/erbB-3 but has only a small stimulatory effect on the proliferation of these cells. By contrast, an erbB-2 monoclonal antibody that binds to the extracellular domain of erbB-2 is potently mitogenic for these cells. SUM-52PE cells were also found, by phosphotyrosine Western blot analysis, to express an inordinately large number of tyrosine phosphoproteins. Direct measurement of phosphotyrosine phosphatase (PTPase) activity in SUM-52PE cell membrane protein revealed 2-3-fold lower levels of PTPase activity compared to other normal and neoplastic breast epithelial cell lines. Thus, SUM-52PE cells exhibit altered growth phenotypes not identified previously in human breast cancer cells. The constitutive activation of erbB-2 and erbB-3 in these cells, coupled with their low, membrane-associated, PTPase activity are likely to play direct roles in driving proliferation of these breast cancer cells. PMID- 8631032 TI - Identification of a superficial bladder tumor-associated glycoform of the carcinoembryonic antigen by monoclonal antibody 19A211. AB - Monoclonal antibody (mAb) 19A211 identifies a superficial bladder cancer associated sialylated epitope expressed on a heterogeneous group of glycoproteins. These glycoproteins consist of a series of cytoplasmic glycoproteins ranging from 90 to 140 kDa present in tumor cells and, at a lower level, in normal urothelial cells, and of a membrane glycoprotein of 200 kDa observed in tumor cells only. To further characterize this antigenic system, we took advantage of the high avidity of mAb 19A211 to produce a rabbit polyclonal antibody by immunizing with antigen bound to mAb 19A211 immunoaffinity beads. The resulting polyclonal antibody specifically reacts with the 200-kDa species and not with the other glycoproteins. The biochemical characterization of this 200 kDa tumor-associated antigen showed that it is highly glycosylated (more than 50% w/w) and is anchored to the membrane via a glycosyl phosphatidylinositol link; these properties are shared with the carcinoembryonic antigen (CEA). Further experiments showed reactivity of two mAbs directed against protein epitopes of CEA with the 200-kDa component of 19A211 antigen. However, in immunohistochemistry studies of 29 colon and 23 bladder tumor specimens, no correlation was observed between expression of 19A211 and CEA antigens. Moreover, in RIAs, the intensity of expression of the 19A211 carbohydrate epitope relative to a CEA protein epitope was found to be significantly lower with CEA purified from a colon cancer cell line compared to CEA from a bladder cancer cell line. On the basis of these results, we conclude that the 200-kDa component of 19A211 antigen is a CEA glycoform preferentially expressed by superficial bladder tumors. PMID- 8631033 TI - Allelic imbalance at NME1 in microdissected primary and metastatic human colorectal carcinomas is frequent but not associated with metastasis to lymph nodes or liver. AB - Allelic imbalance at the NME locus on chromosome 17q21 was analyzed in colorectal cancer patients using a highly polymorphic microsatellite repeat sequence within NME1 itself. Duplicate samples of carcinoma and adjacent normal tissue was obtained by microdissection from 6 to 7-microns paraffin sections of 94 primary carcinomas (treatment years 1979-1993) and available lymph node and liver secondaries. In 55 patients informative (heterozygous) at this locus, allelic imbalance was examined in primary and secondary carcinomas. Microsatellite instability prevented assessment of allelic balance in two cases, and there was no evidence of homozygous loss at NME1 in any case analyzed. Allelic imbalance at the NME locus in carcinomas was frequent (27/53; 51%), and concordant results were obtained between primary carcinoma and secondary deposits in 30 of 33 (91%) cases. Three discordant cases showed allelic imbalance in secondary deposits but not the primary lesion. Although frequent, allelic imbalance at NME1 had no relationship to Dukes' stage at presentation or with subsequent hepatic metastasis, nor with the primary carcinoma site (proximal versus distal), tumor size, or mitotic or apoptotic index. Moreover, neither disease-free nor overall survival differed between patients with carcinomas showing NME1 allelic imbalance and patients with carcinomas that did not. Our results show that although allelic imbalance is frequent at the NME locus in primary and secondary colorectal carcinomas, there is no evidence to link this with clinical or pathological features or with metastatic potential. Microsatellite PCR and microdissection of enriched populations of carcinoma cells allowed uniformly successful analysis of samples from archival formalin-fixed paraffin-embedded tissue up to 15 years old and clear assessment of allelic imbalance in tumor specimens. Target sequences (e.g., microsatellites and minisatellites) up to approximately 200-250 bp may be reliably analyzed for allelic balance, suggesting that this method is of general utility in the genetic analysis of primary and metastatic neoplasia. PMID- 8631034 TI - Vascular endothelial growth factor promotes tumor dissemination by a mechanism distinct from its effect on primary tumor growth. AB - Tumor growth is dependent on new blood vessel formation. Inhibition of vascular endothelial growth factor (VEGF), an endothelial cell mitogen and angiogenic factor secreted by a variety of tumors and tumor cell lines, is sufficient to inhibit primary tumor growth. In the present study, we examined the effect of inhibiting VEGF on tumor cell micrometastasis. A transfectant of A431 (a human epidermoid carcinoma cell line) expressing chloramphenicol acetyltransferase (CAT) was injected s.c. into severe combined immunodeficiency (scid) mice, which were then sacrificed after 6 weeks. The presence of A431 metastases at distant sites was demonstrated by detection of CAT activity in whole-organ lysates. Treatment of animals with VEGF-neutralizing antibodies not only inhibited primary tumor growth but also suppressed metastases, as determined by CAT activity in organ lysates. In experiments to determine the mechanism by which anti-VEGF antibody inhibited metastasis, control animals were sacrificed when their tumors had reached the same size as tumors in VEGF antibody-treated animals. Metastases were uniformly present in these control animals. These findings show that inhibition of VEGF alone is sufficient to prevent tumor growth and dissemination in vivo. The inhibitory effect on metastases appears to be distinct from that on primary tumor growth. PMID- 8631035 TI - Re: G.P. Studzinski and D.C. Moore, sunlight--can it prevent as well as cause cancer? Cancer Res., 55: 4014-4022, 1995. PMID- 8631036 TI - Advances in immunotoxin biology and therapy: a summary of the Fourth International Symposium on Immunotoxins. PMID- 8631038 TI - Brain and atrial natriuretic peptides in patients with ischemic heart disease with and without heart failure. AB - The objective of the study was the evaluation of natriuretic peptides in ischemic heart disease. Atrial and brain peptides (ANP, BNP) were elevated in patients with ischemic heart failure, as compared with patients with angina without over failure, and controls (p < 0.01). BNP/ANP ratio was higher in NYHA class IV than in class III patients (2.67 +/- 0.87 vs. 1.52 +/- 0.59, respectively). Patients in the angina group, in whom elevated BNP or ANP was found, had subclinical systolic or diastolic dysfunction. There was inverse correlation between BNP, ANP and the left-ventricular ejection fraction (each r = 0.78, p < 0.001). We conclude that BNP is elevated as a result of myocardial dysfunction, but not of ischemia and seems to be a better index of disease stage and prognosis than ANP. PMID- 8631037 TI - Prevention of coronary heart disease in clinical practice: a commentary on current treatment patterns in six European countries in relation to published recommendations. AB - Market research surveys can be a source of up-to-date information about current clinical practices. Data from one such survey, Cholesterol Monitor (made available by Merck & Co., Inc., Whitehouse Station, N.J., USA) was examined to ascertain to what extent management of cholesterol in six European countries conforms with the advice of the joint Task Force of the European Society of Cardiology, European Atherosclerosis Society and European Society of Hypertension for prevention of coronary heart disease (CHD). Rates of cholesterol testing in patients with CHD varied from less than 50% in the UK to more than 80% in France and Italy. Across Europe, the average cholesterol levels in surveyed patients was 6 mmol/l, and the average intervention level was 7.5 mmol/l. In all countries, there was evidence of a substantial treatment gap, even among high-risk patients with established CHD. This gap took the form of non-treatment of a proportion of patients whose risk status merited intervention on the basis of expert recommendations and the results of the Scandinavian Simvastatin Survival Study (in which sustained lowering of total and low-density lipoprotein cholesterol reduced total and coronary mortality in patients with baseline total cholesterol as low as 5.5 mmol/l). The findings indicate that a concerted programme of physician education is required if the recommendations of the joint Task Force are to be put into effect, and if the present nontreatment/undertreatment of cholesterol in high-risk patients is to be corrected. PMID- 8631040 TI - Presence of contractile-type smooth muscle cells in the endocardium. AB - A smooth muscle layer exists in the parietal endocardium. Notwithstanding previous pioneer work, little has been known on the phenotypes of these smooth muscles. In humans as in animals, smooth muscles show two distinct phenotypes, the 'synthetic' and 'contractile' states, which can be differentiated on the basis of the expression of two types of myosin heavy chain isoforms, SM1 and SM2. In this study, using SM1- and SM2-specific monoclonal antibodies, we performed immunohistochemical examinations to determine the phenotype of the endocardial smooth muscles. We report here that the endocardial smooth muscles have the contractile phenotype. PMID- 8631041 TI - Power spectral analysis of heart rate variability during upright tilt test: a comparison of patients with syncope and normal subjects. AB - We analyzed heart variability (HRV) response to tilt in 35 patients with a history of neurocardiogenic syncope and in 8 normal volunteers. Frequency domain examination was performed using power spectral analysis of RR variability during resting supine position and during the 256 beats preceding the onset of syncope or completion of 60 min of upright tilt to 70 degrees. Both low (0.05-0.15 Hz) and high frequency (0.15-0.4 Hz) spectral components of HRV increased markedly in all groups as a result of tilting. Statistically significant differences were noted between the patient groups at completion of upright tilting. However, these differences in spectral components between groups were of small magnitude when compared to the overall increase in spectral power occurring in all groups as a result of the tilt and are difficult to correlate clinically with the appearance, or lack of appearance of tilt-induced syncope. Power spectral analysis of HRV, as presently performed, appears to lack discriminative power to detect the rapid and marked changes in sympathovagal modulation known to occur during tilt test in patients with neurocardiogenic syncope. PMID- 8631039 TI - Lipoprotein(a) and oxygen free radicals in survivors of acute myocardial infarction: effects of captopril. AB - Long-term treatment of survivors of an acute myocardial infarction with angiotensin-converting enzyme inhibitors has a beneficial impact on their long term outcome. We tested the hypothesis that captopril could reductively cleave the lipoprotein(a) molecule and in addition act as a scavenger of oxygen free radicals. In a double-blind trial, 20 patients were randomized to receive either captopril 50 mg daily or corresponding placebo. patients were followed for a period of 30 days. Blood samples were drawn prior to randomization and after 30 days of treatment. Plasma concentrations of lipoprotein(a) and malondialdehyde were evaluated. Captopril treatment produced a significant reduction in plasma content of lipoprotein(a) ( < 0.05) and at day 30 the plasma content of lipoprotein(a) was also significantly lower than that in the placebo group (p < 0.05). furthermore, on day 30 plasma concentrations of malondialdehyde, an indicator of oxidative damage, were significantly lower in the captopril group when compared to baseline values and corresponding placebo group values (p < 0.05). The observed effect of captopril treatment on lipoprotein(a) and malondialdehyde might be ascribed to the sulfhydryl group in the captopril molecule. PMID- 8631042 TI - Electropharmacologic characteristics and radiofrequency catheter ablation of sustained ventricular tachycardia in patients without structural heart disease. AB - Twenty-six patients (mean age 39 +/- 17 years) with idiopathic sustained ventricular tachycardia (VT) were included for study. The patients were divided into two groups: group I: 14 patients with VT originating from the right ventricular outflow tract (wide QRS tachycardia with complete left bundle branch block pattern), and group II: 12 patients with VT originating from the left ventricle (wide QRS tachycardia with complete right bundle branch block pattern). Most of the group I patients (11/14) needed isoproterenol to facilitate induction of VT, and were sensitive to both verapamil and adenosine. Eight patients had successful radio-frequency (RF) ablation and were free of VT without any antiarrhythmic drugs. In group II, sustained VT was induced by programmed ventricular stimulation in all the patients (only 3 patients needed isoproterenol for facilitation); verapamil could terminate all the VT but none of the patients responded to adenosine. Eight patients received RF ablation and 6 patients had successful ablation without recurrent tachycardia on a long-term basis. Different sensitivity to adenosine and isoproterenol between right and left ventricular idiopathic VT suggested different underlying mechanisms for both types of VT. The patients who did not receive catheter ablation still had attacks of VT despite antiarrhythmic drug treatment; however, none of these patients had sudden death since the first attack of VT (mean 95 +/- 51 months), suggesting a benign prognosis in idiopathic VT. PMID- 8631043 TI - Does the baseline impedance measurement during radiofrequency catheter ablation influence the likelihood of an impedance rise? AB - Most radiofrequency energy delivery systems provide a baseline (pre-ablation) impedance measurement; however, the application of this value, particularly in avoiding catheter overheating and coagulum formation, has not been described. We evaluated the ability of the product of the power output P and the baseline impedance Z (P x Z) to predict the likelihood of an impedance rise and coagulum formation during radiofrequency energy delivery in 62 consecutive patients undergoing successful catheter ablation of the slow atrioventricular (AV) nodal pathway or an accessory pathway. The mean P x Z during the 114 pulses resulting in an impedance rise was 3,770 +/- 846 W omega; only 42 impedance rises in 14 patients occurred at a P x Z < 3,5000. For comparison, the P x Z during the single radiofrequency pulse that resulted in loss of preexcitation or the slow AV nodal pathway in the entire patient group was 3,118 +/- 590 (p = 0.001) and in only 9 patients was the P x Z > 3,500. this data suggest that adjusting the power during each radiofrequency pulse to maintain the P x Z < 3,500 should enable the operator to avoid most impedance rises. PMID- 8631044 TI - Effects of renal neutral endopeptidase inhibition on sodium excretion, renal hemodynamics and neurohormonal activation in patients with congestive heart failure. AB - We investigated the effects of inhibiting endogenous atrial natriuretic factor (ANF) metabolism on renal hemodynamics, sodium excretion and neurohormones in 12 patients with New York Heart Association functional class II congestive heart failure (CHF) due to left ventricular systolic dysfunction. In a randomized, placebo-controlled, double-blinded fashion, 8 patients received a single oral dose of candoxatril, an inhibitor of renal neutral endopeptidase, and 4 patients received placebo. Candoxatril treatment increased plasma ANF by 70 +/- 71 pg/ml (p < 0.015 vs. placebo) and plasma cGMP by 7.9 +/- 2.7 pmol/ml (p < 0.001 vs. placebo), with maximal effects at 3.5 h. Urinary cGMP more than doubled (p = 0.025 vs. placebo). Candoxatril increased urinary sodium by 2.7 +/- 2.0 mEq/h (p < 0.05 vs. placebo) and significantly elevated filtration fraction with no significant effect on glomerular filtration rate, renal plasma flow or lithium clearance. A significant reduction in aldosterone concentration with a similar trend in plasma renin activity was noted in candoxatril-treated patients. Thus in patients with moderate heart failure, renal neutral endopeptidase inhibition increases urinary sodium excretion. The lack of an effect on renal hemodynamics suggests that this natriuresis results from ANF-mediated inhibition of tubular sodium reabsorption. These findings justify additional investigation into potential clinical benefit of endopeptidase inhibition in patients with CHF. PMID- 8631045 TI - Tolerability and efficacy of antihypertensive treatment with cilazapril in general practice. AB - Data on the safety, tolerability and efficacy of the new angiotensin-converting enzyme (ACE) inhibitor cilazapril were obtained in a multicenter observational study carried out in a general practice setting. A total of 33,841 hypertensive patients were prescribed cilazapril 2.5-5.0 mg per day and followed for an average of 109 days. 28,792 patients (85.1%) had a baseline diastolic blood pressure (BP) > or = 95 mm Hg. 24,649 patients received cilazapril alone. during the observation period the mean blood pressure of these patients fell from 177/105 to 148/87 mm Hg (an average drop delta of 29/18 mm Hg). The response rate (diastolic BP < or = 90 mm Hg and delta > or = 10 mm Hg) in these patients was 78.9%. Adverse events were reported by 7.3% of all patients. In all, 3.7% of patients discontinued cilazapril treatment because of side effects. The most frequently reported side effect (1.5% of cases) was the dry cough typical of ACE inhibitors. The doctors judged the efficacy of cilazapril as very good or good in 89.8% of cases and its tolerability as very good or good in 94.6%. This reflects the good compliance (regular tablet intake) of more than 90% of patients. PMID- 8631046 TI - Effects of intravenous disopyramide on coronary hemodynamics and vasodilator reserve in hypertrophic obstructive cardiomyopathy. AB - Disopyramide reduces the subaortic pressure gradients and improves the clinical symptoms of patients with hypertrophic obstructive cardiomyopathy. Changes in coronary hemodynamics and vasodilator reserve in response to this agent have not been evaluated in such patients. To assess the acute effects of intravenous administration of disopyramide on coronary hemodynamics, microvascular dilatory capacity, and balance between myocardial oxygen supply and demand in hypertrophic obstructive cardiomyopathy, we examined 12 patients using an intravascular Doppler catheter and spectral analysis. Intravenous disopyramide 100 mg over 10 min caused mild increases in heart rate and aortic systolic pressure and a significant fall in left ventricular systolic pressure, resulting in a 13% decrease in the product of heart rate and left ventricular systolic pressure (from 1.13 +/- 0.18 x 10(4) before disopyramide to 0.98 +/- 0.17 x 10(4) beats/min x mm Hg 10 min afterwards; p < 0.05) and a reduction in the resting peak systolic pressure gradients of the left ventricular outflow tract. There was a 14% reduction in coronary blood flow (from 93.5 +/- 13.2 to 80.3 +/- 11.6 ml/min; p < 0.05) with an increase in coronary resistance (from 0.94 +/- 0.16 to 1.23 +/- 0.21 mm Hg/ml/min; p < 0.001). The index of coronary vasodilator reserve remained unchanged. These findings suggest that intravenous disopyramide causes a coronary vasoconstrictive effect without significantly changing the coronary microvascular dilatory capacity, but this effect may not be harmful to the balance between myocardial oxygen supply and demand in patients with hypertrophic obstructive cardiomyopathy. PMID- 8631047 TI - Long-term risk of death, cardiac events and recurrent chest pain in patients with acute chest pain of different origin. AB - The purpose of the study was to describe the prognosis of patients with acute chest pain of different origin, but without myocardial infarction (non-AMI). A total of 204 patients were included. In 56, a definite diagnosis was obtained within 24-48 H of admission. The remaining 148 patients underwent the following examinations: exercise test, myocardial scintigraphy, echocardiography, Holter monitoring, hyperventilation test, oesophago-gastro-duodenoscopy, oesophageal manometry, oesophageal pH monitoring, Bernstein test, physical chest wall examination, bronchial histamine test, chest X-ray and ultrasonic upper abdominal examination. Ischaemic heart disease (IHD) was diagnosed in 64 patients, 81 had gastro-oesophageal disorders, 58 chest wall disorders, 9 pericarditis, 5 pulmonary embolism, 4 pneumonia/pleuritis, 3 pulmonary cancer, 2 dissecting aortic aneurysm, 1 aortic stenosis and 1 herpes zoster. During follow-up of 33 months, 31 of the 64 patients with IHD had a cardiac event (cardiac deaths, non fatal AMI, bypass surgery or PTCA), whereas only 3 event occurred among the 140 patients without IHD (p < 0.00001). However, the frequency of readmissions and of recurrent episodes of chest pain were similar in the 3 major diagnostic groups (NS). To conclude, the high-risk subset of a non-AMI population can be identified by means of non-invasive cardiac examination. The remainder who have other diagnoses are at low risk. However, the morbidity is high with frequent readmissions and recurrent episodes of chest pain and the need for development of strategies with regard to diagnosis and treatment of these patients are emphasized. PMID- 8631048 TI - Antimitochondrial antibodies after acute myocardial infarction. AB - The development of anti-heart mitochondrial antibodies (AHMA) after acute myocardial infarction (AMI) has been previously demonstrated in experimental studies. We performed a prospective study to check the incidence, variation curve and specificity of AHMA and AMI by using two different immunological tests: complement fixation and antihuman globulin consumption. Serial venous blood samples were drawn from the patients as follows: for the first 14 days after the event, once every other day; from 14 to 60 days after the acute event, once a week. The development of AHMA was demonstrated in the sera of 15/35 (45.4%) patients with AMI by complement fixation test using mitochondrial antigen obtained from normal human heart tissue compared to 12/33 (36.3%) by using mitochondrial antigen from the infarcted region. The antibodies exhibited cross reaction with mitochondrial antigens obtained from human skeletal muscle, but did not react with heterologous heart mitochondrial antigens. According to the results of the antihuman globulin consumption test, the AHMA is immunoglobulin M (IgM). None of the patients developed postmyocardial infarction syndrome. PMID- 8631049 TI - Left atrial appendage blood flow determined by transesophageal echocardiography in patients with complete atrioventricular block. AB - Function of the left atrial appendage (LAA) represented by LAA outflow is an important predictor for thrombus formation in patients with nonrheumatic atrial fibrillation, but the pattern of LAA flow in patients with complete atrioventricular (AV) block has rarely been studied. Twenty-one patients with complete AV block and an implanted VVI or VVIR pacemaker were studied with transesophageal echocardiography. The LAA outflow velocity obtained during the ventricular diastolic phase was significantly higher than that obtained during the ventricular systolic phase (41.5 +/- 6.0 vs. 34.9 +/- 9.7 cm/s; p < 0.001). The LAA inflow velocity obtained during the ventricular diastolic phase was also significantly higher that that obtained during the ventricular systolic phase (29.9 +/- 7.8 vs. 26.4 +/- 5.3 cm/s; p < 0.01). In addition, the LAA outflow and inflow velocity time integrals during the ventricular diastolic phase were significantly higher than those during the ventricular systolic phase (4.66 +/- 0.96 vs. 4.08 +/- 1.05 cm, p < 0.01, and 2.81 +/- 0.77 vs. 2.56 +/- 0.65 cm, p < 0.05, respectively). Thus, due to both diastolic augmentation of the LAA flow related to active atrial contraction and minor early diastolic LAA flow formation, left ventricle diastolic function might have some influence on LAA flow. Thus may have implications for the pathogenesis of LAA thrombi in left ventricular dysfunction. PMID- 8631050 TI - Common form of Lyme borreliosis carditis--complete heart block with syncope: report on 3 cases. AB - Third degree atrioventricular (AV) heart block with severe Adams-Stokes attacks in 3 patients with Lyme borreliosis is described. All patients had similar clinical manifestations: previously healthy, they experience syncope as an abrupt onset of the disease. Data on skin changes--erythema migrans--was subsequently obtained, although the patients did not recall being bitten by a tick. Diagnoses were based on clinical manifestations and on positive serologic test results to borrelia. Following AV block returned to sinus rhythm with normal AV conduction in all patients. PMID- 8631051 TI - Echocardiographic follow-up of diphtheric myocarditis. AB - We report the first case of diphtheric myocarditis documented by serial electrocardiography and echocardiography. The electrocardiographic changes preceded the contraction abnormalities by 3 weeks. During a prolonged neurologic disease with extensive polyneuropathy, the patient had only subtle clinical signs of myocarditis. In patients with diphtheria, electrocardiographic changes may warrant repetitive echocardiographic examinations. PMID- 8631052 TI - Sustained nonischemic ventricular tachycardia during dobutamine stress echocardiography. AB - Dobutamine stress echocardiography has become widely accepted as a safe, reliable and cost effective modality for the evaluation of patients with suspected myocardial ischemia or for prognostic stratification and outcome assessment in patients with known coronary artery disease. while the benefits of this means of cardiovascular testing are very clearly apparent, it is important to understand and recognize possible complications. This paper discusses the occurrence of sustained, nonischemic ventricular tachycardia during dobutamine stress echocardiography in the absence of functional, physiologic or anatomic evidence of coronary artery disease or cardiomyopathy. PMID- 8631054 TI - Amiodarone and reversible benign intracranial hypertension. PMID- 8631053 TI - Late complications in traumatic coronary artery fistula: report of a case requiring surgical repair after 8 years. AB - We report the unusual case of a patient with traumatic coronary artery fistula who had been free of symptoms for more than 4 years, but who ultimately required surgical repair 8 years after the trauma due to late complications including angina pectoris, atrial flutter and fibrillation, congestive heart failure, and tricuspid regurgitation. Our findings suggest that early surgical repair should be undertaken in cases of traumatic coronary artery fistula, even if the shunt is minimal and early symptoms are mild. The persistence of, or a slow increase in, shunt flow over the years greatly increases the risk of the ultimate development of life-threatening complications. PMID- 8631055 TI - [Models of focal hypoxia of the central nervous system]. AB - Vascular disease and focal cerebral ischemia still represent the major cause of neurological morbidity and mortality. Mechanisms of hypoxic changes are associated with energy depletion and impairment of biological membranes. Reperfusion after the stroke plays an important role in the development of morphological and functional changes of the nervous tissue. In experiments, different models of focal cerebral ischemia based on the middle cerebral artery occlusion (MCAO) are used. Four main categories of such models are most frequently employed: 1. Temporary intraluminal occlusion of part of the circle of Willis (via internal carotid artery), 2. Abluminal application of the vasoconstrictor peptide (endothelin-1) to the MCA, 3. Tromboembolic models, 4. Microclips. Reliable quantification of morphological changes is also possible. Discussed models are used for testing different types of treatment of the cerebral ischemia, including pharmacological stimulation and blocking of individual membrane receptor systems. PMID- 8631056 TI - [Programmed neural cell death?]. AB - During normal development of the nervous system, up to 50 percent or more neuronal and glial cells die by programmed cell death. In the developing neuron, a continual access to neurotrophic factors is required for survival of the cells. The death may be a passive process, and the absence of neurotrophic factors leads to a loss of metabolic activity and, ultimately to cellular degeneration. Alternatively, apoptosis may be a metabolically active process and neurotrophic factors are necessary to repress the "suicide" response of the cell. The role of intracellular calcium, RNA-synthesis, protein synthesis a gene expression is discussed. PMID- 8631057 TI - [Magnesium and the transport system]. AB - Magnesium is sometimes denoted as "the overlooked cation" for although it is the second most abundant intracellular cation, the study of its homeostasis and regulation has long been neglected. However, at present it is evident that magnesium plays an important intracellular regulatory role. Magnesium transport systems can be divided into two principal categories: paracellular transport systems and transmembrane transport system. Paracellular pathway plays an important role in the intestinal and renal magnesium transports. The movement of magnesium across biological membranes is discussed from the perspectives of the secondary active transport and specific magnesium channels. PMID- 8631058 TI - [Physiologic, biochemical and genetic aspects of malignant hyperthermia]. AB - Malignant hyperthermic syndrome (MHS) is based on a metabolic dysfunction of the skeletal muscle. It is characterized by an elevation of muscle metabolism and rigidity, accompanied by an increase of arterial pCO2, lactate and potassium plasma concentration, and body temperature. In sensitive individuals, MHS can be evoked pharmacologically. To identify substances that evoke this syndrome or those useful for its therapy, MHS is modelled in pigs. The primary defect attributed to MHS is the impairment of sarcoplasmic calcium homeostasis based on a dysfunction of one of calcium ion channels. In some cases, genetic mapping has shown that MHS is related to changes in 19 chromosome (in humans). Abnormal function of the ion channel is probably not sufficient for the expression of MHS. The syndrome manifests only when several modifying factors coincide. PMID- 8631059 TI - [Stochastic models of neuronal activity]. AB - Techniques for modeling of a stochastic activity of neurons are briefly reviewed. Our model is proposed, some experimental results with input Gaussian stochastic processes are discussed, and the concept of e-curves is introduced. PMID- 8631061 TI - [History of the Physiological Society]. PMID- 8631060 TI - [Measurement of hemodynamics in the periodontium and gingiva]. AB - Measurements of hemodynamics in parodontium and gingiva using a reflexive photoelectrical plethysmographic recorder, insensitive to salivation changes, has been described. Registration is accomplished by means of ECG recorder. PMID- 8631062 TI - [The early care of patients with primary brain stem injuries]. PMID- 8631063 TI - [The regularity of the development of bedsores in cerebral diseases and their prevention]. PMID- 8631065 TI - [Relationship between indwelling catheters and bacteriuria]. PMID- 8631064 TI - [An analysis of and epidemic infection of the upper respiratory tract in the neonatal ward]. PMID- 8631066 TI - [Temperature measure with relation to blood circulation changes after rejoining severed fingers]. PMID- 8631067 TI - [The issues of breast feeding and the solving methods after cesarean section]. PMID- 8631069 TI - [Continuing education of collegiate graduate nurses in hospitals]. PMID- 8631068 TI - [Opinion of the evaluation of graded management of nursing in hospital]. PMID- 8631070 TI - [Significance and measures to control the nursing quality index]. PMID- 8631071 TI - [Improving audio-visual education programs in nursing class]. PMID- 8631072 TI - [Analysis of the society and psychology of blood dialysis patients]. PMID- 8631074 TI - [Analysis of articles on mental health care published by three kinds of journals of nursing from 1985 to 1993]. PMID- 8631073 TI - [Techniques of intraosseous infusion]. PMID- 8631075 TI - [Study on Chinese herb incense to disinfect wards' air]. AB - The purpose of this study was to find out a proper way to do the air sterilization in the wards. In this study, Chinese herb-burn-incense was used to different wards. Its effect was compared with routine air sterilization methods such as ultraviolet radiator, formaldehyde, and lactiacid method. The bacterial culture of the air was done. The result indicated that Chinese herb-burn-incense not only had the same effect as routine methods in air sterilization but also had no irritation to the patients. PMID- 8631076 TI - [Management of the burn intensive care unit]. PMID- 8631077 TI - [Emergency care of 95 cases of children with alimentary toxicosis]. PMID- 8631078 TI - [Care of replantation of avulsed scalp]. PMID- 8631079 TI - [Postoperative nutritional support for patients with oral and maxillofacial malignancies]. PMID- 8631080 TI - [To work out and use scaling for evaluation of nursing quality]. PMID- 8631081 TI - [Preliminary research on management of case histories using the nursing process]. PMID- 8631082 TI - [The four step teaching method of primary nursing]. PMID- 8631083 TI - [Application of nursing process to reform of basic nursing teaching]. PMID- 8631084 TI - [Care of Budd-Chiari syndrome with balloon dilatation]. PMID- 8631085 TI - [Care of complications of percutaneous plugging of patent arterial duct]. PMID- 8631086 TI - [Principles and development of oncologic nursing]. PMID- 8631087 TI - [Current opinion of urinary tract infection related to indwelling urethral catheterization]. PMID- 8631088 TI - [Exploration of nursing diagnosis with a cognitive reasoning model]. AB - One of the important topics in theoretic studies on nursing diagnosis, still a new concept in China, is to explore the logical reasoning process from collecting data to making diagnoses. This paper introduced an epistemological model which mentions every step in detail on how to make a nursing diagnosis. The model was built on the basis of analyzing the knowledge structure of nursing diagnosis and applying the methodology of cognitive psychology. PMID- 8631089 TI - [Study on the efficacy of genetically engineered vaccines against hepatitis B for interruption of perinatal transmission]. AB - The infectivity rate of newborn babies who had been borne from HBsAG(+), HBeAg(+) and anti-HBc(+) mothers was very high (85%). 142 babies born in the hospital were divided into three groups, in this study. In the group 1, 57 babies were inoculated with 20 micrograms recombinant DNA vaccinia vaccines against hepatitis B. The injections were given at newborn, 1 month, and 6 months, respectively. In group 2, 41 babies were inoculated with 20 micrograms genetic engineering vaccines against hepatitis B at same time were intervals as group 1. In group 3, 44 newborn babies were inoculated with 10 micrograms as same vaccines as group 2 HBIG plus 1ml (200 U/ml), at same time intervals as group 1. The immune pretection rates of newborn babies in three groups were 88.2%, 85.9% and 100%, respectively. The anti-HBs pasitive conversion rates were 82%, 86% and 98%, respectively. The group 3 was compared with group 1 and 2. Statistical analysis showed the significant differences (P < 0.05). The result showed the immune program of group 3 was superior to that of group 1 and 2, and none of the 44 babies in group 3 were infected. The efficacy of immunization by genetic engineering vaccines were superior to that of blood-derived vaccine. The genetic engineering vaccines against hepatitis B would be more useful for interruption of perinatal transmission of HBV. PMID- 8631090 TI - [OPD care for asthmatic children]. PMID- 8631091 TI - [Total environment protection for moderate and severe grade of acute radiation sickness]. PMID- 8631092 TI - [Application of liquid valium naristillae during induced abortion]. PMID- 8631093 TI - [Coordination with transplantation of cryopreserved vascularized bone allografts]. PMID- 8631094 TI - [Instruction of functional exercise of pillow-pad treated simple compression fracture of thoracic-lumbar spine]. PMID- 8631095 TI - [Applying of the elastic bandage to the radial keratotomy patient]. PMID- 8631096 TI - [Self blood ultraviolet irradiation and oxygenation therapy]. PMID- 8631097 TI - [Management and effect of nurses' extra degree course study]. PMID- 8631098 TI - [Strengthen the reform of teaching management for nurses' evening university]. PMID- 8631099 TI - [Experiences in using electrical audio-visual aids in the teaching of basic nursing]. PMID- 8631100 TI - [Psychological care of opiate dependent patients]. PMID- 8631102 TI - [Nursing management of the operating room with six organ transplantations at the same time]. PMID- 8631101 TI - [Study of subcuticular suture skill for perineotomy and catgut-suture reaction]. AB - The post perineotomy complications such as catgut-suture-reaction, incision infection, and laceration are very common in the hospital. The purpose of this study was to find a better suture skill. The subcuticular suture skill was studied in this study. The relationship between suture skill and catgut suture reaction was analysed. 200 women had vaginal delivery with perineotomy subcuticular suture who were hospitalized during 3 to 10 in 1994 were compared with 399 women with perineotomy routine suture skill who were hospitalized during the same period in 1992 and 1993. The result indicated that the incidence of cat gut suture reaction and secondary suture in the research group were 9%, 7.5% lower than that in control group (P < 0.01). The average time of hospitalization was 5.3 days shorter (P < 0.025). These implicate the future study on perioneotomy suture skill. PMID- 8631104 TI - [Care of renal transplantation of aged patients during surgery]. PMID- 8631105 TI - [Clinical variation and care of typhoid fever]. PMID- 8631103 TI - [Prevention and control of infection after orthotopic heart transpl]. PMID- 8631106 TI - [Nosocomial infection of nephrotic syndrome in childhood]. PMID- 8631107 TI - [Procedures of ultraviolet blood irradiation and oxygenation for newborns with hypoxic-ischemic encephalopathy]. PMID- 8631108 TI - [Study of early functional training of extrinsic ocular muscles after surgery]. PMID- 8631109 TI - [Observation of the effect of early rehabilitation of cerebral vascular diseases]. PMID- 8631110 TI - [The function of the nursing department in infection control in general hospitals]. PMID- 8631111 TI - [Standard evaluation of nursing quality enhance the quality of nursing rounds]. PMID- 8631112 TI - [Talking about the value tendency of nursing]. PMID- 8631113 TI - [Experience in establishing pediatric teaching department in baccalaureate nursing education]. PMID- 8631114 TI - [Computer software to assist teaching of basic nursing]. PMID- 8631115 TI - [The experience of using joint teaching methods in the teaching of nursing ethics]. PMID- 8631116 TI - [Psychotherapy of 172 cases of preoperative children with cleft lip and palate]. PMID- 8631117 TI - [Quantitative measurement of mental symptoms in breast patients after mastectomy]. PMID- 8631118 TI - [Causes and care of postoperative weakness]. PMID- 8631119 TI - [Application of computers in the nursing field]. PMID- 8631120 TI - Phosphorylation of topoisomerase I in L5178Y-S cells is associated with poly(ADP ribose) metabolism. AB - The reason for different phosphorylation of topoisomerase I in two sublines of L5178Y murine lymphoma (LY cells) was investigated. Camptothecin-resistant LY-S cells show increased poly(ADP-ribose) level and lowered topoisomerase I phosphorylation compared to camptothecin-sensitive LY-R cells. In this study diminished phosphorylation of LY-S topoisomerase I was observed for sites recognized by casein kinase 2 but not for those phosphorylated by protein kinase C. Tryptic digests of LY-S topoisomerase I labeled in vitro by casein kinase 2 indicated that phosphorylation was similarly lowered at different sites. Activity of casein kinase 2 measured in nuclear extracts was about 1.7 times lower for LY S than LY-R cells. This difference was diminished or eliminated by increasing casein concentration, diluting the extract or increasing the ionic strength. Activity of poly(ADP-ribose) polymerase was 5.3 times higher in LY-S than in LY-R nuclei. When the activity of the polymerase was inhibited by treatment of LY-S cells with benzamide, casein kinase 2-catalyzed phosphorylation of topoisomerase I increased. This was accompanied by an increase in sensitivity to camptothecin as reflected in the diminished viability of LY-S cells. PMID- 8631121 TI - A comparison of the propensity for gene amplification between near-tetraploid and near-diploid V79 clones resistant to 150 nM methotrexate. AB - Among various 150-nM methotrexate-resistant (MTXr) V79 clones isolated, we found that two near-tetraploid clones as well as a near-diploid clone with amplification in the dihydrofolate reductase (dhfr) gene readily developed resistant to 40 000 nM MTX within 3 months during stepwise increased MTX selection, while two near-diploid clones without gene amplification could not acquire resistance beyond 5000 nM MTX. Then, we studied how the clones increased the resistance to MTX, and compared the propensity for gene amplification among three types of clones. Dot blot analysis showed that the acquisition of the high levels of resistance to MTX observed in two near-tetraploid clones and a near diploid clone with gene amplification was associated with amplification in the dhfr gene. The amplified dhfr gene was overexpressed at mRNA and protein levels in the clones. Southern blot analysis of Hind III- and Eco RI-digested DNA in the clones at the time when they became resistant to 10 000 nM MTX indicated that they amplified the dhfr gene fragments which existed in low amounts in parental V79 cells, and that no gross rearrangement of the amplified dhfr gene was detected. Furthermore, fluorescence in situ hybridization analysis showed that the amplified dhfr gene was located on one chromosome as cluster(s). On the other hand, two near-diploid clones without gene amplification did not show any amplification of the dhfr gene even at 5000 nM-MTX resistant stage. These combined results suggest that the near-tetraploid clone as well as the near diploid clone with the dhfr gene amplification have genomic instability with the propensity for gene amplification during stepwise MTX selection, and have a similar process for the development of the dhfr gene amplification. PMID- 8631123 TI - Cause and effect between concentration-dependent tissue damage and temporary cell proliferation in rat stomach mucosa by NaCl, a stomach tumor promoter. AB - This study was designed to test whether concentration or dose of NaCl was responsible for the initial tissue damage (after 1 min) and resulting temporary cell proliferation at 17 h in stomach mucosa of male F344 rats after gastric intubation of 0.65, 1.3, 2.6 and 3.7 M NaCl. Histological damage was studied by dual staining combining horseradish peroxidase-labeled Griffonia simplicifolia agglutinin-II staining (HRP-GSA-II) and periodic acid cold thionin-Schiff reaction (PATS). Cell proliferation was studied by measuring replicative DNA synthesis with liquid scintillation counting and by BrdU staining. NaCl at the same overall dose of 0.8 g/kg body weight induced different degrees of response depending on the concentration. For 4 ml of 0.65 M NaCl, there was no tissue damage after 1 min nor any increase in replicative DNA synthesis after 17 h in the pyloric mucosa. Administration of 1.3 M NaCl (2 ml), 2.6 M NaCl (1 ml) and 3.7 M NaCl (0.7 ml) induced concentration-dependent damage of the surface mucous cell layer after 1 min and increased replicative DNA synthesis after 17 h (P<0.05). Concentration-dependent increase in replicative DNA synthesis at 17 h was also induced with the same volume (1 ml) of 1.3, 2.6, and 3.7 M NaCl, while a volume-dependent increase in replicative DNA synthesis at 17 h was induced with 0.4, 0.7 and 1 ml of 3.7 M NaCl. However, a greater increase in replicative DNA synthesis was always observed when using higher NaCl concentrations at the same dose. Liquid scintillation counting was well-correlated with BrdU staining. These results suggest that a high concentration of NaCl is responsible for the initial tissue damage and resulting temporary cell proliferation during stomach tumor promotion. PMID- 8631122 TI - Effects of cell proliferation and cell death (apoptosis and necrosis) on the early stages of rat hepatocarcinogenesis. AB - An experiment was performed to investigate whether, during regression of the liver hyperplasia induced by a direct mitogen, apoptosis differentially affects replicated and non-replicated hepatocytes. After a single dose of the direct mitogen lead nitrate (LN), male Wistar rats were given repeated injections of tritiated thymidine, and were killed either 3 days (time of maximal hepatic DNA increase) or 15 days (complete regression of the hyperplasia) after mitogen treatment. Determination of liver DNA radioactivities and labelling indices (LIs) at the two time points revealed an approximately 40% loss in total liver DNA radioactivity, a 20% decrease in the specific activity of DNA, and a 20% reduction in the cell LI. Three days after LN administration 64% of the apoptotic bodies contained thymidine grains in their nuclear fragments. The results indicated that apoptosis affects both hepatocytes that replicated, and those that did not replicate, the former being slightly more sensitive. A second experiment was then performed to investigate whether and to what extent different types of cell death (apoptosis versus necrosis) influence the growth of hepatocytes initiated by a chemical carcinogen. Male Wistar rats were given a single dose of diethylnitrosamine, and 2 weeks thereafter either a single dose of LN, or a necrogenic dose of carbon tetrachloride (CCl4). Bromodeoxyuridine was next infused for 5 days, and some of the animals were killed at this time point, and others after an additional 3 weeks. Administration of CCl4 resulted in an increase in both the average size and the percentage area occupied by placental glutathione S-transferase-positive lesions. In contrast, administration of lead nitrate resulted in a strong reduction (50%) in the number of positive lesions with no remarkable change in the percentage area occupied by them. These differential effects occurred even though comparable LIs were observed in rats treated with the two agents. The results suggest that lead nitrate leads to a loss of initiated hepatocytes, due to the apoptosis that occurs during regression of the LN-induced hyperplasia. PMID- 8631124 TI - Early hepatic changes induced in rats by two hepatocarcinogenic organohalogen pesticides: bromopropylate and DDT. AB - The aim of the present studies was to describe the effect of two organohalogen pesticides: DDT and bromopropylate, on early changes in rat liver, proposed in the literature to be useful endpoints in screening of non-genotoxic hepatocarcinogens and/or liver tumor promoters. We investigated the effects on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and cytochrome CYP2B1-dependent monooxygenase induction. The histological and cytochemical changes in the liver were also recorded. Male Wistar rats received bromopropylate in one, three or five daily oral doses of 125, 250, and 500 mg/kg body wt. day-1. DDT was applied as one, three, and five daily oral doses of 24 mg/kg body wt. day-1 (this dose is close to the mean hepatocarcinogenic dose in male Wistar rats: 34.1 mg/kg body wt. day-1). In the case of both pesticides the early effects observed consisted of hepatomegaly accompanied by an increase in the p-nitroanisole O-demethylase activity and hepatocyte proliferation. Hepatocyte proliferation was elevated during the total experimental period. Vacuolated cytoplasm and evident focal necrosis may suggest that the maximal increase in hepatocyte proliferation, preceding hepatomegaly, is at least partly related to a regenerative liver response to pesticides. In addition to the above-mentioned early changes, the present findings provide new evidence for the occurrence of dose-dependent abnormal mitoses (and c-mitoses) in the hepatocytes of the bromopropylate and DDT treated rats. PMID- 8631125 TI - Fibre-induced lipid peroxidation leads to DNA adduct formation in Salmonella typhimurium TA104 and rat lung fibroblasts. AB - Certain end-products of lipid peroxidation bind to DNA forming a fluorescent chromophore. Incubation of both Salmonella typhimurium TA104 and a rat lung fibroblast cell line, RFL-6, with various types of mineral fibre resulted in a time- and dose-dependent increase in DNA fluorescence. The increase in DNA fluorescence was shown to be directly related to the amount of iron that could be mobilized from the fibre surface using in vitro studies in the absence of cells or bacteria. Crocidolite and man-made vitreous fibre-21 (MMVF-21) mobilized significant quantities of iron and were significantly more active than chrysotile and refactory ceramic fibre-1 (RCF-1). Fibre-induced malondialdehyde-DNA adduct formation, the fluorescent product, was increased by incubating cells with buthionine sulfoximine and ameliorated by co-treatment with N-acetylcysteine, indicating a protective role for glutathione. Similarly, vitamin E was also shown to inhibit DNA adduct formation. These results suggest that mineral fibre-induced lipid peroxidation produced genotoxic products which can diffuse into nucleus and interact with cellular DNA. In conclusion, fibre-induced lipid peroxidation may be a possible mechanism in the genotoxic action of fibrous materials. PMID- 8631126 TI - Increased c-fos mRNA and binding to the AP-1 recognition sequence accompanies the proliferative response to deoxycholate of HT29 cells. AB - To further understand the molecular mechanisms of bile acid-mediated colon tumor promotion, we have examined the possible role of AP-1 activity in this process. The AP-1 complex has been reported to play an important role in control of cell growth. Our studies show that lithocholate, deoxycholate and ursodeoxycholate exhibited marked proliferative effects on a human adenocarcinoma cell line (HT29), while cholate was without effect. The proliferative effects appeared to be confined to narrow concentration windows which differed for the different bile acids. We demonstrate that deoxycholate caused an increase in expression of c-fos mRNA and increased binding to the AP-1 site, effects which were maximum at the concentration at which the bile acid induced the maximum proliferative effect on the cells. Cholate was without effect on AP-1 binding activity. In addition, we show that the AP-1 complex induced by treatment of the cells with the bile acid contained the c-fos protein. This could suggest that prolonged deregulated expression of AP-1 activity in colonic cells by certain bile acids may contribute to tumor promotion in the colon. PMID- 8631128 TI - Autoregulation of human CYP1A1 gene promotor activity in HepG2 and MCF-7 cells. AB - Cytochrome CYP1A1 gene expression, induced by polycyclic aromatic hydrocarbons and dioxins, eg. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is regulated mainly at the level of transcription. Inducible activation of the CYP1A1 promotor is mediated by a ligand-dependent transcription factor dimer complex including the aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) proteins. Additional factors seem to be involved in tissue- and cell-specific modification of the induction process. In the present study HepG2 and MCF-7 cell lines were used to examine a possible cell-specific autoregulation of CYP1A1 promotor function. Chimeric CYP1A1-CAT reporter constructs and a human CYP1A1 cDNA expression plasmid were used in transient co-expression experiments. In HepG2 cells co-expression of increasing amounts of CYP1A1 cDNA significantly down regulated constitutive as well as the TCDD-induced CYP1A1 promotor driven CAT activity. In contrast, co-transfection of MCF-7 cells with a 3-fold molar excess of CYP1A1 cDNA relative to the CYP1A1-CAT reporter construct caused an approximately 2-fold increase in the TCDD-induced CAT activity, whereas no effect was observed on constitutive promotor activity. This autoregulatory mechanism(s) of the human CYP1A1 gene product was independent of specific 5' flanking promotor segments tested. RT-PCR analyses did not indicate any changes in mRNA level of AHR and ARNT in the co-transfection studies. Thus these studies show that the human CYP1A1 gene is exposed to cell-specific autoregulation, probably achieved via different functions of trans-acting factors. PMID- 8631127 TI - Induction of the transcription factor AP-1 in cultured human colon adenocarcinoma cells following exposure to bile acids. AB - We studied the effects of bile acids on inducibility of the transcription factor AP-1 in human colon carcinoma LoVo cells. Firstly, cells were treated with chenodeoxycholic acid and the nuclear extracts from those cells were processed by electrophoretic mobility shift assays to analyze nuclear AP-1 DNA-binding activity. We demonstrated that chenodeoxycholic acid induced AP-1 DNA-binding activity in a dose- and time-dependent fashion. Antibody supershift experiments clearly revealed that the majority of protein components in induced AP-1 DNA binding activity were the products of oncogenes c-fos and c-jun. On the other hand, DNA-binding activity in the nuclear extracts for either NF kappa B, Sp1, or ATF/CREB was not affected by bile acids, suggesting that the effect of bile acids was rather specific for AP-1. Transient transfection experiments supported this notion: expression of the AP-1-luciferase reporter construct was induced by bile acids in a dose-dependent manner, and expression of either reporter construct for NF kappa B, Sp1, or ATF/CREB was not influenced by treatment of the cells with bile acids. We also demonstrated that those bile acids efficiently activated AP-1 dependent promoter in DLD-1 cells, which (as well as LoVo cells), are derived from colon adenocarcinoma, but not in COLO320DM cells which are from colon carcinoid tumor. Thus, we may indicate that bile acids exclusively induce nuclear AP-1 activity in colon adenocarcinoma cells. PMID- 8631129 TI - TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells. AB - Induction of PAI-2 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been studied in human primary hepatocytes, hepatoma HepG2 cells and monocytic U937 cells, extending recent findings in human keratinocytes. PAI-2 represents a serpine-type protease inhibitor with wide-ranging implications in fibrinolysis, extracellular matrix proteolysis, growth factor activation and carcinogenesis. PAI-2 was induced by >10(-9) M TCDD in hepatocytes and HepG2 cells and by >10(-10) M TCDD in U937 cells. In the latter cell line, PAI-2 induction by TCDD and by 12-O tetradecanoyl phorbol-13-acetate (TPA) has been compared. TCDD appeared to be less efficient than TPA as an inducer of PAI-2. In contrast to induction by TPA, PAI-2 induction by TCDD was found to be biphasic, with an early peak of mRNA at 1 3 h and a late peak at 12-24 h. A biphasic response was also seen at the protein level although production of PAI-2 protein lagged behind the corresponding mRNA. PAI-2 is known to contain AP-1 sites, i.e. Jun/Fos protein-binding sites, in its promotor region. Hence, PAI-2 induction by TCDD has originally been conceived to be due to an indirect response, secondary to the induction of Jun/Fos proteins. Therefore, expression of jun/fos genes and their AP-1 activity were studied at the early phase of PAI-2 induction by TCDD. TCDD did not increase mRNA of c-fos, c-jun, junB or junD (in contrast to TPA which markedly increased the expression of c-fos and junB), nor did TCDD increase AP-1 activity. In conclusion, the findings suggest that PAI-2 induction by TCDD is not restricted to human keratinocytes but includes liver cells and monocytic U937 cells. The induction mechanism is complex but the early phase does not appear to involve Jun/Fos proteins. PMID- 8631132 TI - Inhibition by acetylsalicylic acid, a cyclo-oxygenase inhibitor, and p bromophenacylbromide, a phospholipase A2 inhibitor, of both cirrhosis and enzyme altered nodules caused by a choline-deficient, L-amino acid-defined diet in rats. AB - Effects of inhibitors of arachidonic acid (AA) metabolism on the development of fatty liver, cirrhosis, glutathione-S-transferase placental form (GST-P)-positive nodules and the generation of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances (TBARS), caused by a choline-deficient, L-amino acid defined (CDAA) diet, were examined in male Fischer 344 rats by feeding CDAA diets supplemented with the inhibitors for 12 and 30 weeks. Acetylsalicylic acid (ASA) (at doses of 0.1 and 0.2%) and p-bromophenacylbromide (BPB) (0.1 and 0.2%) were used as inhibitors of, respectively, cyclo-oxygenase and phospholipase A2, and quercetin (QU) (0.75 and 1.5%) and nordihydroguaiaretic acid (NDGA) (0.1 and 0.2%) as inhibitors of lipoxygenase. None of the inhibitors affected the development of fatty liver caused by the CDAA diet. ASA at a doe of 0.2% almost completely prevented the appearance of cirrhosis, GST-P-positive nodules, 8-OHdG and TBARS in seven out of 11 (63.7%) rats. BPB at a dose of 0.2% also exerted inhibitory effects on all of these lesions but to a lesser extent than ASA. QU and NDGA exerted inhibitory effects limited to the GST-P-positive nodule case. The results indicate that a perturbed AA metabolism, particularly of the cyclo oxygenase pathway, derived secondarily from depletion of labile methyl groups or phosphatidylcholine, might play key roles in the cirrhosis, hepatocarcinogenesis and oxidative stress caused by a CDAA diet. The results also indicated a possible involvement of the lipoxygenase pathway in hepatocarcinogenic processes. PMID- 8631130 TI - Regulation of mdr1b gene expression in Fischer, Wistar and Sprague-Dawley rats in vivo and in vitro. AB - Administration of 2-acetylaminofluorene (2-AAF) to rats increased mdr1b expression in Fischer, Wistar and Sprague-Dawley rat livers; however, the response was much smaller in Sprague-Dawley livers. To investigate the basis of this difference we further examined the regulation of the mdr1b gene in hepatocytes isolated from Fischer, Sprague-Dawley and Wistar rats. A time dependent increase in basal expression of mdr1b but not mdr2 was observed in hepatocytes isolated from all three strains of rats. After 4 days in culture, a larger increase in mdr1b mRNA levels was observed in Fischer and Wistar rat hepatocytes (3.5- and 4.6-fold respectively) than Sprague-Dawley hepatocytes (2 fold). Treatment of primary hepatocytes with 2-AAF caused an induction of mdr1b expression that varied among the three strains. Notably, Sprague-Dawley hepatocytes were not responsive to 2-AAF. In contrast to the parent compound, the electrophilic metabolites N-hydroxy-2-acetylaminofluorene and N-acetoxy-2 acetylaminofluorene caused a dose-dependent induction of mdr1b expression in both Fischer and Sprague-Dawley hepatocytes, indicating that differences in the metabolic activation of 2-AAF between the strains may account for the differences in mdr1b by 2-AAF. Hepatocytes isolated from all three strains of rats showed an equivalent induction of mdr1b after treatment with cycloheximide. Nuclear run-on assays demonstrated that the increases in mdr1b expression with time in culture and after xenobiotic treatment were due to increased transcription. PMID- 8631131 TI - Effect of acetylator genotype on 3,2'-dimethyl-4-aminobiphenyl induced aberrant crypt foci in the colon of hamsters. AB - Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in both rodent and human carcinogenesis. The colon carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMAB), like other arylamines, undergoes N-acetylation and O-acetylation by polymorphic acetyltransferase (NAT2). In the present study we characterized ACF in hamster colon for the first time and compared the ability of DMAB to induce ACF in homozygous rapid and slow acetylator congenic Syrian hamsters (Bio 1.5/H NAT2r and Bio 1.5/H-NAT2s, respectively), differing only at the NAT2 gene locus and other closely linked loci. The animals received DMAB (75 mg/kg body weight s.c.) or vehicle (PBS/DMSO 1:1) as a control, twice weekly for 2 weeks, then once a week for 4 weeks. Ten weeks after the first injection ACF were observed in the DMAB treated hamsters, but not in the controls. However, the number of ACF was three times higher (P=0.016) in the colons of the NAT2r hamsters compared with the colons of the NAT2s hamsters. In the two congenic hamster lines we also studied the induction of ACF with 1,2-dimethylhydrazine (DMH) treatment, a colon carcinogen not metabolized by NAT2. Hamsters given DMH (25 mg/kg body weight s.c.), once a week for 3 weeks, showed ACF induction in the colon after 10 weeks, but there was no difference between the NAT2r and NAT2s hamsters. Further scanning electron microscopic and histological examination of ACF observed with the light microscope, revealed the same gross morphology and therefore confirmed the basis for the scoring of ACF. The ACF in hamster colons were principle similar to the lesions observed in other species. PMID- 8631133 TI - A prospective cohort study on the relationship between onion and leek consumption, garlic supplement use and the risk of colorectal carcinoma in The Netherlands. AB - The association between onion and leek consumption, garlic supplement use and colon and rectum carcinoma among men and women was evaluated in the Netherlands Cohort Study, a large-scale prospective cohort study on diet and cancer. Onions, leeks, and garlic belong to the Allium genus and contain large amounts of potentially chemopreventive compounds. The Netherlands Cohort Study was started in 1986 among 120 852 men and women, aged 55-69 years. Dietary intake was measured with a 150-item food frequency questionnaire. After 3.3 years of follow up, 150 and 143 incident male and female cases of colon carcinoma, and 93 and 57 cases of rectum carcinoma, respectively, with complete dietary data were available for analysis. Dietary data were available for 1525 men and 1598 women of a randomly selected subcohort, that was followed up to estimate person-time in the entire cohort. In men, the adjusted rate ratios (RRs) in multivariable analysis for colon and rectum carcinoma in the highest compared to the lowest onion consumption categories were 0.87 (95% confidence interval [CI] = 0.48 1.65), and 0.66 (95% CI = 0.28-1.52), respectively. The RRs for proximal colon carcinoma were lower than for distal colon carcinoma. Leek consumption was not associated with colon and rectum carcinoma incidence in men. None of the RRs were significantly different from unity and no trends in the RRs were detected. A lower risk was found for rectum carcinoma in women consuming less than 0.25 onions per day (RR=0.36, 95% CI = 0.13-0.99), but the trend in the RRs was not statistically significant (P = 0.25). All other RRs for colon and rectum carcinoma associated with onion consumption were slightly higher than one. Leek consumption was not associated with colon and rectum carcinoma incidence. The use of garlic supplements was not associated with colon and rectum carcinoma in men and women combined. This study does not support an inverse association between the consumption of onions and leeks, or the use of garlic supplements and the incidence of male and female colon and rectum carcinoma. PMID- 8631134 TI - Separation of 7-methyl- and 7-(2-hydroxyethyl)-guanine adducts in human DNA samples using a combination of TLC and HPLC. AB - We have used a combination of thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) to achieve separation of 32P-postlabelled 7 methylguanine and 7-(2-hydroxyethyl)-guanine adducts. The level of these two adducts was determined in human total white blood cells (mean values 0.7 to 1.5 adducts per 10(7) normal nucleotides) and isolated lymphocytes (mean values 1.1 to 12 adducts per 10(7) normal nucleotides). The separation of these two adducts revealed that the level of 7-(2-hydroxyethyl)-guanine was twice the level of 7 methylguanine adducts in total white blood cells, whereas, in isolated lymphocytes it was at least four times more than the 7-methylguanine adduct. The combined level of these two adducts in the lymphocytes of non-smokers was 1.1 to 8.4 adducts per 10(7) normal nucleotides and in the lymphocytes of smokers, the level was 5.6 to 12 adducts per 10(7) normal nucleotides. We also report detection of three unidentified adducts in the samples analysed, and at least one of these adducts seemed to be related to smoking. The chromatographic behaviour and depurination at neutral pH indicated the probable 7-alkylguanine or 3 alkyladenine nature of these unidentified adducts. PMID- 8631135 TI - Effect of vitamin-antioxidant micronutrients on the frequency of spontaneous and in vitro gamma-ray-induced micronuclei in lymphocytes of donors: the age factor. AB - The effect of prolonged consumption of a vitamin-antioxidant mixture (VAM) on the frequency of spontaneous and in vitro gamma-radiation-induced micronuclei (MN) in peripheral blood lymphocytes in donors of various ages was investigated. Three groups of donors were recruited: (i) 56-83 years old (35 subjects), (ii) 23-30 years old (13 subjects), and (iii) 63-82 years old (12 subjects). Blood was sampled every 4 months for one year in all donors of the three groups. After the first sampling of blood, the donors of groups (i) and (ii) took VAM containing the vitamins A, C, E, as well as beta-carotene, folic acid, and rutin daily for 4 months. After the second blood sampling, the intake of VAM was terminated. The third blood sample was taken 4 months after termination of VAM intake. A part of the blood was exposed to gamma-radiation and the frequency of spontaneous and induced MN in lymphocytes was assayed. The analyses showed that the frequency of spontaneous and in vitro gamma-ray-induced MN in aged donors was significantly higher than that in young donors. No seasonal variations in MN frequency were observed in human lymphocytes during one year. Aged donors showed a statistically significant decrease in spontaneous MN in lymphocytes after a 4 month period of consumption of VAM. The intake of VAM by both aged and young donors promoted a decrease in MN induced lymphocytes in vitro by gamma-radiation. The results of our observations enable the suggestion that consumption of VAM favours a decrease in the chromosome damage produced by endogenous and exogenous factors in human lymphocytes. PMID- 8631136 TI - Tobacco smoke-associated N7-alkylguanine in DNA of larynx tissue and leucocytes. AB - The presence of N7-alkylguanine adducts in DNA was analysed in a group of 46 patients with larynx tumours. All patients were subjected to laryngectomy and the tissues accessible for analysis by (32)P-post-labelling assay were larynx tumour, larynx non-tumour and peripheral blood leucocytes. N7-Alkylguanine adducts were detected in all the studied DNA samples. The average level of N7-alkylguanines was 26.2/10(7) nucleotides in tumour cells, 22.7/10(7) in non-tumour cells and 13.1/10(7) in blood leucocytes. There was significantly higher level of N7 alkylguanines in the larynx tissues in males than in females. The effect of tobacco smoking on DNA adduct levels was shown by an increase in the average levels of N7-alkylguanines in the subject groups classified according to their smoking habits. A moderate age-related increase in levels of N7-alkylguanine was demonstrated in larynx tumour tissue. The levels of N7-alkylguanine adducts in larynx cells were compared with that of aromatic DNA adducts. Pearson correlation coefficients (0.28 for tumour tissue and 0.30 for non-tumour tissue) indicate independent formation and removal of N7-alkylguanine and aromatic DNA adducts resulting from tobacco smoke exposure. PMID- 8631137 TI - Lack of bioavailability of dichlorobenzidine form diarylide azo pigments: molecular dosimetry for hemoglobin and DNA adducts. AB - The hypothetical release of 3,3'-dichlorobenzidine (DCB) from two insoluble azo pigments and from a soluble azo dye was investigated in female Wistar rats for a 4 week treatment with 0.2% (w/w) Colour Index Pigment 13 (PY13) or 0.2% (w/w) Colour Index Pigment Yellow 17 (PY17) in the diet or 0.06% (w/v) Colour Index Direct Red 46 (DR46) in the drinking water. Steady-state DCB-hemoglobin adduct levels were determined by GC/MS with negative chemical ionization as well as DCB DNA adduct levels in the liver by (32)P-postlabelling and compared with the respective adduct levels obtained in animals after treatment for 4 weeks with 0.00024, 0.0012 or 0.006% (w/v) DCB in the drinking water. A dose-proportional increase in adduct levels from 8.1 ng/g hemoglobin and 2.6 ng/g DNA (relative adduct level, RAL, 3.3x10(-9)) to 160 ng/g hemoglobin and 45.4 ng/g DNA (RAL 56.1x10(-9)) was observed in the DCB-treated rats. In rats treated with DR46 total adduct levels of 17.7 ng/g hemoglobin and 5.2 ng/g DNA (RAL 6.4x10(-9))were determined. No hemoglobin of DNA adducts were found in rats treated with PY17 in the diet, at a limit of detection of 0.1 ng/g hemoglobin and 0.08 ng/g DNA (RAL 0.1x10(-9)). In animals treated with PY13 in the diet no adducts or only minimal amounts slightly above the limit of detection could be identified. Taking into consideration that PY13 was contaminated with 0.02% of the respective soluble monoazo compound, it is concluded that the small amounts of DCB detected have been released from the contaminating soluble monoazo compound and not from insoluble PY13. The results of the present study demonstrate the lack of bioavailability of DCB from the diarylide azo pigments PY17 and PY13. PMID- 8631138 TI - Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer? AB - High red meat diets have been linked with risk of sporadic colorectal cancer; but their effects on mutations which occur in this cancer are unknown. G-->A transitions in K-ras occur in colorectal cancer and are characteristic of the effects of alkylating agents such as N-nitroso compounds (NOC). We studied th effect of red meat consumption on faecal NOC levels in eight male volunteers who consumed diets low or high in meat (60 or 600 g/day), as beef, lamb or pork, whilst living in a metabolic suite. Increased intake of red meat induced a significant (P<0.024) 3-fold increase from 40 + or - 7 to ab average of 113 + or 25 microgram/day NOC, a range of exposure in faeces similar to that from tobacco specific NOC in cigarette smoke. THe diets were isoenergetic and contained equal amounts of fat, but concentrations of heterocyclic amines were low. Faecal excretion of the promotor ammonia was significantly increased to 6.5 + or - 1.08 mmol/day. When the high red meat diets were supplemented with 20 g phytate-free wheat bran in six volunteers there was no reduction in NOC levels (mean 138 + or 41 microgram/day NOC), but faecal weight increased. Higher starch and non-starch polysaccharide intakes reduced intraluminal cross-linking in microcapsules (r= 0.77) and reduced faecal pH (r=-0.64). In two volunteers there was no effect of 600 g white meat and fish o faecal NOC (mean low white meat diet 68 + or - 10 microgram/day, high white meat 56 + or -6 microgram/day nor on faecal nitrate, nitrite and iron. Faecal nitrite levels increased on changing from a white to red meat diet (mean high white meat diet 46 + or - 7 mg/day, high red meat diet mean 80 + or - 7 mg/day.) Increased endogenous production of NOC and precursors from increased red meat, but not white meat and fish, consumption may be relevant to the aetiology of colorectal cancer. PMID- 8631139 TI - Effect of sulphur mustard on the initiation and elongation of transcription. AB - Sulphur mustard is a potent alkylating agent that causes severe vesication as well as systemic and genotoxic effects. Despite its long history as a chemical warfare agent, the mechanism of its toxicity remains unknown and no successful pharmacological intervention has yet been found. In this study we have examined the effects of mustard alkylation of DNA on transcriptional processes. Gel mobility shift analysis shows that mustard alkylation of the lac UV5 promoter increases the stability of the promoter-RNA polymerase binary complex. Following formation of the initiation complex and addition of elongation nucleotides, approximately 45% of the RNA polymerase in the initiated complex remained associated with the alkylated promoter, compared to only 7% remaining associated with the unalkylated promoter. For the RNA polymerase able to escape the initiation complex, mustard alkylation of the DNA template resulted in the production of truncated transcripts. Analysis of these truncated transcripts revealed that sulphur mustard alkylates DNA preferentially at 5'-AA, 5'-GG and 5' GNC sequences on the DNA template strand and this is significantly different from the alkylation sites observed with nitrogen mustard. This study represents the first report at the molecular level of sulphur mustard-induced effects on transcriptional processes. PMID- 8631140 TI - Characterization of bacterial cytochrome cd(1)-nitrite reductase as one enzyme responsible for catalysis of nitrosation of secondary amines. AB - Bacterial formation of carcinogenic N-nitroso compounds may play a role in the etiology of human cancer. Biochemical and immunological studies in denitrifying bacteria (Pseudomonas aeruginosa) strongly support the identification of cytochrome cd(1)-nitrite reductase as the enzyme responsible for the catalysis of nitrosation through the production of nitric oxide or NO(+)-like species. Interestingly, electron paramagnetic resonance studies have shown that large quantities of nitric oxide or NO(+)-species were also produced by non denitrifying enterobacteria (Escherichia coli, Proteus morganii). PMID- 8631141 TI - Effects of oxygen radical scavengers on connexins 32 and 26 expression in primary cultures of adult rat hepatocytes. AB - Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear. It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals. In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA). DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not. The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU. Furthermore, extensive GJIC was also observed with DMSO and DMTU. These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression. PMID- 8631142 TI - Iron bound to the lipophilic iron chelator, 8-hydroxyquinoline, causes DNA strand breakage in cultured lung cells. AB - The formation of DNA-strand breaks was studied in cultured human lung cells (A 549) subjected to iron, either in the form of iron(III) citrate or in combination with the metal chelators ethylene diamine tetra-acetic acid (EDTA), nitrilo triacetic acid (NTA), or 8-hydroxyquinoline (8HQ). After 15 min exposure to 5 microM iron(III) citrate or iron chelate, the cellular levels of iron were found to be three times higher in cells subjected to iron-8HQ than in cells subjected to iron(III) citrate, iron-EDTA or iron-NTA. Exposure to iron-8HQ caused extensive DNA-strand breakage, whereas no such breakage was found in cells exposed to iron-EDTA or iron-NTA. The DNA damage caused by iron-8HQ increased with time and dose, and DNA-strand breakage was clearly demonstrable in cells after 15 min exposure to as little as 0.1 microM iron-8HQ. Moreover, iron-8HQ was strongly toxic to the cells and inhibited their growth after exposure. Along with the formation of DNA-strand breaks, the concentration of cellular malondialdehyde increased four-fold after exposure to iron-8HQ and two-fold after exposure to iron-EDTA or iron-NTA, suggesting that reactive oxygen metabolites might be involved in the toxic action. Moreover, both iron-EDTA and iron-NTA caused a considerable hydroxylation of deoxyguanosine (dG) residues in DNA in vitro, whereas iron(III) citrate and iron-8HQ only caused a minor hydroxylation of dG. This points to the possibility that iron-8HQ-mediated DNA-strand breakage in cells might be due to the action of a metal-bound oxyl radical formed from the iron-8HQ complex rather than to the formation of hydroxyl radicals. Altogether, these findings indicate that iron bound to the lipophilic chelator, 8HQ, has strong toxic properties and that it may cause substantial DNA-strand breakage and lipid peroxidation in living cells. PMID- 8631143 TI - Induction of micronuclei and initiation of enzyme-altered foci in the liver of female rats treated with cyproterone acetate, chlormadinone acetate, or megestrol acetate. AB - The synthetic anti-androgen and progestin cyproterone acetate (CPA), recently found to be genotoxic for the liver, and two structurally similar progestins, chlormadinone acetate (CMA) and megestrol acetate (MGA), have been compared for clastogenic and tumor-initiating activities in female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg/kg, CPA induced the maximum increase in the frequency of micronucleated hepatocytes (6.6-fold as compared to controls) when treatment was performed 3 days before partial hepatectomy and cell sampling 2 days later. Under the same experimental conditions the clastogenic potencies of CMA and MGA were 69% and 36% of that of CPA respectively. In the liver foci assay, p.o. dosing with 100 mg/kg CPA once a week for 6 successive weeks induced, as compared to controls, a significant increase in the number and area of gamma-glutamyltranspeptidase-positive foci. At the same dosage schedule the tumor-initiating activity of CMA and MGA was 7- to 10-fold lower than that of CPA. These findings suggest that the 1,2 alpha methylene group, present in CPA but absent in both CMA and MGA, favours the activation to a reactive species and/or hinders the biotransformation to non toxic metabolites. PMID- 8631144 TI - Developmental changes in hepatic activation of 2-amino-3,8-dimethylimidazo[4,5 f]quinoxaline in rabbit. AB - MeIQx, a potent bacterial mutagen formed when meat is cooked, requires metabolic activation to exert its genotoxicity, a reaction catalysed primarily by CYP1A2 in adult mammals. Little is known about mammalian developmental changes in the mutagneic activation of compounds such as MeIQx. In rabbits we have shown previously that expression of CYP1A2 increases with age and is inducible by 3 methylcholanthrene (MC) from as early as 4 days pre-parturition. We have therefore investigated the effect of age on rabbit liver activation of MeIQx (assessed in an Ames test using Salmonella typhimurium TA98) before and after treatment of the animals with MC. MeIQx activation could not be detected using hepatic microsomal fractions from rabbits of <17 days of age. Thereafter activation increased with age to peak in weanling animals. Following MC treatment MeIQx activation was increased, being detectable in samples from rabbits as young as 9-11 days. The inducibility of MeIQx activation increased with age, reaching a plateau between 17 and 35 days. These rates of activation were broadly parallel to the changes in CYP1A2 specific content. These results indicate that the ability of rabbit liver to activate MeIQx is dependent on CYP1A2 activity, the expression of which is developmentally regulated. Although it has been established that human activation of MeIQx is also CYP1A2 dependent, whether a similar situation exists in infant humans has yet to be determined, although there is evidence that CYP1A2-dependent activity reaches a peak in late childhood. PMID- 8631145 TI - Ascorbic acid may protect against human gastric cancer by scavenging mucosal oxygen radicals. AB - High dietary ascorbic acid intake appears to protect against gastric cancer. This may be due to its action as a scavenger of reactive radical species formed in the gastric mucosa, resulting in a reduced level of radical-mediated DNA damage. We have studied 82 patients, of whom 37 had Helicobacter pylori-associated gastritis, a condition which predisposes to gastric cancer. Using electron paramagnetic resonance (EPR) spectroscopy we have demonstrated, for the first time, that ascorbyl radicals are generated in human gastric mucosa, presumably as a result of scavenging of free radicals by ascorbic acid. Quantification of ascorbyl radicals demonstrates that there is a higher concentration in those patients with H.pylori gastritis compared with subjects with normal histology (P < 0.01). We also found gastric mucosal luminol-enhanced chemiluminescence and malondialdehyde concentrations (which are believed to be markers of radical generation and tissue damage) to be higher in patients with H.pylori gastritis compared with those with normal histology (P < 0.001 and P < 0.01 respectively). The observed concentrations of the ascorbyl radical correlate with the level of luminol-enhanced chemiluminescence (r = 0.41, P < 0.001), but not with malondialdehyde concentrations (r = 0.08, P = 0.47). Mucosal ascorbic acid and total vitamin C concentrations did not vary between histological groups, nor did they correlate with mucosal levels of the ascorbyl radical, chemiluminescence or malondialdehyde. These data suggest that ascorbic acid is acting as a scavenger of free radicals generated in human gastric mucosa. The experiments therefore provide direct supportive evidence for the hypothesis that ascorbic acid protects against gastric cancer by scavenging reactive radical species which would otherwise react with DNA, with resultant genetic damage. PMID- 8631146 TI - Identification of two novel cellular genes associated with multistage carcinogenesis of human endocervical cells by mRNA differential display. AB - Carcinogenesis originating from cervical cells has been recognized as a multistage process in which human papillomavirus (HPV) infection and cofactors, such as cigarette smoke, are frequently involved in the development of malignant cancer. However, the molecular mechanism underlying this process remains poorly understood. To identify the cellular genes involved in multistage cervical oncogenesis, we used the mRNA differential display method to analyze primary human endocervical cells (HEN), HPV 16-immortalized cells (HEN-16) and cigarette smoke condensate (CSC)-transformed cells (HEN-16T). Two cDNAs--PA4 and PA9--have been identified and isolated after comparing 8000 cDNAs from HEN, HEN-16 and HEN 16T. Northern blot analysis showed that PA4 was expressed 2- to 3-fold higher in HEN-16 and HEN-16T than in HEN, whereas PA9 was expressed uniquely in HEN. Moreover, the same patterns of PA4 and PA9 expression were found for a second line of HPV 16-immortalized endocervical cells and the corresponding transformed cells. An analysis of cDNA sequences showed that PA4 and PA9 had no homology to nucleotide sequences in Genbank. We suggest that immortalization, but not tumorigenesis, up-regulated a new oncogene, PA4, and down-regulated a new tumor suppressor gene, PA9. These results demonstrated the utility of the human endocervical cell model system and the mRNA differential display method for identifying the genes that may be involved in multistage cervical carcinogenesis. PMID- 8631147 TI - Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes. AB - The involvement of cytochrome P450 in the cytotoxicity and DNA damage-repair induced by N-nitrosodipropylamine (NDPA), N-nitroso-n-butyl-n-propylamine (NBPA), and N-nitrosodibutylamine (NDBA) was investigated in cultured hepatocytes isolated from untreated, phenobarbital (PB)- and pyridine (PYR)-pretreated rats. Pretreatment of rats with PB caused a 10-fold increase in the sensitivity of hepatocytes to the cytotoxic actions of NDPA, NBPA and NDBA as measured by trypan blue exclusion, whereas PYR pretreatment increased the sensitivity of hepatocytes to NDPA and NBPA, but not to NDBA. This elevated sensitivity correlated well with increased 7-pentoxyresorufin depentylase activity catalyzed by P450 2B1 in cultures from PB-pretreated rats and enhanced p-nitrophenol hydroxylase activity of P450 2E1 in cultures of hepatocytes from PYR-pretreated rats. Unscheduled DNA synthesis showed that DNA damage-repair was significantly increased in freshly isolated hepatocytes from PB- and PYR-pretreated rats. With increasing time in culture, however, there was a marked reduction in the DNA damage repair response, concomitant with a decrease in the cytotoxicity of NDPA, NBPA and NDBA in primary cultures of hepatocytes. Coincident with this, a rapid loss in the specific activities of P450 2B1 and 2E1 was detected during the first 48 h in all primary cultures. Although N-nitrosodimethylamine (NDMA), used as a positive control, produced high nuclear grain counts in cultures from PYR-pretreated rats, the toxic effect of NDMA in rat hepatocytes was much weaker than that observed with NDPA, NBPA and NDBA. This result suggests that the type of DNA damage or repair efficiently induced by NDPA, NDBA or NDBA might differ from that due to NDMA. PMID- 8631148 TI - Malignant transformation of human ectocervical cells immortalized by HPV 18: in vitro model of carcinogenesis by cigarette smoke. AB - In addition to the established role of human papillomaviruses (HPVs) in cervical cancer, smoking has been suggested to be an important cofactor. Previously, primary human ectocervical cells immortalized by HPV types 16 and 18 DNA did not form tumors on nude mice. Here, we derived a new line of HPV 18-immortalized ectocervical cells (HEC-18-1), which was also non-tumorigenic. To examine the role of cigarette smoking in the progression of cervical cancer initiated by HPV 18, we adapted these cells to growth in serum and high calcium and treated the cells with cigarette smoke condensate until tumorigenic cells (HEC-18-1C) were produced. Moderate and late passage serum-adapted untreated HEC-18-1 (HEC-18-1S) remained non-tumorigenic. A typical HEC-18-1C tumor was an invasive squamous cell carcinoma, from which we established a clonal line of cells (HEC-18-1CT). Although the physical state of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was affected little, the differentiation of the epithelium derived in organotypic (raft) culture from HEC 18-1CT was altered dramatically. Moderate and late passage HEC-18-1 and HEC-18-1S were reconstructed into mild dysplasia in organotypic (raft) culture. On the other hand, the moderate passage malignantly transformed HEC-18-1CT displayed severe dysplasia/carcinoma in situ in raft culture. We describe here the first direct evidence of the role of cigarette smoke in the progression of HPV initiated carcinogenesis using an in vitro model system. PMID- 8631149 TI - The effect of tamoxifen and two of its non-isomerizable fixed-ring analogs on multistage rat hepatocarcinogenesis. AB - Long-term treatment of breast cancer patients with tamoxifen has prompted concern over potential toxicity of this drug with chronic administration. Since tamoxifen has estrogenic action in the rat liver and estrogenic agents can increase hepatoma incidence in rats, tamoxifen and two non-isomerizable, fixed-ring analogs (FRT1 and FRT2) were evaluated as promoting agents in a two-stage model of hepatocarcinogenesis in female Fischer F344 rats. The rats were subjected to 70% partial hepatectomy and half of the animals were administered the initiating agent, diethylnitrosamine (DEN; 10 mg/kg body wt), while the other half were not initiated. Groups of initiated and uninitiated animals were allowed to recover for 2 weeks and were then administered tamoxifen or one of the fixed-ring analogs admixed into AIN-76A diet at 25, 100 or 250 mg/kg diet. After 6 months of anti estrogen administration the rats were sacrificed and uterine weights, blood levels of anti-estrogen, and liver histopathology were assessed. Uterine weights were decreased 2- to 3-fold by each of the agents, consistent with an anti estrogenic action in the rat. The serum levels in rats administered 250 mg anti estrogen/kg diet for 6 months were 320+/-20 ng/ml for tamoxifen, 320+/-10 for FRT1 and 350+/-20 for FRT2. The liver levels after a 6 month administration of 250 mg anti-estrogen/kg diet were 13 870+/-860 ng/g for tamoxifen, 13 300 +/-860 for FRT1 and 26 900+/-1900 for FRT2. A dose-dependent increase in serum and liver level of each compound was noted when measured at the 6 month time period. The number and percentage of the liver occupied by altered hepatic foci (AHF) were determined by quantitative stereology. A dose-dependent increase above initiated controls was observed in the initiated, tamoxifen-treated rats. Both fixed-ring analogs also increased the number and size of AHF compared with initiated controls, but were less potent than tamoxifen, suggesting that tamoxifen has an intrinsic promoting action in the liver that is independent of its ability to isomerize to more potent estrogenic compounds. In addition, the fixed-ring analogs have a weaker promoting activity in the rat liver than does tamoxifen. This may be due to pharmacokinetic differences at the lower two doses, but it is independent of achieved serum level at the highest dose and hence may reflect differences in intrinsic activity of these compounds. Thus tamoxifen and the two fixed-ring analogs promote the development of rat hepatocarcinogenesis. PMID- 8631150 TI - p53 and Ha-ras mutations in chemically induced hamster buccal pouch carcinomas. AB - Well-differentiated squamous cell carcinomas were induced in hamster buccal pouch epithelium by twice weekly topical applications of N-methyl-N-benzylnitrosamine (MBN) or 7,12-dimethylbenz[a]anthracene (DMBA) over a period of 15 weeks. Each of the 22 tumors induced (14 MBN and eight DMBA) were evaluated by single-strand conformation polymorphism and DNA sequencing to identify mutations in conserved exons (E5-E8) of the p53 tumor suppressor gene and codons 12/13 and 61 of Ha-ras. In addition, Northern blot analysis of 10 MBN tumors and five DMBA tumors was performed to determine whether the mdm-2 gene was overexpressed, p53 mutations were detected in five of 14 (35%) MBN-induced carcinomas and in two of eight (25%) DMBA-induced carcinomas. Ha-ras mutations were detected in three of 14 (21%) MBN-induced carcinomas and in three of eight (37%) DMBA-induced carcinomas. One MBN-induced carcinoma exhibited a mutation in both the p53 and Ha-ras genes. The majority (five of seven of p53/Ha-ras mutations induced by MBN were G->A transitions and two of these occurred at hamster p53 codon 248, which corresponds to human p53 codon 245, a known mutational tumor 'hot spot'. A->T transversion at Ha-ras codon 61 accounted for three of five (60%) DMBA-induced mutations. There was no evidence of mdm-2 overexpression in any of the tumors evaluated. Overall, the results provide additional support for the validity of the hamster buccal pouch model of oral carcinogenesis, as applied to sequential cellular and molecular analysis and cancer chemoprevention studies. PMID- 8631151 TI - High level expression of the multidrug resistance (MDR1) gene in the normal bladder urothelium: a potential involvement in protection against carcinogens? AB - It has been suggested that expression of the P-glycoprotein transmembrane efflux pump (PGP), encoded by the multi-drug resistance (MDR1) gene, may play a role in the protection of epithelial tissues from a variety of local and systemic toxins. We report that in approximately 50% (6/11) of the population, MDR1 messenger RNA levels in the normal urinary epithelium are comparable to those found in the highest expressing tissues in the body, and suggest a role for PGP in the normal bladder urothelium. MDR1 mRNA levels in the normal urothelium do, however, vary over a 60-fold range between individuals, and furthermore are uniformly significantly lower (about 6-fold, P 1000x) magnification. Each sample was initially scanned at low magnification to determine the presence and location of surface abnormalities. Photographs taken at medium and high magnification were of the area most representative of the abnormality. Nine samples (30%) were completely normal at all magnifications. Of the total viewed at low power, 18 (60%) were normal, 11 (37%) revealed ridging and 1 (3%) revealed melting. Of the total viewed at medium power, 14 (47%) were normal, 3 (10%) showed cracking, 2 (7%) showed melting or a combination of ridging and cracking, and 11 (37%) showed ridging. At high power, 10 (34%) were normal, 11 (38%) revealed either melting or a combination of two defects, 5 (17%) showed ridging, and 3 (10%) revealed cracking. Fifteen (50%) of the samples revealed a surface abnormality (cracking or melting) other than ridging. The majority of condoms viewed under scanning electron microscopy revealed surface abnormalities. The implication of the detected abnormalities needs further evaluations. While ridging may represent a minor abnormality associated with condom handling, multiple abnormalities or melting may represent a more significant atypicality. PMID- 8631189 TI - Can grapefruit juice influence ethinylestradiol bioavailability? AB - The effects of grapefruit juice on the bioavailability of 17 alpha ethinylestradiol (EE2) after a single oral administration of 50 micrograms EE2 have been investigated. The pharmacokinetics of EE2 were studied in an open, randomized, cross-over study in which 13 healthy volunteers were administered the drug with herbal tea or grapefruit juice (naringin, 887 mg/ml). In contrast to herbal tea, grapefruit juice increased the peak plasma concentration (Cmax) significantly to 137% (mean; range 64% to 214%, p = 0.0088) and increased the area under plasma concentration-time curve from 0 to 8 hours (AUC0-8) to 128% (mean; range 81% to 180%, p = 0.0186). This study shows that grapefruit juice increases the bioavailable amount of EE2. A possible explanation may be that grapefruit juice inhibits the metabolic degradation of EE2. Whether the increased bioavailability of EE2 following grapefruit juice administration is of clinical importance should be investigated in long-term studies. PMID- 8631193 TI - Optical immunosensors. AB - The development of biochemical detection systems that use antibodies as the recognition element, also known as immunosensors, has generated considerable interest from science and industry alike. This is due to the practically inexhaustible number of applications for which such a technology could conceivable lend itself, such as medicine, process control, and environmental monitoring. The potentially high sensitivity of such systems makes them ideal for situations where the utmost speed and accuracy are demanded. Moreover, there is theoretically no limit to the chemical and biological moieties for which an immunosensor can be manufactured. This critical review emphasizes both the biological and transduction aspects of optical immunosensors. The basic concepts of antibody-antigen interactions in solution are reviewed, and factors affecting the kinetics at the solid-liquid interface are characterized. In addition, the aspects of reagent immobilization, inherent to immunosensor design, are discussed. Finally, a comprehensive overview describing the mechanisms, advantages, and shortcomings of several immunosensing formats such as fiber optic, planar waveguide, surface plasmon resonance, and continuous-flow immunosensor is presented. The future of these sensors, we well as the integration of fluorescence lifetime sensing and fluorescent polarization technologies with immunosensing technologies, is contemplated, posing the question: What place will immunosensors secure in tomorrow's market? PMID- 8631191 TI - Inhibition of ovulation by progestin analogs (agonists vs antagonists): preliminary evidence for different sites and mechanisms of actions. AB - Continuous administration of the antiprogesterone RU486 inhibits ovulation in women and in monkeys; in this regard RU486 may act as a progestin agonist rather than as an antagonist. We compared the site(s) and mechanism(s) of RU486-induced ovulation inhibition with those of levonorgestrel (LNG). Six regularly menstruating cynomolgus monkeys each received placebo, RU486 (1 mg/kg/d) or LNG (2 g/kg/d) i.m. between days (cd) 2-22 of three separate menstrual cycles. Serum levels of estradiol (E2), progesterone (P4), androstenedione, LH and FSH were analyzed by RIAs in daily blood samples. Basal and GnRH-stimulated (1 and 50 g of GnRH i.v. 2 h apart) secretion of LH and FSH was assessed using serial blood samples collected for 12 h on cd 10. Mean cycle length was prolonged by RU486 and LNG treatments from 32 d to 70 d and 52 d, respectively (p < 0.02). Ovulation was inhibited in five of the six primates during RU486, and in all six during LNG treatment. During RU486 treatment, serum E2 levels were similar to those of the control cycle; despite peaks of E2 secretion, no LH peaks were seen. In contrast, E2 concentrations were profoundly suppressed during LNG treatment (p < 0.005). The reduction in serum E2 was accompanied by lower levels of androstenedione, and suppressed ratio of E2/androstenedione (p < 0.02) suggesting both reduced synthesis and aromatization of androgen precursors during administration of LNG. Consequently, LNG treatment was associated with higher levels of serum FSH and LH (p < 0.001; 1-way ANOVA). Similarly, as during the luteal phase of the menstrual cycle, the amplitude of basal LH-pulses was increased during LNG treatment (p < 0.05), whereas RU486 treatment did not affect basal LH secretion. The GnRH stimulated release of LH was similar during the placebo, RU486 and LNG cycles; enhanced release of FSH was seen during administration of LNG. Thus, in the present model system, RU486 seems to inhibit ovulation mainly at the level of hypothalamus, possibly by interfering with the steroidal positive feedback signals from the ovary. However, LNG inhibits ovulation differently, most likely via direct progesterone-like effects on folliculogenesis and the hypothalamus. The pituitary does not appear to be the major site of action(s) of RU486 or LNG. Thus, the differential mechanisms of ovulation inhibition by RU486 and LNG seem to result from lesser intraovarian impact of RU486 as well as dissimilar influences on tonic gonadotropin secretory levels. We conclude that when inhibiting ovulation, RU486 does not act as a progestin agonist, but rather, functions through a hypothalamic mechanism(s), which might be unique to RU486 as a progesterone antagonist. PMID- 8631194 TI - The mathematical basis for imaging cardia electrical function. AB - The essential difficulty of the source-imaging problem in electrocardiology is that the data (body surface potentials) depend continuously on the properties of complex uniquely determined equivalent sources (e.g., epicardial potentials) while such sources do not depend continuously on the data (precluding utility of direct inversion of the operator relating sources and data). This is in distinction to the standard digital tomographic imaging problems in radiology, which superficially resemble the electrocardiographic imaging problem in their requirement for solution of a linear integral equation. From the mathematical standpoint, the electrocardiographic imaging problem requires that an operator with a continuous inverse be constructed from the original operator. This may be accomplished either by slightly perturbing the original operator (the regularization approach), or by performing a calculation on the data (based on inherent source evolution singularities) which allows restriction of the admissible solution domain to a compact set (the topological approach). An understanding of the mathematical questions fundamental to this imaging problem is helpful in assessing its present status and in identifying promising directions for the development of a clinically useful technique. PMID- 8631192 TI - Interaction of oral contraceptive use with the effects of age, exercise habits and other cardiovascular risk modifiers on metabolic risk markers. AB - An analysis was undertaken to determine whether combined oral contraceptive (OC) use interacts with the effects of potential cardiovascular risk modifiers (age, body mass index, cigarette smoking, alcohol intake, exercise habit, family histories of heart disease or diabetes, number of pregnancies and duration of OC use) on blood pressure and lipid, lipoprotein, glucose and insulin risk markers for cardiovascular disease. Relationships between risk modifiers and risk markers were compared between non-users (n = 418) and users of low-estrogen dose OC (n = 925, categorised according to progestin content as monophasic levonorgestrel, triphasic levonorgestrel, norethindrone or desogestrel). OC use diminished the adverse effects of age on glucose tolerance. Aerobic exercise had a particularly beneficial effect on triglyceride levels and OGTT insulin response in OC users. The rise in HDL and HDL2 cholesterol concentrations with alcohol intake seen in non-users was diminished in OC users. Increasing duration of use of a desogestrel combination was associated with increasing HDL cholesterol concentrations. No adverse effects of risk modifiers on metabolic risk markers and blood pressure were augmented by OC use, and some were even diminished. PMID- 8631195 TI - Brain segmentation and white matter lesion detection in MR images. AB - This paper presents a review of methods and techniques that have been proposed for the segmentation of magnetic resonance (MR) images of the brain, with a special emphasis on the segmentation of white matter lesions. First, artifacts affecting MR images (noise, partial volume effect, and shading artifact) are reviewed and methods that have been proposed to correct for these artifacts are discussed. Next, a taxonomy of generic segmentation algorithms is presented, categorized as region-based, edge-based, and classification algorithms. For each category, the applications proposed in the literature are subdivided into 2-D, 3 D, or multimodal approaches. In each case, tables listing authors, bibliographic references, and methods used have been compiled and are presented. This description of segmentation algorithms is followed by a section on techniques proposed specifically for the analysis of white matter lesions. Finally, a section is dedicated to a review and a comparison of validation methods proposed to assess the accuracy and the reliability of the results obtained with various segmentation algorithms. PMID- 8631196 TI - Involvement of oxidative stress in D-xylose-induced cataractogenesis in cultured rat lenses. AB - In order to clarify the involvement of oxidative stress in in vitro sugar-induced cataractogenesis, we examined changes in lipid peroxide, reduced glutathione, vitamin E, and water contents following cataractogenesis in rat lenses cultured with 20 mM D-xylose over a period of 24 h. In D-xylose-treated lenses, an apparent opacity appeared at the equator after 6 h of D-xylose treatment, increases in lipid peroxide and water contents and decreases in reduced glutathione and vitamin E contents occurred with the appearance of opacity. We further examined the effect of the treatment of vitamin E in a liposomal form on cataractogenesis and the changes of lipid peroxide, reduced glutathione, vitamin E, water, and xylitol contents in rat lenses cultured with 20 mM D-xylose for 24 h. This vitamin E treatment significantly prevented not only cataractogenesis, but also an increase in lipid peroxide content and a decrease in vitamin E content in the D-xylose-treated lenses. However, the vitamin E treatment had no effect not only on a decrease in reduced glutathione content, but also on increases in water and xylitol contents, which are known to be induced via aldose reductase, in the D-xylose-treated lenses. These results indicate that not only osmotic stress but also oxidative stress should be involved in cataractogenesis in rat lenses cultured with D-xylose and support the involvement of oxidative stress in in vitro sugar-induced cataractogenesis. PMID- 8631198 TI - Endothelin-like immunoreactivity and receptor binding in the choroid and retina. AB - This study has examined the localisation and receptor-binding of the endothelins in retina and choroid of human and rat origin. Immunoreactivity to anti-ET1 and anti-ET3 was investigated in trypsin digests, frozen sections and ultrathin sections using immunocytochemistry and immunogold labelling techniques. In addition, receptor binding of 125I-ET1 and 125I-ET3 was visualised and quantified using autoradiography and image analysis. Intense immunoreactivity to anti-ET1 and anti-ET3 was observed in the photoreceptor inner segments and in the outer plexiform layer (OPL) of human and rat retina. Ultrastructural localisation using immunogold labelling confirmed the presence of ET1 and ET3 in the photoreceptor cells. In retinal vascular digests, ET1 was visualised in the arteries, arterioles and at the pre-arteriolar sphincters, however, immunoreactivity to anti-ET3 was absent in the retinal vasculature. Both ETA and ETB-type receptor binding sites to 125I-ET1 and 125I-ET3 were detected in the vascular smooth muscle of choroidal and retinal vessels with the former being predominant. Extravascular binding sites of the ETB-type were found in the ganglion cell layer. PMID- 8631197 TI - Calcitonin gene-related peptide induced relaxation of the rabbit iris dilator muscle. AB - Calcitonin gene-related peptide (CGRP) and Substance P (SP) -immunoreactive nerves have been found in the anterior uvea of various mammalian species. Although SP is known to play a major role in control of pupil motility in rabbits, little is known about the effect of CGRP on the iris smooth muscles. We isolated iris sphincter and dilator muscles from rabbit eyes and investigated the mechanical responses and intracellular cyclic AMP (cAMP) levels in these muscles. CGRP (up to 0.1 microM) had no effect on either the resting muscle tone or the amplitude of contraction evoked by field stimulation of the sphincter. On the other hand, CGRP (0.1 microM) relaxed dilator muscle which had been pre contracted by phenylephrine and reduced the amplitude of contraction evoked by field stimulation. These responses were antagonized by CGRP (8-37), a CGRP antagonist. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), dose-dependently inhibited the contraction evoked by field stimulation. However, 3 microM IBMX had no effect on CGRP inhibition of twitch contraction in this preparation. CGRP had little effect on cAMP production in dilator muscle either with or without IBMX. In conclusion, the miosis which occurs during an ocular inflammatory response, when both CGRP and SP are thought to be released from terminals of sensory neurons, results from CGRP relaxation of the dilator and from the strong contractile effect of SP on the sphincter. Adenylate cyclase activation does not seem to be involved in the relaxant effect of CGRP on the dilator. PMID- 8631199 TI - Initiation of impaired outer segment degradation in vivo using an antisense oligonucleotide. AB - This paper describes the first successful in vivo application of antisense DNA technology to induce the accumulation of photoreceptor outer segment derived debris in the retina. An antisense oligonucleotide (CatSC), which was previously demonstrated to be an effective tool to induce debris accumulation in vitro, was injected into the vitreous of pigmented and non-pigmented rats. The animals were euthanased 7 days after the injections. The number of inclusions significantly increased in the RPE layer of Long Evans and RCS-rdy + rats injected with 66 ug of CatSC to 96.2 +/- 13.6 (SD) (p < 0.0003) and 204.2 +/- 39.3 (SD) (p < 0.0001), respectively. The difference between the number of phagosome-like inclusions present in control saline, 6.6 ug of CatSC or 66 ug of sense oligonucleotide (S1) injected animals was not statistically significant. There were no abnormalities observed in the inner layers of the retina but the accumulation of phagosome-like inclusions was accompanied by disorganisation in the apices of outer segments. The large number of inclusions found in CatSC treated animals showed the characteristics of phagosomes containing stacks of undigested photoreceptor outer segment membranes which suggest that the lysosomal digestion process was halted or at least slowed down by the antisense oligonucleotide. PMID- 8631200 TI - Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium. AB - The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM. PMID- 8631201 TI - The effects of growth factors on Tenon's capsule fibroblasts in serum-free culture. AB - This study was performed to develop and improve a completely defined in vitro ocular wound-healing model of fibroblast proliferation for glaucoma filtration surgery. This model is essential for the investigation of protein-sensitive drugs and cytokines. Tenon's capsule fibroblasts in their third passage were incubated overnight, washed free of serum, and fed defined media, Aim V or Clonetics FBM serum-free medium containing platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, or fibronectin at various dilutions and in combinations at optimum concentrations. Proliferation was measured by 3H thymidine incorporation at 1, 3, and 7 days. Morphology was compared to controls fed Minimum Essential Medium + 10% serum. Single factors stimulated the greatest amount of thymidine uptake on day 3. Optimum concentrations were epidermal growth factor at 5 ng/ml, basic fibroblast growth factor at 10 ng/ml and platelet derived growth factor at 20 ng/ml. Identical combinations of factors stimulated nearly twice the thymidine uptake in Clonetics medium as in Aim V. Epidermal growth factor activity was inhibited by either basic fibroblast growth factor or platelet-derived growth factor. Basic fibroblast growth factor and platelet derived growth factor together produced a less than additive effect. The performance of either serum-free medium may be improved by the addition of basic fibroblast growth factor or platelet-derived growth factor. The optimum serum free medium (Clonetics FBM) with growth factors was unable to stimulate proliferation as much as Minimum Essential Medium + 10% NBS, but was successful in maintaining viability during the 7 day test period. PMID- 8631202 TI - Ultrastructure of hyaluronic acid and collagen in the human vitreous. AB - The ultrastructure of hyaluronic acid (HA) and collagen in the human vitreous was investigated. After fixation with 4% glutaraldehyde and 0.1% cetylpyridinium chloride (CPC), sectioned blocks of retina and attached vitreous body were stained with 500 ppm ruthenium red (RR) in 1.25% glutaraldehyde, postfixed with 500 ppm RR in 2% OsO4, and embedded in Epon 812. In addition, collagen II immunostaining was done. Collagen fibrils were coated with amorphous material believed to be HA. No amorphous material was seen in the fibrils in the cortical areas adjacent to the retina, but the amount of material gradually increased toward the center of the vitreous body. By using anticollagen II antibody, we showed the presence of collagen II, often oriented perpendicular to the surface. The central vitreous demonstrated some dense materials that were collagen fibrils packed into bundles of parallel fibrils. In summary, collagen fibrils contained little HA in the cortical areas adjacent to the retina, but they were packed into bundles of parallel fibrils in the central vitreous. PMID- 8631203 TI - The composition of wide-spaced collagen in normal and diseased Descemet's membrane. AB - Descemet's membrane, the specialised basement membrane of the corneal endothelium, contains a form of extracellular matrix described as wide-spaced collagen. In healthy human Descemet's membrane, wide-spaced collagen forms a highly ordered array in a region called the anterior banded zone. However, in corneal endotheliopathies such as Fuchs' endothelial dystrophy and the iridocorneal-endothelial syndrome large amounts of wide-spaced collagen are deposited posterior to Descemet's membrane in a grotesque parody of the anterior banded zone termed a posterior collagenous layer. The purpose of this study was to identify the composition of the wide-spaced collagen found in the Descemet's membrane of normal and diseased human corneas. Tissue from three normal human corneas, three from patients with Fuchs' endothelial dystrophy and five from patients with the iridocorneal-endothelial syndrome was prepared for immuno electron microscopy by freezing or embedding in Lowicryl K4M resin. Immunocytochemistry on ultrathin sections was performed with antibodies to collagen Types I, III, V, VI and VIII, fibronectin, laminin, P component and tenascin. Ultrastructural labelling of the wide-spaced collagen in the anterior banded zone of normal and diseased corneas and also of the wide-spaced collagen in the posterior collagenous layer of all the diseased corneas was demonstrated with antibody to collagen Type VIII. Wide-spaced collagen was not labelled by any of the other antibodies used. Large amounts of Type VIII collagen are present in discrete regions of healthy and diseased Descemet's membrane. The deposition of Type VIII collagen may significantly influence the pathobiology of the corneal endotheliopathies. PMID- 8631204 TI - A longitudinal study of cortical cataracts using retroillumination photographs. AB - The National Eye Institute (NEI) computer planimetry system has been shown to have good reproducibility in assessing the size of cortical cataracts from retroilluminatin photographs. In this study, we determined the usefulness of this system in monitoring cortical cataract changes over time. Using the Neitz Kawara retroillumination camera, retroillumination photographs of cortical cataracts in 81 eyes were obtained every six months for an average of 31 months. As previously described, the cortical cataract outlines were traced in a masked fashion onto transparent plastic overlays, and the tracings then digitized into a Macintosh Quadra computer using a computer scanner. Cortical cataract area was then determined using a specially developed software program. For each eye, the rate of cortical area change was determined by the slope of the regression line fitted to the follow-up measurements. Cataract progression was classified to be significant if the slope exceeded a critical value. Of the 81 eyes, 24 (30%) had significant cataract progression, while 57 (70%) did not progress. This study presents data on progression of the area of cortical cataracts and suggests the usefulness of the NEI computer planimetry system for monitoring such changes from retroillumination photographs. The slope-based test can also play a useful part in longitudinal studies with irregular time intervals and variable number of visits. PMID- 8631205 TI - Changes in GABA metabolism in streptozotocin-induced diabetic rat retinas. AB - We examined the pathogenesis of reduced amplitude in electroretinogram (ERG) oscillatory potentials (OPs) in diabetes, in relation to possible changes in the metabolisms involving retinal amino acid neurotransmitters. With use of streptozotocin diabetic rats, flash ERGs were recorded and quantitative analyses of retinal free amino acids were performed. Immunocytochemical localizations of retinal glycine and GABA were examined. In addition, activities of glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T) were measured. Our results revealed that the amplitudes of OP 1 and OP 2 decreased, and retinal glycine and GABA content significantly increased in the diabetic rats. An increased immunoreactivity of GABA was observed in Muller cells in the diabetic rat retinas, while no apparent changes were found in glycine immunoreactivity. Finally, increased activations of GAD with reduced activities of GABA-T were observed in the diabetic rat retinas. Thus, reduced amplitudes of OPs were associated with changes in content, localization, and enzyme activities related to GABA in the retinas, implying that changes in GABA metabolism can be a candidate for the pathogenesis of the abnormal OPs in diabetes. PMID- 8631206 TI - Pharmacological characterization of endothelin receptors in the rabbit iris sphincter muscle: suggestion for the presence of atypical receptors. AB - Pharmacological profiles of endothelin (ET) receptors in the isolated rabbit iris sphincter were characterized. ET isopeptides caused dose-dependent contraction of the preparation. The respective EC50 values for ET-1, ET-2 and ET-3 were 39.4, 58.0 and 84.3 nM, so that ET-1 was twice as potent as ET-3. Sarafotoxin (SRTX) b, an ET(A)/ET(B) non-selective agonist, caused very potent contraction with an EC50 of 1.13 nM. However, selective ET(B) receptor agonists SRTX-c and IRL 1620 showed no contractile activity up to 1 microM. BQ-123, a selective ET(A) receptor antagonist, shifted the dose-response curves of ET isopeptides to the right. The pA2 value for ET-1 was 5.52 with a slope of 1.06, which is not different from unity, and the pK(B) value for ET-2 was 5.06. Interestingly, very low doses of BQ 123 antagonized responses to ET-3 and SRTX-b, with a Schild plot slope of approximately 0.7 which is significantly different from unity, suggesting receptor heterogeneity. The abscissal intercepts of the Schild plots were -9.29 for ET-3 and -8.53 for SRTX-b. FR 139317, another ET(A) receptor antagonist, also preferentially antagonized responses to ET-3. RES-701-1, a selective ET(B) receptor antagonist, did not shift dose-response curves for ET-1 and ET-3. These results suggest that ET receptors in the rabbit iris sphincter muscle cannot be classified into the ET(A), ET(B) or ET(C) receptor subtypes, so far established. When compared to the established receptor subtypes, ET receptors in this preparation were quite different from the ET(B) receptor, but apparently showed a pharmacological profile most similar to the ET(A) receptor, suggesting the presence of heterogeneous and atypical ET(A) receptors. PMID- 8631207 TI - ERG of form deprivation myopia and drug induced ametropia in chicks. AB - Chick eyes occluded for various periods or treated with various concentrations of kainic acid (KA) or 2-amino-4-phosphonobutylate (APB) during development showed characteristic changes of electroretinography and of refraction. In occluded eyes, oscillatory potential amplitudes (OP-A) were reduced, even with high intensity stimulation, in proportion to duration of occlusion, but b wave amplitude was unchanged, implying functional changes in inner layers of the deprived retina. OP-A reduction after only 1 week of occlusion and reversibility of this change might reflect retinal changes preceding axial elongation. KA was confirmed to induce myopia with axial elongation and APB to induce hyperopia with axial shortening. KA and APB both suppressed OP-A. KA reduced ON and OFF responses, but low-dose APB suppressed only ON responses. Study results suggest that myopia could be induced by changes of inner retina mediating OP attenuation and degradation of ON and OFF responses. This manuscript reports unpublished work that is not currently under consideration for publication elsewhere. PMID- 8631208 TI - Short-term hemodynamic changes in episcleral arteriovenous anastomoses correlate with venous pressure and IOP changes in the albino rabbit. AB - In the present study we have determined the effects of acute diameter changes in the recently discovered episcleral arteriovenous anastomoses (AVA) on episcleral venous pressure and intraocular pressure (IOP) in the rabbit eye. The pressure was measured in episcleral arterioles, AVA and veins (vascular pressure) with a pressure chamber mounted on the tip of a microendoscope. After constriction of the AVA following topical administration (100 micrograms) of epinephrine we observed a decrease in the episcleral vascular pressure as well as in the IOP. Acute widening of the AVA after topical administration of 5mg nitroprusside led to a significant increase in the pressure of arterioles, AVA, veins and IOP which lasted several min. After 0.5mg topical nitroprusside the blood flow in the AVA increased. No significant effects were seen in the intravascular pressures; the IOP had a tendency to decrease. Experimental lowering of the IOP to 10mmHg caused an increase of vascular diameters and of blood flow in the AVA. It is assumed that-at least in drastic hemodynamic disorders-the degree of the AVA-perfusion might influence aqueous humor outflow and IOP due to changes of the episcleral venous pressure. PMID- 8631209 TI - Immunodetection of an arrestin-like protein in human retinal pigment epithelium. AB - The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity. PMID- 8631210 TI - Intravitreal transforming growth factor-beta 2 decreases cellular infiltration in endotoxin-induced ocular inflammation in rabbits. AB - Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which has been identified in normal and inflamed ocular fluids, may play a role in the evolution of inflammatory ocular lesions. In this study we utilized a rabbit model of LPS-induced uveitis to determine if exogenous TGF-beta 2 could alter its course. Recombinant TGF-beta 2 (1-2000 ng), LPS (10 or 20 ng), or TGF-beta 2 (100 ng) plus LPS (10 ng) were injected intravitreally in one eye of a New Zealand white rabbit and the contralateral eye served as a paired control which received an equal volume of vehicle. The uveitic response was assessed by biomicroscopic examination of the anterior uvea and analysis of protein and cells in the aqueous humor. Ocular tissues were processed for histologic, immunohistochemical and in situ hybridization analyses. Rabbits injected with doses of TGF-beta 2 > or = 500 ng developed a mild uveitic response, compared to LPS alone, accompanied by expression of IL-1 beta mRNA and protein in the anterior uvea. Interestingly, rabbits coinjected with LPS (10 ng) and a nonuveitic dose (100 ng) of TGF-beta 2 exhibited a similar increase in ocular vascular permeability, but a decrease in inflammatory cell infiltration into the anterior uvea and aqueous humor (1185 +/- 117 versus 2465 +/- 176; p < 0.05). No evidence of inflammation was observed in eyes injected with 100 ng TGF-beta 2 alone. Similar to other models of inflammation, TGF-beta may interrupt the cascade of events leading to ocular inflammation, thereby suggesting therapeutic potential. PMID- 8631211 TI - Early detection and management of right ventricular infarction: the role of the critical care nurse. AB - Patients with acute inferior myocardial infarction (IMI), complicated by a more extensive right ventricular infarction (RVI), have an increased risk of both complications and mortality. The critical care nurse plays an important role in 1) the early detection of RVI and 2) the management of the common hemodynamic and conduction disturbances that can typically arise. PMID- 8631212 TI - Music therapy: a nursing intervention for the control of pain and anxiety in the ICU: a review of the research literature. AB - Critical care patients experience both pain and anxiety related to their acute illness or injury and some painful treatments. Research on music therapy has shown that it can decrease pain and anxiety in critical care patients. This author suggests practice changes based on the body of research, which investigated the use of music. PMID- 8631213 TI - Peripheral electrical stimulation: titrating neuromuscular blocking agent levels. AB - Objective monitoring of the response to neuromuscular blocking agents is essential to ensure the patient receives the maximum benefit from the drug without harm. Peripheral nerve stimulation using a train-of-four mode of four quick pulses is an intervention the critical care nurse uses at the bedside to monitor the level of neuromuscular blockage for each patient. The nurse can use this objective evaluation and a dosage protocol to titrate effective levels of neuromuscular blocking agents for ventilated patients. PMID- 8631214 TI - Realization impact: nursing assessment and strategies. AB - Dealing with the anguish of family members who stand at the threshold of the awareness that their loved one is in danger of dying requires great ingenuity on the part of the critical care nurse. This article describes a phenomenon called "realization impact," which is a process through which family members proceed before facing the fact that their family member could die. An understanding of the realization impact phenomenon can facilitate transactions (defined as the purposeful interaction toward a common goal) between the critical care nurse and the family members of patients in danger of dying. PMID- 8631215 TI - Readability of written materials: implications for critical care nurses. AB - Written materials are given to patients every day in critical care units. Unfortunately, some of the materials are written at a reading level that is too difficult for patients to read. Patients and families need understandable materials to help them make the difficult decisions they face. PMID- 8631216 TI - Parental participation in treatment decisions for pediatric oncology ICU patients. AB - Ethical dilemmas involving treatment decisions are increasing in frequency as medical technology continues to provide the ability to prolong life. When the dilemmas involve treatment decisions for children, the added dimension of parental participation makes the issue even more complex. The author describes a pilot study showing the extent of documentation of parents' participation in treatment decisions for pediatric oncology ICU patients. PMID- 8631217 TI - The right balance. PMID- 8631218 TI - Thermodilution: an advanced technique for measuring continuous cardiac output. AB - A thermodilution method of obtaining continuous cardiac outputs represents an improvement in the assessment and treatment of critically ill patients. The critical care nurse assists in placement of the pulmonary artery catheter, which includes a thermal filament for continuous readings of cardiac output, and uses the continuous cardiac output readings to make assessment decisions and trouble shoot the equipment. PMID- 8631220 TI - Managing terminal dyspnea: caring for the patient who refuses intubation or ventilation. AB - Dyspnea in a dying patient is one of the most distressing symptoms challenging a critical care nurse. A humane response to terminal dyspnea, which may result when the patient chooses to forego life-sustaining intubation and ventilation, is expected by the patient. The author presents strategies for reducing the distress of dyspnea for patients who are dying when intubation and ventilation are being withheld. PMID- 8631221 TI - Clinical nurse specialist: making the shift from critical care to home care. AB - Because patients are being discharged earlier with more complex medical and nursing needs, now is the time for Critical Care Clinical Nurse Specialists to consider home care as a viable practice arena. The home-care Clinical Nurse Specialist role offers exciting, non-traditional job opportunities that may add a dimension to your practice that you will truly enjoy. PMID- 8631222 TI - The critical care nurse practitioner: an advanced practice role for the critical care nurse. AB - Research provides compelling evidence that supports the current and future utilization of critical care nurse practitioners (CCNPs) in pivotal roles as providers of cost-effective, quality patient-centered care for critically ill and technology-dependent adults. This paper gives a historical perspective and describes the current national need for CCNPs to practice in expanded roles both within and outside the boundaries of intensive care. An existing CCNP program is described. In addition, guidelines for CCNP program development and role implementation are discussed. PMID- 8631219 TI - A review of normal saline instillation: implications for practice. AB - Nurses commonly use normal saline instillation (NSI) as a component of the suctioning procedure. The current research on NSI has not clearly identified many positive aspects of the procedure. Much of the research suggests it may actually be harmful. There has been little investigation into the reasons NSI is used. It is presumed that NSI is used to increase secretion removal when patients have thick endotracheal secretions due to inadequate humidity to the airway. Nurses need to be aware of the potential negative effects of routine NSI as well as alternative methods for maintaining adequate airway humidification. PMID- 8631224 TI - [Risk of HIV infection after needle injuries]. PMID- 8631223 TI - Acid-base disorders in medicine. AB - The practice of internal medicine involves daily exposure to abnormalities of acid-base balance. A wide variety of disease states either predispose patients to develop these conditions or lead to the use of medications that alter renal, gastrointestinal, or pulmonary function and secondarily alter acid-base balance. In addition, primary acid-base disease follows specific forms of renal tubular dysfunction (renal tubular acidosis). We review the acid-base physiologic functions of the kidney and gastrointestinal tract and the current understanding of acid-base pathophysiologic conditions. This includes a review of whole animal and renal tubular physiologic characteristics and a discussion of the current knowledge of the molecular biology of acid-base transport. We stress an approach to diagnosis that relies on knowledge of acid-base physiologic function, and we include discussion of the appropriate treatment of each disorder considered. Finally, we include a discussion of the effects of acidosis and alkalosis on human physiologic functions. PMID- 8631225 TI - [Postoperative follow-up in hepatic echinococcosis]. AB - OBJECTIVE: To determine whether the monitoring of patients who have undergone surgical excision of hydatid cysts of the liver is useful. Furthermore, sonographic and computertomographic criteria of recurrent hydatid disease should be described. PATIENTS AND METHODS: Of 84 patients operated on for hepatic hydatid disease 42 (22 men, 20 women; mean age 44 [25-82] years) were examined between 6 months and 15 years post-operatively (median of 6 years) clinically, serologically (complement-fixation reaction, immunohaemagglutination) and by ultrasound. If the latter was positive, computed tomography was also performed. RESULTS: Recurrence of hydatid disease in the liver was diagnosed in three patients and in the peritoneal cavity in one patient. The demonstration of internal septa by solitary or multiple daughter cysts proved to be a reliable imaging criterion. CONCLUSION: Ultrasonography should be performed as a baseline test after surgical removal of hydatid cysts in the liver and specific antibody titres measured. This will make it easier to distinguish a recurrence from postoperative changes. PMID- 8631226 TI - [Anticytoplasmic antibodies (cANCA) in syphilitic nodules of the lung]. AB - HISTORY AND CLINICAL FINDINGS: A chest radiograph was done in a 75-year-old man with cough productive of a whitish sputum. It showed round foci in the right middle and upper lobes. Serum anticytoplasmic antibody (cANCA) was 1 : 160, suggesting Wegener's granulomatosis. But the pulmonary foci actually increased in size on administration of prednisolone, 10 mg daily. INVESTIGATIONS: As the lung biopsy was not diagnostic, a diagnostic thoracotomy with biopsy was done and the anterior segment of the right middle lobe resected. Histological examination revealed a gumma. Active syphilis was confirmed by a TPHA-Test with a titre of 1 : 40960. TREATMENT AND COURSE: Antisyphilitic treatment was given for 35 days, at first 1 mega (Penicillin E daily for five days, then erythromycin, 1000 mg, for five days, a penicillin rash having occurred. Serial serology showed a continual fall of the lues and cANCA titres. Four months postoperatively the titre in the TPHA test had fallen to 1 : 640, and was nonreactive after five years. cANCA titre fell to 1 : 16 and was negative 42 weeks after resection. CONCLUSION: The connection between pulmonary gumma and the presence of antiplasmatic antibodies remains unclear. Inflammatory changes in the vascular wall, which occur in both Wegener's granulomatosis and tertiary syphilis, may have played a role. PMID- 8631227 TI - [Cat scratch disease. Bartonella henselae antibodies and DNA detection in regional lymphadenopathy]. AB - HISTORY AND CLINICAL FINDINGS: Two hard, pressure-sensitive nodules developed in the lower jaw of a 22-year-old woman. After a dental cause had been excluded, she was treated for suspected tonsillitis with Ceftibuten. Erythrocyte sedimentation rate was increased to 18 mm in the first hour. There were no other significant biochemical findings and fine-needle biopsy of one of the nodules showed nonspecific inflammatory reaction. INVESTIGATIONS: Sonography revealed two lymph nodes, 7 and 22 mm in diameter. Suspected cat scratch disease was confirmed by immunofluorescence with Bartonella (Rochalimaea) henselae and quintana antigens. TREATMENT AND COURSE: After a course of Clarithromycin (250 mg twice daily) for 6 weeks the lymph nodes had shrunk and the overlying skin was thin and discoloured brown. One node was incised and drained and the material examined. Microbiology was negative, but DNA sequencing confirmed Bartonella henselae. As a consequence, Rifampicin was given for 2 months (600 mg daily). Wound healing was very slow and the scar had regressed little after 9 months. PMID- 8631228 TI - [Therapy of pulmonary embolism]. PMID- 8631229 TI - [Zoster and the nervous system]. PMID- 8631230 TI - [Changes in the liquidation right in the revised Federal Health Insurance regulation]. PMID- 8631231 TI - [Environmental medicine evaluation of increased blood levels of hazardous materials in cardiac arrhythmias]. PMID- 8631232 TI - [Databank-supported clinical medicine]. PMID- 8631233 TI - [Apoptosis of T-helper cells is massively increased in AIDS]. PMID- 8631234 TI - [Minimal invasive therapy of gallstones by laparoscopic cholecystostomy]. PMID- 8631236 TI - [Solicitation for excimer laser by the ophthalmologist]. PMID- 8631235 TI - [Occupational medicine aspects related to carcinogenic hazardous substances]. PMID- 8631237 TI - [Indications for transposition osteotomy in coxa valga]. PMID- 8631238 TI - [Benign paroxysmal positional vertigo]. PMID- 8631239 TI - [Sputum gram staining in pneumonia]. PMID- 8631240 TI - [Myopathies in chronic renal failure patients on dialysis]. PMID- 8631241 TI - [Diagnosis and therapy of necrotizing fasciitis. Hyperbaric oxygenation as a supplemental therapy form]. PMID- 8631242 TI - [Transportation of patients under the intra-aortic balloon pumping treatment]. PMID- 8631243 TI - [HIV-associated Pneumocystis carinii pneumonia, primary pentamidine inhalation prophylaxis notwithstanding. Are there differences with Pneumocystis carinii pneumonia without prophylaxis?]. AB - OBJECTIVE: To study the effect of pentamidine aerosol inhalation, known to be effective and well tolerated in the primary prevention of HIV-associated Pneumocystis carinii pneumonia (PCP), on the severity, on clinical and radiological findings and prognosis of break-through PCP. PATIENTS AND METHODS: Data were obtained from the case notes of 17 men (mean age 36 [23-55] years) with HIV-associated PCP, treated between 1989 and 1994, who had received primary prophylaxis with pentamidine aerosol. During the same period 42 patients with HIV associated PCP but no pentamidine prophylaxis (39 men, three women; mean age 36 [21-67] years) were also treated. Bronchoalveolar lavage of the most affected segment was performed. Diagnostic measures and treatment remained constant during the period of observation. RESULTS: The clinical presentation was the same in the two groups. There was also no difference between them regarding sensitivity to the lavage (94 and 98%). However, infiltration of the upper segments was significantly more common in the prophylaxis group (90 and 43%; P < 0.02). There was no statistically significant difference between the two groups in death rate (13 and 7%) and the long-term prognosis after PCP (survival time 18 and 24 months, respectively). CONCLUSION: Primary prophylaxis of PCP had no significant effect other than altering the distribution pattern of the pneumonic infiltrates towards the apical segments. PMID- 8631244 TI - [Endoscopic therapy in acute hemorrhage caused by duodenal diverticula]. AB - HISTORY AND CLINICAL FINDINGS: A 70-year-old previously healthy woman had been feeling nauseous for one day and had passed several liquid tarry stools. A barium meal previously done as an out-patient had shown a duodenal diverticulum 3.5 cm in diameter with marked contrast-medium retention. Her general condition was impaired, her skin pale and cold, while heart rate and blood pressure were normal. Rectal examination confirmed tarry stool and thus suggested upper gastrointestinal bleeding, the contrast-medium retention pointing to the duodenal diverticulum as a likely site. INVESTIGATIONS: Haemoglobin concentration was 9.1 g/dl, the haematocrit 26.6%. Total protein was reduced to 4.4 g/dl. Esophagogastroduodenoscopy (performed about 10 hours after the barium meal) showed erosion at the duodenal bulb and contrast retention in the juxtapapillary diverticulum, but no acute bleeding was discovered. TREATMENT AND COURSE: Repeat endoscopy on the following day revealed acute bleeding (Forrest stage Ia) from an arterial stump in the diverticulum. It was stopped with local injection of adrenaline (6 ml of 1:10,000 solution) and fibrin glue, but the injections had to be repeated twice. Another endoscopy 30 days after the first showed merely a mucosal scar. CONCLUSION: Early endoscopy enables one to make the diagnosis and to provide minimally invasive treatment of bleeding from a duodenal diverticulum. PMID- 8631245 TI - [Lemierre's post-tonsillitis sepsis with meningitis and intravascular consumption coagulopathy as complication of infectious mononucleosis with pansinusitis]. AB - HISTORY AND CLINICAL FINDINGS: 24 days after the onset of infectious mononucleosis, clinically and serologically confirmed, an otherwise healthy 18 year-old schoolboy developed a fulminant septicaemia with acute meningitis and loss of consciousness, consumptive coagulopathy and acute renal failure. INVESTIGATIONS: Computed tomography demonstrated pansinusitis. Lumbar puncture produced purulent cerebrospinal fluid with 11,500 cells/microliters, predominantly granulocytes, protein 205 mg/dl, glucose 19 mg/dl, indicating bacterial meningitis. The suspected diagnosis of posttonsillitis septicaemia (Lemierre's syndrome) was confirmed by repeated demonstration of fusiform gram negative bacteria in anaerobic blood cultures, identified as Fusobacterium necrophorum. Anaerobic CSF culture grew Prevotella bivia of the Bacteroidaceae family. TREATMENT AND COURSE: Both the consumptive coagulopathy and the renal failure were successfully treated and the patient's condition stabilized. The sinuses were surgically drained under high doses of piperacillin/sulbactam and chloramphenicol. Despite the sensitivity of the cultured bacteria to the administered antibiotics the septic temperature continued, but disappeared within 4 days of metronidazole having been added. After 5 weeks of antibiotic treatment, three of them in an intensive care unit, the patient was discharged in good general condition. CONCLUSION: This case illustrates that severe septicaemia caused by rare bacteria may follow an attack of infectious mononucleosis which had taken an uncomplicated course. PMID- 8631246 TI - TGF-beta signals and a pattern in Xenopus laevis endodermal development. AB - We have analyzed two gene products expressed in the early endoderm of Xenopus laevis: Xlhbox-8, a pancreas-specific transcription factor and intestinal fatty acid binding protein (IFABP), a marker of small intestinal epithelium. Expression of the pancreas marker relies on cell signaling mediated by both the TGF-beta and FGF classes of secreted peptide growth factors, whereas, expression of the more posterior small intestinal marker does not. Endodermal explants devoid of mesoderm express both markers in a regionalized manner. Cortical rotation is required for the expression of the more anterior marker, Xlhbox-8, but not for the small intestinal marker, IFABP. These findings suggest that endodermal patterning is dependent, in part, on the same events and signals known to play important roles in mesodermal development. Furthermore, inhibition of TGF-beta signaling in the endoderm leads to ectopic expression of both mesodermal and ectodermal markers, suggesting the TGF-beta signaling may play a general role in the segregation of the three embryonic germ layers. PMID- 8631247 TI - Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. AB - The limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Previous studies have shown that the paired domain-containing transcription factor Pax-3 is expressed in the limb in cells that are precursors for limb muscles (Williams, B. and Ordahl, C.P. (1994) Development 120, 785-796). In splotch (Pax-3-) embryos, the limb muscles fail to develop and cells expressing Pax-3 are no longer found in the limb. In this paper we have analyzed the role of Pax-3 in the migration and subsequent differentiation of limb muscle precursors. By labeling somites adjacent to the prospective forelimb with the lipophilic dye DiI, we have shown that cells derived from these somites do not migrate into the limbs of splotch mice. The failure of limb muscle precursors to invade the limb in splotch mice is associated with the absence of c-met expression in premigratory cells, together with a change in the morphology of the ventral dermomyotome. In addition, we have shown the lateral half of somites derived from day E9.25 splotch embryos can undergo muscle differentiation when grafted into the limb bud stage 20 chick host embryos. Our results indicate that Pax-3 regulates the migration of limb muscle precursors into the limb and is not required for cells in the lateral somite to differentiate into muscle. PMID- 8631249 TI - Clonal analysis of the late flowering fca mutant of Arabidopsis thaliana: cell fate and cell autonomy. AB - Plants that are homozygous for the fca mutation bolt and flower later than wild type (FCA) plants. The mutation has little or no effect on the fate map of the dry seed, except that the central cells give rise to further rosette leaves instead of the bolting stem, cauling leaves and inflorescence. The large and variable sectors affecting the late rosette leaves of fca plants were used to generate an abstract frequency-distance fate map of vegetative growth. The map relates the initiation of leaves in the plant apex to their final arrangements. The map was found to be a shallow dome with phyllotaxy superimposed on its surface. X-irradiation was used to provoke loss of the FCA allele from cells in heterozygous seeds. The resulting fca sectors had no effect on the plant phenotype. Even when L2 and L3 cells at the centre of the meristem could not produce the FCA gene product, bolting and flowering was unaffected. The genotypically fca mutant tissue was incorporated into phenotypically normal stems, cauline leaves and flowers. Possible reasons for the non-autonomous behaviour of the trait are discussed. PMID- 8631248 TI - Expression of type 1 inositol 1,4,5-trisphosphate receptor during axogenesis and synaptic contact in the central and peripheral nervous system of developing rat. AB - Release of intracellular Ca2+ is triggered by the second messenger inositol 1,4,5 trisphosphate, which binds to the inositol 1,4,5-trisphosphate receptor and gates the opening of an intrinsic calcium channel in the endoplasmic reticulum. In order to understand the importance of this mechanism in development, we have examined the distribution of the type 1 inositol 1,4,5-trisphosphate receptor during development, in some areas of the rat brain and spinal cord and in peripheral neurons, using in situ hybridization and immunohistochemistry. In brain, we find that type 1 inositol 1,4,5-trisphosphate receptor is expressed in neurons from very early in development; low levels of expression are first detected after the neurons have migrated to their final positions, when they start to differentiate and begin axonal growth. Increasing levels of expression are observed later in development, during the time of synaptogenesis and dendritic contact. Glial cells do not express type 1 inositol 1,4,5-trisphosphate receptor, except for a transient period of expression, probably by oligodendrocytes, in developing fibre tracts during the onset of myelination. In contrast with the brain, both grey and white matter of the spinal cord express type 1 inositol 1,4,5-trisphosphate receptor throughout development, and it remains present in the adult spinal cord. We also show, for the first time, that type 1 inositol 1,4,5-trisphosphate receptor is expressed in the peripheral nervous system. Strong labelling was observed in the dorsal root ganglia and during development this expression seems to coincide with the onset of axogenesis. These results suggest that type 1 inositol 1,4,5-trisphosphate may be involved in the regulatory mechanism controlling Ca2+ levels in neurons during the periods of cell differentiation, axogenesis and synaptogenesis. PMID- 8631250 TI - Origin of the c-kit-positive interstitial cells in the avian bowel. AB - Interstitial cells of Cajal (ICC) aroused much interest among neuroanatomists at the beginning of the century. These small cells, organized into networks, are intercalated between nerve fibers and muscle cells, and are now considered by many authors to be responsible for the pacemaker activity of the gut. Renewed interest in these cells arose recently when the receptor tyrosine kinase, c-kit, was shown to be associated with their functional activity. The embryonic origin of interstitial cells has remained a controversial issue ever since their discovery. Some authors consider them to be of neural or glial nature and thus of neural crest origin. Others consider them to be of fibroblastic or muscular nature. We have applied the quail-chick marker system to solve this problem. ICC were identified by means of a chicken-c-kit nucleic probe which cross-reacts with the quail c-kit gene product. We constructed chimeric bowels by grafting isotopically quail vagal neural crest into chick embryos at embryonic day 2 (E2). The enteric innervation of the chimeras was then of quail origin. In situ hybridization of the chimeric bowels showed that all the c-kit-positive cells were of the chick type, and therefore belonged to the gut mesenchyme and were not neural crest-derived cells. This observation was confirmed by culturing aneural chick guts on the chorio-allantoic membrane. Typical ICC, as defined at the EM level and by their expression of the c-kit receptor, developed in the gut wall in the complete absence of enteric innervation. One can conclude the ICC are of mesodermal origin and develop independently from enteric neurons with which they later establish anatomical and functional relations. PMID- 8631251 TI - Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype. AB - Considerable evidence has demonstrated that retinoic acid influences the formation of the primary body axis in vertebrates and that this may occur through the regulation of Hox gene expression. In this study, we show that the phenotype induced by exogenous retinoic acid in the zebrafish can also be generated by the overexpression of Hoxa-1 following injection of synthetic RNA into the fertilised egg. The isolation, sequence and expression pattern of the zebrafish Hoxa-1 gene is described. We show that exogenously applied retinoic acid causes the ectopic accumulation of Hoxa-1 message during gastrulation in the hypoblast in the head region. Overexpression of Hoxa-1 following injection of RNA causes abnormal growth of the anterior hindbrain, duplication of Mauthner neurons in rhombomere (r) 2 and fate changes of r2 mesenchymal and neurogenic neural crest. These results are discussed in terms of the role of Hoxa-1 in controlling anterior hindbrain patterning and the relationship between expression of Hoxa-1 and retinoic acid. PMID- 8631252 TI - A test for cell autonomy, based on di-cistronic messenger translation. AB - We have devised a test for cell autonomy of a gene that is switched on ectopically in a clone of cells, allowing us to ask whether the wild-type activity of this gene can influence neighbouring cells. To switch on the test gene, we used the yeast FRT system, and marked the FRT-generated cell clone by co expressing beta-galactosidase. Co-expression is achieved by a stretch of 5' untranslated mRNA from the homeotic gene Ultrabithorax (Ubx), which is inserted between the two coding sequences. We show that this Ubx sequence mediates efficient and reliable di-cistronic mRNA translation in wing imaginal discs of Drosophila. Applying our test to Ubx, we find that ectopic Ubx in wing discs strictly coincides with beta-galactosidase expression. Consequently, wing cells are transformed into cells that appear to be intermediates between wing and haltere cells, contesting the view that homeotic genes act as binary switches. PMID- 8631253 TI - The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination. AB - We have analysed expression of the ecmA and ecmB genes of Dictyostelium by enzymatic double staining using beta-galactosidase and beta-glucuronidase reporter gene constructs. Cells expressing the ecmA gene first appear as scattered cells at the mound stage of development and we show that this is also true for cells expressing the ecmB gene. During tip formation the ecmA-expressing cells move to the apex of the mound, while the ecmB-expressing cells accumulate in the base. The ecmB-expressing cells constitute part of the basal disc if the culminant is formed in situ but are discarded if a migratory slug is formed. During slug migration they are replaced by a band of ecmB-expressing cells, situated in the front half of the prespore zone and tightly apposed to the substratum. When culmination is triggered these cells rapidly move to the back half of the prestalk zone, possibly acting as a point of attachment to the substratum. Ultimately, they are joined by cells at the back of the slug, the rearguard cells, to form the basal disc. Thus, contrary to previous belief, basal disc formation is initiated very early during culmination and occurs by the forward movement of cells located in the anterior of the prespore zone. PMID- 8631254 TI - Analysis of cell movement during the culmination phase of Dictyostelium development. AB - Co-ordinated cell movement of tens of thousands of cells and periodic signals characterise the multicellular development of the cellular slime mould Dictyostelium discoideum. We investigated cell movement by analysing time-lapse video recordings made during the slug stage and the culmination phase of Dictyostelium development. Slugs viewed from the side showed an even, straight forward movement with the tip slightly raised in the air. Slugs that had migrated for a prolonged period of time either culminated or showed a behaviour best described as abortive culmination. Culmination is initiated by a local aggregation of anterior-like cells at the base of the slug at the prestalk prespore boundary, where they form a stationary mass of cells. Prespore cells continue to move forward over this stationary pile and, as a result, are lifted into the air. The stationary group of anterior-like cells thereby end up to the back of the slug. At this point the slug either falls back on the agar surface or continues culmination. If the slug continues to migrate these cells regain motility, move forward to the prespore-prestalk boundary and form a new pile again. In the case of culmination the neutral red stained cells in the pile move to the back of the slug and form a second signalling centre beside the tip. Both centres are characterised by vigorous rotational cell movement. The cells belonging to the basal centre will form the basal disc and the lower cup in the fruiting body. The upper cup will be formed by the prestalk cells rotating most vigorously at the prestalk-prespore boundary. The remaining neutral red stained anterior-like cells in the prespore zone sort either to the upper or lower organising centre in the fruiting body. PMID- 8631255 TI - A muscle-specific variant of microtubule-associated protein 4 (MAP4) is required in myogenesis. AB - Microtubule-associated protein 4 (MAP4) transcripts vary in different mouse tissues, with striated muscle (skeletal and cardiac) expressing 8- and 9-kb transcripts preferentially to the more widely distributed 5.5- and 6.5-kb transcripts (West, R. W., Tenbarge, K. M. and Olmsted, J. B. (1991). J. Biol. Chem. 266, 21886-21896). Cloning of the sequence unique to the muscle transcripts demonstrated that these mRNAs vary from the more ubiquitous ones by a single 3.2 kb coding region insertion within the projection domain of MAP4. During differentiation of the myogenic cell line, C2C12, muscle-specific MAP4 transcripts appear within 24 hours of growth in differentiation medium, and a larger MAP4 isotype (350 X 10(3) Mr) accumulates to high levels by 48 hours of differentiation. In situ hybridization analyses of transcript distribution in mouse embryos demonstrated that muscle-specific transcripts appear early in myogenesis. To block the expression of the muscle-specific MAP4, stable lines of C2C12 were generated bearing an antisense construct with the muscle-specific MAP4 sequence. Myoblast growth was unaffected whereas myotube formation was severely perturbed. Fusion occurred in the absence of the muscle MAP4 isotype, but the multinucleate syncytia were short and apolar, microtubules were disorganized and normal anisotropic myofibrils were absent. The patterns of expression of the muscle-specific transcripts and the antisense experiments indicated that this unique structural form of MAP4 plays a critical role in the formation and maintenance of muscle. PMID- 8631256 TI - Late effects of retinoic acid on neural crest and aspects of rhombomere. AB - We exposed st.10 chicks to retinoic acid (RA), both globally, and locally to individual rhombomeres, to look at its role in specification of various aspects of hindbrain derived morphology. Previous studies have looked at RA exposure at earlier stages, during axial specification. Stage 10 is the time of morphological segmentation of the hindbrain and is just prior to neural crest migration. Rhombomere 4 localised RA injections result in specific alterations of pathways some crest cells that normally migrate to sites of differentiation of neurogenic derivatives. The r4 crest cells that give rise to mesenchymal derivatives are unaffected. In addition, r4 gene expression is also partially altered by RA; within 6 hours of r4 exposure to RA, ectopic expression of Krox-20 is seen in r4 and Hoxb-1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals later in development, they showed an anterior displacement of the facial ganglion in addition to a mis-direction of the extensions of its distal axons and a dramatic decrease in the number of contralateral vestibuloacoustic neurons normally seen in r4. Only this r4 specific neuronal type is affected in r4; the motor neuron projections seem normal in experimental animals. The specificity of this result, combined with the loss of Hoxb-1 expression in r4 and the work by Krumlauf and co-workers showing gain of contralateral neurons co-localised with ectopic Hoxb-1 expression, indicates a role for Hoxb-1 and RA in the specification of this cell type in normal development. These results suggest that RA, at st.10, is able to affect some aspects of segment identity while leaving others unchanged. PMID- 8631257 TI - Polyembryonic development: insect pattern formation in a cellularized environment. AB - THe polyembryonic wasp Copidosoma floridanum produces up to 2000 individuals from a single egg. During the production of individual embryos the original anteroposterior axis of the egg is lost and axial patterning must subsequently be reestablished within each embryo. The mechanism by which this occurs is unknown. In most insects, egg polarity is established during oogenesis and early development takes place in a syncytium. In Drosophila melanogaster, the syncytium is considered essential for establishing the morphogenetic gradients that initiate segmental patterning. However, we found that development of C. floridanum occurs almost exclusively in a cellularized environment. To determine whether the D. melanogaster patterning cascade is conserved in the absence of a syncytium, we analyzed the expression of Even-skipped, Engrailed and Ultrabithorax/Abdominal-A during polyembryonic development. Here we show that in spite of the absence of a syncytium, the elements of the D. melanogaster segmentation hierarchy are conserved. The segment-polarity gene Engrailed and the homeotic genes Ultrabithorax/Abdominal-A are expressed in a conserved pattern relative to D. melanogaster. However, we detect an alteration in the expression of the Even-skipped antigen. Even-skipped is initially expressed in segmentally reiterated stripes and not in the pair-rule pattern as it is in D. melanogaster. We also observe that the expression of these regulatory proteins does not occur during the early proliferative phases of polyembryony. Our results indicate that a syncytium is not required for segmental patterning in this insect. PMID- 8631258 TI - Embryonic patterning mutants of Tribolium castaneum. AB - The identification and analysis of genes controlling segmentation in Drosophila melanogaster has opened the way for understanding similarities and differences in mechanisms of segmentation among the insects. Homologues of Drosophila segmentation genes have been cloned and their expression patterns have been analyzed in a variety of insects, revealing that the patterns of expression of many genes are conserved. Conserved expression patterns do not, however, necessarily reflect conserved gene function. To address gene function, we have conducted a screen for mutations that alter embryonic patterning of the beetle, Tribolium castaneum. One of the mutations isolated, godzilla, affects early steps in the segmentation process in the whole animal, like Drosophila pair-rule mutants. Another mutation, jaws, is novel: it caused both a dramatic homeotic transformation in the thorax and first abdominal segment as well as a deletion of most of the segments of the abdomen. In Tribolium and other intermediated germ band insects, the anterior segments of the embryo are determined in the syncytium of the blastoderm, whereas the abdominal segments proliferated in the cellular environment. Both the godzilla and jaws mutations affect segments that are formed in the syncytium differently from those that are formed after cellularization. These regionally specific phenotypes may reflect the different patterning mechanisms that must be employed by the anterior and posterior regions of an intermediated germ insect. PMID- 8631259 TI - Mos is required for MAP kinase activation and is involved in microtubule organization during meiotic maturation in the mouse. AB - Mos is normally expressed during oocyte meiotic maturation in vertebrates. However, apart from its cytostatic factor (CSF) activity, its precise role during mouse meiosis is still unknown. First, we analyzed its role as a MAP kinase kinase kinase. Mos is synthesized concomitantly with the activation of MAP kinase in mouse oocytes. Moreover, MAP kinase is not activated during meiosis in oocytes from mos -/- mice. This result implies that Mos is necessary for MAP kinase activation in mouse oocytes. Raf-1, another MAP kinase kinase kinase, is already present in immature oocytes, but does not seem to be active when MAP kinase is activated. Moreover, the absence of MAP kinase activation in mos -/- oocytes demonstrates that Raf-1 cannot compensate for the lack of Mos. These results suggest that Raf-1 is not involved in MAP kinase activation. Second, we analyzed the organization of the microtubules and chromosomes in oocytes from mos -/- mice. We observed that during the transition between two meiotic M-phases, the microtubules and chromosomes evolve towards an interphase-like state in mos -/- oocytes, while in the control mos +/- oocytes they remain in an M-phase configuration, as in the wild type. Moreover, after spontaneous activation, the majority of mos -/- oocytes are arrested for at least 10 hours in a third meiotic M-phase where they exhibit monopolar half-spindles. These observations present the first evidence, in intact oocytes, of a role for the Mos/.../MAP kinase cascade in the control of microtubule and chromatin organization during meiosis. PMID- 8631260 TI - In vitro analysis of epiblast tissue potency for hematopoietic cell differentiation. AB - In murine embryogenesis, all cells that will constitute the embryonic structures originate from the epiblast (primitive ectoderm) tissue, the epithelial cell sheet of the gastrulating embryo. The cells of this tissue are totipotent at the beginning of gastrulation, but at the end of this period are specified to particular cell lineages. Thus, it is likely that during murine gastrulation, the potency of epiblast cells that were originally totipotent becomes restricted as development progresses. However, the mechanisms of this process are unknown. We have investigated this process in vitro, focusing on the hematopoietic cell lineage. To detect the hematogenic potency of the epiblast tissue, we established an in vitro culture system in which the hematopoietic cell differentiation of the epiblast tissue was supported by a stromal cell layer. With this culture system, we investigated the process by which this potency becomes spatially and temporally restricted during gastrulation. The results showed that hematogenic potency resides in the entire epiblast of the early- to mid-gastrulating embryo, but becomes restricted to the posterior half of the epiblast at the headfold stage. Furthermore, we showed that this process is altered by exogenous bone morphogenetic protein-4 (BMP-4) or activin A, which may be mesoderm inducers in Xenopus embryogenesis. PMID- 8631261 TI - Dysgenesis of cephalic neural crest derivatives in Pax7-/- mutant mice. AB - Pax7 is a member of the paired box containing gene family. Its expression pattern suggests a function in cephalic neural crest derivatives, skeletal muscle and central nervous system development. To understand the role of Pax7 during mouse embryogenesis, we used the homologous recombination technique in embryonic stem cells and generated Pax7-/- mice. Homozygous animals are born but die shortly afer weaning. They exhibit malformations in facial structures involving the maxilla and nose. Our analysis suggests that the observed phenotype is due to a cephalic neural crest defect. No obvious phenotype could be detected in the central nervous system and skeletal muscle. Functional redundancy between Pax7 and Pax3 is discussed. PMID- 8631262 TI - Allele-specific in situ hybridization (ASISH) analysis: a novel technique which resolves differential allelic usage of H19 within the same cell lineage during human placental development. AB - Precursory studies of H19 transcription during human foetal development have demonstrated maternally derived monoallelic expression. Analyses in extra embryonic tissues, however, have been more equivocal, with discernible levels of expression of the paternal allele of H19 documented in the first trimester placenta. By refining the in situ hybridization technique we have developed an assay to enable the functional imprinting status of H19 to be determined at the cellular level. This assay involves the use of oligonucleotide DNA probes that are able to discriminate between allelic RNA transcripts containing sequence polymorphisms. Biallelic expression of H19 is confined to a subpopulation of cells of the trophoblast lineage, the extravillous cytotrophoblast, while the mesenchymal stroma cells maintain the imprinted pattern of monoallelic expression of H19 throughout placental development. This data demonstrates that the low level of paternal H19 expression previously detected in normal human placenta is not due to a random loss of functional imprinting, but appears to result from a developmentally regulated cell type-specific activation of the paternal allele. In addition, biallelic expression of H19 does not seem to affect the functional imprinting of the insulin-like growth factor II gene, which is monoallelically expressed at relatively high levels in the extra-villous cytotrophoblasts. These results imply that the allelic usage of these two genes in normal human placental development may not be directly analogous to the situation previously documented in the mouse embryo. PMID- 8631263 TI - decapentaplegic, a target gene of the wingless signalling pathway in the Drosophila midgut. AB - dishevelled, shaggy/zeste-white 3 and armadillo are required for transmission of the wingless signal in the Drosophila epidermis. We show that these genes act in the same epistatic order in the embryonic midgut to transmit the wingless signal. In addition to mediating transcriptional stimulation of the homeotic genes Ultrabithorax and labial, they are also required for transcriptional repression of labial by high wingless levels. Efficient labial expression thus only occurs within a window of intermediate wingless pathway activity. Finally, the shaggy/zeste-white 3 mutants revealed that wingless signalling can stimulate decapentaplegic transcription in the absence of Ultrabithorax, identifying decapentaplegic as a target gene of wingless. As decapentaplegic itself is required for wingless expression in the midgut, this represents a positive feed back loop between two cell groups signalling to each other to stimulate each other's signal production. PMID- 8631264 TI - Retinal axon divergence in the optic chiasm: midline cells are unaffected by the albino mutation. AB - The visual pathway in albino animals is abnormal in that there is a smaller number of ipsilaterally projecting retinal ganglion cells. There are two possible sites of gene action that could result in such a defect. The first site is the retina where the amount of pigmentation in the retinal pigment epithelium is correlated with the degree of ipsilateral innervation (La Vail et al. (1978) J. Comp. Neurol. 182, 399-422). The second site is the optic chiasm, the site of retinal axon divergence. We investigated these two possibilities through a combination of in vivo and in vitro techniques. Our results demonstrate that the growth patterns of retinal axons and the cellular composition of the optic chiasm in albino mice are similar to those of normally pigmented mice, consistent with the albino mutation exerting its effects in the retina, and not on the cells from the chiasmatic midline. We directly tested whether the albino mutation affects the chiasm by studying 'chimeric' cultures of retinal explants and chiasm cells isolated from pigmented and albino mice. Crossed and uncrossed axons from pigmented or albino retinal explants display the same amount of differential growth when grown on either pigmented or albino chiasm cells, demonstrating that the albino mutation does not disrupt the signals for retinal axon divergence associated with the albino optic chiasm. Furthermore, in vitro, a greater proportion of albino retinal ganglion cells from ventrotemporal retina, origin of uncrossed axons, behave like crossed cells, suggesting that the albino mutation acts by respecifying the numbers of retinal ganglion cells that cross the chiasmatic midline. PMID- 8631265 TI - A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers. AB - We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula. PMID- 8631266 TI - Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. AB - The totipotent stem cells of the pregastrulation mouse embryo which give rise to all embryonic somatic tissues and germ cells express Oct-4. The expression is downregulated during gastrulation and is thereafter only maintained in the germline lineage. Oct-4/lacZ transgenes were used to determine how this pattern of expression was achieved, and resulted in the identification of two separate regulatory elements. The distal element drives Oct-4 expression in preimplantation embryos, in migratory and postmigratory primordial germ cells but is inactive in cells of the epiblast. In cell lines this element is specifically active in embryonic stem and embryonic germ cells. The proximal element directs the epiblast-specific expression pattern, including downregulation during gastrulation; in cell lines its activity is restricted to epiblast-derived cells. Thus, Oct-4 expression in the germline is regulated separately from epiblast expression. This provides the first marker for the identification of totipotent cells in the embryo, and suggests that expression of Oct-4 in the totipotent cycle is dependent on a set of factors unique to the germline. PMID- 8631267 TI - Rhombomere of origin determines autonomous versus environmentally regulated expression of Hoxa-3 in the avian embryo. AB - We have investigated the pattern and regulation of Hoxa3 expression in the hindbrain and associated neural crest cells in the chick embryo, using whole mount in situ hybridization in conjunction with DiI labeling of neural crest cells and microsurgical manipulations. Hoxa3 is expressed in the neural plate and later in the neural tube with a rostral border of expression corresponding to the boundary between rhombomeres (r) 4 and 5. Initial expression is diffuse and becomes sharp after boundary formation. Hoxa3 exhibits uniform expression within r5 after formation of rhombomeric borders. Cell marking experiments reveal that neural crest cells migrating caudally, but not rostrally, from r5 and caudally from r6 express Hoxa3 in normal embryo. Results from transposition experiments demonstrate that expression of Hoxa3 in r5 neural crest cells is not strictly cell-autonomous. When r5 is transposed with r4 by rostrocaudal rotation of the rhomobomeres, Hoxa3 is expressed in cells migrating lateral to transposed r5 and for a short time, in condensing ganglia, but not by neural crest within the second branchial arch. Since DiI-labeled cells from transposed r5 are present in the second arch, Hoxa3-expressing neural crest cells from r5 appear to down regulate their Hoxa3 expression in their new environment. In contrast, when r6 is transposed to the position of r4 after boundary formation, Hoxa3 is maintained in both migrating neural crest cells and those positioned within the second branchial arch and associated ganglia. These results suggest that Hoxa3 expression is cell-autonomous in r6 and its associated neural crest. Our results suggest that neural crest cells expressing the same Hox gene are not eqivalent; they respond differently to environmental signals and exhibit distinct degrees of cell autonomy depending upon their rhombomere of origin. PMID- 8631268 TI - Regional specification of motoneurons along the anterior-posterior axis is independent of the notochord. AB - Cek8 and low affinity NGF receptor (LNGFR) are expressed at high levels on the chick spinal motoneurons of the brachial and lumbar segments from embryonic day (E) 5 to E7, but weakly on the motoneurons of the non-limb-innervating segments. We determined by means of heterotopic neural tube transplantation, that the expression of these molecules was already intrinsically determined at E2. We used these spatiotemporal specific molecules as markers of motoneuron subpopulations. To analyze how motoneurons acquire regional specification along the anterior posterior (A-P) axis and in the transverse plane, we observed the expression of these molecules on ectopic motoneurons induced by implanting a supernumerary notochord or floor plate at E2. The ectopic motoneurons induced by the graft obtained from either the thoracic or lumbar segments had the same expression profile as the normal motoneurons at each A-P level. These findings suggest that regional specification of motoneurons, at least of Cek8 and LNGFR expression, is independent of the notochord and the floor plate and that the whole neural tube appears to be committed to differentiate into the motoneuron subtypes along the A P axis at the operative stages. PMID- 8631269 TI - An ovomucin-like protein on the surface of migrating primordial germ cells of the chick and rat. AB - A mucin was discovered on the surface of migratory primordial germ cells (PGCs) from chick and rat embryos by means of two monoclonal antibodies. The protein was found to be identical or closely related to ovomucin, a 600 X 10(3) relative molecular mass glycoprotein, and a major constituent of the vitelline membrane of the avian yolk. Based on its resemblance to ovomucin it is referred to as ovomucin-like protein (OLP). The OLP was expressed on PGCs from E3 to E7 female, and from E3 to E12 male chick embryos as the PGCs migrate and colonize the gonadal ridges. After the PGCs have settled in the gonads, they no longer express OLP. In tissue cultures of dissociated cells from E6 gonads, OLP was present only on cells that were positive for PAS staining, the standard histological method to identify PGCs in the chick embryo. Since unfixed PGCs were recognized by the antibodies, at least part of the OLP is localized on the cell surface. The anti OLP antibodies also stained PGCs in the gonads of the rat embryo, showing that the expression of this antigen on PGCs is phylogenetically conserved. Ovomucin isolated from vitelline membrane prevented adhesion of fibroblasts but not PGCs when used a as a substratum in vitro. The anti-adhesive quality of the mucin resides in the sialic acid residues of the carbohydrate side chains. We propose that OLP has a similar anti-adhesive quality as the ovomucin from vitelline membrane, and that this anti-adhesive property is important to prevent precocious adhesion of migrating PGCs to blood vessel walls and to connective tissue in the mesentery as they migrate toward the gonadal ridges. PMID- 8631270 TI - Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system. AB - Interactions between embryonic neural cells generate the specific patterns of connectivity observed in nervous systems. Cell surface carbohydrates have been proposed to function in cellular recognition events guiding such interactions. Carbohydrate-binding proteins (lectins) that recognize specific oligosaccharide ligands in embryonic neural tissue provide a molecular mechanism for carbohydrate mediated cell-cell interactions in neural development. Therefore, we have screened an embryonic Drosophila melanogaster cDNA library, expressed in COS1 cells, for carbohydrate-binding activity. COS1 cells expressing putative Drosophila lectins were identified and recovered based on their adhesion to immobilized preparations of neutral and zwitterionic glycolipids extracted from Drosophila embryos. We have identified an endogenous lectin expressed during Drosophila embryogenesis. The cloned lectin, designated 'gliolectin', possesses a novel protein sequence with a calculated molecular mass of 24,993. When expressed in Drosophila S2 cells, the lectin mediates heterophilic cellular aggregation. In embryos, gliolectin is expressed by a subset of glial cells found at the midline of the developing nervous system. Expression is highest during the formation of the Drosophila embryonic axonal commissures, a process requiring midline glial cell funcion. Immunoprecipitation with a monoclonal antibody against gliolectin yields a protein of Mr=46,600 from Drosophila embryonic membranes, suggesting that post-translational modification of gliolectin is extensive. Epitope- tagged chimericproteins composed of the amino terminal one-half of gliolectin and the Fc region of human IgG bind a small subset of the total glycolipids extracted from Drosophila embryos, demonstrating that the lectin activity of gliolectin can discriminate between oligosaccharide structures. The presence of gliolectin in the developing Drosophila embryonic nervous system further supports a role for cell surface carbohydrates in cell-cell recognition and indicates that the molecular diversity of animal lectins is not yet completely defined. PMID- 8631271 TI - The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development. AB - The differentiation of Drosophila germ cells is a useful model for studying mechanisms of cell specification. We report the identification of a gene, stonewall, that is required for germ cell development. Mutations in stonewall block proper oocyte differentiation and frequently cause the presumptive oocyte to develop as a nurse cell. Eventually, germ cells degenerate apoptotically. Stonewall is a germ cell nuclear protein; Stonewall has a DNA binding domain that shows similarities to the Myb and Adf-1 transcription factors and has other features that suggest that it is a transcription activating factor. We suggest that Stonewall transcriptional regulation is essential in cystocytes for maturation into specialized nurse cells and oocyte. PMID- 8631272 TI - The Drosophila genes crumbs and stardust are involved in the biogenesis of adherens junctions. AB - Morphogenetic movements of epithelia during development underlie the normal elaboration of the final body plan. The tissue integrity critical for these movements is conferred by anchorage of the cytoskeleton by adherens junctions, initially spot and later belt-like, zonular structures, which encircle the apical side of the cell. Loss-of-function mutations in the Drosophila genes crumbs and stardust lead to the loss of cell polarity in most ectodermally derived epithelia, followed in some, such as the epidermis, by extensive apoptosis. Here we show that both mutants fail to establish proper zonulae adherentes in the epidermis. Our results suggest that the two genes are involved in different aspects of this process. Further, they are compatible with the hypothesis that crumbs delimits the apical border, where the zonula adherens usually forms and where Crumbs protein is normally most abundant. In contrast, stardust seems to be required at an earlier stage for the assembly of the spot adherence junctions. In both mutants, the defect observed at the ultrastructural level are preceded by a misdistribution of Armadillo and DE-cadherin, the homologues of beta-catenin and E-cadherin, respectively, which are two constituents of the vertebrate adherens junctions. Strikingly, expansion of the apical membrane domain in epidermal cells by overexpression of crumbs also abolishes the formation of adherens junctions and results in the disruption of tissue integrity, but without loss of membrane polarity. This result supports the view that membrane polarity is independent of the formation of adherens junctions in epidermal cells. PMID- 8631273 TI - The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein. AB - Mutations in the inturned (in) gene result in abnormal wing hair polarity and in many wing cells forming two or more hairs instead of the normal single hair. We have generated genetic mosaics in a number of different experiments and find that the in gene is required in all regions of the wing and that it functions in a cell autonomous fashion. We report the molecular cloning of the in gene, the molecular mapping of in mutations and the isolation and sequencing of an in cDNA clone. The in gene encodes a novel protein whose sequence suggests it will be membrane bound. The ability of an in cDNA, the expression of which is driven by the basal activity of the hsp70 promoter to rescue an in mutation suggests that patterned expression of in is unlikely to play a role in the function of the gene. PMID- 8631274 TI - Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila. AB - In D. melanogaster the binary switch gene Sex-lethal (Sxl) plays a pivotal role in somatic sex determination -- when the Sxl gene is on the female pathway is followed, while the male pathway is followed when the gene is off. In the present study we have asked whether the Sxl gene is present in other species of the genus Drosophila and whether it is subject to a similar sex-specific on-off regulation. Sxl proteins were found in all of the drosophilids examined, and they display a sex-specific pattern of expression. Furthermore, characterization of the Sxl gene in the distant drosophilan relative, D. virilis, reveals that the structure and sequence organization of the gene has been well conserved and that, like melanogaster, alternative RNA processing is responsible for its sex-specific expression. Hence, this posttranscriptional on-off regulatory mechanism probably existed before the separation of the drosophilan and sophophoran subgenera and it seems likely that Sxl functions as a sex determination switch gene in most species in the Drosophila genus. Although alternative splicing appears to be responsible for the on-off regulation of the Sxl gene in D. virilis, this species is unusual in that Sxl proteins are present not only in females but also in males. The D. virilis female and male proteins appear to be identical over most of the length except for the amino-terminal approx. 25 aa which are encoded by the differentially spliced exons. In transcriptionally active polytene chromosomes, the male and female proteins bind to the same cytogenetic loci, including the sites corresponding to the D. virilis Sxl and tra genes. Hence, though the male proteins are able to interact with appropriate target pre-mRNAs, they are apparently incapable of altering the splicing pattern of these pre mRNAs. PMID- 8631275 TI - PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. AB - It has been proposed that the Xenopus homeobox gene, XlHbox8, is involved in endodermal differentiation during pancreatic and duodenal development (Wright, C.V.E., Schnegelsberg, P. and De Robertis, E.M. (1988). Development 105, 787 794). To test this hypothesis directly, gene targeting was used to make two different null mutations in the mouse XlHbox8 homolog, pdx-1. In the first, the second pdx-1 exon, including the homeobox, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N terminus of PDX-1, replacing most of the homeodomain. Neonatal pdx-1 -/- mice are apancreatic, in confirmation of previous reports (Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Nature 371, 606-609). However, the pancreatic buds do form in homozygous mutants, and the dorsal bud undergoes limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. This outgrowth does not contain insulin or amylase-positive cells, but glucagon expressing cells are found. The rostral duodenum shows a local absence of the normal columnar epithelial lining, villi, and Brunner's glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. The PDX-1/beta-galactosidase fusion allele is expressed in pancreatic and duodenal cells in the absence of functional PDX-1, with expression continuing into perinatal stages with similar boundaries and expression levels. These results offer additional insight into the role of pdx-1 in the determination and differentiation of the posterior foregut, particularly regarding the proliferation and differentiation of the pancreatic progenitors. PMID- 8631276 TI - The control of trichome spacing and number in Arabidopsis. AB - Arabidopsis trichomes are single-celled epidermal hairs that serve as a useful model for the study of plant cell differentiation. An examination of the distribution of trichomes early in their development revealed that developing trichomes occur adjacent to another trichome much less frequently than would be expected by chance. Clonal analysis of epidermal cell lineages ruled out a role for cell lineage in generating the observed minimum-distance spacing pattern. Taken together, these results are consistent with a role for lateral inhibition in the control of trichome development. We also report the identification of a new locus, Reduced Trichome Number (RTN), which affects the initiation of trichomes. This locus was initially detected by the reduced number of leaf trichomes on Landsberg erecta plants compared to that on Columbia plants. Quantitative Trait Locus mapping revealed that more than 73% of the variation in trichome number was due to a major locus near erecta on chromosome 2. The reduced number of trichomes conditioned by the Landsberg erecta allele of this locus appeared to be due to an early cessation of trichome initiation. The implications of these observations are discussed with regard to previously published models of trichome development. PMID- 8631277 TI - Predictive value of EEG for outcome and epilepsy following neonatal seizures. AB - The value of the electroencephalogram in predicting outcome and epilepsy was examined in neonates who had experienced EEG-confirmed neonatal seizures. Electroencephalogram, neuroimaging studies, and other clinical variables were systematically analyzed in 81 consecutive neonates with EEG-confirmed seizures. The surviving subjects were followed for a mean of 17 months to determine if they developed post-neonatal seizures (PNS) and abnormal neurodevelopmental outcome. Several EEG variables were correlated with neurodevelopmental outcome and PNS when analyzed with univariate and multivariate statistical analyses. The EEG background activity and the presence of status epilepticus were strong predictors of outcome, but were not associated with PNS. The presence of rhythmic theta alpha bursts was highly favorable for both outcome and PNS. In the interictal EEG, the number of negative sharp waves in the temporal region correlated with outcome and PNS. Clinical variables associated with unfavorable outcomes included an abnormal neonatal neurologic exam and certain seizure etiologies (e.g. cerebral dysgenesis and infections). Global abnormalities on neuroimaging studies were invariably associated with an unfavorable outcome and with the development of PNS in 66% of cases. Using multivariate analysis, prediction of outcome (favorable versus unfavorable) was accurately achieved in 85% of cases when combining EEG variables with neuroimaging and clinical findings. In conclusion, in neonates with EEG-confirmed seizures, the EEG is a useful predictor of outcome, but is a less useful predictor of PNS. PMID- 8631278 TI - Spike detection. I. Correlation and reliability of human experts. AB - A panel of 5 experienced electroencephalographers detected spikes in EEG trials from 40 epilepsy patients and 10 control subjects. 1952 spikes were detected, and detailed attribute scores were recorded. Statistics from the theory of measurement error are utilized to quantify the reliability and difficulty of the study. An extension of the Pearson correlation coefficient, called the detection correlation coefficient, is derived and used in recognition of the fact that readers agree on numerous non-spike regions. Spike perception is modeled with both dichotomous and continuous values. as expected, the study reliability is higher when using the continuous values. Standard sensitivity and specificity definitions are extended and applied to continuous-valued spike perception. A database of "panel scores" was created from the 5 reader scorings by merging spikes within 75 msec on each side. The average inter-reader correlation is 0.79 with a corresponding reliability of 0.95. Average spike attributes are calculated, and the resulting database can serve as a "gold standard" for testing computer algorithms or other readers. PMID- 8631279 TI - Surface mapping of spike potential fields: experienced EEGers vs. computerized analysis. AB - An EEG epileptiform spike focus recorded with scalp electrodes is clinically localized by visual estimation of the point of maximal voltage and the distribution of its surrounding voltages. We compared such estimated voltage maps, drawn by experienced electroencephalographers (EEGers), with a computerized spline interpolation technique employed in the commercially available software package FOCUS. Twenty-two spikes were recorded from 15 patients during long-term continuous EEG monitoring. Maps of voltage distribution from the 28 electrodes surrounding the points of maximum change in slope (the spike maximum) were constructed by the EEGer. The same points of maximum spike and voltage distributions at the 29 electrodes were mapped by computerized spline interpolation and a comparison between the two methods was made. The findings indicate that the computerized spline mapping techniques employed in FOCUS construct voltage maps with similar maxima and distributions as the maps created by experienced EEGers. The dynamics of spike activity, including correlations, are better visualized using the computerized technique than by manual interpretation alone. Its use as a technique for spike localization is accurate and adds information of potential clinical value. PMID- 8631280 TI - Subdural potentials at orbitofrontal and mesial prefrontal areas accompanying anticipation and decision making in humans: a comparison with Bereitschaftspotential. AB - Field potentials associated with the execution of a warned choice Go/No-Go reaction task were recorded from prefrontal supplementary (SMA) and primary motor cortex (MI) by using subdural electrodes in 5 epileptic patients during presurgical evaluation. The choice was between a Go and a No-Go imperative stimulus (S2) in the S1-S2 paradigm. Orbitofrontal and mesial prefrontal areas generated a slow preceding potential before S2 (most likely late CNV), and bilateral mesial prefrontal areas generated a transient potential, most likely related to decision making, upon S2 in both Go and No-Go conditions. In self paced, repetitive movement, the Bereitschaftspotential was seen only at SMA and MI, but not in the prefrontal area. The present result, therefore, suggests that in humans orbitofrontal and mesial frontal areas play an important role in preparation for cognition and in decision making, whereas SMA and MI do so in motor preparation. PMID- 8631281 TI - Re-examination of the evidence for low-dimensional, nonlinear structure in the human electroencephalogram. AB - We have re-examined single channel EEG data obtained from normal human subjects. In the original analysis, calculation of the correlation dimension with the Grassberger-Procaccia algorithm produced results consistent with and interpretation of low-dimensional behavior. The re-examination suggests that the previous indication of low-dimensional structure was an artifact of autocorrelation in the oversampled signal. Calculations with a variant of the Grassberger-Procaccia algorithm modified to be less sensitive to autocorrelations, and comparison with linear gaussian surrogate data, indicate that these data may be more appropriately modeled by linearly filtered noise. Discriminant analysis further indicates that the correlation dimension is a poor discriminator for distinguishing between EEGs recorded at rest and during periods of cognitive activity. It remains possible that the application of other nonlinear measures or the examination of multichannel EEG data may resolve structures not found in these calculations. PMID- 8631282 TI - Ipsilateral independent periodic lateralized epileptiform discharges. AB - We have identified a previously unreported subtype of periodic lateralized epileptiform discharge (PLED) characterized by periodic discharges arising from ipsilateral independent foci. All 4 patients had acute cerebral lesions, and 3 of them had focal motor seizures with secondary generalization. The site of localization of the PLEDs corresponded to the boundaries of the underlying structural lesion or lesions, and this, together with the variable temporal relationship between them, supports a cortical origin for PLEDs associated with underlying lesions. the spatial and temporal independence of these periodic discharges in combination with their association with (1) acute cerebral lesions. (2) altered consciousness and seizures, and (3) resolution with time leads us to propose the term "IpsiIPs" to describe this subtype of PLEDs. PMID- 8631283 TI - Dental pulp response to traumatic injuries--a retrospective analysis with case reports. AB - The reactions of the dental pulp to traumatic injuries can be extremely varied. They range from almost immediate pulp death to long-term slow pulp canal calcification. In this study the pulpal reactions were divided into three types: pulps with a very poor prognosis that required endodontic therapy soon after the tooth was traumatized. Seventy-nine teeth were studied in this category, and all 79 teeth required endodontic therapy; pulps with a moderate prognosis that required endodontic intervention some 18 to 24 months after the traumatic episode. Forty-eight teeth were studied in this category, and 27 of them required endodontic therapy: pulps with a very good prognosis that rarely required endodontic therapy. Fifty-two teeth were studied in this category, and only 2 required endodontic therapy. The prognosis of a particular pulp depends on the degree and type of trauma. PMID- 8631285 TI - Endodontic measurements in digital radiographs acquired by a photostimulable, storage phosphor system. AB - Digora is a newly developed digital system for intraoral radiography utilizing photostimulable storage phosphor image plates. The aim was to test the quality of the system in respect to the visibility of endodontic files and root apices at different exposures. In teeth from five dry mandibular jaw sections, Hedstrom files no. 15 were placed with the file tip at varying distances from the apex. The specimens were covered with soft tissue equivalent material and placed behind a 2-cm thick polymethyl methacrylate cylinder filled with water. Distance from focus to image plate was 30 cm. Exposures were made at 60 kV and 7 mA at 7 timer settings from 16 ms to 1000 ms in 50% increments. All images were viewed from the monitor by each of 8 observers allowed to use available contrast enhancement facilities to obtain subjectively good image quality. Utilizing built-in measurement functions, distances from file tip and root apex, respectively, to a reference line were measured in 0.1 mm. For measurements between file tip and reference line, and root apex and reference line, there was no statistically significant difference between measurements made at the lowest exposure, compared to the second lowest exposure but there was a statistically significant difference compared to the 5 highest. Measurements at the second lowest exposure were not significantly different from those obtained at the 5 highest exposures. Interobserver variability expressed as standard deviation between observers showed the largest value for measurements obtained at the lowest exposure. The mean observed distances between the tip of the file and the root apex were calculated and showed no statistically significant differences between different exposures. We concluded that the Digora intraoral image plate system provided reliable endodontic measurements even at very low exposures. PMID- 8631284 TI - The reliability of electric pulp test after concussion injury. AB - Traumatic injuries to teeth present a difficult diagnostic problem with respect to determining the vitality of the affected teeth. Conventionally, a latency period of 4 to 6 weeks is proposed as the amount of time required for the return of a positive electric pulp test (EPT) response; however, this has been based on empirical data. The purpose of this study was to 1) determine a time frame during which a return of a reliable response to EPT may be measured, and 2) correlate this time frame with pulpal vascular changes that occur after traumatic injuries. Four canines from each of nine ferrets were used for this experiment (18 traumatized and 18 control teeth). A reproducible concussion injury was devised by releasing a known weight at a constant height above the facial surface of the experimental teeth. The EPT response was confirmed by the use of an EMG muscle recorder. Animals were killed at 24, 48 and 72 h and 6, 12 and 28 days after the injury and the teeth analyzed histologically. The histological sections showed an absence of inflammatory cells in both groups; however, according to the NIH image system, an increase in the number of vessels was detected in the experimental group when compared to the controls. In a majority of the cases, a positive response to the EPT returned within 12 days post concussion injury and as early as 10 days. In addition, a significant vascular change was accompanied with the loss of the EPT response. This study provided a reliable method by which concussion injuries may be studied using an animal model. PMID- 8631286 TI - Influence of storage conditions of extracted teeth on dentine removal by a standardised method of filing. AB - Extracted teeth are often stored in a variety of solutions for different lengths of time prior to use in studies on the efficacy of root canal preparation. The aim of this study was to evaluate the effect of duration (12 h, 1, 4 and 15 weeks) and type of storage medium (Saline, Formal-saline, Sodium Hypochlorite and Dry conditions) on dentine removal by standardised filing. Apparatus was constructed to standardise variables inherent in filing. These included the file, the rate, duration and amplitude of its push-pull motion, its alignment with a flat dentine surface, the interfacial force (80 gms) and rate of irrigation. Freshly extracted teeth were immediately stored in one of the 13 combinations of storage media and duration. The teeth were prepared into test specimens by splitting the roots and flattening the root canal surface. Twenty specimens were designated to each combination. Dentine removal was quantified by profilometry using maximum depth at 8 selected points. Statistical analysis of the mean values showed highly significant differences (p<0.001) between the media at 1, 4 and 15 weeks.When comparisons of mean values were made within the storage groups, there were highly significant differences (p<0.001) in dentine removal between 0, 1, 4 and 15 weeks of storage in sodium hypochlorite and saline. There were no significant differences found for storage in formal-saline. In dry conditions, the differences were significant (p<0.01) between 0, 1, 4 and 15 weeks of storage, but were not significant when the 4 weeks values were excluded. PMID- 8631287 TI - Effect of various sealers with gutta-percha as root-end fillings on healing after replantation. AB - This study examined histologically the effect of three sealers used with gutta percha as root-end fillings placed in a replantation model. A total of 14 molar teeth were used in seven monkeys. After extraction, root ends were resected, the canals contaminated with oral bacteria, root-end cavities prepared, and fillings of gutta-percha and sealer placed prior to replantation. After 8 weeks the teeth and surrounding jaw were removed and prepared for histological examination. Six roots filled with gutta-percha plus a fortified zinc oxide-eugenol cement (IRM), 13 with gutta-percha plus zinc oxide-eugenol (EP), and eight with gutta-percha plus Kloropercha, were available for examination. The tissue response to gutta percha with either zinc oxide material was characterized by little or no inflammation of limited extent. In contrast, more severe inflammation was observed against root ends filled with gutta-percha and Kloropercha. Gutta-percha plus IRM or zinc oxide-eugenol had a much more favourable response than that to amalgam reported previously in a similar experiment. PMID- 8631288 TI - Histological suture changes following retraction of the maxillary anterior bone segment after corticotomy. AB - By cephalometric and dental cast analyses, the effects of corticotomy in combination with 45-50 days retraction of the maxillary anterior segment were evaluated in five female Japanese monkeys (Macaca fuscata). In addition, microscopic changes of the related sutures were examined. According to the analyses of the dental casts, the retraction of the maxillary anterior segment in the experimental group was increased when compared with that of the control group. There was no change in occlusal molar relationship. The cephalometric analysis revealed that the "Point A" and "Metal Implant" on the mid-sagittal site between the upper central incisors in the experimental group showed more pronounced retraction than that in two untreated control animals. These results were histologically evaluated by changes of the suture structure: irregularity of functional arrangement of component cells and fibers, an increase in number of cells, resorption and formation of bone, and an enlargement of the suture width. Resorption of tooth roots and alveolar bone, and irregularity of the periodontal ligament were observed. In general, these histological changes appeared more extensive in the control specimens compared with the experimental specimens. The osseous histological changes were mainly observed in the compact bone area, while in the spongy bone area of both control and experimental specimens only minimal changes occurred. These results demonstrated the efficacy of combining a corticotomy procedure with retraction of the maxillary anterior bone segment by an orthodontic force. PMID- 8631289 TI - Multiple dens invaginatus in two brothers. AB - Dens invaginatus is a developmental variation resulting from an alteration in the normal growth pattern of the dental papilla of a tooth. They predominantly occur on maxillary permanent lateral incisors. Their occurrence in several maxillary incisor teeth in an individual has been frequently reported, and the examination of bilateral teeth for the anomaly is well accepted. However, the present case illustrates the importance of examining both maxillary and mandibular incisors in patients who present with a dens invaginatus, in addition to other members of the family. PMID- 8631290 TI - Effect of wavelength and bandwidth on the clinical reliability of laser Doppler recordings. AB - The aim of this study was to investigate the effect of wavelength and bandwidth on laser Doppler flowmeter signals from vital and root-filled teeth, and to establish their sensitivity and specificity. Twenty human subjects were recruited, each with a vital tooth and the contralateral tooth root filled but not restored apart from the palatal access cavity. Readings were taken from these teeth for 3 min at 20 Hz for each of 3.1 kHz, 14.9 kHz and 22.1 kHz bandwidths using a modified laser Doppler blood flow monitor which permitted simultaneous recording using 810 nm and 633 nm light sources with a probe of four optical fibres with 0.25 mm separation. Ten traces from each combination of variables was examined by 10 trained observers who indicated if the traces came from vital or root-filled teeth judged by the amplitude and regularity of pulsatility of the trace. From the accuracy of their replies, sensitivity and specificity were calculated. Median flux values were higher for vital teeth than for root-filled teeth and for the 810 nm wavelength than for the 633 nm wavelength. Flux values increased with wider bandwidth using the 810 nm light source. With the 633 nm light source, the highest flux values were recorded using the 3.1 kHz bandwidth. Using the Mann-Whitney U test, there was a highly significant difference between readings from vital and root-filled teeth for the 3.1 kHz/810 nm wave length combination (p<0.003) and a significant difference for the 3.1 kHz/633 nm wavelength group (p<0.02). Comparison of other groups showed no significant difference (p>0.05). The 810 nm wavelength showed good sensitivity but poor specificity at 14.9 and 22.1 kHz bandwidths. The 633 nm wavelength showed good specificity, but poor sensitivity, at 14.9 and 22.1 kHz bandwidths. The 3.1 kHz bandwidth showed the best sensitivity and specificity for both wavelengths. Sensitivity and specificity were increased if the results of fast Fourier analysis were considered in addition to observations of flux values and pulsatility of traces. The 810 nm/3.1 kHz combination offered the greatest sensitivity and specificity as a test to distinguish between root-filled and vital teeth. This combination was best when the results of Fourier analysis were considered in addition to visual observations. PMID- 8631291 TI - Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. AB - Cytokinesis in a plant cell is accomplished by the formation of a cell plate in the center of the phragmoplast. Little is known of the molecular events associated with this process. In this study, we report the identification of a dynamin-like protein from soybean and demonstrate that this protein is associated with the formation of the cell plate. Plant dynamin-like (PDL) protein contains 610 amino acids showing high homology with other members of the dynamin protein family. Western blot experiments demonstrated that it is associated with the non ionic detergent-resistant fraction of membranes. Indirect immunofluorescence microscopy localized PDL to the cell plate in dividing soybean root tip cells. Double labeling experiments demonstrated that, unlike phragmoplast microtubules which are concentrated on the periphery of the forming plate, PDL is located across the whole width of the newly formed cell plate. Based on the temporal and spatial organization of PDL in the phragmoplast, we termed this protein 'phragmoplastin'. The data suggest that phragmoplastin may be associated with exocytic vesicles that are depositing cell plate material during cytokinesis in the plant cell. PMID- 8631292 TI - A new stress protein: synthesis of Schizosaccharomyces pombe UDP- Glc:glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability. AB - We have identified and begun the characterization of the gene encoding UDP Glc:glycoprotein glucosyltransferase in Schizosaccharomyces pombe. This gene, here designated gpt1, codes for a polypeptide having a signal peptide of 18 amino acids followed by 1429 amino acids with no transmembrane domain, as expected for a soluble protein of the endoplasmic reticulum (ER). The C-terminal tetrapeptide PDEL most probably corresponds to a novel ER retention signal in this fission yeast. Synthesis of the corresponding mRNA was induced 2- to 9-fold by conditions known to affect glycoprotein folding in the ER (e.g. heat shock, culture in the presence of a Ca2+ionophore, 2-mercaptoethanol or inhibitors of protein N glycosylation such as tunicamycin or 2-deoxyglucose). This is the first evidence obtained in vivo that supports the proposed involvement of the enzyme in the quality control of glycoprotein folding in the ER. Thus far, the said involvement was inferred solely from the ability of the enzyme to glucosylate misfolded but not native glycoproteins in cell-free assays. The gpt1 gene was disrupted and gpt1- cells were found to be viable. Moreover, no significant differences in the growth rate patterns at 18, 28 or 39 degrees C or in cell morphology between gpt1+ and gpt1- cells were observed, although they differed slightly in size. PMID- 8631293 TI - Gsh-1, an orphan Hox gene, is required for normal pituitary development. AB - The anterior pituitary regulates the function of multiple organ systems as well as body growth, and in turn is controlled by peptides released by the hypothalamus. We find that mutation of the Gsh-1 homeobox gene results in pleiotropic effects on pituitary development and function. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced numbers of growth hormone- and prolactin-producing cells. Moreover, the pituitary content of a subset of pituitary hormones, including growth hormone, prolactin and luteinizing hormone, is significantly decreased. The hypothalamus, although morphologically normal, is also perturbed in mutants. The gsh-1 gene is shown to be essential for growth hormone-releasing hormone (GHRH) gene expression in the arcuate nucleus of the hypothalamus. Further, sequence and electrophoretic mobility shift data suggest the Gsh-1 and GHRH genes as potential targets regulated by the Gsh-1-encoded protein. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis. PMID- 8631294 TI - GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity. AB - We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity. PMID- 8631295 TI - The delta psi- and Hsp70/MIM44-dependent reaction cycle driving early steps of protein import into mitochondria. AB - New steps in the reaction cycle that drives protein translocation into the mitochondrial matrix have been defined. The membrane potential (delta psi)- and the mtHsp70/MIM44-dependent import machinery cooperate in the transfer of the presequence across the inner membrane. Translocation intermediates, arrested at a stage where only the presequence could form a complex with mtHsp70, still required delta psi for further import. Delta psi at this stage prevented retrograde movement, since mtHsp70 did not bind to the presequence with sufficient affinity. In contrast, mature regions of incoming chains adjacent to the presequence were bound by mtHsp70 tightly enough to stabilize them in the matrix. Cycling of the mtHsp70 on and off incoming chains is a continuous process in the presence of matrix ATP. Both MIM44-bound and free forms of mtHsp70 were found in association with the incoming chains. These data are consistent with a reaction pathway in which the mtHsp70/MIM44 complex acts as a molecular ratchet on the cis side of the inner membrane to drive protein translocation into the matrix. PMID- 8631296 TI - Association of the fusion protein NSF with clathrin-coated vesicle membranes. AB - N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion. PMID- 8631297 TI - Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. AB - The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called 'der' for 'degradation in the ER'. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER. PMID- 8631298 TI - Hsp60-independent protein folding in the matrix of yeast mitochondria. AB - Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro studies, which had demonstrated that only a subset of proteins require chaperones for efficient folding. PMID- 8631299 TI - The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. AB - p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation. PMID- 8631300 TI - EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3 kinase. AB - Overexpression of a TPA-insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal-dependent changes in aKPClambda were observed for a PDGF receptor add-back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3-kinase, and were inhibited by wortmannin, an inhibitor of PI3-kinase. Furthermore, an N-terminal fragment of the catalytic subunit of PI3-kinase, p110alpha, inhibited aPKClambda-dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3-kinase. PMID- 8631301 TI - Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state. AB - The receptor-associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)-gamma response. The effects of expressing kinase negative JAK mutant proteins on signal transduction in response to IFN-gamma in wild-type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase negative JAK1 can sustain substantial IFN-gamma-inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase-negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN-gamma inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild-type cells, kinase-negative JAK2 acts as a dominant-negative inhibitor of the IFN-gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN-gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN-gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor-JAK complex may again depend on JAK2. Interestingly, a JAK1-dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state. PMID- 8631302 TI - Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation. AB - Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation. PMID- 8631303 TI - Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase. AB - The Tpl-2 protein serine/threonine kinase was originally identified, in a C terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats. The kinase domain of Tpl-2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl 2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl-2 and Tpl-2deltaC (a C-terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK-1 and SEK-1, in vitro. Furthermore, transfection of Tpl-2 into COS-1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK-1 and SAP kinase (JNK), which are substrates for MEK-1 and SEK-1, respectively. Tpl-2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl-2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK-1. PMID- 8631305 TI - B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor. AB - The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached. PMID- 8631304 TI - Specific loss of apoptotic but not cell-cycle arrest function in a human tumor derived p53 mutant. AB - The p53 tumor-suppressor gene product is frequently inactivated in malignancies by point mutation. Although most tumor-derived p53 mutants show loss of sequence specific transcriptional activation, some mutants have been identified which retain this activity. One such mutant, p53175P, is defective for the suppression of transformation in rodent cells, despite retaining the ability to suppress the growth of p53-null human cells. We now demonstrate that p53175P can induce a cell cycle arrest in appropriate cell types but shows loss of apoptotic function. Our results therefore support a direct role of p53 transcriptional activation in mediating a cell-cycle arrest and demonstrate that such activity is not sufficient for the full apoptotic response. These data suggest that either p53 can induce apoptosis through a transcriptionally independent mechanism, a function lost by p53175P, or that this mutant has specifically lost the ability to activate genes which contribute to cell death, despite activation of genes responsible for the G1 arrest. This dissociation of the cell-cycle arrest and apoptotic activities of p53 indicates that inactivation of p53 apoptotic function without concomitant loss of growth inhibition can suffice to relieve p53 dependent tumor-suppression in vivo and thereby contribute to tumor development. PMID- 8631306 TI - A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins. AB - Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis. PMID- 8631308 TI - Constitutive activation of NF-kappa B is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. AB - Human T-cell leukemia virus type I (HTLV-I) encodes a 40 kDa trans-acting protein, Tax, that regulates transcription of both the proviral and cellular genes, and can transform rat fibroblasts. To determine the functional importance of its trans-acting capacities in cell transformation, we have examined two representative pathways of transcriptional activation--HTLV-I long terminal repeat (LTR) mediated and NF-kappa B dependent--by mutational analysis of Tax. In contrast to a previous report, mutants lacking the ability to activate an NF kappaB-dependent promoter failed to transform rat fibroblasts, whereas a mutation which abolishes the HTLV-I LTR-mediated trans-activation demonstrated a wild-type capacity for cell transformation. Stable expression of Tax competent for transformation caused enhanced DNA binding of NF-kappa B in rat fibroblasts. We also demonstrate that stable co-expression of the NFKB2 precursor, known as a member of the I kappa B proteins, with wild-type Tax blocked transformation as well as eliminated aberrant NF-kappaB activation by Tax without interference with the HTLV-I LTR-mediated trans-activation. Our results indicate that constitutive activation of NF-kappa B is essential for Tax-mediated transformation of rat fibroblasts. PMID- 8631307 TI - Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. AB - The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase. PMID- 8631309 TI - Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo. AB - Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo. PMID- 8631310 TI - Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes. AB - The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability. PMID- 8631311 TI - Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome. AB - In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5' end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555-2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha-sarcin loop, the site of interaction of elongation factors EF-Tu and EF-G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940-1965 region of domain IV, around the site of affinity labeling by the 3' end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390-2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA. PMID- 8631312 TI - Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. AB - Initiation of RNA-dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV-1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV-1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo- and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV-1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV-1 RNA, HIV-1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer-template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication. PMID- 8631313 TI - A leucine zipper motif determines different functions in a DNA replication protein. AB - RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors. PMID- 8631314 TI - ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. AB - During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes. PMID- 8631315 TI - Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing. AB - In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2 chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation induced chromosome damage and cytotoxicity. PMID- 8631316 TI - A novel dystrophin/utrophin-associated protein is an enzymatically inactive member of the phosphoglucomutase superfamily. AB - A 60-kDa protein localised in adherens-type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin [Belkin, A. M. & Burridge, K. (1995) J. Biol. Chem. 270, 6328-6337]. In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins. The PGM-related protein (PGM-RP), which contains 506 amino acids (55.6 kDa), is smaller than PGM1 (566 amino acids, 61 kDa). The active site consensus sequences of prokaryotic and eukaryotic mutases are not conserved in PGM-RP, a finding consistent with the lack of enzymatic activity of PGM-RP in vitro, and the absence of a phosphorylated intermediate in vivo. The organisation of the PGM RP gene is essentially identical to that of PGM1. We propose that the PGM-RP gene, which we have mapped to human chromosome 9qcen-q13, evolved from the PGM1 gene, and encodes a protein with a structural rather than an enzymatic role. PGM RP is expressed predominantly in muscle with the highest levels in smooth muscle. The significance of the interaction between dystrophin/utrophin and an increasing number of cytoplasmic proteins including PGM-RP remains to be explored. PMID- 8631317 TI - Cucumber T-complex protein. Molecular cloning, bacterial expression and characterization within a 22-S cytosolic complex in cotyledons and hypocotyls. AB - T-complex protein (TCP) found in mammalian cells and yeast has been proposed as cytosolic folding machinery. We report here the cloning and initial characterization of a plant TCP cDNA. CSTCP-1 cDNA prepared from mRNA of cotyledons of germinating cucumber seeds encodes a polypeptide composed of 535 amino acid residues. The 59157-Da protein exhibits only 28% identity to both TCP 1p from yeast or and its homolog in Arabidopsis thaliana. Antibodies raised against the bacterially expressed plant protein were used to analyze the intracellular localization of TCP in two different plant tissues: fat-degrading non-dividing cotyledons and meristematic hypocotyls during seed germination. Cell fractionations included differential centrifugation and sedimentation of large complexes at 23000O x g for 4h. The latter fraction was further fractionated by sedimentation velocity centrifugation. This enrichment was required to detect by Western blotting cytosolic 59-kDa species as constituents of 22-S particles. From hypocotyls, a preparation of T-complex was obtained which consisted almost exclusively of proteins in the molecular range of 57-62 kDa. Likewise, the radioactive Cucumis sativus TCP-1 synthesized from CSTCP-1 mRNA in vitro using reticulocyte lysate was shown to migrate as a 61-kDa species. PMID- 8631318 TI - NMR data show that the carcinogen N-2-acetylaminofluorene stabilises an intermediate of -2 frameshift mutagenesis in a region of high mutation frequency. AB - The heteroduplex, D(ACCGGCGCCACA) . d(TGTGG-CCGGT), containing two bulged bases, a cytosine and the guanine G7, either unmodified or modified with the carcinogen N-2-acetylaminofluorene, have been studied by NMR as models of slipped-mutagenic intermediates (SMI). The melting temperature of the modified heteroduplex is strongly increased compared with that of the unmodified heteroduplex. NMR studies have shown that all the bases of the unmodified heteroduplex are stacked within the helix, without any disruption of the sequential connectivities. The two strands are in a B-like conformation. Nevertheless, exchangeable-proton studies have revealed that base pairing is very weak, or even lacking, over two base pairs apart from the bulge. Concerning the modified heteroduplex, no B-like connectivity is observed in the G5-C9 segment. Moreover, the cytosine C8 is rejected outside the helix, whereas the N-2-acetylaminofluorene moiety is inserted within the helix. The G5.C18, C6.G17 and C9.G16 bases are remarkably stable when the temperature is increased, in agreement with the high melting temperature. Some small unassigned peaks reveal the presence of the minor conformation in equilibrium. The strong stabilisation of the N-2 acetylaminofluorene-modified heteroduplex compared with the unmodified duplex is in agreement with the high N-2-acetylaminofluorene-induced mutation frequency compared with the spontaneous frequency and with the hypothesis of mutagenesis occurring during replication. PMID- 8631319 TI - The NeuAc(alpha-2,6)-Gal/GalNAc-binding lectin from elderberry (Sambucus nigra) bark, a type-2 ribosome-inactivating protein with an unusual specificity and structure. AB - The cDNA encoding the NeuAc(alpha-2,6)Gal/GalNAc binding lectin from elderberry (Sambucus nigra) bark (SNAI) was isolated from a cDNA library constructed with mRNA from the bark. Sequence analysis of this lectin cDNA revealed a striking similarity to the previously sequenced type-2 ribosome-inactivating proteins from Ricinus communis and Abrus precatorius. Molecular modelling of SNAI further indicated that its structure closely resembles that of ricin. Since SNAI strongly inhibits cell-free protein synthesis in a rabbit reticulocyte lysate it presumably is a type-2 ribosome-inactivating protein. However, SNAI differs from all previously described type-2 ribosome-inactivating proteins by its specificity towards NeuAc(alpha-2,6)Gal/GalNAc and its unusual molecular structure. PMID- 8631321 TI - Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins. AB - Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed P1.1Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome 11 of Saccharomyces cerevisiae. The human protein migrates on a polyacrylamide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but mRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differentiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength giving rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and p1O5Mcm. PMID- 8631320 TI - Cloning and sequencing of a cDNA encoding a larval-pupal-specific cuticular protein in Tenebrio molitor (Insecta, Coleoptera). Developmental expression and effect of a juvenile hormone analogue. AB - A cDNA clone encoding a larval-pupal cuticular protein, named TMLPCP-22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenebrio molitor. Northern-blot and in situ hybridization analyses showed that the expression of TMLPCP-22 is regulated in a stage-specific and tissue-specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP-22 mRNA was expressed again, confirming its larval-pupal specificity. PMID- 8631322 TI - Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA binding proteins. AB - We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A. This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction. The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity. Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions. Dhel II is able to unwind long stretches of DNA, but with decreased efficiency. Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 10-fold on substrates greater than 200 nucleotides. In particular, the mitochondrial single-stranded DNA binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II. These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II. PMID- 8631323 TI - Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity. AB - Pokeweed antiviral protein (PAP) and the A-chain of ricin (RTA) are two members of a family of ribosome-inactivating proteins (RIPS) that are characterised by their ability to catalytically depurinate eukaryotic ribosomes, a modification that makes the ribosomes incapable of protein synthesis. In contrast to RTA, PAP can also inactivate prokaryotic ribosomes. In order to investigate the reason for this differing ribosome specificity, a series of PAP/RTA hybrid proteins was prepared to test for their ability to depurinate prokaryotic and eukaryotic ribosomes. Information from the X-ray structures of RTA and PAP was used to design gross polypeptide switches and specific peptide insertions. Initial gross polypeptide swaps created hybrids that had altered ribosome inactivation properties. Preliminary results suggest that the carboxy-terminus of the RIPs (PAP 219-262) does not contribute to ribosome recognition, whereas polypeptide swaps in the amino-terminal half of the proteins did affect ribosome inactivation. Structural examination identified three loop regions that were different in both structure and composition within the amino-terminal region. Directed substitution of RTA sequences into PAP at these sites, however, had little effect on the ribosome inactivation characteristics of the mutant PAPs, suggesting that the loops were not crucial for prokaryotic ribosome recognition. On the basis of these results we have identified regions of RIP primary sequence that may be important in ribosome recognition. The implications of this work are discussed. PMID- 8631324 TI - Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,. AB - This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K( 1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = 50.0 +/- 6.2 J . mol(-1) . K(-1). No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II. PMID- 8631326 TI - Isolation from spleen of a 57-kDa protein substrate of the tyrosine kinase Lyn. Identification as a protein related to protein disulfide-isomerase and localisation of the phosphorylation sites. AB - A 57-kDa protein (p57) has been purified to homogeneity from a microsomal fraction of rat spleen. It is specifically and efficiently phosphorylated by the Src-like tyrosine kinase Lyn purified from the same source with a Km of 0.34 microM. The tyrosine kinases c-Fgr, Fyn, C-terminal Src kinase and p72syk, as well as the Ser/Thr-specific cAMP-dependent protein kinase and protein kinases CK1 and CK2 do not phosphorylate p57. C-terminal Src kinase, which acts to down regulate the Src-like protein-tyrosine kinases, almost completely prevents the protein phosphorylation catalysed by Lyn. Protein mass fingerprinting with tryptic fragments identified p57 as a protein related to protein disulfide isomerase which belongs to the superfamily of Cys-Gly-His-Cys-containing sequences. Lyn phosphorylates tyrosine residues Y444, Y453 and Y466 which are located in a highly acidic region of the protein at the C-terminus. Upon phosphorylation, p57 forms a complex with Lyn which can be immunoprecipitated with anti-Lyn IgG. The association which occurs between the phosphorylated substrate and the SH2 domain of the kinase is consistent with the suggested 'processive phosphorylation' model, which implies that a primary phosphorylation site of the substrate binds to the SH2 domain of the enzyme and triggers the phosphorylation at secondary site(s). PMID- 8631325 TI - Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli. AB - UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.-L. & Chang, H.-Y. (1993) FEBS Lett. 329, 153-158] and it is proposed that these liver and muscle forms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised. Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration. PMID- 8631327 TI - Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,. AB - Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed. PMID- 8631328 TI - Activation of human neutrophil procollagenase by stromelysin 2. AB - Neutrophil procollagenase (MMP-8) was efficiently activated by incubation with active stromelysin 2 (MMP-10). A single-step activation mechanism involving the cleavage of the Gly78-Phe79 peptide bond at the end of the propeptide domain was observed. Determination of the collagenolytic activity revealed the generation of active neutrophil collagenase displaying high specific activity. When compared with the specific activity following mercurial activation, which generates active collagenase by autoproteolytic cleavage of either Phe79-Met8O or Met8O-Leu81 peptide bonds [Blaser, J., Knauper, V., Osthues, A., Reinke, H. & Tschesche, H. (1991) Eur J. Biochem. 202, 1223-1230], the specific activity of the stromelysin 2-activated enzyme was considerably higher. Thus, human neutrophil procollagenase was 'superactivated' by stromelysin 2, as was recently shown for the stromelysin 1-activated enzyme [Knauper, V., Wilhelm, S. M., Seperack, P. K., De Clerck, Y. A., Langley, K. E., Osthues, A. & Tschesche, H. 1993 a) Biochem. J. 295, 581 586]. PMID- 8631329 TI - Purification and characterization of an intracellular catalase-peroxidase from Penicillium simplicissimum. AB - The first dimeric catalase-peroxidase of eucaryotic origin, an intracellular hydroperoxidase from Penicillium simplicissimum which exhibited both catalase and peroxidase activities, has been isolated. The enzyme has an apparent molecular mass of about 170 kDa and is composed of two identical subunits. The purified protein has a pH optimum for catalase activity at 6.4 and for peroxidase at 5.4. Both activities are inhibited by cyanide and azide whereas 3-amino-1,2,4-triazole has no effect. 3,3'-Diaminobenzidine, 3,3'-dimethoxybenzidine, guaiacol, 2,6 dimethoxyphenol and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) all serve as substrates. The optical spectrum of the purified enzyme shows a Soret band at 407 nm. Reduction by dithionite results in the disappearance of the Soret band and formation of three absorption maxima at 440, 562 and 595 nm. The prosthetic group was identified as a protoheme IX and EPR spectroscopy revealed the presence of a histidine residue as proximal ligand. In addition to the catalase-peroxidase, an atypical catalase which is active over a broad pH range was also partially purified from P. simplicissimum. This catalase is located in the periplasm and contains a chlorin-type heme as prosthetic group. PMID- 8631330 TI - Isolation and structures of glycoprotein-derived free oligosaccharides from the unfertilized eggs of Scyliorhinus caniculus. Characterization of the sequences galactose(alpha 1-4)galactose(beta 1-3)-N-acetylglucosamine and N acetylneuraminic acid(alpha 2-6)galactose(beta 1-3)-N-acetylglucosamine. AB - As previously reported [Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H. & Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K. & Inoue, Y. (1989) J. Biol. Chem. 264, 18520-185261, the unfertilized eggs of two different species of fresh-water fish, Plecoglossus altivelis and Tribodolon hakonensis, contain relatively large amounts of free sialooligosaccharides. These oligosaccharides were found to derive from glycophosphoproteins, owing to the activity of a peptide - N4-(N-acetyl-beta-D glucosaminyl)asparagine amidase [Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1992) J. Biol. Chem. 267, 24287-24296; Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114]. Here we describe a new type of free oligosaccharides, isolated from unfertilized eggs of Scyliorhinus caniculus. From the structural analysis, based upon 1H-NMR spectroscopy, the following glycan units are proposed.[Formula: see text] PMID- 8631331 TI - Influence of the signal sequence and chaperone SecB on the interaction between precursor protein prePhoE and phospholipids. AB - To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 [corrected] nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers. It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB. Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers. The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB prePhoE complex upon binding of the precursor to the bilayer. The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made. PMID- 8631332 TI - Molecular characterisation of plant endoplasmic reticulum. Identification of protein disulfide-isomerase as the major reticuloplasmin. AB - Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase. Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein. PMID- 8631333 TI - Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system. AB - The effect of different cellular free-energy states on the uptake of methyl alpha D-glucopyranoside, an analogue of glucose, by the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular [ATP]/[ADP] ratio was varied by changing the expression of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show that the initial uptake rate was decreased under conditions of lowered intracellular [ATP]/[ADP] ratios, irrespective of which method was used to change the cellular energy state. When either the expression of the atp operon was changed or 2,4 dinitrophenol was added to wild-type cells, the relationship between initial phosphotransferase uptake rate and the logarithm of the [ATP]/[ADP] ratio was approximately linear. These results suggest that the cellular free-energy state, as reflected in the intracellular [ATPI]/[ADP] ratio, plays an important role in regulating the activity of the phosphotransferase system. PMID- 8631334 TI - Mitochondrial sulfide oxidation in Arenicola marina. Evidence for alternative electron pathways. AB - Sulfide is oxidized in the mitochondria of the lugworm Arenicola marina. Mitochondrial sulfide oxidation is coupled with oxygen consumption and with an equimolar production of thiosulfate [Volkel, S. & Grieshaber, M. K. (1994) Mar. Biol. 118, 137-147]. Mitochondrial respiration in the presence of malate (or succinate) and ADP but without sulfide could be completely inhibited by rotenone, antimycin, cyanide, and sulfide. Only 40% inhibition was achieved by salicylhydroxamic acid. Sulfide oxidation (with sulfide as the only substrate) was fully inhibited by antimycin and by salicylhydroxamic acid but not by rotenone or sulfide. Moreover, sulfide oxidation was 3-4-fold less sensitive to cyanide as compared to normal respiration. The data indicate that sulfide oxidation in A. marina is linked to the respiratory electron transport chain. We suggest that electrons from sulfide enter the respiratory chain via ubiquinone or at the ubiquinol-cytochrome-c oxidoreductase. At sulfide concentrations higher than 10 microM, the cytochrome-c oxidase is blocked and electrons from sulfide are transferred to oxygen via an alternative terminal oxidase. PMID- 8631335 TI - Acetaldehyde mediates the synchronization of sustained glycolytic oscillations in populations of yeast cells. AB - In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long-standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180 degrees out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 microM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 microM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells. PMID- 8631336 TI - Activation of gene transcription by prostacyclin analogues is mediated by the peroxisome-proliferators-activated receptor (PPAR). AB - Xenobiotic amphipathic carboxylates, known collectively as hypolipidemic peroxisome proliferators (e.g., aryloxyalkanoic acids), or native long-chain fatty acids induce liver peroxisome proliferation and other biological activities. This broad spectrum of effects results from modulation of transcription of specific genes mediated by binding of peroxisome-proliferators activated receptors (PPAR) to respective sequence-specific promoter elements (PPRE). The broad specificity and relatively low potency of reported hypolipidemic peroxisome proliferators prompted us to search for specific highly potent peroxisome proliferators. Here we report that stable prostacyclin analogues may act in such a manner. mPPAR alpha-mediated expression of a reporter gene linked to the peroxisomal rat acyl-CoA oxidase promoter was dose-dependently induced by carbaprostacyclin and iloprost. The ED50 for carbaprostacyclin was 25 nM, and carbaprostacyclin was therefore 25-fold and 200-fold more effective than the most potent xenobiotic (5,18,11,14-eicosatetraynoic acid) and native (arachidonic acid) inducers, respectively. Induction was further increased by cotransfecting the cells with mPPAR alpha and an expression vector for retinoic acid-X-receptor. PPAR-mediated activation of gene expression by prostacyclin analogues was specific for PPAR and was not observed using other members of the superfamily. No activation of gene expression was induced by other prostaglandins or leukotrienes at concentrations 100-fold higher than those of the prostacyclin analogues. Induction of gene expression by prostacyclin analogues was inhibited in cells transfected with the long-chain-acyl-CoA synthase, indicating that the acidic form of prostacyclin, rather than the respective CoA derivative or a metabolite derived thereof, serves as the activator of the PPAR/PPRE transduction pathway. Hence, PPAR-mediated modulation of gene transcription by prostacyclins may form the basis for their novel role as regulators of gene expression. Xenobiotic hypolipidemic peroxisome proliferators and native long-chain fatty acids seem to exploit the PPAR/PPRE transduction pathway used by prostacyclin. PMID- 8631337 TI - Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. AB - The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97 amino-acid protein is cysteine-rich and has a typical signal sequence for secretion. Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures. The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus. PMID- 8631338 TI - Comparison of self-sustained sequence-replication reaction systems. AB - The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments. PMID- 8631339 TI - Structure and activity of a chimeric interleukin-8-melanoma-growth-stimulatory activity protein. AB - A 72-amino-acid chimeric protein, Chi1, was constructed from the N-terminal part of interleukin 8, IL-8-(1-53), and the C-terminal part of melanoma growth stimulatory activity, MGSA-(54-72). Chi1 protein showed receptor-binding specificity and biological activity similar, but not identical to IL-8 and decidedly different from MGSA. The structure of Chi1 was determined in solution by two-dimensional NMR and molecular-dynamics calculations. The structure resembled the structures of MGSA and IL-8 closely, containing a triple-stranded beta-sheet in the IL-8 part and an amphipathic alpha-helix in the MGSA part. Chi1 formed dimers at millimolar concentrations via the first strand from the N terminus, analogous to IL-8 and MGSA. In contrast to the latter molecules, however, the alpha-helix of Chi1 did not pack against the beta-sheet part, but was an independent structural element. This structural difference could be explained mainly by the modulation of hydrophobic interactions between the helix and the rest of the protein in Chi1 as compared to IL-8 and MGSA. It is concluded that tight helix packing is not required for receptor binding and biological activity of Chi1. PMID- 8631340 TI - 31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration. AB - 31P-NMR spectra of NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross-polarization, magic angle spinning and high-power proton-decoupling on both lyophilized and hydrated samples. Previous studies on the lyophilized complex of L. casei dihydrofolate reductase with NADPH and methotrexate, measuring the isotropic shifts and principal components of the chemical shift tensors, have shown that the 2' phosphate group of bound NADPH exists as a mixture of the dianionic and monoanionic states [Gerothanassis, I. P, Barrie, P. J., Birdsall, B. & Feeney, J. (1994) Eur J. Biochem. 226, 211-218]. In the present study on hydrated samples, the characterization of the isotropic shift and chemical shift tensors of the 2' phosphate signal indicates that the 2'-phosphate is almost exclusively in the dianionic state. This is in agreement with earlier 31P-NMR studies in solution [Feeney, J., Birdsall, B., Roberts, G. C. K. & Burgen, A. S. V. (1975) Nature 257, 564-566]. In experiments examining progressively hydrated (6%, 12%, 15%, by mass) samples, the observed signals become increasingly narrower probably because the microenvironments of the 31P nuclei become more homogeneous upon sample hydration. Chemical exchange between mobile water molecules and bound protons close to individual sites on NADPH has been indirectly monitored on a hydrated sample (15% water, by mass) using a pulse sequence proposed by Harbison and coworkers [Harbison, G. S., Roberts, J. E., Herzfeld, J. & Griffin, R. G. (1988) J. Am. Chem. Soc. 110, 7221-7223]. In this experiment, the two diphosphate signals are totally suppressed while the 2'-phosphate phosphorus signal remains: this indicates a significant polarization of the 2'-phosphate nuclei from protons in exchange with those of mobile water molecules. PMID- 8631341 TI - Nisin Z, mutant nisin Z and lacticin 481 interactions with anionic lipids correlate with antimicrobial activity. A monolayer study. AB - Monomolecular layers of lipids at the air/water interface have been used as a model membrane to study membrane interactions of the lantibiotic nisin. The natural lantibiotics nisin A and nisin Z proved to have a high affinity for the anionic lipids phosphatidylglycerol and bis(phosphatidyl)glycerol (cardiolipin). The interaction with zwitterionic phopholipids or neutral lipids is very low at surface pressures higher than 32 mN/m. Nisin, nisin mutants and lacticin 481 show a remarkable correlation between anti-microbial activity and anionic lipid interaction. The results indicate that primarily the N-terminal part (residues 1 22) penetrates into the lipid phase. Reduction of the flexibility at positions 20 21 has a negative effect on monolayer interaction and activity. The C-terminal part is probably responsible for ionic interactions of nisin in monomeric or oligomeric form with anionic lipids. In mixtures of anionic and zwitterionic lipids maximal interactions are found at approximately 70 mol/100 mol anionic lipid. Gram-positive bacteria, which form the main target for nisin, are characterized by a high content of anionic lipids in the membrane. Monolayers formed of lipid extracts of bacteria sensitive to nisin were more strongly penetrated than those of bacteria relatively insensitive to nisin. PMID- 8631342 TI - Antigenic regions of human chromogranin A and their topographic relationships with structural/functional domains. AB - Chromogranin A is a protein contained in the secretory granules of many neuroendocrine cells. The linear antigenic sites of human chromogranin A were studied by examining the cross-reaction of polyclonal and monoclonal anti chromogranin A antibodies with native chromogranin A and with synthetic peptides encompassing most of the chromogranin A sequence. Chromogranin A residues 1-20, 47-67, 107-158, 254-297, 331-375, and 395-419 were found to be poorly or not antigenic, while residues 25-46, 163-210, 231-253, 298-314 and 68-106, 222-230, 315-330, 376-394 were found to contain weak and strong antigenic sites, respectively. Residues 68-70 (GAK) and 81-90 (GFEDELSEVL) were strongly recognized by two mouse mAbs (B4E11 and A11, respectively). Since mAb A11 has been previously used for immunohistochemical analysis of chromogranin-A-producing tissues from different species and for in vivo imaging of chromogranin-A-positive endocrine tumors, these results imply that at least part of the 81-90 region is surface-exposed in cryostat tissue sections as well as in vivo. The results may help in selecting new antibodies with improved affinity and immunogenicity for in vivo targeting of chromogranin-A-producing tumors. PMID- 8631343 TI - Microenvironmental changes in platelet membranes induced by the interaction of fibrinogen-derived peptide ligands with platelet integrins. AB - A few studies have confirmed the influence of peptides containing either the RGD or dodecapeptide H-12-V (HHLGGAKQAGDV) sequence on cell membrane structure and function. In order to consider previous findings and to explore microenvironmental changes associated with the interaction of these two fibrinogen-derived peptides with platelet membranes, we employed fluorescence quenching and electron paramagnetic resonance techniques to monitor the possible alterations in platelet membrane dynamics induced by RGDS and H-12-V. The interaction of RGDS with platelet membranes resulted in reduced values of the h+1/ho parameter in both 5-doxylstearic acid and 16-doxylstearic acid spectra indicating a significant rigidification of the membrane lipid bilayer. Otherwise, the fibrinogen-derived peptide that contained the gamma chain C-terminal sequence H-12-V had a fluidizing effect on the platelet membrane lipid bilayer. The labelling of platelet membranes with 1-anilino-8-naphthalenesulphonate (ANS) enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and ANS embedded into the membrane lipid bilayer. As RGDS interacts with platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. The opposite alterations were recorded upon interaction of platelet membranes with H-12-V. Furthermore, a small shift towards longer wavelengths, which accompanies the spectra of ANS in control platelet membranes, vanishes during the interaction with the peptide H-12-V. This observation can be accounted for by a decrease in the polarity of the ANS environment, and may suggest an enhanced contact of the membrane tryptophan with phospholipid fatty acids. Thus, the data indicate that after the action of H-12-V on platelet membrane receptors, the membrane tryptophan residues become exposed to the external environment and the quenchable fraction of membrane tryptophan becomes smaller. The increase (a) in the relative rotational correlation time (tau c) of 4 (ethoxyfluorophosphinyloxy)-2,2,6,6-tetramethylpiperidine- 1-oxyl (ethoxyfluorophosphinyloxy-TEMPO) and (b) in the hw/hs ratio in the spectra of 4 maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (maleimido-TEMPO) indicate that under these conditions there is an effective immobilization of some domains located on the hydrated surface of membrane proteins and mobilization of those domains buried inside the membrane protein molecules. The interaction of RGDS with platelet membrane integrins resulted in contrary effects, as compared to H 12-V. In conclusion, our spectroscopic data indicate that these two fibrinogen derived peptides induce opposite effects in the dynamics of platelet membrane components. PMID- 8631344 TI - Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans. AB - Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material. A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts. PMID- 8631345 TI - The RNA component of mitochondrial ribonuclease P from Aspergillus nidulans. AB - Several RNA molecules that copurified with Aspergillus nidulans mitochondrial ribonuclease (RNase) P were identified [Lee, Y C., Lee, B. J., Hwang, D. S. & Kang, H. S. (1996) Eur J. Biochem. 235, 289-296], and their partial sequences were determined. Using an oligonucleotide probe, we cloned and mapped the gene encoding this putative RNA component of RNase P (RNase P-RNA), situated between URFA3 (unidentified reading frame A3) and cobA (apocytochrome b) genes in the mitochondrial genome of A. nidulans. The gene is extremely (A+T)-rich and contains two regions of sequence similarity conserved among the known mitochondrial RNase P-RNAs and the eubacterial RNase P-RNAs. The determination of 5' and 3' termini by primer extension and sequencing indicated that the length of the RNA transcript is 232 nucleotides. Northern-blot analysis revealed that its only subcellular location was the mitochondria. Two RNase P-RNA fragments of 110 nucleotides and 80 nucleotides, each containing one of the two conserved regions, could be recovered from the nuclease-treated enzyme without significant loss of activity. The sizes of these fragments appeared to be the minimum lengths required for the vitro activity of the enzyme. PMID- 8631346 TI - Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis. AB - The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found. PMID- 8631347 TI - Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor. AB - Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI (1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin. PMID- 8631348 TI - A molecular map of the interactions between titin and myosin-binding protein C. Implications for sarcomeric assembly in familial hypertrophic cardiomyopathy. AB - The thick filaments of vertebrate striated muscles contain with myosin a number of accessory proteins of the intracellular immunoglobulin superfamily, which are localized in a distinct pattern of stripes 43 nm apart. The specific localization of these proteins is believed to be due partly to their interaction with the giant muscle protein titin (also called connectin), which spans the entire sarcomere and may act as a molecular ruler. We have used recombinant fragments of titin covering the thick filament region to investigate their interaction with myosin-binding protein C (MyBP-C) from skeletal and cardiac muscle. The interaction of titin and MYBP-C is directed by a subset of titin immunoglobulin domains that are specific for the C-region of the thick filament, supporting the ruler hypothesis for the myosin-binding proteins. The interaction of recombinant titin with overlapping fragments of human cardiac MyBP-C maps the titin-binding site within the C-terminal region, which is deleted in patients suffering from the chromosome-11-associated form of familial hypertrophic cardiomyopathy. This disorder is therefore likely to be the result of thick-filament misassembly by abolishing the ternary interaction of titin, myosin and MyBP-C. PMID- 8631349 TI - Interaction between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 as revealed by chemical cross-linking. AB - The interaction between photosystem I (PS I) and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was investigated by covalent cross linking in the presence of a hydrophilic cross-linker, N- ethyl-3-(3 diaminopropyl)carbodiimide. Under the experimental conditions employed, five distinct cross-linking products of flavodoxin and PS I subunits are formed. Immunoblot analyses show that these species are the result of cross-linking of flavodoxin to PsaC, PsaD, an unidentified low-molecular-mass PS I polypeptide, and a 15-kDa subunit. The latter has been indirectly identified as the PsaF subunit. Analysis of the interaction of flavodoxin with PS I from a psaE mutant indicates that the PsaE subunit is required for correct complex formation between flavodoxin and PS I, although this subunit is not directly cross-linked to flavodoxin. In addition, the cross-linking products of PsaD with PsaC and PsaL, and PsaE with PsaF, are observed. The covalent complex of flavodoxin and PS I is shown to be fully inhibited with respect to electron transfer to soluble flavodoxin, ferredoxin or ferredoxin:NADP+ oxidoreductase. PMID- 8631350 TI - Expression and characterization of recombinant alpha-galactosidase in baculovirus infected insect cells. AB - A cDNA encoding coffee bean alpha-galactosidase was subcloned into baculovirus expression vectors, pVL-1393 and pAc-GP67B, for intracellular and extracellular expression in Spodoptera frugiperda (Sf9) insect cells, respectively. The expressed protein (recombinant alpha-galactosidase) was immunologically reactive with antisera raised against its native counterpart isolated from coffee beans and was biologically active towards the substrate p-nitrophenyl alpha galactopyranoside. The subcellular distribution of recombinant alpha galactosidase expressed from different vectors was analyzed by Western blotting, immunofluorescent labeling, and electron microscopy. In addition, recombinant alpha-galactosidase was compared to the native enzyme with respect to glycosylation, thermostability, and pH profile. Furthermore, a recombinant alpha galactosidase molecule with a His6 tag at its C-terminus was constructed by an overlap PCR method so that the enzyme expressed in Sf9 cells can be purified by a simple affinity chromatography procedure. PMID- 8631351 TI - Phosphorylation of plant proteins and the identification of protein-tyrosine kinase activity in maize seedlings. AB - Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s). PMID- 8631353 TI - hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system. AB - In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus. PMID- 8631352 TI - Glutathione reductase and glutamate dehydrogenase of Plasmodium falciparum, the causative agent of tropical malaria. AB - The use of glutathione reductase inhibitors in chemotherapy is the raison d'etre for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first beta strand and the diphosphate fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P.falciparum glutathione reductase as a drug target are discussed. PMID- 8631354 TI - NMR structural studies of DNA decamer duplex containing the Dewar photoproduct of thymidylyl(3'-- >5')thymidine. Conformational changes of the oligonucleotide duplex by photoconversion of a (6-4) adduct to its Dewar valence isomer. AB - The single-stranded deoxynucleotide decamer containing a site-specific Dewar valence isomer of the (6-4) adduct of thymidylyl (3'-->5')-thymidine was generated by direct photolysis of d(CGCATTACGC) with UV-B and UV-C irradiation. The conformation of the Dewar-photomodified deoxyoligonucleotide duplex, (C1-G2 C3-A4-T5[DW]T6-A7-C8-G9-C10) . (G11-C12-G13-T14-A15-A16-T17-G18+ ++-C19-G20), has been studied by one- and two-dimensional NMR spectroscopy. While the eight of the ten complementary nucleotides form Watson-Crick-type hydrogen bonding, the 5'-TpT 3' bases of the Dewar lesion show no evidence of complementary hydrogen bonding formation to each other. The Dewar covalent linkage for the adjacent pyrimidine base leads to unusual base stacking, which is different from that of normal B DNA. Unusual NOEs indicate that the formation of a Dewar photoproduct in the B DNA duplex is likely to alter its local and global structures. Also, detailed NMR data show that the base pairing and stacking of the Dewar-photoproduct-containing decamer duplex differ from that of the (6-4)-adduct-containing decamer duplex, suggesting that isomerization of the (6-4) adduct to its Dewar form induces a substantial change in the structure of the oligonucleotide duplex. PMID- 8631355 TI - Asp7O in the peripheral anionic site of human butyrylcholinesterase. AB - The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat was the same for wild-type and D70G mutant, being 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase. PMID- 8631356 TI - Alternative splicing of the pyruvate kinase M gene in a minigene system. AB - The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10. PMID- 8631358 TI - Three-dimensional structure of the lantibiotic nisin in the presence of membrane mimetic micelles of dodecylphosphocholine and of sodium dodecylsulphate. AB - The lantibiotic nisin is a cationic, polycyclic bacteriocin of 34 residues, including several unusual dehydro residues and thioether-bridged lanthionines. The primary target of its antimicrobial action is the cytoplasmic membrane. Therefore the conformation of nisin when bound to membrane-mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was determined with high-resolution NMR spectroscopy. Structures were calculated on the basis of NMR-derived constraints with the distance-geometry program DIANA and were further refined by restrained energy minimization using X-PLOR. The conformation of nisin complexed to both types of micelles is the same, irrespective of the different polar head-groups of the detergents. The structure consists of two structured domains: an N-terminal domain (residues 3-19) containing three lanthionine rings, A, B and C; and a C-terminal domain (residues 22-28) containing two intertwined lanthionine rings numbered D and E. These domains are flanked by regions showing structural variability. Both domains are clearly amphipathic, a property characteristic for membrane-interacting polypeptides. The structures of the ring systems are better defined than those of the linear segments. The four-residue rings B, D and E of nisin all show a beta turn structure, which is closed by the thioether linkage. The backbones of the rings B and D form type 11 beta-turns. Ring E resembles a type I beta-turn. Preceding the intertwined rings D (residues 23-26) and E (25-28) another type-II beta-turn is found formed by the residues 21-24, so that the C-terminal domain consists of three consecutive beta-turns. The structures of nisin in the micellar systems differ significantly from the previously determined (and now partially recalculated) structure in aqueous solution [van de Ven, F. J. M., van den Hooven, H. W., Konings, R. N. H. & Hilbers, C. W. (1991) Eur J. Biochem. 202, 1181-1188] in the first lanthionine ring around dehydroalanine 5. PMID- 8631357 TI - Human biliverdin IXalpha reductase is a zinc-metalloprotein. Characterization of purified and Escherichia coli expressed enzymes. AB - Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity. A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A) rich RNA, and appeared, by Southern blot analysis, to be the product of a single copy gene. Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity. PMID- 8631359 TI - Surface location and orientation of the lantibiotic nisin bound to membrane mimicking micelles of dodecylphosphocholine and of sodium dodecylsulphate. AB - The interaction of nisin, a membrane-interacting cationic polypeptide, with membrane-mimicking micelles of zwitterionic dodecylphosphocholine and of anionic sodium dodecylsulphate was studied. Direct contacts have been established through the observation of NOEs between nisin and micelle protons. Spin-labeled DOXYL stearic acids were incorporated into the two micellar systems. From the paramagnetic broadening effects induced in the 1H-NMR spectrum of nisin it is concluded that the molecule is localized at the surface of the micelles. The interactions of nisin with zwitterionic and with anionic micelles resemble each other as do the nisin conformations [van den Hooven, H. W., Doeland, C. C. M., van de Kamp, M., Konings, R. N. H., Hilbers, C. W. & van de Ven, F. J. M. (1995) Eur J. Biochem. 235, 382-393]. The hydrophobic residues are immersed into the micelles and oriented towards the center, whereas the more polar or charged residues have an outward orientation. The micellar systems are considered to model the first step in the mechanism of antimicrobial action of nisin, this step is the binding of nisin to the cytoplasmic membrane of target bacteria. Detailed information on this initial binding step is obtained. Hydrophobic and electrostatic interactions appear to be involved in the nisin-micelle contacts. It is suggested that subtilin, a lantibiotic structurally related to nisin, has a comparable membrane interaction surface. PMID- 8631360 TI - Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides. AB - The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone. PMID- 8631361 TI - Oxidative inactivation of human 5-lipoxygenase in phosphatidylcholine vesicles. AB - Human 5-lipoxygenase is a non-heme iron protein which possesses 5-oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the substrate for the reaction, on O2 concentration and on exposure to phospholipids and calcium. The apparent first-order rate constant for enzyme inactivation (k(in)) was 0.6 min( 1) during the oxygenation of arachidonic acid in air-saturated buffer containing phosphatidylcholine vesicles and Ca2+. The rate of enzyme inactivation was dependent on the substrate for the reaction and was about threefold slower during the oxygenation of 5,8-icosadienoic acid and 12(S)-hydroxyicosatetraenoic acid compared with arachidonic acid. Lowering the 02 concentration to 60 microM during the oxygenation of arachidonic acid also caused a 2.5-fold decrease in k(in) without affecting the initial rate of the reaction resulting in an increase in both 5-hydroperoxyicosatetraenoic acid (5-HPETE) and leukotriene A4 accumulation. The concentration of 02 for half-maximal activity (initial rate and product accumulation) was approximately 10 microM. In contrast, the activity and the rate of inactivation during the leukotriene A4 synthase reaction with exogenous 5 HPETE (k(in)=2.0 min(-1) were independent of 02 concentration. A rapid inactivation of the enzyme was also observed during aerobic incubation with phosphatidylcholine vesicles and Ca2+ in the absence of substrate, with a sequential loss of the oxygenase (t1/2 = 0.5 min) and pseudoperoxidase (t1/2 = 7 min) activities. Protection against this turnover-independent inactivation was observed in the presence of the selective reversible 5-lipoxygenase inhibitor L 739,010 ([1S, 5R] 3-cyano-1-(3-furyl)-6-(6-[3-(3 alpha-hydroxy-6,8-dioxyabicyclo [3.2.11 octanyl)] pyridin-2-ylmethoxy) naphthalene) and by prior treatment of vesicles with sodium borohydride and, to a lesser extent, by glutathione peroxidase. The results show that the inactivation of 5-lipoxygenase in phospholipid vesicles is dependent on the structure of the unsaturated fatty acid substrate for the reaction, on the concentration of oxygen and on a turnover independent oxidation at the active-site leading to the sequential loss of the oxygenase and pseudoperoxidase activities of the enzyme. PMID- 8631363 TI - O-glycosylated species of natural human tumor-necrosis factor-alpha. AB - Tumor-necrosis factor-alpha, produced by human B-cell lymphoblastoid cell line BALL-1, was expressed as four protein bands on SDS/PAGE analysis. It may have been glycosylated, based on the fact that the heavier two of the four bands disappeared after neuraminidase treatment. Sugar composition analyses revealed that the tumor necrosis factor-alpha contained galactose, N-acetylgalactosamine and N-acetylneuraminic acid as sugar components. To prepare sugar chains, tumor necrosis factor-alpha was treated with alkaline borodeuteride and the oligosaccharide-alditols liberated were fractionated by gel-filtration chromatography on a Bio-Gel P-4 column, followed by normal-phase HPLC. Three oligosaccharide-alditols were obtained, and the structures of two of them were identified by methylation analysis and exoglycosidase digestion. The structures of these oligosaccharide-alditols were Gal beta 1-3(NeuAc alpha 2-6)GalNAcol and Gal beta 1-3GalNAcol (GalNAcol, N-acetylgalactosaminitol). The structure of the remaining oligosaccharide-alditol was determined to be NeuAc alpha 2-3Gal1 3GalNAcol by composition and methylation analyses. About 20% of tumor necrosis factor-alpha was found to be 0-glycosylated, based on the results of the sugar composition and structure analyses. An amino acid sequence analysis of the glycosylated peptides was performed after Staphylococcus aureus V8 protease digestion of tumor necrosis factor-alpha had been completed, and it was proved that the 0-glycosylation site of tumor necrosis factor-alpha was Ser 4. PMID- 8631362 TI - The cloning of a rapidly evolving seminal-vesicle-transcribed gene encoding the major clot-forming protein of mouse semen. AB - Approximately 30 kb of the mouse genome, containing the gene for a major seminal vesicle transcript, has been cloned. The gene was identified by the similarity to members of a family with rapidly evolving genes that includes the gene encoding the major clot protein in rat semen, SVS II, and the human semenogelin genes. The nucleotide sequence of 16.9 kb was determined; this sequence encompasses the gene of 2215 bp plus 9-kb and 5.6-kb regions flanking the 5' and 3' ends of the gene. The transcription unit is divided into three exons, of which the first encodes the signal peptide, the second the secreted protein, while the third exon contains 3'-nontranslated nucleotides only. The transcript encodes a protein of 375 amino acid residues, including a signal peptide of 22 residues. The secreted polypeptide is a protein of Mr 38442 and is similar in sequence but smaller than the major clot-forming protein of rat semen, SVS II. It is highly charged at pH 7 and it has an isoelectric point of 10.68. The central part of the protein consists of tandem repeats that might serve as a substrate for transglutaminase. PMID- 8631364 TI - Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides. AB - The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion. PMID- 8631365 TI - Hormonal regulation of aldose reductase in rat ovary during the estrous cycle. AB - The physiological roles of aldose reductase [alditol:NAD(P)+1-oxidoreductase] have not been fully elucidated yet, although it has been implicated in the pathogenesis of diabetic complications. In the rat ovary we found remarkable changes in the enzyme level during the 4-day estrous cycle. After diestrus, the activity and protein content of aldose reductase increased to the maximum level on proestrous morning and rapidly fell off to the lowest level on the early morning of estrus. At this time its mRNA level in the ovary was significantly decreased compared with that on the morning of proestrus. Immunohistochemical staining of the diestrous ovary demonstrated localization of the enzyme protein in the granulosa cells and in the oocytes. At the end of proestrus when its level was low, immunoreactive aldose reductase in the granulosa cells was localized preferentially to the antrum side, with lesser staining in the cells lining the follicles. Administration of chlorpromazine to the rats on proestrus significantly restored the enzyme level on the following morning of the expected estrus. This effect of chlorpromazine was abolished when human chorionic gonadotropin was administered to the chlorpromazine-treated rats. When chlorpromazine was administered to the rats treated with bromocriptine, an inhibitor of pituitary prolactin secretion, aldose reductase activity in the ovary was significantly elevated compared with that in the rats treated with chlorpromazine alone. These findings suggest that in the rat ovary it is under hormonal regulation during the estrous cycle. The enzyme may possess a new functional role in the reproductive system of the female rat, which can be disordered under diabetic conditions. PMID- 8631366 TI - Kinetic evidence for the existence of a rate-limiting step in the reaction of ferric hemoproteins with anionic ligands. AB - The kinetics of azide and fluroide binding to various monomeric and tetrameric ferric hemoproteins (sperm whale Mb, isolated alpha and beta chains of human Hb reacted with p-chloromercuribenzoate, dromeday, ox and human Hb) has been investigated (at pH 6.5 and 20 degrees C over a large range (20 microM to 2 M) of ligand concentration. It has been observed that the pseuo-first-order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptomatic concentration-independent value typical for each hemoprotein. This behavior, which has been detected only by an investigation covering an unusually large range of ligand concentrations appears to be independent of the ionic strength, and it underlies the existence of a rate-limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo- first-order rate constant becomes faster than a given value characteristic of the specific hemoprotein. Such a behavior is not observed in the case of fluroide binding probably because the pesudo- first-order rate constant for this ligand is much slower and never attains a value faster than that of the rate-limiting step. In general terms, this feature should involve a conformational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into the heme pocket and its reaction with the ferric iron. PMID- 8631367 TI - The molecular basis for the natural resistance of the cytochrome bc1 complex from strobilurin-producing basidiomycetes to center Qp inhibitors. AB - Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)-beta methoxyacrylate inhibitors of the ubiquinol oxidation center(center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers. To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)-beta-methoxyacrylates. The latter exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E) beta-methoxyacrylates. PMID- 8631368 TI - Effects of pancreatic spasmolytic Polypeptide (PSP) on epithelial cell function. AB - Trefoil peptides are expressed near endodermal ulcerations and may modulate epithelial repair. The trefoil pancreatic spasmolytic polypeptide (PSP) was tested for growth activity in vitro on epithelial cells and in vivo following intragastric or intravenous infusion in parenterally fed intact rats. Ion transport was assessed as changes in short-circuit current in rat intestine and adenocarcinoma cells in Ussing chambers. PSP stimulated growth of MCF-7 and Colo 357 cells, but only in the presence of extracellular glutathione (GSH). The effect was attenuated by GSH depletion with buthionine sulphoximine, even in GSH containing media. When GSH-reduced PSP was carboxymethylated with iodoacetic acid, it still depended on extracellular GSH for its growth effect. Intestinal epithelial proliferation in rats was not affected by either intravenous or intraluminal infusion. PSP had no effect on basal or stimulated ion flux in rat jejunum or epithelial monolayers. The peptide did not compete with 125I-labeled epidermal growth factor for its receptor. [14C]Iodoacetamide treatment of PSP, followed by prolonged tryptic digestion yielded predominantly a 14C-labeled tetrapeptide fragment containing Cys1O4, with a lesser quantity of a 14C-labeled 15-amino-acid peptide containing Cys95 (molar ratio 15:1). GSH may predominantly reduce the Cys6-Cys1O4 terminal disulphide bond in PSP. We conclude that some epithelia may exhibit a growth response to PSP if extracellular GSH is present. Reduction of PSP by GSH is not necessary for this response, suggesting that the trefoil receptor or its signal transduction is GSH sensitive. PSP could assist wound healing by interactions with epithelial cells exposed concurrently to a local high GSH concentration. PMID- 8631369 TI - Diradylglycerol formation in cholecystokinin-stimulated rabbit pancreatic acini. Assessment of precursor phospholipids by means of molecular species analysis. AB - The aim of the present study was to assess the origin of the 1,2-diradylglycerols produced during prolonged hormonal stimulation of rabbit pancreatic acini by comparison of their relative molecular species composition with that of the major acinar phospholipids. Both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) consisted of 1,2-diacyl as well as 1-alk-1-2 acyl species. In contrast, phosphatidylinositol (PtdIns), phosphatidylserine and phosphatidic acid existed only in the 1,2-diacyl form. Acinar cells did not contain detectable amounts of 1-alkyl-2-acyl phospholipids. Similarly, the acinar 1,2-diradylglycerol fraction consisted of 1,2-diacylglycerols and 1-alk-1-enyl-2 acylglycerols. Mass 1,2-diradylglycerol measurements revealed that prolonged stimulation with cholecystokinin resulted in a marked and sustained increase in acinar 1,2-diradylglycerol content. Based on the relative amounts of the 1,2 diacyl species present in both the 1,2-diradylglycerol fraction and the individual phospholipids, it is calculated that under control conditions 60% of the 1,2-diacylglycerols originate from PtdCho and 40% from PtdIns, whereas under stimulatory conditions 53% is calculated to be derived from PtdCho, 46% from PtdIns and 1% from PtdEtn. Likewise, it is calculated that in control as well as stimulated acini 100% of the 1-alk-l-enyl-2-acylglycerols originate from plasmenylcholine. Further evidence in favour of the idea that at least a considerable part of the 1,2-diacylglycerols produced during prolonged hormonal stimulation originate from inositolphospholipids is provided by the observation that labeling of phosphatidylinositol 4,5-bisphosphate with inorganic phosphate reached isotopic equilibrium markedly faster under stimulatory conditions as compared to the control situation, which is in agreement with an elevated turnover rate. The data presented support the idea that PtdCho and inositolphospholipids are the major precursors in basal and stimulated 1,2 diradylglycerol production in rabbit pancreatic acini. PMID- 8631370 TI - Site-directed mutagenesis of conserved charged residues in the helical region of the human C5a receptor. Arg2O6 determines high-affinity binding sites of C5a receptor. AB - The human C5a receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg2O6, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282-->Ala) or partial loss of ligand binding capacity (Arg175-->Ala, Arg2O6-->Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln2O6]C5aR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (<10%) of high-affinity sites (Kd approximately 2 nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of G alpha-16. The number of high-affinity sites of wild-type and [Gln2O6]C5aR in 293 cells could be up-regulated by coexpression of Gi alpha-2 and down-regulated by GTP[gamma S]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg2O6 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human C5aR by affecting the intracellular G-protein coupling. PMID- 8631371 TI - Functional analysis of human/chicken transferrin receptor chimeras indicates that the carboxy-terminal region is important for ligand binding. AB - Chimeric human/chicken transferrin receptors have been constructed using the polymerase chain reaction. Different regions of the 671-residue external domain of the human transferrin receptor were replaced by the corresponding sequences from the chicken transferrin receptor. As chicken transferrin receptors do not bind human transferrin, functional analysis of such chimeric receptors provides an approach to define the ligand-binding site of the human transferrin receptor. Four of 16 chimeric human/chicken transferrin receptors expressed in chick embryo fibroblasts were efficiently transported to the plasma membrane and displayed on the cell surface. Studies of the four chimeric receptors indicated that binding of human transferrin was abolished if the carboxy terminal 192 amino acids of the human transferrin receptor (residues 569-760) were replaced with the corresponding region from the chicken transferrin receptor. Further, a chimeric receptor in which the carboxy-terminal 72 residues were derived from the chicken transferrin receptor exhibited a 16-fold decrease in binding affinity for human transferrin. In contrast, analysis of the other two chimeric receptors showed that 340 amino acids of the human transferrin receptor external domain more proximal to the transmembrane region (residues 151-490) could be replaced with the corresponding region from the chicken transferrin receptor without loss of high-affinity ligand binding. In contrast, two mAbs against the human transferrin receptor external domain, B3/25 and D65.3, that do not compete with transferrin binding, do not bind the chimeric transferrin receptors in which the membrane proximal part is replaced by chicken sequences, while they do bind the two other chimeric transferrin receptors with high affinity. These data indicate that sequence differences in the carboxy-terminal region of human and chicken transferrin receptor external domains are important for the species specificity of transferrin binding and imply that this portion of the human transferrin receptor is critical for ligand binding. PMID- 8631372 TI - Involvement of an arachidonic-acid-dependent pathway in the interferon-beta mediated expression of C202 gene in Ehrlich-ascites-tumor cells. AB - We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon beta (IFN-beta) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN-beta transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p-bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2, or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN-beta-inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase-catalysed pathways may be a mechanism by which second messengers for IFN-beta-mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN-beta-based activation of C202 gene expression. PMID- 8631373 TI - Regulation of p42 mitogen-activated-protein kinase activity by protein phosphatase 2A under conditions of growth inhibition by epidermal growth factor in A431 cells. AB - Epidermal growth factor (EGF), which plays an important role in the growth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. In a previous study aimed at dissecting the biochemical events leading to this dual action in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen-activated protein (MAP) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Conversely, an early peak of MAP kinase activation, that rapidly falls below the basal level, is observed in the presence of a growth-inhibitory concentration of EGF. To assess the mechanism of the p42 MAP kinase inactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A in this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the phosphatase 2A activity was completely reversed concurrently with MAP kinase inactivation, after 40 min of treatment with 10 nM EGF. Conversely, in cells treated with 1 pM EGF, phosphatase 2A activity remained below the control level during all the time of the treatment, in association with a sustained MAP kinase activation. These results suggest that MAP kinase inactivation is closely related to phosphatase 2A activation. We then investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long-term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affect the EGF-induced MAP kinase regulation, indicating that mitogen-induced protein phosphatases are not, or are only slightly, required in this regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with differential mechanisms of p42 MAP kinase regulation catalysed by the serine/threonine phosphatase 2A. PMID- 8631375 TI - Detection and identification of human pathogenic Leishmania and Trypanosoma species by hybridization of PCR-amplified mini-exon repeats. AB - A single pair of PCR primers within a conserved region of the mini-exon repeat was used to amplify the repeats from 10 species of pathogenic Leishmania belonging to four major clinical groups and also from three species of Trypanosoma. Oligonucleotide hybridization probes for the detection and identification of the PCR-amplified repeats were constructed from alignments of mini-exon intron and intergenic sequences. The probes generated from mini-exon intergenic regions of the L. (V.) braziliensis, L. (L.) donovani, and L. (L.) mexicana species hybridized specifically to their cognate groups without discriminating between the species within the groups. The probes for L. (L.) major and L. (L.) aethiopica were species-specific, while the L. (L.) tropica probe also hybridized with the L. (L.) aethiopica mini-exon repeat. The mini-exon intron-derived probes for T. cruzi, T. rangeli, and T. brucei were species specific. This method involving the detection of specific PCR-amplified products produced using a single primer set represents a novel sensitive and specific assay for multiple trypanosomatid species and groups. PMID- 8631376 TI - Sodium beta-artelinate--a new potential gametocytocide. AB - The water-soluble artemisinin analogue sodium beta-artelinate, a fast-acting blood schizontocide, was evaluated for gametocytocidal action against simian malaria Plasmodium cynomolgi B, and a single dose of the compound has been found to be an effective gametocytocide by both oral and intravenous routes. The compound was able to sterilize the circulating gametocytes in rhesus monkey, resulting in loss of mosquito infectivity and oocyst development in the Anopheles stephensi. However, no sporontocidal action has been observed with this compound. PMID- 8631374 TI - Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva. AB - The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the possible roles for these molecules in host cell invasion is discussed. PMID- 8631377 TI - Cellular immune responses of jirds to extracts of life cycle stages and adult excretory secretory products during the early development of Brugia pahangi. AB - The Brugia-jird model of lymphatic filariasis was used to examine the induction of cellular immune responses during the early premicrofilaremic phases of the infection. The intensity of the pulmonary granulomatous inflammatory response (PGRN) was determined by measuring granuloma areas around Sepharose beads coated with parasite extracts which were embolized in the lungs of jirds prior to necropsy. Necropsies were performed at 7, 14, 28, 56, and 150 days postinfection (DPI). These time points correspond to specific developmental changes in the life cycle. Lymphocyte blastogenesis assays were performed using cells from draining renal lymph nodes and splenocytes at 14 and 150 DPI. Soluble extracts of third stage larvae (L3), fourth stage larvae (L4), adult females, adult males, microfilariae (MF), and excretory secretory products (ES) of males and females were used in both measurements of cellular responsiveness. A marked granulomatous response to parasite extracts peaked at 7 DPI or 14 DPI followed by a gradual decrease to a hyporesponsive state at 120 DPI. The response of renal lymph node cells also was significantly elevated at 14 DPI and significantly decreased at > 150 DPI. The splenocyte responses were erratic and did not follow this pattern. Significant differences in PGRN responses to somatic extract preparations were not seen during the early stages of the infection (7, 14, 28 DPI), but those to MF and L3 were significantly less at 56 and 120 DPI. Although PGRN responses to ES followed a similar pattern, these were less than those to the somatic extract. The data indicated that a rapid, intense cell-mediated inflammatory response is induced early during a primary infection and that this response is rapidly downregulated. This downregulation begins prior to the maturation of adult parasites and microfilarial production. The early phase of the cellular response appears to be compartmentalized in that this response was consistently observed in the renal lymph nodes but not in the spleen. Soluble protein components of the parasites responsible for these responses are likely multiple and shared by all life cycle stages. PMID- 8631378 TI - Strongyloides stercoralis: eosinophil-dependent immune-mediated killing of third stage larvae in BALB/cByJ mice. AB - Challenge worm survival was significantly reduced when BALB/cByJ mice were vaccinated against Strongyloides stercoralis infective third stage larvae (L3) regardless of whether the challenge infections consisted of systemically migrating L3 or L3 implanted in diffusion chambers. The only cell type that increased in number in diffusion chambers in immunized mice, 1 week after booster immunizations, was the eosinophil, and maximal levels of eosinophils were coincident with parasite killing. Mice were treated with mAb to eliminate IL-5 or granulocytes to assess the role that eosinophils play in larval killing. Treated animals showed no decrease in immunity when challenge infections consisted of systemically migrating L3 administered 3 weeks after booster immunizations. Eosinophil numbers in immunized mice decreased to control levels when measured 3 weeks post-booster immunization, both in diffusion chambers and in the peripheral blood, whereas they were elevated at 1 week after booster immunizations. Direct contact between host cells and L3 was, however, still required for larval killing in immunized hosts 3 weeks after booster immunizations. Elimination of eosinophils by treatment with mAb to IL-5 or granulocytes significantly reduced protective immunity, when L3 were implanted in diffusion chambers at 1 and 3 weeks post-booster. However, as systemically migrating L3 were still killed in immunized, eosinophil-depleted animals, other cell types may play a role in larval destruction. Two human eosinophil granule products were found to be toxic for host-adapted L3+, but had no effect on infective L3, indicating that host adapted larvae are possible targets for eosinophil-mediated destruction of third stage larvae. These findings suggest that inactivation of eosinophils by mAb treatment abolishes protective immunity to L3 contained within diffusion chambers and that small numbers of eosinophils are sufficient for immune-mediated killing of S. stercoralis L3. PMID- 8631379 TI - Strongyloides stercoralis: role of antibody and complement in immunity to the third stage of larvae in BALB/cByJ mice. AB - Mice immunized against Strongyloides stercoralis L3 were shown to kill greater than 90% of challenge larvae contained within diffusion chambers. The objective of the present study was to identify the host components responsible for immunity. Serum from unprotected, control mice and protected, immune mice in doses of 25-500 microliters was transferred into naive mice at the same time and location as larval challenge. Transfer of as little as 50 microliters of immune serum was able to confer protective immunity. The serum-transferred immunity was ablated by excluding cells from the larval microenvironment or by depleting granulocytes through monoclonal antibody treatment in the recipient mice. Specific antibody isotypes were isolated using protein G and isotype-specific affinity columns. The resulting transfer experiments identified IgM as the isotype responsible for protective immunity to S. stercoralis L3. Antibody binding studies in vivo were performed and only IgM bound to the surface of infective L3 and host-derived L3 (L3+) in immune animals. Elevated levels of C3 were also found bound to the surface of L3/L3+ in immune mice. Cobra venom factor treatment of immunized mice to deplete complement completely eliminated C3 binding to the surface of L3/L3+ and ablated immunity. Therefore, IgM, complement, and granulocytes are necessary for immune elimination of S. stercoralis L3/L3+. Identification of antigens recognized by IgM may help select possible vaccine candidates. PMID- 8631380 TI - Trypanosoma cruzi: the Tc-85 surface glycoprotein shed by trypomastigotes bears a modified glycosylphosphatidylinositol anchor. AB - Tc-85, an 85-kDa surface glycoprotein specific for the trypomastigote stage of Trypanosoma cruzi, has been implicated in the invasion of host cells by the parasite. Radioactive palmitic acid was incorporated into Tc-85 immunoprecipitated from the culture medium with the H1A10 monoclonal antibody, suggesting that shedding occurs with Tc-85 bearing its GPI anchor. In contrast to the glycoprotein remaining in the parasites, the glycosylphosphatidylinositol moiety in shed Tc-85 is resistant to phosphatidylinositol phospholipase C and becomes susceptible to the enzyme following alkali treatment. An alkylglycerol was identified by thin layer chromatography of an ether extract after the enzymatic reaction. Resistance to cleavage by phospholipase C is due to fatty acid esterification of the inositol residue in shed Tc-85. This is the first example of inositol modification in anchors from a glycoprotein of Trypanosoma cruzi. PMID- 8631381 TI - Why is man an unsuitable reservoir for the transmission of Leishmania major? AB - Leishmania major strains cause human cutaneous leishmaniasis in various arid regions in the Old World. Nevertheless, there is apparently no anthroponotic transmission even when the incidence of cases is very high and the involvement of reservoir animals is obligatory. To investigate this phenomenon we compared the development of L. major in Phlebotomus papatasi artificially infected with parasites in human, rabbit, or sand rat blood. The parasites, sandflies, and reservoir rodents came originally from the Jordan Valley. Following meals of promastigotes in human blood, parasites were retained in 48.0% and heavy infections developed in 6.6% of the sandflies. Such meals with sand rat blood resulted in 76.9% infected, including 58.5% heavily infected flies, whereas rabbit blood produced intermediate results. Similar results were obtained when infections were initiated with amastigotes and the infection rates were significantly different. Only flies with heavy infections are considered as potential transmitters of leishmaniasis. The adverse effect of human blood was attributed to the erythrocytes after similar experiments in which sandflies ingested promastigotes either with human erythrocytes and rabbit plasma or rabbit erythrocytes and human plasma. Amastigotes of an Israeli strain of L. major died in medium containing 50% human blood. Also, addition of 20% human blood to growth media of parasites from Israel, Kenya, or Turkemenistan caused mortality of 70 to 80% of the initial inoculum in 24 hr. At that time there was a fivefold increase in the number of Israeli parasites cultured with sand rat blood. These results imply that the vector potential and the chances of transmission are drastically decreased when man is the source of L. major parasites. PMID- 8631382 TI - Some observations on the biting behavior of Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus and their implications for malaria control. AB - Studies were carried out in three villages in western Kenya on the biting behavior of Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus. Blood feeding behavior and departure from houses were studied under the impact of permethrin-impregnated eaves-sisal curtains. Only 2-13% of the female vector population was collected biting before 2200 hr. Over 90% of the villagers went to bed by 2100 hr. An. funestus was 6.6-8.2 times more likely to bite people indoors than outdoors, while An. gambiae s.l. females were only 2 times as likely. Under the influence of permethrin-impregnated sisal curtains placed under the eaves of village houses, there was a marked egress of blood-fed An. funestus and An. gambiae s.s. Permethrin seems to have induced exophily of half-gravid female An. gambiae s.s. While An. gambiae s.s. remained highly anthropophagic under the impact of permethrin, An. funestus shifted to feeding more on cattle. An arabiensis were largely zoophilic. Our results underline the difficulties of controlling An. gambiae s.s., the principal African malaria vector. New strategies must be found to control this vector. PMID- 8631383 TI - Amblyomma variegatum (Acari: Ixodidae): mechanism and control of arbovirus secretion in tick saliva. AB - Saliva is considered to be the conduit by which pathogens are transmitted from blood-sucking arthropod vectors to their vertebrate hosts, but supporting evidence for this is fragmentary. To determine if Thogoto (THO) virus, a tick borne member of the influenza virus family, is transmitted via tick saliva, and whether virus replication is a prerequisite for such transmission, two experimental conditions were compared: (1) "biological transmission" and (2) "mechanical transmission." In (1), THO virus was allowed to infect and replicate in a natural vector, Amblyomma variegatum: virus was detected in saliva collected from 3/22 (14%) ticks. In (2), virus was inoculated directly into the hemocoel with the drug used to induce salivation and saliva was collected immediately to preclude the possibility of virus replication: virus was detected in saliva collected from 31/170 (18%) ticks. The results demonstrate that THO virus is secreted in tick saliva and that virus can pass from the hemolymph to the salivary glands independently of viral replication within the tick. The comparatively low numbers of ticks that yielded virus-positive saliva samples together with the results from assays of serial saliva samples suggested that virus secretion may not be a continuous process during salivation. Ticks in which THO virus had established an infection showed an impaired secretory response compared with uninfected ticks and ticks used for mechanical transmission. PMID- 8631384 TI - Echinococcus multilocularis: microsatellite polymorphism in U1 snRNA genes. PMID- 8631385 TI - Brugia pahangi: the cuticular glutathione peroxidase (gp29) protects heterologous membranes from lipid peroxidation. PMID- 8631387 TI - Arresting the mitotic oscillator and the control of cell proliferation: insights from a cascade model for cdc2 kinase activation. AB - We consider a minimal cascade model previously proposed for the mitotic oscillator driving the embryonic cell division cycle. The model is based on a bicyclic phosphorylation-dephosphorylation cascade involving cyclin and cdc2 kinase. By constructing stability diagrams showing domains of periodic behavior as a function of the maximum rates of the kinases and phosphatases involved in the two cycles of the cascade, we investigate the role of these converter enzymes in the oscillatory mechanism. Oscillations occur when the balance of kinase and phosphatase rates in each cycle is in a range bounded by two critical values. The results suggest ways to arrest the mitotic oscillator by altering the maximum rates of the converter enzymes. These results bear on the control of cell proliferation. PMID- 8631388 TI - 17beta-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes. AB - Effects of 17beta-estradiol (E2) in vitro on Na-dependent Ca2+ efflux from, and depolarization-induced Ca2+ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E2 (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca2+ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca2+ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca2+ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E2, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca2+ transport mechanisms in nerve endings. PMID- 8631389 TI - Effect of progesterone and estradiol benzoate on superoxide dismutase activity in the brain of male rats. AB - The activities of mitochondrial, manganese-containing superoxide dismutase (MnSOD) and cytoplasmic, copper-zinc-containing superoxide dismutase (CuZnSOD) were measured in subcellular fractions of whole brain homogenates prepared from intact and gonadectomized (GDX) male rats, untreated or treated subcutaneously (sc) with a single dose of 2 mg progesterone (P) and/or 5 micrograms estradiol benzoate (EB). Neither MnSOD nor CuZnSOD was affected by the removal of the testes. Similarly, CuZnSOD activity was steady following systemic administration of P and/or EB to intact and GDX animals 2 h or 24 h prior to sacrifice. On the other hand, both P and EB suppressed MnSOD in the brain of either intact or GDX rats. These results suggest involvement of P and EB in the control of MnSOD activity in the brain of male rats. PMID- 8631386 TI - On the physiology of metazoa. AB - The problem on integration and control of the various processes of the metazoan organism is a major challenge to the physiologist. The traditional research strategy in dealing with the problem is neuron-oriented and its roots extend back into the last century when knowledge of hormones was lacking. In the present article, the traditional strategy is analyzed in the light of available data and its logical basis is questioned. Different levels of communication are supposed to occur in the animal or human body. Circulating hormones are responsible for the highest level of communication that occurs between organs or tissues. The central concept in the article is that regulation of circulating hormones constitutes a higher level of control relative to regulation of intercellular hormones. This is regardless of whether the latter occurs in the nervous system or elsewhere. The approach is utilized in defining the mechanism that integrates and controls the part processes of the body. The mechanism is defined as endothelial; the vascular endothelial system is the controlling part and the nervous system is one of the subordinate parts. Thanks to the new approach, meaningful biological explanations of major psychiatric disorders are now possible. PMID- 8631391 TI - Marked depression of radiation-induced emesis by olfactory bulbectomy or pre exposure using low doses. AB - Acute emetic response to relatively low-doses of X-irradiation on suncus (Suncus murinus) was examined. The behaviors recorded for each subject, using a video cassette recorder system, were (1) the number of emesis, during exposure to a dose of 3.0 Gy; (2) emesis threshold. Results showed that the emetic threshold was observed at 0.85 Gy, and the number of radioemesis during exposure was 29. However, the observed threshold dose became 2.22 Gy following olfactory bulbectomy. The emetic number decreased significantly (p <0.01), and reached a value one-fourth of the sham-control. The bulbectomized suncus showed a resistance to X-irradiation. Furthermore, I examined whether the animals could also acquire radio-resistance when they were subjected to a brief of dose X-rays (0.3 Gy) prior to a exposure to 3.0 Gy. Results showed that brief pre-exposure increased the observed threshold, a pattern that was exactly the same as shown in the bulbectomized animals. Increasing the pre-exposure dose further to 0.45-0.60 Gy, however, resulted in the complete disappearance of the effect. These results suggest that only the mice pre-irradiated with 0.30 Gy acquired resistance to radiation-induced emesis. PMID- 8631390 TI - Renal vascular reactivity to ATP in hyper- and hypothyroid rats. AB - The effects of adenosine triphosphate (ATP) on the renal vasculature of isolated kidneys from control, hyper- and hypothyroid rats were characterized. ATP responsiveness was evaluated in basal tone and in raised tone (phenylephrine 10( 6) M) preparations. These responses were compared with those obtained with barium chloride or sodium nitroprusside (SNP), used respectively as nonreceptor agonist for vasoconstriction or vasodilation. In preparations at basal tone, ATP produced dose-related vasoconstriction, which was increased in hyperthyroid kidneys, and was severely attenuated in kidneys from hypothyroid rats. In raised tone preparations from control rats ATP produced a dual response: vasoconstriction at low doses, which declined with increasing doses to give way to vasodilator responses; biphasic responses were found in some kidneys. Hyperthyroid kidneys showed increased pressor responses and a vasodilator response similar to those seen in kidneys from control rats. However, in hypothyroid kidneys the vasodilator response was abolished. The responses to barium chloride and to SNP were significantly increased and decreased in hyper- and hypothyroid kidneys, respectively; vasoconstrictor responses to SNP were also found in hypothyroid kidneys. Hence the abnormal responses to ATP observed in both thyroid dysfunctions may be partially explained by unspecific alterations in the contractile machinery of the renal vasculature in these kidneys. However, ATP responsiveness (vasoconstriction at low tone and vasodilation at raised tone) was more severly affected in hypothyroid kidneys, suggesting that purinergic (P2X and P2Y) receptor activity may be decreased in these organs. PMID- 8631392 TI - Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926. AB - Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64 +/- 0.18 (m +/- SE, n = 11) and 3.12 +/- 0.24 (n = 11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14 +/- 3% (n = 34) and 22 +/- 13% (n =10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12 +/- 0.04 (n = 11) and 0.07 +/- 0.01 nmol/min/mg cell protein (n = 11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC. PMID- 8631393 TI - L-arginine and pancreatic islet blood flow in anesthetized rats. AB - The aim of the present study was to see if L-arginine, which induces insulin release and is a precursor of the endothelial-derived relaxing factor nitric oxide, affects whole pancreatic and/or islet blood flow. For this purpose, anesthetized male Sprague-Dawley rats were injected intravenously with either saline or L-arginine (25, 100 or 250 mg/kg body weight). All doses of arginine caused a slight increase in blood glucose concentration, while the highest dose (250 mg/kg body weight) also increased insulin concentration. However, no changes in either mean arterial blood pressure, whole pancreatic or islet blood flow could be discerned with any of the doses of arginine used. It is concluded that insulin release is not necessarily associated with an increased islet blood perfusion. PMID- 8631394 TI - Chronic sensory denervation reduces thrombin-stimulated endothelin release from aortic endothelial cells. AB - The long-term (trophic) influence of perivascular nerves on the endothelium was investigated by measuring changes in thrombin-stimulated release of the potent vasoconstrictor, endothelin, after selective chronic denervation. Rat pups were treated with either guanethidine or capsaicin to destroy sympathetic or sensory nerves, respectively. The abdominal aortas from the rats at three months of age (5 pooled per experiment) were incubated with 4U thrombin/ml in medium for 24 h at 37 degrees C, and the amount of endothelin released from the preparation determined by immunoassay. After neonatal sensory denervation there was a significant reduction in the thrombin-stimulated release of endothelin compared to the controls (0.012 +/ -0.012 (4) compared to 0.063 +/- 0.012 (6), pmol/cm2/24 h, p < 0.02). There was no change in endothelin release after sympathetic denervation. In summary, sensory nerves play a trophic role in the expression of endothelin in endothelial cells of the intima. PMID- 8631395 TI - Cl/HCO(-3) exchanger is operative in isolated enterocytes from rat jejunum. AB - Enterocytes isolated from rat jejunum were tested for the existence of a Cl-/HCO( 3) exchange, previously evidenced in basolateral membrane vesicles but not in brush border. Cells were found to retain functional integrity and transport capabilities long enough to allow Cl- fluxes to be measured. Both efflux and uptake experiments indicate that a Cl-/HCO(-3) antiport, inhibited by 4,4' diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), is functional under resting conditions. PMID- 8631396 TI - Development of post-hatching melatonin rhythm in zebra finches (Poephila guttata). AB - We examined levels of melatonin in the pineal, eyes and plasma over a 24 h period during development in the altricial zebra finch. Beginning as early as 2 days after hatching there was a distinct 24 h rhythm in melatonin in the pineal and plasma. Beginning at day seven after hatching there was also a 24 h rhythm present in the eyes. In the pineal and eyes the amplitude of the 24 h rhythm increased with age. In contrast, the amplitude of the plasma melatonin rhythm at 2 days was already within the range of adults and did not increase with age. These results confirm and expand earlier findings in the European starling and parallel those from precocial birds indicating that the circadian system is already competent at or shortly after hatching even in atricial birds. PMID- 8631398 TI - Results of completed clinical trials for gastric cancer. PMID- 8631397 TI - Seasonal pattern of melatonin excretion in humans: relationship to daylength variation rate and geomagnetic field fluctuations. AB - In order to investigate the influence of various environmental parameters on melatonin excretion, the night-time urinary melatonin excretion of 16 healthy volunteers was measured in samples collected monthly over a period of one year. No significant interindividual differences were detected in the monthly rate of change of melatonin excretion. A seasonal bimodal pattern did, however, emerge. Peak values were observed in June and November. In these months a combination of high daylength stability and low values of the vertical component of the geomagnetic field was recorded. Trough values were found in April and August October when low daylength stability was combined with high values of the vertical component of the geomagnetic field. We propose that the daylength variation rate, and the fluctuations of the vertical component of the geomagnetic field, interact to induce the changes in melatonin secretion which signalize the different seasons in humans. PMID- 8631399 TI - Open European multicentre gastric cancer trials. PMID- 8631400 TI - Possible items to be studied in future randomized studies for gastric cancer. PMID- 8631401 TI - p53 mutations and overexpressions in Japanese breast cancer. AB - Mutations in the p53 gene were analysed in 50 cases with breast cancer by PCR SSCP and direct DNA sequencing. Mutations were found in 10 of 50 cases (20%) including a case with Tis, and loss of the normal allele was found in five of these cases. Eight of the 10 mutations occurred within highly conserved domains, but no mutational hot spots were found. GC to AT transitions were the most frequent mutations (six of 10 cases), while mutations at CpG dinucleotides were found in three cases (30%). AT to TA transversions were the second most frequent mutation (20%). The level of expression of p53 protein was assessed by Western blot analysis in 62 cases. Overexpression of p53 protein was detected in 21 of 62 cases (34%) including a case with Tis. There was a strong correlation between missense mutations in the p53 gene and overexpression of p53 protein. Overexpression of p53 protein was closely associated with increasing tumour size and ER-negative status among the various factors investigated, suggesting that p53 overexpression may reflect the lack of differentiation in breast cancer. PMID- 8631402 TI - The prognostic significance of breast tumour microcalcifications. AB - The prognostic significance of breast tumour microcalcifications has shown, so far, contradictory results. We compared two groups of breast cancer patients: in the first group, the patients showed mammographic and histological microcalcifications, whereas the second group did not. The prognostic indices for both groups included the size of the primary tumour, the number of positive axillary nodes, the histological type, the grade, the hormone-receptor status and the menopausal status. An increased number of cases with both oestrogen and progesterone receptor-positive tumours was found in the group with microcalcifications, which is a favorable prognostic sign for these patients. For the remaining prognostic indices there was no difference between the two groups. PMID- 8631403 TI - The satisfaction and savings of early discharge with drain in situ following axillary lymphadenectomy in the treatment of breast cancer. AB - A pilot study was performed to determine if early discharge with the drain in situ following axillary lymphadenectomy is feasible and safe. One hundred and one women who had axillary lymphadenectomy as part of their breast cancer treatment were studied. Ninety-six were offered early discharge with the axillary drain in situ, five were unsuitable for early discharge. The cohort who opted for early discharge (n = 39) had a lower rate of seroma formation (18% vs 34%) and a reduction in median hospital stay of 5 days. Patient satisfaction was high in both groups and there were no complications associated with early discharge or drain displacement. Early discharge after axillary lymphadenectomy with the axillary drain in situ is safe, feasible, popular with patients and offers considerable resource savings. PMID- 8631404 TI - Patterns of clinical metastasis in breast cancer: an analysis of 100 patients. AB - Many patients diagnosed with breast cancer will develop metastases and these have diverse presentations. We have reviewed 100 consecutive patients who have died with metastatic breast cancer, to determine the frequency, sites and mode of presentation of recurrent disease. The commonest site of failure was loco regional (n = 61), this usually presented with a mass, but a minority of patients also complained of pain. Bone metastases developed in 60 patients and produced bone pain, pathological fracture (n = 6) or cord compression (n = 5). Pulmonary metastases producing shortness of breath were diagnosed in 34 patients and were asymptomatic in a further 10. Intra-abdominal metastases were found at some time in 23 patients, most commonly in the liver (n = 20) and the majority complained of epigastric pain (n = 17). Brain metastases occurred in 23 patients and produced a wide range of symptoms including those of a space-occupying lesion (n = 10), cranial nerve palsy (n = 7), diabetes insipidus (n = 3), focal limb weakness (n = 2) and meningitis (n = 1). Three patients had choroid metastases producing reduced visual acuity. Recurrent breast carcinoma can present in a variety of ways, therefore any new symptom or sign should be considered to represent recurrence until proved otherwise. PMID- 8631405 TI - A series of carcinoid tumours of the breast. AB - Eight patients with a carcinoid tumour of the breast were treated between 1985 and 1992. All were reviewed for their history, physical examination, mammographic and ultrasound features. Staining methods according to Grimelius, with H&E and Neurone Specific Enolase were repeated, and all were positive. In only one patient was a positive axillary lymph node found. No recurrences were seen during follow-up. In agreement with the available literature there seems to be a less aggressive behaviour of carcinoid tumours of the breast in patients over 65 years of age. When a carcinoid tumour is suspected in this age group, the diagnosis can be confirmed by (immuno)histological analysis of a needle biopsy. If the diagnosis of carcinoid is confirmed, a limited operation, without an axillary lymph node dissection, may be considered. PMID- 8631406 TI - Expression of p53 and its relationship with human papillomavirus in penile carcinomas. AB - We studied p53 over-expression in a series of 42 primary penile carcinomas (seven verrucous carcinomas, 14 well-differentiated, 15 moderately-differentiated and six poorly-differentiated squamous cell carcinomas) from Chinese patients using the p53 protein specific mouse monoclonal antibody DO-7 on paraffin sections. p53 protein was detected in 40% (17 cases) of the tumours. The p53 staining was not observed in the penile warts (n = 6) and verrucous carcinomas (n = 7). Positive p53 staining was identified only in the less differentiated tumour cells in the periphery of the tumour cell nests in all the cases. The non-invasive dysplastic epithelium next to the tumours could also be positive for p53 protein (three out of 10 cases in which the dysplastic epithelium adjacent to the tumour was adequately sampled). Furthermore, 100% of the human papillomavirus (HPV)-positive cases showed positive p53 staining. It is concluded that p53 over-expression is present in penile squamous cell carcinomas and adjacent non-invasive tumour cells. An inverse correlation between HPV and p53 gene mutation is not observed in penile cancers. PMID- 8631408 TI - Medial thigh myocutaneous flap for covering extended hemipelvectomy. AB - When a large tumour involves the buttock and anterolateral upper thigh, modifications to the classical hemipelvectomy may be required for its removal. Herein, we report the use of a medial myocutaneous flap for coverage of soft tissue defects produced by such procedures. PMID- 8631407 TI - Resectability of retroperitoneal sarcomas: a matter of surgical technique? AB - The management of retroperitoneal sarcomas has been hampered by the difficulty in complete resection, the resectability rate in the literature being about 53%. In a review of the last 88 consecutive patients with retroperitoneal sarcomas the resectability rate was 95%. At a mean follow-up of 48 months, the local recurrence rate was 17% following wide resection and 59% following local excision (P = 0.0002). For patients with minimum follow-up of 5 years, the local recurrence rate was 39% for those with primary tumours and 57% for those referred with local recurrence. Local recurrence diminished the rate of long-term survival. The 5- and 10-year survival rates for the primary retroperitoneal sarcomas (n = 55) were 66% and 57% and for those referred with locally recurrent sarcoma (n = 33) 57% and 26%, respectively. The 5-year survival rate varied significantly with the grade of the tumour, from 88% for Grade I to 44% for Grade III tumours (P = 0.006). In conclusion, with modern surgical techniques the resectability rate of retroperitoneal sarcomas is about 95%, and the survival rate of the primary tumours approximates that of the primary soft tissue sarcomas of the extremities. PMID- 8631409 TI - Results of irradiation treatment in patients with non-resectable oesophageal cancer. AB - Forty-eight patients with non-resectable cancer of the oesophagus and oesophagogastric junction (Group A: Stage I/II, 32; Group B: Stage III/IV, 16) underwent intraluminal Iridium-192 high dose-rate afterloading therapy (5-7 Gy/session, total dose: 5-21 Gy, mean: 12.4 Gy) and external beam irradiation (Karnofsky > or = 80% 50-60 Gy/2 Gy per day; Karnofsky 60-79%: 30 Gy/3 Gy per day). Durable satisfactory palliation (intake of at least semi-solid food) was demonstrated in 96% of patients. The mean survival for group A was 19.1 months and that for group B, 6.9 months, with a 12-month survival rate of 66% (group A) and 0% (group B) (P < 0.001). Local tumour response and complication rate were significantly dose-related with a predicted response rate of 70.5%, and a complication rate of 50% at ERD 129.3 Gy. PMID- 8631410 TI - Serum thymidine kinase in colorectal neoplasia. AB - Thymidine kinase (TK), an enzyme known to be associated with DNA synthesis, has been measured in the serum of patients with asymptomatic colorectal adenomas (n = 40), asymptomatic colorectal carcinoma (n = 21) and patients known to have hepatic metastases form colorectal tumours (n = 33); enzyme levels have been compared with an age-matched group of asymptomatic people with no evidence of colorectal neoplasia at screening colonoscopy (n = 26). TK activity in patients with metastatic disease (median 4.23; range 2.03-14.12 pmol/ml/h) and in patients with adenomas (median 2.33; range 1.59-8.73 pmol/ml/h) was significantly higher than in the normal controls (median 2.04; range 1.29-5.40 pmol/ml/h). However TK activity in patients with asymptomatic cancer (median 1.85; range 1.00-4.50 pmol/ml/h) was not significantly different from the control group. PMID- 8631412 TI - Survival and local recurrence after anterior resection and abdominoperineal excision for rectal cancer. AB - The aim of this retrospective study is to compare the outcome of abdominoperineal excision (APE) and anterior resection (AR) for rectal cancer in 136 patients. Local recurrence rates and 5-year survival probabilities were estimated for the AR and APE group. Further comparisons were carried out between hand-sewn and stapled anastomoses after AR, and between patients after AR and APE for tumours 2 to 6 cm from the dentate line. Local recurrence after AR occurred in 14% and after APE in 10% of these cases. Five-year survival probabilities and local recurrence frequencies showed no statistically significant difference (P > 0.05). Local recurrence rates were 13.5% after hand-sewn anastomoses and 15% after the stapled procedure. No statistically significant difference was observed in the 5 year survival and recurrence rate (P > 0.05). Seventy-four of 136 patients had tumours located 2 to 6 cm from the dentata line. Local recurrence occurred in 21% after AR and 5% after APE, showing a statistically significant difference in frequency (P < 0.05). No significant difference was found in cumulative 5-year survival probabilities. APE for advanced low rectal cancer showed a significant reduction in local recurrences compared to AR. PMID- 8631411 TI - Prediction of recurrence in B-C stages of colorectal cancer by p53 nuclear overexpression in comparison with standard pathological features. AB - This study investigated the predictive value of p53 nuclear overexpression on recurrence of colorectal adenocarcinomas compared with established prognostic pathological features. Sixty-one paraffin-embedded sections from primary tumours were examined by immunohistochemistry. Specific nuclear staining was detected in 27 (44.2%) cases. Positivity was more frequent in tumours with venous invasion (76.9%) (P = 0.06) and in rectal cancer (68.4%) (P = 0.06). After a median observation time of 46 months, p53-positive tumours exhibited a higher percentage of recurrence (40.7% vs 11.7%) (P = 0.03), and a higher likelihood of relapse at 5-year follow-up (46% vs 13%) (P = 0.006). Among the pathological variables analysed, only the extent of bowel wall invasion showed a relationship with recurrence. After adjustment for the other covariates in a Cox's regression model, p53 overexpression was the only factor showing independent prognostic significance (hazard ratio: 4.96; 95% Confidence Interval (CI): 1.47-16.71) (P = 0.012). The results of this study show that nuclear p53 protein overexpression has higher predictive value than standard pathological variables. PMID- 8631413 TI - Cancer of the rectum--palliative endoscopic treatment. AB - Three methods of palliative endoscopic treatment of malignant strictures of the rectum are presented: laser therapy and its combination with the implantation of either plastic prostheses, or self-expanding metal stents. The aim of all procedures is to avoid a colostomy at least in older patients with shorter life expectancy and incurable tumours. The additional application of a stent maintains the luminal patency and prevents the repetition of laser therapy. Since 1988 we have treated 60 patients with these techniques. First, two to six sessions of laser therapy were necessary for recanalization of the stenosis. In the time following this the patients returned two to eight times for regular laser therapy before they died from the carcinoma. In spite of laser therapy one patient developed an obstruction and another suffered from an iatrogenic perforation of the rectal cancer. In both cases a stoma was fashioned. To avoid repetitive laser sessions we successfully inserted a plastic endoprosthesis in seven cases. In another two cases the implantation failed because of perforation and the patients had to undergo surgery. For the next 10 cases we have used flexible self expanding metal stents. Serious complications or signs of re-obstruction were not observed until the patients' death. The survival time ranged from 2 to 25 months. PMID- 8631414 TI - Local effects of colorectal cancer are well palliated by endoscopic laser therapy. AB - We review the results of treating the local effects of 26 cases of inoperable colorectal cancer with Nd:YAG laser. There were 16 men and 10 women of mean age 75, with 17 (65%) rectal, eight (31%) sigmoid and one (4%) ascending colon cancer. They presented with symptoms primarily of obstruction in 12 patients (46%), bleeding in 10 (39%) and diarrhoea in four (15%). Initial therapy to relieve symptoms required a mean of 1.5 (1-3) laser treatments over a mean of 1.5 (1-2) weeks. Twelve patients (46%) had total relief and 12 (46%) had partial relief. Sixteen patients received follow-up maintenance therapy, with laser treatments performed over a mean interval of 7.3 (1-20) weeks. One died at first follow-up treatment; all but two of the others were well maintained by laser treatment alone. Three patients (12%) suffered complications, with two deaths (8%), one due to cardiac failure and the other due to stercoral perforation of the colon. Four patients remained alive after a mean follow-up period of 51 (9 84) months. The mean survival of the others was 5 (0-23) months. Laser palliation for colorectal cancer is efficacious and relatively safe, allows improved quality of survival in 92% of patients after initial treatment, and, alone, can satisfactorily keep patients relatively free of local symptoms in 88% of patients surviving into the follow-up period. PMID- 8631417 TI - Breast cancer management--a look forward. A view of breast cancer management in the United Kingdom in 1995 and the year 2000. AB - This paper endeavours to contrast the modes of presentation and techniques used in diagnosis and management policies of a 40-year-old woman with breast cancer at present, and as it is likely to be in the year 2000. An attempt is made to review the situation as it occurs across the UK at present, followed by an attempt to forecast how a similar patient is likely to be managed in the year 2000. The nature of the task inevitably involves generalizations, suppositions, hypotheses, informed guesswork and sheer speculation. The challenge is to stimulate thought and discussion among breast clinicians, so that appropriate and desirable changes may be anticipated, and consensus sought in utilising resources to move toward them. PMID- 8631415 TI - Advanced colorectal cancer treated with combined 5-fluorouracil and folinic acid: the experience within a surgical department. AB - Thirty-seven patients with advanced colorectal cancer were treated with fluorouracil (5-FU) and folinic acid (FA) (Jan 1990-Dec 1992). Clinical assessment and administration of chemotherapy was incorporated as part of the daily work load of a busy general surgical unit. Records were available for all 37 patients and showed that 13 patients (Treatment failures, Group B) failed to receive more than 3 monthly cycles of treatment, while the remaining 24 received 6 or greater cycles (Treatment completed, Group A). There was no survival advantage demonstrated for the complete study cohort (n = 37) when compared to an historical group (n = 1038) of untreated patients. Median survival in Group A (14.2 months) was significantly greater (chi-squared, P < 0.0001) than survival in Group B (6.7 months). Toxicity was common with 43% experiencing mouth ulcers or stomatitis (13% severe). Three per cent had dose-limiting diarrhoea and myelotoxicity was minimal. There were six partial responses and 16 patients had no change in their disease status while on treatment. Current regimen of 5-FU/FA are well-tolerated with low toxicity but show no survival advantage for advanced colorectal cancer. However, these regimens may be administered within the confines of general surgical practice. PMID- 8631416 TI - A look back--the contribution of Victor Riddell to the technique of extrafascial axillary lymph node dissection. PMID- 8631418 TI - Surgical video review--breast surgery. PMID- 8631420 TI - Update on urology--prostate cancer. 4--Treatment of local disease. PMID- 8631419 TI - Paediatric surgical oncology. 6--Neuroblastoma. PMID- 8631422 TI - Primary osteosarcoma of the breast. AB - Osteosarcoma is a rare primary breast tumour. We report on a 55-year-old woman who presented with a left breast lump and a mammogram suggestive of carcinoma. At lumpectomy, a 3 cm mass was removed. Histology was consistent with primary osteosarcoma. There was satisfactory surgical clearance and no evidence of metastasis. She received four courses of adjuvant post-operative chemotherapy and remains free of disease at 14 months from diagnosis. PMID- 8631421 TI - Various features and surgical approach of solitary pancreatic metastasis from renal cell carcinoma. AB - In this paper we report three cases of solitary pancreatic metastasis from renal cell carcinoma (RCC), treated surgically. Various features and the surgical approach of these metastases are discussed with references to the 33 previous published cases collected in the literature. Having eliminated widespread distant metastases, it is reasonable to restrict surgical resection of the pancreas to selected patients having a single synchronous or metachronous metastasis, or those having several unilateral metastatic foci. At any rate a careful long-term follow-up for patients with a past history of RCC is mandatory. PMID- 8631423 TI - Splenic hamartoma: a case report. AB - A case of splenic hamartoma is presented that was incidentally discovered in a 55 year-old male during a work-up to determine the aetiology of hypertension. Diagnostic imaging procedures were suggestive of a splenic hamartoma. Histologically, this was found to be of the pulposal type with haemosiderin and calcification foci and many eosinophil leukocytes. The patient had an uneventful recovery. A review of the pertinent references is included, together with a brief discussion about the occurrence of splenic hamartoma in the context of tuberous sclerosis. PMID- 8631424 TI - Induction chemotherapy via hepatic artery for gallbladder carcinoma. AB - The prognosis after surgery for carcinoma of the gallbladder remains poor. Treatment failure is frequently due to loco-regional recurrence in the adjacent liver and regional lymph nodes. We report a case of gallbladder carcinoma with proven involvement of the cystic duct node (Nevin stage IV). Pre-operative intra arterial induction chemotherapy using two cycles of cisplatin, 5-fluorouracil, doxorubicin and mitomycin C was administered via the common hepatic artery. A radical cholecystectomy was performed 4 weeks later, and histological examination of the resected specimen showed a near total response, with no residual nodal disease. The patient remains well and free of disease 3 years later. Intra arterial induction chemotherapy warrants further evaluation. PMID- 8631425 TI - A case report of mucoepidermoid carcinoma of the parotid gland developing after radioiodine therapy for thyroid carcinoma. AB - This is a report on a 19-year-old female who developed a low grade T2 N0 M0 mucoepidermoid carcinoma of the right parotid gland 3 years and 5 months after the post-operative treatment of 100 mCi of radioactive iodine (131I) for a papillary thyroid carcinoma. The parotid tumour appeared during the patient's pregnancy. There are few reports of salivary gland cancer developing after radioiodine therapy for thyroid carcinoma and it is hoped that this report may stimulate others to investigate this association further in order to clarify the risk of secondary malignancies after radioiodine therapy. PMID- 8631426 TI - Post-gastrectomy gastronomy. PMID- 8631428 TI - The impact of managed care. PMID- 8631427 TI - Reducing employee turnover. PMID- 8631429 TI - Complement your talents. PMID- 8631430 TI - Measuring and marketing esthetic dentistry. PMID- 8631431 TI - Establishing an ideal post space in compromised teeth. PMID- 8631432 TI - Functional-intermediate transitional restorations: an approach to temporization. PMID- 8631433 TI - Double-string technique for final impressions--practical applications. PMID- 8631434 TI - Salaries big bite of overhead pie. PMID- 8631435 TI - Controlling delivery appointment anxiety: focus on interchangeable approved provisional methods. PMID- 8631436 TI - Exceed your patients' expectations--have your lab become part of your dental team. PMID- 8631437 TI - The difficult combination fixed/removable case. PMID- 8631438 TI - The successful mandibular implant denture--part one. PMID- 8631439 TI - Increasing hygiene productivity. PMID- 8631441 TI - How to boost your profession. PMID- 8631440 TI - Sexual peril in today's dental practice. PMID- 8631442 TI - The wave of the future. PMID- 8631443 TI - Accelerate your practice through patient reactivation. PMID- 8631444 TI - Cutting back. PMID- 8631445 TI - Seven keys (pearls) in reconstruction. PMID- 8631447 TI - The impact of bioesthetics on the face, smile and teeth. PMID- 8631446 TI - Repair of four fractured incisors in a multiple-unit, maxillary splint using porcelain veneers. PMID- 8631448 TI - Porcelain veneers: chairside color correction. PMID- 8631450 TI - Lesson from our recent history--and a voice from our past. PMID- 8631451 TI - Practice ownership transition requires a contingency plan. PMID- 8631449 TI - Computers playing major roles in dental offices. PMID- 8631452 TI - The 'new math' of managed-care dentistry. PMID- 8631454 TI - Is your associate profitable? PMID- 8631453 TI - Office newsletters: the steps they forgot to tell you. PMID- 8631455 TI - Factors affecting practice value. PMID- 8631456 TI - The esthetic management of dental implants. PMID- 8631457 TI - The sinus-elevation surgical procedure to support implant prostheses. PMID- 8631458 TI - Root coverage utilizing the subepithelial connective tissue graft. PMID- 8631459 TI - Periodontal regeneration. PMID- 8631460 TI - Use computers to segment your patient base. PMID- 8631461 TI - Rethinking the concept of marketing professional services. PMID- 8631462 TI - How to get the loan you want. PMID- 8631463 TI - Before you sign. PMID- 8631464 TI - An investment plan for all seasons. PMID- 8631465 TI - Staff leasing: once a gimmick, now an option. PMID- 8631466 TI - Implant maintenance: a chairside test for real-time monitoring. PMID- 8631467 TI - Restoring Class V caries or defective restorations with sort-tissue autografts. PMID- 8631468 TI - Microabrasion--a conservative approach to removing surface staining. PMID- 8631469 TI - Periodontics as an elective procedure. PMID- 8631470 TI - Practice building--it's really all in your head. PMID- 8631471 TI - Are you prepared to retire? PMID- 8631473 TI - Create a bond of trust in only two minutes. PMID- 8631472 TI - Does waiting too long try your patients? PMID- 8631474 TI - Building your farm team. PMID- 8631475 TI - Internal marketing begins on the telephone. PMID- 8631476 TI - Setting up for success. PMID- 8631477 TI - A letter system to boost your practice. PMID- 8631478 TI - Twelve reasons to add dental videography to your practice. PMID- 8631479 TI - Dual-arch, closed-mouth impression technique. PMID- 8631480 TI - Lower denture impression technique--when all else has failed! PMID- 8631481 TI - What you were not taught about shade selection. PMID- 8631482 TI - Cosmetic provisional veneers. PMID- 8631483 TI - Steps for staff meeting success. PMID- 8631484 TI - For sale: starter practice. PMID- 8631486 TI - Practice reinvention emphasizes new services. PMID- 8631485 TI - A great practice builder. PMID- 8631488 TI - When does it make sense for new dentists to buy a practice? PMID- 8631487 TI - The myth of patient education. PMID- 8631489 TI - Air-abrasive microdentistry: a new perspective on restorative dentistry. PMID- 8631490 TI - Lasers and laminates. PMID- 8631491 TI - Apical membrane turnover is accelerated near cell-cell contacts in an embryonic epithelium. AB - During embryogenesis, the killifish Fundulus heteroclitus forms a monolayered tight epithelium called the enveloping layer (EVL). These epithelial cells have been shown to rearrange during epiboly, as they spread to cover the large yolk cell. Membrane remodeling by exocytosis and endocytosis is important in establishing and maintaining the apical-basolateral polarity of many epithelial cells and is a necessary component of epithelial rearrangements, as cells constantly break contacts and reform tight junctions. To study these phenomena in Fundulus heteroclitus embryos, we labeled the apical membranes of EVL cells with fluorescent lectins and lipids and followed membrane dynamics. Apical membrane components were found to be highly immobilized, allowing us to observe localized sites of apical membrane turnover in situ, over the period of several days. We found that apical membrane turnover in the EVL cells of post-epiboly killifish embryos is accelerated at cell-cell contacts, in a peripheral band of apical membrane which closely borders circumferential tight junctions. Moreover, this turnover rate is increased during epiboly, when the cells are actively rearranging. To investigate whether this increased membrane turnover may be related to the mechanical forces experienced by the rearranging EVL cells, post- epiboly embryos, whose EVL cells no longer rearrange, were subjected to mechanical deformation. In these manipulated embryos, apical membrane turnover was accelerated at cell-cell contacts in EVL cells which experienced externally applied mechanical tension. These results suggest that local mechanical tension may modulate regional apical membrane turnover within EVL cells during the process of epiboly. PMID- 8631492 TI - Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: identification by mRNA differential display and the role of DNA replication in zygotic gene activation. AB - Zygotic gene activation (ZGA) definitely occurs by the 2-cell stage in the mouse embryo. Analysis of protein synthesis by two-dimensional gel electrophoresis reveals a class of genes whose expression transiently increases in the 2-cell embryo. Although the paucity of biological material has prevented a systematic identification of these genes, the mRNA differential display method circumvents this problem. Using this approach we find a transient increase in the mRNA abundance of the translation initiation factor eIF-4C that is inhibited by alpha amanitin and correlated with a transient increase in the relative rate of protein synthesis for eIF-4C. We confirm the transient increase in eIF-4C mRNA abundance by a reverse transcription-PCR-based assay using eIF-4C-specific primers. The first round of DNA replication seems critical for eIF-4C expression, since addition of aphidicolin prior to S phase in the 1-cell embryo inhibits the magnitude of the increase in eIF-4C expression. Aphidicolin treatment also inhibits the synthesis of an accepted marker for ZGA, the transcription requiring complex (TRC), which is also transiently expressed during the 2-cell stage. Incubating late 1-cell/early 2-cell embryos in medium containing aphidicolin reveals that the second round of DNA replication is not required for the increase in eIF-4C expression but DNA replication is required for the decrease in both eIF 4C expression and TRC synthesis. The decrease in eIF-4C expression, however, does not require cytokinesis or mitosis, since it occurs when 2-cell embryos are cultured in the presence of cytochalasin D or nocodazole, respectively. Changes in chromatin structure may be involved in the decrease in both eIF-4C and TRC expression, since neither decrease occurs when 2-cell embryos are cultured in trapoxin, which is a specific and irreversible inhibitor of histone deacetylase. Results of these experiments suggest that the first round of DNA replication is permissive with respect to ZGA and that the second round is repressive. PMID- 8631493 TI - The cAMP receptor subtype cAR2 is restricted to a subset of prestalk cells during Dictyostelium development and displays unexpected DIF-1 responsiveness. AB - Dictyostelium discoidium cells express a family of cell surface cAMP receptors, and these G-protein-coupled receptors are each expressed with unique spatial and temporal patterns. One of these receptors, cAR2, is present during the postaggregative stages of development and our previous work suggests that it is preferentially expressed in prestalk cells. We report here the isolation of the promoter for carB, the gene which encodes cAR2. Using this fragment to generate a carB::lacZ, gene fusion construct, we investigated carB expression in detail. Expression is first detected at the tight aggregate stage and subsequently in a pattern reminiscent of the prestalk-specific gene ecmA. There are subtle differences, however, with, ecmA being expressed significantly in the anterior like cells of the migrating pseudoplasmodium and in the basal disc and lower cup supporting the sorus during terminal development. carB is not expressed in any of these places. The presence of these different prestalk cell subtypes was confirmed by double indirect immunofluorescence using anti-cAR2 and anti-beta galactosidase antibodies. While virtually all cAR2-expressing cells also express ecmA::lacZ, a substantial fraction of ecmA::lacZ-positive cells do not express cAR2. We also found the regulation of carB gene expression to differ from that of ecmA. carB expression is induced in vitro by extracellular cAMP, but surprisingly, not by DIF-1, a soluble molecule thought to be essential for the initiation of prestalk differentiation. Thus, cAR2 appears to be a cAMP receptor present on a restricted subset of prestalk cells and whose expression does not respond typically to the prestalk inducer DIF-1. DIF-1 sensitivity may, therefore, not be characteristic of all early prestalk differentiation. PMID- 8631494 TI - Initial cell type divergence in Dictyostelium is independent of DIF-1. AB - Prespore and prestalk cells can be distinguished within aggregates of Dictyostelium by the expression of well-characterized cell type-specific genes. Fusion of the tagB regulatory region to Escherichia coli beta-galactosidase revealed that this prestalk specific gene marks the differentiation of the initial prestalk cell population, PST-1. The reporter gene was expressed normally in tagB- mutant cells despite the fact that they do not accumulate measurable levels of DIF-I, a morphogen that was previously implicated in prestalk differentiation. In an independent experimental system, wild-type cells respond to the addition of DIF-I by induction of the prestalk marker ecmA and repression of the prespore marker cotB. We found that DIF-1 did not affect the expression of the tagB or carB genes, both of which are prestalk specific and essential for PST A cell differentiation. We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates mainly at later stages. PMID- 8631495 TI - Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. AB - The vascular smooth muscle cells of coronary arteries are distinguished from those of the proximal aorta by a number of structural and functional criteria which may include an increased propensity for atherosclerotic transformation. At present, the source of this variation between smooth muscle subpopulations is uncertain. Whilst smooth muscle of the proximal aorta is thought to be derived from neural crest, the origin of coronary vascular smooth muscle remains uncharacterized. We have previously shown that precursors of the coronary vasculature enter the tubular heart on the same day as the epicardial mantle starts to envelop the myocardium and that coronary vessels form by ingrowth of these migratory precursors and not by outgrowth from the aorta (Mikawa and Fischman, 1992). To study the origin of coronary smooth muscle cells, the proepicardial organ, from which epicardial cells arise, was tagged with either a vital dye (DiI) or replication-defective retroviruses encoding beta galactosidase. Cellular lineage marking was achieved by either direct targeting of putative vasculogenic cells in the proepicardium in ovo or tagging dissected proepicardial cells in vitro followed by transplantation to stage-matched host embryos. Monitoring of tagged cells during coronary vasculogenesis indicate incorporation of proepicardial-derived cells into three vessel-associated populations; coronary smooth muscle, perivascular connective tissue, and endothelial cells. Immunoconfocal microscopy identified both endothelial and smooth muscle cell populations within the proepicardial organ. The results demonstrate that: (1) the proepicardium contains a progenitor population of coronary smooth muscle cells that migrates into the heart along with ingrowth of the epicardium and (2) prior to the migration, the coronary smooth muscle lineage is established. PMID- 8631496 TI - A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. AB - In the Drosophila nervous system, lateral inhibition regulates commitment to a neural fate by preventing neighbouring cells from developing alike. This signalling process is mediated by two transmembrane proteins-Notch as receptor and Delta as its ligand. The Delta-related protein Serrate also acts as a Notch ligand in Drosophila, but in a different developmental process that organizes patterning of the wing. We have previously shown that lateral inhibition operates at early stages of neurogenesis in vertebrates, via genes homologous to Drosophila Delta and Notch. We report here the cloning of a chick Serrate homologue, C-Serrate-1. This gene is expressed in the central nervous system, as well as in the cranial placodes, nephric epithelium, vascular system, and distal limb-bud mesenchyme. In most of these sites, its expression is associated with expression of C-Notch-1 and C- Delta-1. All three genes are expressed in the ventricular zone of the hindbrain and spinal cord, throughout the period when neurons are being born. Within this zone, C-Delta-1 and C-Serrate-1 are expressed in complementary subsets of nondividing cells that appear to be nascent neurons: C- Serrate-1 expression is restricted to specific locations along the dorsoventral axis, forming narrow bands extending from the anterior hindbrain to the tail. Our observations strongly suggest that Delta-Notch signalling delivers lateral inhibition not only early but throughout vertebrate neurogenesis to regulate neuronal commitment, and that Serrate-Notch signalling may act similarly in this process. By analogy with its role in Drosophila wing patterning, C Serrate-1 may also have a role in organising the dorso-ventral pattern of the neural tube. We argue that signalling via Notch maintains neurogenesis, both in vertebrates and in flies, by keeping a proportion of the neuroepithelial cells in an uncommitted stem-cell-like state. PMID- 8631497 TI - Antibodies to the Type II TGFbeta receptor block cell activation and migration during atrioventricular cushion transformation in the heart. AB - Epithelial-mesenchymal transformation is a critical event in the development of many organ systems including the heart. Descriptive studies have implicated a number of factors in mediating this transformation, including transforming growth factor beta (TGFbeta). We now report that disruption of a TGFbeta signal transduction complex by antibodies directed against the Type II TGFbeta receptor blocks both the endocardial cell activation and subsequent migration that constitute transformation in the chick atrioventricular (AV) cushion. The Type II receptor was localized to both endothelial and endocardial cells of the chick embryo. Incubation of AV cushion explants from Stage 14, 16, and 18 embryos with antibody resulted in a blockade of AV endocardial cell transformation by greater than 50% as measured by mesenchyme formation. Similarly, the appearance of procollagen Type I, a marker of endocardial cell transformation, was blocked. In addition, within 2 hr after the incubation of activated Stage 18 explants with Type II antibody the rate of migration of transformed cells was decreased by 50%. These data suggest that TGFbeta acts directly on AV cushion endocardial cells to stimulate epithelial-mesenchymal transformation and that TGFbeta mediates at least two distinct components of AV cushion transformation, activation and migration. PMID- 8631499 TI - A cAMP regulated K+-selective channel from the sea urchin sperm plasma membrane. AB - Ion channels are deeply involved in sperm physiology. In sea urchin sperm cyclic nucleotide levels increase during quimotaxis and in the acrosome reaction (AR). Although cyclic nucleotides are second messengers known to directly or indirectly modulate ion channels, it is not clear how they modulate sperm responses to the egg outer layer. Here, we describe a cAMP regulated K+-selective channel from sea urchin sperm plasma membranes fused into planar bilayers that may have a role during sea urchin sperm quimotaxis and/or the AR. Its single channel conductance in 100 mM KCl is 103 pS. In bi-ionic experiments, the channel displayed a K+/Na+ permeability ratio (PK+/PNa+) of approximately 5. Thus, in sea water its reversal potential would be approximately -13 mV and channel opening would depolarize spermatozoa. The channel has low open probability (Po = 0.8 +/- 0.2% at 0 mV applied voltage) and weak voltage dependence. Channel activity is reversibly up regulated by cAMP in the cis bilayer side, but not by cGMP. This modulation followed a single Langmuir isotherm with an apparent kd of 200 microM. At this concentration the channel open probability at 0 mV increased up to 11- fold. TEA+ blocked the channel only from the trans side. Also Ba2+ in trans blocked the channel in a voltage-dependent manner. PMID- 8631498 TI - The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis. AB - The GATA family of nuclear factors has been implicated in the regulation of cell type-specific transcription. We report the isolation of the Xenopus GATA-4 and GATA-6 cDNA clones and characterize the expression patterns of the xGATA-4/5/6 genes. By comparing the sequence of the cDNAs with those previously reported from chick and mammalian sources, we conclude that each is conserved across vertebrate evolution as a distinct gene product. Each gene is expressed in differentiated adult heart and gut, but maintains distinct transcript patterns in various other adult tissues. During embryogenesis, each gene displays a similar overlapping distribution of transcripts localized throughout the developing cardiogenic region. The xGATA-4 gene can be detected in dorsal cardiac progenitor rudiments prior to migration. Axis disruption experiments were used to demonstrate that transcription of these genes is intimately associated with the specification of cardiac progenitors. Ectopic expression of each gene is specifically capable of activating during embryogenesis the transcription of the cardiac genes encoding actin and myosin heavy chain alpha. The data are consistent with a primary role for the GATA-4/5/6 genes in regulating heart development. PMID- 8631500 TI - Biphasic activation of Fyn kinase upon fertilization of the sea urchin egg. AB - Fertilization results in the tyrosine phosphorylation of several egg proteins within minutes of sperm-egg binding and inhibitor studies have shown that tyrosine kinase activity is required for many aspects of egg activation. The present study demonstrates the presence of p59c-fyn kinase in the sea urchin egg and examines the effect of fertilization on the activity of this enzyme. Fertilization had little effect on Fyn kinase activity during the first 2 min after insemination; however, activity had increased approximately eightfold between 5 and 15 min postinsemination. This initial, rapid increase in kinase activity was followed by a period of slightly elevated kinase activity, which was two- to threefold higher than that in the unfertilized egg. Bindin, as well as various parthenogenic agents known to activate the calcium- and pH-mediated pathways of egg activation, failed to elicit any change in enzyme activity, indicating that activation of the kinase required sperm-induced egg activation. However, phorbol ester treatment did induce a slow increase in kinase activity within 30 to 60 min of administration. These findings indicate that the p59fyn kinase is activated within minutes of fertilization and may play a role in the egg activation process. PMID- 8631501 TI - A nuclear protein regulated during the transition from active to quiescent phenotype in cultured endothelial cells. AB - Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with the FUS gene product, TLS (Translocated in Liposarcoma; Crozat et al, 1993; Rabbits et al., 1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis. PMID- 8631502 TI - The Drosophila rhomboid protein is concentrated in patches at the apical cell surface. AB - Patterned expression of the Drosophila rhomboid (rho) gene is thought to promote signaling by the EGF receptor (EGFR) in specific cell types. In this report we examine the subcellular localization of the Rhomboid protein (Rho) which is predicted to be an integral membrane protein. At the light level, immunocytochemical staining for Rho reveals a small number of large patches (or plaques) at or near the apical cell surface. In some cells Rho plaques colocalize with Armadillo at adherens junctions, while in other cells plaques are only found basal to the adherens junction. Immunoelectron microscopy reveals that Rho plaques are composed of a highly localized patch of plasma membrane and a densely staining underlying structure. Concentration of Rho in distinct plaques depends on a balance of synthesis and membrane recycling since increasing the amount of rho expression or blocking membrane recycling leads to more uniform cell surface labeling. A limiting cellular component also appears to be required for concentrating Rho in plaques. We discuss clustering of Rho in plasma membrane patches with respect to the proposed role of Rho in promoting EGF-R signaling. PMID- 8631503 TI - Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis. AB - Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer- extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis. PMID- 8631504 TI - Novel postfertilization inward Ca2+ current in ascidian eggs ensuring a calcium entry throughout meiosis. AB - The conductance change after fertilization in the oocyte of the ascidian Ciona intestinalis has been followed by the whole cell patch-clamp technique. Two new inward currents, which are absent in unfertilized eggs, are elicited by hyperpolarization from a holding potential of +20 mV, which is the resting potential soon after fertilization. These currents reach their maximum level during the first meiotic division cycle, and then decrease in intensity, becoming almost undetectable at the 2-cell stage. These currents are most easily seen at high concentrations of barium. At least one, and likely both, of these currents appears to be carried by Ca ions. One of the currents is blocked by low concentrations of gadolinium; the other one is blocked by higher concentrations, although gadolinium at these levels does not block fertilization and the associated early depolarizing jump of the eggs. Thus these currents are not carried by channels that mediate the fertilization current. However, gadolinium blocks normal transition to 2-cell stage and blocks current oscillations synchronous to free calcium oscillations that occur normally in eggs around meiosis II. The electrical signature of calcium-release activated currents, taken together with these findings, suggests that these inward currents ensure a calcium entry pathway throughout meiosis. A plausible function of these currents may be to refill the Ca stores that are depleted after fertilization and that are required to progress into mitotic cell division. This interpretation is reinforced by experiments on unfertilized eggs with intracellular Ca stores depleted by thapsigargin, where both the newly described currents are observed. PMID- 8631505 TI - An accumulation of p34cdc2 at the end of mouse oocyte growth correlates with the acquisition of meiotic competence. AB - Growing incompetent mouse oocytes released from follicular cells are unable to spontaneously resume meiosis in vitro. To identify the reasons for meiotic incompetence in these cells, the levels of p34cdc2/cyclin B kinase and p42MAPK between incompetent and competent oocytes were compared. p34cdc2 was present at very low levels in incompetent oocytes and accumulated abruptly at the time of meiotic competence acquisition. By contrast, cyclin B and p42MAPK were present at similar concentrations in both types of oocytes. Okadaic acid induced centrosome phosphorylation and meiotic reinitiation in incompetent oocytes, without inducing an increase in p34cdc2 concentration. However, the p34cdc2 present in incompetent oocytes was activated and all events following germinal vesicle breakdown were induced up to the formation of a metaphase I spindle including p42MAPK activation, sustained increase in p34cdc2 kinase activity, and translational activation of a dormant mRNA. We suggest that a threshold level of p34cdc2 has to be reached for meiotic reinitiation to be spontaneously triggered: competence is restricted at a point preceding MPF activation. Whatever the mechanism involved in this restriction point, i.e., subthreshold concentration of p34cdc2 and/or lack of an activator or presence of an inhibitor, it is bypassed by okadaic acid. Downstream of this point meiosis progresses up to metaphase 1, even though p34cdc2 concentration remains low. PMID- 8631506 TI - Multiple actions of stem cell factor in neural crest cell differentiation in vitro. AB - The neural crest is a transient tissue of the vertebrate embryo that gives rise to most primary sensory neurons and pigment cells in the adult organism, among other cell types and tissues. Many neural crest cells are pluripotent in the sense that their progeny can generate more than one phenotype. The presence of pluripotent neural crest cell-derived cells at sites of terminal differentiation suggests that location-specific cues from the embryonic environment, such as growth factors, are involved in directing their survival, proliferation, and cell type specification. We have therefore examined the influences of one pertinent growth factor, stem cell factor (SCF), on neural crest cell development by in vitro colony assay in a serum-free culture medium. SCF showed three major effects. (1) SCF is trophic for early neural crest cells, that is, either pluripotent cells and/or their more mature progeny. This effect occurs only if SCF is present throughout the culture period, and it is not observed when a neurotrophin is present in addition to SCF. (2) More colonies contain sensory neuron precursors in the presence of SCF. This effect is neutralized by NGF and neurotrophin-3 (NT-3), but not by brain-derived neurotrophic factor (BDNF). (3) The combination of SCF and any neurotrophin tested (NGF, BDNF, NT-3) is trophic for melanogenic cells, whereas SCF alone does not detectably affect melanogenesis. This suggests either that both types of factor are required for melanotrophic action or that melanogenic cells become dependent on neurotrophins after exposure to SCF. Our observation that SCF is required during the first half of the culture period only, and NGF during the second half only, indicates the latter possibility. Whereas coat color changes in the mouse mutants W (c-kit defect) and Steel (SCF defect) and several in vivo and in vitro studies by other investigators have shown previously that SCF is melanotrophic, they also indicated the requirement of an additional factor, or factors, in melanogenesis. Our data suggest that SCF affects neural crest cell development at multiple levels and that survival of melanogenic cells is mediated by a combination of SCF and a neurotropin, rather than by SCF alone. PMID- 8631508 TI - The role of FGF-3 in early inner ear development: an analysis in normal and kreisler mutant mice. AB - The development of the otic placode is believed to depend on an inductive signal from the adjacent hindbrain. A candidate for this signal is FGF-3 (Int-2), which is expressed in the hindbrain adjacent to the future ear in rhombomeres 5 and 6 (r5 and r6). However, in vitro tests (Represa et al. (1991), Nature 353, 561-563) conflict with findings from FGF-3 knockout mice (Mansour et al. (1993), Development 117, 13-28). The former suggest that FGF-3 from the hindbrain is required to induce formation of the otocyst, while the latter imply that FGF-3 is required only in the later process of otocyst differentiation. We find that in normal embryos at early stages the gene is expressed not only in r5 and r6, but also in most of the hindbrain anterior to this and in the head ectoderm in the prospective otic placode region. In kreisler mutant embryos, however, there is no heightened expression in r5 and r6, but the early patch of expression in the prospective otic placode ectoderm is still seen and the otic vesicle still forms at nearly the normal place. Subsequent malformations of the inner ear in kreisler and in FGF-3 knockout mice are similar, involving failure of the development of the endolymphatic appendage. These findings argue that FGF-3 is not required as an inductive signal for invagination of the otic placode to form a vesicle, whose future site is already marked out independently of any localized FGF-3 signal from r5 and r6. FGF-3 does, however, appear to be required for a correct pattern of differentiation within the vesicle. PMID- 8631507 TI - Abnormal microenvironmental signals underlie intestinal aganglionosis in Dominant megacolon mutant mice. AB - Dominant megacolon (Dom) is a mutation in an uncharacterized murine gene which is associated with intestinal aganglionosis and the focal absence of melanocytes in heterozygous animals. The phenotype of Dom/+ heterozygotes is similar to the lethal spotted and piebald lethal mutations, which are due to defects in endothelin-mediated intercellular signals. In this study, the DbetaH-nlacZ transgenic marker for enteric neural crest cells is used to study the distribution of enteric neurons and their precursors in Dom/+ mice and embryos. Vagal neural crest-derived cells in wild-type embryos colonize the gut in a cranial-to-caudal progression. In Dom/+ embryos, colonization was retarded from the earliest stages examined (embryonic Day 11.0), including progression through the small intestine. The early onset of this defect contrasts with impaired neural crest colonization associated with the lethal spotted and piebald lethal mutations which manifest only in the large intestine. Analysis of Dom/+ - +/+ aggregation chimeras indicated that defective colonization is not an autonomous (intrinsic) property of Dom/+ neuroblasts, but like lethal spotted and piebald lethal, the Dominant megacolon mutation directly or indirectly affects microenvironmental signals which influence the migration, proliferation, and/or survival of enteric neural crest cells. PMID- 8631510 TI - Purkinje cell lineage and the topographic organization of the cerebellar cortex: a view from X inactivation mosaics. AB - We utilized a strain of mice, derived from a radiation mutagenesis experiment and carrying an activity-attenuated allele of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD), to analyze the development of the cell lineage leading to cerebellar Purkinje neurons. Due to random X inactivation during early embryonic development, X- linked genes can be used to distinguish between clonally related populations of cells in X inactivation mosaics. Following histochemical staining for G6PD activity, the numeric proportions of Purkinje cells expressing either the wild-type or the mutant enzyme and the spatial distribution of these cellular phenotypes and their relation to anatomically and genetically defined cerebellar compartments were analyzed. Our data suggest that cerebellar Purkinje neurons originate from a limited pool of some 129 precursors. The size of this pool is different from the one derived from chimeric mice, allowing us to deduce the relative timing of Purkinje cell lineage restriction. Our data also show that Purkinje neurons of distinct lineage are extensively intermingled within the cerebellar cortex. Together, these findings suggest both a role for cell-cell communication in the development of genetically defined cerebellar compartments and a temporal window during which such cellular interactions may take place. PMID- 8631509 TI - Mechanism of an evolutionary change in muscle cell differentiation in ascidians with different modes of development. AB - We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression. PMID- 8631512 TI - Cleavage pattern and mesentoblast formation in Acanthochiton crinitus (Polyplacophora, Mollusca). AB - In characteristic spiralian embryos the mesentoblast is the stem cell of the mesodermal bands. It is a derivative of the dorsal quadrant. At least in gastropod molluscs, the ancestral form for the specification of the dorsal quadrant out of four initially equal quadrants is by centralization of one of the four macromeres after the separation of the presumptive ecto- and entoblast cells. Then this macromere is induced by the animal micromeres to produce the mesentoblast. In this paper it is shown that in the embryo of the polyplacophoran Acanthochiton crinitus, specification of the dorsal quadrant and formation of the mesentoblast exactly follow the same pattern. After deletion of the first quartet of micromeres none of the macromeres is centralized, no mesentoblast is formed, and the embryo remains radially symmetrical. Apparently, the mechanism for the specification of the dorsal quadrant and the formation of the mesentoblast has been conserved during the evolution of the main molluscan taxa. It has been discussed whether this mechanism might be a plesiomorphous property, characteristic of less derived spiralian phyla. PMID- 8631511 TI - Myogenic determination factor expression in the developing avian limb bud: an RT PCR analysis. AB - This study examines the time of appearance of myogenic determination factor (MDF) transcripts in developing chick limbs and other embryonic tissues using reverse transcription-polymerase chain reaction (RT-PCR). In this assay, PCR amplification profiles of cDNA samples from stage 15-26 limb buds are compared to those of E11 heart, a tissue operationally defined as containing background levels of MDF transcripts which are thought to be nonfunctional. Myf 5 and MRF 4 transcripts are detected in stage 15 forelimbs, which is the earliest stage limb in which myogenic precursors have been detected following their migration from the somite. In contrast, MyoD and myogenin transcripts do not appear until about 1.5 days later, closer to the time of cytological muscle differentiation. A survey of MyoD, myf 5, and MRF 4 transcripts in other embryonic tissues reveals that MyoD transcripts are distributed in a highly skeletal muscle-specific pattern. In contrast, myf 5 and MRF 4 mRNAs are detected at significant levels in embryonic tissues that do not contain muscle or muscle precursor cells such as stage 9-15 neural tubes and stage 9- 12 lateral plates, while these transcripts are found at very low levels in E11 heart and liver. The RT-PCR results are compared to those from in situ hybridization experiments as well as from cell culture assays of the myogenic potential of early limb cells. PMID- 8631514 TI - Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures. AB - Bone morphogenetic proteins (BMPs) induce ectopic cartilage and bone when implanted intramuscularly in adult rats. Expression data suggest that BMPs signal skeletal development in embryos. An important question is which cells are targets of BMP signaling in adult and embryonic tissues. Here, we examined the effect of BMP-2 on micromass cultures of chick limb bud mesenchyme. We report that BMP-2 promotes formation of cartilage and simultaneously inhibits development of muscle cells. To follow the fate of presumptive muscle cells, we replaced chick somites with quail somites at the wing level and made micromass cultures from the chimeric wings. In untreated cultures, quail cells formed muscle but not cartilage. In BMP-2-treated cultures, quail cells disappeared altogether. This suggests that BMP-2 may simultaneously promote cartilage differentiation and reduce the presumptive myogenic cell populations in regions of skeletal development. PMID- 8631515 TI - Will he grow out of it? PMID- 8631513 TI - Primary sequence and developmental expression pattern of mRNAs and protein for an alpha1 subunit of the sodium pump cloned from the neural plate of Xenopus laevis. AB - Expression of a catalytic alpha subunit of the sodium pump was followed in early Xenopus embryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for alpha1 subunit isoforms were cloned from a neural plate stage Xenopus library and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for alpha1 subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed alpha1, transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, alpha1 mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, alpha1 mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations. PMID- 8631516 TI - Autism and medical disorders: a review of the literature. AB - The authors reviewed all the population studies on autism published in the English language with particular reference to the rate of medical disorders. Seven studies met criteria for inclusion in the survey. The mean of possibly autism-related medical disorders in persons with autism across these studies was 24.4%. There was a trend for higher rates of medical disorders among subjects with severe mental retardation. The evidence in respect of atypical autism was equivocal, and the overall prevalence of medical disorders in this group was similar to that found in typical autism. PMID- 8631517 TI - Neuropathological study of a case of autistic syndrome with severe mental retardation. AB - Infantile autism is a syndrome of unknown aetiology and unknown neuro-anatomic substrate. The authors report a histological study of the brain of a well documented 16-year-old female with autistic syndrome and severe mental retardation, using direct microscopic examination of the whole brain. The major findings are low brain weight, a thin corpus callosum and ventricular dilatation. No abnormalities were found in the hippocampus or cerebellum. Excessive axonal elimination during brain development is hypothesized. The relations of hypothetical developmental events with the clinical features of autistic syndrome are discussed. PMID- 8631518 TI - Vision, cognition and developmental characteristics of girls and women with Rett syndrome. AB - Forty-two females with Rett syndrome, aged 2.5 to 47 years, were assessed with the Teller Acuity Cards and a new version of the Fagan test for age 2 years and above, and their parents were interviewed about the children's communication skills. The visual function of the subjects indicated arrested development, and they scored significantly lower on the Fagan test than a normal comparison group. Their visual processing and memory deteriorated somewhat with age, while those of the comparison group showed a slight increase. Both age at onset of Rett syndrome symptomatology and speech measures were inversely correlated with visual processing and memory, indicating that age at recession may have differential consequences for different functions. Among the subjects, persistent looking was associated with low cognitive function. The results have implications for intervention, and demonstrate that the paradigm of preferential looking may be useful in cognitive assessment of females with Rett syndrome. PMID- 8631519 TI - A randomised controlled trial of different intensities of physiotherapy and different goal-setting procedures in 44 children with cerebral palsy. AB - Forty-four children aged 3 to 11 years with quadriplegic cerebral palsy were prospectively stratified and randomised into four treatment groups. The acquisition of motor skills was assessed in a 2 x 2 factorial design using the Gross Motor Function Measure. The two factors were conventional amounts of physiotherapy vs intensive amounts of physiotherapy, and the use of broad, generalised aims vs the use of specific measurable goals directed at motor skill acquisition. 82% of the children improved. Over the two-week period, intensive physiotherapy produced a slightly greater effect than conventional physiotherapy but the factor more strongly associated with increased motor skill acquisition was the use of specific measurable goals. PMID- 8631521 TI - The maturation of motor dexterity: or why Johnny can't go any faster. AB - The speed of alternating movements at the ankle, metacarpophalangeal and wrist joints in 11 healthy children and 13 adults doubled between age 3 and 11 years, despite a 32-fold increase in limb-segment inertia produced by the doubling in limb length over the same period. The data for the children showed little or no practice effect. The speeds for the adults, though faster than those for the children, were more widely dispersed, indicating the possibility that training might increase the speed of the slowest adult. The findings are consistent with a previous report demonstrating a parallel increase in the speed of calf muscles over the first 10 years of life and it is inferred that the increase in dexterity at the wrist and metacarpophalangeal joints also depends on an increase in muscle speed with age. Muscle maturation may impose a rate-limiting envelope for all motor tasks which is particularly evident in rapidly alternating movements. These findings have implications for training in sport and music and for the understanding of motor delay, clumsiness and speech difficulties. PMID- 8631520 TI - Energy consumption in children with spina bifida and cerebral palsy: a comparative study. AB - The authors looked for differences in the energy expenditure patterns of ambulant children with cerebral palsy and spina bifida. Oxygen consumption was measured according to type of cerebral palsy or level of spina bifida lesion, and in healthy children. The rate of oxygen consumption (mL/kg/min) was significantly higher in the children with diplegia than in those with hemiplegia or with spina bifida or the healthy children. Oxygen cost (mL/kg/m) was significantly higher and velocity was significantly slower in all the groups with disability than in the healthy children. The reason children with diplegia consumed more oxygen than other children when walking may be that their abnormal equilibrium reactions impaired their balance and their ability to control their walking speed. PMID- 8631522 TI - Establishing normal values of moving two-point discrimination in children and adolescents. AB - To establish normal values of moving two-point discrimination (2pd) in children and adolescents, bilateral median and ulnar nerve distribution in 313 subjects aged 4 to 18 years was evaluated. A moving 2pd of 2 to 3mm for both the median and ulnar nerve distributions bilaterally was found in 270 subjects. 2% (N = 5) had moving 2pds of 4mm for both right and left median nerve distributions. There was a significant increase in mean age with 2, 3 and 4mm of moving 2pd for median and ulnar nerves bilaterally. There was no significant difference between sexes for ulnar nerves bilaterally or left median nerve. A borderline difference was found between the sexes for the right median nerve. Multivariate logistic regression revealed age as a significant predictor of discrimination for all variables. Significantly more subjects had a moving 2pd of 4mm in the ulnar nerve distribution than in the median nerve distribution. PMID- 8631523 TI - Clinical and molecular pathological features of severe childhood autosomal recessive muscular dystrophy in Saudi Arabia. AB - The clinical, biochemical and histochemical features of 14 patients (nine females and five males) with severe childhood autosomal recessive muscular dystrophy (SCARMD) seen at a tertiary hospital in Riyadh from 1982 to 1993 are described. Onset was at 3 to 9 (median 3) years and four of five children aged > 12 years lost ambulation. Five of the eight pairs of parents were closely consanguineous. The mean creatine kinase was 20 times the upper normal limit. Histochemistry of muscle showed dystrophic features in all cases, and dystrophin was positive in all cases examined (N = 6). Three patients (two girls and a boy) were deficient in adhalin, the 50-kDa dystorphin-associated glycoprotein. A boy aged 13 years had rapidly progressing disease. Another boy of the same age (from a family characterized by early onset and slower progression) had normal dystrophin and adhalin. The clinical features conformed with previous observations from Sudan, North Africa and Qatar in the Arabian Peninsula. The disease is common in Saudi Arabia and seems to be more prevalent than Duchenne muscular dystrophy. PMID- 8631524 TI - Delayed development of sensorineural hearing loss after neonatal hyperbilirubinemia: a case report with brain magnetic resonance imaging. AB - Sensorineural hearing loss has long been known to be a clinical consequence of kernicterus. Brainstem auditory evoked potentials (BAEPs) that occur in hyperbilirubinemic infants, can be reversed in the neonatal period by exchange transfusion. The case was reported in an infant with neonatal hyperbilirubinemia from hemolysis due to glucose-6-phosphate dehydrogenase (G6PD) deficiency and napthalene exposure. BAEPs showed that the baby had normal hearing at 30 decibels at 13 days of age, after exchange transfusions, but had developed profound bilateral sensorineural hearing loss by 7 months of age. The brain magnetic resonance imaging (MRI) findings at 7 months are also presented. PMID- 8631525 TI - The treatment of infantile spasms by paediatric neurologists in the UK and Ireland. PMID- 8631527 TI - Nursing home 'excess' adds to Medicaid costs. PMID- 8631526 TI - Iodine and brain development. PMID- 8631529 TI - American Heart Association 68th scientific sessions, Anaheim, CA. PMID- 8631528 TI - A wake-up call for caution. If insomnia is the patient's problem, is over-the counter melatonin the cure? PMID- 8631530 TI - Difficult dementia: six steps to control problem behaviors. AB - For patients with a confirmed diagnosis of dementia, your challenge is to promote a quality life during their remaining years. This task often includes managing problem behaviors. A systematic approach starts with pinpointing the nature of the specific behavior, reviewing possible physical and emotional stressors, and checking for coexisting affective or psychotic disorders. It often helps to reduce environmental stimulation and to simplify the patient's tasks. Drug therapy with an antipsychotic or benzodiazepine is indicated if a clear-cut behavioral strategy has not proven fully effective, the behavior has been well documented, and the behavior presents a clear danger to the patient or others or prevents necessary care from occurring. PMID- 8631531 TI - Preventing falls: how to identify risk factors, reduce complications. AB - Falls are a common problem of old age and are associated with considerable morbidity. Falling itself is not a diagnosis but a symptom of multiple underlying diseases, the effects of certain medications on homeostasis, and/or environmental hazards or obstacles that interfere with safe mobility. Preventing falls requires a systematic diagnostic approach focused on identifying and reducing risk factors. Specific preventive strategies include treating underlying medical conditions, prescribing an exercise program to improve mobility, removing fall hazards in the home, and taking steps to minimize the fear of falling. PMID- 8631532 TI - Painful rash on the right cheek. PMID- 8631533 TI - Nursing home rules can lead to abuses, too. PMID- 8631534 TI - Do we know the best therapy for early endometrial cancer? PMID- 8631535 TI - Endometrial carcinoma--relative effectiveness of adjuvant irradiation vs therapy reserved for relapse. AB - Fifty-four patients with recurrent endometrial carcinoma were identified from a retrospective review of charts of 304 endometrial cancer patients seen between 1983 and 1989 at our center. A review was undertaken to identify the patterns of relapse, to determine the outcome of salvage treatment, to examine the factors predictive of effective salvage, and, if salvage is effective, to assess an alternative strategy to routine adjuvant postoperative pelvic radiotherapy. Forty percent of the entire recurrent population are long-term survivors. Of the 54 relapsing patients, primary therapy had been surgery alone in 32 and surgery and adjuvant radiotherapy (rt) in 22. Isolated pelvic recurrence was the predominate relapse site in those who had not received adjuvant pelvic RT as primary therapy (23 of 32 or 72%). Distant relapse predominated in those who received adjuvant RT (17 or 22 or 77%). Twenty-eight (54%) failed in the pelvis alone, and 26 (46%) had a component of distant failure. Of the 28 with isolated pelvic relapse, 16 had vaginal mucosal disease involvement only and 12 had disease in the parametrium and/or the pelvic sidewall. With a minimum follow-up for the survivors of 5 years, 21 of the 28 with isolated pelvic relapse received radical radiotherapy and 14 or 67% had maintained pelvic control until death or last follow-up. Eleven of 14 (79%) with disease confined to the mucosa had pelvic control, whereas only 3 of 7 (43%) with extramucosal disease were controlled. No patient experienced major treatment-related toxicity. Tumor size, anatomic extent of pelvic recurrence, RT dose, and disease-free interval were examined for prognostic significance for pelvic control and survival by univariate analysis. Only anatomic extent of pelvic recurrence showed a nonstatistically significant trend as a predictor for control with P = 0.08. In conclusion, a significant proportion of patients with disease recurrence confined to the pelvis can be rendered disease-free long-term with maintained pelvic control. A reexamination of the role of routine adjuvant pelvic RT is therefore undertaken in light of these data. PMID- 8631536 TI - Radical-hysterectomy: personal reflections. PMID- 8631537 TI - Purification and characterization of hexosaminidase from human uterine cervical carcinoma. AB - Normal human uterine cervical tissue and uterine cervical carcinoma tissue were collected and subjected to fractionation of hexosaminidase isoenzymes Hex A, Hex B, and Hex I using DEAE-cellulose anion-exchange chromatography. Hex A was found to be the major isoenzyme in control tissues, whereas Hex B was the major isoenzyme in carcinoma tissues. These two major isoenzyme fractions were first purified using heparin-Sepharose 4B affinity chromatography and then subjected to further purification on epsilon-ACMA-Sepharose 4B. The purified isoenzymes were found to be homogeneous by polyacrylamide gel electrophoresis. The Hex A and Hex B isoenzymes obtained from control and carcinoma patients were characterized. Hex A from control and carcinoma patients exhibited more or less similar properties. However, the Hex B isoenzyme from carcinoma patients exhibited some characteristic variations in pH, temperature, substrate concentration optima, and isoelectric point compared with the control. From these observations it may be inferred that altered properties of the Hex B isoenzyme fraction from carcinoma patients may be the reason for the elevated activity of hexosaminidase in carcinoma of the uterine cervix. PMID- 8631538 TI - Preoperative evaluation of D-dimer and CA 125 levels in differentiating benign from malignant ovarian masses. AB - Plasma levels of D-dimer (DD) and CA 125 were measured preoperatively in 121 patients with ovarian masses submitted to laparotomy. DD and CA 125 levels were higher in the 56 patients with epithelial ovarian cancer than in the 65 patients with benign ovarian disease (P < 0.0001). The logistic regression procedure showed that both DD assay (cutoff 416 ng/ml) and CA 125 assay (cutoff 65 U/ml) were significant predictive variables for malignancy (P = 0.0001). The concordance between predicted probabilities and observed responses was 75.7% for DD, 72.1% for CA 125, and 90.8% for Dd and CA 125. It is worth noting that DD was increased ( > 416 ng/ml) in 73% of FIGO stage I patients, whereas CA 125 was above 65 U/ml in only 33.3%. Sensitivity, specificity, positive predictive value, and negative predictive value of the tests in differentiating benign from malignant ovarian masses were as follows: 76.8, 93.8, 91.5, and 82.4%, respectively, for CA 125; 94.6, 76.9, 77.9, and 94.3%, respectively, for the combination CA 125 or DD; and 73.2, 100.0, 100.0, and 81.3%, respectively, for the combination CA 125 and DD. The combination CA 125 and DD seems to be a useful diagnostic tool to differentiate benign from malignant ovarian masses. Elevated preoperative levels of both antigens are invariably associated with a postsurgical diagnosis of epithelial ovarian cancer. PMID- 8631539 TI - Novel phosphonium salts display in vitro and in vivo cytotoxic activity against human ovarian cancer cell lines. AB - Phosphonium salts are part of a class of lipophilic cationic molecules that accumulate preferentially in mitochondria and inhibit the growth of human and rodent carcinoma cells in vitro and in animal models. The delocalized cations tested previously such as dequalinium have exhibited considerable cross resistance against multiple drug-resistant cells expressing gp 170. In order to overcome this cross resistance, we have developed two novel phosphonium salts which contain haloalkyl moieties with potential protein alkylating capabilities. 3-Chloropropyltris(4-dimethylaminophenyl)phosphonium chloride (APPCL) and 3 iodopropyltris(4-dimethylaminophenyl)phosphonium iodide (APPI) are more lipophilic than other phosphonium salts described to date. By comparing the 50% inhibitory concentration (IC50) values for the A2780 human ovarian carcinoma parental line to a multiple drug-resistant variant (A2780-DR), the degree of cross resistance (IC50 for A2780-DR/IC50 for A2780 Parental) were found to be 494 for doxorubicin, but only 2.7 for APPCL. Similarly, the degree of cross resistance using a cisplatin-resistant variant (IC50 for A2780-CR/IC50 for A2780 Parental) was 30 for cisplatin, but only 2.2 for APPCL. APPCL is also active in vitro against UCI 101 (IC50 = 80 nM), an ovarian carcinoma line isolated from a patient who had failed chemotherapy with taxol, doxorubicin, and high-dose cisplatin. The cytotoxicity of APPI was comparable to that of APPCL with an IC50 ranging from 16.7 to 83.0 nM for a panel of seven cell lines. When administered intraperitoneally at a total dose of 46 mg/kg over 15 days, APPCL increased the median lifespan of nude mice bearing UCI 101, from a control value of 48.0 to 92.5 days (P < 0.0061). The median survival of the APPI-treated mice was 55 days. A total of 37.5% of the APPCL-treated group and 12.5% of the APPI-treated group were long-term survivors: sacrifice of these mice on Day 180 and subsequent histology showed no evidence of disease. Exposure to APPCL and APPI caused mitochondrial damage to UCI 101 cells at sublethal doses in vitro, as shown by morphological damage observed with transmission electron microscopy. APPCL appears to decrease the uptake of rhodamine 123 by mitochondria, suggesting that mitochondria may be significant targets or initial reservoirs for this agent. In conclusion, APPI and APPCL show promising anticancer activity against a variety of human ovarian carcinoma cell lines warranting further investigation. PMID- 8631540 TI - Concordance of DNA ploidy pattern as measured by flow cytometry in primary, metastatic, and persistent ovarian carcinoma. AB - In an attempt to evaluate the stability of DNA content in ovarian carcinoma, 66 tumor specimens from 25 patients with stage III disease were analyzed by flow cytometry. For all patients, both primary tumor and omental metastasis were available, and for 16 patients the persistent tumor found at second-look operation was also available. The concordance of the tumor DNA content in the primary tumor and in the corresponding omental metastasis was 72%; the concordance in persistent tumor at second-look operation was 63%. When diploid and aneuploid tumors with very low DNA index were considered together, the concordance of the DNA content in primary and metastatic tumors reached 84%. According to our data, if DNA ploidy is used to differentiate patients with a favorable prognosis (DNA diploid and DNA aneuploid with low DNA index) from those with an unfavorable prognosis (DNA aneuploid with high DNA index), a 16% risk of misclassification appears unacceptable in prospective studies. Hence, the assessment of two specimens (preferably the primary lesion and a metastatic site) to determine the most abnormal DNA ploidy result would reduce the risk of underclassification of tumor. PMID- 8631541 TI - Human papillomavirus infection in Hong Kong Chinese women with normal and abnormal cervix--detection by polymerase chain reaction method on cervical scrapes. AB - Detection of human papillomavirus (HPV) was accomplished by utilizing polymerase chain reaction methods on cervical scrapes from a total of 488 Hong Kong Chinese women attending the colposcopy, general, and antenatal clinics. From colposcopy clinic, 10% of normal cervix, 31% of condyloma, 56, 50, and 57% of CIN I, CIN II, and CIN III, respectively, and 88% of squamous cell carcinoma contain HPV. The overall detection rate is 30%. The most prevalent type is 16. Four percent of scrapes from normal cervix of patients attending general gynecology clinic and none from antenatal clinic show HPV. The detection rate appears to be more related to the types of cervical lesions than to the age of the patients. PMID- 8631542 TI - High-grade endometrial carcinoma in secretory endometrium in young women: a report of five cases. AB - Five cases of high-grade carcinoma occurring adjacent to secretory endometrium are described. One of the patients had the Lynch II syndrome, the others appeared to be sporadic. None of these cases appeared to be related to hyperestrogen. The presenting symptom in four was abnormal vaginal bleeding, and the other case was found incidentally in the course of investigations for infertility. There were delays in diagnosis due to the patients not seeking medical attention for abnormal bleeding, a reluctance of clinicians to perform dilatation and curettage, and difficulty in pathologic interpretation of the curettings. The interpretive problems related to extremely small volumes of tumor, difficulty in distinguishing carcinoma from menstrual endometrium, and difficulty in determining whether a carcinoma was endometrial rather or endocervical in origin. The tumors were aggressive, manifest by their high grade and stage. The importance of these cases is to impress upon clinicians and pathologists that endometrial carcinoma may occur in young women without any of the usual risk factors. PMID- 8631543 TI - Elevated concentrations of antiestrogen binding sites in membrane fractions of human ovarian tumors. AB - The density and affinity of tamoxifen (TAM) binding sites in isolated plasma membrane fractions from human ovarian tissue and ovarian tumors was measured. TAM binding in both membrane preparations was specific, saturable, and had a high affinity (KD < 1 nM). The density of TAM binding sites in the tumors was at least three times higher than that in the normal ovaries, whereas the affinity of TAM binding sites in the tumors was lower than in normal ovaries. The relatively large number of TAM binding sites that are distinct from estrogen receptors in ovarian tumors suggest that TAM alone and in combination with other chemotherapy could be used with advantage in ovarian cancer. PMID- 8631544 TI - Incidence and effect on survival of abdominal wall metastases at trocar or puncture sites following laparoscopy or paracentesis in women with ovarian cancer. AB - The aim of this retrospective study was to examine the incidence and prognostic significance of abdominal wall metastases in patients with ovarian cancer present at the primary debulking at the entry sites of previous laparoscopy or paracentesis. The clinical records of 219 patients were studied. In 7 of 43 patients (16%) who had undergone laparoscopy and 3 of 30 patients (10%) who had undergone paracentesis previous to the primary debulking, an abdominal wall metastasis had developed at the entry sites. All metastases occurred in patients with FIGO stage IIIC-IV including ascites. Survival analysis using the Cox proportional hazards model showed that after adjustment for age, FIGO stage, histology, grade, ascites, and residual disease after primary debulking, the presence of abdominal wall metastases in the entry sites of previous laparoscopy or paracentesis was negatively, although not statistical significantly, correlated with survival (P = 0.14). PMID- 8631545 TI - Histologic transformation of benign endometriosis to early epithelial ovarian cancer. AB - Between 1975 and 1990, 79 patients with Stage I epithelial ovarian cancer were treated at Massachusetts General Hospital. Patients were identified from the tumor registry and medical records were retrospectively reviewed. Pathological slides were evaluated for the presence of endometriosis, specifically looking for malignancy arising in endometriosis. Evidence of endometriosis was found in 22 of the 79 cases (28%). In the 23 cases of endometrioid histology, 9 cases (39%) were associated with endometriosis and, in the 17 cases of clear cell tumors, 7 (41%) were associated with endometriosis. All 8 cases of mixed histology had clear cell and/or endometrioid components and 4 cases (50%) were associated with endometriosis. Endometrioid adenocarcinoma accounted for 41% of the tumors associated with endometriosis, clear cell carcinoma 31%, mixed (endometrioid and/or clear cell types) 18%, and other types 9%. Among the 22 patients with associated endometriosis, we found 7 carcinomas (32%) arising in endometriosis. In these 7 cases a spectrum of benign and atypical endometriosis with a transition to clear cell or endometrioid adenocarcinoma were identified. These premalignant changes were characterized by cytologic atypia and architectural proliferation. Endometriosis was frequently encountered among patients with Stage I epithelial ovarian cancer of endometrioid and clear cell histologies. Endometriosis may play a role in the pathogenesis of some early stage malignant ovarian epithelial neoplasms. PMID- 8631547 TI - Paclitaxel is only a weak radiosensitizer of human cervical carcinoma cell lines. AB - Two human squamous cell cervical carcinoma cell lines, C-33A (HTB 31) and MS751 (HTB 34), were exposed to either paclitaxel alone or paclitaxel for 24 hr followed by graded doses of Cs-137 radiation. Each was then analyzed for both clonogenic survival and alterations to cell cycle progression. No radiosensitization or affect on the cell cycle was seen using 1 x 10(-9) M paclitaxel. Each line was equally sensitive to the drug with approximately 50% cell lethality seen after 1 x 10(-8) M of paclitaxel. This concentration of paclitaxel also produced substantial G2M arrest, seen immediately after drug exposure and lasting up to 2 days. Gamma radiation delivered during the time of G2M arrest showed only a small degree of radiosensitization by paclitaxel for the relatively radioresistant MS751 line at 4 Gy (SF4 = 16.0 +/- 3.2% --> 5.7 +/- 1.1%, P = 0.049) but no sensitization using radiation doses of conventional fraction size [sensitizer enhancement ratios 1.1 (0.80-1.40) and 1.3 (0.95-1.65) for the C-33A and MS751 cell lines, respectively]. It is concluded that paclitaxel produces only a modest radiosensitization effect, indicating that this compound will have limited benefit as a radiosensitizer for the treatment of cervical cancer. PMID- 8631546 TI - Growth suppression of a cervical cancer cell line (TMCC-1) by the human wild-type p53 gene. AB - To investigate the effects of human wild-type p53 expression on the proliferation of cervical carcinoma cells, a plasmid, pMO7-hp53, which contains a full-length cDNA of the human wild-type p53 (wt-p53) gene, was transfected into a cell line (TMCC-1) derived from an endocervical type, human papilloma virus-positive adenocarcinoma of the uterine cervix. The exogenous wt-p53 expression induced growth suppression, morphological changes, and loss of anchorage-independent growth of the tumor cells. As the wt-p53 gene apparently plays a negative role in growth regulation of cervical carcinoma cells, this gene may possibly be of some use for treating subjects with a cervical carcinoma. PMID- 8631548 TI - Immunodetection of squamous cell carcinoma-associated variant of nuclear protein. AB - A new monoclonal antibody, MAb C63.3, was developed by immunizing mice with the high molecular weight fraction of the cytoplasmic proteins from the cervical squamous cell carcinoma. In Western blotting, MAb C63.3 reacted with cytoplasmic and chromatin antigens expressed by cervical and vulvar squamous cell carcinomas and much weaker with normal cervical tissue and skin cells. Different molecular variants of C63.3 chromatin antigens were found in normal tissues (heavy variants, M(r) 65,000 and 59,000) compared to squamous cell carcinomas (light variant, M(r) 48,000). Diagnostic value of the cancer-associated M(r) 48,000 antigen is discussed. MAb C63.3 was internalized by cells and bound to the chromatin which suggests that antigen(s) C63.3 might represent a receptor for a yet unknown ligand (growth factor). It is suggested that the high molecular weight C63.3 antigens play a role in maintaining the nonmalignant phenotype. PMID- 8631549 TI - Beta-core fragment (beta-core/UGF/UGP), a tumor marker: a 7-year report. AB - In 1988 we published three papers describing immunoassay results for urine beta core fragment as a marker of gynecological cancers. Many other papers have been published since, and three commercial immunoassays have been established. beta Core fragment is called beta-core, UGF, or UGP by different commercial vendors. To avoid confusion we call it beta-core/UGF/UGP here. In this 7-year report, we compare the three commercial assays, establish cutoff limits, and use the Ciba Corning kit for two large studies. The first was a retrospective study, measuring beta-core/UGF/UGP in gynecological cancer and control urines accumulated in our freezers (n = 486). The second is a first prospective study, testing over a 16 month period beta-core/UGF/UGP levels in urines of all new patients attending the Gynecology Oncology Clinic (n = 548). In the retrospective study, elevated beta core/UGF/UGP levels ( > 1.9 ng/ml) were detected in 11% of urines from healthy individuals (n = 132), in 11% from women with benign gynecological disease (n = 196), in 44% from cervical cancer (n = 68), 56% from ovarian cancer (n = 54), and 47% from endometrial cancer (n = 38). Altogether, beta-core/UGF/UGP levels were elevated in 50% of 170 samples from gynecological cancers. Overall, sensitivity increased with advancing stage of malignancy. Sensitivity was 28% for stage I, 50% for stage II, 47% for stage III, and 68% for stage IV malignancies. In the prospective study very similar results were recorded. Elevated beta-core/UGF/UGP levels ( > 1.9 ng/ml) were detected in 11% of urines from healthy individuals (n = 99), 11% from individuals with benign gynecological disease (n = 196), 7% from women with carcinoma in situ (n = 28), in 42% of samples from cervical cancer (n = 69), 56% from ovarian cancer (n = 59), and 52% from endometrial cancer. Altogether, beta-core/UGF/UGP levels were elevated in 48% of 225 gynecological cancer samples. Overall, sensitivity increased with advancing stage of malignancy. Sensitivity was 29% for stage I, 66% for stage II, 60% for stage III, and 77% for stage IV malignancies. In both studies sensitivity for beta core/UGF/UGP increased with advancing stage of disease. Sensitivity for cervical and endometrial cancers was slightly lower than that for ovarian malignancies. This difference may be due to the preponderance of advanced-stage-disease patients in the ovarian cancer group. beta-Core/UGF/UGP may be a general stage dependent marker for all gynecological cancers. The same false-positive results and very similar sensitivity values were found in a retrospective and a prospective study. They confirm each other, and suggest a definitive false positive rate and sensitivity of this tumor marker for gynecological cancers. PMID- 8631550 TI - The 72-kDa metalloproteinase immunostaining in cervical carcinoma: relationship with lymph nodal involvement. AB - OBJECTIVE: In the present study we detected 72-kDa metalloproteinase expression in our series of early stage cervical carcinomas and analyzed the relationship between 72-kDa metalloproteinase staining and risk of nodal involvement with the goal of identifying a parameter useful in predicting the metastatic potential of lesions. MATERIALS AND METHODS: The medical records of 34 patients with FIGO stage I squamous cell cervical carcinoma who had undergone primary radical surgery with systematic pelvic and para-aortic lymphadenectomy (Piver's type III) at the Institute of Gynecologic and Obstetrics, Ancona University, between January 1988 and January 1993 were recruited from our series of 57 consecutive cases and reviewed. Any characteristic that could be relevant for prognosis was recorded in all of the cases: histologic grade of differentiation, tumor size, lymphatic spread, and adjuvant radiotherapy. Immunohistochemical staining was performed on serial sections of tumors using the avidin-biotin complex technique (Vector Laboratories, Burlingame, CA). The affinity-purified rabbit anti-72-kDa metalloproteinase antibody was used. Positive staining was expressed as a percentage of positive cells per 10(3) counted neoplastic cells (the 72-kDa metalloproteinase index). RESULTS: The tissue 72-kDa metalloproteinase immunoreactivity was diffusely expressed in all cervical carcinomas (ranging from 8.6 to 51.9%, with a median of 17.8%) and showed a significant relationship with respect to lymphatic spread. In the presence of lymph nodal involvement, the 72 kDa metalloproteinase index was significantly higher than in the absence of nodal metastasis (32.9 +/- 12.2% versus 18.1 +/- 9.0%, means +/- standard deviations with P = 0.001); a significant relationship was also observed between the 72-kDa metalloproteinase index and the number of positive nodes (r = 0.8, with P = 0.01). No significant relationship was defined with respect to the other prognostic parameters. The Cox proportional hazard analysis showed a significant relationship between the 72-kDa metalloproteinase index and disease-free survival (P < 0.0001) that was independent of tumor size, nodal involvement, and lymphvascular space invasion. CONCLUSIONS: Although the small numbers do not allow any definitive conclusion, a significant relationship between the 72-kDa metalloproteinase index and the risk of lymphatic spread is defined in early stage cervical carcinoma. The 72-kDa metalloproteinase immunostaining seems to have a prognostic value, suggesting the possibility of an association between neoplastic aggressiveness and 72-kDa metalloproteinase expression. PMID- 8631551 TI - Glutathione peroxidase activity in endometrium: effects of sex hormones and cancer. AB - The aim of this study was to determine whether glutathione peroxidase (GSH-Px) activity in endometrial tissue is regulated by sex hormones and to compare the GSH-Px activity of normal and cancerous endometrium. The localization of GSH-Px in human normal endometrium and endometrial cancer was determined immunohistochemically. GSH-Px activity was assayed in endometrial tissue obtained from women with endometrial cancer and age-matched controls, as well as in rat uterine tissue. GSH-Px immunoreactivity was localized in the glandular epithelium of normal human endometrium and reached a maximum in the late proliferative and early secretory phases of the menstrual cycle. In spayed rats, uterine GSH-Px activity was significantly increased by exogenous estrogen (P < 0.01) and significantly reduced by exogenous progesterone (P < 0.01). GSH-Px activity in endometrial cancer tissue was significantly higher (P < 0.01) than that in endometrial tissue from age-matched healthy controls. Among endometrial cancer tissues, a significant increase in GSH-Px activity was associated with well differentiated rather than moderately or poorly differentiated adenocarcinoma (P < 0.01), with slight rather than marked myometrial invasion (P < 0.01), and with the presence of concurrent endometrial hyperplasia (P < 0.01). These results show that endometrial GSH-Px activity is regulated by sex hormones, being stimulated by estrogen and suppressed by progesterone, and that the level of GSH-Px activity in endometrial cancer tissue may be a significant prognostic factor. PMID- 8631552 TI - Expression of epidermal growth factor receptor in carcinoma of the cervix. AB - Increased expression of the epidermal growth factor receptor (EGFR) gene has been shown in a large number of tumors, generally indicating a more aggressive biological behavior of cancers than those with low or normal expression. The role of EGFR in the tumorigenesis of the uterine cervix has been poorly understood and controversial. In order to explore the relationship between EGFR status and cervical carcinoma, tissues were analyzed from 40 patients, each of whom had invasive cervical carcinoma prior to treatment, 20 patients with cervical intraepithelial neoplasia (CIN) and 10 control cases who underwent hysterectomy due to benign gynecological disease at Yonsei University College of Medicine. We measured EGFR with an enzyme-linked immunosorbent assay which was a sandwich type using a mouse monoclonal capture antibody and a rabbit antiserum as detector. Patients with invasive cervical cancer were found to have significantly higher median EGFR expression than either the patients with CIN (P = 0.002) or the control (P = 0.001), respectively. However, there was no significant difference in EGFR status between CIN and the control groups. Overexpression of EGFR was found in 29 of 40 (72.5%) invasive cervical cancers and in 5 of 20 (25%) CIN patients. In invasive cervical cancer, no significant difference in EGFR levels was noted when stratified according to age, menopausal status, histological cell type, or clinical stage. With regard to tumor size, lesions of 4 cm and larger had significantly higher receptor levels than those lesions under 4 cm (P = 0.003). Even though quantitative EGFR status did not correlate with other prognostic parameters except tumor size, our results were consistent with the concept that EGFR may play an important role in malignant transformation and tumorigenesis in cervical cancer. PMID- 8631553 TI - Treatment of recurrent adenocarcinoma of the endometrium with pelvic exenteration. AB - Women with recurrent endometrial carcinoma are usually not considered candidates for pelvic exenteration. To assess the efficacy of this procedure, the records of all patients undergoing pelvic exenteration for adenocarcinoma of the endometrium at four institutions from 1955 through 1988 were reviewed. Of the 31 procedures performed, 7 were for primary therapy and 4 were judged to be palliative in nature and were excluded from analysis. Of the 20 patients with recurrent endometrial cancer who underwent exenteration with curative intent, all had previously received pelvic radiotherapy, 14 as part of their primary treatment and 6 as part of the treatment of recurrent disease. Six of 20 patients also received chemotherapy or hormonal therapy prior to exenteration. The median patient age was 65 years (range 44-79 years). At most recent follow-up, 8 patients were alive and disease free, 2 were alive with disease, 6 had died of disease, and 4 had died of other causes. The median follow-up of living patients is 89 months. Twelve of 20 patients experienced major complications, the most common of which was neovaginal flap necrosis. Of the 20 patients, 1 patient (5%) died in 1963 of surgical complications. The Kaplan-Meier estimate of 5-year disease-free survival is 45%. Pelvic exenteration can produce an acceptable rate of disease-free survival in highly selected patients with local recurrence of endometrial adenocarcinoma who have exhausted other treatment modalities. PMID- 8631554 TI - A single institution experience with weekly intramuscular methotrexate for nonmetastatic gestational trophoblastic disease. AB - The purpose of this study is to report our experience with weekly intramuscular methotrexate for nonmetastatic gestational trophoblastic disease. Treatment was begun at a dose of 40 mg/m2 and escalated weekly by 5-10 mg/m2, depending upon response and tolerance, to a maximum dose of 60 mg/m2. Remission was induced with weekly intramuscular methotrexate alone in 12 (60%) of 20 patients in 2-12 (median 8) weeks. The remaining 7 patients had a complete response to alternate chemotherapy. There were no major toxicities. Although the results of the present study are less favorable in terms of response, the overall published results using weekly intramuscular methotrexate for nonmetastatic gestational trophoblastic disease indicate that it is comparable in efficacy to other first line treatments while having the advantages of convenience, low cost, and low toxicity. PMID- 8631555 TI - Determination of the appropriate fraction number and size of the HDR brachytherapy for cervical cancer. AB - Based on the linear-quadratic model, we have made two isoeffect tables for transforming the traditional low dose rate (LDR) point A doses at 20, 30, 40, 50, 60, and 70 Gy to those of the high dose rate (HDR) dose per fraction. HDR fractions ranged from 1 to 12, with corresponding sizes for each fraction. We also propose the therapeutic gain ratio (TGR) method for determining the appropriate fraction number of HDR brachytherapy in cervical cancer. TGR is defined as addition of the calculated biological therapeutic difference with the supposed physical therapeutic difference of HDR brachytherapy. Through the TGR method, we predicted that after 2 to 3, 2 to 4, and 4 to 7 fractions of HDR treatments, the tumor control rate and complication rate would be equivalent to those of LDR point A doses of 30, 40, and 70 Gy, respectively. The TGR is affected by many factors, such as the equivalent total dose of LDR, dose rate of LDR, HDR fraction number, T1/2, and differences between LDR and HDR in the dose in critical organs. The TGR method might explain why a low fraction number of HDR can be used in clinical practice. We may use this principle to replace the traditional trial-and-error method for transcribing the relationship between LDR and HDR treatments. PMID- 8631557 TI - Loss of heterozygosity of chromosome 3p sequences is an infrequent event in endometrial cancer. AB - The genetic events associated with endometrial cancer are at present poorly understood. Frequent loss of heterozygosity (LOH) in a particular chromosomal region is often indicative of the involvement of a tumor suppressor gene. Previous studies are in disagreement over the involvement of a tumor suppressor gene(s) on the short arm of chromosome 3 in endometrial tumorigenesis. A set of 75 endometrial carcinomas was examined for the presence of LOH using 10 microsatellite repeat polymorphisms which are localized to chromosome 3p. In addition, these tumors were examined for the presence of replication errors (RER). Eleven of the 64 RER-negative tumors (17.2%) displayed LOH at one or more loci on chromosome 3p. The highest frequency of LOH at a single marker was 10.8% (4/37) at the locus D3S1312. The tumors investigated did not suggest that there was any common region of deletion. There was a significant increase in the frequency of 3p LOH in high-grade versus low-grade endometrioid adenocarcinomas (P < 0.05). Our results indicate that it is unlikely that a tumor suppressor gene on the short arm of chromosome 3 plays a major role in endometrial tumorigenesis. PMID- 8631556 TI - Estrogen receptor expression is a common feature of ovarian borderline tumors. AB - PURPOSE: The presence of estrogen receptor (ER) and its therapeutic significance in ovarian borderline tumors (OBT) have not been established. We recently observed a response to tamoxifen therapy given empirically to a patient with unresectable, recurrent serous borderline tumor (SBT). In view of this observation the present study was undertaken to assess ER expression in 51 cases of OBT. MATERIALS AND METHODS: ER expression was determined retrospectively, using an immunohistochemical method on formalin-fixed, paraffin-embedded specimens, from 35 cases of SBTs, 6 cases of mucinous mullerian (MMBT), and 10 cases of mucinous intestinal borderline tumors (MIBT). ER was considered positive if > 5% of tumor epithelial cell nuclei were immunostained. Both SBTs and mucinous borderline tumors (MBTs) were included to determine the influence of histologic type on ER expression. RESULTS: The patients ranged in age from 25 to 77 years (median 43 years for SBTs, 36 years for MMBTs, and 37 years for MIBTs). The stage distribution for the SBTs was stage I in 27 patients (77%), stage II in 4 patients (11.5%), and stage III in 4 patients (11.5%). All patients with MBTs were stage I. ER expression was observed in the majority of cases and correlated with histologic type: 94% (33/35) of SBTs and 100% (6/6) of MMBTs were ER positive compared to 0% (0/10) of MIBTs (P < 0.01). In the SBT category the presence of ER did not correlate significantly with stage or age. In addition, ER was positive in all four SBT implants (including one involved lymph node) and two recurrent SBTs analyzed. CONCLUSION: ER expression is a common feature of SBT and MMBT, but not MIBT. The relevance of ER expression in the pathogenesis and treatment of OBTs requires further investigation. PMID- 8631558 TI - Carcinomatous meningitis complicating cervical cancer: a clinicopathologic study and literature review. AB - Patients with gynecologic malignancies may develop metastases throughout the neuraxis. Cervix-related carcinomatous meningitis is a distinctly unusual clinical event with only two previous cases reported in the English medical literature. We review clinical, radiographic, and pathologic findings of a woman with advanced adenocarcinoma of the uterine cervix, whose course was complicated by leptomeningeal metastases. Carcinomatous meningitis occurs in the setting of rapidly advancing systemic disease and represents a terminal complication of cervical cancer. PMID- 8631559 TI - Coincidental renal cell and endometrial carcinoma: a case report. AB - A case of renal cell carcinoma and coexistent endometrial carcinoma is reported. The renal tumor in the lower pole of left kidney was detected by staging preoperative ultrasound scan and was confirmed to be a multilocular cystic renal cell carcinoma. There were two separate foci of moderately differentiated endometrial cancer in the endometrial cavity. Microscopic tumor deposits were also found in the left ovary and the right obturator pelvic lymph nodes. The multicentric involvement of endometrial cavity is uncommon and even rarer is the association with renal cell carcinoma. To the best of our knowledge, this is the first report of an association between renal cell carcinoma and endometrial cancer. This case demonstrates the importance of complete preoperative investigation before any definitive surgery for cancer. Without the abdominal ultrasound scanning, the asymptomatic renal cell carcinoma would probably have been undetected. PMID- 8631561 TI - Characteristics relating to ovarian cancer: implication for prevention and detection. PMID- 8631560 TI - Transitional cell carcinoma of the endometrium. AB - Only one case of endometrial transitional cell carcinoma (TCC) has been previously reported in the literature. We report a second case. In both the previously reported case and in this case there was a separate focus of TCC in the adnexa. Both patients were treated with adjuvant whole pelvic radiation and are alive and without evidence of disease at 5 years and 15 months, respectively. Study of more cases of endometrial TCC is needed to determine whether this type of endometrial carcinoma has a more favorable response to radiation therapy than other types of endometrial carcinoma with extrauterine spread. PMID- 8631562 TI - Invasive adenocarcinoma of the cervix following Lletz for adenocarcinoma in situ. PMID- 8631563 TI - Primary extrauterine mullerian adenosarcoma of the peritoneum. PMID- 8631564 TI - Iatrogenic atrial septal defect following balloon mitral valvuloplasty. PMID- 8631565 TI - Echocardiography in acute myocardial infarction: today and tomorrow. PMID- 8631566 TI - Serum troponin-T in acute myocardial infarction. AB - This study was carried out to assess the utility of serum troponin-T estimation in the diagnosis of acute myocardial infarction (AMI). One hundred and twenty two consecutive patients presenting with Q wave myocardial infarction were included in the study. In patients with AMI, serum troponin-T level was significantly elevated (4.21 +/- 3.49 ng/ml) on the first day of AMI (normal = 0.1 ng/ml). The elevated serum levels were detected even on the second (4.82 +/- 3.01 ng/ml) and the 3rd day (5.83 +/- 3.83 ng/ml) of AMI. Our results thus indicate that serum troponin-T is elevated twenty eight folds on the first day of AMI and the levels remain elevated as long as the third day. Hence, troponin-T can be used as sensitive biochemical marker of AMI, both in the immediate as well as in the early phase of myocardial infarction. PMID- 8631568 TI - Type and significance of fetal arrhythmias. AB - Fetal echocardiography was undertaken in 350 high risk pregnant females between 14 to 36 weeks of gestation at our institution. Significant fetal arrhythmias were diagnosed with the help of M-mode and pulsed Doppler echocardiography in 24 pregnant females. Three categories of arrhythmias were observed: (i) atrial or ventricular extrasystoles in 17, (ii) supraventricular tachycardia in 4, and (iii) congenital complete atrioventricular block in 3. The first category of arrhythmia was benign in 100 percent of cases with complete resolution before or just after birth. The second category was completely treatable with antiarrhythmic drugs given to the mother, thus preventing complications of heart failure and hydrops. In the third category, one fetus who had associated severe structural cardiac malformation, died in utero. The other two are doing well postnatally and are on medical follow up. Recognition of arrhythmias and appropriate prenatal treatment will prevent intrauterine death and also help avoid unnecessary operative or premature deliveries. PMID- 8631567 TI - Effect of atrial septal defect created during balloon mitral valvuloplasty on calculation of cardiac output and mitral valve area. AB - We studied the effect of atrial septal defect (ASD), produced during transseptal puncture, on estimation of cardiac output (CO) and mitral valve area (MVA), after successful balloon mitral valvuloplasty (BMV) using the Inoue balloon in 20 patients. Oximetry run, pressure gradients, thermodilution CO and MVA were measured initially while temporarily occluding the ASD by partially inflating the Inoue balloon catheter. Measurements were repeated after withdrawing the balloon catheter into the right atrium. Post-BMV cardiac output and MVA were similar in both the situations (4.52 +/- 1.37 L/min vs 4.50 +/- 1.19 L/min; 1.89 +/- 0.4 cm2 vs 1.93 +/- 0.38 cm2 respectively, p = ns). Only 2 patients showed a step up at atrial level on oximetry (9% and 16% respectively) but did not have significantly different CO or MVA, both with ASD occluded or otherwise. We conclude that the magnitude of ASD created during BMV by Inoue balloon technique is small and does not significantly affect the estimation of CO or MVA if the septal puncture is done in the fossa ovalis area. PMID- 8631570 TI - Radiofrequency ablation of idiopathic ventricular tachycardia. AB - Eleven patients (9 male, 2 female) with ventricular tachycardia (VT) and structurally normal heart underwent radiofrequency (RF) ablation of VT focus. The detailed electrophysiological mapping localized the VT focus in the left ventricular (LV) apicoseptal region in 5, LV posteroseptal region in 2, right ventricular (RV) outflow tract in 2 and RV midseptal and inflow regions in one patient each. The technique of endocardial activation mapping during VT, pacemapping and the presence of His-Purkinje potential (for LV focus) were used to identify the precise site of delivery of RF energy. The procedure was successful in 9 patients. There was no complication. The mean fluoroscopic time was 45 +/- 21 (range 20-120) minutes. RF ablation is effective and may be considered as therapy of choice for patients with ventricular tachycardia and normal heart. PMID- 8631569 TI - Percutaneous rotational atherectomy in peripheral vascular disease for lesions unsuitable for balloon angioplasty. AB - Percutaneous rotational atherectomy (Rotablator), a high speed (> 140,000 RPM) rotational burr was used to relieve 90-99 percent obstruction in 3 superficial femoral and 2 axillary arteries. These patients had severe claudication in respective extremities. In 4 patients, the lesion was considered to be unsuitable for balloon angioplasty and one patient underwent rotational atherectomy after failure to cross the lesion with balloon catheter. The burr size used ranged from 1.5 to 2.5 mm. After rotablation, the stenosis was reduced from 94.6 +/- 4.5 percent to 42 +/- 8.4 percent. The residual narrowing was further reduced by adjunctive balloon angioplasty to 14 +/- 5.5 percent (p < 0.001). Except for hemoglobinuria in one patient, there were no complications. All patients had good distal pulsations and were relieved of their claudication. On follow-up of 5-18 months, there has been no restenosis. Thus, our preliminary experience suggests that rotational atherectomy is safe and produces gratifying results in patients with peripheral vascular disease having lesions unsuitable for primary balloon angioplasty. PMID- 8631571 TI - Elective stenting for type B and C lesions: immediate results and follow-up angiographic restenosis. AB - From January to December 1995, 73 out of 174 patients with coronary artery disease underwent elective stenting for type B and C lesions. The age ranged from 35 to 73 years (mean +/- SD : 52.1 +/- 12.6) and the majority (91.7%) were males. Of the 74 vessels treated, the target vessel was LAD in 49 (66.4%), LCx in 13 (17.6%), RCA in 8 (10.8%) and SVG in 4 (5.2%). Based upon the ACC/AHA task force classification, 58 (79.5%) patients had type B1, 9 (12.3%) B2 and 6 (8.2%) had type C lesions. A total of 89 stents were deployed to treat 76 lesions with a range of 1 to 3 stents per lesion. A single stent was required for 67 lesions, 2 stents for 8 and 3 stents for 2 lesions. The stents used were Wiktor (29), Palmaz Schatz (26), Gianturco-Roubin (24), Microstent (6) and Freedom (4), depending upon the anatomical and morphological characteristics with the lesion. Using high pressure strategy, the stents were deployed successfully in all (100%) with a reduction in luminal diameter stenosis from 92 +/- 5.4 to -5 +/- 6 percent. There was no subacute stent thrombosis despite nonusability of oral anticoagulation in 95.9 percent patients. None had any major complication in the form of acute myocardial infarction, need for emergency bypass graft surgery or death. Minor complications were encountered in 9 (12.3%) patients. At a mean follow-up of 26 +/- 14 weeks, 74 percent of the patients were asymptomatic. Out of 31 patients who had completed 6 months after the procedure, repeat angiography was performed in 29 (93.5%) at a mean duration of 29 +/- 6 weeks. The angiographic restenosis was found in 6 (20.7%) patients. In conclusion, type B and C lesions can be treated successfully using elective stenting with excellent immediate results and clinical outcome. Angiographic restenosis, which develops in about one-fifth of patients, appears to be much lower than reported after balloon angioplasty for these complex lesions. PMID- 8631572 TI - Double outlet right ventricle with restrictive ventricular septal defect. PMID- 8631573 TI - Doppler, pulsus and electrical alternans following prosthetic valve thrombosis. PMID- 8631574 TI - Congenital partial absence of left pericardium: demonstration by computed tomography after artificial pneumothorax. PMID- 8631575 TI - Transient tuberculous constrictive pericarditis. PMID- 8631576 TI - AICD implantation and it's implications for the anaesthesiologist. PMID- 8631577 TI - Palliation by balloon atrial septostomy in total anomalous pulmonary venous connection with restrictive interatrial communication. PMID- 8631578 TI - Percutaneous coronary revascularization versus coronary bypass surgery in multivessel disease. PMID- 8631580 TI - Biology of tumor cell invasion: interplay of cell adhesion and matrix degradation. AB - Invasion of malignant cells requires altered cellular interaction with extracellular matrix. Integrin-type cell adhesion receptors play an important role in this process. Integrin-related cell biological phenomena explain cancer cell migration, and recent developments in the field have made it possible to propose that integrins are also involved in the penetration through basement membranes and other molecular barriers. Finally, malignant melanoma has been used here as an example to speculate on the function of each integrin in light of information from different experimental models. PMID- 8631579 TI - Unusual ECG tracing, unusual cause. PMID- 8631581 TI - Continuing increase in incidence of germ-cell testis cancer in young adults: experience from Connecticut, USA, 1935-1992. AB - The current study is designed to examine long-term trends by histologic types of testis cancer in Connecticut. A regression model was used to identify age, period, or cohort as determinants of the time-trend on histologic types of testis cancer. The results from this descriptive epidemiologic study show that the overall age-adjusted incidence rate of testis cancer has increased 3.5-fold in Connecticut during the past nearly 60 years of cancer registration. The rates for seminoma and non-seminoma have been increasing since the mid-1950s and increase in a similar manner for those aged 15 to 49. The largest increase was observed in the age groups 20 to 44 for seminoma and 15 to 34 for non-seminoma. The observed increase was limited to whites. The results from age-period-cohort modeling suggest that the observed increase in seminoma before 1950s could be largely attributable to a period effect, while the increase for cohorts born after about 1910 both for seminoma and for non-seminoma are mainly explained by a strong birth-cohort effect. Therefore, the observed increase in germ-cell testis cancer in this population is likely to continue in the coming years. Thus far, the proposed hypotheses, such as exposure to DES in utero, earlier lifetime exposure to viruses, trauma or unusual amounts of heat to the testis, cannot adequately explain the observed incidence patterns of testis cancer. Analytical epidemiologic studies with large sample size are urgently needed to examine the risk factors responsible for the increase. PMID- 8631582 TI - c-myc oncogene expression and cell proliferation in mixed oligo-astrocytoma. AB - Mixed gliomas (oligo-astrocytomas) are brain tumours with an admixture of 2 different cell populations: astrocytes and oligodendroglia. On the basis of histological features and behaviour, these tumours are classified as low-grade mixed gliomas (MG) and malignant mixed gliomas (MMG). We have studied the relationship between c-myc protein expression and cellular proliferation in this class of tumours. Using antibody c-33 for c-myc and PC-10 for the proliferating cell nuclear antigen (PCNA), immunohistochemistry was performed on 14 MG and 9 MMG. PCNA was increased in MMG as compared to MG in both astrocytic and oligodendroglial areas. However, more c-myc-positive cells were seen only in the astrocyte areas of MMG. Analysis of the relationship of c-myc and PCNA suggests that the correlation of c-myc with cellular proliferation is dependent on tissue type and differentiation status. PMID- 8631583 TI - Intragenic mutations of the p16(INK4), p15(INK4B) and p18 genes in primary non small-cell lung cancers. AB - The p15(INK4B), p16(INK4) and p18 genes are members of the gene family coding for inhibitors of cyclin-dependent kinases 4 and 6. p15(INK4B) and p16(INK4) are located at 9p21, a chromosomal region frequently deleted in many human neoplasms. To examine the role of these 3 genes in lung carcinogenesis, somatic mutations within the genes were analyzed by single-strand conformation polymorphism and DNA sequencing in 71 non-small-cell lung cancer (NSCLC) samples. Six somatic mutations in the p16(INK4) gene and 3 cases with a polymorphic allele were observed. Loss of heterozygosity in the p18 gene was found in 1 sample. We did not find any intragenic mutations in the p15(INK4B) or p18 genes. We conclude that p16(INK4) mutations play a role in the formation of some NSCLCs, whereas the involvement of p15(INK4B) and p18 is uncommon. PMID- 8631585 TI - HTLV-I and HTLV-II infections in subjects at risk for HIV-I infection from southeastern Italy (Apulia region). AB - To assess the prevalence of HTLV-I and HTLV-II infections in different groups at risk for HIV-I infection, a study on 867 subjects was carried out by means of serological and PCR analyses. Serum specimens were collected from 268 intravenous drug users (IVDU), 66 homosexual men, 248 subjects with sexually transmitted diseases (STD), 105 thalassemics and 180 hemophiliacs. Sera from 3 IVDU and a sample from an STD patient were confirmed as HTLV-II seropositive; a thalassemic patient was seropositive for HTLV-I; a homosexual man, though confirmed as HTLV I/II-seroreactive, could not be typed by serological methods. No hemophiliac was found to be HTLV-I/II-reactive. All 3 HTLV-II-seroreactive IVDU and the HTLV-I infected thalassemic were confirmed by PCR; an additional sample from an IVDU, indeterminate by Western blot, was confirmed to be positive for HTLV-II by PCR. Subtyping of HTLV-II samples indicated the presence of II/b subtype in all 4 cases. Up to now, the reservoir for HTLV-II infection in southeastern Italy is mainly represented by IVDU, while HTLV-I infection seems to be sporadic. PMID- 8631586 TI - Lung cancer mortality among males of Catalonia and Spain compared with other European countries between 1975-1977 and 1987-1989. AB - This study compares the lung cancer mortality rates among males in the years 1975 1977 and 1987-1989 in Catalonia and Spain with other European countries selected for their geographical proximity. Adjusted calculations using the direct method have been made for male lung cancer mortality. Adjusted truncated rates for the age groups 0-44, 45-64 and more than 65 years were also calculated, as well as percent differences between the mortality rates of each period studied. Lung cancer mortality rates for males in Catalonia and Spain show relative increments of 46.58% and 52.41%, respectively. In contrast, lung cancer mortality in countries such as England-Wales and Switzerland have decreased. Moreover, the 0 44 year age group in Catalonia and Spain shows the absolute highest rates in the 1987-1989 period among all the countries analyzed. The marked contrast of the lung cancer mortality rate evolution in Catalonia and Spain compared with some other European countries and the considerable increase of the mortality in younger age groups indicate the need to intensify lung cancer control measures. PMID- 8631584 TI - Biliary glycoprotein (BGP) expression on T cells and on a natural-killer-cell sub population. AB - Human T and natural-killer (NK) cells, that are thought to be the major cytotoxic effector-cell populations in the defence against neoplastic cells, were isolated from blood and decidua in order to analyze their expression of carcinoembronic antigen-(CEA)-family-member proteins. Biliary glycoprotein (BGP,CD66a) was the only member of the carcinoembryonic antigen family detected. While freshly isolated T-cells expressed low amounts of BGP, freshly isolated NK cells were negative. After in vitro stimulation for 3 days, T cells up-regulated their BGP expression and a sub-group of NK cells (CD16- CD56+), known to predominate in decidua revealed de novo expression of BGP. In contrast, stimulated CD16+ CD56+ NK cells, which occur exclusively in the blood, remained negative. The expression of BGP could be shown on the protein level by using a panel of 12 well-defined MAbs and on the transcription level in rt-PCR and subsequent oligonucleotide hybridization. Interestingly, rIL-2-stimulated T cells expressed 3-fold higher levels of BGP compared with those seen after stimulation with phytohemagglutinin (PHA). PHA, on the other hand, induced a higher expression of HLA-DR, an activation marker of T cells. The differential regulation implies a distinct function of BGP and HLA-DR. PMID- 8631587 TI - Comparative analyses of bone marrow micrometastases in breast and gastric cancer. AB - This study is a comparative analysis of the prevalence, absolute number and aggregation status of bone marrow micro-metastases (BMM) between breast (n=234) and gastric (n=102) cancer patients based on a standardized number of 1 X 10(6) bone marrow-derived cells per patient. Additionally, expression of the epithelial cell adhesion molecule E-cadherin was analyzed on disseminated tumor cells. A positive BMM status was demonstrated in 88/234 breast and 45/102 gastric cancer patients. The presence of CK18+ cells positively correlated with parameters of advanced tumor progression in breast, but not in gastric cancer. Interestingly, 25.2% of the node-negative patients already had micrometastatic cells in the bone marrow at diagnosis. Regarding the absolute number of CK18+ cells and the frequency of CK18+ cell clusters, no significant difference was found between the 2 tumor types. However, clusters consisting of more than 10 CK18+ cells (type II clusters) were present exclusively in breast cancer patients. Additionally, co expression of CK18 and E-cadherin was detectable in 15/21 micrometastases positive breast but in only 1/9 gastric cancer patients. While prevalence of micrometastatic cells in bone marrow is discussed as an early indicator for systemic disease, aggregation status and a certain antigen profile might be indicative for site-specific differences in the manifestation pattern of solid metastases. PMID- 8631588 TI - Deletion mapping of chromosome region 9p21-p22 surrounding the CDKN2 locus in melanoma. AB - The cyclin-dependent kinase-4 inhibitor gene CDKN2, localized at chromosome region 9p21, has been shown to be a familial melanoma gene, though we found that mutations of it are rare in uncultured sporadic melanomas. To determine Whether the region of allelic loss at 9p21 frequently observed in sporadic melanomas includes the CDKN2 locus, new polymorphic microsatellite probes were isolated from the genomic segments surrounding the CDKN2 gene and used for the study of loss of heterozygosity (LOH) in melanoma. The LOH study of matched uncultured tumor-constitutional DNA pairs from 66 metastatic cutaneous and 19 primary uveal melanomas showed that 63% and 32% of the respective tumors suffered allelic loss in the 9p21 region. Two regions of common losses which did not include the CDKN2 locus were observed: in a region of common loss near the D9S157 locus, telomeric to the CDKN2 locus, deletions were observed in 51% of informative cases; in the other region of common loss, near the D9S171 locus, centromeric to the CDKN2 locus, deletions were observed in 47% of informative cases. At the D9S974 locus, located within 20 kb of the CDKN2 gene, deletions were observed in 43% of informative cases. Homozygous deletions of the CDKN2 locus were observed in 8 cases of cutaneous melanoma and 2 cases of uveal melanoma; mutations in CDKN2 exon 2 were found in 2 of the 46 cases with allelic deletion in 9p21. Our results support the following conclusions: (i) somatic mutation of the CDKN2 gene is rare in sporadic melanomas with allelic loss at 9p21; (ii) homozygous loss is more frequent than mutation of the CDKN2 gene in sporadic melanomas; (iii) at 9p21-p23 genes other than CDKN2 may be involved in the development of sporadic melanomas. PMID- 8631589 TI - Can papilloma virus testing be used to improve cervical cancer screening? AB - This report investigates different options for using human papillomavirus (HPV) testing in cervical cancer prevention. These options are evaluated by a stochastic model of the progression of pre-malignancy and its relationship to HPV infection. Three screening policies are compared: 2 based on cytological screening, with or without HPV testing, and 1 in which HPV testing is the primary screening method. A policy of HPV testing for women with mildly abnormal smears would have little effect on the overall incidence of invasive cancer when compared with a policy of repeat cytology, provided follow-up is efficient. Moreover, the potential value of HPV testing as a primary screening method is strongly dependent on the proportion of neoplasias that are HPV-negative. Important factors in assessing the future role of HPV testing would be cost effectiveness and benefits from improved compliance. PMID- 8631590 TI - Maternal and perinatal factors associated with non-Hodgkin's lymphoma among children. AB - This nested case-control study based on 1.7 million live births in Sweden explores the associations between maternal and perinatal factors and the occurrence of childhood non-Hodgkin's lymphoma (NHL). The National Swedish Cancer Registry ascertained 168 cases in successive birth cohorts from 1973 through 1989 recorded in the Swedish Medical Birth Registry. From the nationwide Birth Registry, 5 controls without NHL and alive at the date the case was diagnosed were randomly selected from the pool of children, with each case matched by gender, birth year and birth month. Standardized information on selected maternal and perinatal factors up to one month after delivery were recorded in the Medical Birth Registry. Mothers of children with NHL were more likely than mothers of controls to have undergone Cesarean section [Odds ratio (OR) 1.6] and to have been exposed to paracervical anesthesia during delivery (OR 1.8). Children with NHL were more likely than controls to have endocrine-metabolic disorders (OR 3.3). This study is one of the largest focusing on the etiology of childhood NHL. Most of the maternal and perinatal characteristics studied did not markedly affect risk for childhood NHL, which may be due to maternal and perinatal factors not included in these data or to exposures later in life. PMID- 8631591 TI - Characterization of p53 gene mutations in basal-cell carcinomas: comparison between sun-exposed and less-exposed skin areas. AB - Mutations in the p53 gene in 32 basal-cell carcinomas (BCC) developed in Japanese patients were identified by the polymerase chain reaction and single-strand conformation polymorphism analysis, followed by sequencing the DNA. Among 16 BCC developed in continuously sun-exposed areas, 6 tumors showed 7 base substitutions, most of which were G:C to A:T transitions, mainly at the dipyrimidine sites. Seven out of 16 BCC developed in less-exposed areas showed 8 base substitutions, but the majority (75.0%) of them were transversions. These results suggest that the mutation in the p53 gene plays a significant role in the tumorigenesis of BCC developed in less-exposed areas as well as those in sun exposed areas in Japanese patients. There must be therefore causative factors other than UV irradiation for BCC in less-exposed areas. PMID- 8631592 TI - Neoplastic cells of Hodgkin's disease show differences in EBV expression between Kenya and Italy. AB - The Epstein-Barr Virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease (HD). However, the association of EBV with this disease varies greatly from series to series and from country to country. Epidemiological studies have shown differences in HD occurring in different parts of the world. In particular, it has been reported that HD in developing countries differs from HD in Western countries in terms of epidemiological, pathological and clinical characteristics. These discrepancies among populations suggest an interaction with environmental factors and a direct role of different etiological agents. At present, there are no data on the frequency of association of EBV with HD in equatorial Africa. In this study, a large series of HD cases have been collected at the University of Nairobi, Kenya, and at the Universities of Bologna and Siena, Italy. The cases have been reviewed and classified according to the REAL Classification and the presence of EBV has been assessed by in situ hybridization (ISH). A statistical difference in EBV expression was found between HD from Kenya and HD from Italy. EBV-positive neoplastic cells were detected in 92% of Kenyan cases, whereas only 48% of Italian cases showed EBER1/2 positivity in the neoplastic cells. Our results suggest that, in Kenya, EBV plays a more direct role in the pathogenesis of HD, as it does for endemic Burkitt lymphoma. PMID- 8631593 TI - Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis. AB - Studies aimed at elucidating the function of the protein synthesis factor eukaryotic initiation factor 4E (elF-4E) have demonstrated that overexpression of this protein results in marked cell phenotypic and proliferative changes, including neoplastic transformation of cells. These data suggest that elF-4E may somehow participate in the development and progression of tumors in vivo. In order to determine how elF-4E exerts its transforming effects, we examined vascular permeability factor (VPF) levels in cells transfected with an elF-4E vector. Cells overexpressing elF-4E showed an increase in intracellular, and an average 130-fold increase in secreted VPF protein levels (CHO 0.13+/-0.12 ng/ml; CHO-4E 20.5+/-12.5 ng/ml) over control cells. HUVEC growth induction revealed these VPF levels to be biologically active. Northern analysis revealed no difference in VPF transcript between the 2 cell lines. Polysome analysis showed that the VPF message in elF-4E-transfected cells was associated with the heavy polysomal regions, whereas the VPF message was associated with light polysomes in control cells. These data strongly suggest that enhanced VPF expression is achieved through translational regulation rather than transcriptional regulation in cells overexpressing elF-4E. This indicates that elF-4E-induced VPF expression may be an important factor in some forms of tumor angiogenesis and development. PMID- 8631594 TI - Differentiation of U-937 promonocytic cells with mitomycin C or cis diamminedichloroplatinum II. AB - Administration of 0.3 microM mitomycin C (MMC) or 2.0 microM cis diamminedichloroplatinum II (CDDP) decreased the growth activity and induced the differentiation of U-937 human promonocytic cells, as shown by nitroblue tetrazolium reduction and an increase in surface expression of the leukocyte integrins CD11b/CD18 and CD11c/CD18. Expression of these differentiation markers started to be significant at 48 hr of treatment. These concentrations resulted in little cell damage (determined by Trypan blue exclusion) and slightly induced apoptosis (determined by DNA degradation and changes in nuclear morphology). The treatments induced a transient increase in c-fos and c-jun mRNA levels, with maximum values at 1-6 hr; a transient increase in collagenase mRNA level, with a maximum value at 48 hr; and a progressive increase in vimentin and lamin A and C mRNA levels. These changes were qualitatively similar to those produced by 12-0 tetradecanoylphorbol 13-acetate. CDDP and MMC also caused a transient increase of total AP-1 binding activity, as determined by gel retardation assays. The drugs produced an early transient activation (3-6 hr) of membrane-bound protein kinase C, followed by a later activation (48 hr) of both the membrane and the cytosolic enzyme. These results suggest that protein kinase C and AP-1-dependent gene expression could be involved in myeloid cell differentiation by alkylating agents. PMID- 8631595 TI - Colon-cancer cell variants producing regressive tumors in syngeneic rats, unlike variants yielding progressive tumors, attach to interstitial collagens through integrin alpha2beta1. AB - Nine clones of tumor cells, derived from a single rat colon carcinoma, were analyzed for their adhesive properties and in vivo growth patterns. Four clones (denoted REG) gave rise to regressively growing tumors. Cells from the 4 REG clones attached significantly better to collagen types I and III than did cells from the 5 clones (denoted PRO) which grew progressively in vivo. In contrast, REG and PRO clones did not differ in their attachment to collagen type IV, laminin or fibronectin. The attachment of REG cells to collagen was dependent on Mg2+, but not Ca2+. Monospecific rabbit IgG to rat integrin beta 1-chain inhibited REG cell attachment to collagen, demonstrating involvement of a beta 1 integrin in this process. PRO and REG cells expressed an underglycosylated beta 1 chain (Mr approximately 105,000) that was somewhat smaller than beta 1-chains described previously on rat fibroblasts and hepatocytes (Mr approximately 115,000). Monoclonal IgG to rat integrin alpha 2 beta 1, but not to alpha 1 beta 1, readily inhibited REG cell attachment to collagen, demonstrating the involvement of integrin alpha 2 beta 1. However, beta 1 and alpha 2 integrin subunits were found in purified glycoproteins from both PRO and REG cells. This suggests that alpha 2 beta 1 integrin is expressed by both cell variants, but is functional as a collagen receptor on REG cells only. In this system of tumor-cell variants, the clear-cut differences in attachment to interstitial collagens of the 9 clones suggest a possible relationship between this attachment and the capacity to induce progressive or regressive tumors. PMID- 8631596 TI - Interleukin 1 (IL-1) production is not essential for acquired resistance of human A375 melanoma cells to anti-proliferative effect of IL-1. AB - The proliferation of human melanoma cell line A375-6 is inhibited by interleukin l (IL-l). However, the cells acquired resistance to IL-l after a long period of culture. We have reported that 2 resistant subclones, A375-R8 and -R19, produced IL-l alpha constitutively and that IL-l induced IL-6 production in an autocrine manner. Therefore, we supposed that IL-l alpha production renders the cells resistant to IL-l. To investigate the relationship between IL-l alpha production and IL-l resistance, we transfected the IL-l alpha expression plasmid to the IL-l sensitive clone, A375-6, and the anti-sense mRNA expression plasmid to IL-l resistant cells, A375-R8 and -R19. A375-6MS, a transfectant of mature IL-l alpha expression plasmid, expressed IL-l alpha mRNA and produced IL-l activity at a level comparable to the resistant cells. The transfectant also produced IL-6 and exhibited augmented expression of Mn-SOD mRNA. However, IL-l sensitivity of this transfectant was not affected. With respect to sensitivity to anti-proliferative effects of other cytokines, such as IL-6 and TNF alpha, there was no difference between the transfectant and parent cells. Although A375-R8PH10 and -R19PH10, transfectants of IL-l alpha anti-sense mRNA expression plasmid, exhibited a decrease in the level of IL-l production, their IL-l sensitivity did not differ from parent cells. These results, therefore, suggest that IL-l alpha production is not essential or sufficient for the acquisition of resistance to the anti proliferative effect of IL-l. PMID- 8631597 TI - Varying patterns of expression of insulin-like growth factors I and II and their receptors in murine mammary adenocarcinomas of different metastasizing ability. AB - We studied the expression of insulin-like growth factors I (IGF-I) and II (IGF II) and their receptors (IGF-R) in 2 related murine mammary adenocarcinoma in vivo lines, M3 and MM3, with different metastasizing ability. We further investigated the effects of IGFs on the secretion of a key enzyme in the metastatic cascade, the urokinase-type plasminogen activator (uPA) in M3 and MM3 cells. M3 is a spontaneous mammary tumor originated in BALB/c mice, with a 40% incidence of lung metastases. MM3 variant, obtained by successive s.c. implants of M3 lung metastases into syngeneic mice, shows a 95% incidence of lung metastases. Similar levels of expression of IGF-I protein were found in M3 and MM3 tumors, whereas IGF-II expression was 4-fold higher im MM3. RNAse protection assays showed similar levels of IGF-I mRNA in M3 and MM3 tumors and revealed a 4 fold increase in IGF-II transcripts in MM3 tumors compared with M3. Authentic IGF I and II messages were also found in primary cultures of M3 and MM3 cells. IGF-I mRNA levels were similar in both cultures and, as described for solid tumors a 5 fold increase in IGF-II message was detected in MM3 cells. The presence of type I and mannose-6-phosphate (Man-6P)/type II IGF-R was demonstrated in both M3 and MM3 tumors. A 2-fold increase of type I IGF-R was detected in MM3 tumors compared with M3. Man-6P/type II IGF-R levels were 2-fold lower in MM3 tumors than in M3. As observed in tumor membranes, type I IGF-R concentrations were higher and Man 6P/type II IGF-R lower in cultures of MM3 epithelial cells compared with MM3 cells. In addition, we found that IGF-I enhanced secreted uPA activity in both M3 and MM3 cells while IGF-II only stimulated uPA secretion in MM3 cells. PMID- 8631598 TI - Different mechanisms are responsible for c-jun mRNA induction by cisplatin and ultraviolet light. AB - Ultraviolet light (UV) and different DNA-damaging agents are known to induce AP-l transcription-factor activity. Whereas UV induction appears to be triggered by events at the cell membrane, the mechanism of AP-l activation by alkylating or platinating agents is not known. We have here examined the effect of cisplatin on AP-l activity in RPMI-8322 melanoma cells. Cisplatin was found to induce binding of nuclear proteins to TRE elements from the c-jun and collagenase-gene promoters, and was also found to induce activation of a c-jun-promoter reporter construct. Compared with stimulation by UV, cisplatin stimulation of c-jun promoter activity was found to be less sensitive to a dominant negative mutant of Raf-I protein kinase. Furthermore, whereas UV treatment resulted in strong MAP kinase activation, cisplatin treatment resulted only in a weak and transient increase. These data suggest that the Raf-MAPK pathway is of minor importance for the induction of c-jun-promoter activity by cisplatin. Finally, we report that cisplatin induction of c-jun in RPMI-8322 cells was blocked by herbimycin A, an inhibitor of Src-family tyrosine kinases. In contrast, UV induction of c-jun was not blocked by herbimycin A. In conclusion, our data strongly suggest that UV and cisplatin induction of c-jun mRNA in RPMI-8322 melanoma cells occur by distinct mechanisms. PMID- 8631599 TI - Effects of sandostatin, alone and in combination with surgical castration, on pancreatic carcinogenesis in rats and hamsters. AB - In a previous short-term study (4 months) we found that Sandostatin, when administered prophylactically, inhibited growth of putative pre-neoplastic ductular lesions induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine (BOP), but not of acinar lesions induced in rat pancreas by azaserine. The present long-term (12 months) study was carried out to investigate the effects of Sandostatin (3 microgram/day), alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and BOP-treated hamsters. In order to mimic therapy in humans, treatment of the animals started 4 months after the last injection with carcinogen, when (pre)neoplastic lesions had already developed. After treatment with Sandostatin for 8 months, rats developed fewer pancreatic atypical acinar cell nodules and tumours than those not treated with Sandostatin. Moreover, multiplicity of (pre)neoplastic acinar lesions was also lower in orchiectomized rats than in intact rats. However, Sandostatin treatment did not enhance the inhibitory effect of surgical castration on pancreatic carcinogenesis in rats. In hamsters that were both orchiectomized and treated with Sandostatin, the development of borderline lesions was significantly inhibited, whereas such an effect was not present in hamsters that were either surgically castrated or treated with Sandostatin alone. In Sandostatin-treated hamsters a significantly lower number of microcarcinomas was found than in hamsters not treated with Sandostatin. The present findings suggest that Sandostatin, particularly in combination with surgical castration, might be of therapeutic value for treatment of ductular pancreatic tumours. PMID- 8631600 TI - Functional role of sialyl Lewis X and fibronectin-derived RGDS peptide analogue on tumor-cell arrest in lungs followed by extravasation. AB - Our study demonstrates that synthetic sialyl Lewis X (SLex) as a ligand for selectins and fibronectin-derived RGDS peptide analogue [Ar(DRGDS)3] inhibits lung metastases produced by i.v. co-injection of B16-BL6 melanoma cells. To investigate the inhibitory mechanisms in a living animal, we performed positron emission tomography (PET) analysis after i.v. injection of [2-18F]2-fluoro-2v deoxy-D-glucose-labeled tumor cells with or without liposomal SLex or Ar(DRGDS)3. The real-time PET measurement for the first 120 min, started immediately after injection, showed that tumor-cell arrest, i.e., accumulation in the target organ (lung) was remarkably inhibited by liposomal SLex, but not inhibited by Ar(DRGDS)3 or liposomal Me-SLex, which is not recognized by selectins. In contrast, Ar(DRGDS)3 inhibited the invasion of B16-BL6 cells into reconstituted basement membrane (Matrigel) following tumor arrest, whereas SLex- or Me-SLex entrapped liposomes did not affect tumor invasion. In the metastatic processes containing tumor-cell lodgement and arrest in the target organ followed by extravasation (invasion), SLex resulted in the inhibition of initial arrest of tumor cells, presumably tumor-endothelium interaction, while Ar(DRGDS)3 achieved inhibition of tumor invasion into basement membrane at later steps of the cascade, consequently leading to inhibition of metastasis. Thus, tumor-cell arrest in lungs in the metastatic processes must be precisely and properly controlled by different adhesion molecules at different stages, which are similar to those observed in leukocyte-endothelium interaction. PMID- 8631601 TI - Growth arrest and suppression of tumorigenicity of bladder-carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9. AB - Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression. PMID- 8631602 TI - Anti-L-selectin monoclonal antibody treatment in mice enhances tumor growth by preventing CTL sensitization in peripheral lymph nodes draining the tumor area. AB - To examine the in vivo contribution of L-selectin in the sensitization of tumor specific CTL, we investigated the effects of treatment with the anti-L-selectin monoclonal antibody (MAb) MEL-14 on the immune response to Moloney-murine sarcoma virus (M-MSV)-induced tumors, which exhibit spontaneous regression following generation of a strong virus-specific CTL response. Daily systemic administration of MEL-14 for 10 days to M-MSV-injected mice gave rise to larger sarcomas that persisted for a longer time, compared with those arising in control mice injected with virus only. The enhanced tumor growth could not be attributed to cytotoxic activity on leukocytes by MEL-14 since no reduction in the total cell number was detected in peripheral blood and spleen of MAb-treated mice. Evaluation of the immunological response in MAb-treated animals revealed a strong reduction in the generation of virus-specific CTL precursors (CTLp) in tumor-draining peripheral lymph nodes (PLN) 10 and 15 days after M-MSV injection, while in spleen, where lymphocyte localization is independent of L-selectin expression, CTLp generation was only delayed. By day 20, when tumors had begun to regress, the CTLp number showed a marked increase in both spleen and local PLN, where naive recirculating CTL could now enter because L-selectin was no longer down-regulated or blocked by the injected MAb. Our findings indicate that functional inactivation of L selectin by MEL-14 treatment prevented migration of naive L-selectin+CTL through high endothelial venules (HEV) and their accumulation in PLN draining the tumor area, thereby precluding the initiation of a tumor-specific CTL response that takes place primarily at this site. PMID- 8631603 TI - Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line. AB - This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase alpha mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase beta. PMID- 8631604 TI - Overexpression of the proto-oncogene/translation factor 4E in breast-carcinoma cell lines. AB - The expression of the proto-oncogene, translation factor elF-4E, was examined in breast-cell lines: 5 carcinomas and 2 normal. At the protein level, elF-4E was 10 times higher in the carcinoma lines than in normal cells, which is comparable to the level found in breast-cancer biopsies. The elevation appears to be due to increased transcription, since the elF-4E mRNA was correspondingly increased. These results demonstrate that cells isolated from naturally occurring breast carcinomas maintain an elevated expression of the factor. Turnover rates for elF 4E (mRNA and protein) were determined for normal and cancer cells. We found that elF-4e mRNA is relatively stable during an 8-hr incubation with Actinomycin D, but the half-life of the protein is fairly short (approximately 4.5 hr). This suggests that, in proliferating cells, elF-4E may be turning over rapidly, possibly to fine-tune the changes in translation rates which occur during the cell cycle. PMID- 8631605 TI - Colonization of human lung grafts in SCID-hu mice by human colon carcinoma cells. AB - Human colon carcinoma cell lines were examined in a colonization assay using SCID hu mice engrafted with human fetal lung (HFL) tissues. Cell lines SW620 and COLO 320DM, derived from metastatic tumors, colonized HFL grafts after i.v. injection into SCID-hu mice. Cell lines SW480 and T34 initiated from primary colon tumors were unable to colonize HFL grafts. The ability to colonize HFL grafts but not mouse lungs of SCID-hu-L mice correctly reflects the clinical origin of these human colon carcinoma cell lines. Expression of a number of cell adhesion molecules was examined on SW480, SW620 and in vivo selected highly aggressive variants of SW620. NCAM and integrin alpha 3 expressed on the surface of SW480 cells were lost from metastatic cells, while carbohydrate ligands sialyl Lewis x and a, previously shown to be upregulated in metastatic colorectal tumors, were expressed at higher levels on colonizing cells. Unlike SW480, SW620 and its in vivo selected variants expressed RNA for calcium binding protein calbindin-D28K, a neuroendocrine marker. Acquisition of neuroendocrine features might be of potential importance in the development of the metastatic phenotype. PMID- 8631606 TI - Somatostatin analog RC-160 inhibits the growth of human osteosarcomas in nude mice. AB - We investigated the effects of the potent somatostatin analog RC-160 on the growth of human osteosarcoma cell lines SK-ES-1 and MNNG/HOS, transplanted into nude mice or cultured in vitro. Growth of SK-ES-1 and MNNG/HOS tumors in nude mice was significantly inhibited after 4 weeks of treatment with daily s.c. injections of 100 micrograms RC-160, as measured by a reduction in tumor volume and weight. Histologically, the number of mitotic cells was decreased in the groups treated with RC-160. In mice bearing either tumor model, administration of RC-160 significantly decreased serum growth hormone and insulin-like growth factor I (IGF-I) levels. Specific high-affinity receptors for somatostatin and epidermal growth factor were found on membranes of MNNG/HOS tumors but not on SK ES-1 tumors. Receptor analyses also demonstrated high-affinity binding sites for IGF-I on membranes of both tumors. In cell cultures, the proliferation rate of MNNG/HOS cells, but not of SK-ES-1, was significantly suppressed by RC-160. Our findings demonstrate that RC-160 can significantly inhibit the growth of SK-ES-1 and MNNG/HOS osteosarcomas in mice. PMID- 8631607 TI - Customized implant abutment copings to achieve biologic, mechanical, and esthetic objectives. AB - Fixed restorations for patients with implant-supported abutments frequently produce an unsatisfactory mechanical, biologic, or esthetic compromise. Pure titanium transmucosal abutments have biologic advantages, and factory-milled abutments for the comparable implant interface have mechanical advantages. The ultimate restorative objective is to satisfy the patient's desire for an esthetic restoration. This can generally be accomplished with precisely positioned implants; however, compromised locations need to be corrected with modified abutments. The use of customized copings fabricated by casting to pure titanium abutments can satisfy all three criteria, and may in some situations be the restorative procedure of choice. The clinical and laboratory procedures involved in achieving these criteria are described in detail. PMID- 8631608 TI - The measurement of molar furcation defect fill using digital computer technology- report on a new technique. AB - Recent advances in digital imaging technology have opened up new horizons for dental researchers. This study demonstrates the efficacy of a new technique for measuring regeneration in surgically created molar furcal defects. The investigators evaluated histologic material from a recently completed animal study using five adult baboons. In the animal study, surgically created molar furcation defects were treated using the principles of guided tissue regeneration. From the histologic data of one animal a computer calculated the volume of new bone, connective tissue, epithelium, and cementum as a percentage of the original defect size. The results of this study indicated that digital imaging technology is a useful research tool for determining the volume of defect fill in surgically treated Grade II molar furcation defects in the baboon animal model. PMID- 8631610 TI - Histologic evaluation of a sinus elevation procedure: a clinical report. AB - A histologic assay of the grafting material associated with the sinus elevation procedure would provide insight into the quality and quantity of vital bone at the implant-bone interface. This case report documented for the first time the sequential healing process of a sinus graft in the same patient at 4, 8, 12, and 20 months. Histology of trephine-obtained core samples showed that in a sinus grafted with a mixture of xenograft (80%) and autograft (20%), 12 to 20 months was required for remodeling to vital bone. The study also demonstrated that a significant amount of vital, mature bone was generated by this procedure. Quantification of the resulting bone and comparison with other grafting techniques should be the next phase of continuing research efforts. PMID- 8631609 TI - Is there an anterior loop of the inferior alveolar nerve? AB - The inferior alveolar nerve has an anterior ramification with two terminal branches, the mental nerve and the anterior plexus (or "incisor nerve"). The mental nerve is described to have a superior, lateral, and posterior course formed as a loop. The length of the loop is reported to be between 3 and 7 mm. In the present study, dissection of the ramification of the inferior alveolar neurovascular bundle was performed unilaterally in 58 patients. In 43 cases the loop was 1.0 mm long. In addition, the anatomy of the ramification with the mental and incisor canals frequently diverged in other respects from prevalent descriptions in the literature. PMID- 8631611 TI - Fixed bonded prosthodontics: a 10-year follow-up report. Part II. Clinical assessment. AB - The survival rates of resin-bonded fixed partial dentures, bonded splints, and bonded combination splints and fixed partial dentures (splints with pontics replacing one or two missing teeth) were determined. For statistical analysis, the splints and splint-prostheses were combined into one group. The average age of all devices combined was 5.7 years. Eleven of 145 resin-bonded fixed partial dentures failed; the survival rate was 83.0%. There was no statistically significant difference between the survival rates of prostheses, whether bonded to prepared teeth or unprepared teeth. Fifteen of 98 splints and splint prostheses failed. The survival rate of splints and splint-prostheses bonded to prepared teeth (71.6%) was higher than that of splints and splint-prostheses bonded to unprepared teeth (53.6%). To determine the continuing relevance of resin-bonded prostheses, the number of instances in which implant dentures could have been used was calculated. Implant treatment would have been possible in only 14 or 98 cases. PMID- 8631613 TI - The use of a slowly resorbable collagen barrier in the regeneration of bone in deep wide defects: a case report. AB - This paper presents a case in which a resorbable collagen membrane and a collagen based spacemaker and root surface conditioning were used in a guided tissue regeneration procedure. There was no need to disturb the healing potential of the underlying selected cells with an early reentry procedure. Clinical postoperative findings at the time of surgery and in an 18-month follow-up and the differences in density of the mineralized tissues, as detected by standardized series of juxtagingival radiographs and during surgical reentry, showed encouraging results in bone regeneration and soft tissue management. PMID- 8631612 TI - Stent marker materials for computerized tomograph-assisted implant planning. AB - This article analyzes different materials incorporated as markers in stents worn by patients undergoing a multiplanar reformatted computerized tomography (MRCT) study of the dental arch for implant placement planning. Forty-five patients were scanned with marked stents. The type of material used for stent markers was evaluated for visibility; ease of analysis in relation to the alveolar ridge crest and adjacent teeth, other markers, or restorations; and relative to its applicability as a presurgical guide. The advantages or disadvantages of each type of marker were discussed. The clinician should be aware of the variety of marker materials available and of the advantages and disadvantages associated with each to optimize the use of the multiplanar reformatted computerized tomography in implant placement planning. PMID- 8631614 TI - Superficial bone resorption: a negative or positive factor in wound healing? AB - A review of the literature indicates that one of the major elements of the healing pattern after periodontal surgery is the growth of granulation tissue originating from the periphery of the surgical wound, the periodontal ligament, and the underlying bone. In the past, the periosteum has been given great importance in the process of tissue regeneration; in the current study the authors examined the possible results of removal of the periosteum, which would leave bare bone as the only source of tissue regeneration. The review of a previous study involving perforations to the cortical plate to promote communication between marrow spaces and the receptor bed surface indicated the potential of bone regeneration through the endosteum. The purpose of this article was to stimulate studies devoted to a better understanding of bone potential in periodontal regeneration. PMID- 8631615 TI - Bone formation in the goat maxillary sinus induced by absorbable collagen sponge implants impregnated with recombinant human bone morphogenetic protein-2. AB - This study assessed the efficacy, safety, and technical feasibility of inducing bone formation in an animal model of maxillary sinus floor augmentation using recombinant human bone morphogenetic protein-2 (rhBMP-2) impregnated on an absorbable collagen sponge (ACS). Bilateral antral maxillary sinus floor elevation procedures were surgically performed in six adult female Alpine-Saanen goats. Bone formation in response to the implant was evaluated using sequential radiographs, computerized tomography, and gross pathologic and histologic analysis performed at necropsy. Computerized tomographic scans documented nonosseous radiopacity in both sinuses postimplantation. Sinuses implanted with the rhBMP-2/ACS subsequently demonstrated increasing radiopacity local to the implant site, while radiopacity of the negative control sinuses remained unchanged or decreased. The results demonstrated the ability of an rhBMP-2/ACS implant to induce substantive new bone formation within the maxillary sinus of goats without adverse sequelae. The rhBMP-2/ACS composite implant may represent an acceptable alternative to traditional bone grafts and bone substitutes for maxillary sinus floor augmentation procedures in humans. PMID- 8631616 TI - A new method for determining corneal epithelial barrier to fluorescein in humans. AB - PURPOSE: To derive the value of corneal epithelial barrier to fluorescein in humans from experiments in which the fluorophore in instilled in a single drop. METHOD: A commercial scanning fluorophotometer, the Fluorotron Master, was used to scan through an anterior segment. It could not resolve the tear film from the cornea, but in the early stages of measurement, the tear component predominated. After 20 minutes, the remaining fluorescein was washed out of conjunctival sac, and the amount that penetrated into the cornea was estimated. Because the resolution was not sufficient to estimate the concentrations, the total masses in the tear film and in the cornea derived from the area under the profile were used to calculate the epithelial permeability. A value for the tear film thickness had to be assumed. RESULTS: The technique led to reproducible values for epithelial permeability-a range of 2:1 in a subject. High concentrations of fluorescein were required to achieve a sufficient penetration into the cornea, and this led to error in estimating the tear film concentration of the dye. A method is presented that corrects for this effect. For 17 normal subjects, the corrected permeability was 0.15 nm/second, and the tear turnover was 0.15/minute ( +/- 0.016, standard error.) CONCLUSIONS: This technique is convenient and yields two useful physiological parameters from a single clinical procedure. The permeability values are considerably higher than those found by previous workers, and the source of the discrepancy is discussed. PMID- 8631617 TI - Apoptosis in the corneal epithelium. AB - PURPOSE: To determine the viability of corneal surface cells and to determine whether apoptosis is present in the corneal epithelium of the rabbit. METHODS: The viability of epithelial surface cells was examined using a calcein-ethidium assay. In this two-color fluorescence assay, viable cells fluoresce green, and the nuclei of nonviable cells fluoresce red. The number of nonviable cells on the normal corneal epithelial surface was quantified. Surface and shed cells also were examined from shear-stressed corneas. The TUNEL technique (TdT-mediated dUTP nick-end labeling) was used to detect DNA fragmentation characteristic of apoptotic cells. RESULTS: The calcein-ethidium viability assay revealed that the normal epithelial surface is composed mainly of viable cells, with nonviable cells making up a small percentage of the whole. There was an increase in the density of nonviable cells from the periphery of the epithelial surface to the center, In flatmounts of the cornea, TUNEL labeling demonstrated that a small percentage of surface cells exhibited DNA fragmentation, whereas cross-sections revealed that DNA-fragmented cells were found exclusively on the epithelial surface. Epithelial cells from shear-stressed corneas showed an increased number of apoptotic cells. CONCLUSIONS: The normal epithelial surface consists mainly of viable cells, with only a small percentage of nonviable cells and apoptotic cells. The results suggest that nonviable epithelial cells are shed after terminal differentiation, whereas viable cells can be shed by classical apoptosis with the formation of blebs. Thus, there appears to be more than one mechanism for removal of cells from the corneal surface. PMID- 8631618 TI - Direct noninvasive measurement of tear oxygen tension beneath gas-permeable contact lenses in rabbits. AB - PURPOSE: To develop a noninvasive measurement of tear oxygen tension and to determine tear oxygen tension and calculated oxygen flux rates during wear of contact lenses of varying oxygen transmissibility (Dk/L). METHODS: The basis of the tear oxygen tension (PO2) measurement is phosphorescence quenching of Pd-meso tetra-(4-carboxyphenyl) porphine by oxygen. A fiber-optic bundle, positioned approximately 2 mm from a cuvette or from the corneal surface, delivers an excitation flash (< 4 microseconds, 539 +/- 23 nm) and collects the phosphorescence emission (> 645 nm) of the dye at 1 MHz for 2 ms. Porphine (500 microM) + 1% albumin is instilled directly into the tears of a sedated rabbit, and phosphorescence is measured before and after contact lens insertion. RESULTS: Phosphorescence intensity decay lifetimes are related inversely to PO2 and follow the Stern-Volmer relationship. Calibration control experiments revealed a temperature effect on lifetime of 1% per degree, dye concentration independence over a 100-fold dilution, and no significant pH effect between 6.8 and 7.9. Phosphorescence lifetimes indicated that tear PO2 beneath contact lenses reached steady state within 3 to 8 minutes. Mean steady state tear PO2 (mm Hg +/- SE; N > 7) beneath contact lenses was: polymethylmethacrylate (Dk/L = 0), tear PO2 = 0.58 +/- 0.22; Oxyflow f30 (Dk/L = 15.3), tear PO2 = 27.7 +/- 3.4; Fluorex 700 Dk/L = 30), tear PO2 = 52.5 +/- 3.8; Oxyflow 151 (Dk/L = 51.9), tear PO2 = 78.7 +/- 23.9. Steady state tear PO2 beneath an Oxyflow f30 after application of topical anesthetic and 10 minutes of lens wear was 41 +/- 1.2 mm Hg, significantly higher than without anesthetic. CONCLUSIONS: Phosphorescence-based measurements allow the direct, sensitive, and noninvasive assessment of oxygen availability beneath a contact lens. Oxygen levels beneath permeable contact lenses found by direct measurement are significantly lower than what mathematical models predict. The measured values, however, are close to PO2 estimates using the equivalent oxygen percentage (EOP) technique. Tear PO2 measurements may be used to examine metabolic disturbances as shown by reduced oxygen consumption rates after application of topical anesthetic. PMID- 8631619 TI - Adreno-cholinergic modulation of junctional communications between the pigmented and nonpigmented layers of the ciliary body epithelium. AB - PURPOSE: Cell-to-cell communications between the epithelial layers of the ciliary body may be critical for aqueous humor production. The aim of this study was to identify pharmacologic agents that affect this path. METHODS: Whole New Zealand rabbit ciliary bodies were mounted in Ussing-type chambers with Ca2+(-)free and Ca2+(-)rich Tyrode's in the nonpigmented (NPE; aqueous) and pigmented (PE; serosa) epithelial side hemichambers, respectively. The NPE of the PE were then permeabilized, either selectively to monovalent ions with amphotericin B or nonselectively to small solutes with digitonin. Resultant active transport activities were tracked as short circuit currents (ISCS). RESULTS: Permeabilization of the NPE with either 10 microM amphotericin B or 10 micro M digitonin led to an aqueous-to-serosa-positive ISC. This ISC was inhibited by serosal-side ouabain and heptanol, indicating movement of Na+ from permeabilized NPE to the PE by the interlayer junctional path, followed by PE-to-serosa active Na+ transport. Permeabilization of the PE with amphotericin B elicited an ISC in the opposite direction, This ISC was abolished by aqueous-side ouabain and by heptanol, consistent with sequential PE to NPE Na+ translocation, followed by active, NPE-to-aqueous transport. Acetylcholine, epinephrine, norepinephrine, and the alpha 1-adrenergic agonist phenylephrine, but not brominidine, an alpha 2 adrenergic agonist, each caused an approximately 50% reduction of these currents. The inhibitions were fully dependent on serosal-side Ca2+ and were blocked by one calmodulin inhibitor, trifluoperazine, but not by another, calmidazolium. CONCLUSIONS: The above observations provide evidence that cholinergic or alpha 1 adrenergic activation of the PE causes Ca2+(-)dependent inhibition of the NPE-PE junctional path. A triflouperazine-sensitive entity, which may be distinct from calmodulin, is involved in the inhibition. PMID- 8631620 TI - Beta-adrenergic stimulation of Na+, K+, Cl- cotransport in fetal nonpigmented ciliary epithelial cells. AB - PURPOSE: The effects of adrenergic agonists and antagonists on Na+, K+, Cl- cotransport in fetal human nonpigmented ciliary epithelial (NPE) cells were investigated. METHODS: 86Rb+ as a marker for K+ was used to study ouabain insensitive, bumetanide-sensitive 86Rb+ uptake in cultured NPE monolayers. Cyclic adenosine monophosphate (cAMP) formation in NPE cells was determined by radioimmunoassay. RESULTS: 1 microM isoproterenol caused a 1.65-fold stimulation in Na+,K+,Cl cotransport measured as bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake. The half-maximal concentration for this effect was 6.4 nM, with maximal stimulation at 100 nM isoproterenol. Epinephrine stimulated Na+, K+, Cl- cotransport similarly to isoproterenol, whereas norepinephrine stimulated at much higher concentrations (half-maximal effective concentration = 1.4 microM). Stimulation of Na+, K+, Cl- cotransport by 1 microM isoproterenol was inhibited completely by the beta 2-adrenergic antagonist ICI-118,551 at 100 nM, with a half maximal inhibitory concentration of 5 nM. Neither atenolol, a beta 1-specific adrenergic antagonist, prazosin, an alpha 1-adrenergic antagonist, nor yohimbine, an alpha 2-specific antagonist, was as effective. These four antagonists inhibited isoproterenol-stimulated cAMP formation with potencies similar to those observed against stimulated Na+, K+, Cl- cotransport. The hypotensive adrenergic antagonist timolol, propranolol, and betaxolol also inhibited Na+, K+, Cl- cotransport stimulated by isoproterenol in the order timolol > propranolol > betaxolol. Na+, K+, Cl- cotransport could be maintained in a stimulated state for at least 2 hours in the presence of agonist, but activity returned to basal levels within 20 minutes of isoproterenol removal. Adrenergic stimulation of Na+, K+, Cl- cotransport was blocked 80% to 85% by 70 microM H-89, a protein kinase A inhibitor. CONCLUSIONS: These data suggest that beta 2-adrenergic receptor activation results in increased cAMP formation and sustained stimulation of Na+, K+, Cl- cotransport in fetal human NPE cells. Protein kinase A activation is required for maximal stimulation of Na+, K+, Cl- cotransport by adrenergic agonists. PMID- 8631621 TI - Endothelin-induced changes of second messengers in cultured human ciliary muscle cells. AB - PURPOSE: To characterize the pharmacology of endothelin-induced changes in phospholipase C (PLC) activity, intracellular calcium concentration ([Ca2+]i) and cyclic adenosine monophosphate (cAMP) in cultured human ciliary muscle (HCM) cells. METHODS: Changes in PLC activity of HCM cells were determined by production of [3H] inositol phosphates. [Ca2+]i was determined by single-cell dynamic fluorescence ratio imaging. Radioimmunoassays were used to determine cAMP and prostaglandin E2 (PGE2) concentrations. RESULTS: Endothelin-1 (ET-1) stimulated PLC (mean EC50 = 335 pM) and activated calcium mobilization in HCM cells. These effects were mediated by the endothelin ETA receptor subtype because at ETB receptor-selective agonist, sarafotoxin S6c, was ineffective. Additionally, effects of ET-1 were inhibited by pretreatment with a selective ETA agonist, BQ610 (mean pKi = 9.96 for PLC). ET-1 also stimulated the production of PGE2 (mean EC50 = 12.0 nM) and cAMP (mean EC50 = 5.2 nM) by these cells. PGE2 appeared to mediate the stimulatory effect of ET-1 on adenylyl cyclase because blockade of ET-1-induced PGE2 production by 10 microM indomethacin also completely blocked the ET-1-activated cAMP production. CONCLUSIONS: ET-1 stimulated PLC and increased [Ca2+]i in HCM calls by the ETA receptor subtype. ET 1 also increased cAMP production, an effect likely mediated by the enhanced production of prostaglandins. PMID- 8631622 TI - Receptor binding and biologic activity of synthetic ET-1 peptides in the retinal pericyte. AB - PURPOSE: The purpose of this study was to examine the effect of synthetic endothelin (ET)-1 peptides with antigenic potential for binding and biologic activity using an in vitro model of microvascular pericytes. METHODS: All possible sequential hexapeptide fragments of endothelins -1, -2, and -3 were synthesized on polyethylene rods and tested for reactivity with antibodies for ET 1 and ET-3. The most highly antigenic peptide ET-1[3-8] and the least antigenic ET-1[1-6], which gave low reactivity in the enzyme-linked immunosorbent assay (ELISA), were synthesized. The C-terminal hexapeptide ET-1[16-21], which has been reported as an ETB receptor agonist but which gave no reactivity in the ELISA, also was investigated. The synthesized ET analogues were tested for receptor binding, inositol phosphate generation, and mitogenesis in bovine retinal pericytes. RESULTS: ET-1[16-21] partially inhibited both [125I]-ET-3 binding at high concentrations, as did ET-1[1-6]. In contrast, ET-1[3-8] displaced labeled ET-1 binding but not labeled ET-3 binding. None of the peptides had any significant mitogenic or second-messenger responses when compared to those elicited by the full ET-1 molecule. CONCLUSIONS: The result of the current work shows that the sequence, ET[3-8], is involved in isopeptide-specific binding to ETA receptors, but it suggests that other regions of the molecule are necessary for full bioactivity in microvascular pericytes. PMID- 8631623 TI - Expression and downregulation of the GABAergic phenotype in explants of cultured rabbit retina. AB - PURPOSE: To study the morphologic and neurochemical development of the rabbit retina in explant culture. METHODS: Explants of retina from newborn rabbits were cultured in defined medium in the absence of serum or soluble growth factors. The morphology of the explant and the neurochemical development of the GABAergic system were examined by light microscopy, autoradiography, and immunohistochemistry for 7 days and compared to those of the postnatal rabbit retina in vivo. RESULTS: Cultured explants from newborn rabbit retina develop and maintain well-defined plexiform and cellular layers up to 7 days. Exogenous 3H gamma-aminobutyric acid (GABA) and antibodies against GABA labeled a population of horizontal, amacrine and displaced amacrine cells in the ganglion cell layer during the first 3 days in culture. After 4 days in culture, the extent of uptake and immunolabeling was diminished among all three cell types, but labeled horizontal cells were markedly rare. At 7 days in culture, uptake and GABA-like immunoreactivity could not be detected in horizontal cells, but antibodies to calbindin-D reacted with horizontal and amacrine cells in the appropriate retinal layers. Peanut agglutinin lectin binding studies revealed a mosaic of cone photoreceptor inner segments indistinguishable from that of neonatal retina in vivo. CONCLUSIONS: The experiments show that the maturation of cellular layers and the developmental expression of the GABAergic phenotype can be observed in retinal explants cultured under chemically defined conditions. Histochemical evidence is presented that indicates cultured explants of newborn rabbit retinas express markers of the GABAergic phenotype in a manner consistent with that observed in vivo. The authors show that horizontal cells continue to survive in culture after the diminution in GABA immunoreactivity. PMID- 8631624 TI - Temporal and spatial regulation of integrin vitronectin receptor mRNAs in the embryonic chick retina. AB - PURPOSE: To identify integrin vitronectin receptor subunit mRNAs in the developing avian retina and to track their expression. METHODS: Reverse transcription-polymerase chain reaction was used to identify integrin vitronectin receptor subunit mRNAs expressed in embryonic chick retina. cDNA clones encoding the beta 5 subunit were isolated and sequenced. Expression patterns of mRNAs encoding alpha v, beta 3, and beta 5 subunits were analyzed using northern analysis and in situ hybridization. RESULTS: Integrin beta 1, beta 3, and beta 5 subunit mRNA were identified in embryonic day 6 chick retina. The sequence of chicken beta 5 was 77% identical to that of human beta 5, and sequences with known signaling functions were highly conserved. Integrin alpha v, beta 3, and beta 5 mRNAs were expressed throughout the development of the embryonic retina, with the highest levels per retina observed around the embryonic day 9. In situ hybridization showed that both beta 3 and beta 5 were expressed throughout the developing retina, particularly in undifferentiated neuroepithelial precursor cells. At later times, beta 3 was expressed uniformly throughout the retina, whereas beta 5 expression was highest in a band throughout the central retina. CONCLUSIONS: The strong conservation of sequences with known signaling functions in chicken beta 5 suggests that it functions in a manner similar to human beta 5. Spatial expression patterns of vitronectin receptor subunits at early times of development point to a range of possible functions beyond axon outgrowth, including retinoblast proliferation, adhesion, and migration. PMID- 8631626 TI - Increased cell genesis in retinal pigment epithelium of perinatal vitiligo mutant mice. AB - PURPOSE: To compare cell proliferation in vitiligo and control mouse retinal pigment epithelium (RPE) perinatally. METHODS: C57BL/6J-mivit/mivit mice and congenic +/+ controls were injected once with bromodeoxyuridine 1 hour before they were killed between embryonic day 18 and postnatal day 8. Wholemounts of Carnoy-fixed posterior eyecups, minus lens and neural retina, were stained immunohistochemically to detect DNA synthesis (bromodeoxyuridine incorporation) and mitotic cells (R3 antibody binding). Cells were counted in carefully controlled sampling sites, and total RPE area and face-view cell areas were calculated. Retinal pigment epithelial cell heights were measured on light and electron micrographs. RESULTS: Total surface areas of the mutant and control RPE monolayer were similar (I.E., RPE wholemount area was normal), but cell number was approximately doubled in the mutant RPE. By postnatal day 6, mutant cells had approximately 70% the face-view area as controls, but their heights were increased approximately 80%, so that cell volumes were near normal despite the higher packing density. Regional differences in cell size in the control RPE were absent in the mutant specimens. The mutant RPE showed an increased bromodeoxyuridine labeling index, as well as an absolute increase in the number of cells engaged in DNA synthesis and in mitosis. CONCLUSIONS: Cell genesis in the vitiligo RPE is quantitatively abnormal perinatally, well before the neural retina has been recognized to display functional or morphologic defects. Cells are being generated at an abnormally high rate, so that twice the normal number of cells are packed into a RPE of normal total area. PMID- 8631625 TI - Pigment epithelial and retinal phenotypes in the vitiligo mivit, mutant mouse. AB - PURPOSE: To describe the abnormal phenotype in retinal pigment epithelium (RPE) and neural retina of vitiligo mutant mice from embryonic stages to old age. METHODS: Eyes of wild-type controls and congenic vitiligo mutants were examined by light and electron microscopy from embryonic day (E) 12 to 2 years of age. The amount and distribution of pigment in the RPE was studied in wholemounts. RESULTS: Earliest phenotypic expression of mivit is seen in the RPE, which is abnormally multilayered dorsally at E12 to E13, and contains both hyperpigmented and hypopigmented patches. Postnatally, most RPE cells have abnormally short, compact, apical microvilli not containing melanosomes and not interdigitating with rod outer segments (ROS). Rod outer segments begin to degenerate relatively late, at approximately postnatal day (P) 30, and fragments accumulate in the subretinal space; photoreceptor nuclei decrease in number progressively from approximately P60 to P500. Retinal detachment, more prominent than in most other retinal degenerations, begins as ROS break up. Additional unusual events are the appearance of macrophage-like cells in the subretinal space by P21 to P60 and extensive shedding of photoreceptor nuclei across the external limiting membrane and into the subretinal space from approximately P180 to P500. Photoreceptor cell degeneration follows a radial gradient, more severe centrally, and is more advanced superiorly than inferiorly. By 2 years, almost all rod and cone cells are gone, and the residual neural retina is invaded by heavily pigmented cells. CONCLUSIONS: The initial ocular target of the mivit gene is the RPE, which is abnormal for many weeks before photoreceptor cells differentiate and become demonstrably affected. The authors hypothesize that the slowly progressive photoreceptor cell degeneration is secondary to abnormal function of the RPE. This mutation serves to refocus attention on critical influences of the RPE on function and maintenance of photoreceptor cells. PMID- 8631627 TI - Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells. AB - PURPOSE: K+ channels in the retinal pigment epithelium (RPE) play a number of important roles, including the establishment of membrane potential, the transport of K+ between the subretinal space and choroid, and the generation of the c-wave of the electroretinogram. Previous studies on amphibian RPE demonstrated that these functions are likely served by an inwardly rectifying K+ channel. The aim of this study was to characterize inwardly rectifying K+ channels in cultured and freshly isolated adult human RPE (hRPE) cells. METHODS: Single cells were dispersed enzymatically from primary cultures of adult hRPE or from fresh adult hRPE-choroid. Ionic currents were recorded using either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: In 5 mM external K+, roughly 20% of cultured hRPE cells exhibited a strong inwardly rectifying K+ conductance that passed inward but little outward current. This conductance increased when [K+]o was increased and exhibited a voltage-dependent block by external Na+ at negative potentials. In contrast, all freshly isolated hRPE cells exhibited a mild inwardly rectifying K+ conductance that mediated substantial outward current at physiological voltages. This conductance decreased when [K+]o was increased and showed no voltage-dependent block by external Na+. CONCLUSION: The authors conclude that fresh hRPE cells express a mild inwardly rectifying K+ conductance. The operation of this conductance at physiological voltages makes it a likely candidate for the resting K+ conductances of the apical and basolateral membranes. Cultured hRPE cells express a functionally different channel type that may reflect a change in phenotype. PMID- 8631628 TI - Retinal blood flow changes in type I diabetes. A long-term follow-up study. AB - PURPOSE: The authors previously reported that blood speeds in the retinal arteries were significantly lower in patients with type I diabetes than in controls without diabetes. The purpose of this long-term, follow-up study was to characterize the natural course of changes in blood speed and blood flow in these patients. METHODS: Twenty-four patients were followed up with serial annual measurements of the blood flow in a temporal retinal artery using the bidirectional laser Doppler technique and monochromatic photography. The follow up period ranged from 2 to 6 years (mean, 3.8 years). Using standardized color fundus photography and fluorescein angiography, a retinopathy score was generated for each eye studied. Linear regression analysis was used to compute the slope of the change in retinal blood flow for each patient during the follow-up period. RESULTS: Retinal blood flow slopes were negative in 15 patients and positive in 9 patients. Multiple linear regression analysis showed that the retinal blood flow slopes were significantly related to the retinal blood flow measured at entry to the study and to the median duration of diabetes during the follow-up period (R2 = 0.56; P = 0.0002). There was a positive correlation between the retinal blood flow slopes and the median retinopathy score during the follow-up period (P = 0.47; P = 0.02). CONCLUSIONS: As duration of diabetes becomes longer and retinopathy becomes more severe, there is a transition from negative to positive retinal blood flow slopes. This bimodal relationship between the change in retinal blood flow and the duration of diabetes reflects the complex pathologic alterations that occur in the diabetic retina. PMID- 8631629 TI - Diabetic-like retinopathy ameliorated with the aldose reductase inhibitor WAY 121,509. AB - PURPOSE: To evaluate the efficacy of WAY-121,509, a potent new aldose reductase inhibitor (ARI), in preventing the retinopathy that develops in the galactose-fed rat model of diabetic ocular complications. METHODS: Sprague-Dawley rats were randomized into treatment and duration groups and fed diets with either 50% starch of 50% galactose with or without WAY-121,509 (25 mg/kg body weight per day). Progression of cataracts was monitored by slit-lamp biomicroscopy. After duration of 4, 8, 16, and 24 months, levels of plasma glucose and glycated hemoglobin, as well as erythrocyte and retinal galactose and galactitol, were measured in rats in each group. Retinal vasculatures of the 24-month rats were isolated by elastase digestion and analyzed by computer-assisted morphometry. RESULTS: Mature, diabetic-like cataracts developed within 5 weeks in all the galactose-fed, untreated rats, but only nonprogressive anterior cortical opacities were present in lenses of 85% of the ARI-treated galactosemic animals after 3 months. Plasma glucose remained the same in all groups. Erythrocyte and retinal galactose and glycated (galactosylated) hemoglobin were elevated with galactosemia and were unaffected by ARI treatment. Erythrocyte and retinal galactitol levels were decreased by 91% and 95%, respectively, with inhibitor treatment. At 24 months, capillary length, width, density, the number of microaneurysms, and the percent of capillary length involved in intraretinal microvascular abnormalities, expressed as hypercellular channels with diameters > 20 microns, were significantly increased by galactosemia and were attenuated in the galactose-fed, ARI-treated group. CONCLUSIONS: A dose of WAY-121,509 sufficient to reduce retinal polyol levels by 95% ameliorated the development of galactose-induced cataracts and diabetic-like retinopathy but was insufficient to prevent early lens opacifications or all the diabetic-like retinal microangiopathies. PMID- 8631630 TI - Depth-dependent forward light scattering by donor lenses. AB - PURPOSE: To determine quantitatively forward light scattering in the human lens as a function of depth. To use this to explain the psychophysical result, verified earlier in vitro, showing total light scattering in the human lens to decrease with scattering angle according to an approximate power law (power -2). METHODS: The amount of light scattered by donor lenses (n = 15; age range, 43 to 82 years) from a 1 mm x 0.1 mm white slit beam was measured as a function of depth in the lens for seven angles from 10 degrees to 165 degrees and for four wavelengths from 400 to 700 nm. Absolute values for light scattering (Rayleigh ratios) were derived. RESULTS: Light scattering of the total lens corresponded quantitatively to psychophysically determined in vivo stray light data. Powers were approximately -2.2. An important source of forward scattered light is located superficially at the anterior and the posterior poles. Nuclear forward light scattering varied over 2 log units, more or less in line with clinical assessment of nuclear opacity (LOCS III NO score). Nuclear powers were approximately -1.4. CONCLUSIONS: In vitro forward light scattering of donor lenses as a whole corresponded with in vivo data, but different depths in the lens contributed differently. Studies on functionally relevant light scattering by the human lens proteins should be conducted to explain true (in vivo) lenticular light scattering. PMID- 8631631 TI - Glutathione S-transferase M1 genotype and age-related cataracts. Lack of association in an Italian population. AB - PURPOSE: To investigate possible associations between the gene number and allelic forms of glutathione S-transferase M1 (GSTM1) and the occurrence of nucleic and cortical age-related cataracts. METHODS: Patients with cortical cataract, nuclear cataract, mixed and cortical cataract, and no cataract were sytematically selected from subjects evaluated in the Italian-American Study of the Natural History of Age-Related Cataract. The patients were typed for the A, B, and null alleles of GSTM1 using a variation of the amplification refractory mutation system. RESULTS: Forty-nine percent of patients (50/102) with cortical cataracts, 45% (13/29) with nuclear cataracts, 51% (36/71) with mixed nuclear and cortical cataracts, and 50% of controls (49/98) were homozygous for the null GSTM1 allele. Twenty-five percent of patients (26/102) with cortical cataracts, 24% (7/29) with nuclear cataracts, 31% with mixed nuclear and cortical cataracts, and 27% of controls (26/98) displayed only the A allele for GSTM1. Twenty-four percent of patients (24/102) with cortical cataract, 24% (7/29) with nuclear cataracts, 14% (10/71) with mixed nuclear and cortical cataract, and 18% of controls showed only the B allele for GSTM1. Two percent of patients (2/102) with cortical cataracts, 7% (2/29) with nuclear cataracts, 4% (3/71) with mixed nuclear and cortical cataracts, and 5% of controls (5/98) showed both A and B alleles for GSTM1. CONCLUSIONS: No associations between the GSTM1 alleles, including the null allele, and cataracts were detected in this study. PMID- 8631632 TI - Analysis of cytomegalovirus infection and replication in acinar epithelial cells of the rat lacrimal gland. AB - PURPOSE: The objectives of this investigation were threefold: to advance understanding of the nature and impact of herpesvirus infection in the lacrimal gland; to determine the influence of gender and sex hormones on viral infectivity and replication capacity in lacrimal tissue; and to compare the susceptibilities of lacrimal, submandibular, and parotid cells to viral invasion. METHODS: Acinar epithelial cells were isolated from lacrimal or salivary glands from intact, orchiectomized, ovariectomized, or sham-operated rats, cultured on Matrigel in serum-free media, and briefly exposed to rat cytomegalovirus (RCMV). Cells were then incubated for varying time intervals, and RCMV titers and secretory component (SC) levels in media or cell extracts were measured by plaque assay or radioimmunoassay. Exocrine glands also were obtained from rats after RCMV inoculation in vivo and were analyzed for viral infection. RESULTS: These findings demonstrated that RCMV invades the rat lacrimal gland after intravenous or intraperitoneal viral inoculation; RCMV infects and undergoes a time-, dose-, strain-, and gender-dependent replication in acinar epithelial cells from rat lacrimal tissue; the magnitude of RCMV replication in acinar epithelial cells in vitro may, in fact, induce an acute increase in the cellular production of SC; and the extent of viral replication in lacrimal and salivary gland epithelial cells displays distinct, tissue-specific variations. CONCLUSIONS: The herpesvirus RCMV invades and replicates in acinar epithelial cells from the rat lacrimal gland. The magnitude of the viral infection may be significantly influenced by gender and alterations in the hormonal environment. PMID- 8631633 TI - Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats. AB - PURPOSE: Inducible nitric oxide (NO) synthase (iNOS) has been implicated in the pathogenesis of endotoxin-induced uveitis (EIU). This study was undertaken to localize the cells, in the eye, which express iNOS during EIU in the rat. METHODS: EIU was induced in Lewis rats by a single foot pad injection of 150 micrograms lipopolysaccharide (LPS) from Salmonella typhimurium. At different time intervals after LPS injection, the authors evaluated ocular inflammation (slit lamp observation), iNOS localization by in situ hybridization, and comparison of OX-42- and ED1-positive cell appearance and of glial response by specific immunohistochemistry. RESULTS: iNOS mRNA was not detected in the iris ciliary body nor in the retina of control rats. It was detected strongly in the epithelial cells of the iris-ciliary body at 6 hours and also in stromal cells of the ciliary processes at 16 hours after LPS injection. In the neuroretina, iNOS mRNA was observed in the inner layers 16 hours after LPS injection. iNOS-positive cells were also present on the vitreous at this time. At 6 and approximately 16 hours after LPS injection, immunohistochemistry experiments revealed a large number of OX-42- and ED1-positive cells (microglia, macrophages, or polymorphonuclear leukocytes) colocalized in part with some iNOS-positive cells in the ciliary body and in the retina. Furthermore, expression of iNOS in Muller cells cannot be excluded. CONCLUSIONS: These observations confirm that subcutaneous injection of endotoxin dramatically induces NOS mRNA expression in the eye, and they demonstrate that epithelial cells of the iris-ciliary body and cells infiltrating the anterior segment of the eye and the retina are the major source of NO. These results support the hypothesis that both inflammatory and resident ocular cells are involved in iNOS expression during EIU. PMID- 8631634 TI - Intraocular pressure measurement in cynomolgus monkeys. Tono-Pen versus manometry. AB - PURPOSE: In living cynomolgus monkey eyes, the authors compared manometrically set and measured intraocular pressure (IOP) with simultaneous IOP readings obtained with the Tono-Pen (TP), a handheld applanation tonometer based on the Mackay-Marg principle. METHODS: In three pentobarbital-anesthetized cynomolgus monkeys, IOP was set and measured manometrically after anterior chamber cannulation through the peripheral cornea with a 26-gauge needle connected to a vertically adjustable reservoir and a pressure transducer. Intraocular pressure was raised in approximately 5 mm Hg steps from 5 mm Hg to 60 mm Hg and then lowered in 5 mm Hg steps to 5 mm Hg, with TP measurements taken at each increment and decrement in open and stopcock modes. RESULTS: Linear regression analysis of TP on manometric readings for grouped data from all six eyes, with each data point representing the average of all the TP readings from one eye at each manometric pressure setting, showed a slope 0.692 +/- 0.016 and 0.683 +/- 0.023 (both significantly different from 1; P < 0.001), intercept 1.21 +/- 0.60 and 1.64 +/- 0.82 mm Hg (both significantly different from 0.0, P < 0.05), and correlation coefficient 0.981 and 0.96 in open stopcock and closed stopcock mode, respectively. There were no striking differences when the data were analyzed for individual eyes or animals, for open versus closed stopcock manometry, or for increasing versus decreasing manometric IOP. CONCLUSIONS: The TP provides reproducible measurements of IOP in cynomolgus monkeys, with measurement accuracy dependent on the generation of an appropriate calibration curve. PMID- 8631635 TI - Dexamethasone-cyclodextrin-polymer co-complexes in aqueous eye drops. Aqueous humor pharmacokinetics in humans. AB - PURPOSE: To test an aqueous eye drop solution containing a high concentration of dexamethasone in a cyclodextrin-based drug delivery system. This system increases both drug solubility in aqueous eye drops and drug permeability into the eye, through drug cyclodextrin-polymer co-complexes. METHODS: 2-hydroxypropyl-beta cyclodextrin is a water-soluble oligosaccharide that can be used to dissolve lipophilic drugs, such as dexamethasone, in aqueous solutions. Co-complexation with a polymer further increases the solubility and increases drug permeability through biologic membranes. Eye drops containing dexamethasone (0.32% and 0.67%), 2-hydroxypropyl-beta-cyclodextrin, and polymer were given to patients before cataract surgery, and the resultant dexamethasone concentration was measured from aqueous humor samples. RESULTS: The dexamethasone-cyclodextrin drops give a significantly higher concentration of dexamethasone in aqueous humor than dexamethasone alcohol 0.1% (Maxidex). Heating of the dexamethasone-cyclodextrin polymer co-complexes appears to enhance the permeability of the drug into the eye. CONCLUSIONS: The cyclodextrin-based drug delivery system enhances both the solubility of dexamethasone in aqueous eye drops and the permeability of the drug into the human eye. Dexamethasone concentration levels in the human aqueous humor exceed those reported with currently available steroid eye drops. PMID- 8631636 TI - Human trabecular meshwork cell survival is dependent on perfusion rate. AB - PURPOSE: To determine whether suppression of flow may be detrimental to trabecular cell survival and to the morphologic characteristics of the trabecular meshwork. METHODS: The anterior segments of normal human eye bank eyes were placed in perfusion organ culture. The effect of various perfusion rates of culture medium, and of the constant flow and constant pressure methods of delivery of culture medium, were studied. Trabecular cell survival was determined by quantitation of cell nuclei in histologic sections and by morphologic observation. RESULTS: Trabecular meshworks with perfusion rates of 1 microliter/minute and higher had significantly more trabecular cells than meshworks with lower perfusion rates. A significant loss of trabecular cells was found in meshworks cultured with the constant pressure technique when compared with fellow eyes cultured with the constant flow of medium. Those constant pressure cultured meshworks with surviving cells had higher flow rates than those with necrotic cells. CONCLUSIONS: A minimum perfusion rate of 1 microliter/minute is required for trabecular cell survival in perfusion organ culture. Constant pressure perfusion of medium is unsuccessful in maintaining trabecular cells in long-term culture if low perfusion rates occur. Constant flow appears to mimic the in vivo situation more closely. PMID- 8631637 TI - Toxoplasmic retinochoroiditis in the hamster. PMID- 8631638 TI - Myopia. The nature versus nurture debate goes on. PMID- 8631639 TI - Measurement of intraocular pressure by telemetry in conscious, unrestrained rabbits. AB - PURPOSE: The aim of this study was to develop a system for the continuous recording of the intraocular pressure (IOP) in rabbits maintained in their normal environment. A telemetric system originally designed for the measurement of cardiovascular parameters in unrestrained, conscious laboratory animals was adapted for this purpose. METHODS: Experiments were performed in adult albino female rabbits. The transmitter was placed under the skin in the neck region. Its catheter was tunneled subcutaneously to the superior conjunctival sac and inserted into the midvitreous. Correlation with the IOP in the anterior chamber was performed by pneumatonography measurement and by manometric pressure perfusion in the implanted rabbits. Transitory increase in IOP was induced by a rapid intravenous injection of 20 ml/kg of 5% glucose. Timolol maleate (0.5%) or saline was administered (50 microliters) in the instrumented eye before the intravenous glucose injection. RESULTS: The intraocular catheter remained patent and was well tolerated for at least 2 months. A constant circadian rhythm of IOP was recorded as previously reported. Intraocular pressure measurements were highly correlated to pneumatonographic and to manometric measurements, indicating the accuracy and reliability of the recording system. A significant inhibition of the IOP increases following the intravenous injection of glucose was induced by 0.5% timolol treatment when compared to saline instillation. CONCLUSIONS: The continuous recording of IOP by our telemetric method represents a breakthrough for studying the effect of various pharmacologic agents in conscious, unrestrained rabbits under physiological conditions that have not been possible with previously described methods. PMID- 8631640 TI - Continuous measurement of intraocular pressure in rabbits by telemetry. AB - PURPOSE: Intraocular pressure (IOP) is dynamic and can be influenced by the use of tonometers. The authors developed a technique to implant a telemetric pressure transducer to measure IOP continuously in unrestrained rabbits. METHODS: A commercial telemetric pressure transducer was implanted subcutaneously on the dorsal neck, between the scapulae, of pigmented rabbits. A fluid-filled catheter that conducts pressure to the transducer was routed to the orbit subcutaneously and implanted in the anterior chamber through a limbal opening. The transducer measured pressure and broadcast this information by amplitude modulation radio to a receiver in the animal's cage. Data were recorded at a rate of 50 or 100 samples per second for 60 seconds to study transient changes in IOP to tonometry, or for 15 seconds every 2.5 minutes to study circadian behavior of IOP. RESULTS: Intraocular pressure was recorded from seven rabbits for 180 to 370 days. Within 3 to 8 days of implant surgery, IOP began to follow a circadian rhythm: IOP decreased after room lights were turned on at 00:00 CT and increased after they were turned off at 12:00 CT. (The term CT refers to circadian time and begins at 00:00 CT when lights are turned on.) The maximum difference in IOP between light and dark phases ranged from 6.4 mm Hg to 16.6 mm Hg. When the lighting cycle was advanced by 6 hours, the time of nocturnal rise in IOP also advanced, but it did so gradually over 10 to 14 days. CONCLUSIONS: The implanted pressure transducer provides a convenient, preinvasive method to measure and study IOP in unrestrained experimental animals. This technique will be used to study circadian variations in IOP, the effects of environmental stimuli, and the oculohypotensive effects of therapeutic agents. PMID- 8631641 TI - Lipopolysaccharide outer core is a ligand for corneal cell binding and ingestion of Pseudomonas aeruginosa. AB - PURPOSE: Pseudomonas aeruginosa has been observed to be adherent to and inside epithelial cells during experimental corneal infection. The authors identified bacterial ligands involved in adherence and entry of P. aeruginosa into corneal epithelial cells. METHODS: In vitro gentamicin survival assays were used to determine the intracellular survival of a panel of P. aeruginosa mutants. Strains (10(6) to 10(7) colony-forming units) were added to primary cultures of rabbit corneal epithelial cells (approximately 10(5)/well) for 3 hours, nonadherent bacteria were washed away, and extracellular bacteria were killed with gentamicin. The antibiotic was then washed away, and epithelial cells were lysed with 0.5% Triton X-100 to release internalized bacteria. Bacterial association (sum of bound and internalized bacteria) was measured by the omission of gentamicin. Similar assays were carried out with whole mouse eyes in situ. RESULTS: A lipopolysaccharide core with an exposed terminal glucose residue was found to be necessary for maximal association and entry of P. aeruginosa into corneal cells. Bacterial pili and flagella were not involved. Mutants of P. aeruginosa strains that do not produce an LPS core with a terminal glucose residue had a significantly lower level of association with (approximately 50%) and ingestion by ( > 90%, P < 0.01) corneal cells than did strains with this characteristic. Complementation of the LPS productions defect by plasmid-borne DNA returned association and ingestion to near parental levels. Lipopolysaccharides and delipidated oligosaccharides with a terminal glucose residue in the core inhibited bacterial association and entry into corneal cells. Experiments using P. aeruginosa LPS mutants and corneal cells on whole mouse eyes confirmed the role of the LPS core in cellular entry. CONCLUSIONS: Corneal epithelial cells bind and internalized P. aeruginosa by the exposed LPS core. PMID- 8631642 TI - Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Beta-chemokine synthesis in corneal cells. AB - PURPOSE: To determine whether human corneal epithelial cells and keratocytes produce chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) protein and monocyte chemotactic protein-1 (MCP-1) after exposure to proinflammatory cytokines interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either human recombinant IL-1 alpha or TNF-alpha. At selected time intervals after exposure, culture supernatants were removed and assayed for RANTES and MCP 1 by the enzyme-linked immunosorbent assay. Total RNA was extracted from the cell cultures, and steady state mRNA levels were measured by reverse transcriptase polymerase chain reaction. RESULTS: Exposure to keratocytes to either IL-1 alpha or TNF-alpha resulted in > 100-fold increases in RANTES protein secretion and > 150-fold increases in MCP-1 protein secretion, as well as in rapid sustained increases in intracellular levels of their corresponding mRNAs. Exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not stimulate MCP-1 secretion nor intracellular levels of MCP-1 mRNA. Epithelial cells also failed to secrete RANTES protein even through the two inducing cytokines did stimulate increased expression of RANTES mRNA. CONCLUSIONS: These results suggest that RANTES and MCP-1 gene expression in human keratocytes differs markedly from their expression in human corneal epithelial cells and that the stroma of the cornea may be more important than the epithelium in recruitment of mononuclear leukocytes responsible for cell-mediated immunity. PMID- 8631643 TI - Extracellular matrix alterations in human corneas with bullous keratopathy. AB - PURPOSE: To uncover abnormalities of extracellular matrix (ECM) distribution in human corneas with pseudophakic and aphakic bullous keratopathy (PBK/ABK). METHODS: Indirect immunofluorescence with antibodies to 27 ECM components was used on frozen sections of 14 normal and 20 PBK/ABK corneas. RESULTS: Fibrillar deposits of an antiadhesive glycoprotein tenascin in the anterior and posterior stroma, epithelial basement membrane (BM), bullae and subepithelial fibrosis (SEF) areas, and posterior collagenous layer (PCL) were revealed in disease corneas. Tenascin in midstroma, which was observed in some cases, correlated with decreased visual acuity. In normal central corneas, tenascin was never found. Other major ECM abnormalities in PBK/ABK corneas compared to normals included: discontinuous epithelial BM straining for laminin-1 (alpha 1 beta 1 gamma 1), entactin/nidogen and fibronectin; accumulation of fibronectin and alpha 1-alpha 2 type IV collagen on the endothelial face of the Descemet's membrane; and abnormal deposition of stromal ECM (tenascin, fibronectin, decorin, types I, III, V, VI, VIII, XII, XIV collagen) and BM components (type IV, collagen, perlecan, bamacan, laminin-1, entactin-nidogen, fibronectin) in SEF areas and in PCL. CONCLUSIONS: The study provides a molecular description of an ongoing fibrosis on the epithelial, stomal, and endothelial levels in PBK/ABK corneas. These fibrotic changes may follow initial endothelial damage after cataract surgery, may be caused by the upregulation of fibrogenic cytokines, and may play a significant role in the progression of bullous keratopathy. PMID- 8631644 TI - Comparison of clinical and laboratory parameters for systemic lupus erythematosus activity in Israelis versus Europeans. AB - The clinical activity of systemic lupus erythematosus (SLE) may be influenced by ethnic and environmental factors. The Consensus group on Activity Criteria for SLE attempted in a multicenter study to determine well-accepted criteria for disease activity. Of the 704 randomly assigned patients, 41 (5.8%) were from Israel. A detailed history including epidemiological, clinical and laboratory data was recorded. Significant differences were found between Israeli and European patients in the occurrence of Raynaud's phenomenon [12 (30%) vs. 358 (51%), respectively (P < 0.05)] and skin vasculitis [6 (15%) vs. 216 (34%), respectively (P < 0.05)]. In addition to those clinical differences, significant differences were also found in the occurrence of VDRL, low complement levels and lupus anticoagulant. Additional differences were found in some laboratory data, indicating differences in the sensitivity of the various laboratories. We conclude that the differences between the Israeli and European groups in the clinical data can be attributed mainly to environmental factors (weather, viruses), and in the laboratory data to ethnic differences (e.g., HLA) and to the different diagnostic methods used. PMID- 8631646 TI - The effects of in utero diagnostic X-irradiation on the development of preschool age children. AB - High doses of X-irradiation may affect the developing human embryo and fetus, causing brain, eye, skeletal and other defects. Although the doses used in diagnostic irradiation are not considered to be high enough to cause fetal anomalies, it is unknown whether they affect the long-term development of the in utero exposed children. Using the Bayley or McCarthy developmental scales we examined 52 children born to mothers exposed to diagnostic X-irradiation of the abdomen and/or pelvis during the first trimester of pregnancy, 60 children born to mothers exposed during pregnancy (58 in the first trimester) to X-rays in areas other than the abdomen or pelvis, and 62 normal children without maternal exposure to X-rays during pregnancy. All children were aged 1-5 years at examination. Parental socioeconomic status was the same in all three groups. There were no differences in the results of the medical and neurological examination, and in the motor or congnitive scores among three groups. Embryonic or fetal exposure to X-irradiation in doses below 5 rads does not seem to have any effect on development during childhood. PMID- 8631645 TI - Sodium-lithium countertransport: a predictor of diabetic nephropathy in Jewish insulin-dependent diabetes mellitus patients of different ethnic origin? AB - Diabetic nephropathy in Jewish insulin-dependent diabetes mellitus (IDDM) patients has been found to correlate to their ethnic origin. It has also been found that increased sodium-lithium countertransport (SLC) in erythrocytes, as a genetic marker for essential hypertension, may identify those patients at risk for diabetic nephropathy. The purpose of this study was to investigate a possible correlation between this genetic marker and the ethnic origin of Jewish IDDM patients and their parents and the risk for developing diabetic nephropathy. Although SLC was slightly increased in IDDM patients with microalbuminuria, SLC was not correlated with the existence of diabetic nephropathy nor with the ethnic origin and blood pressure of these Jewish IDDM patients. Thus, other genetic factors may play a role in the different prevalence of diabetic nephropathy in Jewish IDDM patients of different ethnic origin. PMID- 8631648 TI - Osteoarthritis, the hazard of living longer: arthroscopy vs. conservative treatment. AB - The lengthening of the average life span has led to an increase in degenerative diseases, among them osteoarthritis. Arthroscopy, which has been used extensively to decrease pain and inflammation and increase the range of motion, has recently been challenged as the treatment of choice. Several studies have found that joint lavage, which is a less costly procedure, provides better solutions to joint disorders. In Israel, where resources for health care are limited, physicians must also be guided by cost-benefit and cost-effectiveness. Since there is limited benefit to be gained from arthroscopy in cases where there is no mechanical damage to the knee, we suggest that nonsurgical, conservative methods be employed in treating the majority of cases of osteoarthritis. PMID- 8631647 TI - Delivery of the very low birthweight breech: what is the best way for the baby? AB - A retrospective analysis was done to determine whether vaginally vs. cesarean section-born breech infants in the very low birthweight range are at increased risk for morbidity and mortality. Eighty-three viable singleton breech infants weighing 700-1,600 g, who were delivered in our medical center during the period 1980 through 1993, were followed for up to 5 years of age. Analysis of data after correction for weight, gestational age and other confounding variables such as antepartum complications revealed that survival rates in the 700-1,000 g group were similar in both routes of delivery. In the 1,001-1,600 g group, survival rate was 60.9% after vaginal delivery, as compared to 100% after cesarean section (P < 0.01). No difference was demonstrated in long-term outcome between the vaginal and cesarean groups, although the population was too small to draw statistical conclusions. Considering the limitation of such a retrospective analysis, our data identified a trend that supports prophylactic cesarean section in cases of pre-term breech infants weighing 1,000-1,600 g. PMID- 8631649 TI - Hexane cardiotoxicity--an experimental study. AB - Ventricular arrhythmia has been postulated as a possible cause of death in young black children who abuse volatile substances, primarily benzine and certain glues that contain n-hexane. A series of protocols were designed to determine the effect of n-hexane on myocardial function and morphology in male laboratory rats. In the first protocol, experiments were designed to study the effect of n-hexane in initiating ventricular fibrillation and in modifying myocardial magnesium and potassium levels. The results showed that the thresholds for ventricular fibrillation and myocardial magnesium and potassium levels were reduced compared to control values. In the second protocol, n-hexane-treated rats were supplemented with intragastric administration of magnesium and potassium salts. The outcome of the experiments indicated that although the myocardial magnesium and potassium levels were corrected, the threshold for ventricular fibrillation remained low compared to control values. In the third protocol, experiments were designed to examine myocardial morphology by electron microscopy. Cellular changes were observed in the myocardium as a result of administering n-hexane. These cellular changes were considered to be responsible for the decreased threshold for ventricular fibrillation. The present study indicates that n hexane, a constituent of benzine and certain glues, is cardiotoxic. PMID- 8631650 TI - The effect of cyclophosphamide on rat blastocysts in vitro and their subsequent development following transfer to pseudopregnant rats. AB - These investigations were undertaken to assess the effects of cyclophosphamide (CPA) on rat blastocysts. In the first set of experiments, blastocysts were exposed to 100 micrograms/ml CPA in culture for 48 h. In the second experiment, blastocysts were cultured for 48 h following injection of 20 or 40 mg/kg of CPA to pregnant rats on day 4 of gestation. In the third experiment, blastocysts were cultured for 2 h in the presence of 100 micrograms/ml of CPA and then transferred to pseudopregnant rats. The results from the first experiment revealed a clear toxic effect of CPA on blastocysts following culture. A similar effect occurred when blastocysts were harvested and cultured following in vivo injections of CPA. In the third experiment a clear embryotoxic effect (larger number of resorptions and retarded embryos in comparison to control) was found on day 13 of gestation. The origin of activation of CPA by the blastocysts could not be ascertained from the above results. PMID- 8631651 TI - Bicycle helmets in Israel: observed change in usage following a nationwide campaign. AB - Bicycle-related head injuries are an important cause of injury and death among bicycle riders. The use of bicycle helmets could reduce the rate of serious head trauma among bicyclists involved in accidents. A nationwide survey was conducted in Israel to determine the usage of such helmets. This survey preceded a media campaign encouraging the use of bicycle helmets. A second survey compared the rates of helmet usage following the media campaign with those rates prior to the campaign. A modest but significant increase in the use of bicycle helmets was observed. In order to further increase this rate, additional educational campaigns are needed and possibly the enactment of legislation. PMID- 8631652 TI - Sudden death in the Gulf War. PMID- 8631653 TI - Public health in Jewish lore Medicus Judaicus: the physician in Jewish lore rights and duties. PMID- 8631654 TI - Jacques L. Monod, Francois J. Jacob and Andre M. Lwoff--introducers of new dimensions in cellular genetics and molecular biology. PMID- 8631655 TI - Ovulation induction in the era of recombinant gonadotropins and GnRH analogues. PMID- 8631656 TI - Gene replacement strategies for cancer. AB - The strategy for the inactivation or replacement of cancer-causing genes is analogous to the classic concept of gene therapy for replacement of defective or nonfunctioning genes. The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes. Regional administration of viral vectors expressing wildtype p53 and antisense K-ras prevents growth for tumors with the specific genetic lesions in orthotopic tumor models and mediates regression of large established tumors. These studies provide a rationale for a new clinical protocol recently approved by the United States National Institutes of Health (NIH) Recombinant DNA Advisory Committee and the Federal Drug Administration (FDA) to replace a defective p53 gene with intratumor injection of recombinant retrovirus expressing normal p53 and to inactivate mutant K-ras by expression of antisense K-ras mRNA. If these agents are efficacious, their lack of toxicity may provide a sufficiently high therapeutic index such that they could be used as an adjuvant to surgery to treat patients with earlier stages of cancer or as prevention for second primary cancers for individuals with genetic abnormalities in premalignant lesions. Although much research needs to be done, the possibility of specific gene targeting with a high therapeutic index makes this a promising area for investigation. PMID- 8631657 TI - Sudden death among the Israeli civilian population during the Gulf War--incidence and mechanisms. AB - The Gulf War in January 1991 provided a unique opportunity to evaluate the influence of acute stress on the incidence of sudden cardiac death (SCD) in the Israeli civilian population. Pursuing this intriguing issue seemed warranted as we documented and reported a pronounced increase in the incidence of acute myocardial infarctions during that period. The purpose of the present study therefore was to document the incidence of SCD in different regions of Israel during a 10 day period preceding the Gulf War and a similar period following its onset, and to try to identify a possible correlation between the intensity of threat and the incidence of SCD. Sixty-eight cases during the Gulf War study period were compared to 213 cases in 5 control periods; overall 281 cases of SCD were documented. A rise in the incidence of SCD during the first 10 days of the war as compared to previous periods was noted but did not reach statistical significance. No correlation was demonstrated for SCD incidence among different regions in relation to the intensity of threat. Mechanisms by which acute stress precipitates an acute coronary syndrome are discussed, and an explanation for the lack of a statistically significant difference in SCD incidence is offered. PMID- 8631658 TI - Isolation of monoclonal antibodies reacting with the core component of lipopolysaccharide from Rhizobium leguminosarum strain 3841 and mutant derivatives. AB - Monoclonal antibodies reacting with the core oligosaccharide or lipid A component of Rhizobium lipopolysaccharide (LPS) could be useful for the elucidation of the structure and biosynthesis of this group of macromolecules. Mutant derivatives of Rhizobium leguminosarum 3841 with LPS structures lacking the major O-antigen moiety were used as immunogens, and eight antibodies were selected for further study. All the antibodies reacted with the fast-migrating species known as LPS-2 following gel electrophoresis of Rhizobium cell extracts. For four of these antibodies, reactivity with affinity-purified LPS was lost after mild acid hydrolysis, indicating that they probably recognized the core oligosaccharide component. The four other antibodies still reacted with acid-treated LPS and may recognize the lipid A moiety, which is stable to mild acid hydrolysis. The pattern of antibody staining after gel electrophoresis revealed differences in LPS-2 epitope structure between each of the mutants and the wild type. Furthermore, for each of the mutants the antibodies crossreacted with a minor band that migrated more slowly than LPS-2; we have termed this more slowly migrating form LPS-3. The majority of the antibodies also reacted with LPS from strain CE109, a derivative of Rhizobium etli CE3, confirming that the LPS core antigens can be relatively conserved between strains of different Rhizobium species. One of the antibodies isolated in this study (JIM 32) was unusual because it appeared to react with all forms of LPS from strain 3841 (namely, LPS 1, LPS-2, and LPS-3). Furthermore, JIM 32 reacted positively with the LPS from many strains of Rhizobium tested (excluding the Rhizobium meliloti subgroup). JIM 32 did not react with representative strains from Bradyrhizobium, Azorhizobium or other related bacterial species. PMID- 8631659 TI - Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis. AB - The cloning and sequencing of the gene encoding porphypain, a cysteine proteinase previously isolated from detergent extracts of the Porphyromonas gingivalis W12 cell surface, are described. The prtP gene encoded a unique protein of 1,732 amino acids, including a putative signal sequence for protein secretion. The predicted molecular mass for the mature protein was 186 kDa, which was close to the observed molecular mass of 180 kDa. There was one copy of prtP in the genomes of seven P. gingivalis strains examined. The gene was located 5' to a region with a high degree of homology to the insertion element IS1126 in P. gingivalis W12. The PrtP protein had regions of high homology to HagA, a hemagglutinin of P. gingivalis, and to several purported proteinases of P. gingivalis that have Arg-X specificity. A detailed comparison of genes encoding the latter and cpgR suggested that rgp-1, prpR1, prtR, agp, cpgR, and possibly prtH were derived from identical genetic loci. Although an rgp-1-like locus was detected in seven P. gingivalis strains by Southern blot analyses, agp and cpgR were not detected, not even in the strains from which they were originally isolated. In addition, at least 20 copies of a repeat region common to PrtP, the Rgp-1-like proteins, and HagA were observed in each of the seven genomes examined. The repeat region hybridization patterns for strains W83 and W50 were very similar, and they were identical for strains 381 and ATCC 33277, providing further evidence that these strains are closely related genetically. PMID- 8631660 TI - An extracellular factor regulates expression of sdiA, a transcriptional activator of cell division genes in Escherichia coli. AB - The sdiA gene codes for a protein that regulates expression of the ftsQAZ cluster of essential cell division genes of Escherichia coli. SdiA up-regulates the ftsQ2p promoter that initiates transcription into the ftsQAZ cluster. In this paper, we report that expression of sdiA is itself regulated by a factor that is released into the growth medium by E. coli. When medium that had previously supported growth of E. coli (conditioned medium) was used to support growth of an indicator E. coli strain that contained an sdiA-lacZ transcriptional reporter, there was a 50 to 80% decrease in sdiA expression as monitored by beta galactosidase activity. The down-regulation of PsdiA was associated with a decrease in expression of the SdiA target promoter ftsQ2p, as monitored by expression of an ftsQ2p-lacZ transcriptional fusion. An effect of conditioned medium on ftsQ2p expression was not seen when the wild-type sdiA gene was disrupted by insertional mutagenesis, indicating that the effect on ftsQ2p expression was secondary to the down-regulation of PsdiA. Conditioned medium had no effect on expression of Plac, PrpoS, or several other promoters associated with the ftsQAZ gene cluster (ftsQ1p and ftsZ1-4p). This suggests that the response is specific for PsdiA and for promoters that are regulated by the sdiA gene product and that cell-to-cell signalling may play a role in regulating expression of this group of genes. PMID- 8631661 TI - Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase. AB - A transcriptional analysis of the phosphatidylinositol-specific phospholipase C (plcA) gene of Bacillus thuringiensis indicated that its transcription was activated at the onset of the stationary phase in B. thuringiensis but was not activated in B. subtilis. The B. thuringiensis gene encoding a transcriptional activator required for plcA expression was cloned by using a B. subtilis strain carrying a chromosomal plcA'-'lacZ fusion as a heterologous host for selection. This trans activator (designated PlcR) is a protein of a calculated molecular weight of 33,762 which appears to be distantly related to PreL and NprA, regulator proteins enhancing transcription of neutral protease genes during the stationary phase of a Lactobacillus sp. and B. stearothermophilus, respectively. plcR gene transcription was analyzed in B. thuringiensis and in B. subtilis. PlcR positively regulated its own transcription at the onset of the stationary phase. There is a highly conserved DNA sequence (17 bp) 34 nucleotides upstream from the plcR transcriptional start site and 49 nucleotides upstream from the plcA transcriptional start site. As PlcR positively regulates its own transcription and plcA transcription, this conserved DNA sequence may be the specific recognition target for PlcR activation. PMID- 8631662 TI - Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants. AB - The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes. PMID- 8631663 TI - Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics. AB - Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria. PMID- 8631664 TI - Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products. AB - Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped. PMID- 8631665 TI - Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma S. AB - sigma S is an alternate sigma factor which functions with RNA polymerase to activate transcription of genes that are involved in a number of stress responses, including stationary-phase survival and osmoprotection. The similarity of the sigma S protein to sigma D (Escherichia coli's major sigma factor) in the regions thought to recognize and bind promoter sequences suggests that sigma S- and sigma D-associated RNA polymerases recognize promoter DNA in a similar manner. However, no promoter recognition sequence for sigma S holoenzyme (E sigma S) has been identified. An apparent conservation of cytosine nucleotides was noted in the -35 region of several sigma S-dependent promoters. Site-directed mutagenesis and reporter gene fusions were used to investigate the importance of the -35 cytosine nucleotides for sigma S-dependent transcription. Substitution of cytosine nucleotides for thymidine at the -35 site of the sigma D-dependent proU promoter effectively abolished transcription by E sigma D but allowed E sigma S to direct transcription from the mutant promoter. Inclusion of the sigma D consensus -10 hexamer strengthened transcription by E sigma S, demonstrating that both E sigma D and E sigma S can recognize the same -10 sequences. Conversely, replacement of -35 site cytosine nucleotides with thymidine in the sigma S dependent osmY promoter reduced transcription by E sigma S and increased transcription by E sigma D. Our data suggest that DNA sequences in the -35 region function as part of a discriminator mechanism to shift transcription between E sigma D and E sigma S. PMID- 8631666 TI - Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk. AB - To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components. PMID- 8631667 TI - Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus. AB - Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495 508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species. PMID- 8631668 TI - Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis. AB - A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric division. The development programs in these two cells involve the compartmentalized activities of sigma E in the larger mother cell and of sigma F in the smaller prespore. Activation of sigma E requires expression of the sigma F-directed gene spoIIR. By immunofluorescence microscopy of a strain containing a spoIIR-lacZ fusion, we have shown that spoIIR is transcribed exclusively in the prespore. By placing spoIIR under the control of PspoIIE, it was possible to express spoIIR before the spore septum was formed. Strains containing the PspoIIE-spoIIR construct activated sigma E only in the mother cell in organisms that underwent the asymmetric sporulation division. Thus, compartmentalization of sigma E activity did not require the compartmentalization of spoIIR expression. Nor did the compartmentalization of sigma E require SpoIIAA, SpoIIAB, sigma F, or sigma F-dependent transcription, all of which are required for prespore-specific gene expression. It is inferred that although sigma F and sigma E direct compartmentalized gene expression, neither of these sigma factors, nor the genes under their control, directs the process of compartmentalization. PMID- 8631669 TI - Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis. AB - Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins. PMID- 8631671 TI - Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity. AB - Ferric siderophores, vitamin B12, and group B colicins are taken up through the outer membranes of Escherichia coli cells by an energy-coupled process. Energy from the cytoplasmic membrane is transferred to the outer membrane with the aid of the Ton system, consisting of the proteins TonB, ExbB, and ExbD. In this paper we describe two point mutations which inactivate ExbD. One mutation close to the N-terminal end of ExbD is located in the cytoplasmic membrane, and the other mutation close to the C-terminal end is located in the periplasm. E. coli CHO3, carrying a chromosomal exbD mutation in which leucine at position 132 was replaced by glutamine, was devoid of all Ton-related activities. A plasmid encoded ExbD derivative, in which aspartate at position 25, the only changed amino acid in the predicted membrane-spanning region of ExbD, was replaced by asparagine, failed to restore the Ton activities of strain CHO3 and negatively complemented ExbD+ strains, indicating an interaction of this mutated ExbD with wild-type ExbD or with another component. This component was shown to be ExbB. ExbB that was labeled with 6 histidine residues at its C-terminal end and that bound to a nickel-nitrilotriacetic acid agarose column retained ExbD and TonB specifically; both were eluted with the ExbB labeled with 6 histidine residues, demonstrating interaction of ExbB with ExbD and TonB. These data further support the concept that TonB, ExbB, and ExbD form a complex in which the energized conformation of TonB opens the channels in the outer membrane receptor proteins. PMID- 8631672 TI - A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo. AB - Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism. Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6 biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc). These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo. We report here the cloning and sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization. DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304 S) in glycerol kinase. The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined. Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43. However, IIIGlc activates the mutant enzyme, which could not be predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2.5 nm from the IIIGlc binding site (M. Feese, D. W. Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. USA 91:3544-3548, 1994). It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A. S. Kamath Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross, J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization. PMID- 8631670 TI - Surveying a supercoil domain by using the gamma delta resolution system in Salmonella typhimurium. AB - A genetic system was developed to investigate the supercoil structure of bacterial chromosomes. New res-carrying transposons were derived from MudI1734 (MudJr1 and MudJr2) and Tn10 (Tn10dGn). The MudJr1 and MudJr2 elements each have a res site in opposite orientation so that when paired with a Tn10dGn element in the same chromosome, one MudJr res site will be ordered as a direct repeat. Deletion formation was studied in a nonessential region (approximately 100 kb) that extends from the his operon through the cob operon. Strains with a MudJr insertion in the cobT gene at the 5' end of the cob operon plus a Tn10dGn insertion positioned either clockwise or counterclockwise from cobT were exposed to a burst of RES protein. Following a pulse of resolvase expression, deletion formation was monitored by scoring the loss of the Lac+ phenotype or by loss of tetracycline resistance. In exponentially growing populations, deletion products appeared quickly in some cells (in 10 min) but also occurred more than an hour after RES induction. The frequency of deletion (y) diminished with increasing distance (x) between res sites. Results from 15 deletion intervals fit the exponential equation y = 120 . 10(-0.02x). We found that res sites can be plectonemically interwound over long distances ( > 100 kb) and that barriers to supercoil diffusion are placed stochastically within the 43- to 45-min region of the chromosome. PMID- 8631673 TI - Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain. AB - Deletion of the tetA(L) chromosomal region of Bacillus subtilis in a strain designated JC112 increased the strain's sensitivity to low tetracycline concentrations. It also resulted in phenotypic changes that correlate with the previously found role of TetA(L) in mediating electrogenic NA+/H+ antiport. Growth of JC112 was impaired relative to that of the wild type at both pH 7.0 and 8.3; Na(+)- and K(+)-dependent pH homeostases were impaired at alkaline pH. The phenotype of JC112 was complemented by plasmid-borne tetA(L) and related tet(K) genes; the antiport activity conferred by the tet(K) gene had an apparently higher preference for K+ over Na+ than that conferred by tetA(L). The data were consistent with TetA(L) being the major Na+(K+)/H+ antiporter involved in pH homeostasis in B. subtilis as well as a significant Na+ extrusion system. The phenotype of JC112 was much more pronounced than that of an earlier transposition mutant, JC111, with a disruption in the putative tetA(L) promoter region. Northern (RNA) blot analysis of tetA(L) RNA from wild-type and JC111 strains revealed the same patterns. That JC111 nevertheless exhibited some Na+ and alkali sensitivity may be accounted for by disruption of regulatory features that, in the wild type, allow increased tetA(L) expression under specific conditions of pH and monovalent cation concentration. Evidence for several different regulatory effects emerged from studies of lacZ expression from the transposon of JC111 and from a tetA(L)-lacZ translational fusion introduced into the amyE locus of wild type and JC112 strains. PMID- 8631674 TI - Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis. AB - A variety of Mycobacterium species contained the 5-deazaflavin coenzyme known as F420. Mycobacterium smegmatis was found to have a glucose-6-phosphate dehydrogenase that was dependent on F420 as an electron acceptor and which did not utilize NAD or NADP. The enzyme was purified by ammonium sulfate fractionation, phenyl-Sepharose column chromatography, F420-ether-linked aminohexyl-Sepharose 4B affinity chromatography, and quaternary aminoethyl Sephadex column chromatography, and the sequence of the first 26 N-terminal amino acids has been determined. The response of enzyme activity to a range of pHs revealed a two-peak pattern, with maxima at pH 5.5 and 8.0. The apparent Km values for F420 and glucose-6-phosphate were, respectively, 0.004 and 1.6 mM. The apparent native and subunit molecular masses were 78,000 and approximately 40,000 Da, respectively. PMID- 8631676 TI - Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis. AB - Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces. PMID- 8631677 TI - Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation. AB - Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl CoA and Pi are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands. PMID- 8631675 TI - Analysis of a gene that suppresses the morphological defect of bald mutants of Streptomyces griseus. AB - When present in multiple copies, orf1590 restored sporulation to class IIIA bald mutants of Streptomyces griseus, which form sporulation septa and thick spore walls prematurely. The orf1590 alleles from class IIIA bald mutants restored sporulation upon introduction at a high copy number into those same mutants, and the nucleotide sequence of one of these alleles was identical to that of the wild type strain. We conclude that overexpression of orf1590 suppresses the defect in class IIIA bald mutants. Previous nucleotide sequence and transcript analyses suggested that orf1590 could encode two related proteins, P56 and P49.5, from nested coding sequences. A mutation that prevented the synthesis of P56 without altering the coding sequence for P49.5 eliminated the function of orf1590, as did amino acid substitutions in the putative helix-turn-helix domain located at the N terminus of P56 and absent from P49.5. To determine the coding capacity of orf1590, we analyzed translational fusions between orf1590 and the neo gene from Tn5. Measurement of the expression of fusions to the wild-type and mutant alleles of orf1590 indicated that P56 was the sole product of orf1590 during vegetative growth. Attempts to generate a nonfunctional frameshift mutation in orf1590 were unsuccessful in the absence of a second-site bald mutation, suggesting that orf1590 may be required during vegetative growth by preventing early sporulation. Our results are consistent with the hypothesis that P56 at a high level delays the premature synthesis of sporulation septa and spore walls in class IIIA mutants. PMID- 8631678 TI - Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities. AB - Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants. PMID- 8631679 TI - Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. AB - The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in combination with a diffusible signal compound, N-(3-oxohexanoyl) homoserine lactone, termed the autoinducer. We have synthesized a set of autoinducer analogs. Many analogs with alterations in the acyl side chain showed evidence of binding to LuxR. Some appeared to bind with an affinity similar to that of the autoinducer, but none showed a higher affinity, and many did not bind as tightly as the autoinducer. For the most part, compounds with substitutions in the homoserine lactone ring did not show evidence of binding to LuxR. The exceptions were compounds with a homocysteine thiolactone ring in place of the homoserine lactone ring. Many but not all of the analogs showing evidence of LuxR binding had some ability to activate the luminescence genes. None were as active as the autoinducer. While most showed little ability to induce luminescence, a few analogs with rather conservative substitutions had appreciable activity. Under the conditions we employed, some of the analogs showing little or no ability to induce luminescence were inhibitors of the autoinducer. PMID- 8631680 TI - Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15. AB - N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination. PMID- 8631681 TI - The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin. AB - The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium. PMID- 8631683 TI - Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth. AB - lambda's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions. Related work has demonstrated that Int protein expression depends on the rare AGA tRNA. Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death. Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion. These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene. This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena. It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision. PMID- 8631682 TI - Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201. AB - Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis. PMID- 8631684 TI - Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation. AB - Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin. PMID- 8631686 TI - Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium. AB - Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions. PMID- 8631685 TI - maoB, a gene that encodes a positive regulator of the monoamine oxidase gene (maoA) in Escherichia coli. AB - The structural gene for copper- and topa quinone-containing monoamine oxidase (maoA) and an unknown amine oxidase gene have been located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that the unknown gene was located within a 1.1-kb cloned fragment adjacent to the maoA gene. The nucleotide sequence of this fragment was determined, and a single open reading frame (maoB) consisting of 903 bp was found. The gene encoded a polypeptide with a predicted molecular mass of 34,619 Da which was correlated with the migration on a sodium dodecyl sulfate-polyacrylamide gel. The predicted amino acid sequence of the MaoB protein was identical to the NH2-terminal amino acid sequence derived by Edman degradation of the protein synthesized under the self-promoter. No homology of the nucleotide sequence of maoB to the sequences of any reported genes was found. However, the amino acid sequence of MaoB showed a high level of homology with respect to the helix-turn-helix motif of the AraC family in its C terminus. The homology search and disruption of maoA on the chromosome led to the conclusion that MaoB is a transcriptional activator of maoA but not an amine oxidase. The consensus sequence of the cyclic AMP-cyclic AMP receptor protein complex binding domain was adjacent to the putative promoter for the maoB gene. By use of lac gene fusions with the maoA and maoB genes, we showed that the maoA gene is regulated by tyramine and MaoB and that the expression of the maoB gene is subject to catabolite repression. Thus, it seems likely that tyramine and the MaoB protein activate the transcription of maoA by binding to the regulatory region of the maoA gene. PMID- 8631687 TI - Characterization of the flagellar hook length control protein fliK of Salmonella typhimurium and Escherichia coli. AB - During flagellar morphogenesis in Salmonella typhimurium and Escherichia coli, the fliK gene product is responsible for hook length control. A previous study (M. Homma, T. Iino, and R. M. Macnab, J. Bacteriol. 170:2221-2228, 1988) had suggested that the fliK gene may generate two products; we have confirmed that both proteins are products of the fliK gene and have eliminated several possible explanations for the two forms. We have determined the DNA sequence of the fliK gene in both bacterial species. The deduced amino acid sequences of the wild-type FliK proteins of S. typhimurium and E. coli correspond to molecular masses of 41,748 and 39,246 Da, respectively, and are fairly hydrophilic. Alignment of the sequences gives an identity level of 50%, which is low for homologous flagellar proteins from S. typhimurium and E. coli; the C-terminal sequence is the most highly conserved part (71% identity in the last 154 amino acids). The central and C-terminal regions are rich in proline and glutamine residues, respectively. Linker insertion mutagenesis of the conserved C-terminal region completely abolished motility, whereas disruption of the less conserved N-terminal and central regions had little or no effect. We suggest that the N-terminal (or N terminal and central) and C-terminal regions may constitute domains. For several reasons, we consider it unlikely that FliK is functioning as a molecular ruler for determining hook length and conclude that it is probably employing a novel mechanism. PMID- 8631688 TI - Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. AB - Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin. PMID- 8631689 TI - Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide. AB - The gene cluster of Streptococcus pneumoniae coding for the type 3 capsular polysaccharide contains four genes (cap3ABCD). A DNA fragment containing the cap3A gene was amplified by PCR and cloned under the control of a T7 RNA polymerase-dependent promoter. Overexpression of this gene in Escherichia coli resulted both in a 47-kDa protein in the cytoplasm of isopropyl-beta-D thiogalactopyranoside-induced bacteria and in high levels of UDP-glucose dehydrogenase activity. These data demonstrate, in a direct experimental way, that cap3A encodes the UDP-glucose dehydrogenase of pneumococcus type 3. PMID- 8631690 TI - Perturbation of nifT expression in Klebsiella pneumoniae has limited effect on nitrogen fixation. AB - In the nitrogenase system of Klebsiella pneumoniae, nifT is located between nifDK, the structural genes for dinitrogenase, and nifY, whose product is involved in nitrogenase maturation. It is, therefore, a reasonable hypothesis that the NifT protein might also have a role in the maturation of nitrogenase. However, the phenotypic characterization of nifT and nifT-overexpressing strains for effects on the regulation, maturation, and activity of nitrogenase identified no properties that were distinct from those of the wild type. We conclude that the K. pneumoniae NifT protein is not essential for nitrogen fixation under the conditions examined. PMID- 8631691 TI - A halotolerant mutant of Saccharomyces cerevisiae. AB - FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID- 8631692 TI - DNA binding of the Bordetella pertussis H1 homolog alters in vitro DNA flexibility. AB - BpH1, the Bordetella pertussis H1 homolog, interacts with chromosomal DNA. With DNase I protection assays, we demonstrate in this study that BpH1 binds DNA in a nonspecific manner and that it may cover DNA fragments from end to end. Although the binding was shown to be nonspecific, preferential binding sites and sites resistant to BpH1 binding were identified within and upstream of the pertussis toxin promoter sequence. In the presence of DNA ligase, BpH1 favored the formation of multimeric DNA fragments of various sizes and prevented ring closures, suggesting a diminished flexibility of the DNA fragments and thus indicating that BpH1 acts as a macromolecular crowding agent. PMID- 8631693 TI - Site-specific proteolysis of the Escherichia coli SecA protein in vivo. AB - A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252. Chromosomal wild-type secA was replaced by these secA constructs. Simultaneous expression of TEV protease led to cleavage of both SecA derivatives. In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites. Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo. PMID- 8631694 TI - Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli. AB - Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min. PMID- 8631695 TI - Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae. AB - The ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae. We report here, however, that strains with null mutations in the ERG24 gene can grow on defined synthetic media in aerobic conditions. These sterol mutants produce ignosterol (ergosta-8,14-dienol) as the principal sterol, with no traces of ergosterol. In addition, we mapped the ERG24 gene to chromosome XIV between the MET2 and SEC2 genes. Our results indicate that ignosterol can be a suitable sterol for aerobic growth of S. cerevisiae on synthetic media and that inactivation of ERG24 is only conditionally lethal. PMID- 8631696 TI - A novel member of the cspA family of genes that is induced by cold shock in Escherichia coli. AB - Escherichia coli contains a major cold shock protein, CspA (or CS7.4), whose production is predominantly induced at low temperatures. This bacterium is known to possess five additional genes, each encoding a protein highly similar to CspA (referred to as the CspA family). Here we identified a gene that encodes a cold shock-inducible analog of CspA and CspB. This newly cloned cspG gene is located at 22 min on the E. coli genetic map, apart from the other cspA family genes. Its gene product (70 amino acids) is 73 and 77% identical to CspA (70 amino acids) and CspB (71 amino acids), respectively. Analyses of a cspG-lacZ transcriptional fusion and Northern (RNA) hybridization revealed that cspG is a low-temperature responsive gene. Its low-temperature-inducible promoters were determined, and the results indicated that the cspG sequence is highly similar to both the cspA and cspB sequences not only in the coding regions but also in the 5'-upstream noncoding regions surrounding their own promoters. PMID- 8631697 TI - The RuvABC proteins and Holliday junction processing in Escherichia coli. PMID- 8631698 TI - Rifampin-induced initiation of chromosome replication in dnaR-deficient Escherichia coli cells. AB - The dnaR130 mutant of Escherichia coli, which was thermosensitive in initiation of chromosome replication, was capable of thermoresistant DNA synthesis in the presence of rifampin at a low concentration that allowed almost normal RNA synthesis. The DNA synthesis in the presence of the drug depended on protein synthesis at the high temperature. The protein synthesis in the dnaR-deficient cells provided a potential for thermoresistant DNA synthesis to be induced at a high dose of the drug that almost completely prevented RNA synthesis. The induced synthesis was synchronously initiated from oriC and proceeded semiconservatively toward terC. The replication depended on the dnaA function, which was essential for normal initiation of replication from oriC. The capability for drug-induced replication was abolished by certain rifampin resistance mutations in the beta subunit of RNA polymerase. Thus, the drug can induce the dnaA-dependent initiation of replication in the dnaR-deficient cells through its effect on RNA polymerase. This result implies that the dnaR product is involved in the transcription obligatory for the initiation of replication of the bacterial chromosome. PMID- 8631699 TI - Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli. AB - Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position 41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism. PMID- 8631700 TI - The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair. AB - The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication. PMID- 8631701 TI - Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N acetylglucosamine transferase (RfaK). AB - A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1- >4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core. PMID- 8631703 TI - A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24. AB - An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Arg-p-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp. An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer. The enzyme had an isoelectric point of 4.7. It is optimally active at pH 9.0. The Km and Vmax of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 micromol/min/mg, respectively. The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III. The Km and Vmax for Arg-Arg-MNA were 0.019 mM and 145 micromol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II. The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors. The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III. These results indicate that DAP BI may be a new type of the DAP family. PMID- 8631702 TI - Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. AB - The live attenuated bacillus Calmette-Guerin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection. PMID- 8631704 TI - A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli. AB - The motor that drives the flagellar filament of Escherichia coli contains three "switch" proteins (FliG, FliM, and FliN) that together determine the direction of rotation. Each is required, in addition, for flagellar assembly and for torque generation. These proteins interact in the Saccharomyces cerevisiae two-hybrid system: FliG interacts with FliM, FliM interacts with itself, and FliM interacts with FliN. The interaction between FliG and FliM has been subjected to mutational analysis. FliG (fused to the GAL4 DNA-binding domain) and FliM (fused to a GAL4 transcription activation domain) together activate transcription of a GAL4 dependent lacZ reporter gene. DNA encoding FliG was mutagenized by error-prone amplification with Taq polymerase, mutant fliG genes were cloned (as DNA-binding domain-fliG gene fusions) in S. cerevisiae by gap repair of plasmid DNA, and mutants exhibiting an interaction defect were isolated in a two-hybrid screen. The mutations were each mapped to the first, second, or last third of the fliG gene by multifragment cloning in vivo and then identified by DNA sequencing. In this way, we identified 18 interaction-defective and 15 silent (non-interaction defective) fliG mutations. Several residues within the middle third of FliG are strongly involved in the FliG-FliM interaction, while residues near the N or C terminus are less important. This clustering, when compared with results of previous studies, suggests that the FliG-FliM interaction plays a central role in switching. PMID- 8631705 TI - Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri. AB - Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high pressure liquid chromatography with indirect photometric detection. Two thiols, 2 mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation. PMID- 8631706 TI - Regulation of VanB-type vancomycin resistance gene expression by the VanS(B)-VanR (B) two-component regulatory system in Enterococcus faecalis V583. AB - Acquired VanA- and VanB-type glycopeptide resistance in enterococci is due to synthesis of modified peptidoglycan precursors terminating in D-lactate. As opposed to VanA-type strains which are resistant to both vancomycin and teicoplanin, VanB-type strains remain teicoplanin susceptible. We have determined the sequence of a 7,160-bp DNA fragment associated with VanB-type resistance in Enterococcus faecalis V583 that contains seven open reading frames. The distal part encoded the VanH (B), VanB, and VanX (B) proteins that are highly similar to the putative VanH, VanA, and VanX proteins responsible for VanA-type resistance. Upstream from the structural genes for these proteins were the vanY(B) gene encoding a D,D-carboxypeptidase and an open reading frame vanW with an unknown function. The proximal part of the gene cluster coded for the apparent VanS(B) VanR (B) two-component regulatory system. VanR (B) was related to response regulators of the OmpR subclass, and VanS (B) was related to membrane-associated histidine protein kinases. Analysis of transcriptional fusions with a reporter gene and promoter mapping indicated that the VanR B-VanS B two-component regulatory system activates a promoter located immediately downstream from the vanS B gene. Vancomycin, but not teicoplanin, was an inducer, which explains teicoplanin susceptibility of VanB-type enterococci. PMID- 8631707 TI - Flagellin A is essential for the virulence of Vibrio anguillarum. AB - A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host. PMID- 8631708 TI - Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin. AB - We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin. PMID- 8631710 TI - Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants. AB - The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tu3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration behavior of EpiC depended on the oxidation state of the enzyme, indicating the formation of an intramolecular disulfide bridge between C-274 and C-321. The cysteine residues in the motifs WC-274YG and C-321HG of EpiC are conserved in all lantibiotic enzymes of the C type (so-called LanC proteins) and in the CylM protein. Mutated epiC genes from S. epidermidis epiC mutants were cloned and expressed in E. coli. Sequence analysis revealed that the mutations occurred in the two motifs -S-X-X-X-G-X-X-G- and -N-X-G-X-A-H G-X-X-G-, which are conserved in all LanC proteins. For the investigation of EpiC EpiA interactions, precursor peptide EpiA was coupled to N-hydroxysuccinimide activated Sepharose High Performance Material (HiTrap). Under reducing conditions, EpiC was retarded on the EpiA-HiTrap column. In the incubation experiments, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides. PMID- 8631709 TI - Topological characterization of the essential Escherichia coli cell division protein FtsN. AB - Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane spanning domains for the function of this protein. Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal. Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus. To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made. Deletion of the N terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function. Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane spanning domain from MalG restored subcellular localization and function. These N terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function. It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm. FtsQ and FtsI were also analyzed. PMID- 8631711 TI - Surface display of a functional single-chain Fv antibody on staphylococci. AB - Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1(kappa) [IgG1(kappa)] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed. PMID- 8631712 TI - Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli. AB - We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs. PMID- 8631713 TI - p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon. AB - Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta 2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1. PMID- 8631714 TI - Identification of TonB homologs in the family Enterobacteriaceae and evidence for conservation of TonB-dependent energy transduction complexes. AB - The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli requires the TonB-dependent energy transduction system. A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilitate structure and function studies. In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-negative organisms was examined. Cross-species reactivity patterns obtained against TonB homologs of known sequence were used to refine epitope mapping, with some epitopes ultimately confirmed by inhibition experiments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the family Enterobacteriaceae (including E. coli), including previously unidentified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species. These homologs were also detected by a polyclonal alpha-TrpC-TonB serum that additionally recognized the known Yersinia enterocolitica TonB homolog and a putative TonB homolog in Edwardsiella tarda. These antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify potential TonB homologs in several other nonenteric gram-negative species. In vivo chemical cross-linking experiments demonstrated that in addition to TonB, auxiliary components of the TonB-dependent energy transduction system are broadly conserved in members of the family Enterobacteriaceae, suggesting that the TonB system represents a common system for high-affinity active transport across the gram-negative outer membrane. PMID- 8631716 TI - Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides. AB - Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c2 family of electron transport proteins (isocytochrome c2). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of approximately40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes. PMID- 8631715 TI - Regulators of aerobic and anaerobic respiration in Bacillus subtilis. AB - Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis. PMID- 8631717 TI - Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli. AB - The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase. PMID- 8631718 TI - Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis. AB - We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants. PMID- 8631719 TI - Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa. AB - Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP ribosyltransferase activity of the 53-kDa form of exoenzyme S. PMID- 8631720 TI - Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein. AB - The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear. PMID- 8631721 TI - Positive autoregulation of vipR expression in ViaB region-encoded Vi antigen of Salmonella typhi. AB - vipR was the first gene in the ViaB region found to be involved in production of the Vi capsular antigen in Salmonella typhi and to be transcribed in the same direction as nine downstream genes. To investigate its function, we constructed a nonpolar vipR mutant by inserting a cat cartridge into the vipR gene on the chromosome. Inactivation of the gene decreased its transcriptional level and the levels of the downstream genes. When a functional vipR gene on a low-copy-number plasmid was introduced into the vipR mutant, the transcriptional levels of the chromosomal genes were restored. These results indicate that the vipR gene product acts as a transcriptional activator for the downstream genes. Interestingly, the vipR gene also was positively regulated by its own product. DNA-binding studies showed that protein extract that contained the VipR product specifically retarded the mobility of a 451-bp BspHI-XbaI fragment upstream of the vipR gene. In addition, complementation analysis indicated that the vipR gene was cotranscribed with a least two downstream genes, vipA and vipB. These findings suggest that the VipR product may interact with the regulatory region upstream of the vipR gene and induce operonic mRNA synthesis that includes the vipR and downstream genes. PMID- 8631722 TI - Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins. AB - Neisseria gonorrhoeae is capable of iron utilization from human transferrin in a receptor-mediated event. Transferrin-binding protein 1 (Tbp1) and Tbp2 have been implicated in transferrin receptor function, but their specific roles in transferrin binding and transferrin iron utilization have not yet been defined. We utilized specific gonococcal mutants lacking Tbp1 or Tbp2 to assess the relative transferrin-binding properties of each protein independently of the other. The apparent affinities of the wild-type transferrin receptor and of Tbp1 and Tbp2 individually were much higher than previously estimated for the gonococcal receptor and similar to the estimates for the mammalian transferrin receptor. The binding parameters of both of the mutants were distinct from those of the parent, which expressed two transferrin-binding sites. Tbp2 discriminated between ferrated transferrin and apotransferrin, while Tbp1 did not. Results of transferrin-binding affinity purification, and protease accessibility experiments were consistent with the hypothesis that Tbp1 and Tbp2 interact in the wild-type strain, although both proteins were capable of binding to transferrin independently when separated in the mutants. The presence of Tbp1 partially protected Tbp2 from trypsin proteolysis, and Tbp2 also protected Tbp1 from trypsin exposure. Addition of transferrin to wild-type but not mutant cells protected Tbp1 from trypsin but increased the trypsin susceptibility of Tbp2. These observations indicate that Tbp1 and Tbp2 function together in the wild-type strain to evoke binding conformations that are distinct from those expressed by the mutants lacking either protein. PMID- 8631724 TI - Phylogenetic analysis of Metabacterium polyspora: clues to the evolutionary origin of daughter cell production in Epulopiscium species, the largest bacteria. AB - It is rare that there are molecular clues to the evolutionary origin of developmental traits. We have encountered an evolutionary juxtaposition that may explain the origin of the unique replicative morphology of Epulopiscium spp., the largest known bacteria, which reproduce by the internal production of multiple live offspring. We report here a 16S rRNA-based phylogenetic analysis of Metabacterium polyspora, a multiple-endospore-forming, uncultivated inhabitant of guinea pig cecum. Cells of M. polyspora were harvested from cecum contents by sedimentation in a Ficoll gradient and lysed. The bacterial 16S rRNA genes of this lysate were amplified by PCR. Sequence analysis of the cloned PCR products revealed two dominant, closely related 16S rRNA types. In situ hybridization of cecum contents with fluorescently labeled oligonucleotides, diagnostic of these two sequences, showed that they represent distinct strains of M. polyspora. Phylogenetic analyses of the sequences showed that M. polyspora is closely related to Epulopiscium spp. On the basis of this result and other correlations, we propose that the process of sporulation was modified in a predecessor of Epulopiscium spp. to produce live offspring instead of quiescent endospores. PMID- 8631723 TI - Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. AB - The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane. PMID- 8631725 TI - Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required. AB - Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events. PMID- 8631726 TI - Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli? AB - treA and osmY expression and RpoS protein levels were investigated in glucose limited continuous culture. The level of induction of these stationary-phase markers became as high during growth at a D of 0.1 to 0.2 h(-1) as in carbon starved batch cultures but only in rpoS+ bacteria. The stress protectant trehalose was actually produced at higher levels at low growth rates than in stationary-phase cultures. The pattern of induction of RpoS-dependent activities could be separated from those regulated by cyclic AMP (cAMP) or endoinduction, and the induction occurred at extreme glucose limitation. Escherichia coli turns to a protective stationary-phase response when nutrient levels fall below approximately 10(-7) M glucose, which is insufficient to saturate scavenger transporters regulated by cAMP plus endoinducers, and this response is optimally expressed at 10(-6) M glucose. The high-level induction of protective functions also explains the maintenance energy requirement of bacterial growth at low dilution rates. PMID- 8631727 TI - Carboxylation of epoxides to beta-keto acids in cell extracts of Xanthobacter strain Py2. AB - A novel enzymatic reaction involved in the metabolism of aliphatic epoxides by Xanthobacter strain Py2 is described. Cell extracts catalyzed the CO2-dependent carboxylation of propylene oxide (epoxypropane) to form acetoacetate and beta hydroxybutyrate. The time courses of acetoacetate and beta-hydroxybutyrate formaton indicate that acetoacetate is the primary product of propylene oxide carboxylation and that beta-hydroxybutyrate is a secondary product formed by the reduction of acetoacetate. Analogous C5 carboxylation products were identified with 1,2-epoxybutane as the substrate. In the absence of CO2, propylene oxide and 1,2-epoxybutane were isomerized to form acetone and methyl ethyl ketone, respectively, as dead-end products. The carboxylation of short-chain epoxides to beta-keto acids is proposed to serve as the physiological reaction for the metabolism of aliphatic epoxides in Xanthobacter strain Py2. PMID- 8631728 TI - The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain. AB - Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR. Here we show that the C terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition. PMID- 8631729 TI - Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium. AB - purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6 phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine. PMID- 8631730 TI - Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA. AB - Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions. PMID- 8631731 TI - Proteins responsible for lysogenic conversion caused by coliphages N15 and phi80 are highly homologous. AB - Lysogenic conversion caused by lambdoid bacteriophage phi80 and that caused by coliphage N15 have similar characteristics, suggesting that similarities in their cor genes and Cor proteins are responsible for this effect. Here we present the nucleotide sequence of the N15 cor gene. The N15 cor gene homolog was found in the phi80 cor region, but in the opposite direction of that of the open reading frame to which the phi80 cor gene had previously been assigned (M. Matsumoto, N. Ichikawa, S. Tanaka, T. Morita, and A. Matsushiro, Jpn. J. Genet. 60:475-483, 1985). PMID- 8631732 TI - A deduced Thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains. AB - The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes. PMID- 8631733 TI - A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation. AB - A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well established. Whether ATP is required for synthesis of the first phosphodiester bond or at a slightly later stage in synthesis of nascent transcripts, however, remains controversial. Goodrich and Tjian (Goodrich, J.A., and Tjian, R. (1994) Cell 77, 145-156) recently proposed that synthesis of the first phosphodiester bond of promoter specific transcripts by RNA polymerase II is independent of ATP and general transcription factors TFIIE and TFIIH. Here we investigate this model. Taken together, our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation. First, we observe that synthesis of the first phosphodiester bond of transcripts initiated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that, in this enzyme system, ATP-dependent activation of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts. Finally, we demonstrate that the activated initiation complex is unstable and decays rapidly to an inactive state in the presence of the inhibitor ATP-gammaS (adenosine 5'-O (thio)triphosphate), even during reiterative synthesis of abortive transcripts. PMID- 8631734 TI - Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. AB - The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo. PMID- 8631735 TI - Hyperphosphorylation of nucleoplasmin facilitates Xenopus sperm decondensation at fertilization. AB - Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus. PMID- 8631736 TI - Doc2 enhances Ca2+-dependent exocytosis from PC12 cells. AB - We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+ dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells. PMID- 8631737 TI - Alteration of the cystic fibrosis transmembrane conductance regulator folding pathway. AB - The cellular phenotype of the most common cystic fibrosis-causing mutation, deletion of phenylalanine 508 (deltaF508) in the amino-terminal nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), is the inability of the mutant protein to fold and transit to the apical membrane of affected epithelial cells. Expressed NBD1s were purified and folded in vitro into soluble monomers capable of binding nucleotide. Here we report that the deltaF508 mutation has little effect on the thermodynamic stability of the folded NBD1. The deltaG(0)(D,GdnHCl) is 15.5 kJ/mol for the wild type NBD1 and 14.4 kJ/mol for NBD1deltaF. In contrast, the mutation significantly reduces the folding yield at a variety of temperatures, indicating that Phe-508 makes crucial contacts during the folding process, but plays little role in stabilization of the native state. Under conditions that approximate the efficiency of maturation in vivo, the rate off-pathway is significantly increased by the disease causing mutation. These results establish a molecular mechanism for most cases of cystic fibrosis and provide insight into the complex processes by which primary sequence encodes the three-dimensional structure. PMID- 8631738 TI - Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C: its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin. AB - Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+ dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser306 and Ser313. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane. PMID- 8631739 TI - Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS mediated assembly of [2Fe-2S] clusters into apo-SoxR. AB - SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli. The active protein isolated from E. coli contains a pair of [2Fe-2S] clusters per SoxR dimer. We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro. Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron sulfur clusters into the apoprotein. Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min. Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear [2Fe-2S] clusters were restored by both the enzymatic and nonenzymatic reconstitutions. A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of [2Fe-2S] centers, even in the presence of NifS. Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR. Moreover, the catalytic generation of [2Fe-2S] centers extends the known specificity of this enzyme beyond that already shown for [4Fe-4S] centers. Catalytic generation of [2Fe-2S] containing SoxR could allow for rapid activation of this transcription factor in vivo. PMID- 8631740 TI - Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone. AB - Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome. PMID- 8631741 TI - The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+ ATPase. AB - The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase. PMID- 8631742 TI - Characterization of an interleukin 6 cytokine family antagonist protein from a marine sponge, Callyspongia sp. AB - An inhibitor of IL-6 binding to the human hepatoma line HepG2 and myeloma cell line U266 was identified in a saline extract of the marine sponge, Callyspongia sp. Functional activity, measured through the increase in haptoglobin production by HepG2 cells stimulated with IL-6, could be strongly inhibited by the extract. Similarly, IL-6-induced production of IgM by the B cell line SKW6.4 was substantially reduced. In neither cell line was there evidence of toxicity produced by the extract. Other sponges of the Callyspongia species were found to contain analogous activity. The activity was destroyed by trypsin treatment or boiling of the extract, suggesting that the inhibition is due to a protein. When the binding of IL-6 to its receptor complex was dissected in vitro, inhibition of binding of IL-6 to soluble receptor by the extract was not detected, but binding of the IL-6-sIL-6R complex to soluble gp130 was inhibited in a dose-dependent fashion. This was borne out in cellular assays since the extract inhibited activation of HepG2 cells stimulated with oncostatin M or leukemia inhibitory factor, cytokines which also use gp130 for signal transduction. These results suggest that the Callyspongia extract contains a protein which blocks the interaction of the IL-6 family of cytokines with their signal transduction moiety, gp130. Elucidation of the structure and mode of action of such a protein would be helpful in designing gp130 antagonists to inhibit the functions of this cytokine family, overproduction of which has been associated with cancer and pathologies of autoimmune disease and AIDS. PMID- 8631743 TI - Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability. AB - Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2 dependent mismatch repair. PMID- 8631744 TI - Introduction of selectin-like binding specificity into a homologous mannose binding protein. AB - The structures of the ligand-binding C-type carbohydrate-recognition domains of selectin cell adhesion molecules and of mannose-binding proteins (MBPs) are similar to each other even though these proteins bind very different carbohydrate ligands. Our current understanding of ligand binding by E-selectin is based on structural studies of unliganded E-selectin and of MBP-carbohydrate complexes, combined with results from mutagenesis of E-selectin. Five regions of E-selectin that differ in sequence from the corresponding regions of MBP have been introduced into the carbohydrate-recognition domain of MBP. Four of the changes have little effect on ligand binding. Insertion of one stretch of positively charged amino acids alters the sugar binding selectivity of the domain so that it now binds HL-60 cells and serum albumin derivatized with sialyl-Lewis X tetrasaccharide, thus mimicking the properties of E-selectin. PMID- 8631745 TI - Cleavage of the alpha6A subunit is essential for activation of the alpha6Abeta1 integrin by phorbol 12-myristate 13-acetate. AB - The alpha6 integrin subunit is proteolytically cleaved during biosynthesis in a covalently associated heavy and light chain. To examine the importance of cleavage for the function of the alpha6 subunit, we introduced mutations in the cDNA encoding the RKKR (876-879) sequence, the presumed cleavage site, in which either one or two basic residues were replaced by glycine. Wild-type and mutant alpha6A cDNAs (alpha6GKKR, alpha6RKKG and alpha6RGGR) were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1, at levels comparable to that of the wild-type alpha6Abeta1. A single alpha6A polypeptide chain (150 kDa) was precipitated from surface-labeled alpha6GKKR, alpha6RKKG, and alpha6-RGGR transfectants, while the separate heavy (120 kDa) and light chains (31 or 30 kDa) were precipitated from the wild-type alpha6RKKR transfectant. Thus, a change in the RKKR sequence prevents cleavage of alpha6. After activation by the anti-beta1 stimulatory mAb TS2/16 both cleaved and uncleaved alpha6Abeta1 integrins bound and spread on laminin-1. Remarkably, the phorbol ester phorbol 12-myristate 13-acetate, which activates wild-type alpha6Abeta1 to bind to laminin-1, did not activate uncleaved alpha6Abeta1. We conclude that uncleaved alpha6Abeta1 is capable of ligand binding and transducing outside/in signals, like wild-type alpha6A-beta1. However, inside/out signaling is affected. It appears that cleavage of alpha6 is required to generate the proper conformation in alpha6 that enables affinity modulation of the alpha6A beta1 receptor by phorbol 12-myristate 13-acetate. PMID- 8631746 TI - Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel. AB - In contrast to excitable tissues where calcium channels are well characterized, the nature of the B lymphocyte calcium channel is unresolved. Here, we demonstrate by single cell analysis of freshly isolated rat B cells that the anti immunoglobulin (Ig)-induced calcium influx takes place through a channel which shares pharmacologic and serologic properties with the L-type calcium channel found in excitable tissues. It is sensitive to the dihydropyridines nicardipine and Bay K 8644, to calciseptine, and to an anti-peptide antibody raised against the alpha1 subunit of the L-type calcium channel, but is voltage-insensitive. Anti-alpha1 and anti-alpha2 antibodies stain B but not T lymphocytes. Application of a cGMP agonist, measurement of cGMP levels in anti-Ig-stimulated B cells, and examining the effect of a guanylyl cyclase inhibitor on the anti-Ig response show that cGMP mediates the influx. This possibly involves a cGMP-dependent protein kinase. The anti-Ig-induced response is not abolished by prior treatment of B cells with a high dose of thapsigargin. These findings undermine the widely held belief of a categorical divide between excitable and non-excitable tissue calcium channels, demonstrate the limitations of the capacitative calcium influx theory, and point to a distinction between the calcium response mechanisms utilized by B and T lymphocytes. PMID- 8631747 TI - Exoloop 3 of the luteinizing hormone/choriogonadotropin receptor. Lys583 is essential and irreplaceable for human choriogonadotropin (hCG)-dependent receptor activation but not for high affinity hCG binding. AB - The luteinizing hormone/choriogonadotropin (CG) receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated C-terminal half of 303 amino acids. The N-terminal half is capable of high affinity hormone binding whereas the C-terminal half is capable of low affinity hormone binding and receptor activation. However, the precise location of the receptor activation site is currently unknown. We present evidence for the first time that Lys583 of exoloop 3 is crucial and irreplaceable for receptor activation to induce cAMP synthesis. Exoloop 3 is comprised of 11 amino acids and flanked by two Lys residues, Lys573 and Lys583, that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys583 did not affect the high affinity human CG binding, but they resulted in the complete loss of cAMP synthesis induced by human CG. Ala substitutions of the other amino acids in exoloop 3 did not make such a dramatic impact on cAMP induction. The Ala scan revealed two distinct groups of amino acids in terms of their importance in cAMP induction, one group being more important than the other. Interestingly, these two groups of amino acids are arranged in an alternate sequence. This result suggests a specific structure similar to a beta-like structure for exoloop 3. PMID- 8631748 TI - Diastereoisomers of cytolysins, a novel class of potent antibacterial peptides. AB - An amphipathic alpha-helical structure is considered to be a prerequisite for the lytic activity of most short linear cytolytic polypeptides that act on both mammalian cells and bacteria. This structure allows them also to exert diverse pathological and pharmacological effects, presumably by mimicking protein components that are involved in membrane-related events. In this study D-amino acid-incorporated analogues (diastereomers) of the cytolysin pardaxin, which is active against mammalian cells and bacteria, were synthesized and structurally and functionally characterized. We demonstrate that the diastereomers do not retain the alpha-helical structure, which in turn abolishes their cytotoxic effects on mammalian cells. However, they retain a high antibacterial activity, which is expressed in a complete lysis of the bacteria, as revealed by negative staining electron microscopy. The disruption of the alpha-helical structure should prevent the diastereomer analogues from permeating the bacterial wall by forming transmembrane pores but rather by dissolving the membrane as a detergent. These findings open the way for a new strategy in developing a novel class of highly potent antibacterial polypeptides for the treatment of infectious diseases, due to the increasing resistance of bacteria to the available antibacterial drugs. PMID- 8631749 TI - Heme iron reduction and catalysis by a nitric oxide synthase heterodimer containing one reductase and two oxygenase domains. AB - Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2',5' ADP affinity chromatography. The heterodimer catalyzed NADPH-dependent NO synthesis from L-arginine at a rate of 52 +/- 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine. PMID- 8631750 TI - Acute depression of mitochondrial protein synthesis during anoxia: contributions of oxygen sensing, matrix acidification, and redox state. AB - Mitochondrial protein synthesis is acutely depressed during anoxia-induced quiescence in embryos of Artemia franciscana. Oxygen deprivation is accompanied in vivo by a dramatic drop in extramitochondrial pH, and both of these alterations strongly inhibit protein synthesis in isolated mitochondria. Here we show that the oxygen dependence is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depressed protein synthesis within 5 min and resulted in a 77% reduction after 1 h, aerobic incubations with saturating concentrations of cyanide or antimycin A had little effect during the first 20 min and only a modest effect after 1 h (36 and 20% reductions, respectively). Yet the mitochondrial NAD(P)H pools were fully reduced after 2-3 min with all three treatments. This cyanide- and antimycin-insensitive but hypoxia-sensitive pattern of protein synthesis depression suggests the presence of a molecular oxygen sensor within the mitochondrion. Second, we show for the first time that acidification of extramitochondrial pH exerts inhibition on protein synthesis specifically through changes in matrix pH. Matrix pH was 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient was abolished with nigericin, the extramitochondrial pH optimum for protein synthesis displayed an alkaline shift of approximately 0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion. PMID- 8631751 TI - Identification of a novel cysteine string protein variant and expression of cysteine string proteins in non-neuronal cells. AB - Cysteine string proteins (Csps) are synaptic vesicle proteins thought to be involved in calcium-dependent neurotransmitter release at nerve endings. Here, we report the cloning of two Csp variants, termed Csp1 and Csp2, from bovine adrenal medullary chromaffin cells. The bovine Csp1 appears to be the homologue of rat brain Csp, sharing 95% identity at the amino acid level. The nucleotide sequence of csp2 is identical with that of csp1 except for a 72-base insert which introduces a stop codon into the coding sequence, which would be predicted to result in a truncated protein 3.3 kDa smaller than Csp1. Furthermore, polymerase chain reaction analysis detected homologues of Csp1 and Csp2 in rat kidney, liver, pancreas, spleen, lung, and adrenal gland. Expression of Csps in non neuronal tissues was confirmed by Northern blotting and by immunoblotting with anti-Csp1 antiserum which also demonstrated expression of both full-length and truncated Csps in spleen. The widespread tissue distribution is inconsistent with a role of Csps as specific regulators of presynaptic calcium channels as previously proposed. We suggest that Csps may have a more general role in membrane traffic in non-neuronal as well as neuronal cells. PMID- 8631752 TI - Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase) AB - Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3 kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton. PMID- 8631753 TI - Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor. AB - The NF-kappaB and NF-IL6 elements have previously been shown to play an important role in regulation of both the mouse and human interleukin-6 gene. Between these two elements lies a G/C-rich sequence, which contains three repeats of the element CCACC, protein binding to which has not been previously characterized. In this study we demonstrate that the transcription factor Sp1 binds to these repeats and plays an important role in basal and in inducible expression of the murine interleukin-6 gene. PMID- 8631754 TI - Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin. AB - Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin. PMID- 8631755 TI - Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes. AB - CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype. However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions. To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal). Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta. Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta. Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta. Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression. PMID- 8631757 TI - Isolation and in vitro characterization of CheZ suppressors for the Escherichia coli chemotactic response regulator mutant CheYN23D. AB - The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein. We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sann, M.G., Swanson, R.V., Bourret, R.B., and Simon, M.I. (1995) Mol. Microbiol. 15, 1069-79). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY. PMID- 8631756 TI - Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain. PMID- 8631758 TI - Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha. AB - The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14 3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein. PMID- 8631759 TI - Functional expression of Escherichia coli endonuclease IV in apurinic endonuclease-deficient yeast. AB - Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of abasic (AP) sites or oxidative DNA strand breaks. Yeast lacking Apn1 (apn1-) are hypersensitive to simple alkylating agents (which produce many AP sites) and to oxidants and display an elevated spontaneous mutation rate due to endogenous damages. We explored whether the prokaryotic repair enzyme could substitute for its yeast counterpart. Plasmid constructs were generated that expressed endonuclease IV at 1/20 to 10-fold the AP endonuclease activity of wild-type yeast; some of these plasmids expressed hybrid forms of endonuclease IV equipped with the C-terminal nuclear localization signal of Apn1. Although hybrid endonuclease IV-Apn1 (but not native endonuclease IV) was selectively localized to the yeast nucleus, expression of this chimeric protein at 25% of the normal Apn1 level did not restore alkylation or oxidant resistance to apn1- yeast, but it did partially counteract the mutator phenotype of apn1- yeast. Expression of either the hybrid protein or native endonuclease IV at approximately 10 times the wild-type Apn1 levels restored wild-type resistance to methyl methanesulfonate and near-wild-type H2O2 resistance. High level expression of native endonuclease IV also restored the normal spontaneous mutation rate to apn1- yeast. These data place limits on the amounts of AP endonuclease activity necessary for repair of DNA damages caused by both endogenous and environmental agents and point to a direct role of spontaneous AP sites as potentially mutagenic lesions. PMID- 8631760 TI - Nerve growth factor stimulates the tyrosine phosphorylation of endogenous Crk-II and augments its association with p130Cas in PC-12 cells. AB - The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells. PMID- 8631761 TI - O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator. AB - DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator. PMID- 8631762 TI - Site-directed mutagenesis of recombinant sulfite oxidase: identification of cysteine 207 as a ligand of molybdenum. AB - Each of the four cysteines in rat sulfite oxidase was altered by site-directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli. Three of the replacements proved to be silent mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity. The C207S mutation was also generated in cloned human sulfite oxidase. The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis. The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation of the peak at 350 nm and a lesser decrease in absorbance from 450 600 nm as compared with the native human molybdenum domain. The molybdenum and molybdopterin contents of the two samples were comparable. These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal. PMID- 8631763 TI - FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria. AB - Respiratory defective mutants of Saccharomyces cerevisiae previously assigned to complementation group G178 are characterized by an abnormally low ratio of FAD/FMN in mitochondria. A nuclear gene, designated FLX1, was selected from a yeast genomic library, based on its ability to confer wild-type growth properties to a representative G178 mutant. Genetic evidence has confirmed that the flavin nucleotide imbalance of G178 mutants is caused by mutations in FLX1. The sequence of FLX1 is identical to a reading frame recently reported to be present on yeast chromosome IX (GenBank Z47047). The sequence and tripartite repeat structure of the FLX1 product (Flx1p) indicate it is a member of a protein family consisting of mitochondrial substrate and nucleotide carriers. In yeast, FAD synthetase is present in the soluble cytoplasmic protein fraction but not in mitochondria. Riboflavin kinase, the preceding enzyme in flavin biosynthesis, is present in both subcellular fractions. The absence of FAD synthetase in mitochondria implies that FAD is imported from the cytoplasm. The lower concentration of mitochondrial FAD in flx1 mutants suggests that Flx1p is involved in flavin transport, a role that is also supported by biochemical evidence indicating more efficient flux of FAD across mitochondrial membrane vesicles prepared from wild-type strains than membrane vesicles from flx1 mutants. PMID- 8631764 TI - Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid by 19F NMR. AB - Measurements of free calcium ion concentration in the sarcoplasmic reticulum ([Ca2+]SR) and an evaluation of its relationship to changes in cytosolic free calcium and energy state of the cell, as well as heterogeneity of the SR calcium pool, were performed using 19F NMR in Langendorff perfused rabbit hearts loaded with acetoxymethyl ester of 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N' tetraacetic acid. We report a base-line time-average [Ca2+]SR value of 1.5 mM (n = 13) in the beating heart, similar to the value measured at diastole. We further report that [Ca2+]SR decreases by approximately 30% at the start of systole and that there is no evidence of spacial heterogeneity in [Ca2+]SR during the contraction cycle. However, there appears to be a heterogeneous response to SR calcium channel release activator (caffeine) and SR calcium-ATPase inhibitor (cyclopiazonic acid), consistent with studies suggesting that there are subpopulations of SR. Raising cytosolic free calcium by depolarizing the cell with 30 mM extracellular KCl, resulted in an increase in [Ca2+]SR; however, the calcium gradient was unchanged. Lowering cell phosphorylation potential, which would reduce the free energy available for the SR Ca2+-ATPase, leads to a decrease in the calcium gradient across the SR, but this reduced gradient was primarily due to an increase in cytosolic free calcium and not a net release of SR calcium. PMID- 8631765 TI - A novel cytoplasmic hemimethylated oriC binding activity. AB - Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity. This activity appears to be located in the cytoplasm rather than in membrane fractions. It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC. These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site. The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity. The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect thte relative levels of this binding activity. A possible role in sequestration of the newly replicated hemimethylated origin is discussed. PMID- 8631766 TI - Induction of inducible nitric-oxide synthase by the heterotrimeric G protein Galpha13. AB - While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents. PMID- 8631767 TI - Apoptosis induced by transforming growth factor-beta in fetal hepatocyte primary cultures: involvement of reactive oxygen intermediates. AB - Transforming growth factor-beta (TGF-beta), a growth regulator of fetal hepatocytes in primary culture, also regulates death of these cells. Dose response analysis showed that the TGF-beta concentration needed to induce hepatocyte death (2.5 ng/ml) was 5 times that needed to inhibit growth in these cells (0.5 ng/ml). In response to TGF-beta, hepatocytes induced DNA fragmentation and the appearance of nuclei with a DNA content lower than 2C (diploid content), typical of a programmed cell death model. TGF-beta-induced apoptosis in fetal hepatocytes was preceded by an induction of reactive oxygen species production and a decrease in the glutathione intracellular content, indicating that this factor induces oxidative stress in fetal hepatocytes. Studies performed to analyze levels of c-fos mRNA, a gene whose expression is modulated by redox state, demonstrated that only high, apoptotic concentrations of TGF-beta (2.5 ng/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of tert-butyl hydroperoxide. Gel mobility shift assays showed that the c-fos induced expression was coincident with an increase in AP-1 activity. Finally, cell death induced by TGF-beta in fetal hepatocytes was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta in these cells. In summary, the results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta in fetal hepatocytes. PMID- 8631768 TI - Selective screening of a large phage display library of plasminogen activator inhibitor 1 mutants to localize interaction sites with either thrombin or the variable region 1 of tissue-type plasminogen activator. AB - Phage display technology has been exploited to study in detail the interaction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged "loop" of tissue-type plasminogen activator (t-PA), denoted variable region 1 (VR1). For this purpose, a PAI-1 mutant phage library was used that served as a reservoir of PAI-1 proteins potentially deficient in the interaction with either VR1 or thrombin. A stringent two-step selection procedure was developed. (i) A negative selection was performed by incubating the pComb3/PAI-1 mutant library with an excess of a thrombin mutant with its VR1 domain substituted with that of t-PA (thrombin-VR1). (ii) The remaining phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody. Four consecutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant phages of approximately 50-fold. Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR1, revealed the following mutations. Four independent variants share a mutation of the P4' residue (Glu350 --> Lys). Nine independent PAI-1 variants share a substitution of P1' (Met347 --> Lys), whereas three others share a P2 substitution (Ala345 --> Asp). Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residue is essential for the interaction with the VR1 domain, consistent with the data of Madison et al. (Madison, E.L., Goldsmith, E.J., Gething, M.J., Sambrook, J.F., and Gerard, R.D. (1990) J. Biol. Chem. 265, 21423-21426), whereas the P1' and P2 residues confer thrombin specificity. Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interaction with either thrombin or VR1. This study demonstrates that phage technology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequate selection scheme is devised. PMID- 8631769 TI - The accessibility of yeast ribosomal protein L1 as probed by proteolysis and site directed mutagenesis is different in intact 60 and 80 S ribosome. AB - Accessible regions of protein L1 in intact 60 and 80 S ribosomes from Saccharomyces cerevisiae were first detected by controlled proteolysis. The N terminal region of L1 in either 60 S or 80 S particles, was inaccessible to proteases, but the central and C-terminal regions were accessible. The accessibility of the central region differed depending on the ribosome state. These regions were further examined by determination of the chemical reactivity of specific cysteine residues introduced into these regions by site-directed mutagenesis. All cysteine mutant proteins were capable of binding yeast 5 S rRNA in vitro and the ribosomes containing the mutant proteins were functional in vivo. Residues Cys-257 and Cys-275 were modified in both the 60 and 80 S ribosomes but the modification rates were different in the two ribosome states. Both residues Cys-62 and Cys-286 were inaccessible in 80 S or 60 S ribosomes. Taken together, the present study identified several accessible regions of L1 in intact ribosomes and further showed that the accessibility of some of the regions was altered upon ribosomal subunit association. The most likely interpretation of these results is that the conformation of the ribosomal protein L1 was altered upon ribosomal subunit association. PMID- 8631770 TI - Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs. AB - Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3' hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex. PMID- 8631771 TI - Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. AB - Most members of the Bcl-2 protein family of apoptosis regulating proteins contain two evolutionarily conserved domains, termed BH1 and BH2. Both BH1 and BH2 in the Bcl-2 protein are required for its function as an inhibitor of cell death and for heterodimerization with the proapoptotic protein Bax. In this report, we mapped the region in Bax required for heterodimerization with Bcl-2 and homodimerization with Bax, using yeast two-hybrid and in vitro protein-protein interaction assays. Neither the BH1 nor the BH2 domain of Bax was required for binding to the wild type Bcl-2 and Bax proteins. Moreover, Bax (deltaBH1) and Bax (deltaBH2) mutant proteins bound efficiently to themselves and each other, further confirming the lack of requirement for BH1 and BH2 for Bax/Bax homodimerization. Bax/Bax homodimerization was not dependent on the inclusion of the NH2-terminal 58 amino acids of the Bax protein in each dimerization partner, unlike Bcl-2/Bcl-2 homodimers which involve head-to-tail interactions between the region of Bcl-2 where BH1 and BH2 resides, and an NH2-terminal domain in Bcl-2 that contains another domain BH4 which is conserved among antiapoptotic members of the Bcl-2 family. Similarly, heterodimerization with Bcl-2 occurred without the NH2 terminal domain of either Bax or Bcl-2, suggesting a tail-to-tail interaction. The essential region in Bax required for both homodimerization with Bax and heterodimerization with Bcl-2 was mapped to residues 59-101. This region in Bax contains a stretch of 15 amino acids that is highly homologous in several members of the Bcl-2 protein family, suggesting the existence of a novel functional domain which we have termed BH3. Deletion of this 15-amino acid region abolished the ability of Bax to dimerize with itself and to heterodimerize with Bcl-2. The findings suggest that the structural features of Bax and Bcl-2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino-acid sequence similarity. PMID- 8631772 TI - Active human cytomegalovirus protease is a dimer. AB - The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 microM in 10% glycerol and 0.55 microM in 20% glycerol at 30 degrees C and pH 7.5. PMID- 8631773 TI - Molecular cloning of a developmentally regulated N-acetylgalactosamine alpha2,6 sialyltransferase specific for sialylated glycoconjugates. AB - A cDNA encoding a novel sialyltransferase has been isolated employing the polymerase chain reaction using degenerate primers to conserved regions of the sialylmotif that is present in all eukaryotic members of the sialyltransferase gene family examined to date. The cDNA sequence revealed an open reading frame coding for 305 amino acids, making it the shortest sialyltransferase cloned to date. This open reading frame predicts all the characteristic structural features of other sialyltransferases including a type II membrane protein topology and both sialylmotifs, one centrally located and the second in the carboxyl-terminal portion of the cDNA. When compared with all other sialyltransferase cDNAs, the predicted amino acid sequence displays the lowest homology in the sialyltransferase gene family. Northern analysis shows this sialyltransferase to be developmentally regulated in brain with expression persisting through adulthood in spleen, kidney, and lung. Stable transfection of the full-length cDNA in the human kidney carcinoma cell line 293 produced an active sialyltransferase with marked specificity for the sialoside, Neu5Ac-alpha2,3Gal beta1,3GalNAc and glycoconjugates carrying the same sequence such as G(M1b) and fetuin. The disialylated tetrasaccharide formed by reacting the sialyltransferase with the aforementioned sialoside was analyzed by one- and two-dimensional 1H and 13C NMR spectroscopy and was shown to be the Neu5Ac-alpha2,3Gal-beta1,3(Neu5Ac alpha2,6)GalNAc sialoside. This indicates that the enzyme is a GalNAc alpha-2,6 sialyltransferase. Since two other ST6GalNAc sialyltransferase cDNAs have been isolated, this sialyltransferase has been designated ST6GalNAc III. Of these three, ST6GalNAc III displays the most restricted acceptor specificity and is the only sialyltransferase cloned to date capable of forming the developmentally regulated ganglioside G(D1alpha) from G(M1b). PMID- 8631774 TI - Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion. AB - Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate cytokines that act locally and influence functional aspects of many cell types. We have previously defined functional epithelial receptors for interferon-gamma, interleukin (IL)-4, and a recently described IL-4-like cytokine IL-13. In this study, we examine the ion transport properties of T84 cells, a crypt-like epithelial cell line, following exposure to IL-4 and IL-13. Basolateral exposure of epithelial monolayers to both IL-4 and IL-13 attenuated epithelial barrier function and increased paracellular flux of a dextran marker by greater than 65% in a dose- and time-dependent fashion. Stimulated Cl- secretion, as measured by epithelial short circuit current, however, was diminished only by IL-4 and not IL 13, demonstrating cytokine specificity in this epithelial function. Decreased Cl- secretion following IL-4 exposure was associated with diminished Cl- channel activity and IL-4 pretreatment of epithelia decreased expression of the cystic fibrosis transmembrane regulator. Finally, stimulated fluid transport across cultured epithelia was diminished following exposure to IL-4, but not IL-13. These results indicate that while post-receptor signaling events induced by IL-13 and IL-4 may be similar, end point function is cytokine-specific. PMID- 8631775 TI - Detection of a physical and functional interaction between Csk and Lck which involves the SH2 domain of Csk and is mediated by autophosphorylation of Lck on tyrosine 394. AB - The COOH-terminal Src kinase (Csk) is responsible for the phosphorylation of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases. Up to now, no stable binding of Csk to Src kinases has been detected. We therefore decided to analyze this interaction using two systems which allow detection of transient interaction. We produced and purified recombinant proteins in the glutathione S-transferase prokaryotic expression system. First, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its substrates Lck, a lymphocyte-specific member of the Src family. This interaction requires the autophosphorylation of Lck on tyrosine 394 (the phosphorylation of which is correlated with an increase of the kinase activity) and involves a functional Csk SH2 domain. Second, using the yeast two-hybrid system, we confirmed in vivo the physical interaction between Csk and Lck. Furthermore, in vitro we showed that autophosphorylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk. These findings might explain the mechanism(s) by which Csk interacts with most of Src kinases to down-regulate their kinase activity. PMID- 8631776 TI - Bovine hemoglobin cross-linked through the beta chains: functional and structural aspects. AB - 2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch. The reaction of bovine Hb with NFPLP leads to a cross-linkage between the beta subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into alpha beta dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross link of beta chains may be formed between lysine 82 of beta2 and the N terminus of methionine 2 of beta1 or between the two lysine 82 residues of both beta2 chains. Digestion of modified beta globin chains and isolation of the cross linked peptide have showed that NFPLP cross-links Met-beta2 and Lys-beta82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute. PMID- 8631777 TI - Blocking the Ca2+-induced conformational transitions in calmodulin with disulfide bonds. AB - Calcium-dependent regulation of intracellular processes is mediated by proteins that on binding Ca2+ assume a new conformation, which enables them to bind to their specific target proteins and to modulate their function. Calmodulin (CaM) and troponin C, the two best characterized Ca2+-regulatory proteins, are members of the family of Ca2+-binding proteins utilizing the helix-loop-helix structural motif (EF-hand). Herzberg, Moult, and James (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca2+-induced conformational transition in troponin C involves opening of the interface between the alpha-helical segments in the N-terminal domain of this protein. Here we have tested the hypothesis that a similar transition is the key Ca2+-induced regulatory event in calmodulin. Using site-directed mutagenesis we have substituted cysteine residues for Gln41 and Lys75 (CaM41/75) or Ile85 and Leu112 (CaM85/112) in the N-terminal and C-terminal domains, respectively, of human liver calmodulin. Based on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca2+-free conformation of the protein, thus blocking the putative Ca2+-induced transition. We found that intramolecular disulfide bonds are readily formed in both mutants causing a decrease in affinity for Ca2+ and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin. The regulatory activity is fully recovered in CaM41/75 and partially recovered in CaM85/112 upon reduction of the disulfide bonds with dithiothreitol and blocking the Cys residues by carboxyamidomethylation or cyanylation. These results indicate that the Ca2+ induced opening of the interfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult James model. PMID- 8631778 TI - LEC14, a dominant Chinese hamster ovary glycosylation mutant expresses complex N glycans with a new N-acetylglucosamine residue in the core region. AB - The Chinese hamster ovary cell (CHO) glycosylation mutant, LEC14, was previously selected for resistance to pea lectin (Pisum sativum agglutinin) and shown to behave dominantly. The lectin resistance properties of LEC14 cells are related to, but distinct from, those of LEC18, a dominant Chinese hamster ovary mutant that synthesizes complex N-glycans with a novel O-6-linked GlcNAc residue in the core region (Raju, T.S., Ray, M., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302). Detailed structural studies of a complex N-glycan fraction from LEC14 cells have revealed yet another novel modification of the core region. [3H]Glc-labeled LEC14 cellular glycopeptides were desialylated, and the fraction that did not bind to concanavalin A-Sepharose was found to have an increased proportion of species that bound to tomato-agarose, and to ricin-agarose. 1H NMR spectroscopy and methylation linkage analysis of the tomato and ricin-bound fractions purified from approximately 10(10) LEC14 cells showed they were complex N-glycans containing a 2,3,6-trisubstituted core Man residue. To examine the core region more closely, these N-glycans were digested with mixtures of beta-D galactosidases and N-acetyl-beta-D-glucosaminidases to obtain core glycopeptides. The latter were largely unbound by concanavalin A-Sepharose or pea lectin agarose. 1H NMR spectroscopy and electrospray ionization-mass spectrometry showed that the LEC14 core glycopeptides contain a new GlcNAc residue that substitutes the core beta(1-4)-Man residue at O-2 to give the following novel, N-linked core structure. [structure: see text] PMID- 8631779 TI - Identification of the high affinity receptor binding region in human immunoglobulin E. AB - We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition. PMID- 8631780 TI - A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyl diacylglycerol. AB - The sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol is associated with the thylakoid membranes of many photosynthetic organisms. Previously, genes involved in sulfolipid biosynthesis have been characterized only in the purple bacterium Rhodobacter sphaeroides. Unlike plants and cyanobacteria, photosynthesis in this bacterium is anoxygenic due to the lack of a water splitting photosystem II. To test the function of sulfolipid in an organism with oxygenic photosynthesis, we isolated and inactivated a sulfolipid gene of the cyanobacterium Synechococcus sp. PCC7942. Extensive analysis of the sulfolipid-deficient null mutant revealed subtle changes in photosynthesis related biochemistry of O2. In addition, a slight increase in the variable room temperature chlorophyll fluorescence yield was observed. Regardless of these changes, it seems unlikely that sulfolipid is an essential constituent of a functional competent water oxidase or the core antenna complex of photosystem II. However, reduced growth of the mutant under phosphate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids under phosphate-limiting growth conditions. PMID- 8631781 TI - Synergistic induction of osteocalcin gene expression: identification of a bipartite element conferring fibroblast growth factor 2 and cyclic AMP responsiveness in the rat osteocalcin promoter. AB - Fibroblast growth factors (FGFs) are important regulators of calvarial osteoblast growth and differentiation. We have studied the regulation of the osteoblast specific gene osteocalcin (OC) by FGF2 in phenotypically immature MC3T3-E1 calvarial osteoblastic cells. FGF2 markedly induces OC mRNA accumulation in MC3T3 E1 cells in the presence of forskolin (FSK). Similarly, OC promoter activity (luciferase reporter) is up-regulated 6-10-fold by FGF2/FSK or by FGF2/8-bromo cyclic AMP. Half-maximal induction of OC promoter activity occurs at 1 nM FGF2. By 5' deletion analysis and dinucleotide point mutations, we map one component of this FGF2/FSK response to a GCAGTCA motif in the region -144 to -138 relative to the OC transcription initiation site. The OC promoter region -154 to -90 confers FGF2/FSK responsiveness on the Rous sarcoma virus minimal promoter. By 3' and internal deletion analyses, the region between -90 to -99 is also found to be necessary for FGF2/FSK synergy (encodes a PuGGTCA motif previously identified as a component of FSK induction). A DNA binding activity that recognizes the region 148 to -125 of the rat OC promoter is induced in crude nuclear extracts from MC3T3-E1 cells treated with FGF2 or FGF2/FSK. This binding activity is sequence specific and does not recognize the TCAGTCA DNA cognate of AP1. Members of the ATF, Fos, and Jun family are not immunologically detected in this inducible DNA binding activity. However, transient co-expression of ATF3 but not ATF2 selectively attenuates the FGF2 component of induction. Thus, a novel FGF2 regulated DNA-protein interaction in the OC promoter participates in the transcriptional control of OC expression by FGF and cyclic AMP in MC3T3-E1 calvarial osteoblasts. PMID- 8631782 TI - Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa. AB - Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined. PMID- 8631783 TI - A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. AB - We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes. PMID- 8631784 TI - Tissue-specific expression and dietary regulation of chimeric mitochondrial 3 hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice. AB - We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium , and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice. PMID- 8631785 TI - Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family: functional expression requires C-terminal truncation. AB - The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not. PMID- 8631786 TI - Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95vav, in platelets. AB - The vav proto-oncogene product, p95vav or Vav, is primarily expressed in hematopoietic cells and has been shown to be a substrate for tyrosine kinases. Although its function is unknown, Vav shares a region of homology with DBL, an exchange factor for the Rho family of GTP-binding proteins. The presence of this domain and the observation that cells transformed with Vav display prominent stress fibers and focal adhesions similar to those that are observed in RhoA transformed cells suggests that Vav may play a role in regulating the actin cytoskeleton. We have, therefore, examined Vav phosphorylation in platelets, which undergo dramatic cytoskeletal reorganization in response to agonists. Two potent platelet agonists, thrombin (via its G protein-coupled receptor) and collagen (via its interaction with the alpha2beta1 integrin), caused Vav to become phosphorylated on tyrosine. Weaker platelet agonists, including ADP, epinephrine and the thromboxane A2 analog, U46619, did not. The phosphorylation of Vav in response to thrombin was maximal within 15 s and was unaffected by aspirin, inhibitors of aggregation, or the presence of the ADP scavenger, apyrase. Vav phosphorylation was also observed when platelets became adherent to immobilized collagen (via integrin alpha2beta1), fibronectin (via integrin alpha5beta1), and fibrinogen (via integrin alphaIIbbeta3). These results show that Vav phosphorylation by tyrosine kinases 1) occurs during platelet activation by potent agonists, 2) also occurs when platelets adhere to biologically relevant matrix proteins, 3) requires neither platelet aggregation nor the release of secondary agonists such as ADP and TxA2, and 4) can be initiated by at least some members of two additional classes of receptors, G protein-coupled receptors and integrins, providing further evidence that both of these can couple to tyrosine kinases. PMID- 8631787 TI - Molecular cloning and functional expression of murine JE (monocyte chemoattractant protein 1) and murine macrophage inflammatory protein 1alpha receptors: evidence for two closely linked C-C chemokine receptors on chromosome 9. AB - We have isolated cDNA clones that encode two closely related, murine C-C chemokine receptors. Both receptors are members of the G-protein-coupled, seven transmembrane domain family of receptors and are most closely related to the human monocyte chemoattractant protein 1 receptor. Expression of each of the receptors was detected in murine monocyte/macrophage cell lines, but not in nonhematopoietic lines. Expression of these receptors in Xenopus oocytes revealed that one receptor signaled in response to low nanomolar concentrations of murine JE, whereas the second receptor was activated by murine macrophage inflammatory protein (MIP) 1alpha and the human chemokines MIP-1beta and RANTES. Binding studies revealed high affinity binding of radiolabeled mJE to the mJE receptor and murine MIP-1alpha to the second receptor. Chromosomal localization indicated that the two receptor genes were clustered within 80 kilobases of each other on mouse chromosome 9. Creation of receptor chimeras suggested that the amino terminus was critically involved in mediating signal transduction and ligand specificity of the mJE receptor, but not the mMIP-1alpha receptor. The identification and cloning of two functional murine chemokine receptors provides important new tools for investigating the roles of these potent cytokines in vivo. PMID- 8631788 TI - Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase. AB - Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase. Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio 5'-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical. PMID- 8631789 TI - Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers. AB - The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12 depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10. PMID- 8631790 TI - Two distinct pathways for histamine H2 receptor down-regulation. H2 Leu124 --> Ala receptor mutant provides evidence for a cAMP-independent action of H2 agonists. AB - Pretreatment of Chinese hamster ovary cells expressing the histamine H2 receptor (CHOrH2 cells) with histamine resulted in a time-dependent (t1/2 approximately 7 h) and dose-dependent (EC50=18 nM) H2 receptor down-regulation measured as [125I]iodoaminopotentidine binding (44+/-10% down-regulation). Pretreatment of CHOrH2 cells with cholera toxin or forskolin also led to H2 receptor down regulation. Forskolin time-dependently (t1/2 approximately 7 h) and dose dependently (EC50 = 0.3 microM) induced H2 receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H2 receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O. Lameh, J., Hogger, P., and Sadee, W. (1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H2 receptor from the Gs-protein, we introduced the Leu124 --> Ala mutation in the second intracellular loop of the H2 receptor. The H2 Leu124 --> Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH2 cells, H2 receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP independent pathway. PMID- 8631791 TI - A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane. AB - Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transmembrane segment that inserts into the endoplasmic reticulum membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal endoplasmic reticulum insertion signals. PMID- 8631792 TI - Granulocyte-macrophage colony-stimulating factor provokes RAS activation and transcription of c-fos through different modes of signaling. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF) provokes a proliferative response and induction of early-response genes such as c-fos in target cells. It also induces rapid tyrosine phosphorylation of cellular proteins, including the beta subunit (betac) of its functional receptor. However, locations and functions of phosphorylated tyrosine residues within the betac are unclear. To elucidate the mechanism of the human GM-CSF receptor signal transduction, mutational analyses were made of the cytoplasmic domain of the beta c, using murine BA/F3 cells. Deletion of the conserved box 1 motif resulted in loss of tyrosine phosphorylation of the betac, thereby indicating an essential role for this motif in activating the tyrosine kinase which phosphorylates betac. A C-terminal truncated mutant at position 589 activated the c-fos promoter, and this activation was diminished by a substitution at tyrosine 577 (Tyr577). However, the same substitution in the full-length betac did not completely abrogate the c-fos promoter activation, hence, redundant signaling pathways probably exist. When we analyzed signaling molecules functioning downstream of the beta-c we found that Tyr577 is essential for Shc phosphorylation, while tyrosine phosphorylation of PTP1D was mediated through Tyr577 as well as through other site(s). We suggest that GM-CSF stimulates at least two modes of signals leading to Ras activation, an event which ultimately gives rise to promoter activation of c-fos. PMID- 8631793 TI - Biochemical and biophysical analyses of recombinant forms of human topoisomerase I. AB - Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells. Each protein is inhibited by camptothecin, topotecan, and 9-aminocamptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine. The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration. Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory. The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different. Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended. A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1 174 (approximately 21 kDa) of Topo I are largely if not completely unfolded. This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity. PMID- 8631795 TI - Slow binding inhibition of phospho-N-acetylmuramyl-pentapeptide-translocase (Escherichia coli) by mureidomycin A. AB - Enzymes of the membrane cycle of reactions in bacterial peptidoglycan biosynthesis remain as unexploited potential targets for antibacterial agents. The first of these enzymes, phospho-N-acetylmuramyl-pentapeptide-translocase (EC 2.7.8.13), has been overexpresed in Escherichia coli and solubilized from particulate fractions. The work of W.A. Weppner and F.C. Neuhaus ((1977) J. Biol. Chem. 252, 2296-303) has been extended to establish a usable routine fluorescence based continuous assay for solubilized preparations. This assay has been used in the characterization of the natural product, mureidomycin A as a potent slow binding inhibitor of the enzyme with Ki and Ki* of 36 nM and 2 nM, respectively. PMID- 8631794 TI - The domain organization of human topoisomerase I. AB - Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies. PMID- 8631796 TI - Localization of Ca2+ extrusion sites in pancreatic acinar cells. AB - We have investigated the localization of Ca2+ extrusion sites in mouse pancreatic acinar cells. Employing a new technique, in which high resolution localization of cellular Ca2+ exit is achieved by confocal microscopy and a Ca2+-sensitive fluorescent probe coupled to heavy dextran to slow down diffusion of extracellular Ca2+, it is shown directly that the secretory pole (secretory granule area) is the major site for Ca2+ extrusion following agonist stimulation. This Ca2+ extrusion appears not to be a consequence of exocytosis, as assessment of secretion under our experimental conditions (low external Ca2+ concentration, room temperature) using the technique of monitoring quinacrine fluorescence shows little loss of secretory granules in spite of sustained Ca2+ exit. We conclude that Ca2+ is primarily extruded by Ca2+ pumps from the secretory pole and propose that this process is useful for maintaining a high Ca2+ concentration in the acinar lumen, which is necessary for promotion of endocytosis. PMID- 8631797 TI - An immunological approach reveals biological differences between the two NDF/heregulin receptors, ErbB-3 and ErbB-4. AB - The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB 3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB 3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB 2, is essential for transmission of NDF signals through ErbB-3. PMID- 8631798 TI - Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi synthetic antibody phage display library. AB - This report describes the construction of leucine zipper-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers. A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library. Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments. The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts. The bivalent (scFv)2 fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis. Fos and Jun homodimer (scFv)2 antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv)2 Fos-Jun heterodimers. These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity. PMID- 8631800 TI - Axonal amyloid precursor protein expressed by neurons in vitro is present in a membrane fraction with caveolae-like properties. AB - In cortical neurons differentiating in vitro, transmembrane amyloid precursor protein (APP) is distributed in two pools. Whereas the first pool is present in all cell compartments, the second pool is highly enriched in the axon and cell body. In an earlier study we demonstrated that this second pool, referred to as axonal-APP (Ax-APP), is present in the vicinity of the plasma membrane and colocalizes only partially with clathrin (Allinquant, B., Moya, K.L., Bouillot, C., and Prochiantz, A. (1994) J. Neurosci. 14, 6842-6854). In this report, using immunocytochemical and fractionation techniques we demonstrate that Ax-APP is present in microdomains enriched in the glypiated glycoprotein F3. The F3/Ax-APP microdomains are resistant to nonionic detergents and sediment at low density on a sucrose gradient. The two latter properties are reminiscent of those of caveolae, a type of plasmalemmal vesicle found in several cell types, but not previously described in the nervous system due to the absence of caveolin in neurons. The presence of Ax-APP in caveolae-like vesicles raises the possibility that APP serves as a transmembrane signaling molecule for GPI-linked glycoproteins. In addition, our data support new hypotheses on the endocytic pathways leading to the production of the amyloidogenic betaA4 peptide. PMID- 8631799 TI - A family of genes coding for two serologically distinct chicken interferons. AB - Southern blot analysis and screening of a genomic lambda phage library with the previously cloned chicken interferon (IFN) cDNA indicated that the chicken genome contains at least 10 IFN genes. A particularly strongly hybridizing phage clone that we analyzed in more detail carried a head to tail arrangement of three intron-less IFN genes that differed from each other and from the cloned chicken IFN cDNA by only a few base changes. The primary translation products of these three IFN genes consist of 193 amino acids, and the mature proteins are composed of 162 amino acids. All three genes of this IFN family, designated IFN1, yielded active chicken IFN when expressed individually in transfected COS7 cells. A weakly hybridizing phage clone contained an additional intron-less chicken IFN gene, designated IFN2, whose product was 57% identical to chicken IFN1. Southern blot analysis suggested that the chicken genome contains a single IFN2 gene. The primary translation product of IFN2 consists of 203 amino acids, and the mature protein is composed of 176 amino acids. Purified recombinant chicken IFN2 from Escherichia coli had a specific antiviral activity of about 10(6) units/mg, which was about 20-fold lower than that of chicken IFN1 purified in parallel. The antiviral activity of chicken IFN2 from E. coli or from transfected COS7 cells could not be neutralized by antiserum to recombinant chicken IFN1. Thus, like mammals, the chicken has a large number of type I IFN genes that code for at least two serologically distinct antiviral activities. PMID- 8631801 TI - Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells. AB - Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5' phosphosulfate was 0.20 microM. PMID- 8631802 TI - The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins. AB - Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process. The first step, which is to dock the NLS containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-alpha. Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-alpha and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box. We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding. Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1alpha/importin-alpha and hSrp1/karyopherin alpha/NPI-1, respectively, and show considerable sequence divergence from each other. We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence. PMID- 8631803 TI - Signal transduction through the beta1 integrin family surface adhesion molecules VLA-4 and VLA-5 of human B-cell precursors activates CD19 receptor-associated protein-tyrosine kinases. AB - We demonstrate that the CD19 receptor associates with the beta1 family integrin receptors on human B-cell precursors as well as mature B-lymphocytes, and engagement of the beta1 family integrin receptors with monoclonal antibody homoconjugates leads to rapid activation of the CD19-associated protein-tyrosine kinases (PTK) and results in hyperphosphorylation of CD19 on tyrosine residues. Our findings prompt the hypothesis that homoconjugate-induced integrin clustering may effect the approximation and, by intermolecular cross-phosphorylation, activation of the CD19-associated PTK and subsequent tyrosine phosphorylation of the CD19 receptor. The ability of the beta1 family integrin receptors to transmit a biochemical signal triggering the CD19-linked multifunctional PTK pathway provides a possible explanation for the pleiotropic biologic responses generated though adhesive VLA-4- and VLA-5-mediated contacts. PMID- 8631805 TI - Activated neu induces rapid tumor progression. AB - Expression of the activated neu oncogene in transgenic mice has been associated with both the synchronous (single-step) and the stochastic (multistep) transformation of the mammary epithelium. To determine the basis for these conflicting observations, additional strains of transgenic mice carrying the activated neu oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were produced. Activated neu transgene expression, as measured by in situ hybridization and ribonuclease protection assays, resulted in rapid conversion of the normal mammary epithelium to malignant phenotype in three independent strains of mice. Expression of the transgene in male mice led to epithelial hyperplasia of the epididymis and male infertility but not malignancy. These results indicate that tissue context is an important parameter in malignant progression and that expression of appropriate levels of activated neu is sufficient for rapid production of mammary tumors in transgenic mice. PMID- 8631804 TI - Mutations in the cytoplasmic domain of the integrin beta1 chain indicate a role for endocytosis factors in bacterial internalization. AB - Mutations that result in defective beta1-integrin focal adhesion formation were analyzed for effects on bacterial internalization. Mutations in the cytoplasmic domain of the beta1 chain that disrupt the sequence NPIY resulted in integrins deficient in bacterial uptake. Other mutations in the beta1 chain that reduced cytoskeletal association showed enhanced bacterial uptake. Replacement of the NPIY sequence of the beta1 subunit by the endocytosis internalization sequence PPGY resulted in integrin receptors highly proficient in bacterial internalization, yet severely defective in focal contact localization. Electron microscopy indicated that coated structures associated specifically with bacteria binding beta1-integrins, with an apparent recruitment of coated pits from ventral cell surfaces to apical surfaces corresponding to nascent bacterial phagosomes. Clathrin inhibition studies indicated a role for the adaptor molecule AP2 as well as clathrin in integrin-mediated bacterial internalization. These results indicate that association of beta1-integrins with the cytoskeleton at focal contacts interferes with integrin-mediated bacterial internalization. Also, although actin polymerization is required for bacterial uptake, clathrin is probably involved in bacterial uptake promoted by beta-1-integrins. PMID- 8631806 TI - Characterization of carbohydrate-binding protein p33/41: relation with annexin IV, molecular basis of the doublet forms (p33 and p41), and modulation of the carbohydrate binding activity by phospholipids. AB - A protein, p33/41, expressed in bovine kidney and many other tissues was identified as a lectin which binds to sialoglycoproteins and glycosaminoglycans in a calcium-dependent manner. Partial amino acid sequences of p33/41 are highly homologous to those of calcium/phospholipid-binding annexin protein, annexin IV (endonexin), p33/41 exhibited similar calcium/phospholipid-binding activity (Kojima, K., Ogawa, H., Seno, N., Yamamoto, K., Irimura, T., Osawa, T., and Matsumoto, I. (1993) J. Biol. Chem. 267, 20536-20539). To further characterize p33/41, we cloned the p33/41 cDNA and characterized the recombinant protein encoded by this cDNA. Oligonucleotide probes were synthesized based on partial amino acid sequences of p33/41 and used for screening. A p33/41 cDNA clone was isolated encoding a protein of 319 amino acids with a calculated molecular mass of 35,769 Da. The deduced amino acid sequence was identical to that of bovine annexin IV except for one amino acid substitution. The recombinant protein gave two 33-kDa (p33) and 41-kDa (p41) bands on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and only one 33-kDa band under reducing conditions, as did the native protein. Mass spectrometric analysis combined with site-directed mutagenesis of each of the four cysteine residues of the recombinant protein revealed that p41 is a dimer of p33 cross-linked at Cys-198 via a disulfide bond. The recombinant protein bound to columns of heparin and fetuin glycopeptides in a calcium dependent manner and to phospholipid vesicles composed of phosphatidylserine (PS)/phosphatidylcholine (PC), phosphatidylethanolamine (PE)/PC or phosphatidylinositol (PI)/PC. Furthermore, concurrent binding assays showed that the binding of the recombinant protein to phospholipid vesicles was not affected by heparin, whereas that to heparin was influenced by the phospholipid composition of the vesicles; the highest binding was observed with vesicles composed of PE/PC. These results suggest that p33/41 binds two types of ligands via different sites and that phospholipids modulate the carbohydrate binding activity of p33/41. PMID- 8631807 TI - Mouse T cell membrane proteins Rt6-1 and Rt6-2 are arginine/protein mono(ADPribosyl)transferases and share secondary structure motifs with ADP ribosylating bacterial toxins. AB - Mono ADP-ribosylation is a posttranslational protein modification that has been implicated in the regulation of key biological functions in bacteria as well as in animals. Recently, the first cDNAs for eucaryotic mono(ADPribosyl)transferases were cloned and found to exhibit significant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6. In this paper we describe secondary structure analyses of Rt6 and related proteins and show conserved structure motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreover, we have expressed soluble mouse Rt6-1 and Rt6-2 gene products in which C-terminal tags (FLAG-His6) replace the native glycosylphosphatidylinositol anchor signal sequences. Purified recombinant Rt6-2, but not Rt6-1, shows NAD+ glycohydrolase activity, which is inhibited by the arginine analogue agmatine. Immunoprecipitation of recombinant Rt6-1 and Rt6-2 with anti-FLAG M2 antibody followed by incubation with [32P]NAD+ leads to rapid and covalent incorporation of radioactivity into the light chain of the M2 antibody. The bound label is resistant to treatment with HgCl2 but sensitive to NH2OH, characteristic of arginine-linked ADP-ribosylation. These results demonstrate that Rt6-1 and RT6-2 possess the enzymatic activities typical for NAD+-dependent arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31). They are the first such enzymes to be molecularly characterized in the immune system. PMID- 8631808 TI - Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa. AB - Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25. PMID- 8631809 TI - Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway. AB - The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner. PMID- 8631810 TI - Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. AB - A lysophospholipase was purified 506-fold from rat liver supernatant. The preparation gave a single 24-kDa protein band on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8. The purified enzyme was used for the preparation of antibody and peptide sequencing. A cDNA clone was isolated by screening a rat liver lambda gt11 cDNA library with the antibody, followed by the selection of further extended clones from a lambda gt10 library. The isolated cDNA was 2,362 base pairs in length and contained an open reading frame encoding 230 amino acids with a Mr of 24,708. The peptide sequences determined were found in the reading frame. When the cDNA was expressed in Escherichia coli cells as the beta-galactosidase fusion, lysophosphatidylcholine hydrolyzing activity was markedly increased. The deduced amino acid sequence showed significant similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase. The three sequences contained the GXSXG consensus at similar positions. The transcript was found in various tissues with the following order of abundance: spleen, heart, kidney, brain, lung, stomach, and testis = liver. In contrast, the enzyme protein was abundant in the following order: testis, liver, kidney, heart, stomach, lung, brain, and spleen. Thus the mRNA abundance disagreed with the level of the enzyme protein in liver, testis, and spleen. When HL-60 cells were induced to differentiate into granulocytes with dimethyl sulfoxide, the 24-kDa lysophospholipase protein increased significantly, but the mRNA abundance remained essentially unchanged. Thus a posttranscriptional control mechanism is present for the regulation of 24-kDa lysophospholipase. PMID- 8631811 TI - A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization. AB - Structural studies of human immunodeficiency virus type 1 (HIV-1) integrase have been impeded by the low solubility of the protein. By systematic replacement of hydrophobic residues, we previously identified a single amino acid change (F185K) that dramatically improved the solubility of the catalytic domain of HIV-1 integrase and enabled the structure to be determined by x-ray crystallography. We have introduced the same mutation into full-length HIV-1 integrase. The resulting recombinant protein is soluble and fully active in vitro, whereas, HIV-1 carrying the mutation is replication-defective due to improper virus assembly. Analysis of the recombinant protein by gel filtration and sedimentation equilibrium demonstrate a dimer-tetramer self-association. We find that the regions involved in multimerization map to both the catalytic core and carboxyl-terminal domains. The dramatically improved solubility of this protein make it a good candidate for structural studies. PMID- 8631812 TI - The effect of amino acid substitutions in the conserved aromatic region of subunit II of cytochrome c oxidase in Saccharomyces cerevisiae. AB - Mitochondrial encoded subunit II of cytochrome c oxidase carries the metal center, which acts as the initial acceptor of electrons from cytochrome c. Among the conserved features of this protein is a region in which five aromatic and three non-aromatic amino acids are conserved in a wide variety of organisms. This aromatic region has been postulated to be involved in transfer of electrons from the copper center in subunit II to the remaining metal centers of cytochrome oxidase in subunit I. To test the functional importance of two conserved, aromatic tryptophan residues and one conserved, non-aromatic glycine residue, yeast strains with alterations at these positions were characterized. The strains with altered codons were tested for their ability to carry out cellular respiration, for their growth rates on non-fermentable carbon sources, and for their cytochrome c oxidase activity. The results demonstrate that the aromatic character of the tryptophan residues appears necessary for subunit II function, while the conserved glycine can be replaced with other, small, uncharged residues. PMID- 8631813 TI - Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. AB - The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma. PMID- 8631814 TI - There are three distinct forms of bombesin. Identification of [Leu13]bombesin, [Phe13]bombesin, and [Ser3,Arg10,Phe13]bombesin in the frog Bombina orientalis. AB - Amphibian bombesin is the prototypic peptide that defines the bombesin-like peptide family. In this paper we show that in the frog Bombina orientalis, there are actually 3 distinct forms of bombesin, and each of these peptides is an agonist with differing affinities for the known bombesin receptors. Oligonucleotides complementary to the 5'- and 3'-untranslated regions of the bombesin mRNA were used to amplify bombesin-related cDNAs from the skin, brain, and gut of B. orientalis. Three classes of cDNAs were found. One class encoded the previously characterized form of bombesin which has a Leu at position 13 ([Leu13]bombesin). The other two classes, respectively, encoded new bombesin-like peptides which we have designated as [Phe13]bombesin and [Ser3,Arg10,Phe13]bombesin ([SAP]bombesin). The existence of [SAP]bombesin in skin was confirmed by tandem mass spectrometry. Polymerase chain reaction analysis of genomic DNA showed the mRNAs for [Leu13]bombesin, [Phe13]bombesin, and [SAP]bombesin most likely arise from separate genes. Polymerase chain reaction analysis showed different patterns of tissue-specific expression for each form. [Leu13]Bombesin and [SAP]bombesin were predominantly expressed in skin, brain, and gut; [Phe13]bombesin was expressed only in brain, and [Leu13]bombesin predominated in oocytes. [SAP]Bombesin contained a cleavage site between residues 4 and 5, which if used would yield the peptide [SAP]bombesin(5 14) which has the sequence [Gln3,Arg6]neuromedin B. Thus a frog homolog of NMB could derive from the [SAP]bombesin prohormone. [Phe13]Bombesin, [SAP]bombesin, and [SAP]bombesin(5-14) were synthesized and their affinities for the mammalian bombesin-like peptide (GRP and NMB) receptors determined. These peptides acted as agonists for the GRP and NMB receptors, with relative potencies for the GRP receptor of [Leu13]bombesin > [Phe13]bombesin > [SAP]bombesin(5-14) > [SAP]bombesin and for the NMB receptor of [Phe13]bombesin > [SAP]bombesin(5-14) > [Leu13]bombesin > [SAP]bombesin. None of these peptides demonstrated high affinity binding for the BRS-3 receptor. The different receptor affinities and tissue distribution of these peptides suggests distinct physiologic roles and raises the possibility of as yet uncharacterized mammalian homologs of these new amphibian peptides. PMID- 8631815 TI - Carboxy-terminal vesicular stomatitis virus G protein-tagged intestinal Na+ dependent glucose cotransporter (SGLT1): maintenance of surface expression and global transport function with selective perturbation of transport kinetics and polarized expression. AB - The Na+-dependent glucose transporter (SGLT1) mediates absorption of luminal glucose by the intestine. However, available intestinal cell lines that recapitulate a monolayer phenotype only express SGLT1 at low levels. Thus, to facilitate studies of the biology of SGLT1 function in epithelial monolayers, we engineered an epitope-tagged construct containing the YTDIEMNRLGK sequence (from the vesicular stomatitis virus G protein). The tag was placed at the carboxyl terminus since this is the least conserved portion of SGLT1. Transiently transfected COS-1 cells demonstrated surface expression of the immunoreactive protein and enhanced Na+-dependent glucose uptake that was phloridzin-sensitive (a specific competitive inhibitor of SGLT1). However, subsequent detailed analyses of epitope-tagged SGLT1 using stably transfected clones derived from the Caco-2 human intestinal epithelial cell line revealed substantial effects of the epitope on critical functions of SGLT1. When compared with native SGLT1 transfectants, the apparent Km for sugar transport was increased 23-fold (313 microM to 7.37 mM for native versus epitope-tagged SGLT1). In contrast, the apparent KNa for epitope-tagged SGLT1 was similar to that for native SGLT1. Permeabilization studies indicated that the C-terminal epitope tag was intracellular and thus could not directly disrupt extracellular ligand-binding sites. Immunolocalization and functional assays designed to detect polarized surface expression indicated that epitope tagging resulted in loss of apical targeting and enrichment of basolateral expression. Functional isolation of the small apical pool of epitope-tagged SGLT1 (by selective inhibition of basolateral epitope-tagged SGLT1) revealed that, despite the documented kinetic alterations in sugar transport, epitope-tagged SGLT1 could promote absorptive Na+ currents. These data show that 1) the C terminus of SGLT1 is intracellular; 2) disruption of protein structure by addition of a C-terminal tag leads to selective modifications of SGLT1 function; 3) the kinetics of sugar transport can be altered independently of influences on the Na+-binding site of SGLT1; and 4) the weak basolateral targeting sequence present within the epitope tag is dominant over endogenous SGLT1 apical targeting information and can direct polytopic membrane protein localization. The data also caution that subtle effects of foreign sequences must be considered when epitope tagging polytopic membrane proteins. PMID- 8631816 TI - Characterization of distinct nuclear and mitochondrial forms of human deoxyuridine triphosphate nucleotidohydrolase. AB - Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2 terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2 terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame. PMID- 8631817 TI - Identification of a consensus cyclin-dependent kinase phosphorylation site unique to the nuclear form of human deoxyuridine triphosphate nucleotidohydrolase. AB - In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996) J. Biol. Chem. 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339-353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [32P]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2). Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2). Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo. PMID- 8631818 TI - A disulfide-bonded dimer of the Golgi beta-galactoside alpha2,6-sialyltransferase is catalytically inactive yet still retains the ability to bind galactose. AB - The alpha2,6-sialyltransferase is a terminal glycosyltransferase localized in the trans Golgi and trans Golgi network. Here we show that 30% of the total rat liver Golgi alpha2,6-sialyltransferase forms a disulfide-bonded 100-kDa species that can be converted to the 50-kDa monomer form of the enzyme upon reduction. Limited proteolysis of both enzyme forms demonstrates that the 100-kDa species is a disulfide-bonded homodimer of the alpha2,6-sialyltransferase. The alpha2,6 sialyltransferase disulfide-bonded dimer is found in bovine liver Golgi membranes and in Golgi membranes prepared and solubilized in the presence of 100 mM iodoacetamide, suggesting that it is not unique to rat liver or formed aberrantly upon membrane lysis. The dimer form of the enzyme possesses no significant catalytic activity and has a much lower affinity for CDP-hexanolamine-agarose compared with the monomer form. In contrast, both the alpha2,6-sialyltransferase monomer and the disulfide-bonded dimer bind strongly to galactose and galactose terminated substrates. These results suggest that the alpha2,6-sialyltransferase disulfide-bonded dimer lacks catalytic activity due to a weak affinity for its sugar nucleotide donor, CMP-NeuAc, and that this catalytically inactive form of the enzyme may act as a galactose-specific lectin in the Golgi. PMID- 8631819 TI - Inhibition of macrophage scavenger receptor activity by tumor necrosis factor alpha is transcriptionally and post-transcriptionally regulated. AB - Regulation of expression of the scavenger receptor is thought to play a critical role in the accumulation of lipid by macrophages in atherosclerosis. Tumor necrosis factor-alpha (TNF-alpha) has been shown to suppress macrophage scavenger receptor function (van Lenten, B.J., and Fogelman, A.M. (1992) J. Immunol. 148, 112-6). However, the mechanism by which it does so is unknown. We evaluated the mechanism by which TNF-alpha inhibited macrophage scavenger receptor surface expression and binding of acetylated low density lipoprotein (aLDL). Binding of aLDL to phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages was suppressed by TNF-alpha in a dose-dependent manner. Inhibition of aLDL binding was paralleled by a reduction of macrophage scavenger receptor protein as detected by the Western blot. TNF-alpha partially decreased macrophage scavenger receptor mRNA steady state levels in PMA-differentiated THP-1 macrophages, a result that was confirmed by reverse transcription-polymerase chain reaction. PMA increased the luciferase activity driven by the macrophage scavenger receptor promoter in the transfected cells, whereas TNF-alpha partially reduced luciferase activity. However, macrophage scavenger receptor mRNA half-life was dramatically reduced in cells treated with TNF-alpha relative to untreated cells. Reduction in macrophage scavenger receptor message in response to TNF-alpha was dependent on new protein synthesis because it was blocked by cycloheximide. These results indicate that TNF-alpha regulates macrophage scavenger receptor expression in PMA differentiated THP-1 macrophages by transcriptional and post-transcriptional mechanisms but principally by destabilization of macrophage scavenger receptor mRNA. PMID- 8631820 TI - Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes. AB - The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F. (1994) Plant Mol. Biol. 26, 1867 1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway. PMID- 8631821 TI - Structural and functional characterization of the human CD36 gene promoter: identification of a proximal PEBP2/CBF site. AB - CD36 is a cell surface glycoprotein composed of a single polypeptide chain, which interacts with thrombospondin, collagens type I and IV, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and erythrocytes parasitized with Plasmodium falciparum. Its expression is restricted to a few cell types, including monocyte/macrophages. In these cells, CD36 is involved in phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low density lipoprotein. To study the molecular mechanisms that control the transcription of the CD36 gene in monocytic cells we have isolated and analyzed the CD36 promoter. Transient expression experiments of 5'-deletion fragments of the CD36 promoter coupled to luciferase demonstrated that as few as 158 base pairs upstream from the transcription initiation site were sufficient to direct the monocyte-specific transcription of the reporter gene. Within the above region, the fragment spanning nucleotides -158 to -90 was required for optimal transcription in monocytic cells. Biochemical analysis of the region -158/-90 revealed a binding site for transcription factors of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family at position -103. Disruption of the PEBP2/CBF site markedly diminished the role of the PEBP2/CBF factors in the constitutive transcription of the CD36 gene. The involvement of members of the PEBP2/CBF family in chromosome translocations associated with acute myeloid leukemia, and in the transcriptional regulation of the myeloid-specific genes encoding for myeloperoxidase, elastase, and the colony-stimulating factor receptor, highlights the relevance of the regulation of the CD36 gene promoter in monocytic cells by members of the PEBP2/CBF family. PMID- 8631822 TI - The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. AB - Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111 165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity. PMID- 8631823 TI - Adhesion through the interaction of lymphocyte function-associated antigen-1 with intracellular adhesion molecule-1 induces tyrosine phosphorylation of p130cas and its association with c-CrkII. AB - The B-lymphoblastoid cell line JY undergoes homotypic aggregation in a lymphocyte function-associated antigen-1 (LFA-1)-mediated, intracellular adhesion molecule-1 (ICAM-1)-dependent manner when stimulated with phorbol 12-myristate 13-acetate or anti-LFA-1 antibodies. Under conditions that lead to cell aggregation, we observed rapid tyrosine phosphorylation of p130cas, a protein previously identified to be phosphorylated on tyrosine in both v-src- and v-crk-transformed cells. Phosphorylation of p130cas was dependent on binding of LFA-1 to its ligand, ICAM-1, as demonstrated by the use of anti-ICAM-1 antibodies. Several observations suggest that this event may be an important step in the signaling pathway initiated by LFA-1. p130cas phosphorylation was rapidly reversible upon disengagement of the LFA-1-ICAM-1 complex and required cell adhesion since binding of phorbol 12-myristate 13-acetate-stimulated JY cells to purified ICAM-1 or cross-linking of either LFA-1 or ICAM-1 was not sufficient to induce phosphorylation of p130cas. The integrin-stimulated phosphorylation of p130cas created binding sites that were recognized in vitro by the SH2 domain of c-CrkII, a key adaptor protein involved in cell differentiation and transformation. Moreover, we also showed that the LFA-1-stimulated tyrosine phosphorylation of p130cas induces the formation of a p130cas.CrkII and p130cas.CrkL complex in intact cells. This observation suggests that adhesion mediated by the interaction of LFA-1 and ICAM-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interaction via SH2 domains, a key process shared with growth factor signaling pathways. PMID- 8631824 TI - Isoform diversity of dystrobrevin, the murine 87-kDa postsynaptic protein. AB - Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins. PMID- 8631825 TI - Transforming growth factor-beta1 modulates p107 function in myeloid cells: correlation with cell cycle progression. AB - Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of hematopoietic cell growth. Here we report that TGF-beta1 signals inhibition of IL 3-dependent 32D-123 murine myeloid cell growth by modulating the activities of cyclin E and cyclin-dependent kinase 2 (cdk2) proteins and their complex formation in the G1 phase of the cell cycle. Whereas the cyclin E protein was hyperphosphorylated in TGF-beta1 treated cells, TGF-beta1 decreased both the phosphorylation of cdk2 and the kinase activity of the cyclin E-cdk2 complex. Decreased cyclin E-cdk2 kinase activity correlated with decreased phosphorylation of the retinoblastoma-related protein p107. In support of these observations, transient overexpression of p107 inhibited the proliferation of the myeloid cells, and expression of antisense oligodeoxynucleotides to p107 mRNA blocked TGF beta1 inhibition of myeloid cell growth. Furthermore, as reported previously, in 32D-123 TGF-beta1 treated cells, c-Myc protein expression was decreased. TGF beta1 increased the binding of p107 to the transcription factor E2F, leading to decreased c-Myc protein levels. p107 inhibited E2F transactivation activity and was also found to bind the c-Myc protein, suggesting p107 negative regulation of c-Myc protein function. These studies demonstrate the modulation of p107 function by TGF-beta1 and suggest a novel mechanism by which TGF-beta1 blocks cell cycle progression in myeloid cells. PMID- 8631826 TI - Biosynthesis of the linkage region of the mycobacterial cell wall. AB - The "core" structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes, consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan, mAG), which, in turn, is linked to peptidoglycan via a special linkage unit, -alpha-L-Rhap(1-->3)-D GlcNAc-P-. Little is known of the biosynthesis of this complex, although it is the site of action of several common anti-tuberculosis drugs. Isolated cell membranes of Mycobacterium smegmatis catalyzed the incorporation of [14C]GlcNAc from UDP-[14C]GlcNAc into two glycolipids (1 and 2) and of [14C]Rha from TDP [14C]Rha into glycolipid 2. These products were characterized as polyprenol-P-P GlcNAc (glycolipid 1) and polyprenol-P-P-GlcNAc-Rha (glycolipid 2) based on sensitivity of synthesis to tunicamycin, chromatographic characterization of the products of mild acid hydrolysis, and mass spectral analysis of the glycosyl and polyprenyl units. Glycolipids 1 and 2 were shown to be precursors of the linkage unit in polymerized cell wall. The inclusion in the assays of UDP-[14C]Galp and a preparation of cell walls allowed the incorporation of [14C]Gal into two further glycolipids (3 and 4). Preliminary evidence indicates a precursor-product relationship among glycolipids 1, 2, 3, and 4. Thus, the first steps in the biosynthesis of the mycobacterial cell wall involve synthesis of the linkage disaccharide on a polyprenyl-P-P carrier followed by growth of the galactan unit. Assays are thus defined for the screening of new anti-tuberculosis drugs active against cell wall synthesis. PMID- 8631827 TI - Requirement for phosphatidylinositol 3'-kinase activity in platelet-derived growth factor-stimulated tyrosine phosphorylation of p125 focal adhesion kinase and paxillin. AB - The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr 740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3' kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin. PMID- 8631828 TI - Three N-terminal variants of the AE2 Cl-/HCO3- exchanger are encoded by mRNAs transcribed from alternative promoters. AB - Multiple AE2 Cl-/HCO3- exchanger mRNAs have been identified in rat. To determine the genetic basis for these mRNAs and whether they encode different variants of the exchanger, we used both rapid amplification of cDNA ends and S1 nuclease protection protocols and examined the organization of the gene. mRNAs encoding three N-terminal variants of AE2 (AE2a, AE2b, and AE2c) were identified and shown to be transcribed from alternative promoters. The AE2a transcription unit consists of 23 exons, with exons 1 and 2 containing 5'-untranslated sequence and the first 17 codons. The first exon of AE2b is located in intron 2; it contains 5'-untranslated sequence and an alternative 3-amino acid N-terminal coding sequence and is spliced to exon 3. The first exon of AE2c is located in intron 5; it consists of 5'-untranslated sequence and is spliced to exon 6, which contains the translation initiation codon corresponding to Met-200 of AE2a. Northern analysis shows that AE2a is expressed in all tissues, AE2b exhibits a more restricted distribution with highest levels in stomach, and AE2c is expressed only in stomach. Thus, the use of alternative promoters leads to the production of three N-terminal variants of AE2 that exhibit tissue-specific patterns of expression. PMID- 8631829 TI - Role of IkappaBalpha ubiquitination in signal-induced activation of NFkappaB in vivo. AB - In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus. PMID- 8631830 TI - Overproduction of DNA cytosine methyltransferases causes methylation and C --> T mutations at non-canonical sites. AB - Multicopy clones of Escherichia coli cytosine methyltransferases Dcm and EcoRII methylase (M. EcoRII) cause an approximately 50-fold increase in C --> T mutations at their canonical site of methylation, 5'-CmeCAGG (meC is 5 methylcytosine). These plasmids also cause transition mutations at the second cytosine in the sequences CCGGG at approximately 10-fold lower frequency. Similarly, M. HpaII was found to cause a significant increase in C --> T mutations at a CCAG site, in addition to causing mutations at its canonical site of methylation, CCGG. Using a plasmid that substantially overproduces M. EcoRII, in vivo methylation at CCSGG (S is C or G) and other non-canonical sites could be detected using a gel electrophoretic assay. There is a direct correlation between the level of M. EcoRII activity in cells, the extent of methylation at non canonical sites and frequency of mutations at these same sites. Overproduction of M. EcoRII in cells also causes degradation of DNA and induction of the SOS response. In vitro, M. EcoRII methylates an oligonucleotide duplex containing a CCGGG site at a slow rate, suggesting that overproduction of the enzyme is essential for significant amounts of such methylation to occur. Together these results show that cytosine methyltransferases occasionally methylate cellular DNA at non-canonical sites and suggest that in E. coli, methylation-specific restriction systems and sequence specificity of the DNA mismatch correction systems may have evolved to accommodate this fact. These results also suggest that mutational effects of cytosine methyltransferases may be much broader than previously imagined. PMID- 8631832 TI - Potent low molecular weight substrates for protein-tyrosine phosphatase. AB - The ability of protein-tyrosine phosphatases (PTPases) to catalyze the hydrolysis of simple aromatic phosphates has been recognized for some time. However, these compounds are significantly poorer substrates than their peptide-based counterparts containing phosphotyrosine. Consequently, the effort to create potent PTPase substrates has predominantly focused on the use of peptidic carriers to deliver the phosphotyrosine moiety to the enzyme active site. We now report the synthesis and evaluation of several low molecular weight aromatic phosphates that serve as robust substrates for the rat PTPase, PTP1. We initially surveyed the ability of PTP1 to catalyze the hydrolysis of a variety of phenyl phosphate structural variants. Sterically demanding substituents positioned ortho and (to a lesser extent) meta to the phosphate group severely compromise the ability of these species to serve as phosphatase substrates. However, both benzylic and negatively charged substituents para to the hydrolyzable phosphate dramatically promote hydrolytic efficiency, which appears to be augmented through a dramatic enhancement in the affinity of the substrate for PTP1. The best substrate examined in this study exhibits a Km of 16 +/- 3 microM. In addition, it serves as an inhibitor of the PTP1-catalyzed hydrolysis of p-nitrophenyl phosphate with a Ki of 4.9 +/- 0.7 microM. The extraordinary structural simplicity of this compound, as well as those of several others described herein, provides a promising starting point for the design of potent PTPase inhibitors. PMID- 8631831 TI - Examining rhodopsin folding and assembly through expression of polypeptide fragments. AB - Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsin. Both of the pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin. PMID- 8631833 TI - GABP mediates insulin-increased prolactin gene transcription. AB - The insulin-response element from the prolactin gene is identical to the Ets binding site, and dominant-negative Ets protein inhibits insulin-increased prolactin gene expression. Immunoblotting identified the Ets-related transcription factor GABP in nuclear extracts from GH cells. Expression of GABP alpha and GABP beta 1 squelches insulin-increased prolactin gene expression. GABP alpha and GABP beta 1 bind the insulin-response element of the prolactin promoter, and anti-GABP alpha and anti-GABP beta 1 antibodies supershift a species seen with nuclear extracts from GH cells. GABP alpha immunoprecipitated from insulin-treated, 32P-labeled GH cells was phosphorylated 3-fold more than GABP alpha from control cells. There was no increase in phosphorylation of GABP beta in response to insulin. Mitogen-activated protein (MAP) kinase activity is increased 10-fold in insulin-treated GH4 cells. MAP kinase immunoprecipitated from control cells does not phosphorylate GABP alpha while MAP kinase immunoprecipitated from insulin-treated cells shows substantial phosphorylation of GABP alpha. These studies suggest that GABP mediates insulin-increased transcription of the prolactin gene. GABP may be regulated by MAP kinase phosphorylation. PMID- 8631834 TI - Induction of NFAT-mediated transcription by Gq-coupled receptors in lymphoid and non-lymphoid cells. AB - The nuclear factor of activated T cells (NFAT) was discovered as an inducible transcription factor activated by antigen stimulation of the T cell receptor in lymphocytes. Stimulation of NFAT-mediated transcription is now reported in both lymphoid and non-lymphoid cells following activation of a neurotransmitter receptor. Carbachol induces robust luciferase responses in Jurkat and pheochromocytoma PC12 cells expressing an NFAT-luciferase reporter construct and a Gq-coupled m3 muscarinic receptor. Cyclosporin blocks this response in PC12 cells, as in Jurkat cells. In PC12 cells expressing a Gi-coupled m2 muscarinic receptor, carbachol induces NFAT-mediated luciferase activity that is strictly dependent upon co-expression of a chimeric G alpha q/alpha i subunit, which confers Gq-effector coupling on Gi-linked receptors. These findings suggest that neurotransmitters, autacoids, or hormones acting on Gq-protein-coupled receptors may serve as physiological stimulators of NFAT in lymphoid and non-lymphoid cells. PMID- 8631835 TI - The mammalian degenerin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Caenorhabditis elegans. AB - Mutations of the degenerins (deg-1, mec-4, mec-10) are the major known causes of hereditary neurodegeneration in the nematode Caenorhabditis elegans. We cloned a neuronal degenerin (MDEG) from human and rat brain. MDEG is an amiloride sensitive cation channel permeable for Na+, K+, and Li+. This channel is activated by the same mutations which cause neurodegeneration in C. elegans. Like the hyperactive C. elegans degenerin mutants, constitutively active mutants of MDEG cause cell death, suggesting that gain of function of this novel neuronal ion channel might be involved in human forms of neurodegeneration. PMID- 8631836 TI - Vitamin A2 bound to cellular retinol-binding protein as ultraviolet filter in the eye lens of the gecko Lygodactylus picturatus. AB - The yellow eye lenses of the diurnal gecko Lygodactylus picturatus contain, in addition to the usual crystallins, a monomeric protein with a molecular mass of 16kDa. It comprises 6-8% of the total water-soluble lens proteins. We here identify it as a novel type of crystallin, most closely related with cellular retinol-binding protein I (CRBP I). Because of its tiny size, we designate it as iota-crystallin. The typical endogenous ligand of CRBP is all-trans-retinol. In the gecko lens, however, the ligand of iota-crystallin turns out to be 3 dehydroretinol (vitamin A2), which causes the yellow color of this lens. The iota crystallin.3-dehydroretinol complex absorbs shortwave radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against ultraviolet damage. Whereas other crystallins have been recruited from stress proteins and metabolic enzymes, iota-crystallin represents a completely new class of taxon-specific lens proteins. Also, its ligand 3-dehydroretinol represents a novel type of lens pigment. PMID- 8631837 TI - The expression and regulation of STATs during 3T3-L1 adipocyte differentiation. AB - STATs (Signal Transducers and Activators of Transcription) comprise a family of transcription factors that reside in the cytoplasm of resting cells. In response to a variety of stimuli, STATs become tyrosine-phosphorylated and translocate to the nucleus where they mediate transcriptional regulation. We have used the 3T3 L1 murine cell line to examine the expression of STAT proteins as a function of their differentiation into adipocytes. The expression of STATs 1, 3, and 5, but not of STAT 6, is markedly elevated in adipocytes as compared with their fibroblast precursors. Exposure of 3T3-L1 preadipocytes to tumor necrosis factor alpha (TNF alpha) blocks their differentiation into adipocytes. Therefore, we examined STAT expression as a function of differentiation in the presence of this cytokine. The expression of STATs 1 and 5 is markedly attenuated in the presence of TNF alpha, whereas STAT 3 expression is unaffected by this treatment. Only STAT 1 is down-regulated by TNF alpha in fully differentiated cells. Thus, although the expression of STATs 1, 3, and 5 is markedly enhanced upon differentiation, only STAT 5 expression is tightly correlated with the adipocyte phenotype. These data suggest that STAT 5, and possibly STAT 1, could be potential inducers of tissue-specific genes, which contribute to the development and maintenance of the adipocyte phenotype. PMID- 8631838 TI - Expression of biologically active fusion genes encoding the common alpha subunit and the follicle-stimulating hormone beta subunit. Role of a linker sequence. AB - The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common alpha subunit noncovalently linked to a hormone specific beta subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications, and signal transduction. Structure-function studies of follicle-stimulating hormone (FSH) and the other glycoprotein hormones are often hampered by mutagenesis induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure-activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH beta subunit to the amino end of the alpha subunit in the presence or absence of a linker sequence. In the absence of the CTP linker, the secretion rate was decreased over 3-fold. Unexpectedly, however, receptor binding/signal transduction was unaffected by the absence of the linker. These data show that the single-chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same. PMID- 8631839 TI - Conformational properties of substrate proteins bound to a molecular chaperone alpha-crystallin. AB - alpha-Crystallin, the major protein of the ocular lens, acts as a molecular chaperone by suppressing the nonspecific aggregation of damaged proteins. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we prepared a tryptophan-free mutant of human alpha A crystallin and assessed the conformation of thermally destabilized proteins captured by this chaperone using fluorescence spectroscopy. The fluorescence emission characteristics of bound substrates (rhodanese and gamma-crystallin) and the results of fluorescence quenching experiments indicate that the proteins captured by alpha-crystallin are characterized by a very low degree of unfolding. In particular, the structure of rhodanese bound to alpha A-crystallin appears to be considerably more native-like compared to that of the enzyme bound to the chaperonin GroEL. We postulate that alpha-crystallin (and likely other small heat shock proteins) recognize preferentially the aggregation-prone conformers that occur very early on the denaturation pathway. With its ability to capture and stabilize these early non-native structures, alpha-crystallin appears to be uniquely well suited to chaperone the transparency properties of the ocular lens. PMID- 8631841 TI - Glycation and glycoxidation of histones by ADP-ribose. AB - The reaction of long lived proteins with reducing sugars has been implicated in the pathophysiology of aging and age-related diseases. A likely intranuclear source of reducing sugar is ADP-ribose, which is generated following DNA damage from the turnover of ADP-ribose polymers. In this study, ADP-ribose has been shown to be a potent histone glycation and glycoxidation agent in vitro. Incubation of ADP-ribose with histones H1, H2A, H2B, and H4 at pH 7.5 resulted in the formation of ketoamine glycation conjugates. Incubation of histone H1 with ADP-ribose also rapidly resulted in the formation of protein carboxymethyllysine residues, protein-protein cross-links, and highly fluorescent products with properties similar to the advanced glycosylation end product pentosidine. The formation of glycoxidation products was related to the degradation of ketoamine glycation conjugates by two different pathways. One pathway resulted in the formation of protein carboxymethyllysine residues and release of an ADP moiety containing a glyceric acid fragment. A second pathway resulted in the release of ADP, and it is postulated that this pathway is involved in the formation of histone-histone cross-links and fluorescent advanced glycosylation end products. PMID- 8631840 TI - Biophysical characterization of a recombinant soluble interleukin 2 receptor (Tac). Evidence for a monomeric structure. AB - The interleukin 2 receptor (IL2R) plays a prominent role in the biology of T cells, B cells, and NK cells during activation. Of the three chains described, the alpha-chain of the receptor (Tac; IL2R alpha; CD25) is the most subject to regulation and is shed from the surface of activated cells to generate a soluble form in serum and tissues. Conflicting results have been reported on the native structure of soluble Tac, suggesting variously a monomer, a dimer, or higher noncovalent forms, spawning different models for its mechanism of action. We similarly show a large M(r)(app) by HPLC sieving chromatography, suggesting a tetrameric form. However, stoichiometry-ordered size (SOS) analysis of antibody antigen complexes indicated only a single epitope per Tac molecule, compatible with a monomeric form. This larger M(r)(app) also conflicted with prior in vivo data showing rapid filtration of soluble Tac through the renal glomerulus that was not expected of a larger complex. Using different solvents, denaturants, and columns in the chromatography suggested that the elevated M(r)(app) values were an artifact of solute-column interactions, termed "ionic exclusion", rather than reflecting larger native structures. Analytical ultracentrifugation using a new type of analysis specific to glycoproteins demonstrated monomeric masses under all salt conditions with no tendency to form dimers or higher aggregates. Finally, circular dichroism spectroscopy showed no salt-dependent changes to suggest conformational alterations that might correlate with mobility changes on high pressure liquid chromatography. We conclude therefore that Tac is monomeric under physiologic conditions. Assessments of higher molecular weight for the purified soluble protein by other methods may be explained by the highly acidic nature of the molecule, which hampers matrix penetration with chromatographic media and by the high carbohydrate content and low partial specific volumes that accelerate the molecule in sedimentation media relative to pure protein standards. PMID- 8631842 TI - Molecular cloning and expression of cDNA encoding rat brain cytosolic acyl coenzyme A thioester hydrolase. AB - The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced. A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme. Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase. These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT. PMID- 8631843 TI - Evidence of post-transcriptional regulation of U6 small nuclear RNA. AB - Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dose-dependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or half life were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA half-life. Down-regulation of native U6 RNA levels was transient, with recovery noted within 48-96 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or half-lives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dose-dependent decrease in U6 RNA half-life. These studies suggest a hitherto unrecognized U6-specific intracellular regulatory mechanism, through which over-accumulation of U6 small nuclear RNA is prevented. PMID- 8631844 TI - Structural basis of extended spectrum TEM beta-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants. AB - The E166Y and the E166Y/R164S TEM-1 beta-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-A resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalosporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the omega-loop. These structural observations strongly suggest that the link between the position of the omega-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased omega-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparisons of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis. PMID- 8631845 TI - Glut4 is targeted to specific vesicles in adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue. AB - Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4 containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells. PMID- 8631846 TI - Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase. Structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site. AB - Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1 4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site. PMID- 8631847 TI - Differential 9-cis-retinoic acid-dependent transcriptional activation by murine retinoid X receptor alpha (RXR alpha) and RXR beta. Role of cell type and RXR domains. AB - The 9-cis-retinoic acid (9cRA)-inducible enhancer of the rat cellular retinol binding protein type II gene (CRBP II) was shown to be differentially regulated by the murine retinoid X receptor alpha (RXR alpha) as compared with RXR beta. Transient transfection assays performed in NIH 3T3 fibroblast cells demonstrated that RXR alpha yielded a high level of 9cRA-dependent transcription of a reporter gene linked to the CRBP II enhancer, when compared with RXR beta. This effect was cell type-dependent, since both receptors elicited comparable transcriptional activation of the same reporter in P19 embryonal carcinoma cells. To further explore the structural determinants responsible for the differences between these two receptors, a series of chimeric receptor constructs were made. Co transfection assays utilizing these chimeras demonstrated that both the N terminus and the hinge region connecting the DNA binding domain with the ligand binding domain of RXR alpha were responsible for the high level of 9cRA-dependent transcription observed in NIH 3T3 cells, Furthermore, the hinge region of RXR alpha was shown to be necessary to repress, in the absence of hormone, the transcriptional activation function located in the N-terminal domain of RXR alpha. These results stress the importance of functional links between different RXR domains and suggest an RXR subtype and cell type-dependent specificity in the control of the 9cRA response. PMID- 8631848 TI - Modulation of interferon (IFN)-inducible gene expression by retinoic acid. Up regulation of STAT1 protein in IFN-unresponsive cells. AB - Interferons (IFN) and retinoids failed to inhibit the growth of a number of breast tumor cell lines. However, a combination of these two biological response modifiers significantly suppressed the cell growth at pharmacologically achievable doses. The molecular basis for such enhancement was investigated in MCF-7, a breast tumor cell line resistant to growth inhibition by IFN-beta. Pretreatment of cells with retinoic acid (RA) for 16 h followed by IFN-beta, but not the converse, induced cytotoxic effects in the cells. Continuous presence of RA was not necessary, although it enhanced the degree of cell death when present. Further analyses revealed that IFN-beta failed to activate IFN-stimulated gene transcription. However, IFN-beta strongly up-regulated the gene expression in RA pretreated cells. Both IFN-beta- and IFN-gamma-inducible gene expression were enhanced via a modulation of the transcriptional factor IFN-stimulated gene factors-3 and GAF binding to respective cognate regulatory elements. STAT1 was undetectable in these cells prior to RA treatment. RA increased the levels of this crucial regulator, thereby restoring IFN responses. Thus, RA augmentation of STAT1 may be an early step in the cooperative anti-tumor effects of IFN and RA. PMID- 8631849 TI - Amino acid residue at codon 268 determines both activity and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases. In vitro mutagenesis study. AB - Histo-blood group A transferase produces A antigens and transfers GalNAc to the acceptor substrate, H structures of glycolipids and glycoproteins. B transferase transfers galactose in place of GalNAc to the same acceptor substrate to synthesize B antigens. We have previously identified four amino acid substitutions between human A and B transferases. Out of these four, substitutions at the last two positions (codons 266 and 268) were found to be crucial for the different donor nucleotide-sugar specificities between A and B transferases as analyzed by gene transfer of chimeric A-B transferase genes. In the present study, we have in vitro mutagenized codon 268 of these two transferase cDNA expression constructs (glycine and alanine in A and B transferases, respectively) and produced substitution constructs with every possible amino acid residue at this position. We examined the activity and specificity of each construct by gene transfer followed by immunodetection of A and B antigens and in vitro enzymatic assay. Amino acid substitution constructs on the A transferase backbone with alanine, serine, and cysteine expressed enzymes with A and B transferase activities. Weak A activity was detected with histidine and phenylalanine constructs while weak B activity was detected with asparagine and threonine constructs. All the other amino acid substitutions at codon 268 on the A transferase backbone showed neither A nor B activity. The glycine construct on the B transferase backbone expressed both A and B transferase activities. Some substitution constructs on the B transferase backbone maintained B activity while some other substitutions abolished the activity. These results show that the side chain of the amino acid residue at 268 of the human A and B transferases is responsible for determining both activity and nucleotide-sugar donor substrate specificity and strongly suggest its direct involvement in the recognition of and binding to the sugar moiety of the nucleotide-sugars. PMID- 8631850 TI - RANTES and MCP-3 antagonists bind multiple chemokine receptors. AB - Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within 4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines. In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1 (9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines, the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH2-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine antagonists. PMID- 8631851 TI - Construction of a form of the MoFe protein of nitrogenase that accepts electrons from the Fe protein but does not reduce substrate. AB - The direction of electron flow through nitrogenase is generally believed to be from the Fe protein to the P-clusters to the FeMo cofactor and then to substrate. In order to examine oxidation states of the P-clusters that might be involved in this pathway, we have constructed a form of the MoFe protein that contains a species called the MoFe cluster (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1995) Biochemistry 33, 11842-11849) in place of FeMo cofactor. This MoFe cluster containing protein was purified, and the presence of the cluster was confirmed by reisolation of the MoFe cluster followed by EPR spectroscopy. The protein does not reduce protons or acetylene, however, upon the addition of the Fe protein and MgATP, MgATP hydrolysis occurs at a rate 28% of the wild-type protein. As isolated in the presence of excess dithionite the MoFe cluster-containing protein is EPR silent. Upon addition of the Fe protein and MgATP a g = 1.94 EPR signal develops that integrates to about 1 spin per P-cluster. This signal only develops when both the Fe protein and MgATP are added and it arises from the P-clusters. PMID- 8631852 TI - Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross talk between aryl hydrocarbon- and estrogen-mediated signaling. AB - Aryl hydrocarbon receptor (AhR) ligands have diverse biological effects including striking antiestrogenic activity. We have investigated at the molecular level the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We show that the previously documented TCDD-mediated decrease in estradiol-inducible gene products such as cathepsin D (cat D) is due to a sharp decline in mRNA accumulation despite any change in estrogen receptor (ER) mRNA levels. The decline in cat D mRNA level is most likely due to a decrease in transcription of the cat D gene since TCDD blocks the ability of ER to transactivate from an estrogen response element. AhR is required for this activity as TCDD is no longer antiestrogenic in a mutant cell line that is deficient in functional AhR. We provide evidence that the loss of transactivation potential by ER in the presence of TCDD is due to a sharp decrease in its ability to bind to an estrogen response element. Reciprocally, estradiol treatment blocked TCDD-induced accumulation of CYP1A1 mRNA and AhR-mediated activation of the CYP1A1 promoter. This is due to the ability of liganded ER to interfere with the binding of AhR to the xenobiotic response element. These results provide a molecular mechanism for the antiestrogenic effects of TCDD and demonstrate the presence of a two-way crosstalk between the intracellular signaling pathways involving estrogens and aryl hydrocarbons. PMID- 8631853 TI - Programming the Rous sarcoma virus protease to cleave new substrate sequences. AB - The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally. PMID- 8631854 TI - A non-cholinergic transmitter, pituitary adenylate cyclase-activating polypeptide, utilizes a novel mechanism to evoke catecholamine secretion in rat adrenal chromaffin cells. AB - Pituitary adenylate cyclase-activating polypeptide (PACAP) is the most potent non cholinergic neurotransmitter to stimulate catecholamine secretion from rat chromaffin cells; however, the mechanism of action is not clear. We used amperometric detection of exocytosis and indo-1 monitoring of [Ca2+]i to identify PACAP actions in cultured chromaffin cells. PACAP (100 nM) required external Ca2+ to evoke secretion. However, unlike nicotine and KCl which caused immediate and relatively brief secretion, PACAP has a latency of 6.8 +/- 0.96 s to the first secretory response and secretion continued for up to 2 min. PACAP elevation of [Ca2+]i showed similar latency and often remained above base line for several minutes following a brief exposure. ZnCl2 (100 microM) selectively inhibited PACAP-stimulated secretion and [Ca2+]i with little effect on nicotine-evoked responses. Nifedipine (10 microM) had little effect on PACAP-evoked secretion but inhibited nicotine-evoked secretion by more than 80%, while omega-conotoxin (100 nM) failed to affect either agonist. PACAP-stimulated cAMP levels required 5 s to significantly increase, consistent with the latency of exocytotic and Ca2+ responses. Forskolin (10 microM) caused responses similar to PACAP. PACAP-evoked exocytosis was blocked by the protein kinase A inhibitor adenosine 3'5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS). These data showed that PACAP stimulates exocytosis by a mechanism distinctly different from cholinergic transmitters that appears to involve cAMP-mediated Ca2+ influx. Differences in receptor coupling mechanisms and pharmacology of Ca2+ entry stimulated by cholinergic and peptidergic agonists support the idea that the peptidergic system maintains catecholamine secretion under conditions where the cholinergic system desensitizes or otherwise fails. PMID- 8631855 TI - Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene. AB - The activity of the apical membrane Na+/H+ exchanger NHE3 isoform of renal or intestinal epithelial cells is chronically regulated by a wide variety of stimuli, including acidosis, cAMP, glucocorticoids, and thyroid hormone. To understand the molecular mechanisms responsible for long term regulation of this cation transporter, we have isolated and determined the structure of this gene from a rat genomic library. The Nh3 gene spans > 40 kilobases and contains 17 exons that are flanked by typical splice donor and acceptor sequences at the exon intron boundaries. The transcription initiation site was mapped by S1 nuclease protection analyses of mRNA from rat kidney and intestine. Multiple start sites were clustered between nucleotides -100 and -96 relative to the translation initiation codon. An atypical TATA-box and CCAAT-box are centered 30 and 147 nucleotides, respectively, upstream of the predominant transcription initiation site. Sequence analysis of approximately 1.4 kilobases of the 5'-flanking promoter region also revealed the presence of other putative cis-acting elements recognized by various transcription factors (e.g. AP-1, AP-2, C/EBP, NF-I, OCT 1/OTF-1, PEA3, Sp1, glucocorticoid, and thyroid hormone receptors), some of which may participate in the chronic regulation of this gene. The glucocorticoid responsiveness of the Nhe3 gene was assessed by fusing its 5' regulatory region to the firefly luciferase reporter gene and then by measuring the expression of the chimeric gene in transiently transfected renal epithelial OK and LLC-PK1 cells. Glucocorticoid treatment significantly increased the luciferase activity of the chimeric gene in both cell lines, thereby indicating that glucocorticoid regulation of Nhe3 is mediated primarily by a transcriptional mechanism. PMID- 8631856 TI - Target cell-specific DNA transfer mediated by a chimeric multidomain protein. Novel non-viral gene delivery system. AB - Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains: 1) an ErbB-2 -specific single chain antibody confers target cell specificity, 2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enable sequence-specific high affinity binding to DNA. Carrier protein purified from bacterial lysates displayed both ErbB-2-specific and DNA sequence-specific binding in vitro. Complexes which formed spontaneously by the interaction of the fusion protein with a luciferase reporter gene construct carrying a GAL4-specific recognition sequence, after condensation of the DNA and compensation of excess negative charge with poly-L-lysine were able to transfect ErbB-2-expressing cells in vitro in a cell-specific manner. Transient expression of the luciferase gene driven by the SV40 early promoter was observed and correlates with the amount of carrier protein in the complex. Truncated forms of the carrier protein lacking either the cell recognition domain or the translocation domain failed to facilitate efficient DNA transfer. PMID- 8631857 TI - The rBAT gene is responsible for L-cystine uptake via the b0,(+)-like amino acid transport system in a "renal proximal tubular" cell line (OK cells). AB - Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillaron, J., Chillon, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barcelo, P., Estivill, X., Zorzano, A., Nunes, V., and Palacin, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacin, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+) like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line. PMID- 8631858 TI - Interaction of ascorbate and alpha-tocopherol in resealed human erythrocyte ghosts. Transmembrane electron transfer and protection from lipid peroxidation. AB - A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythrocyte ghosts. Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F2-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F2-isoprostane formation due to extravesicular ferricyanide. This protection was associated with a rapid transmembrane oxidation of intravesicular ascorbate by extravesicular ferricyanide. Transmembrane electron transfer, which was measured indirectly as ascorbate-dependent ferricyanide reduction, correlated with the content of alpha-tocopherol in the ghost membrane in several respects. First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous alpha-tocopherol in the ghost membrane. Second, when exogenous alpha-tocopherol was incorporated into the ghost membrane during the resealing step, subsequent ferricyanide reduction was enhanced. Last, incubation of intact erythrocytes with soybean phospholipid liposomes, followed by resealed ghost preparation, caused a proportional decrease in both the membrane content of alpha-tocopherol and in ferricyanide reduction. Incorporation of exogenous alpha tocopherol during resealing of ghosts prepared from liposome-treated cells completely restored the ferricyanide-reducing capacity of the ghosts. These results suggest that the transmembrane transfer of ascorbate-derived electrons in erythrocyte ghosts is dependent in part on alpha-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell. PMID- 8631859 TI - The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation. AB - Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling. PMID- 8631861 TI - Protein kinase CK2 mutants defective in substrate recognition. Purification and kinetic analysis. AB - Five mutants of protein kinase CK2 alpha subunit in which altogether 14 basic residues were singly to quadruply replaced by alanines (K74A,K75A,K76A,K77A; K79A, R80A,K83A; R191A,R195A,K198A; R228A; and R278A, K279A,R280A) have been purified to near homogeneity either as such or after addition of the recombinant beta subunit. By this latter procedure five mutated tetrameric holoenzymes were obtained as judged from their subunit composition, sedimentation coefficient on sucrose gradient ultracentrifugation, and increased activity toward a specific peptide substrate as compared with the isolated alpha subunits. The kinetic constants and the phosphorylation efficiencies (V(max)/Km) of all the mutants with the parent peptide RRRADDSDDDDD and a series of derivatives, in which individual aspartic acids were replaced by alanines, have been determined. Three mutants, namely K74A,K75A,K76A,K77A; K79A, R80A,K83A; and R191A,R195A, K198A, display dramatically lower phosphorylation efficiency and 8-50-fold higher Km values with the parent peptide, symptomatic of reduced attitude to bind the peptide substrate as compared with CK2 wild type. Such differences either disappear or are attentuated if the mutants R191A,R195A, K198A; K79A,R80A,K83A; and K74A,K75A, K76A,K77A are assayed with the peptides RRRADDSADDDD, RRRADDSDDADD, and RRRADDSDDDAA, respectively. In contrast, the phosphorylation efficiencies of the other substituted peptides decrease more markedly with these mutants than with CK2 wild type. These data show that one or more of the basic residues clustered in the 191-198, 79-83, and 74-77 sequences are implicated in the recognition of the acidic determinants at positions +1, +3, and +4/+5, respectively, and that if these residues are mutated, the relevance of the other acidic residues surrounding serine is increased. In contrast the other two mutants, namely R228A and R278A,K279A, R280A, display with all the peptides V(max) values higher than CK2 wild type, counterbalanced however by somewhat higher Km values. It can be concluded from these data that all five mutations performed are compatible with the reconstitution of tetrameric holoenzyme, but all of them influence the enzymatic efficiency of CK2 to different extents. Although the basic residues mutated in the 74-77, 79-83, and 191-198 sequences are clearly implicated in substrate recognition by interacting with acidic determinants at variable positions downstream from serine, the other basic residues seem to play a more elusive and/or indirect role in catalysis. PMID- 8631860 TI - Effects of aminofluorene and acetylaminofluorene DNA adducts on transcriptional elongation by RNA polymerase II. AB - A prominent model for the mechanism of transcription-coupled DNA repair proposes that an arrested RNA polymerase directs the nucleotide excision repair complex to the transcription-blocking lesion. The specific role for RNA polymerase II in this mechanism can be examined by comparing the extent of polymerase arrest with the extent of transcription-coupled repair for a specific DNA lesion. Previously we reported that a cyclobutane pyrimidine dimer that is repaired preferentially in transcribed genes is a strong block to transcript elongation by RNA pol II (Donahue, B.A., Yin, S., Taylor, J.-S., Reines, D., and Hanawalt, P. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8502-8506). Here we report the extent of RNA polymerase II arrest by the C-8 guanine DNA adduct formed by N-2-aminofluorene, a lesion that does not appear to be preferentially repaired. Templates for an in vitro transcription assay were constructed with either an N-2-aminofluorene adduct or the helix-distorting N-2-acetylaminofluorene adduct situated at a specific site downstream from the major late promoter of adenovirus. Consistent with the model for transcription-coupled repair, an aminofluorene adduct located on the transcribed strand was a weak pause site for RNA polymerase II. An acetylaminofluorene adduct located on the transcribed strand was an absolute block to transcriptional elongation. Either adduct located on the nontranscribed strand enhanced polymerase arrest at a nearby sequence-specific pause site. PMID- 8631862 TI - The interaction between apolipoprotein E and Alzheimer's amyloid beta-peptide is dependent on beta-peptide conformation. AB - An important feature of Alzheimer's disease (AD) is the cerebral deposition of amyloid. The main component of the amyloid is a 39-44-amino acid residue protein called amyloid beta (A beta), which also exists as a normal protein in biological fluids, known as soluble A beta. A major risk factor for late-onset AD is the inheritance of the apolipoprotein (apo) E4 isotype of apoE. How apoE is involved in the pathogenesis of AD is unclear; however, evidence exists for a direct apoE/A beta interaction. We and others have shown that apoE copurifies with A beta from AD amyloid plaques and that under certain in vitro conditions apoE promotes a beta-sheet structure in A beta peptides. Currently we document the high affinity binding of A beta peptides to both human recombinant apoE3 and -E4 with a KD of 20 nM. This interaction is greatly influenced by the conformational state of the A beta peptide used. Furthermore, we show that the fibril modulating effect of apoE is also influenced by the initial secondary structure of the A beta peptide. The preferential binding of apoE to A beta peptides with a beta sheet conformation can in part explain the copurification of A beta and apoE from AD amyloid plaques. PMID- 8631863 TI - Direct association between the Ret receptor tyrosine kinase and the Src homology 2-containing adapter protein Grb7. AB - Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell. PMID- 8631864 TI - Characterization of an alternatively spliced GM2 activator protein, GM2A protein. An activator protein which stimulates the enzymatic hydrolysis of N acetylneuraminic acid, but not N-acetylgalactosamine, from GM2. AB - GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2. We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J. M. (1992) Biochem. J. 282, 807-813). GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schroder, M., Suzuki, K., and Sandhoff, K. (1991) FEBS Lett. 289, 260-264). GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus. Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein. We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector. The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by beta hexosaminidase A. Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase. These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109. The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway. PMID- 8631865 TI - Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds. AB - Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins. PMID- 8631866 TI - Activation of the ATP-sensitive K+ channel by long chain acyl-CoA. A role in modulation of pancreatic beta-cell glucose sensitivity. AB - Long-term exposure to elevated levels of long chain free fatty acids decreases glucose-induced insulin secretion from pancreatic islets and clonal pancreatic beta-cells. The mechanism for this loss of glucose sensitivity is at present not known. In this study, we evaluated the possibility that increases in long chain acyl-CoA esters (LC-CoA), the metabolically active form of free fatty acids, might mediate the loss of glucose sensitivity. We observed that cellular levels of LC-CoA increased more than 100% in response to overnight incubation with 0.5 mM palmitic acid complexed to albumin. In the same studies, the total CoA pool increased by about 40%. Patch-clamp studies demonstrated that saturated and unsaturated LC-CoA, but not malonyl-CoA or free CoASH, induced a rapid and slowly reversible opening of ATP-sensitive K+ channels. The effect was concentration dependent between 10 nM and 1 microM. These findings indicate that the ATP regulated K/ channels is a sensitive target for LC-CoA and suggest that high levels of LC-CoA, which accumulate in response to hyperglycemia or prolonged exposure to free fatty acids, may prevent channel closure and contribute to the development of beta-cell glucose insensitivity. PMID- 8631867 TI - A relA/spoT homologous gene from Streptomyces coelicolor A3(2) controls antibiotic biosynthetic genes. AB - A 0.972-kilobase pair DNA fragment from Streptomyces lividans that induces the production of the blue-pigmented antibiotic actinorhodine in S. lividans when cloned on a multicopy plasmid has led to the isolation of a 4-kilobase pair DNA fragment from Streptomyces coelicolor containing homologous sequence. Computer assisted analysis of the DNA sequence revealed three putative open reading frames (ORFs), ORF1, ORF2, and ORF3. ORF2 extends beyond the sequenced DNA fragment, and its deduced product shares no similarities with any other known proteins in the data bases. ORF3 is also truncated, and its 41-amino acid C-terminal product is identical to the S. coelicolor adenine phosphoribosyltransferase. The 847-amino acid ORF1 protein, with a predicted molecular mass of 94.2 kDa, strongly resembled the relA and spoT gene products from Escherichia coli and the homologs from Vibrio sp. strain S14, Haemophilus influenzae, Streptococcus equisimilis H46A, and Mycoplasma genitalium. Unlike these proteins, the ORF1 amino acid sequence analysis revealed the presence of a putative ATP/GTP-binding domain. A mutant was generated by deleting most of the ORF1 gene that showed an actinorhodine-nonproducing phenotype, while undecylprodigiosin and the calcium dependent antibiotic were unaffected. The mutant strain grew at a much lower rate than the wild-type strain, and spore formation was delayed. When the gene was propagated on a low copy number vector, not only was actinorhodine production restored, but actinorhodine and undecylprodigiosin production was enhanced in both the mutant and wild-type and morphological differentiation returned to wild type characteristics. (p)ppGpp synthetase activity was not detected in purified ribosomes from the ORF1-deleted mutant, while it was restored by complementation of this strain. PMID- 8631868 TI - Complete amino acid sequence and identification of the platelet glycoprotein Ib binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca. AB - Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743 750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb binding site resides on the beta and not on the alpha subunit based on an enzyme linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72). PMID- 8631869 TI - The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. AB - During a search for novel drugs possessing analgesic properties but devoid of the psychotropic effects of marijuana, a group of molecules designated as nonclassical cannabinoids was synthesized by Pfizer. Of these nonclassical cannabinoids CP-55,940 has received the most attention principally because it was used as the high affinity radioligand during the discovery and characterization of the G-protein-coupled cannabinoid receptor. In an effort to obtain information on the stereoelectronic requirements at the cannabinoid receptor active site, we have studied the conformational properties of CP-55,940 using a combination of solution NMR and computer modeling methods. Our data show that for the most energetically favored conformation, (i) the aromatic phenol ring is perpendicular to the cyclohexane ring, and the phenolic O-H bond is coplanar with the aromatic ring and points away from the cyclohexyl ring; ii) the dimethylheptyl chain adopts one of four preferred conformations in all of which the chain is almost perpendicular to the phenol ring; and iii) an intramolecular H-bond between the phenolic and hydroxypropyl groups allows all three hydroxyl groups of CP-55,940 to be oriented toward the upper face of the molecule. Such an orientation by the OH groups may be a characteristic requirement for cannabimimetic activity. PMID- 8631870 TI - Dynamics and environment of mitochondrial water as detected by 1H NMR. AB - The dynamics and environment of water in suspensions of isolated rat liver mitochondria have been investigated by 1H NMR. NMR longitudinal and transversal relaxation times (T1 and T2) were measured in the resuspension medium (2.65 s and 44.57 ms) and in mitochondrial suspensions (1.74 s and 23.14 s), respectively. Results showed monoexponential relaxation in both cases, suggesting a fast water exchange across the inner mitochondrial membrane. Ferromagnetically induced shift of the extramitochondrial water with nonpermeant ferromagnetic particles revealed no detectable water signal from the intramitochondrial compartment, confirming the fast exchange case. Simulations on a two-compartment model indicated that the intramitochondrial water residence time has an upper limit of approximately 100 microseconds. Calculated intramitochondrial relaxation times revealed that the intramitochondrial environment has an apparent viscosity 30 times larger than the resuspension medium and 15 times larger than the cytosol of erythrocytes. The higher apparent viscosity of the mitochondrial matrix could account for reductions of more than one order of magnitude in the diffusion coefficient of water and other substrates, limitations in the rate of enzymatic reactions which are diffusion controlled and a more favorable formation of multienzyme complexes. PMID- 8631871 TI - The isolation and characterization of a novel corticostatin/defensin-like peptide from the kidney. AB - We report the isolation and characterization of RK-1, a novel peptide found in the kidney. RK-1 is related to the corticostatin/defensins and has the sequence MPC-SCKKYCDPWEVIDGSCGLFNSKYCCREK but differs from the very cationic corticostatins/defensins in having only one arginine and a calculated charge at pH 7 of +1. Like some myeloid corticostatin/defensins RK-1 inhibits the growth of Escherichia coli. Since corticostatin/defensins effect ion flux in responsive epithelia we used volume changes in villus enterocytes as a model system to study the effects of RK-1 on ion channels in epithelial cells. At concentrations > or = 10(-9) M RK-1 decreased enterocyte volume in a dose-dependent manner through a pathway that requires extracellular calcium and is inhibited by niguldipine, a dihydropyridine-sensitive "L"-type Ca(2+)-channel blocker. In other assay systems for corticostatin-defensins, such as the inhibition of adrenocorticotropin stimulated steroidogenesis, or cell lysis, RK-1 was inactive or only weakly active. These results demonstrate the existence of a novel system of biologically active peptides in the kidney represented by RK-1 which is antimicrobial and can activate epithelial ion channels in vitro. PMID- 8631872 TI - Lactosylceramide stimulates Ras-GTP loading, kinases (MEK, Raf), p44 mitogen activated protein kinase, and c-fos expression in human aortic smooth muscle cells. AB - Previously, our laboratory has shown that lactosylceramide (LacCer) can serve as a mitogenic agent in the proliferation of aortic smooth muscle cells "a hallmark in the pathogenesis of atherosclerosis" (Chatterjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554-561). Here we report a novel aspect of LacCer-mediated signal transduction. We demonstrate that LacCer (10 microM) can stimulate the phosphorylation of mitogen-activated protein (MAP) kinase p44MAPK to phosphorylated p44MAPK in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipitated MAP kinase revealed that within 5 min of incubation of cells with LacCer there was a 3.5-fold increase in the activity of p44MAPK. This continued up to 10 min of incubation; thereafter, the MAP kinase activity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44MAPK was phosphorylated by LacCer. Incubation of cells with ceramide and glucosylceramide did not significantly stimulate p44MAPK activity. Preincubation with tyrphostin (20 microM; a potent and specific inhibitor of tyrosine kinase) markedly inhibited the LacCer mediated stimulation in p44MAPK activity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulated (7 fold) the loading of GTP on Ras. Concomitantly, LacCer stimulated the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fold) in these cells as compared to control. In summary, one of the biochemical mechanisms in LacCer mediated induction in the proliferation of aortic smooth muscle cells may involve Ras-GTP loading, activation of the kinase cascade (MEK, Raf, p44MAPK), and c-fos expression. PMID- 8631873 TI - The 4-kDa nuclear-encoded PetM polypeptide of the chloroplast cytochrome b6f complex. Nucleic acid and protein sequences, targeting signals, transmembrane topology. AB - The 4-kDa subunit of cytochrome b6f complex encoded by the nuclear PetM gene in Chlamydomonas reinhardtii has been characterized. 38 of the 39 residues of the mature protein have been established by Edman degradation, a cDNA clone encoding the complete precursor has been isolated and sequenced, and a 0.6-kb transcript detected. The deduced amino acid sequence of the precursor includes an N-terminal transit peptide of 60 amino acids with stromal targeting features. Examination of the sequence suggests that PetM spans the membrane as a single transmembrane alpha-helix, which is supported by its non-extractability following dissociating treatments. When PetM and PetG, another small subunit of the b6f complex, are folded into alpha-helices, an array of identical residues becomes apparent. Proteolysis data, charge distribution, and homology with PetG are consistent with a lumenal localization of the N terminus of PetM. PMID- 8631874 TI - Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. AB - The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5' flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5' deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning -634 to -531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (-540) motif, AP-1 sites (-533, 79), a NF-kappa B (-600) consensus sequence, and a GT box (-52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activate protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/est and AP-1 sites and is MEK1-independent. PMID- 8631875 TI - The MalT-dependent and malZ-encoded maltodextrin glucosidase of Escherichia coli can be converted into a dextrinyltransferase by a single mutation. AB - malZ is a member of the mal regulon. It is controlled by MatT, the transcriptional activator of the maltose system. MalZ has been purified and identified as an enzyme hydrolyzing maltotriose and longer maltodextrins to glucose and maltose. MalZ is dispensable for growth on maltose or maltodextrins. Mutants lacking amylomaltase (encoded by malQ), the major maltose utilizing enzyme, cannot grow on maltose, maltotriose, or maltotetraose, despite the fact that they contain an effective transport system and MalZ. From such a malQ mutant a pseudorevertant was isolated that was able to grow on maltose. The suppressor mutation was mapped in malZ. The mutant gene was cloned. It contained a Trp to Cys exchange at position 292 of the deduced protein sequence. Surprisingly, the purified mutant enzyme was still unable to hydrolyze maltose as was the wild type enzyme, while both were able to release glucose from maltodextrins. However, the mutant enzyme had gained the ability to transfer dextrinyl moieties to glucose, maltose, and other maltodextrins. Thus, it had gained an activity associated with amylomaltase. It was the MalZ292-associated transferase reaction that allowed the utilization of maltose. In addition, we discovered that mutant and wild type enzyme alike were highly active as gamma-cyclodextrinases. PMID- 8631876 TI - The role of the C-terminal domain of I kappa B alpha in protein degradation and stabilization. AB - In the present study, the role of the I kappa B alpha C terminus in NF-kappa B/I kappa B alpha regulation was examined in NIH 3T3 cells engineered to inducibly express wild type or mutated human I kappa B alpha proteins under the control of the tetracycline responsive promoter. Deletion studies demonstrated that the last C-terminal 30 amino acids (amino acids (aa) 288 to aa 317, deleted in I kappa B alpha delta 3), including most of the PEST domain, were dispensable for I kappa B alpha function. However, deletions from aa 261 to 317 or aa 269 to 317 (I kappa B alpha delta 1 and I kappa B alpha delta 2 respectively), lacked the ability to dissociate NF-kappa B/DNA complexes in vitro and were unable to inhibit NF-kappa B dependent transcription. Moreover, I kappa B alpha delta 1 and I kappa B alpha delta 2 mutants were resistant to inducer-mediated degradation. Analysis of I kappa B alpha deletions in the presence of protein synthesis inhibitors revealed that, independently of stimulation, I kappa B alpha delta 1 and I kappa B alpha delta 2 had a half-life four times shorter than wild type I kappa B alpha and the interaction of I kappa B alpha delta 1 and I kappa B alpha delta 2 with p65 was dramatically decreased in vivo as measured by co-immunoprecipitation. Interestingly, protease inhibitors which blocked inducer-mediated degradation of I kappa B alpha also stabilized the turnover of I kappa B alpha delta 1 and I kappa B alpha delta 2. Based on these studies, we propose that in the absence of stimulation, the C-terminal domain between aa 269 and 287 may play a role to protect I kappa B alpha from a constitutive protease activity. PMID- 8631877 TI - AdipoQ is a novel adipose-specific gene dysregulated in obesity. AB - Adipose differentiation is accompanied by changes in cellular morphology, a dramatic accumulation of intracellular lipid and activation of a specific program of gene expression. Using an mRNA differential display technique, we have isolated a novel adipose cDNA, termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids with a secretory signal sequence at the amino terminus, a collagenous region (Gly-X-Y repeats), and a globular domain. The globular domain of adipoQ shares significant homology with subunits of complement factor C1q, collagen alpha 1(X), and the brain-specific factor cerebellin. The expression of adipoQ is highly specific to adipose tissue in both mouse and rat. Expression of adipoQ is observed exclusively in mature fat cells as the stromal vascular fraction of fat tissue does not contain adipoQ mRNA. In cultured 3T3 F442A and 3T3-L1 preadipocytes, hormone-induced differentiation dramatically increases the level of expression for adipoQ. Furthermore, the expression of adipoQ mRNA is significantly reduced in the adipose tissues from obese mice and humans. Whereas the biological function of this polypeptide is presently unknown, the tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue. PMID- 8631878 TI - Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5' phosphate decarboxylase. AB - Human UMP synthase is a bifunctional protein containing two separate catalytic domains, orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5' phosphate decarboxylase (EC 4.1.1.23). These studies address the question of why the last two reactions in pyrimidine nucleotide synthesis are catalyzed by a bifunctional enzyme in mammalian cells, but by two separate enzymes in microorganisms. From existing data on subunit associations of the respective enzymes and calculations showing the molar concentration of enzyme to be far lower in mammalian cells than in microorganisms, we hypothesize that the covalent union in UMP synthase stabilizes the domains containing the respective catalytic centers. Evidence supporting this hypothesis comes from studies of stability of enzyme activity in vitro, at physiological concentrations, of UMP synthase, the two isolated catalytic domains prepared by site-directed mutagenesis of UMP synthase, and the yeast ODCase. The two engineered domains have activities very similar to the native UMP synthase, but unlike the bifunctional protein, the domains are quite unstable under conditions promoting the dissociated monomer. PMID- 8631879 TI - Arrest of subunit folding and assembly of nicotinic acetylcholine receptors in cultured muscle cells by dithiothreitol. AB - In this study we have used cultured muscle cells to investigate the role of disulfide bond formation in the sequence of molecular events leading to nicotinic acetylcholine receptor (AChR) assembly and surface expression. We have observed that disulfide bond formation in newly synthesized AChR alpha-subunits occurs 5 20 min after translation and that this modification can be blocked by dithiothreitol (DTT), a membrane-permeant thiol-reducing agent. DTT treatment was found to arrest AChR alpha-subunit conformational maturation, assembly, and appearance on the cell surface, showing that these events are dependent on prior formation of disulfide bonds. Subunits prevented from maturation by the reducing agent do not irreversibly misfold or aggregate, since upon removal of DTT, AChR alpha-subunits undergo formation of disulfide bonds and resume folding, oligomerization, and surface expression. We have previously found that nascent alpha-subunits form transient complexes with the molecular chaperone calnexin immediately after subunit synthesis (Gelman, M.S., Chang, W., Thomas, D. Y., Bergeron, J. J. M., and Prives, J. M. (1995) J. Biol. Chem. 270, 15085-15092) and have now observed that both the formation and the subsequent dissociation of these complexes are unaffected by DTT treatment. Thus, alpha-subunits appear to dissociate from calnexin independently of their undergoing disulfide bond formation and achieving conformational maturation. This finding together with the absence of irreversible misfolding of DTT-arrested alpha-subunits suggests that calnexin may act to prevent misfolding by aiding in the initial folding events and is not an essential participant in the late stages of alpha-subunit maturation. PMID- 8631880 TI - Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83. AB - The precursor of matrix metalloproteinase 3 (MMP-3/ stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His82-Phe83 bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His82 or Phe83 to Arg to obtain a stable MMP 3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45 kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha 2-macroglobulin. However, the SH group of Cys75 of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys75. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr85, whose activity and substrate specificity were similar to those of the 46-kDa MMp 3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3. PMID- 8631881 TI - A novel family of Cys-Cys, His-Cys zinc finger transcription factors expressed in developing nervous system and pituitary gland. AB - A screen designed to identify proteins that specifically bind to retinoic acid response elements resulted in the identification of a rat cDNA encoding a novel protein containing six Cys-Cys, His-Cys zinc fingers. This gene is expressed in a restricted fashion exhibiting distinct temporal and spatial patterns in the developing nervous system, primarily brain, spinal cord, sensory ganglia, retina, and nasal epithelia, as well as in the pituitary, and is referred to as neural zinc finger factor 1 (NZF-1). NZF-1 binds specifically to a cis-regulatory element of the beta-retinoic acid receptor (RAR beta) gene, as well as to other related DNA elements, including two in the upstream enhancer region of the mouse Pit-1 gene. In heterologous cells, NZF-1 activates transcription from promoters containing specific binding sequences and can synergize with other factors, such as Pit-1, to regulate gene expression. These results suggest that NZF-1 may exert regulatory roles in the developing and mature nervous system and in the pituitary gland. Identification of a second mouse gene highly homologous to NZF-1, encoded by a distinct genomic locus, reveals a dispersed gene family encoding proteins containing Cys-Cys, His-Cys motifs. PMID- 8631882 TI - Proprotein-processing endoprotease furin decreases regulated secretory pathway specific proteins in the pancreatic beta cell line MIN6. AB - Prohormone convertases PC2 and PC3, yeast Kex2-family endoproteases specific to the regulated secretory pathway, cleave proinsulin to insulin in the secretory granules of pancreatic beta cells. The well-differentiated beta cell line MIN6 expresses PC2 and PC3 and another regulated secretory pathway-specific protein chromogranin A. Furin, another yeast Kex2 endoprotease, exists in the trans-Golgi networks of many cell types. The beta cell line RINm5F (a cell line that is less differentiated than the MIN6 cell line) does not express the regulated pathway specific proteins, but strongly expresses furin. We suspected that furin expression may cause the decrement of regulated secretory pathway-specific proteins. To test this hypothesis, we expressed a furin cDNA with a metallothionein promoter in MIN6 cells. With Zn2+ stimulation of furin expression, the messages of PC2, PC3, and chromogranin A decreased, and the processing of proinsulin to mature insulin became less efficient. The furin expressing MIN6 cells exhibited less insulin content and weakened insulin secretion in response to a high glucose concentration. The conditioned medium from furin-expressing MIN6 cells also exerted a decrease of PC2 and PC3 expression in unaltered MIN6 cells. Thus, proteins cleaved by furin inside the cells or by truncated furin shed into the culture medium appear to cause decreased PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion in MIN6 cells. PMID- 8631883 TI - Cloning of human Stat5B. Reconstitution of interleukin-2-induced Stat5A and Stat5B DNA binding activity in COS-7 cells. AB - We have isolated a second human Stat5 cDNA, Stat5B, and demonstrated that the genes encoding both Stat5A and Stat5B are located at chromosome 17q11.2. Both genes were constitutively transcribed in peripheral blood lymphocytes. By using specific antisera, we demonstrated that both Stat5A and Stat5B are activated by interleukin-2 (IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia cells, and human T cell lymphotropic virus type I-transformed MT-2 T cells. In COS-7 cells, which constitutively express the Janus family tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B DNA binding activities was dependent on the coexpression of Jak3 along with the IL-2 receptor beta chain and the common cytokine receptor gamma-chain. This IL-2-induced Stat5 activation was dependent on the presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2 receptor beta chain, indicating that either of these two tyrosines can serve as a docking site. Moreover, we demonstrated that human Stat5 activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in Stat5B, indicating that these tyrosines are required for dimerization. The COS-7 reconstitution system described herein provides a valuable assay for further elucidation of the IL-2-activated JAK-STAT pathway. PMID- 8631885 TI - A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. AB - By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells < 4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with < 6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions. PMID- 8631884 TI - Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study. AB - Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+. PMID- 8631886 TI - Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4. AB - We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis. PMID- 8631887 TI - Purification of a soluble UmuD'C complex from Escherichia coli. Cooperative binding of UmuD'C to single-stranded DNA. AB - The Escherichia coli UmuD' and UmuC proteins play essential roles in SOS-induced mutagenesis. Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein. We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA. The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD' (12 kDa) molecules. In contrast to its inability to bind to double-stranded DNA, UmuD'C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization. A Hill coefficient, n = 3, characterizes the binding of UmuD'C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA. The apparent equilibrium binding constant of UmuD'C to single-stranded DNA is approximately 300 nM. Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative. Binding of UmuD'C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction. The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remains bound to DNA in the presence of RecA. PMID- 8631888 TI - Phosphoinositide 3-kinase and p72syk noncovalently associate with the low affinity Fc gamma receptor on human platelets through an immunoreceptor tyrosine based activation motif. Reconstitution with synthetic phosphopeptides. AB - Previously, we have demonstrated that the cytoplasmic tyrosine kinase p72syk is coupled to the platelet Fc receptor for IgG (Fc gamma RIIA) (Chacko, G. W., Duchemin, A. M., Coggeshall, K. M., Osborne, J. M., Brandt, J. T., and Anderson, C. L. (1994) J. Biol. Chem. 269, 32435-32440). Further analysis of the platelet activation by Fc gamma RIIA demonstrated that Fc gamma RIIA is also inducibly coupled to the serine/threonine and lipid kinase, phosphoinositide 3-kinase (PI 3 K). activation of platelets with anti-Fc gamma RIIA antibodies resulted in the noncovalent association of PI 3-K with Fc gamma RIIA as well as an increase in Fc gamma RIIA-associated PI 3-K activity. Binding of both p72syk and PI 3-K to Fc gamma RIIA was reconstituted with synthetic phosphopeptides corresponding to the sequence of the atypical immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of Fc gamma RIIA. Our findings demonstrate that coupling of both p72syk and PI. 3-K activities to Fc gamma RIIA is regulated by tyrosine phosphorylation of the ITAM, and we speculate that p72syk might act as an adapter to recruit PI 3-K to activated Fc gamma RIIA. PMID- 8631889 TI - A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone beta subunit promoter in gonadotropes of transgenic mice. AB - Analysis of luteinizing hormone (LH) beta subunit promoters from a broad range of species including teleosts and humans revealed strict conservation of a sequence homologous to the steroidogenic factor-1 (SF-1) regulatory element of cytochrome P-450 steroid hydroxylase genes. Interaction between SF-1 and this putative response element in the bovine LH beta promoter was confirmed by electrophoretic mobility shift assays. Furthermore, cotransfection of alpha T3-1 cells with an expression vector encoding SF-1 induced binding site-dependent transcription from the bovine LH beta promoter. Physiological significance of the LH beta SF-1 consensus sequence was established using transgenic mice containing either the wild type bovine promoter or a promoter with a site-specific mutation of this site. Mutation of the SF-1 binding site nearly eliminated promoter activity, and the mutant transgene remained inactive following induction of gonadotropin releasing hormone accomplished by castrating male and female mice. Thus, increases of gonadotropin-releasing hormone within a physiological range did not compensate for the loss of the SF-1 binding site. Together, these findings indicate that the SF-1 binding site is a key regulator of LH beta promoter activity in vivo and implicate SF-1 as at least one of the transcription factors that acts through this site. PMID- 8631890 TI - Genetic characterization of Clostridium botulinum type A containing silent type B neurotoxin gene sequences. AB - A recent study detected genes encoding type B botulinum neurotoxin in some type A strains of Clostridium botulinum that exhibit no type B toxin activity. In this study, we investigated the presence, structure, linkage, and organization of genes encoding botulinum neurotoxin (BoNT) and other components of the progenitor complex. Sequence analysis showed that the silent BoNT/B gene is highly related to that from authentic proteolytic type B C. botulinum. However, a stop signal and deletions were found within the sequence. A non-toxin nonhemagglutinin gene (NTNH) was mapped immediately upstream of both the BoNT/A and silent BoNT/B genes. Significantly the NTNH gene adjacent to the defective BoNT/B gene was "chimeric, " the 5'- and 3'-regions of the gene had high homology with corresponding regions of the type B NTNH gene, while the 471-amino acid sequence in the central region was identical to NTNH of type A. Hemagglutinin genes HA-33 and HA-II were not found adjacent to the NTNH/A gene, but instead there was an unidentified open reading frame previously reported in strains of C. botulinum types E and F. By contrast HA-II, HA-33, and NTNH genes were located immediately upstream of the silent BoNT/B gene. Pulsed-field gel electrophoretic analysis of chromosomal DNA digests indicated the distance between type A and B gene clusters to be less than 40 kilobases. PMID- 8631891 TI - Characterization of terminal sialic acid linkages on human thymocytes. Correlation between lectin-binding phenotype and sialyltransferase expression. AB - T cell surface sialylation changes during maturation in the thymus. We have previously demonstrated increased expression of mRNA encoding the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase in mature medullary human thymocytes, compared with immature cortical thymocytes. For this enzyme, increased expression of transferase mRNA correlated with increased sialylation of O-glycans. We have now examined the pattern of expression in the human thymus of two additional sialyltransferases, the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6N) and the Gal beta 1,3/4GlcNAc alpha 2,3-sialyltransferase (ST3N). The patterns of mRNA expression were compared with the pattern of binding of two sialic acid-specific plant lectins, Sambucus nigra agglutinin and Maackia amurensis agglutinin, which preferentially recognize alpha 2,6- and alpha 2,3-linked sialic acids, respectively, on N-glycans. By in situ hybridization, mRNA encoding ST3N was detected uniformly throughout the thymus. All thymocytes bound M. amurensis agglutinin, demonstrating a direct correlation between the level of ST3N mRNA expression and cell-surface glycosylation. In contrast, mRNA encoding ST6N was also expressed uniformly throughout the thymus; however, only mature (CD3hi) medullary thymocytes bound S. nigra agglutinin. On mature thymocytes, S. nigra agglutinin appeared to bind primarily to the cell-surface glycoprotein CD45; since only the mature thymocytes expressed the CD45RA isoform, while both mature and immature populations expressed the CD45R0 isoform, CD45RA may be a preferred substrate for ST6N. These results demonstrate that glycoprotein sialylation is tightly regulated during T cell development and that the developmentally regulated expression of specific oligosaccharide structures on the cell surface may be influenced by expression of both the relevant glycosyltransferase and specific acceptor substrates. PMID- 8631892 TI - DNA recognition by normal and oncogenic thyroid hormone receptors. Unexpected diversity in half-site specificity controlled by non-zinc-finger determinants. AB - The nuclear hormone receptors regulate target gene expression in response to hormones of extracellular origin. The DNA binding specificity of these receptors therefore plays the critical role of defining the precise repertoire of target genes that respond to a given hormone. We report here an analysis of the DNA binding specificity of the thyroid hormone receptor (c-ErbA protein) and that of an oncogenic derivative, the v-ErbA protein. These otherwise closely similar proteins exhibit quite divergent DNA sequence specificities at multiple positions within the DNA binding site. The thyroid hormone receptor (c-ErbA protein exhibits a particularly broad DNA specificity, whereas the v-ErbA protein is comparatively quite specific. Intriguingly, these differences in DNA recognition largely map to an N-terminal receptor domain not traditionally implicated in DNA binding, and are further influenced by heterodimer formation with retinoid X receptors. We propose that the N terminus of nuclear hormone receptors plays an critical role in DNA recognition by altering the conformation of the receptor domains that make the actual base-specific contacts. PMID- 8631894 TI - Outside-in integrin signal transduction. Alpha IIb beta 3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation. AB - alpha IIb beta 3-(GP IIb IIIa) is the most abundant integrin expressed on platelets and plays a critical role in platelet aggregation and normal hemostasis. In response to platelet stimulation by agonists such as thrombin, alpha IIb beta 3 becomes a receptor for the adhesive proteins fibrinogen, von Willebrand factor, vitronectin, and fibronectin. Binding of extracellular matrix ligands allows the integrin to transmit a signal to the inside of the cell, but the exact mechanisms whereby integrins transduce these signals remains unclear. In this paper we demonstrate that the beta 3 subunit of alpha IIb beta 3 was phosphorylated on tyrosine residues in response to thrombin-induced platelet aggregation. However, tyrosine phosphorylation was not observed when platelets were stimulated by thrombin in the presence of an inhibitor of aggregation. Phosphotyrosine was only detected when platelets were solubilized under protein denaturing conditions. A peptide corresponding to residues 740-762 of the beta 3 cytoplasmic domain was capable of binding the signaling proteins SHC and GRB2. GRB2 binding occurred only when both tyrosine residues (Tyr-747 and Tyr-759) were phosphorylated. SHC binding also occurred to a peptide monophosphorylated at Tyr 759. The data suggest that tyrosine phosphorylation of an integrin beta subunit may be important in initiating outside-in signaling cascades by inducing association of signaling components directly with the integrin. PMID- 8631893 TI - Multiple Ca(2+)-calmodulin-dependent protein kinase kinases from rat brain. Purification, regulation by Ca(2+)-calmodulin, and partial amino acid sequence. AB - We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms. PMID- 8631895 TI - Processing and activation of CMH-1 by granzyme B. AB - Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1 beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp198-Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp23-Ala24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro sugge st that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing. PMID- 8631896 TI - Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair. AB - Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38. In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi. In the in vitro nucleotide excision repair (NER) system that consists entirely of purified proteins, we show that neither TFIIHi nor a mixture of purified Rad3 and Rad25 proteins is active in NER but that the combination of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damaged DNA. These results provide the first evidence for a direct requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision step of NER. The NER efficacy of TFIIH is greatly diminished or abolished upon substitution of Rad3 with the rad3 Arg-48 mutant protein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA. Our results further indicate that the carboxyl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro. These studies reveal the differential requirement of Rad3 DNA helicase and CTD kinase activities in damage specific incision versus RNA polymerase II transcription. PMID- 8631897 TI - Serum response factor mediates AP-1-dependent induction of the skeletal alpha actin promoter in ventricular myocytes. AB - "Fetal" gene transcription, including activation of the skeletal alpha-actin (SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic ligands. Induction of the promoter by transforming growth factor beta or norepinephrine requires serum response factor (SRF) and TEF-1; expression is inhibited by YY1. We and others postulated that immediate-early transcription factors might couple trophic signals to this fetal program. However, multiple Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to SREs, nor physical interaction between Fos/Jun and SRF, was detected in mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene via SRF, without detectable binding to the promoter or to SRF. PMID- 8631898 TI - Phosphorylation of microtubule-associated proteins MAP2 and MAP4 by the protein kinase p110mark. Phosphorylation sites and regulation of microtubule dynamics. AB - The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells. PMID- 8631899 TI - Simultaneous but independent activation of adenylate cyclase and glycosylphosphatidylinositol-phospholipase C under stress conditions in Trypanosoma brucei. AB - Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled. PMID- 8631901 TI - Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae. AB - Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp. PMID- 8631900 TI - Molecular expression of the alpha-chemokine rabbit GRO in Escherichia coli and characterization of its production by lung cells in vitro and in vivo. AB - GRO proteins are alpha-chemokine cytokines that attract neutrophils and stimulate the growth of a variety of cells. Previously, we observed that rabbit alveolar macrophages transcribe the genes for at least two GRO homologues. In order to study the role of GRO cytokines in lung inflammation, we cloned the predominant rabbit GRO cDNA (RabGRO) from alveolar macrophages, expressed bioactive recombinant protein (rRabGRO) in Escherichia coli, and developed a sensitive and specific enzyme-linked immunosorbent assay for RabGRO protein. We found that rabbit AM express and secrete GRO in vitro in response to both exogenous (e.g. lipopolysaccharide, heat-killed Staphylococcus aureus, and crystalline silica) and endogenous inflammatory stimuli (e.g. tumor necrosis factor-alpha) as determined by both radioimmunoprecipitation and enzyme-linked immunosorbent assay. Biologically significant amounts of GRO are present in vivo in the bronchoalveolar lavage fluid of rabbits with E. coli pneumonia; by in situ hybridization, GRO mRNA is detectable in infiltrating pulmonary leukocytes and bronchial epithelial cells. These results indicate that GRO chemokines are likely to be important mediators of the inflammatory response that accompanies acute infectious processes in the lungs. PMID- 8631902 TI - Cloning by metabolic interference in yeast and enzymatic characterization of Arabidopsis thaliana sterol delta 7-reductase. AB - Reduction of the delta 7 double bond of sterols, a key biosynthetic step in higher eukaryotes, is lacking in lower eukaryotes like the yeast Saccharomyces cerevisiae, leading to terminal sterols with a delta 5,7-conjugated diene structure. Genes encoding two sterol reductases involved, respectively, in the reduction of sterol delta 14 and delta 24(28) double bonds have been cloned to date, but no sequence information was available on the enzyme responsible for delta 7-bond reduction. This study presents the cloning of the NADPH-sterol delta 7-reductase (delta 7-red) from Arabidopsis thaliana, based on a metabolic interference approach in yeast. The principle is the functional expression of a plant cDNA library in the yeast strain FY1679-28C tolerant to sterol modifications and the selection of clones resistant to the polyene fungicide nystatin. The toxicity of this compound is dependent on the presence of delta 5,7 unsaturated sterols in the yeast plasma membrane. One clone out of 10(5) transformants exhibits a cDNA-dependent alteration of cell sterol composition. The 1290-base pair cDNA open reading frame was isolated and sequenced. The corresponding protein presents a significant sequence similarity with yeast delta 14- and delta 24(28)-reductases and with human lamin B receptor. The coding sequence was extracted by polymerase chain reaction and inserted into a galactose inducible yeast expression vector to optimize expression. Analysis using transformed wild type yeast or sterol altered mutants, indicated that delta 5,7 ergosta- and cholesta-sterols are efficiently reduced in vivo, regardless of the structural variations on the side chain. No reductase activity was observed toward the delta 14 or the delta 5 positions of sterols. In vivo extensive delta 7-reduction of the free and esterified pools of sterols was observed upon induction of the enzyme. Ergosterol present before induction was reduced into ergosta-5,22-dieneol, whereas ergosta-5-eneol is the new end product of sterol neosynthesis, indicating that the yeast delta 22 desaturase may be no longer active on C-7-saturated sterols. In vitro tests indicated that delta 7-reductase activity is preferentially associated with the endoplasmic reticulum membrane and confirmed the previous finding that NADPH is the reducing agent. PMID- 8631903 TI - Purification and properties of a phosphatidic acid-preferring phospholipase A1 from bovine testis. Examination of the molecular basis of its activation. AB - We recently identified a cytosolic phospholipase A1 activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in Triton X-100 mixed micelle assay system (Higgs, H.N., and Glomset J.A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9574-9578). In the present work we purified the bovine testis enzyme 14,000-fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl-analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC50 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds. PMID- 8631904 TI - Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity. AB - NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs. PMID- 8631905 TI - A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells. AB - The human intestinal fatty acid binding protein (IFABP) binds long-chain fatty acids in vitro, but its intracellular function has remained speculative. A polymorphism in the gene that encodes IFABP results in an alanine (Ala54) to threonine (Thr54) substitution at codon 54 that alters the in vitro binding affinity of the protein for long-chain fatty acids. To identify potential functional variability between Ala54 and Thr54 IFABP, we established permanently transfected Caco-2 cell lines that express either Ala54 or Thr54 IFABP. We found that Caco-2 cells expressing Thr54 IFABP transport long-chain fatty acids and secrete triglycerides to a greater degree than Caco-2 cells expressing Ala54 IFABP. These results provide the first demonstration that IFABP participates in the intracellular transport of long-chain fatty acids. In addition, the observed increase in transport of fatty acids across cells expressing Thr54 IFABP suggests a plausible physiologic mechanism for our prior observation that Pima Indians with a Thr54 IFABP genotype have increased post-absorptive lipid oxidation rates and are more insulin-resistant than Pimas with a Ala54 IFABP genotype. PMID- 8631906 TI - A lysosomal cysteine proteinase from Dictyostelium discoideum contains N acetylglucosamine-1-phosphate bound to serine but not mannose-6-phosphate on N linked oligosaccharides. AB - Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-alpha-1-P residues in phosphodiester linkage to serine. We extended these studies and, in contrast to earlier reports, found that proteinase-1 contains 7.5 mol of Fuc, 8 mol of Man, 2 mol of Xyl, and 30 mol of GlcNAc per calculated mol of protein but no Man-6-P residues. The protein binds to concanavalin A and wheat germ agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose but little of the GlcNAc. beta-Elimination under reducing conditions released only GlcNAc-alpha-1-P. There was no evidence for the release of disaccharides or of fucitol. A rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-alpha-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-alpha-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and alpha-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: Glc NAc-alpha-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-alpha-1-P-specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-alpha-1-P specific monoclonal antibody but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-alpha-1 P-specific antibody. An exception to this is beta-N-acetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications. PMID- 8631908 TI - Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity. AB - We have examined vitamin D receptor (VDR), thyroid hormone receptor (TR), and retinoid X receptor beta (RXR beta) binding to vitamin D response elements (VDREs), two thyroid hormone response elements (TREs) (DR4 and F2), and a retinoic acid response element (DR5). VDR/RXR bound well to the VDREs and to DR4 and DR5 using the electrophoretic mobility shift assay. Surprisingly, VDR/RXR also bound well to F2, which contains half-sites arranged as an inverted palindrome. In co-transfection experiments using CV-1 cells, we observed that VDR repressed basal transcription in the absence of ligand on DR3 and osteopontin VDREs and F2, but had no effect on DR4 or DR5. VDR selectively mediated ligand dependent transcription on only VDREs. VDR also exhibited dominant negative activity as it blocked triiodothyronine (T3)-mediated transcriptional activity on DR4 and F2. These results demonstrate that VDR/RXR heterodimers can bind promiscuously to a wide range of hormone response elements, including inverted palindromes. Moreover, they show that unliganded VDRs, similar to TRs and retinoic acid receptors, can repress basal transcription. Last, they also suggest a novel repressor function of VDR on T3-mediated transcription which may be significant in tissues where VDR and TR are co-expressed. PMID- 8631907 TI - Desmosomal cadherin binding domains of plakoglobin. AB - Plakoglobin is a major component of both desmosomes and adherens junctions. At these sites it binds to the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and cytoskeletal binding functions. Plakoglobin also forms distinct cytosolic protein complexes that function in pathways of tumor suppression and cell fate determination. Recent studies in Xenopus suggest that cadherins inhibit the signaling functions of plakoglobin presumably by sequestering this protein at the membrane and depleting its cytosolic pool. To understand the reciprocal regulation between desmosomal cadherins (desmoglein and desmocollin) and plakoglobin, we have sought to identify the binding domains involved in the formation of these protein complexes. Plakoglobin comprises 13 central repeats flanked by amino-terminal and carboxyl-terminal domains. Our results show that repeats 1-4 are involved in binding desmoglein-1. In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of either end of the central repeat domain. The binding sites for two adherens junction components, alpha-catenin and classical cadherins, overlap these sites. Competition among these proteins for binding sites on plakoglobin may therefore account for the distinct composition of adherens junctions and desmosomes. PMID- 8631909 TI - Cell-specific induction of distinct oncogenes of the Jun family is responsible for differential regulation of collagenase gene expression by transforming growth factor-beta in fibroblasts and keratinocytes. AB - Transforming growth factor-beta (TGF-beta) plays a major role in regulating connective tissue deposition by controlling both extracellular matrix production and degradation. In this study, we show that TGF-beta transcriptionally represses both basal and tumor necrosis factor-alpha-induced collagenase (matrix metalloprotease-1) gene expression in dermal fibroblasts in culture, whereas it activates its expression in epidermal keratinocytes. We demonstrate that this differential effect of TGF-beta on collagenase gene expression is due to a cell type-specific induction of distinct oncogenes of the Jun family, which participate in the formation of AP-1 complexes with different trans-activating properties. Specifically, our data indicate that the inhibitory effect of TGF beta in fibroblasts is likely to be mediated by jun-B, based on the following observations: (a) TGF-beta induces high levels of jun-B expression and (b) over expression of jun-B mimics TGF-beta effect in inhibiting basal collagenase promoter activity and preventing tumor necrosis factor-alpha-induced trans activation of the collagenase promoter. In contrast, TGF-beta induction of collagenase gene expression in keratinocytes is preceded by transient elevation of c-jun proto-oncogene expression. Over-expression of c-jun leads to trans activation of the collagenase promoter in both cell types, suggesting that c-jun is a ubiquitous inducer of collagenase gene expression. Transfection of keratinocytes with an antisense c-jun construct together with a collagenase promoter/reporter gene construct inhibits basal and TGF-beta-induced up regulation of the collagenase promoter activity, implying that c-jun mediates TGF beta effect in this cell type. Collectively, our data suggest differential signaling pathways for TGF-beta in dermal fibroblasts and epidermal keratinocytes, leading to cell type-specific induction of two AP-1 components with opposite transcriptional activities. PMID- 8631910 TI - Dithiocarbamates trigger differentiation and induction of CD11c gene through AP-1 in the myeloid lineage. AB - It has recently been shown that the alteration of the cell-redox status affects the transcription factor expression and activity. Dithiocarbamates (DTCs) are potent antioxidant agents that can switch the expression of genes dependent on the activation of the transcription factors AP-1 and NF kappa B. In this study, we show that these agents triggered the expression of genes involved in myeloid differentiation of the promonocytic U-937 cell line. DTCs promoted differentiation-associated changes that included the surface up-regulation of beta 2-integrins (CD11a-c/CD18), cell growth arrest concomitant with transferrin receptor (CD71) down-modulation, induction of the nonspecific esterase enzyme, and a rapid drop in the mRNA levels of c-myc. A further analysis, focused on the molecular mechanisms leading to the activation of CD11c expression, revealed that the pyrrolidine derivative of DTC (PDTC) increased CD11c mRNA levels and augmented its gene promoter activity. Transfection experiments with reporter constructs harboring different promoter regions of CD11c gene, indicated the presence of a functional DTC-responsive region located between positions -160 and +40 of the promoter. Gel retardation assays revealed that the PDTC-induced DNA protein complexes were restricted to members of the Fos and Jun families that bound to an AP-1 site located at position -60 from the transcription start site. A role for this site was confirmed by in vitro mutagenesis experiments that indicated the functional importance of this site for the CD11c gene transcriptional activation in response to PDTC. The effect of DTCs on myeloid cell differentiation supports a possible role for these agents in the therapy of some bone marrow-derived malignancies. PMID- 8631911 TI - Association of Rab1B with GDP-dissociation inhibitor (GDI) is required for recycling but not initial membrane targeting of the Rab protein. AB - We have identified the Rab1B effector-domain mutant (D44N) that, when geranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell-free systems or intact cells, fails to form detectable complexes with GDP-dissociation inhibitors (GDIs). GDI-Rab complexes were collected on anti-FLAG affinity beads after incubating recombinant geranylgeranylated Rab1B with FLAG epitope-tagged GDI in vitro, or transiently coexpressing Myc-tagged Rab1B with FLAG-GDI-alpha or FLAG-GDI-2 in human embryonal kidney 293 cells. [3H]Mevalonate labeling and immunoprecipitation studies confirmed that the inability of Myc-Rab1BD44N to associate with GDI in vivo was not due to failure of the mutant to undergo geranylgeranylation. Immunofluorescence localization and immunoblot analysis of subcellular fractions indicated that expressed Myc-Rab1BD44N was efficiently delivered to intracellular membranes in 293 cells. This was confirmed when the fate of the prenylated pool of Rab1BD44N in 293 cells was traced by labeling the geranylgeranyl groups attached to the nascent protein with [3H]meval onate. However, in contrast to the prenylated Rab1BWT, which was distributed in both the membrane and soluble fractions, the prenylated Rab1BD44N was completely absent from the cytosol. Overexpression of Myc-Rab1BD44N did not impair ER --> Golgi glycoprotein trafficking in 293 cells, which was assessed by monitoring the Golgi dependent processing of coexpressed beta-amyloid precursor protein. The current findings suggest that nascent prenylated Rab1B can be delivered to intracellular membranes in intact cells without forming a stable complex with GDI, but that recycling of prenylated Rab1B to the cytosolic compartment is absolutely dependent on GDI interaction. PMID- 8631912 TI - Two cytoplasmic domains of mammalian adenylyl cyclase form a Gs alpha- and forskolin-activated enzyme in vitro. AB - Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2). The first cytoplasmic domain of type I enzyme (IC1) and the second cytoplasmic domain of type II enzyme (IIC2-delta 3, a construct in which 36 N-terminal amino acids of the C2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in Gs alpha- and forskolin-activated enzyme activity. The turnover number for Gs alpha- and forskolin-stimulated enzyme activity of the complex between IC1 and IIC2-delta 3 was 8.2 s-1. The concentration of IIC2-delta 3 to achieve half-maximal activation of IC1 was 0.8 and 1.3 microM when stimulated by forskolin and Gs alpha, respectively. The concentration of IIC2 delta 3 needed to complex with IC1 was reduced 10-fold (0.08 microM) when the enzyme was activated by both forskolin and Gs alpha, suggesting that Gs alpha and forskolin increased the affinity of the two cytoplasmic domains for each other. PMID- 8631913 TI - Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. AB - Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole. PMID- 8631914 TI - Wortmannin-sensitive trafficking pathways in Chinese hamster ovary cells. Differential effects on endocytosis and lysosomal sorting. AB - Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors. The catalytic subunit of the p85/p110 PI 3'-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole. This suggests that PI 3'-kinases may play analogous roles in mammalian cells. We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3'-kinase. Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition of the p85/p110 PI 3'-kinase in intact cells. The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate. Experiments with fluorescent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin. Wortmannin altered the morphology of the recycling compartment, which was more vesiculated than in untreated cells. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected. In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins. Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3'-kinase activity or to redistribute transferrin receptors. Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes. PMID- 8631915 TI - Protein-tyrosine phosphatase-mediated decrease of epidermal growth factor and platelet-derived growth factor receptor tyrosine phosphorylation in high cell density cultures. AB - Contact-induced growth inhibition is a characteristic feature of normal cells grown in monolayer. The importance of reversible tyrosine phosphorylation in mitogenic signaling, together with earlier reports of increased levels of protein tyrosine phosphatases (PTPs) in densely cultured cells, has led to the proposal that PTPs may be involved in mediating contact inhibition of cell growth. We have compared net levels of ligand-induced tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in mink lung epithelial cells cultured under sparse or dense conditions. The levels of net tyrosine phosphorylation of the stimulated EGF receptor was found to be more than 4-fold higher in sparse cultures. This difference was greatly reduced when cells were pretreated with the PTP inhibitor phenyl arsine oxide. Monitoring of dephosphorylation rates in vivo of the stimulated EGF receptors revealed increased EGF receptor-directed PTP activity in dense cultures. The platelet-derived growth factor beta-receptor, expressed in stably transfected porcine aortic endothelial cells, also displayed lower levels of ligand induced net tyrosine phosphorylation in cells from dense cultures. This density-dependent difference in tyrosine phosphorylation was reduced by pretreatment of cultures with the PTP inhibitor orthovanadate. A PTP-mediated decrease of the in vivo net levels of ligand induced tyrosine phosphorylation of EGF and platelet-derived growth factor receptors in cells at high density have thus been demonstrated. Loss of this previously unnoticed regulatory pathway may be involved in cellular transformation. PMID- 8631916 TI - Construction of a combinatorial IgE library from an allergic patient. Isolation and characterization of human IgE Fabs with specificity for the major timothy grass pollen allergen, Phl p 5. AB - To characterize human IgE antibodies with specificity for a major allergen at the molecular level, we have constructed an IgE combinatorial library from a grass pollen allergic patient. cDNAs coding for IgE heavy chain fragments and for light chains were reverse-transcribed and polymerase chain reaction-amplified from RNA of peripheral blood lymphocytes and randomly combined in plasmid pComb3H to yield a combinatorial library of 5 x 10(7) primary clones. IgE Fabs with specificity for Phl p 5, a major timothy grass pollen allergen, were isolated by panning. Sequence analysis showed that the 4 of the Fabs used the same heavy chain fragments which had combined with different kappa light chains. Soluble recombinant IgE Fabs were purified by affinity chromatography to Phl p 5 and, like natural IgE antibodies, cross-reacted with group 5 allergens from different grass species. The described approach should facilitate studies on the molecular interaction between IgE antibodies and allergens and encourages the consideration of specific IgE Fabs that are capable of interfering with allergen-IgE binding as potential therapeutic tools. PMID- 8631917 TI - Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis. AB - Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12 myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase. PMID- 8631918 TI - Stimulation by parathyroid hormone of interleukin-6 and leukemia inhibitory factor expression in osteoblasts is an immediate-early gene response induced by cAMP signal transduction. AB - Parathyroid hormone and other agents that stimulate bone resorption function, at least in part, by inducing osteoblasts to secrete cytokines that stimulate osteoclast differentiation and activity. We previously demonstrated that parathyroid hormone induces expression by osteoblasts of interleukin-6 and leukemia inhibitory factor without affecting the 16 other cytokines that were examined. We also showed that stimulation of osteoclast activity by parathyroid hormone is dependent on activation of the cAMP signal transduction pathway and secretion of interleukin-6 by osteoblasts. In the current study, we demonstrate that the rapid and transient stimulation of interleukin-6 and leukemia inhibitory factor is inhibited by actinomycin D and superinduced by protein synthesis inhibitors, the classical characteristics of an immediate-early gene response. Moreover, activation of cAMP signal transduction by parathyroid hormone and parathyroid hormone-related protein is necessary and sufficient to induce both interleukin-6 and leukemia inhibitory factor. In addition, cAMP analogues as well as vasoactive intestinal peptide and isoproterenol, two neuropeptides that stimulate bone resorption by activating cAMP signal transduction in osteoblasts, also induce interleukin-6 and leukemia inhibitory factor in these cells. Taken together with our previous results, this study suggests that interleukin-6 is crucial for stimulation of bone resorption not only by parathyroid hormone, but also by parathyroid hormone-related protein, vasoactive intestinal peptide, and beta-adrenergic agonists, like isoproterenol. PMID- 8631919 TI - Phosphorylation-independent stimulation of DNA topoisomerase II alpha activity. AB - It has been suggested that casein kinase II phosphorylates DNA topoisomerase II alpha (topo II alpha) in mouse FM3A cells, by comparison of phosphopeptide maps of topo II alpha labeled in intact cells and of topo II alpha phosphorylated by various kinases in vitro. The phosphorylation of purified topo II alpha by casein kinase II, which attached a maximum of two phosphate groups per topo II alpha molecule, had no effect on the activity of topo II alpha. Dephosphorylation of purified topo II alpha by potato acid phosphatase, which almost completely dephosphorylated the topo II alpha, did not reduce the activity of topo II alpha. The incubation itself, regardless of phosphorylation or dephosphorylation status, stimulated the enzyme activity in both reactions. Topo II alpha activity was stimulated by incubation in a medium containing low concentrations of glycerol but not in that containing high concentrations of glycerol, such as the 50% in which purified topo II alpha is stored. The stimulation of topo II alpha activity by incubation was dependent on the concentration of topo II alpha, requiring a relatively high concentration of topo II alpha. PMID- 8631920 TI - Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli. AB - beta-Ketoacyl-acyl carrier protein (ACP) synthase III (the fabH gene product) condenses acetyl-CoA with malonyl-ACP to initiate fatty acid biosynthesis in the dissociated, type II fatty acid synthase systems typified by Escherichia coli. The accumulation of malonyl-acyl carrier protein (ACP) following the inhibition of a reconstituted fatty acid synthase system by acyl-ACP implicated synthase III (FabH) as a target for acyl-ACP regulation (Heath, R. J., and Rock, C. O. (1996) J. Biol. Chem. 271, 1833-1836); therefore, the FabH protein was purified and its biochemical and regulatory properties examined. FabH exhibited a Km of 40 microM for acetyl-CoA and 5 microM for malonyl-ACP. FabH also accepted other acyl-CoAs as primers with the rank order of activity being acetyl-CoA approximately propionyl-CoA >> butyryl-CoA. FabH utilized neither hexanoyl-CoA nor octanoyl CoA. Acyl-ACPs suppressed Fabh activity, and their potency increased with increasing acyl chain length between 12 and 20 carbon atoms. Nonesterified ACP was not an inhibitor. Acyl-ACP inhibition kinetics were mixed with respect to acetyl-CoA, but were competitive with malonyl-ACP, indicating that acyl-ACPs decrease FabH activity by binding to either the free enzyme or the acyl-enzyme intermediate. These data support the concept that the inhibition of chain initiation at the beta-ketoacyl-ACP synthase III step contributes to the attenuation of fatty acid biosynthesis by acyl-ACP. PMID- 8631921 TI - Functional characterization of the YUR1, KTR1, and KTR2 genes as members of the yeast KRE2/MNT1 mannosyltransferase gene family. AB - Eukaryotic glycan structures are progressively elaborated in the secretory pathway. Following the addition of a core N-linked carbohydrate in the endoplasmic reticulum, glycoproteins move to the Golgi complex where the elongation of O-linked sugar chains and processing of complex N-linked oligosaccharide structures take place. In order to better define how such post translational modifications occur, we have been studying a yeast gene family in which at least one member, KRE2/MNT1, is involved in protein glycosylation. The family currently contains five other members: YUR1, KTR1, KTR2, KTR3 and KTR4 (Mallet, L., Bussereau, F., and Jacquet, M. (1994) Yeast 10, 819-831). All encode putative type II membrane proteins with a short cytoplasmic N terminus, a membrane-spanning region, and a highly conserved catalytic lumenal domain. Kre2p/Mnt1p is a alpha 1,2-mannosyltransferase involved in O- and N-linked glycosylation (Hausler, A., Ballou, L., Ballou, C.E., and Robbins, P.W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6846-6850); however, the role of the other proteins has not yet been established. We have carried out a functional analysis of Ktr1p, Ktr2p, and Yur1p. By in vitro assays, Ktr1p, Ktr2p, and Yur1p have been shown to be mannosyltransferase but, in vivo, do not appear to be involved in O glycosylation. Examination of the electrophoretic mobility of the N-linked modified protein invertase in null mutant strains indicates that Ktr1p, Ktr2p, and Yur1p are involved in N-linked glycosylation, possibly as redundant enzymes. As found with Kre2p (Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H. (1992) Genetics 130, 273-283), Ktr1p, Ktr2p, and Yur1p also seem to be implicated in the glycosylation of cell wall mannoproteins, since yeast cells containing different gene disruptions become K1 killer toxin-resistant. Immunofluorescence microscopy reveals that like Kre2p; Ktr1p, Ktr2p and Yur1p are localized in the Golgi complex. PMID- 8631922 TI - Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. AB - The signal transduction cascade which initiates transmembraneous influx of Ca2+ into endothelial cells in response to the discharge of intracellular Ca2+ stores is thought to involve a step sensitive to tyrosine kinase inhibition. We investigated the interrelationship between Ca2+ signaling and protein tyrosine phosphorylation following cell stimulation with either the receptor-dependent agonist, bradykinin, or the protein-tyrosine phosphatase inhibitor, phenylarsine oxide. In cultured human endothelial cells phenylarsine oxide instigated a concentration-dependent increase in the intracellular concentration of free Ca2+ ([Ca2+]i). This increase in [Ca2+]i was not associated with the tyrosine phosphorylation of phospholipase C gamma, enhanced formation of inositol 1,4,5 trisphosphate, or the rapid depletion of intracellularly stored Ca2+ but was coincident with the enhanced and prolonged tyrosine phosphorylation of a number of cytoskeletal proteins. In bradykinin-stimulated cells the tyrosine phosphorylation of the same cytoskeletal proteins (most notably 85- and 100-kDa proteins) was transient when cells were stimulated in the presence of extracellular Ca2+, was maintained under Ca2+-free conditions, and was reversed following readdition of extracellular Ca2+. These data suggest that the tyrosine phosphorylation of 2 cytoskeletal proteins is determined by the level of Ca2+ present in intracellular stores thus indicating a critical role for tyrosine phosphorylation in the control of capacitative Ca2+ entry in endothelial cells. PMID- 8631923 TI - Integrin alpha v beta 5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin. AB - The macrophage colony-stimulating factor (M-CSF) is able to induce the expression of the alpha v beta 5 integrin receptor on the surface of cultured human macrophages (De Nichilo, M. O., and Burns, G. F. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2517-2521). In the present study, we establish that the adhesion of M CSF-treated macrophages to vitronectin is mediated by the integrin alpha v beta 5, and show by indirect immunofluorescence analysis that alpha v beta 5 and the cytoskeletal protein paxillin localize to focal contacts upon adhesion to vitronectin. Immunoprecipitation and Western blot analysis revealed that M-CSF treated macrophages do not express focal adhesion kinase (FAK), thereby providing direct evidence for integrin-dependent localization of paxillin to focal contacts in the absence of FAK expression. Investigation of paxillin phosphorylation by two-dimensional phosphoamino acid analysis indicates that paxillin is 99% phosphorylated on serine residue(s) in response to vitronectin adhesion, and only 1% phosphorylated on tyrosine. Stimulation of protein kinase C (PKC) activity with the phorbol ester phorbol 12-myristate 13-acetate enhances paxillin phosphorylation, while two selective inhibitors of PKC, GF109203X and chelerythrine chloride, effectively block the phosphorylation of paxillin induced in response to vitronectin adhesion. Taken together, these data demonstrate that in M-CSF-treated macrophages adherent to vitronectin, paxillin localizes to focal contacts in the absence of FAK expression and is predominantly phosphorylated on serine residue(s) in a PKC-dependent manner. PMID- 8631924 TI - Chondrocyte matrix metalloproteinase-8. Human articular chondrocytes express neutrophil collagenase. AB - This study confirms that normal human articular chondrocytes express neutrophil collagenase or matrix metalloproteinase-8 (MMP-8), a gene product previously thought to be expressed exclusively by neutrophil leukocytes. Both MMP-8 protein and mRNA were present in articular cartilages collected from normal human donors. Cartilage extracts were assayed by immunoblotting and by analysis of enzymatic activity on gelatin-substrate gels. Latent MMP-8 extracted from cartilage has a molecular mass of 55 kDa; active MMP-8 was identified at 46 and 42 kDa. In the absence of a reducing agent, MMP-8 migrated in a high molecular mass complex above 200 kDa. Northern blotting results demonstrated the expression of MMP-8 in chondrocytes, which could be up-regulated by stimulation with interleukin-1 beta. In addition, reverse transcription-polymerase chain reaction using nested primers and in situ hybridization revealed the presence of MMP-8 mRNA in chondrocytes. The presence of both MMP-8 protein and message in cartilage supports the concept that neutrophil collagenase could be the enzyme described as "aggrecanase". PMID- 8631925 TI - Repression of transforming growth factor beta 1 protein by antisense oligonucleotide-induced increase of adrenal cell differentiated functions. AB - Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To determine whether this peptide plays an autocrine role in BAC, cells were transfected with 10 microM unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF beta 1 mRNA in an attempt to inhibit TGF beta 1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of 32P-labeled TGF beta 1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF beta 1 AON uptake reached a plateau after 8 h of transfection (16% of the radioactivity added) and remained fairly constant for at least 24 h. In contrast, the uptake of TGF beta 1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then declined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF beta 1 AON was still high with about 8% of the radioactivity added, whereas that of TGF beta 1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF beta 1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF beta 1 AON nor SON modified TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caused the disappearance of TGF beta 1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF beta 1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF beta 1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF beta 1 AON. PMID- 8631926 TI - Cloning and functional expression of a human eosinophil CC chemokine receptor. PMID- 8631927 TI - The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f. AB - Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schurmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx. PMID- 8631928 TI - Receptor and membrane interaction sites on Gbeta. A receptor-derived peptide binds to the carboxyl terminus. AB - The functional organization of Gbetagamma is poorly understood. Regions of bovine brain Gbetagamma that interact with a photoaffinity derivative of an alpha2 adrenergic receptor-derived peptide from the third intracellular loop (diazopyruvoyl-modified peptide Q (DAP-Q)) and a hydrophobic membrane probe (3 trifluoromethyl-3-(m-iodophenyl)diazirine (TID)) were examined. We previously showed that DAP-Q cross-links to specific, competable sites on both the alpha and beta subunits of Go/Gi but not on the gamma subunit and that betagamma subunit was required for stimulation of Go/Gi GTPase activity (Taylor, J. M., Jacob Mosier, G. G., Lawton, R. G., Remmers, A. E., and Neubig, R. R. (1994) J. Biol. Chem. 269, 27618-27624). Similarly, we show here that the membrane-associated photoprobe [125I]TID labels alpha and beta but not gamma. We have now mapped the sites of incorporation of DAP-Q and TID into the beta subunit. TID labels both the 14-kDa amino-terminal and the 23-kDa carboxyl-terminal fragments from a partial tryptic digest of beta while DAP-Q labels only the carboxyl-terminal fragment. Further mapping with endopeptidase Lys C reveals substantial labeling of multiple fragments by TID while DAP-Q labels predominantly a approximately 6 kDa fragment within the carboxyl-terminal 60 amino acids of beta1. Thus, regions within the 7th (or possibly 6th) WD-40 repeat of the beta subunit of G protein interact with the receptor-derived peptide while membrane interaction involves multiple sites throughout the beta subunit. PMID- 8631929 TI - Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase. AB - The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme. PMID- 8631930 TI - Copper and calcium binding motifs in the extracellular domains of fibroblast growth factor receptors. AB - High affinity fibroblast growth factor (FGF) receptors contain a cluster of acidic amino acids in their extracellular domains that is reminiscent of the calcium binding domains of some cell adhesion molecules. Based on this observation, we used a calcium blotting technique to show that FGFR-1 binds calcium and that calcium binding is not observed in a mutagenized form of the receptor that lacks the acidic box region. The acidic box also binds other divalent cations, including copper. This latter interaction appears unique since the binding of copper to FGFR-1 mediates the binding of the receptor to immobilized heparin. While this observation may help explain the angiogenic properties of copper, divalent cation binding to FGF receptors may also mediate the interaction between FGF receptors, cell adhesion molecules and other proteoglycan components of the extracellular matrix. PMID- 8631931 TI - Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase. AB - The c-kit receptor tyrosine kinase (KIT) is constitutively activated in three different types of neoplastic mast cell lines by naturally occurring mutations that result in substitutions of Val or Tyr for Asp814 in the phosphotransferase domain. In an effort to characterize the role of the Asp814 residue, we have investigated the properties of mutant KITs in which the Asp814 residue was deleted or mutated to a series of other amino acids. With the exception of rare instances, mutant KITs with substitutions of Asp814 were found to be constitutively phosphorylated on tyrosine and activated in the absence of the ligand, stem cell factor (SCF), whereas a deletion mutant lacking Asp814 (KITDel Asp-814) did not exhibit tyrosine phosphorylation and activation even after treatment with SCF. In addition to constitutive activation, furthermore, both highly activated substitution mutants (KITVal-814 and KITTyr-814) and modestly activated substitution mutants (KITGly-814 and KITHis-814) were continuously degraded in the absence of SCF, whereas wild-type KIT (KITWild) required SCF stimulation to undergo degradation. These results suggested that the Asp814 residue may play a crucial role in regulating enzymatic activity and expression of KIT and that various types of mutations at the Asp814 residue may generate oncogenic protein with constitutive activation and degradation. PMID- 8631932 TI - Seven helix chemoattractant receptors transiently stimulate mitogen-activated protein kinase in Dictyostelium. Role of heterotrimeric G proteins. AB - Mitogen-activated protein (MAP) kinases are involved in controlling a cell's responses to a variety of stimuli and can be activated by both protein tyrosine kinase and G protein-coupled receptors. It was shown previously that Dictyostelium MAP kinase ERK2 is required for normal activation of adenylyl cyclase and erk2 null cells are aggregation-deficient. In this manuscript, we show that the Dictyostelium MAP kinase ERK2 is rapidly and transiently activated in response to the chemoattractant cAMP. This response requires cAMP receptors, but is independent of the coupled G alpha2 subunit and the only known G beta subunit. These data indicate that ligand-mediated receptor activation of adenylyl cyclase requires two receptor-dependent pathways, one of which requires heterotrimeric G proteins, including G alpha2 and the only known G beta subunit, and the second of which requires ERK2. Our results suggest that ERK2 may be activated by a novel receptor-mediated pathway. PMID- 8631933 TI - Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation. AB - Exposure of mammalian cells in culture to the anti-inflammatory drugs sodium salicylate or indomethacin results in activation of heat shock factor 1 (HSF1) DNA binding activity. We have previously shown that the drug-induced HSF1 becomes associated with the heat shock elements of the hsp70 promoter, yet transcription of the hsp70 gene is not induced (Jurivich, D. A., Sistonen, L., Kroes, R. A., and Morimoto, R. I. (1992) Science 255, 1243-1245). In this study, we have examined the basis for uncoupling the heat shock transcriptional response. Comparison of heat shock and drug-induced forms of HSF1 has revealed that the transcriptionally inert drug-induced HSF1 is constitutively but not inducibly serine-phosphorylated, whereas heat shock-induced HSF1 is both constitutively and inducibly serine-phosphorylated. The transcriptionally inert intermediate represented by drug-induced HSF1 can be converted to the transcriptionally active state by a subsequent exposure to heat shock. The only detectable change in HSF1 is the acquisition of inducible serine phosphorylation. These data reveal that acquisition of the trimeric DNA binding state of HSF1 is independent of and precedes inducible phosphorylation and furthermore that inducible phosphorylation correlates with transcriptional activation. PMID- 8631934 TI - B-Myb expression in vascular smooth muscle cells occurs in a cell cycle-dependent fashion and down-regulates promoter activity of type I collagen genes. AB - The members of the Myb family of transcription factors are defined by homology in the DNA-binding domain; all bind the Myb-binding site (MBS) sequence (YG(A/G)C(A/C/G)GTT(G/A)). Here we report that cultured bovine vascular smooth muscle cells (SMCs) express B-myb. Levels of B-myb RNA found in exponential growth were reduced dramatically in serum-deprived quiescent SMCs; B-myb mRNA levels increased in the cell cycle during the late G1 to S phase transition following restimulation with serum, epidermal growth factor, or phorbol ester plus insulin-like growth factor-1. Changes in the rate of B-myb gene transcription could account for part of the observed increase following serum addition. Treatment of SMC cultures with actinomycin D indicated a >4-h half-life for B-myb mRNA during the S phase of the cell cycle. Cotransfection of either a bovine or human B-myb expression vector down-regulated the activity of a multimerized MBS element-driven reporter construct in SMCs. Putative MBS elements were detected upstream of the promoters of the two chains of type I collagen, which we have found to be expressed inversely with growth state of the SMC (Kindy, M. S., Chang, C.-J., and Sonenshein, G. E. (1988) J. Biol. Chem. 263, 11426-11430). In cotransfection experiments, B-myb expression down-regulated the promoter activity of alpha1(I) and alpha2(I) collagen constructs an average of 92 and 82%, respectively. Thus, B-myb represents a potential link in the observed inverse relationship between collagen gene expression and growth of vascular SMCs. PMID- 8631936 TI - Further evidence for the structure of the subtilisin propeptide and for its interactions with mature subtilisin. AB - Evidence is presented for some secondary structure, very likely alpha-helical, of the propeptide of subtilisin E in aqueous salt solution, as well as for strong intermolecular interactions between the propeptide and the mature sequence both in the processed and unprocessed states (i.e. in prosubtilisin). Prosubtilisin is shown to exist as a dimer according to size exclusion high performance liquid chromatography under nondenaturing conditions; that dimer may be on the autoprocessing pathway. According to such a model, the prosequence of one prosubtilisin molecule is the template for the refolding of the mature sequence of the second, and, in turn, the hydrolytic process is intermolecular as well. Support for such an intermolecular folding model also includes potent slow binding inhibition of subtilisin by the propeptide, specific proteolysis of the propeptide by subtilisin, and evidence for intermolecular processing under a variety of conditions. PMID- 8631935 TI - A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors. AB - The maize VP1 protein is a seed-specific regulator of gene expression that effects the expression of a subset of abscisic acid (ABA)-regulated genes that are expressed during the maturation program of the seed. In addition, VP1 has pleiotropic effects on seed development that are not related to ABA. In transient expression assays, VP1 has been shown to transactivate gene expression through at least two distinct promoter elements: the G boxes from the ABA-inducible wheat Em gene and the SphI box from the maize C1 gene. We have investigated how VP1 can transactivate gene expression through diverse promoter elements by analyzing its association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the DNA binding activity of EmBP-1 in a gel retardation assay. This enhancing activity has also been observed on transcription factors as diverse as Opaque-2, Max, Sp1, and NF-kappaB. Deletion of a small but highly conserved region (BR2) in VP1 eliminates the enhancement in vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid fragment from VP1 sandwiched between the maltose-binding protein and LacZ can confer the enhancement function to this fusion protein in vitro. A weak and relatively nonspecific interaction between BR2 and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects. PMID- 8631937 TI - Expression of the transcription factor, Spi-1 (PU.1), in differentiating murine erythroleukemia cells Is regulated post-transcriptionally. Evidence for differential stability of transcription factor mRNAs following inducer exposure. AB - Increased expression of the transcription factor Spi-1 (PU.1) results from retroviral insertion in nearly all Friend spleen focus-forming virus-transformed murine erythroleukemia cell lines and exposure of these cells to Me2SO, induces their differentiation and decreases Spi-1 mRNA level by 4-5-fold. While these results suggest that alterations in Spi-1 expression have significant effects on erythroblast growth and differentiation, neither the cause nor the effect of the decrease in Spi-1 expression that follows Me2SO exposure has been established. The experiments described here demonstrate that the effect of inducers on Spi-1 expression is regulated post-transcriptionally. Nuclear run-off transcriptions demonstrated that Spi-1 transcription was not decreased following Me2SO exposure. Additionally, expression of a recombinant Spi-1 mRNA under transcriptional control of a constitutively active Rous sarcoma virus promoter was regulated identically to endogenous Spi-1 mRNA. The ability of Me2SO to destabilize Spi-1 mRNA was selective, as the stability of the erythroid transcription factors GATA 1 and NF-E2 were not similarly effected. The effect of Me2SO on the stability of Spi-1 mRNA provides a novel means of altering gene expression in these cells and is likely to have significance for the differentiation of these cells. PMID- 8631938 TI - Spermidine release from xenopus oocytes. Electrodiffusion through a membrane channel. AB - The mechanism of spermidine release from Xenopus oocytes was examined by measuring release of radioactive [3H]spermidine under different ionic conditions, and under voltage-clamp. In normal solution (2 mM K+), the efflux rate is less than 1% per hour, and is stimulated approximately 2-fold by inclusion of Ca2+ (1 mm) in the incubation medium. Spermidine efflux is stimulated approximately 10 fold in high [K+] (KD98) solution. In KD98 solution, efflux is strongly inhibited by divalent cations (Ki for Ba2+ block of spermidine efflux is approximately 0.1 mM), but not by tetraethylammonium ions or verapamil. Spermidine efflux rates were not different between control oocytes and those expressing HRK1 inward rectifier K+ (Kir) channels. When the membrane potential was clamped, either by changing external [K+] in oocytes expressing HRK1, or by 2-microelectrode voltage clamp, spermidine efflux was shown to be strongly dependent on voltage, as expected for a simple electrodiffusive process, where spermidine3+ is the effluxing species. This result argues against spermidine diffusing out as an uncharged species, or in exchange for similarly charged counterions. These results are the first conclusive demonstration of a simple electrodiffusive pathway for spermidine efflux from cells. PMID- 8631939 TI - Golgi localization and in vivo activity of a mammalian glycosyltransferase (human beta1,4-galactosyltransferase) in yeast. AB - Gene fusions encoding the membrane anchor region of yeast alpha1, 2 mannosyltransferase (Mnt1p) fused to human beta1, 4-galactosyltransferase (Gal Tf) were constructed and expressed in the yeast Saccharomyces cerevisiae. Fusion proteins containing 82 or only 36 N-terminal residues of Mnt1p were produced and quantitatively N-glycosylated; glycosyl chains were shown to contain alpha1,6-, but not alpha1,3-mannose determinants, a structure typical for an early Golgi compartment. A final Golgi localization of both fusions was confirmed by sucrose gradient fractionations, in which Gal-Tf activity cofractionated with Golgi Mnt1p activity, as well as by immunocytological localization experiments using a monoclonal anti-Gal-Tf antibody. In an in vitro Gal-Tf enzymatic assay the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had comparable Km values for UDP-Gal (about 45 microM). To demonstrate in vivo activity of the Mnt1/Gal-Tf fusion the encoding plasmids were transformed in an alg1 mutant, which at the non-permissive temperature transfers short (GlcNAc)2 glycosyl chains to proteins. Using specific lectins the addition of galactose to several yeast proteins in transformants could be detected. These results demonstrate that Gal-Tf, a mammalian glycosyltransferase, is functional in the molecular environment of the yeast Golgi, indicating conservation between yeast and human cells. The in vivo function of human Gal-Tf indicates that the yeast Golgi is accessible for UDP-Gal and suggests strategies for the construction of yeast strains, in which desired glycoforms of heterologous proteins are produced. PMID- 8631940 TI - Spectral analysis of lactoperoxidase. Evidence for a common heme in mammalian peroxidases. AB - The identity of the non-extractable heme of mammalian lactoperoxidase (LPO) has remained unsolved for over 40 years. Accepted possibilities include a constrained heme b or an 8-thiomethylene-modified heme b. Recent studies of myeloperoxidase (MPO) (Fenna, R., Zeng, J., and Davey, C. (1995) Arch. Biochem. Biophys. 316, 653 656; Taylor, K. L., Strobel, F., Yue, K. T., Ram, P., Pohl, J., Woods, A. S., and Kinkade, J. M., Jr. (1995) Arch. Biochem. Biophys. 316, 635-642) suggest possible prosthetic group similarities between MPO and LPO. To address heme identity for LPO, we used comparative magnetic circular dichroism (MCD) spectroscopy of LPO versus myoglobin (Mb), horseradish peroxidase (HRP), and MPO. MCD spectra of native Fe3+-LPO and Fe3+-CN--LPO are approximately 10 nm red shifted from analogous forms of Mb and HRP, including the formate-Mb adduct. MCD spectra of native LPO and MPO are opposite in sign, and MCD spectra of their cyanoadducts also differ. These data indicate the LPO heme is distinct from heme b of Mb and HRP as well as from "heme m" of MPO. From this work and literature analysis, we suggest that the non-extractable "heme l" of LPO has the two vinyl groups of heme b but lacks the 2-sulfonium-vinyl linkage of heme m. The observed red shifts in LPO spectra may derive from ester linkages to protein as for MPO. Strong spectral analogies between LPO and mammalian peroxidases (e.g. from saliva, eosinophils, thyroid, intestine) indicate similar prosthetic heme moieties. PMID- 8631941 TI - Crystallographic evidence that the F2 kringle catalytic domain linker of prothrombin does not cover the fibrinogen recognition exosite. AB - The 2.6-A x-ray crystal structure of bovine alpha-thrombin in complex with rhodniin, a protein inhibitor isolated from the bug Rhodnius prolixus, has been solved and refined. The structure has enabled us to trace the N-terminal part of the 49-residue A-chain of bovine alpha-thrombin for the first time, which is fixed in a U-shaped loop on the molecular surface opposite the active site canyon. Model building shows that the 25 amino acid residues that link the A chain and F2 kringle cannot run through the fibrinogen recognition exosite. This demonstrates that this fibrinogen recognition exosite is available in prothrombin and meizothrombin. PMID- 8631942 TI - The catalytic cysteine and histidine in the plant acyl-acyl carrier protein thioesterases. AB - The plant acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of their utilities in the genetic engineering of plant seed oils. Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis, suggesting that these enzymes activate the hydrolysis of the thioester bond using the same basic catalytic machinery as those of proteases and lipases. To identify the cysteine and histidine residues that are critical in catalysis we substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine (Cys-320) to an Ala or a Ser, and three conserved histidines (His-140, His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a substantial loss of enzyme activity. However, only C320A and H285A completely inactivated the enzyme, indicating that these two residues are essential for catalysis. Considerable activity (>60%) still remained when Cys 320 was converted to a Ser, but this mutant (C320S) displayed a reversed sensitivity toward thiol or serine hydroxyl inhibitors compared with the wild type enzyme. A pH optimal study demonstrates that while the wild-type enzyme has the highest activity between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to higher pH and a significant increase of activity around pH 12. This result suggests that Arg-285 (pKa 12) is deprotonated at high pH, thus partially mimicking the role of His-285 for proton abstraction in the wild-type enzyme. We conclude that the Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can attack the thioester bond of the substrate 12:0 ACP, assisted by His 285. Because plant TEs are highly conserved in length and sequence and the residues investigated here are completely conserved in all available TEs, it is reasonable to believe that homologues of Cys-320 and His-285 are present in the active sites of all plant acyl-ACP TEs. PMID- 8631943 TI - trans-Activation by the hnRNP K protein involves an increase in RNA synthesis from the reporter genes. AB - The function of many of the pre-mRNA-binding proteins in mRNA biogenesis is unclear. We have analyzed the biochemical function of the hnRNP K protein by using a mouse cDNA clone. A previous study indicated that the expression of hnRNP K activates c-myc promoter in transient transfection assays. We show that the expression of hnRNP K results in a trans-activation of a variety of RNA polymerase II promoters. The trans-activation function depends on the sequences of hnRNP K that are also necessary for RNA binding. However, the RNA binding motifs are not sufficient for trans-activation. We could identify a mutant that bound RNA in vitro but was impaired in its ability to trans-activate the reporter genes. The trans-activation was not a result of the stabilization of the reporter mRNA, because hnRNP K increased the steady-state level of the reporter mRNA without altering its decay rate. By doing nuclear run-on assays, we provide evidence that the hnRNP K protein trans-activates the reporter genes by increasing the level of transcription. PMID- 8631944 TI - Expression of novel secreted isoforms of human immunoglobulin E proteins. AB - Four human IgE isoforms produced by alternative splicing of the epsilon primary transcript were expressed as chimeric mouse/human anti 5-dimethylamino-1 naphthalenesulfonyl antibodies in the murine myeloma cell line Sp2/0. The four isoforms include the classic secreted form and three novel isoforms with altered carboxyl termini. All of these isoforms lack the transmembrane region encoded by the M1/M1' exon and are therefore predicted to be secreted proteins. When expressed in Sp2/0 cells, three of the IgE isoforms are assembled into complete molecules of two Ig heavy chains and two Ig light chains, whereas the fourth isoform is predominately assembled into half-molecules of one Ig heavy chain and one Ig light chain. All four isoforms are secreted with similar kinetics. In contrast, when the isoform containing the C epsilon4 domain joined directly to the M2 exon (IgE grande) is expressed in the J558L cell line, it is degraded intracellularly, suggesting a cell line-dependent regulation of secretion. These data show that these novel isoforms of human IgE, predicted to occur from in vivo and in vitro mRNA analysis, can be produced and secreted by mammalian cells. The different forms of IgE may have physiologically relevant but distinct roles in human IgE-mediated immune inflammation. The availability of purified recombinant human IgE isoforms makes it possible to analyze the functional differences among them. PMID- 8631945 TI - Altered structural and mechanistic properties of mutant dihydropteridine reductases. AB - Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position. PMID- 8631946 TI - Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile. AB - Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles. Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme. Recombinant thiaminase I functions as a monomer with a Km for thiamin of 3.7 +/- 0.6 microM and a kcat of 34 s-1. Electrospray ionization Fourier transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42,127, 42,198, and 42,255 due to added Ala and Gly-Ala at the amino terminus. Similar analysis of the 4-amino-2 methyl-6-chloropyrimidine inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro79 and Thr177. Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys113 as the active site nucleophile. PMID- 8631948 TI - Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart. AB - A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 degrees C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R, 15S) epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology. PMID- 8631947 TI - Disrupted signaling in a mutant J2E cell line that shows enhanced viability, but does not proliferate or differentiate, with erythropoietin. AB - The immature erythroid J2E cell line proliferates and terminally differentiates following erythropoietin stimulation. In contrast, the mutant J2E-NR clone does not respond to erythropoietin by either proliferating or differentiating. Here we show that erythropoietin can act as a viability factor for both the J2E and J2E NR lines, indicating that erythropoietin-initiated maturation is separable from the prevention of cell death. The inability of J2E-NR cells to mature in response to erythropoietin was not due to a defect in the erythropoietin receptor sequence, although surface receptor numbers were reduced. Both the receptor and Janus kinase 2 were phosphorylated after erythropoietin stimulation of J2E-NR cells. However, protein interactions with the erythropoietin receptor and Grb2 were restricted in the mutant cells. Subsequent investigation of several other signaling molecules exposed numerous alterations in J2E-NR cells; phosphorylation changes to phosphatidylinositol 3-kinase, phospholipase Cgamma, p120 GAP, and mitogen-activated protein kinases (p42 and p44) observed in erythropoietin stimulated J2E cells were not seen in the J2E-NR line. These data indicate that some pathways activated during erythropoietin-induced differentiation may not be essential for the prevention of apoptosis. PMID- 8631949 TI - An intronic enhancer essential for tissue-specific expression of the aldolase B transgenes. AB - Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost. PMID- 8631950 TI - Binding and hydrolysis of TNP-ATP by Escherichia coli F1-ATPase. AB - It had previously been suggested that Vmax hydrolysis rate of 2', 3'-O-(2,4,6 trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) by F1-ATPase required filling of only two catalytic sites on the enzyme (Grubmeyer, C., and Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727), whereas recently it was shown that Vmax rate of ATP hydrolysis requires that all three catalytic sites are filled (Weber, J., Wilke-Mounts, S., Lee, R. S. F., Grell, E., and Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133). To resolve this apparent discrepancy, we measured equilibrium binding and hydrolysis of MgTNP-ATP under identical conditions, using betaY331W mutant Escherichia coli F1-ATPase, in which the genetically engineered tryptophan provides a direct fluorescent probe of catalytic site occupancy. We found that MgTNP-ATP hydrolysis at Vmax rate did require filling of all three catalytic sites, but in contrast to the situation with MgATP, "bisite hydrolysis" of MgTNP-ATP amounted to a substantial fraction (approximately 40%) of Vmax. Binding of MgTNP-ATP to the three catalytic sites showed strong binding cooperativity (Kd1 < 1 nm, Kd2 = 23 nm, Kd3 = 1.4 microM). Free TNP-ATP (i.e. in presence of EDTA) bound to all three catalytic sites with lower affinity but was not hydrolyzed. These data emphasize that the presence of Mg2+ is critical for cooperativity of substrate binding, formation of the very high affinity first catalytic site, and hydrolytic activity in F1-ATPases and that these three properties are strongly correlated. PMID- 8631951 TI - Artificial chaperone-assisted refolding of carbonic anhydrase B. AB - We recently reported a new approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and a cyclodextrin (Rozema, D., and Gellman, S. H. (1995) J. Am. Chem. Soc. 117, 2373-2374). Here, we provide a detailed study of carbonic anhydrase B (CAB) refolding assisted by these "artificial chaperones." When CAB is heated in the presence of a competent detergent, or when guanidinium-denatured CAB is diluted to nondenaturing guanidinium concentration in the presence of such a detergent, the detergent forms a complex with the non-native protein, thereby preventing aggregation. CAB is unable to refold from the detergent-complexed state, but folding can be induced by introduction of a cyclodextrin, which strips the detergent away from the protein. Use of artificial chaperones provides excellent yields of reactivated CAB under conditions that lead to little or no reactivation in the absence of the refolding assistants. Our studies show that the detergent can capture the unfolded protein even at submicellar concentrations, but that not all CAB-detergent complexes lead efficiently to refolded enzyme upon introduction of the stripping agent. Effective refolding appears to require that detergent stripping occur as rapidly as possible; intrinsically slow methods of detergent removal (dialysis or use of macroscopic adsorbents) are less effective than cyclodextrin at inducing renaturation upon detergent removal. The detailed characterization of artificial chaperone-assisted CAB refolding reported here should guide the application of this strategy to other proteins. PMID- 8631952 TI - Dyrk, a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII. AB - The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions. PMID- 8631953 TI - Investigation of the calcium-mediated association between the carbohydrate head groups of galactosylceramide and galactosylceramide I3 sulfate by electrospray ionization mass spectrometry. AB - Calcium has been shown previously to cause aggregation of phosphatidylcholine/cholesterol liposomes containing galactosylceramide (GalCer) with similar liposomes containing cerebroside sulfate (galactosylceramide I3 sulfate) (CBS), suggesting that it mediates a carbohydrate-carbohydrate association between these two glycolipids. In order to determine if such an association occurs, the noncovalent complexes formed on addition of calcium chloride to GalCer and CBS in methanol were examined by positive and negative ion spray mass spectrometry. Monomeric Ca2+ complexes of both lipids were observed. In addition, Ca2+ also caused oligomerization of GalCer. Oligomerization of CBS anion was not seen, but dimers would not have been observed, as they would be neutral. However, Ca2+ caused heterotypic complexation of GalCer and CBS. Although these heterotypic complexes were of low abundance in methanol compared with the other monomeric and homotypic oligomeric positive ions formed at low declustering potentials, the heterotypic dimer [GalCer.CBS.Ca2+-H]+ had the greatest stability of all oligomers formed and was the only one to survive at high declustering potentials. Na+ did not cause oligomerization of GalCer in methanol indicating that the complexes of GalCer with Ca2+ are not caused by van der Waals interactions between the lipid moieties. GalCer and CBS are present in high concentrations in myelin. This Ca2+-mediated carbohydrate-carbohydrate interaction, which can bridge apposing bilayers, may be involved in adhesion of the extracellular surfaces of the myelin sheath. PMID- 8631954 TI - Dietary calorie restriction in mice induces carbamyl phosphate synthetase I gene transcription tissue specifically. AB - Dietary calorie restriction (CR) delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. Here, we present the novel finding that chronic reduction of dietary calories by 50% without changing the intake of dietary protein induced the activity of mouse hepatic carbamyl phosphate synthetase I (CpsI) 5-fold. In liver, CpsI protein, mRNA, and gene transcription were each stimulated by approximately 3-fold. Thus, CR increased both the rate of gene transcription and the specific activity of the enzyme. Short-term feeding studies demonstrated that higher cpsI expression was due to CR and not consumption of more dietary protein. Intestinal CpsI activity was stimulated 2-fold, while its mRNA level did not change, suggesting enzyme activity or translation efficiency was stimulated. CpsI catalyzes the conversion of metabolic ammonia to carbamyl phosphate, the rate-limiting step in urea biosynthesis. cpsI induction suggests there is a shift in the metabolism of calorie-restricted animals toward protein catabolism. CpsI induction likely facilitates metabolic detoxification of ammonia, a strong neurotoxin. Enhanced protein turnover and metabolic detoxification may extend life span. Physiologic similarities between calorie-restricted and hibernating animals suggest the effects of CR may be part of a spectrum of adaptive responses that include hibernation. PMID- 8631955 TI - Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders. AB - Urine extracts from children diagnosed with generalized peroxisomal disorders were screened by continuous flow-negative ion fast atom bombardment-mass spectrometry. In 45 of 60 children with generalized peroxisomal disorders, we observed one or more intense ions (m/z 489, 505, 461, and others) that are infrequently found in children with cholestatic liver disease or normal children. Compounds giving rise to these ions were isolated using reverse phase and anion exchange chromatography. After appropriate derivatization and/or methanolysis the compounds were analyzed using capillary gas chromatography-mass spectrometry. The major compounds were found to be 12,13-dihydroxy-9-octadecenoic acid and 9,10 dihydroxy-12-octadecenoic acid, with one of the hydroxyl groups in glycosidic linkage with glucuronic acid. Minor compounds were glucuronic acid conjugates of 9,10-dihydroxy-octadecanoic acid, and 12,13-dihydroxy-6,9-, 15,16-dihydroxy-9,12 , and 9, 10-dihydroxy-12,15-octadecadienoic acids. A series of hexadecanoic, hexadecenoic, and hexadecadienoic acid glucuronides which appear to be beta oxidation products of the C18 fatty acids were also observed, with the major species being 10, 11-dihydroxy-7-hexadecenoic acid glucuronide. In all, 16 C16 and C18 dihydroxy fatty acids were identified by gas chromatography-mass spectrometry. A series of at least 11 trihydroxy fatty acids was also observed but not fully characterized. Measurement of these compounds may prove to be useful in the diagnosis of some peroxisomal disorders. PMID- 8631956 TI - Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product. AB - The Caenorhabditis elegans cell death gene, ced-3, encodes one of the two proteins required for apoptosis in this organism. The primary sequence similarities between Ced-3 and the mammalian interleukin-1beta converting enzyme (ICE) suggest that these two proteins may have functionally similar active sites and that Ced-3 may function as a cysteine protease. Here we report that in vitro transcribed and translated Ced-3 protein (p56) underwent rapid processing to smaller fragments. Replacement of the predicted active site cysteine of Ced-3 with serine (C364S) prevented the generation of smaller proteolytic fragments, suggesting that the processing might be an autocatalytic process. Peptide aldehydes with aspartic acid at the P1 position blocked Ced-3 autocatalysis. Furthermore, the protease inhibition profile of Ced-3 was similar to the profile reported for ICE. These functional data demonstrate that Ced-3 is an Asp dependent cysteine protease with substrate specificity similar to that of ICE. Aurintricarboxylic acid, an inhibitor of apoptosis in mammalian cells, blocked Ced-3 autocatalytic activity, suggesting that an aurintricarboxylic acid sensitive Ced-3/ICE-related protease might be involved in the apoptosis pathway(s) in mammalian cells. PMID- 8631957 TI - Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study. AB - Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled 35S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation). PMID- 8631958 TI - Human cytomegalovirus immediate-early protein IE2 tethers a transcriptional repression domain to p53. AB - The IE2 gene of human cytomegalovirus has been implicated in the development of coronary restenosis, and the gene product appears to inhibit p53-dependent transactivation. Here we describe an analysis of the IE2-p53 interaction. Repression of p53 function by IE2 requires two separable domains of IE2. The N terminus of IE2 interacts with p53. IE2 has little effect on the ability of p53 to bind specific DNA sequences. Reduction of the transactivation activity of p53 is caused by a transcriptional repression function contributed by the C-terminal domain of IE2. These findings suggest that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53. PMID- 8631959 TI - Different fate of a single reporter protein containing KDEL or KKXX targeting signals stably expressed in mammalian cells. AB - In mammalian cells, resident luminal and type I transmembrane proteins of the endoplasmic reticulum usually contain KDEL and KKXX at the carboxyl terminus. These sequences induce retrieval from compartments located downstream in the secretory pathway. It has been suggested that the retrieval may occur from multiple sites, ranging from the intermediate compartment to the trans-Golgi network. To compare the retrieval of luminal and type I membrane proteins, we have used different forms of a single reporter, the human CD8 glycoprotein, stably expressed in FRT cells. Metabolic labeling and oligosaccharide analysis show that the mechanism based on the KDEL signal is leaky. With time, the KDEL containing CD8 form reaches the trans/trans-Golgi network compartments, where the protein is terminally glycosylated. At this stage, the retrieval mechanism stops being effective and the protein is consequently secreted. Conversely, the mechanism based on the KKXX signal guarantees that most of the KKXX-containing CD8 form resides in the endoplasmic reticulum, little in the Golgi complex and undetectable levels at the plasma membrane. The O-glycosylation of this protein comprises for the vast majority the sole addition of peptide-bound GalNAc that occurs in an early Golgi compartment. PMID- 8631960 TI - The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. AB - We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin. The KI values associated with the formation of the first reversible complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53 +/- 0. 06 and 0.61 +/- 0.08 microM, respectively; the rate constants for the forward isomerization, k2, into the second reversible complex (EI*) were 0.97 +/- 0.99 and 0.11 +/- 0.01 s-1, respectively, and rates of the reverse isomerization from EI*, k-2, were 0.031 +/- 0.004 and 0.0082 +/- 0.0008 s 1, respectively. Indomethacin was estimated to form the EI complex with the same affinity for both PGHS1 and PGHS2, 10.0 +/- 2.8 microM and 11.2 +/- 2.0 microM, respectively, and dissociate from EI* at approximately the same rate 0.0011 +/- 0.0002 s-1 and 0.0031 +/- 0.0003 s-1, respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 +/- 0.08 s-1 as compared with 0.034 +/- 0.004 s-1. These results show that the overall affinity for the inhibition of PGHS1 versus PGHS2 was 30-fold greater for indomethacin (KI* = 0.032 +/- 0.005 and 1.02 +/- 0.08 microM, respectively) and 3 fold greater for flurbiprofen (KI* = 0.017 +/- 0.002 and 0.045 +/- 0.004 microM, respectively). The results also show that for both PGHS1 and PGHS2, flurbiprofen was bound tighter to the initial EI complex than indomethacin; however, the rate of dissociation from EI* was slower for indomethacin than flurbiprofen. The rate of the forward isomerization to EI* is primarily responsible for the selectivity of both NSAIDs for PGHS1. This analysis shows the quantitative importance of the different kinetic parameters upon the overall binding affinity of these NSAIDs and should greatly assist in our understanding of the structural interactions that promote enzyme-inhibitor binding. PMID- 8631962 TI - Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91, and STAT3 p92 in polymorphonuclear leukocytes. AB - Granulocyte-macrophage colony-stimulating factor (GM-CSF), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although GM-CSF receptor is devoid of tyrosine kinase enzymatic activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF in PMN. In the present study we demonstrate that also the signal transducers and activators of transcription (STAT) family members STAT1 p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF stimulation and physically associated with both GM-CSF receptor beta common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and p93fes catalytic activity. We also demonstrate that the association of the GM-CSF receptor beta common subunit with JAK2 is ligand-dependent. Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN. PMID- 8631961 TI - A splice variant of the ITF-2 transcript encodes a transcription factor that inhibits MyoD activity. AB - Proteins of the basic helix-loop-helix (bHLH) family are transcription factors that bind DNA containing the E box motif (CANNTG) found in the promoters of many muscle-specific genes. ITF-2 is a bHLH protein with widespread expression that is thought to form active heterodimers with MyoD, a muscle-specific bHLH transcription factor. We have isolated cDNAs derived from two alternatively spliced forms of mouse ITF-2, termed MITF-2A and -2B. These proteins differ in their N termini. Neither MITF-2A nor -2B transactivated the cardiac alpha-actin promoter, which contains an E box, when transfected into nonmuscle cells. In fact, MITF-2B inhibited MyoD activation of the cardiac alpha-actin promoter. This inhibitory activity required the N-terminal 83 amino acids since MITF-2A showed no inhibitory activity, and a mutant MITF-2B with deletion of the N-terminal 83 amino acids failed to inhibit MyoD-mediated transcriptional activation. MyoD activity was also inhibited by Id, a HLH protein, and this inhibition was reversed by the addition of excess E12 or MITF-2A. However, the inhibition of MyoD activity by MITF-2B was not reversed with E12 or MITF-2A. While Id is thought to inhibit MyoD by binding and sequestering potential dimerization partners, MITF-2B appears to inhibit MyoD activity by forming an inactive heterodimer with MyoD. Thus, differentially spliced transcripts of mouse ITF-2 encode different proteins that appear to dimerize with MyoD and activate or repress transcription. PMID- 8631963 TI - Inositol 1,4,5-trisphosphate slowly converts its receptor to a state of higher affinity in sheep cerebellum membranes. AB - Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale. PMID- 8631964 TI - Differential storage of prolactin, granins (Chromogranin B and secretogranin II), and constitutive secretory markers in rat pituitary GH4C1 cells. AB - The rat pituitary cell line GH4C1 secretes granins (chromogranin B and secretogranin II) and prolactin by the regulated secretory pathway. The intracellular storage of prolactin is preferentially induced by hormone treatment with estradiol, insulin, and epidermal growth factor. The goal of this study was to determine the effect of hormone treatment on storage of granins and constitutive secretory markers. The granins were efficiently stored in both hormone-treated and -untreated cells (17% of total secreted in 4 h). Secreted alkaline phosphatase (SEAP), a truncated membrane protein that would not be expected to enter secretory granules, and glycosaminoglycan, a marker for the constitutive secretory pathway, exhibited 70 80% secretion under both conditions. In comparison, the relative prolactin secretion was 31 and 68% from hormone treated and -untreated cells, respectively. Phorbol ester and KCl stimulated prolactin secretion 2.3-fold from untreated cells and 5. 5-fold from hormone treated cells. In contrast, SEAP secretion was stimulated 1.5-fold from both treated and untreated cells, consistent with secretion by the constitutive secretory pathway. Stimulated secretion of granins was detected from both hormone treated and -untreated cells. These results suggest that granin and prolactin storage are differentially regulated and that the constitutive secretory pathway is not affected by hormone treatment. PMID- 8631965 TI - Regulatory elements that control transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces cerevisiae OLE1 gene. AB - In Saccharomyces cerevisiae, unsaturated fatty acids are formed from saturated acyl-CoA precursors by Ole1p, a delta-9 fatty acid desaturase. OLE1 mRNA levels are differentially regulated by the addition of saturated or unsaturated fatty acids to the growth medium. One component of this regulation system involves the control of OLE1 transcription. Saturated fatty acids induce a 1.6-fold increase in transcription activity, whereas a large family of unsaturated fatty acids repress OLE1 transcription as much as 60-fold. A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty acid regulated (FAR) region approximately 580 bp upstream of the start codon that is essential for transcription activation and unsaturated fatty acid repression. Deletion of an 88-bp sequence within that region resulted in a complete loss in transcription activation and unsaturated fatty acid regulation. The 111-bp FAR element strongly activates transcription and confers unsaturated fatty acid regulation on a heterologous CYC1 promoter test plasmid. Essential elements required for unsaturated fatty acid repression of OLE1 were found in the 5 and 3 region of the 111-bp sequence. The FAR element-mediated activation and fatty acid repression of transcription was found to be closely tied to fatty acyl-CoA metabolism. Two fatty acid activation genes, FAA1 and FAA4, were found to be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences. Disruption of either gene results in reduced levels of unsaturated fatty acid repression; disruption of both genes completely blocks the regulatory response. Acyl-CoA binding protein (ACBP) plays a role in determining the level of FAR element activated transcription. Disruption of the ACBP gene causes a >5 fold activation of OLE1 transcription and a similar increase in OLE1 mRNA levels. Unsaturated fatty acid repression of OLE1 transcription, however, is not affected by the disrupted ACBP gene. These studies show that promoter elements responsible for unsaturated fatty acid-mediated transcription repression are tightly linked to OLE1 activation sequences and that OLE1 transcription levels are closely tied to acyl-CoA metabolism. PMID- 8631966 TI - Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform. AB - Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP. PMID- 8631967 TI - Integral repeats and a continuous coiled coil are required for binding of striated muscle tropomyosin to the regulated actin filament. AB - Tropomyosin is a coiled-coil protein that binds along the length of filamentous actin and contains sequence repeats that correspond to actin monomers in the filament. Analysis of striated muscle alpha-tropomyosin mutants in which internal sequence has been deleted or replaced with non-tropomyosin sequence showed that the following parameters are important for high affinity, cooperative binding of tropomyosin-troponin to actin. 1) Tropomyosin must be a coiled coil along its entire length. 2) An integral number of repeats corresponding to the actin monomers along its length is more important than the total number. 3) In comparison, the actin affinity is relatively insensitive to changes in the sequence of the internal regions of tropomyosin. The results suggest that the internal sequence repeats function as weakly interacting spacers to allow proper alignment of the ends on the regulated actin filament. PMID- 8631968 TI - Age-related decline in mitogen-activated protein kinase activity in epidermal growth factor-stimulated rat hepatocytes. AB - A number of studies have demonstrated that the proliferative capacity of cells declines with aging. In particular, epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats. Growth factor stimulation activates a genetic program in large part regulated by a family of mitogen-activated protein kinases (MAPK) that phosphorylate and thereby activate transcription factors involved in controlling the expression of proliferation-associated genes. In the present study, we compared the activation of the extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) MAPK in EGF-stimulated hepatocytes derived from young (6-month) and aged (24-month) rats. JNK activity was not appreciably altered by EGF treatment of cells from either age group. In contrast, ERK2 was highly activated by EGF treatment, but the magnitude of activation was significantly lower in hepatocytes of aged animals compared to those of young animals (7-fold versus 20-fold, respectively). The reduced ERK2 activity in response to EGF was associated with decreased c-fos and c-jun mRNA expression and lower levels of AP-1 transcription factor DNA binding activity in the aged hepatocytes. Finally, the basal expression of MAPK phosphatase 1, a MAPK-regulated gene involved in regulating MAPK activity, was higher in aged hepatocytes. Taken together, these findings suggest that an alteration in the balance between MAP kinase-phosphatase activities could contribute to the age-related decline in proliferative capacity. PMID- 8631969 TI - Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase. AB - The lpcA locus has been identified in Escherichia coli K12 novobiocin supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS. PMID- 8631970 TI - Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. AB - Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for epithelial cells in vitro. It appears likely that HGF participates in tissue regeneration following hepatic and renal injury in vivo. The activity of HGF is localized to the injured tissues by a proteolytic activation system; HGF remains as an inactive single-chain form in the normal state and is converted to an active heterodimeric form in response to tissue injury. A protease responsible for this conversion is induced in the injured liver, but it has not yet been identified. We have previously purified and characterized HGF activator (HGFA), a serum-derived serine protease that efficiently activates single-chain HGF in vitro. In this study, we found that the HGF-converting activity in the injured liver was inhibited by an anti-HGFA antibody. We also found that the active form of HGFA was generated exclusively in the injured tissues. Thus, it appears likely that HGFA is the key enzyme that regulates the activity of HGF in the injured tissues. We also analyzed the heparin binding properties of the precursor and mature forms of HGFA. HGFA had a weak affinity for heparin near the physiological salt concentration in its precursor form but acquired a strong affinity for heparin upon activation that is linked to blood coagulation. This property may ensure the local action of this enzyme at the site of tissue injury. PMID- 8631971 TI - Co-evolution of ligand-receptor pairs in the vasopressin/oxytocin superfamily of bioactive peptides. AB - In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides. PMID- 8631972 TI - Cooperative oxygen binding to scapharca inaequivalvis hemoglobin in the crystal. AB - Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient nH = 1.45 and an oxygen pressure at half saturation p50 = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species. PMID- 8631973 TI - Influence of cluster formation of acidic phospholipids on decrease in the affinity for ATP of DnaA protein. AB - DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We examined the influence of artificial mixed membrane composed of synthetic acidic (phosphate) lipid and basic (ammonium) lipid on the affinity of DnaA protein for ATP. Two sets of acidic and basic lipids with distinguishable numbers of hydrophobic alkyl chains were devised. Synthetic membranes made of the sole acidic lipid but not the basic bilayers inhibited the ATP binding to DnaA protein and stimulated the release of ATP from the ATP-DnaA complex. The basic bilayer forming compounds served as the matrix for the guest acidic lipids. Acidic lipids dispersed in the basic matrix membrane had little effect on ATP binding and on ATP release. Conversely, acidic lipids forming cluster structures in the mixed artificial membranes inhibited the ATP binding and stimulated the release of ATP. These observations suggest that in mixed lipid bilayers, a cluster structure of acidic lipids seems to be an important parameter to decrease the affinity of DnaA protein for ATP. PMID- 8631974 TI - Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. AB - A murine model of ornithine transcarbamylase (OTC) deficiency was used in this study to evaluate the efficacy of recombinant adenoviruses for correcting the metabolic defect in liver. Recombinant adenoviruses deleted in E1 and containing a human OTC cDNA expressed little functional OTC enzyme in vivo and had no observable impact on the underlying metabolic abnormalities of the OTC-deficient mouse (i.e. elevated urinary orotate and serum glutamine). E1-deleted vectors were improved through the use of the strong constitutive promoter from cytomegalovirus driving the normal murine homolog of OTC cDNA and the ablation of E2a with a temperature-sensitive mutation. Infusion of this improved vector into the mouse model was associated with a complete normalization of liver OTC enzyme activity that persisted for at least 2 months with complete but transient correction in serum glutamine and urine orotic acid. These studies illustrate the utility of improved adenoviral vectors in the treatment of liver metabolic disease. PMID- 8631975 TI - Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets. AB - Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response. PMID- 8631976 TI - A binding site for thrombin in the apple 1 domain of factor XI. AB - Previously we defined a binding site for high molecular weight kininogen (HK) in the A1 domain of factor XI (FXI). Since thrombin can activate FXI and HK inhibits the activation of FXI by thrombin, we have identified a thrombin binding site in FXI. Both the recombinant A1 domain (Glu1-Ser90) and a synthetic peptide (Phe56 Ser86) containing the HK binding site inhibited FXI activation by thrombin. Both a monoclonal antibody, 5F7, recognizing the A1 domain, and the rA1 domain were shown to be competitive inhibitors of thrombin-catalyzed FXI activation. The peptides Ala45-Arg54 and Val59-Arg70 acted synergistically to inhibit FXI activation by thrombin. Mutant rA1 domain constructs (Val64 --> Ala and Ile77 --> Ala), which do not inhibit FXI binding to HK, retain full capacity to inhibit FXI activation by thrombin. The peptide Ala45-Arg54 inhibited thrombin-catalyzed FXI activation, whereas it had no effect on FXI binding to HK. In contrast, the peptide Asn72-Leu83 (which inhibited FXI binding to HK) did not inhibit FXI activation by thrombin. Thus, a thrombin binding site exists in the A1 domain of FXI spanning residues Ala45-Arg70 that is contiguous with but separate and distinct from the HK binding site. These sites may regulate which ligand is bound to FXI and through which pathway FXI is activated. PMID- 8631977 TI - The IgG binding site of human FcgammaRIIIB receptor involves CC' and FG loops of the membrane-proximal domain. AB - Fc gamma receptors for the Fc part of IgG are the mediators for antibody effector functions. FcgammaRIII and FcgammaRII are low affinity receptors that, through the interaction with immune complexes, initiate a variety of immunological responses, such as phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators. We set out to define the IgG binding site on human FcgammaRIII. We assumed that potential beta-turns in Ig-like domains are the most probable determinants for ligand binding, and chimeric FcgammaRIIIB/FcepsilonRI receptors as well as single residue mutants were constructed in these regions of FcgammaRIIIB. Substitution of four amino acids in the membrane-proximal domain (Gln126, Arg156, Lys162, Val164) resulted in decreased binding of human IgG1. Lys162 and Val164 were found also to be crucial for the interaction with the IgG-binding inhibitory monoclonal antibody 3G8. In a putative three-dimensional model constructed in this study, these residues map on the CC loop (Gln126), on F beta-sheet (Arg156), and on the FG loop (Lys162, Val164). Our data are consistent with the study about human FcgammaRII (Hulett, M. D., Witort, E., Brinkworth, R. I., McKenzie, I. F. C., and Hogarth, P. M. (1994) J. Biol. Chem. 269, 15287-15293), suggesting that common structural determinants, i.e. FG loop or the GFC surface of the membrane-proximal domain, can be involved in interactions with IgG by both low affinity receptor classes FcgammaRII and FcgammaRIII. PMID- 8631978 TI - Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. AB - Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions. PMID- 8631980 TI - Structural states of the nucleosome. AB - The nucleosome is the fundamental component of the eukaryotic chromosome, participating in the packaging of DNA and in the regulation of gene expression. Its numerous interactions imply a structural dynamism. Previous biophysical studies under limited sets of conditions have not been able to reconcile structural differences and transitions observed. We have determined a series of nucleosome conformations over a >10,000-fold range in salt concentration using a combination of biochemical methods, spectroscopic electron microscopy, and three dimensional reconstruction techniques for randomly oriented single particles. This study indicates several ionic strength-dependent nucleosome conformations and also reconciles the differences between currently existing divergent models for the nucleosome. At low ionic environments, the particle appears highly elongated, becoming more compact and prolate ellipsoidal as ionic strength is increased to 10 mm NaCl. At 30 mM NaCl, the particle exhibits a spheroidal conformation. As ionic strength is increased to 150 mM NaCl, the nucleosome conformation changes and becomes oblate. Above 450 mM NaCl, the structure becomes highly elongated again. The result of this study is a unifying concept in which the three-dimensional structure of the nucleosome is inferred to be dynamic in response to ionic interactions and in accord with biochemical and genetic studies. PMID- 8631979 TI - Diphtheria toxin-mediated ablation of parietal cells in the stomach of transgenic mice. AB - The self-renewing epithelial populations present in the gastric units of the mouse stomach are descended from a multipotent stem cell and undergo an orderly migration-associated differentiation followed by apoptosis. The steady state census of the three principal cell types (acid-producing parietal cells, mucus producing pit cells, and pepsinogen and intrinsic factor-producing zymogenic cells) is accurately controlled, despite marked differences in the rates of migration of each lineage. A transgenic mouse model has been created to define functional interrelationships between the proliferation, differentiation, and death programs of these lineages. Nucleotides -1035 to +24 of the noncatalytic beta subunit gene of mouse H+/K+-ATPase were used to direct expression of an attenuated diphtheria toxin A subunit in the parietal cell lineage. These transcriptional regulatory elements are not active in members of the pit and zymogenic lineages. Stomachs, prepared from postnatal day 28-80 transgenic mice and their normal littermates, were subjected to single- and multilabel immunohistochemical studies as well as qualitative and quantitative light and electron microscopic morphologic analyses. The toxin produced complete ablation of differentiated parietal cells. Loss of parietal cells was accompanied by a 5 fold increase in the number of undifferentiated granule-free cells located in the proliferative compartment of gastric units. This amplified population of granule free cells included the multipotent stem cell as well as committed precursors of the pit and zymogenic lineages. Loss of mature parietal cells was also associated with (i) a block in the differentiation program of the zymogenic lineage with an accumulation of pre-neck cells and a depletion of their neck and mature zymogenic cell descendants, and (ii) an approximately 2-fold amplification of pit cells. These findings are consistent with the notion that epithelial homeostasis within gastric units is maintained by instructive interactions between their different cell lineages. Unlike pit and zymogenic cells, parietal cells complete their differentiation in the gastric unit's proliferative compartment before undergoing a bipolar migration along the unit. Thus, the mature parietal cell is in a strategic position to influence decision-making among gastric epithelial cell precursors and to modulate the migration-associated terminal differentiation programs of the pit and zymogenic lineages. PMID- 8631981 TI - Expression cloning of a human GT3 synthase. GD3 AND GT3 are synthesized by a single enzyme. AB - Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second alpha-2,8 sialic acid to GD3, NeuNAcalpha2-->8NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, thus forming GT3, NeuNAcalpha2-->8NeuNAcalpha2-->8NeuNAc alpha2-->3Galbeta1--> 4Glc- >Cer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes GD3 synthase. The newly identified enzyme was therefore named GD3/GT3 synthase (GD3/GT3ST). GD3/GT3ST synthesized GT3 most efficiently when GM3, NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, was incubated as an acceptor, indicating that GD3/GT3ST is a polysialyltransferase that can transfer more than one sialic acid residue via alpha-2,8 linkage to gangliosides. Moreover, a longer period of incubation of GD3 with GD3/GT3ST produced a significant amount of GT3 and higher polysialogangliosides. Among various cell lines expressing GD3/GT3ST, higher polysialogangliosides including GT3 were detected only in cell lines where the amount of GD3/GT3 mRNA is sufficiently high. The expression of GD3/GT3ST mRNA among human tissues is highly restricted to fetal and adult brains. The GD3/GT3ST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues. PMID- 8631982 TI - Mechanism of digeranylgeranylation of Rab proteins. Formation of a complex between monogeranylgeranyl-Rab and Rab escort protein. AB - Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates. PMID- 8631983 TI - Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP ribose signaling pathway. AB - Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+ mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5 trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+ mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger. PMID- 8631984 TI - Insertion of the 70-kDa peroxisomal membrane protein into peroxisomal membranes in vivo and in vitro. AB - Biosynthesis and intracellular transport of 70-kda peroxisomal membrane protein (pmp70) has been studied in rat hepatoma, h-4-ii-e cells. Pulse-chase analysis showed that a newly synthesized 35S-PMP70 first appeared in the cytosolic fraction and was then transported into the peroxisomal fraction. The half-life of 35S-PMP70 in the cytosolic fraction was approximately 3 min. Integration of 35S PMP70 into membranes occurred in the peroxisomal fraction and was completed within 30 min. No proteolytic processing of 35S-PMP70 was observed. An in vitro import system was reconstituted to characterize the insertion mechanism of PMP70 into peroxisomes. Peroxisomes isolated from rat liver were incubated at 26 degrees C with [35S]methionine-labeled in vitro translation products of PMP70 mRNA in the presence of the cytosolic fraction. The peroxisomes were reisolated and insertion of 35S-PMP70 into the membrane was analyzed using a Na2CO3 procedure. The binding and insertion of 35S-PMP70 were dependent on temperature and incubation time and was specific for peroxisomes. Pretreatment of peroxisomes with trypsin and chymotrypsin almost abolished the binding and insertion of 35S PMP70. The translation products contained several truncated 35S-PMP70s. The NH2 terminally truncated 35S-PMP70s, with a molecular mass greater than 50 kDa, bound to and inserted into peroxisomal membranes, whereas truncated 35S-PMP70s smaller than 45 kDa did not. These results suggest that PMP70 is post-translationally transported to peroxisomes without processing and inserted into peroxisomal membranes by a specific mechanism in which a proteinaceous receptor and a certain internal sequence of PMP70 are involved. PMID- 8631985 TI - Plasma membrane calcium pump isoform 4a has a longer calmodulin-binding domain than 4b. AB - Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+ calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49 residue calmodulin-binding region. PMID- 8631986 TI - Intra-Golgi transport inhibition by megalomicin. AB - Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra Golgi transport inhibition. PMID- 8631987 TI - cDNA cloning and characterization of murine transcriptional enhancer factor-1 related protein 1, a transcription factor that binds to the M-CAT motif. AB - The M-CAT motif is a cis-regulatory DNA sequence that is essential for muscle specific transcription of several genes. Previously, we had shown that both muscle-specific (A1) and ubiquitous (A2) factors bind to an essential M-CAT motif in the myosin heavy chain beta gene and that the ubiquitous factor is transcriptional enhancer factor (TEF)-1. Here we report the isolation of mouse cDNAs encoding two forms (a and b) of a TEF-1-related protein, TEFR1. The TEFR1a cDNA encodes a 427-amino acid protein. The coding region of TEFR1b is identical to 1a in both nucleotide and predicted amino acid sequence except for the absence of 43 amino acids downstream of the TEA DNA-binding domain. Three TEFR1 transcripts (approximately 7, approximately 3.5, and approximately 2 kilobase pairs) are enriched in differentiated skeletal muscle (myotubes) relative to undifferentiated skeletal muscle (myoblasts) and non-muscle cells in culture. In situ hybridization analysis indicated that TEFR1 transcripts are enriched in the skeletal muscle lineage during mouse embryogenesis. Transient expression of fusion proteins of TEFR1 and the yeast GAL4 DNA-binding domain in cell lines activated the expression of chloramphenicol acetyltransferase (CAT) reporter constructs containing GAL4 binding sites, indicating that TEFR1 contains an activation domain. An anti-TEFR1 polyclonal antibody supershifted the muscle specific M-CAT.A1 factor complex in gel mobility shift assays, suggesting that TEFR1 is a major component of this complex. Our results suggest that TEFR1 might play a role in the embryonic development of skeletal muscle in the mouse. PMID- 8631988 TI - Cloning and regulation of cornifin beta, a new member of the cornifin/spr family. Suppression by retinoic acid receptor-selective retinoids. AB - In this study, we describe the isolation and characterization of a cDNA clone C12 that encodes a new member of the cornifin/small proline-rich protein (spr) family, which we have named cornifin beta. C12 encodes a 1.1-kilobase pair mRNA and a 24.3-kDa cytosolic protein with a high proline content (19%). Its total amino acid sequence exhibits a 37-66% identity while the first 30 amino acids at the amino terminus are 87% identical to that of members of the cornifin family. At its carboxyl terminus, cornifin beta contains 21 tandem repeats of an octapeptide. Cornifin beta expression is restricted to several squamous epithelia. It is highly expressed in esophagus, tongue, and oral mucosa but, in contrast to cornifin alpha, is not detectable in the epidermis. Both retinoic acid and a retinoid selective for the nuclear retinoic acid receptors were very potent suppressors of cornifin beta expression while an analog selective for the nuclear retinoid X receptors was much less effective, suggesting that a specific retinoid signaling pathway is involved in this suppression. Cornifin beta can function through some of its Gln residues as an amine acceptor in transglutaminase-catalyzed cross-linking reactions. These results indicate that cornifin beta functions as a cross-linked envelope precursor. PMID- 8631989 TI - Identification of a novel domain in the aryl hydrocarbon receptor required for DNA binding. AB - The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the AHR nuclear translocator protein (ARNT). Both proteins possess basic helix-loop-helix motifs. ARNT binds to the side of the xenobiotic responsive element (XRE) that resembles an E-box (the sequence recognized by the majority of other basic helix-loop-helix proteins), whereas AHR binds to the side of the XRE that does not conform to the E-box sequence. The basic region of ARNT closely resembles those of other E-box binding proteins, whereas the "nominal basic region" of AHR (amino acids 27 39), although required for XRE binding, deviates from this consensus. By extensive mutational analysis it is shown here that an additional block of amino acids of AHR (from tyrosine 9 to lysine 20) that contains a highly basic segment is required for XRE binding and transcriptional activation. Deletion of the first nine amino acids negates XRE binding. Substitution of either tyrosine 9 or arginine 14 with alanine eliminates XRE binding, whereas alanine substitutions at certain other sites within the block reduce but do not eliminate binding. The reported absence of the first nine amino acids in the purified protein may therefore be artifactual. These results suggest that the amino acids of AHR involved in binding to the XRE constitute a novel DNA-binding domain, comprising amino acids located within and amino-terminal to the nominal basic region. PMID- 8631990 TI - Phosphorylation of cholecystokinin receptors expressed on Chinese hamster ovary cells. Similarities and differences relative to native pancreatic acinar cell receptors. AB - Phosphorylation of G protein-coupled receptors is an established mechanism for desensitization in response to agonist stimulation. We previously reported phosphorylation of the pancreatic acinar cell cholecystokinin (CCK) receptor and the establishment of two-dimensional phosphopeptide mapping of its sites of phosphorylation (Ozcelebi, F., and Miller, L. J. (1995) J. Biol. Chem. 270, 3435 3441). Here, we use similar techniques to map sites of phosphorylation of the same receptor expressed on a stable receptor-bearing Chinese hamster ovary (CHO) CCKR cell line. Like the native cell, the CHO-CCKR cell receptor was phosphorylated in response to agonist stimulation in a concentration-dependent manner; however, the time course was quite different. CHO-CCKR cell receptor phosphorylation increased progressively to a plateau after 15 min, while in the acinar cell it peaks within 2 min and returns to baseline over this interval. There were distinct qualitative and quantitative differences in the sites of phosphorylation of the two receptor systems. One site previously attributed to action of a staurosporine-insensitive kinase in the acinar cell was absent in the CHO-CCKR cell. Site-directed mutagenesis was utilized to eliminate predicted sites of protein kinase C action, but only two of four such sites affected the phosphopeptide map of this receptor. Chemical and radiochemical sequencing were performed on these and other phosphopeptides which were present in both the CHO CCKR cells and agonist-stimulated pancreatic acinar cells to provide direct evidence for the phosphorylation sites actually utilized. Thus, these data support the usefulness and limitations of a model cell system in studying receptor phosphorylation and desensitization. PMID- 8631991 TI - The Ras-related GTPase Rac1 binds tubulin. AB - The Ras-related Rho family are involved in controlling actin-based changes in cell morphology. Microinjection of Rac1, RhoA, and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fiber formation, and filopodia formation, respectively. To identify target proteins involved in these signaling pathways cell extracts immobilized on nitrocellulose have been probed with [gamma 32P]GTP-labeled Rac1, RhoA, and Cdc42Hs. We have identified two 55-kDa brain proteins which bind Rac1 but not RhoA or Cdc42Hs. These 55-kDa proteins were abundant, had pI values of around 5.5, and could be purified by Q-Sepharose chromatography. The characteristics on two-dimensional gel analysis suggested the proteins comprised alpha- and beta-tubulin. Indeed, beta-tubulin specific antibodies detected one of the purified 55-kDa proteins. Rac1 bound pure tubulin (purified by cycles of polymerization and depolymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, did not significantly affect the ability of Rac1 to interact with tubulin while the "effector-site" mutant D38A prevented interaction. These results suggest that the Rac1-tubulin interaction may play a role in Rac1 function. PMID- 8631992 TI - The CD40L promoter contains nuclear factor of activated T cells-binding motifs which require AP-1 binding for activation of transcription. AB - Four nuclear factor of activated T cells (NF-AT) binding motifs were found in the murine CD40 ligand promoter. Electrophoretic mobility shift assays using 18-base pair (bp) long oligonucleotides corresponding to the proximal site and nuclear extracts from activated T cells revealed two complexes which were inhibited by cyclosporin A and contained NF-ATc and NF-ATp. Neither complex contained AP-1 proteins. Multimers of the 18-bp oligonucleotides were not active in transient transfection assays using luciferase reporter gene constructs. In contrast, a 30 bp long oligonucleotide bound AP-1 proteins in addition to NF-AT proteins and its multimers strongly induced luciferase gene expression. These results suggested that NF-AT proteins play an important role in the expression of the CD40L gene and that their transcriptional activity requires AP-1 binding. PMID- 8631993 TI - Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases. AB - The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization. PMID- 8631994 TI - Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein. AB - We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication. PMID- 8631995 TI - Selective degradation of accumulated secretory proteins in the endoplasmic reticulum. A possible clearance pathway for abnormal tropoelastin. AB - The specific pathway of tropoelastin secretion was investigated in fetal calf ligamentum nuchae (FCL) cells using brefeldin A (BFA) to disrupt the secretory pathway. Electron microscopic studies of BFA-treated FCL cells showed ultrastructural changes consistent with the reported effects of BFA on intracellular organelles. When FCL cells were labeled with [3H]leucine in the presence of BFA, radiolabeled tropoelastin was not secreted, nor was there an intracellular accumulation of the protein. In contrast, fibronectin accumulated within the cells in the presence of BFA. Northern analysis of mRNA levels in FCL cells showed that the message for tropoelastin was unaffected by BFA treatment. Pulse chase experiments conducted in the presence of BFA demonstrated that the tropoelastin retained within the cells was rapidly degraded. Ammonium chloride, nocodazole, and cycloheximide had no effect on the degradation of tropoelastin, indicating that the degradation did not involve the endosome/lysosome pathway, movement via microtubules, or a short-lived protein, respectively. Incubation of FCL cells with BFA in the presence of N-acetyl-Leu-Leu-norleucinal, however, allowed tropoelastin to steadily accumulate in the cells. Cells pulsed in the presence of BFA alone showed that tropoelastin initially accumulates within the cells for approximately 1 h prior to being degraded, thus indicating that a critical threshold of tropoelastin must be reached before degradation can occur. Results from this study provide evidence for selective degradation of a soluble secreted protein by a cysteine protease following retention of the protein in the endoplasmic reticulum. PMID- 8631996 TI - A novel cytoplasmic dual specificity protein tyrosine phosphatase implicated in muscle and neuronal differentiation. AB - Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing phosphotyrosine, dsPTPs can hydrolyze phosphoserine/threonine-containing substrates and have been shown to dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6, from rat hippocampus. rVH6 contains the conserved dsPTP active site sequence, VXVHCX2GX2RSX5AY(L/I)M, and exhibits phosphatase activity against activated MAPK. In PC12 cells, rVH6 mRNA is induced during nerve growth factor-mediated differentiation but not during insulin or epidermal growth factor mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly expressed in proliferating cells and declines rapidly during differentiation. rVH6 expression correlates with the inability of fibroblast growth factor to stimulate MAPK activity in proliferating but not in differentiating MM14 cells. rVH6 protein localizes to the cytoplasm and is the first dsPTP to be localized outside the nucleus. This novel subcellular localization may expose rVH6 to potential substrates that differ from nuclear dsPTPs substrates. PMID- 8631997 TI - Microtubule-associated protein-dependent binding of phagosomes to microtubules. AB - In macrophages, phagosome movement is microtubule-dependent. Microtubules are a prerequisite for phagosome maturation because they facilitate interactions between phagosomes and organelles of the endocytic pathway. We have established an in vitro assay that measures the binding of purified phagosomes to microtubules. This binding depends on the presence of membrane proteins, most likely integral to the surface of phagosomes, and on macrophage cytosol. The cytosolic binding factor can interact with microtubules prior to the addition of phagosomes to the assay, suggesting that it is a microtubule-associated protein (MAP). Consistent with this, depletion of MAPs from the cytosol by microtubule affinity removes all binding activity. Microtubule motor proteins show no binding activity, whereas a crude MAP preparation is sufficient to support binding and to restore full binding activity to MAP-depleted cytosol. We show that the activating MAP factor is a heat-sensitive protein(s) that migrates at around 150 kDa by gel filtration. PMID- 8631998 TI - Cytosine deaminase gene as a positive selection marker. AB - Cytosine deaminase (EC 3.5.4.1), a non-mammalian enzyme, catalyzes the deamination of cytosine and 5-fluorocytosine to form uracil and 5-fluorouracil, respectively. Eukaryotic cells have been genetically modified with a bacterial cytosine deaminase gene to express a functional enzyme. When the genetically modified cells are combined with 5-fluorocytosine, it creates a potent negative selection system, which may have important applications in cancer gene therapy. In this paper, we introduce a novel positive selection method based upon the expression of the cytosine deaminase gene. This method utilizes inhibitors in the pyrimidine de novo synthesis pathway to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. Thus, only cells expressing the cytosine deaminase gene can be rescued in a positive selection medium. PMID- 8631999 TI - Differential heparin inhibition of skeletal muscle alpha-dystroglycan binding to laminins. AB - The laminin binding properties of alpha-dystroglycan purified from rabbit skeletal muscle membranes were examined. In a solid phase microtiter assay, 125I laminin (laminin-1) bound to purified alpha-dystroglycan in a specific and saturable manner with a half-maximal concentration of 8 nM. The binding of 125I- alpha-dystroglycan to native laminin and merosin (a mixture of laminin-2 and -4) was also compared using the solid phase assay. The absolute binding of 125I- alpha-dystroglycan to laminin (6955 +/- 250 cpm/well) was similar to that measured for merosin (7440 +/- 970 cpm/well). However, inclusion of 1 mg/ml heparin in the incubation medium inhibited 125I-alpha-dystroglycan binding to laminin by 84 +/- 4.3% but inhibited 125I-alpha-dystroglycan binding to merosin by only 17 +/- 5.2%. Similar results were obtained with heparan sulfate, while de N-sulfated heparin, hyaluronic acid, and chondroitin sulfate had no differential effect. These results were confirmed by iodinated laminin and merosin overlay of electrophoretically separated and blotted dystrophin-glycoprotein complex. In contrast to the results obtained with skeletal muscle alpha-dystroglycan, both laminin and merosin binding to purified brain alpha-dystroglycan was significantly inhibited by heparin. Our data support the possibility that one or more heparan sulfate proteoglycans may specifically modulate the interaction of alpha-dystroglycan with different extracellular matrix proteins in skeletal muscle. PMID- 8632000 TI - Purification and characterization of UEF3, a novel factor involved in the regulation of the urokinase and other AP-1 controlled promoters. AB - Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters. PMID- 8632001 TI - Alterations in nucleosome core structure in linker histone-depleted chromatin. AB - We have previously shown that the sequential arrangement of histone-DNA contacts is essentially the same in the nucleosomal core of sea urchin sperm nuclei, where chromatin is highly condensed and repressed, and in nuclei from lily bud sepals or yeast, where chromatin is highly active in transcription and replication and is significantly or completely depleted of histone H1. However, the difference in the strength of some histone-DNA contacts has not been understood or discussed. In this work, we demonstrate that some of these differences are due to a conformational change in the nucleosomal core. We show that the nucleosomal core in linker histone-depleted chromatin is in a different conformational state compared with the nucleosomal core in folded chromatin or in isolated core nucleosomes. This conformational state is characterized by altered strengths in the histone H4 and H2A/H2B contacts with the regions of sharply bent nucleosomal DNA around sites +/-1 and +/-4 and site +/-5, respectively. We demonstrate that this conformation, which we call the "stretched nucleosome," is a general feature of unfolded linker histone-depleted chromatin and may occur during chromatin activation. Our results suggest that this nucleosome structural alteration does not depend on chromatin sources and histone variants studied in this work. In addition, we show that this alteration is reversible and is caused by the stretching of linker DNA during chromatin unfolding. PMID- 8632003 TI - L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation. AB - The subcellular localization of l-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A4 > LDH-A3B) with the peroxisomes comprising relatively more LDH-A3B than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal beta-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the beta-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA beta-oxidation system could be verified in a modified beta-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mm) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD+/NADH levels and maintain the flux of fatty acids through the peroxisomal beta-oxidation spiral. PMID- 8632002 TI - The yeast VRG4 gene is required for normal Golgi functions and defines a new family of related genes. AB - Sodium vanadate is an effective agent for the enrichment of yeast mutants with defects in glycosylation steps that occur in the Golgi complex (Ballou, L., Hitzeman, R. A., Lewis, M. S., and Ballou, C. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3209-3212). We isolated and screened vanadate-resistant glycosylation mutants in the budding yeast, Saccharomyces cerevisiae, to identify any that may be defective in the secretory pathway, since changes in normal glycosylation may reflect defects within the secretory pathway. We identified one such mutant, allelic to vrg4/van2, that is defective in processes that occur specifically in the Golgi complex. Protein secreted from vrg4 mutants lacks the outer chain glycosylation that is normally extended during passage through the Golgi. This mutant fails to retrieve soluble endoplasmic reticulum proteins from the Golgi and accumulates the Golgi-specific biosynthetic intermediate of the vacuolar protein, carboxypeptidase Y. Analyses of intracellular membranes by staining with the fluorescent lipophilic dye, DiOC6, and by electron microscopy reveals a dramatic alteration in the membrane morphology of vrg4 mutant cells. The VRG4 gene encodes a 36.9-kDa membrane protein that is essential for cell viability. A sequence homology search has identified five related genes, establishing that VRG4 is a founding member of a family of structurally similar genes. Taken together, these results suggest that the VRG4 gene plays an important role in regulating Golgi functions and in maintaining the normal organization of intracellular membranes. PMID- 8632004 TI - The tyrosine phosphatase PTP1C associates with Vav, Grb2, and mSos1 in hematopoietic cells. AB - The association of the murine motheaten phenotype of severe hemopoietic dysregulation with loss of PTP1C tyrosine phosphatase activity indicates a critical role for this SH2 domain-containing phosphotyrosine phosphatase in the regulation of hemopoietic cell growth and differentiation. To explore the molecular basis for PTP1C effects on hematopoiesis, we have investigated the possibility that this enzyme interacts with the product of the Vav proto oncogene, a putative guanine nucleotide exchange factor expressed exclusively in hemopoietic cells. Our data indicate that PTP1C physically associates with Vav in murine spleen cells and in EL4 T lymphoma and P815 mastocytoma cells, and that this interaction is increased following mitogenic stimulation and the induction of both PTP1C and Vav tyrosine phosphorylation. The results also reveal tyrosine phosphatase activity to be present in Vav immunoprecipitates from stimulated splenic and P815 cells and suggest that a major portion of total cellular PTP1C catalytic activity is associated with Vav. As Vav-associated tyrosine phosphatase activity was not detected in PTP1C-deficient motheaten splenic cells, it appears that PTP1C accounts for most, if not all, Vav-coprecipitable tyrosine phosphatase activity in normal cells. The data also demonstrate the capacity of the Vav SH2 domain alone to bind to PTP1C in activated P815 cells, but suggest a role for the two Vav SH3 domains in enhancing this interaction. In addition, the results reveal PTP1C association with two other molecules implicated in Ras activation, the Grb2 adaptor protein and mSos1, a GTP/GDP exchanger for Ras. PTP1C therefore has the capacity to bind and potentially modulate various signaling effectors involved in activation of Ras or Ras-related proteins, and, accordingly, regulation of Ras activation represents a possible mechanism whereby PTP1C influences hemopoietic cellular responses. PMID- 8632005 TI - Phosphorylation of caveolin by src tyrosine kinases. The alpha-isoform of caveolin is selectively phosphorylated by v-Src in vivo. AB - Caveolae are flask-shaped plasma membrane specializations that are thought to exist in most cell types. A 22-kDa protein, caveolin, is an integral membrane component of caveolae membranes in vivo. Previous studies have demonstrated that caveolin is phosphorylated on tyrosine by oncogenic viral Src (v-Src) and that caveolin is physically associated as a hetero-oligomeric complex with normal cellular Src (c-Src) and other Src family tyrosine kinases. Caveolin contains eight conserved tyrosine residues that may serve as potential substrates for Src. Here, we have begun to study the phosphorylation of caveolin by Src family tyrosine kinases both in vitro and in vivo. Using purified recombinant components, we first reconstituted the phosphorylation of caveolin by Src kinase in vitro. Microsequencing of Src-phosphorylated caveolin revealed that phosphorylation occurs within the extreme N-terminal region of full-length caveolin between residues 6 and 26. This region contains three tyrosine residues at positions 6, 14, and 25. Deletion mutagenesis demonstrates that caveolin residues 1-21 are sufficient to support this phosphorylation event, implicating tyrosine 6 and/or 14. In vitro phosphorylation of caveolin-derived synthetic peptides and site-directed mutagenesis directly show that tyrosine 14 is the principal substrate for Src kinase. In support of these observations, tyrosine 14 is the only tyrosine residue within caveolin that bears any resemblance to the known recognition motifs for Src family tyrosine kinases. In order to confirm or refute the relevance of these in vitro studies, we next analyzed the tyrosine phosphorylation of endogenous caveolin in v-Src transformed NIH 3T3 cells. In vivo, two isoforms of caveolin are known to exist: alpha-caveolin contains residues 1-178 and beta-caveolin contains residues 32-178. Only alpha-caveolin underwent tyrosine phosphorylation in v-Src transformed NIH 3T3 cells, although beta-caveolin is well expressed in these cells. As beta-caveolin lacks residues 1 31 (and therefore tyrosine 14), these in vivo studies directly demonstrate the validity of our in vitro studies. Because alpha- and beta-caveolin are known to assume a distinct but overlapping subcellular distribution within a single cell, v-Src phosphorylation of alpha-caveolin may only affect a subpopulation of caveolae that contain alpha-caveolin. PMID- 8632006 TI - Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa Is required for its binding to cofactor VIIIa. AB - This report describes the analysis of a novel mutant human factor IX protein from a patient with hemophilia B (factor IX activity <1%; factor IX antigen 45%). Enzymatic amplification of all eight exons of the factor IX gene followed by direct sequence analysis reveals a single nucleotide change (a guanine --> adenine transition) in exon 2 at nucleotide 6409 which results in a glycine --> arginine substitution at amino acid 12 in the gamma-carboxyglutamic acid rich (Gla) domain of the mature protein. Factor IX was isolated by immunoaffinity chromatography from plasma obtained from the proband. The purified protein is indistinguishable from normal factor IX by polyacrylamide gel electrophoresis. Characterization of the variant in purified component assays reveals that it is activated normally by its physiologic activator factor XIa, but its phospholipid dependent activation by the factor VIIa-tissue factor complex is diminished. In the presence of phospholipid and 5 mM Ca2+, the activities of variant and normal plasma-derived factor IX are similar; however, in the presence of activated factor VIIIa (intrinsic tenase complex), the normal augmentation of the cleavage of the specific substrate of factor IX, factor X, is not observed. The determination of the association constants for normal and variant factor IXa with factor VIIIa shows that the affinity of the activated variant factor IX for the cofactor factor VIIIa is 172-fold lower than normal. Competition studies using active site-inactivated factor IXas in the intrinsic tenase complex confirm that the defect in the variant protein is in its binding to factor VIIIa. We conclude that the structural integrity of the Gla domain of human factor IX is critical for the normal binding of factor IXa to factor VIIIa in the intrinsic tenase complex. In addition, a glycine at amino acid 12 is necessary for normal activation of factor IX by the factor VIIa-tissue factor complex. PMID- 8632007 TI - The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences. AB - We describe the genomic organization and functional characterization of the mouse gene encoding vascular endothelial growth factor (VEGF), a polypeptide implicated in embryonic vascular development and postnatal angiogenesis. The coding region for mouse VEGF is interrupted by seven introns and encompasses approximately 14 kilobases. Organization of exons suggests that, similar to the human VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino acid isoforms, but does not predict a fourth VEGF isoform corresponding to human VEGF206. Approximately 1. 2 kilobases of 5'-flanking region have been sequenced, and primer extension analysis identified a single major transcription initiation site, notably lacking TATA or CCAT consensus sequences. The 5'-flanking region is sufficient to promote a 7-fold induction of basal transcription. The genomic region encoding the 3'-untranslated region was determined by Northern and nuclease mapping analysis. Investigation of mRNA sequences responsible for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima, D. T. , Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208) revealed that the 3' untranslated region was sufficient to trigger the rapid turnover of a normally long-lived reporter mRNA in vitro. These data and reagents will allow the molecular and genetic analysis of mechanisms that control the developmental and pathological expression of VEGF. PMID- 8632008 TI - Transformation of NIH 3T3 cells by HER3 or HER4 receptors requires the presence of HER1 or HER2. AB - Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF. PMID- 8632009 TI - DNA binding specificity of the CCAAT/enhancer-binding protein transcription factor family. AB - CCAAT/enhancer-binding protein (C/EBP) transcription factor family members are related by a high degree of amino acid sequence identity to the basic leucine zipper DNA-binding domain and show distinct but overlapping patterns of tissue- and stage-restricted expression. Although C/EBPalpha and C/EBPbeta have been shown to recognize a consensus sequence derived from regulatory elements in virus and acute-phase response genes, the potential for more subtle differences in the binding preference of the C/EBP family has not been previously addressed. The consensus sequence of C/EBPdelta has not been reported. By using the method of polymerase chain reaction-mediated random site selection to assess the DNA binding specificity of the C/EBP family in an unbiased manner, we demonstrated the sequence preferences for C/EBP family members. With small variations, these C/EBP family members showed similar sequence preferences, and the consensus sequence was identified as RTTGCGYAAY (R = A or G, and Y = C or T). The phosphorylation of C/EBPdelta by casein kinase II increased the binding activity, but did not affect the binding specificity, whereas it was reported that the phosphorylation of C/EBPalpha and C/EBPbeta decreased the binding affinity. The specificity of action of C/EBP family members may be derived from the characteristics of each factor, including the expression profiles, the DNA binding affinities, the cofactors, and so on, in addition to the DNA binding specificities. PMID- 8632010 TI - Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans. AB - The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII RCAr) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., Rodriguez-Boulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids. We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the Km for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-O-sulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate. PMID- 8632012 TI - Identification of a novel receptor kinase that phosphorylates a phospholipase C linked muscarinic receptor. AB - Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied beta-adrenergic receptor/adenylate cyclase pathway, in which receptor specific phosphorylation is known to be mediated by beta-adrenergic receptor kinase ( beta-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for beta-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser345-Leu463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30-60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparin-Sepharose. The purified protein kinase represented an approximately 18,600-fold purification and was a single polypeptide with a molecular weight of approximately 40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, beta-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase. PMID- 8632011 TI - The role of cytosolic calcium in chronic adaptation to phosphate depletion in opossum kidney cells. AB - Chronic dietary phosphate restriction is associated with up-regulation of sodium dependent phosphate (Na/Pi) cotransport by renal proximal tubular epithelial cells in association with increases in Na/Pi cotransporter mRNA and protein. We investigated whether changes in cytosolic calcium mediate this adaptive response in opossum kidney cells, a continuous line of renal epithelial cells. After 24 h of phosphate depletion, steady-state cytosolic calcium levels were increased; this increase was observed at physiologic levels of phosphate restriction and was prevented by the calcium channel blocker verapamil. Chronic phosphate depletion was also associated with parallel increases in Na/Pi cotransport activity, Na/Pi cotransporter mRNA, and Na/Pi cotransporter protein, all of which were blocked in verapamil-treated cells. Actinomycin D, at a dose that prevented the increase in NaPi-4 mRNA during phosphate depletion, also prevented the increase in Na/Pi cotransport activity. Incubation with the calcium ionophore ionomycin or A23187 reproduced the increase in Na/Pi cotransporter mRNA in phosphate-replete cells. Conversely, chelation of cytosolic calcium by quin-2/AM prevented the increase in Na/Pi cotransporter mRNA in phosphate-depleted cells. The effect of an increase in cytosolic calcium was specific for the Na/Pi cotransporter as mRNA levels for the sodium-dependent glucose transporter were not affected. Our observations suggest that chronic phosphate restriction increases steady-state cytosolic calcium, which, in turn, increases transcription of Na/Pi cotransporter mRNA, thereby stimulating Na/Pi cotransport activity. PMID- 8632013 TI - Allosteric regulation of the thermostability and DNA binding activity of human p53 by specific interacting proteins. CRC Cell Transformation Group. AB - Conformational stability is a prerequisite for the physiological activity of the tumor suppressor protein p53. p53 protein can be allosterically activated for DNA binding by phosphorylation or through noncovalent interaction with proteins such as DnaK, the Escherichia coli homologue of the heat shock protein Hsp70. We present in vitro evidence for a rapid temperature-dependent change in the conformation and tetrameric nature of wild-type p53 upon incubation at 37 degrees C, which correlates with a permanent loss in DNA binding activity. We show that p53 is allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37 degrees C by binding of two classes of ligands to regulatory sites on the N and C terminus of the molecule through which an intrinsic instability of p53 is neutralized. Deletion of the domain conferring instability at the C terminus is sufficient to confer enhanced stability to the total protein. DnaK binding to the C terminus can profoundly protect p53 at 37 degrees C from a temperature-dependent loss of the DNA binding activity but does not renature or activate denatured p53. In contrast, another activator of the DNA binding activity of latent p53, the monoclonal antibody PAb421, which also interacts with the C terminus of the protein, is not able to protect p53 from thermal denaturation. Two monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, do not activate the latent DNA binding function of p53 but can protect the p53 wild-type conformation at 37 degrees C. Thus, activation of the DNA binding function of p53 is not synonymous with protection from thermal denaturation, and therefore, both of these pathways may be used in cells to control the physiological activity of p53. The protection of p53 conformation from heat denaturation by interacting proteins suggests a novel mechanism by which p53 function could be regulated in vivo. PMID- 8632014 TI - Membrane topology of the 12- and the 25-kDa subunits of the mammalian signal peptidase complex. AB - The cleavage of signal sequences of secretory and membrane proteins by the signal peptidase complex occurs in the lumen of the endoplasmic reticulum. Mammalian signal peptidase consists of five subunits. Four have been cloned, SPC18, SPC21, SPC22/23, and SPC25, of which all but SPC25 have been demonstrated to be single spanning membrane proteins exposed to the lumen of the endoplasmic reticulum. We have determined the cDNA sequence of the remaining 12-kDa subunit (SPC12) as well as the membrane topologies of SPC12 and SPC25 in rough microsomes. Both polypeptides span the membrane twice with their N and C termini facing the cytosol and contain only very small, if any, lumenal domains. Therefore, SPC12 and SPC25 are likely to be involved in processes other than the enzymatic cleavage of the signal sequence. PMID- 8632015 TI - Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. AB - DREF, a transcription regulatory factor which specifically binds to the promoter activating element DRE (DNA replication-related element) of DNA replication related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknullt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences. PMID- 8632016 TI - Differentiation and cell surface expression of transforming growth factor-beta receptors are regulated by interaction with matrix collagen in murine osteoblastic cells. AB - Although transforming growth factor (TGF)-beta enhances bone formation, it inhibits the differentiation of osteoblasts. To clarify the regulatory mechanism of osteoblastic differentiation and TGF-beta actions, the relationship among differentiation, TGF-beta actions, and matrix protein synthesis was examined using murine osteoblast-like MC3T3-E1 cells. Alkaline phosphatase (ALP) activity continued to increase during long-term cultures, and the increase was closely associated with a reduction in cell surface TGF-beta receptors competent to bind TGF-beta. Both the stimulation of proteoglycan synthesis and the inhibition of ALP activity by TGF-beta were also suppressed. Collagen synthesis inhibitors and an anti-alpha2beta1 integrin blocking antibody blocked the changes in ALP activity and TGF-beta receptors, and a DGEA peptide that interferes binding of collagen to alpha2beta1 integrin also blocked the increase in ALP activity. Furthermore, when MC3T3-E1 cells were cultured on extracellular matrix layers obtained from these cells, all the differentiation-associated changes could be observed without collagen production, and the extracellular matrix-induced differentiation was also blocked by an anti-alpha2beta1 integrin antibody. These results demonstrate that the interaction of cell surface alpha2beta1 integrin with matrix collagen synthesized by osteoblasts themselves is involved in the osteoblastic differentiation and the reduction in cell surface receptors and actions of TGF-beta. It is suggested that matrix collagen synthesized under the stimulation by TGF-beta plays an important role in the regulation of osteoblastic differentiation and TGF-beta actions by differentiation-associated down regulation of TGF-beta receptors. PMID- 8632017 TI - Purification and characterization of the Ca2+-ATPase of Flavobacterium odoratum. AB - The P-type Ca2+-ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized. Inside-out membrane vesicles were extracted with C12E8, followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography. The purified Ca2+-ATPase consists of a single polypeptide. It migrates electrophoretically with an apparent molecular mass of 60,000 Da, consistent with the phosphorylation pattern originally reported in membrane vesicles. This single polypeptide is functional and capable of calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and reverse phosphorylation. Maximum hydrolysis activity occurs at pH 8.0, with a specific activity of approximately 75 micromol of ATP hydrolyzed min-1 mg-1 protein. The purified Ca2+-ATPase has an apparent Km for calcium of 1.5 microM and for ATP of 90 microM. Vanadate strongly inhibits the activity with an IC50 of 0.6 microM. The prokaryotic Ca2+-ATPase is insensitive to the SR Ca2+-ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by [gamma 32P]ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by Pi in both the presence and absence of calcium. PMID- 8632018 TI - Xenobiotic responsive element-mediated transcriptional activation in the UDP glucuronosyltransferase family 1 gene complex. AB - We have isolated genomic DNA clones containing rat UDP-glucuronosyltransferase family 1 (UGT1) sequences and have shown drug-responsive and tissue-specific alternative expression of multiple first exons (Emi, Y., Ikushiro, S., and Iyanagi, T. (1995) J. Biochem. (Tokyo) 117, 392-399). The UGT1 locus encodes at least nine UGT1 isoforms. UGT1A1 is a major 3-methylcholanthrene (MC)-inducible form in rat liver. In this report, we have identified a cis-acting element necessary for transcriptional activation of UGT1A1 in hepatocytes. A promoter region was fused to a chloramphenicol acetyltransferase gene, and the resultant construct was transiently transfected into hepatocytes. A DNA fragment carrying 1,100 nucleotides derived from the 5'-flanking region of the UGT1A1 gene was enough for MC induction. Unidirectional deletion of this region revealed that there existed one xenobiotic responsive element (XRE), TGCGTG, between -134 and 129. When a single base substitution was introduced into the XRE, MC-induced expression of the UGT1A1 gene was completely abolished. In addition, an XRE deleted construct failed to respond to MC. Gel mobility shift assays showed MC inducible binding of the nuclear aromatic hydrocarbon receptor-ligand complex to this motif. Gel shift-coupled DNase I protection analyses revealed that the GCGTG core sequence was a target site of the liganded aromatic hydrocarbon receptor. These results suggest that the XRE participates in induction of the rat UGT1A1 gene by MC. PMID- 8632019 TI - Effects of hypothermic storage on the vascular endothelium: a scanning electron microscope study of morphological change in human vein. AB - OBJECTIVE: The aims of this study were: i) to identify morphological changes occurring in the endothelium of human umbilical veins subjected to the typical storage procedures used in transplantation and ii) to determine the relative efficacy of preservation solutions containing intra and extracellular levels of sodium and potassium. EXPERIMENTAL DESIGN: Prospective. PROCEDURE: Scanning electron micrographs were taken pre and post cold hypoxic storage of human umbilical veins for 3 or 16 hours. RESULTS: Cold preservation resulted in severe cell detachment with subsequent loss of monolayer continuity and exposure of thrombogenic basal membrane components (highly significant after only 3 hours of cold storage, Kruskal-Wallis, p < 0.01). The morphological alterations culminated in EC with spherical shapes. Cytoplasmic membranes presented an increased number of microvilli and intercellular processes, followed by microvillous swelling and surface blebbing as damage increased. Bleb detachment was seen in severely damaged specimens. However, morphological preservation was not significantly affected by the duration of hypoxia or the ionic balance of the solution tested. CONCLUSIONS: The present results demonstrate that even short periods (3 hours) of cold storage without revascularization cause significant morphological damage to the endothelium. The ionic composition of the preservation solution did not significantly affect the process. The morphological changes seen in this study could explain storage-related problems such as loss of normal vascular permeability and increased thrombogenicity, problems often associated with transplantation procedures. PMID- 8632020 TI - Preservation of cell organelles during storage of human atrial tissue in the University of Wisconsin solution. AB - AIM OF THE STUDY: The University of Wisconsin storage solution (UW) (E.I. du Pont de Nemours, Wilmington, DE) has been successful in extending the storage period using some model systems of donor heart preservation for cardiac transplantation. The ability of UW to preserve human cardiac cell organelle (sarcoplasmic reticulum, mitochondria and sarcolemmal) membrane composition (enzyme activity, protein, cholesterol and phospholipid content) was compared to St. Thomas's Hospital Solution (ST) and saline. METHODS: Human atrial appendages were stored at 4 degrees C for 24 h in saline, ST or UW or not stored (controls) and the cell organelles isolated. Each fraction was assayed for enzyme activity (mitochondria: azide sensitive Ca2+ ATPase, cytochrome C oxidase; sarcolemmal membrane: Na+K+ ATPase, p-nitrophenylphosphatase; sarcoplasmic reticulum: CA2+ uptake, Ca2+ ATPase, NADPH cytochrome C reductase), protein, cholesterol and phospholipid content. RESULTS: "Protein yield" proved to be the most sensitive marker for cell organelle preservation. Only the sarcolemmal membrane showed no decrease in either enzyme activities of "protein yield" after storage in saline, ST or UW. Mitochondria showed no decrease in enzyme activities but a decrease in "protein yield" after storage in all 3 solutions. The "protein yield" of sarcoplasmic reticulum was significantly reduced after storage in UW, saline and ST. No correlation could be drawn between cholesterol and phospholipid content and the preservation of cell organelle function. CONCLUSIONS: It is possible to distinguish between the ability of solutions to preserve the membrane composition of human cardiac tissue during hypothermic storage. Using simple assays to assess preservation provides preliminary screening for a superior solution which can then be used in more complicated transplantation models to more fully assess cardiac function. PMID- 8632021 TI - Long-term results after conservative aortic valve repair for aortic regurgitation with ventricular septal defect. AB - Aortic valve repair was performed in 61 patients having aortic regurgitation (AR) associated with ventricular septal defect (VSD). Preoperative regurgitation was classified according to the Sellers' classification and grade I was in 3 patients, grade II in 19, grade III in 34, and grade IV in 5 patients. Forty-six patients underwent only plication of aortic valve, and in 15 patients the commissuroplasty was added to reinforce the free edge of the cusp. Furthermore, aortoplasty (aortic annuloplasty) was performed in 11. Actuarial survival rate and freedom from reoperation rate were 91.6% and 87.2%, respectively at 10 years after the initial repair. The rate of freedom from deterioration for 22 patients showing grade I or II of AR before the initial operation was 86.4% at 1 year, 76.7% at 5 and 10 years, while the rate for 36 patients with grade III or more severe AR was 91.7% at 1 year, 77.4% at 5 years and 63.9% at 10 years. The rate at 10 years after operation between the two groups was different significantly (p < 0.05). Twenty among 22 patients having grade I or II of AR before operation were in NYHA class I, while 23 of 30 survivors with grade III or IV of AR were in class I, 6 in class II, and 1 in class III. Eight patients received reoperation and four of them underwent repeated conservative aortic valve repair, and one received aortic valve replacement at reoperation. Five of 7 survivors with reoperation improved in NYHA class I and 2 in class II. These results suggested that careful follow-up was required especially for the first 10 years after the initial operation in patients showing preoperative severe AR and receiving aortic repair. PMID- 8632022 TI - Luke warm blood cardioplegia for CAB surgery in patients with severely impaired LV function. Improved results. AB - Ward blood cardioplegia (WBC) has recently been reported to improve myocardial protection in adult open heart surgery, especially in high risk cases. However, WBC has been reported to have some disadvantages including narrow safety margins concerning brain and kidney perfusion. We therefore modified our technique to utilize luke-warm blood cardioplegia (LWBC). We carried out 470 open heart procedures using luke-warm cardioplegia (anterograde + retrograde perfusion) from 1/2/1991 - 30/9/1992; 94 had LVEF < 30% and form the basis of this study. Other major risk factors in this group included: > 70 yrs - 26 patients, L main > 50% - 14 patients, emergencies - 11 patients, redo's - 3 patients. Eightyone patients underwent CAB only; 3 had additional MVR, 3 additional closure acute VSD of whom one underwent additional LV aneurysmectomy, one additional AVR; 4 patients underwent AVR only, and 2 MVR. Average number of grafts/patient for the 81 isolated CAB's was 4.5. IABP was necessary postbypass in 4 patients (9 emergencies were on IABP support at time of operation). Thirty day mortality was 3 patients (3.2%). Late mortality was 5 cases. These results are superior to those achieved using cold protection and warm blood cardioplegia. LWBC is a safe method for myocardial protection in patients undergoing CAB, particularly when LV function is severely compromised. PMID- 8632023 TI - Heart transplantation after repair of postinfarction ventricular septal defect. AB - A 68 year-old man underwent surgical repair of a ventricular septal defect following an acute myocardial infarction. Recurrent interventricular septal rupture with significant left-to-right shunting led to progressive deterioration in cardiac function and intractable heart failure. The patient underwent orthotopic heart transplantation three months after his initial operation, and he is clinically well sixteen months after transplantation. PMID- 8632024 TI - Postinfarction cardiac rupture after coronary revascularization. AB - Rupture of the myocardium is a catastrophic complication of acute myocardial infarction. Coronary revascularization has been increasingly utilized for the treatment of postinfarction angina. Further, myocardial infarction is a well known complication of coronary revascularization. Nonetheless, cardiac rupture after coronary revascularization has been rarely described. Described herein are three such cases with a review of the literature. PMID- 8632025 TI - Simultaneous Bentall's procedure and sternal turnover in a patient with Marfan syndrome. AB - A patient with Marfan syndrome with ascending aortic aneurysm, aortic regurgitation and pectus excavatum was treated with simultaneous Bentall's procedure and sternal turnover. Partial sternal necrosis and mediastinitis developed 12 days after surgery. After debridement of about one-third of turnovered sterno-costal complex (SC), successful salvage of the remaining two thirds of SC and sterilization of the infected aortic Dacron graft were accomplished with pedicled omental flap. The 7 previously published cases of simultaneous Bentall's procedure and sternal turnover are reviewed. PMID- 8632026 TI - Concurrent right atrial myxoma and malignant lymphoma. AB - Atrial myxomas are the most common primary tumor of the heart. We report an unusual case where an incidentally found right atrial myxoma was associated with a malignant lymphoma. Surgical management of the concurrent problems is discussed as well as a review of pertinent literature and efficacy of diagnostic modalities. PMID- 8632027 TI - A patch technique for reconstruction of stenotic pulmonary bifurcation in a Rastelli-type operation after palliations for cyanotic congenital heart disease. A report of two cases. AB - A Rastelli-type operation was successfully performed on two patients with cyanotic congenital heart disease with pulmonary artesia (PA) and severe stenosis or obstruction of the pulmonary bifurcation, following the improvement of a pulmonary artery index (PA index) after palliative systemic pulmonary shunts. It is a point that stenotic or obstructive pulmonary bifurcation was reconstructed by a patch technique. An extracardiac conduit was used a combination of a bicuspid valved pericardial roll and a prosthetic vessel graft. In a 5-year-old male with an extreme type of tetralogy of Fallot associated with PA and patent ductus arteriosus (PDA), the enlargement of stenotic pulmonary bifurcation was attempted using a brimshaped equine pericardial patch in order to get sufficient suture margin and be safely anastomosed with a conduit. In a 6-year-old female with complete transposition of the great arteries, PA, PDA and right aortic arch (RAA), an obstructive pulmonary bifurcation behind the RAA was enlarged using a brimshaped equine pericardial patch. The attachment of the conduit was shifted to the left pulmonary artery to avoid compression of an extracardiac conduit and pulmonary artery behind RAA. Reconstruction of the pulmonary bifurcation using an equine pericardial patch is useful to anastomose an extracardiac conduit and the stenotic or obstructive pulmonary bifurcation. PMID- 8632028 TI - Situs inversus and myocardial revascularization. Case report. AB - This report describes an internal mammary artery by-pass grafting to the anterior descending coronary artery in a man with an unstable postinfarction angor pectoris and a "situs inversus totalis" condition. The association of "situs inversus totalis" and atherosclerotic coronary artery disease has the same incidence as in normal people. To the authors' knowledge, this is the second case in medical literature of coronary artery by-pass surgery with internal mammary artery graft in a patient with "situs inversus totalis". PMID- 8632029 TI - Incorporation of fibronectin-impregnated vascular prostheses in the pig. Microscope study. AB - An interface near the endothelial extracellular matrix is necessary to augment and maintain endothelial cell attachment. The use of plasma lectins constitutes one of the present lines of research designed to improve this interface. We studied the incorporation of 2 series of arterial prostheses with a diameter of 4 mm and a mean length of 9 cm. They were implanted in the carotid arteries of adult Europig minipigs. Prostheses were of two types: polytetrafluoro-ethylene (PTFE) and knitted Dacron. Two series of 12 pigs each were used. One was explanted at 3 months and the other at six. Each pig was grafted with one prosthesis impregnated with the plasma components of diluted Fibrogel and one non impregnated prosthesis which served as control. The explanted prostheses and adjacent parts of the carotid were prepared for light or scanning electron microscopy. Proximal, median and distal segments were cut and embedded in resin. Collagen distribution was revealed by Milligan's trichrome stain, and fibrin distribution by Picro-Mallory staining. Macroscopic examination showed discrete periprosthetic adhesion for impregnated prostheses and complete adhesion for non impregnated prostheses. Scanning electron microscopy revealed a median endothelial cell coating on impregnated grafts whereas the only endothelial cells on non-impregnated grafts, were perianastomotic. On impregnated grafts, Milligan's trichrome staining revealed an even collagen distribution. The walls of non-impregnated grafts exhibited capillary cell infiltrations with breaches in the outer structures. In impregnated prostheses, the absence of such breaches enabled us to postulate that their incorporation was better than that of the non impregnated grafts. The minipig model was hard to handle because of the aggressiveness engendered by restricted feeding designed to limit weight increases. In general, however, we may justifiably conclude that in this model, the use of plasma lectins improved prosthetic incorporation. PMID- 8632030 TI - Sartorius myoplasty for the treatment of infected groins with vascular grafts. AB - INTRODUCTION: Vascular graft infection is a rare (0.8-2.6%) but serious complication of reconstructive vascular surgery, that comprises limb-threatening when located in the groin. PURPOSE: Review our experience in the treatment of infected groin wounds after femoral surgery by using sartorius myoplasty. MATERIALS AND METHODS: Between January 1989 and October 1993, 13 patients with infected groin wounds involving vascular graft (Grade III of Szilagyi) were treated with local gently debridement, 10% povidone iodide lavage for 5 days, local and systemic antibodies for 14 days and in situ change of the infected graft followed by sartorius myoplasty, detaching its origin from superior anterior iliac spine and fixing it to inguinal ligament, except in one case where tissue cultures were negative and the graft was not changed. Follow-up was done using clinical and echographical criteria at 3, 6, 12 months after operation, and annually. RESULTS: Hospital mean stage was 10 days (8-13). The mean follow-up was 36 months (12-65). There was only one immediate complication from hemorrhage that needed surgery. The patient whose graft was not changed developed an abscess in the prosthetic bed 12 weeks after treatment. It was necessary to change it after debridement and antibiotics. No mortality was related to this technique. CONCLUSIONS: Sartorius myoplasty, with detaching and origin transposition, after in situ change of infected graft, is an excellent therapeutic option for reconstruction and treatment for infected groins with vascular grafts because it is easy to perform and offers very good results in long-term follow-up. PMID- 8632031 TI - Bilateral common iliac artery aneurysms secondary to fibromuscular dysplasia accompanied with a coronary aneurysm. A case report. AB - Fibromuscular dysplasia (FMD) is found in various arteries but the common iliac arteries and the coronary arteries are seldom involved. A 49-year-old woman was referred to our hospital with a pulsatile mass in her right lower quadrant. She had subdural hemorrhage in a postpartum state at the age of 27. After admission angiography diagnosed the bilateral iliac artery aneurysms and coronary angiography revealed a coronary artery aneurysm, the size of which was 12 mm x 19 mm in the region of the left main trunk. The bilateral iliac artery aneurysms were resected and Y-shaped vascular prosthesis was replaced. Microscopic sections of the aneurysm showed remarkable decrease of elastic fibers and thinning of the media, but no increase occurred in the smooth muscle. Histopathological diagnosis was FMD (periarterial fibroplasia). To our knowledge, no patient with a iliac arterial aneurysm caused by FMD has been reported and only a few cases with coronary arterial FMD have been described. This is the first report of iliac arterial aneurysm due to FMD. Since this case has multiple aneurysms, a long-term follow-up is definitely required, especially focusing on the coronary aneurysm. PMID- 8632032 TI - Ruptured aneurysms of the internal iliac artery. Report of two cases. AB - Solitary internal iliac artery aneurysms are very rare. Their localisation deep in the pelvis makes diagnosis difficult. Therefore they are not detected until they reach a significant size and produce symptoms. These symptoms are due to compression on the adjacent structures or their rupture. Duplex scan, computerized tomography, and arteriography are the most useful diagnostic procedures. The correct surgical therapy consists of the ligature of the internal iliac artery and endoaneurysmorraphy with obliteration of the tributary branches. We report herein two cases of aneurysms of the internal iliac artery that made their clinical debut with signs of rupture. PMID- 8632034 TI - Resection of the aorta for leiomyosarcoma of the inferior vena cava. AB - Two cases of infra renal vena cava leiomyosarcoma are reported. Their locoregional extension resulted in a joint resection of the aorta and the vena cava. The follow-up of the two cases is of respectively 34 months and 32 months. We think that large resections are necessary, due to the microscopic characteristics of such tumors in order to try to improve the prognosis. PMID- 8632033 TI - Hepatic artery aneurysm: an ever present danger. AB - Hepatic artery aneurysms are infrequent vascular lesions, difficult to diagnose preoperatively manifesting themselves usually by acute rupture. We report our experience in both diagnosis and surgical treatment of 3 cases. The selective angiograph of the celiac tripod and of superior mesenteric artery, is an indispensable means able, not only to put forward a certain diagnosis, but also to supply precious notions on the possibilities of compensation on the side of the collateral circle and of consequence, to suggest operative tactics. Surgical management may range from the simple binding to the reconstruction of the hepatic arterial axis by means of prosthesis grafts and if possible to the simple excision and termino-terminal regraft. PMID- 8632035 TI - How to deal with carotid sling and to prevent its complications. PMID- 8632036 TI - Right post pneumonectomy pleural empyema caused by an esophagopleural fistula due to an esophageal carcinoma. AB - A case report of a patient with pleural empyema resulting from esophagopleural fistula following perforation of squamous cell carcinoma of the esophagus is presented. The patient had previously received a right pneumonectomy for bronchogenic carcinoma with the adjunct of postoperative radiotherapy. Description of the case, differential diagnosis and therapy of esophagopleural fistulas (EPF) as well as a review of the Literature is described and discussed. PMID- 8632037 TI - Bilateral intralobar pulmonary sequestration. AB - A 34-year-old male presented with an infected intralobar pulmonary sequestration of the left lower lobe. Aortography revealed bilateral anomalous systemic arteries, originating in the lower level of the descending thoracic aorta, to the lower lobe on each side. The portion of the right lower lobe, which was perfused by the anomalous systemic artery was seen otherwise normal in anatomy without any recognizable sequestered lung tissue. The patient underwent a left postero lateral thoracotomy on June 22, 1994. Each aberrant artery was recognized to take off from a common branch of the descending aorta at the level of the diaphragm. A left lower lobectomy with division of the left aberrant artery as well as ligation of the right anomalous artery were done. A postoperative pulmonary perfusion scan depicted normal uptake of radioactivity in the right lower lobe, suggesting normal pulmonary arterial perfusion to the area receiving previously the anomalous systemic arterial flow. An anomalous systemic artery perfusing an otherwise normal lung can be classified as one of the forms of intralobar pulmonary sequestration and could be ligated without resection of the involved area of the lung. PMID- 8632038 TI - Subcutaneous jugulofemoral bypass: a simple surgical option for palliation of superior vena cava obstruction. AB - BACKGROUND: Percutaneous placement of an intraluminal stent is usually a successful intervention for the disabling symptoms of Superior Vena Cava (SVC) obstruction. However, on occasion this may not be feasible and, as malignant disease is responsible for 90% of cases, the morbidity associated with median sternotomy or thoracotomy usually precludes surgical bypass. OBJECTIVE: To achieve good palliation of the symptoms of SVC obstruction by surgical bypass without performing sternotomy or thoracotomy. PATIENTS: Two patients with SVC obstruction secondary to lung cancer and a third after radiochemotherapy for malignant mediastinal teratoma. In all patients intraluminal stenting was considered but was not possible. METHODS: Jugulofemoral bypass was performed using long saphenous vein which was tunnelled subcutaneously from the femoral to the jugular vein. RESULTS: One patient required wound exploration for haemorrhage. Good palliation was achieved in all patients. One patient died 3 months post-operatively from lung cancer and the remaining two are alive without symptoms at 13 months and 6 weeks postoperatively. CONCLUSIONS: Though the majority of patients with SVC obstruction can be treated with non-surgical methods, subcutaneous jugulofemoral bypass may provide good palliation if these are not feasible. PMID- 8632039 TI - Infection, infidelity, and informed patients. PMID- 8632040 TI - Gag clauses in managed care. PMID- 8632041 TI - Acute renal failure in an elderly man taking warfarin. PMID- 8632042 TI - Lyme disease overdiagnosis: causes and cure. PMID- 8632044 TI - Case in point. Emphysematous cystitis. PMID- 8632043 TI - Management after a first myocardial infarction. AB - Treadmill exercise testing is the most important risk-stratifying technique, because of its ability to assess residual ischemia, left ventricular dysfunction, and a tendency toward arrhythmias. Cessation of smoking is the most important lifestyle change. Among prophylactic medications, aspirin can be considered for most patients, beta-blockers for many, and ACE inhibitors for some. PMID- 8632047 TI - Unexpected nephropathy. PMID- 8632046 TI - Chest pain and ST-segment elevation in a young man with cancer. PMID- 8632045 TI - Recurrent miscarriage: causes and management. AB - The workup is a challenge, as genetic, anatomic, endocrine, or immunologic factors may be involved. Although 60% of patients will have a successful pregnancy even without intervention, correctable causes should be sought. PMID- 8632048 TI - Recurrent gastrointestinal bleeding in a 21-year-old man. PMID- 8632049 TI - Osteoporosis: taking a fresh look. AB - Risk factors are being refined; old ones are being dropped and new ones are being added. Early detection-before fracture occurs-with bone-density assessment represents another important advance. Several strategies are now available to inhibit bone loss, and means are in prospect to promote bone formation directly. Of these, alendronate and slow-release fluoride appear to have particular promise. PMID- 8632051 TI - Recognizing post-polio syndrome. AB - The disorder consists of fatigue accompanied by new muscle weakness and muscle pain or, for patients whose acute polio had included bulbar involvement, new difficulty in swallowing or change in voice. The epidemiology remains unclear, fueling anxiety among polio survivors. Yet its course is not drastically progressive, and impairment is usually limited. PMID- 8632050 TI - Caring for the terminally Ill. AB - More than 50% of dying patients do not receive adequate symptomatic relief. Fear of hastening death is the primary reason for physicians' reluctance to prescribe high-dose pain medication. Yet the ethical principle of "double effect" clearly states that palliative care which results in respiratory depression is justified as long as the goal of management is relief of suffering, rather than death. PMID- 8632052 TI - Application of 6-hydroxydopamine into the fatpads surrounding the draining lymph nodes exacerbates adjuvant-induced arthritis. AB - Adjuvant-induced arthritis (AA) was examined in Lewis rats following local injection of 6-hydroxydopamine (6-OHDA) into the fatpads of the popliteal and inguinal lymph nodes which drain the hindlimbs (DLN). This method of 6-OHDA treatment resulted in noradrenergic (NA) denervation of DLN, spleen, and other organs in the peritoneal cavity, while sparing NA nerve fibers in the hindlimbs. Sympathectomy exacerbated the inflammation and osteopathic destruction of arthritic joints. Significant increases in dorsoplantar width in arthritic rats following denervation were observed by day 27 following immunization compared to nondenervated arthritic animals. Radiographic evaluation on day 27 after immunization confirmed the inflammation of soft tissue and revealed deterioration of bones of the ankle joint in both AA groups compared with the control groups; more extensive joint damage was apparent in arthritic rats following denervation compared to nondenervated arthritic rats. These findings suggest that the NA innervation of DLN and spleen (and possibly other organs of the peritoneal cavity) plays a regulatory role in the expression of AA. These data supports the hypothesis that absence of NA innervation in lymphoid organs during initiation, onset, and progression of the disease results in exacerbation of AA. PMID- 8632053 TI - Lymphocyte traffic changes induced by monolateral vagal denervation in mouse thymus and peripheral lymphoid organs. AB - In this report we show that after monolateral vagal denervation (vagotomy), performed at the cervical level, a transient effect, lasting about 24h, was produced on lymphocyte release from mouse thymus to peripheral lymphoid organs (spleen and lymph nodes). Labelling thymocytes in situ with fluorescein isothiocyanate (FITC) we note that the export of immature cells, CD4+CD8+, double positive (DP), and double negative, CD4-CD8- (DN), from the thymus was consistently increased 24 and 48 h after vagotomy. Double staining with anti-L3T4 (CD4) and anti-mouse CD8alpha showed that the number of DP and DN cells was significantly higher in both spleen and lymph nodes of vagotomized mice compared to controls (sham-operated), whereas the percentage of CD4+CD8- and CD8+CD4-, single positives (SP), was decreased. Considering thymic cellularity and apoptotic values, we exclude the non-specific effect of stress and suggest that this phenomenon could be in part due to a transient lack of the facilitating influence exerted by vagal efferent fibers on lymphocyte traffic at the cortico medullary junction of the thymic gland, where mature cells, SP, leave the thymus to enter systemic circulation. PMID- 8632054 TI - Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase. AB - This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases. PMID- 8632055 TI - Immunoreactive S100 proteins of blood immunocytes and brain cells. AB - Brain S100, an acidic protein with Ca2+-binding and neurotrophic properties, may be involved in the genesis of neurodegenerative diseases. Based on sharing of common antigens between the immune and nervous systems, we performed a comparative analysis of S100 in blood immunocytes (lymphocytes and monocytes) and brain cells. By using polyclonal antibodies to S100, an immunoreactive S100 was detected in human blood immunocytes and U373 human astrocytoma cells. The U373 cells contained a much higher level of S100 as compared to immunocytes, both cell types being compared at 1 X 10(6) cell concentration. Through protein immunoblotting, the immunocyte antigen was compared with pure S100 of bovine brain (authentic sample) and S100 of U373 cells and brain cells (human and mouse brain). The monomeric form of immunocyte-derived S100 was a low molecular mass (12-14kDa) protein, but slightly larger than authentic S100 (10.5 kDa) The S100 of U373 cells and brain cells was mainly a polymer (60-100 kDa), although the brain cells also showed a low molecular mass (10.5 kDa) band that corresponded to authentic S100. The molecular mass differences suggest that peripheral blood immunocytes contain an immunoreactive S100 that differs in size but is antigenically related to brain S100 family. PMID- 8632056 TI - Expression of high- and low-affinity neurotrophin receptors on human transformed B lymphocytes. AB - We performed high-sensitivity flow cytometry and Western blotting to study the expression of the low-affinity NGF receptor (p75NGFR) and of the transmembrane tyrosine kinase (Trk) family of high-affinity receptors for the different neurotrophic factors on Epstein-Barr virus (EBV)-transformed human B lymphocytes. Reverse transcriptase (RT) polymerase chain reaction (PCR) with single and multiple sets of primers (multiplex RT-PCR) was used to survey the repertoire of neurotrophin receptor transcripts in this cell line. We demonstrated that transformed B cells express detectable levels of Trk b and its mRNA. Conversely, negative results were obtained for p75NGFR, Trk a, and Trk c. Exposure of EBV transformed B lymphocytes to brain-derived neurotrophic factor (BDNF) triggered the phosphorylation of Trk b, as demonstrated by Western blots of cell lysates probed with monoclonal antibody against phosphotyrosine. PMID- 8632057 TI - Immunoregulatory effects of interferon-beta and interacting cytokines on human vascular endothelial cells. Implications for multiple sclerosis autoimmune diseases. AB - The mechanism(s) of action responsible for the anti-inflammatory effects mediated by interferon (IFN)-beta are still elusive although suggestions include anti viral effects, the enhancement of natural killer (NK) or suppressor T cell activity and opposition to the effects of inflammatory cytokines. As vascular endothelial cells are active participants in inflammatory and demyelinating processes, we decided to examine the effects of IFN-beta on the expression of major histocompatibility complex (MHC) gene products and intercellular adhesion molecule (ICAM)-1 on human vascular endothelial cells (ECs). Human umbilical ECs demonstrated constitutive expression of ICAM-1 and MHC class I molecules but did not express MHC class II molecules. Basal expression of ICAM-1 molecules was enhanced by TNF alpha and to a lesser extent by IFN-beta, but was not affected by IFN-gamma. MHC class I expression on ECs was enhanced by IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha. Furthermore, a synergistic effect was observed to combinations of these interacting cytokines. Incubation of ECs with IFN-gamma, but not IFN-beta, induced class II expression in a dose dependent manner. Moreover, co-incubation of ECs with IFN-beta and IFN-gamma resulted in significant down-regulation of class II molecules expression which was directly dependent on IFN-beta concentration. Northern blot analysis of DR alpha and Beta 2-microglobulin mRNA expression suggested that cytokine-mediated regulation of MHC molecules is at the transcriptional level, while modulation of ICAM-1 expression appears to be at the transcriptional as well as post-transcriptional level. Thus, our study demonstrated that IFN-beta and interacting cytokines exert complex immunoregulatory effects on endothelial cells with differential modulatory effects on various cell surface markers. Understanding the biological significance of these immunomodulatory effects mediated by IFN-beta may have important implications for cytokine-based strategies in the treatment of inflammatory and autoimmune diseases. PMID- 8632059 TI - Neutropenia in a patient receiving intravenous immune globulin. AB - A child with Guillain-Barre syndrome treated with intravenous immune globulin (IVIG) developed neutropenia (absolute neutrophil count = 390), which resolved 3 days after completion of the therapy. Potential mechanisms for the development of neutropenia during the use of IVIG therapy are discussed. In this case, testing of the IVIG used revealed the presence of a high concentration of anti-neutrophil antibodies compared to other samples. It is recommended that white blood cell and neutrophil counts be monitored daily during the use of such therapy. PMID- 8632058 TI - Virus encoding an encephalitogenic peptide protects mice from experimental allergic encephalomyelitis. AB - The association of viral infections with autoimmune central nervous system (CNS) diseases such as post-infectious encephalomyelitis and possibly multiple sclerosis (MS) prompted the investigation to understand how virus infection could modulate autoimmune responses. Recombinant vaccinia viruses encoding an encephalitogenic portion of myelin basic protein (MBP) were evaluated in an animal model for human demyelinating disease, experimental allergic encephalomyelitis (EAE). We have determined that mice vaccinated with recombinant viruses encoding an encephalitogenic region of MBP were protected from EAE. In vivo depletion of CD8+ T cells did not abrogate this protection, suggesting lack of regulation by this cell type. These studies demonstrate that virus infection may be a means to modulated immune responsiveness to CNS disease. PMID- 8632060 TI - Modulation of acetylcholine receptor function in TE671 (rhabdomyosarcoma) cells by non-AChR ligands: possible relevance to seronegative myasthenia gravis. AB - The acetylcholine receptor (AChR) is the main target antigen in myasthenia gravis (MG), but about 15% of patients with typical immunologically mediated MG do not have detectable anti-AChR antibodies. Previous studies showed that plasma from these 'seronegative' patients (SNMG) reduced AChR function in the human AChR expressing TE671 cell line, and it was proposed that SNMG plasmas may act indirectly via phosphorylation of AChR. We show here that substances such as the beta 2-adrenergic agonist, salbutamol, calcitonin-gene-related-peptide (CGRP), and cholera toxin, that increase intracellular cAMP via binding to specific cell surface receptors, reduced AChR function in TE671 cells. Moreover, non-specific activation of cell surface proteins by lectins achieved similar results. These observations lead us to hypothesise that SNMG immunoglobulins act in TE671 cells by cross-linking of specific cell surface antigen(s) resulting in generation of intracellular cAMP and/or other second messengers. The role of such antibodies at the neuromuscular junction in vivo could be reduction in AChR function by desensitization and/or damage to the postsynaptic membrane following complement activation. PMID- 8632061 TI - Serum and cerebrospinal fluid levels of soluble adhesion molecules in multiple sclerosis: predominant intrathecal release of vascular cell adhesion molecule-1. AB - Activated cerebral vascular endothelial cells express leukocyte, vascular cell, and intracellular adhesion molecules (E-selectin, VCAM-1 and ICAM-1) which facilitate leukocyte adhesion to endothelium and migration into inflammatory lesions. Paired serum and cerebrospinal fluid (CSF) levels of soluble (s) E selectin, sVCAM-1 and sICAM-1 were determined by ELISA in patients with clinically definite MS in relapse, and patients with other inflammatory (IND) and non-inflammatory neurological disease (NIND). CSF levels of sVCAM-1 and sICAM-1 were significantly increased in MS patients compared to IND and NIND patients. Elevation of CSF sVCAM-1 in MS patients was the most marked finding (P = 0.0001) and an increased sVCAM-1 index indicated that this was due to intrathecal release of sVCAM-1. There were no differences in serum and CSF sE-selectin levels between the study groups. Measurement of the sVCAM-1 index may provide a marker of disease activity in patients with clinically definite MS. PMID- 8632062 TI - Pentoxifylline, a phosphodiesterase inhibitor, induces immune deviation in patients with multiple sclerosis. AB - The outcome of immune responses can be predicted by the lymphokine production pattern of the participating cells. Cytokines of the T helper type 1 (Th1) cells mediate inflammatory responses and delayed-type hypersensitivity (DTH), whereas Th2-like T cells predominantly produce cytokines, which stimulate antibody production by B cells. Immunoregulatory therapy of autoimmune diseases with unknown antigens may be achieved by inhibiting the production of inflammatory cytokines and induction of protective cytokines of Th2-like T cells. To determine the immunoregulatory capacity of the phosphodiesterase inhibitor pentoxifylline (PTX), which is known to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), this drug was used in mitogen and antigen-stimulated lymphocyte cultures as well as in patients with multiple sclerosis. PTX significantly decreased TNF-alpha and interleukin-12 (IL-12), whereas it increased IL-4 and IL-10 production. In addition, PTX inhibited cell proliferation, which was associated with a marked reduction in CD25 (IL-2 receptor alpha-chain) and CD54 (intercellular adhesion molecule-1; ICAM-1) expression. Increasing doses of PTX significantly reduced TNF-alpha and IL-12 mRNA expression of blood mononuclear cells, but increased IL-4 and IL-10 expression in eight patients with relapsing-remitting multiple sclerosis. These results indicate that PTX modulates immune reactions favouring a Th2-like response and may therefore be useful for the treatment of autoimmune diseases with a dominant Th1-like T cell response. PMID- 8632063 TI - Murine encephalitogenic lymphoid cells induce nitric oxide synthase in primary astrocytes. AB - A cytokine-inducible form of nitric oxide synthase (iNOS), capable of producing large quantities of nitric oxide (NO), can be induced in many cell types. We demonstrate that conditioned medium from encephalitogenic myelin basic protein sensitized lymphoid cells (MBP-CM) induces the expression of iNOS in primary cultures of murine astrocytes in a time- and concentration-dependent manner. iNOS mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) as early as 3 h post-exposure. Accumulation of nitrite into the astrocyte culture medium, an indirect measure of NO, was measurable 3 h post-exposure, plateaued at 24 h, and was prevented by the simultaneous administration of the NOS inhibitors, L-N(G)-nitroarginine methyl ester, N(G)-nitro-L-arginine or aminoguanidine. Astrocyte expression of iNOS protein, detected by immunohistochemistry and immunoprecipitation/Western blot, was prevented by inhibitors of RNA or protein metabolism, consistent with its dependence on de novo protein synthesis. PMID- 8632064 TI - Copolymer 1 inhibits chronic relapsing experimental allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice and interferes with PLP specific T cell responses. AB - Copolymer 1 (Cop 1) is a synthetic amino acid copolymer effective in suppression of experimental allergic encephalomyelitis (EAE) and developed as a candidate drug for multiple sclerosis (MS). In the present study, we induced chronic relapsing (CR)-EAE in (SJL/J X BALB/c)F1 mice by either whole spinal cord homogenate or two synthetic peptides of proteolipid protein (PLP), p139-151 and p178-191. When Cop 1 was added to the encephalitogenic inoculum, mice were almost completely resistant to disease induction. T cell lines to p139-151 and p178-191 were specific to these peptides. Their antigen-specific responses were inhibited by Cop 1 in a dose-dependent manner, while their polyclonal response to the superantigen staphylococcal enterotoxin A (SEA) was not affected by Cop 1. Using biotinylated PLP derivatives, we demonstrated that the two PLP peptides bound to I-A(s) molecules, and that their binding was completely inhibited by unlabelled Cop 1. Furthermore, Cop 1 could displace the PLP peptides from the MHC binding site. These results support the potential of Cop 1 as a broad-spectrum drug for MS. PMID- 8632065 TI - Cutaneous manifestations and consequences of smoking. AB - Cigarette smoking is strongly linked to serious internal diseases such as cancer, cardiovascular disease, and lung disease. However, the external manifestations and consequences of smoking are relatively unknown. Although generally less ominous, the cutaneous manifestations of smoking may be associated with significant morbidity. This article reviews the known adverse effects on the skin of smoking. PMID- 8632066 TI - Expression of a glycoprotein of the carcinoembryonic antigen family in normal and neoplastic sebaceous glands. Limited role of carcinoembryonic antigen as a sweat gland marker. AB - BACKGROUND: Carcinoembryonic antigen (CEA) is a well-known marker for sweat gland differentiation in adnexal neoplasms. OBJECTIVE: The aim of this study was to examine the expression of glycoproteins of the CEA family, that is, CEA-180, nonspecific cross-reacting antigens (NCAs), and biliary glycoprotein (BGP), in sebaceous glands and in neoplasms with sebaceous differentiation. METHODS: Normal adult and fetal skin, hyperplasias, hamartomas, and neoplasms with sebaceous or follicular differentiation were stained immunohistochemically with a panel of polyclonal and monoclonal antibodies highly specific for CEA-180, NCAs, and BGP. Double immunostaining was performed to correlate the CEA expression with that of epithelial membrane antigen (EMA), a glycoprotein consistently found in differentiating sebocytes. RESULTS: Whereas sweat glands coexpressed CEA, NCAs, BGP, and EMA, sebaceous glands were exclusively labeled with the antibodies recognizing BGP or EMA. Staining of the sebaceous glands was restricted to mature sebocytes, sparing immature cells. At the ultrastructural level immunoreactivity for BGP and EMA was demonstrable in the golgi area, in small vesicles, and along the cell membranes. During fetal development BGP was not found until the sebaceous glands matured. The expression of BGP and EMA was highly conserved in reactive, hamartomatous, and neoplastic proliferations of adnexal structures with sebaceous differentiation. CONCLUSION: The expression of BGP, a CEA glycoprotein, in differentiating sebocytes accounts for the reactivity of many anti-CEA antibodies with sebaceous glands and thus disqualifies the CEA family as a monospecific marker for sweat gland differentiation. PMID- 8632067 TI - Behcet's disease. Report of twenty-five patients from the United States with prominent mucocutaneous involvement. AB - BACKGROUND: Behcet's disease is a multisystem disease that is rare in the United States. OBJECTIVE: The purpose of our study was to assess the characteristics and treatment of a series of patients with Behcet's disease in the United States. METHODS: A retrospective clinical review of 25 patients with Behcet's disease was performed, and histopathologic findings and therapeutic modalities were reviewed. RESULTS: All patients had oral and genital aphthae, and 22 of 25 patients had cutaneous lesions consistent with Behcet's disease. Eight of 25 patients had relatively severe systemic disease. Nine of 14 biopsy specimens showed a neutrophilic vascular reaction. Our therapeutic "ladder" included aggressive topical and intralesional corticosteroids, colchicine, dapsone, methotrexate, and thalidomide; we reserved systemic corticosteroids and immunosuppressive medications for severe ocular or severe systemic disease. CONCLUSION: This series of patients with Behcet's disease was characterized by patients with prominent mucocutaneous involvement and a low prevalence of ocular involvement. These findings may be attributed to patient selection from referral to a university dermatology clinic. PMID- 8632068 TI - Pigmented basal cell carcinoma in Hispanics. AB - BACKGROUND: Pigmented basal cell carcinoma (PBCC) may occasionally be misdiagnosed as melanoma. In the Hispanic population, PBCC is common. OBJECTIVE: We attempted to determine the prevalence of PBCC in a Hispanic population. METHODS: A randomized, blinded, retrospective study was designed to assess histologic slides for the presence of microscopic pigment. Basal cell carcinoma (BCCs) from 30 patients with a Hispanic surname were compared histologically with BCCs from 30 patients with a northern European surname. In the prospective phase of the study, 15 Hispanic and 44 non-Hispanic patients with clinically suspected BCC or PBCC completed a questionnaire about their ethnic background and skin type to determine whether PBCC is more common in Hispanics. RESULTS: Pigment was identified twice as frequently in BCCs from patients with a Hispanic surname than in BCCs from patients with a northern European surname. In the prospective clinical study, 66% of clinically diagnosed PBCCs were found in Hispanic patients, whereas only 11% of nonpigmented BCCs came from Hispanic patients (p < 0.01). CONCLUSION: In patients with a BCC, PBCCs are more common in Hispanics than non-Hispanics. This may reflect an increased incidence of PBCCs in the Hispanic population. PMID- 8632070 TI - The occurrence of atopic dermatitis in north Europe: an international questionnaire study. AB - BACKGROUND: Atopic dermatitis is a common multifactorial disease that seems to be increasing in frequency. OBJECTIVE: Our purpose was to determine the increased prevalence of atopic dermatitis in North Europe. METHODS: Approximately 3000 7 year-old children in Denmark, Germany, and Sweden were enrolled in a cross sectional questionnaire study that was conducted in the autumn of 1992 with common protocols and standardized procedures. RESULTS: The response rate was 90%. The frequency of atopic dermatitis was calculated to be 15.6% (95% confidence interval 14.2% to 17%) with some regional differences. Girls more often had flexural eczema and outnumbered boys in a ratio of 1.3:1.0. Boys more often had a personal history of asthma, whereas girls more often had a family history of asthma. CONCLUSION: The increasing prevalence of atopic dermatitis has been confirmed. This simple questionnaire model works well, is cost effective, and is sufficiently sensitive and specific to conduct large-scale epidemiologic investigations in school children. PMID- 8632069 TI - Cutaneous extravascular necrotizing granuloma (Winkelmann granuloma): confirmation of the association with systemic disease. AB - BACKGROUND: An unusual palisading granuloma has been described in patients with immunoreactive diseases. Multiple names have been given to this lesion. OBJECTIVE: Our aim was to verify whether a distinct palisading granuloma can be used as a marker for systemic disease. We also propose unifying nomenclature. METHODS: Thirty-four biopsy specimens from 22 patients were selected for study on the basis of histologic criteria. The medical histories of these patients were subsequently reviewed for clinical information. RESULTS: At least 21 of the 22 patients with cutaneous extravascular necrotizing granuloma had evidence of an underlying immunoreactive systemic illness. In each, the systemic disease preceded or was diagnosed concurrently with the cutaneous lesions. CONCLUSION: The cutaneous extravascular necrotizing granuloma has unique clinical and histologic features. In a great majority of cases, a systemic immunoreactive disease is present. PMID- 8632071 TI - Nail matrix nevi: a clinical and histopathologic study of twenty-two patients. AB - BACKGROUND: Because most dermatologists do not regularly perform biopsies of longitudinal melanonychia, even when the pigmentation presents as a single band, the true prevalence of nail matrix nevi is unknown. OBJECTIVE: Our purpose was to determine the prevalence of nail matrix nevi in white patients with longitudinal melanonychia involving a single digit and to determine whether longitudinal melanonychia caused by a nail matrix nevus can be clinically distinguished from longitudinal melanonychia from other causes. METHODS: From January 1989 to December 1994 we performed a nail biopsy on 100 of 128 consecutive white patients who had a single band of "idiopathic" longitudinal melanonychia. RESULTS: A nail matrix nevus was detected in 22 patients. A junctional nevus was found in 19 specimens and a compound nevus in three specimens. CONCLUSION: Nail matrix nevi in Caucasian patients are uncommon but not exceptional. The number of nevi presenting with longitudinal melanonychia exceeded that of melanoma. The diagnosis of nail matrix nevi is impossible clinically and always requires histopathologic study. The pathologic features of nail matrix nevi are similar to those of skin nevi except for their architectural pattern, which reflects the peculiar anatomy of the nail unit. PMID- 8632072 TI - Lymphocyte counts of patients who have had skin cancer. AB - BACKGROUND: Investigations of lymphocyte counts in patients with skin cancer have given conflicting results, possibly because homogeneous groups of patients were not studied. OBJECTIVE: Our purpose was to measure lymphocyte counts in patients with skin cancer to determine whether any abnormalities were associated with the number of cancers removed and to determine whether a lymphocyte count could identify patients at risk of the development of large numbers of cancers. METHODS: Apparently otherwise normal patients who had histologically confirmed skin cancers removed were studied. One group consisted of patients who had one skin cancer removed but had not had another within a minimum of 5 years. The other group consisted of patients who had had three or more skin cancers. Standard flow cytometry was used to determine the total lymphocyte count, CD4 (helper cell) count, and CD8 (cytotoxic cell) count. RESULTS: Ninety-six patients with multiple skin cancers, and 24 with one skin cancer were studied. Only basal cell carcinomas (BCCs) were removed from 84 patients and the results from this homogeneous group were as follows: women had a higher CD4 cell count than men (p < 0.05); patients with 20 or more BCCs had a lower lymphocyte count (p < 0.01); and patients with one BCC had a higher CD4/CD8 ratio than those who had multiple BCCs (p < 0.05). CONCLUSION: Differences were found between men and women, as well as between subgroups of patients with skin cancer. However, the range of lymphocyte counts was large and it was not possible to determine a threshold below which patients had a worse prognosis. A lymphocyte count is not a reliable way of predicting which patients will have a large number of skin cancers. PMID- 8632073 TI - IgG anti-50 kd lymphocyte membrane peptide antibody in patients with Sezary syndrome. AB - BACKGROUND: Recent evidence suggests that as cutaneous T-cell lymphoma progresses, cell-mediated immunity is reduced and humoral responses are augmented. OBJECTIVE: The present study was designed to compare the IgG response in Sezary syndrome with that in mycosis fungoides. METHODS: The IgG antilymphocyte response was studied in six patients with Sezary syndrome and in 11 patients with mycosis fungoides by means of immunoblot analysis, enzyme-linked immunosorbent assay, immunohistochemistry, and flow cytometry. RESULTS: An IgG antilymphocyte response to a 50 kd peptide was seen in five of the six patients with Sezary syndrome; however, none of the 11 patients with mycosis fungoides expressed this response. CONCLUSIONS: An enhanced IgG immune response to 50 kd lymphocyte peptide may be helpful in identifying disease progression in patients with cutaneous T-cell lymphoma. PMID- 8632074 TI - Colchicine for epidermolysis bullosa acquisita. AB - BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a chronic subepidermal blistering disease that is difficult to treat. Recently one patient with severe EBA was described who responded dramatically to colchicine. OBJECTIVE: Our purpose was to determine the efficacy of colchicine in the treatment of EBA. METHODS: Four patients with severe EBA refractory to conventional therapy were treated with colchicine 0.6 to 1.5 mg a day for up to 4 years. RESULTS: In all four patients the lessening of skin fragility and the decrease in spontaneous blister formation were dramatic; few side effects were noted. CONCLUSION: Colchicine should be considered in the treatment of EBA. PMID- 8632075 TI - A double-blind, randomized, placebo-controlled evaluation of short-term treatment with oral itraconazole in patients with tinea versicolor. AB - BACKGROUND: The use of short-term oral azoles is an alternative to topical therapy in patients with tinea versicolor. OBJECTIVE: We compared the efficacy and safety of oral itraconazole with that of placebo in 36 patients with mycologically proven tinea versicolor. METHODS: Patients were randomly assigned to 7 days of treatment with either itraconazole, 200 mg once daily, or placebo. A potassium hydroxide examination and assessment of scaling, erythema, pruritus, and global condition were performed at baseline and at 4 weeks after treatment. RESULTS: The itraconazole-treated group demonstrated significant improvement over both baseline (p < 0.01) and placebo (p < 0.02) in scaling, erythema, and pruritus. Sixty-seven percent of itraconazole-treated patients were free of symptoms at week 5, as compared with 12% of placebo-treated patients. Ninety-four percent of itraconazole-treated patients were considered to be healed or markedly improved at the study's end point compared with 6% of placebo-treated patients (p < 0.01). A total of 89% in the itraconazole-treated group had a negative potassium hydroxide examination at the follow-up visit compared with 6% in the placebo-treated group (p < 0.01). There was a single report of a possibly treatment-related adverse event in each treatment group. CONCLUSION: Short-term treatment with itraconazole is effective and well tolerated in the management of tinea versicolor. PMID- 8632076 TI - Botulinum A exotoxin use in clinical dermatology. AB - Botulinum toxin (BT) prevents the release of acetylcholine at the neuromuscular junction and produces reversible paralysis of striated muscle. This effect was initially used for ophthalmologic indications, particularly strabismus. The idea of using the toxin for purely cosmetic purposes arose from the observation that therapy for facial dystonias markedly decreased lines and wrinkles in the treated areas. The ease of application, high safety profile, and impressive results from the cosmetic use of BT have led to its rapid acceptance by many clinicians. As use of BT increases and its indications expand, an understanding of this therapeutic modality is timely. This article discusses the pharmacologic aspects of BT as well as the methods for administration as they relate to its cosmetic use. PMID- 8632077 TI - Favorable prognostic factors in recurrent and metastatic melanoma. AB - Survival among patients with recurrent and metastatic melanoma varies widely. Several clinical and pathologic variables correlate with improved survival. Awareness of these favorable prognostic characteristics should assist in patient counseling and help identify those who may benefit from more aggressive therapeutic intervention. PMID- 8632078 TI - Kaposi's sarcoma: advances and perspectives. AB - Kaposi's sarcoma (KS) is an unusual neoplasm that has proved to be an enigma in many ways since its original description by Kaposi in 1872. Its epidemiology has stimulated tremendous interest, amplified markedly in 1981 when it became known as an original defining part of the complex of immune disorders now known as AIDS. The cell of origin, etiology, and therapy for both AIDS-associated and AIDS unassociated KS continue as matters of intense investigation. In fact, whether it is a reactive hyperplasia or a true malignancy is still a matter of debate, as is the concept of multicentricity versus metastases. Epidemiologic studies suggest that a separate agent apart from HIV-1 may cause KS. A newly postulated KS associated herpes virus may be linked. The role of the HIV-1 tat gene product, basic fibroblast growth factor, scatter factor, oncostatin M, and other factors that regulate the growth of KS cells are discussed, as well as therapeutic options. PMID- 8632080 TI - Proceedings from the national conference to develop a national skin cancer agenda. American Academy of Dermatology and Centers for Disease Control and Prevention, April 8-10, 1995. PMID- 8632081 TI - Guidelines of care for dermatomyositis. American Academy of Dermatology. PMID- 8632079 TI - Surgical pearl: extra-rapid tourniquet for single digit surgery. PMID- 8632082 TI - Guidelines of care for cutaneous lupus erythematosus. American Academy of Dermatology. PMID- 8632083 TI - Melanoma Self-examination Day: Melanoma Monday, May 1, 1995. PMID- 8632084 TI - The incidence of malignant melanoma in the United States: issues as we approach the 21st century. AB - The risk of malignant melanoma developing in an American in the United States has now reached 1 in 87 (up more than 1800% since the 1930s). This rising incidence of malignant melanoma is, in fact, real because (1) it is not due to increased surveillance; (2) it is not due to better cancer-counting methods in general; (3) it is not due to changes in histologic diagnostic criteria; (4) it is being noted worldwide; and (5) most importantly, despite rising survival percentages, the mortality rate from malignant melanoma also continues to rise. On the basis of these trends, incidence rates for malignant melanoma will continue to rise for at least the next 10 to 20 years, although the demographics of those affected may change. Effective programs to improve public and professional education must be developed to enhance early clinical detection and behavioral changes. An establishment of a National Melanoma Registry is needed to more effectively assess the magnitude and impact of future incidence and the success of prevention program efforts into the next century. PMID- 8632085 TI - Ultrapulse CO2 laser ablation of xanthelasma. PMID- 8632086 TI - Severe granulomatous reaction and facial ulceration occurring after subcutaneous silicone injection. PMID- 8632087 TI - IgA pemphigus: report of a case with immunoelectron localization of bound IgA in the skin. PMID- 8632088 TI - Recurrent exacerbation of acne by granulocyte colony-stimulating factor administration. PMID- 8632089 TI - Bullous solar elastosis. PMID- 8632090 TI - Progressive symmetric erythrokeratodermia. PMID- 8632091 TI - Hair darkening and new growth associated with etretinate therapy. PMID- 8632092 TI - Melanocytic nevi with focal atypical epithelioid cell components. PMID- 8632093 TI - A case of deep tufted angioma. PMID- 8632095 TI - Drug-induced progesterone dermatitis. PMID- 8632094 TI - Clinical trials of the new antifungal drug therapies. PMID- 8632096 TI - The long-term impact on nursing students of participating in international education. AB - Internationalizing the nursing curriculum is essential to the education of the nurse professional prepared for the rapidly changing challenges of the 21st century. Despite recognition of this essential need, the long-term effects of international education on nursing students have not been examined. The purpose of this study was to describe the long-term impact of study abroad experiences on baccalaureate graduates. Using a descriptive survey design, data were collected from 27 alumni (88 per cent response rate) who completed the International Education Survey. Although the impact was found to decrease over time, respondents reported the highest impact in enhanced international perspective and increased personal development; lower impact was reported in the professional nurse role and intellectual development dimensions. Students who participated in longer programs (12 to 16 weeks) reported higher long-term impact than those participating in 3- to 4-week programs. Respondents' age at the time of the international education was positively correlated with personal development. No association was found between the respondents' year in college in which they participated and reported long-term impact. This study is a contribution to the efforts of those who are committed to making nursing education relevant in a global society. PMID- 8632097 TI - Transforming curriculum for a nursing leadership course: a collaborative approach. AB - The purpose of this project was to strengthen the transformational leadership competence of students by incorporating perceptions of all course participants in the curriculum of a nursing leadership course. Students and faculty collectively assessed each other's ability to communicate, associate, sanction, delegate, initiate, and achieve external legitimacy by completing a Leadership Performance Competence Profile. Profile results guided the teaching strategies and activities used during the course. At the beginning of the course the sanction competence was perceived as a mutual weakness; at the end of the course perceptions of all six leadership competencies were stronger. End-of-course student satisfaction was also examined. This project suggests collaborative teaching strategies can enhance perceptions of competence and satisfaction of course participants. PMID- 8632098 TI - Interdisciplinary education and practice. American Association of Colleges of Nursing. PMID- 8632100 TI - Nurse executive. Opportunities in the land of plenty. PMID- 8632099 TI - Critical caring: can faculty do it? PMID- 8632101 TI - Professional practice. Feel free. PMID- 8632103 TI - Research. Research traditions--a decade of progress. PMID- 8632102 TI - Public policy. A values orientation to health care policy. PMID- 8632104 TI - The budget process in schools of nursing: a primer for the novice administrator. AB - All administrators are expected to be competent in budget and financial management. Novice administrators of schools of nursing are expected to know about the budgetary process, budgeting techniques, and the various types of budgets that can be used, such as the open-ended budget, incremental budget, alternate-level budget, quota budget, formula budget, intramural budget, zero based budget, and cost center budget. In addition, administrators are expected to know what key questions need to be asked about how the budget is structured and revenue sources and how to manage and evaluate their budgets. PMID- 8632105 TI - Past-present-future messages for nursing leadership. AB - Past-present-future messages for nursing leadership were derived from the personal perspectives of contemporary outstanding nurses in the United States. Based on professional life histories provided by 40 participating members of the American Academy of Nursing in its early years, a composite profile of these contemporary nurse leaders emerged. From their stories, rich in discovery of meaning, past-present-future messages are posited with futuristic hints to guide the education of nursing leaders into the 21st century. PMID- 8632106 TI - Roles and responsibilities of clinical nurse researchers. AB - A follow-up survey of 142 nurse researchers employed in clinical settings (NRECS) was conducted 10 years after the first one conducted by Knafl, Bevis, and Kirchhoff in which only 34 individuals qualified for inclusion. An 80-item questionnaire included items about the structure of the position, processes used, variables that may influence outcomes, and outside activities. When ineligible persons were excluded, the response rate was 75 per cent. Most commonly NRECS had positions in clinical settings only (55.7 per cent), offices (75.5 per cent), some staff (72.6 per cent), and secretarial support (52.8 per cent), and they usually reported to the chief nurse executives (71.7 per cent). Although the majority of NRECS reported responsibility for research activities, the average time spent on research is only 50 per cent. Most (82 per cent) have a nursing research committee, but NRECS also sit on other research-related committees in the department or hospital. Details about salary, responsibilities, and processes will be helpful to those preparing themselves or others for this role, for those who wish to start such a position for themselves or another, or for those in the role wanting to know how other NRECS perform. PMID- 8632107 TI - Constraints that nursing program administrators encounter in promoting faculty change and development. AB - The purpose of this study was to investigate the constraints that nursing program administrators encounter in promoting faculty change and development. The underlying theoretical framework was based on the concepts of change and innovation presented by Lunde and Hartung, Downs and Mohr, and Kanter. Administrators of baccalaureate schools of nursing (n = 271) who were members of the American Association of Colleges of Nursing participated in this study. All participants answered a questionnaire on types of development activities provided, beliefs regarding faculty development, and constraints in providing faculty development. Descriptive statistics, cross-tabulation, and regression analysis were used to analyze data. Development activities reported ranged from sabbaticals to providing centers for faculty development. Lack of research skills in master's degree--prepared faculty was determined to be the greatest perceived limitation. Available resources, perceived faculty responsibility for own development, and perceived faculty interest were found to be predictive of provision of development activities. Results of this study suggest that availability of resources affects faculty development as does the perception by administration that faculty need to show interest and assume personal responsibility for professional development. PMID- 8632108 TI - Nurses' attitudes toward caring for patients with acquired immunodeficiency syndrome. AB - The purpose of this study was to examine nurses' attitudes toward caring for patients with acquired immunodeficiency syndrome (AIDS) and factors that might be related to these attitudes. One hundred thirty-eight (138) registered nurses responded to an anonymous mail survey. The survey instrument assessed the subjects' attitudes toward caring for AIDS patients along five dimensions: (1) fears and concerns relating to caring for AIDS patients, (2) attitudes toward health care utilization by AIDS patients, (3) attitudes toward caring for terminally ill patients, (4) attitudes toward homosexuality, and (5) attitudes of significant others toward AIDS patients. The data obtained were analyzed using frequency distributions, independent t tests, and analysis of variance. Results of this study indicated that educational background is not related to nurses' attitudes toward caring for patients with AIDS; however, nurses who have had experience caring for patients with AIDS have more positive attitudes towards health care utilization by AIDS patients (P < .05) and more positive attitudes toward homosexuality (P < .04) than nurses who have not cared for AIDS patients. PMID- 8632109 TI - The lack of influence of long-term potassium citrate and calcium citrate treatment in total body aluminum burden in patients with functioning kidneys. AB - BACKGROUND: It has been suggested that citrate salts might enhance aluminum (Al) absorption from a normal diet, posing a threat of Al toxicity even in subjects with normal renal function. We have recently reported that in normal subjects and patients with moderate renal failure, short-term treatment with tricalcium dicitrate (Ca3Cit2) does not significantly change urinary and serum Al levels. However, we have not assessed total body Al stores in patients on long-term citrate treatment. OBJECTIVE: The objective of this study was to ascertain body content of Al non-invasively using the increment in serum and urinary Al following the intravenous administration of deferoxamine (DFO) in patients with kidney stones and osteoporotic women undergoing long-term treatment with potassium citrate (K3Cit) or Ca3Cit2, respectively. METHODS: Ten patients with calcium nephrolithiasis and five with osteoporosis who were maintained on potassium citrate (40 mEq/day or more) or calcium citrate 800 mg calcium/day (40 mEq citrate) for 2 to 8 years, respectively, and 16 normal volunteers without a history of regular aluminum-containing antacid use participated in the study. All participants completed the 8 days of study, during which they were maintained on their regular home diet. Urinary Al excretion was measured during a two-day baseline before (Days 5, 6) and for 1 day (Day 7) immediately following a single intravenous dose of DFO (40 mg/kg). Blood for Al was obtained before DFO administration, and at 2, 5 and 24 hours following the start of the infusion. RESULTS: The median 24-hour urinary Al excretion (microgram/day) at baseline versus post-DFO value was 15.9 vs. 44.4 in the normal subjects and 13.3 vs. 35.7 in the patients. These values were all within normal limits and did not change significantly following DFO infusion (p = 0.003 and p = 0.0001, respectively). The median change of 17.1 micrograms/day in urinary Al in the normal subjects was not significantly different from the 18.7 micrograms/day change measured in the patient group (p = 0.30). Similarly, no change in the mean serum Al was detected at any time following the DFO infusion, either in the patient or control group (patients 4.1 to 4.3 ng/ml, controls 7.4 to 4.6 ng/ml). CONCLUSION: The results suggest that abnormal total body retention of Al does not occur during long-term citrate treatment in patients with functioning kidneys. PMID- 8632111 TI - Association of macronutrients and energy intake with hypertension. AB - Hypertension, a major public health problem, becomes more prevalent during aging. Epidemiological studies suggest that environmental factors such as nutrition may play a major role in blood pressure (BP) regulation. It is generally accepted that obesity and sodium/alcohol consumption are important factors, and many believe that calcium, magnesium and potassium consumption are regulatory as well. Less emphasis has been placed on whether macronutrients influence blood pressure significantly. This review focused on the ability of excess calories and consumption of carbohydrates, fats, and proteins to regulate blood pressure. PMID- 8632110 TI - Vitamin and mineral deficiencies which may predispose to glucose intolerance of pregnancy. AB - There is an increased requirement for nutrients in normal pregnancy, not only due to increased demand, but also increased loss. There is also an increased insulin resistant state during pregnancy mediated by the placental anti-insulin hormones estrogen, progesterone, human somatomammotropin; the pituitary hormone prolactin; and the adrenal hormone, cortisol. If the maternal pancreas cannot increase production of insulin of sustain normoglycemia despite these anti-insulin hormones, gestational diabetes occurs. Gestational diabetes is associated with excessive nutrient losses due to glycosuria. Specific nutrient deficiencies of chromium, magnesium, potassium and pyridoxine may potentiate the tendency towards hyperglycemia in gestational diabetic women because each of these four deficiencies causes impairment of pancreatic insulin production. This review describes the pathophysiology of the hyperglycemia and the nutrient loss in gestational diabetes and further postulates the mechanism whereby vitamin/mineral supplementation may be useful to prevent or ameliorate pregnancy-related glucose intolerance. PMID- 8632112 TI - Serum ionized magnesium and other electrolytes in the antenatal period of human pregnancy. AB - OBJECTIVE: To determine: 1) the concentrations and fractions of serum ionized magnesium (IMg2+) in normal antenatal patients and 2) whether they are altered by concentrations of other cations and serum proteins. DESIGN: A cross sectional study design. SETTING: An urban community hospital obstetrics clinic. SUBJECTS: 144 normal antenatal patients divided into four gestational age groups: (1) 25 at 6 to 12 weeks, (2) 55 at 13 to 20 weeks, (3) 37 at 21 to 27 weeks, and (4) 27 at 28 to 38 weeks. INTERVENTIONS: Utilizing ion selective electrodes, we determined concentrations and fractions of ionized magnesium, concentrations of ionized calcium, sodium, and potassium, and the pH level in the venous serum of the patients. Total magnesium (TMg), total calcium, total protein, albumin, and inorganic phosphorus were measured. MEASURES OF OUTCOME: The mean concentrations and fractions were compared between the groups. Correlations between concentrations or fractions of Mg and concentrations of the other cations or proteins were determined. RESULTS: All values are means+/-SD. IMg2+ concentrations are 0.53+/-0.03 mmol/L in the first trimester and are lowest (0.49+/-0.05 mmol/L, p<0.001) by the third trimester. The percent of ionized magnesium remains at approximately 66% in each antenatal group. Although mean Mg concentrations fall with advancing gestational age, the main determinant of the IMg2+ concentration in an individual patient is her TMg concentration. CONCLUSIONS: IMg2+ concentrations and fractions remain within a very narrow range during pregnancy and are minimally affected by physiologic hemodilution. We hypothesize that mobilization of intracellular or bone stores of magnesium helps maintain serum concentrations, thus placing a stress on magnesium balance. PMID- 8632113 TI - The ion-selective magnesium electrode: a new tool for clinicians and investigators. PMID- 8632114 TI - Vitamin E and vitamin E-quinone levels in red blood cells and plasma of newborn infants and their mothers. AB - OBJECTIVE: Vitamin E is a physiological antioxidant and protects cell membranes from oxidative damage. This study has determined whether vitamin E level in RBC of newborns has any relationship with its level in their mothers. We have also examined levels of vitamin E and vitamin E-quinone, an oxidized product of vitamin E, in paired samples of red blood cells (RBC) and plasma of newborns and their mothers. METHODS: Blood was collected from 26 mothers and their full-term placental cords at delivery. Vitamin E and vitamin E-quinone levels were determined in RBC and plasma by HPLC. RESULTS: Newborn-plasma had significantly lower vitamin E levels compared with maternal-plasma both when expressed as nmole/ml (5.5+/-0.4 vs 26.1+/-1.1, p = 0.0001) or nmole/mumole total lipids (1.9+/-0.1 vs 2.6+/-0.1, p = 0.0001). Vitamin E level in the newborn-RBC was similar to that of maternal-RBC when expressed as nmole/ml packed cells (2.77+/ 0.14 vs 2.95+/-0.13), but was significantly lower when expressed as nmole/mumole total lipids (0.56+/-0.03 vs 0.64+/-0.04, p = 0.03) from that of maternal-RBC. Vitamin E-quinone levels are significantly elevated in newborns compared with their mothers both in RBC (29.4+/-2.1 vs 24.1+/-1.2, p = 0.04) and plasma (39.9+/ 5.3 vs 25.3+/-4.2, p = 0.006) when expressed as nmole/mmole total lipids but not when expressed as nmole/ml. There was a significant correlation of vitamin E between newborn-plasma and newborn-RBC (r = 0.65, p = 0.0002 for nmole per ml packed RBC;r = 0.63, p = 0.0007 for nmole per mumole total lipids). The relationship between maternal plasma and newborn plasma was significant when vitamin E was normalized with nmole/mumole total lipid (r = 0.54, p = 0.007 but not when expressed as nmole/ml (r = 0.09, p = 0.64). However, vitamin E in the RBC of maternal and newborn had significant correlation when expressed as per ml packed cells (r = 0.61, p = 0.001) and per total lipid (r = 0.46, p = 0.02). There was no relationship of vitamin E-quinone levels between RBC and plasma of newborns and their mothers. CONCLUSIONS: Elevated blood levels of vitamin E quinone suggest increased oxidative stress and utilization of vitamin E in newborns compared to their mothers. Because vitamin E levels in RBC of newborns are lower and significantly related to vitamin E levels in RBC of their mothers, an increase in vitamin E supplementation to mothers during pregnancy may increase vitamin E levels in the newborn and help impede the effect of extrauterine oxygen toxicity. PMID- 8632116 TI - A measure of stages of change in fruit and vegetable consumption among fourth- and fifth-grade school children: reliability and validity. AB - OBJECTIVE: We developed, pilot-tested, and field-applied a stages of change questionnaire regarding fruit and vegetable (F&V) consumption among fourth- and fifth-grade students. METHODS: The design included cross-sectional assessment of internal consistency and construct validity, and 2-week and 7-week longitudinal assessment of test-retest reliability. Subjects included 134 students from one school for pilot-testing and 252 from two schools for field application. Thirty two questions from McConnaughy et al's stages of change questionnaire were adapted and pilot-tested; minor revisions were made for field application. Statistical analyses included principal components analysis to identify subscales; cluster analysis to identify subgroups within students; Cronbach's alpha coefficient to assess internal consistencies; Pearson product-moment correlations to assess test-retest reliabilities; and oneway ANOVA's by F&V stages of change clusters with actual F&V consumption, F&V self-efficacy subscales, F&V preferences, and F&V outcome expectations subscales to determine construct validity. RESULTS: Principal components analysis from the field application indicated two subscales (precontemplation and beyond precontemplation) accounting for 39.5% of variance. Cluster analysis indicated 6 interpretable clusters; 2 (n = 63) provided responses inconsistent with the stages of change theory and 4 (n = 189) provided responses consistent with the theory. Internal consistencies and test-retest reliabilities were acceptable. Students in the "beyond precontemplation" clusters had higher levels of self efficacy and outcome expectations regarding eating F&V. CONCLUSIONS: Measuring stages of change other than the precontemplation stage in F&V consumption among elementary school children is problematic. Perhaps the theoretical concept does not apply to children, or elementary school children lack the ability to comprehend the questions measuring the concept, or the approach used was not entirely appropriate. PMID- 8632115 TI - Fat intake of women during normal pregnancy: relationship with maternal and neonatal essential fatty acid status. AB - OBJECTIVE: To study the relationships between the essential fatty acid (EFA) composition of maternal diet, maternal EFA status and the EFA status of healthy newborn infants. METHODS: A prospective longitudinal study was performed in which 176 pregnant women completed a food frequency questionnaire (FFQ) before 13, at 22, and at 32 weeks of gestation, so that changes in nutrient intake throughout pregnancy could be recorded. Around 22 weeks, a dietary history was performed and a maternal blood sample was collected. Immediately after delivery, a blood sample from the umbilical vein and a piece of the umbilical cord were collected. Fatty acid compositions were determined for phospholipids (PLs) isolated from maternal and umbilical plasma and from umbilical vein and artery vessel walls. RESULTS: No significant differences in the mean daily intake of total fat, saturated fat, mono-unsaturated fat, polyunsaturated fat and linoleic acid (18:2(n-6), LA) were observed between the three trimesters. Maternal LA intake was positively associated with LA levels in maternal and umbilical plasma and negatively associated with 20:3(n-9), 18:3(n-3), 20:4(n-3) and 22:5(n-3) levels in maternal plasma, with 20:5(n-3) levels in umbilical plasma and with 22:6(n-3) levels in umbilical vein vessel walls. Significant positive correlations for almost all (n 6) and (n-3) fatty acids were observed between maternal and umbilical plasma levels. CONCLUSIONS: The maternal dietary fat composition appears to be consistent during pregnancy. A high maternal LA intake may have a lowering effect on the maternal as well as on the neonatal (n-3) fatty acid status. Finally, neonatal EFA status is strongly related to maternal EFA status. PMID- 8632117 TI - "Weight cycling" and mortality: how do the epidemiologists explain the role of intentional weight loss? AB - In the past 5 years, four prospective, epidemiologic studies of "weight cycling" found that mortality is higher for persons with unstable body weight than for persons whose body weight is relatively stable [1-3,5]. These findings have generated considerable interest and controversy in both the scientific community and the lay press because of the belief that the weight fluctuations were caused by intentional weight loss. None of these studies, however, collected information that would indicate whether the weight changes were intentional or unintentional. This review examines the reasons given by the authors of these studies to support the inference that intentional weight loss caused the increase in mortality. The authors acknowledged that weight loss can be caused by preexisting illness, and they usually made efforts to control for the confounding effects of illness in their analyses. However, they generally concluded that the weight fluctuations resulted from unsuccessful dieting, but no data were presented on the incidence or causes of weight fluctuation in the populations from which the samples were drawn. In none of the studies did the authors recommend that obese persons stop trying to lose weight. The reader is cautioned to await further studies in which intentionality of weight loss is directly assessed before concluding that dieting to reduce weight increases the risk of mortality. PMID- 8632118 TI - Associations between obesity, breakfast-time food habits and intake of energy and nutrients in a group of elderly Madrid residents. AB - OBJECTIVE: The objective of this study was to gain more knowledge about the breakfast habits of different groups of elderly people and to investigate the differences in breakfast habits between overweight and normal weight elderly subjects. METHODS: A study was made of the food preferences, dietary habits and the intake of energy and nutrients at breakfast in a group of 122 elderly Spanish people (65 men and 57 women) aged 75.7+/-8.7 years. Study participants were divided into two groups: overweight and obese subjects (O) with a body mass index (BMI)>or=25 kg/m2 (58% of the population), and normal weight subjects (NW) with a BMI<25 kg/m2 (42% of the population). The members of each group were further divided into subgroups according to age (>or=80 years of age (Y) andor=5 cups/day only in those who curtailed caffeinated coffee on advice of a physician or for heart/circulatory problems. CONCLUSIONS: Curtailing of caffeinated coffee in this adult cohort was primarily due to health concerns, but few of those who curtailed caffeinated coffee attribute the change to the advice of a physician. PMID- 8632124 TI - Grieving the death of an adult child. AB - Little attention has been paid to the experiences of older parents whose children die during their adult years. Parents feel unsupported and uninformed by medical professionals, and excluded from the decision-making process related to their adult children's illnesses and death. Parents derived comfort and a sense of usefulness in maintaining and strengthening their relationship with surviving grandchildren. PMID- 8632126 TI - Support groups for visitors of residents in nursing homes. AB - Contrary to popular myth, family members expressed the desire to be involved with loved ones residing in nursing homes. Open and continuous communication between the nursing staff and family members reduces anxiety and promotes beneficial results to nursing home residents. Although family members want to be kept informed about any changes in their loved one's status, they find it difficult to deal with mental deterioration and need additional support from the nursing staff. PMID- 8632125 TI - Old problem, different approach: alternatives to physical restraints. AB - Individualized care, resident assessment by a multidisciplinary team, and rehabilitation enhance functional independence in nursing home residents. Creativity in choosing alternatives to restraints was important in successful restraint reduction. Residents free of restraints had higher ADL levels, were more continent, and sustained fewer serious injuries, all of which are important quality of life indicators. PMID- 8632127 TI - An application of transcutaneous electrical nerve stimulation to control pain in the elderly. PMID- 8632128 TI - Social support and depression in widows and widowers. AB - The purpose of this descriptive study was to examine the relationships between social support and depression and the amount of social support used by 31 widows and 35 widowers. Participants completed the Personal Resource Questionnaire, the Beck Depression Inventory, and a demographic questionnaire. A significant negative relationship was observed between perceived social support and depression. There was no difference in the amount of depression experienced by widows and widowers. There were no significant differences in the two groups' perceptions of support received from their environment or in the number of supportive contacts identified in their social networks. PMID- 8632129 TI - Body positioning and the elderly with adult respiratory distress syndrome: implications for nursing care. AB - Elderly patients with lung diseases also experience age-related pulmonary structural changes. Because of the age-related pulmonary structural changes, this age group requires specialized nursing interventions. Therapeutic body positioning may lead to increased arterial oxygen tension and decreased supplemental oxygen needs. PMID- 8632130 TI - Images of aging. PMID- 8632131 TI - Giving seems to be an essential part of one's sense of meaning. Do you know of ways to facilitate giving opportunities among the frail elderly? PMID- 8632132 TI - Older adults and sexuality: a multidimensional perspective. AB - The purpose of this study was to describe the multidimensional nature of sexuality in men and women age 55 and older and compare these men and women on selected dimensions of sexuality. The community-based sample of 161 older adults (men = 69, women = 92) completed a 77-item, anonymous questionnaire. Gender differences were found for selected demographics, health and health-related variables, and selected aspects (sexual interest, participation, and satisfaction) of sexual activities. MANOVA yielded a significant multivariate F indicating men and women differed on the combination of seven dependent variables (self-esteem, intimacy, and sexual knowledge, attitudes, interest, participation, and satisfaction) while univariate Fs did not yield significant gender differences on each of the dependent variables. Study results described a multidimensional view of sexuality and aging which can be used to increase nurses' understanding of selected gender differences of older adults' sexuality. PMID- 8632133 TI - Morphometric video analysis of the engine-driven nickel-titanium Lightspeed instrument system. AB - Two hundred and sixteen Lightspeed instruments were evaluated microscopically for the presence of corrosion, surface debris, and alloy defects. The instruments were assessed morphometrically for consistency of physical design and dimensions by measuring and analyzing eight parameters of the instrument pilot tips, heads, and shafts. Results from visual inspection showed that none of the instruments were corroded; 23 presented surface porosities, and 17 had sharp strips of alloy. Data obtained by morphometric analysis indicated the mean diameter of the head of only 7 of 18 sizes met the +/- 0.02 mm allowable tolerance set forth by the American Dental Association (ADA) Specification No. 28. Observation and video analysis indicated that instruments of the same size adhere to the same basic design, but that morphometric variations do exist. The visual and intersize analysis indicated that the Lightspeed is not an instrument of any one determined shape that changes only in diameter. Rather, it is a series of instruments that show gradual shifts in both size and shape as the instrument size increases. Lightspeed instruments are a new type of nickel-titanium endodontic instrument that cannot be evaluated using the standards proposed by the American National Standards Institute/ADA Specification No. 28 for files and reamers. PMID- 8632134 TI - Influence of particle size of hydroxyapatite as a capping agent on cell proliferation of cultured fibroblasts. AB - The influence of particle size of biomaterials on cell reaction and cell proliferation was studied by means of coculturing fibroblasts with calcium phosphate ceramics. Both dense and porous hydroxyapatite ceramics obtained under different sintering conditions were selected, and two particle sizes of 300 and 40 micrometers were used to examine cell reaction. The four materials were each cocultured with dental pulp-derived fibroblasts for 7 days. Cell reaction was observed by phase-contrast microscopy and scanning electron microscopy. Cell proliferation was measured by counting the number of trypsinized cells in 7-day old culture. On the seventh day, the dense 300-micrometer particles of hydroxyapatite were completely covered by cultured cells that had proliferated on the dish surface. On the other hand, the porous and dense 40-micrometer particles were captured or gathered by the cultured cells, which seemed not to proliferate. The porous 300-micrometer particles were accompanied by numerous small broken pieces on the dish surface, and the cells proliferated only around the large particles. From these results, the dense 300-micrometer particles of hydroxyapatite can be considered the most appropriate biomaterial. PMID- 8632136 TI - In vitro cytotoxicity and dentin permeability of HEMA. AB - An in vitro diffusion chamber was used to measure the diffusion of 2-hydroxyethyl methacrylate (HEMA) through etched human dentin disks. Concentrations of HEMA, which diffused through dentin, were measured by ultraviolet spectroscopy, and the effect of initial HEMA concentration, dentin thickness, and back pressure on diffusion were assessed. The cytotoxicity of HEMA was determined using BALB/c 3T3 mouse fibroblasts in direct contact with HEMA for 12 or 24 h. HEMA diffused rapidly through dentin under all conditions, but increased thickness, back pressure, or decreased initial concentration all reduced diffusion. The permeability coefficient of HEMA was approximately 0.0003 cm/min, and diffusion through 0.5 mm of dentin reduced the HEMA concentration by a factor of approximately 6,000 (with 10 cm of H2O back pressure). It was concluded that the risk of acute cytotoxicity to HEMA through dentin was probably low, but that decreased dentin thickness, lack of polymerization, or extended exposure times might increase the risk significantly. PMID- 8632135 TI - Interleukin-1 alpha stimulates interstitial collagenase gene expression in human dental pulp fibroblast. AB - Interstitial collagenase (matrix metalloprotease-1) is a member of a family of matrix metalloproteases and is thought to play a role in degradation of the extracellular components, such as collagens in normal extracellular matrix remodeling and in many disease processes. We examined interstitial collagenase mRNA expression in human dental pulp fibroblast cultures by Northern blot analysis. These cells did not express interstitial collagenase mRNA in an unstimulated condition. Inflammatory cytokines, such as interleukin-1 alpha, induced interstitial collagenase mRNA expression in these cells. The interstitial collagenase mRNA levels began to increase after 2-h exposure, reaching a maximum after 8 h, then dropping to the unstimulated level at 48 h. These effects were observed in a dose-dependent manner in a dose range of 0.1 to 10 ng/ml. Transforming growth factor-beta reduced the levels of interstitial collagenase mRNA expression that were induced by interleukin-1 alpha. These observations suggest that interstitial collagenase mRNA expression in human dental pulp fibroblasts is regulated by the inflammatory cytokines and that interstitial collagenase may play a role in tissue degradation in inflamed dental pulp. PMID- 8632137 TI - A commercially available cell culture device modified for dentin barrier tests. AB - The suitability of a dentin barrier test based on a commercially available cell culture chamber was evaluated by testing the cytotoxicity of dental cements. The two chambers of the culture device as produced are separated by a membrane. This was replaced by a bovine dentin disk (500 micrometers thick). Mouse fibroblasts were grown on the "pulpal" side of the dentin for 24 h; test materials were then placed into the "cavity" side of the upper chamber. The number of viable cells was determined after 24 h. After exposure to zinc phosphate cement at a powder/liquid ratio of 2:1, approximately 100% of cells survived. A ratio of 1:1 yielded 81% survival. Only 24% and 28% of the cells survived after exposure to Ketac Fil and Ketac Silver, respectively. The light-curing glass ionomer cement (vitrebond) and zinc oxide-eugenol killed all cells. These results agree with those obtained from a previous study, wherein the dentin barrier test device was constructed in our laboratory. PMID- 8632138 TI - Tensile and tear properties of dental dam. AB - The tensile and tear properties of highly extensible latex are sensitive to specimen shape. Three specimen shapes (ASTM D412 Die C dumbbell tensile specimen, rectangular tensile specimen with 1.74 mm hole, and ASTM D624 Die C tear specimen) were evaluated for proposed ANSI/ADA specification #90 for dental dams. Fresh and aged dental dams from two manufacturers (Aseptico and Hygenic) in three weights (thin, medium, and heavy) and from two other manufacturers (Ivory and Ivoclar) in one weight (medium) were tested. Means and standard deviations of 10 specimens for tensile strength (MPa), elongation (%), and tear strength (kN/m) are included herein. Data were analyzed by analysis of variance. Means were compared by a Tukey-Kramer interval calculated at the 0.05 significance level. The use of the dumbbell and tear specimens for the evaluation of dental dam should be reconsidered. The rectangular specimen with a hole is recommended for use in the proposed specification because of its sensitivity to condition (fresh versus aged) and manufacturer. PMID- 8632139 TI - The antimicrobial effect within dentinal tubules of four root canal sealers. AB - The aim of this study was to investigate four root canal sealers--Pulp Canal Sealer EWT, Sealapex, AH26, and Ketac-Endo--for their antibacterial effects within dentinal tubules. Sterile saline served as a control. Sixty-six standardized bovine root specimens were infected with Enterococcus faecalis following smear layer removal. The materials were placed in the lumina, and six specimens from each group were stored for 24 h (48 h for AH26) and 7 days, after which dentin samples were taken from within the lumina using ISO 023 to 035 burs. Powder samples were incubated and the quantity of bacteria present assessed using spectrophotometry. All sealers showed antibacterial activity at 24 h, except Ketac-Endo. The activity of Pulp Canal Sealer EWT was similar at 24 h and 7 days. Sealapex had greater antibacterial effect at 7 days than it did at 24 h. The strongest effects were demonstrated by AH26. PMID- 8632140 TI - An in vivo comparison of gradient and absolute impedance electronic apex locators. AB - Two electronic apex locators based on the gradient (Apit) and absolute (Odontometer) impedance principles were evaluated. Distances of file tips to apical foramina were determined in vivo with the electronic apex locators and subsequently verified after extraction. Mean differences between file tips and apical foramina were +0.14 +/- 0.27 mm and -0.36 +/- 0.71 mm for the Apit and Odontometer, respectively. These differences were statistically significant (p < 0.001). the Apit's and Odontometer's results ranged from +0.85 to -0.65 mm and from +0.35 to 2.45 mm from the apical foramen, respectively. Ninety-three and 73% of the findings for the Apit and the Odontometer, respectively, were within the +/- 0.5 mm range, whereas 100% and 86%, respectively, were within the +/- 1.0 mm range. Thirty-three and 43% of the results for the Apit and the Odontometer, respectively, were 1.0 mm or less coronal to the apical foramen. The Apit tended to yield the more reliable results because of the narrower range. PMID- 8632141 TI - Incomplete healing (scar tissue) after periapical surgery--radiographic findings 8 to 12 years after treatment. AB - Twenty-four cases treated by periapical surgery--which 2 to 6 yr after surgery were classified radiographically as incomplete healings (scar tissue)--were further followed, extending the observation period to 8 to 12 yr. One case healed completely, 1 failed, and 22 were still recorded in the same healing group. All the 22 scar tissue cases were characterized by a reduction of the defect in bone. In 13 cases, continuous periodontal structures were seen and the defect thereby separated from the root. The findings support the conclusion that cases clearly showing features of incomplete healing (scar tissue) at the regular follow-up 1 yr after surgery can be regarded as successes. They need not be recorded for further systematic control. A general follow-up program for apicectomy cases is suggested. PMID- 8632142 TI - Endodontic retreatment of unusually long maxillary central incisors. AB - Anatomical and morphological variations in teeth may affect the success and prognosis of endodontic therapy. The reported data regarding such variations in the maxillary central incisors are limited. This article presents a case of endodontic retreatment in unusually long maxillary central incisors. PMID- 8632143 TI - Molecular cloning, expression, and chromosomal localization of a human brain specific Na(+)-dependent inorganic phosphate cotransporter. AB - We describe the molecular cloning of a cDNA encoding a human brain Na(+) dependent inorganic phosphate (P(i)) cotransporter (hBNPI). The nucleotide and deduced amino acid sequences of hBNPI reveal a protein of 560 amino acids with six to eight putative transmembrane segments. hBNPI shares a high degree of homology with other Na(+)-dependent inorganic P(i) cotransporters, including those found in rat brain and human and rabbit kidney. Expression of hBNPI in COS 1 cells results in Na(+)-dependent P(i) uptake. Northern blot analysis demonstrates that hBNPI mRNA is expressed predominantly in brain and most abundantly in neuron-enriched regions such as the amygdala and hippocampus. Moderate levels of expression are also observed in glia-enriched areas such as the corpus callosum, and low levels are observed in the substantia nigra, subthalamic nuclei, and thalamus. In situ hybridization histochemistry reveals relatively high levels of hBNPI mRNA in pyramidal neurons of the cerebral cortex and hippocampus and in granule neurons of dentate gyrus. The level of hBNPI mRNA is quite low in fetal compared with adult human brain, suggesting developmental regulation of hBNPI gene expression. Southern analyses of nine eukaryotic genomic DNAs probed under stringent conditions with hBNPI cDNA revealed that the hBNPI gene is highly conserved during vertebrate evolution and that each gene is most likely present as a single copy. Using fluorescent in situ hybridization, we localized hBNPI to the long arm of chromosome 19 (19q13) in close proximity to the late-onset familial Alzheimer's disease locus. PMID- 8632144 TI - Generation and characterisation of stable cell lines expressing recombinant human N-methyl-D-aspartate receptor subtypes. AB - Transfection of mouse L(tk-) cells with human N-methyl-D-aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist D, L-(epsilon)-2-[3H]amino-4-propyl-5 phosphono-3-pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6-5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors. PMID- 8632146 TI - A tau promoter region without neuronal specificity. AB - The microtubule-associated protein tau is produced from a 6-kb mRNA expressed primarily in neurons. A 2-kb tau mRNA has also been characterized, which produces a tau isoform that localizes to the nucleus, and an 8-kb mRNA is expressed in the PNS. Mapping and sequencing of the human tau gene start showed that it has an unusually GC-rich 5'-untranslated region coded by a single untranslated exon (designated -1). Primer extensions and expression assays indicated that upstream of exon -1 is a promoter that is not neuron specific. This region contains consensus binding sites for transcription factors AP2, Sp1, and GCF. PMID- 8632145 TI - The neuroendocrine proteins secretogranin II and III are regionally conserved and coordinately expressed with proopiomelanocortin in Xenopus intermediate pituitary. AB - Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretary granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans-Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15- and 35-fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30-fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family. PMID- 8632147 TI - Induction of proenkephalin gene expression in cultured bovine chromaffin cells is dependent on protein synthesis of AP-1 proteins. AB - In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c-fos, c-jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE-2 element of the proenkephalin promoter was sufficient for basal and second messenger-induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE-2 and AP-1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP-1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c fos, but not of c-jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP-1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells. PMID- 8632148 TI - Promoter region and alternatively spliced exons of the rat mu-opioid receptor gene. AB - The actions of exogenous and endogenous opioids are mediated by at least three different opioid receptors, called mu, kappa, and delta. Recently, we have detected a new variant of the rat mu-opioid receptor, which we termed rMOR1B and which differs from rMOR1 (now also called rMOR1A) in the amino acid sequence at the C-terminus. Both isoforms were proposed to be splicing variants of the same gene. To elucidate the molecular mechanism leading to the formation of the new variant, the exon/intron structure of the rat mu-opioid receptor gene in the respective area has been determined by analyzing a genomic P1 phage clone. In addition, we have investigated the putative promoter region of this gene. The present study revealed that rMOR1B is generated by an alternative splicing event whereby a previously unknown exon will be placed behind exon 3 to form rMOR1B mRNA, which is separated from the latter by an intron. Therefore, this new exon has to be called exon 4, whereas the former exon 4, which encodes the C-terminus of MOR1A, now becomes exon 5. Examination of the putative rat promoter region revealed a high degree of nucleotide sequence homology to the mouse gene. Using an RNase protection approach, one single transcription initiation site could be located at 230 bp upstream of the translation start. This is similar to the situation in the mouse, where four major transcription start sites were reported to lie close together around 270 bp upstream of the protein coding region. PMID- 8632149 TI - Brain-derived neurotrophic factor stimulates AP-1 and cyclic AMP-responsive element dependent transcriptional activity in central nervous system neurons. AB - Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1 dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons. PMID- 8632150 TI - Ras-regulated hypophosphorylation of the retinoblastoma protein mediates neuronal differentiation in PC12 cells. AB - To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12-O-tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells. PMID- 8632151 TI - Denervation, but not decentralization, reduces nerve growth factor content of the mesenteric artery. AB - In the present study we applied an improved nerve growth factor (NGF) extraction method to examine the effects of denervation and sympathetic decentralization on NGF levels in vascular tissue. Adult male Wistar Kyoto rats underwent mesenteric arterial denervation or splanchnic nerve transection. Four days after operation, animals were killed, and the mesenteric artery and coeliac-superior mesenteric ganglia were removed. The arterial adventitia was stripped from the media to measure NGF levels in nerve and smooth muscle separately. A high concentration of NGF was detected in the normal artery, 90% of which was in the adventitial layer. Surgical denervation significantly reduced the NGF levels in the artery and ganglia by 78 and 71%, respectively. However, within the artery the level of NGF was reduced in the adventitia but not in the media. Thus, the large reduction of NGF content resulted from the loss of nerve plexus from the artery. In contrast, decentralization did not alter the NGF content in the artery, in either the adventitia or media. Our results are in marked contrast to previous studies reporting elevated levels of NGF following denervation. This discrepancy is explained by the ability of our new procedure to extract much greater amounts of NGF from the tissue. PMID- 8632152 TI - Amyloid precursor protein metabolism in primary cell cultures of neurons, astrocytes, and microglia. AB - Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid beta peptide (A beta) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease. APP is highly expressed in the brain. To assess the source of cerebral A beta, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments. We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons. The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia. The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more A beta than astrocytes or microglia. PMID- 8632153 TI - Stimulation of cell elongation in teleost rod photoreceptors by distinct protein kinase inhibitors. AB - The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN 62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC. PMID- 8632154 TI - Transduction mechanisms involved in thrombin receptor-induced nerve growth factor secretion and cell division in primary cultures of astrocytes. AB - In astrocytes, thrombin and thrombin receptor-activating peptide (TRAP-14), a 14 amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [3H]-thymidine incorporation into DNA (EC50 = 1 nM and +650% at 100 nM for thrombin; EC50 = 3 microM and +600% at 100 microM for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 nM thrombin or 100 microM TRAP-14). The [3H] thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as Gi or G(o), is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP 14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms. PMID- 8632155 TI - Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress. AB - Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H2O2). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-(13)C2, 6,6-(2)H2] glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H2O2, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H2O2 caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H2O2 exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H2O2-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H2O2 detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions. PMID- 8632156 TI - A nonneuronal isoform of cell adhesion molecule L1: tissue-specific expression and functional analysis. AB - The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1. PMID- 8632158 TI - Induction of the serotonin1A receptor in neuronal cells during prolonged stress and degeneration. AB - Neuronal migration in brain is followed by differentiation of committed neurons and simultaneous apoptosis of uncommitted preneuronal cells due to a limiting supply of trophic factors and nutrients. We have dissected differentiation and apoptosis by designing a simple in vitro model for this nutrient deprivation using engineered neuronal cell lines stably transfected with a promoterless segment (G-21) of the intronless human serotonin1A receptor (5-HT1A-R) gene. Despite the use of widely different heterologous promoters (cytomegalovirus and Rous sarcoma virus) for the stable expression of G-21, a dramatic increase in expression of the 5-HT(1A)-R (five- to 15-fold) and its mRNA was always observed during degeneration and apoptosis of nutrient-deprived neuronal cells. Involvement in this induction of a 170-bp 5'-end untranslated sequence (5'-UT) (tail end of the 500-bp natural promoter) of G-21 was confirmed by stable transfection of neuronal cells with an SV-40 promoter-driven construct harboring the 5'-UT and the reporter chloramphenicol acetyltransferase (CAT) cDNA. Presence of the 5'-UT resulted in a threefold increase in CAT expression during nutrient deprivation in randomly chosen clones. The induction was also observed in the endogenous 5-HT1A-R, expressed by embryonic day 16 mouse hippocampal neurons, subsequent to nutrient deprivation and onset of degeneration. A trophic role of the 5-HT1A-R has been suggested in earlier studies. Considering the example of protective heat shock proteins, which are induced during various types of stress, our results suggest that stressed neuronal cells undergoing degeneration and apoptosis synthesize increased levels of 5-HT1A-R as a final attempt to survive. PMID- 8632157 TI - A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity. AB - The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity. PMID- 8632159 TI - Extracellular striatal dopamine and glutamate after decortication and kainate receptor stimulation, as measured by microdialysis. AB - Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 microM concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so-called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study. PMID- 8632160 TI - Phorbol ester-enhanced noradrenaline secretion correlates with the presence and activity of protein kinase C-alpha in human SH-SY5Y neuroblastoma cells. AB - The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes alpha, epsilon, and zeta on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC(50) 0.6 microM) but not 100 mM (K+)-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and (K+) evoked release after pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) for 8 min, with IC50 values of 0.7 and 2.4 microM, respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC alpha and PKC-epsilon but not PKC-zeta. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin 1, conditions that caused down-regulation of PKC-alpha, but not PKC-epsilon or zeta. The PKC inhibitor Go-6976 (2 microM), which has been shown to inhibit selectively PKC-alpha and beta in vitro, also inhibited the TPA enhancement of carbachol- and (K+)-evoked NA release by > 50%. These data suggest that in SH SY5Y cells, the ability of TPA to enhance carbachol- and (K+)-evoked NA secretion is due to activation of PKC-alpha. PMID- 8632161 TI - Interaction of NMDA and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells. AB - To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (+/-)-3-(2-carboxypiperazin-4 yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by approximately 50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors. PMID- 8632162 TI - Coupling of astroglial alpha 2-adrenoreceptors to second messenger pathways. AB - We have investigated which alpha 2-receptor subtypes are expressed in cultured cortical astroglia, and their coupling to second messengers. Binding assays using [3H]rauwolscine showed a very low number of alpha 2 receptors in the astrocytic cultures. Treatment of cultures with dibutyryl cyclic AMP (dBcAMP) increased significantly the number of receptors. The RNase protection assay was used to investigate which receptor subtype the cells express. The alpha 2B message was expressed at a low level in both treated and untreated cells, the levels of mRNA for the alpha 2A/D subtype were up-regulated significantly in cells treated with dBcAMP and no expression of mRNA for the alpha 2C subtype was detected. The alpha 2 agonist dexmedetomidine inhibited forskolin-induced increases in cyclic AMP both in treated and untreated cultures in a pertussis toxin-dependent manner. This effect was abolished by the alpha 2-receptor antagonist rauwolscine. Selective alpha 2-receptor agonists dexmedetomidine, clonidine, and UK14,304 all increased intracellular calcium only in dBcAMP-treated cells. The antagonist rauwolscine abolished this effect. Ca2+ responses were also seen in the absence of extracellular Ca2+ and they were inhibited by the phospholipase C inhibitor U 73122, suggesting that astroglial alpha 2 receptors are coupled to the inositol phospholipid pathway. We therefore also tested the effect of dexmedetomidine directly on inositol 1,4,5-trisphosphate accumulation. A significant increase was seen that was blocked by the antagonist rauwolscine and, as expected, by U-73122. In short, the results demonstrate that the alpha 2 receptors in astroglia are coupled to multiple second messenger pathways. They are up-regulated in cells treated with dBcAMP, which simultaneously assume a process-bearing morphology. If this morphological change reflects some in vivo process such as reactive gliosis, the up-regulation of alpha 2-receptor expression could mean an adaptive change in astrocytic responses to a common neurotransmitter, noradrenaline. PMID- 8632163 TI - The phosphoinositide signal transduction system is impaired in bipolar affective disorder brain. AB - The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration dependently activated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and by NaF. GTP gamma S-stimulated [3H]Pl hydrolysis was markedly lower (50%) at all tested concentrations (0.3-10 microM GTP gamma S) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTP gamma S in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTP gamma S-stimulated [3H]Pl hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl 1,3-dicarboxylic acid, serotonin, and ATP each activated [3H]Pl hydrolysis above that obtained with GTP gamma S alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTP gamma S alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [3H]Pl hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder. PMID- 8632164 TI - Apolipoprotein E and low-density lipoprotein binding and internalization in primary cultures of rat astrocytes: isoform-specific alterations. AB - Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 >> E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human 125I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a KD of 1.8 nM and a Bmax of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37 degrees C confirmed that astrocytes bind, internalize, and degrade 125I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with 125I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. 125I-ApoE2 binding was significantly lower than that of the other 125I-apoE isoforms in both cell types. 125I-ApoE4 binding was similar to that of 125I-apoE3 in both astrocytes and neurons. On the other hand, 125I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles. PMID- 8632165 TI - Cellular mechanisms of protection by metabotropic glutamate receptors during anoxia and nitric oxide toxicity. AB - Metabotropic glutamate receptors, nitric oxide (NO), and the signal transduction pathways of protein kinase C (PKC) and protein kinase A (PKA) can independently alter ischemic-induced neuronal cell death. We therefore examined whether the protective effects of metabotropic glutamate receptors during anoxia and NO toxicity were mediated through the cellular pathways of PKC or PKA in primary hippocampal neurons. Pretreatment with the metabotropic glutamate receptor agonists (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, (1S,3R)-1 aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), and L(+)-2-amino-4 phosphonobutyric acid (L-AP4) 1 h before anoxia or NO exposure increased hippocampal neuronal cell survival from approximately 30 to 70%. In addition, posttreatment with 1S,3R-ACPD or L-AP4 up to 6 h following an insult attenuated anoxic- or NO-induced neurodegeneration. In contrast, treatment with L-(+)-2 amino-3-phosphonopropionic acid, an antagonist of the metabotropic glutamate receptor, did not significantly alter neuronal survival during anoxia or NO exposure. Protection by the ACPD-sensitive metabotropic receptors, such as the subtypes mGluR1 alpha, mGluR2, and mGluR5, appears to be dependent on the modulation of PKC activity. In contrast, L-AP4-sensitive metabotropic glutamate receptors, such as the subtype mGluR4, may increase neuronal survival through PKA rather than PKC. Thus, activation of specific metabotropic glutamate receptors is protective during anoxia and NO toxicity, but the signal transduction pathways mediating protection differ among the metabotropic glutamate receptor subtypes. PMID- 8632166 TI - Brain expression of apolipoproteins E, J, and A-I in Alzheimer's disease. AB - Inheritance of the epsilon 4 allele of apolipoprotein (apo) E is associated with increased risk of Alzheimer's disease (AD) and with increased beta-amyloid peptide (A beta) deposition in the cortex. Apo E is a member of a family of exchangeable apos, characterized by the presence of amphipathic alpha-helical segments that allow these molecules to act as surfactants on the surface of lipoprotein particles. Two members of this family, apo E and apo J, have been shown to bind soluble A beta, and both are associated with senile plaques in the AD cortex. We now have studied the pattern of brain apo expression and found that five members of this class are present: apo A-I, A-IV,D,E, and J. By contrast, apos A-II, B, and C-II were not detectable. Immunohistochemistry revealed that, in addition to apo E and apo J, apo A-I immunostained occasional senile plaques in AD cortex. Immunoblot analysis showed no difference in the relative amounts of any of these apos in tissue homogenates of frontal lobe from AD or control patients. Comparison by APO E genotype showed no differences in the amount of apo E in brain among APO E epsilon 3/3, epsilon 3/4, or epsilon 4/4 individuals; however, a significant decrease in the amount of apo J was associated with the APO E epsilon 4 allele. No differences in apo J levels were detected in CSF samples of AD subjects. We propose that several members of the exchangeable apo family may interact with A beta deposits in senile plaques through common amphipathic alpha-helical domains. Competition among these molecules for binding of A beta or A beta aggregates may influence the deposition of A beta in senile plaques. PMID- 8632167 TI - Evaluation of cathepsins D and G and EC 3.4.24.15 as candidate beta-secretase proteases using peptide and amyloid precursor protein substrates. AB - No single protease has emerged that possesses all the expected properties for beta-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of beta-amyloid peptide. We have isolated and purified a brain derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of beta-amyloid peptide. Its molecular size of 55-60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate beta-amyloid peptide production. We have also tested the ability of these enzymes to form carboxyl-terminal fragments from full length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM --> NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of beta amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate beta-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM --> NL peptide and does not generate carboxy terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as beta-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases. PMID- 8632168 TI - Evidence disputing the link between seizure activity and high extracellular glutamate. AB - As seizures in experimental models can be induced by the activation and suppressed by the inhibition of glutamate receptors, it is often proposed that a high extracellular glutamate level subsequent to excessive presynaptic release and/or altered glutamate uptake is epileptogenic. The purpose of this study was to ascertain the link between seizure activity and high extracellular glutamate. To assist the detection of any putative rise in extracellular glutamate during seizures, microdialysis was coupled to enzyme-amperometric detection of glutamate, which provides maximal sensitivity and time resolution. Electrical activity and field potential were also recorded through the dialysis membrane to confirm that epileptic activity was present at the sampling site. No increase in dialysate glutamate content was detected during picrotoxin-induced seizures, even when the K+ concentration in the perfusion medium was raised to 50% above that measured previously during paroxysmal activity. In addition, sustained inhibition of glutamate uptake by L-trans-pyrrolidine-2,4-dicarboxylate increased the extracellular glutamate level > 20-fold but did not produce electrophysiological changes indicative of excessive excitation. These findings indicate that seizures are not necessarily accompanied by an increased extracellular glutamate level and that increased glutamatergic excitation in epilepsy may result from other abnormalities such as increased density of glutamate receptors, enhanced activation subsequent to reduced modulation, or sprouting of glutamatergic synapses. PMID- 8632169 TI - Beta-dystroglycan: subcellular localisation in rat brain and detection of a novel immunologically related, postsynaptic density-enriched protein. AB - The distribution of a glycoprotein component of the muscle dystrophin complex, beta-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that beta-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle beta-dystroglycan) abolished reactivity against both beta-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of beta dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species. PMID- 8632170 TI - Intrasynaptosomal free calcium concentration during rat brain development: effects of hypoxia, aglycaemia, and ischaemia. AB - The effects of hypoxia, aglycaemia, and hypoxia-aglycaemia on intrasynaptosomal free Ca2+ concentration ([Ca2+]i) have been investigated in rat brain synaptosomes prepared from animals aged 5, 10, 15, 20, 25, and 60 days. After 60 min of hypoxia there was no significant difference, when compared with controls, in basal [Ca2+]i or [Ca2+]i following depolarisation in all of the ages studied. Following 60 min of aglycaemia there was no significant difference from controls in [Ca2+]i of synaptosomes prepared from pups of < or = 20 days, although a significant rise in [Ca2+]i was seen in preparations from animals > 20 days old. Sixty minutes of hypoxia-aglycaemia led to a significant rise in [Ca2+]i only in preparations from animals 15-60 days old. With both aglycaemia and hypoxia aglycaemia a progressive increase in the magnitude of the rise in [Ca2+]i was seen with development. These data suggest increases in [Ca2+]i in adult nerve terminals following prolonged aglycaemia and hypoxia-aglycaemia but no change following prolonged hypoxia. In contrast, no significant changes in [Ca2+]i values were apparent in neonatal nerve terminals under any of these conditions. In control synaptosomes with glucose and oxygen freely available, a decrease in resting and depolarised [Ca2+]i during development was seen, suggesting a change in calcium homeostasis within the nerve terminal as the brain develops. It is suggested that the mechanism underlying the relative resistance to ischaemic damage of neonatal brain as compared with adult brain may be related to the regulation of calcium at the nerve ending. PMID- 8632171 TI - Amyloid beta protein primes cultured rat microglial cells for an enhanced phorbol 12-myristate 13-acetate-induced respiratory burst activity. AB - Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid beta peptide (A beta), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide induced production of nitric oxide was altered in cultures previously incubated with synthetic A beta (1-40) for 24 h. In addition, no direct stimulatory effect of A beta (1-40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in A beta (1-40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, A beta(1-40) may merely act as a priming rather than a triggering stimulus. PMID- 8632172 TI - Acute and prolonged alterations in brain free magnesium following fluid percussion-induced brain trauma in rats. AB - Several studies have reported declines in brain total and free magnesium concentration after a traumatic insult to the CNS. Although the evidence suggests that this magnesium decline is associated with eventual neurologic outcome after trauma, the duration of free magnesium decline and its impact on related bioenergetic variables are relatively unknown. The present study has therefore used phosphorus magnetic resonance spectroscopy to determine the length of time that free magnesium remains suppressed after traumatic brain injury in rats. Immediately after the traumatic event, brain intracellular free magnesium declined to < 60% of preinjury values and remained significantly depressed (50 +/ 8%; p < 0.001) for 5 days before recovering to preinjury levels by day 8. Cytosolic phosphorylation ratio and mitochondrial oxidative capacity also significantly decreased (p = 0.008) and increased (p = 0.002), respectively, after trauma. However, unlike the time of maximum magnesium change, the maximum changes in these bioenergetic variables occurred at 16-24 h after trauma and thereafter remained stable until after the magnesium had recovered. We conclude that free magnesium decline after trauma precedes changes in bioenergetic variables. Furthermore, therapies targeted at reestablishing magnesium homeostasis after trauma may require administration over a 1-week period. PMID- 8632173 TI - Enolase activity and isoenzyme distribution in human brain regions and tumors. AB - The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of alpha alpha-, alpha gamma, and gamma gamma-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type alpha alpha-enolase was the predominant isoenzyme, and alpha gamma-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of alpha alpha-enolase and a lower proportion of gamma gamma-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of alpha alpha-enolase and lowest proportion of gamma gamma-enolase. PMID- 8632174 TI - A one-dimensional (proton and phosphorus) and two-dimensional (proton) in vivo NMR spectroscopic study of reversible global cerebral ischemia. AB - The suitability of two-dimensional (2D) proton spectroscopy for monitoring, in vivo, the changes in levels of brain metabolites induced by cerebral ischemia was investigated in an experimental model of 30-min reversible ischemia induced by four-vessel occlusion in the rat. The resulting data were compared with those obtained by one-dimensional (1D) proton and phosphorus spectroscopy. Phosphorus spectra obtained during ischemia showed significant drops in levels of phosphocreatine (-73%), beta-ATP (-60%), and intracellular pH (to 6.30) and an increase in inorganic phosphate level (905%). 1D and 2D proton spectra showed decreases in the N-acetylaspartate/creatine-phosphocreatine ratio that were not significantly different [-21% (1D) and -32% (2D)]. Similarly, the increases in lactate/creatine-phosphocreatine ratio were not significantly different [2,546% (1D) and 3,020% (2D)]. 2D spectroscopy also indicated a decrease in aspartate ( 66%) and an increase in the inositol-choline derivative (+124%) pools during ischemia and an increase in alanine pool (+516%) during reperfusion. The glutamate-glutamine pool and taurine content did not change significantly during ischemia but decreased during reperfusion. The glucose level transiently decreased (-67%) during ischemia and increased immediately after (+261%). The levels of all the metabolites investigated returned to control values within 175 min after ischemia. 2D spectroscopy seems to be a reliable method of monitoring the changes in levels of cerebral compounds known to be involved in ischemia. PMID- 8632175 TI - Induction of urokinase-type plasminogen activator in rat facial nucleus by axotomy of the facial nerve. AB - The response of plasminogen activator activity in the CNS to peripheral nerve axotomy was examined in vivo. After transection of the rat facial nerve, a transient increase in plasminogen activator activity was observed in the facial nucleus on the operated side with maximal activity 3-5 days after lesion. This activity was inhibited by the urokinase-specific inhibitor amiloride but not by antibodies against tissue plasminogen activator. The molecular mass of the induced form of plasminogen activator was estimated to be approximately 48 kDa. An in vitro assay of plasminogen hydrolysis also demonstrated an increase in amiloride-sensitive plasminogen activator activity in facial nerve extracts following facial nerve axotomy. These data indicate that the plasminogen activator activity induced in the facial nucleus following axotomy of facial motoneurons is of the urokinase type. It is suggested that the urokinase-type plasminogen activator might play a role in the events accompanying injury and regeneration in the facial nucleus following motoneuron lesion. PMID- 8632176 TI - Oxidative stress, hypoxia, and ischemia-like conditions increase the release of endogenous amino acids by distinct mechanisms in cultured retinal cells. AB - The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe(2+)-induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca2+ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca(2+)-independent mechanism. The increased release observed in N methyl-D-glucamine+ medium suggested the reversal of the Na+-dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na(+)-dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [3H]arachidonic acid and free radicals seems to affect the Na+ dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca2+]i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism. PMID- 8632177 TI - Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-62 inhibits adrenal medullary chromaffin cell functions independent of its action on the kinase. AB - KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells. PMID- 8632178 TI - Binding of 2',3'-cyclic nucleotide 3'-phosphodiesterase to myelin: an in vitro study. AB - The binding of 2', 3'-cyclic nucleotide 3'-phosphodiesterase isoform 1 (CNP1) to myelin and its association with cytoskeletal elements of the sheath have been characterized with in vitro synthesized polypeptides and purified myelin. We have previously shown that the cysteine residue present in the carboxy-terminal CXXX box of CNP1 is isoprenylated, and that both C15 farnesyl and C20 geranylgeranyl isoprenoids can serve as substrates for the modification. Here, we have mutated the CXXX box to obtain selectively farnesylated CNP1 or geranyl- geranylated CNP1 and found that these two modified forms of CNP1 behave identically in all of the assays performed. Isoprenylation is essential but not sufficient for the binding of in vitro synthesized CNP1 to purified myelin, because a control nonmyelin protein is isoprenylated, yet unable to bind to myelin. In our assay, membrane bound CNP1 partitions quantitatively into the nonionic detergent-insoluble phase of myelin, suggesting that CNP1 binds to cytoskeletal elements within myelin. However, isoprenylated CNP1 fails to bind to the cytoskeletal matrix isolated from myelin by detergent treatment, implying that both detergent-soluble and insoluble myelin components are involved in the binding of CNP1. A model for the interactions between CNP1 and myelin is presented, consistent with models proposed for other isoprenylated proteins. PMID- 8632179 TI - Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures. AB - Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin-1 beta (IL-1 beta) or the protein kinase C activator phorbol 12 myristate 13-acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1 beta. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1 beta. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor alpha and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1 beta, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS 2 mRNA. Furthermore NS-398, a specific inhibitor of cyclooxygenase-2, blocked > 80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors. PMID- 8632180 TI - Regulation of GTP cyclohydrolase I gene expression and tetrahydrobiopterin content in cultured sympathetic neurons by leukemia inhibitory factor and ciliary neurotrophic factor. AB - Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase. (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3-7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78-96% reduction in GTPCH mRNA content relative to beta actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype. PMID- 8632181 TI - Flesinoxan dose-dependently reduces extracellular 5-hydroxytryptamine (5-HT) in rat median raphe and dorsal hippocampus through activation of 5-HT1A receptors. AB - The effects of systemic administration of the serotonin (5-hydroxytryptamine) 5 HT1A receptor agonists flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin on extracellular 5-HT were measured using microdialysis probes in both median raphe nucleus and dorsal hippocampus. Both 5-HT1A agonists dose-dependently decreased dialysate 5-HT levels from both brain regions. The effects of flesinoxan in the median raphe (0.3 mg/kg) and dorsal hippocampus (1.0 mg/kg) could be blocked by the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N (2-pyridyl)cyclohexane carboxamide trihydrochloride (WAY 100,635) at a dose of 0.05 mg/kg s.c. The antagonist itself had no effect at this dosage. Local perfusion of flesinoxan for 30 min through the dialysis probe into the median raphe region at concentrations of 20, 100, and 1,000 nM resulted in a significant decrease in dialysate 5-HT content from both regions. The effect of 100 nM flesinoxan could be blocked by coperfusion of 1,000 nM WAY 100,635. The data indicate that flesinoxan is a potent 5-HT1A receptor agonist and also support the notion that somatodendritic 5-HT1A autoreceptors regulate both terminal and somatodendritic 5-HT release. PMID- 8632182 TI - Ganglioside-induced adherence of botulinum and tetanus neurotoxins to adducin. AB - Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an approximately 116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the approximately 116-kDa protein more than that with GT1b. A purified preparation of the water-soluble approximately 116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the approximately 116-kDa protein. These sequences matched with the data bank sequences for beta-adducin, a cytoskeletal protein. The carboxy terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s). PMID- 8632184 TI - Effect of hypoxia on Na(+)-K(+)-Cl- cotransport in cultured brain capillary endothelial cells of the rat. AB - The effect of hypoxia on Na+, K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring 86Rb+ uptake as a tracer for K+. RBECs expressed both Na+, K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na+, K(+)-ATPase activity by 38.9%, whereas it significantly increased Na(+)-K(+) Cl- cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by approximately 65%) along with a complete inhibition of Na+, K(+)-ATPase and a coordinated increase of Na(+)-K(+)-Cl- cotransport activity (up to 100% above control values). Oligomycin augmentation of Na(+)-K(+)-Cl- cotransport activity was not observed in the presence of 2-deoxy-D-glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na+, K(+)-ATPase activity is reduced, RBECs have the ability to increase K+ uptake through Na(+)-K(+)-Cl cotransport. PMID- 8632183 TI - The high-affinity sulphonylurea receptor regulates KATP channels in nerve terminals of the rat motor cortex. AB - The coexpression of sulphonylurea binding sites and ATP-sensitive K+(KATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive KATP channels and charybdotoxin sensitive, large-conductance calcium-activated K+ BKCa channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and KATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the KATP channel may serve to limit glutamate release under conditions of metabolic stress. PMID- 8632185 TI - Altered glycosaminoglycan chain structure in a variant of the C2 mouse muscle cell line. AB - Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of 35SO4 into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation. PMID- 8632186 TI - Modulation of 45Ca2+ influx into cells stably expressing recombinant human NMDA receptors by ligands acting at distinct recognition sites. AB - A 45Ca2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45Ca2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45Ca2+ influx by five glutamate site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45Ca2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45Ca2+ influx into cells expressing NR1a/NR2A receptors (IC50 = 408 microM) and NR1a/NR2B receptors (EC50 = 37.3 microM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC50 = 0.099 microM) compared with NR1a/NR2A receptors (IC50 = 164 microM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg2+ and Zn2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45Ca2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells. PMID- 8632187 TI - Identification, purification, and characterization of the molecular forms of Aplysia californica peptidylglycine alpha-amidating enzyme. AB - Peptidylglycine alpha-amidating enzyme (PAM; EC 1.14.17.3) is responsible for the conversion of peptides with a COOH-terminal glycine into alpha-amidated peptides, a posttranslational modification often required for biological activity and/or increased stability. Such an activity able to convert the model peptide D-Tyr-Val Gly into D-Tyr-Val-amide was found to be present in the marine mollusk Aplysia californica. Examination of this amidating activity as well as its immunoreactivity demonstrates that (1) it can be found mainly in the atrial gland, heart, and CNS but is barely detectable in the hepatopancreas and gonads, (2) it requires as essential cofactors copper, molecular oxygen, and ascorbate, and (3) it exists in at least two molecular forms, a soluble and a membrane-bound form. Purification of this activity from the atrial gland was accomplished using Cu(2+)-chelating Sepharose, gel permeation, and hydroxyapatite chromatography. In addition, using polyclonal antibodies raised against various parts of the rat amidating enzyme, we demonstrate that numerous immunologically recognized regions are conserved in both the soluble and membrane-bound Aplysia californica PAM. PMID- 8632188 TI - Chronic in vivo sodium azide infusion induces selective and stable inhibition of cytochrome c oxidase. AB - The effect of chronic subcutaneous infusion of sodium azide on the activity of mitochondrial respiratory chain enzymes was investigated in Sprague-Dawley rats. Treatment with approximately 1 mg/kg/h sodium azide induced chronic, partial inhibition of cytochrome c oxidase, whereas the activities of respiratory complexes I and III were not significantly affected. The inhibition of cytochrome c oxidase was evident by 7 days after infusion began, and the effect was stable for at least 3 weeks. The selectivity of azide for cytochrome c oxidase is discussed in the context of other findings of azide effects on enzymes. The results of the present study indicate that the sodium azide infusion paradigm described here provides a useful tool for the evaluation of selective and stable cytochrome oxidase inhibition in vivo. PMID- 8632189 TI - NMR studies of Pi-containing extracellular and cytoplasmic compartments in brain. AB - The inorganic phosphate (Pi) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of Pi. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated Pi spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from infinity to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three compartment model containing a mixture of Pi buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the Pi peaks arising from the three compartments with different pH values if each peak made up 10-35% of total Pi area. In vivo, we identified the plasma compartment by intraarterial infusion of Pi. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that Pi compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2 DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger Pi peaks represented intracellular spaces. PMID- 8632191 TI - Central regional profile of dental hygiene students. AB - PURPOSE: This study was designed to identify personal, familial, academic, and employment characteristics of dental hygiene students from the central region of the United States to develop a comprehensive student profile for research and recruitment purposes. METHODS: In fall 1990, 25 dental hygiene program directors in ten states in the central region of the United States and 475 students enrolled in these programs were mailed surveys to develop program and student profiles. Data were analyzed using measures of central tendency and frequency. RESULTS: Directors and students from 24 of 25 dental hygiene programs responded. Data are reported from these 24 directors and 422 of 464 (91%) of the students enrolled in the programs. Dental hygiene students in the central region of the United States are similar in personal, parental, and academic backgrounds to the general population of college students nationally. Unlike the general college population, the regional dental hygiene population tends to be singularly female. Students chose to major in dental hygiene for economic and personal reasons. The availability of employment opportunities, the flexibility to work full- or part time, and the prospects of making a better income and becoming a professional influenced their decision. Students identified dentists, dental hygienists, and relatives as being influential in their becoming interested in the dental hygiene profession. Previous dental office employment experience also influenced many students in their career choice. CONCLUSION: The study findings provide information for dental hygiene educators and others interested in student characteristics and factors that influence the selection of dental hygiene as a career. PMID- 8632190 TI - Allelic variation of human serotonin transporter gene expression. AB - Mood, emotion, cognition, and motor functions as well as circadian and neuroendocrine rhythms, including food intake, sleep, and reproductive activity, are modulated by the midbrain raphe serotonin (5-HT) system. By directing the magnitude and duration of postsynaptic responses, carrier-facilitated 5-HT transport into and release from the presynaptic neuron are essential for the fine tuning of serotonergic neurotransmission. Interest in the mechanism of environmental factor-, disease-, and therapy-induced modification of 5-HT transporter (5-HTT) function and its impact on early brain development, event related synaptic plasticity, and neurodegeneration is widespread and intensifying. We have recently characterized the human and murine 5-HTT genes and performed functional analyses of their 5'-flanking regulatory regions. A tandemly repeated sequence associated with the transcriptional apparatus of the human 5 HTT gene displays a complex secondary structure, represses promoter activity in nonserotonergic neuronal cells, and contains positive regulatory components. We now report a novel polymorphism of this repetitive element and provide evidence for allele-dependent differential 5-HTT promoter activity. Allelic variation in 5 HTT-related functions may play a role in the expression and modulation of complex traits and behavior. PMID- 8632192 TI - Survey of ethnicity and academic preparation of faculty in dental hygiene programs. AB - PURPOSE: The purpose of this study was to determine numbers of minority faculty in dental hygiene programs and to examine the academic preparation of faculty members who are dental hygienists. METHODS: A survey of ethnicity and academic preparation of full- and part-time faculty was sent to directors of all accredited U.S. dental hygiene programs in November 1991. Equal Employment Opportunities Commission (EEOC) categories and definitions were used for ethnic minorities. Professional minorities were defined as male dental hygienists and female dentists. Survey data were analyzed using chi-square and ANOVA for unequal cell frequencies. RESULTS: One hundred twenty-six (62%) of the 203 surveys were returned. Only 41 (32%) of the programs reported one or more ethnic minorities among the full- or part-time faculty. Of the 1,025 dental hygienists employed full- or part-time as faculty, there were 59 ethnic minorities and 10 males. The majority of the dental hygiene programs that had dentists as full- or part-time faculty employed white male dentists. The most common minority among dentists was white female. Most dental hygiene program director respondents (66%) reported no minority faculty in their programs. Of the dental hygiene programs that did have minority faculty, most had only one member of one minority group. No significant differences in educational preparation were found between ethnic minority and white dental hygiene faculty. Significant differences in academic preparation of dental hygiene faculty were found among programs in different educational settings and, for full-time faculty, by degree awarded program graduates. CONCLUSION: The limited numbers of minority faculty in dental hygiene programs restricts the number of dental hygiene students who have access to minorities as professional role models. To increase cultural diversity in dental hygiene education, efforts are needed to recruit more minority students and to encourage them to pursue advanced degrees for careers as dental hygiene educators. PMID- 8632193 TI - Components of nonresponse in mail surveys of dental hygienists. AB - PURPOSE: This study was designed to investigate the reporting of nonresponse in mail survey research and to determine if nonresponse as a result of inaccurate addresses varied by sampling frame. Nonresponse bias is defined as researchers' inability to contact some members of a sample, or the refusal of some sample members to provide information. METHODS: Published survey research articles in the Journal of Dental Hygiene from 1988 through 1992 were reviewed for nonresponse information. Thirty-nine articles described studies that met the requirements of being a mail survey of dental hygiene subjects. All 39 articles included information about the population base and the number of subjects in the sample. Descriptive statistics were used to analyze the data. RESULTS: Response rates ranging from 33% to 92% were reported in 37 articles. Only 15 of the 37 articles included information on the ability to contact subjects. The percentage of subjects who could not be contacted ranged from 1.1% to 13.3%. Professional organization membership rosters provided the most accurate mailing lists, followed by school class rosters. Least accurate were licensure lists and magazine lists. CONCLUSION: More attention to research design may help dental hygiene researchers recognize the impact of nonresponse bias. PMID- 8632194 TI - Attitudes of Iowa dental hygienists regarding self-regulation and practice. AB - PURPOSE: This study investigated Iowa dental hygienists' perceptions about education, practice, and licensure regarding issues related to self-regulation. Furthermore, it was the purpose of the study to determine if these perceptions were statistically related to membership in the American Dental Hygienists' Association (ADHA). METHODS: In October 1992, 150 Iowa dental hygienists were randomly selected to participate in a mail survey. The questionnaire included items on self-regulation, licensure, practice, supervision, delivery systems, and independent practice. Responses were analyzed by frequency distributions and the chi-square statistic. RESULTS: Ninety responses were received, representing 80% of the 112 subjects with valid Iowa addresses, but only 60% of the selected sample. Of the 36 variables analyzed, only two were statistically significantly different by ADHA membership. Thus, data were combined and reported for all respondents. The majority of Iowa dental hygienists (71.6%) believed dental hygiene was pursuing self-regulation to improve quality of care. They wanted more dental hygiene representation on licensure boards and almost 50% wanted separate boards. The majority of respondents favored control of dental hygiene practice and 70% wanted alternatives to the current dental hygiene care delivery system. Most respondents stated general supervision and independent practice would increase access to oral healthcare without increasing patients' risk of health problems. CONCLUSIONS: The majority of Iowa dental hygienists support the dental hygiene profession's goal of self-regulation. ADHA members and nonmembers have similar attitudes and opinions about dental hygiene self-regulation, licensure, delivery systems, and practice. PMID- 8632196 TI - Oral healthcare of Native Americans of the Plains States. PMID- 8632195 TI - Updating a measure of dental anxiety: reliability, validity, and norms. AB - PURPOSE: The Corah Dental Anxiety Scale was introduced in 1969. Since then, changes in dental practice and language usage have dated the scale. The purposes of this study were to (1) slightly modify the scale, (2) assess its reliability and validity, and (3) develop norms for the revised version. The scale modifications recognized the roles of dental hygienists and female dentists in the dental office. Two variations of the first item also were constructed. One asked about going to the dentist "for a checkup?; the other asked about going "because of a toothache." METHODS: The revised instrument was included in face-to face interviews with 662 dentate adults living in the Detroit, Michigan, area. Reliability was estimated by Cronbach's alpha. Validity was examined by correlating the revised scale with another measure of dental anxiety and measures of conceptually related variables. RESULTS: Cronbach's alpha coefficient was 0.82 for the checkup version of the scale, 0.79 for the toothache version, and 0.82 for a scale including both variations of the first item, indicating adequate reliability. Dental anxiety correlated positively with history of dental problems (r = .13, .09, and .10 for the checkup, toothache, and combined versions, respectively), negatively with frequency of preventive dental visits (r = -.28, .22, and -.25), and positively with another measure of dental anxiety (r = .61, .59, and .62), supporting the validity of the scales. CONCLUSIONS: The checkup version of the revised scale is recommended for future use. The Dental Anxiety Scale as modified continues to be a reliable and valid instrument 20 years after its introduction. PMID- 8632197 TI - Utah's parent, teacher, and physician sealant awareness surveys. AB - PURPOSE: The Year 2000 Oral Health Objective 13.8 reads, "Increase to at least 50% the proportion of children who have received protective sealants on the occlusal surfaces of permanent molar teeth." In order to develop the next phase of Utah's continuing sealant promotion program directed towards meeting this objective, the Dental Health Bureau surveyed parents, teachers, and physicians in 1992. METHODS: Parents of 3,355 first- and sixth-grade students in a stratified random sample of 25 Utah urban and rural schools were surveyed to determine sealant awareness and placement. In 20 elementary urban and rural schools, 471 teachers were surveyed to determine their source of dental health knowledge and their dental unit subject matter. Five hundred ninety-six physicians were surveyed to determine their knowledge of sealants. The results of the parent and teacher surveys were analyzed using chi-square to determine statistically significant differences between urban and rural respondents. The physician results are reported in percentages. RESULTS: The parental response rate was 32%, teacher response rate was 37%, and physician response rate was 44%. Between 73% and 81% of the parents knew about sealants and 40% to 47% have had sealants placed on a child's teeth. The most frequently mentioned source of knowledge about sealants was the dentist or dental hygienist. Fewer rural than urban parents had heard about dental sealants and had insurance that covered sealants. Of the teachers who taught dental health, most taught brushing/flossing, sugar avoidance, and annual dental visits. Only a few taught about sealants. Most of the teachers received their information from a Dental Health Bureau newsletter or from their dentist or dental hygienist. When questioned about their knowledge of sealants, 68% of the physicians indicated they were unfamiliar with them. Eighty four percent requested professional information and 65% requested patient information. CONCLUSIONS: The dental hygienist as a preventive healthcare professional is a frequent source of sealant information. Parents, physicians, and teachers need up-to-date oral health information. Dental hygienists can be instrumental in achieving the Year 2000 Oral Health Objective on dental sealants by promoting awareness in the public sector and by placing dental sealants. PMID- 8632198 TI - Oral health behaviors in medical patients with diabetes mellitus. AB - PURPOSE: Periodontal disease is common among patients with noninsulin dependent diabetes mellitus (NIDDM) and insulin dependent diabetes mellitus (IDDM) and interferes with diabetic control. However, no study has examined predictors of oral health behavior among patients with diabetes in a medical setting. This study describes self-reported oral hygiene among primary care patients with IDDM and NIDDM, evaluating age, race, gender, psychosocial stress, family dysfunction, and other health predictors of preventive oral behaviors. METHODS: A written survey of oral health behaviors, psychological stress, and family function was conducted among 390 out of 473 possible primary care dentate patients with diabetes (IDDM=81; NIDDM=309). Fasting blood sugar and hemoglobin A1C also were obtained. Data were collected between January 1989 and June 1991, and analyzed using Mantel-Haenszel's chi-square and logistic regression. RESULTS: Patients with IDDM with severe family dysfunction were less likely to brush, floss, or visit a dentist regularly than those with better reported family function. Whites with NIDDM were more likely to practice these oral health behaviors than were blacks. Female gender correlated with flossing in the group of patients with IDDM, and with brushing in both groups. Patients with NIDDM who exercised were also more likely to visit the dentist annually. Age was positively associated with a history of periodontal disease in the group with IDDM. CONCLUSIONS: These data document for the first time the relationship between family dysfunction and oral health practices of patients with IDDM, and corroborate the known associations among white race, female gender, and oral hygiene. This information could be used to coordinate diabetic oral health promotion between primary care physicians and oral health professionals. PMID- 8632201 TI - Change and research. PMID- 8632200 TI - Effect of repeated sterilization and ultrasonic cleaning on curet blades. AB - PURPOSE: Instrument cleaning and sterilization are important steps in infection control. This study investigated the effect of repeated ultrasonic cleaning, cycles of autoclave or chemiclave, on the surface and cutting edge of stainless steel and carbon steel curets. METHODS: Eight carbon steel and six stainless steel Columbia #13/14 curets were cleaned or sterilized in a series of treatments by one of the following methods: ultrasonic cleaning, chemiclave sterilization, and autoclave sterilization. In addition, two carbon steel instruments were placed in an anticorrosive dip followed by autoclave. The blades of the curets were examined and photographed with the scanning electron microscope (SEM) at 200 X and 1000 X for baseline and after 2, 4, 8, 16, and 32 treatment cycles. Photographs were evaluated for visible change in surface appearance by four examiners independently. Photographs for each treatment time were compared to the baseline (pretreatment) photographs for the same instrument. They were scored as (1) no visible change; (2) slight change; or (3) moderate to major change. Changes observed were: surface pitting, corrosion products as additions to the surface, edge deterioration, or loss of structure. RESULTS: Stainless steel curets showed slight or no change after the three treatments. Carbon steel curets were affected by all treatments. The chemiclave produced slight change. The autoclave produced slight change after the fourth treatment cycle and moderate to major change after the eighth cycle. Those carbon steel instruments treated by anticorrosive dip followed by the autoclave showed pronounced change after 16 or 32 cycles. The most varied results were from the ultrasonic cleaner; one blade showed moderate to major change after only four cycles, the other showed slight change at that point and major change only after 32 cycles. CONCLUSION: With stainless steel curets, ultrasonic cleanings or sterilization with autoclave or chemiclave can be used without visibly affecting the cutting edge. With carbon steel instruments, chemiclave is the least damaging sterilization method followed by anticorrosive treatment before autoclaving. Use of the autoclave without the anticorrosive pretreatment or use of an ultrasonic cleaner negatively affects the integrity of the surface and cutting edge of these instruments. PMID- 8632199 TI - Interpersonal communications skills for dental hygiene students: a pilot training program. AB - PURPOSE: The educational preparation of most oral health professionals specifically has not addressed professional-patient interaction skills. The purpose of this pilot research project was to determine if the Carkhuff model of communication skills training would improve the interpersonal communication skills of junior dental hygiene students. METHODS: The pilot training program was designed to assess and improve interpersonal communication skills using an experimental pretest-posttest design. In Spring 1990, twenty-four baccalaureate dental hygiene students were randomly divided into experimental and control groups. The experimental group completed twenty (20) hours of interpersonal skills training based on Carkhuff's Human Resources Development Model. Video demonstrations, patient simulations, and role modeling were used to enhance learning and retention. Data were collected using three instruments: 1. "Acting" patient video interviews were evaluated by three independent raters to assess change in skilled behaviors. 2. A self-report instrument and a scenario/response option questionnaire were completed to examine change in knowledge of communication skills. 3. A self-report emotional empathy score determined the effects of training on the dimension of empathy. These instruments were administered pretraining, post-training, and one year later to assess differences in behavior, knowledge, and empathy due to the training and maturation. Data analysis included a two-way analysis of variance with repeated measures, frequency distributions, means, standard deviations, and Chronbach's coefficient alpha. A Mann Whitney U-test also was calculated to examine the differences in the distribution of the change scores over time. RESULTS: Post-train results demonstrated significant differences in knowledge but not in behavior. One-year retention scores showed a significant improvement (p<.01) in both knowledge and behavior by the experimental group when compared to the control group. Empathic responsiveness was not related to the training since both groups progressed modestly but at different rates over the 15-month project. CONCLUSIONS: It is important that dental hygiene practitioners have the requisite interpersonal skills to facilitate the partnership between each patient and client in attaining and maintaining optimal oral health. The results of this pilot study support the recommendation that interpersonal communication skills training become an integral part of the dental hygiene curriculum. However, further studies on this aspect of education should be completed in order to verify the results of this study and provide further support for changes in the education and socialization process of dental hygiene professionals. PMID- 8632203 TI - Self-report of oral health services provided by nurses'aides in nursing homes. AB - PURPOSE: The purpose of this investigation was to identify the oral healthcare role of nurses' aides in nursing homes. Nurses' aides were asked to report what preventive and referral oral health services they provided, factors that encourage and discourage performance of these services, and their perceived knowledge of oral hygiene care procedures. METHODS: A random sample of 40% (n = 14) of nursing home facilities in southeastern Virginia was selected. A 14-item questionnaire was administered to 121 nurses' aides employed at 11 of these nursing home facilities during April, May, and June of 1991. Frequency and percentages were analyzed for discrete, nominally, and ordinally scaled data. RESULTS: Eleven of the 14 nursing homes selected agreed to participate, representing 32% of the nursing homes in the southeastern region of Virginia. Ninety-eight out of 121 questionnaires were completed and analyzed. Nurses' aides in nursing home facilities typically reported providing preventive oral health services such as mouthrinsing (71%), toothbrushing (63%), and denture cleaning procedures (37%) for nursing home residents. The majority of nurses' aides indicated that patient cooperativeness was a major factor that encouraged (71%) or discouraged (88%) the performance of oral health services. Aides typically reported suspicious and abnormal findings detected in residents' mouths to the nurse in charge of the shift (97 5). The majority of nurses' aides rated their knowledge of mouthrinses for residents with teeth (99%), denture cleaning (99%), toothbrushing (97%), fluoride mouthrinses for residences with teeth (90%), mouth checks for residents with teeth (91%), and flossing (61%) as adequate or excellent. However, a large percentage of nurses' aides rated their knowledge of saliva substitutes (45%) and flossing (39%) as poor. CONCLUSION: This study indicated that nurses' aides generally provide daily oral hygiene services to nursing home residents. Thus, the role of the dental hygienist should be to outline specific educational and curricular guidelines and conduct training programs for nurses' aides and to develop instructional materials to be used in nursing homes. Future dental hygiene graduates must be prepared to meet the needs of oral hygiene education for nurses' aides. PMID- 8632204 TI - Emphasis on communication. PMID- 8632202 TI - The effectiveness of a low-chemical, low-protein medical glove to prevent or reduce dermatological problems. AB - PURPOSE: This study investigated the effectiveness of a low-chemical, low protein, powder-free latex medical glove to reduce or prevent hand dermatitis in a group of oral healthcare workers who had a self-reported history of hand irritation from medical glove use. METHODS: Dental hygienists who had previously reported dermatological conditions from routine use of medical gloves were invited to participate in this study to test a new medical glove. All subjects completed baseline information and 85 subjects participated in the study. Subjects wore the gloves routinely for six weeks unless complications developed. Postuse questionnaires were sent to the 85 participants. Dermatological signs and symptoms were measured at both times and subjects' perceptions of change in dermatological conditions were measured postuse. Data were analyzed by frequency distributions, paired t-test, and McNemar's chi-square statistic. RESULTS: For the 85 participants, the reduction in number of dermatological signs and symptoms was statistically significant (t = 3.46; p = 0.001) from a mean of 2.5 (sd = 2.8) to a mean of 1.3 (sd = 2.6) symptoms. A statistically significantly (chi-square = 7/8; p = 0.005) greater proportion of subjects were symptom-free at postuse (61.2%) than at baseline (41.2%). Almost 90% of the oral healthcare workers perceived that the number of dermatological signs and symptoms, the intensity of signs and symptoms, and the area involved stayed the same or decreased. CONCLUSIONS: The medical gloves used in this study were effective in reducing the number of dermatological problems associated with routine medical glove use by oral healthcare workers with self-reported histories of problems with medical gloves. PMID- 8632205 TI - Quality assurance concepts and skill development: results of a national study. AB - PURPOSE: Knowledge of quality assurance concepts and development of evaluation skills are essential to demonstrating professional accountability. Evaluation skills include self-assessment and peer review, both of which need to be specifically taught and practiced. The purpose of this study was twofold: 1) to determine if quality assurance concepts were taught and opportunities to develop evaluation skills were provided to associate and baccalaureate dental hygiene graduates, and 2) to determine if differences exist between these two groups. METHODS: In fall 1993, a 90-item survey instrument based on quality assurance concepts, skills, and the dental hygiene process and standards of care was validated and administered to a national stratified random sample of 1993 graduates (n=500). Responses were analyzed using descriptive statistics, chi square, and the Wilcoxon rank sum test. For all statistical procedures, the .05 level of significance was established. RESULTS: A 65% response rate was obtained. No graduate reported having a specific course on quality assurance. Significant differences were found between the two groups, with a higher percentage of baccalaureate graduates reporting that self-assessment and peer evaluation concepts were taught and opportunities to practice peer review skills provided. No differences were found regarding opportunities to practice self-assessment. Perhaps more important, less than 44% of both groups reported experience in peer review and 66% in self-assessment activities related to specific courses. CONCLUSION: In order to better prepare practitioners to be accountable for care provided to the public, quality assurance concepts and opportunities to develop skills must be incorporated into the curricula for all dental hygiene students. PMID- 8632206 TI - Career promotional activities of Iowa dental hygienists. AB - PURPOSE: The purpose of this research was to investigate the relationship between Iowa dental hygienists' career promotional behaviors and (1) attitudes about career satisfaction, (2) beliefs about the job market, (3) membership in the American Dental Hygienists' Association, (4) demographics, and (5) employment characteristics. METHODS: In February 1993, questionnaires were mailed to a stratified random sample of 467 Iowa dental hygienists licensed and residing in the state of Iowa. Participation in four career promotional behaviors was ascertained as well as several independent variables. The data were analyzed with frequency distributions, Pearson's correlation coefficients, and chi-square statistics. RESULTS: An adjusted response rate of 92.3% was achieved (N=423 respondents). The majority of respondents (79.1%) recommended a career in dental hygiene, but fewer than 20% participated in a dental hygiene career day, gave an undesignated monetary gift to a dental hygiene education program, or donated to a dental hygiene scholarship fund. The independent variables were not related consistently to each of the career promotional activities. CONCLUSIONS: It appears that career promotional behaviors are multifaceted and require additional research to elucidate the interrelationships and enhance coordinated approaches to dental hygiene career recruitment. PMID- 8632207 TI - Anti-infective drug therapy: implications for the lactating mother and nursing infant. PMID- 8632208 TI - Fermentable carbohydrates elevate plasma enteroglucagon but high viscosity is also necessary to stimulate small bowel mucosal cell proliferation in rats. AB - Enteroglucagon is a collective term for a small family of peptides derived from proglucagon by post-translational processing in the L-cells of the distal small intestine and colon. There is evidence that it inhibits gastric secretion, and high levels of enteroglucagon occur in plasma during intestinal adaptation, which suggests that it may also function as a trophic factor for the intestine. Certain types of soluble non-starch polysaccharide (dietary fiber) stimulate the release of enteroglucagon in rats but the mechanism is unknown. In this study we explored the importance of the viscosity and fermentability of nonabsorbed carbohydrates as determinants of plasma enteroglucagon and mucosal cell proliferation in the distal ileum of rats. Replacement of cellulose (10 g/kg) with guar gum in a semisynthetic diet led to a prompt and sustained rise in plasma enteroglucagon concentrations. Our initial hypothesis that this was a consequence of delayed nutrient absorption was disproven by the fact that hydroxypropylmethylcellulose (HPMC), a viscous but nonfermentable polysaccharide, had no effect on plasma enteroglucagon under the same conditions. In contrast, the nondigestible disaccharide lactitol led to a prolonged rise in plasma enteroglucagon, similar to that observed with guar gum. Lactitol is nonviscous, but highly fermentable, and we conclude that fermentable carbohydrate is an important stimulus for the release of enteroglucagon under our experimental conditions. There was no evidence that enteroglucagon released by this mechanism exerted trophic effects on the distal small intestinal mucosa. PMID- 8632210 TI - Dietary alpha-tocopherol attenuates the impact of gamma-tocotrienol on hepatic 3 hydroxy-3-methylglutaryl coenzyme A reductase activity in chickens. AB - The concentration-dependent impact of gamma-tocotrienol on serum cholesterol can be traced to the posttranscriptional down-regulation of 3-hydroxy-3 methylglutaryl coenzyme A reductase activity. gamma-Tocotrienol also suppresses tumor growth. Palmvitee, the tocopherol and tocotrienol-rich fraction of palm oil, is the sole commercial source of gamma-tocotrienol. Contrary to the universal findings of the efficacy of gamma-tocotrienol there are conflicting reports of the impact of Palmvitee on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, serum cholesterol concentrations and tumor development. These conflicting reports led us to examine the impact of alpha-tocopherol on the cholesterol-suppressive action of gamma-tocotrienol. Control and experimental diets were fed to groups of White Leghorn chickens (n = 10) for 26 d. The control diet was supplemented with 21 nmol alpha-tocopherol/g. All experimental diets provided 141 nmol of blended tocols/g diet. The alpha-tocopherol and gamma tocotrienol concentrations of the experimental diets ranged from 21 to 141 and 0 to 120 nmol/g, respectively. We now report that including alpha-tocopherol in tocol blends containing adequate gamma-tocotrienol to suppress 3-hydroxy-3 methylglutaryl coenzyme A reductase activity results in an attenuation of the tocotrienol action (P < 0.001). A summary of results from studies utilizing different Palmvitee preparations shows that effective preparations consist of 15 20% alpha-tocopherol and approximately 60% gamma- (and delta-) tocotrienol, whereas less effective preparations consist of > or = 30% alpha-tocopherol and 45% gamma- (and delta-) tocotrienol. PMID- 8632209 TI - Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats. AB - The effects of dietary soybean protein on lipogenic enzyme gene expression in livers of genetically fatty rats (Wistar fatty) have been investigated. When Wistar fatty rats and their lean littermates (7-8-wk old) were fed a casein or soybean protein isolate diet containing hydrogenated fat (4% hydrogenated fat plus 1% corn oil) or corn oil (5%) for 3 wk, the hepatic messenger RNA concentrations and activities of lipogenic enzymes were significantly lower in rats fed soybean protein than in those fed casein, regardless of genotype or dietary fat. The conversion rates of thyroxine to triiodothyronine by liver microsomes and plasma triiodothyronine concentrations were lower in the fatty rats than in the lean rats and were significantly greater in rats fed soybean protein than in those fed casein. Conversely, plasma and liver triacylglycerol concentrations were lower in soybean protein-fed fatty and lean rats than in those fed casein. The body weight was less in the fatty rats fed soybean protein than in those fed casein after 3 wk of feeding. Moreover, dietary polyunsaturated fatty acids suppressed lipogenic enzyme gene expression in the lean rats but did not in the fatty rats. Dietary soybean protein appeared to be useful for the reduction of obesity. PMID- 8632211 TI - Nutritional state and the swelling-induced inhibition of proteolysis in perfused rat liver. AB - Recent work indicates that cell volume is an important regulator of proteolysis in liver. The antiproteolytic effects of insulin and some amino acids (e.g., glutamine and glycine) are mediated by increases in cell volume. The purpose of the present study was to assess the role of nutritional state in the cell volume dependent regulation of proteolysis in isolated perfused rat liver. In rats that had been prelabeled by an intraperitoneal injection of L-[4,5-3H]leucine 16-20 h prior to the perfusion experiment, hepatic proteolysis was studied by determination of [3H]label release into effluent perfusate. In separate perfusion experiments [3H]inulin and [14C]urea, acting as markers for extracellular and the sum of extra- plus intracellular space, were employed for determination of effector-induced cell volume changes. Proteolysis in the perfused rat liver was inhibited by insulin-like growth factor-I (IGF-1) and taurocholic acid. Both agonists increased the intracellular water space. The nutritional state of the livers had marked influence on the hormone- and amino acid-dependent regulation of proteolysis. In livers from food-deprived rats for 24 h, the swelling responses to glycine, glutamine and alanine were enhanced, whereas the insulin- and IGF-1-induced increases of cell volume were diminished. A stronger inhibition of proteolysis was observed in livers from food-deprived rats upon addition of the amino acids, whereas the insulin- and IGF-1-mediated inhibition of proteolysis was attenuated. Independent of the nutritional state, a close relationship between the cellular hydration state and the corresponding inhibition of proteolysis was observed, regardless of whether cell volume was modified by amino acids, hormones, hypoosmotic exposure or bile acids. We conclude that the nutritional state markedly modifies the swelling potency of amino acids and hormones in liver and by this means affects proteolysis. PMID- 8632213 TI - Amylopectin starch induces nonreversible insulin resistance in rats. AB - Starches that are high in amylopectin are digested and absorbed more quickly than starches with a high amylose content and produce insulin resistance in rats during long-term feeding. The aim of this study was to determine whether amylopectin-induced insulin resistance could be prevented or reversed by a period of high amylose feeding. We employed a randomized design in which two groups of rats were fed either the high amylose and then the high amylopectin diet for two consecutive 8-wk periods or vice versa (high amylopectin and then high amylose). Four other groups were fed either a high amylose or a high amylopectin diet for 8 or 16 wk. All rats were fed two 10-g meals per day (300 kJ/d), and insulin sensitivity was assessed by intravenous glucose tolerance test (IVGTT) after 8 or 16 wk of feeding. We found no difference in glucose tolerance between any group at any time point. Insulin responses, however, were 50% higher (P < 0.01) after 16 wk of high amylopectin feeding [area under the plasma insulin curve (AUC) = 18.1 +/- 1.4 nmol.L-1 x 15 min] compared with high amylose feeding (AUC = 13.0 +/ 1.2 nmol.L-1 x 15 min). The two groups which received both diets developed a similar degree of insulin resistance, equivalent to that after 16 wk of high amylopectin feeding. The findings suggest that amylopectin-induced insulin resistance cannot be reversed or prevented by either a subsequent or previous period of amylose feeding. Taken together, the data suggest that the nature of starch in the Western diet influences the development of noninsulin-dependent diabetes mellitus in humans. PMID- 8632214 TI - Dietary energy restriction in mice negatively regulates hepatic glucose-regulated protein 78 (GRP78) expression at the posttranscriptional level. AB - Dietary energy restriction delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. We showed previously that 50% energy restriction in mice reduces hepatic expression of glucose-regulated protein mRNA by 50 to 80%. Changes in glucose-regulated protein 78 (GRP78) levels can either decrease or increase the rate of secretion of specific proteins. Therefore, energy restriction probably produces a global change in the spectrum of proteins secreted by the liver. These studies were initiated to investigate the molecular basis for the negative regulation of the gene. By use of transfection and nuclear run-on techniques, the strong induction of GRP78 gene transcription in cultured cells subjected to acute, extreme glucose deprivation has been well characterized. However, negative regulation of GRP78 gene expression in vivo by energy restriction is not as well understood. In our studies, a reduction in GRP78 protein levels determined using Western blotting closely paralleled a reduction in hepatic GRP78 mRNA measured by Northern and dot blotting. In each case the changes were statistically significant. This close correspondence indicates that energy restriction does not influence the translation rate or the stability of GRP78 protein. No statistically significant difference in the rate of transcription of the gene was detected in energy restricted mice by use of transcription run-on assays. These results strongly suggest that energy restriction results in destabilization of GRP78 mRNA, thereby repressing hepatic expression of the gene. PMID- 8632212 TI - Hepatic cytochrome P450 and UDP-glucuronosyl transferase are affected by five sources of dietary fiber in germ-free rats. AB - The influence of dietary fiber on xenobiotic-metabolizing enzymes (XME) was assessed using germ-free rats fed inulin and other sources of fiber (wheat bran, carrot, cocoa and oat). The consumption of cocoa fiber greatly modified the hepatic cytochrome P450 isoenzymatic profile, causing a strong enhancement of 1A2 and 2B1/B2 forms, concomitant with a significant decrease of the constitutive form 2C11, compared with all of the other types of fiber. Moreover, rats fed the cocoa fiber diet had a higher specific activity of hepatic UDP-glucuronosyl transferase than their carrot fiber- and wheat bran-fed counterparts. Intestinal UDP-glucuronosyl transferase was unaffected by the type of ingested fiber. Diet composition also did not alter the specific activity of glutathione-S-transferase in the liver, small intestine, or colon. Using earlier results obtained in heteroxenic rats, we show that intestinal microflora plays a key role in some of the effects of fiber on XME, although this is not a necessary prerequisite for all of the liver alterations. PMID- 8632215 TI - Exogenous nucleotides alter the proliferation, differentiation and apoptosis of human small intestinal epithelium. AB - The turnover of intestinal epithelial cells is a finely regulated process extending from undifferentiated crypt stem cells to terminally differentiated villus cells. The final phase of this maturation process is apoptosis and extrusion. Recent studies have shown that programmed cell death (PCD) occurs not only in senescent cells and in rapidly developing tissues but also in response to cellular stress preventing damaged cells from entering uncontrolled proliferation without repair. This study examined the role of exogenous nucleotides on cell proliferation, differentiation and apoptosis in organ cultures of human fetal small intestine. The addition of adenosine monophosphate (AMP), a putative stress reactant, to the culture media resulted in the suppression of crypt cell proliferation followed by the restoration of differentiation and the induction of apoptosis across a broad range of villus epithelium when compared with controls without added nucleotide. In contrast, the addition to cytidine monophosphate (CMP) to the culture media did not increase apoptosis, despite the nucleotide being taken up by the fetal small intestine in a similar dose- and time-dependent manner to AMP. Furthermore, Bax mRNA, an apoptosis-inducer gene, was increased with addition of AMP, suggesting that the induction of apoptosis may be channeled through the p53 pathway. These results suggest that a specific exogenous nucleotide, AMP, may have an important role in controlling the dynamic balance of cellular turnover in the developing human small intestine. PMID- 8632216 TI - Vitamin A supplementation fails to reduce incidence of acute respiratory illness and diarrhea in preschool-age Indonesian children. AB - Vitamin A supplementation of populations of vitamin A-deficient preschool-age children has been shown to reduce childhood mortality, but the primary preventive effects of such supplements on childhood infectious diseases have not been carefully evaluated. We conducted an individually randomized, placebo-controlled, double-masked trial among 1,407 Indonesian preschool-age children, to measure the effects of high dose vitamin A on acute respiratory and diarrheal illnesses. Signs and symptoms of morbidity were monitored using every other day home surveillance by trained interviewers. High dose vitamin A supplements increased the incidence of acute respiratory illnesses (ARI) by 8%, and acute lower respiratory illnesses (ALRI) by 39%. These detrimental effects on acute lower respiratory illnesses were most marked in children with adequate nutritional status (rate ratio 1.83, 95% confidence interval 1.257-2.669). In contrast, vitamin A tended to be protective of ALRI in chronically malnourished children (rate ratio 0.71, 95% confidence interval 0.375-1.331). There was no overall effect of high-dose vitamin A supplements on the incidence of diarrheal disease (rate ratio 1.06, 95% confidence interval 0.920-1.225). However, we found a significant interaction between supplementation and age: vitamin A increased the incidence of diarrhea in children < 30 mo of age, but tended to reduce the incidence in older children. The finding of a significant adverse effect of vitamin A supplements in adequately nourished children highlights the need to review the criteria for selecting populations of preschool-age children for vitamin A supplementation. PMID- 8632217 TI - Zinc supplementation reduces the incidence of persistent diarrhea and dysentery among low socioeconomic children in India. AB - Persistent diarrhea (PD) and dysentery (DD) account for most diarrhea-associated deaths among children in developing countries. Zinc deficiency can cause stunting and impaired immune function, both of which are risk factors for these diarrheal illnesses. We investigated the effect of zinc supplementation on the incidence of PD and DD in a community-based, double-blind randomized trial in children 6-35 mo of age. Increase over baseline in plasma zinc concentrations in the supplemented group compared with a control group (3.61 vs. 0.009 mumol.L-1), indicated successful supplementation. The overall reductions in the zinc supplemented group of 21% in the incidence of PD (95% CI -6 to 42%) and 14% in the incidence of dysentery (95% CI -15 to 36%) were not significant. There was a significant interaction of treatment effect with baseline plasma zinc concentration and age for PD and with gender for DD. In the zinc-supplemented group compared with the control group, the incidence of PD was reduced by 73% (P < 0.05; 95% CI 34 to 91%) in children with a baseline zinc < 7.65 mumol.L-1 and by 49% (P < 0.05; 95%CI 24 to 66%) in children > 11 mo of age. Zinc supplementation resulted in a 38% (P < 0.05 95%CI 8 to 59%) reduction in the incidence of DD in boys. There was no effect on PD among children 6-11 mo old or on DD in girls. In conclusion, zinc supplementation had a significant impact on the incidence of persistent diarrhea in children > 1 y old and in children with low plasma zinc, as well as on dysentery in boys. These findings may have important implications for reducing diarrhea-related morbidity and mortality. PMID- 8632218 TI - Vitamin A status of Indonesian children infected with Ascaris lumbricoides after dosing with vitamin A supplements and albendazole. AB - In developing countries, both marginal vitamin A status and intestinal helminths are common among children. Indonesian children (n = 309, 0.6-6.6 y), known to be infected with Ascaris lumbricoides, were randomized into six different treatment groups (A-F). The treatments included 210 mumol vitamin A supplement and a dose of 400 mg albendazole (5-propylthio-1H-benzimidazol-2-yl carbamic acid methyl ester) administered orally either at the same health visit (Groups B and F) or at different contact times during a 1-mo period (groups A, C, D and E). Vitamin A status was assessed both before and 3-4 wk after the treatments by the modified relative dose response (MRDR) test. Vitamin A supplementation was most important in improving the vitamin A status (P < 0.0001) of these children, whereas treatment for ascariasis alone (P = 0.370) and the statistical interaction between treatment for ascariasis and vitamin A (P = 0.752) were not. Serum retinol concentrations marginally improved (P = 0.051) in two of the groups that received vitamin A and albendazole but not in the third group that received vitamin A only. The MRDR test proved a better discriminator of the effects of these treatments on vitamin A status than changes in serum retinol concentrations. PMID- 8632219 TI - Products based on a high fiber barley genotype, but not on common barley or oats, lower postprandial glucose and insulin responses in healthy humans. AB - Postprandial blood glucose and insulin responses to cereal products made from common barley, oats or a barley genotype containing elevated levels of beta glucans were evaluated in nine healthy subjects. Porridges were made from commercial Swedish whole-meal barley or oat flours, and a mixed whole-meal porridge using the high fiber barley genotype and commercial Swedish common barley (50:50). Also studied were two types of flour-based bread products composed of high fiber barley and common barley in ratios of 50:50 or 80:20, respectively. The common oat and barley porridges produced postprandial glucose and insulin responses similar to the white wheat bread reference, suggesting that the naturally occurring dietary fiber in these whole-meal flours has no impact on the glucose tolerance. In contrast, all high fiber barley products induced significantly lower responses than did the reference product, with the glycemic and insulin indices ranging from 57 to 72 or 42 to 72%, respectively. It is concluded that "lente" products of high sensory quality can be prepared from a barley genotype with an elevated content of soluble dietary fiber. The glycemic index of these products compares favorably with that of products made from common cereals, suggesting their use as a potential component of diets for patients with diabetes and hyperlipidemia, and for individuals predisposed to metabolic disease. PMID- 8632221 TI - Dietary Aspergillus niger phytase increases iron absorption in humans. AB - Phytate is an inhibitor of iron absorption that can be removed before the intestinal site of absorption by microbial phytase, thereby increasing iron absorption from a meal. The effects of two kinds of dietary phytase, cereal phytase and microbial phytase from Aspergillus niger, on iron absorption were investigated. Iron absorption was measured from single meals containing white wheat rolls supplemented with wheat bran with or without phytase activity (expt. 1) and phytase-deactivated wheat bran with or without addition of microbial phytase from A. niger (expt. 2). Each experiment had 10 subjects and two different radio iron tracers: 55Fe and 59Fe were used for comparison of the absorption from the test meals in each experiment. No differences in iron absorption were found between meals containing wheat brain with or without phytase activity. Addition of microbial phytase to the meal containing phytase deactivated wheat bran increased iron absorption from 14.3 +/- 2.6% to 26.1 +/- 3.8% (P < 0.0001). Two pH optima, one at pH 2.0 and one at pH 6.0, were found for A. niger phytase at 37 degrees C, but activity occurred at all pH values between 1.0 and 7.5. The results suggest that effective and complete degradation of phytate occurred in the stomach when A. niger phytase was given with the meal. This may be explained by high activity of microbial phytase at physiological pH conditions of the stomach, whereas wheat phytase has a different pH optimum. PMID- 8632220 TI - Dietary iron supplementation does not aggravate experimental malaria in young rats. AB - The hypotheses that iron-deficient hosts are less susceptible to severe malaria and that iron supplementation aggravates infection have been supported by some clinical and experimental evidence. In the present study, the course of Plasmodium berghei infection was monitored in an experimental model of dietary iron deficiency and iron supplementation. Weanling Wistar rats were fed purified diets with different iron concentrations: 20 mg/kg (Group D, n = 24), 50 mg/kg (Group N, n = 24) and 100 mg/kg (Group S, n = 12). After 15 d, rats from Group D were anemic (mean hemoglobin 81 g/l). The next day, 12 rats from Group D (thereafter Group DS) and 12 rats from Group N (thereafter Group NS) were transferred to the same iron-supplemented diet as in Group S, whereas the remaining animals (Groups D, N and S) were maintained on the original diets for further 14 d. At that time, 9 rats from each group were inoculated intraperitoneally with 10(6) erythrocytic parasites (P. berghei ANKA strain), whereas 3 rats from each group remained as noninfected controls. All animals were killed 14 d after inoculation, when significantly lower levels of hemoglobin, serum iron and percent transferrin saturation were found in infected animals from Group D compared with all other groups. However, the time course of parasitemias was similar in all groups. These data indicate that the development of P. berghei was neither suppressed by iron deficiency nor enhanced by iron supplementation in this model. Furthermore, iron repletion during infection did produce a noticeable improvement of hematological variables in previously iron-deficient animals. PMID- 8632222 TI - Digestibilities of energy, protein, fat and nonstarch polysaccharides in a low fiber diet and diets containing coarse or fine whole meal rye are comparable in rats and humans. AB - The apparent digestibility of energy, protein, fat and nonstarch polysaccharides (NSP) of a low fiber diet and two high fiber diets containing coarse or fine whole meal rye bread was studied in experiments with humans and rats. Human subjects consumed the experimental diets for 3 wk each in a 3 x 3 cross over design. For the rat diets, duplicate portions of the foods consumed by the human subjects were mixed together, freeze dried and ground. There was a good agreement in the digestibility of energy (humans: 94.7 +/- 0.9, 91.2 +/- 1.2 and 91.6 +/- 1.4%; rats: 95.0 +/- 0.8, 92.5 +/- 1.4 and 91.7 +/- 1.8%) and fat (humans: 95.2 +/- 1.5, 94.4 +/- 1.0 and 94.8 +/- 2.5%, rats: 95.4 +/- 0.9, 94.0 +/- 0.4 and 94.0 +/- 0.4%) for the low fiber diet and the diets containing coarse or fine whole meal bread, respectively. Apparent and true digestibility of protein was consistently lower (P < 0.0001) in humans (apparent digestibility: 90.6 +/- 1.5, 86.2 +/- 1.4 and 86.3 +/- 2.3%; true digestibility: 95.1 +/- 1.5, 90.7 +/- 1.4 and 90.8 +/- 2.2%) than in rats (apparent digestibility: 92.3 +/- 1.1, 89.4 +/- 0.9 and 88.9 +/- 1.0%; true digestibility: 98.3 +/- 1.1, 94.9 +/- 0.9 and 94.2 +/ 1.0%) for all three diets. The digestibility of NSP tended to be lower (P < 0.066) in rats than in humans for the diet containing fine whole meal bread (rats: 59.6 +/- 8.0%, humans: 68.0 +/- 5.2%) and the low fiber diet (rats: 72.1 +/- 10.8%; humans: 80.5 +/- 7.1%), whereas it was similar in both species for the diet containing the coarse whole meal bread (rats: 66.1 +/- 6.0%; humans: 65.8 +/ 9.3%). In spite of some differences in digestibility values, our results suggest that the rat is a suitable model for humans to predict digestibility of nutrients in mixed diets containing cereal fiber sources. PMID- 8632223 TI - Biliary manganese excretion in conscious rats is affected by acute and chronic manganese intake but not by dietary fat. AB - We hypothesized that biliary excretion of manganese would be sensitive to acute and chronic variations in manganese and fat intakes. In the acute study, we gavaged rats with solutions containing 54Mn with either 0, 0.2, 1 or 10 mg Mn as MnCl2. We collected bile from unanesthesized rats that were simultaneously reinfused with bile acids. Total manganese excretion (from 0.5 to 6.5 h after dosing) was proportional to the acute doses (approximately 3.4% of doses). In the chronic study, weanling rats were fed diets containing 5 or 20 g corn oil/g diet and 0.49 or 72 micrograms Mn/g diet for 8 wk and then deprived of food for 12 h before bile collection. Manganese-deficient animals excreted only 0.7% as much manganese in bile as manganese-replete animals, but this reduction was not sufficient to prevent 50-80% reduction of tissue manganese concentrations. Moreover, biliary manganese excretion (calculated for 24 h) by both manganese deficient and manganese-replete rats (deprived of food for previous 12 h) accounted for only 1% of their manganese intake on the previous day. Dietary fat and manganese concentrations had few effects on excretion of total or individual bile acids. Ours is the first report of biliary excretion of orally administered manganese by conscious rats. PMID- 8632224 TI - beta-Carotene absorption and cleavage in rats is affected by the vitamin A concentration of the diet. AB - The purpose of this study was to examine whether intestinal beta-carotene cleavage activity, measured with the dioxygenase assay, is affected by vitamin A intake and whether this in vitro activity is a determinant of beta-carotene cleavage in vivo, measured in lymph-cannulated rats. Six groups of 10-20 rats were fed a diet with a low, normal or high retinyl palmitate concentration (120 RE, 1200 RE and 12,000 RE per kg, respectively) for 14 to 18 wk, either supplemented or not with 50 mg beta-carotene/kg in the last 6 wk. Intestinal dioxygenase activity was 90% higher (P < 0.05) in the animals fed the unsupplemented low vitamin A diet than in the animals fed the unsupplemented high vitamin A diet, whereas in beta-carotene-supplemented rats intestinal dioxygenase activity was significantly lower than in unsupplemented rats. The molar ratio between retinyl esters and beta-carotene in lymph collected over 8 h after a single intestinal dose of beta-carotene (250 micrograms) to beta-carotene unsupplemented rats fed the three levels of vitamin A was correlated with intestinal dioxygenase activity (r = 0.66, P = 0.003). Dioxygenase activity in the liver was not affected by the vitamin A concentration of the diet but was 70% higher in the beta-carotene-supplemented rats. Based on the difference in liver vitamin A contents between beta-carotene-supplemented and unsupplemented rats we estimated beta-carotene conversion factors of 9:1 for the rats fed the high vitamin A diet and 4:1 for the rats fed the normal and low vitamin A diets. Intestinal beta-carotene cleavage activity is higher in vitamin A-deficient rats than in rats with a high intake of either vitamin A or beta-carotene. The intestinal dioxygenase activity as measured in vitro is an adequate indicator of in vivo beta-carotene cleavage activity. PMID- 8632225 TI - Dietary antibiotics decrease taurine loss in cats fed a canned heat-processed diet. AB - In a crossover design, cats were fed a canned heat-processed diet (18 g dry matter/kg initial body wt) either with (+) or without (-) antibiotics [a mixture of penicillin G, procaine (25 mg/18 g diet) and tetracycline (50 mg/18 g diet)]. The (-/+) group received no antibiotics during the first 5-wk period and received antibiotics during the second 5-wk period; the (+/-) group received the reverse. Plasma, whole blood, urinary and fecal concentrations of taurine, fecal bile acid excretion and cholyltaurine hydrolase activities were measured. Consumption of antibiotics for 5 wk resulted in a lower rate of depletion of plasma taurine. Taurine concentrations decreased more over the first 5 wk in cats in the (-/+) group than in cats in the (+/-) group [from 116 +/- 26 to 26 +/- 6 mumol/L (-/+) and from 109 +/- 6 to 77 +/- 7 mumol/L (+/-) for plasma, and from 546 +/- 8 to 292 +/- 29 mumol/L (-/+) and from 560 +/- 11 to 431 +/- 20 mumol/L (+/-) for whole blood]. Urinary total taurine excretions during the 5th week were 54 mumol/d for the (-/+) group and 135 mumol/d for the (+/-) group (pooled SEM, +/- 13). Fecal total taurine excretions during the 5th week were 184 and 53 mumol/d for the (-/+) and (+/-) groups, respectively, (pooled SEM +/- 9). Most of the fecal taurine was unconjugated (free). Fecal bile acid excretions during the 5th week were 235 +/- 18 and 106 +/- 11 mumol/d for the (-/+) and (+/-) groups, respectively. Dietary antibiotics suppressed fecal cholyltaurine hydrolase activity of cats. Fecal cholyltaurine hydrolase activities during the 5th week were 279 +/- 54 and 42 +/- 10 nmol cholic acid released.min-1.g dry feces-1 in the (-/+) and (+/-) groups, respectively. After the crossover, mean values for the groups were reversed, showing that the observed changes were due to the antibiotic treatment. These results support the hypothesis that the dietary taurine requirement of cats is largely determined by the extent of microbial degradation of taurine in the gastrointestinal tract. PMID- 8632227 TI - Riboflavin-binding protein induces early death of chicken embryos. AB - Maternal riboflavin-binding protein (RfBP) mediates the deposition of riboflavin in avian eggs. A strain of Single-Comb White Leghorn chicken, genetically unable to synthesize RfBP, produces eggs deficient in riboflavin. Embryos in these eggs die on or about 13 of incubation, with severe hypoglycemia and impaired fatty acid oxidation. Injection of free riboflavin prior to incubation allows these embryos to survive and hatch normally. The egg whites of eggs from the RfBP deficient strain were injected with different relative amounts of riboflavin and RfBP to determine their effects on the developing embryo. Injection of protein bound riboflavin resulted in a substantial number of the embryos (27%) surviving until the end of the experiment (d 19) with an average weight of 13.7 g, demonstrating that some embryos were able to use the bound riboflavin. In control groups injected with riboflavin alone, 10 of 17 embryos survived until d 19 with an average weight 18.5 g, whereas those injected with saline died between d 10 and 13 with an average weight of 3.8 g. A fourth group, injected with apo-RfBP, produced embryos with an average weight of 0.24 g. This early death suggests that the protein scavenges the small amount of riboflavin normally available to the early embryo. Further studies showed that titration of RfBP-binding sites with exogenous riboflavin lessens this effect. These studies show that although riboflavin-binding protein is essential for the deposition of riboflavin in eggs, its presence in excess can diminish the availability of riboflavin to the chicken embryo. PMID- 8632226 TI - Muscle and liver protein synthesis adapt efficiently to food deprivation and refeeding in 12-month-old rats. AB - Our aim was to analyze mechanisms involved in the adaptation of protein metabolism to food deprivation and refeeding in adult rats. Twelve-month-old rats, which had been food-deprived for 113 h and refed for 6 h, were injected subcutaneously with a flooding dose of valine (with 50% [1-13C]-L-valine) to measure in vivo protein synthesis in tibialis anterior, soleus and liver. Protein and RNA contents were also measured. In both muscles, protein mass was maintained during food deprivation. Due to a drop in protein synthetic capacity (Cs), total and myofibrillar protein synthesis rates were reduced in food-deprived rats and were not stimulated by a 6-h refeeding. In contrast, protein levels were maintained lower than RNA levels in liver during food deprivation, and Cs was higher than in fed rats. Protein synthesis rates and ribosomal efficiency were reduced in food-deprived rats. Due to maintenance of protein synthetic capacity, there was a rapid stimulation of liver protein synthesis with refeeding, which induced a significant rise in protein mass (also related to an inhibition of protein degradation). In conclusion, coordinated responses of liver and muscles allowed a sparing of muscle proteins during food deprivation and a rapid recovery of liver proteins during refeeding. Control of ribosome quantity could play a critical role in these adaptations in tissue protein synthesis in adult rats. PMID- 8632228 TI - Methionine and 2-hydroxy-4-methylthiobutanoic acid are transported by distinct Na(+)-dependent and H(+)-dependent systems in the brush border membrane of the chick intestinal epithelium. AB - The pathways that facilitate the uptake of L-methionine (L-Met), D-methionine (D Met) and L-2-hydroxy-4-methylthiobutanoic acid (L-HMB) were characterized in chick intestinal brush border membrane vesicles. A model of high affinity transport (Km approximating 0.1 mmol/L) converged to the data obtained for 35S-L Met uptake under Na(+)-gradient and Na(+)-free conditions. The maximal velocity of 35S-L-Met transport was almost threefold greater in the presence of a Na(+) gradient. Concentrations (100 mmol/L) of D-Met, the L-isomers of neutral amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid completely inhibited 35S L-Met transport. A model of low affinity competitive inhibition (Ki approximately 17 mmol/L) described D-Met inhibition of 35S-L-Met transport. These data indicate that L- and D-Met are transported by a broad specificity system B type transporter. Maximal rates of 3H-L-HMB uptake were obtained under conditions of an internally directed H(+)-gradient (pHin = 7.5, pHout = 5.5). A model of intermediate affinity transport (Km approximately 1 mmol/L) described H(+) dependent 3H-L-HMB uptake across the vesicles. The data from this and other studies indicate that a H(+)-dependent, nonstereo-specific system facilitates the uptake of the hydroxy analogues of linear amino acids, including HMB. PMID- 8632229 TI - Phylloquinone and menaquinone-4 distribution in rats: synthesis rather than uptake determines menaquinone-4 organ concentrations. AB - To clarify the origin of organ menaquinone-4 (MK-4), the distributions of phylloquinone and MK-4 were investigated in rats fed diets containing phylloquinone, MK-4 or menadione (1.1, 2.2 and 31 mumol/kg diet, respectively, 6 rats per group). Warfarin (2 x 1 mg/kg subcutaneously) was given (3 rats per group) to study the effect of vitamin K cycle blockage. In rats fed phylloquinone the vitamin accumulated mainly in liver and heart. Additionally, the diet resulted in significantly higher organ MK-4 concentrations compared with the vitamin K-deficient controls. The epoxide of MK-4 also was significantly higher in some organs. The MK-4 diet increased MK-4 concentration primarily in the heart, liver and lung. Rats fed menadione had significantly higher MK-4 and MK-4 epoxide concentrations in all organs examined. The greatest accumulations were in nonhepatic organs, particularly the pancreas, salivary gland and brain. Generally, liver and plasma had low MK-4 concentrations. Warfarin treatment lowered significantly the MK-4 concentrations, whereas MK-4 epoxide accumulated. The study shows the following: 1) dietary phylloquinone is accumulated mainly in the heart and liver, 2) the MK-4 accumulation in nonhepatic organs is due to synthesis rather than uptake and 3) MK-4 rather than phylloquinone may be the functional vitamin in nonhepatic organs. PMID- 8632230 TI - Moderate food restriction abolishes the pregnancy-associated rise in serum growth hormone and decreases serum insulin-like growth factor-I (IGF-I) concentrations without altering IGF-I mRNA expression in rats. AB - Pregnancy-associated growth of maternal and fetal tissues is likely mediated by insulin-like growth factors (IGF). To study the effect of food restriction during pregnancy on the IGF system, pregnant rats either had free access to food (control) or were fed 60% of control food intake. Serum and liver samples were obtained throughout gestation. Serum IGF-I and growth hormone (GH) concentrations were measured by RIA, and IGF binding proteins (IGFBP) were characterized by Western ligand blotting and gel filtration chromatography. Weight gain of control dams was nearly twice that of restricted dams. Litter size was not significantly different; however, fetuses and placentas of restricted dams were 20% smaller than those of controls. Serum IGF-I concentrations on d 20 were 35 and 23% of d 5 concentrations in control and restricted pregnant rats, respectively. However, hepatic IGF-I mRNA did not differ between the treatment groups. A pregnancy associated rise in serum GH was observed in control but not food-restricted dams. Insulin-like growth factor binding protein with apparent molecular retention of 38-42 kDa (IGFBP-3), 29-31 kDa and 24 kDa were apparent in serum of pregnant dams in early gestation; however, IGFBP-3 was no longer detected by Western ligand blot by d 15 of gestation. The decline in IGFBP-3 was accompanied by an increase in IGFBP-1 and -2, particularly in the serum of food-restricted dams. These data suggest that in food-restricted pregnant rats, serum IGF-I and IGF-I mRNA are insensitive to serum GH concentrations and are not regulated at the level of transcription. PMID- 8632231 TI - Fermentation of carbohydrate in rat ileal excreta is enhanced with cecal inocula compared with fecal inocula. AB - The differential fermentative capacities of microflora from two regions of the large bowel and how fermentation was altered by prior exposure of the microflora to the substrate to be fermented were studied using an in vitro fermentation system. Bacterial inocula were prepared from cecal contents and feces from three groups of rats fed purified diets containing 100 g/kg dietary fiber from canned peas or psyllium seed husk, or a nonpurified diet containing 170 g/kg dietary fiber. The substrate for all fermentations was ileal excreta from colectomized rats fed a purified diet containing 100 g/kg dietary fiber from canned peas. Anaerobic fermentations were conducted for 0, 3, 6, 12, 24, 48 and 96 h. Sugars of the unfermented polysaccharides were measured by gas chromatography following acid hydrolysis; disappearance was the measure of fermentation. Independent of inoculum source, > 90% of the starch and arabinose and 75% of the uronic acids, but < 30% of non-starch glucose (the measure of cellulose), were fermented by 24 h. Cecal inocula fermented arabinose and uronic acids more quickly (P < 0.05) and fermented more (P < 0.05) non-starch glucose than fecal inocula. Inocula adapted to psyllium seed husk fermented starch faster (P < 0.05) and non-starch glucose, arabinose and uronic acids more slowly (P < 0.05) than inocula adapted to peas or nonpurified diet. Bacterial efficiency of carbohydrate utilization, the increase in muramic acid/mole carbohydrate fermented, was greatest (P < 0.05) with cecal inocula adapted to peas. We conclude that using cecal microflora as the inoculum source provides a more accurate index of fermentation during transit through the large bowel and that noncellulosic and storage polysaccharides of the plant cell wall are utilized before cellulose. PMID- 8632232 TI - Obese pigs fed a high cholesterol diet from birth to 2 months are less susceptible than lean pigs to atherosclerosis. AB - Dietary cholesterol in infancy may alter cholesterol metabolism and the propensity to develop atherosclerosis. This study examined the effects of a 1% cholesterol diet (HC) vs. a no-cholesterol diet (NC) during the first 2 mo of life on pigs selectively bred for leanness or obesity. Three lean and three obese pigs received the no-cholesterol diet, and four lean and four obese pigs received the 1% cholesterol diet from d 1. Lean and obese pigs fed the no-cholesterol diet showed no increase in serum lipid concentrations, nor did they develop atherosclerosis. Obese pigs fed the 1% cholesterol diet developed significantly higher serum total cholesterol (TC) and high density lipoprotein cholesterol (HDL C) at 35 d than lean pigs fed the 1% cholesterol diet. By d 55, only HDL-C remained significantly higher in the obese pigs, resulting in a higher (P < 0.1) TC/HDL-C ratio in the lean pigs. Atherosclerotic plaque formation in the aorta was more extensive in the lean pigs. Cholesterol synthesis measured in vivo and at termination was equally suppressed in lean and obese pigs fed the 1% cholesterol compared with pigs fed the no-cholesterol diet. We conclude that genetic differences in the response of these lean and obese pigs to a high cholesterol diet render obese pigs less susceptible to atherosclerosis despite higher serum TC concentrations. The persistent elevation of HDL-C in obese pigs is the most likely mechanism of protection. PMID- 8632235 TI - Maintaining the standards of surgical training. PMID- 8632233 TI - The protein efficiency ratios of 30:70 mixtures of animal:vegetable protein are similar or higher than those of the animal foods alone. AB - Animal foods in general are considered to be foods with high protein qualities, although their qualities are not always similar because of differences in essential amino acids. The purpose of this study was to compare the protein quality of different animal foods and of their mixtures with vegetable foods, mainly cereals, at the 30:70 animal:vegetable protein proportion with experiments performed under the same conditions. The animal foods were eggs, beef, pork, barbecued lamb, chicken, ham, sausage and milk powder. The vegetable foods used in the mixtures were rice, lime-treated corn flour, wheat flour and cooked black beans. The protein concentrations in the raw and cooked materials were analyzed. The protein efficiency ratios (PER) and digestibilities were determined in Fisher 344 weanling rats. Based on the corrected PER, the foods with the best protein quality were egg (3.24), sirloin beef (3.16), lamb (3.11) and chicken breast (3.07), which were significantly different (P < 0.05) from milk powder (2.88) and beef liver and beef round (2.81 and 2.70, respectively). The ham (2.63) and the pork loin (2.57) had a similar protein quality to that of casein (2.50). The lowest protein quality was found in sausages (2.14). In most of the mixtures of animal and vegetable protein (30:70), the PER was similar to or higher than that of the animal food alone. Beans were the vegetable food that showed the lowest response to the addition of animal food. The conclusion of the study is that some 30:70 mixtures of animal:vegetable protein, such as chicken, beef round and pork with cereals could be utilized for regular meals because of their high PER and low cost. PMID- 8632234 TI - Soybean protease inhibitors and pancreatic carcinogenesis. PMID- 8632236 TI - Reconstruction of the severely atrophic edentulous maxilla using Le Fort I osteotomy with simultaneous bone graft and implant placement. AB - PURPOSE: This study evaluated the outcome of patients who underwent simultaneous Le Fort I osteotomy, an interpositional bone graft, and implant placement for reconstruction of the severely atrophic edentulous maxilla. PATIENTS AND METHODS: Twenty patients operated by the same surgeon were included in the study. Patients were followed annually with clinical and radiographic examinations for an average of 33 months. RESULTS: One hundred thirty-nine implants were initially inserted in the bone grafts at the time of Le Fort I osteotomy. Twenty-five implants (18%) failed to osseointegrate. Seventeen of the 25 implants lost were from three patients. The most significant prognostic factor appeared to be the thickness of the atrophic maxillary ridge. Twelve of the 20 patients completed prosthetic restoration, with an average follow-up of 21 months after loading. No implants were lost after loading, and all prostheses have remained stable. CONCLUSIONS: This method of reconstruction of the severely atrophic maxilla achieves an implant survival rate of 82% while correcting the unfavorable maxillomandibular relationship commonly seen in these patients. PMID- 8632237 TI - Modification of the modified condylotomy. AB - PURPOSE: This article describes the results of modification of the condylotomy procedure for treating painful internal derangements of the temporomandibular joint. MATERIALS AND METHODS: Data on postoperative disc position, joint space, and pain were evaluated in patients who underwent either the original modified condylotomy or the newly modified procedure. RESULTS: Less anterior and inferior sag of the condyle was produced by the new technique. Pain relief and disc reduction were about the same with both procedures. CONCLUSIONS: The new modifications make the operation quicker and easier, while producing outcomes similar to those obtained with the original modified procedure. PMID- 8632238 TI - Computer-assisted mandibular condyle positioning in orthognathic surgery. AB - PURPOSE: The correct placement of the mandibular condyle in the glenoid fossa during the osteosynthesis after the sagittal ramus osteotomy is often a problem. This article describes a computer-assisted positioning device. METHODS: The procedure is based on the use of a three-dimensional optical localizer to establish the condylar segment position. The operative procedure is only slightly different from the usual. RESULTS: The system is very easy to use, it does not require preoperative data acquisition, and the accuracy obtained is in fractions of a millimeter. PMID- 8632239 TI - The auriculomastoid fasciocutaneous island flap: a new flap for orofacial reconstruction. AB - PURPOSE: Various designs for previously reported postauricular skin flaps have the disadvantages of being two-stage procedures, providing a limited pedicle, or requiring microvascular anastomoses. To overcome such problems, a new auriculomastoid fasciocutaneous (AMFC) island flap for orofacial reconstruction has been developed. This article presents the technique and reports the clinical results. PATIENTS AND METHODS: A long ipsilateral AMFC island flap pedicled by the parietotemporal fascia and based on the parietal branches of the superficial temporal artery, the occipital artery, and the postauricular artery was designed. Twenty-five of these island flaps were used to reconstruct intraoral or external facial defects. RESULTS: The fasciocutaneous flap could be extended to reach any orofacial defect as a single or compound design in a one-stage subcutaneous procedure. Compound types of AMFC island flaps, including scalp, parietal bone, or the parietotemporal fascia were performed successfully based on single vessels. The flap has the characteristics of providing thin and pliable skin, a good color match to the face, and restored sensitivity. The donor defect is designed to be closed directly and concealed behind the ear without ear deformity. CONCLUSIONS: This flap is very useful in orofacial reconstruction because the skin quality equals that of the radial forearm flap, without the need for microvascular anastomoses, with many additional advantages and various modifications of design. PMID- 8632240 TI - Endoscopic forehead lift: technique and case presentations. AB - PURPOSE: The advent of the endoscopic forehead lift has provided an alternative to the conventional open approach. This article describes the basic technique, with some modifications, and reports three clinical cases. RESULTS: The subperiosteal forehead technique rejuvenates the upper third of the face with no scalp resection, minimal risks of hypesthesia, limited risk of alopecia, reduced tissue trauma, small camouflaged scars, less bleeding and edema, improved postoperative comfort and faster recovery compared with the standard open techniques. CONCLUSIONS: The endoscopic subperiosteal forehead lift is a useful technique for providing rejuvenation of the upper third of the face. It reduces or eliminates forehead rhytids by eliminating the reflex contracture of the frontalis and contributes to softening of the vertical glabellar rhytids. Longitudinal studies will be required to assess the effectiveness of this technique compared with open techniques. PMID- 8632241 TI - Methohexital infusion technique for conscious sedation. AB - PURPOSE: The purpose of this study was to evaluate a methohexital infusion technique for conscious sedation in oral surgical procedures. Patients were evaluated for recall, comfort, recovery, and surgeon's estimate of cooperation. MATERIALS AND METHODS: Twenty adult (mean age, 29 years) ASA I or II patients requiring various dentoalveolar procedures were entered into the study. Administration of 1.5 micrograms/kg fentanyl and 1 mg midazolam were given until sedation was achieved. An infusion of methohexital was started using 50 micrograms/kg/min and increased as needed to 75 micrograms/kg/min. Postoperatively, visual analog scales were used to evaluate the efficacy of the technique. RESULTS: The average total amount of methohexital infused was 257 mg for an average surgical time of 88 minutes. Recovery times were short and uneventful. Patients were cleared for discharge in under 35 minutes. No cardiac or respiratory side effects were noted other than a mild increase ( < 15%) in heart rate. Shivering and hiccoughing were noted in two and four cases, respectively. Recall of surgical events reported by patients was low and patient cooperation was high as reported by surgeons. CONCLUSION: The continuous infusion of methohexital for conscious sedation has been shown to be safe, effective, inexpensive, and well accepted by patients and surgeons. PMID- 8632242 TI - Intralesional vinblastine injections for the treatment of oral Kaposi's sarcoma: report of 10 patients with 2-year follow-up. AB - PURPOSE: This study evaluated the use of intralesional vinblastine injections for the treatment of the intraoral lesions of Kaposi's sarcoma. PATIENTS AND METHODS: Eighteen patients were treated with up to three intralesional injections of 0.1 mg/cc vinblastine. RESULTS: All lesions responded to the local injections; 40% required one injection; 31%, two injections; and 29%, three injections. Large, exophytic lesions usually required the multiple injections. No complications were encountered, and the patients tolerated the protocol well. Ten patients were followed for 24 months. Eight died of their general disease during this period. Four patients developed new intraoral lesions, which were treated with, and responded favorably to, the three-injection protocol. CONCLUSION: Intralesional vinblastine injections are an effective and useful treatment alternative for human immunodeficiency virus (HIV)-associated intraoral Kaposi's sarcoma. PMID- 8632244 TI - Mandibular lengthening by distraction osteogenesis using osseointegrated implants and an intraoral device: a preliminary report. AB - PURPOSE: This study investigated an approach to distraction osteogenesis of the mandible using osseointegrated implants and an intraoral device. MATERIALS AND METHODS: Five adult dogs were used for this experiment. After the extraction of the left mandibular premolar and molar teeth, two osseointegrated implants were placed. Abutment connection, attachment of the intraoral distraction device, and an osteotomy in the region between the implants were performed 3 months after implantation. The distraction was done at rate of 1 mm/day for 10 consecutive days to elongate the mandible 10 mm. The animals were killed 2, 3, and 4 weeks after the distraction was completed, and radiographic and histologic examinations were done. RESULTS: The longer the time after completion of distraction, the more uniform the new bone that was observed radiographically and histologically in the gap created by the distraction. The titanium implants remained stable during the course of mandibular lengthening. CONCLUSION: An intraoral device using osseointegrated dental implants can serve as a mechanism for distraction osteogenesis in the maxillofacial skeleton. PMID- 8632243 TI - Ultrasonic root-end preparation: clinical and radiographic evaluation of results. AB - PURPOSE: This study evaluated the results of periradicular surgery performed using ultrasonic root-end preparation. PATIENTS AND METHODS: The course of healing during observation periods ranging from 6 months to 3 years was examined in 157 teeth. RESULTS: One hundred forty-five teeth were successfully treated, for a success rate of 92.4%. CONCLUSION: It was concluded that the ultrasonic root-end preparation technique for periradicular surgery is a simple procedure that provides excellent results. PMID- 8632245 TI - Functional stability of sagittal split ramus osteotomies: effects of positional screw size and placement configuration. AB - PURPOSE: This study examined the effects of positional screw size and placement configuration on osteotomy site stability in a sagittal split ramal osteotomy model. MATERIALS AND METHODS: A reproducible analog of a sagittal split ramus osteotomy was developed to facilitate equitable comparisons of fixation stability. The distal segment of each analog was advanced by 7 mm and fixed bilaterally with 2.0-mm or 2.4-mm cortical screws applied in either a linear or a triangular configuration. Reduced analogs were placed in a straining frame, and simulated masticatory loads wee applied alternatively to the mandibular first molars. Ensuring osteotomy site displacements were acquired and registered by displacement transducers attached to a computer-based data acquisition program. A coordinate transformation procedure was used to convert the component displacements captured by the individual transducers into a common "instability factor" to reflect fixation stability for each construct and loading condition. RESULTS: Between configurations, the magnitude of interfragmentary displacement was less when the screws were applied in a triangular than in a linear configuration (P < or = .0001). Within configurations, 2.4-mm cortical screws provided greater stability than 2.0-mm screws. The stability profiles of the 2.0 mm screws, applied in a linear configuration at the superior border, were very similar regardless of whether two or three screws were used. The loads at which the linear systems became unstable were significantly higher (P < or = .0001) than for the triangular systems. Despite the twofold or higher difference in the mean instability factor of the individual test constructs, the SDs for each construct were only one tenth to one fifth of the corresponding means. CONCLUSIONS: None of the internal screw systems were grossly unstable when used to stabilize ramal osteotomies. Other conditions, including quality and strength of investing bone, being equal, cortical screws placed in a triangular configuration provide greater stability than screws placed in a linear configuration. Osteotomy sites stabilized by 2.4-mm screws are more stable than those stabilized by 2.0-mm screws. It does not matter whether two or three screws are applied at the superior border in a linear configuration, provided that the outer screws are an inch apart. The model system has a very good measurement precision and has important implications in optimizing fixation device design and placement configuration. PMID- 8632246 TI - Retention of asymptomatic bone plates used for orthognathic surgery and facial fractures. AB - Over the past three decades there has been an improvement in biomaterials that has virtually replaced the use of stainless steel with titanium and its alloys. Based on the possibility of causing corrosion, toxicity, hypersensitivity, and stress protection, stainless steel should not be considered as a permanent implant in maxillofacial fixation. However, because of their superior corrosion resistance, noncarcinogenicity, hyposensitivity, nontoxicity, and excellent tissue compatibility, titanium and Ti-6Al-4V may be retained as permanent implants in maxillofacial fixation. The stress protection offered by such devices, and the possible complications associated with their removal, also support this concept. Finally, the costs incurred in removal do not justify the benefits derived. PMID- 8632247 TI - Removal of asymptomatic bone plates used for orthognathic surgery and facial fractures. PMID- 8632248 TI - Economic analysis and its application to oral and maxillofacial surgery. AB - PURPOSE: Recent clinical publications have increasingly emphasized comparison of cost with benefits in such areas such as drug therapies, surgical procedures, and prophylaxis regimes within certain populations. In the past, cost analysis was based principally on the comparative market price of new treatment compared with standard therapy. Benefits were assessed solely in terms of objective clinical and imaging improvement. Now, issues such as quality of life, early return to occupation, and subjective symptoms of pain and discomfort caused by a treatment are also being critically evaluated. Addressing these latter issues, however, is often complicated and expensive. This article reviews some terms and principles of cost analysis, cost effectiveness, and cost-benefit analysis. Examples are given of recent attempts to quantify costs and benefits for individuals, hospitals, health organizations, and society as a whole. Guidelines are suggested concerning how these studies can be applied to oral and maxillofacial surgery. PMID- 8632249 TI - Adenomatoid hyperplasia of the palate in an Asian child. PMID- 8632250 TI - Arthroscopic diagnosis and treatment of temporomandibular joint synovial chondromatosis: report of a case. PMID- 8632251 TI - Nail gun injury to the maxillofacial region: report of a case. PMID- 8632252 TI - Adenomatoid odontogenic tumor arising in a calcifying odontogenic cyst. PMID- 8632253 TI - Necrotizing fasciitis of the cervical region in an AIDS patient: report of a case. PMID- 8632254 TI - Concurrent osteochondroma of the mandibular condyle and ipsilateral cranial base resulting in temperomandibular joint ankylosis: report of a case and review of the literature. PMID- 8632255 TI - Squamous cell carcinoma arising in association with an orthokeratinized odontogenic keratocyst. Report of a case. PMID- 8632256 TI - Technique for vertical positioning of the maxilla after Le Fort osteotomy. PMID- 8632257 TI - Tranexamic acid rinses in anticoagulated patients. PMID- 8632258 TI - The safety and efficacy of thoracoscopic lung biopsy for diagnosis and treatment in infants and children. AB - Thoracoscopic techniques were used to perform lung biopsies and limited resections in 36 consecutively treated cases. Biopsies were performed for interstitial lung disease in 27 cases, presumed metastatic lesions in 5, and cavitary lesions in 4. Histological diagnosis was obtained in 35 of the 36 cases, and therapy was directly affected by the results in 30 of 36 cases. There were no postoperative complications, and the average hospital stay for patients admitted the morning of surgery was less than 2 days. Limited thoracoscopic resection provides a safe and effective means for diagnosing and treating parenchymal disease of the lung. PMID- 8632259 TI - Continuous thoracic epidural infusions for postoperative analgesia after pectus deformity repair. AB - PURPOSE: To determine whether continuous epidural analgesia after repair of a pectus deformity is a viable and safe alternative to high-dose narcotics in children. METHODS: Data were collected prospectively for 19 children (4 to 17 years of age; 15 boys, 4 girls) who underwent pectus excavatum (14) or carinatum (5) repair between June 1, 1991 and July 1, 1994. Seventeen had a thoracic epidural catheter placed for postoperative pain control and two did not. The epidural catheter was routinely plead preoperatively by the anesthesiologist at the T3-T8 level, after induction of general anesthesia. Epidural catheters were test-dosed with local anesthesia alone or in combination with fentanyl, and afterward a continuous epidural infusion was maintained on the floor. Postoperative pain was assessed by nursing and house staff on the Wong-Baker scale, with adjustment of the dose rate or analgesic medication as appropriate. RESULTS: All patients had extubation before leaving the operating room and were sent to the general pediatrics ward after leaving the recovery room. The average duration of the epidural was 69 hours (range, 20 to 116 hours). Sixteen patients received their test epidural dose preoperatively, and one patient had his in the recovery room. Fifteen epidural initially were dosed with bupivicaine (1 to 2 mg/kg) alone or in combination with fentanyl (1 to 2 micrograms/kg). Two patients received initial doses of lidocaine (1 to 1.5 micrograms/kg). Ten of 17 patients received fentanyl (1 microgram/kg/h) with bupivicaine (0.5 to 1.0 mg/kg/h) in the epidural as their maintenance medication, and the remainder received bupivicaine alone at the same dosage rate. Eight of 17 patients required additional intermittent supplemental narcotics, with an average of two doses of intravenous morphine per day (0.1 mg/kg) over the first 3 postoperative days. In contrast, the two patients who did not have an epidural catheter for pain control required high-dose intravenous morphine (0.2 mg/kg) every 2 to 3 hours for the first 3 to 4 postoperative days. No catheter-related complications occurred. CONCLUSION: Thoracic epidural analgesia was completely successful in nine (53%) children who underwent repair of pectus deformity, and effectively reduced the intravenous narcotic demand in the other eight. Pain control was excellent, and no catheter related complications were encountered. The data show that this method of analgesia in children is a safe and attractive alternative to intravenous narcotics, and eliminates the potential disadvantages of sedation and respiratory compromise. PMID- 8632260 TI - A comparison of laparoscopic and traditional open splenectomy in childhood. AB - This study aimed to determine whether laparoscopic splenectomy is more advantageous than open splenectomy in pediatric patients. Data from 61 patients treated between June 1983 and September 1994 were reviewed. Length of hospitalization, hospital costs, operating time, and postoperative complications were evaluated. Forty-seven patients had open splenectomy. Nineteen of these underwent concomitant procedures. Fourteen patients had laparoscopic splenectomy, and four had concomitant cholecystectomy. The data show a trend toward a 1-day reduction in hospital stay associated with laparoscopic splenectomy (P < .02). Operating time was 83% longer for the laparoscopic approach (P < .001), and operating costs were almost $3,000 more (P < .001) than for open splenectomy. The total hospital cost also was greater for laparoscopic procedures (P < .1), primarily reflective of a more than $3,000 difference for splenectomy alone (P < .02). Two of the fourteen laparoscopic patients (14%) had complications. One patient with Evan's syndrome had pneumonia that required antibiotics. Another patient required conversion to an open procedure because of poorly controlled hemorrhage from a short gastric vessel. Twelve of the open splenectomy patients (25%) had complications: atelectasis (3), fever (4), wound infection (2), pneumonia (1), laryngospasm (1), and pancreatitis (1). The authors conclude that laparoscopic splenectomy is a safe but currently more expensive alternative to open splenectomy, primarily because of the use of disposable instruments. Benefits include a shorter hospital stay, no greater risk of postoperative complications, and subjective improvement in the cosmetic result. Disadvantages include increased operating time and cost. Evaluation of larger series will be needed to determine the significance of the difference in complication rates between the two procedures. PMID- 8632262 TI - Meconium obstruction in markedly premature infant. AB - Markedly premature infants may present with intestinal obstruction and perforation secondary to inspissated meconium in the absence of cystic fibrosis. Between 1990 and 1994, 13 patients were treated for intestinal obstruction secondary to inspissated meconium. The average birth weight was 760 g. Prenatal and postnatal risk factors were identified, and included intrauterine growth retardation, maternal hypertension, prolonged administration of tocolytics, patent ductus arteriosus, hyaline membrane disease, and intraventricular hemorrhage. Stooling was absent or infrequent during the first 2 weeks of life. Surgical presentation consisted of distension and/or perforation between days 2 and 17 of life. Twelve patients required operative intervention. Findings invariably included one or more obstructing meconium plugs with proximal distension and frequent necrosis of the dilated segments. Surgical options consisted of resection or enterotomy, accompanied by primary closure or by distal irrigation and exteriorization. Irrigation led to iatrogenic bowel injury in two patients. One patient was managed successfully with oral and rectal gastrograffin and oral acetylcysteine. Ten patients were discharged, all of whom had normal stooling patterns and tested negatively for cystic fibrosis. Three patients died, two from the primary disease. The markedly premature infant is at risk for obstruction and eventual perforation secondary to meconium plugs, presumably formed in conjunction with intestinal dysmotility. Prompt diagnosis and timely intervention require a high index of suspicion, attention to stooling patterns and abdominal examinations, and screening radiographs when indicated. PMID- 8632261 TI - Recurrence of immune thrombocytopenic purpura after splenectomy. AB - Immune thrombocytopenic purpura (ITP) frequently leads to splenectomy. Accessory spleens maybe found in a variety of locations, and may not be readily apparent. Retained accessory splenic tissue can lead to recurrent ITP, which, this report (involving multiple relapses in a single patient) demonstrates. PMID- 8632263 TI - A brief account of the founding of the American Pediatric Surgical association. PMID- 8632265 TI - Acalculous cholecystitis in children. AB - Acalculous cholecystitis (AC) is a rare disease in children, and its spectrum has not been well established. Twenty-five children with AC were identified (treated between 1970 and 1994) by retrospective clinical and pathological review. The authors recognized two distinct forms of this disease: acute (duration of symptoms < 1 month) and chronic (duration > 3 months). Thirteen children had acute AC. Seventy-five percent were males; the age range was from 2 months to 20 years. Of these cases, six occurred in the immediate postoperative period, five were in association with a systemic medical illness, and two had an infectious cause (Salmonella). The mean time of onset of symptoms ranged from 4 to 30 days after surgery or hospitalization (mean, 16 days). All children presented with fever, right-upper-quadrant pain, and vomiting. Other manifestations included jaundice (38%) and right-upper-quadrant mass (23%). Most had leukocytosis (76%) and abnormal liver function test results (62%). Ultrasonography was the most commonly used radiological test, and all 10 cases tested met the ultrasonographic criteria for acute AC. Cholecystectomy was performed in nine children, and pathological examination confirmed cholecystitis. No postoperative complications occurred. The other four children were managed nonoperatively with intravenous antibiotics. One died, but the other three recovered fully. Twelve children had chronic AC. Sixty-seven percent were females; the age range was 7 to 18 years. All presented with chronic symptoms of right-upper-quadrant pain and nausea or vomiting. The leukocyte count and results of liver function tests were normal. Seventy-five percent had evidence of abnormal gallbladder function (noted by a radionuclide hepatobiliary scan or cholecystography). All children in this group underwent cholecystectomy, with pathological confirmation of chronic inflammation. No complications occurred, and all patients had complete resolution of symptoms. The authors conclude that AC in children occurs in two distinct patterns. The acute and chronic forms differ in their clinical setting and presentation. Cholecystectomy is effective treatment of AC, although there may be a role for nonoperative management in selected cases. PMID- 8632264 TI - The inflammatory response in pediatric biliary disease: macrophage phenotype and distribution. AB - PURPOSE: Extrahepatic biliary obstruction in infants and children leads to ductal hyperplasia and portal fibrosis. Inflammatory mediators responsible for increased cellular proliferation and matrix deposition are hypothesized to result from the intrahepatic recruitment and activation of lymphocytes and macrophages (M phi). The authors previously showed components of this mechanism in studies that demonstrated increased adhesion molecule expression in biliary atresia, as well as evidence of altered hepatic M phi function during the course of experimental cholestatic liver injury. Therefore they sought determine the expression of macrophage receptor markers CD68 and CD14 in pediatric biliary disease. METHODS: Sixteen liver specimens were snap-frozen and cryosectioned onto polylysine-coated slides. Sections were stained with murine monoclonal antibodies to CD68 (resident M phi) and CD14 (monocyte-M phi lipopolysaccharide [LPS] receptor) glycoproteins. The sections were analyzed using a semiquantitative scale of proliferation and were position-graded from 0 to 3 (maximal). RESULTS: Blinded analysis showed that marked proliferation of CD68-positive cells occurred in five of the six patients with biliary atresia (BA) and in one patient who had severe cholestasis. Normal perisinusoidal liver M phi were found in specimens from patients with hepatitis (2), choledochal cyst (1), and congenital hepatic fibrosis (1). Similarly, expression of CD14 periportal M phi was found only in patients with BA or cholestasis (1.9 +/- 0.3 [mean +/- SEM]) and was absent in other diseases. Strong sinusoidal expression of CD14 was evident in all patients who had extrahepatic biliary obstruction. An early biopsy specimen from a premature infant with BA did not show cholestasis, fibrosis, CD68 Mo proliferation, or CD14 expression; however, another biopsy specimen, obtained further in the course of jaundice showed the progressive development of all features. CONCLUSION: These findings suggest proliferation of resident M phi in association with cholestasis. The presence of the LPS receptor on periportal cells during cholestatic liver injury points to a potential source of cytokines responsible for the inflammatory reaction of biliary atresia. PMID- 8632266 TI - Islet Autotransplantation after total pancreatectomy in a child. AB - Islet autotransplantation can prevent surgically induced diabetes after total pancreatectomy in adults; however, the efficacy of this procedure has not been established in children. The authors report the case of a 12-year-old boy who underwent total pancreatectomy and islet autotransplantation for intractable pain caused by idiopathic chronic pancreatitis. Islets were prepared from the excised pancreas by collagenase digestion and mechanical dispersion. The resultant preparation, containing 109,500 islets, was injected into the recipient's liver via the portal vein. No complication from the pancreatectomy or transplant occurred. Postoperatively, the patient had complete relief of abdominal pain. He remained insulin-independent, with normal fasting blood glucose and hemoglobin A1c levels, for 21/2 years. Preoperatively, the acute insulin response and the rate of glucose disappearance (Kg) were 213 microU/mL and 2.14% (respectively) after intravenous administration of 20 g of glucose. Although lower than pretransplantation values, both insulin response and Kg remained normal at 4 months (88 microU/mL; Kg, 1.01%); however, these decreased further, to below normal, by 2 years posttransplantation (10 microU/mL; Kg, 0.67%). Two-and-a-half years after transplantation, fasting hyperglycemia (> 200 mg/dL) was evident, and the patient was begun on exogenous insulin. Five years posttransplantation he remains insulin-dependent with a fasting serum C-peptide level of 0.20 ng/mL, which increased to 0.35 ng/mL in response to intravenous arginine, indicating sustained islet function. During the documented decreases in insulin secretion and Kg posttransplantation, the patient's body weight increased by 65% (from 34 to 56 kg) as a result of normal growth; the number of transplanted islets relative to body mass decreased accordingly, from 3,200 to 1,950 islets per kilogram of body weight. In this case, the number of islets transplanted likely could not meet the increased insulin demands of the larger body mass. Thus, exogenous insulin supplementation was needed to prevent hyperglycemia. In conclusion, insulin independence was initially established in a child by islet autotransplantation after total pancreatectomy. The failure of the islets to maintain normoglycemia long-term suggests that a sufficient number must be transplanted (to meet the demands of normal growth and development) for sustained insulin independence. PMID- 8632268 TI - In utero lung growth of fetal sheep with diaphragmatic hernia and tracheal stenosis. AB - The observation that tracheal ligation produces pulmonary hyperplasia even in animals with surgically induced diaphragmatic hernia (DH) has led to rapid application of the technique to human fetuses with DH. The aim of this study was to determine how rapidly fetal lung volume increases after creation of a high grade tracheal stenosis in fetal sheep with surgically created DH. Twenty-three fetal sheep were prepared with a left thoracotomy at 90 days' gestational. Six had creation of a DH with tracheal stenosis (DHTS) over an 18-gauge cannula, which was then removed. Ten had DH alone, and seven control animals (CT) had a thoracotomy without DH. Thirty days later, vascular and tracheal loop catheters were inserted in all animals and tunneled out the ewes' flank. Between 125 and 140 days' gestation, lung volumes and lung liquid production were measured in awake, unanesthetized animals using a standard double-marker dilution technique. Average lung volumes (in milliliters) were 150.9 +/- 13.9 for CT, 29.3 +/- 4.4 for DH, and 414.5 +/- 88 for DHTS (p < 0.01). Mean lung liquid production varied from 6.00 +/- 2.23 mL/h in DH animals before 130 days to 16.69 +/- 8.29 mL/h in DHTS animals after 135 days' gestation. DH animals had lower lung liquid production (8.51 +/- 1.4 mL/h) than CT (12.4 +/- 0.8 mL/h) or DHTS animals (12.4 +/- 2.2 mL/h)(P < .01). The rate constant gamma (h-1) for lung liquid production was significantly higher in DH animals than in either CT or DHTS animals (P < .01). Tracheal stenosis in this model causes rapid lung growth before 130 days' gestation. The authors speculate that short periods of incomplete stenosis might reverse the pulmonary hypoplasia associated with DH. To achieve this goal, the timing and duration of treatment and the optimal degree of stenosis must be defined. PMID- 8632267 TI - Endogenous nitric oxide and pulmonary vascular tone in the neonate. AB - PURPOSE: In newborns, inhaled nitric oxide (NO) has been shown to ameliorate increased pulmonary vascular resistance (PVR) precipitated by hypoxia. The role of endogenous NO production in this response is not clear. The contribution of endogenous NO to resting PVR in normoxic newborns also has not been well studied. The authors used an isolated, in situ, neonatal piglet lung-perfusion model, devoid of systemic detractors in which endogenous NO could be selectively inhibited, to determine whether (1) endogenous NO plays a role in the maintenance of PVR with normoxia, (2) endogenous NO plays a role in the response to hypoxia, and (c) inhaled NO can reverse changes induced by inhibition of endogenous NO. METHODS: Sixteen neonatal piglets underwent occlusive tracheostomy and pressure cycled ventilation. After heparinization and ligation of the ductus arteriosus, left atrial and pulmonary arterial cannulation were performed, without ischemia, via a median sternotomy. The aorta was ligated, and lung perfusion was set at 80 mL/kg/min via an extracorporeal membrane oxygenation circuit. Hematocrit (40% to 45%), pH (7.37 to 7.44), Pco2 (35 to 40 mm Hg), and peak inspiratory pressures (20 mm Hg) were constant. Pulmonary artery pressure (PPA), left atrial pressure (PLA), and circuit flow (QPA) were recorded continuously. PVR calculated as follows: PVR[(dynes x seconds x cm(-5)) x 1,000] = [(PPA-PLA/(QPA x 1,000/60)] x 1,332. The experimental animals were ventilated with normoxic gas (FIO2, 0.21), followed by hypoxic gas (FIO2, 0.07), returned to normoxia, and then divided into two groups of eight animals each. One group remained normoxemic (FIO2, 0.21; SPA02, 100%) while the other group was made hypoxemic by ventilation with hypoxic gas (FIO2, 0.07; SPA02, 50%). Endogenous NO was suppressed with L-arginine-N omega methyl ester (L-NAME) at 40 mg/kg in both groups. Inhaled NO was given at 40 ppm in both groups. Analysis of variance for repeated measures was used to test for statistical significance. RESULTS: Baseline normoxic PVR (3,403 +/- 1,169) was increased significantly by hypoxia (6,524 +/- 1,018, P < .01) and was fully resorted to baseline by normoxia (3,497 +/- 1,079; P = NS). In normoxic animals, inhibition of endogenous NO production by L-NAME increased PVR to levels similar to those seen during hypoxic stress (6,345 +/- 1,441, P < .01). Replacement of endogenous NO by inhaled NO reversed PVR to normoxic baseline values (3,986 +/- 1,363, P = NS). In hypoxic animals, inhibition of endogenous NO production by L-NAME also increased PVR from hypoxic baseline (9,655 +/- 1,642, P < .01). Replacement of endogenous NO by inhaled NO reversed PVR to hypoxic baseline (6,450 +/- 1,796, P = NS). CONCLUSION: In this piglet model, endogenous NO is important in the regulation of pulmonary vascular tone during both normoxia and hypoxia. Inhaled NO completely reversed the elevations in PVR caused by inhibition of endogenous NO and may normalize PVR in diseases in which the production of endogenous NO is compromised. PMID- 8632269 TI - Sonographic predictors of survival in fetal diaphragmatic hernia. AB - The authors studied the predictive value of detailed fetal sonographic parameters on outcome for 55 patients with prenatally diagnosed congenital diaphragmatic hernia managed at an ECMO center. Their sonographic assessment included gestational age at time of diagnosis, polyhydramnios (largest amniotic fluid pocket diameter), presence of liver and/or stomach herniation, and abdominal circumference at the level of the umbilical cord. They measured the right lung two-dimensional area at the level of the atria as an estimate of lung size and mediastinal shift. The ratio of right lung area to head circumference (LHR) was calculated to minimize lung size differences owing to gestational age. The principal outcome variable was survival. The overall survival rate was 65%. If the diagnosis was made after 25 weeks' gestation, the survival rate was 100% (12 of 12); the rate was 56% if the diagnosis was made at or before 25 weeks (P < .005). All five neonates with an LHR of less than 0.6 died; the survival rate was 100% for those whose LHR was greater than 1.35; and those with an LHR between 0.6 and 1.35 had a 61% survival rate (P < .001). The survival rate for those whose liver was not herniated was 100% (10 of 10); herniation of the liver decreased the survival rate to 56% (P < .05). Stomach position, polyhydramos, and abdominal circumference were not found to be useful survival predictors. No prenatal sonographic parameter was absolutely predictive of postnatal death except very small right lung size, which was present in only 5 of the 55 patients. Survival is highly likely if the liver is not herniated into the thorax and/or the right lung is large. PMID- 8632270 TI - Spontaneous resolution of antenatally diagnosed adrenal masses. AB - Four infants had adrenal masses detected prenatally, through ultrasonography, between 18 and 30 weeks' gestation. Two were predominantly cystic, and two were solid. The sizes ranged from 0.8 to 1.5 cm. There were no associated prenatal maternal complications or stress factors. The urine vanillylmandelic acid levels at birth were normal in three infants. Two infants had a documented decrease in mass size at birth (compared with the last intrauterine study). All masses had a progressive decrease in size after birth, and ultrasound results were normal at 6 to 12 weeks of age. All four patients are well, with normal study results, at 2 to 5 years of age. PMID- 8632271 TI - Prenatal diagnosis and the pediatric surgeon: the impact of prenatal consultation on perinatal management. AB - PURPOSE: Pediatric surgeons are increasingly called on by obstetrical colleagues to counsel parents about the implications of a prenatal ultrasound finding. Our understanding of the natural history of many prenatally diagnosed surgical conditions has grown significantly in recent years. Whether prenatal surgical consultation can influence perinatal course had not been investigated. METHODS: During an 21-month period, 12,865 prenatal ultrasound studies were performed on a total of 4,551 patients, and 221 prenatal surgical consultations were obtained through a newly established fetal treatment program at a tertiary care prenatal diagnostic center. To evaluate the impact of prenatal pediatric surgical consultation on perinatal course, the authors reviewed changes in management including termination of pregnancy, in utero intervention, and altered site, mode, or timing of delivery. RESULTS: Two hundred twenty-one fetuses were referred for consultation; their 234 congenital anomalies included genitourinary (36%), thoracic (16%), intraabdominal (14.5%), abdominal wall (10.6%), neurological (9%), skeletal (6%), and head and neck (2.5%) defects; 2.5% had tumors and 2.5% were twin pregnancies. Pregnancy was terminated in 9.5% of cases, because of patient request, chromosomal abnormality, or dismal prognosis. In 3.6%, the decision to terminate was changed as a result of consultation. Site of delivery was changed as a result of consultation in 37% to facilitate postnatal evaluation and initiate immediate treatment. Mode of delivery was changed in 6.8% to prevent dystocia, hemorrhage into a tumor, as in sacrococcygeal teratoma, or to provide an emergency airway, as in cervical teratoma. The timing of delivery was changed in 4.5% to avoid further damage to fetal organs in cases of obstructive uropathy, gastroschisis, sacrococcygeal teratoma with high-output failure, and hydrocephalus. Five percent (11) underwent treatment in utero for fetal hydrothorax, obstructive uropathy, twin-twin transfusion syndrome, or lymphangioma. The overall perinatal mortality rate was 2.5%. CONCLUSION: Prenatal pediatric surgical consultation may have a significant impact on the perinatal management of the fetus with a surgically correctable congenital anomaly. Providing obstetric colleagues and families with valuable insight into the surgical management of anomalies allows fetal intervention when appropriate, and delivery in an appropriate setting, by the safest mode of delivery, and at the gestational age appropriate to minimize effects of the anomaly. PMID- 8632272 TI - Ultrasonography as an adjunct in the diagnosis of acute appendicitis: a 4-year experience. AB - This study was designed to evaluate the sensitivity and specificity of abdominal ultrasonography as a diagnostic modality in a large series of children who presented with possible appendicitis. From August 1990 to July 1994, 452 children (203 boys, 249 girls) with an average age of 11 years (range, 1 to 20 years) underwent graded compression ultrasonography of the right lower quadrant of the abdomen for the evaluation of possible appendicitis. In the first 18 months of the study all patients with the possible diagnosis of appendicitis (group I; 180 patients) had abdominal ultrasonography after members of the surgical team evaluated and documented their findings in the medical record. In the second study period (30 months), abdominal ultrasonography was recommended only when the clinical diagnosis of acute appendicitis was equivocal (group II; 272 patients). Abdominal ultrasonography was performed using the graded compression technique with a 5.0-MHz linear array transducer. A positive ultrasound study for appendicitis was defined as the presence of an enlarged noncompressible appendix with an outer wall to outer wall diameter of greater than 6 mm, the presence of a complex mass, or the presence of an appendicolith. The sonographic data were correlated with surgical and pathological findings. Appendicitis was confirmed in 112 of the 452 cases. In 17 of these, the appendix was perforated. In the overall group of 452 children, abdominal ultrasonography had a sensitivity of 90%, specificity of 96%, and accuracy of 95%. There was no significant morbidity in the 11 patients with a false-negative study result. All 11 patients had an uncomplicated appendectomy. There were 11 false-positive results; 10 of these patients had a negative laparotomy result (negative laparatomy rate, 8.9%). For the two groups, the sensitivity and specificity of ultrasonography in the diagnosis of appendicitis were equivalent (group 1: 88% sensitivity, 96% specificity; group 2: 92% sensitivity, 97% specificity). On the basis of the high sensitivity and specificity rates, ultrasonography of the appendix can be a useful adjunct to standard examination in the diagnosis of acute appendicitis. PMID- 8632273 TI - Abdominal tuberculosis in children: review of 26 cases. AB - The protean clinical manifestations and varied complications of abdominal tuberculosis continue to challenge the diagnostic acumen and therapeutic skills of all physicians. Although abdominal tuberculosis in children has not been common in the United States over the past 2 decades, the authors found 26 case reports for the period 1980-1993. Three clinical patterns were evident: intestinal (13) peritoneal (9), and asymptomatic with incidental calcifications apparent on abdominal radiographs (4). The diagnosis was suspected for only 23% of these cases, which emphasizes the nonspecific symptomatology caused by this extrapulmonary manifestation and the need for a high index of suspicion to make a prompt diagnosis. In this study, 24 of the 26 (91%) were of Hispanic origin; the other two were indo-Chinese, another high-risk group. Most patients (88%) had a positive PPD skin test result. Mycobacteria were isolated from 15 of 21 (71.4%) cultures, with M bovis in 80% and M tuberculosis in 20%. Antituberculous chemotherapy is the mainstay of treatment; surgery is reserved for tissue diagnosis in cases of peritoneal tuberculosis and for the management of complications of intestinal tuberculosis. The response to chemotherapy usually is excellent, and long-term sequelae are uncommon. It appears that steroids do not decrease the incidence or degree of fibrosis in intestinal tuberculosis. PMID- 8632274 TI - Medullary thyroid carcinoma in children with multiple endocrine neoplasia types 2A and 2B. AB - Recently it has become possible to identify persons who have multiple endocrine neoplasia (MEN) syndrome types 2A and 2B based on the presence of missense mutations in the RET protooncogene. Kindred members who have inherited these syndromes can be identified before clinical or biochemical evidence of medullary thyroid carcinoma (MTC) develops, the malignancy that occurs in all affected patients. It is not known whether prophylactic removal of the thyroid gland early in childhood, based on a positive genetic test result, has a better clinical outcome than that associated with thyroidectomy after MTC is diagnosed clinically or biochemically. The authors' goal was to determine the long-term outcome for patients with MEN 2A and 2B who had thyroidectomy for MTC during childhood. These results were compared with those of patients who had prophylactic removal of the thyroid gland after the genetic diagnosis of MEN 2A was established. The hospital records of 49 children with MEN 2A or 2B were reviewed. Each patient had thyroidectomy for MTC before 16 years of age. The mean age at the time of operation was 10 years, and the mean follow-up period for those who had surgery before the availability of direct DNA genetic testing was 9.8 years. The indications for surgery included an elevated basal or stimulated plasma calcitonin level, a positive genetic test result, a thyroid mass, family history of MTC, or a phenotype diagnostic of MEN 2B. All children for whom the diagnosis of MEN 2A was established by direct genetic testing had thyroidectomy within the last 2 years. Of the 11 patients with MEN 2B who underwent thyroidectomy during childhood, 10 had MTC, and only 3 (27%) remain free of disease after the mean follow-up period of 11 years. One patient died, and seven are alive with persistent MTC. Among the 24 patients with MEN 2A who had their thyroid glands removed because of a family history of MTC or because of biochemical evidence of the disease, 5 (21%) have persistent or recurrent MTC after the mean follow-up period of 9.3 years. In four of these, the MTC was confined to the thyroid gland at the time of thyroidectomy. Of the 14 children who had thyroidectomy based on direct DNA testing, MTC was present in 11. Only four had elevated levels of stimulated plasma calcitonin before surgery. None had lymph node metastasis or surgical complications. The authors conclude that a significant number of patients with MEN 2A or 2B who undergo thyroidectomy in childhood for MTC have persistent or recurrent disease long-term. The genetic diagnosis of patients with these syndromes may allow for prophylactic surgery before the development of biochemical or clinical evidence of MTC. This approach is safe, but longer clinical follow-up will be necessary to confirm that MTC has been cured. PMID- 8632275 TI - Renal cell carcinoma in childhood and adolescence: a retrospective survey for prognostic factors in 22 cases. AB - To identify prognostic factors for renal carcinoma in young patients, a retrospective analysis was performed of 22 patients (< or = 21 years of age) with histologically verified renal cell carcinoma. Demographic, staging, and treatment variables were collected in a database, and their effect on survival was determined using Kaplan-Meier probability distribution. The median age was 15.5 years (range, 3 to 21 years), and the male:female ratio was 13:9. Only three patients were black. Histopathologic examination showed 15 clear cell tumors, 4 mixed cell type, 2 papillary, and one well-differentiated adenocarcinoma. The median size of the primary tumor was 10 cm (range, 5 to 20). There were seven patients with stage I tumors, one with stage II, and 14 with stage IV. Complete resection of the primary tumor was accomplished in 12 patients. The overall 5 year survival rate was 30% (confidence interval, 20% to 40%). The 5-year survival rate was better for patients who had complete resection of the primary tumor (60% v 10%). Unresectability was associated with involvement of nodes and/or occurrence of metastases, thus an independent effect of complete resection on survival could not be demonstrated. The data showed that age, tumor size, location, and histology were not predictors of outcome; tumor stage and complete surgical resection were the only meaningful prognostic factors. The presentation of renal cell carcinoma as a localized or systemic disease may reflect a twofold biological behavior. In the first group, the disease is curable with resection, in the second, it is unaffected by surgery or adjuvant therapy. In light of the very low incidence of this renal malignancy in childhood, prospective multicenter studies will be required to improve the poor therapeutic results. PMID- 8632276 TI - Childhood pleuropulmonary blastoma: caution against nonoperative management of congenital lung cysts. AB - Pulmonary blastoma is a rare and aggressive malignant tumor that affects children and adults. Recently a 3-year-old boy with a 2-year history of bilateral unilocular pulmonary cysts was transferred for evaluation of a cough and high spiking fever. A chest radiogram showed left pulmonary consolidation with pleural effusion, but thoracentesis was unsuccessful. Computerized tomography (CT) was suggestive of a pulmonary abscess, but CT-guided drainage did not yield any purulent fluid. Percutaneous biopsies were performed, and the cytology showed malignant cells. During thoracotomy, a large tumor involving the left lower lobe and pleural space was found, and a biopsy was performed. A frozen section showed blastemal and mesenchymal components devoid of neoplastic epithelium, consistent with the pleural variant of pulmonary blastoma. A left lower lobectomy, with tumor decortication of the pleural space, achieved total gross tumor removal. The child received aggressive multiagent chemotherapy, and midway through it he underwent elective excision of the opposite lung cyst. It has been 17 months since the lobectomy; he is off chemotherapy and has no evidence of disease. A review of the literature showed that a large number of pediatric pulmonary blastomas are associated with cystic lung disease. Because total tumor removal offers the only chance of a good long-term outcome, surgical excision or close follow-up of pulmonary cysts in children is strongly recommended. PMID- 8632277 TI - Esophageal atresia: past, present, and future. PMID- 8632278 TI - Extremity sarcomas: an analysis of prognostic factors from the Intergroup Rhabdomyosarcoma Study III. AB - PURPOSE: Prognostic factors for extremity sarcomas have been reported previously, after analysis of Intergroup Rhabdomyosarcomas Studies (IRS) I and II. This report reviews the experience of IRS III (1984-1992), in light of these reported factors, and the pretreatment factors used in the staging system currently being evaluated in IRS IV. The results of treatment of extremity sarcomas in IRS III are reported. METHODS: The charts of all patients entered in IRS III with an extremity-site tumor were reviewed. This group included patients with shoulder girdle and buttock sites. All patients were treated according to IRS III protocols. Survival rates were estimated by the method of Kaplan and Meier, and comparisons among groups of patients were made using a log-rank test. A multivariate analysis was performed to analyze all pretreatment factors that were significant by univariate analysis. RESULTS: Of the 189 patients entered in IRS III with extremity sites, 88 (47%) had the most common alveolar histology. Fifty nine patients were in group I at the time of presentation (completely resected disease), 48 in group II (microscopic residual disease), 36 in group III (gross residual disease), and 46 in group IV (metastatic disease). By univariate analysis, the significant prognostic factors affecting survival were clinical group, age at time of diagnosis, tumor size, distant metastases, nodal metastases, and local and distant recurrence. By multivariate analysis of pretreatment factors, age at time of diagnosis, nodal metastases, and distant metastases were significant prognostic factors affecting survival; tumor size approached significance. Both the clinical group system and pretreatment staging system (Lawrence/Gehan) predicted significant differences in survival between groups of patients. The lower survival rate among group II and III node-negative patients with a distal tumor (who, with more extensive surgery, could have been in group I) in comparison to group I patients with a distal lesion, approached significance. In patients without distant metastases, survival was significantly different in those patients that had negative nodes from those in whom nodes were not biopsied. CONCLUSION: This review confirms that both clinical grouping and the new pretreatment staging system used in IRS IV can predict the likelihood of survival of children with extremity sarcomas. By multivariate analysis of the elements included in the staging system, nodal metastases, distant metastases, and tumor size were useful in predicting survival. In addition, age was a significant predictor. This study confirms previous suggestions that complete excision with gross and microscopically negative margins is preferable in the treatment of children with extremity rhabdomyosarcomas. PMID- 8632279 TI - Cardiac tamponade in the pediatric oncology population: treatment by percutaneous catheter drainage. AB - PURPOSE: The treatment of pericardial effusion resulting in cardiac tamponade has undergone an evolution in recent years, with the use of less invasive drainage methods in selected cases. To determine optimal therapy for pediatric oncology patients with pericardial effusion and tamponade, the authors reviewed their institutional experience with percutaneous catheter drainage. METHODS: Patient records and operative reports were reviewed, and nine patients were identified who met clinical and echocardiographic criteria of cardiac tamponade and were treated with percutaneous pericardial catheter drainage. RESULTS: The median age at time of diagnosis was 14 years (range, 5 months to 19 years), and the male:female ratio was 7:3. Underlying malignancies included acute myeloblastic leukemia in three, acute lymphoblastic leukemia in one, and Hodgkin's disease, B cell lymphoma, medulloblastoma, desmoplastic small round cell tumor, and rhabdomyosarcoma in one each. EIght patients (89%) were receiving granulocyte colony-stimulating factor (GCSF) during the period when tamponade developed. All patients had a large or moderate-to-large pericardial effusion and right ventricular collapse with hemodynamic compromise on echocardiography, and two patients (22%) also had pericardial thickening. In nine patients, percutaneous catheter drainage was performed intraoperatively and under fluoroscopic or echocardiographic guidance. A median of 300 mL (range, 82 to 500 mL) of fluid was removed from the pericardial sac during the initial drainage, and cytology was positive in one (6%). Complete echocardiographic resolution was observed in eight patients (89%); a small posterior component persisted in one patient but was not significant hemodynamically. The catheters remained in place for a median of 5 days (range, 1 to 35 days) while repeat aspirations were performed. Tamponade resolved in all patients, and one died of overwhelming systemic sepsis. The survival period was 10 to 22 months, and tamponade or the drainage procedure did not contribute to death. Four patients remain alive after 4 month to 7 years of follow-up. CONCLUSION: Cardiac tamponade was effectively treated in all patients and did not recur with percutaneous catheter drainage alone. THere was no evidence of pericardial loculation or infection despite pancytopenia being prevalent with underlying illness and chemotherapy. Percutaneous catheter drainage is an effective treatment for pediatric oncology patients with pericardial tamponade. Because of its simplicity in comparison to move invasive techniques, initial treatment with percutaneous drainage should be considered in this patient population. PMID- 8632280 TI - Ascaris lumbricoides infestation as a cause of intestinal obstruction in children: experience with 87 cases. AB - PURPOSE: The purpose of this study is to describe the occurrence and management of bowel obstruction caused by Ascaris lumbricoides, a common parasite in warm climates that affects children with limited socioeconomic means. METHODS: Eighty seven patients with intestinal infestation owing to Ascaris lumbricoides were treated in the past 10 years (1984-1994). There were 48 (55.2%) girls and 39 (44.8%) boys. The mean age was 4.6 years, with peak occurrence at 2 years of age. Half the patients had a history of passing worms by mouth or anus. The majority of patients, 64 (73.5%), presented with a subacute clinical course; 23 (26.5%) had acute presentation, with severe abdominal pain, fever, dehydratation, vomiting, and abdominal distension and required vigorous fluid resuscitation and emergency surgical intervention. Diagnosis was achieved with plain abdominal roentgenograms, which showed a "whirlpool" pattern of intraluminal worms in most cases. RESULTS: Six patients had been incorrectly diagnosed as having appendicitis; two cases had appendicitis owing to Ascaris in the cecum and distal ileum. The majority of cases with a subacute presentation respond to medical (anthelmintic) management using oral administration of racine oil and piperazine. Of the 23 patients taken to the operating room, 11 required external "milking" of the obstructing bolus of worms from the ileum into the colon, six required intestinal resection and end-to-end anastomosis, six had an appendectomy, and three needed an enterotomy to manually extract the worms. In one case, initial management consisted of an ileostomy because of intraoperative instability owing to sepsis. Subsequently, after stabilization and treatment with anthelmintic agents, closure of the stoma with an end-to-end ileocolostomy was performed. There was no significant postoperative morbidity or mortality. CONCLUSION: These observations suggest that physicians should have a high index of suspicion for parasitic infestation in warm climates where economically deprived children present with symptoms of intestinal obstruction. Ascaris lumbricoides may be the cause of these events in endemic areas. Oral piperazine and racine oil can successfully resolve most subacute cases; however, aggressive resuscitation and prompt surgical intervention in patients with intestinal obstruction result in a satisfactory outcome. PMID- 8632281 TI - Does posterior sagittal anorectoplasty in patients with high imperforate anus provide superior fecal continence? AB - The posterior sagittal anorectoplasty (PSARP) has become the primary surgical procedure for patients with high imperforate anus. Very few careful long-term follow-up studies have established the superiority of the PSARP procedure over other surgical repair techniques. The authors' goal was to evaluate the operation and to identify the factors associated with improved continence. The medical records, operative reports, and radiographs of 53 patients (46 male, 7 female) who underwent PSARP for high imperforate anus at Children's Hospital and Medical Center (CHMC) between 1982 and 1990 were reviewed retrospectively. Subjective follow-up data were collected by telephone questionnaire, assessing habits indicative of stool continence, and a "fecal continence score" (FCS) was calculated for each patient. A prospective, 7-day diary assessing similar patterns of fecal continence was completed by each patient's family. The mean age of the patients studied was 8.0 years, and the mean follow-up period was 6.8 years. The telephone questionnaire was completed for 48 patients (94%). Toilet training for bowel continence was successful in 20 patients (42%) and occasionally successful in another 20 patients (42%), but 8 patients (16%) had no awareness of impending stool. Forty-five (94%) were physically active, but 16 (33%) reported social problems related to offending odor. The mean fecal continence score for all patients was 3.0 +/- 1.4 (5=excellent, completely continent), which was nearly identical to published scores for the other types of surgical repair. The FCS did not improve with age. The parents' responses to the telephone questionnaire matched the results obtained from the prospectively collected continence diary data. The authors' results for PSARP in patients with high imperforate anus do not differ substantially from those achieved by other repair techniques. Previously cited prognostic factors such as fistula anatomy and sacral anomalies did not appear to alter the functional results in the authors' series. Aggressive postoperative bowel management should be anticipated in all patients who have high imperforate anus and may benefit those who otherwise would achieve less satisfactory continence. PMID- 8632282 TI - Economics and education: impact on pediatric surgery in the next decade. PMID- 8632283 TI - One-stage versus two-stage Soave pull-through for Hirschsprung's disease in the first year of life. AB - Several investigators have reported good results after a one-stage Soave procedure without a stoma for infants with Hirschsprung's disease. The authors reviewed their concurrent experience with the one- and two-stage approaches, comparing the two groups with respect to rate of complications and clinical outcome. Over a 3-year period, 36 infants with colonic Hirschsprung's disease presenting in the first year of life were treated with a Soave pull-through. Thirteen had a one-stage pull-through, and 23 had a two-stage procedure using an initial stoma. There was no difference with respect to median age at time of diagnosis, median follow-up period, length of aganglionosis, or male:female ratio between the groups. The incidences of major complications such as small bowel obstruction, segmental or acquired aganglionosis, anastomotic leak, and malabsorption were equal between the two groups. However, 13% of the two-stage patients required revision of the stoma. All major complications in the one-stage group were in those who weighed less than 4 kg at the time of surgery. Minor complications such as wound infection, perianal excoriation, and need for repeated dilatation were similar between the groups, but minor stoma-related complications (prolapse or retraction) occurred in 26% of the two-stage infants. When complications were stratified using a more sophisticated scale of severity, no significant difference was found between the groups. The overall complication rate was 1.5 events per patient in the one-stage group and 2.0 events per patient in the two-stage group. This small difference was related to the presence of a stoma in the two-stage group. Overall, 10 of 12 survivors in the one-stage group and 22 of 23 in the two-stage group were doing well, with normal bowel function noted on long-term follow-up (mean period, of 14 and 19 months, respectively). Both one- and two-stage approaches were associated with a significant complication rate, although long-term outcome was excellent in both groups. The higher complication rate in the two-stage group was attributable to the presence of a stoma. For small infants, it may be beneficial to delay the one-stage pull through until weight exceeds 4 kg. PMID- 8632284 TI - Intestinal bypass of the esophagus. AB - Esophageal replacement by a segment of isoperistaltic ileum with cecum or by transverse or left colon will allow near-normal swallowing for many years. The authors reviewed the course of 59 children who had bypass of their entire esophagus and of four whose distal esophagus was resected and replaced. The follow-up period ranges from 1 to 37 years; in 36 cases, it exceeds 5 years. Thirty children had caustic strictures and 25 had either isolated esophageal atresia or atresia with fistula. Two children with esophageal injury caused by foreign body ingestion and two with congenital strictures also required complete bypass. Four patients required resection and replacement of the distal esophagus only; two had acquired strictures from gastroesophageal reflux, one had varices, and one had a teratoma involving the esophagus. A retrosternal isoperistaltic ileocolic segment is our preference for complete esophageal replacement. Forty eight patients underwent esophageal reconstruction with this procedure. The esophagus damaged by caustic ingestion was left in place in all patients, without any subsequent problem. The authors have not used the distal esophagus for anastomosis in patients with atresia, because this segment may be abnormal; and, in any case, an isoperistaltic cologastric anastomosis does not reflux. The right or left colon or jejunum was used in the other cases. Three children lost an interposed intestinal segment from necrosis even though the bowel appeared to be well vascularized at the end of the operation. Each patient had successful reconstruction using another type of interposition. An intrathoracic leak occurred in one infant. A cervical anastomotic leak developed in 11 children, and a stricture in 13. Strictures were more common in patients who had caustic burns. Three patients required surgery for adhesive intestinal obstruction. A redundant colon transplant with ulceration, and the herniation of an ileal segment into the pleural cavity with obstruction prompted reoperation in two other patients. There were two deaths early in the series, one of which was secondary to postoperative respiratory arrest. The other death occurred in a child who had a caustic pharyngeal burn and chronic aspiration. All patients were seen in our office recently, or they or their parents were interviewed by phone. All of them are taking all of their nutrition by mouth. Forty-three of the 61 survivors have had no difficulty with swallowing. One required reoperation to enlarge the thoracic inlet. Seventeen other have mild dysphagia that does not require treatment. The patients with esophageal atresia or atresia and fistula consistently have not grown as well as those who required replacement for an acquired condition or injury. PMID- 8632285 TI - Save the child's esophagus: management of major disruption after repair of esophageal atresia. AB - PURPOSE: Given the bias that the native esophagus is the best conduit between the oropharynx and the stomach, the authors report a "conservative" approach to massive esophageal leak, which may be considered "radical" by others. Major disruption of the anastomosis after primary repair of esophageal atresia is a recognized and feared complication. Historically, management has been the performance of cervical esophagostomy and gastrostomy. The aim of this report is to describe the authors' approach to this difficult and serious complication. METHODS: A 15-year retrospective analysis was performed of all patients having esophageal atresia. Data collection focused on the management of all patients with clinically significant esophageal disruption. Radiographically detected (clinically asymptomatic) leaks were managed by continuation of drainage by thoracostomy tubes already in place and are not included. Reoperative thoracotomies were performed, which included primary repair (2), placement of pleural patch alone (2), pleural patch with intercostal muscle flap buttress (2), and operative debridement and drainage alone (1). RESULTS: It was noted that seven patients had clinically significant esophageal disruption requiring reoperation, with circumferential disruptions ranging from 15% to 85%. Presentation included persistent pleural collection (4) and pneumothorax (3). Both patients who underwent primary repair had no evidence of leakage on follow up esophagograms, neither did one with a pleural patch alone and one with an intercostal muscle flap. Five of the seven patients were tolerating oral feedings at the time of follow-up (range, 6 months to 8 years). One of the two others (both currently inpatients), has a recurrent leak associated with mediastinitis, and the other (who had primary repair) has a presumed neurological impairment of eating. CONCLUSION: Clinically significant disruption of primary esophageal repair should not warrant a cervical esophagostomy and placement of a gastrostomy tube, thus precluding eventual use of the native esophagus. The authors have shown that management by reoperation with primary repair, intercostal muscle flap with or without pleural patch, and/or drainage allows the patient to maintain the native esophagus and yields a generally good outcome after a prolonged healing time. PMID- 8632286 TI - Transanastomotic feeding tubes in repair of esophageal atresia. AB - To avoid the need for a gastrostomy and parenteral nutrition during the 7- to 10 day healing period after esophageal anastomosis, the authors modified their technique for esophageal atresia repair to include placement of a transanastomotic feeding tube. A SILASTIC transanastomotic feeding tube and early enteral nutrition was used for 19 of 23 consecutively treated patients after repair of esophageal atresia and tracheoesophageal fistula. One of the 19 patients had recurrent fistula and another had an anastomotic leak. Five patients had significant gastroesophageal reflux (noted on barium esophagram), and four had strictures that required dilatation. Parenteral nutrition was necessary for only two patients. The authors conclude that transanastomotic feeding tubes and early enteral nutrition are safe and effective, reduce costs, and do not appear to increase the incidence of anastomotic leaks, strictures, or gastroesophageal reflux. PMID- 8632288 TI - Early improvement in intestinal function after isoperistaltic bowel lengthening. AB - Isoperistaltic bowel lengthening (the Bianchi procedure) has been used increasingly in the management of infants and children with short bowel syndrome. Although clinical improvement is observed frequently, few studies document the early effects of the Bianchi procedure on nutrient absorption and transit time. Five infants and children (aged 3 months to 4 years) with profound short bowel syndrome (< 50 cm of small bowel) underwent isoperistaltic bowel lengthening (10 to 40 cm) when their bowel was greater than 3 cm in diameter. One to 2 weeks preoperatively, the following were obtained for each patient: 24-hour stool counts, transit time (charcoal), intestinal clearance of barium, and nutrient absorption (fat balance and D-xylose). The studies were repeated 1 and 6 months postoperatively. The mean stool count per 24 hours decreased from eight preoperatively to four and three at 1 and 6 months postoperatively. Transit time increased from 52 minutes to 135 and 205 minutes, and clearance of barium improved from 4.5 hours to 2.4 and 2.6 hours, respectively. Results of D-xylose absorption and dietary fat balance studies, both abnormal preoperatively, also normalized at 1 and 6 months. These data show that the Bianchi procedure provides short- and intermediate-term improvement in intestinal and nutrient absorption, which should allow more rapid weaning from parenteral nutrition. PMID- 8632287 TI - Delayed effects of epidermal growth factor after extensive small bowel resection. AB - Epidermal growth factor (EGF) is produced in the gastrointestinal tract and has been shown to have a transient stimulatory effect on mucosal growth and uptake of glutamine. This study investigated the delayed effects of EGF on mucosal brush border membrane enzymes and glutamine uptake after extensive small bowel resection. Twenty-four New Zealand White rabbits underwent a 50% to 60% midjejunoileal enterectomy. One group of 12 had a subcutaneous osmotic pump inserted, delivering EGF at 0.3 microgram/kg/h for the first 7 postoperative days. The other group of 12 served as controls. Six rabbits from each group were killed at 3 weeks, and the remaining 12 were killed at 6 weeks. Six additional rabbits served asd nonsurgical controls. There was a twofold increase in mucosal dry weight at 3 weeks without EGF, and an almost fourfold increase with EGF, over control rabbits. This effect of EGF on the mucosa persisted for 6 weeks. Enzymatic activity per gram of protein in each group of rabbits was similar between the four groups of rabbits, although maltase activity increased approximately fourfold over that of nonoperative control animals. However, enzyme capacity of maltase and aminooligopeptidase (AOP) increased threefold and twofold (respectively) at 3 weeks without EGF, and sixfold and fourfold with EGF. Functional capacity is a measure of the load of nutrients that the intestine can digest and absorb, and therefore the derivable benefit to the animal. Glutamine uptake capacity increased 60% in 3 weeks without EGF, then declined by 6 weeks. However with EGF it increased 200% by 3 weeks, and further increased 400% by 6 weeks over control levels. The authors conclude that EGF markedly increases the functional capacity of the small intestine in rabbits that have undergone extensive small bowel resection; this effect persists for up to 6 weeks after a small initial dosage of EGF. PMID- 8632289 TI - Hypertonic saline improves brain resuscitation in a pediatric model of head injury and hemorrhagic shock. AB - INTRODUCTION: Brain injury accompanied by hypovolemic shock is a frequent cause of death in multiply injured children. Hypertonic saline (HTS) has been shown to return hemodynamics to normal in adult models, without increasing intracranial pressure (ICP) as seen with crystalloids. To assess fluid resuscitation, the authors evaluated HTS versus lactated Ringer's solution (LR) with respect to hemodynamics and cerebrovascular hemoglobin oxygen saturation (Sco2) in anesthetized, head-injured, 1-month-old piglets. METHODS: Group 1 (n = 6) was studied for 3.5 hours after a cryogenic brain injury and no shock. Groups 2 and 3 had cryogenic brain injury followed by hemorrhagic shock, in which mean arterial pressure (MAP) was reduced to 40 to 50 mm Hg and maintained for 30 minutes. Group 2 (n = 5) was then resuscitated with 1 mL of 7.5% HTS per 1 mL of blood loss. Group 3 (n = 6) was resuscitated with 3 mL of LR per 1 mL of blood loss. Sco2 was determined by near-infrared spectroscopy in the injured region of the brain. All data were analyzed using analysis of variance with repeated measures. RESULTS: MAP, ICP, temperature, serum sodium, and cardiac output (CO) were similar in all groups during baseline and between groups 2 and 3 during shock. After resuscitation, MAP, CO, and core temperature were similar in all three groups, and serum sodium was increased in the HTS group (by 29%). Sco2 increased transiently after cryogenic injury in all groups, then gradually decreased to below baseline. After shock, Sco2 decreased precipitously in group 2 and 3. After resuscitation, Sco2 was different in the two resuscitation groups, increasing in the HTS group, above baseline values, but remaining below baseline values in the LR group (P < .002). ICP was lowered by HTS resuscitation and increased by LR resuscitation (P < .002) CONCLUSION: In our model of head injury and shock, resuscitation with either HTS or LR restored MAP and CO to control levels. However, during shock, the injured brain was severely deoxygenated, and administration of HTS restored cerebral oxygenation whereas LR did not, reflecting improved cerebral resuscitation by HTS without elevating ICP. The data suggest that HTS is a better resuscitation fluid than LR in head-injured children with hemorrhagic shock. PMID- 8632290 TI - The outcome for children with blunt trauma is best at a pediatric trauma center. AB - PURPOSE: The mortality rate for pediatric trauma patients cared for in adult trauma centers has been shown, by means of TRISS methodology, not to differ significantly from that of the Major Trauma Outcome Study (MTOS). The question remains, however, whether the outcome of injured children is better in a designated pediatric trauma center (DPTC). The authors' hypothesis is that outcome is better at a DPTC. METHODS: The records of 1,797 children (0 to 15 years of age) admitted to a DPTC between 1987 and 1993 were reviewed. TRISS methodology was used to calculate probability of survival for outcome comparison with the MTOS. The data also was compared with outcome in relation to the admitting Glasgow Coma Score (GCS) reported in the National Pediatric Trauma Registry (NPTR). RESULTS: The outcome of all children at this DPTC had a Z score of +1.4199 (P > .1). The Z score of children admitted because of penetrating trauma (PT, n = 460) did not differ significantly from that of the MTOS. However, the children admitted because of blunt trauma (BT, n = 1,337) had a Z score of +3.3501 (M score = .90), which is significantly better than that of the MTOS (P < .001). The BT population with an ISS of > or = 9 (n = 149) had a Z score of +2.8686 (P < .005) (M = .95). By GCS comparison, the BT group had a outcome similar to that reported in the NPTR. Head injury was the cause of death for 26 (84%) of the 31 PT deaths and 20 (83%) of the 24 BT deaths (three of the remaining four had associated severe head injury). Only 1 of 24 (4%) BT liver injuries and 5 (21%) of 24 BT splenic injuries required surgical intervention. This low incidence of liver and splenic surgical invention is similar to that reported by other DPTCs, but for children treated at adult centers the rates are 37% to 58% and 43% to 53% for liver & splenic surgical intervention, respectively. CONCLUSION: Children with BT have a significantly better outcome at a DPTC; the outcome for children with PT does not differ. Successful nonoperative treatment of blunt abdominal injuries is more likely to occur at a DPTC than at adult trauma centers "with pediatric committment." Thus, children with blunt injuries should be taken to a DPTC, when available. PMID- 8632291 TI - Hospital reimbursement for pediatric trauma care. AB - PURPOSE: Despite the proven efficacy of pediatric trauma centers, their continued development is threatened by the perception that their cost exceeds the reimbursement for their services. The authors reviewed actual reimbursement for a group of pediatric trauma patients and compared with that for a group of appendectomy patients chosen to reflect the authors' surgical population at large. METHODS: The records of 209 consecutively treated trauma patients and 37 age-matched appendectomy patients treated in 1992 and 1993 were reviewed. Trauma patients were divided into two groups: moderate injury (ISS < or = 9; n = 134) and serious injury (ISS > or = 10; n = 75). RESULTS: Hospital bills for the appendectomy patients were reimbursed at 72% of charges and 112% of costs. Payment was received at a mean of 36 days (range, 9 to 62 days) after discharge. Reimbursement for moderately injured patients was 104% of charges and 137% of costs and was received at a mean of 81 days (range, 3 to 270 days) after discharge. Six months postdischarge, reimbursement for seriously injured patients was 63% of charges and 86% of costs. Reimbursement was slow for some children who sustained severe injury, but as legal actions brought by patient's families were completed, open accounts were settled, and revenue in both groups totaled 76% of charges and 103% of costs 18 months postdischarge. CONCLUSION: Hospital reimbursement for care at a level I pediatric trauma center exceeds 75% of charges and 100% of costs, no different from the overall rate for the general hospital surgical population. Analysis of reimbursement rates for trauma patients may be time-dependent. PMID- 8632292 TI - Death from pelvic fracture: children are different. AB - This study compares outcome from pelvic fractures in children with that of adults. Data for 23,700 children registered in the National Pediatric Trauma Registry (NPTR) were compared with those of 10,720 adults recorded over 5 years in the registry of our level I trauma center. Patients were categorized by open versus closed fracture and by fracture type as defined by a modification of the Key and Conwell system. Outcome was evaluated by mortality rate and incidence of fracture-induced fatal exsanguination. The 722 pelvic fractures recorded in the NPTR represent 3% of the population and is half the frequency represented by the 532 adults evaluated (P < .001). The overall mortality rate was 5% for children and 17% for adults. Two children died of fracture-related exsanguination; there were 18 such deaths among the adults. Pelvic ring disruption was encountered more commonly among adults, and was associated with a significantly higher mortality rate. Patients with initial hemodynamic instability were more likely to die, although children less so than adults. The authors conclude that children do not die of pelvic fracture-associated hemorrhage as often as adults. Massive blood loss in the child occurs most commonly from solid visceral injury rather than from pelvic vascular disruption. PMID- 8632293 TI - Indicators of genitourinary tract injury or anomaly in cases of pediatric blunt trauma. AB - PURPOSE: Microscopic hematuria (> or = 20) RBCs per high-power field [HPF] has been used frequently as an indicator for genitourinary (GU) tract injury in pediatric cases of blunt trauma. The aim of this study was (1) to determine whether a certain threshold of microscopic hematuria was associated with GU tract injury in our patient population, and (2) to identify additional factors warranting evaluation of the GU tract. METHODS: The records of 100 patients under 18 years of age whose discharge diagnosis in the trauma registry included hematuria or GU tract injury were reviewed retrospectively (1989 through 1993). The following data were collected: age, sex, mechanism of injury, physical findings, associated injuries, urinalysis results, radiographic study results, disposition, and outcome. Data were analyzed using the SPSS program. RESULTS: The majority of children were victims of motor vehicle accidents. All but one study patient had an intravenous pyelogram and/or computed tomography scan. Twenty seven patients had GU tract injuries or previously unrecognized congenital anomalies (9 contusions, 5 lacerations, 1 vascular pedicle injury, 4 bladder injuries, 3 urethral/vaginal tears, 5 anomalies). Twenty-seven percent (3 of 11) of children with minor injuries and 25% (2 of 8) of those with major injuries had microscopic hematuria of less than 20 RBCs/HPF. Mechanism of injury and hypotenison were not associated with GU tract injury. One third of the patients with isolated chest or abdominal injuries, and 50% of those with combined chest/abdominal injuries had GU tract injuries or anomalies identified. Pelvic fracture was associated with GU tract injury or anomaly in 50% of cases (P < 0.02). CONCLUSION: The authors found that (1) a threshold of > or = 20 RBCs/HPF as an indication for radiograph evaluation would have missed 28% of cases with GU tract injuries or occult anomalies, and (2) pelvic fractures and abdominal/chest injuries help to identify patients who require evaluation of the GU tract. The need for GU tract evaluation in pediatric trauma patients is based as much on clinical judgment as on the presence of hematuria. PMID- 8632294 TI - Lower cytokine release by fetal porcine platelets: a possible explanation for reduced inflammation after fetal wounding. AB - Fetal dermal wound healing is unique because of its rapidity, minimal inflammation, and lack of scarring. Cytokines such as transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) evoke an inflammatory response and scarring when applied to fetal wounds. Because adult and fetal platelet counts are comparable, the aim of this study was to test the hypothesis that the minimal inflammatory response seen in the fetus is attributable to differences in the serum content of cytokines released by fetal platelets. Using Yorkshire swine, blood was collected from 10 adults and 10 fetuses at day 60 of gestation (fullterm, 114 days). Platelets were isolated from anticoagulated blood and examined by transmission electron microscopy. Serum was analyzed for PDGF-AB and TGF-beta 2 by enzyme-linked immunosorbent assay (ELISA), and TGF-beta 1 by 125I radioimmunoassay. TGF-beta samples were assayed with and without prior acid activation to determine the total TGF-beta and the biologically active form of the cytokine. Electron microscopy of adult and fetal platelets showed no gross structural differences. Alpha granules, which contain cytokines as well as procoagulant factors, were present in similar quantities and with the same degree of homogeneity. The cytokines analyzed were present in all the adult and fetal sera tested. However, PDGF-AB was present in significantly lower concentrations in the fetus (383 +/- 72 pg/mL v 972 +/- 185 pg/mL in the adult; P<.05). In addition, the fetal samples contained lower amounts of TGF-beta 1 (13,895 +/- 1,770 v 29,864 +/- 5,050 pg/mL; P < .05) and TGF-beta 2 (6,758 +/- 734 v 13,407 +/- 1,395 pg/mL; P < .05). The majority of TGF-beta was in latent form; the adult sera contained significantly more active TGF-beta 1 and active TGF-beta 2 than the fetal sera. The ratios of active TGF-beta 1 to active TGF-beta 2 were similar for the adult (22.3) and fetus (18.5). However the ratio of total TGF-beta 1 to total TGF-beta 2 was significantly lower for the fetus (2.26 v 7.69). The authors conclude that although no gross differences in platelet ultrastructure were noted, fetal porcine platelets release lower quantities of cytokines into serum. This lower serum cytokine content and the relative concentrations of TGF-beta 1 of TGF-beta 2 may explain, in part, the minimal inflammation and sparse fibrosis characteristic of fetal wounds. These observations provide further insight into the unique fetal response to wounding and may offer alternative avenues to modulate the postnatal wound healing response. PMID- 8632295 TI - Lack of intestinal pacemaker (C-KIT-positive) cells in infantile hypertrophic pyloric stenosis. AB - The pathogenesis of infantile hypertrophic pyloric stenosis (IHPS) is not well understood. Recent studies have shown that the protonocogene c-kit is essential for the development or maintenance of autonomic gut motility, and also show that the c-kit gene protein product (C-KIT) positive cells in the mammalian gut are responsible for intestinal pacemaker activity. This study examines cells in the pyloric muscles of 23 patients (16 with IHPS, 7 controls) for the presence of the C-KIT (C-KIT+), using immunohistochemical techniques with antihuman C-KIT sera. In the controls, many C-KIT immunoreactive (IR+) cells were observed in the muscle layers. The myenteric plexuses were demarcated by a moderate number of C KIT-IR+ cells. However, in the IHPS patients, C-KIT-IR were either absent or significantly reduced. No C-KIT-IR+ cells were found around the myenteric plexuses. These findings suggest that a lack of c-kit expression (as an indicator of intestinal pacemaker activity) in the hypertrophic pyloric smooth muscles may be an important factor in the pathogenesis of IHPS. PMID- 8632296 TI - Inhibition of cerebral neurogenic vasodilation by L-glutamine and nitric oxide synthase inhibitors and its reversal by L-citrulline. AB - The possibility that L-citrulline is able to reverse inhibition of neurogenic vasodilation in isolated porcine cerebral arteries produced by nitric oxide synthase (NOS) inhibitors and Gln was examined by using in vitro tissue bath techniques. The results indicated that transmural nerve stimulation elicited a frequency-dependent and tetrodotoxin-sensitive vasodilation in isolated ring segments of arteries with or without endothelial cells. The dilation was abolished by L-N-omega-nitro-L-Arg and L-N-omega-L-Arg methyl ester and was completely reversed by L-citrulline, but not by D-citrulline. In parallel studies, the transmural nerve stimulation-induced relaxation was blocked in part by Gln. The blockade was completely reversed by L-citrulline and L-Arg, but not by their D-enantiomers. The time courses of relaxation after L-citrulline reversal of inhibition of vasodilation produced by Gln and NOS inhibitors were identical to that of relaxation in the control. The residual relaxation after L citrulline reversal was abolished by L-NNA and L-N-omega-nitro-L-Arg. At 1 mM, L glutamate, tau-aminobutyric acid or NH4Cl did not block transmural nerve stimulation-induced relaxation, nor did Gln inhibit sodium nitroprusside-induced relaxation or neuronal NOS activity. These results provide pharmacological evidence that cerebral perivascular nerves can recycle L-citrulline to L-Arg for synthesizing nitric oxide to induce neurogenic vasodilation. Although the exact mechanism of action of Gln in inhibiting nitric oxidergic neurovascular transmission remains undetermined, Gln does not seem to act by releasing ammonium ions, inhibiting NOS or modifying the nitric oxide-cyclic GMP pathway. Cerebral metabolism of Gln may play an important role in regulating nitric oxidergic neurogenic vasodilation. PMID- 8632297 TI - I2-imidazoline binding sites: relationship with different monoamine oxidase domains and identification of histidine residues mediating ligand binding regulation by H+1. AB - We have shown that I2-imidazoline binding sites (I2BSs) are located on both monoamine oxidases A (MAO-A) and B (MAO-B) and are selectively regulated by H+ and K+ in vitro. In the present study we used chemical modifying agents to investigate the localization of I2BSs with respect to different MAO domains and the mechanisms of ligand binding regulation by K+ and H+. In mitochondrial or solubilized preparations from rabbit kidney and liver, modification of cysteine residues, which are critical for MAO activity, did not affect [3H]idazoxan binding, indicating that I2BS is not associated to the cysteine-containing flavin adenine dinucleotide (FAD) prosthetic group or to the catalytic site of MAOs. Among various chemical modifying agents, only diethylpyrocarbonate and 4 bromophenacyl bromide, two histidine modifying agents, inhibited [3H]idazoxan binding to I2BS. The pH profile of diethylpyrocarbonate effect was consistent with the specific modification of histidine residues. In protection experiments, the effect of diethylpyrocarbonate was not prevented in the presence of saturating concentrations of amiloride, guanabenz or KCl, suggesting that these residues are not located within the ligand or K+ binding sites. In contrast, histidine residues appear to be within a MAO domain involved in regulation of [3H]idazoxan binding by H+. Indeed, the pH-dependent increase in [3H]idazoxan binding was fully abolished after treatment of solubilized material with diethylpyrocarbonate. In conclusion, our results show that MAO I2BSs are not located within the flavin adenine dinucleotide prosthetic group or the catalytic site. Histidine(s) residue(s) involved in the regulation of ligand binding to I2BS by H+ also has been identified. PMID- 8632298 TI - Activation of cyclic AMP-dependent protein kinase reverses tolerance of a nucleoside transporter to ethanol. AB - Adenosine mediates some of the acute and chronic effects of ethanol in neural cells. In cultured NG108-15 cells, ethanol inhibits adenosine uptake via a specific facilitative nucleoside transporter leading to an increase in extracellular adenosine, activation of adenosine A2 receptors and increases in intracellular cyclic AMP (cAMP). After chronic ethanol exposure, an adaptive decrease in receptor-stimulated cAMP levels occurs. Additionally, the transporter becomes insensitive to rechallenge with ethanol and adenosine uptake is not inhibited. cAMP levels are decreased in cells chronically exposed to ethanol and we show here that cAMP-dependent kinase (PKA) activity in cellular homogenates also is decreased. Therefore, decreased cAMP-dependent phosphorylation may be responsible for loss of ethanol sensitivity. To test this hypothesis, NG108-15 cells were treated with agents that alter PKA activity and the ethanol sensitivity of adenosine transport was measured. In naive cells, decreasing PKA activity with the cAMP antagonist, Rp-adenosine-3',5'-cyclic phosphorothioate, resulted in ethanol-insensitive adenosine uptake. This effect was blocked by the phosphatase inhibitor, okadaic acid. These results suggest that loss of ethanol sensitivity is correlated with decreased PKA activity. Therefore, stimulating PKA activity in chronically treated cells should restore sensitivity of adenosine uptake to inhibition by ethanol. Indeed, the cAMP agonist, Sp-adenosine-3',5' cyclic phosphorothioate, restored ethanol sensitivity of transport in cells treated chronically with ethanol. Our results suggest that ethanol sensitivity of adenosine transport is regulated by PKA and protein phosphatase activities in NG108-15 cells. Moreover, the effects of chronic ethanol exposure on adenosine transport can be reversed by activating PKA. PMID- 8632299 TI - Triazolam biotransformation by human liver microsomes in vitro: effects of metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole. AB - Biotransformation of the triazolobenzodiazepine triazolam to its hydroxylated metabolites, alpha-hydroxy (OH)- and 4-OH-triazolam, was studied in vitro using microsomal preparations of human liver. Mean values of Vmax (10.3 nM/min/mg of protein) and Km (304 microM) for the 4-OH pathway exceeded values for the alpha OH pathway (2.4 and 74, respectively). However the mean Vmax/Km ratios for the two pathways were nearly identical, indicating that both contribute approximately equally to intrinsic clearance. Ketoconazole was a powerful inhibitor of triazolam biotransformation, having mean competitive Ki values of 0.006 and 0.023 microM for the alpha-OH and 4-OH pathways. This is consistent with the role of P450-3A isoforms in mediating triazolam biotransformation. The serotonin2 antagonist antidepressant nefazodone inhibited the alpha-OH and 4-OH pathways (Ki = 0.6 and 1.7 microM, respectively), but with considerably less activity than ketoconazole. Among six selective serotonin reuptake-inhibitor antidepressants, norfluoxetine was the most potent inhibitor (Ki = 2.7 and 8.0 microM) and fluoxetine itself was the weakest (Ki = 7.0 and 44.3 microM). In a double-blind clinical pharmacokinetic-pharmacodynamic study, administration of triazolam (0.125 mg) preceded by ketoconazole, compared to triazolam preceded by placebo, produced a nearly 9-fold reduction in apparent oral clearance of triazolam (41 vs. 337 ml/min) and a 4-fold prolongation of half-life (13.5 vs. 3.4 hr). Pharmacodynamic testing indicated enhancement of electroencephalographic beta activity, and enhanced decrements in digit-symbol substitution test performance, attributable to coadministration of ketoconazole. Plasma ketoconazole concentrations measured in the clinical study ranged from 0.02 microgram/ml (projected minimum) to 4.95 micrograms/ml (maximum). An in vitro-in vivo scaling model, using these plasma ketoconazole concentrations together with liver partition ratios and the in vitro Ki values, predicted a decrement of triazolam clearance due to ketoconazole coadministration that was consistent with the 88% decrement in clearance actually observed in vivo. PMID- 8632300 TI - BMS-180448, a novel glyburide-reversible cardioprotective agent, enhances postischemic recovery of contractile function in dogs. AB - BMS-180448 has been found to retain the cardioprotective potency of its chemically related analog, cromakalim, although having significantly less peripheral vasodilating activity. The effect of the ATP-sensitive potassium channel opener, BMS-180448, on postischemic recovery of function (segmental shortening) was determined in open chested, anesthetized dogs instrumented with ultrasonic crystals. The plasma concentration of the effective and ineffective doses of BMS-180448 was compared to concentrations used in isolated rat hearts. BMS-180448 was given as a total dose of 4.2, 1.4 or 0.5 mg/kg over 30 min, starting 15 min before ischemia. Ischemia was initiated by a complete occlusion of the left anterior descending coronary artery for 15 min. Reperfusion was maintained for 3 hr and segmental shortening was measured. During ischemia, systolic bulging was observed in the ischemic region in drug- and vehicle-treated groups. Upon reperfusion, some contractile functional recovery was observed in vehicle-treated controls within minutes, but quickly decreased so that slight bulging was observed up to 3 hr into reperfusion. High dose BMS-180448 significantly improved the recovery of contractile function such that, by 3 hr after reperfusion, segmental shortening had recovered to 60% of base line. The 1.4-mg/kg dose also significantly improved reperfusion function, but 0.5 mg/kg of BMS-180448 was without effect. None of the doses of BMS-180448 significantly affected peripheral hemodynamic status or collateral blood flow. The plasma concentration of the 1.4-mg/kg dose was approximately 3 microM during ischemia. In isolated rat hearts, BMS-180448 significantly increased postischemic function at 3 microM and higher concentrations, which agrees with the dog data. BMS-180448 was protective in a dose-dependent manner in a canine model of stunned myocardium, and the concentrations necessary for protection are similar to that for rats. PMID- 8632302 TI - The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2a adenosine receptor antagonist. AB - We have characterized the in vitro pharmacological profile of the new potent and selective A2a adenosine receptor antagonist SCH 58261 [7-(2-phenylethyl)-5-amino 2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine]. In binding studies on rat and bovine brain tissues, SCH 58261 showed affinity in the low nanomolar range at A2a adenosine striatal receptors and good A2a adenosine vs. A1 adenosine selectivity (about 50- to 100-fold in rat and bovine brain, respectively). SCH 58261 did not show affinity for either the A3 adenosine receptor or other receptors at concentrations up to 1 microM. Saturation experiments on rat A1 and A2a adenosine receptors indicated the competitive nature of the antagonism. SCH 58261 antagonized competitively the effects induced by the A2a adenosine-selective agonist CGS 21680 (2-[4-(2-carboxyethyl)-phenethyl amino]-5'-N- ethylcarboxamidoadenosine) in two functional assays, such as inhibition of rabbit platelet aggregation and porcine coronary artery relaxation. Specifically, the compound showed pA2 values of 7.9 and 9.5, respectively. SCH 58261 (300 nM) failed to antagonize 5'-N-ethylcarboxamidoadenosine-induced vasorelaxation in the isolated guinea pig aorta, a response mediated by A2b adenosine receptors. Likewise, at the same concentration, the compound weakly inhibited the A1 adenosine-mediated negative chronotropic effect induced by 2 chloro-N6-cyclopentyladenosine in the isolated rat atria. These data show that SCH 58261 is a potent and selective non-xanthine A2a adenosine antagonist which has competitive properties in biological responses mediated by this receptor subtype. The compound is of interest for investigating the biological role of A2a adenosine receptors and deserves further attention to clarify the therapeutic potential of A2a antagonists. PMID- 8632301 TI - Pharmacological characterization of in vivo properties of putative mixed 5-HT1A agonist/5-HT2A/2C antagonist anxiolytics. I. Antipunishment effects in the pigeon. AB - A conflict procedure in pigeons was used to characterize the antipunishment effects of the putative mixed 5-hydroxytryptamine (5-HT)1A agonist/5-HT2A/2C antagonists WY 50,324, CGS 18102A, LEK 8804 and FG 5974 and to further investigate interactions between the antipunishment effects of the 5-HT1A agonists buspirone and 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] administered in combination with the mixed 5-HT2A/2C antagonist ritanserin and the alpha 1 antagonist prazosin. The 5-HT1A agonists, buspirone and 8-OH-DPAT, which lack affinity for 5-HT2A/2C receptors, produced dose-related increases in punished responding. Of the compounds with a mixed binding profile, only WY 50,324 showed effects that were comparable to those observed after 8-OH-DPAT, whereas FG 5974 and CGS 18102A exhibited limited effects on punished responding, and LEK 8804 was ineffective. Administration of a relatively low, behaviorally active dose of ritanserin (0.16 mg/kg) significantly enhanced the potency of 8-OH DPAT and buspirone to increase punished responding from 8 to 50-fold without altering their effects on unpunished responding. Importantly, ritanserin failed to increase the number of doses of 8-OH-DPAT that significantly increased punished responding. In contrast, prazosin (2.5 mg/kg) significantly enhanced the potency and increased the number of doses of buspirone exerting significant effects on punished responding, but did not alter the effects of 8-OH-DPAT. Taken together, the results neither explain the suggested greater efficacy in producing anxiolytic effects of compounds with putative mixed 5-HT1A agonist and 5-HT2A/2C antagonist properties, nor confirm a proposed interaction between alpha1 adrenoreceptors and 5-HT1A agonists in preclinical tests of anxiolytic activity. PMID- 8632303 TI - The quaternary transformation products of N-(3-chloropropyl)-4-piperidinyl diphenylacetate and N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) have differential affinity for subtypes of the muscarinic receptor. AB - A 3-chloropropylamine derivative (N-(3-chloropropyl)-4-piperidinyl diphenylactate) of the selective muscarinic antagonist N,N-dimethyl-4-piperidinyl diphenylacetate was synthesized and its conversion to a stable azetidinium ion and interaction with muscarinic receptors was investigated. When dissolved in aqueous solution at pH 7.4, N-(3-chloropropyl)-4-piperidinyl diphenylactate formed a stable azetidinium ion with a half-time of approximately 3.6 hr. The selectivity of the azetidinium ion for native M1, M2 and M3 subtypes of the muscarinic receptors was investigated in competitive binding experiments on the hippocampus, heart and submaxillary gland of rats, respectively, using N [3H]methylscopolamine as the radioligand. The azetidinium ion exhibited equivalent high affinities for the M1 and M3 mucarinic receptor subtypes (KD = approximately 5 nM), but 10-fold lower affinity for the M2 muscarinic receptor subtype (KD = 44 nM). Similar competitive binding experiments were carried out on Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor. In these experiments, the azetidinium ion exhibited similar high affinities for the M1, M3, M4 and M5 muscarinic receptor subtypes (KD = approximately 2.4 nM), but approximately 14-fold lower affinity for the M2 muscarinic receptor subtype (KD = 34 nM). In contrast to the azetidinium ion, the parent N-(3-chloropropyl)-4-piperidinyl diphenylactate compound was 130-fold less potent. An analogous series of experiments were carried out with the aziridinium ion derived from the muscarinic receptor alkylating agent, N-(2-chloroethyl)-4 piperidinyl diphenylactate. For these binding experiments, the incubations were carried out at 0 degrees C to prevent the aziridinium ion from alkylating muscarinic receptors. The aziridinium ion was found to have equivalent high affinities for the M1, M3, M4 and M5 subtypes of the muscarinic receptor (KD = approximately 6.6 nM), but about 11-fold lower affinity for the M2 muscarinic receptor subtype (KD = 72 nM). Our results suggest that 3-haloalkylamine derivatives of 4-piperidinyl diphenylactate may be candidate prodrugs that may penetrate into brain and form azetidinium ions that have a long-lasting central anticholinergic effect. PMID- 8632304 TI - Role of imidazoline receptors in the cardiovascular actions of moxonidine, rilmenidine and clonidine in conscious rabbits. AB - The present study in conscious rabbits with intracisternal (i.c.) catheters sought to determine the relative contribution of the I1 subtype of imidazoline receptors (IR) and alpha 2 adrenoceptors to the hypotensive effects of rilmenidine, clonidine and moxonidine with an I1-IR/alpha 2 adrenoceptor antagonist efaroxan and a specific alpha 2 adrenoceptor antagonist 2 methoxyidazoxan (2-MI). The alpha 2 adrenoceptor antagonist effect of efaroxan was compared with 2-MI by performing cumulative dose-response curves in the presence of alpha-methyldopa (400 micrograms/kg i.c.). 2-MI was 5.6 times more potent than efaroxan at reversing 75% of the hypotension elicited by alpha methyldopa (P < .025). This dose ratio was used to match doses of efaroxan and 2 MI for similar alpha 2 adrenoceptor blockade. The effects of efaroxan (4.1, 13, 41 micrograms/kg i.c.) and 2-MI (0.74, 2.3, 7.4 micrograms/kg i.c.) were investigated on a single i.c. dose of rilmenidine (12 micrograms/kg), clonidine (0.75 microgram/kg) and moxonidine (0.51 microgram/kg). These doses of the antihypertensive agents, which were determined from cumulative dose-response curves, produce 90% of the maximum hypotension. Efaroxan was more effective at reversing the hypotension induced by moxonidine and rilmenidine than was 2-MI (P < .01). These findings suggest that rilmenidine and moxonidine act predominantly via IR. By contrast, 2-MI was more effective at reversing the clonidine-induced hypotension than was efaroxan (P < .001), suggesting that clonidine acts mainly via alpha 2 adrenoceptors in conscious normotensive rabbits. Thus, a higher selectivity of the second generation agents moxonidine and rilmenidine for I1-IR over alpha 2 adrenoceptors, compared with the first generation agent clonidine, appears to be necessary for this effect to be manifested in their hypotensive actions. PMID- 8632305 TI - Oral biological activities of spontaneous nitric oxide releasers are accounted for by their nitric oxide-releasing rates and oral absorption manners. AB - We examined whether p.o. biological activities of (+/-)-(E)-4-ethyl-2-[(E) hydroxyimino]-5-nitro-3-hexenamide (FK409), a spontaneous nitric oxide (NO) releaser, and the derivatives, i.e., (+/-)-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5 nitro-3-hexenyl]-3 - pyridinecarboxamide (FR144420) and (+/-)-N-[(E)-4-ethyl-3 [(Z)-hydroxyimino]-6-methyl-5- nitro-3-heptenyl]-3-pyridinecarboxamide (FR146801), could be accounted for by their NO-releasing rates and p.o. absorption manners. These compounds spontaneously released NO with the rates in the rank order of FK409 > FR144420 > FR146801. Total contribution of these drugs as NO donors in vivo was almost the same from the determination of urinary nitrite/nitrate (NOx) levels after p.o. administration of the compounds at 10 mg/kg to rats. Plasma NOx level after p.o. administration of FK409 at 10 mg/kg to rats reached maximal level at 120 min, and decreased gradually. On the other hand, plasma NOx levels time-dependently increased during 360 min after p.o. administration of FR144420 and FR146801 at the same dose. FK409 and FR144420 showed hypotensive effects immediately after p.o. administration at 10 mg/kg to rats, and the maximum response of FR144420 was less and the duration of the effect was longer than those of FK409, respectively. On the other hand, FR146801, which is most stable in solution, did not show any significant hypotensive effect during 240 min after p.o. administration at the same dose. In conclusion, the response and the duration of biological activity after p.o. administration of three spontaneous NO releasers can be closely accounted for by their NO-releasing rates and p.o. absorption manners. PMID- 8632306 TI - Effects of somatostatin and related peptides on the membrane potential and input resistance of rat ventral subicular neurons, in vitro. AB - The effects of bath-applied somatostatin (SS), and related peptides on the membrane potential and input resistance of 117 ventral subicular neurons were investigated by intracellular recording in rat brain slices. Electrophysiological properties, which included burst-firing in response to depolarizing current pulses, indicated that the neurons studied were of the pyramidal type. For the 89 cells analyzed quantitatively, membrane potential was -69.1 +/- 0.3 mV (mean +/- S.E.) and input resistance was 23.9 +/- 0.5 megohms. SS (5 microM) caused a hyperpolarization of 3.4 +/- 0.3 mV (n = 9) and reduced input resistance by 16 +/ 3.1% (n = 6). SS D-Trp8, somatostatin, octreotide, CGP 23996 and MK 678 shared these effects, but somatostatin was inactive. SS effects persisted when bathing solutions contained tetrodotoxin, reduced calcium and elevated magnesium concentrations and when both of these treatments were combined. They were unaltered by antagonists at gamma-aminobutyric acid receptors or at ionotropic glutamate receptors. The effects of MK 678, SS, SS D-Trp8 and somatostatin were concentration-dependent, and these peptides were equipotent at 500 nM and at 5 microM. For MK 678, the EC50 was 316 nM for the hyperpolarization and 90 nM for the reduction in input resistance. We conclude that SS acts directly on pyramidal neurons of the rat subiculum to cause a hyperpolarization and a decrease in input resistance. We suggest that these effects are mediated by the SSTR2 receptor subtype. PMID- 8632307 TI - Calcium sensitization in perfused beating guinea pig heart by a positive inotropic agent MCI-154. AB - We investigated the effects of MCI-154, a positive inotropic agent that increases the myofilament response to Ca++, on Ca++ transients, left ventricular (LV) function and phosphodiesterase (PDE) III activity of guinea pig heart, and compared them with the effects of pimobendan and milrinone. In Langendorff guinea pig hearts loaded with a fluorescent Ca++ probe indo-1, LV pressure and Ca++ transient were measured simultaneously. MCI-154 (10(-10)-10(-6) M) increased LV developed pressure, +dP/dt and -dP/dt with a reduction of LV end-diastolic pressure, although it did not affect coronary flow. The positive inotropic activity of MCI-154 was more potent than that of pimobendan and milrinone; EC50 values (concentrations for increasing +dP/dt by 50% from base line) were 4.31, 41.5 and 294 x 10(-9) M, respectively. The positive inotropic effect of MCI-154 was accompanied by the increase in systolic, diastolic and amplitude of indo-1 fluorescence ratio. The relative increase in Ca++ transients against the increase in LV contractility produced by MCI-154, however, was significantly less than that by increasing perfusate [Ca++], pimobendan or milrinone. MCI-154 (3 x 10(-7) 10(-4) M) inhibited the activity of PDE III isolated from guinea pig LV tissues, but the inhibitory effect of MCI-154 was less than pimobendan and milrinone; IC50 values were 10.1, 3.5 and 2.4 x 10(-6) M, respectively. These findings suggest that MCI-154 exerts a positive inotropic effect mainly through Ca(++)-sensitizing action in intact beating whole hearts. PMID- 8632308 TI - Relative analgesic potency of mu, delta and kappa opioids after spinal administration in amphibians. AB - The analgesic effects of 12 opioid agonists in amphibians were measured using the acetic acid test. Spinal administration of dermorphin, [D-Ala2,NMePhe4,Gly-ol] enkephalin, fentanyl and morphine (mu opioids); [D-Ser2,Leu5-Thr6]-enkephalin, [D Ala2,D-Leu5]-enkephalin, [D-Pen2,D-Pen5]-enkephalin and deltorphin (delta opioids); and Cl977 [(5R)-(544 alpha,744 alpha,845 beta)-N-methyl-N-[7-(1 pyrrolidinyl)-1- oxaspiro[4,5]dec-8yl]-4-benzofuranacetamide monohydrochloride, bremazocine), U50488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methanesulfonate) and nalorphine (kappa opioids) produced a dose-dependent and long-lasting analgesia in the Northern grass frog, Rana pipiens. With all opioids, time course experiments showed that this analgesic effect lasted for at least 4 hr, with no untoward effects observed within each dosage range used. The analgesic effects of the 12 opioids were blocked by systemic naltrexone pretreatment. Comparison of dose-response curves demonstrated that the rank order of potency was such that, in general, mu opioids > delta opioids > kappa opioids. Dose-response curves obtained in the presence of a fixed dose of naltrexone showed the greatest shift for [D-Ser2,Leu5-Thr6] enkephalin, less so for [D-Ala2,NMePhe4,Gly-ol]-enkephalin and the least shift for bremazocine. ED50 values for mu and delta opioids in the amphibian acetic acid test were significantly correlated to ED50 values of the same opioids reported in the literature for the rodent TF test. These results show that a spinal site of opioid analgesia is present in amphibians and supports the utility of this alternative, nonmammalian model for studies of opioid analgesia and pain research. PMID- 8632309 TI - Buprenorphine's physical dependence potential: antagonist-precipitated withdrawal in humans. AB - Buprenorphine is a partial mu opioid agonist with demonstrated efficacy in the treatment of opioid dependence. One potential advantage of buprenorphine over full mu opioid agonists is its reported low physical dependence profile. This study systematically examined physical dependence produced by maintenance with a clinically relevant dose of buprenorphine using antagonist challenge procedures. In this residential laboratory study, eight opioid-dependent volunteers maintained on 8 mg/day of sublingual buprenorphine were each challenged on independent occasions with placebo, i.m. naloxone (0.3, 1.0, 3.0 and 10.0 mg/70 kg) and p.o. naltrexone (0.3, 1.0 and 3.0 mg/70 kg) 14 hr after their daily buprenorphine dose using a repeated measures, cross-over design. Both naloxone and naltrexone precipitated time- and dose-dependent withdrawal, as evidenced by changes in subject-rated, observer-rated and physiological measures. Significant precipitated withdrawal occurred at 3.0 and 10 mg/70 kg i.m. of naloxone and 3.0 mg/70 kg p.o. of naltrexone. These results indicate that buprenorphine maintenance produces physical dependence and that i.m. naloxone and p.o. naltrexone produce equivalent effects in withdrawal precipitation under these conditions. Findings have implications for selection of antagonist doses for use in formulating combination agonist/antagonist medications and for use in transition of drug abusers from buprenorphine to antagonist maintenance therapies. PMID- 8632311 TI - Selective inhibition by a class III antiarrhythmic agent, E-4031, of the negative chronotropic response to parasympathetic stimulation in anesthetized dogs. AB - To investigate the influence of a class III antiarrhythmic agent, E-4031, on the vagus control of the heart, we studied the effects of E-4031 on the chronotropic, dromotropic and inotropic responses to parasympathetic stimulation in the autonomically decentralized hearts of the open-chest, anesthetized dogs. E-4031 (0.01-3 mumol/kg i.v.) decreased heart rate dose-dependently without affecting atrioventricular (AV) conduction time, first derivative of "a" wave component of the right atrial pressure (RA dP/dt), maximum rate of the right ventricular pressure development (RV +dP/dt) and arterial blood pressure. When cervical vagus stimulation decreased the heart rate, RA dP/dt and RV +dP/dt and prolonged the AV conduction time, E-4031 antagonized the negative chronotropic response in a dose dependent manner but affected neither dromotropic nor atrial inotropic responses. E-4031 at a high dose of 3 mumol/kg i.v. attenuated the ventricular inotropic response. ID50 for the chronotropism was 0.20 +/- 0.05 mumol/kg. Stimulation of the selective intracardiac parasympathetic nerves to the sinoatrial nodal area decreased the heart rate and RA dP/dt without a dromotropic response. E-4031 antagonized the negative chronotropic response to the stimulation but not the inotropic response. Stimulation of the selective intracardiac parasympathetic nerves to the AV nodal area prolonged the AV conduction time without a chronotropic response. E-4031 at a high dose of 3 mumol/kg i.v. attenuated the negative dromotropic response to the stimulation by 35 +/- 10%. These results suggest that E-4031 preferentially blocks the negative chronotropic response to vagus stimulation without significantly affecting other cardiac responses at a site distal to the muscarinic receptor in the heart in situ. PMID- 8632310 TI - The role of nitric oxide in dizocilpine-induced impairment of spontaneous alternation behavior in mice. AB - We investigated the role played by nitric oxide in the dizocilpine-induced impairment of both spontaneous alternation behavior in a Y-maze and of performance in a multiple-trial passive avoidance task in mice. Dizocilpine (0.1 mg/kg) impaired the spontaneous alternation behavior and the retention of passive avoidance without affecting acquisition in the multiple-trial passive avoidance task. NG-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, dose-dependently impaired the spontaneous alternation behavior, but had no effect on either the acquisition or retention of passive avoidance. NG-nitro-D arginine methylester had no effect on either task. The inhibitory effect of L NAME on the spontaneous alternation behavior was completely reversed by the coadministration of L-arginine. Pretreatment with L-arginine ameliorated the dizocilpine-induced impairment of spontaneous alternation behavior, but not the impairment of the retention of passive avoidance. S-Nitroso-N acetylpenicillamine, a generator of NO, completely inhibited the dizocilpine induced impairment of spontaneous alternation behavior. Finally, the impairment of spontaneous alternation behavior caused by dizocilpine was significantly diminished by pretreatment with dibutyryl cyclic GMP. These results suggest that, although N-methyl-D-aspartate receptors play a critical role in both spatial working memory and long-term memory processes assessed by spontaneous alternation behavior and the passive avoidance, respectively, different neuronal mechanisms may be involved in these two processes. Further, it is suggested that the NO/cyclic GMP system may play a role in spatial working memory. PMID- 8632312 TI - Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist. AB - Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1 induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported. PMID- 8632313 TI - Nicotine-induced inhibition in medial septum involves activation of presynaptic nicotinic cholinergic receptors on gamma-aminobutyric acid-containing neurons. AB - Neuronal responses to drugs acting on nicotinic cholinergic receptors (nAChRs) were examined in the rat medial septal area by using an in vivo extracellular single-unit recording technique. In the medial septal area, iontophorectically applied nicotine inhibited neuronal activity in 45% of the neurons, but had no effect on the remaining neurons. Dihydro-beta-erythroidine application to neurons in the medial septal area not only blocked nicotine-induced inhibition, but also reduced spontaneous neuronal activity of the neurons. When Mg++ was applied iontophoretically to block presynaptic neurotransmitter release, a significant reduction in spontaneous neural activity also was observed. No further reduction of spontaneous activity by dihydro-beta-erythroidine occurred in the presence of Mg++, suggesting an apparent tonic excitatory input to the majority of neurons in the medial septal area under the control of presynaptic nAChRs. Mg++ abolished the nicotine-induced inhibition in the medial septal area without having an effect on nicotine-induced inhibition in the cerebellum. Thus, these data provide evidence that the inhibitory effects of nicotine in the medial septum are due to an action on presynaptic nAChRs, controlling the release of an inhibitory neurotransmitter. Of the medial septal neurons which showed no response to nicotine, nicotine produced excitation in 21% of the cells after Mg++ application, indicating that nicotine can have a direct action on postsynaptic nAChRs, in addition to its presynaptic action, in the medial septum. Finally, application of the gamma-aminobutyric acid antagonist bicuculline reduced the nicotine-induced inhibition on the majority of medial septal neurons tested, but was without effect on the inhibition produced by nicotine on cerebellar Purkinje neurons. Consequently, it can be concluded that the nicotine-induced inhibition in the medial septum is the result of gamma-aminobutyric acid release due to its action on presynaptic nAChRs present on gamma-aminobutyric acid-containing terminals. PMID- 8632314 TI - Characterization of 8-(N-methylisopropyl)amino-N6-(5'-endohydroxy- endonorbornyl) 9-methyladenine (WRC-0571), a highly potent and selective, non-xanthine antagonist of A1 adenosine receptors. AB - Previous studies in our laboratory identified N6-endonorbornyl-9-methyladenine (N 0861) as a highly selective (100-fold) A1-adenosine receptor antagonist (KB = 500 nM). However, its moderate potency limits the degree of A1-receptor blockade that can be achieved by systemically administered N-0861. Structure activity studies were undertaken to invent a compound that had greater affinity for the A1 adenosine receptors than N-0861. C8-N-methylisopropylamino-N6-5'-endohydroxy-N 0861 (WRC-0571) inhibited [3H]-N6-cyclohexyladenosine (CHA) binding to guinea pig A1-receptors with a Ki value of 1.1 nM. WRC-0571 was 200-fold less potent at inhibiting [3H]-5'-N-ethylcarboxamidoadenosine binding to bovine A2a receptors (Ki = 234 nM). WRC-0571 also inhibited the binding of radioligands to cloned human A1, A2a and A3 adenosine receptors with affinities of 1.7, 105 and 7940 nM, respectively. Thus in human adenosine receptors, WRC-0571 is 62-fold selective for the A1 vs. A2a and 4670-fold selective for the A1 vs. A3 receptors; WRC-0571 is therefore the most A1 vs. A3 selective compound yet described. In guinea pig isolated atria, WRC-0571 antagonized the A1-mediated negative inotropic responses to 5'-N-ethylcarboxamidoadenosine (NECA) with a KB of 3.4 nM. WRC-0571 was more than 2500-fold less potent at antagonizing NECA-induced A2b-mediated relaxation in guinea pig aorta. In anesthetized rats WRC-0571 antagonized adenosine-induced bradycardia at concentrations as low as 1 nmol/kg but failed to antagonize A2 mediated hindquarter vasodilation at concentrations up to 10,000 nmol/kg. WRC 0571 is orally active at concentrations as low as 0.3 mumol/kg. WRC-0571 is therefore a highly potent, highly selective antagonist of A1-adenosine receptors both in vitro and in vivo. PMID- 8632316 TI - Epibatidine: a nicotinic acetylcholine receptor agonist releases monoaminergic neurotransmitters: in vitro and in vivo evidence in rats. AB - The effect of the neuronal acetylcholine-gated ion channel receptor agonist (+/-) epibatidine was studied on neurotransmitter release in vitro and motor behavior in vivo. (+/-)-Epibatidine (3-300 nM) caused a concentration- and calcium dependent release of [3H]-dopamine from striatal slices and [3H]-norepinephrine release from hippocampal and thalamic slices. (+/-)-Epibatidine-induced neurotransmitter release was inhibited in all three regions by mecamylamine (3 microM). In contrast, D-tubocurarine (10-100 microM) inhibited only (+/-) epibatidine-induced [3H]-norepinephrine release from the hippocampus and the thalamus. Conversely, dihydro beta-erythroidine (3-100 microM) inhibited (+/-) epibatidine-induced [3H]-dopamine release in the striatum without significantly altering [3H]-norepinephrine release from either the hippocampus or the thalamus. This is consistent with the observation that different nAChRs modulate dopamine release as compared with norepinephrine release. The effect of (+/-)-epibatidine on both [3H]-dopamine and [3H]-norepinephrine release was tetrodotoxin-sensitive, suggesting the involvement of sodium channels. (+/-)-Epibatidine (1-3 micrograms/kg s.c.) produced ipsilateral turning in the unilaterally [6(OH)-DA] lesioned rat. This effect was mimicked by (-)-nicotine (0.35 mg/kg s.c.). Both (+/-)-epibatidine- and (-)-nicotine-induced turning were significantly inhibited by mecamylamine (3 mg/kg s.c.), indicating that the turning response was mediated by nAChRs. (+/-)-Epibatidine also increased locomotor activity in a dose dependent manner. (+/-)-Epibatidine-induced hyperactivity was blocked by D1 and D2 receptor antagonists, SCH 23390 and eticlopride, respectively, suggesting that both dopamine receptor subtypes might be required for the locomotor effect of (+/ )-epibatidine. These results demonstrate that (+/-)-epibatidine displays nAChR agonist activity in the rat CNS and that certain effects are mediated via nAChR stimulated catecholamine release and subsequent activation of corresponding receptors. PMID- 8632315 TI - Characterization of the antinociceptive properties of cimetidine and a structural analog. AB - The antinociceptive and pharmacological properties of the H2 receptor antagonist cimetidine and a novel cimetidine analog, SKF92374, were characterized. On both the hot-plate and tail-flick nociceptive tests, cimetidine and SKF92374 induced complete, dose-related analgesic responses when injected into the lateral ventricle of rats. SKF92374 showed strong similarities to cimetidine in analgesic efficacy, slope of dose-response curves and chemical structure, suggesting that these compounds share a common analgesic mechanism. In contrast, histamine induced submaximal antinociceptive effects, and the H3 antagonist thioperamide, a known HA-releasing drug, had little or no analgesic effects. Compared with cimetidine, SKF92374 showed very weak activity (400-fold lower affinity) on H2 receptors in vitro (isolated guinea pig atrium) and in vivo (rat gastric secretion). In addition, SKF92374 (100 microM) had neither agonist nor antagonist action on guinea pig ileum H1 receptors. SKF92374 was also a weak competitive antagonist of N alpha-methylhistamine-induced inhibition of electrically induced contractions of the guinea pig ileum (Kd = 5.2 microM), an H3 receptor-mediated response. Autoradiographic binding assays in guinea pig brain confirmed a weak antagonism of H3 receptors by SKF92374. The compound (up to 10 microM) also had no effect on unpurified rat brain histamine N-methyltransferase activity. These results support the hypothesis that cimetidine induces analgesia by a novel brain mechanism unrelated to H1, H2 or H3 receptors. PMID- 8632317 TI - Modulation of seizure activity in mice by metabotropic glutamate receptor ligands. AB - The anticonvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in different seizure models by use of intracerebroventricular infusion. The mGluR1a antagonist/mGluR2 agonist, (S)-4 carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] dose-dependently antagonized pentylenetetrazol- and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-2-carboxylate (DMCM)-induced clonic convulsions in mice with ED50 values of 400 and 180 nmol/mice, respectively. A modest increase in electrical seizure threshold was observed in mice injected with (S)-4C3HPG. No effect on seizures induced by systemic administration of N-methyl-D-aspartate was observed by prior intracerebroventricular infusion of (S)-4C3HPG. The more selective (but less potent) mGluR1a antagonist, (S)-4-carboxyphenylglycine, was a weak anticonvulsant in similar seizure models with the exception of convulsions induced by electrical stimulation. (+)-alpha-Methyl-4-carboxyphenylglycine showed no anticonvulsant activity in any of the models examined. Agonists of mGluRs which are particularly potent at mGluR2, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine and (1S,3R)-1 aminocyclopentane dicarboxylic acid significantly protected against sound-induced convulsions in DBA/2 mice and DMCM-induced seizures in mice but were inactive against seizures induced by administration of pentylenetetrazol or by electrical stimulation. These data suggest that mGluR ligands modulate seizure activity in mice and this effect may be mediated via antagonism of mGluR1a-type as well as via activation of mGluR2-type mGluRs. PMID- 8632318 TI - Discriminative stimulus effects of nalbuphine in rhesus monkeys. AB - Three rhesus monkeys discriminated between 0.178 mg/kg of nalbuphine and saline while responding under a fixed-ratio 5 schedule of stimulus-shock termination. Nalbuphine produced dose-related increases in drug-lever responding with > or = 90% of responses occurring on the drug lever at doses larger than 0.1 mg/kg. The duration of action of the discriminative stimulus effects of nalbuphine was less than 5.25 hr. Rank order potency of compounds that substituted for the nalbuphine discriminative stimulus (i.e., > or = 90% responding on the nalbuphine lever) in all three subjects was fentanyl > butorphanol > methadone > morphine. Compounds that did not substitute completely in all monkeys included the kappa agonists ethylketocyclazocine, enadoline, spiradoline and U-50,488 and the nonopioids cocaine, d-amphetamine, clonidine, ketamine and phencyclidine. Naltrexone antagonized the discriminative stimulus effects of nalbuphine, shifting the nalbuphine dose-effect curve in a manner that was consistent with mu receptor mediation. Results from the current study demonstrate that, in rhesus monkeys, the discriminative stimulus effects of nalbuphine are mediated by mu opioid receptors. Although there is evidence suggesting that nalbuphine has kappa agonist effects (e.g., subjective effects in humans), results from several studies, including the current study, strongly suggest that in rhesus monkeys nalbuphine does not exert agonist actions at kappa receptors. Moreover, these data indicate that differences in behavioral effects between nalbuphine and prototypic mu opioids (e.g., morphine) probably result from differences in activity (e.g., efficacy) at mu receptors rather than any kappa agonist actions of nalbuphine. PMID- 8632319 TI - Differential genetic mediation of sensitivity to morphine in genetic models of opiate antinociception: influence of nociceptive assay. AB - Several genetic mouse models of opiate sensitivity have been identified or produced in an attempt to investigate mechanisms underlying individual variation in responses to opiate drugs like morphine. The major models in use presently are the DBA/2 (DBA) versus C57BL/6 (C57) inbred strains, the recombinantly inbred CXBK strain, and mouse lines selectively bred for high- and low-magnitude antinociception after swim stress (HA and LA lines, respectively) or levorphanol administration (HAR and LAR lines, respectively). The hot-plate test, an assay of acute, thermal nociception, was used in the selection of the HA/LA and HAR/LAR lines, and has largely been used to characterize the differential opiate sensitivity of the DBA (high) and C57 (low) strains and the deficient sensitivity of the CXBK strain. There exist, however, many other nociceptive assays used with murine subjects; the most common are the tail-flick/withdrawal test, the acetic acid abdominal constriction test and the formalin test. In the present experiment, baseline nociceptive sensitivities and morphine antinociceptive dose response relationships (0.1-10 mg/kg i.p. or s.c.) were investigated in mice of all four genetic models and in all four major nociceptive assays, with identical parameters. Results indicate a high degree of dissociation between different genetic models, which suggests that these strains differ in their nociceptive and antinociceptive sensitivities due to the effects of very different genetic and physiological mechanisms. In addition, the present findings suggest that morphine inhibits different modalities of nociception via separate mechanisms that can be genetically dissociated and independently altered. Strikingly, in HA/LA and HAR/LAR mice, we find that an inverse relationship exists with respect to morphine antinociceptive sensitivity in the hot-plate and acetic acid abdominal constriction tests, respectively. PMID- 8632320 TI - kappa-Opioid receptor agonists prevent sensitization to the conditioned rewarding effects of cocaine. AB - A place preference conditioning procedure was used to examine the influence of kappa-opioid receptor ligands upon the development of sensitization to the conditioned rewarding effects of cocaine. Previous exposure to cocaine (10-20 mg/kg; i.p.; days 1-5) resulted in an enhancement of the conditioned rewarding effects of this agent, e.g., sensitization. Thus, doses of cocaine (5.0-10.0 mg/kg; i.p.) that failed to produce place preferences in control rats produced significant place preferences in cocaine-experienced animals. In animals that had received the kappa-agonist U50,488H (5.0 mg/kg; s.c.) in combination with the repeated cocaine treatment regimen, no enhancement of cocaine-induced place conditioning was seen. Similarly, the kappa-agonist U69593 administered on days 1 to 5 or only on days 3 to 5 of the cocaine treatment regimen prevented the enhanced response to cocaine. This effect occurred after either systemic (0.04 0.16 mg/kg; s.c.) or intracerebroventricular (1.0 mg) treatment and was abolished by the kappa-opioid receptor antagonist, nor-binaltorphimine. In contrast to its effects when administered in combination with cocaine, prior administration of U69593, alone, failed to modify the conditioned response to cocaine. Microdialysis studies revealed a marked elevation of extracellular dopamine levels within the ventral striatum after repeated cocaine administration. In animals that had received U69593 in combination with cocaine, no elevation of dopamine was seen. These data demonstrate that sensitization develops to the conditioned rewarding effects of cocaine and that the activation of central nervous system kappa-opioid receptors prevents the development of this phenomenon. An involvement of the mesolimbic dopamine system in mediating the interaction of kappa-agonists with cocaine is suggested. PMID- 8632321 TI - Effects of lipid A on calcium homeostasis in renal proximal tubules. AB - It is clear that lipopolysaccharides (LPS) are responsible for the multiorgan failure often associated with endotoxemia. However, little is known of the direct effects of LPS on kidney cells. We examined the effects of lipid A, the biologically active component of LPS, on rat proximal tubule Ca++ homeostasis. Lipid A produced a rapid, transient, concentration-dependent rise in intracellular Ca++ concentration, [Ca++]i, as monitored by fura-2. At 50 micrograms/ml [Ca++]i rose to 138 +/- 12 nM (n = 4) above basal [Ca++]i levels. The response to lipid A was not significantly inhibited by chelating extracellular Ca++ with EGTA (5 mM). However, the rise in [Ca++]i was significantly inhibited by 8-(N,N-dimethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride) and thapsigargin (17 +/- 7 nM and 13 +/- 9 nM rise, respectively; P < .05). These data indicate that the rise in [Ca++]i induced by lipid A is due to release of intracellular stores, and not extracellular influx. We also examined the role of inositol 1,4,5-trisphosphate in the lipid A response. Lipid A caused a time-dependent increase in inositol 1,4,5-trisphosphate that paralleled the rise in [Ca++]i, suggesting the release in [Ca++]i is through an inositol 1,4,5-trisphosphate-mediated release of intracellular stores. The ability of lipid A to alter Ca++ homeostasis suggests a potential for LPS to directly alter proximal tubule physiology and renal function in vivo. PMID- 8632322 TI - Mechanisms for the hepatic uptake and biliary excretion of tributylmethylammonium: studies with rat liver plasma membrane vesicles. AB - Hepatic organic cation transport in vitro, using tetraethylammonium (TEA) as a substrate, consists of at least two steps: sinusoidal uptake is stimulated by an inside-negative membrane potential and canalicular membrane transport is mediated by organic cation:H+ exchange (Moseley et al., 1992b). In vivo, however, TEA is poorly excreted into bile. In contrast, larger, more hydrophobic organic cations, such as tributylmethylammonium (TBuMA), undergo significant hepatobiliary excretion. To better characterize hepatic organic cation transport, TBuMA transport was examined in rat canalicular liver plasma membrane (cLPM) and basolateral liver plasma membrane (bILPM) vesicles. In cLPM vesicles, under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [3H]TBuMA uptake consistent with electroneutral TBuMA:H+ exchange; H(+)-dependent [3H]TBuMA uptake was not the result of a H+ diffusion potential. In the absence of a H+ gradient, intravesicular TBuMA trans-stimulated [3H]TBuMA uptake. Substrates for renal and hepatic organic cation:H+ exchange cis-inhibited H(+) dependent [3H]TBuMA uptake. No ATP-dependent [3H]TBuMA uptake was detected in cLPM vesicles, and the P-glycoprotein substrate, daunomycin, did not cis-inhibit H(+)-dependent [3H]TBuMA uptake. Carrier-mediated [3H]TBuMA uptake exhibited saturability (Km of 0.5 mM and Vmax of 0.5 nmol/mg prot/5 s). In bILPM vesicles, in contrast, a valinomycin-induced intravesicular-negative K+ diffusion potential stimulated [3H]TBuMA uptake. These findings suggest that hepatic transport of TBuMA is similar to TEA but fundamentally different from that of P-glycoprotein substrates, indicating the involvement of at least two separate processes for the hepatobiliary excretion of organic cationic drugs. PMID- 8632323 TI - Cocaine concentration-effect relationship in the presence and absence of lidocaine: evidence of competitive binding between cocaine and lidocaine. AB - To better understand the interaction between cocaine and lidocaine, we studied the cocaine's concentration-effect relationship for action potential duration (APD) and the rate of rise of phase 0 of the action potential (Vmax) of canine papillary muscle in the presence and absence of lidocaine. We measured APD and Vmax during programmed stimulation and superfusion with normal Tyrode's, 30 microM cocaine and 30 microM cocaine + 30 microM lidocaine. Using two microelectrodes, we simultaneously recorded action potentials from two sites during programmed stimulation and measured the conduction velocity and effective refractory period during exposure to normal Tyrode's, cocaine and cocaine + lidocaine. Cocaine with or without lidocaine delayed the plateau of the APD restitution curve. At 1000 msec cycle length, the addition of 30 microM lidocaine to the superfusate containing 30 microM cocaine shortened the time constant for reactivation of Vmax from 514 +/- 63 to 234 +/- 28 msec (P < .01). Lidocaine also improved the conduction velocity decreased by cocaine, but did not significantly change the effective refractory period. The configuration of cocaine concentration-effect curve for APD was biphasic. For cocaine concentrations < 100 microM, APD progressively shortened prolonged with increasing concentrations. As cocaine concentrations increased > 100 microM, APD progressively shortened. The addition of lidocaine to the superfusate with cocaine > 100 microM tended to attenuate the progressive APD shortening due to cocaine. Lidocaine shifted the curve correlating cocaine concentration and reduction of Vmax rightward, but preserved Emax at cocaine concentration > 225 microM. These findings suggest competitive antagonism between cocaine and lidocaine at a single sodium channel receptor. CONCLUSION: lidocaine displaces cocaine from the sodium channel receptor through competitive binding. Lidocaine may prove to be beneficial in reversing cocaine-induced slowing of ventricular conduction. PMID- 8632324 TI - Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding. AB - Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol. PMID- 8632325 TI - Investigation of brain sites mediating cannabinoid-induced antinociception in rats: evidence supporting periaqueductal gray involvement. AB - The purpose of this study was to elucidate brain areas that mediate cannabinoid induced antinociception as assessed in the tail-flick test. Intracerebroventricular administration of the prototypical cannabinoid, delta 9 tetrahydrocannabinol, and the potent bicyclic analog, CP-55,940, produced antinociception at ED50 values of 373 and 64 nmol/rat, respectively. Hypothermic and cataleptic effects also were observed after i.c.v. administration of CP 55,940, but not delta 9-tetrahydrocannabinol. In contrast, the endogenous cannabinoid, anandamide, failed to elicit any apparent pharmacological effects. Administration of CP-55,940 into the caudate putamen produced catalepsy, but failed to produce either antinociception or hypothermia. Micro-injection of CP 55,940 into the ventrolateral aspect of the periaqueductal gray (PAG), in the region of the dorsal raphe, produced antinociception (ED50 dose = 28 nmol/rat), catalepsy and hypothermia. CP-56,667, the inactive stereoisomer of CP-55,940, failed to produce any effects when injected into the same site. Additional studies demonstrated that pertussis toxin completely prevented the pharmacological effects of CP-55,940 when both agents were administered into the posterior ventrolateral PAG. In contrast, dibutyryl-cAMP failed to attenuate cannabinoid-induced antinociception. Finally, CP-55,940 administered into either the posterior dorsolateral or the anterior ventrolateral areas of the PAG was without effect. These results indicate that the antinociceptive and cataleptic effects of cannabinoids in the PAG are dose-related, exhibit regional specificity and are enantioselective. Moreover, the complete prevention of these pharmacological effects by pertussis toxin pretreatment in the PAG is consistent with the involvement of G proteins. These findings suggest that the posterior ventrolateral PAG may be an important brain area for the antinociceptive and cataleptic effects of the cannabinoids. PMID- 8632326 TI - A three-lever operant procedure differentiates the stimulus effects of R(-)-MDA from S(+)-MDA. AB - 1-(3,4-Methylenedioxyphenyl)-2-aminopropane (MDA) produces an effect in humans that is somewhat similar to both a hallucinogen and a central stimulant. We have previously shown that R(-)-MDA but not S(+)-MDA produces a stimulus effect in animals similar to that of the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2 aminopropane (DOM), whereas S(+)-MDA but not R(-)-MDA produces a stimulus effect similar to that of the stimulant amphetamine. Others have suggested that the stimulus effects of the two MDA isomers may be much more similar than our results would indicate. To help clarify this issue, we have shown that rats (n = 8) can be trained to discriminate 1.25 mg/kg of R(-)-MDA (ED50 = 0.99 mg/kg) from 1.25 mg/kg of S(+)-MDA (ED50 = 0.80 mg/kg) versus 0.9% saline with use of a three lever operant procedure. To accomplish this task it was necessary to institute a separation of 4 days between injections of the isomers. In tests of stimulus substitution, the administration of various doses of DOM (ED50 = 0.47 mg/kg), S(+)-DOM (ED50 = 2.23 mg/kg) and mescaline (ED50 = 16.04 mg/kg) produced dose related responding on the R(-)-MDA-appropriate lever. In contrast, the injection of various doses of S(+)-amphetamine (ED50 = 0.46 mg/kg) and cocaine (ED50 = 6.40 mg/kg) produced dose-related responding on the S(+)-MDA-appropriate lever. The administration of racemic MDA, at twice the isomer training dose, resulted in the animals dividing their responses closely between the R(-)-MDA- and S(+)-MDA designated levers. In antagonism tests, the serotonin-2 antagonist pirenperone blocked the stimulus effect of 1.25 mg/kg of R(-)-MDA but had no effect on the stimulus effect of 1.25 mg/kg of S(+)-MDA. Taken together, these data support the contention that each optical isomer of MDA can produce a markedly different stimulus effect. PMID- 8632327 TI - Effect of lipid-free total parenteral nutrition on hepatic drug conjugation in rats. AB - The effect of total parenteral nutrition (TPN) on drug conjugation in male Sprague-Dawley rats was examined using a nutrition solution composed of amino acids and glucose. The overall disposition of acetaminophen including the formation kinetics of the sulfate and glucuronide metabolites was used as an in vivo probe. Selected drug metabolizing enzyme activities also were examined in vitro. TPN, 200 kcal/kg/day, was administered by continuous i.v. infusion for 14 days and changes elicited were compared to control animals allowed free access to rat chow. TPN decreased the total clearance of acetaminophen by 34% and the formation clearance to acetaminophen sulfate by 47%. The formation clearance of acetaminophen to acetaminophen glucuronide was unaffected by TPN. Cytochrome P450 concentration and oxidative demethylase activity toward p-nitroanisole were decreased in parallel, 47 and 53%, respectively, and UDP-glucuronosyltransferase activity with p-nitrophenol and acetaminophen as the acceptor aglycones was decreased 44 and 25%, respectively in the animals receiving TPN. Sulfotransferase activity toward both p-nitrophenol and acetaminophen decreased 28% in animals receiving TPN vs. ad libitum rat chow. Administration of the parenteral nutrition solution as a continuous enteral infusion via a doudenal catheter slightly decreased p-nitroanisole demethylase activity (26%), but had no other significant effects on either cytochrome P450 concentration or on drug conjugating enzyme activities determined in vitro. These results show that parenteral nutrition administered i.v. depresses drug conjugation and suggest that alterations in both hepatic oxidative and conjugative biotransformation arising from total parenteral nutrition are largely attributable to bypassing the intestinal route for nutrient intake. PMID- 8632328 TI - Nonpeptide endothelin receptor antagonists. VI: Pharmacological characterization of SB 217242, a potent and highly bioavailable endothelin receptor antagonist. AB - This study describes the pharmacological characterization of SB 217242, a highly potent orally bioavailable nonpeptide antagonist of both endothelin type A (ETA) and endothelin type B (ETB) receptors. In human cloned ETA and ETB receptors, SB 217242 produced concentration-dependent displacement of [125]I-endothelin-1 (ET 1) in both receptor subtypes with Ki values of 1.1 and 111 nM, respectively. SB 217242 produced concentration-dependent, parallel rightward shifts in the ET-1 concentration-response curves in rat isolated aorta and human isolated pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 4.4 and 5.0 nM, respectively. SB 217242 was 4-, 62- and 125-fold more potent as an ETA receptor antagonist than the previously reported compounds BQ-123, PD 142893 and Ro 46-2005, respectively. SB 217242 (10 microM) did not produce significant effects against contraction produced by other vasoactive agents. SB 217242 produced concentration-dependent antagonism of responses produced by ETB receptor activation as demonstrated by antagonism of sarafotoxin S6c-mediated contraction in the rabbit isolated pulmonary artery with a Kb value of 352 nM. In vitro cell monolayers of Caco-2 cells had high permeability to SB 217242. In vivo pharmacokinetics in the rat confirmed that SB 217242 was rapidly absorbed from the gastrointestinal tract with a bioavailability of 66%. The SB 217242 plasma half-life in rats after intraduodenal administration was 3.3 hr, with a systemic clearance of 27.3 ml/min/kg. Orally administered SB 217242 (0.3-30 mg/kg) produced dose-dependent inhibition of the pressor response to exogenous ET-1 in conscious rats; with a dose of 30 mg/kg p.o., inhibition was observed for at least 5.5 hr. The present study demonstrates that SB 217242 is a highly potent antagonist of both ETA and ETB receptors. In addition, SB 217242 has high in vitro permeability and high oral bioavailability. SB 217242 represents a new orally active pharmacological tool that should assist in the elucidation of the chronic role of endothelin in pathophysiology. PMID- 8632329 TI - Cooperative contributions of cholinergic and NMDA receptors in the presynaptic control of dopamine release from synaptosomes of the rat striatum. AB - In the presence of magnesium, although ineffective alone, N-methyl-D-aspartate (NMDA, 10(-3) M plus glycine 10(-6) M) stimulated the release of [3H]-dopamine ([3H]-DA) continuously synthesized from [3H]-tyrosine when applied with ACh, the amplitude of the NMDA response being dependent on the ACh concentration. Experiments performed with nicotine, oxotremorine and the antagonists hexamethonium and atropine indicated that both muscarinic and nicotinic receptors are involved in the permissive effect of ACh on the NMDA response. Data obtained in the absence of magnesium or with increasing concentrations of magnesium revealed that the permissive effect of ACh on the NMDA-evoked release of [3H]-DA results from removal of the magnesium block of NMDA receptors. The NMDA-evoked release of [3H]-DA observed in the presence of ACh, nicotine or oxotremorine (10( 3) M) was blocked by either of the protein kinase C inhibitors staurosporine (10( 8) M) and chelerythrine (5 x 10(-7) M). However, these drugs were without effect on responses induced by ACh, nicotine or oxotremorine alone and by NMDA (10(-3) M, in the absence of magnesium). Supporting further the involvement of a protein kinase C activation in the permissive effects of ACh or the cholinergic agonists, NMDA (10(-3) M) stimulated the release of [3H]-DA in the presence of both magnesium and phorbol 12-myristate 13-acetate (10(-6) M) or 1 -oleoyl-2-acetyl glycerol (10(-4) M), and the NMDA response was markedly potentiated by ionomycin (10(-7) M) used at a concentration that stimulated [3H]-DA release to about the same degree as ACh (10(-4) M). Therefore, besides their depolarizing action, ACh, nicotine and oxotremorine could eliminate the magnesium block of NMDA receptors by activation of protein kinase C. PMID- 8632331 TI - Combined potassium and calcium channel blocking activities as a basis for antiarrhythmic efficacy with low proarrhythmic risk: experimental profile of BRL 32872. AB - In the search for novel antiarrhythmic agents, compounds with a diversity of electrophysiological actions have been suggested to result in treatments with potentially improved efficacy but with reduced proarrhythmic risk. To test this hypothesis, the antiarrhythmic versus proarrhythmic profile of BRL-32872, a novel agent with combined potassium and calcium channel blocking activity, was assessed in two different in vivo models of ventricular arrhythmia. Furthermore, the effects of potassium and calcium channel antagonists given either alone or in combination were assessed in the same models. Dogs with myocardial infarction received intravenously either vehicle, BRL-32872, the class III antiarrhythmic agent, E-4031, verapamil or a combination of E-4031 with verapamil (n = 8 per group). Ventricular tachyarrhythmias were induced by programmed electrical stimulation (PES). BRL-32872 (0.1, 0.3, 1.0 mg/kg) significantly increased QTc interval (from 387 +/- 10 to 462 +/- 19 msec.sec-1/2 at 1.0 mg/kg, P < .01). Ventricular effective refractory periods were increased in normal and infarcted areas (P < .01). Similar effects were observed with E-4031 (0.1, 0.3, 1.0 mg/kg). Verapamil (0.03, 0.1, 0.3 mg/kg) reduced heart rate, mean arterial pressure and, to a lesser extent, (+)dP/dtmax. Verapamil did not change QTc interval and ventricular effective refractory periods, but increased PR interval (P < .001). PES-induced tachyarrhythmias were not changed by vehicle or increasing doses of verapamil. E-4031 reduced the severity of arrhythmias from sustained ventricular tachycardia (VT) to nonsustained VT (7 dogs at 1.0 mg/kg, P = .013 vs. vehicle). BRL-32872 (0.1 and 0.3 mg/kg) suppressed the induction of sustained VT in six dogs (P = .02 vs. vehicle). In the presence of BRL-32872, 1.0 mg/kg, five dogs became noninducible to PES (P = .013 vs. vehicle). Combination of E-4031 (0.1 mg/kg) with verapamil provided a degree of protection that was similar to that observed with BRL-32872. In a second model, the proarrhythmic potential of BRL 32872 was assessed in anesthetized rabbits sensitized to develop torsades de pointes (TdP). BRL-32872 was compared with the class III antiarrhythmic agents, E 4031, dofetilide, clofilium and RP-58866. The pure class III antiarrhythmic agents induced TdP in 50 to 90% of the rabbits, and prolonged QT interval by 20 to 50%. BRL-32872 (10 micrograms/kg/min) increased QT interval by 35 +/- 5%, but did not promote TdP. In additional experiments, verapamil reduced the incidence of TdP induced by E-4031. These results show that BRL-32872 is a potent antiarrhythmic compound in a model of PES-induced arrhythmias and induces fewer proarrhythmic events than typical class III antiarrhythmic agents. The effects observed with BRL-32872 suggest that a compound with a combination of potassium (class III) and calcium (class IV) channel antagonistic properties might constitute a novel antiarrhythmic agent with reduced proarrhythmic risk. PMID- 8632330 TI - Assessment of the involvement of central nervous system and peripheral opioid receptors in the immunomodulatory effects of acute morphine treatment in rats. AB - The present study assessed the involvement of opioid receptors both in the central nervous system (CNS) and in the periphery (i.e., on immunocytes) in the immune alterations produced by acute morphine treatment in rats. The first experiment showed that the in vitro suppressive effects of morphine on the mitogen-stimulated proliferation of splenic and blood lymphocytes are produced only by a very high concentration of morphine and are not naltrexone-reversible. These results suggest that the in vitro immunomodulatory effects of morphine are not mediated by classical opioid receptors on immunocytes. A second experiment showed that s.c. doses of N-methylnaltrexone that do not gain access to the CNS, as determined by the tail-withdrawal assay, do not antagonize the suppressive effects of a single, s.c. injection of morphine on the mitogen-stimulated proliferation of splenic and blood lymphocytes, splenic natural killer cell activity and the production of interferon-gamma by stimulated splenocytes. Only a high s.c. dose of N-methylnaltrexone that does gain access to the CNS, as determined by the tail-withdrawal assay, blocks morphine's immunomodulatory effects. A third experiment demonstrated that N-methylnaltrexone is 4 to 5 log units more potent in antagonizing most of the immune alterations produced by a single, s.c. injection of morphine when administered i.c.v. than s.c. Taken together, the results of the present study strongly suggest that CNS opioid receptors play an important role in the immune alterations produced by acute morphine treatment in rats. PMID- 8632332 TI - Endothelin-induced nociception in mice: mediation by ETA and ETB receptors. AB - Endothelins (ET-1, ET-2 or ET-3) or endothelin precursors (big-ET-1[1-38], big-ET 2[1-37] or big-ET-3[1-41]) injected i.p. in mice have previously been shown to elicit a characteristic nociceptive behavioral response. In this study, we investigated the endothelin receptor type (ETA or ETB) that mediates this behavioral response. Mice were injected i.p. with ET-1, ET-2, ET-3, big-ET-1[1 38], big-ET-2[1-37], big-ET-3[1-41], sarafotoxin S6a, sarafotoxin S6b, sarafotoxin S6c, ET-1 with Ala substitutions for Cys3 and Cys11 or His-Leu-Asp Ile-Ile-Trp, and quantal dose-response curves were obtained for each of the compounds (except the latter). Co-administration of enzyme inhibitors with the big-endothelins was used to establish the requisite conversion to endothelins and big-ET-1[22-38], big-ET-2[22-37] and ET-3[22-41] amide, and the ETA-selective antagonist cyclo[-D-Asp-Pro-D-Val-Leu-D-Trp-] was used to determine receptor specificity. The ED50 values were 2.9, 3.3 and 23.9 micrograms/kg i.p. for ET-1, ET-2 and ET-3, respectively, 0.6, 0.6 and 13.1 micrograms/kg i.p. for sarafotoxin S6a, sarafotoxin S6b and sarafotoxin S6c, respectively, and 5.3 micrograms/kg i.p. for ET-1 with Ala substitutions for Cys3 and Cys11. Big-ET-1[22-38], big-ET 2[22-37], big-ET-3[22-41] amide and ET-C produced less than 25% effect up to 2000 micrograms/kg. The big-ET-1-induced effects were blocked by the enzyme inhibitors phosphoramidon and thiorphan (ID50 = 0.9 mg/kg) but not by ubenimex (bestatin), captopril or perindopril. Cyclo[-D-Asp-Pro-D-Val-Leu-D-Trp-] blocked ET-1- and ET 2-induced effects but not ET-3-, ACh- or phenyl-p-quinone-induced effects. These results suggest that endothelin-induced nociceptive behavioral response in mice can be mediated via both ET receptor types, ETA and ETB. Further, the ET-1 carboxy-terminal hexapeptide is insufficient to produce the effect, and the Cys3 Cys11 disulfide bridge of ET-1 is not required. PMID- 8632333 TI - Estradiol increases cyclic adenosine monophosphate in rat pulmonary vascular smooth muscle cells by a nongenomic mechanism. AB - Estrogen, like other steroids, may induce rapid nongenomic cellular effects. We studied the effect on intracellular cAMP of short-term exposure (5 min) of cultured rat pulmonary vascular smooth muscle cells (VSMC) to estradiol 17 beta. At confluence, VSMC were incubated in phosphate buffer saline for 1 hr before exposure to different hormones. The reaction was stopped with 0.1 N HCl and cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The 5-min incubation with estradiol 17 beta (0.3-30 microM) significantly increased basal intracellular cAMP in a concentration-dependent manner. The stimulatory effect of estradiol on cAMP was time-dependent, increasing with prolonged exposure to the hormone, and was not affected by the protein synthesis inhibitor, actinomycin D (5 micrograms/ml), at 5 and 30 min. Comparable concentrations of testosterone or estradiol 17 alpha had no significant effect on cAMP. The estrogen receptor partial agonist, tamoxifen also significantly increased basal cAMP in a concentration-dependent manner, but inhibited the effect of estradiol. Furthermore, forskolin elicited a concentration-dependent increase in cAMP (396.6 +/- 53% at 10 microM concentration), which was significantly potentiated in presence of estradiol. The effect of estradiol is unlikely to be mediated by G protein activation, because the G protein inhibitor, pertussis toxin (100 ng/ml), did not significantly affect estradiol-induced increase in cAMP. Removal of Ca++ from the incubation medium inhibited the stimulatory effect of estradiol 17 beta suggesting that estradiol may increase pulmonary VSMC cAMP via a Ca(++)-dependent pathway. We suggest that the effect of estradiol 17 beta in these experiments is nongenomic in nature, and is possibly mediated by direct interaction of the hormone with specific membrane binding sites. PMID- 8632334 TI - Identification of the human cytochromes P450 responsible for the in vitro formation of the major oxidative metabolites of the antipsychotic agent olanzapine. AB - The formation kinetics of 2-hydroxymethyl olanzapine (2-OH olanzapine), 4'-N oxide olanzapine (N-O olanzapine) and 4'-N-desmethyl olanzapine (NdM olanzapine) were analyzed in vitro. Biphasic kinetics were observed for formation of 2-OH and NdM olanzapine. The high-affinity enzyme responsible for 2-OH olanzapine formation by two human liver samples exhibited an intrinsic clearance (CLint) of 0.2 microliter/min/mg. NdM olanzapine formation by two human liver samples exhibited a CLint of 1.0 microliter/min/mg for the high affinity enzyme. The formation of N-O olanzapine was linear up to 300 microM olanzapine, yielding a CLint of 0.32 to 1.70 microliters/min/mg. The formation of 7-hydroxy olanzapine (7-OH olanzapine) exhibited an apparent Km of 24.2 microM. The rates of 2-OH olanzapine formation correlated with CYP2D6 levels and activity, and it was formed to the greatest extent by cDNA-expressed CYP2D6. N-O olanzapine formation correlated with human liver flavin-containing monooxygenase (FMO3) levels and activity. NdM olanzapine and 7-OH olanzapine formation correlated with CYP1A2 catalytic activities and they were formed to the greatest extent by expressed CYP1A2. These results suggest that CYP1A2 catalyzes NdM olanzapine and 7-OH olanzapine formation, CYP2D6 catalyzes 2-OH olanzapine formation and FMO3 catalyzes N-O olanzapine formation. PMID- 8632335 TI - Low level hyperbaric antagonism of diazepam's locomotor depressant and anticonvulsant properties in mice. AB - Exposure to 12 atmospheres absolute (12 ATA) helium oxygen gas (heliox) (low level hyperbaric exposure) antagonizes the behavioral effects of ethanol and n propanol, but not morphine. These and other results indicate that the mechanism of the antagonism is direct (pharmacodynamic) and selective. Our study further investigates the selectivity of low level hyperbaric antagonism by testing its effectiveness against diazepam, a high affinity binding drug that acts via allosteric modulation of GABAA receptors. C57BL/6J mice received injections i.p. of vehicle or diazepam, and were then exposed to 1 ATA air, 1 ATA heliox or 12 ATA heliox. Exposure to 12 ATA heliox antagonized the locomotor depressant effect of 4 and 6 mg/kg, but not 8 mg/kg diazepam. Hyperbaric exposure also antagonized the anticonvulsant effect of 8 and 24 mg/kg, but not 4 mg/kg, diazepam vs. 300 mg/kg isoniazid. Exposure to 12 ATA heliox did not significantly affect blood concentrations of diazepam or its metabolite n-desmethyl diazepam. The pharmacological characteristics of the antagonism (direct, surmountable, rightward shift in diazepam's dose-response curve) closely matched those seen in previous studies for hyperbaric antagonism of ethanol. The results add to the evidence that low level hyperbaric exposure is a direct, mechanistic antagonist that selectively antagonizes drugs that act via perturbation or allosteric modulation of receptor function. Moreover, the results suggest that allosteric coupling pathways, which transduce binding events on ligand-gated ion channels, may represent initial sites of action for ethanol. PMID- 8632336 TI - Saturable disposition of taurine in the rat cerebrospinal fluid. AB - Recently, we described a saturable Na(+)-dependent taurine transporter in the choroid plexus, the blood-CSF barrier (Chung et al., 1994). The goal of this study was to determine whether this transporter plays a role in the in vivo elimination of taurine from the CSF. 3H-taurine and 14C-inulin were injected into the lateral ventricle of anesthetized rats, and the concentrations of the radiolabeled compounds in the CSF were determined. The apparent clearance of taurine from the CSF was greater than the estimated CSF bulk flow (P < .005), which indicates that there is a clearance process in addition to the CSF bulk flow. Taurine distribution into the choroid plexus was at least 10-fold higher than that found in other brain areas. The apparent clearance of 3H-taurine and the distribution of taurine into the choroid plexus and cerebral cortex were dose dependent. The Michaelis-Menten rate constant estimated from the in vivo elimination study (40 +/- 25 microM) is in the range of that obtained for taurine uptake in isolated choroid plexus tissue slices (137 +/- 67 microM). Both alpha- and beta-alanine decreased the clearance of taurine from the CSF as well as the distribution of taurine into the choroid plexus via inhibitory effects (either direct or indirect) on the Na(+)-taurine transporter in the choroid plexus. These data suggest that the previously characterized taurine transporter in the choroid plexus plays a role in the in vivo saturable disposition of taurine in the CSF. PMID- 8632338 TI - A G protein, not cyclic AMP, mediates effects of VIP on the inwardly rectifying K+ channels in endothelial cells. AB - Because some endothelial cells contain a high density of functional vasoactive intestinal peptide (VIP) receptors, it is possible that in some cases, relaxation of blood vessels by VIP is mediated by endothelium. We showed earlier that VIP inhibited inwardly rectifying K+ currents (IKin) in cultured bovine pulmonary artery endothelial cells. Our studies now provide both direct and indirect evidence that activation of these receptors does not occur through an elevation of cAMP level in these cells. Isoproterenol increased cAMP in endothelial cells from 30% to 35% over the basal levels. In contrast, VIP did not elevate cAMP in endothelial cells and even decreased it in some instances. In whole-cell patch clamp experiments, isoproterenol weakly inhibited the IKin (about 80% less than VIP). The magnitudes of effects evoked by other activators of the cAMP cascade (forskolin, cAMP analogs) on this current were intermediate between those of VIP and isoproterenol. Although cAMP elevation can reduce the IKin current in endothelial cells, it is not responsible for the inhibitory effect of VIP on this current. We demonstrated that VIP receptors interact with the IKin channels through a G protein. Guanosine 5'-(3'-O-thiotriphosphate, a nonhydrolyzable GTP analog, or cholera toxin inhibited these channels in a manner similar to inhibition by VIP. The activity of the IKin channels was pertussis toxin insensitive. Furthermore, guanosine-5'-O-(2-thiodiphosphate) blocked the VIP receptor-mediated effect on the IKin. Our results suggest that VIP receptors couple to IKin channels through a G protein. PMID- 8632337 TI - Urine acidification affects the activity of the organic base transporter in a nonstereoselective manner. AB - This investigation determined 1) the effect of urine acidification on renal clearance (Clrenal), total systemic clearance (Cltotal) and nonrenal clearance (Clnonrenal) of pindolol, 2) whether urine acidification affected the stereoselectivity of pindolol excretion and 3) the pharmacodynamic effects that may result from changes in the activity of the organic base transporter. The Clrenal, Cltotal and Clnonrenal values of pindolol isomers were determined during pindolol administration (10 mg twice daily; control phase) and during pindolol administration (10 mg twice daily) with NH4Cl, a systemic and urinary acidifier, (1.5 g every 6 hr). Eight healthy males (22-33 yr) randomly received this therapy for 3 days on two occasions. On day 4, urine and plasma were collected over 24 hr. R-(+) pindolol Clrenal values during control and NH4Cl were 203 +/- 82 and 480 +/- 248 ml/min, respectively (P = .03). S-(-) pindolol Clrenal values during control and NH4Cl were 279 +/- 81 and 593 +/- 294 ml/min, respectively (P = .005). NH4Cl increased R-(+) pindolol Clrenal by 173% +/- 136% (P = .003) and S-( ) pindolol Clrenal by 127% +/- 105% (P = .03). Stereoselective renal excretion of pindolol was unaffected by NH4Cl; the R(+)/S(-) pindolol Clrenal ratio was unchanged from control to NH4Cl (0.74 +/- 0.23 to 0.81 +/- 0.10, P = NS, respectively). NH4Cl, however, affected pindolol Clnonrenal in a stereoselective fashion; R-(+) pindolol Clnonrenal values increased (641 +/- 241 to 851 +/- 251 ml/min; P = .02), whereas S-(-) pindolol Clnonrenal values remained constant (354 +/- 116 vs. 370 +/- 213 ml/min). Changes in pindolol clearance values resulted in a significant reduction in beta-blocking activity assessed by isoproterenol testing. We conclude that increasing the urine proton gradient can increase the Clrenal value of organic bases by 2-fold in a manner that is not stereoselective. NH4Cl, however, did increase the Clnonrenal value of pindolol in a stereoselective manner. These data, therefore, indicate that the administration of a urine-acidifying agent can greatly enhance the elimination of organic bases and ultimately reduce the pharmacologic activity of the organic base. PMID- 8632339 TI - Regulation of 5-hydroxytryptamine release from rat midbrain raphe nuclei by 5 hydroxytryptamine1D receptors: effect of tetrodotoxin, G protein inactivation and long-term antidepressant administration. AB - Our study was undertaken to characterize the functional properties of 5 hydroxytryptamine (5-HT)1D receptors in the rat midbrain raphe nuclei. In a first series of experiments, designed to assess whether 5-HT1D receptors are coupled to Gi/o proteins, the intracerebral injection of pertussis toxin into the dorsal raphe as well as incubation of midbrain raphe slices with the alkylating agent N ethyl-maleimide (NEM) reduced the efficacy of the 5-HT1B/1D agonist sumatriptan to inhibit the electrically evoked overflow of [3H]5-HT from preloaded slices. Furthermore, preincubation with NEM also reduced the efficacy with which the 5 HT1B/1D antagonist GR 127935 enhanced evoked overflow of [3H]5-HT. These results indicate that, in rat midbrain raphe nuclei, 5-HT1D receptors are linked to Gi/o proteins. In an attempt to determine whether 5-HT1D receptors are located on 5-HT neurons, the inhibitory effect of sumatriptan and of the nonselective 5-HT agonist 5-carboxyamidotryptamine on K(+)-evoked overflow of [3H]5-HT was assessed in the presence of the Na+ channel blocker tetrodotoxin. Neither the inhibitory effect of sumatriptan nor that of 5-carboxyamidotryptamine were reduced by the addition of tetrodotoxin to the superfusion medium, suggesting that these 5-HT1D receptors are located on 5-HT neurons and may be considered autoreceptors. In a third series of experiments, rats were treated for 21 days either with the selective 5-HT reuptake inhibitor paroxetine (10 mg/kg/day, s.c.) or the reversible type A monoamine oxidase inhibitor befloxatone (0.75 mg/kg/day, s.c.) and superfusion experiments were performed after a 48-hr washout period. 5-HT1D receptors, similarly to 5-HT1A autoreceptors, desensitize after long-term treatment with a selective 5-HT reuptake inhibitor or a reversible type A monoamine oxidase inhibitor because the efficacy of sumatriptan and of 8-OH-DPAT to inhibit the electrically evoked overflow of [3H]5-HT was reduced after the administration of either drug. PMID- 8632340 TI - Determinants of in vivo activity of neutral endopeptidase 3.4.24.11 and angiotensin converting enzyme inhibitors. AB - Simultaneous inhibition of neutral endopeptidase EC 3.4.24.11 (NEP) and angiotensin converting enzyme (ACE) by equimolar doses (100 mumol/kg i.v.) of SQ 28603 (N-[2-(mercaptomethyl)-1-oxo-3- phenylpropyl]-beta-alanine) and captopril increased sodium excretion by 888 +/- 377 microEq/3 hr and significantly lowered blood pressure by -6 +/- 2 mm Hg in conscious cynomolgus monkeys. This rate of sodium excretion was not significantly different from that elicited by 100 mumol/kg i.v. of SQ 28603 alone (1453 +/- 315 microEq/3 hr). In addition, the natriuretic response to captopril plus SQ 28603 was potentiated by infusion of 10 pmol/kg/min of human atrial natriuretic peptide (hANP 99-126) despite a reduction in renal perfusion pressure from 100 +/- 2 to 86 +/- 2 mm Hg. Lower doses (0.3 to 3 mumol/kg i.v.) of SQ 28603 that had no effect on blood pressure or renal function potentiated the natriuretic, urinary cyclic guanosine monophosphate and atrial natriuretic peptide responses without affecting the depressor activity of 0.3 nmol/kg i.v. of hANP 99-126. The potentiation of the natriuretic activity of 0.3 nmol/kg of hANP 99-126 by 1 or 3 mumol/kg of SQ 28603 was not significantly affected by the addition of equimolar doses of captopril. These results confirmed that the renal responses to the combined inhibitors resulted from NEP inhibition. In contrast, the depressor activity of the combined inhibitors was dependent on the level of ACE inhibition and was not significantly affected by either infusion of hANP 99-126 or prior sodium loading. Therefore, the vascular responses to combined NEP and ACE inhibitors did not necessarily depend upon increases in circulating atrial natriuretic peptide or reductions in angiotensin II levels. The unique profile of renal and vascular responses to combined NEP and ACE inhibition suggested that dual NEP/ACE inhibitors may be useful for the treatment of cardiovascular disorders. PMID- 8632341 TI - Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells. AB - The effect of somatostatin on Bombesin-induced contraction of isolated rabbit colonic smooth muscle cells was examined. Preincubation of muscle cells with somatostatin 10(-6) M inhibited bombesin-induced contraction. To characterize somatostatin receptors, muscle cells (10(5) cells/tube) were incubated at 24 degrees C with 125I-Tyr0-SS-28. Binding reached a plateau at 60 sec and was reversible by addition of excess synthetic SS-28. Scatchard analysis revealed high and low affinity bindings sites (Ka = 0.48 +/- 0.01 and 40 +/- 13 (nM +/- S.E.), 1830 +/- 433 and 65820 +/- 13183 receptors/cell +/- S.E.). Inhibition of 125I-Tyr0-SS-28 binding was possible with biologically active analogs of somatostatin, indicating the specificity of the receptors to somatostatin. Binding of 125I-Tyr0-SS-28 was inhibited by GTP gamma s, a nonhydrolysable analog of guanosine 5'-triphosphate, whereas adenosine 5'-triphosphate at a high concentration (100 microM) slightly inhibited the binding. Further, pretreatment of muscle cells with pertussis toxin at 37 degrees C abolished binding of 125I Tyr0-SS-28, although pretreatment of cells with cholera toxin had no effect. Inasmuch as Gi protein is postulated as a signal protein, muscle cells were labeled with 3H-methionine, before stimulation with Bombesin (10(-6) M), in the presence and absence of somatostatin (10(-6) M). The cells were then lysed and Gi was precipitated by a Gi specific antibody. Gi synthesis was stimulated by bombesin at 60 sec and somatostatin inhibited it (6114 +/- 986 vs. 2998 +/- 841 cpm +/- S.E., P < .05). These data suggest that colonic smooth muscle cells contain specific receptor for somatostatin-28 and that somatostatin reverses bombesin-induced contraction regulated by Gi-type G protein. PMID- 8632342 TI - Pharmacologic characterization of the human 5-hydroxytryptamine2B receptor: evidence for species differences. AB - The 5-Hydroxytryptamine2B (5-HT2B) receptor was cloned originally from rat stomach fundus and its pharmacology was determined to be consistent with that of the receptor responsible for contraction of rat fundal tissue in response to 5 HT. Recently, the cloning of the human homolog of the 5-HT2B receptor has been reported and, in this study, we report a detailed pharmacological characterization of this human receptor. The cloned human 5-HT2B receptor has high affinity for [3H]5-HT (Kd = 10.6 +/- 1.5 nM), and the pharmacology of this receptor matches closely the rat 5-HT2B receptor, consistent with the structural relatedness of these two proteins. Most compounds tested show no difference in affinity for the human or rat receptors. There were, however, groups of compounds that discriminated between the human and rat 5-HT2B receptors. Examples include certain ergolines such as methysergide and mesulergine, which have higher affinity for the human than for the rat receptor. Similarly, certain benzoylpiperidines, e.g., ketanserin, pirenperone and pipamperone, and the antipsychotics clozapine and olanzapine have higher affinity for the human 5-HT2B receptor. These pharmacological findings reinforce the desirability of having the human forms of receptors when considering drug actions. PMID- 8632343 TI - Effects of metabolites of leukotriene B4 on human neutrophil migration and cytosolic calcium levels. AB - Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11 dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7 double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization. PMID- 8632344 TI - Noncompetitive NMDA channel blockade during waking intensely stimulates NREM delta. AB - We previously found that subanesthetic doses of ketamine administered during the dark (active) period (DP) in rats strongly increased the integrated amplitude of the delta (1-4 Hz) electroencephalogram (EEG) in subsequent nonrapid eye movement (NREM) sleep. Here, we injected MK-801 into adult male Sprague-Dawley rats to test the hypothesis that such delta stimulation is characteristic of drugs that noncompetitively block the cation channel gated by the N-methyl-D-aspartate (NMDA) receptor. Injections of 0.3 and 0.5 mg/kg MK-801 in the middle of the DP produced waking intoxication for approximately 3 hr. In the following light period, NREM delta integrated amplitude was markedly increased in every rat (mean 55% increase after 0.5 mg/kg). A separate control experiment with 3-hr sleep deprivation in the mid-DP showed that the delta stimulation could not be attributed to sleep loss during MK-801 intoxication. Mechanisms by which NMDA cation channel blockade might stimulate NREM delta include a compensatory (homeostatic) sleep response to the metabolic, receptor or other neuronal effects of cation channel blockade; pathologic EEG slowing caused by neurotoxicity (in which case NREM delta might provide a noninvasive index of neurotoxic vacuolization); or a persistent, direct action of the drug or its metabolites on delta-generating systems. Questions of mechanism gain interest because of the magnitude of these pharmacologic effects on the sleep EEG component (delta) thought to be correlated with brain recuperative processes. In addition, our findings add to growing evidence implicating excitatory amino acid systems in sleep regulation. PMID- 8632345 TI - Dexamethasone selective inhibition of acute opioid physical dependence in isolated tissues. AB - The effect of dexamethasone on acute opiate withdrawal induced by mu, kappa and delta receptor agonists was investigated in vitro. After a 4-min in vitro exposure to morphine (less selective mu agonist), D-Ala2-N-methyl-Phe4-Gly5-ol) enkephalin (DAGO; highly selective mu agonist) and trans(+/-)-3,4-dichloro-N methyl-N-[2(1-pyrrolidynyl)cyclohexyl]- benzeneacetamide (U50-488H; highly selective kappa agonist) a strong contracture of guinea pig isolated ileum was observed after the addition of naloxone. This effect was also observed when rabbit isolated jejunum was pretreated with deltorphin (highly selective delta agonist). Dexamethasone treatment before or after the opioid agonists tested was capable of both preventing and reverting the naloxone-induced contracture after exposure to mu opiate agonists morphine and DAGO in a concentration- and time dependent fashion. Also, the steroid reduced naloxone-induced contracture after the exposure to U50-488H only when injected before the kappa opiate agonist. Finally, it did not affect the naloxone contracture after exposure to deltorphin. Pretreatment with RU-38486, a glucocorticoid receptor antagonist, inhibited dexamethasone antagonism on responses to both mu and kappa agonists, whereas pretreatment with cycloheximide, a protein synthesis inhibitor, blocked only the antagonistic effects of dexamethasone on responses to the mu opioid agonists. Overall, these data indicate that dexamethasone induces significant effects on mu mediated opiate with-drawal in vitro, which suggest an important functional interaction between corticosteroids and the opioid system primarily at the mu receptor level. The ability of RU-38486 and cycloheximide to block dexamethasone effects indicates that the steroid interference on mu-mediated withdrawal involves a protein synthesis-dependent mechanism via glucocorticoid receptor. PMID- 8632346 TI - Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic-3',5'-guanosine monophosphate-stimulated phosphodiesterase to reverse hypoxic pulmonary vasoconstriction in the perfused rat lung. AB - Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was shown to reverse the hypoxic pressor response (HPR) in the isolated, blood-perfused rat lung model. EHNA, an adenosine deaminase inhibitor, showed reversal of the HPR in a dose-dependent manner (EC50 = 129 +/- 30 microM). We found that the reversal of HPR by EHNA was not mediated by the adenosine receptors because the EHNA effect was not blocked by the adenosine receptor antagonist, 8-p-sulfophenyl-theophylline (67 microM; n = 6). Pretreatment with a cy-clic-3',5'-adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, Rp-adenosine-3',5'-cyclic monophosphorothioate (0.5 mM; n = 4), blocked EHNA reversal of the HPR. As an alternative mechanism of action, EHNA inhibition of cyclic nucleotide phosphodiesterase(s) isozymes was studied in endothelium intact and denuded pulmonary arteries. Using anion-exchange chromatography the cyclic nucleotide phosphodiesterase (PDE) separated into predominantly PDE families 2 and a mixture of 3 and 4. DEAE fractions showing cAMP hydrolysis activated by 5 microM cyclic-3',5'-guanosine monophosphate (cGMP) had a Km for cAMP of 6.3 microM and an apparent Kact for cGMP of 1.4 microM. EHNA was shown to inhibit PDE2 competitively. In intact vessels, the IC50 for EHNA was 3.3 microM using 0.03 microM [3H]-cAMP substrate assayed in the presence of 2 microM cGMP and in denuded vessels 3.7 microM at 0.03 microM [3H]-cAMP substrate in the presence of 5 microM cGMP. Fractions in which cAMP hydrolysis was inhibited or not affected by 5 microM cGMP (PDE3 and 4, respectively) showed an IC50 of > 200 microM for EHNA. We conclude that reversal of the hypoxic pressor response by EHNA in the isolated, perfused rat lung model occurs with a mechanism involving in part inhibition of smooth muscle PDE2. PMID- 8632347 TI - Three-day exposure to low-dose ethanol alters guanine nucleotide binding protein expression in the developing rat hippocampus. AB - Alcohol-related birth defects result from acute and chronic insults that perturb sequential developmental programs. The molecular targets of EtOH include G protein coupled signal transduction pathways. In order to test the hypothesis that G-proteins are involved in EtOH-induced hippocampal teratogenesis, rat pups were administered 3.3 g/kg.day of EtOH on postnatal days (PN) 5 to 7 using the pup-in-a-cup model of third trimester "binge" exposure. This exposure paradigm produced a selective 40% decrease in the 52 kDa isoform of the stimulatory form of the heterotrimeric guanine nucleotide binding protein (G alpha s) in the hippocampus on PN 7 with no significant changes in the levels of G alpha i or G alpha o. Immunohistochemistry demonstrated that this decrease occurred in the somas of both hippocampal pyramidal cells and granule cells of the dentate gyrus. Computer-assisted cell counting indicates that this decrease was not due to pyramidal cell death on PN 7. Northern and slot blot analysis demonstrated a 30% decrease in G alpha s messenger RNA in the hippocampus. These results suggest that EtOHs teratogenic effects in the hippocampus may involve disruption of G alpha s-coupled signal transduction pathways, which are critical for normal synaptogenesis, neurotransmitter signaling and the integration of these signals with growth factor signaling pathways. PMID- 8632348 TI - Arbutin: mechanism of its depigmenting action in human melanocyte culture. AB - Arbutin, a naturally occurring beta-D-glucopyranoside of hydroquinone, is effective in the topical treatment of various cutaneous hyperpigmentations characterized by hyperactive melanocyte function. We examined the mechanism of its depigmenting action in human melanocyte cultures. Arbutin inhibited the tyrosinase activity of cultured human melanocytes at noncytotoxic concentrations. It did not affect the expression of tyrosinase mRNA. Melanin production was inhibited significantly by arbutin, as determined by measuring eumelanin radicals with an electron spin resonance spectrometer. The study of the kinetics and mechanism for inhibition of tyrosinase confirms the reversibility of arbutin as a competitive inhibitor of this enzyme. The utilization of L-tyrosine or L-dopa as the substrate suggests a mechanism involving competition with arbutin for the L tyrosine binding site at the active site of tyrosinase. These results suggest that the depigmenting mechanism of arbutin in humans involves inhibition of melanosomal tyrosinase activity, rather than suppression of the expression and synthesis of tyrosinase. PMID- 8632349 TI - Galactose-modified streptavidin-GC4 antifibrin monoclonal antibody conjugates: application for two-step thrombus/embolus imaging. AB - Diagnostic and therapeutic procedures utilizing the high affinity streptavidin (SA)/biotin system are being investigated for in vivo use. We are developing a rapid two-step imaging technique for the diagnosis of deep venous thrombosis and pulmonary embolism. The optimal SA-bound targeting moiety would circulate adequately for sufficient lesion accumulation, but nonbound reagent would clear in a reasonably short time before the injection of radiolabeled biotin. The objective of this study was to cross-link SA and galactose-modified SA to GC4 antifibrin monoclonal antibody and to study the pharmacokinetics and biodistribution of the radiolabeled GC4-SA conjugates after injection into rabbits. A cross-linking method was developed for the synthesis of the GC4-SA conjugates via the addition reaction of sulfhydryl containing SA derivatives with maleimide-GC4. In vivo, radiolabeled trigalactose modified SA-GC4 exhibited a much faster blood clearance compared to mono-galactose modified GC4-SA or GC4-SA containing no galactose. PMID- 8632350 TI - Cannabinoid receptor proteins are increased in Jurkat, human T-cell line after mitogen activation. AB - Brain-type cannabinoid receptor (CB1) mRNA has been demonstrated in several peripheral tissues; however, the function of this message is not clear. In the present study, we examined the levels of CB1 mRNA and receptor protein in stimulated immune cells to link message and protein expression with cell activation. Consistent with previous reports, immune cell lines from human and mouse were positive by reverse transcription-polymerase chain reaction for CB1 mRNA; however, one cell line, Jurkat, was only weakly positive. Mitogen activation of Jurkat cells, however, increased CB1 mRNA within 2 hr after stimulation and equilibrium binding studies, using Jurkat membranes, showed [3H]CP55,940 specific binding was negative early after mitogen stimulation but positive at 40 hr poststimulation. To investigate whether this observed increase in CB1 mRNA and specific binding activity was associated with expression of the CB1 protein, polyclonal antibodies were produced to a fusion protein consisting of glutathione S-transferase and a 342 amino acid portion (residues 33 through 374) of the CB1 protein. Western blotting analysis showed expression of several immunoreactive proteins on membranes from mitogen-activated Jurkat cells, but not on membranes from unstimulated cells. These results demonstrate a link between the level of CB1 mRNA and surface protein in activated immune cells, suggesting the possibility of a functional role of CB1 in immune cell activation. PMID- 8632351 TI - Differential coupling of rat D2 dopamine receptor isoforms expressed in Spodoptera frugiperda insect cells. AB - By using a baculovirus expression system, the two isoforms of the rat D2 dopamine receptor were expressed at densities ranging up to 15 pmol/mg of protein. D2L and D2S dopamine receptors expressed in aline of Spodoptera frugiperda (Sf9) insect cells Sf9cells, displayed high affinity for the antagonists spiroperidol and (+) butaclamol and the agonist N-propylnorapomorphine. Antisera raised against the D2 receptor immunoprecipitated binding sites for a radiolabeled D2 antagonist from solubilized extracts of infected Sf9cells. In immunoblots of Sf9cells infected with recombinant D2 baculovirus, these antisera recognized a major species of protein of approximately 46 kDa. Photoaffinity-labeling of infected Sf9cells using N-(p-azido-m-[125I]iodophenethyl)spiperone also identified a protein of this size, suggesting that D2 receptors expressed in Sf9cells are largely unglycosylated. In cells expressing receptors at a density greater than 1 pmol/mg, GTP-sensitive, high-affinity binding of agonists was not detected in studies of the inhibition of the binding of a radiolabeled D2 antagonist. When expression levels were under 1 pmol/mg, the binding of agonists was sensitive to the addition of guanine nucleotides, indicating that D2 receptors were coupled to endogenous G proteins. Endogenous G proteins enable both isoforms of D2 receptors to couple to the inhibition of adenylyl cyclase activity. The high-affinity state of the D2 receptor was directly measured using a radiolabeled agonist. Although the density of receptors increased with longer times after infection, the density of high-affinity sites reached a maximum of approximately 40 fmol/mg 30 to 36 hr after infection. Coexpression of D2 receptors and G protein subunits in Sf9cells dramatically increased the density of high-affinity sites, whereas the total density of receptors was unchanged, confirming that D2 receptors in Sf9 cells can exist in the high-affinity-coupled state, but that appropriate G proteins are expressed at relatively low levels. The density of D2S receptors converted to a coupled, agonist-preferring state when coexpressed with G proteins subunits (alpha i1, beta 1 and gamma 2) was 5 times greater than that of D2L receptors expressed under the same conditions, consistent with the hypothesis that D2 dopamine receptor isoforms differentially couple to alpha i1. PMID- 8632353 TI - Differential contribution of descending controls to the antinociceptive actions of kappa and mu opioids: an analysis of formalin-evoked C-fos expression. AB - In this study, the effect of intracerebroventricular (icv) administration of (5R) (5 alpha, 7 alpha, 8 beta)-N-methyl-N-[7-(1-pyrrolindinyl)-1- oxaspiro[4,5]dec-8 yl]-4-benzofurnacetamide monohydrochloride (Cl-977) on pain behaviors and on spinal cord fos-like immunoreactivity (FLI) evoked by unilateral formalin injection into the hindpaw of rats was examined. Intracerebroventricular administration of Cl-977 (0.13-13.00 nmol) produced a dose-dependent inhibition of formalin-evoked pain behaviors, with significant inhibition after 1.30, 4.40 and 13.00 nmol. The estimated ED50 for icv Cl-977 inhibition of formalin-evoked behaviors was 0.95 nmol and the Emax was 53%. The inhibitory effect of 4.40 nmol of icv Cl-977 on formalin-evoked behaviors was prevented by either pretreatment with the kappa selective antagonist nor-binaltorphimine (10 or 100 nmol) or coadministration of the opiate receptor antagonist, naloxone (30 nmol). The lowest dose of icv Cl-977 tested (0.13 nmol) produced a 50% reduction in FLI in the superficial laminae but did not inhibit the expression of FLI in any other regions of the spinal cord. The fos-inhibitory effect of low-dose icv Cl-977 in the superficial cord was reversed by coadministration of naloxone (30 nmol). Higher doses of icv Cl-977 that suppressed formalin-evoked behaviors did not inhibit the expression of FLI in any region of the spinal cord. Finally, neither the inhibitory effect of 4.40 nmol Cl-977 on formalin-evoked behaviors nor the formalin-evoked pattern of FLI expression in the spinal cord of rats treated with this dose of Cl-977 was affected by lesions of the dorsolateral funiculus. These results provide the first evidence that supraspinal kappa receptor-mediated antinociception is not dependent on the integrity of the dorsolateral funiculus and may be mediated exclusively at the supraspinal level, suggesting that there are multiple mechanisms through which opioids can evoke antinociceptive effects. PMID- 8632352 TI - Effects of single and short-term administration of clonidine on hypothalamic pituitary somatotropic function of the adult male rat: an in situ hybridization study. AB - The effects of the alpha-2 adrenoceptor agonist clonidine (CLO) on the growth hormone (GH) regulatory neuronal systems, growth hormone-releasing hormone (GHRH) and somatostatin (SS), were studied in adult male rats given a single or a short term administration (1, 3 and 6 days) of the drug. Acute administration of CLO significantly decreased hypothalamic GHRH content [leaving unaltered GHRH messenger RNA (mRNA) levels] and increased plasma GH levels; hypothalamic SS content/mRNA levels and pituitary GH content/mRNA levels remained unchanged. In 1 and 3-day CLO-treated rats, by contrast, decreased hypothalamic GHRH content was coupled with a significant reduction in GHRH mRNA levels. In these rats, pituitary GH content and mRNA levels were also significantly increased, whereas hypothalamic SS content and mRNA levels remained unaltered. In 6-day CLO-treated rats, hypothalamic GHRH content and mRNA levels were still significantly reduced, plasma GH levels were increased, but to a lesser extent than in 1- and 3-day CLO treated rats, and pituitary GH content and mRNA reverted to control levels. Hypothalamic SS content and mRNA levels remained unaltered. These results indicate that 1) functional activation of alpha-2 adrenergic receptors by CLO increases GHRH release from the hypothalamus, 2) CLO, via GHRH, increases GH secretion and biosynthesis, which in turn feeds back in the hypothalamus to reduce GHRH biosynthesis, and 3) reduction of hypothalamic GH-stimulatory activity tones down the initial pituitary somatotropic hyperfunction. Unaltered hypothalamic SS content and mRNA levels in all CLO-treated rats suggests that the somatostatinergic system is less sensitive than the GHRH system to changes in circulating GH levels. PMID- 8632354 TI - Prolonged beta adrenoceptor stimulation up-regulates cAMP phosphodiesterase activity in human monocytes by increasing mRNA and protein for phosphodiesterases 4A and 4B. AB - Human peripheral blood monocytes were treated for 4 h with a combination of the beta-agonist salbutamol (3 microM) and the low-Km cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (30 microM) to produce a prolonged elevation of cAMP and consequent increase in PDE activity. After this treatment, isozyme-selective PDE inhibitors were used to characterize the cAMP PDE profiles of high-speed supernatants before and after DEAE-Sepharose column chromatography. These experiments, in which total soluble PDE activity was increased by 58%, showed that the increased PDE activity is due to up-regulation of PDE4 and that at least two of the four subtypes are up-regulated. Experiments in whole cells demonstrated that this relatively modest increase in PDE4 activity has significant functional consequences, reducing cAMP accumulation in response to both PGE2 and lower, though not maximal, concentrations of rolipram. Further characterization of PDE4 subtype expression in control and treated monocytes, using polymerase chain reaction and Western blotting with subtype-specific peptide antibodies, showed that resting monocytes express both mRNA and protein for PDE4A, PDE4B and PDE4D. The amount of message for PDE4A and PDE4B appeared to increase upon up-regulation, whereas mRNA for PDE4D was not detected in treated cells. Western blots showed increases in the amount of protein for both PDE4A and PDE4B after treatment. We conclude that the PDE4 subtypes are differentially regulated upon prolonged exposure to elevated cAMP, with the consequence that the PDE4 profiles of control and treated cells differ not only in total activity but also in the relative proportions of the subtypes represented. PMID- 8632355 TI - Pharmacological and immunological characterization of N-methyl-D-aspartate receptors in human NT2-N neurons. AB - NT2 cells are a clonal line of human teratocarcinoma cells that exhibit N-methyl D-aspartate (NMDA) receptor-mediated excitotoxicity after terminal differentiation into NT2-N neurons. In this study, we used modulation of glutamate excitotoxicity to characterize the pharmacological properties and specific antibodies to determine the individual subunits of NMDA receptors expressed by NT2-N neurons. The glycine site antagonist 7-chlorokynurenic acid completely blocked glutamate toxicity in a dose-dependent manner. Histamine and the polyamine agonists spermine and spermidine enhanced glutamate toxicity in a dose-dependent manner consistent with expression of an NR1-NR2B combination of subunits. The efficacy of polyamine agonists suggests the expression of one or more splice variants of the NR1 subunit that lack the putative surface loop encoded by exon 5. Surprisingly, the putative inverse agonists diaminodecane and diaminododecane also enhanced toxicity in a dose-dependent manner. The antagonists arcaine and ifenprodil completely blocked glutamate toxicity in NT2-N cells. The atypical antagonist ifenprodil inhibited toxicity with a uniformly high affinity characteristic of interaction with the NR1-NR2B combination of subunits. Expression of both NR1 and NR2 subunits were detected by Western blot analysis. Neither protein was detectable in undifferentiated cells. In contrast, 70-fold lower levels of the NR2A subunit were detected in both differentiated and undifferentiated cells. The pharmacological and immunological results indicate that a functional NR1-NR2B combination of subunits is expressed by NT2-N neurons. Despite the immunological detection of NR2A subunit, no functional combination of NR1 and NR2A subunits could be demonstrated. PMID- 8632356 TI - Homologous desensitization of thromboxane A2 receptor in 1321N1 human astrocytoma cells. AB - The desensitization mechanisms that regulate the response to thromboxane A2 (TXA2) were investigated in 1321N1 human astrocytoma cells. Exposure of the cells to 9,11-epithio-11, 12-methanothromboxane A2 (STA2), a stable TXA2 receptor agonist, at a concentration of 1 microM for 30 to 45 min resulted in about a 50% decrease in subsequent STA2-stimulated phosphoinositide hydrolysis and Ca2+ mobilization. However, exposure to STA2 for 0 to 5 hr did not change the binding of [3H]SQ29548, a TXA2 receptor antagonist. Because STA2-induced GTPase activation decreased and the GTP sensitivity in inhibition of [3H]SQ29548 binding by STA2 disappeared after the cells had been exposed to STA2 for 30 min, the TXA2 receptor desensitization during the short-term might result from G-protein receptor uncoupling. STA2-induced desensitization was specific for the TXA2 receptor and homologous, because SQ29548 suppressed the desensitization and STA2 pretreatment did not affect the response to carbachol, a muscarinic cholinergic receptor agonist. Exposure to STA2 for 24 hr decreased [3H]SQ29548 binding sites to 20 to 30% of control and abolished STA2-stimulated phosphoinositide hydrolysis, indicating that long-term desensitization might induce down regulation of the TXA2 receptor. However, exposure to STA2 for 1 to 24 hr did not change the level of TXA2 receptor mRNA. These results show that homologous desensitization of the TXA2 receptor in human astrocytoma cells can be divided into two stages; the early stage involves uncoupling of receptors from G-proteins and the late stage involves a loss of receptors in cells. The mRNA levels may not be controlled by stimulation of the TXA2 receptor. PMID- 8632357 TI - Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line. AB - The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i. PMID- 8632358 TI - Methylenedioxymethamphetamine-induced serotonin deficits are followed by partial recovery over a 52-week period. Part I: Synaptosomal uptake and tissue concentrations. AB - The effects of a high dose methylenedioxymethamphetamine (MDMA) regimen on the serotonin (5-HT) system were evaluated over a 52-wk period. MDMA was administered to rats (20 mg/kg) 8 times at 12-hr intervals. Tissue concentrations of dopamine (DA) and 5-HT, and synaptosomal uptake of 3H-5-HT and 3H-DA were measured at 2, 8, 16, 32 or 52 wk posttreatment. Synaptosomal uptake of 3H-5-HT (hippocampus) was decreased at 2 and 8 wk, but not at 16, 32 or 52 wk after drug. 5-HT tissue concentrations were measured in frontal cortex, frontal-parietal cortex, occipital-temporal cortex, nucleus accumbens/olfactory tubercle, striatum, amygdala, hippocampus, septum, hypothalamus, ventral tegmentum/substantia nigra. Two weeks after MDMA treatment, all regions showed decreased 5-HT tissue concentrations except septum. Recovery over the 52-wk interval was noted for all depleted regions, but the rate and degree of recovery was region dependent. Frontal-parietal cortex, occipital-temporal cortex and hippocampus showed the least recovery, with significant depletions at 52 wk posttreatment. Hypothalamus showed an increase in 5-HT tissue concentrations relative to age-matched controls at 52 wk. These results indicate that a high-dose MDMA regimen results in long lasting depletions of serotonin. The rate and degree of recovery of serotonin tissue concentrations seen over the 52-wk test period is region specific. PMID- 8632360 TI - Research in everyday life. PMID- 8632359 TI - Methylenedioxymethamphetamine-induced serotonin deficits are followed by partial recovery over a 52-week period. Part II: Radioligand binding and autoradiography studies. AB - In our study, age-matched Holtzman Sprague-Dawley rats (275-300 g) received injections with either saline (0.9%) or 3,4-methylenedioxymethamphetamine (MDMA; 20 mg/kg free base, s.c) twice daily for 4 days and allowed to recover for 2, 8, 16, 32 and 52 wk after the final injection before death. Radioligand binding studies with 125I-RTI-55 to dopamine uptake sites in striatal homogenates showed no effect of MDMA on the density of dopamine uptake sites. In contrast, saturation binding studies with 125I-RTI-55 to 5-HT uptake sites in hippocampal and frontal-parietal homogenates showed a significant reduction in the number of uptake sites at 2 wk after MDMA treatment (34 and 25%, respectively of controls). By 16 wk, a partial recovery in the number of 5-HT uptake sites was observed in both tissues; however, only a full recovery of serotonin uptake sites was observed in hippocampus at the end of 52 wk. In more detailed studies using autoradiography with 125I-RTI-55, recovery of serotonin uptake sites varied from region to region. In particular, recovery of 5-HT uptake sites in cerebral cortex was observed to follow a rostral-caudal gradient. In addition, recovery of 5-HT uptake site in hippocampus also followed a rostral-caudal gradient. Different rates of recovery of 5-HT uptake sites were also observed for cingulate cortex, laterodorsal thalamus and ventromedial hypothalamus. No effect of MDMA was observed over lateral hypothalamus, substantia nigra and ventral tegmental area, or over serotonergic cell bodies such as dorsal raphe and median raphe. In conclusion, our study is consistent with previous studies describing the selective neurotoxicity of MDMA for serotonin neurons and presents evidence showing the rate of recovery of 5-HT uptake sites varies according to region and that recovery of 5-HT uptake sites in neocortex and hippocampus follows a rostral caudal gradient. PMID- 8632362 TI - Developing a quality indicator logbook for the postanesthesia care unit setting. AB - In the interest of quality care, it is important for health care practitioners to examine practice and identify methods for improvement. Gathering data is one way to impact changes in practice. Recognizing that a simple, efficient method for gathering data was needed, a logbook was developed to replace the more time consuming quality assurance form currently used at our northwest institution. Four incidents particular to the PACU were identified and included in the logbook. The goal of gathering risk indicator data about frequently occuring incidents was to make information available to nurses and adjunct health teams interested in quality assurance and improvement. PMID- 8632361 TI - An integrative review of ambulatory postanesthesia nursing patient outcome research: 1982 to 1993. AB - Specialty nursing practice tends to define itself by a set of competencies believed to be essential by the practitioner. A specialty practice is rarely the result of patient outcome research. Although nurses have been caring for postoperative patients since the early 1900s, a review of published ambulatory postanesthesia patient outcome nursing research has not been reported. As part of a larger study, an integrative review of ambulatory postanesthesia research was conducted to investigate adherence to the scientific method and to examine whether reported research findings link nursing process with patient outcome. Data were collected from identified ambulatory postanesthesia research studies published between 1982 and 1993. The tables of contents from 16 nursing journals were examined to identify published studies. The sample for the integrative review consisted of 28 studies meeting inclusion criteria. Data from the 28 studies were collected independently by two researchers on Ambulatory Post Anesthesia Research Literature Instrument (APARLI) coding sheets developed for the review. Findings indicated that the scientific method was used with varied levels of sophistication. The linkage of nursing process with patient outcome did not appear explicitly in the research studies reviewed. PMID- 8632363 TI - This looks like malignant hyperthermia! AB - Based on the content of this article, the reader should be able to (1) describe the symptoms of malignant hyperthermia (MH); (2) describe the use of dantrolene in the treatment of MH; (3) explain the history of the Malignant Hyperthermia Association of the United States; (4) list the recommended contents of an MH emergency cart; and (5) explain how to plan a mock MH crisis drill. PMID- 8632364 TI - Test-taking techniques. AB - This article discusses test-taking techniques that may be helpful to nurses scheduled to take the postanesthesia nurse certification examination or ambulatory perianesthesia nurse certification examination in November 1995. PMID- 8632365 TI - Investigating the reliability and validity of the infrared tympanic thermometer: a learning experience. AB - This series of articles to date has discussed points that readers should be looking for when reading and critiquing a researcher article. This article presents a brief report of a research project investigating the reliability and validity of the infrared tympanic thermometer (ITT) in afebrile subjects. Using four subjects and 40 temperature diads, the ability of the ITT to predict the subjects' temperatures obtained with a mercury thermometer was very poor (regression coefficient = .39). Test-retest reliability using the same data set resulted in an equally poor correlation coefficient (r = .19). A critique of the methodology of the study is provided as a learning experience. PMID- 8632366 TI - From adversary to advocate: an application of transformational leadership. AB - Traditional management and leadership skills are not as effective in an environment where there is constant change. The successful manager is the one who can offer transformational leadership to the staff. This is demonstrated by a real situation in which abstract principles were applied to an actual perioperative setting. PMID- 8632367 TI - A matter of time. PMID- 8632368 TI - "Rewarming hypothermic postanesthesia patients: a comparison between a water coil warming blanket and a forced-air warming blanket". PMID- 8632369 TI - Perioperative oxygen saturation levels of pediatric patients. AB - Airway compromise is of immediate concern for any patient after surgery but more so for pediatric patients because they are subject to more acute airway changes than adults. The purpose of this study was to identify those pediatric post surgical patients in an acute care setting at risk for oxygen saturation less than 90% during transport from the OR to the PACU. A descriptive, correlational design was employed using a convenience sample of 45 male and female pediatric patients between the ages of 0 months and 16 years who underwent elective surgery with general anesthesia lasting longer than 1 hour. Data were collected on each pediatric subject at four points in time (1) in the preoperative holding area (2) in the OR, (3) during transport to the PACU, and (4) on arrival to and during the stay in the PACU. Results showed that more than 50% of the sample (N = 45) desaturated to oxygen levels below 95%. Specifically, 48% desaturated between 90% to 95% and 8% desaturated below 90%. Pediatric patients age 2 years and younger showed the greatest risk for desaturation below 90%. In addition, as transport time increased, the number of subjects with oxygen saturation levels below 95% increased. PMID- 8632371 TI - Development and use of videotaped instruction for preoperative education of the ambulatory gynecological patient. AB - The current trend of ambulatory surgery has reemphasized the need for preoperative patient education. As patients assume more responsibility for self care, innovative strategies must be used to provide the short-term knowledge that is needed. The development and use of a preoperative videotape is discussed, with Orem's Self-Care Deficit Theory used as a nursing theory framework. The intervention was piloted on four subjects who expressed positive evaluation of the alternative educational strategy. PMID- 8632370 TI - Ondansetron: perioperative use of a serotonin receptor antagonist for the prevention and treatment of nausea and vomiting. AB - Postoperative nausea and vomiting (PONV) are common side effects after surgery and have numerous patient factors and etiologies. Although self-limiting, PONV is not without risks and complications. In the past numerous antiemetics have been used successfully in the management of PONV; however, these drugs are associated with adverse effects. Ondansetron is a serotonin receptor antagonist that is effective in preventing and treating PONV. It is believed that ondansetron binds at the serotonin receptor both in the vagal afferents of the gastrointestinal tract and in the chemoreceptor trigger zone. The reported side effects from ondansetron are minor compared with those of the more commonly used antiemetics such as droperidol and metoclopramide and include headache, dizziness, musculoskeletal pain, drowsiness and sedation, and shivers. PMID- 8632372 TI - The qualitative-quantitative dichotomy. AB - The article presents information about the qualitative and quantitative differences in research approaches. A comparison of these research approaches is offered as an educational tool to increase understanding of why both approaches are needed to build a body of knowledge specific to perianesthesia nursing. PMID- 8632373 TI - Positive responses to difficult behavior. PMID- 8632374 TI - Advance directives and the Patient Self-Determination Act: what is a nurse to do? AB - Since the Patient Self-Determination Act was enacted in 1991, asking patients whether they have advance directives (eg, a Living Will and a Durable Power of Attorney for Health Care Decisions) has become part of the admission procedures at health care facilities. However, questions may still remain about what exactly is the role of the health care provider in this part of the admission process. In many health care settings, the nurse has the responsibility to ask the patient about advance directives. If this occurs during preparation for surgery or other medical procedures, it adds responsibility for the nurse in an already hectic time. This article defines advance directives and examines their place in the admission process. The article further addresses to what extent health care providers are responsible for discussing patients' rights and advance directives and suggests methods that may facilitate the nurse's role in this responsibility. PMID- 8632375 TI - Reading research critically: results--bivariate regression analysis. AB - Regression is an analysis tool used in much of the research we read, and the terminology can be daunting. In reality, bivariate regression analysis is simply an extension of correlational analysis. This article was written to help the reader understand bivariate regression analysis. PMID- 8632376 TI - The new managerial consciousness. AB - In a world of constant change, working "smarter" does not solve the overwhelming sense of being out of control that most managers seem to experience. Rather than work smarter, the issue is to work differently and to examine the management myths that set expectations for a type of managerial competency and performance that no longer works in a constantly changing world. Management principles and practices developed in and for a world that had some predictability are not translatable to a world that has limited if any predictability. A comparison of the myths and the realities may offer some explanation for those managers trying to survive the constant "white water" of change. PMID- 8632377 TI - Conversations in ethics: exploring the literature. AB - Because of changes in the healthcare industry, many questions have surfaced concerning ethical dilemmas. We, on the ethics committee, thought it a good idea to publish some updated and informative literature concerning these dilemmas. PMID- 8632378 TI - What should I write about? PMID- 8632379 TI - Major limb amputation in adults, Zaria, Nigeria. AB - Over a period of 10 years, a total of 320 limb amputations were performed on adults at the Ahmadu Bello University Hospital, Nigeria. The major indication for upper limb amputation was trauma and post-fracture splintage gangrene (57%). In the lower limb the most common indication for amputation was advanced squamous cell carcinoma of the skin involving the bone. There was no case of peripheral vascular disease in these patients other than diabetic ulcers. The fitting of prosthesis was uncommon. Most of these amputations were certainly preventable. The need is for early and effective management of fractures, and the excision and grafting of non-healing leg ulcers before they become chronic. PMID- 8632380 TI - Prospective evaluation of the Glasgow Aneurysm Score. AB - The Glasgow Aneurysm Score (GAS), developed and published by the same authors, is a clinical prognostic scoring system that predicts mortality when operating on either intact or ruptured abdominal aortic aneurysms (AAA) taking into account these clinical criteria: patient's age, shock at presentation, myocardial disease, cerebrovascular disease and renal disease. The GAS was prospectively evaluated by studying 320 consecutive patients with AAA who were operated on at Glasgow, Aberdeen and Inverness, Scotland, in the period between January 1990 and May 1993. Logistic regression analysis showed very similar results to the original analysis used in developing the score. Age, shock, myocardial disease and renal disease were highly significant. Although it was not significant, cerebrovascular disease weight was not significantly different to its value in the original analysis. The mortality correlated well with the values of the score and ranged from 0% for scores below '70 GAS' to 80% for scores over '95 GAS'. The GAS appears, therefore, to be a reliable clinical predicative tool in foretelling the outcome of operating on AAA in terms of operative in-hospital mortality. PMID- 8632381 TI - An audit of percutaneous transluminal angioplasties in the treatment of lower limb ischaemia in a district general hospital over a period of 10 years. PMID- 8632382 TI - Major lower limb amputation for vascular disease in the Grampian area: the outcome of rehabilitation. AB - The outcome of lower limb amputation in the Grampian Region has been studied. In the years 1990-1991, 93 patients had 104 amputations for vascular disease. Rehabilitation was supervised by a multidisciplinary team. The amputation level was: unilateral and below-knee (BKA), 55 patients; unilateral and above-knee (AKA), 27 patients; and bilateral (BA) in 11 patients. At a median follow-up of 27 months, survivors who had had a limb fitted were sent a questionnaire to assess their physical mobility. The response rate was 95%. There were 33 (60%) survivors in the BKA group. Fourteen patients (25%) indicated good mobility and seven (13%) indicated fair mobility. There were five (19%) survivors in the AKA group, only one of whom described fair mobility with a prosthetic limb. There were 5 (46%) survivors of BA. Two described good and one fair mobility. Although vascular amputees have a high mortality rate, physical mobility tends to be good in those fitted with a prosthesis after BKA. PMID- 8632383 TI - Erythrocyte sedimentation rate and C reactive protein in the assessment of suspected bone infection--are they reliable indices? AB - The child who presents limping or with pain in a limb in the absence of a clear cause is a common problem, yet one which must be taken seriously. In the absence of trauma, infection involving the bone or joint must be carefully excluded clinically and often with the aid of further investigations. Preliminary investigations usually involve measurement of the while cell count (WCC), Erythrocyte Sedimentation Rate (ESR), and C Reactive Protein (CRP) as these are regarded as sensitive indicators for the presence of infection. Radiographs of the appropriate areas may be taken to assess for soft tissue swelling, chronic bone changes or evidence of trauma. The following case report demonstrates that normality of these parameters cannot be used to confidently exclude suppurative osteomyelitis and highlights the value of follow-up in apparently self-limiting conditions. PMID- 8632384 TI - Thoracoscopic resection of oesophageal diverticulum: a case report. AB - The introduction of video-assisted surgery has revolutionized thoracic surgery. The main attraction is the avoidance of a large thoracotomy incision and its associated morbidity. We present the case of a 55-year-old woman with an oesophageal diverticulum presenting with troublesome dysphagia. A thoracoscopic assisted oesophageal diverticulectomy was performed. Post-operative recovery was uneventful, and only two doses of intramuscular opiate analgesic were required. The patient remained asymptomatic at a follow-up of 3 months. PMID- 8632385 TI - A simple method of closure of a laparoscopic trocar site. AB - Trocar site herniation is a recognized complication of laparoscopic surgery. Omental herniation, and more importantly small-intestinal herniation with incarceration and obstruction, has been documented in surgical literature, occurring particularly at the large trocar sites (> or = 10 mm) that were not sutured at operation. Secure closure of the trocar site fascial wound is often technically difficult, being hampered by the limited size of skin incision, the depth of the subcutaneous fatty layer, and the concern about placing deep sutures blindly after the abdomen has been decompressed. We describe a simple technique to effect this closure securely and safely, and briefly discuss its other applications and limitations. PMID- 8632386 TI - A simple device for circumcision. AB - Several sizes of plastic cap were designed and made to fit the vertex of the penile glans. The foreskin is prepared in the usual manner, the glans capped with the specially made plastic cap and the foreskin pulled forwards over the cap. A crushing or non-crushing forceps is applied across the foreskin. The plastic cap separates the glans from the clamp, and its unwanted portion is cut with a knife without fear of injuries in the glans. We find this method to be safe even if it is used by a surgeon with limited experience. PMID- 8632387 TI - A simplified and safe method of open gastrostomy. AB - The establishment of a gastrostomy by the percutaneous endoscopic route (PEG) is now accepted as a safe and easy procedure which can be performed under combined sedation and infiltration of local anaesthetic at the chosen site on the anterior abdominal wall. However, surgeons are still not infrequently called upon to perform an open gastrostomy in a selected group of patients, namely: (1) those with extensive laryngeal and pharyngeal tumours in whom it is impossible to pass even a paediatric gastroscope; (2) neonates with oesophageal atresia. Various forms of open gastrostomy have been described through the years. The Foley and dePezzer catheters are commonly used for such gastrostomies. These latex catheters are prone to problems such as tube migration (causing intestinal obstruction), fractures and balloon rupture. The mushroom catheter used in the PEG procedure is easier to manage and lasts longer without requiring replacement. It has a retaining disc at its skin exit site, preventing migration, and can also be closed to prevent leakage of gastric contents. We report a quick and easy way of performing an open gastrostomy utilizing the advantages of the mushroom catheter where the wound and the gastrostomy site are also separate. PMID- 8632388 TI - V-Y plasty for Dupuytren's contracture of the palm. PMID- 8632389 TI - An audit of early wound infection after elective orthopaedic surgery. AB - The incidence of early post-operative wound infection was studied prospectively in 1053 patients undergoing elective orthopaedic procedures over a 3-month period. The study was repeated in 1131 patients a year later, 6 months after the hospital had moved to new premises. A clinical definition of wound infection identified disturbingly high sepsis rates. A total of 44% of all infections occurred after discharge from hospital. Despite the large sample, there was no significant difference in the rate of early wound infection between the two periods (7.85 and 6.82%). There was no significant difference in infection rates between theatres with and without laminar air flow. In the majority (35 out of 53) of minor infections, bacteriological confirmation was not available because no microbiological specimens were received. Conversely, there were five negative swabs out of 16 wounds defined clinically as major infections. We conclude that, where the rate of bacteriological confirmation of wound infection is low, the use of a clinical definition gives higher audited sepsis rates. Large audit samples are required to demonstrate differences as a result of a changed practice. PMID- 8632390 TI - Radiation exposure of orthopaedic trainees: quantifying the risk. AB - Interest in radiation protection issues has recently grown within the medical profession. Several investigators have examined the radiation exposure of orthopaedic surgeons using X-rays during trauma surgery, and found that doses received are within acceptable limits. These studies however, have been performed over short time-periods or have been confined to single procedures only. In this paper, we report the results of monitoring of the total exposure across all procedures of a group of orthopaedic surgeons over a 4-month period. Doses received were all below the International Commission on Radiological Protection (ICRP) recommended limits. While these results are reassuring, they do not represent grounds for complacency. Continued vigilance will be essential as work practices continue to change. In orthopaedics, in contrast with other specialties, the limiting dose is that to the hands. In view of this finding, extremity dosimetry for surgeons regularly using X-rays in theatre should be considered. PMID- 8632391 TI - The effects of weapons: surgical challenge and medical dilemma. PMID- 8632392 TI - Gasless laparoscopy. PMID- 8632393 TI - Observations on Barclay's elephant. AB - This account attempts to trace the fate of the skeleton of an elephant that was gifted by George Ballingall to Dr John Barclay, one of the most important teachers of Anatomy in Edinburgh during the early 19th century. In his will, Barclay gifted his human and comparative anatomy collections, including the elephant, to the Royal College of Surgeons of Edinburgh on two conditions, that a hall should be built to house the collection, and that it should be associated with his name in perpetuity. In the 1830s, the comparative collection, but particularly the skeleton of the elephant, was the pride of the College. Unfortunately, interest in the comparative material rapidly diminished, and, due to constraints on space, while the elephant's skull was retained the rest of the skeleton was disposed of. An unpublished poem written at the time of the Burke trial, in 1829, testifies to the fact that Barclay's elephant was closely associated in the minds of the public with the activities of Dr Robert Knox, the then Conservator of the College museum. PMID- 8632394 TI - Contamination of evacuated pneumoperitoneum air following laparoscopic cholecystectomy: preliminary results from a prospective study. AB - A prospective microbiological analysis of evacuated pneumoperitoneum air in 20 patients following laparoscopic cholecystectomy for benign gallbladder disease is reported. In six patients (30%) a positive culture was obtained, out of which five had bowel derived organisms. Bile specimens taken at the same time as the air specimens were similarly cultured but failed to yield any organisms. There were no post-operative septic complications. There were no deaths. The initial results suggest that the evacuated peritoneal air is a potential source of contamination to the theatre staff and environment. This must be borne in mind when advocating laparoscopic procedures for treatment of high-risk patients. PMID- 8632395 TI - Blunt traumatic rupture of the thoracic aorta: an epidemiological perspective. AB - A study was conducted from 1 July 1991 to 30 June 1994 to determine the incidence of blunt traumatic rupture of the thoracic aorta (RTA) in a defined area of inner metropolitan Sydney. The study group consisted of 30 subjects with RTA following falls, rail or road accidents, who were taken by ambulance to the regional trauma centre or directly to forensic pathology. The incidence of RTA in the resident population of 330,000 was in the range 20-30/10(6)/year. There was one survivor in the series, with 24 scene deaths and five deaths en route to hospital. Road accidents were responsible for 57% of incidents. RTA was found in 36% of the fatalities that occurred as a result of car and motorbike accidents. This was 5 times more common than that observed in pedestrian deaths (P < 0.005). Twenty seven subjects (90%) had at least one co-existent critical or lethal injury. Outcome may be improved by increasing awareness of the high prevalence of RTA in shocked car- and motor-bike-accident victims and stressing the importance of rapid transport of such cases to an appropriate hospital. PMID- 8632396 TI - Murphy's sign, acute cholecystitis and elderly people. AB - The presentation of acute abdomen in elderly people differs from that in younger patients. We retrospectively assessed how the presence or absence of Murphy's sign affected initial diagnosis of acute cholecystitis in elderly patients. In the presence of Murphy's sign, diagnostic accuracy for acute cholecystitis was 80% dropping to 34% when the sign was negative. The positive predictive value of the test in elderly people was 0.58, with a sensitivity of 0.48 and a specificity of 0.79. In elderly patients, a positive Murphy's sign is useful, but a negative sign should be treated with caution and other diagnostic tests and promptly. There should be cautious interpretation of classical signs in elderly patients. PMID- 8632397 TI - Putting a finger on the deep inguinal ring. AB - The clinical distinction between direct and indirect inguinal hernias is often made by determining whether digital pressure over the deep inguinal ring is able to control the hernia. In 25 consecutive patients having inguinal hernia operations, the positions of the mid-inguinal point and the mid-point of the inguinal ligament were determined pre-operatively and compared with the position of the deep inguinal ring measured at operation. Neither the mid-inguinal point nor the mid-point of the inguinal ligament correctly predicted the position of the deep inguinal ring (the mean position of the deep inguinal ring was found to be 0.52 cm lateral to the mid-inguinal point and 0.46 cm medial to the mid-point of the inguinal ligament). If the position of the deep inguinal ring cannot be accurately determined using fixed landmarks, it is unlikely that direct and indirect inguinal hernias can be distinguished by clinical examination. PMID- 8632398 TI - Right-sided low inguinal pain in young women. AB - Young women with right iliac fossa pain are commonly referred to general surgeons as possible acute appendicitis. The differential diagnosis that includes pelvic visceral disease may be very difficult to determine clinically, especially when the history and physical signs are equivocal. We believe that diagnostic accuracy may be improved by eliciting precisely the site of abdominal pain. Right-sided low inguinal pain may be referred from the female pelvic viscera in the same way as testicular pain may be referred to the groin, as these viscera derive their autonomic nerve supply from the T10-L2, mainly T12-L1, spinal segments in both sexes. There are no previous reports of the value of the symptom of right-sided low inguinal pain in differentiating pelvic visceral disease from acute appendicitis in young women. This paper reports a prospective study of the discriminant value of this symptom in such patients. PMID- 8632399 TI - Anastomoses involving the colon and rectum: an 8-year experience. AB - From January 1987 to May 1995, 214 anastomoses involving the rectum or colon were fashioned using single-layer, interrupted serosubmucosal 3/0 braided polyamide. There were six deaths, one clinically evident anastomotic leak, and one wound infection. These results lend further support to the use of a single layer of serosubmucosal sutures for anastomoses involving the colon and rectum. PMID- 8632400 TI - Distal penile lymphoedema following hypospadias repair: a case report. AB - A patient who developed isolated distal penile lymphoedema 10 years following hypospadias repair is reported. The classification and causes of genital lymphoedema and possible reasons for its occurrence in this case are discussed. PMID- 8632401 TI - Ante-natal testicular torsion: only one cause of the testicular regression syndrome? AB - It has been suggested that the most likely cause of the Testicular Regression Syndrome (TRS) is ante-natal torsion of the testis. As testicular torsion is twice as common on the left this theory cannot explain the incidence of right sided or bilateral cases. From a 5-year retrospective surgical and pathological review, we confirmed that the left testis was the most commonly affected, that boys with TRS tended to be delivered closer to term, and that frequently both testes were present at birth, but one or both subsequently vanished. We also found that direct trauma can produce histological findings indistinguishable from TRS. Close to term, fetal testes are liable to be intrascrotal and therefore susceptible to direct trauma. As the left testis descends into the scrotum at an earlier stage than the right, it is therefore at greater risk of injury. Since the findings of TRS can be produced by direct trauma, we suggest that intra partum trauma may predispose to the TRS. PMID- 8632403 TI - Bis(31/31') ([CYS(31), Trp(32), Nva(34)] NPY-(31-36)): a specific NPY Y-1 receptor antagonist. PMID- 8632402 TI - Selectively cytotoxic diterpenes from Euphorbia poisonii. AB - Bioactivity-guided fractionation of the latex of Euphorbia poisonii Pax. (Euphorbiaceae) led to the isolation and characterization of a new tigliane diterpene, 12-deoxyphorbol 13-(9,10-methylene)undecanoate (3), together with five known diterpenes (1,2,4-6). When evaluated for cytotoxicity in a panel of six human solid tumor cell lines, the diterpene esters, 1-3, 5, and 6, were selectively cytotoxic for the human kidney carcinoma (A-498) cell line with potencies for 2 and 3 exceeding that of adriamycin by ten thousand times. Details of the isolations, structural analyses, and cytotoxic activities are described. PMID- 8632404 TI - (2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)- pentane-1,5-dioic acid: a potent and selective antagonist for metabotropic glutamate receptors negatively linked to adenylate cyclase. PMID- 8632405 TI - Novel 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors. AB - Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved. PMID- 8632406 TI - In vivo biodistribution, pharmacokinetic parameters, and brain uptake of 5-halo-y methoxy(or ethoxy)-5,6-dihydro-3'-azido-3'-deoxythymidine diastereomers as potential prodrugs of 3'-azido-3'-deoxythymidine. AB - A new class of 5-halo-6-alkoxy-5,6-dihydro-3'-azido-3'-deoxythymidine diastereomers (5-x-6-OR -5,6-dihydro-AZTs; X = I, Br, Cl; R = Me, Et) were evaluated as potential anti-AIDS prodrugs of 3'-azido-3'-deoxythimidine (AZT). In vivo regeneration of AZT from these 5-X-6-OR-5,6-dihydro-AZTs was examined in Balb/c mice after intravenous tail vein injection. The (5R,6R)- and (5S,6S)-5 bromo(or iodo)-6-methoxy-5,6-dihydro derivatives of AZT (BMAZT, IMAZT) were rapidly converted to AZT, resulting in AZT plasma concentrations after a 144 micromol/kg dose similar to those after an equivalent dose (144 microg/kg, 38.5 mg/kg) of AZT, whereas AZT was not detectable by HPLC after the same dose of the chloro diastereomer (5R,6R)-CMAZT. The interaction of AZT and the 5-X-6-methoxy 5,6dihydro-AZT diastereomers with the 6-[(4-nitrobenzyl)thio]-9-beta-D ribofuranosylpurine equilibrative-sensitive nucleoside transporter in murine erythrocytes was also studied. The (5R,6R)- and (5S,6S)-5-X-6-OMe-5,6-dihydro-AZT diastereomers demonstrated a high affinity (K(i) = 0.2-0.5 mM) for the transporter relative to AZT (K(i) = 1.3 mM), with the exception of (5S,6R)-5 chloro-6-methoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (CMAZT) which has a K(i) value larger than 1.5 mM. [2-(14)C]-Labeled (5R,6R)- and (5S,6S)-5-bromo-6 methoxy(or ethoxy)- 5,6-dihydro-3'-azido-3'-deoxythymidines were synthesized by the regiospecific addition of methyl hypobromite or ethyl hypobromite to the 5,6 olefinic bond of [2-(14)C]-AZT in high radiochemical yield [(5R,6R)-BMAZT, 48%, and (5S,6S)-BMAZT, 33%; (5R,6R)-BEAZT, 61%, and (5S,6S)-BEAZT, 15%), high radiochemical purity (>98%), and high specific activity (56mCi/mmol)]. The amounts of radioactivity in mouse brain after iv injection of [2-(14))C]-labeled (5R,6R)-BMAZT, (5S,6S)-BMAZT, or (5R,6R)-BEAZT were 2-4 fold higher that that for [2-(14)C]-AZT (P < 0.05). The radioactivity remaining in blood after dosing with these 5-bromo-6-alkoxy-5,6-dihydro-AZTs was up to 20-fold higher than after injection of [2-(14)C]-AZT at longer time intervals after injection. The amounts of radioactivity present in femoral bone following injection of [2(-14)C]-AZT, or these 5-bromo-6-alkoxy-5,6-dihydro-AZTs, were similar. Subcellular and regional distributions of [2-(14)C]-labeled AZT, (5R,6R)-BMAZT, or (5R,6R)-BEAZT in mouse brain after jugular vein injection did not show preferential concentration in any particular subcellular fraction nor a marked preferential regional localization for either AZT or these 5,6-dihydro prodrugs of AZT. PMID- 8632407 TI - Tetrahydronaphthalenes: influence of heterocyclic substituents on inhibition of steroid enzymes P450 arom and P450 17. AB - In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1 tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and 3-alkyl substituted 4,5 dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) (TXA(2)) synthase. The test compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of 110 [rp of aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other hand some of these compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) = 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m) testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17: K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM, K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM). PMID- 8632408 TI - Controlled modification of acidity in cholecystokinin B receptor antagonists: N (1,4-benzodiazepin-3-yl)-N'-[3-(tetrazol-5-ylamino) phenyl]ureas. AB - The design, synthesis, and biological activity of a novel series of CCK-B receptor antagonists (1) which incorporate a tetrazol-5-ylamino functionality attached to the phenyl ring of the arylurea moiety of L-365,260 are described. In these compounds, the acidity of the tetrazole was gradually modified by utilization of simple conformational constraints, and X-ray crystallographic data were obtained to support the conformational depenence of the pK(a) of the aminotetrazoles. Compounds to emerge from the present work such as 1f and 2c,d are among the highest affinity and, in the case of 1f, most selective (CCK-A/CCK B, 37 000) antagonists so far reported for this receptor. The C(5)-cyclohexyl compound 2c (L-736,380) dose-dependently inhibited gastric acid secretion in anesthetized rats (ID(50), 0.064 mg/kg) and ex vivo binding of [(125)I]CCK-8S in BKTO mice brain membranes (ED(50), 1.7 mg/kg) and is one of the most potent acidic CCK-B receptor antagonists yet described. PMID- 8632409 TI - Comparative molecular field analysis-based prediction of drug affinities at recombinant D1A dopamine receptors. AB - Determination of quantitative structure-activity relationship (QSAR) for affinity at particular dopamine (DA) receptors has become an even greater priority with the cloning of five DA receptor subtypes. The use of agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in QSAR presents a unique opportunity for accuracy and precision in measurement of biological values. Bound conformations of 11 agonists (for which both affinity data at the recombinant D1A DA receptor and stereochemical configurations were available) were determined by alignment with a template compound, SKF38393, which shows high affinity and selectivity for D1A receptors and is fairly rigid in structure. These aligned structures suggested a 3-point pharmacophore map (one cationic nitrogen and two electronegative centers) of the D1A DA receptor. This map shows both similarities and differences when compared with a previously reported D2 DA receptor pharmacophore map based on biological data from rat brain and with a recently published map of the native D1 DA receptor using several semirigid compounds. Log(1/K(d)) values at recombinant D1A DA receptors were used as the target property for a CoMFA (comparative molecular field analysis) of the 11 aligned structures. The resulting CoMFA model yielded a cross-validated r(2)(q(2)) value of 0.829 and a simple r(2) = 0.96. In contrast, when a CoMFA model was developed for 10 of these compounds using striatal D1 K(d) values, the q(2) value was reduced to 0.178. These results are consistent with the idea that drug affinity data obtained from clonal cells expressing recombinant receptors may be superior to that obtained using heterogeneous mixtures of native receptors prepared from brain membranes. The predictive utility of the CoMFA model was evaluated using several high-affinity dopamine agonists and m- and p-tyramine, two compounds with a single hydroxyl group on the aromatic ring. Predictions were fairly accurate for all compounds but the two tyramines. PMID- 8632410 TI - Synthesis, biological evaluation, and quantitative receptor docking simulations of 2-[(acylamino)ethyl]-1,4-benzodiazepines as novel tifluadom-like ligands with high affinity and selectivity for kappa-opioid receptors. AB - The synthesis and biological evaluation of a series of 2-substituted 5-phenyl-1,4 benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a kappa-opioid agonist and a cholecystokinin-A (CCK A) antagonist, are reported. The radioligand binding models used in these studies were [(125)I](BH)-CCK-8 in rat pancreas (CCK-A), [(3)H]-(MENLE(28,31))-cck-8 in guinea pig cerebral cortex (CCK-B), and [(3)H]U-69593 (kappa(1)), [(3)H]DAMGO (mu), and [(3)H]DADLE (delta) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for kappa-opioid receptors. In particular, the 2-thienyl derivative 7A(X = H) with a K(i) = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig kappa (kappa(1))-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure-affinity relationships in this class of receptors. PMID- 8632411 TI - Synthesis of non-nucleoside analogs of toyocamycin, sangivamycin, and ++thiosangivamycin: influence of various 7-substituents on antiviral activity. AB - A number of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5 carboxamide, and -5-thiocarboxamide derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1). Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole (1) with triethyl orthoformate followed by alkylation via the sodium salt method with a variety of alkylating agents furnished the corresponding 1-substituted pyrroles 2a-k. Ring annulation was achieved with methanolic ammonia affording the 7-substituted 4 amino-6-bromopyrrolo++-[2,3-d]pyrimidine-5-carbonitrile derivatives 3a-k. Debromination of 3a-k, via catalytic hydrogenation, gave the corresponding 7 substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile analogs 4a-j,l. A selective reduction of 4-amino-6-bromo-7-allylpyrrolo[2,3-d]-pyrimidine-5-carbon ril e (3k) in zinc and acetic acid furnished 4-amino-7-allylpyrrolo-[2,3 d]pyrimidine-5-carbonitrile (4k). Conventional functional group transformations involving the 5-cyano group of 4 furnished the 5-carboxamide derivatives 5a-1 and the 5-thio-amide analogs 6a-l. A similar transformation of the aglycone of toyocamycin (4m) furnished the corresponding aglycone of thiosangivamycin (6m). Several of the new compounds (4-6a-ej-l) were evaluated for their ability to inhibit the growth of L1210 murine leukemic cells. Whereas a number of the carboxamide (5) and thioamide (6) derivatives had modest activity, the corresponding nitrile analogs (4) were all inactive. All compounds were tested for activity against HCMV and HSV-1. The non-nucleoside nitrile analogs 4a-m and carboxamide derivatives 5a-l were, with a few exceptions, essentially inactive against HCMV and HSV-1 and relatively nontoxic. In direct contrast, nearly all of the thioamide derivates 6a-1, including the aglycone of thiosangivamycin (6m), were good inhibitors of HCMV and HSV-1. Most were noncytotoxic in their antiviral concentration range. Cytotoxicity which was observed appeared to be a consequence of DNA synthesis inhibition. Several of these compounds, such as 6b,e, were particularly interesting inhibitors of HCMV with IC(50)'s ranging from 0.1 to 1.3 muM. The antiviral activity of both compounds was well separated from cytotoxicity in KB, HFF, and L1210 cells. PMID- 8632412 TI - Structure-activity relationships among 2-substituted 5,6-dichloro-, 4,6-dichloro , and 4,5-dichloro-1-[(2-hydroxyethoxy) methyl]- and -1-[(1,3-dihydroxy-2 propoxy) methyl]benzimidazoles. AB - The sodium salt of 2,5,6-trichlorobenzimidazole (8a) was condensed with [2 (benzyloxy)ethoxy]-methyl chloride (9) and [1,3-bis(benzyloxy)-2-propoxy]methyl chloride (18) to provide the corresponding protected acyclic nucleosides 10a and 19a, which on debenzylation afforded 2,5,6-trichloro-1-[(2 hydroxyethoxy)methyl]benzimidazole (11a) and 2,5,6-trichloro-1-[(1,3-dihydroxy-2 propoxy)methyl] benzimidazole (20a), respectively. A similar condensation of 2,4,6-trichlorobenzimidazole (2a) and 2,4,5-trichlorobenzimidazole (7a) followed by debenzylation yielded 11b, 20b, 11c, and 20c, respectively. A nucleophilic displacement of the 2-chloro group of 11a-c and 20a-c with liquid ammonia, methylamine, dimethylamine, and thiourea furnished several interesting 2 substituted compounds in good yields, e.g., 12-14(a-e), 21-23(a-e), 15-17, and 24 26. Alkylation of the 2-thio analogs 15-17 and 24-26 with benzyl chloride furnished the 2-alkylthio acyclic nucleosides 12d-14d and 21d-23d. Desulfurization of 15 and 24 with Raney Ni furnished 5,6-dichloro-1[(2 hydroxyethoxy)methyl]benzimidazole (12e) and 5,6-dichloro-1-[1,3-dihydroxy-2 propoxy)methyl]benzimidazole (21e), respectively (acyclic analog of 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole). Similarly the dihalo compounds 13e, 14e, and 23e were prepared in moderate yields from the 2-thio analogs 16,17, and 26. Treatment of 2-bromo-5,6-dichlorobenzimidazole (8b) with 27 and 30 gave the protected acyclic compounds 28a and 31a, which on deacetylation with sodium carbonate and potassium cyanide yielded 2-bromo-5,6-dichloro-1-[(2 hydroxyethoxy)methyl]benzimidazole (29a) and 2-bromo-5,6-dichloro-1-[(1,3 dihydroxy-2-propoxy)methyl]benzimidazole (32a), respectively, in moderate yields. The 2-bromo-4,6-dichlorobenzimidazole and 2-bromo-,5-dichlorbenzimidazole analogs 29b,c and 32b,c were prepared in a similar manner. Compounds were tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and for cytotoxicity. In marked contrast to the ribosylbenzimidazoles, none of the acyclic analogs were specific and potent inhibitors of HCMV. Only the 2-thiobenzyl analogs 12d, 13d, 14d, and 23d and the 2-Br analogs 32a,b were active, but activity was not well separated from cytotoxicity. The lack of specific and potent antiviral activity strongly suggests that these acyclic nucleoside analogs are not phosphorylated by HCMV or HSV-1 gene products and that the ribosylbenzimidazoles do not require phosphorylation for antiviral activity. PMID- 8632413 TI - High-affinity inhibitors of dihydrofolate reductase: antimicrobial and anticancer activities of 7,8-dialkyl-1,3-diaminopyrrolo[3,2-f]quinazolines with small molecular size. AB - A series of 7,8-dialkylpyrrolo[3,2-f]quinazolines were prepared as inhibitors of dihydrofolate reductase (DHFR). On the basis of an apparent inverse relationship between compound size and antifungal activity, the compounds were designed to be relatively small and compact. Inhibitor design was aided by GRID analysis of the three-dimensional structure of Candida albicans DHFR, which suggested that relatively small, branched alkyl groups at the 7- and 8-positions of the pyrroloquinazoline ring system would provide optimal interactions with a hydrophobic region of the protein. The compounds were potent inhibitors of fungal and human DHFR, with K(i) values as low as 7.1 and 0.1 pM, respectively, and were highly active against C. albicans and an array of tumor cell lines. In contrast to known lipophilic inhibitors of DHFR such as trimetrexate and piritrexim, members of this series of pyrroloquinazolines were not susceptible to P glycoprotein-mediated multidrug resistance and also showed significant distribution into lung and brain tissue. The compounds were active in lung and brain tumor models and displayed in vivo activity against Pneumocystis carinii and C. albicans. PMID- 8632414 TI - Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase. AB - To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6 quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3 substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro 2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration. PMID- 8632415 TI - Tyrosine kinase inhibitors. 9. Synthesis and evaluation of fused tricyclic quinazoline analogues as ATP site inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor. AB - Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2 (dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure activity relationship studies previously developed for the 4-[(3 bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR. PMID- 8632416 TI - Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine. AB - For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells. PMID- 8632417 TI - 3-and 4-substituted 4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides as potassium channel openers: synthesis, pharmacological evaluation, and structure-activity relationships. AB - 4-N-Substituted and -unsubstituted 3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e] 1,2,4-thiadiazine 1,1-dioxides were synthesized and tested vs diazoxide and selected 3-alykl- and 3-(alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine 1,1 dioxides as potassium channel openers on pancreatic and vascular tissues. Several 4-N-unsubstituted 3-(alkylamino)pyridothiadiazines and some 3-(alkylamino)-7 chlorobenzothiadiazines were found to be more potent than diazoxide for the inhibition of the insulin-releasing process. Moreover, the 3 (alkylamino)pyridothiadiazines appeared to be more selective for the pancreatic than for the vascular tissue. By means of the pharmacological results obtained on pancreatic B-cells, structure--activity relationships were deduced and a pharmacophoric model for the interaction of these drugs with their receptor site associated to the pancreatic K(ATP) channel was proposed. According to their selectivity for the B-cell (endocrine tissue) vs the vascular (smooth muscle tissue) ionic channel, selected 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4, thiadiazine 1,1-dioxides may serve as pharmacological tools in studying the K(ATP) channels ("pancreatic-like" K(ATP) channels) in other tissues. PMID- 8632418 TI - Guanine, pyrazolo[3,4-d]pyrimidine, and triazolo[4,5-d]pyrimidine (8-azaguanine) phosphonate acyclic derivatives as inhibitors of purine nucleoside phosphorylase. AB - Phosphonate acyclic derivates of guanines, pyrazolo[3,4-d]pyrimidines, and triazolo[4,5-d]-pyrimidines (8-azaguanines) are inhibitors of the enzyme purine nucleoside phosphorylase (PNPase) with Ki' values ranging from 0.05 to 1.6 microM. These compounds are enzymatically stable congeners of the potent PNPase inhibitor acyclovir diphosphate (53). PMID- 8632419 TI - Azole endothelin antagonists. 1. A receptor model explains an unusual structure activity profile. AB - The pseudotetrapeptide FR-139317 is a potent and highly selective antagonist of the endothelin-A (ET(A)) receptor; however, its peptidic nature leads to poor oral absorption characteristics which make it an unlikely drug candidate. In an attempt to improve these properties, we have replaced a portion of the amide bond framework of FR-139317 with a heterocyclic surrogate. The resultant analogs are also ET(A)-selective antagonists, but show a structure-activity profile substantially different from that of the peptidic series, particularly with regard to the requirements for the side chain group that has been incorporated into the heterocycle. The nature of the heterocycle itself also has profound effects on the activity of the compounds. Both of these surprising results can be rationalized through examination of a 3D model of ET ligand--receptor binding that has previously been developed in our laboratories. PMID- 8632420 TI - Azole endothelin antagonists. 2. Structure-activity studies. AB - Structure-activity studies have been performed in an attempt to improve the potency of a novel series of azole-based endothelin-A (ET(A)) selective antagonists. Modifications of the hydrophobic group on the terminal urea produced substantial effects on receptor affinity; in particular, the choice of cyclohexyl or arylureas led to substantial improvements in activity. Conformational restriction of these groups provides an additional benefit. N-Methylation of the indole moiety which is part of the heterocyclic dipeptide surrogate also improves potency. The effects of these two modifications appear to be synergistic, with the best of the resultant doubly modified analogs (e.g. 14q, 15y, and 15ff) exhibiting an 80-200-fold improvement over the original leads. PMID- 8632421 TI - Azole endothelin antagonists. 3. Using delta log P as a tool to improve absorption. AB - The oral absorption profile of a family of azole-based ET(A)-selective antagonists has been improved through a rational series of structural modifications which were suggested by analysis of the physicochemical parameter delta log P. Comparison of urea 2 with a series of well-absorbed compounds using delta log P analysis suggested that 2 has an excess capacity for forming hydrogen bonds with solvent. A series of urea modifications were explored as a means of reducing H-bonding capacity while maintaining affinity for the ET(A)-receptor. The correlation between delta log P values and absorption in an intraduodenal (id) bioavailability model was good; this strategy uncovered replacements for each of the urea NH groups which simultaneously improve both potency and drug absorption. A combination of these optimized modifications produces carbamate 16h, a highly-selective ET(A) antagonist with a potency/bioavailability profile consistent with an oral route of administration. PMID- 8632422 TI - Substituted 2,5'-Bi-1H-benzimidazoles: topoisomerase I inhibition and cytotoxicity. AB - Several 2'-aryl-5-substituted-2,5'bi-1H-benzimidazole derivatives were synthesized and evaluated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell line RPMI 8402. This study focused on 18 2,5' bi-1H-benzimidazole derivatives which contained either a 5-cyano, a 5 (aminocarbonyl), or a 5-(4-methylpiperazinyl) group. Among these bibenzimidazoles, the pharmacological activity of 2'-phenyl derivatives and the influence of the different positional isomers of either a 2'-tolyl group or a 2' naphthyl moiety on cytotoxicity and topoisomerase I inhibitory activity were determined. PMID- 8632423 TI - Homologs of idoxifene: variation of estrogen receptor binding and calmodulin antagonism with chain length. AB - A series of homologs of idoxifene [1a, (E)-1-[4-(N-pyrrolidinoethoxy)phenyl]-1-(4 iodophenyl)-2-phenyl-1-butene ] and selected homologs of 4-iodotamoxifen [2a,(E) 1-[4-(N-dimethylamino)-ethoxy]phenyl]-1-(4-iodophenyl)-2-phenyl -1-butene] with the side chain (CH(2))(n) varying in length from n=3 (1b,2b) to n=10(1i,2i) have been synthesized and tested for antagonism of of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to rat uterine estrogen receptor. Compared with 1a (IC(50) =1.5 microM), the homologs showed a progressive increase in calmodulin antagonism with a maximum inhibition at n=7-9 (1f-h)(IC(50)=0.2 microM), declining at n=10 (1i) to IC(50) =1.6 microM. In the pyrrolidino series, estrogen receptor binding affinity peaked at n=3 (1b, RBA= 23; estradiol = 100), declining by n=10 (1i) to RBA = 0.4, but the homolog n=8 (1g, RBA = 3.5) was still comparable to tamoxifen (RBA = 3.9). A similar pattern of activity was seen for the dimethylamino counterparts. These compounds represent a new class of antiestrogens with potent calmodulin antagonism. PMID- 8632424 TI - Synthesis and pharmacologic evaluation of 2-endo-amino-3-exo isopropylbicyclo[2.2.1]heptane: a potent imidazoline1 receptor specific agent. PMID- 8632425 TI - Endogenous natriuretic factors. 5. Synthesis and biological activity of a natriuretic metabolite of diltiazem and its derivatives. AB - In our search for endogenous natriuretic factors from human uremic urine, we have previously identified a new metabolite of the drug diltiazem (Murray et al. Life Sci. 1995, 57, 2145-2161). The structure of this metabolite, (+)-(2S,3S)-3 hydroxy-5-(2-hydroxyethyl)-2,3-dihydro-2-(4-methoxyphenyl) -1,5-benzothiazepin 4(5H)-one (LLU-beta1; 2), was proved by unequivocal synthesis from a diltiazem synthon. The synthetic material also proved to be natriuretic as had the urinary isolate. Given the acetylation at C-3 in diltiazem, the 3-monoacetate (8) and diacetate (3) derivatives of 2 were prepared. The 4-nor-keto (6) derivative of 2 was also synthesized. Only the parent 2 induced natriuresis over a range of doses without accompanying kaliuretic activity at some doses. PMID- 8632426 TI - Synthesis and pharmacology of potential cocaine antagonists. 2. Structure activity relationship studies of aromatic ring-substituted methylphenidate analogs. AB - As part of a program to develop medications which can block the binding of cocaine to the dopamine transporter, yet spare dopamine uptake, a series of aromatic ring-substituted methylphenidate derivatives was synthesized and tested for inhibitory potency in [3H]WIN 35,428 binding and [3H]dopamine uptake assays using rat striatal tissue. Synthesis was accomplished by alkylation of 2 bromopyridine with anions derived from various substituted phenylacetonitriles. In most cases, erythro compounds were markedly less potent than the corresponding (+/-)-threo-methylphenidate (TMP; Ritalin) derivatives. The ortho-substituted compounds were much less potent than the corresponding meta- and/or para substituted derivatives. The most potent compound against [3H]WIN 35,428 binding, m-bromo-TMP, was 20-fold more potent than the parent compound, whereas the most potent compound against [3H]dopamine uptake, m,p-dichloro-TMP, was 32-fold more potent. Threo derivatives with m- or p-halo substituents were more potent than TMP, while electron-donating substituents caused little change or small loss of potency. All of the derivatives had Hill coefficients approaching unity, except m,p-dichloro-TMP, which had an nH of 2.0. Although the potency of the (+/-) methylphenidate derivatives in the two assays was highly correlated (R2 = 0.986), the compounds m-chloro-,m-methyl-, and p-iodo-TMP were 4-5-fold more potent at inhibiting [3H]-WIN 35,428 binding than [3H]dopamine uptake (cocaine has a ratio of 2.3). These and other compounds may be promising candidates for further testing as potential partial agonists or antagonists of cocaine. PMID- 8632428 TI - [125I]iodoproxyfan and related compounds: a reversible radioligand and novel classes of antagonists with high affinity and selectivity for the histamine H3 receptor. AB - The synthesis and biological evaluation of new histamine H3 receptor antagonists with an iodinated aryl partial structure are described as part of an extensive research program to find model compounds for the development of a new radioligand with high H3 receptor affinity and specific activity. All compounds were tested for their H3 receptor antagonist activity in a [3H]-histamine-release assay with synaptosomes from rat cerebral cortex. The new leads with potent H3 receptor antagonist activity belong to a series of derivatives of 3-(1H-imidazol-4 yl)propanol with carbamate (4-7), ester (8-16), and ether (17-22) as functional groups. Structure-activity relationships are discussed. The most active compound in the functional test (-log Ki = 8.3) and in binding studies with [3H]-(R)-alpha methylhistamine on rat cerebral cortex (-log Ki = 9.0) in vitro was 3-(1H imidazol-4-yl)propyl (4-iodophenyl)methyl ether (iodoproxyfan, 19) exhibiting no central H3 receptor antagonist activity in vivo. The potency of iodoproxyfan is more than 300 times lower at H1, H2, alpha1, alpha2, beta1, 5-HT2A, 5-HT3, and M3 receptors than at histamine H3 receptors. Because of the high potency and selectivity of 19, this compound has also been prepared in the [125I]-iodinated form by a nucleophilic halogen exchange reaction using the corresponding bromo derivative 22 as a precursor. The newly prepared [125I]iodoproxyfan (23) possesses advantageous pharmacological properties and fulfills all criteria of a useful radioligand. PMID- 8632427 TI - Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors. AB - An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournie-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R1)CONHCH(R1')CON(R)CH(R2')COOH have been synthesized. The introduction of well-selected beta-branched chains in positions R1 and R1', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile Tyr] and 22 [N-[(2S)-2-mercapto-3-phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S1' and S2' subsites, whereas additional interactions with the S1 binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10(-5) mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE. PMID- 8632429 TI - Molecular modeling of azole antifungal agents active against Candida albicans. 1. A comparative molecular field analysis study. AB - A series of 56 azole antifungal agents belonging to chemically diverse families related to bifonazole, one of the antimycotic drugs of clinical use, were investigated using the comparative molecular field analysis (CoMFA) paradigm. The studied compounds, which have been already synthesized and reported to be active in vitro against Candida albicans, were divided into a training set and a test set. The training set consisted of 40 molecules from all the different structural classes. Due to the lack of experimental structural data on these derivatives, molecular mechanics techniques were used to obtain putative active conformations for all the compounds. the correctness of this molecular modeling work was confirmed a posteriori by comparison with structural data of the analog 2w obtained by X-ray crystallographic analysis (Massa, S.; et al. Eur. J. Med. Chem. 1992, 27, 495-502). Two different alignment rules of the training set molecules were used in this study and are based on the assumption that according to published results on azole antifungal agents, all the studied compounds exert their inhibitory activity through the coordination of their azole moiety to the protoporphyrin iron atom of the fungal lanosterol 14alpha-demethylase enzyme. The predictive ability of each resultant CoMFA model was evaluated using a test set consisting of 16 representative compounds that belong to all the different structural classes. The best 3D-quantitative structure-activity relationship model found yields significant cross-validated, conventional, and predictive r2 values equal to 0.57, 0.95, and 0.69, respectively. The average absolute error of predictions of this model is 0.30 log units, and the structural moieties of the studied antifungal agents which are thought to contribute to the biological activity were identified. The predictive capability of this model could be exploited in further synthetic studies on antifungal azoles. Furthermore, the results obtained by using two different alignments of the inhibitors suggest that the binding mode of these molecules involves both a coordination to the iron protoporphyrin atom and an additional, likewise relevant, hydrophobic interaction with the active site. PMID- 8632430 TI - Nuclear magnetic resonance and molecular modeling study on mycophenolic acid: implications for binding to inosine monophosphate dehydrogenase. AB - The conformation of the sodium salt of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase (IMPD), derived from 1D DIFNOE and 2D ROESY experiments in water and molecular dynamics (MD) is described. The hexenoic acid side chain conformation consistent with the NMR data was similar to that seen in the X-ray structure of MPA. The solution conformation was applied in a molecular modeling study in order to explore the potential features of enzyme binding. Our results, based on striking similarities in molecular volume and electrostatic isopotential between MPA and cofactor NAD+, lead to the suggestion that MPA is capable of binding to the nicotinamide site of IMPD and mimicking the NAD+ inverse regulation of the enzyme. In addition, our proposed model is in good agreement with the observed high affinity of the dinucleotide analogues thiazole- and selenazole-4-carboxamide adenine dinucleotide to IMPD. PMID- 8632431 TI - Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 17. Structure-activity relationships of several series of compounds derived from N-chlorosulfonyl isocyanate. AB - Several series of acyl-CoA:cholesterol O-acyltransferase inhibitors were prepared by the stepwise addition of nitrogen, oxygen, and sulfur nucleophiles to N chlorosulfonyl isocyanate. The (aminosulfonyl)ureas 3-44 were the most potent inhibitors in vitro, with several compounds having IC50 values < 1 microM. Although the other series of compounds were not as potent in vitro, many compounds did display good in vivo activity in cholesterol-fed rats. Several of the oxysulfonyl carbamates (including CI-999, 115) showed excellent lipid lowering activity in the chronic in vivo screen, demonstrating significant cholesterol lowering in a pre-established hypercholesterolemic state. PMID- 8632432 TI - Cationic [99mTcIII(DIARS)2(SR)2]+ complexes as potential myocardial perfusion imaging agents (DIARS = o-phenylenebis(dimethylarsine);SR- = thiolate). AB - Reduction-substitution reactions on [99mTcO4]- with both o phenylenebis(dimethylarsine) (DIARS) and various thiols produce a series of monocationic [99Tc(DIARS)2(SR)2]+ complexes. Addition of [99gTcO4]- to the above reaction mixtures allows the characterization of the "carrier-added" complexes by means of reverse-phase high-performance liquid chromatography with radiometric and optical detection systems. The identity of the [99mTc(DIARS)2(SR)2]+ complexes is confirmed by fast atom bombardment mass spectroscopy; equivalence of the [99gTc(DIARS)2-(SR)2]+ and [99mTc(DIARS)2(SR)2]+ species is demonstrated by identical HPLC retention times. All the [99mTc(DIARS)2(SR)2]+ complexes tested accumulate in the myocardium of Sprague-Dawley rats with an average uptake of 1.5 2.0% of injected dose/g at 30 min. Thus, as designed, these nonreducible Tc(III) complexes do not exhibit the rapid myocardial washout observed for reducible Tc(III) complexes. These [99mTc(DIARS)2(SR)2]+ complexes also exhibit an initially high liver uptake, but the presence of ether groups within the thiolate ligands causes this liver uptake to decrease over time without affecting the heart uptake, thereby improving the heart/liver ratio. PMID- 8632433 TI - Potent inhibitors of acyl-CoA:cholesterol acyltransferase. 2. Structure-activity relationships of novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amides. AB - Novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) and lower serum total cholesterol in cholesterol-fed rats. Among the synthesized compounds, N-(2,2,4,6-tetramethyl-2,3 dihydrobenzofuran-7-yl)amide derivatives showed potent ACAT inhibitory activity. The synthesis and structure-activity relationships of these compounds are described. A methyl group at position 6 of the 2,3-dihydrobenzofuran moiety was important for potent ACAT inhibitory activity. In the series of N-(2,2,4,6 tetramethyl-2,3-dihydrobenzofuran-7-yl) amides, lipophilicity of the acyl moiety was necessary for the potent ACAT inhibitory activity. The highly lipophilic acid amides N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)-2,2- dimethyldodecanamide (10) and 6-(4-chlorophenoxy)-N-(2,2,4,6-tetramethyl-2,3 dihydrobenzofuran-7-y l)-2,2-dimethyloctanamide (50) showed potent activity. Introduction of a dimethylamino group at position 5 of the 2,3-dihydrobenzofuran moiety resulted in highly potent activity. The most potent compound, N-[5 (dimethylamino)-2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl ]-2,2 dimethyldodecanamide (13, TEI-6620), showed highly potent ACAT inhibitory activity (rabbit small intestine IC50 = 0.020 microM, rabbit liver IC50 = 0.009 microM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 0.030 microM), extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (71% at a dose of 0.3 mg/kg/day po), and good bioavailability in fed dogs (Cmax = 2.68 microg/mL at 1 h, 10 mg/kg po). PMID- 8632434 TI - Lipophilic antifolates as agents against opportunistic infections. 1. Agents superior to trimetrexate and piritrexim against Toxoplasma gondii and Pneumocystis carinii in in vitro evaluations. AB - 2,4-Diaminopteridines (21 compounds) and 2,4-diamino-5-methyl-5-deazapteridines (34 compounds) along with three 2,4-diamino-5-unsubstituted-5-deazapteridines and four 2,4-diaminoquinazolines, each with an aryl groups attached to the 6-position of the heterocyclic moiety through a two-atom bridge (either CH2NH, CH2N(CH3),CH2S, or CH2CH2), were synthesized and evaluated as inhibitors of the growth of Toxoplasma gondii in culture and as inhibitors of dihydrofolate reductase enzymes from T. gondii, Pneumocystis carinii, and rat liver. Exceptionally high levels of combined potency and selectivity as growth inhibitors of T. gondii and as inhibitors of the microbial enzymes relative to the mammalian enzyme were found among the 5-methyl-5-deazapteridines but not for the other heterocyclic types. Thirty of the 34 5-methyl-5-deaza compounds gave growth inhibition IC50 values lower than that of pyrimethamine (0.4 microM) with 14 compounds below 0.1 microM, values that compare favorably with those for piritrexim and trimetrexate (both near 0.02 microM). As inhibitors of T gondii DHFR, all but three of the 34 5-methyl-5-deaza compounds gave IC50 values in the order of magnitude with those of piritrexim (0.017 microM) and trimetrexate (0.010 microM), and 17 compounds of this group gave IC50 values versus P. carinii DHFR similarly comparable with those of piritrexim (0.031 microM) and trimetrexate (0.042 microM). Thirteen of these congeners gave both T. gondii growth inhibition and DHFR inhibition IC50 values of 0.10 microM or less, thus indicating facile penetration of the cell membrane. Eleven of these inhibitors of both T. gondii growth and DHFR have selectivity ratios (IC50 rat liver divided by IC50 T. gondii) of 5 or greater for the parasite DHFR. The highest selectivity ratio of nearly 100 belongs to the 5-methyl-5-deaza compound whose 6-substituent is CH2CH2C6H3(OCH3)2-2,5. This compound is over 10(3)-fold more selective for T. gondii DHFR than bridge homologue piritrexim (selectivity ratio 0.088), a compound now in clinical trials. The candidate with CH2NHC6H3(CH3)2-2,5 in the 6 position gave the highest P. carinii DHFR selectivity ratio of 4.0, which is about 60-fold more selective than trimetrexate (0.071) and 80-fold more selective than piritrexim (0.048) toward this enzyme. The 10 best compounds with respect to potency and selectivity includes six compounds bearing 2,5-disubstituted phenyl groups in the side chain (with little, if any, difference in effects of methyl, methoxy, or ethoxy), two side chains bearing 1-naphthyl groups, and two with 5,6,7,8-tetrahydro-1-naphthyl groups. Bridge groups represented in the 10 choice compounds are CH2NH, CH2N(CH3), CH2CH2, and CH2S. The high levels of both potency and selectivity among these agents suggest that in vivo studies now underway may lead to agents that could replace trimetrexate and piritrexim in treatment of toxoplasmosis and P. carinii pneumonia. PMID- 8632435 TI - An NMR, CD, molecular dynamics, and fluorometric study of the conformation of the bradykinin antagonist B-9340 in water and in aqueous micellar solutions. AB - A detailed NMR, CD, fluorometry, and molecular modeling study of a novel bradykinin antagonist B-9340, containing a novel amino acid D-Igl (alpha-(2 indanyl)glycine) at position 7, was carried out. The sequence of B-9340 is D-Arg0 Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D- Igl7-Oic8-Arg9, where Hyp is hydroxyproline, Thi is beta-(2-thienyl)alanine, and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid. The CD results exhibit a striking effect of SDS on the spectrum of the BK antagonist, indicating that interaction with the surfactant induces a folded peptide structure. The interaction of this antagonist with phosphatidylinositol was monitored by fluorometry, indicating that the interaction of the peptide with the lipid is cooperative, and gives a Hill coefficient of 2.3. The two dimensional proton NMR measurements indicate that B-9340 has no stable secondary structure in water solution and contains about 10-15% cis peptide bonds arising from Pro2, Hyp3, and Oic8. In SDS micelles, NMR reveals the existence of two beta turns based on a number of medium-range connectivities that were useful for molecular modeling. The actual molecular modeling and dynamic runs were performed on B-9340 in an environment consisting of a layer of octyl sulfate anions and water. Ther results indicate that the structure of B-9340 in a micellar environment is characterized by a nonideal betaII-turn comprising residues Pro2 to Thi5, a nonideal betaII'-turn comprising residues Ser6-Arg9, and broad folding in the middle part of the molecule. The structure is stabilized by several hydrogen bonds and by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9, whereas the middle part of the peptide is buried in the micelle. The structure is deposited as Brookhaven PDB file 1 BDK. PMID- 8632436 TI - Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases. Synthesis, biological activity, mechanism of action, and computer aided studies. AB - With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones. Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H furo[2,3-h]quinolin-2-one (10). These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism. The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration. In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8. The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9 olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA. In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored. These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark. They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy. Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP. On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases. PMID- 8632438 TI - Synthesis and biological evaluation of N-acetylneuraminic acid-based rotavirus inhibitors. AB - Rotavirus can cause severe gastrointestinal disease, especially in infants and young children, and is particularly prevalent in Third-World countries. Therefore, the development of potential inhibitors of this virus is of great interest. The present study describes the synthesis and in vitro biological evaluation of a number of N-acetylneuraminic acid-based compounds as potential rotavirus inhibitors. Our data suggests that it is indeed possible to inhibit adhesion of the virus, and hence in vitro replication, with carbohydrate-based molecules, although this inhibition does appear to be strain dependent. PMID- 8632437 TI - Synthesis, chromatographic resolution, and anti-human immunodeficiency virus activity of (+/-)-calanolide A and its enantiomers. AB - The anti-HIV agent (+/-)-calanolide A (1) has been synthesized in a five-step approach starting with phloroglucinol [-->5-->6-->11-->18-->(+/-)-1], which includes Pechmann reaction, Friedel-Crafts acylation, chromenylation with 4,4 dimethoxy-2-methylbutan-2-ol, cyclization, and Luche reduction. Cyclization of chromene 11 to chromanone 18 was achieved by employing either acetaldehyde diethyl acetal or paraldehyde in the presence of trifluoroacetic acid and pyridine or PPTS. Luche reduction of chromanone 18 at lower temperature preferably yielded (+/-)-1. Reduction of chromone 12, synthesized by Kostanecki Robinson reaction from chromene 11, failed to afford (+/-)-1. The synthetic (+/-) 1 has been chromatographically resolved into its optically active forms, (+)- and (-)-1. The anti-HIV activities for synthetic (+/-)-1, as well as resultant (+)- and (-)-1, have been determined. Only (+)-1 accounted for anti-HIV activity, which was similar to the data reported for the natural product, and (-)-1 was inactive. PMID- 8632439 TI - Synthesis and antiviral activity of pyranosylphosphonic acid nucleotide analogues. AB - Pyranosyl nucleotide analogues have been designed so that the intramolecular base to phosphorus distance closely approximates that of natural nucleotides. This was achieved by attaching the phosphorus directly at the anomeric position and the base at the 4-position of the carbohydrate. A series of compounds incorporating natural bases and having this novel structure were made via a short synthesis starting from commercially available glycals. Addition of triisopropyl phosphite to the glycals furnished alpha- and beta-2-enopyranosylphosphonates which were then substituted with the heterocycle using Mitsunobu chemistry. Deprotection afforded the 2',3'-unsaturated isonucleotide analogue. In some cases the deprotection sequence induced double-bond migration leading to the 1',2' unsaturated derivative. NMR spectroscopic structural analysis established an axial preference for the base and an equatorial preference for the beta phosphorus which results in intramolecular base to phosphorus distances within 1 A of that of natural nucleotides. All of the deprotected compounds were screened for inhibition of HCMV, HSV-2, and HIV replication. Several compounds inhibited HCMV and HSV-2, the most potent of which was the unsaturated cytosine analogue 18 (HCMV IC50 = 10 microM, HSV-2 IC50 = 85 microM). None of the compounds were cytotoxic at the highest dose (1 mM) tested. None of the compounds were inhibitory to HIV. PMID- 8632440 TI - Novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonists: synthesis and structure-activity relationships of 6-(1H-imidazol-1-yl)-7-nitro 2,3(1H,4H)-pyrido[2,3-b]pyrazinedione and related compounds. AB - We have synthesized and evaluated azaquinoxalinediones 3a-c for their activity in inhibiting [3H]AMPA binding from rat whole brain. It was found that the azaquinoxalinedione nucleus functions as a bioisostere for quinoxalinedione in AMPA receptor binding. The detailed structure-activity relationships of 6- and/or 7-substituted 2,3(1H,4H)-pyrido[2,3-b]pyrazinedione derivatives 4, 7-1-, 13, 15 and 16 showed some differences in comparison with those of the corresponding substituted quinoxalinediones, including 6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H) quinoxalinedione (1) (YM90K). The X-ray study exhibited that conformation of the 7-nitro group of 1.HCl was nearly coplanar with the quinoxaline ring, whereas the 6-imidazol-1-yl group was rotated with respect to the aromatic ring. From the glycine site on NMDA receptor binding study, it is indicated that bulkiness of 6 substituents on pyridopyrazinediones may be responsible for the selectivity against the glycine site. Among the series of azaquinoxalinediones, 6-(1H imidazol-1-yl)-7-nitro-2,3(1H,4H)-pyrido[2,3-b]pyrazinedione (8c) exhibited a combination of the best affinity to AMPA receptors with a Ki value of 0.14 microM and selectivity against the glycine site (no affinity at 10 microM). In vivo, 8c also protected against sound-induced seizure in DBA/2 mice (minimum effective dose, 10 mg/kg ip). PMID- 8632441 TI - Studies on selectin blocker. 1. Structure-activity relationships of sialyl Lewis X analogs. AB - As part of our studies of selectin blockers, we prepared 1-deoxy-3'-O-sulfo LeX analogs (1-3), 1-deoxy-3'-O-phosphono LeX analogs (4), and 1-deoxy sLeX analogs (5-7), and examined their inhibitory activities against natural ligand (sLeX) binding to E-selectin, P-selectin, and L-selectin. The 1-deoxy sLeX 5 was up to 20 times more potent an inhibitor than the sLeX tetrasaccharide toward P- and L selectin binding. This indicates that the modification of the 1 or 2 position of sLeX is useful in the design of a more potent selectin blocker. PMID- 8632443 TI - Electron cryo-microscopy of vitrified bulk biological specimens: ideal and real structures of water-lipid phases. AB - Lipid-water mixtures were studied by X-ray cryo-diffraction in order to assess the structural changes during freezing. We show that the water of aqueous lipid phases, in the concentration range of 10-30% (water weight/total weight), is vitrified by high-pressure freezing. Vitrified lipid phases can be cryo-sectioned and imaged by electron cryo-microscopy. Both the ideal or average and the real or local structures of the lipid mixtures can be studied at a resolution better than 2 nm. While the average structure of the lipid phases is in good agreement with that determined by X-ray diffraction, the local structure reveals features that might play an important role in the function of biological membranes such as in endo- and exocytosis. PMID- 8632442 TI - Probing nuclear ultrastructure by electron spectroscopic imaging. AB - Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20-30 nm. This complexity hinders the study of structure-function relationships within the mammalian nucleus. Element-specific mapping using the energy-filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of strains relative to unstained material when biochemical-specific staining protocols, such as EDTA-regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogenous. ESI shows substantial promise for the investigation of structure-function relationships in biological specimens. PMID- 8632444 TI - Morphological studies of pseudowollastonite for biomedical application. AB - Pseudowollastonite ceramic (psW) composed of CaO.SiO2 was found to be bioactive in a simulated body fluid environment. The chemical reaction initiated at the material surface resulted in hydroxyapatite (HA) formation. These bone-bonding properties are essential for securing the necessary physico-chemical integration of the material with living bone. Materials behaving in this way can be considered for potential biomedical application as bone tissue substitute for a natural bone repair or replacement as implant. A mechanism of hydroxyapatite formation on pseudowollastonite ceramics surface was investigated during exposure to a stimulated body fluid (SBF) for a period of 3 weeks. Morphology and structure of the surface product and its original substrate was examined by thin film X-ray diffraction, scanning electron microscopy and high-resolution transmission electron microscopy. HA crystals were found to form on an amorphous silica intermediate layer. (100) lattice planes of HA were resolved and identified. Concentration of ions in the SBF and pH of the SBF were monitored throughout the exposure. Additional pH measurements were made at the interface of psW with SBF. The HA formation occurred when there was a sudden increase of pH from 7.25 to 10.5 at the interface of psW with SBF as a result of ionic exchange between 2H+ and Ca2+ within the psW network. This ionic exchange transformed the psW crystals into an amorphous silica phase. The appropriate pH and the ion concentrations were essential for partial dissolution of the amorphous silica phase and subsequent precipitation of a Ca-P rich phase which then transformed to HA. PMID- 8632445 TI - Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy. AB - Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image. PMID- 8632446 TI - Fluorescent porphyrin counterstaining of chromatin DNA in conjunction with immunofluorescence methods using FITC-labelled antibodies. AB - This work describes the use of two cationic porphyrins for fluorescent DNA counterstaining of HeLa cells also stained by means of indirect immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Staining HeLa cells with meso-tetra(4-N-methylpyridyl)porphine (T4MPyP) and meso-tetra(p-N-trimethylanilinium)-porphine (TMAP), both used at 5 x 10(-6) M, gives rise to a deep red emission of chromatin from interphase nuclei and mitotic chromosomes when the cells are excited with blue (490 nm) light. The red-fluorescing chromatin contrasted very well with the yellowish-green emission from FITC-immunofluorescent staining. No significant difference in chromatin fluorescence found with either T4MPyP or TMAP was detected. Counterstaining with the porphyrins could be carried out as a separate step after immunolabeling or, more simply, by their inclusion in the mounting medium. Spectral analysis demonstrated that the fluorescent emission maximum of T4MPyP was at 665 nm and that the intensity of the fluorescent emission showed a considerable increase in the presence of DNA. PMID- 8632447 TI - Dispersion, aberration and deconvolution in multi-wavelength fluorescence images. AB - The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image. PMID- 8632448 TI - Alternative arrangements of the protein chain are possible for the adenovirus single-stranded DNA binding protein. AB - A second crystal form of the C-terminal domain of the adenovirus single-stranded DNA binding protein crystallizes in space group P2(1)2(1)2(1) with a=61.0 angstrom, b=91.2 angstrom and c=149.4 angstrom. The crystals contain two molecules per asymmetric unit and diffract to a maximum resolution of 3.0 angstrom. The crystal is composed of infinite chains of molecules along the crystallographic 2(1) axis parallel to c. The principal intermolecular interaction is a hooking of the C-terminal 17 residues of one molecule onto the next molecule in the protein chain. Adjacent molecules in the chain are rotated approximately 90 degrees with respect to their neighbours. The difference in relative orientation of adjacent molecules between the two crystal forms of the protein implies a degree of flexibility in the protein chain that would facilitate DNA binding. PMID- 8632449 TI - Structure, interactions and dynamics of PRD1 virus II. Organization of the viral membrane and DNA. AB - Structure, dynamics and stability of the membrane and double-stranded (ds) DNA genome packaged within the native PRD1 virion have been probed by laser Raman spectroscopy. The Raman signature of PRD1 is complex, but exhibits distinctive marker bands diagnostic of the internal lipid bilayer and dsDNA. The Raman markers demonstrate, respectively, a liquid crystalline lipid phase(L(alpha)) and B DNA conformation throughout the temperature range (5 degrees to 50 degrees) of virion stability. Despite the absence of large scale lipid phase transitions or DNA melting, small temperature-dependent changes in the Raman markers of lipid and DNA are detected, indicating coupling between their structures. Minor deviations of DNA from the canonical B form are imposed by the membrane. The Raman markers indicate further that base stacking and phosphate group interactions of the packaged PRD1 genome differ from those of unpackaged PRD1 DNA. Specific Raman band perturbations are proposed as indicators of DNA-membrane interaction. Hydrogen-deuterium exchange kinetics of packaged and unpackaged PRD1 DNA are indistinguishable, demonstrating that base imino and amino protons are not affected significantly by either the condensation or membrane enclosure associated with DNA packaging. This contrasts with the significant acceleration of base exchanges detected in the packaged DNA of bacteriophage P22, which lacks a viral membrane. The distinctive H-->2H exchange profile of the PRD1 genome, the absence of packaging-induced acceleration of exchange kinetics and the apparent direct interaction between DNA and phospholipids suggest a specific role for the viral membrane in PRD1 assembly. We propose a "membrane-surface-catalyzed" model for dsDNA condensation and organization within the PRD1 virion. PMID- 8632450 TI - The three-dimensional structure of two mutants of the signal transduction protein CheY suggest its molecular activation mechanism. AB - The three-dimensional crystal structures of the single mutant M17G and the triple mutant F14G-S15G-M17G of the response regulator protein CheY have been determined to 2.3 and 1.9 angstrom, respectively. Both mutants bind the essential Mg2+ cation as determined by the changes in stability, but binding does not cause the intrinsic fluorescence quenching of W58 observed in the wild-type protein. The loop beta4-alpha4 appears to be very flexible in both mutants and helix alpha4, which starts at N94 in the native Mg2+-CheY and at K91 in the native apo-CheY, starts in both mutants at residue K92. The side-chain of K109 appears to be more mobile because of the space freed by the M17G mutation. In the triple mutant the main chain of K109 and adjacent residues (loop beta5-alpha5) is displaced almost by 2 angstrom affecting the main chain at residues T87 to E89 (C terminus of beta4). The triple mutant structure has a Mg2+ bound at the active site, but although the Mg2+ coordination is similar to that of the native Mg2+-CheY, the structural consequences of the metal binding are quite different. It seems that the mutations have disrupted the mechanism of movement transmission observed in the native protein. We suggest that the side-chain of K109, packed between V86, A88 and M17 in the native protein, slides forwards and backwards upon activation and deactivation dragging the main chain at the loop beta5-alpha5 and triggering larger movements at the functional surface of the protein. PMID- 8632451 TI - Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. AB - The delta-endotoxin CytB, found in parasporal inclusions of Bacillus thuringiensis subspecies kyushuensis, is a membrane pore-forming protein which is lethal to the larvae of Dipteran insects and broadly cytolytic in vitro. The crystal structure of CytB in the protoxin form has been determined by isomorphous replacement using heavy-atom derivatives of both the wild-type protein and an engineered cysteine mutant. The atomic model comprising residues 19 to 245 and 28 bound water molecules has been refined at 2.6 angstrom resolution to a crystallographic R-factor of 19.7% and a free R-factor of 26.1%. CytB has a single domain of alpha/beta architecture but a novel connectivity comprising two outer layers of alpha-helix hairpins wrapped around a mixed beta-sheet. In the protoxin form, CytB is a dimer linked by the intertwined N-terminal strands in a continuous, 12-stranded beta-sheet. Proteolytic processing cleaves the intertwined beta-strands to release the active CytB as a monomer, as well as removing the C-terminal tail to uncover the three-layered core. The homologous toxin CytA should show the same fold. Mutations in CytA that inhibit expression map to the dimer contacts and to the tip of helix pair A-B in contact with the sheet, apparently preventing correct folding. Mutations that inhibit toxicity map to the edge of the beta-sheet adjoining the helix pair C-D and to the sheet face, while mutations on the helix surfaces have no effect. Therefore segments forming the sheet, rather than the amphiphilic but short helices, are responsible for membrane binding and pore formation. A conformational change is postulated by which the helix pair C-D peels away from the sheet to lie on the membrane surface, while the sheet region rearranges to form an oligomeric trans-membrane pore. PMID- 8632453 TI - Implications of N and C-terminal proximity for protein folding. AB - We have investigated the proximity of the N and C termini in protein structures, and developed a model to test the theoretical possibility that proteins fold with their termini closely associated. On average, the distance between the termini is not significantly different from what would be expected based on chance. However, the theoretical model indicated that it is possible to greatly decrease the N-to C terminal distance by allowing small (approximately six amino acid residues) solvent-accessible terminal fragments to move. Subsequent to this distance minimization method, more than 90% of the proteins studied had smaller-than expected N-to-C distances, but only minor structural modification. PMID- 8632452 TI - Structure of the cysteine-rich intestinal protein, CRIP. AB - LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha helix appears to be packed more tightly against the preceding antiparallel beta sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif. PMID- 8632454 TI - Comparison of the (30-51, 14-38) two-disulphide folding intermediates of the homologous proteins dendrotoxin K and bovine pancreatic trypsin inhibitor by two dimensional 1H nuclear magnetic resonance. AB - The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each intermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bonds. This is thought to be a consequence of the extreme stability of the native BPTI conformation. The current study addresses the question of whether the native-like conformation would be populated significantly at the two-disulphide stage in disulphide refolding if the final structure is less stable than in the case of BPTI. Dendrotoxin K from black mamba venom provides a good model to test this, since it contains the BPTI fold and was shown to fold predominantly via the same pathway, but its native conformation is stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refolding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and disulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respectively, has been determined by 1H NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. The analysis reveals that the dendrotoxin K intermediate adopts a partly-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-folded conformation of the BPTI intermediate with just the Cys30-Cys-51 disulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native conformation of the dendrotoxin K intermediate, relative to BPTI, appears to reduce the cooperativeity of the folding process. PMID- 8632455 TI - Local conformational signals and the statistical thermodynamics of collapsed helical proteins. AB - We investigate the role that local conformational tendencies can have in guiding the folding of helical proteins, using simple statistical mechanical models. The theory provides a synthesis of classical models of the helix-coil transition in polymers with an approximate treatment of the effects of excluded volume, confinement, and packing alignment of the helices based on a free energy function. The theory studies the consequences of signals encoded locally in the sequence as stabilization energies associated with three types of local structure: native helical conformations, native non-helical conformations, and native helix caps or start-stop signals. The role of randomness in the energies of conformations due to tertiary interactions is also studied vis-a-vis the difficulty of conformational search. The thermal behavior of the model is presented for realistic values of the conformational signal energies, which can be estimated from experimental studies on peptide fragments. Estimates are made for the relative contribution of local signals and specific tertiary interactions to the folding stability gap. PMID- 8632456 TI - Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. AB - The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP independent lac UV5 promoter by O1 at its natural position increased up to 50 fold in the presence of an optimally positioned auxiliary O(id)). Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo. Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor. PMID- 8632457 TI - Linker histone-dependent DNA structure in linear mononucleosomes. AB - We have examined the binding of the linker histone H5 (LH) to mononucleosomes. Mononucleosomes reconstituted on short DNA fragments display a series of discrete bands on a gel corresponding to various nucleosome positions along the DNA. When a series of engineered H5s with differing extents of the C-terminal tail are bound to these mononucleosomes, the electrophoretic mobilities of the resulting complexes are altered. Not only is there a general increase in mobility upon complex formation, but there is a reduction in the differences in mobility of the most distal nucleosomes. The complexes were also visualized by electronmicroscopy. From these two complementary studies, we conclude the following. (1) Entering and exiting DNAs are uncrossed in the LH-free particles, despite a DNA wrapping of 1.65 to 1.7 turns around the histone core. This results from a bending of the entering and exiting DNA away from each other and the histone surface, presumably as a consequence of electrostatic repulsion. This confirms and extends conclusions derived from our recent examination of the same particles in 3D through cryo-electron microscopy. (2) Binding of the globular domain of H5 increases DNA wrapping to 1.8 to 1.9 turns, but fails to induce a crossing due to an accentuation of the bends. (3) The C-terminal tail of H5 bridges entering and exiting DNAs together into a four-stranded stem over a distance of about 30 bp. The occurrence of such a stem may introduce constraints on models of the 30 nm chromatin fiber. PMID- 8632459 TI - Topological complexity of SV40 minichromosomes. AB - During attempts to measure the extent to which the proteins of simian virus 40 (SV40) minichromosomes restrain the ability of SV40 DNA to alter its twist in response to temperature changes, we found that temperature-shift-induced linking number changes are not reversible for isolated minichromosomes, suggesting that such changes, both in isolated minichromosomes and in cells, may be a consequence of structural alterations in chromatin proteins rather than of simple changes in DNA twist. We also found that the SV40 minichromosome pool is composed of subpopulations that display different responses to temperature shifts. For example, the linking number of DNA in newly replicated minichromosomes is more responsive to in vivo temperature changes than is the linking number of DNA in bulk minichromosomes. In addition, the linking number profiles of both isolated and intracellular minichromosomes change during the course of infection. These observations emphasize the topological complexity of SV40 minichromosomes and encourage caution in the interpretation of experiments carried out on bulk minichromosomes. PMID- 8632458 TI - RNase P from a photosynthetic organelle contains an RNA homologous to the cyanobacterial counterpart. AB - RNase P is a ribonucleoprotein enzyme in all organisms and organelles investigated so far, with the exception of chloroplasts where no enzyme associated RNA has been detected to date. As an approach to answer the question whether an RNA component is present in RNase P from photosynthetic organelles, we have used a phylogenetically oriented strategy and searched for RNase P RNA in a postulated intermediate in plastid evolution, the cyanelle of Cyanophora paradoxa. We have detected a 351 nucleotide long RNA similar to cyanobacterial RNase P RNAs, with a proposed secondary structure that closely resembles a bacterial consensus. The RNA is encoded on the cyanelle genome and copurifies with enzyme activity. The RNA is not catalytically active by itself, but the activity of the cyanelle RNase P holoenzyme is destroyed by nuclease treatment, indicating an essential role of the RNA. Hence cyanelle RNase P, combining properties of bacterial and eukaryotic enzymes, occupies an intermediate position in RNA enzyme evolution. The first description of an RNA component in RNase P from a photosynthetic organelle might thus be an important step towards an understanding of plastid RNase P structure and function. PMID- 8632460 TI - Weak and strong states of kinesin and ncd. AB - Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.003 micro M, K(ncd)d approximately 0.24 micro M), whilst the ADp state was the weakest binding (K(kin)d approximately 11.32 micro M, K(ncd)d approximately 12.02 micro M). In ATP, the motor. ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3 thiotriphosphate) there is a shift towards tighter binding (K(kin)d approximately 4.23 micro M, K(ncd)d approximately 2.34 micro M), consistent with a tight binding motor. ATP-like state being enriched. In the presence of non-hydrolysable analogue beta,gamma-imidoadenosine 5'-triphosphate the binding is still tighter (K(kin)d approximately <0.27 micro M, K(ncd)d approximately 0.21 micro M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesis and ncd switch from weak to strong binding via ADP release, and back again via ADP trapping. In a two-state model, the transition from weak to strong binding is force-generating. PMID- 8632461 TI - Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis. AB - Although both light chain amyloidosis (AL) and deposition disease (LCDD) involve the aggregation of light chain V(L) domains into highly insoluble deposits, the factors which determine both disease onset and type (amyloid fibrils (AL) or granular deposits (LCDD)) are not clear. Previously, we showed that the AL associated replacement Arg61 --> Asn, introduced as a point mutation into the kappa V(L) domain REI, greatly destabilizes the domain and renders it susceptible to the formation of ordered, fibril-like aggregates in vitro. The importance of Arg61 for stability may be due to the role of this residue in making a key, conserved salt bridge with Asp82 located on an adjacent loop. Here we show that an Asp82 --> Ile replacement, recently identified in a V(L) associated with LCDD, also highly destabilizes REI as a point mutation and makes it susceptible to in vitro aggregate formation. The D82I aggregate, however, is dramatically different in morphology from aggregates obtained from amyloid-associated mutants, suggesting that specific amino acid residue changes can control not only the onset of aggregation disease but also aggregate morphology and disease type. PMID- 8632462 TI - Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly. AB - Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit. PMID- 8632463 TI - Identification of functional regions of the Nun transcription termination protein of phage HK022 and the N antitermination protein of phage lambda using hybrid nun N genes. AB - Phages lambda and HK022 express proteins N and Nun, respectively, each of which acts with a number of Escherichia coli host Nus factors at lambda NUT RNA sites, to influence transcription elongation. The lambda nut sites, nearly identical sequences located downstream of the early promoters, pL and pR, were first identified as cis-acting signals required for the action of N in forming termination-resistant transcription complexes. Surprisingly, the Nun protein, resembling N and expressed by another lambdoid phage, HK022, also acts with Nus proteins to terminate specifically transcription initiating at pL and pR near the lambda nut sites. Based on structural considerations of the amino acid sequences, we have constructed nine hybrid N-nun genes and used these hybrids to identify functional regions of the N and Nun proteins. Three classes of hybrid gene products were identified: (1) those that, like N, support antitermination, (2) those that, like Nun, terminate transcription, and (3) those that block N action but do not terminate transcription. We find that, similar to N, the amino terminal portion of Nun is involved in RNA recognition. The more carboxy portions influence transcription elongation, antitermination (N) and termination (Nun). Depending on the derivations of the more carboxy regions, hybrids with either the N or Nun amino portions support either termination or antitermination. The activity of a hybrid protein may be influenced by the host strain depending on the nature of the rpoC locus, a locus encoding the beta' subunit of RNA polymerase. One of the hybrid proteins blocks antitermination when the rpoC locus is wild-type. The same hybrid in the presence of the rpoC100 mutation, which alters the beta' subunit, has antitermination activity. This result supports the argument that the beta' subunit plays an essential role in determining the progress of transcription elongation. PMID- 8632464 TI - The Agaricus bisporus hypA gene encodes a hydrophobin and specifically accumulates in peel tissue of mushroom caps during fruit body development. AB - Differential screening of a cDNA library was used to clone genes that are specifically expressed during mushroom development in the basidiomycete Agaricus bisporus. One of the isolated genes encodes a polypeptide of 112 amino acid residues and belongs to the fungal gene family encoding hydrophobins. This gene, hypA, has the characteristic pattern of eight cysteine residues at conserved positions and a hydrophobicity pattern that is very similar to class I hydrophobins. Elucidation of the genomic structure of hypA led to the identification of a second copy, hypC, located downstream of hypA. Although at a much lower level, hypC is like hypA specifically expressed on fruit bodies. The hypA mRNA level is transiently increased ten days after fruit body induction and expression appears to be associated with rapid expansion of the mushroom caps. In mushroom caps, very high concentrations of hypA messengers were found in the (outer) peel tissue, where they accumulate to more than 60% of the total mRNA mass. The corresponding protein with a molecular mass of 8 to 9 kDa was purified from this peel tissue and was identified by N-terminal sequencing. Our results suggest that HYPA forms a protective hydrophobic layer instrumental in cap formation. PMID- 8632465 TI - Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii. AB - We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex. Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence. Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cox1 termination codon is created by post transcriptional RNA processing or editing. Western analysis revealed an A. castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria. These observations indicate that although A. castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 "fusion" protein. Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region. Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A. castellanii COX2 may undergo additional post-translational processing to its final form. PMID- 8632466 TI - Hyperthermostable surface layer protein tetrabrachion from the archaebacterium Staphylothermus marinus: evidence for the presence of a right-handed coiled coil derived from the primary structure. AB - The scaffold of the surface layer covering the hyperthermophilic archaebacterium Staphylothermus marinus is formed by an extended filiform glycoprotein complex, tetrabrachion, which is anchored in the cell membrane at one end of a 70 nm stalk and branches at the other end into four arms of 24 nm length. The arms from a canopy-like meshwork by end-to-end contacts, enclosing a "quasi-periplasmic space". The primary structure of the complex, obtained by an approach based entirely on the polymerase chain reaction, shows that the light and the heavy chains are encoded in this order in a single gene and are generated by internal proteolytic cleavage. One light chain associates with the N-terminal part of a heavy chain to form one of the four arms of the complex, comprising about 1000 residues. Following a glycine-rich linker of about ten residues, the C-terminal 500 residues of the four heavy chains converge to form a four-stranded parallel coiled coil, which ends in a transmembrane segment. The sequence of the coiled coil is exceptional in that the heptad repeat of hydrophobic residues typical for left-handed coiled coils shifts to an undecad repeat after an internal proline residue, indicating that the C-terminal part of the sequence forms a right-handed coiled coil. Such a periodicity has not been detected in coiled coils to date. The almost flawless pattern of aliphatic residues, mainly leucine and isoleucine, throughout the hydrophobic core of the stalk provide one explanation for its exceptional stability. PMID- 8632467 TI - The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism. AB - The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively. The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture. PMID- 8632468 TI - Solution structure of a pyrimidine-purine-pyrimidine triplex containing the sequence-specific intercalating non-natural base D3. AB - We have used NMR spectroscopy to study a pyrimidine-purine-pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3 benzamido)phenylimidazole (D3), in the third strand. The D3 base has been previously shown to specifically recognize T-A and C-G base-pairs via intercalation on the 3' side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D3 base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D3 base stack on the bases of all three strands and mimic a triplet. The conformation of the D3 base and its sequence specificity are discussed. PMID- 8632469 TI - Analysis of the low frequency normal modes of the T-state of aspartate transcarbamylase. AB - Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed. PMID- 8632471 TI - Stability changes upon mutation of solvent-accessible residues in proteins evaluated by database-derived potentials. AB - The stability changes in peptides and proteins caused by the substitution of a single amino acid, which can be measured experimentally by the change in folding free energy, are evaluated here using effective potentials derived from known protein structures. The analysis is focused on mutations of residues that are accessible to the solvent. These represent in total 106 mutations, introduced at different sites in barnase, bacteriophage T4 lysozyme and chymotrypsin inhibitor 2, and in a synthetic helical peptide. Assuming that the mutations do not modify the backbone structure, the changes in folding free energies are computed using various types of database-derived potentials and are compared with the measured ones. Distance-dependent residue-residue potentials are found to be inadequate for estimating the stability changes caused by these mutations, as they are dominated by hydrophobic interactions, which do not play an essential role at the protein surface. On the contrary, the potentials based on backbone torsion angle propensities yield quite good results. Indeed, for a subset of 96 out of the 106 mutations, the computed and measured changes in folding free energy correlate with a linear correlation coefficient of 0.87. Moreover, the ten mutations that are excluded from the correlation either seem to cause modifications of the backbone structure or to involve strong hydrophobic interactions, which are atypical for solvent-accessible residues. We find furthermore that raising the ionic strength of the solvent used for measuring the changes in folding free energies improves the correlation, as it tends to mask the electrostatic interactions. When adding to these 106 mutations 44 mutations performed in staphylococcal nuclease and chemotactic protein, which were first discarded because some of them were suspected to affect the backbone conformation or the denatured state, the correlation between measured and computed folding free energy changes remains quite good: the correlation coefficient is 0.86 for 135 out of the 150 mutations. The success of the backbone torsion potentials in predicting stability changes indicates that the approximations made for deriving these potentials are adequate. It suggests moreover that the local interactions along the chain dominate at the protein surface. PMID- 8632470 TI - Characterization of the electrostatic perturbation of a catalytic site (Cys)-S /(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants. AB - We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2' dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2' dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S /(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2 pyridyl disulfide reactivity probe and the enzyme active centre region. PMID- 8632472 TI - Crystal structure of the complex of the variable domain of antibody D1.3 and turkey egg white lysozyme: a novel conformational change in antibody CDR-L3 selects for antigen. AB - The crystal structure of the Fv fragment of the murine monoclonal anti-lysozyme antibody D1.3, complexed with turkey egg-white lysozyme (TEL), is presented. D1.3 (IgG1, kappa) is a secondary response antibody specific for hen egg-white lysozyme (HEL). TEL and HEL are homologous and differ in amino acid sequence in the antibody-antigen interface only at position 121. The side-chain of HEL residue Gln121 makes a pair of hydrogen bonds to main-chain atoms of the antibody light chain. In the D1.3-TEL structure, TEL residue His121 makes only one hydrogen bond with the light chain as a result of 129 degree and 145 degree change in peptide torsion angles for residues Trp92 and Ser93. Probably as a consequence of this conformational change, the D1.3-TEL association occurs at a much slower rate than the D1.3-HEL association. The D1.3-TEL complex is destabilized with respect to the D1.3-HEL interaction by the loss of two hydrogen bonds, exclusively due to the substitution of histidine for glutamine. While antibodies of secondary responses are indeed highly specific for antigen, this work demonstrates that by undergoing subtle conformational change antibodies can still recognize mutated protein antigens, albeit at a cost to affinity. PMID- 8632473 TI - Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor Rho. AB - The function of transcription termination factor Rho from Escherichia coli is dependent upon its ability to bind to specific sites on nascent RNA molecules. The roles of 19 individual amino acid residues (Ile49 to Ser67) in and near a phylogenetically conserved sequence segment of Rho that is similar to the RNP1 motif found in many RNA-binding proteins were examined by testing the phenotypic consequences of mutational changes that were introduced into rho by a random sequence cassette mutagenesis procedure. The tests of each mutant included the ability of the cells to survive at 42 degrees C in the absence of wild-type rho, the efficiency of termination at a Rho-dependent terminator (lambdatR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA. The results revealed that residues in the RNP1-like sequence of DGFGFLR (residues 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were particularly sensitive to mutation: Asp60, Phe62 and Arg66. The properties of the mutants are consistent with a secondary structure model, derived from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 and Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 accessible on the same face of the beta-structure for interaction with RNA. PMID- 8632474 TI - Residues in the RNP1-like sequence motif of Rho protein are involved in RNA binding affinity and discrimination. AB - The termination of transcription in Escherichia coli by action of Rho factor is dependent on the ability of this homohexameric protein to make productive interactions with the nascent RNA molecule to be terminated. The roles of two residues in a phylogenetically conserved sequence motif in the RNA-binding domain of Rho, Asp60 and Phe62, were analyzed by studies of the biochemical properties of pure mutant proteins. F62S Rho had greatly reduced affinity for lambda cro RNA, very poor ability to terminate transcription in vitro by itself and only partial termination activity (at a level consistent with its in vivo defect) in the presence of NusG. D60G Rho had a high affinity for lambda cro RNA but a much lower ability to discriminate against RNA molecules lacking cis-acting Rho utilization sequences, and a reduced efficiency of termination that was not improved by NusG. These results indicate a major role for Phe62 in stabilizing the binding of Rho to RNA through hydrophobic interactions, while Asp60 provides an electrostatic repulsive force that allows a rapid dissociation of non productive complexes with RNA. PMID- 8632475 TI - Chromatin structure of the yeast URA3 gene at high resolution provides insight into structure and positioning of nucleosomes in the chromosomal context. AB - To characterize nucleosome structure and positioning in the chromosomal context, the chromatin structure of the whole URA3 gene was studied in the genome and in a minichromosome by testing the accessibility of DNA to micrococcal nuclease and DNase I. The cutting patterns and hence the chromatin structures were almost indistinguishable in the genome and in the minichromosomes. The only notable exception was enhanced cutting between nucleosomes U3/U4 and U4/U5 in the minichromosomes. The results demonstrate that there is no severe constraint acting from outside the URA3 gene in chromosomes and minichromosomes. While low resolution mapping showed six regions with a positioned nucleosome (U1 to U6), each region resolved in a complex pattern consistent with multiple overlapping positions. Some regions (U1, U4, U5 and U6) showed multiple positions with a dominant rotational setting (DNase I pattern), while U2 showed positioning within 10 bp but with no defined rotational setting, demonstrating that nucleosome positions were not in phase and not coordinately regulated. Reduced DNase I cutting from about 50 bp form the 5' end towards 3' end was common to all nucleosome regions. This polarity has been observed on isolated core particles. The results demonstrate that the DNase I pattern observed in vitro indeed reflects a structural property of nucleosomes in the chromosomal context. It is emphasized that despite the local heterogeneity revealed by high-resolution mapping, the low-resolution map is a reasonably accurate representation of the chromatin structure. PMID- 8632477 TI - M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties. AB - A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5 MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence. PMID- 8632476 TI - Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs. AB - We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals. PMID- 8632478 TI - ATPase activity of the type IC restriction-modification system EcoR124II. AB - We have investigated the ATPase activity of the type IC restriction-modification (R-M) system EcoR124II. As with all type I R-M systems EcoR 124II requires ATP hydrolysis to cut DNA. We determined the KM for ATP to be 10(-5) to 10(-4) M. By measuring ATP hydrolysis under different conditions and by simultaneously monitoring DNA restriction, methylation and ATP hydrolysis we propose that the order of events during restriction is: (1) binding of EcoR124II to a non methylated recognition sequence, (2) start of DNA-dependent ATP hydrolysis which continues even after restriction is complete, (3) restriction of DNA, (4) methylation of the product. Non-cleavable DNA substrates, such as recognition site containing oligonucleotides, also support ATP hydrolysis. Methylation can also occur prior to ATP hydrolysis and prevent DNA degradation. PMID- 8632479 TI - Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations. AB - Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG1G2G3-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF monomodified substrates, dissociation of UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication. PMID- 8632480 TI - The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties. AB - In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation. PMID- 8632482 TI - Additional therapy after prostatectomy: implications for patient counseling. PMID- 8632481 TI - Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity. AB - A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex. PMID- 8632483 TI - Assessing p53 status in breast cancer prognosis: where should you put the thermometer if you think your p53 is sick? PMID- 8632484 TI - Gender may render women at risk for lung cancer. PMID- 8632485 TI - Beta carotene fails to prevent cancer in two major studies; CARET intervention stopped. PMID- 8632486 TI - DNA test for bladder cancer hits a high mark. PMID- 8632487 TI - HER-2/neu gene bumped into the limelight. PMID- 8632488 TI - Council giving voice to minority cancer patients. PMID- 8632489 TI - Antibody-enzyme conjugates for cancer therapy. AB - The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed "antibody-directed enzyme prodrug therapy" (ADEPT), has attracted considerable interest since the concept was first described in 1987. Prodrug forms of both clinically used anticancer agents and novel cytotoxic compounds have been developed to take advantage of potential prodrug-generating technology employing a variety of enzymes with widely differing substrate specificities. A particular advantage of the ADEPT approach is that it may allow the use of extremely potent agents such as nitrogen mustards and palytoxin, which are too toxic to be readily used in conventional chemotherapy. Preliminary studies using an antibody-enzyme conjugate constructed with a bacterial enzyme and a murine monoclonal antibody not only have established the value of the ADEPT technique, but also have highlighted the potential problem of immunogenicity of proteins of nonhuman origin. This problem has been tackled in the first instance by the use of immunosuppressive agents, but long-term solutions are being investigated in the development of second-generation ADEPT systems, including the development of human antibody-human enzyme fusion proteins and catalytic antibodies. Such improvements, coupled with further refinement of the prodrug-drug element of the system and the wide variety of antibody-enzyme-drug combinations available, should mean that ADEPT-based approaches will form an important element of the search for the anticancer drugs of the future. PMID- 8632490 TI - Follow-up prostate cancer treatments after radical prostatectomy: a population based study. AB - BACKGROUND: Radical prostatectomy is one of the most commonly used curative procedures for the treatment of localized prostate cancer. The probability that a patient will undergo additional cancer therapy after this procedure is largely unknown. PURPOSE: The objective was to determine the likelihood of additional cancer therapy after radical prostatectomy. METHODS: Data for this study were derived from a linked dataset that combined information from the Surveillance, Epidemiology, and End Results Program and Medicare hospital and physician claims. Records were included in this study if patient histories met the following criteria: (a) residing in Connecticut, Washington (Seattle-Puget Sound), or Georgia (Metropolitan Atlanta); (b) having been diagnosed with prostate cancer during the period from January 1, 1985, through December 31, 1991; (c) undergoing radical prostatectomy by December 31, 1992; and (d) having no evidence of other types of cancer. Patients were considered to have had additional cancer therapy if they had had radiation therapy, orchiectomy, and/or androgen-deprivation therapy by injection after radical prostatectomy. The interval between the initial treatment and any follow-up treatment was calculated from the date of radical prostatectomy to the 1st day of the follow-up cancer therapy. All presented probabilities are based on Kaplan-Meier estimates. RESULTS: The study population consisted of 3494 Medicare patients, 3173 of whom underwent radical prostatectomy within 3 months of prostate cancer diagnosis. Although radical prostatectomy is often reserved for localized cancer, less than 60% (1934) of patients whose records were included in this study had organ-confined disease, according to final surgical pathology. Overall, the 5-year cumulative incidence of having any additional cancer treatment after radical prostatectomy reached 34.9% (95% confidence interval [CI] = 31.5%-38.5%). For patients with pathologically organ-confined cancer, the 5-year cumulative incidence was 24.3% (95% CI = 20.0%-29.3%) overall and ranged from 15.6% (95% CI = 9.7%-24.5%) for well-differentiated cancer (Gleason scores 2-4) to 41.5% (95% CI = 27.9%-58.4%) for poorly differentiated cancer (Gleason scores 8-10). The corresponding figures for pathologically regional cancer were 22.7% (95% CI = 12.0%-40.5%) and 68.1% (95% CI = 58.7%-77.1%). CONCLUSION: Further treatment of prostate cancer was done in about one third of patients who had had a radical prostatectomy with curative intent and in about one quarter of patients who were found to have organ-confined disease. IMPLICATIONS: Given the common requirement for follow-up cancer treatments after radical prostatectomy and the uncertainties about the effectiveness of the various follow-up treatment strategies, further investigation of these treatments is warranted. PMID- 8632492 TI - Differences in lung cancer risk between men and women: examination of the evidence. AB - BACKGROUND: Lung cancer incidence is gradually leveling off in U.S. men but is continuing to rise in U.S. women. This increase in U.S. women exceeds that expected from a slower decline of smoking among women. Recent epidemiologic and biochemical studies suggest gender differences in susceptibility to tobacco carcinogens. PURPOSE: We conducted an up-to-date, more in-depth evaluation of our earlier observation of a potential gender difference in relative risk (RR) of lung cancer due to smoking. We added information from several additional case and control subjects and included more precise histologic classification of the cancer type, accurate quantitation of smoke exposure, and adjustments for body size. METHODS: The present investigation was a part of an ongoing hospital-based, case-control study by the American Health Foundation. It included data from 1889 case subjects (1108 males and 781 females) with lung cancer of squamous/epidermoid, small-cell/oat cell, large-cell, and adenocarcinoma types and 2070 control subjects (1122 males and 948 females) with diseases unrelated to smoking. The case and control subjects were admitted to participating hospitals from 1981 to 1994 and were pair-matched by age, sex, hospital, and the time of hospital admission. Ex-smokers and non-Caucasians were excluded from analyses to avoid confounding. The RRs and 95% confidence intervals were estimated from adjusted odds ratios (ORs) by use of unconditional multiple logistic regression analysis, and statistical significance was determined by two-sided tests. The ORs for major histologic types were estimated at increasing levels of exposure to cigarette smoke. RESULTS: Our results indicated that women were more likely to be never-smokers than men, particularly those with the squamous/epidermoid-type cancer (8.3% for women versus 2.9% for men 55 years old or older). Men started smoking earlier, reported inhaling more deeply, and smoked more cigarettes per day than women. In contrast, dose-response ORs over cumulative exposure to cigarette smoking were 1.2-fold to 1.7-fold higher in women than in men for the three major histologic types; these differences were more pronounced for small cell/oat cell carcinomas and adenocarcinomas than for squamous/epidermoid carcinomas. Adjustments for weight, height, or body mass index did not alter the ORs. CONCLUSIONS: These results confirm our earlier finding that the ORs for major lung cancer types are consistently higher for women than for men at every level of exposure to cigarette smoke. Furthermore, this gender difference cannot be explained by differences in base-line exposure, smoking history, or body size, but it is likely due to the higher susceptibility to tobacco carcinogens in women. PMID- 8632491 TI - The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. AB - BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) have been detected in a wide variety of human cancers. In breast cancer, the presence of p53 gene alterations has been associated with worse prognosis. PURPOSE: We compared a complementary DNA (cDNA)-based sequencing method and an immunohistochemical (IHC) method for their abilities to detect p53 mutations in breast cancer specimens. In addition, we determined the prognostic value of information obtained when these two methods were used. METHODS: Specimens from 316 primary breast tumors were evaluated for the presence of mutant p53 protein by use of the mouse monoclonal antibody Pab 1801 (that recognizes both wild-type and mutant forms of p53) and standard IHC methods. In addition, the entire coding region of p53 genes expressed in these tumors was screened for mutations by combining reverse transcription, the polymerase chain reaction, and DNA sequencing. Probabilities for overall survival (OS), breast cancer-corrected survival (BCCS; death from breast cancer is the considered event), and relapse free survival (RFS) were estimated by use of the Kaplan-Meier method, and survival curves for different patient subgroups were compared by use of the logrank method. All reported P values are from two-sided tests. RESULTS: Sixty nine (22%) of 316 tumors had p53 gene mutations detected by the cDNA-based sequencing method; only 31 (45%) of these mutations were located in evolutionarily conserved portions of the p53 coding region. Sixty-four tumors (20% of the total) had elevated levels of p53 protein as detected by IHC, suggesting the presence of mutations. Of the sequencing-positive tumors (i.e., p53 mutant), 23 exhibited negative IHC reactions, indicating that IHC failed to detect 33% of the mutations. Furthermore, 19 of the IHC-positive tumors were sequencing negative (i.e., p53 wild-type), suggesting a 30% false-positive frequency with IHC. Four tumors (1.3% of the total) could not be analyzed by the cDNA-based sequencing method, and three tumors (1% of the total) could not be analyzed by IHC. The 5-year estimates for RFS, BCCS, and OS were significantly shorter for patients with p53 sequencing-positive tumors than for patients with sequencing-negative tumors (P = .001, P = .01, and P = .0003, respectively). Patients with IHC-positive tumors showed reduced survival in all three categories when compared with those with IHC-negative tumors, but the differences were not statistically significant. CONCLUSIONS: Use of a cDNA-based sequencing method to determine the status of the p53 gene in primary breast cancers yielded better prognostic information than IHC performed with the Pab 1801 monoclonal antibody. PMID- 8632494 TI - Interleukin 1 (IL-1)-dependent melanoma hepatic metastasis in vivo; increased endothelial adherence by IL-1-induced mannose receptors and growth factor production in vitro. AB - BACKGROUND: The growth of cancer cells in inflammatory tissue is often observed. This can be the result of favorable conditions for endothelial cell adherence and/or increased production of local growth factors. PURPOSE: The role of the proinflammatory cytokine interleukin 1 (IL-1) in the prometastatic and growth promoting environment of inflammation was studied in vivo, and the mechanism of cytokine action was studied in vitro as well. METHODS: Systemic inflammation was induced by the intravenous injection of IL-1 beta or lipopolysaccharide (LPS), and the hepatic metastasizing ability of B16 melanoma (B16) cells following intrasplenic injection was studied. IL-1 receptor blockade was accomplished with the use of the IL-1 receptor antagonist (IL-1Ra). In vitro, IL-1Ra was used to assess the mechanism for prometastasis and growth promotion of cultured hepatic sinusoidal endothelium stimulated with LPS. RESULTS: There was a statistically significant (P < .01) enhancement in the parameters of hepatic metastasis when B16 cells were injected intrasplenically either 4 hours after IL-1 injection or 6 or 12 hours after LPS injection. IL-1Ra pretreatment reduced IL-1-induced enhancement of metastasis by 73%-87% and completely inhibited the augmentation of metastasis following LPS injection. In vitro, the adherence of melanoma cells to LPS-treated endothelium increased nearly twofold but was completely abrogated when IL-1Ra was added before LPS. Similar to melanoma adherence, a 2.5-fold increase (P < .05) in functional mannose receptors was observed with LPS treatment but was prevented by the addition of IL-1Ra did not affect basal mannose-receptor activity in unstimulated epithelium. Mannose-receptor activity and B16 cell adherence significantly correlated (r = .9) with LPS treatment. Conditioned medium from LPS-stimulated epithelium augmented B16 cell proliferation compared with control conditioned medium (P < .01). Production of B16 cell growth factor(s) was markedly reduced (P < .01) when IL-1Ra was added. CONCLUSIONS: These results demonstrate that systemic inflammation induces an enhancement of melanoma cell metastasis and growth by IL-1-dependent mechanisms in vivo. In vitro, the mechanism(s) is consistent with IL-1-mediated increase in expression of mannose receptors and production of tumor cell growth factor(s) from the endothelium. IMPLICATIONS: Given the multiple and complex cytokine cascade induced in vivo and in vitro during LPS-induced systemic inflammation, IL 1 plays a strategic role. Since IL-1Ra is without side effects in humans, studies on intraoperative infusion of IL-1Ra during tumor resection may be indicated. PMID- 8632495 TI - False-positive results in clinical trials: multiple significance tests and the problem of unreported comparisons. PMID- 8632493 TI - gamma-Glutamyl transpeptidase mediation of tumor glutathione utilization in vivo. AB - BACKGROUND: Glutathione is a tripeptide used by cells to protect against oxidative and free radical damage. It may also be involved in biochemical mechanisms that cause some tumors to become resistant to anticancer drugs. gamma Glutamyl transpeptidase (GGTP) is a membrane-bound enzyme that cleaves extracellular glutathione, providing cells with amino acids necessary for intracellular synthesis of this compound. Increased expression of GGTP has been found in a number of human tumors; however, few studies have examined the contribution of GGTP to tumor glutathione metabolism in vivo. PURPOSE: Our goals were to study the utilization of host glutathione by 3-methylcholanthrene (MCA) induced sarcomas grown in rats and to evaluate the involvement of tumor GGTP in this process. METHODS: The left ovaries of 21 female Fischer 344 rats were isolated by laparotomy and placed in subcutaneous positions through stab wounds in the abdominal wall. A 3-mm cube of MCA sarcoma was then sutured to each of the isolated ovaries. The MCA implants obliterated the ovarian tissue, yielding isolated tumors with one arterial supply (the ovarian artery) and one draining vein (the ovarian vein, referred to as the tumor vein). After 2 weeks of tumor growth, blood was drawn from the tumor vein, the inferior vena cava (IVC), and the aorta of 16 animals. Glutathione and cysteine concentrations in plasma samples from this blood were determined by high-performance liquid chromatography and used to calculate glutathione and cysteine utilization ratios for the tumor and the systemic circulations ([(concentration aorta-concentration tumor vein)/concentration aorta] x 100 and [(concentration aorta-concentration IVC)/concentration aorta ] x 100, respectively). The utilization ratios from these control animals were compared with those from acivicin (AT-125; an irreversible GGTP inhibitor)-treated rats (the remaining five animals). Data are presented as mean +/- standard deviation; reported P values are from two-tailed tests of statistical significance. RESULTS: In the control animals, glutathione and cysteine concentrations were significantly lower in the tumor vein (3.55 +/- 1.9 and 5.69 +/- 2.8 microM, respectively) and in the IVC (5.65 +/- 2.3 and 7.17 +/- 2.4 microM, respectively) than in the artery (12.48 +/- 5.7 and 12.33 +/- 5.9 microM, respectively; all P values < .05). In addition, the glutathione utilization ratio was significantly higher for the tumor circulation than for the systemic circulation (69% +/- 14% versus 52% +/- 14%; P < .003). The combined glutathione and cysteine utilization ratio was also significantly higher for the tumor circulation than for the systemic circulation (116% +/- 35% versus 88% +/- 28%; P < .02). Treatment with AT-125 lowered the tumor glutathione utilization ratio significantly (45% +/- 12% for treated animals versus 69% +/- 14% for control animals; P < .005). CONCLUSIONS: Our results show that glutathione and cysteine in the host circulation are used by MCA sarcomas. The significant reduction in tumor utilization of serum glutathione after treatment with AT-125, a GGTP inhibitor, indicates that GGTP is important in tumor glutathione metabolism. PMID- 8632496 TI - Re: Smoking, treatment for Hodgkin's disease, and subsequent lung cancer risk. PMID- 8632497 TI - Re: Population-based study of tamoxifen therapy and subsequent ovarian, endometrial, and breast cancers. PMID- 8632498 TI - Outbreak of obstructive ventilatory impairment associated with consumption of Sauropus androgynus vegetable. AB - BACKGROUND: Forty four individuals, suffering from temporary insomnia and poor appetite followed by progressive difficulty breathing after four weeks or more ingestion of the Sauropus androgynus or Sabah vegetable, were reported to the National Poison Center of Taiwan by physicians between August 23, 1994 and August 25, 1995. OBJECTIVE: A telephone questionnaire survey was designed and conducted to collect demographic data, information about use of the vegetable, past medical history and clinical presentation. Laboratory data were obtained from their physicians as available. RESULTS: Forty one patients, predominantly women, 43 +/- 11 years old, were identified in our survey. They reported a variety of sources and preparation methods for the vegetable. Difficulty breathing, identified in 36 cases, was the clinical hallmark. Twenty people gave a history of dyspnea delayed until 44 +/- 40 days after discontinuing vegetable consumption. Laboratory evidence of obstructive ventilatory impairment (FEV1/FVC 56 +/- 12%, FEV1 31 +/- 6%, PaO2 71 +/- 15%) was observed in 12 cases tested. An open lung biopsy performed in a demonstration case disclosed bronchiolitis obliterans organizing pneumonia. CONCLUSION: In this case series of 41 victims, we have identified a severe pulmonary effect and hypothesize that it is related to consumption of sauropus androgynus vegetable. Papaverine has been previously identified in this vegetable but is unlikely to be responsible for the full range of toxicity seen. PMID- 8632499 TI - Chloral hydrate toxicity from oral and intravenous administration. AB - BACKGROUND: Overdose from enteric chloral hydrate results in cardiovascular and central nervous system symptoms. CASE REPORTS: This case series compares and contrasts two cases of oral chloral hydrate overdose with two cases of accidental i.v. administration. Whereas ingestion of 219 mg/kg of chloral hydrate resulted in transient bigeminy, ingestion of up to 960 mg/kg caused torsades de pointes and ventricular fibrillation which were effectively treated with defibrillation and a beta blocker. I.V. administration in humans does not appear previously documented. Two cases of i.v. administration of a therapeutic chloral hydrate dose resulted in central nervous system depression and minimal local effects at the injection site. CONCLUSIONS: Given the high bioavailability of oral chloral hydrate the major determinant of cardiotoxicity may be the dose rather than the route of administration. Cardiac arrhythmias due to chloral hydrate appear to be responsive to beta blocker therapy. PMID- 8632500 TI - Lead poisoning diagnosed by abdominal X-rays. AB - BACKGROUND: Adult lead poisoning due to ingested lead is uncommon. CASE REPORT: A 27-year-old woman had abdominal pain with radiodensities of the bowel contents and a blood lead of 70 micrograms/dL. A traditional medicine, Deshi Dewa, consumed as a fertility pill was incriminated. CONCLUSION: Densities on abdominal X-ray may indicate ingested lead. PMID- 8632501 TI - Acute hexogen poisoning after occupational exposure. AB - BACKGROUND: Hexogen (cyclonite, RDX) nitrate explosive is an infrequent cause of poisoning. CASE REPORT: A 42-year-old man with no prior history of epilepsy experienced grand mal seizures after sieving fine hexogen (RDX) powder for four hours in an ammunition plant. Physical examination was normal on arrival at the emergency room but recurrent seizures occurred six hour after admission. EEG, CT scan and MRI were normal and the patient recovered uneventfully. CONCLUSIONS: The available toxicological data on this rare occupational poisoning are reviewed. PMID- 8632503 TI - Silver products for medical indications: risk-benefit assessment. AB - BACKGROUND: Legitimate medicinal use of silver-containing products has dramatically diminished over the last several decades. Recently, however, some manufacturers have begun to enthusiastically promote oral colloidal silver proteins as mineral supplements and for prevention and treatment of many diseases. Indiscriminate use of silver products can lead to toxicity such as argyria. OBJECTIVE: To assist health care professionals in a risk versus benefit assessment of over-the-counter silver-containing products, we herein examine the following issues: historical uses, chemistry, pharmacology, clinical toxicology, case reports of adverse events in the literature, and the recent promotion of over-the-counter silver products. Other sources of silver exposure (including environmental and dietary) and EPA exposure standards are discussed. A list of currently available silver products is provided for easy reference and screening. CONCLUSIONS: We emphasize the lack of established effectiveness and potential toxicity of these products. PMID- 8632502 TI - Plasma and urine concentrations of atropine after the ingestion of cooked deadly nightshade berries. AB - BACKGROUND: Adult intoxications due to ingestion of deadly nightshade berries is uncommon. CASE REPORTS: Collective intoxication of eight persons occurred after accidental ingestion of ripened Atropa belladonna berries. Three of the four adults displayed delirious states with visual hallucinations; one patient fell into a coma and required mechanical ventilation. Four children and one adult exhibited mild peripheral anticholinergic symptoms. Kinetic data were obtained on the three hospitalized adults. DISCUSSION: The optimal intensive care for such patients is discussed. PMID- 8632504 TI - Thioridazine cardiotoxicity. PMID- 8632505 TI - Systemic pulmonary toxicity--a puzzler. PMID- 8632506 TI - Treatment of cardiac membrane stabilizing dysrhythmias. PMID- 8632507 TI - An outbreak of pulmonary poisoning. PMID- 8632508 TI - Pulmonary toxicity following exposure to an aerosolized leather protector. AB - BACKGROUND: An aerosol spray for leather protection was reformulated to remove trichloroethane. The new formulation contained isobutane, n-heptane, ethyl acetate and fluoroaliphatics. RETROSPECTIVE REVIEW: Thirty-nine patients reported symptoms to the regional poison center. Respiratory symptoms developed within hours of exposure. Most symptoms resolved within two days. Abnormal pulmonary function tests, including obstructive disease or diminished diffusing capacity, were demonstrated in three of the four tested patients. CONCLUSIONS: The mechanism for the pulmonary toxicity has not been determined. PMID- 8632509 TI - Reformulated leather protectors: safer for the ozone than the public. PMID- 8632510 TI - An evaluation of unleaded petrol as a harm reduction strategy for petrol sniffers in an aboriginal community. AB - OBJECTIVE: In mid 1989, leaded petrol was replaced by unleaded petrol to reduce lead toxicity in petrol sniffers in Maningrida, a remote Aboriginal community in Northern Australia. RETROSPECTIVE REVIEWS: Hospital admissions between 1987 and 1992 due to petrol sniffing were compared for Maningrida and a community using only leaded petrol. RESULTS: Admissions from Maningrida decreased significantly following introduction of unleaded petrol (chi 2 on 2df = 22.25, p < 0.001). Lead and hydrocarbon exposures were also compared for three groups from Maningrida (27 sniffers using only unleaded petrol; 15 exsniffers and 13 nonsniffers) and 24 individuals admitted to hospital for petrol sniffing related illness from other communities using only leaded petrol. Median blood lead levels for hospitalized sniffers (using only leaded petrol). Maningrida sniffers (using only unleaded petrol), exsniffers and nonsniffers were 5.06, 1.87, 1.24 and 0.17 microM/L respectively. There were significant differences between blood lead level, delta aminolevulinic acid dehydratase activity, and free erythrocyte protoporphyrin for sniffers of leaded and unleaded petrol, whereas these indices were not significantly different for current sniffers and exsniffers in Maningrida. Hydrocarbons were only detectable in the blood of active sniffers (toluene < or = 0.5 micrograms/mL; benzene < or = 0.17 micrograms/mL blood; n-hexane not detected). CONCLUSIONS: The elimination of tetraethyl lead from petrol resulted in a significant decrease in hospitalization of petrol sniffers. PMID- 8632512 TI - Clinical and sociodemographic features of acute carbamate and organophosphate poisoning: a study of 70 adult patients in north Jordan. AB - OBJECTIVE: To define the sociodemographic and clinical aspects of organic phosphate and carbamate poisoning. DESIGN: The records of 70 adults (33 males and 37 females) with carbamate or organophosphate intoxication admitted to a North Jordan Teaching Hospital over a five-year period were reviewed retrospectively. These patients represented 10% of all drug overdoses admitted over the same period. RESULTS: The most cases occurred in the 15-19 year-old age group and the female to male ratio was 1.1:1. Carbamate intoxication was more than twice as common as organophosphate intoxication. Two thirds (64%) of the patients intended to commit suicide, 26% were due to accidental ingestion and the remaining 10% were from occupational exposure. Muscarinic manifestations were the predominant clinical feature followed by central nervous system and then nicotinic manifestations. Low grade fever, not related to infection, was observed in 49% of the patients and respiratory difficulty in 47%, of which 11% required assisted ventilation. Twenty-nine percent of the patients presented with coma. Three patients died for a hospital mortality of 4%. CONCLUSIONS: The widespread use of carbamates and organophosphates as household pesticides and the lack of adequate regulations controlling their sale and application has encouraged teenagers to prefer them as a modality of attempted suicide. This source of poisoning has become a major health problem in some developing countries. PMID- 8632511 TI - Urine toxicology screens in drivers suspected of driving while impaired from drugs. AB - OBJECTIVE: Police departments, in conjunction with the National Highway Traffic Safety Administration, have developed a standardized evaluation aimed at identifying drivers impaired by drugs other than ethanol. These evaluations are performed by specially trained police officers known as Drug Recognition Experts. METHODS: We retrospectively reviewed the evaluations of 242 drivers detained for driving while impaired in the City and County of Denver from January 1, 1988 to June 30, 1990. RESULTS: All drivers had urine toxicology screens performed, which were positive for a mean 1.2 +/- 0.9 SD (range zero to four) for drugs having the potential for causing driving impairment. The 193/242 urine screens (79.8%) testing positive showed the following drugs: cannabis 162 (66.9%), stimulants (including cocaine metabolites) 80 (33.1%), depressants (benzodiazepines and barbiturates) 24 (9.9%), narcotics 12 (5.0%), inhalants (toluene) 1 (0.4%), hallucinogens (LSD) 1 (0.4%), and other 3 (1.2%). Drug Recognition Experts, based on their initial evaluation, were able to predict correctly some or all of the drugs found on the urine screens in 178/242 (73.6%) of cases. Overall agreement between the Drug Recognition Experts opinions and urine screen results had a kappa value (p < 0.05) of 0.41. CONCLUSIONS: There was a high rate (79.8%) of positive urine toxicology screens in drivers suspected of nonethanol drug impairment. In most cases, Drug Recognition Experts were able to reliably predict the results of these screens. PMID- 8632513 TI - Disaster preparedness of poison control centers in the United States. AB - OBJECTIVE: To assess the state of disaster readiness of poison control centers, a survey questionnaire was sent to all 96 institutional poison control center members of the American Association of Poison Control Centers in the US, both certified and noncertified programs. DESIGN: The data reported are the results and responses from 76 of 96 (79.2%) poison control centers. RESULTS: Fifty-four percent of responding centers have written disaster plans, with 25% having drills to practice the plans. Of the centers that do not have a written plan, the majority have policies and procedures in place to address physical plant damage, increased phone traffic, loss of phone systems and malfunction of computers. Eighty-six percent of respondents have a back up generator, and 82% have an uninterruptable power supply in place. Fifty-four percent have a back up phone system and 33% have cellular phone capacity. Forty-six percent of responding centers have arrangements with other agencies in the event of a disaster. Only half of the managing directors of the responding centers believe their center can meet the public's needs in the event of a disaster. PMID- 8632514 TI - Disaster planning--our finest hour? PMID- 8632515 TI - Predictors of carbon monoxide and hydrogen cyanide exposure in smoke inhalation patients. AB - OBJECTIVE: A prospective study of civilian (nonfirefighter) smoke inhalation patients was carried out to test the hypotheses that: 1) absorption of carbon monoxide and hydrogen cyanide from smoke can be predicted by clinical examination and historical data; and, more specifically 2) a history of exposure to burning synthetic polymers is an important predictor of systemic cyanide levels. METHODS: The study was conducted over a three-year period at six urban hospitals. Patients with or without burns who were exposed to smoke within five hours of hospital arrival were sampled for carboxyhemoglobin, whole blood cyanide, urine cotinine and urine creatinine. Controls consisted of a smaller group of smoking status matched, nonsmoke-exposed burn patients. ANALYSIS: Historical information was obtained on SMOKING status, FIRETYPE (structural vs other), MATERIAL burned (natural vs synthetic) and LAGTIME (from exposure to sampling). A smoke inhalation SCORE (0-10) was assigned to each case, based on physical examination findings and changes on chest X ray, and carboxyhemoglobin and cyanide levels were entered into various multivariate linear regression models. RESULTS: A total of 40 cases and 9 controls were recruited, ranging in age from 15 to 92 years. Thirty-four cases were discharged alive and six expired in-hospital. Observed carboxyhemoglobin levels ranged from 1.2% to 41.6% in cases (mean 8.6%), and from 0.5 to 7.3% in controls (mean 2.9%). Observed cyanide levels ranged from nondetectable (< 0.05 micrograms/mL) to 2.79 micrograms/mL in cases (mean 0.25 micrograms/mL), and from nondetectable to 0.11 micrograms/mL in controls (mean 0.03 micrograms/mL). Among cases, linear regression models explained up to 35% of the observed variance in carboxyhemoglobin levels (p < 0.001) and up to 48% of the variance in cyanide levels (p = 0.0001). CONCLUSIONS: SCORE was the strongest predictor of both carboxyhemoglobin and cyanide levels; LAGTIME also explained significant variance for [log-transformed] carboxyhemoglobin. Historical factors, such as FIRETYPE, MATERIAL, and SMOKING status, did not explain significant variance in most of the statistical models employed. PMID- 8632516 TI - The effect of hypertonic sodium bicarbonate on QRS duration in rats poisoned with chloroquine. AB - OBJECTIVE: To determine efficacy of hypertonic sodium bicarbonate in narrowing QRS prolongation produced by chloroquine. DESIGN: Randomized, controlled animal experiment using an accepted rat model of sodium channel blockade. METHODS: Hypotension and widening of QRS complexes (lead II) of the ECG were produced in 16 rats by administration of a total of 87 mg/kg chloroquine intravenously over 20 minutes. Eight rats were treated with 6 mL/kg 1 M sodium bicarbonate intravenously over two minutes beginning ten minutes into the chloroquine infusion. Serial measurements of QRS duration and systolic blood pressure were obtained for 30 minutes. RESULTS: QRS intervals narrowed more rapidly in animals receiving sodium bicarbonate (p = .045), although the difference in mean rate of narrowing between groups was modest at only .23 msec/min. Because of large variances, no statistically significant differences could be demonstrated in systolic blood pressure. CONCLUSIONS: Hypertonic sodium bicarbonate partially reversed sodium channel blockade and resultant QRS interval prolongation produced by chloroquine in rats. These data should be interpreted with caution, given the need to extrapolate to humans and the modest effect of sodium bicarbonate on QRS narrowing. PMID- 8632517 TI - In vitro competitive inhibition of plasma cholinesterase by cocaine: normal and variant genotypes. AB - OBJECTIVE: To determine the inhibitory constant, Ki, of cocaine for a number of the different genetic variants of human plasma cholinesterase. DESIGN: In vitro analysis of plasma cholinesterase activity in the presence of cocaine as a competitive inhibitor. METHODS: Six normal (UU) control sera and seven sera with the following plasma cholinesterase genotypes were assayed: AA, UA, AS, UF, US, AF and SS. Plasma cholinesterase activity was determined in the samples by colorimetric measurement of propionylthiocholine metabolism over a range of concentrations. Competitive activities were then determined in the presence of varying concentrations of cocaine. Double reciprocal plots (1/v vs 1/S) were used to calculate Km and Vmax for propionylthiocholine, and Ki for cocaine for each genotype. RESULTS: The variant forms of the plasma cholinesterase had high cocaine Ki values--all were approximately ten times greater than the Ki for normal plasma cholinesterase. CONCLUSIONS: Since the inhibitory constant is an indirect measure of an enzyme's affinity for a competing substrate, a high Ki for cocaine at recreational or therapeutic concentrations would translate into a longer in vivo half-life. Our results support the growing evidence that low plasma cholinesterase activity predisposes to cocaine toxicity. PMID- 8632518 TI - Multiple-dose activated charcoal in an accidental vancomycin overdose. AB - BACKGROUND: Multiple-dose activated charcoal may enhance the enterocapillary clearance of vancomycin. CASE REPORT: A 17-day-old female neonate born with congenital meningomyelocele and Arnold-Chiari malformation was iatrogenically overdosed with a 500 mg intravenous bolus of vancomycin during a shunt operation. The Red Man's Syndrome developed within minutes, characterized by sudden hypotension, skin rash and cyanosis. Serum vancomycin level at one hour after the injection was 165.7 micrograms/mL, as measured by an enzyme immunoassay method (EMIT). Multiple dose activated charcoal, 1 g/kg, was first given five hours after injection, and continued every four hours for 12 doses. The half-life of vancomycin during charcoal administration was calculated to be 9.4 h or less than the reported 13.4-33.7 h half-life in normal neonates. The neonate's renal function tests and brainstem auditory responses remained normal. CONCLUSIONS: Gastrointestinal dialysis with multiple-dose activated charcoal without cathartics appeared to shorten the elimination half-life of vancomycin. PMID- 8632519 TI - Two outbreaks of acute Tung Nut (Aleurites fordii) poisoning. AB - OBJECTIVE: Aleurites fordii, widely distributed in the Southeastern US, Taiwan, mainland China, Japan and India, is commonly known as Tung Nut, Tung Oil Tree or Chinawood Oil Tree. The seeds are the most toxic part. CASE REPORTS: We report two outbreaks of Aleurites fordii poisoning, occurring on November 27, 1992 and November 29, 1994. Thirty-five elementary school students and 29 senior high school students misidentified Aleurites fordii seeds as chestnuts and ingested variable amounts. METHODS: We conducted a survey by questionnaire to supplement the hospital record information. Simple descriptive statistics and Chi-square (Fisher's exact) tests were calculated. RESULTS: The three most common symptoms of the patients were vomiting, abdominal pain and diarrhea. The more serious clinical presentations occurred in younger victims. Our information suggests that food attenuates intestinal irritation perhaps by delaying absorption of the toxic principle. With symptomatic treatment, all of the symptoms and signs subsided within one to two days. CONCLUSIONS: Aleurites fordii can be cultivated and is easily accessible to the community and schools. Public education about the toxicity of Tung Nut seeds in areas of ready availability may reduce the chance of misidentification and subsequent poisoning. PMID- 8632520 TI - "Profiting" from vegetables. PMID- 8632521 TI - Erythromycin-induced acute pancreatitis. AB - BACKGROUND: Pancreatitis due to ingestion of erythromycin is rare. CASE REPORT: A 20-year-old woman took erythromycin 3 g and acetaminophen 6 g before a tooth extraction. Forty minutes later, she experienced severe abdominal pain, nausea, and vomiting. The serum amylase and lipase were elevated. She recovered from the pancreatitis without sequelae after supportive treatment. CONCLUSION: This is the fourth reported case of erythromycin-induced acute pancreatitis in the English literature. PMID- 8632522 TI - Suicidal chloral hydrate poisoning. AB - BACKGROUND: The cumulative reports of chlorate hydrate toxicity suggest an unacceptable risk for this commonly used sleep aid. CASE REPORT: A 29-year-old male was admitted after ingestion of 70 g of chloral hydrate. He was hypotensive and hypothermic. Spontaneous respiration was insufficient. Fluid resuscitation did not restore adequate blood pressure levels. Low dose catecholamine infusion resulted in ventricular arrhythmias. The patient was treated with combined hemoperfusion and hemodialysis during an eight hour period. During this time, serum concentrations of trichlorethanol fell. Blood pressure and heart rate increased and consciousness was regained without signs of neurological deficits. CONCLUSIONS: Although outcome was favorable, this case report illustrates the potentially high acute toxicity of chloral hydrate and supports its removal from the market. PMID- 8632523 TI - Urodynamic and clinical effects of terazosin therapy in patients with symptomatic benign prostatic hyperplasia. AB - PURPOSE: We evaluated the urodynamic and clinical effects of terazosin in patients with symptomatic benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: A total of 45 patients who participated in a multicenter trial was evaluated with urodynamic pressure-flow studies before and after 26 weeks of treatment. RESULTS: Maximum flow rate and symptom score improved significantly in 22 patients with and 11 without bladder outlet obstruction who completed 26 weeks of treatment. In patients with bladder outlet obstruction the condition was significantly reduced and in patients without obstruction, significant urodynamic changes could not be detected. CONCLUSIONS: Terazosin treatment results in symptomatic relief and improved urinary flow in patients with and without bladder outlet obstruction, and in significant improvement in patients with urodynamically proved obstruction. PMID- 8632524 TI - This month in investigative urology. Commentary on nitric oxide in interstitial cystitis. PMID- 8632525 TI - Renal vein occlusion: a review. AB - PURPOSE: The different etiologies of renal occlusion are reviewed. A special category, division of the left renal vein in abdominal aortic surgery, is also discussed in the review. MATERIALS AND METHODS: The various diagnostic modalities used in cases of renal vein occlusion include excretory urography, ultrasound, nuclear scan, angiography, venography, computerized tomography and magnetic resonance imaging. The main goals of therapy in this condition should be conserve renal parenchyma and to protect renal function. RESULTS: The principal mode of treatment is medical and includes correction fluid and eletrocyte imbalance, dialysis, antihypertensive drugs, anticoagulation and in certain cases thrombolysis. CONCLUSIONS: Renal vein occlusion in adults is usually a result of the vein thrombosis which is frequently associated with the nephrotic syndrome. The anatomy of renal vascularization is of primary importance in understanding its pathophysiological responses and the clinical and diagnostic presentation of patients with this condition. The reaction of the kidney to its vein occlusion is determined by the balance between the acuteness of the disease, extent of the development of collateral circulation, involvement of 1 or both kidneys and the origin of the underlying disease. Renal vein occlusion is generally a complication of some other condition but may also be a primary disease. PMID- 8632526 TI - Laparoscopic radical nephrectomy for renal tumor: the Washington University experience. AB - PURPOSE: We report our experience with laparoscopic radical nephrectomy in 17 consecutive patients with renal tumors. MATERIALS AND METHODS: The clinical data on 17 consecutive patients undergoing laparoscopic radical nephrectomy were reviewed. Of the patients 12 with stage pT1 or pT2 renal cell carcinoma 7 cm. in diameter or smaller undergoing laparoscopic radical nephrectomy were compared to 12 undergoing open radical nephrectomy for stage pT1 or pT2 renal cell carcinoma 6 cm. in diameter or smaller. RESULTS: Among the 17 patients undergoing laparoscopic radical nephrectomy average operative time was 6.9 hours (range 4.5 to 9) and average estimated blood loss was 105 cc (range 50 to 600). Average weight of the surgical specimen was 402 gm. (range 190 to 1,100). In 12 of 16 patients in whom laparoscopic radical nephrectomy was completed the specimen was removed intact. The patients required an average of 24 mg. morphine sulfate equivalent (range 2 to 220) for postoperative pain. Average hospital stay was 4.5 days (range 3 to 11) and average interval to resume normal activities was 3.5 weeks (range 2 to 4). The 12 patients in the open and laparoscopic radical nephrectomy groups were similar with respect to age, American Society of Anesthesiologists score and interval surgery. Laparoscopic radical nephrectomy required significantly more operative time than open radical nephrectomy (6.9 versus 2.2 hours, respectively). However, the laparoscopic radical nephrectomy group compared to the open radical nephrectomy group had significantly less postoperative pain (24 versus 40 mg. morphine sulfate equivalent required for postoperative analgesia), shorter interval to resuming oral intake (1 versus 3 days), more rapid discharge from the hospital (4.5 versus 8.4 days) and more rapid return to normal activities (3.5 versus 5.1 weeks). The laparoscopic nephrectomy group also fully recovered more rapidly than the open surgical group (5.8 versus 39 weeks). To date, during a 4-year period there was no retroperitoneal recurrence or seeding of port site. CONCLUSIONS: Laparoscopic radical nephrectomy is a lengthy and demanding procedure. However, it affords patients with renal cell carcinoma a markedly improved postoperative course while accomplishing the necessary surgical goals. PMID- 8632527 TI - Clinical implications of clinically insignificant store fragments after extracorporeal shock wave lithotripsy. AB - PURPOSE: We determined the natural history and clinical significance of small, asymptomatic, noninfection related stone fragments after extracorporeal shock wave lithotripsy (ESWL). MATERIALS AND METHODS: We prospectively followed 160 patients with 4 mm. or less asymptomatic calcium oxalate/phosphate stone fragments after ESWL for 1.6 to 88.8 months (mean 23) to stone-free status, censorship or intervention. Kaplan-Meier estimates of probability to anatomical stone-free, decreased or stable status were determined as well as the probability of symptomatic episodes or required urological intervention. RESULTS: Stone-free status or a decreased, stable or increased amount of residual stone occurred in 38 (23.8%), 26 (16.3%), 67 (41.9%) and 29 (18.1%) of the 160 patients, respectively. At 5 years after ESWL the probability of a stone-free, stone-free or decreased status, or stone-free, decreased or stable status was 0.36, 0.53, and 0.80, respectively. A total of 91 patients (56.9%) remained asymptomatic while 69 (43.1%) had a symptomatic episode or required intervention 1.6 to 85.4 months (mean 26) after ESWL (probability estimated at 0.71 at 5 years). CONCLUSIONS: While patients with small noninfection related stone fragments after ESWL may be followed expectantly, a significantly number will require intervention or have symptomatic episodes within 2 years. The term clinically insignificant applied to any residual stone after ESWL is likely a misnomer. PMID- 8632529 TI - Radical nephrectomy for renal cell carcinoma 30 mm. or less: long-term follow results. AB - PURPOSE: We investigated the clinical and pathological features, and long-term followup of patients with radical nephrectomy for renal cell carcinoma of 30 mm. or smaller and a normal contralateral kidney. MATERIALS AND METHODS: Between 1973 and 1993, 74 patients 29 to 83 years old (average age 60) underwent radical nephrectomy for renal cell carcinoma of 30 mm. or smaller. Average followup was 101 months (range 10 to 236). Of the 74 tumors 21 were stage pT1, 36 stage pT2 and 12 stage pT3 (all 69 stag N0 to NxM0), while 5 were stages pT1 to 3, N1 or M1. A total of 27 patients died during follow-up. Of 11 patients who died of disease progression 5 had metastatic disease initially. RESULTS: Overall survival rates were 73% at 5 years, 55% at 10 years and 38% at 20 years. Cancer-specific survival rates without the 5 patients with stage N1 or M1 disease were 91% at 5 years, and 80% at 10 and 20 years. For the group with nonmultifocal renal cell carcinoma, stage NO to Nx and/or MO, the cancer-specific survival rates were 93% at 5 years, and 81% at 10 and 20 years. For the group with multifocal renal cell carcinoma, stage NO to Nx and/or MO, the cancer-specific survival rate was 75% at 5, 10, and 20 years. There was a significant survival difference between the nonmultifocal and multifocal renal cell carcinoma groups at 5 years (p<0.05) but not at 10 and 20 years. CONCLUSIONS: Small renal cell carcinoma is definitively not a benign disease and a tumor of 30 mm. or smaller does not mean there is no risk of metastatic or multifocal disease. PMID- 8632528 TI - Intraoperative evaluation of renal cell carcinoma: a prospective study of the role of ultrasonography and histopathological frozen sections. AB - PURPOSE: Nephron sparing surgery is being performed increasingly for treatment of renal cell carcinoma, including in select patients with a normal contralateral kidney. The number of tumors in the involved kidney (single versus multiple) and presence or absence of perinephric fat involvement (pathological stage T1 to 2 versus T3A) are important prognostic factors. In a prospective study we evaluated the accuracy of intraoperative histopathological frozen section analysis of renal capsular biopsies for assessing local tumor stage, and the accuracy of intraoperative ultrasonography for assessing tumor focality. MATERIALS AND METHODS: Intraoperative frozen section biopsies and ultrasonography were compared with information obtained from preoperative computerized tomography (CT), intraoperative inspection of the kidney by the surgeon and permanent histopathological specimens. RESULTS: We evaluated 99 patients (102 kidneys) with localized sold renal masses undergoing either radical nephrectomy (48) or nephron sparing surgery (54). Final pathological analysis revealed 95 renal cell carcinomas (stage T3A in 24), 6 oncocytomas and 1 angiomyolipoma. Multiple tumors were detected in 18 of 102 kidneys overall. Frozen section analysis identified 87% of the stage T3A lesions with no false-positive results, compared to CT, which only identified 67%. Ultrasonography identified 14 of 18 multifocal tumors (78%) and was not more accurate than the combination of CT and intraoperative inspection. However, during nephron sparing surgery ultrasonography was useful to localize the intrarenal extent of tumors (17 cases). CONCLUSIONS: Our results clarify the role of intraoperative ultrasonography and frozen section analysis in patients undergoing nephron sparing surgery for renal cell carcinoma. Frozen section analysis may be useful in select patients with small peripheral tumors who are under consideration for elective nephron sparing surgery. PMID- 8632531 TI - Influence of continent ileal urinary diversion on vitamin B12 absorption. AB - PURPOSE: Because vitamin B12 is mainly absorbed in the terminal ileum, we evaluated potential absorption deficiencies in continent ileal reservoirs. MATERIALS AND METHODS: Eight to 25 months (mean 13) after surgery we evaluated 25 patients with a Kock pouch and 29 with an ileal neobladder. A Schilling test and red blood count were done, and serum levels of vitamin B12 and folic acid were determined. RESULTS: Absorptive capacity was decreased in 20 of the 25 Kock pouch patients but none of the 29 ileal neobladder patients. Four patients in each group had low vitamin B12 levels. No patient had megaloblastic anemia. Folic acid levels were normal in all patients. CONCLUSIONS: A loss of 50 cm. of terminal ileum seems to be the critical margin sufficient vitamin B12 absorption. PMID- 8632530 TI - Simplified uretero-intestinal implantation in continent cutaneous urinary diversion using ileovalvular segment as afferent loop and appendix as continent outlet. AB - PURPOSE: We performed continent cutaneous urinary diversion with implantation of ureters as in ileal loop diversion to reduce the risk of ureterointestinal implantation stricture. MATERIALS AND METHODS: Four patients underwent colonic pouch and 11 underwent ileocecal continent urinary diversion the ileocecal segment used as an afferent loop and the sphincter-like function of the ileocecal valve used as an antireflux mechanism. RESULTS: All preoperatively undilated renal units (26 of 30) remained postoperatively undilated. Of the 4 preoperatively dilated renal units 3 also showed postoperatively improvement (mean followup 8 months). However, after 3 to 14 months transient reflux occurred during contraction waves in the reservoir in 6 renal units. CONCLUSIONS: Our modified technique simplifies ureteral reimplantation and appears to diminish the postoperative complication of anastomotic structure. The solution to the problem of occasional reflux may be reinforcement of the ileocecal valve. PMID- 8632532 TI - Bone mineral and related biochemical variables in patients with Kock ileal reservoir or Bricker conduit for urinary diversion. AB - PURPOSE: Bone material content was studied with single photon absorptiometry and dual energy x-ray absorptiometry , and the various biochemical parameters related to bone metabolism in patients with Kock reservoirs or Bricker conduits for urinary diversion. MATERIALS AND METHODS: We examined 34 patients with Kock ileal reservoirs to the skin (29) or urethra (5) and 14 with Bricker conduits 2 to 17 years after urinary diversion. Bone mineral density was measured in the radius with single photon absorptiometry, and in th femur, lumbar spine and whole body with dual x-ray absorptiometry. Serum concentrations of 25-hydroxyvitamin D, 1,25 dihydroxyvitamin D, osteocalcin, parathyroid hormone, bone specific alkaline phosphatase and ionized calcium were determined. Arterial blood gases were analyzed. RESULTS: The mean values for bone mineral density did not differ from age-matched controls in either group and no signs of decrease were observed with followup. Vitamin D serum concentration values remained within normal limits in all patients. Most patients had normal blood gas values. Mean values for osteocalcin, parathyroid hormone, bone specific alkaline phosphatase and ionized calcium also were within normal limits, although a few patients had elevated osteocalcin values indicating increased bone turnover. CONCLUSION: Urinary diversion with a Kock reservoir or Bricker conduit did not cause bone demineralization of significant changes in different markers of bone metabolism in patients examined 2 to 17 years after urinary diversion. PMID- 8632533 TI - Desire to void and force of micturition in patients with intestinal bladders. AB - PURPOSE: We attempted to determine how patients with an orthotopic bladder perceive the desire to void and the force achieved to evacuate the bladder. MATERIALS AND METHODS: A total of 24 men who had undergone post-cystectomy bladder substitution (ileocecal in 12, sigmoid in 6 and ileal in 6) was evaluated subjectively an objectively by pressure-flow study 1 to 3 years postoperatively. RESULTS: Desire to void was felt at the base of the penis or in the perineum by 20 men (83%). Abdominal pressure contributed to intra-reservoir pressure by 51 to 54% in ileocecal, 20 to 24% in sigmoid and 23 to 25% in ileal neobladders. CONCLUSIONS: Patients perceive the desire to void when drops of urine leak into the proximal urethra from an overfilled neobladder. Urine is evacuated mainly by abdominal straining for ileal neobladders, mainly by contraction for sigmoid neobladders, and by approximately equal contributions of contradiction and straining for ileocecal neobladders. PMID- 8632534 TI - The long-term study of continent cutaneous urinary reservoirs and neobladders. PMID- 8632535 TI - Ureteral frozen section analysis during cystectomy: a reassessment. AB - PURPOSE: Frozen section analysis of the distal ureteral margins is commonly performed at cystectomy to exclude involvement of tumor in retained ureter. We reviewed our experience with 101 consecutive cystectomies to determine the contemporary incidence and clinical significance of the urothelial abnormalities detected by frozen section analysis performed at operation. METHODS AND MATERIALS: The pathology reports for 101 patients treated with nerve sparing cystectomy between 1982 and 1989 were reviewed. Frozen section and final ureteral analyses were compared. RESULTS: Of the patients 8% had evidence of a urothelial abnormality ranging from mild atypia to frank carcinoma in situ involving the distal ureters on frozen section. Only 4 patients had documented carcinoma in situ at the final margin and all 4 ultimately died of disease. The frozen section false-positive and false-negative rates were 2 and 6%, respectively. In 6 patients with ureteral urothelial abnormalities documented on frozen section ureterointestinal anastomosis was performed despite persistent abnormalities at the ureteral margins, frequently after multiple frozen analyses failed to clear the margins definitively. None of the 6 patients in this group experienced upper tract recurrence during a mean followup of 41 months. CONCLUSIONS: These data suggest that routine frozen section analysis of the ureteral margins at cystectomy may not be necessary for most patinets undergoing cystectomy. PMID- 8632536 TI - The quality of life during intravesical bacillus Calmette-Guerin therapy. AB - PURPOSE: In the treatment of cancer the subjective evaluation of therapy induced side effects and quality of life must be considered. Adjuvant intravesical bacillus Calmette-Guerin (BCG) immunotherapy is regarded as highly effective but associated with significant side effects. To clarify whether these side effects significantly impact on patient satisfaction with life, a prospective survey of the health related quality of life and side effects was performed during an intravesical BCG treatment course. MATERIALS AND METHODS: Symptoms and side effects were recorded daily during the 6-week instillation period in 30 patients. The quality of life was determined before, during and after intravesical therapy with the assistance of a self-reported questionnaire. RESULTS: Due to a detailed subjective assessment of side effects, the course of local symptoms could be characterized with great reliability. Although side effects occurred, satisfaction with life in the patients studied was high and was not impaired during the treatment. The incidence of side effects correlated well with patient subjective evaluation of quality of life. CONCLUSIONS: Assessment of side effects and quality of life, as presented in this pilot study, can assist in the quantification of localized symptoms in intravesical therapy could contribute to the comparability of clinical studies. With regard to intravesical BCG immunotherapy, our results suggest that the specific side effects do not seriously impair satisfaction with life in these patients. PMID- 8632537 TI - Neoadjuvant combined microwave induced local hyperthermia and topical chemotherapy versus chemotherapy alone for superficial bladder cancer. AB - PURPOSE: We evaluated the effectiveness of local bladder hyperthermia and intravesical chemotherapy compared to intravesical chemotherapy alone in the treatment of superficial transitional cell carcinoma. MATERIALS AND METHODS: A new system designed to deliver simultaneously local bladder hyperthermia and intravesical chemotherapy has been developed at our institute. The system consists of a computerized 915 MHz. microwave source that directly heats the bladder walls (within a temperature range of 42.5 to 45.5C) using a transurethral catheter. From February 1989 to December 1993, 52 patients 44 to 81 years old (mean age 64.3) with superficial stages Ta to T1, grades 1 to 3 transitional cell carcinoma of the bladder were selected for neoadjuvant intracavitary treatment. Tumors were left intact as marker lesions. Of the patients 29 were randomly assigned to receive combined neoadjuvant intravesical chemotherapy and local hyperthermia (group 1), while 23 received intravesical chemotherapy alone (group 2). The treatment protocol included multiple sessions performed on an outpatient basis. Mitomycin C (40 mg. in 50 cc distilled water) was used for intravesical chemotherapy in both groups. All patients underwent transurethral resection of residual tumors and of all suspicious areas 7 to 10 days after completion of treatment. Only a complete response was considered for statistical analysis. RESULTS: A pathological complete response was documented in 19 cases (66%) in group 1 and 5 (22%) in group 2 (chi-square p< 0.01). CONCLUSIONS: According to these preliminary data, microwave induced hyperthermia combined with local intravesical chemotherapy seems to be a feasible, safe and promising approach for neoadjuvant and minimally invasive treatment of superficial bladder cancer. PMID- 8632539 TI - New data from abroad about intravesical therapy of bladder cancer--how does it play in Peoria? PMID- 8632538 TI - The effect of intravesical mitomycin C on recurrence of newly diagnosed superficial bladder cancer: a further report with 7 years of follow up. AB - PURPOSE: We determined the role, if any, of 1 and 5 instillations of intravesical mitomycin C in the treatment of newly diagnosed superficial bladder cancer. MATERIALS AND METHODS: A multicenter randomized clinical trial was done involving 502 patients with newly diagnosed superficial bladder cancer. After complete transurethral resection patients with newly diagnosed superficial bladder cancer. After complete resection patients were randomized into 1 of 3 treatment arms: no further treatment, 1 instillation of mitomycin C at resection and 1 instillation at resection and at 3-month intervals for 1 year (total 5 instillations). The dose of mitomycin C used was 40 mg./40 ml. water. End points were interval to first superficial recurrence, recurrence rate (defined as the number of positive cystoscopies per year) and progression-free interval rate (progression defined as the development of muscle invasive or metastatic disease, or death from bladder cancer). RESULTS: After median followup of 7 years 1 and 5 instillations of mitomycin C resulted in decreased recurrence rates and increased recurrence-free interval. The benefit of mitomycin C was observed in patients at low, medium and high risk for subsequent recurrence. There was suggestive but not conclusive evidence that 5 instillations of mitomycin C offered a slight advantage over 1 instillation. CONCLUSIONS: Our analysis confirms the positive benefit of mitomycin C to decrease the number of subsequent recurrences and increase the recurrence-free interval. PMID- 8632540 TI - Optimal delivery of perioperative chemotherapy: preliminary results of a randomized, prospective, comparative trial of preoperative and postoperative chemotherapy for invasive bladder carcinoma. AB - PURPOSE: We performed a planned interim analysis of a randomized trial comparing initial to postoperative chemotherapy for bladder cancer. The purpose of our analysis was to detect early evidence of survival differences, tolerance to therapy influenced by the sequence of treatment, predictive value of clinical state and influence of methotrexate, vinblastine, doxorubucin and cisplatin (M VAC) on bladder resectability. MATERIALS AND METHODS: A total of 100 consecutive patients were randomized to receive 2 M-VAC courses before and 3 courses after surgery (group 1) or 5 adjuvant M-VAC courses following cystectomy (group 2). Survival, clinical response, clinical and pathological stage, and toxicity were evaluated in this second timed interim analysis. RESULTS: Of all patients 70% received at least 4 M-VAC courses. Overall survival at 31.7 months (range 1.8 to 87.7) was similar in groups 1 (60%) and 2 (63%), and independent of clinical stage. Preoperative clinical staging accurately identified patients at high risk for recurrence, while 37 of the 48 group 2 patients (77%) were considered at high risk by pathological staging (P3b, P4a, node-positive and unresectable disease). Comparison of pathological stage revealed that 14 of the 51 group 1 patients (28%) achieved stage P0 while only 1 of the 48 group 2 patients (2%) had P0 disease at surgery (p= 0.00043). Disease was unresectable in 3 group 1 (6%) and 8 group 2 patients (17%, p= 0.09). Tolerance to treatment was not significantly different in the 2 study arms. CONCLUSIONS: No survival advantage was noted between neoadjuvant and adjuvant M-VAC in our interim analysis. However, results suggest that M-VAC chemotherapy may be effective in increasing the resectability of localized bladder cancer and may contribute to organ preservation. Clinical stage was a reliable predictor of pathological findings at surgery. Future studies can use clinical staging to determine therapy before surgery for the select stages that we treated. Identification of the subset likely to achieve complete pathological remission will permit the selection of patients for organ preservation strategies. PMID- 8632541 TI - Perioperative chemotherapy for invasive bladder cancer--what should we tell our patients? PMID- 8632542 TI - Risk factors for bladder tumors in spinal cord injury patients. AB - PURPOSE: We evaluated risk factors for the development of bladder tumors in spinal cord injury patients. MATERIALS AND METHODS: A retrospective review was done of all bladder tumors at 1 institution with matched controls for 7 years. RESULTS: We identified 17 malignant and 2 benign tumors. Indwelling bladder catheters and a history of bladder stones were statistically significant risk factors. Four patients with negative biopsies underwent repeat biopsy due to suspicious cytology and cancer was found. CONCLUSIONS: An indwelling urinary catheter and a history of bladder stones are statistically significant risk factors. Cytology and biopsy are complimentary in the evaluation of urothelial malignancy in this population. A high index of suspicion and thorough evaluation are needed in spinal cord injury patients. PMID- 8632543 TI - The outcome of patients with classic bladder exstrophy in adult life. AB - PURPOSE: We determined the outcome of 20 older adult bladder exstrophy patients regarding urinary continence, sexual function, fertility and psychosocial integration. MATERIALS AND METHODS: A total of 16 men and 4 women completed an anonymous questionnaire and the charts were reviewed for medical history. RESULTS: Of 9 patients who void spontaneously 6 are dry for 4 hours and 3 for 2 hours. Among the remaining 11 patients 5 are dry on clean intermittent catheterization, 3 who had undergone uterosigmoidostomy are dry for more than 4 hours and 3 had an incontinent stoma. Four men and 2 women are married, including 2 men and 1 women who had a total of 7 children. Ten of 16 men (63%) reported that they ejaculate a few cubic centimeters in volume, 3 ejaculate only a few drops and 3 have no ejaculation (2 of whom underwent cystectomy). Semen analyzed in 4 patients (3 with azoospermia and 1 with oligospermia) and average volume of ejaculate was 0.4 cc (range 0.2 to 1). All women reported regular and normal menstrual periods. A total of 15 patients experienced normal erections that were described as satisfactory by 8 and not satisfactory by 6 due to a small penis, with dorsal chordee in 1. Of the 16 men 12 (75%) experienced satisfactory orgasms while 10 had participated in sexual intercourse with complete partner satisfaction in 9. Half of the men and all women describe intimate relationships as serious and longterm. Of the 20 patients 15 (75%) achieved a high level of education. CONCLUSIONS: Bladder exstrophy patients generally achieve good results but the fertility of most men is in doubt. PMID- 8632544 TI - Magnetic resonance imaging after intraurethral collagen injected for stress urinary incontinence. AB - PURPOSE: Magnetic resonance imaging (MRI) was performed to evaluate the results of intraurethral collagen injected stress urinary incontinence. MATERIALS AND METHODS: A total of 32 women underwent MRI of the pelvis at a median of 12 months after the last injection. The appearance, volume and position of the intraurethral collagen were assessed by 1 radiologist blinded to the outcome, and these findings were compared to clinical data to identify predictive features of success. RESULTS: Intraurethral collagen was easily imaged by MRI and appeared as a hyperintense focus within the wall of the urethra. Neither volume nor position of retained intraurethral collagen was predictive of clinical outcome (p= 0.80 and p= 0.32, respectively). The volume of injected intraurethral collagen strongly correlated with the retained volume in clinically successful and failed cases (Pearson's r= 0.64 and r= 0.90, respectively). No evidence of local or remote pathological conditions resulting from intraurethral collagen injection was identified. CONCLUSIONS: The position and volume of intraurethral collagen were not predictive of clinical outcome. While MRI is not recommended fro routine investigation, it is an excellent research modality for assessing the fate of intraurethral collagen injection. PMID- 8632545 TI - Post-prostatectomy incontinence: urodynamic findings and treatment outcomes. AB - PURPOSE: We examined urodynamic findings and treatment outcomes in a large population of men with post-prostatectomy incontinence. MATERIALS AND METHODS: A total of 215 men was referred for evaluation and treatment of significant post prostatectomy incontinence. Urodynamic evaluation consisted of provocation multichannel medium fill cystometry with vigorous attempts to demonstrate incontinence. Treatment was directed by the results of the urodynamic study. A pad scoring system was used to gauge the severity of incontinence before and after treatment. RESULTS: Based on the results of urodynamic studies 40% of the men had genuine stress incontinence alone and approximately 60% had a major component of bladder dysfunction contributing to incontinence. Treatment results of 135 men demonstrated a significant decrease in pad score (p<0.001) for those treated with anticholinergics, those undergoing artificial sphincter insertion and those treated pharmacologically before sphincter placement. CONCLUSIONS: In our large series most men with prostatectomy incontinence did not have genuine stress incontinence alone. Thus, urodynamic studies are critical, not only to define cause of incontinence but to direct effective therapy. PMID- 8632546 TI - The artificial urinary sphincter for post-radical prostatectomy incontinence: impact on urinary symptoms and quality of life. AB - PURPOSE: We addressed the impact of the artificial urinary sphincter on the health related quality of life and urinary symptoms in men with post-radical prostatectomy incontinence. MATERIALS AND METHODS: A total of 30 men with an AMS800 artificial urinary sphincter following radical prostatectomy responded to a questionnaire dealing with the impact of the symptoms on activities of daily living and quality of life. We compared these results to those of 31 patients who underwent radical prostatectomy but did not require an artificial urinary sphincter. RESULTS: Incontinence was minimal in both groups. Irritative symptoms were noted in the artificial urinary sphincter group, as well as some impairment in activities of daily living. No significant differences were noted with respect to quality of life. CONCLUSIONS: The artificial urinary sphincter is an effective form of therapy for post-radical prostatectomy incontinence but irritative voiding symptoms occur, which tend to limit activities of daily living. PMID- 8632547 TI - The endoscopic fascial sling for treatment of female urinary stress incontinence. AB - PURPOSE: We describe a new technique, the endoscopic fascial sling, for the treatment of female urinary stress incontinence. MATERIALS AND METHODS: A total of 22 women 25 to 84 years old with urinary stress incontinence underwent this procedure. RESULTS: There were no urethral erosions or wound infections using the fascial strip. Of the women 16 (73%) are cured of incontinence and 4 (18%) are improved (use pads less frequently than preoperatively), and the procedure failed in 2 (9%). CONCLUSIONS: The endoscopic fascial sling offers a new alternative for the treatment of female stress incontinence, combining the reliability of sling procedures and the decreased morbidity of needle suspension techniques. PMID- 8632548 TI - The role of clinical research in the diagnosis and treatment of urinary incontinence. PMID- 8632549 TI - Penile vascular assessment using color duplex sonography in men with Peyronie's disease. AB - PURPOSE: We assessed penile vasculature in men with Peyronie's disease using color duplex ultrasound. MATERIALS AND METHODS: A total of 99 men with Peyronie's disease underwent duplex ultrasound with 60 mg. intracavernous papaverine to gain an understanding of penile vasculature and its correlation to erectile rigidity. Patients were stratified into groups according to duplex ultrasound vascular parameters and the presence or absence of impotence (that is rigidity adequate for intromission). RESULTS: Of 97 men 31 (32%) complained of impotence, 8 of 99 (8%) had evidence of corporeal veno-occlusive dysfunction on duplex ultrasound (defined as end diastolic flow velocity greater than 4.5 cm. per second) and 43% had a history of vascular risk factors that may have contributed to erectile insufficiency. Impotent patients had decreased peak systolic flow velocity, increased end diastolic flow velocity and higher a percent of vascular risk factors (p= 0.0006, 0.027 and 0.0004, respectively) compared to potent patients. CONCLUSIONS: Duplex ultrasound provides a dynamic noninvasive functional assessment of penile vasculature in Peyronie's disease. Although corporeal veno occlusive dysfunction has been considered the primary vascular etiology of erectile dysfunction associated with Peyronie's disease, arterial insufficiency is a major contributor, which is best detected before definitive therapy. PMID- 8632550 TI - Correlation of nocturnal penile tumescence monitoring duplex ultrasonography and infusion cavernosometry for the diagnosis of erectile dysfunction. AB - PURPOSE: Nocturnal penile tumescence monitoring was compared to penile duplex ultrasonography and pharmaco-infusion cavernosometry in 50 cases of erectile dysfunction. MATERIALS AND METHODS: Nocturnal penile tumescence was evaluated in all patients as normal or abnormal according to standard general criteria. The results were compared to penile duplex ultrasonography parameters (peak systolic velocity, normal greater than 35 cm. per second, and diastolic velocity, normal less than 5 cm. per second), and to the flow rate needed to maintain erection (normal less than 15 ml. per minute) with pharmaco-infusion cavernosometry. RESULTS: Of the 50 patients 26 had normal nocturnal penile tumescence, including 25 (96%) with normal penile velocity, 18 (69%) with normal penile diastolic velocity and 22 (85%) with normal flow to maintain erection. On the other hand, 24 men had abnormal nocturnal penile tumescence of whom 7 (29%) had abnormal penile blood flow velocity, 17 (71%) had abnormal diastolic flow velocity and 18 (75%) had high flow rate to maintain erection. CONCLUSIONS: Normal nocturnal penile tumescence appears to correlate well with normal systolic blood velocity and cavernosometry but poorly with diastolic blood velocity. On the other hand, a low correlation exists between abnormal nocturnal penile tumescence and abnormal diastolic blood flow or abnormal cavernosometry. Furthermore, no correlation exists between abnormal nocturnal penile tumescence and abnormal systolic blood flow. According to this observation we presume that nocturnal penile tumescence, penile duplex and infusion cavernosometry should be performed to achieve a reasonably accurate diagnosis. PMID- 8632552 TI - Impotence. PMID- 8632551 TI - Minimally invasive therapies in the treatment of erectile dysfunction in anticoagulated cases: a study of satisfaction and safety. AB - PURPOSE: We evaluated patient satisfaction with and the safety of vacuum therapy and self-injection during warfarin treatment of impotent men. MATERIALS AND METHODS: In a 24-week prospective study 33 patients were assigned to vacuum therapy or intracavernous injections with crossover at 12 weeks. Patients maintained diaries, and were followed with physical examinations, coagulation studies and questionnaires. RESULTS: Of the 33 patients 26 completed the study with 706 vacuum applications (mean 1.9 weekly) and 605 injections (mean 1.6 weekly). There are 11 acute minor complications with vacuum therapy (petechiae that resolved spontaneously) and no chronic complications. Only quality of climax was diminished with vacuum therapy. Self-injection resulted in acute minor complications (3 ecchymoses and 1 prolonged erection requiring intervention) and 1 chronic complication (corporeal fibrosis with mild curvature). CONCLUSIONS: The adverse effects of vacuum therapy and intracavernous self-injection in patients on warfarin do not exceed the rate in the general urological population. These therapies appear to be safe in patients receiving warfarin. PMID- 8632553 TI - Quality of semen after extracorporeal shock wave lithotripsy for lower urethral stones. AB - PURPOSE: We investigated whether extracorporeal shock lithotripsy (ESWL) of stones in the distal ureter can cause decreased in male fertility. MATERIALS AND METHODS: In vivo and in vitro examinations were performed to clarify possible changes in semen after ESWL of lower urethral stones. For the in vivo examination semen from 10 patients was examined on the day before and 3 days after ESWL. For the in vitro examination semen from 12 healty volunteers was divided into 2 portions after ejaculation, with half immediately examined according to World Health Organization criteria and half directly exposed to the ESWL focus. RESULTS: The in vitro and in vivo examinations revealed a decrease in sperm density and sperm motility after ESWL. Macroscopic analysis revealed hemospermia in 30% of the patients after ESWL compared to 0% before ESWL. In addition, after ESWL all patients had microscopic hemospermia compared to 10% before ESWL. CONCLUSIONS: The in vivo and in vitro examinations demonstrated deterioration in semen quality. Sperm density and sperm motility returned to normal 3 months after ESWL, indicating at least a transitory decrease in male fertility may occur. PMID- 8632554 TI - Questionnaire-based outcomes study of nononcological post-vasectomy complications. AB - PURPOSE: We conducted an outcomes analysis to determine the incidence of post vasectomy complications. MATERIALS AND METHODS: A questionnaire (154 questions) addressing post-vasectomy complications, incidence of post-vasectomy scrotal pain and quality of life issues was sent to 470 patients. Followup telephone surveys were made. RESULTS: A total of 182 patients (42.3%) responded. Mean follow-up was 4.8 years. The most common complication was post-vasectomy scrotal pain in 34 men (18.7%), which adversely affected the quality of life in 4 (2.2%). In retrospect, 71.4% of the men were satisfied with the decision for vasectomy, 19.3% had equivocal feelings and 9.3% were dissatisfied. CONCLUSIONS: Chronic scrotal pain is the most common post-vasectomy complication that may adversely affect quality of life in men undergoing vasectomy. PMID- 8632555 TI - Seminal improvement following repair of ultrasound detected subclinical varicoceles. AB - PURPOSE: We determined whether repair of subclinical varicoceles detected by scrotal duplex ultrasonography results in significant seminal improvement and identified the best ultrasonographic criteria to use in the selection of patients for subclinical varicocelectomy. MATERIALS AND METHODS: Of 256 consecutive infertile men being evaluated by physical examination and color duplex scrotal ultrasonography 76 underwent varicocele repair and were followed with serial semen analyses. All subclinical varicoceles were confirmed by venography. The outcome of varicocelectomy was determined by changes in total motile sperm count and compared among patients with different clinical grades of varicoceles and ultrasonographically measured in veins sizes. RESULTS: A significant overlap was observed between ultrasonographically measured venous diameter and clinical grade of varicocele. There was no correlation between venous diameter and postoperative outcome when controlled for clinical grade. Significant postoperative improvement in semen parameters was noted in 67% of patients with clinical and only 41% with subclinical varicocelectomy (p<0.05). The best ultrasonographic cutoff to predict a positive outcome after subclinical varicocelectomy was venous diameter greater than 3mm. Patients with larger clinical varicoceles had greater postoperative seminal improvement than those with small or subclinical varicoceles regardless of baseline sperm count. CONCLUSION: Varicocele size has a direct impact on the probability and amount of seminal improvement after varicocelectomy. Outcome following subclinical varicocelectomy is significantly less than after repair of clinical varicoceles. Although 41% of patients with subclinical varicoceles had significant postoperative improvement in semen parameters, an equal number were worse postoperatively and, thus, mean sperm count was unchanged for the group. The results of our study suggest that subclinical varicocelectomy is of questionable benefit. PMID- 8632557 TI - Vasectomy and extracorporeal shock wave lithotripsy hurt-limit varicocelectomy procedures--To transurethral resection or not? PMID- 8632556 TI - Semen parameters before and after transurethral surgery for ejaculatory duct obstruction. AB - PURPOSE: We examined how transurethral resection of the ejaculatory ducts, performed for infertility, affects semen quality in patients with azoospermia and oligo-asthenospermia. MATERIALS AND METHODS: A retrospective review was done of 46 cases of transurethral resection of the ejaculatory ducts for ejaculatory duct obstruction, confirmed by transrectal ultrasound. Clinical course and semen quality were assessed by semen parameter indications. RESULTS: In 65% of the patients transurethral resection of the ejaculatory ducts improved semen quality (greater than a 50% increase in total motile sperm count) and 20% initiated a pregnancy an average of 6.1 months postoperatively. Statistically significant increases in total motile sperm count were achieved in men with azoospermia and those treated for oligo-asthenospermic indications; the improvement also was shown to be sustainable. Complications occurred in 20% of the men. CONCLUSIONS: Significant and durable semen quality improvement can be achieved after transurethral resection of the ejaculatory ducts for all surgical indications. In most unsuccessful cases the reason for failure is unclear. PMID- 8632558 TI - Primary chemotherapy in patients with nonseminomatous germ cell tumors of the testis and biological disease only after orchiectomy. AB - PURPOSE: We assessed the efficacy of primary chemotherapy in patients with nonseminomatous germ cell tumors of the testis and elevated serum tumor markers as the only evidence of disease after orchiectomy. MATERIALS AND METHODS: We analyzed the outcome of 20 patients with biological disease only who received cisplatin-based (16) or carboplatin-based (4) chemotherapy as primary treatment following orchiectomy. RESULTS: Serum tumor markers returned to normal levels in all 20 patients. One patient required subsequent surgery for recurrent retroperitoneal mature teratoma. Two patients experienced a relapse with active disease, 1 of whom died of progressive germ cell tumor. Of the patients 19 remained free of disease 18 to 116 months after the end of treatment. CONCLUSIONS: Since results with primary retroperitoneal lymph node dissection suggest that elevated serum tumor markers usually reflect systemic metastases rather than retroperitoneal disease, primary chemotherapy seems to be the most appropriate strategy to consider in patients with biological disease only following orchiectomy. PMID- 8632559 TI - Determination of prostatic volume with transrectal ultrasound: A study of intra observer and interobserver variation. AB - PURPOSE: We assessed the intra-observer and interobserver variation in measurements of prostatic volume using transrectal ultrasound. MATERIALS AND METHODS: Two volume estimations were performed by 1 observer in 40 patients and by 2 observers in 75. RESULTS: Mean prostatic volumes measured ranged from 18.9 to 87.0 ml. (median 39.8) for 1 observer and from 15.5 to 95.4 ml. (median 42.4) for 2 observers. The mean difference plus or minus standard deviation and limits of agreement between the paired volume estimations were 0.1 +/- 3.3 ml. and -6.5 +/- 6.7 ml., respectively, for 1 observer, and -0.3 +/- 5.3 ml and -10.9 to 10.3 ml., respectively, for 2 observers. The mean difference was 9.8% for 2 observers. The mean difference was 9.8% for 2 observers compared to 4.6% for 1. CONCLUSIONS: The results show that there is considerable variation in repeated measurements of prostatic volume and the variation is greatest for 2 observers compared to 1 observer. PMID- 8632560 TI - Transurethral marsupialization of a medial prostatic cyst in patients with prostatitis-like symptoms. AB - PURPOSE: The results of transurethral marsupialization as treatment for medial prostatic cysts were assessed. MATERIALS AND METHODS: Between June 1992 and August 1994 we performed transrectal ultrasound on 704 patients with symptoms of bladder outlet obstruction or lower urinary tract symptoms and a medical prostatic cyst was found in 34 (5%). Transurethral marsupialization of the cyst via incision of the prostatic floor under transrectal ultrasound guidance was performed in 18 patients. Followup was 12 to 25 months (mean 18). RESULTS: Patients with a medial prostatic cyst complained of prostatitis-like symptoms (77%), scrotal pain (62%), impaired micturition (32%), small volume ejaculation (35%), painful ejaculation (24%), hemospermia (24%) and infertility (12%). After marsupialization of the cyst, symptoms resolved completely in 14 patients (78%), improved in 17 (94%) and did not improve in only 1 (6%). No complications of this procedure were noted. The 16 patients who did not undergo surgery still complain of prostatitis-like symptoms without evidence of bacterial prostatitis. CONCLUSIONS: We believe that a medial prostatic cyst can cause prostatitis-like symptoms and that marsupialization of the cyst can provide symptom relief in the majority of patients. PMID- 8632562 TI - Prostate disease potpourri--measurement reliability, therapeutic effectiveness and pathophysiological diagnosis. PMID- 8632561 TI - Differential diagnosis of prostatism: A 12-year retrospective analysis of symptoms, urodynamics and satisfaction with therapy. AB - PURPOSE: We determined the incidence of voiding symptoms, urodynamic etiology and satisfaction with therapy in a large cohort of men with prostatism during a 12 year period. MATERIALS AND METHODS: We retrospectively analyzed the records of 2,845 consecutive men who underwent urodynamic evaluation between January 1982 and December 1994. Patients were divided into groups 1 and 2 according to the years of study (between 1982 and 1988, and between 1989 and 1994, respectively). Parameters of evaluation included prevalence and distribution of voiding symptoms, urodynamic etiology of symptoms and satisfaction with therapy (medical or surgical). RESULTS: There was 843 evaluable patients 50 to 94 years old (mean age 63.2) Group 2 patients were younger, and had a 22% higher prevalence of nocturia and a 12% higher prevalence of daytime frequency. The prevalence of all other symptoms was the same in both groups. On urodynamics 523 patients (62%) had demonstrable evidence of bladder outlet obstruction of whom 345 (66%) had concomitant detrusor instability. Of the 843 patients 647 (77%) had detrusor instability, which was the sole diagnosis in 199 (24%). We noted low pressure/low flow in 137 patients (16%) and impaired detrusor contractility in 152 (17%), including 57 (7%) in whom the latter condition was the only diagnosis. Urodynamic findings remained the same during the entire 12-year period. Global satisfaction and symptomatic improvement were better with surgical than medical therapy, although the degree of satisfaction was independent of the urodynamic etiology of symptoms. CONCLUSIONS: Symptomatic men with prostatism are presenting with a greater prevalence of significant nocturia and daytime frequency than in the past with no change in urodynamic findings. In addition, patient level of satisfaction remains greater with surgical than medical therapy regardless of the urodynamic presence of bladder outlet obstruction. PMID- 8632563 TI - Cost consequences of surveillance, medical management or surgery for benign prostatic hyperplasia. AB - PURPOSE: The cost consequences of alternative treatment modalities for benign prostatic hyperplasia (BPH) were investigated. The present lifetime costs of watchful waiting, medical management and surgery alone and in various combinations were estimated for a synthetic cohort of men comprised of 5 age groupings. MATERIALS AND METHODS: Synthetic cohort models were constructed to follow men at different ages "analytically" for specific intervals and to calculate the cumulative health care costs associated with alternative BPH treatment regimens during those periods. These models accounted explicitly for survival probabilities, the use of different types of health care services and products, price changes for those services and products, failure rates of some therapies and a discount factor needed to compute the present value of the cost streams. The models were implemented with hospital discharge and other data on BPH incident cases in the state of Florida in approximately 1989. RESULTS: The addition of medical management to the mix of therapies is likely to increase overall health care spending on BPH treatment, perhaps by a considerable amount. The cost-effectiveness of each type of BPH therapy differs by the age of the patient at which it is first initiated. All other parameters being equal, surgery appears to be more cost-effective at younger patient ages, while medical management has a cost advantage at older ages. CONCLUSIONS: The cost implications of alternative BPH therapies are substantial, and warrant more detailed consideration by clinicians and health policy specialists. PMID- 8632564 TI - Japanese men have smaller prostate volumes but comparable urinary flow rates relative to American men: results of community based studies in 2 countries. AB - PURPOSE: We compared prostate volume and peak urinary flow rate in Japanese and American men 40 to 79 years old. MATERIALS AND METHODS: Prostate volume and peak urinary flow rate were measured in eligible Japanese men and results were compared to those of a randomly selected American cohort. RESULTS: Mean prostate volume plus or minus standard deviation averaged 20.3 +/- 10.6 ml. in Japanese and 29.6 +/- 13.4 ml. in American men, while predicted cross-sectional increases with age decade were 1.5 and 5.5 ml., respectively. Peak urinary flow rate was higher but the decrease with increasing age was greater in Japanese men. CONCLUSIONS: Prostate volume is larger and the increase with age is more pronounced in American than in Japanese men. However, Japanese men may have a higher peak urinary flow rate and greater cross-sectional decrease with age. PMID- 8632565 TI - Benign prostatic hyperplasia. PMID- 8632566 TI - The effects of ejaculation on serum prostate specific antigen. AB - PURPOSE: We evaluated the correlation of prostate specific antigen (PSA) and ejaculation in patients with symptomatic benign prostatic hyperplasia and an active sexual life. MATERIALS AND METHODS: In a study of 40 patients 50 to 60 years old (mean age 55) with prostatic symptoms serum PSA was evaluated before as well as 1 and 7 days after ejaculation. Due to clinical significance of PSA in diagnosis and monitoring of prostate cancer, we included men at risk age and with an active sex life. RESULTS: The results were compared to those of a control group of 10 asymptomatic (without coitus) men 50 to 60 years old (mean age 55 years). There were no statistically significant differences in PSA levels before and after ejaculation or between the groups. These results suggested that there was no physiological relationship between ejaculation and PSA level. CONCLUSIONS: Based on our data we conclude that sexual activity does not preclude the use of PSA to screen men for prostatic cancer. PMID- 8632567 TI - Prostate specific antigen is metabolized in the liver. AB - PURPOSE: The site of metabolism of prostate specific antigen (PSA) was determined. MATERIALS AND METHODS: In a prospective study, during clinically indicated left and right heart catheterizations for various cardiac diseases in 12 men (mean age 62.5 +/- 8.3 years, standard deviation), selective blood samples were obtained from the infra-renal, infra-hepatic and supra-hepatic inferior vena cava, renal vein, superior vena cava, pulmonary artery and femoral artery. Mean PSA concentration was calculated for all vascular sites. Using a paired Student t test, the mean difference between the afferent and efferent PSA concentrations across the renal, hepatic, pulmonary and pelvic circulation was calculated. RESULTS: The hepatic gradient between the infra-hepatic and suprahepatic inferior vena cava showed the greatest decrease (0.11 +/- 0.16 ng./ml. or 8.3%) in PSA concentration and was statistically significant (p = 0.04). A smaller decrease across the pulmonary circulation was statistically insignificant. No decrease in the PSA concentration was noted across the renal circulation. The PSA concentration increased significantly (0.19 +/- 0.18 ng./ml. or 16.3%, p = 0.003) across the pelvic circulation, confirming the release of PSA from the prostate. CONCLUSIONS: PSA is released from the prostate. The kidneys and lungs do not have a significant role in elimination of PSA, and the liver appears to be the most likely site of its metabolism. Although our sample size is small and the PSA range is narrow, our results strongly support these conclusions. PMID- 8632568 TI - Standard versus age-specific prostate specific antigen reference ranges among men with clinically localized prostate cancer: A pathological analysis. AB - PURPOSE: Age-specific prostate specific antigen (PSA) references ranges have been suggested to account for the age-dependent nature of the serum PSA concentration. It has been hypothesized that reference ranges of 0 to 2.5 ng./ml.serum PSA (40 49 years), 0 to 3.5 ng./ml. (50-59 years), 0 to 4.5 ng./ml. (60 to 69 years) and 0 to 6.5 ng./ml. (70 to 79 years) would detect fewer (potentially insignificant) prostate cancers in older men and more (potentially curable) cancers in younger men. MATERIALS AND METHODS: To investigate the pathological stage of tumors that would be affected by the use of age-specific PSA references ranges, we reviewed the medical records for 4,597 men with clinically localized (stage T1c, T2, or T3a) prostate cancer, with an average age of 62 +/- 7 years (range 38 to 76), who underwent radical prostatectomy between 1984 and 1994 at our institutions. Favorable pathological results were defined as organ-confined disease or capsular perforation with a Gleason score of less than 7, and unfavorable pathological results were defined as capsular perforation with a Gleason score of 7 or more, seminal vesicle invasion or lymph node involvement. RESULTS: Overall, 18% of the men had PSA levels less than the standard PSA reference range (4.0 ng./ml.) compared to 22% when using the age-specific ranges. There were 74 more cancers detected in men younger than 60 years with the use of age-specific ranges, of which 81% had favorable pathological results. Among the men 60 years or older, 191 of 252 cancers (76%) not detected by using age specific ranges less than 3% were also stage T1c and 95% of these undetected T1c cancers were of favorable pathological status. Of those cancers not detected in older men with the age specific ranges were of favorable pathological status. Of those cancers not detected in older men with age-specific ranges less than 3% were also stage T1c and 95% of these undetected T1c cancers were of favorable pathological status. Age-specific PSA reference ranges increased the potential for detection of prostate cancer by 18% in the younger men and decreased the detection by 22% in the older men. CONCLUSIONS: Among these men with clinically localized prostate cancer, age specific PSA references ranges increased the detection of more potentially curable tumors in young men and decreased the detection of less advanced tumors in the older men compared to the standard reference range of 4.0 ng./ml. Among older men with nonpalpable (stage T1c) tumors age-specific PSA references ranges would have detected fewer tumors. However, 95% of these "missed" tumors would have had favorable pathological findings. PMID- 8632569 TI - Plasma neuroendocrine markers in patients with benign prostatic hyperplasia and prostatic carcinoma. AB - PURPOSE: Approximately 50% of all malignant prostatic tumors contain neuroendocrine cells, which cannot be attributed to small cell prostatic carcinoma or carcinoid-like tumors, and which represent only 1 to 2% of all prostatic malignancies. Only limited data are available concerning the plasma levels of neuroendocrine markers in patients with prostatic tumors. Therefore, we determine the incidence of high plasma levels of neuroendocrine markers in patients with benign and malignant prostatic disease. MATERIALS AND METHODS: The presence of elevated plasma neuropeptide levels was investigated in 135 patients with prostatic carcinoma and 28 with benign prostatic hyperplasia. Plasma chromogranin A, neurone-specific enolase, substance P, calcitonin, somatostatin, neurotensin and bombesin levels were analyzed by immunoassays, and were compared to clinical and pathological stages of disease. Plasma prostatic acid phosphatase and prostate specific antigen levels were also determined. All patients were followed for at least 2 years after inclusion in the study. RESULTS: Significantly elevated levels of chromogranin A were detected in 15% of patients with prostatic carcinoma before any treatment. During hormone resistant prostate cancer progression plasma chromogranin A and neuron-specific enolase levels were elevated in 55% and 30% of the patients, respectively. In patients with stage D3 disease survival curves were generated by the Kaplan-Meier method, and log rank analysis revealed a statistically significant difference between groups positive and negative for chromogranin A. Substance P and bombesin were also occasionally elevated in prostatic tumors. Determination of neuroendocrine differentiation by neuron-specific enolase or chromogranin A immunoassays was not helpful in the prediction of progressive localized prostatic carcinoma. CONCLUSIONS: Future studies of plasma neuropeptide levels should confirm whether these parameters can be used as prognostic markers during late progression of prostatic carcinoma or for the selection of patients suitable for evaluation of new antineoplastic drugs to be active against neuroendocrine tumors. PMID- 8632570 TI - Preoperative prediction of tumor heterogeneity and recurrence after radical prostatectomy for localized prostatic carcinoma with digital rectal, examination prostate specific antigen and the results of 6 systematic biopsies. AB - PURPOSE: Digital rectal examination, preoperative serum prostate specific antigen (PSA) concentration and results of 6 ultrasound guided systematic sextant biopsies in 257 consecutive patients with clinical stages T2 and T1c prostatic carcinoma were evaluated for their use in predicting pathological stage and tumor recurrence. MATERIALS AND METHODS: Each of the 257 consecutive specimens was examined using the 3 mm. step section technique. Results of preoperative digital rectal examination, PSA and 6 systematic sextant biopsies were correlated with pathological stage, margin status and postoperative PSA during a mean followup of 2 years. Patients were considered to have disease progression based on elevated PSA level by a supersensitive assay. RESULTS: Digital rectal examination could not predict pathological stage and tumor recurrence. Preoperative PSA concentration, number of positive biopsies and tumor grade in the biopsy specimens correlated well with pathological stage. The best predictor of tumor recurrence was the biopsy result. However, a precise prediction of outcome (87% probability of being PSA negative versus 0%) was possible only in a third of the patients if the biopsy results were used. Use of preoperative PSA concentration did not improve this probability. CONCLUSIONS: Preoperative PSA concentration and/or biopsy results correlate significantly with pathological stage and margin status. Precise prediction of tumor recurrence is possible in only approximately a third of the patients with clinical stage T2 prostatic carcinoma. PMID- 8632571 TI - Clinical efficacy of bone alkaline phosphatase and prostate specific antigen in the diagnosis of bone metastasis in prostate cancer. AB - PURPOSE: We investigated the usefulness of bone alkaline phosphatase isoenzyme and prostate specific antigen (PSA) determined by radioimmunoassay to predict bone scan evidence of metastasis in newly diagnosed untreated and treated prostate cancer. MATERIALS AND METHODS: We analyzed bone alkaline enzyme concentrations in 350 men, including 150 controls, 100 with benign prostatic hyperplasia and 100 with prostate cancer (52 with stages T1 to 4, M0 and 48 with stages T1 to 4, M1 to 4). We also analyzed bone alkaline phosphatase enzyme concentrations in 61 stages T1 to 4, M0 prostate cancer cases during followup after radical prostatectomy or hormonal therapy, and 17 had clinical progression (9 with local, 5 with lymph node and 3 with bone metastases). Simultaneously, we analyzed PSA concentrations. RESULTS: Average bone alkaline phosphatase enzyme levels were 12, 11.1 and 10.0 ng./ml. in the control, benign prostatic hyperplasia and stage M0 prostate cancer groups, respectively (p not significant), and 83.2 ng./ml. in patients with stage M1 to 4 disease (p<0.001). Considering that to diagnose bone metastasis the cutoff for bone alkaline phosphatase enzyme and PSA is 30 ng./ml. and 100 ng./ml., respectively, clinical effectiveness was 93.7% and 81.8%, respectively. Finally, measurement of both substances at the same time increased clinical effectiveness to 97.9%. During followup a bone alkaline phosphatase enzyme level that becomes greater than 30 ng./ml. (0% in the local and lymphatic progression groups, and 100% in the bone metastasis group) indicates the need to perform a bone scan. CONCLUSIONS: We recommend the clinical use of bone alkaline phosphatase enzyme determined by radioimmunoassay and PSA measurement for the diagnosis of bone metastases and progression of prostate cancer because of the good sensitivity and specificity. PMID- 8632572 TI - A new long acting formulation of the luteinizing hormone-releasing hormone analogue goserelin: results of studies in prostate cancer. AB - PURPOSE: To assess the pharmacodynamic equivalence of the new 10.8 mg. goserelin depot with the current 3.6 mg. depot 3 studies were performed in patients with advanced prostate cancer. MATERIALS AND METHODS: In 2 comparative studies 160 patients were randomized for dosing every 12 weeks using the 10.8 mg. depot or every 4 weeks using the 3.6 mg. depot. In the noncomparative study 35 patients received the 10.8 mg. depot. Blood sampling for serum testosterone and evaluation of toxicity was done during the 48-week study period. RESULTS: Serum testosterone profiles of the 10.8 and 3.6 mg. goserlin depots were similar with testosterone levels decreasing into the castrate range by day 21 after depot administration. The safety profile of 10.8 mg. goserelin is comparable to that of the current monthly depot with the main side effects related to androgen deprivation. CONCLUSIONS: The new long acting depot was pharmacologically equivalent, and well tolerated locally and systemically, and will offer added convenience to patients and health care personnel. PMID- 8632573 TI - Prostate cancer--staging and prognosis. PMID- 8632574 TI - Randomized study of neoadjuvant testicular androgen ablation therapy before radical prostatectomy in men with clinically localized prostate cancer. AB - PURPOSE: We determined whether 12 weeks of neoadjuvant testicular androgen ablation therapy using a luteinizing hormone-releasing hormone agonist could improve pathological outcomes in men undergoing radical retropubic prostatectomy for clinically localized (stages T1C, T2A and T2B) prostatic carcinoma. MATERIALS AND METHODS: A total of 56 participants was randomized to receive either monthly injections of a luteinizing hormone-releasing hormone agonist at 4-week intervals followed by radical retropubic prostatectomy (28) or to undergo immediate radical retropubic prostatectomy alone (28). Operations were performed via similar technique and all prostatic specimens were processed histologically in their entirety. RESULTS: There was no improvement in pathological outcome using luteinizing hormone-releasing hormone agonist preoperatively compared to surgery alone. Of 28 men undergoing immediate radical retropubic prostatectomy 23 had organ-confined (17) or specimen-confined (6) disease versus 22 of 28 who received luteinizing hormone-releasing hormone neoadjuvant therapy for 12 weeks preoperatively (16 with organ-confined and 6 with specimen-confined disease, p = 1.00). In addition, when the study population was analyzed by pretreatment prostate specific antigen (PSA) levels (10 ng./ml. or less, or greater than 10 ng./ml/) there was also no difference in pathological outcome (p = 0.65 for PSA greater than 10 and p = 0.32 for PSA less than 10). CONCLUSIONS: Neoadjuvant androgen ablation therapy for 12 weeks before radical prostatectomy in patients with clinically localized adenocarcinoma of the prostate does not result in improved pathological outcomes. PMID- 8632575 TI - Prediction of capsular perforation and seminal vesicle invasion in prostate cancer. AB - PURPOSE: Capsular perforation and seminal vesicle invasion are unfavorable prognostic factors in prostate cancer. Accurate preoperative prediction of these factors would be clinically useful for planning treatment, especially in patients being considered for radiation therapy, nerve sparing radical prostatectomy and watchful waiting. However, current methods are imprecise at predicting the presence and extent of these factors. We determined which combination of commonly available preoperative variables provides the best prediction of capsular perforation and seminal vesicle invasion of patients with clinically localized prostate cancer. MATERIALS AND METHODS: We reviewed the preoperative medical records and biopsy findings from 314 patients with clinical stages T1cN0M0 to T2cN0M0 cancer who underwent radical retropubic prostatectomy and bilateral pelvic lymphadenectomy between September 1991 and June 1993. Radical prostatectomy specimens were embedded and evaluated by whole mount sections. RESULTS: Capsular perforation was observed in 104 patients (33.1%) and seminal vesicle invasion was noted in 46(14.6%). Preoperative variables predictive of capsular perforation and seminal vesicle invasion on univariate analysis were serum prostate specific antigen (PSA) concentration, clinical, stage, Gleason primary and secondary patterns, Gleason score, nuclear grade, perineural invasion and percent cancer in the biopsy specimens. On multivariate analysis, independent prognostic factors for capsular perforation and seminal vesicle invasion were PSA, Gleason score and percent cancer in the biopsy specimens. CONCLUSIONS: The combination of serum PSA concentration, Gleason score and percent cancer in the biopsy specimens provides the best prediction of capsular perforation and seminal vesicle invasion. Models based on this combination of factors may be clinically use to stratify patients for nonoperative treatment. PMID- 8632577 TI - The everting suture: A new technical aid for ureteral nipple construction. AB - PURPOSE: A new technique using an everting suture to construct a ureteral nipple is described. MATERIALS AND METHODS: The method was used to create 28 ureteral nipples. A 2-zero silk suture is passed through the ureteral wall, transfixing the lumen, and is tied 5 cm. away from the ureteral wall. The ends are cut 10 cm. long. The suture is grasped within the ureteral lumen and with traction the ureter is everted in 1 step without trauma. RESULTS: The suture in smooth 1-step atraumatic eversion of the ureter in all cases. CONCLUSIONS: With this technique a well formed nipple of the desired length can be constructed. PMID- 8632576 TI - Catecholamine release caused by carbon dioxide insufflation during laparoscopic surgery. AB - PURPOSE: We evaluated plasma catecholamine levels during pneumoperitoneum in laparoscopic surgery. MATERIALS AND METHODS: Plasma epinephrine and norepinephrine were evaluated in 29 patients who underwent laparoscopic retroperitoneal surgery in a half lateral decubitus position (group 1) or laparoscopic varicocelectomy in a Trendelenburg position (group 2). RESULTS: The levels of epinephrine and norepinephrine increased significantly 5 minutes after carbon dioxide insufflation compared to levels after Veress needle insertion and just before insufflation. The elevation of catecholamine levels during laparoscopic procedures was greater in group 1. CONCLUSIONS: Our results indicate that carbon dioxide insufflation may cause catecholamine release during laparoscopic surgery. Careful monitoring of hemodynamics is mandatory at the beginning of the procedure. PMID- 8632578 TI - Fogarty catheter extraction of unusual urethral foreign bodies. AB - PURPOSE: The removal of multiple obstructing foreign bodies from the urethra of an acutely ill, elderly man is describe. MATERIALS AND METHODS: A 4F Fogarty catheter under fluoroscopic guidance was used to manipulate, with slow traction, several clarified foreign bodies from the bulbar urethra to the meatus for extraction. RESULTS: The urethra was cleared rapidly, allowing drainage of retained, infected urine via a Foley catheter. Inspection of the objects revealed incrusted, friable masses consistent with nut shells. CONCLUSIONS: Some obstructive urethral foreign bodies can be expediently removed in the emergency setting with a fluoroscope and a Fogarty catheter in a well lubricated urethra. Self-insertion of foreign bodies into the urethra usually accompanies a psychiatric co-diagnosis, which should also be evaluated. PMID- 8632579 TI - New use of Foley catheter for precise vesicourethral anastomosis during radical retropubic prostatectomy. AB - PURPOSE: We describe a new method of using a Foley catheter to assist vesicourethral anastomosis during radical retropubic prostatectomy. MATERIALS AND METHODS: A total of 81 patients underwent radical retropubic prostatectomy with this technique. Followup ranged from 4 to 48 months. Peri-catheter urethrograms were performed at 3 weeks. Patients were evaluated specifically for bladder neck contracture, urinary continence and prolonged catheterization. RESULTS: Bladder neck contracture, occurred in 4.9% of the patients and 87.6% were completely continent of urine. Only 1 patient required extended postoperative catheterization. CONCLUSIONS: Use of a Foley catheter for vesicourethral anastomosis is consistent and simple, and provided good surgical results in our experience. PMID- 8632581 TI - Laparoscopically assisted heminephrectomy of a horseshoe kidney: a case report. PMID- 8632580 TI - Results of the treatment of neurogenic bladder dysfunction in spinal cord injury by sacral posterior root rhizotomy and anterior sacral root stimulation. AB - PURPOSE: We evaluated the results of treatment of neurogenic bladder dysfunction in spinal cord injury by sacral posterior root rhizotomy and anterior sacral root stimulation using the Finetech-Brindley stimulator. MATERIALS AND METHODS: In 52 patients with spinal cord lesions and urological problems due to hyperreflexia of the bladder complete posterior sacral root rhizotomy was performed and a Finetech Brindley sacral anterior root stimulator was implanted. All patients were evaluated and followed with a strict protocol. A minimal 6-month followup is available in 47 cases. RESULTS: Complete continence was achieved in 43 of the 47 patients with 6 months of followup. A significant increase in bladder capacity was attained in all patients. Residual urine significantly decreased, resulting in a decreased incidence of urinary tract infections. In 2 patients upper tract dilatation resolved. In 3 patients rhizotomy was incomplete and higher sectioning of the roots was necessary. One implant had to be removed because of infection. CONCLUSIONS: The treatment of neurogenic bladder dysfunction in spinal cord injury by anterior sacral root stimulation with the Finetech-Brindley stimulator in combination with sacral posterior root rhizotomy provides excellent results with limited morbidity. PMID- 8632583 TI - Spontaneous regression of primary and metastatic renal cell carcinoma. PMID- 8632582 TI - Ballooning of the duodenal segment after a pancreas-kidney transplantation. PMID- 8632584 TI - Hydronephrosis and hydroureter with extremely high levels of serum carbohydrate antigens 19-9 and SPan-1: a case report. PMID- 8632585 TI - Sliding inguinal hernia containing the ureter: a case report. PMID- 8632588 TI - Inverted papilloma: an unusual recurrent, multiple and multifocal lesion. PMID- 8632587 TI - Pregnancy in a patient with an ileal substitute bladder followed by severe destabilization of the pelvic support. PMID- 8632586 TI - Retroperitoneal chloroma presenting with uremia, bilateral ureteral obstruction and encasement of the great vessels. PMID- 8632589 TI - Priapism revealing ARG 506 to GLN factor V mutation. PMID- 8632590 TI - Discordance between the site of primary nonseminomatous germ cell tumor of the testis and retroperitoneal metastatic disease secondary to anomalous venous drainage of the testis. PMID- 8632591 TI - Re: Buccal mucosal urethral replacement. PMID- 8632592 TI - Re: Osteoporosis as a complication of orchiectomy in 2 elderly men with prostatic cancer. PMID- 8632593 TI - Re: The surgical correction of buried penis: A new technique. PMID- 8632594 TI - Re: Selection of optimal prostate specific antigen cutoffs for early detection of prostate cancer: receiver operating characteristic curves. PMID- 8632595 TI - Segmental multicystic kidney and ipsilateral duplication anomalies. AB - PURPOSE: We characterize segmental multicystic dysplasia, in which there is a duplex collecting system with multicystic changes in the upper pole. MATERIALS AND METHODS: Three neonates with an abdominal mass and/or prenatal diagnosis of a multicystic kidney were evaluated. Postnatal ultrasound showed a classic multicystic kidney and small ipsilateral orthotopic ureterocele in all cases. All patients had ipsilateral grade V lower pole reflux and a renal scan showed functioning lower pole systems. RESULTS: One patient underwent bilateral ureteroneocystostomy and another underwent upper pole nephrectomy. There has been reduction in the size of the segmental multicystic kidney in 2 patients and decrease in reflux grade in 2 (followup 2 to 5 years). CONCLUSIONS: Newborns with an apparent multicystic kidney should undergo a voiding cystourethrogram and renal scan to confirm the diagnosis. Segmental multicystic kidney can be followed nonoperatively in most cases because the cysts tend to involute. PMID- 8632596 TI - Continent urinary diversion using preputial penile or clitoral skin flap. AB - PURPOSE: We report our efforts to create an ideal continent urinary catheterizable stoma. MATERIALS AND METHODS: Our approach includes the creation of a continent stoma using a preputial penile island skin flap in male patients and a preputial clitoral island skin flap in female patients. The flaps are transposed suprapubically, tubularized and implanted in a nonrefluxing manner into the bladder or other urinary reservoir. RESULTS: From 1994 to April 1995 the technique was performed in 14 male patients 4 months to 42 years old and 7 female patients 4 to 14 years old. Early results were satisfactory at 2 to 12 months of follow up (mean 6). CONCLUSIONS: This technique could be an alternative to other methods of urinary continent diversion. PMID- 8632597 TI - Common sheath reimplantation yields excellent results in the treatment of vesicoureteral reflux in duplicated collecting systems. AB - PURPOSE: We evaluated our 10-year experience with the surgical treatment of vesicoureteral reflux in uncomplicated duplicated collecting systems. MATERIALS AND METHODS: Between 1984 and 1994, 54 refluxing renal units (8 bilateral) in 37 female and 9 male patients required surgery. Patient age ranged from 7 months to 17 years (average 4.9 at surgery). Postoperative followup (average 14.2 months) included voiding cystourethrography and renal sonography or excretory urography. RESULTS: Common sheath ureteral reimplantation via an intravesical approach was performed in 48 of the 54 refluxing renal units. Of the remaining 6 renal units detrussorrhaphy was performed in 4, and ureteroureterostomy combined with ureteral reimplantation and partial lower pole nephrectomy were done in 1 each. Two treated renal units had persistent postoperative vesicoureteral reflux, which resolved after subureteral polytetrafluoroethylene (Teflon) injection. No renal had postoperative hydronephrosis. Contralateral reflux was identified in 1 patient who underwent unilateral reimplantation. Our overall success rate was 96% for the surgical correction of vesicoureteral reflux in uncomplicated duplicated collecting systems. Common sheath reimplantation had a 98% success rate. CONCLUSIONS: Although a duplicated collecting system increases the risk for surgical treatment, the presence of a duplication anomaly does not adversely affect surgical outcome. Modifications of procedures commonly performed in the surgical treatment of single system reflux to accommodate common sheath reimplantation have excellent surgical results with minimal morbidity. PMID- 8632598 TI - Nephrogenic adenoma occurring in an augmented bladder. PMID- 8632599 TI - Vesical neck reconstruction in patients with the exstrophy-epispadias complex. AB - PURPOSE: We evaluated the factors critical in achieving urinary medicine continence in patients with the exstrophy-epispadias complex. MATERIALS AND METHODS: A total of 51 patients with epispadias and 33 with classic bladder exstrophy underwent vesical neck reconstruction. Patient records were reviewed to identify factors associated with achievement of continence, including timing of bladder closure and urethroplasty, effect of bladder capacity before and after vesical reconstruction, and effect of enterocystoplasty. RESULTS: Complete urinary continence was achieved in 42 of the 51 patients with epispadias (82%) and in 23 of the 33 with exstrophy (70%). Delayed bladder closure did not affect the ability to gain continence but increased the likelihood of subsequent enterocystoplasty (12 of 19 patients, 63%) compared to early bladder closure (5 of 14 patients, 36%). CONCLUSIONS: Preliminary urethroplasty did not enhance urinary control or reduce the need for enterocystoplasty. Bladder capacity before vesical neck reconstruction did not predict the need for enterocystoplasty or ultimate continence status in individuals. Adequate bladder capacity after vesical neck reconstruction was an important determinant of urinary continence. Approximately half of the patients bladder exstrophy (17 of 33) required augmentation cystoplasty to achieve urinary continence. PMID- 8632600 TI - Complete duplication of the bladder and urethra in the coronal plane: case report. PMID- 8632602 TI - Priapism associated with sickle cell hemoglobinopathy in children: long-term effects on potency. AB - PURPOSE: Children with sickle cell priapism have traditionally been treated conservatively with surgery done as a last resort. Only sparse subjective data are available on the long-term assessment of potency in these patients. MATERIALS AND METHODS: We retrospectively reviewed the charts of all pediatric patients with sickle cell priapism who presented to Children's Hospital of Michigan between 1972 and 1992, and subsequently assessed erectile capabilities subjectively by questionnaire and objectively by RigiScan. RESULTS: Of the 15 patients interviewed 5 had undergone shunt procedures. The return of potency tended to vary inversely with patient age at onset and duration of priapism. CONCLUSIONS: Shunts performed within 48 hours, especially in postpubertal children, seemed more likely to preserve potency. PMID- 8632601 TI - The Malone antegrade colonic enema enhances the quality of life in children undergoing urological incontinence procedures. AB - PURPOSE: Functional alterations of the gastrointestinal and genitourinary tracts, and physical limitations in children with spina bifida, imperforate anus and spinal cord injury challenge the ability to have independent fecal and urinary continence. Urologists have successfully helped these patients achieve urinary continence. We report our experience with the antegrade colonic enema procedure, which allows select individuals to achieve continence of stool, enhancing quality of life. MATERIALS AND METHODS: Since December 1992, 18 antegrade colonic enema procedures were performed in 12 female and 6 male patients 5 to 31 years old of whom 14 had spina bifida, 2 had imperforate anus and 2 had spinal cord injury. Simultaneous urological continence procedures were performed in 8 patients, including appendicovesicostomy in 4, augmentation cystoplasty in 2 and augmentation cystoplasty plus an ileal Mitrofanoff procedure in 2. Four patients previously underwent urological reconstruction. RESULTS: In 24 months of followup (average 6.6) all patients with a functioning stoma remained continent of stool and 17 were continent of urine. Complications related to the antegrade colonic enema procedure occurred in 4 children (22%) of whom 3 required further surgery. Three patients (17%) had minor stomal stenosis. CONCLUSIONS: The antegrade colonic enema procedure is easily performed and it should be considered for any child with significant physical limitations and/or refractory fecal incontinence before urological continence promoting procedures are done. PMID- 8632603 TI - Painless scrotal masses in the pediatric population: prevalence and age distribution of different pathological conditions--A 10 year retrospective multicenter study. AB - PURPOSE: In the pediatric population a broad spectrum of intrascrotal pathology ranging from congenital to neoplastic lesions present as a painless scrotal mass. The aim of our 10-year retrospective study was to review 71 pediatric cases of a painless scrotal mass to determine the overall and age specific prevalence of diseases manifesting as such masses. MATERIALS AND METHODS: From 1980-1991, 71 patients 1 day to 16 years old with a painless scrotal mass underwent evaluation using 7.5 or 10 MHz. ultrasound probes with transverse and longitudinal sections. RESULTS: The painless scrotal mass was testicular in 61 cases (86%, 28 neoplasms, 27 congenital malformations, and 6 posttraumatic/inflammatory lesions) and extratesticular in 10 (14%, 5 neoplasms, 2 hematoceles, 2 pachyvaginitis and 1 sebaceous cyst). Patient age distribution showed 2 peaks at 0 to 1-year and 13 to 14-year intervals. More than a third of the painless scrotal masses (24 cases) were found during the first year of life, predominantly congenital anomalies (in utero torsion) and neoplasia. A total of 45 patients (63%) underwent surgery (orchiectomy in 39 and conservative treatment in 6) and a pathognomonic echo pattern allowed nonsurgical treatment in 26 (37%). CONCLUSIONS: Testicular ultrasound proved to be highly reliable in differentiating intratesticular from extratesticular lesions but it demonstrated poor specificity because of extensive overlap between benign and malignant pathologies. Therefore, testicular ultrasound changed the management of a few select cases of a painless scrotal mass (epididymal cysts/spermatoceles and in utero torsion). PMID- 8632604 TI - Fibromatosis of the spermatic cord in a child: case report. PMID- 8632605 TI - Urinary nitric oxide synthase activity and cyclic GMP levels are decreased with interstitial cystitis and increased with urinary tract infections. AB - PURPOSE: Since urinary nitric oxide synthase (NOS) activity correlates with certain disease process affecting the urinary tract and since nitric oxide increases cyclic GMP levels by activating guanylyl cyclase, urinary particulate NOS activity and cyclic GMP levels are evaluated in female patients with interstitial cystitis (IC) and compared with those from female controls and female patients with urinary tract infections (UTIs). MATERIALS AND METHODS: Urinary NOS activity is measured as the formation of [(14)C]-L-citrulline from [(14)C]-L-arginine, and urinary cyclic GMP levels are measured by an [(125)I] radioimmunoassay. RESULTS: Female patients with IC have significantly less NOS activity in their urine pellet particulate fractions than female control females UTIs, 2.3 +/- 1.0, 14 +/- 3.0, and 120 +/- 10 pmol. citrulline formed/min./mg. protein. Urinary cyclic GMP levels are significantly lower in IC patients than in female controls or females with UTIs: 0.50 +/- 0.06, 0.82 +/- 0.14. and 3.72 +/- 0.81 micromol. cyclic GMP/g. creatinine. CONCLUSIONS: Regulation of urinary NOS activity with subsequent changes in nitric oxide and cyclic GMP may be an important determinant of symptoms and immunologic responses to UTIs and IC. PMID- 8632606 TI - High levels of soluble p55-TNF receptors in seminal and prostatic fluids of normal and infertile men. AB - PURPOSE: To study the role of tumor necrosis factor (TNF) in the male reproductive systems by examining the occurrence, source, and possible functional significance of soluble TNF receptors in seminal fluids of normal and infertile men. MATERIALS AND METHODS: Concentrations of soluble TNF receptors (p55-sTNF-R and p75-sTNF-R) were measured by ELISA in human sera, seminal fluids, prostatic fluid and fluid obtained from an epididymal spermatocele. RESULTS: The level of p55-sTNF-R in seminal fluids of normospermic men was approximately equal to 20 fold higher than in normal serum (13.9 +/- 6.9 ng./ml. versus 0.7 +/- 0.2 ng./ml.). In contrast, p75-sTNF-R, which occurs in serum at amounts higher than p55-sTNF-R, was almost indiscernible in the seminal fluids (<0.18 +/- 0.28 ng./ml. versus 1.9 +/- 0.6 ng./ml. in sera). Concentrations of p55-sTNF-R in seminal fluids of oligoasthenospermic and azoospermic men were similar to those of normospermic men (15.6 +/- 8.5 ng./ml. and 14.9 +/- 6.5 ng./ml., respectively). Higher p55-sTNF-R concentrations were found in prostatic fluids and first split ejaculates (39.8 +/- 1.2 ng./ml. and 32 +/- 1.7 ng./ml., respectively), while second split ejaculates and the fluid from an epididymal spermatocele were found to contain p55-sTNF-R at lower levels (10.8 +/- 1 ng./ml. and 1 ng./ml., respectively). CONCLUSIONS: These findings suggest intense local biosynthesis of p55-sTNF-R in the prostate occurring independently of spermatogenesis. Possible functional implications are: 1) shielding of spermatozoa from the inhibitory effect of TNF in the female reproductive tract; 2) a role for TNF in the normal physiology of the prostate; and 3) blocking TNF mediated immune response in the prostate, which may have bearings on the development of prostatic hypertrophy or cancer. PMID- 8632607 TI - Desensitization of muscarinic receptor-coupled inositol phospholipid hydrolysis in human detrusor cultured smooth cells. AB - PURPOSE: The aim of this study was to investigate the desensitization characteristics of muscarinic M3 receptors in primary cultures of human detrusor smooth muscle cells. MATERIALS AND METHODS: Cell cultures were prepared from cold cup pinch biopsies of the human detrusor muscles by explant culture methods. Accumulation of (3)H-inositol phosphates was measured on confluent monolayers as described previously in detail. Desensitization was achieved by preincubating the cells with carbachol or histamine for 5 to 60 minutes. RESULTS: Carbachol induced a concentration-dependent increase in phosphoinositide turnover in naive cells, the response being rapid and evident after only a 30-second exposure to the agonist. Preincubation of the cells with carbachol produced a concentration dependent decrease in the inositol phosphate response to a second challenge with carbachol. Preexposure to carbachol for only 5 minutes prior to a rechallenge reduced the mean size of response of the second stimulation to 49% of that observed in naive cells. Preexposure of the cells to histamine did not alter the response of the cells to a subsequent challenge with carbachol and vice versa. CONCLUSIONS: The muscarinic receptors retained by human detrusor smooth muscle cells in culture are susceptible to a desensitization of the carbachol-induced increase phosphoinositide turnover observed in these cells. This desensitization is rapid, and the results indicate that it is homologous and does not occur via a postreceptor mechanism but at the level of the receptor itself. PMID- 8632608 TI - Loss of heterozygosity at the p53, RB, DCC and APC tumor suppressor gene loci in human bladder cancer. AB - PURPOSE: Allelic losses within several tumor suppressor genes have been detected frequently in various types of human cancer. We investigated the roles and possible interactions of the tumor suppressor genes p53, Rb, DCC and APC in bladder cancer. MATERIALS AND METHODS: Loss of heterozygosity (LOH) of these 4 genes was examined in 45 human bladder cancers by polymerase chain reaction and restriction fragment length polymorphism assay. RESULTS: Of the evaluated cases, LOH was seen at P53 in 38%, at Rb in 22%, at DCC in 36% and at APC in 6% of tumors. Loss of heterozygosity at p53 and Rb was predominantly observed in high grade (grade 3) and/or invasive (T2 or greater) tumors, whereas LOH at DCC was present irrespective of tumor grade and stage. Allelic losses at either p53, Rb, DCC or APC were seen in 82% of high grade tumors, but in only 21% of low grade (grade 1 and 2) tumors (p<0.005). Similarly, 71% of invasive tumors had LOH at one or more loci compared with 20% of superficial (Ta and T1) tumors (p<0.005). Interestingly, p53-LOH and Rb-LOH were often observed simultaneously in the same tumor. CONCLUSIONS: These results suggest that loss of the p53, Rb and/or DCC genes is involved in most of the late and some of the early steps of bladder carcinogenesis. PMID- 8632609 TI - 1,25-dihydroxycholecalciferol enhances the expression of MHC class II antigens and intercellular adhesion molecule-1 by human renal tubular epithelial cells. AB - PURPOSE: Beside its role in calcium and phosphorus metabolism, 1,25 dihydroxycholecalciferol (1,25-D3) exerts multiple effects on cytokine and major histocompatibility complex (MHC) class II expression in monocytes and lymphocytes. In different renal diseases tubular epithelial cells express MHC class II molecules and cell adhesion molecules,, such as intracellular adhesion molecule-1 (ICAM-1). Therefore, modulation of MHC class II and ICAM-1 expression in renal tubular epithelial cells by 1,25-D3 may be relevant to lymphocyte adhesion to tubular epithelial cells and immune mediated renal injury. However, the expression of MHC class II antigens and cellular adhesion molecules by renal tubular epithelial cells in response to 1,25-D3 has not been investigated. MATERIALS AND METHODS: We generated human renal tubular epithelial cells and SV40 transfected tubular epithelial cells to investigate immune modulation of 1,25 on renal epithelial cells. Major histocompatibility complex class II molecules and ICAM-1 were detected by a specific enzyme linked immunoassay. RESULTS: We found a dose-dependent increase of both constitutive and induced MHC class II and ICAM-1 expression in tubular epithelial cells stimulated with 1,25-D3. Dose-dependent stimulation of MHC class II and ICAM-1 expression was not restricted to primary human renal tubular epithelial cells but was also detected in SV40 transfected cells. CONCLUSIONS: Expression of MHC class II and ICAM-1 is crucial for antigen presentation by and lymphocyte adhesion to renal tubular epithelial cells. Modulatory effects of 1,25-D3 on immune accessory function of renal tubular epithelial cells may be of clinical significance in renal diseases. PMID- 8632610 TI - Effects of Y-26763, a novel K-channel opener, on electrical responses of smooth muscles in the guinea pig bladder. AB - PURPOSE: The effects of Y-26763, a novel K-channel opener, on spontaneous and carbachol-induced electrical responses of bladder smooth muscle in guinea pig were studied. MATERIALS AND METHODS: Intracellular microelectrodes were used to record membrane electrical responses from the detrusor smooth muscle of guinea pig. RESULTS: In most cells studied, the membranes were spontaneously active with continuous or periodic bursts of spike discharge which were sensitive to nicardipine, a Ca++ -antagonist. Y-26763 inhibited the spike discharges with or without hyperpolarization of the membrane; the spike inhibition appeared at lower concentrations (>10(-8 M.) than the hyperpolarization (>10(-7) M.). The effects of Y-26763 were antagonized by glibenclamide, which suggested an involvement of ATP-sensitive K-channels. Carbachol increased the discharge frequency of spikes, with (>10(-6)M.) or without (10(-7) M.) depolarization of the membrane. Y-26763 (>10(-7) M.) inhibited the spike generation but not the depolarization induced by carbachol (10(-6)M.) in a concentration-dependent manner. CONCLUSIONS: Y-26763 may directly prevent electrical excitability of bladder smooth muscles by inhibiting spike discharges and also by hyperpolarizing the membrane. The results suggest that this K-channel opener could have clinical applications for the treatment of unstable bladders. PMID- 8632611 TI - Canine bladder blood flow and oxygenation: changes induced by filling, contraction and outlet obstruction. AB - PURPOSE: To study changes in bladder blood flow and oxygenation associated filling, contraction and outlet obstruction. MATERIALS AND METHODS: Intravesical pressure, bladder flow, bladder wall oxygen tension, iliac artery blood flow and systemic blood pressure were measured simultaneously in anesthetized dogs (N = 18). RESULTS: In the empty bladder, blood flow and oxygen tension in the bladder were greater than at the dome with and without outlet obstruction. Bladder filling caused a significant decrease in bladder wall blood flow and oxygen tension with or without outlet obstruction. Spontaneous bladder contractions resulted in a marked decrease in bladder wall perfusion in the obstructed bladder but not in the unobstructed bladder. Pelvic nerve stimulation produced strong bladder contractions associated with significant drop in bladder wall perfusion and bladder oxygenation in both the open and closed bladder neck models. Little change was noted after stimulation of the hypogastric nerve. CONCLUSIONS: Bladder distention and contraction, especially against a closed bladder neck, induce significant ischemia and hypoxia of the bladder wall. These findings may be important in the pathophysiology of a variety of common clinical problems. PMID- 8632612 TI - Protective effect of Tadenan on bladder function secondary to partial outlet obstruction. AB - PURPOSE: Tadenan (DEBAT, Paris, France) is a pharmaceutical agent used in the treatment of benign prostatic hyperplasia (BPH). The specific aim of this study was to determine if pretreatment of rabbits with Tadenan reduced either the hypertrophic response of the bladder to partial outlet obstruction or the accompanying contractile dysfunction. MATERIALS AND METHODS: Twenty-five male New Zealand rabbits (3 to 5 kg.) were separated into 5 groups of 5 rabbits each. Each rabbit in groups 1,2, and 3 received Tadenan orally at 1, 10 and 100 mg./kg./day for 3 weeks. Group 4 received vehicle only (peanut oil); Group 5 were controls. The bladders were evaluated (in vitro studies) after 2 weeks of obstruction. RESULTS: 1) Tadenan did not reduce the effect of partial outlet obstruction on bladder mass. 2) Tadenan pretreatment resulted in a significant protective effect on the contractile responses to field stimulation, bethanechol and KCl. CONCLUSIONS: These results clearly demonstrate that Tadenan pretreatment protected the bladder from the contractile dysfunctions induced by partial outlet obstruction. PMID- 8632613 TI - Neurogenic modulation of urethral resistance in the guinea pig. AB - PURPOSE: The resistance offered to urinary flow by the urethra is one of the factors determining the course of micturition. It was the aim of the present work to study the dependence of urethral resistance on the degree of relaxation of the urethra. MATERIALS AND METHODS: Experiments were done in the guinea pig. Ten animals were used. In 5 animals saline was forced through the (unrelaxed) urethra at imposed flow rates in the range of 1.1 to 43.0 ml. per minute while the urethral pressure was measured. Second degree polynomials were fitted to the pressure/flow data. In the other 5 animals micturition contractions were evoked and pressure/flow plots were derived from the measured signals. A straight line was fitted to the lowest pressure values at each flow rate in these plots. These pressure values represent the most relaxed state of the urethra in these voidings. RESULTS: The pressures measured in the unrelaxed urethra were much higher than the pressures measured during voiding in the same flow rate range, but the intercepts of the mathematical equations fitted to the pressure/flow data on the pressure axis were not significantly different in the 2 groups. CONCLUSIONS: The unrelaxed urethra has a much "steeper" pressure/flow characteristic than the relaxed urethra. However, the urethral closing pressure, that is, the intercept of the pressure/flow characteristic on the pressure axis, does not depend on the state of relaxation of the urethra. PMID- 8632614 TI - Intravesical electrical stimulation induces a prolonged decrease in micturition threshold volume in the rat. AB - PURPOSE: Intravesical electrical stimulation (IVES) has been used clinically to treat patients with voiding disorders. The aim of the present experimental study was to obtain objective evidence of a modulation of the micturition reflex by intravesical electrical stimulation (IVES). MATERIALS AND METHODS: Forty-one female rats, anesthetized by alpha-chloralose were used for the experiments. Intravesical electrical stimulation was given by a catheter electrode in the bladder (5 minutes of continuous stimulation at 20 Hz, 7 to 11 mA). The effect was evaluated by the change in cystometric micturition threshold volume. RESULTS: The threshold volume of the micturition reflex decreased significantly to 82% of controls after IVES (p<0.001; n=31). The effect was reversible and lasted for about 1 hour. The decrease was prevented by a transient blockade of the bladder nerves during IVES. CONCLUSIONS: Intravesical electrical stimulation induced a prolonged modulation of the micturition reflex in anesthetized rats. The effect was due to activation of bladder mechanoreceptor afferents and remained long after the period of stimulation. It is proposed that the modulation was due to a prolonged enhancement of excitatory synaptic transmission in the central micturition reflex pathway. Such a modulation may underlie the curative effect of IVES in certain voiding disorders. PMID- 8632615 TI - Prostanoid production in rabbit corpus cavernosum: I. regulation by oxygen tension. AB - PURPOSE: To investigate the effects of oxygen tension on prostanoid synthesis in rabbit penile corpus cavernosum tissue (RCC) in organ culture. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were incubated in organ culture media under varying oxygen conditions (0%, 12% and 21% oxygen), in the presence or absence of acetylcholine and arachidonate stimulation. Prostanoids were measured in collected media by radioimmunoassay. Prostaglandin H synthase (PGHS) protein levels and mRNA PGHS expression were measured under both 0% and 21% oxygen conditions. RESULTS: Basal and acetylcholine-stimulated PGI2 release was progressively diminished as a function of diminishing oxygen tension (pO2 from approximately 165 to 25 mm.Hg). The basal and stimulated production of other prostanoids, thromboxane A2, PGF2alpha, and PGE2, was also significantly inhibited under 0% oxygen (approximately 25 mm.Hg) conditions. However, incubation under 0% oxygen did not alter PGHS protein levels nor mRNA PGHS expression. Cavernosal strips incubated under 0% oxygen but supplemented with exogenous arachidonate (10 microM.) maintained significantly lower PGI2 production than tissues exposed to 21% oxygen (approximately 165 mm.Hg). CONCLUSIONS: These data demonstrate that oxygen tension regulates prostaglandin production in corporal tissue. The reduction in prostanoid production during hypoxia can be attributed to inhibition of PGHS activity rather than the expression of the enzyme. In view of the role of PGI2 as an inhibitor of platelet aggregation and white cell-endothelial adhesion, our findings may provide mechanistic insight into the alteration in corporal blood homeostasis ischemic hypoxic priapism. PMID- 8632617 TI - Intracellular calcium modulators for cardiac muscle in pathological conditions. AB - This is a brief review of agents that stabilize calcium release from the sarcoplasmic reticulum in cardiac muscle. An excess intracellular calcium concentration (calcium overload) is a common feature in a variety of cardiac cell injuries. Calcium overload elicits diastolic and systolic failure, and is involved in the genesis of arrhythmias. These abnormalities appear in part to be caused by the spontaneous release of calcium ions from the sarcoplasmic reticulum. Previous efforts to treat calcium overload were made with the intention to decrease the total intracellular content of calcium ions. However, such procedures would result in a decrease in contractility. Agents that stabilized calcium release from the sarcoplasmic reticulum may therefore be useful to correct abnormalities in calcium overload. In this review, after briefly describing intracellular calcium homeostasis, strategies against calcium overload, especially those involving magnesium ion, ryanodine, caffeine, dantrolene, phenytoin, R56865, KT361 and flunarizine will be discussed. PMID- 8632618 TI - The role of accumulation of sodium and calcium on contractile failure of the hypoxic/reoxygenated heart. AB - The present study was undertaken to determine whether myocardial energy or ion levels are related to oxygen-replenishment-induced recovery of cardiac contractile force after hypoxia. Isolated rat hearts were perfused for 3 to 40 min under hypoxic conditions, followed by 45 min of reoxygenation. Hypoxia induced a cessation of cardiac contractile force, a rise in resting tension, a decrease in high energy phosphates, and an increase in lactate. Myocardial ATP, creatine phosphate (CP) and lactate reached steady-state levels after 15, 10 and 5 min of hypoxia, respectively. Hypoxic conditions in the present study also caused an increase in sodium content and a decrease in potassium content, but not changes in calcium content, along with a prolonged hypoxic period. When the hearts were perfused for more than 25 min under hypoxic conditions, no recovery of contractile force was observed following 45-min of reoxygenation. Hypoxic perfusion for more than 25 min induced an accumulation of tissue sodium content approximately 3 fold higher than the pre-hypoxic value at the end of hypoxia, and also induced a marked increase in myocardial calcium content upon reoxygenation. When tissue sodium content accumulated by less than 300% of the pre-hypoxic value, cardiac contractile function was partially reversed by reoxygenation and calcium-overload was not observed. The recovery of post-hypoxic cardiac contractility correlated with tissue sodium content during hypoxia rather than with myocardial high energy phosphate content at the end of hypoxia. These results suggest that accumulation of tissue sodium content in the hypoxic myocardium and calcium content in the reoxygenated myocardium may be indicative of hypoxia/reoxygenation-induced cardiac contractile failure. PMID- 8632619 TI - Persistence of balloon-induced arterial injury with hyperlipidemia despite gemfibrozil. AB - Restenosis after balloon dilitation of atherosclerotic arteries reflects migration and proliferation of vascular smooth muscle cells and infiltration of monocyte/macrophages. Hypercholesterolemia may contribute to this phenomenon. Accordingly, we used the lipid-lowering agent gemfibrozil to determine whether potentially detrimental effects of hypercholesterolemia on vascular remodeling after mechanical injury could be attenuated. New Zealand white rabbits fed either a chow diet (control), a 0.25% cholesterol-enriched diet, or a 0.25% cholesterol enriched diet supplemented with gemfibrozil (0.05%, 0.1%, or 0.02%) for one week were subjected to balloon-induced carotid injury and maintained on the same diet for an additional 4 weeks. Histology of the vascular wall was then characterized. Plasma triglycerides before and 4 weeks after injury did not change in any of the treatment groups (p = 0.24). Plasma cholesterol increased in all animals receiving the high cholesterol diet, and the increases remained unaffected by supplementation with gemfibrozil. In control rabbits, intimal thickening area [intima (mm2)/(intima + media (mm2))] 4 weeks after injury was 27.0 +/- 7.7% (n = 16). Values were the same in hypercholesterolemic rabbits (29.7 +/- 11.8%, n = 12; p = ns). However, in 16% the lumen was completely occluded by thrombus and intimal thickening could not be quantified. In hypercholesterolemic rabbits given gemfibrozil, intimal thickening was increased by 33% compared with controls (35.9 +/- 11.6%, n = 39, pound 0.05) and by 21% compared with hypercholesterolemic animals not given gemfibrozil (p = ns). None had thrombotic luminal occlusion. Macrophages detected immunohistochemically were only modest in number in vessels from control animals. In vessels from hypercholesterolemic animals and from animals whose diets were supplemented with gemfibrozil, macrophages were increased in number in both intima and media. Thus, gemfibrozil did not appear to attenuate processes implicated in restenosis. Its attenuation of thrombotic occlusion may be related to effects we have noted it exerts on fibrinolytic systems independent of lipid metabolism. PMID- 8632616 TI - Testicular tissue oxygen pressure. AB - PURPOSE: We evaluate the feasibility of continuous, intratesticular tissue oxygen pressure (IT-pO2) measurement under various conditions. MATERIALS AND METHODS: Flexible, polarographic microcatheter systems (1.5 F) for continuous pO2 measurements have been used. The measurements were conducted on 37 adult male albino rats. The probes could be placed intratesticularly under controlled temperatures by microsurgical techniques. The IT-pO2 of the ipsilateral and contralateral testicle was determined after unilateral torsion. Furthermore the influence of intratesticular temperature on IT-pO2 was determined. RESULTS: Intratesticular pO2 was found to be 21 +/- 5 mm.Hg under normal conditions. Under complete ischemia, pO2 dropped within 3 to 5 minutes to a level of approximately 1 mm.Hg. A counterclockwise unilateral testicular torsion of 720 degrees led to a decrease of the IT-pO2 within 5 to 7 minutes to 5 +/- 1.5 mm.Hg. After detorsion the IT-pO2 recovered within 15 minutes, while the IT-pO2 of the contralateral testicle remained unchanged. Simple surgical exposure of the testicle led to a reversible decrease of the intratesticular pO2 to 7 +/- 2.5 mm.Hg correlating with an intratesticular temperature decrease from approximately 31.5 to 23.5 C. CONCLUSIONS: The measurements are feasible and may provide a continuous intraoperative assessment of intratesticular oxygen pressure. The contralateral IT-pO2 after unilateral torsion remained unchanged. The IT-pO2 and testicular blood flow depend, if the utilization of oxygen is unchanged, on intratesticular temperature. PMID- 8632620 TI - The visual estimation of intraoperative myocardial ischemia. AB - We report a case who had myocardial ischemia after release of the aortic cross clamp during mitral valve replacement. Myocardial ischemia was visually estimated by use of intraoperative MCE (myocardial contrast echocardiography). After appropriate treatment the ischemic area disappeared and the patient showed a good postoperative course. MCE is a useful method with which to detect visually the wash out pattern of cardioplegia from myocardium after reperfusion during open heart surgery. PMID- 8632621 TI - Percutaneous transvenous mitral commissurotomy in patients with mitral stenosis and coexistent hyperthyroidism. AB - Percutaneous transvenous mitral commissurotomy (PTMC) was performed successfully without complications in 3 patients with severe mitral stenosis and hyperthyroidism. All 3 patients had pliable, noncalcified mitral valves. One patient who had been treated with methimazole for 6 months was still in a hyperthyroid state when she presented with intractable congestive heart failure and was found to have severe mitral stenosis. The heart failure improved immediately after PTMC, but the patient remained in New York Heart Association functional class 2 until a euthyroid state was achieved with I131 therapy. In the other 2 patients, hyperthyroidism was unsuspected at the time of PTMC. Unexpectedly suboptimal symptom improvement led to the diagnosis of hyperthyroidism 1 month after the intervention. In all 3 patients, PTMC resulted in an immediate hemodynamic and clinical improvement. However, complete clinical improvement occurred only when euthyroid state was achieved after antithyroid treatment. The present study suggests that PTMC is a safe and effective intervention modality in patients with coexisting hyperthyroidism and severe mitral stenosis. The procedure may be considered a therapeutic option in patients with hyperthyroidism and severe mitral stenosis. PMID- 8632623 TI - Flow-dependent regulation of gene expression in vascular endothelial cells. AB - Vascular endothelial cells are constantly exposed to wall shear stress generated by blood flow. Endothelial cells act as mechanoceptors sensing and responding to shear stress, and play a role in flow-dependent phenomena such as angiogenesis, vascular remodeling and atherosclerosis. Numerous recent studies have demonstrated that endothelial cell functions change in response to shear stress, and that the responses are often accompanied by changes in related gene expression. More recently there has been evidence that genes known to be regulated by shear stress may have a common cis-element (shear stress responsive element; SSRE) in their promoter regions. A molecular mechanism for endothelial cell responses to mechanical stress is close to being elucidated. In this paper, shear-stress-mediated regulation of endothelial gene expression is reviewed. PMID- 8632622 TI - Cardiogenic shock following recombinant alpha-2b interferon therapy for chronic hepatitis C. A case report. AB - A 57-year-old woman with chronic hepatitis C was treated with alpha-2b interferon (IFN). Forty-five days after the initiation of IFN therapy, she developed cardiogenic shock. Acute perimyocarditis as a cause of cardiogenic shock was clinically suspected by the findings of complete atrioventricular block, regional wall motion abnormality and pericardial effusion. Since IFN therapy may induce cardiogenic shock in some patients, it is important to carefully monitor patients under treatment with IFN for abnormal cardiac signs. PMID- 8632624 TI - Accelerated ST-segment reduction after thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) compared to urokinase. AB - Effects of therapy with urokinase (UK) and with recombinant tissue plasminogen activator (rtPA) were compared in patients with acute myocardial infarction (AMI). To achieve homogenous therapeutic conditions the comparison was restricted to patients having their first AMI and to cases of clinically successful thrombolytic therapy (defined by non-invasive criteria, such as a 50% decrease in elevated ST-segment in the worst load of a 12 lead ECG within 300 min after onset of thrombolytic therapy, complete pain resolution during thrombolytic therapy, and later confirmed by angiography 10 days after AMI). Effects of UK and rtPA on continuous multilead ST-segment analysis and cardiac proteins (creatine kinase and its isoenzyme CK-MB, aspartate transaminase and hydroxybutyrate dehydrogenase) were analyzed during 24 hours following onset of therapy. Continuous ST analysis showed a faster resolution of the elevated ST-segments after thrombolytic therapy with rtPA than with UK(p < 0.01). Accelerated idioventricular rhythms (p < 0.05) occurred sooner following rtPA than UK treatment. The wash-out of creatine kinase was increased (p < 0.01) after rtPA. Although both drugs induced comparable, angiographically controlled reperfusion, the results suggest that the process of reperfusion was accelerated during thrombolysis with rtPA compared to UK. Thrombolytic therapy of AMI with rtPA may hence improve myocardial salvage. PMID- 8632625 TI - The mechanism of sympathovagal imbalance in patients with myocardial ischemia. AB - To investigate the mechanism of sympathovagal imbalance due to myocardial ischemia, we studied 42 consecutive patients undergoing successful percutaneous transluminal coronary angioplasty by correlating frequency domain and time domain measures of heart rate variability with parameters such as echocardiography, stress thallium scanning and radionuclide angiography before, immediately after and 2 months after the procedure. Of these, 20 patients (Group N) had normal and 22 patients (Group A) had abnormal regional wall motion. A control group of 20 healthy subjects (Group C) underwent echocardiography and examination of heart rate variability twice at 2-month intervals to check for spontaneous variations. At baseline, frequency domain measures such as low and high frequency power and time domain measures such as SDANN index (the mean of the standard deviations of the average of RR intervals) were lower in Group A than in Groups N and C, whereas no differences were detectable in ultra low and very low frequency, total power, SDNN index (the mean of the standard deviations of the mean of normal RR intervals), and r-MSSD (the root mean square of successive RR differences). There was high association between the diastolic wall stress index and both high frequency (r = -0.82) and low frequency power (r = -0.77). There were similar findings for the systolic wall stress index (r = -0.72 for high frequency and r = -0.64 for low frequency power). After successful coronary angioplasty, regional wall motion, left ventricular wall stress indices and all measures of heart rate variability were unchanged in Group N. In Group A the mean summed segment score improved from 15.9 +/- 2.6 to 12.2 +/- 1.7 (p < 0.0001), and mean low frequency, mean high frequency power (logarithmic units), and SDANN index (msec) increased from 6.10 +/- 0.23 to 6.36 +/- 0.28 (p < 0.005), from 5.36 +/- to 0.40 to 5.70 +/ 0.39 (p < 0.01) and from 70 +/- 18 to 83 +/- 18 (p < 0.01) respectively. In addition, low and high frequency power and SDANN index, lower at baseline in Group A than in the other two groups, were comparable in the three groups after coronary angioplasty. The evolution of diastolic and systolic wall stress indices paralleled that of the above three parameters. In conclusion, diastolic and systolic wall stress indices, in addition to segmental left ventricular dysfunction, were synergistically involved in determining sympathovagal imbalance in patients with significant coronary artery disease; the reversal of left ventricular dysfunction and wall stress indices improves the profile of heart rate variability. Alterations in cardiac geometry and wall stress influence mainly the discharge of afferent sympathetic and efferent parasympathetic innervations and also principally the long-term heart rate variations instead of short-term modulation. PMID- 8632627 TI - Clinical characteristics and EPS-guided therapy in 142 cases of sustained ventricular tachycardia. AB - One hundred and forty-two consecutive patients with sustained monomorphic ventricular tachycardia (VT) were investigated. Only 26.1% of VTs were associated with ischemic heart disease (IHD). The induction rate of sustained VT upon electrophysiologic study (EPS) was 82.9% in patients with IHD and 65.3% in non IHD. Of 76 inducible sustained VTs, pharmacologic therapy was finally selected in 35 cases, ablative therapy in 25 and surgical therapy in 12. Long-term prognosis was compared between groups divided according to type of ventricular arrhythmia induced at final EPS after antiarrhythmic therapy as follows: Group A: complete suppression of VT, Group B: clinical or non-clinical nonsustained VT, Group C: clinical sustained VT. The event rate in IHD was 6.3% in Group A, 44.4% in Group B and 100% in Group C. In non-IHD, the event rate was 24.0%, 25.0% and 75.0% (Groups A, B and C, respectively). Complete suppression of VT showed a good prognosis in IHD, however, a slightly higher recurrence rate was observed in non IHD. In ablative therapy, some recurrences and sudden deaths were observed in spite of complete suppression of both VTs in both the IHD and non-IHD groups. Review of the efficacy of antiarrhythmic procedures is recommended during the follow-up period. PMID- 8632626 TI - Prognostic indicators of major cardiac events in patients with asymptomatic coronary artery disease. AB - We investigated the role of myocardial ischemia in acute myocardial infarction and cardiac death in 253 patients with asymptomatic coronary disease (206 men, 47 women, mean age: 55 +/- 8 years). Patients were divided into two groups: those with angina pectoris with no history of myocardial infarction (AP group, 93 patients) and those with a history of myocardial infarction (MI group, 160 patients). We also examined the usefulness of exercise electrocardiographic and Holter electrocardiographic findings as prognostic indicators of cardiac events. After 24-hour Holter electrocardiograms were obtained in both groups, patients were assigned to subgroups with or without silent myocardial ischemia (SMI) based on the presence or absence of transient ST-segment depression. Prognostic indicators were evaluated by multiple regression analysis. Cardiac events occurred in 26 (10.3%) of 253 patients; in 6 patients these events were fatal. The incidence of cardiac events was significantly higher in the SMI group than in the non-SMI group (16.4% versus 5.6%, p < 0.05). SMI was identified as a significant prognostic indicator in the overall population (p = 0.0088), as were the number of diseased coronary arteries in the AP group (p = 0.0152), and SMI (p = 0.0022) in the MI group. There were 3 deaths related to cardiac events in each group. The mean time from onset of angina pectoris to death was 73 +/- 41 months compared with 33 +/- 43 months in the MI group. Our findings suggest that the severity of the coronary lesion and SMI were important predictors of major cardiac events, and that the mechanism of the onset of cardiac events was different in the AP and MI groups. PMID- 8632628 TI - Abnormal function of platelet G proteins in long QT syndrome. AB - Several hypotheses have been proposed for the pathophysiology of the congenital long QT syndrome, however, the underlying mechanism has not yet been elucidated. This study evaluated G protein function in patients with congenital long QT syndrome (LQTS) and compared it with that of normal subjects. Platelet-rich plasma was collected and the cyclic AMP (cAMP) level of platelets was measured in three conditions utilizing radioimmunoassay: the basal state (Basal cAMP), after stimulation by PGE1 (PGE1-cAMP), and after stimulation by PGE1 followed by inhibition by adrenaline (Adr-cAMP), and the results were compared between 7 LQTS patients and 10 healthy volunteers (control). Gs function was defined as (PGE1 cAMP)/(Basal cAMP) and Gi function as ?(PGE1-cAMP)-(Adr-cAMP)?/(PGE1-cAMP). Basal cAMP was lower in patients than in the controls: 2.9 +/- 0.6 pmol/ 10(8) cells vs. 4.2 +/- 0.7 pmol/10(8) cells (p < 0.05). The increase in cAMP after PGE1 was similar in the two groups but the peak was lower in the patients: 16.8 +/- 6.2 pmol/10(8) cells vs. 24.8 +/- 7.4 pmol/10(8) cells (PGE1-cAMP). After addition of adrenaline, cAMP decreased to 14.2 +/- 5.8 pmol/10(8) cells vs. 16.2 +/- 7.6 pmol/10(8) cells and the change was significantly smaller in the patients than in the controls: 0.17 +/- 0.12 vs. 0.38 +/- 0.16 (p < 0.05). Basal cAMP was weakly correlated with sinus cycle length (r = -0.48, p > 0.3) and QTc was correlated with Gs function (r = 0.52, p > 0.3) but not with Gi function. Patients with associated Torsade de Pointes had a significantly lower Gi function compared to those without (p < 0.05). In LQTS patients, G protein function was abnormal and the abnormality was associated with clinical characteristics of long QT syndrome. The relationship between the abnormal G protein function and the regulation of the repolarization of the ventricular myocardium needs to be studied further. PMID- 8632629 TI - Clinical and angiographic analysis of congenital coronary artery fistulae in adulthood. Is there any new trend? AB - Fifteen (2.1%) patients were diagnosed as having congenital coronary artery fistula (CAF) in a consecutive series of 704 adult patients undergoing selective coronary arteriography; the incidence was astonishingly higher than previous observations. The presentation of clinical symptoms and the electrocardiographic changes at rest and/or after exercise testing probably attributable to the CAF were observed unexpectedly often in spite of the fact that the magnitude of the shunt seemed not to be significant. With respect to the anatomy of the CAF, the incidence of origination from plural coronary arteries and that of fistulation into the left ventricle were also unexpectedly high. These observations presented a striking contrast to those of previous reports, but we were unable to determine the reason. In four cases, the CAF was ligated either electively or concurrently with mitral valve surgery, and the results were satisfactory. Taking these circumstances into consideration, we should not minimize the impact of a CAF with a seemingly small shunt. PMID- 8632630 TI - Pediatric blood lead testing in Kansas, 1994 (continuing education credit). PMID- 8632631 TI - Non-Hodgkin's lymphoma on the increase (continuing education credit). PMID- 8632632 TI - Evaluation of the reperfusion syndrome after liver ischemia in the rat. AB - Hepatic surgery in man often requires a transient interruption of the blood flow to the liver. After the vascular declamping the hepatic reperfusion induces a group of phenomena commonly called "reperfusion injuries." The aim of this study was to evaluate the presence and effect of vasoactive agents that could induce the acute pulmonary arterial hypertension which contributes to reperfusion injury. Wistar rats were used. The hepatic ischemia was induced by crossclamping the whole hepatic hilus for 20, 40, and 60 min. In control experiments a sham operation was performed. Blood samples were collected from the suprahepatic inferior vena cava. Strips of the main pulmonary artery were set up in an isolated organ bath and tested for the response to noradrenaline, adrenaline, KCl, and plasma samples. Plasma levels of catecholamines were determined by high performance liquid chromatography. Plasma concentration of noradrenaline significantly increased from 1.6 +/- 0.4 (control) to 10.8 +/- 2.9 ng.ml-1 and adrenaline concentration rose from 2.7 +/- 0.7 to 38.7 +/- 7.6 ng.ml-1 after ischemia. Noradrenaline potency, compared to control values, significantly increased after prolonged liver ischemia. The plasma samples collected after prolonged liver ischemia caused a greater contraction of the pulmonary artery than from control plasma. This contraction is partially inhibited by phentolamine. We conclude that hepatic ischemia modifies the response of the pulmonary artery to exogenous noradrenaline. At the same time it induces an increase in the plasma levels of adrenaline and noradrenaline. The resulting combined effect may cause the pulmonary hypertension which has been observed in reperfusion injury. PMID- 8632633 TI - Delayed repair: the role of glutathione in a rat incisional wound model. AB - Glutathione is a low molecular weight tripeptide that is a major intracellular antioxidant, modulates DNA synthesis, and may regulate signal transduction mechanisms. Our previous studies in rats suggested that intracellular stores of glutathione were sensitive to skin ischemia and, therefore, may regulate the early temporal course of wound healing. A 4-cm incision was placed on a rat's back and in vivo wound strength was measured over time. Animals were depleted of glutathione using L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of the enzyme gamma-glutamylcysteine synthetase. Some animals were treated in combination with allopurinol/BSO or with allopurinol alone. The data demonstrated at 4 days that BSO treatment produced a fourfold reduction in glutathione (3.51 +/- 1.78) over baseline (16.15 +/- 2.18) levels and twofold reduction (5.0 +/- 1.1) over untreated sham controls (11.1 +/- 2.3) (P < 0.05). Allopurinol provided no protection to glutathione levels. BSO treatment alone reduced wound burst strength compared to the other groups (P < 0.05). Allopurinol treatment enhanced wound strength over sham controls and BSO groups at 9 days after wounding (P < 0.05). Hydroxyproline content in wounds accumulated faster by Day 4 in the BSO treatment groups compared to sham controls (P < 0.05), whereas the BSO-treatment groups had lower hydroxyproline levels measured at Day 6 (P < 0.05). These data provide the first evidence that wound healing is related to the temporal course of glutathione metabolism. The effect may not be related to oxidant stress since allopurinol provided enhanced wound burst strength without protecting wound glutathione levels. PMID- 8632634 TI - Determinants of pancreatic microcirculation in acute pancreatitis in rats. AB - The aim of the study was to evaluate the effects of arterial hypotension, high volume crystalloid resuscitation, and isovolemic hemodilution on pancreatic microvascular perfusion during acute pancreatitis. Using intravital microscopy, pancreatic functional capillary density was analyzed in rats 1 and 2 hr after onset of acute pancreatitis. Pancreatic microvascular perfusion in acute pancreatitis was characterized by a (-62%) significant reduction of functional capillary density predominantly in perinecrotic but also in nonnecrotic tissue ( 43%). Pancreatic microvascular perfusion failure was aggravated by arterial hypotension but attenuated by treatment with high-volume crystalloid resuscitation. Isovolemic hemodilution was found superior to high-volume crystalloid resuscitation in maintaining pancreatic functional capillary density and therefore has the best potential in preserving tissue integrity and thereby limiting progression of disease. This study underlines the importance of early fluid resuscitation/hemodilution in patients presenting with acute pancreatitis. PMID- 8632636 TI - PGG-glucan, a leukocyte-specific immunostimulant, does not potentiate GVHD or allograft rejection. AB - PGG-glucan is an immunomodulator which can enhance the host response to infection. Phase I/II clinical trials have documented the safety and potential efficacy of this compound to reduce postoperative infectious complications in high risk surgical patients. Organ transplant recipients may benefit from this drug due to their high rates of postoperative infectious complications. A rat cardiac rejection model (ACI --> Lew) and a mouse skin graft model (C3H/HeJ --> B6AF1/J) were used with four treatment arms (control, Cyclosporine A (CsA), antilymphocyte serum (ALS), and CsA + ALS with and without PGG-glucan). Small intestinal allografts (Lew --> LBNF1) were performed in rats to evaluate GVHD. In the mouse GVHD model, donor splenocytes were given to irradiated recipients (C57BL/6 --> B6AF1), with and without PGG-glucan treatment. There was no difference in survival between PGG-glucan treatment and placebo for the control, CsA, and CsA + ALS groups in rat cardiac recipients. Recipients receiving ALS and treated with PGG-glucan survived a median of 42.5 days versus 63.5 days for those ALS-treated animals not receiving PGG-glucan (P = 0.045). In the remaining groups there was no difference in survival between PGG-glucan-treated groups and the control groups. PGG-glucan did not shorten survival in three of four treatment groups in the rat cardiac rejection model. High dose ALS with PGG-glucan did result in a marginal decrease in survival in cardiac allograft recipients. If the one outlying animal with indefinite survival is excluded, the difference is not statistically significant (P = 0.098). These results show that even though PGG glucan has immunostimulatory properties, it does not significantly potentiate rejection or GVHD in these animal models. This preliminary work may be important in determining whether PGG-glucan can be safely given to immunosuppressed organ transplant recipients to reduce postoperative infectious complications. PMID- 8632635 TI - Increase of mtDNA-binding proteins and mitochondrial mRNAs in regenerating liver. AB - In order to elucidate the mechanism of energy supply for liver regeneration after partial hepatectomy, we investigated mtDNA replication and transcription in regenerating rat liver. Changes of mtDNA-binding proteins, mtDNA, and mitochondrial mRNAs were monitored at 0, 12, 24, 48, 96, and 168 hr after the operation by gel mobility shift assay and Southern and Northern blot analyses, respectively. We focused on mtDNA-binding proteins specific for four different sequence elements possibly involved in regulation of mtDNA replication and transcription. Amounts of all the proteins sharply increased to maximum (4- to 10 fold of the preoperative level) 12 hr after partial hepatectomy and then decreased for 24 hr. After 24 hr, the amount of the respective binding protein changed diversely. Mitochondrial mRNA levels per gram tissue weight were unchanged during the first 12 hr after partial hepatectomy, but dramatically increased to maximum (4-fold) at 24 hr. In contrast, mtDNA content, expressed as a ratio of the nuclear cytochrome c1 gene, was unchanged during the first 48 hr and then started to increase, reaching maximum (1.5-fold) at 4 days. These results suggest that the energy supply in the early stage of the liver regeneration following hepatectomy is achieved mainly through enhancement of mtDNA transcription in which the mtDNA-binding proteins probably play regulatory roles. PMID- 8632637 TI - A surgical model to increase the intestinal absorptive surface: intestinal lengthening and growing neomucosa in the same approach. AB - Despite numerous investigations short bowel syndrome (SBS) is still an unsolved clinical problem. The purpose of this study was to develop a new surgical approach that combines both intestinal lengthening and growing neomucosa. The operative principle is based on a longitudinal division of an intestinal segment antimesenterically and a formation of two intestinal tubes out of bowel halves anastomosing one cutting edge to the incised mucosal midline and the other to the antimesenteric side of the constructed first intestinal tube, so that a common wall between the two hemiloops was created which leads to grow neomucosa. This procedure was applied to 11 rabbits. The hemiloops and neomucosa were investigated for its feasibility and morphologic characteristics. Gross and microscopic examinations at 8 weeks revealed that all the intestinal tubes were viable and patent. No anastomotic leakage was present. In all animals, the serosal surfaces of the common walls were covered with neomucosa that resembles the mucosa of the existing intestine morphologically; there was no significant difference in villus height, villus width at base, crypt depth, number of villi per unit serosal length, villus surface, and villus surface per unit serosa. The growth of neomucosa was completely a result of epithelialization without any evidence of contraction. The quantity and rate of neomucosal growth seems unaffected by intestinal segment length. Finally a two times increase in intestinal length, whereas the diameter halved and a statistically significant increase (P < 0.001) in total villus surface via growing neomucosa was obtained. This model might be useful in the management of SBS when supported by clinical and functional investigations. PMID- 8632638 TI - Ischemia-induced peripheral arterial vasospasm role of alpha 1- and alpha 2 adrenoceptors. AB - Ischemia produces functional and structural alterations in peripheral blood vessels. Our study was designed to investigate alpha-adrenoceptor modifications induced by prolonged ischemia and their role in enhancing the contractile response during reperfusion in canine femoral arteries. Unilateral vessel ischemia was created by double crossclamping a femoral artery and was maintained for 3 hr. Reperfusion was allowed for 1 hr. The contralateral femoral artery was utilized as a control. The vessels were harvested for contractile and binding studies. Isometric tension studies revealed that the magnitude of the maximum contractile response to norepinephrine (NE), potassium chloride (KCl), and methoxamine was 671 +/- 82 mg/mm2 versus 245 +/- 43 mg/mm2 (P < 0.01), 485 +/- 46 mg/mm2 versus 229 +/- 62 mg/mm2 (P < 0.05), and 486 +/- 88 mg/mm2 versus 126 +/- 38 mg/mm2 (P < 0.01), respectively for ischemic and nonischemic femoral arteries. EC50 values showed that ischemic vessels were more sensitive to NE, KCl, and methoxamine (P < 0.05). Sodium nitroprusside induced concentration-dependent and complete relaxation in all arterial rings (100% relaxation) but the ischemic femoral artery showed less sensitivity (P < 0.05). Binding studies showed that the number of binding sites (Bmax) for [3H]Prazosin and [3H]Rauwolscine were significantly increased in ischemic versus nonischemic vessels (1261 +/- 95 fmole/mg versus 704 +/- 113 fmole/mg, P < 0.03, and 490 +/- 86 fmole/mg versus 175 +/- 15 fmole/mg, P < 0.04, respectively). Furthermore, a significant decrease in affinity (Kd) for [3H]Prazosin and [3H]Rauwolscine was observed in ischemic versus nonischemic tissues (9.4 +/- 1.7 nM versus 4 +/- 0.2 nM, P < 0.02, and 6.4 +/- 1.5 nM versus 1.8 +/- 0.6 nM, P < 0.01, respectively). The increase in canine femoral artery vasoreactivity, following prolonged ischemia, seems to be also due to an increased density and functional activity of alpha-adrenoceptors expressed by the ischemic arterial smooth muscle cells. PMID- 8632639 TI - Thymopentin modulates Th1 and Th2 cytokine response and host survival in experimental injury. AB - OBJECTIVE: To investigate the impact of thymopentin (Thy) on mortality and in vivo cytokine release in an animal model of gut-derived sepsis which includes different combinations of allogeneic blood transfusion (T) and burn injury plus bacterial gavage (BG). DESIGN: Randomly controlled experiments. MATERIAL: Two hundred sixteen Balb/c (H-2d) and 50 C3H/HeJ (H-2k) mice. INTERVENTIONS: In the first study 60 Balb/c mice were given Thy (1 mg/kg). The same day of therapy onset, 40 mice were transfused with allogeneic blood (from C3H/HeJ mice). The remaining 20 mice received aliquots of saline. Five days post-T, 20 of the 40 transfused mice were subjected to a 20% TBSA thermal injury and simultaneous gavage with 1 x 10(9) Escherichia coli and the other 20 mice underwent a sham burn. The 20 nontransfused mice also received a 20% burn plus bacterial gavage. In all animals Thy was administered for 15 days. Three control groups (n = 20 each) entered the same protocol design, but they did not receive Thy. In the second study 96 animals were randomized to six groups (n = 16 each) according to the above experimental design. Animals were sacrificed by exsanguination after burn or 5 days post-T in nonburned mice to measure TNF-alpha, IL-2, and IL-4 plasma levels. RESULTS: The highest mortality (70%) occurred when T was combined with BG. Thy significantly reduced mortality in both groups that underwent BG, regardless of the association with T. TNF-alpha was detectable in 30% of the tested samples, IL-2 in 50%, and IL-4 in 70%. Thy significantly reduced the levels of IL-4 and increased the production of IL-2. CONCLUSIONS: The protective effect of Thy in this experimental model may be mediated by modulation of cytokine release. PMID- 8632640 TI - Effect of the somatostatin analogue octreotide on experimental pancreatitis in rats. AB - Somatostatin and its analogue octreotide have a profound inhibitory effect on the endocrine and exocrine secretions of the pancreas, stomach, and small intestine. Previous studies have been inconclusive about the possible therapeutic effect of somatostatin and its analogues in the treatment of pancreatitis. This study assessed the effect of the long acting somatostatin analogue, octreotide, in two models of experimental pancreatitis in rats. Necrotizing pancreatitis was induced by pancreatic injection of 5 ml taurocholate, 5% in male Wistar rats. In a second model mild edematous pancreatitis was induced by intravenous injection of caerulein at a supramaximal dose, 6 micrograms/kg/hr, for 5 hr. Compared to untreated rats, treatment with octreotide either prior to or following the induction of necrotizing pancreatitis resulted in less hypocalcemia (P < 0.05) and acidosis (P < 0.05), and prevented the increase in pancreatic weight (P < 0.05). Amylase levels remained high. After 20 days, there was less pancreatic damage, lower mortality rates (P < 0.05), and increase in body weight (P < 0.05). In the model of milder pancreatitis, octreotide treatment attenuated the increase in pancreatic weight (P < 0.05) and pathological damage (P < 0.05). We concluded that the somatostatin analogue octreotide has a beneficial effect in the treatment of experimental acute pancreatitis. PMID- 8632641 TI - Amelioration by Kupffer cell blockade in hepatic damage induced by cold preservation with subsequent plasma-supplemented perfusion. AB - The effects of Kupffer cell blockade with gadolinium chloride (GdCl3) on hepatic injury in the cold preservation with subsequent perfusion was investigated. The condition of the liver grafts which were cold-preserved in University of Wisconsin solution was evaluated by measuring the uptake rates of indocyanine green and hyaluronic acid (IUR and HUR), two indices of liver damage. Perfusion injuries after cold storage occurred in 24-hr-preserved liver and progressed further in 36-hr-preserved liver. GdCl3 prevented the decrease in IUR and ameliorated perfusion injuries in the 36-hr-preserved liver, although GdCl3 did not inhibit the decrease in HUR. The HURs were remarkably decreased in the livers stored even for 6 hr, for which the cold-preserved livers were quite viable, whereas IURs were significantly decreased in livers stored for 24 hr or more when perfusion injury was apparently developed. These results suggest that the IUR is an indicator of hepatic perfusion injury in preserved livers and that Kupffer cell blockade may be a promising strategy for the prevention of hepatic injury after cold preservation with subsequent perfusion. PMID- 8632642 TI - Thromboxane A2 in preservation-reperfusion injury: the effect of thromboxane A2 synthetase inhibitor. AB - It has been suggested that thromboxane A2 (TXA) plays important roles in preservation/reperfusion organ injury. In this report, we investigated the prostanoid release from the liver and the effect of a selective TXA synthetase inhibitor (E)-3-[p-(1H-imidazol-yl-methyl)-phenyl]-2-propenoic acid, OKY046) during cold preservation and after reperfusion. Rat livers were preserved in lactated Ringer's solution at 4 degrees C for 2, 4, and 6 hr and perfused with oxygenated Krebs-Henseleit buffer using recirculating perfusion system, and prostanoids were measured during cold preservation and after reperfusion. OKY046 and a novel TXA receptor antagonist [(9,11), (11,12)-Dideoxa-9a, 11a-dimethyl methano-11,12-methano-13,14-dihydro-13-aza-14-oxo-15-cyclo pentyl-16,17,18,19,20 pentanor-15-epi-TXA, ONO3708] were added into the preservation solution and perfusate. Along with the preservation time, both the production and release of TXA was observed to increase; however, almost all the produced TXA was stored in the liver tissue. Afterwards, the stored TXA was released into perfusate in 15 min after reperfusion. OKY046 significantly decreased both the production and release of TXA. In addition, OKY046 improved the histological damage and trypan blue uptake of liver cells. Our results demonstrate that TXA, stored in the liver during preservation, might therefore be a potential trigger of reperfusion injury, and as a result, OKY046 reduces reperfusion injury by decreasing the production of TXA during preservation. PMID- 8632643 TI - Selective glutathione repletion with oral oxothiazolidine carboxylate (OTZ) in the radiated tumor-bearing rat. AB - Oxothiazolidine carboxylate (OTZ) is a cysteine prodrug which augments intracellular glutathione (GSH) levels. We examined the effects of oral OTZ on tumor and host tissue reduced GSH levels in fasting and radiated models of GSH depletion. In addition, we studied the tumor's ability to utilize OTZ via the enzyme, oxoprolinase. Fischer 344 rats (n = 40) were implanted with MCA sarcoma and studied at 10% tumor burden. Treatment consisted of 10 mmol/kg OTZ or buffer orally. After a 24-hr fast, 16 animals were treated and tumor, kidney, jejunal, and colonic mucosa were collected after 4 hr. Significant increases in GSH with OTZ (n = 8) vs buffer (n = 8) were seen in kidney (5.6 +/- 0.4 vs 4.3 +/- 0.9; P < 0.01), jejunum (13.8 +/- 1.3 vs 12.1 +/- 1.1; P < 0.05), and colon (6.7 +/- 1.2 vs 5.3 +/- 0.6; P < 0.05), but not tumor (8.9 +/- 2.4 vs 10.6 +/- 1.4; P = 0.12). Sixteen animals were treated 4 hr before and 18 hr following 1100 cGy of abdominal radiation and at 4 days, tumor, jejunal, and colonic mucosa were collected. Significant increases in GSH with OTZ (n = 8) vs buffer (n = 8) were noted in jejunum (9.3 +/- 1.1 vs 7.5 +/- 1.8; P < 0.05) and colon (5.6 +/- 1.1 vs 4.3 +/- 0.9; P < 0.05) but not tumor (8.4 +/- 1.6 vs 7.6 +/- 1.4; P = 0.34). To determine tissue oxoprolinase activity, tumor, kidney, liver, jejunal, and colonic mucosa were collected from 8 animals. Oxoprolinase activity was highest in the kidney (814 +/- 145) with no difference noted between liver and tumor (280 +/- 117 and 324 +/- 137, respectively). Oral OTZ selectively increases reduced GSH levels in normal tissues compared to tumor following fasting and whole abdominal radiation. This increase does not appear to be due to a differential activity of oxoprolinase. OTZ may have a role in protection against toxicity associated with oxidative injury by selective repletion of normal host tissue GSH levels. PMID- 8632644 TI - Effect of rat mesenchymal stem cells on development of abdominal adhesions after surgery. AB - One of the common and most serious side effects of abdominal surgery is the formation of adhesions within the peritoneal cavity during healing. Efforts to prevent adhesion formation have concentrated on inhibiting the inflammatory response, inhibiting the formation or encouraging the lysis of fibrin, and protection of the damaged serosal surface. We are interested in regenerating the serosal surface by providing a source of mesothelial progenitor cells. Rats were divided into groups of 10 each. Abdominal adhesions were created by removing a circle of peritoneum and suturing it back into place. Two weeks later the rats were euthanized and the adhesions scored on a scale of 0-5. A population of mesenchymal stem cells (MSCs) isolated from the skeletal muscle of neonatal rats was tested. The cells were grown in primary culture to expand the population and then trypsinized and frozen at -80 degrees C. They are then thawed and grown in secondary culture before use. The control group were injected with saline i.p. immediately after surgery. The experimental groups received (1) 1.4 X 10(6) MSCs, (2) 5 X 10(6) MSCs, (3) 7.5 X 10(6) dead MSCs, (4) 5 X 10(6) rat smooth muscle cells immediately post-op, and (5) 5 X 10(6) MSCs 4-6 hours after surgery. Only live MSCs given immediately after surgery by i.p. injection significantly decreased the adhesion scores of the rats (mean score of 3.5 vs 0.9). MSCs injected i.p. 4-6 hours after surgery actually increased the adhesion scores (3.5 vs 4.7), and rat smooth muscle cells injected i.p. immediately after surgery had no effect on adhesions. The exact mechanism of action of the MSCs is unknown at this time. However, we postulate that the MSCs have the capacity to differentiate into mesothelial cells capable of repopulating the injured mesothelium. PMID- 8632645 TI - A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes. AB - A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart. PMID- 8632647 TI - Acquired immunity to TNF: effects on intestinal wound healing. AB - Increased concentrations of tumor necrosis factor (TNF) damage normal tissue and produce a shock-like syndrome--changes that can be prevented with anti-body specific antisera. These findings suggest that TNF-stimulated immunity should protect normal tissue and promote wound healing. To test this hypothesis, 30 Fischer 344 rats (150-200 g) were serially immunized against TNF (20 micrograms/kg). Convalescent sera assayed (micro-ELISA) for circulating antibodies revealed titers (2.54 +/- 0.08 au) significantly higher (P < 0.00001) in immunized animals than in nonimmunized controls (0.11 +/- 0.06 au). Following this, 10 immunized (Group I), 10 nonimmunized (Group II), and 10 control rats underwent partial cecectomy with primary anastomosis. Animals from Groups I and II received TNF (25 micrograms/kg) while controls received saline intravenously on Postoperative Days 1, 3, and 5. Animals were then sacrificed to determine: (1) hydroxyproline content of the anastomosis, (2) mitochondrial respiratory control ratio, and (3) pyruvate dehydrogenase activity of the muscle. We found that (1) exposure to increased concentrations of TNF (Group II) depresses (P < 0.01) biologic markers of wound healing and (2) acquired immunity to TNF (Group I) eliminates this response. In conclusion, acquired immunity to TNF protects the healing intestinal anastomosis from the effects of exposure to increased levels of TNF. PMID- 8632646 TI - Chronic pulmonary hypertension in sheep: temporal progression of lesions. AB - Scant data exist on the evolution of the lesions of pulmonary hypertension. This study establishes a model in sheep in which the left upper lobe (LUL) was rendered hypertensive by a systemic-pulmonary shunt while the rest of the pulmonary circulation remained normotensive. By examining lung tissue at 2 months and 1 1/2 years after shunting, we sought the temporal progression of pulmonary hypertensive lesions. In the hypertensive LULs (n = 5), many vascular lesions were seen in contrast to the absence of lesions in both the contralateral normotensive lungs (n = 5) and the "control" lungs from sheep which underwent thoracotomy without shunting (n = 5). Vascular necrosis and vasculitis were present after 2 months (P < 0.01) but disappeared after 1 1/2 years. In contrast, intimal thickening was present after 1 1/2 years (n = 2, P < 0.01) but not significantly after 2 months. These intimal lesions often demonstrated increased cellularity staining positively for factor VIII. Plexiform lesions were present at 2 months (P < 0.05) but were more profuse after 1 1/2 years (P < 0.01). These findings are consistent with an early vascular injury and a later remodeling or reparative process in hemodynamic pulmonary hypertension. PMID- 8632648 TI - Luminal cholecystokinin and gastrin cause gallbladder contraction. AB - Cholecystokinin-8 placed in the gallbladder lumen causes gallbladder contraction by a neurally mediated, tetrodotoxin-sensitive mechanism. We wished to determine whether other cholecystokinin-like peptides in the gallbladder lumen cause contraction and whether this response is inhibited by cholecystokinin-receptor antagonists. In this study, we measured gallbladder contraction induced by cholecystokinin-like peptides or by hepatic bile placed in the gallbladder lumen. Isolated gallbladders were suspended in an organ bath while luminal hormones were infused. Gallbladder contraction was measured by continuous monitoring of luminal pressure. Cholecystokinin-8, cholecystokinin-5, and gastrin-17 caused dose related gallbladder contraction with similar potency when placed in the lumen. Each stimulated 70-80% maximal contraction at a luminal concentration of 10(-6) M. Cholecystokinin-receptor antagonists CR1409 and loxiglumide partially inhibited contraction caused by luminal cholecystokinin-8. Bile from fed animals, but not from fasted animals, stimulated gallbladder contraction to 36 +/- 4% of maximal when placed in the gallbladder lumen. We conclude that cholecystokinin and gastrin peptides in the gallbladder lumen cause contraction. This may be mediated through receptors of the cholecystokinin-B type, possibly on intrinsic nerves. Bile from fed animals also contains substances that stimulate gallbladder contraction when bile is placed in the gallbladder lumen. These findings suggest intrinsic nerves participate in the postprandial gallbladder response to cholecystokinin. PMID- 8632649 TI - In vivo immunosuppressive effect and the decreasing expression of MHC class II antigens by purine nucleoside phosphorylase inhibitor 8-amino-9-benzylguanine in recipient tissues of canine renal allograft rejection. AB - 8-Amino-9-benzylguanine (8-ABG), a potent purine nucleoside phosphorylase inhibitor, was administered to dogs for 10 days following single renal transplantation. A significant prolongation of graft survival was observed in the groups treated with 100 and 150 mg kg/day of 8-ABG per os compared with the control group that was not treated with any immunosuppressant. Expression of major histocompatibility complex (MHC) class II antigens (Ag) was investigated in the normal kidney(s) and renal allografts of mongrel dogs after single renal transplantation. The 8-ABG was administered to the normal and renal allografted dogs and no detectable MHC class II Ag in the normal kidneys was found. During acute rejection, the MHC class II Ag was expressed on the renal tubular epithelium and glomerular vascular endothelium in graft kidneys. The intensity of the MHC class II Ag expression was correlated to the severity of rejection. This abnormal expression of MHC class II Ag on allograft kidney was suppressed by 8 ABG treatment. Our results suggest that MHC class II Ag expression can be induced on the renal allografts during acute rejection. This abnormal expression of MHC class II Ag may serve as a specific index for diagnosis of kidney allograft rejection. That 8-ABG can suppress abnormal expression of MHC class Ag on allografted kidney and prolong graft survival indicates that 8-ABG may provide an alternative approach for the development of a potential new immunosuppressant. PMID- 8632650 TI - Effect of subtotal pancreatectomy on the rate of liver regeneration: the role of hepatic stimulator substance. AB - The endogenous signals controlling liver regeneration are becoming rapidly defined. One of the key findings during liver regeneration following partial hepatectomy is the appearance of peripheral blood factors that stimulate DNA synthesis in the parenchymal cells. Hepatopoietin A, or hepatocyte growth factor, has been described as a complete mitogen for hepatocytes, one of its major tissue sources being the pancreas. The changes in the rate of DNA synthesis (incorporation of [3H] thymidine into DNA) in both subtotally pancreatectomized partially hepatectomized (Group A) and partially hepatectomized (Group B) male rats were determined at different postoperation times. DNA synthesis in Group A was increased progressively, reaching a maximum at 40 hr after operation in contrast to the 24- and 32-hr peaks obtained in nonpancreatectomized rats (Group B). However, liver regeneration in Group A was significantly lower than in Group B. To evaluate the effects of hepatic stimulator substance (HSS) on liver regeneration, the time course of HSS biological activity during the liver regeneration process was determined in the livers of Group A rats. A concurrent change of HSS activity and rate of liver regeneration was observed. These results support the hypothesis that HSS biological activity may be delayed but not suppressed by subtotal pancreatectomy. PMID- 8632651 TI - Peritoneal interleukin-8 in acute appendicitis. AB - Interleukin-8 (IL-8) is a chemoattractant that is highly selective for neutrophils. This study was designed to investigate the presence of IL-8 in peritoneal fluid of patients with acute appendicitis. The clinical circumstances underlying the secretion of IL-8 by mesothelium and its mechanism of activation have not been defined. In an in vitro model for bacterial peritonitis the role of bacteria in activating human mesothelial cells to secrete IL-8 was studied. Cultured human mesothelium was incubated with various species of pathogenic bacteria, isolated from peritoneal exudate fluids of patients with appendicitis. The amount of IL-8 secreted by the cultured mesothelial cells was determined in an IL-8 ELISA, as IL-8 was present in the original peritoneal fluid of these patients. Peritoneal fluids from patients with a perforated appendix were found to contain a significantly higher concentration of IL-8 compared to peritoneal fluids from patients with nonperforating appendicitis (121.6 (57.8) ng/ml versus 0.2 (0.07) ng/ml, respectively; mean (SEM), P < or = 0.01). Species of Bacteroides and Fusobacterium necrophorum induced IL-8 secretion from cultured mesothelial monolayers to levels comparable to those found in peritoneal fluids in vivo. Heat-killed bacteria and bacterial supernatant were also able to stimulate mesothelium to secrete IL-8. The results suggest that in the early phase of bacterial peritonitis the influx of PMN is regulated by bacteria-induced IL-8 secretion by the mesothelium lining the peritoneal cavity. PMID- 8632653 TI - Microtubules regulate pulmonary vascular smooth muscle contraction. AB - Microtubules are ubiquitous in eukaryotic cells. However, the role of microtubules in the mechanisms of pulmonary vascular smooth contraction has not previously been described. The purpose of this study was to examine the effect of microtubular inhibition (vinblastine) on the following mechanisms of pulmonary vascular smooth muscle contraction in rats using isolated pulmonary artery rings: (1) receptor-independent, calcium-dependent contraction via smooth muscle cell depolarization (response to KCl); (2) receptor-dependent, calcium-dependent contraction via alpha 1-adrenergic receptor stimulation (response to phenylephrine, PE); (3) receptor-dependent, calcium-independent contraction via thromboxane A2 receptor stimulation (response to the thromboxane mimetic, U 46619). Rats were studied 4 days after administration of vinblastine (750 micrograms/kg i.v.). Concentration-response curves were generated for KCl (5 mM to 100 mM) and for PE and U-46619 (10(-9) to 10(-4) M) (n = 8 rings/4 rats per group). Saline injected rats were controls. Pulmonary vascular smooth muscle contraction by calcium-dependent and -independent mechanisms was significantly increased following microtubular inhibition. These findings suggest that microtubules have an important role in the response of pulmonary vascular smooth muscle to vasoconstricting agonists. PMID- 8632652 TI - Attenuation of multiple organ dysfunction in a chronic sheep model by the 21 aminosteroid U74389G. AB - Animal studies have shown that 21-aminosteroids have beneficial effects on cell and organ functions in several acute models of traumatic, hemorrhagic, and septic shock. However, it is not known if the 21-aminosteroid U74389G has any beneficial effect on organ functions in a recently developed chronic sheep model of multiple organ dysfunction after trauma. Furthermore, it is not known whether this drug has any effect on in vivo leukocyte function in this animal model. To study this, anesthetized animals were subjected to hemorrhagic shock (2 hr at a mean arterial blood pressure of 50 mmHg) and femoral reaming at Day 0. The following 5 days, endotoxin (ET; 0.75 micrograms/kg BW) and zymosan-activated plasma (ZAP; 20 ml/animal) were given every 12 hr. During the third phase (Days 6-10), the animals were merely observed. This kind of model resulted in progressive organ dysfunction indicated by increased cardiac output, decreased systemic vascular resistance, an increase of plasma-sorbitoldehydrogenase, impaired bilirubin metabolism, and impaired renal and lung function in nontreated animals. Animals receiving U74389G (3 mg/kg BW) during resuscitation from hemorrhagic shock and each time before ET/ZAP administration showed less severe organ dysfunction. Furthermore, U74389G showed beneficial effects on lung function, although it had no effect on accumulation of leukocytes in the lung or on the chemiluminescence response of isolated leukocytes from bronchoalveolar lavage fluid. These results suggest that U74389G may be a useful therapeutic agent in the prevention of multiple organ dysfunction after hemorrhagic and traumatic shock. PMID- 8632654 TI - Current concepts in the development of cultured skin replacements. AB - Current tissue-culture techniques permit the rapid expansion of keratinocyte populations, such that an area of cultured epithelium equivalent to that of the surface of an adult can be obtained from an initial small skin biopsy. Unfortunately, technical obstacles have delayed the widespread clinical use of multilayered sheets of epithelium. These factors include difficulties in preparing and transferring fragile cultured epithelial sheets, as well as frequent unsatisfactory "take" of cultured grafts on the wound bed. As greater understanding of the complex interactions of cells and matrix evolves, so have new techniques in the field of cultured keratinocytes for grafting. We have utilized an animal model that allows us to examine some of these new methods and the factors which influence graft take. It has become clear that adhesion properties of keratinocytes, early delivery of proliferative keratinocytes to the wound, the development of dermal replacements, and improved delivery systems for keratinocytes are important factors which must be considered for the optimal provision of skin replacements. PMID- 8632655 TI - Confirmation of spurious hypoxemia using continuous blood gas analysis in a patient with chronic myelogenous leukemia. AB - Patients with extreme leukocytosis or thrombocytosis who have hypoxemia on arterial blood gas analysis may demonstrate normal oxygen saturation using pulse oximetry. The most commonly invoked explanation for this phenomenon is oxygen consumption in the blood gas sample prior to analysis. However, others have challenged the premise that the hypoxemia is spurious. We describe a patient with extreme leukocytosis and hypoxemia in whom normoxia was confirmed by continuous blood gas analysis. PMID- 8632656 TI - Apoptosis in myelodysplasia: a paradox or paradigm. AB - A growing body of evidence indicates that certain diseases are associated with increased apoptosis or inhibition of apoptosis. Among them, the myelodysplastic syndromes (MDS) are unique in that apoptosis is related, in apparently opposite directions, to the various facets of MDS. Ineffective hematopoiesis may be related to excessive intramedullary cell death via apoptosis and leukemic transformation conceivably results from escape from the apoptotic control. Future studies should be directed to define cellular susceptibility to and circumvention from apoptotic control mechanism(s). PMID- 8632657 TI - Postremission therapy with two different dose regimens of cytarabine in adults with acute myelogenous leukemia. AB - Sixty-seven out of 105 (64%) adults with de novo acute myelogenous leukemia (AML), achieving complete remission after induction chemotherapy, entered two successive postremission treatment protocols. Between 1987 and 1989, 35 patients received an intermediate dose of cytarabine (IDAC) along with other drugs. Between 1990 and 1993, 32 patients received high dose cytarabine (HIDAC) with similar other drugs. Patients treated with IDAC had a median survival of 13.8 months (95% CI 11.2-23.1 months) and a 2 year survival of 34.3 +/- 8.0%. Patients receiving HIDAC had a median survival of 35.5 months (95% CI, lower limit 29.8 months) and a 2 year survival of 71.6 +/- 9.4% (P < 0.002). The 2 year actuarial leukemia-free survival (LFS) was 17.8 +/- 6.6% in the IDAC group and 67.3 +/- 10.0% months in the HIDAC group (P = 0.004). The HIDAC group had a significant 2 year survival advantage over the IDAC group only in patients younger than 45 years. The 2 year survival in the first group was 83.3 +/- 10.8% versus 23.5 +/- 10.3% in the IDAC group (P = 0.0001). In patients older than 45 years, no significant differences in 2 year survival was noticed (52.9 +/- 15.78 versus 44.4 +/- 11.7, P = 0.8). Censoring the 21 patients who underwent bone marrow transplantation (BMT) at BMT did not change significantly the survival analysis of the patients in each group. This study is consistent with previous reports favoring HIDAC intensification in the postremission treatment of young patients with AML. PMID- 8632658 TI - Combination chemotherapy of carboplatin and cytosine arabinoside for high-risk leukemia: a pilot study. AB - Fourteen patients with high-risk leukemia (six with relapsed AML, three with relapsed ALL, one with AML-M0, four with CML in myeloid blastic crisis) were treated with a combination chemotherapy of carboplatin (200-300 mg/m2/day) and cytosine arabinoside (100 mg/m2/day) by 24 h continuous infusion for 5-7 days. Five patients (35.7%) achieved complete remission including two patients complicated with myelofibrosis (one with AML-M0 and one with CML in myelo megakaryocytic crisis). Thirteen patients had nausea and vomiting, five patients had severe, prolonged neutropenia for which it was necessary to administer granulocyte colony-stimulating factor and six patients had severe thrombocytopenia. We concluded that this regimen is effective for the treatment of high-risk leukemia. PMID- 8632659 TI - Complex hypodiploidy in acute myeloid leukaemia: a United Kingdom Cancer Cytogenetics Group study. AB - Forty cases of acute myeloid leukaemia with 41 hypodiploid clones were investigated in a collaborative study. Cases with -5, -7, -X or -Y either alone or in association with an established translocation were excluded. Karyotypes were reviewed in all cases and bone marrow or blood morphology was reviewed in 22 cases. Twenty-six cases were very complex (more than five abnormal chromosomes), 14 cases were complex (two to five abnormal chromosomes) and only one case was simple (one abnormal chromosome). Chromosomes 5, 7, 17 and 18 were involved in a significantly greater number of cases than expected. Only five cases had normal chromosomes 5 and 7. Chromosomes 10, X and Y were involved in significantly fewer cases than expected. Ten cases had ring chromosomes and 18 showed clonal evolution. Patients were aged between 19 and 90, median 61 years. Evidence of myelodysplasia was found on morphological review in 18/22 cases, and on clinical features in a further five cases. There was a high proportion of cases with FAB M6, 'erythroleukaemia' (9/31 cases). Only 4/16 patients treated with cytotoxic therapy achieved remission. Median survival was 2.5 months (35 patients). Survival was slightly better for patients with normal chromosomes 5 and 7, and for those with simpler karyotypes, but this was not statistically significant. This study confirms the association between hypodiploidy, complex karyotype, abnormalities of chromosomes 5 and 7 and a poor prognosis. PMID- 8632660 TI - Increased proliferation of eosinophil clusters in myelodysplastic syndromes. AB - A patient with myelodysplastic syndromes (MDS) developed eosinophilia during treatment with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). To study the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in nine patients and four healthy controls. Eosinophil clusters increased significantly in the patients (P < 0.01) compared with controls, but eosinophil colonies were not different between controls and MDS patients. In addition, the eosinophil clusters were significantly increased with rhGM-CSF in MDS patients compared with controls, although serum GM-CSF concentrations were similar in both groups. These results suggest that eosinophil clusters are increased in MDS either through abnormal progenitor proliferation or hypersensitivity to GM-CSF. PMID- 8632661 TI - Fluorescence in situ hybridization provides evidence for two-step rearrangement in a masked Ph chromosome formation. AB - A chronic myelogenous leukemia (CML) patient with a masked Ph chromosome due to the translocation (9;10;22)(q34;q24;q11) is reported. Banding analysis showed a 9q+ chromosome typical of standard t(9;22)(q34;q11), and fluorescence in situ hybridization studies confirmed the involvement of a chromosome 10 in the masked Ph formation and also the presence of 3' ABL-DNA sequences in the der(22). This complex rearrangement could be explained by two consecutive translocations: the first, a standard t(9;22) (q34;q11), the second, a translocation between a chromosome 10 and the der(22) with a breakpoint in sequences derived from chromosome 9 telomeric to the ABL gene. By reverse transcription polymerase chain reaction (RT-PCR), we studied the BCR/ABL transcript junction: a chimeric m-RNA b3-a2, indicating a breakpoint within the major breakpoint cluster region, was found. PMID- 8632662 TI - Prognostic value of rhodamine-efflux and MDR-1/P-170 expression in childhood acute leukemia. AB - The evidence that mechanisms other than P-170 expression may influence its "pump" and the retention/efflux of chemotherapeutic agents, prompted us to investigate the value of a functional multidrug resistance (MDR) assay in a series of childhood acute leukemia samples. Forty acute leukemia cases, mainly of lymphoid origin (ALL), were evaluated for MDR expression using a functional test based on rhodamine-123 efflux (Rhd-E). This was correlated with the quantification of P 170 external epitopes based on the positivity with the 4E3.16 and MRK16 monoclonal antibodies (MAbs). When compared with the status of the disease and response to treatment, the mean (m) Rhd-E value was significantly lower in patients at diagnosis (m = 7.1% versus m = 22.4% at relapse) and in patients who achieved a complete remission (m = 8.81% versus 31.5% in resistant cases). In the 22 samples analyzed, an overall correlation was found between the functional assay and the P-170 expression (r = 0.6), despite the much lower level of MDR positivity recognized by the immunocytometric method (m = 0.78% and 0.9% in cases at diagnosis versus m = 3.7% and 4.1% at relapse, with the 4E3.16 and MRK16 MoAbs). These data suggest that the assessment of the clinical impact of MDR expression in pediatric ALL should be based on methodological approaches capable of providing information extended to the P-170 pump function, rather then only on its gene and protein expression. PMID- 8632664 TI - A cardiotonic steroid bufalin-like factor in human plasma induces leukemia cell differentiation. AB - A Na+,K(+)-ATPase inhibitor, bufalin, has been shown previously to induce leukemia cell differentiation. The presence of a circulating Na+,K(+)-ATPase inhibitor has been proposed in mammals. The aim of this study was to explore an endogenous bufalin-like factor that induces leukemia cell differentiation. We found a fraction, designated as fraction A, obtained from human plasma extract that inhibits the growth of several human-derived leukemia cell lines. The effect of the fraction was retained after protease digestion or heat treatment. Murine leukemia cells and ouabain-resistant cells, which are insensitive to bufalin, appeared to be refractory to fraction A in terms of growth inhibition. Fraction A also induced functional and morphological maturation in THP-1 cells. Fraction A was recognized by anti-bufalin anti-serum and inhibited 3H-bufalin binding to K562 cells. These findings suggest that fraction A shows a similar behavior to that of bufalin on leukemia cells by inhibiting Na+,K(+)-ATPase. We propose that an endogenous Na+,K(+)-ATPase inhibitor in human plasma may play a role in cell differentiation. PMID- 8632663 TI - Megakaryocytic differentiation of a leukemic cell line, MC3, by phorbol ester: induction of glycoprotein IIb/IIIa and effects on expression of IL-6, IL-6 receptor, mpl and GATA genes. AB - We investigated megakaryocytic differentiation in a newly-established Ph1 positive leukemic cell line, MC3, which showed tri-lineage immunophenotypes (myeloid antigens2+, CD19(1+) and CD41a1+) and was positive for CD34 and CD38. TPA induced MC3 cells to differentiate to an early stage of megakaryocyte lineage exhibiting an increase in the expression of platelet glycoproteins (GP) IIb/IIIa (CD41a), and an increase in cell size and nuclear ploidy. TPA treatment also enhanced the expression of GPIIb mRNA, and induced the expression of interleukin 6 (IL-6) and its receptor mRNAs, while it did not induce transcripts of the genes IL-11 and mpl ligand, and further decreased the transcript of the mpl gene. Consistent with these findings, MC3 cells treated with TPA showed an increased expression of GATA-1, but not GATA-3 transcripts, whereas those without TPA treatment expressed only the GATA-2 transcript. These results provide an insight into the study for the regulatory mechanism of megakaryocytopoiesis and leukemic cell differentiation. PMID- 8632666 TI - The first intron of the BCR gene contains two minor alternative exons. AB - In Philadelphia chromosome (Ph1) positive leukemias, the BCR gene is fused to the ABL gene. The resulting chimeric BCR-ABL oncoproteins are thought to play a central role in the pathogenesis of these diseases. We previously described two exons that can be spliced alternatively to the second BCR exon in place of the first exon to form minor messages. In this paper, we localize the alternative exons to a 4.1 kb BglII fragment in the 5' region of the large first intron of the BCR gene. This genomic structure is of interest because of its analogy to the organization of the ABL gene and because this part of the gene is not affected by the breakpoints occurring in Ph1-positive acute lymphoblastic leukemia (ALL). Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the alternative messages in all cases of chronic myelogenous leukemia (CML) tested, including seven samples in the chronic phase, five in the accelerated phase and nine in the acute phase, as well as in the majority of other samples studied. These findings suggest a functional role for the variant transcripts. PMID- 8632665 TI - Induction of differentiation in erythroleukemic K562 cells by gamma-irradiation. AB - Various agents have been shown to induce differentiation in neoplastic cells. The present study aimed at investigating comparable phenomena induced by high doses of gamma-irradiation in the presence of physiological factors. The erythroleukemic K562 cells were gamma-irradiated or treated with cytosine arabinoside (Ara-C), and examined for cell size, protein content, acetylcholinesterase (AChE)-activity and hemoglobin synthesis in relation to mitotic activity. At doses above 10 Gy, differentiation was induced, as recognized by elevated AChE-activity, accompanied by an increase in cell size and protein content and cessation of cell proliferation. Moreover, irradiation, as well as Ara-C, induced hemoglobin synthesis when cultures were supplemented with hemin prior to treatment. It is suggested that the basic mechanisms of differentiation induction are similar for ionizing radiation and certain chemical agents and are related to continued growth of essential cytoplasmic constituents during inhibition of mitotic activity. PMID- 8632667 TI - Clonal dominance of RadLV-induced lymphomas. AB - RadLV-induced leukemogenesis begins with the emergence of a pleioclonal population of preleukemic (PL) cells, which subsequently gives rise to a monoclonal lymphoma. We have recently found that the pleioclonal-->monoclonal transition may occur early during the PL latency and long before the eruption of a full blown lymphoma. We sought to find out what causes one PL clone to become dominant. Our working hypothesis was that a dominant clone(s) at the PL stage has the ability to inhibit the development of other, recessive clones. Since some premalignant characteristics of a progenitor clone are probably maintained in the descending lymphoma, we studied whether tumors that developed after injection of a high dose (HD) of PL cells were dominant over tumors that developed after injection of a limiting dose (LD) of PL cells. To identify dominant clones, HD and LD lymphomas were mixed in a co-culture and the outgrowth of one clone over the other was determined by T beta-TCR rearrangement analysis. A checker-board combination of seven lymphomas revealed a dominance hierarchy scale. Lymphomas induced directly by the virus (without transfer) were dominant over both HD and LD lymphomas. High dose lymphomas were dominant over LD lymphomas and LD lymphomas were always recessive. The speed at which a dominant lymphoma outgrew the co-culture suggested that dominance is acquired through the ability of the prevailing cells to actively suppress the growth of recessive cells. PMID- 8632668 TI - Ultrastructural investigation of circulating villous lymphoid cells: a tool in the differential diagnosis of splenic lymphoma with villous lymphocytes. AB - Ultrastructural examination of circulating lymphoid cells was performed in three cases of splenic lymphoma with circulating lymphocytes (SLVL) in order to define morphological features helpful to distinguish this lymphoma from hairy cell leukemia (HCL). The samples for ultrastructural investigation were obtained by Ficoll sedimentation from peripheral blood and routinely processed for electron microscopy. The ultrastructural features examined were: morphology of villi, morphology of nuclei, presence of nucleoli, distribution of heterochromatin, type of cytoplasmic organelles, presence of specific intracytoplasmic structures such as the ribosome-lamella complex, lysosome-like bodies and perinuclear microfibrils. Our results and a careful review of the literature seemed to confirm that SLVL has electron microscopic features typical enough to be relevant in the differential diagnosis with HCL. PMID- 8632669 TI - The sensitivity of chronic lymphocytic leukaemia lymphocytes to irradiation in vitro. AB - The inhibition of [3H]-thymidine incorporation into the DNA of mitogen-stimulated chronic lymphocytic leukaemia lymphocytes by chlorambucil or gamma-irradiation in vitro was measured in a series of patients, some of whom were untreated, some treated and some who were showing resistance to first-line or second-line treatment. There was evidence of resistance to irradiation developing in parallel with that to chlorambucil. The resistance to chlorambucil in chronic lymphocytic leukaemia (CLL) is not necessarily due to altered drug transport or metabolism but to a more fundamental process affecting DNA damage. PMID- 8632670 TI - Expression of Id2 and Id3 mRNA in human lymphocytes. AB - Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate. PMID- 8632671 TI - Decreased expression of both Fc gamma RII and Fc gamma RIII mRNA in leukemic granulocytes. AB - Morphologically mature granulocytes from patients with chronic myeloid leukemia exhibit a defect in internalization of heat-aggregated IgG. In this study, we investigate the status of the steady-state levels of the mRNA for the two receptors for IgG, Fc gamma RII and Fc gamma RIII, as a step towards understanding the molecular basis of the defect and in turn the discordant maturation of the leukemic cells. Our data show that the mRNA for both receptors is lower in the leukemic cells relative to the normal cells. This may be one of the causes for the defective endocytosis. PMID- 8632672 TI - Tissue factor expression in human leukemic cells. AB - Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mo) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mo demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL 60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation. PMID- 8632673 TI - Deciphering the pathogenesis of coagulation dysfunction in leukemia. PMID- 8632674 TI - Inhibition of human leukaemia 60 cell growth by S-D-lactoylglutathione in vitro. Mediation by metabolism to N-D-lactoylcysteine and induction of apoptosis. AB - The inhibition of human leukaemia 60 cell growth by S-D-lactoylglutathione in vitro is mediated by the inhibtion of de novo pyridimine synthesis. When S-D lactoylglutathione was added to human leukaemia 60 cells in culture, it was hydrolysed by thiolesterase activity to reduced glutathione and D-lactate but also converted to N-D-lactoylcysteinylglycine and N-D-lactoylcysteine by gamma glutamyl transferase and dipeptidase. The N-D-lactoylcysteine inhibited human leukaemia 60 cell growth: the median growth inhibitory concentration IC(50) value was 46.7 +/ -0.9 (N=30) and the median toxic concentration TC(50) value was 103 +/- 1 microM. Other N-(R)2-hydroxyacylcysteine derivatives, N-D-mandelylcysteine and N-L-glyceroylcysteine, were less effective inhibitors of human leukaemia 60 cell growth, whereas N-D-lactoylcysteine ethyl ester was more effective: the IC(50) value was 16.5 +/- 1.5 microM(N=8). Cytotoxic concentrations of S-D lactoylglutathione-induced apoptosis in human leukaemia 60 cells. The S-D lactoylglutathione was not toxic to peripheral human lymphocytes at the same concentrations but rather induced growth arrest. The expected mechanism of action of N-D-lactoylcysteine is inhibition of dihydro-orotase, which is particularly susceptible to inhibition by cysteine derivatives. PMID- 8632675 TI - Effects of recombinant human granulocyte colony-stimulating factor on the growth potential of two murine myeloid leukemias. AB - We investigated the in vitro and in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the proliferation of two murine leukemic cell lines. The rhG-CSF stimulated leukemic colony formation of the promyelocytic leukemic cell line L-8801 in methylcellulose culture and increased the number of L-8801 cells in liquid culture. However, rhG-CSF treatment prolonged the median survival period of mice implanted with L-8801 cells and the emergence of the leukemic blast cells in peripheral blood. Meanwhile, rhG-CSF had no influence on that of the megakaryoblastic leukemic cells L-8057 and failed to prolong the median survival period of L-8057 leukemic mice. Receptor binding analysis revealed that L-8801 cells expressed a G-CSF receptor (Kd=125 pM, 479 binding sites/cell) and L-8057 cells had no G-CSF receptors. Then, we examined the growth potential of these cells. The median survival period was longer for mice implanted with L-8801 cells cultured with rhG-CSF for 72 h in vitro than for cells grown without rhG-CSF. Furthermore, the median survival period of mice implanted with spleen cells from L-8801 leukemic mice treated with rhG-CSF was prolonged compared with those from leukemic mice without rhG-CSF. In contrast, there was no effect of rhG-CSF on the growth potential of the spleen from L-8057 leukemic mice. The results of our present study demonstrate that rhG-CSF reduced the growth of L-8801 leukemic cells in vitro and in vivo mediated through G-CSF receptors, thereby suppressing the development of leukemia. PMID- 8632677 TI - Modulation of surface TNF expression by human leukaemic cells alters their sensitivity to exogenous TNF. AB - In this study, U937 leukaemic cells underwent apoptotic cell death following exposure to TNF. Pre-incubation of cells for 48 h with VitD(3) (10(-8)M) induced resistance to TNF, whereas incubation with tau-IFN or GM-CSF increased susceptibility to TNF. Resistance to exogenous TNF (exTNF) following culture with VitD(3) was associated with increased expression of endogenous TNF (enTNF). The TNF inhibitors pentoxifylline(PTF) and dichloroisocoumarin (DCI) inhibited TNF synthesis by U937 cells and abrogated the increase in resistance to TNF seen with VitD(3). The tau-IFN increased TNF expression, whereas GM-CSF had little effect. The data show that the sensitivity of leukaemic cells to exTNF can be modulated by cytokines. The protective effect of VitD(3) is mediated in part by directly upregulating enTNF synthesis. PMID- 8632676 TI - Effects of fludarabine and gemcitabine on human acute myeloid leukemia cell line HL 60: direct comparison of cytotoxicity and cellular Ara-C uptake enhancement. AB - This study was designed to compare the effects of fludarabine and gemcitabine on cytosine arabinoside (Ara-C) uptake and retention, and their specific cytotoxicity on HL 60 human acute myeloid leukemia cells. The leukemic blasts were exposed to either drug at equimolar concentrations (10 microM) for 3 h and further incubated with Ara-C (5 microM), added immediately (day 0) or after an interval of 24 h in cells were kept in a drug free medium (day 1). On day 0, leukemic cells exposed to fludarabine 10 microM had a significant (P<0.01) increase in Ara-C uptake (297 +/- 11 pmol/10(7) cells) with respect to control cells (not pre-treated: 195 +/- 10 pmol/10 (7) cells). After treatment of leukemic cells with fludarabine, cytoplasmic Ara-C peaked after 180 min of exposure, as well as nuclear bound Ara-C. At the same time, a significant decrease in the number of S-phase leukemic cells, consistent with depressed [3 H]TdR uptake was observed. Although on day 0 gemcitabine 10 microM did not have potentiating effects on Ara-C uptake, it showed a high degree of intrinsic cytotoxicity as a single agent(clear from cell cycle distribution, [3H]TdR uptake, plating efficiency (PE) data and percentage of apoptotic cells). Cells exposed to gemcitabine, on the other hand, showed on day 1 a significant increase in Ara-C uptake (2.4 x control values in the cytoplasmic and 3x in the nuclear fractions) and a reduced number of S-phase blasts, [3H]TdR uptake and PEs, as well as an increased apoptotic cell death. Evidently, it is possible to modulate Ara-C uptake by leukemic cells with gemcitabine. Although this effect is similar to that demonstrated with fludarabine, its kinetics and time of efficacy are different and also, because of its intrinsic higher cytoxicity and lack of important side effects, gemcitabine could be considered a suitable candidate for Ara-C association therapy in acute leukemia. PMID- 8632678 TI - The novel immunomodulator, Linomide, stimulates interleukin-2-induced human natural killer (NK) cell and PHA-stimulated T cell proliferation from normal donors. AB - Donor-derived cell-mediated immunotherapy has been shown to be an effective tool for reinduction of remission in chronic myeloid leukaemia (CML) patients who have relapsed post-bone marrow transplantation (BMT). Linomide, quinoline-3 carboxamine (LS 2616), is a new immunomodulator shown to increase the number of NK precursors in mice in addition to upregulating the quantity of CD56(+), CD3(-) and CD16(+) NK cells in the peripheral blood of patients following autologous BMT (ABMT). We investigated the in vitro effects of Linomide on NK activity of normal human donors. Large granular lymphocytes (LGLs) and NK cells were incubated overnight with Linomide (0.02-4.8 mg/ml), recombinant human interleukin-2 (IL-2, 75 IU/ml), or a combination of both. Linomide, at 0.02-0.3 mg/ml, augmented IL-2 induced proliferation of LGLs and NK cells in an inversely proportional manner. In contrast, Linomide at 0.6-4.8 mg/ml inhibited IL-2-induced proliferation of LGLs and NK cells in a dose-dependent manner. Linomide was able to potentiate phytohemaglutinin-induced CD3(+) cell proliferation. In addition, supernatants derived from Linomide treated CD3(+) T cells were able to mimic the direct stimulatory effect of Linomide on activated NK cell proliferation. These supernatants were found to have low levels of tissue necrosis factor-alpha (TNF alpha) and interleukin-1 (IL-1) and therefore Linomide stimulation of NK and T cell proliferation may be due to its inhibitory effect on the secretion of these cytokines by activated CD3(+) T cells. Linomide had no effect on cytotoxicity nor on the phenotypic expression of resting and IL-2-activated LGLs or NK cells. In view of our results, Linomide could possibly play a potential role in adoptive cell-mediated immunotherapy post-BMT. PMID- 8632679 TI - Effects of endogenous interleukin 1 on blast cells derived from acute myelogenous leukemia patients. AB - The in vitro effects of interleukin 1 (IL1) secreted by acute myelogenous leukemia (AML) blasts (termed endogenous IL 1 secretion) were investigated. Interleukin 1-dependent AML blast functions were inhibited during in vitro culture either by IL1-specific neutralizing antibodies (anti-IL1 alpha and anti IL1 beta) or by the IL1 receptor antagonist (IL1RA). Endogenous IL1 secretion showed a wide variation between individual patients, but despite this variation IL1 inhibition significantly decreased both spontaneous blast proliferation and spontaneous blast secretion of IL1 alpha, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha and interleukin 6. In contrast to spontaneous blast proliferation, in the presence of exogenous hematopoietic growth factors (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, tumor necrosis factor alpha, stem cell factor), IL1 inhibition caused either increased or decreased AML blast proliferation depending on the individual patient. When AML blasts were cultured with stem cell factor plus granulocyte-macrophage colony stimulating factor, IL1 inhibition significantly increased AML blast proliferation. Thus, IL1 is important for regulation of AML blast proliferation and cytokine secretion independent of the level of endogenous IL1 secretion, but the final effect of IL1 is highly dependent on the cytokine network in the AML blast microenvironment. PMID- 8632681 TI - Parvovirus B19 infection infrequently involved in children and adults with myelodysplastic syndrome. AB - Parvovirus B19 infection has occasionally been reported to mimic myelodysplastic syndrome (MDS) or to cause worsening of anemia in MDS. We examined the presence of parvovirus DNA in a series of children (n=19) and adults (n=39) with a diagnosis of MDS. The series of adults included only refractory anemia (RA) and RA with ring sideroblasts (RARS). Investigation for parvovirus B19 DNA in bone marrow cells was performed employing the nested form of the polymerase chain reaction (PCR). Only a 51-year-old male with RA tested positive for parvovirus DNA. Serial examinations demonstrated the disappearance of parvovirus DNA from the bone marrow. We conclude that parvovirus infection may only rarely mimic MDS or be a superimposed infection in childhood MDS or in RA and RARS in adults. PMID- 8632680 TI - Levels of CAF7 (CD98) expression correlate with the complete remission duration in childhood acute leukemia. AB - The levels of expression of the activation antigen CD98 were studied by mAB CAF7 in 51 newly diagnosed consecutive cases of childhood acute lymphoblastic leukemia aged from 1 to 13 years. The mean follow-up was 8 months. A wide range of CAF7 expression was observed, the highest mean fluorescence intensity exceeding the lowest by 20 times. No correlation was revealed between CAF7 cell surface density on the one hand and sex, age, WBC, platelet count, LDH level, FAB groups and immunophenotypes on the other. A positive association between the levels of CAF7 expression and the complete remission (CR) duration was observed. The group of CAF7(low) patients had a significantly shorter CR duration compared to the CAF7(intermediate) and CAF7(high) cases (P=0.0099). Half of the CAF7(low) patients did not respond to the induction therapy and failed to achieve remission. These correlations were clearly marked in common ALL (cALL), which was usually considered to have a favorable outcome. All CAF7(low) cALL cases had a significantly shorter CR duration (P=0.027). Thus CAF7 appears to provide additional information on the biological characteristics of childhood ALL and may have prognostic value regarding the response to therapy and remission duration. PMID- 8632682 TI - Detection of c-myc oncogene amplification in a CML blastic phase patient with double minute chromosomes. AB - Double minute chromosomes (dmin) are relatively rare in leukemias. Cytogenetic analysis of blood cells from a woman with blastic phase chronic myelogenous leukemia (BC-CML) showed numerous dmin chromosomes and complex abnormalities including a Philadelphia (ph(1))-chromosome. Oncogene amplification in hematopoietic malignancies is also rare. Using PCR, we retrospectively investigated the extent of c-myc gene amplification in DNA extracted from stored blood smears from the patient. To qualify the PCR products, the beta-globin gene was used as the internal reference gene and it was co-amplified with the c-myc gene. The extent of amplified c-myc was about 6.8-fold. This finding suggests that the c-myc gene was amplified in dmin and that the gene amplification contributes to the progression to acute leukemia or rapid growth of leukemic cells. PMID- 8632683 TI - Fc gamma RII and Fc gamma RIII on normal leukaemic granulocytes: a flow cytometry and northern analysis. AB - Morphologically mature granulocytes from patients with chronic myeloid leukaemia (CML) exhibit a defect in internalization of heat-aggregated IgG. In order to investigate this defect at the molecular level and, in turn, the discordant maturation of these granulocytes, we compared the expression of Fc gamma RII and Fc gamma RIII, the two receptors for IgG on the surface of granulocytes, in normal and CML samples. Our flow cytometric data show that the number of granulocytes expressing Fc gamma RIII is lowered in CML patients to near half that in normal individuals, with a simultaneous decrease in the steady-state levels of the mRNA for Fc gamma RIII. Mean fluorescence intensity (MFI), an indicator of the number of receptors per cell, varies widely for Fc gamma RIII in normal individuals whereas it is more localized and lowered in granulocytes from CML patients. The number of granulocytes positive for Fc gamma RII is also significantly lowered in CML samples compared to the normals, which exhibit a wide variation in the number of cells positive for the receptor, even though their nRNA levels do not vary much. The CML granulocytes, in general, exhibit lowered levels of the steady-state mRNA for Fc gamma RII. The MFI for the surface expression of Fc gamma RII is only marginally different between the two cell types. Our data indicate that the morphologically homogeneous population of CML granulocytes actually consists of at least two types of cells, one which expresses the Fc receptors and one which does not. PMID- 8632685 TI - Using health status measures with the seriously mentally ill in health services research. AB - Changes in the delivery of mental health care have prompted interest in using generic health status measures to test the effect of system change on those receiving treatment. Of special concern are those with serious and persistent mental illness who may be neglected when cost containment efforts reduce the availability of treatment services. This population may be affected by these changes, which might go undetected if investigators use scales that measure only pathology and not the full spectrum of well-being. The purpose of this study was to test the feasibility of using self-report health status measures with this population, to describe the psychometric properties of the scales, and to report the health status scores of a random sample of the Medicaid psychiatrically disabled population. We found that the four health status scales had adequate psychometric properties, that score variability was high, the distributions normal, and that patterns of association with more traditional clinical measures were of the expected size and direction. One scale, General Health Perceptions, had reliability and item-to-score correlation below acceptable levels. PMID- 8632684 TI - Why do physicians prefer to withdraw some forms of life support over others? Intrinsic attributes of life-sustaining treatments are associated with physicians' preferences. AB - Some physicians caring for critically ill patients have preferences for withdrawing some forms of life support over others, even after the decision to withdraw life support has already been made. Past research has attempted to explain these preferences by variations in clinical circumstances. The authors wondered whether differences in the forms of life support themselves might be important, and whether these differences would reveal implicit goals that physicians attempt to achieve. Four hundred fifty-six university-affiliated internists were surveyed and their rank-ordered preferences for withdrawing eight different forms of life support were assessed. The authors then sought to explain these preferences on the basis of intrinsic characteristics of the eight forms of life support determined by an expert panel of critical care physicians. In general, the physicians studied prefer to withdraw forms of life support that are scarce, expensive, invasive, artificial, unnatural, emotionally taxing, high technology, and rapidly fatal when withdrawn. They prefer not to withdraw forms of therapy that require continuous rather than intermittent administration, and forms of therapy that cause pain when withdrawn. Even when a decision has been made to withdraw life-sustaining treatment from a patient, many physicians have preferences for the manner in which this is accomplished. These preferences may reflect perceived intrinsic characteristics of different forms of life support that are consistent across physicians. PMID- 8632686 TI - Comparing the use of diagnostic tests in Canadian and US hospitals. AB - Although Americans pay much more for a day in the hospital than Canadians, we know little about whether inpatient physician practice patterns might explain some of this difference. The authors compared the utilization of all diagnostic imaging (plain radiographs, computed tomography (CT) and magnetic resonance imaging (MRI) scanning, ultrasound, nuclear medicine and vascular studies) and selected laboratory tests (hematology, basic biochemistry, and advanced biochemistry) for all patients discharged with selected medical and surgical diagnoses in 1990 and 1991 from four university hospitals and four community hospitals in Canada (n = 6,491) and the United States (n = 7,980). Overall, US medical patients received 22% more diagnostic tests than their Canadian counterparts (544.2 relative value units [RVUs] vs. 446.5 RVUs in Canada, P < 0.001), which was mainly the result of higher radiology use. Although mean radiology use was 40% higher in the United States (370.0 vs. 264.5 RVUs in Canada, P < 0.05), there was little difference in the use of laboratory tests between countries (174.2 vs. 182.4 RVUs in Canada, P = 0.3). Within radiology, only CT and MRI use differed significantly between countries (US patients received 119% more tests than Canadians). These findings were consistent after adjustments for age, gender, diagnosis-related group, and university status. Differences in test use between countries were mainly the result of more testing among the US elderly than counterparts in Canada. Among surgical patients, there was little difference between countries for radiology (76.3 vs. 67.3 RVUs in Canada, P < 0.05) and laboratory (83.6 vs. 91.4 RVUs in Canada, P < 0.05). Comparable inpatients admitted to US hospitals received more diagnostic tests than their Canadian counterparts even in hospitals with similar availability of technology. Differences between countries were larger for high-cost tests than for lower-cost tests. Much of the difference in test use is explained by more intensive use for the elderly in the United States. PMID- 8632687 TI - Attitudes of medical students and primary care physicians regarding input of older and younger patients in medical decisions. AB - This study was designed to determine whether medical students, residents, and fully trained physicians differ in their attitudes toward decision-making input by older and younger patients, whether they believe that physicians should have greater input than patients in medical decisions, whether physicians grant different decision-making authority to younger versus older patients, and whether physician gender affects attitudes toward patient input in medical decisions. Respondents (n = 818) were first- (n = 311) and third-year (n = 227) medical students and family practice and internal medicine residents (n = 120) and faculty (n = 160) from the University of Kansas Medical Center (n = 367) and The Ohio State University Hospital (n = 451) who completed the Beisecker Locus of Authority: Geriatrics Scale to assess attitudes regarding involvement in medical decision making for either a 25- or 75-year-old patient. Respondents were alternately assigned to one of the two patient age vignettes. Analyses included descriptive statistics, t tests, and four-way analysis of variance. Ninety percent of respondents believed that physicians should have greater input in decisions than patients. Female respondents advocated greater patient input than male respondents. As training and experience increased beyond medical school, there was an increased tendency toward belief in physician-only decision making. For the older patient, residents advocated the most patient input and faculty advocated the least. Level of training influenced belief in patient input when its interaction with patient age and institution were examined. PMID- 8632688 TI - The effects of having a regular doctor on access to primary care. AB - The authors assessed the relationship between having a regular doctor and access to care, as measured by a set of preventive and primary care utilization indicators recommended by the Institute of Medicine. The 1987 National Medical Expenditure Survey was used in the analyses (n = 30,012). The results of the regression analyses suggest that individuals with any type of regular source of care had better access than those without a regular source of care. Persons with a regular doctor had better access to primary care than those with a regular site but no regular doctor. However, the apparent advantage of having a regular doctor over a regular site disappeared when only those individuals reporting a physician's office, clinic, or health maintenance organization as their regular source of care were compared. These results suggest that policies that promote the doctor-patient relationship will increase access, although the gains may be negligible for individuals who use mainstream primary care sites (physician's office, clinic, or health maintenance organization) versus sites such as walk-in clinics or emergency rooms. PMID- 8632689 TI - Factors that determine the treatment for local and regional prostate cancer. AB - This article assesses the significance of comorbid and nonclinical factors in type of treatment received by elderly male patients with local-regional stage prostate cancer. Multivariate analysis of data from the Virginia Cancer Registry was linked to Medicare claim files, the Area Resource File, and 1990 Census Data. The type of initial treatment received was studied in 3117 men with local regional staged prostate cancer diagnosed from 1985 to 1989. The frequency of surgical and radiation therapy for prostate cancer rose between 1985 and 1989 (12.5% to 18.5% for surgery, P < 0.001; 25% to 32% for radiation, P < 0.001). Age was the most important predictor of therapeutic choice; no therapy was given to 26% of men 65 to 69 years old versus 63% of men 85 years or older P < 0.001). Race, residence (rural versus urban), and comorbidity were also strong factors in predicting initial therapy. Using logistic regression, three treatment alternatives were evaluated. Age (odds ratio [OR] .51; 99% confidence interval [CI] = .43, .60), comorbidity (OR .72; 99% CI .63, .82), income (OR 1.14; 99% CI 1.01, 1.28), residence (OR .65; 99% CI .48, .87), diagnosis year (OR 1.15; 99% CI 1.07, 1.23) all were associated independently with treatment versus no treatment. For surgery versus radiation, age (OR .40; 99% CI .27, .57), race (OR 2.92; 99% CI 1.65, 5.15) and education (OR 1.75; 99% CI 1.31, 2.34) were significant factors. For hormonal/orchiectomy versus surgery/radiation, age (OR 5.19; 99% CI 3.84, 7.01), comorbidity (OR 1.28; 99% CI 1.03, 1.58), distance to radiation oncologist (OR .89; 99% CI .80, .99), and diagnosis year (OR .89; 99% CI .79, 1.00) were significant. The number of men receiving surgical and radiation treatments for prostate cancer increased between 1985 and 1989. During that period, age consistently played a significant role in all therapeutic decisions. Other factors, such as comorbidity, race, socioeconomic status, and distance, also were important considerations, depending on the treatment alternative. PMID- 8632690 TI - Assessing the health of the nation. The predictive validity of a preference-based measure and self-rated health. AB - Health-related quality-of-life (HRQOL) measures are becoming increasingly important for evaluating the effectiveness of medical interventions and assessing the health of populations. Preference-based instruments, a subset of HRQOL measures, allow comparisons of overall health status in populations and in clinical settings, and are suitable for economic analyses; but validity studies have used selected samples, mostly examining morbidity. This study compared the performance of a preference-based instrument with self-rated health in predicting subsequent self-rated health, hospitalization, and mortality in a national cohort. A version of the Health Utility Index (HUI), constructed from questions in the 1982 to 1984 National Health and Examination Survey I Epidemiologic Follow up Study (NHEFS), was used to develop scores for the 1982 to 1984 survey sample. The relationship between both the NHEFS-HUI and self-rated health in 1982 to 1984, and subsequent decline in self-rated health, hospitalizations, and mortality experienced by 1987 were examined using survival analyses. The analyses adjusted for sociodemographic variables (age, sex, race, education, and income), medical conditions, and smoking status reported at the 1982 to 1984 NHEFS interview. Results indicated that NHEFS-HUI and self-rated health scores were worse in older persons, persons with one or more medical conditions, African Americans, and those with less education and lower incomes. The effects of all 19 chronic conditions and smoking were reflected in lower self-rated health scores, whereas the NHEFS-HUI did not capture the effects of two of the conditions or smoking status. Both measures made independent contributions to predicting hospitalizations and mortality by 1987; in addition, the NHEFS-HUI predicted decline in subsequent self-rated health. The NHEFS-HUI also predicted health outcomes in the subgroup of those in initial excellent or very good self-rated health. A preference-based instrument demonstrated predictive validity in three relevant domains of health status outcomes across all sociodemographic groups examined in this cohort. Self-rated health was better able to capture concurrent decrements in health associated with certain chronic illnesses and smoking. It is concluded that preference-based measures capturing both functional status and health perceptions should be incorporated explicitly into national surveys to assess the health of populations. PMID- 8632691 TI - The severity classification system for acquired immunodeficiency syndrome hospitalizations. Association with survival after discharge and inpatient resource use. AB - The Severity Classification for AIDS Hospitalizations (SCAH) was applied to a longitudinal person-based data set of Maryland adult residents diagnosed with acquired immunodeficiency syndrome (AIDS) between 1983 and 1989 to predict long term survival. In contrast to other AIDS severity measures, SCAH can be applied to administrative data bases for analyses of large populations. Although SCAH was created to predict inpatient mortality using cross-sectional hospital discharge data, the models used in this study show SCAH stage at first AIDS hospitalization to predict long-term survival in persons with AIDS, even after adjusting for sociodemographic and treatment variables. Additional models in the study show SCAH stage at first hospitalization has a strong association with inpatient length of stay and associated charges, making it useful for health care resource planning. PMID- 8632692 TI - Health-related quality of life of diabetic Pima Indians. AB - This study was designed to measure the health-related quality of life of a sample of diabetic Pima Indians. Health status questionnaires were administered to 54 diabetic Pima Indians attending an outpatient pharmacy in southern Arizona. Health-related quality of life was assessed using the SF-36 Health Survey. Internal consistencies of the eight multi-item scales of the SF-36 were estimated. Nonparametric analyses were performed to determine relationships between mean SF-36 scale scores and various clinical and demographic variables. Favorable internal consistencies for the multi-item scales were observed, with alpha coefficients ranging from 0.63 to 0.91. SF-36 scale scores were not influenced by sex or education level. Age was significantly associated with four of the eight dimensions. Indicators of glycemic control were not significantly associated with any SF-36 multi-item scale scores. Subjects with more comorbid chronic conditions had significantly lower SF-36 scores. If the goal of maximizing health status of American Indians is to be reached, health-related quality-of-life assessment should be used as a means to monitor progress toward that goal. Further evaluation of the SF-36 or other health status/quality-of-life instruments in this population should be undertaken. PMID- 8632693 TI - Protective action of isoquinolinesulfonamides in in vitro models of neuronal apoptosis. AB - We have previously reported that a group of isoquinolinesulfonamide kinase inhibitors (H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; H8, N-[2 (methylamino)ethyl]-5-isoquinolinesulfonamide; and H9, N-2-(aminoethyl)-5 isoquinolinesulfonamide) prevents apoptosis triggered in neurons by an inhibition of phosphatases 1 and 2A with okadaic acid, and by inhibition of protein kinase C with staurosporine and chelerythrine. In the present study we assessed the capability of isoquinolinesulfonamides (IQS) to prevent different types of apoptosis in primary cultures of cerebellar granule neurons. We induced apoptosis by: a) serum removal, b) potassium deficiency, c) serum removal + potassium deficiency, and d) beta-amyloid peptide (beta AP). The IQS prevented apoptosis in all the above models. Thus, it appears that the IQS-sensitive pathway is a common mechanism in different types of neuronal apoptosis, and that it might be used as a target in the development of novel neuroprotective drugs. PMID- 8632695 TI - Up-regulation of rat adrenomedullin gene expression by endotoxin: relation to nitric oxide synthesis. AB - The effect of bacterial endotoxin (LPS) on adrenomedullin (AM) gene expression was investigated in cultured rat aortic vascular smooth muscle (VSM) cells and in tissues from anesthetized rats. The addition of LPS together with interferon gamma to VSM cells resulted in a marked increase in the abundance of AM mRNA as well as the appearance of mRNA for the inducible isoform of nitric oxide (NO) synthase (iNOS). Intravenous injection of LPS into rats also increased AM mRNA abundance and induced iNOS mRNA in lung, heart, liver, and kidney. AM significantly enhanced NO synthesis evoked by LPS and interferon-gamma in cultured VSM cells. These data suggest that AM may contribute to circulatory failure during endotoxin shock, in part, by modulating NO synthesis. PMID- 8632694 TI - Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells. AB - O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6 fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5 pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2 fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2 fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6 fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro substitution at the 4- or 3-position of the benzyl group were comparable to O6 benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT. PMID- 8632697 TI - Age-related changes before and after imposition of hemodynamic stress in the mammalian heart. AB - This review focusses on the following issues: how the mammalian heart grows and ages; age-related changes in the mammalian heart before and after imposition of hemodynamic stress; and antiaging modulation in the mammalian heart. The heart and other organs grow and age together in the whole body, and interactions occur between these organs. Therefore, the age-related changes at the molecular and cellular level in the in vivo heart are the summation of the changes of the heart per se and the effects of other organs or tissues on the heart. Furthermore, myocytes grow and age under the influence of age-related changes in other myocytes and other types of cells in the myocardial tissue through autocrine or paracrine mechanisms, because myocytes are exposed to many biologically active substances which are released from those cells. Since hypertension and ischemia are very common hemodynamic events in aged hearts, the characteristics in aged hearts are discussed in terms of responses to hypertension or ischemia. The induction of proto-oncogenes and heat shock protein genes in response to milder hemodynamic stress such as pressure-overload and ischemia is diminished in aged hearts. However, the aged heart can respond to more severe stress to a level similar to that of young-adult hearts. Therefore, the senescent heart is characterized by its attenuated adaptation to hemodynamic stress and by its ability to adapt to limited environmental changes. Several interventions have antiaging effects on the heart at the molecular and cellular level. PMID- 8632696 TI - Tyrosine kinase inhibition suppresses angiotensin contraction in hypertensive and normotensive small resistance arteries. AB - Protein tyrosine kinases (PTKs) may influence vascular resistance, which is controlled primarily at the level of small arteries and arterioles. This study evaluates these kinases in resistance arteries from spontaneously hypertensive (SHR) and normotensive (WKY) rats using the protein tyrosine kinase inhibitor, tyrphostin-25. Gracilis muscle arteries (90-160 um, internal diameter) were mounted on a resistance vessel myograph and contractile responses to phenylephrine (PE) and angiotensin II (AII) were measured in the presence and absence of tyrphostin-25 (0.02-20 uM). Tyrphostin-25 inhibited AII, but not PE contractions and was less potent in the hypertensive arteries. This demonstrates for the first time that PTKs contribute to the contractile activity of resistance arteries from both hypertensive and normotensive rats. Further, results confirm in small arteries that AII-induced contractions are PTK-dependent and that arteries from hypertensive rats are hyporesponsive to PTK inhibition. PMID- 8632698 TI - Sequential estrogen and progesterone (P) followed by P withdrawal increases the level of oxytocin messenger ribonucleic acid in the hypothalamic paraventricular nucleus of the male rat. AB - We previously reported that alterations in gonadal steroid hormones can influence oxytocin (OT) messenger ribonucleic acid (mRNA) expression in the hypothalamic paraventricular nucleus (PVN) of the steroid-treated ovariectomized rat, the pregnant rat, and the lactating rat. Specifically, OT mRNA in the PVN of the female rat was increased by sequential estrogen and progesterone (P) followed by withdrawal of P. In this study we investigated whether this same steroid paradigm increases OT mRNA levels in the castrate rat. Castrate rats were administered either sequential estrogen and P followed by P withdrawal or no steroid treatment prior to sacrifice. PVN OT mRNA was measured by Northern blot hybridization and pituitary OT peptide content by radioimmunoassay (RIA). The steroid-treated rats had increased OT mRNA levels compared to the sham treated rats (p < 0.04), but pituitary OT peptide content was not significantly altered. We conclude that sequential estrogen and P followed by P withdrawal increases PVN OT mRNA in the castrate, as well as the ovariectomized, rat. PMID- 8632699 TI - Cellular mechanism of angiotensin II-induced atrial natriuretic peptide release in rat right atrial tissue. AB - This study presents an investigation of the mechanism of angiotensin II (Ang II) induced atrial natriuretic peptide (ANP) release in superfused sliced right atria of rats. Ang II (0.1 microM) enhanced ANP release by 49%. This phenomenon was significantly blocked by (Sara1-Ileu 8) Ang II (1 microM) and losartan (0.1 microM). The use of neomycin (100 microM), a phospholipase-C inhibitor completely suppressed the effect of Ang II on ANP increase. To elucidate the intracellular mechanism of ANP released by Ang II, the role of protein kinase C (PKC) was determined by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and phorbol ester : 4-beta-phorbol 12-myristate-13-acetate (PMA). We observed that PMA (0.1 microM) stimulated ANP release whereas H-7 (10 microM), an inhibitor of PKC in the presence of Ang II, prevented ANP increase. The role of calcium was also evaluated with 8-(N-N-diethylamino)-ocytyl-3,4,5, trimethoxy-benzoate (TMB-8) (10 microM) and N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7) (10 microM), which completely inhibited ANP release by Ang II. Pre-treatment with diltiazem (10 microM), an antagonist of the Ca++ channel, did not prevent ANP increase due to Ang II, but A23187 (5 microM) enhanced ANP release by Ang II. These results suggest that PKC and intracellular calcium play an important role in ANP release under the influence of Ang II in rat atrial tissue. PMID- 8632701 TI - Taurine accelerates the regression of hypercholesterolemia in stroke-prone spontaneously hypertensive rats. AB - The effects of taurine on the regression of pre-established hypercholesterolemia were examined in stroke-prone spontaneously hypertensive rats (SHRSP). Hypercholesterolemia was induced by feeding a hypercholesterolemic diet to SHRSP for 30 days. Then, the diet was switched to normal chow with or without 3% taurine, and the effects were followed up for another 30 days. During regression serum cholesterol level was rapidly decreased, and was accelerated by taurine. A similar accelerated decrease in cholesterol content by taurine was seen also in tissues including the liver, intestine, and aorta. In the liver, acyl CoA:cholesterol acyltransferase (ACAT) activity was significantly low in the taurine-supplemented group, parallel with the hepatic cholesteryl ester content. On the other hand, hepatic cholesterol 7 alpha-hydoxylase activity maintained a higher level in the taurine-supplemented group. These results showed that taurine accelerates the regression of hypercholesterolemia, and suggested that this effect is related to the increase in cholesterol catabolism to bile acid through the enhancement of 7 alpha-hydoxylase activity. PMID- 8632700 TI - Lipoprotein metabolism in human peritoneal cells. AB - The feasibility of using human cells isolated from peritoneal dialysis effluent as a model for studying lipoprotein and cholesterol metabolism was investigated. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (acetyl-LDL) by saturable, high affinity receptor-mediated processes. Positive correlations of the percentage of macrophage cells with degradation rates of LDL (r = 0.742; p < 0.05) and acetyl-LDL (r = 0.931; p < 0.01) indicated that macrophage cells significantly contributed to lipoprotein degradation. LDL receptor-mediated degradation was calcium dependent, and sensitive to pronase and chloroquine treatments. The receptor exhibited specificity for lipoproteins containing apolipoprotein B (apoB) or apolipoprotein E (apoE). Exposure of cells to LDL for 24 hrs significantly down-regulated LDL receptor-mediated degradation. Acetyl-LDL receptor-mediated degradation was calcium independent, inhibited by chloroquine, and was sensitive to pronase and fucoidin treatments. The scavenger receptor exhibited specificity for only acetyl-LDL. These results demonstrate that human peritoneal cells can provide a source of human tissue macrophages suitable for studies of cholesterol and lipoprotein metabolism and offer the opportunity for comparison of metabolic characteristics of in vivo maturated macrophages with available macrophage-like cell lines. PMID- 8632702 TI - High-fat diet induces aggressive behavior in male mice and rats. AB - The present study investigated whether dietary fat increases aggressive behavior in male mice and rats. High fat consumption may elevate circulating estrogen levels and estrogens, in turn, are associated with various non-reproductive behaviors, such as male aggression. The animals were assigned to two groups including those consuming a diet high in polyunsaturated fats (43% calories from fat) and those consuming a low-fat diet (16% calories from fat). Each male animal was housed with two females for three weeks. The male mice and rats were then confronted with an intruder kept on a medium-fat feed. The latency to first aggressive encounter was significantly shorter among the male animals kept on a high-fat diet than those males kept on a low-fat diet. Furthermore, the time spent exhibiting aggression was longer in the high-fat groups. Serum levels of estradiol (E2) were elevated by 2-fold in the male animals consuming a high-fat diet, when compared with the male animals kept on a low-fat diet. These findings suggest that dietary fat can increase aggressive behavior in male mice and rats, possibly by elevating circulating E2 levels. PMID- 8632703 TI - In vivo binding of [11C]SKF 75670 and [11C]SKF 82957 in rat brain: two dopamine D 1 receptor agonist ligands. AB - The high affinity benzazepine D1 agonists SKF 75670 and SKF 82957 labeled with C 11 were evaluated in vivo in rats as potential radioligands for imaging dopamine D1 receptors with positron emission tomography (PET). Their in vivo pharmacological profile revealed selective binding for both tracers in rat brain regions rich in D1 receptors such as the caudate-putamen. The more lipophilic [11C]SKF 82957 (6-chloro-[11C]SKF 75670) showed a higher brain uptake (more than 2-fold up to 30 min), higher specific uptake in the striatum and higher signal-to noise ratio (striatum-to-cerebellum = 3.2 +/- 0.4 for [11C]SKF 75670 and 9.7 +/- 2.5 for [11C]SKF 82957 at 60 min post-injection) as compared to [11C]SKF 75670. Both radiotracers exhibited high specificity and selectivity for D1 receptors, since only D1 competitors but not the D2 antagonist sulpiride or the 5-HT2 antagonist ritanserin reduced significantly their binding the striatum with [11C]SKF 75670 or the striatum and olfactory tubercles with [11C]SKF 82957. Previous reports have shown that only D1 agonists can recognize the functional high-affinity state from the low-affinity state of D1 receptors. [11C]SKF 75670 and especially [11C]SKF 82957 are D1 agonist radioligands that can potentially be used to study in vivo the functional high-affinity state of D1 receptors using PET. PMID- 8632704 TI - Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity. AB - Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles. PMID- 8632705 TI - Tissue distribution of ibogaine after intraperitoneal and subcutaneous administration. AB - The distribution of the putative anti-addictive substance ibogaine was measured in plasma, brain, kidney, liver and fat after ip and sc administration in rats. One hr after ip dosing (40 mg/kg), drug levels ranged from 106 ng/ml (plasma) to 11,308 ng/g (fat), with significantly higher values after sc administration of the same dose. Drug levels were 10-20 fold lower 12 hr after the same dose. These results suggest that: 1) ibogaine is subject to a substantial "first pass" effect after ip dosing, demonstrated by higher drug levels following the sc route, 2) ibogaine shows a large accumulation in adipose tissue, consistent with its lipophilic nature, and 3) persistence of the drug in fat may contribute to a long duration of action. PMID- 8632706 TI - Pharmacokinetic and pharmacodynamic optimization of intraperitoneal chemotherapy. AB - Intraperitoneal drug administration has been utilized to increase chemotherapeutic exposure to tumors confined to the peritoneal cavity, such as those found in ovarian, gastric, colo-rectal and mesotheliomal cancers. Extensive pre-clinical and clinical experimentation has been conducted to assess the pharmacokinetic and therapeutic benefits of this mode of therapy. Pharmacokinetic studies have shown that the barrier function of the peritoneal membrane may be utilized to produce large, favorable concentration gradients between peritoneal perfusate and blood. However, most clinical studies so far have demonstrated minimal increases in drug efficacy or decreases in drug toxicities from intraperitoneal drug therapy alone. This paper reviews the application of adjunctive therapies that have been rationally conceived to optimize intraperitoneal drug therapy through the alteration of antineoplastic pharmacokinetics and pharmacodynamics. PMID- 8632707 TI - Is enzyme release a sign of irreversible injury of cardiomyocytes? AB - The amount of creatine kinase (CK) release (percent of releasable CK) and the amount of irreversibly injured cardiomyocytes evaluated by counting trypan blue stained nuclei (percent of total) was investigated in isolated perfused rat hearts under various conditions: Intermittent contractive depression by low calcium (0.5 mM) and by administration of BDM (10 mM) as well as by anoxia/reoxygenation. For comparison severe injury induced by calcium paradox was also studied. CK release amounted to 0.5% to 3% (controls 15 to 105 min) and to 3 to 5% for the interventions and about 40% for calcium paradox. Irreversibly injured myocytes amounted to 0.1 to 0.3% and 0.3 to 0.5% respectively and to about 40% in calcium paradox. Thus, the percentage of enzyme release exceeded the percentage of irreversibly injured cells by more than one order of magnitude under all experimental conditions, including controls, except for calcium paradox where the percentages were the same. We conclude that cytosolic enzymes can be released to substantial amounts without irreversible injury of cardiomyocytes under various conditions, and only with severe membrane lesions (Ca paradox) enzyme release reflects irreversibly injury. PMID- 8632708 TI - HLA-DR antigen and interleukin-2 receptor expression on primary-cultured human hepatic macrophages in relation to liver cirrhosis and hepatitis virus infection. AB - We analyzed primary-cultured human hepatic macrophages (HHMphi) from 12 patients with non-cirrhotic and cirrhotic livers for cell surface expression of HLA-DR antigen and interleukin-2 receptor (IL-2R). Compared to the relatively abundant HLA-DR antigen, IL-2R expression was generally low. No significant difference was observed between HLA-DR antigen expression nor IL-2 receptor expression. HHMphi from patients with serum hepatitis viral markers, however, expressed significantly more HLA-DR antigen than did HHMphi of patients without viral markers, which suggest a possible role of HHMphi as antigen-presenting cells (APC) in viral hepatitis. This direct, quantitative measurement of cell surface molecule expression on hepatic macrophages of human may provide an important clue to the pathophysiology of human liver disorders. PMID- 8632710 TI - Chronic anabolic-androgenic steroid treatment affects brain GABA(A) receptor gated chloride ion transport. AB - Previous research in this laboratory has shown that chronic treatment of adult male rats with an anabolic-androgenic steroid (AAS) produced anxiolytic behavior and increased the functional response of cortical gamma-aminobutyric acid(A) (GABA(A)) receptors. The experiments reported here were aimed at further characterizing the effect of chronic AAS exposure on cerebral cortical GABA(A) receptors. Adult male rats were injected with dianabol (1,4-androstadien-17alpha methyl-17beta-ol-3-one; 10 mg/kg/day, SC) for 4 weeks. A significant decrease in ventral prostate gland weight was found after 2 weeks of dianabol, and returned to control levels 3 and 10 days after steroid discontinuation. Testicular weights decreased throughout the treatment period but reached statistical significance only during the withdrawal period. Serum 3alpha-androstanediol level was marginally increased afer 2 weeks of dianabol injection, and was significantly decreased at 3 and 10 days after withdrawal. GABA-stimulated 36chloride (Cl-) influx in cortical synaptoneurosomes was increased in animals treated with dianabol for 2 and 4 weeks, and remained elevated 3 days after dianabol withdrawal, returning to control levels at withdrawal day 10. The increase in receptor efficacy was associated with a transient increase in receptor sensitivity (inverse of EC50), apparent after 2 weeks of AAS treatment and at withdrawal day 3. In a follow-up experiment, metabolites of dianabol were tested for the in vitro efficacy in potentiating GABA-stimulated Cl- transport. Only 3alpha-androstanedial and androsterone were found to have potent stimulatory effects. The 3beta-reduced metabolites were inactive, as were metabolites that contained a methyl group at the 17alpha position. These results point to significant facilitative effects of dianabol treatment on brain GABA(A) receptors via the metabolic formation of neuroactive steroids. PMID- 8632711 TI - Comparative dopaminergic and muscarinic antagonist activity of clozapine and haloperidol. AB - Clozapine is an atypical antipsychotic drug, and its dopamine and muscarinic antagonist activity has been compared with haloperidol in rodents. Elevation in rat striatal acetylcholine (ACh) and mice cerebellar cGMP has been used as an agonist response for oxotremorine and quinpirole. Pretreatment with clozapine significantly blocked oxotremorine-induced elevation in striatal ACh (p<0.01) and cerebellar cGMP(p<0.05). At the same doses, clozapine had no significant effect on quinpirole-induced increases in ACh and cGMP levels. Pretreatment with haloperidol significantly antagonized quinpirole-induced elevation in striatal ACh (p<0.01) and cerebellar cGMP(p<0.05), and haloperidol had no significant effect on oxotremorine-induced agonist responses. Thus, clozapine is antimuscarinic at a dose level that lacks dopamine antagonist properties, whereas haloperidol is a dopamine antagonist and lacks antimuscarinic activity. The atypical neuroleptic profile of clozapine may be due to its high antimuscarinic and low antidopaminergic activity. PMID- 8632709 TI - Characterization of nitric oxide dependent changes in carbohydrate hepatic metabolism during septic shock. AB - The role of nitric oxide in the alterations of liver carbohydrate metabolism during septic shock has been studied in fed and starved animals injected with bacterial lipopolysaccharide (LPS). One h after LPS injection an hyperglycemic peak was observed followed by hypoglycemia when the plasma nitric oxide concentration increased. However, in animals pharmacologically treated with nitric oxide donors only hypoglycemia was observed. In isolated hepatocytes from LPS treated rats an impairment of the gluconeogenic flux was observed accompanied by a decrease in the mRNA levels of the glucose transporter GLUT-2 and phosphoenolpyruvate carboxykinase, at the time that increased the mRNA levels of the inducible form of nitric oxide synthase. These results suggest that part of the effects observed in response to LPS challenge are due to early signaling molecules (cytokines and other factors molecules) whereas other effects can be attributed to nitric oxide synthesis which in turn has specific effects on hepatic metabolism. PMID- 8632712 TI - Purification and characterization of angiotensin II-generating chymase from hamster cheek pouch. AB - Hamster cheek pouch vascular tissues contain an angiotensin II-forming enzyme which is inhibited by chymostatin but not by any angiotensin-converting enzyme inhibitors. The enzyme was purified to apparent homogeneity by gel filtration and heparin-Sepharose affinity chromatography. The molecular mass estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis was 28 kDa and the optimum pH was between 7.5 and 9.0. The angiotensin II-forming activity was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by aprotinin. The N-terminal sequence showed high homology with chymases from various species. Thus, the angiotensin II-generating enzyme obtained from hamster cheek pouch vessels is a chymase. PMID- 8632713 TI - Protein kinase C-alpha regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells. AB - In a previous study (Am. J. Pathol. 1994, 145: 1265-1270) we found rat coronary vascular smooth muscle cell (SMC) proliferation and apoptosis to be regulated by protein kinase C (PKC). In the present study we analysed whether selective depletion of alpha isozyme of PKC would affect SMC proliferation and/or apoptosis. First, using Western blot technique, it was determined that the rat SMC express alpha, delta, epsilon and zeta isozymes of PKC. The selective depletion of PKC-alpha in SMC was achieved by exposing cells to antisense oligodeoxynucleotide to mRNA for PKC-alpha (AS-PKC-alpha). The effect of AS-PKC alpha on SMC proliferation was analysed by measurement of 3H-thymidine incorporation. The results indicated that a single dose of AS-PKC-alpha at a concentration of 10-100microM caused long-lasting (for at least 4 days) inhibition (up to 55%) of 3H-thymidine incorporation by SMC. This observation indirectly demonstrates that PKC-alpha regulates SMC proliferation. However, it was not possible to induce a significant level of apoptosis in SMC exposed even to the highest dose of AS-PKC-alpha. These data, in conjunction with the previously shown induction of apoptosis in SMC by calphostin C, suggests that another isozyme of PKC is likely to be involved in regulation of SMC apoptosis. Finally, we observed that induction of apoptosis via PKC-dependent mechanism is prevented by supplementing the culture medium with serum. This shows striking similarity with the regulation of apoptosis by the c-myc-dependent pathway. In conclusion, PKC-alpha joins the group of proteins such as c-myc, proliferating cell nuclear antigen and cdc2 kinase which may be therapeutical targets, for antisense oligodeoxynucleotides, in order to prevent SMC hyperplasia. PMID- 8632714 TI - Mechanisms involved in the relaxant effect of estrogens on rat aorta strips. AB - The effects of estrogens 17beta-estradiol (17beta-E2), 17alpha-estradiol (17alpha E2) and diethylstilbestrol (DES) on CaCl2 (3mM)-induced contractions on rat aorta strips have been assayed. Both 17alpha-E2 and DES, but not the 17beta-E2 relaxed and inhibited the contraction induced by CaCl2. The antiestrogen tamoxifen (0.1, 1 and 3 microM) antagonizes, in a concentration-dependent way, the relaxant effect of 17alpha-E2 but the relaxation induced by DES is only significantly antagonized with 3 microM of tamoxifen. Cycloheximide (0.1 and 0.3 mM) does not modify the 17alpha-E2 or DES effects. However, the inhibitors of cAMP-dependent protein kinase TPCK (1 microM) and Rp-cAMPS (10 microM) inhibit the relaxation induced by 17alpha-E2 and DES. The elimination of endothelium by rubbing, significantly inhibits the effect of DES but does not modify the effect of 17alpha-E2. Our results suggest that estrogen-induced relaxation is a non-genomic effect possibly or presumably produced by activation of estrogenic receptors and mediated by cAMP. The DES-effect is partially endothelium-dependent but the effect of 17alpha-E2 is independent of endothelium. PMID- 8632715 TI - Effects of endothelin on mitogen-activated protein kinase activity and protein synthesis in isolated adult feline cardiac myocytes. AB - The growth-promoting effects of endothelin-1 (ET-1) were examined in adult heart cells. The activity of mitogen-activated protein kinases (MAPKs) was measured in cytosolic extracts of isolated adult feline cardiac myocytes incubated with and without ET-1. Kinase activity was assessed by phosphorylation of the exogenous substrate, myelin basic protein. ET-1 stimulated the activity of MAPK up to 4 fold, with peak activation occurring between five and ten minutes after addition of ET-1. Polyclonal antisera raised against a 14-amino acid sequence of the erk-2 gene product, a MAPK isoform, identified two major bands in cytosolic extracts of the cardiac myocytes. Partial purification of kinase activities using Mono Q anion-exchange chromatography demonstrated two major peaks of myelin basic protein kinase activity. Subsequent immunoblots of the eluted fractions demonstrated that the immunoreactive bands observed in the cytosolic extracts eluted in those fractions possessing kinase activity. Overnight pretreatment of the cardiac myocytes with 100 ng/ml pertussis toxin inhibited the ET-1 stimulated increase in MAPK activity by 50 - 70%, but did not alter stimulation by 100 nM phorbol 12-myristate 13-acetate (PMA). These data suggest that stimulation of MAPK by ET-1 may be mediated by more than one pathway. MAPK has been shown to be activated in the intracellular transmission of growth factor signals. Indicative of a growth effect in this adult heart cell model, myocytes exposed to increasing concentrations of ET-1 demonstrated a dose dependent increase in [3H] phenylalanine incorporation into cellular protein. This response was blocked by staurosporine and partially inhibited by pretreatment with pertussis toxin, again suggesting the possible involvement of multiple early signals. These data from isolated adult cardiac myocytes further support the hypothesis that ET-1 has a role in the regulation of cardiac growth. PMID- 8632716 TI - Effect of thyroidectomy on photoreceptor cells in adult rat retina. AB - To evaluate the effect of thyroid hormone on the retina in the adult male rat, morphometric and autoradiographic studies were made. Four days after an intravenous pulse injection of L-[3H]-leucine into adult male rats, the eyeballs were removed and the autoradiogram made. Two weeks after thyroidectomy, the length of photoreceptor cell inner segments was significantly decreased, but that of the outer segments did not change. Daily supplement of T4 (100 microg/kg sc twice for 2 days) before L-[3H]-leucine injection did not restore the decrease in the length. The silver grains of radioactive material distributed in the outer segment were divided into 3 equal portions. Thyroidectomy significantly increased the number of grains in the basal portion near the inner layer indicating a reduction in the renewal rate of the outer segment, but the T4 supplement did not restore the reduction in the renewal rate. These findings suggest that thyroid hormone is required to maintain the rate of renewal of the photoreceptor cell outer segment but the hormone supplement did not rapidly restore the renewal rate. PMID- 8632717 TI - Evaluation of the mechanism of endothelial dysfunction in the genetically diabetic BB rat. AB - Endothelial dysfunction is known to occur in chemically-induced animal models of diabetes. The BB diabetic rat is a genetic diabetes-prone model which more closely resembles Type I diabetes mellitus. In this study, we examined the role of superoxide anion radical and cyclooxygenase activity on endothelial dysfunction in aorta of the spontaneous diabetic BB rat. Vascular endothelial function was studied in vitro in aortic rings from 8-wk diabetic rats and age matched nondiabetic littermates. There was no alteration in reactivity to norepinephrine as a result of diabetes. Relaxation to acetylcholine (but not nitroglycerin) was impaired in diabetic rings. Relaxation to acetylcholine was abolished by 100 microM L-nitroarginine but unaltered by an equimolar concentration of aminoguanidine (an inducible nitric oxide synthase inhibitor) in both control and diabetic rings. Incubation with 10 microM indomethacin did not alter relaxation to acetylcholine in either control or diabetic rings. In contrast, addition of 20 U/ml superoxide dismutase enhanced relaxation to acetylcholine in diabetic rings but had no effect on relaxation to acetylcholine in control rings. Thus, nitric oxide-mediated, endothelium-dependent relaxation is diminished in aortic rings of the genetic diabetic BB rat. Furthermore, superoxide anion radicals but not cyclooxygenase products play an important role in endothelial dysfunction in this genetic diabetic model. PMID- 8632718 TI - K1 and K3 capsular antigens of Klebsiella induce tumor necrosis factor activities. AB - Capsular polysaccharide antigens isolated from Klebsiella pneumoniae sero-type 1 (K1) and sero-type 3 (K3) could induce tumor necrosis factor-alpha in ICR mice. K1 and K3 capsular antigens were found to be non-toxic by brine shrimp bioassay. When injected into Ehrlich ascites tumor-bearing mice, both K1 and K3 capsular antigens exhibited significant suppression in the growth of tumor cells. The significance of these observations is discussed. PMID- 8632719 TI - Modulation of NMDA and dopaminergic neurotransmissions by sigma ligands: possible implications for the treatment of psychiatric disorders. AB - Sigma (sigma) receptors, improperly classified as belonging to the opiate receptor family when discovered in 1976, were subsequently confused with phencyclidine binding sites for several years. It's only recently, with the emergence of new selective ligands that their functional significance could be meaningfully addressed. Several subtypes of sigma receptors are present in high densities in the limbic structures as well as in motor-related areas of the CNS. Different lines of evidence suggest that a major role for sigma receptors might be to regulate the activity of the glutamatergic system via the modulation one of its subtype of receptor, the NMDA receptor. This modulation of the glutamatergic system could in turn interfere with the dopaminergic neurotransmission with which, however, sigma ligands could also interact directly. The potential involvement of sigma receptors in schizophrenia has been considered ever since their discovery. The initial suggestion to this respect emerged from the observation that several of the earliest sigma ligands induced psychotomimetic symptoms such as delusions, hallucinations and depersonalization. This link was later reinforced with the demonstration that several neuroleptics, such as haloperidol, have a high affinity for sigma receptors, whereas, some new molecules with a high affinity for sigma receptors, but a low affinity for dopaminergic receptors demonstrated a "neuroleptic-like" pharmacological profile. However, the therapeutic efficacy of selective sigma ligands in schizophrenia has not yet been established and it has even been suggested that sigma receptors might be responsible for some side effects of the classical neuroleptics. The possible implication of sigma receptors in affective disorders has also been suggested by reports showing that some antidepressant drugs have a high affinity for sigma receptors and that long-term treatments with anti- depressant drugs, even with those devoid of affinity for sigma receptors, modify their binding characteristics. In conclusion, indirect evidence suggests possible etiological and/or therapeutic roles for sigma receptors in some psychiatric disorders. However, despite several attempts, no clear indications of a therapeutic efficacy of sigma ligands has yet emerged. More selective ligands and fundamental studies on the respective role of the different subtypes of sigma receptors are needed before clear concepts can be formulated. p3 PMID- 8632720 TI - Mature human atherosclerotic plaque contains peroxidized phosphatidylcholine as a major lipid peroxide. AB - The initial stage of atherosclerotic plaque formation involves oxidation of the phosphatidyl-choline moiety of low density lipoprotein (LDL) and subsequent uptake by macrophages. Ongoing uptake in developing plaque also may involve oxidized LDL and would require an oxidizing environment in plaque lipids. Atherosclerotic plaque lipids from 12 patients undergoing peripheral vascular procedures were extracted in chloroform: methanol (2:1). This extract was applied to a 25 cm 5 micron silica HPLC column and eluted with a ternary gradient mobile phase utilizing a laser light scattering (ELSD) mass detector. Individual lipid fractions were then analyzed. Cholesterol, both free and esterified, was the most prominent lipid in plaque (104 +/- 74 mg/gm tissue. However, lipid peroxides were present in much higher concentrations (3.52 +/- 2.84 FU X 10(4)/mg phospholipid) and overall level (21.27 +/- 10.10 FU X 10(4)/gm plaque) in the phospholipid component (*p< 0.05). Phosphatidyl-choline (PC) accounted for 63% of the total phospholipid peroxides recovered (6.31 +/- 5.09 mg/gm plaque; *p<0.05). PC and phosphatidylinositol (PI) content were linearly related to lipid peroxide fluorescence (PC; r=0.696; p=0.01) (PI; r=0.809; p=0.001). Lipid peroxides in human atherosclerotic plaque are present primarily in the phospholipid component and phosphatidyl-choline forms the bulk of these peroxides. PC may play an important role in ongoing plaque lipid accumulation. PMID- 8632721 TI - Expression of functional glucagon receptors on lymphoid cells. AB - The objective of the present studies was to determine whether the existence of functional glucagon receptors could be established on lympoid cells. The glucagon receptor, which positively regulates adenylate cyclase, is a member of the superfamily of seven transmembrane domain G-protein coupled receptors. Previously reported specific binding with [125I]-glucagon to a variety of lymphoid and myeloid cell preparations suggests that glucagon receptors are expressed within the immune system. In the present study, Northern analysis of polyA RNA isolated from primary mouse and rat derived lymphoid tissues and lymphoid cell lines EL 4.IL-2, Jurkat E6-1, CH12LX, and BCL1-3B3 cells were probed with a 32P-labeled human hepatic glucagon receptor. Mouse spleen and thymus, rat spleen, and the B cell line, CH12LX, all possessed a single 1.5 kb fragment (BCL1-3B3, 1.4 kb) which hybridized to the glucagon receptor cDNA probe, as compared to mouse liver which exhibited a 2.8 kb fragment. EL-4.IL-2 and Jurkat E6-1 cells possessed a 3.7 kb fragment with an additional 2.75 kb band present in Jurkat E6-1 cells. Treatment of mouse splenocytes and T- and B-lymphoma cells with glucagon (0 - 100 nM) produced a dose-dependent enhancement in intracellular cAMP which was maximal at 5 min post treatment followed by a gradual decline. Direct addition of glucagon to spleen cell cultures over a broad concentration range produced no effect on either lymphoproliferation following stimulation with anti-CD3 mAb, or LPS nor on the antibody forming cell (AFC) response to sRBC. Conversely, glucagon effectively reversed the suppression of the sRBC AFC response produced by delta9 tetrahydocannabinol (delta9-THC), and partially reversed the suppression produced by 2',3'-dideoxyadenosine, both of which are potent inhibitors of adenylate cyclase. These studies confirm the expression of functional glucagon receptors on lymphoid cells. PMID- 8632722 TI - Characterization of nitrobenzylthioinosine binding sites in the mitochondrial fraction of rat testis. AB - The presence of [3H] NBMPR binding sites in the mitochondrial fraction of rat testis is described. The dissociation constant (KD) from saturation studies was 0.16 +/- 0.04 nM. The association and dissociation rate constants (k1 and k-1) were 3.95 +/- 0.57 x 10(8) M(-1) min(-1) and 0.025 +/- 0.002 min(-1), respectively. The number of binding sites was 2,100 +/- 163 fmols/mg protein. [3H] NBMPR binding was inhibited, in a nanomolar range, by NBMPR (KI= 0.23 +/- 0.02 nM), OH-NBMPR (KI= 2.30 +/- 0.55 nM) and HNBTG (KI= 2.58 +/- 0.33 nM). In the micromolar range, adenosine receptor ligands such as PIA (3.46 +/- 1.36 microM), 2-chloroadenosine (18.81 +/- 3.36 microM) and NECA (8.26 +/- 3.90 microM), and mitochondrial benzodiazepine receptor ligands such as Ro 5-4864 (5.15 +/- 1.82 micrmoM and PK 11195 inhibited the specific binding of [3H] NBMPR. These results suggest the existence of a nucleoside transport system in the mitochondrial fraction of rat testis. PMID- 8632723 TI - A new alternative splice variant of the mouse Fas antigen with a deletion in the N-terminal portion of the extracellular domain. AB - The Fas antigen (Fas/APO-1/CD95) has been shown to induce apoptosis when bound to a monoclonal anti-Fas antibody or Fas ligands. Recently, a new soluble human Fas isoform which lacks the transmembrane domain due to alternative splicing has been isolated; however, no mouse Fas isoforms have been reported so far. Analysis of Fas transcripts by RT-PCR detected no Fas transcripts corresponding to the human soluble Fas isoform in mouse thymus, spleen and liver. However, we detected a new isoform with a 117-bp deletion in the second exon in various mouse tissues and cell lines. This isoform, termed truncated Fas (T-Fas), can be generated by alternative splicing and lacks the N-terminal portion of the extracellular domain just after the signal sequence. Since the deletion involves the first cysteine rich motif believed to be necessary for binding to the Fas ligand, the T-Fas protein may lack the ability to induce apoptosis. The expression of T-Fas relative to that of the normal Fas varies considerably among mouse tissues and cell lines, suggesting preferential transcription of the T-Fas isoform in certain cell types. PMID- 8632725 TI - Identification of a region important for human monoamine oxidase B substrate and inhibitor selectivity. AB - Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161 375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10( 8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity. PMID- 8632724 TI - Adaptive changes in adenosine receptors following long-term treatment with the adenosine receptor agonist R-phenylisopropyl adenosine. AB - Changes in brain A1 and A2A receptors and in the corresponding mRNA were studied using quantitative receptor autoradiography and in situ hybridisation. [3H]-DPCPX was used as an antagonist ligand at A1 receptors and [3H]-CGS 21680 as an agonist ligand at A2A receptors. Treatment of rats with the relatively A1 receptor selective adenosine analogue R-PIA (0.3 mg/kg) for 7 days in the presence of the peripherally acting antagonist 8-p-sulfophenyltheophylline (8-PST; 10 mg/kg) caused a decrease in the binding of the A1 receptor ligand, but not in that of the A2A receptor ligand. The effect on A1 receptors was also seen in the presence of 100 microM GTP that decreases agonist binding to insignificant levels. There was no change in either A1 or A2A receptor mRNA. No significant changes were detected following administration of either R-PIA or 8-PST alone. These results thus demonstrate an effect on brain A1 receptors after systemic administration of R-PIA in the presence of a peripherally acting adenosine antagonist, demonstrating that, under these conditions, the agonist reaches receptors in significant amounts. PMID- 8632726 TI - Potent growth inhibitory activity of a novel Ornithogalum cholestane glycoside on human cells: induction of apoptosis in promyelocytic leukemia HL-60 cells. AB - Growth inhibitory activities of a novel 22-homo-23-norcholestane glycoside found in bulbs of Ornithogalum saundersiae were examined in vitro using human promyelocytic leukemia HL-60 cells, human T-lymphocytic leukemia MOLT-4 cells, and mitogen-stimulated human peripheral-blood mononuclear cells (PBMC). The growth of HL-60 cells and MOLT-4 cells was strongly suppressed in the presence of the glycoside; the IC50s of which were 21.0 and 18.0 nM, respectively. Suppressive effect of the glycoside on HL-60 cell growth appears to be mediated partially through induction of apoptosis which was demonstrated by the presence of DNA fragmentation of the leukemic cells. Flow cytometric analysis of glycoside treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G0/G1 cells and a concomitant increase of S and/or G2M cells. The growth inhibiting effect of the glycoside on HL-60 cells was promoted by calcium and was inhibited in the presence of zinc, which support involvement of endonuclease activation in the glycoside-induced apoptosis. The glycoside also inhibited mitogen-stimulated blastogenesis of PBMC, the IC50 of which was 6.2 nM. These results provided the first evidence ever for the potent growth inhibitory activity of Ornithogalum glycoside on human leukemia cell lines and PBMC. PMID- 8632730 TI - American Nurses Association calls proposed Medicare and Medicaid cuts hazardous to patients. PMID- 8632727 TI - Changes in rat brain muscarinic receptors after inhibitory avoidance learning. AB - It is widely accepted that cerebral acetylcholine is necessary for learning and memory, but little is known about the type of muscarinic receptors involved in these functions. To investigate this problem, [3H]-N-methyl-scopolamine which binds to different types of muscarinic receptors, [3H]-Pirenzepine an M1 receptor antagonist, and [3H]-Oxotremorine-M which binds mainly to M2 receptors, were used as ligands to look for possible changes in muscarinic receptor density in neostriatum (NEO), hippocampus (HIP), amygdala (AMY), and temporo-parietal neocortex (CTX), after testing for retention of inhibitory avoidance, trained with high or low footshock intensities. After low reinforcement there was an M1 postsynaptic receptor up-regulation in NEO, HIP, and CTX, and an M2 presynaptic receptor down-regulation in HIP, which suggests a concerted pre- and postsynaptic cholinergic activation in this area. An up-regulation of both M1 and M2 receptors was detected in CTX of low and high footshocked animals, which indicates the presence of a cortical postsynaptic M2 receptor. PMID- 8632728 TI - Modulation of the length of the cell cycle time of MCF-7 human breast cancer cells by melatonin. AB - It has been shown that melatonin has a direct inhibitory effect on the proliferation of MCF-7 human breast cancer cells in culture. In the present work, we studied whether the length of the cell cycle of MCF-7 cells in increased by melatonin. In MCF-7 cells partially synchronized and labelled with [3H]thymidine, melatonin (10(-9)M), added to the culture medium, shifted the period of the labeling index rhythm from 20.36 hours to 23.48 hours. The fact that melatonin significantly increased (p<0.005) the duration of the cell cycle of human breast cancer cells, support the notion that this hormone exerts part of its antitumor effect through a cell-cycle-specific mechanism by delaying the entry of MCF-7 cells into mitosis. PMID- 8632729 TI - Who defines "futile" treatment. PMID- 8632731 TI - Glimepiride for NIDDM. PMID- 8632732 TI - Testosterone patches for hypogonadism. PMID- 8632733 TI - Changing medical organization and the erosion of trust. AB - Trust in medicine contributes to effective communication, cooperation in treatment, and the ability to cope with uncertainties. Social trust in medicine reflects public attitudes and is shaped by media and current events. Interpersonal trust depends on the degree to which patients see their doctors as competent, responsible, and caring. The commercialization of medical care, conflicts of interest, media attention to medical uncertainty and error, and the growth of managed care all challenge trust. Trust is encouraged by patient choice, continuity of care, and encounter time that allows, opportunities for feedback, patient instruction, and patient participation in decisions. An informal inquiry of medical leaders indicates that most believe trust is eroding. Institutions are taking measures to help restore trust: eliciting patient feedback; providing more information for patients are the public; improving staff education and sensitivity training; paying attention to clinicians' interpersonal skills; sponsoring support groups; instituting patient empowerment projects; and focusing on ethics issues. PMID- 8632734 TI - Population-centered and patient-focused purchasing: the U.K. experience. AB - A critical feature of the reforms to the National Health Service (NHS) in the United Kingdom is the separation of responsibility for purchasing and providing health care. In the new structure, health authorities purchase care from a population perspective, and general practitioner fundholders undertake patient focused purchasing. The evidence suggests that each approach has distinctive advantages. There is, however, a risk of fragmentation if the decisions of the different types of purchaser are not coordinated. This risk is compounded by the emergence of hybrid models combining aspects of population-centered and patient focused purchasing. The separation of purchasing from providing has changed the balance of power within the NHS, although whether the benefits of the new system outweigh the costs is a matter of continuing debate. PMID- 8632735 TI - Explaining reduced cancer survival among the disadvantaged. AB - The fact that socially disadvantaged cancer patients face a greater risk of mortality than the advantaged is well recognized but poorly understood. Existing research and a newly completed 10-year survival study suggest that complex interrelations among biological factors, medical interventions, and specific dimensions of social differentiation determine survival differences. Patterns of interrelations among determinants of survival appear compatible with an "economic" model in some forms of cancer and a "cognitive-behavioral" model in others. Findings presented here suggest that improved access to health care will reduce mortality risk among the disadvantaged in at least some malignancies, but will not alone make their survival chances equal to those of the advantaged. PMID- 8632738 TI - The Ethics Committee. We want to know what Mississippi nurses think. PMID- 8632736 TI - The new genetics and women. AB - The U.S. Human Genome Project (HGP) is a federally funded effort to produce a detailed genetic and physical map of all human chromosomes. Because of their central role in reproduction and caregiving, women are likely to be affected differently, and more significantly, by the information the HGP generates. It is important to identify inequities that may emerge from gender differences and to consider ways in which they may be avoided, reduced, or overcome. Although this type of analysis is one of the goals of the Ethical, Legal, and Social Issues Program of the National Center for Human Genome Research, few studies have focused explicitly on the impact of the HGP on women. This article describes the potential impact of the "new genetics" on women. Identification of gender differences as they affect both research and clinical practice and the psychosocial, legal, and ethical implications of the HGP should evoke and inform public discussion and policies that may be generated by these issues. PMID- 8632737 TI - Adult immunization priorities in the United States. AB - Pneumonia and influenza (P&I) are the sixth leading cause of death in the United States. Despite universal coverage under Medicare, one-half to three-quarters of elderly adults fail to get vaccinated against P&I disease. Hepatitis B vaccine is also widely underutilized by adults. Although more than 100 times as many adults as children die from vaccine-preventable disease, the Centers for Disease Control and Prevention (CDC) currently allocates the vast majority of federal immunization funds to childhood programs. Top CDC officials say this is in accordance with the will of the Congress and the President. However, analysis of legislative documents shows that there is no legal bar or restriction to the use of federal funds to support adult immunization. CDC has the authority to use federal immunization funds to enhance adult immunization services, but the agency has yet to make adult immunization a priority. A commentary follows. PMID- 8632739 TI - [Molecular biology of Lewis antigens--histo-blood type antigens and sialyl Lewis antigens as tumor associated antigens]. AB - The biosynthetic pathways of the Lewis histo-blood type antigens, Lewis a (Le(a)) and Lewis b (Le(b)), in correlation with ABH antigen synthesis and the synthesis of sialyl Lewis antigens, sialyl Lewis a (sLe(a)) and sialy Lewis x (sLe(x)), known as tumor associated antigens are described based on the recent molecular biological studies. Individuals are divided by their erythrocyte Lewis antigen phenotypes into three types, Le (a+b-) which has Le(a) antigen but not Le(b) antigen, Le (a-b+) which has Leb but not Le(a), and Le (a-b-) having neither Le(a) nor Leb. It was verified that Le (a-b-) individuals are the homozygotes with the nonfunctional Lewis gene (Le gene) which is inactivated by the missense mutations. Two kinds of the inactivated Le gene alleles were found in the Japanese population, and named le1 and le2. Individuals having a Le (a+b-) or a Le (a-b-) -non-secretor phenotype are the mutants who lack the secretor enzyme (Se enzyme) activity. The Se gene encoding the Se enzyme has been recently cloned and analyzed for the mutation resulting in inactivation of the Se enzyme of the non-secretor individuals. Our Se gene mutant analyses on the Japanese population ensured that the Se gene is responsible for synthesis of the Le(b) antigen. Mutant analyses of the other genes, H gene and FucTVI gene, which are also involved in the synthesis of Lewis antigens are described. We recently demonstrated that the sLe(a) antigen is the product of the Lewis gene since all le/le patients, who are determined as the genuine Lewis negative individuals by Le genotyping, did not express any kinds of type 1 chain Lewis antigens (Le(a), Le(b), and sLe(a)) in their digestive organs. It is, therefore, unuseful to measure the CA19-9 titer of the genuine Lewis negative cancer patients. PMID- 8632740 TI - [Cancer-associated alterations of glycoconjugates--special reference to glycosphingolipids]. AB - Glycoconjugates show the dramatic alterations of the carbohydrate structures in association with oncogenic transformation of the cells. Since glycoconjugates on the cell surfaces are involved in the cellular recognition and response, the structural changes of the carbohydrate in the cancer cells seemed to be closely related to the cell-cell adhesion, cell growth, cell behavior and metastatic property. One of the characteristic changes of glycosphingolipids in association with the oncogenic transformation is oncofetal change, and the other is the molecular mimicry of the glycosphingolipids to those in the cells of immune system. These phenomena indicate how the cancer cells smartly escape the immune surveillance system of the host. In this chapter, cancer-associated changes of glycosphingolipids together with glycolipid synthetase are summarized, and also our recent work on glycosphingolipids in metastatic tumor cells is introduced. PMID- 8632741 TI - [Carbohydrate antigens as cell adhesion molecules]. AB - Some cell surface carbohydrate determinants such as sialyl Lewis A and sialyl Lewis X serve as ligands for the cell adhesion molecules called selectins. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelial cells during the course of hematogenous metastasis of cancer. This review considers primary adhesion of cancer cells to endothelial cells mediated by carbohydrate-selectin interaction followed by the secondary involvement of the classical cell adhesion molecules called integrins in extravasation of cancer cells. Alteration in the glycosyltransferase activities that induce the accelerated synthesis of abnormal carbohydrate determinants on cancer cells is also discussed. PMID- 8632742 TI - [Carbohydrate antigens in carcinoma invasion and metastasis]. AB - Human colorectal carcinomas with increased metastatic potential and with poor prognosis are characterized by high content of sialyl-Lewis X carbohydrate antigen as detected by monoclonal antibody (mAb) FH6. The levels of these carbohydrate antigens apparently increase during colorectal carcinoma progression from nonmetastatic to metastatic tumors and inversely correlate with post operative survival of colon carcinoma patients. Cell lines have been selected for high and low levels of cell surface sialyl-Lewis X antigen using mAb FH6. The high expresser cells adhere strongly to cytokine-activated endothelial cells apparently through E-selectin. The high expresser cells are also adhesive to sections of human livers, although involvement of selectins in this process is unknown. Increased expression of sialyl-Lewis X antigens on the surface of these high expresser variant cells is apparently due to increased alpha 1,3 fucosyltransferase, a biosynthetic enzyme presumable responsible for the final step of the production of sialyl-Lewis X antigens. Human fucosyltransferase VI cDNA has been stably transfected into the low expresser variant cells. alpha 1,3 Fucosyltransferase activity, cell surface sialyl-Lewis X carbohydrate antigen, and adhesion to activated endothelial cells have been showed to increase in these transfectant cells. Furthermore, these cells show dramatic increase in their liver metastatic potential when it is tested by the intrasplenic injection into nude mice. We propose that carbohydrate antigens with unique peripheral epitopes serve as unique molecular phenotypes that determine colorectal cancer metastasis. PMID- 8632743 TI - [Carbohydrate antigens as tumor marker]. AB - Since S. Hakomori's pioneer work, many has studied on carbohydrate chains on cancer. Many monoclonal antibodies are developed and some of them are already evaluated as tumor markers. They are used routinely in daily clinical practice for immunohistochemistry and immunodetection of circulating antigens in patients' sera. However, most of them are not specific to cancers, are negative in early cancers, they are usually elevated in sera of patients with advanced or recurrent cancers and used in monitoring patients. In the major organic sites, positive rates of some serum markers are shown in a table and discussed their usefulness and limitations. Problems of similarities and discrepancies in many commercial systems of sugar chain tumor markers are discussed. Recent topic in the function of sialylated carbohydrate antigens is their strong affinities with cell-adhesion molecules, for that reason, sialyl Lewis A and sialyl Lewis X are thought to be responsible for the distant metastasis of those cancers that produce them. PMID- 8632744 TI - [Functions of mucin molecules in gastrointestinal cancer]. AB - Studies about the function of mucin molecules as surface molecule of adenocarcinoma of gastrointestinal tract had just started, and several important function of mucins had been revealed. In the process of the malignant transformation, class of the expressed mucin core protein and content of glycochain of mucin molecule was changed. Changes of glycoprotein of mucin molecule during transformation affect immunogenesity, tumorigenesity, metastatic ability and sensitivity for anti-cancer drugs. Glycosylated mucin acts important roles during metastatic sequence and prognosis of the gastrointestinal cancer was collerated with expression of immatured mucin of cancer cells. A type of mucin with immature type of glycochain, MUC1, had a protective function for cytotoxicity, e.g. natural killer cell and cytotoxic T-cell, and deglycosilation of MUC1 sensitize cancer cells for cytotoxicity. And MUC1 reduce sensitivity for anti-cancer drugs and MUC1 was glycosilated during process to get resistance for anti-cancer drug. The functions of mucin molecules is not fully revealed, but further studies will indicate importance of mucin molecules in tumor progression. PMID- 8632745 TI - [Expression of carbohydrate antigen in lung cancers]. PMID- 8632746 TI - [Clinical value of carbohydrate antigens, sialyl Lewis-x and sialyl Lewis-a in gastrointestinal cancer]. AB - To evaluate clinical importance of the expression of sialyl Lewis-X (sLe(x)) and sialyl Lewis-a antigen (sLe(a)) in gastrointestinal cancers, we examined immunohistochemically expression of the two antigens in esophageal, gastric, colorectal, and pancreatic cancer. Expression of sLe(x) and sLe(a) were associated with several clinicopathologic features which reflect tumor aggressiveness in esophageal, gastric and colorectal cancer, but not in pancreatic cancer. In esophageal and colorectal cancer, survival rate of the patients with sLe(x) positive tumors was significantly poorer than that of the patients with sLe(x) negative tumors, while in gastric cancer that with sLe(a) positive tumors was significantly poorer than that with sLe(a) negative. Cox's multivariate analysis revealed that sLe(x) expression status was one of the significant discriminants of prognosis in colorectal cancer patients and sLe(a) status in gastric cancer patients. These results suggest that sLe(x) and sLe(a) expression could be involved in aggressiveness of gastrointestinal cancer and might prove to be a potent marker for prognosis in patients with gastric cancer and colorectal cancer. PMID- 8632747 TI - [Biological malignant potential and carbohydrate antigens in breast cancer]. AB - We investigated the association of expression of carbohydrate antigens with the degree of malignancy in breast cancer. When this expression was compared in the cancerous portion and adjacent non-cancerous portion in each of the patients, increased expression of type2 carbohydrate antigens were found in cancerous portions. The prognosis of patients in whom type2 carbohydrate antigens were increased in the cancerous portion was significantly poorer than in patients in whom they were not increased. The experimental studies, using cell lines derived from human breast cancer, resulted that the adhesion of cancer cells to vascular endothelial cells was mediated by sialyl Lewis(x) via E-selectin. Moreover, it was suggested that there were the induction of E-selectin expression on the endothelial cells by cancer cells and secondary adhesional system following the initial adhesion mediated by E-selectin and carbohydrate ligands. Consequently, it appears that the role that carbohydrates play in metastasis as cell adhesion molecules, is the underlying the relationship between the expression of carbohydrates and the prognosis of patients with breast cancer. PMID- 8632748 TI - [Prognosis of colorectal cancer and sialyl-lea antigen]. PMID- 8632749 TI - [Effect of high concentration insulin during glucose clamp on blood amino acids and lipids]. PMID- 8632750 TI - [CD10-positive lymphocytes in human thymoma]. PMID- 8632751 TI - RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl 1H-indole-3-ethanamine hydrochloride), a selective alpha 1A-adrenoceptor antagonist, displays low affinity for functional alpha 1-adrenoceptors in human prostate: implications for adrenoceptor classification. AB - Norepinephrine (NE) contracts smooth muscle cells within the human lower urinary tract (LUT) (bladder neck, prostate, and urethra). Receptor distribution and pharmacological evidence have implicated activation of alpha 1A-adrenoceptors. We disclose the pharmacological properties of the novel, selective alpha 1A adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro- alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053) and examine critically the pharmacological identity of the alpha 1-adrenoceptor mediating contractions to NE in human LUT tissues. In several tissues from rat and cloned adrenoceptors, RS-17053 displayed high affinity for the alpha 1A-adrenoceptor (pKi and pA2 estimates of 9.1-9.9) and a 30-100-fold selectivity over the alpha 1B- and the alpha 1D-adrenoceptor subtypes (pK1 and pA2 estimates of 7.7-7.8). However, in isolated smooth muscle preparations from human LUT tissues, RS-17053 antagonized responses to NE only at high concentrations. Estimates of affinity (pA2) at alpha 1-adrenoceptors mediating NE-induced contractions were 7.5 in prostatic periurethral longitudinal smooth muscle (compared with 8.6 for prazosin), 6.9 in anterior fibromuscular stroma (prazosin, 8.9), and 7.1 in bladder neck (prazosin, 8.5). These findings indicate that contractile responses to NE in human LUT tissues are mediated by a receptor displaying pharmacological properties that are clearly different from those of the defined alpha 1A adrenoceptor and raise the possibility that multiple forms of the alpha 1A adrenoceptor may exist in human LUT that are discriminated by RS-17053. In this regard, the affinity estimates obtained with RS-17053 and other alpha 1 adrenoceptor antagonists in human LUT tissues are identical to those described for the putative alpha 1L-adrenoceptor. PMID- 8632752 TI - The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site. AB - Opioids are cationic compounds that mediate their biological action through three highly homologous receptors (mu, delta, and kappa) known to belong to the G protein-coupled receptor (GPR) family. The third putative transmembrane domain of opioid receptors contains a conserved aspartate residue that is typically found in biogenic amine binding GPRs and is generally believed to form an ion pair with the cationic neurotransmitters. Using site-directed mutagenesis, we investigated the possibility of an identical role for this residue (Asp128) in the mouse delta opioid receptor. Removal of the carboxylate group via an aspartate-to-alanine mutation did not modify binding affinity of a representative set of opioid compounds, including bremazocine, diprenorphine, naloxone, Tyr-D-Thr-Gly-Phe-Leu Thr, [D-Ala2,D-Leu5]enkephalin, cyclic[D-penicillamine2,D penicillamine5]enkephalin, deltorphin II, (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl 2,5-di-methyl-1- piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide, and naltrindole. It nevertheless decreased receptor expression level and affected the binding of three agonists ([D-Ala2,D-Leu5]enkephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr, and (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di- methyl-1-piperazinyl]-3 hydroxybenzyl]-N,N-diethylbenzamide) when the receptor was under Na(+)-induced low affinity state. On the other hand, the aspartate-to-asparagine mutation strongly impaired the binding of all of the above ligands and highlighted differential modes of interaction for alkaloids and peptides. Finally, removal of the homologous carboxylate group in the mouse mu receptor had distinct effects because it dramatically reduced the binding potency of some, but not all, tested ligands. Taken together, these results demonstrate that (i) the direct ligand/receptor interaction previously demonstrated for the beta-adrenergic receptor does not take place in the delta receptor, (ii) Asp128 nevertheless contributes to stabilization of the spatial conformation of the binding pocket, and (iii) these conclusions cannot be extended to the closely related mu receptor. PMID- 8632754 TI - Proteinase-activated receptor-2 in rat aorta: structural requirements for agonist activity of receptor-activating peptides. AB - We measured in rat aorta rings the relaxant activity of a number of peptides derived from the activating sequence (SLIGRL, or PP6) of the proteinase-activated receptor-2 (PAR-2). The relaxant action of PP6-NH2 mimicked the action of low concentrations of trypsin (0.5-1 unit/ml; 1-2 nM), was dependent on an intact endothelium, and was blocked by N-omega-nitro-L-arginine methyl ester but not by N-omega-nitro-D-arginine methyl ester. The relaxant actions of PP6, SLIGRL-NH2 (PP6-NH2), SLIGR (PP5), and SLIGR-NH2 (PP5-NH2) were comparable in magnitude, with relative potencies of PP6-NH2 > or = PP6 > PP5-NH2 > PP5. Peptides lacking either a leucine at position 2 (SAIGRL) or an arginine at position 5 (SLIGAL) exhibited markedly reduced or no relaxant activity; nevertheless, the tetrapeptide LIGR-NH2 exhibited low but detectable intrinsic activity. With the use of reverse-transcriptase/polymerase chain reaction, we documented the presence of PAR-2 mRNA in aorta tissue and determined that the rat aorta amino terminal receptor-activating sequence was the same as that reported for the murine PAR-2 receptor. We concluded that the rat aorta tissue has a PAR-2 receptor that can be activated by peptides as short as four amino acids; the leucine and arginine at positions 2 and 5, respectively, of the proteolytically revealed PAR-2 receptor-activating sequence play key roles in regulating receptor function. PMID- 8632753 TI - Expression cloning of a human brain neuropeptide Y Y2 receptor. AB - The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34 substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34 substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor. PMID- 8632755 TI - Epitope mapping studies with human anti-cytochrome P450 3A antibodies. AB - A subset of patients with hypersensitivity reactions to the aromatic anticonvulsants phenytoin, carbamazepine, and phenobarbital have circulating antibodies that recognize members of the rat cytochrome P450 (CYP) 3A subfamily. These antibodies do not recognize related human CYP3A proteins despite the high degree of structural similarity. To investigate the relationship between P450 mediated drug metabolism and the development of anti-P450 antibodies, we initiated epitope mapping studies by screening a library of fusion proteins constructed from rat CYP3A1 with an anti-CYP3A1-positive patient serum sample. Positive signals from colony lifts were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting, and a 26-amino acid sequence corresponding to amino acids 342-367 of the CYP3A1 protein (NKAPPTY DTVMEMEYLDMVLNETLRL) was identified as containing the epitope recognized by IgG3 antibodies in this serum sample. By subjecting inserts from two clones into a second round of library construction and screening by immunoblot analysis, we further defined the epitope to EYLDMVLNETLRL. Single amino acid deletions identified DMVLNETLRL as the minimum amino acid sequence required for antibody binding. The corresponding sequence in the four human CYP3A proteins differs by only one amino acid (DMVVNETLRL) This amino acid is critical to antibody recognition as immunoreactivity of the L361V mutant is markedly reduced. Anti CYP3A antibodies in nine of nine additional sera also recognized the 13-amino acid epitope; for five of these sera, the minimum antibody binding sequence was DMVLNETLRL. The proximity of this epitope to a region determining substrate specificity may provide the link among reactive metabolite production, hapten formation, and the production of anti-P450 antibodies in anticonvulsant-induced idiosyncratic reactions. PMID- 8632756 TI - Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells. AB - Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2 , 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16 treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis. PMID- 8632758 TI - Expression, induction, and catalytic activity of the ethanol-inducible cytochrome P450 (CYP2E1) in human fetal liver and hepatocytes. AB - The mechanisms responsible for ethanol-mediated teratogenesis have not been resolved. However, possible etiologies include the local formation of the teratogen acetaldehyde or oxygen radicals by fetal ethanol-oxidizing enzymes. As alcohol dehydrogenases are expressed at very low concentrations in human embryonic tissues, the ethanol-inducible P450 enzyme, CYP2E1, could be the sole catalyst of fetal ethanol oxidation. With this in mind, we examined the expression of this P450 in liver samples from fetuses ranging in gestational age from 16 to 24 weeks. Immunoblot analysis of fetal liver microsomes revealed the presence of a protein immunoreactive with CYP2E1 antibodies that exhibited a slightly lower molecular weight than that found in adult liver samples. Embryonic CYP2E1 expression was further confirmed by the reverse transcriptase reaction with RNA from a 19-week gestational fetal liver used as template. Catalytic capabilities of human fetal microsomes were assessed by measurement of the rate of ethanol oxidation to acetaldehyde, which were 12-27% of those exhibited by adult liver microsomes. Immunoinhibition studies with CYP2E1 antibodies revealed that the corresponding antigen was the major catalyst of this reaction in both fetal and adult tissues. We then assessed whether embryonic CYP2E1 was, like the adult enzyme, inducible by xenobiotics. Treatment of primary fetal hepatocyte cultures with either ethanol or clofibrate demonstrated a 2-fold increase in CYP2E1 levels compared with untreated cells. Collectively, our results indicate that CYP2E1 is present in human fetal liver, that the enzyme is functionally similar to CYP2E1 from adults, and that fetal hepatocyte CYP2E1 is inducible in culture by xenobiotics, including ethanol. Because fetal CYP2E1 mediates ethanol metabolism, the enzyme may play a pivotal role in the local production of acetaldehyde and free radicals, both of which have potential deleterious effects on the developing fetus. PMID- 8632757 TI - Cloning of cDNAs encoding the human gamma-aminobutyric acid type A receptor alpha 6 subunit and characterization of the pharmacology of alpha 6-containing receptors. AB - A cDNA encoding the human gamma-aminobutyric acidA (GABAA) receptor alpha 6 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows 91.4% identity with the published rat alpha 6 subunit. In situ hybridization histochemistry reveals the alpha 6 mRNA to be located within the granule cell layer of the human cerebellar cortex. Recombinant human alpha 6 beta gamma 2S GABAA receptors have been expressed in both stably transfected cells and Xenopus oocytes, and the pharmacology of the benzodiazepine binding site has been determined. The recombinant receptor has a diazepam-insensitive pharmacology, with negligible affinity for a number of classic benzodiazepines. A number of compounds that bind to the benzodiazepine site potentiated the GABA response of alpha 6 beta 2 gamma 2 receptors. Most importantly, the classic benzodiazepine antagonist ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate (Ro 15-1788) and the partial inverse agonist ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3 carboxylate (Ro 15-4513) both acted as agonists at the alpha 6 containing receptor. This observation demonstrates definitively that efficacy of benzodiazepine compounds cannot be generalized across receptor subtypes and may also help explain some of the behavioral effects that have been reported for these compounds. PMID- 8632759 TI - Interleukin-1 alpha-induced modulation of topoisomerase I activity and DNA damage: implications in the mechanisms of synergy with camptothecins in vitro and in vivo. AB - Studies have shown that cytokines are directly cytotoxic to tumor cells in vitro and in vivo and that interleukin-1 alpha (IL-1 alpha) potentiates the cytotoxicity of certain clinically active drugs in a number of human tumor cells, including carcinomas of breast and ovary. We found that interleukin-1 alpha potentiated cytotoxicity of camptothecin (4-5-fold) during simultaneous drug exposure in human ovarian NIH:OVCAR-3 cancer cells in vitro. Studies indicated that IL-1 alpha significantly increased topoisomerase I-catalyzed camptothecin induced DNA cleavable complexes in the ovarian cell line, which was not due increased intracellular camptothecin as IL-1 alpha failed to effect cellular uptake of camptothecin. Pretreatment of the ovarian cells with IL-1 alpha did not result in increased expressions of mRNA for the topoisomerase I gene, whereas a small increase (approximately 1.5-fold) in the expression of topoisomerase I protein was observed, suggesting that IL-1 modulated the activity of topoisomerase I for the observed increase in cleavable complex formation. Treatment of human ovarian tumor cells grown as xenografts in nude mice with IL-1 alpha followed by CPT-11 at minimally toxic doses significantly (5-6-fold) enhanced antitumor activity of either agent alone. Because camptothecins are active against solid tumors in vivo, combinations of IL-1 alpha with these active drugs may lead to more effective treatment of ovarian cancers in the clinic. PMID- 8632761 TI - Microtubule effects of welwistatin, a cyanobacterial indolinone that circumvents multiple drug resistance. AB - Welwitindolinones are a family of novel alkaloids recently isolated from the blue green alga Hapalosiphon welwitschii. We demonstrate that incubation of SK-OV-3 human ovarian carcinoma cells and A-10 vascular smooth muscle cells with welwitindolinone C isothiocyanate, now termed welwistatin, results in dose dependent inhibition of cell proliferation, which is correlated with increases in the percentage of cells in mitosis. Treatment of A-10 cells with welwistatin resulted in reversible depletion of cellular microtubules but did not affect microfilaments. Pretreatment of A-10 cells with paclitaxel prevented microtubule depolymerization in response to welwistatin. Welwistatin inhibited the polymerization of purified tubulin in vitro but did not alter the ability of tubulin to bind [3H]colchicine or to hydrolyze GTP. Also, welwistatin did not induce the formation of topoisomerase/DNA complexes. Results of the present study indicate that welwistatin is a new antimicrotubule compound that circumvents multiple drug resistance and so may be useful in the treatment of drug-resistant tumors. PMID- 8632760 TI - Regulation of CYP4A expression in rat by dehydroepiandrosterone and thyroid hormone. AB - Dehydroepiandrosterone (DHEA) is a peroxisome proliferating agent when administered in pharmacological dosages, but it has not been shown to function through the peroxisome proliferator-activated receptor in cell-based assays. Because members of the thyroid hormone/vitamins A and D nuclear receptor subfamily, including PPAR, are known to modulate each other's function in gene expression by heterodimerization, we sought to establish whether DHEA and thyroid hormone interact to regulate several of the hepatic and renal enzymes associated with peroxisome proliferation, i.e., peroxisomal beta-oxidation and microsomal NADPH:cytochrome P450 oxidoreductase and the cytochromes P450 4A. In rats administered exogenous T3 to attain a hyperthyroid state, induction of the three isozymes of CYP4A (4A1, 4A2, and 4A3) by DHEA was suppressed > 60-80% at the mRNA level, with induction of CYP4A2 mRNA being completely inhibited. Nuclear run-on transcription assays indicated that this inhibitory effect was regulated at the level of transcription. Induction of hepatic peroxisomal beta-oxidation by DHEA or the peroxisome proliferator nafenopin was in large part unaffected by treatment of animals with T3 under any condition tested. Microsomal NADPH:cytochrome P450 oxidoreductase activity was induced by either DHEA or T3; cotreatment resulted in an additive induction. When animals were treated with a lower dose of exogenous T3 that rendered the animals slightly hyperthyroid, only induction of hepatic CYP4A2 mRNA by DHEA or nafenopin was significantly inhibited (> 80%) compared with euthyroid control animals. Animals that had been rendered hypothyroid through removal of the thyroid gland showed normal induction of CYP4A genes by DHEA in liver, suggesting that their induction by DHEA was not dependent on the presence of thyroid hormone. The administration of exogenous T3 to thyroidectomized rats in the presence of DHEA potently suppressed hepatic induction of all three genes at the mRNA and protein level. In experiments with cultured rat hepatocytes, physiological concentrations of T3 potently inhibited the induction of CYP4A2 mRNA levels by nafenopin but had little effect on induction of CYP4A1 or 4A3 mRNA. At higher T3 concentrations, the induction of CYP4A1/4A3 mRNA and protein was also inhibited. These results suggest that T3 modulates the expression of CYP4A2 at the level of transcription in physiologically relevant concentrations but that hyperthyroid conditions are required to suppress expression of CYP4A1/4A3 genes. In euthyroid rodent kidney, which only expresses CYP4A2 under either basal or DHEA-induced conditions, near physiological levels of T3 caused potent suppression of peroxisome proliferator dependent induction of CYP4A2 mRNA levels by either DHEA or nafenopin. In thyroidectomized rats, basal expression of CYP4A2 mRNA was decreased relative to euthyroid controls, but DHEA was as effective an inducer of this mRNA as it is in euthyroid rats. As seen in euthyroid rats, T3 administration potently suppressed DHEA induction of CYP4A2 mRNA levels under either basal or induced conditions. Although CYP4A expression was not derepressed in liver or kidneys of hypothyroid animals, our results indicated that the thyroid status of the animal did affect basal expression of CYP4A2, suggesting involvement of thyroid hormone or some other factor regulated by the thyroid gland on its constitutive expression. PMID- 8632762 TI - Effects of expression of a mouse brain L-type calcium channel alpha 1 subunit on secretion from bovine adrenal chromaffin cells. AB - Regulated exocytosis from bovine chromaffin cells is stimulated by the influx of Ca2+ through plasma membrane ion channels that are opened by nicotinic stimulation and/or depolarization. Recently, we developed a novel method that enabled us to investigate the function of a cloned Ca2+ channel type C alpha 1 subunit in forming channels that stimulate exocytosis. In the present study, we demonstrate by immunocytochemistry that bovine chromaffin cells normally express an epitope specific for the type C alpha 1 subunit. We investigated the effects of expression of additional class C alpha 1 subunits (mouse brain clone) on various aspects of secretory function in bovine chromaffin cells by measuring secretion of cotransfected human growth hormone (GH, a reporter for the regulated secretory pathway in the transfected cells). New channels were activated in response to depolarization by both elevated K+ and nicotinic cholinergic agonist. The new channels had their greatest effects when secretion was stimulated suboptimally. Secretion was enhanced only after the first 30 sec of stimulation, and the enhancement extended beyond 5 min of continuous stimulation. In contrast to the endogenous L-type Ca2+ channels, the latency was not decreased by the dihydropyridine L-type Ca2+ channel agonist, Bay K 8644. The findings suggest that (i) the Ca(2+)-sensitive mechanism for triggering or maintaining exocytosis is capable of being saturated by high levels of Ca2+, (ii) secretion caused by nicotinic agonist stimulation can be significantly enhanced by activation of voltage-sensitive Ca2+ channels, and (iii) the effects on secretion of the L-type Ca2+ channels formed on expression of the mouse brain class C alpha 1 subunit are distinct from those of endogenous L-type Ca2+ channels. PMID- 8632763 TI - Trophic effect of exogenous nerve growth factor on rat striatal cholinergic neurons: comparison between intraparenchymal and intraventricular administration. AB - Penetration into the brain is an important consideration in the pharmacological use of neurotrophic factors for the treatment of brain neurodegeneration, e.g., in Alzheimer's disease. Furthermore, intracerebroventricular treatment with nerve growth factor (NGF) has been found to induce side effects, including aberrant sympathetic sprouting and weight loss. Such findings suggest that direct intraparenchymal application of minimal amounts of trophic factors might be therapeutically desirable. We compared the effectiveness of intrastriatal and intracerebroventricular administrations of NGF on striatal cholinergic neurons in adult rats. Daily intrastriatal administration for 1 week of > or = 50 ng of NGF resulted in an increase in mRNA levels for choline acetyltransferase (ChAT) in striatal cholinergic cells to approximately 2-fold over control. A daily intraventricular dose of 4.5 micrograms of NGF was required for a similar response. Both 5 and 50 ng of NGF/day failed to induce an effect on transmembrane protein tyrosine kinase trkA mRNA levels, but injections of 750 or 1500 ng/day of NGF up-regulated trkA mRNA expression to approximately 2-fold of control. NGF delivered intracerebroventricularly failed to induce an observable change in striatal trkA mRNA, even at a dosage of 4.5 micrograms of NGF/day. These quantitative differences in NGF actions were reflected at the level of NGF receptors. Using Western blotting procedures, we found pronounced tyrosine phosphorylation of Trk-type proteins 2 hr after intrastriatal injection of 50 ng of NGF. Maximal responses were seen with either 150 or 750 ng of NGF. For maximal activation of Trks by intraventricular NGF injection, 4.5 micrograms of NGF was required. Taken together, our results strongly favor intraparenchymal injections or infusions of NGF, and possibly other trophic factors, for therapeutical applications to maximize the effects on the targeted neuronal populations and to minimize undesirable side effects. PMID- 8632764 TI - Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up regulate these proteins in human colon carcinoma cells. AB - Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates. PMID- 8632765 TI - Protective effect of transforming growth factor-beta 1 on beta-amyloid neurotoxicity in rat hippocampal neurons. AB - Neurodegeneration associated with Alzheimer's disease is believed to involve toxicity to beta-amyloid (A beta) and related peptides. Treatment of cultured rat hippocampal neurons with A beta 1-40 (1 microM) or the active fragment A beta 25 35 (1 microM) for 5 days led to a approximately 40-50% decrease in neuronal viability. The hydrophilic antioxidant ascorbic acid (300 microM) and the lipophilic antioxidant 2-mercaptoethanol (10 microM) both protected significantly against A beta neurotoxicity. Despite the protective effects of these antioxidants, both acute and chronic treatments with A beta 25-35 did not increase production of superoxide anions, as monitored with the fluorescent probe hydroethidine. Similarly, overexpression of Cu/Zn-superoxide dismutase using adenovirus-mediated gene transfer did not protect against A beta neurotoxicity. A beta neurotoxicity, however, was prevented in cultures infected with a recombinant, replication-defective adenovirus overexpressing the Ca2+ binding protein calbindin D28k. Transforming growth factor-beta 1 (TGF-beta 1) has been shown to protect neurons against both Ca(2+)- and free radical-mediated neuronal degeneration. We found that A beta neurotoxicity was significantly attenuated by single treatments with TGF-beta 1 (0.1-10 ng/ml) and prevented by repetitive treatments (10 ng/ml/day). The protective effects of TGF-beta 1 were associated with a preservation of mitochondrial potential and function, as determined with rhodamine-123-based microfluorimetry. Because both increased oxidative stress and pathophysiological Ca2+ fluxes can impair mitochondrial function, preservation of mitochondrial potential by TGF-beta 1 could be directly associated with its protection against A beta neurotoxicity. The ability of TGF-beta 1 to increase the expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL is discussed in this context. PMID- 8632766 TI - Oct-1 transcription factor is a negative regulator of rat CYP1A1 expression via an octamer sequence in its negative regulatory element. AB - The rat CYP1A1 negative regulatory element (NRE) contains AP-1 and Oct-1 motifs at -808 to -788 bp. The CYP1A1 sequence from -813 to -779 bp and an identical sequence bearing a point mutation in the octamer motif were synthesized. Gel mobility shift assays showed the formation of two complexes with the wild-type CYP1A1 sequence and nuclear extracts from H4IIE and HepG2 hepatoma cells and from rat liver. The formation of the major complex was significantly reduced with the mutant octamer-containing oligomer and was specifically competed by an Oct-1 oligodeoxyribonucleotide. The addition of Oct-1 antibody caused a supershift of the major complex. The presence of the wild-type sequence, but not the mutant octamer sequence, caused a 3-fold decrease in SV40 enhancerless promoter activity in transfected HepG2 cells. Co-transfection of an Oct-1 expression vector with rat CYP1A1 NRE octamer-containing, promoter/reporter gene constructs specifically further decreased promoter activity of the wild-type octamer-containing constructs in HepG2 cells. The results indicate that Oct-1 binds to the rat CYP1A1 promoter NRE and is a negative regulator of rat CYP1A1 expression. PMID- 8632767 TI - Reserpine inhibits amphetamine action in ventral midbrain culture. AB - Although amphetamine releases catecholamines from isolated secretory vesicles, a number of in vivo experiments have indicated that the vesicular amine transport blocker reserpine does not block amphetamine-induced release. To address this paradox, we examined the effect of reserpine on amphetamine-induced dopamine release from postnatal ventral midbrain neurons in culture. These cultures provide a preparation in which intracellular, extracellular, and releasable dopamine pools can be measured simultaneously. We found that 1 microM reserpine for 90 min reduced stimulation-dependent dopamine release by > 95%. In parallel, reserpine reduced amphetamine-induced dopamine release by > 95% compared with cells not exposed to reserpine or by 75% compared with reserpine-treated cultures. This shows that amphetamine acts principally by redistributing dopamine from synaptic vesicles to the cytosol. PMID- 8632769 TI - Intracellular trafficking of the muscarinic acetylcholine receptor: importance of subtype and cell type. AB - Agonist-induced decrease of surface muscarinic receptor number occurs in a number of cell lines. Recent work has suggested that some muscarinic receptor subtypes undergo internalization, whereas others do not. We investigated the agonist induced trafficking of various muscarinic receptor subtypes transfected into CHO cells and compared it with the trafficking of receptors expressed natively in neuronal cells, fibroblasts, or epithelial cells. SH-SY5Y neuroblastoma cells, which express predominantly the m3 receptor subtype, show qualitatively similar changes in surface receptor number in response to agonist stimulation to those occurring in NG108-15 cells, which express predominantly the m4 subtype. The rate constants for internalization, however, were considerably different, indicating that receptors in SH-SY5Y cells show a much faster turnover than those in NG108 15 cells. In the transfected cells, the muscarinic receptor subtypes m1 and m3, which are coupled to second messenger systems via Gq/11, showed little agonist induced loss of surface receptors. In contrast, the muscarinic receptor subtypes m2 and m4, which are coupled via Gi or G(o), showed a substantial loss of surface receptors after treatment with agonist. An interesting implication of this result is that agonist-induced receptor trafficking can still occur efficiently, even at very high receptor densities. Significant agonist-induced internalization also occurs in a fibroblast line (HeLa) and an epithelial cell line (HT29), both of which express predominantly m3 receptors. Our results suggest that the extent and rate of the loss of receptors from the cell surface in response to agonist stimulation are governed by both the receptor subtype and the cell type in which it is expressed. PMID- 8632768 TI - The 1'-substituent on the anilino ring of the antitumor drug amsacrine is a critical element for topoisomerase II inhibition and cytotoxicity. AB - The mechanism of action of the antitumor drug amsacrine involves intercalation of the acridine chromophore into DNA and inhibition of topoisomerase II. The substituent at position 1' on the aniline is believed to be essential to the formation of the topoisomerase II/DNA cleavable complex and therefore to the cytotoxicity of the drug. To further delineate the role of the 1'-substituent, we investigated the effects on topoisomerase II activities of three anilinoacridine derivatives that differ only by the nature of the substituent at position 1'. The results of the cytotoxicity assays performed with cells sensitive (DC-3F) and resistant [DC-3F/9-hydroxy-ellipticine (9-OH-E)] to topoisomerase inhibitors are correlated with the effects of the drugs on topoisomerase II-mediated DNA cleavage in vitro. The influence of topoisomerase II alpha on the mechanism of action of the drugs was examined using resistant DC-3F/9-OH-E cells transfected with a plasmid carrying a wild-type human topoisomerase II alpha cDNA. Depending on the nature of the 1'-substituent of the drugs, the restoration of normal topoisomerase II alpha catalytic activity in human topoisomerase II alpha cDNA transfected DC-3F/9-OH-E cells either does not modify the susceptibility of the cells to the drug or partially reverses the resistance phenotype. The molecular and cellular studies reveal that topoisomerase II alpha is implicated in the cytotoxicity of amsacrine and confirm that the substituent at position 1' on the anilino ring of amsacrine governs the interaction with topoisomerase II. PMID- 8632770 TI - Stimulation of Ca(2+)-dependent membrane currents in Xenopus oocytes by microinjection of pyrimidine nucleotide-glucose conjugates. AB - Microinjection, but not extracellular application, of cytidine-5'-diphosphate-D glucose (CDPG) has been shown to elicit Ca(2+)-dependent currents in Xenopus laevis oocytes. These responses were comparable to those of inositol-1,4,5 trisphosphate (InsP3) in being both rapid and dose dependent. For example, maximal amplitudes of CDPG-induced current were similar (approximately 365 +/- 75 nA at 1 microM CDPG) to those of InsP3. The CDPG currents were insensitive to removal of extracellular Ca2+, indicating the dependence on Ca2+ release from intracellular Ca2+ stores but not on Ca2+ entry through plasma membrane. CDPG induced currents were reduced or abolished by pretreatment with thapsigargin, by injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' tetraacetic acid, or by extracellular perfusion of the Cl- channel blocker niflumic acid but were insensitive to injection of the InsP3 antagonist heparin. These results suggest that CDPG induces Ca2+ discharge from intracellular Ca2+ stores via a mechanism distinct from that of InsP3 in Xenopus oocytes. Another pyrimidine nucleotide-glucose derivative, uridine-5'-diphosphate-alpha-D-glucose, also induced Ca(2+)-dependent currents, but the activity was lower than that of CDPG (maximal amplitude, 272 +/- 62 nA). Other nucleotide-glucose compounds (adenosine-5'-diphosphate-D-glucose, guanosine-5'-diphosphate-D-glucose, and thymidine-5'-diphosphate-D-glucose) had no current responses when injected into oocytes. After injection of CDPG, CDPG-induced Ca2+ release appeared to couple to a Ca2+ entry pathway similar to that coupled to InsP3. These results indicate that pyrimidine nucleotide-glucose conjugates may provide novel pharmacological tools for the study of Ca2+ signaling in oocytes. PMID- 8632771 TI - Interaction of the COOH-terminal domain of the neurotensin receptor with a G protein does not control the phospholipase C activation but is involved in the agonist-induced internalization. AB - The agonist-induced internalization of the neurotensin receptor was studied in transfected Chinese hamster ovary cells expressing either the wild-type or a truncated rat neurotensin receptor, lacking the complete intracellular COOH terminal end. Incubation of cells expressing the wild-type neurotensin receptor in the presence of the peptide resulted in a dramatic decrease in the [3H]neurotensin binding at the cell surface. This disappearance of cell surface binding sites resulted from the internalization of the receptor after the binding of the peptide. The receptor/peptide complexes were internalized in an intracellular compartment resistant to acid washes. The truncated receptor displayed high affinity binding properties for neurotensin in cell homogenates and activated phospholipase C as did the wild-type receptor. However, in cells expressing the truncated receptor, incubation with neurotensin only induced a partial decrease in cell surface binding, and internalization of the bound peptide was also impaired. On cell homogenates, the GTP analogue Gpp(NH)p was found to decrease the affinity of [3H]neurotensin for the wild-type receptor, whereas no similar effect was observed with the truncated receptor. These results show that the intracellular COOH-terminal region of the rat neurotensin receptor is not required for its functional coupling with intracellular G protein but is involved in the shift of the affinity of the receptor for the agonist, which occurs as a consequence of receptor activation and coupling. Because the truncated receptor was shown to internalize poorly, it may be proposed that internalization is not directly related to the activation of G protein but rather is a consequence of modification of receptor affinity, after activation by the agonist. PMID- 8632772 TI - Dopamine D2L receptors stimulate Na+/K(+)-ATPase activity in murine LTK- cells. AB - Ion transport can be regulated by dopamine receptors. D1-like receptors inhibit both Na+/H+ exchange (NHE) and Na+/K(+)-ATPase activity, whereas D2-like receptors stimulate NHE. However, the effect of D2-like receptors on Na+/K(+) ATPase activity is controversial. In renal proximal tubular cells, where several D1-like and D2-like receptors are expressed, D2 agonists have been reported either to have no effect or to act in concert with D1 agonists to inhibit Na+/K(+)-ATPase activity. We therefore studied the effect of D2 receptors on Na+/K(+)-ATPase activity in LTK- cells transfected with a rat D2Long receptor cDNA (maximum receptor density = 0.91 +/- 0.26 pmol/mg protein, dissociation constant = 2.39 +/- 0.79 nM, seven experiments). The activation of D2 receptors in these transfected cells by the selective D2 agonist LY171555 led to the inhibition of forskolin-stimulated cAMP accumulation. In the D2Long-transfected, but not in nontransfected cells, LY171555 caused a concentration-dependent stimulation of Na+/K(+)-ATPase activity (EC50 = 0.55 +/- 0.2 microM, Emax = 28 +/ 6%, six experiments), which was completely blocked by the D2-selective antagonist (-)-sulpiride. The D2-stimulated Na+/K(+)-ATPase activity was not secondary to D2 receptor activation of K+ channels or NHE activity since LY171555 stimulated Na+/K(+)-ATPase activity in D2Long-transfected cells, even when K+ channels were blocked by CsCl and intracellular Na+ was clamped by monensin. The D2-stimulated Na+/K(+)-ATPase activity was blocked by pertussis toxin and mimicked by dideoxyadenosine. We conclude that agonist occupancy of D2Long dopamine receptors stimulates Na+/K(+)-ATPase activity; this effect is mediated by the inhibition of cAMP production and is independent of intracellular Na+ and K+ concentration. PMID- 8632774 TI - Repair of ionizing-radiation damage in the radiation resistant bacterium Deinococcus radiodurans. AB - Deinococcus radiodurans is extremely resistant to the lethal and mutagenic effects of ionizing-radiation and many other physical and chemical agents that damage DNA. This resistance is known to be due to D. radiodurans' extremely proficient DNA repair processes. However, little is known about the precise mechanisms employed by this organism to achieve its efficient repair. In the past two years there has been substantial progress in studies on the repair and tolerance of ionizing radiation damage. Areas of progress include: 1) studies on the importance of the deinococcal recA-gene in repair; 2) characterization of a large number of new ionizing radiation-sensitive strains; 3) newly discovered genetic loci with novel repair-related mutational phenotypes; 4) demonstration of efficient interplasmidic and interchromosomal recombination occurring postirradiation; and 5) recent speculations on the mechanisms of radiation resistance and the driving forces of natural selection for DNA damage resistance in D. radiodurans. PMID- 8632773 TI - Stimulatory regulation of the large-conductance, calcium-activated potassium channel by G proteins in bovine adrenal chromaffin cells. AB - G proteins regulate the electrical activity of various cells through their actions on membrane ion channels. In the present study, the effect of G proteins was examined on unitary, large conductance (BK), Ca(2+)-activated K+ channels measured in excised, inside-out patches of membrane obtained from cultured bovine adrenal chromaffin cells. Cytoplasmic application of either guanosine 5'-O-(3 thiotriphosphate) (GTP gamma S) or AlF-4 to stimulate G proteins resulted in a > 4-fold increase in the open probability of the BK channel measured at +40 mV in the presence of a 1 microM concentration of Ca2+. A similar stimulatory regulation was observed after the addition of an activated, mixed Gi/Go alpha preparation. The increase in the open probability during G protein stimulation was associated with a large reduction in the duration of a long closed state of the channel and could be observed in the presence of a protein kinase inhibitor. The half-maximal voltage required for steady state activation of the BK channel decreased from +63 mV to +48 mV in the presence of GTP gamma S. In addition, the half-maximal Ca2+ concentration required for channel opening was reduced from 11.7 microM in control measurements to 1.3 microM during regulation by GTP gamma S. Thus, G proteins increase the open probability of the chromaffin BK Ca(2+) activated K+ channel by shifting the voltage dependence of channel gating to more negative potentials and by enhancing the affinity of the channel for Ca2+. Stimulatory regulation may provide a compensatory mechanism for decreasing the action potential duration during secretagogue-mediated exocytosis. PMID- 8632775 TI - Amino acid residues affecting the activity and stability of human O6-alkylguanine DNA alkyltransferase. AB - Amino acid residues in the human O6-alkylguanine-DNA alkyltransferase (AGT) were mutated and seventeen of the mutant proteins expressed in the ada- ogt-E. coli strain GWR 109 which is very sensitive to killing by methylating agents because of the absence of endogenous alkyltransferases. Thirteen of the mutations tested (delta-10, delta 1-19, R128A, N137A, H146A, R147A, delta N157, Y158A, E172Q, delta 92-97, Y114E, C145A and E172stop) reduced activity to below detectable levels when crude cell extracts were tested for the ability to remove O6 [3H]methylguanine from 3H-methylated DNA. However, only 4 of these mutations (delta 92-97, Y114E, C145A and E172stop) led to a complete loss of activity when tested for the ability to protect the cells from killing by MNNG. This suggests that the other nine mutations do not lead to the complete inactivation of AGT but produce protein with a reduced activity or in reduced amounts. These results show that none of the residues altered in these mutations (delta 1-10, delta 1-19, R128A, N137A, delta N157, H146A, R147A, Y158A and E172Q) are absolutely essential for AGT activity in protection against killing by MNNG. The stability of the mutant AGT proteins was determined by measuring the half-life of the protein synthesis was blocked. These results indicated that five of mutants that lacked AGT activity when tested in the crude extracts (Y114E, R128A, C145A, delta N157 and Y158A) were stable in the cell showing that the alteration of these residues does greatly reduce AGT activity. The other eight mutants lacking activity in crude extracts (delta 1-10, delta 92-97, E172Q, E172stop, delta 1-19, N137A, H146A and R147A) produced a large decrease in the stability of the AGT protein. This may account for the inability to detect AGT activity in vitro despite the ability to protect from MNNG toxicity in vivo. It is of particular interest that mutation of residues His146, Arg147, Asn137 and Glu172 resulted in unstable AGT proteins active in vivo but not in vitro. The crystal structure of the related Ada-C alkyltransferase suggests the involvement of these residues with the Cys145 acceptor site in a hydrogen bond network that may stabilize the protein and aid in the reaction mechanism. The data presented here support the existence of such an interaction existing in the human AGT and stress its importance in maintaining the configuration of the protein. PMID- 8632778 TI - A modified quantitative polymerase chain reaction assay for measuring gene specific repair of UV photoproducts in human cells. AB - Methods for measuring the induction and repair of ultraviolet (UV) induced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 kilobases (kb) or larger can be obtained using the enzyme-sensitive site (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985). More recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sensitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). However, examination of these reports indicates that the PCR based DNA repair assays lack precision (Govan et al., 1990; Kalinowski et al., 1992; Tornaletti and Pfeifer, 1994; Jennerwein and Eastman, 1991). We report here, the development of a highly precise QPCR DNA repair assay. The assay was used to measure the induction and repair of UV photoproducts in a 2.7 kb genomic fragment containing the human growth hormone (hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly reproducible with an overall coefficient of variation from the mean of about 2.5%. This level of precision enabled the apparent simultaneous resolution of cyclobutane dimer (CPD) and (6-4) photoproduct (6-4PP) induction and repair. PMID- 8632776 TI - Site-specific induction and repair of benzo[a]pyrene diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition. AB - Most genotoxic DNA base modifications localized at key genomic sequences constitute the molecular alterations crucial or mutagenesis and tumorigenesis. We have utilized lesion-rendered inhibition of restriction endonuclease cleavage for the analysis of site-specific DNA damage induced by (+/-)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, anti BPDE) in human genes. The H-ras protooncogene and insulin gene sequences were used as targets for modification in vitro and in vivo. Selective induction of individual facultative bands, resulting from covalent modification of the cognate recognition sites, was observed in modified plasmid DNA for a number of restriction nucleases. The ras gene-specific damage, at the PstI, BstYI, NotI and BstEII recognition sites, was visualized and quantitated in human genomic DNA adducted by anti-BPDE. Repair of lesions at hexanucleotide sequences and/or regions surrounding the restriction site, was assessed as a gradual disappearance of facultative bands in DNA from repair-proficient human fibroblasts exposed to the carcinogen in confluent culture. Efficiency of the PstI site-specific repair was compared at low and high levels of initial damage. Higher genotoxic dose caused a decrease in the extent of adduct removal from the bulk DNA, while the specific site of the ras gene was still subject to fast repair. No measurable PstI site-specific repair was detected in the insulin gene. These results show the region-selective induction of bulky anti-BPDE DNA damage in non-related genomic targets and suggest that repair of these lesions in human cells proceeds with the efficiency tightly controlled at different levels of initial genotoxic load. PMID- 8632777 TI - Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA. AB - Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination. PMID- 8632779 TI - Multiple nuclear localization signals in XPG nuclease. AB - We report here evidence for the mechanism of nuclear localization of XPG nuclease in human cells. Several candidate nuclear localization signal (NLS) peptides have been proposed for XPG protein. We have identified XPG peptides containing functional NLS and a potential nuclear retention signal (NRS) using in situ immunofluorescene localization of transiently expressed beta-galactosidase fusion proteins. Two XPG regions with putative NLS [amino acid (AA) coordinates: NLS-B (AA 1057-1074) and NLS-C (AA 1171-1185)] were each shown to independently localize the beta-gal extensively (> 80%) to the nucleus of HeLa cells. The C terminus peptide containing NLS-C, an NLS conserved evolutionarily between yeasts and humans, also directed sub-localization of beta-galactosidase to intranuclear foci reminiscent of native XPG protein, as well as to peri-nucleolar regions. Peptides in the putative XPG 'NLS domain' (AA approximately 1051-1185) apparently function in concert for nuclear localization and also for retention of XPG in nuclear matrix-associated foci. Evidence presented elsewhere (Park et al., 1995) indicates that the peptide containing NLS-C (AA 1146-1185) also regulates the dynamic localization of XPG in the nucleus following UV-irradiation. PMID- 8632781 TI - How not to act on good advice. PMID- 8632780 TI - Identification of a HeLa mRNA fraction which corrects the mitomycin C sensitivity of irs1 cells. AB - The hamster cell mutant irs1 is defective in its response to DNA lesions caused by a variety of mutagens, particularly cross-linking agents. These cells have been assigned to complementation group 2 of X-ray-sensitive mutants and the mutated gene is called XRCC2(X-ray repair cross complementing). We have identified, by microinjection, a human mRNA fraction which can transiently correct the sensitivity of these cells to cross-linking agents. This fraction contains mRNAs of 3.5 kb (+/- 0.25) including, therefore, the transcript of the XRCC2 gene. PMID- 8632782 TI - Britain caught out by 'unscientific' reactions to Europe's beef crisis. PMID- 8632783 TI - Genome ethics panel comes under the microscope at NIH. PMID- 8632784 TI - BSE a specific bovine disease? PMID- 8632785 TI - Merck, SmithKline and patents. PMID- 8632786 TI - Complementary medicine. PMID- 8632787 TI - Hard evidence. PMID- 8632788 TI - Smoke them out. PMID- 8632789 TI - A new strategy for genome sequencing. AB - Existing approaches to sequencing the human genome are based on the assumption that each region to be sequenced must first be mapped. But there is a simpler strategy in which any number of laboratories can cooperate. PMID- 8632790 TI - How to tell a cell where it is. PMID- 8632791 TI - Cognitive cartography. PMID- 8632792 TI - Fat regulation. Life without neuropeptide Y. PMID- 8632793 TI - Dioxins in diesel exhaust. PMID- 8632794 TI - Redox regulation of cell signalling. PMID- 8632795 TI - Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. AB - Secreted signalling molecules provide cells with positional information that organizes long-range pattern during the development of multicellular animals. Evidence is presented that localized expression of Decapentaplegic instructs cells about their position along the anterior-posterior axis of the Drosophila wing in two distinct ways. One mechanism is based on the local concentration of the secreted protein; the other is based on the ability of the cells to retain an instruction received at an earlier time when their progenitors were in close proximity to the signal. Both mechanisms are involved in axis formation. PMID- 8632796 TI - Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. AB - Neuropeptide Y (NPY), a 36-amino-acid transmitter distributed throughout the nervous system, is thought to function as a central stimulator of feeding behaviour. NPY has also been implicated in the modulation of mood, cerebrocortical excitability, hypothalamic-pituitary signalling, cardiovascular physiology and sympathetic function. However, the biological significance of NPY has been difficult to establish owing to a lack of pharmacological antagonists. We report here that mice deficient for NPY have normal food intake and body weight, and become hyperphagic following food deprivation. Mutant mice decrease their food intake and lose weight, initially to a greater extent than controls, when treated with recombinant leptin. Occasional, mild seizures occur in NPY deficient mice and mutants are more susceptible to seizures induced by a GABA (gamma-aminobutyric acid) antagonist. These results indicate that NPY is not essential for certain feeding responses or leptin actions but is an important modulator of excitability in the central nervous system. PMID- 8632797 TI - Rat spermatogenesis in mouse testis. AB - Recently, transplantation of mouse donor spermatogonial stem cells from a fertile testis to an infertile recipient mouse testis was described. The donor cells established spermatogenesis in the seminiferous tubules of the host, and normal spermatozoa were produced. In the most successful transplants, the recipient mice were fertile and sired up to 80 per cent of progeny from donor cells. Here we examine the feasibility of transplanting spermatogonial stem cells from other species to the mouse seminiferous tubule to generate spermatogenesis. Marked testis cells from transgenic rats were transplanted to the testes of immunodeficient mice, and in all of 10 recipient mice (in 19 of 20 testes), rat spermatogenesis occurred. Epididymides of eight mice were examined, and the three from mice with the longest transplants (> or = 110 days) contained rat spermatozoa with normal morphology. The generation of rat spermatogenesis in mouse testes suggests that spermatogonial stem cells of many species could be transplanted, and opens the possibility of xenogeneic spermatogenesis for other species. PMID- 8632799 TI - Geometric determinants of the place fields of hippocampal neurons. AB - The human hippocampus has been implicated in memory, in particular episodic or declarative memory. In rats, hippocampal lesions cause selective spatial deficits, and hippocampal complex spike cells (place cells) exhibit spatially localized firing, suggesting a role in spatial memory, although broader functions have also been suggested. Here we report the identification of the environmental features controlling the location and shape of the receptive fields (place fields) of the place cells. This was done by recording from the same cell in four rectangular boxes that differed solely in the length of one or both sides. Most of our results are explained by a model in which the place field is formed by the summation of gaussian tuning curves, each oriented perpendicular to a box wall and peaked at a fixed distance from it. PMID- 8632798 TI - A gene complex acting downstream of dpp in Drosophila wing morphogenesis. AB - Localized expression of the transforming growth factor-beta (TGF-beta) homologue decapentaplegic (dpp) is crucial for Drosophila wing development. Here we show that spalt and spalt-related (sal and salr), two closely related genes that encode transcription factors, are expressed in response to dpp in a central territory of the wing imaginal disc, where they are required for the patterning of the wing. They are among the first identified elements that act downstream of dpp in wing development. The phenotypic consequences of misexpression of sal and salr suggest that an important outcome of dpp activity is the subdivision of the wing disc into territories smaller than lineage compartments, through the regulation of transcription-factor-encoding genes such as sal and salr. PMID- 8632800 TI - Associative, bidirectional modifications at the hippocampal mossy fibre-CA3 synapse. AB - Long-term potentiation (LTP) and long-term depression (LTD) are activity dependent changes in synaptic strength that may serve as the cellular mechanisms of information storage in the vertebrate brain. The mossy fibre-CA3 synapse displays NMDA (M-methyl-D-aspartate) receptor-independent forms of LTP and LTD that were thought to be non-associative. Here we report that the mossy fibre-CA3 synapse displays each of the known types of LTD in vivo, including associative, heterosynaptic and homosynaptic LTD. These types of LTD are induced when only two of the three conditions necessary for mossy fibre LTP induction are provided. Because some of these conditions can be provided by convergent CA3 afferents, each type of LTD can be induced in an associative manner, which suggests that LTD is involved in associative information storage. Similar to the induction of NMDA receptor-dependent LTD and LTP at other cortical synapses, mossy fibre LTD occurs when synaptic conditions are insufficient to induce LTP, and both LTP and LTD induction are influenced by previous synaptic activity, consistent with the view that common principles govern activity-dependent plasticity at cortical synapses. PMID- 8632802 TI - Cut2 proteolysis required for sister-chromatid seperation in fission yeast. AB - Although mitotic cyclins are well-known substrates for ubiquitin-mediated proteolysis at the metaphase-anaphase transition, their degradation is not essential for separation of sister chromatids; several lines of evidence suggest that proteolysis of other protein(s) is required, however. Here we report the anaphase-specific proteolysis of the Schizosaccharomyces pombe Cut2 protein, which is essential for sister-chromatid separation. Cut2 is located in the nucleus, where it is concentrated along the short metaphase spindle. The rapid degradation of Cut2 at anaphase requires its amino-terminal region and the activity of Cut9 (ref. 14), a component of the 20S cyclosome/anaphase-promoting complex (APC), which is necessary for cyclin destruction. Expression of non degradable Cut2 blocks sister-chromatid separation but not cell-cycle progression. This defect can be overcome by grafting the N terminus of cyclin B onto the truncated Cut2, demonstrating that the regulated proteolysis of Cut2 is essential for sister-chromatid separation. PMID- 8632803 TI - Ribozyme-catalysed amino-acid transfer reactions. AB - The 'RNA world' hypothesis proposes an early stage in the evolution of life in which both genomic and catalytic functions were fulfilled by RNA. The evolution of RNA-catalysed protein synthesis would have been a necessary step in the transition from such an RNA world to modern protein-dominated biology. For this to have been possible, RNA must be capable of catalysing amide-bond formation using acylated carrier RNA substrates as amino-acid donors. We have used in vitro selection and evolution to isolate ribozymes with acyl transferase activity from a pool of random RNA sequences. One of these acyl transferases with a 5'-amino group transfers an amino acid to itself in a reaction that we propose to be analogous to peptidyl transfer on the ribosome. PMID- 8632801 TI - Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. AB - The receptor-ligand pairs CD28-B7 and CD40-gp39 are essential for the initiation and amplification of T-cell-dependent immune responses. CD28-B7 interactions provide 'second signals' necessary for optimal T-cell activation and IL-2 production, whereas CD40-gp39 signals co-stimulate B-cell, macrophage, endothelial cell and T-cell activation. Nonetheless, blockade of either of these pathways alone is not sufficient to permit engraftment of highly immunogenic allografts. Here we report that simultaneous but not independent blockade of the CD28 and CD40 pathways effectively aborts T-cell clonal expansion in vitro and in vivo, promotes long-term survival of fully allogeneic skin grafts, and inhibits the development of chronic vascular rejection of primarily vascularized cardiac allografts. The requirement for simultaneous blockade of these pathways for effective inhibition of alloimmunity indicates that, although they are interrelated, the CD28 and CD40 pathways are critical independent regulators of T cell-dependent immune responses. PMID- 8632805 TI - More worries about pollution. PMID- 8632804 TI - Simplified hot start PCR. PMID- 8632806 TI - Creutzfeldt-Jakob researchers seek greater access to data on cases. PMID- 8632808 TI - Health agency avoids Congress's axe. PMID- 8632809 TI - Pollutants 'may harm fetal development'. PMID- 8632810 TI - California looks to science partnerships ... PMID- 8632807 TI - MITI turns up heat on research into thermophile genes. PMID- 8632811 TI - Japanese minister takes pay cut to atone for HIV contamination. PMID- 8632812 TI - Questionable judgement. PMID- 8632813 TI - The case for the passive voice. PMID- 8632814 TI - Pioneers of vaccination. PMID- 8632815 TI - School daze. PMID- 8632816 TI - Strengths and weaknesses in memory. PMID- 8632817 TI - Looking for the twilight zone. PMID- 8632819 TI - Plastid in human parasites. PMID- 8632818 TI - Agrin signals at the junction. PMID- 8632821 TI - Ancient DNA and island endemics. PMID- 8632820 TI - The birth of microsatellites. PMID- 8632822 TI - The history of the galaxies. AB - Astronomical observations now reach far enough back in time, in enough depth and detail, to reveal the history of galaxies since their formation. The early Universe contained a network of gas clouds that filled much of the space between the young galaxies, where stars were forming at a high rate. Since then, intergalactic space has been swept clean, and galaxies have continued to convert the dwindling supply of gas slow into stars. PMID- 8632823 TI - Role of the thrombin receptor in development and evidence for a second receptor. AB - Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, endothelial cells, leukocytes and mesenchymal cells. A G-protein coupled receptor that is proteolytically activated by thrombin is a target for drug development aimed at blocking thrombosis, inflammation and proliferation. Here we show that although disruption of the thrombin receptor (tr) gene in mice causes about half of the tr-/- embryos to die at embryonic day 9-10, half survive to become grossly normal adult mice with no bleeding diathesis. Strikingly, tr-/- platelets respond strongly to thrombin, whereas tr-/- fibroblasts lose their ability to respond to thrombin. We conclude that the thrombin receptor plays an unexpected role in embryonic development, suggesting a possible new function for the 'coagulation' proteases themselves. Moreover, a second platelet thrombin receptor exists, and different thrombin receptors have tissue-specific roles. This may allow development of therapeutics that will selectively block thrombin's different cellular actions. PMID- 8632824 TI - Speed of processing in the human visual system. AB - How long does it take for the human visual system to process a complex natural image? Subjectively, recognition of familiar objects and scenes appears to be virtually instantaneous, but measuring this processing time experimentally has proved difficult. Behavioural measures such as reaction times can be used, but these include not only visual processing but also the time required for response execution. However, event-related potentials (ERPs) can sometimes reveal signs of neural processing well before the motor output. Here we use a go/no-go categorization task in which subjects have to decide whether a previously unseen photograph, flashed on for just 20 ms, contains an animal. ERP analysis revealed a frontal negativity specific to no-go trials that develops roughly 150 ms after stimulus onset. We conclude that the visual processing needed to perform this highly demanding task can be achieved in under 150 ms. PMID- 8632825 TI - Target-cell-specific concentration of a metabotropic glutamate receptor in the presynaptic active zone. AB - The probability of synaptic neurotransmitter release from nerve terminals is regulated by presynaptic receptors responding to transmitters released from the same nerve terminal or from terminals of other neurons. The release of glutamate, the major excitatory neurotransmitter, is suppressed by presynaptic autoreceptors. Here we show that a metabotropic glutamate receptor (mGluR7) in the rat hippocampus is restricted to the presynaptic grid, the site of synaptic vesicle fusion. Pyramidal cell terminals presynaptic to mGluR1alpha-expressing interneurons have at least a ten-fold higher level of presynaptic mGluR7 than terminals making synapses with pyramidal cells and other types of interneuron. Distinct levels of mGluR7 are found at different synapses made by individual pyramidal axons or even single boutons. These results raise the possibility that presynaptic neurons could regulate the probability of transmitter release at individual synapses according to the postsynaptic target. PMID- 8632826 TI - Experience-dependent modification of synaptic plasticity in visual cortex. AB - In many regions of the cerebral cortex, Ca2+ influx through NMDA (N-methyl-D aspartate) sensitive glutamate receptors (NMDA receptors) can trigger two forms of synaptic plasticity: long-term depression (LTD) and long-term potentiation (LTP). LTD is induced by low levels of postsynaptic NMDA-receptor activation, for instance in response to low-frequency stimulation, whereas LTP is induced by the stronger activation that occurs following high-frequency stimulation. Theoretical studies have shown that the properties of synaptic LTD and LTP can account for many aspects of experience-dependent plasticity in the developing visual cortex, provided that the LTD-LTP crossover point (the modification threshold, theta(m)) varies as a function of the history of cortical activity. Here we provide direct experimental evidence that the value of theta(m) depends on sensory experience. We find in visual cortex of light-deprived rats that LTP is enhanced and LTD diminished over a range of stimulation frequencies, and that these effects can be reversed by as little as two days of light exposure. Our findings support the idea that a variable synaptic modification threshold allows synaptic weights in neural networks to achieve a stable equilibrium. PMID- 8632827 TI - Genetic modification of heterochromatic association and nuclear organization in Drosophila. AB - Heterochromatin is the highly compact, usually pericentromeric, region of eukaryotic chromosomes. Unlike the more gene-rich euchromatin, heterochromatin remains condensed during interphase, when it is sequestered to the periphery of the nucleus. Here we show, by using fluorescent in situ hybridization to interphase diploid nuclei of Drosophila, that the insertion of heterochromatin into a euchromatic gene, which results in position-effect variegation (PEV), also causes the aberrant association of the gene and its homologous copy with heterochromatin. In correlation with the gene's mutant variegating phenotype, the cytological association of the heterochromatic region is affected by chromosomal distance from heterochromatin and by genic modifiers of PEV. Proteins that are thought to be involved in the formation of heterochromatin can therefore influence the interphase nuclear position of a chromosomal region. This suggests that heterochromatin and proteins involved in its formation provide a structural framework for the interphase nucleus. PMID- 8632828 TI - Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5 bisphosphate. AB - Vinculin, a prominent cytoskeletal protein at cell-substrate adhesions (focal adhesions) and cell-cell adhesions (adherens junctions), interacts with other cytoskeletal proteins, including talin and actin. An intramolecular interaction between the head and tail domains of vinculin masks the binding sites for both proteins. The exposure of cryptic binding sites may be important for promoting focal adhesion assembly. Several agents that induce the formation of focal adhesions act through the GTP-binding protein Rho, which elevates phosphatidylinositol-4,5-bisphosphate (PtdInsP2) levels by activating phosphatidyl-inositol-4-phosphate-5-OH kinase (PtdIns-5-OH kinase). PtdInsP2 regulates several actin-binding proteins, including profilin, gelsolin and alpha actinin, and interacts with vinculin. Here we report that PtdInsP2 dissociates vinculin's head-tail interaction, unmasking its talin- and actin-binding sites. Microinjection of antibodies against PtdInsP2 inhibit assembly of stress fibres and focal adhesions. PMID- 8632829 TI - Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA. AB - The adenovirus L1 unit represents an example of an alternatively spliced precursor messenger (pre-mRNA) where on 5' splice can be jointed to one of two alternative 3' splice sites, producing the 52,55K or the IIIa mRNAs (Fig. 1a). Efficient usage of the distal IIIa 3' splice site requires late viral protein synthesis and is therefore confined to the late phase of virus infection. Here we show that, in extracts from uninfected cells, the classical SR proteins, which are essential splicing factors, inhibit IIIa pre-mRNA splicing by binding to an intronic repressor element and preventing recruitment of the U2 small nuclear ribonucleoprotein particle to the spliceosome. We further show that the viral repressor element has splicing-enhancer activity when appropriately placed in the pre-mRNA. Together, our results demonstrate that SR proteins function as activators or repressors of splicing depending on where on the pre-mRNA they bind. PMID- 8632830 TI - Products and services. PMID- 8632831 TI - The response of neurons and microglia to blast injury in the rat brain. AB - Rats subjected to a single non-penetrative blast were examined for possible neuronal damage and glial reaction by immunohistochemistry and electron microscopy. The most dramatic feature in rats killed between 1 and 14 days after the blast was the widespread response of microglial cells in various parts of the brain in which the cells were hypertrophied and their surface antigens, like complement type three receptors (CR3), were upregulated. The blast wave also induced the vigorous expression of major histocompatibility complex (MHC) class I and II (Ia) antigen. In rats killed 21 days after the blast, the elevated immunoreactivity of microglia had subsided and at 28 days both the microglial external morphology and immunoreactivity were comparable to those of normal animals. In rats killed 4-7 days after the blast, the neurons in the cerebral and cerebellar cortex appeared normal except for the occurrence of some 'darkened' dendrites. The incidence of 'darkened' dendrites was most common in rats killed at day 14 but they were absent at 21 and 28 days. Microglial cells were closely associated with some of the 'darkened' dendrites. Results in this study show that a non-penetrative blast in rats provokes a widespread microglial activation suggesting increased endocytosis and immunological responses. However, it remains uncertain whether such a drastic response was a direct activation of the cells by the blast wave or elicited indirectly by some chemical factors released from the damaged brain tissues. PMID- 8632832 TI - Superoxide anions in the pathogenesis of talc-induced cerebral vasocontraction. AB - We have recently reported that sustained contraction of the canine basilar artery induced by the intrathecal injection of talc (crystallized hydrous magnesium silicate) mimicked delayed vasospasm following subarachnoid haemorrhage. The present study aims to examine the pathomechanism underlying talc-induced vasocontraction, from the viewpoint of free radical theory, which has been established as a cause of delayed vasospasm. We estimated the effects of a prolonged intrathecal infusion of human recombinant Cu/Zn superoxide dismutase (hr SOD) on the contraction of the basilar artery caused by the intrathecal injection of talc in beagle dogs, which were assigned to the three groups: G1, sham operation with saline treatment; G2, talc injection with saline treatment; and G3, talc injection with 2 ml of hr SOD (7 x 10(4) U/ml) treatment. Talc administration resulted in the reduction in the angiographic calibre of the basilar artery by 63 and 61% on days 3 and 7 (G2). The treatment with hr SOD (G3) led to a significant attenuation of talc-induced contraction of the basilar artery on days 3 (P < 0.05 vs. G2) and 7 (P < 0.05 vs. G2). In the basilar artery wall of days 3 and 7 in G2, pathological changes such as myonecrosis, cytoplasmic vacuolation and detached intercellular junctions were observed. However, these pathological changes almost disappeared in G3. The present findings suggest that superoxide anions may initiate and/or mediate talc-induced vasocontraction and subsequent structural damage of the basilar artery. PMID- 8632833 TI - Long-term culture of organotypic multicellular glioma spheroids: a good culture model for studying gliomas. AB - Gliomas, as well as other solid tumours, contain tumour stroma composed of connective tissue, macrophages, capillaries and other non-cellular constituents. Therefore, a homogeneous culture of tumour cells alone, as is often used as a culture model for gliomas, is not ideal to study all aspects of gliomas. In the present study we describe an alternative culture model, i.e. organotypic multicellular spheroids (OMS), that histologically closely resembles the tumour in vivo. Glioma explants, obtained at surgery from five patients, were cultured on agarose to form OMS, which were cultured for up to 16 weeks. At regular intervals, OMS were fixed and histological and immunocytochemical analyses were carried out. The histology as well as the immunocytochemical characteristics of the OMS proved to be almost unchanged after a culture period of 16 weeks. In contrast to monolayer cultures, glial fibrillary acidic protein (GFAP) expression in the OMS is preserved after 16 weeks of culture. However, in OMS from three out of five patients, small GFAP-negative cells appeared in the outer cell layers between 1 and 2 weeks of culture. Furthermore, after about 6 weeks of culture, the capillaries disappeared from the OMS. After prolonged culture, tumour cell heterogeneity, the cellular composition, and the histology of the OMS still closely resembled the tumour in vivo. It is suggested that OMS provide a good long-term culture model for the study of gliomas. PMID- 8632834 TI - Cytolytic effects of autologous lymphokine-activated killer cells on organotypic multicellular spheroids of gliomas in vitro. AB - Knowledge about lymphokine-activated killer (LAK) cell infiltration and LAK cell cytotoxicity is essential to improve the effectiveness of LAK cell therapy against gliomas. In the present study, organotypic multicellular spheroids (OMS) of glioma tissue were used as a culture model to study the effects of LAK cells on gliomas. Compared to tumour cell lines and spheroids derived from tumour cell lines, OMS have several advantages with respect to preservation of tumour cell heterogeneity and the maintenance of the tumour architecture, e.g. capillaries and extracellular matrix. Four glioma specimens, obtained at surgery, were cultured directly on agarose to form OMS, which were then co-cultured with either autologous LAK cells or autologous non-activated peripheral blood lymphocytes (PBLs). After various time periods of co-cultivation, the OMS were fixed and examined both histologically and immunocytochemically. The present results showed that LAK cells infiltrated the OMS completely within 24 h of co-cultivation and severe cellular damage was observed, whereas PBLs infiltrated the OMS poorly and there was only marginal cellular damage. The present study indicates that OMS of gliomas provide an experimental model to investigate the infiltration and cytotoxicity of LAK cells on glioma tissue in vitro. PMID- 8632836 TI - Pathology of subcortical visual centres in relation to cortical degeneration in Alzheimer's disease. AB - Subcortical visual centres such as the lateral geniculate nucleus, the lateral inferior pulvinar and the superior colliculus, together with the primary visual cortex and its adjacent white matter, were studied in 12 Alzheimer brains and five age-matched controls. The periodic acid methenamine technique was used for the demonstration of senile plaques and the Gallyas technique for neurofibrillary tangles and neuritic threads in the neuropil. In the lateral geniculate nucleus and inferior pulvinar, the presence of periodic acid methenamine-positive senile plaques was observed in variable numbers in all Alzheimer cases. In the lateral geniculate nucleus, senile plaques were encountered more often in parvocellular than in magnocellular layers, in the interlaminar zones, in the optic radiation and in the adjacent pre-geniculate nucleus. Gallyas staining did not reveal any neurofibrillary tangles, neuritic threads or neuritic plaques, meaning that in this thalamic region there are mainly amyloid deposits without neuritic degeneration. In the superior colliculus both amyloid and neuritic plaques, as well as neurofibrillary tangles and neuritic threads were encountered in the superficial and deep layers. In the primary visual cortex, all types of senile plaques were observed as well as a rather high number of neurofibrillary lesions in pyramidal neurons, mainly in layers 5 and 6, but also in several types of non pyramidal neurons. In the underlying white matter there was a morphologically heterogeneous population of neurofibrillary tangle-bearing neurons and a considerable number of threads representing degenerating axons, suggesting that degeneration could follow corticosubcortical connections. These data demonstrate that lesions in the primary visual structures and pathways are more prevalent than previously observed and could partly explain the visual disturbances in Alzheimer's disease. PMID- 8632835 TI - Transthyretin expression in medulloblastomas and medulloblastoma cell lines. AB - Transthyretin is a protein crucial to the transport of lipophilic molecules such as thyroid hormones and retinoids. In the central nervous system, large amounts of transthyretin are synthesized by the choroid plexus and are secreted into the cerebrospinal fluid. The choroid plexus is the only site of transthyretin synthesis in the brain. Transthyretin is expressed by most benign and malignant choroid plexus tumours while gliomas and meningiomas do not express transthyretin. Other major sites of transthyretin synthesis are the retinal pigment epithelium and hepatocytes. Medulloblastoma is the prototypical primitive neuroectodermal tumour of the cerebellum and can show multiple lines of differentiation, including the expression of retinal markers. In this study, we examined transthyretin expression both at the RNA and protein level in four medulloblastomas and six medulloblastoma cell lines using Northern and Western blot analysis, reverse transcription polymerase chain reaction (PCR), RNA in situ hybridization, and immunohistochemistry. All four medulloblastomas and five of the six medulloblastoma cell lines expressed transthyretin-mRNA as demonstrated by reverse PCR and in situ hybridization while three medulloblastomas and one cell line were positive on Northern blot. The medulloblastoma with the most abundant RNA expression was transthyretin-immunoreactive on cryosections and the medulloblastoma cell line that was positive on Northern blot also expressed transthyretin at levels detectable by Western blot. No transthyretin immunoreactivity was seen in 16 additional medulloblastomas studied on paraffin sections. These findings indicate that low-level expression of transthyretin-mRNA is common in medulloblastomas and medulloblastoma cell lines. Expression of transthyretin protein occurs rarely but can reach significant levels. Transthyretin expression in medulloblastoma is consistent with retinal pigment epithelium differentiation in medulloblastomas and reflects their pluripotential nature. Furthermore, the potential for transthyretin-immunoreactivity in medulloblastoma should be kept in mind when performing immunohistochemical studies on poorly differentiated cerebellar tumours. PMID- 8632837 TI - Experimental induction of corpora amylacea-like inclusions in rat astroglia. AB - Corpora amylacea (CA) are glycoproteinaceous inclusions that accumulate in the human central nervous system during normal ageing, and to an even greater extent in Alzheimer's disease and other neurodegenerative disorders. They are particularly prominent in subpial and subependymal regions, and are most commonly located within astrocytes and their processes. We previously demonstrated that human CA share many tinctorial and histochemical properties in common with Gomori positive cytoplasmic granules which accumulate in periventricular astrocytes of the ageing vertebrate brain and in rat astroglial cultures exposed to the sulphydryl agent, cysteamine (CSH). In the present study, long-term exposure of neonatal rat astrocyte cultures to CSH resulted in the formation of large spherical, PAS-positive cytoplasmic inclusions which are highly reminiscent of, if not identical to, human CA. As in the case of human CA and Gomori-positive astrocyte granules, the CSH-induced CA-like inclusions exhibit non-enzymatic peroxidase activity and consistent immunolabelling with antibodies directed against the mitochondrial protein, sulphite oxidase. Taken together, our findings suggest that progressive mitochondrial damage and macroautophagy play an important role in the biogenesis of CA (and Gomori-positive granules) in astrocytes of the ageing periventricular brain. PMID- 8632838 TI - Increase of mitochondria in vasa nervorum of cases with mitochondrial myopathy, Kearns-Sayre syndrome, progressive external ophthalmoplegia and MELAS. AB - Previous studies on patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) revealed accumulations of mitochondria in endothelial cells, smooth muscle cells of pial arterioles, and small intracerebral arteries up to 250 microns in diameter; in pericytes of capillaries, endothelial cells, and smooth muscle cells of small blood vessels in skeletal muscle; and according to preliminary results also in endothelial and smooth muscle cells of capillaries and arterioles in sural nerves. These mitochondria do not show the prominent paracrystalline inclusions which are seen in striated muscle fibres and led to the identification of this group of disorders. To corroborate our preliminary findings in peripheral nerves, additional cases have been evaluated morphometrically by electron microscopy including cases in which mitochondrial DNA (mtDNA) mutations have been identified. In fact, an increase of the mean number and an enlargement of the mean cross-sectional area of mitochondria was noted in endothelial and smooth muscle cells of endoneurial and epineurial arterioles, and in endothelial cells and in pericytes of capillaries in sural nerves of the 20 cases with mitochondrial disorders studied. This increase was statistically significant compared to the control group. However, due to heteroplasmia, which is a common feature in mitochondrial disorders, and because of the limited number of measurable blood vessels and cases, no significant differences could be detected between the various types of mitochondrial diseases which were characterized by different point mutations or deletions of mtDNA. Our findings suggest that the mitochondria play a significant role in the pathogenesis not only of myopathic and encephalopathic symptoms, but also in the pathogenesis of peripheral neuropathy which appears to be regularly associated with mitochondrial disorders. PMID- 8632840 TI - Medication-error reporting. Banish a system that blames. PMID- 8632841 TI - Comments that should stay "off the record". PMID- 8632839 TI - Verifying NG tube placement. PMID- 8632842 TI - Medicating tube-fed patients. PMID- 8632843 TI - Managing body-substance exposures. PMID- 8632844 TI - Applying wrist restraints. PMID- 8632845 TI - Compazine. PMID- 8632846 TI - When your patient needs a contrast dye injection. PMID- 8632847 TI - Learning how to cope with tragedy. PMID- 8632848 TI - Written patient-education materials. PMID- 8632849 TI - Actionstat. Bowel obstruction. PMID- 8632850 TI - Your role in a code blue. PMID- 8632851 TI - Analgesics: mapping out pain relief. PMID- 8632852 TI - Full partner in care. PMID- 8632853 TI - Caring for patients with liver cancer. PMID- 8632855 TI - Putting the best foot forward. PMID- 8632857 TI - How to collaborate with nurse practitioners. PMID- 8632856 TI - Witness to success. PMID- 8632854 TI - Fostering farewell: giving children the chance to let go. PMID- 8632858 TI - Avoiding the i.m. route for analgesic administration. PMID- 8632859 TI - Self-test. Understanding infectious and neoplastic disorders. PMID- 8632860 TI - Myths & facts ... about birth control. PMID- 8632861 TI - Bill's birthday gift. PMID- 8632862 TI - Medicare's POS option. AB - Guidelines recently issued by the Health Care Financing Administration (HCFA) explicitly allow health maintenance organizations (HMOs) with a Medicare risk contract to sell a point-of-service (POS) benefit to their Medicare members. For an additional charge, these HMO members will now be allowed to go out-of-network for certain medical services. PMID- 8632865 TI - Will the Internet supplant community health networks? AB - As issues of security, accountability and access are resolved, the Internet will act as the central infrastructure for a global as well as a community health information network. It will help nurses extend their services to the community and to educate specific patient populations. PMID- 8632864 TI - Legal issues in long-term care--Part II. AB - A number of cases involving long-term care (LTC) employees and facilities involve actions that are intentional. Neglect and abuse of the elderly, reckless misconduct and reporting these incidents are discussed. PMID- 8632863 TI - Accreditation, managed care and subacute care. AB - Accreditation promotes continuous quality improvement of systems that affects the quality of patient care delivered, attains desirable clinical and financial outcomes and strategically positions the subacute care provider in the rapidly changing health care delivery system. Thus, as consumers and purchasers of health care plans continue to demand quality care with the best possible outcomes at significant cost savings, accreditation may become the panacea of the future. PMID- 8632866 TI - Establishing an NP-staffed minor emergency area. AB - A nurse practitioner-staffed minor emergency area provides high quality care for approximately 21% of their adult emergency department population. Patients are triaged based on set criteria, allowing for short treatment times. The physical layout, triage criteria and the NPs' scope of practice in the Level I trauma center's ED are detailed. PMID- 8632867 TI - Census variation staffing. AB - A Census Variation Staffing (CVS) model has been used successfully on all nursing units for 4 years. Historical data and nursing hours per patient day (NHPPD) are used to determine the staffing needs of each unit on a daily, shift-by-shift basis. CVS has been heralded as the single largest factor in the hospital's consistent profitability--averaging annual savings of $485,100. PMID- 8632868 TI - Redesigning the RN and NA roles. AB - To ensure optimal utilization of RNs, the nursing care delivery system in a surgical nursing division assessed the tasks that could be delegated to ancillary personnel. Nursing assistants completed competency-based training and after six months, several positive outcomes were evident: dramatic decreases in overtime, improved patient care and increased patient and staff satisfaction. PMID- 8632869 TI - Nurse-managed primary care. AB - A nurse-managed primary care community health center, Abbottsford Community Health Center (ACHC), provides highly accessible, quality, cost-effective care to the residents of the Abbottsford Community. Data revealed that, in comparison to the aggregate family practices in an HMO, ACHC has achieved better outcomes in areas such as emergency department visits, inpatient days and client care costs. The nurse manager's roles and responsibility are critical in achieving this success. PMID- 8632870 TI - CPOM: alleviating the demand for ICU beds. AB - Technology is adapting to health care's changing environment as more acutely ill patients, including those with respiratory risks, are being placed on the general care units. Centralized noninvasive pulse oximetry, a method to measure oxygen saturation, provides a relatively inexpensive, efficient means to serve a broader spectrum of patients. PMID- 8632872 TI - Effect of a professional practice model on autonomy, job satisfaction and turnover. AB - Dissatisfaction and rapid turnover of registered nurses (RNs) challenge nurse administrators. The professional practice model (PPM) can increase the amount of personal control nurses have over their work. Use of a PPM allows innovation, promotes collegial relationships and emphasizes personal responsibility. In this study, facilitating an autonomous climate for RN practice resulted in increased job satisfaction and decreased. PMID- 8632871 TI - Redesigning surgical services. AB - As a result of changing health care reimbursement, a surgical services management team re-evaluated how to do business. Roles of the team members were revised; clinical standards and customer satisfaction also were examined. PMID- 8632874 TI - A service manager model: instituting case management. AB - Using a service manager model as opposed to a traditional nurse case manager archetype fosters multidisciplinary team interaction. Benefits of this approach to the hospital, the staff and the patients are identified. PMID- 8632875 TI - The Caux Round Table principles for business. AB - In 1994, the Caux Round Table in Switzerland developed what is believed to be the first international code of business ethics. Business leaders from Japan, Europe and the United States collaborated to create a set of principles rooted in two basic ideals: kyosei and human dignity. Because of its importance and its applicability to the conduct of today's health care businesses, this entire column is devoted to reprinting these "Principles for Business." PMID- 8632876 TI - Support surfaces--a technology review. AB - Technology assessment performed on the wide range of clinical support systems available highlights the potential difficulties encountered when already utilized clinical therapies become subject to careful scientific scrutiny. The technology assessment committee must look beyond tacitly accepted indications for technology use and instead focus their evaluations on current, published clinical outcomes research. Where this latter clinical research is lacking, the technology assessment committee's goal is to balance the best available evidence of clinical safety and efficacy of a technology against the relative costs of that therapy. PMID- 8632873 TI - Building a case management model in a small community hospital. AB - Facilities moving toward case management can help promote success by borrowing from the experience of others and then making necessary modifications to incorporate the unique needs and characteristics of each institution. The author has used this approach in developing a program well suited for application to a small community hospital. PMID- 8632877 TI - A smaller institution's answer to nursing consultants. PMID- 8632878 TI - Agency nursing: one hospital's experience. PMID- 8632880 TI - The abandonment of the patient. PMID- 8632879 TI - Managing multicultural patient care issues. PMID- 8632882 TI - Nursing report cards needed for acute care settings. PMID- 8632881 TI - Swing shift nurses: more heart attacks. PMID- 8632883 TI - Turning conflict into cooperation: an independent study (continuing education credit). PMID- 8632884 TI - Are you ready for the next legislative session? PMID- 8632887 TI - The school nurse. PMID- 8632886 TI - What do you bring to the profession of nursing? PMID- 8632885 TI - Integrating folk healing systems with contemporary nursing systems: an application of Leininger's theory. PMID- 8632888 TI - Conflict and nursing management. PMID- 8632889 TI - The Cooperative Cardiovascular Project (CCP). PMID- 8632890 TI - Home care: the future. PMID- 8632893 TI - Immortalization of human mammary epithelial cells transfected with mutant p53 (273his). AB - Normal human breast epithelial cells were transfected with expression vectors containing the p53 gene mutated at either codon 143, 175, 248 or 273, or by infection with a recombinant retroviral vector containing the p53 gene mutated at codons 143, 175, 248, or 273. The breast epithelial cells were monitored for extension of in vitro lifespan and immortalization. Expression of some, but not all, p53 mutants resulted in an extension of in vitro lifespan. Experiments with the p53 temperature sensitive mutant 143ala revealed that at 32 degrees C, the nonpermissive temperature, the growth of breast epithelial cells was inhibited. At 37 degrees C, the mutant conformation, there was increased proliferation of cells, resulting in extension of in vitro lifespan. Breast epithelial cells expressing p53 mutant 273his maintained DNA binding and transcriptional activities and one clone immortalized after a period of growth arrest (crisis). The progression of this immortalization event was characterized by the reactivation of telomerase using the telomeric repeat amplification protocol (TRAP), and terminal restriction fragment analysis (TRF). This is the first reported immortalization of human mammary epithelial cells transfected with a mutant 53. PMID- 8632891 TI - Little things mean a lot--my first year as a nurse. PMID- 8632892 TI - Tob, a novel protein that interacts with p185erbB2, is associated with anti proliferative activity. AB - We have molecularly cloned a cDNA for a novel protein termed Tob (Transducer of ErbB-2) that interacts with the c-erbB-2 gene product p185erbB2. Nucleotide sequencing reveals that the Tob protein is a 45 kDa protein that does not contain either SH2 (Src Homology 2) or SH3 domain but is homologous to the previously characterized anti-proliferative gene product BTG-1 at its amino-terminal half. The carboxyl-terminal half of Tob is characterized by the presence of a sequence rich in proline and glutamine and shows no homology to known proteins. Like BTG 1, exogenously expressed Tob is able to suppress growth of NIH3T3 cells, but the growth suppression is hampered by the presence of kinase-active p185erbB2. By using the GST-Tob protein that contains either full length or amino-terminal half of Tob, we show that the carboxyl-terminal half of Tob is relevant to its interaction with p185erbB2. Furthermore, we could co-immunoprecipitate the Tob protein with anti-ErbB-2 antibody, and reciprocally the p185erbB2 with anti-Tob antibodies. These data suggest that p185erbB2 negatively regulates the Tob mediated anti-proliferative pathway through its interaction with Tob, resulting possibly in growth stimulation by p185erbB2. Finally, expression of the Tob mRNA is observed in various cell types and is not correlated with expression of c-erbB 2, suggesting that other receptor-type protein-tyrosine kinases are also involved in the Tob-mediated regulation of cell growth. PMID- 8632894 TI - Reduced phosphotyrosine binding by the v-Src SH2 domain is compatible with wild type transformation. AB - The SH2 domain of v-Src binds phosphotyrosyl-proteins in vivo and in vitro. The function of this domain is necessary for transformation of Rat-2 cells and for morphologically wild-type transformation of chicken embryo fibroblasts (CEF). The phosphate group of phosphotyrosine interacts directly with a conserved arginine residue in the FLVRES motif of the SH2 domain, R175 in v-Src. To examine the role of phosphotyrosine binding in transformation by v-Src, we have characterized the effects of R175 mutations on the transforming ability of v-Src and on the interaction of the v-Src SH2 domain with phosphotyrosyl-proteins. The R175H mutation, and to a lesser extent the R175K mutation, reduced but did not eliminate the binding of phosphotyrosyl-proteins to the v-Src SH2 domain. However neither mutation affected transformation of CEF or Rat-2 cells by v-Src and neither mutation resulted in major changes in the level or pattern of protein tyrosine phosphorylation in transformed CEF. In contrast, the R175E mutant of v Src induced fusiform transformation of CEF and failed to transform Rat-2 cells; the mutant SH2 domain was insoluble when expressed in bacteria, suggesting that the R175E mutation disrupts the structure of the v-Src SH2 domain. We conclude that, although the Arg residue in the FLVRES motif is invariant in most if not all SH2 domains, at position 175 in the v-Src SH2 domain residues other than arginine can support the binding of phosphotyrosyl-proteins, albeit at reduced levels. Furthermore under the expression conditions normally used, that is when v Src is expressed under the control of a retroviral LTR, the reduced binding of phosphotyrosyl-proteins is compatible with wild-type transformation. PMID- 8632895 TI - A 400 kb novel deletion unit centromeric to the BRCA1 gene in sporadic epithelial ovarian cancer. AB - Allelic deletions on chromosome 17q21 in sporadic ovarian cancer are common, suggesting that inactivation of a tumor suppressor gene(s) in that region may be important for the etiology of these tumors. The recently identified BRCA1 gene on 17q21, involved in the development of familial breast/ovarian cancer, could be a candidate. However, inactivating mutations on BRCA1 in sporadic ovarian cancer has been rarely described. Furthermore, the potential relationship of BRCA1 gene to ovarian tumors of borderline malignancy remains also unclear. We constructed a highly detailed deletion map of chromosome 17q21 based on PCR amplification of eight polymorphic tandem repeat markers in a 650 kb area including three BRCA1 intragenic markers. DNA from 52 sporadic ovarian cancers and 26 borderline tumors, together with their corresponding normal control tissues were used. Only one borderline tumor showed loss of heterozygosity at one marker, whereas 65% of invasive ovarian cancers displayed allelic loss in at least one of the markers studied. A common deletion unit, located approximately 60kb centromeric to BRCA1, was revealed. These results suggest that inactivation of the BRCA1 gene may not be responsible for the development of borderline ovarian tumors and that another tumor suppressor gene, located centromeric to the BRCA1 gene, may play a role in sporadic ovarian cancer development. PMID- 8632896 TI - Isolation and characterization of a novel gene expressed in multiple cancers. AB - Using differential display method, we have isolated and characterized a novel gene, N8, encoding an approximately 24 kDa protein. It is located on human chromosome 8q13 region. N8 gene is expressed at high levels in tumor derived cell lines from multiple cancers. It is also expressed at higher levels in lung tumors than normal lung tissue. N8 is also differentially expressed in fetal and adult tissues. In adult, N8 is expressed at high levels in brain, kidney, prostate, pancreas and intestine and at very low levels in lung, liver, hematopoietic cells and gonads. During murine embryonic development N8 is expressed in the epithelium of the intestine, stomach, olfactory epithelium, neuronal layers of retina, kidney and salivary gland. Taken together, these results suggest that N8 may play different roles during embryogenesis and in the adult animals. PMID- 8632897 TI - 17 beta-Estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation. AB - HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF 7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen responsive signal transduction pathways that converge on these enzymes. PMID- 8632898 TI - Human epidermal cancer and accompanying precursors have identical p53 mutations different from p53 mutations in adjacent areas of clonally expanded non neoplastic keratinocytes. AB - Microdissection of biopsies with sequencing of exons 4-8 of the p53 gene permitted precise morphological identification of correlation between mutations and/or loss of heterozygosity, immunoreactivty of p53 and type of squamous neoplasia. Seventy-two specimens from ten lesions of sun-exposed sites including normal epidermis were analysed. Irrespective of p53 immunoreactivity and morphological grade dysplasia, in situ or invasive cancer, in each case, carried the identical mutation indicating that invasive skin cancer and its precursors derive from the same original neoplastic clone. Additionally, morphologically normal epidermis showed some sharply demarcated immunoreactive areas. These never had the same p53 mutation as that of the adjacent tumor, indicating that their mutations were separate events and ruling them out as common precursors of cancer. Non-immunoreactive normal epidermis did not show p53 mutations. Our findings indicate that a large fraction of keratinocytes in sun-exposed human skin carry mutations of p53 and suggest that at least two options exist for such cells (i) innocuous clonal expansion with preserved morphology and normal differentiation or (ii) malignant transformation with the p53 mutation as an early event. Suggestive evidence existed that the p53 mutations were qualitatively different in the two respective groups of lesions. PMID- 8632899 TI - Inhibition of oncogene-mediated transformation by ectopic expression of p21Waf1 in NIH3T3 cells. AB - The p53-regulated p21Waf1 protein is a universal inhibitor of cyclin-dependent kinases (CDKs). To study the potential tumor-suppressive properties of CDK inhibitors, the ability of p21Waf1 to interfere with oncogene-mediated cellular transformation was analysed in the NIH3T3 cell system. Cotransfection of waf1 together with activated ras or several other oncogenes into NIH3T3 cells potently inhibited the formation of transformed foci in a dose-dependent manner. Expression of the CDK-binding N-terminal half of p21Waf1 (N-p21Waf1) was necessary and sufficient to inhibit Ras-induced focus formation. In contrast, expression of the C-terminal domain (C-p21Waf1) had no effect on Ras-induced focus formation. Immunofluorescence analysis revealed that ectopically expressed p21Waf1 and C-p21Waf1 were localized in the nucleus, while N-p21Waf1 was found in the cytoplasm, with the tendency to accumulate around the nuclear membrane. Surprisingly, stable NIH3T3 transfectants expressing ectopic p21Waf1 grew at the same rate and displayed similar cell cycle distribution as NIH3T3 cells transfected with the same vector containing no insert. However, ectopic p21Waf1 expression did inhibit Ras-mediated anchorage-independent colony formation, indicating that p21Waf1 can selectively interfere with oncogene-mediated transformation without affecting NIH3T3 cell growth, at least at the levels of p21Waf1 expression achieved in these experiments. Transient transfection of waf1 into NIH3T3 cells inhibited Ras-induced transcription from a E2F-responsive element but not from a serum-responsive element, indicating that p21Waf1 acts downstream of early transcriptional events induced by Ras but upstream of E2F controlled gene transcription. These results provide evidence that p21Waf1 potently suppresses oncogene-mediated cellular transformation of NIH3T3 cells and that it may do so by inhibiting E2F-driven transcription of S phase genes. PMID- 8632900 TI - Ras-independent activation of Rel-family transcription factors by UVB and TPA in cultured keratinocytes. AB - UV irradiation of mammalian cells results in the activation of transcription factors which mediate induction of early response genes designed to repair and minimise the damage sustained by the cell. Evidence from studies in HeLa cells suggest that UVC regulates NF-kappa B activity via tyrosine kinases and activation of Ras and Raf kinase. In this study we have used a previously characterized TPA-responsive element (VLTRE) that binds Rel/NF-kappa B proteins and a Ras-responsive element (B10 RRE) to analyse the signalling pathway in UVB stimulated gene transcription in cultured keratinocytes. We demonstrate that the tumour promoters TPA and UVB use different signalling intermediates to activate different sets of Rel/NF-kappa B proteins. UVB transactivation is independent of PKC activity but dependent on tyrosine kinase activity where was TPA stimulation requires PKC but not tyrosine kinase activity. Furthermore, neither UVB- nor TPA transactivation is mediated through p21 Ras but both stimuli are dependent on a functional Raf protein. A constitutively active Raf-1 kinase however, was unable to induce transactivation through VLTRE. Thus, Raf has an essential but permissive role in UVB activation of Rel proteins. These findings demonstrate that keratinocytes contain a novel Ras-independent pathway for induction of Rel mediated transcription. PMID- 8632901 TI - Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene. AB - We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein. PMID- 8632902 TI - Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2. AB - We recently showed that EGF and anisomycin activate two kinases, p45 and p55, whose distinguishing feature is that their detection in in-gel kinase assays is enhanced by copolymerised poly-Glu/Tyr or poly-Glu/Phe (Cano E, Hazzalin CA and Mahadevan LC, Mol. Cell. Biol., 20:117-121). Their activation characteristics and sizes are strikingly similar to those of JNK/SAPKs, which are also strongly activated by anisomycin. However, we show here that p45 and p55 are not JNK/SAPKs but murine forms of MAPKAP kinase-2 because: (i) Detection of immunoprecipitated JNK/SAPKs is completely dependent on the presence of c-Jun as substrate in the in gel kinase assays, whereas detection of p45 and p55 is not. (ii) Detection of p45 and p55 activity is enhanced by the presence of poly-Glu/Tyr or poly-Glu/Phe, whereas JNK/SAPKs are not detectable under these conditions. (iii) Although the sizes of the murine JNK/SAPKs and MAPKAP K-2 are similar, human JNK/SAPKs migrate at 45 and 55 kDa whereas human MAPKAP K-2 migrates at 50 kDa; the poly-Glu/Tyr enhanced activity in human cells migrates at 50 KDa. (iv) Purified rabbit muscle MAPKAP K-2 is detectable as two bands of activity on in-gel kinase assays and their detection is enhanced by poly-Glu/Tyr. (v) Finally, the anisomycin activated poly-Glu/Tyr-enhanced p45 and p55 kinases can be immunoprecipitated from murine cells using an anti-MAPKAP K-2 antibody. Thus, EGF- and anisomycin activated p45 and p55 are not JNK/SAPKs but MAPKAP K-2, implying that both these agents activate the p38/RK MAP kinase cascade. PMID- 8632904 TI - TATA-dependent repression of human immunodeficiency virus type-1 transcription by the adenovirus E1A 243R oncoprotein. AB - Human adenovirus E1A oncoprotein activates or represses transcription from a variety of viral and cellular promoters by several complex mechanisms. The E1A products, 289R and 243R, have differential effects on transcription directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Previous reports indicate that repression of HIV-1 LTR-directed gene expression by E1A 243R is mediated through the kappa B enhancer elements located between nucleotides -105 and -82 relative to the transcription initiation start site (+1). Results from this study suggest a novel mechanism for transcriptional repression of the HIV-1 LTR by E1A 243R that is enhancer-independent and that is mediated through basal HIV-1 promoter elements. Transient expression assays, in which 5'-truncated or site-directed mutant HIV-1 LTR-CAT reporters were tested for their response to repression mediated by wild-type or mutant 243R, demonstrate that LTR sequences upstream of -31 relative to the transcription initiation start site (+1) and inclusive of the enhancer elements are dispensable for 243R-mediated repression. The ability of 243R to repress HIV-1 basal promoter activity requires both an intact N-terminus of E1A 243R and the TATA element within the HIV-1 promoter. These results support a novel mechanism for E1A 243R induced transcriptional repression that is enhancer-independent and that targets directly the general transcription machinery. PMID- 8632903 TI - The human tumour suppressor gene p53 is alternatively spliced in normal cells. AB - Alternative splicing affecting the p53 carboxy-terminus has previously been described in mouse but not in normal human cells. We report here the detection in normal human lymphocytes of an alternatively spliced form of human p53 mRNA containing an additional 133 bp exon derived from intron 9. This splice variant encodes a truncated protein of 341 amino-acids including 10 new amino-acids derived from the novel exon. The truncated protein, which lacks part of the p53 tetramerization domain, fails to bind DNA in vitro and has a transcriptional defect in vivo in both yeast and mammalian cells. Quantitative RT-PCR experiments suggest that the alternatively spliced form is only present in significant amounts in quiescent cells. Considering the numerous functions ascribed to the carboxy-terminus of the p53 protein, this splice variant may have important implications for the biological role of p53 in normal cells. PMID- 8632905 TI - Inhibition of growth of estrogen receptor positive and estrogen receptor negative breast cancer cells in culture by AA-etherA, a stable 2-5A derivative. AB - The design, chemical synthesis and biological activities of a nuclease-resistant, nontoxic bioactive 2-5A derivative, AA-etherA [i.e., adenylyl-(2'-5')-adenylyl (2'-2")-9-[(2'-hydroxyethoxy)-methyl]adenine], are described as a new approach to the inhibition of breast cancer cell growth. AA-etherA inhibits DNA replication and cell division of both estrogen receptor positive (MCF-7) and estrogen receptor negative (BT-20) breast cancer cells in culture in a dose-dependent manner. Maximal inhibition in MCF-7 and BT-20 cells was obtained with 100 microM AA-etherA after four days of treatment, with an GI50 of 58 and 37 microM, respectively. AA-etherA is stable in the cytoplasm. Treated cells accumulate within the late G1/early S phase of the cell cycle and then progress only very slowly through S phase. AA-etherA does not activate RNase L, as do 2-5A and other 2-5A derivatives, nor does it increase p68 kinase (PKR) content of the cells. High resolution, two-dimensional protein gel electrophoresis reveals twofold or greater inhibition of synthesis of 92 proteins out of 682 proteins that were reproducibly detected as high quality spots with average rates of synthesis of > or = 20 p.p.m. in untreated cells. The specificity of the effects of AA-etherA on select proteins and its failure to activate RNase L indicate that AA-etherA does not act through a general effect on mRNA translation or stability, but rather inhibits cell proliferation through a block to DNA replication, with a concommitant reduction in the synthesis of specific proteins, some of which may be required for cell cycle transit. Two likely targets to account for the AA etherA inhibition of DNA replication are DNA topoisomerase I, which is inhibited by AA-etherA in other cell lines, and thymidine kinase, which could be inhibited in a manner similar to the effect of acyclovir. These data indicate that 2-5A analogs, particularly bifunctional 2-5A analogs like AA-etherA, will be useful for controlling cancer cell growth. Further development of such 2-5A analogs may provide highly specific compounds for chemotherapy and chemoprevention. PMID- 8632907 TI - Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells. AB - We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium. PMID- 8632906 TI - The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway. AB - Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself. PMID- 8632908 TI - Nerve growth factor-induced accumulation of PC12 cells expressing cyclin D1: evidence for a G1 phase block. AB - The anti-proliferative effect of nerve growth factor (NGF) on the rat pheochromocytoma cell line PC12 has been previously shown to be accompanied by the accumulation of cells in either the G1 phase with a 2c DNA content, or with a 4c DNA content characteristic for G2/M, as evidenced by flow cytometric analysis of DNA distribution using propidium iodide. Herein, these apparently conflicting results are clarified. The present studies indicate that a simple DNA distribution profile obtained by this technique can confound interpretation of the biological effects of NGF on cell-cycle distribution due to the presence of tetraploid cells. Using cyclin D1 and incorporation of bromodeoxyuridine as markers of respectively, G1 and S phase, we show that PC12 cultures can have a considerable amount of tetraploid cells which, when in the G1 phase, have a 4c DNA content and express cyclin D1. During exposure to NGF, this population increases, reflecting the accumulation of cells in the G1 phase of the cell cycle. The data presented, support the possibility that events affecting the expression or action of G1 regulatory proteins may be involved in the molecular mechanism of the anti-mitogenic effect of NGF. PMID- 8632911 TI - Overexpression of the AML1 proto-oncoprotein in NIH3T3 cells leads to neoplastic transformation depending on the DNA-binding and transactivational potencies. AB - The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21) and t(3;21) translocations associated with myelogenous leukemias. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are generated by alternative splicing. Both forms have the runt homology domain that possesses the DNA-binding ability but, unlike AML1b, AML1a lacks a putative transcriptional activation domain downstream of the runt homology domain. By using retroviral infection, we demonstrated that AML1b causes neoplastic transformation of NIH3T3 cells. AML1b-expressing cells form macroscopic colonies in soft agar and induce tumors in nude mice, indicating that AML1 can be a transforming gene when overexpressed in fibroblasts. Both the runt homology domain and the transactivational domain were required to transform NIH3T3 cells. By analysis of deletion mutants, it was shown that an element determining the transactivational potency exists between amino acids 288 and 396 within the region downstream of the runt homology domain. Furthermore, we demonstrated that this region was also required for fibroblast transformation, indicating that the transforming activity of AML1 is correlated with the transactivational potencies. These results suggest a role of AML1 in the regulation of cellular proliferation, as well as myeloid cell differentiation. PMID- 8632909 TI - Differential phosphorylations of Spi-B and Spi-1 transcription factors. AB - Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi 1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities. PMID- 8632910 TI - Different p53 mutations produce distinct effects on the ability of colon carcinoma cells to become blocked at the G1/S boundary after irradiation. AB - The LoVo colon carcinoma cell line that presents two wild type p53 alleles was used as the recipient for a series of transfections with p53 expression vectors coding for wild-type or three different mutants (143ala, 175his or 273his). The parental cell line as well as all clones that had rearranged the plasmid with consequent loss of p53 c-DNA were readily blocked at the G1/S boundary following 10 Gy of irradiation. For each mutation two clones with different levels of mutant protein expression were selected. Confirmation of the integration of the exogenous sequence was obtained by the expression of the mutant m-RNA, established by reverse transcription and DGGE or Southern blot. Flow cytometric measurements of 5-bromodeoxyuridine incorporation revealed a total G1/S block of the 143ala transfectants, similarly to the parental and control transfectant cells, but little or no cell cycle block for the 175his and 273his clones. Although it has been shown in vitro that all three mutations interfere with transcriptional activation by the wild-type protein, not only did we observe p53 protein induction and nuclear accumulation following irradiation, but WAF-1/CIP-1 m-RNA was increased in some of the clones for which the G1/S block was abolished. Our results show that mutant p53 proteins are to some extent submitted to the control of the cellular environment in cancer cells with wild type p53 alleles, but with an efficacy that depends on the mutation. PMID- 8632914 TI - A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein - partial purification and characterization from rat brain. AB - The Rho subfamily, consisting of three members (RhoA, -B and -C), belongs to the small GTP-binding protein superfamily. The Rho subfamily is implicated in regulation of various actin filament-dependent cell functions, such as cell aggregation, cell motility and cytokinesis. The Rho subfamily receives an upstream signal and is converted from the GDP-bound inactive form to the GTP bound active form which transduces a signal to a downstream pathway. This conversion is regulated by GDP/GTP exchange proteins (GEPs) and several GEPs for the Rho subfamily have been identified. The GEPs thus far reported are mainly isolated from the cytosol fraction of various tissues and are not specific for the Rho subfamily. Here we have partially purified a membrane-associated GEP specific for the Rho subfamily (mRho GEP). mRho GEP was extracted from the crude synaptic membrane fraction of rat brain by a combination of detergent and NaCl, and partially purified by several column chromatographies. The partially purified mRho GEP was active on RhoA but was inactive on other small GTP-binding proteins including at least Rac1, Ki-Ras and Rab3A. RhoA undergoes post-translational lipid modifications and mRho GEP required these lipid modifications for its GEP activity. mRho GEP was not active in the presence of Rho GDI, an inhibitory Rho GEP. These results indicate that there is a membrane-associated GEP specific for Rho and suggest that Rho is activated by this GEP on the membranes. PMID- 8632913 TI - Tnk1: a novel intracellular tyrosine kinase gene isolated from human umbilical cord blood CD34+/Lin-/CD38- stem/progenitor cells. AB - Degenerate PCR was employed to identify novel tyrosine kinase genes from an enriched population of human umbilical cord blood hematopoietic stem/progenitor cells. One novel tyrosine kinase gene, designated Tnk1, was cloned. The sequence of the complete Tnk1 coding region predicts a 72 kD protein. Comparison of Tnk1 to available sequences in protein databases reveals that it is most homologous to Ack, an intracellular tyrosine kinase which associates with the GTP-bound form of p21cdc42Hs. Like Ack, Tnk1 consists of an N-terminal kinase domain, a putative SH3 domain immediately C-terminal to the kinase domain, and a proline-rich C terminal region. Analysis of Tnk1 mRNA expression demonstrates that Tnk1 is expressed in all cord blood, bone marrow and adult blood sub-populations, as well as in most of the leukemia cell lines examined (16 of 20). Hybridization to fetal multi-tissue Northern blots detected several different Tnk1 transcripts in all fetal tissues examined. In contrast, a single Tnk1 transcript was detected in only five of 16 adult tissues examined (prostate, testis, ovary, small intestine and colon). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes localized the Tnk1 gene to the short arm of chromosome 17 (17p13.1), near the p53 locus. Thus, Tnk1 is a novel tyrosine kinase that may be involved in signalling pathways utilized broadly during fetal development, more selectively in adult tissues and in cell of the lymphohematopoietic system. PMID- 8632912 TI - The anti-proliferative effect of sulindac and sulindac sulfide on HT-29 colon cancer cells: alterations in tumor suppressor and cell cycle-regulatory proteins. AB - Nonsteroidal anti-inflammatory drugs lower the incidence of and mortality from colon cancer. Sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. We have shown that sulindac and sulindac sulfide reversibly reduce the proliferation rate of HT-29 colon cancer cells, alter their morphology, induce them to accumulate in the G0/G1 phase of the cell cycle, and sulindac sulfide induces cell death by apoptosis. In this study we confirmed that sulindac and sulindac sulfide prevent HT-29 cells from progressing from the G0/G1 into the S phase. This block in cell cycle progression is associated with an initial rise, then an abrupt decrease in the levels of p34cdc2 protein. Sulindac and sulindac sulfide decrease the levels of mitotic cyclins, induce the levels of p21WAF-1/cip1, and reduce the total levels of pRB, with a relative increase in the amount of the underphosphorylated form of pRB in a time- and concentration dependent manner. In addition, these compounds reduce the levels of mutant p53. These responses are not associated with intestinal cell differentiation and occur independent of the ability of these compounds to induce apoptosis. We conclude that sulindac and sulindac sulfide reduce the levels of major components of the molecular cell cycle machinery and alter the levels of several tumor suppressor proteins in a manner consistent with cell cycle quiescence. These mechanisms may be operative in vivo to account, in part, for the anti-neoplastic effects of these compounds. PMID- 8632916 TI - Transcriptional regulation of the c-fms proto-oncogene mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) in murine cell lines. AB - Differentiation of blood cells is paralleled by a timely ordered expression of cytokine receptor genes. We show here that the expression of the c-fms gene which encodes the lineage-specific receptor for macrophage colony-stimulating factor (M CSF or CSF-1) is directly linked to ligand-mediated activation of the receptor for the granulocyte/macrophage colony-stimulating factor (GM-CSF). In interleukin 3 (IL-3) dependent multipotent progenitor cells, FDC-Pmix GMV#2 cells, GM-CSF treatment results in the rapid formation of full-length c-fms transcripts. Surprisingly, this upregulation of c-fms transcripts is also observed in mouse NIH3T3 fibroblasts stably transfected with genes coding for the alpha- and beta subunits of the GM-CSF receptor. These results indicate a direct control by the GM-CSF receptor that takes place regardless of cell differentiation. Furthermore, a 2.1 kb genomic fragment containing the c-fms proximal promoter directs GM-CSF inducible expression of a reporter gene, suggesting a regulation of c-fms gene expression on the transcriptional level. PMID- 8632917 TI - Human Txk: genomic organization, structure and contiguous physical linkage with the Tec gene. AB - Txk is a Tec-family tyrosine kinase expressed in mouse and human T lymphocytes. Among the Tec kinases, Txk is unique in that its amino terminal region does not include a pleckstrin homology domain or other known extended functional region. Txk is encoded at human chromosome 4p12 and at a recognized region of conserved synteny on mouse chromosome 5. The genomic organization of Txk consists of 15 exons with strong exon-intron organizational homology to Btk, the only other Tec family kinase for which the genomic structure is fully known. The human Tec gene also maps to 4p12 and, based on limited studies reported here, possesses organizational homology with Btk and Txk. We have sequenced a continuous region of DNA that contains 3' Tec and 5' Txk exons separated by only a approximately 1.5 kb intergenic region containing the putative promoter region of Txk. The close physical linkage of these Tec-family tyrosine kinases, which are expressed in different hematopoetic cell lineages, suggests their potential for coordinate cis-regulation. PMID- 8632915 TI - Regulation of specific DNA binding by p53: evidence for a role for O glycosylation and charged residues at the carboxy-terminus. AB - The carboxy-terminus of p53 contains a basic region which represses DNA binding, and this repression can be relieved by PAb421, an antibody against the basic region. The EB-1 human cell line contains wild type p53 protein which fails to express the PAb421 epitope and is highly active both in biological assays and in DNA binding assays. We show by wheat germ agglutinin chromatography and galactosyl-transferase labelling that this p53 is O-glycosylated, and that at least one of the sugar residues masks the PAb421 epitope, as demonstrated by recovery of reactivity with PAb421 after digestion of Western blots of EB-1 cell extract with hexosaminidase. A minor population of p53 molecules in EB-1 cells lacks the modification, and there is a correlation between the ability to bind DNA with high affinity and masking of the PAb421 epitope. We also show that strongly positively charged peptides, including short peptides from the basic region of p53, can derepress DNA binding, probably by disruption of an intramolecular interaction involving the basic region. We propose that any intervention which prevents this intramolecular interaction, including addition of bulky residues such as sugar groups, can activate DNA binding by p53. PMID- 8632918 TI - Patterns of macroparasite abundance and aggregation in wildlife populations: a quantitative review. PMID- 8632920 TI - Evolutionary pressures in the spread and persistence of infectious agents in vertebrate populations. AB - Infectious agents have considerable potential to regulate or constrain the population growth of vertebrate hosts in natural habitats. A broad theoretical framework provides many insights into how the biology of the parasite and the demography of the host interact to determine this impact. It may manifest itself as a steady influence over time via stable endemic infection or in a recurrent epidemic fashion, sometimes with unpredictable intervals between epidemics depending on the generation time of the pathogen (time from infection to recovery or host death), its ability to induce lasting immunity and the population growth rate of the host species. Building on these notions, the paper focuses on recent work on the population dynamics of genetically variable pathogen populations and examines the factors that determine the evolution of virulence and the maintenance of genetic diversity in both host and pathogen. Recent research extends conventional theoretical templates to include population genetic elements and the within-host dynamics of the parasite and its interaction with the vertebrate immune system. PMID- 8632921 TI - Parasitic disease in amphibians: control by the regulation of worm burdens. AB - This review considers three case studies based on macroparasites of anurans: (a) natural infections in the permanently-aquatic Xenopus laevis which represent the worm burdens acquired, and the implications for pathology, when hosts are exposed to continuous, year-round, transmission; (b) the desert toad, Scaphiopus couchii, which experiences invasion very briefly each year and provides a simplified system involving only a single significant infection (Pseudodiplorchis americanus); (c) the mesic Bufo bufo which has been the subject of experimental laboratory studies designed to measure the effects of Rhabdias bufonis infection on host growth, physical performance and survival. Experimental manipulation of both Scaphiopus and Bufo provide quantitative data on disease effects of macroparasites, including precise measurements of parasite-induced host mortality. Field data for Xenopus and Scaphiopus show that, despite high initial worm burdens from efficient transmission, infection levels at parasite maturity are modulated below those leading to significant disease. Experimental data for Scaphiopus and Bufo have documented the time-course and magnitude of this decline in intensities, and there is circumstantial evidence for Scaphiopus that this regulation is host-mediated. Immunological studies on Xenopus show that disease effects of the pathogenic Pseudocapillaroides xenopodis are exacerbated in thymectomised hosts and reversed by implantation of thymuses from MHC-compatible donors. Thus, whilst factorial experiments can demonstrate the potential of helminths to cause significant disease and mortality in anuran host-macroparasite interactions, powerful post-invasion regulation of worm burdens appears to exert a strong control of parasite-induced disease in natural host populations. PMID- 8632919 TI - Modelling patterns of parasite aggregation in natural populations: trichostrongylid nematode-ruminant interactions as a case study. AB - The characteristically aggregated frequency distribution of macroparasites in their hosts is a key feature of host-parasite population biology. We begin with a brief review of the theoretical literature concerning parasite aggregation. Though this work has illustrated much about both the sources and impact of parasite aggregation, there is still no definite analysis of both these aspects. We then go on to illustrate the use of one approach to this problem--the construction of Moment Closure Equations (MCEs), which can be used to represent both the mean and second moments (variances and covariances) of the distribution of different parasite stages and phenomenological measures of host immunity. We apply these models to one of the best documented interactions involving free living animal hosts--the interaction between trichostrongylid nematodes and ruminants. The analysis compares patterns of variability in experimental infections of Teladorsagia circumcincta in sheep with the equivalent wildlife situation--the epidemiology of T. circumcincta in a feral population of Soay sheep on St Kilda, Outer Hebrides. We focus on the relationship between mean parasite load and aggregation (inversely measured by the negative binomial parameter, k) for cohorts of hosts. The analysis and empirical data indicate that k tracks the increase and subsequent decline in the mean burden with host age. We discuss this result in terms of the degree of heterogeneity in the impact of host immunity or parasite-induced mortality required to shorten the tail of the parasite distribution (and therefore increase k) in older animals. The model is also used to analyse the relationship between estimated worm and egg counts (since only the latter are often available for wildlife hosts). Finally, we use these results to review directions for future work on the nature and impact of parasite aggregation. PMID- 8632924 TI - Persistence and transmission of tick-borne viruses: Ixodes ricinus and louping ill virus in red grouse populations. AB - The population dynamics of tick-borne disease agents and in particular the mechanisms which influence their persistence are examined with reference to the flavivirus that causes louping-ill in red grouse and sheep. Pockets of infection cause heavy mortality and the infection probably persists as a consequence of immigration of susceptible hosts. Seroprevalence is positively associated with temporal variations in vectors per host, although variation between areas is associated with the abundance of mountain hares. The presence of alternative tick hosts, particularly large mammals, provides additional hosts for increasing tick abundance. Grouse alone can not support the vectors and the pathogen but both can persist when a non-viraemic mammalian host supports the tick population and a sufficiently high number of nymphs bite grouse. These alternative hosts may also amplify virus through non-viraemic transmission by the process of co-feeding, although the relative significance of this has yet to be determined. Another possible route of infection is through the ingestion of vectors when feeding or preening. Trans-ovarial transmission is a potentially important mechanism for virus persistence but has not been recorded with louping-ill and Ixodes ricinus. The influence of non-viraemic hosts, both in the multiplication of vectors and the amplification of virus through non-viraemic transmission are considered significant for virus persistence. PMID- 8632922 TI - Introduction: ecological impact of parasitism on wildlife host populations. PMID- 8632923 TI - Maintenance of a microparasite infecting several host species: rabies in the Serengeti. AB - Whether and how microparasites such as rabies persist in their host populations are among the fundamental questions of infectious disease epidemiology. Rabies is fatal disease of all mammalian species, but not all mammalian species can maintain the infection as reservoirs. The approach to control depends on which of the affected species do act as reservoirs. Bringing together old and new data, we examine here the role of wild and domestic animals in maintaining rabies in the Serengeti region of Tanzania, presenting our findings in two parts. In Part I, we argue that domestic dogs are the likely reservoirs because: (1) rabies has been continuously present in the dog population since its (re)introduction in 1977, whilst (2) wildlife cases have been very rare over this period, despite intensive study of Serengeti carnivores; (3) outbreaks of rabies in wild canids (jackals) elsewhere in Africa (Zimbabwe) have followed, rather than preceded, outbreaks in the dog population; (4) all viruses isolated from wild carnivores in the Serengeti ecosystem (including the Kenyan Masai Mara) are antigenically and genetically indistinguishable from the typical domestic dog strain; (5) dog rabies control in the Serengeti between 1958-77 apparently eliminated the disease from both dogs and wildlife. Having identified dogs as reservoirs, Part II explores some possible mechanisms of maintenance in dog populations. In theory, infection is more likely to be maintained at higher dog densities, and we provide evidence that rabies is maintained in one district with a dog density > 5/km2, but not in two other districts with densities < 1/km2. Because 5 dogs/km2 is much lower than the expected density required for persistence, we go on to investigate the role of atypical infections, showing: (1) from serology, that a substantial proportion of healthy dogs in the Serengeti have detectable serum levels of rabies-specific antibody; (2) from mathematical models that, whilst we cannot be sure what seropositivity means, persistence in low-density dog populations is more likely if seropositives are infectious carriers, rather than slow-incubators or immunes. PMID- 8632925 TI - Wildlife disease and conservation in Hawaii: pathogenicity of avian malaria (Plasmodium relictum) in experimentally infected iiwi (Vestiaria coccinea). PMID- 8632928 TI - Can asphyxiated infants at risk for neonatal seizures be rapidly identified by current high-risk markers? AB - OBJECTIVE: Markers currently used to identify infants at highest risk for perinatal hypoxic-ischemic cerebral injury are insensitive in predicting the subsequent occurrence of neonatal seizures and/or neurodevelopmental sequelae, ie, cerebral palsy. To facilitate therapeutic strategies, early identification of the infant at highest risk for developing seizures secondary to hypoxia ischemia or asphyxia is critical, particularly if novel but potentially toxic therapies currently under experimental investigation become available for clinical use. METHODS: Ninety-six inborn term infants considered at high risk for having neonatal seizures secondary to hypoxia ischemia or asphyxia and admitted to the neonatal intensive care unit directly after labor and delivery were prospectively evaluated. Markers of high risk included the presence of moderate to thick meconium-stained amniotic fluid (MSAF), fetal heart rate (FHRT) abnormalities abruptio placentae, intubation and positive pressure ventilation in the delivery room (DR), chest compressions and epinephrine administration as part of resuscitation, a 5-minute Apgar score of 5 or less, umbilical cord arterial pH of 7.00 or less, and/or a base deficit of -14 mEq/L or more negative. RESULTS: Seizures developed in 5 (5.2%) of the 96 infants. High-risk markers included FHRT abnormalities only (n=36), FHRT abnormalities and MSAF (n=20), MSAF only (n=23), abruptio placentae (n=6), intubation in the DR (n=44), intubation in the neonatal intensive care unit (n=22), chest compressions (n=2), 5-minute Apgar scores of 5 or less (n=21), umbilical cord arterial pH of 7.00 or less (n=21), and base deficits of -14 mEq/L or more negative (n=19). By univariate analysis, significant relationships with seizures were found with Apgar scores, the need for intubation in the DR, umbilical cord arterial pH, and base deficit. Combinations of the identified risk markers showed a strong relationship with seizures with the following odds rations (ORs), 95% confidence limits, sensitivity, specificity, and positive predictive values (PPVs): (1) low cord pH and intubation, OR, 163 (confidence limits, 7.9 and 3343.7); sensitivity, 100%; specificity 94%; and PPV, 50%; (2) low cord pH and low 5-minute Apgar score, OR, 39 (confidence limits, 3.9 and 392.5); sensitivity, 80%; specificity, 91%; and PPV, 33.3%; and (3) low pH, intubation, and low 5-minute Apgar score, OR, 340 (confidence limits, 17.8 and 6480.6); sensitivity, 80%; specificity, 98.8%; and PPV, 80%. CONCLUSIONS: A combination of high-risk postnatal markers, specifically, a low 5-minute Apgar score and intubation in the DR in association with severe fetal acidemia, facilitates the identification within the first hour of life of term infants at highest risk for developing seizures secondary to perinatal asphyxia. PMID- 8632926 TI - Preliminary experience with intestinal transplantation in infants and children. AB - OBJECTIVE: This report discusses the preliminary experience with intestinal transplantation in children at the University of Nebraska Medical Center. PATIENTS: During the past 4 years, 16 intestinal transplants have been performed in infants and children. Thirteen have been combined liver and bowel transplants, and the reminder were isolated intestinal transplants. Nearly half of the patients were younger than 1 year of age at the time of surgery, and the vast majority were younger than 5 years of age. All but one had short bowel syndrome. RESULTS: The 1-year actuarial patient and graft survival rates for recipients of liver and small bowel transplants were 76% and 61%, respectively. Eight of 13 patients who received liver and small bowel transplants remain alive at the time of this writing, with a mean length of follow-up of 263 (range, 7 to 1223) days. Six patients are currently free of total parenteral nutrition. All three patients receiving isolated intestinal transplants are alive and free of parenteral nutrition. The mean length of follow-up is 384 (range, 330 to 450) days. Major complications have included severe infections and rejection. Lymphoproliferative disease, graft-versus-host disease, and chylous ascites have not been major problems. CONCLUSIONS: Although intestinal transplantation is in its infancy, these preliminary results suggest combined liver and bowel transplants and isolated intestinal transplantation may be viable options for some patients with intestinal failure caused by short bowel syndrome or other gastrointestinal disease in whom long-term total parenteral nutrition is not an attractive option. PMID- 8632930 TI - The delivery of immunizations and other preventive services in private practices. AB - OBJECTIVES: To measure the proportion of children cared for in private practices who are fully immunized and have been screened for anemia, tuberculosis (TB), and lead poisoning by 2 years of age. DESIGN: Cross-sectional chart review. SETTING: Fifteen private pediatric practices in central North Carolina (11 chosen randomly). PATIENTS: One thousand thirty-two randomly selected 2-year-old children. MAIN OUTCOME MEASURES: Proportion of children immunized and screened for anemia, TB and lead poisoning by 24 months of age and immunization and screening rates of the practices. RESULTS: Sixty-one percent of the children were fully immunized at 24 months of age; the rates among practices varied widely (38% to 82%). Sixty-eight percent of the children had been screened for anemia, 57% had been screened for TB, and 3% had been screened for lead poisoning. Physicians overestimated the proportions of fully immunized children in their practices by an average of 10% (range, -3% to 17%). The median number of well child visits by 2 years of age was 5 (range, 0 to 14), and only 19% of the entire sample made 8 or more well child visits, the number recommended by the American Academy of Pediatrics in the first 18 months of life. The numbers of well child and non-well child visits were the strongest predictors of complete immunization. Practice characteristics associated with being fully immunized included the use of preventive services prompting sheets (eg, flow sheets) in the medical records, not seeing the same physician for all well child care, and having nurses review patients' immunization status during their visits to the office. CONCLUSIONS: Underimmunization and inadequate screening are significant problems in private pediatric practices in North Carolina. Physicians are unaware of the rates of underimmunization in their offices. PMID- 8632929 TI - The efficacy of nebulized budesonide in dexamethasone-treated outpatients with croup. AB - OBJECTIVE: To determine the added clinical benefit of nebulized budesonide in children with mild to moderate croup treated with 0.6 mg/kg oral dexamethasone. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Emergency department of a tertiary-care pediatric hospital with 47,000 visits per year. PARTICIPANTS: Children 3 months to 5 years of age with a syndrome consisting of hoarseness, inspiratory stridor, and barking cough and a croup score of 3 or greater after at least 15 minutes of mist therapy. Patients were excluded from the study if they had diagnoses of epiglottitis, chronic upper or lower airway disease (not including asthma), or severe croup or had received corticosteroids within the preceding 2 weeks. INTERVENTION: All patients received 0.6 mg/kg oral dexamethasone and were randomly assigned to receive 4 mL (2 mg) of budesonide solution (n=25) or 4 mL of 0.9% saline solution (n=25) by updraft nebulizer with a continuous flow of oxygen at 5 to 6 L/min. MAIN OUTCOME MEASURES: The primary outcome measure was the proportion of patients in each group who had clinically important changes (two points) in the croup score during the 4 hours after treatment. RESULTS: Eighty-four percent (n=21) of the patients who received budesonide had clinically important responses, compared with 56% (n=14) in the placebo group. The number of patients who would need to be treated with nebulized budesonide for one patient to have a clinically important response is four patients. CONCLUSIONS: Despite receiving simultaneous oral dexamethasone, pediatric outpatients with mild to moderate croup have added, clinically important improvement in respiratory symptoms after treatment with budesonide. PMID- 8632927 TI - Pulmonary administration of vasoactive substances by perfluorochemical ventilation. AB - OBJECTIVES: Therapeutic management of respiratory distress syndrome, pneumonia, and pulmonary hypertension includes delivery of biologically active agents to the neonatal lung. However, mechanical abnormalities of the lung, intrapulmonary shunting, ventilation-perfusion mismatching, and elevated surface tension impede effective systemic or intratracheal delivery of agents to the lung during conventional gas ventilation. The objective of this study was to test the hypothesis that perfluorochemical (PFC) liquid ventilation can be used for pulmonary administration of vasoactive drugs (PAD) and to compare these responses to those elicited with intravascular (IV) administration during tidal liquid ventilation. METHODS: Cardiovascular responses of 16 preterm and neonatal lambs to randomized doses of acetylcholine, epinephrine, and priscoline were studied. Physiologic gas exchanged and acid-base balance were maintained using previously described tidal liquid ventilation techniques. In subgroups of animals, the distribution pattern of carbon 1- and choline 14-labeled dipalmitoylphosphatidylcholine (14C-DPPC) in saline and the responses to priscoline after hypoxia-induced pulmonary hypertension and hypoxemia administered during liquid ventilation were studied. RESULTS: Dose-response curves for PAD and IV administration demonstrated progressive, dose-dependent, cholinergic responses to acetylcholine (decreased mean systemic arterial pressure [MAP] and heart rate), sympathomimetic responses to epinephrine (increased MAP and heart rate), and alpha-adrenergic blockade responses to priscoline (decreased MAP and mean pulmonary arterial pressure). Compared with IV administration, PAD of priscoline resulted in a significantly greater decrease in pulmonary relative to systemic arterial pressure; this response was potentiated by hypoxia, reduced pulmonary pressures to near normal values, and improved oxygenation. The 14C-DPPC in saline was distributed relatively homogeneously throughout the lung by PAD, with 80% of the lung pieces receiving amounts of 14C-DPPC with +/-20% of the mean value. CONCLUSIONS: This study demonstrates that vasoactive agents can be delivered to the lung directly by PAD during PFC liquid ventilation. The inherent advantages of this method relate to the physical properties of PFC liquid ventilation as a vehicle (respiratory gas solubility, low surface tension enhancing distribution, and inertness precluding interaction) and physiological properties of the lung as an exchanger. PMID- 8632933 TI - Informing subjects of epidemiologic study results. Children's Cancer Group. AB - OBJECTIVE: To assess the feasibility and process of providing feedback to parents regarding the results of epidemiologic research, in particular to look at the importance and clarity of the information provided, parental reactions to the results, and utilization of the data provided. METHODOLOGY: Parents who participated in an epidemiologic study of pediatric brain tumors (patient and control mothers) were sent a letter summarizing the results of the study and the Parent Study Results Survey to complete and return. The final sample used for analyses was 109 (patient) and 90 (control) mothers. Analyses were conducted to determine differences between patient and control mothers and differences among subsets defined by educational level and vital status of the patient. RESULTS: Mothers rated the importance and clarity of the information very highly, although patient mothers were more likely than control mothers to want more information and a telephone contact. Patient and control mothers were similar in reported sadness, anxiety, and being overwhelmed, but patient mothers felt less satisfied and relieved. Patient mothers expressed feeling more guilt nad anger than control mothers, although even the levels among the patient mothers were only moderate. Close to half of all mothers commented on the inconclusiveness of the study results. Nearly all mothers indicated they would suggest that other parents participate in epidemiologic research. CONCLUSIONS: It is valuable to many parents that they receive information about results of research in which they have participated. We found little evidence of strong negative effects to a detailed feedback letter. We recommend that evaluative data be used to guide the process of informing research participants about study results and that investigators consider making feedback letters a standard part of research protocols. PMID- 8632931 TI - The contribution of missed opportunities to childhood underimmunization in Baltimore. AB - OBJECTIVE: To determine the community-wide incidence of missed opportunities to vaccinate, to describe the clinical settings in which they occur, and to estimate the impact of missed opportunities on immunization coverage. DESIGN AND METHODS: We abstracted outpatient medical records from a random, community-based sample of 2-year-old children whose residence was inner-city Baltimore. The date of each vaccine and the date, diagnoses, and temperature at each visit were collected for 502 children at 98 different provider sites. MAIN OUTCOME MEASURES: Missed opportunities to vaccinate and up-to-date vaccination status. RESULTS: By 24 months of age, 75% of the children had at least one missed opportunity and only 55% were up-to-date for the 4:3:1 series. Missed opportunities occurred at more than one third of eligible visits for each vaccine, including > 20% of preventative care visits. Diagnoses commonly associated with missed opportunities were "well child," otitis media, upper respiratory infection, gastroenteritis, skin infection, and resolving illness. If no missed opportunities had occurred, 73% of the children would have been up-to-date by 24 months. CONCLUSIONS: Missed opportunities occurred commonly at providers serving inner-city children in Baltimore and represent a major factor in underimmunization. Reduction of missed opportunities by accurate screening at all visits and adherence to the contraindication guidelines is a provider-based, low-cost method to increase immunization coverage. PMID- 8632932 TI - Discharging patients with prescriptions instead of medications: sequelae in a teaching hospital. AB - OBJECTIVE: This study measures the incidence of discrepancies among written prescriptions, medication regimens transcribed onto patient discharge instruction sheets (DCIs), and labels on medications dispensed by community pharmacies after discharge of patients from an academic medical center. METHODS: During a 2-month study period, we collected copies of prescriptions and DCIs. We also called care givers after discharge and asked them to read the medication labels that were filled from discharge prescriptions. Care givers were also asked whether they received instruction from community pharmacies. RESULTS: Data were collected on 335 prescriptions for 192 patients. Differences among the prescriptions, DCIs, and medication labels were found for 40 (12%) of the medications prescribed at discharge, representing 19% of the patients studied. Nineteen prescriptions had prescriber errors in dosing frequencies or dosage formulations. Three prescriptions were filled with different medication concentrations or strengths than requested. Prescriptions were altered by the community pharmacists for unexplained reasons in 6 cases, whereas the DCIs and original prescriptions differed in 12 cases. Only 44% of families were counseled about proper medication administration by their pharmacists. CONCLUSIONS: A potential for medication errors exists when pediatric patients are discharged with unfilled prescriptions. The potential may be worsened when discharge instructions are created from a prescription rather than from the label of a dispensed medication. Educational and risk-management efforts should emphasize the importance of writing complete, legible prescriptions and consulting appropriate reference materials to ensure that dose formulations and guidelines are accurate. Whenever possible, prescriptions should be filled before patients are discharged, so that the dispensed medications can be reviewed, and health care providers can provide accurate discharge instructions. PMID- 8632934 TI - Breastfeeding and the working mother: effect of time and temperature of short term storage on proteolysis, lipolysis, and bacterial growth in milk. AB - BACKGROUND: Women who breastfeed have to store expressed milk while at work for later feeding to their infants; however, storage conditions are often not optimal. OBJECTIVE: Top assess microbial growth and stability of milk protein and lipid at 15 degrees C to 38 degrees C for up to 24 hours. METHODS: Sixteen healthy women who breastfed exclusively, either at home (n=11) or who expressed milk for their infants (n=5), were studied during early (1 month) or late (5 to 6 months) lactation. Expressed milk was stored at 15 degrees C, 25 degrees C, and 38 degrees C for 1 to 24 hours for quantitation of pH, proteolysis, and lipolysis; bacterial growth was quantified at 0, 4, 8, and 24 hours of storage. RESULTS: Milk pH decreased 2 units by 24 hours of storage at all temperatures tested. Proteolysis was minimal during milk storage at 15 degrees C or at 25 degrees C for 24 hours and was apparent only after 24 hours of storage at 38 degrees C. Lipolysis was rapid, starting in the first hours of storage and progressing to 8% at 24 hours. Thus, while the greatest increment in proteolysis products was a 40% increase above baseline after 24 hours of storage at 38 degrees C, free fatty acid concentration at this storage time was 440% to 710% higher than in freshly expressed milk. Bacterial growth was restricted mainly to nonpathogens, was minimal at 15 degrees C throughout the 24 hours of storage, was low at 25 degrees C for the first 4 to 8 hours, and was considerably higher at 38 degrees C even during the relatively short period of 4 hours. CONCLUSIONS: Storage of human milk is safe at 15 degrees C for 24 hours, whereas at 25 degrees C it is safe for 4 hours. Milk should not be stored at 38 degrees C. Minimal proteolysis during storage suggests that milk proteins probably maintain their structure and function during short-term storage, while the marked lipolysis might slow bacterial growth during this time. PMID- 8632936 TI - Carbohydrate and alcohol content of 200 oral liquid medications for use in patients receiving ketogenic diets. AB - OBJECTIVE: The ketogenic diet is used in patients with intractable seizure disorders. To maintain a ketotic state, patients on this diet must maintain a strict low-carbohydrate intake. Because these patients often require medication, and because many pharmaceutical products (especially liquid formulations) contain significant quantities of carbohydrates, it is important that each drug product be evaluated before proceeding with the ketogenic diet. The purpose of this study was to compile the carbohydrate content of oral liquid products for patients on or considering a ketogenic diet. METHODS: A list of 200 oral liquid drug products and their manufacturers was compiled using the Children's Health Care-Minneapolis pharmacy purchasing database, the Physicians' Desk Reference, and previously published lists of oral liquid medications. Manufacturers were contacted either by telephone or by letter and asked specifically about the sucrose, fructose, sorbitol, glycerin, and alcohol content of these liquid formulations. RESULTS: The carbohydrate and alcohol content of 200 oral liquid products was determined and shown in an accompanying table. CONCLUSIONS: Many oral liquid medications contain significant amounts of carbohydrate. Tablet and capsule formulations are preferred when possible. To date, it has been difficult to quantify the carbohydrate content of liquid medications because of unavailability of this information. The carbohydrate content of 200 oral liquid medications is provided here to assist health care practitioners, patients, and care givers in designing drug regimens low in carbohydrates for patients on or considering a ketogenic diet. PMID- 8632935 TI - Protein requirements of infants and children: growth during recovery from malnutrition. AB - OBJECTIVE: To evaluate the adequacy of protein intakes now recommended as safe for infants and toddlers. METHODS: Subjects were recovering malnourished infants, age 5.3 to 17.9 months, length age (LA) 2.5 to 6.4 months, weight age (WA) 1.5 to 5.2 months, weight/length (W/L) 78% to 100% of National Center for Health Statistics data; and toddlers age 11.4 to 31.6 months, LA 6.1 to 17.9 months, WA 3.9 to 12.0 months, W/L 79% to 99%. Infants were assigned at random to formulas with 5.5%, 6.7%, or 8.0% energy as 60:40 whey:casein protein. The 5.5% was based on FAO-WHO-UNU safe protein and average energy for ages 2.5 to 6.0 months. Toddlers received 4.7% (recommended for 6 to 18 months), 6.4%, or 8.0%. Identical concentrations (weight/kcal) of other nutrients were maintained; intakes were adjusted weekly to reach, in 90 days, the 50th percentile of weight for a LA 3 months greater than the initial one. RESULTS: Infants consumed 125 +/- 11 (SD), 116 +/- 10, and 126 +/- kcal and 1.7 +/- 0.1, 1.9 +/- 0.2, and 2.5 +/- 0.3 g protein kg-1 . d-1; gained 2.4 +/- 0.7, 2.9 +/- 0.7, and 2.6 +/- 0.5 months in LA, and reached a W/L of 105 +/- 5, 103 +/- 6, and 105 +/- 5% of reference. Sum of four fat-folds (sigma FF) grew 13.1 +/- 6.9, 10.4 +/- 4.8, and 11.7 +/- 5.3 mm to 32.5 +/- 5.2, 31.7 +/- 4.7, and 30.5 +/- 5.5 mm; arm muscle areas (AMA) 57%, 51%, 70% to 1004 +/- 109, 1017 +/- 110, and 1004 +/- 116 mm2, still low; arm fat areas (AFA) 93%, 66%, and 93% to higher-than-normal 598 +/- 105, 610 +/- 101, and 541 +/- 116 mm2. Regression of intake on weight gain estimated energy for maintenance + activity to be 81.0 +/- 7.5 (SEM) kcal . kg-1 . d-1, and cost of gain (storage + metabolic cost) as 7.6 +/- 1.7 kcal/g, with no significant effect of % protein. Toddlers consumed 107 +/- 9, 103 +/- 12, and 105 +/- 10 kcal and 1.3 +/- 0.1, 1.6 +/- 0.2, and 2.1 +/- 0.2 g protein . kg-1 . d-1, gained 3.3 +/- 0.7, 2.9 +/- 0.6, and 3.3 +/- 0.7 months in LA; to a W/L of 102 +/- 1, 102 +/- 3, and 101 +/- 4%. Sigma FF grew 9.2 +/- 4.0, 7.4 +/- 4.3, and 6.0 +/- 3.8 to 28.9 +/- 5.2, 30.5 +/- 3.7, and 27.0 +/- 2.7 mm; AMA 31%, 33%, and 34% to 1121 +/- 115, 1124 +/- 110, and 1117 +/- 120 mm2; AFA 53%, 44%, and 45% to higher-than normal 578 +/- 106, 636 +/- 99, and 569 +/- 68 mm2. Cost of maintenance + activity was 70.8 +/- 3.8 (SEM) kcal . kg-1 . d-1, that of weight gain 9.7 +/- 1.35 kcal/g, with no effect of % protein. CONCLUSIONS: Within age groups, there were no significant protein-related differences in growth. In both infants and toddlers, high-energy intakes resulted in mild obesity, with lean body mass still deficient. Protein intakes two SD below the means in the lowest protein/energy cells, 1.5 g . kg-1 . d-1 for infants and 1.1 g times kg-1 . d-1 for toddlers, should still be safe for nearly all children of comparable biological ages. PMID- 8632937 TI - Molecular basis of phenylketonuria and a correlation between genotype and phenotype in a heterogeneous southeastern US population. AB - OBJECTIVE: To determine the molecular basis of phenylketonuria (PKU) and related hyperphenylalaninemia (HPA) and to establish correlations between phenylalanine hydroxylase (PAH) genotypes and biochemical and clinical phenotypes in an ethnically diverse US population, PAH genotypes were determined in 35 patients with PKU or HPA and 1 carrier from the Medical Genetics Clinic of the Emory University School of Medicine. METHODOLOGY: Patients were identified through Georgia's population-based newborn screening program. PAH genotypes in these individuals were determined from dried blood spots or whole-blood samples using a combination of polymerase chain reaction-mediated amplification, denaturing gradient gel electrophoresis, and direct-sequence analysis. The phenotypic severity of patients with PKU and HPA was based on pretreatment serum phenylalanine (PHE) levels during the neonatal period and on dietary tolerance of PHE later in life. RESULTS: Sixty-eight (96%) of 71 mutant alleles were identified. Major mutations in this population included R408W (11 of 71), I65T (11 of 71), Y414C (6 of 71), L348V (4 of 71), and IVS10 (4 of 71). Five new nucleotide substitutions, E76A (1 of 71), R241L (1 of 71), Q304R (2 of 71), C334S (1 of 71), and R400R (2 of 71) were also detected. Thirty-two of the thirty-five nonrelated patients examined in this study were completely genotyped. Strong correlations were observed between the level of PAH activity predicted from the genotype, when known from previous in vitro expression studies of the mutant proteins, and pretreatment serum PHE levels (r = .841) or clinical severity (Kendall rank-order correlation coefficient, .936). CONCLUSIONS: These results demonstrate strong correlations between PAH genotype and biochemical and clinical phenotypes in this heterogeneous American sample population, extending our previous findings from relatively homogeneous European populations. These correlations further demonstrate the clinical utility of genotype analysis in the treatment of patients with PKU and HPA. PMID- 8632938 TI - A single-sample, subcutaneous gonadotropin-releasing hormone test for central precocious puberty. AB - OBJECTIVE: We compared a rapid, subcutaneous (SQ), single-sample gonadotropin releasing hormone (GnRH) stimulation test with the standard multiple-sample, intravenous (IV) GnRH stimulation test used in the evaluation of central precocious puberty (CPP). METHODS: We evaluated 22 patients presenting with evidence of precocious puberty. GnRH (100 microg) was administered subcutaneously in the clinic setting with single serum luteinizing hormone (LH) measured 40 minutes after injection. A standard IV GnRH stimulation test was performed within 2 weeks, with serum LH obtained at 0, 20, 40, and 60 minutes. LH was assayed by immunochemiluminometric assay. RESULTS: The mean peak LH levels after IV and SQ testing were identical. A significant correlation (r = .88) was found between the LH determined by SQ stimulations and the peak LH determined by IV GnRH testing. CPP was diagnosed (LH, >/- 8 IU/L) by both SQ and IV testing in 7 of 22 patients and was excluded by both tests in 14 of 22 patients. A diagnostic discrepancy between peak IV and SQ results was seen in 1 patient. CONCLUSIONS: We conclude that mean GnRH-stimulated LH levels from rapid SQ and standard IV testing are indistinguishable and that individual LH levels by each method are strongly correlated. A rapid SQ GnRH test is a valid tool for laboratory confirmation of CPP. PMID- 8632939 TI - Evaluation of the effectiveness of a pavement stencil in promoting safe behavior among elementary school children boarding school buses. AB - OBJECTIVE: The majority of school bus-related fatalities among children attending elementary school in the United States occur as children board or alight from buses. Injuries occur during boarding when children enter the street and are struck by buses or other vehicles. This study evaluated the effectiveness of a stencil in the shape of a school bus applied to the pavement at a bus stop in improving safe behaviors at bus stops. Specifically, we assessed the frequency of children running toward the bus as it approached or entered the street. METHODS: Elementary school bus stops with similar roadways, traffic profiles, and numbers of children boarding participated in the study. Stops were randomly assigned to an intervention group, in which children were instructed to remain within a safe area during boarding that was demarcated by a pavement stencil, or an education only group, in which the safe area was demarcated by some existing environmental feature. Both groups received education about safe boarding procedures. Observers rated behavior at each stop daily for 5 consecutive weeks. Data were analyzed as bivariate odds of any unsafe behavior in the education-only group. RESULTS: One hundred forty-five observations from seven bus stops with stencils and 174 daily observations from six education-only stops were completed. Observations of children in the education-only group were twice as likely to show unsafe behavior while waiting (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.3-3.6) and during boarding (OR, 2.1; 95% CI, 1.2-3.9). ORs were significantly higher in the education-only group for boys, girls, and children in grades 4 through 6. When no adult was present, there was a greater likelihood of unsafe behavior among all children in the education-only group while waiting (OR, 16.1; 95% CI, 3.9-72.4) and during boarding (OR, 15.0; 95% CI, 3.2-81.4). The presence of an adult at the stop did not have an independent effect on behavior. Children at education-only stops located on roadways with high traffic volume were more likely to engage in unsafe behavior while waiting (OR, 8.0; 95% CI, 3.8-17.3) and during boarding (OR, 4.9; 95% CI, 2.2-11.0). No differences were observed during boarding between stencil and education-only groups when 10 or more children were at the stops. CONCLUSION: The pavement stencil, when accompanied by education about safe boarding, may represent a cost-effective approach to reducing unsafe behavior at bus stops by children of elementary school age. PMID- 8632940 TI - Sweat chloride concentrations in infants homozygous or heterozygous for F508 cystic fibrosis. AB - OBJECTIVE: To determine whether an adequate volume of sweat could be obtained routinely from infants younger than 6 weeks old and to evaluate sweat chloride levels in infants with known genotype statuses, including heterozygote carriers for cystic fibrosis (CF). METHODOLOGY: Infants were evaluated using pilocarpine iontophoresis and measurement of sweat volume and chloride concentration. The majority of these infants were referred because of newborn screening test results positive for CF based on immunoreactive trypsinogen analysis. DNA analyses for the 3-base pair deletion at codon 508 of the CF transmembrane regulator gene (F508 mutation) were performed whenever possible, and patients with CF were categorized by genotype. RESULTS: Sweat tests were performed successfully (>/-50 mg of sweat) in 99.3% of the infants tested, and there was no difference in the proportion of unsuccessful tests in infants younger than or older than 6 weeks of age. The normal mean +/- SD sweat chloride was 10.6 +/- 5.2 mEq/L (95% confidence interval, 9.9-11.3). Patients with CF who are F508 homozygotes or F508 compound heterozygotes or who have two other non-F508 mutant alleles were shown to have similar sweat chloride levels, with mean values of 99.9, 98.8, and 96.6 mEq/L, respectively. The group of infants who were found to be CF (F508) heterozygote carriers, when compared with the healthy group, had mildly but significantly increased sweat chloride concentrations, with a mean +/- SD of 14.9 +/- 8.4 mEq/L (95% confidence interval, 13.4-16.4). CONCLUSIONS: Quantitative pilocarpine iontophoresis can be used successfully in infants younger than 6 weeks of age who are undergoing routine diagnostic evaluations to follow up newborn screening test results that are positive for CF. The upper limit of normal sweat chloride in infants should be revised to 40 mEq/L (mean + 3 SD of the CF heterozygote carrier group). CF heterozygote carrier infants with one F508 mutant allele show phenotypic manifestations of CF, including subclinical elevations of sweat chloride. PMID- 8632941 TI - Cognition, communication, and hearing in young children with cleft lip and palate and in control children: a longitudinal study. AB - OBJECTIVE: To compare a group of children with cleft lip and palate (CLP) with a group of noncleft, matched control children on measures of cognitive development, speech and language abilities, and audiologic status at 12 and 24 months of age. DESIGN: Using a prospective, longitudinal study design, a group of 16 children with CLP and a group of 16 noncleft control children matched for race, sex, birth order, and socioeconomic status were compared using the Bayley Scales of Infant Development, the Receptive-Expressive Emergent Language Scale, the Sequenced Inventory of Communication Development-Revised (SICD-R), the Preschool Language Scale-Revised (PLS-R), the mean length of utterance, audiometric evaluation, and impedance screening at 12 and 24 months of age. All were free of other congenital abnormalities or known causes of neurodevelopmental dysfunction. RESULTS: Children with CLP had lower mental developmental index (MDI) and psychomotor developmental index scores than control children. They had lower language comprehension scores on the SICD-R and PLS-R and lower expressive language scores on the Receptive-Expressive Emergent Language Scale, SICD-R, and PLS-R than control children. The group with CLP had a significantly higher number of children with abnormal tympanogram results or ventilation tubes at 12 and 24 months. There was a positive correlation between language test scores at 24 months and MDI scores at 24 months (r = .73 to .85). Seventy-five percent of children with CLP who had hearing loss at 12 months had MDI scores more than 1 SD below the mean versus 0% of children with normal hearing. There was a relationship between hearing status at 12 months and comprehension and expressive language scores at 24 months. CONCLUSIONS: Children with CLP had significantly lower scores on tests of cognition, comprehension, and expressive language abilities than matched control children at 12 and 24 months of age. They also had a higher frequency of middle-ear disease and ventilation tubes than control children, although no significant difference in hearing sensitivity was found between groups. Early identification and treatment of these delays may reduce subsequent verbal deficits, hearing loss, and academic difficulties. PMID- 8632942 TI - Evaluating intensive family preservation programs: a methodological review. AB - OBJECTIVES: To determine the adequacy of evaluations of family preservation services (FPS), which are designed to support families and prevent out-of-home placements of children at risk of abuse or neglect, and to assess the effectiveness of FPS at reducing out-of-home placements of children. DATA SOURCES: References published from 1977 to 1993 were identified from a computerized search of databases for English-language publications using the key phrases "family preservation," "child abuse," and "family-based services." Unpublished references were identified by mail or phone from a listing of more than 200 programs in a national directory. SELECTION OF STUDIES: Of 802 references initially identified, 46 program evaluations were reviewed. Ten studies met the following inclusion criteria: (1) evaluated an intensive family preservation program, (2) included outcome data in the report, and (3) used a comparison group. Five were randomized trials, and 5 were quasi-experimental studies (nonrandomized). DATA EXTRACTION: Descriptive information about the programs and evaluations was collected. To determine methodological quality, two independent raters used a 15-item questionnaire to examine the assignment of families to treatment groups, the interventions provided, and the outcomes assessed. A composite score of 11 or greater represented an acceptable study, 6 to 10 represented an adequate study, and 5 or less represented an unacceptable study. RESULTS: Only two studies were rated acceptable, four were adequate, and four were unacceptable. Methodological shortcomings included poorly defined assessment of risk, inadequate descriptions of the interventions provided, and nonblinded determination of the outcomes. Rates of out-of-home placements were 21% to 59% among families who received FPS and 20% to 50% among comparison families. The relative risk of placement was significantly reduced by FPS in only two studies (one randomized trial and one quasi-experimental study). CONCLUSIONS: Despite current widespread use of FPS to prevent out-of-home placements of children, evaluations of FPS are methodologically difficult and show no benefit in reducing rates of out-of-home placements of children at risk of abuse or neglect in 8 of 10 studies. Consistent, methodologically rigorous evaluations are needed to determine the effectiveness of FPS and to guide social policy for high risk children and their families. PMID- 8632943 TI - Validation of a nutritional screen in children with respiratory syncytial virus admitted to an intensive care complex. AB - OBJECTIVE: We sought to validate a nutritional screen that was easy to perform and accurate in identifying pediatric patients at risk for adverse clinical outcomes based on their nutritional status. METHODS: Twenty-five consecutive patients admitted to our pediatric intensive care complex between July 1992 and July 1993 with a primary diagnosis of respiratory syncytial virus infection were evaluated. Nutritional screen parameters included historical (disease and condition), growth (anthropometrics), and laboratory (hemoglobin, lymphocyte count, and albumin) data. Outcome measures included days in the hospital, days of mechanical ventilation, days not fed enterally, and days receiving oxygen. RESULTS: Regression analysis indicated that a score of 5 or less signified a low risk of adverse outcome and a score of greater than 5 signified a high risk. Eleven of 25 patients had low scores, and 14 of 25 had high scores. All outcome measures differed significantly between the low- and high-risk groups: median number of days in the hospital, 7 and 13.5, respectively; median number of days of ventilation, 0 and 8.5, respectively; median number of days without enteral feeding, 3 and 8.5, respectively; and median number of days receiving oxygen, 4 and 20, respectively. CONCLUSIONS: Our nutritional screen identifies patients in the pediatric intensive care complex with respiratory syncytial virus at increased risk for adverse outcome. This screen may be useful in identifying pediatric patients at risk for adverse clinical outcomes from other medical diagnoses. PMID- 8632945 TI - Respiratory characteristics during sleep in healthy small-for- gestational age newborns. AB - OBJECTIVE: Small-for-gestational age (SGA) infants born with intrauterine growth retardation (IUGR) differ from appropriate-for-gestational age (AGA) infants by: a) alterations in a number of neurologic and neurophysiologic characteristics; b) modified heart rate variability during the neonatal period; and c) increased morbidity rates during the first months of life. However, there are no data on the impact of IUGR on respiratory function at birth. METHODS: We studied newborns who were 35 to 36, 37 to 38, and 39 to 41 weeks' conceptional age (CA): 31 were AGA and 26 were SGA. All were clinically and neurologically normal at birth and none exhibited abnormal events during the first year of life. Polygraphic recordings were performed between two meals during the normal postnatal stay in the maternity ward. RESULTS: During both active sleep (AS) and quiet sleep (QS), SGA infants in all CA groups had significantly higher values for the incidence of 2 to 4.9 seconds and 5 to 9.9 seconds central respiratory pauses (RP), the apnea index (AI) [AI=% of nonbreathing time], and the time spent with periodic breathing (PB), as compared with AGA infants. Respiratory frequency was usually similar in SGA and AGA infants. In addition, the trend of age-related respiratory modifications was disturbed in SGA infants, as compared with AGA infants: at 39 to 41 weeks CA, SGA infants had no significant decreases in RP, AI, or PB, and no increase in respiratory frequency. However, between-state differences were similar in both groups. In all AGA and SGA infant groups respiratory frequency seemed to be an individual characteristic: infants who breathed faster during AS breathed faster during QS, and vice-versa. CONCLUSION: Our data demonstrate significant modifications in the establishment of respiratory rhythm control in SGA infants, whereas the patterns of state-related and subject-dependent breathing characteristics were similar in SGA and AGA infants. We speculate that the dysregulation of respiratory function control maturation observed in healthy SGA infants may be related to subtle brainstem modifications attributable to the decreased blood supply and chronic hypoxia associated with IUGR. PMID- 8632944 TI - The relationship between idiopathic mental retardation and maternal smoking during pregnancy. AB - OBJECTIVES: Smoking has been linked to small cognitive, achievement, and behavioral deficits but has not been associated with more severe cognitive impairments. This investigation evaluated the relationship between maternal smoking during pregnancy and idiopathic mental retardation (MR). METHODS: Data on maternal smoking during pregnancy were obtained during face-to-face interviews with the mothers of 221 children with idiopathic MR and the mothers of 400 children attending public school. All children had been born in the five-county metropolitan Atlanta area in 1975 or 1976 and were living in the area when they were 10 years of age. We used exposure odds ratios (ORs) to assess the relationship between maternal smoking and MR, controlling for sex, maternal age at delivery, race, maternal education, economic status, parity, and alcohol use. RESULTS: Maternal smoking during pregnancy was associated with slightly more than a 50% increase in the prevalence of idiopathic MR (adjusted OR, 1.6; 95% confidence interval, 1.0-2.4), and children whose mothers smoked at least one pack a day during pregnancy had more than a 75% increase in the occurrence of idiopathic MR (OR, 1.9; 95% confidence interval, 1.0-3.4). This increase was neither accounted for by other sociodemographic risk factors for MR nor explained by an increase in the prevalence of low birth weight among the children of smokers. CONCLUSIONS: Our data suggest that maternal smoking may be a preventable cause of mental retardation. PMID- 8632947 TI - Infants and young children in orphanages: one view from pediatrics and child psychiatry. AB - A large body of medical knowledge exists that can inform the public policy debate as to whether the current needs and future life prospects of poor children could better be served in orphanages than by continuing safety net programs, such as Aid to Families with Dependent Children, Medicaid, and Supplemental Social Security Income, which maintain children in families. This special article explores a century of pediatric and child psychiatry research covering five areas of potential biologic and social risk to infants and young children in orphanage care: (1) infectious morbidity, (2) nutrition and growth, (3) cognitive development, (4) socioaffective development, and (5) physical and sexual abuse. These data demonstrate that infants and young children are uniquely vulnerable to the medical and psychosocial hazards of institutional care, negative effects that cannot be reduced to a tolerable level even with massive expenditure. Scientific experience consistently shows that, in the short term, orphanage placement puts young children at increased risk of serious infectious illness and delayed language development. In the long term, institutionalization in early childhood increases the likelihood that impoverished children will grow into psychiatrically impaired and economically unproductive adults. PMID- 8632946 TI - Morbidity and mortality in children associated with the use of tobacco products by other people. AB - OBJECTIVE: To evaluate the impact of adult tobacco use on the health of children. DESIGN: A literature review identified relevant research reports. Meta-analysis was used to compute a pooled risk ratio for each condition studied. The risk ratios were combined with data on exposure rates to produce estimates of the population-attributable risk. RESULTS: Each year, among American children, tobacco is associated with an estimated 284 to 360 deaths from lower respiratory tract illnesses and fires initiated by smoking materials, more than 300 fire related injuries, 354,000 to 2.2 million episodes of otitis media, 5200 to 165,000 tympanostomies, 14,000 to 21,000 tonsillectomies and/or adenoidectomies, 529,000 physician visits for asthma, 1.3 to 2 million visits for coughs, and in children younger than 5 years of age, 260,000 to 436,000 episodes of bronchitis and 115,000 to 190,000 episodes of pneumonia. CONCLUSIONS: The use of tobacco products by adults has an enormous adverse impact on the health of children. Although more research is needed in several areas, action to reduce the morbidity and mortality among children should not be delayed. New laws and policies are needed to grant children protection from bodily injury and death attributable to use of tobacco products by others. PMID- 8632948 TI - Making sense of State v Messenger. PMID- 8632950 TI - Facilitated communication. PMID- 8632949 TI - Preliminary experience with intestinal transplantation in infants and children. PMID- 8632953 TI - Nondiscrimination in the care of pediatric patients. Committee++ on Practice and Ambulatory Medicine. American Academy of Pediatrics. PMID- 8632951 TI - Apnea as a presenting sign of hydrocephalus. PMID- 8632954 TI - Nondiscrimination in the care of pediatric patients. Committee on Practice and Ambulatory Medicine. American Academy of Pediatrics. PMID- 8632952 TI - Timing of elective surgery on the genitalia of male children with particular reference to the risks, benefits, and psychological effects of surgery and anesthesia. American Academy of Pediatrics. PMID- 8632955 TI - Committee Report: population-to-pediatrician ratio estimates: a subject review. Committee on Careers and Opportunities. American Academy of Pediatrics. PMID- 8632956 TI - Antenatal exposure to steroids and the additional prophylaxis effect of indomethacin to prevent intraventricular hemorrhage. PMID- 8632957 TI - Colic is pain. PMID- 8632958 TI - More questions than answers. PMID- 8632959 TI - Lead levels, home dust, and proximity to lead smelters. PMID- 8632960 TI - Practice and guidelines. PMID- 8632961 TI - Pharmacologic prevention of coronary artery disease. What do clinical trials show? AB - Can atherosclerotic heart disease be halted or even reversed by aggressive pharmacologic therapy? Studies in recent years have shown that small amounts of plaque regression may be accompanied by considerable reduction in the rate of clinical cardiac events. In this article, the authors examine the results of important studies on lipid-lowering agents, low-dose aspirin, estrogen replacement, and calcium antagonists. PMID- 8632962 TI - Percutaneous revascularization strategies. Usefulness in ischemic coronary artery disease. AB - Current data support the use of revascularization procedures in many patients with ischemic symptoms or silent myocardial ischemia. Patients with extensive disease may benefit even when ischemia cannot be demonstrated. The development of new devices, particularly coronary stents, has broadened the population of patients who can be treated in the cardiac laboratory. Patients with acute myocardial infarction are best treated with direct angioplasty. If thrombolytic therapy is used, rescue angioplasty should probably be done when lysis is unsuccessful. Angioplasty should be used selectively in patients with successful thrombolysis. Both bypass surgery and angioplasty are reasonable initial strategies for patients with stable angina or significant silent ischemia who require coronary revascularization. PMID- 8632963 TI - Influenza in the elderly. Prevention is the best strategy in high-risk populations. AB - Diagnosis of influenza can be difficult in the elderly because of coexisting disease or communication difficulties. However, since the infection often results in complications and may be lethal in these patients, early identification is imperative. Influenza virus vaccine (Fluogen, Fluzone, FluShield) may prevent infection or reduce the severity of complications, even though response may be attenuated in elderly patients. Newer vaccines under investigation may prove to be more efficacious. Education is necessary to improve rates of vaccination, particularly among residents and staff of nursing homes. Antiviral agents are important adjuncts in the fight against influenza, but they are not a substitute for vaccination. PMID- 8632964 TI - Benefits of dietary fiber. Myth or medicine? AB - In recent years, many health claims have been made about dietary and supplemental fiber. However, some reports (eg, those regarding oat bran) have been controversial. A review of scientifically rigorous studies shows that fiber has some preventive or therapeutic benefits in irritable bowel syndrome, diverticulosis, colorectal cancer, diabetes, and hypercholesterolemia. However, it appears to have no direct benefit in patients with inflammatory bowel disease, gallstones, or obesity. The United States has one of the lowest per capita intakes of fiber in the world. Therefore, increasing daily fiber intake either through diet or with supplements is recommended for most Americans. Consumer interest groups should lobby for more fiber-enriched foods. The challenge for education and healthcare professionals alike is to remold the nation's interest in and understanding of dietary fiber. PMID- 8632965 TI - Benign nocturnal leg cramps. Current controversies over use of quinine. AB - Benign nocturnal leg cramps are a relatively common and bothersome complaint, particularly among the elderly. Careful history taking and physical examination can exclude the majority of disorders in the differential diagnosis. Mechanical treatment of an acute muscle cramp involves stretching of the affected muscle. Prophylaxis includes both mechanical and pharmacologic measures. The efficacy of quinine sulfate has been supported in the majority of well-designed studies, but its use is controversial, and the FDA has banned over-the-counter quinine-based products used for leg cramps. Potentially fatal hypersensitivity reactions and thrombocytopenia can occur with use of quinine. PMID- 8632966 TI - Perioperative management of rheumatoid arthritis. Areas of concern for primary care physicians. AB - The presence of rheumatoid arthritis can impose increased risks on patients undergoing surgical procedures. Affected patients often have peripheral joint inflammation, temporomandibular joint disease, laryngeal involvement, or cervical spine disease. Preoperative evaluation should also include assessment of cardiovascular disease, pulmonary disorders, and Sjogren's syndrome. Perioperative control of infection and prophylactic use of antibiotics are particularly important. To avoid wound-healing problems, preoperative discontinuation of therapy with aspirin, nonsteroidal anti-inflammatory drugs, and some slow-acting antirheumatic agents may be necessary. Effective management requires a team approach involving the primary care physician, the surgeon, other subspecialists, and allied healthcare personnel. To prevent unnecessary delays once the patient is hospitalized, preoperative assessment should be done primarily on an outpatient basis. PMID- 8632967 TI - Healthcare--whose business is it? PMID- 8632968 TI - Recurrent streptococcal pharyngitis. Using practical treatment options to interrupt the cycle. AB - Most family physicians have seen cases like this: A child is brought in with pharyngitis, which responds to antibiotic therapy. Soon the mother comes in with fever and a sore throat. She also recovers with therapy, but soon she's back with the child, who has pharyngitis again. Dr Ruoff explains why streptococcal infection recurs and how to assess probability so treatment can be started without waiting for test results. He also discusses alternatives to standard penicillin therapy, some of which may avoid the problems of noncompliance. PMID- 8632969 TI - Prostate-specific antigen testing for prostate cancer. Practical interpretation of values. AB - The mere mention of prostate cancer strikes fear in the hearts of men. Fortunately, prostate-specific antigen (PSA) is now a recognized tumor marker that can indicate but not exclude the presence of this potentially fatal disease. In this article, Drs Randrup and Baum explain the nature of PSA and the practical application of serum PSA determination. Also discussed are three PSA-based strategies for early diagnosis and guidelines for interpreting PSA values in the primary care setting. PMID- 8632970 TI - The solitary pulmonary nodule. Is it malignant or benign? AB - Solitary pulmonary nodules are usually identified on routine chest radiographs in asymptomatic patients. Most nodules have a benign cause, but bronchogenic carcinoma, metastases, and other malignant processes are important causes as well and must be excluded in differential diagnosis. Plain chest radiography, computed tomography, and fine-needle aspiration biopsy are useful diagnostic tools. When a malignant cause cannot be ruled out, the patient's age, smoking history, and nodule size must be considered. Observation by serial radiographs may be the appropriate course for patients who are at low risk for malignancy. For moderate- and high-risk patients, an immediate and more invasive workup is indicated. PMID- 8632971 TI - Iritis. How to recognize and manage a potentially sight-threatening disease. AB - A red eye is a common complaint, often related to benign conditions. However, a red eye in conjunction with symptoms such as photophobia, pain, and decreased visual acuity is an important clue to a much more serious disorder. Iritis is one of these but is reversible with proper use of mydriatic and cycloplegic agents and corticosteroids. Heightened clinical suspicion is necessary for timely diagnosis. The examiner should be skilled in the use of a slit lamp or should refer the patient to an ophthalmologist for immediate evaluation. Once the diagnosis is made, treatment with topical corticosteroids is imperative. Close monitoring is required because overuse of corticosteroids has ominous side effects. PMID- 8632972 TI - Normal sinus heart rates: 60 and 100 bpm are too high. PMID- 8632973 TI - The kindest cut. PMID- 8632974 TI - Controversy over fat content of meat. PMID- 8632975 TI - Stable ischemic heart disease. Using stress and imaging procedures to direct therapy. AB - The predictive value of a screening test depends on its accuracy and the pretest likelihood of disease (i.e, prevalence of the disease in the population). In ischemic heart disease, there are no absolute indications for exercise electrocardiographic testing in asymptomatic patients. However, it is the major noninvasive test for coronary artery disease (CAD). We prefer to use exercise testing whenever possible, because it provides physiologic data that may indicate prognosis and aid in decision making regarding medical treatment. In several situations, exercise assessment combined with radionuclide angiography or echocardiography is indicated for evaluation of patients with CAD. In certain groups of patients, pharmacologic stress testing is preferred. The two methods used most often are dipyridamole administration followed by thallium perfusion imaging and dobutamine administration followed by echocardiography. They have similar sensitivity, specificity, and diagnostic accuracy for CAD and CAD events. Coronary angiography, although invasive, remains the "gold standard" for assessment of CAD and its severity. It is most useful in combination with results of exercise testing. Beta-adrenergic blockers, calcium channel blockers, and nitrates have specific uses in CAD and should be chosen on the basis of their pharmacologic properties. Further details regarding non-pharmacologic and pharmacologic treatment and revascularization strategies in patients with CAD are discussed elsewhere in this symposium. PMID- 8632976 TI - Lifestyle change for coronary artery disease. What to tell patients. AB - Heart-healthy living is a wise choice for anyone, but it assumes even greater importance in persons who have or are at risk for coronary artery disease. Such patients need to know which measures have proved most effective in the prevention and treatment of the disease. This article reviews the data regarding risk factor reduction and offers recommendations for effective lifestyle modifications. PMID- 8632977 TI - Motor learning by field approximation. AB - We investigated how human subjects adapt to forces perturbing the motion of their ams. We found that this kind of learning is based on the capacity of the central nervous system (CNS) to predict and therefore to cancel externally applied perturbing forces. Our experimental results indicate: (i) that the ability of the CNS to compensate for the perturbing forces is restricted to those spatial locations where the perturbations have been experienced by the moving arm. The subjects also are able to compensate for forces experienced at neighboring workspace locations. However, adaptation decays smoothly and quickly with distance from the locations where disturbances had been sensed by the moving limb. (ii) Our experiments also how that the CNS builds an internal model of the external perturbing forces in intrinsic (muscles and / or joints) coordinates. PMID- 8632978 TI - The effects of sequence context on DNA curvature. AB - Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA. PMID- 8632979 TI - Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein. AB - Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins. PMID- 8632980 TI - Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping. AB - Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible. PMID- 8632981 TI - Proteins associated with RNase E in a multicomponent ribonucleolytic complex. AB - The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. PMID- 8632983 TI - A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA. AB - Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation. PMID- 8632982 TI - Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast. AB - Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes. PMID- 8632984 TI - Movement of yeast cortical actin cytoskeleton visualized in vivo. AB - Fusion proteins between the green fluorescent protein (GFP) and the cytoskeleton proteins Act1p (actin), Sac6p (yeast fimbrin homolog), and Abp1p in budding yeast (Saccharomyces cerevisiae) localize to the cortical actin patches. The actin fusions could not function as the sole actin source in yeast, but fusions between the actin-binding proteins Abp1p and Sac6p complement fully the phenotypes associated with their gene deletions. Direct observation in vivo reveals that the actin cortical patches move. Movement of actin patches is constrained to the asymmetric distribution of the patches in growing cells, and this movement is greatly reduced when metabolic inhibitors such as sodium azide are added. Fusion protein-labeled patches are normally distributed during the yeast cell cycle and during mating. In vivo observation made possible the visualization of actin patches during sporulation as well. PMID- 8632985 TI - Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture. AB - Mutations in the endothelin 3 (EDN3) gene severely affect the development of neural crest-derived melanocytes. In this paper, we report the action of EDN3 on neural crest cells in vitro. The presence of EDN3 leads to a large increase in the number of cells, the majority of which eventually differentiate into melanocytes that aggregate to form a reproducible pigmentation pattern. Quantitative analysis of the effect of different culture conditions revealed that EDN3 initially promotes neural crest cell proliferation. This phase of expansion, which can be prolonged for a few weeks if the cells are replaced regularly, is followed by both a decrease in cell proliferation and the onset of melanocytic differentiation. Therefore, EDN3 is a potent mitogen for early neural crest cell precursors that can give rise to melanocytes. PMID- 8632986 TI - Were bowerbirds part of the New Zealand fauna? AB - Bowerbirds (Ptilonorhynchidae) have previously been considered to be confined to the Australo-Papuan continental plate. We provide molecular evidence that the extinct New Zealand Piopio Turnagra capensis is, in fact, a bowerbird. Such a finding is surprising on biogeographical grounds. However, recent molecular evidence on the relationships of the New Zealand moas and kiwis with the Australo Papuan flightless birds suggests the need for a reassessment of current views on the origins of New Zealand's fauna. PMID- 8632987 TI - Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine. AB - Human aging is impacted severely by cardiovascular disease and significantly but less overtly by renal dysfunction. Advanced glycation endproducts (AGEs) have been linked to tissue damage in diabetes and aging, and the AGE inhibitor aminoguanidine (AG) has been shown to inhibit renal and vascular pathology in diabetic animals. In the present study, the effects of AG on aging-related renal and vascular changes and AGE accumulation were studied in nondiabetic female Sprague-Dawley (S-D) and Fischer 344 (F344) rats treated with AG (0.1% in drinking water) for 18 mo. Significant increases in the AGE content in aged cardiac (P < 0.05), aortic (P < 0.005), and renal (P < 0.05) tissues were prevented by AG treatment (P < 0.05 for each tissue). A marked age-linked vasodilatory impairment in response to acetylcholine and nitroglycerine was prevented by AG treatment (P < 0.005), as was an age-related cardiac hypertrophy evident in both strains (P < 0.05). While creatinine clearance was unaffected by aging in these studies, the AGE/ creatinine clearance ratio declined 3-fold in old rats vs. young rats (S-D, P < 0.05; F344, P < 0.01), while it declined significantly less in AG-treated old rats (P < 0.05). In S-D but not in F344 rats, a significant (P < 0.05) age-linked 24% nephron loss was completely prevented by AG treatment, and glomerular sclerosis was markedly suppressed (P < 0.01). Age-related albuminuria and proteinuria were markedly inhibited by AG in both strains (S-D, P < 0.01; F344, P < 0.01). These data suggest that early interference with AGE accumulation by AG treatment may impart significant protection against the progressive cardiovascular and renal decline afflicting the last decades of life. PMID- 8632988 TI - Hippocampal atrophy in recurrent major depression. AB - Hippocampal volumes of subjects with a history of major depressive episodes but currently in remission and with no known medical comorbidity were compared to matched normal controls by using volumetric magnetic resonance images. Subjects with a history of major depression had significantly smaller left and right hippocampal volumes with no differences in total cerebral volumes. The degree of hippocampal volume reduction correlated with total duration of major depression. In addition, large (diameter > or = 4.5 mm)-hippocampal low signal foci (LSF) were found within the hippocampus, and their number also correlated with the total number of days depressed. These results suggest that depression is associated with hippocampal atrophy, perhaps due to a progressive process mediated by glucocorticoid neurotoxicity. PMID- 8632989 TI - A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing. AB - Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA. PMID- 8632990 TI - POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. AB - The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types. PMID- 8632991 TI - Long-time quantum simulation of the primary charge separation in bacterial photosynthesis. AB - Accurate quantum mechanical simulations of the primary charge transfer in photosynthetic reaction centers are reported. The process is modeled by three coupled electronic states corresponding to the photoexcited chlorophyll special pair (donor), the reduced bacteriopheophytin (acceptor), and the reduced accessory chlorophyll (bridge) that interact with a dissipative medium of protein and solvent degrees of freedom. The time evolution of the excited special pair is followed over 17 ps by using a fully quantum mechanical path integral scheme. We find that a free energy of the reduced accessory chlorophyll state approximately equal to 400 cm(-1) lower than that of the excited special pair state yields state populations in agreement with experimental results on wild-type and modified reaction centers. For this energetic configuration electron transfer is a two-step process. PMID- 8632992 TI - Targeted DNA recombination in vivo using an adenovirus carrying the cre recombinase gene. AB - Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in development and disease. The cre-loxP system from bacteriophage P1 has been used in transgenic animals to induce site-specific DNA recombination leading to gene activation or deletion. To regulate the recombination in a spatiotemporally controlled manner, we constructed a recombinant adenoviral vector, Adv/cre, that contained the cre recombinase gene under regulation of the herpes simplex virus thymidine kinase promoter. The efficacy and target specificity of this vector in mediating loxP-dependent recombination were analyzed in mice that had been genetically engineered to contain loxP sites in their genome. After intravenous injection of the Adv/cre vector into adult animals, the liver and spleen showed the highest infectivity of the adenovirus as well as the highest levels of recombination, whereas other tissues such as kidney, lung, and heart had lower levels of infection and recombination. Only trace levels of recombination were detected in the brain. However, when the Adv/cre vector was injected directly into specific regions of the adult brain, including the cerebral cortex, hippocampus, and cerebellum, recombination was detectable at the injection site. Furthermore, when the Adv/cre vector was injected into the forebrains of neonatal mice, the rearranged toxP locus from recombination could be detected in the injected regions for at least 8 weeks. Taken together, these results demonstrate that the Adv/cre vector expressing a functional cre protein is capable of mediating loxP-dependent recombination in various tissues and the recombined gene locus may in some cases be maintained for an extended period. The use of the adenovirus vector expressing cre combined with localized delivery to specific tissues may provide an efficient means to achieve conditional gene expression or knockout with precise spatiotemporal control. PMID- 8632993 TI - A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene. AB - We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene. PMID- 8632994 TI - Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. AB - Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage mediated extracellular matrix proteolysis and tissue invasion. PMID- 8632995 TI - Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. AB - Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function. PMID- 8632996 TI - Isolation of 10 differentially expressed cDNAs in p53-induced apoptosis: activation of the vertebrate homologue of the drosophila seven in absentia gene. AB - We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death. PMID- 8632997 TI - Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region. AB - Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination. PMID- 8632998 TI - STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line. AB - Myeloid leukemia M1 cells can be induced for growth arrest and terminal differentiation into macrophages in response to interleukin 6 (IL-6) or leukemia inhibitory factor (LIF). Recently, a large number of cytokines and growth factors have been shown to activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In the case of IL-6 and LIF, which share a signal transducing receptor gp130, STAT3 is specifically tyrosine phosphorylated and activated by stimulation with each cytokine in various cell types. To know the role of JAK-STAT pathway in M1 differentiation, we have constructed dominant negative forms of STAT3 and established M1 cell lines that constitutively express them. These M1 cells that overexpressed dominant negative forms showed no induction of differentiation-associated markers including Fc gamma receptors, ferritin light chain, and lysozyme after treatment with IL-6. Expression of either c-myb or c-myc was not downregulated. Furthermore, IL-6- and LIF-mediated growth arrest and apoptosis were completely blocked. Thus these findings demonstrate that STAT3 activation is the critical step in a cascade of events that leads to terminal differentiation of M1 cells. PMID- 8632999 TI - CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency. AB - Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI 1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI 1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation. PMID- 8633000 TI - Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen. AB - With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency. PMID- 8633001 TI - Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties. AB - Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording. Nonaxotomized facial motoneurons from wild-type and transgenic mice had similar properties; they had an input resistance of 38 +/- 6 M omega and fired repetitively after injection of positive current pulses. When cells were voltage-clamped at or near their resting membrane potential, alpha-amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), or vasopressin generated sustained inward currents. In transgenic axotomized mice, facial motoneurons could be found located ipsilaterally to the lesion; they had an input resistance of 150 +/- 30 M omega, indicating that they were smaller in size, fired repetitively, and were also responsive to AMPA, NMDA, and vasopressin. Morphological measurements achieved 1 week after the lesion have shown that application of brain-derived neurotrophic factor prevented the reduction in size of axotomized transgenic motoneurons. These data indicate that Bcl2 not only prevents morphological apoptotic death of axotomized neonatal transgenic motoneurons but also permits motoneurons to conserve functional electrophysiological properties. PMID- 8633002 TI - The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons. AB - The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems. PMID- 8633003 TI - Serotonergic modulation of muscle acetylcholine receptors of different subunit composition. AB - Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5 hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. PMID- 8633005 TI - P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans. AB - The P-glycoprotein (Pgp) efflux pump can influence the hepatocellular concentration of xenobiotics that are modulators and substrates of cytochrome P4503A (CYP3A). We tested the hypothesis that Pgp is a determinant of drug inducible expression of CYP3A. The magnitude of CYP3A induction by rifampicin was compared in the human parental colon carcinoma cell line LS 180/WT (wild type) and in two derivative clones overexpressing the human multidrug resistance gene MDR1 (also designated PGY1) because of either drug selection (LS 180/ADR) or transfection with MDRI cDNA (LS 180/MDR). In both MDR1 cDNA-overexpressing clones, rifampicin induction of CYP3A mRNA and protein was decreased and required greater rifampicin concentrations compared with parental cells. The role of Pgp in regulation of CYP3A expression in vivo was analyzed in mice carrying a targeted disruption of the mdr1a mouse gene. Oral treatment with increasing doses of rifampicin resulted in elevated drug levels in the livers of mdr1a (-/-) mice compared with mdr1a (+/+) mice at all doses. Consistent with the enhanced accumulation of rifampicin in mdr1a (-/-) mice, lower doses of rifampicin were required for induction of CYP3A proteins, and the magnitude of CYP3A induction was greater at all doses of rifampicin in mdr1a (-/-) mice compared with mdr1a (+/+) mice. We conclude that Pgp-mediated transport is a critical element influencing the CYP3A inductive response. PMID- 8633004 TI - Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site directed mutagenesis. AB - A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. PMID- 8633007 TI - Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability. AB - In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2 terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors. PMID- 8633008 TI - Electroconvulsive shock and lidocaine reveal rapid consolidation of spatial working memory in the water maze. AB - Head trauma leading to concussion and electroconvulsive shock (ECS) in humans causes amnesia for events that occurred shortly before the injury (retrograde amnesia). The present experiment investigated the amnesic effect of lidocaine and ECS in 25 rats trained on a working memory version of the Morris water task. Each day, the escape platform was moved to a new location; learning was evidenced by a decrease in the latency to find the platform from the first to the second trial. "Consolidation" of this newly encoded spatial engram was disrupted by bilateral inactivation of the dorsal hippocampus with 1 microliter of 4% lidocaine applied as soon as possible after the first trial. When trial 2 was given after recovery from the lidocaine (30 min after the injection), a normal decrease in latency indicated that the new engram was not disrupted. When trial 2 was given under the influence of lidocaine (5 min after injection), absence of latency decrease demonstrated both the success of the inactivation and the importance of hippocampus for the task. To examine the role of events immediately after learning, ECS (30 or 100 mA, 50 Hz, 1.2 sec) was applied 0 sec to 45 sec after a single escape to the new platform location. A 2-h delay between ECS and trial 2 allowed the effects of ECS to dissipate. ECS applied 45 sec or 30 sec after trial 1 caused no retrograde amnesia: escape latencies on trial 2 were the same as in control rats. However, ECS applied 0 sec or 15 sec after trial 1 induced clear retrograde amnesia: escape latencies on trial 2 were no shorter than on trial 1. It is concluded that the consolidation of a newly formed memory for spatial location can only be disrupted by ECS within 30 sec after learning. PMID- 8633006 TI - cemA homologue essential to CO2 transport in the cyanobacterium Synechocystis PCC6803. AB - We have isolated mutants of Synechocystis PCC6803 that grew very slowly in a low sodium medium, which is unfavorable for HCO3(-) transport, and examined two of these mutants (SC1 and SC2) for their ability to take up CO2 and HCO3(-) in the light. The CO2 transport activity of SC1 and SC2 was much lower than that of the wild type (WT), whereas there was no difference between the mutants and the WT in their activity of HCO3(-) transport. A clone containing a 3.9-kilobase-pair insert DNA that transforms both mutants to the WT phenotype was isolated from a genomic library of WT Synechocystis. Sequencing of the insert DNA in the region of mutations in SC1 and SC2 revealed an open reading frame (designated cotA), which showed significant amino-acid sequence homology to cemA encoding a protein found in the inner envelope membrane of chloroplasts. The cotA gene is present in a single copy and was not cotranscribed with any other gene(s). cotA encodes a protein of 247 amino acids containing four transmembrane domains. There was substitution of a single base in SC1 and two bases in SC2 in their cotA genes. A possible role of the cotA gene product in CO2 transport is discussed. PMID- 8633009 TI - Mapping protein-protein interactions by affinity-directed mass spectrometry. AB - A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule molecule contacts, including ligand-receptor and protein-oligonucleotide interactions. PMID- 8633010 TI - Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel. AB - The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121 134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions. PMID- 8633011 TI - Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism. AB - We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event. PMID- 8633012 TI - Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains. AB - Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base. PMID- 8633013 TI - Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. AB - Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly 6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL 3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated. PMID- 8633014 TI - Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. AB - The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor development suggest a fundamental role for this enzyme in the initiation and progression of cancer. A true functional role for DNA-MTase in the neoplastic process would be further substantiated if the target cells affected by the initiating carcinogen exhibit changes in enzyme activity. This hypothesis was addressed by examining DNA-MTase activity in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity was found only in the target alveolar type II cells of the susceptible A/J mouse and caused a marked increase in overall DNA methylation in these cells. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. Increased gene expression was also detected by RNA in situ hybridization in hypertrophic alveolar type II cells of carcinogen-treated A/J mice, indicating that elevated levels of expression may be a biomarker for premalignancy. Enzyme activity increased incrementally during lung cancer progression and coincided with increased expression of the DNA-MTase activity are strongly associated with neoplastic development and constitute a key step in carcinogenesis. The detection of premalignant lung disease through increased DNA-MTase expression and the possibility of blocking the deleterious effects of this change with specific inhibitors will offer new intervention strategies for lung cancer. PMID- 8633015 TI - Immunologic basis of transplant-associated arteriosclerosis. AB - Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation. PMID- 8633016 TI - DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism. AB - Recent biochemical and crystallographic results suggest that a type II DNA topoisomerase acts as an ATP-modulated clamp with two sets of jaws at opposite ends: a DNA-bound enzyme can admit a second DNA through one set of jaws; upon binding ATP, this DNA is passed through an enzyme-mediated opening in the first DNA and expelled from the enzyme through the other set of jaws. Experiments based on the introduction of reversible disulfide links across one dimer interface of yeast DNA topoisomerase II have confirmed this mechanism. The second DNA is found to enter the enzyme through the gate formed by the N-terminal parts of the enzyme and leave it through the gate close to the C termini. PMID- 8633017 TI - Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins. AB - The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG. PMID- 8633018 TI - Homophilic adhesion mediated by the neural cell adhesion molecule involves multiple immunoglobulin domains. AB - The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation. PMID- 8633019 TI - 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. AB - It has previously been argued that the repressor of protein synthesis initiation factor 4E, 4E-BP1, is a direct in vivo target of p42mapk. However, the immunosuppressant rapamycin blocks serum-induced 4E-BP1 phosphorylation and, in parallel, p70s6k activation, with no apparent effect on p42mapk activation. Consistent with this finding, the kinetics of serum-induced 4E-BP1 phosphorylation closely follow those of p70s6k activation rather than those of p42mapk. More striking, insulin, which does not induce p42mapk activation in human 293 cells or Swiss mouse 3T3 cells, induces 4E-BP1 phosphorylation and p70s6k activation in both cell types. Anisomycin, which, like insulin, does not activate p42mapk, promotes a small parallel increase in 4E-BP1 phosphorylation and p70s6k activation. The insulin effect on 4E-BP1 phosphorylation and p70s6k activation in both cell types is blocked by SQ20006, wortmannin, and rapamycin. These three inhibitors have no effect on p42mapk activation induced by phorbol 12 tetradecanoate 13-acetate, though wortmannin partially suppresses both the p70s6k response and the 4E-BP1 response. Finally, in porcine aortic endothelial cells stably transfected with either the wild-type platelet-derived growth factor receptor or a mutant receptor bearing the double point mutation 740F/751F, p42mapk activation in response to platelet-derived growth factor is unimpaired, but increased 4E-BP1 phosphorylation is ablated, as previously reported for p70s6k. The data presented here demonstrate that 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. PMID- 8633020 TI - Metabolism of Alzheimer beta-amyloid precursor protein: regulation by protein kinase A in intact cells and in a cell-free system. AB - Various compounds that affect signal transduction regulate the relative utilization of alternative processing pathways for the beta-amyloid precursor protein (beta APP) in intact cells, increasing the production of nonamyloidogenic soluble beta APP (s beta APP) and decreasing that of amyloidogenic beta-amyloid peptide. In a recent study directed toward elucidating the mechanisms underlying phorbol ester-stimulated s beta APP secretion from cells, it was demonstrated that protein kinase C increases the formation from the trans-Golgi network (TGN) of beta APP-containing secretory vesicles. Here we present evidence that forskolin increases s beta APP production from intact PC12 cells, and protein kinase A stimulates formation from the TGN of beta APP-containing vesicles. Although protein kinase A and protein kinase C converge at the level of formation from the TGN of beta APP-containing vesicles, additional evidence indicates that the regulatory mechanisms involved are distinct. PMID- 8633021 TI - Dominant-negative p53 mutations selected in yeast hit cancer hot spots. AB - Clinically important mutant p53 proteins may be tumorigenic through a dominant negative mechanism or due to a gain-of-function. Examples for both hypotheses have been described; however, it remains unclear to what extent they apply to TP53 mutations in general. Here it is shown that the mutational spectrum of dominant-negative p53 mutants selected in a novel yeast assay correlates tightly with p53 mutations in cancer. Two classes of dominant-negative mutations are described; the more dominant one affects codons that are essential for the stabilization of the DNA-binding surface of the p53 core domain and for the direct interaction of p53 with its DNA binding sites. These results predict that the vast majority of TP53 mutations leading to cancer do so in a dominant negative fashion. PMID- 8633022 TI - Identification of the promoter of the mouse obese gene. AB - Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis regulatory elements. A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box. Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts. When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression. When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active. Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression. Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells. These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene. PMID- 8633023 TI - Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. AB - The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer. PMID- 8633024 TI - Ethinylestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production. AB - Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism by which this protection is mediated is unclear. Since animal studies strongly suggest that production of endothelium derived relaxing factor is enhanced by estradiol, we have examined the effect of estrogens on nitric oxide (NO) synthase (NOS) activity, protein, and mRNA in cultured bovine aortic endothelial cells. In reporter cells rich in guanylate cyclase, it has been observed that long-term treatment (> or = 24 hr) with ethinylestradiol (EE2) dose-dependently increased guanylate cyclase-activating factor activity in the conditioned medium of endothelial cells. However, conversion of L-[14C]arginine to L-[14C]citrulline by endothelial cell homogenate or quantification of nitrite and nitrate released by intact cells in the conditioned medium did not reveal any change in NOS activity induced by EE2 treatment. Similarly, Western and Northern blot analyses did not reveal any change in the endothelial NOS protein and mRNA content in response to EE2. However, EE2 dose- and time-dependently decreased superoxide anion production in the conditioned medium of endothelial cells with an EC50 value (0.1 nM) close to that which increased guanylate cyclase-activating factor activity (0.5 nM). Both of these effects were completely prevented by the antiestrogens tamoxifen and RU54876. Thus, endothelium exposure to estrogens appears to induce a receptor mediated antioxidant effect that enhances the biological activity of endothelium derived NO. These effects could account at least in part for the vascular protective properties of these hormones. PMID- 8633026 TI - Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns. AB - Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer. PMID- 8633025 TI - Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma. AB - High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse. PMID- 8633027 TI - Aromatic amino acid transamination and methionine recycling in trypanosomatids. AB - Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids. PMID- 8633028 TI - Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein. AB - Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity. PMID- 8633029 TI - Foamy virus reverse transcriptase is expressed independently from the Gag protein. AB - In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. PMID- 8633030 TI - Cell density regulates cellular reversal frequency in Myxococcus xanthus. AB - Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting. Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal. In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min. It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M. xanthus. In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups. We found that developmental cells in large groups reverse much less than cells in small groups or as single cells. This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein). Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way. The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation. We also found that M. xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail." PMID- 8633031 TI - Neurocytopathic effects of beta-amyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease. AB - Growing evidence indicates that cells of the mononuclear phagocyte lineage, which includes peripheral blood monocytes (PBM) and tissue macrophages, participate in a variety of neurodestructive events and may play a pivotal role in neurodegenerative conditions such as Alzheimer disease. The present study sought to determine whether exposure of PBM to beta-amyloid peptide (A beta), the major protein of the amyloid fibrils that accumulate in the brain in Alzheimer disease, could induce cytopathic activity in these cells upon their subsequent incubation with neural tissue. PBM were incubated with A beta for 3 days, centrifuged and washed to remove traces of cell-free A beta, and then applied to organotypic cultures of rat brain for varying periods of time. By using a cell-viability assay to quantitate neurocytopathic effect, an increase in the ratio of dead to live cells was detected in cultures containing A beta-stimulated PBM versus control PBM (stimulated with either bovine serum albumin or reverse A beta peptide) as early as 3 days after coculture. The ratio of dead to live cells increased further by 10 days of coculture. By 30 days of coculture, the dead to live cell ratio remained elevated, and the intensity of neurocytopathic effect was such that large areas of brain mass dissociated from the cultures. These results indicate that stimulation of PBM with A beta significantly heightens their neurocytopathic activity and highlight the possibility that inflammatory reactions in the brain play a role in the neurodegeneration that accompanies Alzheimer disease. PMID- 8633032 TI - Retinal glial cell glutamate transporter is coupled to an anionic conductance. AB - Application of L-glutamate to retinal glial (Muller) cells results in an inwardly rectifying current due to the net influx of one positive charge per molecule of glutamate transported into the cell. However, at positive potentials an outward current can be elicited by glutamate. This outward current is eliminated by removal of external chloride ions. Substitution of external chloride with the anions thiocyanate, perchlorate, nitrate, and iodide, which are known to be more permeant at other chloride channels, results in a considerably larger glutamate elicited outward current at positive potentials. The large outward current in external nitrate has the same ionic dependence, apparent affinity for L glutamate, and pharmacology as the glutamate transporter previously reported to exist in these cells. Varying the concentration of external nitrate shifts the reversal potential in a manner consistent with a conductance permeable to nitrate. Together, these results suggest that the glutamate transporter in retinal glial cells is associated with an anionic conductance. This anionic conductance may be important for preventing a reduction in the rate of transport due the depolarization that would otherwise occur as a result of electrogenic glutamate uptake. PMID- 8633033 TI - Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-aging mechanism in rodents. AB - Many studies of caloric restriction (CR) in rodents and lower animals indicate that this nutritional manipulation retards aging processes, as evidenced by increased longevity, reduced pathology, and maintenance of physiological function in a more youthful state. The anti-aging effects of CR are believed to relate, at least in part, to changes in energy metabolism. We are attempting to determine whether similar effects occur in response to CR in nonhuman primates. Core (rectal) body temperature decreased progressively with age from 2 to 30 years in rhesus monkeys fed ad lib (controls) and is reduced by approximately 0.5 degrees C in age-matched monkeys subjected to 6 years of a 30% reduction in caloric intake. A short-term (1 month) 30% restriction of 2.5-year-old monkeys lowered subcutaneous body temperature by 1.0 degrees C. Indirect calorimetry showed that 24-hr energy expenditure was reduced by approximately 24% during short-term CR. The temporal association between reduced body temperature and energy expenditure suggests that reductions in body temperature relate to the induction of an energy conservation mechanism during CR. These reductions in body temperature and energy expenditure are consistent with findings in rodent studies in which aging rate was retarded by CR, now strengthening the possibility that CR may exert beneficial effects in primates analogous to those observed in rodents. PMID- 8633034 TI - Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice. AB - One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1 day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4 8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single positive cells and the maintenance of thymocyte viability during the double positive stage of intrathymic differentiation. PMID- 8633036 TI - Plasmoviruses: nonviral/viral vectors for gene therapy. AB - We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells. PMID- 8633035 TI - The phosphatase activity of carbonic anhydrase III is reversibly regulated by glutathiolation. AB - Carbonic anhydrase isozyme III (CAIII) is unique among the carbonic anhydrases because it demonstrates phosphatase activity. CAIII forms a disulfide link between glutathione and two of its five cysteine residues, a process termed S glutathiolation. Glutathiolation of CAIII occurs in vivo and is increased during aging and under acute oxidative stress. We show that glutathiolation serves to reversibly regulate the phosphatase activity of CAIII. Glutathiolation of Cys-186 is required for phosphatase activity, while glutathiolation of Cys-181 blocks activity. Phosphotyrosine is the preferred substrate, although phosphoserine and phosphothreonine can also be cleaved. Thus, glutathiolation is a reversible covalent modification that can regulate CAIII, a phosphatase that may function in the cellular response to oxidative stress. PMID- 8633038 TI - Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes. AB - Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes. PMID- 8633037 TI - Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5). AB - We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation. PMID- 8633040 TI - Macrophage killing is an essential virulence mechanism of Salmonella typhimurium. AB - Phagocytic cells are a critical line of defense against infection. The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host. A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence. Salmonella is one of these pathogens. Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells. Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages. All four insertions were located in the ompR/envZ regulon. These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells. PMID- 8633039 TI - Converting cancer genes into killer genes. AB - Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known. PMID- 8633041 TI - Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain of peptidyl-tRNA. AB - The mechanism by which elongation factor G (EF-G) catalyzes the translocation of tRNAs and mRNA on the ribosome is not known. The reaction requires GTP, which is hydrolyzed to GDP. Here we show that EF-G from Escherichia coli lacking the G domain still catalyzed partial translocation in that it promoted the transfer of the 3' end of peptidyl-tRNA to the P site on the 50S ribosomal subunit into a puromycin-reactive state in a slow-turnover reaction. In contrast, it did not bring about translocation on the 30S subunit, since (i) deacylated tRNA was not released from the P site and (ii) the A site remained blocked for aminoacyl-tRNA binding during and after partial translocation. The reaction probably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that is not puromycin-reactive [Moazed, D. & Noller, H. F. (1989) Nature (London) 342, 142-148]. In the complete system--i.e., with intact EF-G and GTP--the 50S phase of translocation is rapidly followed by the 30S phase during which the tRNAs together with the mRNA are shifted on the small ribosomal subunit, and GTP is hydrolyzed. As to the mechanism of EF-G function, the results show that the G domain has an important role, presumably exerted through interactions with other domains of EF-G, in the promotion of translocation on the small ribosomal subunit. The G domain's intramolecular interactions are likely to be modulated by GTP binding and hydrolysis. PMID- 8633042 TI - A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane. AB - Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins. PMID- 8633044 TI - Myofibroblasts differentiate from fibroblasts when plated at low density. AB - Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF beta. PMID- 8633043 TI - Pax3 modulates expression of the c-Met receptor during limb muscle development. AB - Pax3 is a transcription factor whose expression has been used as a marker of myogenic precursor cells arising in the lateral somite destined to migrate to and populate the limb musculature. Accruing evidence indicates that the embryologic origins of axial and appendicular muscles are distinct, and limb muscle abnormalities in both mice and humans harboring Pax3 mutations support this distinction. The mechanisms by which Pax3 affects limb muscle development are unknown. The tyrosine kinase receptor for hepatocyte growth factor/scatter factor encoded by the c-met protooncogene is also expressed in limb muscle progenitors and, like Pax-3, is required in the mouse for limb muscle development. Here, we show that c-met expression is markedly reduced in the lateral dermomyotome of Splotch embryos lacking Pax3. We show that Pax3 can stimulate c-met expression in cultured cells, and we identify a potential Pax3 binding site in the human c-MET promoter that may contribute to direct transcriptional regulation. In addition, we have found that several cell lines derived from patients with rhabdomyosarcomas caused by a t(2;13) chromosomal translocation activating PAX3 express c-MET, whereas those rhabdomyosarcoma cell lines examined without the translocation do not. These results are consistent with a model in which Pax3 modulates c-met expression in the lateral dermomyotome, a function that is required for the appropriate migration of these myogenic precursors to the limb where the ligand for c-met (hepatocyte growth factor/scatter factor) is expressed at high levels. PMID- 8633045 TI - Phosphorylation of the fused protein kinase in response to signaling from hedgehog. AB - The hedgehog gene (hh) of Drosophila melanogaster exerts both short- and long range effects on cell patterning during development. The product of hedgehog is a secreted protein that apparently acts by triggering an intra-cellular signaling pathway, but little is known about the details of that pathway. The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase that genetic experiments have implicated in signaling initiated by hedgehog. Here we report that the fused protein is phosphorylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. In cell culture, phosphorylation of fused protein occurs in response to the biologically active form of hedgehog and cannot be blocked by activation of protein kinase A, which is thought to be an antagonist of signaling from hedgehog. These results suggest that fused and protein kinase A function downstream of hedgehog but in parallel pathways that eventually converge distal to fused. The reconstruction of signaling from hedgehog in cell culture should provide further access to the mechanisms by which hedgehog acts. PMID- 8633047 TI - Mixed synapses discovered and mapped throughout mammalian spinal cord. AB - Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system. PMID- 8633046 TI - Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers. AB - A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium. PMID- 8633049 TI - Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP. AB - Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release. PMID- 8633048 TI - Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism. AB - Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-) sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control. PMID- 8633050 TI - A general assay for antibody catalysis using acridone as a fluorescent tag. AB - A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected. PMID- 8633051 TI - A rigid trans-spanning dinitrile ligand. AB - A rigid dinitrile ligand was synthesized from two xanthene units condensed to a naphthalene-1,4,5,8-diimide spacer. The rigidity and C shape of the ligand gave exclusively trans complexes with Pd(II), Ag(I), and Au(I). Evidence for complexation, coordination geometry, and stoichiometry was provided by a combination of 1H NMR, 19F NMR, and IR spectroscopy. The AuBF4 and PdCl2 complexes were shown to have a 1:1 (metal-to-ligand) stoichiometry and the AgBF4 complex was shown to have a 1:2 stoichiometry in solution. The preorganization of the dinitrile ligand resulted in complexes much more stable than their monodentate counterparts. PMID- 8633052 TI - Bond orientational order, molecular motion, and free energy of high-density DNA mesophases. AB - By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This change in order was previously inferred from intermolecular force measurements and is now confirmed by 31P NMR. Controlled reversible swelling and compaction under osmotic stress, spanning a range of densities between approximately 120 mg/ml to approximately 600 mg/ml, allow measurement of the free-energy changes throughout each phase and at the phase transition, essential information for theories of liquid-crystalline states. PMID- 8633053 TI - Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H NHase) induced by its reaction product in Rhodococcus rhodochrous J1. AB - The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates). PMID- 8633054 TI - Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53. AB - Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells. PMID- 8633055 TI - Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24). AB - Plectin, a 500-kDa intermediate filament binding protein, has been proposed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton. To set the basis for future studies on gene regulation, tissue-specific expression, and pathological conditions involving this protein, we have cloned the human plectin gene, determined its coding sequence, and established its genomic organization. The coding sequence contains 32 exons that extend over 32 kb of the human genome. Most of the introns reside within a region encoding the globular N-terminal domain of the molecule, whereas the entire central rod domain and the entire C-terminal globular domain were found to be encoded by single exons of remarkable length, >3 kb and >6 kb, respectively. Overall, the organization of the human plectin gene was strikingly similar to that of human bullous pemphigoid antigen 1 (BPAG1), confirming that both proteins belong to the same gene family. Comparison of the deduced protein sequences for human and rat plectin revealed that they were 93% identical. By using fluorescence in situ hybridization, we have mapped the plectin gene to the long arm of chromosome 8 within the telomeric region. This gene locus (8q24) has previously been implicated in the human blistering skin disease epidermolysis bullosa simplex Ogna. Detailed knowledge of the structure of the plectin gene and its chromosome localization will aid in the elucidation of whether this or any other pathological conditions are linked to alterations in the plectin gene. PMID- 8633056 TI - Potentiation of the bioavailability of daidzin by an extract of Radix puerariae. AB - The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Apparently the antidipsotropic activity of the RP extract cannot be accounted for solely by its daidzin content (22 mg/g). In addition to daidzin, six other isoflavones were identified in the RP extract and quantified--namely, puerarin (160 mg per g of extract), genistin (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). None of these, administered either alone or combined, contributes in any significant way to the antidipsotropic activity of the extract. Plasma daidzin concentration-time curves determined in hamsters administered various doses of pure daidzin or RP extract by i.p.injection indicate that the crude extract daidzin has approximately 10 times greater bioavailability than the pure compound. Reconstruction of the dose-response effects for pure and crude daidzin using bioavailable daidzin rather than administered dose gives a single curve. Synthetic daidzin added to the RP extract acquires the bioavailability of the endogenous daidzin that exists naturally in the extract. These results show that (i) daidzin is the major active principle in methanol extracts of RP, and (ii) additional constituents in the methanol extract of RP assist uptake of daidzin in golden hamsters. PMID- 8633057 TI - The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein. AB - The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes. PMID- 8633059 TI - Mammalian phospholipase D: phosphatidylethanolamine as an essential component. AB - Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn. PMID- 8633058 TI - E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins. AB - Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties. PMID- 8633060 TI - Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor. AB - Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5. PMID- 8633061 TI - Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo. AB - Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations. PMID- 8633062 TI - Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis. AB - The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death. PMID- 8633063 TI - Prediction of the stability of DNA triplexes. AB - We present rules that allow one to predict the stability of DNA pyrimidine.purine.pyrimidine (Y.R.Y) triple helices on the basis of the sequence. The rules were derived from van't Hoff analysis of 23 oligonucleotide triplexes tested at a variety of pH values. To predict the enthalpy of triplex formation (delta H degrees), a simple nearest-neighbor model was found to be sufficient. However, to accurately predict the free energy of the triplex (delta G degrees), a combination model consisting of five parameters was needed. These parameters were (i) the delta G degrees for helix initiation, (ii) the delta G degrees for adding a T-A.T triple, (iii) the delta G degrees for adding a C(+)-G.C triple, (iv) the penalty for adjacent C bases, and (v) the pH dependence of the C(+)-G.C triple's stability. The fitted parameters are highly consistent with thermodynamic data from the basis set, generally predicting both delta H degrees and delta G degrees to within the experimental error. Examination of the parameters points out several interesting features. The combination model predicts that C(+) -G.C. triples are much more stabilizing than T-A.T triples below pH 7.0 and that the stability of the former increases approximately equal to 1 kcal/mol per pH unit as the pH is decreased. Surprisingly though, the most stable sequence is predicted to be a CT repeat, as adjacent C bases partially cancel the stability of one another. The parameters successfully predict tm values from other laboratories, with some interesting exceptions. PMID- 8633064 TI - Design and synthesis of ribonucleic guanidine: a polycationic analog of RNA. AB - Replacement of the phosphodiester linkages of the polyanion RNA with guanidinium linkers (represented by g) provides the polycation ribonucleic guanidine (RNG). An anticipated structure for the triple-helical hybrid [r(Up)9U.r(Ag)9A.r(Up)9U] is presented. A basic strategy for the synthesis of RNG oligomers is described. Synthetic procedures are provided for tetrameric adenosyl RNG [r(Ag)3A]. PMID- 8633066 TI - Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene requires a specific core promoter structure. AB - The terminal deoxynucleotidyltransferase (TdT) gene encodes a template independent DNA polymerase that is expressed exclusively in immature lymphocytes. The TdT promoter lacks a TATA box, but an initiator element (Inr) overlaps the transcription start site. The Inr directs basal transcription and also mediates activated transcription in conjunction with an upstream element called D'. We have begun to address the fundamental question of why the TdT promoter contains an Inr rather than a TATA box. First, we tested the possibility that the TdT promoter lacks a TATA box because the -30 region is needed for the binding of an essential regulator. Mutations were introduced into the -30 region, and the mutants were tested in transient transfection and in vitro transcription assays. The mutations had only minor effects on promoter strength, suggesting that this first hypothesis is incorrect. Next, the effect of inserting a TATA box within the -30 region was tested. Although the TATA box enhanced promoter strength, appropriate regulation appeared to be maintained, as transcription in lymphocytes remained dependent on the D' element. Finally, a promoter variant containing a TATA box at -30, but a mutant Inr, was tested. Surprisingly, transcription from this variant, both in vitro and in vivo, was dramatically reduced. These results suggest that the TdT promoter, and possibly other natural promoters, contain an Inr element because one or more activator proteins that interact with surrounding control elements preferentially function in its presence. PMID- 8633065 TI - Characterization of mouse angiogenin-related protein: implications for functional studies on angiogenin. AB - Angiogenin-related protein (Angrp), the putative product of a recently discovered mouse gene, shares 78% sequence identity with mouse angiogenin (Ang). In the present study, the relationship of Angrp to Ang has been investigated by producing both proteins in bacteria and comparing their functional properties. We find that mouse Ang is potently angiogenic, but Angrp is not, even when assayed at relatively high doses. A deficiency in catalytic capacity, which is essential for the biological activity of Ang, does not appear to underlie Angrp's lack of angiogenicity. In fact, Angrp has somewhat greater ribonucleolytic activity toward tRNA and dinucleotide substrates than does Ang. Instead, an inability to bind cellular receptors is implicated since Angrp does not inhibit Ang-induced angiogenesis. Poor conservation of the Ang receptor recognition sequence 58-69 in Angrp most likely contributes to this defect. However, other substitutions must also influence receptor binding since an Angrp quadruple mutant that is identical to Ang in this segment still lacks both angiogenic activity and the capacity to inhibit Ang. The functional differences between Ang and Angrp, together with evidence presented herein that Angrp is regulated differently than Ang, suggest that the roles of the two proteins in vivo may be quite distinct. PMID- 8633067 TI - Dressed polyions, counterion condensation, and adsorption excess in polyelectrolyte solutions. AB - The phenomenon of Manning-Oosawa counterion condensation is given an explicit statistical mechanical and qualitative basis via a dressed polyelectrolyte formalism in connection with the topology of the electrostatic free-energy surface and is derived explicitly in terms of the adsorption excess of ions about the polyion via the nonlinear Poisson-Boltzmann equation. The approach is closely analogous to the theory of ion binding in micelles. Our results not only elucidate a Poisson-Boltzmann analysis, which shows that a fraction of the counterions lie within a finite volume around the polyion even if the volume of the system tends towards infinity, but also provide a direct link between Manning's theta-the number of condensed counterions for each polyion site-and a statistical thermodynamic quantity, namely, the adsorption excess per monomer. PMID- 8633068 TI - Differences in the RNA binding sites of iron regulatory proteins and potential target diversity. AB - Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system. PMID- 8633070 TI - ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation. AB - ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2. PMID- 8633069 TI - Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity. AB - Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity. PMID- 8633071 TI - DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement. AB - DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations. PMID- 8633072 TI - Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation. AB - Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations. PMID- 8633073 TI - Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon. AB - The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones. PMID- 8633074 TI - Mutation detection with MutH, MutL, and MutS mismatch repair proteins. AB - Escherichia coli methyl-directed mismatch repair is initiated by MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which cleaves at d(GATC) sites in the vicinity of a mismatch. This reaction provides an efficient method for detection of mismatches in heteroduplexes produced by hybridization of genetically distinct sequences after PCR amplification. Multiple examples of transition and transversion mutations, as well as one, two, and three nucleotide insertion/deletion mutants, have been detected in PCR heteroduplexes ranging in size from 400 bp to 2.5 kb. Background cleavage of homoduplexes is largely due to polymerase errors that occur during amplification, and the MutHLS reaction provides an estimate of the incidence of mutant sequences that arise during PCR. PMID- 8633075 TI - Mutator tRNAs are encoded by the Escherichia coli mutator genes mutA and mutC: a novel pathway for mutagenesis. AB - We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1 2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed. PMID- 8633076 TI - Kinetics of cytokine expression during primary human immunodeficiency virus type 1 infection. AB - In the present study, we have determined the kinetics of constitutive expression of a panel of cytokines [interleukin (IL) 2, IL-4, IL-6, IL-10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha)] in sequential peripheral blood mononuclear cell samples from nine individuals with primary human immunodeficiency virus infection. Expression of IL-2 and IL-4 was barely detected in peripheral blood mononuclear cells. However, substantial levels of IL 2 expression were found in mononuclear cells isolated from lymph node. Expression of IL-6 was detected in only three of nine patients, and IL-6 expression was observed when transition from the acute to the chronic phase had already occurred. Expression of IL-10 and TNF-alpha was consistently observed in all patients tested, and levels of both cytokines were either stable or progressively increased over time. Similar to IL-10 and TNF-alpha, IFN-gamma expression was detected in all patients; however, in five of nine patients, IFN-gamma expression peaked very early during primary infection. The early peak in IFN-gamma expression coincided with oligoclonal expansions of CD8+ T cells in five of six patients, and CD8+ T cells mostly accounted for the expression of this cytokine. These results indicate that high levels of expression of proinflammatory cytokines are associated with primary infection and that the cytokine response during this phase of infection is strongly influenced by oligoclonal expansions of CD8+ T cells. PMID- 8633077 TI - Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2). AB - The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus controlling region, is highly responsive to any of several participating factors. PMID- 8633078 TI - Viral dynamics in hepatitis B virus infection. AB - Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapid decline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistently infected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50% daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particles per day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cells in two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitis B antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-lives for virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysis of infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivo and has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study also represents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total daily production of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-life of virus producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infected patients treated for up to 24 weeks. PMID- 8633080 TI - Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. AB - The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1 tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed. PMID- 8633079 TI - Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection. AB - The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes. PMID- 8633081 TI - Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. AB - Cholecystokinin (CCK) secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition of CCK release caused by pancreatic proteases is due to proteolytic inactivation of a CCK-releasing peptide present in intestinal secretion. To purify the putative luminal CCK-releasing factor (LCRF), intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in conscious rats. From these secretions, the peptide was concentrated by ultrafiltration followed by low-pressure reverse-phase chromatography and purified by reverse-phase high-pressure liquid chromatography. Purity was confirmed by high-performance capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially purified fractions strongly stimulated both pancreatic secretion and CCK release while CCK receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for 41 residues as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV. When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose related manner in conscious rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRF-(1-6) abolished the CCK releasing activity of intestinal secretions. These studies demonstrate, to our knowledge, the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release. PMID- 8633082 TI - Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway. AB - The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway. PMID- 8633083 TI - Heterometallic hybrids of homometallic human hemoglobins. AB - Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered. PMID- 8633084 TI - Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor. AB - Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such. PMID- 8633085 TI - Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. AB - Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally. PMID- 8633086 TI - A methylated human 9-kb repetitive sequence on acrocentric chromosomes is homologous to a subtelomeric repeat in chimpanzees. AB - We have implemented an approach for the detection of DNA alterations in cancer by means of computerized analysis of end-labeled genomic fragments, separated in two dimensions. Analysis of two-dimensional patterns of neuroblastoma tumors, prepared by first digesting DNA with the methylation-sensitive restriction enzyme Not I, yielded a multicopy fragment which was detected in some tumor patterns but not in normal controls. Cloning and sequencing of the fragment, isolated from two dimensional gels, yielded a sequence with a strong homology to a subtelomeric sequence in chimpanzees and which was previously reported to be undetectable in humans. Fluorescence in situ hybridization indicated the occurrence of this sequence in normal tissue, for the most part in the satellite regions of acrocentric chromosomes. A product containing this sequence was obtained by telomere-anchored PCR using as a primer an oligonucleotide sequence from the cloned fragment. Our data suggest demethylation of cytosines at the cloned Not I site and in neighboring DNA in some tumors, compared with normal tissue, and suggest a greater similarity between human and chimpanzee subtelomeric sequences than was previously reported. PMID- 8633087 TI - Local densities orthogonal to beta-sheet amide planes: patterns of packing in globular proteins. AB - We have investigated the efficiency of packing by calculating intramolecular packing density above and below peptide planes of internal beta-pleated sheet residues in five globular proteins. The orientation of interest was chosen to allow study of regions that are approximately perpendicular to the faces of beta pleated sheets. In these locations, nonbonded van der Waals packing interactions predominate over hydrogen bonding and solvent interactions. We observed considerable variability in packing densities within these regions, confirming that the interior packing of a protein does not result in uniform occupation of the available space. Patterns of fluctuation in packing density suggest that the regular backbone-to-backbone network of hydrogen bonds is not likely to be interrupted to maximize van der Waals interactions. However, high-density packing tends to occur toward the ends of beta-structure strands where hydrogen bonds are more likely to involve nonpolar side-chain groups or solvent molecules. These features result in internal protein folding with a central low-density core surrounded by a higher-density subsurface shell, consistent with our previous calculations regarding overall protein packing density. PMID- 8633088 TI - Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells. AB - Benzodiazepine (BZA)-5B, a CAAX farnesyl-transferase inhibitor, was previously shown to block the farnesylation of H-Ras and to reverse the transformed morphology of Rat1 cells expressing oncogenic H-RasV12. Non-transformed Rat1 cells were not affected by BZA-5B, suggesting that they produce a form of Ras whose prenylation is not blocked by this compound. The likely candidate is K RasB, which differs from H-Ras primarily in the terminal 24 amino acids. In the current study we examined the effect of BZA-5B on the prenylation of a chimeric oncogenic Ras protein designated H/K-RasBV12, consisting of the first 164 amino acids of H-RasV12 followed by the last 24 amino acids of K-RasB. BZA-5B failed to block the prenylation of this chimera and was thus unable to reverse the transformed morphology of Rat1 cells in which it was expressed. Another potent inhibitor of H-Ras farnesylation, L-739,749, also failed to block prenylation of H/K-RasBV12. Similar results were obtained in transfected cells expressing a widely used version of K-RasBV12 containing a 10-amino acid extension at its NH2 terminus. Neither BZA-5B nor L-739,749 reversed the transformed morphology of cells expressing H/K-RasBV12. The resistance of K-RasB to farnesyltransferase inhibition provides a likely explanation for the resistance of nontransformed cells to the growth inhibitory effects of BZA-5B and L-739,749. PMID- 8633090 TI - Is the lever arm of myosin a molecular elastic element? PMID- 8633089 TI - The neck region of the myosin motor domain acts as a lever arm to generate movement. AB - The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain. PMID- 8633092 TI - On signal sequence polymorphisms and diseases of distribution. AB - We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution. PMID- 8633091 TI - Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene. AB - The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors. PMID- 8633093 TI - High-resolution physical mapping by combined Alu-hybridization/PCR screening: construction of a yeast artificial chromosome map covering 31 centimorgans in 3p21-p14. AB - We describe an integrated approach to large-scale physical mapping using an Alu PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands p14-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of approximately equal to 100 kb. We observe an inverse distribution of Alu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high resolution human physical maps. PMID- 8633094 TI - Systemic versus cartilage-specific expression of a type II collagen-specific T cell epitope determines the level of tolerance and susceptibility to arthritis. AB - Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA. PMID- 8633095 TI - Human immunodeficiency virus (HIV) type 2-mediated inhibition of HIV type 1: a new approach to gene therapy of HIV-infection. AB - Human immunodeficiency virus (HIV) type 2, the second AIDS-associated human retrovirus, differs from HIV-1 in its natural history, infectivity, and pathogenicity, as well as in details of its genomic structure and molecular behavior. We report here that HIV-2 inhibits the replication of HIV-1 at the molecular level. This inhibition was selective, dose-dependent, and nonreciprocal. The closely related simian immunodeficiency provirus also inhibited HIV-1. The selectivity of inhibition was shown by the observation that HIV-2 did not significantly downmodulate the expression of the unrelated murine leukemia virus; neither did the murine leukemia virus markedly affect HIV-1 or HIV-2 expression. Moreover, while HIV-2 potently inhibited HIV-1, the reverse did not happen, thus identifying yet another and remarkable difference between HIV-1 and HIV-2. Mutational analysis of the HIV-2 genome suggested that the inhibition follows a complex pathway, possibly involving multiple genes and redundant mechanisms. Introduction of inactivating mutations into the structural and regulatory/accessory genes did not render the HIV-2 provirus ineffective. Some of the HIV-2 gene defects, such as that of tat and rev genes, were phenotypically transcomplemented by HIV-1. The HIV-2 proviruses with deletions in the putative packaging signal and defective for virus replication were effective in inducing the suppressive phenotype. Though the exact mechanism remains to be defined, the inhibition appeared to be mainly due to an intracellular molecular event because it could not be explained solely on the basis of cell surface receptor mediated interference. The results support the notion that the inhibition likely occurred at the level of viral RNA, possibly involving competition between viral RNAs for some transcriptional factor essential for virus replication. Induction of a cytokine is another possibility. These findings might be relevant to the clinical epidemiological data suggesting that infection with HIV-2 may offer some protection against HIV-1 infection. PMID- 8633096 TI - Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis. AB - Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential. PMID- 8633097 TI - Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells. AB - Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression. PMID- 8633098 TI - BCL2 regulates neural differentiation. AB - A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation. PMID- 8633099 TI - Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and pulsed folding and annealing cage. AB - Free GroEL binds denatured proteins very tightly: it retards the folding of barnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES.nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES.nucleotide complex. Fast-folding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL.denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL.GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination of folding cage and an annealing machine. PMID- 8633101 TI - Biological relevance and consequences of chemical- or metal-induced DNA cross linking. AB - A vast number of chemicals are known to induce mutagenesis and/or carcinogenesis in mammals. Although disruption of cellular nuclear material resulting ultimately in mutagenesis/carcinogenesis can be accomplished by various mechanisms, the search for biomarkers of chemical-induced toxicity continues. This review focuses on the ability of certain metals or chemicals to bind to DNA in a cross-link fashion in whole animal as well as under in vitro conditions. The methodologies currently used to determine DNA cross-linking are described. The biological relevance of the presence of chemical- or metal-induced DNA cross-linking as a measure of carcinogenesis in humans is still under debate, as there is no clear correlation between the disease and the DNA cross-link reaction. PMID- 8633102 TI - Regulation of skeletal muscle blood flow during contractions. AB - Skeletal muscle blood flow increases nearly 20-fold during exercise. Despite experimental work in this area for more than a century, the physiological mechanisms responsible for the regulation of skeletal muscle blood flow during contractions remain relatively unknown. Historically, experiments have focused on identifying chemical factors associated with increased tissue metabolism that also act as vasodilators. However, functional vasodilation cannot be attributed to changes in the local concentration of any single metabolic factor. The rapid increase in muscle blood flow during the initial moments of exercise led others to consider a role for neural mechanisms in the development of functional hyperemia. However, a specific neural pathway has not been identified. Mechanical factors, such as vascular compression and increases in perfusion pressure, also affect vascular resistance and skeletal muscle blood flow. Yet the specific manner in which these mechanical factors interact during muscle contractions is not well understood. The recent determination that arterial feed vessels, upstream from the active tissue and microcirculation, also dilate during muscle contractions has led to the consideration that the vessels are primarily responsible for the regulation of bulk flow to the tissue. Since these vessels are anatomically separated from the active tissue, more complex, indirect regulatory mechanisms must be explored. It is also likely that the specific factors responsible for functional vasodilation and increased muscle blood during exercise differ between muscle fiber types and relative contraction intensities. Consideration of these factors may lead to a better understanding of the regulation of muscle blood flow. PMID- 8633100 TI - Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy. AB - Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers. PMID- 8633103 TI - Tolazoline decreases survival time during microwave-induced lethal heat stress in anesthetized rats. AB - Effects of alpha-adrenergic antagonists have been studied during environmental heating but not during microwave-induced heating. Tolazoline may exert some of its effects via alpha-adrenegic blockade. In the present study, ketamine anesthetized Sprague-Dawley rats were exposed to 2450-MHz microwaves at an average power density of 60 mW/cm2 (whole-body specific absorption rate of approximately 14 W/kg) until lethal temperatures were attained. The effects of tolazoline (10 mg/kg body weight) on physiological responses (including changes in body temperature, heart rate, blood pressure, and respiratory rate) were examined. Survival time was significantly shorter in the tolazoline group than in saline-treated animals. In general, heart rate and blood pressure responses were similar to those that occur during environmental heat stress. Heart rate, however, was significantly elevated in animals that received tolazoline, both before and during terminal microwave exposure. It is possible that changes associated with the elevated heart rate (e.g., less cardiac filling) in tolazoline-treated animals resulted in greater susceptibility to microwave induced heating and the lower survival time. PMID- 8633105 TI - Effects of luteinizing hormone-releasing hormone on pulsatile prolactin secretion in adult hyperprolactinemic female rats. AB - Regulation of prolactin secretion involves complex interactions of multiple inhibitory and stimulatory factors. Among them, luteinizing hormone-releasing hormone (LHRH) has been shown, when analyzed in single samples, to exert both stimulatory and inhibitory influences on prolactin secretion. In the present study, we have further examined the effects of LHRH on prolactin secretion by studying the pulsatile pattern of the hormone after the administration of the neuropeptide. For this purpose, adult sham-operated and pituitary-grafted female rats, exhibiting diestrus smears were bled for 3 hr during the morning (1030 to 1330 hr). Two pulses of LHRH (10 ng/kg body wt) were administered 60 and 120 min after starting the bleeding period. Pituitary grafting increased the mean serum prolactin levels, absolute amplitude of the hormone peaks, and its mean half life, compared with control animals. In sham-operated rats, LHRH administration diminished mean serum prolactin levels, the absolute pulse amplitude and frequency of prolactin peaks. In pituitary-grafted animals, LHRH administration only decreased the pulse frequency of prolactin peaks. These data suggest that LHRH significantly suppressed pulsatile prolactin secretion, and that this effect was blunted by exposure to previously elevated circulating prolactin levels. PMID- 8633104 TI - 1,25-dihydroxyvitamin D3 stimulates phagocytosis but suppresses HLA-DR and CD13 antigen expression in human mononuclear phagocytes. AB - This study investigated the regulatory activity of 1,25-dihydroxyvitamin D3 (1,25 [OH]2D3) on phagocytic cells obtained from normal human peripheral blood. Flow cytometric analysis enabled identification of two discrete populations of cells, one predominantly monocytes ("monocyte" gate) and one containing primarily lymphoid and other cell types ("lymphoid" gate). The monocyte-associated antigens CD13 and CD33 were highly expressed by cells in this monocyte gate and used to monitor this population. Following 5 days of culture, cells in the monocyte gate manifested high phagocytic activity as determined by ingestion of fluorescent carboxylmicrospheres and exhibited high expression of class II HLA-DR products. 1,25-(OH)2D3 profoundly upregulated phagocytic activity while downregulating HLA DR antigen expression on the cells in the monocyte gate. Moreover, 1,25-(OH)2D3 also reduced cell surface CD13 expression on the cells with low but not high phagocytic activity in this gate. Proportional activities by the 1,24-(OH)2D3 and 24,25-(OH)2D3 metabolites indicated the regulatory effects are likely mediated by the 1,25-(OH)2D3 receptor (VDR). Prostaglandin E2 (PGE2), a known modulator of monocyte/macrophage activity also markedly inhibited HLA-DR expression while enhancing the phagocytic activity of cells in the monocyte gate. In contrast to 1,25-(OH)2D3, PGE2 clearly upregulated CD13 expression in cells with high phagocyte activity. Since indomethacin, an inhibitor of PGE2 synthesis, failed to reverse the 1,25-(OH)2D3 induced inhibitory effect on HLA-DR expression, this effect is apparently not mediated through endogenous PGE2 synthesis. Based on these findings we speculate that 1,25-(OH)2D3 may be capable of acting as both an upregulating agent during natural immunity via the enhancement of phagocytosis by monocyte/macrophage populations and as a "downregulator" during acquired immune responses via an inhibitory effect on MHC class II antigen expression by professional antigen-presenting cells. PMID- 8633107 TI - Recombinant human bone morphogenetic protein (rhBMP) induced heterotopic bone development in vivo and in vitro. AB - In vivo, recombinant human bone morphogenetic protein (rhBMP-2) with deactivated bone matrix as a carrier, implanted in muscle in adult rates, induced development of heterotopic bone, including bone marrow. The volume of bone was proportional to the dose of rhBMP-2 in a range of 0.2-150 microg. In vitro, in response to 50 microgram dose range, subcutis- and brain-derived outgrowths differentiated into loosely woven connective tissues composed of spindle-shaped fibroblasts, adipocytes, and cartilage. Muscle-derived connective tissues cultivated first in culture media supplemented with 50 microgram of rhBMP-2 for 72 hr, then enclosed in a diffusion chamber, and immediately transplanted into a rectus abdominous muscle pouch in an autogenic rat for 28 days, induced cartilage development on the inside and transmembrane hetertoptic bone development including bone marrow on the outside. These experiments are interpreted to show that muscle derived connective tissue cells have the competence of embryonic cells to develop de novo in response to BMP in postfetal life. PMID- 8633106 TI - Alterations in the level of phosphotyrosine signal transduction constituents in human parotid tumors. AB - Human parotid tumors were evaluated for the activation of the phosphotyrosine signaling pathway by Western blot, enzyme activity assay, and reverse transcriptase-polymerase chain reaction. Warthin's tumor and mucoepidermoid carcinomas had the greatest level of tyrosine phosphorylated proteins identified in plasma membrane fractions. These tumors, along with pleomorphic adenocarcinoma, showed high levels of membrane expression of the tyrosine kinase receptor, c-erbB-2, and phosphatidylinositol-3-kinase. Expression of the epidermal growth factor receptor was confined to normal tissue. The level of mRNA for c-erb was elevated only in mucoepidermoid carcinomas. Messenger RNA levels for ras were unchanged from control levels in all tumors, while the level of src mRNA was higher in the tumor samples than the normal parotid tissue. The activities of several signal transduction kinases, including protein kinase A and C were elevated in tumor tissue (7.7- to 18.9- and 0.4- to 3.7-fold higher, respectively), relative to surrounding normal tissue. While the level of glandular amylase was reduced (22%-0% of normal levels) in the tumor tissue, epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) content was dramatically higher in the neoplastic tissue (10- to 170-fold and 4.6 to 6.0-fold, respectively). These results suggest that with the presence of elevated levels of EGF, TGFalpha, and the oncoprotein receptor c-erbB-2 in the membrane of parotid tumors, cell proliferation and activation of the phosphotyrosine signal transduction pathway may involve autocrine stimulation through the expression of high levels of growth factor and receptor in the same tissue. PMID- 8633108 TI - Upregulation of apolipoprotein A-IV gene expression in residual ileum after massive small bowel resection requires the biliary secretion in rats. AB - Small bowel resection results in adaptation of residual intestine, but little is known about the postresectional response of apolipoproteins synthesized mainly in the small intestine. We have investigated the postresectional response of apolipoprotein A-1 and A-IV gene expression in residual ileum and assessed the mechanism of response, particularly the role of biliary secretion. Time course of changes in apolipoprotein A-1 and A-IV mRNA levels was examined by Northern blotting in the residual terminal ileum for 24 hr after 85% jejunoileal resection in fasted rats. Localization of these mRNAs was studied using in situ hybridization histochemistry. Effect of biliary diversion on the postresectional response of mRNAs and proteins was estimated by Northern blotting and immunoblotting, respectively. Apolipoprotein A-IV mRNA began increasing at 1 hr postresection, achieved a maximum by 12 hr (5-fold increase) and remained stable to 24 hr, while apolipoprotein A-1 mRNA did not change. Apolipoprotein A-IV mRNA accumulated predominantly in the upper part of ileal villi and increased its intensity postresection, and apolipoprotein A-1 mRNA was detected in the villus base to tip. The postresectional increase in apolipoprotein A-IV MRNA and protein was completely abolished by concurrent biliary diversion. The results suggest that the enterocytes in the ileal villi rapidly adapt to massive small bowel resection by increase in apolipoprotein A-IV gene expression which is mediated by biliary constituents but not luminal nutrients. PMID- 8633109 TI - Effects of dietary calcium and phosphorus on vitamin D metabolism and calcium absorption in hamster. AB - We studied the following responses to restriction of dietary calcium and phosphorus in the growing hamster: (i) serum concentrations of calcium, inorganic phosphorus, magnesium, and vitamin D metabolites; and (ii) calcium transport by ileum. Diets fed were normal calcium with normal or low phosphorus or low calcium with normal or low phosphorus. We found serum 1 alpha,25-dihydroxycalciferol (1,25-[OH]2D) concentration did not differ significantly among the diet groups. Calcium absorption, measured as serosal/mucosal calcium concentration ratio produced by everted ileal sac, was greater in the low calcium, normal phosphorous group than in all other groups. The other groups did not differ from one another in calcium absorption. Feeding the low calcium, normal phosphorus diet increased inorganic phosphorus and magnesium but decreased calcium concentration in serum in comparison with the three other diets. Both low phosphorus diets were without effect on serum calcium, but the low calcium, low phosphorus diet increased serum inorganic phosphorus and magnesium above that of the normal calcium, low phosphorus diet. Ileal calcium absorption in hamster (i) was independent of serum 1,25-(OH)2D concentration; (ii) increased in response to low dietary calcium if dietary phosphorus was normal; and (iii) was independent of dietary calcium, if dietary phosphorus was low. Despite increased calcium absorption, serum calcium was decreased in the low calcium-normal phosphorus group as compared with all other groups. Feeding low calcium diets increased serum inorganic phosphorus and magnesium as compared with feeding the corresponding normal calcium diets (i.e., independently of whether dietary phosphorus content was normal or low). These studies demonstrate that the interrelationships between calcium absorption and vitamin D and mineral metabolism in hamster differ from other mammals. PMID- 8633110 TI - Plasminogen activator inhibitor-1 in nonobese subjects with non-insulin-dependent diabetes mellitus. AB - Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) have been shown to be a risk factor for the development of vascular complications in obese and hyperinsulinemic non-insulin-dependent diabetes (NIDDM) patients. To clarify whether PAI-1 also plays an essential role in the development of such complications in NIDDM patients without obesity or hyperinsulinemia, PAI-1 was analyzed in relation to blood pressure, fasting plasma levels of glucose (FPG), hemoglobin A1C (HbA1c), immunoreactive insulin (F-IRI), C-peptide (CPR), total cholesterol (TC), triglyceride (TGL), and HDL-cholesterol (HDL-C) in 77 NIDDM patients and 10 healthy control subjects. The NIDDM patients were not obese (body mass index [BMI]:<26 kg/m2) or hyperinsulinemic, and BMI in the controls was between 19 and 24 kg/m2. In addition, parameters of insulin secretion reserve, including sigmaIRI, insulinogenic index, and CPR at 5 min after glucagon loading, were evaluated simultaneously. Plasma levels of PAI-1 were higher in the NIDDM group (9.3+/-0.9 ng/ml) than in the controls (4.3+/-0.7 ng/ml;P<0.01). Levels of FPG and HbA1c were also elevated in the NIDDM group (P<0.05 for each), but F-IRI did not differ between the two groups. However, multiple regression analysis revealed no significant correlation in the NIDDM between PAI-1 and F-IRI or the parameters of insulin secretion reserve. Regardless of the presence or absence of vascular complications, PAI-1 did not vary significantly in the NIDDM. These findings suggest that the effects of PAI-1 on the development of diabetic complications in NIDDM patients may not proceed in the same way in those with versus those without obesity or hyperinsulinemia, because no correlation was found between PAI-1 and insulin secretion reserve, while plasma levels of PAI-1 were higher in the NIDDM group than in the controls. PMID- 8633111 TI - Emerging applications of magnetic resonance imaging in the evaluation of articular cartilage. AB - MR imaging rapidly is emerging as a tool of unparalleled power for examining the articular cartilage and other important structures in diarthrodial joints. In addition to delineating the morphology of cartilage, MR imaging is capable of quantifying a variety of compositional and functional parameters relevant to arthritis. Moreover, because MR imaging is a nondestructive technique, multiple parameters can be analyzed in the same region of tissue and frequent serial examinations can be performed on even asymptomatic patients. This offers an unprecedented opportunity to study arthritis in ways not imaginable before and potentially to expand the envelope of MR imaging to include a population of patients for whom imaging has had relatively little to offer. PMID- 8633112 TI - Scintigraphy in the evaluation of arthropathy. AB - Significant progress has been made in radionuclide imaging of bones and joints. This largely is owing to advances in radiopharmaceuticals, particularly the antibodies, and in technology, particularly in the introduction of new computers and multiheaded camera systems resulting in improved imaging. These techniques have applied to the evaluation of articular and para-articular diseases including rheumatoid arthritis, septic arthritis, osteoid osteoma, transient osteoporosis, reflex sympathetic dystrophy, avascular necrosis, and facet joint syndrome. This article reviews scintigraphy in these conditions. PMID- 8633113 TI - Scoring radiographic abnormalities in rheumatoid arthritis. AB - Serial radiologic examinations can demonstrate the initial development and stability or progression of erosions and cartilage damage during the course of rheumatoid arthritis. Measurements of the radiographic progression rate is potentially the most powerful tool available to the individual practitioner and the clinical investigator. This article examines various methods for scoring radiographic abnormalities in rheumatoid arthritis. PMID- 8633114 TI - Magnetic resonance imaging of rheumatoid arthritis. AB - MR imaging is able to demonstrate both the structural changes that occur in rheumatoid arthritis and the inflammatory changes, including synovial proliferation and joint effusion. MR imaging can demonstrate erosion before it is visible on radiographs. Although MR imaging appears to be very helpful in assessing the severity of rheumatoid arthritis and its response to therapy, the optimal technique for this assessment and the ultimate clinical value of MR imaging have yet to be determined. PMID- 8633115 TI - Imaging of osteoarthritis. AB - Osteoarthritis represents the most common joint disorder affecting humans. This article describes the cause of this disease, the radiographic-pathologic correlation, and particularly the radiographic appearance of osteoarthritis in the hip and the knee. PMID- 8633116 TI - Osteonecrosis, transient osteoporosis, and transient bone marrow edema: current concepts. AB - Osteonecrosis, transient osteoporosis, and transient bone marrow edema are closely related diseases that may have an overlapping clinical and radiographic presentation, thus creating difficulty in establishing a diagnosis. Close scrutiny of MR images may aid in distinguishing the pattern of osteonecrosis from other conditions, but in other cases careful clinical and radiologic follow up may be required. The pathogenesis, radiologic diagnosis, and clinical relevance of these conditions are described and reviewed in this article. PMID- 8633117 TI - Septic arthritis. AB - Diagnosis of septic joint can be a problem for both the clinician and the imager. The longer the delay in diagnosis of a septic arthritis, the greater the chance of significant complication. Many imaging modalities are available to the imager and each plays a definite role. In determining the modality of choice, one should choose what is most efficacious for the individual patient. PMID- 8633118 TI - Pigmented villonodular synovitis. AB - Pigmented villonodular synovitis is a benign proliferative disorder of the synovium of uncertain cause. It may involve tendon sheaths, bursae, or joints, the latter occurring as diffuse involvement or a localized nodule. This article reviews the clinical features of the disorder and the imaging features of the disease, concentrating on the findings and utility of MR imaging. PMID- 8633119 TI - Synovial osteochondromatosis. AB - Primary or idiopathic synovial osteochondromatosis is an uncommon condition probably caused by synovial metaplasia and typically resulting in multiple calcified periarticular bodies. Synovial osteochondromatosis also may occur secondary to other joint disorders. This article reviews the pathogenesis, pathologic features, clinical features, and radiologic imaging of synovial osteochondromatosis. The differentiation of synovial osteochondromatosis from other conditions associated with periarticular calcified bodies is discussed. PMID- 8633121 TI - Imaging of arthropathies. Crystal disease. AB - The crystalline arthropathies are a heterogeneous group of biochemical disorders that share the common feature of crystal deposition in and around joints. These metabolic derangements result in a variety of clinical symptoms, radiographic signs, and pathologic abnormalities. The diagnosis of these disorders usually is straightforward when typical articular symptoms are present and characteristic radiographs or joint crystals are obtained. Current concepts regarding crystal associated arthropathies are reviewed along with a discussion of their often distinctive radiographic appearances. PMID- 8633120 TI - Seronegative spondyloarthropathies. AB - The seronegative spondylarthropathies are a group of multisystem inflammatory disorders that share a variety of clinical, radiologic, and genetic features. They include ankylosing spondylitis, psoriatic arthritis, Reiter's disease, and arthritis associated with inflammatory bowel disease. This article reviews these multisystem inflammatory disorders. PMID- 8633122 TI - Amyloidosis and silicone synovitis: updated classification, updated pathophysiology, and synovial articular abnormalities. AB - Amyloidosis is a heterogeneous group of disorders characterized by the extracellular deposition of protein fibrils in organs or tissues. Silicone synovitis, like amyloid arthropathy, is characterized radiographically by multiple subchondral cysts in the absence of cartilage space narrowing. This article examines and compares the classification, pathophysiology, and synovial articular abnormalities associated with amyloidosis and silicone synovitis. PMID- 8633123 TI - Ganglia and cysts around joints. AB - Para-articular cysts frequently are seen on routine imaging examinations. They may be clinically asymptomatic or may cause pain, swelling, or impaired joint function. They often are associated with underlying joint disorders, such as trauma, degeneration, or inflammation. This article discusses the cause, symptoms, and appearance of para-articular cysts on various imaging examinations including arthrography, ultrasound, CT scan, and MR imaging. Specific attention is focused on those cystic masses appearing around the hip, knee, ankle and foot, shoulder, elbow, wrist and hand, spine, temporomandibular joints, and periosteum. PMID- 8633125 TI - Hemochromatosis arthropathy. PMID- 8633124 TI - Inflammatory myopathies: radiologic evaluation. AB - Clinical evaluation of skeletal muscle in patients with inflammatory myopathies (IM) has long been hampered by difficulty in assessing the morphology and functional integrity of skeletal muscles. Advances in radiologic techniques, particularly MR imaging, have improved the ability to manage patients with neuromuscular disease in general and IM in particular. MR imaging can probe beyond the skin to identify focal muscle structural lesions, determine their extent, characterize their composition, direct invasive procedures, and monitor therapies. This article reviews the clinical, pathologic, and imaging aspects of these diseases. PMID- 8633126 TI - Hemophilic arthropathy. PMID- 8633127 TI - HIV arthritis. PMID- 8633128 TI - Lyme arthropathy. PMID- 8633129 TI - Stress fractures. PMID- 8633130 TI - Mammographic halo sign revisited. AB - PURPOSE: To determine whether increased silver halide deposition accounts for some curvilinear areas of hyperlucency (halo signs) that surround breast masses on screen-film mammograms. MATERIALS AND METHODS: Breast images obtained in 43 women (aged 30-67 years; mean, 48.6 years) that showed a halo sign were selected from the authors' teaching files. Optical magnification (x8.0-12.5) of the masses with halos was used to establish the pattern of silver halide deposition in the film emulsion. RESULTS: True radiolucent halos, differentiated from Mach bands by means of the increased silver halide deposition in the film emulsion, were identified in 44 masses in 36 women. Partial true radiolucent halos were identified in 32 (73%) of 44 masses. Thirty-eight (86%) of the 44 masses were cysts; three (7%), fibroadenomas; two (4%), infiltrating ductal carcinomas; and one (2%), axillary lymph node metastasis. True radiolucent halos were more common in growing benign and malignant lesions. CONCLUSION: A halo is not always a perceptual illusion. PMID- 8633131 TI - Primary carcinoma of the lung overlooked at CT: analysis of findings in 14 patients. AB - PURPOSE: To describe the appearances of overlooked lung cancer at computed tomographic (CT) examination and to analyze the reasons for failure to diagnose these lesions. MATERIALS AND METHODS: Fourteen patients with 15 overlooked lung cancers were identified by radiologists at three institutions. Location, shape, and cell type of each cancer were reviewed, and other relevant findings of CT examinations were assessed. RESULTS: The missed tumors manifested as endobronchial lesion (n = 10), solitary parenchymal nodule (n = 2), area of focal peripheral air-space disease (n = 2), or pleural-based thickening (n = 1). Eleven (73%) of the 15 lesions were located in a lower lobe. In six (43%) of 14 patients, major distracting findings were present elsewhere in the thorax. CONCLUSION: Endobronchial location and lower lobe predominance were the most common characteristics of overlooked lung cancer at CT. The presence of unrelated major abnormalities at CT may also have contributed to failure to diagnose the tumor. PMID- 8633132 TI - Missed lung cancer at CT: imaging findings in nine patients. AB - PURPOSE: To assess the imaging findings and course of lung cancers missed on computed tomographic (CT) scans. MATERIALS AND METHODS: A retrospective review of CT scans was performed on nine patients who underwent CT examination for various clinical indications and in whom a lung cancer had been missed. Subspecialty chest radiologists determined whether retrospectively identified lesions were missed due to observer error or technical failure. RESULTS: Five missed tumors were peripheral and four were central in location. All peripheral tumors were less than 3 mm in diameter. The interval from initial examination to detection of peripheral tumors ranged from 8 to 95 months. This interval for central tumors was shorter (3-14 months). Estimated doubling times ranged from 24 to 285 days. CONCLUSION: Small lung cancers that are near the threshold for detectability may be missed at CT. Such failure of detection is attributable primarily to the poor conspicuity of lesions. Retrospective identification at CT raises medicolegal issues. PMID- 8633133 TI - Hypersensitivity pneumonitis: correlation of individual CT patterns with functional abnormalities. AB - PURPOSE: To correlate the pattern and extent of abnormalities on thin-section computed tomographic (CT) scans with pulmonary function test results in subacute and chronic hypersensitivity pneumonitis. MATERIALS AND METHODS: Thin-section scans (1-3 mm collimation) obtained in 22 patients were assessed for pattern of abnormality and extent of disease. CT scores were correlated with functional parameters by using Spearman rank correlation and forward stepwise regression analysis. RESULTS: The most common CT patterns were decreased attenuation and mosaic perfusion (n = 19), ground-glass opacification (n = 18), small nodules (n = 12), and a reticular pattern (n = 8). Areas of decreased attenuation correlated with severity of air trapping indicated by residual volume (r = .58, P < .01), whereas ground-glass opacification and reticulation correlated independently with restrictive lung function. CONCLUSION: Areas of decreased attenuation and mosaic perfusion are an important ancillary CT finding in hypersensitivity pneumonitis, and obstructive functional abnormalities indicate that this phenomenon is caused by bronchiolitis. PMID- 8633134 TI - Non-Hodgkin lymphoma: contribution of chest CT in the initial staging evaluation. AB - PURPOSE: To analyze incremental information derived from routinely obtained chest computed tomographic (CT) scans compared with chest radiographs in newly diagnosed non-Hodgkin lymphoma and the effect of this information on staging and therapy. MATERIALS AND METHODS: Abnormalities on chest radiographs and CT scans obtained at specific sites were prospectively identified in 181 consecutive patients with no previous treatment. When discrepant information was found, the effect on staging and treatment was determined. RESULTS: CT and chest radiographic findings were negative in 99 (55%) patients. CT findings were positive and chest radiograph findings were negative in 17 (9%). Both chest radiograph and CT findings were positive in 65 (36%) patients, 16 with identical sites of disease and 49 with more extensive intrathoracic disease at CT. Most stage changes occurred in the diffuse large cell histologic subtype. CONCLUSION: Although routine chest CT findings increased stage of disease in some patients, it had no effect on initial treatment of newly diagnosed non-Hodgkin lymphoma at this institution. PMID- 8633135 TI - Abdominal iron deposition: metabolism, MR findings, and clinical importance. PMID- 8633136 TI - Ambulatory sclerotherapy for malignant pleural effusions. AB - PURPOSE: To determine the feasibility of ambulatory drainage and sclerotherapy in malignant pleural effusions. MATERIALS AND METHODS: Nineteen consecutive patients with symptomatic malignant pleural effusions were enrolled. None of the patients previously underwent sclerotherapy. A fluoroscopically placed 10.3-F catheter was connected to a closed gravity drainage bag system. Sclerotherapy was performed with bleomycin when daily drainage was less than 100 mL. Radiographic response was graded at 30 days. All patients were examined for symptomatic response and for complications. RESULTS: The tubes remained in place 2-11 days (mean, 5.1 days). Total pleural drainage ranged from 950 to 3,925 mL (mean, 1,647 mL); all 19 patients had improvement of symptoms. At 30 days, 10 (53%) patients had a complete response, five (26%) had a partial response, and four (21%) had progressive disease. CONCLUSION: Ambulatory sclerotherapy is a safe, viable alternative to conventional inpatient treatment of malignant pleural effusions. PMID- 8633137 TI - Alpha 1-antitrypsin deficiency: evaluation of bronchiectasis with CT. AB - PURPOSE: To assess bronchiectasis depicted with computed tomography (CT) in patients with alpha 1-antitrypsin deficiency and to examine associated clinical correlates. MATERIALS AND METHODS: CT scans in 14 patients with alpha 1 antitrypsin deficiency were evaluated by two thoracic radiologists for the presence and extent of bronchiectasis and emphysema. The findings were correlated with numeric infection scores on the basis of symptoms of sputum production and respiratory infection and with a history of conditions that may predispose to development of bronchiectasis. RESULTS: Six (43%) of 14 patients had CT evidence of bronchiectasis. Patients with bronchiectasis had significantly higher infection scores than did patients without bronchiectasis (P < .005). Two patients had diffuse cystic bronchiectasis, and neither reported a history of illness that may have predisposed them to this condition. CONCLUSION: Bronchiectasis may be more common in patients with alpha 1-antitrypsin deficiency than has been previously recognized. The diagnosis of alpha 1-antitrypsin deficiency should be considered in patients with emphysema and diffuse cystic bronchiectasis. PMID- 8633138 TI - Prospective study of a PACS: information flow and clinical action in a medical intensive care unit. AB - PURPOSE: To prospectively compare efficiency and outcome of a standard film-only system with those of a digital picture archiving and communication system (PACS). MATERIALS AND METHODS: The film-only system, which used either analog film or computed radiography (CR) hard copy, was compared with a PACS, which used CR images displayed on a multiviewer in the radiology department and a workstation in the medical intensive care unit. A random sample of nonroutine, bedside chest radiographs was studied. RESULTS: Within 20 minutes of completion of radiography, 246 of 328 (75%) of the images were available at the workstations; it took 1.8 hours for 238 of 317 (75%) of the images to be displayed on the multiviewer. When the workstation was used, the staff did not access the image information earlier, but clinical actions were initiated more promptly in response to imaging findings. Consultation with radiologists decreased from 507 of 561 (90%) images with hard copies to 70 of 249 (28%) with the workstation. CONCLUSION: Use of a PACS improves the delivery of chest images, facilitates the initiation of clinical actions, and decreases input by radiologists. PMID- 8633139 TI - Diffuse adenomyosis: comparison of endovaginal US and MR imaging with histopathologic correlation. AB - PURPOSE: To compare the accuracy of endovaginal ultrasound (US) and magnetic resonance (MR) imaging in the diagnosis of adenomyosis. MATERIALS AND METHODS: The authors prospectively studied 119 consecutive patients undergoing hysterectomy. The endovaginal US scans and MR images were interpreted independently in a double-blind fashion. Imaging findings were compared with those at histopathologic examination. RESULTS: At histopathologic examination, adenomyosis was found in 28 of the 119 patients (24%). Sensitivity and specificity was 89% for endovaginal US and 89% for MR imaging. The positive predictive value was 71% for US and 65% for MR imaging. The negative predictive value was 96% for US and 95% for MR imaging. There was no statistically significant difference between the sensitivities (P = .65) and specificities (P = .75) of endovaginal US and MR imaging. The mean junctional zone (JZ) thickness on MR images in patients with and without proved adenomyosis was 15.0 mm +/- 4.9 and 7.7 mm +/- 3.3, respectively (P < .0001). When receiver operating characteristic curves were applied retrospectively, the optimal JZ value for the diagnosis of adenomyosis with MR imaging was > or = 12 mm. CONCLUSION: Endovaginal US was as accurate as MR imaging in the diagnosis of uterine adenomyosis. Use of a JZ thickness of > or= 12 mm should further optimize the diagnostic accuracy of MR imaging. PMID- 8633140 TI - Clinical evaluation of prostate biopsy parameters: gland volume and elevated prostate-specific antigen level. AB - PURPOSE: To determine whether excess prostate-specific antigen (PSA) improves the accuracy of predicting positive biopsy results. MATERIALS AND METHODS: Results of prostate biopsy in 130 consecutive patients were correlated with transrectal ultrasound (TRUS) findings, digital rectal examination results, serum PSA level, and excess PSA (serum PSA level minus predicted PSA level [prostate volume x 0.12]), alone and in combination. Random sextant biopsy was performed in all patients, as well as directed biopsy of any suspicious lesion seen at TRUS. RESULTS: Excess PSA alone was most accurate in the prediction of positive biopsy results. Use of an excess PSA of > or = 0 ng/mL instead of a serum PSA level of > 4 ng/mL to initiate prostate biopsy increased the positive yield from 49% to 56% and accuracy from 51% to 62%, eliminating 21% of biopsies while maintaining sensitivity at 94% (vs 95% with serum PSA level). CONCLUSION: Use of only excess PSA of > or = 0 ng/mL to initiate prostate biopsy results in the best combination of sensitivity and specificity compared with the other standard parameters. PMID- 8633141 TI - Renal resistive indexes: variability in Doppler US measurement in a healthy population. AB - PURPOSE: To determine the variability in resistive index (RI) in normal kidneys, possible causes of variability, and consequences of reporting a single value. MATERIALS AND METHODS: Doppler ultrasound RI measurements were obtained in the upper, middle, and lower regions of 118 kidneys in 58 healthy subjects (aged 24 70 years; 35 women, 23 men) who subsequently underwent angiography. The effects of sampling a particular parenchymal region, vascular territory, or kidney were assessed. RESULTS: Kidney region, vascular territory, and right versus left kidney had no consistently significant effect (P < or = .05) on RI. Age had a statistically significant effect. RI readings were highly correlated with each other both within a subject and within a kidney. The probability that a single RI value would exceed 0.70 in a healthy 45-year-old subject was 6%; this decreased to 3% when three readings were averaged. CONCLUSION: The variability of RI measurements in a kidney suggests that a number of RI readings should be averaged before a single representative value is reported. PMID- 8633142 TI - Early stage melanoma: lymphoscintigraphy, reproducibility of sentinel node detection, and effectiveness of the intraoperative gamma probe. AB - PURPOSE: To assess the influence of lymphoscintigraphic and intraoperative gamma probe findings on the surgical management of melanoma and to test reproducibility of lymphoscintigraphic findings. MATERIALS AND METHODS: After lymphoscintigraphic identification of the sentinel node, intraoperative gamma probe localization and sentinel lymph node excision were performed in 25 patients. To assess reproducibility, 13 patients underwent lymphoscintigraphy twice within 2-17 days. A modified preparation of technetium-99m sulfur colloid with smaller particles than routinely obtained was injected intradermally around the lesion. Dynamic flow images were obtained at 10 seconds per frame followed by a series of static images obtained every 5 minutes for 30 minutes. RESULTS: A sentinel node was identified in all patients. In eight patients, multiple drainage pathways were seen and surgical management was changed. In 11 of the 13 who underwent lymphoscintigraphy twice, sentinel node identification was reproducible. CONCLUSION: Lymphoscintigraphy is reproducible in detection of the sentinel node and with the surgical probe helps effectively guide surgical management. PMID- 8633144 TI - Effect of rate of contrast medium injection on hepatic enhancement at CT. AB - PURPOSE: To determine the effect of the rate of contrast medium injection on liver enhancement at computed tomography (CT). MATERIALS AND METHODS: Forty-five patients who underwent a follow-up CT examination of the liver were included in five different groups according to the compared rates of contrast material delivery: group A, 2 versus 3 mL/sec; group B, 2 versus 4.5 mL/sec; group C, 3 versus 4.5 mL/sec; group D, 3 versus 6 mL/sec; and group E, 4.5 versus 6 mL/sec. RESULTS: Time to peak enhancement was shorter for the faster rates of injection. In each group, maximum enhancement was nearly identical for the paired examinations (group A, 57 vs 58 HU; group B, 48 vs 47 HU; group C, 55 vs 58 HU; group D, 55 vs 54 HU; group E, 62 vs 61 HU, respectively). Mean enhancement in each group was similar when calculated at 3-second intervals. CONCLUSION: Higher rates of injection shorten the time to peak liver enhancement but have no effect on maximum liver enhancement. PMID- 8633143 TI - Phase II clinical evaluation of Gd-EOB-DTPA: dose, safety aspects, and pulse sequence. AB - PURPOSE: To investigate the efficacy of gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) in the detection of focal liver lesions with respect to dose, side effects, and pulse sequence. MATERIALS AND METHODS: A randomized double-blinded trial was performed in 33 patients with focal solid liver lesions. A bolus of Gd-EOB-DTPA, a liver-specific contrast agent, was intravenously administered at three different doses (12.5, 25, and 50 mumol per kilogram of body weight). Magnetic resonance imaging with different T1 weighted techniques was performed 20 and 45 minutes after administration of Gd EOB-DTPA. Changes in liver signal intensity, lesion-liver contrast-to-noise ration (C/N), detectable liver lesions, side effects, and adverse events were evaluated. RESULTS: Gd-EOB-DTPA significantly (P < .05) increased liver signal intensity and lesion-liver C/N within the dose range tested. Lesion detection was improved 20 and 45 minutes after administration of Gd-EOB-DTPA. A dose of 12.5 mumol was sufficient for the detection of focal liver lesions, and the breath hold, T1-weighted, fast low-angle shot pulse sequence was the most useful. No significant changes in vital signs, clinical laboratory test results, and urinalysis were observed. CONCLUSION: Gd-EOB-DTPA is an efficient, diagnostically useful, and safe contrast agent. PMID- 8633145 TI - Internal carotid artery dissection: MR imaging features and clinical-radiologic correlation. AB - PURPOSE: To analyze the usefulness of magnetic resonance (MR) imaging techniques and diagnostic sensitivity in internal and carotid artery dissection (ICAD) and to correlate these findings with those of other imaging modalities and with clinical signs. MATERIALS AND METHODS: Thirty-one patients with ICAD underwent MR imaging within 2-40 days (mean, 13 days) after onset of clinical symptoms. All patients initially underwent Doppler sonography; 15 patients underwent additional angiographic studies. RESULTS: T1-weighted fat-suppressed MR images most accurately demonstrated intramural hematoma in the internal carotid artery (ICA) in all patients. Kinking or coiling of the ICA was found in nine (29%) patients. Hyperintense signal was seen on T1- and T2-weighted images in the carotid canal or in the cavernous sinus in two-thirds of the patients. Findings characteristic of ICAD were seen on 13 of 16 angiograms. CONCLUSION: MR imaging most accurately demonstrated ICAD. The high rate of kinking and coiling in the carotid artery suggests that these anatomic conditions may be predisposing factors. PMID- 8633146 TI - Intracranial aneurysms: diagnostic accuracy of MR angiography with evaluation of maximum intensity projection and source images. AB - PURPOSE: To determine whether evaluation of source images from magnetic resonance (MR) angiography in addition to maximum-intensity projection (MIP) images improves the detection of aneurysms. MATERIALS AND METHODS: Conventional and MR angiography were performed in 193 patients with various intracranial vascular lesions or normal findings. Images were evaluated in a blinded manner. Two readings were performed 6 weeks apart by evaluating MIP images with and without source images. Results were evaluated with receiver operating characteristic analysis. RESULTS: Sensitivity for the detection of aneurysms increased slightly when source images were included. The detection rate of internal carotid artery aneurysms was most improved with the addition of source images. No statistically significant differences in performance were found between the readings with MIP images alone and with source images. CONCLUSION: Sensitivity may improve with combined reading of nonselective MIP and source images. PMID- 8633147 TI - Cerebral dural arteriovenous fistulas: percutaneous transvenous embolization. AB - PURPOSE: To evaluate angiographic and clinical results in patients with a dural arteriovenous fistula (AVF) who underwent percutaneous transvenous embolization. MATERIALS AND METHODS: Retrospective chart analysis and radiologic studies were performed in 24 patients (aged 20-87 years) with a dural AVF treated with percutaneous transvenous embolization. Lesions were located in the transverse and/or sigmoid or superior sagittal sinus. Clinical follow-up was 3-44 (mean, 10.8) months. RESULTS: After percutaneous transvenous embolization of 24 dural AVFs, there was complete occlusion in 17 patients, important flow reduction in three, and moderate flow reduction in four. Twenty patients were clinically cured, 17 with complete occlusion and three with important flow reduction. In patients with moderate flow reduction, clinical improvement was good in two and moderate in one. One patient remained clinically unchanged. A transient complication was seen in one patient, and a permanent complication was seen in one patient. One patient, whose preexisting clinical status was poor, died. During long-term follow-up, the condition of two patients worsened. CONCLUSION: Percutaneous transvenous embolization appears to be effective in the treatment of dural AVFs. More experience is needed to evaluate long-term results. PMID- 8633148 TI - Cerebral white matter: technical development and clinical applications of effective magnetization transfer (MT) power concepts for high-power, thin section, quantitative MT examinations. AB - PURPOSE: To evaluate white matter disorders with magnetization transfer (MT) techniques. MATERIALS AND METHODS: In 46 healthy volunteers and 46 clinical patients, MT Z spectra were obtained with various continuous-wave-equivalent MT powers (B1CW) and frequency offsets. RESULTS: With B1CW of 270 Hz and 4,000-Hz frequency offset, the MT ratio of normal callosal white matter was 59.2% +/- 1.5 (standard deviation), with less than 5% contribution from direct saturation and spin locking. A small statistically significant (P < .01) regional variation in normal white matter was seen. Plaques in MS patients had a broad (or wide) range of MT ratios; normal appearing white matter had a slightly reduced MT ratio. Vasogenic edema had a minimal effect on MT ratio, and radiation necrosis showed prominent reductions in MT ratio. CONCLUSION: High MT power techniques can expand the dynamic range of MT ratios, maintain a relatively pure MT effect, and be used effectively in MT imaging to evaluate white matter disorders. PMID- 8633149 TI - Lesions of the triangular fibrocartilage complex: MR findings with a three dimensional gradient-recalled-echo sequence. AB - PURPOSE: To evaluate the use of a three-dimensional gradient-recalled-echo (GRE) magnetic resonance (MR) imaging sequence in the depiction of lesions of the triangular fibrocartilage (TFC) complex. MATERIALS AND METHODS: MR images of the TFC complex were evaluated in 31 patients who underwent wrist arthroscopy less than 6 months after MR imaging. The results were compared with the arthroscopic findings. RESULTS: Eleven of 12 full-thickness TFC tears were depicted, but one partial-thickness tear, one abnormal disk, and one normal disk were overstaged. Lesions in the volar and dorsal radioulnar ligament and lesions of the attachments of the TFC complex to the ulna often were overstaged. Lesions of the ulnolunate and ulnotriquetral ligaments often were understaged. CONCLUSION: Imaging with the three-dimensional GRE sequence is reliable in the depiction of TFC tears and the exclusion of tears of components of the TFC complex other than those of the ulnolunate and ulnotriquetral ligaments. PMID- 8633150 TI - Through the "retrospectoscope": a glimpse of missed lung cancer at CT. PMID- 8633151 TI - Uremic leontiasis ossea: "bighead" disease in humans? Radiologic, clinical, and pathologic features. AB - PURPOSE: To describe the clinical, radiologic, and pathologic findings in patients with uremic leontiasis ossea (ULO). MATERIALS AND METHODS: Five patients with renal osteodystrophy developed marked hyperostosis of the facial and cranial bones. Radiologic studies included plain radiography of the skull (n = 5), computed tomography with three-dimensional reconstruction (n = 4), magnetic resonance imaging (n = 3), and fluorine-18 sodium fluoride positron emission tomography (PET) (n = 1). Specimens from bone biopsies (three patients) were examined. RESULTS: Skull and facial alterations were remarkably similar. Numerous nodules of varying attenuation and signal intensity in the widened diploic space suggested brown tumors in different stages of evolution. Biochemical data and PET findings enabled confirmation of markedly increased bone turnover. Bone specimens demonstrated severe osteitis fibrosa. After parathyroidectomy, facial changes in all patients stabilized or improved mildly. CONCLUSION: A similar entity in animals, "bighead" disease, which results from nutritional and uremic secondary hyperparathyroidism, may provide a useful animal model for ULO in humans. Mild forms of this entity may be more common than the scarcity of previous reports suggests. PMID- 8633152 TI - Spinal metastases: indications for and results of percutaneous injection of acrylic surgical cement. AB - PURPOSE: To determine the efficacy of percutaneous vertebroplasty in treating spinal metastases that result in pain or instability. MATERIALS AND METHODS: Thirty-seven patients (20 men, 17 women; aged 33-86 years) underwent 52 percutaneous injections of surgical cement into a vertebra (vertebroplasty) with fluoroscopic guidance in 40 procedures. Vertebroplasty was performed for analgesia in 29 procedures, stabilization of the vertebral column in five procedures, and both in six procedures. RESULTS: Twenty-four of the 33 procedures performed for analgesia that were evaluated resulted in clear improvement; seven, moderate improvement; and two, no improvement. Improvement was stable in 73% of patients at 6 months. In the procedure performed for stabilization, no displacement of treated vertebrae was observed (mean follow-up, 13 months). Three patients had transient radiculopathy due to cement extrusion, and two patients had transient difficulty in swallowing. CONCLUSION: Vertebroplasty of metastases is a minimally invasive procedure that provides immediate and long-term pain relief and contributes to spinal stabilization. PMID- 8633153 TI - Mottle on computed radiographs of the chest in pediatric patients. AB - PURPOSE: To investigate the relationship between radiation exposure and perceived mottle at bedside pediatric chest examinations performed with screen-film and computed radiographic techniques. MATERIALS AND METHODS: In a pediatric intensive care unit, chest radiographs were obtained with both computed radiography (60 radiographs) and a 600-speed screen-film system (14 radiographs). The relative radiation exposure was estimated by using the sensitivity value obtained in the processing of each computed radiograph. Five radiologists assessed the mottle present in the computed radiographs and screen-film images. RESULTS: For computed radiographs, the perceived level of mottle was inversely related to radiation exposure. For the same radiation exposure, the perceived mottle on computed radiographs was significantly higher than that on screen-film images (P < .001 for small cassettes; P < .01 for large cassettes). CONCLUSION: Pediatric computed radiography of the chest requires approximately twice the exposure of a 600-speed screen-film system to attain the same level of mottle. PMID- 8633154 TI - Diagnosis of acute pulmonary embolism: time for a new approach. PMID- 8633155 TI - Neonatal intracranial ischemia and hemorrhage: diagnosis with US, CT, and MR imaging. AB - PURPOSE: To assess the usefulness of ultrasound (US), computed tomography (CT), and magnetic resonance (MR) imaging in the detection of intracranial hemorrhage and ischemia in newborns. MATERIALS AND METHODS: Seventy-six neonates who underwent US within 72 hours of CT or MR examination were studied. Four observers rated images for the presence of germinal matrix hemorrhage (GMH), intraventricular hemorrhage (IPH), extraaxial hemorrhage, and hypoxic-ischemic encephalopathy. RESULTS: In 39% of neonates, CT and MR imaging provided greater confidence than US for the diagnosis or exlusion of neonatal ischemia or hemorrhage. Kappa analysis revealed significantly better interobserver agreement with CT than with US for the detection of GMH, IVH, IPH, and cortical infarction or ischemia (P <.005). Interobserver agreement was significantly better with MR imaging than with US for the detection of GMH, IVH, and cortical infarction or ischemia (P < .005). CONCLUSION: Sensitivity and interobserver agreement are better with MR imaging and CT than with US for the detection of neonatal cortical ischemia or infarction. PMID- 8633157 TI - Acute graft-versus-host disease in children: abdominal CT findings. AB - PURPOSE: To review the computed tomographic (CT) appearance of acute gastrointestinal graft-versus-host disease (GVHD) in children. MATERIALS AND METHODS: Sixteen abdominal CT scans obtained in 12 children with acute gastrointestinal GVHD who underwent allogeneic bone marrow transplantation (BMT) were compared with 16 CT scans obtained in autologous bone marrow recipients not at risk for GVHD (control group). Autopsy findings in six patients with GVHD were compared with CT findings. RESULTS: All CT scans in patients with GVHD showed an abnormally enhanced, thin mucosal layer of bowel wall involving fluid-filled, dilated, poorly opacified bowel loops from the duodenum to the rectum. This corresponded histologically to mucosal destruction and replacement by a thin layer of highly vascular granulation tissue. Bowel wall thickening was often absent. Infiltration of mesenteric fat was seen in 91% of patients The control group showed no similar abnormalities. CONCLUSION: Acute gastrointestinal GVHD characteristically appears on CT scans as multiple, diffuse, fluid-filled bowel loops with a thin, enhancing layer of bowel wall mucosa. Bowel wall thickening often is absent. PMID- 8633156 TI - Clockwise whirlpool sign at color Doppler US: an objective and definite sign of midgut volvulus. AB - PURPOSE: To evaluate the clockwise whirlpool sign at color Doppler ultrasound (US) in the diagnosis of midgut volvulus. MATERIALS AND METHODS: Pediatric patients (160 boys, 76 girls; age range, 0 day to 14 years) with possible midgut volvulus underwent abdominal gray-scale US and color Doppler US. Midgut volvulus was diagnosed by recognition of the whirlpool sign (wrapping of the superior mesenteric vein and the mesentery around the superior mesenteric artery). If the whirlpool rotated clockwise with caudal movement of the transducer, the direction of the whirlpool was defined as clockwise. RESULTS: Color Doppler US showed a whirlpool sign that was clockwise in 12 of 13 patients with surgically confirmed midgut volvulus and counterclockwise in three patients without midgut volvulus. Sensitivity, specificity, and positive predictive value of clockwise whirlpool sign for midgut volvulus were 92%, 100%, and 100%. CONCLUSION: The clockwise whirlpool sign is diagnostic of midgut volvulus. Color Doppler US should be performed as an initial imaging study in children suspected of having midgut volvulus. PMID- 8633158 TI - Color Doppler twinkling artifact in hyperechoic regions. AB - PURPOSE: To investigate a new color Doppler ultrasound (US) artifact that manifested as a rapidly changing mixture of red and blue behind a strongly reflecting structure. MATERIALS AND METHODS: In 140 consecutive patients with parenchymal calcifications seen at US, the presence of color signal was assessed in calcified areas relative to adjacent noncalcified tissue. The artifact, called the twinkling color artifact, was stimulated with various strongly reflecting structures immersed in still water. RESULTS: The artifact was found in 42 parenchymal calcifications. In vitro experiments showed that the twinkling artifact was present in granular structures, whereas no color signal was noted in smooth surfaces. The "twinkling sign" appeared to be generated by a strongly reflecting medium composed of individual reflectors. CONCLUSION: The presence of a color signal close to calcifications should be interpreted with caution, and a flow spectrum should always be recorded to eliminate the twinkling artifact. PMID- 8633159 TI - Malignant melanoma: impact of superficial US on management. AB - PURPOSE: To evaluate the impact of superficial ultrasonography (US) on clinical management of melanoma. MATERIALS AND METHODS: Superficial US in areas at high risk for local recurrence or nodal metastases was performed in 33 patients with cutaneous melanoma. Impact categories were assigned to each US study (n = 55): category 3, US added information that altered clinical management; category 2, US added information that did not change management; category 1, no added information and management unchanged; category 0, not helpful or was misleading. RESULTS: Twenty-two US studies (40%) were category 3: detection of nonpalpable metastases altered surgical therapy (n = 2), demonstration of pharmacodynamic response to chemotherapy (n = 5), and determination of benignancy or malignancy (n = 15). Nine (16%) were category 2: identification of nonpalpable metastases did not alter management. Twenty-two (40%) were category 1: supported clinical impression of no metastases (n = 18) or helped confirm cutaneous, subcutaneous, or nodal metastases (n = 4). Two (4%) were category 0: missed proved metastases. CONCLUSION: Superficial US affected management of melanoma by allowing detection and characterization of masses, guidance of biopsy, and assessment of pharmacodynamic response. PMID- 8633160 TI - Distal radioulnar joint arthrography: simplified technique. AB - Three-compartment arthrography has been advocated in the current literature for evaluation of the intrinsic ligaments of the wrist. Previous techniques for distal radioulnar joint (DRUJ) injection introduce the needle directly into the joint space. As is done in shoulder arthrography, however, the needle tip can be positioned at the distal-radial margin of the ulnar head, allowing it to lie within the capsule. Retrospective review of 45 cases of wrist arthrography with this technique disclosed no complications. PMID- 8633161 TI - Colorectal cancer and ulcerative colitis. PMID- 8633162 TI - Lacrimal canaliculus obstruction: nonsurgical treatment with a newly designed polyurethane stent. AB - A newly designed polyurethane stent was used to successfully treat 30 eyes (in 28 patients) with epiphora resulting from obstruction of the lacrimal canaliculus. After stent placement, epiphora resolved in all eyes. During follow-up, epiphora recurred in 20 eyes, but epiphora did not recur and the lacrimal canaliculus was patent in 12 eyes after the stent was removed. The stent appears to be valuable for nonsurgical treatment of obstruction of the lacrimal canaliculus. PMID- 8633163 TI - Does lymphangiography still have a place in the staging of Hodgkin disease? PMID- 8633164 TI - Is the Mammography Quality Standard Act worth the cost? PMID- 8633165 TI - Is the Mammography Quality Standards Act worth the cost? PMID- 8633166 TI - Is the Mammography Quality Standards Act worth the cost? PMID- 8633167 TI - Percutaneous aspiration thromboembolectomy. PMID- 8633168 TI - Anatomic distribution of pulmonary emboli at pulmonary angiography: implications for cross-sectional imaging. AB - PURPOSE: To determine how often emboli detected angiographically in peripheral pulmonary arterial branches would be missed with cross-sectional imaging. MATERIALS AND METHODS: Seventy-nine of 88 consecutive pulmonary angiograms interpreted as positive for pulmonary emboli were reviewed retrospectively to detect pulmonary emboli. Three angiograms interpreted as negative when reviewed retrospectively were excluded. Findings of 76 angiograms in 76 patients (32 men, 44 women; aged 19-85 years) were correlated with the results of scintigraphy (n = 72) and Doppler ultrasound (n = 60), clinical presentation (n = 76), and follow up with chart review (n = 72). RESULTS: Two hundred five emboli were identified. Nineteen patients had solitary emboli. Sixty emboli were in the upper lobes. The largest arterial branch with pulmonary embolism was lobar in 14 patients, segmental in 38, subsegmental in 20, and smaller in three. CONCLUSION: If cross sectional imaging can depict emboli in only segmental and larger arterial branches, then emboli in 23 of 76 patients (30%) would have been missed with cross-sectional imaging. PMID- 8633169 TI - Human aorta: preliminary results with virtual endoscopy based on three dimensional MR imaging data sets. AB - PURPOSE: To determine the feasibility of use of magnetic resonance (MR) imaging data sets to perform virtual intraarterial endoscopy (VIE). MATERIALS AND METHODS: Seven female and 14 male patients (aged 9-86 years [mean, 42 years]) with various pathologic aortic conditions underwent three-dimensional gadolinium enhanced spoiled gradient-echo MR imaging. With prototype software, VIE postprocessing algorithms (based on ray casting) were applied to the imaging data sets. Findings at conventional angiography were used as the standards of reference. RESULTS: The vessel wall was seen from the inside in each case, and the following pathologic conditions were depicted clearly: the two lumina in a congenial double aortic arch and the single lumen after correction, vessel narrowing in coarctations, characteristics of Leriche syndrome, stenoses, occlusions, and the true and false lumina of dissections. CONCLUSIONS: Limitations of VIE include the image quality of the original data set, the threshold chosen to minimize intraluminal artifacts, and the inherent smoothing of vessel walls. PMID- 8633170 TI - Draped aorta: CT sign of contained leak of aortic aneurysms. AB - PURPOSE: To evaluate the usefulness of a new computed tomographic (CT) sign of "draped aorta" in the diagnosis of a contained leak in an aortic aneurysm. MATERIALS AND METHODS: CT scans were retrospectively evaluated in 10 patients in whom an unidentifiable aortic wall and posterior aspect of the aorta that followed the vertebral contour were seen. CT findings were compared with those at surgery. RESULTS: Seven patients had a deficient aortic wall and a contained leak. Two patients had a deficient wall and a mycotic aneurysm. One patient had a false aneurysm at the aortic graft anastomosis. CONCLUSION: On CT images, draping of the aorta if highly indicative of deficiency of the aortic wall and a contained leak. PMID- 8633171 TI - Can the US examination for lower extremity deep venous thrombosis be abbreviated? A prospective study of 755 examinations. AB - PURPOSE: To determine if the ultrasound (US) survey of the lower extremity for deep venous thrombosis (DVT) can be curtailed without compromising diagnostic efficacy. MATERIALS AND METHODS: The authors performed 755 US examinations in 721 patients (1,024 lower extremities) referred for suspicion of lower extremity DVT. The full lengths of the deep veins were studied, and findings were categorized at five locations: common femoral vein (CFV), proximal superficial femoral vein (CFV), mid-SFV, distal SFV, and popliteal vein (PV). RESULTS: Acute thrombus was seen in one or more veins in 131 (17.4%) of the 755 examinations. DVT isolated to a single vein was seen in 28 (21.4%) of the 131 positive examinations: DVT was limited to the CFV in eight studies (61%), to the SFV in six studies (4.6%), and to the PV in 14 studies (10.7%). CONCLUSIONS: DVT limited to a single vein occurs with sufficient frequency that the US screening survey cannot be abbreviated without loss of diagnostic efficacy. PMID- 8633172 TI - "Black blood" T2-weighted inversion-recovery MR imaging of the heart. AB - PURPOSE: To develop a short-inversion-time inversion-recovery (STIR) magnetic resonance imaging pulse sequence for evaluating the myocardium that is relatively free of flow and motion artifact. MATERIALS AND METHODS: The authors implemented a breath-hold, cardiac-triggered STIR sequence with preparatory radio-frequency pulses to eliminate signal from flowing blood. A segmented rapid acquisition with relaxation enhancement (turbo spin echo) readout was used, with the inversion recovery delay adjusted to null fat. The sequence was implemented at 1.0 and 1.5 T and tested in phantoms, five healthy volunteers, and three patients. RESULTS: Phantom studies confirmed the expected behavior of the sequence. In the volunteers, fat-suppressed images of the heart with STIR contrast were generated in a breath-hold period. Blood in the heart chambers was uniformly nulled, and motion artifacts were effectively suppressed. Focal high signal intensity consistent with edema was seen in two patients with acute myocardial infarction; in a third patient, a paracardiac mass was visualized and sharply demarcated relative to normal myocardium. CONCLUSION: Fast STIR imaging of the heart with effective suppression of flow and motion artifacts was implemented. The approach has much potential for high-contrast imaging in a variety of diseases affecting the heart and mediastinum. PMID- 8633173 TI - Latissimus dorsi muscle: assessment of stimulation after cardiomyoplasty with Doppler US tissue imaging. AB - PURPOSE: To evaluate Doppler ultrasound (US) tissue imaging for assessment of stimulated skeletal muscle contraction. MATERIALS AND METHODS: Seven patients were studied after left latissimus dorsi cardiomyoplasty. Myograft contraction programmed on alternate cardiac cycles was assessed with Doppler US tissue imaging. Beat-to-beat variation in inferior wall motion was assessed by examining peak myograft velocities during 10 muscle-assisted and 10 nonassisted cardiac cycles. The temporal relationship between electrostimulation and myograft contraction, changes in cardiac geometry, and the effect of alterations in stimulation voltage and muscle synchronization were assessed. RESULTS: Significant beat-to-beat variation in velocity profile could be detected in the proximal myograft in six patients (P < .05). Potentiation of infero-posterior wall motion was measurable in five patients (mean peak systolic wall velocity: nonassisted, 2.5 cm.sec-1 +/- 0.5 [standard deviation]; assisted, 7.8 cm.sec-1 +/ 6.3). The response between stimulation voltage and inferoposterior wall velocity was sigmoid. CONCLUSION: Doppler US tissue imaging depicted the effects of myostimulator programming on muscle contraction and ventricular wall motion. PMID- 8633174 TI - Esophagorespiratory fistulae due to esophageal carcinoma: palliation with a covered Gianturco stent. AB - PURPOSE: To evaluate therapeutic effects and complications of a covered Gianturco stent for esophagorespiratory fistulae. MATERIALS AND METHODS: Of 95 patients with esophageal carcinomas, 10 had esophagorespiratory fistulae and were treated with a silicone-covered Gianturco stent. The authors retrospectively assessed patients' food intake capacity and delayed problem of the stent. RESULTS: All fistulae were occluded without immediate complications. Two patients could swallow all foods; four, most foods; three soft foods; and one, only liquids. Clinical problems occurred between 1 and 24 weeks in four patients: reopened fistula (n = 1), recurrent fistula (n = 1) (both patients were successfully treated with another esophageal stent), and dyspnea (n = 2) due to tracheal compression by stent and tracheal invasion by tumor (one patient was treated with a tracheal stent). CONCLUSION: A silicone-covered modified Gianturco stent is effective for palliation of esophagorespiratory fistulae caused by esophageal cancer. Simultaneous use of a tracheal stent is recommended for extrinsic tracheal narrowing by the proximal tip of the stent and invasion by tumor. PMID- 8633175 TI - Complex biliary stones: treatment with a small choledochoscope and laser lithotripsy. AB - PURPOSE: To evaluate the use of a small choledochoscope and laser lithotripsy in the treatment of complex biliary stone disease. MATERIALS AND METHODS: Twenty five consecutive patients with complex biliary stone disease not amenable to surgical therapy, peroral endoscopic removal, or simple percutaneous retrieval techniques underwent 35 stone-removal procedures. A 3.4-mm endoscope and a pulsed dye coumarin green laser were used to pulverize and remove stones through preexisting, mature transhepatic biliary drain tracts (n = 18), t-tube tracts (n = 3), cholecystostomy tube tracts (n = 3), and a hepaticocutaneous enterostomy (n = 1). Procedures in 14 of the 25 patients (56%) were performed on an outpatient basis. RESULTS: Twenty-four of the 25 patients (96%) were cleared of their stone burden and underwent subsequent catheter therapy of strictures or abscesses as necessary. Complications of the stone removal included fever and chills in six patients (24%) and mild bleeding from a bile duct wall during removal of an adherent stone in one patient. CONCLUSION: Use of a small choledochoscope and a coumarin green pulsed dye laser is safe and effective in the management of complex biliary stone disease. PMID- 8633176 TI - Image-guided percutaneous hepatic biopsy: effect of ascites on the complication rate. AB - PURPOSE: To determine if image-guided percutaneous hepatic biopsy is contraindicated in patients with ascites. MATERIALS AND METHODS: The records of 476 patients (173 with ascites and 303 without) who underwent image-guided hepatic biopsy were reviewed retrospectively for number of passes, type of needle, and indications. Coagulopathy was corrected with appropriate blood products before biopsy. Complications were classified as minor (decrease in hematocrit value not necessitating treatment) of major (bleeding that necessitated transfusion or surgery or resulted in death). RESULTS: Major complications occurred in six patients with ascites and 10 without. Minor complications occurred in 10 patients with ascites and 15 without. With ascites, all major complications necessitated blood transfusions but not surgery. Five patients with major complications had a documented moderate or severe amount of perihepatic ascites. without ascites, nine of the 10 patients required blood transfusions and one required surgery. No deaths occurred in either group. CONCLUSION: Perihepatic ascites does not statistically significantly affect the major of minor complication rate of image-guided percutaneous hepatic biopsy. PMID- 8633177 TI - Dysplasia in ulcerative colitis: is radiography adequate for diagnosis? AB - PURPOSE: To determine to usefulness of barium enema examination in detecting dysplasia in patients with ulcerative colitis. MATERIALS AND METHODS: Radiographic findings of 22 areas of dysplasia in 10 patients (seven men, three women; aged 34-81 years at diagnosis) were reviewed. Serial changes in radiographic features of four areas of dysplasia in three patients were retrospectively investigated. RESULTS: Fourteen of 22 areas of dysplasia were shown on radiographs. Dysplasia in the rectum or sigmoid colon was depicted less frequently than that in other segments of the colon. Radiographic features were classified as obvious nodular protrusions (seven lesions), irregular mucosa (five lesions), or nodular protrusions with irregular mucosa (two lesions). Six of the seven areas of dysplasia shown as irregular mucosa were accompanied by minute spiculations in the margin of the colonic lumen. There was no correlation between radiologic features and histologic grade of dysplasia. CONCLUSION: Barium enema examination may be used as a complementary method of cancer surveillance with endoscopy. These methods show about two-thirds of lesions associated with dysplasia. PMID- 8633178 TI - MR cholangiopancreatography after unsuccessful or incomplete ERCP. AB - PURPOSE: To assess the value of magnetic resonance (MR) cholangiopancreatography in patients in whom endoscopic retrograde cholangiopancreatography (ERCP) was unsuccessful or did not completely delineate ductal abnormalities. MATERIALS AND METHODS: MR cholangiopancreatography was performed in 37 consecutive patients because ERCP was unsuccessful (n = 20), postsurgical biliary-enteric anatomy was present (n = 10), or evidence of complete pancreatic duct obstruction was found ar ERCP (n = 7). RESULTS: MR cholangiopancreatography was successful in all patients. Eleven patients had normal MR findings and required no further intervention. Eight patients has abnormalities that were detected with MR but were followed up clinically. Eleven patients subsequently underwent laparotomy, three underwent therapeutic percutaneous transhepatic cholangiography (PTC), two underwent diagnostic (PTC), and one underwent ultrasound-guided biopsy. CONCLUSIONS: MR cholangiopancreatography plays an important role in the care of patients in whom ERCP is unsuccessful or incomplete and when technical difficulties can be anticipated. PMID- 8633179 TI - Pancreas divisum: evaluation with MR cholangiopancreatography. AB - PURPOSE: To determine the usefulness of magnetic resonance (MR) cholangiopancreatography in the diagnosis of pancreas divisum. MATERIALS AND METHODS: In 310 patients, MR imaging was performed with heavily T2-weighted, two dimensional, fast-spin-echo sequences. A body coil was used for 139 patients, and a torso multicoil and high-resolution imaging parameters were used for 171 patients. In 108 patients, correlation with endoscopic retrograde cholangiopancreatography (ERCP) was available. RESULTS: Two hundred sixty-eight examinations (86%) were diagnostic. Pancreas divisum was observed with MR imaging in 25 of 268 cases (9%)(interobserver agreement, 98% [kappa = .88]). Among the 171 examinations performed with a torso multicoil, 10% were nondiagnostic and the rate of detection of pancreas divisum was 12%. Pancreas divisum was depicted in six of the 108 patients who underwent ERCP, and there were no false-negative or false-positive MR imaging results in these patients. CONCLUSION: MR cholangiopancreatography is an accurate tool in the diagnosis of pancreas divisum and could replace ERCP. PMID- 8633180 TI - Catecholamine responsiveness in human lymphocytes evaluated by intracellular free calcium measurements. AB - Intracellular free calcium concentration [f(Ca2+)i] and cyclic adenosine monophosphate (cAMP) in lymphocytes and basal plasma noradrenaline (NA) were measured in nine healthy male subjects (age 22-72 years). Lymphocytes were stimulated with isoproterenol and the plant lectin concanavalin A (Con A). Con A induced a dose dependent increase in f(Ca2+)i without increasing inositol lipid turnover. The mechanism by which Con A mobilized f(Ca2+)i is not clear, but we found that the level of prostaglandin E2, a metabolite from arachidonic acid, increased after stimulation with Con A, indicating the possibility of arachidonic acid released by phospholipase A2. Isoproterenol inhibited the Con A-induced calcium mobilization in a dose dependent manner in lymphocytes stimulated with both isoproterenol and Con A. This inhibitory effect of isoproterenol is most probably mediated via cAMP. Both isoproterenol induced increase in cAMP and inhibition of calcium mobilization were significantly correlated to basal plasma NA. Taken together our data indicate that the lymphocyte response to a specific stimulus may depend not only on the strength of this stimulus, but also on the level of sympathetic nerve activity. PMID- 8633181 TI - The effect of menopause and hormone replacement therapy on bone alkaline phosphatase. AB - We evaluated two different assays for the determination of bone-specific alkaline phosphatase (B-AP). One assay was a direct method using a two-site IRMA (AP(Hyb)). The other assay determined B-AP indirectly after precipitation with lectin (AP(BM)). The assays were compared to serum osteocalcin (levels) and total AP in 20 premenopausal women and in 40 early postmenopausal women before and after 9 months of treatment with hormone replacement therapy (HRT) (n = 20) or placebo (n = 20). Serum osteocalcin correlated with serum AP(Hyb) (r = 0.45), and with serum AP(BM) (r = 0.33) (both p < 0.001). The correlation between AP(Hyb) and AP(BM) was moderate, r = 0.71 (p < 0.001). When comparing postmenopausal to premenopausal women, serum osteocalcin had a t score (mean +/- SEM) of 2.30 +/- 0.29 followed by total AP(T-AP) (1.20 +/- 0.28), AP(Hyb) (1.05 +/- 0.33) and AP(BM) (0.64 +/- 0.21) (paired t-test: p < 0.01 for osteocalcin vs. other markers). After 9 months of HRT all markers had declined significantly to premenopausal levels (mean +/- SEM): osteocalcin with 44.5% +/- 6.1%; AP(BM) with 32.1% +/- 5.6%; AP(Hyb) with 36.4% +/- 4.4% and T-AP with 24.4% +/- 2.9%; p < 0.001. Of the markers, only serum osteocalcin correlated significantly with the rate of bone loss in the placebo group (r = 0.52, p < 0.02). We conclude that both assays for B-AP reflect bone turnover in postmenopausal women with and without HRT. Under controlled conditions they did not show any advantage of osteocalcin in diagnosing increased bone turnover or monitoring the effect of an anti-resorptive therapy in postmenopausal women. PMID- 8633182 TI - Studies of the release and turnover of a human neutrophil lipocalin. AB - A 24-kDa protein was purified from human neutrophil extracts and shown to be the newly discovered neutrophil gelatinase-associated lipocalin (NGAL), based on structural and immunochemical data. A specific enzyme-linked immunosorbent assay (ELISA) was developed for the determination of NGAL in human plasma and tissue fluids. Normal human plasma contains 72 micrograms l-1 of NGAL (range 40-109 micrograms l-1) in two main forms, monomer and dimer. 35S-methionine metabolic studies of human neutrophils showed that granulocyte macrophagecolony-stimulating factor (GMCSF) stimulated significant synthesis and secretion of NGAL in a dose- and time-dependent fashion. NGAL was rapidly released as monomer and dimer on incubation of heparinized whole blood with opsonized yeast, reaching a plateau corresponding to about 35% of total cell content after 30 min. Following intravenous injection of 125-iodine labelled NGAL there was a more rapid initial clearance of the monomeric than of the dimeric form; t1/2 10 min vs. 20 min. During the second phase the two forms cleared at similar rates. Severe acute peritonitis was accompanied by a 10-fold increase in NGAL plasma levels and the NGAL level in peritoneal exudates, which reached about 40 mg l-1. There was a good linear correlation between the concentrations of NGAL, leucocyte elastase and NP4 (neutrophil proteinase 4 = P3). PMID- 8633183 TI - Ethanol affects leukotriene generation and leukotriene-induced functional responses in human polymorphonuclear granulocytes. AB - Since ethanol has been shown to inhibit the inflammatory response, we evaluated whether ethanol affected generation of leukotrienes in polymorphonuclear granulocytes (PMN) in vitro. Using the calcium ionophore A23187 as stimulus, the leukotriene B4 (LTB4) and leukotriene C4 (LTC4) generation were dose-dependently impaired by ethanol. No significant difference in the levels of the omega oxidized metabolites was observed. However, the total LTB4 production (LTB4 plus omega-oxidized metabolites) was significantly decreased in the samples treated with ethanol. Furthermore, ethanol also modulated LTB4-induced functional responses. PMN aggregation, oxidative metabolism and elastase release were all inhibited in the presence of 1% ethanol (to 74 +/- 15%, 50 +/- 4% and 57 +/- 3% of controls, respectively). However, ethanol had no effect on intracellular calcium mobilization or on the change of the PMN membrane potential induced by either LTB4 or A23187. Thus, a possible mechanism for the reduced functional PMN responses in the presence of ethanol might be impaired generation of leukotrienes, but it is conceivable that ethanol impairs also other steps of the stimulus response coupling since the LTB4-induced functional responses were inhibited. PMID- 8633184 TI - The human cystatin C gene promoter: functional analysis and identification of heterogeneous mRNA. AB - Human cystatin C is a low molecular weight protein involved in the control of human cysteine proteinase activity as well as microbial cysteine proteinase activity threatening the integrity of tissues. The gene for cystatin C is located on the short arm of chromosome 20, spans 6.5 kb and has three exons. To understand the mechanisms for the expression of cystatin C at the transcriptional level we mapped the 5' boundary of mRNA transcripts and studied the 5'-region of the cystatin C gene in a transient expression system with chimeric constructs utilizing various fragments of 1.1 kb of the 5'-flanking region coupled to the gene for human growth hormone. Mapping of the 5'-end of human cystatin C mRNA from placenta and seminal vesicles (low to medium versus high cystatin C expression, respectively) identified three major transcription initiation sites (positions -75, -78 and -80, A of initiation ATG as +1) and three minor sites (positions -98, -101 and -103). The relative amounts of different mRNA species were approximately the same in these two tissues. Functional analysis of the 5' region in cultured HeLa cells revealed one region (positions -279 to -156) with a strong positive effect on transcription and comprising three identical tandemly arranged GC-rich sequences. PMID- 8633185 TI - Biological variation in bone-derived biochemical markers in serum. AB - Analytical, within-subject and between-subject components of variation have been determined for total alkaline phosphatase (ALP) and its bone isoform, osteocalcin, and tartrate-resistant acid phosphatase (TR-ACP) in serum specimens from a cohort of 10 healthy subjects over a 1-month period. From these data, we have calculated the desirable analytical imprecisions, the indices of individuality, the critical differences for significant change detection, and the number of specimens required to estimate the homeostatic setpoint of an individual. For total ALP and its bone isoform, most of the observed variability was biologic, whereas osteocalcin and TR-ACP also had relatively high analytical variability. Total and bone ALP were also the parameters showing the lowest within-subject variability, whereas TR-ACP showed the lowest interindividual fluctuation. Osteocalcin and bone ALP had marked individuality, showing that the use of population-based reference limits is inadequate for their interpretation. The applicable differences required for two results to be significantly different (p < or = 0.05) are bone ALP: 20%; osteocalcin: 29%; and TR-ACP: 35%. The results demonstrate that the biochemical markers of bone turnover studied are of limited use to screen for metabolic bone disorders, but can be useful adjuncts for monitoring groups of patients in longitudinal studies. PMID- 8633186 TI - Promoter-mediated, dexamethasone-induced increase in cystatin C production by HeLa cells. AB - Cystatin C, an efficient inhibitor of cysteine proteinases, is present in all investigated human extracellular fluids. Dexamethasone caused a significant and dose-dependent increase in the cystatin C secretion of cultivated HeLa cells up to a maximal increase of 80% at 10(-6) mol l-1 dexamethasone. Increased production of cystatin C was also observed at lower concentrations, suggesting that glucocorticoids might play a physiological role in the production of cystatin C. The effect of dexamethasone on the cystatin C gene expression was also studied in a transient transfection expression system using chimeric plasmid constructs of the cystatin C gene promoter (positions -2 to -1084) coupled to the structural gene for human growth hormone (hGH). In this system, a small, but statistically significant, increase in hGH secretion was also observed upon dexamethasone treatment, suggesting that the glucocorticoid-induced increase in secretion of cystatin C is due to a promoter-mediated increase in transcription of the cystatin C gene. PMID- 8633187 TI - Effect of felodipine on renal haemodynamics and tubular sodium handling after single-dose cyclosporin infusion in renal transplant recipients treated with azathioprine and prednisolone. AB - A total of 25 renal transplant recipients, treated solely with prednisolone and azathioprine, were investigated in a randomized, double-blind, placebo controlled, cross-over study. The effect of a single oral dose of felodipine 5 mg or placebo on: glomerular filtration rate (GFR); renal plasma flow (RPF); renal vascular resistance (RVR); renal tubular sodium and water handling, measured by the lithium clearance technique; plasma levels of angiotensin II (AngII), aldosterone (Aldo), atrial natriuretic factor (ANF) and arginine vasopressin (AVP); blood pressure (BP), and heart rate (HR) was studied before, during, and after an intravenous infusion of cyclosporin (CyA). Three consecutive clearance periods were performed, each lasting 1 h. During the second period, CyA (0.75 mg kg-1 body weight) was infused. Before infusion of CyA, felodipine caused a significant rise (6.7%) in RPF and lowered RVR, but did not change GFR significantly. The rise in RPF was abolished by infusion of CyA. After infusion, both GFR (7.8%) and RPF (9.4%) were significantly higher and RVR lower after felodipine than after placebo. Proximal tubular output and total sodium excretion were higher on the felodipine day before and after, but not during CyA infusion. In all three periods felodipine reduced both systolic and diastolic BP. In conclusion, a single dose of felodipine increases RPF and decreases blood pressure in renal transplant recipients not treated with CyA. Although some of these changes are abolished by an acute intravenous infusion of CyA, the effects of felodipine are present again also during the 1st hour after the infusion and thereby indicate at least in part some renal protective effect of felodipine. It is suggested that a higher dose of felodipine might also have been preventive against CyA renal side-effects during the acute infusion. PMID- 8633188 TI - The natriuretic effect of lithium in man: is the proximal tubule involved? AB - The possible role of the proximal tubule in the natriuresis which follows the administration of small doses of lithium, as used in lithium clearance studies, was investigated in 12 healthy males on a fixed sodium intake. Subjects were given placebo tablets, or 100 mg or 600 mg of lithium carbonate; renal function was assessed 3-6 h later. The 600-mg dose of lithium carbonate caused a 50-60% increase in sodium excretion, whereas the 100-mg dose was without effect. Creatinine clearance, used as an index of glomerular filtration rate, was unaffected by either dose. Three indices of end-proximal fluid delivery were used simultaneously: urine flow rate during suppression of vasopressin secretion (Vmax), phosphate clearance and lithium clearance (the latter only on the days on which lithium was administered). No effect of either dose of lithium on Vmax or phosphate clearance was evident; nor was there a difference between values for lithium clearance following the two doses. We conclude that administration of the standard 600-mg test dose of lithium carbonate does not affect proximal tubular function. PMID- 8633189 TI - Lymphocyte subset composition in peripheral blood from normal subjects may be influenced by both spleen size and plasma norepinephrine. AB - The aim of the present study was twofold: (a) to confirm a previously observed negative relationship between plasma catecholamines and the percentage of natural killer (NK) cells in peripheral blood from resting human subjects, and (b) to examine the relationship between the size of the spleen and the composition of circulating lymphocyte subsets in resting subjects. A total of 14 young healthy male subjects were investigated in a supine resting position. Lymphocyte subset composition was determined with two-colour flow cytometry, and lymphocyte subsets were expressed as percentages of mononuclear cells. Spleen size was evaluated with ultrasonography. Plasma catecholamines were determined. Plasma norepinephrine and the percentage of NK-cells (CD3-CD56+) were negatively correlated (rs = -0.62, p = 0.019). The CD4/CD8 ratio and plasma norepinephrine were positively correlated (rs = 0.57, p = 0.037) and the major part of this correlation was due to a correlation between plasma norepinephrine and the percentage of CD4+ cells. The percentage of NK cells (CD3-CD56+) was predicted by a multiple regression model including the percentage of CD8+ cells and the spleen index, a measure of spleen size (r = 0.93, p < 0.001). The correlation in the resting state between plasma norepinephrine and the percentage of NK cells (negative correlation) on the one hand and the CD4/CD8 ratio (positive correlation) on the other contrasts with the acute mobilizing effects of epinephrine, isoproterenol and exercise on lymphocyte subsets. These relationships remain unexplained, but results accord with the hypothesis that catecholamines may have dual effects on lymphocyte subsets. The results support the view that the spleen may have a depot function for NK cells. PMID- 8633190 TI - Blood reticulocyte count and plasma lactate dehydrogenase activity are positively related to the free fatty acid/albumin ratio in geriatric patients. AB - The objective of this study was to investigate the in vivo relationship between plasma free fatty acid (FFA)/albumin molar ratio and indicators of cellular damage. A case series study was carried out in 20 geriatric patients in a stable clinical condition. Their plasma albumin concentration was in the range 26-42 g l 1. There was a significant positive correlation between the FFA/albumin ratio and (a) reticulocyte count (r = 0.61, p = 0.006), (b) lactate dehydrogenase activity (r = 0.69, p = 0.002), and (c) haptoglobin concentration (r = 0.46, p = 0.05). The haemoglobin concentration was inversely related to relative reticulocyte count (r = -0.55, p = 0.01). Absolute and relative reticulocyte counts were positively associated (r = 0.92, p < 0.0001). The results are in accordance with the contention that a high FFA/albumin ratio in vivo may elicit cellular damage. Further studies are required to elucidate to what extent a high FFA/albumin ratio might be causally related to diseases. PMID- 8633191 TI - Nordihydroguaiaretic acid blocks IL-2-independent lymphocyte proliferation and enhances responses to PPD. AB - The authors examined the mechanism by which the non-specific lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibits human lymphocyte proliferative responses and compared its effects with those of cyclosporine (CsA). It was found that CsA-resistant proliferative responses induced by direct PK-C activators such as mitogenic concentrations of the phorbol ester TPA or the putative antitumour agent bryostatin 1 (bryo 1) were inhibited in a concentration dependent manner by NDGA (IC50 = 2 microM). In contrast, CsA-sensitive IL-2 dependent proliferative responses induced by PHA, anti-CD3 or the purified protein derivative (PPD) of M. tuberculosis were not significantly inhibited by NDGA concentrations as high as 8 microM. The expression of the IL-2R by lymphocytes was also resistant to NDGA concentrations that effectively blocked the mitogenic effects of TPA or bryostatin, but could be inhibited by higher concentrations of NDGA (IC50 = 8 microM). In addition, NDGA, but not CsA, blocked the production of IL-6 by human mononuclear cells. Furthermore, PPD-induced proliferation was significantly enhanced by NDGA. These data would suggest that NDGA at concentrations below 8 microM selectively inhibits IL-2-independent proliferation. NDGA's ability to inhibit IL-6 while enhancing the proliferative response to PPD may indicate an anti-inflammatory therapeutic potential of antioxidants in mycobacterial infections. PMID- 8633192 TI - Expression and characterization of a divalent chimeric anti-human CD3 single chain antibody. AB - Murine anti-CD3 monoclonal antibodies (MoAbs) are used in clinical practice for immunosuppression. However, there are two major drawbacks to this treatment: the associated cytokine release syndrome and human anti-mouse antibody response. To overcome these side-effects, the authors generated a chimeric anti-human CD3 single chain antibody, scUCHT1. It is an IgM variant of UCHT1, a mouse IgG1 MoAb directed against human CD3. scUCHT1 consists of the light and heavy variable chain binding domains of UCHT1 and a human IgM Fc region (CH2 to CH4). scUCHT1 was produced by COS-7 and SP2/0 transfectants, and mainly assembled in a dimeric form. It retained the binding specificity and affinity of the parental MoAb UCHT1. In contrast to UCHT1, scUCHT1 did not induce T-cell proliferation and cytokine release (TNF-alpha and IFN-gamma) in in vitro assays. These results suggest that the engineered chimeric anti-CD3 single chain antibody (scUCHT1) may be useful in clinical immunosuppressive treatment. PMID- 8633193 TI - Conformation of the T-cell antigen receptor-beta chain C-domain contributes to V beta 3 epitope recognition by monoclonal antibody KJ25. AB - The clonotypic T-cell antigen receptor (TCR)-beta chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V-domain of TCR-beta (V beta 3) chain, but its epitope requirements are unknown. In this study of TCR-alpha beta chain interactions using chimeric recombinant TCR-beta chains, the authors found that partial substitution of the C beta-domain with that of interleukin-2 receptor alpha chain (Tac) sequences led to the loss of TCR-V beta 3 epitope recognition by KJ25. These results suggest that epitope recognition of the TCR-V beta 3 by KJ25 MoAb is dependent not only on the V-domain, but also on the close contact with the extracellular C-domain which influences the conformation and epitope recognition of the V beta 3-region. This may not be unique to V beta 3 and may be a general feature of TCR-beta protein folding. PMID- 8633194 TI - Sharing of four DR-beta sequence motifs between HLA-DRB1*1601 and DRB1*1101 correlates with frequent degenerate T-cell recognition of HA306-320 peptide complexed to these two molecules. AB - This paper shows that the seven HA306-320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA-DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by V beta-J beta usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306-320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306-320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306-320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. beta 85-86, beta 67-71, beta 57 and beta 28-31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules. PMID- 8633195 TI - Role of monocytes in the up-regulation of the early activation marker CD69 on B and T murine lymphocytes induced by microbial mitogens. AB - CD69 is an early marker of lymphoid cell activation. The authors report on an up regulation of CD69 in splenic B and T cells of C57Bl/6 mice after administration of lipopolysaccharide (LPS) or microbial immunosuppressive/mitogenic (ISM) proteins produced by C. albicans (p43) and African Swine Fever Virus (p36). This up-regulation of CD69 was observed 6 and 24 h after mitogenic treatments. The same pattern of increased CD69 expression was observed in the lymph nodes of mice treated with p43 or LPS, whereas p36 treatment failed to induce increased CD69 expression in this organ. Intracellular calcium mobilization was induced in splenic B and T lymphocytes after incubation of total spleen cells with LPS, p43 or p36. This increase was higher in B than in T cells. Increased calcium mobilization was also seen in lymph node B cells after incubation with p43 or p36 and in lymph node T cells after p43 stimulation. Up-regulation of CD69 expression on B and T cells was also observed after in vitro stimulation of spleen cells with the three mitogens used. Similar results were obtained with culture supernatants of macrophage/monocyte (M phi) cells activated with LPS (LPS/M phi CS). Stimulation of M phi cells with LPS or with the ISM proteins is demonstrated by the increased production of nitrites by these cells. The increased in vitro expression of CD69 was, however, not abolished by monoclonal antibodies to M phi cytokines such as IL-6, IL-10 or TNF alpha. No increased expression of CD69 was found in vitro on purified B or T cells, even when mixed upon stimulation with p43, p36, LPS or with LPS/M phi CS. However, an increase in the expression of CD69 was observed on B cells co-cultured with M phi cells after treatment with LPS or p36. All three mitogens failed to induce increased CD69 expression on cultured T cells mixed with M phi cells. PMID- 8633196 TI - Kinetics, localization and isotype profile of antibody responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins. AB - The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built-in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B-cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen specific IgG (IgG-SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG-SC in the spleen, persisted for several weeks and, with time, IgG-SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG-SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG-SC in LN, spleen and BM suggesting that iscoms induce a potent B cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund's complete adjuvant, TiterMax or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses. PMID- 8633197 TI - Both murine SAA1 and SAA2 yield AA amyloid in alveolar hydatid cyst-infected mice. AB - Amyloid susceptible C57BL/6 and partially amyloid resistant A/J mice, infected intraperitoneally with 250 alveolar hydatid cyst (AHC), the larval stage of a cestode parasite Echinococcus multilocularis, develop multiple organ amyloid deposits at approximately 1 and 4 weeks post infection (p.i.), respectively. Pooled spleens and livers from each mouse strain, at 8 and 10 weeks p.i., were used for the purification of protein AA utilizing a HiLoad Superdex 200 column equilibrated with 5 M guanidine-HCl. Protein AA from each mouse strain was separated on 16% Tris-tricine SDS-PAGE gels and immunoblotted with monospecific rabbit anti-mouse AA IgG; five and six immunoreactive AA subspecies were detected in the C57BL/6 and A/J materials, respectively. N-Terminal amino acid sequence analysis was performed on the bulk column-purified protein AA as well as on the electroblotted AA subspecies from each mouse strain. The results show a mixture of serum amyloid A1 (SAA1) and (SAA2)-derived AA protein from each mouse strain; SAA1-derived AA, although alluded to, has never been demonstrated as tissue deposits in mice. These findings suggest that the intense and persistent inflammatory processes in AHC-infected mice may have induced conversion of weakly amyloidogenic SAA1 to AA. This conversion could be detected by amino acid sequencing of electrophoretically separated AA subspecies. PMID- 8633198 TI - Intranasal inoculation of Bordetella bronchiseptica in mice induces long-lasting antibody and T-cell mediated immune responses. AB - Humoral and cellular immune responses were analysed in mice inoculated intranasally with Bordetella bronchiseptica. After infection, the number of bacteria that colonized the respiratory tract of the mice increased during the first day and decreased thereafter. Total IgG levels increased as early as 14 days after infection and decreased with time after infection, whereas total IgA and IgM levels were lower but remained stable. Specific antibodies to the bacteria were mainly IgG2a and IgA and persisted up to 10 months after infection. Some of these specific antibodies were directed against adenylate cyclase haemolysin, the bacterial factor that had been shown to be necessary for initiation of infection. The proliferation of Bordetella bronchiseptica-reactive spleen cells occurred during the acute phase of infection. T cells from infected mice produced increasing amounts of IFN gamma and IL-2 after infection. Although very low levels of IL-10 were produced, no IL-4 was detected after bacterial stimulation in vitro. These results suggest that Bordetella bronchiseptica infection induces primarily a Th1-type T-cell response. Importantly, the authors demonstrated that antibody and T-cell responses directed against bacterial determinants of the virulent strain and to purified adenylate cyclase-haemolysin were long-lasting. This observation could be due to the fact that Bordetella bronchiseptica may persist intracellularly in the host as it was demonstrated in vitro. PMID- 8633199 TI - Distribution and nucleotide biases of the somatic hypermutations in the functional kappa light chain gene of a human follicular lymphoma line. AB - The immunoglobulin kappa chain gene of human lymphoma cell line HF-1.3.4 was partially sequenced from the 3' end of the leader exon 2.0 kb downstream. The sequenced stretch of DNA included 1.5 kb of the non-coding JK region 3' to the JK2 element of the mature gene. Among the known VK germline genes the closet relative was KV328, which was 91% homologous to HF-1.3.4. In the 1.5 kb JK region homology with JK allele of Whitehurst et al. (allele 2) was 89%, with the allele of Hieter et al. (allele 1) 87%. The vast majority of the differences located in the leader intron, the VJ exon or 0.6 kb 3' to the exon, a localization characteristic of somatic hypermutations of immunoglobulin genes. Another indication that most of the differences observed were due to somatic hypermutations is that the 153 bp stretch of the kappa constant gene (CK) sequenced from the mRNA was 100% homologous with the published CK sequence. The most differences between the JK region sequence and that of Whitehurst et al. probably represent somatic mutations: 43% were transversions, 55% transitions and 2% deletions. In the non-coding JK region transversions of C.G to G.C rather than to A.T were heavily over-represented. This is possibly a feature of B-cell hypermutations in humans and mice. PMID- 8633200 TI - Long-lasting immune response induced by recombinant bacillus Calmette-Guerin (BCG) secretion system. AB - The recombinant bacillus Calmette-Guerin (rBCG) secretion system utilizing an extracellular alpha antigen of Mycobacterium kansasii (alpha-K) was characterized biochemically and immunologically. The human immunodeficiency virus type 1 (HIV 1) p17gag B cell epitope fused to alpha-K was secreted in extremely large amounts. At least three mice out of seven inoculated with rBCG generated high titres of antibody to the epitope. The long-lasting antibody production persisted more than 14 months. PMID- 8633201 TI - Chicken joint amyloid protein is of the AA-type. I. Characterization of the amyloid protein. AB - Amyloid fibrils were extracted from deposits in joint tissue of heavy breed layers with spontaneous amyloid arthropathy and characterized as being of the AA type. Amino acid sequencing revealed a pattern quite similar to duck AA. Acute phase sera of chicken experimentally injected with Enterococcus faecalis showed a SAA-protein like band cross reacting with anti-chicken AA in immunoblot. PMID- 8633203 TI - The effect of strenuous exercise, calorie deficiency and sleep deprivation on white blood cells, plasma immunoglobulins and cytokines. AB - Moderate exercise appears to stimulate the immune system, but there is good evidence that intense exercise can cause immune deficiency. In the present study the authors examined the effect of continuous physical exercise (35% of VO2 max), calorie deficiency and sleep deprivation on the immune system of young men participating in a 5-7 days military training course. There was a two-three fold increase of neutrophils from day 1, the values remained high and decreased slightly at the end of the course. Monocyte counts also increased with a pattern similar to that of neutrophils. Eosinophils decreased to 30% of control and lymphocyte numbers decreased by 30-40%. All the major subgroups (CD4 T cells, CD8 T cells, B cells, NK cells) were reduced. Neutrophil function, as tested by measuring chemotaxis, was significantly stimulated during the first days of the course, in particular in the group with the lowest calorie intake. The mitogenic response of lymphocytes to PHA and Con A was variable, ranging from stimulation during one course to no effect in another course. Serum levels of immunoglobulins decreased significantly during the course. IgG was reduced by 6-7%, IgA by 10-20% and IgM by 20-35%. The authors found no changes of interleukin 1, 2 and 4 during the course, but a (12-20%) reduction (P less than 0.01) of interleukin 6, and an increase (P less than 0.01) of granulocyte-macrophage colony stimulating factor. Altogether the results from the ranger course present a mixed-up picture. The non specific phagocyte-related immunity was enhanced. On the other hand, the data indicate that even a moderate physical activity, around the clock, caused significant suppression of a number of parameters reflecting the status of the specific, lymphocyte-related immunity. It is noteworthy, however, that there was no significantly increased infection rate during the course or in the first 4-5 weeks thereafter. PMID- 8633202 TI - Experimental human Plasmodium falciparum infections: longitudinal analysis of lymphocyte responses with particular reference to gamma delta T cells. AB - The kinetics of the gamma delta T-cell response was analysed in the context of the overall haematological response in subjects experimentally infected with sporozoites of Plasmodium falciparum. Numbers of gamma delta and alpha beta T cells and NK cells declined markedly during infection to reach minimum values 12 13 days post-infection when the patients were ill. This decline commenced from the beginning of the erythrocytic cycle and well before parasites could be detected microscopically and clinical symptoms developed. Platelet numbers also declined. In vivo activation of gamma delta T cells was evident with sequential up-regulation of the activation markers CD69 and HLA-DR. gamma delta T cell numbers were highest after treatment with the majority being CD4-CD8-, HLA-DR+ and showing reduced CD45RA expression. Contrary to some published observations gamma delta T-cell percentages remained within the normal range. Little evidence of upregulation of activation or memory markers was observed in the alpha beta T cell population. In vitro proliferative responses to malaria antigen which involve gamma delta T cells were lost as the infection progressed and the lymphocyte count declined but these could be restored with the addition of exogenous IL-2 to cultures. The authors findings are consistent with a protective and/or immunomodulatory role for gamma delta T cells in malaria. PMID- 8633204 TI - Peptide antibiotics: holy or heretic grails of innate immunity? AB - In the last 2 years (1994-95), two symposium volumes and three reviews have been published that were fully devoted to peptide antibiotics (antibacterial peptides or antimicrobial peptides). Since the field has been growing rapidly, this review is largely a follow-up of new results published in the last 2 years. Sequencing of the 16S RNA of the small ribosomal subunit indicate that the microbial world is much larger than generally appreciated. The importance of the natural flora is stressed and its effect on the evolution of peptide antibiotics and immunity in general is discussed. PMID- 8633205 TI - Cloning and sequencing of an MPB70 homologue corresponding to MPB83 from Mycobacterium bovis BCG. AB - MPB70 is secreted in high concentrations by Mycobacterium bovis BCG substrain Tokyo (BCG Tokyo), but little by substrains Pasteur (BCG Pasteur) and M. tuberculosis. The gene encoding a MPB70 homologue secreted by BCG Tokyo was found at the upstream region of the gene encoding MPB70, with approximately 2.3 kilobase pairs (kbp) spacing: the same gene was also found in BCG Pasteur. This gene was cloned and sequenced from BCG Tokyo. The DNA sequence which contained a 663 base pair (bp) open reading frame beginning at position 1 and ending with a TAA codon at position 661 was found. Its theoretical molecular mass was calculated to be 22.068 kDa. This gene was highly homologous to the coding region of mpb70 and the deduced amino acid sequence was very similar to MPB83 reported by Harboe et al. It was speculated that the gene the authors characterized probably corresponded to the mpb83 gene. PMID- 8633206 TI - Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. AB - The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70. PMID- 8633207 TI - Augmented antigen presentation by mouse Ia + T clone cells BK-BI-2.6.O4.1 mediated by transferrin receptors. AB - The murine T clone cells BK-BI-2.6.O4.1 (BI/O4.1) synthesize and express MHC class II molecules constitutively. BI/O4.1 cells are able to present various protein antigens to antigen-specific CD4 + T cells. However, a 10-fold higher concentration of antigen is needed to activate specific T cells to lymphokine secretion by BI/O4.1 cells in comparison with spleen cells or with the more homogeneous population of bone marrow-derived macrophages (BMMph). The authors tested whether the reduced antigen presentation potential of BI/O4.1 cells was augmented by transferrin-mediated uptake of the model antigen ovalbumin (OVA) coupled to human ferric transferrin. It was shown that 240-fold less OVA was sufficient to induce proliferation of an OVA-specific T-cell clone when the conjugate and not native OVA was used. The presence of ferric TF in the cultures competitively inhibited this effect of the conjugate. A similar shift in the dose response curve to lower doses of antigen was induced by the conjugate when B lymphoma cells were used as antigen-presenting cells. BMMph and P388D1 cells processed and presented the conjugate with similar efficiency as native OVA, although both cell types exposed transferrin receptors. These data suggest that the reduced antigen presentation potential of BI/O4.1 T clone cells is due to the inefficient uptake of OVA by pinocytosis and delivery into the processing compartment. PMID- 8633208 TI - Enhanced T lymphocyte expression of LFA-1, ICAM-1, and the TNF receptor family member OX40 in HgCl2-induced systemic autoimmunity. AB - Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl2, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl2. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4+ and CD8+ cells. Compared to control rats, HgCl2 exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1 + cells were significantly increased in all CD45RC-defined subsets of CD4+ and CD8+ cells. Within the CD4 + CD45RC10 T lymphocyte population, HgCl2-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4 + CD45RC10 blast cells of HgCl2-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26 + cells. The results indicate that induction of autoimmunity by HgCl2 in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4+ CD45RC10 cells, which may be caused by a direct effect of HgCl2 on these cells, and may precipitate further activation of T and B lymphocytes by HgCl2. PMID- 8633209 TI - T-cell receptor diversity expressed by CD4+ T cells activated by primary allogeneic HLA-DR stimulation: estimation of the degree of CDR3 diversity. AB - In order to analyse the diversity of T-cell receptors (TCRs) expressed by the T cell population activated by allogeneic HLA-DR stimulation, TCR beta cDNA was synthesized from mRNA of human CD4+ T cells that had been stimulated in a primary mixed lymphocyte reaction (MLR). The TCR beta cDNA was amplified by the polymerase chain reaction (PCR), subjected to bacterial cloning, and sequenced from V beta through J beta. Twenty-six different V beta and 10 different J beta segments were detected among 56 randomly selected cDNA clones. Occurrences of V beta 17.1 and J beta 1.5 were higher than those found in the CD4+ T-cell population activated with a CD3-specific antibody. A total of 53 different CDR3 sequences, two of them occurring more than once, were detected among the 56 cDNA clones. In order to estimate the degree of CDR3 diversity, amino acid similarity in the CDR3 region of the cDNA was calculated and compared with those of the anti CD3-activated T-cell sequences as well as those of various published T-cell clone sequences, each directed to either alloantigens or single antigenic peptides. It was found that the similarity score among CDR3 sequences obtained from the MLR (56.4 +/- 10.3) was comparable to those of anti-CD3-activated T cells (55.7 +/- 10.7) and those of T-cell clones directed toward alloantigens (range, 48.4 +/- 12.4-59.4 +/- 13.1), but significantly smaller than those of T-cell clones directed toward single antigenic peptides such as those derived from myelin basic protein (75.6 +/- 17.9) and cytochrome c (76.9 +/- 20.5). These results provide quantitative proof that TCRs of T cells activated by primary allogeneic HLA-DR stimulation have a larger diversity than those recognizing single antigenic peptides. PMID- 8633210 TI - Establishment and characterization of RAG-2 deficient non-obese diabetic mice. AB - The authors have established a new immunodeficient mouse strain on the genetic background of the diabetes prone non-obese diabetic (NOD) mouse. A deletion mutant of the RAG-2 gene was back crossed 10 generations onto the NOD/Bom strain background. The homozygous NODrag-2-/- mice lack functionally mature B and T lymphocytes and do not develop insulitis or diabetes throughout life. In contrast, heterozygous NODrag-2+/- develop both insulitis and diabetes with an incidence similar to the wild type NOD mice. In transfer experiments, spleen cells from diabetic NOD donors were found to transfer disease to NODrag-2-/- recipients similar to what has been previously observed in transfer to irradiated NOD recipients or to immunodeficient NOD-scid/scid mice. While resembling the recently established NOD-scid/scid mice in many respects, the NODrag-2-/- mice represents an advantageous model for reconstitution of the pathogenesis of murine IDDM as it does not produce any endogenous, mature T or B lymphocytes. PMID- 8633211 TI - Identification and characterization of specific receptor for interleukin-8 from the surface of human monocytes. AB - A specific receptor for interleukin-8 has been identified on the surface of human monocytes using 125I IL-8 as a probe. A binding kinetic pattern shows that saturation was attained after 90 min and that the receptor was distinct from the receptors of other cytokines (IL-L alpha, IL-2, TNF alpha, GMCSF) and FMLP. Scatchard analysis of the binding data shows that 7000-10,000 receptors /monocyte are present with an equilibrium Kd 7 x 10(-9) M. By immunoblot, the receptor for IL-8 showed a sharp band with approximate M.W. 59 kD, consistent with the M.W. of IL-8 receptor of neutrophils. In Boyden Chamber, monocytes migrated towards IL-8 and the cytokine was observed to induce transient rise of intracellular Ca+2 in the cells. Thus, identification of functionally active IL-8 receptor in monocyte may be helpful for understanding its possible role during inflammation. PMID- 8633212 TI - Multiple levels of MHC class I down-regulation by ras oncogenes. AB - A number of tumours and oncogene transformed cells displayed reduced MHC class I surface expression which seemed to enable their escape from immune surveillance. To test whether oncogenic activation is directly involved in suppressing MHC class I expression, a model of inducible oncogene expression was chosen. Mouse fibroblasts transfected with different oncogenes expressed under the control of the dexamethasone-inducible MMTV promoter were analysed in the presence and absence of hormone for the mRNA and protein expression of MHC class I molecules as well as the respective oncogenes. Immunofluorescence analyses demonstrated an inverse association of MHC class I and oncogene expression after dexamethasone stimulation, independent of the type of oncogene causing transformation. Hormone mediated induction of oncogene expression caused down-regulation of all H-2 loci. Kinetic experiments using MMTV c-Ha-ras(A) transfectants revealed that down regulation of MHC class I surface expression was preceded by a dexamethasone induced change of morphology, anchorage-independent growth, and an increase of the ras protein p 21. Parallel monitoring of mRNA expression demonstrated a time dependent up-regulation of ras specific transcripts, which was associated with differential regulation of MHC class I heavy and light chain transcripts. Beta2 microglobulin transcripts were transiently suppressed, whereas MHC class I heavy chain transcripts remained unaffected. To investigate the mechanisms of oncogene mediated down-regulation of MHC class I expression, H-2 promoter transfections and a nuclear run on assays were performed. In MMTV c-Ha-ras(A) cells, neither alterations of the H-2 promoter activity nor of the transcriptional activity of H 2 antigens was observed in the presence of dexamethasone, whereas both could be up-regulated by interferon-gamma treatment. These data suggest that oncogene mediated transformation is directly associated with MHC class I down-regulation, but that complex interactions affecting MHC class I heavy and light chain genes at the transcriptional and/or post-transcriptional level are involved in this process. PMID- 8633213 TI - In vivo activation of T-cell induction into the primed phenotype and programmed cell death by staphylococcal enterotoxin B. AB - The authors demonstrate that SEB immunization activates V beta 8+ T cells and induces the acquisition of the primed phenotype as defined previously by low MEL 14 and high Pgp-1 expression. SEB-activated spleen CD4+ and CD8V beta 8+ T cells have different population dynamics and regulate the expression of MEL-14 and Pgp 1 differentially, suggesting that the SEB-MHC class II complex preferentially activates CD4V beta 8+ T cells. Interestingly, at day 3 after SEB immunization, V beta 8+ T cells expressing low, but not high, levels of MEL-14 undergo apoptosis, indicating that T-cell activation is a prerequisite for triggering programmed cell death. These results might help to trace antigen-reactive cells to the activated or primed pool, as well as to identify those cells which will undergo programmed cell death. PMID- 8633214 TI - Experimental AA-amyloidosis in mice is inhibited by treatment with the anti rheumatic drug tenidap. AB - In a mouse model for induction of experimental AA-amyloidosis, treatment with tenidap was shown to inhibit the development of amyloidosis. Studies have shown that the drug inhibits the cytokines interleukin (IL)-6, IL-1 and tumour necrosis factor alpha (TNF-alpha) which are known to stimulate hepatocytes to synthesize acute phase proteins (APPs). The APP serum amyloid protein (SAA) is the precursor for amyloid protein AA and tenidap treatment reduces the serum levels of SAA in mouse and humans. It is suggested that reduction of SAA levels reduces the risk of AA fibril formation and thus the development of amyloidosis. PMID- 8633215 TI - Mapping of the epitope recognized by non-specific cytotoxic cells: determination of the fine specificity using synthetic peptides. AB - NCC recognize a conserved target cell antigen (NKTag) expressed on protozoan parasites and on transformed tumour cells. In the present study, synthetic peptides corresponding to N-terminal, C-terminal and internal NKTag(deduced) amino acid sequences were tested for binding and inhibition of NCC lysis of sensitive target cells. A 20-mer peptide equivalent to amino acids (aa) nos. 55 74 specifically inhibited NCC lysis of human EBV transformed target cells (IM-9). Inhibitory effects were nonreversible and concentration dependent, and 30 min pre incubation produced optimum inhibition. The inhibitory 20-mer peptide was truncated into 17, 14, 10, 9 and 6-mer peptides and tested for inhibition of cytotoxicity. All produced almost complete inhibition except the 6-mer which had no activity. The NKTag sequence required for NCC binding (minimally) consisted of seven amino acids [aa nos 68-74 (ARG-ASN-LEU-THR-PHE-ILE-LEU-)]. The specificity of inhibition and the distribution of target cells expressing NKTag was determined. A 14-mer peptide composed of aa nos 61-74 inhibited lysis of HL-60, IM-9, DAUDI, YAC-1, U937 and NC-37 target cells. Flanking peptides (aa nos 35-54 and 75-94) were negative. Biotinylated aa nos 61-74 bound to NCC effector cells. The recognition requirements for aa sequence versus aa content were determined. Randomization of the aa in the cognate 9-mer obliterated the inhibitory effects. The 17-mer (cognate) synthetic peptide inhibited conjugate formation between NCC and IM-9 targets. These data demonstrate that NCC recognize a conserved antigen determinant on susceptible target cells consisting of a minimum of 7-9 amino acids in the N-terminal region of NKTag. PMID- 8633217 TI - Inhibition of immunoglobulin production in vitro by IgG and F(ab')2 fragments, but not by the Fc portion. AB - Antibody-secreting B cells were measured as plaque-forming cells (PFC) in a modified haemolysis-in-gel assay, using protein A coupled sheep erythrocytes as targets. Human lymphocytes from blood (PBL), bone marrow or spleen were stimulated in vitro by various polyclonal B-cell activators and incubated with intravenous immunoglobulin (IVIG) or peptide fragments of IVIG. IgG and IgM production from PBL and bone marrow cells, measured as PFC, was inhibited more than 50% by IVIG 2.5 mg/ml, compared to controls without IVIG. Inhibition of the IgG and IgM response of spleen cells by IVIG varied depending on the stimuli. Using Staphylococcus aureus protein A (SPA), inhibition was almost 90% (P < 0.001). The inhibition of the IgG and IgM responses to lipopolysaccharide from Escherichia coli (LPS) were 70% (P < 0.01) and 28% (P < 0.05), respectively. IgG stimulation by pokeweed mitogen (PWM) was inhibited by 57% (P < 0.01), but the IgM response was inhibited only by the higher IVIG concentration of 5.0 mg/ml. In mixed lymphocyte cultures of spleen cells, IgG and IgM production were inhibited by more than 60% (P < 0.05). The effect of IgG, IgG-F(ab')2 and IgG-Fc on LPS or PWM-stimulated spleen cells were compared, using equimolar concentrations of the various preparations. IgG- and IgM-producing PFC were significantly (P < 0.05) inhibited in a dose-dependent fashion by IgG and F(ab')2, but not by Fc. LPS induced IgG and IgM production was inhibited also when IgG and F(ab')2 were added up to 48 h after the stimulator. A comparison of IgG, F(ab')2 and Fc products from different companies showed that all IgG and F(ab')2 preparations significantly inhibited IgG and IgM production of LPS-stimulated spleen cells. No significant inhibition was obtained with any of the purified Fc products. PMID- 8633216 TI - Brush border enzymes in evaluation of acute small bowel rejection. AB - The change in activity of three disaccharidase enzymes (maltase, sucrase and lactase) was determined according to the method of Dahlqvist during acute rejection in non-immunosuppressed piglet small bowel grafts. In addition, two brush border enzymes, lactase and aminopeptidase, were stained with monoclonal antibodies. Diminishing disaccharidase activity was an early event during rejection. Diminution began 2 days before distinct morphological changes were seen in the mucosal biopsies. Evaluation of disaccharidase activity can thus be used as a confirmatory method in detecting rejection. Reduction in immunohistological staining of lactase and aminopeptidase with monoclonal antibodies and changes in mucosal morphology were observed to progress simultaneously. PMID- 8633219 TI - Different percentages of peripheral blood gamma delta + T cells in healthy individuals from different areas of the world. AB - The frequency of gamma delta + T cells in the peripheral blood of 26 Turkish, 24 Swedish, 35 Japanese and 14 "Asian' (non-Japanese) healthy blood donors and healthy volunteers were investigated by flow cytometry. In the Turkish group, 9.3% (median value) of the CD3+ peripheral blood T cells expressed the gamma delta T cell receptor. A similar level of gamma delta + T cells was found in the non-Japanese "Asian' healthy volunteers (9.2%), while significantly lower values were detected in the Swedish (4.2%) and Japanese (4.5%) groups. These dramatic differences in normally occurring gamma delta + T cells in different groups of healthy individuals were further reflected by a low incidence of > 10% gamma delta + T cells in the Swedish (0/24) and Japanese (6/35) groups compared to the Turkish (12/26) and "Asian' (5/14) groups. The described gamma delta + T cell differences between distinct ethnic groups are thus likely to be a consequence of environmental factors, but additional genetic influences cannot be ruled out. The present study demonstrates the potential importance of the ethnic origin and environmental history of subjects examined in studies of gamma delta + T cells disease relations. PMID- 8633218 TI - Characterization of autoantibodies to natural killer cells in HIV-infected patients. AB - Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Auto-antibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/microliters) and antibody negative (141/microliters) patients (P = 0.3). However, pre incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells (P = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo. There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo. PMID- 8633220 TI - Accumulation of cadmium, copper and zinc in the liver of some passerine species wintering in central Norway. AB - The concentration (mg kg-1 dry weight) of cadmium (Cd), copper (Cu) and zinc (Zn) in the liver of Parus palustris, P. montanus, P. major, Carduelis chloris and Pyrrhula pyrrhula was determined in birds collected in October-March, 1992-1995, in Central Norway. This study is especially focused on interspecific and age dependent variations. The metal concentrations in liver are generally higher for adults than for juveniles, and there is an accumulation of Cd in the Parus species during winter. There is a positive correlation between Cd-Cu and Cu-Zn, but not for Cd-Zn. Juvenile P. montanus and P. major from a subalpine area had a higher accumulation of Cd and Cu than juvenile P. palustris from lowland areas, probably as an effect of a higher consumption of willow (Salix spp., known to have a high level of cadmium) seeds and insects living on willows, and a relatively high copper level in the ground of the subalpine area. PMID- 8633221 TI - The three Rs and biomedical research. PMID- 8633222 TI - Immunological tolerance. PMID- 8633223 TI - Immunological tolerance. PMID- 8633224 TI - Koch keeps new watch on infections. PMID- 8633225 TI - U.S. beefs up CDC's capabilities. PMID- 8633226 TI - Malaria hideout found in new mothers. PMID- 8633227 TI - Environmental estrogens. New yeast study finds strength in numbers. PMID- 8633229 TI - Chlamydia linked to atherosclerosis. PMID- 8633228 TI - SIV data raise concern on oral-sex risk. PMID- 8633230 TI - New dynamic duo: PET, MRI, joined for the first time. PMID- 8633231 TI - Astronomers probe creation by measuring isotopes. PMID- 8633232 TI - To see a world in 80 kilograms of rock. PMID- 8633233 TI - The "bio" in biochemistry: protein folding inside and outside the cell. PMID- 8633234 TI - A new turn (or two) for twist. PMID- 8633235 TI - Environmental estrogens: can two "alrights" make a wrong? PMID- 8633236 TI - Far-infrared hydrogen lasers in the peculiar star MWC 349A. AB - Far-infrared hydrogen recombination lines H15 alpha (169.4 micrometers), H12 alpha (88.8 micrometers), and H10 alpha (52.5 micrometers) were detected in the peculiar luminous star MWC 349A from the Kuiper Airborne Observatory. Here it is shown that at least H15 alpha is strongly amplified, with the probable amplification factor being greater than or about equal to 10(3) and a brightness temperature that is greater than or about equal to 10(7) kelvin. The other two lines also show signs of amplification, although to a lesser degree. Beyond H10 alpha the amplification apparently vanishes. The newly detected amplified lines fall into the laser wavelength domain. These lasers, as well as the previously detected hydrogen masers, may originate in the photoionized circumstellar disk of MWC 349A and constrain the disk's physics and structure. PMID- 8633237 TI - Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. AB - The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAFII31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAFII31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAFII31 but nevertheless inhibits the p53-TAFII31 interaction. PMID- 8633238 TI - Evidence that Spt6p controls chromatin structure by a direct interaction with histones. AB - Genetic analysis has implicated SPT6, an essential gene of Saccharomyces cerevisiae, in the control of chromatin structure. Mutations in SPT6 and particular mutations in histone genes are able to overcome transcriptional defects in strains lacking the Snf/Swi protein complex. Here it is shown that an spt6 mutation causes changes in chromatin structure in vivo. In addition, both in vivo and in vitro experiments provide evidence that Spt6p interacts directly with histones and primarily with histone H3. Consistent with these findings, Spt6p is capable of nucleosome assembly in vitro. PMID- 8633240 TI - twist: a myogenic switch in Drosophila. AB - Somatic muscle is derived from a subset of embryonic mesoderm. In Drosophila, Twist (Twi), a basic helix-loop-helix transcription factor, is a candidate regulator of mesodermal differentiation and myogenesis. Altering amounts of Twist after gastrulation revealed that high levels of Twist are required for somatic myogenesis and block the formation of other mesodermal derivatives. Expression of twist in the ectoderm drives these cells into myogenesis. Thus, after an initial role in gastrulation, twist regulates mesodermal differentiation and propels a specific subset of mesodermal cells into somatic myogenesis. Vertebrate homologs of twist may also participate in the subdivision of mesoderm. PMID- 8633241 TI - Structural evidence for functional domains in the rat hippocampus. AB - The hippocampus has two major outputs: multisynaptic pathways to the cerebral cortex and a massive descending projection directly to the lateral septal part of the basal ganglia. Here it is shown that the descending output is organized in such a way that different hippocampal regions map in an orderly way onto hypothalamic systems mediating the expression of different classes of goal oriented behavior. This mapping is characterized by a unidirectional hippocampo lateral septal projection and then by bidirectional lateral septo-hypothalamic projections, all topographically organized. The connectional evidence predicts that information processing in different regions of the hippocampus selectively influences the expression of different classes of behavior. PMID- 8633239 TI - Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist. AB - The myogenic basic helix-loop-helix (bHLH) and MEF2 transcription factors are expressed in the myotome of developing somites and cooperatively activate skeletal muscle gene expression. The bHLH protein Twist is expressed throughout the epithelial somite and is subsequently excluded from the myotome. Ectopically expressed mouse Twist (Mtwist) was shown to inhibit myogenesis by blocking DNA binding by MyoD, by titrating E proteins, and by inhibiting trans-activation by MEF2. For inhibition of MEF2, Mtwist required heterodimerization with E proteins and an intact basic domain and carboxyl-terminus. Thus, Mtwist inhibits both families of myogenic regulators and may regulate myotome formation temporally or spatially. PMID- 8633242 TI - Infection and AIDS in adult macaques after nontraumatic oral exposure to cell free SIV. AB - Unprotected receptive anal intercourse is a well-recognized risk factor for infection with human immunodeficiency virus-type 1 (HIV-1). Isolated human case reports have implicated HIV-1 transmission by oral-genital exposure. Adult macaques exposed nontraumatically to cell-free simian immunodeficiency virus (SIV) through the oral route became infected and developed acquired immunodeficiency syndrome (AIDS). The minimal virus dose needed to achieve systemic infection after oral exposure was 6000 times lower than the minimal dose required to achieve systemic infection after rectal exposure. Thus, unprotected receptive oral intercourse, even in the absence of mucosal lesions, should be added to the list of risk behaviors for HIV-1 transmission. PMID- 8633243 TI - Synergistic activation of estrogen receptor with combinations of environmental chemicals. AB - Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses. PMID- 8633244 TI - Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. AB - Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn-/- mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i. PMID- 8633245 TI - A subfamily of P-type ATPases with aminophospholipid transporting activity. AB - The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation. Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced. The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases. Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family. PMID- 8633246 TI - Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. AB - Molecular chaperones in the eukaryotic cytosol were shown to interact differently with chemically denatured proteins and their newly translated counterparts. During refolding from denaturant, actin partitioned freely between 70-kilodalton heat shock protein, the bulk cytosol, and the chaperonin TCP1-ring complex. In contrast, during cell-free translation, the chaperones were recruited to the elongating polypeptide and protected it from exposure to the bulk cytosol during folding. Posttranslational cycling between chaperone-bound and free states was observed with subunits of oligomeric proteins and with aberrant polypeptides; this cycling allowed the subunits to assemble and the aberrant polypeptides to be degraded. Thus, folding, oligomerization, and degradation are linked hierarchically to ensure the correct fate of newly synthesized polypeptides. PMID- 8633248 TI - Preclinical evaluation of CPT-11 and its active metabolite SN-38. AB - CPT-11 (irinotecan) is a water-soluble analogue of camptothecin (CPT), an antitumor drug extracted from the Chinese tree Camptotheca acuminata. SN-38 is an active metabolite of CPT-11 that contributes significantly to its activity. The antitumor effects of CPT-11 and SN-38 are exerted through a novel mechanism of action; inhibition of DNA topoisomerase I. CPT-11 and its metabolite have demonstrated potent inhibitory activity against a variety of cancer cell lines in vitro and against several murine and human tumors grafted in mice in vivo, including those that express multidrug resistance. CPT-11 has also shown synergistic activity in combination with 5-fluorouracil and cisplatin in vitro. No irreversible or unusual toxicities were observed with CPT-11 in animal toxicity studies. In summary, the preclinical profile of CPT-11 confirmed this drug to be an attractive candidate for clinical development. PMID- 8633247 TI - Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. AB - Women are particularly susceptible to malaria during first and second pregnancies, even though they may have developed immunity over years of residence in endemic areas. Plasmodium falciparum-infected red blood cells (IRBCs) were obtained from human placentas. These IRBCs bound to purified chondroitin sulfate A (CSA) but not to other extracellular matrix proteins or to other known IRBC receptors. IRBCs from nonpregnant donors did not bind to CSA. Placental IRBCs adhered to sections of fresh-frozen human placenta with an anatomic distribution similar to that of naturally infected placentas, and this adhesion was competitively inhibited by purified CSA. Thus, adhesion to CSA appears to select for a subpopulation of parasites that causes maternal malaria. PMID- 8633249 TI - CPT-11: an original spectrum of clinical activity. AB - Due to its novel mechanism of action, CPT-11 (irinotecan) has significant in vitro activity against a variety of solid tumors, including those particularly resistant to other cytotoxic agents. This activity has been confirmed in clinical trials of single-agent CPT-11 conducted in Japan, Europe, and the United States. In chemotherapy-naive patients with advanced non-small cell lung cancer, a response rate of 32% to 34% has been shown in Japan with CPT-11 monotherapy, although this has been improved to within the range of 43% to 54% using CPT-11 in combination with cisplatin. Prior chemotherapy appears to reduce the response rate substantially in this setting, although the mechanisms of cross-resistance are unknown. CPT-11 is also active in small cell lung cancer, with a single agent response rate of 47% in patients previously treated with cisplatin. As might be expected, CPT-11 is more active when combined with cisplatin as first-line chemotherapy for small cell lung cancer, with Japanese investigators reporting an average response rate of 85%. Consistent results from studies of colorectal cancer in Japan, the United States, and Europe, indicate that CPT-11 is active as a single agent in patients who have developed progressive disease following 5 fluorouracil (5-FU)-based treatment. In patients with metastatic colorectal cancer, approximately 18% to 27% of patients with 5-FU refractory disease and 15% to 32% of patients who are chemotherapy-naive respond to single agent therapy. Efforts to combine CPT-11 with 5-FU with or without folinic acid are ongoing. There is less clinical experience with CPT-11 in the treatment of other solid tumors, but activity has been reported in phase II trials of patients with squamous cell carcinoma of the uterine cervix or skin, and in those with cancer of the ovary, stomach, or pancreas and in patients with lymphoma. While notable objective response rates have been reported for single agent CPT-11, the precise role of this drug in the treatment of patients with solid tumors has yet to be defined, especially as part of first-line therapy. CPT-11 appears to be one of the most exciting new drugs to reach clinical development in the past decade, and its seemingly wide spectrum of clinical activity suggests that it may have a substantial impact on the treatment of many of the most common epithelial malignancies. PMID- 8633250 TI - CPT-11: clinical experience in phase I studies. AB - Phase I studies of CPT-11 (irinotecan) have been conducted in Europe, the United States, and Japan to determine the maximum tolerated dose (MTD) and the most appropriate intravenous administration schedule for further evaluation in phase II investigations. Diarrhea and/or neutropenia were the major dose-limiting toxicities in all the phase I studies. In Japanese and US investigations, the CPT 11 MTD was defined as 240 to 250 mg/m2 using a once every 3 weeks schedule and 100 and 150 mg/m2 using a weekly schedule. The weekly intermittent schedule was associated with greater dose intensity and was thus chosen in Japan (100 mg/2 every week) and in the United States (150 mg/m2/wk for 4 weeks followed by 2 weeks rest) for further evaluation in phase II studies. Preliminary results of an ongoing US study showed that CPT-11 could be administered in doses of at least 175 mg/m2 on an every other week basis. In European studies, the MTD was 90 to 100 mg/m2/d with IV infusion over 3 consecutive days every 3 weeks and 100 to 115 mg/m2 with a weekly infusion for 3 weeks every 4 weeks. European experience with a single infusion every 3 weeks showed diarrhea to be dose limiting at 350 mg/m2, but concomitant administration of high-dose loperamide allowed administration of CPT-11 at doses of up to 600 mg/m2. This latter schedule was better tolerated, achieved the highest dose intensity, and was considered to confer greater convenience in an outpatient setting when compared with the other European regimens. European phase II studies were therefore commenced using a CPT-11 schedule comprising a single intravenous infusion every 3 weeks at a dose of 350 mg/m2. PMID- 8633251 TI - Eukaryotic DNA topoisomerase I: genome gatekeeper and its intruders, camptothecins. AB - Topoisomerase I enzymes are ubiquitous and play a pivotal role in DNA transcription, replication, and repair. The eukaryotic form of this enzyme is highly conserved and its inhibition leads to accumulation of DNA strand breaks ('cleavable complexes') and ultimately cell death. An understanding of the role of eukaryotic topoisomerase I has led researchers to identify this enzyme as a potential target for anticancer therapy. Indeed, topoisomerase I is inhibited by samptothecin (isolated from a plant extract), and derivatives of this agent are being developed with improved physicochemical and pharmacologic characteristics. These agents may provide a new dimension to chemotherapy through their novel mechanism of action. PMID- 8633252 TI - CPT-11 in the treatment of colorectal cancer: clinical efficacy and safety profile. AB - CPT-11 (irinotecan) is a promising anticancer agent with a novel mechanism of action dependent on the inhibition of the DNA eukaryotic enzyme, topoisomerase I. The clinical utility of CPT-11 in advanced colorectal cancer has been documented in more than 400 patients recruited in phase II clinical trials in Europe, Japan, and United States. Among 178 eligible patients in a multicenter European study, the overall response rate to CPT-11 on a once-every-3-weeks regimen was 18%, and the median duration of response was 9.1 months. Thirty-two percent of the patients had no evidence of disease progression at 6 months. These results were similar in chemotherapy-naive and pretreated patients. These findings are consistent with the results of other studies conducted in Japan and the United States in which a weekly CPT-11 regimen was associated with response rates of 15% to 32% in chemotherapy-naive or pretreated patients. The principal adverse events of CPT-11 are neutropenia and delayed diarrhea, which in the European studies developed as grade 3 or 4 toxicity in 21% and 12% of the cycles (47% and 38% of patients), respectively. Neutropenia did not appear to be cumulative, with total recovery by day 22 in most cases. Loperamide was considered the most effective agent for controlling delayed diarrhea. Other adverse events included an early cholinergic-like syndrome (consisting of diaphoresis, early diarrhea, and abdominal cramps), nausea and vomiting, fatigue, and alopecia. In conclusion, CPT 11 has shown promising antitumor activity in the treatment of patients with advanced colorectal cancer, including those refractory to 5-fluorouracil (5-FU) based regimens, suggesting no cross-resistance to 5-FU. CPT-11 appears to have activity similar to that of 5-FU in first-line treatment and, moreover, remains active after failure of 5-FU therapy. The specific gastrointestinal toxicity is manageable, and a better control of this type of toxicity is expected in the future. CPT-11 would therefore appear a welcome addition to the oncology armamentarium for this difficult-to-treat malignancy. PMID- 8633253 TI - Role of chemotherapy for advanced colorectal cancer: new opportunities. AB - Since the late 1950s, 5-fluorouracil (5-FU) has remained the most effective chemotherapeutic agent in the treatment of advanced colorectal cancer, although response rates to 5-FU monotherapy are typically no more than 15%. However, efforts at improving the response to this agent have involved its administration with biochemical-modulating agents and in protracted infusion schedules. The combination of 5-FU with folinic acid has evinced the most promising results, with an approximate twofold increase in response rate compared with monotherapy (23% versus 11%). Preliminary evidence also suggests further benefits when this regimen is administered by continuous infusion. These results support the current clinical practice of using 5-FU plus folinic acid as first-line therapy in advanced colorectal cancer. However, for those patients refractory to a 5-FU based chemotherapy, there is no established effective treatment option. Current approaches to enhance second-line therapy involve biochemical modulation of 5-FU and prolongation of its administration schedule or the use of new antitumor agents such as CPT-11 (irinotecan) and oxaliplatin. Among the new agents in development, CPT-11 has demonstrated promising antitumor activity in phase II studies of patients with advanced colorectal cancer, achieving response rates of 15% to 32% and 17% to 25% in chemotherapy-naive and pretreated patients, respectively. Prior disease progression on 5-FU does not affect the response to CPT-11, indicating no cross-resistance between the two agents. This suggests that CPT-11 is a promising new second-line agent for this difficult-to-treat disease. Additional studies will address the benefit of CPT-11 alone in second-line treatment and determine the role of CPT-11 in combination with thymidylate synthase inhibitors in first-line treatment. PMID- 8633254 TI - Relevant parameters for describing the oxygenation status of solid tumors. AB - AIM: Several studies using different methods of pO2 measurement have shown that in many tumor entities tissue oxygenation is quite heterogeneous and thus cannot be sufficiently described by a single value. This study presents those distribution parameters which are mandatory to describe the complete oxygenation status of a solid tumor. METHODS: In determining the oxygenation status different aspects of relevancy have to be taken into account. First, several parameters are necessary to characterize the location, the spread, and the shape of the pO2 distribution from a statistical point of view (statistical parameters). In addition, parameters for the description of the biological relevance of the tumor oxygenation have to be considered (biological parameters). These parameters reflect the role of oxygenation for the metabolic microenvironment and the biological behavior of the tumor as well as the significance under therapeutic and diagnostic aspects. Finally, some parameters are necessary to assess the quality of pO2 measurement itself (quality parameters). RESULTS: By combining all pO2 readings from one tumor a statistical distribution is obtained, which is in many tumors asymmetrically shifted towards low pO2 values. In order to describe the location, spread, and shape of this distribution the following parameters should be stated: median, mean, 10% and 90% percentile, minimum, maximum and 10 to 90% interpercentile range. For data presentation these values can be combined in a box-whiskers plot. In order to reflect the importance of tumor oxygenation under diagnostic, therapeutic or cell-biological aspects the fraction of pO2 readings < or = 2.5 mm Hg, < or = 5 mm Hg, and < or = 10 mm Hg should be stated. Where negative pO2 readings occur in a study, the fraction of pO2 readings < 0 mm Hg as well as the most negative pO2 value should be presented in order to assess the quality of the pO2 measurement. CONCLUSIONS: For a comprehensive description of the oxygenation status of solid tumors the following distribution parameters should be stated: median, mean, 10% and 90% percentile, minimum, maximum, 10 to 90% interpercentile range, fraction of pO2 readings < 0 mm Hg, < or = 2.5 mm Hg, < or = 5 mm Hg, and < or = 10 mm Hg as well as the most negative pO2 reading. PMID- 8633255 TI - [Surgery versus radiotherapy in Ewing's sarcoma with good prognosis. Analysis of the CESS-86 data]. AB - PURPOSE: The evaluation of radiotherapy and surgery as exclusive local treatment in comparably selected subgroups of patients with Ewing's sarcoma on the basis of the CESS 86-data. PATIENTS AND METHODS: In the German multicenter Ewing's sarcoma study CESS 86, treatment consisted of four 9-week-courses of VACA- or VAIA chemotherapy plus local therapy. VACA (vincristine, actinomycin D, cyclophosphamide, adriamycin) was given in low-risk extremity tumors with a tumor volume below 100 cm3. High-risk patients with central lesions or a tumor volume > 100 cm3 received VAIA (ifosfamide instead of cyclophosphamide). Local therapy started after one complete chemotherapy course in week 10. Based on an individual decision in each patient, local therapy was either radical surgery or resection plus postoperative irradiation with 45 Gy or definitive radiotherapy with 60 Gy. Because of poor results with radiotherapy in a preceding study, it was intended to restrict irradiation to patients with small lesions. RESULTS: Hundred and seventy-seven protocol patients were recruited from January 1986 through June 1991 and 176 received local therapy: 39 underwent radical surgery, 44 received definitive radiotherapy and 93 were treated with resection and postoperative irradiation. The median tumor volume was higher in patients with radiotherapy as compared to combined local treatment or radical surgery, 156 cm3 versus 140 cm3 versus 102 cm3. The overall 5-year survival after radiotherapy and surgery was nearly identical, 63% versus 67% for the whole group 75% versus 65% in tumors < 100 cm3 volume and 65% versus 67% in tumors with 100 cm3 to 600 cm3 volume, respectively. CONCLUSIONS: With regard to tumor volume, the most important single prognostic factor in Ewing's sarcoma, irradiated patients were poorer selected than surgically treated patients despite the fact that a selection of good-risk patients for radiotherapy was intended. The nearly identical survival figures after surgery and radiotherapy suggest that radiotherapy is as effective as surgery if selection of patients is comparable. PMID- 8633256 TI - [Radiotherapy of regional lymph nodes compared to retroperitoneal lymphadenectomy in the treatment of seminomas. Retrospective analysis of 161 patients]. AB - BACKGROUND: Retroperitoneal lymphadenectomy is obsolete for treatment of seminomas, radiation therapy is the treatment of choice. As no literature is available about a comparison of both methods, we refer to data of the University of Magdeburg. PATIENTS AND METHODS: Hundred and sixty-one patients with seminoma of stage I and II were retrospectively analysed. They were treated at the University of Magdeburg between 1975 and 1991 by radiation therapy of regional lymph nodes or by retroperitoneal lymphadenectomy. After high semicastration, 98 patients were irradiated, 63 patients received a retroperitoneal lymphadenectomy. Twenty-one patients were treated by adjuvant chemotherapy, too. RESULTS: The 5 year survival-rates according to Kaplan-Meier were 96% for stage I, 85% for stage IIA, 92% for state IIB, and 68% for stage IIC. The overall survival rates for all stages were 95% after 2 years, 92% after 5 years, and 89% after 10 years. Relapses located retroperitoneally occurred significantly more often after retroperitoneal lymphadenectomy (9.5%) compared with radiation therapy (2.0%), relapses outside the operation situs or radiation fields, respectively, were registrated at the same frequency (4.8% and 7.1%, respectively). Disease-free survival rates decreased significantly with increasing stages (p < 0.001, Wilcoxon-test). Relapses increased from 4.1% for stage I up to 58.3% for stage IIC. CONCLUSION: After semicastration for primary treatment of seminomas radiation therapy of the regional lymph nodes is the treatment of choice. Retroperitoneal lymphadenectomy is obsolete. PMID- 8633258 TI - [Radiobiologic in-vivo investigation of angiogenesis in the yolk sac vascular system of chicken embryos]. AB - PURPOSE: The aim of this study was to investigate whether the yolk sac blood vessel system of chick embryos represents a useful model of in-vivo measurements of angiogenesis in radiobiology. MATERIAL AND METHODS: The fertilized eggs of the crossbreeding White Plymouth Rox X Sussex were used for the study of the extraembryonic vascularisation after irradiation with different fractionation and dosage. On day 3 of incubation an oval window was cut into the shell and then the area vasculosa was irradiated with 1.0 cm in diameter. Forty-eight hours after irradiation in-vivo photographs were taken for quantitative evaluation of blood vessel density. RESULTS: Irradiation from 2.0 to 8.0 Gy in a single fraction leads to a slight decrease in vascular density. After a single fraction of 10.0 Gy a distinct increase in vascular density occurs. Fractionated irradiation leads to a considerable clear increasing of vascular density in comparison with the single fraction and same total dose. CONCLUSION: Our model of the fertilized egg enables in-vivo measurements of angiogenesis in radiobiology. PMID- 8633257 TI - [5-fluorouracil: cause of a fatal myocardial infarction in combined radiochemotherapy?]. AB - PURPOSE: A 53-year-old female patient was treated with combined radiochemotherapy with 5-fluorouracil (5-FU) because of adenocarcinoma of the lung. She died after the 2nd day of the first course on fatal myocardial infarction. The histological evaluation of the heart revealed no severe chronic fibrosis. In our opinion the myocardial infarction was partly 5-fluorouracil-related in this case. PATIENTS AND METHODS: Published case reports on this theme were reviewed and discussed. RESULTS: The review of the literature showed a 5-FU associated cardiotoxicity in 98 cases. Most of the patients were without evidence of pre-existing myocardial disorders. The discussion about the reasons of the 5-FU-associated cardiotoxicity is still going on. Cardiotoxicity will rise up in 1.1% to 4.5% of the patients treated with 5-FU. Patients with a history of cardiac disease were at significantly increased risk for 5-FU-induced cardiotoxicity. CONCLUSION: In high risk-patients 5-FU should not be given without electrocardiographic monitoring. The continuous infusion is better than a bolus treatment. PMID- 8633259 TI - Evaluation of rectal shielding in a Henschke system applicator. AB - PURPOSE: To assess the effectiveness of avoid shielding in the Henschke intracavitary gynaecologic 3-channel applicator. MATERIAL AND METHODS: An acrylic phantom was used with our locally modified 3-channel Henschke applicator so that standard treatments with caesium-137 sources could be simulated. Thermoluminescent dosimeters were used to measure point A and rectal doses with and without ovoid shielding to assess the benefits of this shielding. Unshielded measurements were also compared to our planning computer calculations to assess its accuracy. The thermoluminescent dosimeters were calibrated against a caesium teletherapy unit. An estimate of shielding effect produced by the ovoid shielding when using iridium-192 wire was also determined. RESULTS: Doses received at points in the plane containing the rectal point as defined by the ICRU in its report 38 [4] show a reduction ranging from 5% to 15% over the area measured due to the ovoid shielding. As expected this benefit is more pronounced when using iridium-192 sources. Given the steep dose gradients, good agreement is found between computed and measured values of point A doses. CONCLUSION: While ovoid shielding will never provide a large reduction in rectal dose using caesium-137 sources, our results indicate that it can be a worthwhile option when using the Henschke applicator. PMID- 8633260 TI - [Radiation load on the skin using a silicone-coated polyamide wound dressing during photon and electron radiotherapy]. AB - BACKGROUND: Silicone-coated polyamide wound dressing is frequently used for the supportive treatment in patients with radiation induced skin lesions. The use of this kind of dressing during radiotherapy with high energy beams shifts the dose built-up effect towards the skin surface. Thus the dose delivered to the skin increases. The present work quantifies changes of the skin dose by a commercial silicon-coated polyamide wound dressing. The dependence on the beam quality and on different treatment techniques is investigated. PATIENTS AND METHODS: Measurements were performed with photon (60Co, 6 MV, 42 MV) and electron (7 MeV, 20 MeV, 40 MeV) beams using thin LiF thermoluminescence dosimeters (TLD) in a perspex phantom. The beams were directed perpendicularly to the phantom surface. For 60Co and 6 MV photon beams the skin dose was evaluated in vivo at different beam arrangements and at a given reference dose. RESULTS: For 60Co, 6 MV and 42 MV photon beams wound dressing caused a dose increase on the surface of the perspex phantom by a factor of 1.65, 1.39 and 1.33 respectively. Using oblique or rotational techniques for 60Co and 6 MV photon irradiation the wound dressing increased the skin dose but less compared to perpendicular beam direction. For electron beams the skin dose is relatively high (from 84% to 92%) and an increase by a dressing has no clinical relevance (factor 1.03 to 1.05). CONCLUSION: The silicone-coated polyamide wound dressing causes no relevant skin dose increase during radiation treatment with electron beams and can be left on the skin during irradiation. During radiation treatment with photon beams like 60Co and 6 MV the protective procedure should be adapted to skin changes, in case of strong skin reactions a removal during the time of irradiation should be considered. PMID- 8633261 TI - [Supraglottic partial laryngectomy with or without postoperative radiotherapy in supraglottic carcinomas]. PMID- 8633262 TI - [Randomized multicenter ESHO-study: Hyperthermia improves results in recurrent and metastatic malignant melanoma]. PMID- 8633263 TI - [Concurrent radiochemotherapy of primary squamous cell carcinoma of the vulva]. PMID- 8633264 TI - [Long-term results after conservative surgery of hypothalamic/ chiasmatic astrocytomas in childhood]. PMID- 8633265 TI - [Improvement of therapy and the quality of radiotherapy in Hodgkin's disease in the USA in 1973 versus 1983]. PMID- 8633266 TI - [Do perioperative blood transfusions influence the prognosis of patients with colorectal cancer?]. PMID- 8633267 TI - [Anal cancer: current results of the EORTC and RTOG/ECOG studies]. PMID- 8633269 TI - [Combined effect of Paclitaxel and ionizing radiation. Experimental data of in vitro and in-vivo models]. PMID- 8633268 TI - [Preclinical and clinical results in the simultaneous radiochemotherapy of head and neck tumors with Paclitaxel]. PMID- 8633270 TI - [Paclitaxel and simultaneous radiotherapy in stage-III non-small-cell bronchial carcinoma]. PMID- 8633272 TI - Surgical advances in implant dentistry. PMID- 8633271 TI - [Taxol and simultaneous radiotherapy in stage IIIA and IIIB, locally advanced, non-small-cell bronchial carcinoma. Phase-I clinical trial]. PMID- 8633273 TI - Prevention and management of oral surgery-related complications. PMID- 8633274 TI - Closed lock of the temporomandibular joint. PMID- 8633276 TI - Waiver of co-payments or deductibles--is it legal? PMID- 8633275 TI - Dental detection of spousal abuse. PMID- 8633277 TI - Parameters of care: a structured method for improving care. PMID- 8633278 TI - TDA legislative accomplishments: the last ten years. PMID- 8633279 TI - A little help from our friends... PMID- 8633280 TI - ADA Council on Membership addresses local needs and concerns. PMID- 8633281 TI - New trends show employee dentists increasing in number. PMID- 8633282 TI - Dispensing prescription drugs to patients--a response from the Pharmacy Board. PMID- 8633283 TI - Carbon dioxide laser surgical therapy for the management of oral leukoplakia: a case report. PMID- 8633284 TI - The new supplemental fluoride dosage schedule. PMID- 8633285 TI - Components of a successful practice. PMID- 8633286 TI - The patient's perspective. PMID- 8633287 TI - The Texas "employment-at-will" doctrine and related employment issues. PMID- 8633288 TI - The 1994 C.T. Rowland Award Orthodontic Case Report. Malocclusion. PMID- 8633289 TI - How carcinogens cause cancer. AB - Several concepts have been formed regarding the origin of primary malignancies, but only recently has a theory on second primary cancers evolved. The aim of this report is to review the literature regarding current concepts associated with carcinogenesis, tobacco and alcohol, oral premalignancy, field cancerization and cancer prevention. PMID- 8633290 TI - Smoking and wound healing: a review. AB - It is now generally accepted that smoking impairs wound healing. This article briefly reviews wound healing and a number of local and systemic responses to smoking which may have deleterious effects on wound healing. It is important that dentists fully understand the deleterious effects of smoking on wound healing and that they fully explain to their patients who smoke the compromised tissue response and surgical results that can be anticipated. PMID- 8633291 TI - Recent graduates' evaluation of their dental oncology curriculation. AB - A survey of recent dental school graduates regarding their preparation for and provision of dental care for cancer patients in their practices suggests several trends. Although an extensive educational effort has brought about an introduction of dental students to medically compromised patients, over 90% of the recent graduates surveyed recommended maintaining or increasing curriculum time devoted to each of twelve didactic and clinical dental oncology areas considered. The majority of dentists surveyed evaluated their dental school educational experience as weak in five (biopsy techniques, treatment modalities, maxillofacial prosthetics, nutrition for cancer patients, and patient support sources) of the twelve didactic and clinical areas considered. Not one of the twelve areas was evaluated as strong by a majority of the dentists. It is apparent that accommodation to the suggestions of practicing dentists must be made if general dentists are to provide high quality, comprehensive dental care for cancer patients. PMID- 8633292 TI - Prescribing nicotine substitutes for tobacco cessation: considerations and contraindications. AB - This article deals with the topic of nicotine replacement drugs and their use in smoking cessation. Questions frequently arise as to their prescription by dentists. Provided patients are carefully selected on the basis of medical and psychological profiles, there is no ethical or legal contraindication to dentists prescribing these aids. This article reviews the current literature and answers commonly asked questions concerning these effective drugs. PMID- 8633293 TI - What are you doing about oral cancer? Report of a survey of Texas dentists. PMID- 8633294 TI - Oral aspects of chemotherapy: patient information. PMID- 8633295 TI - Trends in tobacco use. AB - The production and use of tobacco have been accepted parts of the American life style for over 500 years. Tobacco use in the United States has gone through many stages over the years. The present article reviews the trends of tobacco use over the last 100 years and the public health strategies recently initiated to control tobacco use and promote public health. Finally, suggestions regarding the dentist's role in limiting tobacco use and promoting public health are presented. PMID- 8633297 TI - Utilization review organizations. PMID- 8633296 TI - The ability of disability coverage. PMID- 8633298 TI - Rights. PMID- 8633299 TI - Leadership. PMID- 8633300 TI - Advances in the use of guided tissue regeneration for localized ridge augmentation in combination with dental implants. AB - Osseointegrated dental implants placed into adequate bone enjoy a high success rate. However, placing implants into resorbed alveolar ridges can result in fixtures that are not optimally positioned or must be placed in insufficient bone, thus reducing their rate of success. Recently, techniques for bone regeneration developed for use around teeth have been applied to implant dentistry. Guided Tissue Regeneration (GTR), also called Guided Bone Regeneration (GBR), uses thin membranes which act as barriers to soft tissue ingrowth. This approach for regenerating bone around implants is described in detail in this paper. Initial approaches and modifications are covered as are specific details of current techniques. PMID- 8633301 TI - Notes on the history of dental radiology. PMID- 8633303 TI - Intraoral digital radiography. PMID- 8633302 TI - Modernizing your office for oral radiography. PMID- 8633304 TI - Diagnostic imaging for dental implant assessment. PMID- 8633305 TI - Practical imaging of the temporomandibular joint. PMID- 8633306 TI - Specialized radiographic techniques as an aid to the diagnosis of maxillofacial lesions: a series of case studies. PMID- 8633307 TI - Guidelines for prescribing dental radiographs. United States Food and Drug Administration. PMID- 8633309 TI - A message from the Texas Bureau of Radiation Control. PMID- 8633308 TI - X rays: what is the risk? PMID- 8633310 TI - Is managed care in your future? PMID- 8633311 TI - Illegal remuneration. PMID- 8633312 TI - The status of oral and maxillofacial radiology in American dentistry. PMID- 8633313 TI - The 1994 survey of Texas dentists--final report. PMID- 8633314 TI - The evaluation of a reported allergic reaction to an amide local anesthetic: a case report. PMID- 8633315 TI - How to increase treatment plan acceptance. PMID- 8633316 TI - Patient records--do yours comply with State law? PMID- 8633317 TI - Changes in the Texas controlled substance schedule. PMID- 8633318 TI - When does it make sense for a new dentist to buy a practice? PMID- 8633320 TI - Ultrasonics in periodontal therapy--the paradigm has changed. PMID- 8633319 TI - Consent for a minor. PMID- 8633321 TI - Nursing report cards: are Texas nurses ready? PMID- 8633322 TI - Streamlining, yet reaching out: a report from the CMG Task Force. PMID- 8633323 TI - Nursing report card: a new quality indicators yardstick. PMID- 8633324 TI - [Medicine, physicians and mass media--dissatisfaction and misunderstandings]. PMID- 8633325 TI - [Folic acid and pregnancy--certain knowledge, uncertain practice]. PMID- 8633326 TI - [Microvascular reconstruction in the jaw and the oral cavity. A medical progress]. PMID- 8633327 TI - [Breast cancer]. PMID- 8633328 TI - [Tumor size and histological grading of stage 1 breast cancer. Prognostic and therapeutic significance]. AB - Systemic adjuvant therapy can improve the prognosis for breast cancer patients. In node negative disease most patients have a good prognosis with local therapy only. Therefore, identification of subgroups with higher risk of relapse is warranted in order to avoid overtreatment of a large number of patients. A total of 399 tumours from patients without axillary metastases, operated on in the years 1964-72, were measured preoperatively and graded according to a modified Scharff-Bloom-Richardson scheme. Patients with T2 (UICC) tumours and histologic grade 2 and 3, had a 3.3-fold relative risk of mortality from breast cancer compared with the rest of the patients. These patients should receive adjuvant systemic therapy. PMID- 8633329 TI - [Free vascular grafts in reconstructions in the head and neck region]. AB - Since 1989, 40 free-tissue grafts were used in 39 patients to repair defects following major head and neck ablative surgery (n = 32) and for reconstruction of the mandible because of osteoradionecrosis or trauma (n = 7). The radial forearm flap was used in 17 patients (five including a segment of radius), the lateral arm flap in seven, fibula in five and a segment of ileum for restoration of the hypopharynx in 11 cases. Three radial forearm flaps and the bone in one lateral arm flap failed. Better functional and cosmetic results seem to be obtained with free flaps than with other reconstructive techniques. PMID- 8633330 TI - [Craniofacial surgery]. AB - Craniofacial surgery is the term given to surgical techniques which allow combined access to the neurocranium and the facial skeleton. These techniques are applied primarily for treatment of fractures of the frontal/nasal/orbital regions, tumours of the orbits and the anterior cranial base, and congenital malformations, including simple or complex craniofacial synostoses, e.g. Crouzon and Apert syndromes. Indications for surgical treatment and the timing of the surgery are discussed. In addition, the most common surgical techniques are described and some clinical cases are presented. PMID- 8633331 TI - [Classification of amyloidosis]. AB - Amyloidosis is a collective term for a heterogeneous group of disorders characterized by deposition of a fibrillar, proteinaceous material, amyloid, in various tissues and organs. Increasing knowledge about the different proteins that constitute the amyloid fibrils has made it possible to classify amyloidosis by the fibril protein, which appears more rational than the traditional classification by its clinical symptoms. A group of experts on amyloidosis met in Oslo in 1990 and agreed upon a nomenclature and classification based on the chemical properties of the amyloid fibrils. PMID- 8633332 TI - [Human T-cell lymphotrophic virus type I and T-cell lymphoma]. AB - Human T-cell lymphotropic virus type I is an oncogenic retrovirus, endemic in Southwestern Japan, the Caribbean, some parts of Africa and Central and South America. The virus is etiologically associated with adult T-cell leukemia/lymphoma and a myelopathy called tropical spastic paraparesis or HTLV-I associated myelopathy. Transmission of the virus is almost identical to that of HIV. The latency period before onset of clinical symptoms can last from a few years (tropical spastic paraparesis) up to several decades (adult T-cell leukemia/lymphoma). Four different clinicopathological subtypes of the T-cell neoplasia are known, and in this article we describe two patients with the subtype lymphoma. PMID- 8633333 TI - [Guillain-Barre syndrome. Variation on the theme]. AB - Guillain-Barre syndrome, or acute inflammatory demyelinating polyneuropathy, is a frequent cause of acute onset of flaccid paresis and areflexia. Electrophysiological studies show demyelination, sometimes with varying degrees of axonal degeneration. In some cases axonal degeneration apparently develops rapidly, without signs of primary demyelination. However, it is still a matter of discussion whether a pure axonal form of Guillain-Barre syndrome exists, or whether the axonal degeneration is secondary to demyelination. We report on clinical and electrophysiological findings in three patients with variants of Guillain-Barre syndrome. These include pure demyelination, combined demyelination and axonal degeneration and possible primary axonal degeneration. Electrophysiological studies can differentiate between the variants of Guillain Barre syndrome, and thus give indications of pathogenesis and prognosis. PMID- 8633334 TI - [Postoperative bacteriuria and urinary tract infections in gynecological patients]. AB - This prospective study was carried out to establish the risk of postoperative asymptomatic bacteriuria developing into a symptomatic urinary tract infection if left untreated. Among 454 women who underwent routine gynaecological surgery using transurethral catheters for a mean of 1.4 days, 243 (53.5%) had postoperative bacteriuria and 74 (16.3%) developed urinary tract infection. Positive urine cultures showed a low predictive value for infection, even when combined with the presence of pyuria. Among 174 women discharged with asymptomatic bacteriuria, 36 (20.7%) developed urinary tract infection, of which all but two were caused by Gram-negative organisms or enterococci. A set of recommendations for handling postoperative bacteriuria is proposed. PMID- 8633335 TI - [Painless femoral neck fracture in a patient with multiple sclerosis]. AB - We describe a patient with multiple sclerosis who suffered a painless hip fracture. She was admitted to the hospital three weeks after falling on her left hip. She had been unable to walk after the trauma, but had had no pain. The condition was initially misinterpreted as multiple sclerosis schub. X-ray examination showed fracture of the left femoral neck, and by the time she was admitted to hospital the patient was paretic. She has improved substantially after an operation involving cementless total hip arthroplasty. Painless fractures in patients with multiple sclerosis may be confused with exacerbations of their neurological disease. PMID- 8633336 TI - [Folate deficiency, cancer and congenital abnormalities. Is there a connection?]. AB - The biochemical role of folate is in the interconversion of one-carbon units in intermediary metabolism; a process in which a methyl group is formed de novo. The methyl group is subsequently transferred to adenosylmethionine, which is an important methyl donor in the methylation of DNA. A negative correlation exists between the intake of folate in pregnancy and the occurrence of neural tube defects and certain malignant brain tumours in children. Numerous clinical studies have pointed to an association between folate status in adults and both the occurrence of cancer and the premalignant changes, cervical dysplasia, bronchial metaplasia, and colorectal adenomas. Folate deficiency may cause chromosomal damage, due to impaired DNA synthesis or repair. Moreover, decreased production of adenosylmethionine may influence the expression of developmental genes and of oncogenes and/or tumour suppressor genes through disturbed methylation of DNA. PMID- 8633337 TI - [Press coverage of medical issues]. AB - We assessed the coverage of medical issues in six Norwegian newspapers during the period January - March 1995. A majority of the articles and a minority of the letters presented neutral information about the national health system. 13% of the articles and 61% of the letters were, in our opinion, critical. Only 2% and 5%, respectively, conveyed unequivocally positive attitudes. In at least three out of 11 incidents, the journalists violated the ethical rules commonly accepted by members of the press for criticism of named health workers. In 503 major articles overall correctness of the medical information, as judged by the authors, was 85% and varied between newspapers (top 98%, bottom 70%). We conclude that health workers are more likely to be criticised than praised. The credibility of the medical information varied from one newspaper to another. PMID- 8633338 TI - [Evaluation of health information in newspapers and brochures. What can we believe?]. AB - Health service users need reliable information to be able to make informed decisions. We evaluated the scientific quality of information presented at a large health fair in Oslo in 1995 and health-related articles in five newspapers during one week in the spring of 1995. The brochures scored consistently low on the index used to measure scientific quality. It was generally impossible to distinguish opinions from facts, or to assess the validity and consistency of the evidence underlying the conveyed information. Important consequences (benefits, risks and costs) were rarely assessed. Health-related articles in the lay press also scored low on the index. Minor changes could have led to marked improvement of some of the articles. It is important to improve the ability of journalists and producers of brochures to appraise their work in a critical light. PMID- 8633340 TI - [Physicians and the mass media. Opinion of physicians on the coverage of medicine, health policy and the medical profession by the mass media]. AB - Every fourth physician disapproves of mass media coverage of health policy questions. Four out of ten physicians are dissatisfied with the way scientific medical issues are covered, and seven out of ten are dissatisfied with articles and programmes about the medical profession. The physicians' dissatisfaction was predicted by a low level of perceived job autonomy and a high level of perceived unrealistic expectations from patients, families, superiors and politicians. The doctors' disapproval of press coverage of the medical profession increased with perceived stress. The dissatisfaction was clearly greater among younger than among older physicians. Internists and surgeons tended to be more dissatisfied than physicians in other specialties. Dissatisfaction with mass media was not, however, a question of personal grudge: respondents who felt that they themselves had been unfairly spoken of by the media did not disapprove of the media coverage of health issue, or of the medical profession in general, to any greater degree than did doctors who had no such personal complaints. PMID- 8633339 TI - [Physicians pilloried? On physicians' blunders and blundering physicians in Norwegian tabloids]. AB - Doctors frequently claim that the tabloid press focuses too much on malpractice cases and that the identity of the individual physicians is also revealed. A systematic review of two Norwegian tabloids over six months in 1994 showed that articles on malpractice occurred less often than expected. We found 1.6 articles each week, on average. In addition, the doctors were identified in only three of 42 articles. The situation leading to the complaint was commented by the doctor involved in one article, while criticism from a colleague who was not directly involved was included in two of 42 articles. The results indicate that doctors need to be trained to handle the press. PMID- 8633341 TI - [What are the possibilities for public health care in a system based on physicians' patient lists?]. AB - In Norway, primary health care is characterised by decentralisation of both care of patients and general public health services to municipal level. In all but the larger municipalities, both these services are carried out by the same primary health care doctors. In order to strengthen the quality of the doctor-patient relationship, and the associated responsibility, four municipalities are testing out a system where all inhabitants are placed on a specific doctor's list. Since the design of the test primarily emphasises the doctor-patient relationship, many community health doctors and planners are concerned about how this will affect local public health tasks. The authors discuss the experiences from these four municipalities as regards the impact on public health tasks and suggest new ways of organising public health at municipal level based on the doctors' overall responsibility for their listed patients. PMID- 8633342 TI - [Doping legislation]. PMID- 8633343 TI - [Diagnostic imaging of pulmonary embolism]. PMID- 8633345 TI - [How well does a dying person hear?]. PMID- 8633344 TI - [Familial hypercholesterolemia in children]. PMID- 8633346 TI - [Only nerves?]. PMID- 8633347 TI - [Physicians are no longer always at work...]. PMID- 8633349 TI - [Stay clear from the quacks]. PMID- 8633348 TI - [Misleading advertising on a new NSAID]. PMID- 8633350 TI - Looking at the Visible Man. World's first digital human body reference is now available on the Internet. PMID- 8633351 TI - A perspective on change. PMID- 8633352 TI - Hospital cost shifting: surgeon found items marked up as much as 14,000%. PMID- 8633353 TI - When you are first at the scene of an accident. PMID- 8633354 TI - Embracing change. PMID- 8633355 TI - Practical tips for appearing before the State Board of Nursing. PMID- 8633356 TI - Nursing care for the prevention of deep vein thrombosis. AB - 1. Deep vein thrombosis (DVT) and pulmonary embolism (PE) are major health problems that often result in significant postsurgical morbidity and mortality. 2. To prevent DVT, patient care includes graduated compression stockings or the use of a pneumatic compression device, and administration of the correct dose of anticoagulation agent (heparin or LMWH). 3. Taken together, the various drug therapies and physical interventions can clearly prevent DVT. Careful evaluation of the patient's risk factors, along with a monitored postoperative therapy can minimize the morbidity and mortality of this "unseen" condition. PMID- 8633357 TI - Sharing our world: perioperative nurses have the vision. AB - 1. Perioperative nurses must bridge the information gap and eliminate public misconceptions about surgery and OR nursing. Fear of the unknown realm of surgery and televised inaccuracies about surgical procedures magnify the lack of awareness of health care consumers about surgery and the holistic nursing care perioperative nurses provide for their patients. 2. There is a national need for more aggressive general promotion of health care awareness and education. This need can be successfully facilitated at the grassroots level with minimal efforts from members of our surgical departments. 3. One effective way to solve this problem is to invite health care consumers into our facilities. PMID- 8633358 TI - What causes human cancer? AB - 1. Lung cancer is the leading cause of cancer death in American men and has now surpassed breast cancer as the leading cause of cancer death among American women. 2. Heavy alcohol consumption is associated with increased cancer risks. 3. Intercourse at an early age and multiple sexual partners have been linked to an increased risk of cervical cancer. 4. The most widespread environmental carcinogen, accounting for the majority of superficial skin cancers and some types of melanoma, is ultraviolet radiation from the sun. PMID- 8633359 TI - When the OR manager is task focused rather than people focused. AB - Being an effective manager requires a delicate balance of "task focused" and "people focused" managerial actions. Even when the manager has a reason to be focused on tasks, employees should be unlikely to find lack of "people focus." Morale, creativity, and initiative will decrease on the unit, the manager's power and authority may be usurped, and the manager may feel lonely and unsupported by staff. This lack of support for the manager also may be demonstrated in written evaluations or by the employees going to the manager's superior to complain. PMID- 8633360 TI - Metastasis as a cause of pain following total hip arthroplasty: a case report. PMID- 8633362 TI - Sources of cancer data and their accuracy. PMID- 8633361 TI - Burden of proof in a civil case. PMID- 8633363 TI - Management of infected hip arthroplasty. AB - 1. Infected hip arthroplasties can be successfully managed with the appropriate surgical treatment and a long-term antimicrobial therapy. 2. The type of surgical treatment must be based on considerations of various prognostic indications such as the infective agent, the duration of the infection, the stability of the implant component, and the quality of the bone stock and the soft tissue. 3. The majority of infections in artificial hip joints occur by direct inoculation during surgery or by contamination of a local hematoma that arose after surgery. PMID- 8633364 TI - Evaluation of the thromboxane A2 synthetase inhibitor OKY-046 in a warm ischemia reperfusion rat model. AB - The pathophysiology of ischemia-reperfusion renal injury is mediated, in part, by the generation of the vasoconstricting prostanoid thromboxane A2 (TXA2). This study was undertaken to evaluate the renoprotective effects, as well as the optimal timing and dosage, of a selective thromboxane synthetase inhibitor, OKY 046, in a unilateral nephrectomized, 60 min ischemia, 72 hr reperfusion, rodent model. Forty-one rats were subjected to right nephrectomy only (group A), or right nephrectomy with 60 min of left renal ischemia and treatment with inactive vehicle only (group B), or 2 mg/kg or 4 mg/kg of OKY-046 administered intravenously before (groups C and D) or after (groups E and F) pedicle clamping. Outcome variables included animal survival; change in kidney weight; 0, 24, and 72 hr plasma creatinine (CR); urea nitrogen (BUN); thromboxane B2 (TXB2) and 6 keto prostaglandin F(1alpha) (6 kPGF(2alpha)) levels; creatinine clearance (CRCL); and histologic evidence of renal injury. Animal survival and postperfusion kidney weight were not significantly different among the groups. However, renal functional parameters were significantly improved with the 2 mg/kg dose of OKY-046 administered after renal ischemia. (group B 72 hr Cr= 8.01 +/- 1.1 mg% vs. group E=3.99 +/- 1.5 mg%, and group B 72 hr BUN=241.3 +/- 32.8 mg% vs. group E=52.6 +/- 22.5 mg%). The CRCL was also improved in group E vs. group B, although these results did not reach statistical significance (group B=0.069 ml/min vs. group E=0.194 ml/ min). The 24 hr TXB2 levels were significantly increased in group B (0 hr=754.1 +/- 219.4 pg/ml vs. 24 hr=2055.9 +/- 550.0 pg/ml), and pre- or posttreatment with OKY-046 abrogated this increase (group C 0 hr=517.1 +/- 80.9 pg/ml vs. 24 hr=384.7 +/- 251.5 pg/ml, and group E 0 hr=781.6 +/- 390.4 pg/ml vs. 24 hr=183.0 +/- 81.4 pg/ml). The 24 hr 6 kPGF(1alpha) levels decreased in all groups, whereas 72 hr 6 kPGF(1alpha) levels increased above baseline in groups A, C, and E, but not in group B. These data demonstrate the beneficial effects of thromboxane A2 synthesis inhibition in the setting of ischemia-reperfusion injury and suggest that this renoprotection correlates with late vasodilatory prostanoid synthesis. PMID- 8633365 TI - Abnormal in vivo response to sodium nitroprusside after porcine single lung transplantation. AB - The chronic increase of pulmonary vascular resistance after lung transplantation is only partly due to an active increase in baseline vasomotor tone, but the nature of the acute pulmonary hypertension after ischemia and reperfusion is not known. We studied the effects of sodium nitroprusside on pulmonary hemodynamics during reperfusion in porcine left lung allotransplants. In twelve pigs (weight: 18 to 24 kg) pulmonary arteries of the native and the transplanted lung were cannulated for right-heart bypass. The total blood flow was 2 L/min. Flow distribution between the lungs was measured at equal mean pulmonary artery pressure, and pulmonary vascular resistance at equal and constant flow-i.e., 1 L/min to each lung. After baseline measurements sodium nitroprusside (1, 3, and 9 microg/kg/min) was administered to six animals (SNP group). The control group (n=6) received an equal amount of the vehicle. After 30 min of discontinuation of the drug infusion, the schedule was repeated. In the transplanted lung, pulmonary vascular resistance decreased in all animals during the first hour of reperfusion. During the second drug infusion pulmonary vascular resistance was significantly lower in the SNP group compared with the control group only at the highest infusion rate of the drug (9 microg/kg/min), which also induced a 44% decrease in systemic vascular resistance. Arterial oxygen tension remained comparable in the two groups throughout the study. Our data suggest that other factors besides active vasoconstriction may contribute to the acute increase of pulmonary vascular resistance after lung transplantation. PMID- 8633366 TI - Prevention of chronic rejection by donor-specific blood transfusion in a new model of chronic cardiac allograft rejection. AB - Chronic graft rejection is now a major barrier to the long-term survival of cardiac transplants. A major hallmark of chronic rejection is intimal thickening of arteries in the graft leading to vascular occlusion. Current animal models of chronic rejection generally utilize immunosuppression to prevent acute rejection of grafts disparate at major histocompatibility antigens or graft disparities involving minor histocompatibility antigens alone. In the present communication we describe a new model of chronic rejection involving grafting of PVG-R23 hearts into PVG-RT1(u) recipients. The R23 hearts, which differ from the RT1(u) recipients at class II MHC, are rejected with a chronic time course and demonstrate extensive severe vascular myointimal proliferation within the coronary arteries. Furthermore we demonstrate for the first time that donor specific blood transfusion can prevent chronic rejection and the intimal thickening of the coronary arteries. PMID- 8633367 TI - The PAF antagonist TCV-309 reduces graft PMN infiltration and enhances early function of 24-hour-preserved rat kidneys with long warm ischemia. AB - The purpose of this study was to investigate whether treatment with TCV-309, a PAF antagonist, improves life-sustaining function of renal grafts that have suffered warm ischemia (WI) prior to cold storage (CS) and whether TCV-309 influences leukocyte sequestration in tissues. Syngeneic kidneys with 20 min of WI and 24 hr of CS were transplanted into bilateral nephrectomized rats. In the treated group, TCV-309 was administered (i.v. 1 mg/kg) 5 min before reperfusion. Rats in the control group received saline. On day 14, 80% rats survived in the treated group, which was higher than the controls (0%). At 24 hr of reperfusion, myeloperoxidase (MPO) activity, a marker enzyme for PMNs, in the treated kidney was significantly lower than the controls, but did not differ from the normal values. The MPO activity in the controls was higher than the normal values. In conclusion, the PAF antagonist improves posttransplant function of rat kidneys subjected to a period of WI and CS. PMNs are involved in postischemic renal injury, which is, at least partially, mediated by PAF. The effectiveness of PAF antagonist in treatment of recipients may lead to its clinical application in transplantation of ischemically injured kidneys. PMID- 8633368 TI - Effect of tacrolimus on hemodynamics and absorption of experimental small intestinal transplants. AB - We have previously reported the adverse effects of cyclosporine on small intestine transplant physiology. In this study, we report for the first time the effect of tacrolimus (FK) on graft intestinal blood flow and intramural distribution, vascular resistance, and absorptive function. Isogeneic small intestine transplantation was performed in Lewis rats. Animals were grouped based upon the following treatment schedules: no treatment for 1 week in group 1; 0.6 ml/kg/day i.m. polyethylene glycol (PEG) for 1 week in group 2; 2 mg/kg/day i.m. FK for 1 week in group 3; 0.6 ml/kg/day PEG for 1 week and then 0.3 ml/kg/day for 5 weeks in group 4; 2 mg/kg/day FK for 1 week and then 1 mg/kg/day for 5 weeks in group 5. Group 6 was the same as in group 5 but FK was withdrawn for 1 week prior to assessment. Maltose absorption was measured to evaluate graft absorptive function. Blood flow and its intramural distribution to mucosal and serosal/muscularis layers were determined using the radioactive microsphere technique. Perfusion pressure was measured to calculate vascular resistance. One week of FK administration in group 3 did not change graft hemodynamics and absorption significantly. Prolonged FK treatment up to 6 weeks in group 5 resulted in a significant increase in mucosal vascular resistance (71.0 +/- 9.6 versus 47.7 +/- 6.7 U/g, P<0.01) and significant decreases in mucosal blood flow (1.14 +/- O.15 versus 1.69 +/- 0.24 ml/g/min, P<0.01) and maltose absorption (30 min after loading. 155.4 +/- 26.9 versus 216.4 +/- 29.6, P<0.01; 60 min after loading: 172.9 +/- 24.5 versus 229.1 +/- 32.6 glucose mg/dl P<0.01). The serosal/muscularis layer remained relatively unaffected. Withdrawal of FK for 1 week after prolonged treatment in group 6 resulted in restorations of all parameters measured to normal ranges. We conclude that a short course of FK is safe, but prolonged FK administration has harmful effects on the hemodynamics and function of small intestinal transplants. Complete recovery is achieved when FK is discontinued. PMID- 8633369 TI - Small bowel allograft rejection detected by serum intestinal fatty acid-binding protein is reversible. AB - We hypothesized that, following experimental small bowel transplantation, immunosuppressive therapy initiated on the day of the initial rise in serum intestinal fatty acid-binding protein (I-FABP) would result in graft salvage. In previously published work, we showed that I-FABP was not detectable in the serum of isografted Lewis rats, but could be measured in the peripheral circulation during small bowel allograft rejection. A clinically useful method to monitor trans- planted allografts for rejection should detect the problem early in its evolution so that treatment to reverse the process would salvage a functional organ. Lewis rats served as recipients of LBNF1 out-of-continuity small bowel allografts and were studied in two groups: group I (control) received no immunosuppression and group II received cyclosporine (CsA, 15 mg/kg/d, p.o.) when I-FABP rose to > or = 80 ng/ml. Serum I-FABP was measured daily until the time of sacrifice. Full-thickness graft biopsies were obtained on postoperative days 3 (baseline), 6 or 7 (elevated I-FABP), 10, and 14 (sacrifice). Following transplantation baseline serum I-FABP (day 2 or 3) averaged < or = 10.0 ng/ml. I FABP remained at baseline through day 5 (range 0-50 ng/ml) in all animals and then rose abruptly on either day 6 or 7 (range 86-150 ng/ml; P < 0.001 vs. baseline). Histology on day 6 or 7 revealed a mild-to-moderate cellular rejection. Cyclosporine therapy reversed the rejection reaction and restored the bowel to normal histology. Serum I-FABP returned to baseline. In untreated animals, serum I-FABP remained elevated for several days and then returned to baseline levels coincident with fulminant rejection and mucosal sloughing. I-FABP was released into the peripheral circulation early in the evolution of acute rejection in this model of small bowel transplantation. Immunosuppressive therapy initiated when elevated levels of I-FABP were detected in the serum resulted in graft salvage. Cyclosporine immunotherapy consistently reversed rejection in this model. This article represents the first report of salvage of small bowel allografts when immunosuppressive therapy was instituted prospectively on the basis of a serum marker. Immunoreactive I-FABP appears to hold significant potential as a biochemical screening tool for acute rejection occurring In small bowell allografts. PMID- 8633370 TI - T and B cell responsiveness to donor class I MHC molecules and peptides in long survivors with kidney allografts. AB - LEW rats with long-surviving (> 100 days) (DA x LEW)F1 kidney allografts were generated by treating the recipients with cyclosporine for 14 days after grafting. All rats were monitored after transplantation for the development of antibodies to intact donor class I MHC molecules. Cyclosporine completely suppressed the early antibody response to intact DA class I MHC molecules in all 19 LEW rats. However, 17 of the 19 rats developed antibodies between four and six weeks after grafting-i.e., between two and four weeks after the cessation of cyclosporine therapy, and maintained high levels of antibody to the donor class I molecules in spite of the long-term presence of the allograft. The 2 rats that did not produce antibodies to donor class I MHC molecules, along with one of the 17 that did produce antibodies, were immunized with a synthetic peptide corresponding to a region of the DA class I MHC molecule known to be recognized by LEW CD4+ T cells via the indirect recognition pathway. All 3 long survivors developed self APC-dependent CD4+ T cell proliferation to the immunizing donor peptides, and strong antibody responses to these peptides. However, none of these long survivors suffered rejection episodes as a consequence of the peptide immunization. In one of the two long-surviving rats without antibodies to intact donor class I MHC molecules at the time of peptide priming, the peptide priming resulted in the prompt development of strong antibodies to intact donor class I molecules. However, the other of these 2 rats did not produce such antibodies after peptide priming. Thus in this model of kidney allograft tolerance, with long-term exposure of the recipient's immune system to donor antigens without evidence of rejection, none of the animals develops tolerance for the indirect T cell recognition of donor class I MHC antigens. In occasional animals, B cells potentially reactive to intact donor class I molecules are present and are adequately exposed to antigen but are quiescent because of the absence of T cell help, perhaps as a consequence of reversible T cell suppression or anergy. In other occasional animals, B cell nonreactivity (anergy or tolerance) to intact donor class I molecules appears to develop. PMID- 8633371 TI - Acute graft rejection of human fetal pancreas allografts using donor-specific human peripheral blood lymphocytes in the SCID mouse. AB - Transplantation of human fetal pancreas (HFP) is being considered as a potential treatment for insulin-dependent diabetes mellitus (IDDM). Therefore, it is necessary to have an experimental model of HFP-specific allograft rejection in order to understand all the fators that contribute to allograft rejection, and in which to test potential immunomodulatory protocols. The severe combined immunodeficient (SCID) mouse provides such a model. Previously, it has been reported that human allograft rejection can be observed in SCID mice engrafted with human lymphocytes. However, graft rejection is inconsistent and depends on both the number of lymphocytes injected and on the activation state. Here, we describe a model in which SCID mice are injected intraperitoneally with donor specific lymphocytes generated by an in vitro culture period with irradiated donor splenocytes. Injection of the donor-sensitized PBL results in an acute rejection of HFP allografts (as early as 4 days post-transplant). This model does not require the establishment of chimerism. in the SCID mice, as demonstrated by the lack of detectable human CD45 cells in the peripheral blood of rejecting mice. Allograft rejection was due to human CD4+ and CD8+ cells, as determined by immunohistochemical analysis of graft-infiltrating cells. The advantages of this model include the potential to specifically manipulate either the phenotype of the responding cells or the mechanism in which the responding population is generated. This model can provide a rapid method to test the efficacy of immunomodulatory regimens designed to protect allografts from an acute rejection response. PMID- 8633372 TI - Detection of IGA anti-OKT3 antibodies in OKT3-treated transplant recipients. AB - Murine OKT3 monoclonal antibodies function as an immunosuppressant drug for organ transplant recipients. A contraindication to retreatment may develop, however, because a high proportion of OKT3-treated patients form anti-OKT3 antibodies. Previous data have shown that only antiidiotypic IgG antibodies can negate the beneficial effect of the drug. Eighty-two OKT3-treated transplanted patients were tested by ELISA for IgG, IgK and IgA anti-OKT3 antibodies and compared with 200 controls. The anti-OKT3 antibody-positive sera were screened additionally by flow cytometry for the presence of antiidiotypic activity by measuring Ortho OKT3-FITC activity on a CD3-positive cell line, Jurkat, before and after incubation with serial dilutions of patient and control sera. Forty-four of 82 patients developed antibodies to OKT3, 20 manifested IgG, 20 produced both IgG and IgA, and 4 IgA only. We never detected IgM anti-OKT3. Of the 44 anti-OKT3-positive patient sera, 25 showed antiidiotypic specificity. Two IgG/IgA anti-OKT3-positive patient sera were IgG-depleted by Protein G. Both continued to exhibit antiidiotypic IgA activity. IgA anti-OKT3 was associated with low serum OKT3 levels and lack of ability of OKT3 to lower total CD3 cell numbers to therapeutic levels. This is the first report of IgA anti-OKT3 antibody in transplant recipients. Isotype IgA anti-OKT3 was observed in 54% of the patients whose sera tested positive for anti OM by ELISA. The IgG/ IgA anti-OKT3-positive patient sera tested continued to exhibit antiidiotypic OKT3 reactivity when depleted of IgG. We urge that OKT3 treated patients be monitored routinely for IgA anti-OKT3 antibodies to avoid the expense and potential complications of retreatment with this drug in sensitized patients. PMID- 8633373 TI - The effect of pravastatin on acute rejection after kidney transplantation--a pilot study. AB - Hyperlipidemia is an important complication of kidney transplantation affecting up to 74% of recipients. HMG-CoA reductase inhibitors are reported to provide safe and effective treatment for this problem. A recent study suggests that pravastatin, an HMG-CoA reductase inhibitor, also decreases the incidence of both clinically severe acute rejection episodes and natural killer cell cytotoxicity after orthotopic heart transplantation. We have performed a prospective randomized pilot study of the effect of pravastatin on these same parameters after cadaveric kidney transplantation. Graft recipients were randomized to receive pravastatin after transplantation or no pravastatin (24 patients in each group) in addition to routine cyclosporine and prednisone immunosuppression. Lipid levels, acute rejection episodes and serial natural killer cell cytotoxicities were followed for 4 months after the transplant. At the end of the study period, pravastatin had successfully controlled mean total cholesterol levels (202.6 +/- 9.3 vs. 236.5 +/- 11.9 mg/dl, P < 0.02), LDL levels (107.9 +/- 6.6 vs.149.6 +/- 10.7 mg/dl, P < 0.002), and triglyceride levels (118.8 +/- 14.2 vs. 157.2 +/- 13.8 mg/dl, P < 0.05). In addition, the pravastatin-treated group experienced a reduction in the incidence of biopsy-proven acute rejection episodes (25% vs. 58%, P = 0.01), the incidence of multiple rejections episodes (P < 0.05), and the use of both pulse methylprednisolone (P = 0.01) and OKT3 (P = 0.02). Mean natural killer cell cytotoxicity was similarly reduced (11.3 +/- 1.6 vs. 20.0 +/- 2.0% lysis of K562 target cells, P < 0.002). These data suggest that pravastatin exerts an additional immunosuppressive effect in kidney transplant recipients treated with cyclosporine-based immunosuppression. PMID- 8633374 TI - Diabetes mellitus after renal transplantation: characteristics, outcome, and risk factors. AB - The incidence and risk factors of posttransplant diabetes mellitus were evaluated in 1325 consecutive renal transplant recipients. Thirty-three (2.5%) patients developed diabetes mellitus requiring insulin therapy. Onset occurred a mean of 5.7 +/- 1.5 months following transplantation. The patients were compared with 33 paired-control kidney recipients. The patients were significantly older than the controls (46.8 +/- 1.9 vs. 40.6 +/- 2.1 years) (P<0.05), and chronic renal failure was more often related to interstitial nephritis (P<0.05). A family history of diabetes mellitus, the body mass index, ethnic origin, HLA phenotype, and the total doses of steroids and cyclosporine were similar in the two groups. The number of patients with at least one rejection episode was significantly higher among the diabetic patients (21 versus 9) but the number of episodes was similar. Diabetes occurred a mean of 1.1 +/- 0.3 months following rejection treatment. Intravenous pulsed prednisolone was always used for anti-rejection therapy. Insulin was withdrawn in 16 cases after a mean of 4 +/- 1 months, independently of steroid dosage reductions. Actuarial patient and graft survival rates were not significantly different, although 6-year outcome tended to be better in the controls (86% versus 93% for patient survival and 67% versus 93% for graft survival). This study suggests that pulsed steroid therapy might be the critical factor in the onset of posttransplant diabetes and that the risk is increased in older patients with chronic interstitial nephrititis. PMID- 8633375 TI - Patient death after renal transplantation--an analysis of its role in graft outcome. AB - Whether patient deaths among renal transplant recipients should be counted as transplant failures when they occur while the transplant is still functioning is a controversial issue. Analyses of more than 45,000 first cadaver transplants reported to the UNOS Scientific Renal Transplant Registry between October 1987 and March 1995 showed that deaths among transplant recipients were strongly associated with the patient's age and presence of insulin-dependent diabetes. Other factors associated with poor early graft function were also associated with a significantly increased risk of death. Deaths within the first 5 years increased from 9% of recipients aged 2-15 to 30% of those over age 45 (P < 0.001). Deaths with a functioning graft occurred in 2% of the youngest patients and 13% in the older age group (P < 0.001). Among diabetics, 32% died within 5 years (12% with a functioning graft) compared with 10% (6% with graft function) of patients with glomerulonephritis (P < 0.001). When the transplanted kidney failed to function immediately or the patient required dialysis during the first week after transplant, 30% of patients died within 5 years compared with 20% when the graft functioned (P < 0.001). There was no difference in the percentage of deaths with a functioning graft. We conclude that deaths among renal transplant recipients follow an expected pattern in which the likelihood of death increases with age and diabetes. PMID- 8633376 TI - A pilot study of ribavirin therapy for recurrent hepatitis C virus infection after liver transplantation. AB - Ribavirin is a guanosine analogue that normalizes serum liver enzymes in most nontransplant patients with chronic hepatitis C virus (HCV) infection. We conducted an uncontrolled pilot study of ribavirin in 9 liver transplantation recipients that had persistently elevated liver enzymes, active hepatitis by liver biopsy, and HCV RNA in serum by polymerase chain reaction. Ribavirin was given orally at dosages of 800-1200 mg per day for 3 mo. All 9 patients promptly responded to ribavirin: mean (+/- SD) ALT decreased from 392 +/- 377 IU/L immediately before treatment to 199 +/- 185 and 68 +/- 37 IU/L after 1 and 12 weeks of treatment, respectively, complete normalization of enzymes occurred in 4 patients. None of the patients cleared the virus from their serum during therapy, and biochemical relapse occurred in all patients 4 +/- 4.2 weeks after cessation of therapy. The hepatitis activity index of liver biopsy specimens obtained before and at the cessation of therapy was similar. Ribavirin treatment was resumed in 4 patients because of increasing fatigue (2 patients), rising bilirubin (3), or increasing necroinflammation on liver biopsy (2); the biochemical response to the second course of therapy was similar to the first course in all 4 patients. Ribavirin caused reversible hemolysis in all patients, including symptomatic anemia in 3 patients that resolved after reduction of drug dosage. These results suggest that ribavirin may be of benefit in the treatment of HCV infection after liver transplantation. Further studies are needed to determine the optimal dosage and duration of therapy. PMID- 8633377 TI - Transplantation for end stage liver disease related to alpha 1 antitrypsin. AB - Alpha 1 antitrypsin deficiency (AT) is an autosomal recessive disease associated with chronic liver disease in adults and children and emphysema in adults. The disease is one of the most common inherited disorders of the Caucasian population of North Europe and North America and is the most common genetic reason for pediatric orthotopic liver transplantation (OLTx), although it is a rare indication in adults. The natural history of the disease is unpredictable and the pathogenesis of the liver injury unclear. Thirty-five patients with histologically apparent alpha 1 AT accumulation in the liver (22 adults, 13 children) have been transplanted in this center. Clinical features were correlated with the pretransplant phenotype, serum alpha 1 antitrypsin levels and potential precipitating factors. All children were PiZZ homozygotes, most of whom had presented with neonatal hepatitis. The majority of adult patients were heterozygotes presenting with portal hypertension and liver cirrhosis. Current one-year posttransplant survival figures are 73% for adults and 87.5% for children. Replacement of the cirrhotic liver results in acquisition of the donor phenotype, a rise in serum levels of alpha 1 antitrypsin, and apparent prevention of associated disease. PMID- 8633378 TI - Prevention of bone loss in cardiac transplant recipients. A comparison of biphosphonates and vitamin D. AB - Bone mineral density is already abnormally reduced at the moment of cardiac transplantation and bone loss occurs at an impressive rate in the first postoperative year. The aim of the study was to compare two prophylactic medical regimens as to their efficacy in mitigating bone loss after transplantation. Forty-eight consecutive recipients were randomized to receive either alternating calcium carbonate and disodium etidronate (group A) or a daily supplement of calcium carbonate and alphacalcidol (group B). Bone mineral density measurements were performed immediately before hospital discharge and 6, 12, and 24 months after surgery using dual energy X-ray absorptiometry. Clinical events were recorded and roentgenograms of the spine were performed postoperatively and 1 and 2 years later. In both treatment groups bone loss remained significant at the level of the lumbar spine in the first postoperative year (P<0.005) and at the level of the femoral neck in the first (P<0.005) and the second (P<0.06) year after transplantation. Six months after transplantation, however, patients receiving alphacalcidol had a significant reduction in bone loss at the level of the lumbar spine (P=0.047) and at the level of the femoral neck (P=0.043). At the level of the femoral neck this decrease in bone loss was even more pronounced in the second postoperative year (P<0.001). In the group of patients treated with disodium etidronate, 4 recipients needed additional hospitalizations for treatment of symptomatic fractures at the level of the lumbar spine or the femoral neck. No such events happened in recipients receiving vitamin D supplements. Prophylactic administration of calcium carbonate and alphacalcidol after cardiac transplantation reduces bone loss and seems to decrease osteoporotic complications. PMID- 8633380 TI - Clinically relevant doses and blood levels produce experimental cyclosporine nephrotoxicity when combined with nitric oxide inhibition. AB - Cyclosporine (CsA) administration and nitric oxide (NO) blockade promote similar chronic renal hemodynamic alterations in rats. We evaluated various clinical CsA doses under conditions of NO blockade using L-NAME (N-nitro L-arginine methyl ester). Groups of Sprague-Dawley rats kept on a normal salt (+NaCl) or low-salt ( NaCl) diet were given CsA 7.5 mg/kg, 2.5 mg/kg, or vehicle (VH) for 21 days. CsA or VH treatment was preceded by one week of L-NAME and continued for three weeks. Inulin clearance, CsA blood level, and weekly blood pressure change were assessed at 28 days. Marked CsA dose dependent reductions in GFR in -NaCl animals (P < 0.01 versus VH + L-NAME) and +NaCl animals (P < 0.05 versus VH + L-NAME, +NaCl) as well as blood pressure elevations (P < 0.01 versus VH + L-NAME at 28 days) occurred in groups concurrently treated with CsA and L-NAME. In addition, Impaired renal function and morphologic lesions in rats (CsA 2.5 mg/kg) receiving L-NAME or CsA alone demonstrated CsA blood levels within the therapeutic range of human renal transplant patients. VH groups treated with L-NAME alone produced blood pressure elevations but were spared of renal functional or morphological alterations. Primary renal morphologic lesions in CsA treated animals included proximal tubule collapse and vacuolization and, less frequently, interstitial edema and vacuolization of interstitial cells. Unique to rats treated simultaneously with CsA and L-NAME were vascular abnormalities consisting of endothelial and arteriolar medial hyperplasia and occasional acute medial necrosis. In conclusion, acute CsA nephrotoxicity can be enhanced by simultaneous NO blockade, suggesting NO has a protective effect in CsA-induced nephropathy. These results can be achieved with a drug exposure profile that correlates with clinical therapy. PMID- 8633379 TI - Hepatic Retransplantation--an analysis of risk factors associated with outcome. AB - Hepatic retransplantation is controversial because the results are inferior to primary transplants and organs are so scarce. To determine the factors that are associated with poor outcome within the first year following retransplantation, we performed a multivariate analysis, using stepwise logistic regression, of 418 hepatic retransplantations performed at a single institution from November 1987 to December 1993. The minimum follow-up was 1 year. Seven variables were found to be independently associated with subsequent graft failure (defined as either patient death or retransplantation): donor age (odds ratio 2.2 for each 10-year increase over age 45, 95% CI 1.3 to 3.7), female donor sex (odds ratio 1.7, 95% CI 1.05 to 2.7), recipient age (odds ratio 1.6 for each 10-year increase over age 45,95% CI 1.2 to 2.8), need for preoperative mechanical ventilation (odds ratio 1.8, 95% CI 1.1 to 2.9), pretransplant serum creatinine (odds ratio 1.24 for each increase of 1 mg/dl, 95% CI 1.1 to 1.4), pretransplant total serum bilirubin (odds ratio 1.4 for each 10-mg/dl increase over 15 mg/dl, 95% CI 1.1 to 1.8), and the primary immunosuppressant, using tacrolimus as the reference category (odds ratio for cyclosporine-based immunosuppression 3.9, 95% CI 2.3 to 6.8). Although not part of the logistic regression model, the timing of retransplantation was also found to be important, with the overall probability of failure increasing from 0.58 on day 0 to a peak of 0.8 on day 38 and decreasing slowly after that. The implications of these results regarding the appropriateness of retransplantation are discussed. PMID- 8633381 TI - Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model. AB - The serious shortage of available donor organs for patients with end stage organ failure who are in need of solid organ transplantation has led to a heightened interest in xenotransplantation. The major barrier to successful discordant xenotransplantation is hyperacute rejection. Hyperacute rejection results from the deposition of preformed antibodies that activate complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement -- however, these molecules are species-restricted and therefore are ineffective at inhibiting activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in the pig-to-human combination, F1 offspring were generated from a transgenic founder animal that was engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was detected in the hearts and kidneys of these transgenic F1 animals, similar to expression levels in human kidney tissue. The hCD59 was expressed on both large vessel and capillary endothelium. Ex vivo perfusion experiments, using human blood as the perfusate, were performed with transgenic porcine hearts and kidneys to evaluate the ability of hCD59 to inhibit hyperacute rejection. These experiments demonstrated that transgenic organs expressing hCD69 resisted hyperacute rejection, as measured by increased organ function for both the hearts and the kidneys, as compared with control pig organs. Hearts from hCD59 expressing animals demonstrated a five-fold prolongation in function compared with controls, 109.8 +/- 20.7 min versus 21.2 +/- 2.9 min (P = 0.164). The hCD59 expressing kidneys also demonstrated significantly prolonged function at 157.8 +/ 27.0 min compared with 60.0 +/- 6.1 min for controls (P = 0.0174). Deposition of C9 neoantigen In the vasculature of porcine organs perfused with human blood was markedly reduced in organs expressing hCD59. These studies demonstrate that C5b-9 plays an important role in hyperacute rejection of a porcine organ perfused with human blood and suggest that donor pigs transgenic for hCD59 may be an integral component of successful clinical xenotransplantation. PMID- 8633382 TI - The effect of combination splenectomy and low-dose FK506 therapy on graft survival after liver allograft transplantation in rats. AB - The effect of splenectomy on allograft survival was investigated using orthotopic liver transplantation in a rat experimental model (ACI rat liver grafted to LEW rat). Control rats without any immunosuppressive treatment died, on average, 10.4 +/- 1.4 days after operation. Splenectomy alone somewhat prolonged the survival (13.4 +/- 2.0 days), and low-dose FK506 therapy moderately prolonged it (22.7 +/- 7 days). The graft survival period was significantly prolonged (39.7 +/- 6.3 days) when them two treatments were combined. The elevation of cytotoxic antiallograft antibodies was suppressed by splenectomy but not by low-dose FK506 therapy. The development of jaundice was moderately suppressed by FK506 but not by splenectomy. There was no difference between the pattern of body weight decline in either of them two groups and that in control rats. When these two treatments were combined at the same time, the elevation of cytotoxic antibodies, development of jaundice and decline of body weight were suppressed. These data indicate that B cells play an important role in the acute rejection of the rat liver allograft at least partially via production of cytotoxic antiallograft antibody. Splenectomy or other immunosuppressive methods affecting B cells can be a supplement for immunosuppression when using reduced-dose FK506. PMID- 8633383 TI - Recovery of blood mononuclear cell calcineurin activity segregates two populations of renal transplant patients with different sensitivities to cyclosporine inhibition. AB - In vitro studies have shown that the immunosuppressive property of cyclosporine (CsA) depends on its ability to inhibit the phosphatase activity of calcineurin, a critical enzyme for T cell activation. Here we sought to investigate whether measurement of calcineurin activity in peripheral blood mononuclear cells (PBMC) from 30 renal transplant patients given CsA as a part of their immunosuppressive regimen would help in optimizing CsA therapy. We first documented that in PBMC from these patients complete inhibition of calcineurin phosphatase activity by in vitro addition of CsA occurs at concentrations that are easily achieved in vivo for a dose as low as 3 mg/kg/day orally, which corresponds to trough CsA blood levels of 100-150 ng/ml. However, ex vivo, at a blood CsA trough level of 250 ng/ml, calcineurin activity in PBMC was only inhibited from 40% to 70% as compared with controls. Patients on higher doses of CsA had a further inhibition of baseline calcineurin activity, although a complete suppression was never reached. A significant correlation was found between trough CsA concentration and the basal calcineurin activity (r=0.48; P=0.0085). To clarify the relationship between the daily exposure of patients to CsA and changes in the enzyme activity of calcineurin, we then correlated the pharmacokinetic profile of CsA in these patients with different CsA dosing (<4, 4-6, >6-8, >8 mg/kg/day) with the profile of calcineurin activity at different intervals from dosing. Each of the above CsA doses suddenly reduced calcineurin activity, with a nadir at 2 hr after maximum blood concentration. The degree of the inhibition was not a function of peak CsA blood levels. In all patients, CsA blood level returned to basal values 10 hr after dosing. By contrast, only in 50-70% of patients (depending on the dose) did calcineurin activity return to baseline at the same time point after dosing. In summary we have shown that (1) inhibition of calcineurin activity measured ex vivo in PBMC taken from CsA-treated transplanted recipients reflects the blood CsA trough level; (2) after CsA the time-course of inhibition of enzyme activity is relatively independent from CsA pharmacokinetics; (3) the rate of recovery of calcineurin activity 10 hr after CsA dosing segregates two populations of transplanted recipients -- one with complete recovery of the enzyme activity and another that never returns to the baseline calcineurin level. PMID- 8633384 TI - Recipient cells expressing single donor MHC locus products can substitute for donor-specific transfusion in the induction of transplantation tolerance when pretreatment is combined with anti-Cd4 monoclonal antibody. Evidence for a vital role of Cd4+ T cells in the induction of tolerance to class I molecules. AB - Syngeneic cells expressing single donor MHC locus products have been shown to induce specific immunological hyporesponsiveness, but not tolerance, to an allograft. In this study we have attempted to potentiate the beneficial effect of pretreatment with single donor MHC locus products by the addition of anti-Cd4(2) monoclonal antibody to the pretreatment protocol. We show that pretreatment with recipient L cells expressing the products of a single donor locus (K, D, or IA) can induce tolerance to a C57BL/10 (H2b) cardiac allograft in C3H/He (H2k) mice, when given in combination with the depleting anti-Cd4 monoclonal antibody YTA 3.1.2. Both the induction and maintenance phases of tolerance were found to be antigen-specific. Cells expressing donor class II antigens, IA(b) were found to be most effective. Interestingly, donor class I molecules were also found to be capable of inducing specific unresponsiveness in combination with anti-Cd4, provided an optimal antigenic load was delivered at the time of pretreatment. Pretreatment with cells expressing donor class I and anti-Cd8 monoclonal antibody had no beneficial effect on graft survival. These data show that single donor MHC locus products can induce immunologic tolerance to fully MHC and minor histocompatibility antigen-mismatched heart grafts when given under the cover of anti-Cd4 mAb. They also show that Cd4+ T cells play an important role in the induction of specific unresponsiveness to class I alloantigen in vivo and suggest that the blockade of T cells capable of recognizing class I alloantigen presented indirectly is important in the induction of tolerance. PMID- 8633385 TI - The use of magnetic beads coated with soluble HLA class I or class II proteins in antibody screening and for specificity determinations of donor-reactive antibodies. AB - An adequate method that will permit rapid specificity determination of donor reactive antibodies is urgently needed. Such a method could also be used for monitoring the presence of HLA antibodies in panel scanning. We here describe a method using magnetic beads coated with soluble HLA antigens that can be directly added to patient serum for efficient absorption of HLA antibodies. The entire procedure takes 45 min, and can therefore be easily adopted for use in acute crossmatching situations. Furthermore since no live cells are required for identification of alloantibodies in the screening for HLA specific panel-reactive antibodies, it can be used as an ideal complement for screening of panel-reactive antibodies. Binding of antibodies to the antigen-coated beads is easily visualized using flow cytometry. A new method for quick purification of soluble MA antigens from a pool of lymphocytes or thrombocytes is also presented. PMID- 8633386 TI - Renal allograft rejection--in situ demonstration of cytotoxic intratubular cells. AB - A nonisotopic in situ hybridization method to detect perforin mRNA was developed in cytospin preparations of IL-2-stimulated normal human lymphocytes and applied to formalin-fixed acutely rejected renal transplant material. Individual cells expressing perforin mRNA were localized in severely damaged tubular areas, and a number of these cells appeared to be located inside the tubular basement membrane in close association with tubular epithelial cells. Immunoperoxidase staining in acetone-fixed cryostat sections of acutely rejected kidney confirmed that a considerable proportion of infiltrating cells was CD8+; many of these were in an intratubular location. In addition, perforin protein was identified in individual cells in similar locations to perforin mRNA-positive cells. Again, some intratubular cells were identified. Our findings illustrate that these cells can be fully activated with definite cytotoxic potential. Previously we have demonstrated that T lymphocytes proliferate within the tubular compartment during tubulitis, a characteristic condition in acute renal allograft rejection, and that there is associated tubular epithelial cell proliferation. In this study we think that we have further clarified the consequences of invasion of tubules by lymphoid cells. Our in situ hybridization method in rapid and convenient and may be applied to archival material. PMID- 8633387 TI - Successful renal transplantation from two donors with methanol intoxication. AB - Two patients with acute methanol intoxication are reported, one with acute renal failure. Both were declared brain-dead and kidneys were harvested at 80 and 130 hr after hospital admission. All four kidneys were transplanted and subsequently functioned well. In both donors who had received ethanol treatment, thrombocytopenia was present. The reluctance to use kidneys from such donors and from donors with acute renal failure before harvesting is discussed. Waiting lists for renal transplantation are growing and there is a world-wide shortage of cadaver organs. We were recently surprised to find reluctance to consider two local patients dying from methanol intoxication as suitable organ donors, and we report the outcome of four kidneys transplanted from these donors. We were unable to find any similar cases reported in the English literature. PMID- 8633388 TI - A case report of pregnancy in renal transplant recipient treated with FK506 (tacrolimus). PMID- 8633389 TI - Improved islet isolation from rat pancreas using 35% bovine serum albumin in combination with Dextran gradient separation. AB - We have developed a gradient separation technique in which albumin-coated (35% BSA) rat islets are isolated in Dextran (BSA/D). Versus Dextran alone (DA), BSA/D increases mean islet yield from 850 +/- 115 to 1222 +/- 210 islets/donor (mean +/ SD, P=.001). With numbers of donors held constant, the time to euglycemia was significantly shorter in syngenic recipients of BSA/D than DA islets (5.7 +/- 4.8 versus 14.2 +/- 5.6 days, P=.02). However, when the number of islets were held constant, differences in time to cure were not statistically significantly different (P>.19): with 1200 islets, 3.8 +/- 2.8 (DSA/D) versus 9.2 +/- 8.2 (DA) days; with 750 islets, 18.3 +/- 5.8 (BSA/D) versus 18.0 +/- 4.5 (DA) days. In all groups, posttransplant day 1 glucose was lowest with the BSA/D technique. Thus BSA/D isolation gives a higher yield with at least equal viability. We recommend the BSA/D technique for its efficiency and economy. PMID- 8633390 TI - Undergrading of prostate cancer biopsies: a paradox inherent in all biologic bivariate distributions. PMID- 8633391 TI - Pediatric urolithiasis: medical and surgical management. PMID- 8633392 TI - Prognostic value of p53 in muscle-invasive bladder cancer treated with preoperative radiotherapy. AB - OBJECTIVES: The relationship of p53 mutations as analyzed immunohistochemically to radiation response and therapeutic outcome was examined in a cohort of 301 patients with muscle-invasive transitional cell carcinoma of the bladder treated relatively uniformly with preoperative radiotherapy (50 Gy in 25 fractions) 4 to 6 weeks prior to radical cystectomy. METHODS: Adequate formalin-fixed paraffin embedded archival tissue for the immunohistochemical staining of p53 using antibody DO1 was obtained in 109 patients. The median follow-up for those living was 91 months. RESULTS: Overall, p53 staining was positive in 56% of the cases, with 60% positive in Stage T2 (n = 48), 42% in Stage T3a (n = 31), and 63% in Stage T3b (n = 30). Overexpression of p53 did not correlate with actuarial local control, distant metastasis freedom, disease freedom, or overall survival. However, significant associations were seen when these analyses were limited to patients with clinical Stage T3b disease. In this subgroup, the actuarial 5-year rates for patients with p53 positively and negatively stained tumors were 55% and 100%, respectively, for distant metastasis freedom (P = 0.01), 51% and 91% for disease freedom (P = 0.04), and 32% and 91% for overall survival (P = 0.006). Cox proportional hazards models that included p53 staining and other prognostic factors of significance in the univariate analyses revealed p53 to be independently predictive of survival for patients with Stage T3b disease. CONCLUSIONS: The prognostic value of p53 immunostaining rested with Stage T3b patients. Although no correlations were found with radiation response, p53 positivity in this subgroup was associated with a higher rate of distant metastasis and reduced overall survival. For these patients, p53 negativity would indicate that aggressive local treatment (that is, preoperative radiotherapy and cystectomy) is sufficient, whereas p53 positivity would indicate that multiagent chemotherapy is required because of the increased risk of distant metastasis. PMID- 8633393 TI - Laparoscopic and retroperitoneoscopic repair of ureteropelvic junction obstruction. AB - OBJECTIVES: The aim of this study was to evaluate laparoscopic and retroperitoneoscopic pyeloplasty and to compare the efficacy of dismembered and nondismembered techniques. METHODS: Since May 1993, a modified laparoscopic transperitoneal (14 patients) and a retroperitoneoscopic approach (3 patients) have been used for the management of ureteropelvic junction obstruction. In 7 patients aberrant vessels were encountered; 1 patient had a horseshoe kidney. Surgical repair was achieved by dismembered pyeloplasty (8 patients), nondismembered Fenger-plasty (longitudinal incision, transverse closure; 3 patients), transaction and reanastomosis of the renal pelvis (1 patient), ureterolysis and displacement of crossing vessels (4 patients). RESULTS: In 1 patient dismembered pyeloplasty could not be scheduled because of cardiovascular problems. A minimal transient lesion of the sympathetic nerve was observed postoperatively in 1 patient and pulmonary embolism in another. The operative time in dismembered pyeloplasty was between 240 and 360 minutes (mean, 280); the results were good in all patients. Equally good results were obtained with nondismembered Fenger-plasty, and the operating time was shorter (120 to 180 minutes). Ureterolysis was found to have a failure rate of 50%. CONCLUSIONS: Laparoscopic dismembered pyeloplasty yielded good results but it is too complicated to become a standard procedure. Nondismembered Fenger-plasty, which also showed good results, is more suitable for laparoscopy and retroperitoneoscopy. The indications for this technique should be defined more precisely as more experience is being collected. The results of ureterolysis when used as a single measure were poor, and, therefore, this technique should be abandoned. PMID- 8633394 TI - Long-term follow-up of Acucise incision of ureteropelvic junction obstruction and ureteral strictures. AB - OBJECTIVES: There are few data on the long-term effectiveness of various endoureterotomy procedures. One such technique for the treatment of ureteral strictures and ureteropelvic junction (UPJ) obstruction utilizes a recently developed ureteral cutting balloon catheter, Acucise. This device may be used under fluoroscopic guidance alone, which significantly reduces operating time. The purpose of this study is to determine the long-term efficacy of the Acucise balloon in treatment of ureteral strictures and UPJ obstruction. METHODS: Fifteen patients have been treated with the Acucise balloon, with an average follow-up of 21.6 months. Five of the patients have been followed for more than 2 years, and 11 of the patients had greater than 1 year follow-up. RESULTS: The overall success rate, defined as resolution of obstruction radiographically or disappearance of symptoms, or both, was 73%, with only 4 overt failures. All but one of the procedures were completed in 45 minutes or less, and 13 of the 15 patients were treated as outpatients. There were two significant complications. Seventy-five percent (3 of 4) of the treatment failures occurred within the first 4 months following original procedure. Of the 4 total failures. 2 patients had ureteral strictures greater than 2 cm in length, which were likely ischemic in nature secondary to previous surgeries. One patient, with a primary UPJ obstruction, was found to have a crossing vessel at subsequent open pyeloplasty. CONCLUSIONS: The Acucise cutting balloon offers the urologist a rapid and effective alternative for the management of ureteral strictures and UPJ obstruction. In our experience, early results (3 to 4 months) are usually indicative of long-term success. Proper patient selection may further improve long-term results of this simple, innovative technique. PMID- 8633395 TI - Routine CT scan in cystectomy patients: does it change management? AB - OBJECTIVES: To evaluate the practical use of routine computed tomography (CT) scan in changing management of patients with muscle-invasive bladder cancer who are candidates for cystectomy. METHODS: One hundred five patients (52 with tumors confined to the bladder and 53 with extravesical spread) were evaluated for cystectomy. The presence of nodal metastasis at surgery or biopsy was correlated with preoperative CT findings. RESULTS: The CT scan was abnormal (nodes > 1.5 cm in size) in 32% and normal (nodes < 1.5 cm) in 68% of 28 patients with positive nodes. Of 52 cases with T2 tumors, CT scan was abnormal in 6, 14% (1 of 7) with positive and 11% (5 of 45) with negative nodes, whereas of 53 with T3-4 tumors, 15 had abnormal scans, 38% (8 of 21) with positive and 22% (7 of 32) with negative nodes. CT scan changed management (a biopsy deferred surgery) in 2 (2%) of the 105 cases. CONCLUSIONS: A routine CT scan was not helpful in management of operable T2 tumors but might change therapy in selected patients with T3-4 tumors who are considered for cystectomy. PMID- 8633396 TI - The international prostate symptom score: physician versus self-administration in the quantification of symptomatology. AB - OBJECTIVES: The International Prostate Symptom Score (IPSS), originally known as the American Urological Association Score, is the most commonly used scoring system for the quantification of benign prostatic hyperplasia symptoms. One area that has remained unexplored is the stability of the questionnaire construct between different modes of administration. The objective of this study was to measure differences in IPSS when administered by the physician versus patient self-administration. The impact of order of administration was also examined. METHODS: Sixty-four patients completed two IPSS questionnaires during the same office visit, one self-administered and the other by physician interview. The influence of order of administration was examined by randomly allocating patients to either self-administration or physician-administration first. Total symptom scores (questions 1 to 7) and quality of life scores (question 8) were compared between the two modes of administration. Multivariate analysis of variance was performed to assess the effect of age, medical history (obtained from the IPSS form), order of administration, and physician (A and B) on the observed differences. RESULTS: The distribution of differences was similar between the two orders of administration. Overall, 26 patients had a higher total score with self administration, 30 with physician-administration, and 8 had identical scores on both. Mean total symptom scores and quality of life assessments by physician- versus patient-administered questionnaires were similar (10.9 versus 10.4 and 1.8 versus 2.2, respectively). No statistically significant difference was observed. The order of administration did not show statistical significance (P > 0.05) by multivariate analysis. As well, no interaction was seen between the aforementioned variables. CONCLUSIONS: There is no difference in the information obtained if patients self-administer the questionnaire as opposed to physician administration. PMID- 8633397 TI - Is one single prostate biopsy helpful for choosing a medical treatment of benign prostatic hyperplasia? A quantitative computerized morphometric study. AB - OBJECTIVES: Individual differences in proportion of stroma, epithelium, and luminal space components prostatic hyperplasia (BPH) may explain the differences in clinical outcome of the patients treated with alpha-reductase inhibitors or alpha-blocking agents and other alternative treatments. Knowledge of the individual proportions of these elements may orient the clinician toward different therapeutic approaches. To determine whether a single prostate biopsy is representative of the whole adenoma, using quantitative morphometry, we have compared the percentage of smooth muscle cells and glandular cells in one prostate needle biopsy and in the corresponding prostate adenoma removed by open surgery. METHODS: Quantification was made using a computerized image analysis system and immunohistochemical staining (actin antiactin for the smooth muscle cells and anti-prostate-specific antigen (PSA) for the epithelial cells) in 14 patients who underwent retropubic prostatectomy. RESULTS: Mean percentage of smooth muscle fibers, glandular epithelium, and glandular lumina in the prostate biopsy were, respectively, 34% (range, 20% to 42%, SD 5.9), 29% (range, 13% to 42%, SD 7.2), and 20% (range, 13% to 30%, SD 4.8). In the corresponding prostate adenoma, they were, respectively, 38% (range, 28% to 45%, SD 4.7), 32% (range, 25% to 40%, SD 4.5), and 19% (range, 13% to 34%, SD 6.1). The mean percentages of epithelial or glandular cells in the prostate biopsy and the corresponding adenoma were not statistically different (P > 0.05), whereas those for the smooth muscle cell percentage were different (P = 0.02). However, a statistically significant correlation between the whole adenoma and the needle biopsy sample was found in the percentage of smooth muscle cells (P = 0.028). Analyzing the morphometric data in conjunction with the serum PSA level and the volume of the adenoma, we found a statistically significant positive correlation between the volume of the adenoma and the ratio (percentage of epithelial cells/serum PSA level) (P = 0.009, r = 0.67). CONCLUSIONS: Major differences are found in the primary tissue composition of different hyperplastic prostates. Computerized histologic quantification of the different components of BPH in the entire adenoma and a needle biopsy demonstrate that a single prostate biopsy is representative of the entire prostate adenoma. Morphometric data from a single biopsy of the prostate adenoma in combination with the serum PSA level and the volume of the prostate adenoma could therefore help to orient medical treatment of BPH by patient selection based on the knowledge of the distribution of the various components of BPH. PMID- 8633398 TI - A multicenter, randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of terazosin in the treatment of benign prostatic hyperplasia. AB - OBJECTIVES: This study was designed to evaluate the safety and efficacy of the selective alpha 1-adrenoceptor blocker terazosin in the treatment of benign prostatic hyperplasia (BPH). METHODS: Two hundred twenty-four patients aged 50 to 80 years, who had a diagnosis of BPH based on medical history, physical examination, and digital palpation, were recruited from 11 different sites between January 1992 and January 1994. The study consisted of a screening phase, a placebo phase, a double-blind dose-titration phase, and a double-blind maintenance phase. RESULTS: Of the patients recruited, 164 entered the double blind phase and of these 134 were evaluable. Only 11 patients withdrew because of an adverse event, 7 in the terazosin and 4 in the placebo group. Compared to placebo, terazosin significantly increased peak and mean urine flow rates without significantly affecting voided volume or postvoid residual volume. It significantly improved both the obstructive and irritative symptoms associated with BPH. Fifty-one patients from the terazosin group reported a total of 120 adverse events compared with 83 reported by 42 patients in the placebo group. The majority of these events were mild to moderate. Seventeen terazosin-treated patients reported hypotension-related adverse events and 4 withdrew from the study. However, concurrent treatment with antihypertensive agents did not affect the blood pressure response of the terazosin group. CONCLUSIONS: Overall, this study showed terazosin to be safe and effective in relieving the signs and symptoms of BPH and should be considered as a treatment alternative. PMID- 8633399 TI - Physiologic (intraindividual) variation of serum prostate-specific antigen in 814 men from a screening population. AB - OBJECTIVES: We measured the intraindividual variation of prostate-specific antigen (PSA) in the serum of healthy men screened for prostate cancer. METHODS: We used a fully automated PSA assay system (ACS: 180 assay) to evaluate a screening population of 814 men (mean age, 63.3 years; range, 50 to 79 years) without documented prostate cancer or prostate surgery. A second blood sample was drawn 15 to 183 days after the first specimen (mean, 80 days). RESULTS: In the ACS PSA ranges of 0 to 7.2 ng/mL, 7.3 to 17.9 ng/mL, and 18.0 ng/mL or greater (O to 4 ng/mL, 4 to 10 ng/mL, and 10.0 ng/mL or greater by the Tandem-R assay), the mean coefficient of variation of the first and second blood drawn was 20%, 12%, and 10%, respectively. In 435 men whose first blood samples were measured twice for PSA difference (interassay or run-to-run variation), the intraindividual variation in the range of 0 to 7.2 ng/mL was significantly larger than the interassay variation, which was also true the 7.3 to 17.9 ng/mL range. In the range of 0 to 7.2 ng/mL, 251 of 695 (36%) showed a 20% or greater relative increase and 69 of 695 (10%) showed a 1.3 ng/mL (0.75 ng/mL by the Tandem-R assay) or greater absolute increase of PSA at the second blood sample. CONCLUSIONS: We conclude that in the low ranges of PSA concentrations, one should consider the possibility of substantial intraindividual variation when interpreting serial PSA measurements. PMID- 8633400 TI - Diagnostic yield of repeated transrectal ultrasound-guided biopsies stratified by specific histopathologic diagnoses and prostate specific antigen levels. AB - OBJECTIVES: To determine the diagnostic yield of secondary and tertiary transrectal ultrasound (TRUS)-guided biopsies of the prostate in men suspected of having carcinoma of the prostate because of an elevated serum prostate-specific antigen (PSA) level or an abnormal digital rectal examination (DRE). METHODS: The pathology database at the Dallas Veterans Affairs Medical Center was retrospectively searched for patients who had undergone at least two TRUS-guided biopsies of the prostate within a 6-month time span. Pertinent demographic data, serum PSA, outcomes of the two (or more) biopsies stratified in six distinct histopathologic diagnoses, and Gleason grade if carcinoma of the prostate was identified, were entered into a database and analyzed. RESULTS: A total of 123 men had at least two TRUS-guided biopsies, of which 22 had three biopsies. Mean age of this group was 68.5 +/- 0.51 (SE), and mean PSA was 11.5 +/- 1.07 (SE). Of 123 patients, 28 had a positive second biopsy following a negative first biopsy, for a positive biopsy rate of 23%. Only 2 of 22 patients who underwent a third biopsy were found to have carcinoma of the prostate, for a positive biopsy rate of 9%. The positive biopsy rate for the second biopsy was 19% (3 of 16) if the PSA was 4.0 ng/mL or less, 15% (10 of 66) if the PSA was between 4 and 10.0 ng/mL independent of the DRE findings, and 37% (15 of 41) if the PSA was 10.0 ng/mL or higher. Benign prostatic hyperplasia (59 of 123 [48%]) and atypia (38 of 123 [31%]) were the most common histopathologic diagnoses on the first biopsy, and the positive re-biopsy rates were similar for these two groups (25% versus 21 %). CONCLUSIONS: An overall positive biopsy rate of 23% in our retrospective series of 123 men with a mean PSA of 11.5 ng/mL warrants the performance of a second biopsy independent of the histopathologic diagnosis made on the first (negative) biopsy, if the outcome of such biopsy would have therapeutic consequences for the patient. This policy should not be restricted to men with a PSA above the cutoff level of 4.0 ng/mL alone. Patients with atypia should be pursued aggressively, as even on a third biopsy the positive biopsy rate was 29%. PMID- 8633402 TI - Performance measurement in prostate cancer care: beyond report cards. AB - OBJECTIVES: Review and analyze various approaches to performance measurement in health care, demonstrating the value of provider-initiated performance measurement in which ongoing monitoring of both processes and outcomes of care coupled with the use of clinical guidelines enhances performance improvement efforts. Describe some of the issues and findings associated with the use of such a methodology in prostate cancer care. METHODS: Literature review and case study. RESULTS: There are a number of significant limitations in the use of a "report card" methodology to improve quality and efficiency in health care. The complementary approach of combining "instrument panels" and clinical guidelines within an overall continuous quality improvement framework appears to have resulted in improved clinical outcomes and reduced costs in a six-physician urology group located in a heavily managed-care penetrated market. CONCLUSIONS: Performance measurement tools are integral to efforts to improve outcomes and efficiency in health care. Providers of care might consider adapting the kind of performance improvement methodology described in this article. Practice benefits including improved clinical and economic outcomes are likely to follow. PMID- 8633401 TI - Transperineal prostate needle biopsy guided by transurethral ultrasound in patients without a rectum. AB - OBJECTIVES: Patients with elevated prostate-specific antigen (PSA) and no access to the rectum present a diagnostic challenge to the urologist. This study was undertaken to determine the efficacy of transperineal prostate biopsy using transurethral ultrasound guidance for the detection of prostate cancer. METHODS: Five men status post either total colectomy or abdominoperineal resection (age range: 58 to 73 years, mean age 65.8 years) were referred to us for the evaluation of an elevated PSA (range: 5.6 to 21.4 ng/dL, mean 16.1 ng/dL). Seven procedures were performed utilizing transurethral ultrasound to guide transperineal prostate biopsies in these men. RESULTS: Biopsy results revealed benign prostatic hyperplasia in 4 procedures and prostate cancer in 3 procedures. CONCLUSIONS: Transurethral ultrasound enables the practitioner to perform accurate sonographic assessment and precise biopsy of the prostate in such patients. PMID- 8633403 TI - Determining prognosis of clinically localized prostate cancer by immunohistochemical detection of mutant p53. AB - OBJECTIVES: Mutations of the p53 tumor suppressor gene can result in unregulated cellular growth and have been implicated in numerous malignancies. The objective of this study was to determine whether the detection of mutant p53 by immunohistochemical staining is predictive of progression in clinically localized adenocarcinoma of the prostate. METHODS: Immunohistochemical staining for mutant p53 was performed on 40 formalin-fixed radical prostatectomy specimens. Benign glands in the sections served as controls. Immunoreactivity (IR) was categorized semi-quantitatively from 0 to 4+ (0 = no IR, 1+ = 1 % to 10%, 2+ = 11% to 40%, 3+ = 41 % to 70%, 4+ = 71 % to 100%). Results were then compared to Gleason score, Stage (T2 versus T3), surgical margins, lymph node and seminal vesicle involvement, age, race, preoperative prostate-specific antigen (PSA), and biochemical progression. Biochemical progression was defined as a persistently elevated postoperative PSA of 0.2 ng/mL or greater. RESULTS: Thirty-two of the 40 tumors (80%) stained for mutant p53. None of the tumors that did not stain progressed, whereas 20 of 32 (62.5%) of the tumors that did stain progressed, with an overall mean followup of 50.8 months. Immunoreactivity did not correlate with any of the known prognostic variables but did have statistically significant correlation with progression by all three statistical methods used (Fisher's exact test, logistic regression, and log-rank test). CONCLUSIONS: Strict quality control and newer antigen retrieval techniques reveal p53 abnormalities in many prostate cancers. Immunohistochemical detection of mutant p53 appears to be an independent predictor of progression. These data suggest potential utility of p53 as a preoperative prognostic indicator in localized prostate cancer. PMID- 8633404 TI - Implications of prostate micrometastases in pelvic lymph nodes: an archival tissue study. AB - OBJECTIVES: In the United States, radical retropubic prostatectomy for adenocarcinoma usually includes a staging pelvic lymphadenectomy. If frozen section analysis of the lymph nodes fails to reveal any evidence of metastases, the prostate is removed. We have previously noted that as many as 56% of patients undergoing radical prostatectomy demonstrate rising serum prostate-specific antigen (PSA) levels by 4 years postoperatively. This report was designed to determine whether micrometastases undetectable by conventional pathologic methods: could have accounted for these biochemical failures. METHODS: A retrospective analysis of formalin-fixed paraffin-embedded pelvic lymph node material was undertaken using a reverse transcription-polymerase chain reaction (RT-PCR)-based assay designed to amplify messenger RNA from PSA. All specimens were obtained from a group of 57 patients with prostate cancer who had undergone staging pelvic lymphadenectomy at the time of radical prostatectomy, and whose long-term follow-up was known. RESULTS: Although all of these nodes appeared to be free of tumor by conventional pathologic methods, a RT-PCR assay was used to identify evidence of prostate metastases in 44% of evaluable samples. Of these, 14 of 16 went on to manifest rising serum PSA values by 5 years postoperatively. CONCLUSIONS: These results suggest that molecular staging of pelvic lymph nodes prior to planned therapy for clinically organ-confined prostate cancer may better distinguish between patients with local disease and those for whom local therapy alone will not be curative. To our knowledge, this is the first large-scale retrospective gene expression study published. PMID- 8633405 TI - Calcitonin receptor mRNA expression in the human prostate. AB - OBJECTIVES: A subpopulation of prostate neuroendocrine (NE) cells contain calcitonin (CT). It has been postulated that CT-producing cells in the prostate account for the high CT level in the semen, and may be involved in the regulation of other epithelial cells via a paracrine mechanism. The presence of CT binding sites in the plasma membrane fraction of prostate tissue has been demonstrated by radioligand binding assay. In the present study, we investigated the CT receptor gene expression in the human prostate, a key component of the autocrine/paracrine loop in the CT functional pathway. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate the CT receptor mRNA expression in normal prostate tissue. Subsequent DNA sequencing was used to verify RT-PCR amplified products and to determine the isoform of the receptor. To define the location of the CT receptor expression, nonradioactive in situ hybridization was performed with a digoxigenin-labeled probe complementary to the coding region of the CT receptor mRNA. A polyclonal antibody against CT was used to reveal the CT-secreting cells in the prostate. RESULTS: CT receptor MRNA expression was detected in the prostate tissue. Further analysis of the DNA sequence showed that CT receptor expressed in the prostate was the isoform without a 16-amino acid insert in the first intracellular domain. In situ hybridization revealed that CT receptor was present in the prostate NE cells. Immunocytochemical staining of mirror image sections showed that some CT secreting cells also expressed CT receptor. CONCLUSIONS: CT receptor expression in the prostate, a key component in the CT functional pathway, is located in subsets of dispersed NE cells (CT secreting and CT nonsecreting), which indicates that prostate CT may play an important role in the autocrine/paracrine regulation of the prostate NE system. PMID- 8633406 TI - Predicting the need for adjuvant systemic therapy in patients receiving postprostatectomy irradiation. AB - OBJECTIVES: This study was initiated to determine if clinical, pathologic, or biochemical variables available in patients receiving postprostatectomy irradiation could be predictive of treatment outcomes, including the need for subsequent systemic therapy. METHODS: Between January 1992 and January 1995, 50 patients received external beam irradiation for a nonzero or rising postprostatectomy prostate-specific antigen (PSA) level. The median PSA values preoperatively and preradiation were 24 and 2.7 ng/mL, respectively. At the time of treatment, no patient had evidence of disseminated disease. The patients received a combination of axial and noncoplanar irradiation to an average total dose of 65 Gy at 1.8 Gy fractions. Univariate analysis and multivariate logistic regression were performed to identify factors predictive of outcome. RESULTS: Treatment was well tolerated with no grade 3 or higher gastrointestinal or genitourinary complications. A decline in the serum PSA during irradiation occurred in 82% of the patients. During radiation, 13 patients (26%) had a complete response (PSA less than 0.05 ng/mL), 56% had a partial response, and 18% had a rise in their PSA level. With a median follow-up of 16 months, 50% of all patients have an undetectable level of PSA. Twenty-six percent of patients have had evidence of biochemical progression. Seventeen percent of responding patients and 67% of nonresponders have evidence of progression (P= 0.002). On univariate analysis, other significant prognostic factors included clinical stage (P < 0.01), the preradiation PSA level (P < 0.05), and N stage (P < 0.01). On stepwise multivariate logistic analysis, a predictive model was generated from which treatment outcome could be predicted with 80% accuracy (percent correct classification) and an 85% sensitivity. CONCLUSIONS: The outcome of patients with an elevated postprostatectomy PSA level who receive irradiation can be accurately predicted using a combination of clinical stage, preradiation PSA value, Gleason score, and the response to treatment. In this way, patients who do not require additional treatment (that is, hormonal) will avoid the unnecessary cost and toxicity. PMID- 8633407 TI - Effect of pelvic radiotherapy for prostate cancer on bowel, bladder, and sexual function: the patient's perspective. AB - OBJECTIVES: To document the effects of pelvic radiotherapy on bowel, bladder, and sexual function, as reported by the patient. METHODS: A confidential questionnaire was distributed to 202 prostate cancer patients. Mean age was 70 years (range, 49 to 87) and mean follow-up was 33 months (range, 12 to 72). Radiation was given by a standard four field box technique on a linear accelerator to 60 to 66 Gy over 6.5 weeks. Treatment was limited to the prostate and seminal vesicles for small well-differentiated tumors, but otherwise included internal and external iliac nodes. RESULTS: Responses were obtained from 192 patients (95%). No or mild change in bowel function was reported by 64% of patients, 25% reported moderate change, and 11% reported severe change. Rectal urgency was a concern for 20%, and 5% reported fecal soiling in the preceding 6 months. Hematochezia at least once a week was reported by 9% and daily by 5%. Frequent hematochezia decreased from 17% in the second and third post-treatment years to 4% after 3 years (P = 0.02). Transfusions or laser surgery for proctitis was required by 4%. No patient required a colostomy for rectal complications. Urinary stream was unchanged or improved for 83%. Nocturia was unchanged or improved in 70%. Some form of protection for urinary incontinence was required by 2%, and 0.5% noted frequent hematuria and 2% moderate to severe dysuria. Potency prior to radiotherapy was reported by 82% and was lost following radiotherapy in 35%. Technical factors, including treatment volumes and total dose, did not correlate to the risk of moderate or severe complications. CONCLUSIONS: The sequelae of pelvic radiotherapy as viewed from the patient's perspective reveal a major alteration in bowel function in 11%, significant bladder symptoms in 4%, and loss of potency in 35%. PMID- 8633408 TI - Cryosurgical ablation of the prostate: two-year prostate-specific antigen and biopsy results. AB - OBJECTIVES: Percutaneous cryosurgical ablation of the prostate (CSAP) was performed on patients with localized or locally advanced adenocarcinoma of the prostate. To assess local disease control, post-treatment biopsy and serum prostate-specific antigen (PSA) levels were obtained at 3 and 24 months post treatment. METHODS: From June 1990 through May 1994, CSAP was performed 448 times on 383 patients under Institutional Review Board protocols. A urethral warming catheter was used for all procedures. A total of 239 patients were followed for a minimum of 21 months after treatment. None of this group had received prior local treatment. The group consisted of patients who were newly diagnosed and treated solely with cryotherapy (virgin); the remainder had been on androgen deprivation therapy (ADT) prior to CSAP. RESULTS: Biopsies were obtained from 114 patients at 21 months or more after treatment. In the virgin group, 79% had a negative biopsy after one or more treatments, and 88% of the ADT group are negative after one or more treatments. Overall, 69% had a negative biopsy after one treatment and 82% had a negative biopsy following one or more CSAP treatments. Of a group of 163 patients, PSA data were evaluable at 21 months or more after treatment. In the virgin group, 60% had a PSA 0.4 ng/mL or less, and 77% had a PSA 1.0 ng/mL or less. In the ADT group, 40% had a PSA 0.4 ng/mL or less, and 69% had a PSA value of 1.0 ng/m Lor less. Complications were minimal, the most common one being urethral tissue sloughing, which occurred in 10% of patients. CONCLUSIONS: CSAP appears to be effective in obtaining local control as measured by biopsy and PSA 21 months or more post-treatment. When retrospectively comparing our results with recently published radiotherapy series, CSAP was more effective in obtaining nadir PSA values 1.0 ng/mL or less and negative biopsies at 21 months or more after treatment. PMID- 8633409 TI - Comparison of subcapsular and total orchiectomy for treatment of metastatic prostate cancer. AB - OBJECTIVES: To determine whether subcapsular orchiectomy provides suboptimal treatment of metastatic prostate cancer when used to avoid the psychologic consequences of the empty scrotum that results from total orchiectomy. METHODS: We compared testosterone and prostate-specific antigen levels and survival of 37 patients who underwent total orchiectomy and 37 patients who underwent subcapsular orchiectomy for metastatic prostate cancer. RESULTS: The two groups of 37 patients were similar by clinical parameters. Postoperatively, testosterone levels were 21 +/- 11 ng/dL for subcapsular versus 21 +/- 9 ng/dL for total orchiectomy patients. Tumor response was similar in the two groups when assessed by prostate-specific antigen measured 3 weeks, 6 months, and 1, 2, and 3 years postoperatively. Survival was similar when assessed using Kaplan-Meier analysis (P = 0.76). CONCLUSIONS: Subcapsular orchiectomy is a viable option for treatment of metastatic prostate cancer. PMID- 8633410 TI - Quantitative technetium-99M dimercaptosuccinic acid renal scanning in children. AB - OBJECTIVES: To develop a method for assessing absolute renal function (as well as morphology) in children, using gamma camera imaging of intravenously injected technetium-99m (99mTc) dimercaptosuccinic acid (DMSA). METHODS: Forty-five children (ages 1 week to 10 years; mean, 2.0 years) were imaged using a planar technique in which not only the appearance but the absolute amount of intravenously administered 99mTc DMSA taken up by the kidneys was calculated for each child and compared with contemporaneous determinations of creatinine clearance. RESULTS: There was a close correlation between the absolute DMSA uptake and creatinine clearance (r = 0.752). CONCLUSIONS: Renal function in children, expressed as creatinine clearance, can be accurately estimated by measuring absolute DMSA uptake with planar gamma camera imaging using 99mTc DMSA. PMID- 8633411 TI - Synchronous bilateral Wilms' tumor in a neonate. AB - OBJECTIVES: Wilms' tumor is the most common solid abdominal tumor of childhood. Wilms' tumor is extremely rare in the neonate, with less than 20 well-documented cases. No bilateral lesions in neonates have been described in the literature. METHODS: We report on a neonate who presented with synchronous bilateral Wilms' tumors. RESULTS: Child underwent bilateral partial nephrectomy, followed by standard chemotherapy, and is now well 2 years after completion of the therapy without any evidence for recurrent disease. CONCLUSIONS: Wilms' tumor should be included in the differential diagnosis of the solid renal mass in the neonate. PMID- 8633412 TI - Hematuria in a newborn infant caused by bladder hemangioma. AB - A newborn boy had gross hematuria caused by hemangiomas of the bladder. The sonographic examination and the voiding cystourethrogram showed an irregular and thick bladder wall with a lesion extending into the vesical lumen. Cystoscopic examination and biopsy of the lesions were diagnostic of hemangioma. Immediately after lesion fulguration, the hematuria disappeared, and 3 weeks later the follow up ultrasound examination showed resolution of the lesion. At 1 year, the patient's urinalysis and ultrasound scan were normal. To our knowledge, this is the first reported case of hematuria in a newborn caused by a bleeding hemangioma of the bladder. PMID- 8633413 TI - A new minimally invasive open pelvic lymphadenectomy surgical technique for the staging of prostate cancer. AB - We report a new method for lymphadenectomy, the minilaparotomy (inguinal) pelvic lymph node dissection (MLPLND), and compare it with laparoscopic pelvic lymph node dissection (LPLND) in terms of cost, effectiveness, operation time and morbidity. We reviewed a series of 111 consecutive patients: 51 had MLPLND and 60 had LPLND. All patients had proved adenocarcinoma of the prostate by biopsy. Of the MLPLND patients, only 1 had to stay overnight in the hospital, and all left within 24 hours. Pelvic lymphadenectomy consisted of nodal removal along the internal iliac vessels and the external iliac vein, and nodes of the obturator foramen. A total of 14% of the patients had disease involving the lymph nodes. The cost of MLPLND was 50% of the cost of LPLND, with no interoperative or postoperative morbidity. This new operation can be performed thoroughly an inexpensively in approximately 35 minutes, with little or no morbidity. Since the drawbacks of laparoscopic techniques associated with instrument costs and the learning curve for this technically difficult operation are eliminated, staging pelvic lymphadenectomy can be performed routinely on a wider variety of patients with potential metastatic disease. Currently, we recommend MLPLND to any patient with a tumor of Gleason score 7 or higher or a serum prostate-specific antigen value of 15 ng/mL or higher. PMID- 8633414 TI - Treatment of recurrent vesicourethral anastomotic stricture after radical prostatectomy with endourethroplasty. AB - We report our experience with successful treatment of 2 cases of severe recurrent vesicourethral anastomotic stricture after radical prostatectomy with endourethroplasty. Both patients had multiple failures of conventional treatments but have been free of stricture recurrence after endourethroplasty with 11 and 25 months follow-up, respectively. Follow-up urethroscopy showed open anastomotic segments with epithelialization after endourethroplasty in both patients. The patient who was continent prior to endourethroplasty remained continent afterward. PMID- 8633415 TI - Pelvic steal syndrome. PMID- 8633416 TI - Rhabdomyolysis and acute renal failure following radical perineal prostatectomy. AB - Rhabdomyolysis and acute renal failure can be a rare but serious complication resulting from compromising operative positions following some urologic and gynecologic procedures. We report a lethal case of rhabdomyolysis following radical perineal prostatectomy. The possibility of this complication must be considered prior to performing any operation in the exaggerated lithotomy position. Prevention, early diagnosis, and aggressive treatment are the keys to a successful recovery. PMID- 8633417 TI - Management of forgotten or retained indwelling ureteral stents. AB - Five patients presented with forgotten or retained stents, and a plan of management for this complication is described. Stents were in place from 1 to 7 years. We developed an algorithm for evaluating and treating these complications. All patients should have intravenous urogram to determine function and to identify any obstruction. If there is no stent encrustation, a simple extraction under fluoroscopic control can be attempted. If significant stent calcification is present, extracorporeal shock-wave lithotripsy may be tried first. Open procedures are reserved for those patients with more than 3 mm of stent encrustation extending throughout the length of the stent, or with large volume upper tract calcification. For minimally calcified stents or for those stents with upper curls that will not straighten out on gentle traction, percutaneous extraction can be attempted in the radiology suite. If this is unsuccessful, then percutaneous nephrostolithotomy is the next step. PMID- 8633419 TI - Fungal bezoar and bladder rupture secondary to candida tropicalis. AB - Candidal urinary tract infections typically occur in a host with compromised immune function. Although usually associated with aerobic bacterial infections, emphysematous cystitis occasionally complicates fungal infections of the lower urinary tract, especially in diabetics. Another uncommon occurrence is formation of a "fungus ball" leading to obstructive uropathy. We present a case of bladder rupture in a patient with emphysematous cystitis and obstructing fungal bezoar caused by Candida tropicalis. Various factors predispose to fungal urinary tract infection, including diabetes mellitus, neurogenic bladder, antibiotic usage, and the presence of an indwelling urinary catheter. PMID- 8633418 TI - Mast cell and substance P-positive nerve involvement in a patient with both irritable bowel syndrome and interstitial cystitis. AB - Many patients with interstitial cystitis (IC) also have irritable bowel syndrome (IBS), both of which occur overwhelmingly in women, are characterized by pain, and worsen under stress. Bladder and colon biopsies of a female patient with both IC and IBS were evaluated immunohistochemically. There were 40 +/- 10 mast cells (MC)/mm2 (normal, less than 10) in the bladder, which were degranulated. The colon contained 148 +/- 11 MC/mm2 (normal, less than 50), mostly close to numerous substance P (SP)-positive nerves. Histamine, methylhistamine, and the unique MC enzyme tryptase were evaluated in 24-hour urine during two flare-ups. These results may help explain the concurrent presentation and the painful nature of these syndromes. PMID- 8633420 TI - Annular constriction of the rectum secondary to transitional cell carcinoma of the bladder. AB - Recurrent transitional cell carcinoma of the bladder is often a locally progressive disease. Regional lymph node metastasis is a frequent finding and may be the first sign of recurrence. Involvement of the gastrointestinal tract is rare, but metastasis to the colon, small intestine, and stomach have been reported. Herein, we present a case of recurrent transitional cell carcinoma of the bladder manifesting as an annular constricting mass of the rectum, and review the literature regarding this unusual lesion. PMID- 8633421 TI - Use of neodymium: yttrium-aluminum-garnet laser for removal of a congenital posterior urethral polyp in a 3-year-old child: a case report and review of the literature. AB - Congenital posterior urethral polyps are an uncommon but well-described disease process known to cause hematuria or bladder outlet obstruction in boys. Conventional treatment has included transurethral resection or open cystotomy for complete removal of the polyp. We report the first use of the neodymium:yttrium aluminum-garnet laser for endoscopic excision of a posterior urethral polyp in a 3-year-old boy. PMID- 8633422 TI - Neurofibromatosis with involvement of the prostate gland. AB - Urologic manifestations of neurofibromatosis type 1 are rare. The most common urologic area of involvement has been the urinary bladder. Prostate involvement in generalized neurofibromatosis has been noted previously in only 10 patients, 3 of whom were adults. Of these 3, 1 had a neurofibroma, 1 had a malignant schwannoma, and 1 patient died before tissue diagnosis could be obtained. The patient discussed in this report is the second known adult patient with biopsy proven neurofibroma of the prostate gland. In addition, this is the first reported use of endorectal magnetic resonance imaging of the prostate to localize and specify the extent of neurofibromas in these patients. PMID- 8633424 TI - Cost analysis and the practicing radiologist/manager: an introduction to managerial accounting. AB - Cost analysis is inherently one of the most tedious tasks falling on the shoulders of any manager. In today's world, whether in a service business such as radiology or medicine or in a product line such as car manufacturing, accurate cost analysis is critical to all aspects of management: marketing, competitive strategy, quality control, human resource management, accounting (financial), and operations management, to name but a few. This is a topic that we will explore with the intention of giving the radiologist/manager the understanding and the basic skills to use cost analysis efficiently, making sure that major financial decisions are being made with adequate cost information, and showing that cost accounting is really managerial accounting in that it pays little attention to the bottom line of financial statements but places much more emphasis on equipping managers with the information to determine budgets, prices, salaries, and incentives and influences capital budgeting decisions through an understanding of product profitability rather than firm profitability. PMID- 8633423 TI - Usefulness of ultrasound contrast medium in perineal sonography for visualization of bladder neck funneling--first observations. AB - OBJECTIVES: To assess the efficacy of ultrasound medium when imaging bladder neck anatomy with perineal ultrasound. METHODS: In 10 female patients with urinary stress or stress-urge incontinence, a new echogenic contrast medium (Echovist) was intravesically administered and perineal ultrasound performed. The examination was done with the women in the upright position both without and with ultrasound contrast medium at rest and during pressing, and the pictures of the bladder base, bladder neck, and urethra were compared. RESULTS: With the patient in the upright position, the ultrasound contrast medium enters the urethra during pressing and bladder neck funneling is identified more accurately than without contrast medium. With Echovist, bladder neck funneling was detected in 9 of the 10 cases but without it in only 4 cases. CONCLUSIONS: The use of ultrasound contrast medium results in a better visualization of the bladder neck anatomy. Bladder neck funneling and urinary leakage are seen more distinctly, and this improves the diagnostic reliability in female urinary stress incontinence. This pilot study supports the necessity for further investigations in the use of ultrasound contrast medium as a diagnostic improvement of perineal ultrasound. PMID- 8633425 TI - The origins of health services research in radiology: a review. PMID- 8633426 TI - Errors in judgment. PMID- 8633428 TI - Efficient searching for specific resources on the World-Wide Web: creation of a search server for radiologists. AB - OBJECTIVE: Our objective was to develop an efficient way to search for radiology information on the World-Wide Web (WWW). MATERIALS AND METHODS: With the Harvest Information Discovery and Access System and a DECstation 3000 computer, each week we gather and update WWW documents that relate to radiology. To limit the size of the gathered documents, certain data (such as image files) are omitted. To date, we have gathered from 20 main radiology sites. By accessing our server, thousands of documents can be efficiently searched for specific radiology information. RESULTS: At no charge, individuals with access to the WWW can request a search of our server for specific radiology information. We return to the requester in HyperText Markup Language a document with results of the search included as embedded pointers. Our server address, or uniform resource locator, is http://glimpse.cs.arizona.edu/radiology.html. CONCLUSION: Use of a server dedicated to radiology is an efficient way to search the internet for radiology resources, such as teaching files. We provide access to many sites with one search, and we select those sites to ensure that only relevant and timely information is gathered. We also index that information. PMID- 8633427 TI - Using the World-Wide Web to train and certify physicians in the safe use of fluoroscopy. AB - OBJECTIVE: We sought to create a computer-generated certification examination that concurrently tests and educates fluoroscopy users. MATERIALS AND METHODS: A trial examination was placed on the Internet for area physicians who requested fluoroscopic privileges at our institution. The name and address of each individual who achieves a passing score is automatically sent by e-mail to the examination administrator. RESULTS: Physicians expressed widespread satisfaction with this certification process. The difference in number of attempts and score of the first attempt between groups of radiologists and nonradiologists favored the radiologists (p approximately .03 in both cases) when analyzed using an unpaired t test. CONCLUSION: This examination ensures understanding of the procedural parameters that contribute to skin injury in patients and affords an easy approach to self-education. PMID- 8633429 TI - Pancreaticobiliary manifestations of AIDS. AB - The pancreas and the biliary tract are frequent sites of infectious, Inflammatory, and neoplastic disease in patients with HIV infection. However, the symptoms of pancreaticobiliary involvement may be relatively mild so that the prevalence of these disorders is probably underestimated. An appreciation of the imaging findings of HIV-associated pancreaticobiliary disorders is important because involvement of these organs may be the only criterion that establishes the diagnosis of AIDS (Table 1). PMID- 8633430 TI - Giant ulcers in the ileum and colon caused by cytomegalovirus in patients with AIDS. PMID- 8633431 TI - Primary Aspergillus osteomyelitis in the tibia of an immunosuppressed man. PMID- 8633432 TI - Toxoplasmosis of the corpus callosum: another butterfly. PMID- 8633433 TI - Isolated renal mucormycosis in two patients with AIDS. PMID- 8633434 TI - Current status of MR cholangiopancreatography. AB - MR cholangiopancreatography (MRCP) is an exciting new application of MR imaging that combines the advantages of projectional and cross-sectional imaging techniques. The technique of MRCP is in its infancy, and it is not yet known whether it will replace traditional techniques such as diagnostic ERCP or remain an adjunctive diagnostic tool. This article critically reviews the various techniques advocated for MRCP and discusses the potential role of MRCP in evaluating the pathology of the biliary tract and pancreas. PMID- 8633435 TI - MR cholangiopancreatography using HASTE (half-Fourier acquisition single-shot turbo spin-echo) sequences. AB - OBJECTIVE: Images of breath-hold MR cholangiopancreatography (MRCP) using HASTE (half-Fourier acquisition single-shot turbo spin-echo) sequences were taken in healthy volunteers. The technique was then evaluated as a noninvasive alternative to direct cholangiopancreatography in patients with pancreaticobiliary diseases. SUBJECTS AND METHODS: Forty healthy volunteers and 56 patients with various pancreaticobiliary diseases were examined by MRCP using HASTE with 128 echo train lengths on a 1.5-T MR unit. A body phased-array coll was used for data collection. Imaging times were 2 sec for the single-slice technique with a 20-mm slice thickness and 18 sec for sequential acquisition by the multislice technique with a 5-mm slice thickness (effective TE, 87 msec). We used the healthy volunteers to determine our ability to detect normal structures. The results obtained by HASTE for both patient groups were correlated with imaging by percutaneous transhepatic cholangiography or endoscopic retrograde cholangiopancreatography. RESULTS: In all healthy volunteers, HASTE-MRCP showed both the common bile duct and the main pancreatic duct. Cystic ducts were visualized in 88% of these volunteers by HASTE-MRCP, and branches of pancreatic ducts were visualized in 75% by HASTE-MRCP. The diameter and length of dilated or stenotic ducts seen on HASTE-MRCP were correlated with percutaneous transhepatic cholangiography or endoscopic retrograde cholangiopancreatography images in 56 diseased patients. Not only the position of stenosis or dilatation but also the distal portion of the stenosis was visualized by HASTE-MRCP. CONCLUSION: Breath hold HASTE-MRCP with a phased-array multicoil consistently allows for high quality images of both normal and diseased pancreaticobiliary tracts. This technique can be used as a noninvasive screening method for pancreaticobiliary diseases in the majority of patients. PMID- 8633436 TI - Hemobilia after a gunshot injury to the liver. PMID- 8633437 TI - Timing of parenchymal enhancement on dual-phase dynamic helical CT of the liver: how long does the hepatic arterial phase predominate? AB - OBJECTIVE: Dual-phase dynamic helical CT is now being used to detect and characterize benign and malignant hypervascular lesions in the liver. The purpose of this study is to define the timing and degree of parenchymal enhancement of normal liver during the hepatic arterial phase. SUBJECTS AND METHODS: This prospective study included 102 patients with known or suspected hypervascular hepatic lesions who underwent dual-phase helical CT. After unenhanced CT scanning, we injected iopamidol (Isovue 300; Bracco Diagnostics, Princeton, NJ) at 3 ml/sec for 120 ml, then at 2 ml/sec for 55-60 ml. Scan delay for the hepatic arterial phase was 25 sec and for the portal venous phase was 76 sec. Section thickness was 7 mm and pitch was 1:1. Operator-defined regions of interest were obtained from all three phases. RESULTS: Mean unenhanced attenuation of the liver was 51 +/- 12 H. The liver revealed progressive enhancement during the hepatic arterial phase as follows: an increase of 10 H occurred at a mean time of 33 +/- 4 sec, 20 H at 39 +/- 6 sec, 30 H at 44 +/- 8 sec, 40 H at 46 +/- 6 sec, and 50 H at 48 +/- 5 sec. At 20 H and 30 H of enhancement, we found a statistically significant difference (p < .01) for the mean times of men and women. Mean peak enhancement during the portal venous phase was 89 +/- 23 H. CONCLUSIONS: Because the hepatic arterial contribution to liver perfusion is approximately 30%, parenchymal enhancement greater than approximately 30% of peak might indicate portal venous predominance. In our study, this percentage corresponded to an increase of approximately 30 H. Therefore, detection of hypervascular lesions in the hepatic arterial phase may be compromised when imaging lasts longer than approximately 44 sec after the initiation of contrast material injection because 44 sec was the mean time for 30 H of enhancement in our series. However, variability between patients was marked, particularly between men and women. Furthermore, the data suggests that the hepatic arterial phase may be relatively brief and that it may be difficult to image properly using current helical CT technology. PMID- 8633438 TI - Variability of consecutive in vivo MR flow measurements in the main portal vein. AB - OBJECTIVE: The variability of consecutive cine phase-contrast MR flow measurements could significantly affect their use for clinical decisions, especially during provocative testing. The purposes of this study were to determine the normal variability of flow and consecutive flow measurements in the main portal vein on MR images and to determine how intraobserver variability, interobserver variability, and MR imager variability affect these measurements. SUBJECTS AND METHODS: MR flow measurements were acquired four consecutive times at the same location in the main portal vein of 12 subjects and three consecutive times at the same location in a nonpulsatile vessel model. All acquisitions were completed within 10 min. All main portal vein MR data sets were evaluated manually in a blinded review by two independent observers during three separate sessions spaced a mean of 4.5 weeks apart. Flow model data sets were evaluated during a single session by one observer. Variabilities were subsequently calculated by a components-of-variance analysis and by the coefficient of variation (SD/mean x 100). RESULTS: Of the total variance, 90% was due to flow variability among subjects (intersubject), 6% to flow variability within one subject (intrasubject), 2% to intraobserver variability, and 2% to interobserver variability. The coefficient of variation of consecutive MR portal vein flow measurements within a single subject was 11% +/- 5% (range, 3-23%). Intra- and interobserver variabilities were 5% +/- 2% (range, 1-11%) and 4% +/- 4% (range, 0 17%), respectively. MR imager variability was 1% +/- 1% (range, 0-2%). CONCLUSION: The mean variability of consecutive cine phase-contrast MR flow measurements in the main portal vein is 11% +/- 5% and could affect research and clinical protocols that employ this technique. PMID- 8633439 TI - Primary dissecting aneurysm of the hepatic artery: sonographic, CT, and angiographic findings. PMID- 8633440 TI - Unenhanced helical CT of ureteral stones: incidence of associated urinary tract findings. AB - OBJECTIVE: The objective of this study was to determine the incidence of urinary tract findings associated with ureteral stones on unenhanced helical CT scans of patients with acute renal colic. MATERIALS AND METHODS: One hundred forty-one consecutive patients with suspected renal colic were referred by the emergency department for a helical CT scan that was obtained without oral or IV contrast. The CT scans of 54 of these patients were interpreted as positive for ureteral calculi, and these CT scans were reviewed retrospectively. The size and location of each ureteral calculus and of any concurrent urinary tract calculi were recorded. The presence or absence of hydronephrosis, hydroureter, perinephric edema or soft-tissue stranding, and periureteral edema was also noted. RESULTS: We reviewed the original 5-mm axial images from the 54 CT scans. Calculi were present in the proximal, mid, and distal ureter in eight, four, and 14 patients respectively, and at the ureterovesicular junction in 28 patients. Only two patients had more than one ureteral calculus, and none had a contralateral ureteral calculus. Concurrent renal parenchymal and/or nonobstructing calculi in the renal pelvis were seen in five patients with proximal, mid, or distal ureteral stones and in 16 patients with ureterovesicular junction stones. Hydronephrosis was found in 37 patients, hydroureter in 36 patients, and perinephric soft-tissue changes in 35 patients. Periureteral edema could not be evaluated in the 28 patients with ureterovesicular junction calculi because of the adjacent bladder, but periureteral edema was clearly seen immediately adjacent to the ureteral stone in 17 of the other 26 patients. Only two of the 54 patients had no evidence of hydronephrosis, hydroureter, or perinephric soft tissue changes. CONCLUSION: In patients with ureteral calculi imaged with unenhanced CT for acute renal colic, associated findings included hydronephrosis, hydroureter, perinephric soft-tissue changes, and periureteral edema. These common findings provided supportive evidence that an acute obstructive process was present. PMID- 8633441 TI - Traumatic epididymitis: evaluation with color Doppler sonography. AB - OBJECTIVE: We performed this study to determine whether scrotal trauma can cause hyperemia of the epididymis. This diagnosis is helpful because traumatic epididymitis can be treated conservatively. MATERIALS AND METHODS: We retrospectively reviewed color Doppler and gray-scale sonograms of five patients who had suffered trauma to the scrotum that resulted in epididymal hyperemia, which we called traumatic epididymitis. We also reviewed the presentation and management of each patient. RESULTS: Color Doppler sonography revealed focal (one patient) and diffuse (four patients) hyperemia. Gray-scale images revealed epididymal enlargement in all patients. These findings were indistinguishable from those of infectious epididymitis by sonography. One patient also had hyperemia of the testis. Four of the five patients were managed conservatively; the other underwent surgical exploration for a coexisting testicular rupture. CONCLUSION: Careful evaluation of the epididymis with both gray-scale and color Doppler sonography should be part of every sonographic survey of the scrotum for blunt trauma. Traumatic epididymitis, which may be noted on color Doppler images, should not be confused with infectious epididymitis. Surgery is not necessary unless another injury requires it. PMID- 8633442 TI - Translabial sonography in evaluating the lower female urogenital tract. AB - Transvaginal sonography has become an invaluable technique for examining the uterus, adnexa, and other nongynecologic structures in the pelvis because it provides better spatial resolution than transabdominal sonography [1]. Transvaginal sonography is a technique that complements manometric urodynamic examination by permitting a precise study of periurethral soft tissue. However, the principal disadvantage of transvaginal sonography is the distortion it provokes in vesicourethral anatomy [2]. Furthermore, transvaginal sonography cannot be used in small girls, virgins, and women with narrow vaginas, nor can it be used in assessing incontinence and other voiding dysfunctions because of direct effects on the physiology of the urinary tract caused by the probe itself [3]. Translabial sonography, which we have routinely used since 1990 [4], is an excellent alternative means of examination for these patients and for disorders of the lower urogenital tract. We have used translabial sonography in different types of incontinence. To learn more about the physiology of micturition, using translabial sonography, we let patients micturate not only in a recumbent position but also standing while straining and coughing. The latter position simulates a provocative cystometry. In this paper, we describe the translabial sonographic technique and some of the disorders we have encountered. PMID- 8633444 TI - Is routine double-gloving worthwhile during interventional radiologic procedures? PMID- 8633443 TI - Prostatic biopsy after proctocolectomy: a transgluteal, CT-guided approach. PMID- 8633445 TI - Needle localization in MR-guided biopsy and aspiration: effects of field strength, sequence design, and magnetic field orientation. AB - OBJECTIVE: The purpose of this investigation was to evaluate the accuracy of MR Imaging for needle depiction at 0.2 and 1.5 T with multiple pulse sequences and needle orientations. The goal was to provide a framework for biopsy approach and imaging technique parameter selection that will ensure the safety and accuracy of MR-guided procedures. MATERIALS AND METHODS: Eight titanium and stainless steel alloy MR-compatible biopsy devices were immersed in fluid phantoms and placed into 1.5- and 0.2-T MR systems used for clinical imaging. Spin-echo, turbo spin echo, and gradient-echo images were obtained with the needle shafts of the biopsy devices placed parallel to, perpendicular to, and at angles of 30 degrees and 60 degrees relative to the static magnetic field of the scanner. All images were obtained with the frequency-encoding direction parallel to and perpendicular to the needle shaft. Needle width and tip position were measured from images on a freestanding workstation, and the apparent tip position was compared with that obtained by direct measurement. The difference between these values was calculated for each needle type, imaging sequence, frequency-encoding direction, and needle orientation. RESULTS: Artifactual widening was much more apparent at 1.5 T than at 0.2 T, as was error in determining needle tip position. Artifacts at both field strengths were most pronounced with gradient-echo sequences, less so with turbo spin-echo sequences, and least of all with spin-echo sequences. For spin-echo and turbo spin-echo sequences, when the frequency-encoding axis was perpendicular to the needle shaft, the apparent width of the needle was larger, but error in needle tip position was smaller. Artifacts were much less apparent, but error in tip position increased, as the orientation of the needle shaft became more parallel to the direction of the magnetic field. CONCLUSION: Specific measurements differed with field strength, but needle tip localization within 1 mm was obtained at both 0.2 and 1.5 T with the appropriate frequency-encoding direction, pulse sequence, and imaging parameters. Orientation of the needle parallel to the magnetic field significantly reduced the apparent width of the needle at both field strengths but also decreased the accuracy of needle tip position localization. PMID- 8633446 TI - Sequential helical CT angiography of aortoiliac disease. AB - OBJECTIVE: The purpose of this work was to study aortoiliac disease with sequential helical CT angiography. SUBJECTS AND METHODS: Sequential helical CT angiography combines two successive helical sets for data acquisition obtained during two successive bolus injections of IV contrast material and two breath holds. Twenty-eight patients with aneurysm and 11 with occlusive disease had CT angiography. Of those 39 patients, 18 also had conventional catheter angiography. For each of the 39 patients, a CT angiogram of three segments of the aorta and 13 arteries was assessed, including the suprarenal, juxtarenal, and infrarenal aorta; celiac axis; superior and inferior mesenteric arteries; and pairs of renal, common iliac, hypogastric, external iliac, and common femoral arteries. In 18 patients undergoing both CT and conventional angiography, the appearance of these vessels was graded as occlusive (grade 0), severely stenotic (grade 1), moderately stenotic (grade 2), mildly stenotic (grade 3), normal (grade 4), ectatic (grade 5), and aneurysmal (grade 6). RESULTS: Of the 624 arteries expected to be opacified in 39 patients, 585 (94%) were actually imaged with CT angiography. In the 18 patients who had both CT angiography and catheter angiography, the two studies were in complete agreement in 243 (90%) of 269 arteries. In 13 vessels (5%), CT angiography produced an image that was one grade higher-and in 11 vessels (4%), one grade lower-than conventional angiography. In two vessels, a two-grade difference was noted. The independent readings matched on the 0-6 scale in 95% of the evaluations. An additional 5% of the readings differed by one unit. Compared with conventional angiography, CT angiography of clinically significant (> or = 85%) narrowing (grades 0 and 1) and aneurysm (grade 6) yielded sensitivity of 93%, specificity of 96%, and accuracy of 95%. CONCLUSION: Sequential helical CT angiography of the abdomen can provide sufficient vascular detail to allow evaluation of expanded vascular territories. The technique can allow accurate assessment of both stenotic and aneurysmal disease of the aorta and the iliac arteries. PMID- 8633447 TI - Intravascular sonographic evaluation of iliac artery angioplasty: what is the mechanism of angioplasty and can intravascular sonography predict clinical outcome? AB - OBJECTIVE: The purpose of this study was to establish the arterial responses of the iliac artery after percutaneous transluminal angioplasty (PTA), using intravascular sonography, and to correlate intravascular sonography parameters with clinical outcome. SUBJECTS AND METHODS: The study included 21 patients studied with intravascular sonography before and after PTA of the iliac artery. Distinction was made between intravascular sonography cross sections collected from the common iliac and those from the external iliac artery. First, qualitative and quantitative intravascular sonography data obtained at the most stenotic site were compared with data derived from all corresponding cross sections of the dilated segment. Second, the predictive value of intravascular sonography parameters for the patient outcome was assessed. RESULTS: The free lumen and media-bounded areas seen in the common iliac artery were larger than those seen in the external iliac artery. Qualitative and quantitative effects of PTA observed with intravascular sonography on the two types of artery were not different. Vascular damage occurred in 81% of the patients. The frequency of vascular damage at the most stenotic site was slightly lower than in each dilated segment studied. The reduction in area stenosis after intervention was associated with an increase in the free lumen and media-bounded areas, whereas the plaque area reduced only slightly. The increase in the free lumen and media-bounded areas and the decrease in the plaque area at the most stenotic site after intervention were larger than the mean values. Qualitative data seen with intravascular sonography at the most stenotic site before and after intervention were not predictive of the patient outcome. In patients with an uneventful outcome after intervention, the free lumen area measured at the most stenotic site after PTA was larger and the area stenosis was smaller than in patients with a failure. CONCLUSION: This study of intravascular sonography established that although the common iliac artery is larger than the external iliac artery, the qualitative and quantitative effects of PTA in both types of artery were similar. The size of the free lumen area and the degree of stenosis seen with intravascular sonography after PTA at the most stenotic site may be predictive of a patient outcome. PMID- 8633448 TI - Accuracy of sonography in the evaluation of calf deep vein thrombosis in both postoperative surveillance and symptomatic patients. AB - OBJECTIVE: The purpose of this study was to determine the accuracy and to assess the interobserver variability of compression sonography (CS) in detecting isolated calf deep vein thrombosis (DVT) in postoperative and symptomatic patients without clinical or contrast venographic indication of previous lower extremity DVT. SUBJECTS AND METHODS: A prospective double-blinded study was undertaken on 287 consecutive legs using contrast venography (CV) as the gold standard. One hundred seventy-nine legs were from the postoperative group, and 108 legs were from the symptomatic group. The CS technique was used to diagnose DVT, and color Doppler imaging was used only as a road map. Calves were examined while the patient was sitting. Also, interobserver agreement was measured in the CS evaluation of calf veins of another group of 54 symptomatic legs. RESULTS: Forty (22%) CVs and 13 (7%) CS examinations for calf assessment were indeterminate in the postoperative group as compared with 33 (30%) and 10 (9%) in the symptomatic group. The sensitivity, specificity, negative predictive value, and positive predictive value of CS in detecting calf DVT with or without extension to above-the-calf veins were 92% (confidence interval [Cl], 82-100%), 100%, 98% (Cl, 95-100%), and 100%, respectively, in the postoperative group and 86% (Cl, 72-100%), 96% (Cl, 90-100%), 94% (Cl, 87-100%), and 90% (Cl, 77-100%), respectively, in the symptomatic group. The sensitivity of CS was 92% (Cl, 81 100%) in the postoperative and 88% (CL, 65-100%) in the symptomatic group with isolated calf DVT. Of all calf muscular branch DVTs, 52% were detected by CS alone as compared with 21% seen on CV alone. The kappa value for the CS assessment of isolated calf vein DVTs was 0.60. CONCLUSION: Sonography is a highly accurate test with acceptable reproducibility for the detection of DVT in calf veins in both postoperative and symptomatic patients. Technically adequate calf assessment can be achieved in the majority of patients if the calf examination is performed with the patient sitting. PMID- 8633449 TI - Sonographic imaging of calf veins in patients with suspected deep vein thrombosis: a cost-effective strategy? PMID- 8633450 TI - Accuracy of high-resolution CT in identifying chronic pulmonary thromboembolic disease. AB - OBJECTIVE: The aims of this study were to determine the reliability of the high resolution CT (HRCT) appearance of the lung parenchyma in distinguishing patients with chronic pulmonary thromboembolism (CPTE) from those with other pulmonary diseases and to compare HRCT with radionuclide scanning. SUBJECTS AND METHODS: Sixty-seven patients for whom HRCT scans were available for review were included in the study. Twenty-eight had proven pulmonary arterial hypertension (PAH), 17 cases of which were caused by CPTE, and 39 had other pulmonary abnormalities. Diagnosis based on the HRCT appearance was attempted by two radiologists, who independently evaluated pulmonary parenchyma for a mosaic pattern of variable attenuation, for a measurable disparity in the size of pulmonary vessels, and for the presence of peripheral scars. HRCT findings were compared with radionuclide scan findings and pulmonary angiography findings. RESULTS: For both readers (readers 1 and 2), sensitivity (94% and 100%, respectively) and specificity (96% and 98%, respectively) were high for distinguishing patients with CPTE from patients with other pulmonary abnormalities, including those with nonthromboembolic PAH. The average ratios of segmental vessel size were 2.2 for patients with CPTE and 1.1 for those with nonthromboembolic diseases. Mosaic attenuation was identified in all patients with CPTE but was also seen in 22% (reader 1) and 14% (reader 2) of patients with no evidence of CPTE. Radionuclide scans revealed a high probability for pulmonary emboli for all but one patient with CPTE but also revealed a high probability for three patients who had no emboli. CONCLUSION: HRCT findings of disparity in the size of segmental vessels and a mosaic pattern of variable attenuation reliably distinguished patients with CPTE from those with nonthromboembolic PAH and from those with other pulmonary abnormalities. In addition, HRCT was more specific than radionuclide scanning in identifying patients with CPTE. PMID- 8633451 TI - The hepatopulmonary syndrome: radiologic findings in 10 patients. AB - OBJECTIVE: The purpose of this study was to review the radiologic manifestations of the hepatopulmonary syndrome. MATERIALS AND METHODS: We retrospectively reviewed clinical records, chest radiographs, 99m Tc-macroaggregated albumin (MAA) perfusion lung scans, chest CT scans, and pulmonary angiograms of 10 patients with proven hepatopulmonary syndrome. RESULTS: Chest radiographs showed basilar, medium-sized (1.5-3.0 mm) nodular or reticulonodular opacities in all cases. CT was done in eight cases and showed basilar dilatation of lung vessels with a larger than normal number of visible branches. The vascular basis for these opacities was best appreciated on conventional CT scans of 10-mm sections. No individual arteriovenous malformations were seen on CT scans. High-resolution CT scans showed no evidence of interstitial fibrosis. 99mTc-MAA perfusion lung imaging, done in seven patients, showed pulmonary arteriovenous shunting in five. Contrast echocardiography confirmed intrapulmonary shunting in these five patients. Pulmonary angiography, done in four cases, showed subtle distal vascular dilatation in two and moderate dilatation with early venous filling in two but did not reveal any individual arteriovenous malformations. CONCLUSION: Chest radiographs in hepatopulmonary syndrome usually show bibasilar nodular or reticulonodular opacities. Conventional CT shows that these opacities represent dilated lung vessels. High-resolution CT is useful in excluding pulmonary fibrosis or emphysema as the cause of these opacities. 99mTc-MMA perfusion imaging or contrast echocardiography can be used to confirm intrapulmonary arteriovenous shunting. PMID- 8633452 TI - Three-dimensional gadolinium-enhanced MR angiography of the thoracic aorta. AB - OBJECTIVE: Our objective was to evaluate image quality and preliminary clinical experience with three-dimensional gadolinium-enhanced MR angiography of the thoracic aorta. SUBJECTS AND METHODS: Ninety patients with suspected thoracic aorta pathology underwent 97 MR examinations at 1.5 T with a 4-min, three dimensional spoiled gradient-echo techniques. Gadolinium infusion was timed for maximum arterial contrast during acquisition of the central portion of K-space. No ECG gating or breath-holding was used. All MR examinations were evaluated retrospectively for intravascular signal-to-noise ratio (SNR). In 30 of the 90 patients, results from surgery (n = 11), angiography (n = 12), or both (n = 7) were available. Four radiologists who were unaware of the angiographic or surgical findings assessed each of these 30 examinations for three types of pathology: dissection, coarctation, or aneurysm. The observers also assessed aortic branch vessel patency and vascular anomalies in the 19 patients who had angiographic correlation. RESULTS: Image quality (determined as SNR) was highest in the aortic arch, upper descending thoracic aorta, and upper abdominal aorta. We saw a small reduction in the SNR in the ascending aorta and lower descending thoracic aorta (p < .0001), attributable to cardiac and respiratory motion. Image quality was not affected by slow flow. MR imaging correctly diagnosed pathology in all 30 patients with angiographic or surgical correlation, including eight dissections, three coarctations, and 10 aneurysms. The type of the dissection was correctly determined in all eight patients. Stenoses of major branch vessel origins were detected with a sensitivity of 90% (95% bayesian confidence interval, 99-63%) and a specificity of 96% (95% bayesian confidence interval, 99 89%) in the 19 patients with angiographic correlation. Five vascular anomalies, including an aberrant right subclavian artery, a bovine arch, and three accessory renal arteries, were correctly identified. CONCLUSION: Three-dimensional gadolinium-enhanced MR angiography has the potential to accurately diagnose aortic dissection, coarctation, and aneurysm. It does not require ECG gating or breath-holding and thereby extends the diagnostic utility of MR imaging for the thoracic aorta. PMID- 8633453 TI - Three-dimensional respiratory-gated MR angiography of coronary arteries: comparison with conventional coronary angiography. AB - OBJECTIVE: MR coronary angiography is most often performed using two-dimensional techniques. Although three-dimensional (3D) acquisitions do have important advantages, they take too long for a single breath-hold and are thus susceptible to respiratory motion artifacts. The purpose of this study was to investigate the accuracy of a unique respiratory-gated 3D MR angiographic technique in identifying the proximal coronary arteries in patients suspected of having coronary artery disease. In addition, we investigated the capability of this technique to detect proximal stenoses. SUBJECTS AND METHODS: We performed a prospective blinded study in 20 patients who were referred for conventional coronary angiography. A cardiac-gated 3D gradient-echo sequence with fat suppression was used. Retrospective respiratory gating was performed using navigator echoes of the diaphragm position. Using multiplanar reformatting, two independent readers blindly analyzed the data sets for visualization of major coronary arteries, lengths of imaged segments, and detection of significant stenoses (> 50% occlusion of the luminal diameter by conventional angiography). RESULTS: Seventy-seven of 80 (96%) coronary arteries were positively identified. In one patient, an anomalous coronary anatomy was readily identified and confirmed by conventional angiography. The average lengths of the imaged segments of the right, left main, left anterior descending, and left circumflex coronary arteries were 58 +/- 13 mm, 9 +/- 5 mm, 59 +/- 16 mm, and 24 +/- 10 mm, respectively. Overall sensitivity for the detection of stenoses was low (38%), with a specificity of 95%. Interobserver agreement was 0.92, with a kappa value of 0.65. CONCLUSION: Respiratory-gated 3D MR angiography allows accurate identification of proximal coronary arteries and may be valuable for 3D imaging of coronary anomalies. Further technical improvements are required to enhance the value of the technique in detecting stenoses. PMID- 8633454 TI - Complications of lung transplantation: radiologic findings. AB - The first clinically successful lung transplantation was performed in 1983. Since that time, more than 2700 transplants have been recorded by the International Lung Transplant Registry [1]. Lung transplantation is currently limited to patients with endstage lung disease and a life expectancy of less than 18 months [1]. Unilateral lung transplantation is the most commonly performed procedure. Bilateral transplantation generally is reserved for patients with pulmonary sepsis. One-year survival after transplantation is currently 80-90%, and 5-year survival is estimated at 50% [1]. Early detection and treatment of the complications of lung transplantation are critical to decrease patient morbidity and mortality [2-4]. This article reviews the radiologic findings of the most common complications of lung transplantation, using our experience with 85 patients. PMID- 8633455 TI - Sonographic signs of breast implant rupture. AB - OBJECTIVE: This study evaluated sonography as a screening test for breast implant rupture and developed diagnostic criteria for implant rupture. SUBJECTS AND METHODS: Women contemplating implant removal were evaluated prospectively with sonography. Implants were classified as normal, indeterminate, or ruptured. Individual sonographic signs were analyzed for their statistic association with implant rupture. To measure the degree of interobserver variation, static images were evaluated by two different observers. RESULTS: Of 236 implants evaluated, surgical confirmation was available in 78, 22 of which were ruptured and 56, intact. Echogenic noise, multiple discontinuous parallel linear echoes, and echodense aggregates in the implant lumen were statistically associated with rupture. Based on sonographic findings, we classified 34 implants as intact (at surgery: 31 intact, 3 ruptured). We classified 19 as ruptured (at surgery: 11 ruptured, 8 intact). Of the 25 implants we classified as indeterminate, 17 were intact and 8 were ruptured at surgery. Therefore, sonography had a positive predictive value of 58%, a negative predictive value of 91%, a sensitivity of 50%, and a specificity of 55%. Receiver operating characteristic analysis suggests a learning curve effect and no significant interobserver variation. CONCLUSIONS: A normal sonographic result is highly predictive of an intact implant. Thus, sonography is useful in evaluating symptomatic women or women concerned about implant rupture. An indeterminate sonographic result suggests the need for further testing. PMID- 8633456 TI - Complex radial folds versus subtle signs of intracapsular rupture of breast implants: MR findings with surgical correlation. AB - Detection of intracapsular rupture of silicone breast prostheses using MR imaging is often performed by identifying the "linguine sign" [1]. The linguine sign is easily differentiated from simple radial folds that are seen in intact implants. However, more subtle signs of intracapsular rupture, including undulating subcapsular lines and the "teardrop sign," are less often recognized [2-5] and may prove difficult for the less experienced radiologist to differentiate from complex radial folds of intact implants. In this essay, we illustrate the MR imaging findings of complex radial folds in intact implants and compare them with findings of incomplete shell collapse in ruptured implants in a surgically confirmed series of explanted silicone breast prostheses. PMID- 8633457 TI - Core needle biopsy of synchronous ipsilateral breast lesions: impact on treatment. AB - OBJECTIVE: The purpose of this study was to evaluate the role of core biopsy in the diagnosis of multiple synchronous ipsilateral breast lesions and to determine the impact of this information on patients' management. MATERIALS AND METHODS: Of 371 patients who underwent core-needle breast biopsy under stereotaxic (n = 278) or sonographic (n = 93) guidance, 20 (5%) underwent core biopsy of two mammographically separate lesions in the ipsilateral breast on the same date. Fourteen of these 20 patients subsequently underwent surgery. We retrospectively reviewed the medical, radiographic, and histopathologic records in these 14 patients and in 91 patients with single mammographic lesions diagnosed as carcinoma by means of core biopsy during the same period. RESULTS: In 11 patients, core biopsy revealed two sites of carcinoma. Core biopsy findings in these 11 patients were two areas of infiltrating ductal carcinoma (n = 5), one infiltrating ductal carcinoma and one infiltrating lobular carcinoma (n = 2), one infiltrating ductal carcinoma and one ductal carcinoma in situ (n = 1), and two foci of ductal carcinoma in situ (n = 3). All 11 patients with two core biopsy proven foci of carcinoma underwent mastectomy. Patients were significantly more likely to be treated with mastectomy if core biopsy revealed two rather than one site of carcinoma (100% versus 38%, p < .001). CONCLUSION: Core-needle biopsy is useful in diagnosing multiple synchronous ipsilateral breast lesions. By showing whether carcinoma is present in one or more sites in the breast, core biopsy can provide information of critical importance in making treatment decisions. PMID- 8633458 TI - Extrasynovial spaces of the cruciate ligaments: anatomy, MR imaging, and diagnostic implications. AB - OBJECTIVE: The purpose of our study was to define the anatomy of the extrasynovial space that cruciate ligaments occupy by examining the pattern on MR imaging of normal fluid distribution in the joints around the cruciate ligaments and correlating this distribution with histologic analysis of synovial reflections around the cruciate ligaments. MATERIALS AND METHODS: MR images of five cadaveric knees were obtained serially after larger and larger amounts of contrast material were injected into the joint space. The patterns of fluid distribution around cruciate ligaments were noted. In two other cadaveric knees, the synovial sheath around the anterior cruciate ligament was injected directly with contrast material under CT guidance. Anatomic and histologic correlation was made with findings on corresponding MR images. RESULTS: The pattern of fluid distribution is bounded by the synovial reflections around cruciate ligaments. When maximum joint distention is achieved, fluid almost surrounds the cruciate ligaments. The area without fluid is a triangular space between the anterior and posterior cruciate ligaments that appears on the midsagittal image. This triangular space of the cruciate ligaments is an extrasynovial space within which both the anterior cruciate ligament and the posterior cruciate ligament reside. CONCLUSION: The overlying synovial membrane of the cruciate ligaments does not normally allow joint fluid to enter the substance of the ligaments or the triangular space of the cruciate ligaments. Therefore, fluid collections seen on MR imaging in these extrasynovial spaces or structures likely arises from injury to the cruciate ligaments. PMID- 8633459 TI - Evaluation of femoral prosthetic loosening using CT imaging. PMID- 8633460 TI - Power Doppler sonography in the assessment of musculoskeletal fluid collections. AB - OBJECTIVE: Power Doppler sonography is a relatively new technique that has been shown to depict hyperemia associated with musculoskeletal inflammatory disease. We performed this study to evaluate the ability of power Doppler sonography to differentiate musculoskeletal fluid collections of varying etiologies. SUBJECTS AND METHODS: Gray-scale and power Doppler sonography were performed on 39 patients with joint effusions or appendicular fluid collections. Blood flow (hyperemia) in the soft tissues adjacent to the fluid collections was subjectively analyzed and graded on a scale of 1 to 4 (1, normal flow; 2-4, increasing degrees of hyperemia). All fluid collections were aspirated within 24 hr of the sonographic examination. We found 31 joint effusions and 12 periarticular collections with appropriate imaging and pathologic correlation. RESULTS: Adjacent to 36 effusions and fluid collections, we saw moderate or marked hyperemia. Thirty-five of the 36 had an inflammatory or neoplastic cause, including 15 infected collections. One fluid collection had a degenerative etiology (subdeltoid bursitis secondary to supraspinatus tendon tear). Adjacent to the seven remaining effusions and fluid collections, we saw normal or mildly increased hyperemia; none of these collections had an inflammatory etiology. CONCLUSION: Power Doppler sonography helps distinguish inflammatory and infectious musculoskeletal fluid collections from those that are noninflammatory, and it may help guide the decision to perform diagnostic aspiration. Power Doppler sonography does not reliably differentiate between inflammatory collections of infectious and noninfectious origin because collections of either origin may significantly increase adjacent soft-tissue perfusion. PMID- 8633461 TI - Radiographic appearance of follicular bronchitis in children. AB - OBJECTIVE: The purpose of this study was to present the first radiographic description of a newly described disease in children, follicular bronchitis. MATERIALS AND METHODS: We retrospectively reviewed the medical history and radiographs of eight children with biopsy evidence of follicular bronchitis. RESULTS: All eight infants had findings on initial radiographs that were consistent with viral inflammatory disease. The clinical features of follicular bronchitis started by 6-8 weeks old and peaked between about 2 and 3 years old. After several months of the disease, the infants' radiographs showed a more obvious interstitial pattern. When these children were approximately 3 years old, the radiographic findings began to return to normal. Four children have been followed up for at least 8 years. By that age, the clinical symptoms of respiratory disease have disappeared. All four children tested after they were 8 years old had abnormal results of pulmonary function tests. CONCLUSION: The combination of unique clinical features associated with the radiographic appearances we describe should allow radiologists to suggest the diagnosis of follicular bronchitis. PMID- 8633462 TI - Power Doppler sonographic pattern of acute pyelonephritis in children: comparison with CT. AB - OBJECTIVE: Focal areas of decreased perfusion may be shown by power Doppler sonography in children with acute pyelonephritis. The purposes of this study were to assess the ability of power Doppler sonography to reveal acute pyelonephritis and to compare the sonographic images with enhanced CT images. SUBJECTS AND METHODS: We performed B-mode sonography, power Doppler sonography, and enhanced CT (reference method) of the kidneys of 30 children with symptoms suggesting upper urinary tract infection. All imaging studies were obtained within 24 hr of admission of each child to our hospital. Power Doppler sonography was performed with the same equipment and the same settings for all children. Imaging studies were performed before the results of urine cultures were obtained. Triangular areas of decreased perfusion visible on both longitudinal and axial scans were considered indicative of acute pyelonephritis on power Doppler sonographic images. On CT images, areas of decreased attenuation of the renal parenchyma visible immediately after IV injection of iodinated contrast agent or areas of increased attenuation on delayed scans were considered indicative of acute pyelonephritis. Power Doppler sonography and CT were compared for each renal pole (n = 120). RESULTS: For 17 (89%) of the 19 patients with CT-proven acute pyelonephritis, power Doppler sonography diagnosed this condition on the correct side. Seventeen (77%) of the 22 poles showing acute pyelonephritis on CT scans were also revealed by power Doppler sonography. The two patients in whom acute pyelonephritis was not revealed by power Doppler sonography were an obese 15-year old girl and a 7-year-old boy with right upper pole pyelonephritis. This boy was one of the first patients to be included in the study. Also, in an obese 11-year old girl, a false-positive indication of pyelonephritis was given by power Doppler sonography. CONCLUSION: Power Doppler sonography seems to be significantly more sensitive than conventional sonography for the detection of acute pyelonephritis in children. This noninvasive technique should be able to replace CT or dimercaptosuccinic acid scintigraphy in many children with urinary tract infections. PMID- 8633463 TI - Gadopentetate dimeglumine-enhanced MR imaging of subdural hematoma in an abused infant. PMID- 8633464 TI - Desmoplastic cerebral astrocytoma of infancy: report and review of the imaging characteristics. PMID- 8633465 TI - Normal thymus extending between the right brachiocephalic vein and the innominate artery. PMID- 8633466 TI - Comparison among sonography, double-tracer subtraction scintigraphy, and double phase scintigraphy in the detection of parathyroid lesions. AB - OBJECTIVE: This study prospectively evaluated the sensitivity of high-resolution sonography compared with double-tracer 201Tl-99mTc scintigraphy (Tl-Tc) subtractive scintigraphy and double-phase 99mTc-sestamibi (Tc-MIBI) scintigraphy prior to surgery in the assessment of patients with primary hyperparathyroidism in a geographic region where areas of endemic thyroid goiter are present. SUBJECTS AND METHODS: Sonography and scintigraphy were used as first-step imaging procedures in 73 patients with primary hyperparathyroidism. In 30 (41%) of 73 cases, we found an association with a thyroid abnormality. We compared sonography with double-tracer Tl-Tc scintigraphy in 41 cases, with Tc-MIBI scintigraphy in 22 other cases, and with both scintigraphic studies in 10 other cases. RESULTS: Surgery demonstrated 68 solitary parathyroid lesions (66 adenomas, one hyperplasia, and one carcinoma), two adenomas in two patients, and multiple hyperplastic glands in two patients for a total of seven lesions. In one case no abnormal parathyroid gland was found. Overall sensitivity of sonography, Tl-Tc, and Tc-MIBI scintigraphy was 85%, 62%, and 82%, respectively. In patients with concomitant thyroid disease, the sensitivity of sonography, dual-tracer Tl-Tc, and Tc-MIBI was 77%, 67%, and 80%, respectively. CONCLUSION: Our study proves that sonography and scintigraphy are equally able to detect parathyroid lesions before surgery in patients with concomitant thyroid diseases. In patients without thyroid abnormalities, detection rates of sonography and Tc-MIBI do not show any statistical difference, and the detection rate of Tl-Tc is significantly inferior to that of sonography. Sonography alone should be used as the first step for localization of abnormal parathyroid glands prior to surgery, and Tc-MIBI scintigraphy should be used as the second step when sonography is negative. PMID- 8633467 TI - Sestamibi SPECT scintigraphy for detection of postoperative hyperfunctional parathyroid glands. PMID- 8633468 TI - Dynamic MR imaging of the temporomandibular joint in patients with arthrosis: relationship between contrast enhancement of the posterior disk attachment and joint pain. AB - OBJECTIVE: The purpose of this study was to determine the relationship between joint pain, disk position, and the degree of contrast enhancement of the posterior disk attachment in patients with temporomandibular joint arthrosis using dynamic contrast-enhanced MR imaging. SUBJECTS AND METHODS: T1- and T2 weighted spin-echo and spoiled gradient-recalled acquisition in the steady state MR imaging was performed in 36 healthy volunteers and 105 patients who were asymptomatic or who had joint pain, muscular pain, or both. On T1-weighted images, the position of the disk was classified as normal and anteriorly displaced. Next, on sagittal spoiled gradient-recalled acquisition in the steady state MR images, signal intensity from teh posterior disk attachment was measured. Time-intensity curves of the signal-intensity ratio versus the time after contrast administration were obtained in each case. Diagnostic accuracy of enhancement among the clinical symptoms was assessed using receiver operating characteristic curves. Imaging findings of contrast enhancement were correlated with pain and disk position. RESULTS: In quantitative measurement, a rapid enhancement pattern was seen most frequently in patients with joint-pain, whereas relatively gradual enhancement was seen in the healthy subjects and in patients who were asymptomatic or who had muscular pain. The mean peak-signal-intensity ratio of the group with joint pain was significantly higher than that of the other groups (p < .001). In the group with joint pain, anterior disk displacement without reduction was strongly associated with the mean signal-intensity ratio (p < .01). With regard to receiver operating characteristic analysis, the group with joint pain showed significantly greater accuracy than did the other groups (p < .001). CONCLUSION: Our results suggest that prominent contrast enhancement of the posterior disk attachment on spoiled gradient-recalled acquisition in the steady state MR images may help differentiate intraarticular from extraarticular causes of pain in and around the temporomandibular joint. In the group with joint pain, anterior disk displacement without reduction was closely associated with prominent enhancement. PMID- 8633469 TI - MR imaging of the parotid gland in Sjogren's syndrome: a proposal for new diagnostic criteria. AB - OBJECTIVE: Because classic diagnostic techniques for sjogren's syndrome (SS) are invasive and require radiation exposure, a noninvasive diagnostic method might benefit patients with SS. The purpose of this study was to determine whether quantitative analysis of MR images of the parotid gland can differentiate patients with SS from normal subjects. SUBJECTS AND METHODS: We performed quantitative analysis of MR images of the parotid gland for 40 patients with definite and probable SS, for 30 normal subjects matched by age and sex to the SS patients, and for 10 patients with parotid inflammation. MR images of teh parotid gland were assessed by calculating standard deviations (SD) of signal intensity. RESULTS: SD of signal intensity exclusively (p < .0001) separated MR images of the parotid glands of patients with definite SS from those of normal subjects. SD of signal intensity for the parotid glands of patients with probable SS also were significantly higher (p < .001) than those of normal subjects. Changes in signal intensity were specific for SS and did not occur with parotid inflammation. Tentative categorization on the basis of signal intensity patterns on MR images of the parotid glands of SS patients showed a high correlation with the results of labial gland biopsy (r = .834) and sialography (r = .936). CONCLUSION: Taken together, these results conclusively indicate that quantitative analysis of MR images for SD of signal intensity is useful method for diagnosing SS and can replace classic methods, which are invasive. PMID- 8633470 TI - Cesare Gianturco, 1905-1995. PMID- 8633471 TI - Isolated enlargement of intramammary lymph nodes. PMID- 8633472 TI - The concentric-ring sign revisited. PMID- 8633473 TI - Presence of superficial siderosis assists in the diagnosis of myxopapillary ependymoma. PMID- 8633474 TI - Re: Voiding cystourethrography in boys. PMID- 8633475 TI - Workup of carotid calcifications. PMID- 8633476 TI - Additional technique for repositioning central venous catheters. PMID- 8633477 TI - Diaphragmatic desmoid tumor. PMID- 8633478 TI - Re: Bronchopleural-subarachnoid fistula manifesting as intracranial gas on CT scans. PMID- 8633480 TI - Magnetic pull from outer space. PMID- 8633479 TI - Nonencapsulated ancient schwannoma of the renal sinus. PMID- 8633481 TI - Extraluminal appendicolith in perforating appendicitis. PMID- 8633482 TI - Small-bowel hemangiomatosis in a patient with Maffucci's and blue-rubber-bleb nevus syndromes. PMID- 8633483 TI - Intracranial dissemination of a pituitary adenoma: presentation as an unusual mass in the cerebellopontine angle. PMID- 8633484 TI - Nonulcer dyspepsia: Helicobactor pylori or idiopathy? PMID- 8633485 TI - Lessons from the follow-up of patients with large colorectal adenomas: be or not be, that is the question. PMID- 8633486 TI - A review of immune modifier therapy for inflammatory bowel disease: azathioprine, 6-mercaptopurine, cyclosporine, and methotrexate. PMID- 8633487 TI - Malignancy in Crohn's disease. AB - The risk for the development of malignancy in Crohn's disease is not as well defined as it is with ulcerative colitis. The risk for the development of small bowel adenocarcinoma is greater in patients with Crohn's disease than in the general population although the magnitude of this increased risk is unclear. Risk factors associated with the development of small bowel carcinoma in Crohn's disease include male sex, duration of disease, associated fistulous disease, and the presence of surgically excluded loops of bowel. Crohn's colitis has been associated with an increased risk of colorectal carcinoma in patients with long standing colitis, strictures, fistulae, and right-sided colonic disease. Secondary gastrointestinal carcinomas are rare in Crohn's disease. This article reviews the current literature regarding the association of various malignancies in Crohn's disease. Although concentrating on population based and case-control studies, it includes referral center studies as well as case reports. PMID- 8633488 TI - Long-term outcome of triple therapy in Helicobacter pylori-related nonulcer dyspepsia: a prospective controlled assessment. AB - OBJECTIVE: To ascertain whether triple therapy alters the history of Helicobacter pylori (HP)-related nonulcer dyspepsia (NUD). METHODS: Forty-one young (<45 yr) dyspeptic patients were confirmed to be HP-related NUD by serology, rapid urease test, and antral biopsy. Endoscopy excluded the presence of ulcer. These cases were randomly plotted into control (n = 21) and triple therapy (n = 20) groups. In the former group, H2 blocker was given for 2 months and then intermittent antisecretory agents for up to 1 yr. In the latter group, 20 patients received a course of triple therapy and then were treated like the control group. The symptom scores (range: 0-10) were collected on enrollment, and at the end of 2nd, 6th, and 12th months. Each case had serial tests of HP IgG ELISA titer on start, at weeks 2, 4, and 8, at the 6th month, and at the end of the 1st yr. The second endoscopy was done in the 9th wk for eradication survey, and the third endoscopy, at the end of the 1st yr to resurvey the HP status. The histological gradings of biopsy specimens, sampled on each endoscopy, were compared. RESULTS: In the triple therapy group, the rate of eradication of HP was 75%. At he end of the 2nd month, the HP-eradicated cases of the triple therapy group improved the symptom score more significantly then the noneradicated cases (2.42+/-1.37 vs.4.76+/ 1.58, p <0.001). At the ends of the 6th month and 1st yr, the symptom scores of the eradicated cases improved more significantly than those of the control group (6th month, 0.61+/-1.18 vs.] 2.66+/-2.06; 1st yr, 0.82+/-1.17 vs.] 3.56+/-2.89, p <0.001). The decline trend of ELISA titers occurred only in eradicated cases and became significantly evident from the 4th wk (0.30+/-0.15 vs.] 0.49+/-0.07, p <0.05) and thereafter. Both acute and chronic pathological grading was improved in the triple group at the end of the 1st yr (acute, 1.95-0.46; chronic, 1.9 0.92; p <0,01) CONCLUSION: Compared with control therapy at 1 yr, triple therapy showed greater symptomatic, serological, and histological improvements. Therefore, triple therapy is beneficial to symptomatic HP-related NUD. PMID- 8633489 TI - Metachronous colon cancer in persons who have had a large adenomatous polyp. AB - OBJECTIVE: To determine, among persons who have had a large colon polyp, the risk of subsequent colon cancer at a site distant from that polyp. METHODS: Follow-up was done for 226 persons at the Mayo Clinic who had had a > or = 1-cm polyp demonstrated on barium enema between 1965 and 1970 and for whom yearly colon surveillance examination was recommended. Information was collected from Mayo Clinic records and from contact with patients, physicians, and other hospitals regarding the results of surveillance examinations and the development of colon cancer. Colon surveillance was routinely done at the Mayo Clinic using the technique of single contrast barium enema with vigorous manual fluoroscopic examination and proctoscopy. The expected rate of colorectal cancer (CRC) was calculated based on previously published rates for this community. RESULTS: Patients received, on average, four colon examinations in addition to the examination that discovered the index polyp. During 2126 person-years of follow up, 16 persons developed a colon cancer at a location other than the site of the index polyp, in comparison with 4.0 expected cases, for a standardized incidence ratio of 4.0 (95% CI,2.3, 6.4). The cancers were large (mean 4.5cm) at presentation, and eight of the 16 cancers had been preceded within 3 yr by at least one negative barium enema. CONCLUSIONS: The rate to develop colon cancer in persons who have had a large colon polyp es about 4 times the expected rate, suggesting that such persons should be considered for aggressive colonoscopic surveillance. The failure to detect early cancer or its precursors by surveillance barium enema is probably explained by inherent insensitivity of single contrast barium enema. PMID- 8633490 TI - Genetic counseling in an extended attenauted familial adenomatous polyposis kindred. AB - OBJECTIVES: To provide DNA-based genetic counseling to family members in the direct genetic lineage of a family fulfilling phenotypical criteria for the autosomal, dominantly inherited, attenuated familial adenomatous polyp (AFAP) syndrome. This enabled highly targeted cancer risk estimation based on cancer phenotype in concert with the presence or absence of the adenomatous polyposis coli (APC) germline mutation. Management recommendations could then be fully responsive to this syndrome's natural history. METHODS: Detailed family history with pathology verification of colonic polyps and cancer was performed on an extended AFAP kindred. Endoscopic gastrointestinal examinations enabled detailed knowledge of the syndrome's upper and lower gastrointestinal tract phenotype. Molecular genetic evaluation of DNA led to the identification of the APC germline mutation which co-segregated with the phenotype. RESULTS: Forty-two members of this extended AFAP family underwent DNA testing, wherein 27 were found to harbor the APC germline mutation,thereby enabling precision in their genetic counseling. Anecdotal examples of this counseling experience, with particular attention to psychological reactions, as well as concerns about such issues as insurance and employer discrimination, have been described. CONCLUSIONS: When DNA-based testing is offered to AFAP family members, genetic counselors must compassionately consider patients' psychological concerns when providing detailed risk status and available surveillance and management programs. PMID- 8633491 TI - Prognostic criteria in Clostridium difficile colitis. AB - OBJECTIVE: To determine the prognostic factors in Clostridium difficile (CD) colitis. METHODS: We conducted a retrospective study of proven cases of CD colitis in l8 months. Seventy six patients (from a 605-bed community hospital in the Bronx, NY) with proven CD colitis were studied. Mortality in patients with CD colitis was also examined. RESULTS: Seventy six patients with proven CD colitis were admitted between January 1993 and June 1994. Eighteen patients died during the same admission. Upon admission, serum albumin was less than 25 g/L in 12 (20.6%) of the survivors and in eight (44%) of the deceased patients (p <0.05). A fall in serum albumin levels was noted with the onset of symptoms of CD colitis in those who survived as well as in those who died, with a greater fall of 11.2 g/L (range 10-20 g/L) in patients who died compared with a fall of 6 g/L (range 5 10 g/L)in those who survived (p <0.05). Use of more than three antibiotics was noted in 13 (72%) of those who died and in 18 (31%) of those who survived (p <0.05). Persistence of CD cytotoxin 7 or more days after initiation of treatment was present in 14 (77%) of those who died and in eight (13%) of the survivors (p <0.01). Duration of hospitalization correlated with the development of CD colitis (35.89 vs 11.7 days) with no significant difference between survivors and deceased patients with CD colitis. Factors such as age, sex, residence, past medical history score, mean score of presenting complaints of CD colitis, history of prior episodes CD colitis, and mean number of recurrent episodes showed no difference in mortality. CONCLUSION: Factors predictive of an increased mortality in patients with CD colitis include a serum albumin of less than 25 g/L on admission, a fall in serum albumin level of greater than 11 g/L at the onset of symptoms of CD colitis, use of three or more antibiotics, and persistence of positive CD cytotoxin in the stool after completion of 7 or more days of treatment. PMID- 8633492 TI - Endoscopic transpapillary bile duct biopsy without sphincterotomy for diagnosing biliary strictures: a prospective comparative study with bile and brush cytology. AB - OBJECTIVES: Endoscopic transpapillary bile duct biopsy has a high sensitivity for detection of malignant biliary strictures, but is commonly performed after endoscopic sphincterotomy. We performed transpapillary biopsy without sphincterotomy, using a recently developed, malleable biopsy forceps, and prospectively studied the usefulness of this diagnostic procedure, compared with that of bile and brush cytology. METHODS: We succeeded in transpapillary biopsy without sphincterotomy in 45 (87%) of 52 patients. In 43 patients with biliary strictures (31 malignant, 12 benign) who successfully underwent all endoscopic samplings by bile aspiration, brushing, and biopsy, the diagnostic value of these three sampling methods was compared. RESULTS: Transpapillary biopsy (81%) had a significantly higher level of sensitivity for malignancy than bile (32%) and brush (48%) cytology. Transpapillary biopsy was more sensitive for bile duct cancer (88%) than for pancreatic cancer (71%), as were cytology techniques. No false positives were found in any of the three sampling methods. No complications accompanied the endoscopic procedures. CONCLUSIONS: Transpapillary bile duct biopsy without sphincterotomy is a simple, safe, and effective technique for diagnosing biliary stricture. We recommend that this technique be performed routinely at initial endoscopic retrograde cholangiopancreatography for patients with a stricture or filling defect of the extrahepatic bile duct. PMID- 8633493 TI - Peroral laser lithotripsy of difficult intrahepatic and extrahepatic bile duct stones: laser effectiveness using an automatic stone-tissue discrimination system. AB - OBJECTIVES: The use of laser lithotripsy with an integrated stone-tissue discrimination system is an ambitious treatment modality for bile duct stone fragmentation. The aim of our prospective study was to determine the effectiveness and safety of the laser system and to find whether it reduced the need for choledochoscopy. METHODS: Thirty patients with complicated bile duct stones were treated perorally with a flashlamp-pulsed Rhodamine-6G dye laser and an automatic stone-tissue discrimination system. Initial treatment sessions were performed under fluoroscopic guidance in each patient and switched to choledochoscopic control if the stone could not be approached properly. RESULTS: Eighteen of 19 patients with extrahepatic bile stones were treated under fluoroscopic control; 17 of 19 patients were successfully treated through laser therapy. In nine of the patients with intrahepatic stones (n = 11), choledochoscopy was necessary for sufficient laser lithotripsy; seven of those patients became stone-free. Twenty-four of 30 patients (80%) were stone-free after sole laser therapy. Combined with other methods, the overall success rate was 27/30 (90%). Therapy-related mortality was 0%. CONCLUSIONS: Laser lithotripsy is effective and safe. The stone-tissue discrimination system facilitates therapy under fluoroscopic control and precludes the need for choledochoscopy, which is highly significant (p <0.001) if the calculi are extrahepatically located. PMID- 8633495 TI - Hyperventilation and esophageal dysmotility in patients with noncardiac chest pain. AB - OBJECTIVES: Hyperventilation is known to cause esophageal spasm, but the importance of this interaction in clinical practice is unknown. In the present study, we report the effects of hyperventilation provocation on esophageal motility in a consecutive series of patients with noncardiac chest pain. METHODS: In a prospective observational study design, 46 consecutive patients with normal coronary angiograms were studied. Esophageal motility was recorded at rest and after voluntary over-breathing at 40 breaths/min for 3 min. RESULTS: Hyperventilation was associated with a significant fall in mean distal peristaltic amplitude [66 (SD 28) to 55 (SD 24) mm Hg, p <0.001] and mean duration [2.9 (SD 0.7) to 2.6 (SD 0.9) s,p = 0.02]. It induced diffuse spasm in two (4%) patients, and nonspecific motility disorders in 10 (22%). Chest pain was reproduced in seven (15%) patients, but in none did this coincide with an important change in peristaltic amplitude, duration, or frequency. CONCLUSION: Hyperventilation has important effects on esophageal motility, and manometrists should be aware of these before recommending that anxious patients overbreathe to help relaxation during clinical studies. Although overbreathing is a common source of dysmotility, it rarely produces chest pain via its effects on the esophagus. PMID- 8633494 TI - The effect of Helicobacter pylori infection on gastric emptying of digestible and indigestible solids in patients with nonulcer dyspepsia. AB - OBJECTIVE: There is no general agreement with regard to the effect of Helicobacter pylori infection on gastric emptying in patients with nonulcer dyspepsia. METHODS: We performed the 14C urea breath test as well as simultaneous recordings of scintigraphic gastic emptying and gastric clearance of radiopaque markers to determine the effect of Helicobacter pylori infection on gastric emptying of digestible and indigestible solids in nonulcer dyspepsia patients. RESULTS: Sixty patients, 30 males and 30 females, were enrolled in the study. There were 22 patients (36.7%) without and 38 patients (63.3%) with H. pylori infection. Twenty-four of the 60 patients (40%) showed normal and 36 patients (60%) showed delayed gastric emptying of digestible solids. In addition, 21 patients (35%) showed normal and 39 patients (63%) showed delayed gastric emptying of indigestible solids. There was no correlation between delayed gastric emptying of digestible and indigestible solids (p > 0.05, difference not significant). Although not statistically significant, the incidence of delayed indigestible solid emptying was higher than that of delayed digestible solid emptying (65% vs. 60%). Among different patterns of gastric emptying, no difference was noted between the patients with and without H. pylori infection. CONCLUSION: Delayed gastric emptying of indigestible solids is as common as delayed gastric emptying of digestible solids in nonulcer dyspepsia patients. The status of H. Pylori infection appeared not to influence the incidence of delayed gastric emptying of digestible and indigestible solids. PMID- 8633496 TI - Impaired small intestinal peristaltic reflexes and sensory thresholds are independent functional disturbances in patients with chronic unexplained dyspepsia. AB - OBJECTIVE: To study disturbances of gastrointestinal motility and afferent (sensory) dysfunction in functional (unexplained) dyspepsia, and the interrelationships between motility and sensory dysfunction. METHODS: Twelve patients with functional dyspepsia and 12 controls matched for age and gender were studied. Intestinal perception thresholds were tested by a standardized stepwise distension procedure in the third portion of the duodenum with a barostat device. Small intestinal motility was measured with a low compliance perfusion system proximal and distal to the distending balloon. RESULTS: First perception of duodenal balloon distension occurred at significantly (p <0.01) lower pressures in patients (23 +/- 3 mm Hg, mean +/- SEM) than in healthy controls (31 +/- 3 mm Hg). Patients had a lower maximal intestinal pain tolerance than controls (31 +/- 2 mm Hg vs. 39 +/- 1 mm Hg, p <0.05). Duodenal distension inhibited intestinal motility distal to the distending balloon (peristaltic reflex) more often in health controls (11/12) than in patients with functional dyspepsia (5/12, p <0.05). These alterations of small intestinal motility occurred at pressure values below the perception thresholds, and disturbed motility responses were not associated with perception thresholds. CONCLUSION: Disturbed peristaltic reflexes and decreased sensory thresholds for perception of intestinal sensations are prevalent but may be independent abnormalities in patients with unexplained dyspepsia. PMID- 8633497 TI - Reproducibility and physiological characteristics of 24-hour ambulatory esophangeal manometry/pH-metry. AB - OBJECTIVES: To assess the reproducibility and physiological characteristics of ambulatory esophageal manometry/pH-metry. METHODS: Ten healthy volunteers were studied on two separate occasions (2 wk apart) using the Synectics Microdigitrapper System. The pH recording failed in one subject; thus, pH data were analyzed in nine, and motility measurements in all 10 subjects. RESULTS: A high degree of reproducibility was established for the five pH and seven manometry variables assessed. Using the paired t test and Wilcoxon signed rank test to compare the two sets of data, all p values were >0.05. Narrow 95% confidence intervals containing the zero measures were also obtained on the differences of the data from both sessions for all variables. Better correlation coefficients were obtained for the motility parameters than for the pH parameters. As would be expected, reflux occurred more often in the upright than in the supine position. Furthermore, esophageal contractions were of higher amplitude and longer duration in the distal esophagus, and the frequency of propulsive contractions sequences was higher during meals and in the upright position than in the supine position. CONCLUSIONS: This study demonstrates the reproducibility of ambulatory esophageal manometry/pH-metry, suggesting that this technology may be useful in assessing the effect of pharmacological intervention on esophageal motor function. PMID- 8633498 TI - Alcoholism is associated with hepatitis C but not hepatitis B in an urban population. AB - OBJECTIVES: Previous studies have suggested an association of viral hepatitis with alcoholism, although the role of confounding risk factors (e.g. i.v. drug use) has not been adequately excluded. We therefore compared the seroprevalences of hepatitis B and C in alcoholic patients to that of a nonalcoholic control group. METHODS: Hepatitis B surface antigen, hepatitis B core antibody, hepatitis B surface antibody, and hepatitis C virus antibody testing (second generation ELISA and a confirmatory recombinant immunoblot assay) was performed in 150 consecutive alcoholics admitted for detoxification and in 166 randomly selected patients attending a general medical clinic who were screened for alcoholism. RESULTS: Hepatitis B and C seropositivities in actively drinking alcoholics are 49.3 and 35.3%, respectively, and were significantly associated with a history of i.v. drug abuse. Out of 166 general medicine clinics patients, 93 were classified as nonalcoholic (by both self-report and collateral verification), 46 patients had a history of alcoholism , and 27 were indeterminate. In the subgroup of patients without known viral hepatitis risk factors, there was no significant difference in hepatitis B seropositivity among nonalcoholic general medicine clinic patients, alcoholic general medicine clinic patients, and alcoholic patients admitted for detoxification (22.1%, 30.3%, and 27.6%, respectively). In contrast, anti-HCV recombinant immunoblot assay seropositivity in alcohol patients admitted for detoxification without risk factors was significantly greater than in nonalcoholic general medicine patients without risk factors (10 vs 0%, p >0.01). Stepwise logistic regression analysis revealed that alcoholism requiring detoxification was a significant risk factor for hepatitis C but not for hepatitis B seropositivity. CONCLUSIONS: The increased seroprevalence of hepatitis C in actively drinking alcoholic patients without known risk factors suggests that alcoholism, in some way, is a predisposing factor for HCV infection. PMID- 8633499 TI - Clinical significance of serum hepatitis C virus titers in patients with chronic type C hepatitis. AB - OBJECTIVES: To investigate the relationship between serum hepatitis C virus (HCV) titers and clinicopathological characteristics of chronic type C hepatitis. METHODS: Serum HCV cDNA concentrations were determined by a competitive polymerase chain reaction assay in 60 Taiwanese patients with chronic type C hepatitis. RESULTS: The concentration of serum HCV cDNA ranged between 10(2) and 10(8) copies/mL. The titers of serum HCV (logarithmic transformed copies of HCV cDNA/mL serum) were not significantly correlated with clinicopathological characteristics with respect to either sex of the patients, source of infection, higher serum ALT level (>150 IU/L) or histological severity. In contrast, serum HCV titers were significantly higher in patients with age above 50 yr and with type 1b HCV infection. Moreover, the influence of advancing age on serum HCV titer was genotype-independent by multivariate analysis. CONCLUSIONS: These results suggest that advancing age and genotype are both important determinants of HCV viremia and that the pathogenesis of HCV infection might not be caused by direct cytotoxicity of the virus. PMID- 8633500 TI - Ursodeoxycholic acid and methotrexate for primary sclerosing cholangitis: a pilot study. AB - OBJECTIVE: Ursodeoxycholic acid (UDCA) and methotrexate (MTX) are both undergoing evaluation for the treatment of patients with primary sclerosing cholangitis (PSC). In this pilot study, we sought to study the safety and estimate of efficacy of a combination of these two drugs administered over a 2-yr period in patients with PSC. METHODS: Nineteen patients with well defined PSC were entered prospectively into a pilot study with anticipation of 2-yr follow-up. The patients received UDCA (13-15 mg/kg/day)in divided doses in conjunction with MTX (0.25 mg/kg/wk). The results of treatment were compared with a concurrently studied, but not randomized, group of 10 patients receiving UDCA alone. At entry, the two groups were comparable with respect to age, sex, liver biochemistries, and histological stage when available. RESULTS: During this period, five patients treated with the combination of UDCA and MTX were severed from the study (three referred for transplantation, one death from small bowel cancer, and one with pre existing, high ileostomy output who withdrew voluntarily). MTX was discontinued by the investigators in an additional five patients (hair loss in three, pulmonary problems in two). There was no change in fatigue or itching compared with baseline in the group receiving the UDCA/MTX combination. Changes in biochemistries from baseline values were not different in the group receiving UDCA and MTX compared with the group receiving UDCA alone. Furthermore, after MTX was withdrawn and UDCA was continued, there was no clear evidence of further biochemical change. The use of MTX in combination with UDCA was associated with toxicity without any further improvement in liver biochemistries compared with the use of UDCA alone. CONCLUSION: This pilot study found no evidence to support the use of MTX in combination with UDCA for patients with PSC. PMID- 8633501 TI - Eosinophilia in primary biliary cirrhosis. AB - OBJECTIVES: Recent studies have shown the occurrence of eosinophilia in patients with primary biliary cirrhosis (PBC). To examine whether eosinophilia is indeed a distinctive feature of PBC, we performed extensive leukocyte differential analysis using a highly sophisticated hematology instrument. We also investigated the relationship between eosinophil dynamics and clinical features of PBC including the effects of ursodeoxycholic acid (UDCA) treatment. METHODS: A flow cytometry-based blood cell analyzer (Technicon H6000) was used to examine peripheral blood eosinophil counts in 38 patients with PBC and 131 patients with various liver deseases. We also performed eosinophil quantitation in 19 PBC patients before and after administration of UDCA for 4 wk. RESULTS: Patients with PBC had significantly higher relative and absolute eosinophil counts when compared with other liver diseases (5.7 +/- 0.5% [p < 0.0001, mean +/- SEM] and 312 +/- 26 cells/microliter [p < 0.01], respectively). Twenty-one of 38 PBC patients (55%) exhibited relative eosinophilia. In patients with PBC, the eosinophil count was: 1) significantly higher in those with early histological stages (stage I-II, 6.5 +/- 0.5% vs stage III-IV, 4.4 +/- 0.7%,p < 0.05), 2) positively correlated with basophil count (p < 0.01), serum IgA levels (p < 0.05), and the degree of eosinophil infiltration in the portal tract (p < 0.01), and 3) markedly reduced by UDCA treatment (before: 5.9 +/- 0.7%, 307 +/- 37 cells/microliter; after: 2.8 +/- 0.03% [p < 0.001], 162 +/- 26 cells/microliter ?p < 0.001]). CONCLUSIONS: Eosinophilia is a common and distinctive feature of patients with PBC. UDCA ameliorates eosinophilia as well as liver function tests in PBC patients. Eosinophilia may be useful as one of the initial clues in the diagnosis of PBC, especially in its early stage. PMID- 8633502 TI - The usefulness of ultrasonic microprobe imaging for endoscopic variceal ligation. AB - OBJECTIVES: This study investigated whether ultrasonic microprobe (UMP) imaging was useful in judging the therapeutic effect of endoscopic variceal (EVL) on varices as well as assessing varices and surrounding vessels before and after EVL. METHODS: UMP imaging was performed repeatedly safely and easily via the biopsy channel of an endoscope in 20 patients with esophageal varices. Using this modality in combination with our method of "intensive ligation," we treated esophageal varices successfully. RESULTS: Before EVL, UMP imaging displayed esophageal varices, periesophageal collaterals, and the azygous vein as echo-free lumens. In 15 of 20 patients, UMP imaging clearly detected the perforating vein, one of the optimal ligation sites for EVL, connecting the varix and collateral vein. Just after EVL, UMP imaging revealed a hyperechoic change ("snow ball" sign or "snow channel" sign), indicating blood flow stasis in the esophageal varices. On subsequent EVL, UMP imaging showed the cross-sectional area of varices was undetectable (occluded) or partially detectaable. After repeated EVL (2.9 treatment sessions on average), UMP imaging, as well as endoscopy, indicated that almost all varices were eradicated (undetectable). However, UMP imaging demonstrated that neither collaterals nor the azygous vein had significantly changed in size. CONCLUSIONS: This modern diagnostic modality provided a clinical adjunct to current endoscopy when observing esophageal varices and surrounding vessels before and after EVL to determine the need for additional therapy. PMID- 8633503 TI - Abnormalities of gastric emptying in portal hypertension. AB - OBJECTIVES: To investigate: 1) the rate of gastric emptying of portal hypertensive patients and 2) whether alterations in gastric emptying play any role in the development of portal hypertensive gastropathy. METHODS: Fifty patients (37 with esophageal varices) with cirrhosis and seven with extrahepatic portal hypertension underwent upper GI endoscopy followed by radionuclide gastric emptying studies using a semi-solid meal. Twenty-six patients also under went corrected wedged hepatic venous pressure measurement. Sixteen normal subjects underwent gastric emptying studies only. RESULTS: Varices were completely obliterated by sclerotherapy in 17 patients and were patent in 27. Thirty-seven patients had portal hypertensive gastropathy, 25 of whom had mild changes and 12 severe. No significant difference in gastric emptying was observed between patients with mild and severe portal hypertensive gastropathy and between those with portal hypertensive gastropathy and a normal gastric mucosa. There was no significant difference in gastric emptying between normal subjects and portal hypertensive patients although the latter group showed a tendency for faster gastric emptying. No difference in the rate of gastric emptying was observed between portal hypertensive patients with intrahepatic and extrahepatic pathology. However, patients with esophageal varices (patent and obliterated) emptied their stomachs significantly faster than those without (p = 0.01). There was no correlation between the rate of gastric emptying and corrected wedged hepatic venous pressure. CONCLUSIONS: It would appear that, although alterations in gastric emptying are common in portal hypertension, gastric emptying does not appear to play a causative role in the mucosal changes characteristic of portal hypertensive gastropathy. PMID- 8633504 TI - Activity of gastric mucosal nitric oxide synthase in portal hypertensive gastropathy. AB - OBJECTIVE: The importance of portal hypertensive gastropathy, as a potentially bleeding lesion in cirrhotics with portal hypertension, has recently been appreciated. Histologically, dilation of the mucosal and submucosal vessels of the stomach is noted in this entity. The possibility of nitric oxide acting as a mediator for this mucosal vascular dilation has not been explored. METHODS: We determined, in a group of 10 male cirrhotic patients with esophageal varices and endoscopic changes consistent with severe portal hypertensive gastropathy (Group A), the gastric mucosal nitric oxide synthase activity. This was determined by measuring the rate of conversion of [3H]-arginine to [3H]-citrulline. Serum levels of nitrates and nitrites, the end products of nitric oxide, were also measured. The results were compared with those of a group of 10 male controls with no liver disease (Group B). RESULTS: Gastric mucosal constitutive and inducible nitric oxide synthase levels were significantly higher in group A (125.4 +/- 4.3 and 259.7 +/- 5.5 pmol/mg protein/minute, respectively) than in group B (88 +/- 8.6 and 130.8 +/- 6.6 pmol/mg protein/minute, respectively) ( p < 0.002 and < 0.0001, respectively). Serum nitrate/nitrite levels were 30.1 +/- 3.2 nmol/ml in group A and 15.5 +/- 0.09 nmol/ml in group B (p < 0.001). CONCLUSIONS: We conclude that the significantly increased gastric mucosal nitric oxide synthase activity, in patients with portal hypertensive gastropathy, suggests an important role for nitric oxide in the pathogenesis of this mucosal lesion. PMID- 8633505 TI - Dorsocranial liver resection and direct hepatoatrial anastomosis for hepatic venous outflow obstruction: long-term outcome and functional results. AB - OBJECTIVES: The management of hepatic venous outflow obstruction, usually known as Budd-Chiari syndrome, remains complex despite a variety of treatments. METHODS: We present the results from 16 patients with Budd-Chiari syndrome who underwent dorsocranial liver resection and direct hepatoatrial anastomosis over a 10-yr period. The inferior caval vein was occluded in 10 patients. RESULTS: Operative mortality was 12.5% (2/16). During a mean follow-up of 7.2 yr, three patients required reoperation, two of whom had veno-occlusive disease. The late mortality was 14% (2/14). Clinical status improved in terms of: abdominal pain, 81 versus 14%; lower limb edema, 56 versus 14%; hematemesis, 19% versus 0%; hepatomegaly, 94 versus 36%; esophageal varices, 56 versus 7%; splenomegaly, 56 versus 21%; and ascites, 87 versus 29%. Mean serum bilirubin (micromol/l) fell from 40.2 to 27.1 (p = 0.005), and serum albumin remained unchanged. A patent hepatoatrial anastomosis was demonstrated in 10/12 survivors. The actuarial survival rate was 74.2% at 5 and 10 yr. CONCLUSIONS: Thus, in patients with outflow obstruction of the major hepatic veins, transcaval dorsocranial liver resection and direct hepatoatrial anastomosis recreate an adequate hepatic runoff. Moreover, this procedure is appropriate for patients with occlusion of the inferior caval vein, obviates or defers the need for liver transplantation, and prevents recurrence of thrombosis. PMID- 8633506 TI - Sequential evaluation of portal venous hemodynamics by Doppler ultrasound in patients with severe acute hepatitis. AB - OBJECTIVES: Portal hypertension may develop in patients with severe acute hepatitis. Sequential changes of portal venous hemodynamics in acute hepatitis is not well understood. This study evaluated portal hemodynamic changes and prognostic values in patients with severe, acute hepatitis. METHODS: Doppler studies, liver function tests, and virology studies were done in the inclusion, the 3rd month, and the 6th month for patients with severe, acute hepatitis. An indocyanine green clearance was done in the inclusion. Doppler portal hemodynamic studies were done in the hilar area by an average of two measurements. RESULTS: A total of 88 consecutive patients was included. Nine of them died. On initial study, fatalities were generally older patients with more delayed indocyanine green clearance, lower portal vein velocity, lower albumin values, higher bilirubin values, longer prothrombin time, and ascites. Using stepwise logistic regression, portal blood flow and prothrombin time were the two independence prognostic factors. By multiple linear regression, portal blood flow was associated with ascites, and average portal blood velocity was associated with bilirubin. During the hospital days, transient, depressed portal blood velocities followed by a hyperdynamic stage were found in survivors. The portal vein velocity changes for fatalities either were kept at a lower level or had a declining pattern. CONCLUSIONS: Doppler ultrasound detects portal hemodynamic changes for patients with severe, acute hepatitis. Sequential portal hemodynamic studies will be helpful for evaluating patients with severe, acute hepatitis. PMID- 8633507 TI - Occult GI bleeding in the alcoholic. AB - Consecutive admissions (1020) for alcohol detoxification were evaluated. Twenty two (2.2%) of the 990 patients without overt bleeding tested positive for the presence of fecal occult blood. Subsequent endoscopic evaluation of this population revealed a substantial prevalence of peptic ulcer disease (23.8%) and premalignant colonic neoplasia (31.6%). Upper GI mucosal inflammation was present in all patients. As in the general population, occult blood loss in the stool of alcoholics appears to be an important marker for colorectal neoplasia and to be of clinical significance in the prevention and early detection of colon cancer. Fecal occult blood should not be attributed to alcohol ingestion without the exclusion of coexistent pathology. PMID- 8633508 TI - Is portal hypertension due to liver cirrhosis a major factor in the development of portal hypertensive gastropathy? AB - OBJECTIVES: The gastric mucosa of patients with portal hypertension frequently manifests changes in its appearance that are readily identifiable by endoscopy. Many of these can be sources of bleeding, and some imply the presence of systemic disease. Although portal hypertension is critical in development of portal hypertensive gastropathy (PHG), the role that other factors might play in its pathogenesis is uncertain. METHODS: Four groups of subjects were studied prospectively: 37 with portal hypertension due to cirrhosis, 26 noncirrhotic subjects with portal hypertension due to extrahepatic portal vein obstruction (PVO), nine cirrhotic patients with extrahepatic PVO, and 57 control subjects. The diagnosis in each case was based on a combination of clinical data, needle liver biopsy, ultrasonography, splenoportography, and upper GI endoscopy. RESULTS: Snake skin, scarlatina rash, diffuse hyperemia, and diffuse bleeding were frequent endoscopic gastric findings in cirrhotic patients. These findings were seen less frequently in noncirrhotic patients with portal hypertension due to PVO than in cirrhotic patients (p< 0.0001). The highest incidence was seen in cirrhotic patients with PVO (P< 0.001). Positive correlations existed among the endoscopic findings, the clinical estimate of the cirrhosis severity (Child-Pugh grade), and the size and appearance of esophageal varices (Beppu score). No endoscopic findings of the gastric mucosa enabled one to distinguish between groups. Hypergastrinemia was present in cirrhotics with and without PVO but not in noncirrhotic patients with portal hypertension resulting from isolated PVO. CONCLUSION: These findings suggest that the endoscopic findings of PHG are affected by the severity of the underlying liver disease and the presence or absence of coexisting PVO. There is no association between PHG and the presence of gastric varices. Thus, the development of the gastric lesions characteristic of PHG requires not only portal hypertension but also some other consequence of parenchymal liver disease. PMID- 8633510 TI - Clinicocolonoscopic profile of colonic tuberculosis. AB - BACKGROUND: Colonic tuberculosis is common in developing countries. However, its diagnosis is difficult. Nevertheless, colonoscopy and biopsy examination have shown promising results. METHODS: We evaluated the clinical spectrum and colonoscopic features of 62 patients with colonic tuberculosis. RESULTS: Abdominal pain, fever, anorexia, weight loss, and change in bowel habit were seen in more than 50% of the patients. Massive rectal bleeding was frequently (13%) observed. Colonoscopy revealed strictures in 17, deformed ileocecal valve in 34, ulcers in 52, nodules in 49, polypoidal lesions in three, and fibrous bands in five patients. Segmental tuberculosis, lesions mimicking carcinoma, and multiple site involvement were observed in 19%, 20%, and 50% of the patients, respectively. Histopathologically, well formed granulomas were seen in 27, collections of epithelioid cells in 18, and chronic, nonspecific inflammatory changes in 17 of the cases. Acid-fast bacilli could not be isolated from any of the patients. All of the patients responded to the anti-tubercular treatment. Follow-up colonoscopy in 22 patients demonstrated regression of lesions. CONCLUSIONS: Colonic tuberculosis is common in India. Our findings indicate that colonoscopy is useful for its diagnosis. However, histopathology many not always be helpful. Therefore, in a given clinical and colonosopic setting, a therapeutic trial may be indicated. PMID- 8633509 TI - Intravenous load of fructose and fructose 1,6-diphosphate: effects on uricemia in patients with nonalcoholic liver disease. AB - OBJECTIVES: The i.v. load of fructose causes a significantly higher adenosine triphosphate (ATP) degradation and uric acid production in cirrhotic patients than in healthy controls. Resynthesis of ATP from adenosine diphosphate (ADP) may be facilitated by the phosphorylated compound fructose 1,6-diphosphate, which is used as energy support in parenteral nutrition. The aim of our research was to evaluate: 1) The 1-h uricemic effect of i.v. fructose (0.5 g/kg body weight) in 10 healthy controls and in 78 patients with differenct stages of non-alcoholic chronic liver damage associated or not with malnutrition or hepatocellular carcinoma; and 2) the effect of fructose 1,6-diphosphate (5 g/50 ml) administered i.v. after fructose infusion on the induced uricemia in a subgroup of 13 patients with well compensated cirrhosis. RESULTS AND CONCLUSIONS: The increase of uricemia above the basal level after fructose infusion was significantly higher (p < 0.01) in cirrhotics (3 mg/dl) than in controls (1.2 mg/dl) and in patients with chronic hepatitis (1.9 mg/dl) and was completely reversed by fructose 1,6 diphosphate in the patients tested. Neither Child-Pugh classes of cirrhosis nor malnutrition (present in about 50% of the patients) or hepatocarcinoma significantly affected the fructose-induced uricemia. Therefore, the fructose test efficiently differentiates cirrhotics from chronic hepatitis patients and healthy subjects, but it does not distinguish the various stages of the progression of cirrhosis or its complications. PMID- 8633512 TI - Multiple rings of the esophagus associated with gastroesophageal reflux [case report]. PMID- 8633511 TI - Selective blockade of cholecystokinin type B receptors with L-365,260 does not impair gallbladder contraction in normal humans. AB - OBJECTIVES: To evaluate the effect of selective blockade of type B cholecystokinin receptors on gall bladder contraction in normal humans and to compare methods for quantitative analysis of gall bladder contraction. METHODS: L 365,260, a novel, nonpeptide cholecystokinin antagonist shown to be selective for type B cholecystokinin receptors, was administered every 6 h over a 5-7 day period. Plasma levels of L-365,260 were determined by high pressure liquid chromatography. Gallbladder contraction after a standardized fatty meal was measured by ultrasonography, and results were calculated by ellipsoid or sum of cylinders methods. RESULTS: L-365,260 levels were comparable to levels in earlier studies demonstrating inhibition of pentagastrin-stimulated acid secretion in normal subjects and blockade of anxiogenic effects of cholecystokinin injections in patients with panic disorder. Regardless of the method used for estimating gallbladder size, none of the L-365,260 doses studied inhibited gallbladder contraction. Gallbladder size was most consistently estimated by the ellipsoid method using measurements normalized to individual values for minimum and maximum gallbladder dimensions. CONCLUSIONS: Multiple oral doses of L-365,260 do not alter ultrasonographically assessed gallbladder contraction at doses shown to be clinically effective in earlier studies. Despite being more difficult to implement, the sum of cylinders method for estimating gall bladder size offers no advantage over the ellipsoid method. PMID- 8633513 TI - Pancreatitis secondary to percutaneous liver biopsy-associated hemobilia. AB - Hemobilia refers to hemorrhage in the biliary tree and is most commonly associated with accidental and iatrogenic trauma. Rarely has pancreatitis been reported in association with hemobilia and never as a consequence of percutaneous liver biopsy-induced hemobilia. We report the case of a 64-yr-old man who presented with pancreatitis 6 days after a percutaneous liver biopsy. Within 24 h of admission, he developed hematochezia. Emergency endoscopy was performed, and with a side-viewing duodenoscope, blood and clot were clearly seen oozing from the papilla. The origin of bleeding was identified angiographically as a pseudoaneurysm of the right hepatic artery. Bleeding stopped and pancreatitis resolved after angiographic embolization of the hepatic artery pseudoaneurysm. A review of the English language literature reveals eight well-documented cases of pancreatitis associated with hemobilia, including the current report. Seven cases were associated with ruptured hepatic artery aneurysms and one case with hemorrhagic acalculous cholecystitis. Six of the patients received appropriate therapy to stop bleeding and recovered uneventfully. Pancreatitis should be recognized as a potential important complication of hemobilia. In addition, hemobilia should be considered in the differential diagnosis for those patients with apparent biliary or idiopathic acute pancreatitis and no stones seen on ultrasonography. When bleeding is stopped via surgical or radiological methods, the clinical course of hemobilia-associated pancreatitis appears to be benign. PMID- 8633514 TI - A fatal Sunday brunch: Amanita mushroom poisoning in a Gulf Coast family. AB - Mushroom poisoning with resultant liver toxicity often occurs in experienced collectors. The increasing popularity of natural foods has lead to an increased incidence of mushroom poisoning among the general population. This report describes a grandmother with over 30 yr of self-taught expertise in selecting and preparing wild mushrooms, who prepared a mushroom dish that led to the poisoning of herself and three family members. A 3-yr-old child died of fulminant hepatic failure, and the others recovered completely. The clinical presentation, course, and management of patients with mushroom poisoning are discussed. PMID- 8633515 TI - Bleeding from polypoid colonic arteriovenous malformations. AB - The endoscopic appearance of vascular ectasias has been well characterized. We present two cases of bleeding polypoid colonic arteriovenous malformations. PMID- 8633516 TI - K-ras mutation in a tubular adenoma originating at an ileostomy in a familial adenomatous polyposis patient. AB - Genetic alterations in a tubular adenoma with severe dysplasia arising in a Brooke ileostomy of a familial adenomatous polyposis patient were analyzed. Clinical and morphological characteristics suggest that ileal mucosa progressed to colonic metaplasia and then to dysplastic adenoma. Such changes at ileostomy sites are rare, and little is known about the associated genetic alterations. To determine whether metaplastic epithelium progression to adenoma in the ileum is subject to the same mutations identified in colon carcinogenesis, we evaluated somatic genetic alterations associated with sporadic colorectal cancer development. Sequences examined included mutation cluster regions of the p53 tumor suppressor gene and the k-ras oncogene. Using polymerase chain reaction and DNA sequencing, we identified a point mutation at codon 12 of the K-ras oncogene. To our knowledge, this is the first report of a ras mutation occurring in a tumor originating from ileal mucosa. PMID- 8633517 TI - Cystic duct perforation as a complication of endoscopic stone extraction. PMID- 8633518 TI - Endoscopic stenting of gallbladder for symptomatic cholelithiasis in patients with end-stage liver disease awaiting orthotopic liver transplantation. AB - Cholecystectomy in patients with advanced cirrhosis is associated with excessive morbidity and mortality. Symptomatic cholelithiasis in patients awaiting orthotopic liver transplantation poses unique management issues. Three patients with end-stage liver disease, severe enough to warrant hepatic transplantation, developed symptoms and complications of cholelithiasis requiring intervention. Each underwent placement of a biliary stent from gallbladder to duodenum at endoscopic retrograde cholangiopancreatogram. In each case, biliary symptoms and complications ceased after stent placement. Two patients remained symptom-free until they underwent successful liver transplantation, and the third awaits transplant and is currently free of symptoms of cholelithiasis. Endoscopic placement of gallbladder stents represents a temporizing alternative for the management of patients with symptomatic cholelithiasis awaiting hepatic transplantation. PMID- 8633519 TI - Gastric gland heterotopia with extensive lymphoid stroma: a gastric lymphoepithelial cyst. AB - Benign submucosal epithelial lesions of the stomach are rare. Their morphological aspect and pathogenesis and hence classification are unclear. The literature on this subject consists mainly of case reports describing lesions such as hamartomas, gastric gland heterotopia, duplication, submucosal cystic glands, gastritis cystica profunda, and adenomyoma. We report a patient presenting with a lymphoepithelial cyst, a lesion hitherto not yet described in the stomach. This lesion was an incidental finding in a surgical specimen from a patient during surgery for gastric adenocarcinoma. The cyst was submucosal, located at a distance from the tumor, and differs from classic "gastric gland heterotopia" by the presence of extensive lymphoid stroma. PMID- 8633520 TI - Primary esophageal lymphoma in AIDS presenting as a nonhealing esophageal ulcer. PMID- 8633521 TI - Early occurrence of acute fatty liver in pregnancy. AB - We report a case of acute fatty liver of pregnancy characterized by its early occurrence in the 26th wk of a twin pregnancy. Usually, this illness begins only late in the third trimester, although onset of disease as early as 26 wk has already been reported. The death of both fetuses also illustrates the high mortality rate and the necessity for prompt diagnosis and treatment. PMID- 8633522 TI - Emphysematous gastritis. AB - Emphysematous gastritis is a rare infectious disease of the stomach. A patient with fatal emphysematous gastritis associated with empyema thoracis is reported. The diagnosis was suspected as a result of a plain skiagram of the abdomen and was confirmed on CT scan of the abdomen. PMID- 8633523 TI - Isolated hepatic mucormycosis in an immunocompetent child. AB - A case of isolated localized hepatic mucormycosis in an immunocompetent 3 1/2-yr old girl with concomitant acute toxoplasmosis is described. Mucormycosis is rare in immunocompetent patients, and hepatic mucormycosis has so far been described only in the context of disseminated disease. The infection resolved spontaneously without surgical debridement and/or appropriate medical therapy with amphotericen B. PMID- 8633524 TI - Pancreatic somatostatin-secreting gangliocytic paraganglioma with lymph node metastases. AB - Gangliocytic paraganglioma (GPG) with local lymph node metastasis was found in the pancreas of a 74-yr-old female who presented with diarrhea, steatorrhea, vomiting, nausea, and abdominal pain. A Whipple procedure led to a complete resolution of these symptoms and a return of an elevated stomatostatin level to normal. This is the first description of GPG in this location and the first endocrinologically active GPG. PMID- 8633525 TI - Adenocarcinoma in an enterovesiculocutaneous fistula. PMID- 8633526 TI - Gastric granulocytic sarcoma as a cause of acute upper gastrointestinal bleeding. PMID- 8633527 TI - Isolated magnesium malabsorption in a 10-year-old boy. AB - Isolated magnesium malabsorption (congenital hypomagnesemia) has been reported in approximately 30 patients worldwide. Patients typically present by 6 months of age with convulsions and diarrhea. We report an unusual case of isolated magnesium malabsorption in an older boy with no diarrhea. PMID- 8633528 TI - Colonoscopy evaluations: justification by cost? AB - The type of colonic imaging (radiological vs colonoscopic) for evaluating symptomatic patients without evidence of bleeding in both an efficacious and cost conserving manner has become a very debated issue. In a randomized, controlled clinical trial, the authors hoped to examine the prevalence of neoplasm and the effectiveness and cost-effectiveness of initial diagnostic strategies of colonoscopy versus flexible sigmoidoscopy and air contrast barium enema in patients without evidence of intestinal bleeding. One hundred forty-nine patients over the age of 40 with symptoms suggestive a colonic disease without evidence of bleeding (no hematechezia, negative test for fecal occult blood, and normal serum hemoglobin) were randomized to undergo either initial colonoscopy or flexible sigmoidoscopy plus barium enema. Patients with incomplete lower GI tests were referred for the corresponding alternative imaging modality. Cost analyses using sensitivity analysis were performed. Baseline information with respect to age, race, sex, inpatient status, reason for referral, mean weight loss, hemoglobin, blood urea nitrogen, and albumin were similar in both groups. Eighteen patients (24%) who initially received air contrast barium enema and flexible sigmoidoscopy then required colonoscopy, whereas only five patients (6%) who initially underwent colonoscopy first required air contrast barium enema plus flexible sigmoidoscopy. The study found that: a) The prevalence of cancer in the study was low (one of 149 patients); b) initial colonoscopy detected more persons with adenomas than that of air contrast barium enema plus flexible sigmoidoscopy (23 of 75 patients vs 13 of 74 patients, odds radio, 2.07, CI,0.90-4.92; this approached significance); and c) air contrast barium enema plus flexible sigmoidoscopy detected more diverticulosis (46 of 74 patients vs 31 of 75 patients, odds ratio, 0.41, 95% CI, 0.21-0.87). The significant conclusions were that patients undergoing flexible sigmoidoscopy plus air contrast barium enema were more likely to undergo alternative procedures and that sensitivity analysis suggested that, for most areas in the United States, initial colonoscopy would be more cost-effective for the outcome of detection of adenomas (1). PMID- 8633529 TI - Stenting for choledocholithiasis: temporizing or therapeutic? AB - Maxton and colleagues report their experience using biliary endoprostheses for treatment of failed common bile duct stone clearance after sphincterotomy. Of 283 patients with choledocholithiasis referred to their tertiary facility, 85 failed to have their ducts cleared with the first ERCP. There were 21 male and 64 female subjects; mean age was 77.5 yr. Clinical presentations were jaundice (39), cholangitis (23), and biliary colic and/or abnormal liver blood tests in the remainder. The patients were characterized as "elderly and ill with either jaundice or cholangitis present in almost 75%." Follow-up data were obtained for all patients. ERCP was first performed using a duodenoscope with a 3.2-mm instrument channel. A 7-French double pigtail stent was placed in each of the 85 patients with retained stones. Subsequent ERCP were performed at 2- to 3-month intervals using a therapeutic duodenoscope (4.2-mm instrument channel). A second stent was placed if stones remained in the bile duct after repeated extraction attempts. Patients deemed too frail and elderly for frequent ERCP had their first stent left in place, with stent exchanges and attempts at stone extraction every 6-12 months. Mechanical lithotripsy was used in 23 patients, extracorporeal shock wave lithotripsy (ESWL) in 11, and dissolution therapy via nasobiliary catheter in 10. Acute illnesses resolved in 84 of 85 patients, with significant decreases in bilirubin and alkaline phosphatase levels by the second ERCP. Six patients died with temporary stents in situ, one form a respiratory arrest the day of ERCP; the other deaths were unrelated to ERCP or choledocholithiasis. Fifty of the remaining 79 patients had successful stone clearance; 68% of these required two ERCP, 20% three ERCP, 6% four ERCP, and, in another 6%, a total of five ERCP were required before their ducts were free of stones. Seven cases of cholangitis among these 50 patients were treated successfully with i.v. antibiotics, fluids, and "early" stent replacement. Twenty-six patients had long term biliary drainage with the original stents in situ over 12 months. Four of these patients were among the six deaths, all unrelated to biliary stones or ERCP. Three patients were referred for surgical stone removal. The authors conclude that placement of a single 7-French stent after failure to clear common duct stones is safe, provides affective biliary drainage, can prevent the need for urgent surgical intervention, and allows for transfer of sick patients to centers of expertise. Further attempts at bile duct clearance were successful in most cases. PMID- 8633530 TI - Yield pressure: a new concept in the evaluation of GERD? AB - This study evaluates the yield pressure at the gastroesophageal junction in a group of 73 patients undergoing diagnostic endoscopy and in another group of 82 patients during the course of manometry for suspected GERD. The group included 17 patients who had previously undergone a successful Nissen fundoplication and eight patients who had a failed Nissen fundoplication. Air is insufflated into the stomach, and a water perfused pressure transducer is used to detect intragastric pressure. The pressure at which the cardia was seen to open at endoscopy, or when a common cavity phenomenon was detected at manometry, was taken as the opening pressure. Yield pressure was calculated as the difference between the opening pressure of the cardia and the resting gastric pressure. Results indicated a significant decrease in yield pressure in 65 patients with esophagitis compared with 65 patients with no evidence of reflux or esophagitis. A significant inverse correlation was found between yield pressure and the size of the hiatus hernia noted in these patients. There was also a correlation between valvular appearance of the cardia at endoscopy and the yield pressure. A progressive decrease in yield pressure occurred with an increasing deterioration in the physical appearance of the valve. There was no significant relationship found between yield pressure and lower esophageal sphincter (LES) pressure or intra-abdominal length. A small but significant relationship was found between yield pressure and acid exposure of the lower esophagus. The 17 patients with a successful Nissen showed a significantly increased yield pressure to supranormal values. In contrast, the eight patients with a failed Nissen had yield pressures within the range of the patients with esophagitis. In eight patients, yield pressure was measured by both manometry and endoscopy and showed no significant differences between the two methods. PMID- 8633531 TI - Some aspects of the screening for colorectal cancer. PMID- 8633532 TI - Dysplasia and carcinoma in small colorectal polyps. PMID- 8633533 TI - Hemobilia and Bernard-Soulier syndrome. PMID- 8633534 TI - Cutaneous hypersensitivity reaction to mesalazine. PMID- 8633535 TI - Hematoma of the rectus abdominis muscle and sheath. PMID- 8633536 TI - Cell kinetics of the gastric mucosa of patients treated with omeprazole. PMID- 8633537 TI - Colonic perforation with volume laxatives. PMID- 8633538 TI - Malignant melanomas in the small intestine: a study of 103 patients. AB - OBJECTIVES: Malignant melanoma shows an unusual predilection to metastasize to the small intestine. A proportion of small bowel melanomas occur without history of an antecedent primary. We evaluated a group of patients with malignant melanoma in the small intestine to further our understanding of this disease. METHODS: We reviewed 103 cases of malignant melanoma in the small intestine (77 surgical resections and 26 autopsies) accessioned at the Armed Forces Institute of Pathology between 1945 and 1991 for demographic, chronological, and pathological features. RESULTS: Mean age at time of primary was 45.6 yr for surgical and 34.1 yr for autopsy cases (p = 0.01). Mean age at time of small intestinal involvement was 52.2 yr for surgical and 42.7 yr for autopsies (p = 0.03). Primary lesions preceded intestinal disease by an average of 5.6 yr for surgical and 2.1 yr for autopsies. The age distribution of surgical patients with and without known primary melanomas at the time of small intestinal melanoma was not significantly different. The same was true for autopsy patients. Using regression analysis, the linear relationship of age at primary melanoma (AAP) on age at small intestinal melanoma (AASI) was given by AAP = 2.30 + 0.85 (AASI), and that of AASI on AAP was given by AASI = 3.94 + 1.02 (AAP) (r = 0.93 and p < 0.0001 for both regressions). CONCLUSIONS: Our data and results support the concept that small bowel involvement by melanoma, even without a known primary, is most probably metastatic. The age at which an unknown primary occurred in cases of intestinal melanoma, or the age at which intestinal metastasis may appear in cases with known primary melanoma, can be estimated. There appear to be two subsets of primary melanoma: one that occurs among younger patients and is more aggressive with rapid metastasis and early death and one that occurs among older patients, is more indolent, and metastasizes less rapidly. PMID- 8633540 TI - Hepatic hemodynamics in a patient with nodular regenerative hyperplasia. AB - Nodular regenerative hyperplasia of the liver is an uncommon condition. Approximately 50% of these patients develop portal hypertension. Few previous reports document the site of increased resistance to blood flow within the liver in this disorder. We measured Doppler waveform patterns of the right hepatic vein by pulsed Doppler ultrasonography and portal, wedged hepatic, and free hepatic venous pressure by intravenous catheter before and after splenectomy in a 47-yr old woman with nodular regenerative hyperplasia who presented with portal hypertension and pancytopenia. Nodular regenerative hyperplasia was histologically confirmed. Pre- and postoperative measures indicated a marked difference between wedged hepatic venous pressure and free hepatic venous pressure, whereas there was little difference between portal venous pressure and wedged hepatic venous pressure. Doppler waveform patterns of the right hepatic vein showed an unclear pulsatile flow pattern with a decreasing reversed phase. The above data suggest that portal hypertension in nodular regenerative hyperplasia is primarily sinusoidal, similar to that seen with cirrhosis. PMID- 8633539 TI - Pathology and cellular kinetics of gallbladder with an anomalous junction of the pancreaticobiliary duct. AB - OBJECTIVES: Anomalous junction of the pancreaticobiliary duct (AJPBD) is thought to be an important risk factor for gallbladder carcinoma in Japan. In this report, we examine histopathology and cellular kinetics of gallbladder mucosae of patients with AJPBD and the possible risk of gallbladder carcinoma. METHODS: We examined 62 gallbladders from patients with AJPBD (group A), 16 gallbladder carcinomas from patients with AJPBD (group B), 60 gallbladder carcinomas from patients without AJPBD (group C), and six normal gallbladders from patients without AJPBD (group D). Histopathology, mucosal heights, and proliferative cell nuclear antigen-labeling index were obtained from routinely processed tissue specimens. RESULTS: The incidence of hyperplastic changes in group A and in the noncancerous regions (NCRs) of group B was greater than in the NCRs of group C (p < 0.05). The incidence of dysplastic changes in the NCRs of group B was greater than in the NCRs of group C (p < 0.05). The mucosal heights in group A and in the NCRs of group B were higher than in the NCRs of group C (p < 0.05). A high proliferative cell nuclear antigen-labeling index was observed in group A and in the NCRs of group B, where hyperplastic changes were frequently observed. CONCLUSIONS: These results suggest that a sequence of hyperplastic changes with a corresponding increase in cellular kinetics with progression through dysplasia to carcinoma may be important in carcinogenesis in gallbladders of patients with AJPBD. AJPBD itself may be a possible risk for gallbladder carcinoma. PMID- 8633541 TI - Autoimmune hepatitis in a patient with sickle cell disease. AB - In this report, we describe the case of a 28-yr-old woman with sickle cell anemia who presented with acute hepatic failure manifested by anorexia, malaise, painless jaundice, elevated aminotransferase activities, and severe coagulopathy. Liver biopsy revealed changes consistent with autoimmune hepatitis. Treatment with corticosteroids and azathioprine was followed by improvement in biochemical liver test results. The literature on sickle cell-associated liver diseases is reviewed. PMID- 8633542 TI - Minimally invasive management of acute superior mesenteric artery occlusion: combined urokinase and laparoscopic therapy. AB - Acute superior mesenteric arterial occlusion is a medical emergency mandating prompt diagnosis and therapy. Traditional management includes exploratory laparotomy with possible embolectomy or bowel resection. Unfortunately, these patients are often frail and elderly and tolerate open laparotomy poorly. In this report, we describe a patient with acute superior mesenteric artery occlusion successfully managed with a minimally invasive approach. The acute arterial occlusion was managed with intra-arterial fibrinolytic therapy, and bowel viability was assessed with diagnostic laparoscopy. This combined approach of arteriographic and laparoscopic therapy avoided exploratory laparotomy, and we believe that it is an important therapeutic option in select patients with acute mesenteric ischemia. PMID- 8633543 TI - Pilot study of the use of naltrexone to treat the severe pruritus of cholestatic liver disease. PMID- 8633544 TI - Caroli's syndrome in twin sisters. PMID- 8633545 TI - Prolonged survival in fibrosing cholestatic hepatitis with long-term ganciclovir therapy. AB - A 45-yr-old man underwent liver transplantation for cirrhosis due to hepatitis B and developed recurrent infection. Serial liver biopsies revealed fibrosing cholestatic hepatitis, an entity that is associated with rapid graft failure, and this was treated with long-term intravenous ganciclovir therapy. The patient is alive and well 2 yr after transplantation, despite the presence of well established cirrhosis and a marked accumulation of intrahepatic hepatitis B surface and core antigens. It is postulated that partial reduction of viral replication resulted in an incomplete syndrome in which rapid graft failure did not occur, but progressive fibrosis developed. Our case suggests that newer nucleoside analogues that provide a greater degree of inhibition to hepatitis B virus replication may greatly improve the outcome of patients with recurrent infection after liver transplantation. PMID- 8633546 TI - Long-term survival in a patient with verrucous carcinoma of the esophagus. AB - A 66-yr-old white male with a long-standing history of gastroesophageal reflux and Barrett's esophagus developed squamous cell dysplasia proximal to the site of the metaplastic epithelium. Two months later, he presented with progressive dysphagia. Upper endoscopy revealed near obliteration of the lumen from a large friable mass in the distal esophagus. Repeat endoscopic biopsies revealed areas of focal dysplasia but were inconclusive for the presence of malignancy. At surgery, a large inflammatory fibrotic mass was resected that was confirmed histologically to be a verrucous squamous cell carcinoma. Twenty-two months after the resection, there is no evidence of tumor recurrence. The case and relevant literature is discussed. PMID- 8633547 TI - Recurrent acute pancreatitis: an additional manifestation of the "wandering spleen" syndrome". AB - A case of "wandering spleen" associated with recurrent pancreatitis and diagnosed with computed tomography (CT), is reported. The entity was also associated with a rotated, distended stomach, gastric outlet obstruction secondary to extrinsic compression of the duodenum, and partial small bowel obstruction secondary to extrinsic compression of a mobile, distended cecum that lay under the right diaphragm. PMID- 8633549 TI - Cullen's sign secondary to intra-abdominal non-Hodgkin's lymphoma. PMID- 8633548 TI - Mesalamine-induced lung toxicity. AB - Lung toxicity associated with 5-aminosalicylate (5-ASA) agents is a rare entity. We report the case of a 32-yr-old woman with ulcerative colitis who developed progressive shortness of breath while taking one of the 5-ASA drugs, oral mesalamine. Bilateral pulmonary infiltrates, peripheral eosinophilia, and histological findings consistent with acute pneumonitis characterized the lung injury. Although the differential diagnosis is broad, mesalamine-induced lung damage must be considered in patients who develop unexplained respiratory symptoms while taking this agent. PMID- 8633550 TI - Prostatic carcinoma simulating a rectal leiomyoma. PMID- 8633551 TI - Basal cell carcinoma of the anus. PMID- 8633552 TI - A case of esophageal perforation due to a pill-induced ulcer successfully treated with conservative measures. PMID- 8633553 TI - Colon cancer with a high serum alpha-fetoprotein level. AB - We report on a patient with colon cancer and a very high serum alpha-fetoprotein level. This 75-yr-old man presented with right lower quadrant abdominal pain. An abdominal CT scan as well as ultrasonography showed a tumor in the cecum. Serum alpha-fetoprotein level was extremely high (3,070 ng/ml). At laparotomy, a large mass was found in the cecum, and a right hemicolectomy was performed. Histological examination, including immunohistochemical study, showed an adenocarcinoma of the colon producing alpha-fetoprotein. PMID- 8633555 TI - Primary malignant melanoma of the esophagus. AB - A case of primary malignant melanoma of the esophagus is presented, followed by a review of the literature. This uncommon tumor generally manifests itself as a pedunculated, polypoid lesion in the middle-lower third of the esophagus. The histological diagnosis is usually made post-resection. The treatment of choice is surgical resection. The advanced stage of the disease at the time of presentation and the aggressive biological behavior of the tumor result in a dismal prognosis. PMID- 8633554 TI - Choledochocele-containing stones. AB - The case of a 68-yr-old woman with a rare type of Alonso-Lej's type III choledochal cyst (choledochocele) is described. This patient was admitted to the hospital because of right upper quadrant pain. Endoscopy revealed a bulge of the papilla of Vater resembling a submucosal tumor. Endoscopic retrograde cholangiopancreatography demonstrated cystic dilation of the terminal portion of the common bile duct. In addition, small stones were located in the gallbladder, the common bile duct, and the cystic lesion. Cholecystectomy, resection of the cystic lesion, and papilloplasty were performed. Histologically, the interior wall of the cyst was lined with biliary mucosa and demonstrated no evidence of malignancy. We reviewed 63 reported cases of choledochocele in Japan and 65 reported cases in the English literature. The clinical features of choledochoceles were similar in both groups, and they were associated with a minimal risk of malignant degeneration. These findings may have important implications for treatment. PMID- 8633556 TI - New options for spontaneous bacterial prophylaxis: costly or cost effective? PMID- 8633557 TI - Is having alpha-1-antitrypsin deficiency necessarily bad for your liver? PMID- 8633558 TI - Endoscopic esophageal manometry. PMID- 8633559 TI - Re: Barrett's esophagus and colonic neoplasms. PMID- 8633560 TI - Re: Anorectal inflammatory conditions. PMID- 8633561 TI - Gallstone opacification during cholelitholytic treatment: the effect of radiopaque contrast media. PMID- 8633562 TI - Pancreatic abscess secondary to central venous catheter infection. PMID- 8633563 TI - The HLA DRB1*0301-DQB1*0201 haplotype confers protection against inflammatory bowel disease. PMID- 8633564 TI - Contemporaneous diagnosis of Crohn's disease and adenocarcinoma of the jejunum. PMID- 8633565 TI - Low eradication rates of Helicobacter pylori with omeprazole plus amoxycillin combination in a Turkish population. PMID- 8633566 TI - Prostaglandins, NSAIDS, and GI mucosal integrity: can we identify patients at risk of NSAID-induced gastric injury? PMID- 8633567 TI - Nodular regenerative hyperplasia, dysplasia, and hepatocellular carcinoma. PMID- 8633568 TI - Gastric premalignancy and cancer screening in high-risk patients. PMID- 8633569 TI - Genetic markers and premalignancy. PMID- 8633570 TI - Principles of screening and surveillance. PMID- 8633571 TI - Screening high-risk groups for colorectal neoplasia. PMID- 8633572 TI - Early detection of neoplasia of the esophagus and gastroesophageal junction. AB - Recommendations for preventing adenocarcinoma: The standard definition of Barrett's epithelium should be the presence of intestinalized mucosa in the lower esophagus. Patients in this category should be considered for inclusion in a screening program for the detection of dysplasia or carcinoma. Those who are a poor operative risk should not be screened if the detection of an end-point such as high-grade dysplasia or intramucosal carcinoma will still not lead to resection. In some centers, however, alternative experimental methods of mucosal ablation may be available. The endpoint for screening is invasive or intramucosal carcinoma (or--in centers with a very low operative mortality--high-grade dysplasia). These should lead to consideration of surgery or, in specialized centers and as part of controlled studies, newer alternative modes of epithelial ablation. Intermediate markers, e.g., use of aneuploidy, gene markers, or their products, are at present experimental. Screening should be carried out annually or, possibly, biennially. This screening should utilize a standard protocol with an endoscope capable of obtaining large-particle biopsies. Four quadrant biopsies should be taken about every 2 cm, beginning 2 cm above the proximal limit of the gastric rugae, continuing until unequivocally in squamous mucosa, and following any tongues of glandular epithelium. Recommendations for prevention of squamous carcinoma: In high-risk populations, esophageal cytology, possibly supplemented by tests for blood in the stomach, appear most useful. Repeated screening may be necessary to detect early invasive or preinvasive (dysplastic) tumors. Although preliminary results from dietary intervention studies have yet to show a statistically significant decrease, if trends continue these will reach significance and may be the best overall method of cancer prevention. Measures to reduce smoking and drinking are to be encouraged, but their effectiveness is questionable. For patients who are positive on screening, endoscopy (possibly with Lugol's iodine) may provide the best indication of harboring underlying carcinoma or dysplasia. Endoscopic resection will play an increasing role in treatment. With the exception of tylosis, most other predisposing conditions are unlikely to be cost effective. PMID- 8633573 TI - Premalignant and early malignant lesions in the gastrointestinal tract: definitions, terminology, and problems. AB - The definition of a "premalignant" lesion is discussed, together with its relationship to dysplastic change. The definition and description of dysplasia are given. Systems for grading dysplasia are set out, and the clinical implications of these changes are discussed. Although the "dysplasia terminology" in current use remains the gold standard for clinical practice, a series of more recent developments, including the discovery of flat and depressed adenomas, the possibility of de novo carcinoma throughout the gastrointestinal tract, as well as advances in molecular biology, raise the question of whether this terminology will be adequate in future. Other terminologies exist that provide an alternative viewpoint, but none is satisfactory. PMID- 8633574 TI - Prostaglandin E2 content in residual gastric juice reflects endoscopic damage to the gastric mucosa after naproxen sodium administration. AB - OBJECTIVES: The diagnostic potential of residual gastric juice for development of naproxen sodium-induced mucosal damage has not been explored. We studied prostaglandin E2 (PGE2) content in residual gastric juice before and after naproxen sodium administration and assessed relationships with endoscopic mucosal damage. METHODS: Thirty volunteers received the recommended over-the-counter dose (660 mg/day) of naproxen sodium or placebo in this 7-day, double-blind, endoscopically controlled, cross-over study. RESULTS: PGE2 concentration in gastric juice did not increase after placebo; naproxen significantly reduced PGE2 concentration (p < 0.001). In three subjects in whom no endoscopic changes occurred after naproxen administration, PGE2 concentration increased by 14%. In seven subjects who developed hemorrhagic changes, PGE2 concentration declined by 50% (p = 0.08). In 20 subjects who developed numerous hemorrhagic and erosive changes within the antral mucosa, PGE2 concentration declined by 70% (p < 0.001). The starting PGE2 value in subjects with severe mucosal hemorrhagic and erosive changes after naproxen was almost eightfold higher than in subjects who did not develop mucosal damage (p = 0.007) and 67% higher than in subjects with hemorrhagic changes only (p = 0.25). CONCLUSION: PGE2 measurement in residual gastric juice, aspirated before and after nonsteroidal antiinflammatory drug treatment, may be useful in monitoring mucosal damage and may identify patients who are likely to develop endoscopic mucosal changes. PMID- 8633575 TI - Hepatocellular carcinoma and nodular regenerative hyperplasia: possible pathogenetic relationship. AB - OBJECTIVE: In a recent review of hepatocellular carcinoma (HCC) in North American residents, we were surprised to learn that 42.6% of these tumors in the 1980-1993 consultation files of the Armed Forces Institute of Pathology had arisen in noncirrhotic livers. We subsequently noted that the nonneoplastic livers of a number of these had nodular regenerative hyperplasia (NRH), a condition that has been associated with liver cell dysplasia, a putative premalignant lesion. To investigate the possibility that NRH might be a precursor of HCC, we studied those cases in which there was an association of HCC and NRH and examined the possible role of portal vein obstruction in NRH occurring in livers with HCC. METHODS: Subjects were selected based on study criteria and histological slides, clinical/autopsy records were reviewed, and features of neoplastic and nonneoplastic liver were noted. Simple statistical comparisons were made between the groups with and without NRH with respect to defined variables. RESULTS: Of 804 patients suitable for study, 342 were noncirrhotic, and 23 of these had NRH. Mean age of patients with NRH was 65 +/- 13.6 (SD) yr. Seventeen of these (73.9%) had liver cell dysplasia, and 16 (69.6%) had portal venous invasion. Liver cell dysplasia occurred in a significantly greater proportion of those with NRH than those without (p < 0.01), but there was no significant difference between both groups with regard to portal venous invasion. Three patients (13%) had received chemotherapy and/or radiotherapy before diagnosis of NRH. CONCLUSIONS: These findings may be due to the development of HCC within the dysplastic foci that occur in livers with NRH, but the findings do not exclude the converse possibility that NRH may also develop in a noncirrhotic liver with HCC, secondary to portal venous invasion with portal vein occlusion. The temporal relationship between HCC and NRH is probably determined in each case by the particular interaction of multiple pathogenetic factors. Among patients with HCC, factors other than the portal vein obstruction by tumor invasion may play a role in the pathogenesis of NRH. PMID- 8633576 TI - Long-term follow-up of older patients with iron deficiency anemia after a negative GI evaluation. AB - OBJECTIVE: To determine the clinical outcome of a cohort of patients over the age of 50 yr with iron deficiency anemia in whom a source was not identified during a prior GI evaluation. METHODS: A cohort of 69 patients (43 men, 26 women) with a mean age of 68 yr (range 50-89 yr) who previously had a negative GI evaluation for documented iron deficiency anemia was followed for an average of 39 months (range 2-89 months). RESULTS: In 49 patients (71%), the iron deficiency anemia resolved. All remained stable during the follow-up period except five patients in whom iron deficiency recurred (from 18 months to 5 yr later). Subsequent GI evaluations were negative, and iron deficiency resolved with iron supplementation in all five patients. In 16 patients (23%), a mild chronic anemia persisted during the follow-up period. None of these patients required transfusions, and 12 of the 16 had concomitant medical illnesses that likely contributed to an anemia of chronic disease. Only four patients (6%) had a persistent anemia severe enough to require intermittent transfusions. However, in three of these patients, the anemia was likely secondary to chronic disease (chronic renal failure in two patients, metastatic carcinoma in one patient). Therefore, only one patient (1%) had consistent iron deficiency anemia severe enough to require intermittent transfusions, which was found to be secondary to duodenal angiodysplasia during a subsequent GI evaluation. CONCLUSION: The prognosis of iron deficiency anemia in older patients after a negative GI evaluation is favorable. The anemia resolves and remains stable in the majority of patients after iron replacement. Subsequent GI evaluations should be reserved for those patients whose anemia is refractory to iron replacement and in whom there are not concomitant medical illnesses contributing to an anemia of chronic disease. PMID- 8633577 TI - Periodic rectal motor activity: the intrinsic colonic gatekeeper? AB - BACKGROUND: Rectal motor activity is incompletely understood. The aim of this study was to characterize the patterns of rectal motor activity and to examine their diurnal variation and their relationships to proximal colonic activity and to meals. METHODS: We performed a 30-h ambulatory motility study by recording pressure activity at multiple sites in the colon in 18 normal subjects. RESULTS: During 288 h of recording, discrete bursts of tonic and phasic activity were seen in the rectum of all subjects at night and during the day lasting > or = 3 min, with a predominant frequency of 3 waves/min: periodic rectal motor activity (PRMA). Nocturnally, the number of cycles and the proportion of time occupied by this activity were greater (p < 0.001) and the inter-cycle interval was shorter (p < 0.008) compared with daytime, but the cycle duration was similar. Only 4 versus 5% (nocturnal vs daytime) of cycles propagated aborad, whereas 36 versus 14% (p < 0.01) propagated retrogradely, 16 versus 47% (p < 0.01) occurred simultaneously, and 44 versus 34% were confined to the rectum. There was considerable intra- and intersubject variability. PRMA was not related to meals or to anal motor activity, but 81% of nocturnal and 94% of daytime cycles occurred within 5 min of a motor event in the more proximal colon. CONCLUSIONS: PRMA is a characteristic feature of the normal rectum and is more frequent at night. The temporal association with motor events in the proximal colon suggests that PRMA is triggered by the arrival of stool or gas in the rectum. Because most cycles are either segmental or are propagated retrogradely, PRMA may serve as an intrinsic braking mechanism that prevents untimely flow of colonic contents, particularly during sleep. PMID- 8633578 TI - Symptom duration in patients with irritable bowel syndrome. AB - OBJECTIVES: The etiology and natural history of irritable bowel syndrome (IBS) is poorly understood. We compared rectal sensory thresholds and compliance, SCL-90 scores, and follow-up questionnaires among normal controls, patients with longstanding ( > 5 y) disease (L-IBS), and patients with recent onset ( > 2 y) disease (R-IBS). The onset of symptoms in R-IBS was related to specific events such as infection (n = 10), surgery (n = 5), and stress (n = 4), but no specific event could be identified in six patients. METHODS: A diagnosis of IBS was made using Manning criteria ( > or = 3) and clinical grounds. Psychological data were obtained by psychometrics (SCL-90) scores. Rectal wall compliance and thresholds for the sensation of stool and discomfort were evaluated using the electronic barostat. RESULTS: The mean thresholds for phasic and ramp distention were similar for R-IBS and L-IBS groups for the perception of stool and discomfort. When compared with normals, the mean stool thresholds for phasic distention were significantly lower for L-IBS and R-IBS groups. SCL-90 scores were significantly increased in L-IBS in the mean phobia score (45 R-IBS vs 61 L-IBS), anxiety score (49 R-IBS vs 63 L-IBS), paranoia score (44 R-IBS vs 60 L-IBS), and hostility score (47 R-IBS vs 61 L-IBS) (all p < 0.05). On follow-up questionnaire, 60% of R IBS versus 46% of L-IBS patients indicated that their symptoms had improved (p < 0.05). R-IBS patients also experienced fewer episodes of abdominal pain per week at follow-up than L-IBS patients (3.9 +/- 1.0 vs 8.5 +/- 1.7, respectively) (p < 0.05). CONCLUSIONS: Our findings suggest that IBS patients with short symptom duration and fewer psychological symptoms have a better prognosis than patients with a long history of IBS and associated psychological distress, although long term prospective studies are needed. PMID- 8633579 TI - Increased urine catecholamines and cortisol in women with irritable bowel syndrome. AB - OBJECTIVES: There are few data on the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis in individuals with chronic GI symptoms. The current study was designed to describe and compare urine catecholamine (norepinephrine, epinephrine) and cortisol levels in women diagnosed with irritable bowel syndrome (IBS-patients), women who report similar symptom levels but had not sought health care services (IBS-nonpatients; IBS-NP), and asymptomatic (control) women. METHODS: Seventy-three women (24 IBS; 24 IBS-NP; 25 controls) were interviewed for demographic, GI, gynecological, and psychological data and then followed for two menstrual cycles with a daily health diary. Urine samples were obtained in the evening and morning at specific phases across two menstrual cycles. RESULTS: Women in the IBS group had significantly higher PM and AM urine norepinephrine levels. Urine epinephrine and cortisol levels were also generally higher in women with IBS. Differences in neuroendocrine indicators of arousal were not accounted for by differences in demographic variables, lifestyle characteristics, menstrual distress, or average daily measures of anxiety or depression. CONCLUSIONS: Increases in indicators of sympathetic nervous system activation in women seeking health care for IBS may reflect greater symptom distress or may contribute to increased symptom distress. PMID- 8633580 TI - Clinical features of type III (mixed) paraesophageal hernia. AB - OBJECTIVES: The clinical presentation of patients with a paraesophageal hernia is poorly understood. The aim of this study was to evaluate the progression of symptoms in patients with type III paraesophageal hernia. METHODS: We evaluated 25 patients (mean age 66 yr) with a type III paraesophageal hernia, who then had laparoscopic surgical repair. The patients characterized their symptoms as 1) chronic or 2) prompting evaluation (progressive or new). We defined postprandial distress as chest or epigastric pain, shortness of breath, or nausea or vomiting during or shortly after meals. RESULTS: We identified three distinct types of chronic symptoms: heartburn only (36%), heartburn and postprandial distress (32%), and postprandial distress only (32%). Twenty-three patients (92%) reported postprandial distress as the symptom prompting evaluation compared with only 10 patients (40%) reporting heartburn. Eight patients (32%) never had heartburn. Postprandial distress was their only symptom. Laparoscopic repair of the paraesophageal hernia resolved postprandial distress in 74% and improved symptoms in the remaining 26% of patients (mean follow-up 12 months). CONCLUSIONS: Post prandial distress is the most prominent symptom in patients with a type III paraesophageal hernia. Most patients had chronic symptoms of a sliding hiatal hernia but later featured more pronounced postprandial distress. However, one third of the patients never experienced significant heartburn. A type III paraesophageal hernia should be suspected in patients, with or without heartburn, who develop new or progressive symptoms of postprandial distress. PMID- 8633581 TI - Chloroquine for mild to moderately active ulcerative colitis: comparison with sulfasalazine. AB - Chloroquine and hydroxychloroquine have been used successfully in a number of immunological diseases because of their immunomodulatory effect. Preliminary studies have shown them to be effective in the treatment of idiopathic ulcerative colitis. We assessed the efficacy or oral chloroquine in the treatment of idiopathic ulcerative colitis of mild to moderate severity in a randomized, controlled, blinded trial. Forty patients were assigned to receive chloroquine phosphate (500 mg/day) or sulfasalazine (3 g/day). The outcome of therapy was monitored at the end of 2 wk and 4 wk by symptom assessment and sigmoidoscopic examination. An objective scoring system was used for the evaluation, and patients were categorized into those showing complete remission, partially responding, and nonresponders. There was a sequential improvement in the individual scores as well as total activity score in both groups at 2 wk and 4 wk. With chloroquine, 12 patients (60%) showed complete remission at 4 wk, whereas six (30%) had a partial response. With sulfasalazine, 11 patients (55%) had complete remission at 4 wk, and eight (40%) showed partial response. There was no significant difference in the response to the two drugs (p > 0.05). Five (25%) patients in the chloroquine group had side effects, compared with eight (40%) in the sulfasalazine group (p > 0.05). All side effects were minor and transient, not requiring withdrawal of the drug. We conclude that short-duration chloroquine therapy is a safe and effective alternative to sulfasalazine in the treatment of mild to moderately active ulcerative colitis. PMID- 8633582 TI - Perianal Crohn's disease in children and adolescents. AB - OBJECTIVES: To review the frequency, severity, course, and outcome of perianal Crohn's disease in children and adolescents. METHODS: Records of 141 children and adolescents with Crohn's disease were reviewed. Of these, 18 patients (eight female, 10 male) between 9 and 18 yr of age (nine blacks and nine whites) had significant perianal disease. The disease was classified on the basis external appearance, degree of inflammation, presence of infection or abscess formation, spread to vulva, loss of perineal body, and occurrence of anal canal stricture as being mild, moderate, or severe. RESULTS: Eight patients had severe involvement, whereas moderate and mild degree of perianal disease was seen in six and four patients, respectively. Disease localization was as follows: pancolitis, 12; ileocolitis, four; and left colitis, two. Therapy with Azulfidine or 5-amino salicylic acid preparation, local and systemic steroids, metronidazole, immunosuppressives, and enteral and/or parenteral nutrition was used in various combinations. Surgery was necessary in eight patients: in four for management of anal canal stricture, incision and drainage of abscess in two, total proctocolectomy in one, and fecal diversion in an additional patient. Over a follow-up period ranging from 18 months to 15 yr, nine patients had partial improvement, whereas in the rest, the disease either did not improve or worsened. CONCLUSIONS: Severe, mutilating perianal Crohn's disease, causing significant tissue destruction, occurs in both sexes and is extremely refractory to treatment in the majority of patients. The outcome in mild and moderate disease is better. PMID- 8633583 TI - Fecal lactoferrin as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins. AB - OBJECTIVES: 1) To investigate which neutrophil-derived proteins in feces most accurately reflect disease activity in inflammatory bowel disease. 2) To examine the extracellular release of these proteins by activated neutrophils and their stability in feces by in vitro study. METHODS: We studied 41 patients (91 samples) with ulcerative colitis (UC), 34 patients (105 samples) with Crohn's disease (CD), and 25 control subjects. Fecal levels of lactoferrin (Lf), polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase (MPO), and lysozyme (Lys) were measured by ELISA. We also measured fecal hemoglobin (Hb) and alpha 1-antitrypsin (alpha 1-AT), useful markers of disease activity in UC and CD, respectively. For the in vitro study, blood samples were stimulated with phorbol myristate acetate or latex beads. For the assessment of stability, homogenized stool samples were stored at 4 degrees C, 25 degrees C, and 37 degrees C for various periods. RESULTS: 1) Fecal Lf, PMN-E, MPO, and Lys concentrations were significantly increased in the active phase of the disease compared to the inactive phase in both UC and CD. 2) Fecal Lf, PMN-E, MPO, and Lys concentrations correlated significantly with fecal Hb concentration in UC, whereas fecal Lf, PMN-E, and MPO concentrations correlated significantly with alpha 1-AT concentration in CD. In UC, fecal Lf, PMN-E, MPO, and Lys concentrations were high in 15, 9, 14, and 14 samples, respectively, of 25 samples with normal Hb concentration. In CD, fecal Lf, PMN-E, and MPO concentrations were high in 19, 10, and 16 samples, respectively, of 30 samples with normal alpha 1-AT concentration. 3) The extracellular release of Lf was the most efficient and this molecule was the most stable in feces. CONCLUSIONS: Both our clinical and our in vitro studies suggested that Lf is the most suitable of these proteins to use as neutrophil-derived fecal marker of inflammation for clinical application. PMID- 8633584 TI - Bismuth-based combination therapy for Helicobacter pylori-associated peptic ulcer disease (metronidazole for eradication, ranitidine for pain). AB - OBJECTIVES: 180 Helicobacter pylori-positive patients with peptic ulcer disease were randomly allocated to double-blind placebo-controlled treatment with one of four anti-H. pylori regimens consisting of bismuth subnitrate suspension (B), oxytetracycline (OT), metronidazole (M)/metronidazole placebo, or ranitidine (R)/ranitidine placebo. METHODS: Regimen 1: B 150 mg q.i.d., OT 500 mg q.i.d., M 400 mg t.i.d. for 10 days and R 300 mg b.i.d. for 4 wk. Regimen 2: same as regimen 1 except ranitidine. Regimen 3: same as regimen 1 except metronidazole. Regimen 4: same as regimen 1 except metronidazole and ranitidine. Gastroscopy and 14C-urea breath test were performed 4 wk after cessation of therapy, and breath test six months after cessation. RESULTS: According to intention-to-treat analysis, H. pylori eradication rates were 96%, 91%, 20%, and 9% with regimens 1, 2, 3, and 4, respectively. Comparing regimens 1+2 and 3+4, the eradication rates with and without metronidazole were 93% and 14%, respectively (p < 0.0001). Metronidazole increased the occurrence of diarrhea and abdominal pain. Comparing regimens 1+3 with 2+4 ranitidine did not influence H. pylori eradication (58% with and 50% without ranitidine; p = 0.37) or ulcer healing (93% with and 90% without ranitidine; p = 0.72) significantly, but reduced the occurrence of pain (p < 0.01). Six months after treatment, three patients who were H. pylori negative at 4 wk had become positive. These three had all received metronidazole placebo. H. pylori status remained negative in the other 85 patients. CONCLUSIONS: H. pylori eradication with this triple therapy is critically dependent on metronidazole. Adding ranitidine reduces the occurrence of abdominal pain during such therapy. PMID- 8633585 TI - Comparison of rapid serological tests (FlexSure HP and QuickVue) with conventional ELISA for detection of Helicobacter pylori infection. AB - BACKGROUND: There is a need for accurate and rapid tests for Helicobacter pylori infection especially since the recent National Institutes of Health Consensus Development Conference on H. pylori in peptic ulcer disease charged the medical community with treating H. pylori infection in all patients with H. pylori and ulcer disease. METHODS: We prospectively compared a simple, rapid serological test (FlexSure HP, SmithKline Diagnostics) for the detection of serum IgG antibodies against H. pylori with another rapid test (QuickVue, Quidel) and two enzyme immunoassays (HM-CAP, Enteric Products, and PyloriStat, BioWhittaker). Serum samples from 551 individuals including both symptomatic patients (196) and asymptomatic volunteers (355) were tested for the presence of IgG antibodies against H. pylori. The presence or absence of active H. pylori infections was determined using the [13C]-urea breath test. RESULTS: All of the serological tests performed well. FlexSure HP had calculated sensitivity, specificity, and accuracy of 94.4, 87.6, and 91.1%, respectively, relative to the urea breath test. In 49 of the 551 samples, the urea breath test and FlexSure HP did not agree. Those samples were tested with HM-CAP immunoassay to confirm presence or absence of IgG antibodies against H. pylori. After the resolution of the discordant results, the sensitivity, specificity, and accuracy of FlexSure HP were 96.0, 95.1, and 95.6%, respectively, and were comparable to HM-CAP and PyloriStat. FlexSure HP was compared with histology or culture in 75 cases, and the accuracy was 100%. FlexSure HP and QuickVue were compared using 200 serum samples. FlexSure HP was more specific (88.7 vs 79.4%) and accurate (91 vs 84%) than QuickVue (p < 0.05 for both), relative to the urea breath test with discordant samples unresolved. FlexSure HP was also simpler to use, easier to interpret, and faster than QuickVue. FlexSure HP required no sample dilution, one reagent, four steps, and 5 min to complete. CONCLUSION: FlexSure HP is an excellent option for in-office tests for the physician who desires immediate results or for small laboratories that do not have the volume of H. pylori testing to justify ELISA test formats. PMID- 8633586 TI - Prevalence of CagA-bearing Helicobacter pylori strains detected by the anti-CagA assay in patients with peptic ulcer disease and in controls. AB - OBJECTIVE: Cytotoxin-associated gene (CagA)-bearing Helicobacter pylori strains have been associated with significant gastroduodenal pathologies. We have performed a study to evaluate the prevalence of CagA-bearing strains in a group of H. pylori-positive peptic ulcer disease and non-ulcer dyspepsia (NUD) patients, and healthy asymptomatic controls. METHOD: Two hundred ninety-seven peptic ulcer disease, 45 NUD subjects, and 200 asymptomatic controls were studied. The newly developed anti-CagA antibody assay was used for the purpose of this study. The assay was performed by a conventional three-step enzyme-linked immunosorbent assay (ELISA) to detect the concentration of anti-CagA antibody present in the tested sera against the recombinant CagA 17/12 fusion protein. The final results were expressed with reference to a standard curve constructed from pooled CagA+ sera. Anti-CagA antibody assay reproducibility was assessed by intraplate and interplate variations. RESULTS: The mean intraplate and interplate variations were 8.0% and 11.2%, respectively. Anti-CagA antibody was present in 165/197 (84%) duodenal ulcer disease, 80/100 (80%) gastric ulcer disease, 25/45 (55.6%) NUD subjects, and 29/100 (29%) asymptomatic controls. The ulcer disease subjects were significantly more likely than the NUD subjects and the asymptomatic controls to have a positive anti-CagA antibody assay ( p < 0.005 and p < 0.001, respectively). Moreover, the NUD subjects were more likely to be anti CagA+ antibody than the asymptomatic controls (p < 0.005). CONCLUSIONS: This newly developed anti-CagA antibody assay was highly reproducible. Anti-CagA antibody positivity was present in a significantly higher percentage of peptic ulcer disease subjects than in non-ulcer and asymptomatic healthy controls. Thus, anti-CagA antibody can be used as a clinical marker for peptic ulceration. PMID- 8633587 TI - Helicobacter pylori serology in patients with chronic gastritis. AB - OBJECTIVES: Helicobacter pylori (Hp) infection is known to cause several gastroduodenal diseases. In patients with non-ulcer dyspepsia, we assessed the link between Hp infection and gastric mucosal inflammation, as well as the influence of Hp and inflammation on the serum levels of anti-Hp antibodies (IgG), pepsinogen A (PGA), pepsinogen C (PGC), and gastrin. METHODS: Entering the study were 221 patients with non-ulcer dyspepsia, all of whom underwent upper gastrointestinal endoscopy. RESULTS: Of the 221 patients investigated, 135 (61%) were Hp positive. The higher the bacterial load, the worse the associated gastritis, the gastric antrum and body being considered. All of the serological indices studied were found to be influenced by gastritis. Serum IgG satisfactorily discriminated between Hp-positive and Hp-negative subjects, with a sensitivity of 84% and a specificity of 86%. PGC, PGA, and gastrin were less accurate. Only PGC only found to be correlated with Hp load. The product of IgG and PGC improved the diagnostic accuracy of IgG alone. CONCLUSIONS: Hp infection, frequently found in patients with non-ulcer dyspepsia, is associated with gastric mucosal inflammation; of the indices studied, serum IgG and PGC most accurately indicated Hp infection, and their product may be proposed as an aid in diagnosing Hp infection in dyspeptic patients. PMID- 8633588 TI - Helicobacter pylori infection is the major risk factor for atrophic gastritis. AB - OBJECTIVE: To evaluate the association of both Helicobacter pylori (Hp) infection and advancing age with increased prevalence of atrophic gastritis. METHODS: Two hundred and thirty-eight subjects who had no esophagitis, peptic ulcers, or malignancies in the upper gastrointestinal tract were divided into three groups according to age: group A, < 30 yr; group B, 30-49 yr; group C, > or = 50 yr. Two biopsy specimens were obtained from the lesser curvature of the antrum and two from the anterior and posterior walls of the fundus to assess the degree of gastritis and histological evidence of Hp infection. Hp infection was evaluated by Giemsa staining and serum IgG antibodies. Serum gastrin (SG) and pepsinogen (PG) were determined by radioimmunoassay. RESULTS: In all age groups, the prevalence of atrophic gastritis was significantly more common in subjects with evidence of Hp infection. In Hp-positive subjects, the prevalence of atrophic gastritis increased with advancing age. Atrophic gastritis was extremely rare, regardless of age, in Hp-uninfected patients. SG increased, and PG I and the PG I:II ratio decreased with age in Hp-positive subjects. This trend was not apparent in Hp-negative subjects. CONCLUSION: Our results suggest that Hp infection is a stronger predictor than advancing age for atrophic gastritis. PMID- 8633589 TI - Distribution of inflammation and atrophy in the stomach of Helicobacter pylori positive and -negative patients with chronic gastritis. AB - OBJECTIVES: To investigate the extent of inflammation and atrophy in the stomach of Helicobacter pylori-positive and -negative patients with chronic gastritis. METHODS: Endoscopy with biopsies from the lesser curvatures of the antrum, angulus, middle body, and the greater curvature of the middle body of the stomach was performed in 59 patients with histologically confirmed chronic gastritis. The extent of atrophic gastritis was assessed endoscopically as well histologically. H. pylori status was assessed by histology as well as enzyme-linked immunosorbent assay. The histological severity of chronic and acute inflammation, glandular atrophy, and intestinal metaplasia was assessed according to the Sydney system. RESULTS: In H. pylori-positive patients, H. pylori was evenly distributed throughout the stomach when the extent of atrophic gastritis was limited to the antrum and the lesser curvature of the body, but disappeared from the antrum of patients with more extensive atrophic gastritis. The severity of acute and chronic inflammation at the greater curvature of the body increased with the extension of atrophic gastritis. In H. pylori-negative patients, the severity of chronic inflammation at the greater curvature of the body was significantly higher in patients with extensive atrophic gastritis than in those with a lesser extent of atrophic gastritis. CONCLUSION: At the greater curvature of the body, the development of atrophy is closely associated with the increase in the severity of inflammation, which is more marked in H. pylori-positive patients. PMID- 8633590 TI - Electrically evoked cerebral potentials during esophageal distension at perception and pain threshold. AB - OBJECTIVE: To investigate esophageal sensory function in normal volunteers. METHODS: The method of evoking cerebral potentials (EPs) after electrical stimulation of the esophagus was applied in eight healthy male volunteers, 20-40 yr old. Electrical stimulation was obtained via bipolar Ag/AgCl electrodes attached to a latex balloon mounted on a catheter. The balloon was inflated stepwise to perception and pain thresholds. At the perception threshold for balloon distension, the intensity of electrical stimulation was increased stepwise to perception threshold for electrical stimulation, and subsequently to 2-fold, 4-fold, and 8-fold perception threshold. EPs were recorded from the vertex (Cz) and forehead (Fz, reference) and compared between the increasing electrical stimulation intensities and between perception and pain thresholds for mechanical stimulation. RESULTS: The results show that amplitudes of EPs significantly increased with increasing electrical stimulation intensities, whereas latencies of EPs were not affected. Latencies and amplitudes of EPs were comparable at perception and pain thresholds for esophageal distension. CONCLUSIONS: The study suggests that repetitive electrical stimulation of the human esophagus is predominantly mediated via vagal A-delta fibers. The unaltered EPs at the perception and pain thresholds suggest that vagal A-delta fibers are not involved in mediation of pain. The findings support the view that painful and nonpainful stimuli are mediated via different visceral afferents between the esophagus and brain in humans. PMID- 8633591 TI - The incidence of protein C deficiency in thrombosis-related portal hypertension. AB - OBJECTIVE: To know the incidence of protein C deficiency associated with noncirrhotic, thrombosis-related portal hypertension. METHODS: Thirty-six patients were studied who had thrombosis-related portal hypertension diagnosed by means of hepatic venography or abdominal echocardiography or during abdominal surgery. Liver disease was excluded in 20 patients based on normal liver function tests and normal histology on liver biopsy. At the time of protein C assays, these patients were not receiving oral anticoagulation, and, in those recently diagnosed, the assays were performed more than 14 days after the last thrombotic event. Antigenic and functional assays for protein C were performed by ELISA and chromogenic assay, respectively. RESULTS: We found 11 patients with protein C deficiency who had a median age of 28 yr (range 19-55 yr) at time of diagnosis. Five patients had a history of systemic thromboembolism, and upper GI bleeding was the most frequent symptom related to portal hypertension (six cases). Antigenic protein C levels were measured in nine of the 11 patients (mean 31.88%, range 10-49%). Functional protein C level was assayed for all 11 patients (mean 40.90%, range 15-58%). After diagnosis, all patients received oral anticoagulants (ideally International Normalized Ratio: 2-3). CONCLUSION: We suggest that protein C screening should be performed in patients with thrombosis-related portal hypertension. PMID- 8633592 TI - Short segment Barrett's esophagus: clinical and histological features, associated endoscopic findings, and association with gastric intestinal metaplasia. AB - OBJECTIVES: To prospectively determine the clinical features, associated esophageal endoscopic lesions, associated gastric intestinal metaplasia, and prevalence of dysplasia and adenocarcinoma of short segment Barrett's. METHODS: All patients undergoing upper endoscopy over a 5-month period were scrutinized for endoscopic features suggestive of short segment Barrett's, and, if present, multiple biopsies were obtained from the suspicious areas. Prevalence of gastric intestinal metaplasia was determined by obtaining biopsies from the antrum, body, and cardia. RESULTS: Two hundred thirty seven patients were examined. Short segment Barrett's was suspected in only 42 patients, and traditional Barrett's was noted in 45 patients. Short segment Barrett's was confirmed by biopsy in 48%. Clinical presentation of short segment Barrett's was that of typical or complicated gastroesophageal reflux disease in 53%. A hiatal hernia was the most common associated esophageal endoscopic finding; however, none of the endoscopic findings differed significantly from findings of patients who did not have short segment Barrett's. Diagnosis of short segment Barrett's required histological analysis. A significant difference was noted in the prevalence of intestinal metaplasia between the esophagus and stomach in patients with Barrett's. No dysplasia or adenocarcinoma was detected in patients with short segment Barrett's. CONCLUSIONS: Short segment Barrett's is a frequent finding in patients undergoing upper endoscopy. All patients with short tongues or patches of red mucosa lying less than 2 cm above the esophagogastric junction should be biopsied to exclude short segment Barrett's. Large scale endoscopic and histological surveillance studies along with long-term follow-up are required to clarify short segment Barrett's prevalence and cancer risk. PMID- 8633594 TI - The galactose elimination capacity test: a study of the technique based on the analysis of 868 measurements. AB - OBJECTIVES: Our objective in this study was to analyze the correspondence of galactose concentration-on-time-decay curve to theoretical assumptions and the confidence limits of the determination of galactose elimination capacity. METHODS: We analyzed a retrospective series of 868 galactose elimination tests, performed on subjects with and without liver disease. Zero-order kinetics of galactose elimination was tested by comparison of the residual variance of linear regression with that obtained after quadratic transformation. The uncertainty in determination of galactose elimination capacity was calculated on the regression line by computing the 95% confidence limits of the estimate. RESULTS: The time course of galactose concentration suggested an initial uneven distribution, and the first (20-min) data point deviated significantly from the regression. The galactose decay curve in plasma rejected linearity in 13% of tests; after exclusion of the first data-point, linearity was rejected in only 3% of cases. The 95% confidence interval of galactose elimination capacity was on average +/- 16%, but in individual tests it was as large as +/- 60-80%. The uncertainty of the test was not affected by linearity. It was larger, with poor fitting of the experimental data on the regression of galactose concentration on time, low number of data points, and low galactose elimination. It was maintained within +/ 20% only when residual variance was > or = 2% of total variance (nearly 50% of tests). CONCLUSION: The methodology for the determination of galactose elimination capacity leads to considerable uncertainty as to the final result, which must be considered whenever the test is used for clinical purposes in the decision-making process. It tends to be larger in patients with advanced disease and can be accurately calculated so as to contribute to a proper evaluation of the test result. PMID- 8633593 TI - Characteristics of patients with benign gastric outlet obstruction requiring surgery after endoscopic balloon dilation. AB - OBJECTIVES: The aim of this study is to identify factors that will predict which patients with benign gastric outlet obstruction will not respond to endoscopic balloon dilation, in a long-term observation. METHODS: Over a 51-month period, 42 patients with benign gastric outlet obstruction received endoscopic balloon dilation. The "through-the-scope" technique with the aid of a guide wire was used for dilation. Fifteen factors were analyzed prospectively in 40 patients. Multivariate analysis was used to find the independent factors of the failure of treatment. RESULTS: The median follow-up period was 23 months (range 1-51 months). Twenty-eight (67%) patients achieved sustained improvement, and 14 (33%) patients underwent surgery. The overall symptom-free rates in 12, 24, 36, and 48 months were 85.3%, 78%, 68.8%, and 68.8%, respectively. The independent prognostic factor for failure of treatment was the need for more than two courses of endoscopic balloon dilation to relieve symptoms (odds ratio, 6.857; 95% confidence interval, 1,031-45,606). CONCLUSIONS: Endoscopic balloon dilation for the treatment of benign gastric outlet obstructions is an effective alternative to surgery. Patient who needs more than two courses of endoscopic balloon dilation to relieve symptoms should receive surgery. PMID- 8633595 TI - Pericardial effusion and left ventricular function in acute pancreatitis. AB - OBJECTIVES: To establish the prevalence of pericardial effusion and to evaluate left ventricular function in patients with acute pancreatitis. METHODS: Twenty one consecutive acute pancreatitis patients were studied. In 15 patients, the pancreatitis was of biliary origin and was attributable to other causes in the remaining six; eight patients had severe pancreatitis and 13 a mild disease. Using M-mode and B-mode echocardiography, pericardial effusion and left ventricular function were evaluated in all of the patients within 48 h of pain onset. All patients underwent an electrocardiogram and a standard chest x-ray. In 12 patients, the same parameters were evaluated 1 month after clinical recovery. Twenty healthy subjects, comparable for sex and age, have been studied as controls. RESULTS: Left ventricular function was similar in patients with acute pancreatitis and in healthy subjects; it was also similar both in patients with biliary and nonbiliary pancreatitis as well as in patients with severe pancreatitis and in those with mild disease. In the 12 patients in whom echocardiography was performed on hospital admission and one month after clinical recovery, no changes of left ventricular function were observed. Two patients with a mild form and one with a severe disease had pericardial effusion. One patient had negative T waves at electrocardiogram and an apical asynergy at echocardiography; these alterations disappeared after recovery. Chest x-ray revealed pleural effusion in seven patients with severe pancreatitis, whereas no alterations of cardiac silhouette were found. Acute abdominal fluid collections were also detected in seven of eight patients with severe pancreatitis, by contrast-enhanced computed tomography. CONCLUSIONS: The presence of pericardial effusion or left ventricular asynergy may be observed occasionally at echocardiography in acute pancreatitis patients; these findings, in contrast to the presence of pleural and abdominal effusions, seem to be unrelated to the severity of the disease. PMID- 8633597 TI - Alcohol consumption and coronary heart disease: good habits may be more important than just good wine. PMID- 8633596 TI - Alcohol consumption and myocardial infarction: a case-control study in France and Northern Ireland. AB - The effect of alcohol consumption was assessed in 561 men with myocardial infarction and 643 healthy controls recruited from France and Northern Ireland between 1988 and 1991 in the ECTIM Study (Enquete Cas-Temoins de I'Infarctus du Myocarde). In total, patients consumed less wine than did controls, while non wine-derived alcohol consumption did not differ significantly. After adjustment for cardiovascular risk factors and country of recruitment by logistic regression, alcohol consumption displayed a protective effect against myocardial infarction, the magnitude of which was comparable in both countries. This effect, which was essentially due to wine consumption in France and to nonwine consumption in Northern Ireland, was largely attenuated by the introduction of high density lipoprotein cholesterol into the model. Thus, both wine and nonwine consumption appear to exert a protective effect against myocardial infarction which is partly mediated through an increase in high density lipoprotein cholesterol. PMID- 8633598 TI - Sex difference in high density lipoprotein cholesterol in six countries. AB - It is known that women have higher levels of high density lipoprotein (HDL) cholesterol than men. The authors examined the association between HDL cholesterol and biologic sex in 8,631 women and 10,690 men aged 45-54 years from six countries studied between 1972 and 1989. The variation in the sex difference for HDL cholesterol was significant; the smallest difference (0.06 mmol/liter) was seen in China and the largest (0.40 mmol/liter) in Canada. Adjustment for differences in body mass index, smoking, alcohol use, and heart rate reduced but did not eliminate the variability. The sex difference in HDL cholesterol levels, usually assumed to be due to biologic factors, differs across cultures and may be related to environmental factors. PMID- 8633599 TI - White blood cell counts in persons aged 65 years or more from the Cardiovascular Health Study. Correlations with baseline clinical and demographic characteristics. AB - A higher white blood cell (WBC) count has been shown to be a risk factor for myocardial infarction and stroke in middle-aged populations. This study evaluated the relation between baseline WBC count and other risk factors, as well as subclinical and prevalent disease, in the Cardiovascular Health Study, an epidemiologic study of coronary heart disease and stroke in 5,201 persons aged 65 years or older. Baseline data were collected over a 12-month period in 1989-1990. WBC counts were statistically significantly higher in people with prevalent and subclinical atherosclerotic cardiovascular disease than in those who were free of disease. WBC counts correlated (p < 0.01) positively with coagulation factors, measures of glucose metabolism, creatinine, smoking, and triglycerides. In contrast, WBC counts correlated negatively with high density lipoprotein cholesterol, forced expiratory volume, forced vital capacity, and height. The correlations between WBC counts and risk factors were similar in both the entire cohort and the subgroup of persons who had never smoked. The authors conclude that WBC counts in the elderly are associated with prevalent and subclinical atherosclerotic cardiovascular disease, as well as its risk factors. PMID- 8633601 TI - Adult height and risk of breast cancer among white women in a case-control study. AB - Data from a hospital-based case-control study were analyzed to evaluate the relation of adult height to the risk of breast cancer among white women. The authors compared 5,358 newly diagnosed breast cancer cases and 4,555 controls interviewed from 1976 to 1992 in hospitals located mainly in the United States. Overall, there was no association between stature and risk of breast cancer. In comparison with women whose heights were less than 62 inches (< 158 cm), the adjusted odds ratios were 1.1 (95% confidence interval (Cl) 0.9-1.2), 1.0 (95% Cl 0.9-1.2), 1.0 (95% Cl 0.9-1.1), and 1.0 (95% Cl 0.8-1.2) for women with heights of 62-63, 64-65, 66-67, and > or = 68 inches (equivalent to 158-160, 163-165, 168 170, and > or =173 cm), respectively. There was no consistent evidence of modification of the effect of height by other risk factors. The results suggest that adult stature in white women is not related to the risk of breast cancer. PMID- 8633600 TI - Brain tumor risk in children in relation to use of electric blankets and water bed heaters. Results from the United States West Coast Childhood Brain Tumor Study. AB - The possible relation between the occurrence of brain tumors in children and exposure to electric blankets or electrically heated water beds was investigated in a multicenter, population-based case-control study conducted on the West Coast of the United States. Information on maternal exposure during pregnancy or direct exposure to the subject child was collected by in-person interview from the mothers of 540 case children and 801 control children. Cases were 19 years of age or younger and were diagnosed between 1984 and 1991. Controls were recruited using a random digit dialing procedure. The risk of brain tumor occurrence from in utero exposure to either electric blankets (odds ratio (OR) = 0.9, 95% confidence interval (Cl) 0.6-1.2) or heated water beds (OR = 0.9, 95% Cl 0.6-1.3) was not elevated. Brain cancer risk did not vary by use in any trimester of pregnancy, and children with mothers who reported use throughout their pregnancy had no increased risk. Similar results were observed for exposure to the child, in that no association between brain cancer and use of electric blankets (OR = 1.0, 95% Cl 0.6-1.7) or heated water beds (OR = 1.2, 95% Cl 0.7-2.0) was observed. Risks did not vary significantly by age, sex, race, socioeconomic status, or histologic category for either in utero exposure or child's exposure. This study provides no evidence to support the hypothesis that there is a relation between brain cancer occurrence in children and 50-/60-Hz magnetic field exposure from the use of electric blankets and heated water beds. PMID- 8633602 TI - Falls in the elderly: a prospective study of risk factors and risk profiles. AB - In this prospective study, the authors determined intrinsic risk factors for falls and recurrent falls and constructed a risk profile that indicated the relative contribution of each risk factor and also estimated the probabilities of falls and recurrent falls. In 1992, over a 28-week period, falls were recorded among 354 elderly subjects aged 70 years or over who were living in homes or apartments for the elderly in Amsterdam and the vicinity. During the study period, 251 falls were reported by 126 subjects (36%), and recurrent falls (> or =2 falls) were reported by 57 subjects (16%). Associations of falls and recurrent falls with potential risk factors were identified in logistic regression models. Mobility impairment regarding one or more of the tested items (i.e., impairment of balance, leg-extension strength, and gait) was associated with falls (adjusted odds ratio (OR) =2.6) and was strongly associated with recurrent falls (OR = 5.0). Dizziness upon standing was associated with falls (OR = 2.1) and recurrent falls (OR = 2.1). However, several risk factors were associated with recurrent falls only: history of stroke (OR = 3.4), poor mental state (OR = 2.4), and postural hypotension (OR = 2.0). The authors constructed a risk profile for recurrent falls that included the five risk factors mentioned above. Inclusion of all risk factors in the profile implied an 84% probability of recurrent falls over a period of 28 weeks, compared with 3% when no risk factor was present. The probability of recurrent falls ranged only from 11% to 29% when predicted by number of falls occurring in the previous year. Physical activity, use of high risk medication, and the use of vitamin D3, which was randomly allocated to the participants, were not strongly related to either falls or recurrent falls. In conclusion, a large range of probabilities of falls, especially of recurrent falls, was estimated by the risk profiles, in which mobility impairment was the major risk factor. Recurrent fallers may therefore be especially amenable to prevention based on mobility improvement. PMID- 8633603 TI - Negative effect of a short interpregnancy interval on birth weight following loss of an infant to sudden infant death syndrome. AB - It was hypothesized that a short interpregnancy interval immediately following the birth of an infant that had succumbed to sudden infant death syndrome (SIDS) (and no other cause of death) would be associated with a reduced mean birth weight in the next infant. Mothers who had given birth to two children in the state of Oregon between 1975 and 1984 and whose first child had died in infancy from either SIDS (n = 84) or some other cause (n = 305) were identified from vital records. A multiple regression analysis in which adjustment was made for possible confounding variables (including the birth weight of the deceased child) was conducted. When the firstborn child had succumbed to SIDS, the mean birth weight of the next baby was 314 g (2,978 g vs. 3,292 g, p = 0.04) lower when the interpregnancy interval was less than 6 months versus greater than 6 months. In contrast, a less-than-6-month interval had a slightly positive effect (60 g) on the mean birth weight of the next baby when the firstborn child had died due to a cause other than SIDS. These results suggest that parents who have lost a child to SIDS may wish to delay a new pregnancy for at least 6 months. PMID- 8633604 TI - Effect of not breastfeeding on the risk of diarrheal and respiratory mortality in children under 2 years of age in Metro Cebu, The Philippines. AB - The effects of not breastfeeding on mortality due to diarrhea and acute lower respiratory infection (ALRI) in children under 2 years of age were examined using data from a 1988-1991 longitudinal study of 9,942 children in Metro Cebu, The Philippines. Cox regression methods were used to study the magnitude of the risks, possible interactions with birth weight and nutritional status, and the effect of additional confounding factors. Not breastfeeding had a greater effect on diarrheal mortality than on ALRI mortality. In the first 6 months of life, failing to initiate breastfeeding or ceasing to breastfeed resulted in an 8- to 10-fold increase in the rate of diarrheal mortality. The rate of mortality associated with both ALRI and diarrhea was increased nearly six times by not breastfeeding, but the rate of ALRI mortality alone was not increased. The data also suggested that the risk of mortality associated with not breastfeeding was greater for low birth weight infants and infants whose mothers had little formal education. After age 6 months, the protective effects of breastfeeding dropped dramatically. These findings underscore the importance of promoting breastfeeding, especially during the first 6 months of life, and of targeting high risk groups such as low birth weight babies and those of low socioeconomic status. PMID- 8633605 TI - Public health impact of various risk factors for acute otitis media in northern Finland. AB - The aim of this study was to assess the excess risk attributable to alterable risk factors for acute otitis media in Finnish children, including day care attendance, parental smoking, and a short duration of breastfeeding. Data on a population-based cohort of 2,512 children were gathered from medical records and questionnaires from 1985 to 1988. Excess (attributable) fractions for the risk factors were calculated among 825 children (target population) followed for 2 years, from a dynamic logistic model fitted to the entire cohort (estimation data). In theory, one child out of every five affected in the exposed population would have escaped otitis media completely if he/she had been moved from nursery day care to home care, and two out of every five affected could have escaped recurrent episodes in this way. The corresponding figures for family day care were lower: one and two children out of every six affected, respectively. Cessation of parental smoking and breastfeeding would have smaller effects. The impacts were more modest in the whole population. Nevertheless, approximately 14% of all of the otitis media episodes would have been avoided if all of the children had been cared for at home. These figures are hypothetical, since it is unlikely that use of day care outside the home can be avoided altogether, but they offer further evidence of the notable role of day care attendance as a risk factor for otitis media. PMID- 8633606 TI - Seroprevalence of immunoglobulin G, M, and A antibodies to Helicobacter pylori in an unselected Danish population. AB - The seroprevalences of increased levels of immunoglobulin G (lgG), M (lgM), and A (lgA) antibodies to Helicobacter pylori were assessed by enzyme-linked immunosorbent assay techniques in 3,589 Danes who participated in a population study in Copenhagen County in 1982. A total of 33.9% of the study population had one or more classes of increased antibodies to H. pylori. Increased levels of lgG, lgM, and lgA antibodies to H. pylori were seen in 25.9% (95% confidence interval (Cl) 24.5-27.3), 4.5% (95% Cl 2.2-7.0), and 12.0% (95% Cl 10.9-13.1) of the participants, respectively. Women were significantly more likely than men to be seropositive for lgM antibodies (Mantel-Haenszel summary odds ratio = 1.85, 95% Cl 1.34-2.57). Seropositivity for lgM antibodies to H. pylori was found less often with increasing age. An lgG antibody response was not seen in 23.7% of cases with overall increased antibodies to H. pylori. Increased levels of lgG or lgA antibodies were more frequent in people with a history of peptic ulcer disease. Seroprevalences of increased H. pylori antibodies are high in unselected populations. Primary H. pylori infections are contracted at all ages, but infection rates decline with age. Inclusion of measurements of lgA and lgM antibody levels in future screening for H. pylori may improve the diagnostic sensitivity of serologic analyses. PMID- 8633608 TI - Re: "Incidence of thyroid cancer in New Caledonia, South Pacific, during 1985 1992". PMID- 8633607 TI - Case series analysis of adverse reactions to vaccines: a comparative evaluation. AB - A modified cohort method has been proposed for estimating the relative incidence of rare adverse reactions after vaccination. The method requires only a sample of the cases, thus avoiding the need for following large population cohorts or selecting controls. This case series method has statistical power equivalent to that of the full cohort method when the risk periods after vaccination are short and vaccine coverage is high. The method also eliminates confounding by variables associated with both the outcome and avoidance of vaccination. In this paper, the cohort, case-control, and case series methods are reviewed, and their underlying assumptions and performances are compared. Theoretical results are illustrated using data on febrile convulsions after measles-mumps-rubella vaccination in the United Kingdom. PMID- 8633609 TI - Re: "Validation studies using an alloyed gold standard". PMID- 8633610 TI - Re: "Association of education with reported age of onset and severity of Alzheimer's disease at presentation: implications for the use of clinical samples". PMID- 8633611 TI - Re: "Mortality following conjugal bereavement and the effects of a shared environment". PMID- 8633612 TI - Re: "No association between serum ferritin and asymptomatic carotid atherosclerosis. The Atherosclerosis Risk in Communities (ARIC) Study". PMID- 8633613 TI - Re: "Public drinking water contamination and birth outcomes". PMID- 8633614 TI - Contraception, unintended pregnancies, and sexually transmitted diseases: why isn't a simple solution possible? PMID- 8633615 TI - Case-control studies of screening efficacy: the use of persons newly diagnosed with cancer who later sustain an unfavorable outcome. PMID- 8633617 TI - Relation of maternal race to the risk of preterm, non-low birth weight infants: a population study. AB - The authors used 1982-1983 Illinois vital records and 1980 US Census income data to determine the contribution of maternal race to the risk of preterm (< 260 days), non-low birth weight (> 2,500 g) infants. This older cohort was chosen to avoid the confounding effect of cocaine associated with its increased local availability after 1985. In Chicago, the unadjusted preterm, non-low birth weight rate was 14% for African Americans (n = 43,059) compared with 9% for Mexican Americans (n = 10,397) and 7% for whites (n = 26,152) (odds ratio = 1.7, 95% confidence interval 1.6-1.8; odds ratio = 2.4, 95% confidence interval 2.3-2.5, respectively). In logistic models that included maternal sociodemographic and prenatal care variables, the adjusted odds ratio of preterm, non-low birth weight for. African Americans compared with Mexican Americans and whites was 1.6 (95% confidence interval 1.4-1.8) and 1.5 (95% confidence interval 1.2-1.7), respectively. The authors conclude that some factor closely linked to the African American race, not underserved minority status per se, is a fundamental cause of preterm, non-low birth weight. PMID- 8633616 TI - Genetic and environmental influences on insulin levels and the insulin resistance syndrome: an analysis of women twins. AB - Multiple factors may determine insulin resistance and the insulin resistance syndrome. The contributions of genes and environment to the distribution of fasting insulin levels and to the associations of fasting insulin with elements of the syndrome were evaluated in the second examination of the Kaiser Permanente Women Twins Study (Oakland, California, 1989-1990). Subjects included 556 white women (165 monozygous twin pairs, 113 dizygous pairs; 455 women with normal glucose tolerance, 75 with impaired glucose tolerance, and 26 with non-insulin dependent diabetes by World Health Organization criteria). The intraclass correlation coefficients for log fasting insulin for monozygous and dizygous twin pairs were 0.64 and 0.40, respectively. After adjustment for age, behavioral factors, and body mass index, the estimated classic heritability was 0.53 (p = 0.003). Commingling analysis of fasting insulin indicated the presence of four distributions (p < 0.001), consistent with at least one, and perhaps two, genes influencing this trait. In an unmatched multiple regression model among women from monozygous twin pairs only, log fasting insulin was independently associated with body mass index (p < 0.0001), waist/hip ratio (p = 0.02), and glucose intolerance (p = 0.04), but not with triglycerides, high density lipoprotein cholesterol, or hypertension. After removal of genetic influences by analysis of monozygous intrapair differences, only body mass index (p < 0.0001) remained independently related to fasting insulin. The authors conclude that, in addition to significant genetic influences on fasting insulin, environmental or behavioral factors (particularly nongenetic variation in obesity) are important determinants of fasting insulin and the insulin resistance syndrome. PMID- 8633618 TI - Sex and time trends in cardiovascular disease incidence and mortality: the Framingham Heart Study, 1950-1989. AB - Variations in cardiovascular disease mortality between sexes, over time, and across regions point to population differences in the biologic, behavioral, and environmental factors influencing cardiovascular health. The authors examined 20 year trends in risk factors, incidence, and mortality among women and men in Framingham, Massachusetts, who were members of the Framingham Heart Study and aged 50-59 years in 1950, 1960, and 1970. The incidence declined 21% between the female cohorts (p < 0.01 for trend) with the greatest decline occurring between the 1950 and 1960 cohorts. The 20-year incidence declined only 6% between the male cohorts despite an 18% decline (p < 0.05 for trend) during the first 10 years of follow-up. Cardiovascular disease mortality declined 59% between the female cohorts and 53% between the male cohorts (both p < 0.001 for trend). The largest mortality declines occurred between the 1950 and 1960 female cohorts during the second 10 years of follow-up and between the 1960 and 1970 male cohorts during both follow-up periods. Obesity, hypercholesterolemia, and high blood pressure were significantly lower at baseline and 10 years later in the 1970 female cohort compared with the 1950 cohort (all p < 0.001). Smoking and high blood pressure were significantly lower at baseline and 10 years later in the 1970 male cohort compared with the 1950 cohort (both p < 0.001). More than half of the 51% decline in coronary heart disease mortality observed in women between 1950 and 1989 and one third to one half of the 44% decline observed in men could be attributed to improvements in risk factors in the 1970 cohorts. PMID- 8633620 TI - Occupational risk factors for prostate cancer: results from a case-control study in Montreal, Quebec, Canada. AB - A population-based case-control study of cancer and occupation was carried out in Montreal, Canada. Between 1979 and 1986, 449 pathologically confirmed cases of prostate cancer were interviewed, as well as 1,550 cancer controls and 533 population controls. Job histories were evaluated by a team of chemist/hygienists using a checklist of 294 workplace chemicals. After preliminary evaluation, 17 occupations, 11 industries, and 27 substances were selected for multivariate logistic regression analyses to estimate the odds ratio between each occupational circumstance and prostate cancer with control for potential confounders. There was moderate support for risk due to the following occupations: electrical power workers, water transport workers, aircraft fabricators, metal product fabricators, structural metal erectors, and railway transport workers. The following substances exhibited moderately strong associations: metallic dust, liquid fuel combustion products, lubricating oils and greases, and polyaromatic hydrocarbons from coal. While the population attributable risk, estimated at between 12% and 21% for these occupational exposures, may be an overestimate due to our method of analysis, even if the true attributable fraction were in the range of 5-10%, this represents an important public health issue. PMID- 8633619 TI - Change and secular trends in physical activity patterns in young adults: a seven year longitudinal follow-up in the Coronary Artery Risk Development in Young Adults Study (CARDIA). AB - Levels and changes in self-reported physical activity over a 7-year period were examined to determine tracking and to estimate the proportion of total cohort change attributable to secular trends. A population-based sample of 2,328 men and 2,787 women aged 18-30 years at baseline (52% black and 48% white) from Birmingham, Alabama, Chicago, Illinois, Minneapolis, Minnesota, and Oakland, California, were examined four times between 1985-1986 and 1992-1993. The intraclass correlation for up to four measures was 0.57 for the entire sample, varying between 0.57 for white men and 0.42 for black women, indicating a moderate tendency for tracking. The energy expenditure in physical activity at each examination was greatest in black men and, compared with black men, about 5% less in white men, 30% less in white women, and 50% less in black women. The total cohort decrease in mean physical activity was approximately 30% in each race-sex group. The secular trend accounted for 38% of the total cohort change in black men, 43% in black women, 52% in white men, and 81% in white women. Physical activity declined sharply during the early years of adulthood, partly because of secular trend. Young adults are therefore an important target group for physical activity promotion programs to reverse individual and populationwide declines prior to middle age. PMID- 8633621 TI - Risk factors for non-Hodgkin's lymphomas in acquired immunodeficiency syndrome (AIDS) AB - The possibility that an agent in addition to the human immunodeficiency virus (HIV) may contribute to the etiology of non-Hodgkin's lymphoma in persons with acquired immunodeficiency syndrome (AIDS) was studied using participants from the Multicenter AIDS Cohort Study (MACS) of homosexual and bisexual men enrolled in 1984-1985 and also in 1987-1991. A nested case-control analysis was conducted. The primary source of information on potential exposures and characteristics of the participants was the baseline study entry interview that was conducted prior to the development of AIDS. A total of 84 cases of non-Hodgkin's lymphoma were identified and compared with 527 participants who developed AIDS but had no evidence of cancer. The groups were similar for most sociodemographic characteristics as well as sexual activity and past history of antecedent illnesses. Although the non-Hodgkin's lymphoma cases reported less frequent use of recreational drugs and cigarettes compared with other persons with AIDS, these differences were not significant. Non-Hodgkin's lymphoma cases reported more frequent intake of aspirin during the week before the interview. However, there were no differences between the comparison groups for long-term aspirin intake or intake of other analgesics. The absence of any specific and strong association between non-Hodgkin's lymphoma and the various behavior-related activities and exposures considered in this analysis suggests that these factors are not related to a second agent in the etiology of HIV-induced non-Hodgkin's lymphoma. The possibility that a very common agent in this study population or that differences in the nature of the immune dysfunction resulting from HIV infection could act as a cofactor for HIV-induced non-Hodgkin's lymphoma cannot be excluded. PMID- 8633622 TI - Predictors of mortality in the Amsterdam cohort of human immunodeficiency virus (HIV)-positive and HIV-negative drug users. AB - The impact of human immunodeficiency virus (HIV) infection and other risk factors on mortality was studied in a cohort of Dutch injection drug users and drug users who did not inject. Participants were recruited between 1985 and 1992 and followed up through 1993. Vital status was ascertained through repeat visit information, supplemented by population register data. A total of 77 deaths were recorded among 632 drug users, for a mortality rate per 1,000 person-years of 7 for HIV-negative noninjection drug users, 18 for HIV-negative injection drug users, and 64 for HIV-positive injection drug users. In multivariate analyses, limited to injection drug users, a positive HIV serostatus, age above 40 years, and using benzodiazepines several times daily were significantly associated with an elevated risk of death, both for death from all causes and for death preceding acquired immunodeficiency syndrome (AIDS) diagnosis (pre-AIDS). For pre-AIDS death, the adjusted relative risk associated with HIV infection was 2.2 (95% confidence interval 1.3-3.7). Only 38% of HIV-infected injection drug users who died were diagnosed with AIDS. However, 76% of HIV-infected injection drug users who died without AIDS diagnosis had evidence of immunosuppression (CD4 count < 500/microliters). Daily use of methadone and participation in needle and syringe exchange schemes were not associated with lower mortality rates. This study illustrates in a group of injection drug users with a 30% HIV seroprevalence and a high background mortality the profound influence on mortality that HIV infection has gained. PMID- 8633623 TI - Use of semiquantitative food frequency questionnaires to estimate the distribution of usual intake. AB - The authors consider whether semiquantitative food frequency questionnaires can be used to survey a population to estimate the distribution of usual intake. They take as an assumption that, if they were possible to obtain, the mean of many food records or recalls would be an accurate representation of an individual's usual diet. They then assume that nutrient intake as measured by a questionnaire follows a linear regression model when regressed against the usual intake of that nutrient. If the coefficients in this regression relation were known, then the distribution of usual intake could be constructed from the responses to the questionnaire. Since one generally does not know the values of the coefficients, they need to be estimated from a calibration study in which respondents complete the questionnaire together with multiple food records or recalls. This can be done either through an internal subset of the data or through an independent external study. With an internal substudy, the authors find that food frequency questionnaires typically provide little information about the distribution of usual intake in addition to that obtained from the multiple records or recalls in the substudy. When the substudy is external, if it is small then having very large numbers of subjects completing food frequency questionnaires in the survey is no more efficient than having a few subjects completing food records or recalls. However, if the external substudy is large and accurately characterizes the relation between the questionnaire response and usual intake, food frequency questionnaires can provide a cost-efficient way of estimating the distribution of usual intake. These results do not apply to the different problem of correcting relative risks for the effects of measurement error. PMID- 8633624 TI - Re: "Hypothesis: low serum cholesterol, suicide, and interleukin-2". PMID- 8633625 TI - Re: "Meta-analysis of Pap test accuracy". PMID- 8633626 TI - Ultrasonographic measurement of amniotic fluid volume in normal diamniotic twin pregnancies. AB - OBJECTIVE: Our purpose was to determine the amniotic fluid volume in normal diamniotic twins. STUDY DESIGN: The single amniotic fluid index for both twin members, the maximum depth and width of each twin's largest pocket, were measured every 4 to 6 weeks between 15 and 40 weeks in 91 normal diamniotic twin gestations. The two-diameter pocket in each twin was determined as its largest pocket's depth multiplied by its width. Normal twin gestations were defined as those with <20% birth weight discordance, appropriate for gestational age, no fetal anomalies, delivery at > or = 37 weeks, and normal newborns. Amniotic fluid index values, depths, and two-diameter pockets were stratified into 2-week intervals and transformed into base 10 logarithms because of their nongaussian distributions. The correlations of these measurement with gestational age were evaluated. Their means and 90%, 95%, and 98% confidence intervals were determined. RESULTS: The amniotic fluid index changed significantly with gestational age. However, depths and two-diameter pockets did not. The amniotic fluid index rose from 15 to 24 weeks, plateaued until 36 weeks, and then declined. The 90%, 95%, and 98% confidence intervals for each twin's depth were 2.4 to 7.9, 2.1 to 8.8, and 1.9 to 10 cm, respectively (mean 4.3 cm). These respective confidence intervals for each twin's two-diameter pocket were 8 to 44, 7 to 52, and 5 to 63 cm2 (mean 19 cm2). The amniotic fluid index confidence interval curves were plotted from the log (amniotic fluid index)=0.8276 + 0.01675x-0.0000001900x4, R2=0.78, p=0.002 (where x is gestational age). CONCLUSION: The amniotic fluid volume in normal diamniotic twin pregnancies was established ultrasonographically. Only the amniotic fluid index changed significantly with gestational age. These findings may have significance in the clinical management of twin gestation. PMID- 8633627 TI - Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia. AB - OBJECTIVE: Nitric oxide is a potent vasorelaxant produced by endothelial cells. We tested the hypothesis that urinary and perhaps plasma nitric oxide metabolites would be reduced in women with preeclampsia. STUDY DESIGN: Plasma and urine from 14 women meeting strict clinical criteria for the diagnosis of preeclampsia and 20 normal nulliparous women were assayed for the stable metabolites of nitric oxide, nitrate and nitrite. RESULT: There was no significant difference of plasma concentrations of nitrate and nitrite between women with preeclampsia and women with normal pregnancies (32.7 +/- 3.1 vs 25.8 +/- 2.4 micromol/L). Plasma creatinine levels were elevated in women with preeclampsia (0.85 +/- 0.09 vs 0.66 +/- 0.02 mg/dl, p<0.01), indicating a reduced glomerular filtration rate. Urine concentrations of nitrate and nitrite normalized by creatinine excretion were significantly lower in women with preeclampsia compared with normal pregnant women (0.37 +/- 0.06 vs 0.69 +/- 0.11 micromol of nitrite per milligram creatinine, p. <0.05). CONCLUSIONS: Our study using concomitant measurement of plasma and urine nitrate and nitrite suggests a reduced production of nitric oxide in women with preeclampsia compared with normal pregnant women. PMID- 8633628 TI - Antiplatelet antibody testing in thrombocytopenic pregnant women. AB - OBJECTIVE: The purpose of the study was to attempt to distinguish pregnant women with gestational thrombocytopenia from those with idiopathic immune thrombocytopenia by eight different platelet antibody assays. STUDY DESIGN: Sera from pregnant women with presumed gestational thrombocytopenia (n = 160) and idiopathic immune thrombocytopenia (n=90) were prospectively tested for indirect and platelet-associated immunoglobulins G and M and complement C3, as well as for serotonin release. After the results were analyzed, a subset of patients were subsequently analyzed for circulating antiplatelet antibody directed against platelet membrane glycoprotein GPIIb/IIIa. RESULTS: Indirect immunoglobulin G was significantly greater in the 85 women with idiopathic immune thrombocytopenia than in the 129 women with gestational thrombocytopenia (p<0.001). Platelet associated immunoglobulin G was elevated in the majority of women, both those with gestational thrombocytopenia and those with idiopathic immune thrombocytopenia. There were also no statistically significant difference in the values for platelet-associated C3 or indirect immunoglobulin M and C3. Levels of platelet-associated immunoglobulin M showed a tendency to be higher in women with gestational thrombocytopenia (p=0.04), as did the values in the serotonin release assay (p=0.06). CONCLUSION: Our data demonstrate that patients with gestational thrombocytopenia had surprisingly high levels of platelet-associated immunoglobulin despite mild thrombocytopenia. Comparison of a relatively large number of patients with idiopathic immune thrombocytopenia and gestational thrombocytopenia indicates that women with idiopathic immune thrombocytopenia cannot be distinguished from those with gestational thrombocytopenia by means of one or more of the prototypic platelet antiglobulin tests currently in use. Our preliminary data with glycoprotein-specific assays indicate that they may be more useful. PMID- 8633629 TI - Systemic vascular resistance index determined by thoracic electrical bioimpedance predicts the risk for maternal hypotension during regional anesthesia for cesarean delivery. AB - OBJECTIVE: Our purpose was to evaluate the predictive value of the baseline systemic vascular resistance index for the development of maternal hypotension during regional anesthesia for cesarean delivery. STUDY DESIGN: Patients receiving a standardized spinal or epidural anesthetic for nonemergency cesarean delivery were studied prospectively. Hemodynamic data were obtained noninvasively with an NCCOM-3 cardiac output monitor (Bomed Medical Manufacturing, Irvine, Calif.), which uses thoracic electrical bioimpedance to estimate stroke volume and cardiac output. Measurements obtained were indexed to body surface area. The systemic vascular resistance index was calculated from mean arterial pressure and thoracic electrical bioimpedance-derived cardiac index. Hemodynamic data obtained were analyzed to identify statistically significant predictors of maternal hypotension. RESULTS: Maternal hypotension occurred in 24 of 42 (57%) patients studied. The incidence of hypotension did not differ between the types of anesthesia: spinal 17 of 274 (62%) versus epidural 7 of 15 (47%, p=0.48). The mean interval to the onset of hypotension was 12.2 minutes (SD 2.2 minutes, range 2 to 24 minutes). Mean (SD) baseline maternal systolic blood pressure was higher in patients who had hypotension (145 torr [4]) than those who did not (129 torr [4], p=0.01). The mean (SD) baseline systemic vascular resistance index was higher in patients who had hypotension (633 [SD 36] dyne . cm . sec-5/m2) than those who did not (454 [SD 29] dyne . cm . sec-5/m2; p =0.001). With receiver operator characteristic curves, a baseline systemic vascular resistance index of 500 had a sensitivity of 83%, a specificity of 78%, a positive predictive value of 83%, and a negative predictive value of 78% for maternal hypotension (odds ratio 17.5, 95% confidence interval 3.1 to 109.4). A baseline systolic blood pressure of 140 torr had a sensitivity and specificity of 42% and 72%, respectively (odds ratio 1.9, 95% confidence interval 0.4 to 8.8). CONCLUSIONS: Baseline systemic vascular resistance index obtained by noninvasive cardiac output monitoring with thoracic electrical bioimpedance and systolic blood pressure are useful to predict the risk for maternal hypotension with regional anesthesia. Patients with increased baseline systemic vascular resistance index or systolic blood pressure are at increased risk for hypotension. PMID- 8633630 TI - Cocaine's effect on plasma oxytocin concentrations in the baboon during late pregnancy. AB - OBJECTIVE: The hypothesis for this investigation was that intravenous cocaine would result in an elevation of maternal plasma oxytocin levels in the baboon during late pregnancy. STUDY DESIGN: Five gravid chronically instrumented baboons had timed arterial blood samples obtained before and after an intravenous bolus cocaine infusion at least 5 days after surgery. Plasma oxytocin concentrations were measured by specific radioimmunoassays, and baseline samples were compared with postcocaine samples. RESULTS: The plasma oxytocin concentrations were significantly elevated at all sampling times after the 1.0 mg/kg cocaine dose (p<0.05). CONCLUSION: The increased oxytocin concentration after cocaine use may be responsible for the increased incidence of preterm labor. PMID- 8633631 TI - Myocardial and cerebral oxygen delivery are not adversely affected by cocaine administration to early-gestation fetal sheep. AB - OBJECTIVES: Cocaine administration to near-term pregnant sheep causes fetal hypoxemia, but oxygen delivery to the heart and brain are preserved because of increased blood flow. We hypothesized that cocaine administration during earlier fetal gestation impairs oxygen delivery to the heart and brain. STUDY DESIGN: Ten pregnant ewes and fetuses at 0.7 term gestation underwent surgical instrumentation. After 48 hours of recovery fetal blood pressure, heart rate, cerebral and myocardial blood flow, and arterial oxygen content were determined before and during cocaine administration to the ewe. RESULTS: Fetal hypoxemia was not noted in these animals. Fetal myocardial blood flow increased from 220 +/- 100 ml per 100 gm per minute to 349 +/- 183 ml per 100 gm per minute (p=0.03), and oxygen delivery increased from 16 +/- 5 ml of oxygen per 100 gm per minute to 22 +/- ml of oxygen per 100 gm per minute (p=0.02). Fetal cerebral blood flow and oxygen delivery remained unchanged. CONCLUSION: Cerebral and myocardial oxygen delivery are unimpeded by maternal cocaine administration in 0.7 term gestation ovine fetuses. PMID- 8633632 TI - The relationship between pregnancy and sexual risk taking. AB - OBJECTIVES: We attempted to determine whether risks of acquiring sexually transmitted diseases differ between pregnant and nonpregnant women. STUDY DESIGN: Women attending clinics in Brooklyn (332 pregnant and 1069 nonpregnant) were interviewed and tested for Trichomonas vaginalis and Chlamydia trachomatis. Independent-sample t tests were conducted via SPSSX (SPSS Inc., Chicago) to assess differences in risk behavior across pregnancy status. RESULTS: In the pregnant sample 17.2% had positive test results for chlamydia and 23.4% had T vaginalis. In the nonpregnant women the rates were 10.9% and 17.7%, respectively (p<0.01). Pregnant respondents used condoms less consistently than nonpregnant women (p<0.01). Although nonpregnant women reported a higher frequency of sexual activity and more sexual partners in the previous month, the strength of those relationships was weak. CONCLUSION: We have found that pregnancy does not represent a time of reduced sexual risks. The differences in self-reported risk, with the exception of consistency of condom use, all showed very weak indexes of strength. Providers of obstetric services should incorporate "safer sex" messages into routine prenatal care. PMID- 8633633 TI - Receiver-operator characteristic curves for the ultrasonographic prediction of small-for-gestational-age fetuses in low-risk pregnancies. AB - OBJECTIVE: Our purpose was to assess the accuracy of third-trimester ultrasonographic biometry in the diagnosis of small-for-gestational-age fetuses in a low-risk obstetric population. STUDY DESIGN: A total of 1000 low-risk pregnancies were prospectively examined at 31 weeks' gestation. The diagnostic accuracy of the fetal abdominal circumference and estimated fetal weight according to 24 formulas in the literature were evaluated by the use of receiver operator characteristic curves. RESULTS: The incidence of a birth weight <10th percentile was 8.2%. The diagnostic accuracies of abdominal circumference and estimated fetal weight were remarkably similar. None of the 24 formulas performed significantly better than the measurement of the abdominal circumference. At a specificity of 90%, 46% of infants with a birth weight <10th percentile and five of six cases with adverse perinatal outcomes were predicted. CONCLUSION: In a low risk obstetric population third-trimester ultrasonographic biometry has limited value in predicting small-for-gestational-age fetuses, and estimation of fetal weight does not carry an advantage over measurement of the abdominal circumference. The optimal cutoff value remains uncertain. However, by accepting a 10% false-positive rate, half of small-for-gestational-age fetuses and most perinatal complications could be recognized. PMID- 8633634 TI - Effects of estrogen on nitric oxide biosynthesis and vasorelaxant activity in sheep uterine and renal arteries in vitro. AB - OBJECTIVES: Our purpose was to determine whether estrogen alters the relaxation responses to bradykinin and superoxide dismutase of the uterine and renal arteries and to determine the role of nitric oxide in that response. STUDY DESIGN: Ten nulliparous, ovariectomized nonpregnant sheep received either estradiol-17beta or vehicle solution. In vitro studies evaluating vasorelaxation were done with either bradykinin or superoxide dismutase. The nitric oxide inhibitor nomega-nitro-L-arginine methyl ester was used to determine the role of nitric oxide in this process. Nitric oxide synthase activity was assessed by measuring citrulline generation. RESULTS: We found a dose dependency of relaxation to bradykinin and superoxide dismutase. Estrogen enhanced this response in uterine arteries. Estrogen increased citrulline generation in uterine but not renal arteries. Nomega-nitro-L-arginine methyl ester blocked relaxation responses and citrulline generation in both arteries. CONCLUSION: In nonpregnant sheep we found that nitric oxide release and nitric oxide synthase activity is enhanced by estrogen in the uterine arteries but not in the renal arteries. Increases in nitric oxide synthase activity may be important in the hyperemic response of the uterus during estrus. PMID- 8633635 TI - Daily and hourly temporal association between delta4-androstenedione-induced preterm myometrial contractions and maternal plasma estradiol and oxytocin concentrations in the 0.8 gestation rhesus monkey. AB - OBJECTIVE: Our purpose was to investigate the temporal relationship between delta4-androstenedione-induced preterm switching of myometrial activity patterns from contractures to contractions and maternal plasma estradiol and oxytocin concentrations in the 0.8 gestation rhesus monkey. STUDY DESIGN: Eight rhesus monkeys (132 to 136 days' gestation) were instrumented under halothane with femoral artery and vein catheters and uterine electromyogram electrodes. At 138 to 142 days' gestation baseline maternal femoral artery blood samples for estradiol and oxytocin measurement were taken at 30-minute intervals for 7 hours, starting 2 hours before the onset of darkness. The day after baseline sampling a continuous intravenous delta4-androstenedione infusion (0.3 mg . kg-1 .hr-1 in 10% intralipid at 0.25 ml . hr-1) was started in four monkeys, while four monkeys were infused intravenously with intralipid alone. The sampling regimen was then repeated at 1 and 3 days after the start of the delta4-androstenedione or intralipid infusion. Contractions were counted and estradiol and oxytocin were measured by radioimmunoassay. RESULTS: Androstenedione promoted a premature nocturnal increase in myometrial contractions in conjunction with an increase in maternal plasma concentrations of estradiol and oxytocin, which were of similar magnitude to those measured in spontaneous term labor. The increase in maternal estradiol preceded the increase in maternal oxytocin levels and myometrial contractions. The onset of the increase in maternal plasma oxytocin was closely associated with the appearance of myometrial contractions after delta4 androstenedione treatment. In contrast, no sustained premature contractions or changes in estradiol and oxytocin occurred in intralipid-treated monkeys. CONCLUSIONS: We conclude that in the 0.8 gestation rhesus monkey (1) the increase in maternal plasma estradiol precedes the increase in maternal plasma oxytocin after delta4-androstenedione treatment and (2) delta4-androstenedione-induced preterm myometrial contractions are closely associated in time with physiologic increases in maternal plasma oxytocin concentrations. PMID- 8633636 TI - The calcium-dependent nitric oxide production of human vascular endothelial cells in preeclampsia. AB - OBJECTIVE: Nitric oxide is an important vasodilator, and in this study we studied whether the calcium-dependent nitric oxide production capacity of human umbilical vein endothelial cells was affected by preeclampsia. STUDY DESIGN: Human umbilical vein endothelial cells were isolated from 11 preeclamptic and 10 normotensive pregnancies. The maximal calcium ionophore A23187-stimulated nitric oxide production capacity was measured as accumulation of nitrate and nitrite into the culture medium, and it was related to the number of viable endothelial cells by measurement of their mitochondrial dehydrogenase activity. RESULTS: The cell number-related nitric oxide production capacity was similar in preeclamptic and normotensive pregnancies. The total nitric oxide production of cells from preeclamptic pregnancies was significantly lower (p <0.001). This difference, however, was mainly caused by larger amount of viable endothelial cells recovered from normotensive pregnancies. CONCLUSION: The maximal calcium-dependent nitric oxide production capacity of individual human umbilical vein endothelial cells is not affected by preeclampsia. PMID- 8633637 TI - Long-term effects of multiple pregnancies on cardiac dimensions and systolic and diastolic function. AB - OBJECTIVE: This study evaluated whether the recurring volume loading and hormonal changes in multiple pregnancies might have some cumulative effect on heart size and function. STUDY DESIGN: Echocardiograms were performed on 20 healthy women with at least 4 (mean 5.2) term pregnancies; 20 healthy, age-matched, nulliparous women served as controls. RESULTS: There were no significant differences in chamber dimension, systolic or diastolic function, valvular incompetence, or heart rate between the groups. There was a small, but significant, prolongation in deceleration time of the E wave in the multiparous women. CONCLUSION: These findings show that the human heart is generally able to repeatedly adapt to multiple episodes of volume overload in pregnancy without lasting detrimental structural or functional changes. PMID- 8633639 TI - Maternal serum analyte levels after first-trimester multifetal pregnancy reduction. AB - OBJECTIVE: Our purpose was to assess the effect of multifetal pregnancy reduction on maternal serum levels of analytes used for screening low-risk women for fetal chromosome abnormalities and neural tube defects. STUDY DESIGN: Peripheral blood samples were obtained between 15.09 and 20.9 weeks' gestation from 10 consecutive women who had undergone first-trimester multifetal pregnancy reduction. The samples were assayed for levels of alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol. Analyte concentrations were interpreted within our maternal serum screening program. RESULTS: Levels of alpha-fetoprotein were significantly elevated in all samples. In each pregnancy levels of human chorionic gonadotropin and unconjugated estriol were consistent with the number of continuing gestations. CONCLUSIONS: First-trimester multifetal reduction does not alter second-trimester levels of human chorionic gonadotropin and unconjugated estriol. Further study is needed to determine whether these analytes could be used to screen pregnancies for fetal chromosome abnormalities after first-trimester multifetal reduction. PMID- 8633638 TI - Cervical ripening in humans: potential roles of estrogen, progesterone, and insulin-like growth factor-I. AB - OBJECTIVE: During pregnancy in humans a gradual connective tissue remodeling takes place in the cervix. The aim of this study was to examine a possible relationship between the action of gonadal steroids and growth factors and the biochemically identifiable changes in connective tissues during cervical ripening. STUDY DESIGN: Cervical biopsy specimens and serum samples were taken from 20 term pregnant and 20 nonpregnant menstruating women. Estrogen receptors and progesterone receptors were measured with enzyme immunoassays. The messenger ribonucleic acid levels for estrogen receptors, progesterone receptors, and insulin-like growth factor-I were determined by solution hybridization with human complementary deoxyribonucleic acid probes. The concentration of collagen and its solubility by pepsin digestion were measured. Statistical evaluations were done with the Student t test. RESULTS: In term pregnancy the estrogen receptor level decreased to 14% and the progesterone receptor level to 24% of nonpregnant levels (p <0.001 and p <0.01). The insulin-like growth factor-I messenger ribonucleic acid level increased 400% (p <0.01), whereas the messenger ribonucleic acid levels for estrogen receptors and progesterone receptors were unchanged. The changes coincided with a twofold decrease in collagen concentration (hydroxyproline) and a twofold increase in collagen solubility. CONCLUSION: Estrogen receptors and progesterone receptors are present in human cervix. A significant down-regulation of estrogen receptors and progesterone receptors and a fourfold increase in the insulin-like growth factor-I messenger ribonucleic acid level were registered in term pregnant cervix. These findings coincided with the remodeling of the cervical connective tissue. PMID- 8633640 TI - The anatomy of the nerves of the uterus. PMID- 8633641 TI - Tropical benzocaine: does it alleviate pain? Who knows? PMID- 8633642 TI - Effect of corticosteroid use before preterm delivery: outcome of periventricular hemorrhage. PMID- 8633644 TI - Use of intravenous immunoglobulin to prevent recurrent spontaneous abortion. PMID- 8633643 TI - Is it the severity of dysplasia or is it the presence or absence of dysplasia in cone biopsy specimens that predicts residual dysplasia in hysterectomy? PMID- 8633646 TI - Summary of analysis of published articles. January 1 through December 31, 1995. PMID- 8633645 TI - Neonatal outcome after preterm delivery for preeclampsia. PMID- 8633647 TI - Chorionic villus sampling safety. Report of World Health Organization/EURO meeting in association with the Seventh International Conference on Early Prenatal Diagnosis of Genetic Diseases, Tel-Aviv, Israel, May 21, 1994. AB - Accumulated experience of 138,996 cases of chorionic villus sampling shows that chorionic villus sampling is a safe procedure with an associated fetal loss rate comparable to that of amniocentesis. The chorionic villus sampling registry shows that chorionic villus sampling is currently performed primarily between 9 and 12 weeks' gestation and carried no increased risk of limb reduction defects: the overall incidence of limb reduction defects after chorionic villus sampling is 5.2 to 5.7 per 10,000, compared with 4.8 to 5.97 per 10,000 in the general population. Analysis of the pattern distribution of limb defects after chorionic villus sampling revealed no difference from the pattern in the general population. This applies specifically to transverse limb defects. Together with the overall incidence of limb reduction defects, these data provide no evidence for any risk for congenital malformation determined by chorionic villus sampling. Because chorionic villus sampling is currently performed generally after 8 completed weeks of pregnancy, few data are available for analysis of complications related to earlier procedures. Avoiding early chorionic villus sampling also excludes sampling in cases of early fetal death, which can be diagnosed reliably by ultrasonography at 9 weeks of pregnancy. PMID- 8633648 TI - The modified biophysical profile: antepartum testing in the 1990s. AB - OBJECTIVE: Our goal was to determine the false-negative and false-positive rates of antepartum testing by use of the modified biophysical profile. STUDY DESIGN: From Jan. 1, 1990, through Dec. 31, 1994, antepartum testing results were gathered prospectively and tabulated monthly. For 1 year, 1991, detailed intrapartum and neonatal data were collected from all women admitted and delivered as a result of an abnormal antepartum test result. RESULTS: The false negative rate of the antepartum testing protocol was 0.8 per 1000 women tested. Sixty percent of those delivered because of an abnormal antepartum test had no evidence of short-term or long-term fetal compromise. False-positive test results led to preterm delivery in 1.5% of those tested before term. CONCLUSION: The false-negative rate of the modified biophysical profile is lower than that of the nonstress test and compares favorably with the false-negative rates of the contraction stress test and the complete biophysical profile. Iatrogenic prematurity resulting from intervention for false positive test results occurred in 1.5% of women tested before 37 weeks. PMID- 8633649 TI - Fetal RhD genotyping in fetal cells flow sorted from maternal blood. AB - OBJECTIVE: The aim of this study was to determine the accuracy of noninvasive fetal RhD genotyping by fetal cell isolation from maternal blood. STUDY DESIGN: Candidate fetal cells from 18 pregnant women (one twin gestation) were flow sorted. Polymerase chain reaction amplification of a 261 bp fragment of the RhD gene was performed on sorted fetal cells. The presence of amplified product was considered predictive of the Rhd-positive genotype in the fetus. RESULTS: Sixteen of the 19 fetal RhD genotypes were correctly predicted in fetal cells isolated from maternal blood (10 were Rh positive, 6 were Rh negative). In 3 cases no amplification products were detected in RhD-positive fetuses. The association between presence of the fragment and RhD-positive genotype was significant (p=0.003, Fisher's exact test). CONCLUSIONS: Noninvasive prenatal diagnosis of the fetal RhD genotype is feasible. Absence of amplification products in the reaction requires confirmation that fetal material is present. Improvements in fetal cell purity and yield should increase diagnostic accuracy, although the current protocol has a positive predictive value of 100% and a negative predictive value of 67%. PMID- 8633650 TI - The safety of calcium channel blockers in human pregnancy: a prospective, multicenter cohort study. AB - OBJECTIVE: Our purpose was to examine the potential teratogenicity of calcium channel blockers. STUDY DESIGN: Six teratogen information services prospectively collected and followed up 78 women with first-trimester exposure to calcium channel blockers. Pregnancy outcome was compared (by paired t text of chi2 analysis) with that of a control group matched for maternal age and smoking. RESULTS: There was no increase in major malformation (2/66=3.0% [calcium channel blockers] vs 0% [nonteratogenic controls], p=0.27); a fivefold increase was ruled out (baseline 2%, alpha = 0.05, beta = 0.20). The defects reported were attributable to maternal diabetes or coingestion of teratogens. The increase in preterm delivery 28% [calcium channel blockers] vs 9% [nonteratogenic controls], p=0.003), attributed to maternal disease by stepwise regression, was the most important factor responsible for the observed decrease in birth weight (mean -334 gm vs nonteratogenic controls, p=0.08). CONCLUSION: This study suggests that calcium channel blockers do not represent a major teratogenic risk. PMID- 8633651 TI - Is fetal neurologic and physical development accelerated in preeclampsia? AB - OBJECTIVE: Our objective was to determine whether the Ballard score, a maturity score for neonatal neuromuscular and physical development, is more advanced in preterm infants of preeclamptic women than in controls. STUDY DESIGN: A matched cohort study design was used. One hundred women with strictly defined preeclampsia (new-onset hypertension, proteinuria, and hyperuricemia) were matched for gestational age, race, and gender to 100 normotensive women with preterm delivery. All patients had an assigned antenatal gestational age based on ultrasonography before 24 weeks. The gestational age, based on antenatal ultrasonography and last menstrual period, was compared with the Ballard score given at the time of neonatal physical examination within the first 12 hours after delivery. The difference in gestational age between the Ballard score and antenatal ultrasonography (Ballard score - ultrasonography) was calculated for each patient. Results are expressed as median and range and are compared with a Student t test. RESULTS: The mean gestational age at delivery by antenatal ultrasonography in patients with severe preeclampsia was 32.06 +/- 2.74 and 32.03 +/- 2.70 weeks, respectively. The median difference between scores in patients with severe preeclampsia and normal patient were 1.3 +/- 1.8 and 1.5 +/- 1.6 weeks, respectively (p = 0.41). CONCLUSION: On the basis of criteria defined by the Ballard score, preeclampsia was not associated with accelerated fetal neurologic and physical development. PMID- 8633652 TI - Neonatal survival and disability rate at age 18 months for infants born between 23 and 28 weeks of gestation. AB - OBJECTIVE: Our purpose was to determine gestational age-specific outcomes of infants born in a period of surfactant use. STUDY DESIGN: All 465 consecutive births between 23 and 28 weeks' gestation in a tertiary center from 1987 to 1992 were analyzed prospectively. At 18 months' corrected age, 217 of 254 (85%) survivors were evaluated. RESULTS: From 1987 and 1988 to 1991 and 1992 there was an increase in survival for infants born at 24 weeks (from 0% to 33% p = 0.17), 25 to 26 weeks (38% to 71%, p < 0.005), and 27 to 28 weeks (66% to 84%, p < 0.05). At each weekly interval from 24 to 28 weeks of gestation the respective incidence of normality was 44%, 71%, 57%, 76% and 72% (not significant) and the respective mean developmental quotient was 91 +/- 17, 89 +/- 25, 90 +/- 24, 96 +/ 15 and 96 +/- 14 (not significant). CONCLUSIONS: Gestational age was strongly associated with outcome in terms of survival. Overall, 70% of children followed up were developing within the normal range. PMID- 8633653 TI - Second-trimester echogenic bowel and intraamniotic bleeding: association between fetal bowel echogenicity and amniotic fluid spectrophotometry at 410 nm. AB - OBJECTIVE: Our purpose was to determine whether the presence of heme pigments in amniotic fluid is associated with the ultrasonographic findings of increased fetal bowel echogenicity in the second trimester. STUDY DESIGN: Spectrophotometric analysis of amniotic fluid for optical density at 410 nm was prospectively performed to study the presence of heme pigments in (1) 104 pregnancies undergoing second-trimester amniocentesis for routine cytogenetic indications and (2) in 14 pregnancies undergoing amniocentesis for prenatal karyotyping because of fetal strongly echogenic bowel. In the routine amniocentesis group the fetal small bowel echogenicity was assessed immediately before amniocentesis and classified as nonechogenic (n = 64), mildly echogenic (n = 36), or hyperechogenic (n = 4) with the fetal iliac wing and liver used as references. Only amniotic fluid specimens that were obtained at the first attempt and that were not blood-stained were included in this study, with the first milliliter being discarded in all samples. RESULTS: In the routine amniocentesis group abnormal amniotic fluid optical density readings were significantly more frequent in fetuses with increased bowel echogenicity compared with those with nonechogenic bowel (8/40 [20%] vs 3/64 [5%], respectively; p < 0.001). In the hyperchogenic bowel group abnormal amniotic fluid optical density readings were found in four samples (29%). Overall, 12 of 54 fetuses (22%) with increased bowel echogenicity had a detectable peak at 410 nm. Three of the 12 (25%) fetuses with echogenic bowel and positive readings for hemoglobin were chromosomally abnormal. CONCLUSIONS: Fetal small bowel echogenicity is associated with the presence of heme pigments in amniotic fluid as determined by amniotic fluid optical density at 410 nm. Swallowing of amniotic fluid after intraamniotic bleeding seems implicated in the etiology of second-trimester echogenic bowel in both euploid and aneuploid fetuses. PMID- 8633654 TI - Stretching of fetal membranes increases the concentration of interleukin-8 and collagenase activity. AB - OBJECTIVE: The aim of this study was to determine whether stretching of fetal membranes can increase interleukin-8 concentration and collagenase activity. STUDY DESIGN: Strips of whole fetal membranes, amnion, or muscles of the lower uterine segment were stretched for 2 or 4 hours. Interleukin-8 and collagenase activity were measured in homogenized control and stretched samples. Immunohistochemical staining for interleukin-8 was carried out. RESULTS: The interleukin-8 concentration increased significantly after the whole fetal membranes were stretched for 2 and 4 hours (p <0.0007 and 0.001, respectively). Also, stretching of amnion and muscles of the lower uterine segment for 2 and 4 hours increased the concentration of interleukin-8 significantly (p <0.0007 after 2 and 4 hours, respectively). Collagenase activity was significantly increased after stretching of amnion, amniochorion, and muscles of the lower uterine segment for 4 hours (p <0.0007, 0.006, and 0.0007, respectively). After stretching, samples were darkly stained for interleukin-8 compared with control nonstretched samples. CONCLUSION: Stretching of amnion, amniochorion, and muscles of the lower uterine segment increased interleukin-8 production and collagenase activity. PMID- 8633655 TI - Risk of fetal mosaicism when placental mosaicism is diagnosed by chorionic villus sampling. AB - OBJECTIVE: Our purpose was to determine the risk of fetal mosaicism when placental mosaicism is found on chorionic villus sampling. STUDY DESIGN: We present a case of mosaic trisomy 22 detected on chorionic villus sampling and subsequently found in the fetus. A review of comprehensive chorionic villus sampling studies with emphasis on follow-up for fetal mosaicism was conducted. RESULTS: Among 13 studies reviewed, 469 cases of placental mosaicism are presented; fetal mosaicism was found in 50 (10.7%). Factors associated with fetal mosaicism are (1) mosaicism on mesenchymal core culture and (2) type of chromosome abnormality involved--specifically, marker chromosomes (26.7%) and common autosomal trisomies (19.0%). Amniocentesis predicted fetal genotype in 93% to 100% of cases of placental mosaicism, depending on the cell type in which mosaicism was diagnosed. CONCLUSIONS: Although mosaicism is usually confined to the placenta, the fetus is involved in about 10% cases. Patients should be counseled about this risk and the accuracy of follow-up amniocentesis. PMID- 8633657 TI - Cardiac oxygenation by extracorporeal membrane oxygenation in exteriorized fetal lambs. AB - OBJECTIVE: The purpose of this study was to determine the degree of cardiac oxygenation produced by different routes of extracorporeal membrane oxygenation in fetal lambs submerged in warm saline solution. STUDY DESIGN: Seven fetal lambs ranging in age from 113 to 133 days of gestation were delivered by cesarean section and oxygenated with extracorporeal membrane oxygenation. To maintain the patency of the ductus arteriosus, prostaglandin E1 was continuously infused intravenously to the fetus. Initially the extracorporeal membrane oxygenation route was from the right atrium to the carotid artery. Then the extracorporeal membrane oxygenation route was changed to flow from the right atrium to the umbilical vein. The fetus was kept in a warm saline solution bath, and the fetal circulation was maintained. Extracorporeal membrane oxygenation flow ranged between 100 and 200 ml/min throughout the experiment. Simultaneous blood samples were taken during both types of extracorporeal membrane oxygenation from the following points in the fetal circulation: premembrane (least oxygenated blood leaving the fetus from the right atrium), postmembrane (oxygenated blood returning to the fetus), the carotid artery, and the left ventricle. The respiratory gases and pH of each sample were measured. Six fetuses received nonradioactive colored microspheres injected into the oxygenated blood returning to the fetus flow before returning to the fetuses during both types of extracorporeal membrane oxygenation. After the animals were killed, microspheres were counted in the myocardium separately taken from the right and left atria and the right and left ventricles to determine cardiac blood flow. RESULTS: During right atrium to carotid artery extracorporeal membrane oxygenation, left ventricle PO2 remained low as postmembrane PO2 increased; these values were not significantly correlated (r = 0.234, p = 0.61). During right atrium to umbilical vein extracorporeal membrane oxygenation, left ventricle and postmembrane PO2 exhibited a significant positive correlation (r = 0.855, p = 0.014). When the extracorporeal membrane oxygenation route was switched from the right atrium to carotid artery to the right atrium to umbilical vein, there was a significant increase in left ventricle PO2 and a decrease in left ventricle PCO2, whereas the respiratory gases and pH remained unchanged at other sites in the circulation. Microsphere counts were consistently higher during right atrium to umbilical vein extracorporeal membrane oxygenation than during right atrium to carotid artery extracorporeal membrane oxygenation in all four samples from different parts of myocardium (p < 0.001 by paired t test). CONCLUSION: More effective cardiac oxygenation is provided by right atrium to umbilical vein extracorporeal membrane oxygenation than by right atrium to carotid artery extracorporeal membrane oxygenation. PMID- 8633656 TI - Mechanism and rate of placental transfer of zalcitabine (2',3'-dideoxycytidine) in Macaca nemestrina. AB - OBJECTIVE: Our purpose was to determine whether the ant-human immunodeficiency virus drug zalcitabine (2',3'-dideoxycytidine) is actively transferred across the placenta in the near-term Macaca nemestrina. STUDY DESIGN: Constant rate infusions of zalcitabine (1.31 microg/min/kg) and antipyrine (66.7 microg/min/kg) were administered to the dam through the femoral vein (n = 4) or to the fetus through the carotid artery (n = 3) in a randomized cross-over design. Zalcitabine was also administred at a 10-fold higher infusion rate to the dam (n = 3). The effect of zidovudine on the transplacental transfer of zalcitabine was studied by coinfusing the two drugs to the dam (n = 2). Samples from maternal plasma, fetal plasma, and amniotic fluid were collected at regular intervals during the 30-hour infusions. RESULTS: The maternal-fetal transfer clearance of zalcitabine (0.41 +/ 0.16 ml/min/kg) was not significantly different from the fetal-maternal transfer clearance of the drug (0.66 +/ 0.30 ml/min/kg). Moreover, the steady-state fetal maternal plasma concentration ratios of zalcitabine after the low 0.58 +/- 0.06) and high (0.66 +/- 0.10) infusion rates to the dam were similar. This ratio was not substantially changed (0.69) when zalcitabine was coadministered with zidovudine. The placental transfer rate of zalcitabine was 11% (+/-4%) that of antipyrine. CONCLUSION: The placental transfer of zalcitabine is passive and unaffected by simultaneous administration of zidovudine. In Macaca nemestrina the average fetal exposure to zalcitabine is approximately 60% of the maternal exposure. PMID- 8633659 TI - Antenatal ultrasonographic findings differentiating complete from partial agenesis of the corpus callosum. AB - Four cases of complete (three) and partial (one) agenesis were evaluated ultrasonographically. The frontal lobe/biparietal diameter ratio were evaluated in 113 normal fetuses and compared with those ratios in fetuses with corpus callosum agenesis. In the presence of the classic ultrasonographic features of agenesis of the corpus callosum, frontal lobe shortening, along with absence of the cavum septi pellucidi, might contribute to the diagnosis of complete agenesis of the corpus callosum and distinguish it from partial agenesis. PMID- 8633658 TI - Enrichment of fetal trophoblast cells from the maternal peripheral blood followed by detection of fetal deoxyribonucleic acid with a nested X/Y polymerase chain reaction. AB - OBJECTIVE: Fetal cells circulate in the maternal blood during early pregnancy. Because these cells are rare, noninvasive prenatal diagnosis from fetal cells can be achieved only after efficient enrichment procedures. Our aim was to develop a two-step enrichment procedure to isolate trophoblast cells from 20 ml of peripheral blood. STUDY DESIGN: Blood was obtained from pregnant women between 6 and 15 weeks of gestation, before invasive procedures were performed. After enrichment, the success of isolating fetal cells was determined by amplification of Y chromosome sequences. RESULTS: A highly specific X/Y polymerase chain reaction was established, sensitive enough to detect X and Y chromosome-specific sequences in one single cell and in one male among 100,000 female cells. Sex determination by polymerase chain reaction was compared with results from conventional karyotyping. The success rate was 91.7%. CONCLUSION: Enrichment of trophoblast cells from maternal blood as described here might be useful for early noninvasive prenatal diagnosis. PMID- 8633661 TI - Compound presentation resulting from the forward-roll technique of external cephalic version: a possible mechanism. AB - Limited reports exist about the frequency of compound presentation after external cephalic version. A compound hand and foot presentation is reported after external cephalic version in a woman desiring vaginal birth after cesarean delivery. We report a mechanism by which a forward roll during external cephalic version may predispose an infant in complete breech position to a compound foot presentation. PMID- 8633660 TI - Spontaneous closure of the human fetal ductus arteriosus--A cause of fetal congestive heart failure. AB - OBJECTIVE: Closure of the fetal ductus arteriosus, which is usually due to nonsteroidal antiinflammatory agents, may be detrimental. Therefore prenatal and postnatal clinical and echocardiographic findings in four human fetuses with spontaneous ductus arteriosus occlusion are reported. STUDY DESIGN: Echocardiographic and clinical data were retrospectively analyzed. RESULTS: Spontaneous closure of the ductus arterious was discovered in four fetuses (gestational age 34 to 38 weeks). No mother had received nonsteroidal antiinflammatory agents. Enlargement of the right heart and pulmonary arteries and tricuspid and pulmonary regurgitation were present in all cases. Two fetuses had right ventricular hypertension. Postnatally their right ventricular function recovered promptly. The others had severe right heart failure with abnormal umbilical venous pulsations. After immediate delivery none had signs of persistent pulmonary hypertension. However, they have echocardiographic evidence of right ventricular dysfunction 2 to 6 months after delivery. CONCLUSIONS: Occlusion of the fetal ductus arteriosus may also occur in the absence of treatment with nonsteroidal antiinflammatory agents. Immediate delivery resulted in good clinical outcome, although right ventricular dysfunction may persist. PMID- 8633662 TI - Rapid aneuploid diagnosis of high-risk fetuses by fluorescence in situ hybridization. AB - OBJECTIVE: Our purpose was to develop fluorescence in situ hybridization to repetitive chromosome-specific sequences to detect chromosome aneuploidy faster than hybridization to unique targets or karyotyping. STUDY DESIGN: Aneuploidy involving chromosomes 13, 18, 21, X, and Y comprises 70% of chromosome abnormalities in 10- to 12-week fetuses, 95% of the phenotypically significant newborn chromosome abnormalities. Our improved 8-hour protocol used repetitive probes to label and count the number of these centromeric chromosome domains. RESULTS: This protocol correctly determined chromosome 13, 18, and 21 status in 50 of 50 unselected direct amniocyte samples and found abnormal patterns in 27 of 27 archived trisomy 21 cases. Altogether karyotyping confirmed 744 of 745 chromosome-specific repetitive sequence test results. CONCLUSION: This protocol rapidly tests abnormal fetuses and newborn infants in whom diagnosis is made at the initiation of labor or before urgent surgery when a cytogenetic result cannot be completed. PMID- 8633663 TI - Molecular genetic etiology of twin reversed arterial perfusion sequence. AB - OBJECTIVE: Our purpose was to determine whether the twin reversed arterial perfusion sequence (acardiac anomaly) results from fertilization of the first or second polar body. STUDY DESIGN: Placental or fetal tissue was obtained from nine twin sets discordant for twin reversed arterial perfusion. After deoxyribonucleic acid extraction, the polymerase chain reaction was used to amplify five polymorphic microsatellite repeats. The products were differentiated by polyacrylamide gel electrophoresis, and patterns were compared within twin sets. RESULTS: Deoxyribonucleic acid fingerprinting patterns were identical in all twin sets for all primer pairs. It is calculated that the chance that any of the acardiac twins resulted from fertilization of either the first or second polar body is <4% and the chance that they all resulted from polar body fertilization is <0.001%. CONCLUSION: Twins discordant for the twin reversed arterial perfusion sequence anomaly are monozygous. Our results exclude polar body fertilization as a likely cause of this condition. PMID- 8633664 TI - Placental transfer of milrinone in the nonhuman primate (baboon). AB - Milrinone is an inotropic agent for short-term intravenous use in the management of congestive heart failure. The purpose of this article is to question the previously reported lack of transplacental transfer of milrinone. Loading and continuous intravenous doses, considered to be therapeutic in humans, were administered to four near-term baboons. Transplacental passage was documented, with a maternal/fetal serum ratio of approximately 4:1 found during the 3 hours of infusion. PMID- 8633666 TI - Violence and gynecologic health in women <50 years old. AB - OBJECTIVE: Our purpose was to examine, in women aged 18 to 49, the relationship of violent experiences (child abuse, violent crime, spouse abuse) to gynecologic problems. STUDY DESIGN: Data from 1599 participants in a randomized, sociodemographically representative telephone survey of U.S. women were used. Gynecologic problems wer measured by self-report of receiving a physician's diagnosis of severe menstrual problems, a sexually transmitted disease, or a urinary tract infection. Statistical analyses used were the chi2 test and multiple logistic regression. RESULTS: A total of 31.5% of participants reported a diagnosis of gynecologic problems in the past 5 years. Those with problems were more likely to report childhood abuse, violent crime victimization, and spouse abuse. In logistic regression models controlling for sociodemographic factors and access to medical care, violence events remained significantly associated with gynecologic problems. CONCLUSIONS: A history of gynecologic problems may indicate that a patient has been harmed by violence. However, all women are at risk of being harmed by violence, and a violence history should be obtained as part of the intake protocol for all patients. Physicians need to be prepared to provide information about local community services, including counseling, domestic violence centers, shelters, and advocacy groups. PMID- 8633667 TI - Endometrial ablation: a series of 568 patients treated over an 11-year period. AB - OBJECTIVE: Our purpose was to retrospectively review the intraoperative and long term outcomes of 568 patients with abnormal uterine bleeding who were treated by endometrial ablation over an 11-year period. STUDY DESIGN: From 1893 to 1994, 401 endometrial ablations were performed with the neodymium-yttrium-aluminum-garnet laser and another 167 patients were treated by electrosurgery. The majority of the patients were treated for irregular, heavy menses. Fifty-seven had ablation because of abnormal bleeding associated with a serious medical disorder, 12 with a bleeding diathesis, and 50 with morbid obesity. All patients had preoperative endometrial sampling that demonstrated benign histology. Nineteen patients had submucous myomas that were resected at the time of hysteroscopic ablation. All patients received preoperative and postoperative suppression. The minimum follow up period was 1 year. RESULTS: The average operative time was 32.5 minutes. The mean hospital stay was 8 hours. Four patients who received 32% dextran 70 in dextrose (Hyskon) as the distending medium had pulmonary edema postoperatively. One case of endometritis was also detected. No uterine perforations were observed. Amenorrhea developed in 58% of the patients, 34% reported light or normal menstrual flow, and 8% did not respond (continued heavy flow). CONCLUSION: This study represents one of the largest published series of endometrial ablation, with a mean follow-up of 4.5 years. It demonstrates that hysteroscopic endometrial ablation is a reliable, safe alternative to hysterectomy for the surgical management of abnormal uterine bleeding. PMID- 8633665 TI - Hormone replacement therapy, hormone levels, and lipoprotein cholesterol concentrations in elderly women. AB - OBJECTIVE: Our purpose was to assess the relationships of lipid and lipoprotein cholesterol levels to hormone replacement therapy and hormone levels in elderly women. STUDY DESIGN: A sample of 292 postmenopausal women 55 to 99 years old (mean 76 years) was drawn from Leisure World Laguna Hills, California, an upper middle-class, white independent-living population. We compared 84 women receiving unopposed estrogen replacement therapy and 38 women taking combination hormone replacement therapy with 170 women who had never used hormone replacement therapy. Nonparametric tests for differences in lipid and lipoprotein cholesterol levels among groups and multiple stepwise regression models were used. RESULTS: Estrogen users (with and without progestin) had lower total and low-density lipoprotein cholesterol and higher high-density lipoprotein and high-density lipoprotein subfraction types 2, 2a, and 2b cholesterol levels. High density lipoprotein type 3 subfractions were lower in combination hormone replacement therapy users but higher in unopposed estrogen users relative to nonusers. The conjugated equine estrogen dose was negatively correlated with total (p = 0.0009) and low-density lipoprotein cholesterol (p <0.0001) levels and positively correlated to high-density lipoprotein cholesterol (p = 0.002) and its subfractions. The medroxyprogesterone acetate dose showed no consistent effect on cholesterol levels. CONCLUSION: The associations found here reaffirm the significant role of estrogen replacement therapy on lipid and lipoprotein cholesterol levels and provide no evidence of a reduction in the beneficial effect of estrogen with the addition of a progestational agent to the replacement regimen. PMID- 8633668 TI - Increased levels of redox-active iron in follicular fluid: a possible cause of free radical-mediated infertility in beta-thalassemia major. AB - OBJECTIVE: Our purpose was to investigate the follicular fluid parameters associated with redox activity and the consequent production of the deleterious hydroxyl radical in beta-thalassemia major. STUDY DESIGN: The levels of ferritin, total iron, total copper, and redox-active iron were measured in follicular fluid aspirated from three follicles during three consecutive ovum pickups from a patient with beta-thalassemia major and were compared with the levels in nine follicles aspirates from nine healthy control patients. The redox activity in the follicular fluid samples was monitored by the extent of follicular fluid-mediated deoxyribonucleic acid degradation and salicylate hydroxylation. RESULTS: Total iron and ferritin concentrations were elevated in thalassemic follicular fluid samples compared with control samples (6.7 fold, and 53.3-fold, respectively), whereas the total copper concentration was similar. Thalassemic follicular fluid samples exhibited a marked increase of redox activity, indicating a higher potential of free radical production leading to deoxyribonucleic acid degradation. Likewise, free radical-induced conversion of salicylate to dihydroxybenzoic acid derivatives was enhanced in the thalassemic follicular fluid samples compared with controls (2,3-dihydroxybenzoic acid: 67.7 +/- 22 vs 20.3 +/- 12.9 ng/mg protein; 2,5-dihydroxybenzoic acid: 101.6 +/- 25.9 vs 4.42 +/ 2.7 ng/mg protein). CONCLUSIONS: The increased level of redox activity found in the follicular fluid from a patient with beta-thalassemia major focuses the attention on the small fraction of redox-active iron ions as mediators of free radical production, inducing tissue injury and possibly contributing to impairment of reproduction in these patients. PMID- 8633669 TI - Hormonal treatment for bleeding irregularities in Norplant implant users. AB - OBJECTIVE: Our purpose was to evaluate whether prolonged or irregular bleeding during Norplant implant use could be alleviated with the use of oral hormonal medication. STUDY DESIGN: One hundred fifty users of the Norplant levonorgestrel contraceptive implant with prolonged or frequent bleeding were enrolled in this prospective, randomized, comparative study and assigned to one of three treatment groups for 20 days: ethinyl estradiol 50 microg, an oral contraceptive (50 microg ethinyl estradiol and 250 microg levonogestrel), and placebo. Total days of bleeding during treatment and length of the bleeding-free interval were analyzed. RESULTS: Women treated with the levonorgestrel-ethinyl estradiol pill bled an average of 2.6 days during treatment compared with 5.4 and 12.3 days in the ethinyl estradiol and placebo groups, respectively. Differences between both hormonal groups and placebo were significant (p <0.00001); moreover, the combined pill was more effective than ethinyl estradiol along (p <0.0001). CONCLUSION: The combined pill proved to be an excellent palliative treatment and is a more practical approach because of availability at all clinic sites. PMID- 8633670 TI - Can cervical cancer be prevented by a see, screen, and treat program? A pilot study. AB - OBJECTIVE: Our purpose was to determine the feasibility of providing a cervical screening facility to the underprivileged communities through an educational program and a mobile clinic in which cytologic smears could be taken, screened immediately, and, when appropriate, the patients treated on site with minimal delay. STUDY DESIGN: A prospective study was conducted in two parts on 5045 patients living in squatter areas around Cape Town, South Africa. The patients were educated about cervical cancer and its prevention and were offered a free Papanicolaou smear taken in a fully equipped mobile clinic. These were immediately stained and processed. Patients diagnosed cytologically as having high-grade squamous intraepithelial lesions were assessed colposcopically and, when indicated, immediately treated by large loop excision of the transformation zone under local anesthesia. RESULTS: In phase 1, colposcopy was done in the nearest colposcopy clinic, 20 km from the screening site. The defaulter rate was 66%. In phase 2, colposcopy and treatment were offered on site. A total of 97% of patients referred for colposcopy attended the clinic, and all patients requiring treatment have been adequately treated. CONCLUSION: With a rapid turnaround time for the reporting of cytologic results and given a colposcopy and treatment facility available located at the screening site at the time women receive their results, the majority of women will undergo colposcopy and treatment. PMID- 8633671 TI - Second primary cancers after vulvar and vaginal cancers. AB - OBJECTIVE: Our objective was to examine the occurrence of second primary cancers after vaginal and vulvar cancers. STUDY DESIGN: Women in whom cancers of the vagina (in situ, n=461; invasive, n=888) and vulva (in situ, n=2898; invasive, n=2685) were diagnosed between 1973 and 1988 were identified from nine population based cancer registries. Subjects were followed through 1989 for the development of a subsequent primary cancer. RESULTS: We found increased risks of all second cancers combined among women with cancer of the vulva (observed/expected in situ = 1.5; observed/expected invasive = 1.3) and vagina observed/expected invasive = 1.2). Most of the excess second cancers were smoking related (e.g., cancers of the lung, buccal cavity and pharynx, esophagus, nasal cavity and larynx) or related to infection with human papillomavirus (e.g., cervix, vulva, vagina, and anus). CONCLUSION: These associations indicate that the follow-up care of women with cancers of the vulva and vagina should involve efforts to promote smoking cessation. The data are also consistent with a common sexually related cause for cancers of the cervix, vulva, vagina, and anus. PMID- 8633672 TI - An incremental dosing protocol for women with severe vaginal trichomoniasis and adverse reaction to metronidazole. AB - OBJECTIVE: Our purpose was to develop and test an incremental dosing protocol for women with adverse reaction to metronidazole and severe symptomatic Trichomonas vaginitis. STUDY DESIGN: Two women with documented Trichomonas infection and presumed metronidazole allergy were initially treated with a number of alternative methods without success. With persistent severe symptoms associated with their infection, these women were admitted to the hospital and underwent an intravenous incremental metronidazole dosing protocol. RESULTS: Both patients were successfully treated without adverse event. They are both symptom-free and apparently cured several months after treatment. CONCLUSION: This protocol offers a new therapeutic option to women with adverse metronidazole reactions and severe symptomatic Trichomonas vaginitis resistant to treatment with nonmetronidazole therapy. PMID- 8633674 TI - Ultraradical debulking of epithelial ovarian cancer with the ultrasonic surgical aspirator: a prospective randomized trial. AB - OBJECTIVE: Our purpose was to evaluate the effectiveness, benefits, and potential side effects of the use of the cavitational ultrasonic surgical aspirator for cytoreduction of advanced ovarian cancer. STUDY DESIGN: Forty patients with stage IIC to IV ovarian carcinoma were randomized to undergo ultraradical cytoreductive surgery with (group 1, 20 patients) or without (group 2, 20 patients) the use of the CUSA 200 cavitational ultrasonic surgical aspirator (Valleylab, Boulder, Colo). Preoperative and postoperative information and follow-up data were collected prospectively and compared between both groups. RESULTS: The amount of residual tumor after debulking surgery, the duration of the operation, and the rate of perioperative complications were comparable in both study groups. The amount of perioperative blood loss was significantly lower (1450 ml vs 1650 ml, p=0.02) in group 1. These patients had less morbidity (p<0.05), and could be discharged from the hospital significantly sooner (14 days vs 16 days, p=0.031). CA 125 levels 1 and 2 months after surgery were significantly lower in the group with cavitational ultrasonic surgical aspirator (p<0.01 and p<0.01, respectively), suggesting more successful cytoreduction. However, this is not reflected in a better disease-free or overall survival. CONCLUSION: Cavitational ultrasonic surgical aspirator-assisted cytoreductive surgery of advanced ovarian carcinoma is more effective in eradicating disease, reduces morbidity, and is cost beneficial. PMID- 8633673 TI - Conization for cervical intraepithelial neoplasia is followed by disappearance of human papillomavirus deoxyribonucleic acid and a decline in serum and cervical mucus antibodies against human papillomavirus antigens. AB - OBJECTIVE: Our purpose was to investigate whether conization for cervical intraepithelial neoplasia eliminates human papillomavirus deoxyribonucleic acid and effects the levels of serum and cervical mucus antibodies against human papillomavirus antigens. STUDY DESIGN: Analysis of paired cervical brush and serum samples taken from 23 women with cervical intraepithelial neoplasia before and 16 to 27 months after conization was performed for presence of human papillomavirus deoxyribonucleic acid by polymerase chain reaction and for human papillomavirus antibodies by enzyme-linked immunosorbent assay. RESULTS: Four women had recurrent cervical intraepithelial neoplasia, whereas 19 women were disease free. Eighteen of 23 women were positive for human papillomavirus deoxyribonucleic acid before treatment. At follow-up only the 4 women with recurrent cervical intraepithelial neoplasia were positive. Serum immunoglobulin G levels and A levels and immunoglobulin A levels in cervical mucus against most of the tested human papillomavirus antigens had declined at follow-up. CONCLUSIONS: Human papillomavirus deoxyribonucleic acid was regularly eliminated and human papillomavirus antibody levels, especially local immunoglobulin A, declined after efficient treatment, suggesting that conization may be effective for treating the underlying human papillomavirus infection. PMID- 8633675 TI - Selective photosensitizer distribution in vulvar condyloma acuminatum after topical application of 5-aminolevulinic acid. AB - OBJECTIVE: Our purpose was to determine the feasibility of selective photosensitization of vulvar condylomas by use of tropical application of 5 aminolevulinic acid. STUDY DESIGN: In vivo fluorescence was assessed and biopsy specimens of condylomas were taken for fluorescence microscopy in 24 patients at different times after application of 2.5% 5-aminolevulinic acid ointment or 20% 5 aminolevulinic acid cream. RESULTS: Both in vivo fluorescence imaging and fluorescence microscopy showed selective fluorescence of condylomas of the labia minora and vestibule only within short time intervals, because fluorescence of poorly keratinized normal epithelium was induced by both 5-aminolevulinic acid formulations. In non-hair-bearing skin, lesional fluorescence remained highly selective. Fluorescence microscopy showed that 90 minutes after drug application peak selectivity in epithelial lesional fluorescence was significantly higher with 2.5% 5-aminolevulinic acid ointment (4.5 +/- 0.9) than it was with 20% cream (2.1 +/- 0.2). CONCLUSION: Selective fluorescence of vulvar condyloma acuminatum can be induced by nonselective topical 5-aminolevulinic acid application. Studies evaluating selective photodynamic destruction of condylomas are justified. PMID- 8633676 TI - Antimullerian hormone as a serum marker of granulosa cell tumorsof the ovary: comparative study with serum alpha-inhibin and estradiol. AB - OBJECTIVE: Our purpose was to evaluate serum antimullerian hormone as a marker for granulosa cell tumors. STUDY DESIGN: Serum antimullerian hormone concentrations were determined in 16 patients with an adult-type granulosa cell tumor; in female patients with ovarian adenocarcinoma, benign ovarian cysts, or extraovarian cancers; and in normal premenopausal and postmenopausal women. Serum antimullerian hormone, alpha-inhibin, and estradiol levels were compared in 10 patients with a granulosa cell tumor during 6 to 47 months of follow-up. RESULTS: Serum antimullerian hormone was undetectable in normal postmenopausal women and was <5 micrograms/L in premenopausal women. Normal serum levels were found in patients with ovarian cancers or cysts or with extraovarian cancers. Levels were between 6.8 and 117.9 microg/L in eight of nine patients with a progressive granulosa cell tumor. In the remaining case antimullerian hormone, alpha-inhibin and estradiol concentrations were normal. Serum antimullerian hormone and alpha inhibin levels became elevated at least 11 months before the recurrence was clinically detectable. During clinical remission serum antimullerian hormone, beta-inhibin, and estradiol were normal in most cases. CONCLUSION: Serum antimullerian hormone is a sensitive, specific, reliable marker of adult-type granulosa cell tumors and is useful to evaluate the efficacy of treatment and to detect recurrences early. PMID- 8633677 TI - Trial of labor in patients with a previous lower uterine vertical cesarean section. AB - OBJECTIVE: Our purpose was to determine the efficacy and safety of a trial of labor in patients previously delivered at least once by a lower uterine vertical cesarean section. STUDY DESIGN: A retrospective review was performed at a single tertiary perinatal center, The University of Florida Health Science Center, Jacksonville. The medical records of all patients with a previous low vertical cesarean section who underwent a trial of labor during a 72-month period from January 1988 until December 1993 were reviewed. The medical records of the next two patients who did not have a prior uterine incision admitted to labor and delivery after the index case served as the controls. The duration and outcome of labor, including mode of delivery, maternal and perinatal morbidity, and birth trauma were evaluated. RESULTS: Of 77 patients with a previous low vertical cesarean incision, 11 (14.3%) had a repeat operation compared with 14 of 154 patients (9.0%) in the no previous cesarean section group (not significant). No differences were noted in the incidences of operative vaginal deliveries or prolonged duration of the first or second stages of labor, or in the rate or maximum dose of oxytocin infusion between the two groups. One patient in the previous cesarean section group had uterine rupture. The incidence of umbilical artery pH < or = 7.20 was similar. No difference in the number of infants with 1- or 5-minute Apgar scores < or = 7 was noted. CONCLUSION: A trial of labor in women with previous low vertical cesarean sections results in an acceptable rate of vaginal delivery and appears safe for both mother and fetus. PMID- 8633678 TI - Fetal fibronectin in vaginal specimens predicts preterm delivery and very-low birth-weight infants. AB - OBJECTIVE: The purpose of this study was to evaluate the association of vaginal fetal fibronectin expression to risk of preterm delivery and delivery of very-low birth-weight infants. STUDY DESIGN: Vaginal secretions were obtained from women between 22 and 35 weeks' pregnant with minimal cervical dilation (< or = 2 cm) and threatened preterm delivery. The secretions were analyzed for the presence of fetal fibronectin. Other clinical information including cervical dimensions, uterine activity, serum C-reactive protein concentration, vaginal pH, evidence of vaginal or systemic infection, and vaginal bleeding were also obtained. RESULTS: Of the 112 patients recruited, 40 (35.7%) were delivered prematurely (<37 weeks). For prediction of preterm delivery, the fetal fibronectin test result had a sensitivity, specificity, and positive and negative predictive values of 67.5, 90.3, 79.4, and 83.3%, respectively (odds ratio 19.3, p < 0.0001). Women with a positive fetal fibronectin test had a nearly 13-fold increased probability of being delivered of an infant weighing <1500 gm than did women with a negative fetal fibronectin test (32.4% vs 2.5%, p<0.0001). Categoric analysis and multiple logistic regression demonstrated that fetal fibronectin was an independent risk factor for prediction of preterm delivery and birth weight <1500 gm. CONCLUSION: Vaginal fetal fibronectin expression is an independent risk factor for preterm delivery and birth of very-low-birth-weight infants in symptomatic women with intact amniotic membranes and minimal cervical dilatation. PMID- 8633679 TI - Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum. AB - OBJECTIVE: We tested the hypothesis that serum free (nonesterified) fatty acid and triglyceride concentrations are increased in nulliparous women with preeclampsia relative to women with uncomplicated pregnancies and that these lipids decrease post partum, consistent with the known resolution of clinical symptoms. The relationships between serum concentrations of these lipids and the lipid peroxidation metabolite malondialdehyde were also examined. STUDY DESIGN: Predelivery and 24 to 48 hour postpartum venous blood samples were collected from eight women with preeclampsia and nine women with uncomplicated pregnancies after an 8- to 10-hour fast. Sera were analyzed for concentrations of triglycerides, free fatty acids, total cholesterol, high-density lipoprotein cholesterol, low density lipoprotein cholesterol, and malondialdehyde. RESULTS: Antepartum serum triglyceride and free fatty acid concentrations were increased approximately twofold in women with preeclampsia relative to uncomplicated pregnancies (p <0.02 and 0.004, respectively). Total, high-density lipoprotein, and low-density lipoprotein cholesterol concentrations did not differ between groups. Concentrations of all lipids decreased significantly in both groups within 48 hours post partum. However, triglyceride and free fatty acid concentrations remained higher in women with preeclampsia (p<0.006, both variables). Triglyceride and free fatty acid concentrations correlated positively, both ante partum (R2 0.42, p<0.01) and post partum (R2 0.39, p<0.02). Antepartum concentrations of malondialdehyde were 50% higher in women with preeclampsia (p<0.01) and decreased post partum (p <0.02) but did not decrease in controls (p = 0.07). Antepartum serum triglycerides and free fatty acids correlated positively with malondialdehyde concentrations (R2 0.38, p <0.02, both cases). CONCLUSION: Triglycerides and free fatty acids, but not cholesterol, are increased in preeclampsia and correlate with the lipid peroxidation metabolite malondialdehyde. We speculate that these interactions may contribute to endothelial cell dysfunction in preeclampsia. PMID- 8633680 TI - Gestational pyelonephritis--associated Escherichia coli isolates represent a nonrandom, closely related population. AB - OBJECTIVE: A select group of Escherichia coli strains known as uropathogenic cause pyelonephritis in nonpregnant individuals. We investigated whether Escherichia coli from gestational pyelonephritis represent a random population or possess common uropathogenic characteristics. STUDY DESIGN: Repetitive element sequence-based polymerase chain reaction, plasmid profiles, hemolysin, and O serotypes were assayed from Escherichia coli isolates of 57 pregnant patients with acute pyelonephritis at different gestational ages. RESULTS: The majority of the first trimester isolates fell primarily into repetitive element sequence based patterns 1 and 3 and O6, O15, and O75 serotypes. Second-trimester isolates had multiple patterns with high-frequency repetitive element sequence-based polymerase chain reaction 1 and 5 and an unknown (OX) serotype. Pattern 3, predominantly O75 serotype, was found primarily among third-trimester isolates. CONCLUSION: It is likely that Escherichia coli associated with acute pyelonephritis during different trimesters of pregnancy represents nonrandom closely related isolates, and some of these strains may be characteristic in pregnant patients only. PMID- 8633681 TI - Can a "snapshot" sagittal view of the cervix by transvaginal ultrasonography predict active preterm labor? AB - OBJECTIVE: Our purpose was to test the hypothesis that wedging of the cervical internal os determined by transvaginal ultrasonography is associated with premature labor and delivery. STUDY DESIGN: Seventy patients admitted to the hospital for threatened preterm labor were evaluated by transvaginal ultrasonography before institution of therapy. Bivariate and logistic regression analyses were performed to determine the variables that made a significant contribution to the prediction of preterm delivery. RESULTS: Preterm delivery was significantly associated with the presence of cervical wedging, as noted on cervical scan, and with short cervical length. A history of previous preterm delivery was of marginal significance as a predictor of preterm delivery (p=0.09). Preterm delivery was not significantly correlated with age, previous voluntary termination of pregnancy, gestational age at the time of study, previous normal spontaneous vaginal delivery, or tocolytic therapy. Use of wedging as a diagnostic test for the prediction of preterm delivery yielded a sensitivity of 100% a specificity of 74.5%, a positive predictive value of 59.4%, and a negative predictive value of 100%. CONCLUSION: The presence of wedging and shorter cervical length was suggestive of true preterm labor requiring aggressive management. A transvaginal ultrasonographic "snapshot" view of the cervix seems to be a more reliable method to evaluate the cervix in patients with threatened premature labor than are uterine contractions alone. PMID- 8633683 TI - The intolerance of aloneness. PMID- 8633682 TI - The clinical significance of ultransonographically detected subchorionic hemorrhages. AB - OBJECTIVES: The null hypothesis is that there is no difference in outcome when pregnancies with ultrasonographically documented subchorionic hemorrhages are compared with those without these hemorrhages. STUDY DESIGN: We performed a case control study, utilizing our computerized ultrasonographic database. Cases were matched with two or three controls in two separate control groups. Matching criteria were maternal age, gestational age at scan, and invasive procedures (chorionic villus sampling or amniocentesis). General exclusion criteria were absence of fetal heart motion and fetal anomalies. Presence of subchorionic hemorrhage was an exclusion criterion for both control groups; however, in addition, presence of vaginal bleeding was a further criterion for one of the two. Statistical analysis was performed with chi2 analysis and Yates' correction. Odd ratios and 95% confidence intervals were calculated. RESULTS: There was no difference in maternal characteristics between the cases and controls. The incidence of subchorionic hemorrhage was 1.3%. There was an increased risk of miscarriage (odds ratio 2.8, 95% confidence interval 1.7 to 7.4), stillbirth (4.5, 1.5 to 13.2), abruptio placentae (11.2, 2.7 to 46.4), and preterm labor (2.6, 1.5 to 4.6) when cases were compared with controls without subchorionic hemorrhage or bleeding. These risks were also increased in comparison with the control group with bleeding, except with respect to miscarriage. In this case the risks were similar in both cases and controls but increased with respect to the controls without bleeding. The mean birth weight was lower in the cases than in both control groups. CONCLUSION: The presence of an ultransonographically detected subchorionic hemorrhage increases the risk of miscarriage, stillbirth, abruptio placentae, and preterm labor. The presence of bleeding alone appears to increase the risk of miscarriage. It is unclear whether the subchorionic hemorrhage is causative or whether it is simply a sign of an underlying process that produces these negative effects. PMID- 8633684 TI - Images in neuroscience. Neuroimaging, IX. Functional magnetic resonance imaging. PMID- 8633685 TI - The borderline patient's intolerance of aloneness: insecure attachments and therapist availability. AB - OBJECTIVE: This article describes the clinical and theoretical significance of intolerance of aloneness for patients with borderline personality disorder. It is intended to make their treatment more effective and less burdensome. METHOD: Clinical observations from the author's more than 9,000 hours of psychotherapeutic work and 500 psychotherapy consultations with borderline patients are synthesized with findings of relevant empirical studies and attachment theory. RESULTS: Intolerance of aloneness is a deficit that is associated with the borderline patient's typical clinging and attention-seeking or detached forms of attachment. Suggestions are given for ways in which clinicians can respond to these dysfunctional attachment behaviors to diminish the patient's feared aloneness without encouraging unnecessary regressions. A framework for understanding the long-term attachment processes required to correct this deficit is offered. CONCLUSIONS: Intolerance of aloneness is a core deficit in borderline patients that can become less handicapping with reliable, but not excessive, responsiveness of the therapist. PMID- 8633686 TI - Clozapine for treatment-refractory mania. AB - OBJECTIVE: The efficacy of clozapine for treatment-resistant mania was examined in a prospective trial for patients with bipolar or schizoaffective disorder. METHOD: The subjects were 25 acutely manic patients with either bipolar disorder (N = 10) or schizoaffective disorder-bipolar subtype (N = 15) for whom lithium, anticonvulsants, and neuroleptics had been ineffective, had produced intolerable side effects, or both. After a 7-day washout, the patients were treated with clozapine monotherapy. They were evaluated over 13 weeks with the Young Mania Rating Scale and the Brief Psychiatric Rating Scale (BPRS). RESULTS: Of the 25 patients, 18 (72%) exhibited marked improvement on the Young Mania Rating Scale, and eight (32%) exhibited marked improvement on the BPRS. The bipolar patients as compared to schizo-affective patients, and the nonrapid as compared to rapid cyclers, had significantly greater improvement in total BPRS score. CONCLUSIONS: These results suggest that clozapine is an effective therapy for treatment resistant bipolar and schizoaffective mania. PMID- 8633687 TI - Relation of serum valproate concentration to response in mania. AB - OBJECTIVE: This study was designed to determine the relation of valproate serum levels to clinical improvement and development of adverse effects in hospitalized patients with acute mania. The initial fixed-dose escalation design, the monotherapy with divalproex, and the control of variables that is possible only with hospitalized patients reduced the confounding factors present in most outpatient studies of serum level-response relationships. METHOD: Sixty-five hospitalized patients who met the Research Diagnostic Criteria for bipolar disorder with mania were treated with divalproex, 750 mg/day for 2 days and then 1,000 mg/day on days 3-5; the dosage was subsequently adjusted as clinically indicated for the remainder of the 21-day study. Manic symptoms were assessed with the Mania Rating Scale, which is derived from the Schedule for Affective Disorders and Schizophrenia. RESULTS: At day 5, patients with serum valproate levels > or = 45 micrograms/ml were two to seven times as likely as patients with levels < 45 micrograms/ml to show 20% or greater improvement in scores on the manic syndrome subscale, the behavior and ideation subscale, elevated mood, increased activity, motor hyperactivity, and psychosis. Endpoint analyses yielded similar results. Adverse experiences characteristic of divalproex treatment were disproportionately associated with serum levels > or = 125 micrograms/ml. CONCLUSIONS: Acutely manic patients treated with divalproex who have valproate serum levels between 45 and 100-125 micrograms/ml are much more likely to have efficacious and well-tolerated responses than patients with lower or higher levels of valproate. PMID- 8633688 TI - Outcome of intensive inpatient treatment for combat-related posttraumatic stress disorder. AB - OBJECTIVE: This study analyzed the outcome of a 4-month intensive inpatient program for combat-related posttraumatic stress disorder (PTSD) among Vietnam veterans. METHOD: The subjects were 51 male veterans with PTSD who completed the inpatient treatment program. Comprehensive measures of PTSD and psychiatric symptoms, as well as social functioning, were assessed at admission, discharge, and 6, 12, and 18 months after discharge. RESULTS: The overall study group showed an increase in symptoms from admission to follow-up and a decrease in violent actions and thoughts and legal problems. Family and interpersonal relationships and overall morale were improved at discharge but then returned to pretreatment levels at 18 months. Patient evaluations also indicated that the program affected morale and interpersonal relationships but not symptoms. CONCLUSIONS: The chronic nature of combat-related PTSD among Vietnam veterans is evident. The study raises the possibility that long-term intensive inpatient treatment is not effective, and other forms of treatment should be considered after rigorous study of such variables as length of stay, trauma versus rehabilitation focus, and patient characteristics. PMID- 8633689 TI - Posttraumatic stress symptoms following forensic dental identification: Mt. Carmel, Waco, Texas. AB - OBJECTIVE: This study was conducted to determine risk factors for posttraumatic stress in medical care professionals who perform postmortem identifications. METHOD: Thirty-one dentists (29 men and two women) who had identified the dead from the fire at the Branch Davidian compound in April 1993 were compared to 47 dentists (45 men and two women) who lived in the area but had not identified any of these remains. Posttraumatic symptoms in both groups were measured by using the Impact of Event Scale and the Brief Symptom Inventory. For the remains handlers only, the subjective distress of handling remains and the social support received during the procedure were reported. RESULTS: Higher scores on the Impact of Event Scale intrusion subscale, the overall Impact of Event Scale, and the obsessive-compulsive subscale of the Brief Symptom Inventory were found for the remains handlers than for the comparison group. Within the remains handler group, distress was significantly related to the hours of exposure to the remains, prior experience handling remains, age, and the support received from spouses and co workers during the identifications. CONCLUSIONS: Posttraumatic stress symptoms can be expected in some health professionals who perform postmortem identifications. Prior experience and social support may mitigate some of these responses. PMID- 8633690 TI - Quality of life for patients with obsessive-compulsive disorder. AB - OBJECTIVE: The health-related quality of life of patients with obsessive compulsive disorder was compared to published norms for the general U.S. population and for patients with either depressive disorders or diabetes. METHOD: Sixty medication-free outpatients with moderate to severe obsessive-compulsive disorder were evaluated by using the Structured Clinical Interview for DSM-III-R and the Yale-Brown Obsessive Compulsive Scale. Health-related quality of life was measured with the self-rated Medical Outcomes Study 36-Item Short-Form Health Survey. RESULTS: The instrumental role performance and social functioning of the patients with obsessive-compulsive disorder were worse than those of the general population and of diabetes patients. The more severe the obsessive-compulsive disorder, the lower were the patients' social functioning scores, even after depression ratings were controlled for; scores on instrumental role performance did not correlate with severity of obsessive-compulsive disorder. The ratings of the obsessive-compulsive disorder patients on physical health domains resembled those of the general population and exceeded those of the diabetes patients. The general health and physical health ratings of the obsessive-compulsive disorder patients exceeded those of the depressed patients. In mental health domains, after adjustment for differences in gender distribution, quality of life ratings were similar for the patients with obsessive-compulsive disorder and those with depressive disorders. CONCLUSIONS: Moderate to severe obsessive-compulsive disorder is associated with impaired social functioning and impaired instrumental role performance, but only impairment in social functioning is linearly related to severity of obsessive-compulsive disorder. PMID- 8633691 TI - Amphetamine use: return of an old scourge in a consultation psychiatry setting. AB - OBJECTIVE: Recreational use of amphetamine and its analogues has increased considerably during the last decade. The aim of this study was to determine possible demographic characteristics of amphetamine users and nonusers who were seen in psychiatric consultations on inpatient medical and surgery wards. METHOD: The authors examined data from 2,983 patients admitted to the University of California, San Diego, Medical Center and seen by consultation-liaison psychiatrists from January 1989 to June 1995. Amphetamine use was identified by the results of toxicological screening or patients' self-reports. RESULTS: Throughout the interval covered by the analysis, 8.7% of the patients with consultations were current amphetamine users. The percentage of users decreased from 9.1% in 1989 to 4.5% in 1992 but increased after that to reach 15.6% in the first half of 1995. The users were generally younger than the nonusers; they were typically male, white, single, uninsured, and unemployed. In comparison with nonusers, the users were more likely to have a past psychiatric history (outpatient treatment or inpatient admission) and a family history of psychiatric disorder. The users were more often admitted because of suicide attempts, had a higher probability of being HIV-positive, and were overrepresented in the psychiatric consultations requested by the departments of infectious diseases, obstetrics/gynecology, and trauma surgery. CONCLUSIONS: The high prevalence of amphetamine-using patients referred for psychiatric consultation is striking. Although young white men were especially likely to be users, there were users in all age groups, in nearly all hospital wards, and with a variety of psychiatric symptoms. PMID- 8633693 TI - Sex differences in neuroanatomical and clinical correlations in schizophrenia. AB - OBJECTIVE: The purpose of this study was to investigate sex differences in relationships between brain and behavior in schizophrenia. METHOD: Frontal lobe, temporal lobe, and whole brain volumes were obtained from magnetic resonance images of 91 patients with schizophrenia (54 men and 37 women) and 114 healthy comparison subjects (62 men and 52 women). Four independent symptom scales, based on the following symptom clusters, were derived from clinical data: negative, disorganization, Schneiderian hallucinations-delusions, and suspicion-hostility. Regression analyses incorporating the four clinical scales and neuroanatomical volumes were performed to investigate possible interactions between brain region and sex. RESULTS: Significant interactive effects of sex and frontal lobe volume were found in regression analyses of the disorganization and suspicion-hostility symptom scales. In men, higher frontal lobe volume was associated with milder severity of disorganization but was not correlated with severity of suspicion hostility. In women, higher frontal lobe volume was associated with more severe disorganization as well as more severe suspicion-hostility. No associations were found between brain volume and severity of negative or Schneiderian symptoms. CONCLUSIONS: Differences between male and female patients were observed in the relationships between frontal lobe volume and two of the four clinical dimensions examined. These findings suggest that aspects of the neuropathological basis for some symptoms of schizophrenia may be sexually dimorphic. PMID- 8633692 TI - Injection drug use and risk of HIV transmission among homeless men with mental illness. AB - OBJECTIVE: The high seroprevalence of HIV that has been reported among homeless individuals with mental illness indicates an urgent need to examine HIV risk behavior in this population. METHOD: Injection drug use and sexual behavior were assessed in comprehensive interviews with 218 homeless mentally ill men in a New York City shelter. First, the proportion of men who had injected drugs was established. Then, among those who had injected drugs, the injection drug use behaviors associated with HIV transmission (i.e., whether they had ever engaged in high-risk behaviors and had ever engaged in risk-reduction behaviors) and their current sexual risk behaviors were examined. RESULTS: Fifty (23%) of the 218 men had injected drugs. Among these 50, the great majority had engaged in high-risk behaviors, including sharing needles (66%) and using shooting galleries (64%). Few had engaged in risk-reduction behaviors, such as cleaning needles with bleach (22%) and using a needle exchange program (2%). In the past 6 months alone, the majority of the injection drug users had had unprotected sex with women (48%) or with men (10%). CONCLUSIONS: This study documents a high lifetime prevalence of injection drug use in a group of homeless men with mental illness. The men who had injected drugs reported injection drug use and sexual behaviors with high risk of HIV transmission and gave scant evidence of risk-reduction behaviors. These individuals may fall between service systems and may be difficult to reach but, nonetheless, must be included in efforts to prevent transmission of HIV infection. PMID- 8633694 TI - Clinical characteristics of Kraepelinian schizophrenia: replication and extension of previous findings. AB - OBJECTIVE: Subtypologies of schizophrenia based on cross-sectional criteria, such as the nomenclature of the DSMs, have not been successful in identifying valid diagnostic subgroups among patients with schizophrenia. A subtypology that uses criteria to classify individuals on the basis of longitudinal deficits in self care may identify a more valid subgroup of schizophrenic patients. METHOD: This study describes the clinical characteristics of a group of schizophrenic patients identified on the basis of a longitudinal criterion: at least 5 years of continuous and complete dependence on others for obtaining and maintaining the basic necessities of life, including food, clothing, and shelter. RESULTS: Sixty one "Kraepelinian" schizophrenic inpatients, when compared to 80 non-Kraepelinian schizophrenic inpatients who were similar in years of illness, age, and education, demonstrated more severe negative symptoms and more severe formal thought disorder; yet the severity of their delusions, hallucinations, and bizarre behavior did not differ significantly. None of the Kraepelinian patients and eight non-Kraepelinian patients met DSM-III-R criteria for schizoaffective disorder. CONCLUSIONS: Data from this replication study suggest that Kraepelinian schizophrenic patients, identified on the basis of a longitudinal course characterized by severe dysfunctions in self-care, may represent an alternative, and possibly more valid, method of subtyping schizophrenia. PMID- 8633695 TI - When does amphetamine-induced psychosis become schizophrenia? PMID- 8633696 TI - Images in Psychiatry. The Burgholzli Clinic. PMID- 8633697 TI - Differential effect of clozapine on weight: a controlled study. AB - OBJECTIVE: This study examined whether clozapine induces more weight gain than haloperidol and whether weight gain is related to clinical improvement. METHOD: The weight and symptoms of 39 outpatients with schizophrenia who were randomly assigned to double-blind treatment with either clozapine or haloperidol were assessed. The weight and symptoms of 33 of the patients who chose to take clozapine during a 1-year follow-up after the study ended were also assessed. RESULTS: The patients treated with clozapine gained significantly more weight over baseline (7%) than the haloperidol-treated patients (1%). Weight gain was not significantly correlated with improvements in either positive or negative symptoms. Fifty-eight percent of the patients followed for 1 year gained at least 10% over their baseline weight. CONCLUSIONS: Weight gain is an important side effect of clozapine and is unrelated to the drug's differential antipsychotic efficacy. PMID- 8633699 TI - Prospective evaluation of panic potentiation following 35% CO2 challenge in nonclinical subjects. AB - OBJECTIVE: The authors examined the effect of panic provocation on the subsequent development of panic attacks and panic disorder in nonclinical subjects with no history of spontaneous panic. METHOD: Sixty-two subjects who had completed a study examining fearful responses to a single vital capacity inhalation of 35% CO2 were reevaluated 1 year following the challenge test. RESULTS: Challenge induced panic was not related to the later development of panic or panic disorder. According to the Structured Clinical Interview for DSM-III-R--Non Patient Edition, none of the subjects met DSM-III-R criteria for panic disorder and only six subjects reported spontaneous panic during the year after panic provocation. Of the six subjects who experienced spontaneous panic, two had panicked in response to the CO2 challenge. CONCLUSIONS: The experimental provocation of panic in nonclinical subjects appears to be a safe research paradigm for exploring the psychopathogenicity of panic disorder. PMID- 8633698 TI - Serum levels of clozapine and norclozapine in patients treated with selective serotonin reuptake inhibitors. AB - OBJECTIVE: The selective serotonin reuptake inhibitor (SSRI) fluoxetine can increase serum levels of clozapine and norclozapine, but effects of other SSRIs are unknown. Thus, the authors evaluated interactions of clozapine with fluoxetine, paroxetine, and sertraline. METHOD: Serum clozapine and norclozapine concentrations were assayed in 80 psychiatric patients, matched for age and clozapine dose, given clozapine (mean dose = 279 mg/day) alone or with fluoxetine (mean dose = 39.3 mg/day), paroxetine (mean = 31.2 mg/day), or sertraline (mean = 92.5 mg/ day). Each patient's dose of clozapine was stable for at least a month before serum sampling. RESULTS: Concentrations of clozapine plus norclozapine averaged 43% higher, and the risk of levels higher than 1000 ng/ml was 10-fold greater (25%), in the patients taking SSRIs, with minor differences between patients taking the individual SSRIs. CONCLUSIONS: SSRIs can increase circulating concentrations of clozapine and norclozapine, sometimes to potentially toxic levels. PMID- 8633700 TI - Defense mechanisms and adjustment in normal adolescents. AB - OBJECTIVE: This study compared self-perception of defense patterns with objective and independent ratings of general adjustment in normal adolescents. METHOD: Self perception of defense utilization by 140 high school students was measured by an adolescent version of Bond's Defense Style Questionnaire. The DSM-III-R Global Assessment of Functioning Scale was used by clinicians in determining general adjustment. RESULTS: Defense style and adjustment were significantly related; greater maturity of defense style was associated with better global adjustment, and conversely, greater immaturity of defense style was associated with a lower level of functioning. CONCLUSIONS: Adolescents' self-perceived defense patterns have conscious correlates associated with general adjustment. This study extends the validity of the defense mechanism paradigm to the adolescent age group. PMID- 8633701 TI - Prediction of 3-year outcome of treated alcoholics by an empirically derived multivariate typology. AB - OBJECTIVE: This study examined whether an empirically derived multivariate typology can predict outcome in treated alcoholics. METHOD: Two hundred fifty nine hospitalized male alcoholics were divided into two subtypes by means of cluster analysis of clinical factors. Type A showed the features of sporadic late onset alcoholism, whereas type B manifested the characteristics of familial early onset alcoholism. The prospective 3-year outcomes in the two subtypes were compared. RESULTS: The follow-up rate was 83%. The mortality and abstinence rates for type A patients (N = 98) were 15.3% and 32.7%, respectively, and for type B patients (N = 116), 23.3% and 18.1%, respectively. The age-corrected relative mortality risk and relative abstinence rate for type A patients compared with type B patients were 0.59 and 1.60, respectively. CONCLUSIONS: Familial early onset alcoholism has a poor outcome in Japan, and this multivariate typology has high predictive validity. PMID- 8633703 TI - Conversion disorder with pseudohallucinations. PMID- 8633702 TI - How well does the psychiatry residency in-training examination predict performance on the American Board of Psychiatry and Neurology. Part I. Examination? AB - OBJECTIVE: Scores on the American College of Psychiatrists' Psychiatry Residency In-Training Examination (PRITE) were correlated with scores on the American Board of Psychiatry and Neurology (ABPN) Part I examination in psychiatry. METHOD: Pearson correlations between 1992 PRITE scores and 1994 ABPN Part I psychiatry scores were calculated. Analyses were based on 701 examinees. RESULTS: There was a moderate correlation between scores on the PRITE and ABPN Part I examination in psychiatry and a low correlation of neurology scores. CONCLUSIONS: These correlations were observed even though the two examinations differ in their purposes, content outlines, development, and administration. PMID- 8633704 TI - Use of SPECT to evaluate postcardiotomy delirium. PMID- 8633705 TI - Inositol-induced mania? PMID- 8633706 TI - Clozapine-induced parotitis: an immunological cause? PMID- 8633708 TI - Clozapine treatment for a veteran with comorbid psychosis and PTSD. PMID- 8633707 TI - Elevated clozapine levels after fluvoxamine initiation. PMID- 8633710 TI - Valproic acid for panic disorder associated with multiple sclerosis. PMID- 8633709 TI - Delirium caused by tacrine and ibuprofen interaction. PMID- 8633711 TI - Tardive dyskinesia induced by risperidone? PMID- 8633712 TI - Mood alterations and tramadol. PMID- 8633713 TI - Critique of DSM-IV: associated features of neuroleptic-induced parkinsonism. PMID- 8633714 TI - "Smart drugs": a caution to everybody. PMID- 8633715 TI - Gambling in Minnesota. PMID- 8633716 TI - Predicting inpatient violence. PMID- 8633717 TI - Previous neurobiological study of Holocaust survivors. PMID- 8633718 TI - Further exploration of gender differences in personality disorders. PMID- 8633719 TI - On psychotic major depression. PMID- 8633720 TI - Sexual abuse and borderline personality disorder. PMID- 8633721 TI - What's in a name? PMID- 8633723 TI - School and seasonal affective disorder. PMID- 8633722 TI - What's in a name? PMID- 8633724 TI - Reviewing DSM-IV. PMID- 8633725 TI - Position statement on training needs in addiction psychiatry. AB - Psychiatry has only recently developed training in the area of substance-related disorders. Nevertheless, these illnesses are highly prevalent and are associated with substantial morbidity and mortality. Patients with these disorders often do not encounter or have access to effective treatment. Because of this, the American Psychiatric Association recommends developing improved training to assure that 1) the basic psychiatric residency addresses essential aspects of addiction prevention and treatment, 2) addiction psychiatry fellowships are strengthened to provide needed manpower for consultation, academic teaching, and research, and 3) psychiatrists are trained to provide leadership for the multidisciplinary teams characteristic of this field. This statement was drafted by the Committee on Training and Education in Addiction Psychiatry. It was approved by the APA Assembly in November 1995 and by the Board of Trustees in December 1995. PMID- 8633726 TI - Position statement on substance-related disorders. AB - Substance-related disorders are widespread among the general public and are often accompanied by other psychiatric disorders. However, historical social stigma and other factors have led to underdiagnosis and limited access to care. Cost effective treatment is best delivered in a comprehensive, flexible continuum of services, which should be accessible on the same basis as other medical care. The American Psychiatric Association should continue to promote access to care, high quality treatment, education, training, research, parity in third-party coverage, and equal treatment for patients suffering from addictive disorders. This statement was drafted by the Task Force on Psychiatric Services for Addicted Patients. It was approved by the Assembly in November 1995 and by the Board of Trustees in December 1995. PMID- 8633727 TI - Preventing tobacco use--the youth access trap. PMID- 8633728 TI - The amphibole hypothesis of asbestos-related cancer--gone but not forgotten. PMID- 8633729 TI - Topics for our times: don't inhale--reflections on Garbage! PMID- 8633730 TI - Public hospitals and health care reform: choices and challenges. PMID- 8633731 TI - The Washington Heights-Inwood Healthy Heart Program: a 6-year report from a disadvantaged urban setting. AB - OBJECTIVES: This report summarizes 6 years of experience in a large community based cardiovascular disease prevention program in a predominately minority, urban setting. METHODS: The program seeks to reduce cardiovascular disease risk factors in an area of approximately 240,000 people in New York, NY; this population includes many Latino immigrants of low educational attainment and socioeconomic status. All program materials were in Spanish and English and at a low literacy level. RESULTS: Major elements that achieved high levels of reach and support were a marketing campaign promoting low-fat milk, exercise clubs, and a Spanish-language smoking cessation video. Program elements that did not meet expectations or were abandoned were school-based smoking prevention initiatives, cholesterol screening, and efforts to involve local physicians. At the end of 6 years, the program was transferred to a local community organization. CONCLUSIONS: Conclusions are that it is feasible to implement a complex cardiovascular disease prevention program in a socially disadvantaged urban community; that additional evaluation research is needed; that such programs can be transferred from an academic center to a community organization; and that such programs are unlikely to be sustained effectively without external resources. PMID- 8633732 TI - Who administers? Who cares? Medical administrative and clinical employment in the United States and Canada. AB - OBJECTIVES: We compared US and Canadian health administration costs using national medical care employment data for both countries. METHODS: Data from census surveys on hospital, nursing home, and outpatient employment in the United States (1968 to 1993) and Canada (1971 and 1986) were analyzed. RESULTS: Between 1968 and 1993, US medical care employment grew from 3.976 to 10.308 million full time equivalents. Administration grew from 0.719 to 2.792 million full-time equivalents, or from 18.1% to 27.1% of the total employment. In 1986, the United States deployed 33,666 health care full-time equivalent personnel per million population, and Canada deployed 31,529. The US excess was all administrative; Canada employed more clinical personnel, especially registered nurses. Between 1971 and 1986, hospital employment per capita grew 29% in the United States (mostly because of administrative growth) and fell 14% in Canada. In 1986, Canadian hospitals still employed more clinical staff per million. Outpatient employment was larger and grew faster in the United States. Per capita nursing home employment was substantially higher in Canada. CONCLUSIONS: If US hospitals and outpatient facilities adopted Canada's staffing patterns, 1,407,000 fewer managers and clerks would be necessary. Despite lower medical spending, Canadians receive slightly more nursing and other clinical care than Americans, as measured by labor inputs. PMID- 8633734 TI - The elimination of selected chronic diseases in a population: the compression and expansion of morbidity. AB - OBJECTIVES: This study evaluates the effect of eliminating a specific disease on the mortality, long-term disability, and overall health status of a population. Primarily, it examines whether elimination leads to a compression of morbidity. METHODS: The Sullivan method was used to calculate disability-free life expectancy. Cause-deleted disability prevalence was estimated with a multiple logistic regression model that used data from the Dutch National Survey of General Practice. Cause-deleted probabilities of dying were derived with the cause-elimination life-table technique, assuming independence among competing causes of death. RESULTS: Eliminating disabling nonfatal diseases such as arthritis/back complaints results in a decline in life expectancy with disability -that is, an absolute compression of morbidity. Eliminating highly fatal diseases such as cancer leads to an increase in the number of years and the proportion of life with disability--that is, a relative expansion of morbidity. CONCLUSIONS: While eliminating fatal diseases leads to an increase in disability-free life expectancy, life expectancy with disability may increase as well. This represents an increasing burden to society. On the other hand, eliminating nonfatal disabling diseases leads to absolute compression of morbidity. PMID- 8633733 TI - Occupational exposure to chrysotile asbestos and cancer risk: a review of the amphibole hypothesis. AB - OBJECTIVES: This article examines the credibility and policy implications of the "amphibole hypothesis," which postulates that (1) the mesotheliomas observed among workers exposed to chrysotile asbestos may be explained by confounding exposures to amphiboles, and (2) chrysotile may have lower carcinogenic potency than amphiboles. METHODS: A critical review was conducted of the lung burden, epidemiologic, toxicologic, and mechanistic studies that provide the basis for the amphibole hypothesis. RESULTS: Mechanistic and lung burden studies do not provide convincing evidence for the amphibole hypothesis. Toxicologic and epidemiologic studies provide strong evidence that chrysotile is associated with an increased risk of lung cancer and mesothelioma. Chrysotile may be less potent than some amphiboles for inducing mesotheliomas, but there is little evidence to indicate lower lung cancer risk. CONCLUSIONS: Given the evidence of a significant lung cancer risk, the lack of conclusive evidence for the amphibole hypothesis, and the fact that workers are generally exposed to a mixture of fibers, we conclude that it is prudent to treat chrysotile with virtually the same level of concern as the amphibole forms of asbestos. PMID- 8633735 TI - Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992. AB - OBJECTIVES: This study sought to describe prevalence rates of chronic gynecological conditions and correlates of these conditions in a representative sample of US women. METHODS: National Health Interview Survey data from 1984 through 1992 for women aged 18 to 50 were used. RESULTS: The estimated annual prevalence rate for the reported presence of one or more gynecological conditions was 97.1 per 1,000 women. Menstrual disorders were most common, with an annual prevalence rate of 53.0 per 1,000 women. Adnexal conditions and fibroids were the next most common conditions, with rates per 1,000 women of 16.6 and 9.2, respectively. Prolapse, endometriosis, and fibroids were the conditions most likely to lead to hysterectomy within the year prior to the interview. More than three quarters (77.1%) of women with gynecological conditions had talked with a doctor in the previous year concerning their condition, and 28.8% reported spending 1 or more days in bed in the previous year because of their condition. CONCLUSIONS: Nearly a tenth of American women aged 18 to 50 report having one or more chronic gynecological conditions annually, the most common being disorders of menstruation. PMID- 8633736 TI - The prevalence of self-reported peptic ulcer in the United States. AB - OBJECTIVES: The purpose of this study was to draw a current picture of the sociodemographic characteristics of peptic ulcer in the United States. METHODS: During the National Health Interview Survey of 1989, a special questionnaire on digestive diseases was administered to 41,457 randomly selected individuals. Data were retrieved from public use tapes provided by the National Center for Health Statistics. Odds ratios were calculated by logistic regression after adjustment for sample weights in the survey. RESULTS: Of adult US residents, 10% reported having physician-diagnosed ulcer disease, and one third of these individuals reported having an ulcer in the past year. Old age, short education, low family income, being a veteran, and smoking acted as significant and independent risk factors. Gastric and duodenal ulcer occurred in both sexes equally often. Duodenal ulcer was more common in Whites than non-Whites, while gastric ulcer was more common in non-Whites. CONCLUSIONS: The age-related rise and socioeconomic gradients of peptic ulcer represent the historic scars of previous infection rates with Helicobacter pylori. The racial variations reflect different ages at the time of first infection; younger and older age at the acquisition of H. pylori appear to be associated with gastric and duodenal ulcer, respectively. PMID- 8633738 TI - Smoking cessation in young adults: age at initiation of cigarette smoking and other suspected influences. AB - OBJECTIVES: Previous research has suggested that early smoking initiation predicts longer duration of smoking, heavier daily consumption, and increased chances of nicotine dependence. This report set out to estimate the relationship between smoking cessation and age of initiation, as well as nicotine dependence, sex, race, and education. METHODS: A sample of 1007 young adults was randomly selected from a large health maintenance organization in southeast Michigan. Hazard ratios of quitting associated with age at smoking initiation were estimated among 414 persons who smoked daily for 1 month or more. RESULTS: With potential confounders controlled for, the likelihood of cessation was significantly higher in smokers who initiated smoking after age 13. The hazard ratio for quitting associated with smoking initiation at ages 14 to 16 was 1.6 and with initiation at or after age 17 was 2.0, compared with initiation at or before 13 years of age. Factors that decreased the likelihood of cessation were nicotine dependence and low education. CONCLUSIONS: Public policy to discourage early smoking, if it succeeds in delaying the initiation of smoking, might contribute to the reduction of smoking-related mortality and morbidity by increasing the potential for quitting. PMID- 8633737 TI - Preventing cardiovascular disease through community-based risk reduction: the Bootheel Heart Health Project. AB - OBJECTIVES: The purpose of this study was to determine whether a community-based risk reduction project affected behavioral risk factors for cardiovascular disease. METHODS: Community-based activities (e.g., exercise groups, healthy cooking demonstrations, blood pressure and cholesterol screenings, and cardiovascular disease education) were conducted in six southeastern Missouri counties. Evaluation involved population-based, cross-sectional samples of adult residents of the state and the intervention region. Weighted prevalence estimates were calculated for self-reported physical inactivity, cigarette smoking, consumption of fruits and vegetables, overweight, and cholesterol screening. RESULTS: Physical inactivity decreased within the intervention region, that is, in communities where heart health coalitions were developed and among respondents who were aware of these coalitions. In addition, the prevalence rates for reports of cholesterol screening within the past 2 years were higher for respondents in areas with coalitions and among persons who were aware of the coalitions. CONCLUSIONS: Even with modest resources, community-based interventions show promise in reducing self-reported risk for cardiovascular disease within a relatively brief period. PMID- 8633739 TI - Youth access to tobacco: the effects of age, gender, vending machine locks, and "it's the law" programs. AB - OBJECTIVES: This study evaluated the influence of age, gender, vending machine lockout devices, and tobacco industry-sponsored voluntary compliance programs ("It's the Law" programs) on underage youths' ability to purchase tobacco. METHODS: Twelve youths made 480 attempts to purchase tobacco in Massachusetts from over-the-counter retailers and vending machines with and without remote control lockout devices. Half the vendors were participating in It's the Law programs. RESULTS: In communities with no requirements for lockout devices, illegal sales were far more likely from vending machines than from over-the counter sources (odds ratio [OR] = 5.9, 95% confidence interval [CI] = 3.3, 10.3). Locks on vending machines made them equivalent to over-the-counter sources in terms of illegal sales to youths. Vendors participating in It's the Law programs were as likely to make illegal sales as nonparticipants (OR = 0.87, 95% CI = 0.57, 1.35). Girls and youths 16 years of age and older were more successful at purchasing tobacco. CONCLUSIONS: The It's the Law programs are ineffective in preventing illegal sales. While locks made vending machines equivalent to over the-counter sources in their compliance with the law, they are not a substitute for law enforcement. PMID- 8633741 TI - Smoking prevalence in US birth cohorts: the influence of gender and education. AB - OBJECTIVES: To assess long-term trends in cigarette smoking according to the combined influence of sex and education, this study examined smoking prevalence in successive US birth cohorts. METHODS: Data from nationally representative surveys were examined to assess smoking prevalence for six successive 10-year birth cohorts stratified by race or ethnicity, sex, and educational attainment. RESULTS: Substantial declines in smoking prevalence were found among men who had a high school education or more, regardless of race or ethnicity, and slight declines among women of the same educational background were revealed. However, little change was found in smoking prevalence among men of all race/ethnic groups with less than a high school education, and large increases were found among women with the same years of schooling, especially if they were White or African American. CONCLUSIONS: These data suggest that persons of low educational attainment have yet to benefit from policies and education about the health consequences of cigarette smoking. PMID- 8633740 TI - Is smoking associated with depression and anxiety in teenagers? AB - OBJECTIVES: An association of smoking with depression and anxiety has been documented in adult smokers. This study examines this association in a representative group of teenage smokers. METHODS: A two-stage cluster sample of secondary school students in Victoria, Australia, were surveyed by using a computerized questionnaire, which included a 7-day retrospective diary for tobacco use and a structured psychiatric interview. RESULTS: Subjects reporting high levels of depression and anxiety were twice as likely to be smokers after the potential confounders of year level, sex, alcohol use, and parental smoking were controlled for. Regular smokers were almost twice as likely as occasional smokers to report high levels of depression and anxiety. In a stratified analysis, an association between regular smoking and psychiatric morbidity was found in girls of all ages but for boys only in the youngest group. CONCLUSIONS: The cross-sectional association is consistent with the use of smoking by teenage girls as self-medication for depression and anxiety. Therefore, future health promotional campaigns might consider strategies that attend to perceived psychological benefits of smoking. PMID- 8633742 TI - Temporal variation in drinking water turbidity and diagnosed gastroenteritis in Milwaukee. AB - Daily counts of diagnosed gastroenteritis (gastrointestinal events) in Milwaukee County, Wisconsin, from January 1992 through April 1993 were compared with reported daily turbidity from the two drinking water treatment plants serving the county. Turbidity in both plants was associated with an increased number of gastrointestinal events even after exclusion of a major documented outbreak of cryptosporidiosis. During the 434-day period prior to the outbreak, an increase in turbidity of 0.5 nephelometric turbidity units at one of the plants was associated with relative risks for gastrointestinal events of 2.35 among children (95% confidence interval [CI] = 1.34, 4.12) and 1.17 among adults (95% CI = 0.91, 1.52). PMID- 8633743 TI - Preconceptional and prenatal multivitamin-mineral supplement use in the 1988 National Maternal and Infant Health Survey. AB - This paper examines the prevalence of multivitamin-mineral supplement use before and during pregnancy, as well as predictors of nonuse, in 9953 women who delivered live infants in the 1988 National Maternal and Infant Health Survey. Ninety-seven percent of the women were advised to take multivitamin-mineral supplements in prenatal care. Sixty-seven percent of Black mothers took supplements during pregnancy, as compared with 84% of White mothers. Multivariate analysis revealed that Black mothers; mothers who are less educated, younger, unmarried, and non-smokers; and mothers who participate in Women, Infants, and Children programs are at elevated risk for nonuse. These data help identify groups in need of supplementation guidance. PMID- 8633744 TI - The validity of hospital administrative data in monitoring variations in breast cancer surgery. AB - To assess the validity of using hospital administrative data to measure variations in surgery for early-stage breast cancer, ICD-9-CM coded information was compared with corresponding tumor registry data for 1293 breast cancer patients undergoing lumpectomy or mastectomy at a tertiary referral center from January 1989 to October 1993. Relative to "gold standard" tumor registry data, the administrative data proved 83.4% sensitive and 80.4% specific in identifying women with localized disease who would be potential candidates for lumpectomy. The proportion of women with localized disease undergoing lumpectomy in groups defined by race and insurance status was nearly identical, whichever data were used. Administrative data, which is often readily and publicly available, may be useful in studying variations in breast cancer treatment in key demographic groups. PMID- 8633745 TI - An education program for parents of children with asthma: differences in attendance between smoking and nonsmoking parents. AB - We studied smoking status in relation to parental attendance at an asthma education program for child patients of a health maintenance organization. Nonattendance rates were 24%, 42%, and 78% in nonsmoking, one-smoker, and two-or more-smoker families, respectively, and 33% overall. Only the number of smokers (odds ratio [OR] = 3.1; 95% confidence interval [CI] = 1.8, 5.3) and perceived adverse impact of asthma on the family (OR = 0.4; 95% CI = 0.2, 0.9) were retained in a multivariate model that correctly classified 73% of families; demographic characteristics, frequency of asthma symptoms, and health care use were rejected. There was a tendency for smoking parents to deny that their child had asthma (17% among families with two or more smokers; 9% among nonsmoking families). Asthma education programs may fail to involve parents who smoke. PMID- 8633746 TI - Maternal smoking during pregnancy and the risk of congenital urinary tract anomalies. AB - To study maternal smoking during pregnancy and the risk of congenital urinary tract anomalies, we interviewed mothers of 118 affected infants born to residents of western Washington State during 1990 and 1991 and mothers of 369 control infants randomly selected from those without birth defects delivered during those years in five hospitals in King County, Washington. Maternal smoking was associated with an increased risk of congenital urinary tract anomalies in offspring (adjusted odds ratio [OR] = 2.3; 95% confidence interval [CI] = 1.2, 4.5). This risk was higher among light smokers (1-1000 cigarettes during the pregnancy) (OR = 3.7; 95% CI = 1.7, 8.6) than among heavy smokers (OR = 1.4; 95% CI = 0.6, 3.3). Our results corroborate previous findings and support the hypothesis of a causal relation. PMID- 8633747 TI - How long will today's new adolescent smoker be addicted to cigarettes? AB - This study estimated the expected smoking duration for young smokers who have started recently. Data from National Health Interview Surveys were combined to model the ages at which smoking prevalence will decline to various percentages of the peak smoking prevalence for each successive birth cohort. Smoking-cessation ages were then estimated for the males and females born from 1975 through 1979. The median cessation age for those in this cohort who start smoking as adolescents is expected to be 33 years for males and 37 years for females. Thus, 50% of these adolescent males may smoke for at least 16 years and 50% of these adolescent females may smoke for at least 20 years, based on a median age of initiation of 16 to 17 years. Despite the decline in the median age of US smokers who quit, these data predict that smoking will be a long-term addiction for many adolescents who start now. PMID- 8633749 TI - The injection of crack cocaine among Chicago drug users. PMID- 8633748 TI - Temporarily detained: tuberculous alcoholics in Seattle, 1949 through 1960. AB - Repeatedly noncompliant tuberculosis patients (who are often homeless or substance users) are once again being forcibly detained. Health officials intend that confinement be used only when "less restrictive alternatives" have failed. Past programs of detention can inform current efforts. In 1949, Seattle's Firland Sanatorium established a locked ward. Although initially intended only for active public health threats, the ward was eventually used to maintain order among Firland's alcoholic patients. That is, the staff detained alcoholics--regardless of their infectivity or compliance with medications--for breaking sanatorium rules. In this manner, maintaining institutional order became a legitimate reason for invoking public health powers. Although new detention regulations strive to protect patients' civil liberties, attention must also be paid to the day-to-day implementation of coercive measures. When public health language is used to justify administrative or institutional requirements, disadvantaged patients may be stigmatized. PMID- 8633750 TI - The hazards of epidemiology, continued. PMID- 8633751 TI - Clarifying the issues in tuberculosis control. PMID- 8633752 TI - A call for the destruction of smallpox virus stores. PMID- 8633753 TI - The implications of CDC's research on organizational downsizing. PMID- 8633754 TI - A monoclonal immunoassay for the coplanar polychlorinated biphenyls. AB - Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants with diverse toxic, teratogenic, reproductive, immunotoxic, and tumorigenic effects. Three of the least abundant of the 209 PCB isomers (congeners) are the most toxic and most difficult to quantify. These are 3,4,3',4'-tetrachlorobiphenyl, 3,4,3',4',5'-pentachlorobiphenyl, and 3,4,5,3',4',5'-hexachlorobiphenyl (IU-PAC No. 77, 126, and 169, respectively). An immunizing hapten was designed to retain the 3,4,3',4' chlorine-substitution pattern and coplanarity characteristic of these toxic congeners. The optimal competitors for immunoassay were weaker binding distinctive single-ring fragments of the PCBs. A monoclonal antibody designated S2B1 was derived and used in direct (antibody-capture) competitive enzyme immunoassays (EIAs). The EIAs are highly specific for non-ortho substituted congeners and do not recognize the more prevalent but much less toxic noncoplanar PCB congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8 tetrachlorodibenzofuran, or dichlorobenzenes. Hapten and competitor design for this assay suggests a basis for development of sensitive EIAs for other classes of PCB congeners. PMID- 8633755 TI - XPS and TOF-SIMS microanalysis of a peptide/polymer drug delivery device. AB - The localization of a peptide drug dispersed in a solid matrix of hydroxypropyl cellulose (HPC) was determined at micrometer lateral resolution using secondary ion mass spectrometry (SIMS)/ion microscopy. Leuprolide formulated as a sustained release drug delivery device has been selected as a model compound for this investigation. One key facet of this study was to attempt to understand the distribution and ultimate bioavailability of the peptide dispersed in an inert polymer matrix. The results reported in this paper demonstrate that the lateral distribution of leuprolide along the surfaces of cross sections prepared from different polymer formulations is different. Ion microscopy directly measures the lateral distribution of protonated molecular ions as well as specific fragment peaks and provides a direct method of determining peptide distributions in polymers. Ion images of leuprolide dispersed in HPC demonstrate that the peptide distribution is critically dependent on polymer composition. The mass spectrometry results augment quantitative X-ray photoelectron (XPS) measurement of C and N levels in different polymer/peptide formulations. The combination of XPS and TOF-SIMS techniques provides a powerful method for determining the distribution of peptides in polymer matrices. PMID- 8633756 TI - Synthesis and use of quaternized polyethylenimine-coated zirconia for high performance anion-exchange chromatography. AB - The synthesis of an alkali-stable strong anion-exchange stationary phase by deposition of polyethylenimine (PEI), followed by cross-linking and quaternization, onto porous zirconia particles is described. Physical characterization of quaternized PEI-zirconia and PEI-zirconia shows that 50% and 24% of the amine groups are cross-linked, respectively. A plot of log k' versus log (competing ion concentration) is linear for three homopeptides, suggesting that ion exchange is the primary mechanism of retention on quaternized PEI zirconia. Column efficiency for two 2,4-dinitrophenyl amino acids increased by 80% upon increasing the temperature from 50 degrees C to 100 degrees C. The hydrophobicity of quaternized PEI-zirconia was studied using a homologous series of p-alkoxybenzoic acids. For quaternized PEI-zirconia and PEI-zirconia, we found that the free energy of transfer of a methylene unit from the mobile phase to the stationary phase was -2.0 and -0.90 kJ/mol, respectively. The free energy of transfer of a methylene unit on quaternized PEI-zirconia is similar to that of a typical ODS phase (-2.4 kJ/mol). A van't Hoff plot for the above two 2,4 dinitrophenyl amino acids showed that the enthalpies of transfer are exothermic and fairly large (approximately -14 kJ/mol). Isocratic separations on quaternized PEI-zirconia of inorganic and organic anions are presented. Quaternized PEI zirconia, quaternary amine-functionalized silica, and PEI-zirconia are compared chromatographically. Quaternized PEI-zirconia is more efficient than the silica based phase in the separation of benzoic acid derivatives but slightly less efficient than PEI-zirconia. The major virtue of quaternized PEI-zirconia is that it is chemically stable in the pH range of 1-13 and is also stable at temperatures up to 100 degrees C. PMID- 8633757 TI - Selective detection in RP-HPLC of Tyr-, Trp-, and sulfur-containing peptides by pulsed amperometry at platinum. AB - A technique for electrochemical detection of Trp-, Tyr-, and sulfur-containing peptides, using a two-step potential waveform at a platinum wall-jet electrode, has been developed. The detection is fully compatible with reversed-phase HPLC employing gradients of acetonitrile in water/trifluoroacetic acid. At approximately +1.2 V (first potential) versus Ag/AgCl, Trp-, Tyr-, and Cys containing peptides are predominantly detected, while at +1.4 - 1.6 V, Met- and (Cys)2-containing peptides are additionally detected. The electrode surface is cleaned by the second potential (+2.0 V). the linearity is at least 2 orders of magnitude. The sensitivity is in the picomole range. By using postcolumn electrochemical conversion, the selectivity toward Met and Cys-containing peptides can be enhanced. Applications are shown for the determination of caseinomacropeptide (6.6 kDa) and a tryptic map of beta-casein. PMID- 8633758 TI - Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement. AB - Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator. PMID- 8633759 TI - Tandem reflectron time-of-flight mass spectrometer utilizing photodissociation. AB - A tandem time-of-flight (TOF) mass spectrometer has been designed to obtain complete MS/MS spectra from compounds eluting from a gas chromatograph. This application requires high spectral generation rate, unit mass resolution for both precursor selection and product spectra, and efficient ion utilization. These objectives are achieved by reflectron TOF mass separation in both stages and laser photoinduced dissociation as the ion fragmentation method. Careful timing of the laser pulse relative to ion extraction allows ions of a single m/z value up to m/z 1000 to be photodissociated while ions with adjacent m/z values are essentially unaffected. The convergent foci of the ion packet and laser pulse results in ion fragmentation efficiencies as high as 79%. An ion gate prevents the nonselected precursor ions from convoluting the product spectra. Product spectra can be generated at the maximum laser repetition rate (currently 200 Hz). To achieve unit mass resolution for all product m/z values simultaneously, a novel reflectron was designed for the second TOF stage. PMID- 8633760 TI - C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration dependent digestions. AB - The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments. PMID- 8633761 TI - Structural characterization of phospholipids by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. AB - Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry provides for structural analysis of the principal biological phospholipids: glycerophosphatidylcholine, -ethanolamine, serine, and -inositol. Both positive and negative molecular or quasimolecular ions are generated in high abundance. Isolated molecular ions may be collisionally activated in the source side of a dual trap mass analyzer, yielding fragments serving to identify the polar head group (positive ion mode) and fatty acid side chains (negative ion mode). Azimuthal quadrupolar excitation following collisionally activated dissociation refocuses productions close to the solenoid axis; subsequent transfer of product ions to the analyzer ion trap allows for high-resolution mass analysis. Cyro-cooling of the sample probe with liquid nitrogen greatly reduces matrix adduction encountered in the negative ion mode. PMID- 8633762 TI - Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions. AB - By utilizing delayed pulsed ion extraction of ions generated via the matrix assisted laser desorption/ionization (MALDI) technique, fast (< 320 ns) metastable ion fragmentation is observed for both peptide and protein analytes in the ion source of a linear time-of-flight mass spectrometer. Small peptides such as the oxidized B chain of bovine insulin exhibit fragmentation at the amide linking bond between peptide residues. Overlapping sequence information is provided by fragmentation from both the C- and N-terminal ends of the peptide (cn , yn-, and z*n-type fragment ions). Larger proteins can also exhibit a wealth of sequence specific fragment ions in favorable cases. One example is cytochrome c, which undergoes substantial (approximately 80%) fast fragmentation at the amide bonds along the amino acid backbone of the protein. Only amide bond cleavages initiating from the C-terminal end (cn fragments) are observed. The observed fragmentation pattern provides a significant amount of potential sequence information for these molecules. External mass calibration of the intact protonated molecular ions is demonstrated with mass accuracies typically around 100 ppm. Mass accuracies for the observed fragment ions ranged from +/- 0.20 Da for the smaller peptides studied (i.e., oxidized B chain of bovine insulin) to +/ 0.38 Da for the largest protein studied (cytochrome c), based upon the known sequences. PMID- 8633763 TI - Use of the derivatizing agent 4-aminobenzoic acid 2-(diethylamino)ethyl ester for high-sensitivity detection of oligosaccharides by electrospray ionization mass spectrometry. AB - A method for the high-sensitivity detection of oligosaccharides by electrospray ionization mass spectrometry (ESI-MS) is reported. The method involves the chemical derivatization with 4-aminobenzoic acid 2-(diethylamino)-ethyl ester (ABDEAE). This derivative, which contains a 2-(diethylamino)ethyl group, having a high proton affinity, enhances the ionization efficiency of analytes in the positive ESI mode. Experiments using maltohexaose as a model oligosaccharide revealed that derivatization with ABDEAE gave a remarkably large increase in molecular ion abundance. Using a mixture of acetonitrile, 2-methoxyethanol, 2 propanol, and water (1:1:1:1 v/v/v/v) as solvent for ESI, ABDEAE-derivatized maltohexaose could be detected at a level of 10 fmol. This represents a 5000-fold improvement in sensitivity over underivatized maltohexaose. ESI tandem mass spectrometry of the ABDEAE-derivatized maltohexaose provides structural information at the low-picomole level. In this spectrum, 1,5X' and 0,2A" series of sequence ions, arising from ring cleavage, were observed as the predominant ions. PMID- 8633764 TI - The nucleic acid ligand. A new tool for molecular recognition. PMID- 8633765 TI - Structural study of electrolysis-induced degradation of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2. AB - Growth hormone releasing peptide (GHRP, sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) is a synthetic hexapeptide under consideration for transdermal iontophoretic drug delivery. Cyclic voltammetry, controlled-potential electrolysis, HPLC/UV analysis, LC/MS/MS analysis, and EPR spin-trapping studies indicate that the electrolysis-induced oxidative degradation of GHRP is likely to be mediated by electrogenerated oxygen radicals from the electrolysis of water. Within 2 h and up to 2.5 V versus an Ag/AgCl reference electrode, the peptide backbone remains largely intact. The chemical modifications are selectively on imidazole (histidine) and indole (tryptophan). Strategies for alleviating the electrolysis induced degradation of GHRP are proposed. PMID- 8633766 TI - Attomole biomolecule mass analysis by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance. AB - Significantly improved sensitivity for analysis of biomolecules by MALDI FT-ICR mass spectrometry is achieved by (i) microscope-monitored sample deposition onto a small indentation on the probe tip and (ii) multiple remeasurement of ions from a single laser shot. A simple modification to the solids probe tip allows for microdeposition of a few amols of analyte onto small indentation spots previously aligned with the laser beam. Ion multiple remeasurement of the same ion packet enhances the signal-to-noise ratio and thus extends the achievable FT-ICR MS detection limit. We demonstrate that FT-ICR can be used to detect parent and structurally significant fragment ions of peptides and phospholipids at low amol amounts. Positive ion mass spectra for approximately 90 amol of a mixture of angiotensin II and bradykinin, approximately 40 amol of dipalmitoylglycerophosphatidylcholine, and approximately 8 amol of substance P constitute the lowest reported detection limits to date for FT-ICR mass analysis of MALDI-generated ions. PMID- 8633767 TI - An integrated analytical method for determination of polychlorinated aryl methyl sulfone metabolites and polychlorinated hydrocarbon contaminants in biological matrices. AB - A simple column chromatography method was developed for separation and cleanup in the determination of chlorinated hydrocarbon contaminants and their methyl sulfone (MeSO2-) metabolites in biological tissues. The method was validated for determination of 11 polychlorinated biphenyls (PCBs), 15 tetra- to heptachloro 3- and 4-MeSO2-PCBs, 3-MeSO2-DDE, and tris(4-chlorophenyl)-methanol spiked to herring gull egg, smelt, and polar bear liver and adipose tissue using gas chromatography with electron-capture detection (GC-ECD). The overall mean recovery relative to the internal standard was 103% +/- 8%, independent of analyte, substrate type, and lipid extract weights up to approximately 0.7 g. Precision of replicate analyses of individual congeners was good. There were no significant residual biogenic or xenobiotic interferences in the aryl methyl sulfone fraction of any substrate. Sensitivity and linearity of molar response of MeSO2-PCBs and MeSO2-DDE was tested for ECD and electron-capture negative ion mass spectrometry monitoring the total ion current (TIC) and the molecular ion (SIM). The mean practical quantitation limit among MeSO2-PCBs and 3-MeSO2-DDE was lowest for SIM (2.1 +/- 0.9 pg) and similar for ECD and TIC (24.2 +/- 4.6 and 44.4 +/- 17.1 pg, respectively). Response factors were linear above the practical quantitation limit to at least the nanogram level for all three techniques. In spite of superior sensitivity, there was more inherent variability in the response factors for SIM (approximately 18%-56% CV) than for ECD (approximately 7%-12% CV) or TIC (approximately 11%-18% CV); therefore, ECD or TIC is recommended for quantitative analysis. PMID- 8633768 TI - End-label, free-solution capillary electrophoresis of highly charged oligosaccharides. AB - The effect of fluorescent tags on the separation of negatively charged oligosaccharides, derived from a partially hydrolyzed kappa-carrageenan, was studied. When the charge-to-friction ratio of oligosaccharides is increased by the end-label, the migration order is from smaller to larger oligomers, and the resolution of larger oligomers could be improved by using a sieving medium. The migration order can be entirely reversed when the charge-to-friction ratio of the solute is decreased by the end-label. The experimental electrophoretic mobilities obtained in this work are in excellent agreement with the recently reported theoretical model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780). The maximum number of separated oligomers (Mmax) as a function of applied voltage and injection time was also studied, but no strong dependencies were found. Resolution between small oligomers could be significantly improved by following this separation principle. PMID- 8633769 TI - Extraction of serum and urine calcium with ion exchange membrane filters for isotope enrichment determination using thermal ionization mass spectrometry. AB - The extraction of calcium from water, serum, and urine using Bio-Rex 25 mm ion exchange membrane filters was compared with the oxalate precipitation procedure currently used in our laboratory. Total recoveries of a known quantity of calcium loaded onto the membrane filters for water, serum and urine were as follows: (a) cation exchange filter, 85%, 74%, and 66%; (b) Chelex, 65%, 98%, and 20%; and (c) oxalate precipitation, 93%, 100%, and 96%, respectively. Regression analysis for precipitation versus ion exchange isotope ratio measurements of standards prepared using highly enriched calcium-44 showed slopes of unity. An improvement of automated sample analysis was observed for water and urine calcium samples extracted with ion exchange filters. PMID- 8633770 TI - On-line Raman spectroscopy of ribonucleotides preconcentrated by capillary isotachophoresis. AB - Normal Raman spectroscopy is used as an on-line detector for capillary isotachophoresis (ITP) of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate in phosphate buffers. Preconcentration is from a 1 x 10(-2) M phosphate buffer (pH 7.5) into a leading electrolyte of 0.1 M KCl or Na2SO4, with a terminating electrolyte of 0.1 M 4-morpholinepropane-sulfonic acid. The ribonucleotides are concentrated to above 10(-2) M at the detection window, allowing measurement of Raman spectra with 1 s integration, from starting concentrations of 5 x 10(-6) M or higher. PMID- 8633771 TI - Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy. AB - We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3 naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3 naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis. PMID- 8633773 TI - Real-time detection and quantification of DNA hybridization by an optical biosensor. AB - The use of an optical biosensor, the resonant mirror, for direct and rapid detection of DNA-DNA hybridization has been demonstrated. Biotinylated oligonucleotide probes were immobilized on the sensor surface, via streptavidin, and the hybridization of a complementary target oligonucleotide (40-mer) was monitored in real time. The interaction at the sensor surface was shown to be sequence specific under conditions of low stringency. Regeneration of the surface immobilized probe was possible, allowing reuse without a significant loss of hybridization activity. A comparison of probes indicated that the relative position of complementary sequence and the length of probe affected the hybridization response obtained. The potential of the sensor for quantitation of a hybridized DNA target was investigated. From radiolabeling data, the lowest amount of hybridized target sequence which could be determined directly was 19.9 fmol/mm2 (263 pg/mm2) of sensor surface. The dependence of the sensor response on the concentration of probe and target oligonucleotide was established. Utilizing the assay as an end-point determination method, the lowest detectable concentration of target oligonucleotide (40-mer) was 9.2 nM. This compares favorably to other sensor methods described previously without the requirement for labels. PMID- 8633772 TI - Development of an efficient amino acid sequencing method using fluorescent Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate. AB - In this paper, a new method is described for N-terminal amino acid sequencing of peptides using the fluorescent reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3 benzoxadiazol-4-yl isothiocyanate (DBD-NCS). Sequence determination is carried out by identifying thiazolinone (TZ) amino acids, which are generally unstable and difficult to detect. The employed system can easily and quickly derive TZ amino acids using the Edman reaction with DBD-NCS; these amino acids are also stable enough to be efficiently detected by high-performance liquid chromatography. Resultant detection limits for DBD-TZ amino acids range from 50 fmol to a sub-picomole level (S/N = 3). This system successfully analyzed sequences of Leu5-enkephalin (25 pmol) and angiotensin I (100 pmol) using fluorometric detection at 524 nm with excitation at 387 nm. PMID- 8633774 TI - Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme coding DNA fragments. AB - A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover. PMID- 8633775 TI - Applicability of factor analysis in solid state NMR. AB - Factor analysis has been used to deduce the composition of multicomponent magic angle spinning NMR spectra comprised of overlapping isotropic peaks. The technique has been used to determine the number of constituents present and was combined with a target transformation minimization procedure to identify the component MAS spectra. The new analysis procedure is compared to the conventional least-squares approach and is found to be superior in all cases. PMID- 8633776 TI - Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions. AB - Laser desorption time-of-flight mass spectra of peptides and proteins, as well as of lower molecular weight analytes, have been obtained by using a pulsed nitrogen UV laser (337 nm) to irradiate mixtures of 2-150 microns graphite particles and solutions of the analytes in glycerol. Protonated analytes as well as abundant alkali cation adducts were observed. Carbon cluster ions, Cn+, typically had a low abundance but dominated the mass spectrum at elevated laser powers. In spectra of a cytochrome c tryptic digest, all but one of the tryptic peptides were easily observed. Spectra of low molecular weight analytes dissolved in glycerol are very similar to FAB spectra of the same glycerol solution with added alkali salts. However, in many peptide and protein spectra, glycerol ion abundances are very low, and the alkali ions dominate the spectra at low mass. These spectra may correspond to wet and dry surface desorption conditions, respectively. The best spectra of the larger molecules were observed under dry conditions. In these initial experiments, we have obtained a sensitivity in the pico- to nanomole range and a mass resolution of about 300. The signal intensity is as good as that in conventional MALDI, and under optimal conditions, few background peaks appear, even at low mass. PMID- 8633777 TI - Rapid tryptic mapping using enzymatically active mass spectrometer probe tips. AB - A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. PMID- 8633778 TI - Tandem mass spectrometry of herbicide residues in lipid-rich tissue. AB - A tandem mass spectrometry procedure, originally developed for bacterial biofilms was adapted for the identification of herbicide residues in lipid-rich tissue of amphipods collected from microcosms in a prairie wetland. For this application, the amounts of tissue employed (less than 1 mg wet weight), and detection of target analytes at picogram levels, were similar to the values reported for bacterial biofilms. Described is an application of the technique for the identification of residues of the herbicide S-2,3,3-trichloroallyl diisopropyl thiocarbamate (triallate; trade name Avadex-BW). For amphipods collected from microcosms exposed to the herbicide 2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid methyl ester (diclofop-methyl, trade name Hoe Grass), there were detectable levels of only the hydrolysis product, diclofop acid, in the lipid-rich tissue. Other transformation products reported for bacterial biofilms were not observed in the amphipods. PMID- 8633779 TI - Determination of N-nitrosodimethylamine at part-per-trillion levels in drinking waters and contaminated groundwaters. AB - The carcinogen N-nitrosodimethylamine (NDMA) may be quantitated routinely at ultratrace (ng/L) levels in drinking water or contaminated groundwater. The aqueous sample is passed through a preconditioned Empore C18 filter disk to remove neutral nonpolar species and then extracted continuously overnight with highest purity dichloromethane. The latter is then concentrated to 1 mL, and a large aliquot (up to 200 microL) is loaded onto a dual-stage carbon sorbent trap, after which the solvent is removed with ultrapure helium. The concentrated residues are then injected onto a gas chromatographic column using a short-path thermal desorber. NDMA is selectively detected using a chemiluminescent nitrogen detector (CLND) operated in its nitrosamine-selective mode. The reporting limit for this procedure, evaluated using two independent statistically unbiased protocols, is 2 ng of NDMA/L. A related procedure, employing an automatic sampler instead of the short-path thermal desorber, provides convenient analysis of heavily contaminated samples and exhibits a reporting limit (same protocols cited previously) of 110 ng of NDMA/L. When the two methods are used together in a "two tiered" protocol, NDMA concentrations spanning 4 orders of magnitude (ng/L to microgram/L levels) may be measured routinely. The low-level procedure employing only the short-path thermal desorber was applied successfully to three sources of drinking water, where NDMA concentrations ranged between 2 and 10 ng of NDMA/L. The two-tiered protocol was applied to a series of contaminated groundwaters whose NMDA concentrations ranged between approximately 10-7000 ng of NDMA/L. The results agreed with those obtained from an independent collaborating laboratory, which used a different analytical procedure. PMID- 8633780 TI - Population distributions and intralaboratory reproducibility for fat-soluble vitamin-related compounds in human serum. AB - Concentrations of alpha- and beta-carotene, lycopene, beta-cryptoxanthin, zeaxanthin, lutein, alpha-, gamma-, and delta-tocopherol, retinol, and retinyl palmitate have been determined in over 1400 human sera from two epidemiological studies. Complete adult population distributions for these analytes, and for a chromatographically defined "total carotenoids" component, are detailed as data defined histograms. The distributions for retinol and alpha-tocopherol concentrations are much narrower than those for the other analytes. Information provided by analysis of control samples facilitated intercomparison of the two studies and provided univariate and bivariate estimates of the intra-laboratory measurement reproducibility. The utility of an analytical measurement is shown to be dependent on both the measurement uncertainty and the distribution of the analyte within the population. PMID- 8633781 TI - Structural identification of the degradation products of the antitumor peptide antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11). AB - The basic hexapeptide antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) was degraded in acid and alkaline media. In acid solution, only one degradation product is found whereas in alkaline solution at least six products are formed. These compounds were analytically characterized and structurally identified by reversed-phase high-performance liquid chromatography, capillary electrophoresis, liquid chromatography/mass spectrometry, fast atom bombardment tandem mass spectrometry, optical rotation analysis, and chiral gas chromatography. The product formed in acidic solution is the terminally deamidated antagonist [Arg6,D Trp7,9,MePhe8]substance P (6-11); this product was also found in alkaline degradation mixtures. Other important degradation products originate from racemization of the amino acid residue L-Met, formation of ornithine from Arg, and the oxidation of Met to its sulfoxide form. PMID- 8633782 TI - Analyzing for cryptosporidium. EPA scrambles to refine an analytical method. PMID- 8633783 TI - Multicomponent kinetic determinations using artificial neural networks. AB - Neural networks were successfully used for multicomponent kinetic determinations of species with rate constant ratios approaching unity without the aid of spectral discrimination. The ensuing method relies on two inputs describing the profile of the kinetic curve for each mixture, which is obtained by preprocessing kinetic data using nonlinear least-squares regression. A straightforward network architecture (2:4s:21) was used to resolve mixtures of 2- and 3-chlorophenol; the trained network estimated the concentrations of both components in the mixture with a relative standard error of prediction of approximately 5%, which is much lower than that obtained with Kalman filtering. The effect of some variables such as the rate constant and analyte concentration ratios on the proposed multicomponent determination is discussed. PMID- 8633784 TI - Improved fiber-optic chemical sensor for penicillin. AB - An optical penicillin biosensor is described, based on the enzyme penicillinase. The sensor is fabricated by selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibers. The penicillin-sensitive matrices are fabricated by immobilizing the enzyme as micrometer-sized particles in a polymer hydrogel with a covalently bound pH indicator. An array of penicillin-sensitive and pH-sensitive matrices are fabricated on the same fiber. This array allows for the simultaneous, independent measurement of pH and penicillin. Independent measurement of the two analytes allows penicillin to be quantitated in the presence of a concurrent pH change. An analysis was conducted of enzyme kinetic parameters in order to model the penicillin response of the sensor at all pH values. This analysis accounts for the varying activity of the immobilized penicillinase at different pH values. The sensor detects penicillin in the range 0.25-10.0 mM in the pH range 6.2-7.5. The sensor was used to quantify penicillin concentration produced during a Penicillium chrysogenum fermentation. PMID- 8633785 TI - Artificial neural networks for multicomponent kinetic determinations. AB - An artificial neural network (ANN) procedure that uses the scores of a principal component model as input data was tested for calibration in the resolution of binary mixtures from kinetic measurements. The results thus obtained are compared with those provided by partial least-squares (PLS) regression and principal component regression (PCR). The ANN was first applied to simulated single wavelength kinetic curves. The effect of experimental variability was considered by assuming rate constants to fit a normal distribution curve. An amount of instrumental noise was also added to the simulated curves. Both linear and nonlinear systems were tested. Non-linearity was assumed to result from interactions between analytes and modeled by introducing a multiplicative term in the rate equation. The results provided by the three methods on linear systems were comparable; in the presence of interactions between analytes, however, the ANN method clearly outperformed the other two. The ANN method was also used to resolve mixtures of Fe(III), Co(II), and Zn(II) by displacement from their EGTA complexes with 4-(2-pyridylazo)resorcinol (PAR) using a stopped-flow injection assembly including a diode array detector. Preliminary experiments revealed the Co(II) and Zn(II) displacement reactions to be pseudo-first-order and that of Fe(III) to be a multistep process that departed from the linear behavior of the other two. Again, the results obtained with ANN were better than those provided by PCR and PLS. PMID- 8633786 TI - Electrochemical detection of histamine and 5-hydroxytryptamine at isolated mast cells. AB - The electrochemical oxidation of histamine has been investigated as an analytical tool. In a physiological buffer, histamine is oxidized at carbon fiber microelectrodes at potentials close to the background in a chemically irreversible process. Cylindrical carbon fiber electrodes were used as amperometric detectors for histamine separated with a reversed-phase capillary column, and detection limits of 240 amol were achieved. Electrodes with beveled tips were used as real-time sensors by monitoring with repetitive cyclic voltammograms at a scan rate of 800 V/s with a 16.7-ms repetition rate, and detection limits of 1.4 microM were achieved. Both techniques were used to probe histamine and 5-hydroxytryptamine (5-HT) stored in rat peritoneal mast cells. The content in single cells was measured by capillary HPLC, and both substances were found in single cells. Although the analysis revealed a large cell-to-cell variation in the amount of histamine and 5-HT, the average amount was 150 and 4 fmol of histamine and 5-HT, respectively. Release of histamine and 5-HT was measured with the electrode placed 1 microm from the cell surface. Release was observed as a series of sharp concentration spikes, consistent with corelease of the two substances from individual vesicles following exocytosis. PMID- 8633787 TI - Noncovalent protein--oligonucleotide interactions monitored by matrix-assisted laser desorption/ionization mass spectrometry. AB - Positive ion mode matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to explore nonspecific interactions between proteins and oligonucleotides. The formation of noncovalent complexes showed correlation with the type of oligonucleotide bases and with the amino acid composition of the proteins. Among the four DNA homooligomers, abundant protein-nucleic acid complexes were detected for pd(T)n, whereas negligible attachment was evident for pd(A)n, pd(C)n, and pd(G)n. Mixed base sequence nucleic acids (pd(AGCTCAGCTT) and d(TTAGCAGCTT) also showed affinity to Arg-Lys. The protein affinity of pd(T)n turned out to be nonspecific and produced a larger variety of complexes when the number of basic residues in the protein was increased. Complexation of pd(T)n with small basic dipeptides (Arg-Lys or His-His) led to significant improvement in the mass resolution for positive ions. For example, the mass resolution of the pd(T)20/Arg-Lys complex exhibited about 4 times improvement over pd(T)20 alone. The protein--oligonucleotide interactions were also pH and matrix dependent. Lowering the pH from its original value (pH = 1.7) led to diminishing complex related signal, whereas increasing the pH resulted in the appearance of a larger variety of complexes. 2,5-Dihydroxybenzoic acid matrix demonstrated much greater tendency to produce complex ions than did the three other matrix materials we tested. A possible explanation of the observed phenomena was based on pH controlled ion pair formation between oligonucleotides and proteins. PMID- 8633788 TI - A microscale electrospray interface for on-line, capillary liquid chromatography/tandem mass spectrometry of complex peptide mixtures. AB - A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at flow rates < 1 microL/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a flame-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 microm and an opening less than 5 microm in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union. Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide production spectra (MS/MS) using real-time, automated instrument control procedures. PMID- 8633790 TI - Probe-immobilized affinity chromatography/mass spectrometry. AB - A new technique has been developed that provides affinity separations directly on the surface of a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) probe. This strategy provides both in situ identification of biomolecules through biospecific antibody/antigen interactions and molecular weight information in a rapid and facile manner. Our technique, which we call probe affinity MS, directly couples the high selectivity of immobilized affinity chromatography with the sensitivity of MALDI-MS. As this technique permits the rapid identification of antigens, ligands, and other compounds from complex biological mixtures, we believe that it will be useful in a wide range of applications in virtually all fields of the life sciences. PMID- 8633789 TI - Quantitation of metal isotope ratios by laser desorption time-of-flight mass spectrometry. AB - Laser desorption time-of-flight mass spectrometry (LD/TOF-MS) is evaluated for the determination of stable metal isotope ratios. The isotope ratios of five metal ions (Cu, Ca, Mg, Fe, Zn) in atomic absorption standard solutions and two metal ions (Ca, Mg) in human serum samples are determined. With an existing LD/TOF-MS instrument we show that the technique can overcome the difficulties of the most commonly used methods for measuring metal isotope ratios: (1) all metals are ionizable without surface treatment, thus overcoming the major drawback of thermal ionization mass spectrometry (TIMS); (2) there is no matrix involved to interfere with the metal ion detection, thus overcoming the major disadvantage of inductively coupled plasma mass spectrometry (ICPMS); (3) there is no interference from hydride ions, a major disadvantage of fast atom bombardment secondary ionization mass spectrometry; (4) a mixture of metals can be detected simultaneously using a single laser wavelength, overcoming the major disadvantage of resonance ionization mass spectrometry; (5) accuracy and precision comparable to ICPMS can be achieved with the current instrumentation; (6) precision comparable to TIMS is feasible; and most importantly (7) high precision can be achieved on very small quantities of material because the LD/TOF-MS instrument permits all masses to be monitored simultaneously and very small differences in isotope ratio can be detected. PMID- 8633791 TI - Use of non-cross-linked polyacrylamide for four-color DNA sequencing by capillary electrophoresis separation of fragments up to 640 bases in length in two hours. AB - Four-color DNA cycle sequencing was performed on an M13mp18 template using dye labeled primers. Sequencing fragments were separated by capillary electrophoresis at 60 degrees C and at an electric field of 150 V/cm. The sieving medium was 5%T, non-cross-linked polyacrylamide in 7 M urea. The use of high temperature for the separation reduces formation of secondary structures in the sequencing fragments, generating a sequence that is free of compressions without the use of strongly denaturing gels. The use of high temperatures also increases the separation rate compared with room-temperature operation. Fragments up to 640 bases are separated in less than 2 h. PMID- 8633792 TI - Unified view of kinetic-based analytical methods with emphasis on ruggedness. A review. AB - All analytical determinations can be grouped into two general categories, namely equilibrium-based and transient-based methods. This is an important grouping because most conventional approaches to transient-based methods are much less rugged than their equilibrium-based counterparts. As a result it is necessary to control variables within much narrower tolerances for transient-based methods than equilibrium-based methods to obtain similar degrees of reliability. This paper reviews measurement and data-processing methods developed to reduce effects of variables on transient-based methods, with emphasis on a general approach that is applicable to a wide variety of methodologies. The approach emphasized is identified as a pseudo-equilibrium method. In this method, transient data are used to compute signals that would be measured if all processes that affect the measurement could be monitored to equilibrium. Results included show that the pseudo-equilibrium method is applicable to the three most common types of responses from the transient phases of chemical and physico-chemical processes. Data included show 10- to 100-fold improvements in ruggedness relative to conventional measurement and data-processing options. PMID- 8633793 TI - Slurry preparation by high-pressure homogenization for cadmium, copper and lead determination in cervine liver and kidney by electrothermal atomic absorption spectrometry. AB - Homogenization with a flat valve homogenizer in combination with high-speed blending was evaluated for the preparation of slurries suitable for the ETAAS determination of cadmium, copper and lead concentrations in six SRMs and in frozen cervine liver and kidney. Fresh tissue (approximately 2 g) or powdered SRM (approximately 0.1 g) was dispersed, at high speed, in 20 ml of ethanol-water (1 + 9 v/v) containing 0.25% m/m tetramethylammonium hydroxide. The resulting suspension was passed through a high-pressure flat valve homogenizer. Determinations performed on the resulting homogenate, provided estimates for Cd, Pb and Cu concentrations that were within 27, 23 and 18% of the certified values, respectively, for the six SRMs. In all instances, the experimental results did not differ significantly from the certified values. For frozen tissues there was good agreement between the concentrations as determined by slurry homogenization ETAAS and conventional digestion-ICP-MS. In addition, no significant differences were detected between the slopes of the calibration curves for external standards and standard additions to homogenized sample (SRMs or fresh tissue). Moreover, replicate determinations of analyte concentrations in slurries at various times post-preparation did not detect any segregation of the homogenates during 6 d. For these matrices at least, short-term sample storage had no discernible effect on the analyte apparent concentrations. The applicability of the process was limited only by the levels of contaminating Pb and Cu introduced into the sample by the homogenizer. PMID- 8633794 TI - Potassium ion-selective optodes based on the calix[6]arene hexaester and application in human serum assay. AB - Plasticized PVC membranes incorporating the hexaester of calix[6]arene, potassium tetrakis(4-chlorophenyl) borate and a neutral H+-selective chromo-ionophore (ETH 5294) have been used as reversible sensing devices for optical determination of potassium ion in pH-buffer solutions. At pH 9.0, the absorbance response of the optode shows a good correlation with the theoretically derived formula in the range from 1 x 10(-6) to 1 x 10(-2) mol l(-1) potassium ion. Typical response times (t95) in the samples are 3 min, the relative standard deviation for repeated measurements being within 0.5%. The selectivity of this potassium selective membrane allows its application for the reversible optical detection of potassium concentration in human serum samples. PMID- 8633795 TI - Detection of foot-and-mouth disease viral sequences in various fluids and tissues during persistence of the virus in cattle. AB - OBJECTIVE: To assess whether foot-and-mouth disease virus (FMDV)-specific sequences could be identified in tissues from persistently virus-infected animals. DESIGN: Cattle with experimentally induced persistent FMDV infections were slaughtered at 750 days after viral exposure. Experimentally infected pigs were slaughtered at 28 days after FMDV inoculation. Postmortem specimens were asceptically removed. ANIMALS: Three bovids and 3 pigs were studied, as well as 1 control animal for each species. PROCEDURE: Various tissues were examined for the presence of FMDV-specific sequences by dot-blot hybridization assay, using a molecularly cloned FMDV cDNA corresponding to the polymerase coding region. RESULTS: The FMDV-specific genomic sequences were only detected in RNA from spleen, lung, larynx, tonsils, pancreas, liver, esophagus, and WBC of bovids. CONCLUSIONS: It was established that, at late stages of the persistent infection, when virus isolation was not possible, cattle may carry FMDV-specific sequences in different tissues. Retention of viral sequences could not be demonstrated in specimens from experimentally infected swine, 28 days after viral inoculation. PMID- 8633796 TI - Serum haptoglobin concentrations in a population of feedlot cattle. AB - OBJECTIVE: To determine serum haptoglobin concentrations in a population of feedlot cattle and evaluate their usefulness in predicting subsequent clinical respiratory tract disease. DESIGN: Prospective longitudinal study. ANIMALS: 366 beef calves. PROCEDURE: Serum samples were obtained at feedlot entry and 40 and 65 days on feed (DOF). Calves were observed daily for clinical signs of respiratory tract disease. The lungs of 144 of the calves were evaluated at slaughter for the presence of gross lesions of pneumonia. RESULTS: 58% of the calves had detectable serum haptoglobin concentration in at least 1 sample. The proportion of calves with detectable haptoglobin were similar at each sample collection time. A higher proportion of the calves had values > 10 mg/dl at 40 DOF. The proportion of calves observed with clinical disease during the 10-day period after the 40 DOF sample increased (P < 0.10) as serum haptoglobin concentration increased. At 65 DOF, calves with serum haptoglobin value > 10 mg/dl had a higher (P < 0.05) rate of subsequent clinical respiratory tract disease than did calves with lower values. The proportion of calves with gross pulmonary lesions slaughter increased (P < 0.05) from 39% among calves without detectable serum haptoglobin concentration in any of the 3 samples to 63% among calves with at least 1 observed value > 10 mg/dl. CONCLUSIONS: We observed associations between serum haptoglobin concentration and subsequent clinical respiratory tract disease and pulmonary lesions at slaughter. However, serum haptoglobin concentration alone is not adequate for prediction of clinical disease. CLINICAL RELEVANCE: The usefulness for cross-sectional sampling of serum haptoglobin concentration as a diagnostic tool for clinical respiratory tract disease in feedlot cattle appears to be limited. PMID- 8633797 TI - Comparison of two techniques of narcotic-induced anesthesia for use during recording of magnetic motor evoked potentials in dogs. AB - OBJECTIVE: To compare 2 types of narcotic-induced anesthesia for recording of transcranial magnetic motor evoked potentials (TMMEP) in dogs. DESIGN: The effect of different doses of sufentanil and midazolam and of sufentanil and nitrous oxide on onset latencies and peak-to-peak, amplitudes of TMMEP was evaluated and compared. ANIMALS: 18 neurologically normal dogs. PROCEDURE: Premedication with droperidol and fentanyl. Induction and maintenance of anesthesia either with sufentanil and midazolam or with sufentanil and nitrous oxide. Recording of TMMEP from the extensor carpi radialis muscle of the forelimb and from the cranial tibial muscle of the hind limb. RESULTS: Both types of narcotic anesthesia induced dose-dependent suppression of TMMEP; compared with baseline recordings, latencies increased, amplitudes decreased, and reproducibility became poorer with increasing dose of the anesthetics. Using surgical-depth doses of the anesthetics, TMMEP could still be recorded in all dogs with sufentanil and nitrous oxide, but not with sufentanil and midazolam anesthesia. CONCLUSIONS: Sufentanil and nitrous oxide anesthesia was superior to sufentanil and midazolam anesthesia for TMMEP recording. CLINICAL RELEVANCE: In small animal medicine, and in dogs in particular, spinal cord diseases are among the most frequently encountered neurologic disorders. The development of techniques for recording TMMEP in anesthetized dogs allows noninvasive evaluation of transmission along descending motor pathways of the spinal cord. PMID- 8633798 TI - Helicobacter mustelae-associated hypergastrinemia in ferrets (Mustela putorius furo). AB - OBJECTIVE: To determine whether ferrets naturally infected with Helicobacter mustelae were hypergastrinemic, compared with ferrets that were specific-pathogen free (SPF) for H mustelae. DESIGN: Plasma gastrin concentrations in H mustelae infected and SPF ferrets were measured at 3 time points and compared to determine whether H mustelae was associated with hypergastrinemia. ANIMALS: 21 H mustelae infected ferrets and 10 SPF ferrets. PROCEDURE: The H mustelae status of the ferrets was confirmed prior to commencement of the study. Gastric endoscopy was used to obtain gastric mucosal pinch biopsy specimens that were processed for rapid-urease assay, microaerophilic culturing, and histologic evaluation. Plasma gastrin concentrations were determined at 3 time points: baseline after a 12-hour nonfeeding period, and 30 and 60 minutes after oral administration of a standardized meal. Gastrin was measured by radioimmunoassay. RESULTS: The results for the H mustelae-infected group (mean +/- SEM pg/ml) were: baseline, 54.4 +/- 2.56; 30 minutes, 94.5 +/- 6.05; and 60 minutes, 82.6 +/- 5.73. The SPF group results were: baseline, 55.8 +/- 7.35; 30 minutes, 80.8 +/- 5.77; and 60 minutes, 59.7 +/- 4.95. There was a significant (P < 0.01) difference at the 60-minute time point between the 2 groups of animals. The H mustelae group had a 17% higher mean gastrin value at 30 minutes. CONCLUSIONS: Helicobacter mustelae is associated with hypergastrinemia in ferrets. CLINICAL RELEVANCE: Helicobacter induced hypergastrinemia may be related to the pathogenesis of peptic ulcer disease in ferrets. PMID- 8633799 TI - Reactivity of purified complement component 3b with bovine neutrophils and modulation of complement receptor 1. AB - OBJECTIVE: To study binding of purified complement component C3b to bovine blood and mammary neutrophils (PMN) after various treatments and determine their ability to modulate receptor numbers. DESIGN: Cell isolation, activation, and flow cytometric studies. ANIMALS: Healthy lactating Holstein cattle. PROCEDURE: Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytometric assays using fluorescein isothiocyanate labeled C3b were developed to evaluate binding to PMN complement receptor 1. Multiple substances were tested to determine their overall effect on C3b binding to PMN. Blood and milk PMN were isolated by differential centrifugation and exposed to optimal concentrations of recombinant human C5a, formyl-methyl leucyl phenylalanine, recombinant bovine interferon-gamma, variable concentrations of phorbol myristate acetate (0.01 to 100 ng), calcium ionophore A23187, serum opsonized zymosan, zymosan-activated serum (ZAS), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ng). Additionally, mammary and blood PMN were preincubated in skim milk and whey. RESULTS: Variable concentrations of phorbol myristate acetate caused a dose-dependent increase in percentage of PMN binding C3b, and increased the amount of C3b bound per cell. Significant increases were observed after PMN treatment with calcium ionophore, serum opsonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydrocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activation during migration into the mammary gland. Binding of C3b was inhibited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elicited larger responses than those observed for mammary PMN. CONCLUSIONS: Modulation of complement receptors on bovine PMN is possible. Additionally, significant difference between the level of binding of C3b to blood and milk PMN, with milk PMN having higher binding, may be attributable to migration of PMN into the mammary gland, causing increased receptor expression. CLINICAL RELEVANCE: Contribution to a greater understanding of the role of complement in bovine immunologic systems, leading to testing for in vivo enhancement of bovine immune responses to invading pathogens. PMID- 8633800 TI - Induction of protective immunity against transmissible gastroenteritis virus after exposure of neonatal pigs to porcine respiratory coronavirus. AB - OBJECTIVE: To test the ability of porcine respiratory coronavirus (PRCV) to induce protective immunity to antigenically related transmissible gastroenteritis virus (TGEV) in neonatal pigs. DESIGN: Neonatal pigs were exposed to PRCV when they were 2, 4, or 6 days old and challenge-exposed to virulent TGEV at 10 days of age. ANIMALS: 34 hysterectomy-derived, colostrum-deprived pigs. PROCEDURE: After challenge exposure, clinical signs were observed, body weight, antibody response, and virus shedding were measured, and mortality was determined. RESULTS: After exposure to PRCV, principals had a slightly slower rate of weight gain than did controls; with 1 exception (a PRCV-exposed pig that was dyspneic for 1 day), principals and controls remained clinically normal until shortly after challenge exposure, when all pigs became listless and anorectic and developed watery diarrhea. However, by day 3, most of the pigs that had been exposed to PRCV when they were either 2 or 4 days old began to recover and most (15/18) survived. Conversely, the clinical condition of most of the other pigs worsened and most (14/16) died. Pigs exposed to PRCV when they were 2 or 4 days old also differed from all other pigs in that they had serum virus-neutralizing antibodies for PRCV and TGEV at the time of challenge exposure. CONCLUSIONS: The PRCV can induce protective immunity to TGEV in neonatal pigs and such immunity develops at or about 6 days after exposure to PRCV. Moreover, protective immunity may be coincident with the appearance of virus-neutralizing antibody. CLINICAL RELEVANCE: Exposure to PRCV should enhance a TGE herd vaccination program. PMID- 8633801 TI - Antigenic and restriction enzyme analysis of Campylobacter spp associated with abortion in sheep. AB - OBJECTIVE: To determine the scope of strain variations among Campylobacter spp associated with abortion in sheep. DESIGN: To examine Campylobacter spp isolated from cases of abortion for biochemical, antigenic, and genetic differences. SAMPLE POPULATION: 15 isolates of Campylobacter spp isolated from cases of abortion during a single lambing season. PROCEDURE: Isolates were examined, using biochemical tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins (OMP), and DNA restriction enzyme analysis (REA). RESULTS: Eight strain variants were detected among the 15 isolates. 14 of the isolates were C jejuni, 13 of which were biotype I and 1 biotype II, and the remaining isolate was identified as C fetus subsp fetus. Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis OMP patterns had distinctive profiles for C fetus subsp fetus and C jejuni biogroup-II isolates and 3 variants within the C jejuni biogroup-I isolates. Examination of REA patterns of DNA from the 15 isolates digested with Cfo I indicated clear differences correlating with species and biogroups and 4 REA variants among biotype-I isolates. CONCLUSIONS: Marked antigenic and genetic heterogeneity of Campylobacter isolates were associated with ovine abortion within a defined geographic area. CLINICAL RELEVANCE: Representatives of differing OMP and REA profile groups should be considered for incorporation in vaccines to optimize protection in this region and possibly other geographic areas. PMID- 8633802 TI - Reliability of a single fecal egg per gram determination as a measure of individual and herd values for trichostrongyle nematodes of cattle. AB - OBJECTIVES: To determine precision of a single fecal egg count, to evaluate the necessity of repeated testing to ascertain the number of parasite eggs found per gram of fecal material (EPG) of a given animal, and to infer the number of cattle that must be sampled to estimate the mean EPG value of a given herd. DESIGN: Calves born in 4 successive years were tested for 3 consecutive days in May, July, and September to determine their fecal EPG value. Results were analyzed to determine important sources of variance in the EPG values. ANIMALS: 427 Black Angus calves. PROCEDURES: Nematode EPG values were determined by zinc sulfate flotation of fecal samples taken from the rectum. Variance components were estimated by restricted maximal likelihood procedures. RESULTS: The largest source of variation in fecal egg counts arose from differences among calves. Variation associated with sampling within calf was larger than that associated with year of sampling or day when samples were taken. Repeatability of EPG determinations was between 0.4 and 0.7. CONCLUSIONS: Collection of 3 replicate samples reduced variance associated with a single calf mean by 25 to 30%. Additional replication would further reduce the variance, but at a diminishing rate. To accurately estimate mean EPG for a herd, a randomly drawn sample should be similar in composition to the herd and include at least 1 animal from the high EPG group, which often constitutes 15 to 20% of the calves. To ensure that, on average, 95% of the samples drawn contain 1 or more animals with high EPG, the sample must include 15 to 20 animals within the grazing group under study. PMID- 8633803 TI - Effect of heartworm infection on fade of norepinephrine-induced constriction in canine pulmonary vein. AB - OBJECTIVE: To test the possible role of endothelial cells in mediating fade of norepinephrine-induced constriction and the effect of heartworm infection on these responses. DESIGN: Rings of pulmonary vein from control and heartworm infected dogs were constricted with norepinephrine (10(-5.5)M) and followed over 65 minutes. Time profiles were established by measuring active tension every 2 minutes for the first 10 minutes, then every 5 minutes for 15 minutes, then every 10 minutes for 40 minutes. Time profiles were done in pulmonary vein rings with and without endothelial cells, and in the presence and absence of N-nitro-L arginine methyl ester (L-NAME; nitric oxide synthase inhibitor), mefenamic acid (cyclooxygenase inhibitor), or methylene blue (guanylate cyclase inhibitor). ANIMALS: 12 noninfected control and 11 heartworm-infected dogs. RESULTS: Pulmonary vein constricted with norepinephrine spontaneously loses tension (fades) over time. Fade was not different between control and heartworm-infected dogs. In pulmonary vein from control dogs, methylene blue decreased fade while L NAME and mefenamic acid did not. In pulmonary vein from heartworm-infected dogs, L-NAME and methylene blue significantly decreased fade, but mefenamic acid did not. CONCLUSION: Nitric oxide, but not cyclooxygenase products, mediates fade of norepinephrine-induced constriction in pulmonary vein from heartworm-infected dogs. In control dogs, neither nitric oxide nor cyclooxygenase products appear to be involved in fade. We conclude that in canine pulmonary vein, fade of norepinephrine-induced constriction is mediated, in part, by endothelial cells. CLINICAL RELEVANCE: Altered production of endothelium-derived relaxing factors may be important in the pathogenesis of heartworm disease. PMID- 8633804 TI - Distribution and appearance of elastic fibers in the dermis of clinically normal dogs and dogs with solar dermatitis and other dermatoses. AB - OBJECTIVE: To determine the distribution and amount of elastic fibers in the dermis of clinically normal dogs and dogs with dermatoses, particularly solar dermatitis. DESIGN: Skin specimens from 7 anatomic sites were obtained from 19 clinically normal dogs after euthanasia to evaluate the normal distribution of elastic fibers. Biopsy specimens also were obtained from 34 dogs with dermatoses, including 16 with solar dermatitis. Tissue sections were stained with H&E, Verhoeff-van Gieson, and periodic acid-Schiff. ANIMALS: 19 clinically normal dogs and 34 dogs with dermatoses. PROCEDURE: Numbers of elastic fibers were graded subjectively. Comparisons between clinically normal dogs and dogs with dermatoses were made. RESULTS: Normal elastic fibers were present in low numbers in the dermis of adult dogs, regardless of anatomic site or presence or severity of dermatitis. Condensed elastotic material was visualized in only 2 dogs with solar dermatitis. In both dogs, the elastotic material was Verhoeff-van Gieson and periodic acid-Schiff stain positive but was not visible with H&E stain. The most frequent histopathologic finding in the dermis of dogs with solar dermatitis was superficial dermal fibrosis. CONCLUSIONS: The dermis of clinically normal dogs does not contain abundant elastic fibers. Alterations of elastic fibers in dogs with solar dermatitis are rare. Superficial dermal fibrosis may be a better indicator of solar damage. PMID- 8633805 TI - Use of enzyme-linked immunosorbent assay and radioimmunoassay to determine serum and urine dexamethasone concentrations in thoroughbreds after intravenous administration of the steroid. AB - OBJECTIVE: To develop a simple and sensitive ELISA for detection of dexamethasone in horse serum and urine. SAMPLE POPULATION: Blood and urine samples from 3 thoroughbred mares. PROCEDURE: A dexamethasone oxime was prepared and conjugated to hemocyanin, bovine serum albumin and to horseradish peroxidase. One- and two step double-antibody ELISA methods, as well as a radioimmunoassay method, were performed. The one-step ELISA was used to test urine from 3 Thoroughbred mares injected with 5 mg of dexamethasone, IV. RESULTS: The ELISA could detect dexamethasone in the range of 0.01 to 50 ng/ml, with intra- and interassay variations of 8.92 and 9.42%, respectively. Serum dexamethasone concentration reached a peak of 20 to 35 ng/ml 15 minutes after steroid administration and decreased to 1 ng/ml in 2.5 hours. Urine dexamethasone concentration 18 to 50 ng/ml 1 to 2 hours after drug administration and decreased to 1 ng/ml at 10 hours. CONCLUSION: The developed assay is sensitive as well as simple for detecting dexamethasone in horse serum and urine, and is comparable to radioimmunoassay. CLINICAL RELEVANCE: This method can be useful for screening samples from racehorses, because it is sensitive and does not require sample preparation or sophisticated equipment. PMID- 8633806 TI - Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate after intra-articular administration in horses. AB - OBJECTIVE--To document plasma, urine, and synovial fluid disposition of 2 common intra-articularly administered steroid preparations, methylprednisolone acetate (MPA) and isoflupredone acetate (IPA). DESIGN--Descriptive investigation. SAMPLE POPULATION--100 mg of MPA or 4 mg of IPA was administered to 2 groups of 4 healthy sound radiographically normal female horses. PROCEDURE--Blood samples were collected at time 0 (before) and 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration of the designated steroid. Complete urine collection for measurement of designated steroid was accomplished by use of occluding 28-F balloon catheters. Synovial fluid samples were aseptically aspirated from the injected and contralateral uninjected tarsocrural joint at time 0 and 8, 24, 48, 240, and 672 hours after administration of the designated steroid. All samples were screened by ELISA to detect parent drug or metabolite equivalent, with a sensitivity of 2.5 ng/ml for MPA and 0.1 ng/ml for IPA. If drug was detected by ELISA in the plasma or synovial fluid, the samples were further quantified and specified, using HPLC with a lower limit of quantification (10 ng/ml). RESULTS- Between 2 and 12 hours after administration, plasma contained < 10 ng of MPA or IPA/ml (parent drug or metabolite equivalent), as intermittently detected by ELISA. Parent drug or metabolite equivalent was detected in the urine for 24 and 72 hours after injection of IPA and MPA, respectively. Synovial fluid from the contralateral joint contained no detectable MPA or IPA at any sample collection time. Median half-life for MPA, as detected by HPLC, was 10.3 hours (range, 6.1 to 10.6) in the synovial space. Median half-life for methylprednisolone, as detected by HPLC, was 10.4 (range, 9.9 to 32.1) hours. CONCLUSIONS--Both steroids appeared to be rapidly hydrolyzed to their respective ester forms, as detected by HPLC. The ELISA appeared to be a useful screening tool for detection of corticosteroids in this variety of body fluids. PMID- 8633807 TI - Analgesic, hemodynamic, and respiratory effects of caudal epidurally administered xylazine hydrochloride solution in mares. AB - OBJECTIVE: To examine effects of 0.25 mg of xylazine/kg of body weight diluted to a total volume of 6 ml/450 kg with sterile 0.9% NaCl, administered into the epidural space of the sacrococcygeal joint on perineal analgesia, sedation, ataxia, and respiratory and cardiovascular function in standing mares. DESIGN: Randomized, blinded study, using xylazine (treatment) and 0.9% NaCl (controls). At least 2 weeks elapsed between the treatments. ANIMALS: Eight healthy mares. PROCEDURE: Blood samples were drawn. Systemic hemodynamics were determined, including cardiac output and pulmonary arterial, systemic arterial, and right atrial pressures. Two-way ANOVA with repeated measures was used to detect significant (P < 0.05) differences between mean scores of analgesia, sedation, ataxia, and cardiorespiratory variables before and during a 3-hour testing period. Analgesia was determined by lack of sensory perception to electrical stimulation at the perineal dermatome and no response to needle prick stimulation extending from coccyx to S3 dermatomes. Sedation was determined by head ptosis. RESULTS: Epidurally administered xylazine induced variable bilateral caudal analgesia extending from coccyx to S3, with minimal sedation, ataxia, and cardiovascular and respiratory depression in standing mares. Analgesia was attained at 15 +/- 6 minutes and lasted for 165 to over 180 minutes. Heart and respiratory rates, systolic, diastolic, and mean arterial blood pressure, PCV, hemoglobin concentration, arterial oxygen content, and oxygen transport were decreased after xylazine, but not 0.9% NaCl, treatment. Cardiac output, stroke volume, mean right atrial pressure, mean pulmonary artery pressure, systemic vascular resistance, pulmonary vascular resistance, arterial and mixed venous pH and gas tensions (PO2 and PCO2), oxygen consumption, blood temperature, and rectal temperature did not change significantly (P < 0.05) after epidural administration of xylazine or 0.9% NaCl. CONCLUSIONS: Caudal epidurally administered xylazine (0.25 mg/kg in 6 ml of 0.9% NaCl) can be given safely to induce prolonged (>2 hours) caudal analgesia with minimal sedation, ataxia, and circulatory and respiratory disturbances in conscious, standing mares. PMID- 8633808 TI - Effect of oral administration of flunixin meglumine on the inflammatory response to endotoxin in heifers. AB - OBJECTIVE: To compare the effect of oral and IV administrations of flunixin meglumine on the endotoxin-induced inflammatory response in heifers. DESIGN: The study was conducted in 2 experimental sets in which heifers were exposed to low IV doses of Escherichia coli endotoxin. Within each set, heifers were allocated to 3 treatment groups; pretreatment with flunixin meglumine orally and IV prior to endotoxin administration, or endotoxin administration only. The dose of flunixin used was the recommended therapeutic dose in cattle. ANIMALS: 11 clinically normal heifers weighing from 400 to 640 kg. PROCEDURE: A permanent cannula was inserted into the jugular vein on the day prior to the experiment. Blood samples were collected regularly during the experiment and analyzed for the content of prostaglandin F2 alpha metabolite, cortisol, blood mononuclear cells, and polymorphonuclear neutrophilic leukocytes and rectal temperature was measured. RESULTS: Endotoxin administration caused clinical signs and hematologic changes characteristic of endotoxemia in cattle. Flunixin administered orally prior to experimentally induced endotoxemia exerted an effect equal to that after its IV administration. Significant increases in rectal temperature and prostaglandin F2 alpha metabolite concentrations after administration of endotoxin were abrogated when the heifers were pretreated with flunixin, irrespective of route of administration. Cortisol concentrations were lower after pretreatment with flunixin. However, flunixin did not prevent the decrease in blood mononuclear cells and polymorphonuclear neutrophilic leukocytes seen after endotoxin administration. CONCLUSION: Owing to no major difference in the inflammatory response between oral and IV flunixin dosing, flunixin granules may be an alternative to parenteral use in bovine practice. PMID- 8633809 TI - Intratumoral administration of carboplatin for treatment of squamous cell carcinomas of the nasal plane in cats. AB - OBJECTIVE: To develop a slow-release carboplatin formulation for intratumoral administration to cats. DESIGN: Preliminary study to analyze pharmacokinetic effects of purified sesame oil in the carboplatin formulation for intratumoral administration, and a second study to evaluate the efficacy and toxicosis of intratumoral administration of carboplatin in purified sesame oil. ANIMALS: 23 cats with squamous cell carcinomas of the nasal plane. PROCEDURE: Eight cats with advanced-stage tumors were submitted to intratumoral administration of 100 mg of carboplatin/m2 of body surface area, with or without purified sesame oil, using a two-period, cross-over design. Fifteen additional cats were treated by intratumoral administration of carboplatin in purified sesame oil. Four weekly intratumoral chemotherapy injections of carboplatin in purified sesame oil at a dosage of 1.5 mg/cm3 of tissue were given. RESULTS: Purified sesame oil in the formulation significantly reduced systemic exposure to carboplatin and drug leakage from the sites of injection. Cumulative effects of repeated intratumoral administrations on plasma concentrations of carboplatin were not observed. Systemic toxicosis was not observed, and local toxicosis was minimal. Healing of ulcerated lesions was not compromised. Rates of complete clinical tumor clearance and complete response were 67 and 73.3%, respectively. Product-limit estimates of mean progression-free survival times was 16 +/- 3.3 months. The 1-year progression-free survival rate was 55.1 +/- 13%. Local recurrence was observed in 7 cats; 4 had marginal tumor recurrence, and 3 had in-field and marginal tumor recurrence. CONCLUSIONS: Intratumoral carboplatin chemotherapy is safe and effective for cats with squamous cell carcinoma of the nasal plane. Future studies to improve treatment efficacy could include evaluation of increased dose intensity as well as combination of this modality with radiotherapy. CLINICAL RELEVANCE: Intratumoral administration of carboplatin in a water-sesame-oil emulsion was found to be a practical and effective new treatment for facial squamous cell carcinomas in cats. PMID- 8633810 TI - Role of prostaglandins and enteric nerves in Escherichia coli heat-stable enterotoxin (STa)-induced intestinal secretion in pigs. AB - OBJECTIVE: To examine the role of prostaglandins and enteric nerves in mediating intestinal secretion induced by enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) in pig jejunum and distal portion of the colon. ANIMALS: Two- to 3-week-old suckling crossbred pigs were studied. DESIGN: Changes in ion transport in response to mucosal addition of E coli STa to jejunal and colonic tissues were studied in the presence and absence of inhibitors. PROCEDURE: Flat sheets of muscle-stripped proximal portions of the jejunum and distal portions of the colon were mounted in Ussing chambers equipped to measure changes in short circuit current (Isc), a measure of active ion transport. Tissues were exposed to 200 ng of STa/ml administered to mucosal solutions, and subsequent changes in Isc were recorded. RESULTS: In control tissues, changes in Isc induced by STa in the distal colon were significantly greater (21.4 +/- 4 muA/cm2) than those observed in the jejunum (14.0 +/- 2 muA/cm2). When either segment was exposed to the neurotoxin, tetrodotoxin, or to the nitric oxide synthase inhibitor, N(G)-nitro-L arginine-methyl ester, Isc responses to STa were unchanged, suggesting no involvement of submucosal nerves in mediating STa-induced secretion. When tissues from the distal portion of the colon and jejunum were pretreated with piroxicam, a prostaglandin synthesis inhibitor, the STa-induced Isc response was significantly reduced by 52 and 57%, respectively. CONCLUSIONS: These results indicate that the pig jejunum and distal portion of the colon are sensitive to the secretory actions of enterotoxigenic E coli STa, and that the responses are mediated, in part, by release of prostaglandins. PMID- 8633811 TI - Lipid peroxidation in free skin grafts in rats. AB - OBJECTIVE: To evaluate accumulation of products of lipid peroxidation in free skin grafts in rats over 10 days after grafting. DESIGN: Prospective analysis. ANIMALS: 30 adult Sprague-Dawley rats. PROCEDURE: Free skin grafts were applied to 1 hemithorax of 30 rats (2 groups, n = 15/ group), the other hemithorax acting as a nongrafted control. Biopsy specimens were taken from the nongrafted side of all rats immediately before and from the nongrafted and grafted sides immediately after the procedure. Biopsy specimens of both sides of the thorax were performed on days 1, 3, and 7 after grafting in group-1 rats, and on days 2, 4, and 10 after grafting in group-2 rats. Thiobarbituric acid (TBA) analysis for malondialdehyde and other aldehydic products (TBA-reactive substances) was used to determine lipid peroxidation. Concentration of TBA-reactive substances was determined by absorbance spectrophotometry at a wavelength of 532 nm. RESULTS: Accumulation of products of lipid peroxidation, reflected by increase in absorbance, continued to increase over the 10 days of this study. Difference in absorption between the nongraft and graft biopsy specimens was significant (P = 0.0011). Absorbance on days 3 and 4 was significantly higher than control and day 0 values (P < or = 0.05). Absorbance on days 7 and 10 was significantly higher than control, day-0, and day-1 values (P < or = 0.05). Rate of increase in absorption was maximal at day 4 and rapidly decreased thereafter. CONCLUSION: Accumulation of lipid peroxidation products in skin grafts may be best explained by oxygen-derived free radical-induced injury attributable to postischemic reperfusion. Maximal rate of accumulation corresponded to the known time of graft recirculation and revascularization. CLINICAL RELEVANCE: Predictability of free radical damage may allow timely pharmacologic intervention to reduce radical formation and ameliorate effects of peroxidation. Such intervention may help increase free graft survival or mitigate the effects of vascular compromise to axial pattern flaps. PMID- 8633812 TI - Efficacy dose determination study of tilmicosin phosphate in feed for control of pneumonia caused by Actinobacillus pleuropneumoniae in swine. AB - OBJECTIVE: To determine the effective dosage of tilmicosin phosphate when fed to pigs for the control of pneumonia attributable to Actinobacillus pleuropneumoniae. DESIGN: Randomized complete block design, with initial weight as the blocking factor. ANIMALS: Seeder pigs were used to infect clinically normal male and female pigs weighing between 13.6 and 36.3 kg at each of 4 trials. PROCEDURE: Five doses of tilmicosin phosphate (0, 100, 200, 300, and 400 micrograms/g) were fed to pigs for 21 days. Pigs received experimental feeds 7 days before the seeder pigs were placed into pens. Feeding continued for an additional 14 days, with seeder pigs removed 3 to 8 days after placement. All pigs were euthanatized and necropsied, with lung bacterial flora and percentages of pneumonic involvement determined. RESULTS: Improvement in clinical impression score, daily rectal temperature, and weight gain were seen for all doses of tilmicosin, compared with controls. For the same variables, tilmicosin administered at 200 to 400 micrograms/g resulted in improvements over the 100 micrograms/g dose. CONCLUSIONS: Data indicate that tilmicosin phosphate fed to pigs at 200 to 400 micrograms/g is effective in controlling and preventing A pleuropneumoniae-induced pneumonia, when administered in feed for 21 days. PMID- 8633813 TI - Clinical field trials with tilmicosin phosphate in feed for the control of naturally acquired pneumonia caused by Actinobacillus pleuropneumoniae and Pasteurella multocida in swine. AB - OBJECTIVE: To determine and evaluate the efficacy of the dose range of tilmicosin phosphate fed to pigs for control of pneumonia attributable to Actinobacillus pleuropneumoniae during episodes of clinical disease in commercial herds. DESIGN: 12 trials were run in 9 geographic locations in herds with a history of pneumonia caused by A pleuropneumoniae. ANIMALS: Clinically normal male and female pigs of various body weights. PROCEDURE: Two doses of tilmicosin phosphate (200 and 400 micrograms/g) and a 0 dose were administered in the feed for 21 days. Variables for determining efficacy were daily independent composite clinical impression score, individual pig weight, mortality, percentage of pneumonic involvement, and frequency of isolation of bacterial pathogens. RESULTS: Medicated pigs had significantly lower mortality attributed to pneumonia than did nonmedicated pigs. In trials with confirmed pneumonia caused by A pleuropneumoniae or Pasteurella multocida, weight gain, feed conversion, and clinical impression scores were significantly improved in the pigs receiving 200 or 400 micrograms/g of tilmicosin, compared with nonmedicated pigs. CONCLUSIONS: The clinical field trials reported here confirm that tilmicosin in the feed at 200 micrograms/g is effective for control of swine pneumonia attributable to A pleuropneumoniae or P multocida. CLINICAL RELEVANCE: Under the moderate natural challenge conditions encountered, tilmicosin at 400 micrograms/g was not different from tilmicosin at 200 micrograms/g. PMID- 8633814 TI - Transcatheter modification of the atrioventricular node in dogs, using radiofrequency energy. AB - OBJECTIVE: To develop a protocol for reliably inducing atrioventricular (AV) block (ideally first- or second-degree), using radiofrequency energy. DESIGN: An electrosurgical unit was coupled to an ammeter, which was connected to the distal pole of an electrode catheter positioned at the AV node. Control settings had previously been calibrated to the power output in a circuit with a 100-ohm resistance. ANIMALS: 10 clinically normal dogs. PROCEDURE: Transcatheter AV nodal modification was attempted, using progressive power applications of 10 to 20 W for progressive durations of 10 to 30 seconds. Atrioventricular nodal conduction and refractivity were measured before and 20 minutes and 1 month after ablation. Electrocardiograms were monitored throughout the 1-month period. RESULTS: Eight of the 10 dogs developed complete AV block, I developed stable 2:1 AV block, and another had no long-term change in AV nodal conduction. Four dogs attained their maximal degree of AV block in 2 to 5 days. Three of these had no AV nodal conduction changes until 2 to 4 days after ablation. CONCLUSIONS: An electrosurgical unit can be economically modified for radiofrequency transcatheter ablation. Stable, incomplete AV block was rarely induced using this protocol, whereas complete AV block often developed. A major finding was frequent delay between energy delivery to the AV nodal region and induction of AV block. CLINICAL RELEVANCE: Induction of complete AV block using this technique, followed by permanent pacemaker placement, is an effective alternative to long-term antiarrhythmic treatment in animals with chronic atrial arrhythmias. Transcatheter ablation could be used to treat other forms of tachycardia, as it is in human medicine. PMID- 8633815 TI - Nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine in patients with HIV-1 infection. A randomized, double-blind, placebo-controlled trial. National Institute of Allergy and Infectious Diseases AIDS Clinical Trials Group Protocol 241 Investigators. AB - OBJECTIVE: To study the addition of a third human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor, nevirapine, to the combination of zidovudine and didanosine. DESIGN: A 48-week, randomized, double-blind, placebo controlled trial at 16 AIDS (acquired immunodeficiency syndrome) Clinical Trials Units. PATIENTS: 398 adults who had HIV-1 infection, had 350 or fewer CD4+ T lymphocytes/mm3, and had had more than 6 months of previous nucleoside therapy. INTERVENTION: 1) Either nevirapine or placebo (200 mg/d for 2 weeks, then 400 mg/d thereafter) and 2) open-label zidovudine (600 mg/d) and didanosine (400 mg/d for patients weighing > or = 60 kg). MEASUREMENTS: CD4+ T lymphocyte counts, time to first HIV-1 disease progression event or death, adverse events, and nevirapine levels in plasma samples taken at random were measured in all patients. Plasma levels of HIV-1 RNA HIV-1 infectivity titer in peripheral blood mononuclear cells; serum p24 antigen levels; and plasma levels of zidovudine and didanosine were measured in patients enrolled at half the study sites. RESULTS: After 48 weeks of study treatment the patients assigned to the triple-combination regimen (nevirapine, zidovudine, and didanosine) had an 18% higher mean absolute CD4 cell count (95% Cl, 7% to 29%; P = 0.001), a 0.32 log10 lower mean infectious HIV-1 titer in peripheral blood mononuclear cells (Cl, 0.05 to 0.59 log10 infectious units per million cells; P = 0.023), and a 0.25 log10 lower mean plasma HIV-1 RNA level (Cl, 0.03 to 0.48 log10 RNA copies/mL; P = 0.028) than did patients assigned to the double-combination regimen (zidovudine and didanosine). Severe rashes were more common among patients assigned to receive the triple combination (9% compared with 2%; P = 0.002). Risk for disease progression did not differ between the two groups (relative hazard of the triple-combination group, 1.24 [Cl, 0.75 to 2.06]; P > 0.2), although the study had only moderate power to detect a major difference. CONCLUSIONS: Adding nevirapine to zidovudine and didanosine improved the long-term immunologic and virologic effects of therapy and was associated with severe rash among the patients studied, who had had extensive previous therapy. These results support 1) the continuing development of combinations of more than two antiretroviral drugs to increase and prolong HIV 1 suppression and 2) the potential utility of nevirapine in combination regimens. PMID- 8633816 TI - Survival in HIV-infected patients who have received zidovudine: comparison of combination therapy with sequential monotherapy and continued zidovudine monotherapy. Multicenter AIDS Cohort Study Group. AB - BACKGROUND: Among patients who begin receiving zidovudine during intermediate stage human immunodeficiency virus (HIV) infection, it is unclear whether changing to combination therapy (adding didanosine or zalcitabine) or sequential monotherapy (changing to didanosine or zalcitabine) significantly improves survival. OBJECTIVE: To determine, among patients who began receiving zidovudine during intermediate-stage HIV infection, the differential effects of changing to combination therapy (zidovudine with didanosine or zalcitabine) or sequential monotherapy (with didanosine or zalcitabine) or continuing zidovudine monotherapy. PATIENTS: 1077 HIV-seropositive men in the Multicenter AIDS (acquired immunodeficiency syndrome) Cohort Study who began receiving zidovudine before an AIDS-defining illness developed. SETTING: University-affiliated clinics in Baltimore, Chicago, Los Angeles, and Pittsburgh. DESIGN: Longitudinal cohort study, Treatment groups and important prognostic variables were modeled as time dependent covariates in Cox proportional hazards models. MEASUREMENTS: Progression to AIDS and death. RESULTS: Compared with patients receiving continued zidovudine monotherapy, patients receiving combination therapy had a 45% improvement in survival (relative risk, 0.55 [95% Cl, 0.41 to 0.74; P < 0.001]) and patients who changed to sequential monotherapy had a 32% improvement in survival (relative risk, 0.68 [Cl, 0.52 to 0.89; P = 0.005]). In the landmark analyses, the median prolongation of survival associated with changing therapy was, at best, 3 to 6 months. Survival curves converged at 3.5 years for the 50 cells/mm3 disease-stage landmark, at 4.4 years for the 100 cells/mm3 landmark and at 4.9 years for the 150 cells/mm3 landmark. Mortality within these periods was 100%, regardless of treatment group or landmark. CONCLUSIONS: For patients who began receiving zidovudine during intermediate-stage disease, changing to either combination therapy or sequential monotherapy was associated with a statistically significant survival benefit compared with continuation of zidovudine monotherapy. The absolute increase in survival was modest, however, and long-term survival remained poor. Simultaneous time-dependent adjustment for changes in therapy and in important prognostic variables is necessary to derive relatively unbiased estimates of treatment effects in observational studies of HIV infection. PMID- 8633817 TI - The effect of high-dose saquinavir on viral load and CD4+ T-cell counts in HIV infected patients. AB - OBJECTIVE: To evaluate the efficacy and safety of high-dose therapy with the human immunodeficiency virus (HIV) protease inhibitor saquinavir and to establish the duration of the effect of this therapy. DESIGN: Open-label study. SETTING: Clinical research referral center. PATIENTS: 40 adults with human immunodeficiency virus type 1 (HIV-1) infection and CD4+ T-cell counts of 200 to 500 cells/mm3. INTERVENTION: Monotherapy with 3600 mg or 7200 mg of saquinavir per day, in six divided doses, for 24 weeks. MEASUREMENTS: Patients were monitored for adverse events and were evaluated monthly for CD4+ T-cell count, HIV-1 viral load (as measured by reverse transcriptase polymerase chain reaction [PCR] for plasma HIV RNA levels), immune-complex-disassociated p24 antigen levels, peripheral blood mononuclear cell viral DNA levels (as measured by PCR), and resistance mutations to saquinavir. Quantitative peripheral blood mononuclear cell cultures were also done every 2 months. RESULTS: The low-dose saquinavir regimen (3600 mg/d) resulted in a maximal mean decrease in plasma HIV RNA levels of 1.06 log RNA copies/mL of plasma and a mean maximal increase in CD4 counts of 72 cells/mm3. At week 24, the plasma HIV RNA level remained 0.48 log RNA copies/mL of plasma lower than baseline (P < 0.001) and the CD4 count remained 31 cells/mm3 higher than baseline (P = 0.165). The high-dose saquinavir regimen (7200 mg/d) produced a mean maximal decrease in the plasma HIV RNA level of 1.34 log RNA copies/mL of plasma and a mean maximal increase in CD4 count of 121 cells/mm3. At week 24, the plasma HIV RNA level remained 0.85 log RNA copies/mL of plasma lower than baseline (P < 0.001) and the CD4 count remained 82 cells/mm3 higher than baseline (P = 0.002). The high-dose regimen produced a greater reduction in plasma HIV RNA level (P = 0.08), a greater reduction in peripheral blood mononuclear cell cultures (P = 0.008), and a greater increase in CD4 count (P = 0.002) than did the low-dose regimen. Higher plasma drug concentrations in individual patients correlated with greater reductions in plasma HIV RNA levels over the two doses. Nine patients receiving the low-dose regimen and four patients receiving the high-dose regimen developed key saquinavir resistance mutations. Adverse reactions, most commonly gastrointestinal problems and elevated serum aminotransferase levels, were more common in patients receiving the high-dose regimen, but most adverse events were mild and all were reversible. CONCLUSION: Saquinavir is a potent antiviral agent that has a favorable toxicity profile at high doses. Higher doses produce a greater and more durable suppression of viral load and elevation in CD4+ T-cell counts and may delay the development of resistance mutations. Therapy with high-dose saquinavir alone or in combination with other antiretroviral agents should be investigated further. PMID- 8633818 TI - Exercise training in patients with heart failure. A randomized, controlled trial. AB - OBJECTIVE: To assess the benefit of exercise training in patients with heart failure caused by left ventricular systolic dysfunction and to further describe the physiologic changes associated with exercise training in these patients. DESIGN: Randomized, controlled trial. SETTING: Urban outpatient clinic. PATIENTS: 40 men with compensated heart failure who were receiving standard medical therapy were randomly assigned to an exercise-training group or to a control group that did not exercise. Fifteen of the 21 patients assigned to exercise training and 14 of the 19 patients assigned to the control group completed the study. INTERVENTION: Patients assigned to exercise training participated in a program of three exercise sessions per week for 24 weeks. MEASUREMENTS: Symptom-limited exercise tests with gas exchange analysis done just before randomization, at week 12, and at week 24. RESULTS: At week 24, the following changes (mean +/- SE) were seen in patients in the exercise group and patients in the control group, respectively; exercise duration, 2.8 +/- 0.6 minutes and 0.5 +/- 0.5 minutes; peak oxygen consumption (VO2), 231 +/- 54 L/min and 58 +/- 38 L/min; peak ventilation, 12 +/- 3 L/min and -4 +/- 3 L/min; peak heart rate, 10 +/- 4 beats/min and -2 +/- 4 beats/min; and peak power output, 20 +/- 6 W and 2 +/- 5 W. Differences between the increases occurring in the exercise group and the changes occurring in the control group were significant (P < 0.05). Among patients in the exercise group, 85% of the increase in peak VO2 occurred by week 12, and 46% of the increase in peak VO2 was caused by the increase in peak heart rate. CONCLUSION: Exercise training does not appear to be contraindicated in patients with compensated heart failure. Exercise training improved exercise tolerance, as measured by increases in peak VO2, exercise duration, and power output. This improved exercise tolerance was caused in part by an increase in peak heart rate. PMID- 8633819 TI - Persistent hepatitis C viremia predicts late relapse after sustained response to interferon-alpha in chronic hepatitis C. TriVeneto Viral Hepatitis Group. AB - OBJECTIVE: To define long-term outcome in patients with chronic hepatitis C who remain viremic after sustained biochemical response to interferon-alpha therapy. DESIGN: Prospective evaluation of an outpatient cohort. SETTING: University hospital. PATIENTS: 107 patients with chronic hepatitis C who maintained normal aminotransferase levels as long as 12 months after interferon-alpha therapy. Patients were followed prospectively for an additional 6 to 36 months. MEASUREMENTS: Aminotransferase levels were monitored at 3-month intervals. Serum hepatitis C virus (HCV) RNA was tested by polymerase chain reaction before therapy, at the end of therapy, and 12 months after therapy. The HCV genotype was defined by spot hybridization using serum specimens obtained before treatment. RESULTS: Hepatitis C virus RNA was detected in 27 (25%) patients with sustained biochemical response; 80 (75%) patients were negative for HCV RNA. Patients positive for HCV RNA were older (P < 0.001), had received a smaller interferon alpha dose (P = 0.02), and were more frequently infected with HCV genotype 2 (P < 0.01). Liver histologic findings were active in 57% of patients positive for HCV RNA, despite normal alanine aminotransferase levels, compared with only 12% of patients who were negative for HCV RNA (P = 0.01). The estimated probability of hepatitis relapse by 4 years after therapy was 53% in viremic patients and 0% in patients negative for HCV RNA (P < 0.001). CONCLUSION: Patients with chronic hepatitis C should be tested for serum HCV RNA 1 year after a sustained biochemical response to interferon-alpha therapy to determine whether the response is complete and permanent. PMID- 8633820 TI - Vasopressin administration in refractory cardiac arrest. AB - BACKGROUND: Successful outcomes after cardiopulmonary resuscitation remain disappointingly infrequent, in animal studies, administration of exogenous vasopressin during closed- and open-chest cardiopulmonary resuscitation has recently been shown to be more effective than optimal doses of epinephrine in improving vital organ blood flow. OBJECTIVE: To describe the clinical effects and outcomes of administering vasopressin to patients in cardiac arrest refractory to current medical therapies. DESIGN: Case reports. SETTING: University hospital. PATIENTS: 8 adults with in-hospital cardiac arrest. INTERVENTIONS: After intravenous epinephrine (administered according to American Heart Association guidelines) and defibrillation efforts had failed, patients in cardiac arrest who were having cardiopulmonary resuscitation received 40 U of vasopressin intravenously and then defibrillation. MEASUREMENTS: Return of spontaneous circulation and hospital discharge rates. RESULTS: After administration of vasopressin, spontaneous circulation was promptly restored in all patients. Three patients were discharged from the hospital with intact neurologic function; the other five lived for between 30 minutes and 82 hours. CONCLUSION: In the presence of ventricular fibrillation with severe hypoxia and acidosis, vasopressin seems to be more potent and effective than adrenergic vasopressors for restoring spontaneous cardiovascular function. These results do not justify the widespread use of vasopressin for refractory cardiac arrest. However, on the basis of these cases, further studies comparing vasopressin with epinephrine are warranted in an effort to improve the currently dismal prognosis of patients after cardiac arrest. PMID- 8633821 TI - Human herpesvirus-6 in transplantation: an emerging pathogen. AB - BACKGROUND: Human herpesvirus-6 can be an opportunistic pathogen in transplant recipients. PURPOSE: To summarize the epidemiologic and clinical aspects of human herpesvirus-6 infection, the immunologic basis of the pathogenicity of human herpesvirus-6, and the management of human herpesvirus-6 infection in transplant recipients. DATA SOURCES: English-language articles identified through a MEDLINE search from 1986 (when human herpesvirus-6 was discovered) to the present, bibliographies of identified articles, and recognized texts. STUDY SELECTION: Reports on human herpesvirus-6 infections in bone marrow transplant recipients and in solid organ transplant recipients. DATA EXTRACTION: Data on the virology, detection, epidemiology, clinical features, and treatment of human herpesvirus-6 infection were manually abstracted from the indicated sources and summarized. Data quality and validity were independently assessed by both authors. DATA SYNTHESIS: Human herpesvirus-6 infection occurred in 38% to 60% of bone marrow transplant recipients and 31% to 55% of solid organ transplant recipients, usually 2 to 4 weeks after transplantation. Human herpesvirus-6 has two variants, designated variant A and variant B; transplant recipients were infected almost exclusively with variant B. Bone marrow suppression, interstitial pneumonitis, and encephalitis were the most commonly reported types of clinical disease caused by human herpesvirus-6. The marrow-suppressive effect of human herpesvirus-6 ranged from transient or self-limited bone marrow suppression to chronic or fatal aplastic anemia; bone marrow suppression was thought to be partially cytokine mediated. Because it can depress cell-mediated immunity, human herpesvirus-6 may facilitate superinfection by other pathogens. Human herpesvirus-6 resembles cytomegalovirus in its antiviral susceptibilities: It is resistant to acyclovir but susceptible to ganciclovir and foscarnet. Prophylaxis of human herpesvirus-6 infection is feasible in transplant recipients, but this issue must be studied further. CONCLUSION: Human herpesvirus-6 can be a pathogen in transplant recipients. Prompt recognition of disease associated with human herpesvirus-6 is important because this virus is susceptible to currently available antiviral agents. Future research should focus on delineating the complete clinical spectrum, immunologic sequelae, and efficacy of prophylactic strategies for human herpesvirus-6 infection in transplant recipients. PMID- 8633822 TI - "Failure to thrive" in older adults. AB - The term "failure to thrive" is frequently used to describe older adults whose independence is declining. The term was exported from pediatrics in the 1970s and is used to describe older adults with various concurrent chronic diseases, functional impairments, or both. Despite this heterogeneity, failure to thrive has had its own international Classification of Diseases, Ninth Revision (ICD-9) code since 1979 and has been approached as a clinically meaningful diagnosis in many review articles. This conceptual framework, however, can create barriers to proper evaluation and management. The most worrisome of these barriers is the reinforcement of both fatalism and intellectual laziness, which need to be balanced with a deconstructionist approach, wherein the major areas of impairment are identified and quantified and have their interactions considered. Four syndromes known to be individually predictive of adverse outcomes in older adults are repeatedly cited as prevalent in patients with failure to thrive: impaired physical functioning, malnutrition, depression, and cognitive impairment. The differential diagnosis of contributors to each of these syndromes includes the other three syndromes, and multiple contributors often exist concurrently. Some of these contributors are unmodifiable, some are easily modifiable, and some are potentially modifiable but only with the use of resource-intensive strategies, initial interventions should be directed at easily remediable contributors in the hope of improving overall functional status, because a single contributor may simultaneously influence several other syndromes that conspire to create the phenotype of failure to thrive. How aggressively should more resource-intensive strategies for less easily modifiable contributors be pursued? This is a central clinical, ethical, and policy issue in geriatric medicine that cannot be settled without better process and outcome data. This paper examines the medical etymology of failure to thrive and proposes a rational approach to evaluation and management that is based on the limited medical literature. PMID- 8633823 TI - 100 apples divided by 15 red herrings: a cautionary tale from the mid-19th century on comparing hospital mortality rates. AB - In 1863, Florence Nightingale argued that London hospitals were dangerous, especially compared with provincial facilities. She bolstered this contention with statistics published in William Farr's Registrar-General report which claimed that 24 London hospitals had mortality rates exceeding 90%, whereas rural hospitals had an average mortality rate of 13%. Farr had calculated mortality rates by dividing the total number of patients who died throughout the year by the number of inpatients on a single day. When calculated as the annual number of deaths divided by the total number of inpatients during the year, the mortality rate of London hospitals was 10%. A raucous debate erupted in the London medical press over how best to calculate hospital mortality rates. Critics claimed that Farr had not adjusted for differences in severity of illness between urban and rural hospitals and that his figures would mislead the public. Farr and Nightingale, in turn, criticized the poor quality of hospital data. This story reinforces the need to understand the methodologic derivation of statistics intended to compare provider quality. PMID- 8633824 TI - Protease inhibitors for HIV infection. PMID- 8633825 TI - Breast cancer susceptibility genes: current challenges and future promises. PMID- 8633826 TI - As I was dying. An examination of classic literature and dying. PMID- 8633827 TI - Interferon-gamma and management of infectious diseases. PMID- 8633828 TI - Prophylaxis of pneumocystis pneumonia and toxoplasmosis. PMID- 8633829 TI - The effectiveness of early treatment with "second-line" antirheumatic drugs. A randomized, controlled trial. AB - OBJECTIVE: To compare two therapeutic strategies for patients with recent-onset rheumatoid arthritis. DESIGN: Open, randomized clinical trial. SETTING: Outpatient clinics of six clinical centers. PATIENTS: 238 consecutive patients with recently diagnosed rheumatoid arthritis. INTERVENTIONS: Delayed or immediate introduction of treatment with slow-acting antirheumatic drugs (SAARDs). MEASUREMENTS: Primary end points were functional disability, pain, joint score, and erythrocyte sedimentation rate at 6 and 12 months and progression of radiologic abnormalities at 12 months. RESULTS: Statistically significant advantages at 12 months for patients receiving the SAARD strategy (immediate treatment with SAARDs) with regard to all primary end points that may be clinically important are indicated by the differences in improvements from baseline and their 95% CIs. These differences were 0.3 (95% CI, 0.2 to 0.6) for disability (range, 0 to 3), 10 mm (CI, 1 to 19 mm) for pain (range, 0 to 100 mm), 39 (CI, 4 to 74) for joint score (range, 0 to 534), and 11 mm/h (CI, 3 to 19 mm/h) for erythrocyte sedimentation rate (range, 1 to 140 mm/h), all in favor of SAARD treatment. The SAARD strategy also appears to be advantageous at 6 months. Radiologic abnormalities progressed at an equal rate in the SAARD and the non SAARD groups; the difference in progression (range, 0 to 448) was 1 (CI, -3 to 5). Analyses were based on the intention-to-treat principle and thus included 29% of patients in the non-SAARD group who discontinued the non-SAARD treatment strategy; treatment was usually discontinued because of insufficient effectiveness. The SAARD strategy including two alternative SAARDs could not be continued by 8% of patients, usually because of adverse reactions. CONCLUSIONS: Early introduction of SAARDs may be more beneficial than delayed introduction for patients with recently diagnosed rheumatoid arthritis. PMID- 8633830 TI - Growth hormone replacement in healthy older men improves body composition but not functional ability. AB - OBJECTIVE: To determine whether growth hormone replacement in older men improves functional ability. DESIGN: Randomized, controlled, double-blind trial. SETTING: General community. PATIENTS: 52 healthy men older than 69 years of age with well preserved functional ability but low baseline insulin-like growth factor 1 levels. INTERVENTION: Growth hormone (0.03 mg/kg of body weight) or placebo given three times a week for 6 months. MEASUREMENTS: Body composition, knee and hand grip muscle strength, systemic endurance, and cognitive function. RESULTS: The participants' mean age was 75.0 years (range, 70 to 85 years). At 6 months, lean mass had increased on average by 4.3% in the growth hormone group and had decreased by 0.1% in the placebo group, a difference of 4.4 percentage points (95% CI, 2.1 to 6.8 percentage points). Fat mass decreased by an average of 13.1% in the growth hormone group and by 0.3% in the placebo group, a difference of 12.8 percentage points (CI, 8.6 to 17.0 percentage points). No statistically or clinically significant differences were seen between the groups in knee or hand grip strength or in systemic endurance. The mean Trails B score in the growth hormone group improved by 8.5 seconds, whereas scores in the placebo group deteriorated by 5.0 seconds, a difference of 13.5 seconds (CI, 3.1 seconds to 23.9 seconds; P = 0.01). However, the growth hormone group's score on the Mini Mental Status Examination deteriorated by 0.4, whereas the placebo group's score improved by 0.2, a difference of 0.6 (P = 0.11). The two treatment groups had almost identical scores on the Digit Symbol Substitution Test (P > 0.2). Twenty six men in the growth hormone group had 48 incidents of side effects, and 26 placebo recipients had 14 incidents of side effects (P = 0.002). Dose reduction was required in 26% of the growth hormone recipients and in none of the placebo recipients (P < 0.001). CONCLUSIONS: Physiologic doses of growth hormone given for 6 months to healthy older men with well-preserved functional abilities increased lean tissue mass and decreased fat mass. Although body composition improved with growth hormone use, functional ability did not improve. Side effects occurred frequently. PMID- 8633831 TI - Once-daily aminoglycoside dosing in immunocompetent adults: a meta-analysis. AB - OBJECTIVE: To compare the efficacy, nephrotoxicity, and ototoxicity of once-daily aminoglycoside dosing with those of standard aminoglycoside regimens in immuno competent adults. DATA SOURCES: A structured MEDLINE search from 1966 to April 1995 using the keywords aminoglycosides, drug administration schedule, and adult; bibliographic searching of review articles, position papers, and references of the selected articles; contact with primary authors of selected articles to obtain information not in the published reports and lists of potentially relevant articles. STUDY SELECTION: Randomized, controlled trials that 1) compared an intravenous once-daily aminoglycoside regimen with a standard aminoglycoside regimen in infected immunocompetent adults and 2) examined efficacy, mortality, or toxicity. DATA EXTRACTION: For each selected study, two independent reviewers assessed methodologic quality and abstracted data. The heterogeneity of individual study risk ratios was assessed and data were pooled using a random effects model. RESULTS: Forty-two studies were reviewed for possible inclusion. Thirteen independent studies met the selection criteria, and their results were pooled. The trials had a mean methodologic quality score of 0.69 (range, 0.50 to 0.91). Heterogeneity exists among the individual risk ratios for clinical cure (P = 0.07); significant heterogeneity does not exist for the other outcomes. For the pooled efficacy outcomes, the risk ratio for bacteriologic cure is 1.02 (95% CI, 0.99 to 1.05), and the risk ratio for mortality is 0.91 (CI, 0.63 to 1.31). For the pooled toxicity outcomes, the risk ratio for nephrotoxicity is 0.87 (CI, 0.60 to 1.26), and the risk ratio for ototoxicity is 0.67 (CI, 0.35 to 1.28). CONCLUSIONS: Standard and once-daily aminoglycoside dosing regimens are equivalent with regard to bacteriologic cure, and once-daily dosing shows a trend toward reduced mortality and toxicity. However, additional studies are needed for more precise estimates of mortality and toxicity risk ratios. The equivalency of the dosing regimens, the ease of administration, reduced nursing time, and reduced variability in the timing of drug administration that are associated with once-daily dosing may mean that the once-daily regimen is clinically advantageous. PMID- 8633833 TI - Colonic bacillary angiomatosis. PMID- 8633832 TI - Pennyroyal toxicity: measurement of toxic metabolite levels in two cases and review of the literature. AB - BACKGROUND: Pennyroyal is a widely available herb that has long been used as an abortifacient despite its potentially lethal hepatotoxic effects. However, quantitative data for pennyroyal constituents and their metabolites in humans have not been previously reported. OBJECTIVES: To quantify pennyroyal metabolites in human overdose, to correlate these findings with clinical variables, and to place these findings in the context of previously reported cases of pennyroyal toxicity. DESIGN: Clinical case series of pennyroyal ingestions; quantification of pennyroyal metabolites by gas chromatography and mass spectrometry; qualitative detection of protein-bound adducts of the metabolites of pennyroyal constituents in human liver by Western blot assay; and review of the literature based on a search of MEDLINE, Index Medicus, and the reference citations of all available publications. RESULTS: We report four cases of pennyroyal ingestion. One patient died, one received N-acetylcysteine, and two ingested minimally toxic amounts of pennyroyal and were not treated with N-acetylcysteine. In the fatal case, postmortem examination of a serum sample, which had been obtained 72 hours after the acute ingestion, identified 18 ng of pulegone per mL and 1 ng of menthofuran per mL. In a serum sample from the patient treated with N acetylcysteine, which had been obtained 10 hours after ingestion, the menthofuran level was 40 ng/mL. Review of 18 previous case reports of pennyroyal ingestion documented moderate to severe toxicity in patients who had been exposed to at least 10 mL of pennyroyal oil. CONCLUSION: Pennyroyal continues to be an herbal toxin of public health importance. Data on human metabolites may provide new insights into the toxic mechanisms and treatment of pennyroyal poisoning, including the potential role of N-acetylcysteine. Better understanding of the toxicity of pennyroyal may also lead to stricter control of and more restricted access to the herb. PMID- 8633834 TI - Detection of a clonal BCL2 gene rearrangement in tissues from a patient with Whipple disease. PMID- 8633835 TI - Checklist of information for inclusion in reports of clinical trials. The Asilomar Working Group on Recommendations for Reporting of Clinical Trials in the Biomedical Literature. PMID- 8633836 TI - Reported complications of silicone gel breast implants: an epidemiologic review. AB - PURPOSE: To review the range of local and systemic complications attributed to silicone breast implants and to evaluate the epidemiologic literature on these complications. DATA SOURCES: Epidemiologic studies of the potential risks of silicone breast implants identified by MEDLINE search and literature review. STUDY SELECTION: Epidemiologic studies with cohort, case-control, and cross sectional designs. When epidemiologic studies were unavailable (as for estimates of local complications), case series were reviewed. DATA EXTRACTION: Epidemiologic studies evaluated for methodologic quality, including such characteristics as study design, sample size and selection, determination of silicone exposure and outcome variables, and duration of follow-up. DATA SYNTHESIS: The epidemiologic literature on the potential complications of silicone breast implants has concentrated primarily on connective tissue disorders and cancer. Estimated of the true incidence of local complications, such as rupture, capsular contracture, and breast pain, are unavailable. Studies of scleroderma and other defined connective tissue diseases suggest that implant recipients have no substantially increased risk for these disorders; however, the epidemiologic literature is insufficient to rule out an association between breast implants and connective tissue disease-like syndromes. Overall, the rate of breast cancer does not seem to be increased in women with silicone breast implants. However, the risk to women as they reach the postmenopausal years in not yet known. CONCLUSIONS: Information is insufficient to adequately advise women who currently have or are seeking to obtain breast implants about the overall risk of these devices. No epidemiologic study has indicated that the rate of well-defined connective tissue disease or breast cancer has greatly increased in women with silicone breast implants, but no studies have ruled out a moderately increased risk for these diseases. No studies have adequately addressed the crucial issue of local complications such as rupture and capsular contracture, although evidence increasingly points to a higher risk for rupture as implants age. PMID- 8633837 TI - Plasma cytokines, cytokine antagonists, and disease progression in African women infected with HIV-1. AB - OBJECTIVES: To examine the relation of circulating cytokines and cytokine antagonists to the progression of human immunodeficiency virus type 1 (HIV-1) disease. DESIGN: Cross-sectional analysis. SETTING: An ambulatory acquired immunodeficiency syndrome (AIDS) research clinic in Kinshasa, Zaire. PATIENTS: 48 women with AIDS, 51 women with HIV infection who were clinically asymptomatic, and 11 female controls who did not have HIV infection, all from Zaire. MEASUREMENTS: Plasma levels of interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha), interleukin-6, interleukin-8, interferon-gamma, interleukin-1beta receptor antagonist (interleukin-1Ra), and TNF soluble receptor p55 (TNFsRp55) were assayed by specific radioimmunoassays. Plasma levels of interferon-gamma were assayed by commercial enzyme-linked immunosorbent assay. The Wilcoxon rank sum test was used to assess the significance of mean and median differences between groups. RESULTS: Of the 48 patients with AIDS, circulating interleukin 1beta was detected in 2, TNF-alpha in 4, interleukin-6 in 3, and interleukin-8 in 12. None of these factors were seen in any of the 11 controls. Median values of interleukin-1beta (320 pg/mL), TNF-alpha (210 pg/mL), and interleukin-8 (750 pg/mL) were elevated in HIV-infected asymptomatic patients compared with patients with AIDS (2-, 2.6-, and 18.7-fold higher, respectively; P < 0.001). Interleukin 1Ra and TNFsRp55 levels were substantially higher than interleukin-1beta and TNF alpha levels in HIV-infected asymptomatic patients (73- and 14-fold, respectively) and were higher than those in patients with AIDS (17.8- and 1.74 fold, respectively). CONCLUSION: High circulating levels of the proinflammatory cytokines interleukin-1beta and TNF-alpha, combined with an excess of their natural inhibitors interleukin-1Ra and TNF-sRp55, were seen in clinically asymptomatic HIV-1-positive African women but not in African women with AIDS or in HIV-negative controls. Circulating cytokine antagonists may play a clinical role in modulating cytokine-associated symptoms in the early phases of HIV infection. PMID- 8633838 TI - Noncardiac surgery in the cardiac patient: what is the question? AB - Before having major noncardiac surgery, patients with known or suspected coronary artery disease frequently have noninvasive cardiac testing to better define their cardiac risk. The rationale for this approach is that prophylactic coronary revascularization will significantly reduce the number of adverse cardiac events. No randomized studies support this conclusion. Furthermore, recent studies have suggested that adverse cardiac events result from postoperative stress and excess catecholamine levels, which cause an imbalance between myocardial oxygen supply and demand. Plaque rupture in this setting, if it occurs, is secondary and not primary, in contrast to its pivotal role in spontaneous myocardial infarction. Therefore, improved clinical outcomes are more likely to result from preventing excess oxygen demand after surgery rather than from deciding which tests optimally predict adverse events. The exception is the patient with a clinical syndrome consistent with existing plaque rupture who requires active therapy for the cardiac disease independent of the need for noncardiac surgery. Otherwise, the tests should be skipped and the patient cleared. PMID- 8633839 TI - Screening for cardiac disease in patients having noncardiac surgery. AB - The preoperative evaluation of the cardiac patient having noncardiac surgery offers an opportunity to identify occult and further define known cardiovascular disease to modify both perioperative and long-term care. The baseline probability of cardiovascular disease should initially be assessed using clinical variables and identifying unstable symptoms, including unstable angina and congestive heart failure. The decision about whether to obtain noninvasive testing to further define cardiovascular status should be made on the basis of the testing's potential to modify perioperative care, the prior probability of advanced coronary disease based on clinical history, and the magnitude of the surgical procedure. Noninvasive testing is best done in selected patients who are at moderate clinical risk. Otherwise, testing loses its predictive value because of a high incidence of false-negative and false-positive results. Quantitative imaging can also be used to identify those patients in whom coronary angiography is indicated. The value of coronary revascularization before noncardiac surgery has not been studied in a randomized, prospective manner, but several cohort studies have suggested that patients who survive coronary artery bypass grafting have decreased risk during subsequent noncardiac surgery. Given the potential short-term increase in morbidity from two surgical procedures, it is prudent to reserve coronary revascularization before noncardiac surgery for those patients in whom it is associated with improved long-term survival. If coronary revascularization is reserved for these patients, then the overall evaluation should prove cost-effective from the perspective of both perioperative and long term cardiovascular care. PMID- 8633840 TI - Rheumatoid arthritis: treat now, not later! PMID- 8633841 TI - Effects of famiciclovir in acute herpes zoster. PMID- 8633842 TI - Renal impairment associated with losartan. PMID- 8633843 TI - Drug legalization, harm reduction, and drug policy. PMID- 8633844 TI - Drug legalization, harm reduction, and drug policy. PMID- 8633845 TI - Drug legalization, harm reduction, and drug policy. PMID- 8633846 TI - Drug legalization, harm reduction, and drug policy. PMID- 8633847 TI - Drug legalization, harm reduction, and drug policy. PMID- 8633848 TI - Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture. AB - In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle. PMID- 8633849 TI - Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors. AB - In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels. PMID- 8633850 TI - Development of an oligonucleotide probe for Aureobasidium pullulans based on the small-subunit rRNA gene. AB - Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonizes leaf surfaces and has potential as a biocontrol agent of pathogens. To assess the feasibility of rRNA as a target for A. pullulans-specific oligonucleotide probes, we compared the nucleotide sequences of the small-subunit rRNA (18S) genes of 12 geographically diverse A. pullulans strains. Extreme sequence conservation was observed. The consensus A. pullulans sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 A. pullulans strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum was also identified. These probes have potential in monitoring and quantifying fungi in leaf surface and other microbial communities. PMID- 8633851 TI - Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. AB - A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm. PMID- 8633852 TI - Isolation of Geobacter species from diverse sedimentary environments. AB - In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process. PMID- 8633853 TI - Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition. AB - To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b) cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua. PMID- 8633854 TI - Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications. AB - The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The delta ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the delta ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the delta ath1 strain may make this strain useful in commercial applications, including baking and brewing. PMID- 8633855 TI - The lactose transporter in Leuconostoc lactis is a new member of the LacS subfamily of galactoside-pentose-hexuronide translocators. AB - The gene encoding the lactose transport protein (lacS) of Leuconostoc lactis NZ6009 has been cloned from its native lactose plasmid, pNZ63, by functional complementation of lactose permease-deficient Escherichia coli mutants. Nucleotide sequence analysis revealed an open reading frame with the capacity to encode a protein of 639 amino acids which had limited but significant identity to the lactose transport carriers (LacS) of Streptococcus thermophilus (34.5%) and Lactobacillus bulgaricus (35.6%). This similarity was present both in the amino terminal hydrophobic carrier domain, which is homologous to the E. coli melibiose transporter, and in the carboxy-terminal enzyme IIA-like regulatory domain. The flanking regions of DNA surrounding lacS were also sequenced. Preceding the lacS gene was a small open reading frame in the same orientation encoding a deduced 95 amino-acid protein with a sequence similar to the amino-terminal portion of beta galactosidase I from Bacillus stearothermophilus. The lacS gene was separated from the downstream beta-galactosidase genes (lacLM) by 2 kb of DNA containing an IS3-like insertion sequence, which is a novel arrangement for lac genes in comparison with that in other lactic acid bacteria. The lacS gene was cloned in an E. coli-Streptococcus shuttle vector and was expressed both in a lacS deletion derivative of S. thermophilus and in a pNZ63-cured strain, L. lactis NZ6091. The role of the LacS protein was confirmed by uptake assays in which substantial uptake of radiolabeled lactose or galactose was observed with L. lactis or S. thermophilus plasmids harboring an intact lacS gene. Furthermore, galactose uptake was observed in NZ6091, suggesting the presence of at least one more transport system for galactose in L. lactis. PMID- 8633856 TI - Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora. AB - The pathways of short-chain fatty acid (SCFA; acetate, propionate, and butyrate) formation from glucose were determined for the human fecal microbial communities of two subjects. The pathways were identified by radioisotope analysis of the SCFA and CO2 obtained after incubation of fecal suspensions with glucose under 20% CO2 with [1-14C]glucose, [3,4-14C]glucose, or 14CO2. Acetate was chemically degraded to learn the labeling of the methyl and carboxyl carbons. The labeling of CO2 and acetate showed that the major route of glucose catabolism was the Embden-Meyerhof-Parnas pathway, with production of CO2 from pyruvate carboxyl carbon. Labeling of the methyl and carboxyl carbons of acetate by 14CO2 or [3,4 14C]glucose proved that acetate was formed from CO2 by the Wood-Ljungdahl pathway. CO2 reduction accounted for about one-third of the acetate formed by suspensions from subject 1 and about one-fourth of the acetate formed by suspensions from subject 2. Propionate was formed by a CO2 fixation pathway, and butyrate was formed by classical routes of acetyl-S coenzyme A condensation. The amount of CO2 formed from [1-14C] glucose and acetate labeling patterns obtained with the other 14C precursors indicated that the Entner-Doudoroff, transketolase transaldolase, and heterolactic pathways were not significant. Fermentation of cabbage cellulose by subject 1 followed the same pathways as were used for glucose. The results with suspensions from subject 2 suggested that some radioactive acetate was formed from the C-3 of glucose by the Bifidobacterium pathway. PMID- 8633857 TI - Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes. AB - The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium. PMID- 8633858 TI - Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici. AB - The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated. PMID- 8633859 TI - Model for the combined effects of temperature, pH, and sodium lactate on growth rates of Listeria innocua in broth and Bologna-type sausages. AB - A modified Monod equation was successfully applied to describe the maximum specific growth rate of Listeria innocua in a broth model in the presence of various concentrations of sodium lactate or NaCl. The combined effects of temperature and pH were assessed by translating the parameters of the modified Monod equation mu(m), alpha, and p') as functions of pH and/or temperature. As a result, the area in which the growth rate could be predicted was extended to include as a variable not only the salt concentration but also pH and temperature. The number of parameters needed to describe the experimental data was thereby reduced from 48 to 4 (NaCl) and from 42 to 5 (sodium lactate). The decline in the goodness of fit that accompanied the reduction in the number of parameters was within statistically acceptable ranges. The resulting model was compared with a polynomial fit, and it was proposed that the former was more suitable for the purpose of this study. The broth model for sodium lactate was evaluated with Bologna-type sausages. Because of the "worst-case" design of the broth model, it was necessary to reestimate one or all parameters to obtain a good description of the growth rate of L. innocua in the meat product. However, the simplicity of the model and the practical usefulness of its parameters offer considerable prospects for its use in predictive microbiology. PMID- 8633861 TI - Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme. AB - Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74 kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism. PMID- 8633860 TI - Characterization of 16S rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide oxidizing bacteria. AB - Oil field bacteria were characterized by cloning and sequencing of PCR-amplified 16S rRNA genes. A variety of gram-negative, sulfate-reducing bacteria was detected (16 members of the family Desulfovibrionaceae and 8 members of the family Desulfobacteriaceae). In contrast, a much more limited number of anaerobic, fermentative, or acetogenic bacteria was found (one Clostridium sp., one Eubacterium sp., and one Synergistes sp.). Potential sulfide oxidizers and/or microaerophiles (Thiomicrospira, Arcobacter, Campylobacter, and Oceanospirillum spp.) were also detected. The first two were prominently amplified from uncultured production water DNA and represented 28 and 47% of all clones, respectively. Growth on media containing sulfide as the electron donor and nitrate as the electron acceptor and designed for the isolation of Thiomicrospira spp. gave only significant enrichment of the Campylobacter sp., which was shown to be present in different western Canadian oil fields. This newly discovered sulfide oxidizer may provide a vital link in the oil field sulfur cycle by reoxidizing sulfide formed by microbial sulfate or sulfur reduction. PMID- 8633862 TI - Natural occurrence of Fusarium mycotoxins in field samples from the 1992 Wisconsin corn crop. AB - Analysis of 98 moldy corn samples collected in Wisconsin between November 1992 and January 1993 for Fusarium toxins by various immunochemical assays revealed overall average mycotoxin concentrations of 305.6, 237.7, and 904.3 ng/g for type A trichothecenes (TCTCs), deoxynivalenol (DON)-related type B TCTCs (total DON), and zearalenone (ZE), respectively. A small portion (5.1%) of the samples was found to be contaminated with high levels ( > 1 microgram/g) of type A TCTCs and total DON during the whole survey. Over 40% of the samples had 100 to 1,000 ng of total DON per g, while 17% of the samples had the same levels of type A TCTCs. The analytical data were consistent with those from mycological examinations for the samples in which various toxic Fusarium spp., including F. sporotrichioides, F. poae, and F. graminearum, were found. The samples received in November 1992 had relatively low concentrations of toxin; the average levels of type A TCTCs and total DON were 9.9 and 79 ng/g, respectively. The toxin concentrations became progressively higher in the samples received in December. The average levels for the type A TCTCs and total DON increased to 920 and 335 ng/g, respectively. However, the levels of ZE were higher in the samples collected earlier. The average levels for samples collected in November and late December were 1,195 and 242 ng/g, respectively. Analysis of selected samples by high-performance liquid chromatography monitoring with an enzyme-linked immunosorbent assay revealed that T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and T-2 tetraol (T-2-4ol) were common in these samples. Statistical analysis revealed a weak correlation between the levels of total type A TCTCs and total DON in the samples (r = 0.18, P = 0.09), but a strong correlation between the levels of ZE and total type B TCTCs (r = 0.75, P < 0.0001) was found. The mycotoxin levels of total type A TCTCs, total DON-related type B TCTCs, and ZE in the cobs (5.2, 3.9, and 21 micrograms/g, respectively) were considerably higher than those in the kernels (1.0, 0.5, and 0.5 microgram/g, respectively). The type A toxin levels increased from a range of 14 to 35 ng/g to a range of 110 to 538 ng/g after the moldy corn samples were held at 5 degrees C for 8 days in the laboratory. PMID- 8633863 TI - Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes. AB - Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6 diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples. PMID- 8633864 TI - Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes. AB - In situ hybridization with fluorescent oligonucleotides was used to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors that had been fed for several months with either sucrose or a mixture of volatile fatty acids. Sections of the granules were hybridized with 16S rRNA-targeted oligonucleotide probes for Bacteria, Archaea, specific phylogenetic groups of methanogens, and two syntrophic propionate-oxidizing strains, MPOB and KOPROP1. Cells of the syntrophic strain KOPROP1 were not detected in either type of sludge granules. Hybridizations of the sucrose-fed granules showed an outer layer of mainly bacterial microcolonies with different morphologies. More inwards of these granules, a layer of different methanogenic microcolonies mixed with large colonies of the syntrophic strain MPOB could be detected. The MPOB colonies were intertwined with hydrogen- or formate-consuming methanogens, indicating their syntrophic growth. The granules fed with volatile fatty acids showed an outer layer of mainly bacteria and then a thick layer of Methanosaeta-like methanogens mixed with a few bacteria and a layer of methanogens mixed with syntrophic MPOB microcolonies. The centers of both sludge types consisted of large cavities and methanogenic microcolonies. These results indicate a juxtapositioning of syntrophic bacteria and methanogens and provide additional evidence for a layered microbial architecture of anaerobic granular sludge. PMID- 8633865 TI - Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. AB - A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin. PMID- 8633866 TI - Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. AB - The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods. PMID- 8633868 TI - Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid tolerant mutant which demonstrates increased virulence. AB - The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence. PMID- 8633867 TI - The genes for secretion and maturation of lactococcins are located on the chromosome of Lactococcus lactis IL1403. AB - Southern hybridization and PCR analysis were used to show that Lactococcus lactis IL1403, a plasmid-free strain that does not produce bacteriocin, contains genes on its chromosome that are highly homologous to lcnC and lcnD and encode the lactococcin secretion and maturation system. The lcnC and lcnD homologs on the chromosome of IL1403 were interrupted independently by Campbell-type integrations. Both insertion mutants were unable to secrete active lactococcin. Part of the chromosomal lcnC gene was cloned and sequenced. Only a few nucleotide substitutions occurred, compared with the plasmid-encoded lcnC gene, and these did not lead to changes in the deduced amino acid sequence. No genes homologous to those for lactococcin A, B, or M could be detected in IL1403, and the strain does not produce bacteriocin activity. PMID- 8633869 TI - Xylulose and glucose fermentation by Saccharomyces cerevisiae in chemostat culture. AB - Saccharomyces cerevisiae ATCC 24860 was cultivated in chemostat culture under anoxic conditions with 111.1 mmol of glucose liter-1 alone or with a mixture of 66.7 mmol of xylulose liter-1 and 111.1 mmol of glucose liter-1. The substrate consumption rate was 5.4 mmol g of cells-1 h-1 for glucose, whereas for xylulose it was 1.0 mmol g of cells-1 h-1. The ethanol yield decreased from 0.52 carbon mole of ethanol produced per carbon mole of sugar consumed during the utilization of glucose alone to 0.49 carbon mole produced per carbon mole consumed during the simultaneous utilization of xylulose and glucose, while cell biomass was maintained at 2.04 to 2.10 g liter-1. Xylulose coutilization was accompanied by a shift in product formation from ethanol to acetate and arabinitol. Xylulokinase activity was absent during glucose metabolism but detectable during simultaneous utilization of xylulose and glucose. Xylulose cometabolism resulted in increased in vitro activity of pyruvate decarboxylase and an increased concentration of the intracellular metabolite fructose 1,6-diphosphate without significant changes in the concentrations of 6-phosphogluconate and pyruvate. The results are discussed in relation to (i) altered enzyme activities and (ii) the redox flux of the cell. PMID- 8633870 TI - Isolation of Thermus strains from hot composts (60 to 80 degrees C). AB - High numbers (10(7) to 10(10) cells per g [dry weight]) of heterotrophic, gram negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70 degrees C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80 degrees C) of the composting process. PMID- 8633872 TI - Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal. AB - Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site. C. perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca. 48 km) and 50 nautical miles (ca. 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site. C. perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site. At stations where C. perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm. In some cases, C. perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation. We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site. PMID- 8633873 TI - A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus. AB - Unicellular cyanobacteria of the genus Synechococcus are among the most abundant members of the picoplankton in the open ocean, and their contribution to primary production is considerable. While several isolates have been used for physiological, biochemical, and molecular studies of their unique adaptations to the marine environment, it has become necessary to develop molecular genetic methods for one or more model open-ocean cyanobacteria in order for studies of these organisms and their unique properties to progress. A number of molecular tools for the genetic manipulation of Synechococcus sp. strains WH7803, WH8102, and WH8103 have been developed. These include a plating technique for obtaining isolated colonies at high efficiencies and a conjugation method for introducing both a replicative vector and a suicide vector. In addition, a method for the generation of random, tagged chromosomal insertions (N. Dolganov and A. R. Grossman, J. Bacteriol. 175:7644-7651, 1993; N. F. Tsinoremas, A. K. Kutach, C. A. Strayer, and S. S. Golden, J. Bacteriol. 176:6764-6768, 1994) has been applied to these organisms. PMID- 8633874 TI - Mechanism by which gamma irradiation increases the sensitivity of Salmonella typhimurium ATCC 14028 to heat. AB - Effects of irradiation and heating on survival of Salmonella typhimurium ATCC 14028 were examined by measuring DNA damage and the integrity of the cytoplasmic membrane. S. typhimurium cells fell into two distinct groups following heating: (i) heat-sensitive cells, which were rapidly inactivated at 65 degrees C and (ii) heat-resistant cells, which were only slowly inactivated at 65 degrees C. Radiation sensitivity of S. typhimurium was greater in the presence of air than in the presence of N2 gas (radiation doses required to inactivate 90% of the cells, 0.394 +/- 0.029 in air and 0.561 +/- 0.035 in N2). Recovery of the covalently closed circular form of plasmid pBR322 from S. typhimurium transformants (Ampr Tetr) was decreased by irradiation but not by heating. Heating prior to irradiation significantly decreased the recovery of plasmid DNA without affecting survival of S. typhimurium. Transformability of the recovered plasmid pBR322 was affected by neither irradiation nor heating, and mutation of antibiotic resistance genes was not detected in S. typhimurium. Heating, but not irradiation, caused destabilization of the cytoplasmic membrane, allowing penetration of hydrophobic dye. These results suggest that lethality of heating followed by irradiation for S. typhimurium was additive, reflecting irradiation induced DNA damage and heat-induced membrane destabilization. When irradiation preceded heating in the absence of air, more cells were inactivated than was expected, because of heat-inactivating radiation-damaged DNA. PMID- 8633871 TI - Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. AB - We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation. PMID- 8633875 TI - A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. AB - Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification. PMID- 8633876 TI - Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola. AB - In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose , mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate limiting conditions such as those often found in the rumen. PMID- 8633877 TI - Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin. AB - Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures. PMID- 8633878 TI - Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. AB - Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis. PMID- 8633879 TI - Variacin, a new lanthionine-containing bacteriocin produced by Micrococcus varians: comparison to lacticin 481 of Lactococcus lactis. AB - A new lanthionine-containing bacteriocin, variacin, displaying a broad host range of inhibition against gram-positive food spoilage bacteria, has been identified from two strains of Micrococcus varians isolated from meat fermentations. The new bacteriocin was purified, and its amino-terminal end and total amino acid composition were determined. The structural gene was isolated and analyzed. Variacin is resistant to heat and pH conditions from 2 to 10. Its primary sequence shows significant homology to lacticin 481 to Lactococcus lactis, which is more pronounced for the probacteriocin than for the leader sequence. Variacin, like lacticin 481, contains lanthionine and beta-methyllanthionine residues, but its leader sequence clearly resembles nonlantibiotic leader sequences. In particular, the prepeptide contains glycine residues at positions -1 and -2 of the processing site. PMID- 8633880 TI - Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. AB - Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight. PMID- 8633881 TI - Microbial degradation of hydrochlorofluorocarbons (CHCl2F and CHCl2CF3) in soils and sediments. AB - The ability of microorganisms to degrade trace levels of the hydrochlorofluorocarbons HCFC-21 and HCFC-123 was investigated. Methanotroph linked oxidation of HCFC-21 was observed in aerobic soils, and anaerobic degradation of HCFC-21 occurred in freshwater and salt marsh sediments. Microbial degradation of HCFC-123 was observed in anoxic freshwater and salt marsh sediments, and the recovery of 1,1,1-trifluoro-2-chloroethane indicated the involvement of reductive dechlorination. No degradation of HCFC-123 was observed in aerobic soils. In some experiments, HCFCs were degraded at low (parts per billion) concentrations, raising the possibility that bacteria in nature remove HCFCs from the atmosphere. PMID- 8633882 TI - rpoS regulation of acid, heat, and salt tolerance in Escherichia coli O157:H7. AB - An rpoS mutant (rpoS::pRR10) of Escherichia coli O157:H7 ATCC 43895 was generated. Stationary-phase acid, heat, and salt tolerance was significantly reduced, and starvation-induced acid tolerance did not develop in the mutant. RpoS was also important for survival of E. coli O157:H7 in dry, fermented sausage. PMID- 8633883 TI - Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC). AB - The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707. PMID- 8633884 TI - Nisin stimulates oxygen consumption by Staphylococcus aureus and Escherichia coli. AB - Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane. PMID- 8633885 TI - Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli. AB - Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid. Evidence that plasmid-like structures are formed in H. polymorpha and that their formation is stimulated by DNA damage is presented in this study. PMID- 8633886 TI - Degradation of 2-sec-butyl-4,6-dinitrophenol (dinoseb) by Clostridium bifermentans KMR-1. AB - A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6 dinitrophenol (dinoseb) to a level below the limit of detection by high performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates. KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source. KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2. The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2. During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases. This is the first demonstration of dinoseb degradation by a pure microbial culture. PMID- 8633889 TI - Nurse caring behaviors for persons with acquired immunodeficiency syndrome/human immunodeficiency virus. AB - The purpose of this study was to identify nurse caring behaviors desired by patients with acquired immunodeficiency syndrome (AIDS) or human immunodeficiency virus (HIV) seropositive. Forty-six adults with either a diagnosis of AIDS or HIV seropositive participated in the study. Subjects indicated "treat me as an individual" as the highest scoring item. Themes derived from the data included acceptance, respect, treatment of the person as an individual, and nonjudgmental attitudes of the nurse toward the person with AIDS/HIV. PMID- 8633888 TI - Every nurse should be a patient, every researcher should be a subject. PMID- 8633887 TI - Defective site-specific integration elements are present in the genome of virulent bacteriophage LL-H of Lactobacillus delbrueckii. AB - The phage attachment site, attP, and the integrase-encoding gene, int, are sufficient to promote site-specific integration of the temperate phage mv4 genome into the chromosome of the Lactobacillus delbrueckii host (L. Dupont, B. Boizet Bonhoure, M. Coddeville, F. Auvray, and P. Ritzenthaler, J. Bacteriol. 177:586- 595, 1995). The mv4 genome region containing these elements was compared at the nucleotide and amino acid levels with that of the closely related virulent phage LL-H. Complex DNA rearrangements were identified; a truncated integrase gene and two sites homologous to the mv4 attP site were detected in the genome of the virulent phage LL-H. These observations suggest that the two phages derive from a common temperate ancestor. PMID- 8633890 TI - Accuracy of infant emesis volume assessment. AB - Even though nurses assess volume output for blood, emesis, and stool using visual processing, assessment accuracy has not been studied nor has the application of information-processing theory to visual assessment been shown. The purpose of this investigation was to determine the application of information-processing theory to accuracy in visual assessment of emesis. Nursing students and practicing pediatric nurses (N=109) participated in this nonexperimental study in which 20 randomly selected volumes were presented as visual displays of formula on receiving blankets. Subjects were asked to determine the volume. Findings showed that few displays were assessed accurately (M=2.63), but that subjects who have been taught to use a mental frame of reference in assessment were significantly more accurate. Error increased with the display volume. Additional analysis showed that subject practice role, nature of clinical practice, and number of displays assessed for weight accounted for significant proportions of variance in relative error. These findings are consistent with information processing theory and suggest that nurses who are doing visual assessment should learn a processing method that presents a mental frame of reference with which they compare the observed volume. Further, perceived weight of the object observed should be added to visual processing to increase accuracy. PMID- 8633891 TI - Self-care limitations of persons after acute myocardial infarction. AB - In this descriptive study, the self-care limitations of persons who had experienced their first myocardial infarction were identified during the first 3 months of their convalescent period. The five most frequently occurring limitations identified were patterns of personal and family living that interfere with self-care; intense emotional states, likes or dislikes, overriding interest and concerns; perceptions, meanings, and appraisals not in accord in reality; inability to attend to self with respect to changing conditions; and new, unrecognized requirements for self-care associated with changed health. Assessment of limitation is a framework that nurses might use to evaluate the needs of post myocardial infarction patients. PMID- 8633892 TI - Evaluation of absorbent products for women with mild to moderate urinary incontinence. PMID- 8633893 TI - Humanistic behaviors and primary nursing practice. PMID- 8633894 TI - Conducting research in respondents' homes: benefits, problems, and strategies. AB - In summary, homes are valuable yet underused sites for data collection. Although homes are more frequently used for qualitative research because of the nature of qualitative inquiry requiring few distractions and more reflection, homes also can be used for quantitative studies. One major benefit of collecting data in homes is that of obtaining health-related information when a person is at baseline versus in a crisis situation. With adequate funding, attention to safety, and a mechanism for making and confirming appointments, homes serve as valuable sites for obtaining data. PMID- 8633895 TI - Generalizability of the Preoperative Self-Efficacy Scale. PMID- 8633896 TI - Practice guidelines for the prediction and prevention of pressure ulcers: evaluating the evidence. AB - Clinical practice guidelines for the prediction and prevention of pressure ulcer (Agency for Health Care Policy and Research [AHCPR], 1992) were evaluated in a Canadian, university-affiliated, acute care hospital. Through a prospective study, the prevalence of pressure ulcers was determined, and pressure ulcer incidence was tracked to evaluate the accuracy of the Braden Scale for risk assessment. The prevalence rate for stage II or greater pressure ulcers was 13.6%; the rate was 29.7% when stage I (persistent redness) was included. In evaluating the Braden Scale's accuracy in predicting risk, the findings from this study were less favorable than previous reports. The total Braden score that appeared to have the best balance of sensitivity (67%) and specificity (66%) was 19. Several factors should be considered: The scale was implemented and tested hospitalwide with a wide range of patient diagnoses, age, and severity; the study was a cross-section of an existing population; and the levels of nursing care and type of staff vary between units ranging from critical to long-term care. This study highlights the need for individual settings to evaluate the AHCPR Guidelines for the Prediction and Prevention of Pressure Ulcers. PMID- 8633897 TI - The immune system in patients with renal failure. Part 1: Review of immune function. AB - Patients with renal failure often succumb to infection. Alterations in the immune system of these patients, whether inherent to the disease process or precipitated by therapeutic regimens, undermine the promotion of wellness. Part 1 of this article provides a comprehensive understanding of normal immune system function. Part 2 discusses common immune abnormalities related to renal failure and its treatment. In addition, there is an overview of assessment techniques that can assist nephrology nurses in identifying alterations in a patient's immunologic status and direct care to prevent complications from altered immune function. PMID- 8633898 TI - Part 2: Effects of renal failure and its treatment on the immune system and assessment of immune system function. AB - Patients with renal failure often succumb to infection. Alterations in the immune system of these patients, whether inherent to the disease process or precipitated by therapeutic regimens, undermine the promotion of wellness. This article provides a comprehensive understanding of common immune abnormalities, and assessment techniques that can assist nephrology nurses in identifying problems in a patient's immunologic status and direct care to prevent infections, to treat or prevent organ rejection, and to monitor for signs and symptoms of malignancies. PMID- 8633899 TI - Tuberculosis: an old disease with a new face. AB - Tuberculosis (TB) is a disease that was considered essentially eradicated until a few years ago. Many health care providers in the United States, entering their professions during the time that TB was considered conquered, received little training about the means to control and prevent the spread of TB. Efforts once targeted towards the control and prevention of TB have been directed to other areas, while cases of TB have increased. Today health care providers see increased numbers of persons with TB, along with increasing numbers of persons with TB disease that is proving resistant to many of the drugs that previously were used to successfully treat TB. As health care providers, nurses must be able to properly assess the risks that patients face from TB exposure, and the risks that nurses face when caring for TB patients. The primary means of slowing the spread of TB involves early diagnosis, proper treatment and proper isolation of persons suspected of having TB. The goal of this article is to discuss the basic pathophysiology of TB infection and disease, means of spread of TB, risk factors associated with TB, signs and symptoms of TB and basic nursing care of patients with TB, emphasizing the care that patients with both renal disease and TB require. PMID- 8633900 TI - The human immunodeficiency virus and nephrology nursing: implications for infected clients. AB - HIV infection is a chronic disease that can occur in conjunction with a variety of renal problems. When it does, nephrology nursing care is complicated by the interactive nature of dealing with two complex disease processes. Nephrology nurses already possess the skills needed to deal with this complexity, but may need additional knowledge specific to HIV. This article focuses on HIV infection and nursing care of infected patients in renal care settings. PMID- 8633901 TI - Psychosocial characteristics of CAPD patients and the occurrence of infectious complications. AB - OBJECTIVE: The purpose of this study was to ascertain if a relationship exists between the psychosocial characteristics of the CAPD patient and the occurrence of infectious complications exhibited by the patient. DESIGN: Descriptive. SAMPLE/SETTING: Sixty-five (65) patients from 3 dialysis units in the south eastern United States. The subjects at the time of the study were (a) using CAPD and performing their exchanges using a spike decontamination system, or (b) were previously treated by CAPD using a spike decontamination system. METHODS: Patients completed the Exercise of Self-Care Agency scale (ESCA) and three subscales of the Adjective Check List (ACL). The relationship of the patient's occurrences of infectious complication and the scores on these instruments was evaluated using multiple regression techniques and Cox Hazard Ratio. RESULTS: Of the characteristics studied, only autonomy was found to be significantly related to the incidence of peritonitis, time to the first episode of peritonitis, and the total incidence of infectious complications. None of the psychosocial characteristics studied were significantly related to the occurrence of exit site infections. CONCLUSIONS: The patient's autonomy and factors other than the psychosocial characteristics presented in this study contribute to the occurrence of infectious complications in CAPD patients. Attitudes about ESRD and CAPD expressed by the patients permitted them to be categorized into three groups, which had vastly different degrees of success with CAPD; these categorizations may serve as the basis for further study. PMID- 8633902 TI - Peritonitis in patients on peritoneal dialysis: a review of pathophysiology and treatment. AB - The incidence of peritonitis has decreased in peritoneal dialysis patients because of new delivery systems and connecting devices; however, peritonitis is still of major concern. Peritonitis remains the most common reason for admission to hospitals and is still a primary reason for failure of this method of dialysis. Nephrology nurses need a clear understanding of peritonitis etiology, recommended treatment regimens, side effects of treatment, and complications. This should enable nephrology nurses to provide a higher level of care, and, thus, improve patient well-being. PMID- 8633903 TI - Hepatitis C virus and its impact on transplantation. AB - Hepatitis C virus is now the leading cause of serum hepatitis worldwide, a fact that has serious implications for the fields of dialysis and transplantation. This article describes hepatitis C transmission, diagnosis, clinical features and treatment, and explores the controversies and unique problems that hepatitis C presents to the field of transplantation. PMID- 8633904 TI - Risk of community infections in transplant patients: a literature review. AB - Due to concern over the morbidity and mortality associated with infections in transplant recipient patients, a review of the literature was completed to evaluate the risk of patients acquiring infection from pets and during travel and to improve teaching by transplant teams. There is a paucity of information in the literature regarding such infections in transplant patients, however, reviewing studies involving acquired immune deficiency syndrome (AIDS) patients yielded important data. PMID- 8633905 TI - Cytomegalovirus prophylaxis in kidney transplant recipients. AB - Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in the transplant population. It is important to understand the nature of CMV and its effect on the immunosuppressed host. In the last decade, several prophylactic drugs have been tested in transplant recipients. Most studies found that CMV prophylactic drugs protect transplant recipients to some degree. This article reviews the pathophysiology of CMV, the immunosuppressed host, and prophylaxis. PMID- 8633906 TI - Using whirlpool treatment of a groin infection in a patient on PD. PMID- 8633907 TI - Tuberculosis: infection control measures. PMID- 8633908 TI - Sodium citrate use for anticoagulation in hemodialysis. PMID- 8633909 TI - Epoetin alfa--focus on dialyzability. Case study of the anemic patient. AB - Epoetin alfa therapy has typically been administered at the end of dialysis. A recently completed in vivo study with a small patient population strengthens previous study findings and suggests that this protein can be safely administered during high-flux dialysis without fear of membrane adsorption. Data indicate that successful use of this technique depends on a variety of factors, including the site of administration. If confirmed, these findings could benefit both patients and clinicians. PMID- 8633910 TI - Cadaveric donors infected with hepatitis C virus: ethical issues. PMID- 8633911 TI - Postoperative stay associated with the prognosis of patients with colorectal carcinoma. PMID- 8633912 TI - Postoperative stay associated with prognosis of patients with colorectal cancer. AB - OBJECTIVE: The author's objective was to determine whether the length of postoperative stay for patients after colorectal cancer surgery is associated with prognosis. SUMMARY BACKGROUND DATA: Financial pressure to reduce hospital costs has caused physicians to reduce hospital stays by changes in patient care, which reduce hospital stay but may compromise long-term results. METHODS: Using multivariate analysis, the author examined the relationship between postoperative stay and prognosis in a consecutive series of 341 prospectively studied patients with colorectal cancer undergoing potentially curative surgery. RESULTS: In multivariate analysis, patients staying beyond the median of 11 days had more complications (p=0.000), more left hemicolectomies and procedures with colostomies (p=0.000), were older (p=0.002), and lost more blood (p=0.012) than patients staying less than the median. Disease-free survival was significantly and independently related to Dukes' stage (p=0.000), postoperative stay (p=0.001), and blood transfusion (p=0.011). The mean postoperative stay for the 98 patients who later developed recurrence was 15 days compared to 12 days for the 243 patients who remained disease free (p=0.0008). Cumulative disease-free survival of the 142 patients who stayed more than the median of 11 days was 60% compared to 77% for the 199 patients with shorter stays (p=0.000). CONCLUSIONS: These data indicate that shorter hospital stays do not compromise disease-free survival of patients with colorectal cancer. PMID- 8633913 TI - Protein-sparing therapy after major abdominal surgery: lack of clinical effects. Protein-Sparing Therapy Study Group. AB - OBJECTIVE: A prospective multicenter randomized trial was designed to evaluate the clinical efficacy of postoperative protein-sparing therapy. SUMMARY BACKGROUND DATA: The metabolic effect of postoperative protein-sparing therapy has been shown by several studies, but the clinical utility of this treatment has not been investigated by large prospective trials. METHODS: Six hundred seventy eight patients undergoing major elective abdominal surgery were randomly assigned to receive either protein-sparing therapy after surgery (protein-sparing therapy group) or conventional therapy (control group). The patients were monitored for postoperative complications and mortality. RESULTS: The rate of major postoperative complications was similar in both groups (protein-sparing therapy group, 19.5%; control group, 20.9%; p=0.66) as were the overall postoperative mortality rates (4.7% and 3.5%, respectively; p=0.43). CONCLUSIONS: The present study indicates that routine protein-sparing therapy for patients normonourished or mildly malnourished undergoing major abdominal surgery is not clinically justified. PMID- 8633914 TI - A clinical trial on the prevention of catheter-related sepsis using a new hub model. AB - BACKGROUND: Catheter hub contamination is being increasingly recognized as a source of catheter-related sepsis. The authors have investigated the efficacy of a new hub design in preventing endoluminal catheter contamination and catheter related sepsis arising at the hub. METHODS: Adult surgical and intensive care patients requiring a subclavian catheter for at least 1 week were randomly assigned to receive catheters with standard connectors (control group, n=73) or equipped with a new hub model (new hub group, n=78). Skin, catheter tip, and hub cultures were performed at the time the catheter was withdrawn because therapy was terminated or because of suspicion of sepsis, in which case peripheral blood cultures were taken. RESULTS: Of the 151 patients included, 15 (10%) developed catheter-related sepsis. Catheters were more often withdrawn because suspicion of infection in the control group (42 vs. 19%, p<0.005). Catheter sepsis rate was higher in the control group (16 vs. 4%, p<0.01) because of the low rate of catheter sepsis arising at the hub observed in the new hub group (1 vs. 11%, p<0.01). The prevalence of culture-positive catheter hubs without associated bacteremia (colonization) was higher in the control group (18 vs. 5%, P<0.03). CONCLUSIONS: A new catheter hub has proved to be useful in preventing endoluminal bacterial colonization and catheter-related sepsis in subclavian lines inserted for a mean of 2 weeks. PMID- 8633915 TI - Echo-Doppler evaluation of reverse flow sign in the intrahepatic portal branches after surgery. AB - OBJECTIVE: The author's evaluated the clinical significance of the development of reversed portal flow after abdominal surgery other than portosystemic shunt procedure. SUMMARY BACKGROUND DATA: There have been several reports in regard to reversed portal flow demonstrated by pulsed Doppler ultrasonography, most of which were related to portal hypertension. To the authors' knowledge, this is the first report in which reversed portal flow also is present in patients who have undergone abdominal surgery other than portosystemic shunt procedure. METHODS: Preoperative and postoperative pulsed Doppler ultrasonographic examinations were performed in 126 patients who underwent abdominal surgery. Postoperatively, the portal flow direction was assessed in the right portal branch or the umbilical portion of the left portal branch. RESULTS: Of the 126 patients, reversed portal flow developed in 10 after surgery; 9 of them died of liver failure. CONCLUSIONS: The postoperative development of reversed portal flow is considered to have grave prognostic significance, indicating that the degree of postoperative liver damage is extremely critical. PMID- 8633916 TI - Attitudes and opinions toward surgical research. A survey of surgical residents and their chairpersons. AB - OBJECTIVE: To learn more about how research in academic surgery is viewed by surgical residents and their chairpersons. SUMMARY BACKGROUND DATA: There is a general perception that a productive experience in a basic science laboratory is an important prerequisite for a successful career in academic surgery. METHODS: An anonymous mail survey of 189 surgical residents entering the laboratory and their chairpersons (n=81) was done. Questions included how a laboratory was chosen by the resident, the importance of a basic science laboratory experience as a prerequisite to an academic career, and the perceived goal or goals of the laboratory experience. Data were analyzed by chi square analysis. RESULTS: The response rate from each group was excellent (80% response for residents, 90% from chairpersons). Of the residents surveyed, 78% were men and 22% were women; 51% entered the laboratory after 2 years of clinical training and 34% after 3 years; 84% did their research at their home institution and 91% worked in a surgeon's laboratory; 51% were scheduled to be in the laboratory for 1 year, 41% for 2 years, and 7% for 3 years. Two thirds of the residents were salaried by the surgery department. Both residents (70%) and chairpersons (86%) felt that the best surgical journal was Annals of Surgery. Both groups ranked Science as the top basic science journal. Twenty-four percent of the residents felt their peers offered the best advice in choosing a laboratory compared to 0% of the chairpersons (p<0.01); chairpersons felt they themselves or the program director were better advisors (chairpersons, 44%, vs. residents, 27%; p<0.01). Chairpersons believed that the principal investigators' previous success with residents was the major factor in determining in which laboratory to work; the residents placed more value on their interest in the project. Eighty-nine percent of women requested to go into the laboratory versus 66% of men (p<0.05). Half of the chairpersons and residents believed the faculty felt pressure on them to get grants; however, 71% of postgraduate year (PGY) residents who were PGY3 sensed this pressure compared to 44% of the PGY2 residents (p<0.01). Being in debt did not adversely influence the decision of 77% of these residents to do research. The residents felt more so than did their chairpersons that basic science research was necessary to be a successful academic surgeon (p<0.01). CONCLUSIONS: Although there are some differences in opinions between surgical residents and surgical chairpersons about the value and purpose of basic science research, these differences should be embraced and serve to enhance openness and discussion. Overall, surgical residents viewed the research experience away from clinical surgery as a positive one. The main reason for going into the laboratory was because of a genuine interest in the scientific method and the academic mission. PMID- 8633917 TI - Factors influencing postoperative morbidity, mortality, and survival after resection for hilar cholangiocarcinoma. AB - OBJECTIVE; Morbidity and mortality involved in the resection of hilar cholangiocarcinoma were reviewed retrospectively. The clinicopathologic and laboratory parameters that might influence the patient's survival also were re evaluated. SUMMARY BACKGROUND DATA: Although much progress has been made in the diagnosis and management of hilar cholangiocarcinoma, long-term outlook for most patients remains poor. Surgical resection is usually prohibited because of its local invasiveness, and most patients can only be managed by palliative drainage. Recently, many surgeons have adopted a more aggressive resection with varying degrees of success. Several prognostic factors in bile duct carcinoma have been proposed; however, no reports have specifically focused on resected hilar cholangiocarcinoma and its prognostic survival factors using multivariate analysis. METHODS: The clinical records and pathologic slides of 49 cases with resected hilar cholangiocarcinoma were reviewed retrospectively. Twenty clinical and laboratory parameters were evaluated for their correlation with postoperative morbidity and mortality, whereas 31 variables were evaluated for their significance with postoperative survival. Variables showing statistical significance in the first univariate analysis were included in the following multivariate analysis using stepwise logistic regression test for factors affecting morbidity and mortality and Cox stepwise proportional hazard model for factors influencing survival. RESULTS: There were 5 in-hospital deaths, and the cumulative 5-year survival rate in 44 patients who survived was 14.9%, with a median survival of 14.0 months. Multivariate analysis disclosed that co-existent hepatolithiasis and lower serum asparate aminotransferase levels (<90 U/L) had a significant low incidence of postoperative morbidity, whereas a serum albumin of less than 3 g/dL was the only significant factor affecting mortality. Regarding survival, univariate analysis identified eight significant factors: 1) total bilirubin > or = 10 mg/dL, 2) curative resection, 3) histologic type, 4) perineural invasion, 5) liver invasion, 6) depth of cancer invasion, 7) positive proximal resected margin, and 8) positive surgical margin. However, multivariate analysis disclosed total bilirubin > or = 10 mg/dL, curative resection, and histologic type as the three most significant independent variables. CONCLUSIONS: Surgical resection provides the best survival for hilar cholangiocarcinoma. An adequate nutritional support to increase serum albumin over 3 g/dL is the most important factor to decrease postoperative mortality. Moreover, preoperative biliary drainage to decrease jaundice and a curative resection with adequate surgical margin are recommended if longer survival is anticipated. Patients with well-differentiated adenocarcinoma seem to survive longer compared to those with moderately or poorly differentiated tumors. PMID- 8633918 TI - Sequential changes in the metabolic response in critically injured patients during the first 25 days after blunt trauma. AB - BACKGROUND: Understanding the changes in energy expenditure and body composition is essential for the optimal management of the critically injured, yet these changes have not been quantified within the current context of trauma care. METHODS: Ten critically injured patients (median Injury Severity Score = 35) had measurements of energy expenditure and body composition as soon as they were hemodynamically stable and then every 5 days for 21 days. RESULTS: Resting energy expenditure rose to 55% above predicted and remained elevated throughout the study period. Total energy expenditure was 1.32 X resting energy expenditure. Body fat was oxidized when energy intake was insufficient (r=-0.830, p<0.02). Body water changes closely paralleled body weight changes and were largely accounted for by changes in extracellular water. Over the 21-day study period, there was a loss of 1.62 kg (16%) of total body protein (p<0.0002), of which 1.09 kg (67%) came from skeletal muscle. Intracellular potassium was low (133 +/- 3 mmol/L, p<0.02) but did not deteriorate further after hemodynamic stability had been reached. CONCLUSIONS: These results show that the period of hypermetabolism lasts longer and the protein loss is greater in critically injured patients than previously thought. Most, but not all, the protein is lost from muscle. Fat loss can be prevented and cell composition preserved once hemodynamic stability is achieved. PMID- 8633920 TI - Recovery of fasted and fed gastrointestinal motility after open versus laparoscopic cholecystectomy in dogs. AB - OBJECTIVE: The authors investigate the recovery of gastrointestinal motility in the fed and fasted state after laparoscopic and open cholecystectomy. SUMMARY BACKGROUND DATA: Clinical recovery after laparoscopic cholecystectomy is known to be more rapid than after conventional open cholecystectomy. However, the actual effect of a laparoscopic approach on gastrointestinal motility, particularly fed state motility, is not well investigated. METHODS: Laparoscopic (LAP, n=6) or open (OPEN, n=6) cholecystectomy was performed in 12 dogs. Bipolar recording electrodes were placed on the antrum, small intestine, and the transverse and descending colon, and fasting myoelectric data were recorded after operation. Solid meal gastric emptying studies were performed before surgery and on postoperative days 1 and 2. Transit time studies were performed using 10 radiopaque markers. RESULTS: Gastric emptying was significantly delayed in the OPEN group at 120 minutes on postoperative day 1 compared with pre-operative emptying (p<0.05), but was not delayed on postoperative day 2. Gastric emptying was not delayed in the LAP group after operation. Transit time was the same between groups. Gastric dysrhythmias were more frequent on postoperative day 3 (p<0.05) in the OPEN group. There were no significant differences in the presence, cycle length, or propagation velocity of the migrating motor complex on any postoperative day. Discrete or continuous electrical response activity in the colon was observed by postoperative day 1 in both groups. CONCLUSIONS: Fed-state motility is the only parameter for which laparoscopic cholecystectomy showed an improvement in postoperative recovery. Recovery of fasted gastrointestinal motility in dogs is equally rapid after either operation. PMID- 8633919 TI - Effect of ambient temperature on metabolic rate after thermal injury. AB - OBJECTIVE: The authors describe the effect of ambient temperature on metabolic rate after thermal injury. SUMMARY BACKGROUND DATA: Thermal injury induces a hypermetabolic state, which is reported to increase with the extent of burn. The magnitude of this response is further influenced by ambient temperature. METHODS: The resting energy expenditure was measured by indirect calorimetry at ambient temperatures of 22, 28, 32, and 35 C. It was indexed to a calculated basal metabolic rate in normal volunteers and adult patients with burns involving at least 20% of the total body surface area who had no evidence of systemic infection. These measurements were performed between postburn days 6 and 21. RESULTS: The effect of ambient temperature on metabolic rate was measured in 44 burn patients and 8 normal volunteers. Burn size ranged from 20 to 97% total body surface area with a mean of 44 +/- 18.5% total body surface area. Metabolic rate did not change significantly in control subjects as ambient temperature was varied (p<0.05). Regression analysis showed that burn size and ambient temperature were significant determinants (p<0.01) of metabolic rate in the patients and that together these factors accounted for 55% of the variation observed (df adj. r(2)=0.55) across the range of ambient temperatures studied. Metabolic rate was independent of burn size at ambient temperatures of 32 and 35 C (p<0.02) and increased by a factor of 1.5 X basal metabolic rate. A further increase in metabolic rate, which was positively correlated with burn size, resulted from nonshivering thermogenesis at ambient temperatures 28 and 22 C. The magnitude of this response was greatest at 22 C. CONCLUSIONS: These findings suggest that the hypermetabolic response to thermal injury is maximal in burns as small as 20% total body surface area and that an additional burn size-dependent increase in metabolic rate results from heat loss at ambient temperatures below thermoneutrality. PMID- 8633921 TI - Skeletal muscle glutathione after surgical trauma. AB - OBJECTIVE: The authors investigate the effect of surgical trauma on skeletal muscle concentrations of glutathione in patients undergoing selective abdominal surgery. SUMMARY BACKGROUND DATA: The posttraumatic state is accompanied by characteristic changes in the pattern of free amino acids and a decline of protein synthesis in human skeletal muscle. Glutathione has multiple metabolic functions that are involved in cellular homeostasis. It is unknown how surgical trauma affects the glutathione metabolism of skeletal muscle in surgical patients. METHODS: Eight patients undergoing elective abdominal surgery were investigated. Percutaneous muscle biopsies and blood samples were taken before operation and at 6, 24, and 48 hours after operation. The concentrations of glutathione were determined in muscle tissue, plasma, and whole blood, as well as the concentrations of the related amino acids in muscle and plasma. RESULTS: In skeletal muscle, the levels of both reduced and total glutathione decreased by 40% (p<0.01) at 24 hours and remained low at 48 hours after operation compared with the preoperative values. The glutathione concentration in plasma was 20% lower after operation compared with the concentration before operation (p<0.05). There were no changes at the whole blood levels of glutathione. Tissue glutamate and glutamine decreased significantly after operation (p<0.001), whereas intracellular cysteine and glycine remained unchanged. CONCLUSIONS: Skeletal muscle glutathione deficiency occurs after surgical trauma. This may lead to an increase in the susceptibility to intracellular oxidative injury. PMID- 8633922 TI - A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction. AB - OBJECTIVE: The present study was designed to evaluate whether pre-existing hepatic dysfunction (cirrhosis) leads to increased morbidity and mortality, in part through an inappropriate in vivo tumor necrosis factor-alpha response. SUMMARY BACKGROUND DATA: Vibrio vulnificus is the most commonly isolated member of the noncholera Vibrio sp., responsible for fulminant and frequently fatal septicemia. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from Vibrio sp. infection. However, the underlying mechanism behind this association has not been fully delineated. METHODS: Cirrhosis was induced in C57BL/6 (15 to 20 g) mice using thrice-weekly injections of carbon tetrachloride (CCl4) for 7 weeks. Either a 7.0 to 9.5 X 10(7) (low dose) or a 0.8 to 1.2 X 10(9) colony-forming unit (high dose) of V. vulnificus was administered through a mini-laparotomy incision via transgastric puncture into both cirrhotic and control animals. RESULTS: Mortality in cirrhotic mice to low- and high-dose Vibrio infection was 88% (7/8) and 100% (8/8), respectively, whereas mortality in control animals was 0% (0/8) and 12% (1/8), respectively (p<0.01). Tumor necrosis factor-alpha mRNA could be detected by reverse transcriptase polymerase chain reaction in livers and lungs from infected animals 2 and 4 hours after Vibrio administration in both control and cirrhotic animals. Lung and liver tumor necrosis factor-alpha bioactivity, however, was significantly lower in cirrhotic animals infected with Vibrio when compared with controls. Serum tumor necrosis factor-alpha was only sporadically detected in both groups of Vibrio-infected animals. When cirrhotic mice challenged with a low dose of Vibrio sp. were pretreated with 1.0 mg/kg body weight of a novel tumor necrosis factor-alpha receptor immunoadhesin, the increased mortality was completely prevented. CONCLUSIONS: Cirrhotic mice show increased mortality to Vibrio infection, and this increased mortality is dependent on an in vivo tumor necrosis factor-alpha response. PMID- 8633923 TI - Induction of Fas-mediated apoptosis on circulating lymphocytes by surgical stress. AB - OBJECTIVE: The authors determined whether the decrease in lymphocytes after surgery is related to apoptosis. SUMMARY BACKGROUND DATA: Surgery induces a profound but transient depletion of circulating lymphocytes, However, the mechanism underlying this phenomenon is unclear. METHODS: Peripheral blood mononuclear cells were obtained from 18 patients before and after elective surgery and studied for morphologic and biochemical markers of apoptosis, DNA fragmentation, and Fas expression. RESULTS: The DNA staining of peripheral blood mononuclear cells obtained after surgery, which had been cultured for 24 hours in vitro, showed chromatin condensation and fragmentation of cells into collapsed spheres. Moreover, DNA isolated from these peripheral blood mononuclear cells formed a ladder of oligonucleosomal fragments. However, peripheral blood mononuclear cells obtained before surgery showed neither of these changes. The observation that none of these apoptotic cells ingested latex suggested that they were of lymphocytic origin. Fas-positive lymphocytes increased significantly 2 hours after the start of surgery and returned to preoperative levels by postoperative day 7. Anti-Fas antibody augmented apoptosis, whereas ZB4, a Fas antagonist, inhibited apoptosis in lymphocytes after surgery. CONCLUSIONS: These results indicate that circulating lymphocytes in the early perioperative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery. PMID- 8633924 TI - The experience of the University of Alabama with recurrent melanoma. PMID- 8633925 TI - The effectiveness of Whipple resection in patients with pancreatic cancer at Veterans Affairs (VA) hospitals. PMID- 8633926 TI - "Practice makes perfect" is intuitive; the applicability of this axiom to surgical outcomes has been tenuous at best. PMID- 8633927 TI - The effect of nutrition on intestinal epithelial barrier function. PMID- 8633928 TI - Manually constructed and stapled anastomoses. PMID- 8633929 TI - Gallbladder carcinoma. PMID- 8633930 TI - Neurobehavioral tests are monitoring tools used to improve cardiac surgery outcome. PMID- 8633931 TI - The aortic valve: to dilate, repair, or replace--that is the question. PMID- 8633932 TI - Lateral tunnel suture line variation reduces atrial flutter after the modified Fontan operation. AB - BACKGROUND: Atrial flutter (AFL) is a common postoperative sequela of the modified Fontan operation, or total cavopulmonary connection. We hypothesized that injury to the crista terminalis (CT) by the lateral tunnel suture line contributes to the development of AFL in this setting. This study was designed to determine the effects of alteration of the lateral tunnel suture line, relative to the CT, on the inducibility of AFL in an acute canine model of the modified Fontan operation. METHODS: Adult mongrel dogs (n = 25) underwent a median sternotomy and normothermic cardiopulmonary bypass. In groups 1, 2, and 3, through a right atriotomy, a suture line was placed to simulate the lateral tunnel of the modified Fontan operation (n = 20). The lateral aspect of the suture line ran along the CT in group (n = 10), 5 mm medial to the CT in group 2 (n = 5), and 10 mm anterior to the CT, incorporated into the atriotomy closure, in group 3 (n = 5). In group 4 (n = 5), only the lateral portion of the suture line, along the CT, was placed. Form-fitting 253-point unipolar endocardial mapping electrodes were inserted in the left and right atria via bilateral ventriculotomies. Induction of AFL was then attempted using atrial burst pacing. If sustained AFL could not be induced, isoproterenol was administered and the pacing protocol repeated. Endocardial activation sequence maps of spontaneous rhythm and AFT were constructed. RESULTS: Under baseline conditions, after placement of the suture line, sustained AFL could reproducibly be induced in 8/10 dogs in group 1, 0/5 dogs in group 2, 0/5 dogs in group 3, and 5/5 dogs in group 4 (p < 0.001). After isoproterenol administration, sustained AFL was reproducibly inducible in the remaining 2 dogs in group 1, 4/5 dogs in group 2, and 0/5 dogs in group 3 (p = 0.01). The mean cycle length of AFL was 189 +/- 25 ms in group 1, 136 +/- 8 ms in group 2, and 182 +/- 20 ms in group 4 (p < 0.001). Atrial activation sequence maps, during sinus rhythm, demonstrated a line of conduction block along the lateral portion of the suture line in all cases in groups 1 and 4 and in only those cases in group 2 in which sustained AFL was inducible. During AFL this block facilitated unidirectional conduction, permitting propagation of the reentrant wavefront. Mean conduction velocity along the CT during sinus rhythm was 0.63 +/- 0.10 m/s in group 1, 1.04 +/- 0.17 m/s in group 2, 1.01 +/- 0.12 m/s in group 3, and 0.44 +/- 0.13 m/s in group 4 (p < 0.01). CONCLUSIONS: In an acute canine model of the modified Fontan operation, conduction block imposed by the lateral tunnel suture line is an essential component of the AFL circuit. The inducibility of AFL is increased by suture line placement along the CT. Slow conduction, resulting from injury to the CT, promotes this increased inducibility. Avoidance of the CT may reduce the incidence of AFL in children undergoing the modified Fontan operation. PMID- 8633933 TI - Clinical trial of retrograde warm blood reperfusion versus standard cold topical irrigation of transplanted hearts. AB - BACKGROUND: A prospective, randomized clinical study involving 34 patients undergoing heart transplantation compared myocardial preservation of donor hearts maintained with continuous reperfusion with retrograde warm blood cardioplegia during surgical implantation versus the standard cold topical irrigation. METHODS: Hearts in both groups were arrested with a standard crystalloid solution and maintained in a cold saline solution during transportation. In the retrograde group, cardioplegia was administered through a catheter in the coronary sinus during surgical implantation. An average of 471 +/- 30 mL of hyperkalemic crystalloid solution diluted 1:4 in warm blood from the oxygenator was infused. In the standard group, the heart was kept cold by topical irrigation of cold saline solution and was reperfused only when the ascending aorta was unclamped. RESULTS: Preoperative characteristics of donors and recipients were similar in the two cohorts. Ischemic time average 139 +/- 12 minutes in the retrograde group compared with 130 +/- 11 minutes in the standard group (p = 0.57). Cardiopulmonary bypass time averaged 89 +/- 4 minutes in the retrograde group and 110 +/- 12 minutes in the standard group (p = 0.12). Defibrillation at reperfusion was performed in 4 patients (4/17, 24%) in the retrograde group and 12 patients (12/18, 67%) in the standard group (p = 0.01). There were no deaths in the retrograde group (0/17), whereas in the standard group, 3 patients (3/17) died of early graft failure (p = 0.11). Four early graft failures occurred in the standard group (p = 0.06). Two patients (2/17, 12%) were weaned from bypass with ventricular assist devices in the standard group. The number of subendocardial necrotic cells in the first two weekly endomyocardial biopsy specimens averaged 2.7 +/- 0.8 cells/mm2 in the retrograde group and 5.9 +/- 2.4 cells/mm2 in the standard group (p = 0.12). CONCLUSIONS: Retrograde warm blood reperfusion appears to improve the initial recovery of transplanted hearts. The technique is easy to use and may be a useful approach to graft protection during surgical implantation. PMID- 8633934 TI - Neuronal damage after hypothermic circulatory arrest and retrograde cerebral perfusion in the pig. AB - BACKGROUND: Antegrade and retrograde cerebral perfusion during hypothermic circulatory arrest (HCA) has been reported to provide better brain protection during operation than hypothermic circulatory arrest alone. However, the efficacy of these techniques remains to be fully determined, especially when used for prolonged periods. We used a pig model to evaluate the histopathologic consequences of HCA and the potential benefit of cerebral perfusion during HCA. METHODS: Twenty-two pigs were divided into four groups and exposed to either anesthesia alone, 120 minutes of HCA (15 degrees C), 120 minutes of retrograde cerebral perfusion at 15 degrees C during HCA, or 120 minutes of antegrade cerebral perfusion at 15 degrees C during HCA, and then reperfused for 60 minutes under cardiopulmonary bypass at 37 degrees C. The brains were perfusion fixed at the end of the experiments and examined by light microscopy. RESULTS: There were no morphologic changes in any areas of the brains in the anesthesia group, and very minor changes in some areas of the brains in the antegrade cerebral perfusion. group. Varying severity of neuronal damage was found in the brains of all the pigs in the HCA and retrograde cerebral perfusion groups. The severity of ischemic damage in the brain showed the following descending order: hippocampus (CA4), caudate nucleus, cerebral cortex, putamen, thalamus, Purkinje cells of the cerebellum, pons, and mesencephalic gray matter. In the hippocampus the order of damage was CA4, CA3, polymorphous layer of the dentate gyrus, prosubiculum, CA2, CA1, and granule cell layer of the dentate gyrus. The damage in the retrograde cerebral perfusion group was less severe relative to the HCA group in many areas (no significance except mesencephalic gray matter). CONCLUSIONS: These results demonstrate that the pattern of neuronal damage in pigs subjected to HCA and retrograde cerebral perfusion differs from the traditional pattern in that the caudate nucleus and hippocampal CA4 region are the most vulnerable to ischemia hypoxia. Our results also suggest that antegrade cerebral perfusion prevented ischemic damage to the brain and retrograde cerebral perfusion provided some protection but moderately severe damage occurred. PMID- 8633935 TI - Triiodothyronine therapy lowers the incidence of atrial fibrillation after cardiac operations. AB - BACKGROUND: Cardiopulmonary bypass results in a euthyroid sick state, and recent evidence suggests that perioperative triiodothyronine (T3) supplementation may have hemodynamic benefits. In light of the known effects of thyroid hormone on atrial electrophysiology, we investigated the effects of perioperative T3 supplementation on the incidence of postoperative arrhythmias. METHODS: One hundred forty-two patients with depressed left ventricular function (ejection fraction < 0.40) undergoing coronary artery bypass grafting were randomized to either T3 or placebo treatment groups in a prospective, double-blind fashion. Triiodothyronine was administered as a 0.8 micrograms/kg intravenous bolus at the time of aortic cross-clamp removal followed by an infusion of 0.113 micrograms.kg 1.h-1 for 6 hours. Patients were monitored for the development of arrhythmias during the first 5 postoperative days. RESULTS: The incidence of sinus tachycardia and ventricular arrhythmias were similar between groups. Triiodothyronine-treated patients had a lower incidence of atrial fibrillation (24% versus 46%; p = 0.009), and fewer required cardioversion (0 versus 6; p = 0.012) or anticoagulation (2 versus 10; p = 0.013) during hospitalization. Six patients in the T3 group versus 16 in the placebo group required antiarrhythmic therapy at discharge (p = 0.019). CONCLUSIONS: Perioperative T3 administration decreased the incidence and need for treatment of postoperative atrial fibrillation. PMID- 8633936 TI - Taussig-Bing anomaly: arterial switch versus Kawashima intraventricular repair. AB - BACKGROUND: Current corrective surgical approaches for the Taussig-Bing heart include arterial switch with ventricular septal defect (VSD) closure and intraventricular repair as described by Kawashima. METHODS: Between 1983 and 1994, 20 children underwent intracardiac repair of Taussig-Bing anomaly. Mean age at operation was 17 months (range, 1 week to 9 years). Prior palliation included pulmonary artery band (15) with coarctation repair (8) and atrial septectomy (1). Arterial switch with VSD closure was performed in 16 patients, 10 with anteroposterior great arteries. Kawashima repair was performed in 4 patients, all with side-by-side great arteries. RESULTS: After arterial switch, there was one operative death (6.2%) due to myocardial ischemia and three late deaths (18.7%) due to pulmonary hypertension, gastrointestinal bleeding, and acute lymphocytic leukemia. In the Kawashima repair group there have been no deaths. After arterial switch, 9 patients underwent 11 reoperations for residual coarctation (3), residual pulmonary artery stenosis (2), aortic valve replacement, aortic valvuloplasty, unrecognized VSD, mitral valvuloplasty, mediastinitis, and pacemaker insertion. After Kawashima repair, 1 patient underwent reoperation for baffle stenosis and 1 for an unrecognized VSD. CONCLUSIONS: For children with Taussig-Bing anomaly, the Kawashima intraventricular repair (for side-by-side great arteries) preserves the native aortic valve and avoids coronary dissection. The arterial switch operation with VSD closure can be applied without ventriculotomy to all great artery relationships and allows neonatal repair with or without concomitant coarctation repair. Both techniques yield excellent early and intermediate-term results despite the high rates of prerepair palliation and postrepair reoperation for both groups. PMID- 8633937 TI - Selective management of acute type B aortic dissection: long-term follow-up. AB - BACKGROUND: Since 1985, we have selectively treated acute type B aortic dissections. Initial treatment lowered blood pressure and heart rate. Transesophageal echocardiography and computed tomographic scans were used to diagnose and follow up the patients. Patients were operated on for organ ischemia, pain, hypertension, or increasing subpleural fluid on computed tomographic scan. METHODS: We retrospectively reviewed consecutive patients admitted over a 10-year period to the Mt. Sinai Hospital. RESULTS: From August 1985 to May 1995, 68 patients were seen. Three died soon after admission during initial diagnostic evaluation. Seventeen patients underwent operation without mortality or paraplegia (group 1). Forty-seven of 48 patients treated nonoperatively were discharged; 1 patient died of rupture on day 7 (group 2). Actuarial survival for all 68 patients at 1 and 5 years was 92% +/- 4% and 82% +/ 8%. Group 1 survival was 93% +/- 4% and 68% +/- 5%, and group 2 survival was 90% +/- 6% and 87% +/-14%. There were no differences between groups. Late intervention was required in 2 group 1 patients (12%) and in 12 of 48 group 2 patients (25%), again without mortality or paraplegia. CONCLUSIONS: This experience suggests that selective management of acute type B aortic dissection results in acceptable short-term and long-term survival. Avoiding early operation did not compromise late results. PMID- 8633938 TI - Defining neuropsychological dysfunction after coronary artery bypass grafting. AB - BACKGROUND: Despite the large body of literature documenting the presence of cognitive decline after coronary artery bypass grafting, there is little consensus as to the frequency and extent of cognitive impairment. One potential reason for this lack of agreement is the absence of uniform criteria for assessing cognitive decline. METHODS: Two hundred thirty-two patients underwent cognitive testing the day before operation and were examined before discharge, and at 6 weeks and 6 months after grafting. For comparative purposes, five different sets of criteria were used to define cognitive decline. RESULTS: There was little agreement between the criteria as to which patients declined at each test period. The incidence of decline ranged from 66% to 15.3% before discharge, 34% to 1.1% at 6 weeks, and 19.4% to 3.4% at 6 months. CONCLUSIONS: A large variation in reported incidence of cognitive decline after coronary artery bypass grafting can be attributed to the different criteria used to define cognitive impairment. PMID- 8633939 TI - Comparative experimental study of cerebral protection during aortic arch reconstruction. AB - BACKGROUND: The optimal adjunctive method for cerebral protection during aortic arch repair remains controversial. METHODS: Retrograde cerebral perfusion, selective cerebral perfusion, and hypothermic circulatory arrest were compared in terms of their effect on cerebral function of mongrel dogs using somatosensory evoked potentials. Brain temperatures were held at 20 degrees C for 90 minutes during cerebral perfusion or circulatory arrest and then rewarmed gradually to normal temperature. RESULTS: Somatosensory evoked potentials completely disappeared as soon as retrograde cerebral perfusion or hypothermic circulatory arrest started and did not recover completely. In the selective cerebral perfusion group, it recovered in all cases. Only 2% of cerebral blood flow and about 3% of the cerebral metabolic rate for oxygen were obtained during retrograde cerebral perfusion compared with the preoperative value. The analysis of adenosine triphosphate and water content of the brain supported these results. CONCLUSIONS: Retrograde cerebral perfusion had some advantage for cerebral protection compared with hypothermic circulatory arrest, but could not supply sufficient cerebral blood flow to maintain brain function. Selective cerebral perfusion was the safest method for arch reconstruction that requires cerebral protection for 90 minutes. PMID- 8633940 TI - Aortic valve repair and replacement after balloon aortic valvuloplasty in children. AB - BACKGROUND: Little is known about the incidence, indications, and results of surgical repair or replacement of the aortic valve after balloon aortic valvuloplasty (BAV) for congenital aortic stenosis in children. This study was designed to evaluate patterns of failure requiring operation after BAV for congenital aortic stenosis and to review our experience with successful repair, rather than replacement, of selected aortic valves after BAV. METHODS: From March 1986 to June 1995, 60 patients with congenital aortic stenosis aged 1 day to 27 years (mean +/- standard deviation, 7.3 +/- 6 years) underwent BAV. Twenty-three patients (38%) required operation a mean of 44 +/- 37 months (range, 1 to 110 months) after BAV, because of severe aortic insufficiency in 13 patients and recurrent or residual aortic stenosis in 10 patients. Severe aortic insufficiency was invariably due to avulsion of a cusp from the annulus, with resulting cusp prolapse and insufficiency. Operative intervention consisted of valve replacement in 14 patients and valve repair in 9 patients. Repair techniques included reattachment of an avulsed cusp to the aortic annulus, relief of commissural fusion, and debridement of thickened cusps. RESULTS: Actuarial freedom from surgical intervention after BAV was 88% +/- 4% at 1 year, 70% +/- 6% at 5 years, and 51% +/- 12% at 9 years. The need for aortic valve operation was unrelated to age at the time of BAV, indication for operation (aortic insufficiency versus aortic stenosis), age of operation, or preoperative gradient. All patients survived aortic valve operation; there was one late death at an average follow-up of 27 +/- 20 months (range, 2 to 61 months) after aortic valve operation. Stenosis was well relieved in all patients undergoing valve replacement. The 9 valve repair patients have been followed for 22 +/- 14 months (range, 1 to 47 months). Echocardiographic follow-up of the valve repair patients revealed a mean residual aortic stenosis peak instantaneous gradient of 32 mm Hg and mild aortic insufficiency or less in all patients. CONCLUSIONS: Aortic valve operation is required in 5% to 7% of patients yearly after BAV. The need for operation appears to be unrelated to age at the time of BAV; aortic insufficiency predominates over aortic stenosis as an indication for operative intervention. Valve repair can be applied in some patients after BAV with good intermediate-term results and may delay the need for aortic valve replacement. PMID- 8633941 TI - Reactivity to alpha agonists is heightened in immature porcine pulmonary arteries. AB - BACKGROUND: Pulmonary hypertension after cardiopulmonary bypass is a common problem in pediatric cardiac operations. This study tested the hypothesis that there is a difference between adult and immature pulmonary artery constrictor and dilator responses. METHODS: Reactivity of pulmonary artery ring segments from 22 mature (15 to 19 weeks) and 15 immature pigs (4 to 5 weeks) was tested in a vessel myograph. Potassium as a receptor-independent vasoconstrictor and phenylephrine as an alpha-receptor-mediated vasoconstrictor were used to assess smooth-muscle vasoconstriction. To assess endothelial cell function (nitric oxide production and secretion), we used increasing concentrations of bradykinin or acetylcholine. Sodium nitroprusside was used to produce maximum smooth-muscle relaxation at the end of each experiment. RESULTS: The data demonstrated maturation-independent endothelium and smooth-muscle-mediated vasodilation. Pulmonary artery ring segments from immature pigs had significantly less KCl constriction compared with mature pigs (p < 0.05). In contrast, pulmonary ring segments from immature pigs demonstrated enhanced alpha-receptor-mediated constriction compared with mature pigs. CONCLUSIONS: These data may explain perioperative pulmonary vasoconstriction in pediatric patients. PMID- 8633942 TI - Heparin-coated circuits and aprotinin prime for coronary artery bypass grafting. AB - BACKGROUND: The biocompatibility of an extracorporeal circuit is improved by heparin bonding onto its inner surface. To determine the effect of heparin-coated circuits for cardiopulmonary bypass with aprotinin prime on postoperative recovery and resource utilization, a prospective study was done in 102 patients undergoing coronary artery bypass grafting with full systemic heparinization. METHODS: Patients were randomly allocated to be treated with either a heparin coated circuit (n = 51) or an uncoated circuit (n = 51). Differences in blood loss, need for blood transfusion, morbidity, and intensive care stay were analyzed. RESULTS: No differences in blood loss and need for blood transfusion were found between the groups. The relative risk for adverse events in the heparin-coated group was 0.29 (95% confidence interval ranging from 0.10 to 0.80). Adverse events included myocardial infarction (2 patients in the uncoated group versus 0 in the heparin-coated group), rethoracotomy for excessive bleeding (1 versus 2), rhythm disturbance (7 versus 2), respiratory insufficiency (4 versus 0), and neurologic dysfunction (2 versus 0). The lower incidence of adverse events in the heparin-coated group was associated with a shorter intensive care stay (median, 2 days; range, 2 to 5 days) compared with the uncoated group (median, 3 days; range, 2 to 19 days, p = 0.03). The cost savings of 1 day of intensive care stay counterbalanced the additional costs of heparin coated circuits. CONCLUSIONS: The use of heparin-coated circuits for cardiopulmonary bypass with aprotinin prime resulted in a significant reduction in mobidity in the early postoperative phase and a concomitant decrease in intensive care stay, resulting in important cost savings. PMID- 8633943 TI - Influence of milrinone and norepinephrine on blood flow in canine internal mammary artery grafts. AB - BACKGROUND: Vasoactive agents are frequently needed in patients undergoing myocardial revascularization. The purpose of this study was to examine blood flow in the internal mammary artery (IMA) during infusion of drugs that are commonly used after myocardial revascularization. METHODS: A canine right heart bypass preparation allowed precise control of cardiac output and blood pressure, which were maintained constant during drug infusion to isolate the effect of the drug on the IMA conduit. The IMA was anastomosed to a ligated left anterior descending coronary artery. Electromagnetic flow probes measured IMA graft flow. RESULTS: Norepinephrine (0.1 microgram.kg-1.min-1) alone and when combined with phentolamine (8:5 ratio) did not alter IMA flow. Milrinone increased IMA flow 33% +/- 9%, from 37 +/- 7 to 49 +/- 10 mL/min. All hemodynamic variables were unchanged. CONCLUSIONS: The results suggest that: (1) norepinephrine did not have a deleterious effect on IMA flow and (2) milrinone may be a useful drug in patients undergoing myocardial revascularization by increasing IMA blood flow. PMID- 8633944 TI - Postoperative aprotinin: effect on blood loss and transfusion requirements in cardiac operations. AB - BACKGROUND: Aprotinin has been used increasingly to reduce postoperative blood loss in open heart operations. Although it was reported as safe in earlier studies, the overall safety of prophylactic use has been questioned recently. Because of the potential for complications and the high cost, it will be reasonable to use aprotinin more selectively in the postoperative period. METHODS: We prospectively studied the effect of postoperative low-dose aprotinin (2 million kallikrein inactivator units [280 mg]) on blood loss and transfusion requirements in patients undergoing cardiopulmonary bypass. Seventy-five patients were randomly assigned to three groups: prophylactic high-dose aprotinin (group 1), postoperative aprotinin (group 2), or a nonmedicated control group (group 3). RESULTS: The three groups were comparable in all demographic and operative variables. Postoperative chest tube drainage was significantly decreased in both aprotinin groups compared with that in the control group (295 mL in group 1 and 325 mL in group 2 versus 411 mL in group 3; p < 0.05). No significant difference was seen between the two aprotinin groups. The use of homologous blood products was significantly less in group 1 and group 2 than in group 3 (1.15 +/- 1.13 U and 1.35 +/- 1.30 U versus 2.55 +/- 1.09 U; p < 0.05). CONCLUSIONS: Our results suggest that postoperative aprotinin reduces blood loss and transfusion requirements comparably with prophylactic high-dose aprotinin. Thus, one can restrict its use to patients with excessive postoperative bleeding. PMID- 8633945 TI - Pooled air in open heart operations examined by transesophageal echocardiography. AB - BACKGROUND: The clinical significance of pooled air detected by transesophageal echocardiography during open heart operations is not clear. METHODS: Thirty-eight consecutive patients undergoing an open heart operation or an operation on the ascending aorta were divided into two groups on the basis of the absence (group 1, n = 14) or presence (group 2, n = 24) of pooled air. They were examined for intramyocardial echo contrast, ST segment elevation, conduction disturbances, and regional wall motion abnormalities. RESULTS: Echo contrast was found in no patient in group 1 and 66.7% of group 2 patients (p < 0.001). New regional wall motion abnormalities were detected in no patient in group 1 versus 33.3% of group 2 patients (p < 0.05), and ST segment elevation was seen in 33.3% of group 2 patients versus no group 1 patients (p < 0.05). Intramyocardial cardial echo contrast was newly detected after the appearance of pooled air more frequently in patients with ST segment elevation (p < 0.001). Atrioventricular block and sinus arrest appeared in 3 patients and 2 patients, respectively. Postoperative regional wall motion abnormalities were found in 25.0% of patients and were not closely related to intraoperative echo contrast findings. CONCLUSIONS: Pooled air, which is often detected in open heart operation by means of transesophageal echocardiography, is related to several cardiac events, including ST segment elevation, conduction disturbances, and regional wall motion abnormalities, although most of these are transient. PMID- 8633946 TI - Assessment of right ventricular function in swine using sonomicrometry and conductance. AB - BACKGROUND: Assessment of right ventricular (RV) pressure-volume relations has been hampered by difficulty measuring instantaneous, absolute RV volume. Accordingly, several methods were tested for their ability to reflect relative RV volume and to determine changes in RV contractile state. METHODS: Swine (46 to 54 kg; n = 7) were anesthetized and instrumented to measure instantaneous RV pressure, septal-to-RV free wall diameter (SFWD), RV free wall segment length (FWSL), RV volume via conductance (CV), and pulmonary artery flow, the integral of which was used as the standard for stroke volume. Flow-derived stroke volume was correlated with the systolic change in CV, FWSL, and SFWD in the steady state after incremental volume loading and on a beat-to-beat basis during transient inferior vena caval occlusion. Contractility was altered by calcium and pentobarbital and assessed by preload recruitable stroke work (PRSW). RESULTS: Mean (+/- standard error of the mean) correlations (r) versus stroke volume during steady state conditions were 0.85 +/- 0.04 for FWSL, 0.83 +/- 0.04 for CV, and -0.04 +/- 0.24 for SFWD. Mean r values versus stroke volume during caval occlusions were 0.83 +/- 0.03 for FWSL, 0.85 +/- 0.04 for CV, and -0.03 +/- 0.31 for SFWD. Calcium increased mean PRSW slope compared with control using CV (20.3 +/- 2.6 versus 16.1 +/- 1.9 mm Hg; p < 0.05), and pentobarbital decreased mean PRSW slope compared with control using both CV and FWSL (11.3 +/- 1.0 versus 16.1 +/- 1.9 mm Hg, p < 0.05; and 11.9 +/- 2.1 versus 26.1 +/- 4.0 mm Hg, p < 0.05, respectively). There were no changes in PRSW slope with either calcium or pentobarbital using SFWD. The PRSW function was linear with both FWSL and CV but not with SFWD. CONCLUSIONS: In the normal heart, both FWSL and CV, but not SFWD, accurately reflect relative instantaneous RV volume and are thus useful for determining RV contractility by pressure-volume (pressure-dimension) indices. PMID- 8633947 TI - Surgical treatment of systemic atheroembolism from the thoracic aorta. AB - BACKGROUND: Surgical procedures performed exclusively for atheroembolic events arising from the thoracic aorta rarely have been reported. Presented here are 2 patients who underwent successful operation for these problems. METHODS: The clinical presentation, diagnostic evaluation and surgical approach to 2 patients with different embolic sources in the thoracic aorta are presented. One patient had experienced three strokes and was noted by multiplane transesophageal echocardiography to have protruding atheromas with ulcerations in the transverse arch and origin of the brachiocephalic vessels. The transverse arch was replaced using hypothermic circulatory arrest with individual reimplantation of the brachiocephalic vessels. The second patient presented with "blue toe" syndrome from mobile atheromas in the mid-descending thoracic aorta defined by transesophageal echocardiography. A localized debridement was performed using simple aortic cross-clamping. RESULTS: Both patients had uneventful postoperative courses and had no further atheroembolic events. CONCLUSIONS: When standard diagnostic modalities do not delineate an embolic source for either stroke or peripheral embolization, transesophageal echocardiography is recommended as an excellent means of identifying atheromas in the thoracic aorta that could be the source for emboli. Once these lesions are identified, a surgical procedure should be performed to prevent further embolization. PMID- 8633948 TI - Hyperkalemia alters endothelium-dependent relaxation through non-nitric oxide and noncyclooxygenase pathway: a mechanism for coronary dysfunction due to cardioplegia. AB - BACKGROUND: Reported results of hyperkalemia (cardioplegia or organ preservation solutions) on endothelial function are contradictory. The endothelium-dependent relaxation is related to three major mechanisms: cyclooxygenase, nitric oxide, and endothelium-derived hyperpolarizing factor (K+ channel related). The present study was designed to test the hypothesis that hyperkalemia may alter endothelial function through non-nitric oxide and noncyclooxygenase pathways. METHODS: Porcine coronary artery rings (5 to 10 in each group) were studied in organ chambers under physiologic pressure. After incubation with 20 or 50 mmol/L K+ for 1 hour, the response to substance P, an endothelium-dependent vasorelaxant peptide, in K+ (25 mmol/L)-induced contraction was studied in the presence of the cyclooxygenase inhibitor indomethacin (7 mumol/L), the nitric oxide biosynthesis inhibitor NG-nitro-L-arginine (L-NNA) (300 mumol/L), or the adenosine triphosphate-sensitive K(+)-channel blocker glybenclamide (3 mumol/L) in comparison with control arteries (69.8 +/- 4.6% of K+ contraction). RESULTS: Without exposure to hyperkalemia, indomethacin (with or without glybenclamide) did not alter but L-NNA significantly reduced the relaxation (39.7% +/- 3.7%, p < 0.001). After exposure to K+, the indomethacin- and L-NNA-resistant relaxation was further reduced (7.4% +/- 3.2% for 20 mmol/L K+, p < 0.0001; or 13.5% +/- 8.4% for 50 mmol/L K+, p < 0.05, compared with rings without exposure), whereas the indomethacin- and glybenclamide-resistant relaxation was not altered. Incubation with hyperkalemia (50 mmol/L) also significantly reduced the sensitivity (increased EC50) of the indomethacin- and L-NNA-resistant relaxation (-9.75 +/- 0.06 versus -9.33 +/- 0.04 log M, p < 0.01). CONCLUSIONS: Exposure to hyperkalemia reduces the indomethacin- and L-NNA-resistant, endothelium-dependent (endothelium-derived hyperpolarizing factor-related) relaxation. Our study may suggest a new mechanism of coronary dysfunction after exposure to hyperkalemia and open a new area for protection of coronary endothelium in cardiac surgery and for organ preservation in transplantation surgery. PMID- 8633949 TI - Improved cardiac function after prolonged hypothermic ischemia with the Na+/H+ exchange inhibitor HOE 694. AB - BACKGROUND: Na+/H+ exchange represents an important mechanism for pH regulation in the cardiac cell that, however, may paradoxically mediate tissue damage in the reperfused myocardium. We investigated whether inhibition of the exchanger can protect the heart against damage after prolonged hypothermic storage with the use of the selective inhibitor 3-methylsulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694). METHODS: After equilibration, isolated rabbit hearts were arrested with a 3 minute infusion of modified St. Thomas' cardioplegic solution and subsequently maintained in ischemic arrest at 4 degrees C for 12 hours before reperfusion at 37 degrees C for 60 minutes. Left ventricular function and creatine kinase release were measured at intervals throughout reperfusion. High-energy phosphate and adenine nucleotide content were determined in hearts before cardioplegia, at the end of the 12-hour storage period, and at the end of reperfusion. HOE 694 (1 mumol/L) was administered either with cardioplegia and throughout reperfusion (study 1) or selectively with either cardioplegia or reperfusion only (study 2). RESULTS: In study 1, systolic function in untreated hearts recovered to less than 40% of preischemic values and was associated with a greater than 1,000% percent sustained elevation in left ventricular end-diastolic pressure. In contrast, systolic recovery in HOE 694 treated hearts was significantly accelerated and improved to approximately 80%, whereas left ventricular end-diastolic pressure increased to only 300% of baseline. Significant protection also occurred in those hearts in which HOE 694 was administered only at reperfusion while the drug was less effective if given only during cardioplegia. Creatine kinase release was not significantly affected except in study 2, where it was significantly lower after 60 minutes of reperfusion in hearts where HOE 694 was added at the time of reperfusion. Tissue metabolite content was not affected by drug treatment. CONCLUSIONS: This study shows a marked protective effect of the Na+/H+ exchange inhibitor HOE 694 in rabbit hearts subjected to 12 hours of hypothermic ischemia and strongly suggests that antiport inhibitors could play an effective role in myocardial preservation. PMID- 8633951 TI - Effect of temperature in long-term preservation of vascular endothelial and smooth muscle function. AB - BACKGROUND: In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and a suitable environment for the tissue cells. Without good preservation of the vasculature, progressive deterioration of the blood flow during reperfusion may ultimately lead to the no reflow phenomenon, even though the function of the other cells in the organ may be adequately preserved. The aim of this study was to find the optimal storage temperature for preservation of the vasculature. METHODS: The infrarenal aorta of 126 Sprague-Dawley rats were studied in organ baths: as fresh controls, after 36 hours of storage at 0.5 degrees C, 4 degrees C, 8.5 degrees C, and 22 degrees C in University of Wisconsin solution, and after 36-hour storage followed by transplantation and a lapse of 2 hours, 24 hours, and 7 days. The thromboxane analogue U-46619 was used to test contractility. Acetylcholine was used to elicit endothelium-dependent relaxation (EDR), and papaverine to elicit endothelium independent relaxation. RESULTS: Storing the vessels at 0.5 degree C proved best regarding preservation of contractility, with a nonsignificant decrease, whereas storage at 4 degrees C and 8.5 degrees C resulted in a significant decrease after 36 hours. The contractility did not recover within 24 hours of in vivo reperfusion, but full recovery was seen after 7 days. Regardless of the preservation temperature used, a significant impairment in EDR was seen after 36 hours of storage. Two hours after transplantation, vessels stored at 4 degrees C and 8.5 degrees C showed no significant impairment in EDR, whereas those stored at 0.5 degrees C demonstrated a significant loss of EDR. After 24 hours and after 7 days, EDR was normal in all groups. CONCLUSIONS: Endothelium-dependent relaxing factor function is best preserved at 4 degrees C and 8.5 degrees C, whereas preservation of vascular smooth muscle function is best preserved at 0.5 degrees C. PMID- 8633950 TI - Kinetics of induction and protective effect of heat-shock proteins after cardioplegic arrest. AB - BACKGROUND: Heat-shock proteins are known to enhance cardiac resistance to ischemia. METHODS: To evaluate the kinetics of heat-shock protein 70 in relation to its effect on postischemic recovery of cardiac mechanical (cardiac output) and endothelial function (as percentage increase of coronary flow in response to 5 hydroxytryptamine), isolated rat hearts were subjected to prolonged hypothermic cardioplegic arrest at different intervals ranging from 12 to 96 hours after heat stress (n = 6 in each interval). RESULTS: Immunoblotting showed the maximal level of heat-shock protein 70, 0.65 +/- 0.10 (arbitrary units +/- standard error of the mean), at 24 hours after heat shock and similar values at 26 and 30 hours (p = not significant). Postischemic recovery of cardiac output and endothelial function (percentage of preischemic value +/- standard error of the mean) observed at 24 hours was 74.0 +/- 2.4 and 58.3 +/- 7.2, respectively. Similar values were observed at 26 and 30 hours (p = not significant). CONCLUSIONS: In a protocol mimicking conditions for cardiac transplantation, postischemic recovery of cardiac output and endothelial function was improved when the interval between heat stress and ischemia ranged from 24 to 30 hours. This correlated with an apparently critical amount of heat-shock protein 70. PMID- 8633952 TI - Early in vivo experience with the Hemodynamic Plus St. Jude Medical heart valves in patients with narrowed aortic annulus. AB - BACKGROUND: Small aortic orifice primarily resulted in heart prosthesis mismatch in a significant number of patients. The Hemodynamic Plus (HP) series of St. Jude Medical heart valves represents an interesting innovation, allowing a larger valve orifice area with an equivalent tissue annulus diameter. METHODS: Hemodynamic characteristics of the 21-mm HP St. Jude Medical valve were prospectively compared with those of the standard 21-mm and 23-mm St. Jude Medical valves in three groups of 22 patients. Patients were selected from a database to be rigorously matched for age, sex, body surface area, functional class, underlying lesion, native valve opening area, left ventricular function, and preoperative peak and mean valve gradients. Postoperative evaluation (follow up ranging from 3 to 24 months; mean, 11.5 months) included clinical examination and echocardiographic studies. RESULTS: There was no operative mortality or significant perioperative complications. Short-term clinical follow-up was marked by a complete absence of valve-related complications. Presently, all but 1 patient in the 21-mm HP group and 2 in the 21-mm standard group are in New York Heart Association functional class I. Doppler echocardiography-derived mean and maximal pressure gradients were significantly lower in the 21-mm HP group (8.1 +/ 1.9 and 16.4 +/- 3.4 mm Hg) than in the 21-mm standard group (13.4 +/- 3.9 and 21.2 +/- 4.3 mm Hg; p = 0.002 and p = 0.0004, respectively), confirming the better hemodynamic performance already described in in vitro studies. Pressure gradients did not differ significantly between the 21-mm HP and the 23-mm standard groups. The 21-mm HP valve demonstrated the highest performance index; 0.66 +/- 0.08, compared with 0.49 +/- 0.09 for the 21-mm standard valve (p < 0.001) and 0.59 +/- 0.07 for the 23-mm standard valve (p < 0.001). CONCLUSIONS: In vivo hemodynamic performance of the 21-mm HP valve corresponds closely to that of the 23-mm standard valve and is substantially better than that of the 21-mm standard valve. The 21-mm HP St. Jude Medical valve demonstrates excellent hemodynamic characteristics and can be recommended in normal-sized adult patients with narrow aortic root. This valve will minimize the need for aortic annulus enlargement. PMID- 8633953 TI - Cardiopulmonary bypass, rewarming, and central nervous system dysfunction. AB - BACKGROUND: During cardiopulmonary bypass a nasopharyngeal temperature greater than 38 degrees C at the end of rewarming may indicate cerebral hyperthermia. This could exacerbate an ischemic brain injury incurred during cardiopulmonary bypass. METHODS: In a cohort of 150 aortocoronary bypass patients neuropsychologic test scores of 66 patients whose rewarming temperature exceeded 38 degrees C were compared with those who did not. There were no differences between groups with respect to demographic and intraoperative variables. RESULTS: A trend was seen for hyperthermic patients to do worse on all neuropsychologic tests in the early postoperative period but not at 3-month follow-up. By analysis of covariance hyperthermic patients did worse on the visual reproduction subtest of the Weschler memory scale at 3 months (p = 0.02), but this difference was not found by linear regression (p = 0.10). CONCLUSIONS: We were unable to demonstrate any significant deterioration in patients rewarmed to greater than 38 degrees C in the early postoperative period. The poorer performance in the visual reproduction subtest of the Wechsler memory scale at 3 months in the group rewarmed to more than 38 degrees C is interesting but far from conclusive. Caution with rewarming is still advised pending more in-depth study of this issue. PMID- 8633954 TI - Prediction of outcome after revascularization in patients with poor left ventricular function. AB - BACKGROUND: In patients with poor left ventricular function, the determinants of outcome after revascularization are unknown. METHODS: We studied prospectively 57 patients with stable coronary artery disease and poor left ventricular function (left ventricular ejection fraction, 0.28 +/- 0.04) who underwent coronary artery bypass grafting. Clinical variables were assessed as predictors of outcome in all patients, and preoperative stress thallium-201 scintigraphic data were analysed in 37 patients. RESULTS: The operative mortality was 1.7%. At 12 months after operation, 46 of the 49 survivors were angina-free and 35 had fewer heart failure symptoms, but postoperative left ventricular ejection fraction (0.30 +/- 0.09) did not change significantly. Eighteen survivors had left ventricular ejection fraction improved by 0.05 or more (0.30 +/- 0.03 preoperatively, 0.40 +/- 0.05 postoperatively; p = 0.0001). The adjusted odds ratio of large reversible thallium-201 defects in predicting such outcome was 15 (95% confidence interval, 1.6 to 140), whereas other clinical variables had no predictive value. The transplantation-free 5-year survival was 73%. CONCLUSIONS: In patients with poor left ventricular function, surgical revascularization can be performed safely, with good symptomatic relief and long-term survival. One-year survival and improvement in left ventricular function is better in patients with large reversible defects on preoperative stress thallium-201 scintigraphy. PMID- 8633955 TI - Pulmonary endothelial permeability changes after major lung resection. AB - BACKGROUND: Increased pulmonary endothelial permeability has been proposed as a cause of postpneumonectomy pulmonary edema. This study investigated changes in pulmonary endothelial permeability after major lung resection. METHODS: Lung scintigraphy was performed in 21 men (median age, 66 years; range, 34 to 73 years) after pneumonectomy (10 patients) or lobectomy (11 patients). Pulmonary endothelial permeability was measured by the net pulmonary accumulation of intravenous technetium-99m-labeled albumin, calculated as a ratio of lung:heart radioactivity counts. Pulmonary hemodynamics were monitored continuously by a pulmonary artery catheter, and serum levels of inflammatory cytokines were assayed. RESULTS: The lung:heart radioactivity ratio increased significantly in the initial 8 hours after pneumonectomy but not after lobectomy (p < 0.01). Mean pulmonary artery pressure and pulmonary vascular resistance both increased significantly during pneumonectomy (p < 0.05). The intraoperative increase in mean pulmonary artery pressure was inversely related to preoperative mean pulmonary artery pressure (r = -0.47; p = 0.02). The postoperative change in lung:heart radioactivity ratio to the perioperative increase in pulmonary vascular resistance (r = 0.54; p = 0.02) but not to the increase in mean pulmonary artery pressure (r = 0.14; p > 0.05). Serum interleukin-8 and neutrophil elastase levels were elevated in all patients preoperatively. The postoperative change in lung:heart radioactivity ratio was related to preoperative elastase levels (r = 0.61; p = 0.02). CONCLUSIONS: Pulmonary endothelial permeability appears to be increased after pneumonectomy. Preoperative neutrophil activation and the adaptation of the remaining pulmonary vasculature may be etiologic factors. PMID- 8633956 TI - Endoscopic ultrasound with fine-needle aspiration in the diagnosis and staging of lung cancer. AB - BACKGROUND: Esophageal endoscopic ultrasonographic (EUS) guidance for fine-needle aspiration (FNA) of mediastinal lymph nodes has been introduced only recently. The utility of EUS/FNA in diagnosing and staging bronchogenic carcinoma is unknown. METHODS: After a thoracic computed tomographic scan, 27 patients with known or suspected lung cancer underwent EUS. Accessible abnormal mediastinal lymph nodes were aspirated under EUS guidance. Patients with positive cytologic studies did not undergo further testing, whereas the remaining patients underwent mediastinal exploration. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated for both chest computed tomography and EUS/FNA: RESULTS: Twenty-two of 27 patients had mediastinal adenopathy by computed tomography scan. Sixteen patients had positive findings on EUS, 15 with positive FNA (10 non-small cell lung cancer; 5 small cell lung cancer) and 1 with T4 status. Fourteen patients with positive FNA had lymph nodes sampled at level 5, level 7, or both. Of 11 patients with negative EUS/FNA, 2 had positive findings at operation (sensitivity 89%). The diagnosis of lung cancer was established in 7 patients. CONCLUSIONS: The results showed that EUS/FNA improves the accuracy of computed tomographic scan in the staging of lung cancer. By accessing lymph nodes at levels 5 and 7, EUS/FNA complements mediastinoscopy and is considered the staging modality of choice in these regions. Positive EUS/FNA can obviate the need for further invasive staging. PMID- 8633957 TI - Esophageal perforation: emphasis on management. AB - BACKGROUND: Perforation of the esophagus is a deadly injury that requires expert management for survival. METHODS: We performed a retrospective clinical review of 66 patients treated at Emory University affiliated hospitals for esophageal perforation between 1973 and 1993. RESULTS: Iatrogenic perforations accounted for 48 injuries (73%), barogenic perforations occurred in 12 patients (17%), trauma was causative in 3 (5%), and 3 patients had esophageal infection and other causes. Lower-third injuries occurred in 43 cases (65%), middle third in 14 (21%), and upper third in 9 (14%). Early contained perforations were managed successfully by limiting oral intake and giving parenteral antibiotics in 12 patients. Cervical perforations were drained without attempt at closure of the leak. Perforations with mediastinal or pleural contamination recognized early were managed by primary closure and drainage in 28 patients. Reinforcement of the primary closure using stomach fundus, pleural, diaphragmatic, or pericardial flap was performed in 16 patients. Those perforations that escaped early recognition required thoughtful management, using generous debridement and drainage and sometimes esophageal resection. The esophageal T tube provided control of leaks in 3 of these patients and was a useful adjunct. Using these management principles, we achieved a 76% survival rate for all patients. Six patients with perforations complicating endoesophageal management of esophageal varices were a high-risk subset with an 83% mortality rate. CONCLUSIONS: Esophageal perforation remains an important thoracic emergency. Aggressive operative therapy remains the mainstay for treatment; however, conservative management may be preferred for contained perforations and the esophageal T tube may be used for late perforations. PMID- 8633958 TI - Thromboxane receptor blockade improves oxygenation in an experimental model of acute lung injury. AB - BACKGROUND: Adult respiratory distress syndrome remains a major cause of morbidity and mortality. We investigated the role of thromboxane receptor antagonism in an experimental model of acute lung injury that mimics adult respiratory distress syndrome. METHODS: Three groups of rabbit heart-lung preparations were studied for 30 minutes in an ex vivo blood perfusion/ventilation system. Saline control (SC) lungs received saline solution during the first 20 minutes of study. Injury control (IC) lungs received an oleic acid-ethanol solution during the first 20 minutes. Thromboxane receptor blockade (TRB) lungs received the same injury as IC lungs, but a thromboxane receptor antagonist (SQ30741) was added to the blood perfusate just prior to study. Blood gases were obtained at 10-minute intervals, and tidal volume, pulmonary artery pressure, and lung weight were continuously recorded. Oxygenation was assessed by measuring the percent change in oxygen tension over the 30-minute study period. Tissue samples were collected from all lungs for histologic evaluation. RESULTS: Significant differences were found between SC and IC lungs as well as TRB and IC lungs when comparing pulmonary artery pressure (SC = 33.1 +/- 2.2 mm Hg, TRB = 35.4 +/- 2.1 mm Hg, IC = 60.4 +/- 11.1 mm Hg; p < 0.02) and percent change in oxygenation (SC = -20.6% +/- 10.3%, TRB = -24.2% +/- 9.5%, IC = -57.1% +/- 6.2%; p < 0.03). None of the other variables demonstrated significant differences. CONCLUSIONS: Thromboxane receptor blockade prevents the pulmonary hypertension and the decline in oxygenation seen in an experimental model of acute lung injury that mimics adult respiratory distress syndrome. PMID- 8633959 TI - Mainstem bronchial sleeve resection with pulmonary preservation. AB - BACKGROUND: Resection of a mainstem bronchus with pulmonary preservation is a therapeutic option when disease is limited to the mainstem bronchus. We reviewed our experience with this procedure to determine the operative morbidity, mortality, and long-term outcome. METHODS: From January 1965 through January 1995, 22 patients (13 male, 9 female) underwent circumferential mainstem bronchial sleeve resection without removal of pulmonary parenchyma. Median age was 37 years (range, 12 to 70 years). The right mainstem bronchus was involved in 12 patients and the left, in 10. Nineteen patients (86%) were symptomatic; symptoms included cough in 5, dyspnea in 5, wheeze in 3, hemoptysis in 3, and a combination of these in 3. Conventional tomography was done in 8 patients and identified every lesion. Bronchoscopy was diagnostic in all patients. Resection was for cancer in 15 patients (68%), benign stricture in 5 (23%), and an impacted broncholith in 2 (9%). The cancer was a carcinoid in 9 patients, a mucoepidermoid carcinoma in 3, squamous cell carcinoma in 2, and adenoid cystic carcinoma in 1. Fourteen patients were postsurgically classified as stage IIIA (T3 NO MO) and 1 patient as stage IIIB (T4 N2 M0). The median length of the resected bronchus was 2.0 cm (range, 1.0 to 4.0 cm). Two patients required hilar release maneuvers. The bronchial anastomosis was reinforced with pleura in 10 patients, pericardium in 2, and serratus anterior muscle in 1. RESULTS: There were no operative deaths. Three patients (14%) had postoperative complications. Follow-up was complete and ranged from 6 months to 25.7 years (median follow-up, 10.2 years). Twenty-one patients are currently alive. All patients are asymptomatic except 1 patient, who required a stent for an anastomotic stricture. No patient has had recurrence of cancer. CONCLUSIONS: In properly selected patients, mainstem bronchial sleeve resection with lung preservation can be performed safely and provides excellent relief of symptoms with good long-term survival. PMID- 8633960 TI - Lung volume reduction surgery: lessons learned. AB - BACKGROUND: The concept of lung volume reduction for generalized emphysema was proposed by Brantigan and associates in 1958 and reintroduced by Cooper and colleagues in 1994. The present study presents lessons learned from an 18-month experience. METHODS: From August 1, 1994, to August 1, 1995, 53 patients underwent lung volume reduction at Emory University for generalized emphysema. There were 17 women and 36 men ranging in age from 55 to 75 years. The length of stay ranged from 10 to 59 days. At the time of presentation, 47 patients were receiving oxygen and 35 were receiving steroids. Forty-six patients were operated on using a median sternotomy and 7 through a unilateral thoracotomy. All patients underwent preoperative and postoperative pulmonary rehabilitation. RESULTS: There was one early death and four late deaths. Lessons learned from this group of patients are presented. CONCLUSIONS: Lung volume reduction surgery remains a sea of relatively uncharted waters, with the future direction yet to be determined. PMID- 8633961 TI - Angiogenesis and molecular biologic substaging in patients with stage I non-small cell lung cancer. AB - BACKGROUND: Although complete surgical resection remains the primary treatment for localized stage I non-small cell lung cancer, the cancer recurrence rate is 25% to 40%. If one could identify, a subset of patients using molecular factors that contribute to tumour aggressiveness, one might improve prognosis in this group with additional treatment. High expression of angiogenesis factor viii has been associated with the presence of nodal metastases in breast cancer; here we examined its relation to survival with non-small lung cancer. METHODS: We examined angiogenesis using immunohistochemistry on paraffin blocks of tumour from 275 consecutive patients with stage I non-small cell lung cancer, more than 68 months of follow-up, and a 64% 5-year survival. Angiogenesis was calculated from the microvessel number per ten 200 x microscope fields measured at the tumor periphery, in the center, and in the area of highest concentration. RESULTS: Measurements in the central area were inconsistent due to prominent necrosis. However, microvessel number recorded at the periphery and at the "hottest" are correlated well (r2 = 0.952; p = 0.001), and a significant survival advantage was noted for low-level expression at both areas (peripheral, p = 0.046; "hottest", p = 0.006). Multivariate survival analysis using angiogenesis, protooncogene erbB 2, tumor suppressor gene p53, and the proliferation marker KI-67 defined angiogenesis as the most significant prognostic factor in stage I lung cancer. CONCLUSIONS: This molecular biologic substaging system including angiogenesis for stage I non-small cell lung cancer is independent of routine histopathologic factors and revealed an additive adverse effect with expression of several biologic markers (5-year survival: no marker [n = 51] 81%, 1 marker [n = 82] 71%, 2 markers [n = 84] 54%, and 3 to 4 markers [n = 58] 49%; p = 0.0001). PMID- 8633962 TI - Plasma cell granuloma of the lung: difficulties in diagnosis and prognosis. AB - BACKGROUND: The nature of plasma cell granuloma of the lung is still not well defined. Its diagnosis can be difficult, and its long-term prognosis is uncertain. METHODS: Four patients, aged 4 1/2 to 45 years, had resection of a circumscribed plasma cell granuloma. In 1 of them, it was associated with a carcinoma. The fifth patient was treated by irradiation for an invasive form of plasma cell granuloma, which was diagnosed as malignant lymphoma 1 year later. The plasma cell granulomas had a similar polymorphic pattern and were polyclonal at immunohistochemistry. RESULTS: The 3 patients treated by lobectomy, including the patient with an associated carcinoma, were alive and well at 12, 12, and 11 years postoperatively. The patient treated by pneumonectomy had recurrence 9 years later and died 11 years after operation: irradiation was not effective. The patient with lymphoma was treated by chemotherapy and was alive and in remission 5 years later. CONCLUSIONS: An accurate preoperative diagnosis is difficult. The results of biopsy can be inconclusive, and an associated malignancy can be missed. Complete resection remains the best treatment. The long-term prognosis is unpredictable, and late and fatal recurrences are possible. PMID- 8633964 TI - Extended lung preservation with the use of hibernation trigger factors. AB - BACKGROUND: The complications of preserving lungs for transplantation are well known, with successful transplantation only being assured by preservation times of 5 to 6 hours or less. If a new method of consistent lung preservation could be identified, lung transplantation could be extended to many patients. We have previously reported lung preservation times averaging 14.8 hours using a multiorgan autoperfusion block infused with physiologic saline solution as a model. When plasma from deeply hibernating woodchucks (Marmota monax) or the delta opioid DADLE was infused into the multi-organ block, lung preservation times increased threefold to 45 hours. METHODS: In this study, we examined the effect of infusing plasma containing the hibernation induction trigger molecule on lung preservation for transplantation using a multiorgan autoperfusion block. RESULTS: This study demonstrated that successful orthotopic transplantation of single canine lungs is possible after 24 to 33 hours of preservation when the lung has been maintained with plasma containing the hibernation induction trigger molecule. CONCLUSIONS: Theoretically, hibernation induction trigger could be administered to donors before lung harvest in an effort to extend lung preservation times. PMID- 8633963 TI - Tracheobronchial lacerations after intubation and tracheostomy. AB - BACKGROUND: Although long-term complications of intubation and tracheostomy are well documented, little has been reported on acute complications of airway access techniques. METHODS: Fourteen patients (1 male and 13 female patients) aged 15 to 80 years presented with tracheobronchial lacerations after single-lumen intubation (n = 9), double-lumen intubation (n = 1), or tracheostomy (n = 4). RESULTS: A left bronchial laceration after double-lumen intubation was discovered and repaired intraoperatively. A tracheal laceration after single-lumen intubation was recognized during induction of anesthesia. The remaining 12 were diagnosed within 6 to 126 hours (median, 24 hours) after injury. All patients had mediastinal and subcutaneous emphysema. At endoscopy, 12 injuries were located in the thoracic trachea and 1 in the cervical trachea. Twelve underwent primary repair through a right thoracotomy (n = 11) or left cervicotomy (n = 1), and 1 was treated conservatively. Two patients with tracheostomy injury died postoperatively. All repairs healed well but one. The latter was performed 5 days after the injury; a dehiscence occurred, but healed spontaneously. CONCLUSIONS: We conclude that prognosis of tracheal lacerations depends both on the general health of the patient and on the rapidity of diagnosis and treatment. PMID- 8633965 TI - Preoperative assessment of the high-risk patient for lung resection. AB - BACKGROUND: We wanted to determine if cardiopulmonary exercise testing could better identify the threshold where physiologic function is irreparably impaired for patients with borderline pulmonary function who are being considered for lung cancer resection. METHODS: We performed an open, prospective preoperative trial and a postoperative outcome evaluation with a combined medical, surgical, and exercise physiology evaluation at three university hospitals. All eligible patients had spirometry, lung volume determination, and quantitative perfusion scanning and performed a cardiopulmonary stress test, stair climbing, and a 12 minute walk for distance. Functional status was determined with an Eastern Cooperative Oncology Group score, a dyspnea score, and a cardiopulmonary risk index. RESULTS: We identified 12 patients who met strict criteria for borderline pulmonary function during a 1-year study period. The mean forced expiratory volume in 1 second (FEV1) was 1.38 L (48% of predicted). The mean predicted postoperative FEV1 based on pneumonectomy was 700 mL. Eleven of the patients did the stair climb and 10 passed. All 12 patients achieved a maximum oxygen consumption greater than or equal to 10 mL x kg(-1) x min(-1) (mean value, 13.8 mL x kg(-1) x min(-1)). Thirteen operations were performed on the 12 patients. Nine complications occurred in 7 patients. CONCLUSIONS: Patients with borderline pulmonary function can undergo resection safely if they have an FEV1 equal to or greater than 1.6 L or 40% of its predicted value, a predicted postoperative FEV1 of 700 mL or more, a maximum oxygen consumption of 10 mL x kg(-1) x min(-1) or greater, or stair climbing of three flights or more. Cardiopulmonary stress testing and stair climbing add valuable clinical information for patients with an FEV1 of less than 1.6 L. PMID- 8633966 TI - Penetrating thoracic trauma in a pediatric population. AB - INTRODUCTION: Penetrating thoracic trauma in the pediatric population is increasing at an alarming rate. We sought to describe this population and to define prognostic factors that might be of benefit in the management of these patients. METHODS: We retrospectively reviewed the charts and trauma registry records of 65 patients 18 years of age and younger admitted to an urban level I trauma center with the diagnosis of penetrating thoracic trauma. RESULTS: The majority of the patients were adolescent boys. Injury severity score greater than 25 and a corrected admission pH less than 7.3 were associated with higher mortality and increased need for surgical intervention. Isolated thoracic injury was found to be associated with a high mortality rate. Autotransfused blood was used in 9 of the 65 patients. CONCLUSIONS: Injury severity score and corrected admission pH are independent predictors of mortality and need for operation in the pediatric population with penetrating chest injuries. Penetrating thoracic wounds demand special attention by the trauma team. The use of autotransfusion may be beneficial in pediatric trauma victims. PMID- 8633967 TI - Symptomatic tachydysrhythmias after esophagectomy: incidence and outcome measures. AB - BACKGROUND: Supraventricular tachydysrhythmias (SVT) after esophageal operations for carcinoma occur frequently and may be associated with increased morbidity. Prospective data on the etiology, incidence, and importance of these dysrhythmias are sparse. METHODS: In 100 consecutive patients undergoing esophagectomy without prior history of atrial dysrhythmias or receiving antiarrhythmics, we prospectively examined the effects of predefined risk factors by history and pulmonary function on the 30-day incidence of symptomatic postoperative SVT, need for intensive care unit admission, and mortality rate. RESULTS: Symptomatic postoperative SVT occurred in 13 (13%) of the 100 patients studied at a median of 3 days after operation and was accompanied by hypotension in 9/13 (69%). Univariate correlates of SVT were older age (p = 0.03), perioperative use of theophylline (p = 0.044), and a low carbon monoxide diffusion capacity (measured in 56% of patients) on preoperative pulmonary function. Patients in whom SVT developed had a higher rate of intensive care unit admission (p = 0.0001) and a longer hospital stay (p = 0.036). Although patients in whom SVT developed had a higher (p = 0.013) 30-day mortality rate, SVT was not the direct cause of death. CONCLUSIONS: These prospective data show that the true incidence of symptomatic SVT within 30 days of esophagectomy is lower than previously reported. Occurrence of SVT was associated with significant morbidity. Older age was the strongest predictor of SVT after esophagectomy. In high-risk patients, continued monitoring (48 to 72 hours) and early interventions to decrease the incidence of postoperative SVT may improve overall surgical outcomes. PMID- 8633969 TI - Calcified ball thrombus in the left atrium. AB - We experienced a case of calcified ball thrombus that was fixed to the atrial septum in the left atrium. This patient had no symptoms and no cardiac dysfunction. the thrombus was detected during preoperative work-up of a retroperitoneal tumor. The process of fixation to the atrial septum and calcification is unclear. PMID- 8633968 TI - Transaxillary minithoracotomy versus video-assisted thoracic surgery for spontaneous pneumothorax. AB - BACKGROUND: Although management of spontaneous pneumothorax by video-assisted thoracic surgery (VATS) has generally shown superior clinical results to thoracotomy, management of spontaneous pneumothorax by transaxillary minithoracotomy (TAMT) has also shown good clinical results. The objective of this study was to compare the clinical results of VATS and TAMT in treating spontaneous pneumothorax. METHODS: Sixty-six patients, aged 13 to 81 years, with recurrent, persistent or contralateral spontaneous pneumothorax were involved in this study. Thirty-six patients were treated by VATS and 30 by TAMT. The operating time, the amount of analgesics used on the first postoperative day, the duration of the indwelling chest tube, and the number of recurrences after operation were compared. The follow-up periods of both procedures were from 6 to 24 months. RESULTS: Of the 66 patients, 64 were male and 2 were female. The duration of operation, from start of skin incision to insertion of chest tube, was 91.2 +/- 36.8 minutes in VATS and 86.3 +/- 40.9 minutes in TAMT (p = 0.6061). The amount of analgesics (keptoprofen) used was 1.9 +/- 2.3 ampules in VATS and 2.1 +/- 2.9 ampules in TAMT (p = 0.0883). The duration of indwelling chest tube was 5.0 +/- 4.0 days in VATS and 4.3 +/- 2.1 days in TAMT (p = 0.3707). The number of recurrences after operation was 4 in VATS and none in TAMT. CONCLUSIONS: There were no advantages of VATS over TAMT for management of recurrent, persistent, or contralateral spontaneous pneumothorax in regard to the operating time, the amount of analgesics used on the first postoperative day, the duration of the indwelling chest tube, and the number of postoperative recurrences in patients with apical bullae. PMID- 8633970 TI - Sensitivity to sternotomy wires may cause postoperative pruritus. AB - An eczematous eruption developed on the anterior chest of a 58-year-old woman with known nickel sensitivity after the insertion of nickel-containing sternotomy wires. Her wound was revised with removal of the wires to give immediate and sustained relief from the itch. The electron microscopy and parasternal biopsy histology demonstrating a sarcoidal reaction are discussed. PMID- 8633971 TI - Recurrent cerebral vascular accidents are an indication for ascending aortic endarterectomy. AB - We present a patient with severe pedunculated ascending atherosclerosis associated with recurrent cerebral vascular accidents. We recommend that endarterectomy be considered for patients with recurrent cerebral vascular accidents associated with severe atherosclerosis of the ascending aorta when no other cause is found to explain the symptoms. PMID- 8633972 TI - Development of subneopulmonary obstruction early after arterial switch operation in an adult. AB - In a 19-year-old woman who had previously undergone pulmonary artery banding at the age of 1.5 years, a muscular right ventricular outflow tract obstruction developed 3 days after an arterial switch operation. Although conservative therapy proved successful, prophylactic surgical intervention on the conal septum may be beneficial in preventing the postoperative development of right ventricular outflow tract obstruction. PMID- 8633973 TI - Successful repair of coronary artery-coronary sinus fistula with aneurysm in an adult. AB - We report a very rare case of an adult with coronary artery fistula and aneurysm formation. This fistula was successfully closed with direct suture closures by opening the aneurysm under complete cardiopulmonary bypass. The distal terminated orifice of the fistula, which drained to the coronary sinus, was also closed. Finally, aneurysmorrhaphy with overlapping mattress sutures was performed. The postoperative angiographic study demonstrated normal coronary artery distribution, and the patient was asymptomatic without recurrence at 2 years after the operation. PMID- 8633974 TI - Left subclavian artery bypass graft in complicated arterial switch operation. AB - We report the successful use of left subclavian artery bypass graft in a newborn infant with complete transposition of the great arteries accompanied by an unusual coronary artery pattern, in whom left coronary artery insufficiency developed after the arterial switch operation. This procedure has shown good clinical results, with interesting follow-up angiographic findings 8 months after the operation. PMID- 8633975 TI - Squamous cell carcinoma of the lung: an unusual metastasis to pectoralis muscle. AB - A 44-year-old man with clinical T2 N0 M0 squamous cell carcinoma of the lung presented 4 weeks after left pneumonectomy with a single metastasis to his right pectoralis major muscle. Three years after excision and chemotherapy he remains in remission. Muscle metastasis of carcinoma is an infrequent occurrence. Further investigation into the incidence and treatment is warranted. PMID- 8633977 TI - Successful surgical repair of left atrial dissection after mitral valve replacement. AB - A rare case of left atrial dissection after mitral valve replacement is reported. Low output syndrome developed in the immediate postoperative period. Cardiac catheterization showed marked elevation of the pulmonary wedge pressure, and left ventriculography revealed massive paraprosthetic leakage with left atrial dissection. At the reoperation, the dissecting cavity was successfully closed from inside the left atrium under cardiopulmonary bypass. We consider this complication another variation of an atrioventricular discontinuity after mitral valve replacement. PMID- 8633976 TI - Blunt traumatic rupture of a mitral papillary muscle head. AB - Severe mitral regurgitation developed in a patient after a lateral-impact motor vehicle accident. The papillary muscle head was disrupted without evidence of other myocardial injury. We hypothesize that a dramatic and sudden increase in intrathoracic pressure may have produced the injury. The patient experienced progressive heart failure and underwent successful mitral valve repair 9 days after the accident. PMID- 8633978 TI - Mitral valve replacement in a heart transplant recipient with iatrogenic mitral regurgitation. AB - A 65-year-old cardiac transplant recipient suffered rupture of the mitral valve apparatus during endomyocardial biopsy of the left ventricle. Severe mitral regurgitation resulted, and because of heart failure with progressive clinical deterioration the patient was finally subjected to mitral valve replacement. He had a favorable postoperative course and is now asymptomatic. PMID- 8633979 TI - Histologic appearance of transmyocardial laser channels after 4 1/2 weeks. AB - Preliminary results of clinical studies suggest that transmyocardial laser revascularization is an effective treatment for patients with chronic angina that cannot be treated by other means. The mechanism of this effect remains controversial. We present autopsy results from a patient obtained 4 1/2 weeks after operation that show that the channels do not maintain patency. Further work is needed to determine the frequency of channel patency and its relation to clinical benefit. PMID- 8633980 TI - Retained sponge after thoracotomy that mimicked aspergilloma. AB - A 63-year-old man, who had had operation for the treatment of pulmonary tuberculosis 40 years before the present disorder, was admitted to our hospital with massive hemoptysis. Radiologic examinations showed a mass shadow with a crescent air sign resembling aspergilloma. Operative exploration showed a well encapsulated retained surgical sponge between the middle and lower lobes. A bronchial fistula was present in the lower lobe. The appearance of the crescent air sign was caused by drainage of exudative effusion around the retained sponge. Intrathoracic retained surgical sponges associated with bronchial fistula should be included in the differential diagnosis of patients who have mass shadows with crescent air signs but no evidence of Aspergillus infection, and who have a history of thoracotomy. PMID- 8633981 TI - Pulmonary artery sarcoma mimicking pulmonary embolism: successful surgical intervention. AB - Because of its rarity and the similarity of its presentation to that of pulmonary thromboembolic disease, the diagnosis of pulmonary artery sarcoma is often not considered early in patients presenting with recurrent or chronic pulmonary emboli. We present a case of pulmonary artery sarcoma that was treated as pulmonary embolism for 3 years before surgical resection was carried out. Two years after the resection the patient is well with no clinical or radiologic evidence of recurrent or metastatic disease. PMID- 8633982 TI - Effects of intraaortic balloon pumping on coronary and carotid flow during percutaneous cardiopulmonary support. AB - Previous studies have suggested an improved clinical outcome when percutaneous cardiopulmonary support is combined with intraaortic balloon counterpulsation in patients with cardiogenic shock. We evaluated the effect of combined intraaortic balloon counterpulsation and percutaneous cardiopulmonary support therapy on coronary and cerebral blood flow by Doppler measurements in the coronary and the carotid arteries in a patient with cardiac arrest. During pacemaker stimulation, intraaortic balloon counterpulsation in addition to percutaneous cardiopulmonary support markedly improved coronary and carotid blood flow. Possible mechanisms are discussed. PMID- 8633983 TI - Traumatic rupture of the thoracic aorta during the second trimester of pregnancy. AB - A 36-year-old woman in the second trimester of pregnancy underwent emergent operative repair of a traumatic aortic disruption caused by a motor vehicle accident. Left atrial-to-femoral artery bypass was used to maintain fetal circulation during the cross-clamp period. Her healthy, full-term child was subsequently delivered 3 months later by normal vaginal delivery. PMID- 8633984 TI - Therapy for lung failure using nitric oxide inhalation and surfactant replacement. AB - Nitric oxide inhalation and surfactant replacement therapy are relatively new concepts in the treatment of respiratory failure due to hypoxia and reperfusion injury after lung transplantation. We report on a patient in whom reperfusion injury of the lung developed after resuscitation and implantation of a biventricular assist device for sudden cardiac arrest. Lung failure developed within 12 hours after implantation of the biventricular assist device. Lung function was reestablished using combined therapy of nitric oxide and surfactant. Heart transplantation was performed successfully thereafter. This case indicates the potential role of a combined therapy of nitric oxide and surfactant in acute hypoxic lung failure. PMID- 8633985 TI - Selective cerebral perfusion technique during aortic arch repair in neonates. AB - We describe selective cerebral perfusion techniques for repair of the aortic arch in neonates. These techniques may help protect the brain from ischemic injury caused by a cessation of cerebral perfusion for aortic arch reconstruction in patients with hypoplastic left heart syndrome or interrupted aortic arch. PMID- 8633986 TI - Alternative method for cardiac transplantation: surgical considerations and technical aspects. AB - A simplified technique for complete orthotopic cardiac transplantation is described. The potential technical difficulties and surgical considerations are discussed. PMID- 8633987 TI - Alternative technique for assessment and repair of the mitral valve. AB - Valve repair is preferred over replacement in the management of mitral regurgitation when technically possible. Central to the achievement of a durable result is precise assessment of the anatomic abnormality present before repair, as well as accurate intraoperative evaluation of the adequacy of repair accomplished. Cardioplegic techniques commonly employed permit inspection of the valve in a flaccid, arrested state, which may not accurately reflect its function in the contractile heart. The repair can be tested under normal loading conditions only after separation from cardiopulmonary bypass. An alternative technique of myocardial management employing mild hypothermia, continuous aortic root perfusion, and intermittent fibrillation is described that provides an opportunity to directly examine the valve under physiologic conditions before, during, and after completion of the repair. PMID- 8633988 TI - Simple technique of left heart bypass. AB - Left heart bypass is typically established by left atrial appendage cannulation. We report a technique using inferior pulmonary vein cannulation, which is technically simpler. We have used this technique in 20 cases with reliable venous inflow. PMID- 8633989 TI - Anton P. Chekhov, MD (1860-1904): dual medical and literary careers. AB - This is a story of a physician who was one of the world's greatest short story writers. Anton Pavlovich Chekhov successfully performed dual careers of writing and practicing medicine during a short but productive life. Many biographers overlook Dr Chekhov's role, although his medical training and practice flowed over into his writing, influencing both his subject matter and his style. His stories and plays reveal the insights that Dr Chekhov discovered about the human psyche at work, thus delving into psychosomatic medicine nearly 50 years before its time. Despite orders to stop practicing medicine because of his own deteriorating health, Chekhov devoted himself further to the study of medicine, publishing a landmark study in social medicine that advocated reform of the Russian penal system. Meanwhile, his fictional works earned him the coveted Pushkin prize for the best literary work of the year in 1888. Shortly after celebrating his third wedding anniversary to actress Olga Knipper, Anton Chekhov died at age 44, victim of a nearly lifelong battle with tuberculosis. PMID- 8633990 TI - Multimodality therapy in stage III non-small cell lung cancer. AB - BACKGROUND: Non-small cell lung cancer commonly presents as locally advanced disease. This category of tumors is heterogeneous. Although some patients clearly benefit from operative management alone, the vast majority (more than 90%) will succumb to their disease within 5 years. In the past decade a large clinical research effort has been undertaken in an attempt to improve on this outcome using a combination of chemotherapy, radiotherapy, and operation. METHODS: The English-language literature was reviewed using the headings for lung neoplasms and text words combined modality therapy and multimodality therapy. In addition, the bibliographies of relevant articles were reviewed. Emphasis was placed on prospective randomized trials and large phase II studies. We review the rationale, design, and outcome of these trials, including both operative and nonoperative approaches. RESULTS: Several prospective, randomized trials now demonstrate an advantage to combined modality management over radiotherapy or operation alone when a cisplatin-based chemotherapy regimen is incorporated into the treatment plan. This advantage was seen using both operative and nonoperative approaches. CONCLUSIONS: Combined modality therapy offers an improved outcome for patients with stage III non-small cell lung cancer. Whether both operation and radiotherapy are needed for local control, the best sequence of treatment and the optimal chemotherapy regimen remain to be defined. PMID- 8633991 TI - Normothermic techniques during open heart operations. AB - There has been considerable interest in the use of normothermic techniques during cardiac operations, both as a means of myocardial protection and as a more physiologic environment for other organs during cardiopulmonary bypass. Although a limited number of uncontrolled studies have suggested superior clinical results compared with conventional hypothermic regimens, these claims have not been thoroughly investigated using randomized protocols. The limited available data suggest that the successful use of warm blood cardioplegia requires adequate delivery of the solution to all parts of the myocardium at optimal flow rates to maintain aerobic arrest, so those who advocate the use of normothermic arrest must pay particular attention to ensure that their myocardial protection is effective. The advantages of employing normothermic systemic perfusion in regard to factors such as improved hemodynamic performance and reduced blood loss postoperatively need to be balanced against concerns regarding the inadequacy of cerebral protection offered by this method. PMID- 8633992 TI - Myocardial protection: modern studies. PMID- 8633993 TI - As originally published in 1988: Long-term results of mitral valve surgery in patients with severe pulmonary hypertension. Updated in 1996. AB - Mitral valve surgery was performed in 88 patients with severe pulmonary hypertension (average systolic pulmonary artery pressure, 94.7 +/- 22 mm Hg; range, 70-180 mm Hg) over a 10-year period. Sixty-four patients (73%) were in New York Heart Association Functional Class III or IV. There were 64 valve replacements and 24 open mitral commissurotomies. Operative mortality was 5.6% (5 patients) and was not related to the degree of pulmonary hypertension, surgical procedure performed, or type of valve lesion. A 100% follow-up was obtained, ranging from nine months to 10 years, with a mean of 44 months. Six late cardiac deaths (7.2%) occurred, 5 in patients with valve replacement and 1 in a patient who underwent a commissurotomy. Actuarial survival was 86 +/- 3% at five years and 83 +/- 4% at 10 years. Fourteen patients underwent right ventricular catheterization a mean of 24 months following operation. Systolic pulmonary artery pressure had decreased from a mean preoperative value of 101 +/- 22 to 40.5 +/- 7 mm Hg (p < 0.001). Cardiac index increased by 55% of the preoperative values. Functional status improved markedly; 71 survivors (93%) were in New York Heart Association Class I or II. These results indicate that, in patients with mitral valve lesions and severe pulmonary hypertension, (1) surgical procedures can be performed with an acceptable operative mortality; (2) excellent long-term survival and functional results can be obtained; and (3) pulmonary hypertension decreases significantly after operation. Patients with mitral valve disease may benefit from surgical treatment regardless of the degree of pulmonary hypertension. PMID- 8633994 TI - Evolution of bronchus-first technique. PMID- 8633995 TI - Stable osteosynthesis after median sternotomy. PMID- 8633996 TI - Simplified reinforced sternal closure. PMID- 8633997 TI - Reducing aortic root bleeding. PMID- 8633998 TI - Operation for repair of ventricular septal perforation. PMID- 8633999 TI - Beneficial effects of fibrin glue on esophageal perforation. PMID- 8634000 TI - Two-patch repair of atrioventricular canal. PMID- 8634001 TI - Heparin effect on platelets and fibrinolysis. PMID- 8634002 TI - Simplified technique for aortic annular enlargement during aortic valve replacement. PMID- 8634003 TI - The role of psychotherapy in the treatment of depression: review of two practice guidelines. AB - We review two recent practice guidelines' assessments of the role of psychotherapy in the treatment of major depression in adults. We examine the practice guideline published by the American Psychiatric Association (APA) and that published by the Depression Guideline Panel of the Agency for Health Care Policy and Research. We focus on the guidelines' evaluations of psychotherapies, their statements about the role of psychotherapy in first-line treatment of depression, and the procedures they recommend for choosing among psychotherapies. We argue that the APA guideline understates the value of cognitive, behavioral, brief psychodynamic, and group therapies. Both guidelines understate the value of psychotherapy alone in the treatment of more severely depressed outpatients. The APA guideline overvalues the role of combined psychotherapy-pharmacotherapy regimens, particularly in view of the greater cost of this strategy. The APA guideline also makes recommendations about choosing among psychotherapies that are not well supported by empirical evidence. We conclude with some guidelines for guideline development. PMID- 8634004 TI - The role of psychotherapy in the treatment of depression: review of two practice guidelines. PMID- 8634005 TI - The role of psychotherapy in the treatment of depression: review of two practice guidelines. PMID- 8634006 TI - The role of psychotherapy in the treatment of depression: review of two practice guidelines. PMID- 8634007 TI - Practice guidelines and professional challenges: what psychotherapists need to do. PMID- 8634008 TI - Recovery in geriatric depression. AB - BACKGROUND: Clinical characteristics of depression, age at illness onset, medical burden, disability, cognitive impairment, lack of social support, and poor living conditions may influence the course of depression. This study investigates the timetable of recovery and the role of the above factors in predicting recovery in elderly patients with major depression. METHODS: Recovery was studied in 63 elderly (age >63 years) and 23 younger patients with depression who were followed up for an average of 18.2 months (SD, 13.1 months) under naturalistic treatment conditions. Diagnosis was assigned according to Research Diagnostic Criteria after administration of the Schedule for Affective Disorders and Schizophrenia. The Longitudinal Follow-up Interval Examination was used to identify recovery. RESULTS: The recovery rate of depressed elderly patients was similar to that of younger depressed patients. In the elderly patients, age, antidepressant treatment, age at onset, and chronicity of episode were significantly associated with time to recovery since entry. Among these parameters, late age at onset was the strongest predictor of slow recovery. In younger patients, long time to recovery was predicted by weak social support, younger age, cognitive impairment, and low intensity of antidepressant treatment. In the elderly, the intensity of antidepressant treatment began to decline within 16 weeks from entry and approximately 10 weeks prior to recovery. CONCLUSIONS: These findings challenge the view that geriatric depression has a worse outcome than depression in younger adults. However, depressed patients with onset of first episode in late life may be at higher risk for chronicity. Antidepressant treatment prescribed by clinicians may decline prior to recovery despite evidence that high treatment intensity is effective in preventing relapse. PMID- 8634009 TI - Incidence and correlates of tardive dyskinesia in first episode of schizophrenia. AB - BACKGROUND: There is controversy over whether tardive dyskinesia (TD) is solely a consequence of antipsychotic drug treatment or in part may reflect an intrinsic aspect of the disease process. Pathophysiologic factors could, independently or in concert with drug effects, lead to the development of dyskinetic signs. METHODS: We studied prospectively 118 patients in their first episode of psychosis who were treatment-naive or had less than 12 weeks of antipsychotic drug exposure at study entry. Patients received standardized antipsychotic drug treatment and were evaluated for up to 8 1/2 years with regular assessments of psychopathologic signs and symptoms and side effects. RESULTS: The cumulative incidence of presumptive TD was 6.3% after 1 year of follow-up, 11.5% after 2 years, 13.7% after 3 years, and 17.5% after 4 years. Persistent TD had a cumulative incidence of 4.8% after 1 year, 7.2% after 2 years, and 15.6% after 4 years. Taken individually, both antipsychotic drug dose, entered as a time dependent covariate, and poor response to treatment of the first psychotic episode were significant predicters of time to TD. When antipsychotic drug dose and treatment response were examined together, treatment responders had significantly lower hazards for presumptive TD than nonresponders (hazard ratio, 0.29; 95% confidence interval, 0.09 to 0.97). Dose was a trend-level predicter, with each 100-mg chlorpromazine equivalent unit increase in dose associated with a 5% increase in the hazard of presumptive TD (hazard ratio, 1.05; 95% confidence interval, 0.99 to 1.11). CONCLUSION: Poor response to the treatment of a first episode of psychosis and, to a lesser extent, antipsychotic drug dose are important factors in the development of TD. This suggests that there may be a disease-related vulnerability to TD manifest with antipsychotic drug exposure. Potential pathophysiologic factors might include neurodevelopmentally induced structural neuropathologic characteristics, sensitization of nigrostriatal dopamine neurons, and the induction of glutamatergically mediated neurotoxic effects. PMID- 8634010 TI - Hypothalamic-pituitary-adrenal axis activity in panic disorder. 24-hour secretion of corticotropin and cortisol. AB - BACKGROUND: Oversecretion of corticotropin-releasing hormone and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis may contribute to pathophysiologic processes in panic disorder, but documentation of HPA axis disturbance in panic has been inconsistent. In the current study we examined HPA axis activity in panic disorder over a full circadian cycle, using frequent blood sampling to provide detailed assessment of corticotropin and cortisol secretion. METHODS: Twenty patients with panic disorder and 12 normal control subjects were studied. Blood samples were drawn every 15 minutes for 24 hours and assayed for corticotropin and cortisol levels. RESULTS: Patients with panic disorder had elevated overnight cortisol secretion and greater amplitude of ultradian secretory episodes. Patients who entered the study through clinical referral channels had greater cortisol secretion than those recruited by advertisements. Patients with panic disorder who had a low frequency of panic attacks had elevated daytime corticotropin levels and elevated corticotropin ultradian amplitude. Patients with a high frequency of attacks had shifted corticotropin circadian cycles. CONCLUSIONS: Patients with panic disorder demonstrate subtle alterations in HPA axis activity, characterized by overnight hypercortisolemia and increased activity in ultradian secretory episodes, but HPA axis alterations in panic are modulated by illness severity and treatment seeking. It remains unclear whether HPA axis dysregulation in panic represents a pathogenic defect within the axis itself. Inconsistencies in prior work may reflect the subtlety of the abnormalities seen, differences in clinical characteristics of patients studied, and the use of different probes and measurement contexts. PMID- 8634011 TI - Mental health problems among homeless mothers: relationship to service use and child mental health problems. AB - BACKGROUND: The purpose of this study was to describe the prevalence of psychological distress and probable lifetime mental disorders among homeless mothers, their use of services, and the relationship between maternal and child mental health problems. METHOD: The study involved a cross-sectional assessment of 110 mothers and 157 children living in homeless shelters in Los Angeles County. RESULTS: The majority (72%) of sheltered homeless mothers reported high current psychological distress or symptoms of a probable lifetime major mental illness or substance abuse. However, few mothers (15%) in need of services received mental health care, and the main point of contact for those with a mental health problem was the general medical sector. Mothers with a probable mental disorder were also significantly more likely to have children with either depression or behavior problems. CONCLUSIONS: Homeless mothers have a high level of unmet need for mental health services. The relationship between maternal and child problems underscores the need for homeless family interventions that promote access to psychiatric care for both generations. PMID- 8634012 TI - Psychiatric diagnosis in child and adolescent suicide. AB - BACKGROUND: The age, sex, and ethnic distribution of adolescents who commit suicide is significantly different from that of the general population. The present study was designed to examine psychiatric risk factors and the relationship between them and demographic variables. METHODS: A case-control, psychologic autopsy study of 120 of 170 consecutive subjects (age, <20 years) who committed suicide and 147 community age-, sex-, and ethnic-matched control subjects who had lived in the Greater New York (NY) area. RESULTS: By using parent informants only, 59% of subjects who committed suicide and 23% of control subjects who met DSM-III criteria for a psychiatric diagnosis, 49% and 26%, respectively, had had symptoms for more than 3 years, and 46% and 29%, respectively, had had previous contact with a mental health professional. Best estimate rates, based on multiple informants for these parameters, for suicides only, were 91%, 52%, and 46%, respectively. Previous attempts and mood disorder were major risks factors for both sexes; substance and/or alcohol abuse was a risk factor for males only. Mood disorder was more common in females, substance and/or alcohol abuse occurred exclusively in males (62% of 18- to 19-year-old suicides). The prevalence of a psychiatric diagnosis and, in particular, substance and/or alcohol abuse increased with age. CONCLUSION: A limited range of diagnoses--most commonly a mood disorder alone or in combination with conduct disorder and/or substance abuse--characterizes most suicides among teenagers. PMID- 8634014 TI - Communication disturbances in schizophrenia and mania. AB - BACKGROUND: A "natural language" measure was developed for classifying type and severity of communication disturbance in the speech of psychotic patients by assessing their linguistic reference performance. METHODS: This measure was applied to speech samples of schizophrenic, manic, and nonpsychiatric subjects, and the groups were compared on levels and types of communication failures. RESULTS: The speech of the schizophrenic and manic subjects contained much higher frequencies of each of six types of communication failures than did the speech of the control subjects. Proportions of the different types of unclarity differed among the diagnostic groups. CONCLUSIONS: This method provides a measure of overall severity of communication disturbance, discriminates the speech of schizophrenic and manic subjects from that of nonpsychiatric subjects, and reflects some differences in distribution of types of communication failure in schizophrenic vs manic patients. The measure may be helpful in elucidating cognitive weaknesses underlying psychotic communication failures. PMID- 8634013 TI - Personality disorders predict onset of Axis I disorders and impaired functioning among homosexual men with and at risk of HIV infection. AB - BACKGROUND: A longitudinal study was conducted to investigate whether personality disorders (PDs) increase risk for the development of future Axis I disorders and serious functional impairment among human immunodeficiency virus (HIV) seropositive and HIV-seronegative homosexual men. METHOD: Baseline assessments of PDs, Axis I disorders and symptoms, and Global Assessments of Functioning were conducted with a community sample of 107 (66 HIV-positive and 41 HIV-negative) homosexual men participating in a longitudinal study with semiannual interviews over 3 years. RESULTS: Logistic regression analysis indicated that PDs predicted onset of subsequent Axis I disorders after controlling for both HIV status and lifetime Axis I history (adjusted odds ratio, 4.31; P=.01; 95% confidence interval, 1.39 to 13.32). Of the 21 participants with PDs, 16 (76%) were subsequently diagnosed with Axis I disorders on at least one occasion. By contrast, only 36 (42%) of the 86 participants without PDs were subsequently diagnosed with Axis I disorders. Further, 33% of the participants with PDs, in comparison with only 8% of those without PDs, were assigned Global Assessments of Functioning scores of 50 or lower, indicating serious impairment during the postbaseline study period (adjusted odds ratio, 5.70; P<.005; 95% confidence interval, 1.66 to 19.53). CONCLUSION: Personality disorders may contribute to increased risk for onset of Axis I disorders and serious impairment among homosexual men regardless of HIV serologic status. PMID- 8634015 TI - Frequency of morphological phage descriptions in 1995. AB - At least 4500 bacterial viruses have been examined in the electron microscope since 1959. About 4400 phages (96%) are tailed and only 162 phages (4%) are cubic, filamentous, or pleomorphic. Phages belong to 12 virus families and occur in about 130 bacterial genera. Phages are listed by morphotypes and host genera. Siphoviridae or phages with long, noncontractile tails include about 60% of tailed phages. PMID- 8634016 TI - Envelope gene sequences of human T-lymphotropic virus type 1 in Taiwan. AB - Three major types of HTLV-1 had been proposed, the Melanesian type, the Zairian type, and the cosmopolitan type, which was further divided into subtypes A, B and C, according to the phylogenetic tree constructed from LTR sequences of current HTLV-isolates. In this study, the envelope gene sequences of HTLV-1 from 9 Taiwanese were analyzed. Based on the phylogenetic tree constructed by unweighted pair group method and the sequence homology analysis by GCG computer programs, the envelope gene sequences of HTLV-1 proviruses from these 9 Taiwanese belonged to subtype A or subtype B of the cosmopolitan type and were closely related to HTLV-1 from Japan. Twelve subtype-specific nucleotide variations were deduced from the comparison of complete or partial envelope gene sequences of 16 HTLV-1 isolates of known subtypes as well as those of 9 Taiwanese. These data provided the basis for subtyping the cosmopolitan type of HTLV-1 by amplification of envelope gene sequences and restriction fragment length polymorphism studies. A more extensive survey based upon this proposal was warranted. PMID- 8634017 TI - An appraisal of the banded and paracrystalline cytoplasmic inclusions induced in cymbidium mosaic potexvirus- and odontoglossum ringspot tobamovirus infected orchid cells using confocal laser scanning microscropy. AB - The cymbidium mosaic potexvirus (CyMV) banded inclusions and the odontoglossum ringspot tobamovirus (ORSV) paracrystalline inclusions were studied in flowers and leaves of nine orchid cultivars using the confocal laser scanning microscope. The inclusions varied in length and width and were mostly located adjacent to the cell walls. Some ORSV inclusions fully extended across entire infected cells. We propose that the CyMV banded inclusion was formed from virus aggregates which aligned as periodical stacked layers, appearing as cross bands. The virus aggregates were folded into flexible inclusions, giving rise to various shapes and forms. The ORSV paracrystalline inclusions were observed as needle-like crystals. The confocal laser scanning microscope is an effective tool for the study of the three-dimensional structures of plant virus induced inclusions. PMID- 8634019 TI - Asymptomatic vaginal herpes simplex virus infections in mice: virology and pathohistology. AB - One of the causes of genital tract infections in humans are herpes simplex virus types 1 and 2 (HSV-1, HSV-2). Although primary and recurrent infections can be clinically apparent and in part very serious, many infections are asymptomatic and result only in temporary genital shedding of virus (recurrences). During our investigations of vaginitis, strain IES of HSV-1 produced an asymptomatic infection. Replication in the murine vaginal (vag.) epithelium as well as antibody formation after vag. infection was comparable to those of survivors after infection with highly virulent strains. Titration of liver, spleen, ovaries, adrenal glands spinal cord, or brain after vag. IES infection revealed no virus, whereas after i.p. infection virus could be demonstrated in many organs examined. Histological examination with a DNA probe (in situ hybridisation), HSV antibodies (immunohistochemistry), and haematoxylin and eosin (HE) staining showed only small focal HSV lesions of the vaginal epithelium in early stages of the infection, never exceeding to the subepithelial tissue. Severe infiltrations and ulcerations after infection with highly virulent strains (17syn +, ER-) could never be demonstrated after IES vag. infection. Identical replication rates of both groups of HSV despite much greater areas of epithelial necrosis with the virulent strains may be explained by the large number of virus inactivating granulocytes induced by the virulent strains, thus inactivating the hypothetical higher virus load. PMID- 8634018 TI - Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene. AB - A 75% region of the F gene (between nucleotides 334 and 1682) of Newcastle disease virus (NDV) RNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). PCR products were cleaved by three restriction endonucleases and the positions of thirty cleavage sites were mapped in more than 200 NDV strains. Restrictions site analysis established six major groups of NDV isolates and unique fingerprints of vaccine strains. Group I comprised lentogenic strains isolated mainly from waterfowl with some from chickens. "Old" (prior to 1960s) North American isolates of varying virulence including lentogenic and mesogenic vaccine strains belonged to group II. Group III included two early isolates from the Far East. Early European strains (Herts 33 and Italien) of the first panzootic (starting in the late 1920s) and their descendants with some modifications were placed into group IV. NDV strains isolated during the second panzootic of chickens (starting in the early 1960s) were classified into two groups. Group V included strains originating in imported psittacines and in epizootics of chickens in the early 1970s. Group V1 comprised strains from the Middle East in the late 1960s and later isolates from Asia and Europe. Pigeon paramyxovirus-1 strains that were responsible for the third panzootic formed a distinct subgroup in group V1. Our grouping of NDV strains has confirmed group differences established by monoclonal antibodies. It is concluded that restriction site analysis of F gene PCR amplicons is a relatively fast, simple and reliable method for the differentiation and identification of NDV strains. PMID- 8634020 TI - The association between PrP and infectivity in scrapie and BSE infected mouse brain. AB - The structure of the scrapie agent remains unknown. However, scrapie infectivity tends to co-sediment with an infection specific fraction of the glycoprotein PrP (PrPSc) under conditions which solubilise the normal form of this protein (PrPc); accordingly, PrP has been proposed as a candidate component of the agent. To investigate this further we have been examining a new scrapie-related murine model in conjunction with established scrapie models. A bovine spongiform encephalopathy (BSE) derived murine model has short incubation periods, high infectivity titre and low amounts of PrP deposited in the brain. A membrane fraction from scrapie/BSE infected brain is solubilised with Sarkosyl at pH > or = 9.0. Most PrP is also solubilised. In models of the disease with little deposition of the PrP in the brain, this solubilisation step is particularly effective in reducing the amounts of PrP sedimented from brain extracts. Gradient centrifugation of the sedimented fraction shows further separation of infectivity and the residual PrP. It is concluded that at least some PrPSc in the brain need not be associated directly with infectious agents but is deposited in brain solely as a pathological product of infection. However, a residual sedimentable fraction contains PrP which may be a component of the agent. PMID- 8634021 TI - Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop. AB - It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1 BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp 120. PMID- 8634022 TI - A structural DNA binding protein of African swine fever virus with similarity to bacterial histone-like proteins. AB - Here we describe an African swine fever virus (ASFV) protein encoded by the open reading frame 5-AR that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like DNA binding proteins, integration host factor, and Bacillus phage SPO1 transcription factor, TF1. The ASFV 5-AR gene was cloned by PCR and expressed in E. coli. Monospecific antiserum prepared to the 5-AR bacterial expression product specifically immunoprecipitated a protein of approximately 11.6 kDa from ASFV infected swine macrophages at late times post infection. Additionally, the 5-AR expression product was strongly recognized by ASFV convalescent pig serum, indicating its antigenicity during natural infection. Cloned p11.6 bound both double and single stranded DNA-cellulose columns. Consistent with a DNA binding function, immunoelectronmicroscopy localized p11.6 to the virion nucleoid, To our knowledge, p11.6 is the first bacterial histone-like DNA-binding protein found in an animal virus or eukaryotic cell system. PMID- 8634023 TI - Nucleotide sequence and expression in E. coli of the complete P4 type VP4 from a G2 serotype human rotavirus. AB - The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 in E. colic has not been achieved. In this study we present successful expression in E. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses. PMID- 8634024 TI - Rapid and sensitive polymerase chain reaction based detection and typing of foot and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses. AB - Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype. A primer set corresponding to highly conserved regions of the 2B sequence was selected. By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments. In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region. All results were confirmed by direct sequencing of the PCR product. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion. Furthermore, a very high sensitivity was achieved (less than one PFU). Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures. PMID- 8634025 TI - Human T-cell lymphotropic virus types I and II infections in patients with leukaemia/lymphoma and in subjects with sexually transmitted diseases in Nigeria. AB - Serological assays that distinguish antibodies to human T-cell lymphotropic virus types I (HTLV-I) and type II (HTLV-II), and polymerase chain reaction (PCR) tests were used to investigate association of these two human retroviruses with several well-defined clinical conditions in Nigeria. We compared the frequency of HTLV-I and HTLV-II infections among patients with lymphopholiferative disorders (n=65), individuals with various sexually transmitted diseases (n=40), patients with genitals candidiasis (n=25) and apparently healthy individuals (n=60). Serological analysis of blood samples from all four groups showed that 10 of the 190 (5.3%) individuals tested were confirmed positive for the presence of antibodies to HTLV-I(6) or HTLV-II(4). Using the PCR technique, specific HTLV-I or HTLV-II sequences were amplified from the genomic DNA of 4 of 6 HTLV-I seropositive and 3 of the 4 HTLV-II seropositive individuals respectively. However, sequences of both viruses were amplified from the genomic DNAs of the remaining 3 seropositive individuals. Since one of the 5 sets of primer pairs [SK110(II)/SK111(II)], which is used for specific identification of HTLV-II did not amplify the target sequence from the genomic DNAs of any of the 4 HTLV-II confirmed seropositive individuals in this study, it suggested sequence diversity of these viruses in Nigeria. The virus-infected individuals identified in this study were one (1.5%) of the 65 patients with leukaemia/lymphoma (HTLV-I), 6 of 40 (15.0%) individuals (HTLV-I = 1 , HTLV-II = 3, HTLV-I/II = 2) with sexually transmitted diseases (STD), one of 25(4.0%) subjects with genital candidiasis for HTLV-I and 2 of 60 (33.3%) healthy individuals (one for HTLV-I and one for HTLV I/II). There was a significant difference (P < 0.025) between the prevalence of HTLV-I/II infections among patients with lymphoma/leukaemia and those who attended STD clinic in Ibadan, Nigeria. This study also suggests that while HTLV I and HTLV-II may be important sexually transmitted viruses, they may not be specific aetiological agents of the common lymphoproliferative disorders in Nigeria. PMID- 8634026 TI - Genes for fowl adenovirus CELO penton base and core polypeptides. AB - A 3.5-kilobase DNA fragment of the fowl adenovirus type 1 (CELO), located between map units 31.1 and 39.4 has been determined. The sequence contains the probable CELO equivalents of the IIIa protein, penton base, pVII and pV core protein genes of human adenovirus (HAV). The CELO penton base and major core protein (analog HAV pVII) were found to consist of 514 (56.8 kDa) and 72 amino acids (8.4 kDa), respectively. PMID- 8634029 TI - Towards a universal virus database - progress in the ICTVdB. PMID- 8634028 TI - Sequence conservation and expression of the gene encoding the outer capsid glycoprotein among human group C rotaviruses of global distribution. AB - Group C rotaviruses have been identified recently from fecal samples of children with diarrhea in the United States. Using reverse transcriptasepolymerase chain reaction and sequence analysis, we sequenced gene 8s encoding VP7 from two U.S. strains (RI-1 and RI-2), and eight other strains isolated from patients on four continents, and compared these with the sequences of four published strains. The gene 8s of the 14 strains were remarkably conserved in size and in predicted primary and secondary structures. When the sequences of the human VP7s were compared with that of the prototype porcine Cowden strain, six regions were found variable in both deduced primary and predicted secondary structures, four of which were predicted to be hydrophilic and might determine serotype specificity. Gene 8 of the human S-1 strain was further characterized by expression in recombinant baculoviruses. The expressed product was immunogenic but failed to elicit neutralizing antibodies. Our sequence analysis indicates that all the human strains characterized to date belong to a single G genotype, which may constitute a single G serotype, pending further antigenic analysis. Whether the human strains and the Cowden strain are the same serotype remains to be determined. PMID- 8634030 TI - Quality assurance at the Swan District Hospital. PMID- 8634027 TI - Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome. AB - Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear. PMID- 8634032 TI - Compliance with ACHS standards in Australian hospitals. Australian Council on Healthcare Standards. PMID- 8634031 TI - Cerebrovascular accident patients: an interdisciplinary/multidisciplinary audit. AB - OBJECTIVES: To develop appropriate standards to assess the intervention with cerebrovascular accident (CVA) patients by allied health professionals; to establish baseline data with which subsequent information collected could be compared. METHOD: Retrospective criteria auditing of hospital files was undertaken to evaluate whether the allied health professionals were meeting the expected clinical standards for patients admitted with a diagnosis of CVA. RESULTS: Written documentation in hospital files did not meet expected standards in all criteria and varied between professions. The data obtained provided a baseline against which future results could be measured. It was expected that subsequent evaluations would provide improved results. All departments agreed that meeting clinical standards was important and it was agreed to repeat the audit in one year and to include some outcome standards using patients' perceptions of service provision. PMID- 8634033 TI - Coagulation tests in the emergency department. PMID- 8634034 TI - A review of antipsychotic drug doses. PMID- 8634035 TI - Achieving quality management through organizational values. AB - By focusing on its mission values, a hospital can enhance the quality of patient care, improve staff morale, promote cost effectiveness and achieve a competitive advantage in the hospital market place. Since 1986, St Vincent's Private Hospital has implemented strategies and formed task forces to clarify and apply its values to the hospital's overall direction and the everyday work setting. This work has proved that a concentration on the hospital's mission can result in quality management and can benefit both the hospital's basic 'spirit' and its bottom line. Eight guiding principles were found to be essential in achieving quality management through organizational values. PMID- 8634036 TI - Starting in the middle: social work and quality assurance. PMID- 8634037 TI - Hilltop Lodge: an evaluation of the first fifteen months of the CADE unit, Tamworth. AB - Hilltop Lodge is a 16-bed unit in the grounds of Tamworth Base Hospital in Northern New South Wales, designed for the care of the confused and demented elderly (CADE). We present the results of an audit of the first 15 months of its operation performed by evaluating the course of 12 long-stay patients admitted to the unit. We found that although disruptive behavioural problems decreased and sociability improved, only minimal changes occurred in self-help skills and confusion levels. The unit also provided respite care for the temporary care of similar patients; there were major advantages and disadvantages noted, particularly in the functioning of the unit. PMID- 8634038 TI - A quality assurance audit of Illawarra PADP Scheme home glucose meters and evaluation of their use by patients. AB - A total of 187 home glucose meters have been issued to Illawarra residents by the Program of Aids for Disabled Patients (PADP) Scheme operating out of Port Kembla District Hospital in a 5-year period ending 31 December 1989. These meters, representing a valuable health resource, have been loaned to patients without audit or follow up. Sixteen per cent of meters were unable to be located and 33.2% could not be located using existing PADP records. Of those meters which could be tested, 16.4% were inaccurate; the recipient's technique was inadequate for accurate results in 36.1% of instances. This survey has revealed a significant loss of health resources in addition to inadequate clinical assessment and supervision. PMID- 8634039 TI - Adult discharge planning and nursing home placement: a study of risk factors for quality assurance. AB - OBJECTIVE: A study of two large groups of patients was conducted in a large general hospital, comparing the admission characteristics of those referred to nursing homes and those returning home, to identify factors on admission that would facilitate more rapid discharge planning and placement. METHOD: Discharge planning at the Royal Brisbane Hospital was analysed over 12 months, comparing the admission characteristics of 191 patients placed in nursing homes with 444 patients returning home. RESULTS: Referral of patients to social workers was delayed and poorly co-ordinated, leading to prolongation of the discharge process or placement for nursing home patients. The data indicate that the process can be expedited by attention to readily recognizable factors already present on admission. PMID- 8634040 TI - Incidence trends in oesophageal and proximal gastric carcinoma in Victoria. AB - BACKGROUND: There is clinical evidence that adenocarcinoma of the lower oesophagus is increasing in the Australian society. The population-based cancer registry of Victoria was used to describe the incidence of adenocarcinoma of the oesophagus and gastric cardia. METHODS: Data were obtained from 1982 to 1991 and were analysed using the Poisson regression techniques. RESULTS: In men, a statistically significant annual increase of 9.5% in oesophageal adenocarcinoma and a non-significant increase of 1.6% in adenocarcinoma of the gastric cardia was observed. These increases were balanced by decreases in other histological types found in the oesophagus in men resulting in little change in the overall rate. No significant trends by age or histological type were observed in women. CONCLUSIONS: There is evidence for a rise in adenocarcinoma in men in Victoria. Possible risk factors include Barrett's oesophagus. PMID- 8634041 TI - Incompletely excised basal cell carcinoma: a management dilemma? AB - BACKGROUND: Reported recurrence rates for incompletely excised basal cell cancers (BCC) vary widely (30-67%), and the destructive potential of recurrent BCC is well known. When surgically treated BCC are reported by the pathologist as incompletely excised the surgeon is placed in the dilemma of whether to perform an immediate wider excision, or to reserve further treatment until there is clinical evidence of recurrence. The aim of the present study is to determine if there are any clinical or morphological features which may help in this management dilemma. METHODS: Middlemore Hospital histology records were reviewed. In 1986, 82 out of a total of 723 BCC excised were reported to be incompletely excised. The management and recurrence rates of the incompletely excised BCC were determined from the patient records and telephone follow up where necessary. The clinical and morphological features were correlated with the recurrence rates, with the aim of developing a management strategy. RESULTS: The overall recurrence rate was 30.0%. Median time to recurrence was 18.5 months (range 1.5-55 months). Neither the margin of incomplete resection (deep, lateral or both margins), the site of tumour, the histological variant, the sex of the patient, nor prior treatment had any discernible effect on recurrence rates. CONCLUSIONS: Observation is an acceptable management option in most situations, as only one third of incompletely excised lesions needed further treatment. Most recurrences occurred early and careful follow up of these patients was indicated for at least 3 years. PMID- 8634042 TI - Initial parathyroid exploration: current trends in Australia. AB - BACKGROUND: An Australia-wide multicentre prospective study was undertaken to audit cases of initial parathyroid exploration between 1 January and 31 December 1992. One hundred and sixty-two cases were audited. The audit was designed to study pre-operative biochemical assessment, localization techniques and the technique of exploration. METHOD: A pro forma audit form was circulated to all Australian surgeons known to perform parathyroid surgery. Eleven responded. RESULTS: Free serum ionized calcium (iCa) was measured in 30% of cases, with the majority of pre-operative calcium assays being performed using total serum calcium (tCa). In 40 cases (29%) pre-operative localization techniques were used. These proved inaccurate in 10 cases. The intra-operative technique of localization with methylene blue infusion was used in 11% of cases. All four glands were biopsied in 11% of cases. No deaths were reported and a postoperative morbidity rate of 3.1% was noted. Eighty-eight per cent of patients were discharged within 5 days of surgery with a third going home within 48 h of neck exploration surgery. Only one patient (0.6%) required re-exploration for persistent hypercalcaemia due to a second adenoma. CONCLUSIONS: The surgical treatment of hyperparathyroidism can be regarded as safe, with minimum morbidity in experienced hands. Pre-operative localization studies in initial parathyroid exploration are not indicated. PMID- 8634043 TI - Pyogenic infection of the sacroiliac joint. AB - BACKGROUND: This review was undertaken to examine a collected series of patients with this uncommon condition. METHOD: Six definite and three possible cases of pyogenic infection of the sacroiliac joint are reported. Their manner of presentation, investigations, management and outcome are discussed in conjunction with a review of the literature. The patients were identified by searching the medical records of three hospitals over a 10 year period and the clinical data were analysed retrospectively. RESULTS: All patients were clinically tender over the involved sacroiliac joint and were experiencing fever. Technetium-99m bone scans demonstrated increased uptake in the sacroiliac joint in all patients, although two scans were initially negative. Plain pelvic radiographs, computed tomography scans and white cell counts were generally unhelpful in initially establishing the diagnosis. Blood cultures were positive in eight cases, and Staphylococcus aureus was isolated. There were four women and five men with an average age of 25 years. Six reported recent respiratory tract or skin infections. Two of these had also reported an episode of minor pelvic trauma in the preceding 2 weeks. A further two patients were intravenous drug users. Treatment was bed rest and antibiotics in all cases and marked initial improvement was seen. The outcome was variable, with a significant proportion reporting discomfort on the affected side after heavy exercise many years after the infection. CONCLUSION: The importance of a thorough clinical assessment and suspicion of the diagnosis of this uncommon illness is emphasized. PMID- 8634044 TI - Spinal epidural abscess: a report of nine cases and the use of intra-operative ultrasonography. AB - BACKGROUND: Spinal epidural abscess is an uncommon and dangerous lesion. Once neurological complications occur the damage is often irreversible. METHODS: The clinical presentation, operative findings, management and follow up of nine cases of spinal epidural abscess were reported. Four patients were diabetic and four others were intravenous drug addicts. The last patient had a history of a protracted stay in an intensive care unit complicated by pneumonia and pleural effusion. Ultrasonography was used intraoperatively to guide and to assess the adequacy of drainage and decompression of the epidural abscess. RESULTS: Multiple level laminectomy was necessary and Staphylococcus aureus was the most common organism cultured. None of the five patients presenting with acute complete paralysis regained neurological function. Two of the four patients with incomplete paralysis were able to walk with an aid. CONCLUSIONS: Spinal epidural abscess usually presents late and the prognosis is generally poor. Ultrasound may be useful in determining the extent of the abscess during operation to drain the collection. PMID- 8634045 TI - Genitoperineal gangrene: experience in Singapore. AB - BACKGROUND: The experience with genitoperineal gangrene at the Department of Colorectal Surgery, Singapore General Hospital is documented. METHODS: The results of 12 patients treated during the 5 year period between 1 January 1990 and 31 January 1995 were studied. There were 10 men and two women. The mean age of the patients was 55.2 (range 37-83) years. RESULTS: Perianal pain and swelling were the commonest presentation. However, three patients were admitted in a toxic state with mental confusion. The gangrene progressed from anorectal sepsis in six patients. Five of these patients had diabetes mellitus. Escherichia coli was the commonest organism cultured. Early diagnosis, immediate resuscitation, antibiotics and early aggressive surgery was our management policy. Applying these principles, all except two patients survived (mortality 17%). One had advanced malignancy and the other had extensive burn injuries. CONCLUSIONS: A high index of suspicion should be maintained in patients with diabetes and with anorectal sepsis. Should genitoperineal gangrene develop, aggressive surgery is often successful. PMID- 8634046 TI - Psychological aspects of chronic low back pain. AB - BACKGROUND: Patients with chronic low back pain present physicians with diagnostic and therapeutic problems. Physical treatments tend to have low success rates and it is postulated that this may be because low back pain can be a manifestation of abnormal illness behaviour. METHODS: A structured prospective study determined the prevalence of somatization in a sample of 131 adult patients with chronic low back pain using the Illness Behaviour Questionnaire (IBQ) and the Modified Somatic Perception Questionnaire (MSPQ). The scores on these psychological questionnaires were compared with the blind interpretation of pain distribution drawings and with the results of a mechanical classification of the patient's symptoms and signs. RESULTS: Fifty-four per cent of patients had four or more (out of five) abnormal illness indicators. The MSPQ values for the group were significantly above the control values in the literature. Thirty-two per cent of pain diagrams were thought to be incompatible with an organic cause when assessed by an orthopaedic surgeon and sixty-two per cent when assessed by a psychiatrist. CONCLUSIONS: Psychosocial factors are dominant in the presentation of chronic low back pain in adults and the disorder is not primarily a musculoskeletal one. PMID- 8634047 TI - Surgery in the conflict region of Sri Lanka. AB - BACKGROUND: Medecins Sans Frontieres provides medical support to Batticaloa, a town on the east coast, which is in the conflict region of Sri Lanka. METHODS: Information on the range of pathology, the services provided and the diseases treated was collected from a 25 day assignment in mid-1995. RESULTS: Services and facilities are limited and the amount of work is extensive, as indicated by the 248 patients who had one or more operations and 814 surgical cases who were admitted. Ten per cent of the work was with conflict-injured patients and the remainder covered a wide range of specialties. Complications were surprisingly few. CONCLUSIONS: The surgical support supplied through Medecins Sans Frontieres gives the people of Batticaloa much needed medical care and allows surgeons the opportunity to have an enormously satisfying experience. PMID- 8634048 TI - P53 tumour suppressor gene expression in hyperparathyroidism. AB - BACKGROUND: Mutations of the p53 tumour suppressor gene lead to the loss of control of normal cellular proliferation and differentiation and have been shown to be associated with the development of malignancy. METHOD: Archival paraffin resection specimens from 86 cases of hyperparathyroidism treated surgically using the rabbit polyclonal CMI antibody were investigated to detect p53 immunoreactivity in these sections. RESULTS: Eighteen of the 86 sections examined (21%) showed nuclear immunoreactivity. No correlation was detected between tumour histology and p53 immunoreactivity (P = 0.45), nor was there any correlation between tumour clonality and immunoreactivity (P = 0.54). Multiple endocrine neoplasia type 1 (MEN 1) status did not correlate with p53 immunoreactivity. A significant correlation between p53 immunoreactivity and preparathyroidectomy calcium levels of > 1.5 mmol/L was detected (P < 0.005) although no correlation was noted between p53 immunoreactivity and higher levels of preparathyroidectomy intact parathyroid hormone (PTH) levels. CONCLUSION: A relationship is postulated between abnormal serum calcium regulation and p53 mutation in hypercalcaemic states associated with hyperparathyroidism. PMID- 8634049 TI - Smooth muscle cell migration and proliferation is enhanced in abdominal aortic aneurysms. AB - BACKGROUND: The aetiology of abdominal aortic aneurysms (AAA) is as yet undetermined. Smooth muscle cells (SMC) have been implicated in the pathogenesis of AAA as a result of their ability to produce elastin degrading proteases. The present study was undertaken to examine AAA SMC and aortic occlusive disease (AOD) SMC in terms of their respective migration and proliferation in vitro, in order to identify intrinsic differences between these cells. METHODS: Five AAA specimens, four AOD and five inferior mesenteric artery (IMA) specimens were established in culture. The cultures were examined for the extent and the rate of SMC outgrowth and proliferation. Cells were counted following trypsinization using a haemocytometer. RESULTS: For the AAA explants, the cellular outgrowths were first seen at 6.7 days, after culture initiation, while the corresponding outgrowth in the AOD group required 8.8 days (P < 0.05) and the IMA group 11.4 days (P < 0.05). AAA cells reached confluency at a mean of 22.4 days while AOD SMC required 28.6 days (P < 0.05) and IMA 31 days (P < 0.05). In the first passage, the time for AAA SMC doubling was 5.3 days compared to 6.2 days for AOD (P < 0.05) and 8.1 days for the IMA group (P < 0.05). Greater than 98% of the cells, in both groups, stained positive to SMC alpha-actin. CONCLUSION: From these data it is clear that there are intrinsic differences in cellular kinetics between SMC from the two disease states, supporting the hypothesis that AAA are not the result of atherosclerosis. PMID- 8634050 TI - Laparoscopic cholecystectomy and previous abdominal surgery: a safe technique. AB - Previous abdominal surgery has been cited as a contraindication to the performance of laparoscopic cholecystectomy. The present paper describes a technique whereby a Hasson cannula was introduced into the right iliac fossa by the open technique, using a method similar to an appendicectomy. The laparoscope was then introduced through this port and the safe introduction of other ports was achieved. Any adhesions were divided at this stage. The laparoscope was then moved to the umbilical port and a conventional laparoscopic cholecystectomy performed. In the series of six patients with upper or upper and lower abdominal scars, no patient suffered a complication or required a conversion to laparotomy. However, the operating time was increased from 75 to 105 min. This method involves open introduction of the primary trocar in an area devoid of adhesions. It was shown to be safe and with this method previous abdominal surgery should not be regarded as a contraindication. PMID- 8634051 TI - A simple and safe technique for drainage tube insertion after laparoscopic cholecystectomy. PMID- 8634052 TI - Port attachment without capacitive coupling. PMID- 8634053 TI - Helmets in surgical history. AB - The relevance of historical experience is evident in a consideration of helmets designed for head protection in war, industry, sport and road transport. Modern helmets are designed to minimize the risk of brain damage by penetration and by blunt impact; where facial or ocular injury is likely, facial protection may be provided by visors, goggles or full-face helmets. The effectiveness of helmets should be monitored by studies of actual injuries; historically, surgeons have done this, in war and peace, for centuries. PMID- 8634054 TI - Ectopic ureter as a cause of wetting: the role of laparoscopy in its management. AB - A diagnosis of wetting caused by an ectopic ureter usually can be made from the history because of the characteristic pattern of wetting. Localization of the origin of the ectopic ureter is important in guiding the surgical approach. This is usually not a problem for cases of ectopic ureter arising from a duplex system. However, the single ectopic ureter arising from a small dysplastic and often ectopic kidney may defy a long search. Videolaparoscopy, with its magnifying effect, can confirm the diagnosis, localize the dysplastic kidney and allow its removal using endoscopic equipment. PMID- 8634055 TI - Melanoma of the lip. AB - Melanoma of the lip is a very rare condition that tends to behave in an aggressive manner. Surgery remains the mainstay of treatment. Five patients with melanoma of the lip are reported, illustrating some of the difficulties that may be encountered in its management. Radical local treatment is necessary to minimize the risk of local recurrence, and flap reconstruction will usually be required to achieve acceptable aesthetic and functional results. PMID- 8634056 TI - Goitre causing acute respiratory arrest. AB - A patient reported symptoms of upper airway obstruction for 4 years. His symptoms worsened with a viral upper respiratory infection and he suffered acute respiratory arrest. He was resuscitated and a large goitre with intrathoracic extension which was compressing the trachea was resected. The mechanisms of upper airway compromise by goitre are discussed. The symptoms of the effect of goitre on the airway are reviewed. PMID- 8634058 TI - Recurrence of osteoclast-like giant cell carcinoma of the pancreas after 10 years. PMID- 8634057 TI - Spontaneous haemothorax caused by an internal mammary artery aneurysm. PMID- 8634059 TI - Rubisco rules fall; gene transfer triumphs. AB - The most common form of the CO2-fixing enzyme rubisco is a form I enzyme, heretofore found universally in oxygenic phototrophs (cyanobacteria and plastids) and widely in proteobacteria. Two groups, however (1-4), now report that in dinoflagellate plastids the usual form I rubisco has been replaced by the distantly related form II enzyme, known previously only from anaerobic proteobacteria. This raises the important question of how such an oxygen sensitive rubisco could function in an aerobic organism. Moreover, the dinoflagellate rubisco has unusual molecular properties: it is encoded as a polyprotein, by nuclear (rather than plastid) genes, and these genes contain noncanonical spliceosomal introns. The nuclear location and alpha-proteobacterial affinity of dinoflagellate rubisco genes hint at a possible mitochondrial origin and highlight the extraordinary richness of lateral gene transfers, both between and within organisms, that have occurred during rubisco evolution. PMID- 8634060 TI - Microsatellite instability and a TGF beta receptor: clues to a growth control pathway. AB - Defects of growth inhibitory pathways have an important role in disorders of cell growth and differentiation. The discovery of a mutation in one of the principle components of the transforming growth factor beta (TGF beta) receptor system which is linked to a DNA repair defect(1) represents one possible mechanism of escape from negative regulatory influences acting upon cells. TGF beta is a pre eminent negative growth factor and this article discusses (1) the role of TGF beta in maintaining epithelial homeostasis; (2) how breakdown of inhibitory pathways can promote neoplastic development; (3) the significance of a receptor defect in a negative signalling pathway; and (4) the potential therapeutic sequelae resulting from restoration of cellular responsiveness to TGF beta. PMID- 8634061 TI - Histone H4, the cell cycle and a question of integrity. AB - The N-terminal domain of histone H4 has been implicated in various nuclear functions, including gene silencing and activation and replication-linked chromatin assembly. Many of these have been identified by using h4 mutants in the yeast S. cerevisiae. In a recent paper, Megee et al. use this approach to show that mutants in which all four N-terminal H4 lysines are substituted with glutamines accumulate increased levels of DNA damage. A single lysine, but not an arginine, anywhere in the N-terminal domain suppresses this phenotype. It is suggested that histone H4 plays a role in maintaining genome integrity through the cell cycle, possibly by a mechanism involving lysine acetylation. PMID- 8634062 TI - Melanoma formation in Xiphophorus: a model system for the role of receptor tyrosine kinases in tumorigenesis. AB - Cancer is one of the most frequent fatal human diseases. It is a genetic disease, and molecular analysis of the genes involved revealed that they belong to several distinct classes of molecules, one of which is the receptor tyrosine kinases. Neoplastic transformation is regarded as the result of a multistep process and, in most cases, it is hard to evaluate what the initial events in tumor formation are. What makes it difficult to approach this question is the paucity of animals models for tumorigenesis allowing investigation of the mechanisms leading to uncontrolled cell proliferation. Melanoma formation in Xiphophorus is one of these model systems. Here, overexpression and activation of a receptor tyrosine kinase causes neoplastic transformation of pigment cells. Xiphophorus provides all the advantages of a well-characterized genetic system. In addition, some crucial components of the transformation pathway have been identified at the molecular level. As a vertebrate, Xiphophorus might serve as a model system to aid understanding, in more general terms, of the mechanisms of tumorigenesis in human diseases. PMID- 8634063 TI - DNA packaging and cutting by phage terminases: control in phage T4 by a synaptic mechanism. AB - Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a 'packasome'. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation. PMID- 8634064 TI - Inflammation, reproduction, cancer and all that.... The regulation and role of the inducible prostaglandin synthase. AB - Discovery of a second, inducible prostaglandin synthase provides explanations for many previously puzzling observations, but also raises new questions about prostanoid synthesis. A cis-acting sequence closely related to the cyclic AMP response element has been shown to play a role in both basal and induced prostaglandin synthase 2 gene expression. Aspirin and other currently available non-steroidal anti-inflammatory drugs that inhibit prostaglandin synthase activity do not effectively discriminate between the inducible prostaglandin synthase 2 and constitutive prostaglandin synthase 1 enzymes. Identification of a second prostaglandin synthase, induced by inflammatory stimuli, initiated a search for isoform-specific inhibitors. Use of prostaglandin synthase 2 specific inhibitors and antisense oligonucleotides has led to the suggestion that specific ligands activate alternative pathways of prostanoid production, using one of the prostaglandin synthase isoforms preferentially. The coupling mechanisms by which these pathways are activated in response to alternative stimuli should provide additional routes of intervention in prostanoid production. PMID- 8634066 TI - Developmental regulation of alpha beta T cell antigen receptor assembly in immature CD4+CD8+ thymocytes. AB - Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic alpha beta chains associated with invariant CD3- gamma delta epsilon components and sigma chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of alpha beta TCR complexes on their surfaces expressed by mature CD4+ and CD8+ T cells. Recent evidence demonstrates that low surface TCR density on CD4+CD8+ thymocytes results from the limited survival of a single TCR component within the ER, the TCR alpha chain, which as a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCR alpha proteins in immature CD4+CD8+ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of alpha beta TCR complexes in CD4+CD8+ thymocytes and comment on the functional significance of TCR alpha instability during T cell development. PMID- 8634065 TI - The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules. AB - Chondrocytes are specialised cells which produce and maintain the extracellular matrix of cartilage, a tissue that is resilient and pliant. In vivo, it has to withstand very high compressive loads, and that is explicable in terms of the physico-chemical properties of cartilage-specific macromolecules and with the movement of water and ions within the matrix. The functions of the cartilage specific collagens, aggrecan (a hydrophilic proteoglycan) and hyaluronan are discussed within this context. The structures of cartilage collagens and proteoglycans and their genes are known and a number of informative mutations have been identified. In particular, collagen fibrillogenesis is a complex process which can be altered by mutations whose effects fit what is known about collagen molecular structural functions. In other instances, mutations have indicated new functions for particular molecular domains. As cartilage provides the template for the developing skeleton, mutations in genes for cartilage specific proteins often produce developmental abnormalities. The search for mutations amongst such genes in heritable disorders is being actively pursued by many groups, although mutation and phenotype are not always well correlated, probably because of compensatory mechanisms. The special nature of the chondrocyte is stressed in connection with its cell involvement in osteoarthritis, the most widespread disease of diarthrodial joints. PMID- 8634067 TI - The therapeutic potential of antisense oligonucleotides. AB - Specific inhibition of gene expression by antisense agents provides the basis for rational drug discovery based on molecular targets. Due to the specificity of Watson-Crick base-pair hybridization, antisense oligodeoxynucleotides have been used extensively in attempts to inhibit gene expression in both in vitro and in vivo models. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials against diseases including AIDS and cancer. Although the precise mechanism of action of these drugs has not been clarified, these oligodeoxynucleotides offer considerable promise as novel molecular therapeutics. We review the recent attempts to harness the therapeutic potential of these oligodeoxynucleotides and appraise the near-term prospects for antisense technology. PMID- 8634068 TI - Transformations in null mutants of Hox genes: do they represent intercalary regenerates? AB - In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity. PMID- 8634069 TI - Gamete and immune cell recognition revisited. AB - Fertilization is the result of a series of successful recognition and binding events mediated by gamete surface molecules. Recent advances in the identification and characterization of some of these recognition molecules provide extremely valuable information necessary to understand sperm-egg recognition and subsequent egg activation. We discuss these new data in the context of the model of gamete recognition first proposed by F.R. Lillie in the early part of the 20th century, and revisited periodically in the subsequent literature, which relates fertilization events to those of immune cell recognition and activation events. Here we discuss the principles underlying the molecular recognition and activation mechanisms of gametes and immune cells. PMID- 8634070 TI - Surrogate cells or Trojan horses. The discovery of liposomes. AB - An autobiographical account of the liposome, from the perplexities of a blood smear to the growth of a multi-million pound business. PMID- 8634071 TI - Distalization in insects and amphibians. PMID- 8634073 TI - Synthesis and local anesthetic activity of two homological series of diastereomeric phenylcarbamates. AB - By using stereospecific reactions we prepared two homological series of diastereomeric + cis- and + trans-N,N-dimethyl-2-(2- alkoxyphenylcarbamoyloxy)cyclohexylmethyl-ammonium chlorides with number of carbons in the alkoxy group varying from one to eight. Structure of the prepared compounds was proved by NMR and IR spectroscopy. The principal physico-chemical characteristics, including partition coefficients, were obtained and relative local anesthetic activity was measured using a surface in vivo model. It was found that (1) for both cis- and trans-isomers there is a parabolic relationship between log activity and molecular lipophilicity (as measured by TLC RM and log P values), (2) all drugs prepared for this study use hydrophobic pathway to block inactivated channels, and (3) there is no strict requirement for precise disposition of the functional groups responsible for receptor binding, probably due to conformational flexibility of the sodium channel protein. PMID- 8634072 TI - Second messenger systems as targets for new therapeutic agents: focus on selective phosphodiesterase inhibitors. AB - Second messengers are intracellular substances whose concentration is regulated by the action on their specific receptors of extracellular regulatory molecules globally known as first messengers. In particular, the cyclic nucleotides cAMP and cGMP help in the coordination of cellular functions, mainly through their stimulatory effect on multisubstrate protein kinases. The pharmacological modulation of cyclic nucleotide levels has been classically attained by acting on the first messenger receptors. However, the possibility exists that an intervention on the enzymes responsible for the synthesis or degradation of such second messengers could yield similar effects. The enzymes involved in the degradation of cAMP and cGMP, the so-called cyclic nucleotide phosphodiesterases (PDEs), are a group of at least 7 enzyme families that share the property of hydrolysing the cyclic nucleotides to their corresponding 5-monophosphate counterparts. At the present time, most of the interest on the potential utility of selective PDE inhibitors is focused on drugs capable of inhibiting PDE IV, one of the cAMP specific phosphodiesterases, because the tissue distribution of PDE IV strongly suggests that pathologies related to the central nervous and immune systems, could be treated in this way. PDE IV inhibitors can be divided into three structural classes: 1) structural analogues of rolipram, 2) structural analogues of nitraquazone and 3) structures related to the xanthine nucleus, such as denbufylline. An overview of the structure-activity relationships for these classes of compounds and a summary of their possible therapeutic potential will be given. PMID- 8634074 TI - Isolation, characterization and pharmacological activity of Magydaris pastinacea (Lam) Paol. glucosides. AB - The glycoside fraction of fresh rhizomes from Magydaris pastinacea (Lam.) Paol. was separated from the alcoholic extract using the method of Kobayashi et al. The fraction was found to have six main constituents, the most abundant of which had previously been isolated and identified as 7-O-beta-D-glucopyranosyl-8-(2',3' dihydroxy-3-methyl-butylcoumarin++ +). The present paper describes the separation and characterization of the other constituents, all with coumarin or furancoumarin structures. Pharmacological experiments to test these compounds as platelet antiaggregants are also reported. PMID- 8634075 TI - Polycondensed nitrogen heterocycles. Part 27. Indolo[3,2-c]cinnoline. Synthesis and antileukemic activity. AB - Indolo[3,2-c]cinnolines of type 5, variously substituted either in the indole and in the cinnoline moieties, were prepared in good overall yields, by intramolecular cyclization of indolo derivatives 4. Compounds 5a-d showed a good cytotoxic activity against FLC and K562 leukemic cell lines, both sensitive and multi-drug resistant. PMID- 8634076 TI - N-substituted-2-oxo-(2H)1-benzopyran-3-carboxamide derivatives with analgesic and/or diuretic activities. AB - The synthesis of N-substituted-2-oxo-(2H)1-Benzopyran-3-Carboxamide derivatives starting from semicarbazones or thiosemicarbazones and Carbon Suboxide (ratio 1:2) is described. Some compounds showed an interesting analgesic and/or diuretic activity in mice. PMID- 8634077 TI - Synthesis and anticonvulsant activity of new acylthiosemicarbazides and thiazolidones. AB - A number of 1-(3-phenyl-4(3H)-quinazolinone-2-ylmercaptoacetyl)-4-substituted thiosemicarbazide and 2-(3-phenyl-4(3H)-quinazolinone-2 ylmercaptoacetylhydrazono)-3-sub stituted 4-thiazolidone derivatives were synthesized and evaluated for anticonvulsant activity. Some of the tested compounds showed significant activity against MES and ScMet induced seizures. PMID- 8634078 TI - Preparation and characterisation of polyphosphazene-based controlled release systems for naproxen. AB - A new polyphosphazene polymer suitable for preparation of naproxen controlled release systems has been prepared by non complete substitution of the chlorine at the phosphorus atoms of polydichlorophosphazene with phenylalanine ethyl ester and imidazole. Polymeric disks obtained by solvent evaporation were found to maintain their integrity for months either "in vivo" and "in vitro" after incubation in buffer, although the intrinsic viscosity undergoes a rapid decrease. The release rate of naproxen from the films was found to depend on thickness of the matrixes and amount of entrapped drug. "In vitro" release spikes, corresponding to initial in plasma burst after subcutaneous implantation of the films in rats, have been observed with high concentrations of drug in the matrixes. On the other end decreasing the drug concentrations in the polymer matrixes, the naproxen release approximates a zero order kinetic and a controlled release is obtained for weeks. Scanning electron microscopy showed that the disks giving spikes in drug release are characterised by the presence of naproxen crystals on the matrix surface. "In vivo" studies are demonstrating that the initial naproxen concentration spike is useful in the treatment of acute models of inflammation, while a more constant and lasting release is successful in chronic inflammation models. The degradation profile of this new polyphosphazene as well as preliminary pharmacological data are also reported. PMID- 8634079 TI - Retention behaviour of anti-emetic serotonin antagonists in reversed phase high performance liquid chromatography. AB - Granisetron, Ondansetron and Tropisetron, three 5-HT3 antagonists showing anti emetic activity, were analysed by HPLC on lipophilic stationary phases. The addition of an amine or quaternary ammonium salt to the eluents was a powerful tool in the analysis of these basic substances. The influence on chromatographic parameters of pH, ionic strength and various counter-ions in the aqueous phase as well as of different organic modifiers is discussed. Some of the proposed experimental conditions allow a more strictly partition-based separation mechanism and can produce chromatographic parameters suitable for structure activity studies. These experimental conditions are also suitable for analysis of the considered compounds in pharmaceutical dosage forms or in biological fluids. PMID- 8634080 TI - Chromonyl-aminosalicylic acid derivatives as possible antimycobacterial agents. AB - N-(2H,3H,4H-4-oxo-6-R1-2-R2O-benzopyran-3-ylidenmethyl) derivatives and imino derivatives of 3-, 4-, 5-aminosalicylic acids were prepared. It was found that some of the synthesized derivatives of 4-aminosalicylic acid are as effective against typical and atypical strains of mycobacteria as isoniazid (INH). Interesting activity of some derivatives against yeast was also found. PMID- 8634081 TI - Synthesis and antimicrobial activity of some 2-(2-oxobenzothiazole-3-yl)-1 arylethanone derivatives. AB - The synthesis of new 2-(2-oxobenzothiazole-3-yl)-1-arylethanone derivatives is reported. The structure of these products is supported by their UV, IR and 1H-NMR spectra, as well as by elemental analysis. The new compounds were tested for antimicrobial activity. PMID- 8634082 TI - A possible role in chemical carcinogenesis for epigenetic, heritable changes in gene expression. AB - Although genetic changes are clearly important in the initiation of carcinogenesis, there is reason to think that epigenetic changes may also play a role in the process. A key feature of carcinogenesis is the long latency between exposure to carcinogenic insults and the appearance of malignancy. Thus, if epigenetic changes are to be involved, they must somehow be inherited at each cell division without the continued presence of the carcinogen. I propose that self-perpetuating changes in patterns of gene expression are a plausible mechanism for an epigenetic component of carcinogenesis. Networks of transcription factors that regulate each other's and their own expression are known to control important developmental processes, particularly the determination of entire cell lineages. An inherent property of many such autoregulatory networks is the existence of two very distinct, stable steady states, defined in terms of the concentration of each transcription factor in the network. In this report, I present a model in which an acute carcinogen exposure is postulated to shift such a network from one steady-state to the other, effectively turning on or off the expression of at least one of the genes. Because of the autoregulatory nature of the network, this new steady-state is stably inherited at each cell division. Such changes in gene expression may ultimately contribute to the malignant phenotype if the regulatory network affects genes important in cell-cycle checkpoints, maintenance of genome stability, signal transduction, or other processes that are altered in tumor cells. PMID- 8634083 TI - Alteration of gene expression in rat mammary tumors induced by N-methyl-N nitrosourea. AB - Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU) induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL 60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias. PMID- 8634084 TI - Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene. AB - The role of the hepatitis B virus (HBV) X protein in liver tumorigenesis is unresolved. Transgenic mice harboring the X gene (nt 1376-1840 under the control of the human alpha-1-antitrypsin regulatory elements) (ATX mice) display only minor histopathologic alterations of the liver. To determine if ATX mice are more susceptible to the effects of hepatocarcinogens, 12- to 15-d-old male ATX and control littermate mice were injected with a single dose (2 microgram/g body weight) of diethylnitrosamine (DEN). The animals were killed 6-10 mo after exposure and were analyzed for histological changes in the liver. One hundred percent of the DEN-treated AXT mice developed abnormal liver lesions. Then their liver tissues were compared by stereological analysis with those of non transgenic animals, the ATX mice had a relative twofold increase in the total number of focal lesion and a twofold increase in the incidence of hepatocellular carcinoma. Elevated levels of X protein and p53 protein were not detected in carcinogen-induced nodules or tumors. These results are consistent with a model in which the expression of the HBV X protein potentiates the induction of DEN mediated liver disease. PMID- 8634085 TI - Complete sequence analysis of a gene (OS-9) ubiquitously expressed in human tissues and amplified in sarcomas. AB - Amplification and overexpression of genes involved in cellular growth control occur frequently in human tumors. Using a chromosome microdissection-based hybrid selection strategy, we recently identified two novel genes (OS-9 and OS-4) within 12q13-15, a region frequently amplified in human cancers. We now report further characterization of the full-length OS-9 cDNA sequence consists of 2785 bp from which an open reading frame (ORF) with 667 amino-acid residues was deduced, The predicted polypeptide was water soluble and acidic. We also demonstrate that the OS-9 gene encoded a 2.8-kb mRNA transcribed in all 16 human tissues examined, suggesting that OS-9 is ubiquitously expressed in human tissues. OS-9 was co amplified with CDK4 in three of five sarcoma tissues. Homology analysis of the amino-acid sequence reveals significant similarities between OS-9 and two ORFs deduced from genomic sequences in Caenorhabditis elegans and Saccharomyces cerevisiae. The region of similarity extended over 200 residues (approximately one-third of each ORF), and eight cysteines were conserved in all three ORFs. These observations suggest that this region comprises a functional domain present in a novel evolutionarily conserved gene family defined by OS-9. PMID- 8634086 TI - Frequent mutations of Ki-ras but no mutations of Ha-ras and p53 in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats. AB - Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20-27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G-->A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogeneses and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. PMID- 8634087 TI - Metastasis suppressed, but tumorigenicity and local invasiveness unaffected, in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6. AB - Progression of human melanoma toward increasing malignant behavior is associated with several nonrandom chromosomal aberrations, most commonly involving chromosomes 1, 6, 7, 9, and 10. We previously showed that introduction of human chromosome 6 into the highly metastatic human malignant melanoma cell line C8161 completely suppressed metastasis without altering tumorigenicity (Welch DR, Chen P, Miele ME, et al., Oncogene 9:255-262, 1994). Alterations of chromosome 1 are the most frequent chromosome abnormality observed in melanomas, and they frequently arise late in tumor progression. The purpose of the study presented here was to compare the effects of chromosomes 1 and 6 on malignant melanoma metastasis. By using microcell-mediated chromosome transfer, single copies of neo tagged human chromosomes 1 or 6 were introduced into the human melanoma cell line MelJuSo. The presence of the added chromosome was verified by G banding of karyotypes, fluorescence in situ hybridization, and screening for polymorphic markers on each chromosome. The incidence and number of metastases per lung after intravenous or intradermal injection of parental MelJuSo cells was significantly (P<0.01) greater than those of hybrids containing either chromosome 1 or chromosome 6, although chromosome 1 was a less potent inhibitor of metastasis than chromosome 6. Cultures established from primary tumors and metastases remained neomycin resistant, suggesting that portions of the added chromosomes were retained. These results strengthen the evidence for the presence of a melanoma metastasis suppressor gene on chromosome 6. neo6/MelJuSo hybrids expressed 2.4- to 3.4-fold more of the melanoma differentiation-associated gene mda-6 (previously shown to be identical to WAF1/CIP1/Sdi1/CAP20) than parental metastatic cells. mda-6/WAF1 is among the candidate genes on chromosome 6. These results also demonstrate, for the first time, the existence of metastasis suppressor genes on human chromosome 1, although these genes appear to be less potent than the one encoded on chromosome 6. PMID- 8634088 TI - Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells. AB - The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene-transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c-Ha-ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung modules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v-src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel-coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors. PMID- 8634089 TI - Telomerase activity of normal tissues and neoplasms in rat colon carcinogenesis induced by methylazoxymethanol acetate and its difference from that of human colonic tissues. AB - Telomerase activity in tissues may be related to tumor development, especially malignant conversion, in humans. However, there are few reports about telomeres and telomerase activity in animals. In this study, we examined telomerase activity in rat colon carcinogenesis and in normal rat liver tissue and compared it with that of human colon cancer tissues. This is the first report concerning telomerase activity in rats. F344 rats were used, and colon neoplasms were induced with methylazoxymethanol acetate. There was telomerase activity in not only the induced colon neoplasms but also the colon mucosa and livers of untreated rats, in contrast with the results from normal human somatic tissues in previous reports. Indeed, we also observed negative results in normal human mucosa, despite the positive results in colon-cancer tissues. These findings suggest that there is a difference in the telomerase activities in humans and rats. Because rat telomeres are very long (20-100 kp, average 50 kp) compared with human telomeres (5-15 kp, average 12 kp), the difference in the telomere lengths of rats and humans might be related to their enzyme activities, although this is still unclear. Furthermore, because the inhibition of telomerase has been proposed as a novel cancer therapy for humans, the rat model presented here, in which telomerase is expressed in somatic tissues, may be useful for studies of telomerase inhibition, including inhibition by chemopreventive agents. PMID- 8634090 TI - Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester. AB - Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype. PMID- 8634091 TI - Induction of p53 and p21/WAF1/CIP1 expression by nitric oxide and their association with apoptosis in human cancer cells. AB - In this study, human and rat cancer cells were used to investigate the expression of p53 and p21/WAF1/CIP1 and their association with apoptosis after exposure to nitric oxide (NO). It was found that NO induced nuclear accumulation of p53 protein in a dose- and time-dependent manner. The level of p53 protein was elevated by about fivefold compared with that of mock-treated cells 48 h after exposure to 300 ppm NO. The induction of p53 by NO was found by pulse-chase analysis to be mainly regulated by post-translational modification. The correlation between p53 status and apoptosis induced by NO in human cancer cells was also investigated in this study. We found that apoptosis was easily induced in cells containing wild-type p53 (COLO 205 and Hep G2) after exposure to NO. The p21/WAF1/CIP1 protein was induced by NO in cells containing wild-type p53 (Hep G2) but not in cells without p53 (Hep 3B) or with mutated p53 (HT-29). Our results indicate that wild-type p53 and p21/WAF1/CIP1 expression was elevated in human cancer cells by exposure to NO and suggest that this may eventually promote apoptosis. PMID- 8634092 TI - Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells. AB - Gene-specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene-trans-7,8-dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the beta-globin gene (which is transcriptionally inactive) was measured in cells treated with [3H](+/ )-anti-BPDE, [3H](+/-)-BPD, and [3H](-)-BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily detected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the beta-globin gene (exons 1-2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and beta-globin genes showed a dose response to [3H](-)-BPD. The frequency of BPD-induced lesions in the p53 gene was sixfold to sevenfold greater than in the beta-globin gene and 200- to 300-fold greater than in bulk DNA. The BPD-induced lesion frequency in the beta-globin gene was 30- to 50-fold greater than in bulk DNA. The data indicate that the distribution of BPDE induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate. PMID- 8634093 TI - Interference of benzo[a]pyrene diol epoxide-deoxyguanosine adducts in a GC box with binding of the transcription factor Sp1. AB - Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high affinity binding of Sp1 to non-Gc box sites. PMID- 8634094 TI - Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4. AB - The function of p16INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16INK4 independent of Rb. The transfected p16INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD 1),p16INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16INK4 or expressed reduced amounts of p16INK4 protein. Therefore, p16INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16INK4 is a tumor suppressor gene. PMID- 8634095 TI - Coordinate control of growth and cytokeratin 13 expression by retinoic acid. AB - Retinoic acid (RA) modulates the growth and differentiation of various normal and malignant cells. These effects are most likely mediated by changes in gene expression. Genes whose expression is modulated by RA may be useful as markers of growth responsiveness to retinoids. Using differential cDNA cloning we identified 10 genes regulated by RA in the head and neck squamous cell carcinoma cell line MDA886Ln. Keratin (K) 13 gene expression was the gene expression most related to the degree of sensitivity of growth to RA, as K13 was not expressed in a series of RA-resistant cell lines. Our data suggest that low K13 expression may be mechanistically related to resistance to RA-induced growth inhibition. PMID- 8634096 TI - The medicalization of breastfeeding. PMID- 8634097 TI - Retained placenta and suppressed lactogenesis? PMID- 8634098 TI - Global participatory action research (GLOPAR) PMID- 8634099 TI - Two more indications of Candida. PMID- 8634100 TI - Sexuality and breastfeeding. PMID- 8634101 TI - Cabbage leaf enzymes. PMID- 8634102 TI - Health professionals and breastfeeding counseling: client and provider views. AB - Health care providers are the most influential and trusted source of information about breastfeeding, yet many are neither prepared nor able to provide good breastfeeding counseling to their clients. This paper reports findings on low income mothers' and on providers' perceptions of professional breastfeeding counseling. Data collection included focus group discussions with mothers recruited from public health department clinics in the Southeast USA and who were stratified by age, parity, rural/urban residence and feeding method, and focus groups and individual interviews were conducted with health care providers from the same geographic area. The results of the study indicate a gap between the promotion and support processes for breastfeeding, and point to areas where breastfeeding counseling can be strengthened. PMID- 8634104 TI - Peer counselor program increases breastfeeding rates in Utah Native American WIC population. AB - Lack of breastfeeding promotion and support hinder successful breastfeeding. In this study, a breastfeeding peer counselor program improved both the initiation rate and duration of breastfeeding up to three months postpartum among Native American WIC participants. Trained peer counselors contacted subjects prenatally, and at one, two, and four to six weeks postpartum. Breastfeeding rates for the experimental group were compared to historical controls. Women in the peer counselor group who had complete data for three months (n = 41) had a higher rate of breastfeeding than the control group (n = 67) at initiation (84 percent vs. 70 percent; p = 0.05) and at three months postpartum (49 percent vs. 36 percent; p = 0.08). PMID- 8634103 TI - Working women, maternity entitlements, and breastfeeding: a report from Bangladesh. AB - Early infant feeding practices and facilities available for supporting breastfeeding in workplaces in Dhaka, Bangladesh were studied through interviews with working women (n = 238) with children younger than 30 months of age. Of the women interviewed, 20 percent were aware of the benefits of, and had exclusively breastfed in the first month, 13 percent in the second month, and two percent in the fifth month of employment. The median age of starting complementary feeds was 41 days (range, 1-210) preparatory to resuming work. Total duration of breastfeeding was significantly shorter in mothers who had started these feeds before 41 days of age as compared to those who started later (mean +/- SD) 275 +/ 216 days versus 361 +/- 223 days (p = 0.003). Ninety-nine percent of the mothers were unaware of their maternity entitlements, and only 20 percent had taken breaks for breastfeeding, those breaks being treated as "unofficial." Working women should be informed through health personnel and communication media, about the benefits of exclusive breastfeeding and about maternity entitlements. Provision of facilities to support breastfeeding in the workplace must also be encouraged so that maternal employment does not hamper breastfeeding. PMID- 8634105 TI - Telling the world: low income women and their breastfeeding experiences. AB - This study describes the experiences of 17 educated, low-income, culturally diverse women (10 African-American and seven Latin) who were supported by peer counselors/breastfeeding advocates. In-depth interview data were analyzed, using constant comparative analysis. Study findings indicated that the perception of successful breastfeeding can have an empowering effect on women when support for their endeavors are gender- and culturally appropriate. Five themes emerged as primary descriptors of the experience: Making the Discovery, Seeking a Connection, Comforting Each Other, Becoming Empowered, and Telling the World. The final theme best illustrates a substantive theory derived from the data. PMID- 8634106 TI - Breastfeeding in children's books: reflecting and shaping our values. AB - Children's books reflect and reinforce our society's message that bottlefeeding is the normal way to feed human babies. Even books that include breastfeeding may unwittingly undermine their intended message of support. It is important for lactation consultants to be aware of and help to promote those books that show or describe breastfeeding in a positive light. No book is perfect, and no one book will fit all needs, but a number of helpful books that include breastfeeding are available today. We can help to bring them to the attention of parents and children and to encourage their use in libraries, schools, clinics, childbirth classes, and similar settings. PMID- 8634107 TI - A score sheet for evaluating breastfeeding educational materials. AB - Lactation consultants and other professionals are frequently asked to review or recommend a video, pamphlet, or teaching aid for a specific audience. This article lists positive features that support the art and science of breastfeeding, and identifies errors and concepts detrimental to the course of breastfeeding. Six central messages are detailed: milk supply, comfort during breastfeeding, health of baby and mother, social support, presentation of the message, and packaging, including the financial motivation of the author. PMID- 8634108 TI - Breastfeeding difficulties as a result of tight lingual and labial frena: a case report. AB - A breastfed baby with impending failure to thrive resisted assuming a wide mouthed, flanged-lip position at breast. A lingual frenotomy was only partly successful in correcting his breastfeeding problems. Following a labial frenectomy, the baby was better able to flange his upper lip against the breast. He then began breastfeeding with full efficiency. PMID- 8634109 TI - Minimizing alcohol exposure of the breastfeeding infant. AB - What are the effects of alcohol consumed by breastfeeding women on their nursing infants? The woman's weight, whether she is amenorrheic during lactation, and the age of her baby are important considerations that influence blood alcohol concentrations and the effect of a mother's alcohol consumption. PMID- 8634111 TI - Storage containers for human milk: an issue revisited. PMID- 8634110 TI - Alcohol and breastfeeding. PMID- 8634113 TI - Adventures with IBLCE. PMID- 8634112 TI - Time was...1849. Obstetrics: the science and the art. PMID- 8634114 TI - Early effects of smoke inhalation on alveolar macrophage functions. AB - Alveolar macrophage (AM) dysfunctions have been implicated in the pathogenesis of smoke inhalation lung injury. We investigated the early (within 70 min) effects of smoke inhalation on AM. The cells were recovered by bronchoalveolar lavage from rabbits ventilated with cotton smoke for 5 min followed by O2/room air for 60 min (smoke-exposed) or with room air in place of smoke (control). Smoke injury caused arterial blood carboxyhaemoglobin levels to increase 11-fold and reduced arterial blood PO2 (measured approximately 1 h postinjury) by 25 per cent. Scanning electron micrographs revealed denudation of plasmalemmal pseudopods in smoke-exposed AM. Smoke exposure suppressed both AM adherence to plastic and phagocytosis of opsonized bacteria. Basal superoxide (O2-) production was elevated in smoke-exposed AM, compared with controls, whereas PMA-stimulated O2- production was unaffected. Smoke-exposed AM had reduced basal secretion of tumour necrosis factor-alpha (TNF-alpha), but displayed a greater TNF response to stimulation with LPS than did control cells. LPS-stimulated TNF-alpha releases from control and smoke-exposed AM were suppressed by phosphodiesterase inhibitors pentoxifylline and theophylline, and were enhanced by the lipoxygenase inhibitor, MK886. The early responses of AM to smoke inhalation lung injury are consistent with activation of O2- production and priming of TNF-alpha release, concurrent with a functional down regulation of phagocytosis. PMID- 8634115 TI - Stimulation of human keratinocyte proliferation through growth factor exchanges with dermal fibroblasts in vitro. AB - Progress in biotechnology has led to new therapeutic approaches in various fields of human health care, such as the autologous grafting of cultured epidermal cell sheets on burned patients. These cultures depend on various parameters but growth factors are of paramount importance. Cutaneous cells are known to secrete various growth factors in vivo, although only a few have been identified. The aim of this study was to determine if such factors are secreted from human cutaneous cells in culture, to evaluate their effects on epidermal cell proliferation in vitro and to analyse them on SDS-PAGE. Human skin fibroblasts and keratinocytes were co cultured for 8-10 days using a Costar trans-filter system. Dermo-epidermal cooperation was observed in such a co-culture system through the exchange of secretion products in the culture medium. Epidermal cell growth and metabolic activities were highly stimulated in co-culture (2-fold and 1.5-fold, respectively, P < 0.02) compared to the control. The de novo synthesis of secretion products, notably of a protein of about 40 kDa, was specifically induced in co-culture. The identification of new keratinocyte growth factors could accelerate graftable epidermal sheet production in vitro for human wound coverage and possibly enhance wound healing in vivo. PMID- 8634116 TI - Morphology of glycerol-preserved human cadaver skin. AB - Donor allograft skin preserved in 85 per cent glycerol has been used successfully as a temporary coverage for large burn wounds. The glycerol preservation is a method with low costs and has practical advantages such as antibacterial and virucidal effects. This report shows that the glycerol treatment did not affect the fundamental structural integrity of the skin. Intact keratinocytes and Langerhans cells with their characteristic Birbeck granules were still present in the glycerol-treated skin. After treatment with glycerol, the cells in the prepared epidermal cell suspensions were non-viable. MHC class II positive and CD1a positive cells could still be identified in situ and in the suspension. PMID- 8634117 TI - Magnetic resonance imaging: a new diagnostic aid in the care of high-voltage electrical burns. AB - Magnetic resonance imaging (MRI) can detect and delineate alterations in the hydration properties of tissues such as oedema and necrosis. The distinction between living tissue oedema and frank necrosis is also possible with MRI, by use of a spin-echo (SE) sequence and a fast spin-echo (FSE) sequence with a 1.5 T imager. With this background, the aim of this study was to examine the ability of MRI for early detection of concealed tissue injuries caused by high-voltage electrical burns, an entity not previously explored. Clinical use of MRI examinations in patients with high-voltage injuries admitted to the Burn Unit at Linkoping University Hospital, has resulted in the significant elucidation of the deeper tissue injuries that occur. The T2-weighted images provided substantial information about the localization and amount of muscle necrosis, thus enabling increased surgical precision in the treatment of these high-voltage injury victims. FSE sequences produce T2-weighted images with increased speed of acquisition and/or increased image resolution compared to conventional SE sequence. Two illustrative examples are provided. PMID- 8634119 TI - Epidemiology of childhood burns at the Burn Centre in Brno, Czech Republic. AB - Three hundred and ninety-four children were admitted to the burn centre in Brno over a 3-year period. The overall incidence was 31 children per 100,000 children aged 0-14 years. The majority of patients were aged 1-3 years and almost 80 per cent of them were scalded. Proportionally more boys than girls were injured, in most cases at home in the presence of one or both parents. The highest risk time for injuries was between 16.00 and 18.00 h. Seasonal variations had no significant influence on the increased number of admissions to the burn centre. Sixty-six per cent of the children had minor burns. Four children died of the consequences of burns during the period studied. Social and economic factors had a significant influence on the incidence of childhood burns. PMID- 8634118 TI - Experimental gut-derived endotoxaemia and bacteraemia are reduced by systemic administration of monoclonal anti-LPS antibodies. AB - This study aimed to investigate the effects and mechanisms of action of systemic administration of monoclonal antibodies, anti-endotoxin (HA-1A), in an animal model of gut-origin sepsis. In the first experiment, Balb/c mice were transfused with allogeneic blood (C3H/HeJ mice). Five days post-transfusion the animals were gavaged with 1 x 10(9) Escherichia coli and randomized into three groups (n = 22 each) to receive a sham burn (SB group) or a 20 per cent TBSA thermal injury, immediately followed by the systemic administration of monoclonal antibodies (3 mg/kg) (HA-1A group) or aliquots of sterile saline (Control group). The animal survival rate was observed for 10 days postburn. In the second experiment transfusion and burn injury were reproduced but the mice (n = 8/group) were gavaged with 10(9) E.coli labelled with 111indium oxine. Four hours after the burn the mesenteric lymph nodes, liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of translocation of labelled bacteria measured by the residual radioactivity in the organs. Circulating endotoxin levels were determined by limulus assay. The mortality rate of the HA 1A group (9 per cent) was similar to the SB group (0 per cent) and significantly lower than the control group (59 per cent) (P < 0.05). Both plasma endotoxin levels and degree of bacterial translocation in all extraintestinal tissues were significantly lower (by approximately 50 per cent) in the HA-1A group than in the control group (P < 0.05). Systemic administration of HA-1A exerts a beneficial effect by reducing the circulating levels of endotoxin and by increasing the gut barrier function to translocating microorganisms. PMID- 8634121 TI - Ophthalmological complications as a manifestation of burn injury. AB - Facial burns are a frequent component of the presentation of victims who have sustained thermal trauma, reportedly occurring in 20 per cent of burn patients. Even apparently 'f2p4r' facial injuries might well be associated with significant ocular trauma. A retrospective review of 865 patients admitted to our burn centre showed 22 per cent (192) with facial burns. Ocular involvement, defined as globe or eyelid pathology, was present in 15 per cent (127) of these patients. The aetiology and spectrum of ocular injuries is reviewed with lid burns and subsequent lid contractures, accounting for over 50 per cent of ocular complications. Serious ocular pathology necessitating enucleation occurred in only two patients. The difficulties encountered in performing a complete ophthalmological examination in the presence of facial burns are presented in conjunction with a recommended therapeutic plan. PMID- 8634120 TI - Standardization of the first aid treatment of burn injuries in Vorarlberg, Austria. AB - It is 6 years since a burn unit was installed at the Landeskrankenhaus Feldkirch in Vorarlberg. Before that time, systematic clinical burn care was unknown in this country. Systematic first aid treatment of burn injuries was not known either. According to the principles of quality management, an obligation was felt not only to standardize the burn treatment in the unit itself, but also to make known the principles of first aid to the whole medical staff in our country. This is why we carried out many public relation activities consisting of several lectures to general practitioners and training for the emergency staff, including the lay personnel. The population were even informed about first aid treatment via radio and TV. In 1994, the degree of success in the public relation efforts was examined. During a period of 10 months, all the burn patients were asked about the quality of their first aid treatment. The questionnaires of 72 patients were evaluated, and it was found that the criteria demanded of first aid treatment were met within a range of 77.6-100 per cent. From these figures, it was concluded that public relation activities have been very successful in establishing the first aid care, they have led to a better quality of burn treatment in general and specifically, to a significant reduction in cost. PMID- 8634122 TI - Electrical burns of the mouth: still searching for an answer. AB - This report is a review of the literature and the clinical experience of electrical burns of the mouth in the West Midlands Regional Burns Unit at the Birmingham Accident Hospital, UK, over the past 10 years. There were five patients, four were children, 9 months to 5 years of age, and one adult. All patients had electrical conduction injuries involving the lips and perioral structures with involvement of the oral commissure. Electrical arc and flash burns were not included in this study. One child had a severe electrical injury and posed a difficult management problem. Although a variety of reconstructive procedures are available, the dilemma between conservative treatment and immediate reconstruction persists. Early intraoral splinting has been suggested in the literature. It was not used in any of the patients in this series and, though it may be beneficial, the patient compliance of a paediatric population may be questionable. The incidence of electrical burns has been on the decline, but those affecting the mouth still continue to be a major problem in management. PMID- 8634123 TI - Treatment of a severe alkali burn. AB - The case history of a 20-year-old male patient who sustained an 85 per cent total body surface area alkali burn to his skin, after falling into a caustic lime pit, is reported. Considerable problems regarding the correct estimate of burn wound depth, predominant location of the deepest burn on the posterior half of the body, appropriate wound coverage, and lack of sufficient skin graft donor sites required a complex treatment plan. Excisions to fascia and intradermal debridement were required to achieve an appropriate bed for wound closure. Five per cent mafenide acetate solution (Sulfamylon) was applied to prevent burn wound sepsis. Human allografts and Biobrane were used extensively to achieve temporary wound closure, to provide mechanical protection of freshly autografted wounds, and to prevent desiccation following application of cultured epidermal autografts on to debrided wounds and split thickness skin grafted donor sites. The case illustrates a number of problems associated with the evaluation and treatment of patients suffering severe alkali burns, and demonstrates the implementation of both established and evolving technologies in the management of these injuries. PMID- 8634124 TI - Burn injuries caused by chip-pan fires: the Edinburgh experience. AB - Thirty-two patients were admitted to the South-East Scotland Burn Unit over a 4 year period (1990-94), with burns sustained as a consequence of chip-pan fires. This represented 7 per cent of all admissions to the unit. Fourteen patients came from the Edinburgh city area, and 18 from surrounding countries. There were 14 males and 18 females, and an average age of 51.6 years. The average burn surface area was 4.7 per cent. Eighteen patients had burns to their hands, this being the most common area involved. Fourteen patients were managed conservatively, and 18 needed operative intervention. The patients spent an average of 19.4 days in hospital, and they required 46.3 days for complete healing to take place. Prevention by increased community awareness and widespread education is necessary to minimize the incidence, morbidity and cost of this relatively common and preventable type of burn. PMID- 8634125 TI - Vinylpyridine: an unusual cause of a chemical burn. AB - A case of a chemical burn following cutaneous exposure to vinylpyridine is presented. This unusual injury was distinguished by the delay in onset of pain and erythema, followed by gradual resolution leaving patchy skin discolouration. The properties of vinylpyridine are outlined. This case illustrates the dangers of long-term chemical storage and the importance of proper disposal. PMID- 8634126 TI - Frostbite of the face after recreational misuse of nitrous oxide. AB - Exposure of the skin to nitrous oxide, a liquified gas stored under pressure in a cylinder, can occur in anaesthesiologists and in those involved in recreational misuse of the gas. A case is reported of a man who presented to the emergency department after sniffing nitrous oxide and sustaining frostbite injury to his left cheek. Conservative management of this injury allowed spontaneous separation of the necrotic tissue and healing of the granulating wound by contraction. PMID- 8634127 TI - Chemical burns as assault injuries in Jamaica. AB - A three-fold greater incidence of chemical burn injuries in Jamaican hospitals, compared to burn centres in other industrial countries, underscores the problem of the use of common chemicals for assault weapons in this country. With the increased availability of guns for personal use, many Jamaicans learned the value of carrying household chemicals such as sulphuric acid from batteries or sodium hydroxide obtained from cleaning supplies. Chemicals carried in a container, such as one might carry mace, afforded a means of defence among the lower socioeconomic groups who could not afford handguns. This use of dangerous chemicals for defensive weapons has extended to the use of chemicals for assault. The pattern of chemical injury differs significantly from most reports in the literature in both prevalence and aetiology. This review was prepared to examine these injuries with a view to planning strategies for prevention. PMID- 8634128 TI - Hot spring burns. AB - This case report describes a woman who, while visiting a hot spring, received partial- and full-thickness immersion scald burns of both ankles and heels. The prevention of such accidents is most important; efforts should be made to educate guides and tourists about the potential hazards inherent in these resorts. PMID- 8634129 TI - Visceral injuries, wound infection and sepsis following electrical injuries. AB - Visceral injuries, wound infection and sepsis were investigated in 226 inpatients who sustained electrical burns over a period of 15 years. Four patients who sustained thoracic and abdominal organ injuries were noted in this series. The patients had injuries of the small intestine, stomach, colon and the lung. All the patients received operative treatment. Two of them died of sepsis. Injuries to the internal organs should always be considered following high-voltage injuries, and they should be managed as early as possible. The data concerning wound infection and sepsis following electrical injuries were evaluated in three consecutive 5-year periods. Over this period of 15 years, different antibiotic regimens were used for prophylaxis and treatment. Most patients in the current series had been contaminated or infected by various pathogens prior to admission. Long-lasting administration of prophylactic antibiotics in these patients showed no improvement in controlling the sepsis. After 1987, most of the microorganisms were eliminated following more effective antimicrobial therapy. The progressive decrease in infection frequency of species such as Pseudomonas aeruginosa, Proteus mirabilis and Enterobacter cloacae, appeared to be causally related to the changes in the general therapeutic protocol which included new antibiotics. The infections caused by E. coli and Staphylococcus aureus showed a rather steady state. A marked increase in frequency of negative wound cultures was also noted between the years 1989 and 1993. A gradual decrease in mortality rates was observed from the first to the last 5-year period, whereas mortality rates due to sepsis showed a gradual but slower decline. Sepsis (142 patients comprising 62.8 per cent of the total mortality rate) was the most frequent complication resulting in death. PMID- 8634130 TI - Human papilloma virus proliferation in a healing burn. AB - A 4-year-old boy sustained a superficial burn to his finger at the site of a viral papilloma. The burn was treated conservatively. During re-epithelialization the human papilloma virus (HPV) incorporated its genome in the regenerating cells, leading to a verruca as large as the original burn. Diagnosis was reinforced by a history from the patient's mother. Complete resolution of this complication was achieved with topical therapy. PMID- 8634131 TI - Burn due to a sawdust explosion. AB - Burns due to explosions of various types of inflammable dust have been reported, including coal dust, flour and grain dust. A 33-year-old worker was burned over 33 per cent of his body surface area in a sawdust explosion. Recovery was uneventful. This is the first reported case due to this mechanism as far as we have been able to ascertain. PMID- 8634132 TI - The influence of a severe burn injury on the distribution of carnitine between blood cells. PMID- 8634133 TI - Burns incidence in low-income suburbs of Maputo City, Mozambique. PMID- 8634134 TI - Recent references. PMID- 8634135 TI - Fire fatality study: demographics of fire victims. AB - Injury or death caused by fire is frequent and largely preventable. This study was undertaken to define the populations, locations, times and behaviours associated with fatal fires. Seven hundred and twenty-seven fatalities occurring within the State of New Jersey, between the years 1985 and 1991, were examined retrospectively. Most deaths were attributed to a combination of smoke inhalation and burn injury. Five hundred and seventy-four fatalities occurred in residential fires. Smoking materials were the most common source of ignition for residential fires. More than half of the fatal residential fires started between the hours of 11 p.m. and 7 a.m. Children and the elderly represented a disproportionate percentage of fire victims. Victims under the age of 11 years or over the age of 70 years constituted 22.1 per cent of the state population but 39.5 per cent of all fire fatalities. Fire-prevention efforts should target home fire safety, and should concentrate on children and the elderly. The development of fire-safe smoking materials should be encouraged. PMID- 8634136 TI - Predictors of post-traumatic stress disorder following burns injury. AB - Burns patients were assessed 12 months following their injury to determine the factors that predict development of post-traumatic stress disorder (PTSD). Among 35 patients, 31 per cent suffered PTSD, 29 per cent suffered subclinical PTSD reactions and 40 per cent displayed no PTSD symptoms. Concern over scarring and an avoidant coping style accounted for 61 per cent of the variance in post traumatic stress symptomatology. Only half of the patients reporting PTSD had sought professional assistance. The results indicate that postinjury adjustment factors are critical determinants of PTSD development following burns. Implications for therapeutic interventions are discussed. PMID- 8634137 TI - Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model. AB - The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model. PMID- 8634138 TI - Evidence that SP1 modulates transcriptional activity of the multidrug resistance associated protein gene. AB - In previous studies, we have cloned and sequenced a 5'-end region of the multidrug resistance-associated protein (MRP) gene that contains promoter activity as assessed through transient transfections of constructs contained in a pCAT basic reporter plasmid. In the present study, using a series of deletion mutants, evidence was obtained that the SP1 binding sites contained in the promoter are essential for optimal MRP transcriptional activity. These results were supported by the finding that introduction of site-specific mutations into the wild-type SP1 sequence produced a major reduction in CAT activity. DNase I protection assays also demonstrated that SP1 sites are protected from hydrolysis with proteins from nuclei of a variety of cell lines. Gel mobility-shift assays with proteins extracted from CHO, HeLa, HL60, or HL60/ADR demonstrated the presence of a protein that bound to the wild-type SP1 sequence but not to an SP1 sequence containing site-specific mutations. The mobility shift with nuclear extracts was closely similar to that occurring after incubating purified SP1 protein with wild-type SP1 sequence. DNA supershift experiments with antibody to SP1 strongly suggest that the complexes formed with nuclear extracts contain the SP1 protein. PMID- 8634139 TI - Structural organization, sequence, and expression of the chicken NRAMP1 gene encoding the natural resistance-associated macrophage protein 1. AB - One of the most common causes of food poisoning in humans is salmonellosis, which is frequently caused by ingestion with Salmonella-contaminated poultry products. Several lines of evidence suggest that genetic factors control resistance and susceptibility of chickens to infection with Salmonellae. In the mouse, innate resistance to infection with intracellular pathogens such as Salmonella typhimurium, several species of Mycobacteria, and Leishmania donovani is controlled by the mouse chromosome 1 Nramp1Bcg gene. To investigate the role of NRAMP1 in the differential resistance and susceptibility of chickens to infections with S. typhimurium, we have cloned and characterized cDNA clones corresponding to the chicken NRAMP1 gene. Nucleotide and predicted amino acid sequence analyses indicate that the chicken NRAMP1 polypeptide encodes a 555 amino-acid residue membrane protein with 12 putative transmembrane domains, two N linked glycosylation sites, and an evolutionary conserved consensus transport motif. The peptide sequence identity among chicken, mouse, and human NRAMP1 is 68%. The chicken NRAMP1 gene contains 15 exons and spans 5 kb of genomic DNA. One major and two minor transcription initiation sites were detected using primer extension. Nucleotide sequencing of the promoter region revealed the presence of a classical TATAA element and consensus sequences for binding the myeloid specific PU.1 factor and several lipopolysaccharide (LPS) (NF-IL6 and NF-kappa B) and interferon-gamma (IFN-gamma)-inducible response elements. Similar regulatory elements are found in the promoters of mouse and human NRAMP1. Northern blot analyses revealed NRAMP1 expression in reticuloendothelial organs (spleen and liver), lung, and thymus. As demonstrated in mice and humans, the macrophage is also a major site of NRAMP1 mRNA expression in chickens. However, the high levels of expression detected in chicken thymus contrast with the absence of expression of the mammalian Nramp1 gene in this tissue. PMID- 8634140 TI - Plasmid insertional mutation may confer glucocorticoid responsiveness of cell growth. AB - Culture of CHO.K1, a Chinese hamster ovary cell line, requires no particular care about the glucocorticoid level in media. Cell growth of CHO.K1 is little affected by dexamethasone at concentrations up to 3 microM. A clone of CHO.K1 stably transfected with an expression vector displayed a favored growth in dexamethasone containing media. Ironically, dexamethasone was used to trigger the expression of antisense PCNA from the expression vector to impede the cell growth. Proliferating cell nuclear antigen (PCNA), an auxiliary factor of DNA polymerase delta, is required for cell proliferation. The stable cell clone, designated as B11 had a retarded growth rate as compared to its parental cell. However, the B11 cell growth rate and the cell cycle progression were increased by dexamethasone. The glucocorticoid produced no similar effect on the parental cell or other stable transfectants of the same plasmid. Thus, the stimulatory effect of dexamethasone on B11 has little connection with the expression of antisense PCNA and possibly involves a relevant gene in the B11 genome that was mutated due to the random plasmid insertion. A preliminary effort in identifying the targeted gene was made by using plasmid rescue method, and two plasmids were obtained. The rescued DNA of both plasmids specifically hybridized genomic DNA of the parental cells, and one of these plasmids detected a cellular transcript that was absent in B11 cells, suggesting its potential for further investigation. PMID- 8634141 TI - A G/C-rich DNA-regulatory element controls positive expression of the sea urchin Lytechinus pictus aboral ectoderm-specific LpS1 gene. AB - The LpS1 beta gene of Lytechinus pictus is activated at the late cleavage stage about 12 hr after fertilization. LpS1 beta transcripts accumulate exclusively in aboral ectoderm lineages. LpS1 beta is thus a classic example of a gene whose expression is tightly controlled both temporally and spatially during early development. Previous studies on the LpS1 beta promoter identified two G-string DNA elements, one proximal and one distal to the LpS1 beta transcriptional start site, which bind to an ectoderm-enriched nuclear factor. In this report, we show that the ectoderm G-string factor binds to a G/C-rich region larger than the G string itself and that the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. Two regions of 5'-flanking DNA are required for positive control of LpS1 beta, region I from base pairs -762 to -511, and region II, which includes the G/C-rich element, from base pairs -108 to -61. Region I also contains a mesenchyme cell repressor element. The results indicate that LpS1 beta expression is regulated positively in ectoderm cells and negatively in mesenchyme cells. Similar positive and negative control mechanisms regulate the expression of the related Spec genes of Strongylocentrotus purpuratus, but in this gene family the DNA elements are entirely different. We hypothesize that cis-regulatory elements are evolutionarily dynamic and that many different combinations of DNA elements are capable of given rise to aboral ectoderm-specific expression. PMID- 8634142 TI - An efficient cellular system for mutational analysis of prohormone processing. AB - A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones. PMID- 8634143 TI - Cloning, expression, and chromosomal localization of the rat mitochondrial capsule selenoprotein gene (MCS): the reading frame does not contain potential UGA selenocysteine codons. AB - The mitochondrial capsule selenoprotein (MCS) is a selenium-containing polypeptide. It is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. In this paper, we report the isolation and characterization of the rat MCS cDNA and gene. The cDNA contains a reading frame for a 145-amino-acid protein and it lacks the UGA codons, which have been found in the reading frame of the mouse MCS cDNA and have been presumed to encode the selenocysteine in the amino terminal of the deduced mouse amino acid sequence. The deduced amino acid sequence of the rat and mouse MCS shows a high level of homology (79%). The rat MCS gene contains two exons; the intron sequence interrupts the 5' untranslated sequence at the same position as in the mouse MCS gene. The transcription start site is located 184 bp upstream of the translation start site. Alignment of the 5'-flanking regions of the mouse and rat genes reveals that the first 400 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 73%. This conserved region contains no TATA or CAAT box motifs. Northern blot analysis indicates that the MCS mRNA is detectable only in the testis after day 30 of postnatal development. Moreover, in situ hybridization revealed that the rat MCS gene is mainly expressed in round spermatids. From the analysis of mouse-rat cell hybrids that segregate rat chromosomes, the MCS gene was assigned to rat chromosome 2. PMID- 8634144 TI - Species specificity of ribosomal gene transcription: a factor associated with human RNA polymerase I prevents transcription of mouse rDNA. AB - An intrinsic property of class I gene transcription by RNA polymerase I (Pol I) is the species specificity of the initiation reaction. Previous studies have demonstrated that species-specific rDNA promoter recognition is brought about by a TBP-TAF complex, termed TIF-IB in mouse and SL1 in man. We have compared the ability of affinity-purified TIF-IB and SL1 to direct transcription from the homologous rDNA template both in a reconstituted transcription system and in nuclear extracts prepared from mouse and human cells. We show that Pol I from both species and the individual transcription factors, with the exception of TIF IB/SL1, are functionally interchangeable in the reconstituted transcription system containing purified proteins. In nuclear extracts, however, species specific differences are obvious. Whereas SL1 reprograms a heterologous mouse extract to recognize the human promoter, TIF-IB fails to reprogram a human extract unless it is complemented with mouse Pol I. Crude human, but not mouse, Pol I exhibits species-specific differences that disappear after purification. We propose that in extracts and less purified fractions human Pol I exists as 'holoenzyme' containing associated protein(s) that prevent assembly of TIF-IB directed initiation complexes at the murine rDNA promoter. PMID- 8634145 TI - Promoters for the human beta-hexosaminidase genes, HEXA and HEXB. AB - Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and HEXB mutations that could alter enzyme expression in Tay-Sachs and Sandhoff diseases, respectively. PMID- 8634146 TI - Transcriptional repression of human insulin-like growth factor-II P4 promoter by Wilms' tumor suppressor WT1. AB - The Wilms' tumor suppressor gene wt1 encodes a zinc finger-containing protein that binds to the same DNA sequence as Egr-1, a mitogen-inducible immediate-early gene product that activates transcription. In this study, we investigated whether the human insulin-like growth factor-II (IGF-II) P4 promoter might be a target for transcriptional repression mediated by WT1. Using constructs of the IGF-II P4 promoter linked to the chloramphenicol acetyltransferase gene, we have demonstrated that the WT1 protein represses expression of the IGF-II gene through a GCGGGGGAG response element spanning nucleotides -87 to -65 of the IGF-II P4 promoter. Conversely, we have shown that the Egr-1 activates transcription of the IGF-II gene through the same response element. WT1 and Egr-1 proteins interact directly with the WT1/Egr-1 response element of the IGF-II promoter 4 in gel mobility-shift assays. These findings demonstrate the importance of the WT1/Egr-1 consensus element for the expression of the IGF-II gene in response to positive or negative transcription signals. PMID- 8634147 TI - A large variety of alternatively spliced and differentially expressed mRNAs are encoded by the human acute myeloid leukemia gene AML1. AB - The human chromosome 21 acute myeloid leukemia gene AML1 is frequently rearranged in the leukemia-associated translocations t(8;21) and t(3;21), generating fused proteins containing the amino-terminal part of AML1. In normal blood cells, five size classes (2-8 kb) of AML1 mRNAs have been previously observed. We isolated seven cDNAs corresponding to various AML1 mRNAs. Sequencing revealed that their size differences were mainly due to alternatively spliced 5' and 3' untranslated regions, some of which were vast, exceeding 1.5 kb (5') and 4.3 kb (3'). These untranslated regions contain sequences known to control mRNA translation and stability and seem to modulate AML1 mRNA stability. Further heterogeneity was found in the coding region due to the presence of alternatively spliced stop codon-containing exons. The latter led to production of polypeptides that were smaller than the full-length AML1 protein; they lacked the trans-activation domains but maintained DNA binding and heterodimerization ability. The size of these truncated products was similar to the AML1 segment in the fused t(8;21) and t(3;21) proteins. In thymus, only one mRNA species of 6 kb was detected. Using in situ hybridization, we showed that its expression was confined to the cortical region of the organ. The 6-kb mRNA was also prominent in cultured peripheral blood T cells, and its expression was markedly reduced upon mitogenic activation by phorbol myristate acetate (TPA) plus concanavalin A (ConA). These results and the presence of multiple coding regions flanked by long complex untranslated regions, suggest that AML1 expression is regulated at different levels by several control mechanisms generating the large variety of mRNAs and protein products. PMID- 8634148 TI - Gene expression in a young multigene family: tissue-specific differences in the expression of the human alcohol dehydrogenase genes ADH1, ADH2, and ADH3. AB - Three human alcohol dehydrogenase genes, ADH1, ADH2, and ADH3, were formed by tandem duplications and have diverged in their tissue-specific and developmental expression. Their proximal promoters remain 80-84% identical in sequence, approximately the same degree of identity as at synonymous sites in the coding regions of these three genes. To understand the evolution of tissue specificity, gene expression must be studied in many different cells and tissues. A systematic comparison of their promoters reveals the effects of subtle sequence differences on the binding of nuclear proteins to their cis-acting elements. There are differences in the affinity with which some proteins are bound to altered sites including C/EBP sites, USF/MLTF sites, and the G3T site (which binds Sp1). There are also differences in the sites that are occupied, e.g. CTF/NFI-related sites. These sequence differences are reflected in differences in gene expression in three cell lines. In H4IIE-C3 hepatoma cells, the ADH1 promoter was more active than the ADH2 promoter, and the ADH3 promoter was nearly nonfunctional. In HeLa cells, both ADH1 and ADH2 promoters directed expression; again the ADH3 promoter was extremely weak. None of the three promoters had much activity in CV-1 cells. Coexpression of C/EBP alpha greatly stimulated expression of the ADH1 promoter in HeLa cells and in CV-1 cells, but only weakly stimulated expression in H4IIE-C3 cells. The stimulation of the ADH1 promoter by C/EBP alpha was comparable to that of ADH2, despite the weaker binding to the C/EBP sites that flank the TATA box in ADH1. The ADH3 promoter was not greatly stimulated by C/EBP alpha, despite good binding of C/EBP alpha. These results demonstrate that small differences in the cis-acting elements affect affinity of binding by transcription factors and the pattern of gene expression. PMID- 8634149 TI - A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP. AB - Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells. PMID- 8634150 TI - Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro. AB - Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3' flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global regulation of gene expression such as the position effects associated with translocation of genes to heterochromatic centromere and telomere regions of the chromosomes. PMID- 8634151 TI - Cloning and characterization of the bovine Fas. AB - Fas (APO-1, CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, is a cell membrane protein that mediates programmed cell death (apoptosis). In an effort to characterize the possible role of Fas-mediated apoptosis in some important physiological processes in livestock, bovine Fas (bFas) cDNA was isolated and its nucleotide sequence determined. The predicted amino acid sequence encodes a 323-amino-acid protein that contains a leader peptide, a transmembrane domain, and three domains of cysteine-rich motif within the extracellular region. At the amino acid level, bFas is 57% and 50% identical to human (hFas) and mouse (mFas), respectively. Its expression is abundant in peripheral blood lymphocytes, lung, spleen, thymus, and ovary, but minimal in heart, liver, and brain. A polyclonal anti-bFas serum raised against the carboxy terminal half of cysteine-rich motif I recognized a single 46-kD protein in bovine MDBK cells by Western blot analysis. To investigate the apoptotic activity of bFas, MDBK and bFas-transfected L929 cells were exposed to a monoclonal anti hFas IgM. Unlike other cell culture systems, the antibody failed to trigger cell death in MDBK and bFas transfected L929 cells. PMID- 8634152 TI - Mouse microsomal Class 3 aldehyde dehydrogenase: AHD3 cDNA sequence, inducibility by dioxin and clofibrate, and genetic mapping. AB - We have cloned and sequenced the mouse AHD3 cDNA, which codes for the Class 3 microsomal aldehyde dehydrogenase (ALDH3m). The cDNA is 2,997 bp in length excluding the poly(A)+ tail, and has 5' and 3' non-translated regions of 113 bp and 1,429 bp, respectively. The deduced amino acid sequence consists of 484 amino acids, including the first methionine (Mr = 53,942), and contains a hydrophobic segment at the carboxyl terminus which is the putative membrane anchor. The mouse AHD3 protein was found to be: 95% similar to the rat microsomal ALDH3m protein, 65% identical to the mouse, rat and human cytosolic ALDH3c protein, and <28% similar to the rat Class 1 and Class 2 ALDH and methylmalonate-semialdehyde dehydrogenase proteins. Southern hybridization analysis of mouse cDNA probed with the full-length AHD3 cDNA revealed that the Ahd3 gene likely spans less than a total of 25 kb. The mouse Ahd3 gene is very tightly linked to the Ahd4 gene on chromosome 11. Mouse AHD3 mRNA levels are increased by dioxin in mouse Hepa-1c1c7 hepatoma wild-type (wt) cells but not in the Ah receptor nuclear translocator (ARNT)-defective (c4) mutant line, indicating that the induction process is mediated by the Ah (aromatic hydrocarbon) dioxin-binding receptor. AHD3 mRNA levels are also inducible by clofibrate in both the wt and c4 lines. AHD3 mRNA levels are not elevated in the CYP1A1 metabolism-deficient c37 mutant line or as part of the oxidative stress response found in the untreated 14CoS/14CoS mouse cell line. These data indicate that, although inducible by dioxin, the Ahd3 gene does not qualify as a member of the aromatic hydrocarbon [Ah] gene battery. PMID- 8634153 TI - Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF. AB - cDNA encoding the canine keratinocyte growth factor (KGF) was cloned from normal canine prostate tissue. The authentic canine KGF cDNA sequence, 686 bp in length, spans the protein-coding region and 88 bp of the 5' and 19 bp of the 3' untranslated regions of canine KGF. The predicted amino acid sequence of canine KGF is composed of 194 amino acid residues. Canine KGF exhibits highest homology with the human KGF cDNA and amino acid sequences (95.8% and 97.4%, respectively), while it demonstrates the lowest homology with the rat sequences at 88.0% and 92.3%, respectively. The degrees of homology with mouse cDNA and amino acid sequences are 91.8% and 95.9%, respectively. By using RNase protection assay, KGF was shown to be expressed in normal prostate tissues of both mature and young (5 month-old) dogs. In vitro, the recombinant canine KGF has mitogenic activity on cultured canine epithelial cells, whereas it has no effect on cultured canine prostatic stromal cells. This novel canine KGF cDNA may be a valuable tool in the study of human benign prostatic hyperplasia using the canine prostatic as a model. PMID- 8634154 TI - Quantification of 3'OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay. AB - A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules. The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA. After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules. Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of [32P]dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks). The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis. It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents. Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III. The assay is capable of quantifying first several breaks per 10(5) bp. PMID- 8634155 TI - Cloning and characterization of a cDNA encoding the collagen-binding stress protein hsp47 in zebrafish. AB - Hsp47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. Although hsp47 is coordinately expressed together with several collagen types, and vertebrate embryos are known to express collagen genes in complex spatial and temporal patterns, limited information is available regarding the function or regulation of hsp47 during early embryonic development. We have initiated an examination of hsp47 in the zebrafish, Danio rerio, which offers a number of features that make it attractive as a model developmental system with which to examine the expression and function of hsp47. A polymerase chain reaction (PCR)-based cloning strategy was used to isolate a hsp47 cDNA from an embryonic zebrafish cDNA library. The deduced translation product of the cDNA is a 404-amino-acid polypeptide that is 72% identical to chicken, 64% identical to mouse and rat, and 69% identical to human hsp47. The protein contains a typical hydrophobic signal sequence, an RDEL endoplasmic reticulum retention signal, and a serine protease inhibitor signature sequence, all of which are characteristic of hsp47 in higher vertebrates. Thus, it is likely that hsp47 in zebrafish is also localized to the endoplasmic reticulum and may play a similar role to its counterpart in higher vertebrates. Northern blot analysis revealed that the hsp47 gene is expressed at relatively low levels in embryos during normal development but is strongly induced following exposure to heat shock at the gastrula, midsomitogenesis, 2 day, and 3-day larval stages. The level of induction was much higher than has previously been reported in chicken and mouse cells. PMID- 8634156 TI - The composition and ultrastructure of resin tags in etched dentin. AB - PURPOSE: To examine in vitro the composition of resin tags in etched dentin by SEM and vital staining using Alcian Blue dye. MATERIALS AND METHODS: Bovine and human teeth were used. The dentin surface was exposed using water-irrigated #600 grit SiC paper and etched with varying dilutions of aquaeous solutions of phosphoric and maleic acid. Groups of 10 teeth per acid concentration were embedded in PMMA and Scotchbond Multi-Purpose (SMP) and cylinders of Z100 resin composite were bonded to their surfaces. These teeth were shear tested to failure and the failed surfaces subjected to SEM. Groups of three teeth per acid concentration had SMP and cylinders of Z100 bonded to their surfaces. These teeth were decalcified in concentrated HCL acid. The resultant cylinders and their attached resin tags were either subjected to SEM or stained with Alcian Blue dye. The latter specimens were embedded and prepared for light-microscopic examination. A further three human and three bovine teeth per acid concentration were etched and then prepared for SEM examination. All teeth for SEM were critical point dried. RESULTS: Resin tags were shown to consist of both resin and the glycosaminoglycans (GAG) sheath lining of the dentin tubule. As a result of the SEM observations, three mechanisms are suggested for the formation of resin tags, two of which will probably be found in vivo. PMID- 8634157 TI - Enhanced anticaries efficacy of a 0.243% sodium fluoride/10% xylitol/silica dentifrice: 3-year clinical results. AB - PURPOSE: To evaluate the efficacy of a sodium fluoride (NaF)/silica/xylitol dentifrice compared with that of a positive control NaF/silica dentifrice on caries increments in school children over a 3-year period in an area without an optimal level of fluoride in the drinking water (mean level <0.1 ppm). MATERIALS AND METHODS: A 3-year, double-blind clinical caries study was conducted in 2,630 children initially aged 8-10 years at 17 schools in the San Jose, Costa Rica metropolitan area. Clinical dental examinations were performed at participating schools utilizing portable dental equipment. Caries evaluations employed conventional tactile/visual methodology consisting of artificial light, dental mirrors and single-edge #23 explorers. Children accepted into the study were stratified by age and sex into two balanced groups within each school, and randomly assigned to use either a positive control dentifrice containing 0.243% NaF/silica or a test dentifrice containing 0.234% NaF/silica/10% xylitol. Children were instructed to brush with the assigned dentifrice twice daily. Caries evaluations were conducted at baseline, 2 years, and 3 years. RESULTS: After 3 years, subjects using the 0.234% NaF/silica/10% xylitol dentifrice had statistically significantly reduced decayed/filled surfaces (DFS; -12.3% reduction; P < or = 0.001) and decayed/filled buccal and lingual surfaces (DFS BL; -10.5% reduction; P < or = 0/01). PMID- 8634158 TI - Phosphoric acid as a conditioning agent in the Gluma bonding system. AB - PURPOSE: To investigate the effect of phosphoric acid conditioning gels on compatibility with the Gluma dentin bonding system. MATERIALS AND METHODS: Sheer bond strengths (BS), marginal performance in dentin cavities and microleakage resistance in mixed cavities were used as target parameters to determine the efficacy of Gluma/Pekafill on dentin pretreated with conditioners of 5, 10, 20 or 35% H3PO4 and different amounts of silica (0, 5 and 10%) after 15, 30 60 or 120 second treatment duration. The thicknesses of the hybrid layers were determined along the dentin cavity margin by light microscopy. RESULTS: Neither the phosphoric acid concentration and the silica content nor the conditioning times tested had a significantly different effect on SBS or marginal performance (P > 0.05). There was no relationship between the hybrid layer thickness and the target parameters either. The SBS recorded on dentin were in the same order of magnitude as previously found on acid etched enamel (+/- 17 MPa). The marginal performance and microleakage results after thermocycling proved adequate dentin bonding and cavity sealing. Phosphoric acid etching of dentin is compatible with the gluma bonding system. PMID- 8634159 TI - Ambulant 24-hour blood pressure and heart rate of dentists. AB - PURPOSE: To investigate blood pressure fluctuations in dentists during their daily activities, including dental procedures, in comparison with a non-dentist population. MATERIALS AND METHODS: A pilot study on blood pressure and heart rate fluctuations was performed which involved 26 dentists working at a university clinic. A 24-hour blood pressure registration was obtained using the Oxford Monitoring System. RESULTS: In the dentist group, both blood pressure and heart rate were found to be significantly higher during work than during leisure activities. In the control group, no significant difference in blood pressure was recorded between these periods. This study also showed the feasibility of ambulant 24-hour blood pressure registration on dentists during daily activity. Our results ethically justify a more intensive cardiovascular study involving a larger number of dentists working in private practice in order to establish whether our results are valid for the dental practitioner in general. PMID- 8634160 TI - Influence of light intensity on shear bond strength to dentin. AB - PURPOSE: To investigate the influence of light intensity on dentin bond strength of five dentin bonding systems. MATERIAL AND METHODS: The light intensity used to polymerize specimens was controlled at the levels of 100, 200, 400, 600, 800, and 1000 W/m2 (470 +/- 40 nm). Labial surfaces of freshly extracted lower bovine incisors were ground with #600 grit SiC paper to expose dentin. After primer application, bonding agents were applied and bonded with resin composites. A shear bond strength test was performed and the data were analyzed by a one-way ANOVA followed by Newman-Keuls multiple comparison (P<0.05). RESULTS: Statistical analysis of the data indicated that light intensity affected the bond strength to dentin. The mean bond strength of all bonding systems decreased with lower light intensity. The tendency of decreasing bond strength was different among the test materials and each test material had a threshold light intensity that resulted in statistically the same bond strength obtained using a light intensity of 1000 W/m2. PMID- 8634161 TI - Effect of glass inserts on resin composite wear. AB - PURPOSE: To determine the in vitro wear resistance and microstructural stability of composites in which the beta quartz insert was used. MATERIALS AND METHODS: Three different sized inserts were used in conjunction with P-50 and Herculite XR posterior resin composites. The study was designated to create varying gap dimensions between the insert and wall of the preparation (0.75, 1.25 and 1.50 mm). In addition, one half of the inserts contacted the dentin floor of the cavity whereas the other half rested on 1.0 mm of composite. The controls consisted of resin composite only. Using an in vitro wear testing device, all specimens were subjected to 400,000 cycles of masticatory loading. Upon completion of testing, profilometric tracings and SEM were used to determine relative wear, patterns of wear and microstructural changes. Differences, in wear values were analyzed by two-way ANOVA. RESULTS: The wear values for P-50 and Herculite XR controls were 24.8 and 31.3 micrometers, respectively. No significant differences were detected (P<0.05) in amount of wear between the control specimens and the insert specimens, regardless of size. The same results were obtained regardless of whether the insert rested on dentin or resin composite. SEM evaluations however, revealed excellent interfacial continuity after wear testing. While the inserts did not influence the wear resistance, they may provide an excellent means for maintaining functional occlusion. PMID- 8634163 TI - 4-year clinical study of castable ceramic crowns. AB - PURPOSE: To evaluate the 4-year clinical performance of castable glass ceramic crowns used to restore teeth in the posterior segments. MATERIALS AND METHODS: 101 castable ceramic (Dicor) full++ crown restorations were placed in 61 molar and 40 premolar teeth using a bonded resin cement. RESULTS: After 4 years of clinical performance, 15 of the original 101 restorations were known to have failed with 13 of those failures affecting molar restorations. All serviceable restorations were rated as excellent for color match, margin adaptation, proximal contact and gingival health. Cavosurface margin discoloration received a 93.5% alfa response. The results of this study show that Dicor crowns meet the esthetic and biological requirements for posterior restorations. The incidence of fracture of molar restorations in this study indicated that when used for restoring molars, careful case selection and caution must be employed. PMID- 8634162 TI - Bonding mechanism of VariGlass to dentin. AB - PURPOSE: This study investigated the hypothesis that the new resin-reinforced, light-cured ionomer cement (GIC) can develop mechanical retention by forming a hybrid layer in acid-etched dentin. MATERIALS AND METHODS: Dentin discs were obtained from extracted human third molars and sanded with 320 SiC abrasive paper. One third of the surface was acid etched with 10% maleic acid for 15 seconds, washed and gently air-dried for 5 seconds. ProBond primer from the VariGlass (VG) GIC kit was applied onto the acid-etched surface (A) and another third of the unetched surface (B) for 30 seconds. One third of the surface was not treated (C). VG GIC was then applied onto the ++entire surface of the disc. After 24 hours the discs were fractured along their diameters. One half of the fractured disc was highly polished at the interface and treated with 6N HCl for 30 seconds while the other half of the fractured specimen was left untreated. Both halves were viewed by SEM. In another part of the study, a micro-tensile bond strength (MTBS) test was carried out to compare the acid-etched group vs. the nonetched group. RESULTS: SEM pictures revealed a well defined demineralized, resin-infiltrated zone approximately 3 micrometers in thickness for group (A) for both fractured and polished surface. Dentin surfaces that only received primer (B) showed an irregular zone 0.5 micrometers thick. Such a resin-infiltrated layer was resistant to HCl treatment. A gap was observed between the GIC and dentin in group (C). Polished interfaces appeared to be highly infiltrated. However, fractured interfaces revealed considerable porosity within the demineralized-infiltrated zone. MTBS results were (X +/- SD, MPa): 28.9 +/- 5.8 for the etched group and 24.5 +/- 4.9 for the nonetched group. This difference was statistically significant. PMID- 8634164 TI - Recommending serologic HIV testing for the dental patient. AB - This article provides information for dental clinicians to enable them to make appropriate recommendations to patients needing HIV antibody testing. Information concerning serologic tests for HIV, the decision to/not to test, characteristics of the compassionate HIV counselor, pre-test and posttest counseling are detailed. Competence and confidence in addressing these issues is imperative for the dental clinician. PMID- 8634165 TI - Use of a resin-ionomer in guided tissue regeneration: case reports. AB - The cases presented in this report illustrate: (1) the potential for a fluoride releasing resin-ionomer (Geristore) to be used as a sealant between an e-PTFE membrane (Gore-Tex) and tooth structure during a guided tissue regeneration, and (2) the potential for a resin-ionomer to function as a barrier in guided tissue regeneration. In the first case, the e-PTFE membrane was trimmed to only cover the area to be treated prior to its placement over the lesion. The resin-ionomer was mixed following manufacturers specifications, placed on four corners of the pre-trimmed membrane and luted into place. In the second case, the resin-ionomer alone was placed over the lesion, teased off the lesion with a curette while in a semi-set state, and luted into place with freshly mixed material after it set completely. These cases provide preliminary clinical evidence that a resin ionomer may be used subgingivally for guided tissue regeneration. PMID- 8634166 TI - Disinfection and sterilization of composite polishing instruments. AB - PURPOSE: To evaluate the efficacy of several methods of sterilization and disinfection on two different types of composite finishing and polishing instruments. MATERIALS AND METHODS: The instruments consisted of a proprietary light-cured resin cup impregnated with an abrasive and a silicon dioxide impregnated rubber cup. Chemical and physical methods included treatment with iodophor, synthetic phenol, glutaraldehyde, microwaving, autoclaving, and chemiclaving. RESULTS: Glutaraldehyde (Vital Defense-S) was an effective disinfectant; however, other chemical (cold) disinfectants overall, were not reliable. Autoclaving, chemiclaving, and microwaving all provided to be effective means of sterilization. PMID- 8634167 TI - Lisinopril and diltiazem reduce left ventricular mass without changing blood pressure in normotensive subjects with exaggerated blood pressure response to exercise. AB - OBJECTIVE: To determine whether high left ventricular mass may be reduced by antihypertensive drugs in normotensives with exaggerated blood pressure response to exercise as it occurs in hypertensive patients. DESIGN AND METHODS: Randomized, single blind, controlled parallel study evaluating the influence of placebo; lisinopril 20 mg/day, diltiazem 180 mg/d for 5-6 months on left ventricular mass (LVM), evaluated by echo and on "casual" and 24-h ambulatory blood pressure (24-h BP) in normotensive subjects with exaggerated blood pressure response to exercise (Group I) and in weight--and age--matched mild-moderated hypertensive patients (Group II), all with high left ventricular mass. PATIENTS: Placebo, lisinopril and diltiazem, were administered for 5-6 months in respectively 8+9+9 subjects of Group I and 8+9+10 patients in Group II. RESULTS: Placebo did not change either LVM index or 24-h BP values in Group I and Group II. Diltiazem and lisinopril reduced LVM index in both Groups I and II but 24-h BP values were only reduced in Group II. Lisinopril appeared to be more potent than diltiazem on LVM regression. Slopes of LVM index regression were not different between Groups I and II for each drug. Drug-induced changes of LVM index did not correlate with blood pressure changes. CONCLUSIONS: Drug-induced regression of LVM may be achieved in man (Group I) without any reduction of blood pressure. This may be explained by interference with growth-promoting systems other than with cardiac unloading. Also, the similar pattern of LVM regression that was observed in both Groups I and II suggests that similar underlying mechanisms may be involved in the LVH regression in these two populations. PMID- 8634168 TI - [Implantable cardioverter-defibrillators in patients resuscitated from sudden death and in patients with sustained refractory ventricular tachycardia]. AB - OBJECTIVE: The aim of this study was to evaluate the results of our experience with implantable cardioverter-defibrillator therapy. PATIENT SELECTION: We treated with implantable cardioverter-defibrillator (ICD) 18 patients, 15 male and 3 female, mean age 51 years, ranging from 12 to 76 years, with life threatening ventricular arrhythmias. Eleven patients were resuscitated from cardiac arrest and seven had refractory ventricular tachycardia. The underlying condition was coronary artery disease in 12 patients, dilated cardiomyopathy in two, congenital long QT syndrome in one, mitral regurgitation in one and idiopathic in two patients. METHODS: All patients underwent cardiac catheterization and electrophysiological study before ICD implantation. In the first patient epicardial leads were used, but a transvenous approach was used in the remaining 17 patients. The device was implanted in an abdominal position in all patients. Defibrillation and pacing threshold tests were performed during the implantation procedure and whenever necessary. After implantation, patients were followed up in an outpatient basis, with evaluation of therapy efficacy and reprogramming of the device if required. The mean follow-up time was 16 months, ranging from 1 to 40 months. RESULTS: The implantation was successful and without complications in all patients. Defibrillation threshold was considered within normal range, with an electrode impedance ranging from 40 to 65 Ohms. During the follow-up period no deaths occurred, 8 patients (44%) had episodes of VT or VF that were successfully treated, with shock in six patients, with antitachycardia pacing in one and with both modalities in another patient. Inappropriate shocks were observed in three patients (16%). The device was reprogrammed in five patients. CONCLUSIONS: Our experience with implantable ICD in patients who survived cardiac arrest or with refractory VT has shown a low surgical risk with no mortality. The incidence of ICD discharges was high, with a satisfactory efficacy rate and the number of inappropriate shocks was acceptable. The careful patient selection made possible a good cost/benefit relation. PMID- 8634169 TI - [The 3-year prognosis of patients with suspected coronary disease and a normal myocardial scintigraphy with thallium-dipyridamole]. AB - OBJECTIVES: To assess the 3-year prognosis of patients with suspected coronary artery disease and a normal dipyridamole-thallium scintigram. POPULATION: 43 patients, 16 male and 27 female, mean age 55.8 +/- 9.2 years with suspected coronary artery disease. Forty-two patients had anginal complaints. There was a previous positive treadmill exercise test in 14 patients. All the patients had a normal dipyridamole-thallium SPECT. METHODS: Dipyridamole was infused at a rate of 0.56 mg/kg in 4 minutes followed by 3 minutes of low-level cycloergometer exercise. Two mCi of thallium-201 were injected 3 minutes after the end of dipyridamole infusion. Stress and redistribution SPECT acquisitions were performed respectively 5-10 minutes and 4 hours after thallium-201 injection. The cardiac events during a 3-year follow-up were analysed. RESULTS: No patient had unstable angina, PTCA, CABG or death. One patient suffered an inferior myocardial infarction 28 months after the scintigraphy. The event rate was 0.78% per patient per year. CONCLUSION: Three-year prognosis in patients with suspected coronary artery disease after a normal dipyridamole thallium SPECT is excellent approaching that of the general population. PMID- 8634170 TI - Influence of coronary lesions morphology on treadmill exercise stress testing after acute myocardial infarction. AB - OBJECTIVES: To evaluate the influence of infarct related artery lesion morphology on the exercise stress test performed after a first myocardial infarction. METHODS: We reviewed coronary angiography and the exercise stress test performed before discharge from hospital in 105 consecutive patients (91.4% male, mean age 49.6 +/- 9.2 years) with first acute myocardial infarction. Complex coronary lesions were defined by the presence of one of the following characteristics: ulcers, thrombus, shoulders, irregularities and eccentricity. According to either the existence or absence of complex coronary lesions, two groups were considered: Group I--42 patients with complex coronary lesions, and Group II--47 patients without these characteristics in coronary angiogram. Sixteen pts (14%) were excluded because the infarct related artery was occluded or existence of complex lesions in other coronary artery not related to the infarct. Left ventricular systolic function was analyzed using the "CASS score". Exercise stress test performed between the 10th and the 15th day after myocardial infarction, using Bruce protocol, were reviewed. The following parameters were analyzed: exercise time, number of metabolic equivalent units (METS), maximal heart rate attained, double product variation and number of patients with significant ST segment depression and/or angina. RESULTS: No statistically significant differences between the two groups were obtained as far as age, sex, left ventricular function, number of diseased vessels and lesions severity. From the analysis of ergometric parameters we did not find any difference between the two groups of patients about exercise time (Group A--8.37 +/- 2.6 versus (vs) Group B 8.38 +/- 3.18), METS (Group A--8.22 +/- 2.87 vs Group B--8.13 +/- 2.97), maximal heart rate (Group A--88.2% vs Group B--87.5%) and double product variation (Group A- 14547 +/- 5492 vs Group B--14553 +/- 5387). However, the number of ischemic response (defined by usual criteria of St-segment depression and/or angina) was significantly greater in patients with complex coronary lesions (ST--segment depression: Group A--26 pts vs Group B--19 pts, p < 0.05/Angina: Group A--16 pts vs Group B--4 pts, p < 0.001). Thus, complex coronary lesions are related to a high incidence of residual ischemic phenomena detected by electrocardiographic exercise stress test performed on predischarge period of acute myocardial infarction. Further studies will be necessary to show the prognostic value of particular angiographic characteristics found in coronary plaques. PMID- 8634171 TI - [Echocardiographic evaluation of the aortic and pulmonary valve areas in the newborn infant. Normal patterns]. AB - OBJECTIVES: To determine normal values of the aortic and pulmonary functional valve areas in healthy newborn children. MATERIAL AND METHODS: We prospectively studied 32 newborns (17 boys) who were included in the following criteria: healthy parents, normal pregnancy, eutocic delivery, Apgar index 10 at 5 minutes weight between 2.500 and 4.000 kg, normal physical and echocardiographical examinations. The echocardiographical examination was executed during the first 48 hours of life. We used the continuity equation to calculate the aortic and pulmonary functional valve areas. As a reference we used the anatomic (pi r2) aortic and pulmonary valve areas, calculated in the bidimensional images from the distance measured between the two insertion points of the sigmoid valves. RESULTS: We had echocardiographic images and Doppler registrations of excellent quality, in all the newborn children. The functional pulmonary valve area ranged between 0.30 and 0.50 cm2 (mean +/- SD = 0.41 +/- 0.06) and the anatomical one ranged between 0.29 and 0.49 cm2 (mean +/- SD = 0.49 +/- 0.05), without statistical significance and with a correlation index 0.92. The functional aortic valve area ranged between 0.20 and 0.40 cm2 (mean +/- SD = 0.31 +/- 0.05) and the anatomical ranged between 0.21 and 0.36 cm2 (mean +/- SD = 0.29 +/- 0.05), without statistical significance and with a correlation index 0.91. CONCLUSION: There is a good correlation between the functional aortic and pulmonary valve areas, calculated from the continuity equation, and the anatomical ones. These values will be useful in characterizing the critical stenosis of the newborn child with decreased ventricular function, where the transvalvular gradient is inaccurate in the quantification of the obstruction. PMID- 8634172 TI - [Cardiac thrombosis in myocardial infarct]. PMID- 8634173 TI - [The international relations of the Portuguese Society of Cardiology]. PMID- 8634174 TI - Competency test for basic, low-tech pain control. PMID- 8634176 TI - Pain management--highlighting special populations. PMID- 8634175 TI - Oregon's assisted suicide law. PMID- 8634177 TI - Pain management and psychosocial issues in HIV and AIDS. AB - My goal for this presentation is to provide an overview of the pain experience of patients with AIDS, including focuses on prevalence, common syndromes, and factors that contribute to undertreatment. As a means to dramatize the impact of pain on quality of life, we'll discuss excerpts from a videotape of one of my patients who eloquently expresses the difficulty he had in getting effective pain treatment, and the impact if finally made. Finally, we'll discuss basic management principles, largely adapted from those developed over the past decades for cancer pain, with a special emphasis on issues related to the management of pain in AIDS patients who have a history of substance abuse. PMID- 8634178 TI - Current concepts--pain management and parenteral opioids: an update. PMID- 8634179 TI - Advanced pain & symptom management (a case study-workshop discussion). PMID- 8634180 TI - The vision of hospice and total pain relief. PMID- 8634181 TI - Hospice medicine: a different perspective. PMID- 8634182 TI - Synthesis of p-phosphonomethyl-L-phenylalanine using camphor sultam or D-valine as chiral auxiliaries and its incorporation into integrin sequences. AB - The synthesis of N-Boc-p-phosphonomethyl-L-phenylalanine with two different chiral auxiliaries, camphor sultam or D-valine is described. The preparations have essentially identical properties and have been used to incorporate the amino acid into two integrin peptides as non-hydrolyzable isosteres of phosphotyrosine. PMID- 8634183 TI - Nuclear magnetic resonance studies of thyrotropin-releasing hormone (TRH) and analogues incorporating D-histidine and 4-hydroxy-L-proline. AB - NMR studies have been used to examine conformational effects in thyrotropin releasing hormone (TRH), the epimer incorporating D-His, and their analogues where trans- and cis-4-hydroxy-L-proline replace L-proline (Pro). In all six compounds the observed overall conformation of the major conformer around the Pro His amide bond, and the observed increase of the cis/trans ratio between the conformers when L-His is replaced by D-His, can be accommodated by assuming that a ten-membered ring is formed by hydrogen bonding between the N-H of the Pro carboxamide function and the N pi-atom of the His imidazole nucleus. PMID- 8634184 TI - Synthesis of an AZT-HEPT hybrid and homologous AzddU derivatives. AB - 3'-Azido-2',3'-dideoxyuridines 6 and their corresponding alpha anomers 5 were synthesized by condensation of silylated 6-alkyl and 5,6-dialkyl substituted uracils 2 with methyl 3-azido-5-O-tert-butyldiphenylsilyl-2,3-dideoxy-alpha, beta D-erythro-pentofuranoside (4). Compounds 5 and 6 were treated with tetrabutylammonium fluoride to obtain the deprotected nucleosides 7 and 8, respectively. PMID- 8634186 TI - The current global situation of the HIV/AIDS pandemic. PMID- 8634187 TI - Provisional working estimates of adult HIV prevalence as of end 1994, by country. PMID- 8634185 TI - Acquired immunodeficiency syndrome (AIDS)--data as at 15 December 1995. PMID- 8634188 TI - Influenza. PMID- 8634189 TI - Orthodontic management of a horizontally positioned maxillary lateral incisor. AB - A case report of a patient who had lost her maxillary permanent left central incisor, left cuspid and left first premolar teeth due to a cyst operation, is presented. The maxillary permanent left lateral incisor was in horizontal position high in the vestibule. She was treated with removable and fixed orthodontic appliances. The lateral incisor was uprighted and brought into its normal position in the dental arch. For aesthetics and function missing teeth were replaced with a specially designed prosthesis. PMID- 8634190 TI - The Levandoski Panoramic Analysis in the diagnosis of facial and dental asymmetries. AB - Facial and dental asymmetries in 41 children from 8 to 12 year old were evaluated using the Levandoski Panographic Analysis. A high correlation between the standard facial photograph and linear measurement from panoramic radiograph were found. Varying degrees of facial and mandibular asymmetry were common in 100% of the sample. Significant association between the level of the glenoid fossa and the level of the eye from a frontal point of view were found; eleven patients (36.5%) showed left eye higher accompanied by a higher left fossa. Twenty patients (48.78%) presented with mandibular midline deviation toward the left side. The side with the longer condyle was usually accompanied by a longer coronoid in the ipsilateral side. The results of this study support the usefulness of the Levandoski Panographic analysis in diagnosis of facial and dental asymmetries, and its relation to the findings on the facial photographs. PMID- 8634191 TI - Effect of chlorhexidine gluconate mouth wash on the plaque microflora in children using intra oral appliances. AB - The effect of 0.2% chlorhexidine mouth wash (Hexidine) on the plaque microflora was evaluated on children wearing intra oral removable appliances. Plaque samples were collected from the enamel sections, both primary and permanent, mounted on the removable appliances. These appliances were worn by 12 children for one week. Plaque was allowed to accumulate on the in situ test sites and on the adjacent natural dentition. At the end of the experimental period the plaque microflora associated with the enamel sections were compared with that obtained from lingual and interproximal areas of the lower molar teeth. It was also compared with a control group of 12 children without an appliance. In addition, the effect of 0.2% chlorhexidine mouth wash on the plaque microflora for the next 14 days was also determined on both the groups. Although some quantitative difference was found between the proportion of isolates obtained from the different enamel surfaces, it was not statistically significant. There was a statistically significant increase in the isolates of plaque microflora after the insertion of removable appliance in children which decreased significantly with the use of 10 ml of 0.2% chlorhexidine gluconate mouth wash twice a day. The study indicates that the primary and permanent tooth specimens mounted on the intra-oral device collected plaque microflora similar to that present on the adjacent natural dentition and that chlorhexidine gluconate mouth wash therapy is effective in reducing plaque microflora in children with removable appliances. PMID- 8634192 TI - Microleakage and marginal gap formation of glass ionomer resin restorations. AB - The purpose of this in vitro study was to compare microleakage and marginal gap formation of three light-cured glass ionomer cements (LCGI). Thirty non-carious human premolar teeth were used in this study. A Class V cavity was prepared in the buccal surface of each tooth. The teeth were randomly divided into three experimental groups of 10 teeth each and restored with three LCGI materials. The teeth in each group were processed for microleakage study using 5% methylene blue. Subsequently, each tooth was embedded in acrylic resin and sectioned longitudinally in a buccolingual direction. Examination of the specimens for dye penetration was done. Later, evaluation of marginal gap formation and analysis of adhesion mechanism were done using SEM. The results indicated that LCGI restorations placed in vitro without thermocycling did not consistently prevent microleakage. Vari Glass VLC showed the least leakage. There was no significant difference in the microleakage between the occlusal and gingival margins. Marginal gap formation was found only in one LCGI material. Generally, the presence of a clearly identified ion-exchange layer was not found. PMID- 8634194 TI - Study of cervical pulp horns in human primary molars. AB - This study confirmed that cervical pulp horns exist in human primary molars as the following results were found: 1. Fifteen cervical pulp horns were observed in the 40 extracted primary molars group (37.5%), 2. Ten cervical pulp horns were observed out of the 26 Indian primary molars group (38.5%), 3. Buccal extension of cervical pulp horns is 0.23 mm in the extracted primary molars group and 0.36 mm in the Indian primary molars group on average, 4. Cervical pulp horns of primary molars tend to be presented bilaterally in the same individual, 5. The percent of occurrence of cervical pulp horns in primary molars (37.6%, 38.5%) was lower than that of the permanent molar (77.1%). PMID- 8634193 TI - Artificially formed caries-like lesions around Class II glass ionomer restorations in primary molars. AB - The aim of this study was to investigate the microleakage around Class II glass ionomer restorations in primary molars. Two different glass ionomer cements and one amalgam control were assessed in vitro by acidified gel technique. Assessment of caries-like lesions was performed using polarized light microscopy. The traditional glass ionomer (Chemfil II) with dentin conditioner provided the highest protection against caries attack and the amalgam restorations provided the least. PMID- 8634195 TI - Endodontic treatment of a mature tooth with an abnormal clinical crown. AB - In this report, a case of a mature tooth with abnormal crown morphology, which created plaque retentive areas was presented. The pulp was necrosed due to the periodontal disease, and the anomaly was thought to be an aberration of a talon cusp. Therapy involved the reshaping and the endodontic treatment of tooth. PMID- 8634196 TI - Crown dilaceration of permanent incisors following trauma to their primary predecessors. AB - Crown dilaceration of a permanent tooth constitutes 3% of traumatic injuries to developing teeth. It usually involves the maxillary incisors and less frequently their mandibular counterparts. This report concerns a patient, whose primary mandibular central incisors, following a fall at the age of 14 months, sustained partial avulsion with displacement. The teeth were repositioned by the father, with the aid of a pediatrician, and without the assistance of a dentist. The patient presented at the age of 8 years, and, following clinical and radiographic examination, a diagnosis was made of crown dilaceration with lingual displacement of the incisal third of the permanent mandibular central incisors, and hypoplastic enamel on all but the gingival third of their crowns. In addition to this, a supernumerary tooth was discovered labial to and between the mandibular right permanent central incisor and the mandibular right primary lateral incisor. After a brief review of the literature, the treatment of this patient is described and the restoration of the dilacerated teeth, followed by a discussion on crown dilaceration and issues concerning the restoration of such teeth. PMID- 8634197 TI - Prosthetic rehabilitation of a child with induced anodontia. AB - A case of prosthetic rehabilitation of a five and half year old child with induced anodontia is presented. Complete dentures were provided and patient was followed for a period of 20 months. As the permanent teeth erupt into the oral cavity, regular modifications have to be made on the denture base to facilitate their eruption. PMID- 8634198 TI - Dens invaginatus of a mandibular central incisor: surgical endodontic treatment. AB - Dens invaginatus is a developmental malformation that mostly affects maxillary lateral teeth and mandibular teeth are rarely affected. In this paper a mandibular central incisor with a dens invaginatus is identified as requiring both endodontic and surgical treatment is presented. At one year recall appointment satisfactory clinical and radiographic healing was evident. This case demonstrates that also periapical surgery should be performed in some cases with dens invaginatus for resolution of periapical tissues. PMID- 8634199 TI - Submandibular sialolithiasis with concurrent sialoadenitis in a child. AB - Sialolithiasis is a relatively common condition in adults, but it is rarely observed in children. We report a case in a 7-year-old child with a sialolith in the anterior right Wharton's duct resulting in a submandibular sialoadenitis. Under local anesthesia the calculus was removed by means of a sialodochotomy. Post-operative recovery was uneventful and follow-up examinations showed recovery of the function of the affected gland. PMID- 8634200 TI - Parotid sialolithiasis in a child. AB - The occurrence of sialolithiasis in children is uncommon, while parotid sialolithiasis in children is rare. A case of parotid sialolithiasis arising in a 4-year-old girl is presented. PMID- 8634201 TI - A typical Hallermann-Streiff syndrome in a 3 year old child. AB - Hallermann Streiff syndrome is a rare congenital disorder characterized by dyscephaly, dental anomalies, proportionate nanism, hypotrichosis, cutaneous atrophy limited to the head, bilateral congenital cataracts and bilateral microphthalmia. Despite the marked craniofacial characteristics and oral findings, a relative lack of reports in the dental literature has been noted. In this article, a review of the literature regarding the general clinical features and differential diagnosis is presented. A case of a 3-year-old female is reported with special consideration on the management of her dental problems under general anesthesia. The anesthetic risks of such a procedure as well as the prognosis of her dental development are being discussed. PMID- 8634203 TI - Enamel caries initiation and progression following low fluence (energy) argon laser and fluoride treatment. AB - The aim of this study was to evaluate the effect of low fluence argon laser (AL) and acidulated phosphate fluoride (APF) treatment on caries initiation (CI) and progression (CP) in human enamel. Twenty caries-free molars were divided into tooth quarters. Tooth quarters from each specimen were assigned to one of four groups: 1) Control; 2) AL Only; 3) AL before APF; 4) APF before AL. AL was at 0.25 watts for 10 seconds (12.0 +/- 0.5 J/cm2). APF treatment was with a 1.23% gel for 4 minutes. Lesions were created in two treated, sound enamel windows per tooth quarter with an acidified gel. After CI and CP, sections were obtained and imbibed with water for polarized light study. Mean body of the lesion (BL) depths were determined and compared among groups (ANOVA & DMR). After CI, BL depths were: 189 +/- 29 micrometers for Control, 133 +/- 23 micrometers for AL only; 91 +/- 17 micrometers for AL before APF; and 83 +/- 14 micrometers for APF before AL. After CP, BL depths were: 321 +/- 43 micrometers for Control, 206 +/- 35 micrometers for AL only; 118 +/- 21 micrometers for AL before APF; and 114 +/- 19 micrometers for APF before AL. After CI and CP, argon laser irradiation alone resulted in significant reductions in lesion depth when compared with controls (p<0.05). APF treatment before or after argon laser exposure resulted in a significant reduction in lesion depth when compared with AL alone or control groups (p<0.05). Caries initiation and progression in vitro are affected to a significant extent when low fluence (energy) argon laser irradiation of sound enamel alone or in conjunction with APF treatment is done. This caries-protective effect occurs at an argon laser fluence (energy) that is capable of polymerizing visible light-cured resins. PMID- 8634202 TI - Centronuclear myopathy and nursing pattern caries: management of a 1 year old. AB - The pathognomonic open mouth of centronuclear and other myopathies can also have implications for oral health. These patients are at greater risk for gingivitis, dental caries as a result of the mouth breathing and loss of lip seal. Low tongue posture may impede the lateral expansion of the maxilla resulting in a constricted maxillary arch with resultant posterior crossbites and functional shifts. Excessive molar eruption may also occur, resulting in anterior open bites and increases in the anterior lower vertical height. PMID- 8634204 TI - Modified Twin Blocks: fabrication method and use in a child with a Class II malocclusion. AB - The twin blocks technique was developed by Dr. William Clark of Scotland during the early 1980s. Twin Blocks are an uncomplicated system that incorporates the use of upper and lower bite blocks. These bite blocks reposition the mandible and redirect occlusal forces to achieve rapid correction of malocclusions. They are also comfortable and the patients wear them full-time--including eating time. Occlusal forces transmitted through the dentition provide a constant proprioceptive stimulus to influence the rate of growth and the trabecular structure of the supporting bone. This feature of Twin Blocks means easier and quicker treatment. The occlusal inclined plane is the fundamental functional mechanism of the natural dentition. Twin Blocks are bite blocks that effectively modify the occlusal inclined plane to induce favorably directed occlusal forces by causing a functional mandibular displacement. Upper and lower bite blocks interlock at a 45 degree angle and are designed for full-time wear to take advantage of all functional forces applied to the dentition including the forces of mastication. The patients who were treated with Modified Twin Blocks received the following benefits: 1) large overjets and deep overbites were corrected. 2) Class II molar relationships were changed into Class I, and 3) the profiles of the patients were improved by anterior displacement of mandible. PMID- 8634205 TI - Apexification in the endodontic treatment of pulpless immature teeth: indications and requirements. AB - The problems of performing the apexification technique, the indications and requirements for apexification are discussed. A case report is shown as an example. PMID- 8634206 TI - Relaxation and hypnosis in pediatric dental patients. AB - Relaxation and hypnosis are methods which, may solve the problem of extreme dental anxiety, when all other methods, behavioral or pharmacological may not be used. A simple definition of hypnosis is suggestion and repetition. Suggestion is the process whereby an individual accepts a proposition put to him by another, without having the slightest logical reason for doing so. Relaxation is one method of inducing hypnosis. A case of using hypnosis on an 11-year-old boy is described. PMID- 8634207 TI - The effect of nursing or rampant caries on height, body weight and head circumference. AB - In this study, the effect of nursing bottle caries and rampant caries on height, weight and head circumference was evaluated. For this purpose, 126 children, aged 3 to 5 years old, who have nursing or rampant caries were selected. One hundred twenty-six children with no caries and similar age and sex were matched as a control group. When the children who had rampant or nursing caries were evaluated in the direction of the mean weight, it was corresponding to mean weight between 25th and 50th percentiles. The mean percentile weight for control group were corresponding to mean weight between the 50th and 75th percentiles. Of the nursing or rampant caries children, 7.1% weighed less than 80% of their ideal weight, compared with only 0.7% of the control group children. When the children, who had rampant or nursing caries, were evaluated in the direction of the mean height, it was corresponding to mean height between 10th and 25th percentiles. The mean percentile height for control group were corresponding to mean height between the 25th and 50th percentiles. When the head circumference is evaluated, there was no significant statistical difference between the two groups. Since the height and weight of the control group showed a higher percentile category than the nursing or rampant caries group, (P<0.001), it can be stated that rampant or nursing caries may correlate with adversely affected growth of the body. PMID- 8634209 TI - Children's stress during a restorative dental treatment: assessment using salivary cortisol measurements. AB - Dental environment may be a source of stress for the young patient. Such stressful conditions may provoke fear in anxious children. It is well known that stress produces an activation adrenal steroid secretion. Among the methods for assessing child dental fear, measurement of salivary cortisol level is a simple method, because especially in children, sampling of saliva is easy, and cortisol levels in saliva closely mirror serum free cortisol levels, independent of salivary flow rate. For this study, the salivary cortisol levels of 8 children (mean age 5.6 yr) were measured receiving initial dental treatment. Saliva samples were collected via cotton rolls placed to the floor of the mouth at four stages; prior to treatment, during cavitation, placement of the liner and the restoration. Statistical comparison of the results were done by Student-t test. The increase in salivary cortisol levels during cavitation at the first and secondary appointments were significant (p<0.01 and p<0.05), but not at the second. The other comparisons were not statistically significant (p>0.05). The results of this study suggest that in restorative procedures, mostly it is the cavitation step that creates stress and anxiety in children. Knowledge of the most stressful condition may be helpful for the dentist to prepare the child to treatment steps. PMID- 8634208 TI - Laser and light cured composite resin restorations: in-vitro comparison of isotope and dye penetrations. AB - The extent of marginal leakage in class V composite resin restorations cured by visible light and argon laser was observed, in vitro, using 32 extracted primary and permanent anterior teeth, free of caries and any observable fracture lines. Marginal leakage was observed by both 2% methylene blue dye and phosphorous (P32) isotope methods and their efficacy was also assessed. There was a statistically higher degree of marginal leakage in restorations cured by argon laser as compared to visible light by dye method whereas it was not of statistical significance by isotope method. Irrespective of the type of curing and method of observation, primary teeth showed higher degree of marginal leakage. Additionally cervical margin of all the restorations in both primary and permanent teeth showed higher degree of marginal leakage to that of incisal margin by both curing systems and methods used. The dye method was observed to be superior to isotope method towards assessing the extend of marginal leakage. PMID- 8634210 TI - Subgingival irrigation effects of chlorhexidine or sanguinarine on gingivitis in orthodontic patients. AB - This clinical investigation examined the effect of a single subgingival irrigation of chlorhexidine 0.2% or sanguinarine on gingivitis affecting orthodontically banded first molars in adolescent patients. Eighteen patients with gingivitis participated in the study. Probing depth, papilla bleeding index and plaque index were recorded at four sites for three molars at baseline, 2 weeks and 4 weeks by one investigator. A second investigator irrigated a single application of 3 ml of either chlorhexidine, sanguinarine or saline. The gingival bleeding as determined by papilla bleeding index was almost eliminated in the 4 week period. A reduction of the plaque index and probing depth was observed in all three groups. A significant difference related to probing depth between the effect of saline and chlorhexidine (p<0.01) was noted. PMID- 8634212 TI - Hemisection of a permanent mandibular first molar: a treatment option for a vertically impacted second premolar. AB - Hemisection as the name implies is the division of a tooth and removal of the unwanted portion thereafter. This technique was performed on a mandibular first permanent molar. An additional advantage of the procedure in the present case was that the impacted second premolar got adequate space to erupt into its occlusion without any further surgical sacrifices of the badly involved carious permanent molar or the impacted premolar itself. PMID- 8634211 TI - Periodontal status in childhood and early adolescence: three-year follow up. AB - The 4-year study examined the interrelationship between dental plaque, gingival inflammation and gingival sulcus depth in a 78 children (41 boys, 37 girls, aged 1 to 12 years at initial examination) living in a rural community in Israel. Plaque index (PlI) remained essentially the same throughout the study period for both primary and permanent dentition, while gingival index (GI) and sulcus depth increased (p<0.001 for sulcus depth). Sulcus depth was significantly greater in the older children at each yearly examination (p<0.001). A strong correlation existed between age and sulcus depth at each yearly examination (r=0.644, p<0.001). Comparison of PlI and GI with "delta sulcus" failed to show statistical significance between these parameters. The findings strengthen the hypothesis that the increase in sulcus depth in children and young adolescents is associated primarily with age and to a much lesser degree with inflammatory response. PMID- 8634213 TI - The treatment of a fusion between the maxillary central incisor and supernumerary tooth: report of a case. AB - Fusion has been described as a developmental anomaly characterized by the union of two adjacent teeth. Supernumerary teeth develop as a consequence of the proliferation of epithelial cells from dental lamina. In this paper a fusion between maxillary central incisor and a supernumerary tooth which required endodontic, surgical and periodontal treatment is presented. PMID- 8634214 TI - Salivary caries risk factors in long-term event-free survivors of pediatric malignant diseases. AB - In this study we demonstrated that caries prevalence and Plaque Index in long term event-free pediatric oncology patients are related to Streptococcus mutans, Lactobacillus counts and buffer capacity obtained by chairside saliva tests. The scores showed a significant correlation between the microbiological findings and caries experience in both groups. The results were compared with a control group. A similarity in the results was found between the study and control groups. In a subgroup consisting of children who were diagnosed with cancer maximum two years before oral examinations, no significant differences with a control group was noticed. In this study we did not find any evidence of long-term effects on the studied salivary caries risk factors in children who are long-term event-free after cytotoxic treatment. Chairside tests seem to be useful in this patient group: they provide us information which can contribute to the determination of the individual caries risk, and help to motivate the patient and health care workers to maintain optimal oral hygiene. PMID- 8634215 TI - Synodontia between maxillary central incisor and a supernumerary incisor teeth: a dental, genetic and dermatoglyphic study. AB - This case report contains the dental, genetic and dermatoglyphic findings of a patient showing bilateral maxillary central incisors fused to an extra one and his paternal uncle having macrodontia of central incisor teeth. PMID- 8634217 TI - Genetic polymorphisms of the Caucasus ethnic groups: distribution of some blood group genetic markers (Part II). AB - The compiled data on the distribution of polymorphic blood groups (ABO, Diego, Duffy, Kell-Cellano, Kidd, MN, MNSs, P, Penney, Rh(D), Rh-Hr), secretion ABH antigens in saliva, HLA system (HLA-A, HLA-B, HLA-C, HLA-DR), immunoglobulin (GM1) and other miscellaneous data (phenylthiocarbamide taste, tongue rolling) in the Caucasus are presented. Results of the interpopulation heterogeneity test show that, in spite of the limited territory of the Caucasus, a high level of genetic variability was observed. In terms of gene frequencies, these ethnic groups are approximately equidistant from European and West Asian Populations. PMID- 8634216 TI - Melanotic neuroectodermal tumor of infancy: report of a case. AB - The melanotic neuroectodermal tumor of infancy (MNTI) is an uncommon benign neoplasm frequently detected in young infants, which has a predilection for the head and neck region, particularly maxilla. The purpose of this article is to present a documented case of MNTI and discuss clinical, radiological and histopathological features of the disease. The biological behavior of the tumor is emphasized. PMID- 8634218 TI - Heterogeneity in the distribution of ABO blood groups in Hungary. AB - Estimates of the gene frequencies for the ABO system (i = 0.5593, IA = 0.2989, IB = 0.1418) based on a random sample of more than two hundred thousand voluntary donors from the 19 countries of Hungary are provided. Populations West of the Danube differ significantly from those East of the Danube for the frequencies of i and IB. These data are consistent with previous ones on the HP gene. PMID- 8634220 TI - Genetic polymorphisms of the Caucasus ethnic groups: distribution of some serum protein and red cell enzyme genetic markers (Part I). AB - The compiled data on the distribution of polymorphic serum proteins (HP, C3, GC), red cell enzymes (ESD, GlO1, PGD, AK1, ADA, GPT, PGP, PGM1, ACP1) and also on some monomorphic systems (ALB, CAI, CAII, CP, G6PD, HBA, HBB, IDH1, LDHA, LDHB, MDH1, PEPA, PEPB, PEPC, PGM2, PHI, TF) in the Caucasus are presented. The interpopulation heterogeneity test shows a high level of genetic differentiation in the following loci: HP, GC, ESD, AK1, TF, PGD. Gene frequencies in the Caucasian ethnic groups were found to be approximately equidistant from those of European and West Asian populations, in line with their geographical location. PMID- 8634219 TI - DNA profiling of the VNTR locus D2S44 in the population of Madras City and in the tribal Kotas of the Nilgiri Hills, south India. AB - Allele frequency distribution of the VNTR locus D2S44 was studied in Tamil Nadu (South India) population. Randomly chosen individuals (Tamils of the plains, Madras City; N = 142) were tested for HaeIII-generated polymorphism detectable by probe YNH24, and the allele sizes and frequencies were determined. Heterozygosity (93.6%) observed in the Tamils is comparable to that of other populations; the size and frequency distribution of alleles, however, vary significantly. The most prevalent allele, which ranges from 1.2 to 1.9 kb, appears to be unique to the Tamil Nadu population. No mutation was observed for the D2S44 locus in family material made up by 54 subjects (N = 54) including 37 offspring. DNA polymorphism at D2S44 locus was also studied in the endogamous Kota tribe of the Nilgiri Hills, South India, using enzyme HaeIII and probe YNH24. The Kota group (N = 48) is characterized by a very high frequency (32.3%) of the bin 1197-1352 bp. The DNA profile of the Kotas shows distinct differences from that of the urban population in the plains of Tamil Nadu, South India (Tamils of Madras City). The results have also been compared with the literature available on other world populations. The outcome indicates the uniqueness of the tribal Kotas and warrants the importance of DNA profiling in other tribal, caste/endogamous groups of India. This report incidentally represents the first comprehensive DNA profiling data for the locus D2S44 from India fulfilling the requirement for forensic and other applications. PMID- 8634221 TI - Neurodevelopmental problems in Ghanaian children: Part I. Convulsive disorder. AB - Children with convulsive disorder made up 3% of new patients seen in the paediatric department over a ten year period and 51.5% of children subsequently enrolled in the paediatric neuro-developmental clinic of the Korle Bu Teaching Hospital in Accra, Ghana. Generalised tonic-clonic seizures were the commonest type of seizure (76.5%), followed by complex seizures (14.8%) and temporal lobe seizures (3.4%). Majority of patients had no definable cause and normal neurological examination. 47.7% of electroencephalographs had diagnostic abnormalities. Most patients responded well to conventional antiepileptic drugs (AEDs) leading to discontinuation of drug therapy in 48 (7.1%) patients who remained seizure-free for two or more years. Adverse drug reactions and laboratory test abnormalities were rather uncommon. Discontinuation of antiepileptic therapy after effective long-term control is discussed. PMID- 8634222 TI - Changes in blink rates of Nigerian schizophrenics treated with chlorpromazine. AB - Chlorpromazine was administered over a 4-8 week period to 40 schizophrenic patients who had not received previous neuroleptic drug treatment out of a total of 222 schizophrenic patients seen during the study period. Pre-treatment spontaneous eye blink rate was high in the 40 schizophrenics when compared to 34 normal controls, (22 vs 14 blinks/min respectively, t = 17.04, df = 72, p < 0.001). Clinical improvement in the schizophrenic patients correlated with a significant reduction in their pre-treatment versus post-treatment spontaneous eye blink rate (22 vs 9 blinks/min, t = 18.83, df = 78, p < 0.001). The blink rate in the clinically-well schizophrenic patients did not however, reach the level obtained in the normal controls but rather was lowered significantly (9 vs 14 blinks/min, t = 8.11, df = 72, p < 0.001). Blink rate changes in schizophrenic patients on neuroleptic treatment may serve as a quick bedside assessment of clinical improvement and can also be a pointer to those patients who may have sub clinical drug induced parkinsonism. PMID- 8634223 TI - Advanced maternal age and pregnancy. AB - Advanced maternal age pregnancies are still prevalent due to ignorance and lack of effective contraception. These pregnancies have a poorer maternal and perinatal outcome. PMID- 8634224 TI - Diagnosis of ocular disease in Ibadan by ultrasound. AB - 15 patients with ocular disease, in whom views of posterior segment of the eyes could not be obtained underwent ultrasonography of the eyes to aid in the diagnosis and further management of the patients. In some cases the ultrasound was then compared with the histological diagnosis when the eye was removed and in other cases to the ophthalmoscopic examination when a better view of the posterior segment could be obtained. This was done as a pilot study to ascertain the level of accuracy of our ultrasonic diagnosis and how much it could be depended upon for the very important decision of removal of the eye when indicated. PMID- 8634225 TI - The impact of the Expanded Programme on Immunisation on measles-induced sensorineural hearing loss in the western area of Sierra Leone. AB - Measles was found to be responsible for 46.1% of acquired sensorineural hearing loss in children in a study conducted in the western area of Sierra Leone between 1975 to 1985. 125 children with acquired sensorineural hearing loss were seen and investigated in this study, which was conducted to determine the impact of the Expanded Programme on Immunisation and measles-induced hearing loss. 17 children had sensorineural hearing loss consequent to an attack of measles. A progressive drop in the number of cases seen from 6 in 1986 to 0 in 1989 was observed. During the same period the measles immunisation coverage in Sierra Leone increased from 21% in 1986 to 75% in 1990. Given that measles and some other causes of sensorineural hearing loss were preventable, it was hypothesised that lower incidence of these diseases would result in lower rates of acquired sensorineural hearing loss. This study demonstrated an inverse relationship between the incidence of measles induced sensorineural hearing loss and the increased immunisation coverage when Universal Child Immunisation (UCI) was attained in 1990. PMID- 8634226 TI - Under fives nutritional status in rural communities in western Nigeria. Use of age-standardised weight and mid upper arm circumference. AB - The nutritional status of all children under five years in each of two rural areas in the forest belt of western Nigeria was assessed by visiting overseas medical students. Each child under five was weighed and their mid upper arm circumference (MUAC) measured. One quarter of the children had weights below the third percentile for age (WHO standards). In one village nearly half of 45 children had weights below the third percentile and one sixth had weights further below the median than three standard deviations. Standardised weight scores (against reference standards) tended to be a little higher than, but correlated fairly well (r = 0.67) with standardised MUAC. New amalgamated standards for MUAC from 0 to 60 months are presented. PMID- 8634227 TI - Presentation and outcome of sporadic acute bacterial meningitis in children in the African meningitis belt: recent experience from northern Nigeria highlighting emergent factors in outcome. AB - Sixty-six (19.4%) of 341 acutely ill infants and children (> 1 mo-15 yr old) who had a lumbar puncture (LP) done during an inter-epidemic period had bacterial meningitis (BM). No clinical feature was sufficiently characteristic of the presence of BM. Twenty (30.3%) of the 66 patients with BM lacked typical signs of meningitis at the time of diagnosis whereas 61 (22.2%) of the 275 with other illnesses had signs. Three (4.6%) of the 66 patients with BM were discharged against medical advice, 31 (47%) survived intact and 16 (24.2%) each died or survived with sequelae. Case fatality rate was significantly higher in children with coma, focal extracranial infections, delayed diagnosis of BM after admission, irregular administration of antibiotic drugs and treatment with dexamethasone. Among survivors, sequelae rate was significantly higher in children with delayed presentation, convulsions, coma, and prolonged hospitalisation (> 10 days); sequelae rate in patients with convulsions was significantly higher in those with complex convulsions and convulsions occurring after 24 hours of treatment. Irregular provision of drugs by parents and delay in the diagnosis of BM after admission are emergent factors which, in addition to the well known factors of malnutrition and delayed presentation, further worsen the prognosis of BM. A more liberal policy in the use of LPs in acutely ill children is advocated to reduce the risk of missed diagnosis. PMID- 8634228 TI - The electrocardiogram of pregnant Nigerian women. AB - Data from standard electrocardiographic recording in 41 pregnant Southern Nigerian women and in 39 non-pregnant controls of similar ages are given. The results show a mean of 12 degrees axis deviation between the pregnant and non pregnant subjects, with mean values of 44 +/- 23.00 degrees and 56 +/- 11.00 degrees respectively. Compared to the results in Caucasians, there was greater QRS axis deviation from the 1TM to the 3TM in Africans. Mean heart rate of the Nigerian subjects was lower than that reported for the Caucasians, although it was higher than that of the non-pregnant controls. The durations and amplitudes of the different waves and intervals were greater in the non-pregnant subjects. These values also varied between Caucasians and Africans. The occurrence of the S1Q3T3 pattern was observed; with Q3 and T3 increasing significantly (P < 0.05) in the 3TM. A greater significant (P < 0.05) difference in the occurrence of the T3 pattern was observed between the pregnant and the non-pregnant subjects, showing that the T3 is the most prominent ECG change during pregnancy. PMID- 8634229 TI - Urinary Bilharziasis among Nigerian school children: a study in the Agulu Lake Basin, Anambra State of Nigeria. AB - Following the observation of cases of haematuria among a secondary school population near Agulu Lake, an area not previously regarded as an endemic focus of schistosomiasis, a field study on urinary Bilharziasis was carried out among primary school pupils in the three towns surrounding Agulu Lake which was suspected to be the source of the infection. It was discovered that the area is an endemic focus of Schistosoma haematobium with prevalence rates ranging up to 77.6% among the primary school populations in one school. Schools in the parts of the three towns, (Agulu, Nri and Adazi) closest to the Lake proved to be highly endemic with Agulu having the highest prevalence rate (47.2%), followed by Nri 29.9% and Adazi 2.4%. Males were affected slightly more than females and the prevalence increased steadily in both sexes with age in the primary school population studied (5-15 years). Most of the cases were asymptomatic (54.6%). Macroscopic haematuria was noted to be the most sensitive criteria for clinical diagnosis. Interview of the subjects suggested that other areas outside the zone might also be involved. PMID- 8634231 TI - Is the height/plasma creatinine formula of Schwartz useful as a screening test in detecting Nigeria children with low GFR? AB - A group of 42 children with renal diseases seen at the University College Hospital Ibadan were studied in order to evaluate the usefulness of the height/plasma creatinine formula of Schwartz et al i.e. GFR ml/min/1.73 m2 = 0.55 x Height (cm)/Plasma creatinine (mg/dl) in identifying children with renal impairment. The children were divided into 2 groups of those with GFR as measured by endogenous creatinine clearance (Ccr) < 60 ml/min/1.73 cm2 and those with CCr > 60 ml/min/1.73 cm2. There were 21 children in each group. In detecting patients with Ccr less than 60 ml/min/1.73 m2, Schwartz formula had a sensitivity of 52%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 68%. It is concluded that in detecting patients with impaired renal function who may need more accurate methods of estimating GFR, Schwartz formula has a low sensitivity and therefore may not be useful as a screening method. PMID- 8634230 TI - Open clinical trial of roxithromycin in patients of Plateau Hospitals, Jos in upper and lower respiratory tract infections. AB - An open clinical study to assess the efficacy and tolerance of Roxithromycin 150 mg twice daily was carried out amongst Nigerian patients with upper and lower respiratory tract infections at Plateau Hospital Jos. Twenty-two patients aged between 13 and 86 years comprising of twelve women, seven men and three children completed the study. 18 (81.8%) had bronchopulmonary infections, 3 (13.6%) had tonsillitis and 1 (4.6%) had otitis media. Pathogens isolated included streptococcus Pneumonia (22.7%), Streptococcus pyogenes (13.6%), Bramhella Catarrhalis (9.1%), Haemophilus influenzae (9.1%), Staphylococcus Aureus (4.6%), Klebsiella species (4.6%), Pseudomonas Aeruginosa (4.6%). There was 88.2% bacteriological cure and patients responded fast, with no major adverse reactions. Roxithromycin is therefore concluded to be an effective well tolerated drug for treatment of respiratory tract infections in Nigerians. PMID- 8634232 TI - A retrospective study of oral cysts in Nigerian children. AB - A retrospective analysis of oral cysts that were seen over an 11-year period in children at a dental institution in Nigeria was carried out. In general, oral cysts accounted for only 2.6% of the total biopsied lesions during the period under review. The most common oral cysts were the mucous retention cysts, the gingival cysts of infants and the dentigerous cyst. The commonest sites were the maxilla, the mandible and the floor of the mouth respectively and there was no significant difference in sex preference. Most of the cases were seen in the age group 11-16 years while the least was in the group aged 6-10 years. PMID- 8634233 TI - Foetal macrosomia in Cameroon: prevalence, risk factors and complications. AB - 102 neonates weighing 4000 g and above were compared to a control group with similar characteristics except for the weight. The sex distribution was 74.19% males and 25.81% females. The prevalence of macrosomia was 6.41%. Maternal risk factors were: age above 30 years (p < 0.05); grand multiparity P > 5(p < 0.05); post term (p = 0.001); height above 1.65 m (p < 0.001); term weight of 90 Kg and above (p < 0.001); pregnancy weight gain of 15 Kg and above (p < 0.001). Macrosomic neonates had the following complications: foetal distress, shoulder dystocia, respiratory distress and hypoglycaemia. PMID- 8634234 TI - Bacterial causes of acute osteomyelitis in sickle cell anaemia: changing infection profile. AB - The bacterial causes of osteomyelitis were reviewed on 25 patients with sickle cell anaemia using blood culture and direct wound swab. Age range was 2 to 45 years with a median of 23 years. Klebsiella species were cultured in 45% of the blood samples. Staphylococcus aureus was responsible in 20%, Salmonella species in 8% and Streptococcus pyogenes was cultured in 4% of the samples. From direct wound swab culture, Klebsiella was responsible for 36%, Staphylococcus aureus 28% and coagulase Negative Staphylococcus 24%. Proteus species were responsible for 8% and Salmonella was cultured in 4%. The highest number of patients presenting with osteomyelitis occurred in the second decade. From (culture) sensitivity tests, ceftazidine and Ofloxacine were found to be the most effective antibacterial drugs. The preponderance of Klebsiella indicates a change in the previously accepted pattern of infection in which Salmonella species were considered to be the main causative organism. PMID- 8634235 TI - 113Cd nuclear magnetic resonance studies of cabbage histidinol dehydrogenase. AB - Histidinol dehydrogenase (HDH), a dimeric protein, catalyzes two sequential oxidation reactions to yield L-histidine from L-histidinol via L-histidinal. HDH contains 1 mol of Zn(II) per mol of subunit, and removal of this metal abolishes the enzymatic activity. On substitution of Zn(II) with 113Cd(II), the enzyme ([113Cd]HDH) showed similar catalytic activity. The 113Cd NMR spectra of [113Cd]HDH were measured under various conditions. The 113Cd NMR spectrum of [113Cd]HDH showed a resonance at 110 ppm, which indicates that the metal ion is bound to the protein by a combination of nitrogen and oxygen ligands. 113Cd NMR spectra of [113Cd]HDH were measured as complexes with two substrates (L histidinol and DL-histidinal) and four inhibitors (imidazole, histamine, L histidine, and DL-4-(4-imidazolyl)-3-amino-2-butanone) in the absence and presence of NAD+. Significant shifts of [113Cd]-HDH resonance in the presence of the ligand indicate that the metal ion is located in the catalytic site of HDH and that substrates and inhibitors interact with the metal ion. The role of the metal ion in the HDH reaction is discussed. PMID- 8634236 TI - NMR spectroscopic evidence that helodermin, unlike other members of the secretin/VIP family of peptides, is substantially structured in water. AB - The structure in water and additionally in 50% trifluoroethanol (TFE) solution of helodermin, an amidated peptide consisting of 35 amino acids, was elucidated by 2D 1H NMR spectroscopy initially from H alpha chemical shifts and qualitative NOE data. Detailed structures were calculated from the quantitative NOE data which were used as distance restraints in molecular dynamics and energy minimization calculations. Regions of stable secondary structure were defined from the resulting final peptide conformations using a new fitting program that takes into account the summed RMS differences between all structures for short segments of 2 5 residues in length. This procedure allows a reasonably objective method of defining the edges of stable structure. In contrast to other members of the secretin/VIP family of peptides, helodermin shows a defined secondary structure in water alone and possesses an alpha-helix from Glu-9 to Leu-23 that was further stabilized and slightly extended (Phe-6 to Ala-24) on addition of TFE. The N- and C-termini were unstructured in both solutions. Such features, in particular the observation of a linear helix 18 +/- 2 residues in length, are common to other members of the family and become more pronounced in hydrophobic environments. The data provide further circumstantial evidence that an alpha-helix conformation is necessary for receptor binding. The prolonged physiological action of helodermin, compared to its C-terminal deletion analogues and VIP, is at least in part due to the unusual stable secondary structure. PMID- 8634237 TI - Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue. AB - Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry. Predicted mass from the DNA sequence for the Met processed enzyme was 34 196. A pET3d-IUNH construct expressed in E. coli introduced MetAla instead of MetPro at the N-terminus. Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme. PMID- 8634238 TI - Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. AB - Protozoan parasites rely on the host for purines since they lack a de novo synthetic pathway. Crithidia fasciculata salvages exogenous inosine primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relatively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucleoside hydrolase (IU-NH) from C. fasciculata determined by X-ray crystallography at a nominal resolution of 2.5 A. The enzyme has an open (alpha, beta) structure which differs from the classical dinucleotide binding fold. IU-nucleoside hydrolase is composed of a mixed eight-stranded beta sheet surrounded by six alpha helices and a small C-terminal lobe composed of four alpha helices. Two short antiparallel beta strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of beta strands beta 1 and beta 4. Four aspartate residues are located at the bottom of the cavity in a geometry which suggests interaction with the ribose moiety of the nucleoside. These groups could provide the catalytically important interactions to the ribosyl hydroxyls and the stabilizing anion for the oxycarbonium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates purine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchettini, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The substrate binding site is unlike that from purine nucleoside phosphorylase, phosphoribosyltransferases, or uracil DNA glycosylase and thus represents a novel architecture for general acid base catalysis. This detailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], allows analysis of the interactions leading to catalysis and an explanation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262]. PMID- 8634239 TI - NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48. AB - The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2). PMID- 8634240 TI - Delocalizing trypsin specificity with metal activation. AB - Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25 fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30 fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites. PMID- 8634241 TI - X-ray structures of a designed binding site in trypsin show metal-dependent geometry. AB - The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 A resolution via X-ray crystallography in the absence and presence of the transition metals Zn2+, Ni2+, and Cu2+. The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket. Three histidine side chains, two on trypsin at positions 143 and 151 and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket. Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis. Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc binding proteins. PMID- 8634242 TI - Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases. AB - The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution. The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein. Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium. The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta strands, four parallel and two antiparallel. The core beta-sheet is surrounded by nine alpha-helices. The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix. The active site pocket is funnel-shaped. The narrow opening of the funnel is wide enough for a water molecule to pass. The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle. The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141. The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase. However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate. The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase. The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain. In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family. PMID- 8634243 TI - Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate. AB - Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12). The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit. Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. The model was refined by least-squares analysis to a nominal resolution of 2.1 A and a crystallographic R-factor of 15.4% for all measured X-ray data. As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide. The zinc ions are separated by 3.3 A. The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme. Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide. The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 A from the more solvent-exposed zinc ion. The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket. A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein. This most likely explains the broad substrate specificity exhibited by phosphotriesterase. The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide. PMID- 8634244 TI - Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite. AB - The solution structure of loop A from the hairpin ribozyme found in the minus strand of tobacco ringspot virus satellite has been determined by NMR spectroscopy. The ribozyme consists of two internal loops flanked by short helices: loop A and helices I and II include the substrate and substrate binding site; loop B and helices III and IV are the catalytic domain. Loop A is a symmetric internal loop of eight nucleotides that contains the cleavage site. The 2-amino group of the guanine immediately 3' to the cleavage site is essential for catalysis. NMR results show that this guanine forms a sheared G.A base pair. The cytosine residue immediately 5' to the cleavage site forms an AH+.C base pair with an adenine whose pKa is shifted to 6.2 to allow partial protonation near neutral pH. Although the residues flanking the cleavage site are stacked in an A form pattern, the phosphodiester backbone next to the cleavage site on the 3' side is splayed apart. This places the following base-a uracil-in the expanded major groove. The conformational flexibility and the lack of steric hindrance of the uracil as well as the unoccupied Watson-Crick positions on the sheared G.A base pair can allow loop A to specifically interact with the catalytic domain (loop B) without drastically changing its own conformation. The three-dimensional structure of loop A provides explanations for previously published mutation and structural mapping results. PMID- 8634245 TI - Trypanosomal nucleoside hydrolase. Resonance Raman spectroscopy of a transition state inhibitor complex. AB - The transition state for hydrolysis of the N-ribosidic bond of inosine by nucleoside hydrolase has oxocarbenium character and a protonated leaving group hypoxanthine with an sp2-hybridized C1' of the ribosyl [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788 10795]. These features are incorporated into N-(p-nitrophenyl)-D-riboamidrazone, a transition state analogue which binds with a dissociation constant of 2 nM [Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V. L., & Ganem, B. (1994) Biochemistry 33, 3994-4000]. Resonance Raman and ultraviolet-visible absorbance spectroscopy has established that the inhibitor binds as the neutral, zwitterionic species. The enzyme stabilizes a specific resonance state characterized by the quinonoid form of the p-nitrophenyl group with evidence for ion pairing at the nitro group. Incorporation of 15N into a specific position of the amidrazone reveals that the exo-ribosyl nitrogen bonded to the C1' position carries the proton while that bonded to the p-nitrophenyl carbon is unprotonated. This tautomer carries a distributed positive charge centered at the position analogous to C1' of the ribosyl group at the transition state. The molecular electrostatic potentials for the substrate inosine, the transition state, and the transition state inhibitor are compared at the van der Waals surface of the molecules. The tautomer of the inhibitor bound to the enzyme bears a striking electrostatic resemblance to the transition state determined by kinetic isotope effect analysis. The spectral and resonance Raman properties of free and enzyme bound inhibitor have permitted tautomeric assignment of these species and establish that the enzyme substantially changes the electronic distribution of the bound inhibitor toward that of the enzyme-stabilized transition state. PMID- 8634246 TI - Type I collagen CNBr peptides: species and behavior in solution. AB - The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat denatured samples. PMID- 8634247 TI - Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. AB - Methanol-induced conformational transitions in cytochrome c have been studied by near- and far-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existence of at least two cooperative stages of transition has been shown. At the first stage, the native protein is transformed into an intermediate which has only traces of tertiary structure, but has a native-like secondary structure content and is relatively compact; i.e., it has properties of the molten globule state. On the second stage, the alcohol-induced molten globule is transformed into a more helical state, typical of proteins at high alcohol concentrations. The conditions at which the alcohol-induced molten globule exists (moderately low pH and moderately low dielectric constant) could be similar to those existing near negatively charged membrane surfaces. Consequently, these results might explain how the molten globule state can be achieved under physiological conditions. PMID- 8634248 TI - Phosphorus-31 nuclear magnetic resonance studies on coenzyme binding and specificity in glyceraldehyde-3-phosphate dehydrogenase. AB - Binding of NAD(P)+ to wild type and a series of mutants of the glycolytic NAD dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by 31P NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP+ 2'-phosphate pKa deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pKas ranging from 7.7 for the L187A-P188S mutant to < 5.7 for the D32G L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP+ is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pKa of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pKa, it is suggested that the 2'-phosphate ionization state of bound NADP+ in chloroplastic GAPDH is dianionic. The NADP+ dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D32G-L187A-P188S, are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or conformational change upon binding. PMID- 8634249 TI - Modulation of functional properties of galectin-3 by monoclonal antibodies binding to the non-lectin domains. AB - Galectin-3 is a member of a newly defined family of animal lectins, which is composed of three domains: a small amino-terminal domain, a domain containing repeating elements, and a carboxyl-terminal domain containing the carbohydrate recognition site. Various functions have been described or proposed for this lectin, and it appears that galectin-3 has diverse roles. Murine monoclonal antibodies (MAbs) have been generated from mice hyperimmunized with recombinant human galectin-3 or galectin-3C (the carboxyl-terminal domain), and seven MAbs have been characterized in detail. All MAbs generated against the intact galectin 3 recognize the amino-terminal region of the molecule, as demonstrated by ELISA and immunoblotting using recombinant galectin-3C and galectin-3NR, which contains the amino-terminal domain and all the repeating elements. Their epitopes were all found to be within the first 45 amino acids of galectin-3, as determined by using galectin-3 mutants with a truncated amino-terminal region. However, these MAbs were found to profoundly modulate the lectin activities of galectin-3. The MAb B2C10 inhibited (i) the binding of 125I-labeled galectin-3 to IgE coated on microtiter plates; (ii) the galectin-3's hemagglutination activity; and (iii) galectin-3-induced superoxide production by human neutrophils. Other MAbs, especially A3A12, caused marked potentiation of these activities. The results support our model that the lectin function of galectin-3 is influenced by protein homodimerization resulting from self-association of the amino-terminal region of the molecule. The potentiating activities of some MAbs are probably due to facilitation of dimerization galectin-3, and the inhibitory activity of MAb B2C10 is probably the result of its disruption of the self-association process. PMID- 8634250 TI - Hypertriglyceridemic VLDL decreases plasminogen binding to endothelial cells and surface-localized fibrinolysis. AB - The effect of normo (NTG)- and hypertriglyceridemic (HTG)-VLDL on cultured human umbilical vein endothelial cell (HUVEC) surface-localized fibrinolysis was examined following pre-incubation with NTG-, HTG-VLDL, LDL (1-20 micrograms/mL) or buffer (control). Ligand binding assays, using 125I-labeled tcu-PA, t-PA, or Glu-plasminogen (Glu-Pmg) were carried out in the absence/presence of lipoproteins. Scatchard analyses showed that HTG-VLDL decreased the Bmax for 125I labeled Glu-Pmg ligand binding approximately 35% [(2.11 +/- 0.39)-(1.40 +/- 0.32) x 10(6) sites/cell, p < 0.005] and increased the Kd, app approximately 5-fold (0.32 +/- 0.03 to 1.74 +/- 0.08 microM, p < 0.01), while NTG-VLDL, LDL, and buffer had no effect. 125I-labeled PA ligand binding was unaffected by these lipoproteins. Receptor-bound PA activation of cell-bound 125I-labeled Glu-Pmg was measured by quantitation of either the M(r) 20 kDa light- or M(r) 60 kDa heavy chain of 125I-labeled plasmin, following SDS-PAGE. Kinetic analysis of these data (HTG-VLDL vs controls) indicated that HTG-VLDL decreased the V(max) of tcu-PA- and t-PA-mediated activation of plasminogen approximately 2.7-fold (0.317 +/- 0.023 vs 0.869 +/- 0.068 nM s-1, p < 0.01) and approximately 2.9-fold (0.391 +/- 0.098 vs 1.152 +/- 0.265 nM s-1, p < 0.01), respectively. Increasing concentrations of the HTG-VLDL increased 1/V(max), yielding a series of parallel plots, typical for uncompetitive inhibition with a Ki for inhibition of approximately 10 micrograms/mL. The combined ligand binding and kinetic data best fit an uncompetitive inhibition model in which the binding of the large HTG-VLDL particle to the EC surface may directly affect Glu-Pmg binding and activation, thus contributing to early fibrin deposition and the increased thrombotic risk associated with HTG. PMID- 8634251 TI - Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo. AB - Ixr1 is a yeast HMG-domain protein that binds specifically to DNA adducts formed by the antitumor drug cisplatin. Interruption of the IXR1 gene in yeast desensitizes cells to cisplatin. This effect is unrelated to a natural function of Ixr1, which is to repress the transcription of COX5b. Ixr1 interacts specifically and preferentially with DNA modified by cisplatin. In the present work, Ixr1 was purified from a clone expressed in Escherichia coli. The dissociation constant for Ixr1 binding site-specifically to a 92-bp probe containing a single cis-[Pt(NH3)2{d(GpG)-N7(1) -N7(2)}] intrastrand cross-link was measured to be 2.5 (+/- 0.1) x 10(-7) M, similar to that found for HMG1. Ixr1 binds at least an order of magnitude more tightly to cisplatin-DNA adducts than to unmodified DNA. Hydroxyl radical footprinting revealed that Ixr1 protects an area of platinated DNA that is approximately 15 bp in size and centered at the platinum adduct. The binding of HMG-domain proteins to cisplatin-DNA adducts has been proposed to divert these proteins from their natural DNA-binding sites, disrupting transcription. This hypothesis was tested for Ixr1 in yeast. The protein was not titrated away from the Cox5b promoter sufficiently well to disrupt transcription either of Cox5b mRNA from genomic DNA or of the beta galactosidase gene under control of the promoter in a plasmid DNA transformed into yeast. PMID- 8634252 TI - Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase. AB - Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase. To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated. Adenylate kinase from Escherichia coli consists of three domains: the main body of the enzyme with alpha-helical and beta-sheet secondary structure, and domains that close over the AMP and ATP binding sites. Four single tryptophan mutants were constructed to map distances. Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site. Mutant F86W that is located at the AMP binding site, and mutant S41W that is in the loop that close over AMP, complete the mapping library. Energy transfer was measured between each of these tryptophans and 5-[[2 (acetylamino)ethyl]amino]naphthalene-1-sulfonic acid (AEDANS) covalently bound to the single cysteine residue at position 77, which is located in the main body of adenylate kinase. The distance between the tryptophan of the F137W mutant adenylate kinase and the AEDANS-labeled Cys-77 decreased by 12.1 A upon the binding of the bisubstrate inhibitor P1, P5-bis(5'-adenosyl) pentaphosphate (AP5A). There were only small alterations in the tryptophan to Cys-77-AEDANS distances in the Y133W, F86W, and S41W mutants upon the binding of AP5A, ATP, or AMP, implying that movement of residues 133, 86, and 41 in relation to the Cys-77 residue was minimal. These results suggest that there is significant closure of the ATP binding domain upon the binding of ATP or AP5A. Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain. PMID- 8634254 TI - Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein. AB - The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism. The generalized order parameters for 56 main chain alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic. PMID- 8634253 TI - An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan. AB - The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c. PMID- 8634255 TI - Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center. AB - Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus. FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively. The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state. Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P. The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules. The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl. The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds. From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus. The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1. Compared to Rb. sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca. 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca. 80% for Rb. sphaeroides. The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed. PMID- 8634256 TI - Stopped-flow, laser-flash photolysis studies on the reactions of CO and O2 with the cytochrome caa3 complex from Bacillus subtilis: conservation of electron transfer pathways from cytochrome c to O2. AB - The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1. When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM. Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa3 complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex. PMID- 8634257 TI - Subunit composition of the peripherin/rds-rom-1 disk rim complex from rod photoreceptors: hydrodynamic evidence for a tetrameric quaternary structure. AB - Peripherin/rds and rom-1 are homologous integral membrane protein subunits found as an oligomeric complex at the rim regions of rod and cone photoreceptor outer segment disks. These proteins are essential for the morphogenesis of normal outer segments and have been linked to a variety of human retinal degenerative diseases. Previous studies have suggested that disulfide-linked homodimers of peripherin/rds and rom-1 can associate noncovalently to form higher order structures. We have characterized the hydrodynamic properties of Triton X-100 solubilized peripherin/rds-rom-1 complexes from bovine ROS membranes by gel exclusion chromatography on Sepharose C1-6B and velocity sedimentation through H2O- and D2O-based sucrose gradients. A single hydrodynamic species is observed which has a Stokes radius of 6.2 nm, a sedimentation coefficient (S20,w) of 5.8 S, and a partial specific volume of 0.83 mL/g. From these data the molecular mass of the detergent-peripherin/rds-rom-1 complex is calculated to be 240 kDa. The protein component of this complex is estimated to be 135 kDa, providing direct evidence that the solubilized peripherin/rds-rom-1 complex is a tetramer. The abundance of this complex as measured by competitive ELISA and immunoaffinity purification is approximately 4% of total bovine ROS membrane protein and indicates that peripherin/rds-rom-1 tetramers are present at a relatively high average surface density (ca. 4100/ microns m2) at the rim surfaces of rod outer segment disks. PMID- 8634258 TI - Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus. AB - An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5 alpha grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost [Tolner, B., Ubbink-Kok, T., Poolman, B., & Konings, W. N. (1995) Mol. Microbiol. 18, 123-133], suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+. PMID- 8634259 TI - Transforming growth factor beta (TGF beta)-induced nuclear localization of apolipoprotein J/clusterin in epithelial cells. AB - Apolipoprotein J (apoJ)/clusterin was first identified as an 80 kDa secretory glycoprotein present in most body fluids. It has been implicated in a variety of physiological processes including cellular differentiation and apoptosis. We demonstrate here that in addition to the well characterized secreted form of the protein, there exists an intracellular, nuclear form of apoJ. This intracellular form of the protein is induced to accumulate in the nucleus of two epithelial cell lines (HepG2 and CCL64) in response to treatment with transforming growth factor beta (TGF beta). We demonstrate in vitro that apoJ protein can be translated from two in-frame ATG sites. Initiation from the first ATG encodes for the secretory form of apoJ and initiation from the second ATG, located 33 amino acids downstream of the first and lacking the hydrophobic signal sequence, encodes for a truncated apoJ protein. This shorter form of apoJ is not recognized by microsomes and therefore not glycosylated, and we postulate that it is retained intracellularly and targeted to the nucleus due to the presence of an SV40-like nuclear localization sequence (NLS). This mechanism of nuclear targeting of apoJ occurs in cells since the protein isolated from nuclei of TGF beta-treated cells and the in vitro-translated truncated form are identical by V8 protease analysis. These results suggest that the diverse physiological responses attributed to apoJ may be elicited through a common molecular mechanism involving a previously uncharacterized intracellular form of the protein. PMID- 8634260 TI - Novel mechanism for the activation of rhodopsin kinase: implications for other G protein-coupled receptor kinases (GRK's). AB - ATP, its nonhydrolyzable analogue, AMP-PNP, and albumin were found to promote the dissociation of rhodopsin kinase from rod outer segments (ROS) containing photoactivated-rhodopsin (Rho*). These features were embodied in a protocol for the recovery of rhodopsin kinase from incubations containing ROS which had been subjected to a wide range of treatments. It was found that the supernatants recovered from mixtures containing ATP, rhodopsin kinase, and photolyzed ROS membranes catalyzed a Rho*-independent peptide phosphorylation as well as dark phosphorylation of rhodopsin. The activities of this activated kinase in the two aforementioned assays were 7-8% of the maximum intrinsic activity found in appropriate standard assays (i.e., light-stimulated phosphorylation of rhodopsin and Rho*-dependent peptide phosphorylation). The activated kinase reverted to its inactive resting-state in a time dependent fashion, giving a tau 1/2 of decay of approximately 2 min. The intrinsic activity of kinase as measured by the standard assay, however, remained constant during this decay period. No positive evidence was found to suggest that the interconversion activated kinase <--> inactive kinase occurred by a phosphorylation event. Cumulatively, the results show that the interaction of rhodopsin kinase.ATP complex with Rho* leads to the formation, presumably due to the reorganization of the protein structure, of a soluble active kinase species which reverts to the inactive resting state in a time dependent fashion. PMID- 8634261 TI - Cytidine deaminase complexed to 3-deazacytidine: a "valence buffer" in zinc enzyme catalysis. AB - The cytidine deaminase substrate analog inhibitor 3-deazacytidine binds with its 4-amino group inserted into a site previously identified as a probable binding site for the leaving ammonia group. Binding to this site shifts the pyrimidine ring significantly further from the activated water molecule than the position it occupies in either of two complexes with compounds capable of hydrogen bonding at the 3-position of the ring [Xiang et al. (1995) Biochemistry 34, 4516-4523]. Difference Fourier maps between the deazacytidine, dihydrozebularine, and zebularine--hydrate inhibitor complexes suggest that the ring itself moves successively toward the activated water, leaving the amino group behind in this site as the substrate complex approaches the transition state. They also reveal systematic changes in a single zinc-sulfur bond distance. These correlate with chemical changes expected as the substrate approaches the tetrahedral transition state, in which the zinc-activated hydroxyl group develops maximal negative charge and forms a short hydrogen bond to the neighboring carboxylate group of Glu 104. Empirical bond valence relationships suggest that the Zn-S gamma 132 bond functions throughout the reaction as a "valence buffer" that accommodates changing negative charge on the hydroxyl group. Similar structural features in alcohol dehydrogenase suggest that analogous mechanisms may be a general feature of catalysis by zinc enzymes. PMID- 8634262 TI - (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N acetylenolpyruvylglucosamine reductase (MurB). AB - UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP methyl-N-acetylmuramic acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic acid product to 2,3-dideuterio-2 hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis. PMID- 8634263 TI - Biochemical characterization of pre-beta 1 high-density lipoprotein from human ovarian follicular fluid: evidence for the presence of a lipid core. AB - In order to isolate pre-beta 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre-beta 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double dimension gel electrophoresis. Apo A-I in pre-beta 1 HDL represented 17.6% of total apo A-I. Stokes' radii corresponded to 3.42 nm in follicular fluid pre-beta 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre-beta 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre beta 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidylcholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre-beta 1 HDL total weight. Thus, a lipid core is present in pre-beta 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism. PMID- 8634265 TI - Laser flash absorption spectroscopy study of flavodoxin reduction by photosystem I in Synechococcus sp. PCC 7002. AB - The photoreduction of flavodoxin by trimeric photosystem I, both from the cyanobacterium Synechococcus sp. PCC 7002, was investigated by flash absorption spectroscopy. After addition of flavodoxin in darkness, single flash experiments show that the transient signals change between individual flashes. This behavior is assigned to a progressive accumulation of flavodoxin semiquinone, which is relatively stable under most experimental conditions. Different conditions were devised in order to study the reduction of the oxidized and semiquinone forms of flavodoxin separately. Both processes were identified by their differential spectra measured between 460 and 630 nm. Detailed kinetic characteristics of flavodoxin reduction were obtained at pH 8.0 in the presence of salts. The kinetics of reduction of oxidized flavodoxin displays a single-exponential component. The rate of this component increases with the flavodoxin concentration up to an asymptotic value of about 600 s-1. The semiquinone form of flavodoxin being protonated, this rate corresponds to a rate-limiting reaction which could be either an electron transfer reaction or a protonation reaction. In contrast, the reduction of flavodoxin semiquinone is biphasic. A fast first-order phase with t 1/2 approximately 10 microseconds is interpreted as an electron transfer process within a preformed complex. A dissociation constant of 2.64 microM is calculated for this complex by assuming a simple binding equilibrium between photosystem I and flavodoxin semiquinone. The slower phase observed for semiquinone reduction is concentration dependent, and a second-order rate constant of 1.7 x 10(8) M-1 s-1 is calculated. For both one-electron reduction steps, different optimal salt concentrations are observed indicating slightly different interactions between photosystem I and flavodoxin in its oxidized and semiquinone states. PMID- 8634264 TI - Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor. AB - The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery). Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells. PMID- 8634266 TI - Specific recognition of coiled coils by infrared spectroscopy: analysis of the three structural domains of type III intermediate filament proteins. AB - The central domain of cytoplasmic intermediate filament (IF) proteins from vertebrates contains some 310 residues and forms a double-stranded coiled coil (rod) with a length of about 46 nm. The flanking terminal domains show a high cell type specific variability both in sequence and in length. Using Fourier transform infrared (FTIR) spectroscopy we measured secondary structures of isolated domains of type III and IV IF proteins and of the soluble tetramers and the filaments formed by type III IF proteins. The amide I spectrum of the desmin rod is virtually identical to the spectra of other coiled-coil proteins such as tropomyosin and the myosin rod. All these double-stranded coiled coils reveal spectra distinctly different from classical alpha-helical spectra. The spectrum of coiled coils is a triplet of approximately equally strong bands. One band occurs at normal alpha-helix position, while the other two are found at lower wavenumbers. Theoretical aspects of these findings are discussed in the accompanying paper by W. C. Reisdorf and S. Krimm [(1996) Biochemistry 35, 1383 1386]. The amino-terminal head domain of desmin has a multicomponent spectrum with major fractions of beta-sheet. The carboxy-terminal tail domains of desmin and the neurofilament proteins L and H, the latter in the phosphorylated and in the dephosphorylated forms, have very similar FTIR spectra, indicating mostly random structure. The spectrum of desmin type III protofilaments is very similar to the sum of the spectra of the three isolated domains. Polymerization into filaments seems to induce a small change in secondary structure. PMID- 8634267 TI - Infrared amide I' band of the coiled coil. AB - The Fourier transform infrared (FTIR) spectra of several coiled-coil proteins have been shown to possess unusual features in the amide I' region. Band maxima occur in the vicinity of 1630 cm-1, with component bands at higher frequency. This is well below the observed band at 1650 cm-1 found in standard alpha-helical polypeptides such as poly-L-alanine. Normal mode calculations on models of the coiled-coil structure have been performed to investigate this issue. We find that the observed band profile can be reproduced with very small random variation on the phi, psi of tropomyosin. We believe that the shift to lower frequency is due to additional hydrogen bonding of the solvent accessible backbone CO groups to water. PMID- 8634268 TI - Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor. AB - A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2 (trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4 benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-) [3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-) [3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs. PMID- 8634269 TI - Spectroscopic and mechanistic studies of type-1 and type-2 copper sites in Pseudomonas aeruginosa azurin as obtained by addition of external ligands to mutant His46Gly. AB - The spectroscopic and mechanistic properties of the Cu-containing active site of azurin from Pseudomonas aeruginosa were investigated by the construction of a mutant in which one of the ligands of the metal, His46, was replaced by a glycine. Although the mutation creates a hole in the interior of the protein, the 3D structure of the protein does not change to any appreciable extent. However, the spectroscopic (optical, resonance Raman, EPR) properties of the mutant protein are strongly affected by the mutation. In the presence of external ligands, the properties of the original wild-type protein are restored to a smaller or larger extent, depending on the ligand. It is concluded that the hole created by the mutation, even though it is completely buried inside the protein, can be filled by external ligands, often resulting in the creation of a mixture of so-called type-1 and type-2 copper sites. Also, the redox properties (midpoint potential, kinetics of reduction/oxidation) appeared to be strongly affected by the mutation and the presence of external ligands. The results are compared with previous results obtained on the mutant His117Gly. PMID- 8634272 TI - Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase. AB - Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state. PMID- 8634270 TI - Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage, and DNA supercoiling. AB - Binding of the nonhydrolyzable ATP analogue 5'-adenylyl-beta, gamma imidodiphosphate (ADPNP) to Escherichia coli DNA gyrase can lead to a limited noncatalytic supercoiling of DNA. Here we examine the efficiency of coupling between ADPNP binding and the change in linking number either of positively or negatively supercoiled plasmid DNA or of small DNA circles. The coupling efficiency varies from 100% (delta Lk = -2 per gyrase tetramer, a stoichiometry of 1) with positively supercoiled substrates under certain reaction conditions to an undetectably low value with moderately negatively supercoiled substrates (sigma = -0.046) or small circular substrates. Furthermore, the rate of ADPNP binding to the gyrase-DNA complex is also dependent on the topological state of the DNA; the previously observed slow binding of ADPNP to the complex of gyrase with linear DNA is accelerated 16-fold when the substrate DNA is negatively supercoiled, suggesting a functional interaction between the nucleotide-binding and DNA-binding domains which is independent of the strand-passage process. The implications for the normal ATP-dependent supercoiling reaction of the enzyme are considered and the results discussed in terms of current mechanistic models for DNA gyrase action and the possible in vivo roles of the enzyme. PMID- 8634271 TI - Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N acetylmuramate:L-alanine ligase-catalyzed reaction. AB - In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N acetylmuramyl-L-alanine. A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N acetylmuramyl:L-alanine ligase fusion protein was overproduced in E. coli/pMal::murC upon isopropyl beta-thiogalactoside induction. The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography. Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion exchange chromatographic step. The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L alanine. UDP-N-acetyl[18O]muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [18O]oxygen was transferred from UDP-N-acetyl[18O]muramate to the orthophosphate produced in the reaction. No [18O]oxygen was detected in the adenosine nucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl phosphate linkage during the reaction process. ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid. PMID- 8634273 TI - Evidence favoring molybdenum-carbon bond formation in xanthine oxidase action: 17Q- and 13C-ENDOR and kinetic studies. AB - The reaction mechanism of the molybdoenzyme xanthine oxidase has been further investigated by 13C and 17O ENDOR of molybdenum(V) species and by kinetic studies of exchange of oxygen isotopes. Three EPR signal-giving species were studied: (i) Very Rapid, a transient intermediate in substrate turnover, (ii) Inhibited, the product of an inhibitory side reaction with aldehyde substrates, and (iii) Alloxanthine, a species formed by reaction of reduced enzyme with the inhibitor, alloxanthine. The Very Rapid signal was developed either with [8-13C]xanthine or with 2-oxo-6-methylpurine using enzyme equilibrated with [17O]H2O. The Inhibited signal was developed with 2H13C2HO and the Alloxanthine signal by using [17O]H2O. Estimates of Mo-C distances were made, from the anisotropic components of the 13C couplings, by corrected dipolar coupling calculations and by back-calculation from assumed possible structures. Estimated distances in the Inhibited and Very Rapid species were about 1.9 and less than 2.4 A, respectively. A Mo-C bond in the Inhibited species is very strongly suggested, presumably associated with side on bonding to molybdenum of the carbonyl of the aldehyde substrate. For the Very Rapid species, a Mo-C bond is highly likely. Coupling from a strongly coupled 17O, not in the form of an oxo group, and no coupling from other oxygens was detected in the Very Rapid species. No coupled oxygens were detected in the Alloxanthine species. That the coupled oxygen of the Very Rapid species is the one that appears in the product uric acid molecule was confirmed by new kinetic data. It is concluded that this oxygen of the Very Rapid species does not, as frequently assumed, originate from the oxo group of the oxidized enzyme. A new turnover mechanism is proposed, not involving direct participation of the oxo ligand group, and based on that of Coucouvanis et al. [Coucouvanis, D., Toupadakis, A., Lane, J. D., Koo, S. M., Kim, C. G., Hadjikyriacou, A. (1991) J. Am. Chem. Soc. 113, 5271-5282]. It involves formal addition of the elements of the substrate (e.g., xanthine) across the Mo = S double bond, to give a Mo(VI) species. This is followed by attack of a "buried" water molecule (in the vicinity of molybdenum and perhaps a ligand of it) on the bound substrate carbon, to give an intermediate that on intramolecular one-electron oxidation gives the Very Rapid species. The latter, in keeping with the 13C, 17O, and 33S couplings, is presumed to have the 8-CO group of the uric acid product molecule bonded side-on to molybdenum, with the sulfido molybdenum ligand retained, as in the oxidized enzyme. PMID- 8634274 TI - Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme. AB - The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemeprotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contains heme and H4biopterin, and binds L-arginine and a reductase domain fragment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the reduction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemistry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabilize the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spectral analysis of the dissociation reaction showed that it caused structural changes within the oxygenase domain and exposed the distal side of the heme to solvent, enabling it to bind dithiothreitol as a sixth ligand. Importantly, the oxygenase domain monomers could reassociate into a dimeric form even in the absence of the reductase domain. The reaction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain. PMID- 8634275 TI - Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B. AB - Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis. PMID- 8634276 TI - Metal coordination environment and dynamics in 113cadmium bleomycin: relationship to zinc bleomycin. AB - The 13C chemical shifts of Cd- and ZnBlm A2 are almost identical throughout the entire molecule, suggesting that these structures adopt similar conformations. Nuclear magnetic resonance experiments with 113Cd-bleomycin have defined part of the metal-ligand environment of the molecule. Nitrogen atoms from the primary amine, pyrimidine, and imidazole are bound to 113Cd according to 13C spectra showing 113Cd-13C spin-spin couplings. Bound and free forms of the secondary amine nitrogen may be in equilibrium, as suggested by temperature-dependent 13C studies with Cd-bleomycin. In addition, a number of other carbon resonances are in chemical exchange over the temperature range 5-54 degrees C. The temperature dependence of the line widths of carbon atoms of Zn-bleomycin strongly resembles that of Cd-bleomycin. Examination of the 113Cd resonance as a function of temperature also supports the presence of at least two differently coordinated forms of cadmium in the molecule. According to the position of the 113Cd chemical shift, at most four nitrogen atoms are bound to Cd at low temperature. Titrations of 113Cd-bleomycin with chloride or acetate demonstrate that these anions can bind to major and minor forms of the structure and that a minor species exists which does not associate with chloride. PMID- 8634277 TI - X-ray diffraction analysis of cytochrome P450 2B4 reconstituted into liposomes. AB - Two general models of the membrane topology of microsomal cytochrome P450 have been proposed: (1) deep immersion in the membrane, and (2) a P450cam-like heme domain anchored to the membrane with one or two membrane-spanning helices. Lamellar X-ray diffraction of oriented membrane multilayers was employed to distinguish these alternatives. Cytochrome P450 2B4 was reconstituted into unilamellar phospholipid proteoliposomes (molar protein to lipid ratio 1:90). Sedimentation of the proteoliposomes produced an ordered stack of bilayers with a one-dimensional repeat distance (d) perpendicular to the plane of the bilayer. The stacked multilayers were exposed to an X-ray beam (lambda = 1.54 A) at near grazing incidence, and lamellar diffraction patterns were recorded. With proteoliposome multilayers, up to six diffraction orders could be observed. Their spacing corresponded to a d of 63.6 A, calculated according to Bragg's Law, comprising the lipid bilayer, the projection of the incorporated protein beyond the bilayer, and the intermembrane water layer. With liposome multilayers containing no P450, the observed d was 59.6 A. These data suggest that the increase of distance between successive bilayers in the stack due to the presence of P450 2B4 was only about 4 A. This distance is much less than would be expected with the "N-terminal membrane-anchor" model of the membrane topology, in which the P450 molecules largely extend beyond the surface of the membrane (> or = 35 A). Furthermore, the mass distribution deduced from Fourier synthesis confirms that the protein is deeply immersed in the membrane. PMID- 8634278 TI - Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ. and YD. in photosystem II using wild-type and the D2 Tyr160Phe mutant of Synechocystis 6803. AB - The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ. has been hampered by the simultaneous presence of the much more stable YD., the short lifetime of YZ., and the difficulty in trapping the YZ. radical at low temperature. We present here a method for obtaining an uncontaminated YZ. radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the beta-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ. EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ. radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ., this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ.. We have compared the environments of YZ. and YD. by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ. and YD. differ in line shape and line width. (2) Both YZ. and YD. exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD., indicating the hydrogen bond to be well-defined. In the case of YZ. it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD. possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 A. (4) All of the 1H couplings of YD. are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ. spectrum are broad. The environment surrounding YZ. appears to be more disordered and solvent-accessible. PMID- 8634279 TI - Improved binding of cytochrome P450cam substrate analogues designed to fill extra space in the substrate binding pocket. AB - Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties. PMID- 8634280 TI - Evidence for a slow tertiary relaxation in the reaction of tert-butyl isocyanide with horseradish peroxidase. AB - The kinetics of tert-butyl isocyanide binding to the heme protein horseradish peroxidase (HRP) at 22 degrees C was examined on all time scales, from minutes to picoseconds, in aqueous borate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M-1 s-1, while in Mb it is 4 x 10(3) M-1 s-1. Surprisingly, in view of that difference, picosecond and nanosecond photolyses reveal that once the ligand has reached the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the contact pair) is close to 6 x 10(10) s-1 in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair are also similar: for Mb, 10 x 10(10) s-1, and for HRP, 8.5 x 10(10) s-1. The rate of rebinding from the protein-separated cage is near 4 x 10(6) s-1 in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s-1 in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination not seen in mixing experiments. This bimolecular rate constant varies slightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10(4) M-1 s-1 in two cases, both of which are much faster than is observed in mixing experiments, namely, 0.04 M-1 s-1. In Mb, photolysis and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombination. Similar measurements for the smaller ligand methyl isocyanide revealed no anomalous behavior. The interpretation proposed involves tertiary relaxation after ligand escape, which is significant in blocking the return of the large t-BuNC, but has no apparent effect on smaller ligands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merely hinted at in earlier work involving the T-state binding kinetics of hemoglobin. PMID- 8634281 TI - Molecular dynamics simulations of adipocyte lipid-binding protein: effect of electrostatics and acyl chain unsaturation. AB - Molecular dynamics (MD) simulations have been performed on adipocyte lipid binding protein, using the apo and holo forms, bound with stearic and oleic acid. The contribution of electrostatics to protein dynamics and ligand stabilization was assayed by perturbing the electrostatic charge of Arg106 and Arg126 (positive ->neutral) and the fatty acid (132H) headgroup (negative-->neutral). MD simulations for charged holo forms demonstrated significantly greater electrostatic binding energy and a more stabilized hydrogen bond network than simulations performed using neutral forms. Electrostatics, however, appeared to have little effect on fatty acid behavior, e.g., fluctuation of the dihedral head group; number of dihedral transitions within the acyl chain; and change in the end-to-end distance for fatty acid. Instead, fatty acid behavior appeared to be dictated by the presence or absence of an unsaturated bond within the acyl chain. A significantly greater number of transitions were observed during MD simulations in oleic than stearic acid. In addition, significantly greater fluctuation was observed for oleic acid, within the C2 headgroup and C9 and C11 dihedrals (which lie adjacent to the olefin bond of oleic acid). The dynamic behavior of the acyl chain may thereby be more a property of van der Waals contact, and the degree of acyl chain unsaturation, than a function of electrostatics. In the absence of fatty acid, an increase in distance between guanidino carbon centered atoms of Arg126 and Arg106 was observed during MD simulations of the charged apo form. This effect not observed with the neutral apo form or in any of the holo complexes and, presumbably, was a result of repulsion between the negatively charged arginine sidechains. Conserved waters reflected substantially lower mean square displacement (msd) in all simulations, except the neutral apo form. This suggests that the presence of either charged amino acids or lipid provides increased order for water within the binding pocket. These results provide a dynamic perspective of the interactive nature within the FABP binding pocket regulated in a complex manner by the electrostatics within the binding cavity, acyl chain structure and behavior, and water energetics. PMID- 8634282 TI - Solution structure and backbone dynamics of recombinant Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy. AB - The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225 2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid. PMID- 8634283 TI - 13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase. AB - Side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta 5-3-ketosteroid isomerase (EC 5.3.3.1, homodimer, M(r) = 26.8 kDa) have been examined by measurements of longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz (proton frequencies). The data, analyzed using the model-free formalism, yielded an optimized correlation time for overall molecular rotation (tau m) of 17.9 ns, in agreement with the result (18 ns) of fluorescence anisotropy decay measurements [Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415-7422] and Stokes' law calculation (20 ns). The order parameter of the side chain C epsilon of Tyr-14 (S2 = 0.74), which is a measure of the restriction of its high frequency (nanosecond to picosecond) motion, was significantly lower than that of the backbone C alpha (S2 = 0.82), indicating greater restriction of backbone motion. Upon binding of the steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S2 of the side chain C epsilon increased from 0.74 to 0.86, while that of the backbone C alpha did not change significantly. Thus, in the steroid complex, the amplitude of high frequency side chain motion of Tyr-14 became more restricted than that of its backbone which could lower the entropy barrier to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to kcat were detected by exchange contributions to transverse relaxation of both C epsilon and C alpha. Steroid binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and product release, but decreased the exchange contribution to transverse relaxation of the backbone. PMID- 8634284 TI - Rate-determining steps for tyrosine phosphorylation by the kinase domain of v fps. AB - The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption. PMID- 8634285 TI - Binding of nitric oxide and carbon monoxide to soluble guanylate cyclase as observed with Resonance raman spectroscopy. AB - Resonance Raman spectra have been recorded for the ferrous heme of soluble guanylate cyclase (sGC), the only receptor known thus far for .NO. On the basis of the frequencies of porphyrin core sensitive vibrations in the high frequency region of the Raman spectrum, we conclude that the ferrous heme is five coordinate, high spin, when no exogenous ligands are present. We assign a prominent vibration that occurs at 204 cm-1 in the reduced enzyme to the heme Fe(2+)-proximal histidine stretching vibration. In the .NO bound form of the enzyme, the heme Fe2+ retains a five-coordinate geometry. Assuming that .NO binds to the distal side of the heme, this observation indicates that the weak Fe-His bond breaks when .NO binds. Two isotope-sensitive vibrations are observed in the .NO bound enzyme, one at 1677 cm-1, attributed to the N-O stretching vibration, and one at 525 cm-1, attributed to the Fe-NO stretching vibration. When CO is bound to the ferrous heme, the heme ligation is six-coordinate. From this, we conclude that the Fe-His bond is still intact and that, if cleavage of the Fe proximal ligand bond is necessary for complete activation of sGC, then CO should only weakly activate the enzyme, which has been shown to be the case. In the carbonmonoxy enzyme, the Fe-CO stretching vibration is observed at 472 cm-1 and the Fe-C-O bending vibration is detected at 562 cm-1. These frequencies are the lowest yet observed for the Fe-CO stretching and Fe-C-O bending modes in heme proteins or model systems with imidazole as the proximal ligand and suggest that there is significant negative polarity in the distal pocket. The negative polarity and the low frequency of the Fe-His stretching vibration may account for the very low O2 affinity of sGC. PMID- 8634286 TI - Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A. AB - Four single mutants (P42A, P93A, P114A, and P117A) of bovine pancreatic ribonuclease A (RNase A) in which each mutant has one of the four prolines of RNase A changed to alanine were prepared. The physical properties of these four mutants indicate that their native structure is essentially identical to that of wild-type RNase A. The disulfide-intact forms of these proteins were denatured in guanidine hydrochloride (Gdn.HCl) and then refolded by dilution of the Gdn.HCl. Single-jump folding, single-jump unfolding, and double-jump unfolding/folding stopped-flow experiments were carried out on wild-type and the four proline mutants of RNase A using absorption detection to follow the folding kinetics. The single-jump folding experiments carried out at six different final Gdn.HCl concentrations indicate that the folding rate constants of individual steps for the mutants are similar to those of wild-type RNase A. The Tyr92-Pro93 peptide bond has a cis conformation in native wild-type RNase A, and the results from our double-jump stopped-flow experiments indicate that the Tyr92-Ala93 peptide bond in the P93A mutant of RNase A is also cis in the native state. The existence of two cis peptide bonds (preceding Pro93 and Pro114) in wild-type RNase A is probably due to (as-yet-unidentified) long-range interactions, and such interactions are presumably the origin of a cis peptide bond even when alanine is substituted for Pro93. The data from the double-jump stopped-flow experiments are interpreted in terms of a folding/unfolding model. This model specifies the cis/trans isomerization state of the unfolded species (Uvf, Uf, Um, and Us) at each X-Pro peptide bond. Also, this model confirms the existence of several previously postulated chain-folding initiation sites. PMID- 8634287 TI - pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pKa. AB - The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rRNA group I intron catalyzes a site-specific endonucleolytic cleavage of RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA5 + G-->CCCUCU + GA5. The pH-rate dependence was determined for the reaction of the E.G complex with the oligonucleotide substrate d(CCCUC)r(U)d(A5) [(kcat/Km)S conditions]. Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition by specific monovalent cations. The intrinsic pH-rate dependence is log-linear with slope 1 below pH 7, displays an apparent pKa of 7.6, remains nearly level until pH 8.5, and then begins to fall. Two models to explain the apparent pKa were ruled out: (1) the pKa represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pKa arises from a change in rate-limiting step from a pH-dependent to a pH-independent step. In addition, these models, or others involving a single titration, cannot account for the decrease in activity at high pH. A third, unconventional, model is consistent with all of the data. It involves inactivation of the ribozyme by any of several independent titrations of groups with pKa values considerably higher than the apparent pKa of 7.6. The data are consistent with loss of catalytic function upon release of a proton from any one of 19 independent sites with pKa = 9.4 (the unperturbed pKa of N1 of G and N3 of U in solution). Independent experiments investigating the effect of pH on different reaction steps supported this model and suggested the identity of some of the required protons. This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from the pKa of the titrating bases. PMID- 8634289 TI - Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11. AB - Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved. The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11. Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively. Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein. A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11. An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton. L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N terminal domain. PMID- 8634288 TI - Identification of the Zn(II) site in the copper-responsive yeast transcription factor, AMT1: a conserved Zn module. AB - The N-terminal metal-binding domains of the copper-activated yeast transcription factors, ACE1 and AMT1, bind to specific DNA sequences in a Cu-dependent fashion. Recombinant AMT1 and ACE1 metal-binding domains are isolated as Cu4Zn1-protein complexes. Site-directed mutagenesis of AMT1 was used in this study to map the ligands of the Cu(I) and Zn(II) ions. The results are consistent with the N terminal halves of AMT1 and ACE1 consisting of two independent submodules, one binding a single Zn(II) ion and the second binding the tetracopper cluster. The basis of this conclusion is, first, that mutations of two cysteinyl codons and a histidyl codon in the first 42 residues of AMT1 do not alter DNA binding. In contrast, serine substitutions at four cysteine positions at codons 43, 61, 90, and 98 abolish DNA binding. We demonstrated previously that population of the Zn(II) site in AMT1 does not alter the ability of the protein to bind DNA but bound Cu(I) ions are essential for DNA binding [Thorvaldsen, J. L., et al. (1994) Biochemistry 33, 9566-9577]. Second, mutations in the N-terminal 42 residue segment reduce the Zn(II) content of purified mutant AMT1 molecules. Third, a synthetic peptide consisting of the N-terminal 42 residues in AMT1 forms a stable Zn(II) complex and substitution with Co(II) reveals an electronic spectrum identical to that of the Co-substituted intact Cu4AMT1 protein. 113Cd(II) NMR studies reveal that the divalent metal site consists of ligands provided by three cysteinyl thiolates and a single histydyl imidazole. The sequence homology between AMT1, ACE1, and MAC1 in the N-terminal 42 residues suggests that ACE1 and MAC1 will, likewise, contain N-terminal Zn modules. A 42-residue ACE1 synthetic peptide gives identical metal binding properties to the corresponding AMT1 synthetic peptide. Thus, AMT1 and likely ACE1 consist of two contiguous modules, residues 1-42 forming an independent Zn(II) module and residues 43-110 enfolding a tetracopper cluster. PMID- 8634290 TI - Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation. AB - The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization [Laughrea, M., & Jette, L. (1994) Biochemistry 33, 13464-13474; Skripkin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4945-4949] posits that the 248-270 region of the HIV 1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is the full or at least the core dimerization domain of HIV-1 RNA. Here, we show by nested deletion analysis that the 3' boundary of the HIV-1 dimerization domain is immediately downstream of hairpin 248-270 and that the isolated region 248-271 dimerizes at least as readily as longer RNAs. Among various HIV-1Lai RNA transcripts containing hairpin 248-270, all form two types of dimer, as is implicit in the kissing-loop model. The high-stability dimer resists semidenaturing conditions and the low-stability dimer cannot, which is consistent with the model. At physiological temperatures, low-stability dimers are usually formed, as if dimerization without nucleocapsid proteins corresponded to loop-loop interaction without switching from intra- to interstrand hydrogen bonding. Our results show that the 3' DLS (a sequence immediately 3' from the 5' splice junction and originally thought to be the dimerization domain of the HIV-1 genome) and adjacent nucleotides are not necessary for efficient dimerization of HIV-1Lai RNA at low and high ionic strength. Upstream of hairpin 248-270 exists another "DLS-like" sequence that we name 5' DLS: like the isolated 3' DLS, the isolated 5' DLS forms an apparently nonphysiological structure that can become substantially dimeric at high ionic strength. PMID- 8634291 TI - Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex. AB - The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than 0.1 M KCl, suggesting that electrostatic interactions contribute strongly to complex formation. Ion pair interactions are frequently sensitive to high hydrostatic pressure due to electrostriction effects in the solvated ion state. Application of high pressure to the apoCaM.Neurop complex causes a red shift of the Neurop tryptophan emission center of mass and a reduced residual anisotropy but with insignificant reduction in quantum yield. The transition is smooth, reversible, and apparently two-state with a midpoint pressure of approximately 0.8 kbar. The residual anisotropy, quantum yield, and center of mass of the emission spectrum are consistent with the movement of the tryptophan side chain to a more solvated, slightly less restricted environment upon the pressure-induced transition. The pressure effect is independent of the concentration of the complex. These and other data are consistent with the pressure-induced reorganization being a unimolecular event not requiring dissociation of the complex. A volume change of approximately 66 mL mol-1 and a free energy change of approximately 1.7 kcal mol-1 are associated with the reorganization. The residual interactions maintaining the complex under high pressure are characterized by low standard molar volume and/or high standard free energy changes upon disruption but are destroyed by 200 mM KCl. It is postulated that the main effect of salt on the complex at high pressure is to drive the collapse of the hydrophobic pocket to which the peptide is binding. PMID- 8634292 TI - Tyrosine-272 is involved in the inhibition of protein phosphatase-1 by multiple toxins. AB - Protein phosphatase-1 (PP1) is regulated by interaction with different subunits, which include several inhibitory proteins. It is also potently inhibited by several toxins of diverse origins. Recent work has identified a region near the C terminus of PP1 (residues 274-277) whose modification was shown to moderate okadaic acid binding [Zhang et al. (1994) J. Biol. Chem. 269, 16997-17000]. In this study, the role of this region in toxin binding was explored by site directed mutagenesis. A residue (Tyr-272) was identified whose mutation had dramatic effects on the spectrum of inhibitor sensitivity of PP1. The IC50's of a number of mutants of Tyr-272 toward okadaic acid, tautomycin, calyculin A, microcystin-LR, nodularin, inhibitor-2, and cantharidic acid were determined and compared to that of the wild-type enzyme. The sensitivity of PP1 toward tautomycin and calyculin A was markedly decreased, by as much as 3 orders of magnitude, with lesser effects on okadaic acid and nodularin, and with microcystin-LR and inhibitor-2 being the least affected. These studies show that Tyr-272 is of specific importance for the binding of these inhibitors and provide strong evidence for the postulate that these toxins all bind to a common inhibitor site on PP1. In addition, our studies show that Tyr-272 is not required for catalytic activity. PMID- 8634293 TI - Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids. AB - The mechanism of interaction of phorbol esters with conventional protein kinase Cs was addressed by examining the direct binding of this class of activators to protein kinase C beta II. Binding measurements reveal that the major role of phorbol esters is to increase the affinity of protein kinase C for membranes by several orders of magnitude. The relative increase depends linearly on the mole fraction of phorbol esters in membranes, with the potency illustrated by the finding that 1 mol% phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membrane association by approximately 4 orders of magnitude. For comparison, diacylglycerol (DG), which also activates protein kinase C by increasing the enzyme's membrane affinity, is 2 orders of magnitude less effective than PMA in altering protein kinase C's membrane affinity. The remarkably high-affinity interaction with phorbol esters allowed us to measure the direct binding of protein kinase C to PMA in neutral membranes and, thus, to evaluate the effect of Ca2+ on the phorbol ester interaction in the absence of Ca2+ effects on the enzyme's interaction with acidic lipids. Changing the Ca2+ concentration over 5 orders of magnitude had no effect on the direct interaction of protein kinase C with PMA immobilized in phosphatidylcholine membranes. Thus, the Ca(2+)-binding site for membrane association and the phorbol ester-binding site do not interact allosterically. Lastly, a method that does not have the limitations of the Scatchard plot for analysis of amphitropic proteins was used to determine the dissociation constant of protein kinase C from phorbol esters: expressed relative to membrane lipids, the dissociation constant is 1.5 x 10(-5) mol %. In summary, our data reveal that (1) the direct binding of protein kinase C to phorbol esters, in the absence of interactions with acidic lipids, provides a major contribution to the free energy change involved in the association of protein kinase C with membranes and (2) this interaction is not regulated by Ca2+. PMID- 8634294 TI - Ligand recognition by anti-DNA autoantibodies. Affinity, specificity, and mode of binding. AB - Understanding the molecular basis of DNA recognition by anti-DNA autoantibodies is a key element in defining the role of antibody.DNA complexes in the pathogenesis of the autoimmune disorder systemic lupus erythematosus. As part of our efforts to relate anti-DNA affinity and specificity to antibody structure, and ultimately to disease pathogenesis, we have generated a panel of eight anti DNA mAbs from an autoimmune MRL MpJ-lpr/lpr mouse and have assessed the binding properties of these antibodies. We find that none of our anti-DNA mAbs bind to RNA and only one low-affinity mAb cross-reacts with non-DNA antigens, albeit weakly. None of the mAbs in our panel bind double-stranded DNA exclusively. Antibodies that recognize single-stranded DNA can be categorized into two groups based on their affinity and apparent mode of binding. One group possesses relatively high affinity for oligo(dT) and may recognize single-stranded DNA ligands by accommodating thymine bases in hydrophobic pockets on the antigen binding site. The second group binds more weakly, apparently recognizes single stranded DNA nonspecifically, and in some cases also binds double-stranded DNA. Although different mechanisms are used for binding single- and double-stranded ligands, the mode of DNA recognition appears conserved within groups of antibodies. PMID- 8634295 TI - Glucocorticosteroid receptor dimerization investigated by analysis of receptor binding to glucocorticosteroid responsive elements using a monomer-dimer equilibrium model. AB - The aim of this study was to analyze the role of regions of the glucocorticosteroid receptor (GR) outside the DNA binding domain (DBD) in GR binding and homodimerization efficiencies by using a model according to which GR monomers and dimers are in equilibrium and able to bind to each half-palindromic motif of a GRE. We studied wild-type human GR (hGR), an N-terminal domain deleted mutant (lacking amino acids 1-417), a C-terminal deleted mutant (lacking amino acids 550-777, the main part of the ligand binding domain), and two rat GR derivatives limited to the DNA binding domain and proximal sequences. Specific GR monomer and dimer complexes with 33P-labeled palindromic or half-palindromic GREs were identified by gel-shift and methylation interference experiments. The different complexes were quantified, and the multiple equilibrium constants for their formation were determined. The affinity of the monomer for the GRE was not affected by the deletions of regions outside the DBD. However, the affinity of the dimer for the GRE was clearly increased by the presence of the N-terminal domain and, to a lesser extent, by that of the main part of the C-terminal domain. By using this model, we also obtained a GR dimerization constant in the absence of specific binding to GRE. Dimerization of the DBD was not increased by the presence of only one of the GR terminal domains, but an increase in dimerization efficiency was observed when both domains were present, suggesting a structural synergy between the N- and C-terminal domains in GR homodimerization. PMID- 8634296 TI - Binding of the oxidized, reduced, and radical flavin species to chorismate synthase. An investigation by spectrophotometry, fluorimetry, and electron paramagnetic resonance and electron nuclear double resonance spectroscopy. AB - Chorismate synthase (EC 4.6.1.4) binds oxidized riboflavin-5'-phosphate mononucleotide (FMN) with a KD of 30 microM at 25 degrees C, but in the presence of 5-enolpyruvylshikimate-3-phosphate (EPSP), the KD decreases to ca. 20 nM. Similar effects occur with the substrate analogue (6R)-6-fluoro-EPSP (KD = 36 nM) and chorismate (KD = 540 nM). Fluorescence of oxidized FMN is slightly quenched in the presence of chorismate synthase. Addition of EPSP or the (6R)-6-fluoro analogue causes a shift of the fluorescence from 520 to 495 nm. Chorismate causes no shift in, but a quenching of, the fluorescence emission maximum. In the presence of EPSP, (6R)-6-fluoro-EPSP, or chorismate, the neutral flavinsemiquinone is generated. The electron paramagnetic resonance (EPR) line width of the flavin radical is indicative of a neutral flavinsemiquinone. Frozen solution electron nuclear double resonance (ENDOR) of the radical with (6R)-6 fluoro-EPSP shows a number of proton ENDOR line pairs. The largest splitting is assigned to a hyperfine coupling to the methyl group beta-protons at position 8 of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A perpendicular = 8.07 MHz and A parallel = 9.60 MHz, giving Aiso of 8.58 MHz, consistent with a neutral semiquinone form. The isotropic hfc coupling of the 8 methyl protons with (6R)-6-fluoro-EPSP decreases by about 0.5 MHz when chorismate is bound, indicating that the spin density distribution within the isoalloxazine ring system depends critically on the nature of the ligand. The redox potential of FMN in the presence of chorismate synthase was 95 mV more positive than that of free FMN (at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN. The pH dependence of the redox potential of chorismate synthase-bound FMN exhibits a slope of -30 mV per pH unit between pH 6 and 9, indicating that the two-electron reduction of the flavin is associated with the uptake of one proton; this, and the UV-visible spectrum, is consistent with the reduced flavin being bound to chorismate synthase in its monoanionic form. PMID- 8634297 TI - Engineering specificity for folate into dihydrofolate reductase from Escherichia coli. AB - Despite several similarities in structure and kinetic behavior, the bacterial and vertebrate forms of the enzyme dihydrofolate reductase (DHFR) exhibit differential specificity for folate. In particular, avian DHFR is 400 times more specific for folate than the Escherichia coli reductase. We proposed to enhance the specificity of the E. coli reductase for folate by incorporating discrete elements of vertebrate secondary structure. Two vertebrate loop mutants, VLI and VLII containing 3-7 additional amino acid insertions, were constructed and characterized by using steady-state kinetics, spectrofluorimetric determination of ligand equilibrium dissociation constants, and circular dichroism spectroscopy. Remarkably, the VLI and VLII mutants are kinetically similar to wild-type E. coli reductase when dihydrofolate is the substrate, although VLII exhibits prolonged kinetic hysteresis. Moreover, the VLI dihydrofolate reductase is the first mutant form of E. coli DHFR to display enhanced specificity for folate [(kcat/Km)mutant/(kcat/Km)wt = 13]. A glycine-alanine loop (GAL) mutant was also constructed to test the design principles for the VLI mutant. In this mutant of the VLI reductase, all of the residues from positions 50 to 60, except the strictly conserved amino acids Leu-57 and Arg-60, were converted to either glycine or alanine. A detailed kinetic comparison of the GAL and wild-type reductases revealed that the mutations weaken the binding by both cofactor and substrate by up to 20-fold, but under saturating conditions the enzyme exhibits a kcat value nearly identical to that of the wild type. The rate of hydride transfer is reduced by a factor of 30, with a compensating increase in the dissociation rate for tetrahydrofolate. Although key stabilizing interactions have been sacrificed (it shows no activity toward folate), the maintenance of the correct register between key residues preserves the activity of the enzyme toward its natural substrate. Collectively, neither specific proximal point site mutations nor larger, more distal secondary structural substitutions are sufficient to confer a specificity for folate reduction that matches that observed with the avian enzyme. This is consistent with the hypothesis that the entire protein structure must contribute extensively to the enzyme's specificity. PMID- 8634298 TI - Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay? AB - Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the E1 active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041]. We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmalemide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid. PMID- 8634300 TI - Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction. AB - Although there is considerable evidence that signaling by the erythropoietin (EPO) receptor is initiated when it is dimerized by binding EPO, it has been previously reported that the soluble extracellular domains of the EPO receptor (sEPOR) are not dimerized in the presence of EPO and are able to form only 1:1 complexes with EPO. We have now shown unambiguously by light scattering, sedimentation equilibrium, and titration calorimetry that two molecules of sEPOR can bind to a single EPO monomer but that the binding of the second sEPOR is approximately 1000-fold weaker than that of the first. Because this second binding interaction is quite weak (Kd of approximately 1 microM), the 2:1 sEPOR.EPO complexes are easily dissociated during chromatography (forming the 1:1 complexes reported previously) and cannot be isolated in pure form. Global analysis of the sedimentation equilibrium data has enabled us to determine the binding constants and is consistent with a model in which EPO has two independent binding sites for sEPOR but cannot exclude anticooperative or sequential binding models. The influence of glycosylation of EPO and/or sEPOR on the binding affinities has also been investigated. Titration calorimetry is consistent with the sedimentation data and shows that the weaker binding site has a more negative delta H. The relation of these results to the binding of EPO to membrane-bound receptors and to the phenomenon of apparent high-affinity and low-affinity classes of receptors is discussed. PMID- 8634299 TI - Site-directed mutagenesis of active site glutamate-217 in mouse adenosine deaminase. AB - Mouse adenosine deaminase (ADA) contains an active site glutamate residue at position-217 that is highly conserved in other adenosine and AMP deaminases. Previous research has suggested that proton donation to N-1 of the adenosine ring occurs prior to catalysis and supports the mechanism as proceeding via formation of a tetrahedral intermediate at C-6 of adenosine. The proposed catalytic mechanism of ADA based on the recent elucidations of the crystal structure of this enzyme with transition- and ground-state analogs hypothesized that Glu217 was involved in this proton donation step [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284; Wilson, D. K., & Quiocho, F. A. (1993) Biochemistry 32, 1689-1693]. Site-directed mutagenesis of the equivalent glutamate in human ADA resulted in a dramatic loss of enzyme activity [Bhaumik, D., Medin, J., Gathy, K., & Coleman, M. (1993) J. Biol. Chem. 268, 5464-5470]. To further study the importance of this residue, site-directed mutagenesis was used to create mouse ADA mutants. Glu217 was mutated to Asp, Gly, Gln, and Ser, and all mutants were successfully expressed and purified. Circular dichroism and zinc analysis showed no significant changes in secondary structure or zinc content, respectively, compared to the native protein. The mutants showed only a slight variation in Km but dramatically reduced kcat, less than 0.2% of wild-type activity. UV difference and 13C NMR spectra conclusively demonstrated the failure of any of these mutants to hydrate purine riboside, a reaction carried out by the wild-type enzyme that results in formation of an enzyme-inhibitor complex. Surprisingly, Ki values for binding of the inhibitor to the mutants and to wild type protein are similar, irrespective of whether the inhibitor is hydrated upon binding. These data confirm the importance of Glu217 in catalysis as suggested by the crystal structure of mouse ADA. PMID- 8634301 TI - Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants. AB - High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction. PMID- 8634302 TI - Converting an alcohol to an amine in a cationic lipid dramatically alters the co lipid requirement, cellular transfection activity and the ultrastructure of DNA cytofectin complexes. AB - Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE. PMID- 8634303 TI - Non-phospholipid fusogenic liposomes. AB - We have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%. PMID- 8634304 TI - Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene. AB - The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than alpha-tocopherol or alpha-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings. PMID- 8634305 TI - Chloride transport in human proximal colonic apical membrane vesicles. AB - The mechanism(s) of Cl- transport across the human colonic apical membranes are not well understood. Apical membrane vesicles (AMV) purified from organ donor proximal colonic mucosa and a rapid millipore filtration technique were utilized to study 36Cl- uptake into these vesicles. Outwardly directed OH- and HCO3- gradient stimulated 36Cl- uptake into these vesicles demonstrating a transient accumulation over equilibrium uptake. Voltage clamping the membrane potential of the vesicles or making them inside positive with K+/valinomycin failed to influence chloride uptake, indicating that the conductive Cl- uptake pathway is minimal in proximal colonic AMV. Anion exchange inhibitors, DIDS and SITS (1 mM) inhibited OH- and HCO3- stimulated 36Cl- uptake by approximately 60%. Furosemide also demonstrated a small but significant inhibition of chloride uptake. Amiloride, bumetanide and acetazolamide (1 mM) failed to inhibit 36Cl uptake. HCO3- and pH gradient stimulated 36Cl- uptake exhibited saturation kinetics with an apparent Km for chloride of 4.0 +/- 0.7 mM and Vmax of 17.8 +/- 3.9 nmol/mg per min. Bromide, chloride, nitrate and acetate (50 mM each) inhibited 5 mM 36Cl uptake. Inwardly directed gradients of Na+, K+, or Na+ and K+ did not stimulate 36Cl- uptake into these vesicles, indicating that uptake of Na+ and Cl- in human proximal colonic AMV does not involve Na-Cl or Na-K-2Cl cotransport. The above findings indicate that chloride transport in human proximal colonic AMV involves an electroneutral Cl-HCO3- (OH-) exchange process. In view of the previous demonstration of Na+-H+ antiporter in these vesicles, dual ion exchange mechanism of Na+-H+ and Cl-HCO3- in apical membrane domain of human colonocytes is postulated to be the primary mechanism for NaCl absorption in the human proximal colon. PMID- 8634306 TI - Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. AB - The effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated. The toxin induced carboxyfluorescein leakage from liposomes composed of the choline containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanolamine, egg-yolk phosphatidylserine or phosphatidylglycerol. The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations. The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms. Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of acyl residues of phosphatidylcholine used. The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin. The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl-L-alpha-phosphatidylcholine and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents. These observations suggest that the specific binding site formed by the choline containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin. PMID- 8634307 TI - Temperature-controlled release property of phospholipid vesicles bearing a thermo sensitive polymer. AB - As a novel temperature-sensitive liposome, dioleoylphosphatidylethanolamine vesicles bearing poly(N-isopropylacrylamide), which shows a lower critical solution temperature (LCST) near 32 degrees C, were designed. Poly(N isopropylacrylamide) having long alkyl chains which are anchors to the lipid membranes was prepared by radical copolymerization of N-isopropylacrylamide and octadecyl acrylate using azobisisobutyronitrile as the initiator. The copolymer obtained revealed the LCST at about 30 degrees C in an aqueous solution. Dioleoylphosphatidylethanolamine vesicles coated with the copolymer was prepared and release property of the copolymer-coated vesicles was investigated. While release of calcein encapsulated in the copolymer-coated vesicles was limited below 30 degrees C, the release was drastically enhanced between 30 and 35 degrees C. Complete release from the vesicles was achieved within several seconds at 40 degrees C. This temperature-controlled release property of the vesicles can be attributable to stabilization and destabilization of the vesicle membranes induced by the copolymer fixed on the vesicles below and above the LCST, respectively. Moreover, the fluorometric measurement using dioleoyl-N-(7 nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethan ola mine suggested that the extensive release of calcein observed above the LCST is resulted from the bilayer to HII phase transition of the vesicle membranes. Since LCST of the copolymer is controllable, these vesicles might have potential usefulness as a drug delivery system with high temperature-sensitivity. PMID- 8634308 TI - Gamma-irradiation of liposomes composed of saturated phospholipids: effect of bilayer composition, size, concentration and absorbed dose on chemical degradation and physical destabilization of liposomes. AB - Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), or mixtures of these two phospholipids were exposed to gamma-irradiation in an air environment. Disappearance of the mother compounds was monitored by HPLC analysis. Plotting of the logarithmic values of residual DPPC or DPPG concentration versus irradiation dose resulted in straight lines. The slopes of these lines (overall degradation constants) depended on the type of phospholipids, concentration of the liposomes and the size of the liposomes. Under the chosen conditions, addition of DPPG in DPPC liposomes did not affect the degradation rate constant of DPPC and vice versa. The presence of phosphate buffer (pH 7.4), pH or presence of sodium chloride did not affect the irradiation damage either. Minor changes were found upon analysis of total fatty acids by GLC and upon measurement of water soluble phosphate compounds. These changes were less pronounced than the changes monitored by HPLC of phospholipids, because the HPLC analysis monitored the overall degradation of the liposomal phospholipids. Thin-layer chromatography/fast atom bombardment mass spectrometry (TLC/FAB-MS) analysis of irradiated and non-irradiated DPPC or DPPG provided information on the structure of several degradation products. Degradation routes which include these degradation products are proposed. Gamma irradiation neither affected the size of the liposomes nor the bilayer rigidity as determined by dynamic light scattering and fluorescence anisotropy of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. However, upon gamma irradiation, changes in the melting characteristics of the liposomes were found by differential scanning calorimetry (DSC) measurements. The pre-transition melting enthalpy of the liposomal bilayer decreased or disappeared and the main transition broadened. The changes found in DSC scans correlated qualitatively well with the changes recorded after HPLC analysis of phospholipids. PMID- 8634310 TI - Vanadate inhibits vacuolar H(+)-ATPase-mediated proton transport in chicken kidney microsomes by an ADP-dependent mechanism. AB - Recent reports indicate that vacuolar-type proton ATPases from chicken osteoclasts (Chatterjee et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6257-6261), yeast vacuoles and chromaffin granules (Beltran and Nelson (1992) Acta Physiol. Scand. Suppl. 607, 41-47) can be inhibited by vanadate, albeit at a concentration much higher than that required to inhibit P-type ATPases. We have characterized the mechanism by which vanadate inhibits vacuolar-type ATPase-mediated proton transport by chicken kidney microsomes. The initial rate of proton transport is somewhat less sensitive to vanadate than the total acidification, with IC50 values of 1.58 mM and 0.78 mM vanadate, respectively. The inhibition of both the initial rate and total acidification is noncompetitive with respect to ATP. The inhibition is abolished when ADP is removed by an ATP-regenerating system, and the addition of exogenous ADP increases the vanadate inhibition of proton transport in a synergistic manner, thus demonstrating that inhibition by vanadate is dependent on the presence of ADP and explaining the lower effect of vanadate on the initial rate of acidification. Phosphate protects proton transport activity from inhibition by vanadate. These effects of ADP and phosphate suggest that inhibition by vanadate may involve the formation of a complex with ADP at a nucleotide binding site, possibly at the catalytic site of the enzyme. PMID- 8634309 TI - Effect of serum protein binding on real-time trafficking of liposomes with different charges analyzed by positron emission tomography. AB - Liposomes have been used as carriers of various materials and as tools for gene transfer: for the latter purpose, positively charged liposomes are usually used. To evaluate the stability in the presence of serum and the in vivo behavior of such liposomes as well as those aspects of neutral and negatively charged liposomes, we investigated liposomal agglutinability in the presence of serum, serum protein binding to these liposomes, and real-time liposomal trafficking by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol without or with charged lipid were prepared in the presence of mannitol, and the turbidity change in the presence of serum was determined. Turbidity increase was not observed for so called long-circulating liposomes, i.e., liposomes modified with glucuronic acid or with poly(ethylene glycol), or for negatively charged liposomes containing dicetyl phosphate (DCP), phosphatidylglycerol, or phosphatidylserine. On the contrary, a significant turbidity increase was observed when positively charged liposomes modified with stearylamine, stearyltrimethylammonium chloride or 1,2 dimyristyloxypropyl-3-dimethylhydroxyethyl bromide (DMRIE), which is known as a component of liposomes for gene transfer, were used. These liposomes were found to have bound a high amount of serum proteins after separation of unbound serum proteins by use of a spin column. The liposomal trafficking in vivo was determined for three kinds of liposomes, i.e., liposomes with DMRIE, those with DCP, and those without charged lipids. These liposomes were prepared in the presence of 2-[18F]fluoro-2-deoxy-D-glucose ([2-18F]FDG), and the [2-18F]FDG labeled liposomes were administered to mice to perform PET scans. Positively charged liposomes containing DMRIE showed high accumulation in the liver compared with neutral and negatively charged liposomes. Since DMRIE-liposomes tended to aggregate in the presence of serum, and to be associated with serum protein, these characteristics may lead to the high uptake of DMRIE-liposomes by the liver. PMID- 8634311 TI - Role of membrane fluidity in Epstein Barr virus (EBV) infectivity on Akata cell line. AB - Infection of Epstein Barr virus (EBV) to its host cells is initiated by the attachment of the glycoprotein gp 350/220 to the CR2 molecule. We used the sensitivity at the polar environment of the fluorescent probe Laurdan to study the membrane viscosity distribution from single leaving cells on two lymphoid cell lines Raji and Akata. Lipid analysis on both cells line demonstrated a lower cholesterol to phospholipid molar ratio on Akata than Raji cells. Cell fluidity analysis by Laurdan generalized emission polarization (GP) or by DPH polarization, indicated that membrane viscosity of Akata was lower than Raji cells. This difference was correlated to the increased susceptibility of Akata cells in expressing EBV early antigens (EA) after EBV superinfection. PMID- 8634312 TI - Effects of aging on the microclimate pH of the rat jejunum. AB - The acidic microclimate layer in the vicinity of the cell surface of mammalian jejunum is important for absorption of some nutrients, such as small peptides and folate. The present study was undertaken to investigate the effect of aging on the cell surface pH (microclimate pH) of the jejunum of rats. The microclimate pH was measured in vitro in superfused preparations using single-barreled pH sensitive microelectrodes filled with a liquid ion exchanger. The thickness of the microclimate layer was estimated by reading the distance of microelectrode advancements. The existence of a microclimate pH in the jejunum was confirmed in the senescent rats, but the value of the microclimate pH was significantly higher in the senescent (24 mo) rats (6.52 +/- 0.02) than in the young-adult (6 mo) rats (6.09 +/- 0.01) (P < 0.01). Na+ removal from the perfusate or the addition of amiloride elevated the pH in the senescent rats as well as in the young-adult rats. The microclimate layer was slightly thinner in the senescent rats than in the young-adult rats. The acidity of the microclimate layer of intestinal surface is lower in senescent animals than in the young-adult ones. One of reasons for this is the thinner mucus layer in senescent animals. PMID- 8634313 TI - Endocytosis inhibitors abolish the active transport of polypeptides in the mucosa of the nasal upper concha of the rabbit. AB - An active absorption of polypeptides (elcatonin = CCT; adrenocorticotropic hormone) had been previously observed in the nasal respiratory mucosa of the rabbit. Its saturation kinetics and the parallel absence of a net transfer of other non-polypeptidic organic markers excluded the involvement of a simple pinocytosis. This absorption has been now better localized and further characterized. Unidirectional CCT fluxes (determined with radioimmunoassay) have been concomitantly monitored with transepithelial electric potential difference (Vms). Although the mucosae covering the ectoturbinal A and the lower and upper conchae displayed similar Vms, the active CCT transport was only evidenced in the upper concha. In this region cytochalasin B (which by disassembling actin microfilaments prevents the apical formation of vesicles in epithelial cells) and monensin (which prevents the split of the ligand-receptor complex in the endosomes) both eliminated the net CCT absorption, however, also permanently increasing the passive CCT junctional permeability. Aluminum fluoride (which prevents the fusion of endocytic vesicles into endosomes) and colchicine (which disrupts microtubules along which vesicles move in the cytoplasm) also permanently abolished net CCT transport, without affecting, or shortly and transiently affecting, passive permeability. On the whole these results are in favor of an active CCT transport supported by a specific vesicular transport. PMID- 8634314 TI - Time domain dielectric spectroscopy study of human cells. I. Erythrocytes and ghosts. AB - A method, allowing correction of electrode polarization effect in case of cell suspensions of small volume fraction is proposed. The dielectric behavior of human erythrocytes and erythrocyte ghosts suspensions was studied by time domain dielectric spectroscopy (TDDS) and the estimation of the dielectric constants of cell's structural parts (membrane, cytoplasm) on the basis of suitable models are presented. It was shown that in the case of small volume fraction of cells in suspension, the electrode polarization effect can be taken into account by the additional measurement of the corresponding supernatant. The optimal volume fraction of cells in suspensions in the TDDS measurements was found to be 3%-10%. The erythrocyte and erythrocyte ghost suspensions were found to demonstrate a single dispersion which can be described by the Debye equation. The membrane dielectric constant of different erythrocytes and ghosts was distributed near 5. PMID- 8634315 TI - Rapid transmembrane movement of C6-NBD-labeled phospholipids across the inner membrane of Escherichia coli. AB - In this study we have investigated the transmembrane movement of short chain fluorescently labeled phospholipids across the inner membrane of Escherichia coli. Exogenously added C6-NBD-labeled phospholipids rapidly flip across the inner membrane of E. coli, as was shown by a dithionite reduction assay applied to inverted inner membrane vesicles (IIMV) isolated from wild type E. coli cells. The rate of transmembrane movement of the phospholipid probes incorporated into IIMV is temperature dependent, and shows no phospholipid head group specificity. C6-NBD-labeled phospholipids translocate across the membrane of IIMV incubated at 37 degrees C with a t1/2 of 7 min. After the incorporation into IIMV C6-NBD-PG is partially converted to CL by CL-synthase. If IIMV are pretreated with proteinase K the conversion of this fluorescent probe to C6-NBD-CL is not observed anymore, suggesting that the catalytic domain of CL-synthase is at the cytoplasmic site of the plasma membrane of E. coli. Newly synthesized C6-NBD-CL also flips across the inner membrane although at a slower rate than the other phospholipid probes. The transmembrane movement occurs in both directions and is not influenced by treatment of the IIMV with a sulfhydryl reagent or a proteinase, nor by the presence of ATP, or a deltapH across the membrane of the IIMV. However, the transmembrane movement of the C6-NBD-labeled phospholipid probes is not observed in LUVETs (large unilamellar vesicles made by extrusion technique) prepared of wild type E. coli lipids, indicating that the rapid transmembrane movement of phospholipids across the inner membrane of E. coli is a protein-mediated process. PMID- 8634316 TI - Interaction of the fluorescent probe RH421 with ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. AB - Fluorescence titrations have shown that the voltage-sensitive probe RH421 interacts with the water-soluble protein ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. The probe exhibits significantly different fluorescence excitation spectra in pure lipid and pure protein environments. Experiments with a range of polyamino acids showed interactions of the probe with tyrosine, lysine and arginine residues. At saturating RH421 concentrations (> or = microM) the probe quenches 60-75% of the total tryptophan fluorescence of the Na+,K(+)-ATPase preparation. Inhibition of the hydrolytic activity of the Na+,K(+)-ATPase occurs at RH421 concentrations in the micromolar range. This may be due to a probe-induced change in membrane fluidity. The sensitivity of the probe towards conformational changes of the Na+,K(+)-ATPase decreases hyperbolically as one increases the probe concentration. The decrease in sensitivity correlates well with association of the probe in the vicinity of membrane protein, as measured by tryptophan quenching. These results have important practical consequences for the application of RH421 as a voltage indicator in membrane preparations. Based on these and previously reported results, the fluorescent response of RH421 to the ATP-induced conformational change of the Na+,K+-ATPase is consistent with either a redistribution of dye from the liquid-crystalline lipid matrix into the vicinity of membrane protein or a reorganisation of the lipids surrounding the protein into a more rigid structure caused by the conformational change of the protein. PMID- 8634317 TI - Fluorescence studies of the effect of pH, guanidine hydrochloride and urea on equinatoxin II conformation. AB - The solvent denaturation of equinatoxin II (EqTxII) in aqueous solutions of urea, guanidine hydrochloride (Gu-HCl) and at various pH values was examined by monitoring changes in the protein intrinsic emission fluorescence spectra and in the fluorescence spectra of the added external probe ANS. It has been observed that EqTxII denaturation is reflected in a strong red shift of intrinsic fluorescence emission maxima accompanied by a simultaneous decrease in fluorescence intensity and that guanidine hydrochloride is significantly more powerful denaturant than urea or changing of pH. Comparison of intrinsic fluorescence spectra of EqTxII denatured by one of the three denaturing agents has shown that the fully denatured states of the protein in Gu-HCl and urea are similar and substantially different from those induced by changing of pH. Furthermore, according to the measurements of the ANS-fluorescence in EqTxII solutions as a function of pH the protein exists at pH values below 2.0 in an acid-denatured compact state. PMID- 8634318 TI - Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis. AB - The physicochemical effects of a series of alkanols, alkanediols and glycerol on erythrocyte shape and hemolysis at 4 and 20 degrees C were examined. We calculated the dielectric constant of the incubation medium, Ds, and the dielectric constant of the erythrocyte membrane Dm in the presence of organic solutes. The ratio Ds/Dm = -38.48 at 20 degrees C defines the normal biconcave shape in a medium without hemolytic agents. A decrease in Ds/Dm favors externalization or internalization with consequent hemolysis. Alkanols and alkanediols convert biconcave erythrocytes into echinocytes, which is accompanied by an increase in the projected surface area. Glycerol converts biconcave erythrocytes into stomatocytes, which was accompanied by a marginal decrease in the projected surface area. Progressive externalization in alkanols and alkanediols or internalization in glycerol resulted in a decrease in the projected surface area and the formation of smooth spheres. The degree of shape change induced was related to the degree of hemolysis and the ratio Ds/Dm. A decrease in temperature reduced both the degree of shape change and hemolysis. Our results suggest that physicochemical toxicity may be a result of a temperature dependent hydrophobic interaction between the organic solutes and the membrane and is best interpreted by the ability of the solutes to change Ds and Dm. These results are discussed with respect to the physicochemical constants of the organic solutes. PMID- 8634319 TI - Probing the cytoplasmic LOOP1 domain of the yeast plasma membrane H(+)-ATPase by targeted factor Xa proteolysis. AB - The cytoplasmic domain linking transmembrane segments 2 and 3 (LOOP1) of the yeast H(+)-ATPase was probed by the introduction of unique factor Xa recognition sites. Three sites, I170EGR, I254EGR and I275EGR, representing different structural regions of the LOOP1 domain, were engineered by site-specific mutagenesis of the PMA1 gene. In each case, multiple amino acid substitutions were required to form the factor Xa sites, which enabled an analysis of clustered mutations. Both I170EGR and I275EGR-containing mutants grew at normal rates, but showed prominent growth resistance to hygromycin B and sensitivity to low external pH. The engineered I254EGR site within the predicted beta-strand region produced a recessive lethal phenotype, indicating that mutations G254I and F257R were not tolerated. Mutant I170EGR- and I275EGR-containing enzymes showed relatively normal Km and Vmax values, but they displayed a strong insensitivity to inhibition by vanadate. An I170EGR/I275EGR double mutant was more significantly perturbed showing a reduced Vmax and pronounced vanadate insensitivity. The I170EGR site within the putative alpha-helical stalk region was cleaved to a maximum of 10% by factor Xa under non-denaturing conditions resulting in a characteristic 81 kDa fragment, whereas the I275EGR site, near the end of the beta-strand region, showed about 30-35% cleavage with the appearance of a 70 kDa fragment. A I170EGR/I275EGR double mutant enzyme showed about 55-60% cleavage. The cleavage profile for the mutant enzymes was enhanced under denaturing conditions, but was unaffected by MgATP or MgATP plus vanadate. Cleavage at the I275EGR position had no adverse effects on ATP hydrolysis or proton transport by the H(+)-ATPase making it unlikely that this localized region of LOOP1 influences coupling. Overall, these results suggest that the local region encompassing I275EGR is accessible to factor Xa, while the region around I170EGR appears buried. Although there is no evidence for gross molecular motion at either site, the effects of multiple amino acid substitutions in these regions suggest that the LOOP1 domain is conformationally active, and that perturbations in this domain affect the distribution of conformational intermediates during steady-state catalysis. PMID- 8634320 TI - Differential biodistribution of encapsulated and surface-linked liposomal antigens. AB - The biodistribution of liposomal antigens either encapsulated in or surface linked to liposomes of similar composition was studied over time following intravenous injection and the results analyzed in relation to adjuvanticity. The two formulations were shown to behave very differently in vivo. While encapsulated antigen was rapidly focused to liver and spleen as expected, surface linked antigen exhibited a more disseminated distribution which parallels that of the free protein. In dual-labelling experiments, it was also shown that encapsulated antigen remains associated with its liposomal vehicle in contrast to surface-linked antigen which is rapidly dissociated. This dissociation was apparently neither due to an exchange with plasma lipoproteins nor to a direct action of blood constituents. Besides, it was found that surface-linked antigen was rapidly accumulated in the carcass. We propose that the retention of the surface-linked antigen in the carcass results from a pre-processing of the protein involving more probably mononuclear phagocytes. This pre-processing might in turn favor the dissociation of the protein from the liposomes in a form that allows its dissemination in the whole organism and its interaction with more efficient antigen presenting cells such as for example Langerhans or dendritic cells. PMID- 8634321 TI - [3H]Bafilomycin as a probe for the transmembrane proton channel of the osteoclast vacuolar H(+)-ATPase. AB - Bone resorption by the osteoclast is dependent on acidification of the bone surface by a vacuolar type H+-ATPase (V-ATPase) present in the ruffled membrane of the actively resorbing cell. V-ATPases are a highly conserved family of proton pumps consisting of two functional complexes: a cytoplasmic catalytic sector (VC) and a membrane bound proton channel (VB). Bafilomycin A1, a macrolide antibiotic, is a highly potent inhibitor of V-ATPases, and inhibits bone resorption in vitro in isolated rat calvariae. In order to investigate the binding of bafilomycin to the osteoclast V-ATPase, we used a tritiated bafilomycin which had been prepared by acetylating the 21-hydroxyl group of bafilomycin A1. Osteoclast ruffled membrane vesicles were prepared from purified chicken osteoclasts by differential centrifugation and proton transport in these vesicles was shown to be inhibited by [3H]bafilomycin (IC50 approximately 2 nM). Control membrane vesicles or membrane vesicles partially inhibited with [3H]bafilomycin were solubilized and separated by centrifugation on 15-30% glycerol gradients. V-ATPase activity and reconstitutable proton transport activity could be recovered in high density fractions of the gradient. However, the peak of [3H]bafilomycin radioactivity (>70% of total radioactivity in the gradient) was present in a single peak at lower density. Antibodies against subunits of VC (70, 56 and 40 kDa) reacted only in fractions containing the peak V-ATPase activity whereas an antibody to the 39 kDa subunit of VB reacted both with fractions containing the peak V-ATPase activity but also, and more strongly, in fractions containing the peak [3H]bafilomycin. The fractions in the control gradient corresponding to the peak of [3H]bafilomycin were reconstituted into liposomes and shown to mediate passive bafilomycin A1-inhibitable proton conductance. SDS-PAGE followed by autoradiography, indicated that the bafilomycin was not covalently bound to the V ATPase or the proton channel. Quantification of VB by [3H]bafilomycin binding or by antibody staining suggested an excess of the free proton channel to that of the intact holoenzyme. A corresponding amount of free catalytic sector could not be found in any fraction throughout the isolation procedure of the V-ATPase from the initial homogenate. Thus, in conclusion, bafilomycin inhibits the V-ATPase by binding tightly but non-covalently to the proton channel region of the V-ATPase which appears to be present in excess over the intact holoenzyme in the osteoclast. The possible role of an excess of the proton channel subcomplex in the osteoclast is discussed. PMID- 8634322 TI - Structural organization, ion transport, and energy transduction of P-type ATPases. PMID- 8634323 TI - Ionic channel rundown in excised membrane patches. PMID- 8634324 TI - Regulation of ATP-sensitive potassium channels by phosphorylation. PMID- 8634325 TI - DNA minor groove accessibility in the nucleosome core deduced from specific interactions with DAPI. AB - Equilibrium binding properties of DAPI for purified nucleosome cores were derived from fluorescence intensity enhancement. Both the affinity and the number of sites of the primary, minor groove binding are preserved with respect to the corresponding isolated DNA, suggesting that the capacity for specific interactions of the minor groove may well be exerted in chromatin. PMID- 8634326 TI - Molecular cloning and sequence analysis of the chick melanocortin 1-receptor gene. AB - The chick melanocortin 1-receptor gene was isolated. It is found to be an intronless gene encoding a 314 amino acid protein, sharing 64% identity with mammalian counterparts. A cis-element responsible for melanocyte-specific transcription is found in its 5' upstream region. It is probable that the expression of the receptor in melanocytes is closely correlated with that of melanogenesis-related genes. PMID- 8634328 TI - Genomic structure of carp mitogen-activated protein kinase kinase 1 gene. AB - Carp mitogen-activated protein kinase kinase 1 (cMKK1) gene was isolated from a liver genomic library. The sequence around the exon-intron boundaries and 2 kb of the promoter region were determined. Our data indicate that this gene is composed of 11 exons and 10 introns spanning about 9 kb. Multiple potential transcription initiation sites were located by primer extension analysis. Examination of 2 kb of 5'-flanking sequence revealed potential binding sites for a variety of transcription factors such as E2F, Ets-1, GATA-1, Myb, NF-IL6, Sp1, and NF-kB. PMID- 8634327 TI - A novel zinc finger gene preferentially expressed in the retina and the organ of Corti localizes to human chromosome 12q24.3. AB - A cDNA encoding a novel member of the zinc finger gene family, designated zfOC1, has been cloned from the organ of Corti. This is the first transcriptional regulator cloned from this sensory epithelium. This transcript encodes a peculiar protein composed of 9 zinc finger domains and a few additional amino acids. The deduced polypeptide shares 66% amino acid similarity with MOK-2, another protein of only zinc finger motifs and preferentially expressed in transformed cell lines. Northern blot hybridization analysis reveals that zfOC1 transcripts are predominantly expressed in the retina and the organ of Corti and at lower levels in the stria vascularis, auditory nerve, tongue, cerebellum, small intestine and kidney. The human gene was mapped, using a human x hamster somatic cell hybrid panel and fluorescent in situ hybridization, to chromosome 12q24.3. Because of its relative abundance in sensorineural structures (retina and organ of Corti), this regulatory gene should be considered a candidate for hereditary disorders involving hearing and visual impairments that link to 12q24.3. PMID- 8634330 TI - Molecular cloning of troponin I expressed in fast white muscle of a teleost fish, the Atlantic herring (Clupea harengus L.). AB - By cDNA cloning and 5'-RACE analysis we characterised a Clupea harengus troponin I mRNA expressed in larvae and in adult white (fast) muscle, but not in red (slow) or cardiac muscle. The mRNA encodes a TnI protein of the short chain length (176 residues) N-terminally truncated type previously observed only in tetrapod skeletal muscles. Despite its expression specificity the herring TnI does not particularly resemble the tetrapod fast skeletal muscle isoform in sequence but appears to be outside of the tetrapod TnIfast/Tnslow/TnIcardiac isoform family. Surprisingly, the actin/TnC-binding sequence resembles that of arthropod, rather than tetrapod vertebrate, troponin I's and has, besides, unique features not seen in any other troponin I's, vertebrate or invertebrate. PMID- 8634329 TI - Isolation, characterization, and mapping to human chromosome 11q24-25 of a cDNA encoding a highly conserved putative transmembrane protein, TMC. AB - We report the isolation of a novel human cDNA encoding a putative transmembrane protein, TMC. The predicted protein sequence is highly conserved evolutionarily. The cDNA clone was mapped to human chromosome 11q24-25 by fluorescence in situ hybridization. mRNA expression was observed in all tissues tested with the highest levels in testes and ovary. PMID- 8634331 TI - Cloning and characterization of cDNAs for novel proteins with glutamic acid proline dipeptide tandem repeats. AB - cDNAs with identical 3' sequences containing a hexanucleotide repeat -(GAGCCG)9- were isolated from rat pheochromocytoma and brain cDNA libraries. The cDNA with the longest open reading frame codes for a protein of 24.6 kDa containing a 16 fold -(Glu-Pro)- dipeptide repeat within a glutamate and proline rich region at its deduced C-terminus. cDNAs with the identical 3' sequence and a divergent 5' sequence were isolated from a rat skeletal muscle cDNA library. The latter are predicted to code for a protein of 15.5 kDa with a C-terminal repetitive domain identical to that in the pheochromocytoma and brain cDNAs. The cDNAs recognize a 1.8 kb mRNA species present in a variety of tissues, being particularly abundant in cardiac and skeletal muscle. PMID- 8634333 TI - Complete structure of the murine p36 (annexin II) gene. Identification of mRNAs for both the murine and the human gene with alternatively spliced 5' noncoding exons. AB - p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2 containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited. PMID- 8634332 TI - Identification of a novel myosin heavy chain gene expressed in the rat larynx. AB - Based on reactivity to antibodies against known myosin heavy chains, expression of a novel fast myosin heavy chain (MHC) gene was suspected in the thyroarytenoid (TA) muscle of the rat larynx. The 3' ends of MHC transcripts in the TA were amplified by RT-PCR using a primer to a highly conserved MHC sequence and to the poly(A) tail. The resultant products were cloned and fourteen PCR products were screened by dot-blotting with oligonucleotides specific for known skeletal muscle MHC genes. A clone that reacted weakly to the 2B oligo was sequenced and found to encode a novel fast MHC transcript, termed 2L, that appears to represent an eighth vertebrate skeletal muscle MHC gene. By homology analysis, the 2L sequence is most similar to the extraocular MHC, suggesting a possible evolutionary relationship between MHCs associated with the branchial arches. PMID- 8634334 TI - Somatic hypermutagenesis in immunoglobulin genes. III. Somatic mutations in the chicken light chain locus. AB - We report a new approach based on the Monte Carlo method, to analyze gene conversion. With this, we have examined 235 somatic mutations in the chicken Vlambda gene and found that about 75% of somatic mutations significantly correlated with donor sequences in 25 pseudo Vlambda genes (set C) versus about 25% that did not (set N). The RGYW and TAA consensus sequences of mutational hotspots were earlier revealed in mammalian V genes. Analysis for correlation between somatic mutations in the Vlambda gene and the consensus sequences showed that the somatic mutations of set N were correlating with the consensus sequences (P(W < Wrandom) < 0.01) and the somatic mutations of set C were not. Based on further statistical analysis, we suggest that there must be at least two mechanisms responsible for somatic hypermutagenesis in the Vlambda gene: gene conversion and another, which accounts for the elevated frequencies of somatic mutations at the RGYW and TAA consensus sequences. PMID- 8634335 TI - Transcriptional regulation of c-Fes in myeloid leukemia cells. AB - The c-Fes proto-oncogene encodes a myeloid-specific protein-tyrosine kinase that is expressed preferentially in differentiated myeloid cells, but not in early myeloblast progenitor cells. To examine the basis for the phenotypic expression of c-Fes, the transcription initiation sites of the human c-Fes gene were mapped in myeloid leukemia cells and regulatory elements in the genomic c-Fes sequence were characterized. Two major transcription initiation sites were found in the myeloid leukemia cell line THP-1 which delineated exon 1 to be 72-83 bp. When the activity of the CAT reporter gene under the control of the c-Fes promoter region, untranslated exon 1 and intron 1 was measured in TF-1, K562 and MCF-7 cells, only TF-1 cells exhibited chloramphenicol acetyltransferase activity. In contrast, all cell lines supported reporter gene activity when intron 1 was deleted. Deletion analyses revealed a negative regulatory region in intron 1, which was localized by Southwestern analysis and DNA footprinting to a 14 bp region. This negative regulatory region suppressed reporter CAT activity in K562 and TF-1 cells when inserted downstream to the SV40 early promoter. These results suggest that the tissue-specific expression of c-Fes may result, in part, from the negative regulation of transcription in myeloid and nonmyeloid cells. PMID- 8634336 TI - High levels of mouse thymosin beta4 mRNA in differentiating P19 embryonic cells and during development of cardiovascular tissues. AB - The self assembly of actin and the large number of actin-binding proteins are important in the establishment of cell shape and function during embryogenesis. Thymosin beta4 (Tbeta4) is a small acidic peptide that participates in the regulation of actin polymerization in mammalian cells. In the present work, we report the presence of the mRNA encoding for Tbeta4 in mouse embryonic stem cells and its induction in P1 9 embryonal cells stimulated to differentiate into ectodermal-like (neurons and glia) or mesodermal-like cells (cardiac and skeletal muscle). The induction of Tbeta4, mRNA in P19 cells was confirmed by in situ hybridization analysis of early mouse postimplantation embryos. Noteworthy, we observed an important hybridization signal in several areas of the embryo specially in blood vessels and in heart tissues, suggesting a role for this peptide in angiogenesis. In conclusion, the results presented here demonstrate the expression of Tbeta4 gene during early embryogenesis which immediately suggests an important role for this peptide in developmental processes requiring actin-based functions such as the formation of cardiovascular system. PMID- 8634337 TI - Distinct effects of ATP on transcription complex formation and initiation in a yeast in vitro transcription system. AB - The stages and kinetics of transcription complex formation in a Saccharomyces cerevisiae in vitro transcription system were analysed using the anionic detergent sarkosyl. In contrast to findings from other systems, we were not able to distinguish between a fully formed pre-initiation complex and a 'rapid start' complex to which nucleotides were added. A further increase in resistance of transcription to sarkosyl was only observed 12 min after transcription initiation, by which time elongation was underway. Low concentrations of ATP, dATP or, surprisingly, the non-hydrolysable analogue ATPgammaS selectively stimulated transcription when present during assembly of transcription complexes, although the level of stimulation dropped when ATP was added progressively later. The effect of ATP did not correlate with the kinetics of template commitment, signifying that it functioned at a later stage than this, but prior to the full assembly of sarkosyl-resistant pre-initiation complexes. ATP also altered the sarkosyl resistance of initiating transcription complexes possibly by affecting a rate-limiting step leading to earlier appearance of elongated transcripts. This effect was antagonised by ATPgammaS, thus providing evidence that the stimulatory effect of ATP on pre-initiation complex formation and its effect on the lag between initiation and elongation phases are distinct. PMID- 8634338 TI - Identification of a novel mutation in exon 13 of the LDL receptor gene causing familial hypercholesterolemia in two Spanish families. AB - DNA from 30 unrelated Spanish patients with familial hypercholesterolemia (FH) was studied by single-strand conformation polymorphisms (SSCP)/heteroduplex analysis for mutation detection in exon 13 of low density lipoprotein (LDL) receptor gene. Two patients were found to have an abnormal pattern by heteroduplex analysis, and direct sequencing revealed a C to G substitution at nucleotide position 1965, that results in a Phe to Leu change in codon 634, F634L. We have developed a PCR based assay to detect this mutation in family members. We found three additional F634L mutation carriers, and all of them had high cholesterol levels. Haplotype analysis revealed that all F634L mutation carriers had the same allele determined by TaqI -, StuI +, AvaII +, NcoI -, suggesting the presence of a common ancestor. We report a novel mutation located in exon 13 of the LDL receptor gene that causes FH. We also demonstrate the importance of combining SSCP and heteroduplex analysis to improve mutation detection. PMID- 8634340 TI - Unconjugated bilirubin inhibits in vitro major histocompatibility complex unrestricted cytotoxicity of human lymphocytes. AB - Septic complications have been major problems in the management of patients with obstructive jaundice and neonatal jaundice. This study investigates effects of unconjugated bilirubin on lymphocyte-mediated cytotoxicity against human tumor target cells. In vitro exposure of human peripheral blood lymphocytes (PBL) with bilirubin IX alpha in bovine albumin solution resulted in a dose-dependent decrease of both natural killer activity and antibody dependent cellular cytotoxicity (ADCC) activity. Inhibition of both activities correlated with the amounts of intracellular bilirubin. Expression of cell surface CD16, CD56 antigen, and IL-2 receptor beta chain was unchanged in bilirubin-treated PBL as compared to bilirubin-untreated PBL. When bilirubin-treated PBL were cultured with interleukin-2 (IL-2), a dose-dependent decrease of lymphokine-activated killing activity, ADCC activity, and DNA synthesis was observed. Expression of CD56 antigen and IL-2 receptor alpha chain was unchanged in bilirubin-treated PBL following IL-2 stimulation as compared to bilirubin free control. These results suggest that bilirubin inhibits major histocompatibility complex-unrestricted cytotoxicity in both unstimulated and IL-2 stimulated lymphocytes. These observations may help explain the increased susceptibility to infection observed in hyperbilirubinemic patients. PMID- 8634339 TI - Tumor gangliosides enhance alpha2 beta1 integrin-dependent platelet activation. AB - Gangliosides, sialic acid-containing glycosphingolipids, enhance platelet adhesion to collagen and consequent platelet activation. For example, gangliosides shed by neuroblastoma tumor cells (NBTG) added to a subthreshold (non-activating) concentration (1 microgram/ml) of collagen, cause platelet aggregation (59 +/- 10%) and ATP release (2.3 +/- 0.2 nmol) equivalent to that caused by 10 micrograms/ml collagen alone. Here we report further studies to characterize this effect. Platelet aggregation and ATP release were not induced by NBTG in combination with subthreshold concentrations of adenosine diphosphate, epinephrine, thrombin or arachidonic acid, suggesting that NBTG specifically influences collagen-mediated platelet activation. Maximal platelet aggregation and ATP release required extracellular magnesium and only a short (1 min) preincubation with NBTG, suggesting a collagen receptor-mediated mechanism of this ganglioside activity. Since gangliosides interact with several integrin receptors, we determined whether NBTG influences alpha 2 beta 1, a major integrin collagen receptor on platelets. Incubation of platelets with a monoclonal antibody directed against the alpha 2 chain (5E8) blocked the increase in platelet aggregation (9 +/- 3% vs. 80 +/- 2%) and ATP release ( < 0.2 vs 2.5 +/- 0.1 nmol) induced by NBTG and 1 microgram/ml collagen. Incubation with an antibody to the non-integrin collagen receptor, CD36, or with an isotype control antibody did not abrogate the effect of NBTG. Finally, NBTG and its major component, GD2, enhanced alpha 2 beta 1-mediated platelet adhesion to immobilized collagen in an antibody 5E8-inhibitable manner. These findings implicate the alpha 2 beta 1-collagen interaction as a target of the effect of tumor-derived gangliosides. PMID- 8634341 TI - 'In vitro' amyloid fibril formation from transthyretin: the influence of ions and the amyloidogenicity of TTR variants. AB - The mechanisms of amyloid formation in Familial Amyloidotic Polyneuropathy (FAP) are unknown, as well as the factors determining the development of this pathology. To get some insights into this process, we have first tested a fluorimetric assay with thioflavine T, as a quantitative method for transthyretin (TTR) amyloid estimation, using amyloid isolated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and optimized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation from wild type TTR was compared using a kinetic assay. Under the conditions tested sulphate diminishes the amount of amyloid formed from wild type TTR and in addition appears to promote aggregation of preexisting amyloid fibrils. The relative amyloidogenicity of three TTR variants, TTR Met30, TTR Pro55 and TTR Met119 respectively, was evaluated using a pH dependent assay. It was shown that the Pro55 variant is highly susceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other TTR proteins towards precipitation into amyloid. These results are in agreement with the pathological conditions associated with these mutations. This type of assay has a wide application for testing the influence of other factors, such as therapeutical agents, on amyloid formation. PMID- 8634342 TI - Protein kinases involved in the phosphorylation of human tau protein in transfected COS-1 cells. AB - Human tau phosphorylation has been studied in transfected COS-1 cells. Treatment with okadaic acid alters the electrophoretic mobility of human tau protein transiently expressed in transfected cells, due to an increase in the level of phosphorylation. Treatment with okadaic acid also results in an increased phosphorylation of Alzheimer's disease-type phosphoepitopes. Tau phosphorylation within COS-1 cells is partially inhibited by in vivo treatment with DRB, a protein kinase inhibitor. Double treatment of transfected cells with okadaic acid and DRB reveals that phosphorylation of tau protein at the AT8 epitope is achieved by a DRB-resistant protein kinase which is different from that responsible for tau phosphorylation at the SMI-31 epitope, which appears to be sensitive to DRB. PMID- 8634343 TI - Arachidonic acid metabolism and cell proliferation in rat mammary carcinoma cells treated with indomethacin. AB - To determine the specificity of action of indomethacin as an inhibitor of cyclooxygenase in the mammary epithelium, cell proliferation and levels of PGE2 and LTB4 were quantitated in 13762 MAT rat mammary carcinoma cells treated with 10(-4) to 10(-10) M concentrations of drug. Suppression of proliferation of 13762 MAT cells by indomethacin was associated with reduced levels of both PGE2 and LTB4. The antiproliferative activity of indomethacin in rat mammary carcinoma cells may be modulated through inhibition of both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. PMID- 8634344 TI - Zidovudine and dideoxynucleosides deplete wild-type mitochondrial DNA levels and increase deleted mitochondrial DNA levels in cultured Kearns-Sayre syndrome fibroblasts. AB - Kearns-Sayre syndrome is the most commonly diagnosed mitochondrial cytopathy and produces severe neuromuscular symptoms. The most frequent cause is a mitochondrial DNA deletion that removes a 4977-base pair segment of DNA that includes several genes encoding for respiratory chain subunits. Treatment of AIDS patients with nucleoside analogs has been reported to cause mtDNA depletion and myopathies. Here, we report that azidothymidine, dideoxyguanosine, and dideoxycytidine cause a depletion of wild-type mtDNA while increasing the levels of deleted mitochondria DNA in Kearns-Sayre syndrome fibroblasts. The result of these effects is a large increase in the relative amounts of delta mtDNA in comparison to wild type mtDNA. We found that Kearns-Sayre syndrome fibroblasts are a mixed population of cells with deleted mtDNA comprising from 0 to over 20% of the total mtDNA in individual cells. Treatment of cloned cell lines with dideoxycytidine did not result in increased levels of delta mtDNA. The results suggest that nucleoside analogs may act to increase the average delta mtDNA levels in a mixed population of cells by preferentially inhibiting the proliferation of cells with little or no delta mtDNA. This raises the possibility that modulation of deleted mtDNA levels may occur by similar mechanisms in vivo, in response to the influence of exogenous agents. PMID- 8634345 TI - Co-operative, competitive and non-competitive interactions between modulators of P-glycoprotein. AB - We measured the effects of individual modulators and of pairs of modulators of the multidrug resistance pump, P-glycoprotein, on the accumulation of labelled daunomycin into multidrug-resistant P388 leukemia cells at 37 degrees C and developed a kinetic analysis which enables such data to be modelled in terms of co-operative, competitive or non-competitive interaction between pairs of modulators. The modulators verapamil, cyclosporin and trifluoperazine interacted with P-glycoprotein as single molecules, while vinblastine, mefloquine, dipyridamole, tamoxifen and quinidine displayed Hill numbers close to 2, suggesting that pairs of modulator molecules need to act together in order to bring about effective reversal of P-glycoprotein. When the modulators were presented to P-glycoprotein in pairs, we found examples of both competitive and non-competitive behaviour. We interpret these results on a model in which two modulatory sites exit on the MDR pump. To one of these, mefloquine, vinblastine and tamoxifen bind preferentially; to the other, verapamil, dipyridamole, trifluoperazine and quinidine bind (but mefloquine and tamoxifen only weakly if at all). Cyclosporin A can interact with both sites. PMID- 8634346 TI - Development and validation of chromatographic methods (HPLC and GC) for the determination of the active components (benzocaine, tyrothricin and menthol) of a pharmaceutical preparation. AB - Methods are reported for the determination of tyrothricin and benzocaine by HPLC and menthol by GC in the analysis of throat lozenges (tablets) containing all three compounds. After optimization of the variables involved in both HPLC and GC the methods have been characterized and validated according to the guidelines of the Spanish Pharmacopoeia, and applied to both the monitoring of the manufacturing process and the quality control of the final product. PMID- 8634347 TI - Solid state NMR and extraction studies on "phenyl"-bonded stationary phases used for solid phase extraction. AB - The solid phase extraction (SPE) and elution of [14C]-propranolol from aqueous buffer has been studied for a range of phenyl-bonded SPE materials. Differences were noted in the recovery of the analyte using methanol-water eluents depending upon the manufacturer and whether or not phase had been endcapped. Efficient recoveries of [14C]-propranolol were only achieved when triethylamine was added to the eluting solvent as a competing base. Solid state cross polarisation/magic angle spinning (CP/MAS) NMR spectroscopy was used to characterise the phases further, which revealed differences in endcapping between materials as well as differences in the type and extent of cross-linking. PMID- 8634348 TI - Determination of the optical purity of timolol maleate by proton nuclear magnetic resonance spectroscopy with a chiral Pr(III) shift reagent. AB - A 1H NMR spectroscopic method with chiral shift chelate is presented for the determination of the optical purity of timolol maleate. Optimum experimental conditions were established by studying the interactions of solvents (CCl4, CDCl3), substrate concentration, and the type and concentration of chiral lanthanide chelate (Pr(hfc)3, Eu(hfc)3). Larger induced shift (delta delta) and enantiomeric shift difference (delta delta delta) values, and more detailed spectral differences were obtained with Pr(hfc)3 than with Eu(hfc)3. By monitoring the spectral changes of the resonance signals for the enantiomeric C(CH3)3 protons, suitable conditions for quantitative determinations were found when 0.1 molar equivalents of Pr(hfc)3 were complexed with 0.074 M of substrate. Enantiomeric compositions were calculated from the relative intensities of the enantiomeric -C(CH3)3 proton resonances. Based on the analysis of six synthetic enantiomeric mixtures, the mean +/- SD recovery of (R)-(+)-timolol by the proposed method was 99.5 +/- 1.17% of the amount added. PMID- 8634349 TI - Analysis of pharmaceutical creams: a useful approach based on solid-phase extraction (SPE) and UV spectrophotometry. AB - Solid-phase extraction (SPE) using C-18, diol and ion-exchange sorbents followed by UV spectrophotometric (conventional and derivative mode) assay was applied to the analysis of basic, acidic and neutral drugs commercially available in creams. A representative set of drugs (promethazine, chlorhexidine, benzydamine, ketoprofen, ibuprofen, fentiazac, piroxicam, fluorouracil, crotamiton and hydrocortisone acetate) was selected, and for each drug the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedures were capable of removing interfering cream components (excipients including preservatives) allowing accurate spectrophotometric analyses to be performed. In some applications, derivative spectrophotometry was advantageous over the conventional absorption mode with respect to higher selectivity and versatility. PMID- 8634350 TI - Europium(III) ion probe spectrofluorometric determination of diclofenac sodium. AB - A new method has been devised for the determination of diclofenac sodium in bulk and in pharmaceutical preparations using Eu3+ ions as the Fluorescent probe. The technique was built around the hypersensitive property of the transitions of the fluorescent probe ion, Eu3+, at 616 nm. This is normally a forbidden transition, but the interaction with diclofenac sodium, which contains a carboxylic group, makes the transition allowed and enhances the intensity of its fluorescence emission. The Eu3+ fluorescence emission at 592 nm comes from a non hypersensitive transition and is not affected by ligation. The intensity ratio, R, defined as I592/I616, was used as a measure of the percentage of bound probe ions. Diclofenac and Eu(III) forms a (1:1) molar complex. The relative stability constant of the complex was found to be 10(5). A linear relationship between bound Eu3+ and the concentration of diclofenac sodium was found for concentrations from 10 to 200 micrograms ml-1, with a recovery percentage of 100.22 +/- 2.27. The method shows a good agreement with a spectrophotometric method. PMID- 8634351 TI - Adsorptive stripping voltammetry of nicardipine at a HMDE; determination of trace levels nicardipine in blood and urine. AB - The redox behaviour of nicardipine, a 1,4-dihydropyridine calcium antagonist, has been studied in different media on mercury, glassy carbon, gold and platinum electrodes using various voltammetric techniques. A highly sensitive adsorptive stripping voltammetric method for the determination of nicardipine based on adsorption of the drug onto mercury, followed by differential pulse voltammetric determination of the surface species, is described. All factors (pH, supporting electrolyte, accumulation potential and time, etc.) influencing adsorption as well as voltammetric response are discussed. The application of adsorptive stripping voltammetry at the hanging mercury drop electrode (HMDE) to the determination of trace levels of nicardipine in human urine and blood is illustrated, without an extraction procedure being necessary prior to the voltammetric measurement. A limit of detection of 4.8 ng per ml urine and 34 ng per ml blood is found with a mean recovery of nicardipine in urine and blood of 97%. The mean relative error does not exceed 6.5%. PMID- 8634352 TI - An on-line semi-automated solid-phase extraction procedure for high-performance liquid chromatographic determination of lonidamine in serum. AB - A semi-automated solid-phase extraction procedure on-line with gradient elution reversed-phase chromatography permits the determination of lonidamine and its metabolite in human serum. The average recovery from serum at the 2.5 micrograms ml-1 level was (92.8 +/- 3.4)%. The limit of quantitation for a 100 microliter sample size was 50 ng ml-1. The within-day (n = 5) and between-day (n = 5) relative standard deviations for lonidamine determination in serum samples spiked at the 2.5 micrograms ml-1 level were 2.7% and 4.5%, respectively. PMID- 8634353 TI - High-performance liquid chromatographic determination of bisacodyl in pharmaceutical dosage forms marketed in Australia. AB - HPLC methods have been developed for the assay of bisacodyl in various pharmaceutical forms. The extraction procedures are simple and the HPLC conditions separate bisacodyl from its degradation products. The chromatography was performed using a Merck LiChrospher RP-select B column, a mobile phase of 55% acetonitrile/45% 0.05 M KH2PO4 and detection by UV at 214 nm. PMID- 8634354 TI - Stability, compatibility and plasticizer extraction of miconazole injection added to infusion solutions and stored in PVC containers. AB - The stability of miconazole in various diluents and polyvinyl chloride (PVC) containers was determined and the release of diethylhexyl phthalate (DEHP) from PVC bags into intravenous infusions of miconazole was measured. An injection formulation (80 ml) containing a 1% solution of miconazole with 11.5% of Cremophor EL was added to 250-ml PVC infusion bags containing 5% glucose injection or 0.9% sodium chloride injection, to give an initial nominal miconazole concentration of 2.42 mg ml-1, the mean concentration commonly used in clinical practice. Samples were assayed by stability-indicating high-performance liquid chromatography (HPLC) and the clarity was determined visually. Experiments were conducted to determine whether the stability and compatibility of miconazole would be compromised, and whether DEHP would be leached from PVC bags and PVC administration sets during storage and simulated infusion. There was no substantial loss of miconazole over 2 h simulated infusion irrespective of the diluent, and over 24 h storage irrespective of temperature (2-6 degrees C and 22 26 degrees C). All the solutions initially appeared slightly hazy. Leaching of DEHP was also detected during simulated delivery using PVC bags and PVC administration sets. There was a substantial difference between the amounts of DEHP released from PVC bags and from administration sets, and also between the amounts released in solutions stored in PVC bags at 2-6 degrees C and 22-26 degrees C over 24 h. At the dilution studied, miconazole was visually and chemically stable for up to 24 h. The storage of miconazole solutions in PVC bags seems to be limited by the leaching of DEHP rather than by degradation. To minimize patient exposure to DEHP, miconazole solutions should be infused immediately after their preparation in PVC bags. PMID- 8634355 TI - New specific and sensitive HPLC-assays for ethacrynic acid and its main metabolite--the cysteine conjugate--in biological material. AB - A reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of the loop diuretic ethacrynic acid and its potentially active main metabolite, the ethacrynic acid-cysteine conjugate, in biological material. Simple and rapid sample preparation procedures were established using solid-phase extraction for the parent drug and direct injection after one washing step for the metabolite. HPLC separation was performed on a Spherisorb ODS II (3 microns) analytical column using isocratic elution with different mixtures of mobile phases (phosphoric acid-methanol acetonitrile-tetrahydrofuran or triethylamine buffer-methanol, respectively). The analytes were detected by measuring the UV absorption of the eluate at 275 nm. Stability studies revealed that considerable amounts of ethacrynic acid may be released from the cysteine conjugate unless the urine samples are pH stabilized (pH 3-4). The assay provided high sensitivity with limits of quantification of 20 ng ml-1 for ethacrynic acid in plasma and urine, and 240 ng ml-1 for the cysteine conjugate in urine. All validation parameters were within the required limits. For the presented assays, the applicability to pharmacokinetic studies and routine analyses was proved. PMID- 8634356 TI - A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma. AB - A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with 1/concentration2 weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25-50 micrograms ml-1. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from -4.8% to -9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method. PMID- 8634357 TI - Determination of amphotericin B in cerebrospinal fluid by solid-phase extraction and liquid chromatography. AB - A highly sensitive and reproducible liquid chromatography (LC) method for the determination of Amphotericin B in cerebrospinal fluid has been developed and validated. This LC-based method involves using nystatin as an internal standard and solid-phase extraction for sample preparation, followed by reversed-phase separation monitored by absorbance at 410 nm. The method has a limit of quantification of less than 1 ng ml-1 and excellent precision and accuracy, with both percentage relative standard deviation and percentage relative error less than 10%. The established linearity range was 1-10 ng ml-1 (r2 > 0.99). The extraction recovery of Amphotericin B from the cerebrospinal fluid is higher than 90% over the entire linear range. The method has been successfully employed for studying the penetration of Amphotericin B into the central nervous system in dogs and human. PMID- 8634358 TI - The secretion of propranolol enantiomers in human saliva: evidence for active transport? AB - To study the possible transport routes which may lead to the presence of a drug in saliva, the concentration-time curves of the separate enantiomers of propranolol were measured in human saliva and plasma after oral administration of 10 mg of propranolol hydrochloride. Saliva samples were taken with the Salivette device. Plasma and saliva concentrations of the enantiomers of propranolol were determined by HPLC with fluorescence detection. The transport of propranolol from plasma to the salivary gland appears to be not stereospecific and not saturable. Therefore, there is no indication that the transport of propranolol to the salivary gland is active. The concentrations of both enantiomers of propranolol in saliva, however, were higher than those of both enantiomers in venous plasma. In the past this phenomenon was interpreted as an indication of active transport, but it could be explained by the fact that salivary concentration more closely reflects the central compartment than that of peripheral venous blood. PMID- 8634359 TI - Gas chromatographic/mass spectrometric profiling of luteolin and its metabolites in rat urine and bile. PMID- 8634360 TI - A chiral liquid chromatographic method for the determination of the enantiomers of the racemic triazole antifungal drug (SCH 39304) in human plasma. PMID- 8634361 TI - Determination of trace levels of Dy3+ in Dy(HP-DO3A) by ion-pair liquid chromatography with post-column reaction. PMID- 8634363 TI - A neural model for generating and learning a rapid movement sequence. AB - In this article, a neural model for generating and learning a rapid ballistic movement sequence in two-dimensional (2D) space is presented and evaluated in the light of some considerations about handwriting generation. The model is based on a central nucleus (called a planning space) consisting of a fully connected grid of leaky integrators simulating neurons, and reading an input vector [symbol: see text] (t) which represents the external movement of the end effector. The movement sequencing results in a succession of motor strokes whose instantiation is controlled by the global activation of the planning space as defined by a competitive interaction between the neurons of the grid. Constraints such as spatial accuracy and movement time are exploited for the correct synchronization of the impulse commands. These commands are then fed into a neuromuscular synergy whose output is governed by a delta lognormal equation. Each movement sequence is memorized originally as a symbolic engram representing the sequence of the principal reference points of the 2D movement. These points, called virtual targets, correspond to the targets of each single rapid motor stroke composing the movement sequence. The task during the learning phase is to detect the engram corresponding to a new observed movement; the process is controlled by the dynamics of the neural grid. PMID- 8634362 TI - Adiabatic transformability hypothesis of human locomotion. AB - It is hypothesized that metabolic and mechanical changes in human locomotion associated with changes in speed v are constrained by two attractive strategies: Qmetab = 1 and delta Qmetab/delta v = a positive definite constant. Qmetab = delta Eks-1/ml O2s-1 where delta Eks-1 is the summed increments and decrements per unit time in the translational and rotational kinetic energies of the body's segments and ml O2s-1 is the rate at which chemical energy is dissipated. The expected constancy of delta Qmetab/delta v was derived from an extension of Ehrenfest's adiabatic hypothesis by which transformations (increases, decreases) in locomotion v can be considered as adiabatic, even though the biological conditions are nonconservative and non-rate-limited. The expected significance of Qmetab = 1 was derived from stability considerations of the symmetry per stride of stored and dissipated energy. An experimental evaluation was provided by collecting metabolic and mechanical measures on walking (10 subjects) and running (9 subjects) at progressively greater treadmill speeds but within the aerobic limit. Results revealed that walking was restricted to Qmetab < or = 1, with a nonlinear trajectory in v x Qmetab coordinates shaped by Qmetab = 1 (primarily) and the constancy of delta Qmetab/delta v. Running satisfied Qmetab > 1, with a linear trajectory in v x Qmetab coordinates conforming to delta Qmetab/delta v = a constant, with the constant predicted from invariants in the mechanical space v x delta Eks-1. Results also suggested that the metabolic costs of running might be predictable from measures made in the v x delta Eks-1 space. PMID- 8634364 TI - Two-neuro networks. II. Leaky integrator pacemaker models. AB - The behavior of two pacemaker neurons simulated by leaky integrators and connected reciprocally by synapses was studied. In every case the firing of both neurons phase-locks. The resulting limit cycle may or may not show simultaneous firing of both neurons. When both synapses are excitatory, phase-locking with simultaneous neuronal firing is always present. When one synapse is excitatory and the other inhibitory, phase-locking is also present always, while the neurons may or may not fire simultaneously. For a restricted set of parameters, bistability appears; the initial conditions determine whether or not the limit cycle presents simultaneous firing. When both synapses are inhibitory, the system phase-locks without simultaneous firing for almost every set of parameters. PMID- 8634366 TI - Dynamics of the sit-to-stand movement. AB - The strategies of the sit-to-stand movement are investigated by describing the movement in terms of the topology of an associated phase diagram. Kinematic constraints are applied to describe movement sequences, thus reducing the dimension of the phase space. This dimensional reduction allows us to apply theorems of topological dynamics for two-dimensional systems to arrive at a classification of six possible movement strategies, distinguished by the topology of their corresponding phase portrait. Since movement is treated in terms of topological structure rather than specific trajectories, individual variations are automatically included, and the approach is by nature model independent. Pathological movement is investigated, and this method clarifies how subtle abnormalities in movement lead to difficulties in achieving a stable stance upon rising from a seated position. PMID- 8634365 TI - Transient synaptic redundancy in the developing cerebellum and isostatic random stacking of hard spheres. AB - We propose an automaton for the simulation of the distribution of the number of climbing fibers (CF) making synapses on each Purkinje cell (PC) at the maximum of the synaptic redundancy that exists transiently in the newborn cerebellum. This automaton is based on the hypothesis that the synaptic maximum is limited by topological constraints and can be described by an isostatic random stacking of hard spheres. There is convincing agreement between the simulated distribution of the number of CF axons per Purkinje cell and the distribution experimentally obtained by electrophysiological techniques. PMID- 8634367 TI - Context codes and the effect of noisy learning on a simplified hippocampal CA3 model. AB - This paper investigates how noise affects a minimal computational model of the hippocampus and, in particular, region CA3. The architecture and physiology employed are consistent with the known anatomy and physiology of this region. Here, we use computer simulations to demonstrate and quantify the ability of this model to create context codes in sequential learning problems. These context codes are mediated by local context neurons which are analogous to hippocampal place-coding cells. These local context neurons endow the network with many of its problem-solving abilities. Our results show that the network encodes context on its own and then uses context to solve sequence prediction under ambiguous conditions. Noise during learning affects performance, and it also affects the development of context codes. The relationship between noise and performance in a sequence prediction is simple and corresponds to a disruption of local context neuron firing. As noise exceeds the signal, sequence completion and local context neuron firing are both lost. For the parameters investigated, extra learning trials and slower learning rates do not overcome either of the effects of noise. The results are consistent with the important role played, in this hippocampal model, by local context neurons in sequence prediction and for disambiguation across time. PMID- 8634369 TI - A Markovian formalization of heart rate dynamics evinces a quantum-like hypothesis. AB - Most investigations into heart rate dynamics have emphasized continuous functions, whereas the heart beat itself is a discrete event. We present experimental evidence that by considering this quality, the dynamics may be appreciated as a result of singular dynamics arising out of non-Lipschitz formalisms. Markov process analysis demonstrates that heart beats may then be considered in terms of quantum-like constraints. PMID- 8634368 TI - Model predictions of myoelectrical activity of the small bowel. AB - A mathematical model for the periodic electrical activity of a functional unit of the small intestine is developed. Based on real morphological and electrophysiological data, the model assumes that: the functional unit is an electromyogenic syncytium; the kinetics of L, T-type Ca2+, mixed Ca(2+)-dependent K+, potential sensitive K+ and Cl- channels determines electrical activity of the functional unit; the basic neural circuit, represented by a single cholinergic neurone, provides an excitatory input to the functional unit via receptor-linked L-type Ca2+ channels. Numerical simulation of the model has shown that it is capable of displaying the slow waves and that slight modifications of some of the parameters result in different electrical responses. The effects of the variations of the main parameters have been analyzed for their ability to reproduce various electrical patterns. The results are in good qualitative and quantitative agreement with results of experiments conducted on the small intestine. PMID- 8634370 TI - Is correspondence search in human stereo vision a coarse-to-fine process? AB - One possible strategy for the solution of the correspondence problem of stereo matching is the coarse-to-fine mechanism: The matching process starts with a lowpass-filtered version of the stereogram where only a few, high-contrast image features can be extracted and the probability of false matches is therefore low. In subsequent stages, information from higher spatial frequencies is used gradually to improve the correspondence data obtained on the coarser scales. Coarse-to-fine strategies predict that information from coarse scale is used to disambiguate matching information on finer scales. We have tested this prediction by means of the wallpaper illusion using periodic intensity-profiles with different matching ambiguities on different spatial scale. Our psychophysical experiments show (i) that unambiguous information at coarse scale is not always used to disambiguate finer scale information, (ii) that unambiguous fine-scale information can be used to disambiguate coarse-scale information and (iii) that low spatial frequency is more efficient for disambiguation than higher frequency. We conclude that the human stereo vision system does not always proceed from coarse to fine. As an alternative scheme for scale-space integration, we discuss more symmetric schemes such as maximum likelihood combinations of data from different channels. PMID- 8634371 TI - Transient inhibition of protein synthesis induces expression of proto-oncogenes and stimulates resting cells to enter the cell cycle. AB - There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons. PMID- 8634372 TI - The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines. AB - A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had kappa light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. The nuclear protein(s) detected by Th-10a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. The results also indicated that the 95 kDa nuclear protein was phosphorylated in vivo. PMID- 8634373 TI - The GC factor regulates the expression of the insulin-like growth factor-I receptor. AB - We have transfected a plasmid expressing the transcriptional regulator GC Factor (GCF) into cell lines and have found that the GCF: 1 causes a decrease in the levels of insulin-like growth factor I receptor (IGF-IR) mRNA; 2 causes a decrease in the number of IGF-IRs; and 3 represses the activity of the IGF-IR promoter. In addition, we show that the regulation of IGF-IR expression by GCF plays a physiological role in the control of cellular proliferation in vitro. PMID- 8634374 TI - Purine suppression of proliferation of Sertoli-like TM4 cells in culture. AB - The effect of adenosine and related compounds on the proliferation of cultured TM4 cells, a Sertoli-like cell line, has been examined. Adenosine, as well as A1 and A2 adenosine receptor agonists (cyclohexyladenosine and N6-[2-(3,5 dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine) inhibited cell proliferation. These effects were prevented by 8-cyclopentyl theophylline, 1,3-dimethyl propargylxanthine and caffeine, antagonists at the A1, A2 and both receptors, respectively. The xanthines had no effect by themselves and, consistent with this, the bathing medium was found not to contain detectable levels of adenosine. It is concluded that TM4 cell proliferation can be regulated by receptors for adenosine. PMID- 8634375 TI - Neuropsychologic change after cardiac surgery: a critical review. AB - Studies that have examined neuropsychologic change after cardiac surgery address three main issues: (1) the incidence of cognitive change; (2) the identification of factors that put patients at higher risk; and (3) the evaluation of interventions to prevent these complications. This review attempts to bring together concerns associated with various study designs and to integrate the conclusions from these studies. Thirty-five studies have been examined in this review. Some of the difficulties encountered when quantifying the degree of cognitive change are related to study design, patient sampling, and deficit definition. Additionally, changing patient populations have influenced results reported from different health care settings. Increasing age and longer cardiopulmonary bypass times have been correlated with cognitive decline in a number of studies. Filtration devices and blood gas management techniques have decreased but not eliminated the number of patients who have cognitive decline. Cognitive change exists following cardiac procedures. Identification of a subgroup of patients at high risk for cognitive change has been difficult, possibly due to issues of study design. Design of future studies, which may include intraoperative or pharmacologic interventions, is dependent on identification of this high-risk group. PMID- 8634376 TI - Cerebral emboli and cognitive outcome after cardiac surgery. AB - There have been major advancements in cardiac surgery over the past two decades, with a concomitant decrease in mortality and major morbidity. However, several recent studies have demonstrated that cardiac surgery poses significant risk for negative neurologic and neuropsychologic outcome. Although very few patients die as a result of cardiac surgery, more than two thirds of the patients demonstrate evidence of neuropsychologic dysfunction postoperatively. The mechanisms contributing to post-cardiopulmonary bypass neuropsychologic deficits are uncertain. However, two major interrelated etiologic factors, hypoperfusion and emboli, are suggested as probable culprits. It is important to define the relationship between these two putative mechanisms and postoperative neuropsychologic outcome in order to either prevent the problem or treat the effects of emboli or hypoperfusion. For example, if embolism is the cause of the deficits, increasing cerebral perfusion would deliver more emboli and increase the amount of severity of injury. Conversely, if hypoperfusion is the cause of the injury, then decreasing brain blood flow would increase the likelihood of injury. If both are important, their relative significance must be established, then one prevented and the effects of the other treated. This report discusses the methodology for detecting cerebral emboli during cardiac surgery. The incidence and severity of neuropsychologic deficits after cardiac surgery are discussed, as well as emboli in relation to composition and time of occurrence and their effect on neuropsychologic outcome. PMID- 8634378 TI - Magnetic resonance studies of the effects of cardiovascular surgery on brain metabolism and function. AB - Neurologic and neuropsychologic impairment are important sequelae of cardiac surgery in general and of coronary artery bypass graft surgery in particular. Although estimates of incidence vary, the numbers affected are considerable. Despite the ubiquity of such effects and the general consensus that impairments originate from ischemic injury secondary to microemboli produced during surgery, the nature of the underlying brain injuries remains poorly understood. Precise, and preferably quantitative, definition of the localization and nature of the underlying injuries is a precondition for the rigorous evaluation of the efficacy of prophylactic measures. The ability of magnetic resonance imaging (MRI) to detect surgically related lesions and the course of brain swelling is described, as are potential improvements in imaging sensitivity. Results of an experimental program studying chemical sequelae of surgery in a pig model are presented. MR spectroscopy can provide noninvasive information on the biochemical changes in brain and brain metabolism that permit empirical evaluation of various neuroprotective interventions. Functional MRI provides a means of studying the neuropsychologic mechanisms most often affected by cardiac surgery. Experimental data are presented that demonstrate that two such mechanisms, selective attention and working memory, can be imaged successfully. Perfusion mapping, combined with functional imaging, allows for the quantitative study of flow and functional activation. Applied to structures such as the cingulate, these techniques permit comparison of surgical sequelae with processes such as normal aging. MRI technology offers the possibility of improved anatomic, chemical, and functional definition of the effects of cardiac surgery on the brain. PMID- 8634377 TI - Markers of cerebral ischemia after cardiac surgery. AB - The objective of this review is to provide an overview of the use of biochemical markers for the detection of Central Nervous System (CNS) complications after cardiac surgery and extracorporeal circulation (ECC). A computerized literature search in MEDLINE from 1966 onward was the basis for the references. The literature covering the following biochemical markers is reviewed: adenylkinase, creatine phosphokinase isoenzyme BB (CK-BB), lactate, neuron-specific enolase (NSE), S-100 protein, myelin basic protein, lactate dehydrogenase, aspartate aminotransferase, glutathione, vasointestinal neuropeptide, and 7B2-specific neuropeptide. For clinical purposes, it is necessary to have a biochemical marker that can be measured in blood. Lactate, although a primary marker of anaerobic metabolism, and CK-BB values, calculated from the arterio-internal jugular venous difference, appear to correlate with periods of ischemia during ECC. S-100 protein levels have been shown to correlate with duration of ECC, and when combined with NSE values, could be used to identify patients with CNS dysfunction after cardiac surgery. The use of NSE may be limited by its presence in erythrocytes and platelets because the high levels that can result from hemolysis can render it less specific. Although recently introduced, S-100 protein may have the potential to be a valuable marker for CNS dysfunction after ECC. PMID- 8634380 TI - Neural and endothelial control of the peripheral circulation--implications for anesthesia: Part I. Neural control of the peripheral vasculature. PMID- 8634379 TI - Mechanisms of cerebral ischemia: intracellular cascades and therapeutic interventions. AB - In order to gain insight into the pathophysiology of cerebral ischemia, the focus is on the discrete compartmentalization of neurons and the exquisite homeostasis of the neurochemical, ionic, and molecular environment within these compartments. This review looks at excitotoxic mechanisms of cerebral ischemia spatially by separating presynaptic and postsynaptic events as well as temporally by separating early and late events. Drugs that target these events in the excitotoxic cascade are presented and discussed as potential therapeutic interventions for cerebral ischemia. Despite a better understanding of the mechanisms of cerebral ischemia through a myriad of animal model studies with various "neuroprotective" compounds, the challenge remains to apply this knowledge to the development of compounds that demonstrate neuroprotective efficacy in terms of quality-of-life outcomes in humans. PMID- 8634381 TI - The role of neuromonitoring in cardiovascular surgery. AB - This review describes the techniques currently used for quantitative neurophysiologic measurement during cardiac surgery and their potential impact on clinical outcome. Electroencephalography (EEG) characterizes cerebrocortical neuronal electrical activity and was part of some of the earliest cardiopulmonary bypass procedures, yet today it is not widespread use. Each of the common misunderstandings regarding a supposed limitation of this technology is explained. Its major genuine shortcoming, a lack of selectivity, may now be overcome with the combined use of additional monitoring modalities. The influence of intracranial hemodynamics on observed EEG changes may be determined continuously and noninvasively with transcranial Doppler (TCD) ultrasound. TCD provides an indication of sudden change in either blood flow or vascular resistance as well as the detection of emboli. In addition, the metabolic status of cortical neurons can be monitored by regional cerebral venous oxygen saturation (rCVOS) using noninvasive transcranial near-infrared spectroscopy. The % rCVOS tends to remain remarkably stable over a wide range of temperatures, perfusion pressures, and anesthetic states. Marked change in either direction signifies a serious imbalance between oxygen delivery and consumption. Measurement of rCVOS does not require blood flow, pulsatile or otherwise, so that it offers the only means of monitoring during circulatory arrest. By characterizing the dynamic interplay among cerebral hemodynamics, metabolism, and electrogenesis, these technologies permit the rapid detection and correction of potentially hazardous conditions. PMID- 8634382 TI - Neural and endothelial control of the peripheral circulation--implications for anesthesia: Part II, Endothelium-mediated effects in the normal and diseased circulation. PMID- 8634383 TI - Aortic atheromatosis and risks of cerebral embolization. AB - With the advent of transesophageal echocardiography, aortic atheromatosis has emerged as an important source of cerebral embolization. Mobile atheromatous plaque in the ascending aorta and aortic arch has been shown to constitute a strong and independent risk factor in patients with stroke. In patients undergoing coronary bypass surgery, it is the single most important contributing factor to perioperative neurologic morbidity. Emboli originating in the heart, aorta, and proximal cerebral vasculature have been observed intraoperatively in patients undergoing coronary bypass surgery, especially when aortic clamps are released. The constitution of these emboli is unclear, although an indeterminate fraction undoubtedly represents dislodged atheromatous material. The impact of such embolization in terms of neurologic outcome is currently under investigation. Prevention of embolization from mobile aortic atheroma in patients undergoing cardiac surgery may require modification of surgical technique. Secondary prevention in patients with a history of embolization can only be determined once the natural history of such lesions is established. PMID- 8634384 TI - Cerebrovascular assessment of the high-risk patient: the role of transcranial Doppler ultrasound. AB - With increased attention to the causes and effects of neurologic injury related to cardiopulmonary bypass anesthesia and surgery, multiple modality examination and monitoring of cerebral function and perfusion in the perioperative period may prove to be advantageous. Transcranial Doppler examination and monitoring is inexpensive, noninvasive, safe, provides unique information about the functional status of the intracranial circulation, and complements the duplex Doppler study of the extracranial carotid vessels of the neck for preoperative evaluation of the surgical patient. The transcranial Doppler examination permits quantitation of blood flow velocity of the intracranial vessels, evaluation of autoregulatory capacity and vasomotor reserve, determination of symmetry of flow velocity in the circle of Willis, assessment of collateral circulatory capacity, examination of vessels not accessible to the duplex Doppler and serves as a baseline for intraoperative monitoring and the postoperative examination. Noninvasive, unilateral or bilateral, continuous monitoring of brain blood flow velocity intraoperatively or postoperatively with trending, storage, and correlation with other physiologic variables provides evidence of cerebral perfusion, occurrence and rate of cerebral embolism, and continuous monitoring of therapeutic interventions. A review of the incidence of stroke and neuropsychologic deficit after bypass surgery is focused on parameters amenable to diagnosis using transcranial Doppler. Patient-specific risk factors for neurologic injury derived from previous studies are discussed as well as risk factors that are related to anesthetic and surgical management and equipment. A description of Doppler technology and the correlation of transcranial Doppler findings with angiography and radionucleotide scans establishes the accuracy of the Doppler examination. The preoperative examination, provocative tests of vasomotor reserve, the evaluation of cerebral collateral circulation, and examples of Doppler applications are discussed. PMID- 8634385 TI - Adverse neurologic events: risks of intracardiac versus extracardiac surgery. AB - Intracardiac operations such as valve replacements have typically carried a higher risk (4.2% to 13%) of overt central nervous system outcome, compared with coronary artery bypass grafting (CABG) procedures (0.6% to 5.2%). This is likely owing to the increased risk of macroembolization of air or particulate matter from the surgical field during intracardiac surgery. The periods of highest risk for emboli are during aortic cannulation and especially during release of aortic clamps and weaning from bypass. The number of embolic events measured with transcranial Doppler is significantly higher in patients undergoing valve surgery compared with coronary surgery, particularly during cardiac ejection and immediately after bypass. However, there is current evidence that neurologic risk is increasing in patients undergoing CABG owing to the tendency to operate on older patients with more severe aortic atherosclerosis and cerebrovascular disease. Patients having an intracardiac procedure combined with a CABG procedure may be at particularly high risk for adverse neurologic outcome. For all cardiac surgical patients, there is some cause for optimism in that risk may be minimized by improved assessment (e.g., intraoperative transesophageal or epiaortic echocardiographic scanning of the ascending aorta to identify patients at risk) and monitoring (e.g., detection of embolic phenomena, using transesophageal echocardiography or transcranial Doppler technology). Furthermore, in the future, development and testing of more ideal cerebroprotective drugs may allow amelioration of neurologic injury, either by pretreating all patients at risk, or possibly even by delaying treatment until after the suspected occurrence of an insult. PMID- 8634386 TI - Techniques for avoiding neurologic injury during adult cardiac surgery. AB - The mechanisms and pathophysiology of perioperative neurologic injury are reviewed. The principle mechanisms of the ischemic injury are gaseous and particulate emboli and hypoperfusion caused by cerebrovascular occlusive disease. The contribution of the cardiopulmonary bypass circuit to the development of ischemic injury is discussed. The important role of the surgeon in preventing particulate and gaseous microemboli and macroemboli is described and techniques for avoiding hypoperfusion in the presence of occlusive cerebrovascular disease are presented. Patients at greatest risk of perioperative neurologic injury are elderly patients with atherosclerotic aorta, cerebrovascular disease, and previous neurologic abnormalities. The pump oxygenator may be the cause of neurologic injury by generation of emboli, hypoperfusion, and perfusion accidents. The principles of perfusion safety are presented. Massive air embolization may occur from the heart-lung machine or from the heart. Methods of preventing massive air embolus including techniques of bypass and air removal from the heart are discussed. The management of massive air embolus is thoroughly described. The detection of cerebrovascular disease is discussed and the management of the patient with cerebrovascular disease described. Atherosclerosis of the ascending aorta is a major risk factor for perioperative neurologic injury. Methods of detection and management are thoroughly described. Prevention of embolization from intracardiac debris and clot is described. PMID- 8634387 TI - Normothermic versus hypothermic cardiopulmonary bypass: central nervous system outcomes. AB - The recent advent of "warm heart" surgery has resulted in reexamination of the neuroprotective effects of hypothermia in the setting of cardiopulmonary bypass (CPB). Hypothermia has been shown to confer significant protection in the setting of transient, but not permanent, ischemia. The mechanism of this neuroprotection is unclear at this time. Reduction in cerebral metabolic rate is believed to be less important compared with the effect of hypothermia on the release of excitatory neurotransmitters, catecholamines, or other mediators of cellular injury. It is for this reason that mild hypothermia (33-35 degrees C) is believed to confer significant neuroprotection. Two large randomized trials of warm versus cold heart surgery have been reported. Neither study found a difference in terms of neuropsychologic dysfunction. However, one study identified a threefold increase in strokes in the "warm" patients. The reasons for this difference are not clear; however, there were various differences in technique and patient population that may have been important. There are other reports of large series of patients undergoing normothermic bypass, with no increase in stroke rate over what is reported elsewhere in the literature. To date, the evidence would suggest that neuropsychologic function is not affected by CPB temperature, suggesting that the transient ischemia is not an important mechanism in this injury. Stroke after CPB is usually the result of permanent ischemia, and hypothermia's effect in this setting is minimal. It would seem unlikely that hypothermia offers anything more than modest benefits in the clinical situation where there is no circulatory arrest. PMID- 8634388 TI - Determinants of cerebral perfusion during cardiopulmonary bypass. AB - The risk of postoperative neurologic dysfunction in patients undergoing cardiac surgery remains high despite continued improvements in myocardial protective strategies. Part of this neurologic morbidity can be attributed to patients' increased age and underlying pathology, but other factors adversely affecting cerebral blood flow and cerebral metabolism during cardiopulmonary bypass may also contribute. Particulate microembolization during cardiopulmonary bypass appears to be a major cause of postoperative neurologic dysfunction and the pH stat method of carbon dioxide management during hypothermia may potentiate neurologic damage by allowing a greater embolic load to be delivered to the brain. Echocardiography and transcranial Doppler methods may contribute to reducing the incidence of cerebral embolization by recognizing the timing and number of microemboli. Although hypothermia confers cerebral protection, rewarming may unmask and perhaps potentiate any ischemic damage that occurred with embolization during hypothermia. Both the degree and speed of rewarming may be important factors contributing to the extent of ischemic damage and ultimately neurologic function. In addition, many other factors related to cardiopulmonary bypass can alter cerebral perfusion and metabolism, such as nonpulsatile flow, hemodilution, pressure autoregulation, anesthetic and cerebroprotective drugs, and the neuroimmune response to bypass. In this review, the major factors affecting cerebral blood flow during cardiopulmonary bypass are discussed and their relative importance evaluated with regard to postoperative neurologic function. PMID- 8634389 TI - Hypothermia, circulatory arrest, and the pediatric brain. AB - A review has been conducted of ongoing clinical and laboratory studies of hypothermic circulatory arrest (HCA) and low-flow cardiopulmonary bypass (LFB) at a children's hospital in Boston. A prospective randomized clinical trial of HCA versus LFB has shown a higher incidence of perioperative seizures in patients randomized to HCA. At 1 year of age, neurologic and developmental studies have shown an association between seizures and worse outcome. Longer duration of HCA is associated with a worse score on the Bayley scale assessment of gross and fine motor function in particular, as well as a higher probability of neurologic abnormality. A retrospective review of development after HCA for Senning procedure has shown a correlation between more alkaline pH (alpha-stat strategy) during cooling before HCA and lower developmental score relative to a more acidotic strategy (pH stat). The institutional change to alpha-stat was accompanied by several cases of choreoathetosis after HCA. Currently, patients are being randomized between alpha-stat and pH-stat. Laboratory studies have used a piglet model with assessment of cerebral blood flow and metabolism as well as high-energy phosphates and cerebral pH determined by magnetic resonance spectroscopy. High-energy phosphates are maintained by a flow rate of 50 mL/kg/min but are undetectable after approximately 35 minutes of HCS. A pH-stat is associated with more rapid recovery of high-energy phosphates after HCA than alpha-stat. Recent studies have examined the role of nitric oxide in the causation of brain injury after HCA as well as the potential utility of cerebroplegia in increasing the safe duration of circulatory arrest. PMID- 8634390 TI - Cerebral perfusion and hypothermic circulatory arrest. AB - Since DeBakey's replacement of an aortic arch aneurysm using cardiopulmonary bypass and individual perfusion of the brachiocephalic and carotid arteries, selective cerebral perfusion has been used as an effective method of cerebral protection. Although interest in this technique waned with the evolution of hypothermic circulatory arrest, complications arising from long and challenging aortic procedures have led to a renewed interest in perfusion of the cerebral circulation. During aortic arch surgery, antegrade and retrograde cerebral perfusion techniques have been used in an effort to prolong the "safe" duration during which conventional cardiopulmonary bypass flow to the brain is interrupted. Although the degree to which retrograde cerebral flow is able to perfuse cerebral tissue remains controversial, its use may afford protection through other mechanisms as well. This paper will review techniques, benefits, and limitations of antegrade and retrograde cerebral perfusion and their role in conjunction with hypothermic systemic circulatory arrest. PMID- 8634391 TI - Perfluorocarbon emulsions and cardiopulmonary bypass: a technique for the future. AB - Artificial blood has been sought for a considerable period of time and two major lines of research have led to FDA testing of some possible compounds. The two major types of compounds are polymerized hemoglobin moieties and perfluorocarbon emulsions (PFC). Polymerized hemoglobin preparations have the ability to carry oxygen and release it in a manner similar to the oxyhemoglobin dissociation curve of whole blood. PFCs carry oxygen, nitrogen and carbon dioxide, as well as all other non-polar gases, by enhanced chemical solubility. Therefore, all dissolved gases are available for metabolic utilization and no sinusoidal release curve of oxygen is encountered. Early PFC emulsions had problems with toxicity of the emulsifier and were difficult to get into their emulsion for infusion. Furthermore they were very dilute in the active ingredient for gas transport. Today there are second generation PFCs becoming available that have a 40% concentration of the PFC and therefore the potential for gas transport is greatly increased. The PFC emulsions have a very small size, 0.1 microns, so the surface for gas exchange is massively increased as well as the potential increased for perfusion into areas of potentially sludged erythrocytes. Work with the PFCs has shown them now to be able to carry adequate oxygen to work as blood substitutes. They have shown protection from air embolism in a number of animal and end-organ models. What makes the PFCs unique is their ability to carry/absorb nitrogen and therefore protect from gas embolization. There are data in animal models showing significant cerebral protection in cardiopulmonary bypass models. The new PFCs should sometime in the not-too-distant future be tested in human bypass with assessments of neuropsychiatric dysfunction and stroke. PMID- 8634392 TI - Neuropsychologic dysfunction after cardiac surgery: what is the problem? AB - Twenty years ago Aberg published his seminal studies on the neuropsychologic consequences of cardiopulmonary bypass (CPB). Twenty years later, what is the state of current research on the problem of post-CPB neurologic injury, and what different management techniques have been employed to influence this outcome? This article reviews the definition and assessment of postoperative neuropsychologic dysfunction; epidemiologic data and associated risk factors assessing post-CPB neuropsychologic dysfunction are critically appraised. PMID- 8634393 TI - Neurologic assessment and cardiac surgery. AB - Cerebral injury remains a significant complication of cardiac surgery. This complication is evaluated by clinical means that include a neurologic examination. In this report, the most important components of this type of evaluation are described. The neurologic complications of cardiac surgery can be determined by comparing structured preoperative and postoperative clinical evaluations. The neurologic examination must include a mental state examination, examination of cranial nerves, motor, sensory, and cerebellar systems, examination of gait and station, and deep tendon and primitive reflexes. The purpose of this report is to discuss the relevance of the neurologic examination in the assessment of cerebral injury after cardiac surgery, review the components of a structured neurologic examination, and explore the role of "quantitative" stroke scales as a research tool. PMID- 8634394 TI - [Quo vadis radiology? We need an academy]. PMID- 8634396 TI - [High resolution computed tomography of the lung in neutropenic patients with fever]. AB - PURPOSE: To determine the sensitivity and clinical impact of high-resolution CT (HRCT) of the lung in febrile neutropenic patients under antibiotic therapy. MATERIAL AND METHODS: Chest X-ray and HRCT were prospectively performed to exclude pneumonia in 34 patients (53 examinations) suffering from febrile neutropenia following antitumor therapy. Diagnosis was confirmed by bronchoalveolar lavage or sputum cultures. RESULTS: Chest X-ray showed pneumonia in 13/53 examinations, in 12/13 a micro-organism was found. HRCT demonstrated pneumonia in 39/53, in 31/39 a micro-organism was found. All cases with positive cultures showed suspicious HRCT findings. Changes in antibiotic treatment resulted in findings suspicious for pneumonia and evidence of a new or a just treated micro-organism (chest X-ray 8/53, HRCT 31/53); the search for the source of fever was escalated in cases without evidence of micro-organisms and without suspicion of pneumonia findings 14/53. CONCLUSION: HRCT of the lung exhibits a higher sensitivity than conventional radiographs. Findings resulted in relevant therapeutic consequences. Hence, HRCT is indicated in neutropenic patients with fever and inconspicuous chest X-ray. PMID- 8634395 TI - [Gastrointestinal imaging with hydrosonography and hydro-CT]. AB - AIM: To determine the accuracy of hydrosonography (HUS) und hydro-CT (HCT) for staging colon carcinoma. MATERIAL AND METHODS: 74 patients in whom colorectal carcinoma was suspected were examined. At HUS the colonic wall ist distended by a methylcellulose/water suspension and the carcinoma is enlarged to perform staging of the tumour. HCT is a spiral-CT optimised for parenchymal and vessel contrast. Before the scan is started, up to two litres of fluid are given rectally and spasmolytics are administered to reduce peristalsis. Colorectal carcinomas were classified according to the TNM system and histopathologic correlation was achieved. RESULTS: Out of 43 (HUS) and 39 (HCT) colonic lesions 33 (77%) and 36 (92%), respectively, were diagnosed. T-stage accuracy was 88% (HUS) and 66% (HCT), N-stage accuracy 33% and 46% and M-stage accuracy 88% and 85%, respectively. CONCLUSION: The T-stage of sonographically visible tumours of the colon is determined precisely by HUS. In contrast to predicting lymph node involvement distant metastases are reliably detected by both methods. If performed together, HUS and HCT achieve high diagnostic accuracy for staging carcinoma of the colon. PMID- 8634397 TI - [Diagnosis of floating venous thrombi by spiral CT phlebography]. AB - PURPOSE: Evaluation by means of spiral CT as to whether or not a thrombus in a proximal deep vein of the leg is floating or adherent to the vessel wall. METHODS: Local application of contrast agent into an ipsilateral dorsal foot vein and spiral CT were used to examine 16 consecutive cases with deep venous thrombosis proven at conventional venography; in addition, colour Doppler flow imaging was performed. RESULTS: At conventional venography, 8/16 thrombi appeared to be floating and the remaining 8/16 were adherent to the vessel wall. Spiral-CT showed 15/16 thrombi to be adherent to the vessel wall; the floating thrombus correlated with findings in conventional venography. At colour Doppler flow imaging 3/16 thrombi were considered floating, one of them was discordant to conventional venography. The comparison of conventional venography to spiral-CT demonstrates complete agreement for adherence to vessel wall seen in conventional venography (p = 1,0) and significant discordance in cases with free-floating appearance in conventional venography (p = 0,0002). CONCLUSION: Adherence of thrombi to the wall of the vessel at conventional venography is in agreement with computed tomography. Conventional venography probably overestimates the prevalence of free floating thrombi. PMID- 8634398 TI - [Calculated organ doses and effective dosage for computerized tomography examination of the thorax and abdomen: are these doses realistic?]. AB - PURPOSE: To analyse how far dose calculations for the CT examination of the thorax and abdomen can lead to faulty estimations of organ doses and effective dose due to differences in the topographic anatomy between mathematical phantom and man. METHODS: For the CT examination of the thorax, upper abdomen, pancreas, pelvis and the entire abdomen, organ doses were calculated with conversion factors, first with regard to the topographic relation within the phantom, then with additional regard to the real anatomy of the man. RESULTS: In the phantom, the abdomen lies outside the scan volume in case of CT-examination of the thorax and the whole intestine outside the scan volume in case of CT-examination of the upper abdomen and the pancreas, whereas the entire intestine is directly exposed in case of CT-examination of the pelvis. Dependent on whether dose calculations take real anatomy into account, doses of special organs can differ by a factor greater than 15. The calculated effective doses differ by a factor less than 1.5. CONCLUSIONS: Calculations of organ doses for the CT examination of the thorax and abdomen can lead to considerable errors due to different topographic relations between phantom and man. In contrast the calculated effective dose is realistic. Hence, dose calculations with the help of mathematical phantom is an efficient method to estimate the total radiogenic risk. PMID- 8634399 TI - [A new application of MR tomography of the lung using ultra-short turbo spin echo sequences]. AB - PURPOSE: Development of an improved MR sequence for examining the lung. METHOD: T2 weighted ultra-short turbo spin echo sequences were used in five individuals with variations in echo times, delayed triggering and echo intervals. To reduce movement artifacts all examinations were carried out with ECG and respiratory triggering. The sequences giving optimal image quality were then employed in 19 patients having various pulmonary abnormalities. Image resolutions, artifacts, image contrasts and diagnostic value were then judged by two observers and compared with CT. RESULTS: In the first study, a diastole-triggered UTSE sequence with the shortest echo proved optimal (TE = 90 ms, TR = 2-4 s, echo = 9 ms, turbo factor = 19). In the patient series studied, MRT was inferior to CT with regard to resolution and number of artifacts, but better in respect of contrast and diagnostic value. CONCLUSION: Using UTSE of the lung, MRT can produce images of good quality. Compared with CT, contrast is better with MRT, offering diagnostic advantages for MRT. PMID- 8634400 TI - [Preoperative differential diagnosis of cystic adnexal tumors: double-contrast MRT]. AB - PURPOSE: Preoperative assessment of adnexal lesions as benign or malignant by MRI with gastrointestinal and intravenous contrast. METHODS: 46 patients with benign (n = 42) and malignant (n = 4) cystic adnexal tumours underwent MRI of the pelvis. Transaxial and coronal images were acquired using conventional T1- and T2 weighted SE-sequences after oral administration of superparamagnetic iron oxide particles (Ferristene). Additional T1-weighted SE-images were obtained immediately following gadodiamide (Gd DTPA-BMA) injection. RESULTS: MRI correctly classified the four malignant lesions, whereas nine histologically benign lesions were misdiagnosed as malignant. Intravenous contrast yielded a superior delineation of intratumor architecture. CONCLUSION: Due to exclusion of solid structures. MRI with oral and i.v. contrast enables to dismiss suspected malignity in cystic adnexal lesions. Because of the non-specificity of the macroscopic criteria of dignity, the MR diagnosis "malignity" is of limited value. PMID- 8634402 TI - [Magnetic resonance tomography of vasogenic brain edema in animal experiments]. AB - PURPOSE: To systematically evaluate modern magnetic resonance imaging (MRI) techniques of studying brain oedema, an animal model of vasogenic oedema is needed. MATERIAL AND METHODS: We developed such a model by stereotactically cannulating the caudate nucleus in 11 Wistar-rats and infusing a solution of albumin. The way the oedema developed was imaged by MRI and then correlated histologically (three animals) and macroscopically after infusion of Evans' blue (two animals). RESULTS: The extension of the infusion oedema was the same in both MRI and histology as well as at direct inspection of the specimen after infusion of Evans blue. With continuous acquisition of T1-weighted images after infusion of a 1% solution of Gd-DTPA we were able to demonstrate the dynamics of oedema evolution and bulk flow. CONCLUSION: We believe that this infusion oedema is a suitable model for systematically investigating both signal behaviour and pathogenesis of vasogenic brain oedema by MRI. PMID- 8634401 TI - [Importance of duplex velocity histograms in the diagnosis of liver cirrhosis]. AB - PURPOSE: A prospective study was carried out to determine the diagnostic value of velocity histograms of hepatic veins in patients with liver cirrhosis and healthy subjects. METHODS: The hepatic veins of ten healthy subjects and ten patients with liver cirrhosis were examined by the Doppler method. The velocity histogram measures the velocity variation at a defined area in the duplex tracing. Within this tracing, the peak, mean, mode and velocity range were measured. RESULTS: A velocity range of 0.02 +/- 0.015 m/s was examined in healthy subjects, and a range of 0.06 +/- 0.013 was examined in liver cirrhosis patients (p < 0.001). The other measurement options showed no significant changes between the healthy subjects and the liver cirrhosis patients (p < 0.05). CONCLUSIONS: Until now, differentiation of the hepatic vein tracings was possible only on a qualitative basis. With the assessment of the velocity range it is possible to obtain a more objective differentiation between the duplex tracing in hepatic veins of liver cirrhosis patients and healthy subjects. PMID- 8634403 TI - [Early clinical experiences with MR-guided laser-induced thermotherapy (LITT) of liver metastases in preoperative care]. AB - PURPOSE: To evaluate the LITT-induced changes with the aid of MRT and correlate these with histopathological findings. MATERIAL AND METHODS: Five patients with solitary colorectal liver metastases were treated by means of MR-guided LITT before liver resection. Application time and energy of the Nd:YAG laser (1064 nm) was 10-20 minutes and 4.5-8.8 W. MRT monitoring during the LITT was carried out with temperature-sensitive T1 weighted sequences (FLASH-2-D, turbo FLASH). The extent of the induced necrosis as seen on MR was compared with the unfixed specimen and with the histopathological findings. RESULTS: The extent of necrosis visible by MRT correlated with the histopathological findings with an accuracy of 95.3% +/- 4.2%. Following single treatments (three cases) the metastases suffered a reduction of 24%-55% of their original volume. In two patients a second application produced laser-induced necrosis of 78% and 98% of volume. In these two patients a temperature sound was used for measuring regional heating and showed an exact correlation with MR thermometry. CONCLUSION: The results of pre operative MR-guided LITT indicates the potential of this form of treatment for obtaining reproducible tumor necrosis of liver metastases. PMID- 8634404 TI - [Superselective intra-arterial chemotherapy in therapy-refractory lymph node recurrences of breast carcinoma]. AB - PURPOSE: To demonstrate a superselective intraarterial chemotherapy as a therapeutic alternative in the treatment of previously treated recurrent lymph node metastases in breast cancer. METHODS: 14 patients with recurrent lymph node metastases in cases of breast cancer were presented to be treated by intraarterial chemotherapy of 25 mg mitoxantrone/m2 over a period of 24 hours. In two patients with superclavicular lymph node involvement an intraarterial therapy could not be carried out because of a vascular connection to the anterior spinal artery. Involved lymph stations could be reached in superselective technique by side branches of the subclavian artery. Heparin coverage was given intravenously. Every patient had had surgery, radiation, systemic chemo- and hormonal therapy before and was now graded as inoperable. Therapy indication was given by local tumour-induced symptoms. RESULTS: In the 12 treated cases complete remission was seen in three, partial remission in 4, a steady state in two and a progressive disease in three. There were no complications or severe side effects. CONCLUSION: Intraarterial chemotherapy is an effective and well tolerated treatment in recurrent lymph node metastases in cases of breast cancer even if conventional therapies can no longer be used. PMID- 8634405 TI - [Superselective embolization of tumor nodes in solitary kidney in inoperable patients]. AB - PURPOSE: Evaluation of superselective embolisation of renal tumours in inoperable patients with solitary kidneys. METHODS AND PATIENTS: Eight inoperable patients with solitary kidneys bearing tumour nodules underwent 1-3 superselective embolisation procedures with ethibloc (5x) or polyvinyl alcohol (1x). Renal function was monitored with creatinine levels. Tumour size was controlled every three months by means of sonography. RESULTS: Technical success rate was 100%. In 3/3 patients haematuria could be stopped. Post-embolisation renal function was unchanged in 6 patients and deteriorated in two patients; creatinine level rose to a maximum of 2.2 mg%. We observed no other side effects. Seven of eight patients died during a median follow-up period of 9.3 months (4-18 months); in two cases they died due to their underlying malignant disease. One patient had local tumour progress. CONCLUSIONS: Superselective embolisation of renal tumours in patients with solitary kidneys may be a helpful, well-tolerated therapeutic option in inoperable, symptomatic patients. PMID- 8634407 TI - [Functional spiral CT of the larynx]. AB - Imaging of the larynx by computed tomography has reached a new quality through spiral CT. Its significance for evaluation of vocal cords function is examined with 10 patients with glottic tumours before, during and after radiation therapy. The spiral CT procedure which complements laryngoscopy is described: Gantry tilt 10 degrees caudal, two spiral CT starting at the cricoid cartilage with 3 mm slice thickness and pitch 1, one with flat breathing and one with "1" phonation; overlapping reconstruction with large zoom factor, secondary reconstruction image reformating. PMID- 8634406 TI - [Contrast media in peripheral angiography: does cost reduction mean loss of quality and safety? A reevaluation based on a randomized double-blind comparative study of ioxaglate versus iopromide]. AB - PURPOSE: To evaluate whether meglumine-sodium-ioxaglate (Hexabrix) and iopromide (Ultravist) are identically appropriate for peripheral angiography. Outcome variables were pain, image quality and adverse events. METHODS: Sixty patients were included in a randomised double-blind study. In all patients an intraarterial digital subtraction angiography (i.a. DSA) of iliac and peripheral arteries was performed. RESULTS: Analysis of the study revealed no significant difference between both contrast media in terms of the main and additional outcome variables. In comparison to iopromide, ioxaglate caused milder pain sensations (VAS 4.70 vs. 7.76, p = 0.25). Mild adverse events were observed more frequently in ioxaglate angiography (11% vs. 0%, p = 0.1). CONCLUSION: Both contrast media seem to be appropriate for peripheral angiography using DSA technique. Ioxaglate causes a cost reduction of about 20-35%. However, an increase of mild adverse reactions up to 11% to 15% has to be accepted. PMID- 8634408 TI - [Functional CT imaging of the lung in axial and coronal plane after single lung transplantation]. AB - After single-lung transplantation in a patient suffering from obstructive emphysema lung function parameters worsened during follow-up. To complement the routine high-resolution CT (HRCT) scans acquired in inspiration, additional scans were obtained to evaluate regional lung function. The comparison of HRCT scans acquired in inspiration and in expiration revealed different ventilation conditions of both lungs, continuous acquisition in a single slice (dynamic multiscan acquisition) in the axial and coronal plane demonstrated mediastinal shifting and the movement of the diaphragm during the whole breathing cycle. Both modalities can provide important information concerning regional differences of ventilation after single-lung transplantation. Expiratory HRCT should be applied on a regular basis in the follow-up of patients after single-lung transplantation. The application of axial and coronal dynamic multiscan acquisitions will be helpful in particular clinical conditions, like increasing hyperinflation, mediastinal shifting or bronchial collapse within the region of the anastomosis. PMID- 8634409 TI - [Carbon dioxide as an alternative contrast medium in peripheral angiography]. AB - A closed gas pressure pistol was used in 50 patient CO2 angiography as a supplementary method to conventional injection with liquid contrast medium. These were diagnostic pelvis-leg angiographies (n = 36), therapeutic angiographies (n = 8), haemodialysis fistulas (n = 3), suspected stenosis of a renal transplant artery (n = 1) and suspected renal artery stenosis (n = 1). 246 renal angiography series were performed with CO2. Dosages varied in accordance with the imaged vascular area between 10 ccm;(shunt imaging) and up to 100 cm3 (pelvis-leg angiography), at pressures between 400 mbar in case of haemodialysis fistulas up to 2000 mbar in the pelvis-leg area. Short-term feeling of fullness and even nausea were accompanying symptoms in 4 patients. The image quality was slightly inferior to that of conventional contrast medium images due to an elevated signal to-noise ratio. Injector-monitored CO2 angiographies enabled imaging of the distal aorta or of peripheral vascular sections, imaging of the upper extremity and presentation of kidney transplants in patients with a relative or absolute contraindication to iodised contrast media. PMID- 8634410 TI - Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease. PMID- 8634411 TI - Reactive oxygen intermediates cause rapid release of the interleukin-1 decoy receptor from human myelomonocytic cells. AB - Free radicals play an important role in inflammation. We found that reactive oxygen intermediates (ROI) inhibit interleukin-1beta (IL-1beta) binding on human myelomonocytes. Production of superoxide anion (O2-) by Xanthine (X) and Xanthine Oxidase (XO) or NADPH caused a reduction (48% +/- 15% in 25 experiments) in the IL-1beta binding of polymorphonuclear cells (PMN) and monocytes that was inhibited by superoxide dismutase (SOD). Hydrogen peroxide (H2O2) was only active on monocytes and this effect was prevented by catalase. O2(-)-induced loss of IL 1beta binding on PMN reached half maximum at 5 minutes and peaked after 30 minutes. The reduction of IL-1beta binding was due to reduction of IL-1beta receptors (R) on PMN surface without any change in affinity. ROI-induced reduction of surface IL-1R was not caused by receptor internalization, but rather by the release of a soluble form (45 kD) of the type II decoy R. The action of ROI on IL-1 binding was selective because major histocompatibility complex class I, CD18 and CD16 were unaffected. The O2(-)-induced release of IL-1 decoy R was not affected by protein synthesis inhibitors, but was partially blocked by protease inhibitors. Release of the IL-1 type II decoy R might represent one mechanism by which ROI antagonize and limit the proinflammatory effects of IL-1. PMID- 8634412 TI - Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. AB - Cytopenia after high-dose chemotherapy and autologous stem cell reinfusion is a major cause of morbidity. Ex vivo cultured expansion and differentiation of CD34+ peripheral blood progenitor cells (PBPC) to neutrophil precursors may shorten the neutropenic period further. We explored the use of these ex vivo cultured PBPCs in nine patients with metastatic breast cancer. All underwent PBPC mobilization with cyclophosphamide, VP-16, and G-CSF. Subsequently, they underwent four to five apheresis procedures. One apheresis product from each patient was prepared using the Isolex 300 Magnetic Cell Separation System (Baxter Immunotherapy, Irvine, CA) to obtain CD34+ cells. These cells were then cultured in gas permeable bags containing serum-free X-VIVO 10 (BioWhittaker, Walkersville, MD) medium supplemented with 1% human serum albumin and 100 ng/mL PIXY321. At day 12 of culture the mean fold expansion was 26x with a range of 6 to 64x. One patient's cells did not expand because of a technical difficulty. The final cell product contained an average of 29.3% CD15+ neutrophil precursors with a range of 18.5% to 48.1%. The patients underwent high-dose chemotherapy with cyclophosphamide, carboplatin, and thiotepa. On day 0, the cryopreserved PBPCs were reinfused and on day +1 the 12-day cultured cells were washed, resuspended, and reinfused into eight of nine patients. One patient was not infused with cultured cells. The mean number of cultured cells reinfused was 44.6 x 10(6) cells/kg with a range of 0.8 to 156.6 x 10(6) cells/kg. No toxicity was observed after reinfusion. The eight patients have recovered absolute neutrophil counts > 500/microL on a median of 8 days (range 8 to 10 days); the median platelet transfusion independence occurred on day 10 (range 8 to 12 days) and platelet counts > 50,000/microL were achieved by day 12 (range 9 to 14) for the seven patients whose platelet counts could be determined. Expanded CD34+ selected PBPC can be obtained and safely reinfused into patients. PMID- 8634413 TI - STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. AB - A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis. PMID- 8634414 TI - A pilot study of high-dose interleukin-3 treatment of relapsed follicular small cleaved-cell lymphoma: hematologic, immunologic, and clinical results. AB - The growth stimulatory effects of interleukin-3 (IL-3) on normal hematopoietic progenitor cells are well established, and clinical trials using IL-3 after bone marrow transplantation for various malignancies including lymphomas are frequently conducted. Although the IL-3 receptor is expressed on the surfaces of follicular small cleaved-cell lymphoma (FSCCL) cells, the in vivo effects of IL-3 on FSCCL have not been studied previously. Because our preclinical data suggested that IL-3 may have dose-dependent inhibitory effects on FSCCL cells in vitro, we treated eight FSCCL patients with high-dose IL-3 in an outpatient setting. Each patient received 1 mg/m2 of IL-3 subcutaneously daily for 14 days followed by 7 days without IL-3. After three courses (9 weeks), the patients were evaluated for clinical responses. One patient had a minor response, and four had no responses. Three patients who had progressive disease before IL-3 treatment continued to have progressive disease. In two patients with bone marrow involvement with lymphoma, IL-3 had no effect on FSCCL cells. One patient with peripheral blood involvement with FSCCL cells that expressed IL-3 receptors had temporary growth arrest of the circulating malignant cells. IL-3 significantly increased the absolute neutrophil count in seven patients (87%) but had little effect on the number of normal circulating B cells. There was an increase in the number of circulating natural killer cells and CD8+ cells in four patients. Treatment was very well tolerated; no life-threatening toxicities were observed. The most common toxicities were injected conjunctivae (100%), fever (100%), fatigue (87%), and skin rash (75%). Most of the side effects subsided with the continued use of IL-3. These preliminary results suggest that high-dose IL-3 does not stimulate the growth of FSCCL cells in vivo and, in some instances, may cause growth inhibition. PMID- 8634415 TI - Interferon treatment for chronic hepatitis C infection in hemophiliacs--influence of virus load, genotype, and liver pathology on response. AB - In this study, we assessed the effectiveness of interferon treatment in 31 hemophiliacs with chronic hepatitis C virus (HCV) infection. Interferon alfa-2a (3 MU three times weekly) was administered for 6 months. Response was assessed by both serial alanine transaminase (ALT) and HCV RNA levels measured by a sensitive semiquantitative polymerase chain reaction (PCR) method. HCV genotype was determined by restriction fragment length polymorphism (RFLP), and evidence of changing genotypes during interferon therapy was sought. Severity of liver disease was assessed by both noninvasive and invasive methods, including laparoscopic liver inspection and biopsy. Sustained normalization of ALT levels occurred in eight patients (28%), and seven (24%) became nonviremic as assessed by PCR (<80 HCV/mL). Responders universally cleared HCV RNA within 2 months of starting interferon. Genotype 3a was associated with a favorable response to interferon. No evidence was found for a change in circulating genotype in patients who failed to respond to interferon or who relapsed. This study confirms that response rates to interferon are low in hemophiliacs as compared with other groups with chronic HCV infection. We have also demonstrated that virus load measurement over the first 8 to 12 weeks of treatment is an extremely useful method to identify responders at an early stage. PMID- 8634416 TI - A randomized study of high-dose cytarabine in induction in acute myeloid leukemia. AB - High-dose cytarabine (ara-c) may overcome cytarabine resistance in leukemic blasts. It has been used as a successful salvage and in postremission therapy but not as initial induction treatment. Patients aged 15 to 60 years, presenting with newly diagnosed acute myeloid leukemia (AML) were randomized to receive either high-dose cytarabine, 3 g/m2 12 hourly on days 1, 3, 5, and 7 for 8 doses, daunorubicin 50 mg/m2 days 1 to 3, etoposide 75 mg/m2 days 1 to 7, (HIDAC-3-7) or standard dose cytarabine 100 mg/m2 continuous intravenous infusion for 7 days with daunorubicin and etoposide at the same dose and schedule as above (7-3-7). Patients could receive a second or third induction course if complete remission (CR) was not achieved. All patients received the same postinduction consolidation therapy (5-2-5) for 2 courses. Eligible patients had no prior chemotherapy or myelodysplastic disease. Patients have been followed for a median of 4.5 years. Of 301 patients treated, complete response (CR) was achieved in 71% with HIDAC-3 7 and 74% with 7-3-7. For patients in CR, the estimated median remission duration was 45 months with HIDAC-3-7 and 12 months with 7-3-7 (P = .0005 univariate analysis, P = .0004 multivariate analysis). The estimated percentage of patients relapse free 5 years after achieving a CR was 49% on HIDAC-3-7 and 24% on 7-3-7. Patients in CR tended to survive longer with HIDAC-3-7 but there were no overall survival differences between the two arms. HIDAC-3-7 was associated with significantly more toxicity in induction with more leukopenia, thrombocytopenia, nausea, and vomiting and eye toxicity (all P < .001) but a similar incidence of severe central nervous system and cerebellar toxicity compared to 7-3-7. The consolidation treatment was the same in both arms but caused significantly more leukopenia and thrombocytopenia in patients previously treated with HIDAC-3-7 induction (P < .0001). We conclude that a dose-effect exists for cytarabine in AML and that HIDAC-3-7 prolongs remission duration and disease-free survival and is tolerable when used as initial induction therapy in patients with de novo AML. PMID- 8634417 TI - Accelerated cell-cycling of hematopoietic progenitors by the flt3 ligand that is modulated by transforming growth factor-beta. AB - Although hematopoietic growth factors have been extensively studied as to their roles in recruitment of hematopoietic progenitors from quiescence state to cell division state, little is known of their effects on cell-cycling of progenitors that have already transited from quiescence into active cell-cycling. We examined the effects of the flt3 ligand (FL) on cell-cycling of hematopoietic progenitors in serum-free culture. Results from our serial observations of colony formation and replating experiments suggest that FL enhances the rate of growth of interleukin-3 (IL-3)-dependent colonies by shortening the time for each progenitor in the colonies to divide. Cell-cycle analysis showed that shortening of cell-cycle time induced by FL is mainly because of alteration in the G1 phase that hematopoietic progenitors go through. We next investigated the role of transforming growth factor-beta (TGF-beta) in cell-cycling of progenitors, using TGF-beta protein and TGF-beta antisense oligonucleotides, because mRNA of TGF beta was detected by reverse transcriptase polymerase reaction in blast cells that we used as a source of progenitors. TGF-beta lengthened the time required for IL-3-dependent progenitors to become two daughter cells, whereas the effects of TGF-beta antisense oligonucleotides were opposite to those of TGF-beta. The addition of TGF-beta neutralizing monoclonal antibodies to the cultures resulted in effects similar to those seen with TGF-beta antisense oligonucleotides. DNA studies indicated that both TBF-beta and TGF-beta antisense oligonucleotides change the length of G1 phase of the cell-cycle. TGF-beta abrogated the effects of FL on the growth rate of hematopoietic progenitors, whereas the combination of FL with TGF-beta antisense oligonucleotides exerted additive effects. These data show that FL has the potential to accelerate cell-cycling of hematopoietic progenitors, which is susceptible to the modulation by TGF-beta. PMID- 8634418 TI - The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells. AB - Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells. PMID- 8634420 TI - Inhibition of the erythropoietin-induced erythroid differentiation by granulocyte macrophage colony-stimulating factor in the human UT-7 cell line is not due to a negative regulation of the erythropoietin receptor. AB - The human pluripotent UT-7 cell line is growth factor-dependent for proliferation and differentiation. We have previously shown that (1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) induce a myeloid and erythroid pattern of differentiation, respectively; (2) GM-CSF acts predominantly over Epo for cell differentiation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo receptors (Epo-R) at the mRNA and binding site levels; and (4) in contrast, Epo has no effect on GM-CSF receptor (GM-CSF-R) expression. These results suggested that UT-7 cell commitment or differentiation may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors. To test this hypothesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constructed: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3' sequence placed under the transcriptional control of the viral long terminal repeat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R infected clones (12, 6, 10) and one UT-7/neomycin clone (Neo) were selected in medium containing G418. After growth factor deprivation (18 hours), Epo-Rs were expressed at the same level (approximately 6,000 receptors per cell) in all four clones 12, 6, 10, Neo, and in parental UT-7 cells, and exhibited similar affinity (0.1 to 0.2 nmol/L). Cross-linking experiments showed that Epo is associated with three proteins of about 66, 85, and 100 kD in cells of parental UT-7, as well as in cells of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the cross-linking on parental UT-7 cells, but not on cells of clone 12, demonstrating that more than 90% cell surface Epo-Rs were of murine origin. The presence of GM-CSF significantly reduced the number of Epo-Rs expressed on parental UT-7 cells, but not on cells of clones 12, 10, and 6. HuEpo-R Ab inhibited Epo-induced parental UT-7 cell growth, but not that of cells of clone 12, suggesting that the muEpo-R is able to induce human UT-7 cell proliferation. When cells of clone 12 were switched from a medium containing GM-CSF to one with Epo, cell surface glycophorin A (GPA) was induced, as in parental UT-7 cells without inhibition by the huEpo-R Ab, demonstrating that the muEpo-R is also able to transduce a differentiation signal in human cells. However, in cells of clones 12, 6, 10 and Neo, as well as in parental UT-7 cells, the induction of GPA by Epo was inhibited by GM-CSF. This finding demonstrates that, although GM-CSF does not downregulate muEpo-R binding sites on UT-7/muEpo-R infected clones, it still inhibits the effects of Epo on cell differentation. Therefore, hierarchical regulation induced by growth factors for cell commitment or differntiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels. PMID- 8634419 TI - Expression of HOXC4, HOXC5, and HOXC6 in human lymphoid cell lines, leukemias, and benign and malignant lymphoid tissue. AB - Besides their regulatory role in embryogenesis, homeobox (HOX) genes are expressed in a specific manner in hematopoietic cell lineages, implying a role in the molecular regulation of hematopoiesis. Some HOX C cluster genes are found to be expressed in lymphoid cells of mice and humans. Their function and expression in normal hematopoiesis are still largely unknown. We have studied the mRNA expression of HOXC4, HOXC5, and HOXC6 in several stages of lymphocyte maturation by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNA in situ hybridization (RISH). We examined CD34+/CD38low and CD34+/CD38high cells obtained from normal donor bone marrow (BM), a panel of 19 lymphoid cell lines, several types of leukemias and non-Hodgkin's lymphomas (NHL), and lymphocytes isolated from tonsillar tissue and peripheral blood (PB). HOXC4 and HOXC6 were found to be expressed during maturation in B- and T-lymphoid cells. The expression of each gene was found to be initiated at different cell maturation stages. HOXC4 transcripts were present in CD34+/CD38low cells, which are thought to comprise stem cells and noncommitted progenitor cells, and in subsequent stages to terminally maturated lymphoid cells. HOXC6 expression is initiated in equivalents of prothymocyte and pre-pre-B cell stage and remains present in mature cells. However, HOXC5 is only expressed in neoplastic cell lines and in neoplastic cells of NHL, but not in CD34+ BM cells, nor in resting or activated lymphoid cells isolated from tonsil, PB, or in leukemia cells. In cell lines, weak expression of HOXC5 is initiated in equivalents of pre-B cell and common thymocyte stage and is continuously expressed in mature cell lines. Semi-quantitative RT-PCR showed that expression levels of HOXC5 were much lower than those of HOXC4 and HOXC6; furthermore an increase of expression of HOXC4, HOXC5, and HOXC6 during lymphoid cell differentiation was demonstrated. Thus, mainly mature lymphoid cell lines and neoplastic cells of NHL do express HOXC5, in contrast to the lack of expression in normal lymphoid cells and leukemias. These findings suggest involvement of HOXC5 in lymphomagenesis. PMID- 8634421 TI - A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients. AB - Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral-producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models. PMID- 8634422 TI - Clinical features and studies of erythropoiesis in Israeli Bedouins with congenital dyserythropoietic anemia type I. AB - Congenital dyserythropoietic anemia (CDA) type I is a rare macrocytic anemia of unknown etiology. In the present study, we redefined the clinical and laboratory picture of CDA type I, some of its pathogenic aspects, and the association with thalassemia-like features in 20 patients, all of whom belong to one Bedouin tribal group and are probably descended from a common ancestor. In each case ultrastructural studies of bone marrow (BM) erythroblasts showed the classic morphological findings of CDA type I. Serological tests for CDA type II were negative. The clinical picture was variable, but mostly benign. Some patients displayed elevated hemoglobin A2 levels or high ratio of alpha- to non-alpha- globin. However, neither family studies nor complete sequence analysis of the beta-globin was compatible with beta-thalassemia. Increased erythropoiesis was manifested by a high number of BM erythroid burst-forming units. Serum erythropoietin was also elevated. BM flow cytometry studies demonstrated arrest of erythroid precursors in the S phase of the cell cycle. The ultrastructural morphological features of the erythroid precursors, showing peripheral chromatin condensation, suggest apoptosis. Additional studies are indicated to define the molecular basis of this disease. PMID- 8634423 TI - Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro. AB - Studies of gene regulation during early hematopoiesis and of the regulatory network that controls differentiation and lineage commitment are hampered by difficulties in isolating and growing stem cells and early progenitor cells. These difficulties preclude the application of standard molecular genetic approaches to these problems. As an alternative approach we have introduced a lacZ-containing promoter-trap retrovirus into hematopoietic cells. We used the interleukin-3-dependent mouse myeloid progenitor cell 32D as a model to identify transcriptionally active genes. The frequency of integrations that led to transcription of the lacZ gene was estimated to be 0.5% of all integrations, of which 14% were downregulated on differentiation of 32D cells towards neutrophils. Thus, one in every 1,000 to 2,000 integrations identified a developmentally regulated gene. Cellular DNA sequences upstream of proviral integrations were isolated by inverse polymerase chain reaction. Five were further characterized and we confirmed by RNA expression analysis that they were downregulated on differentiation. Sequence analysis revealed identification of novel genes with sequence similarity to known genes. Considering the high efficiency of retroviral infection, our study shows the feasibility of using promoter-trap vectors to identity and isolate developmentally regulated genes from early hematopoietic progenitors. PMID- 8634424 TI - Deficiency of the Hck and Src tyrosine kinases results in extreme levels of extramedullary hematopoiesis. AB - Expression of the Src-family kinases--Src, Hck, and Fgr--increases dramatically during myeloid cell development. Src-deficient mice exhibit functional abnormalities in only one myeloid cell type, the osteoclast, resulting in impaired bone remodeling and osteopetrosis, while hck-/- or fgr-/- mice have few and subtle myeloid cell deficiencies. To determine whether these limited phenotypes are due to the coexpression of multiple Src-family kinases with overlapping functions, we have intercrossed src-/- mice to hck-/- and fgr-/- mutants to produce double mutants. Two thirds of hck-/- src-/- double mutants die at birth; surviving animals develop a severe form of osteopetrosis, resulting in extreme levels of splenic extramedullary hematopoiesis, anemia, and leukopenia. These hematopoietic defects are caused by abnormalities in the bone marrow environment because hck-/- src-/- mutant stem cells reconstitute a normal hematopoietic system in irradiated wild-type mice. In contrast, fgr-/- src-/- double mutants have no defects beyond those observed in src-/- animals. Cultured normal murine osteoclasts express abundant amounts of Src, Hck, and Fgr and Hck levels are increased in src-/- osteoclasts. These observations suggest that Hck and Src serve partially overlapping functions in osteoclasts and that the expression of Hck in src-/- osteoclasts ameliorates their functional defects. PMID- 8634426 TI - Normal human peripheral blood mononuclear cells mobilized with granulocyte colony stimulating factor have increased osteoclastogenic potential compared to nonmobilized blood. AB - Single-cell suspensions of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood mononuclear cells (G-PBMC) cultured in alpha minimal essential medium (alphaMEM) containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared to be dependent on cell density per surface area, so that 5.8 +/- 1.3 aggregates formed per 1 x 10(5) cells when G-PBMC were cultured at densities greater than or equal to 1 x 10(5) cells/cm2. The frequency of aggregate formation was less than 1 per 10(5) cells when G-PBMC were cultured at densities less than 1 x 10(5) cells/cm2. Once formed, aggregates became adherent within 72 hours, and then, over the course of 21 days, released CD3/CD4/CD25 positive cells into the supernatant. This T-cell production peaked between days 7 and 14, reaching a total of 1,269 +/- 125.9 cells released per aggregate by day 21. Between days 14 and 21, the aggregates also generated macroscopic clusters of adherent mononuclear and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the macroscopic foci coalesced into monolayers. Multinucleated TRAP-positive cells were distinguished from macrophage polykaryons by the absence of CD14 expression and the presence of osteoclast-specific membrane receptors for calcitonin and alphavbeta3 vitronectin. The osteoclast nature of these cells was further demonstrated by their ability to form resorption lacunae on dentine slices. Comparable osteoclast formation was not detected in cultures of normal marrow or normal nonmobilized peripheral blood. PMID- 8634425 TI - Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters. AB - GATA and CACC elements commonly are codistributed within the regulatory domains of a variety of erythroid genes. Using Drosophila S2 cells, the actions of GATA1, Sp1, and erythroid Kruppel-like factor (EKLF) at these elements within model erythroid promoters have been assessed. For each promoter studied (erythroid pyruvate kinase, glycophorin B, and a murine betamaj globin-derived construct, GCT) Sp1 and EKLF each activated transcription despite differences in CACC element sequence, orientation, and positioning. However, GATA1 acted in apparent cooperativity with Sp1 at the pyruvate kinase promoter; with EKLF at the betamaj globin-derived GCT promoter; and with either Sp1 or EKLF at the glycophorin B promoter. Thus, GATA1 may functionally interact with each of these Kruppel-like factors depending on promoter context; and at the GCT promoter, transcriptional activation by GATA1 and EKLF was > or = 10-fold higher than levels attributable to additive effects. The possibility that interactions between these activators may be direct was supported by the specific binding of baculoviral-expressed EKLF to GATA1. This report underlines the likelihood that discrete roles exist for Sp1 and EKLF in erythroid gene activation, and supports a mechanism of direct cooperativity for EKLF and GATA1 as coregulators. PMID- 8634427 TI - Kinetics of factor VIII-von Willebrand factor association. AB - The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2 rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds. PMID- 8634428 TI - The synthesis and localization of alternatively spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes. AB - There are several species of alternatively spliced fibronectin (FN). One of these, FN EIIIB, is primarily present in embryonic and in proliferating and migrating cells and is believed to be important for cell maturation. We have studied the synthesis, localization, and secretion of this FN isoform in isolated guinea pig megakaryocytes, nonmegakaryocytic bone marrow cells, and platelets. There was 7.5 times more general FN in megakaryocytes than in nonmegakaryocytic cells based on the analysis of equivalent amounts of protein. FN EIIIB was detected by Western blotting in megakaryocytes but not in nonmegakaryocytic cells present in bone marrow. Neither megakaryocytes nor platelets secreted FN EIIIB, while megakaryocytes secreted 25.3% +/- 4.6% general FN and platelets secreted about 61% general FN in response to thrombin. Analysis of immunostained cells by confocal microscopy revealed that FN EIIIB had been redistributed to the surface of megakaryocytes in response to thrombin. Synthesis was studied by metabolic labeling, and megakaryocytes were shown to synthesize FN and FN EIIIB. Thus, megakaryocytes and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. The expression of FN EIIIB on the megakaryocyte surface may influence migration and maturation. PMID- 8634429 TI - Morphological and functional changes of coronary vasculature caused by transcellular biosynthesis of sulfidopeptide leukotrienes in isolated heart of rabbit. AB - Morphological and functional modifications occurring in Langendorff rabbit heart preparations perfused with purified human leukocytes (PMNL), as an organ model of sulfidopeptide-leukotrienes (sLT) transcellular biosynthesis, were studied. Coronary perfusion pressure (CPP), monitored as an index of coronary vasospasm, increased by 295% after challenge with the Ca(2+)-ionophore A-23187 (0.5 micromol/L) for 30', accompanied by a significant formation of sLT. Increase in CPP was prevented by PMNL pretreatment with the 5-lipoxygenase inhibitor MK-886 (1 micromol/L) or by heart pretreatment with LTD4-receptor antagonist SKF 104353, indicating a pivotal role of PMNL-derived 5-lipoxygenase (5-LO) products in the observed functional modifications. Similar effects were obtained using granulocyte macrophage-colony stimulating factor-primed PMNL challenged with the tripeptide n-formyl-methionyl-leucyl-phenylalanine. Scanning electron microscopy (SEM) of coronary arteries showed craters on the vessel luminal surface, PMNL adhering to endothelial cells (EC), increased number of microvilli on EC, presence of nonviable, desquamating, fusiform EC. SEM and transmission electron microscopy of myocardial microvessels, showed presence of perivascular and intermuscle edema, presence of activated PMNL and decreased number of patent microvessels. These morphological alterations were significantly blunted by MK 886 or SKF 104353. These data provide evidence of close interaction between PMNL and myocardial EC, resulting in enhanced sLT formation via transcellular biosynthesis, originating from transfer of PMNL-derived LTA4 to EC. These potent proinflammatory autacoids are responsible for coronary vasospasm and the morphological alternations observed. PMID- 8634430 TI - Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates. AB - The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu-MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10( 3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5-fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo occlusive risk. PMID- 8634431 TI - Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase. AB - Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity. PMID- 8634432 TI - Six different cytokines that share GP130 as a receptor subunit, induce serum amyloid A and potentiate the induction of interleukin-6 and the activation of the hypothalamus-pituitary-adrenal axis by interleukin-1. AB - Ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) potentiate the elevation of serum corticosterone induced by suboptimal doses of interleukin-1 (IL-1). CNTF also potentiates IL-1-induced serum IL-6. Here, we report that four other cytokines (leukemia inhibitory factor [LIF], oncostatin M [OSM], interleukin-11 and cardiotrophin-1) also potentiated the elevation of serum corticosterone and IL-6 levels induced by IL-1. Furthermore, all the six cytokines studied induced the acute-phase protein serum amyloid A when administered alone. Because these cytokines differ both in structure and in function, but share gp130 as a subunit of their receptors, these results indicate that signaling through gp130 mediates potentiation of IL-1 activities. The potentiation of IL-1-induced serum corticosterone levels is not a consequence of the increased serum IL-6 observed after IL-1 administration. In fact, in IL-6 deficient mice, IL-1 increased serum corticosterone to a level comparable to that observed in wild-type mice. Thus, either endogenous IL-6 does not mediate IL-1 induced corticosterone increase, or its role may be fulfilled by other cytokines. To the extent that gp130-dependent cytokines may serve this role, they may be important feedback regulators of inflammation through the activation of the hypothalamus-pituitary-adrenal axis and the potentiation of acute-phase protein synthesis. PMID- 8634433 TI - Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells. AB - B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins. PMID- 8634435 TI - Immunoglobulin heavy chain variable region gene usage in bone marrow transplant recipients: lack of somatic mutation indicates a maturational arrest. AB - Many recipients of bone marrow transplant (BMT) make normal amounts of serum immunoglobulin but are deficient in generating specific antibody responses to exogenous stimuli. To determine if abnormal usage of VH genes contributes to this immunodeficiency, the usage of VH genes was determined in peripheral blood B cells of four BMT recipients, two of whom had developed chronic graft versus host disease. The pattern of usage of VH3 or VH4 genes assessed at either 90 days or approximately 1 year after transplant was similar to that observed in healthy subjects and was marked by the over utilization of two elements, one VH3 and one VH4. However, the repertoires of each of the four BMT recipients appeared to be less complex than the repertoires of healthy subjects. The differences were a consequence of the accumulation of somatic mutations among rearrangements in the controls but not in the BMT recipients. The failure to accumulate somatic mutations in rearranged VH genes is consistent with a defect in antigen driven B cell responses. These results indicate the although the VH gene content of the repertoire has normalized by 90 days posttransplant, a maturational arrest in B cell differentiation associated with antigen activation persists for at least 1 year after BMT. PMID- 8634434 TI - Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. AB - Pentraxins, which include C reactive protein (CRP) and serum amyloid P component (SAP), are prototypic acute phase reactants that serve as indicators of inflammatory reactions. Here we report genomic and cDNA cloning of mouse ptx3 (mptx3), a member of the pentraxin gene family and characterize its extrahepatic expression in vitro and in vivo. mptx3 is organized into three exons on chromosome 3: the first (43 aa) and second exon (175 aa) code for the signal peptide and for a protein portion with no high similarity to known sequences the third (203 aa) for a domain related to classical pentraxins, which contains the "pentraxin family signature." Analysis of the N terminal portion predicts a predominantly alpha helical structure, while the pentraxin domain of ptx3 is accommodated comfortably in the tertiary structure fold of SAP. Normal and transformed fibroblasts, undifferentiated and differentiated myoblasts, normal endothelial cells, and mononuclear phagocytes express mptx3 mRNA and release the protein in vitro on exposure to interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)alpha. mptx3 was induced by bacterial lipopolysaccharide in vivo in a variety of organs and, most strongly, in the vascular endothelium of skeletal muscle and heart. Thus, mptx3 shows a distinct pattern of in vivo expression indicative of a significant role in cardiovascular and inflammatory pathology. PMID- 8634436 TI - The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell independent expansion and differentiation of human fetal pro-B cells in vitro. AB - The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow derived CD34+CD19+ pro-B cells were analyzed in a stromal-cell-independent, serum deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34 CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL 7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B-cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro-B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor. PMID- 8634437 TI - Hyaluronan-dependent motility of B cells and leukemic plasma cells in blood, but not of bone marrow plasma cells, in multiple myeloma: alternate use of receptor for hyaluronan-mediated motility (RHAMM) and CD44. AB - We investigated the ability of blood B cells, bone marrow (BM) plasma cells, and terminal leukemic plasma cells (T-PCL) from patients with multiple myeloma (MM) to migrate on extracellular matrix proteins. Hyaluronan (HA), but not collagen type I, collagen type IV, or laminin, promoted migration of MM blood B cells, as determined by time-lapse video microscopy. Between 13% and 20% of MM blood B cells migrated on HA with an average velocity of 19 micron/min, and greater than 75% of MM blood B cells exhibited vigorous cell movement and plasma membrane deformation, as did circulating T-PCL and extraskeletal plasma cells from patients with MM. In contrast, plasma cells obtained from BM of patients with MM lacked motility on all substrates tested and did not exhibit cell membrane protrusions or cellular deformation. MM blood B cells and MM plasma cells from all sources examined expressed the HA-binding receptors receptor for HA-mediated motility (RHAMM) and CD44. On circulating MM B cells, both RHAMM and CD44 participated in HA-binding, indicating their expression ex vivo in an activated conformation. In contrast, for the majority of BM plasma cells in the majority of patients with MM, expression of RHAMM or CD44 was not accompanied by HA binding. A minority of patients did have HA-binding BM plasma cells, involving both RHAMM and CD44, as evidenced by partial blocking with monoclonal antibodies (MoAbs) to RHAMM or to CD44. Despite HA binding by both RHAMM and CD44, migration of MM blood B cells on HA was inhibited by anti-RHAMM but not by anti-CD44 MoAbs, indicating that RHAMM but not CD44 mediates motility on HA. Thus, circulating B and plasma cells in MM exhibit RHAMM- and HA-dependent motile behavior indicative of migratory potential, while BM plasma cells are sessile. We speculate that a subset(s) of circulating B or plasma cells mediates malignant spread in myeloma. PMID- 8634438 TI - Expression of A-myb, but not c-myb and B-myb, is restricted to Burkitt's lymphoma, sIg+ B-acute lymphoblastic leukemia, and a subset of chronic lymphocytic leukemias. AB - The A-myb gene encodes a transcription factor that is related both functionally and structurally to the v-myb oncogene. Following our observations that A-myb is expressed in a restricted subset of normal mature human B lymphocytes, with the phenotype CD38+, CD39-, slgM-, we have now investigated the pattern of A-myb expression in neoplastic B cells representating the whole spectrum of B-cell differentiation and compared it to that of c-myb and B-myb. In a panel of 32 B cell lines, A-myb was very strongly expressed in most Burkitt's lymphoma (BL) cell lines, but weak or negative in 2 pre-B acute lymphoblastic leukemia (ALL), 4 non-Hodgkin's lymphoma (NHL), 6 Epstein-Barr virus-immortalized lymphoblastoid cell lines, and 6 myeloma lines. Protein expression paralleled that of the RNA. We have also investigated A-myb expression in 49 fresh cases of B leukemias. Among 24 ALL, 6 were of the null and 11 of the common type and all these were negative for A-myb expression; on the other hand, all 7 B-ALL cases (slg+), as well as one fresh BL case with bone marrow infiltration, expressed A-myb. A-myb was undetectable in 4 prolymphocytic leukemias (PLL) but was strongly expressed in 5/20 (25%) of chronic lymphocytic leukemia (CLL) samples. In the latter A-myb did not correlate with phenotype or clinical stage. Finally, we have studied the progression of one case of CLL into Richter's syndrome and have found that the Richter's cells expressed about 25-fold less A-myb RNA than the CLL cells from the same patient. The pattern of c-myb and B-myb was clearly distinct from that of A-myb. C-myb and B-myb were expressed in all neoplastic groups, except in CLL cells. Thus, A-myb expression, unlike that of c-myb and B-myb, is restricted to a subset of B-cell neoplasias (in particular BL and slg+B-ALL) representative of a specific stage of B-cell differentiation. This expression may in part reflect expression of A-myb by the normal germinal center B cells that are the normal counterpart of these transformed B cells. The data presented strongly support a role for this transcription factor in B-cell differentiation and perhaps in B cell transformation in some neoplasias. PMID- 8634439 TI - Distribution of 11q23 breakpoints within the MLL breakpoint cluster region in de novo acute leukemia and in treatment-related acute myeloid leukemia: correlation with scaffold attachment regions and topoisomerase II consensus binding sites. AB - A major unresolved question for 11q23 translocations involving MLL is the chromosomal mechanism(s) leading to these translocations. We have mapped breakpoints within the 8.3-kb BamHI breakpoint cluster region in 31 patients with acute lymphoblastic leukemia and acute myeloid leukemia (AML) de novo and in 8 t AML patients. In 23 of 31 leukemia de novo patients, MLL breakpoints mapped to the centromeric half (4.57 kb) of the breakpoint cluster region, whereas those in eight de novo patients mapped to the telomeric half (3.87 kb). In contrast, only two t-AML breakpoints mapped in the centromeric half, whereas six mapped in the telomeric half. The difference in distribution of the leukemia de novo breakpoints is statistically significant (P = .02). A similar difference in distribution of breakpoints between de novo patients and t-AML patients has been reported by others. We identified a low- or weak-affinity scaffold attachment region (SAR) mapping just centromeric to the breakpoint cluster region, and a high-affinity SAR mapping within the telomeric half of the breakpoint cluster region. Using high stringency criteria to define in vitro vertebrate topoisomerase II (topo II) consensus sites, one topo II site mapped adjacent to the telomeric SAR, whereas six mapped within the SAR. Therefore, 74% of leukemia de novo and 25% of t-AML breakpoints map to the centromeric half of the breakpoint cluster region map between the two SARs; in contrast, 26% of the leukemia de novo and 75% of the t-AML patient breakpoints map to the telomeric half of the breakpoint cluster region that contains both the telomeric SAR and the topo II sites. Thus, the chromatin structure of the MLL breakpoint cluster region may be important in determining the distribution of the breakpoints. The data suggest that the mechanism(s) leading to translocations may differ in leukemia de novo and in t-AML. PMID- 8634440 TI - Expression of p13MTCP1 is restricted to mature T-cell proliferations with t(X;14) translocations. AB - T-cell prolymphocytic leukemia (T-PLL), a rare form of mature T-cell leukemias, and ataxia telangiectasia clonal proliferation, a related condition occurring in patients suffering from ataxia telangiectasia, have been associated to translocations involving the 14q32.1 or Xq28 regions, where are located the TCL1 and MTCP1 putative oncogenes, respectively. The MTCP1 gene is involved in the t(X;14)(q28;q11) translocation associated with these T-cell proliferations. Alternative splicing generates type A and B transcripts that potentially encode two entirely distinct proteins; type A transcripts code for a small mitochondrial protein, p8MTCP1, and type B transcripts, containing an additional open reading frame, may code for 107 amino-acid protein, p13MTCP1. The recently cloned TCL1 gene, also involved in translocations and inversions associated with T-cell proliferations, codes for a 14-kD protein that displays significant homology with p13MTCP1. We have generated rabbit antisera against this putative p13MTCP1 protein and screened for expression of p13MTCP1 normal lymphoid tissues and 33 cases of immature and mature lymphoid T-cell proliferations using a sensitive Western blot assay. We also investigated the MTCP1 locus configuration by Southern blot analysis. The p13MTCP1 protein was detected in the three T-cell proliferations with MTCP1 rearrangements because of t(X;14) translocations, but neither in normal resting and activated lymphocytes nor in the other T-cell leukemias. Our data support the hypothesis that p13MTCP1 and p14TCL1 form a new protein family that plays a key role in the pathogenesis of T-PLL and related conditions. PMID- 8634441 TI - Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells. AB - Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF beta1) is also produced by BMSCs and can regulate IL-6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF-beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N-BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF-beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion. PMID- 8634442 TI - Retinoid-induced differentiation of acute promyelocytic leukemia involves PML RARalpha-mediated increase of type II transglutaminase. AB - All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells. PMID- 8634443 TI - Detection of apoptotic cell death by proton nuclear magnetic resonance spectroscopy. AB - Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a direct temporal relationship between the onset of apoptosis in Jurkat T cell lymphoblast cultures and a greater than two-fold increase in the signal intensity of the methylene resonance (at 1.3 ppm) as observed by proton nuclear magnetic resonance spectroscopy (1H NMR). The increase in the methylene resonance intensity was seen when apoptosis was induced by serum deprivation, glucocorticoid, and doxorubicin treatment but not in necrotic (nonapoptotic) cell death. We have found similar changes in a variety of other cell lines undergoing apoptosis including the Hut 78 T-cell leukemia, JY natural killer T-cell leukemia, Daudi B-cell lymphoma, HeLa, and 3T3 fibroblast cell lines. Furthermore, this spectral change was diminished in Bcl-2 overexpressing HL-60 cell cultures treated with doxorubicin, which were relatively resistant to apoptosis, as compared to apoptotic HL-60 cultures. 1H NMR spectroscopy therefore may be useful in detecting apoptotic cell death in vivo. PMID- 8634444 TI - Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine DNA-alkyltransferase. AB - A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony-forming cells of the granulocyte macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas. PMID- 8634445 TI - Novel ex vivo analysis of nonclassical, pleiotropic drug resistance and collateral sensitivity induced by therapy provides a rationale for treatment strategies in chronic lymphocytic leukemia. AB - Extensive research into mechanisms of cytotoxic drug resistance and subsequent clinical trials of drug resistance modifiers have produced few encouraging results. In this report, we analyze 4,400+ ex vivo Differential Staining Cytotoxicity (DiSC) assay drug response results from patients with chronic lymphocytic leukemia (CLL) to investigate the development of drug resistance during treatment. Patients were untreated (n = 216) or previously treated with various cytotoxic agents (n = 188). Data was processed to identify ex vivo resistance (or sensitivity) induced by treating patients with prednisolone, chlorambucil, cyclophosphamide, anthracycline, or fludarabine. Induced resistance was apparently not associated with any one known mechanism. Treatment with chlorambucil induced a 10-fold sensitivity to steroids; cyclophosphamide induced greater resistance to anthracyclines than alkylating agents; anthracyclines induced greatest resistance to chlorambucil, cisplatin, carboplatin, and cladribine. Patients previously treated with at least two regimens were only 2.16 fold more resistant to CLL drugs than untreated patients, but had significantly reduced survival (median survival, 7.9 months compared with 61.1 months for untreated patients). These results suggest that chlorambucil and/or an antimetabolite should be administered before cyclophosphamide or anthracyclines to delay the onset of extensive pleiotropic drug resistance. Because individual differences in drug sensitivity are considerable, specific guidance could be obtained from ex vivo assay results. Furthermore, as a model for investigating drug resistance mechanisms, fresh CLL lymphocytes represent a useful alternative to drug-resistant cell lines. PMID- 8634446 TI - Circulating clonal lymphocytes in myeloma constitute a minor subpopulation of B cells. AB - The mononuclear cells in the blood of myeloma patients have been reported to contain a high proportion of phenotypically abnormal myeloma B lymphocytes. These cells have been proposed to constitute the drug-resistant proliferative myeloma cell compartment. To determine the extent of B lymphocyte involvement, the proportion of clonotypic cells among the CD19-expressing cells from myeloma patients was estimated by quantitative polymerase chain reaction analysis of the third complementarity determining region (CDR3). The results indicate that the B lymphocytes constitute, on average, 6% of blood mononuclear cells, and that only a minor fraction of these are clonally related to the myeloma cells. While the small number of circulating clonal cells is not incompatible with their proposed role as a reservoir of proliferating myeloma progenitors, the majority of the B cells appear not to be clonally related to the myeloma cells. PMID- 8634448 TI - Elevation of cerebrospinal fluid soluble CD27 levels in patients with meningeal localization of lymphoid malignancies. AB - Diagnosis of meningeal localization of lymphoid malignancies by means of cytologic examination of the cerebrospinal fluid (CSF) can be difficult. Thus far no reliable CSF tumor markers have been identified. CD27 is a transmembrane disulfide-linked 55-kD homodimer present on most peripheral blood T cells and on a subset of B cells. CD27 is also expressed on human malignant B cells and high levels of soluble CD27 can be present in the serum of patients with B-cell malignancies. The aim of this study is to determine prospectively the diagnostic value of CSF sCD27 as a tumor marker in patients with meningeal localization of lymphoid malignancies. CSF sCD27 levels were determined by sandwich enzyme-linked immunosorbent assay. The optimal cut-off value using receiver operator characteristics curves was found to be 10 U/mL. sCD27 levels were normal in all 50 control patients (lumbar disc protrusion) and in 39 of 40 samples obtained from patients with either solid tumors or acute myeloid leukemia. Of 104 CSF samples from 70 children with acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL) undergoing routine central nervous system (CNS) staging, sCD27 was false positive and false negative in only one sample each. In 70 samples from 45 patients suspected of meningeal localization of ALL or NHL, the sCD27 test had an excellent sensitivity (100%) and specificity (82%). In 7 patients with positive CSF studied longitudinally, sCD27 levels correlated very well with remission and relapse. sCD27 levels were not nonspecifically increased by the administration of cytostatic drugs. Finally, sCD27 was also elevated in the 4 patients studied with primary central nervous system lymphoma (PCNSL). CSF sCD27 is a promising tumor marker in patients with either meningeal localization of lymphoid malignancies or PCNSL, and can be useful in the differential diagnosis of CNS involvement by either lymphoid malignancies or solid tumors. PMID- 8634447 TI - Effects of novel retinoid X receptor-selective ligands on myeloid leukemia differentiation and proliferation in vitro. AB - The biologic effects of retinoids such as all-trans-retinoic acid (ATRA) and 9 cis-retinoic acid on proliferation and differentiation of hematopoietic cells are mediated by binding and activating two distinct families of transcription factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RARs require heterodimerization with RXRs; in addition, RXRs can form homodimers, which can bind to DNA response elements that are either distinct or the same as those bound by the RAR/RXR heterodimers. Therefore, the two retinoid pathways provide sequences that are specific for effective DNA binding and activation of target genes. We have developed several series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers and RAR/RXR heterodimers. We show here that SR11236 and SR11246, which are RXR-selective analogs, had little ability to inhibit clonal growth and induce differentiation of leukemic cells (HL-60 cells and fresh acute myeloid leukemia cells). However, SR11249, SR11256, and LGD1069, which activated both RXR/RXR homodimers and RAR/RXR heterodimers, could inhibit clonal growth and induce differentiation of HL-60 cells as well as leukemic cells from patients, including those with acute promyelocytic leukemia (APL). This is similar to results observed with RAR/RXR specific ligands. Interestingly, the combination of ATRA and either SR11249, SR11256, or LGD1069 showed synergistic effects in inducing differentiation of HL 60 cells. A retinoid (SR11238) with strong anti-AP-1 activity that did not activate the RARs and RXRs for gene transcription from the response element TREpal was inactive in our assay systems, suggesting that the antiproliferative effects of retinoids on leukemic cells is not mediated by inhibiting the AP-1 pathway. We conclude that the RAR/RXR pathway is more important than RXR/RXR pathway for differentiation and proliferation of acute myeloid leukemic cells, and certain retinoids or combination of retinoids with both RAR and RXR specificities may synergistically enhance the differentiation activity of ATRA, which may be relevant in several clinical situations. PMID- 8634449 TI - Activation of protein kinase Cdelta in human myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine. AB - Treatment of human myeloid leukemia cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with induction of protein kinase activity and early response gene expression. The present studies in ara-C-treated U-937 cells extend these findings by demonstrating activation of a protein kinase that phosphorylates myelin basic protein (MBP). Purification by sequential ion exchange chromatography and gel filtration supports the detection of a 40-kD MBP kinase. Substrate and inhibitor studies further support a pattern similar to that of protein kinase C (PKC) isozymes. Results of N-terminal amino acid sequencing and immunoblot analysis demonstrate detection of a 40-kD catalytic fragment of PKCdelta. The results also demonstrate the activation and cleavage of PKCdelta (1) is inhibited by expression of antiapoptotic proteins, and (2) is induced by camptothecin (CAM) and mitomycin C (MMC). These findings support proteolytic activation of PKCdelta in the cellular response to ara-C and other DNA-damaging agents. PMID- 8634450 TI - Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia. AB - Cytofluorimetric detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at the time of diagnosis in 158 patients with acute myeloid leukemia using the C219 monoclonal antibody (MoAb). In 108 of these cases the JSB1 MoAb was also tested. An improved histogram subtraction analysis, based on curve fitting and statistical test was applied to distinguish antigen-positive from antigen-negative cells. A marker was considered positive when more than 20% of the cells were stained. At onset, P-170 was detected in 43% of cases with C219 and in 73% of cases with JSB1. There was a strict correlation between C219 and JSB1 positivity, as all C219+ cases were also positive for JSB1 MoAb (P < .001). No relationship was found between sex, age, organomegaly, and MDR phenotype. Significant correlation was found between CD7 and both C219 and JSB1 expression (P < .001 and .001, respectively). C219-negative phenotype was more often associated with a normal karyotype (24 of 55 with P = .030). Rhodamine 123 (Rh123) staining and flow cytometry analysis showed a significantly decreased mean fluorescence in 51 C219+ and 38 JSB1+ patients compared to 42 MDR negative ones (P < .001). The rate of first complete remission (CR) differed both between C219+ and C219- cases and between JSB+ and JSB- ones (30.9% v 71.1% and 35.4% v 93.1%, respectively, P < .001). Of the 21 C219+ patients who had yielded a first CR, 19 (90.4%) relapsed, compared with 28 of 64 (43.7%) C219- patients (P < .001). Of the 28 JSB1+ patients in first CR, 17 (60.7%) relapsed relative to 8 (29.6%) of 27 JSBI- ones (P = .021). A higher rate of relapses among MDR+ compared with MDR- patients was observed both for C219 and JSB1 MoAbs taken separately (C219 80% v 44%; JSB1 52% v 27%), with no relationship to age. The survival rates (Kaplan-Meyer method) were significantly shorter both in C219+ patients and in JSB1+ cases (P < .001). Disease-free survival curves followed this same trend. The combination (C219- JSB1+) identified a subset of patients with an intermediate outcome compared to C219 positive cases. The prognostic value of both markers (C219 and JSB1) was confirmed in multivariate analysis. These results suggest that the assessment of MDR phenotype by flow cytometry may be an important predictor of treatment outcome. PMID- 8634452 TI - Macrophage inflammatory protein-1alpha is induced by human immunodeficiency virus infection of monocyte-derived macrophages. AB - Disparate findings have been reported as to whether human immunodeficiency virus (HIV) affects cytokine production by macrophages (MA). We investigated production of different cytokines and of macrophage inflammatory protein (MIP)-1alpha by HIV 1Ba-L- or HIV-1Ada-infected blood-derived MA. Relative to controls, only MIP 1alpha levels increased twofold to > 10-fold in supernatants 2 to 3 weeks postinfection (PI), at the time of maximum virus production; levels of the other chemokines (RANTES, interleukin (IL)-8) and cytokines (IL-1alpha, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1) investigated were not affected. MIP-1alpha mRNA signal assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) was, however, only occasionally greater in cells from infected cultures relative to controls. MIP-1alpha levels in supernatants remained in the same range as in control cultures when more than 10 mmol/L Zidovudine was added 24 hours PI, which indicates involvement of virus replication in the effect. Anti-MIP-1alpha antibody labeling identified a 10% to 25% subset of MA, strongly expressing HLA-DR and CD4, and also stained by anti-IL 6 and anti-TNF-alpha antibodies. Two weeks PI, dual staining showed that the majority of the 5% to 20% cells that were p24+ belonged to the MIP-1alpha+ population, which may define a MA subset capable to better sustain HIV replication. MIP-1alpha induced by HIV replication in MA might play a role in the pathophysiology of HIV infection; in impaired hematopoiesis; or as a CD4+ and CD8+ lymphocyte chemoattractant, by recruiting either or both HIV-susceptible and cytotoxic T lymphocytes to virus replication sites. PMID- 8634451 TI - Cloning of murine gp91phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line. AB - The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full-length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function. PMID- 8634453 TI - Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. AB - CD36 is an 88-kD integral membrane glycoprotein expressed on monocytes, platelets, and certain microvascular endothelium serving distinct cellular functions both as an adhesive receptor for thrombospondin, collagen, and Plasmodium falciparum-infected erythrocytes, and as a scavenger receptor for oxidized low-density lipoprotein and apoptotic neutrophils. In this study, we examined the expression of CD36 during in vitro differentiation of peripheral blood monocytes into culture-derived macrophages. Steady-state mRNA levels of CD36 showed a transient eightfold increase during monocyte-to-macrophage differentiation, peaking at the early macrophage stage (days 3 or 4 in culture), following a gradual decrease back to baseline levels by the mature macrophage stage (days 7 or 8 in culture). Immunoblotting with monoclonal antibodies to CD36 supported this transient, yet significant (8- to 10-fold) increase in total protein levels of CD36. The increased CD36 protein was observed at the plasma membrane, whereas an intracellular pool of CD36 was also detected from day 2 to day 6 in culture through indirect immunofluorescence. A concomitant twofold increase in the cells' ability to bind 125I-thrombospondin at the early macrophage stage (day 4) verified the functional competency of the plasma membrane localized CD36, and supported the presence of an intracellular pool of CD36. The in vitro differentiated macrophages as well as alveolar macrophages remained responsive to macrophage colony-stimulating factor (M-CSF), a known transcriptional regulator of monocyte CD36. The M-CSF-induced macrophages resulted in enhanced foam cell formation, which was inhibitable with monoclonal antibodies to CD36. Thus, the transient expression of CD36 during monocyte-to macrophage differentiation, and the ability of M-CSF to maintain macrophage CD36 at elevated levels, may serve as a critical process in dictating the functional activity of CD36 during inflammatory responses and atherogenesis. PMID- 8634454 TI - Chemoattractant-induced changes in surface expression and redistribution of a functional ligand for P-selectin on neutrophils. AB - Adhesion between platelets and neutrophils is mediated through the interaction of P-selectin on activated platelets with a carbohydrate-containing structure on neutrophils, and occurs under both static and shear conditions. Recent studies using flow chambers have shown that neutrophils become activated after binding to surface-adherent platelets expressing P-selectin. The objective of the present study was to investigate the effect of such activation on the interactions of platelet P-selectin with its ligand on neutrophils. Flow cytometric analyses using P-selectin chimeras revealed that activation induced a rapid and marked reduction in chimera binding, with levels of binding decreased by 71% after 15 minutes of stimulation with the chemotactic agent, FMLP. Using a visual assay of platelet-neutrophil rosetting, we showed that the P-selectin ligand was translocated and clustered at the uropod of neutrophils following the shape changes and polarization induced by chemotactic stimulation. Activated neutrophils bound to surface-adherent platelets also displayed the clustering of P-selectin ligand at the uropod, and these neutrophils detached from the platelets when a shear stress (2 dynes/cm2) was applied through the adhesion chamber. These results indicate that chemotactic stimulation of neutrophils induces changes in the surface expression and distribution of a biologically relevant ligand for P-selectin, and that these changes might influence the adhesive interactions occurring between neutrophils and activated platelets. PMID- 8634455 TI - Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3. AB - Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin-inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells. PMID- 8634456 TI - Abnormal assembly of membrane proteins in erythroid progenitors of patients with beta-thalassemia major. AB - The life threatening anemia in beta-thalassemia major (Cooley's anemia) is characterized by profound intramedullary lysis, the cause of which is incompletely understood. Using marrow obtained from beta thalassemia major patients undergoing allogeneic bone marrow transplantation in Pesaro Italy, it became possible to directly study the mechanism of the intramedullary hemolysis. Based on our previous studies, we hypothesized that the unmatched alpha globin chains would interfere with normal assembly of erythroid precursor membrane proteins. Patient and control erythroid precursors were reacted with monospecific polyclonal rabbit antibodies directed against spectrin, band 3, and band 4.1 and with a monoclonal anti-alpha globin chain antibody. Using laser confocal fluorescence microscopy, normal erythroid precursors show no alpha globin chain accumulation and exhibited uniformly smooth rim fluorescence of the three membrane proteins. In some thalassemic precursors, spectrin appeared to interact with large alpha globin accumulations, and in many of these cells the spectin appeared clumped and discontinuous. Band 4.1 interacted strongly with accumulations of alpha globin in thalassemic precursors to produce bizarrely clumped zones of abnormal band 4.1 distribution. Band 3 was incorporated smoothly into thalassemic erythroblast membranes. However, the proerythroblasts and basophilic erythroblasts were significantly deficient in band 3. Thus, accumulations of alpha globin in beta-thalassemia major colocalized with and disrupt band 4.1 and spectrin assembly into the membrane. The cause of deficient band 3 incorporation into thalassemic proerythroblast membranes remains unknown. These profound membrane alterations would likely contribute to the intramedullary lysis seen in Cooley's anemia. PMID- 8634457 TI - Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution. AB - To test the hypothesis that variations in H- and L-subunit composition in the ferritin shell affect intracellular iron metabolism, we established stable transfectants of mouse erythroleukemia cells overexpressing the H-ferritin subunit. Analyses were performed on individual clones of transfected cells induced to differentiate with hexamethylenbisacetamide (HMBA). The results showed that there was a reduction in the amount of hemoglobin produced, in inverse relationship with the level of H-subunit overexpression. Incorporation of [2 14C]glycine into heme was reduced by 20% t0 30% in the clones overexpressing H ferritin subunit compared with control clone. However, the reduction in hemoglobin production was not reversed by addition of heme precursors (delta aminolevulinic acid or iron) or by hemin itself. A reduced accumulation of beta globin mRNA was also observed, which could account for the impaired hemoglobin synthesis. Furthermore, synthesis of the endogenous L-ferritin subunit was greatly repressed. Gel retardation assays performed on cytoplasmic extracts of transfected cells using an iron-responsive element (IRE) as a probe revealed that in overexpressing cells, the iron-regulatory protein (IRP) had a conformation with a high RNA-binding affinity, thus leading to translational repression of the endogenous L-ferritin synthesis. These data suggest that an increased formation of H-rich isoferritins leads to a rapid chelation of the regulatory iron pool. While the mechanism underlying the reduction in beta-globin mRNA remains to be elucidated, this study provides direct evidence for the role of IRP-mediated regulation of ferritin expression in erythroid cell metabolism. PMID- 8634459 TI - Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency. AB - Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients. PMID- 8634458 TI - Nucleic acid composition of bone marrow mononuclear cells in cobalamin deficiency. AB - Recent studies using anion exclusion chromatography have suggested that uracil is misincorporated into the DNA of patients with megaloblastic anemia to levels detectable by nonradioactive methods. We have investigated the nucleotide composition of DNA from the bone marrow mononuclear cells of eight patients with cobalamin deficiency and compared this with that found in normal subjects. The median level of uracil in the megaloblastic group was 0.082 mol% of cytosine (approx. 0.02 mol% of all bases in DNA), which was similar to that found in the control group (median 0.085 mol% of cytosine) and may be attributable, at least in part, to artefactual deamination of deoxycytidine monophosphate during the DNA hydrolysis. Our findings give no support for the view that, by overwhelming the uracil N-glycosidase mechanism, the degree of uracil misincorporation in megaloblastic anemia is sufficient to increase the steady state level of uracil in the DNA by amounts detectable by nonradioactive methods. Using high performance liquid chromatography, we have also demonstrated normal levels of methylcytosine in the DNA of megaloblastic subjects. PMID- 8634460 TI - Hemoglobin level is linked to growth hormone-dependent proteins in short children. AB - Erythropoiesis was investigated in 32 children wih short stature and in eight children with skeletal dysplasia by studying blood hemoglobin in relation to growth and to serum concentrations of insulin-like growth factor I (IGF-I), IGF binding protein-3 (IGFBP-3), and erythropoietin (EPO) before, during, and after 12 months of recombinant human growth hormone (GH) treatment. Blood hemoglobin concentration was positively correlated with relative body height and with serum IGF-I and IGFBP-3 levels (P = .001 to .02), but not with the concentrations of EPO. The normal age-dependency of hemoglobin was lacking. Hemoglobin levels and their responses to GH treatment were similar in the patients with GH deficiency and those with normal GH secretion. Treatment with GH accelerated growth and elevated the concentrations of hemoglobin, IGF-I, and IGFBP-3. In the eight patients with skeletal dysplasia, body mass increased similarly, but gain in height was less than in the other patients, and the increase in hemoglobin was markedly pronounced. In this group, the correlations between hemoglobin, IGF-I, and IGFBP-3 were extremely close (r = 0.80 to 0.85, P = .031 to .008). These findings are in accord with earlier observations from in vitro and animal studies, and suggest that the GH-IGF axis is involved in the physiologic elevation of hemoglobin levels during childhood. PMID- 8634462 TI - When and where is factor XI activated by thrombin? PMID- 8634463 TI - Intrinsic coagulation, thrombosis, and bleeding. PMID- 8634461 TI - Marrow transplantation for patients with thalassemia: results in class 3 patients. AB - Thalassemia patients can be categorized as class 1 (minimal liver damage and iron overload), class 3 (extensive liver damage from iron overload), and class 2 (intermediate). These categories are prognostic for treatment outcome after marrow transplantation. Class 3 patients have more transplant-related mortality than other patients. This study examines transplantation outcome for class 3 patients. Records were reviewed of 215 patients in class 3 who received transplants in Pesaro from HLA-identical related donors between May 1, 1984 and May 1, 1994. The influence of pretransplant, peritransplant, and posttransplant variables on survival, relapse, and transplant-related mortality was examined by product-limit and proportional-hazards multivariate analysis. Age and conditioning regimen were influential on survival, and regimens with less than 200 mg/kg cyclosporine (CY) were associated with 5-year survival probabilities of .74 and .63 patients younger than 17 years and older patients, respectively. Transfusion history and regimen were influential on rejection with 5 year probabilities of .53 and .24 in patients who received less than or greater than 100 red blood cell transfusions before transplantation and regimens containing less than 200 mg/kg CY. Results of transplantation for patients with advanced thalassemia treatment have improved with the introduction of conditioning regimens with less CY. This has been associated with an increase in rejection (particularly in patients who have received < 100 red blood cell transfusions before transplant). Efforts at reducing the rejection rate by modifying the conditioning regimen should be concentrated on younger patients who have received a small number of transfusions. Patients with thalassemia who have HLA-identical family members should be transplanted before they are in class 3. PMID- 8634464 TI - Detection of the factor V Leiden mutation in a nonselected black population. PMID- 8634465 TI - Benchmarking: a data-oriented look at improving health care performance. AB - Benchmarking uses measures of comparative performance to develop an understanding of what is possible and how others have achieved higher levels of performance. Companies measure each other's performance, identify the best performer out of a group, and then seek to identify and understand the practices that can improve both clinical and administrative operations. When a powerful consortium of Cincinnati businesses began addressing ways to lower the cost of employee care in a project known as the Iameter study, the University of Cincinnati Hospital joined the effort to identify and improve major cost factors. One aspect highlighted by this review is the length of stay in the neonatal intensive care unit (NICU). The results of the study were incorporated into an NICU early discharge program, which has had a number of positive effects in both patient care quality and cost containment. PMID- 8634466 TI - Benchmarks and performance indicators: two tools for evaluating organizational results and continuous quality improvement efforts. AB - Benchmarks are tools that can be compared across companies and industries to measure process output. The key to benchmarking is understanding the composition of the benchmark and whether the benchmarks consist of homogeneous groupings. Performance measures expand the concept of benchmarking and cross organizational boundaries to include factors that are strategically important to organizational success. Incorporating performance measures into a balanced score card will provide a comprehensive tool to evaluate organizational results. PMID- 8634467 TI - Tracking skin integrity: a template for hospital and vendor collaboration. AB - Collaborative endeavors are particularly attractive in a time when resources are under extensive demand, but for organizations to be successful at collaboration, many shared values and qualities are necessary. In 1992, a hospital and a vendor of therapeutic bed surfaces entered into a collaborative partnership to test clinical indicator ability to track skin care outcomes. The vendor contributed major funding for the project, a national database from which clinical indicators on pressure ulcers were determined, and data processing and analysis. The hospital provided project coordination, clinical knowledge, data collectors, and access to patients. The article describes the process, problems, and benefits of collaboration and underscores the need for a trusting psychological climate, strong mutual interest in the project, clearly stated goals with resources to meet them, collaborator consideration of each other as peers, and skill in tension management if collaboration is to be successful. PMID- 8634468 TI - Consumer-based research: using focus groups as a method for evaluating quality of care. AB - In response to the consumer movement, health care organizations have become interested in obtaining feedback through face-to-face contacts with the people using their products and services. Focus groups are one qualitative research method of obtaining rich information within a social context. The article discusses the purpose, design, and methodology of focus groups, and a case example is given. Focus groups can add to existing knowledge about how well services are being delivered or what new products might be developed in the future. PMID- 8634469 TI - A team approach for risk assessment, prevention, and treatment of pressure ulcers in nursing home patients. AB - The article describes a unique decentralized wound care program instituted in a nursing home care unit of a Veterans Affairs hospital. This decentralized, interdisciplinary approach to wound care has been effective in reducing the incidence and prevalence of pressure ulcers in a nursing home population over the last 2 years. Prevention was a major focus of the program. The Braden scale was used to identify patients at risk for pressure ulcer formation; proper measures were introduced to reduce identified risk. This collaborative team effort led to improved patient care and decreased incidence and prevalence of pressure ulcers in this population. PMID- 8634470 TI - Stressful nurses: the effect on patient outcomes. AB - The article describes a study that measured, over a 3-month period, staffing problems, including turnover rates; nurse incidents, including absenteeism, back injuries, and needle sticks; and patient incidents, including falls and medication errors. The self-reported stress of the nurses caring for these patients was recorded over the same 3-month period. Data showed that a relatively strong relationship exists between a hospital unit's Stress Continuum Scale (SCS) and the occurrence of patient incidents. The relationship between the SCS and personal incidents and nurse injuries appears weak, as does the relationship between staff turnover and stress. Lagging staff turnover by 1 month resulted in a moderate association with the SCS, however. PMID- 8634471 TI - Quality management in a school-based nursing center: program development and implementation. AB - As community-based health care services continue to grow, quality management programs for new, nontraditional settings are being developed. The article describes a quality management program implemented in a nursing center in an urban, multicultural high school. The three components of the program- formulation of the quality management plan, the development of written structure, process, and outcome standards, and the development of a monitoring and evaluation plan--are summarized. The application of the principles and practices of quality management to a school-based nursing center has provided challenges, opportunities, and rewards for all involved. PMID- 8634472 TI - Describing and analyzing constipation in acute care. AB - Many patients in acute care hospitals experience constipation, yet the literature on constipation focuses on long-term care and does not provide tools for describing and analyzing bowel management from the perspective of health care professionals or patients. The article describes the development of a bowel management task force at one acute care hospital and the initial steps taken to improve clinical quality in this area. A multifaceted approach was used to collect baseline data on practice, expectations, and problems related to bowel management. Valuable data were obtained from both patients and health care providers that have provided direction for improving clinical outcomes and patient satisfaction. PMID- 8634473 TI - Benchmarking: improving outcomes for the congestive heart failure population. AB - The benchmarking process has been used extensively to evaluate and improve performance in business and industry. There is currently increasing interest in utilizing this process in the health care field to maximize efficiency and improve patient outcomes. The article defines benchmarking in health care and lists characteristics of the benchmarking process. The process of conducting a clinical benchmarking project aimed at improving outcomes for patients with congestive heart failure is described and determined to be an effective means of reducing costs and improving both patient outcomes and quality of care. PMID- 8634474 TI - A patient care team approach to multicultural patient care issues. AB - As the demographics of the United States continue to change to include more foreign nationals, nursing professionals will need to become increasingly aware of multicultural issues. Developing a greater cultural awareness of a particular client population can aid health care providers in improved caregiving. An interdisciplinary multicultural patient care team (IMPCT) was formed to enlarge the scope of nursing practice by educating nurses and other support staff about culturally diverse groups. The purpose of the IMPCT was to promote high-quality care to culturally diverse patients and their families. An IMPCT led by an advanced practice nurse would assist nursing staff in developing cultural awareness and sensitivity and would enhance nurses' ability to provide a holistic approach to nursing care. PMID- 8634475 TI - Effect of disruption of the enniatin synthetase gene on the virulence of Fusarium avenaceum. AB - Production of the phytotoxic compound enniatin has been proposed to play a role during the infection process of plants by enniatin-synthesizing Fusarium species. Enniatins are cyclohexadepsipeptides synthesized by the multifunctional enzyme enniatin synthetase. To test the hypothesis that enniatin contributes to pathogenicity, enniatin-nonproducing mutants were constructed by gene disruption of the enniatin synthetase gene of a virulent Fusarium avenaceum strain. Four independent enniatin nonproducing mutants were characterized that did not express enniatin synthetase, as proved by RNA and protein blot analysis. The virulence on potato tuber tissue of the enniatin-nonproducing strains was significantly reduced compared with the virulence of the parent strain and three enniatin producing transformants. Therefore, we conclude that enniatin production contributes to the virulence of Fusarium avenaceum. PMID- 8634477 TI - Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system. AB - The specific recognition of elicitors produced by plant pathogenic bacteria carrying avirulence (avr) genes is postulated to initiate cellular defense responses in plants expressing corresponding resistance genes. The biochemical functions of most avr genes, however, are not known. A heterologous system was developed to phenotypically express Pseudomonas syringae avr genes in Escherichia coli cells that required the P. syringae hrp cluster. E. coli MC4100 transformants carrying the plasmic-borne P. syringae pv. syringae Pss61 hrp cluster and p. syringae pv. glycinea avrB expressed from a triple lacUV5 promoter gained the ability to elicit the hypersensitive response in soybean cultivars expressing Rpg1 and in an Arabidopsis thaliana accession expressing RPM1. Inactivation of energy transducing or outer membrane components of the hrp encoded secretion system blocked phenotypic expression expression of avrB in E. coli, but deletions abolishing harpinPSS production had little effect on the production of the AvrB phenotype by the E. coli transformants. Phenotypic expression of avrA, AvrPto, avrRpm1, avrRpt2, and avrPph3 in E. coli was also shown to require the hrp cluster. The results indicate that generation of the Avr phenotype in P. syringae strains is specifically dependent on the secretion activities of the hrp cluster. PMID- 8634476 TI - Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development. AB - We report the identification of new molecular markers associated with different stages of Rhizobium-induced nodule development in the legume Medicago truncatula. A cDNA library was constructed from pre-nitrogen-fixing M. Truncatula nodules, and differentially screened with a polymerase chain reaction-amplified subtracted probe. Twenty-nine new families of nodulin cDNA clones, designated MtN1 to MtN29, were thus identified in addition to clones for several known nodulins. All MtN genes were shown by Northern (RNA) hybridization analysis to be induced during nodulation, some of them well before nodule emergence. The MtN genes were classified into three groups depending on their expression kinetics. The expression of three MtN genes showed a limited induction by Nod factors purified from Rhizobium meliloti. Homologies with a variety of proteins were found for the deduced amino acid sequences of 10 of the MtN genes. PMID- 8634478 TI - Cloning and characterization of tek, the gene encoding the major extracellular protein of Pseudomonas solanacearum. AB - Susceptible plants infected by Pseudomonas solanacearum usually will, largely due to extracellular proteins (EXPs) and the high-molecular-mass extracellular polysaccharide (EPS I) this pathogen produces. Circumstantial evidence suggested that a 28-kDa protein, the single most abundant EXP made by P. solanacearum in culture, is associated with production of EPS I, and thus might have a role in pathogenesis. The 28-kDa EXP was purified and, based on its N-terminal amino acid sequence, an oligonucleotide mixture was made and used as a hybridization probe to clone the gene encoding it. DNA sequence analysis suggested that the coding sequence for the 28-kDa EXP is within a gene, designated tek, that encodes a 58 kDa membrane-associated precursor protein that is processed by signal peptidase II during export. Analysis of radiolabeled polypeptides expressed from tek confirmed that it encodes a 58-kDa precursor protein, which is exported out of the cells as a 55-kDa preprotein and processed extracellularly to release the very basic 28-kDa EXP from its C terminus. The position, transcriptional direction, and regulated expression of tek suggest that it is cotranscribed with xpsR, a gene essential for regulating biosynthesis of EPS I, and reinforces the association of the 28-kDa EXP with virulence. However, since P. solanacearum mutants lacking only the 28-kDa EXP produced wild-type amounts of EPS I and were fully virulent, the function of this protein remains unclear. PMID- 8634479 TI - Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP1. AB - Three extracellular serine proteases (Alp1a, Alp1b, Alp2) from Cochliobolus carbonum were purified and characterized. Of eight carbon/protein substrates tested, total protease activity was highest when the fungus was grown on medium containing collagen. Alp1a and Alp1b are members of the trypsin family (EC 3.4.21.4), and Alp2 is a member of the subtilisin family (EC 3.4.21.62). Alp1a, Alp1b, and Alp2 have monomer molecular masses of 25, 30, and 38 kDa respectively. Alp1b is glycosylated, whereas Alp1a is not. The gene encoding Alp1a, ALP1, was isolated using PCR primers based on two amino acid sequences: One obtained directly from the N-terminus of Alp1a and another that is highly conserved in other trypsins. The transcriptional start site was determined using RACE and the intron structure and polyadenylation site were determined from a cDNA clone. An internal fragment of ALP1 was used to create Alp1a null mutants by transformation mediated gene disruption. Total protease activity in the mutants was reduced by 35% to 45%. By chromatographic analysis, the mutants had lost two peaks of UV absorption and the two protease activities corresponding to Alp1a and Alp1b, which, together with the biochemical data, indicates that Alp1a and Alp1b are products of the same gene. The in vitro growth and diseases phenotypes of the ALP1 mutants were distinguishable from the wild-type strain; therefore, ALP1 is not by itself required for pathogenicity. PMID- 8634480 TI - Presence of Ty1-copia group retrotransposon sequences in the potato late blight pathogen Phytophthora infestans. AB - Multiple copies of retrotransposon reverse transcriptase coding sequences were identified in Phytophthora infestans by polymerase chain reaction (PCR) amplification using degenerate primers. The P. infestans sequences belong to the Ty1-copia superfamily, and putative elements from different P. infestans isolates show restriction site polymorphisms. Some contain complete open reading frames while others do not, indicating the presence of potentially active as well as inactive elements. PMID- 8634481 TI - Translocation and exudation of tumor metabolites in crown galled plants. AB - Crown gall tumors are induced on susceptible plants by pathogenic strains of Agrobacterium. These neoplastic plants cells produce metabolites, called opines, which provide a source of nutrients to colonizing agrobacteria. Opine production previously has been shown to influence microbial communities in the immediate vicinity of the tumor. We have obtained evidence for opine translocation to and exudation from distal uninfected regions of the plant host. Grafted plants made from an opine-producing transgenic scion with a wild-type stock, or with a wild type scion and an opine-producing stock accumulate opines in the wild-type portions of the plant. Moreover, opines were detected in root, stem, and leaf tissues of nontransgenic plants on which stem crown galls had been induced by pathogenic strains of Agrobacterium. These plants exuded opines from their roots as a component of their root exudates. Translocation of opines from the tumor to other parts of the plant, and their exudation from roots, indicates that these biologically active compounds are available to opine-catabolizing microbes that have not induced the tumors but are present in the rhizosphere or on portions of the plant distant from the site of the gall. PMID- 8634482 TI - The open ring. A new imaging sign in demyelinating disease. AB - Because demyelinating disease of the brain occasionally presents with large ring enhancing lesions on computed tomography (CT) scans and magnetic resonance images (MRIs), the authors sought to determine whether the ring pattern differed from that found in other common brain lesions with ring enhancement. Published MRI and CT scans of patients with adrenoleukodystrophy (23), and multiple sclerosis or similar demyelinating disorders (21), as well as a variety of tumors (44) and infections (44) matched to the demyelinating lesions by year of publication, in which ring enhancement was evident, were photographed. Photographs without diagnostic identification were presented randomly to two independent observers. The observers rated the contrast enhancement pattern as (1) open ring, with enhancement in the border of the lesion abutting the white matter; (2) closed ring; or (3) uncertain. For all diagnostically certain cases (n = 112), inter rater agreement was excellent (kappa = 0.75). As an average of the two reviewers, scans for 11 of 132 cases were read as uncertain; 89% of adrenoleukodystrophy cases, 41% of the multiple sclerosis cases, 3% of tumors, and 9% of infections were classified as having the open-ring pattern. Overall, 66% of demyelinating lesions had an open-ring pattern compared with 7% of the non-demyelinating lesions (chi2 = 41.2, p < 0.0001). An open-ring pattern of enhancement is more likely to be associated with demyelinating lesions than with nondemyelinating lesions. PMID- 8634483 TI - Quantified volumes of temporal lobe structures in patients with epilepsy. AB - The T1-weighted volumetric magnetic resonance images of 31 patients with intractable temporal lobe epilepsy, and 13 control subjects matched for age and sex, were subjected to semiautomated threshold analysis. The method used proved to be relatively fast and reliable. An index of temporal lobe interhemispheric asymmetry was extracted by thresholding high-signal (white matter) pixels. Patients had significantly more asymmetrical indices for white matter and hippocampal volumes that did control subjects, and the two indices were significantly correlated, providing evidence for the validity of the white matter index. Differences in both indices were consistent with decreased tissue on the side of the focus. In classification analyses a combination of these two indices correctly predicted the side of focus at a greater rate than did either used alone. Findings provide support for the hypothesis that seizure activity is associated with atrophy in both mesial and lateral temporal lobe structures. PMID- 8634484 TI - Magnetic resonance imaging of syrinx associated with intramedullary metastases and leptomeningeal disease. AB - Intramedullary spinal cord metastasis with an associated syrinx diagnosed by magnetic resonance imaging (MR) is described. The patient had documented simultaneous leptomeningeal spread of malignant cells and intramedullary spinal cord metastasis with hyalinized blood vessels, venous dilatation, and cavitation detected by autopsy. Metastasis to the spinal cord is unusual, but well described. Syrinx associated with intramedullary spinal cord metastasis has been detected rarely. MRI of syrinx and intramedullary spinal cord metastasis, and the possible pathogenesis of these lesions are discussed. PMID- 8634486 TI - Computed tomography-negative acute thalamic hematoma. AB - A case of acute thalamic hematoma not detected by computed tomography (CT) but unequivocally diagnosed by magnetic resonance imaging (MRI) is presented. A 79 year-old woman presented with acute left hemiparesis. The results of CT obtained on admission as well as on the seventh hospital day were negative for a hematoma. By contrast, serial MRIs exhibited chronological changes in the relaxation properties characteristics of acute hematoma in the thalamus. The case illustrates that contrary to widespread practice, CT cannot absolutely be relied on for the detection of acute intracerebral hematoma. PMID- 8634485 TI - Evaluating brain death with positron emission tomography: case report on dynamic imaging of 18F-fluorodeoxyglucose activity after intravenous bolus injection. AB - A dynamic positron emission tomography (PET) study of the head was performed over 1 hour after the intravenous bolus administration of 18F-fluorodeoxyglucose (18F FDG) to a 18-year-old patient with the clinical diagnosis of brain death. This dynamic PET study was performed on the seventh day after a severe posttraumatic closed-head injury. No intracerebral uptake or retention of tracer was noted, consistent with a diffuse absence of brain metabolism. A small amount of tracer was noted to slowly rise over time within the sagittal sinus, indicating that visualization of sagittal sinuses on technetium 99m-diethylene triaminepentaacetic acid planar images could provide a falsely negative scintigraphic evaluation for the presence of brain death. It is concluded that PET FDG imaging may be a useful technique in evaluating patients for brain death. PMID- 8634488 TI - Extracranial intravascular vasodilatory response to acetazolamide and magnetic resonance angiography. AB - Cerebral vasodilatory testing provides an important measure of both the hemodynamic significance of arterial occlusive disease and the adequacy of collateral pathways. This study measured the extracranial intravascular volume flow rate response to acetazolamide using phase-contrast magnetic resonance angiography. From 10 patients with unilateral carotid transient ischemic attacks (TIAs), a total of 18 extracranial carotid arteries (10 symptomatic, 8 asymptomatic, 2 occluded) and 19 extracranial vertebral arteries were studied. Patients were free of large-vessel intracranial stenoses, evident areas of ischemic infarction, evident areas of nonspecific white matter change, and hemodynamic or low-flow induction of TIA symptomatology. Asymptomatic carotid volume flow rates rose from 151 +/- 19 (standard error of mean) to 220 +/- 26 ml/min while symptomatic flow rates rose from 106 +/- 22 to 145 +/- 25 ml/min. Dominant vertebral volume flow rates rose from 128 +/- 23 to 160 +/- 22 ml/min while nondominant rates rose from 40 +/- 12 to 61 +/- 15 ml/min. Carotid volume flow rates were inversely proportional to percent stenosis for both baseline (r = 0.51, p < 0.02) and acetazolamide (r = 0.81, p < 0.001) data. Baseline-plus acetazolamide volume flow rate techniques safely measure intravascular vasodilatory responses. Intracranial measurement techniques are being developed to further study cerebrovascular reserve using phase-contrast magnetic resonance angiography. PMID- 8634487 TI - Subacute sclerosing panencephalitis and acquired immunodeficiency syndrome: role of electroencephalography and magnetic resonance imaging. AB - Subacute sclerosing panencephalitis (SSPE) had largely disappeared from the United States because of nearly universal measles vaccination, but it has reemerged in children infected with human immunodeficiency virus (HIV). Two children with SSPE are described. The first was HIV positive and presented with seizures and encephalopathy at the age of 21 months. The second developed myoclonus and dementia at age 4 years; she was not infected with HIV, but her mother had acquired immunodeficiency syndrome. Magnetic resonance imaging findings were nonspecific and could have been compatible with HIV encephalopathy. Electroencephalography was characteristic of SSPE, showing high-voltage, periodic slow-wave complexes and background slowing. The diagnosis of SSPE was confirmed by brain biopsy or high measles antibody titers in the cerebrospinal fluid. PMID- 8634489 TI - Preclinical evaluation of SPECT imaging with 131I-labeled monoclonal antibody SZ39 in nude mice bearing human glioma xenografts. AB - The imaging characteristics of monoclonal antibody SZ39 against glioma were evaluated in glioma-bearing nude mice. Monoclonal antibody SZ39 is a murine IgG2a that reacts with a glycoprotein epitope (molecular weight 180,000), a human glioma-associated membrane antigen. Monoclonal antibody SZ39 was labeled with 131I using a modified chloramine T method. Each glioma-bearing nude mouse was given 50 microCi/40 micrograms of the experimental agent, 131I-labeled monoclonal antibody, or 50 microCi/46 micrograms of a control agent, 131I-labeled monoclonal antibody C50, an antibody against colon cancer. Single-photon emission computed tomography (SPECT) was performed every 24 hours in the first week after administration. Glioma-bearing nude mice were killed in groups of 3 at 24 hours and daily up to 72 hours. The ratio of radioactivity uptake in glioma to normal organs was calculated. After administration of the labeled SZ39, glioma was visualized with SPECT on days 1 to 7, particularly at 72 hours. There was no accumulation of radioactivity in glioma with the labeled C50 antibody. All glioma organ ratios increased with time. At 72 hours, the ratio of glioma to brain was 22.46 and of the other organs was 2.64 on average. SZ39 had a relatively low endocytosis rate and was favorable for 131I labeling. These characteristics were helpful to reduce free 131I in the blood and reduce the uptake by other organs. The results suggest that 131I-labeled SZ39 selectively accumulates in glioma, representing a potential strategy for SPECT imaging of these lesions. PMID- 8634490 TI - Intravascular ultrasound imaging of human cerebral arteries. AB - The aim of this study was to assess the feasibility of imaging cerebral arteries in vitro with intravascular ultrasound and to establish a correlation between echographic images and corresponding histological architecture. Intravascular ultrasound imaging was performed using a 30-MHz, 4.3F ultrasound probe. Twenty two arterial segments were obtained at autopsy from 6 patients and were imaged fresh. Arteries were then processed for histological examination and comparisons were made between echographic and histological findings. The correlation between luminal area measurements as determined histologically and by intravascular ultrasound was tested by linear regression analysis. Intravascular ultrasound demonstrated a three-layered appearance in normal cerebral arteries but not in those affected by severe atherosclerosis. Overall, ultrasound correctly identified the presence of a plaque in 83% of patients. Intravascular ultrasound sensitivity and specificity, respectively, were 100 and 80% for calcium deposits and 83 and 75% for fibrous tissue. Intravascular ultrasound and histological measurements correlated well for the determination of luminal area (r = 0.89). Intravascular ultrasound provides accurate characterization of the arterial lumen and geometry, as well as the presence and histological features of atherosclerotic plaque. Thus, it appears to have a great potential for an earlier and more accurate diagnosis of atherosclerosis and may serve to guide new interventional techniques being utilized in the treatment of cerebrovascular diseases. PMID- 8634491 TI - Magnetic resonance and single-photon emission tomography findings in a pair of twins discordant for Alzheimer's disease. AB - The value of functional and morphological neuroimaging in the early diagnosis of Alzheimer's disease (AD) is still debated. Described here are cerebral perfusion and linear measures of medial temporal lobe atrophy in 2 monozygotic twins discordant for AD who were investigated with technetium 99m-hexamethylpropy leneamineoxime single-photon emission tomography (SPET) and magnetic resonance (MR) imaging. Both showed pathological cortical perfusion findings on SPET, while medical temporal lobe atrophy was present only in the affected twin. MR measures of medial temporal lobe atrophy have greater agreement with clinical data than do SPET measures of cerebral perfusion. Evaluation of atrophy may be useful in the early diagnosis of AD. PMID- 8634493 TI - Detection of vasospasm by transcranial Doppler sonography. The challenges of the anterior and posterior cerebral arteries. AB - Little information exists on the utility of transcranial Doppler sonography (TCD) in detecting anterior (ACA) and posterior cerebral artery (PCA) vasospasm following subarachnoid hemorrhage. During the period at risk for vasospasm, 53 patients with subarachnoid hemorrhage who had technically adequate TCD performed within 24 hours of cerebral angiography, allowing evaluation of 87 ACAs and 84 PCAs, were studied. ACA and PCA vasospasm were defined by mean blood flow velocities of at least 120 cm/sec and at least 90 cm/sec, respectively. For detection of ACA vasospasm, sensitivity was 18% and specificity was 65%. For PCA vasospasm, sensitivity was 48% and specificity was 69%. False-positive findings for occlusion accounted for 12 (92%) of 13 ACA of false-positive results and 7 (37%) of 19 PCA false-positive results, and were most often due to anatomical factors and operator error or inexperience. After exclusion of both true-positive and false-positive findings for occlusion and changes in the diagnostic criterion to at least 130 cm/sec for ACA vasospasm and at least 110 cm/sec for PCA vasospasm, specificity improved for both types of vasospasms (100 and 93%, respectively). However, the sensitivity of TCD to detect ACA and PCA vasospasm is limited by a variety of anatomical, technical, and other factors. It is concluded that TCD is highly specific in detecting both ACA and PCA vasospasm on arteries that can be insonated. PMID- 8634492 TI - Middle cerebral artery velocity correlates with nitroglycerin-induced headache onset. AB - Headache often accompanies treatment with nitroglycerin, but the cerebral hemodynamic effects and the exact mechanism of the headache are incompletely understood. Transcranial Doppler monitoring allows evaluation and monitoring of changes in blood flow velocity in the large intracranial arteries. The objective of this study was to assess middle cerebral artery (MCA) blood flow velocities with transcranial Doppler monitoring in subjects receiving continuous low-dose nitroglycerin intravenously or by patch, and correlate these with clinical headache. Twenty-eight normal adult men received nitroglycerin (0.12 micrograms/kg/min intravenously [n = 14] or 0.6 mg/min by transdermal patch [n = 14]), for up to 120 minutes, with monitoring of clinical headache status (standard 4-point scale), blood pressure, heart rate, end-expiratory PCO2 (CO2), and right MCA velocity. All subjects developed headache (mean time to onset, 34 min), reaching moderate or severe levels in 20. There were no differences in age, weight, mean blood pressure, mean heart rate, or resting end-tidal CO2 between those whose headache reached a moderate to severe level and those whose headache remained mild. MCA velocity decreased from baseline values at all levels of clinical headache (onset, -17%; moderate, -18%; severe, -16%; nitroglycerin stopped, -19%) (p, 0.0001 by t test for each stage of headache). MCA velocity remained decreased at the time of headache resolution (-14%; p < 0.001). Blood pressure, heart rate, and CO2 did not change significantly. There were no differences related to route of nitroglycerin dosing. These data show that continuous low doses of nitroglycerin by patch or intravenously produce headache in normal male subjects. MCA velocities were significantly decreased at headache onset and at all levels of headache severity. Changes in MCA velocity persisted beyond the clinical headache. These results suggest a direct MCA vasodilatory effect of nitroglycerin. This method may also be used to evaluate the intracranial hemodynamic effects of other vasoactive drugs, even in clinical settings. PMID- 8634494 TI - Chagasic granulomatous encephalitis in immunosuppressed patients. Computed tomography and magnetic resonance imaging findings. AB - American trypanosomiasis (Chagas' disease), a zoonosis caused by Trypanosoma cruzi with a high incidence in Latin America, may induce an uncommon form of localized encephalitis termed "chagoma", found in few immunocompromised patients. The computed tomography (CT) and magnetic resonance imaging (MRI) findings of brain chagoma are reported for 3 males (ages 32, 32 and 9 yr), the first 2 infected with human immunodeficiency virus (HIV) and the third with acute lymphoblastic leukemia. Diagnosis was confirmed by biopsy. CT disclosed a single, supratentorial, nodular-shaped lesion that substantially enhanced with contrast material, localized in parietal or frontal lobes. T1-weighted MRI showed hypointense lesions that enhanced with gadolinium-diethylenetriaminepentaacetic acid, corresponding to extensive hyperintense areas on T2-weighted images, producing mass effect. The imaging pattern of brain chagoma presented here is similar to that of cerebral toxoplasmosis and should be considered in the differential diagnosis of an intracerebral mass lesion in immunocompromised patients. PMID- 8634495 TI - Balo's concentric sclerosis. Report of two patients with magnetic resonance imaging follow-up. AB - Balo's concentric sclerosis was diagnosed antemortem in 2 patients, by magnetic resonance (MR) imaging showing striking concentric alternating rings in 1 patient and by characteristic histopathological features in the other. The course of the lesions and the concentric pattern were followed by MR imaging for 3 years and 18 months, respectively. One patient demonstrated spontaneous remission that has not been reported in Balo's disease. Balo's disease may not have a fulminant course as described in the past and the MR appearance of the chronic lesion may resemble that of a chronic multiple sclerosis plaque. PMID- 8634496 TI - The difficulties of learning fingerspelling: an experimental investigation with hearing adult learners. AB - Fingerspelling is used to support sign languages, providing a means by which words without signs may be communicated. As with signing itself, it has often been reported that learners find greater difficulty reading fingerspelling than they do in encoding it. An experiment tested the skills of adult hearing learners of the two-handed fingerspelling system used in British Sign Language. Participants were asked to read video recordings of their own fingerspellings; thus each undertook the reading task at their own spelling speed. Participants were divided into fast and slow spellers. Each group made more errors in the reading than the spelling task and this continued to be the case when read items received a contextual cue to assist recognition. Words with regular and irregular spellings were used as a means to investigate the cognitive processes underlying fingerspelling. Regular words were spelled faster and read more accurately, suggesting that these processes place some reliance on phonological encoding. The implications of these results of the learning and practice of fingerspelling are discussed. PMID- 8634497 TI - Temporal and spectral cue use for initial plosive voicing perception by hearing impaired children and normal-hearing children and adults. AB - The influence of voice-onset time (VOT) and vowel-onset characteristics on the perception of the voicing contrast for initial plosive consonants was examined for hearing-impaired children, and normal-hearing children and adults. Listeners identified spoken 'DAD'--'TAD' stimuli controlled for VOT and vowel onset characteristics. Only six of 16 hearing-impaired children appropriately identified the exemplar DAD and TAD stimuli used as endpoints of VOT continua. For this group of six hearing-impaired children, a longer VOT than for the normal hearing listeners was required to elicit /t/ rather than /d/ percepts. The VOT region of perceptual cross-over in labelling widened progressively from normal hearing adults to normal-hearing children to hearing-impaired children. Generally, longer VOTs were required to yield /t/ perception in the context of the DAD vowel than with the TAD vowel. These 'vowel stem' effects on VOT boundary were inconsistent for the hearing-impaired children, and weaker for the normal hearing children than for the adults. These spoken stimuli produced results for VOT cue use that generally parallel those obtained in studies with synthetic stimuli. PMID- 8634498 TI - Comparison of direct and indirect calculations of laryngeal airway resistance in various voicing conditions. AB - Calculation of laryngeal airway resistance using intraoral pressure divided by airflow has become a widely accepted clinical tool. The estimation is based largely on theoretical assumptions, particularly regarding the relationship between intraoral and subglottal pressure. To determine the estimate's validity, direct measures of airflow and subglottal, intraoral and pharyngeal pressures were obtained for four men and four women. Subjects produced normal, loud, soft and simulated breathy and strained syllable trains. Comparison of direct and indirect calculations of laryngeal airway resistance revealed generally good correspondence, particularly for [pi] syllables, although the estimate's accuracy varied among individual subjects. Some interpretive caution is warranted for the extremes of laryngeal airway resistance, with resistance likely to be underestimated in the strained condition, and overestimated in the breathy condition. PMID- 8634499 TI - Lip function in subjects with upper motor neuron type dysarthria following cerebrovascular accidents. AB - The lip function of 16 speakers with upper motor neuron damage following cerebrovascular accident (CVA), was investigated using instrumental measures. Sixteen, non-neurologically impaired adults matched for age and sex served as controls. The results of the instrumental investigations revealed that the CVA speakers had patterns of lip function that were significantly different from the control speakers. Specifically, on maximum force tasks, the CVA speakers were found to produce significantly lower maximum lip force values. Maximum rate of attempts at lip movement was also slower in the CVA speakers. In addition, the instrumental investigation revealed that the CVA speakers demonstrated a significant decline in pressure over the course of the task involving 10 attempts at maximum lip force. In contrast, the control speakers maintained a consistent level of force throughout the 10 repetitions/attempts. Overall, the findings suggest that the CVA speakers have deficits in maximum lip force, endurance of lip strength and rate of lip movements. Correlations conducted between instrumental measures of maximum lip strength and the perceptual ratings of overall intelligibility, precision of consonants, precision of vowels and length of phonemes revealed no significant relationship. PMID- 8634500 TI - Psychological processes in psychogenic voice disorder. AB - Using Freud's hysterical conversion model as a basis for reviewing the psychological features in psychogenic voice disorder, this paper describes research findings which show that individuals with this condition are not usually suffering from severe psychopathology but tend to be women who are experiencing high levels of stress (commonly associated with interpersonal relationship conflicts, low self-esteem, the burden of responsibility and feelings of powerlessness), and have above-average musculoskeletal tension as well as difficulties in voicing their feelings or views. The review indicates that the psychoanalytic interpretation of psychogenic voice loss continues to have relevance. However, it is suggested that in most cases Freud's hysterical conversion model should be reformulated, reducing the core focus to unconscious conflicts, repression, denial and secondary gains, in order to emphasise the psychosocial causes and maintenance of intrapsychic conflict and physical dysfunction. It is also suggested that a reformulated psychoanalytic interpretation of psychogenic voice loss has a lot in common with a cognitive behavioural conceptual model. A psychoanalytic approach may be valuable when focused on early trauma and repressed experiences or feelings, whereas cognitive behavioural treatment strategies, such as stress management and assertiveness training, appear to be particularly relevant in this condition. The paper concludes by briefly considering some areas of future research. PMID- 8634501 TI - Drawing to communicate: a case report of an adult with global aphasia. AB - Treatment for adults with global aphasia has typically involved the use of verbal treatment methods or alternative communication techniques including communication boards, word lists and notebooks. However, many adults with aphasia are unable to communicate verbally and alternative communication techniques can be limited, as a result of the restricted number and type of concepts that can be adequately depicted and expressed. Another viable means of communication for the globally aphasic adult is drawing. However, few individuals with severe aphasia initiate communication through this modality without specific training. In this case report we present several successful treatment methods that were used to train an adult with global aphasia to communicate more effectively through drawing. Several of his drawings are presented to illustrate the results of training in the use of drawing as an alternative means of communication. PMID- 8634502 TI - Patient education: compliance or emancipation? AB - What is patient education? How do nurses teach? What is the meaning of patient education in the secondary care setting to people who have a chronic illness such as asthma? What is the meaning of patient education to nurses in the secondary care setting? How do the political, economic, social and cultural contexts of secondary care and nursing education influence education of people with a chronic illness? PMID- 8634503 TI - The content and delivery of undergraduate nursing curricula. PMID- 8634504 TI - Family nursing: the case for a nurse consultant. PMID- 8634505 TI - Love and duty: issue of concern for nurses when newly physically disabled persons are discharged into the care of families. AB - Richmond (1990, P.65) writes "spinal cord injury occurs not just to the individual but to the entire family. There exists during crisis the potential for growth for individual family members and the entire family system". This article is written from both personal experience and professional interest because little appears to be written in the nursing literature about the family adjustment following discharge from hospital of a person who has a permanent disability following damage to the spinal cord. PMID- 8634506 TI - Sending beginning nursing students to gerontological units: best introduction to nursing or elder abuse? PMID- 8634507 TI - Invisible regulation. PMID- 8634508 TI - Hospice New Zealand annual conference 1995. July 5, 6, & 7. PMID- 8634509 TI - Margaret Ann Whiting. PMID- 8634510 TI - The little man. PMID- 8634511 TI - The challenge of non-compliance. PMID- 8634512 TI - Making a difference to childbearing families: independent midwifery practice. PMID- 8634513 TI - Cytokines and fever. AB - Fever is one of the best examples of responses to injury and infection which depends on neuroimmune interactions. Cytokines function as mediators of fever, acting locally within damaged tissues, circulating factors and the brain. The primary endogenous pyrogens. IL-1, IL-6 and TNF-alpha act at each of these sites and interact with nervous and endocrine systems to modify host defence responses. Understanding the role and mechanisms of cytokine actions in fever is relevant to many other aspects of neuroimmunology, and host responses to pathological challenges. PMID- 8634514 TI - Interleukin-6 and the skin. AB - Interleukin-6 is a pleiotropic cytokine with numerous biological activities. It is produced by normal constituents of the skin, including epidermal cells, fibroblasts and dermal endothelial cells. It is also synthesized by inflammatory cells infiltrating the skin in various pathological conditions. We reviewed the presumed activities of interleukin-6 in normal skin and in some diseases with cutaneous involvement. The action of some drugs used in dermatology was also considered with regard to the modulation of the cytokine release. It is concluded that several important cellular lineages of the skin release interleukin-6. The cytokine appears to play a crucial role in the pathogenesis of both local and systemic inflammation, tumor development and autoimmune diseases. PMID- 8634515 TI - Guillain-Barre as an autoimmune disease. AB - Guillain-Barre syndrome (GBS) entails a demyelinating process of the peripheral nervous system. The etiopathogenesis of the syndrome is still a matter of debate although considerable progress has been accomplished in the recent years. Abundant evidence has been put forward so as to support the role of the immune system in initiating and perpetuating the ongoing damage culminating in the emergence of the clinically overt syndrome. As such, data on the involvement of the humoral immune pathways add to the information already presented with regard to cell-mediated mechanisms participating in disruption of peripheral nerve. The following review will focus on the current knowledge of these complex mechanisms and the relative significance of each in the pathogenesis of GBS. PMID- 8634516 TI - Retrobulbar fibroblasts from patients with Graves' ophthalmopathy induce downregulation of APO-1 in T lymphocytes and protect T cells from apoptosis during coculture. AB - Retrobulbar fibroblasts are a main target of the immune process in Graves' ophthalmopathy (GO) and have been shown to have unique metabolic qualities. The aim of our study was to analyze the immunoregulatory properties of retrobulbar fibroblasts and particularly whether fibroblasts were able to protect T cells from apoptosis. Retrobulbar fibroblasts from patients with GO spontaneously expressed higher concentrations of HLA class I and HLA class II (p<0.05) than control cells, whereas basal CD54 expression was unimpaired. Stimulation with IFN gamma led to a more pronounced increase in HLA class I, class II and CD54 in autoimmune fibroblasts than in control cells (p<0.05). Fibroblasts from both groups had the capacity to prevent apoptosis in preactivated peripheral T cells during coculture. T cell survival was, however, more pronounced after coculture with retrobulbar fibroblasts than with control cells (p<0.05). Prevention of T cell death was associated with a decreased expression of APO-1 on the T cell surface, whereas the bcl-2 expression of the T cells remained unchanged. Our results suggest that the increased expression of immunoregulatory molecules combined with a pronounced capacity of autoimmune fibroblasts to protect infiltrating T cells from apoptosis might at least partly explain the site selectivity as well as the perpetuation of the extrathyroidal manifestation of Graves' disease. PMID- 8634517 TI - Candida albicans mannan-specific, delayed hypersensitivity down-regulatory CD8+ cells are genetically restricted effectors and their production requires CD4 and I-A expression. AB - There is considerable controversy over the induction and activity of down regulatory cells active in various antigen-specific and antigen-nonspecific systems. We have been studying the nature of such cells in a Candida albicans mannan (MAN)-specific system for some time and report here the requirements for CD4+ and I-A+ cells during the inductive phase for the development of CD8+ effector cells, as well as the requirement for genetic compatibility for effector activity of CD8+ cells. Since we have shown previously that CD8+ down-regulatory cells were present in spleens of MAN-treated mice 4 days following the administration of MAN to naive mice, as determined by their ability to suppress delayed hypersensitivity (DH) when transferred to immunized recipients, we treated mice with monoclonal antibodies specific for CD4 and I-A at various times before, with or after the administration of MAN to assess the role of CD4+ and I A+ cells in the development of the CD8+ effector cell. Both anti-CD4 and anti-I-A given before or up to 30 h after the administration of MAN abrogated the ability of splenocytes from MAN-treated mice to down-regulate MAN-specific DH in immunized recipients. Moreover, transfers of down-regulatory cells between H-2 incompatible strains of mice, specifically CBA/J and BALB/cByJ, provided evidence that the effector cell for the down-regulatory activity was also restricted genetically in its activity. Taken together, the data presented indicate that genetically compatible cells are required for both the inductive and effector stages of down-regulation of MAN-specific DH, suggesting that cell-cell cooperation is required for both stages and that CD4+ cells are required in a pathway leading to the development of the CD8+ effector cell. PMID- 8634518 TI - Establishment and characterization of ovalbumin-specific T cell lines from patients with egg allergy. AB - In order to investigate T cell recognition of allergens in hen egg allergy, we have established 30 ovalbumin (OVA)-specific T cell lines (TCLs) from peripheral blood mononuclear cells of 6 patients with atopic dermatitis, who are positive for IgE antibodies to OVA and clinically allergic to hen egg, and characterized them for their cytokine production pattern. All TCLs we could study were mainly composed of CD4+ T cells. Most TCLs produced significant amounts of interleukin 4 (IL-4) and IL-5 but no or very little interferon gamma on antigen stimulation, suggesting that these TCLs belong to TH2-type T cells. Restriction elements and epitope specificities were further studied on some TCLs. Antibody blocking of the proliferative responses of the TCLs to OVA indicated that HLA-DR are acting as the dominant restriction elements for these TCLs with minor contribution of HLA DQ. By use of 187 overlapping synthetic peptides covering the whole sequence of OVA, at least 3 different T cell epitopes were identified. PMID- 8634519 TI - Group 5 allergens of timothy grass (Phl p 5) bear cross-reacting T cell epitopes with group 1 allergens of rye grass (Lol p 1). AB - Selected human T cell clones reactive with group 5 allergens of timothy grass (Phl p 5) were cross-stimulated in specific proliferation assays with group 1 allergens of rye grass (Lol p 1). Such interspecies cross-reactivities result obviously from structural motifs presented on defined Phl p 5 fragments as shown with recombinant Phl p 5 products. PMID- 8634520 TI - Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli. AB - Der f2 protein in a highly antigenic form was directly expressed in bacteria. Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number. This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells. The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E. coli, when compared to a nonchanged origin material. rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form. The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography. This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture. Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test. PMID- 8634521 TI - Significance of rubber elongation factor as a latex allergen. AB - IgE-mediated hypersensitivity to proteins eluting from surgical gloves and other natural rubber products is a well-recognized allergic condition with special impact to health care workers and sensitized patients. Since rubber elongation factor (REF) has been suggested to be the major latex allergen (Hev b 1) we purified REF from rubber particles using electrophoresis and electroelution and reevaluated the occurrence of IgE antibodies to purified REF. The purified protein was subjected to tryptic digestion, peptide separation and amino acid sequencing. Amino acid sequences of 9 tryptic peptides from the purified 14-kD protein gave 100% homology to REF. In immunoblotting, sera from 4 of 6 latex allergic children with spina bifida or other congenital anomalies exhibited IgE antibodies against REF whereas only 1 serum from the 30 other latex-allergic patients had similar antibodies. In IgE ELISA 8/45 (18%) latex-allergic patients had IgE antibodies to purified REF, whereas 30 (67%) of the same patients had IgE antibodies to purified prohevein, another rubber protein, recently shown to be a major natural rubber latex allergen. The observed overall low frequency of IgE antibodies to purified REF, as measured by two specific IgE assays, indicates that REF is one but not the most significant allergen among the natural rubber latex proteins. PMID- 8634522 TI - Fel d 1-specific IgG antibodies induced by natural exposure have blocking activity in skin tests. AB - Cat-allergic patients frequently have IgG antibodies directed against Fel d 1. The aim of this study was to investigate whether these IgG antibodies influence the results of the skin test. Titrated skin tests were performed with Fel d 1 and IgE and IgG antibody levels were measured in 59 patients with cat allergy. Levels of specific IgG against Fel d 1 ranged from less than 0.25 to 3.5 microgram/ml. By means of a multiple regression analysis it was shown that the amount of specific IgG antibodies contributes significantly to the results of the skin test. Presence of specific IgG against Fel d 1 was accompanied by higher skin thresholds for Fel d 1. In conclusion, this study indicates that even low levels of specific IgG, induced by natural exposure to cat allergens, have a blocking effect on the early phase skin reaction. PMID- 8634523 TI - Purification of mast cells with an improved nonsynchronous flow-through coil planet centrifuge. AB - A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells. PMID- 8634524 TI - Tumor necrosis factor alpha immunoreactivity of rat peritoneal mast cell granules decreases during early secretion induced by compound 48/80: an ultrastructural immunogold morphometric analysis. AB - We used fast (seconds) and ultrafast (milliseconds) microwave energy-assisted chemical fixation protocols, postembedding immunogold staining, and a morphometric analysis to investigate the early morphological changes and the TNF alpha immunoreactivity in the cytoplasmic granules of rat peritoneal mast cells that had been stimulated to secrete by exposure to compound 48/80. Exposure to compound 48/80 induced the development of increased numbers of cytoplasmic granules that exhibited decreased electron density; these granules often also appeared swollen. These granule alterations were accompanied by a significantly decreased proportion of granules that were positive for TNF-alpha immunoreactivity. We also calculated the density of TNF-alpha labeling/mu 2 in both dense (unaltered) and altered granules in specimens. TNF-alpha immunoreactivity was present in dense granules (regardless of whether or not the specimens had been stimulated with compound 48/80) and in cells that were fixed with either fast or ultrafast microwave energy. However, altered granules exhibited a decreased density of TNF-alpha label. These findings show that changes in the immunolocalization and/or density of TNF-alpha immunoreactivity occur very rapidly upon stimulation of rat peritoneal mast cells with compound 48/80. PMID- 8634525 TI - Effect of mast cell modulators on IgE-mediated murine biphasic cutaneous reactions. AB - Hapten-specific and mast cell-dependent biphasic cutaneous reactions were induced by intravenous application of anti DNP-IgE antibodies and a subsequent skin test. These reactions were also demonstrated in SCID mice, which indicates that T cell mediated immunity might not be involved in these IgE-mediated cutaneous reactions. Simultaneous application of anti histaminics did not suppress these reactions significantly, while several immunomodulators, such as azelastine, FK506, and prednisolone, significantly inhibited both early and late phase reactions except for the failure of FK506 to inhibit the early reaction. Anti VCAM-1 antibody and anti-tumor necrosis factor-alpha (TNF alpha) antibody but not anti-IL 5 antibody showed similar suppressive effects on both early and late phase reactions. Mast cell and inflammatory cells other than T cells are thought to play an important role in these IgE-induced biphasic reactions. TNF alpha and/or VCAM-1 are required for tissue accumulation of inflammatory cells in this system. PMID- 8634527 TI - Antigen-induced anaphylactic death in mice. AB - Intravenous injection of bovine serum albumin (BSA) into the BSA/CFA-primed ICR mice specifically induced anaphylactic death within 1 h. The anaphylactic death could not be induced until day 8 after sensitization, and sensitization subsisted for more than 3 months. The response was dose dependent; mice challenged with BSA doses higher or equivalent to 25 microgram developed anaphylactic death. The intravenous route was more effective than the intraperitoneal one, while subcutaneous injection was ineffective. Antigen in any of complete Freund's adjuvant, incomplete Freund's adjuvant or aluminum hydroxide could sensitize the mice to develop anaphylactic death. The combination of antigen and the mouse strain or the gender of the mouse determined the susceptibility of the anaphylactic death. AKR, B10.BR, as well as ICR, strains were susceptible. Antigen of HoGG induced a higher mortality rate than that of GAT or lysozyme. Male mice were more susceptible than female ones. The BSA-induced anaphylactic death could be prevented by pretreating ICR mice with cyproheptadine (histamine and serotonin antagonist) or diphenhydramine (histamine antagonist) and ketanserin (serotonin antagonist). Intravenous injection of saline during anaphylaxis also protected the mice from death. Furthermore, immune serum could transfer the anaphylactic death, and heat (56 degrees C, 4 h) did not destroy its activity. The primary IgG subclass induced by GAT, HoGG or lysozyme was IgG1. There was no qualitative difference in the IgG subclass induced in different strains by different antigens. The IgE class of antibodies was not detectable. These results suggest that there is a non-IgE-mediated anaphylactic death which involves the release of histamine and serotonin that cause the increase of vasopermeability and fatal blood volume loss. PMID- 8634526 TI - Pharmacological characterization of Sephadex-induced oedema in rat paws: predominant role of serotonin and platelet-activating factor. AB - An intravenous injection of Sephadex beads has been used to induce lung inflammation and bronchial hyperreactivity in small animals. In the present study, we injected Sephadex beads (0.3-5.5 mg/paw) into rat paws and followed the resulting inflammation plethysmometrically. Our results show that Sephadex beads induced a significant and dose-dependent increase in the hindpaw volume at 5 min; it was maximal at 30-60 min and declined at 4 h. However, the paw volume remained significantly increased for up to 21 days. The initial 4-hour-oedema was confirmed by histopathology of the paw tissues, but the persistent increase in paw volume was related to a chronic inflammatory (granulomatous) response. The Sephadex-induced oedema was predominantly due to serotonin (5-HT) release since specific antagonists such as methysergide (1 mg/kg) and pizotifen (0.1-2 mg/kg) administered both systemically and locally were able to inhibit the oedema (10 100 microgram/paw) as could pretreatment with compound 48/80. In addition, platelet-activating factor (PAF) was also shown to be involved, since systemic pretreatment using the specific PAF antagonist BN 52021 (1 mg/kg) was able to inhibit the increase in paw volume induced by Sephadex. Effective doses of indomethacin (2 mg/kg), L-NAME (1 mg/kg), pyrilamine (1-2 mg/kg), ondansetron (1 mg/kg) and HOE 140 (1 mg/kg) did not affect the Sephadex-induced oedema, thus ruling out the participation of prostaglandins, nitric oxide, histamine, 5-HT3 receptors and bradykinin in its development. Since the late increases in paw volume induced by Sephadex were reduced by pretreatment of the animals with the immunosuppressive drugs rapamycin and dexamethasone but not cyclosporin, our results also suggested that distinct immunological pathways may be involved in the modulation of the chronic phase of inflammation induced by Sephadex beads in rat paws. PMID- 8634528 TI - Reliability of the determinations of polychlorinated contaminants (biphenyls, dioxins, furans). AB - Precision performance parameters from results of 34 interlaboratory performance studies of polychlorinated aromatic ring compounds (biphenyls, dioxins, and furans) (PCCs) have been recalculated by using the international Union of Pure and Applied Chemistry-1987 harmonized protocol. Most studies of 1052 test samples, 56 analytes, 19 matrixes, and 2 types of detectors (electron capture and mass spectrometers) provide among-laboratories relative standard deviations (RSDRS), that are considerably better than those predicted from the Horwitz equation at fractional concentrations of 10(-5) down to 10(-15). The explanation suggested is that supplying common reference calibration solutions, as was done in many of these studies, does not reflect realistic operating conditions. Furthermore, the ability to repeat, discuss, and reassess aberrant reported values results in underestimating the true RSDR. The commonly reported problems of preparation of standard calibrating solutions, instability of the detection system, and failure to follow quality control instructions and good laboratory practices may be important sources of interlaboratory variability in PCC determinations. PMID- 8634529 TI - Determination of benzocaine in rainbow trout plasma. AB - A liquid chromatographic method is described for analysis of benzocaine (BZ), a proposed fish anesthetic, in rainbow trout plasma. Mean recoveries of BZ from plasma samples fortified at 44-10 100 ng/mL were 96-100%. The method detection limit is 10 ng/mL, and the limit of quantitation is 37 ng/mL. Acetylation of BZ occurs in whole blood after storage at room temperature (i.e., 21 degrees C) for 10 min. However, no acetylation of BZ was detected in plasma samples held at room temperature for 4 h. Mean method precision for plasma samples with incurred BZ residue is similar to that for fortified samples in the same concentration range (relative standard deviations of 0.9 and 1.2%, respectively). PMID- 8634530 TI - Determination of allantoin in biological, cosmetic, and pharmaceutical samples. AB - Methods for determining allantoin in biological, cosmetic, and pharmaceutical samples are reviewed. The methods cover 3 main categories: spectrophotometry, alkalimetric titration, and chromatography (thin-layer and liquid). Comments are given on suitability for different sample types and on further development of methods for allantoin determination. PMID- 8634531 TI - Gradient liquid chromatographic method for determination of chlorhexidine and its degradation products in bulk material. AB - A liquid chromatographic method was developed for determination of chlorhexidine and its degradation products in unformulated drug substance. A nonlinear gradient from 80% 0.1M ammonium acetate buffer, pH 5.0, to 20% buffer over 90 min (balance is acetonitrile) is applied to a 3 microns octadecylsilane bonded-phase column. The drug and some of its degradation products are determined at 230 nm. Of 11 previously identified degradation products, 9 are determined with good precision (relative standard deviation of peak area is < 2%). PMID- 8634532 TI - Liquid chromatographic determination of benzylpenicillin and cloxacillin in animal tissues and its application to a study of the stability at -20 degrees C of spiked and incurred residues of benzylpenicillin in ovine liver. AB - A method was developed for determining benzylpenicillin and cloxacillin in animal tissues. Samples are extracted with acetonitrile, and the extract is cleaned up on a C18 solid-phase extraction (SPE) cartridge, derivatized, and quantitated by liquid chromatography with UV detection at 325 nm. The method was validated on spiked bovine kidney, liver, and muscle tissues. Kidney was spiked at 0.01, 0.05, and 0.20 mg/kg; liver and muscle were spiked at 0.20 mg/kg. Recoveries were 75 100% , with coefficients of variation of 2-7%. The method was further validated on kidney, liver, and muscle tissues from 2 sheep dosed with Aquacaine G suspension (containing benzylpenicillin). Mean levels of benzylpenicillin in these tissues ranged from 0.02 to 4.06 mg/kg, with coefficients of variation of replicate analyses between 3 and 7%. The limit of detection was approximately 0.005 mg/kg for benzylpenicillin and cloxacillin in kidney, liver, and muscle tissues. This method was used to study the stability of both spiked and incurred residues of benzylpenicillin in ovine liver during storage at -20 degrees C for 3 months. Assuming the rate of loss follows a first-order kinetic decay, the mean half-life of benzylpenicillin in liver is 62 days for spiked tissues and 71 days for tissues with incurred residues. PMID- 8634533 TI - Liquid chromatographic determination of mebendazole and its metabolites, aminomebendazole and hydroxymebendazole, in eel muscle tissue. AB - An analytical method is presented for liquid chromatographic (LC) determination of mebendazole (MBZ), hydroxymebendazole (MBZ-OH), and aminomebendazole (MBZ-NH2) in eel muscle tissue. Muscle tissue is extracted with ethyl acetate at pH 7.5. After addition of n-hexane, the extract is cleaned up and concentrated on an aminopropyl solid-phase extraction column. The test solutions are analyzed isocratically on a ChromSpher B LC column with acetonitrile-phosphate buffer, pH 6.2, as mobile phase. Limits of detection and quantitation were 0.7 and 1.1 micrograms/kg, respectively, for MBZ-OH; 1.4 and 2.3 micrograms/kg, respectively, for MBZ; and 1.5 and 2.1 micrograms/kg, respectively, for MBZ-NH2- Interand intraday coefficients of variation were 3.5 and 3.4%, respectively, for MBZ-OH; 2.5 and 3.1%, respectively, for MBZ; and 5.8 and 4.8%, respectively, for MBZ-NH2. Mean recoveries were 90% for MBZ, 74% for MBZ-NH2, and 92% for MBZ-OH. A linear range of applicability of at least 10-1000 micrograms/kg was found for each analyte. Incurred MBZ-NH2 (181.3 micrograms/kg) was identified in eel muscle tissue apart from MBZ (23.7 micrograms/kg) after 48 h exposure in a treatment bath containing MBZ at 1 mg/L. PMID- 8634534 TI - Determination of tilmicosin in bovine milk by liquid chromatography with ultraviolet detection. AB - A method is described for determining tilmicosin in bovine milk by liquid chromatography with ultraviolet (UV) detection. Samples are defatted by centrifugation. The lower skim layer is cleaned on a C18 solid-phase extraction cartridge. The extract is concentrated and analyzed with a reversed-phase phenyl column with UV detection at 280 nm. The method was validated with control milk fortified at 50, 100, and 200 ng/mL. Average recoveries (and intralaboratory coefficients of variation) were 97% (9%), 98% (5%), and 101% (3%), respectively. The limit of quantitation is approximately 20 ng/mL, and the limit of detection is approximately 13 ng/mL. The method was tested on milk from a cow dosed with tilmicosin. PMID- 8634535 TI - Rapid identification and quantitation of clotrimazole, miconazole, and ketokonazole in pharmaceutical creams and ointments by thin-layer chromatography densitometry. AB - Thin-layer chromatography (TLC)-densitometry was used to separate, identify, and quantitate clotrimazole, miconazole, and ketokonazole (alone or combined with other drugs) in various pharmacopoeial or proprietary creams and ointments. Clotrimazole was extracted from the cream or ointment with ethyl alcohol, and miconazole and ketokonazole were extracted with a mixture of equal volumes of chloroform and isopropyl alcohol. Active ingredients were separated from excipients and other drugs by TLC on a precoated silica gel F254 plate with a solvent system of n-hexane-chloroform-methanol-diethylamine (50 + 40 + 10 + 1, v/v). The 3 azoles were well separated and easily identified in this chromatographic system. The separated azoles were visualized under short-wave UV light and quantitated by scanning densitometry at 220 nm by comparing the integrated areas of samples with those of standard (one azole was used as internal standard for the other). Recoveries from samples spiked with known amounts of azoles were excellent. The method was validated further by comparison with official liquid chromatographic methods. PMID- 8634536 TI - Efficacy of a latex agglutination test for rapid identification of Staphylococcus aureus: collaborative study. AB - Fifteen laboratories completed a collaborative study comparing the efficacy of a latex agglutination kit (Aureus Test) with that of AOAC Official Method 987.09 (coagulase test for identification of Staphylococcus aureus). Each laboratory analyzed 240 strains of bacteria, including 160 isolates of S. aureus and 80 isolates of other bacteria. Upon receipt of cultures, collaborators subcultured each isolate on both tryptic soy agar (TSA) and Baird-Parker agar medium (BPA) to determine whether the growth medium has any effect on either method. For cultures grown on TSA, the latex test had sensitivity and specificity rates of 99.2 and 97.1%, respectively, whereas the coagulase test had respective rates of 98.4 and 92.5%. For cultures able to grow on BPA, the latex test had sensitivity and specificity rates of 99.2 and 96.6%, respectively, while the coagulase test had respective rates of 98.3 and 91.3%. By using the McNemar pairwise comparison test of the 2 methods, the false-positive and false-negative rates of the latex test were significantly lower (p < 0.01) than those of the coagulase test for strains grown either on TSA or BPA. The latex agglutination test for identification of S. aureus isolated from foods has been adopted by AOAC INTERNATIONAL. PMID- 8634537 TI - Worldwide survey of fumonisin contamination of corn and corn-based products. AB - As part of a comprehensive risk assessment study for fumonisins, reliable data on exposure of populations to these dietary toxins must be obtained. To assess the extent of worldwide exposure, the published literature on the contamination of food and feed supplies has been reviewed and supplemented with unpublished material from various international sources. Fumonisin contamination of corn and corn-based products occurs in many countries. Animal mycotoxicoses such as equine leukoencephalomalacia and porcine pulmonary edema are caused by heavily contaminated animal feeds. For example, as much as 330 micrograms/g fumonisin B1 (FB1) has been found in swine feed. Although commercially available refined corn products for human consumption are generally contaminated at levels below 1 microgram/g FB1, individual products in certain countries can reach far higher levels. Health risks associated with consumption of these products depend on the extent to which they are consumed in a varied diet. Home-grown corn in certain rural areas, where it also constitutes the staple diet, can be contaminated at > 100 micrograms/g. Consumption of corn contaminated at these high levels has been associated with a high incidence of esophageal cancer in these areas. PMID- 8634538 TI - Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn: AOAC IUPAC Collaborative Study. AB - A liquid chromatographic (LC) method for simultaneous determination of fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) in corn was subjected to a collaborative study involving 12 participants from 10 countries, in which the accuracy and reproducibility characteristics of the method were established. Mean analyte recoveries from corn ranged from 81.1 to 84.2% for FB1 (at a spiking range of 500 to 8000 ng/g), from 75.9 to 81.9% for FB2 (at a spiking range of 200 to 3200 ng/g), and from 75.8 to 86.8% for FB3 (at a spiking range of 100 to 1600 ng/g). The valid data were statistically evaluated after exclusion of outliers. Relative standard deviations for within-laboratory repeatability ranged from 5.8 to 13.2% for FB1, from 7.2 to 17.5% for FB2, and from 8.0 to 17.2% for FB3. Relative standard deviations for between-laboratory reproducibility varied from 13.9 to 22.2% for FB1, from 15.8 to 26.7% for FB2, and from 19.5 to 24.9% for FB3. HORRAT ratios, calculated for the individual toxin analogues, ranged from 0.75 to 1.73. The LC method for determination of fumonisins B1, B2, and B3 in corn (at concentrations of 800-12800 ng total fumonisins/g) has been adopted by AOAC INTERNATIONAL. PMID- 8634539 TI - Liquid chromatographic determination of free glutamic acid in soup, meat product, and Chinese food: interlaboratory study. AB - An interlaboratory study of the liquid chromatographic (LC) determination of free glutamic acid in soup, meat product, and Chinese food was performed. Homogenized food samples were extracted with hot water, filtered, and diluted. Aliquot portions were treated with N,N-dimethyl-2-mer-capto-ethyl-ammonium chloride (DMMAC) and o-phtaldialdehyde (OPA) to convert glutamic acid to a stable fluorescent complex. After LC separation on a reversed-phase C18 column with acetonitrile-phosphate buffer (pH 7.0)-water (80 + 180 + 740, v/v) as mobile phase, glutamic acid peaks were measured fluorometrically (excitation, 340 nm, and emission, 389 and/or 440 nm). Homocysteic acid was used as internal standard. Twelve samples (6 blind duplicates) containing about 0.3-1.3% (w/w) of glutamic acid were analyzed singly by 12 laboratories. Results from one participant were excluded. Repeatability relative standard deviations (RSDr) varied from 1.3 to 4.5%, and reproducibility relative standard deviations (RSDR) ranged from 4.1 to 7.1%. Average recovery of glutamic acid determined at 6 levels was 101.5% (range, 98-106%). PMID- 8634540 TI - Determination of fat, protein, and lactose in raw milk by Fourier transform infrared spectroscopy and by analysis with a conventional filter-based milk analyzer. AB - The accuracy of fat, crude protein (CP), true protein (TP), and lactose determinations of raw milk by Fourier transform infrared (FTIR) spectroscopy and by analysis with a conventional filter-based milk analyzer was assessed in 6 trials performed over a 10-month period. At each trial, 30 bulk milk samples collected from 15 European countries and 11 reconstituted milks made from raw milk components were analyzed. When calibrations were performed with reconstituted milks at each trial, accuracy standard deviations for fat, CP, TP, and lactose were, respectively, 0.050, 0.048, 0.035, and 0.076 g/100 g for the filter instrument and 0.047, 0.046, 0.042, and 0.065 g/100 g for the FTIR instrument. When a single calibration was made instead of calibrations at each trial, accuracy standard deviations increased for the filter instrument to 0.130, 0.119, 0.121, and 0.083 for fat, CP, TP, and lactose, respectively, and for the FTIR instrument to 0.082, 0.053, 0.044, and 0.084 g/100 g. Because the FTIR instrument provides more spectral information related to milk composition than does the filter instrument, single-calibration FTIR analysis of milk samples collected in different seasons is more accurate. Using reconstituted milks, prepared such that there is no correlation between fat, CP, and lactose, provides a more robust calibration than using genuine bulk milk, especially when milks with unusual composition are analyzed. PMID- 8634541 TI - Determination of sugars, starches, and total dietary fiber in selected high consumption foods. AB - A general scheme has been developed to determine sugars, starches, and total dietary fiber (TDF) in half-gram freeze-dried subsamples of various foods (or wet samples containing about 0.5 g dry matter). Duplicate subsamples are extracted for free sugars with 80% methanol, dried, derivatized to their trimethylsilylated oximes or ethers, and quantitated by gas-liquid chromatography (GLC). Residues after 80% methanol extraction are incubated with a solution containing amyloglucosidase in acetate buffer. Hydrolyzates are centrifuged, and duplicate aliquots are removed for glucose determination by GLC. Starch content is calculated as glucose (g/100 g) x 0.9. Remaining hydrolyzates are diluted with 4 volumes of 95% ethanol, left at room temperature for 1 h, and then filtered through glass crucibles matted with Celite filter aid. The weight of dry residues are corrected for residual crude protein and ash, and the resulting values are taken to be the TDF content of a sample. A variety of high-consumption foods selected by the Nutrient Data Laboratory of the Agricultural Research Service were analyzed for carbohydrate fractions by this method. Values for total sugar and dietary fiber were compared with those obtained by a commercial laboratory using different methods. PMID- 8634542 TI - Determination of fruit juice authenticity by capillary gas chromatography with flame ionization detection. AB - A method using capillary gas chromatography with flame ionization detection was developed to determine the addition of high-fructose syrup and beet or cane invert syrup to apple or orange juice. Fingerprint oligosaccharides in these inexpensive sweeteners were not detectable (area < 1000) in pure apple or orange juice. One hundred twenty-three pure apple juice and 60 pure orange juice samples representing growing regions around the world were analyzed. Ten samples were intentionally adulterated with each sweetener at levels of 5, 10, and 15%. The detection limit for each sweetener was 5%. PMID- 8634543 TI - Automated spectrophotometric method using 2,2'-dithiodipyridine acid for determination of cholinesterase in whole blood. AB - An automated method using 2,2'-dithiodipyridine (2-PDS) as chromophore for determination of whole-blood cholinesterase activity was developed. Assay procedures, optimal concentrations of chromophore and substrate, detection limit, precision, backgrounds, and sensitivity of the method were compared with those of an earlier automated method based on the Ellman method and using 5,5'-dithiobis(2 nitrobenzoic acid) (DTNB) as chromophore. The new method has the advantages of automation (resulting in increase throughput rate and decrease in amount of reagents used) and good precision and sensitivity. Sample dilutions also are reduced in the new method because hemoglobin interference is less. PMID- 8634544 TI - Determination of selenium in urine by hydride generation atomic absorption spectrometry. AB - A procedure has been developed for determination of total selenium in urine by hydride generation atomic absorption spectrometry. Mineralization was performed with a nitric acid-perchloric acid mixture on a thermostated digestion block. The method was validated by comparison with the method involving mineralization in a microwave acid digestion bomb containing nitric acid and small amounts of vanadium pentoxide. Se(VI) was reduced to Se(IV) by dissolution in 7N HCl. Sample recoveries, precision studies, and analyses of a certified reference material demonstrated the reliability and accuracy of this technique. Urine samples had selenium concentrations ranging from 4.6 to 50.3 micrograms/L. These values correspond to an average of 54.9 micrograms per person per day total ingested and bioavailable Se in the daily diet. PMID- 8634545 TI - Levels of organochlorine pesticides in Mexican butter. AB - Organochlorine pesticide residues were analyzed in 345 samples of butter purchased from Mexican supermarkets in 1994. Three national brands and one foreign brand were analyzed. Most samples contained residues of gama-HCH (91%), HCB (90%), and p,p'-DDE (88%). Residues of alpha-HCH (63%), p,p'-DDT (42%), beta HCH (38%), o,p'-DDT (17%), heptachlor epoxide (7%), and endosulfane sulfate (3%) were also detected. Mean values of pesticide residues determined were 0.093 mg/kg fat for total HCH and 0.056 mg/kg for total DDT. Mean values of organochlorine pesticide residues in Mexican butter were comparable with those in a foreign brand. PMID- 8634546 TI - Determination of imazalil residues in lemons by gas chromatography with nitrogen phosphorus detection. AB - A simple and reliable method was developed for determination of imazalil residues in whole lemons. Imazalil was extracted from lemon samples with ethyl acetate under basic conditions. The ethyl acetate in the extract was evaporated, and the residue was dissolved in dichloromethane. The solution was applied to a solid phase extraction (diol-bonded silica) cartridge. Imazalil was eluted with methanol and determined by gas chromatography (GC) with nitrogen-phosphorus detection. Recoveries at 4 fortified levels (0.1, 1.0, 2.0, and 5.0 micrograms/g) ranged from 93.3 to 97.8%, with coefficients of variation ranging from 0.2 to 3.0%. The detection limit was 0.05 microgram/g. Fifteen commercial lemon samples were analyzed for Imazalil residues; 10 were positive. Concentrations of imazalil residues ranged from 0.22 to 4.44 micrograms/g. Imazalil-positive samples were confirmed by GC with mass-selective detection (m/z 173, 215, and 217). PMID- 8634547 TI - Estimating analytical variances in measurement of polycyclic aromatic hydrocarbons and application to monitoring contaminants in American lobster (Homarus americanus). AB - A method is presented for estimating replicate polycyclic aromatic hydrocarbon (PAH) concentrations in American lobster (Homarus americanus) digestive gland tissue based on recoveries of added perdeuterated surrogates from a single satisfactory analysis. PAH concentrations demonstrated a large interanimal variance, even in specimens captured at the same time in the same place. Principal component analysis showed that the variability of the total system of biological variables (carapace length, lobster weight, and digestive gland weight) could be adequately summarized by the first principal component alone in each data set. Ranks provide ordered classification of individuals, allowing data analysis by statistical methods for continuous variables (i.e., analysis of variance). PAH concentrations in individual lobsters were generally highly sensitive to animal size, sex, and fishing area. Efficient monitoring would result from analyzing individual animals of a single sex from a study area, using as small a geographical study area as possible, measuring a single biological variable, and using individual specimens of as narrow a size range as possible. PMID- 8634548 TI - Simultaneous determination of ascorbic, dehydroascorbic, isoascorbic, and dehydroisoascorbic acids in meat-based food products by liquid chromatography with postcolumn fluorescence detection: a method extension. AB - A simple and rapid liquid chromatographic (LC) method for determining ascorbic, dehydroascorbic, isoascorbic, and dehydroisoascorbic acids in mostly single component food products was evaluated for use in analysis of multicomponent meat based food products such as TV dinners. Ground-beef samples were used as blanks for repeatability studies. Samples were fortified with 5, 10, 20, and 40 ppm of mixed standards of ascorbic acid and isoascorbic acid. Means of 12 recoveries at 4 levels of fortification were 102.5 and 83.5%, respectively, for ascorbic acid and isoascorbic acid, with coefficients of variation of 6.7 and 15.2%, respectively. TV dinner products (21 samples) from a local grocery store were analyzed for vitamin C content. Samples prepared with a commercial food processor and a food grinder were compared. The commercial food processor was more capable than the food grinder in producing a homogeneous sample, which is critical to the method. PMID- 8634549 TI - Emergence of rapid methods for identifying microbial pathogens in foods. AB - Because of the complexities of food analysis, conventional microbiological methods must use time-consuming enrichment steps for culturing viable bacterial cells in foods. With rapid advancements in technology, however, numerous so called rapid methods were introduced into the field of food microbiology in a relatively short time. Culture methods that were once used to obtain profiles for bacterial identification were simplified or automated. Many microbiological procedures were also streamlined or automated to reduce assay time, labor, and materials. Nucleic acid-based assays are used to identify gene sequences in foodborne bacteria, and antibody-based assays are used in numerous formats to detect bacterial pathogens and toxins in foods. The difficulties of analyzing food, however, remain challenging, and rapid methods need to be evaluated thoroughly before they are used for routine food analysis. PMID- 8634550 TI - Analysis of fermented milk products by near-infrared reflectance spectroscopy. AB - Fat, protein, and total solids in fermented milk were determined by near-infrared reflectance spectroscopy (NIRS). A set of 107 samples from diverse types of fermented milk (whole, skimmed, with added flavors, and without added flavors) were used to calibrate the instrument by modified partial least-square regression. Global calibration, using all samples, was more effective than specific calibrations using fewer samples of each type. Standard errors of a calibration were 0.071 for fat, 0.075 for protein, and 0.083 for total solids. Values of the correlation coefficient square (R2) were 0.997 for fat, 0.981 for protein, and 1.000 for total solids. Calibration was validated with an independent set of 34 samples of the same types. Standard errors of validation were 0.08, 0.14, and 0.25 for fat, protein, and total solids, respectively, and values of R2 for the regression of measurements by NIRS versus reference methods were 1.00 for fat and total solids and 0.94 for protein. When Standard 128 of the International Dairy Federation was used to compare NIRS results with those from reference methods, no significant differences were found (p = 0.05). PMID- 8634551 TI - The legacy of Edward Jenner. PMID- 8634552 TI - Mass polio vaccination. PMID- 8634554 TI - Seriously deficient professional performance. PMID- 8634553 TI - The cardioprotective effects of moderate alcohol consumption. PMID- 8634555 TI - Primary biliary cirrhosis: epidemiology helping the clinician. PMID- 8634556 TI - Algae kills dialysis patients in Brazil. PMID- 8634557 TI - Newborn babies threatened with HIV testing. PMID- 8634558 TI - Doctors voice virus fears over xenotransplants. PMID- 8634559 TI - Fatal outbreak traced to wooden tongue depressors. PMID- 8634560 TI - Albendazole chemotherapy for treatment of diarrhoea in patients with AIDS in Zambia: a randomised double blind controlled trial. AB - OBJECTIVE: To determine the value of short course, high dose albendazole chemotherapy in the treatment of persistent diarrhoea related to HIV in unselected patients in urban Zambia. DESIGN: A randomised double blind placebo controlled trial of albendazole 800 mg twice daily for two weeks. Patients were monitored intensively for one month and followed for up to six months. SETTING: Home care. AIDS services in Lusaka and Ndola. PATIENTS: 174 HIV seropositive patients with persistent diarrhoea (defined as loose but not bloody stools three or more times a day for three weeks or longer). No investigations were undertaken except HIV testing after counselling. MAIN OUTCOME MEASURES: Proportion of time periods during which diarrhoea was experienced after completion of treatment; proportion of patients with full remission after completion of treatment; mortality. RESULTS: The patients taking albendazole had diarrhoea on 29% fewer days than those taking placebo (P < 0.0001) in the two weeks after treatment. The benefit of albendazole was maintained over six months. In patients with a Karnofsky score of 50 to 70 (needing help with activities of daily living and unable to work, but not needing admission to hospital) diarrhoea was reduced by 50%. Remission was obtained in 26% of all patients who received albendazole (P = 0.004 against 9% receiving placebo), and this difference was maintained over six months (log rank test, P = 0.003). Albendazole had no effect on mortality. Minimal adverse effects were noted. CONCLUSIONS: For HIV infected Zambians with diarrhoea of more than three weeks' duration albendazole offers substantial relief from symptoms and may be used empirically, without prior investigation. PMID- 8634561 TI - Role of patients' view of their illness in predicting return to work and functioning after myocardial infarction: longitudinal study. AB - OBJECTIVE: To examine whether patients' initial perceptions of their myocardial infarction predict subsequent attendance at a cardiac rehabilitation course, return to work, disability, and sexual dysfunction. DESIGN: Patients' perceptions of their illness were measured at admission with their first myocardial infarction and at follow up three and six months later. SETTING: Two large teaching hospitals in Auckland, New Zealand. SUBJECTS: 143 consecutive patients aged under 65 with their first myocardial infarction. MAIN OUTCOME MEASURES: Attendance at rehabilitation course; time before returning to work; measures of disability with sickness impact profile questionnaire for sleep and rest, social interaction, recreational activity, and home management; and sexual dysfunction. RESULTS: Attendance at the rehabilitation course was significantly related to a stronger belief during admission that the illness could be cured or controlled (t = 2.08, P = 0.04). Return to work within six weeks was significantly predicted by the perception that the illness would last a short time (t = 2.52, P = 0.01) and have less grave consequences for the patient (t = 2.87, P = 0.005). Patients' belief that their heart disease would have serious consequences was significantly related to later disability in work around the house, recreational activities, and social interaction. A strong illness identity was significantly related to greater sexual dysfunction at both three and six months. CONCLUSIONS: Patients' initial perceptions of illness are important determinants of different aspects of recovery after myocardial infarction. Specific illness perceptions need to be identified at an early stage as a basis for optimising outcomes from rehabilitation programmes. PMID- 8634562 TI - Incidence and prognosis of asthma and wheezing illness from early childhood to age 33 in a national British cohort. AB - OBJECTIVE: To describe the incidence and prognosis of wheezing illness from birth to age 33 and the relation of incidence to perinatal, medical, social, environmental, and lifestyle factors. DESIGN: Prospective longitudinal study. SETTING: England, Scotland and Wales. SUBJECTS: 18,559 people born on 3-9 March 1958. 5801 (31%) contributed information at ages 7, 11, 16, 23, and 33 years. Attrition bias was evaluated using information on 14, 571 (79%) subjects. MAIN OUTCOME MEASURE: History of asthma, wheezy bronchitis, or wheezing obtained from interview with subjects' parents at ages 7, 11, and 16 and reported at interview by subjects at ages 23 and 33. RESULTS: The cumulative incidence of wheezing illness was 18% by age 7, 24% by age 16, and 43% by age 33. Incidence during childhood was strongly and independently associated with pneumonia, hay fever, and eczema. There were weaker independent associations with male sex, third trimester antepartum haemorrhage, whooping cough, recurrent abdominal pain, and migraine. Incidence from age 17 to 33 was associated strongly with active cigarette smoking and a history of hay fever. There were weaker independent associations with female sex, maternal albuminuria during pregnancy, and histories of eczema and migraine. Maternal smoking during pregnancy was weakly and inconsistently related to childhood wheezing but was a stronger and significant independent predictor of incidence after age 16. Among 880 subjects who developed asthma or wheezy bronchitis from birth to age 7, 50% had attacks in the previous year at age 7; 18% at 11, 10% at 16, 10% at 23, and 27% at 33. Relapse at 33 after prolonged remission of childhood wheezing was more common among current smokers and atopic subjects. CONCLUSION: Atopy and active cigarette smoking are major influences on the incidence and recurrence of wheezing during adulthood. PMID- 8634564 TI - Population mixing and the incidence of childhood leukaemias: retrospective comparison in rural areas of New Zealand. PMID- 8634563 TI - Serum high density lipoprotein cholesterol, alcohol, and coronary mortality in male smokers. AB - OBJECTIVE: To determine whether the increase in mortality from coronary heart disease with high concentration (> 1.75 mmol/l) of high density lipoprotein cholesterol could be due to alcohol intake. DESIGN: Cohort study. SETTING: Placebo group of the alpha tocopherol, beta carotene cancer prevention (ATBC) study of south western population in Finland. PARTICIPANTS: 7052 male smokers aged 50-69 years enrolled to the ATBC study in the 1980s. MAIN OUTCOME MEASURES: The relative and absolute rates adjusted for risk factors for clinically or pathologically verified deaths from coronary heart disease for different concentrations of high density lipoprotein cholesterol with and without stratification for alcohol intake. Similar rates were also calculated for different alcohol consumption groups. RESULTS: During the average follow up period of 6.7 years 258 men died from verified coronary heart disease. Coronary death rate steadily decreased with increasing concentration of high density lipoprotein cholesterol until a high concentration. An increase in the rate was observed above 1.75 mmol/l. This increase occurred among those who reported alcohol intake. Mortality was associated with alcohol intake in a J shaped dose response, and those who reported consuming more than five drinks a day (heavy drinkers) had the highest death rate. Mortality was higher in heavy drinkers than in non-drinkers or light or moderate drinkers in all high density lipoprotein categories from 0.91 mmol/l upward. CONCLUSIONS: Mortality from coronary heart disease increases at concentrations of high density lipoprotein cholesterol over 1.75 mmol/l. The mortality was highest among heavy drinkers, but an increase was found among light drinkers also. PMID- 8634565 TI - Rupture of the uterus: a review of 32 cases in a general hospital in Zambia. PMID- 8634566 TI - Effect of inadvertent intradermal administration of high dose percutaneous BCG vaccine. PMID- 8634567 TI - Creating a shared vision of out of hours care: using rapid appraisal methods to create an interagency, community oriented, approach to service development. AB - OBJECTIVES: To undertake a district wide review of out of hours primary health care services and identify the views of users and providers about current arrangements and options for development. DESIGN: A range of qualitative and quantitative survey methods based on rapid appraisal methods, modified to apply to an inner city district. SETTING: Socially deprived, multiethnic district in south east London with a population of over 700,000. MAIN OUTCOME MEASURES: Strengths and weaknesses of current out of hours services and suggestions for developments. RESULTS: Widespread dissatisfaction with current arrangements was identified, with specific problems relating to access, availability, demand for services, and interagency communication. Several areas for development were identified, including the establishment of an out of hours cooperative, multiagency primary care emergency centres, and telephone advice-triage. Many of these are now being planned or piloted. CONCLUSIONS: Rapid appraisal provided a helpful method, enabling partnerships to be established between local agencies and users in relation to service development. The shared understanding and commitment to improving services that resulted is now having a major impact on out of hours care in the district. PMID- 8634569 TI - Why we need observational studies to evaluate the effectiveness of health care. AB - The view is widely held that experimental methods (randomised controlled trials) are the "gold standard" for evaluation and that observational methods (cohort and case control studies) have little or no value. This ignores the limitations of randomised trials, which may prove unnecessary, inappropriate, impossible, or inadequate. Many of the problems of conducting randomised trials could often, in theory, be overcome, but the practical implications for researchers and funding bodies mean that this is often not possible. The false conflict between those who advocate randomised trials in all situations and those who believe observational data provide sufficient evidence needs to be replaced with mutual recognition of the complementary roles of the two approaches. Researchers should be united in their quest for scientific rigour in evaluation, regardless of the method used. PMID- 8634568 TI - Nurses taking on junior doctors' work: a confusion of accountability. AB - The number of hospital based posts in which nurses take over clinical work previously done by junior doctors is growing. Accountability for the scope of such new roles and the standards of practice which apply to them are still unclear. When analysed together and compared, the regulations arising from the professional bodies (GMC and UKCC), civil law concerning certain wrongs to patients, and employment law are sometimes contradictory and hard to interpret. The resulting uncertainties about appropriate management for clinical roles evolving between the professions, coupled with an increasingly litigious public, put the nurses and consultants involved at risk of complaints and of disciplinary and legal action. Drawing on our current research into changing clinical roles at the medical-nursing interface, we suggest strategies to reduce risk. Doctors and nurses should be equal partners in planning and managing these new posts, patients should be informed adequately about the nature of the postholder's role and training, significant changes in the work of such postholders should be formally acknowledged by the employer and relevant insurers, individuals taking up new roles should have access to legal advice and support to cover legal risk, and national regulatory bodies need to work together to harmonise their codes of practice in relation to changing clinical roles between the professions. PMID- 8634570 TI - ABC of urology. Urinary stone disease. PMID- 8634571 TI - Recurrent pelvic endometriosis and bilateral ureteric obstruction associated with hormone replacement therapy. PMID- 8634572 TI - Depression in carers of elderly people living at home. Alcohol misuse could be a factor. PMID- 8634573 TI - Depression in carers of elderly people living at home. Other studies were misrepresented. PMID- 8634574 TI - Depression in carers of elderly people living at home. Carers' characteristics could be risk factors. PMID- 8634575 TI - Developing a policy on osteoporosis. Clinical risk factors alone are insufficient in decision making. PMID- 8634576 TI - Developing a policy on osteoporosis. Dual energy x ray absorptiometry may not be the gold standard. PMID- 8634577 TI - Developing a policy on osteoporosis. Bone densitometry is worth while. PMID- 8634578 TI - High potency parenteral B complex vitamin preparation is still available. PMID- 8634579 TI - Managing the increased workload in anticoagulant clinics. Computerised decision support is valuable. PMID- 8634580 TI - Managing the increased workload in anticoagulant clinics. Clinics led by nurse practitioners can work well. PMID- 8634581 TI - False positive findings of mammography will have psychological consequences. PMID- 8634582 TI - Seasonal variation in deep vein thrombosis. Daily measurements of temperature should have been used. PMID- 8634583 TI - Seasonal variation in deep vein thrombosis. Fatal pulmonary embolism is increased in both autumn and winter. PMID- 8634584 TI - Stridor in rheumatoid arthritis may be caused by laryngeal amyloidosis. PMID- 8634585 TI - Waist circumference remains useful predictor of coronary heart disease. PMID- 8634586 TI - Shackling prisoners in hospital. Causes problems for doctors. PMID- 8634587 TI - Shackling prisoners in hospital. Is a breach of human rights. PMID- 8634588 TI - Shackling prisoners in hospital. Is unnecessary and avoidable. PMID- 8634589 TI - Nocturia may be due to growing old. PMID- 8634590 TI - Children and sport. De-emphasise team sport. PMID- 8634591 TI - Children and sport. Not all schoolchildren prefer team sports. PMID- 8634592 TI - Children and sport. Walking to school has future benefits. PMID- 8634593 TI - Nomogram for number needed to treat will be of limited use. PMID- 8634594 TI - Health care rationing. Oregon asked people about moral values. PMID- 8634595 TI - Boosting women consultants. PMID- 8634596 TI - Health care rationing. Who will be patients' advocate if doctors assume the rationing role? PMID- 8634597 TI - Problems with the penis and prepuce. Preputioplasty should be performed more often. PMID- 8634598 TI - Cause of AIDS. PMID- 8634600 TI - Egyptian doctors held as prisoners of conscience. PMID- 8634599 TI - Chlamydial infection is asymptomatic in England as well as Tanzania. PMID- 8634601 TI - Electronic mail can be protected from computer viruses. PMID- 8634602 TI - The food industry fights for salt. PMID- 8634603 TI - Salt and blood pressure revisited. PMID- 8634604 TI - Whose data are they anyway? PMID- 8634605 TI - Melatonin. PMID- 8634606 TI - Primary HIV-1 infection: a new medical emergency? PMID- 8634607 TI - Breast cancer deaths decline in US. PMID- 8634608 TI - US in the dark on doubling in asthma death rates. PMID- 8634609 TI - Government plan for care of elderly people. PMID- 8634610 TI - US court case starts over eyeless babies. PMID- 8634611 TI - Supermarket offers allergy tests. PMID- 8634612 TI - Intersalt revisited: further analyses of 24 hour sodium excretion and blood pressure within and across populations. Intersalt Cooperative Research Group. AB - OBJECTIVES: To assess further the relation in Intersalt of 24 hour urinary sodium to blood pressure of individuals and populations, and the difference in blood pressure from young adulthood into middle age. DESIGN: Standardised cross sectional study within and across populations. SETTING: 52 population samples in 32 countries. SUBJECTS: 10,074 men and women aged 20-59. MAIN OUTCOME MEASURES: Association of sodium and blood pressure from within population and cross population multiple linear regression analyses with multivariate correction for regression dilution bias. Relation of sample median daily urinary sodium excretion to difference in blood pressure with age. RESULTS: In within population analyses (n = 10,074), individual 24 hour urinary sodium excretion higher by 100 mmol (for example, 170 v 70 mmol) was associated with systolic/diastolic blood pressure higher on average by 3/0 to 6/3 mm Hg (with and without body mass in analyses). Associations were larger at ages 40-59. In cross population analyses (n = 52), sample median 24 hour sodium excretion higher by 100 mmol was associated with median systolic/diastolic pressure higher on average by 5-7/2-4 mm Hg, and estimated mean difference in systolic/diastolic pressure at age 55 compared with age 25 greater by 10-11/6 mm Hg. CONCLUSIONS: The strong, positive association of urinary sodium with systolic pressure of individuals concurs with Intersalt cross population findings and results of other studies. Higher urinary sodium is also associated with substantially greater differences in blood pressure in middle age compared with young adulthood. These results support recommendations for reduction of high salt intake in populations for prevention and control of adverse blood pressure levels. PMID- 8634614 TI - Lung cancer, smoking, and environment: a cohort study of the Danish population. AB - OBJECTIVE: The almost twofold difference in lung cancer incidence between people living in Copenhagen and in rural area of Denmark in the 1980s led to public concern; this study was undertaken to assess the effects of air pollution and occupation on lung cancer in Denmark, with control for smoking habits. DESIGN: Cohort study of national population. SUBJECTS: People aged 30-64 and economically active in 1970 (927,470 men and 486,130 women). MAIN OUTCOME MEASURES: Relative risks for lung cancer estimated with multiplicative Poisson modelling of incidence rates. RESULTS: Differences in smoking habit explained about 60% of the excess lung cancer risk in Copenhagen for men and 90% for women. After control for smoking, workers had double the lung cancer risk of teachers and academics. There was only a small independent effect of region. CONCLUSION: Smoking is the main factor behind the regional differences in lung cancer incidence in Denmark, and occupational risk factors also seem to have an important role. The outdoor air in Copenhagen around 1970 contained on average 50-80 micrograms/m3 of sulphur dioxide, 80-100 micrograms/m3 total suspended particulate matter, and up to 10 ng/m3 benzo(a)pyrene and had peak values of daily smoke of 120 micrograms/m3. Region had only a small effect on incidence of lung cancer int eh present study, which suggests that an influence of outdoor air pollution on lung cancer is identifiable only above this pollution level. PMID- 8634613 TI - Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. AB - OBJECTIVE: To determine the ability of measurements of bone density in women to predict later fractures. DESIGN: Meta-analysis of prospective cohort studies published between 1985 and end of 1994 with a baseline measurement of bone density in women and subsequent follow up for fractures. For comparative purposes, we also reviewed case control studies of hip fractures published between 1990 and 1994. SUBJECTS: Eleven separate study populations with about 90,000 person years of observation time and over 2000 fractures. MAIN OUTCOME MEASURES: Relative risk of fracture for a decrease in bone mineral density of one standard deviation below age adjusted mean. RESULTS: All measuring sites had similar predictive abilities (relative risk 1.5 (95% confidence interval 1.4 to 1.6)) for decrease in bone mineral density except for measurement at spine for predicting vertebral fractures (relative risk 2.3 (1.9 to 2.8)) and measurement at hip for hip fractures (2.6 (2.0 to 3.5)). These results are in accordance with results of case-control studies. Predictive ability of decrease in bone mass was roughly similar to (or, for hip or spine measurements, better than) that of a 1 SD increase in blood pressure for stroke and better than a 1 SD increase in serum cholesterol concentration for cardiovascular disease. CONCLUSIONS: Measurements of bone mineral density can predict fracture risk but cannot identify individuals who will have a fracture. We do not recommend a programme of screening menopausal women for osteoporosis by measuring bone density. PMID- 8634615 TI - Case-control study of evening melatonin concentration in primary insomnia. PMID- 8634616 TI - Costs and cost effectiveness of health checks conducted by nurses in primary care: the Oxcheck study. AB - OBJECTIVE: To measure the costs and cost effectiveness of the Oxcheck cardiovascular risk factor screening and intervention programme. DESIGN: Cost effectiveness analysis of a randomised controlled trial using clinical and economic data taken from the trial. SETTING: Five general practices in Luton and Dunstable, England. SUBJECTS: 2205 patients who attended a health check in 1989 90 and were scheduled for re-examination in 1992-3 (intervention group); 1916 patients who attended their initial health check in 1992-3 (control group). Participants were men and women aged 35-64 years. INTERVENTION: Health check conducted by nurse, with health education and follow up according to degree of risk. MAIN OUTCOME MEASURES: Cost of health check programme; cost per 1% reduction in coronary risk. RESULTS: Health check and follow up cost 29.27 pounds per patient. Estimated programme cost per 1% reduction in coronary risk per participant was between 1.46 pounds and 2.25 pounds; it was nearly twice as much for men as women. CONCLUSIONS: The cost to the practice of implementing Oxcheck style health checks in an average sized practice of 7500 patients would be 47,000 pounds, a proportion of which could be paid for through staff pay reimbursements and Band Three health promotion target payments. This study highlights the considerable difficulties faced when calculating the costs and benefits of a health promotion programme. Economic evaluations should be integrated into the protocols of randomised controlled trials to enable judgments to be made on the relative cost effectiveness of different prevention strategies. PMID- 8634617 TI - Costs and cost effectiveness of cardiovascular screening and intervention: the British family heart study. AB - OBJECTIVE: To measure costs and cost effectiveness of the British family heart study cardiovascular screening and intervention programme. DESIGN: Cost effectiveness analysis of randomised controlled trial. Clinical and resource use data taken from trial and unit cost data from external estimates. SETTING: 13 general practices across Britain. SUBJECTS: 4185 men aged 40-59 and their 2827 partners. INTERVENTION: Nurse led programme using a family centered approach, with follow up according to degree of risk. MAIN OUTCOME MEASURES: Cost of the programme it self; overall short term cost to NHS; cost per 1% reduction in coronary risk at one year. RESULTS: Estimated cost of putting the programme into practice for one year was 63 pounds per person (95% confidence interval 60 pounds to 65 pounds). The overall short term cost to the health service was 77 pounds per man (29 pounds to 124 pounds) but only 13 pounds per woman (-48 pounds to 74 pounds), owing to differences in utilisation of other health service resources. The cost per 1% reduction in risk was 5.08 pounds per man (5.92 pounds including broader health service costs) and 5.78 pounds per woman (1.28 pounds taking into account wider health service savings). CONCLUSIONS: The direct cost of the programme to a four partner practice of 7500 patients would be approximately 58,000 pounds. Annually, 8300 pounds would currently be paid to a practice of this size working to the maximum target on the health promotion bands, plus any additional reimbursement of practice staff salaries for which the practice qualified. The broader short term costs to the NHS may augment these costs for men but offset them considerably for women. PMID- 8634618 TI - What can be concluded from the Oxcheck and British family heart studies: commentary on cost effectiveness analyses. AB - OBJECTIVES: To provide a commentary on the economic evaluations of the Oxcheck and British family heart studies: direct comparison of their relative effectiveness and cost effectiveness; comparisons with other interventions; and consideration of problems encountered. DESIGN: Modelling from cost and effectiveness data to estimate of cost per life year gained. SUBJECTS: Middle aged men and women. INTERVENTIONS: Screening for cardiovascular risk factors followed by appropriate lifestyle advice and drug intervention in general practice, and other primary coronary risk management strategies. MAIN OUTCOME MEASURES: Life years gained; cost per life year gained. RESULTS: Depending on the assumed duration of risk reduction, the programme cost per discounted life year gained ranged from 34,800 pounds for a 1 year duration to 1500 pounds for 20 years for the British family heart study and from 29,300 pounds to 900 pounds for Oxcheck. These figures exclude broader net clinical and cost effects and longer term clinical and cost effects other than coronary mortality. CONCLUSIONS: Despite differences in underlying methods, the estimates in the two economic analyses of the studies can be directly compared. Neither study was large enough to provide precise estimates of the overall net cost. Modelling to cost per life year gained provides more readily interpretable measures. These estimates emphasise the importance of the relatively weak evidence on duration effect. Only if the effect lasts at least five years is the Oxcheck programme likely to be cost effective. The effect must last for about 10 years to justify the extra cost associated with the British family heart study. PMID- 8634619 TI - Diagnosis and management of migraine. PMID- 8634620 TI - Intersalt: hypertension rise with age revisited. PMID- 8634621 TI - Salt--overwhelming evidence but still no action: can a consensus be reached with the food industry? CASH (Consensus Action on Salt and Hypertension) PMID- 8634622 TI - Hyperkalaemic cardiac arrest successfully treated with peritoneal dialysis. PMID- 8634623 TI - ABC of urology. Common paediatric problems. PMID- 8634624 TI - OxDONS syndrome. Edinburgh's orthopaedic trauma unit has similar problems. PMID- 8634625 TI - OxDONS syndrome. Message of article is still valid. PMID- 8634626 TI - OxDONS syndrome. Oxford case may be start of epidemic. PMID- 8634627 TI - Early controlled trials .... but "quasirandom allocation " of treatment was reported in 1930. PMID- 8634628 TI - Early controlled trials. Randomised mental health trial began in 1935... PMID- 8634629 TI - Low serum cholesterol and serotonin metabolism. Results may have been affected by confounding. PMID- 8634630 TI - Low serum cholesterol and serotonin metabolism. Risk of depression is higher in elderly patients with lowest serum cholesterol values. PMID- 8634631 TI - Low serum cholesterol and serotonin metabolism. Other studies have been done in humans and monkeys. PMID- 8634632 TI - Children and sport. De-emphasise team sport. PMID- 8634633 TI - Correct technique is important in cardioversion of atrial fibrillation. PMID- 8634634 TI - Epley's procedure should be used to treat benign positional vertigo. PMID- 8634635 TI - Physicians must not be misled by arguments against screening with densitometry. PMID- 8634636 TI - Education for educating surgeons. Surgeons' knowledge base needs to be increased. PMID- 8634637 TI - Education for educating surgeons. Training for trainers does exist in Britain. PMID- 8634638 TI - Women need to be fully informed about risks of hormone replacement therapy. PMID- 8634639 TI - Education for educating surgeons. College has found strong demand for training programmes. PMID- 8634640 TI - Compliance therapy in psychotic patients. Authors were wrong to compare treatment with no treatment. PMID- 8634641 TI - Compliance therapy in psychotic patients. Many ethical questions arise from study. PMID- 8634642 TI - Disease in children with HIV infection in Abidjan. Authors should have emphasised lack of tuberculosis in HIV positive children. PMID- 8634643 TI - Fewer needlestick injuries than expected occurred during immunisation campaign. PMID- 8634644 TI - Junior doctor's pay rates for additional duty hours have not been increased. PMID- 8634645 TI - Primary care out of hours. Emergency centre in Midlothian is successful. PMID- 8634646 TI - Health promotion in primary care. Scheme has been a disaster. PMID- 8634647 TI - Health promotion in primary care. Scheme should be comprehensively reviewed. PMID- 8634648 TI - Primary care out of hours. Emergency referral letters from deputising doctors need to be improved. PMID- 8634649 TI - Cancer incidence in the National Health and Nutrition Survey I. Follow-up data: diabetes, cholesterol, pulse and physical activity. AB - We examined cancer incidence among 14,407 men and women who were enrolled in the National Health and Nutrition Survey I in the early 1970s and then followed through 1987. We studied 657 male and 593 female cancer cases, using Cox regression. Analyses were conducted for all cancers, lung, colorectal, breast, and prostate cancer. Analyses focused on diabetes, cholesterol, pulse, and physical activity, four risk factors with limited or inconsistent prior evidence. All four risk factors were modestly associated with all cancers for men but not for women. For diabetic men, the rate ratio for all cancers was 1.38 [95% confidence interval (CI) = 1.00-1.91]; the elevated risk was particularly evident for colorectal and prostate cancer. Slight inverse trends of cancer risk with cholesterol were apparent for men but not for women and were diminished compared to prior analyses of these data with less follow-up. Males with the lowest quartile of cholesterol versus the highest had a rate ratio of 1.21 (CI = 0.98 1.51) for all cancers. A modest positive trend between pulse and all cancers was seen for males [rate ratio of 1.27 (CI = 1.04-1.57)] for the highest versus the lowest quartile). The rate ratio for men with the least amount of nonrecreational physical activity was 1.29 (CI = 0.99-1.69). There is some evidence in these data that findings for cholesterol and nonrecreational physical activity could be artifacts of the early effects of disease because they diminished when cases were restricted to those with longer follow-up. PMID- 8634650 TI - Characterization of the lung cancer epidemic in the European Union (1970-1990). AB - To characterize the situation of the lung cancer epidemic in the former European Community countries, we analyzed mortality time trends between 1970 and 1990, and by using Poisson log-linear models, we compared patterns of evolution between Mediterranean and non-Mediterranean countries. To ascertain the course traced by the cohort effect and the inflection points marking shifts in trend in the epidemic, we made use of invariant parameters from age-period-cohort models (net drift, curvature) and restricted slope range for cohort effect. Regarding men, whereas non-Mediterranean countries had already initiated the downward phase of the process, the Mediterranean countries, with the single exception of Italy, proved to be entirely in the development stage. Women evinced a different pattern of evolution in regard to both rate magnitudes and trend behavior. Apart from Spain and Greece, a sharp rise in adjusted and specific rates was observed for all countries. PMID- 8634651 TI - Do the nonsmoking daughters of smokers tend to marry smokers? Implications for epidemiological research on environmental tobacco smoke: the IARC collaborative study. AB - The IARC collaborative study on exposure to environmental tobacco smoke (ETS) involved collecting interview data and biochemical indicators of exposure from 1369 nonsmoking women in 13 centers in 10 countries. Information on childhood and adulthood exposure to other people's smoke and duration of this exposure from both parents and spouse was gathered at the interview. Of the 900 women whose husbands smoked (current or exsmokers), 71.3% had one or both parents who smoked (predominantly the father), whereas among the 277 women married to never-smokers, only 60.3% had at least one parent who smoked. The odds ratio for the daughter of a smoker to marry a smoker was, therefore, 1.64 (95% confidence interval = 1.24 2.17; P > 0.001), and there was an exposure-response relation between the number of years of childhood exposure to ETS from the parents and the likelihood of being married to a smoker. These results show that nonsmoking women married to smokers are more likely to have been exposed to tobacco pollution during their whole life. Because the duration of exposure is known to be important in the genesis of lung cancer, some of the excess risk of lung cancer in nonsmoking women married to smokers may be due exposure to ETS from parents during childhood. PMID- 8634652 TI - Sunburn associated with increased number of nevi in darker as well as lighter skinned adolescents of northern European descent. AB - The associations of sun exposure, sunburn, skin color, and other constitutional characteristics with the density of nevi (2 mm or more in diameter) were assessed in a study of 410 secondary school children ages 14-15 years in Tasmania, Australia. Skin color was estimated by using a chromameter that measures across the visible light spectrum (400-700 nm). Skin color and lifetime history of sunburn were significant predictors of nevus density on the arms and legs of girls and boys and on the shoulders and backs of boys. The nevus density ratios between the highest and lowest exposure groups were 2.85 for the arms and legs of boys (P < 0.01), 2.19 for the arms and legs of girls (P < 0.01), and 1.72 (P = 0.03) for the shoulders and backs of boys. The increase in nevus density appeared to occur at lower levels of lifetime sunburn in children with light or medium skin than it did in children with darker skin. Darker-skinned children with a history of many sunburns ( > or = 11 lifetime sunburns) had a similar number of nevi compared with their lighter-skinned peers. PMID- 8634653 TI - Cutaneous melanoma in women: anatomic distribution in relation to sun exposure and phenotype. AB - An analysis of the relationship between the anatomic site of cutaneous melanoma, sun exposure, and phenotype was conducted in 355 women with histologically confirmed superficial-spreading melanoma and in 935 control subjects. The most frequent site for superficial-spreading melanoma was the leg. However, when major sun-related and phenotype risk factors were examined by site, risk ratios were lowest for melanomas that occurred on the leg. A history of frequent sunburns during elementary or high school, increased number of self-assessed large nevi, and blond hair were more strongly associated with melanoma sites other than the leg. Tumors on the trunk were more likely than tumors at other sites to be associated with histological evidence of a preexisting nevus. Results of this work indicate that associations between melanoma phenotypic factors may differ by anatomic site. PMID- 8634654 TI - Serological precursors of cancer: malignant melanoma, basal and squamous cell skin cancer, and prediagnostic levels of retinol, beta- carotene, lycopene, alpha tocopherol, and selenium. AB - To determine the association between prediagnostic serum levels of retinol, beta carotene, lycopene, alpha-tocopherol, and selenium and the subsequent risk of malignant melanoma, and basal and squamous cell skin cancer, a nested case control study among residents of Washington County, MD, was performed. Cases with melanoma (n = 30), basal cell (n = 32), and squamous cell (n = 37) skin cancer who were admitted to hospital for treatment or biopsy of metastatic lesions were each matched by age, sex, and race with two controls. There were no significant associations between serum micronutrient levels and the risk of subsequent skin cancer. PMID- 8634655 TI - Alcohol intake and breast cancer risk: effect of exposure from 15 years of age. AB - Research regarding the relationship between alcohol intake and breast cancer risk has suggested an association between the two, although the data are inconsistent regarding dose effects and susceptible populations. To clarify these issues, we investigated the association of breast cancer risk with alcohol intake at various ages in a population-based case-control study nested within a screening cohort in Sweden. Subjects were women 40-75 years old who participated in a screening program in central Sweden. Information about personal characteristics, diet, and alcohol intake was obtained by a questionnaire sent out at the invitation to the screening interview and at a supplementary interview conducted among a sample of women who did and did not develop breast cancer. Alcohol intake did not affect breast cancer risk among women under 50 years old. However, among those over 50 years of age, ever-drinking conferred a relative risk of 1.8 (95% confidence interval = 1.2-2.6). Current and former drinkers had similar increases in risk. No particular latent period of alcohol effect was identified, but drinking later in life to have a bigger effect than did drinking earlier in life. PMID- 8634656 TI - A urinary marker of alcohol intake. AB - Previously, one of us (B. K. T.) developed an assay that measures levels of free ethanol and ethanol conjugates in urine and showed that the mean levels of these ethanol markers in confirmed alcoholics were at least 20-fold higher than those levels in control subjects. In this study, we assessed the relationship of these biomarkers with self-reported levels of alcohol intake in a multiethnic sample of Los Angeles County residents who were male and over the age of 35 years (n = 128; 40 non-Hispanic whites, 46 blacks, 17 Chinese, and 25 Japanese). Regardless of race, the mean levels of free, bound, and total (free plus bound) ethanol were lowest in nondrinkers, intermediate in weekly drinkers, and highest in daily drinkers (P = 0.0001 in all three statistical tests of differences in the three biomarkers). Stepwise discriminant analysis showed that of the three potential biomarkers, total ethanol best discriminates between the three classes of drinkers (non, weekly, and daily), and that additional inclusion of either free or bound ethanol in the discriminant function had negligible effect. Overall, mean level of total ethanol was 2.2 times higher in weekly than in nondrinkers; daily drinkers, in turn, showed a 4.2-fold increase in mean total ethanol relative to weekly drinkers. However, there was no correlation between any of the three biomarkers and self-reported level (in grams of ethanol) of average consumption in either weekly or daily drinkers whose mean intake was about 13 and 42 g of ethanol/day, respectively. As the level of urinary free ethanol and ethanol conjugates showed extraordinary differences among racial groups for a given level of self-reported ethanol intake, the data suggest possible interracial differences in the in vivo elimination rate of ethanol; this latter finding needs to be confirmed in larger studies. PMID- 8634657 TI - Endogenous estrogens and risk of breast cancer by estrogen receptor status: a prospective study in postmenopausal women. AB - A positive association between postmenopausal serum levels of total estradiol, percentage of free estradiol, and percentage of estradiol not bound to sex hormone-binding globulin (SHBG) and breast cancer risk was recently reported by the New York University Women's Health Study (P. Toniolo et al., J. Natl. Cancer Inst., 87: 190-197, 1995). Data from this prospective study are used to assess whether the observed associations differ according to estrogen receptor (ER) status of the tumor. Between 1985 and 1991, 7063 postmenopausal women donated blood and completed questionnaires at a large breast cancer screening clinic in New York City. Before 1991, 130 cases of first primary breast cancer were identified by active follow-up of the cohort. For each case, two controls were selected, matching the case on age at first blood donation and length of storage of specimens. Biochemical analyses were performed on sera that had been stored at -80 degrees since sampling. ER information was abstracted from pathology reports. Separate statistical analyses were conducted of ER-positive, ER-negative, and ER unknown groups (53, 23, and 54 matched sets, respectively). In each of the 3 groups, the mean estradiol and the mean percentage of free estradiol were greater (21-28% and 6-7%, respectively) in cases than in controls. Conversely, the mean percentage of estradiol bound to SHBG was 9-12% lower in cases than in controls. The logistic regression coefficients measuring the strength of the association between estradiol and its free and SHBG-bound fractions and breast cancer risk were similar in the ER-positive, ER-negative, and ER-unknown groups. These data suggest that in postmenopausal women, the association of endogenous estrogens with breast cancer risk is independent of the ER status of the tumor. This result is more compatible with the hypothesis of a progression from ER-positive to ER negative tumors than with the hypothesis that ER status identifies two distinct types of breast cancer. PMID- 8634658 TI - Glutathione S-transferase M1 genotype affects aminobiphenyl-hemoglobin adduct levels in white, black and Asian smokers and nonsmokers. AB - Cigarette smoking is the major cause of bladder cancer in men in the United States, and the arylamines contained in cigarettes smoke, including 4-amino biphenyl (4-ABP), are believed to play an important role in the induction of bladder cancer among smokers. N-acetylation, which is catalyzed by the genetically controlled hepatic N-acetyltransferase enzyme displaying two phenotypes (slow versus rapid), is a detoxification pathway for arylamines with regard to bladder carcinogenesis. In Los Angeles, CA, non-Hispanic white (white), black, and Asian males have comparable smoking habits and yet dramatically different risks of bladder cancer (31 of 100,000 in whites, 16 of 100,000 in blacks, and 13 of 100,000 in Chinese and Japanese). Previously, we have demonstrated that the prevalence of slow acetylators (the high-risk phenotype) was highest in whites (54%), intermediate in blacks (34%), and lowest in Asians (14%). We also showed that mean 3- and 4-ABP hemoglobin adduct levels were significantly higher in cigarette smokers relative to nonsmokers, and that the level increased with increasing number of cigarettes smoke/day. Most importantly, slow acetylators consistently exhibited higher mean levels of ABP hemoglobin adducts relative to rapid acetylators, regardless of race and level of cigarette smoking. We assessed 151 residents of Los Angeles County (CA) who were either white, black, or Asian (Chinese or Japanese) and over the age of 30 years for their glutathione S-transferase M1 (GSTM1) genotype (null versus non-null), acetylator phenotype (slow versus rapid), levels of 3- and 4-ABP hemoglobin adducts, and current use of tobacco products. Whites (27%) had the highest prevalence of the highest risk profile (slow acetylator, GSTM1 null), followed by blacks (15%) and Asians (2.7%), and the difference was statistically significant (P = 0.006). Whites also had less than one-half the prevalence of the "protective" profile (rapid acetylator, GSTM1 non-null) relative to blacks and Asians (23 versus 57%; P = 0.0001). Regardless of race and level of cigarette smoking, mean levels of 3- and 4-ABP hemoglobin adducts were higher in subjects possessing the higher risk (GSTM1/acetylator profile. Mean level of 4-ABP hemoglobin adduct (adjusting for race, cigarette smoking, and acetylator phenotype) was significantly higher in subjects possessing the GSTM1-null versus GSTM1-non-null genotype (46.5 versus 36.0 pg/g Hb; P = 0.037). The comparable difference in mean levels of 3-ABP hemoglobin adduct was borderline significant (1.6 versus 1.1 pg/g Hb; P = 0.07). Thus, our results suggest that GSTM1 is involved in the detoxification of 3- and 4-ABP and may contribute to the racial variation in bladder cancer incidence among white, black, and Asian males in Los Angeles, CA. PMID- 8634660 TI - Increased expression of cytokeratins CK8 and CK19 is associated with head and neck carcinogenesis. AB - Malignant transformation is often associated with alterations in the expression of normal differentiation markers, which may serve as intermediate end points in carcinogenesis and cancer prevention. To identify early changes in differentiation markers during head and neck cancer development, we examined the expression of cytokeratins (CK1, CK8, CK13, and CK19) and involucrin by immuohistochemical methods in surgical specimens from 29 head and neck squamous cell carcinoma patients that included, in addition to carcinoma, adjacent dysplastic lesions (17 cases), hyperplastic lesions (21 cases), and adjacent histologically normal tissues (15 cases) and in specimens from 31 subjects with premalignant oral lesions (e.g, oral leukoplakia) without cancer, CK13 and involucrin were detected in all specimens from the cancer patients, and no differences in their expression were found among the different histopathological group. CK8 was detected in only 2.7% (1 of 36) of adjacent normal and hyperplastic tissues but in 58.8% (10 of 17) and 75.9% (22 of 29) of dysplastic and carcinoma tissues. The corresponding figures for CK19 expression in adjacent normal, hyperplastic, dysplastic, and carcinoma tissues were 13.3, 70, 71.4, and 82.1%, respectively. The expression of CK1 was not related to the progression from normal to malignant. In the leukoplakia lesions, CK8 CK13, CK19 and involucrin were detected in 13.8, 100, 74.2 and 100% of the specimens, respectively. These results demonstrate that CK19 expression increases in hyperplastic lesions and continues to be expressed in dyplastic and malignant lesions, whereas CK8 expression in low in adjacent normal and hyperplastic tissues and increases only in dysplastic and malignant lesions. Thus, CK19 and CK8 could be markers of sequential premalignant changes in head and neck carcinogenesis. PMID- 8634659 TI - Effect of prolonged beta-carotene or DL-alpha-tocopheryl acetate supplementation on ornithine decarboxylase activity in human atrophic stomach mucosa. AB - The effect of beta-carotene and DL-alpha-tocopheryl acetate (alpha-TAc) on the activity of ornithine decarboxylase (ODC) in human atrophic stomach mucosa and intestinal metaplasia (IM) was studied in a double-blind intervention trial. Persons (227) with upper gastrointestinal symptoms and/or atrophic gastritis (AG) were examined. It was found that ODC activity in the biopsies of antral mucosa increased gradually from normal mucosa (7.2 +/- 1.8 units) to superficial gastritis (22.7 +/- 5.9 units) and to AG (54.2 +/- 6.9 units). Enzyme activity in cases of IM did not differ from atrophic mucosa without IM (56.1 +/- 8.0 versus 51.4 +/- 5.6 units; P > 0.05). For the intervention trial, 3 groups of 20 patients with AG were studied. Patients were supplemented daily for 1 year with beta-C (20 mg; group 1), alpha-TAc (55 mg; group 2), or placebo (group 3). No significant change in ODC activity was observed in placebo-treated subjects during 1-year follow-up. During the first 3 months, beta-C supplementation resulted in about a 50% decrease in ODC activity in atrophic mucosa. A moderate decrease in ODC activity of approximately 18% was observed after 6 months supplementation with alpha-TAc. The possible role of ODC in gastric carcinogenesis is discussed. PMID- 8634661 TI - Effects of watercress consumption on metabolism of a tobacco-specific lung carcinogen in smokers. AB - Epidemiological studies indicate that vegetable consumption protects against lung cancer in humans, but the protective constituents have not been identified. Phenethyl isothiocyanate (PEITC), which is release upon chewing of watercress (nasturtium officinale), is a chemopreventive agent against lung cancer induced by the tobacco-specific lung carcinogen 4- (methylnitrosamino)-1-(3-pyridyl-1 butanone (NNK) in rats and mice. PEITC inhibits the carcinogenicity of NNK by inhibiting its metabolic activation and thereby increasing the levels of detoxified metabolites excreted in urine. In this study, our goal was to determine whether watercress consumption would modify NNK metabolism in smokers. Eleven smokers maintained constant smoking habits and avoided cruciferous vegetables and other sources of isothiocyanates throughout the study. They donated 24-h urine samples on 3 consecutive days (baseline period). One to 3 days later, they consumed 2 ounces (56.8 g) of watercress at each meal for 3 days and donated 24-h urine samples on each of these days (watercress consumption period). One and 2 weeks later, they again donated 24-h urine samples on 2-3 consecutive days (follow-up periods). The samples were analyzed for two metabolites of NNK; 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-methylnitrosamino)-1-(3 pyridyl)but-1-yl]-beta-omega-D-glucosiduro nic acid (NNAL- Gluc). NNAL-Gluc is believed to be a detoxification product of NNK. The urine samples were also analyzed for PEITC-NAC, a metabolite of PEITC. Minimum exposure to PEITC during the watercress consumption period averaged 19-38 mg/day. Seven of the 11 subjects had increased levels of urinary NNAL plus NNAL-Gluc on days 2 and 3 of the watercress consumption period, compared to the baseline period. Overall, the increase in urinary NNAL plus NNAL-Gluc in this period was significant [mean +/- SD 0.924 +/- 1.12 nmol/24 h (33.5%); P < 0.01]. Urinary levels of NNAl plus NNAL Gluc returned to near baseline levels in the follow-up periods. The percentage of increase in urinary NNAL plus NNAL-Gluc during days 2 and 3 of the watercress consumption period correlated with intake of PEITC during this period, as measured by total urinary PEITC-NAC (r = 0.62; P = 0.04). The results of this study support our hypothesis that PEITC inhibits this oxidative metabolism of NNK in humans, as seen in rodents, and support further development of PEITC as a chemopreventive agent against lung cancer. This is the first study to report an effect of vegetable consumption on metabolism of a lung carcinogen in humans. PMID- 8634663 TI - Food habits and pancreatic cancer: a case-control study of the Francophone community in Montreal, Canada. AB - In a population-based case-control study of pancreatic cancer and nutrition among the Francophone population of Montreal (Quebec, Canada), a total of 179 cases and 239 controls matched for age, sex, and language (French) were interviewed between 1984 and 1988. Data on food habits, methods of food preparation and preservation, and related information were obtained through a questionnaire. The study found an increased risk of pancreatic cancer associated with a high consumption of salt [relative risk (RR) = 4.28; 95% confidence interval (CI) = 2.20-8.36], smoked meat (RR = 4.68; CI = 2.05-10.69), dehydrated food (RR = 3.10; 95% CI = 1.55 6.22), fried food (RR= 3.84; 95% CI = 1.74-8.48), and refined sugar (RR = 2.81; 95% CI = 0.94-8.45). An inverse association was found with the consumption of food with no preservatives or additives (RR = 0.08; 95% CI = 0.01-0.59), raw food (RR = 0.28; 95% CI = 0.10-0.75), and food prepared by presto or high-pressure cooking (RR = 0.35% 95% CI = 0.15-0.81), electricity (RR = 0.30; 95% CI = 0.90), or microwave oven (RR = 0.56; 95% CI = 0.34-0.92). Cooking with firewood was associated with a significantly higher risk for pancreatic cancer (RR = 4.63; 95% CI = 1.15-16.52). The results of this study suggest that food habits may play an important role in the etiology of cancer of the pancreas among French Canadians in Montreal, whereas other food habits may reduce the risk of this disease. PMID- 8634662 TI - Dietary factors and the risk of pancreatic cancer: a case-control study in Shanghai China. AB - In Shanghai, China, age-adjusted incidence rates for pancreatic cancer have increased steadily, beginning in the early 1970s. To examine the effects of diet on this cancer, a population-based case-control study was conduct. Cases (n = 451) were permanent residents of Shanghai, 30-74 years of age, newly diagnosed with pancreatic cancer between October 1, 1990 and June 30, 1993. Decreased cases (19%) were excluded from the study. Controls (n = 1552) were selected among Shanghai residents, frequency matched to cases by gender and age. Information on usual adult dietary intake was obtained by trained interviewers in person, using a food frequency questionnaire. Dietary associations were measured by odds ratios and 95% confidence intervals. Risks of pancreatic cancer were inversely associated with consumption of vegetables (P for trend among men = 0.03; among women = 0.15) and fruits (P among men = 0.02; among women = 0.08). Reductions in risk were related also to intake of dietary fiber and micronutrients abundant in plant sources, such as vitamins C and E and carotene. There was also an inverse association with egg consumption (P for trend among men = 0.08; among women = 0.001). No consistent positive associations were observed with intake of other food groups, including preserved animal foods, fresh red meat, organ meat, poultry, and staple foods. On the other hand, risks increased with frequency of consumption of preserved vegetables and foods that were deep fried, grilled, cured, or smoked, providing clues to the possible role of nitrosamines, polycyclic aromatic hydrocarbons, and heterocyclic aromatic amines. The inverse associations observed with intake of dietary fat and protein in our study were unexpected, although these findings were based on consumptions well below the average intake in Western countries, where most previous studies on pancreatic cancer were conducted. Our results suggest that dietary variations have contributed little to the rising trends of pancreatic cancer in Shanghai. However, given the improving food availability and changing dietary patterns in China, further study of dietary and nutritional risk factors for pancreatic cancer appears warranted. PMID- 8634664 TI - Who uses screening mammography regularly? AB - We evaluated factors associated with the regular use of screening mammography among women presenting for screening. Six thousand two hundred forty-four women ages 55 and older who participated in the 1991-1992 Texas Breast Screening Project were classified as regular or irregular users of mammography according to self-reported mammographic history since 1986. Logistic regression was applied to determine odds ratios of specified factors. Fourteen % were regular users of mammographic screening. Being older, black or Hispanic, receiving regular care from a family doctor, believing in a lower prospect of cure of breast cancer, and lacking health insurance coverage were associated with less regular use of screening mammography. Higher educational level, family history of breast cancer, prior breast biopsy, annual income > or = +35,000, receiving regular care from a gynecologist, believing that life would be difficult with breast cancer, and believing in a greater personal risk for breast cancer were associated with a greater likelihood of regular use (P < 0.01). Among multiple factors associated with regular use of screening mammography, sociodemographic variables associated with regular mammography use are similar to those influencing initial use of screening mammography. Women who are difficult to persuade to obtain mammographic screening may be equally difficult to persuade to adhere to regular use. PMID- 8634665 TI - Microsatellite instability is infrequent in neuroblastoma. AB - Neuroblastoma (NB) is a childhood cancer of the autonomic nervous system. The molecular pathology of NB is not yet well understood. Both amplification of the proto-oncogene N-myc and loss of heterozygosity of several chromosomal loci occur in NB, representing genetic instability. In this study, we examined another type of genetic instability, microsatellite instability. Five chromosomal loci known to exhibit this alteration in colon, gastric, and pancreatic cancers were used in a PCR-based assay to examine 30 matched normal and tumor DNAs, which included all stages of tumor progression. Among these 30, only 2 (7%) manifested microsatellite instability. There was no correlation between the occurrence of microsatellite instability and the amplification of the N-myc gene. These data show that microsatellite instability is infrequent in neuroblastoma tumors. PMID- 8634666 TI - [Effect of thapsigargin and cyclopiazonic acid on Mg2+,ATP-dependent Ca2+ accumulation stimulated by oxalate (phosphate) in permeabilized myometrial cells]. PMID- 8634668 TI - [Immunoreactivity of Clethrionomys species voles at various phases of the population cycle]. PMID- 8634667 TI - [RecA proteins in Escherichia coli: damage to the C-terminal subdomain decreases multimerization of the protein]. PMID- 8634669 TI - [Development of sheep, transgenic in genetic design of alphaS1-casein/chymosin]. PMID- 8634670 TI - [Detection of a Drosophila melanogaster cDNA sequence, homologous to the transcript of the protein disulphide isomerase gene]. PMID- 8634671 TI - [Strategy for using a three-dimensional space in a tropical forest by three species of rats in Vietnam]. PMID- 8634672 TI - [A system of P-M hybrid dysgenesis in a Drosophila melanogaster population native to Altai]. PMID- 8634673 TI - The role of insulin in the control of hepatic fatty acid and glycerolipid metabolism assessed by studies in vivo. AB - The liver secretes triacylglycerol-rich very-low-density lipoproteins (VLDL). Teleologically, it would be expected that during the prandial/early absorptive period the rate of hepatic VLDL secretion would be inhibited, in view of the concomitant secretion of chylomicrons from the gut. Such inhibition would limit the extent and duration of post-prandial hyperlipaemia. Experiments in which the fatty acids of the liver were labelled specifically in rats in vivo show that, during a mean, triacylglycerol secretion was inhibited through a combination of diversion of flux towards phospholipid synthesis, and inhibition of the fractional rate of secretion of triacylglycerol. These adaptations occur even in diabetic rats, indicating that insulin is not obligatorily involved in mediating them. It is suggested that uptake of osmolytes (e.g. amino acids that are cotransported with Na+ ions into hepatocytes) from the portal circulation may result in increased hepatocyte volume and that this, in turn, alters fatty acid and glycerolipid metabolism independently of, but possibly in synergy with, insulin action. PMID- 8634674 TI - Alkaloids from frog skins: selective probes for ion channels and nicotinic receptors. AB - Amphibian skin has provided a wide range of biologically active alkaloids, many of which have unique profiles of pharmacological activity and therapeutic potential. Over three hundred alkaloids have been identified and structures of over a dozen different classes of alkaloids have been elucidated. These include the batrachotoxins, which were shown to be potent and selective activators and ligands for sodium channels, the histrionicotoxins, which were shown to be potent non-competitive blockers and ligands for nicotine receptor channel complexes, the pumiliotoxins and related allo- and homo-pumiliotoxins, which were shown to have myotonic and cardiotonic activity due to effects on sodium channels, and epibatidine, which was shown to have potent antinociceptive activity due to selective agonist activity at nicotinic receptors. These alkaloids are known in nature only in amphibian skin, except for homobatrachotoxin, which was recently identified in feathers and skin of a bird. Further classes of alkaloids from amphibian skin include monocyclic pyrrolidines and piperidines, bicyclic decahydroquinolines, pyrrolizidines, indolizidines, and quinolizidines and tricyclic gephyrotoxins, pyrrolizidine oximes, pseudophrynamines, and coccinellines. These alkaloids also have activity in ion channels. It appears likely that all of the frog skin alkaloids are taken up and sequestered into the skin from the diet, which for such amphibians consists mainly of small arthropods. The pyrrolidines, piperidines, 3,5-disubstituted pyrrolizidines and 3,5-disubstituted indolizidines appear likely to be derived from ants, the coccinellines and related tricyclics from beetles and the pyrrolizidine oximes from millipedes. The origins of the batrachotoxins, histrionicotoxins, pumiliotoxins and epibatidine are of particular interest in view of their remarkable biological activities. PMID- 8634675 TI - Organization and structure of an Onchocerca beta-tubulin gene. AB - In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, alpha and beta. Tubulin is particularly important in helminthic parasites as a target for anthelminthic benzimidazoles, which bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in lambda NM1149 (EcoRI and HindIII). Three clones accounted for the entire gene: one from the EcoRI library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3'-ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5'- and 3'-end clones was done by using a series of oligonucleotides. The sequence of the O. gibsoni beta-tubulin gene was completely determined, revealing an exceptionally complex structure as compared to the known beta-tubulin genes: 5,797 base pairs containing 12 exons and 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein. PMID- 8634676 TI - A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11). AB - An intramolecularly quenched fluorogenic peptide structurally related to Leu enkephalin, Abz-GGDFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127/min and kcat/Km = 42/min microM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragment Abz-GGDFLRR and V-EDDnp accounted for more than 95% of the products which are totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 15 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations. PMID- 8634677 TI - Transforming growth factor beta (TGF beta) activity in urine of patients with glomerulonephritis. AB - Several lines of experimental evidence have shown that transforming growth factor beta (TGF beta) may play major role in glomerular diseases, mediating the inflammatory response through glomerulosclerosis. In the present study we evaluated TGF beta activity in occasional urine samples from 7 normal individuals and from 15 patients (10 with focal glomerular sclerosis and 5 with membranous glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay. Urinary TGF beta activity (reported in relation to urine creatinine concentration, Ucr, mean +/- SD) was higher in patients with focal glomerular sclerosis (mean = 17.32 +/- 15.75/10 micrograms Ucr) and patients with membranous glomerulonephritis (mean = 17.78 +/- 11.53/10 micrograms Ucr) than in normal individuals (mean = 0.8 +/- 0.44/10 micrograms Ucr). We also observed that TGF beta activity in urine from patients with focal glomerular sclerosis correlated with their plasma creatinine levels (r = 0.85), suggesting that TGF beta activity may be correlated with other indices of disease progression. Our data suggest that measurement of urinary TGF beta activity could be a useful noninvasive procedure for the evaluation of renal TGF beta production, which may be useful to assess prognosis and to evaluate therapeutic efficacy in patients with renal disease. PMID- 8634678 TI - Diagnosis of bacterial meningitis and septicemia by serum counterimmunoelectrophoresis. AB - We compare the results obtained by counterimmunoelectrophoresis in samples of serum and cerebrospinal fluid with microbiologic methods for 3,298 patients suspected of bacterial meningitis and/or septicemia at Instituto Adolfo Lutz, Sao Paulo, in a retrospective study of the period from July 1998 to July 1994. Of the 415 patients (12.6% of the total cases studied) who were positive by the serum test, only 249 (7.6% of the total cases studied) were also positive when cerebrospinal fluid was assayed. Thus, 40% of the positives (5.6% of the total) were identifiable by analysis of serum but not of cerebrospinal fluid. Neisseria meningitidis accounted for 77.7% (129) and Haemophilus influenzae for 22.3% (37) of the positive results obtained only when serum was examined. These data show that although sensitivity and specificity of serum counterimmunoelectrophoresis are relatively low compared to cerebrospinal fluid counterimmunoelectrophoresis, the serum test is necessary to complement cerebrospinal fluid counterimmunoelectrophoresis data. PMID- 8634679 TI - The effect of thalidomide on BCG-induced granulomas in mice. AB - Granuloma proliferation is the result of a series of complex biological events in which a variety of cell types and cytokines are involved. Tumor necrosis factor alpha (TNF-alpha) plays a central role. In the present study, we investigated the effect of thalidomide (alpha-N-pthalimidoglutarimide), a selective inhibitor of TNF-alpha synthesis, on granuloma formation during BCG infection in Oncins France 1 (OF-1) mice. Subcutaneous injections of 30 mg/kg body weight of thalidomide daily for 14, 21 or 28 days into the mice resulted in the reduction of the size and total number of liver granulomas. The most striking effect of thalidomide was observed after 28 days, when the total number of liver granulomas was reduced by as much as 40% (P < 0.05). Serum TNF-alpha levels of thalidomide-treated mice were significantly lower (85%) than control mice on day 14 and remained lower (55%) on days 21 and 28. Positive immunohistochemical staining specific for TNF alpha were demonstrable only in well-developed granulomas in which central mononuclear cells presented extensive differentiation into epithelioid cells. Daily administration of thalidomide for 21 to 28 days to the BCG-infected mice inhibited local TNF-alpha expression in well-developed granulomas. The mechanisms by which thalidomide modulates the granuloma proliferation are discussed. PMID- 8634680 TI - Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay. AB - The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of costs and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 micrograms/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 microliters/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H2O2/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r = 0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization potency and low absorbance in ELISA or high absorbance values and low in vivo protection were not included in the correlation analysis the coefficient value was r = 0.88. The correlation coefficient did not improve for all 16 antibothropic sera when a partially purified Bothrops jararaca venom fraction was used to coat the ELISA plates. The results indicate that ELISA could be used to determine the neutralizing potency of anticrotalic venom sera. For the antibothropic venom sera further studies are needed. PMID- 8634681 TI - Reduced humoral immunity in uremic mice genetically selected for high antibody response. AB - Biozzi's Selection IV-A mice, genetically selected for 25 generations for high and low antibody response to sheep red blood cells (SRBC), 2-3 months old, were made uremic by subtotal nephrectomy and characterized for antibody production against the selection antigen. T cell activity was evaluated in vitro by lymphocyte proliferation and interleukin 2 (IL 2) production in response to the super antigen staphylococcal enterotoxin B (SEB). Total and IgM antibody titers (log2) were similar in uremic and non-uremic low responder mice (total antibody: 4.0 +/- 0.6 vs 3.6 +/- 0.6; IgG: 3.0 +/- 0.7 vs 2.4 +/- 0.4), while uremic high responders presented with non-uremic animals (total antibody: 10.8 +/- 1.6 vs 13.0 +/- 0.2; IgG: 10.3 +/- 1.5 vs 11.7 +/- 0.3). T cell proliferation and IL 2 production were similar in uremic and non-uremic groups after SEB stimulation. The results indicate a genetic effect on sensitivity to humoral immune response modulation by uremic status, with deficient antibody production despite a normal T cell proliferative response to mitogen stimulation in short-duration renal failure in high responder mice. PMID- 8634682 TI - Operant discrimination learning in detelencephalated pigeons (Columba livia). AB - Operant discrimination learning was analyzed in pigeons after massive telencephalic lesions. Twenty-one pigeons were divided into three groups: non lesioned (N = 6), sham-lesioned (N = 5) and telencephalon lesioned (N = 10). Lesion surgeries were carried out before any experimental training. Learning procedures were run in the same sequence for all groups and under a food deprivation of 80% of the ad libitum weight. Successive discrimination was programmed by the alteration of red and yellow lights in the right key of a standard operant chamber: the red key was correlated with variable-ratio reinforcement; the yellow key was correlated with extinction. Session were run until steady-state key peck rates were obtained. The following results demonstrate discrimination learning by detelencephalated birds. Response shaping and steady-state rates required a larger number of sessions for lesioned pigeons (P < 0.05). They showed increased response rates in red (26.43 +/- 2.59) and yellow (11.17 +/- 2.86) components as compared to the non-lesioned (red: 16.51 +/ 2.0; yellow: 2.02 +/- 0.64) and sham-lesioned (red: 22.84 +/- 1.77; yellow: 4.72 +/- 1.99) groups (P < 0.05). These data show that telencephalic systems are not essential for operant discrimination learning but play a role in the modulation of discriminative behavior. Subtelencephalic systems appear to be functionally important for the organization and storage of learning. PMID- 8634683 TI - The role of angiotensin AT1 receptors in the diuretic, natriuretic, kaliuretic and blood pressure responses induced by angiotensin II activation of the median preoptic nucleus in conscious rats. AB - We determined the effects of two classical angiotensin II (ANG II) antagonists, [Sar1, Ala8]-ANG II and [Sar1, Thr8]-ANG II, and losartan (a nonpeptide and selective antagonist for the AT1 angiotensin receptors) on diuresis, natriuresis, kaliuresis and arterial blood pressure induced by ANG II administration into the median preoptic nucleus (MnPO) of male Holtzman rats weighing 250-300 g. Urine was collected in rats submitted to a water load (5% body weight) 1 h later. The volume of the drug solutions injected was 0.5 microliters over 10-15 s. Pre treatment with [Sar1, Ala8]-ANG II (12 rats) and [Sar1, Thr8]-ANG II (9 rats), at the dose of 60 ng reduced (13.7 +/- 1.0 vs 11.0 +/0 1.0 and 10.7 +/0 1.2, respectively), whereas losartan (14 rats) at the dose of 160 ng totally blocked (13.7 +/- 1.0 vs 7.6 +/- 1.5) the urine excretion induced by injection o 12 ng of ANG II (14 rats). [Sar1, Ala8]-ANG II impaired Na+ excretion (193 +/- 16 vs 120 +/- 19), whereas [Sar1, Thr8]-ANG II and losartan block Na+ excretion (193 +/- 16 vs 77 +/- 15 and 100 +/- 12, respectively) induced by ANG II. Similar effects induced by ANG II on K+ excretion were observed with [Sar1, Ala8]-ANG II, [Sar1, Thr8]- ANG II, and losartan pretreatment (133 +/- 18 vs 108 +/- 11, 80 +/- 12, and 82 +/- 15, respectively). The same doses as above of [Sar1, Ala8]-ANG II (8 rats), [Sar1, Thr8]-ANG II (8 rats), and losartan (9 rats) blocked the increase in the arterial blood pressure induced by 12 ng of ANG II (12 rats) (32 +/- 4 vs 4 +/- 2, 3.5 +/- 1, and 2 +/- 1, respectively. The results indicate that the AT1 receptor subtype participates in the increases of diuresis, natriuresis, kaliuresis and arterial blood pressure induced by the administration of ANG II into the MnPO. PMID- 8634684 TI - Participation of TNF-alpha and IL-1 in the pathogenesis of cyclophosphamide induced hemorrhagic cystitis. AB - The involvement of cytokines TNF-alpha and IL-1 has been investigated in a mouse model of cyclophosphamide (CYP)-induced hemorrhagic cystitis. Male Swiss mice (25 30 g) received CYP in a single i.p. dose of 100, 200 or 400 mg/kg and were sacrificed 6, 12, 24, 48 and 72 h later. Cystitis was evaluated by determining the changes in bladder wet weight (BW) and plasma protein extravasation (PPE, measured by the Evans blue leakage technique). CYP treatment induced a marked increase in BW and in PPE, which was significant within 6 h and reached maximal values within 12 h (BW, 118%, P < 0.05; N = 11; and PPE, 824%, P < 0.05; N = 11), continuing to be significant until 48 h. Pretreatment of animals with whole anti TNF-alpha serum (25 or 50 microliters diluted in 500 microliters 0.9% saline, i.p., 30 min earlier) caused a significant reduction in the CYP-induced BW increase in 6-h and 12-h cystitis (82% and 91%, respectively, P < 0.05; N = 6) and in the CYP-induced PPE increase (60% and 52%, respectively, P < 0.05; N = 6). In addition, the administration of whole anti-IL- 1 beta serum at the same dose promoted a significant blockage of the CYP-induced increase in BW (47%, P < 0.05; N = 6) and PPE increase (41%, P < 0.05; N = 6) only in 12-h cystitis. The control serum did not modify the effect of CYP. Histopathologic analysis of the bladders from anti-TNF-alpha- and anti-IL-1 beta-pretreated groups revealed a significant reduction of the following parameters compared to the control groups: mucosal erosion, hemorrhage, edema, leukocyte migration, fibrin deposition and ulcerations. These results suggests that TNF-alpha and IL-1 are crucial mediators involved in inflammatory events occurring in CYP-induced hemorrhagic cystitis. PMID- 8634685 TI - Chemical sympathectomy blocks androgen biosynthesis during prepuberty. AB - Twenty-one-day old male Wistar rats were injected subcutaneously with guanethidine (GUA) at doses of 5 and 10 mg kg-1 day-1 for 20 days. Animals were sacrificed by decapitation during the prepubertal (41 days of age) and early pubertal (51 days of age) periods of sexual development. The testes were collected, frozen in liquid N2 and stored at -70 degrees C until determination of testicular progesterone (P), androstenedione (A) and testosterone (T). Higher levels of P (2.18 +/- 0.24 ng/g, control = 1.24 +/- 0.16 ng/g) associated with decreased levels of androgens (A = 0.26 +/- 0.06 ng/g and T = 2.05 +/- 0.19 ng/g; control = 1.86 +/- 0.76 ng/g and 8.48 +/- 1.16 ng/g, respectively) were observed in 10 mg GUA-treated rats of prepubertal age, while only P levels (3.12 +/- 0.51 ng/g, control = 1.73 +/- 0.27 ng/g) were increased in rats of early pubertal age. It is important to note that in 41-day old male rats both 5 and 10 mg were effective in decreasing testicular concentration of testosterone. These results suggest that the sympathetic innervation of the testis is involved in the modulation of androgen biosynthesis, acting through a selective step in the steroid biochemical pathway during the pubertal process and that under the conditions employed the blockage in androgen biosynthesis in the prepubertal stage of sexual maturation in dependent on the dose of GUA. PMID- 8634686 TI - Respiratory mechanics after tube thoracostomy in rats. AB - The purpose of the present study was to determine the mechanical respiratory profile after the insertion of a catheter into the pleural cavity of anesthetized, paralyzed, mechanically ventilated rats, thus stimulating the common use of chest tubes in clinical situations. Using the method of end inflation occlusion during constant inspiratory flow in 7 adult Wistar rats, respiratory system, lung, and chest wall total resistance (0.353 +/- 0.058, 0.260 +/- 0.651, 0.091 +/- 0.012 (mean +/- SD) cmH2O.ml-1.s, respectively), viscous resistance (0.140 +/- 0.007, 0.100 +/- 0.007, 0.040 +/- 0.003 cmH2O.ml-1.s< respectively), and viscoelastic resistance (0.213 +/- 0.017, 0.160 +/- 0.022, 0.053 +/- 0.011 cmH2O.ml-1.s, respectively) as well as respiratory system, lung, and chest wall static elastance (4.51 +/- 0.27, 3.85 +/- 0.28, 0.66 +/- 0.12 cmH2O.ml-1, respectively), and dynamic elastance (5.72 +/- 0.24, 4.76 +/- 0.32, 0.96 +/- 0.17 cmH2O.ml-1, respectively) were not significantly modified after the insertion of a tube into the second right intercostal stage. We conclude that, under the present experimental conditions, a catheter inserted into the pleural space per se is not responsible for any alterations in respiratory mechanics. PMID- 8634688 TI - Population survey of the human FMR1 CGG repeat substructure suggests biased polarity for the loss of AGG interruptions. AB - Both sequence length and sequence content are important parameters in determining stability of the fragile X syndrome CGG repeat. In order to estimate the incidence of uninterrupted CGG repeats in the general population and to gain insight into mechanisms responsible for the loss and acquisition of AGG interruptions, 406 randomly selected FMR1 CGG repeat alleles from four broad ethnic groups were assayed for AGG punctuation. Among the 79 different classes of alleles detected, long uninterrupted tracts of pure repeats were rare in the general population, with only 1/406 or 0.25% found at the instability threshold (34-37 pure CGG repeats). There was no significant difference (P>0.05) in the distribution of alleles with long (>20) pure repeat tracts among the different ethnic groups, suggesting that different ethnic groups should be equally susceptible to the development of the disease. Analysis of an additional 43 alleles with total repeat lengths between 35 and 50 repeats, revealed that highly interrupted CGG repeats alleles (>2 AGG interruptions) occur preferentially at modal repeat lengths in the population, providing confirmatory evidence that the presence of AGG interruptions confers stability. A consideration of length variation of the most 3' tract of pure repeats revealed a bimodal distribution pattern with maxima at approximately 10 and 20 repeats. Only unimodal distributions with maxima 9 or 10 were observed for the 5' tract and middle CGG tract within the FMR1 CGG repeat substructure. These results suggest that the loss of the most 3' AGG interruption or its conversion to CGG is a common event in the human population, occurring by a mechanism which preserves overall repeat length. This bias for loss of the distal-most AGG interruption likely plays an important part in predisposing human alleles to the development of the X syndrome. PMID- 8634687 TI - Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. AB - Robertsonian translocations are the most common structural dicentric rearrangements in humans. The stability of these dicentrics is attributed to the inactivation of one centromere by mechanisms which are currently unknown. The presence and amounts of centromeric proteins (CENPs) differ between the centromeres of the few dicentrics which have been studied, providing a limited understanding of the protein components necessary for centromeric function. However, CENP-C previously has been observed only at the active centromeres in two dicentric chromosomes. In the present investigation, the presence and localizations of several centromeric antigens, CENP-B, -C and -E, have been determined in 12 dicentric Robertsonian translocations. Each translocation was studied initially using in situ hybridization with alpha-satellite DNA probes to determine the active centromere. Subsequent immunofluorescence of monoclonal and polyclonal antibodies generated to various centromeric antigens demonstrated that the protein composition differs at the two centromeres of these dicentric translocations. While CENP-B was present at both active and inactive centromeres, CENP-C and -E were located at active centromeres only in the majority of translocations. These results confirm previous observations of CENP-C at active centromeres and provide the first evidence that CENP-E correlates with active centromeres as well, demonstrating that at least two specific centromeric proteins are required for human centromeric function. PMID- 8634689 TI - Highly conserved 3' UTR and expression pattern of FXR1 points to a divergent gene regulation of FXR1 and FMR1. AB - A search for genes with sequence homologies to the FMR1 gene resulted in the isolation of mouse and human homologues of the recently described FXR1 gene. The mouse FXR1 gene shares amino acid identity and similarity of 99.1% and 99.6%, respectively, with the human FXR1 gene and amino acid identify and similarity of 67.3% and 79.5% respectively, with the mouse FMR1 gene. The 3' untranslated region of the FXR1 gene is extremely conserved between human and mouse. The gene structure of FXR1 is very similar to that of FMR1 and both genes probably originate from a common ancestral gene. In contrast to the previously published localization, we mapped the transcribed gene to chromosome region 3q28. An intronless form of the FXR1 gene, either processed functional homologue or pseudogene was localized to 12q12. Northern blot analysis of the human FXR1 gene revealed an expression pattern of a housekeeping gene with stronger expression in muscle. RNA in situ hybridization to sections of mouse embryo and adult tissues has shown that during embryonic development the mouse FXR1 mRNA is expressed in different tissues, most prominent in skeletal muscle, the gonads and distinct regions of the central nervous system, and that the expression is restricted to proliferating cells. While FMR1 is highly expressed in proliferating spermatogonia, FXR1 is highly expressed in postmeiotic spermatids. PMID- 8634691 TI - Non-disjunction in human sperm: evidence for an effect of increasing paternal age. AB - In humans, the relationship between advancing maternal age and the incidence of trisomy has been long established, but the possible effect of increasing age of the father remains controversial. Using a fluorescence in situ hybridization (FISH) approach to directly examine individual sperm for aneuploidy of the sex chromosomes and chromosome 18, we have analyzed approximately 400,000 sperm from 24 men aged 18-60 years. There was no obvious relationship between increasing age and disomy 18, but the incidence of XY,YY and XX disomy all were significantly elevated among older men. This suggests that older men, like older women, have an increased likelihood of producing aneuploid offspring by comparison with their younger counterparts. PMID- 8634692 TI - Germline mutations in the von Hippel-Lindau disease (VHL) gene in Japanese VHL. Clinical Research Group for VHL in Japan. AB - The von Hippel-Lindau disease (VHL) gene is a putative tumor suppressor gene responsible for VHL, an autosomal dominantly inherited multitumor syndrome. It is also implicated in the development of sporadic tumors including clear cell renal carcinoma and central nervous system hemangioblastoma. To define the molecular basis of VHL patients in Japanese populations, we tested for germline mutations of the VHL gene in 45 unrelated Japanese VHL patients by single-strand conformation polymorphism (SSCP) analysis and Southern blot analysis. We detected 23 (51%) intragenic mutations and three (6.7%) deletions by SSCP analysis and Southern blot respectively. The intragenic mutations consisted of 14 missense mutations, seven microdeletions or insertions and two splice-site mutations. Interestingly, nine of 10 mutations in exon 1 are localized in a short region of 37 nucleotides. Five unique sites of mutation were included, which were not seen in previous studies. Unlike Western VHL patients, nonsense mutations were not found in Japanese VHL patients. If the presence of pheochromocytomas is regarded as phenotypic marker for VHl classification, the mutations found in 22 VHL patients without pheochromocytoma consisted of 11 missense mutations, six microdeletions or insertions, two splice-site alterations and three deletions. The mutations found in four VHL patients with pheochromocytomas consisted of one missense mutation at nucleotide 683 (codon 228), two missense mutations at nucleotide 712 (codon 238) and a novel 20 bp insertion at nucleotide 776 (codon 259). Although the mutations at codon 238 are the mutational hot spot found in Western VHL patients with pheochromocytomas, a 20 bp insertion of original VHL cDNA sequence, from nucleotide 777 to 796, is a unique mutation. Our results suggest that mutations in Japanese VHL patients contain some unique features compared with those in Western patients. VHL gene has a critical role for the etiology in VHL in Japanese populations as well as Western VHL. PMID- 8634690 TI - Oncogenic conversion of a novel orphan nuclear receptor by chromosome translocation. AB - A recurrent t(9;22) (q22;q12) chromosome translocation has been described in extraskeletal myxoid chondrosarcoma (EMC). Fluorescent in situ hybridization experiments performed on one EMC tumour indicated that the chromosome 22 breakpoint occurred in the EWS gene. Northern blot analysis revealed an aberrant EWS transcript which is cloned by a modified RT-PCR procedure. This transcript consists of an in-frame fusion of the 5' end of EWS to a previously unidentified gene, which was named TEC. This fusion transcript was detected in six of eight EMC studied, and three different junction types between the two genes were found. In all junction types, the putative translation product contained the amino terminal transactivation domain of EWS linked to the entire TEC protein. Homology analysis showed that the predicted TEC protein contains a DNA-binding domain characteristic of nuclear receptors. The highest identity scores were observed with the NURR1 family of orphan nuclear receptors. These receptors are involved in the control of cell proliferation and differentiation by modulating the response to growth factors and retinoic acid. This work provides, after the PML/RAR alpha gene fusion, the second example of the oncogenic conversion of a nuclear receptor and the first example involving the orphan subfamily. Analysis of the disturbance induced by the EWS/TEc protein in the nuclear receptor network and their target genes may lead to new approaches for EMC treatment. PMID- 8634693 TI - Stability of the Huntington disease (CAG)n repeat in a late onset form occuring on the Island of Crete. AB - Huntington disease (HD) is an autosomal-dominant disorder of mid-life onset characterized by chorea, dementia, and oculomotor disturbances. Anticipation is commonly seen in HD families, particularly when the disease is inherited through the father. The disorder is associated with an expanded (CAG)n repeat in the IT15 gene that is unstable and tends to increase in size during meiotic transmissions, particularly of paternal origin. We have detected an unusual form of HD on the island of Crete which has distinctly different characteristics. Data from eight families encompassing 48 HD patients, showed a median age at onset 15-20 years later than that for HD occurring worldwide. There is no juvenile cases and no anticipation. DNA analysis in 12 HD patients showed expansion of the (CAG)n repeat the size of which was identical among members of each family or varied by only one unit. The elongated DNA segment was passed stably or contracted during both paternal and maternal transmissions thus indicating that unique molecular mechanisms may be operational in this form of HD. PMID- 8634694 TI - Lowe syndrome, a deficiency of phosphatidylinositol 4,5-bisphosphate 5 phosphatase in the Golgi apparatus. AB - The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by congenital cataracts, renal tubular dysfunction and neurological deficits. The gene responsible for this disorder, OCRL-1, has been cloned and mutations identified in patients. The gene product (ocrl-1) has extensive sequence homology to a 75 kDa inositol polyphosphate 5-phosphatase. We report here that OCRL patients' fibroblasts show no abnormality in inositol polyphosphate 5-phosphatase activity, but are deficient in a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] 5-phosphatase activity localized to the Golgi apparatus. Direct biochemical diagnosis of this human disease should now be possible. PtdIns(4,5)P2 has been implicated in Golgi vesicular transport through its role in the regulation of ADP-ribosylation factor, phospholipase D and actin assembly in the cytoskeleton. The regulation of PtdIns(4,5)P2 levels by PtdIns(4,5)P2 5-phosphatase may, therefore, be important in the modulation of Golgi vesicular transport. Given that the primary defect in OCRL is a deficiency of a Golgi PtdIns(4,5)P2 phosphatase, we hypothesize that the disorder results from dysregulation of Golgi function and in this way causes developmental defects in the lens and abnormal renal and neurological function. PMID- 8634695 TI - Homologous unequal cross-over involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of human cytochrome P450 CYP2D6 gene. AB - The cytochrome P450 enzyme debrisoquine 4-hydroxylase (CYP2D6) metabolizes many different classes of commonly used drugs. In Caucasian populations, 5-10% are classified as poor metabolizers (PM) due to autosomal recessive inheritance of two mutant CYP2D6 null alleles. In contrast, up to 5% may demonstrate ultrarapid metabolism (UM) of debrisoquine caused by inherited amplification of functional CYP2D6 genes in the CYP2D6 locus. Poor metabolizer subjects may develop toxic plasma concentrations and adverse drug reactions, whereas UMs may suffer from therapeutic failure. Moreover, mutant CYP2D6 alleles have been implicated as a predictor of susceptibility for diseases such as cancer and neurological disorders. The break points and molecular mechanisms involved in the generation in the PM-associated CYP2D6(D) gene deletion allele and the UM-related CYP2D6 amplification have not been clarified. Here we demonstrate the presence of a 2.8 kb repeated region (CYP-REP) which flanks the active CYP2D6 gene in the wild type allele. The CYP-REP unit may be itself predispose to homologous unequal cross over and contains the Alu element and a tandem 10 bp direct repeat, which could both serve as hotspots for recombination. The break points of the CYP2D6(D) deletion allele are present within the repeated 2.8 kb region, but the exact positions are non-determinable due to perfect recombination of the misaligned, homologous CYP-REP elements. We also propose that the alleles with multiple copies of CYP2D6, which represent the first example of inherited amplification of an active gene in man, can be explained by unequal cross-over events involving the CYP-REP units. In our model, the CYP2D6 deletion and amplification alleles are reciprocal to each other, generated through homologous unequal recombination of no-allelic CYP-REP elements. PMID- 8634696 TI - Distinct transcription start sites generate two forms of BRCA1 mRNA. AB - Using primer extension and 5' RACE, we have mapped the 5' end of the BRCA1 gene and identified a new 5' exon. Two distinct BRCA1 transcripts differing by the first exons were found; these transcripts were generated by the alternative use of dual promoters and alternative splicing. The expression of the distinct transcripts was examined in four primary tissues (placenta, mammary gland, testis and thymus), six normal or cancer cell lines, four primary breast tumor tissues and four primary ovary tumour tissues. Both transcripts were detected in all the samples studied, with the exon 1a transcript being the major expressed form in mammary gland and the exon 1b transcript in placenta. This suggests that the two transcripts may be expressed in a tissue-specific fashion. The 5' flanking regions of both BRCA1 transcripts were analysed, neither contains a TATA box. Initiator elements, which have been proposed to mediate transcription in TATA less promoters, were found at the transcription initiation sites. Transcription factor binding sites such as Sp1, PEA3, C/EBP, CREB, E4F1 and Pu boxes were identified in the 5' flanking regions of the exon 1a transcript, and Sp1, NF-kB and PEA3 binding sites in the 5' flanking region of the exon 1b transcript. The interactions of these DNA elements with trans-acting factors are likely to modulate the alternative use of the distinct transcription start sites and the expression of the BRCA1 gene. PMID- 8634697 TI - Mouse Brca1: localization sequence analysis and identification of evolutionarily conserved domains. AB - The human genes BRCA1, conferring susceptibility to early-onset breast and ovarian cancer, has recently been isolated. Here we describe isolation of cDNAs, sequence analysis, and genomic localization of the murine homolog, Brac1. The mouse cDNA sequence predicts a protein of 1812 amino acids; a number of small gaps account for the 51 fewer residues in the mouse protein relative to human BRCA1. While the predicted mouse and human proteins display on the whole a high level of homology (58% identity, 73% similarity), the regions of greatest homology are at the respective amino and carboxyl termini. Most reported disease associated missense mutations in human BCRA1 occurred within these more highly conserved terminal regions. A predicted zinc-building RING finger domain near the amino terminus lies within a 50 amino acid stretch that is perfectly conserved in both species. The strong conservation during mammalian evolution argues for the importance of this domain, perhaps mediating a role for BRCA1 in DNA and/or protein binding. We have also identified a conserved highly acidic domain in the carboxyl terminal half of the BCRA1 protein resembling acidic transactivation domains of certain transcription factors. Using an interspecific backcross panel, Brca1 was mapped to a region of mouse chromosome 11 that exhibits conserved linkage with 17q21. The sequence and isolated cDNAs will provide useful reagents for studying the expression of Brca1 in the mouse, and for testing the importance of the evolutionarily conserved domains. PMID- 8634699 TI - Liposome-medicated gene transfer and expression via the skin. AB - A beta-galactosidase gene expression construct was used to investigate the effectiveness of gene delivery and expression when DNA/liposome complexes were topically applied to mouse skin in vivo. DNA was complexed with commercial preparation of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium-methyl-sulp hate (DOTAP) in a ratio of 1:1.6 (w/w). The DNA rapidly penetrated the skin and was expressed in the epidermis, dermis and hair follicles. A DNA concentration of 267 microgram/ml DNA was found to be optimal for efficient transfection. Expression was seen as early as 6 h post-application, persisted at high levels 24 and 48 h post-treatment, but was markedly reduced by 7 days after application. In conclusion, utilising a commercially available liposome preparation, topically applied DNA/liposome complexes can be efficiently delivered and expressed in several cell types within the skin. This simple, non-invasive technique may have implications for a number of gene therapy applications. PMID- 8634698 TI - Murine Brca1: sequence and significance for human missense mutations. AB - We have cloned and sequenced a mouse homologue of the human breast and ovarian cancer susceptibility gene, BRCA1. The predicted mouse Brca1 protein is composed of 1812 amino acids. The murine protein is 60% identical and 72% similar to the human BRCA1 protein. Two regions of high homology have been identified between the two proteins. First is the Cys3-His-Cys4 type zinc-finger domain that is identical between the two proteins. The second region is defined by 115 amino acids near the carboxyl end of the Brca1 protein that is 83% identical to human BRCA1 sequence. Seven of eight amino acids involved in human missense mutations that are associated with the disease were found to be conserved between the two species. In contrast, most of the amino acids that are involved in polymorphic variations were not conserved. We therefore propose that the interspecies conservation of predicted amino acid sequences can be used as an additional criterion to determine the significance of human missense mutations. PMID- 8634700 TI - Allele-specific replication timing in imprinted domains: absence of asynchrony at several loci. AB - Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing. PMID- 8634701 TI - Mutation analysis of the TSC2 gene in an African-American family. AB - Tuberous sclerosis complex is an autosomal dominant disorder with loci on chromosome 9q34 (TSC1) and chromosome 16p13.3 (TSC2). The TSC2 gene has been isolated. To date, only a small number of intragenic deletional and point mutations have been detected, almost exclusively in sporadic (no family history) cases. With the exception of a single parent/offspring pair, there have been no published reports of mutations in extended multigenerational chromosome 16-linked TSC2 families. For our TSC studies we ascertained and sampled a four-generation African-American TSC family that shows a high likelihood for linkage to chromosome 16 (z=1.53). Using single-strand conformation polymorphism analysis we identified a 4590/4591delC mutation in exon 34. The 4590/4591delC causes a frameshift mutation resulting in the creation of a premature stop codon. In addition, we have detected a 542del4 polymorphism in the two partially overlapping polyadenylation signals in exon 40 that segregates in the family. The polymorphism has been detected in six of 72 African-American control chromosomes examined, and has not been detected in 80 Caucasian control chromosomes examined. PMID- 8634702 TI - T-->A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia. AB - Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a 46,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H corticosterone and androstenedione were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T-->A transversion in intron 4,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns 4, 5 and 6 either with or without the T-->A mutation in intron 4. RNase protection assays showed that expression of the vector with normal intron 4 yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T-->A transversion in intron 4 was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient. PMID- 8634703 TI - RBM3, a novel human gene in Xp11.23 with a putative RNA-binding domain. AB - Using cDNA selection with a YAC from the Xp11.2 region, we have identified a novel gene (RBM3) that encodes a polypeptide with high sequence similarity to a group of proteins that bind to RNA. On a YAC contig map, RBM3 is located between OATL1 and GATA1/TFE3 in sub-band Xp11.23, and gives rise to alternatively spliced transcripts in a variety of human tissues. The longest open reading frame encodes a 157 amino acid protein with a predicted molecular weight of 17 kDa. Its putative RNA-binding domain most closely resembles that of two previously characterized human RNA-binding proteins, YRRM, the gene for which has been implicated in azoospermia, and hnRNP G, a glycoprotein, also identified as an auto-antigen. The homology of RMB3 in both sequence and size to an RNA binding protein from maize, AAIP , suggests that it functions in a fundamental pathway conserved from plants to mammals. PMID- 8634704 TI - Breakpoint characterization of the ret/PTC oncogene in human papillary thyroid carcinoma. AB - The ret/PTC oncogene, rearranged form of the ret proto-oncogene (c-ret), has been detected specifically in a minority of papillary thyroid carcinomas. Three forms of the ret/PTC oncogene have been identified; the two most common forms, ret/PTC 1 and ret/PTC-3, both result from a paracentric inversion, of the long arm of chromosome 10. In this study, we have successfully amplified the chimeric introns resulting from these inversions, ranging from 1.4 to 10 kb, from four of five tumors known to contain the ret/PTC-1 oncogene (where c-ret rearranges with the H4 gene), and from 1/1 tumors containing the ret/PTC-3 oncogene (where c-ret rearranges with the ele1 gene). We localized the breakpoints within the chimeric introns using nested PCR, and determined the exact nucleotide sequence at the breakpoint for each tumor. Our results indicate that the breakpoints in c-ret occur at sites distributed across intron 11, where breaks in H4 intron 1 appear more frequently at the 5'- end of the intron. Interestingly, in all tumors that we investigated, the breakpoints occurred at sits of two or three nucleotide matches between the contributing germline sequences. In summary, we describe a simple, convenient way to investigate the ret/PTC breakpoints, and have revealed several common features of the breakpoints which warrant further investigations. PMID- 8634705 TI - Analysis of the OA1 gene reveals mutations in only one-third of patients with X linked ocular albinism. AB - The locus for ocular albinism type 1 (OA1) has been assigned to the Xp22.3 region through both linkage and deletion mapping. The disorder was found to be genetically homogeneous, as all informative families showed convincing linkage data with markers on Xp22.3 and all identified deletions involved in the same region. The OA1 gene recently was cloned and several intragenic deletions were identified in affected individuals. We have characterized the genomic structure of the OA1 gene, which spans approximately 40 kb of genomic DNA and contains nine exons. A highly polymorphic dinucleotide repeat was identified in intron 1, that provides a useful tool for molecular diagnosis. Knowledge of the intron/exon boundaries allowed us to search for point mutations in patients' genomic DNA. All nine exons of the OA1 gene, as well as the 5' and 3' untranslated regions, were scanned for point mutations in PCR-amplified DNA from 60 OA1 patients. The mutations identified include: two frameshifts and a splice site mutation leading to truncated OA1 proteins; a deletion of a threonine codon at position 290; and four missense mutations,two of which involve amino acids located within putative transmembrane domains. Two of the mutations each occur in three apparently unrelated families, consistent with previous observations of a founder effect in OA1. Surprisingly, mutations were detected in only one-third of the patients (21 of 60) ascertained. We postulate that mutations not yet identified in either regulatory elements of the OA1 gene, or in other gene(s) located within the same chromosomal region, may be common cause of X-linked ocular albinism. PMID- 8634706 TI - Analysis of the CMT1A-REP repeat: mapping crossover breakpoints in CMT1A and HNPP. AB - The CMT1A-REP repeat sequence flanks a 1.5 megabase pair (Mb) segment of chromosome 17p11.2-12 which is duplicated in Charcot-Marie-Tooth neuropathy type 1A (CMT1A) and deleted in hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A-REP repeat is proposed to mediate misalignment and unequal crossover resulting in reciprocal chromosomal rearrangements in CMT1A and HNPP. We have constructed a physical map of the proximal and distal CMT1A-REP repeats. Cloned fragments from CMT1A-REP repeat regions are used to determine the size of the repeats and assess regions of homology. The crossover breakpoints were mapped in series of 30 unrelated CMT1A patients and 22 unrelated HNPP patients. The CMT1A-REP repeat spans approximately 27 kilobase pairs and appears to be continuous. Locations of restriction enzyme sites are highly conserved for the proximal and distal CMT1A-REP repeats. All crossovers mapped within the CMT1A REP repeat sequence and heterogeneity for breakpoint location demonstrated. Seventy-seven percent (40 to 52) of CMT1A and HNPP chromosomes contained breakpoints which mapped within a 7.9 kb interval, suggesting the presence of a possible 'hotspot'for recombination in CMT1A-REP. DNA sequence analysis for 4 kb of the interval containing the majority of crossovers revealed over 98% sequence identity between proximal and distal CMT1A-REP repeat sequences. Probes useful for molecular-based diagnosis of CMT1A and HNPP are described. PMID- 8634707 TI - Characterization of the large deletion in the GALC gene found in patients with Krabbe disease. AB - Globoid cell leukodystrophy (GLD) of Krabbe disease results from mutations in the galactocerebrosidase (GALC) gene. Previously, we had identified a large deletion in the GALC gene together with a C to T polymorphism at cDNA position 502 in a significant number of cases of infantile Krabbe disease; however, the deletion breakpoint had not been found. In this paper we show that the deletion is approximately 30 kb starting near the middle of the 12 kb intron 10, and includes all of the coding region through exon 17 plus an additional 9 kb. The deletion junction contains a 4 bp direct repeat and is preceded by sequence identified as belonging to the Alu family of interspersed repetitive elements. Using genomic DNA and a PCR-based test to detect normal and deleted sequences at that location, a large number of patients with all clinical types of GLD were analyzed. Of 21 infantile patients found to be heterozygous for the 502T polymorphism reported previously, 15 had the deletion, one could not tested and five, including a Hmong child, did not have the deletion. Sixteen other infantile patients previously tested were found to be either homozygous (10) or heterozygous (6) for the deletion. In addition, five patients with juvenile and adult GLD were found to be heterozygous for the deletion. In every case tested, the deletion was always found on the same allele as the 502T polymorphism. However, other disease-causing mutations have been found on the 502T allele. With careful genotype analysis these families can receive improved genetic information including patient and carrier identification and preimplantation diagnosis. PMID- 8634708 TI - A gene (SRPX) encoding a sushi-repeat-containing protein is deleted in patients with X-linked retinitis pigmentosa. AB - X-linked retinitis pigmentosa (XLRP) is characterized by retinal degeneration with night blindness and progressive reduction of the visual fields. By linkage and deletion analysis a gene locus (RP3) has been mapped to the short arm of the X chromosome between the genes CYBB and OTC. Analysis of transcript in this region has revealed a gene which is abundantly expressed in human retina and encodes a putative membrane protein with significant homologies to short consensus repeat (SCR/sushi) domains known from selections and complement proteins. The gene termed SRPX (sushi-repeat-containing protein, x chromosome) is deleted in an RP patient who also suffers from chronic granulomatous disease and McLeod syndrome. A 75 kb deletion removing exon 1 of the gene was also found in two brothers of a second XLRP family. However, no further functionally significant mutations were detected by SSCP screening of all 10 exons in 34 unrelated XLRP patients nor by full length RT-PCR sequencing in two RP3 families. The role of this highly conserved retinal gene in the pathogenesis of RP therefore remains to be determined. PMID- 8634709 TI - Identification of a novel gene, ETX1 from Xp21.1, a candidate gene for X-linked retintis pigmentosa (RP3). AB - A novel gene encoding a 2.2 kilobase transcript has been isolated from the Xp21.1 region of the human X chromosome by exon amplification. The gene, called EXT1, spans 80 kilobases and contains 12 exons, at least two of which are alternatively spliced and have predicted products of 464 and 471 amino acids respectively. Conceptual translation of the open reading frames shows one product with a 30 amino acid signal peptide, which is absent from the alternative transcript, followed by three complement control protein domains, a hydrophobic region with a possible role in membrane anchorage and short 17 amino acid putative cytoplasmic carboxyl terminus. An alternative first exon contains a 39 amino acid open reading frame which is rich in serine and threonine residues and contains a potential chondroitin/dermatan sulphate attachment site. Northern analysis showed ETX1 expression within the retina and heart with lower levels in several other tissues. Since ETX1 lies within the region thought to contain the x-linked retinitis pigmentosa (xIRP) gene, RP3, it was screened for mutation within a set of 45 xIRP patients using single strand conformation analysis and/or chemical cleavage of mismatch using reverse transcription/polymerase chain reaction amplification of polyA+RNA from blood cells. Three low frequently variants (17 23Ldel, P225S, S413F) were found in both patients and controls; one of which (P225S) was found in four of 45 unrelated patient chromosomes and one of 178 control chromosomes (p <0.001). The allelic association between P225S and xIRP alleles suggests a common ancestral chromosome bearing the P225S variant and an RP3 mutation at a neighbouring locus. PMID- 8634710 TI - Structural characterization of the FKHR gene and its rearrangement in alveolar rhabdomyosarcoma. AB - The FKHR gene, which contains a forkhead DNA-binding motif, is fused to either PAX3 or PAX7 by the t(2;13) or t(1;13) translocation in alveolar rhabdomyosarcoma,respectively. These tumors express chimeric transcripts encoding the N-terminal portion of either PAX protein fused to the C-terminal portion of FKHR. To understand the structural basis and functional consequences of these translocations, we characterized the wild-type FKHR gene and its rearrangement in alveolar rhabdomyosarcomas. By isolating and analyzing phage, cosmid and YAC clones, we determined that FKHR consists of three exons spanning 140 kb and that several highly similar loci are present in other genomic regions. Exon 1 encodes the N-terminus of the forkhead domain and is embedded within demethylated CpG island. RNA analyses reveal FKHR transcripts initiate from a TATA-less promoter within this island. Exon 2 encodes the C-terminus of the forkhead domain and a transcription activation domain, whereas exon 3 encodes a large 3' untranslated region. The intron 1-exon 2 boundary precisely matches the FHKR fusion point in the chimeric transcripts found in alveolar rhabdomyosarcomas. Using pulsed-field and fluorescence in situ hybridization analyses, we demonstrate that the 130kb FKHR intron 1 is rearranged in t(2;13)-containing alveolar rhabdomyosarcomas. Our findings indicate that FKHR intron 1 provides a large target for DNA rearrangemnt. Rearrangement of this intron with PAX3 produces two important functional consequences: in-frame fusion of N-terminal PAX3 sequences to the FKHR transcriptional activation domain and disruption of the FKHR DNA binding domain. PMID- 8634711 TI - Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3. AB - Genetic linkage studies have indicated that chromosome 14q24.3 harbours a major locus for early-onset (onset age <65 years) Alzheimer's disease (AD3). Positional cloning efforts have identified a novel gene S182 or presenilin 1 as the AD3 gene. We have mapped S182 in the AD3 candidate region between D14S277 and D14S284 defined by genetic linkage studies in the two chromosome 14 linked, early-onset AD families AD/A and AD/B. We have shown that S182 is expressed in lymphoblasts and have determined the complete cDNA in both brain and lymphoblasts by RT-PCR sequencing. S182 is alternatively spliced in both brain and lymphoblasts within a putative phosphorylation site located 5' in the coding region. We identified two novel mutations, Ile143Thr and Gly384la located in, respectively, the second transmembrane domain and in the sixth hydrophilic loop of the putative transmembrane structure of S182. As families AD/A and AD/B have very similar AD phenotype our observation of two mutations in functionally different domains suggest that onset age and severity of AD may not be very helpful predictors of the location of putative S182 mutations. PMID- 8634712 TI - Mutations of the presenilin I gene in families with early-onset Alzheimer's disease. AB - We analyzed 12 families with autosomal dominant early-onset Alzheimer' s disease (EOAD)for mutations in the coding region of the presenilin I (PSNLI) gene corresponding to the AD3 locus on chromosome 14q24.3. A total of eight missense mutations at codons 82, 115, 139, 163, 231, 264, 392, and 410 including six novel mutations, were identified in eight families. Cosegregation of the mutations with EOAD was confirmed in three families, one including 36 affected individuals. This study underlines the great allelic heterogeneity and the large distribution of the mutations within the PSNLI coding region. Our results support the notion that PSNLI is the major gene involved in autosomal dominant EOAD. PMID- 8634713 TI - Imprinting mutations in the Beckwith-Wiedemann syndrome suggested by altered imprinting pattern in the IGF2-H19 domain. AB - Regional regulations of parental imprinting in the IGF2-H19 domain of imprinted genes was studied in the Beckwith-Wiedemann syndrome (BWS). We identified BWS patients who had inherited a normal biparental chromosome complement of the chromosome 11p15.5 region (where IGF2 and H19 reside), but had an altered pattern of allelic methylation of both genes, with the maternal chromosome carrying a parental imprinting pattern. In fibroblasts, IGF2 was expressed from both parental alleles and H19 was not expressed, precisely as predicted from the altered pattern of allelic methylation. Interestingly, DNA replication patterns of the 11p15.5 region remained asynchronous as in controls. Our results therefore provide the first example of the dissociation of regional control of DNA replication from regional control of allelic methylation and expression in imprinting. We suggest that the altered pattern of allelic methylation and expression arises in the germline or in the early embryo from defects in resetting or setting of imprinting in maternal germline. Potential candidate regions for mutations include the previously identified translocation breakpoint clusters and the H19 gene itself. The finding of possible 'imprinting mutations' in BWS raises the prospect of identifying genetic factors that control imprinting in this region. PMID- 8634714 TI - Partial characterization and assignment of the gene for protoporphyrinogen oxidase and variegate porphyria to human chromosome 1q23. AB - Protoporphyrinogen oxidase (PPO) catalyses the conversion of protoporphyrinogen IX to protoporphyrin IX. Variegate porophyria (VP), a low-penetrant, autosomal dominate disorder characterized clinically by skin lesions and neurovisceral attacks, is caused by partial deficiency of this enzyme. Linkage between VP and the alpha-1-antitrypsin gene on chromosome 14 has been reported in VP families from South Africa, where the condition occurs at high frequency due to a founder effect. We have cloned a 4.5 kb genomic DNA fragment containing the entire coding sequence for human PPO. This clone has been used to localize the human PPO gene to chromosome 1q23 by fluorescence in situ hybridization analysis. The VP gene was mapped by linkage analysis, using microsatellite markers spanning the region 1q21-q25.1, in seven British VP families. Multipoint analysis between VP, SPTA1, APOA2 and D1S194 gave the maximum LOD score of 6.62 at APOA2, which has been physically mapped to 1q21-q23. Evidence for significant linkage between VP and markers in the alpha-1-antitrypsin region of chromosome 14 was not obtained. Our results assign the genes for PPO and VP to the same region chromosome 1, indicate that the PPO and VP loci are likely to be the same, and provide evidence against locus heterogeneity in VP. PMID- 8634715 TI - A human recessive neurosensory nonsyndromic hearing impairment locus is potential homologue of murine deafness (dn) locus. AB - A locus for recessive neurosensory nonsyndromic hearing impairment maps to chromosome 9q13-q21 in two regionally separate consanguineous families from India. Each family demonstrates a LOD score greater than 4.5 to this region. D9S15, tightly linked to the Friedreich's ataxia locus, a region that has been defined with over 1 Mb of YAC contig information and several expressed sequences, is one of the flanking markers. In mice, the deafness (dn) locus maps to mouse chromosome 19 and flanking loci are syntenic to human chromosome 9q11-q21. The dn mouse is a potential model for the hearing impairment found in both these families. PMID- 8634716 TI - Linkage of congenital hereditary endothelial dystrophy to chromosome 20. AB - Congenital heredity endothelial dystrophy (CHED) is a rare autosomal dominant disorder of the cornea. We have performed genetic linkage analysis with microsatellite markers on a seven generation British pedigree. Two-point linkage analysis revealed significant linkage of CHED (lod score >3) with seven marker loci mapping to chromosome 20. The highest observed lod score was 7.20 (theta=0.026) with marker D20S114. Multipoint analysis gave a maximum lod score of 9.34 between D20S48 and D20S471. This 2.7cM region lies within 30 cM region recently assigned to posterior polymorphous dystrophy (PPD). PPD and CHED may therefore be allelic, or alternatively it is possible that more than one gene in this region is responsible for these two corneal dystrophies. PMID- 8634717 TI - A gene for monilethrix is closely linked to the type II keratin gene cluster at 12q13. AB - Monilethrix is an uncommon hereditary disorder of hair and nail which produces hair fragility and a variable alopecia. Many of the dystrophic hairs have a unique beaded morphology. Ultrastructural changes suggest a defect in the microfilament structure of the cortex of the hair shaft,and hence the cysteine rich trichocyte keratins are candidate genes. Here, in two families with autosomal dominant monilethrix, we have excluded linkage to the type I keratin gene cluster on chromosome 17q, but show that the disorder is closely linked to the type II keratin cluster on 12q, where genes for basic trichocyte keratins are found. The combined maximum lod score for D12S96 was 12.27 at theta=0.0. This is the first mapping of a primary human hair disorder and the first evidence implicating a defect of the word 'hard' keratins of hair and nail disease. PMID- 8634718 TI - Improved oligonucleotide primer set for molecular diagnosis of X-linked agammaglobulinaemia: predominance of amino acid substitutions in the catalytic domain of Bruton's tyrosine kinase. PMID- 8634720 TI - A novel CAG repeat configuration in the SCA1 gene: implications for the molecular diagnostics of spinocerebellar ataxia type 1. PMID- 8634719 TI - Mutation of the endothelin-receptor B gene in Waardenburg-Hirschsprung disease. PMID- 8634721 TI - A novel Ser156Cys mutation in the tissue inhibitor of metalloproteinases-3 (TIMP3) in Sorsby's fundus dystrophy with unusual clinical features. PMID- 8634722 TI - Interstitial 22q11 microdeletion excluding the ADU breakpoint in a patient with DiGeorge syndrome. PMID- 8634723 TI - Intelligibility and spectral differences in high-pitched vowels. AB - The first formant frequency of most German vowels can be 'oversung' in the sense of vocalizations with pitch frequencies above F1 of normal speech. Investigations of sung and synthesized vowels suggested that, with rising F0, either the vowel loses its identity and its spectral characteristics, or changes in the vocal effort and the speaker group are perceived. This study presents high-pitched vocalizations by untrained men, women and children, apart from singing or shouting. Three main results were found: (i) vowel identity can be maintained at high pitches (F0 = 660-870 Hz); (ii) clear spectral differences in high-pitched vowels are demonstrated; (iii) high pitched vowels can be found within one speaker group, apart from changes in the perceived speaker group. Both the F0 dependence of the lower formants and a possible relationship between phonation and articulation are discussed. PMID- 8634724 TI - Aerodynamic study of velopharyngeal insufficiency before and after logopedic treatment. AB - The aerodynamic bases of articulatory defects which characterize velopharyngeal insufficiency are not yet well defined. The purpose of our investigation has been to establish the type and the severity of aerodynamic alterations correlated with this syndrome before and after logopedic treatment; thus we have exposed the phoneme magnitude of pi:/, produced by 20 control individuals and by 12 patients with velopharyngeal insufficiency, to an aerodynamic study by means of a computerized system (Aerophone II, FJ Electronics) able to record, at the same time, phonatory airflow, intraoral pressure, and sound intensity. The data collected were subjected to statistical analysis by using Student's t test. The results obtained before logopedic treatment documented: (1) a remarkable articulatory distortion during the implosion of magnitude of p by a two-phasic pressure wave with a reduced amplitude compared to the one registered in control group subjects (7.3 +/- 2.8 cm H2O vs. 10.9 +/- 2.7 cm H2O; mean +/- SD; p < 0.01); (2) the presence of a nasal airflow in this same phase (0.13 +/- 0.07 liters/s); (3) peak airflow relative to the explosion of magnitude of p inferior to the one recorded in the control group (0.41 +/- 0.08 liters/s vs. 0.57 +/- 0.11 liters/s; p < 0.01); (4) a mean ratio between the duration of the implosive phase of magnitude of p and that of whole articulatory cycle inferior to the value recorded in the control group (34.8 +/- 2.6% vs. 39.2 +/- 2.3%; p < 0.01). At the end of logopedic treatment the aerodynamic investigation allowed to correlate the improvement of the speech defects due to rehabilitation to precise aerodynamic data: (1) a monophasic pressure wave with a peak (14.9 +/- 4.7 cm H2O) that is higher than the one observed in the control group (p < 0.01) and the one observed in the control group (p < 0.01) and the one observed in patients before therapy (p < 0.01); (2) a significant reduction of the nasal airflow recorded before logopedic treatment (0.04 +/- 0.04 liters/s; p < 0.01); (3) a remarkable increase in peak airflow compared to the one recorded before logopedic treatment (0.93 +/- 0.25 liters/s; p < 0.01); (4) a further reduction of the mean percentage ratio between the duration of the implosive phase of magnitude of p and that of the whole articulatory cycle (29.2 +/- 2.0%; p < 0.01) as compared to the one recorded before logopedic treatment. The results of our investigation underline how many different aerodynamic factors are involved in the pathogenesis of articulatory defects of speech related to velopharyngeal insufficiency, and how they reciprocally interfere; furthermore, our data indicate that logopedic treatment does not always restore physiological conditions, but often facilitates the realization of particular articulatory strategies which are not found in normal conditions. PMID- 8634725 TI - Teaching intonation to young deaf children with the intonation meter. AB - Incorrect production of intonation contours is a common phonatory problem in prelingually, profoundly deaf speakers. To help deaf speakers improve this, a visual display system for teaching intonation has been developed. In this system, called the Intonation Meter, visual feedback of intonation is given as a continuous representation of the pitch contour containing only the perceptually relevant aspects of the intonation pattern. This pitch-contour representation is supposed to facilitate the interpretation of the visual feedback of the pitch contour. A study was carried out, using a Single-Subject Design, in which subjects alternately received intonation training by means of regular methods and intonation training by means of regular methods in which also use was made of the Intonation Meter, to evaluate the effectiveness of the Intonation Meter for teaching intonation to young deaf children. Prelingually profoundly deaf children aged 6 to 7 years and 9 to 11 years participated in this study. The results showed that the 9 to 11 year old children showed most progress when the Intonation meter was used in intonation training whereas 6 to 7 year olds progressed well irrespective of whether or not the Intonation Meter was used, which is in accordance with the theory of a critical period for language learning. Alternatively, it is hypothesized that the cognitive requirements of the visual feedback might be to advanced for very young children to be helpful in learning to produce certain pitch contours. PMID- 8634726 TI - Cul-de-sac hypernasality test with pattern recognition of LPC indices. AB - Acoustic differences between samples of [i], [u], and [a] uttered in nose-open and nose-obstructed condition were studied in 6 women with isolated cleft palate and pathological nasalance scores and 9 healthy women with normal nasalance scores. The speech samples were depicted by 14-component vocal tract area feature vectors obtained by linear prediction and the differences between the samples were studied with a self-organized feature map. Each location on the map corresponds to a certain signal pattern, neighboring locations to similar patterns. The group of healthy subjects differed from the patients for vowels [i] and [u] but not for [a]. In the patients the nose obstruction induced a significant change in the location of these vowel samples on the map. In healthy subjects no such changes were detected. The results agreed with perceived differences between the subjects. PMID- 8634727 TI - Corticosteroids for croup. Reconciling town and gown. PMID- 8634728 TI - Outpatient treatment of croup with nebulized dexamethasone. AB - PURPOSE: To determine if treating children who have acute, moderate croup with nebulized dexamethasone sodium phosphate in the emergency department results in clinical improvement by 4 hours and a decrease in the hospitalization rate. DESIGN: Randomized, double-blind, placebo controlled trial. SETTING: Emergency department, children's hospital. PATIENTS: Fifty-five children with croup who were in moderate respiratory distress after treatment with mist for 30 minutes who met inclusion and exclusion criteria. MEASUREMENTS: Croup score, respiratory rate, heart rate, and oxygen saturation were assessed by one of us (D.W.J. or S.S.) before treatment and 2 and 4 hours after treatment. A staff pediatrician or senior pediatric resident, also "blind" to treatment, decided on admission to or discharge from the hospital. The differences between groups for change in croup score and hospitalization rate after treatment were analyzed. RESULTS: Evaluation of the croup scores disclosed a significant improvement in the dexamethasone treated group compared with the placebo group at 4 hours (P=.005, Mann-Whitney U). However, the hospitalization rate was not statistically different at the end of treatment (33% vs 52%, P=.28) or after 24 hours (48% vs 60%, P=.56, Yates corrected chi(2)). Our study has 80% power to detect a two-thirds reduction in hospitalization rate. Two patients with neutropenia treated with dexamethasone had a clinical course consistent with bacterial tracheitis. CONCLUSIONS: Treatment of moderate croup with nebulized dexamethasone results in clinical improvement within 4 hours. We did not show a decrease in hospitalization rates, although our sample size was only large enough to detect a 67% reduction in the rate of hospitalization. Given the infectious complications and the absence of evidence for a sustained clinical effect, we do not recommend that patients with croup be treated with nebulized dexamethasone. PMID- 8634729 TI - Television viewing as a cause of increasing obesity among children in the United States, 1986-1990. AB - BACKGROUND AND METHODS: The prevalence of obesity among children and adolescents has increased, and television viewing has been suggested as a cause. We examined the relation between hours of television viewed and the prevalence of overweight in 1990, and the incidence and remission of overweight from 1986 to 1990 in a nationally representative cohort of 746 youths aged 10 to 15 years in 1990 whose mothers were 25 to 32 years old. Overweight was defined as a body mass index higher than the 85th percentile for age and gender. RESULTS: We observed a strong dose-response relationship between the prevalence of overweight in 1990 and hours of television viewed. The odds of being overweight were 4.6 (95% confidence interval, 2.2 to 9.6) times greater for youth watching more than 5 hours of television per day compared with those watching 0 to 2 hours. When adjustments were made for previous overweight (in 1986), baseline maternal overweight, socioeconomic status, household structure, ethnicity, and maternal and child aptitude test scores, results were similar (odds ratio, 5.3; 95% confidence interval, 2.3 to 12.1). We also found significant relations between television viewing and increased incidence and decreased remission of overweight during this 4-year period, adjusted for baseline covariates. The adjusted odds of incidence were 8.3 (95% confidence interval, 2.6 to 26.5) times greater for youth watching more than 5 hours of television per day compared with those watching for 0 to 2 hours. Estimates of attributable risk indicate that more than 60% of overweight incidence in this population can be linked to excess television viewing time. CONCLUSION: Television viewing affects overweight among youth, and reductions in viewing time could help prevent this increasingly common chronic health condition. PMID- 8634730 TI - A randomized, controlled effectiveness trial of an AIDS prevention program for low-income African-American youths. AB - BACKGROUND: Some interventions to reduce the risk of the acquired immunodeficiency syndrome (AIDS) that target youths have resulted in short-term increases in self-reported condom use. However, long-term intervention effects have not been assessed. STUDY QUESTION: Can a theoretically and culturally based, AIDS-risk reduction intervention delivered to naturally formed peer groups increase self-reported condom use among African-American early adolescents at 6 and 12 months of follow-up? METHOD: A randomized, controlled trial of a community based intervention delivered in eight weekly sessions involved 76 naturally formed peer groups consisting of 383 (206 intervention and 177 control) African American youths 9 to 15 years of age. A theory-based, culturally and developmentally tailored instrument that assessed perceptions, intentions, and self-reported sexual behaviors was administered to all subjects at baseline (preintervention) and 6 and 12 months later. RESULTS: At baseline, 36% of youths were sexually experienced, and by 12 months of follow-up, 49% were sexually experienced. Self-reported condom use rates were significantly higher among intervention than control youths (85% vs 61%; P<.05) at the 6-month follow-up. However, by 12 months, rates were no longer significantly higher among intervention youths. The intervention impact at 6 months was especially strong among boys (85% vs 57%; P<.05) and among early teens (13 to 15 years old) (95% vs 60%; P<.01). Self-reported condom use intention was also increased among intervention youths at 6 months but not at 12 months. Some perceptions were positively affected at 6 months, but the change did not persist at 12 months. CONCLUSIONS: High rates of sexual intercourse underscore the urgent need for effective AIDS-risk reduction interventions that target low-income urban, African American preteens and early teens. A developmentally and culturally tailored intervention based on social-cognitive theory and delivered to naturally formed peer groups recruited from community settings can increase self-reported condom use. The strong short-term improvements in behaviors and intentions followed by some relapse over longer periods argue for a strengthened program and research focus on sustainability. PMID- 8634732 TI - Improving participation and interrater agreement in scoring Ambulatory Pediatric Association abstracts. How well have we succeeded? AB - OBJECTIVE: To determine whether increasing the number and types of raters affected interrater agreement in scoring abstracts submitted to the Ambulatory Pediatric Association. METHODS: In 1990, all abstracts were rated by each of the 11 members of the board of directors of the Ambulatory Pediatric Association. In 1995, abstracts were reviewed by four to five raters, including eight members of the board of directors, two chairpersons of special interest groups, and 10 regional chairpersons, for a total of 20 potential reviewers. Submissions were divided into the following three categories for review: emergency medicine, behavioral pediatrics, and general pediatrics. Weighted percentage agreement and weighted kappa scores were computed for 1990 and 1995 abstract scores. RESULTS: Between 1990 and 1995, the number of abstracts submitted to the Ambulatory Pediatric Association increased from 246 to 407, the number of reviewers increased from 11 to 20, the weighted percentage agreement between raters remained approximately 79%, and weighted kappa scores remained less than 0.25. Agreement was not significantly better for the emergency medicine and behavioral pediatrics abstracts than for general pediatrics, nor was it better for the raters who reviewed fewer abstracts than those who reviewed many. CONCLUSIONS: The number and expertise of those rating abstracts increased from 1990 to 1995. However, interrater agreement did not change and remained low. Further efforts are needed to improve the interrater agreement. PMID- 8634731 TI - The effect of early discharge and other factors on readmission rates of newborns. AB - OBJECTIVE: To assess the relationship between early discharge, breast feeding, and other factors on hospital readmission of newborns. DESIGN: Retrospective record review. SETTING: An urban, private community hospital. PATIENTS: All newborns born over a 1-year period who were readmitted to the hospital within the first 3 weeks of life (n=117). The control group consisted of a systematic sampling of newborns born over the same period who were not readmitted (n=147). RESULTS: Early discharge, defined as discharge when younger than 24, 36, or 48 hours of age, does not seem to contribute to readmission. However, newborns whose initial stay was longer than 72 hours were at significantly lower risk for readmission (P=.02, chi(2)). Factors in the initial hospitalization associated with readmission included vaginal delivery and length of stay less than 72 hours (difference, 12 percentage points; 95% confidence interval [CI], 4% to 20%; P=.005), need for performance of a complete blood count (CBC) (difference, 16 percentage points; CI 6% to 26%; P=.002), presence of jaundice (difference, 17 percentage points; CI, 5% to 29% P=.005), and gestational age 37 weeks or less (difference, 10 percentage points; CI, 2% to 18%; P=.02), discharge weight less than 3 kg (difference, 11 percentage points; CI, 0 to 22%; P=.05). However, almost all newborns delivered vaginally were discharged within less than 72 hours, so our ability to comment on the independent effect of delivery mode on readmission is limited. A trend toward significance was noted between breast feeding and readmission (difference, 9 percentage points; CI, 0% to 18%; P=.07). However, when only vaginal deliveries were considered, this association was statistically significant (difference, 13 percentage points; CI, 4% to 22%; P=.02). A significant association was noted between breast-feeding and jaundice or dehydration. Of babies admitted with jaundice or dehydration, 94% were breast fed, compared with 67% of babies admitted with neither jaundice nor dehydration (difference, 27 percentage points; CI, 13% to 41%; P<.001). CONCLUSIONS: These findings strongly suggest that early discharge (at younger than 24, 36, or 48 hours of age) from the hospital is not associated with hospital readmission within the first 3 weeks of life. Factors associated with readmission included breast-feeding, vaginal delivery and length of stay less than 72 hours, jaundice or need for a CBC, gestational age of 37 weeks or less, and discharge weight less than 3 kg. PMID- 8634733 TI - Welfare reform and children's health. PMID- 8634734 TI - The cycle of violence. Revisited 6 years later. AB - OBJECTIVE: To assess the long-term effects of child abuse and neglect on delinquency, adult criminality, and violent criminal behavior. DESIGN: Prospective cohorts design that matched cases of abuse and neglect with those of controls. Follow-up data collection at 22 to 26 years following abuse or neglect. SETTING: A metropolitan area in the Midwest. PARTICIPANTS: Substantiated cases of child abuse or neglect identified from court records that were obtained from 1967 through 1971 (n=908). Comparison group matched on the date of birth, race, sex, and approximate social class (n=667). The current mean age was 32.5 years. MAIN OUTCOME MEASURES: Official arrest records for nontraffic and violent crimes collected from local, state, and federal law enforcement agencies through mid 1994. RESULTS: Childhood victims of abuse or neglect were more likely than controls to have a juvenile or adult arrest for any nontraffic offense (49% vs 38%) and for a violent crime (18% vs 14%). Victims of physical abuse and neglected children were more likely to be arrested for violence (odds ratios: 1.9 and 1.6, respectively), after controlling for age, race, and sex. CONCLUSIONS: Childhood abuse and neglect have a significant impact on the likelihood of arrest for delinquency, adult criminality, and violence. By the age of 32 years, almost half of the victims of abuse and neglect were arrested for a nontraffic offense. By responding to incidents of child abuse and neglect, health care professionals can play an important role in preventing future violence. More attention must be paid to childhood victims of neglect and to differences in the consequences of abuse and neglect by gender and race or ethnicity. PMID- 8634735 TI - Therapy for acute otitis media. Preference of parents for oral or parenteral antibiotic. AB - OBJECTIVE: To determine if parents prefer single-dose intramuscular (IM) therapy or standard 10-day oral therapy for treatment of acute otitis media (AOM). DESIGN: Parents were asked their preference at the time their child was enrolled in a randomized controlled trial comparing the clinical efficacy of single-dose IM ceftriaxone sodium with 10 days of oral amoxicillin and clavulanate potassium for AOM. Additional information was collected at days 3 to 5 and 14 to 16 after the initiation of the therapy. SETTING: Primarily private practices; 15 sites. PATIENTS: For this study, 648 children aged 3 months to 6 years were randomly assigned to receive IM (n=327) or oral (n=321) therapy. RESULTS: The groups were equivalent in all measured sociodemographic factors. At the time of enrollment, 85% of parents expressed a preference for single-dose IM therapy. At days 3 to 5, no differences were reported in days children were absent from school or day care, parental absence from work, or loss of sleep by children. However, more parents with children in the IM therapy group than in the oral therapy group reported loss of sleep by the parent (35% vs 26%, P=.02, chi(2)). At days 14 to 16, more parents with children in the IM group reported being "very satisfied" with the antibiotic (65%) compared with parents whose children were assigned to the oral therapy group (38%, P<.001). In comparing current therapy to past oral therapy for AOM, 71% of the parents with children in the IM therapy group reported more satisfaction with current therapy, in contrast to 21% of parents with children in the oral therapy group (P<.001). Of the parents, 83% indicated they would prefer single-dose IM therapy for AOM in the future. CONCLUSION: All of the parents prefer single-dose IM therapy for AOM over standard 10-day oral therapy. PMID- 8634736 TI - Pediatric injury hospitalization in Hispanic children and non-Hispanic white children in southern California. AB - OBJECTIVE: To compare the incidence and causes of injury requiring hospitalization or resulting in death or both between Hispanic children and non Hispanic white children. DESIGN: Population-based surveillance of children younger than 15 years residing in eight Orange County cities and communities who were hospitalized or died of injuries sustained during 1991 and 1992. SETTING: Eight hospitals and the coroner's office in central Orange County, California. PARTICIPANTS: Study population was 213,906 children residing in the study area. Forty-nine percent were Hispanic, 37% were non-Hispanic white, 12% were Asian or Pacific Islander, and 3% were of other racial origin. RESULTS: A total of 1361 severe injuries were identified (crude annual rate was 318 per 100,000 population). The crude incidence rate ratio comparing Hispanics and non-Hispanic whites was 1.82. After adjustment for census block group, Hispanic children had a 60% higher injury rate and incidence rate ratios of more than 2 for pedestrian injuries, asphyxia, aspirations, foreign-body ingestions, and poisonings. CONCLUSIONS: Hispanic children had higher injury rates than non-Hispanic white children, even when controlling for census block group. These rate differences may be related to differences in exposure to various causes of injury, injury prevention practices, parenting practices, family size, and language. Injury rate differences by ethnicity that address specific injury hazards must be explored to guide prevention efforts. More culturally and linguistically appropriate interventions are needed to provide injury prevention programs to the Hispanic population. PMID- 8634738 TI - Childhood victims of violence. Hospital utilization by children with intentional injuries. AB - OBJECTIVE: To describe the utilization of hospital resources by children admitted with intentional injuries. METHODS: A sample of medical records for patients 14 years old and younger admitted to a pediatric tertiary care teaching hospital with a level I pediatric trauma center between January 1, 1991, and December 31, 1992, with intentional injuries (external cause codes E950 to E969) and unintentional injuries (E800 to E949) were reviewed after identification from the trauma center and hospital discharge registries. RESULTS: Of 1495 patients admitted for injuries, 95 had intentional injuries. Among these, 36% were caused by child abuse, 37% were caused by assaults, and the remainder were associated with suicide attempts. Compared with all unintentionally injured patients, those with intentional injuries were similar in gender and race but were significantly older (P<.001). Compared with a randomly selected sample of unintentionally injured patients matched for age, gender, and race, intentionally injured patients had longer mean hospital stays (P<.001), had more medical consultations (P<.001), were more likely to be discharged to sites other than home (P<.001), and had higher hospital charges (P=.007). While intentionally injured children had higher Injury Severity Scores (P=.002), their longer hospital stays were independent of injury severity. CONCLUSIONS: Intentionally injured children use more hospital resources and consequently incur higher hospital charges than those with unintentional injuries. Injury acuity contributes to this phenomenon, as do complex social needs. These data suggest that efforts directed at preventing intentional injuries will significantly affect injury-related health care costs. PMID- 8634739 TI - Variation in patient charges for vaccines and well-child care. AB - BACKGROUND: Several state and federal programs have attempted to boost immunization rates by reducing or eliminating provider vaccine costs. The relation between patient vaccine and well-child visit charges and vaccine financing systems is unknown. OBJECTIVES: To determine patient charges for vaccines and well-child visits in three states with varying vaccine financing systems and to examine the effects of a short-term reduction in provider vaccine costs. DESIGN: Cross-sectional survey study of a random sample of physicians in three states. PARTICIPANTS: A total of 2797 pediatricians and family physicians in North Carolina, Texas, and Massachusetts were surveyed. MAIN OUTCOME MEASURES: Current charges to patients for diphtheria-tetanus-pertussis vaccine (DTP), measles-mumps-rubella vaccine, Haemophilus influenzae type b vaccine (Hib), and combined DPT-Hib vaccine for well-child visits; changes in charges over the previous 8 months. RESULTS: Response rate was 62%. Vaccine and well-child visit charges were comparable in North Carolina and Texas. Massachusetts' average charges for well-child visits were higher than in the other states, although vaccine charges were lower; with the use of combined DPT-Hib vaccine, total simulated charges for vaccines and well-child care during the first 6 months of life averaged only 10% less in Massachusetts vs Texas and North Carolina. Neither regional variation in cost of living nor Medicaid reimbursement rates explained this difference. CONCLUSIONS: The average cost and composition of charges for well-child care in Massachusetts, a state with universal purchase of vaccines, compared with the other states, warrant further study to explore whether physicians shift costs to other preventive services to compensate for lower allowable immunization charges. If such cost shifting occurs, current federal immunization initiatives that lower or eliminate provider cost may not provide increased access to preventive services. PMID- 8634737 TI - Recent corticosteroid use and the risk of complicated varicella in otherwise immunocompetent children. AB - OBJECTIVE: To determine whether recent corticosteroid use was associated with an increased risk of complicated varicella-zoster virus infection in otherwise immunocompetent children. STUDY DESIGN: A case-control study design was used because the outcome of interest, complicated varicella-zoster virus infection, is rare. SETTING: Cases and controls were selected from the population of children aged 2 months to 18 years admitted to two hospitals, between January 1979 and July 1994 in one and between January 1974 and July 1994 in the other, with diagnosis codes that indicated chickenpox. POPULATION: Cases were defined as children with invasive varicella-zoster virus infection or associated invasive bacterial infection. Controls were defined as children with uncomplicated varicella admitted for elective surgery, fracture or burn management, psychiatric or social evaluation, treatment of simple dehydration, or evaluation of fever or rash not yet diagnosed. Exclusions included varicella-zoster virus infection in neonates and immunocompromised children. METHODS: A priori criteria were formulated on the basis of a comprehensive literature review to define complicated varicella-zoster virus infection. Recent corticosteroid exposure was defined as corticosteroid use of any sort within 30 days of onset of the chickenpox rash. Data were abstracted by medical chart review. RESULTS: In total, 167 cases and 134 controls were identified. Only three children (two cases and one control) had a history of recent corticosteroid therapy. Recent corticosteroid exposure was therefore not statistically associated with an increased risk of complicated varicella-zoster virus infection (odds ratio, 1.6; 95% confidence interval, 0.2 to 16.9). No differences between cases and controls were found in sex, history of asthma, or length of hospital stay. The mean age of cases was greater than that of controls (6.0 vs 4.7 years; P<.01). CONCLUSIONS: Recent corticosteroid therapy in otherwise immunocompetent children does not appear to be associated with a statistically increased risk of complicated varicella. A conservative estimate of risk, using the upper limit of the 95% confidence interval, is markedly lower than previously published risk estimates. PMID- 8634740 TI - Radiological case of the month. Diaphragmatic hernia with abnormal migration of the kidney. PMID- 8634741 TI - Picture of the month. Human bite marks. PMID- 8634742 TI - Pathological case of the month. Primary hyperparathyroidism. PMID- 8634743 TI - The effect of the pediatric clerkship on medical student attitudes toward pediatrics at 11 medical schools. AB - OBJECTIVES: To investigate how the pediatric clerkship affected student attitudes toward pediatrics, and to determine if correlations existed between changes in attitudes toward pediatrics and in ratings of certain aspects of the clerkship with an increased interest in a pediatric career. METHODS: A one-page survey measuring interest in a career in pediatrics and agreement or disagreement with seven statements about pediatrics was administered at the beginning and end of the pediatric clerkship at 11 medical schools for the 1992-1993 academic year. RESULTS: The proportion of students with a strong interest in a pediatric career increased from 6.7% before the clerkship to 15.2% after the clerkship (for women, 11% to 22%; for men, 4% to 11%). Attitudes toward pediatrics were more favorable at the end vs the beginning of the clerkship. The change that correlated best with change in interest in a pediatrics career was agreement that children are enjoyable to work with. Of the eight aspects of the clerkship rated, the patients worked with on the ward received the most positive mean score. The item that correlated best with increased career interest was a positive feeling toward the ward residents. CONCLUSION: The recent trend for women to have a greater interest in careers in pediatrics than men is continuing. Finding ways to make students more comfortable when they interact with children and improving the teaching skills of residents could improve recruitment of medical students into pediatrics. PMID- 8634744 TI - Congenital lymphocytic choriomeningitis virus infection. PMID- 8634745 TI - Aortic dissection in a 5-year old girl with Marfan's syndrome. PMID- 8634746 TI - Heterogeneity of cerebral palsy. PMID- 8634747 TI - ARCHIVES supports child abuse. PMID- 8634748 TI - Investigating abuse in the asymptomatic twin. PMID- 8634749 TI - Medicaid medicine. PMID- 8634751 TI - Determination of the two diastereoisomers of lobaplatin (D-19466) in plasma ultrafiltrate of cancer patients with a normal or an impaired kidney or liver function by high-performance liquid chromatography with ultraviolet detection. AB - Lobaplatin consists of two diastereoisomers, LP-D1 and LP-D2. Being a new cytostatic agent it represents platinum compounds of the third generation and is active in several in vitro tumor models of murine and human origin. To determine the pharmacokinetics of LP-D1 and LP-D2 in cancer patients with and without a normal kidney and liver function, an HPLC procedure was developed and validated. Plasma ultrafiltrate samples were injected into the HPLC system after solid-phase extraction. The standard curves of LP-D1 and LP-D2 in plasma ultrafiltrate were linear over the range 0.071-9.100 and 0.067-8.639 microM, respectively. The recovery from plasma ultrafiltrate was 84% for both diastereoisomers. The within day accuracy ranged from 98.1 to 100.3% for LP-D1 and from 96.5 to 106% for LP D2. The between-day accuracy ranged from 99.2 to 101.5% fro LP-D1 and 97.7 to and < or = 6.5% for LP-D2, respectively. For pharmacokinetic purposes the method proved to be sufficiently sensitive, specific and accurate for analysing clinical samples. PMID- 8634750 TI - Assessments on the digestibility of oxidized compounds from [1-14C] linoleic acid using a combination of chromatographic techniques. AB - The aim of this study was to evaluate the digestibility coefficients of different groups of oxidized fatty acids by applying a methodology based on chromatographic techniques. After thermoxidative treatment, oxidized labelled linoleic acid was isolated and included at 1% in the experimental diets. Male 250-300 g Wistar rats were fed ad lib for seven days. Lipids extracted from diets and faeces were analyzed using a combination of chromatographic methods and radioactivity measurements to determine the specific digestibilities of four groups of altered fatty acids: oxidized monomers non-polar dimers, oxidized dimers and polymers. Mean digestibility coefficients of oxidized monomers, dimers and polymers were 91.0, 74.5 and 69.8%, respectively. In contrast, non-polar dimers were poorly absorbed. The presence of unaltered labelled fatty acids in faeces indicated that structural modifications may have been taken place prior to absorption, and oxidized fatty acids seem to be the main compounds affected. PMID- 8634752 TI - Rapid, specific and sensitive method for isoniazid determination in serum. AB - An original simple, specific and rapid high-performance liquid chromatographic assay for the determination of isoniazid (INH) in human serum is presented. The drug was extracted from the serum by protein precipitation with 30% (w/v) trichloroacetic acid, then the drug was reacted with the coupling reagent, trans cinnamaldehyde, to form a derivative absorbing at 340 nm. A 20-microliters aliquot was injected into the chromatograph after neutralization with 1 M KOH solution. A liquid chromatograph equipped with a reversed-phase 30-microns C18 precolumn linked to a 4-microns C18 analytical column was used. The drug was eluted with a mixture of acetonitrile-water-triethylamine-acetic acid (400:600:2:1, v/v), pH value was 5 +/- 1. Flow-rate and wavelength were set at 1 ml/min and 340 nm, respectively. The extraction recoveries from human serum averaged 100% for INH at concentrations of 1, 2 and 4 mg/l. The coefficients of variation for three different concentrations for INH in serum in the within-day study varied between 1.2 and 3.5%, whereas those in the day-to-day study varied between 2.8 and 4.3%. PMID- 8634753 TI - Manual and automated (robotic) high-performance liquid chromatography methods for the determination of mycophenolic acid and its glucuronide conjugate in human plasma. AB - A manual and an automated (Zymark PyTechnology robot) HPLC method for simultaneous determination of plasma mycophenolic acid (MPA) and its glucuronide conjugate (MPAG) are described here. Both methods are reproducible and accurate, and both are equivalent in all respects, including quantification limits (MPA, 0.100 microgram/ml; MPAG, 4.00 micrograms/ml), range (using 0.05-0.5 ml of plasma: MPA, 0.0500-20.0 micrograms/aliquot; MPAG, 2.00-200 micrograms/aliquot), precision, and accuracy. MPA and MPAG were stable under the conditions used with both methods. Results from aliquots of paired control samples, analyzed by the manual method over three years at six analytical laboratories, showed excellent agreement in precision and accuracy. PMID- 8634754 TI - Direct determination of zolpidem and its main metabolites in urine using capillary electrophoresis with laser-induced fluorescence detection. AB - Zolpidem is a new sleep inducer belonging to the imidazopyridine class. We wish to report a method for the determination of zolpidem and its main metabolites in urine without extraction using capillary electrophoresis with UV laser-induced fluorescence detection with a He-Cd laser. A 10-nl sample of urine can be directly applied to the capillary. The separation is carried out within 10 min, and the limit of detection is 2 ng/ml. This procedure is very simple and fast. No organic solvents are necessary. PMID- 8634755 TI - Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis. AB - A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with beta cyclodextrin as the chiral selector. The analytes were detected using UV laser induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(-)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple. PMID- 8634756 TI - Simultaneous determination of oxalic and citric acids in urine by high performance liquid chromatography. AB - A simple method for the simultaneous determination of oxalic and citric acids had been developed using reversed-phase HPLC. An aqueous solution containing potassium dihydrogen phosphate (0.25%) and tetrabutylammonium hydrogen sulphate (2.5 mmol) at pH 2.0 was used as mobile phase. Under these conditions both the components were well resolved and detected at 210 nm. The recovery for oxalic and citric acids was 97% and 102%, respectively. The method presented here was applied to urine specimens of a large number of urolithic patients and control subjects. Because of the simplicity of the method its application provides better means of monitoring the concentration of oxalic and citric acids in the formation of renal calculi. PMID- 8634757 TI - Simultaneous determination of serotonin, N-acetylserotonin and melatonin in the pineal gland of the juvenile golden hamster by high-performance liquid chromatography with electrochemical detection. AB - A simple and simultaneous determination of melatonin and its precursors, serotonin (5-HT) and N-acetylserotonin, was achieved by reversed-phase high performance liquid chromatography with electrochemical detection. The addition of an ion-pairing agent, sodium 1-octanesulfonate, to the chromatographic mobile phase caused an increase of the retention time of 5-HT, and resulted in the successful simultaneous resolution of these three indoleamines. This method was used to quantitate these indoleamines in the pineal gland of juvenile golden hamsters. PMID- 8634758 TI - Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection. AB - An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tryptophan and four metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, kynurenic acid and 3 hydroxyanthranilic acid) in human serum is described. This new method, which uses both isocratic elution and two on-line connected programmable ultraviolet and spectrofluorimetric detectors, allows the determination of these metabolites, in the physiological ranges, with satisfying specificity and sensitivity within 30 min. PMID- 8634759 TI - Rapid reversed-phase high-performance liquid chromatographic method for the assay of urinary 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid and confirmation of use of cannabis derivatives. AB - The main active cannabis (marijuana and hashish) derivative delta 9 tetrahydrocannabinol is, in vivo, transformed and excreted mainly as 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide. The method presented here allows the confirmation of the presence of THC-COOH by means of a basic hydrolysis, solid-phase extraction clean-up on reversed-phase (RP) disposable cartridges followed by analysis on a C8 RP column and UV detection; the mobile phase used was a 55% acetonitrile solution in acid phosphate buffer. Over 600 samples both from drug addicts in therapeutic communities and subjects who were not on any drugs therapy were analysed. This method was precise with a linearity range from 10 to more than 500 ng/ml [the lower limit proposed by the National Institute on Drug Abuse (NIDA) for cannabinoid confirmation method is 15 ng/ml]. The sample preparation is simple and fast, allowing the analysis of large numbers of samples. Perfect correlation was observed between data from the HPLC method and a fluorescence polarization immunoassay screening method. The THC-COOH metabolite was found to constitute 30% of all the cannabinoids excreted in urine of abusers. PMID- 8634760 TI - Selective extraction of beta-blockers from biological fluids by column-switching high-performance liquid chromatography using an internal-surface phenylboronic acid precolumn. AB - A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of beta-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 microliters) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed briefly, the selectively retained beta-blockers were eluted with methanol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol. PMID- 8634761 TI - Antibody fragment separations by capillary zone electrophoresis. AB - This study investigated methods of improving the separation and identification of an IgA antibody, McPC603, and its pepsin fragments. The problem presented by purification of antibody fragments (Fabs) and the antibody light chain required accurate and informative analysis of highly hydrophobic proteins, which can polymerize and fold to form secondary structures. Capillary zone electrophoresis (CZE) permits the separation of peptides and small proteins by a method which is orthogonal to the traditional method of reversed-phase HPLC. To facilitate planned studies of the antibody's biological activity, our buffer composition was kept as simple as possible. During CZE analysis, if the buffer pH is below the isoelectric point of the protein, or the protein is large (with a heterogeneous distribution of surface charges), it can irreversibly blind to the capillary wall unless the capillary is coated. We found that C1-coatings in RP-capillaries at pH 9.5 adequately prevented the antibody fragments from binding to the wall. However, the coating did not remain stable at such high pH, so different conditions were sought. We achieved adequate separations in several buffers at nearly physiological pH, in a bare silica capillary which had been coated once with a soluble cationic polymer coating (Micro-Coat applied during column conditioning). Antibody electropherograms changed depending on the type of inorganic buffer salt used in a separation. Phosphate binds to the antigen binding site of the IgA with low affinity, and interesting effects were observed in separations using phosphate buffer. These effects will de discussed. PMID- 8634762 TI - Determination of (R)-(+)- and (S)-(-)-isomers of thiopentone in plasma by chiral high-performance liquid chromatography. AB - A method for the determination of R-(+)- and (S)-(-)-isomers of thiopentone in plasma was developed. Following liquid-liquid extraction, the separation of enantiomers of thiopentone and the internal standard (racemic ketamine) was achieved by high-performance liquid chromatography on an alpha1-acid glycoprotein (AGP) column with ultraviolet detection at 280 nm. The mobile phase consisted of 20 mM KH2PO4 buffer-2-propanol-methanol (93.5:5.0:1.5) at pH 5.0. The flow-rate was 0.9 ml/min. The limit of quantification for each isomer was approximately 10 ng/ml. The assay is suitable for pharmacokinetic studies of (R)-(+)- and (S)-(-) isomers of thiopentone, following usual bolus intravenous clinical doses of the racemic drug. PMID- 8634763 TI - Rapid high-performance liquid chromatographic assay for atovaquone. AB - A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Toxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C6 column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 micrograms/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 micrograms/ml and 10.3% at 1 microgram/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay. PMID- 8634764 TI - Sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of vinorelbine plasma concentrations. AB - A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of vinorelbine in plasma is described. The technique was derived from that published by Debal for an assay of vinorelbine in cell culture medium. The modifications concern the preparation procedure for plasma samples (a two-step liquid-liquid extraction from plasma is described), optimization of the mobile phase composition, and use of a single C18 column. These changes resulted in an improved sensitivity and reproducibility of the assay and led to its feasibility for clinical pharmacokinetic studies. The range of the assay is 2 to 1000 ng/ml. PMID- 8634765 TI - Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry. AB - We devised a sensitive and simple method for determining cyanide and its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 mumol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 mumol/ml for cyanide and from 0.01 to 1.0 mumol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using the method, the blood levels of two victims who had died from cyanide poisoning were determined. PMID- 8634766 TI - Sensitive and selective assay for fentanyl using gas chromatography with mass selective detection. AB - A modified gas chromatographic assay, using mass-selective detection, has been developed for the quantitation of fentanyl in swine serum. Fentanyl and sufentanil, the internal standard, were extracted using a single-step liquid liquid extraction with dichloromethane. Sensitivity and selectivity were improved by using electron-impact ionization (EI) in the selected-ion monitoring (SIM) mode, where fentanyl and sufentanil were monitored using the fragment ions at m/z 245 and 289, respectively. The limit of quantitation (LOQ) is 0.05 ng/ml, using 1 ml of sample, with a C.V. of 10.8% and a signal-to-noise ratio of 29. Standard curves were linear (r2 = 0.999) over the working range of 0.05-1/5 ng/ml, using 1/y2 as a weighting factor. Recoveries averaged 69.8 +/- 4.7%, 91.0 +/- 13.0% and 90.9 +/- 10.3% at serum concentrations of 1.5, 0.5 and 0.1 ng/ml, respectively. Intra- and inter-day variances, were < 12% at 0.1 ng/ml, and < 10% at concentrations of 0.5, 1 and 1.5 ng/ml. Bias was 6.2% at the LOQ and < or = 12.8% at every other standard curve concentration. Applicability of the assay is demonstrated for the pharmacokinetic study of transdermally administered fentanyl in a postoperative swine. PMID- 8634767 TI - Simultaneous determination of zolpidem and zopiclone in human plasma by gas chromatography-nitrogen-phosphorus detection. AB - A simple, specific and selective method for the simultaneous determination of zolpidem and zopiclone in human plasma is described. After a liquid-liquid extraction, the extract is injected into a capillary gas chromatograph with an OV 1 fused-silica column coupled to a nitrogen-phosphorus detector. The detection limits are 1 and 2 ng/ml for zolpidem and zopiclone, respectively. The method described is reproducible and linear over a range of concentrations, rendering it suitable for use for pharmacokinetic studies or toxicological evaluations. Absolute identification of the chromatographed compounds is accomplished by gas chromatography--mass spectrometry in both electron-impact and positive-ion chemical ionisation modes. PMID- 8634768 TI - Determination of trovafloxacin, a new quinolone antibiotic, in biological samples by reversed-phase high-performance liquid chromatography. AB - A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C18 column with a mobile phase consisting of 0.04 M H3PO4-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 microgram/ml and the calibration curves were linear over a concentration range of 0.1 to 20.0 micrograms/ml (r2 = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 micrograms/ml. Human serum samples could be stored for up to 12 months at -20 degrees C and urine samples could be stored up to 18 months at -80 degrees C. PMID- 8634769 TI - Direct analysis of salicylic acid, salicyl acyl glucuronide, salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography. AB - A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C18 column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3-4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2-20 micrograms/ml and linearity was observed from 0.1-200 micrograms/ml and 5-2000 micrograms/ml for SA in plasma and urine, respectively. The method was validated to 0.2 microgram/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates. PMID- 8634770 TI - High-performance liquid chromatographic method for guanylhydrazone compounds. AB - A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 micrograms/ml using a 100 microliters injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats. PMID- 8634771 TI - Stereospecific determination of tiaprofenic acid in plasma: problems with drug degradation. AB - A sensitive stereospecific high-performance liquid chromatographic assay for the quantification of tiaprofenic acid in human plasma was developed. The procedure involved extraction of tiaprofenic acid from acidified plasma into hexanediethyl ether (8:2, v/v). Stereospecific separation was achieved with a prepacked alpha1 acid glycoprotein column without derivatization. The mobile phase consisted of 2% 2-propanol in 0.01 M phosphate buffer, pH 6.5. Tiaprofenic acid was detected at 317 nm. The limit of quantification was found to be 25 ng/ml for each enantiomer using a 0.5 ml plasma sample. The assay was reproducible and accurate to be applied to the stereoselective pharmacokinetic analysis of tiaprofenic acid in plasma. Because of photoinstability of tiaprofenic acid plasma sampling and sample extraction should be performed under light protection. PMID- 8634773 TI - Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography. AB - An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. didanosine was analyzed using a 150 mm x 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20 degrees C, 4 degrees C and -20 degrees C. PMID- 8634772 TI - Simplified high-performance liquid chromatographic method for the determination of citalopram and desmethylcitalopram in serum without interference from commonly used psychotropic drugs and their metabolites. AB - A simplified method for the determination of racemic citalopram and its main metabolite desmethylcitalopram in serum using HPLC was developed. The compounds were extracted with heptane-isoamyl alcohol (98:2) and subsequently transferred into phosphate buffer pH 2.5 for direct injection into the HPLC apparatus. The analytes were separated with an acetonitrile-phosphate buffer, pH 2.5 tetraethylamine mobile phase on a C18 column and measured by UV detection at 240 nm. Within the typical range of serum concentrations (30-100 ng/ml) the inter-day variation was < 6% for both compounds. Possible analytical interference from a number of commonly coadministered psychoactive drugs and their metabolites was studied by extracting sera from patients receiving these drugs. Interference was not a problem for the developed method. PMID- 8634774 TI - Chromatographic properties of cytosine, cytidine and their synthetic analogues. AB - A direct reversed-phase high-performance liquid chromatographic (RP-HPLC) assay was used for the study of the effects of methanol concentration, pH, flow-rate of the mobile phase and column temperature on the retention of the natural nuclei acid components cytosine and cytidine and their synthetic 1-beta-D arabinofuranosyl, 5-aza and 6-aza analogues. The pKa values were also determined. The greatest changes were observed with changes in pH. The relationship between the capacity factors and the hydrophobicity of the compounds studied was also investigated. PMID- 8634776 TI - High-performance liquid chromatographic determination of granisetron in human plasma. AB - This paper describes a high-performance liquid chromatographic method (HPLC) with fluorometric detection for the analysis of granisetron in plasma. The detection is performed at 305 nm for excitation and 365 nm for emission. The method involves sample clean-up by liquid-liquid extraction. N-(1-Naphthyl) ethylenediamine dihydrochloride is used as internal standard. Toluene and phosphate buffer were added to 0.5 ml of plasma added to the internal standard. After extraction, the organic layer was separated and then evaporated to dryness. The residue was reconstituted in eluent mixture. An aliquot was injected onto the HPLC column, Spherisorb CN, equilibrated with an eluent mixture constituted by acetonitrile-phosphate buffer (pH 4.5) (15:85). The proposed technique is reproducible, selective, reliable, and sensitive. Linear detector responses were observed for the calibration curve standards in the range of 0.50 to 100 ng/ml. Extraction recovery from plasma proved to be more than 90%. Precision expressed as C.V. was in the range 2 to 8%. As low as 0.3 ng of granisetron per ml of plasma can be measured with good accuracy. The method has been validated, and stability tests under various conditions have been performed. Its sensitivity is adequate for pharmacokinetic studies. PMID- 8634775 TI - High-performance liquid chromatographic determination of primaquine and carboxyprimaquine concentrations in plasma and blood cells in Plasmodium vivax malaria cases following chronic dosage with primaquine. AB - A reversed-phase HPLC method using acetonitrile-methanol-1 M perchloric acid water (30:9:1:95, v/v) at a flow-rate of 1.5 ml/min on a mu-Bondapak C18 column with UV detection at 254 nm was developed for the separation of primaquine, its major metabolite carboxyprimaquine and other metabolites such as N acetylprimaquine, 4-hydroxyprimaquine, 5-hydroxyprimaquine, 5-hydroxy-6 methoxyprimaquine, demethylprimaquine and 6-methoxyprimaquine, and also other antimalarials. The calibration graphs were linear in the range 0.025-100 micrograms/ml for primaquine and 4-1000 micrograms/ml for carboxyprimaquine. The within-day and day-to-day coefficients of variation averaged 3.65 and 6.95%, respectively, for primaquine and 3.0 and 7.52%, respectively for carboxyprimaquine in plasma. The extraction recoveries for primaquine and carboxyprimaquine were 89 and 83%, respectively. The mean carboxyprimaquine concentration was much higher in plasma and blood cells of Plasmodium vivax patients than that in plasma from healthy subjects. The carboxyprimaquine level was also higher in blood cells than plasma whereas the primaquine concentration was the same in both cases. PMID- 8634777 TI - Current activities involving economic, regulatory, and practice issues in molecular genetic testing. PMID- 8634778 TI - Introduction to apoptosis. PMID- 8634779 TI - Papillomavirus, p53 alteration, and primary carcinoma of the vulva. AB - Twenty-nine samples from 28 cases of vulvar squamous cell carcinoma, of which 13 fulfilled the criteria of the bowenoid subtype (mean age 45 years, range 31-68) and 16 of the usual subtype of invasive squamous cell carcinoma (ISCC) (mean age 67.5 years, range 34-83) were investigated for human papillomavirus (HPV) DNA, TP53 alterations, and mdm2 and bcl-2 gene product deregulation. Microscopically all the bowenoid subtype cases (group I) showed a high-grade intraepithelial (VIN 3, carcinoma in situ) lesion associated with early invasive carcinoma in six cases and overt invasive carcinoma in one. By contrast, no evidence of early carcinoma was present in the ISCCs (group II). By in situ hybridization and/or Southern blot hybridization or polymerase chain reaction (PCR), HPV DNA was detected in all cases of group I and in four of 16 cases (25%) of group II, two only by Southern blot after PCR. By single-strand conformation polymorphism and immunocytochemistry only wild-type TP53 and absence of detectable p53 product, respectively, were found in all cases of group I, i.e., in high-risk HPV-positive carcinomas, whereas mutations and/or p53 overexpression accounted for 75% in group II, i.e., in mainly HPV-negative carcinomas. The TP53 gene mutations observed in invasive carcinomas were significantly related to node-positive cases (p = 0.04). Taken together and in agreement with in vitro data, these results support the view that an alteration of TP53, gained either by interaction with viral oncoproteins or by somatic mutations, is a crucial event in the pathogenesis of vulvar carcinomas, but that TP53 mutations are mainly associated with disease progression. Finally, a preliminary immunocytochemical analysis seems to speak against the possible involvement of both MDM2 and BCL-2 gene products in the development of vulvar carcinoma. PMID- 8634780 TI - Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive silver stained SSCP analysis. AB - p53 mutations are the most common genetic abnormality in humans tumors, but their clinical significance remains to be precisely elucidated. Conventional single strand conformation polymorphism (SSCP) analysis, a well-established technique for detecting p53 mutations, uses radioactively labeled polymerase chain reaction (PCR) products, which migrate abnormally in the presence of mutations. We performed radioactive PCR-SSCP analysis in a series of 30 formalin-fixed, paraffin-embedded ovarian carcinomas and two cell lines (SW480 and Caov4) harboring known homozygous p53 mutations and compared the results with nonradioactive silver-stained SSCP. The purpose was to assess whether nonradioactive SSCP is suitable for detecting p53 mutations in a rapid, sensitive, cost-effective fashion, without the need of radioactive isotopes. We accomplished PCR amplification of p53 exons 5 through 8 in 26 carcinomas, and radioactive SSCP detected p53 mutations in 13 tumors; three mutations were localized in exon 5, six in exon 6, two in exon 7, and two in exon 8. All mutations were correctly identified with nonradioactive SSCP, except for one exon 8 mutation. To establish the sensitivity of nonradioactive SSCP, DNA samples of SW480 and Caov4 were mixed with increasing amounts (0-90%) of normal DNA and subjected to PCR-SSCP analysis. Mutations were detected until the concentration of SW480 and Caov4 was 15% and 10%, respectively, of the total sample. The results of our investigation demonstrate that nonradioactive silver-stained SSCP is a sensitive, rapid, and simple technique to detect p53 mutations, even in formalin-fixed tissues, and could be easily used to investigate large series of patients to assess the clinical significance of p53 mutations in human tumors. PMID- 8634781 TI - Contribution of p53 gene alterations to development of metastatic forms of follicular thyroid carcinoma. AB - Alterations of p53 suppressor gene as a possible indicator of the metastatic potential of thyroid carcinomas were evaluated in a cohort of 45 thyroid carcinomas. Well-differentiated papillary and follicular carcinomas were evaluated; the poorly differentiated and the undifferentiated forms were excluded from the studies. Tumors were divided into two groups: those giving no metastasis for > 10 years and those developing metastasis within 5 years. Gene alterations were tested by immunocytochemical detection of p53 gene expression and by determining loss of heterozygosity (LOH). Considering the two methods together, p53 damages were observed in two out of 11 papillary carcinomas without metastasis (18.1%), one out of nine papillary carcinomas with metastasis (11.1%), two out of 14 follicular carcinomas without metastasis (14.2%), and five out of 11 follicular carcinomas with metastasis (45.4%). Statistical X2 test showed significantly (p = 0.05) only between follicular carcinomas with and without metastasis thus p53 damage may have an impact for metastatic potential of follicular thyroid carcinomas. PMID- 8634782 TI - Clonal analysis of colorectal tumors using K-ras and p53 gene mutations as markers. AB - Mutations to the K-ras oncogene and p53 tumor suppressor gene are two of the most common genetic lesions in human cancers. In the present study we examined the clonality of colorectal tumors with respect to each of these genetic alterations. Screening for mutations was carried out using the polymerase chain reaction-based technique of single-strand conformation polymorphism. Eleven primary colorectal adenocarcinomas and two secondary adenocarcinomas were analyzed at four different sites within the tumor. Involved pericolic lymph nodes were collected from nine of these cases, a metastatic deposit in the liver was obtained in one case, and adjacent adenomatous lesions were collected in two cases. Seven tumors contained mutations in either the K-ras or p53 genes. In all cases, DNA derived from multiple sites within an individual tumor or metastatic deposits arising from that tumor showed the same pattern of gene mutation. Immunohistochemical staining for p53 protein overexpression also showed similar patterns of reactivity within individual tumors and their metastatic deposits. These results suggest that the major clonal expansion of colorectal carcinomas occurs after the acquisition of mutations in these genes. Our results also indicate that sampling errors are unlikely to occur in molecular studies aimed at defining the role of these genes in colorectal cancer progression. PMID- 8634783 TI - A universal method for the mutational analysis of K-ras and p53 gene in non-small cell lung cancer using formalin-fixed paraffin-embedded tissue. AB - The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes. PMID- 8634784 TI - Use of fluorescence in situ hybridization (FISH) in the diagnosis of DiGeorge sequence and related diseases. AB - The proximal portion of human chromosome 22q has been implicated in the pathogenesis of a clinically diverse group of conditions including DiGeorge sequence (DGS), velocardiofacial syndrome, and CHARGE association as well as isolated conotruncal heart anomalies. Frequently, overlap in the clinical presentation of these syndromes occurs and, recently, the presence of microdeletions on chromosome 22q11.2 with varying frequencies has been demonstrated in these syndromes. Using fluorescence in situ hybridization (FISH), we assessed 20 consecutive patients who were cytogenetically and clinically evaluated for a suspected syndrome that could be due to a microdeletion of chromosome 22q11.2. After cytogenetic testing and full clinical evaluation, we compared the results by FISH with the final clinical diagnosis and karyotype results. We found that microdeletions of 22q11.2 were detected in three of the five patients who were evaluated for DGS. The three cases with microdeletions appeared clinically to have DGS while the two negative cases were more atypical. High-resolution banding techniques did not detect a microdeletion in any of the cases; however, one of the 20 patients had a translocation between chromosomes 13 and 22. This patient also had a microdeletion of 22q11.2 detected by FISH and clinical features of DGS. None of the patients who were evaluated for disorders related to DGS showed microdeletions. We conclude that FISH is a useful, easily applied technique for the diagnosis of 22q11.2 microdeletion syndromes, particularly DGS. This test may also be useful in genetic counseling and in both prenatal and postnatal diagnoses. PMID- 8634786 TI - Rapid identification of mixed up bladder biopsy specimens using polymorphic microsatellite markers. AB - During standard registration of incoming surgery specimens, loss of registration numbers may occur. In our laboratory two series of small bladder biopsies (numbered I-V and I-VI, respectively), obtained from two patients, were given the same laboratory registration number. The biopsies were of similar size and embedded in paraffin. Thus, five pairs of Roman-numbered paraffin blocks had the same laboratory registration numbers. The histological findings in several biopsies were similar, some showing carcinoma in situ. Only from biopsy number VI was the identity retained, and this specimen could be used as a reference. We used polymerase chain reaction (PCR)-driven microsatellite marker analysis to identify the specimens using five different microsatellite markers. Within 48 h, two different banding patterns were revealed, allowing us to distinguish the two series. In addition, in one biopsy which showed carcinoma in situ of the bladder, microsatellite instability was observed while in none of this patient's other biopsies containing carcinoma in situ could this phenomenon be detected, which may indicate intratumor heterogeneity or multifocality. In conclusion, it is possible to solve the problem of mixing up small paraffin-embedded biopsies by using microsatellite marker PCR. PMID- 8634785 TI - Insulin-like growth factor II gene expression by congenital mesoblastic nephroma. AB - We studied the expression of insulin-like growth factor II (IGF2) and Wilms' tumor gene (WT1) in nine cases of congenital mesoblastic nephroma (CMN) and five cases of first trimester fetal kidneys by in situ hybridization. Our aim was to determine their site of expression and to correlate their histogenetic relationship to those of other childhood renal tumors. Our results showed that all nine cases of CMN (classic, mixed, and cellular) contained abundant IGF2 but not WT1 transcripts. The IGF2 transcripts were diffusely distributed over the tumor cells. These findings suggest that CMN is derived from primitive mesenchymal nephrogenic cells and have a potential to differentiate into a stromal cell lineage. PMID- 8634787 TI - [Pleural effusion associated with lung cancer. Problems of classification according to TNM categories]. PMID- 8634788 TI - [Resistance of Mycobacterium tuberculosis in the province of Castellon]. AB - We conduced a cross-sectional study to determine the prevalence of resistant mycobacteria in our setting. All patients in whom M. tuberculosis had been isolated in cultures of clinical samples (119) between January 1992 and December 1993 took part in the study. Canetti's method of percentages was used for the study of sensitivity to the following drugs: isocyanide, rifampicin, streptomycin, ethambutol and para-aminosalicylic acid. Overall resistance of M. tuberculosis was 9.24%. Specifically, we found resistance to isocyanide in 5.9%, to streptomycin in 4.20%, to rifampicin in 5.04% and to ethambutol in 1.68%. Resistance was primary in 7% and secondary in 21.05%; 2% showed primary resistance to isocyanide, 1% to isocyanide and rifampicin, 2% to streptomycin, 1% to ethambutol and 1% to streptomycin and rifampicin. These resistance indices are in keeping with those published for other areas of Spain. PMID- 8634789 TI - [Efficacy and tolerance of the treatment of tuberculosis in the aged]. AB - We analyzed 55 confirmed cases of tuberculosis in patients over 65, a sample that amounted to 9% of all patients seen in our practice over a period of 5 years. Mean age was 72.4 and the male/female ratio was 4/1. The most frequently associated diseases were tobacco addiction (49%), chronic obstructive pulmonary disease (33%), alcoholism (25%) and prior diagnosis of tuberculosis (20%). Lung involvement was the most common clinical presentation (76%), followed by pleural (9%) and skeletal (7%) involvement. The clinical picture was non specific, with 13% remaining asymptomatic. Cough was the most frequent symptom (45%) and unilateral apical fibrosis with ulceration was the most frequent radiological finding. Pleural discharge and cavitation were demonstrated in 14 and 22%, respectively. Scarring was visible on X-rays in 44%. The tuberculin test was positive in 88% of the cases in which it was performed. Mean delay in diagnosis was 3.4 months; 62% were diagnosed by sputum test, 11% by culture, and 27% histology. In 4% death was directly caused by tuberculosis. Three patients withdrew from treatment, in one case treatment failed, and there was one relapse detected at follow-up. We observed adverse side effects in 33%, and found no statistically significant differences between the 2 therapeutic protocols used (2 months RHS/7 months RH and 2 months RHZS/4 months RH). The incidence of tuberculosis among the elderly is low in our practice and the entity behaves much as it does in the rest of the adult population. Both the efficacy and tolerance of treatment can be considered optimal. PMID- 8634790 TI - [Analysis of proliferating cell nuclear antigen (PCNA) expression in 24 cases of primary non-small cell pulmonary carcinomas and correlation with survival]. AB - Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein involved in DNA replication that is believed to provide an indication of proliferation in some neoplasms. This study analyzes PCNA expression in 24 cases of primary non small cell lung cancer using monoclonal PC-10 antibodies in paraffin embedded material. We found significant inter- and intra-tumoral variations in PCNA expression, and no statistically significant relation between the amount of PCNA expression and the size and location of tumors, index of mitosis, histological tumor type or patient age. We found a statistically significant relation (r = 0.47; p < 0.05) between survival and amount of PCNA expression in a sample of 19 cases, but no statistically significant differences in survival related to whether PCNA expression was slight (0-25), moderate (25%-50%) or high (> 50%), and no prognostic value for degree of PCNA expression. PMID- 8634791 TI - [Usefulness of transthoracic pulmonary aspiration biopsy in lung abscess secondary to neoplasm]. AB - OBJECTIVE: To evaluate the usefulness of transthoracic needle biopsy (TNB) for the diagnosis of bronchial carcinoma underlying lung abscess (LA), and to determine the bacteriology of lung abscess secondary to neoplasm (LASN). PATIENTS AND METHOD: One hundred thirteen consecutive patients diagnosed of LA were enrolled. Radiologically guided TNB was performed on all patients using 20-22 G needles. Microbiological and cytological samples were processed. Fiberoptic bronchoscopy (FB) was performed if there were risk factors for lung cancer. TNB diagnosed cases were compared with the remaining cases of LA. RESULTS: Neoplasia was found in 21 LA patients. TNB samples provided diagnostic information in 15 cases, there were 2 false negatives, and no cytology sample was processed in 4 cases. Diagnosis was based on FB in 17 cases. All neoplasias were diagnosed with one technique or the other. TNB culture was positive in 90% (19/21) of the LASN patients, H. influenzae being the most frequently isolated bacterium. The number of cultures that presented a single microbe was significantly greater (p < 0.02) among LASN patients (14/19 versus 33/79). These patients also had significantly more aerobic bacteria (19/19 versus 45/79; p < 0.001) and fewer anaerobies (4/19 versus 52/79; p < 0.001) than did the primary LA patients. CONCLUSIONS: 1) TNB is highly useful for arriving at a bacteriologic diagnosis of LASN and in associated cancer. 2) TNB complements FB for the diagnosis of neoplasia underlying LA and helps to reduce the number of unnecessary thoracotomies. 3) A great variety of germs, particularly aerobic bacteria, are implicated in LASN. PMID- 8634792 TI - [Results of a smoking cessation program among Spanish pulmonologists and thoracic surgeons]. AB - We evaluated the efficacy of a smoking cessation program using 16-hours nicotine patches offered by mail to physicians belonging to the Spanish Pneumological and Thoracic Surgery Society. Fifty-five subjects (21 women) with a mean age of 40 underwent treatment with 16-hours nicotine patches with different dose-time applications dependent on Fagerstrom test scores. Abstinence, which was verified by measuring carbon monoxide in expired air, was 23.6% after 6 months of follow up. We observed a low rate of mild side effects. PMID- 8634793 TI - [Requisites for accreditation of teaching units in pneumology]. PMID- 8634794 TI - [Bilateral pleural effusion and rheumatoid arthritis. Diagnostic value of pleural fluid cytology]. AB - We present a 69 years old male patient diagnosed of rheumatoid arthritis (RA) with signs of pleuropulmonar disease. The diagnose of RA was done of 49 years and since then treated with non steroid antiinflammatory drugs and during the acute phases of RA with steroid drugs. The pleural effusions showed an exudate with pseudochilothorax criteria with acid pH acid low glucose concentrations. The cytological study of the effusion demonstrated the presence of characteristic mononuclear cells. PMID- 8634795 TI - [Portable oxygen therapy in the Madrid area]. AB - Portable oxygen therapy using liquid oxygen has been available in the Madrid area since 1992 as part of a government program. The prescription of liquid oxygen theoretically requires the careful selection of patients who will benefit from a portable source of oxygen, as well as the performance of a series of treadmill tests with and without supplemental oxygen in order to assess the benefit derived from this expensive means of delivering therapy. As no studies had been done of whether these criteria for prescribing liquid oxygen were being met, we conducted telephone interviews with patients residing in the Madrid area who had portable oxygen sources at their disposal. Of a total of 190 patients, whose names were provided by oxygen supply companies, 145 could be evaluated. Liquid oxygen was not being used by 17%. Stress tests had not been performed before prescription of liquid oxygen in 65%. In conclusion, it can be suspected that a high percentage of patients receive liquid oxygen who do not meet the criteria for prescription and who have not performed the recommended tests, and that compliance is low. PMID- 8634796 TI - [Bronchiolitis associated with ulcerative colitis]. AB - Inflammatory bowel disease (IBD) only occasionally affects the lung. Noteworthy among the various pulmonary entities related to IBD are chronic bronchitis and bronchiectasis, though reports of bronchiolitis, obliterants bronchiolitis with organizing pneumonia and other interstitial diseases have also been published. Given the rarity of such pneumopathy, we report a case of bronchiolitis in a patient with a prior diagnosis of ulcerative colitis. We discuss the radiological findings and the results of lung function testing, emphasizin the utility of corticosteroid treatment, which usually leads to a good outcome in pneumopathy secondary to IBD. PMID- 8634797 TI - [Polymyalgia rheumatica presenting pulmonary epidermoid carcinoma]. PMID- 8634798 TI - [Castleman's disease presenting as a calcified mediastinal mass]. PMID- 8634799 TI - [Spontaneous pneumothorax as initial manifestation of small-cell bronchogenic carcinoma]. PMID- 8634800 TI - [Recurrent hemoptysis as a presentation form of thoracic actinomycosis]. PMID- 8634801 TI - [Hyaluronic acid in bronchoalveolar lavage of healthy subjects]. PMID- 8634802 TI - Glycolic acid peels. PMID- 8634803 TI - Clinical and histological effects of glycolic acid at different concentrations and pH levels. AB - BACKGROUND: Much has been said about the effects of glycolic acid with little scientific evidence to substantiate the findings. OBJECTIVE. This study reports on the clinical and histological effects of glycolic acid at pH levels 3.25, 3.80, and 4.40, and at 3.25%, 6.50%, 9.75%, and 13.00% on ichthyotic/xerotic skin. METHODS: Product treatment consisted of a 2-week washout period followed by 3 weeks of product application (BID) with A 1-week regression period. Shave biopsies and clinical evaluations for dryness, moisturization, and transepidermal water loss were made at baseline, 1, 2, and 3 weeks of use, and at the regression period. RESULTS: Clinically, ichthyotic/xerotic skin was normalized with histologic evidence of stratum corneum thinning, viable epidermal thickening, and marked increases in glycosaminoglycan and collagen content. CONCLUSION: All pH levels and concentrations demonstrated significant improvement in the condition of the skin with trends implying that increasing the pH increases efficacy. PMID- 8634805 TI - Photoprotective and antiinflammatory effects of topical glycolic acid. AB - BACKGROUND: Concerns about photosensitizing potential of alpha hydroxy acids have been expressed. A previous study, however, reported topical glycolic acid showing the opposite potential, that is, photoprotective. This study was designed to test the antiinflammatory and photoprotective capabilities of glycolic acid. OBJECTIVE: The effects of short-wave ultraviolet light (UVB) on skin treated with glycolic acid were evaluated in two different studies at two different locations. METHODS: In the first study the antiinflammatory potential of topical glycolic acid was tested on erythematous templates on the backs of human volunteers. Erythema was induced by exposure to three times the minimum erythema dose (MED) of UVB. Glycolic acid cream in an oil-in-water vehicle at 12% partially neutralized with ammonium hydroxide to a pH of 4.2 was applied to the template beginning 4 hours postirradiation four times a day. A second template on the same subject was used as a vehicle control. After 48 hours a marked reduction of erythema was noted when compared with the vehicle control site. In the second study, four test sites were exposed to UVB light in the following manner. Site 1 was a nontreated control site and was used to establish the MED for the subjects being tested; site 2 was also exposed to a MED series but was treated 24 hours postirradiation for 7 days with two glycolic acid-based products (cleanser and oil-free moisture lotion, both containing 8.0% glycolic acid at a pH of 3.25); site 3 was treated first with the two glycolic acid-based formulas for 3 weeks prior to being exposed to UVB light; and site 4 was treated as outlined in site 3, with the inclusion that the site was chemically peeled for 6 minutes (with a 50% glycolic solution at a pH of 2.75) 15 minutes prior to UVB exposure. RESULTS: When UVB-burned skin was treated with glycolic acid daily for 7 days (site 2), a 16% reduction in irritation was observed compared to nontreated skin (site 1), implying that skin healed sooner when treated with glycolic acid. When a comparison of nontreated skin was made to skin treated with glycolic acid for 3 weeks prior to UVB exposure (site 1 vs site 3), a sun protection factor (SPF) of 2.4 was achieved. When a comparison of skin treated for 3 weeks was made to skin treated for 3 weeks and chemically peeled (site 3 vs site 4) the data implied that the chemical peel reduced the SPF value of skin treated with glycolic by approximately 50%, however, an SPF trend of 1.7 was still obtained when compared with untreated skin. CONCLUSIONS. The studies demonstrated that topical glycolic acid provides a photoprotective effect to pretreated skin yielding an SPF of approximately 2.4. In addition, when glycolic acid is applied to irradiated skin, it accelerates resolution of erythema. The data obtained from both studies support the hypothesis that glycolic acids acts as an antioxidant. PMID- 8634804 TI - Comparison of the effect of various chemical peeling agents in a mini-pig model. AB - BACKGROUND: With the advent of newer chemical peels, there is now a wide range of peeling agents that can be applied on specific patients. OBJECTIVE: The purpose of this study was to closely examine the more common chemical peeling agents at different concentrations. METHODS: The study methods were carried out by thoroughly cleansing the skin surface with acetone. Different concentrations of the chemical peels were applied on different skin areas (2 x 2 cm each) and left on the skin for 15 minutes: phenol-Bakers, 25%, 50%, 75%, 88%; trichloroacetic acid, 25%, 50%, 75%; glycolic acid, 50%, 70%; and pyruvic acid, 50%, 100%. Serial biopsies were taken from each peeling site at 1, 7, and 21 days post-peel. Biopsies were then evaluated for epidermal changes, inflammation, and collagen deposition. RESULTS: The results show that Bakers phenol peel caused the most inflammation and nonspecific reaction, and in addition, a proportionate amount of new collagen deposition. Plus, increasing concentrations of phenol and TCA caused increasing amount of epidermal sloughing and inflammation after 1 day post-peel. The extent of reaction from the phenol and TCA was directly proportional to the collagen deposition at 21 days. CONCLUSIONS: The glycolic acid and pyruvic acid caused minimal nonspecific reaction. However, the collagen deposition caused by the glycolic acid and pyruvic acid was disproportionately increased suggesting a direct stimulatory effect by the two agents. PMID- 8634806 TI - Glycolic acid modulation of collagen production in human skin fibroblast cultures in vitro. AB - BACKGROUND: Glycolic acid has been used extensively for the treatment of photoaging and wrinkles. Suggestions have been made that glycolic acid may have specific dermal effects, although biochemical studies are limited. OBJECTIVE: This study's purpose was to examine the effect of glycolic acid on the radioactively labeled collagen production in human skin fibroblasts in culture. METHODS: Normal dermal fibroblasts were grown to semi-confluence and incubated in the presence of glycolic acid for 24 hours. Radioactive proline was added to the cultures. Using a specific amino acid assay, the amount of radioactive hydroxyproline was measured and was used as an accurate index of collagen production. RESULTS: Results show that glycolic acid caused an elevated collage production in the fibroblasts. CONCLUSION: These results demonstrate a specific stimulatory effect by the glycolic acid and could explain some of the positive benefits from the clinical use of glycolic acid. PMID- 8634807 TI - The combination of glycolic acid and hydroquinone or kojic acid for the treatment of melasma and related conditions. AB - BACKGROUND: Melasma continues to be a difficult problem. Although the cause is genetic, the condition is aggravated with sunlight, birth control pills, and pregnancy. Although hydroquinone is effective and has been available for years, a new product, kojic acid, has the advantage of being pharmaceutically more stable and, also, a tyrosinase inhibitor. OBJECTIVE: To evaluate on melasma and related conditions two similar formulations of glycolic acid/hydroquinone and glycolic acid/kojic acid. The therapeutic index of the two formulations is examined. METHODS: Thirty-nine patients were treated with kojic acid on one side of the face and hydroquinone in a similar vehicle on the other side of the face. The results were documented by a clinical investigator and with Wood's light examination combined with ultraviolet light photography. RESULTS: Fifty-one percent of the patients responded equally to hydroquinone and kojic acid. Twenty eight percent had a more dramatic reduction in pigment on the kojic acid side; whereas 21% had a more dramatic improvement with the hydroquinone formulation. These results were not statistically different. The kojic acid preparation was more irritating. CONCLUSION: Both glycolic acid/kojic acid and glycolic acid/hydroquinone topical skin care products are highly effective in reducing the pigment in melasma patients. Both formulations should be available to the dermatologist to satisfy the patient's preferences. PMID- 8634808 TI - Short contact 70% glycolic acid peels as a treatment for photodamaged skin. A pilot study. AB - BACKGROUND: Glycolic acid has been one of the more commonly used alpha hydroxy acids for the treatment of photodamaged skin. Its value as a quick "skin refreshing" peeling agent has been widely touted. This type of peel differs from a conventional therapeutic peel (eg, phenol, trichloroacetic acid, or a longer time exposure alpha hydroxy acid peel) in that there is little skin reaction and patients can go about their daily routine without concern. OBJECTIVE: To assess the potential value of glycolic acid-based refresher peels as a cosmetic procedure. METHODS: Twelve healthy subjects with at least a moderate degree of photodamage were treated with monthly serial 70% glycolic acid peels over a period of 4 months. In addition to the "peels," six subjects were randomized to a 10% glycolic acid-based moisturizer twice daily. Patients were evaluated monthly and graded on a clinical scale using objective measures. RESULTS: No conclusive differences were noted on histologic evaluation. Ninety percent (9/10) of patients felt that overall they noticed significant improvement, however, there was no distinction between the two treatment options. The improvement in fine wrinkling and pigmentation was primarily seen in the patients who additionally received 10% glycolic emollient twice daily. CONCLUSION: In this limited pilot study, no specific benefit could be assigned to the concomitant use of monthly glycolic acid refresher "peels" in the treatment of photodamaged skin. PMID- 8634809 TI - Clinical improvement of photoaged skin with 50% glycolic acid. A double-blind vehicle-controlled study. AB - BACKGROUND: Although there is increasing interest in the use of glycolic acid in the treatment of photoaged skin, to our knowledge, no controlled study has been done to assess the efficacy or the mode of this agent. OBJECTIVE: The purpose of this study was to determine whether 50% glycolic acid can improve photoaged skin and to study the histological basis for this improvement. METHODS: Forty-one volunteers were recruited into this double-blind vehicle-controlled study. Glycolic acid (50%) or vehicle was applied topically for 5 minutes to one side of the face, forearms, and hands, once weekly for 4 weeks. Punch biopsies were taken at pretherapy and at 5 weeks for histologic study. RESULTS: Significant improvement noted included decrease in rough texture and fine wrinkling, fewer solar keratoses, and a slight lightening of solar lentigines. Histology showed thinning of the stratum corneum, granular layer enhancement, and epidermal thickening. Some specimens showed an increase in collagen thickness in the dermis. CONCLUSION: The results of this study demonstrate that the application of 50% glycolic acid peels improves mild photoaging of the skin. PMID- 8634810 TI - A histological comparison of 50% and 70% glycolic acid peels using solutions with various pHs. AB - BACKGROUND: Seventy percent glycolic acid solutions are being commonly used as superficial chemical peeling agents. The pH of these solutions ranges from 0.08 to 2.75. The histologic effects of these various pH solutions on human skin have not been studied. OBJECTIVE: The histologic effects of several commercially available glycolic acid solutions at various pHs were examined. METHODS: Test areas of seven glycolic acid solutions were applied to facial skin of two patients. The skin was not prepped for a peel prior to the application of the acid. The solution was left in place for 30 minutes, then neutralized. After 48 hours, a 2-mm punch biopsy was performed and examined histologically. RESULTS: The peeling solutions with a pH below 2 demonstrated the potential to induce crusting and necrosis, which was not seen with the partially neutralized solutions with a pH above 2. The higher concentration acids (70%) created more tissue damage than the lower concentration (50%) when comparing solutions with free acid. CONCLUSION: This study demonstrates that chemical peeling with a 70% free glycolic acid creates more tissue damage than a free glycolic acid. When using a 70% glycolic acid solution, the lower pH products (below pH 2) create more necrosis than the partially neutralized products with a pH above 2. At this time there is no evidence that creating necrosis leads to a more favorable result of the peel. Therefore, the use of partially neutralized glycolic acid solutions seems prudent, since they have a better safety profile than low pH solutions, which contain only free glycolic acid. PMID- 8634811 TI - An alpha hydroxy acid derivative suitable for sensitive skin. AB - BACKGROUND: Individuals with sensitive skin may not tolerate some products formulated with alpha hydroxy acids (AHAs) due to unacceptable levels of stinging and irritation. OBJECTIVE: To develop a neutral molecule that is well tolerated by individuals with sensitive skin without stinging or irritation, yet retains the beneficial effects of AHAs. METHODS: A neutral amide derivative, methoxypropylgluconamide (MPG), was invented. Formulations containing MPG (15%) were tested by cummulative irritation, repeat insult patch testing, and lactic acid stinging assay. Bilateral comparison clinical and histological studies of xerosis and photoaging assessments were performed. RESULTS: MPG was nonirritating and nonsensitizing. Clinically significant improvements in xerosis and photoaging including uneven pigmentation were seen. Improvement in the morphology of the stratum corneum epidermis and in the amount of glycosaminoglycans in the epidermis and dermis were seen histologically. CONCLUSIONS: A molecule was invented (MPG) that could be formulated in a neutral vehicle to avoid skin irritation and produced improvements in xerosis and photoaging. PMID- 8634813 TI - Amelanotic malignant melanoma disguised by Darier's disease (keratosis follicularis) PMID- 8634812 TI - Round table discussion of alpha hydroxy acids. PMID- 8634814 TI - Acrally occurring dermatofibrosarcoma protuberans in children and adults. PMID- 8634815 TI - Lipid profiles, alopecia, and coronary disease: any relationship? PMID- 8634816 TI - Mantle radiation induced symmetrical basal cell carcinomas over the acromioclavicular joints. PMID- 8634817 TI - Improving razor blade biopsy techniques. PMID- 8634819 TI - A redefinition of male pattern baldness. PMID- 8634818 TI - Eccrine spiradenoma, not poroma. PMID- 8634820 TI - Trends in tattooing. PMID- 8634821 TI - Cryosurgery of actinic keratoses: the patient's perspective. PMID- 8634822 TI - Synthesis and post-coital contraceptive activity of ether and ester analogues of 2,3-diaryl-2H-1-benzopyrans. AB - Ether and ester analogues of 2,3-diaryl-2H-1-benzopyrans have been synthesised and tested for their pregnancy inhibiting activity in immature rats. Some of the compounds exhibit potent activity. Structure-activity relationship relative to the hydroxy analogue has been discussed. In general, esters were found to be better inhibitory agents. PMID- 8634823 TI - Specific inhibitors for the glycolytic enzymes of Trypanosoma brucei. PMID- 8634824 TI - 1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogs of CC-1065 and the duocarmycins: synthesis and evaluation. AB - An extensive study of analogs of the potent antitumor antibiotics CC-1065 and the duocarmycins which incorporate the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4 one (CBI) alkylation subunit are detailed. In contrast to early speculation, deep seated modifications in the CC-1065 and duocarmycin alkylation subunits are well tolerated and the CBI-based analogs proved to be potent cytotoxic agents and efficacious antitumor compounds. Full details of studies defining a direct relationship between functional stability and in vitro cytotoxic potency are described. As such, the readily accessible CBI-based analogs were found to be four times more stable and four times more potent than the corresponding analogs containing the authentic CPI alkylation subunit of CC-1065 and comparable in potency to agents containing the authentic alkylation subunit of duocarmycin SA. Similarly, the CBI-based agents alkylate DNA with an unaltered sequence selectivity at an enhanced rate and with a greater efficiency than the corresponding CPI analog and were comparable to the corresponding analog incorporating the duocarmycin SA alkylation subunit. Systematic and extensive modifications and simplifications in the DNA binding subunits attached to CBI were explored with the comparisons of both enantiomers of CC-1065 and the duocarmycins 2 and 3 with enantiomers of 18-24, 25-29, 57-61, 62-65, 66-68, 72, 73, 78 and 79. PMID- 8634825 TI - Synthesis of a glycopeptide carrying a N-linked core pentasaccharide. AB - A glycopeptide carrying a pentasaccharide core structure of asparagine-linked glycoproteins was synthesized. The synthesis of the carbohydrate part was performed starting from monosaccharide components in an unambiguous manner. The resultant pentaglycosyl azide was reduced into corresponding glycosyl amine and coupled with an aspartic acid derivative to furnish an Asn-linked oligosaccharide in a protected form. Subsequent coupling with a dipeptide, followed by deprotection gave the target compound. PMID- 8634826 TI - Molecular dynamics simulations of m3-muscarinic receptor activation and QSAR analysis. AB - Molecular dynamics simulations of the rat m3-muscarinic seven-helix-bundle receptor models were performed on the free, agonist-bound and antagonist-bound forms. A comparative structural/dynamics analysis was performed in order to explain the perturbations induced by the functionally different ligands when binding to their target receptor. Theoretical quantitative structure-activity relationship models were developed; a good correlation was obtained between the interaction energies of the minimized average ligand-receptor complexes and the pharmacological affinities of the considered ligands. The consistency obtained between the structural rearrangement of the transmembrane seven-helix-bundle models considered and the experimental pharmacological efficacies and affinities of the ligands constitutes an important validation of the 3-D models proposed and allows the inference of the mechanism of ligand-induced or mutation-induced receptor activation at the molecular level. PMID- 8634827 TI - 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: oxime ether analogs of pravastatin. AB - Pravastatin, a potent anti-hypercholesteremic drug, has been developed by Bristol Myers Squibb for treatment of hypercholesterolemia and other related diseases. Several structurally related compounds (SQ 31,554, SQ 31,879, SQ 31,947, SQ 32,391, SQ 32,770, SQ 32,390 and SQ 32,469) modified at the 3-position of the hexahydronaphthalene ring system of pravastatin were prepared in the course of developing the basic reagents for a radioimmunoassay of the parent drug. The biological activity of these analogs was comparable to pravastatin itself. Indeed, one member of this series was found to be several times more potent than pravastatin. PMID- 8634828 TI - Inhibition of rat liver mitochondrial monoamine oxidase by hydrazine-thiazole derivatives: structure-activity relationships. AB - The purpose of this research is to study the relationship between chemical structure and inhibitory activity of some hydrazine-thiazole derivatives on rat liver mitochondria monoamine oxidase (MAO). Forty-five compounds belonging to three series of hydrazine-thiazole derivatives, with either alkylic or arylic substituents in the thiazole ring, were tested. The highest inhibitory activity was observed with piperonyl derivatives 25 and 40, which contain a 4-methyl group in the thiazole nucleus. The structure-activity relationship of MAO inhibitors was established in relation to hydrophobic, electronic and steric hindrance parameters. A mechanism of enzyme inhibition was proposed based on the calculation of HOMO energies. PMID- 8634829 TI - Oligonucleotide analogues containing 4'-C-(hydroxymethyl)uridine: synthesis, evaluation and mass spectrometric analysis. AB - 2',3'-Di-O-tert-butyldimethylsilyl-4'-C-(hydroxymethyl)uridine was synthesized and converted into the phosphoramidite building blocks 9 and 13. Novel oligodeoxynucleotide analogues containing 4'-C-hydroxymethyl linked phosphodiester internucleoside linkages and 3'-hydroxyl linked phosphodiester internucleotide linkages were synthesized on an automated DNA-synthesizer. The latter modification introduced an additional 4'-C-hydroxymethyl functionality. Oligodeoxynucleotides with one or two modifications in the middle or in the ends of 17-mers, 15-mers and 14-mers have been evaluated with respect to hybridization properties and enzymatic stability. Compared to unmodified oligomers, 3'-end modified oligodeoxynucleotides were stabilized towards 3'-exonucleolytic degradation, but showed moderately to strongly lowered hybridization properties towards complementary DNA. However, more promising results were obtained in melting experiments with complementary RNA where only small decreases in melting temperature were detected. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to identify products from syntheses of the modified oligodeoxynucleotide analogues. PMID- 8634830 TI - Separation of alpha-adrenergic and imidazoline/guanidinium receptive sites (IGRS) activity in a series of imidazoline analogues of cirazoline. AB - To characterize the structure-activity relationship between alpha 1-adrenergic receptors and the family of imidazoline/guanidinium receptive sites (IGRS), we synthesized and characterized a series of analogues of cirazoline, an imidazoline with high affinity for alpha 1-adrenergic receptors and IGRS. Analysis of potency, affinity and efficacy of the synthesized molecules indicate different structure-activity relationships for IGRS and alpha-adrenergic receptors. Cirazoline exhibits a 25-fold higher affinity for IGRS (pKi 7.9) than for alpha 1 adrenergic receptors. Replacement of the cyclopropyl ring with an isopropoxy group resulted in a molecule that was 20-fold more selective for alpha 1 adrenergic receptors than for IGRS, i.e. a 500-fold increase in selectivity relative to cirazoline. The unsubstituted derivative 3 and the methyl and allyl substituted analogues 4 and 12 are of particular interest: compounds 3 and 4 recognize IGRS with high affinity (pKi 7.83 and 8.17) and high selectivity (398 and 123) with respect to the alpha 1-adrenergic receptor; compound 12 also recognizes IGRS with high affinity (pKi 8.08) and high selectivity (228 and 138) with respect to the alpha 2B and alpha 2C-adrenergic receptor subtypes. Thanks to their IGRS selectivity, these compounds represent novel and valuable pharmacological tools for the characterization and elucidation of the physiological role of these novel sites. PMID- 8634831 TI - The biological activity of cyclic bis(bibenzyls): a rational approach. AB - The biological activities reported for marchantin A (1), a natural cyclic bis(bibenzyl), were studied in comparison with cepharanthine (2), a therapeutically useful bisbenzylisoquinoline alkaloid. Based on the examination of steric, electrostatic, and hydrophobic similarity, as well as on the comparison of biological activities, the similar therapeutic properties of 1 and 2 can be attributed to binding on a common receptor. The wide range of activity of 1 can be interpreted by a mechanism of action based on a calcium binding. PMID- 8634832 TI - Design, synthesis, and biological evaluation of isocyanurate-based antifungal and macrolide antibiotic conjugates: iron transport-mediated drug delivery. AB - The syntheses and preliminary biological evaluation of conjugates of a synthetic isocyanurate-based trihydroxamate siderophore with two antifungal agents, 5-FU (conjugate 9) and norneoenactin (conjugate 12), and a macrolide antibiotic, erythromycylamine (conjugate 18), are described. A 19F NMR study was used to determine the hydrolytic stability of conjugate 9 under assay conditions. Preliminary biological studies with ferric complexes of conjugates 9 and 12 indicated that these antifungal agents are recognized by Candida and perhaps are actively transported into the cell by the siderophore-transport mechanisms. While conjugate 18 did not show any significant antibacterial activity, presumably due to size restriction, the 5-FU conjugate 9 appeared to be moderately active against a variety of Gram-positive strains, and was more active than the 5-FC control against some strains of Staphylococcus. PMID- 8634833 TI - New 1,2,6-thiadiazine dioxide acyclonucleosides: synthesis and antiviral evaluation. AB - New acyclonucleosides derived from 1,2,6-thiadiazine dioxide systems have been synthesized. Lipase-mediated deacylation procedure was used to obtain the deprotected derivatives. All the newly prepared compounds were tested as antiviral agents, but none of them showed significant activity. PMID- 8634834 TI - Structure-affinity relationships of baclofen and 3-heteroaromatic analogues. AB - Substituting a furan, a thiophene, a benzo[b]furan, a benzo[b]thiophene, or a quinoline ring for the p-chlorophenyl moiety of baclofen has led to GABAB ligands with different affinities depending on the nature of the heteroaromatic ring, and on the nature and position of its substituent. As steric effects cannot account for all the affinity variations, we have studied the lipophilic and electronic properties of baclofen and selected 3-heteroaromatic analogues, gaining insight into the structural features necessary for GABAB affinity. Centrifugal partition chromatography (CPC) has been used to measure octan-1-ol water distribution coefficients, while ab initio molecular orbital (MO) calculations were performed to study electronic properties. PMID- 8634835 TI - A behaviorally selective class of thiophene-containing benzodiazepine receptor ligands. AB - In a continued effort to probe the role of the aromatic rings in classical 1,4 benzodiazepine (BDZ) ligand pharmacology, a series of new thiophene-containing benzodiazepine receptor (BDZR) ligands were synthesized. As a first step in determining the binding profile and selectivity to BDZR functional subtypes, the affinities in two central nervous system (CNS) regions, cerebellum, in which a single 'Type I' BDZR could be labeled; and spinal cord, in which we have previously demonstrated some receptor heterogeneity, were determined. These compounds were also assessed for their compliance with a recently developed three dimensional pharmacophore for recognition and activation of the 'Type I' BDZR, using the techniques of computational chemistry. The computations showed all ligands synthesized fulfilled the minimum requirements for recognition, further validating the current pharmacophore. Using the criteria for activation, the new ligands were all predicted to be agonists at the cerebellar 'Type I' BDZR. Since the compounds showed reasonable affinity, the behavioral profile of one of them at five in vivo endpoints was determined. This compound demonstrated more behavioral selectivity than the typical 1,4-BDZ ligand. While they fulfilled the requirements for agonist activity at the 'Type I' BDZR, these ligands showed significantly greater delocalization in the electron density distribution in the lowest unoccupied molecular orbital (LUMO), so that either aromatic ring could serve as an electron accepting site, not just the one comparable to the more classical BDZR agonist, flunitrazepam. It is possible that the ability of the second ring in the tested compound (5a) to also function as an electron acceptor can affect the recognition and activation of other receptor types leading to the more discriminate behavioral profile of this thiophene analog compared to flunitrazepam. PMID- 8634836 TI - NMR structure of d(CGCA3T3GCG)2:tren-microgonotropen-b:Zn(II) complex and solution studies of metal ion complexes of tren-microgonotropen-b interacting with DNA. PMID- 8634838 TI - Analysis of autogenous vein femoral-infrapopliteal bypass for limb salvage in the elderly. AB - The hypothesis that older patients undergoing femoral-infrapopliteal bypass have a similar outcome as a matched younger group of patients undergoing the same operation was tested. Seventy-six femoral-infrapopliteal autogenous saphenous vein bypasses for critical limb ischemia were performed from 1985 to 1990. By using the life-table method, the primary and secondary patency, limb salvage and survival rates are analyzed and compared for older and younger age groups. Forty cases (53%) were performed in an elderly group, defined as age 70 or older. At 4 years, there was no significant difference between age groups in limb salvage and patency rates. However, operative mortality for the older age group was 12%, compared with 0% in the younger group (P = 0.0004). Thus, femoral-infrapopliteal autogenous vein bypass can be performed with comparable limb salvage and patency rates for an older age group, but the risk of operative mortality appears to be increased with age. PMID- 8634837 TI - Thrombolytic therapy for acute deep venous thrombosis: how much is enough? AB - Twenty-eight patients treated with thrombolytic therapy for acute deep venous thrombosis were monitored prospectively with non-invasive testing every 12-24h during treatment to evaluate thrombus response and whether duration of therapy was appropriate. Some 75% (21 of 28) of patients demonstrated improvement with lytic therapy with 36% (10 of 28) demonstrating complete lysis; 95% of responders (20 of 21) initiated lysis within 24h. Some 33% (7 of 21) of all responders and 64% (7 of 11) of those having partial lysis had treatment terminated during thrombus resolution but before maximal lysis. Non-invasive testing indicated that thrombolytic therapy for acute deep venous thrombosis is frequently terminated before maximal lysis of the thrombus. Monitoring thrombus response with venous duplex imaging should be part of the treatment strategy of deep venous thrombosis if thrombolytic therapy is used. This approach should increase efficacy and potentially reduce complications of thrombolytic therapy for acute deep venous thrombosis. PMID- 8634839 TI - Upper extremity thromboembolism after axillary-axillary bypass grafting. AB - Two patients experienced upper extremity thromboembolism after axillary-axillary bypass grafting (AxAG) for symptomatic subclavian artery stenosis. The first patient, a 67-year-old male, presented with left upper extremity thromboembolism 3 years after AxAG with 8 mm externally support PTFE. An arteriogram revealed a patent AxAG, thrombus in the proximal left subclavian arterial stump just distal to its occlusion, and multiple digital artery emboli. The patient was treated with warfarin for 8 months, with resolution of symptoms. The second patient, a 57 year-old male, occluded his AxAG (8 mm knitted Dacron) with minimal return of symptoms. Non-operative treatment was elected and 4 years later the patient presented with right upper extremity (donor side) thromboembolism. Arteriography revealed occlusion of the AxAG, radial artery, and digital arteries of the index, long and ring fingers. Thrombolytic therapy of the right arm was undertaken with minimal improvement. Subsequent detachment of the AxAG and placement of an interposition reversed saphenous vein graft was performed. Both patients continue to be asymptomatic during follow-up of 4.7 and 2.0 years, respectively. PMID- 8634840 TI - Adult respiratory distress syndrome after cardiac surgery. AB - Adult respiratory distress syndrome, characterized by high permeability pulmonary oedema caused by endothelial cell damage, resulting in refractory hypoxemia, has a very high mortality. Cardiopulmonary bypass is said to be responsible for the development of adult respiratory distress syndrome after cardiac surgery. The present study was performed in order to identify predicting and aetiological factors of adult respiratory distress syndrome and multiple organ failure after cardiac surgery. Between January 1984 and December 1993, 3848 patients underwent cardiac surgery with cardiopulmonary bypass in the authors' institution, and were analysed in a retrospective manner. The operations performed were 3444 coronary artery bypass grafts (CABG), 267 valve and 137 combined (CABG + valve) procedures. The incidence of adult respiratory distress syndrome was 1.0% (38 of 3848) with an overall mortality rate of 68.4% (26 patients); 24 of these died from multiple organ failure. Multivariate regression analysis identified hypertension, current smoking, emergency surgery, preoperative New York Heart Association (NYHA) class 3 and 4, low postoperative cardiac output and left ventricular ejection fraction < 40% as significant, independent predictors for adult respiratory distress syndrome. Combined cardiac surgery and diffuse coronary disease were also significant predictors; cardiopulmonary bypass time was not. Thirty-six of the 38 patients that later developed adult respiratory distress syndrome had low postoperative cardiac output, 12 requiring intra-aortic balloon pump support. The remaining two had severe hypotension caused by postoperative bleeding. Twenty-six adult respiratory two had severe hypotension caused by postoperative bleeding. Twenty-six adult respiratory distress syndrome patients (68%) had confirmed gastrointestinal complication (e.g. intestinal ischaemia). Adult respiratory distress syndrome is a rare complication after cardiac surgery but is associated with a very high mortality. Preoperative predictors were identified. Cardiopulmonary bypass alone was not found to be an important factor. Postoperative low cardiac output leading to splanchnic hypoperfusion may be the most important single factor in developing adult respiratory distress syndrome after cardiac surgery. PMID- 8634841 TI - Durability of emergency coronary artery bypass for complications of failed angioplasty. AB - Although emergency coronary artery bypass for complications of percutaneous transluminal coronary angioplasty (PTCA) has proved to be a relatively successful 'bail-out' procedure, little is known about the durability of revascularization under these potentially disastrous circumstances. The authors therefore retrospectively examined their results with this procedure. Emergency coronary artery bypass for complications of PTCA was performed in 112 patients between 1 January 1984 and 19 May 1992. Fifteen patients underwent PTCA for acute myocardial infarction. Eleven patients (9.8%) were stable, and underwent emergency coronary artery bypass after PTCA because of suboptimal angiographic results from percutaneous transluminal coronary angioplasty. None of these stable patients died. The remainder of the patients underwent emergency coronary artery bypass after PTCA because of ongoing documented ischemia, including cardiac arrest requiring cardiopulmonary resuscitation during transit to the operating room in 11 patients (9.8%) and preoperative intra-aortic counterpulsation in 24 (21.4%). The average number of coronary arteries bypassed at emergency coronary artery bypass was 2.2, and 19 patients (17%) received at least one mammary artery conduit. The perioperative incidence of myocardial infarction was 8.9% (10/112), and the operative mortality rate 8% (9/112). During follow-up, which averaged 55 months, the survival rate (including operative mortality) was 85% while 98% of patients experienced freedom from reoperative coronary bypass, 89% experienced freedom from myocardial infarction (including postoperative) and 90% experienced freedom from subsequent catheterization or PTCA. In conclusion, emergency coronary artery bypass for PTCA complications successfully avoids subsequent untoward cardiac events. When compared with published results of PTCA without emergency coronary artery bypass, emergency coronary bypass is more reliable for avoiding subsequent cardiac catheterization (with or without PTCA) than PTCA alone. PMID- 8634843 TI - The present status of endoluminal stented grafts for the treatment of aneurysms, traumatic injuries and arterial occlusions. PMID- 8634844 TI - Natural history of allograft coronary arteriopathy: a retrospective study of 54 patients over a 8 1/2-year period. AB - A total of 288 patients underwent heart transplantation at Loyola University Medical Center over a 8 1/2-year period starting in March, 1984. Of these patients, 54 were identified as having allograft coronary arteriopathy. Diagnosis was made on the basis of abnormal findings on coronary angiography in 44 patients; either an autopsy or explanted heart revealed the diagnosis in the remainder. A total of 279 abnormal lesions were identified by coronary angiography, and over 70% of these stenoses were discrete and located in large epicardial vessels. The left ventricular ed-diastolic pressure demonstrated a steady rise over time (2.5 mmHg/year), and also correlated with the degree of allograft coronary arteriopathy stenosis. The rate of stenosis progression was 33% per year from the time of initial allograft coronary arteriopathy development. Actuarial survival rate was lower than that of patients with arteriopathy. In conclusion, first, the majority of allograft coronary arteriopathy stenoses detected on coronary angiography are discrete, involve large epicardial vessels, and progress rapidly over time. Second, left ventricular end-diastolic pressure seems to reflect the degree of allograft coronary arteriopathy stenosis, and may prove to be a useful indicator of left ventricular dysfunction related to allograft coronary arteriopathy. Third, patients with allograft coronary arteriopathy show a decreased actuarial survival rate. PMID- 8634842 TI - Impact of preoperative risk and perioperative morbidity on ICU stay following coronary bypass surgery. AB - Prolonged intensive care unit treatment (> 3 days) contributes to increased health costs and resource utilization. In order to devise strategies to limit intensive care unit stay, and provide cost-effective medical care, it is necessary to identify the pre- and perioperative risk factors of prolonged treatment. Over 100 potential risk variables were collected prospectively in 889 consecutive patients undergoing isolated coronary bypass surgery between 1990 and 1992. The incidence of intensive care unit therapy lasting > 3 days was 6.8%. Univariate statistics identified 23 pre- and perioperative variables that were potential contributors to prolonged intensive care unit therapy. However, multivariate analysis of preoperative risk variables identified only recent myocardial infarction (within 30 days of surgery) and continued preoperative smoking (within 30 days of surgery) to be independent risk factors. Only 6.3% of patients without preoperative myocardial infarction and 6.1% of non-smokers required prolonged intensive care unit treatment, compared with 14.8% of patients with preoperative myocardial infarction (P = 0.01) and 10.1% of smokers (P = 0.07). When multivariate analysis was repeated with both pre- and perioperative variables, only ischemic morbidity (inotropes, myocardial infarction and low output syndrome; 138 patients) and non-ischemic morbidity (infection, stroke or bleeding; 37 patients) predicted prolonged intensive care unit treatment. Intensive care unit treatment for > 3 days occurred in 26.8% of patients with ischemic morbidity compared with 3.2% of patients without ischemic morbidity (P = 0.001). Prolonged intensive care stay occurred in 32.4% of patients who suffered non-ischemic complications compared with 5.7% of patients who did not suffer these complications. The multiple logistic regression analysis odds ratio for ischemic morbidity was 7.4 (95% c.i. 4.0-13.4) compared with 4.8 (95% c.i. 1.9 10.1) for non-ischemic morbidity. Strategies designed to reduce the incidence of prolonged intensive care unit treatment should include prevention of stroke, infection and bleeding. However, the greatest reduction of intensive care unit utilization would be mediated by prevention of ventricular dysfunction secondary to myocardial ischemia or inadequate myocardial preservation. PMID- 8634846 TI - Pericardial mesothelioma presenting as left atrial thrombus in a patient with mitral stenosis. AB - Primary pericardial mesothelioma is rare and remains a diagnostic and therapeutic challenge. Pericardial mesothelioma usually occurs independently of intracardiac lesions and infiltrates adjacent tissues superficially. An extremely rare case of malignant pericardial mesothelioma complicated by prior mitral valve surgery is reported. The tumour invaded the left atrium and mimicked a thrombus. PMID- 8634845 TI - Mechanical circulatory assistance in paediatric patients with cardiac failure. AB - Extracorporeal membrane oxygenation (ECMO) was initially developed for respiratory failure. Its use, however, has evolved into an excellent method of preoperative and postoperative support in the treatment of infants and children with acquired and congenital heart disease. Along with ECMO, the left ventricular assist device (LVAD) and the intraaortic balloon pump (IABP) have also found a place in the management of paediatric patients with heart failure. This report documents 15 patients who were treated with one or a combination of these mechanical devices, either preoperatively or postoperatively. There is a 74% survival rate and the long-term outcome has been excellent in most cases. The use of heparin-coated devices and tight regulation of heparin has allowed the transfer of infants and children from standard cardiopulmonary bypass to assist devices in the operating room. Mechanical devices are an essential adjunct for the preoperative and postoperative treatment of infants and children with cardiac disease. PMID- 8634847 TI - Surgical therapy for Wolff-Parkinson-White syndrome in patients with bronchial asthma. AB - The surgical therapy for Wolff-Parkinson-White syndrome in patients with bronchial asthma was studied. Between 1974 and 1992, 447 patients with Wolff Parkinson-White syndrome were treated, seven of whom had associated severe bronchial asthma. Supraventricular tachyarrhythmias were induced on occasion by bronchodilating agents such as beta-receptor agonists or theophylline preparations. High-dose steroids were required for acute bronchospasm in three patients, despite possibly leading to cardiac dysfunction. Beta-blockers therapy for Wolff-Parkinson-White syndrome can induce bronchospasm; in patients with Wolff-Parkinson-White syndrome and bronchial asthma, pharmacological agents used to treat one condition may exacerbate the other. Therefore, a non-pharmacological therapy was performed, namely surgical division of the accessory conduction pathway via an endocardial approach, in all seven asthmatic patients with Wolff Parkinson- White syndrome. In all cases, division of the accessory conduction pathway resulted in disappearance of the delta wave, and there were no further tachyarrhythmias either at rest or after administration of bronchodilators. Since surgery, bronchial asthma has been effectively controlled with standard drug therapy in all patients. Non-pharmacological therapy is recommended for patients with Wolff-Parkinson-White syndrome and bronchial asthma. PMID- 8634848 TI - Combined congenital pericardial and diaphragmatic defects: a case report. AB - This report describes the case of a 62-year-old woman who had a preoperative diagnosis of mitral valve insufficiency. At operation her heart was covered by fat which was identified as omental tissue. The pathogenesis, clinical presentation and management of this disorder are presented. PMID- 8634849 TI - Isolated apical intracavitary left ventricular abscess in a normal heart: a rare complication of Streptococcus milleri endocarditis. AB - A 31-year-old patient without past history of cardiac disease presented with an atypical form of Streptococcus milleri endocarditis. The disease progressed in an unusually aggressive manner, with abscess formation in the apex of the left ventricular cavity. The cardiac valves functioned normally and were not affected by the disease. PMID- 8634851 TI - Carotid endarterectomy in patients with contralateral carotid occlusion: review of a 10-year experience. AB - A total of 116 carotid endarterectomies were performed in patients with a totally occluded opposite internal carotid artery over a 10-year period from 1983 until 1992. The average age of patients was 66.4 years; 75% were men and 25% were women. The average degree of stenosis on the operated side was 76.7%. Twenty-one patients (18.1%) had had a documented previous stroke referrable to the side of the occlusion; 22 had a neurologic deficit attributable to the occluded vessel at the time of preoperative evaluation. Indications for surgery included transient ischemic attacks in 35 (30.2%), ipsilateral stroke in 10 (8.6%), amaurosis fugax in 11 (9.5%), and high-grade asymptomatic stenosis in 60 (51.7%). Forty-eight percent of the procedures were performed using local anesthesia, with intraluminal shunts inserted in all except one patient. The combined 30-day mortality and stroke morbidity in this population was 4.3%, which is comparable with a combined stroke and death rate of 4.0% among 956 patients without contralateral carotid occlusion undergoing endarterectomy during this period. This experience suggests that endarterectomy can be performed safely in the patient with internal carotid occlusion and is an important mechanism for the prevention of stroke. PMID- 8634850 TI - Preservation of the pelvic circulation during infrarenal aortic surgery. AB - Paraplegia, ischemia of the colon, and gluteal necrosis are uncommon but devastating sequelae of surgery of the infrarenal aorta. These complications are ischemic in nature, secondary to the following technical maneuvers, individually or in combination: bilateral occlusion of the hypogastric arteries; division of a patent inferior mesenteric artery; or proximal end-to-end aortic to common femoral artery bypass grafting accompanied by stenosis of the external iliac arteries. The etiology of paraplegia after infrarenal aortic surgery is of particular interest since it now appears that it is more likely due to interruption of flow to lumbosacral branches of the hypogastric arteries supplying the conus of the spinal cord and/or to division of a low-lying 'conus medullaris artery' rather than to occlusion of the higher-lying great radicularis artery of Adamkiewicz. Knowledge of the pelvic circulation to the colon, buttocks, and terminal spinal cord allows the surgeon prophylactically to avoid or reconstruct critical branches during operations on the infrarenal aorta. While rare, severe complications cannot be completely eliminated; hopefully their incidence can be reduced by an understanding of their etiology. PMID- 8634853 TI - Syme amputation: results in patients with severe forefoot ischemia. AB - Since 1980, 35 patients (age range 40-77 years) with severe forefoot ischemia have undergone a unilateral Syme amputation. Thirty-one patients (89%) were diabetic. The indication for amputation was either extensive forefoot gangrene or ulceration too advanced for a digital or transmetatarsal amputation. Twenty-two amputations (63%) were immediately preceded by either percutaneous transluminal angioplasty (four) or a bypass procedure to the popliteal artery (five) or an infrapopliteal artery (13). Primary healing occurred in 19 (86%) of 22 amputations immediately preceded by revascularization and in 10 (77%) of 13 limbs undergoing amputation alone. Twenty-eight (97%) of the 29 patients with successful primary healing were successfully rehabilitated. Rehabilitation rendered 13 patients fully ambulatory, 15 ambulatory with intermittent use of a walker or cane and one unable to walk. At follow-up of four months to 13 years (mean 42 months), the cumulative ambulatory rate at 1, 3, and 5 years was 92, 80 and 80%, respectively. Syme amputation allows a return to a functional ambulatory status in a high percentage of patients with severe forefoot ischemia. These findings suggest that Syme amputation is an acceptable option in dysvascular patients with severe forefoot ischemia. PMID- 8634852 TI - Somatosensory evoked potential monitoring during carotid surgery. AB - Controversy exists over the value of intraoperative monitoring and shunting in patients undergoing carotid endarterectomy. Although it is widely believed that contralateral carotid occlusion and previous stroke mandate intraoperative shunting, the susceptibility of these two groups of patients to cerebral ischemia during carotid artery endarterectomy is not well defined. Somatosensory evoked potentials (SSEPs) were monitored in 113 carotid artery endarterectomy patients. Of these, 32 (28.3%) had a previous stroke, 24 (21.2%) had a contralateral carotid occlusion and 33 (29.2%) were diabetic. There were no deaths and only one perioperative stroke (0.9%). Cerebral ischemia occurred in 14 patients (12.4%). Six of these patients had a contralateral carotid occlusion. Some 29 patients (25.7%) were shunted, including 10 with contralateral carotid occlusions that did not have major SSEP changes. In the latter half of the study, 14 patients with contralateral carotid occlusions were selectively shunted (six shunted, eight not shunted) with no neurological complications. Thirty-two patients with prior strokes were selectively shunted (nine shunted, 23 not shunted); of these, one shunted patient undergoing combined carotid artery endarterectomy and coronary artery bypass grafting had a perioperative stroke. Intraoperative monitoring with SSEPs accurately identifies cerebral ischemia secondary to carotid clamping as well as patients requiring shunts. With use of intraoperative SSEP monitoring, selective shunting may be safely performed in patients with a contralateral carotid occlusion or a previous stroke. PMID- 8634854 TI - Screening for abdominal aortic aneurysm in the Jeddah area, western Saudi Arabia. AB - Atherosclerosis and its complications are associated with high morbidity and mortality in the elderly. One of these complications is abdominal aortic aneurysm which may be prevented from rupturing if diagnosed early. Screening for aortic aneurysm was carried out in Jeddah, which is in the western region of Saudi Arabia, to identify the magnitude of this problem. Three groups were studied: patients with hypertension (n = 101), patients with peripheral vascular disease (n = 71) and a third group of a controls (n = 220). The mean (range) age of the whole sample was 66.0 (60-80) years. Evidence of aortic aneurysm was found in seven participants: five in the peripheral vascular disease group (7.0%), one in the hypertensive group (1.0%) and one in the controls (0.5%). In view of the expected increase in the elderly population of Saudi Arabia, as a result of improvements in health care which have recently been achieved, it is expected that a similar increase in the incidence of abdominal aortic aneurysm may occur. Routine screening for abdominal aortic aneurysm in the elderly, especially in those with peripheral vascular disease, may be worthwhile. PMID- 8634855 TI - A comparative study of the effect of autologous platelet-rich plasma and fresh autologous whole blood on haemostasis after cardiac surgery. AB - The effects of fresh autologous platelet-rich plasma and autologous whole blood on haemostasis after cardiopulmonary bypass were examined in adult cardiac surgery patients. Platelet count, adenosine diphosphate 10 microM maximum aggregation rate and clotting Factor VIII were greater in the platelet-rich plasma group (n = 11) than in the whole blood group (n = 8) after platelet-rich plasma or whole blood reinfusion. Blood loss after heparin neutralization was less in the platelet-rich plasma group than in the whole blood group. Blood loss from heparin neutralization to 12h after surgery was correlated with platelet count, fibrinogen and ADP aggregation rate. The number of patients who required homologous blood transfusion was less in the platelet-rich plasma group. In conclusion, the reinfusion of autologous platelet-rich plasma improves haemostasis after cardiopulmonary bypass, and may enable surgery to be performed without homologous blood transfusion. PMID- 8634856 TI - Role of vascular endoscopy in aortoiliac and femoropopliteal thromboendarterectomy. AB - Over the last few years the technique of thromboendarterectomy in the aortoiliac and femoropopliteal position has experienced a renaissance. Nearly 15 years ago thromboendarterectomy was found to have an increased early occlusion rate, especially in the femoropopliteal position (8-10%), and a high late complication of myointimal hyperplasia with subsequent stenosis. Bypass procedures were preferred to thromboendarterectomy because of these reasons. A prospective study was started in Lippstadt after good results of a retrospective study from the University of Ulm (1970-1990), in which angioscopy was used as a means of intraoperative assessment during thromboendarterectomy, were reported. Some 24 aortoiliac, 11 ileofemoral and 26 femoropopliteal semiclosed thromboendarterectomies were admitted to the study. Several technical problems including intimal flaps and incomplete obliteration were detected and treated immediately under direct visualization through the angioscope. During a mean follow-up of 10 months only one patient developed an ileofemoral occlusion. No deep infection or mortality was recorded. Angioscopy reduces the early postoperative occlusion rate after thromboendarterectomy. PMID- 8634857 TI - Beneficial effects of hypertonic mannitol in acute ischemia--reperfusion injuries in humans. AB - Acute ischemia-reperfusion of extremities is characterized by edema, compartment syndrome and neuromuscular dysfunction. Intravenous hypertonic mannitol has been shown to be of benefit in several experimental models. The authors' 5-year experience with the use of hypertonic mannitol and the treatment of acute ischemia reperfusion injuries in humans has been reviewed. Some of 186 patients with acute arterial occlusion following thromboembolism (149) and trauma (37) were treated. Hypertonic mannitol (25 g intravenous bolus followed by 5-10 g intravenous/h) was given perioperatively. Length of preoperative ischemia varied from 1 to 24h. Some of 57.5% of patients had preoperative neuromuscular dysfunction. Following revascularization, limb salvage was obtained in 97.7% of surviving patients and neuromuscular dysfunction improved in 89%. Overall, 15% required fasciotomy. The mortality rate was 3.2%. These data suggest that hypertonic mannitol may have some protective effect in acute ischemia-reperfusion injuries of human extremities. It may decrease the need for fasciotomy and minimize neuromuscular dysfunction. PMID- 8634858 TI - Suspected in utero infection in a boran heifer experimentally infected with Trypanosoma vivax. PMID- 8634859 TI - Use of Suratex for field diagnosis of patent and non-patent Trypanosoma evansi infections in camels. PMID- 8634860 TI - Lameness in dairy cattle. PMID- 8634861 TI - Brain-stem evoked responses as a diagnostic tool for deafness; a neurophysiological test with potential? PMID- 8634862 TI - Aetiology, prevalence and diagnosis of deafness in dogs and cats. AB - Peripheral deafness may be inherited or acquired, congenital or later-onset, and sensorineural or conductive. The most commonly observed forms are inherited congenital sensorineural, acquired later-onset sensorineural (ototoxicity, presbycusis) and acquired later-onset conductive (chronic otitis externa/media). In most dog and cat breeds inherited congenital sensorineural deafness results from perinatal degeneration of the stria vascularis, the vascular bed of the outer wall of the cochlear duct, which leads to hair cell degeneration. The strial degeneration appears to result from the absence of melanocytes, but their function in this structure is unknown. Ototoxicity may result from any of a large number of drugs and chemicals that directly or indirectly destroy cochlear hair cells. The effects are dose-dependent and in rare cases reversible. The most commonly recognized ototoxic drugs are the aminoglycoside antibiotics. Presbycusis, the ageing-related progressive hearing loss unattributable to other causes, is sensorineural but may also include mechanical changes in the tympanum and ossicles. Hearing aids may be accepted by some dogs as long as some residual function remains. Breeds reported to have been affected by congenital sensorineural deafness are listed and those with the highest prevalence are noted. Methods for diagnosis of deafness are described. PMID- 8634863 TI - Human and bovine tuberculosis--new threats from an old disease. PMID- 8634864 TI - Human and bovine tuberculosis in the Monze District of Zambia--a cross-sectional study. AB - One hundred and seventy-six randomly selected rural households in the Monze District of Zambia were interviewed; 103 of these presented cattle for tuberculin testing. Of the 2226 cattle tested, 165 (7.4%) were positive reactors; 33% of herds contained positive animals. Risk of a positive reaction varied with an animal's age and body condition. Cattle in larger herds were more likely to give positive reactions. Ten households reported a human case of tuberculosis (TB) during the preceding 12 months; the herds or these households were six times more likely to have a tuberculin-positive animal than herds in households without a reported human TB case. PMID- 8634865 TI - Bloodless castration of lambs: results of a questionnaire. AB - Interviews of nine flockmasters who used bloodless castrators to castrate lambs revealed wide variation in the size of castrator used and the techniques employed. Eight flockmasters used inappropriately sized instruments which were cumbersome to operate and probably applied excessive crushing pressures over an unnecessarily large area of the lambs' scrotums. The methods of restraining lambs were often inefficient in terms of labour requirement. In 11 of 15 flocks, some lambs were not properly castrated and in six flocks, instances of injury and death attributed to castration were reported. Evaluation of bloodless castrators showed that the crushing pressure applied by castrators even those of the same size could vary by over 100%. The requirement for further research into bloodless methods of castration is highlighted as is the urgent need for training for operators. PMID- 8634867 TI - Castration--a kinder cut? PMID- 8634866 TI - Sole haemorrhages in dairy heifers managed under different underfoot and environmental conditions. AB - Sole haemorrhages associated with laminitis were studied in 30 Holstein heifers in one herd housed either indoors on concrete or out of doors on a dry lot. Examinations were at 4-week intervals during a 15-month period from approximately 13 months of age to 2 months after calving. At 13 months of age, sole haemorrhages were present in 77% of the heifers. Five heifers, affected by clinical laminitis shortly after calving, developed early sole ulcers. Indoor housed heifers had a greater number of and more severe haemorrhages than heifers managed in dry lots (P < 0.001). In both groups, hind claws were more affected than front claws (P < 0.001). Lateral claws were more affected in hind limbs (P < 0.001),whereas medial claws had more sole haemorrhages in front limbs (P < 0.01). Heifers managed in dry lots had more sole haemorrhages in right claws than in left claws (P < 0.01). The number and severity of haemorrhages differed among the six zones of the sole (P < 0.001). Overall, the abaxial white zone (zone 2) had the greatest number of haemorrhages, followed by the white zone at the toe (zone 0), the bulb (zone 5) and the sole-bulb junction (zone 4), respectively. Few haemorrhages occurred in the apex of the sole (zone 1) and the axial groove (zone 3). Zone 0 was more severely affected in heifers managed in dry lots (P < 0.001), whereas zones 2, 4 and 5 had greater numbers of haemorrhages in heifers housed indoors (P < 0.01). Time had no effect on total haemorrhages scores, but the effect of management was significant for zones 0 and 5. Total haemorrhage scores for zone 0 were greater in heifers managed in dry lots (P < 0.05), whereas zone 5 was more affected in indoor-housed heifers (P < 0.01). This study shows that sole haemorrhages do occur in the claws of dairy heifers managed either indoors or in dry lots, and permanent damage to the claws of these young cattle may already have occurred before they reach maturity. PMID- 8634868 TI - Fallibility of faecal consistency as a criterion of success in the evaluation of oral fluid therapy for calf diarrhoea. AB - It is often said that the success of oral rehydration in humans depends on the adequacy of the improvement in the composition and volume of extracellular fluid, not reduction of faecal output. Indeed, the latter may increase initially. Such increases do not prevent the treatment from being effective but they may, falsely, undermine its acceptability to patients or those caring for them. This paper provides data to show that standard oral rehydration solutions used to treat experimentally induced calf diarrhoea procure identical improvements in plasma volume during the first 48 h, whether faeces improve or not, and those calves whose faecal consistency improved actually showed greater deterioration of extracellular fluid volume. While it is important for this to be appreciated by clinicians and explained to owners, it is absolutely imperative that those responsible for the approval of new therapeutic products for registration understand and accept that faecal consistency offers no reliable insight into the effectiveness of oral rehydration therapy for calf diarrhoea. It was, however, interesting that there was some relationship with correction of acidosis--perhaps because some of the contributing factors arise from colonic dysfunction. PMID- 8634869 TI - Detection of fibrin in canine neoplasia. AB - A murine monoclonal antibody, designated 1H10, produced using a human fibrin related immunogen, was shown to bind avidly to dog fibrin, but not to dog fibrinogen. Using immunofluorescence, fibrin was detected in canine gastric adenocarcinoma and in mixed tissue from a mammary tumour. No fibrin could be detected in bronchogenic carcinoma tissue. PMID- 8634871 TI - Reflections on a half century and more in the practice of allergy. PMID- 8634870 TI - Sexual maturity in British breeds of goat kids. AB - Eight male kids of British Sannen and one each of the British Alpine and British Toggenberg breed, born between the last week of February and first week of March 1990, were used. From the age of 12 weeks, they were monitored every week for body and testicular growth and sexual development, until they attained sexual maturity. The development of the sexual behaviour was a gradual process. From 12 weeks of age, the body weight increased at a constant rate. Testicular development, however, showed a biphasic pattern; the testis size increased rapidly between 12 and 20 weeks of age and was followed by a period of slow growth. Sexual maturity occurred at a mean age of 172.7 +/- 4.47 days, and at a mean body weight of 31.25 +/- 1.50 kg when the mean values of scrotal circumference, testis diameter and paired testes width were 24.13 +/- 0.47 mm, 54.01 +/- 1.26 mm and 88.31 +/- 1.61 mm, respectively. In most kids, the quality of the first ejaculate was good, although it varied widely between animals. It appeared that sexual maturity in British goat kids occurs at a younger age than in different breeds in other parts of the world that have been reported in the literature. PMID- 8634872 TI - Insulin allergy. PMID- 8634873 TI - Recurrent headaches: what every allergist should know. AB - OBJECTIVE: To provide the allergist information regarding the recognition, diagnosis, classification, and management of headaches. DATA SOURCES: Literature and relevant articles pertaining to various types of headache are reviewed and the clinical experience of the authors is presented. CONCLUSIONS: After reading this article, the allergist should know the various causes of headache, recognize the warning signs of serious neurologic disease, and determine whether allergy or adverse food reactions are playing a role. PMID- 8634874 TI - Cough productive of casts. AB - Plastic bronchitis is a rare disorder characterized by the formation and, sometimes dramatic expectoration of long, branching bronchial casts. DIsorders associated with bronchial casts either produce an increase in the volume or viscosity of secretions, such as allergic bronchopulmonary aspergillosis, asthma, cystic fibrosis, pneumonia, and chronic bronchitis; or there are obstructions or structural abnormalities that decrease mucous clearance, such as bronchiectasis. The diagnostic evaluation should include serum laboratory studies, specimen cultures, skin tests, chest radiograph and CT scan, pulmonary function tests, and bronchoscopy. Therapy should be directed towards the underlying disorder and should also include maneuvers to facilitate removal of casts to prevent complications, such as secondary pneumonia. PMID- 8634875 TI - Adrenal suppression among asthmatic children receiving chronic therapy with inhaled corticosteroid with and without spacer device. AB - BACKGROUND: Inhaled corticosteroids have become a first-line treatment for chronic asthma. It has been shown that inhaled corticosteroids can have a measurable effect on the hypothalamic-pituitary-adrenal axis in asthmatic children. OBJECTIVE: To investigate the prevalence of adrenal suppression among asthmatic children receiving chronic therapy with low to moderate doses (up to 1000 micrograms) of inhaled beclomethasone dipropionate via a metered dose inhaler (MDI) and via MDI attached to a spacer device (MDI-spacer). METHODS: The study included 39 asthmatic children currently undergoing therapy; 24 received beclomethasone dipropionate by MDI attached to a spacer, and 15 directly by MDI. All the patients had been treated for at least 4 months. Another 21 children were normal controls. The 24-hour urinary free cortisol excretion was measured to evaluate hypothalamic-pituitary-adrenal axis function. RESULTS: Seven of 15 (47%) patients from the MDI group had reduced 24 hour-urinary free cortisol excretion and 2 of 24 (8%) in the MDI-spacer group (P = .006). The mean 24-hour urinary free cortisol excretion of the MDI group was 0.0185 +/- 0.0089 microgram/gram creatinine, and the MDI-spacer and the control groups were, 0.0290 +/- 0.0138 microgram/gram creatinine and 0.0270 +/- 0.0118 microgram/gram creatinine, respectively, (P = 0.37, f = 3.51 ANOVA). CONCLUSION: Chronic inhalation of low to moderate doses of corticosteroids is associated with adrenal suppression in some asthmatic children. This side effect is more common among patients inhaling directly from the MDI and is less frequent when a large volume spacer is attached to the MDI. PMID- 8634876 TI - Sensitization to airborne allergens in children with respiratory symptoms. AB - BACKGROUND: Allergy is one of the most common causes of respiratory symptoms in children and youth. OBJECTIVE: Evaluate the presence and the type of allergic sensitization in a paediatric population with respiratory symptoms. METHODS: We studied 564 consecutive children, 5 months to 17 years of age, with a male to female ratio (M/F) = 1.4, referred to our outpatient clinic in a 12-month period retrospectively. Patients were arbitrarily divided into four groups (grs) according to their age: gr1 = 5 months to 4 years old (181 patients), gr2 = 4 to 7 years (201 patients), gr3 = 7 to 10 years (96 patients), and gr4 = 10 to 17 years (86 patients). Sensitization to house dust mites, pollens, animal dander, and molds was determined by skin prick testing. RESULTS: Sensitization to at least one class of allergen occurred in 304 of the 564 patients (53.9%, M/F ratio = 2.0); the percentage of allergic patients increased with age as follows: 29.8% (54 patients) of the patients in gr1, 55.2% (111 patients) in gr2, 68.8% (66 patients) in gr3 and 84.9% (73 patients) in gr4 (chi(2) = 84.1, P < .01). In the entire allergic population and in gr1 to gr3, the most common positive allergic reaction was to house dust mites (P < .01, chi(2) test each comparison). In contrast, gr4 patients showed a nearly equal percentage of sensitization to pollens and to house dust mites (79.5% and 78.1% respectively) (chi(2) = 0.0, P = >.1). Sensitization to only one class of allergen occurred in 51.3% of the allergic patients and the percentage of these monosensitized patients tended to decrease from gr1 to gr4 (chi(2) = 15.2, P < .1). In the monosensitized group, sensitization to house dust mites was the most frequent in gr1 to gr3 (age <10 years) as in the whole sample. In gr4, the frequency of sensitization to house dust mites was similar to that of sensitization to pollens. On the contrary, within the patient group sensitized to two or more allergens (polysensitized patients), sensitization to house dust mites was as frequent as sensitization to pollens already in gr2 as compared with monosensitized patients. CONCLUSIONS: In children with respiratory symptoms, the percentage of allergic individuals was high and increased with the age of the patients. This phenomenon was associated with an age-related enhancement in the ratio of polysensitized to monosensitized patients and with an age-related increase in the frequency of sensitization to seasonal allergens (ie, pollens). PMID- 8634877 TI - A calibration program for Rotorod samplers. AB - BACKGROUND: Despite widespread use in the United States, the need for calibrating Rotorod Samplers on a regular basis has not been demonstrated. OBJECTIVE: The purpose of this present investigation was to inspect Rotorods that had been operating in the field to determine whether they conformed to the manufacturer's operating specifications. METHODS: Allergists who own Rotorods voluntarily returned their devices to the manufacturer for calibration over the course of two successive winters. The speed of each device's motor was measured with a stroboscopic tachometer. RESULTS: Of the 89 devices examined, ten were found to be operating at the manufacturer's exact speed specification (2400 RPM) and another 31 were within 5%. The remaining devices deviated by more than 5% from the prescribed speed or possessed motors that would not operate. CONCLUSIONS: The authors underscore the importance of calibrating Rotorod Samplers to maintain data quality. PMID- 8634878 TI - Effects of semprex-D and diphenhydramine on learning in young adults with seasonal allergic rhinitis. AB - OBJECTIVES: The purpose of this study was to test the hypothesis that learning ability is impaired in patients with seasonal allergic rhinitis relative to untreated individuals and to evaluate a combination compound (acrivastine 8 mg + pseudoephedrine 60 mg) for attenuation of the learning impairment in these patients. BACKGROUND: In a previous study employing the same method it was shown that young children (10 to 12 yrs) suffering from seasonal allergic rhinitis performed significantly worse on tests of learning and using knowledge after acute treatment with a sedating antihistamine (diphenhydramine 50 mg) or placebo as compared with nontreated healthy controls. This effect was partially reversed by treatment with loratadine. METHODS: Sixty-seven young adults suffering from seasonal allergic rhinitis and 28 matched controls were trained on didactic simulation for three consecutive days. Atopic subjects were treated differentially during training according to a double-blind, randomized, parallel group design with either diphenhydramine hydrochloride 50 mg, a combination compound (acrivastine 8 mg + pseudoephedrine 60 mg, A + P), or placebo, administered qd. After training, all atopic subjects were maintained on A + P treatment for 14 days at which time all groups returned for examination. RESULTS: Mean performance at the end of training was worse for all atopic subjects combined compared with normal subjects. Subjects treated with diphenhydramine performed significantly worse than either normals (P < .001) or those treated with A + P (P < .001). At the examination, the diphenhydramine group's performance differed significantly from those of the normal (P < .001) and A + P groups (P < .001). CONCLUSION: The study supports our previous finding that allergy symptoms reduce learning ability which is further reduced learning ability which is further reduced by diphenhydramine. Atopic subjects with allergies treated with acrivastine + pseudoephedrine learned as well as normal subjects. PMID- 8634880 TI - Short-term effects of extermination and cleaning on cockroach allergen Bla g 2 in settled dust. AB - BACKGROUND: Although cockroach allergen is an important indoor allergen in poor urban environments, no studies on the effectiveness of measures to reduce allergen levels in indoor environments have yet been published. METHODS: As a model of home extermination, we studied cockroach allergen levels in an urban dormitory that was chronically infested with German cockroaches and that underwent semiannual extermination. Dust samples were collected from 18 bedrooms and 5 kitchens located in the dormitory and were analyzed for Bla g 2 using an immunoassay. RESULTS: We detected allergen in almost every bedroom, with median levels ranging from 3.0 U/g (units/g) in settled dust on the bed, 4.0 U/g on the carpeted floor, and 2.8 U/g in closets. In the kitchen, we found somewhat higher levels, 10.8 U/g on the floor and 2.8 U/g in floor cabinets. Repeated measures of floor dust were similar (median 4.4 and 4.0 U/g) despite weekly vacuum cleaning. In the 2 weeks before extermination, median levels were 5.2 U/g and in the 2 weeks following extermination and regular vacuuming, median levels fell to 0.95 U/g. CONCLUSIONS: We concluded that successful allergen abatement could be accomplished in cockroach infested indoor environments using routine extermination and vacuuming. PMID- 8634881 TI - Asthma mortality in Israel 1971-1990. AB - BACKGROUND: One of the enigmas in asthma is that our improved understanding of the pathophysiology and the introduction of new modalities to treat asthma has not led to a parallel decrease in asthma mortality. On the contrary, morbidity and mortality from asthma have increased since the mid-seventies in most of the western countries. This trend of increased asthma mortality rate was not observed in Israel in a survey that examined the changes in asthma mortality rate in Israel during 1960-1986. A small but statistically insignificant increase in asthma mortality rate during the last years of that survey solicited for reexamination and extension of the survey. OBJECTIVES: The purpose of the present study was to evaluate the changes in the asthma mortality rate in Israel during the years 1971-1990. METHODS: We extracted the statistical data on asthma mortality rate in Israel during the 1971-1990 period. Because of the small numbers each year and the variations in asthma mortality rate between years, the data were analyzed after grouping the asthma mortality rate into 5-year periods. RESULTS: The rates, expressed as deaths per 100,000 population per year, were 0.43, 0.18, 0.39, and 0.40 in the 5- to 34-year-old group for the periods 1971 1975. 1976-1980, 1981-1985, and 1986-1990, respectively. We found a statistically significant increase in asthma mortality rate during the 1981-1985 and 1986-1990 periods as compared with 1975-1980 in the young (<34 years old) population. The increase in asthma mortality rate was greater among males. The mortality rate in the older population (35-64 year olds) decreased during 1976-80 as compared with 1971-1975 but did not change thereafter. The rates were 10.4, 4.8, 4.5, and 4.4 cases per 100,000 for 1971-1975, 1976-80, 1981-85, and 1986-90, respectively. CONCLUSIONS: Asthma mortality rate increased in Israel in the young age group (5 34 years) during 1980-1990. This is similar to reports from many other countries with advanced medical care systems. The decrease of asthma mortality rate in 1976 80 probably reflect the general improvement in medical care in Israel during these years. PMID- 8634879 TI - Systemic allergy to endogenous insulin during therapy with recombinant DNA (rDNA) insulin. AB - BACKGROUND: Clinically significant allergic reactions with insulin therapy are known to occur. There have been rare reports of allergic reactions to endogenously secreted insulin manifested as insulin resistance. No reports of systemic or local allergic reactions to endogenous insulin have previously been cited, and no immunologic reactions to endogenous insulin have been reported during therapy with recombinant (rDNA) insulin. METHODS: We report a case in which the patient, a 28-year-old black woman who initially presented with gestational onset diabetes but postpartum continued to require insulin, developed generalized allergic reactions during therapy with subcutaneously injected rDNA insulin. Similar reactions occurred with sulfonylurea therapy. She was unable to tolerate any pharmacologic therapy for diabetes without concurrent use of at least 10 mg of prednisone per day. RESULTS: Skin testing with the insulin preparations were positive, while skin testing to the sulfonylurea hypoglycemic agents were negative. IgE antibodies to insulin where present in high titer. Oral challenge to sulfonylurea hypoglycemic agents produced generalized urticarial reactions coinciding with time of peak insulin secretion. Oral challenge to other medications containing sulfa produced no adverse reaction. Biphasic hypersensitivity reactions occurred during attempts at desensitization which were futile without simultaneous glucocorticoid therapy. CONCLUSIONS: This is the first report of local and systemic allergic reactions to endogenously secreted insulin in association with rDNA insulin therapy. Although immunologic complications with rDNA therapy appear less frequently than with insulin preparations, this case illustrates the need for continued awareness for potential allergic complications occurring with rDNA insulin therapy. PMID- 8634882 TI - Myocardial infarction as the presenting manifestation of systemic lupus erythematosus with antiphosphatidylserine antibodies. AB - BACKGROUND: The presence of antiphospholipid antibodies has been associated with thrombotic events in systemic lupus erythematosus and other diseases. Antiphosphatidylserine antibodies have been shown more recently to have clinical implications being noted in thrombocytopenia, positive VDRL test, prolonged partial thromboplastin time, and recurrent fetal loss. OBJECTIVE: To report a patient with systemic lupus erythematosus who presented with a myocardial infarction resulting from a single clot in the distal left anterior descending coronary artery and to discuss the merits of evaluating such patients for the presence of antiphospholipid antibodies. METHODS: Case report and review of the literature. RESULTS: A 55-year-old woman with no risk factors for coronary artery disease presented with a myocardial infarction. A distal left anterior descending coronary artery clot was seen on angiography. Evaluation for the presence of a hypercoagulable state revealed evidence for systemic lupus erythematosus. Although lupus anti-coagulant and anticardiolipin antibody titers were negative, antiphosphatidylserine IgA antibodies were detected. CONCLUSION: Because antiphosphatidylserine antibodies of the IgA isotype have been shown to be associated with thrombosis and other manifestations of systemic lupus erythematosus, a complete evaluation for antiphospholipid antibodies should include testing for all three isotypes. PMID- 8634883 TI - Coexistent anaphylaxis to Diptera and Hymenoptera. AB - BACKGROUND: Anaphylaxis to the bite of Diptera and specifically the bite of the Tabanidae family (horsefly) have been sparsely documented. The coexistent hypersensitivity to both the order Diptera and Hymenoptera has not been documented. METHODS: We present a patient who experienced anaphylaxis to both insect species. Venom skin testing and RAST revealed sensitivity to several members of the Hymenoptera order. Prick, intradermal and RAST with whole body extracts of Tabanidae species is also documented in this patient. Twenty patients who are sensitive to Hymenoptera and have been bitten by horseflies but have had no reaction to the horsefly bite were used as controls. RESULTS: An anaphylactic reaction to horsefly bite has been documented in a 56-year-old white male. This patient also demonstrated evidence of anaphylactic reaction to Hymenoptera envenomation. In controls consisting of 20 patients with Hymenoptera sensitivity, there was no clinical history of reaction to horsefly bite despite the presence of positive prick and/or positive intradermal tests and/or positive RAST to mixed Tabanidae species extract. CONCLUSIONS: Skin testing to horsefly by prick and/or intradermal testing using whole body insect extract is not useful in making a diagnosis of Tabanidae hypersensitivity. RAST using Tabanidae species as antigen is similarly useless in making a diagnosis of Tabanidae hypersensitivity. In vivo and in vitro diagnosis of horsefly hypersensitivity may be achieved when the salivary gland antigen of the horsefly becomes available. PMID- 8634884 TI - Trends in asthma therapy in the United States: 1965-1992. AB - BACKGROUND: Many recent studies indicate an increasing morbidity and mortality of asthma in the past two decades. This study uses data from the National Disease and Therapeutic Index (NDTI) to document and analyze trends in drug therapy for asthma in the United States from 1965 through 1992. METHODS: The NDTI maintains a continuous rotating national sampling of approximately 1% of US physicians in office-based practice proportionately representative of practicing generalists and specialists who report issuance of drugs in treatment by diagnosis for all patient encounters for a period of two days every 3 months. Annual summaries of five demographic categories and 14 drug categories, characterizing the asthma patient-physician encounters as percent of visits for the 28-year period of 1965 through 1992 are analyzed and characterized. RESULTS: Physician visits for asthma treatment have shifted somewhat from generalists to specialists in internal medicine and pediatrics. Allergists treat a significant proportion of the asthmatic population. Most patients are seen in the office. There has been no significant change in rates of inpatient visits. Age distribution of the population of patient visits for asthma has been stable, but there is a steady drop in ratio of males to females. Since the mid-1970s, inhaled adrenergic bronchodilator prescriptions have been issued at a markedly increasing rate. Concurrently, issuance of xanthines and oral adrenergic drugs also rose dramatically but then decreased beginning in the mid-1980s. Corticosteroids are used in 15% to 20% of visits, but only recently has the inhaled route of administration shown prominence. Allergen immunotherapy for asthma has decreased more than 10-fold. Cromolyn is prescribed infrequently. CONCLUSIONS: Major changes have occurred in drug treatment by physicians for asthma in the US since 1965. Bronchodilating drugs predominate, and they are being prescribed in more effective forms at a generally increasing rate. Corticosteroid use has increased at a slower rate and in smaller proportion of patient-visits, while allergen immunotherapy has dramatically declined. The male-to-female ratio of asthmatic patients who visit doctors for treatment appears to be decreasing. PMID- 8634885 TI - Practice parameters for the diagnosis and management of immunodeficiency. The Clinical and Laboratory Immunology Committee of the American Academy of Allergy, Asthma, and Immunology (CLIC-AAAAI) AB - In this brief review, only the most useful immunologic tests available for defining host defects that lead to susceptibility to infection have been emphasized. It should be pointed out that those evaluations and tests ordered by the physician will rule out the vast majority of the currently recognized defects. Finally, it is important that any patients identified as abnormal by these screening tests be characterized as fully as possible in centers specializing in these diseases before therapy is initiated, since what may appear to be a simple diagnosis on the surface may be an indicator of more complex underlying problems. PMID- 8634886 TI - Allergy diagnosis. PMID- 8634887 TI - Anaphylaxis following ingestion of carmine. PMID- 8634888 TI - Diagnosis of latex allergy. PMID- 8634889 TI - A European perspective. PMID- 8634890 TI - Dental treatment needs of a cohort of Royal Air Force recruits over 5 years. AB - The paper reports the dental status of a cohort of Royal Air Force (RAF) recruits in 1988 and details the work required to render and maintain them dentally fit for their first 5 years in the service. The mean DMFT (7.3) at inspection in 1988 increased by 0.8 whilst the mean number of restorations placed was 4.0. Endodontic therapy was required by 13.4 percent of the recruits and crowns were provided for 9.6 percent. The recruits required twice as many restorations in their first year in the service to render them dentally fit as were required in any subsequent year to maintain fitness, this work needed 58 minutes of dental officers' time in their first year and 43 minutes per year thereafter. The number of dentists seen or courses of treatment undertaken did not have a significant effect on the number of restorations provided. The prevalence and incidence of caries in this group appeared to justify the RAF's policy of annual inspections but the number of carious lesions which did not progress far enough to require restoration after 5 years indicated that a preventive approach to treatment planning is important to avoid overtreatment. PMID- 8634891 TI - Patient perceptions regarding the risks of morbidity and complications of lower third molar removal. AB - The risks associated with third molar surgery are well established. Current philosophy stresses co-development of treatment plans with patients, who need to understand the complications and risks of possible treatment. This understanding was therefore investigated at an initial outpatient appointment (n = 91) after a verbal explanation and immediately prior to surgery (n = 73), by means of visual analogue scales to assess patients' perceptions of the likelihood of each outcome. Most patients (98 percent) responding at an initial appointment (t1) felt that they had been given enough information regarding reasons for removal but the proportion was lower (88 percent) for patients responding at the time of surgery (t2). This reduction was also found for information regarding post operative pain, swelling, trismus and nerve damage. Patients estimated the likelihood of pain as t1 = 70 percent, t2 = 80 percent, swelling t1 = 76 percent, t2 = 75 percent, trismus t1 = 74, t2 = 73 percent, temporary nerve damage: transient labial t1 = 46 percent, t2 = 33 percent, transient lingual t1 = 51 percent, t2 = 41 percent, and permanent nerve damage: labial t1 = 13 percent, t2 = 7 percent, lingual t1 = 14 percent, t2 = 9 percent. At t1 and t2 the mean perceived likelihood for pain, swelling and trismus were significantly higher than the known prevalence of these sequelae. The mean values for temporary lingual and labial anaesthesia were considerably higher (P < 0.05) than the reported prevalence. It was concluded that patients overestimated the likelihood of post operative complications and that these estimates varied with time. The validity of patients' estimates might be improved by verbal explanation supplemented by provision of written information. PMID- 8634892 TI - The development of a socio-dental measure of dental impacts on daily living. AB - This study outlines the development of a socio-dental method which includes measures of the impacts of oral health status on the quality of daily living. The measure, the Dental Impact on Daily Living (DIDL), has five dimensions: Comfort, Appearance, Pain, Performance and Eating Restriction (CAPPER). DIDL differs from other socio-dental indicators in that it assesses the dental impacts on daily living, the relative importance respondents attribute to each impact dimension and their oral status. Weighting is assigned by every person through an easy to use scale using a sliding arrow that the subject moves. DIDL generates an impact score for each dimension. In addition, since impacts do not usually occur separately, a single total impact score is also estimated to assess total oral impacts. The instrument was tested using item-analysis, factor analysis, validation and reliability tests. The measure was tested in Brazil on a sample of 662 people, aged 35 to 44 years, of two social classes and both genders. Three different levels of oral status were used: high DMFT, medium DMFT and low DMFT. Estimating the relative importance people attributed to the different dimensions was important as shown by the fact that differences between sub-groups were found. DIDL attempts to assess how oral health is perceived by individuals and how the mouth and teeth affect people. Combined with clinical status measures, DIDL should prove a valuable tool to assess need. PMID- 8634894 TI - The responsibilities of the food industry in promoting oral health. PMID- 8634893 TI - Oral health and health related behaviours among three-year-old children born to first and second generation Pakistani mothers in Bradford, UK. AB - Many young Muslim children whose families originated from the Indian sub continent have poorer levels of oral health than their indigenous White peers, but it is unclear whether and to what extent subsequent generations may be similarly disadvantaged. This study aimed to compare dental health and associated behavioural attributes among United Kingdom-born three-year-old children of first and second generation Pakistani Muslim mothers resident in areas of social deprivation in Bradford, West Yorkshire. Multi-lingual interviews completed a home-based, semi-structured questionnaire, and a dental examination was conducted at a subsequent visit on 226 children, 117 with Pakistan-born mothers and 109 born in the UK. When the two generation groups were compared, no differences were observed in infant feeding practices, oral hygiene routines or dental attendance patterns, in mothers' attendance at ante-natal classes, or their knowledge of caries prevention. However, a higher proportion of UK-born mothers were able to speak English and reported that they had attended a dentist themselves, had received advice there, and were better informed of categories exempt from dental charges. A 24-hour retrospective qualitative dietary recall indicated that children of UK-born mothers consumed, on average, more frequent intakes of foods classified in the 'bread and cereal' group and of sweet drinks at meal times. While there were no significant differences in the proportion who were cavity free, children of UK-born mothers had higher mean caries experience (dmft=2.30) compared with those of Pakistan-born mothers (dmft=1.38). It was concluded that the total burden of dental caries experience among a subsequent generation of Pakistani children was higher than that of the previous generation. Poor dental health is likely to remain a challenge in this community for the foreseeable future. PMID- 8634895 TI - Toothbrushing frequency between the ages of 12 and 18 years-- longitudinal prospective studies of Finnish adolescents. AB - The purpose of this study was to analyse the establishment of the toothbrushing frequency as a healthy habit by Finnish adolescents between 12 and 18 years of age. The data were collected as part of the nation-wide research programme, the Adolescent Health and Lifestyle Survey. All Finns born in 1968 with birthdays on 20-25 July formed the sample (N=1106). Questionnaires were mailed to the whole sample in February 1981 (12 years of age), -83 (14 years), -85 (16 years) and -87 (18 years). The rate of return for all four questionnaires was 62 percent (six year follow-up) and for two questionnaires (two-year follow-up studies) 79 percent (12 and 14 years), 72 percent (14 and 16 years) and 68 percent (16 and 18 years). Those adolescents who brushed their teeth more than once a day at age 12, were more stable in their behaviour during the following six years than those who brushed their teeth less often. Over two thirds of the respondents, both boys and girls, indicated one or more changes in their toothbrushing frequency in the four questionnaires during the six-year follow-up. The rate of improvement in toothbrushing frequency was higher among girls than boys, and the rate of deterioration in toothbrushing frequency was higher among boys than girls. One sixth of the adolescents still showed a deterioration in their brushing frequency between the ages of 16 and 18. It may be concluded that although toothbrushing frequency will be gradually adopted as a health habit when the adolescents mature between 12 and 18 years, only half the girls and one fifth of the boys adopt the recommended frequency. Health education should still improve performance in those groups which have not established the recommended toothbrushing frequency. PMID- 8634896 TI - Economic assessment of a six-year project with extensive use of dental hygienists in the dental care of children: a pilot study. AB - The aim of the study was to assess the economic efficiency of an alternative division of labour based on an extensive use of dental hygienists combined with a reduced input of dentists. A test clinic was compared to a conventionally run public clinic in respect of dental and financial effects in the dental care of children. The study comprised 80 adolescents, born in 1975, in their 13th to 18th years. The assessment of economic efficiency was based on a cost/benefit analysis on the clinic level, in which the cost was defined as the difference between the test clinic and the control clinic in yearly running variable costs per child, and the benefit as the difference in the yearly caries increment multiplied by a value factor. The results showed statistically significant, lower caries incidence in the test group. Regression analyses, estimating a caries incidence function gave statistically significant explanation values for the variables "Caries prevalence" at 13 years of age' and 'Clinic'. The cost/benefit analysis showed a benefit/cost ratio of 1.48. The division of labour at the test clinic is discussed as a possible main factor for the outcome and suggests further experiments on a larger scale. The study can be regarded as a pilot, intended to be followed by a comprehensive study using a larger number of patients of different ages and more control clinics. PMID- 8634897 TI - The current status of dental health education in the training of midwives and health visitors. AB - In some areas of the United Kingdom, the dental health of young children is deteriorating, with levels of disease reverting to those of ten years ago. The recent enquiry into the education and training of personnel auxiliary to dentistry recognised that other health care staff working in primary care have an important role to play in the education of parents with young children and should be trained accordingly. This study sought to investigate, by questionnaire survey, the current status of dental health in the education of midwives and health visitors. It was found that the majority of courses in health visiting but only two-thirds of those in midwifery now include dental health in their curricula. The use of inappropriately qualified tutors may place these professionals at a disadvantage in providing advice on the prevention of oral disease to parents and prospective parents. PMID- 8634898 TI - Dental caries and dental fluorosis among 4-, 6-, 12- and 15-year-old children in kindergartens and public schools in Kuwait. AB - The purpose of the present study was 1) to describe the occurrence of dental caries and fluorosis among children in kindergartens and public schools in Kuwait, and determine their need for dental health care, 2) to describe changes in caries prevalence and experience from 1982 to 1993 and 3) to provide a baseline for the evaluation of the preventive oral health programmes starting in Kuwait in 1994. The study population comprised 3,500 4-, 6-, 12- and 15-year-old children in kindergartens and public schools in Kuwait, selected by stratified cluster sampling. Dental caries was scored by surface in accordance with WHO criteria, and dental fluorosis was registered by Dean's index (modified). The mean deft was 4.6 at age 4 years, and 6.2 at age 6; 8 and 11 percent of the decay had been treated by extractions or fillings in the two respective age groups. Among the 6-, 12- and 15-year-old children DMFT was 0.2, 2.6 and 3.6 respectively; 12 and 14 percent of the decayed teeth of 12- and 15-year-old children had been treated. As to the 4- and 6-year-old children, 19 and 9 percent were caries-free in the deciduous teeth, while 86, 21 and 14 percent of the 6-, 12- and 15-year-old children were caries-free in the permanent teeth. Among the 4 year-old children 47 percent had caries in the front teeth. None of the children had severe dental fluorosis, but 6 percent at 12 and 15 years showed mild to moderate, but manifest, dental fluorosis. The survey revealed that caries experience as well as caries prevalence had increased since 1982, although there was a marked increase in the number of filled tooth surfaces in all age cohorts. However, in 1993 the DMFT of 12-year-old children was still well within WHO's global goal for the year 2000, whereas the prevalence of dental caries among 6 year-old was considerably higher than the WHO goal. PMID- 8634899 TI - The effect of fluoridation and social class on caries experience in 5-year-old Newcastle children in 1994 compared with results over the previous 18 years. AB - In 1994, the dental health of 327 5-year-old children who had lived in continuously fluoridated (at 1.0 mg/IF-) Newcastle was compared with the dental health of 335 children of the same age in non-fluoridated (less than 0.1 mg/IF-) south east Northumberland. The caries prevalance in social class groups I + II, III, IV + V children was compared both within and between the two areas. The prevalence of dental caries in the three social groupings I + II, III, and IV + V (and the mean dmft) respectively, was 23 percent (0.59), 39 percent (1.21) and 31 percent (1.17) in the fluoridated area, and 38 percent (1.46), 47 percent (2.04) and 62 percent (2.74) in the non-fluoridated area. Differences between the fluoridated and non-fluoridated areas were observed in all social class groupings but the greatest difference in the percentage of children with decay experience occurred in social groups IV + V. There has been a fall in caries experience in all social groups in both the fluoridated and non-fluoridated areas since a previous survey in 1987. However, this reduction was not enough to obviate the disadvantage of social background. Reduction in caries experience especially amongst social classes I and II has meant that the power of water fluoridation to reduce caries experience has diminished. Refinement of the instruments for classifying socio-economic position has the potential to demonstrate greater degrees of inequality than the crude measures such as occupational class as used in this study. PMID- 8634901 TI - A biologically consistent model for comparing molecular phylogenies. AB - In the framework of the problem of combining different gene trees into a unique species phylogeny, a model for duplication/speciation/loss events along the evolutionary tree is introduced. The model is employed for embedding a phylogeny tree into another one via the so-called duplication/speciation principle requiring that the gene duplicated evolves in such a way that any of the contemporary species involved bears only one of the gene copies diverged. The number of biologically meaningful elements in the embedding result (duplications, losses, information gaps) is considered a (asymmetric) dissimilarity measure between the trees. The model duplication concept is compared with that one defined previously in terms of a mapping procedure for the trees. A graph theoretic reformulation of the measure is derived. PMID- 8634900 TI - The dental caries experience of 14-year-old children in the United Kingdom. Surveys coordinated by the British Association for the Study of Community Dentistry in 1994/95. AB - This paper reports the results of standardised clinical survey examinations of 127,481 14-year-old children across the United Kingdom. These 1994/95 coordinated surveys are the latest in a series which seeks to monitor the dental health of children and contribute to assessments of the provision of dental services. The criteria and conventions of the British Association for the Study of Community Dentistry trainers' pack 1992/93 were used. Representative samples were drawn from participating health authorities and boards and caries was diagnosed at the caries into dentine (D3) threshold using a visual method without radiography, fibre-optic transillumination or compressed air. The results demonstrated a wide variation in prevalence across the UK, the mean D3MFT for the former English regions (of the National Health Service) and the other UK 'territories' ranging from 1.2 (South West Thames) to 4.1 (Northern Ireland). The overall mean D3MFT across the UK was 1.93 (D3T = 0.63, MT = 0.15, FT = 1.15). Overall, 57 percent of children had evidence of (dentinal) caries experience (D3MFT > 0), although means ranged from 42 percent (South Wales Thames) to 81 percent (Northern Ireland). The inclusion of data from Northern Ireland for the first time in this series confirms that dental caries prevalence is higher in the province than in other areas of the UK. Trends since the 1990/91 surveys can be assessed for Great Britain. These demonstrate an overall fall in D3MFT of 20 percent since 1990/91 (from 2.30 to 1.85). This is, however, due to the 35 percent reduction in the number of fillings (1.68 to 1.09) which was partially offset by an increase in unrestored dentinal decay of 41 percent (0.44 to 0.62). These findings (taken together with results from regions reporting in 1986/87) demonstrate that although overall improvements have continued in this age group, the rate of improvement has slowed and that changes in the pattern of provision of care for those with disease experience continue to give some cause for concern. On average, across the UK, only 59 percent of the dentinal caries experience identified by survey examinations of permanent teeth was seen as fillings (range 48 to 69 percent). PMID- 8634902 TI - A simplified proof of the NP- and MAX SNP-hardness of multiple sequence tree alignment. AB - We give a simple proof which shows that the multiple sequence tree alignment problem from molecular biology is both NP-complete and MAX SNP-hard. Our proof of MAX SNP-hardness is simpler than that given previously by Wang and Jiang. These results suggest that it is unlikely that the multiple sequence tree alignment problem has polynomial-time algorithms that produce either optimal solutions or approximate solutions whose cost may be arbitrarily close to optimal. PMID- 8634903 TI - Hen's teeth and whale's feet: generalized characters and their compatibility. AB - We propose a new model of computation for deriving phylogenetic trees based upon a generalization of qualitative characters. The model we propose is based upon recent experimental research in molecular biology. We show that the general case of determining perfect compatibility of generalized ordered characters is an NP complete problem, but can be solved in polynomial time for a special case. PMID- 8634904 TI - Constructing lattice models of protein chains with side groups. AB - An algorithm to construct lattice models of polymers with side chains is presented. A search for the global minimum of the error function for a given lattice-to-chain orientation is done by dynamic programming, making the search both fast and complete. Application of the algorithm is illustrated by constructing lattice models for 12 proteins of different sizes and structural types. PMID- 8634905 TI - Heterogeneous molecular biology databases. PMID- 8634906 TI - Beyond the information maze. AB - The exponentially increasing volume of scientific information is threatening to overwhelm the budgets and storage capacities of the world's academic libraries. At the same time, new information technologies are poised to revolutionize the publication infrastructure of science. In evaluating these new technologies we must pay attention to their scalability properties: how well will they perform as the amount of information they are handling increases by orders of magnitude? This article identifies some scalability criteria and evaluates some of the current information technologies with respect to them. It ends by suggesting future directions for these technologies. PMID- 8634907 TI - Characterizing heterogeneous molecular biology database systems. AB - Molecular biology data are distributed among multiple databases. Although containing related data, these databases are often isolated and are characterized by various degrees of heterogeneity: they usually represent different views (schemas) of the scientific domain and are implemented using different data management systems. Currently, several systems support managing data in heterogeneous molecular biology databases. Lack of clear criteria for characterizing such systems precludes comprehensive evaluations of these systems or determining their relationships in terms of shared goals and facilities. In this paper, we propose criteria that would facilitate characterizing, evaluating, and comparing heterogeneous molecular biology database systems. PMID- 8634908 TI - Challenges in integrating biological data sources. AB - Scientific data of importance to biologists reside in a number of different data sources, such as GenBank, GSDB, SWISS-PROT, EMBL, and OMIM, among many others. Some of these data sources are conventional databases implemented using database management systems (DBMSs) and others are structured files maintained in a number of different formats (e.g., ASN.1 and ACE). In addition, software packages such as sequence analysis packages (e.g., BLAST and FASTA) produce data and can therefore be viewed as data sources. To counter the increasing dispersion and heterogeneity of data, different approaches to integrating these data sources are appearing throughout the bioinformatics community. This paper surveys the technical challenges to integration, classifies the approaches, and critiques the available tools and methodologies. PMID- 8634909 TI - A strategy for database interoperation. AB - To realize the full potential of biological databases (DBs) requires more than the interactive, hypertext flavor of database interoperation that is now so popular in the bioinformatics community. Interoperation based on declarative queries to multiple network-accessible databases will support analyses and investigations that are orders of magnitude faster and more powerful than what can be accomplished through interactive navigation. I present a vision of the capabilities that a query-based interoperation infrastructure should provide, and identify assumptions underlying, and requirements of, this vision. I then propose an architecture for query-based interoperation that includes a number of novel components of an information infrastructure for molecular biology. These components include a knowledge base that describes relationships among the conceptualizations used in different biological databases, a module that can determine the DBs that are relevant to a particular query, a module that can translate a query and its results from one conceptualization to another, a collection of DB drivers that provide uniform physical access to different database management systems, a suite of translators that can interconvert among different database schema languages, and a database that describes the network location and access methods for biological databases. A number of the components are translators that bridge the heterogeneities that exist between biological DBs at several different levels, including the conceptual level, the data model, the query language, and data formats. PMID- 8634910 TI - Interaction between Escherichia coli RNase P RNA and the discriminator base results in slow product release. AB - We suggested previously that a purine at the discriminator base position in a tRNA precursor interacts with the well-conserved U294 in M1 RNA, the catalytic subunit of Escherichia coli RNase P. Here we investigated this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precursors to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA. Our data suggest that the discriminator base interacts with the residue at position 294 in M1 RNA. Although product release is a rate-limiting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of product release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the discriminator base position. The results showed that the mutant M1 RNA harboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. The combined data suggest a functional interaction between the discriminator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RNA as well as in cleavage site selection. PMID- 8634911 TI - Mutational analysis of the DST element in tobacco cells and transgenic plants: identification of residues critical for mRNA instability. AB - DST (downstream element), an approximately 40-base sequence derived from the 3' untranslated region (UTR) of SAUR (small auxin up RNA) genes, represents one of only a few sequence elements that have been demonstrated directly to target transcripts for rapid decay in plant cells. Substitution mutations were made in conserved regions of the DST element containing the sequences ATAGAT and GTA, which are invariant among several SAUR genes. The mutant DST elements were inserted into the 3' UTR of a beta-globin reporter gene and then assessed for their ability to destabilize the reporter transcript in stably transformed BY-2 tobacco cells. Their effect on reporter mRNA accumulation in both intact transgenic tobacco plants and stably transformed BY-2 cells was also measured. Five- and six-base substitutions in the ATAGAT and GTA regions of DST, respectively, resulted in inactivation of the element as an instability determinant in all systems tested. Smaller, two-base substitution mutations within the ATAGAT and GTA regions had varying effects on DST function in BY-2 cells, ranging from little or no effect to significant increases in reporter mRNA half-life and accumulation. In contrast, all two-base substitution mutations tested resulted in inactivation of DST in intact tobacco leaves. Together, these results indicate that bases within both the ATAGAT and GTA regions of DST are required for its function as an mRNA instability determinant in both BY-2 cells and leaves of transgenic plants, and that the sequence requirements for DST to function in leaves are more stringent. PMID- 8634912 TI - Regulated ribosomal frameshifting by an RNA-protein interaction. AB - Ribosomal frameshifting is a translational mechanism used as an essential step in the replication cycle of retroviruses. Programmed frameshifting in retroviral translation involves two sequence elements: A heptanucleotide slippery sequence which induces a low basal level of frameshifting and a downstream RNA structure as an enhancer of the process. The precise mechanism of function of these downstream elements is still unclear, but their effect does not solely depend on their stability. Likewise, the possibility that frameshifting could be controlled by specific proteins that bind to these elements and enable or modulate their effects has yet not been substantiated. The RNA hairpin of the HIV-1 gag-pol frameshift cassette was replaced by the iron-responsive element (IRE) from ferritin mRNA, a stem-loop structure that binds iron regulatory proteins (IRPs) in dependence of the iron status of the cell. When a lacZ/luciferase reporter construct was expressed in transfected BHK-21 cells, the IRE or a point-mutated version that is unable to bind IRPs were found to functionally substitute for the HIV-1 hairpin. When cells were treated with the iron chelator desferrioxamine to stimulate IRP binding to the wild-type IRE, frameshift activity was specifically and strongly augmented by protein binding Our data establish that frameshifting can be regulated in a reversible fashion by mRNA-binding proteins. PMID- 8634913 TI - The U18 snRNA is not essential for pre-rRNA processing in Xenopus laevis. AB - The U18 small nuclear RNA (snRNA) is one of several newly discovered intron encoded nucleolar RNAs whose function is unknown. We have studied the accumulation and function of the U18 snRNA in oocytes of the vertebrate, Xenopus laevis. The U18 snRNA contains 13 nt complementary to a highly conserved sequence in 28S ribosomal RNA (rRNA). Three oligonucleotides, selected to contain all or some of the complementary sequence, deplete the U18 snRNA upon injection into Xenopus oocytes. Injection of two of the oligonucleotides has no effect on pre rRNA processing or ribosome transport. Injection of the third oligonucleotide does interrupt pre-18S rRNA processing, but this is due to coincidental simultaneous depletion of the U22 snRNA. The U18 snRNA is the first nucleolar snRNA that is not essential for ribosome biogenesis in vertebrates. PMID- 8634914 TI - Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome dependent guanosine triphosphatase. AB - Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles. PMID- 8634915 TI - An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon. AB - The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7 nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon. PMID- 8634916 TI - Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB. AB - On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM. PMID- 8634918 TI - Functional analysis of an intron 3' splice site in Caenorhabditis elegans. AB - Caenorhabditis elegans introns typically lack both branch point and polypyrimidine tract consensus sequences utilized in other organisms for intron recognition. However, most introns have an unusually long, highly conserved consensus, UUUCAG/R, at the 3' splice site. This site can be recognized even when the -1G is changed to A. To determine how the 3' splice site is defined, we tested mutations in the sequence UUUCAA/A at the 3' splice site of the first intron of a test gene in vivo. Replacement of individual U's with A's or C's compromised splicing. When the sequence UUUCAA/AAG was tested, splicing occurred following both the -1A and the +3G, indicating that both UUUC and the AG contain 3' splice site information. When the sequence UUUCAA/AAA was tested, all splicing occurred following the -1A, suggesting that the UUUC contains sufficient information in the absence of an AG to specify the location of the splice site. In support of this idea, when point mutations were introduced into the UUUC, unspliced RNAs accumulated. Surprisingly, RNAs containing the mutant intron often contained the second, nonmutated intron as well, suggesting that interference with splicing of one intron can interfere with splicing of a second intron in the same pre-mRNA. The majority of these unspliced RNAs were degraded by the system responsible for degradation of transcripts containing nonsense mutations (smg), even though the intron contained no nonsense codon. PMID- 8634917 TI - A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. AB - In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction. PMID- 8634919 TI - Prevention of nitrate tolerance with concomitant administration of hydralazine. AB - Organic nitrates have frequently been used for the treatment of patients with congestive heart failure (CHF). Nitrate tolerance has been identified as a major limitation of this therapy preventing a continuous effect. In the past several years, an effort has been made to develop strategies to prevent nitrate tolerance. Recent animal experimentation has demonstrated the prevention of nitrate tolerance with a concomitant administration of hydralazine. A later study demonstrated similar results in a patient with chronic CHF. The use of oral hydralazine (75 mg qid) resulted in prevention of early development of nitrate tolerance and attenuation of nitrate mediated hemodynamic effects which occurred within the first several hours after initiation of nitroglycerin therapy in the control group not receiving hydralazine. Recent data obtained in in vitro studies have demonstrated enhancement of vascular superoxide production secondary to exposure to nitrates which may be an important mechanism leading to nitrate tolerance. These same studies demonstrated a strong antioxidant effect of hydralazine reducing superoxide formation and preventing nitrate tolerance. Both animal and human experiments have demonstrated prevention of nitrate tolerance with a concomitant use of hydralazine. In vitro studies suggest that hydralazine, by virtue of its antioxidant effect, prevents the nitrate-mediated formation of vascular superoxide, and thus prevents nitrate tolerance. These data provide an explanation for the benefit demonstrated in the V-HeFT studies with the combination of isosorbide dinitrate and hydralazine in the treatment of patients with chronic CHF, and provide a support for the use of this therapeutic regimen in the treatment of patients with chronic heart failure. PMID- 8634920 TI - Potential problems with intermittent nitrate therapy. AB - The organic nitrates continue to be widely used in clinical practice. The development of tolerance to these agents is now a well-recognized problem, although the use of a nitrate-free interval or compounds with specialized pharmacokinetic profiles can overcome this difficulty. Despite this success, prospective studies of intermittent transdermal nitroglycerin have suggested that the nitrate-free interval may be associated with two potential problems. The first is the development of crescendo angina during the nitrate-free interval. Such unstable clinical events occurring during the nitrate-free interval have been termed 'rebound angina' and are reminiscent of ischemic events reported in the munitions industry, where workers are exposed to high nitroglycerin concentrations. Intermittent nitroglycerin therapy has also been reported to cause a decrease in treadmill walking time to the onset of angina. This has been suggested from observations made in clinical trials of intermittent transdermal nitroglycerin therapy and was recently confirmed in a study from our laboratory, where the withdrawal of transdermal nitroglycerin was associated with a reduction in tread-mill exercise performance for a period of at least 6 h. Although rebound ischemia has been reported, this has not been a consistent observation in all clinical trials of intermittent nitrate therapy. Furthermore, it is not at all clear that a decrease in exercise tolerance, which would usually be occurring in the evening hours, is of any clinical importance. Nevertheless, these are potentially important observations that warrant further study and may provide further insight into the pathophysiology of nitrate tolerance. PMID- 8634921 TI - Nitrate tolerance--problems both new and old. AB - Nitrate tolerance has been recognized for many years but only recently has it been shown to be an important clinical problem. Tolerance develops very rapidly during oral, transdermal or intravenous nitrate administration and treatment strategies designed to provide therapeutic effects throughout 24 h each day are associated with marked attenuation of nitrate effects. The mechanisms responsible for tolerance are not clearly understood. The hypotheses put forward to explain this phenomenon include reduction of sulfhydryl groups in vascular smooth muscle with resultant diminished nitric oxide production, neurohormonal activation, plasma volume expansion, and superoxide anion production. Attempts to prevent or reverse tolerance with sulfhydryl group donors, angiotensin-converting enzyme inhibitors, and hydralazine have not been uniformly successful and such approaches are impractical. The beneficial nitrate effect can be preserved by appropriate dosing which provides a period of many hours of nitrate washout during each 24 h period. PMID- 8634922 TI - Therapy of angina pectoris with long-acting nitrates: which agent and when? AB - Nitroglycerin and the long-acting nitrates are effective antianginal agents that have been used in clinical medicine for over 100 years. These drugs are reliable, safe, familiar to clinicians, inexpensive, and easy to use. Side effects are limited to headache and postural hypotensive symptoms. Nitrate tolerance or attenuation, -ie, loss of, or decrease in, nitrate efficacy with repeated dosing is common and represents the major drawback to chronic therapy. Carefully designed dosing regimens and/or appropriate use of nitrate formulations (to include a nitrate-free period each day) will decrease or eliminate the problem of nitrate tolerance. In head-on comparative studies, nitrates appear to be as effective as beta-blockers or calcium channel blockers in the monotherapy of chronic angina. Ideal patient characteristics for nitrate therapy include: predictably favourable response of chest pain to sublingual nitroglycerin; angina episodes suggestive of coronary vaso-constriction or spasm; left ventricular systolic dysfunction; symptoms of congestive heart failure (systolic or diastolic dysfunction). PMID- 8634923 TI - High-dose cytarabine and mitoxantrone as salvage therapy for refractory non Hodgkin's lymphoma. AB - BACKGROUND: High-dose cytarabine (ara-C) alone or in combination with mitoxantrone each has shown to be active in therapeutic trials of refractory non Hodgkin's lymphoma (NHL). In this study, we administered these two drugs to 14 patients with advanced and refractory NHL. METHODS: Ara-C was administered at a dosage of 3 gm/sqm for 2-hour intravenous infusion every 12 hours from day 1 to day 4 (8 doses), and mitoxantrone was given at a dosage of 6 mg/sqm/day for 1 hour intravenous infusion from day 1 to day 5. The clinical efficacy and toxicity were assessed by WH0 criteria. RESULTS: Four patients (28%) attained complete remission (CR) and 2 had partial remission (PR). Of the 4 CR patients, the remission lasted 5 months in one patient and 4 months in another. The remaining 2 patients had CR of only 1.3 months. Myelosup-pression with subsequent infection was the major toxicity of this regimen. Severe neutropenia (<1,000/uL) lasted for an average of 20 days, and thrombocytopenia (<50,000/uL) 24 days. Nonmyeloid toxicities included 100% alopecia, 93% stomatitis, 43% hepatotoxicity, 36% dermatitis, 28% CNS toxicity and 7% chemical conjunctivitis. CONCLUSIONS: A proportion of refractory NHL patients will respond to high-dose ara-C + mitoxantrone, despite that severe myelosuppression is frequently encountered. PMID- 8634925 TI - Urinary PCO2 for hemodynamically unstable patients. AB - BACKGROUND: Gastric intramural pH (pHi) derived from gastric PCO2 has been successfully used to assess splanchnic ischemia for patients with unstable hemodynamics, but with some limitations. Urinary bladder, also an easily accessible hollow viscus, should provide as a useful route for the same purpose. However, no study has used urinary PCO2 to evaluate the adequacy of perfusion in critically ill patients. METHODS: Fifty patients admitted to intensive care unit were included and divided into hemodynamically stable and unstable groups. Several parameters such as arterial pressure, dopamine dosage, heart rate, serum lactate, arterial blood gas, urinary PCO2, and concentrations of Na, K and Cl in urine were measured. Patients with some other renal or pre-renal conditions that might affect urinary PCO2 were excluded. RESULTS: Urinary PCO2 was markedly higher (78.6 +/- 9.9 vs. 43.1 +/- 1.7 mmHg, p < 0.0001) in unstable group. Serum anion gap level, dopamine dosage and heart rate were significantly higher and PaO2/FiO2 ratio as well as mean arterial pressure was lower in unstable group. Serum lactate, arterial pH and other parameters failed to distinguish between groups. Dopamine dosage significantly correlated with urinary PCO2 (r = 0.5357, p = 0.0149) in unstable group. CONCLUSIONS: With careful selection of patients, urinary PCO2 can effectively differentiate hemodynamically unstable patients from stable ones. It also correlates significantly with dopamine dosage in patients with unstable hemodynamics. PMID- 8634924 TI - Ultrasound-guided fine needle aspiration biopsy of small pulmonary nodules abutting to the chest wall. AB - BACKGROUND: Ultrasound (US)-guided needle biopsies in peripheral pulmonary lesions are widely applied. Most pulmonary lesions had a large size. We report our results in evaluating the availability of US-guided fine needle aspiration biopsy (US-guided FNAB) in diagnosing small pulmonary nodules abutting to the chest wall. METHODS: Forty patients, whose chest radiographs showed peripheral pulmonary nodules of diameter smaller than 3.0cm, received chest sonographic examination and US-guided FNAB. Of those, six patients had no definite diagnoses; the remaining 34 patients with pulmonary nodules, we divided into three groups. There were four tiny nodules of diameters smaller than 1.0cm, 11 nodules between 1.1 and 2.0cm, and 19 nodules between 2.1 and 3.0cm. We evaluated the diagnostic yields and rates of complication in relation to the size or nature of the nodule. RESULTS: In the enrolled 40 patients, six were lost to surveillance and had indeterminate diagnoses. Of the remaining 34 patients with proven pathological diagnoses and compatible clinical conditions, the diagnostic yields of US-guided FNAB achieved 88% (30/34) which include 96% (26/27) in malignancies and 57% (4/7) in benign nodules. According to the nodular size, the diagnostic rate achieved 100% (4/4) in tiny nodules, 91% (10/11) in nodules between 1.1 and 2.0cm, and 84% (16/19) in nodules between 2.1 and 3.0cm. Only one patient with a 2.5-cm nodule developed pneumothorax after the US-guided FNAB procedure. CONCLUSIONS: US-guided FNAB is a useful and safe diagnostic tool for small pulmonary nodules abutting to the chest wall. The nodular nature, but not size, affects the diagnostic yields and rates of complication. PMID- 8634927 TI - Treatment of unstable tibial fractures with interlocking nail versus Ender nail: a prospective evaluation. AB - BACKGROUND: For unstable tibial fractures, there are many methods of surgical treatment. The efficacy of the Ender nail and the interlocking nail in the treatment of such fractures is examined. METHODS: One hundred and seven cases of unstable tibial shaft fractures were collected for the prospective study. Randomly, 61 tibia were fixed with interlocking nails and 46, with Ender nails. The mean follow-up period was 30.5 (23 to 40) months. The results of the different treatments were compared. RESULTS: In the group with interlocking nails, the average blood loss was 265 cc; operation time was 61 minutes; hospital days were 10.4; and union time was 15.1 weeks for closed fractures and 17 weeks for Winquist-Hansen type III and IV fractures. In the group with Ender nails, the average blood loss was 135 cc, operation time was 32 minutes, hospital days were 8.3 days, and union time was 17.6 weeks for closed fractures and 22.5 weeks for Winquist-Hansen type III and IV fractures. Student t-test revealed statistically significant difference between the groups in all of the data described above. However, treatment with the different nails showed no significant difference in results for open type I and II fractures. CONCLUSIONS: Ender nail still has its superior usefulness in some aspects of treatment of less comminuted unstable tibial shaft fractures, but for the more comminuted unstable tibial shaft fractures, the interlocking nail is undoubtedly better used. PMID- 8634926 TI - Prognostic factors of primary aldosteronism. AB - BACKGROUND: Primary aldosteronism (PA) is a rare but potentially curable cause of hypertension. Between October 1982 and November 1994, 30 patients of PA received unilateral adrenalectomy with a long-term follow up (mean:60.3 months). Nineteen (63.3%) cases were cured (Group 1); 11(36.7%) cases were improved (Group 2). The purpose of this study was to determine prognostic factors after surgery in patients with PA. METHODS: A retrospective analysis was performed regarding age and sex of the patients, duration of hypertension, family history of hypertension, preoperative blood pressure, plasma aldosterone concentration (PAC), plasma renin activity (PRA) and efficacy of spironolactone on blood pressure between both groups. End-organs (including kidney, heart, retina and brain) involvement was evaluated and compared. Adrenalectomy and renal biopsy specimen for pathology were similarly evaluated. RESULTS: The duration of hypertension was longer in Group 2 than in Group 1 (8.18 +/- 4.94 vs 5.21 +/- 4.24 years). The efficacy of spironolactone on blood pressure (BP) was positive in 81.8% of Group 1 and 16.7% of Group 2. Adrenal cortical adenoma in 24 cases with a cure rate of 70.8% (17/24) and adrenal cortical macronodular hyperplasia in 6 cases with a cure rate of 33.3% (2/6) were noted. Group 2 had more end organs involvement than Group 1. The severity of histopathological change of the renal biopsy was similar. CONCLUSIONS: This study suggests that preoperative response of blood pressure to spironolactone administration predicts the postoperative prognosis of hypertension in patient with PA. Long duration of hypertension and involvement of two or more end-organs were poor prognostic factors. Excellent results can be achieved by unilateral adrenalectomy in adrenal cortical adenoma and fair results, in adrenal cortical macronodular hyperplasia. PMID- 8634928 TI - The clinical evaluation of an outbreak of aseptic meningitis in children. AB - BACKGROUND: Aseptic meningitis is a common disease in children; The present report describes the clinical and laboratory studies of 62 children involved in an outbreak of aseptic meningitis. METHODS: Sixty-two children with aseptic meningitis having been hospitalized from February to June 1993 were evaluated. The clinical data were obtained by review of the medical records. Initial cerebrospinal fluid (CSF) laboratory values were recorded, and CSF specimens were submitted to determine the viral etiology. RESULTS: Thirty-three percent of the cases were aged 2-5 years. Echovirus 30 was isolated from CSF specimens in 17 children, this is the only virus isolated from CSF during this period. The illness was characterized by transient fever, headache and vomiting. There was wide variation in the number of leukocyte from 14 to 2333 cell/mm3 in the spinal fluid. CSF protein ranged from 19 mg/dL to 114 mg/dL, and CSF sugar was normal in all specimens. Virus isolation was related to the cell count in CSF. There was no significant relationship between CSF cell count and protein content (r = 0.162, p = 0.2237). Hospital stay ranged from 1 to 14 days with an average of 3.9 days. CONCLUSIONS. The disease was mild and self-limiting without death or complication. Enterovirus isolation from CSF can substantially establish the diagnosis and the subsequent management, therefore, early discharge is expectable. PMID- 8634929 TI - Intravenous omeprazole prevents rebleeding in peptic ulcer patients with a non bleeding visible vessel: a preliminary report of a randomized controlled study. AB - BACKGROUND: In patients with peptic ulcer bleeding, acid can enhance platelet disaggregation and cause lysis of the clot. Omeprazole, a potent acid suppressor, may be helpful in reducing their rebleeding rate. METHODS: Between March and October 1994, 40 patients with a non-bleeding, visible vessel (NBVV) at ulcer bases were enrolled for study. They were randomized into four groups. Group 1 (n = 10) patients received cimetidine 300mg intravenously (i.v.) q6h; Group II (n = 10) patients received heater probe thermocoagulation therapy and cimetidine 300mg i.v. q6h; Group III (n = 10) patients received omeprazole 40mg intravenous bolus initially followed by 40mg i.v. qd and Group IV (n = 10) patients received omeprazole 40mg intravenous bolus initially followed by 40mg i.v. q12h. Endoscopic examination was done for follow-up, daily, for 1-3 days. RESULTS: Preliminary results showed that the age, initial hemoglobin, ulcer size as well as NBVV size in Groups I-IV were not significantly different (p < 0.05). The rebleeding rates were 40% in Group I, 20% in Group II, 20% in Group III and 0% in Group IV, (p < 0.05 when Group IV is compared with Group I). CONCLUSIONS: Intravenous omeprazole 40mg given q12h can reduce the rebleeding rate of patients with a NBVV. PMID- 8634930 TI - Late-onset idiopathic hypoparathyroidism with thymoma: a case report. AB - A 62-year-old male was admitted because of numbness and twitching of both hands. Hypocalcemia with positive Trousseau's sign was noted. Chest X-ray and computed tomography (CT) showed an anterior mediastinal mass. Skull X-ray and whole body bone scan could not rule out bony metastasis to the left parietal bone, causing an anterior mediastinal tumor with bony metastasis to be suspected initially. Median sternotomy and extended thymectomy were done, and Stage II thymoma with negative calcitonin staining was noted. However, hypocalcemia persisted after thymectomy and the results of pre-operative and post-operative intact-parathyroid hormone (intact-PTH) were less than the detection limit (<13.3 pg/ml). Tumor markers and gallium tumor scan were all negative. Brain CT disclosed calcification over the bilateral basal ganglia and bilateral dentate nuclei of the cerebellum; the supposed metastatic osteolytic lesions of parietal bone were considered to result from pacchionion arachnoid granulation tissues. The coexistence of late-onset idiopathic hypoparathyroidism and thymoma has not been reported before. Long-term replacement therapy with vitamin D and calcium was necessary for this case. PMID- 8634932 TI - Trihexyphenidyl abuse in schizophrenic patient: a case report. AB - Trihexyphenidyl is a synthetic anticholinergic used in psychiatric patient for the relief of neuroleptic-induced extrapyramidal symptoms. It has been reported to have mood elevating, euphorigenic and socially-stimulating effects. The case presented is of anti-cholinergic abuse by a chronic schizophrenic who abused trihexyphenidyl, up to 200 mg per day, to achieve an euphoric effect. The drug was partly prescribed in psychiatric clinics, but mostly bought in the drugstore. The discontinuance of trihexyphenidyl produced anxiety, which was relieved by anxiolytics. The patient also feigned extrapyramidal symptoms to get anticholinergic injections during drug abstinence. A high dose of trihexyphenidyl may precipitate anticholinergic toxic psychosis and interfere with the therapeutic effects of antipsychotics by impeding their absorption. Given a large number of patients receiving this medication, clinicians should be alert when prescribing this drug, especially for patients who have abuse potential. As this agent has never been under public surveillance and is available in local pharmacies, the health authorities should be concerned with its abuse potential. PMID- 8634931 TI - Acute isoniazid intoxication: a case report. AB - Since its introduction in 1952, isoniazid has remained one of the drugs of choice in the treatment and prophylaxis of tuberculosis. In populations with a high prevalence rate of tuberculosis or suicide rate, acute ingestion of isoniazid has occasionally been reported. Acute intoxication by isoniazid is known to cause symptoms of seizures, metabolic acidosis, coma, and even death. These clinical symptoms, however, are not well recognized by physicians in Taiwan, even though the prevalence rate of tuberculosis is relatively high here. This report concerns the case of a 25-year-old female with a past history of tuberculosis who presented with the symptoms of refractory seizures, metabolic acidosis and deep coma after intentional ingestion of some unknown drug. Although implicating agents were not recognized initially, she was successfully revived with basic resuscitation, anticonvulsants and correction of metabolic acidosis. A review of her history revealed that her ingestion of five grams of isoniazid in this case was responsible for the entire clinical spectrum. Given easy access to isoniazid in Taiwan, a diagnosis of isoniazid poisoning should always be considered in patients who present with the classical symptoms of refractory seizures, metabolic acidosis and coma. PMID- 8634933 TI - Pregnancy following in vitro fertilization and embryo transfer by microsurgical epididymal sperm aspiration from a patient with congenital absence of the vas deferens: a case report. AB - Congenital absence of the vas deferens (CAVD) has been considered a virtually untreatable cause of male infertility. Men with this condition have been shown on testicular biopsy to have adequate spermatogenesis, and are theoretically producing sperm capable of fertilizing an oocyte. Yet epididymal transit was thought to be essential for the maturation of spermatozoa and development of their fertilizing ability since the characteristics of sperm motility improve as the sperm passes through the cauda. However recent studies in man have shown that spermatozoa aspirated from the obstructed caput epididymis and ductuli efferentia are, in fact, capable of fertilization in vitro. Microsurgical epididymal sperm aspiration (MESA) from the proximal region (caput) of the epididymis, obtained 0.5 x 10(6) sperm per ml, following washing and direct swim-up. Twelve oocytes were inseminated and three embryos were generated for transfer. The patient conceived and delivered a healthy female baby weighting 2838 gm, on March 3, 1994. This is the first documentation in Taiwan of live birth resulting from MESA from a patient with CAVD combined with in vitro fertilization and embryo transfer. PMID- 8634934 TI - Selective IgA deficiency and anaphylactoid transfusion reaction: a case report. AB - Anaphylactoid transfusion reaction following the administration of incompatible blood products can be life threatening, but accounts for only a very small proportion of all transfusion reactions. One of the causes of anaphylactoid transfusion reaction is the reaction of patient's anti-immunoglobulin A(IgA) with plasma IgA which is usually present in transfused blood. We report a case who had anaphylactoid transfusion reactions after transfusion of only a few milliliters of packed RBC from 3 consecutive donors, in spite of compatible pretransfusion crossmatches. The patient's serum revealed IgA deficiency accompanied by high titer anti-IgA. The family study showed that one of her sons had partial IgA deficiency accompanied by anti-IgA2. In conclusion, although selective IgA deficiency is rare in Taiwan, it may still occasionally result in severe anaphylactoid transfusion reactions. PMID- 8634935 TI - Interleukin-1 alpha stimulates osteoclast formation from peripheral blood monocytes and increases osteoclastic activity. AB - BACKGROUND: Interleukin-1 (IL-1) is produced by monocytes and marrow stromal cells and can stimulate bone resorption in tissue culture. In addition, IL-1 can induce osteoclast-like cell formation in long-term bone marrow culture. However, whether IL-1 can increase osteoclast formation by stimulating fusion of peripheral blood monocytes is not clear. In addition, the precise effect of IL-1 on osteoclastic activity is still not well known. METHODS: Using the bioassay of osteoclast formation, osteoclast-free fetal calvariae were harvested from 14-day timed-pregnant Sprague-Dawley rats and cultured in BGJb medium for 10 days. On the 10th day, mononuclear cells were obtained from peripheral blood of 8-week-old female Sprague-Dawley rats through cardiac puncture and Ficoll-Paque density gradient separation method and co-cultured with osteoclast-free developing bone explant. On the same day, various concentrations of IL-1 alpha (1,10 or 100 U/ml) were added daily in the experimental dishes while only BGJb medium was added in the controls. In addition, 45Ca release assay and quantitation of nuclei per osteoclast were performed to evaluate the effect of IL-1 alpha on osteoclastic activity. RESULTS: Ten days after adding monocytes, there were 14.25 osteoclasts in the control, without IL-1 alpha; with 1 U of IL-1 alpha, there were 14.75 osteoclasts; with 10 U of IL-1 alpha, there were 18.00 osteoclasts; with 100 U of IL-1 alpha, there were 20.75 osteoclasts. In addition, IL-1 alpha stimulated the release of 45Ca and increased the number of average nuclei per osteoclast dose dependently, indicating a significant increase in bone resorption. CONCLUSIONS: IL-1 alpha could increase osteoclast formation by stimulating the fusion of peripheral blood monocytes. In addition, IL-1 alpha is a potent stimulator of osteoclastic activity. PMID- 8634936 TI - Effects of colony-stimulating factors on the all-trans retinoic acid-induced differentiation of acute promyelocytic leukemic cells. AB - BACKGROUND: NB4, a cell line derived from a patient with t(15;17) acute promyelocytic leukemia (APL) that undergoes granulocytic differentiation when treated with pharmacological doses of all-trans retinoic acid (ATRA), was used as a model for induction of differentiation. In this study, we examined the interaction of colony-stimulating factors (CSF) and ATRA in affecting the proliferation and differentiation of NB4 cells. METHODS: Nitroblue tetrazolium (NBT) reduction was used as a functional marker of leukemia cell differentiation. The number of viable cells was counted by trypan blue exclusion test. RESULTS: Proliferation of NB4 cells increased when exposed to 10(-9)M of ATRA, but reduced progressively when exposed to ATRA at the concentrations of 10(-8)M to 10(-6)M. After culture for 5 days, NBT-positive cell was not detectable in the control cultures with medium alone, but its percentage apparently increased to 84% at 10( 7)M ATRA. Granulocyte (G)-CSF per se had no effect on the granulocytic differentiation of NB4 cells, but it could enhance the NBT reduction when used in combination with various concentrations (10(-9)M -10(-6)M) of ATRA. Interleukin (IL)-3 or granulocyte-macrophage-CSF (GM-CSF) alone also had no effect on the NBT reduction in NB4 cells. However, when combined with ATRA, both caused a slight suppression of NBT reduction. No synergistic effect was noted between IL-3 and G CSF on the ATRA-induced granulocytic differentiation. CONCLUSIONS: G-CSF, but not IL-3 or GM-CSF, can enhance the differentiating activity of ATRA. Further investigations are necessary to evaluate its clinical use. PMID- 8634937 TI - Counseling patients about allergic rhinitis. AB - Allergic rhinitis affects 10% to 30% of Americans. Intranasal corticosteroids are safe and effective in managing allergic rhinitis without systemic effects. Pharmacists should educate patients about the proper use of intranasal corticosteroids and advise them to avoid exposure to allergens. PMID- 8634938 TI - Technology and automation in pharmaceutical care. AB - Pharmacy computer systems in the future will help pharmacists assess patients' medication needs, evaluate drug therapy, and manage patient information. Technology companies are developing systems for automatic dispensing and for distributing medications from nurses' stations in hospitals and long-term care facilities. Bar codes are being used in medication dispensing, verifying prescriptions, selecting patient brochures, and screening for drug interactions. Pharmacies in the future will rely on information networks to store, manage, and communicate patient information. Drug use review databases offer highly developed drug therapy screening. PMID- 8634939 TI - The role of technology in patient care. PMID- 8634940 TI - Understanding homeopathy. AB - Interest in homeopathy is growing because of the costs and impersonality of modern medical care and the increase in infectious diseases that do not respond to allopathic medicine. Extreme dilutions are the main reason many scientists doubt the effectiveness of homeopathic products. Scientists have petitioned the Food and Drug Administration to hold homeopathic products to the same standards as other drugs. While some studies support the efficacy of homeopathy, controlled clinical studies determining effective levels of dilution for these substances are needed. PMID- 8634941 TI - An atmosphere of mistrust. PMID- 8634942 TI - Chain pharmacists' attitudes on and awareness of domestic abuse. AB - Little has been written to specifically educate pharmacists on the subject of spousal or partner abuse. This project was undertaken to determine whether pharmacists have received training in domestic abuse and to ascertain their attitudes toward participating in abuse intervention. Two hundred twenty-four pharmacists from two chain pharmacies in Arizona received a questionnaire, and 121 (54%) responded. Most (97.5%) had received no training on domestic abuse intervention techniques and did not feel adequately prepared to intervene in a potential situation. Respondents were divided on the extent to which pharmacists should be involved in intervention. Pharmacists who graduated before 1980 were less likely to agree that intervention was an important activity for pharmacists (p < 0.05). More women than men would feel comfortable helping abused patients if they knew more about intervention. More women than men agreed that pharmacists should keep information about domestic abuse on hand (p < 0.025), but most respondents believed that information should be available in pharmacies to give to patients. Opportunities exist for pharmacists to provide educational materials to patients on domestic abuse and abuse intervention. PMID- 8634943 TI - The pharmacist as self-care advisor. AB - A patient's decision to purchase a nonprescription product is driven by several factors, including the manufacturer's advertising claims, the advice of friends or relatives, and past experience with the product. Pharmacists must be well versed in product- and patient-related factors governing the appropriate use of nonprescription products in order to make appropriate recommendations to their patients and to direct them to a physician or other health care professional when necessary. PMID- 8634944 TI - Sensitivity of fungi to nikkomycin Z. AB - Nikkomycin Z inhibited colony radial extension (Kr) and hyphal density in Geotrichum candidum, Trichoderma koningii, Mucor plumbeus, and Fusarium oxysporum, although the level of inhibition varied with the fungal species and was influenced by the concentration of nutrients in the culture medium. While G. candidum and M. plumbeus were sensitive to the inhibitory effects of nikkomycin Z, T. koningii and F. oxysporum were relatively tolerant of concentrations of the antibiotic up to 100 mumol. Nikkomycin Z also caused significant decreases in rates of hyphal extension (Emax) and swelling and bursting of hyphal apices in G. candidum and M. plumbeus. The sensitivity of G. candidum and M. plumbeus to the effects of the antibiotic was due in part to their ability to remove nikkomycin from a liquid culture medium; the nikkomycin-tolerant species T. koningii and F. oxysporum were unable to transport this nucleoside dipeptide antibiotic. Although both G. candidum and F. oxysporum appeared capable of removing the di- and tripeptides, dialanine and trialanine, from a liquid medium, rate of dipeptide uptake was greater in F. oxysporum, while rate of tripeptide uptake was greater in G. candidum. However, overall rate of uptake of peptides was greater in G. candidum than in F. oxysporum. PMID- 8634945 TI - The Phytophthora sojae genome contains tandem repeat sequences which vary from strain to strain. AB - The oomycete Phytophthora sojae causes root and stem rot of soybean. In P. sojae, 37 cultivarspecific physiological races have been reported with different reactions against 13 single dominant resistance genes in soybean. Recent genetic studies have demonstrated that the P. sojae-soybean interaction follows Flor's gene-for-gene model. Genomic subtraction was carried out to isolate race-specific DNA sequences possibly including avirulence genes. DNA from a race 1 isolate was subtraction-enriched with the DNAs from race 19 and race 22 isolates. The enriched DNAs were used to differentially screen a P. sojae genomic library. Characterization of the clones obtained identified repetitive sequences with variable copy numbers among isolates. Five of these repetitive sequences were tandemly repeated and were localized on single chromosomes. The sixth corresponded to the ribosomal RNA genes. These observations suggest that gene amplification could contribute to the generation of genetic diversity in P. sojae. Enriched sequences corresponding to avirulence genes were not detected. PMID- 8634946 TI - Genetic differentiation in the rice blast fungus revealed by the distribution of the Fosbury retrotransposon. AB - Repetitive DNA sequences genetically differentiate certain host-specific forms of the plant pathogenic fungus Pyricularia grisea. For example, high copy numbers of a sequence designated MGR586 are conserved in isolates that infect rice. In this report, we describe the molecular characterization of another repetitive DNA sequence designated fosbury. Restriction mapping and DNA sequence analysis show that fosbury is a member of a long terminalrepeat (LTR)-containing retrotransposon family, and gel blot hybridization analysis suggests that, like MGR586, fosbury is preferentially found in isolates that infect rice. This supports the view that rice pathogens comprise a genetically distinct form of P. grisea. We also investigated the distribution of fosbury and MGR586 and found that these elements are sometimes associated in the genome, and gel blot hybridization analysis shows that restriction sites flanking both of these elements resolve rice pathogens into similar clonal lineage groups. We conclude that the transposition of repetitive DNA sequences plays an important role in generating DNA fingerprint variation in the rice blast fungus. PMID- 8634947 TI - Dibutyryl cyclic AMP-enhanced germ tube formation in exponentially growing Candida albicans cells. AB - Competence in germ tube formation for exponential phase cells of several Candida albicans was low (less than 14%) in 0.1 M phosphate buffer (pH 6.8) containing 1 mM proline. Addition of 5 mM dibutyryl cyclic AMP (cAMP), a lipophilic cAMP analog, to the induction medium increased germ tube formation up to 50%. This enhancement occurred only when the cells were grown in medium lacking glucose or fructose as a carbon source. N-Acetylglucosamine-induced germ tube formation was enhanced by dibutyryl cAMP under similar conditions, but the enhancement was only slight. Cyclic GMP (cGMP) or AMP did not enhance germ tube formation, while dibutyryl cGMP, theophylline, and butyric acid were inhibitory. These results show that dibutyryl cAMP stimulation of the germ tube formation depends on the growth phase and the nutritional status of the cell. PMID- 8634948 TI - Standards, accreditation and proficiency testing in the management of blood and blood products in hospitals. PMID- 8634949 TI - Selling guidelines door to door. PMID- 8634950 TI - Violence sells. PMID- 8634951 TI - Bucking the trend. PMID- 8634952 TI - Sustaining development. PMID- 8634953 TI - Future of CMAJ may depend on publishing research. PMID- 8634954 TI - Wheelchair drivers sidewalk menace? PMID- 8634955 TI - Lyme disease may be uncommon in BC. PMID- 8634956 TI - Neonatal circumcision revisited. Fetus and Newborn Committee, Canadian Paediatric Society. AB - OBJECTIVE: To assist physicians in providing guidance to parents regarding neonatal circumcision. OPTIONS: Whether to recommend the routine circumcision of newborn male infants. OUTCOMES: Costs and complications of neonatal circumcision, the incidence of urinary tract infections, sexually transmitted diseases and cancer of the penis in circumcised and uncircumcised males, and of cervical cancer in their partners, and the costs of treating these diseases. EVIDENCE: The literature on circumcision was reviewed by the Fetus and Newborn Committee of the Canadian Paediatric Society. During extensive discussion at meetings of the committee over a 24-month period, the strength of the evidence was carefully weighed and the perspective of the committee developed. VALUES: The literature was assessed to determine whether neonatal circumcision improves the health of boys and men and is a cost-effective approach to preventing penile problems and associated urinary tract conditions. Religious and personal values were not included in the assessment. BENEFITS, HARMS AND COSTS: The effect of neonatal circumcision on the incidence of urinary tract infection, sexually transmitted diseases, cancer of the penis, cervical cancer and penile problems; the complications of circumcision; and estimates of the costs of neonatal circumcision and of the treatment of later penile conditions, urinary tract infections and complications of circumcision. RECOMMENDATION: Circumcision of newborns should not be routinely performed. VALIDATION: This recommendation is in keeping with previous statements on neonatal circumcision by the Canadian Paediatric Society and the American Academy of Pediatrics. The statement was reviewed by the Infectious Disease Committee of the Canadian Paediatric Society. The Board of Directors of the Canadian Paediatric Society has reviewed its content and approved it for publication. SPONSOR: This is an official statement of the Canadian Paediatric Society. No external financial support has been received by the Canadian Paediatric Society, or its members, for any portion of the statement's preparation. PMID- 8634958 TI - Integrated delivery systems: has their time come in Canada? AB - In the 1990s every Canadian province is struggling to reduce health care expenditures without jeopardizing access to health care or quality of care. The authors propose a new model for health care delivery: the Canadian Integrated Delivery System (CIDS). A CIDS is a network of health care organizations; it would provide, or arrange to provide, a coordinated continuum of services to a defined population and would be held clinically and fiscally accountable for the outcomes in and health status of that population. A CIDS would serve 100,000 to 2 million people; the care it would provide would be funded on a capitation basis. For providers, there would be explicit financial incentives to minimize costs. At the same time, service quality and consumer choice of primary care practitioner would be maintained. Primary care physicians and specialists would work with other health care service providers to offer a full spectrum of care. CIDS providers would form strategic alliances with community agencies, hospitals, the private sector and other health care services not managed by the CIDS, as needed. For physicians, affiliation with a CIDS that provided strong clinical leadership could be beneficial to their income stability and autonomy. Pilot projects of this model in several communities would determine whether this concept is feasible in the Canadian health care context. PMID- 8634957 TI - Antenatal psychosocial risk factors associated with adverse postpartum family outcomes. AB - OBJECTIVE: To determine the strength of the association between antenatal psychosocial risk factors and adverse postpartum outcomes in the family, such as assault of women by their partner, child abuse, postpartum depression, marital dysfunction and physical illness. DATA SOURCES: MEDLINE, Cinahl, Famli, Psych Abstracts and the Oxford Database of Perinatal Trials were searched from relevant articles published from Jan. 1, 1980, to Dec. 31, 1993, with the use of MeSH terms "depression, involutional," "child abuse," "child neglect," "domestic violence," "family," "marital adjustment," "family health," "newborn health," "child health," "physical illness," "social support," "psychosocial risk," "prediction," "risk factors," "obstetrics" and "prenatal care." Further articles were identified from bibliographies. STUDY SELECTION: Of the 370 articles identified through the search, 118 were included for review. Studies were included if they examined the association between psychosocial risk factors and the outcomes of interest. Articles were excluded if they were reviews of poor quality or they had one or more of the following features: insufficient description of the sample, a high attrition rate, a lack of standardized outcome measures, outcomes other than the ones of interest or results that had already been reported in a previous study. DATA EXTRACTION: The strength of evidence of each study was evaluated. On the basis of the evidence, each risk factor was assigned a rating of the strength of its association with each of the postpartum outcomes. The ratings were class A (good evidence of association), class B (fair evidence) and class C (no clear evidence). Of the 129 antenatal psychosocial risk factors studied, 15 were found to have a class A association with at least one of the postpartum outcomes. DATA SYNTHESIS: Child abuse and abuse of the mother by her partner were most strongly correlated (class A evidence) with a history of lack of social support, recent life stressors, psychiatric disturbance in the mother and an unwanted pregnancy. Child abuse was also strongly associated with a history of childhood violence in the mother or her partner, previous child abuse by the mother's partner, a poor relationship between the mother and her parents, low self-esteem in the mother and lack of attendance at prenatal classes. Postpartum abuse of the mother was also associated with a history of abuse of the mother, prenatal care not started until the third trimester and alcohol or drug abuse by the mother or her partner (class A evidence). Child abuse had a fair (class B) association with poor marital adjustment or satisfaction, current or past abuse of the mother and alcohol or drug abuse by the mother or her partner. There was class B evidence supporting an association between abuse of the mother and poor marital adjustment, traditional sex-role expectations, a history of childhood violence in the mother or her partner and low self-esteem in the mother. Postpartum depression was most strongly associated with poor marital adjustment, recent life stressors, antepartum depression (class A evidence), but was also associated with lack of social support, abuse of the mother and a history of psychiatric disorder in the mother (class B evidence). Marital dysfunction was associated with poor marital adjustment before the birth and traditional sex-role expectations (class A evidence), and physical illness was correlated with recent life stressors (class B evidence). CONCLUSIONS: Psychosocial risk factors during the antenatal period may herald postpartum morbidity. Research is required to determine whether detection of these risk factors may lead to interventions that improve postpartum family outcomes. PMID- 8634959 TI - Legal and ethical issues in genetic testing and counseling for susceptibility to breast, ovarian and colon cancer. AB - The prediction of susceptibility to heritable breast, ovarian and colon cancer raises important legal and ethical concerns. Health care professionals have a duty to disclose sufficient information to enable patients to make informed decisions. They must also safeguard the confidentiality of patient data. These duties may come into conflict if a positive finding in one patient implies that family members are also at risk. A legal distinction is made between a breach of confidentiality and the legitimate sharing of information in a patient's interest or to prevent harm to a third party. Physicians also have a fiduciary duty to warn. Other issues concern the legal liability assumed by genetic counsellors, whose disclosures may influence decisions about childbearing, for example, and the risk of socioeconomic discrimination faced by people with a known genetic susceptibility. Traditional ethical orientations and principals may be applied to these and other questions, but feminist ethics will likely have particular importance in the development of an ethical stance toward testing and counseling for heritable breast and ovarian cancer. PMID- 8634961 TI - Reducing demand for physician visits through public education: a look at the pilot cold-and-flu campaign in London, Ontario. AB - OBJECTIVE: To estimate the effect of the Ontario Ministry of Health's pilot public-education campaign launched in London, Ont., on Jan. 15, 1994, to reduce the number of visits to physicians' offices because of cold and flu symptoms. DESIGN: Before-after comparison of claims to the Ontario Health Insurance Plan. OUTCOME MEASURES: Physician billings for visits because of cold and flu symptoms and total billings for all types of visits during the 2 months before and after the start of the campaign, and during the same two periods in the previous year, in London and in the rest of Ontario. RESULTS: By the time the campaign was started, much of the cold and flu season was already over for that winter. Still, the decrease in billings for visits because of cold and flu symptoms in the 2 months after the campaign was introduced was 6% greater in London than in the rest of Ontario. There was virtually no difference in total billings between London and the rest of the province during the same period. CONCLUSION: The modest relative reduction in physician billings for visits because of cold or flu symptoms in London following the introduction of the public-education campaign may have been due to the intervention as well as to other factors. PMID- 8634962 TI - Understanding the costs of asthma: the next step. PMID- 8634963 TI - A measured breath: new techniques in pulmonary imaging and diagnosis. AB - The development by Dr. Hans Pasterkamp and his team at the University of Manitoba, Winnipeg, of computer software for acoustic imaging of the chest originated in the need for a noninvasive, nonthreatening way to obtain information about lung function and lung disease in infants and children. Pasterkamp's team is developing a single computer program with potential applications in three areas: the measurement of lung sounds in addition to lung function, the multiple-site mapping of chest sounds to help identify the site of disease, and the assessment of upper airways, with potential use in the diagnosis of obstructive sleep apnea. Computer-assisted acoustic imaging promises to augment and enhance more traditional methods of pulmonary testing. PMID- 8634960 TI - Direct and indirect costs of asthma in Canada, 1990. AB - OBJECTIVE: To calculate the direct and indirect costs of asthma in Canada. DESIGN: Cost-of-illness study. SETTING: Canada. PATIENTS: All Canadians receiving inpatient or outpatient care for asthma in 1990. OUTCOME MEASURES: Direct costs incurred by inpatient care, emergency services, physician and nursing services, ambulance use, drugs and devices, outpatient diagnostic tests, research and education. Indirect costs from productivity loss due to absence from work, inability to to perform housekeeping activities, need to care for children with asthma who were absent from school, time spent travelling and waiting for medical care, and premature death from asthma. All costs are in 1990 Canadian dollars. RESULTS: Depending on assumptions, the total cost of asthma was estimated to be between $504 million and $648 million. Direct costs were $306 million. The single largest component of direct costs was the cost of drugs ($124 million). The largest component of indirect costs was illness-related disability ($76 million). CONCLUSIONS: Annual costs of treating asthma are comparable to the individual cost of infectious diseases, hematological diseases, congenital defects, perinatal illnesses, home care and ambulance services. Asthma costs may increase in the future, given current morbidity and mortality trends. Further evaluation of the effectiveness and cost-effectiveness of available asthma interventions in addition to aggregate cost data are required to determine whether resource allocation for the treatment of asthma can be improved. PMID- 8634964 TI - Goya's gratitude. PMID- 8634965 TI - When patient care is shared, who is the most responsible physician? AB - Ottawa lawyer Karen Capen examines the case of five Ontario physicians who faced charges of professional misconduct after a patient they cared for died in 1988. The investigation, which focused on the concept of "most responsible physician," serves as a cautionary tale for all doctors who share the care of a patient with colleagues. PMID- 8634966 TI - Letter to an ethicist: resuscitative interventions. AB - Few issues raise more questions for physicians than the resuscitation of seriously ill patients. In the following exchange of letters, Dr. John Quinlan discusses two difficult cases involving patient resuscitation, while Dr. William Cook responds by referring to the Joint Statement on Resuscitative Interventions that was approved last year by the CMA and several other health care organizations. PMID- 8634967 TI - In Italy, a medical degree often means unemployment or underemployment. AB - Italy's per capita concentration of physicians is the highest in the Western World, and many of them have to struggle to earn a living as they compete for a decreasing number of prestigious hospital positions. Many Italian doctors have more than one job, and some never find work in medicine. PMID- 8634969 TI - The ethics of eating a drug-company donut. AB - How does a physician make medical decisions? Is medical ethics a field that shifts according to the views of individual doctors? A physician who recently completed his residency muses on some of the ethical decision making he witnessed during his training. PMID- 8634968 TI - The lost generation: flood doors open as large numbers of Canadian FPs head south. AB - Newly trained physicians are leaving Canada in record numbers, a trend that is worrying medical educators and people who track physician migration. As American recruiters skim off many of our well-trained and cost-conscious young family physicians, concern is growing about whether a generation of young doctors will be lost to Canada, even though many underserviced areas need their skills. PMID- 8634970 TI - Telecare acting as an "electronic grandmother" for New Brunswickers. AB - A telephone information line is easing demand at the emergency departments of two hospitals in Moncton, NB. Although information lines aren't new, this program is different because nurses are using computer software that provides protocols for more than 900 areas of patient complaint. It helps them direct patients to the most appropriate care, which may range from treatment at home to an immediate visit to an emergency department. PMID- 8634971 TI - Canadian patient played key role in uncovering secrets about early-onset Alzheimer's disease. AB - Last June, the University of Toronto announced that Canadian scientists and a team of international researchers had discovered the gene responsible for most cases of early-onset Alzheimer's disease. One of the key players in that discovery had died just 3 months earlier. Frances Hodge, who participated in a battery of tests for the 20 years she lived with the disease, helped lead researchers to gene S182--and an ember of hope for future generations. PMID- 8634972 TI - Clean-air video will interest MDs who treat patients with allergies, respiratory problems. AB - Canada Mortgage and Housing Corporation wants physicians to help raise public awareness abut the importance of indoor air quality to health. Physicians across Canada are being sent a colourful poster that calls attention to indoor air pollution and ways to make a home healthier, included are order forms for the Clean Air Guide and a companion video, This Clean House. PMID- 8634973 TI - The sad and tragic life of Typhoid Mary. AB - As society grapples with contemporary moral questions raised by the barring of HIV-infected people from jobs and even crossing some national borders, it is probably useful to re-examine the case of Typhoid Mary. The case of Mary Mallon shows how an earlier age resolved the conflict that arises when society's right to protect itself from unnecessary exposure to disease impinges on the liberty of individual citizens. PMID- 8634974 TI - MedicAlert Foundation turns 35, issues warning to MDs about lookalike bracelets. AB - As it celebrates the 35th anniversary of its founding, the Canadian MedicAlert Foundation wants to educate patients about the importance of using genuine MedicAlert bracelets, or idents, that give physicians access to medical information in emergency situations through a toll-free hot line. The foundation also wants physicians to ensure that patients' medical records contain accurate and up-to-date information. PMID- 8634975 TI - Seeing medicine through opera glasses. AB - Few opera buffs stop to consider how the illnesses suffered by opera's great heroes and heroines mirrored the medical treatments and public attitudes of their time. A Toronto physician and his wife, an English professor and literary critic, have written a book exploring how opera presents illnesses such as tuberculosis and syphilis. PMID- 8634976 TI - Drawing the line on genetic intervention in humans. AB - Because the science of genetics can have such profound effects on medicine and mankind, society must define the characteristics of a moral framework within which to make decisions about genetic issues. University of Manitoba medical student Deepak Kaura, who claimed third prize in CMAJ's 1995 Logie Medical Ethics Essay Contest, examines the ethics of genetic intervention in humans. PMID- 8634977 TI - As number of patients grows, so do critical problems facing MDs who provide dialysis. AB - The growth in the number of dialysis patients in Canada is causing a critical shortage of resources. The low transplantation rate and an increase in the number of patients, combined with insufficient funding to expand facilities, threatens access to the expensive but life-sustaining therapy. Michel Martin, who recently underwent a kidney transplant, looks at the situation facing dialysis patients in Quebec and Ontario. PMID- 8634978 TI - Proceedings of the 5th National Conference on Gynecologic Cancers of the American Cancer Society. Washington, DC, April 6-8, 1995. PMID- 8634979 TI - The economics of cancer care. AB - Considerable attention has been paid to the high cost of cancer care. These medical services are expensive, accounting for 10% of total health care expenditures. Factors contributing to high costs include (1) the likelihood that anticancer treatments will lead to costly medical complications: (2) intensive research and development necessary to ensure rapid introduction of a broad array of treatment options; and (3) specialized facilities required for delivery of care. Such efforts as rationing of care and utilizing practice guidelines have been ineffective in controlling costs. To realize savings, we must develop new therapies with sufficient specificity so that anticancer interventions do not impair the patient's general health. PMID- 8634980 TI - The epidemiology of cervical carcinogenesis. AB - Epidemiologic and laboratory data suggest that cervical cancer typically arises from a series of causal steps. Each step can be studied separately in the hope of better etiologic understanding and improved cancer prevention. The earliest identified etiologic step is infection of young women with specific types of venereally transmissible human papillomaviruses (HPVs). Cervical HPV infections often lead to low grade squamous intraepithelial lesions (mildly abnormal Pap smears). Human papillomavirus infections and their associated lesions are extremely common among young, sexually active women. The infections typically resolve spontaneously even at the molecular level within months to a few years. Uncommonly, HPV infections and/or low grade lesions persist and progress to high grade lesions. The risk factors for progression are mainly unknown but include HPV type and intensity, cell-mediated immunity, and reproductive factors. Nutritional factors or co-infection with other pathogens may also be involved at this apparently critical etiologic step between common low grade and uncommon high grade intraepithelial lesions. Except for advancing age, no epidemiologic risk factors have been found for the next step between high grade intraepithelial lesions and invasive cancer. At the molecular level, invasion is associated with integration of viral DNA. Based on worldwide research, the steps in cervical carcinogenesis appear to be fundamentally the same everywhere, with a central role for HPV infection. The importance of etiologic cofactors like smoking, however, may vary by region. PMID- 8634981 TI - Molecular biology of cervical cancer and its precursors. AB - Cervical cancer develops from well-defined precursor lesions referred to as either cervical intraepithelial neoplasia or squamous intraepithelial lesions. It is now known that specific types of human papillomaviruses (HPV) are the principal etiologic agents for both cervical cancer and its precursors. The high oncogenic-risk HPV types associated with invasive cervical cancer produce two oncoproteins, designated E6 and E7, which interact with endogenous cell cycle regulatory proteins, including p53 and Rb. The interaction of virally derived and endogenous cellular proteins converges in deregulation of cell cycle progression and appears to be critical for the development of cervical cancers. However, the development of cervical cancer is a multistep process that cannot be explained simply by infection with specific types of HPV. One additional event that appears to play a role in tumor progression is integration of HPV DNA into the host genome. Integration of HPV DNA frequently disrupts the E2 open reading frames, resulting in overexpression of the E6 and E7 oncoproteins and possibly causing genomic instability. Additional cofactors and mutational events may be important in the pathogenesis of invasive cervical cancers and may include chromosomal rearrangements, loss of constitutional heterozygosity, and proto-oncogene activation. PMID- 8634982 TI - Impact of the Bethesda System. AB - BACKGROUND: Although the Bethesda System provides concise, specific descriptions for classifying cervical cytology, its usefulness has been questioned in the United States and abroad. The author attempted to evaluate the impact of the Bethesda System on cervical screening and subsequent management decisions. METHODS: A MedLine search provided statistical information for comparison of screening procedures and results before and since introduction of the Bethesda System. The effect of the Bethesda System was evaluated by specimen adequacy, inclusion of human papillomavirus changes in the squamous intraepithelial lesion (SIL) category, and effect on the number of abnormal Pap smears. RESULTS: By 1992, 85% of cytology laboratories were using Bethesda system terminology. Diagnosis and treatment of patients with atypical squamous cells of undetermined significance (ASCUS) and low grade squamous intraepithelial lesions were reviewed. In women with ASCUS smears, follow-up studies showed that between 5% and 13% actually had high grade cervical lesions. For patients with a cytologic diagnosis of low grade SIL, one study found an 18.6% incidence of high grade lesions demonstrated by colposcopically directed cervical biopsy. An estimated 2% increase has occurred in the number of women with abnormal Pap smear results who require additional evaluation costing more than $1 billion. CONCLUSIONS: The Bethesda System has improved the quality of Pap smear diagnosis in the United States, but the number of women with abnormal Pap smear results has increased. Although this increase is partly due to the terminology of the Bethesda System, other factors play a role. Whether this has improved cancer prevention is unknown. PMID- 8634983 TI - Screening. The next century. AB - BACKGROUND: Screening for cervical cancer and its precursors has traditionally been performed by Papanicolaou smear. As cost considerations have become more important and as developing countries have begun to initiate screening programs, other approaches to screening have been considered. METHODS: Proposed new methods potentially useful for screening for cervical cancer and its precursors were reviewed. RESULTS: Cervical cancer screening using unaided or aided visual approaches, human papillomavirus DNA testing and typing, cervical photography, and automated Pap smear screening instruments are all potentially valuable when used alone or in combination, depending on the country. CONCLUSIONS: New techniques will make it possible to screen high risk, low-income populations for cervical cancer and its precursors and to do so more cost-effectively than is possible using conventional Western approaches. In addition, the latest technology can enhance the screening methods traditionally used in industrialized countries and can increase the positive and negative predictive value of the screening method. PMID- 8634984 TI - Management of patients with high grade squamous intraepithelial lesions. AB - BACKGROUND: The advent of modern electrosurgical equipment has allowed for outpatient excisional procedures rather than ablative therapy for patients with high grade squamous intraepithelial lesions (HGSILs). METHODS: The advantages and disadvantages of the commonly used ablative and excisional techniques for treatment of HGSILs were reviewed. RESULTS: Electrofulguration or cryotherapy for HGSILs smaller than 2.5 cm results in a 90% cure rate after a single treatment, but the cure rate drops to 50% for treatment of large HGSILs. Carbon dioxide laser therapy has resulted in high cure rates, but the necessary equipment is expensive. Excisional techniques for HGSILs of any size and distribution provide for a greater than 90% cure rate after a single session. Complication rates arising from use of excisional techniques vary from 5% to 20%, depending on the method used; ablative techniques have a uniformly low complication rate (2%). The risk of inadvertently treating underdiagnosed invasive cancer is reduced with excisional but not with ablative methods. Of the excisional methods used to treat HGSILs, the loop electrosurgical excision procedure (LEEP) appears to be the most cost-effective. CONCLUSIONS: The use of ablative techniques for small ectocervical HGSILs may be appropriate provided invasive cancer has been ruled out and the patient can be followed up long term. Loop electrosurgical excision procedure is the preferred excisional technique for lesions larger than 2.5 cm. The "see-and-treat" approach used with LEEP must be limited to only those lesions that are unequivocally verified by an expert in colposcopy and cytology. In questionable cases, the disease should be histologically ascertained by endocervical curettage and punch biopsies before therapy is undertaken. PMID- 8634985 TI - Patterns of care for invasive cervical cancer. Results of a national survey of 1984 and 1990. AB - BACKGROUND: The American College of Surgeons conducted a national patient care and evaluation study of invasive cervical cancer to measure any changes in patterns of care for the years 1984 and 1990. METHODS: Hospitals with cancer programs were invited to submit data on up to 25 consecutive patients with newly diagnosed invasive cervical cancer for each of the two study years. Data were obtained from 684 hospitals on 5904 patients diagnosed in 1984 and from 700 hospitals on 5817 patients diagnosed in 1990. A long term study of patients diagnosed in 1984 was compared with a short term study of patients diagnosed in 1990. Survival data were described only for patients diagnosed in 1984. RESULTS: Of a total of 11,721 patients, 59.4% were diagnosed and treated at the reporting institution in 1984 and 54.8% in 1990. The remaining patients were referred for treatment after diagnosis elsewhere. The diagnosis was established by cervical biopsy for 69.8% of patients, by conization alone for 9.3%, and by both procedures for 11.8%. The histopathologic diagnoses were squamous cell carcinoma (79.8%), adenocarcinoma (15.8%), and other (4.4%). The stage distributions were as follows: IA, 15.9%; IB, 36.8%; IIA, 8.2%; IIB, 15.5%; IIIA, 2.5%; IIIB, 13.3%; IVA, 2.6%; and IVB, 5.2%. The stage was listed as unknown for 20.3% of patients. Patients were treated with surgery alone (29.2%), radiation alone (40.7%), chemotherapy alone (0.7%), or combination therapy (21.5%), and 7.9% received no treatment at the reporting institution. The overall survival for patients diagnosed in 1984 was 68.3%. Survival by stage in this group was as follows: IA, 93.7%; IB, 80.0%; IIA, 67.2%; IIB, 64.7%; III, 37.9%; and IV, 11.3%. CONCLUSIONS: These data indicate that invasive cervical cancer is highly curable when diagnosed early. During the 5-year period, stage distributions were similar, the use of extended hysterectomy increased, and gynecologic oncologists were more often the primary surgeons. The use of radiation alone decreased. PMID- 8634986 TI - Is there really a difference in survival of women with squamous cell carcinoma, adenocarcinoma, and adenosquamous cell carcinoma of the cervix? AB - BACKGROUND: The authors' aim was to assess whether there is a difference in biologic behavior and survival in comparing adenocarcinoma (AdCA), squamous cell carcinoma (SCC), and adenosquamous carcinoma (Ad/SC) of the cervix. METHODS: Cancer registrars at 703 hospitals submitted anonymous data on 11,157 patients with cervical cancer diagnosed and/or treated in 1984 and 1990 for a Patient Care Evaluation Study of the American College of Surgeons. Among these patients, 9351 (83.8%) had SCC; 1405 (12.6%), AdCA; and 401 (3.6%), Ad/SC cancers. There were no significant changes in percentages of the different histologic types between the study years 1984 and 1990, nor was the patient distribution different regarding age, race/ethnicity, and socioeconomic background for each histologic group. Furthermore, the distribution of patients who had had a hysterectomy did not change between 1984 and 1990. RESULTS: A larger percent of patients with SCC (63.8%) than those with Ad/SC (59.8%) or AdCA (50.2%) had tumors larger than 3 cm at greatest dimension. Early stage patients (IA, IB, IIA) often were treated by hysterectomy alone (45.5%) or combined with radiation (21.1%). The remaining patients (21.9%) received radiation alone. Of the patients with clinical stage I disease, 7.6% of Ad/CA patients, 15.5% of Ad/SC patients and 12.6% of SCC patients had positive nodes. Although patients with SCC had higher survival rates for all four clinical stages (I-IV), the differences were only significant for Stage II patients. Patients with clinical stage IB SCC and AdCA treated by surgery alone were found to have significantly better survival rates (93.1% and 94.6% at 5 years, respectively) than women treated by either radiation alone or a combination of surgery and radiation (P < 0.001, both histologic comparisons). For women with Ad/SC tumors, however, the 5-year survival rate was 87.3% for those receiving combined treatment compared with those receiving surgery alone (69.2%) or radiation alone (79.2%). However, these survival curves were not significantly different (P = 0.496). One hundred six patients with positive nodes were available for analysis. The 5-year survival rate of patients with SCC and positive nodes was 76.1%. Surprisingly, patients with Ad/SC and positive nodes had the highest 5-year survival rate (85.7%), whereas, women with AdCA and positive nodes had a sharply reduced 5-year survival rate (33.3%). The curves were significantly different (P < 0.01). For patients with clinical stage I, the risk factors for age, tumor size, nodal status, histologic features, and treatment were analyzed with Cox's multivariate regression. In this analysis, subset IB, greater tumor size, age 80 or older, and positive nodal status were each independently significant for poorer survival. Patients who were treated by surgery alone had a significantly better survival than patients who had other types of treatment or no treatment. Histologic characteristics had no significant effect on survival. In the analysis of patients with pathologic stage I disease, those with SCC had significantly poorer survival and those with Ad/SC had significantly better survival than patients with Ad/CA. Positive nodes had no significant independent effect on survival. In another analysis, tissue type was not found to be an important factor in recurrence time. CONCLUSIONS: 1. Ad/CA and Ad/SC tumors were found to represent 12.6% and 3.6%, respectively, of a large series (N = 11,157) of cervical cancers diagnosed in 1984 and 1990 and reported to the Commission on Cancer of the American College of Surgeons. 2. Two thirds of women with early clinical stage disease (IA, IB, IIA) had hysterectomy as all or part of their primary therapy. 3. No significant differences were found in 5-year survival among the three tissue types in any clinical stage except American Joint Committee on Cancer stage II. PMID- 8634987 TI - Chemoprevention trials and surrogate end point biomarkers in the cervix. AB - Cervical cancer is the second most common malignancy in women worldwide and remains a significant health problem for women, especially minority and underserved women. Despite an understanding of the epidemiologic risks, the screening Papanicolaou smear, and morbid and costly treatment, overall survival remains 40%. New strategies, based on the clinical and molecular aspects of cervical carcinogenesis, are desperately needed. Chemoprevention refers to the use of chemical agents to prevent or delay the development of cancer in healthy populations. Chemoprevention studies have several unique features that distinguish them from classic chemotherapeutic trials; these features touch on several disciplines and weave knowledge of the biology of carcinogenesis into the trial design. In the design of chemoprevention trials, four factors are important: high risk cohorts must be identified; suitable medications must be selected; study designs should include Phases I, II, and III; and studies should include the use of surrogate end point biomarkers. Surrogate end point biomarkers are sought because the cancer develops over a long period of time, and studies of chemopreventives would require a huge number of subjects followed for many years. Surrogate end point biomarkers serve as alternative end points for examination of the efficacy of chemopreventives in tissue. High risk cohorts include women with cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL). Nutritional studies have helped define micronutrients of interest (folate, carotenoids, vitamin C, vitamin E). Other medications of interest include retinoids (4-hydroxyphenylretinamide [4-HPR], retinyl acetate gel, topical all trans-retinoic acid), polyamine synthesis inhibitors (alpha difluoromethylornithine [DFMO]), and nonsteroidal anti-inflammatory drugs (ibuprofen). Phase I chemoprevention studies of the cervix have tested retinyl acetate gel and all-trans-retinoic acid. Phase II trials of all-trans-retinoic acid, beta-carotene, and folic acid have been and are being carried out, whereas Phase III trials of all-trans-retinoic acid have been completed and have shown significant regression of CIN 2 but not CIN 3. Phase I studies of DFMO and Phase II studies of DFMO and 4-HPR are underway. Surrogate end point biomarkers under study include (1) quantitative cytology and histopathology; (2) human papillomavirus type testing; (3) biologic measures of proliferation, regulation, differentiation, and genomic instability; and 4) fluorescence spectroscopic emission. Clinical trials with biologic end points will contribute to our understanding of the neoplastic process and hence aid us in developing new preventive and therapeutic strategies. PMID- 8634988 TI - Prognostic factors of early stage cervical cancer treated by radical hysterectomy. AB - BACKGROUND: This study was performed to identify pathologic and clinical features that best correlate with lymph node metastasis and disease free survival among patients with Stage I and II cervical cancer treated by radical hysterectomy. METHODS: Three hundred-seventy patients with complete clinical information and pathologic material, including cone and cervical biopsies, were selected for analysis. Of these patients, 301 with clinical stages I and II disease were the subject of this paper. The results of patients with microinvasive carcinoma of the cervix, as defined by the Society of Gynecologic Oncologists (depth of invasion < or = 3 mm and no lymph node vascular space invasion), were reported previously and excluded from this analysis. Patients with small cell carcinoma of the cervix were found to have a very poor prognosis (disease free 5-year survival of 36%) and were also excluded from this analysis (Sevin BU, Nadji M, Metkoch MW, Lu Y, Averette HE. Unpublished data, 1995). Variables studied were patient age, weight, race, marital status, and economic status; tumor size; depth of invasion; lymph node-vascular space involvement; cell type; tumor grade; lymph node metastasis; and number of lymph nodes removed. The influence of these variables on survival was examined by univariate analysis with use of Cox's regression model and the log rank test for comparison of survival curves. RESULTS: Factors that predict disease free survival, ranked by degree of significance, were depth of invasion, tumor size, lymph node-vascular space invasion, number of positive nodes, tumor volume, clinical stage, and tumor extension to the vagina or surgical margins. CONCLUSIONS: Radical hysterectomy and bilateral lymphadenectomy is standard therapy for patients with Stage IB and IIA carcinoma of the cervix. A variety of surgically defined risk factors predict 5-year disease free survival, and many of these factors are related. Identification of independent risk factors requires a multivariate analysis of data. PMID- 8634989 TI - Operative laparoscopy and the gynecologic oncologist. Commentary and review. AB - BACKGROUND: A rapid evolution in technology and surgical applications of endoscopy have occurred over the past 5 years. Surgical procedures once thought impossible except through large abdominal incisions are being performed with the use of laparoscopic surgical techniques. Laparoendoscopic techniques have limitations as well as advantages over conventional surgical approaches. METHODS: The medical literature as it relates to laparoscopy and gynecologic oncology was reviewed. RESULTS: Procedures performed through the laparoscope include total hysterectomies, bilateral oophorectomies, pelvic and periaortic lymphadenectomies, omentectomies, colostomies, bowel resections, oophoropexies, and pelvic lid constructions as well as radical hysterectomies and ovarian cancer debulking procedures. These techniques are gaining popularity among gynecologic oncologists, and studies of individual case reports have been followed by studies involving a series of patients. Numerous limiting factors exist, however, foremost among these being the wide variability of endoscopic skills among surgeons and lack of objective long term data supporting the efficacy and safety of these techniques. CONCLUSION: Application of endoscopic techniques in gynecologic oncology procedures is occurring rapidly and is driven partly by market economy forces. Many gynecologic oncologists, however, do not have the necessary endoscopic skills and experience with which to perform such procedures. For these physicians to remain sufficiently qualified, fellowship training programs must encompass formal training curricula in endoscopic surgery, and such programs should often include the faculty as well. Formal and organized credentialing of laparoscopic cancer surgical expertise will ensure a minimum safe level of skills. PMID- 8634991 TI - Familial ovarian cancer. Update and clinical applications. AB - BACKGROUND: Genetics plays a role in all cancers. Evidence exists for the presence of inherited genes associated with the development of ovarian cancer in three familial ovarian cancer syndromes: a site-specific ovarian cancer syndrome, a breast/ovarian cancer syndrome, and an ovarian cancer syndrome associated with hereditary nonpolyposis colorectal cancer. METHODS AND RESULTS: The authors present an updated summary of recent advances within the field of ovarian cancer genetics and examine the extent to which this genetic information, at both an epidemiologic and molecular level, may be used to identify a subset of women who are likely to be at increased risk of developing ovarian cancer. In addition, the extent to which these data may be used to define methods of prevention or treatment for women at risk is discussed. CONCLUSION: Women who are members of high risk ovarian cancer families should receive genetic screening and medical follow-up in an effort to reduce their overall chances of morbidity and death associated with the development of ovarian and other cancers. The construction of cancer family registries will help to identify women at risk and facilitate their entry into clinical trials and screening programs for ovarian cancer. PMID- 8634990 TI - The molecular basis of ovarian cancer. AB - BACKGROUND: Molecular genetic studies of ovarian cancer have been limited by the inaccessible location of the ovary, the advanced stage of tumors available for analysis, and the lack of a well-defined precursor lesion. However, genetic alterations important in ovarian tumorigenesis have been identified recently. METHODS: Molecular genetic evaluation of ovarian cancer primarily has utilized mutation analysis, immunohistochemical techniques, and loss of heterozygosity (LOH) studies. RESULTS: Overexpression of the HER-2/neu oncogene is present in approximately one third of ovarian cancers and is associated with poor prognosis. Mutations of the K-ras oncogene have been identified in a similar proportion of mucinous ovarian tumors, including borderline tumors. The study authors as well as others have frequently detected LOH on chromosome 17, including the p53 and BRCA1 loci, and at 17p3.3 and 1717q22-23. Genetic linkage analysis indicates that the majority of inherited ovarian cancers are caused by mutations in the BRCA1 gene. Mutations in mismatch repair genes have been identified in ovarian cancers that occur as part of the hereditary nonpolyposis colon cancer syndrome. CONCLUSIONS: Sporadic ovarian tumors are the end result of a complex pathway involving multiple oncogenes and tumor suppressor genes, including HER-2/neu, K ras, p53, BRCA1, and additional tumor suppressor genes on chromosome 17. The majority of inherited ovarian cancers are due to mutations in the BRCA1 gene, which appears to be a tumor suppressor gene. It is hoped that an increased understanding of the molecular basis of ovarian cancer will lead to advances in prevention, diagnosis, and treatment. PMID- 8634992 TI - Toward an optimal algorithm for ovarian cancer screening with longitudinal tumor markers. AB - Stored samples from women in the Stockholm screening study were reassayed for CA125II (Centocor, Malvern, PA) and OVX1. The postmenopausal women older than age 50 without ovarian cancer were randomly split into a training set to develop a screening test based on longitudinal marker levels and a second set to validate the test. The CA125II data from each woman is summarized by the slope and intercept from a linear regression of log(CA125II) on time since first sample. The slope versus the intercept for the training set and the ovarian cancer cases formed a bivariate scatter plot. A curve was drawn on the scatter plot that separated most of the women with ovarian cancer from all other women; it delineated a screening test. The specificity of this test was examined on the validation set with a specificity of 99.8%. Bayes' theorem was used to calculate the risk of ovarian cancer (ROC) based on the intercept, slope, and assay variability. It is important to account for assay variability because it can produce large slopes over short periods of time. The maximum risk, which identified 83% (5 of 6) of the ovarian cancers detected within a year of last assay, was applied as a test to the training set and confirmed a high specificity of 99.7%. With this specificity and sensitivity, the ROC algorithm using the CA125II assay has an estimated positive predictive value of 16%, substantially greater than the positive predictive value based on a single assay. Further study is planned to confirm the sensitivity of this approach. PMID- 8634993 TI - Ovarian cancer screening. The role of ultrasound in early detection. AB - BACKGROUND: Because of the urgent need for effective techniques with which to detect organ-confined curable ovarian cancers, efforts have been focused on early detection. Current ovarian cancer screening trials have been hampered by deficiencies in our knowledge of the molecular and biologic events leading to ovarian tumorigenesis. The lack of early ovarian cancer symptoms and the intraperitoneal location of the ovaries contribute to the dilemma of early diagnosis. METHODS: Real-time ultrasound screening is aimed at detecting the earliest possible architectural alterations in the ovary indicative of neoplastic growth. Color Doppler imaging detects early alterations in ovarian blood flow that accompany tumorigenesis. RESULTS: To be effective, these modalities must diagnose asymptomatic curable Stage I ovarian cancers and improve ovarian disease specific survival. Because of the relatively low prevalence of ovarian cancer in the general population, investigators have targeted women at increased risk of ovarian cancer either based on their increasing age or their family histories of cancer. Some of these studies have been underway since the late 1980s and have already demonstrated the potential usefulness and limitations of current sonographic techniques used for screening. CONCLUSIONS: Ultrasound screening for ovarian cancer has not demonstrated adequate sensitivity and specificity, thus it is not used widely outside of the clinical trial setting. Current clinical guidelines for ovarian cancer screening as well as exploratory methods for use in early ovarian cancer detection are presented. PMID- 8634994 TI - Monoclonal antibodies used in the detection and treatment of epithelial ovarian cancer. AB - Conventional therapy for epithelial ovarian cancer, including aggressive cytoreductive surgery followed by combination chemotherapy regimens, has failed to reduce the number of deaths caused by this disease, which remains the most lethal of gynecologic malignancies. Monoclonal antibodies, which offer the promise of high selectivity for detection and therapy, may be targeted to tumor associated antigens, growth factors, receptors, or oncogenes. They may be used alone as immunotherapeutic agents or conjugated to chemotherapeutic drugs, toxins, or radionuclides. Radioimmunoconjugates may also be used for preoperative or intraoperative tumor localization. The authors focused on the clinical utility, technical limitations, and potential of monoclonal antibodies in the detection and treatment of epithelial ovarian cancer with emphasis on radioimmunodetection and radioimmunotherapy. PMID- 8634995 TI - Promising new therapies in the treatment of advanced ovarian cancer. AB - Advanced stage ovarian cancer is the most lethal gynecologic cancer. Despite initial response rates of 60-80% with platinum-based chemotherapy, more than 75% of women with this malignancy die of complications associated with this disease. There is a pressing need to find new chemotherapeutic agents for patients with advanced ovarian cancer. Phase II studies have identified paclitaxel as the most active drug in ovarian cancer since the introduction of cisplatin in the 1970s. Phase III studies will define the role of paclitaxel as initial therapy. Camptothecins (topotecan, CPT-11, 9-amino-camptothecin) inhibit topoisomerase I. CPT-11 and topotecan have shown activity in Phase II trials. Gemcitabine, a pyrimidine antimetabolite, has shown activity in Phase II trials. Other promising drugs (docetaxel, treosulfan) are under investigation. Modulation of drug resistance is being explored in Phase I/II studies. Clinical trials have been initiated with buthionine-sulfoximine, an inhibitor of glutathione biosynthesis, which decreases the ability of resistant cells to inactivate platinum compounds and alkylating agents. Cyclosporin has been shown to increase cisplatin cytotoxicity. Phase I trials have demonstrated the feasibility of combining cyclosporin and cisplatin. Phase II trials of cyclosporin analogs (PSC 833) and paclitaxel in refractory ovarian cancer are ongoing. Promising leads in drug development should provide new therapies for patients with ovarian cancer. Further research in the modulation of drug resistance may identify new mechanisms or strategies with which to prevent the emergence of drug resistance. PMID- 8634996 TI - Molecular basis of endometrial cancer. AB - BACKGROUND: Most human cancers are thought to arise from alterations in oncogenes and tumor suppressor genes. METHODS: Molecular techniques have been used to identify specific genetic alterations in endometrial cancers. RESULTS: Overexpression of the HER-2/neu oncogene occurs in 10% of endometrial cancers and correlates with poor survival. Alterations in other receptor tyrosine kinases (c fms and epidermal growth factor receptor) also occur in some cases. The c-myc oncogene, which encodes a nuclear transcription factor, also may be overexpressed in some invasive cancers. Mutations in the K-ras oncogene occur in 10% and in 20 30% of American and Japanese endometrial cancers, respectively. K-ras mutations also have been observed in endometrial hyperplasias, and this may represent an early event in the development of some cancers. Mutation of the p53 tumor suppressor gene, with resultant overexpression of mutant p53 protein, occurs in 20% of endometrial adenocarcinomas. Overexpression of p53 is associated with advanced stage and poor survival. Because p53 mutations do not occur frequently in endometrial hyperplasias, this may be a relatively late event in endometrial carcinogenesis. Recent studies have shown that mutations occur in microsatellite sequences in some endometrial cancers. Because microsatellite instability in hereditary nonpolyposis colon cancer has been found to be caused by mutations in DNA repair genes, similar mutations are being sought in endometrial cancers. CONCLUSIONS: Although several molecular alterations have been identified, the molecular pathogenesis of endometrial cancer remains poorly understood. PMID- 8634997 TI - Preoperative evaluation and staging of endometrial cancer. AB - Preoperative examination of a patient for whom a diagnosis of endometrial cancer has been made by office biopsy or dilatation and curettage includes careful history taking and physical examination, with emphasis on the pelvic examination, blood tests, and imaging evaluations of the pelvis, abdomen and chest, and other specific studies related to medical operability. The primary approach to endometrial cancer is surgery, which has been used for staging and treatment since the adoption of the Federation of Obstetrics and Gynecology system in 1988. Pilot studies and the Gynecologic Oncology Group have researched this system extensively and have emphasized the findings that could be defined only by pathologic study of the uterus, adnexa, retroperitoneal lymph nodes, and peritoneal cytologic findings. Preoperative endocervical evaluation is no longer necessary unless gross invasion of the cervix is suspected. However, initial histologic findings can identify patients at high risk, that is, those with high grade adenocarcinomas, clear cell carcinomas, adenosquamous carcinomas, and papillary serous adenocarcinomas. Intraoperative pathologic evaluation of the uterus by frozen section, which reveals depth of invasion into the myometrium and correlation with tumor grade, can identify patients for whom lymph node sampling should be performed. PMID- 8634998 TI - Endometrial cancer. Management of high risk and recurrence including the tamoxifen controversy. AB - Risk factors for endometrial cancer include obesity, nulligravidity, late menopause, and anovulatory states. Although diabetes is highly associated with endometrial cancer, hypertension is not an independent variable when correction is made for other factors. Exogenous estrogen increases the risk by at least four times, and smoking is a significant factor. Screening of asymptomatic women may be useful among high risk patients. In addition, racial influence on virulence has recently been identified. Most recurrences of endometrial cancer are identified within 3 years of initial diagnosis. Predictors include ploidy, histologic grade, histologic type, receptor status, and stage. Treatment of recurrence is individualized based on tumor location and receptor status and may involve surgery, radiation therapy, hormonal therapy, or cytotoxic chemotherapy. Tamoxifen has been shown to improve survival among subsets of patients with breast cancer in all stages. A comprehensive literature review and meta-analyses, however, verified an increased risk of endometrial cancer among tamoxifen-treated patients compared with control subjects that may equal the cancer risk from exogenous estrogen exposure. Screening techniques include sonographic assessment of endometrial thickening and vascular patterns, hysteroscopy, and endometrial sampling. A subendometrial cystic proliferation can confuse radiographic evaluation of endometrium, leading to unindicated curettage. A disproportionate incidence of high grade lesions has been reported; however, tamoxifen should not be withheld from patients with breast cancer. PMID- 8634999 TI - Nutritional aspects of breast cancer. AB - Nutrition, in its broadest sense, plays a clear role in breast cancer, identified through its relationship to such known risk factors as rate of growth and development, age at menarche, adult height, postmenopausal body weight, and fat distribution. The relative importance of daily diet is hotly debated. There is no agreement on the relative importance of nutrition's effect, the responsible food groups and nutrients, nor the age or ages at which it can be influential. Evidence linking breast cancer and diet comes from human ecologic and observational studies as well as a body of experimental feeding studies in rats and mice. The population studies, including increasing numbers of migrant studies, and the animal experiments suggest that high-fat diets promote breast cancer, particularly among older women. Studies that compare diets of persons with and without breast cancer and those that record breast cancer incidence among women with different dietary histories have been inconclusive enough as to warrant randomized intervention trials. Recognition of differences between the risk factors for premenopausal and postmenopausal women and increasing insight into the ages at which known risk factors exert their influence have helped in the design of these trials. Human feeding studies and the trials themselves are identifying metabolic changes and their biomarkers as well as biomarkers for changes in fat and nutrient intakes. The Women's Health Initiative Dietary Modification Trial was designed to show whether a sustained (9-year) low-fat, high-fruit-and-vegetable eating pattern will lower the incidence of breast cancer among women who will be between ages 59 and 88 when the trial ends, the time of peak incidence. Meanwhile, the Women's Intervention Nutrition Study will learn in approximately 5 years whether a low-fat, adjuvant eating pattern will improve the prognoses of postmenopausal women with early and moderate stage breast cancer and whether tamoxifen should be used as at least part of their treatment. The results of these trials will have important applications for both public health and the provision of medical care. PMID- 8635000 TI - Hormones and breast cancer. AB - The exact nature of the association between hormones and the development of breast cancer remains uncertain. Studies of endogenous hormone levels and breast cancer suggest a possible role of estrogens and androgens in the cause of breast cancer. Numerous studies have been conducted to assess the risk of breast cancer among women exposed to exogenous hormones. Several studies of women who have used oral contraceptives have shown a twofold increase in risk for the onset of breast cancer at an early age associated with 10 years of oral contraceptive use. One of the largest studies, the Cancer and Steroid Hormone Study, found no association between breast cancer and oral contraceptive use for women up to the age of 54. A meta-analysis combining the results of 31 published studies of the association between hormone replacement therapy and breast cancer revealed no increased risk of breast cancer associated with ever-use (risk ratio [RR], 1.02; 95% confidence interval [CI], 0.93-1.12). Use of oral contraceptives for more than 10 years was associated with a small increased risk (RR, 1.23; 95% CI, 1.08-1.40), but few studies have examined very long term use. A recent study of pregnancy and breast cancer outcome found no adverse influence of pregnancy shortly before or after diagnosis of breast cancer on prognosis. The results of a wide range of studies should be considered when weighing the benefits and risks of hormone use among women at increased risk of breast cancer or with a history of breast cancer. PMID- 8635001 TI - Controversies in breast cancer screening. AB - Because breast cancer is the most common cancer occurring in women in the United States, early detection of breast cancer through screening mammography, physician clinical examination, and breast self-examination has been recommended. However, despite admonitions to physicians and patients to be aggressive in their screening efforts, there has been continued controversy regarding appropriate guidelines for screening. In December 1993, the National Cancer Institute announced that it could no longer recommend routine mammographic screening for women age 40-49 years, which it had previously supported along with other medical organizations. This change in policy, along with data from the Canadian National Breast Screening Study showing an increased mortality rate for screened women, created confusion for physicians and patients alike. The controversy about screening guidelines has created many practical concerns for the physicians involved in the primary health care of women. In the current paper, the author discussed the development of screening guidelines and the current recommendations of various medical organizations and reviewed the data from studies supporting and challenging the current guidelines, with a focus on screening guidelines for women age 40-49 and the elderly. Recommendations are made for physicians on how to communicate with patients regarding screening controversies. PMID- 8635002 TI - Reconstruction after mastectomy. AB - Although reconstruction after mastectomy offers an opportunity for cosmetic rehabilitation that should make mastectomy more acceptable and contribute to overall rehabilitation, the procedure is relatively underutilized. The best cosmetic results usually come from breast conservation rather than from mastectomy and subsequent reconstruction, and most small (T0-T2) cancers can be treated by means of breast-conserving measures. The surgeon who is performing the mastectomy plays a key role in explaining reconstruction to the patient and encouraging her to consider the process. Surgeon- and patient-related factors contribute to under-utilization. Physician assessment of the results of reconstruction, particularly with use of implants, tends to be less favorable than that of the patient. Surgeons may overemphasize the inadequacies of the results and patients may be overwhelmed by the diagnosis and array of decisions that must be made. Immediate reconstruction poses little risk of treatment delay or limitation. Reconstruction after mastectomy does not interfere with follow-up for recurrence. Choices for reconstruction have been limited by the withdrawal of silicone implants from the market. The availability of reconstruction has encouraged the inappropriate use of mastectomy for low risk disease. Prophylactic mastectomies and reconstruction should be performed for appropriate indications. To be effective, prophylactic mastectomy must include the nipple areolar complex. The availability of genetic testing to define very high risk groups brings into question the adequacy of protection offered by this procedure. Whereas prophylaxis in humans for premalignant mastopathy appears to be nearly complete, mastectomy appears to offer little protection in a rodent carcinogen model. The effectiveness of mastectomy for prophylaxis in a genetically high risk human population is unknown. PMID- 8635003 TI - Replacement therapy for breast cancer survivors. A pilot study. AB - BACKGROUND: Traditionally, breast cancer survivors were not considered as candidates for hormone replacement therapy (HRT) because of the possibility that an occult metastatic site of disease might be activated, thus negatively influencing the outcome for the patient. METHODS: A retrospective review of 77 breast cancer survivors who have taken HRT was conducted. RESULTS: Seven recurrences were reported among the 77 patients studied in-depth, with correlations to stage, age, and node and receptor status. There have been no recurrences among the 33 additional patients who were placed on the study after the completion of this analysis. CONCLUSIONS: No significant adverse outcome was detected in this group of breast cancer survivors receiving HRT. Given the established benefits of HRT, a reappraisal of this subject is necessary, and a prospective randomized trial is essential. PMID- 8635004 TI - Gestational trophoblastic disease. AB - BACKGROUND: Gestational trophoblastic disease consists of a group of interrelated diseases, including molar pregnancy, placental site trophoblastic tumor, and choriocarcinoma. METHODS: Advances in the diagnosis and management of gestational trophoblastic diseases over the past 5 years were reviewed. RESULTS: Molar pregnancy is now categorized as complete or partial on the basis of gross and microscopic histopathologic and karyotypic findings. Early detection of persistent gestational trophoblastic tumor (GTT) depends on careful postmolar gonadotropin follow-up and consideration of the diagnosis for any woman of reproductive age with unexplained gynecologic and/or systemic symptoms. Triple therapy with methotrexate, actinomycin D, and cyclophosphamide was once the preferred treatment for patients with high risk metastatic GTT but induced remission in only about 50%. Treatment with etoposide, methotrexate, actinomycin D, cyclophosphamide, and vincristine is now the preferred regimen for treatment of high risk metastatic GTT and has been shown to induce remission in about 70% of patients. CONCLUSIONS: Important advances have been made in the diagnosis and treatment of patients with gestational trophoblastic disease, and patients can be reassured that they can anticipate normal reproductive functioning. PMID- 8635005 TI - Ovarian cancer screening. AB - BACKGROUND: The three most extensively evaluated screening methods for ovarian cancer are pelvic examination, serum CA 125, and transvaginal sonography (TVS). The lack of sensitivity of pelvic examination and serum CA 125 has limited their use in ovarian cancer screening. Currently, the most effective screening method for ovarian cancer is TVS. METHODS: Transvaginal sonography was performed with a standard ultrasound unit and a 5.0 MHz vaginal transducer. Each ovary was measured in three dimensions and ovarian volume was calculated using the prolate ellipsoid formula (L x H x W x 0.523). An ovarian volume greater than or equal to 20 cm3 in premenopausal women and greater than or equal to 10 cm3 in postmenopausal women was considered abnormal. Also, any internal papillary projection from the tumor wall was considered abnormal. A patient with an abnormal screen had a repeat TVS in 4-6 weeks. Women with a persisting abnormality on TVS underwent pelvic examination, serum CA 125 determination, Doppler flow sonography, and tumor morphologic indexing before operative tumor removal. RESULTS: Eighty-five hundred asymptomatic women underwent TVS screening. One hundred twenty-one of these women had a persisting abnormality and underwent tumor removal. Fifty-seven patients had serous cystadenomas and eight had primary ovarian cancers. Six patients had Stage IA ovarian cancer, one had Stage IIC ovarian cancer, and one had Stage IIIB ovarian cancer. Only one of these patients had palpable ovarian enlargement on clinical examination and one had an elevated serum CA 125. All patients are alive and well 4-61 months after conventional therapy. The direct cost of TVS screening was highest during the initial years of the program and fell progressively to $30/screen during the 4th year of the study. Worldwide, more than 14,000 women have been screened using ultrasonography, and 19 ovarian cancers have been detected. More than 20,000 patient-screening-years have been accrued, and there have been no deaths from primary ovarian cancer in the screened population. CONCLUSIONS: Transvaginal sonography screening causes a decrease in stage at detection and a decrease in case-specific mortality. Further study is needed to determine if annual TVS screening will significantly reduce ovarian cancer mortality. The cost for TVS screening is reasonable and is well within the range of that reported for other screening tests. PMID- 8635006 TI - Ovarian cancer screening. The use of serial complementary tumor markers to improve sensitivity and specificity for early detection. AB - BACKGROUND: The use of serum tumor markers for the early detection of ovarian cancer has been limited because of their low sensitivity and low positive predictive value. CA 125 levels are elevated in only about one half of women with Stage I ovarian cancer, thus researchers have focused on using the serial measurement of complementary markers to improve the sensitivity, specificity, and positive predictive value of this approach for screening. METHODS: Multiple serum markers have been analyzed in women with early stage epithelial ovarian cancer. CA 125, CA 15-3, C19-9, CA 54-61, CA 72-4, CEA, HMFG2, IL-6, IL-10, LSA, M-CSF, NB70K, OVX1, PLAP, TAG72, TNF, TPA, and UGTF have been studied alone and in combination in this setting. Complementarity and logistic regression analyses have been performed to assess those markers with the highest likelihood of improving sensitivity and specificity for early detection. Serial analysis of a second-generation CA 125 measuring the intercept (initial level) and slope (change of levels over time) can be used to discriminate malignant cases from benign and normal cases. RESULTS: Analyses have shown that the serial measurement of the new, more sensitive CA 125 has a high sensitivity (83%), specificity (99.7%), and positive predictive value (16%) for the early detection of ovarian cancer. OVX1 used in combination with CA 125 provides the best complementarity. Serial measurements of the two markers have sensitivities in the range of that for transvaginal ultrasonography. CONCLUSION: The serial measurement of complementary serum markers can improve the use of marker screening for epithelial ovarian cancer. With the use of several different methods of analysis, it has been shown that this approach improves the sensitivity, specificity, and positive predictive value of serum markers CA 125 and OVX1. A procedure that measures complementary serum markers over time can be used as a primary screening technique followed by transvaginal ultrasonography. This could provide a cost effective means of early detection and could significantly decrease the probability of surgical intervention for false-positive test results. PMID- 8635007 TI - Mammographic screening of women aged 40-49 years. Benefit, risk, and cost considerations. AB - A recently published meta-analysis of seven randomized trials showed a statistically significant 24% reduction in mortality rate with the use of mammographic screening for women age 40-49 years. The benefit would have been even greater if screening had been performed at shorter annual intervals with the most up-to-date mammographic equipment. Additionally, these trials underestimated benefit due to inclusion of breast cancer deaths among study group women who refused to undergo screening. In contrast to these documented benefits, no woman has ever been shown to have developed breast cancer as a result of mammography, even with multiple examinations at doses many times higher than the current 0.25 cGy from a two-view-per-breast examination. The possibility of low dose risk has been theorized based on excess rates of cancer among populations that have received doses of 100 to more than 1000 cGy, such as atomic-bomb survivors. If there is a risk, it is negligible or nonexistent compared with the screening benefit. The total cost of screening is a summation of the following costs: (1) screening mammogram plus down-stream costs of supplementary mammographic views and/or ultrasound for further evaluation of some screenees; (2) mammographic follow-up; and (3) core or excisional biopsy for those women in whom a suspicious abnormality persists after workup. Based on a range of procedural costs, a 30% reduction in breast cancer deaths due to incidence screening of women age 40-49 will cost $6,930.00-$13,413.00 per year of life expectancy gained. PMID- 8635008 TI - The Canadian National Breast Screening Study. Why? What next? And so what? AB - Why the National Breast Screening Study (NBSS)? In 1979, after reviewing the Breast Cancer Detection Demonstration Projects (BCDDP), the Beahrs Working Group made 11 recommendations. The NBSS protocol reflected a number of these recommendations, particularly the evaluation of screening women age 40-49 and the still unanswered question of the incremental benefit of mammography versus physical examination of the breasts among women age 50-59. Three years after publication of NBSS's 7-year results and in light of other published evidence from screening trials (as opposed to observational studies), it is reasonable to recommend screening with mammography and physical examination every 2 years for women age 50-59. In contrast, it is not reasonable to offer screening mammography to women age 40-49 other than in the context of a controlled trial, an opportunity currently available in the United Kingdom. The Beahrs recommendation, that "physical examinations should be continued in the Breast Cancer Detection Demonstration Projects as a routine screening modality for all ages," remains justifiable as long as so many women age 40-49 are having mammograms performed, given the relatively poor sensitivity of mammography in this age group. Criticism of the NBSS, mainly by radiologists, will continue until the NBSS yields results that support its critics' belief in the efficacy of screening. To date, responses to critics of the NBSS have focused on correcting misinformation and clarifying NBSS procedures. Useful critical commentary should await the results of a 10-year NBSS follow-up and the U.S. National Cancer Institute-sponsored meta-analysis of screening trials. Rigorous critical scrutiny should be directed at all trials. PMID- 8635009 TI - The role of laparoscopy in gynecologic oncology. AB - The use of laparoscopy in the management of gynecologic malignancies has significantly increased over the last 5 years. The safety and adequacy of pelvic and para-aortic lymphadenectomy has been established by several investigators. Patients with early carcinoma of the cervix are now undergoing Schauta (radical vaginal) hysterectomy after laparoscopic lymphadenectomy. Patients with carcinoma of the endometrium are treated by laparoscopically assisted vaginal hysterectomy in conjunction with laparoscopic pelvic and para-aortic lymphadenectomy. Staging and second-look procedures are now being performed laparoscopically in patients with carcinoma of the ovary. The Gynecologic Oncology Group is currently investigating the role of laparoscopic surgery for patients with carcinoma of the cervix, endometrium, and ovary in four Phase II trials. A randomized Phase III trial comparing laparoscopy to laparotomy for patients with carcinoma of the endometrium will begin soon. PMID- 8635010 TI - After treatment. Psychosocial issues in gynecologic cancer survivorship. AB - The woman treated for gynecologic cancer must contend with a challenging group of issues in her efforts to recover psychologically in the months and years after treatment: chance of dying, changed self-perception, treatment-related menopause, impaired or lost fertility, sexual dysfunction, and relationship problems must all be addressed. Psychological and sexual problems after treatment and quality of-life issues for the gynecologic cancer patient have been the subjects of increasing research in the past few years, and a brief literature review is provided. However, the literature contains few descriptive studies regarding the relevant clinical issues faced by the woman treated for gynecologic cancer who has been free of disease for a period of months to years, particularly the younger patient. As the pool of successfully treated women grows, these issues will merit further exploration. The oncology clinician can play a continuing role in offering support, suggestions, and referral in the years of treatment follow up. An overview of the clinical issues relevant to the gynecologic cancer survivor is provided, with reference to the literature and with suggestions for clinical management for the oncology clinician. PMID- 8635011 TI - Longitudinal evaluation of a breast cancer training module. Preliminary results. AB - BACKGROUND: In 1993, the University of Texas M.D. Anderson Cancer Center's Department of Clinical Cancer Prevention began a longitudinal evaluation of its efforts to improve early detection and prevention of breast cancer by training health care professionals in special assessment skills, including how to determine risk and how to teach patients to detect breast cancer early. METHODS: Sixty-three nurses enrolled in the 1-week training module were studied in this descriptive analysis. Their evaluation incorporated not only pretests and post tests of knowledge and clinical performance but also an employer survey, pretraining and posttraining patient logs, and an assessment of clinical skills in each participant's practice setting. RESULTS: Students made statistically significant gains in measures of general knowledge, clinical knowledge and performance, and routine practice of risk assessment, risk counseling, and history taking and physical examination of old (i.e., familiar) patients. Furthermore, the number of patients screened, patients referred, and cancer diagnoses made increased significantly over time, not only between pretraining and posttraining at 6 and 12 months, but also between 6 and 12 months' posttraining. CONCLUSIONS: After learning and practicing new skills, participants successfully incorporated and maintained them and increased referrals and cancers detected, thereby improving the effectiveness of cancer screening. PMID- 8635012 TI - New fears in gynecologic cancer. AB - BACKGROUND: A review of the psychosocial oncology literature indicates the paucity of psychological services available in early detection programs, yet there is an increasing awareness of the need to attend to the psychosocial distress of women whose mothers have had ovarian cancer. The explosion of information transmitted to the public through the news media about ovarian cancer, early detection, and genetic susceptibility should heighten the scientific community's sensitivity to the apprehensions of women who are first degree relatives of women with ovarian cancer. METHOD: The emotional fragility of participants in the early detection ovarian cancer screening program at Yale University is addressed through an initial counseling session. An open-ended, unstructured interview was designed to enable participants to express their concerns about ovarian cancer. This experience is being evaluated. RESULTS: Through psychosocial assessment and evaluation, the authors observed that life cycle events, unresolved episodic grief, cognitive adaptation to loss, and interpersonal relationships contribute to the importance of total care and medical responsibility for the gynecologic cancer patient. Psychosocial counseling has been an integral component of the Yale Early Detection Program since its inception. Preliminary results suggest the necessity of continued counseling intervention to address women's increased anxieties about gynecologic cancers. CONCLUSION: Special attention must be paid to the psychological needs of vulnerable subjects in disease-screening programs. Medical providers of early detection screening should rigorously explore the psychosocial aspects of early detection and provide appropriate intervention to meet women's needs. PMID- 8635013 TI - The borderland between benign and malignant surface epithelial ovarian tumors. Current controversy over the nature and nomenclature of "borderline" ovarian tumors. AB - In the early 1970s, the International Federation of Gynecology and Obstetrics (FIGO) and the World Health Organization (WHO) adopted the terms "borderline malignancy" and "carcinoma of low malignant potential" in their classifications of surface epithelial tumors of the ovary in order to denote a subset of patients with a significantly more favorable prognosis than those with the "usual" surface epithelial carcinomas. Subsequently, a considerable clinicopathologic body of literature has arisen concerning borderline tumors, particularly the serous and mucinous types. Some of them, particularly advanced stage borderline tumors, have been purported to cause significant illness and death. However, some investigators have impugned their malignant nature, especially in Stage I disease, and blame the suggested poor prognosis in advanced cases on a paucity of accurate morbidity and mortality data and ambiguity in current histopathologic terminology; to address the latter, they have proposed to remove any connotation of malignancy by replacing the aforementioned terms with designations such as "atypical proliferating (serous or mucinous) tumor." Another soon-to-be proposed classification will use the terminology "borderline tumors" as a generic group without destructive invasion but with subdivisions into tumors with "epithelial atypia" and those with "intraepithelial carcinoma." The clinical and therapeutic implications of accurate diagnosis of ovarian borderline tumors mandate additional investigation to elucidate their true prognosis; indeed, further dialogue is necessary to arrive at a nosologic system to reflect that biologic behavior. However, until a consensus has been reached, the pathologic diagnosis should reflect, at some point, the currently sanctioned FIGO/WHO classification of surface epithelial ovarian tumors to obviate any misunderstanding that could lead to patient mismanagement. PMID- 8635014 TI - Innovations in brachytherapy in gynecologic oncology. AB - With the discovery of radium by Curie in 1898, researchers recognized that this unique radionuclide had specific biologic properties that were applicable to treating patients with cancer. In the beginning, the radium sources were placed within cavities as independent sources and, when needles were available, implanted into tissues. The first combination of brachytherapy, technologies with external-beam radiation therapy was reported by Wright at the Memorial Sloan Kettering Cancer Center in New York in 1914 in the treatment of a patient with cervical cancer. Next, there was a rapid implementation of brachytherapy in the treatment of cancer by intracavitary placement of radionuclides, interstitial implantation technologies, and systemic administrations. With the development of new radionuclides, including cesium-137, cobalt-60, iridium-192, iodine-125, palladium-103, ruthenium-109, strontium-90, iodine-131, and californium-225, which had varying types of radiation emissions appropriate when properly selected in treatment of cancer, there was a rapid development of innovative technologies to treat all malignancies, especially gynecologic cancer. The evolution of events brought forth new applicators and techniques that allowed for better distribution of the radiation dosage within the tumor being treated, safer use of radionuclides, and the development of computer programs allowing for varying source applications and dose distributions within the volume implanted. PMID- 8635015 TI - Altered fractionation and extended-field irradiation of carcinoma of the cervix. AB - Conventional radiotherapy for treatment of carcinoma of the cervix consists of five daily fractions of 1.80-2.00 Gy, 5 days/week, up to 40.00-50.00 Gy, followed by intracavitary brachytherapy to complete a paracentral dose ranging from 70 to 90 Gy and a lateral pelvic wall dose ranging from 50 to 60 Gy. This results in tumor control in the pelvis for at least 91% of patients with Stage IB disease. However, survival rates at 5-year follow-up have ranged from 49% to 69% for patients with Stage III disease and 25% to 34% for patients with Stage IVA disease. Attempts to improve clinical results for patients with more advanced stages of disease have included hypofractionated and hyperfractionated radiotherapy, accelerated fractionation, and concomitant boost. However, no improvement of tumor control or survival has been obtained. Hyperfractionated external pelvic radiation remains undefined as a method of improving results. Extended-field prophylactic irradiation of the paraaortic lymph nodes has shown significant value in a study group setting. Early diagnosis and prevention continue to be the most promising approaches in the control of carcinoma of the cervix. PMID- 8635016 TI - Management of vulvar cancer. AB - BACKGROUND: Current therapy of vulvar malignancies is reviewed with emphasis on screening, etiology, diagnosis, staging, and treatment of preinvasive and invasive cancer. METHODS: Screening procedures, etiologic possibilities, diagnostic techniques, staging implications, and treatment approaches are discussed in detail. RESULTS: All malignancies of the vulva should be detected at an early stage, when they are most amenable to curative therapy. Regular examination of all women and increased efforts to educate patients should in time reduce patient and physician delay in diagnosis. The cause of the disease remains unclear, because the precursor state has not been defined accurately. The impetus to perform more conservative surgery has been accompanied by the realization of the severe psychosexual sequelae associated with radical vulvectomy. High risk prognostic factors include number of positive groin lymph nodes and diameter of the primary lesion. Diameter of the largest metastasis, intracapsular versus extracapsular nodal tumor location, laterality of spread, and deep groin nodal spread may be predictors of survival. CONCLUSIONS: The overall incidence of vulvar malignancies will not be changed until the pathophysiology of the disease is better understood. Improved survival will depend on earlier and more accurate diagnosis and treatment, including use of radiation therapy. PMID- 8635017 TI - Life and death decisions in the intensive care unit. AB - More than 250,000 women will die of cancer in the United States this year, almost 10% of which are due to gynecologic malignancies. Many of these women will have received care in the intensive care unit (ICU). With important advances in medical technology and the advent of an expanded pharmacologic armamentarium, our ability to maintain life has increased greatly over the past few years. However, this phenomenon has been associated with great emotional and financial cost. It is estimated that ICU charges totaled almost 10% of the $810 billion spent on health care in the United States in 1992. Because 6-month survival rates for patients with cancer admitted to an ICU are the lowest of any disease subgroup (23.7%), we must critically evaluate the role of the ICU in the care of these patients. Decisions regarding admission to an ICU, level of care, and termination of care must take into account patient and family wishes, a reasonable estimation of the reversibility of the acute disease process in question, and the natural history of the underlying disease. Many prognostic scoring systems have been devised to estimate the probability of death among adult ICU patients; however, most of these systems were developed with data from trauma patients rather than from patients with an underlying malignancy, and none are capable of predicting which patient will die. Decisions concerning level of care in the ICU will necessarily involve medical as well as ethical considerations and are best made with a team approach. PMID- 8635018 TI - Gynecologic cancer among the socioeconomically disadvantaged. AB - It is difficult to discern the true dimensions of the relationship between poverty and gynecologic cancer. In well designed studies of patients with gynecologic cancers, demographic stratification usually is performed based on race/ethnicity, age, or geographic locale, but not on economic class. The unstated assumption of many of these reports is that women of color, inhabitants of rural communities, and older women are all poor. Although it is true that these populations are overrepresented among the poor, unless the variable of economic class is specifically evaluated, the broad nature of the problem may go unappreciated: the status of gynecologic cancers among the poor is primarily a reflection of a deeply rooted structural problem in the U.S. economy, the reverberations of which are experienced by all women who cannot afford regular health care. When women are poor and have gynecologic cancers, they often seek orthodox health care only after the symptoms have become unbearable. Explanations of this phenomenon include underlying feelings of pessimism, fatalism, or low self-esteem; faith in a belief system that does not regard the physician as the person to whom one goes for prevention or treatment of diseases; inaccessibility of health care facilities; experiences interpreted as degrading once health care facilities are accessed; high risk behavior and inability to pay. Programs that find effective ways around structural and functional problems of daily life and that respect and understand cultural norms have the best chance of finding temporary solutions to this national problem. PMID- 8635019 TI - Professional negligence in the management of cancer pain. A case for urgent reforms. PMID- 8635020 TI - Elimination of dose limiting toxicities of cisplatin, 5-fluorouracil, and leucovorin using a weekly 24-hour infusion schedule for the treatment of patients with nasopharyngeal carcinoma. AB - BACKGROUND: Cisplatin, 5-flourouracil (5-FU), and leucovorin (PFL) chemotherapy has been reported to be effective in the treatment of cancers but severe mucositis or neutropenia are dose limiting toxicities. This Phase II study evaluated the anticancer effect and the toxicities of a new weekly 24-hour infusional PFL chemotherapy in patients with nasopharyngeal carcinoma (NPC). METHODS: Forty-two patients with stage IV NPC were studied. Cisplatin 25 mg/m2/d, 5-FU 2200 mg/m2/d, and leucovorin 120 mg/m2/d were adminstered weekly by 24-hour intravenous continuous infusion in an outpatient setting. Clinical response and toxicity were evaluated weekly. RESULTS: The complete response rate (CR) was 30% and the partial response (PR) rate 60% in the localized previously untreated group. The CR rate was 22.7% and PR rate 45.5% in local recurrent/metastatic group. The overall response rate was 79%. Eighty-one percent of patients who had no previous chemotherapy and 67% of patients who had previous chemotherapy responded to weekly PFL. There were no dose limiting toxicities. No patient had grade 3 or 4 mucositis or neutropenia. Thirty-two patients (76%) had no oral mucositis. Seven patients (17%) had grade 1 mucositis and 3 patients (7%) had grade 2 mucositis. CONCLUSIONS: Elimination of dose limiting toxicities is possible using a weekly 24-hour infusion schedule of PFL chemotherapy while retaining significant anticancer activity as demonstrated in these patients with advanced NPC. To discover whether this schedule is superior to cisplatin and 5-FU or other PFL chemotherapy regimens requires further investigation. PMID- 8635021 TI - Clinicopathologic evaluation of immunohistochemical E-cadherin expression in human gastric carcinomas. AB - BACKGROUND: E-cadherin plays a crucial role in cell-cell adhesion in epithelial tissues. Recent studies have shown a correlation between decreased E-cadherin expression and cancer cell detachment. METHODS: The expression of E-cadherin was immunohistochemically analyzed using antihuman E-cadherin antibody in 121 cases of human gastric carcinoma. RESULTS: In noncancerous areas, the epithelial cells, including those with intestinal metaplasia, were stained positively in the plasma membrane. In contrast, E-cadherin expression of the cancer cells varied from case to case in primary and secondary sites. Tumors with a decrease in E-cadherin occurred significantly more frequently in undifferentiated adenocarcinoma (P < 0.05) and scirrhous type (P < 0.01). The rate of E-cadherin-negative tumors was higher in patients with peritoneal metastasis (P < 0.01) or in those with distant lymph node metastasis (P < 0.01), though the tumors with liver metastasis had relatively positive E-cadherin expression. Patterns of initial recurrence had similar results. Reduction or loss of E-cadherin expression correlated with shorter survival in patients after curative operation regardless of stage of disease. CONCLUSIONS: The decreased E-cadherin expression correlates with dedifferentiation, infiltrative tumor growth, distant metastasis, and poor survival for patients with gastric carcinoma. Thus, immunohistochemical study of E-cadherin may have clinicopathologic value for patients with gastric carcinoma. PMID- 8635022 TI - Epirubicin-Lipiodol chemotherapy versus 131iodine-Lipiodol radiotherapy in the treatment of unresectable hepatocellular carcinoma. AB - BACKGROUND: Arterially administered iodized oil (Lipiodol) is selectively retained by hepatocellular carcinomas (HCCs), and has been used as a vehicle for delivery of therapeutic agents to these tumors. This study compared the efficacy of Lipiodol-targeted epirubicin chemotherapy with Lipiodol-131I radiotherapy. METHODS: Ninety-five patients with unresectable HCC confined to the liver were administered either Lipiodol-epirubicin emulsion (n = 69; 61 cirrhotics; Okuda tumor Stage I, 14; II, 37; III, 18; epirubicin dose, 75 mg/m2) or Lipiodol-131I (131I) (n = 26; 18 cirrhotics; Okuda tumor Stage I, 6; II, 19; III, 1; dose 750 1050 MBq). The last 28 patients (17 epirubicin, 11 131I) were treated within a prospective randomized trial. Bolus drug or isotope was injected into the hepatic artery by transfemoral cannulation. Lipiodol and 131I uptake were gauged by 10th day computed tomography and 48-hour scintiscan. Treatments were repeated two monthly when indicated. RESULTS: Tumor size at 2 months remained static or diminished partially in 21 of 38 epirubicin recipients (55%) and 15/22 131I recipients (68%). Actuarial survival at 6, 12, and 24 months was 40%, 25%, and 6% with epirubicin, and 58%, 25%, and 0% with 131I; 30-day mortality was 11% and 15%, respectively. Comparison with historic controls indicated survival benefit in Stages I and II. Similar findings were recorded in the 28 patients in the randomized trial. CONCLUSIONS: Patients with unresectable HCC receiving Lipiodol epirubicin or Lipiodol-131I show good tumor localization, acceptable toxicity, and comparable survival benefit at 6 and 12 months with either modality. PMID- 8635023 TI - Detection of hepatitis C virus RNA in hepatocellular carcinoma by in situ hybridization. AB - BACKGROUND: Hepatocellular carcinoma frequently is associated with chronic hepatitis C virus (HCV) infection. The presence of HCV in hepatocellular carcinoma has been detected by reverse-transcription polymerase chain reaction of antigenomic HCV RNA, a tissue-specific replicative form of the virus. Now, however, this method of detecting the presence of HCV has been invalidated by reports of antigenomic RNA in the blood or in peripheral blood mononuclear cells. METHODS: In situ hybridization of HCV RNA was conducted with digoxigenin-labeled cDNA from the core region on surgical specimens of noncancerous and cancerous areas from 12 patients with chronic hepatitis C with or without cirrhosis associated with hepatocellular carcinoma. Several control experiments were also performed, including RNase digestion before hybridization, hybridization with the use of a negative control, and immunohistochemical staining of HCV-core protein. RESULTS: The in situ hybridization showed positive signals both in noncancerous and cancerous areas of the liver tissue in eight cases. Positive signals were confined to neoplastic cells and nonneoplastic hepatocytes. There were fewer HCV positive cells in the cancerous area than in the surrounding noncancerous area. CONCLUSIONS: In situ detection of HCV presents direct evidence of HCV infection in the neoplastic cells of hepatocellular carcinoma and suggests that neoplastic cells may lose their affinity for HCV in the course of malignant transformation. PMID- 8635024 TI - Inflammatory myofibroblastic tumor of the larynx. A clinicopathologic study of eight cases simulating a malignant spindle cell neoplasm. AB - BACKGROUND: Inflammatory myofibroblastic tumors of the larynx are uncommon lesions that easily may be misinterpreted as malignant epithelial or mesenchymal spindle cell neoplasms. METHODS: Eight cases of laryngeal inflammatory myofibroblastic tumors were identified from the files of the Otolaryngic Tumor Registry--Armed Forces Institute of Pathology. Clinical records and follow-up were available in all cases. The light microscopic features (hematoxylin and eosin and special histochemical stains) were evaluated in all cases; immunohistochemical analysis was performed in the seven cases with available paraffin blocks; in four cases ultrastructural analysis was done. RESULTS: The patients included five males and three females ranging in age from 19-69 years (median, 59 years). Presenting symptoms included hoarseness, dysphonia, or rapidly progressive stridor with the duration of symptoms ranging from 10 days to 4 months. The most common site of involvement was the true vocal cord. The lesions appeared as polypoid or pedunculated masses. Histologically, the cellularity of the lesions varied, consisting of spindle-shaped to stellate cells with no consistently discernible growth pattern, in a fibromyxoid stroma that included a mixed inflammatory cell infiltrate. Features suggesting a malignant cellular infiltrate were not present. The spindle-shaped cells had consistent immunoreactivity with vimentin, muscle specific actin, and smooth muscle actin. Ultrastructurally, intracytoplasmic microfilaments were identified. In seven of the patients, conservative but complete excision of the lesion was curative; these patients have been free of disease over periods ranging from 12 to 36 months. In one patient, the lesion recurred twice over a 2-year period and ultimately required a total laryngectomy. This patient died of unrelated causes. CONCLUSIONS: Inflammatory myofibroblastic tumors of the larynx are unusual benign proliferative lesions. Conservative surgical management is advocated and is curative. Recurrence is rare, but metastases disease or death attributable to these lesions is not. PMID- 8635025 TI - Prospective study of combination chemotherapy with cyclophosphamide, doxorubicin, and cisplatin for unresectable or metastatic malignant pleural mesothelioma. AB - BACKGROUND: This study was designed to determine the efficacy and side effects of a combination of cyclophosphamide (C), doxorubicin (D), and cisplatin (P) in patients with inoperable, unresectable, or metastatic malignant pleural mesothelioma. METHODS: Twenty-three patients with unresectable or metastatic malignant pleural mesothelioma were entered onto the study. The median age was 62 years (range, 42-74 years); there were 20 males and 3 females; the median performance status was 1 (Zubrod's scale). The histologic types included epithelial (14 patients), sarcomatoid (4 patients), unclassified (4 patients), and mixed type (1 patient). Twenty patients were known to have been exposed to asbestos and 3 were not. All patients were treated with the following starting dose of chemotherapy: a cycle comprised of C, 500 mg/m2 intravenously, day 1; D, 50 mg/m2 intravenously, day 1; and P, 80 mg/m2 intravenously, day 1 every 3 weeks. The cisplatin dose was reduced to 50 mg/m2 for the subsequent courses. For the assessment of tumor response, all patients had computed tomography scans of the chest after each three cycles of chemotherapy. RESULTS: Overall, 7 of 23 patients (30%) had partial responses (durations of responses [weeks]: 158+, 91+, 70+, 41+, 40, 39, 25), three had minor responses, and 14 had stable or progressive disease. One partial responder later underwent surgical resection and no viable tumors cells were found in the pathologic specimen. All patients have stopped treatment, and eight are still alive. The most common side effect was granulocytopenia (grade 4, 52%; grade 3, 17%). Other hematologic side effects were modest. Nonhematologic side effects included mild to moderate nausea and vomiting, neutropenic fever (three patients), peripheral neuropathy (one patient), and congestive heart failure (one patient). The overall median duration of survival was 60 weeks. CONCLUSION: Combination chemotherapy with CDP was well tolerated and had significant activity against unresectable or metastatic malignant pleural mesothelioma. The median duration of responses was 60 weeks; however, the survival rate was far from satisfactory. Continued development of new approaches including the biologic understanding of tumor development and testing new agents is warranted. PMID- 8635026 TI - Acute promyelocytic leukemia and pregnancy. A case report. AB - BACKGROUND: Acute promyelocytic leukemia (APL) is an uncommon form of acute myeloid leukemia usually associated with disseminated intravascular coagulation (DIC). Pregnancy in patients with APL requires special consideration to maximize the probability of survival of both mother and fetus. METHODS: A patient with APL diagnosed during pregnancy who developed DIC is described. Obstetric and oncologic management of this difficult patient is discussed, and a pertinent literature review of pregnancy in APL is presented. RESULTS: Of 23 pregnancies in patients with APL reported in the literature (including the present patient), 19 yielded live births, including 8 of 12 who received chemotherapy during late pregnancy and all 3 patients who received all-trans-retinoic acid (ATRA) during late pregnancy. Chemotherapy or ATRA induced complete remission in 72% of treated patients. CONCLUSIONS: Proper management of pregnant patients with APL usually results in a live birth and complete remission of the mother's leukemia, despite the potentially devastating consequences of DIC, which is present at diagnosis in most patients. PMID- 8635027 TI - Desmoplasia and neurotropism. Prognostic variables in patients with stage I melanoma. AB - BACKGROUND: Desmoplastic melanomas with and without neurotropism are thought to be more clinically aggressive than other melanomas of comparable thickness. This study evaluates the prognostic significance of desmoplasia and neurotropism in Patients with Stage I cutaneous melanoma completely excised at the initial presentation of disease and prospectively studied for a minimum of 8 years. METHODS: Desmoplasia and neurotropism were evaluated as single prognostic predictors in survival outcome of cutaneous Stage I melanomas and as variables in the University of Pennsylvania Pigmented Lesion Study Group 8-year survival model for Stage I melanoma. In addition, the clinical presentation and follow-up of melanomas with desmoplasia and/or neurotropism was compared with that of other types of cutaneous Stage I melanoma in patients also followed for a minimum of 8 years. RESULTS: Neurotropism was associated with a statistically significant decrease in survival in patients with melanomas with desmoplasia. A decrease in survival also was observed in other types of melanoma with neurotropism, but the difference was not statistically significant. Melanomas with neurotropism had a statistically significant increase in local recurrence. Desmoplasia was not associated with a statistically significant decrease in survival. CONCLUSION: Desmoplasia is not associated with a statistically significant decrease in the prognosis of patients with primary cutaneous Stage I melanoma. The more clinically aggressive behavior of desmoplastic melanomas observed in previous studies may be secondary to initial misdiagnosis and/or inadequate margin assessment of these lesions. Neurotropism, however, is associated with a statistically significant decrease in survival in patients with desmoplastic melanomas and is most likely associated with decreased reduces patient survival in other melanoma types. Neurotropism is also related to an increase in the frequency of local recurrence of melanoma. PMID- 8635029 TI - A review of 40 patients with extraskeletal osteosarcoma. AB - BACKGROUND: Extraskeletal osteosarcomas are rare malignancies that account for about 1% of all soft tissue sarcomas. Few large series have been reported. METHODS: Clinical records and histologic slides of all patients with extraskeletal osteosarcomas treated at the Mayo Clinic between 1915 and 1988 were reviewed. RESULTS: The study group consisted of 40 patients, most of whom presented in the sixth and seventh decades of life (mean age, 50.7 years). There was a male predominance (male-to-female ratio, 1.9:1). The lower limbs most commonly were involved (68%), usually the thigh and buttock regions. Ninety-three percent of tumors presented as an enlarging soft tissue mass, with a history of trauma in nine patients. In nine patients, the lesions were initially interpreted histologically as benign, most commonly as myositis ossificans. Histologically, all were high grade osteosarcomas. Multiple local recurrences (45%) are a feature of this tumor. All recurrences occurred within 3 years. Distant metastasis (65%) is also common and is usually to the lungs (81%). Radical resections appear to be the best option for local control, with resection of the pulmonary metastasis occasionally producing a cure. By univariate analysis of Kaplan-Meier survival curves, the patients with predominantly chondroblastic tumors fared better than those with predominantly osteoblastic tumors (P = 0.03). Analysis of survival differences of the three main subtypes together (osteoblastic, chondroblastic, and fibroblastic) was not significant. A small-sized initial lesion did not equate with better survival. Seventy-three percent died of the disease, with a mean follow-up of 5.9 years. CONCLUSION: Extraskeletal osteosarcoma is a high grade malignant tumor associated with a 5-year survival rate of 37% (95% confidence interval, 28%-59%). Local recurrences and distant metastasis are common and usually occur by 3 years after excision. Patients with the chondroblastic subtype survive longer than those with the osteoblastic subtype. PMID- 8635028 TI - A phase II trial of paclitaxel in patients with advanced soft tissue sarcomas. A Southwest Oncology Group study. AB - BACKGROUND: The objectives of this Phase II trial of paclitaxel were to estimate the response rate and to define the toxicities of paclitaxel administered with recombinant granulocyte-colony stimulating factor in patients with advanced soft tissue sarcomas. METHODS: Patients were eligible if they had a histologic diagnosis of unresectable, recurrent or metastatic soft tissue sarcoma and had had no prior chemotherapy or radiotherapy. Paclitaxel at 250 mg/m2 was given by continuous intravenous infusion over 24 hours every 21 days. Doses were modified in subsequent courses based on nadir counts. Granulocyte-colony stimulating factor was given at 5 micrograms/kg subcutaneously days 3-18. RESULTS: Forty eight patients were treated; 1 patient had a complete response and 5 had partial responses for an overall response rate of 12.5% (95% confidence interval, 4.7% 25.3%). Thirty-eight of the 48 patients experienced grade 4 toxicities, with most of these life-threatening toxicities being hematologic. No deaths were attributed to therapy. CONCLUSIONS: At the tested dose and schedule paclitaxel has antitumor activity approximating that of decarbazine in soft tissue sarcomas. Whether paclitaxel would be more effective administered in a longer infusion or with a chemosensitizer remains to be tested in this group of heterogeneous neoplasms. PMID- 8635030 TI - Local relapse and contralateral tumor rates in patients with breast cancer treated with conservative surgery and radiotherapy (Institut Gustave Roussy 1970 1982). IGR Breast Cancer Group. AB - BACKGROUND: Breast conservation is now established treatment for patients with small breast cancers. The authors reviewed a large series of patients with long term follow-up who underwent conservative treatment. Clinical and pathologic factors were analyzed to identify patients at an increased risk of relapse in the breast (local relapse) or development of a contralateral tumor. METHODS: Seven hundred fifty-seven patients with unilateral invasive breast cancer (T0-2, N0-1, M0) were treated conservatively (wide local excision and radiotherapy) at the Institut Gustave-Roussy between 1970 and 1982. The median follow-up was 9 years. The risk of local relapse or development of a contralateral tumor (as first event) was studied by univariate analysis for the main clinical, pathologic, and treatment factors. Those found to be significant were entered into a Cox proportional regression analysis. RESULTS: Fifty-one patients relapsed in the treated breast (actuarial local relapse rates at 5 and 10 years were 5% and 8%, respectively) and 34 in the contralateral breast (actuarial contralateral tumor rates at 5 and 10 years were 3% and 6%, respectively). Multivariate analysis of the risk factors for local relapse showed that only age younger than 40 years (P < 0.02) or inadequate surgical excisioin (P < 0.02) were significant. No particular risk factors for contralateral tumor development were identified. CONCLUSIONS: Overall, for most patients, the risk of local relapse or of developing a contralateral tumor was low. A small number of young patients with inadequately excised tumors are at higher risk of local relapse, need more meticulous surgery, and may merit higher dose radiotherapy. PMID- 8635031 TI - Predictors of recurrence for patients with small (one centimeter or less) localized breast cancer (T1a,b N0 M0). AB - BACKGROUND: The frequency of small (< or = 1 cm) axillary lymph node negative invasive breast cancers (T1a,b N0 M0) is increasing because of wider implementation of breast cancer screening. Identification of prognostic factors for these patients has been based largely on retrospective pathology review. The authors analyzed histologic factors recorded in the original pathology reports to determine predictors of recurrence for patients with T1a,b N0 M0 breast cancer. METHODS: Two hundred eighteen patients were studied. Potential prognostic factors including measured millimeter tumor size in three dimensions, histologic grade, nuclear grade, and presence or absence of lymphatic vessel invasion were documented prospectively in routine surgical pathology reports of a large community (nonuniversity based) hospital. Follow-up was performed annually by the tumor registry. RESULTS: With a median follow-up of 6.9 years (range, 3-15.8 years), overall recurrence free survival was 93%. Poor nuclear grade (hazard ratio, 5.8; 95% confidence interval, 1.70-19.82; P = 0.004) and lymphatic vessel invasion (hazard ratio, 4.6; 95% confidence interval, 1.34-15.61; P = 0.01) were independent predictors of recurrence. Only 10% of patients had cancers with both poor nuclear grade and lymphatic vessel invasion and their 67% 7-year recurrence free survival (RFS) rate was significantly lower than the 92% RFS rate observed for patients with one of these two factors (P = 0.007) and the 99% RFS for patients with neither poor risk factor (P = 0.0001). CONCLUSIONS: The combination of poor nuclear grade and lymphatic vessel invasion identifies a very small subset (10%) of patients with T1a,b N0 M0 breast cancer with a significant relapse risk that warrants consideration of adjuvant systemic therapy. However, the majority of patients with T1a,b N0 M0 breast cancer have an exceptionally good prognosis. PMID- 8635032 TI - Increasing patient involvement in choosing treatment for early breast cancer. AB - BACKGROUND: This investigation examined factors affecting patient involvement in consultations to decide local treatment for early breast cancer and the effectiveness of two methods of preconsultation education aimed at increasing patient participation in these discussions. METHODS: Sixty patients with Stage I or II breast cancer (1) were pretested on their knowledge about breast cancer treatment and optimism for the future, (2) were randomly assigned to one of two methods for preconsultation education: interactive multimedia program or brochure, (3) completed knowledge and optimism measures, (4) consulted with a medical oncologist, radiation oncologist, and general surgeon, and (5) completed self-report measures assessing their involvement in the consultations and control over decision-making. The consultations were audiorecorded and analyzed to identify behavioral indicators of patient involvement (question-asking, opinion giving, and expressing concern) and physician utterances encouraging patient participation. RESULTS: College-educated patients younger than 65 years of age were more active participants in these consultations than were older, less educated patients. In addition, patients showed more involvement when they interacted with physicians who encouraged and facilitated patient participation. The method of education did not affect patient involvement although patients tended to learn more about breast cancer treatment after using the multimedia program than after reading the brochure. CONCLUSIONS: Although patients vary in their expressiveness, physicians may be able to increase patient participation in deciding treatment by using patient-centered behavior. Also, preconsultation education appears to be an effective clinical strategy for helping patients gain an accurate understanding of their treatment options before meeting with physicians. PMID- 8635033 TI - Pattern of failure in patients with inflammatory breast cancer treated by alternating radiotherapy and chemotherapy. AB - BACKGROUND: Patients with inflammatory breast cancer have a high risk of developing a local recurrence and/or distant metastases. Treatment with combined chemotherapy and locoregional radiotherapy contributes to a decrease in both risks. This study presents treatment results and evaluates the pattern of failure when an alternating chemoradiotherapy schedule is used. METHODS: One hundred twenty-five patients with nonmetastatic inflammatory breast cancer were treated with an alternating schedule of radiotherapy and chemotherapy. All women recruited were younger than 70 years of age and had a T4d, histologically proven infiltrating carcinoma with N0 to N2 axillary disease. The protocol consisted of three cycles of induction chemotherapy with doxorubicin, vincristine, cyclophosphamide, methotrexate, and 5-fluorouracil followed by three series of locoregional radiotherapy, delivering a total dose of 65-75 Gy to the breast tumor. Five additional cycles of chemotherapy with 5 fluorouracil/doxorubicin/cyclophosphamide were to be administered in between the first two and after the third radiotherapy course. A 1-week gap was respected between each course of chemotherapy and each series of radiotherapy. RESULTS: Toxicity was moderate and this strategy proved feasible although most of the patients only received six instead of the eight planned cycles of chemotherapy. Eighty-two percent of the patients achieved a complete response at the end of the treatment. The cumulative 5-year local failure and distant metastasis rates were 27% and 53%, respectively. Assuming competing events, local failures, contralateral recurrences, and distant metastases were the first site of failure in 18%, 5%, and 38% of patients, respectively. The 5-year overall and disease free survival rates were 50% and 38%, respectively. The main prognostic factor was tumor size. CONCLUSIONS: Alternating high doses of radiotherapy and chemotherapy is a feasible treatment schedule and permits breast conservation. Disease free survival is comparable to that of recently published series. As the main causes of failure are distant metastases, higher dose chemotherapy should be evaluated, in an attempt to further improve overall survival. PMID- 8635034 TI - Selected food intake and risk of vulvar cancer. AB - BACKGROUND: A case-control study was conducted to analyze the association between body mass, selected indicator food intake, and vulvar cancer risk. METHODS: The patients included in this report were 125 women aged 80 years or younger with histologically confirmed diagnosis of invasive vulvar cancer who were admitted to a network of general and teaching hospitals in the greater Milan area. Control subjects were 541 patients admitted to teaching and general hospitals in Milan for acute conditions. RESULTS: The risk of vulvar cancer was inversely related to green vegetable and carrot consumption, the corresponding multivariate relative risks for lowest versus highest levels of intake being 2.0 (95% confidence interval [CI], 1.2-3.4) and 1.4 (95% CI, 0.9-2.2). The trend in risk was significant for green vegetables. No consistent association emerged between milk, meat, liver, alcohol and coffee consumption and risk of vulvar cancer. In comparison with leaner women, the relative risks of vulvar cancer were 1.8, 1.9, 2.8, and 2.9 in progressively higher quintiles of the body mass index, and the trend in risk was significant. CONCLUSIONS: These data indicate that the risk of vulvar cancer is related to a number of nutritional and dietary factors. This is of particular interest, because vulvar cancer is a relatively rare neoplasm, whose etiology is still poorly understood, and on which only a few epidemiologic studies have been conducted. PMID- 8635035 TI - The possible role of bcl-2 expression in the progression of tumors of the uterine cervix. AB - BACKGROUND: A close link between human papillomavirus (HPV) and the development of uterine cervical neoplasias has been proposed. However, other cofactors also are required for malignant transformation. METHODS: Forty-six cervical intraepithelial neoplasias (CIN) I/II, 75 CIN III, and 60 invasive squamous cell carcinomas (ISCC) were investigated by immunohistochemical staining for bcl-2 protein (bcl-2), bax protein (Bax), estrogen receptor (ER), and progesterone receptor (PR). The presence of HPV-DNA was examined using the polymerase chain reaction assay. RESULTS: bcl-2 immunoreactivity was found in 17 of 46 (37%) CIN I/II, 48 of 75 (64%) CIN III, and 12 of 60 (20%) ISCC, the positivity in CIN III being significantly higher than in CIN I/II or ISCC (P < 0.004, P = 0.0001). The bcl-2 immunostaining pattern could be subdivided into two groups, basal type and diffuse type, with the incidence of the latter being clearly increased, in line with tumor progression. In the Bax or HPV-DNA positive groups, bcl-2 positive cases in CIN I/II were in the minority, whereas in CIN III they constituted the majority. In ISCC, bcl-2 positive was significantly lower than negative cases, not being associated with Bax and HPV-DNA. Estrogen receptor and PR immunoreactivity were rare. CONCLUSIONS: These results indicate that bcl-2 may play an important role in a relatively early stage of cervical tumorigenesis, in association with Bax expression and HPV infection. PMID- 8635036 TI - Serum basic fibroblast growth factor in men with and without prostate carcinoma. AB - BACKGROUND: Angiogenesis is essential for the growth of neoplasms. Increased vascularity has been associated with human prostatic carcinoma stage and has been shown to offer prognostic information. Basic fibroblast growth factor (bFGF) is a potent angiogenic inducer expressed in malignant prostate tissue. In this investigation, serum bFGF levels were measured in men with and without prostate cancer. METHODS: Serum bFGF was measured using a commercial enzyme-linked immunosorbent assay in archival serum from men with various clinical stages of prostate carcinoma. Sera from men with negative systematic sector biopsies and serum prostate specific antigen (PSA) less than 2.0 ng/ml served as controls. Prostate volume was measured with transrectal ultrasound. RESULTS: Serum bFGF level was significantly higher in men with prostate carcinoma compared with those without (P < 0.0007). Among 44 men (11 with carcinoma) with a serum PSA less than 4.0 ng/ml, only bFGF level was associated with carcinoma (P = 0.008). Using a cutoff of 1.0 pg/ml, bFGF afforded a sensitivity of 83% and specificity of 44% in this group. There was no association between bFGF levels and clinical stage, Gleason score, or prostate volume. CONCLUSIONS: These data demonstrate that serum levels of bFGF are elevated in most men with prostatic carcinoma. Although association with stage was not observed, the finding of significant elevation in most men with carcinoma who have "normal" serum PSA levels may indicate diagnostic utility for this analyte. PMID- 8635037 TI - Association of chronic lymphocytic thyroiditis and thyroid papillary carcinoma. A study of surgical cases among Japanese, and white and African Americans. AB - BACKGROUND: An association between lymphocytic thyroiditis and thyroid papillary carcinoma is still controversial. To determine a definite statistical relation, a histopathologic study was performed on tissues from in three races, because there is a racial and age-related difference in the susceptibility to thyroiditis. METHODS: The prevalence and severity of thyroiditis combined with adenomatous goiter, follicular adenoma, or papillary carcinoma was defined by examination of surgically resected materials from Japanese (626 patients), and white and African Americans (330 and 90 patients, respectively). RESULTS: The prevalence of lymphocytic infiltrates, which are indicative of autoimmune thyroiditis, was significantly higher in patients with papillary carcinoma than in patients with adenomatous goiter or follicular adenoma among Japanese females (63.0%) and males (50.0%), white females (76.0%), and African American females (46.2%). Lymphocyte infiltration into the follicular adenoma or papillary carcinoma correlated with the severity of combined thyroiditis. CONCLUSION: An association between chronic lymphocytic thyroiditis and papillary carcinoma was confirmed in the Japanese, and white and African American populations. The possibility of autoimmune thyroiditis as a predisposing factor for papillary thyroid carcinoma, is suggested. PMID- 8635039 TI - Randomized, double-blind comparison of a prochlorperazine-based versus a metoclopramide-based antiemetic regimen in patients undergoing autologous bone marrow transplantation. AB - BACKGROUND: Highly emetogenic combination alkylator therapy is routinely used in autologous bone marrow transplantation for treatment of eligible patients with solid tumors. Antiemetic therapy remains less than optimal in this setting. METHODS: One hundred twenty-six patients with cancer receiving high dose cisplatin, cyclophosphamide, and carmustine with autologous bone marrow support were randomized to receive one of four double-blinded antiemetic regimens: 4-day continuous infusion prochlorperazine (6 mg/m2 intravenous [i.v.] loading dose followed by 1.5 mg/m2/hour) or metoclopramide (80 mg/m2 iv loading dose followed by 20 mg/m2/hr) each with either dronabinol 5 mg/m2 or placebo capsules for two doses before carmustine on the last day of chemotherapy. All subjects received scheduled lorazepam and diphenhydramine throughout the 4-day study period. Efficacy was measured by the Emetic Process Rating Scale and the Rhodes Index of Nausea and Vomiting (INV) Form 2. RESULTS: One hundred six patients completed the study and were fully evaluable. The median number of emetic episodes on the metoclopramide study arm were: 1 (0-7, day -6), 1 (0-6, day -5), 2 (0-9, day -4), and 2 (0-10, with dronabinol day -3) or 2 (0-7, no dronabinol day -3) and on the prochlorperazine study arm were: 4 (0-12, day -6), 0 (0-8, day -5), 0 (0-12, day 4) and 2.5 (0-9, with dronabinol day -3) or 2 (0-12, no dronabinol day -3). Metoclopramide was significantly better on the first day of therapy (day -6, P < .002) and prochlorperazine was significantly better on the third day of therapy (day -4, P < 0.002). There was no significant difference among any of the four arms on the last day of chemotherapy (day -3), or when the median number of emetic episodes over the total study period were compared. The patients' assessment of nausea, vomiting, and retching on the INV Form 2 was consistent with the observer ratings. Toxicities requiring dose reduction or discontinuation of antiemetic drugs included diarrhea, cardiac arrhythmias, sedation, anxiety, and akathisia. CONCLUSIONS: Both metoclopramide and prochlorperazine in combination with lorazepam and diphenhydramine offer good control of nausea and vomiting although the sedation and low risk for cardiac toxicity limit the regimen to an inpatient setting with close monitoring. No regimen was clearly superior during the entire treatment period but prochlorperazine offered more consistent control after the first day. PMID- 8635038 TI - Recombinant human erythropoietin for the correction of cancer associated anemia with and without concomitant cytotoxic chemotherapy. AB - BACKGROUND: Chronic anemia is a common complication in patients with cancer, especially in those with advanced disease or who are under intensive chemotherapy. Because homologous blood transfusions involve some hazards, the safety and efficacy of recombinant human erythropoietin (r-HuEPO) in the treatment of anemic patients with cancer with and without concomitant chemotherapy were studied. METHODS: One-hundred two cancer patients with hemoglobin less than 11 g/dl, ferritin greater than 30 micrograms/l, and creatinine < 220 mumol/l were enrolled in the study, 94 were eligible for efficacy evaluation. Sixty-eight patients received chemotherapy (CT group) and 26 had no cytotoxic cancer treatment (NT group). Recombinant human erythropoietin was administered subcutaneously at a dose of 150 U/kg three times per week for 6 weeks; in nonresponders the dose was doubled for the subsequent 6 weeks. Response was defined as the achievement of a hemoglobin increase of 2g/dl. Clinical and laboratory parameters, including serum erythropoietin (EPO) levels, performance status, and quality of life, were investigated at baseline and monitored at regular intervals thereafter. RESULTS: Response was achieved by 52% and 62% of CT and NT patients, respectively. The highest response rates were observed in patients with lung cancer or with a histology of squamous cell carcinoma (both 80%). In responding patients, the symptoms of anemia subsided. They no longer needed blood transfusions after 4 weeks of therapy; and both their performance status and quality of life improved significantly. The NT patients achieved slightly more favorable results on lower weekly doses: 450 U/kg/week in NT versus 570 U/kg/week in CT patients. Serum EPO levels were higher in nonresponders at baseline and further increased during the course of treatment. Recombinant human erythropoietin was well tolerated by all patients. CONCLUSION: This multicenter study in a large patient collective shows that r-HuEPO treatment represents a safe and effective means to increase the red cell mass and eliminate the need for blood transfusions in approximately 50% of the patients with chronic anemia of cancer. Responding patients not only have increased levels of hemoglobin, but their performance status also improves significantly, and they enjoy a significantly enhanced quality of life. PMID- 8635040 TI - Hemolytic uremic syndrome after high dose chemotherapy with autologous stem cell support. AB - BACKGROUND: Chemotherapy intensification may lead to new forms of toxicity such as hemolytic uremic syndrome. METHODS: Three patients are described who developed this complication 4 to 6 months after high dose chemotherapy followed by autologous stem cell support. The literature on this subject is reviewed. RESULTS: One patient was conditioned with BEAC (carmustine, etoposide, cytosine arabinoside, and cyclophosphamide) and received autologous bone marrow. The other two underwent triple peripheral stem cell transplantation after conditioning with CTC (carboplatin, cyclophosphamide, and thiotepa). Symptoms were hypertension, microangiopathic hemolytic anemia, thrombocytopenia, and renal insufficiency. One patient had a retinal vein thrombosis. One patient died of a cardiac arrest shortly after the diagnosis was made. The remaining two achieved a partial remission: one with fresh frozen plasma without plasmapheresis and fresh frozen plasma, but improved on high dose intravenous immunoglobulin and vincristine. CONCLUSIONS: Hemolytic uremic syndrome is a serious complication of the more intensive chemotherapy made possible by stem cell support. Because of the rapidly growing indications for this approach, an increase in this type of vascular complication is expected. PMID- 8635041 TI - Completeness of hospital cancer case reporting from the SEER Program of the National Cancer Institute. AB - BACKGROUND: To ascertain the quality of data entering a population-based reporting system, an essential requirement is to study levels of completeness of case-ascertainment and reporting. This study represents an effort to quantify completeness of case reporting in the SEER (Surveillance, Epidemiology, and End Results) Program of the National Cancer Institute. METHODS: Hospitals in each of the participating SEER areas were stratified according to their annual hospital cancer caseload for the year 1987. Within each caseload stratum, a random sample of hospitals was selected for inclusion in this study. Files in the medical record, pathology, and radiation oncology departments in each hospital were reviewed for SEER reportable cases. These cases were then matched against SEER case listings to identify unreported cases. RESULTS: The crude estimated completeness of reporting for 1987 in the six participating SEER areas was 97.7% and the registry-caseload standardized rate was 96.8%. Variation was noted by SEER registry, hospital cancer caseload, and casefinding source (hospital department). Three-quarters of unreported cases were of invasive disease and one fourth were in situ, primarily of the cervix uteri. CONCLUSIONS: There is variation in completeness of casefinding among SEER registries, hospital size, and hospital department source. Additional factors that appear to be related to case ascertainment are cancer site or type and who performs the casefinding function (hospital registry or central registry staff). PMID- 8635042 TI - Frequent expression of P-glycoprotein/MDR1 by nasal T-cell lymphoma cells. AB - BACKGROUND: Lethal midline granuloma is now considered to be a malignant lymphoma derived from peripheral T cells or from natural killer cells. The therapeutic outcome of nasal T-cell lymphoma (NL) treated by conventional chemotherapy for non-Hodgkin's lymphoma is poor, although some patients have a good response to radiotherapy. To clarify the mechanisms of drug resistance, the expression of P glycoprotein (P-gp)/MDR1, which is the product of the multidrug resistance (MDR) 1 gene, and MDR3 mRNA in NL cells, were examined. METHODS: Ten Japanese patients with NL were studied. Nine of these patients were examined before therapy. P glycoprotein expression and phenotypes of lymphoma cells were examined by immunohistochemical staining using UIC2 as an anti-P-gp monoclonal antibody. In one case, the Rhodamine-123 efflux test was performed. MDR1 and MDR3 mRNA were detected by reverse transcription polymerase chain reaction. RESULTS: Nine of the 10 patients were P-gp positive. In one of nine, functional P-gp expression was observed. MDR1 mRNA was detected in all seven examined patients with P-gp positive NLs, whereas MDR3 mRNA was negative. Retrospectively, patients who received chemotherapy alone had poorer outcome than those treated by combination chemotherapy after irradiation. CONCLUSION: The poor prognosis for patients with NL treated with chemotherapy may be explained by P-gp expression of the NL cells. PMID- 8635043 TI - Successful treatment of hepatosplenic candidiasis through repeated cycles of chemotherapy and neutropenia. AB - BACKGROUND: Hepatosplenic candidiasis (HSC) or chronic disseminated candidiasis is an increasingly recognized problem in patients with cancer. Whether patients with HSC should continue to receive antineoplastic therapy, which may cause neutropenia with the risk for progressive HSC or breakthrough fungemia, can be a major dilemma. Patients with HSC at the National Cancer Institute continue antineoplastic therapy, when possible during antifungal therapy for HSC, despite repeated bouts of neutropenia. Therefore, whether this strategy resulted in breakthrough fungemia or progression of HSC was investigated. METHODS: All patients consecutively treated at the National Cancer Institute at the Warren Grant Magnuson Clinical Center from 1982-1992 for HSC were prospectively studied for therapeutic and outcome variables of antifungal and antineoplastic management. Each case was summarized on a time-event line to quantify the duration of simultaneous periods of antineoplastic therapy and antifungal therapy (AFT). RESULTS: Sixteen patients (median age, 22 years) with HSC were studied. Eleven patients had relapsed tumor and 5 had newly diagnosed tumor. During antifungal therapy for HSC, 12 of 16 patients were neutropenic for a median of 10 days (range, 6-91 days) and 11 were profoundly neutropenic for a median of 13 days (range, 1-55 days). Hepatosplenic candidiasis was successfully treated with complete antifungal response in 12 patients and a partial response in 2; 2 patients continued to receive AFT. No patient had breakthrough fungemia and two patients had progression of HSC, only one episode of which occurred during neutropenia. CONCLUSIONS: Hepatosplenic candidiasis in patients with cancer may be treated successfully under careful observation through repeated courses of chemotherapy-induced neutropenia without progression of hepatosplenic candidiasis or breakthrough fungemia. PMID- 8635044 TI - Screening for neuroblastoma in North America. Preliminary results of a pathology review from the Quebec Project. AB - BACKGROUND: The Quebec Neuroblastoma Screening Project was initiated to assess clinical and biologic aspects of neuroblastomas detected by screening infants born in the province of Quebec from May 1, 1989, to April 30, 1994. METHODS: Infants were screened for preclinical detection of neuroblastoma by determination of catecholamine metabolites, vanillylmandelic acid (VMA), and homovanillic acid (HVA). Patients with tumors discovered through this screening as well as patients in the same birth cohort with clinically detected tumors were referred to Quebec Oncology Centers for further investigation, diagnosis, and treatment. Pathology specimens were submitted to Childrens Hospital Los Angeles for central review. Tumors were histopathologically classified according to the Shimada system. RESULTS: As of August, 1993, 340,000 infants were screened at 3 weeks and 245,000 of them were retested at 6 months of age. Thirty-one tumors were detected through this screening and removed. Histologic material was available for 27 cases: 14 were detected at 3 weeks of age and 13 at 6 months of age. Twenty-six patients had tumors with favorable histology (FH), and one patient had a Stage I tumor with unfavorable histology (UH). At the time of this writing, all mass screening patients are alive, including one child with relapsed disease. During this period, 48 tumors were detected clinically in the same birth cohort, 40 of which were evaluated histologically. Of these 40 cases, 28 of 29 tumors diagnosed in patients up to age 12 months indicated an FH, whereas 9 of 11 tumors diagnosed in patients older than age 12 months indicated a UH. All patients with FH tumors are alive including a child with relapsed disease. The single patient with UH diagnosed before age 12 months died of disease. Of the nine patients with UH diagnosed after age 12 months, four died of disease, one relapsed, and four are alive (including one treated with bone marrow transplantation) after variable follow-up periods. CONCLUSIONS: The tumors detected by mass screening, similar to those tumors detected through clinical examination before age 12 months, were predominantly FH with good prognosis. However, those tumors that were missed by screening and were detected clinically after the patient was 12 months of age were predominantly UH, with serious clinical problems. This subgroup of patients not detectable by the current screening system presents an immediate and important clinical challenge that should be addressed in future studies. PMID- 8635045 TI - Two embryonal cancers after in vitro fertilization. AB - BACKGROUND: In vitro fertilization is not considered to be associated with an increased rate of pediatric malignancies, and only three have been reported in the literature. Two additional rare pediatric tumors in children conceived through this technique are reported. METHODS: Two children 12 and 18 months of age, developed hepatoblastoma and clear cell sarcoma of the kidney, respectively. They were both products of uneventful pregnancies induced by in vitro fertilization. No other environmental, prenatal, or family factor was found. RESULTS: The first child died after a failed remission induction with cisplatin, doxorubicin, and vincristine, whereas the second child is alive with no evidence of disease 18 months after diagnosis and treatment according to NWTS protocol. CONCLUSIONS: A possible association between in vitro fertilization and pediatric malignancies is suggested. PMID- 8635046 TI - Oncologists and primary care physicians' attitudes toward pain control and morphine prescribing in France. AB - BACKGROUND: Two representative samples of primary care physicians (N = 600) and medical oncologists (N = 300) in France were surveyed about their attitudes toward and knowledge about cancer pain management. METHODS: The survey was conducted by telephone with a questionnaire based on a model developed by the University of Wisconsin-Madison Pain Research Group. It was designed to assess physicians' estimates of the prevalence of pain among patients with cancer, their practice in prescribing analgesics, their training in cancer pain management, and the quality of care received by cancer patients in their own practice and in France. RESULTS: Barriers to adequate cancer pain management are prevalent and consistently more common among primary care physicians than among medical oncologists. Although 85% of primary care physicians and 93% of medical oncologists express satisfaction with their own ability to manage cancer pain, 76% of primary care physicians and 50% of medical oncologists report being reluctant to prescribe morphine for cancer pain. Both groups cite fear of side effects as their main reason to hesitate to prescribe morphine. Concerns about the risk of tolerance (odds ratio [OR], 1.15-2.52), perceptions that other effective drugs are available (OR, 1.11-2.41), perceptions that morphine has a poor image in public opinion (OR, 0.96-2.07), and the constraints of prescription forms (OR, 1.12-2.26) contribute significantly to physicians' infrequent prescription of morphine, as are being female (OR, 1.01-2.03) and being an older oncologist (OR, 1.09-2.51). CONCLUSIONS: This study (1) confirms the existence among French physicians of attitudinal barriers and knowledge deficits previously reported in other countries that can impede cancer pain management, (2) identifies new barriers to the proper prescription of morphine for cancer pain control, and (3) reveals discrepancies in physicians' attitudes and knowledge about pain control which suggest a need for the systematic evaluation of cancer patients' care. PMID- 8635047 TI - Epithelial noncarcinoid tumors and tumor-like lesions of the appendix. PMID- 8635048 TI - Malignant melanoma: primary surgical management (excision and node dissection) based upon pathology and staging. PMID- 8635049 TI - Pathologic findings from the National Surgical Adjuvant Breast Project (NSABP) protocol B-17: intraductal carcinoma (ductal carcinoma in situ) PMID- 8635050 TI - Axillary lymph nodes and breast cancer: a review. AB - BACKGROUND: The value of surgical staging and treatment of the axillary lymph nodes with either surgery or radiotherapy in the initial management of patients with Stage I or II invasive breast cancer is controversial. METHODS: A review of retrospective and prospective clinical studies was performed to assess the risks of axillary lymph node involvement and the effectiveness and morbidity of various treatment options. RESULTS: The risk of axillary lymph node involvement is substantial for most patients, even those with small tumors. The morbidity resulting from a careful Level I/II axillary dissection or moderate-dose axillary radiotherapy is limited. Such treatment is highly effective in preventing axillary recurrence. The symptoms resulting from axillary failure can be controlled in many, but not all, patients. The available data suggest, but do not prove, that the initial use of effective axillary treatment may result in a small improvement in long term outcome in some patient subgroups. CONCLUSIONS: Most patients should be treated with either axillary surgery or irradiation. Highly selected subgroups of patients may have such low risks of involvement that specific axillary treatment is of little value. However, such subgroups have not yet been well defined. Treatment approaches that do not involve specific axillary treatment should be considered investigational at present, and the patients should be informed as to their potential risks. Prospective clinical studies of these issues should be pursued. PMID- 8635051 TI - Detection of human papillomavirus DNA sequences in oral squamous cell carcinomas and their relation to p53 and proliferating cell nuclear antigen expression. AB - BACKGROUND: The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. METHODS: Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. RESULTS: Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Human papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-16 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. CONCLUSIONS: These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes. PMID- 8635052 TI - High prevalence of human papillomavirus in squamous cell carcinoma and matched normal esophageal mucosa: assessment by polymerase chain reaction. AB - BACKGROUND: Studies using DNA technology have reported the presence of human papillomavirus (HPV) DNA in esophageal carcinomas, suggesting that it could play a role in the pathogenesis of this tumor. In the present study, in addition to DNA from neoplasms, normal mucosa was screened for viral DNA, assuming that this would increase HPV detection substantially. METHODS: Seventeen patients with esophageal carcinoma and 10 control subjects were studied. In 8 of the patients, normal mucosa was also available. Polymerase chain reaction (PCR) was performed using primers for the E6 region of HPV-16 and HPV-18. Koilocytosis, a commonly accepted histopathologic marker of viral infection, was studied, and results were correlated with PCR findings. RESULTS: DNA from neoplastic lesions was positive for HPV-16 and HPV-18 in 8 of 16 (50%) and in 3 of 16 (18.8%), respectively. When tumor tissue and normal mucosa were available, PCR results were 3 of 8 (37.5%), 5 of 8 (62.5%), and 8 of 8 (100%) for HPV-16, in tumor, normal mucosa, and both. For HPV-18, results were 0 of 8 (0%), 5 of 8 (62.5%), and 5 of 8 (62.5%), respectively. In comparison with tumor samples, positivity in normal mucosa was increased for HPV-18 and for both viral genotypes (P = 0.01). No amplification was obtained in the control group. Koilocytosis was present in 33% of the cases. CONCLUSIONS: These results suggested a high prevalence of HPV in esophageal carcinoma. The detection rate is significantly higher in normal mucosa specimens, suggesting that infection probably antedates tumor development. Koilocytosis was substantially less sensitive than PCR. PMID- 8635053 TI - Sialosyl Tn antigen expression is associated with the prognosis of patients with advanced gastric cancer. AB - BACKGROUND: Several studies have revealed a correlation between sialosyl Tn antigen (STN) and certain clinicopathologic features of various cancers, and that STN is an independent prognostic factor. However, the clinical significance of the expression of STN in gastric cancer has not been reported. Thus, the purpose of this study was to evaluate immunohistochemically the clinical significance of expression of STN in gastric cancer. METHODS: The expression of STN in surgically resected specimens of human gastric cancer was evaluated immunohistochemically using a monoclonal antibody (TKH-2), in 60 patients whose serum STN levels were measured and in 54 patients with advanced cancer who had been followed for more than 5 years after gastrectomy. The correlations between the level of STN expression and clinicopathologic factors were analyzed. The staining intensity was graded as follows: (-), less than 5% of the cancer cells expressed STN; (+), 5-50%; (++), more than 50%. RESULTS: Sialosyl TN antigen staining was detected mainly on the cell membrane, in the cytoplasm, and in the luminal contents, and 57.2% of the 60 specimens expressed STN, whereas the corresponding value for positive serum levels was 15%. A higher percentage of advanced tumors expressed STN than did the early cases, but the difference was not statistically significant. All cases with strong staining, the (++) cases, were advanced cases either with lymph node metastases or with cancer invading in or beyond the muscle layer proper. The expression of STN appeared to be related to the clinical stage, the extent of cancer invasion, and the presence of lymph node metastases. Sialosyl TN antigen was detected in the serum in less than 6% of the patients whose tumors were (-) or (+) for STN expression, and in 86.7% of the patients whose tumors expressed high levels of STN (++). The estimated 5-year survival in advanced cases (Stage III) was significantly better in those with negative STN expression than in those with positive STN expression (P < 0.01). CONCLUSIONS: These results suggest that STN may be a useful marker associated with the prognosis of patients with advanced gastric cancer. PMID- 8635054 TI - Resection and adjuvant chemotherapy of pulmonary blastoma: a case report. AB - BACKGROUND: Pulmonary blastoma is a rare malignancy treated primarily with surgery. Chemotherapy has proven effective in some cases, and has been used in adjuvant fashion in pediatric patients. METHOD: This is a case report of a 40 year-old woman treated with surgical resection, and because of the high risk of recurrence, adjuvant chemotherapy was also given. RESULTS: At the time of this writing, the patient has been in continued remission since surgery in October, 1993. CONCLUSION: This rare entity is difficult to diagnose noninvasively. The limited number of case reports makes therapeutic decisions difficult. To the authors' knowledge, this case is the first reported adult treated with adjuvant chemotherapy. A central registry of cases would be helpful to physicians treating patients with this disease. PMID- 8635055 TI - Mucinous cystadenoma of the lung. AB - BACKGROUND: Mucinous cystadenoma is an unusual pulmonary tumor that must be distinguished from mucinous cystic carcinoma and mucinous cystic tumors of borderline malignancy. METHODS: This study of two cases was performed to characterize mucinous cystadenoma clinically and immunohistochemically, using proliferation markers (proliferating cell nuclear antigen [PCNA], MIB1) and carcinoembryonic antigen expression. RESULTS: Pathologic examination in each instance showed unilocular cysts containing abundant clear mucus. The cysts were lined by tall mucinous epithelium, with absence of cytologic atypia and invasive growth. Proliferation markers using immunohistochemical methods showed less than 10% and 5% of labeled nuclei, respectively. Carcinoembryonic antigen immunostaining in both cases was negative. Patients remained free from recurrence for at least 2 years after surgery. CONCLUSIONS: Mucinous cystadenoma of the lung appears to be a benign neoplasm because of its clinical course and immunohistochemical low expression of proliferation markers such as PCNA and MIB1. PMID- 8635056 TI - Clonal changes in inflammatory pseudotumor of the lung: a case report. AB - BACKGROUND: Pulmonary inflammatory pseudotumor, also known as plasma cell granuloma among many other names, is widely believed to be an inflammatory or reactive lesion rather than a neoplasm, although its pathogenesis is still controversial. METHODS: Cytogenetic analysis was performed on a lung mass that showed typical clinical and pathologic features of inflammatory pseudotumor. Ultrastructural and immunohistochemical studies were performed in addition to routine histologic examination. RESULTS: Cytogenetic study of the lesion revealed clonal anomalies of t(1;2)(q21;p23) and del(4)(q27). The patient, a 30-year-old woman, presented with an asymptomatic but enlarging right lower lobe mass for which partial right lower lobectomy was performed. The lung mass was well circumscribed radiographically and grossly. Microscopically, it was characterized by a loosely arranged spindle cell proliferation with prominent plasma cell infiltration. Fibroblastic and myofibroblastic differentiation of the spindle cells was demonstrated by ultrastructural and immunohistochemical studies. CONCLUSION: To the authors' knowledge, this is the first report of clonal cytogenetic changes in a clinically and pathologically typical case of inflammatory pseudotumor in the lung. This finding suggests that pulmonary inflammatory pseudotumor might be a true neoplasm rather than a purely inflammatory or reactive lesion. PMID- 8635057 TI - Chondrosarcoma of the jaw and facial bones. AB - BACKGROUND: Osteosarcomas of the jaw frequently have chondroblastic differentiation, causing confusion with chondrosarcomas. METHOD: Clinicopathologic features and results of treatment were analyzed for a series of 56 patients (27 males and 29 females from 1.5 to 88 years of age) with chondrosarcoma of jaw and facial bones. Twelve patients (21.4%) were younger than 20 years. RESULTS: The major symptom was nasal obstruction or a painless mass; the median interval from the first symptom until initial treatment was 1 year. Of the 56 chondrosarcomas, 25(44.6%) involved the alveolar portion of the maxilla and maxillary sinus; 23 (41.1%) involved the nasal septum, ethmoid, and sphenoid; 6 (10.7%) involved the mandible; and 2 (3.6%) involved the nasal tip. Of the 19 patients with radiographic studies, 15 (78.9%) had an expanding soft tissue mass with varied matrix calcification and destruction of bone and 2 had a purely lytic lesion. The lesion was difficult to assess in the two others. Most tumors had a lobulated growth pattern of hyaline cartilage. Hypercellularity, nuclear pleomorphism, and binucleation were common features. Forty-three tumors were grade 1, 13 were grade 2, and none were grade 3. Modalities of treatment were known for 51 of the 56 patients. Forty-six patients (90.2%) had surgical treatment, 2 (3.9%) had combination radiation therapy and chemotherapy, 1 (2%) had radiation therapy alone, and 2 (3.9%) had biopsy only. Follow-up adequate for analysis was obtained for 42 patients. Of these, 14 (33.3%) had local recurrence; uncontrolled recurrence developed in 9 (21.4%) patients. No distant metastases were documented. Overall actuarial survival at 5, 10, and 15 years was 80.7%, 65.3%, and 56%, respectively. Survival was analyzed for location, size, and histologic grade of tumor. No statistically significant differences were found. CONCLUSIONS: Chondrosarcomas of the jaw and facial bones are extremely rare, locally aggressive tumors. PMID- 8635058 TI - Evolution of bone densitometry in patients with myeloma treated with conventional or intensive therapy. AB - BACKGROUND: Demineralization is a common hallmark of multiple myeloma (MM) that can be evaluated by dual-energy X-ray absorptiometry (DEXA). The evolution of lumbar and whole body bone density were investigated by DEXA in patients with MM treated by conventional or intensive therapy supported by autologous blood stem cell transplantation. METHODS: Sixty six patients younger than 66 years with MM were randomly assigned to either conventional (30 patients, Group A) or intensive therapy supported by autologous blood stem cell transplantation (36 patients, Group B). For all patients, lumbar bone mineral density (BMD) was measured by DEXA at diagnosis and 13.2 +/- 4.2 months after the initiation of treatment. Whole body examinations were performed in 45 patients; in addition to whole body BMD, independent BMD values were recorded for various skeletal sites. RESULTS: At diagnosis, mean lumbar Z score (lumbar mean BMD value) was low (-1.24 +/- 1.45) without any significant difference between the 2 groups. Under treatment, lumbar BMD increased 0.7% in Group A and 4.6% in Group B (P = 0.02). This difference was mainly related to nonresponders in group A who featured a lumbar BMD change of 3.9%, whereas patients in remission in both groups displayed a 4.1% increase (P < 0.001). There was a correlation between the variation of lumbar BMD and the decrease of the serum or urinary monoclonal component (r = 0.34, P = 0.006). After intensive therapy, increase of lumbar BMD was higher in men than in women (7.2% vs. 1%, P = 0.005) perhaps because of variations in hormonal status in women. Unexpectedly, whole body BMD decreased in responders (-3%) because of a decrease in appendicular BMD outweighing the increase in axial BMD. This suggests a redistribution from cortical to cancellous bone in patients with MM responsive to chemotherapy. CONCLUSION: Bone densitometry is a marker of treatment response that may be particularly useful in nonsecretory and light chain MM. Moreover, it provides new information on bone remodeling in patients treated for MM, which may have therapeutic consequences. PMID- 8635059 TI - Myeloid markers in adult acute lymphocytic leukemia. Correlations with patient and disease characteristics and with prognosis. AB - BACKGROUND: The expression of myeloid markers on lymphoblasts has been associated with adverse outcome in acute lymphocytic leukemia (ALL). The purpose of the study was to analyze the experience with adults treated at the University of Texas M. D. Anderson Cancer Center with myeloid marker- (MY) positive ALL in relation to patient and disease characteristics, response to therapy, and prognosis. METHODS: Since 1988, 64 of 162 adults (40%) with newly diagnosed ALL referred to our service had MY-positive ALL. Their characteristics and outcomes were compared with the 98 patients with MY-negative ALL. Patients were treated with the vincristine-doxorubicin-dexamethasone (VAD) regimens. RESULTS: Patients with MY-positive ALL were significantly older (median ages, 47 years vs. 33 years; P = 0.03), had a higher incidence of CD34 antigen expression (59% vs. 36%; P < 0.01), and a lower incidence of common acute leukemia antigen expression (50% vs. 71%; P < 0.01), serum alkaline phosphatase elevation (58% vs. 83%; P < 0.01), and thrombocytopenia at diagnosis (49% vs. 69%; P = 0.02). Myeloid marker positivity, as expected, was significantly higher in null cell ALL (82%), and significantly lower in mature B-cell ALL (17%) (P < 0.01). Forty-one of 64 MY positive patients achieved complete remission (CR) after induction therapy compared with 76 of 98 patients MY-negative disease (CR rate 64% vs. 78%; P = 0.06). With a median follow-up of 45 months, no statistical differences were observed in remission duration or survival between MY-positive and MY-negative patients, overall, and within immunophenotypic subsets (T-cell vs. others), or among subgroups with single marker (CD13, CD14, CD33, CD34) positivity. The 3 year remission duration rates were 32% for MY-negative and 40% for MY-positive patients (P not significant), and 3-year survival rates were 26% and 31%, respectively (P not significant). CONCLUSIONS: With VAD therapy, myeloid marker positivity is not associated with significant differences in prognosis in adult ALL. PMID- 8635060 TI - Familial cutaneous malignant melanoma and tumors of the nervous system. A hereditary cancer syndrome. AB - BACKGROUND: Excessive risk of cutaneous melanoma as a second cancer has been associated with benign or malignant tumors of the nervous system. Cutaneous melanoma and nervous system tumors may independently aggregate in families. There are, however, no previous reports of increased likelihood of tumors of the nervous system in families of patients with cutaneous melanoma or--of cutaneous melanoma in families with tumors of the nervous system. METHODS: The occurrence of nervous system tumors as second cancers was examined in a series of 904 patients with cutaneous melanoma and/or their family members. RESULTS: Fifteen families with 17 members with cutaneous melanoma from this series had one or more additional relatives with tumors of the nervous system, including astrocytoma, medulloblastoma, glioblastoma multiforme, ependymoma, glioma, meningioma, and acoustic neurilemmoma. Another subgroup of 10 patients with cutaneous melanoma had either a meningioma (n = 9) or acoustic neurilemmoma (n = 1) as a second tumor. The pattern of atypical melanocytic nevi occurring in the majority (20/25) of cutaneous melanoma patients in our series and in additional first degree relatives of 9 of 11 of the affected families, has been previously associated with the Familial Atypical Multiple Mole-Melanoma syndrome. CONCLUSIONS: This unusual familial and personal occurrence of tumors, showing differentiation toward tissues of the neural crest, neuroepithelium, and/or mesenchymal derivation, supports a putative association with a hereditary cancer susceptibility trait. PMID- 8635061 TI - The impact of mammography in 1096 consecutive patients with breast cancer, 1979 1993: equal value for patients younger and older than age 50 years. AB - BACKGROUND: Although the benefit of screening mammography in healthy women younger than age 50 remains controversial, few studies have addressed the impact of mammography over time and by patient age, on means of diagnosis and stage of disease among women with breast cancer. METHODS: One thousand ninety-six consecutive patients with operable breast cancer (both invasive and noninvasive intraductal) treated in this practice between 1979 and 1993 were stratified by primary means of diagnosis (patient, physician, or mammography), age < 50 years, 50 years and older), and time period (1979-83, 1984-88, and 1989-93). Tumor size, lymph node status, and tumor type were compared among these groups. RESULTS: (1) Mammography was the primary means of diagnosis in 28.8% of younger and 42.4% of older patients treated most recently (1989-1993), significantly more often than in the past (P < 0.0005). (2) For cancers diagnosed primarily by either patients or physicians, there was not significant change over time (for either younger or older patients) in clinical presentation, tumor size, lymph node status, or histology. (3) For cancers diagnosed primarily by mammogram, i) 95% of patient self-exams and 56% of physician exams were negative, ii) tumor size was significantly smaller (P < 0.00005), iii) lymph nodes more often were negative (P = 0.0002), and d) histology was more likely to be either in-situ or microinvasive (P < 0.00005). These findings were equally true for younger and older patients. CONCLUSION: Increased use of mammography, rather than improvement in patient or physician breast examination, explains the progressively earlier stage of breast cancers found in recent years, a benefit equally apparent in patients younger and older than age 50. PMID- 8635062 TI - Sociodemographic factors associated with the diagnostic staging of breast cancer in southern Italy. AB - BACKGROUND: There are marked regional differences in breast cancer mortality rates in Italy, probably linked to factors such as diagnostic delay, therapeutic strategies, and biologic and sociodemographic differences. To investigate a possible link between sociodemographic factors (e.g. age, education, and residence) and delay in the diagnosis of breast cancer, data were evaluated from all such patients from our Institute living in the Campania Region of Southern Italy for 1991-1993. METHODS: Patients were grouped into Tis-T1/N0-N+ versus T2 4/N0-N+ and the variables examined were age (< 40, 41-50, 51-60, > 60 years), education (< or = 5 vs. > 5 school years) and residence (urban vs. rural). An analysis was made using the Pearson's Chi-square test and the multiple logistic regression. RESULTS: Statistically significant differences were found for both residence (P = 0.04) and education level (P = 0.03) in the older than 60 years age group, but only for residence (P = 0.03) in the 51-60 years age group. The risks according to Mantel-Haenszel were 1.28 for education (P = 0.08) and 1.32 for residence in rural municipalities (P = 0.05). The odds ratio for residence in rural municipalities, adjusted by education and by the education-residence interaction, was 2.26 (95% confidence interval [CI], 1.12-4.54) in the 51-60 years age group and 1.74 (95% CI, 1.01-3.00) in the older than 60 years age group. CONCLUSIONS: These data clearly indicate that residents of rural municipalities, as well as poorly educated subjects, are more likely than their respective counter-parts to have a delayed diagnosis of breast cancer. PMID- 8635063 TI - Clear cell adenosquamous carcinoma of the cervix. An aggressive tumor associated with human papillomavirus-18. AB - BACKGROUND: It is well recognized that adenocarcinomas and adenosquamous carcinomas of the cervix are frequently associated with human papillomavirus (HPV)-16 or -18. However, few studies have investigated associations between histologic variants of these tumors and specific types of HPV. METHODS: Eleven cases of cervical adenosquamous carcinoma with an unusual histologic appearance were characterized using histochemical and immunohistochemical stains. Sections were tested for the presence of HPV DNA using the polymerase chain reaction (PCR) and type specific primers for HPV-16 and -18. Clinical outcome was determined from a chart review. RESULTS: All tumors were histologically characterized by the presence of sheets of cohesive cells with prominent cell borders and a vacuolated or clear cytoplasm containing large amounts of glycogen. All tumors had focal gland formation and stained positive with mucicarmine stain. Using PCR, HPV-18 DNA was identified in all cases. The youngest patient was 24 years old and the oldest 74 years (mean, 43 years). Eight (73%) of the 11 patients have developed recurrent disease with a mean follow-up until recurrence of 9.5 months (range, 3 22 months). Seven (64%) of the 11 patients have died of their cervical tumors. Of the five patients with Stage IB disease, three (60%) have died of their cervical tumors. CONCLUSIONS: A subset of invasive cervical adenosquamous carcinoma associated with HPV-18 that has a distinctive histologic appearance and an aggressive clinical course is described. The term "clear cell adenosquamous carcinoma" is proposed for this unique variant of invasive cervical carcinoma. PMID- 8635064 TI - Operator variability in disease detection and grading by colposcopy in patients with mild dysplastic smears. AB - BACKGROUND: To determine interoperator variability in the colposcopic evaluation of patients with mild dysplastic smears, retrospective comparison of colposcopy and biopsy results in 856 patients examined by 11 colposcopists in the Outpatient Colposcopy Clinic of "L. Mangiagalli" Institute, Milano, was performed. METHODS: The patients underwent a complete colposcopic assessment and target biopsy on suspect areas if a screening smear showed mild dysplasia. Colposcopic findings and disease detection rate for each operator were compared. RESULTS: There was no significant (P > 0.05) difference in the recording of abnormal colposcopic findings. Significant differences were found, however, in abnormal transformation zone grading (P < 0.001), biopsy rate (P < 0.05), and squamocolumnar junction visualization (P < 0.005). There was no significant difference in overall disease detection (P > 0.5) but there was a statistically significant, inverse relation (r = -0.6, P < 0.01) between the high grade (cervical intraepithelial neoplasia grades 2 and 3) and low grade (human papillomavirus/cervical intraepithelial neoplasia grade 1) detection rate for each operator. CONCLUSIONS: High grade disease detection in patients with mild dysplastic smears is influenced the subjectivity of the colposcopic examination. This should be considered when planning optimal management for patients with mild dysplasia. PMID- 8635065 TI - Secondary cytoreductive surgery for recurrent ovarian cancer. A prospective study. AB - BACKGROUND: The prognosis for patients with recurrent epithelial ovarian cancer is poor. Most are treated with salvage chemotherapy. The role of secondary cytoreductive surgery is controversial. Hence, this prospective study was undertaken to determine the feasibility and benefit of secondary cytoreductive surgery before the administration of salvage chemotherapy. METHODS: Between 1990 and 1994, 36 patients with recurrent epithelial ovarian cancer underwent secondary surgical cytoreduction. All had prior primary cytoreductive surgery, platin-based chemotherapy, and had relapsed at least 6 months after completion of primary treatment. The goal was the excision of all macroscopic disease before initiation of chemotherapy or radiation therapy. Statistical analysis was undertaken to determine which clinical and pathologic variables influenced the feasibility of complete excision as well as morbidity, mortality, survival benefit, and quality of life resulting from secondary cytoreductive surgery. RESULTS: Thirty (83.0%) patients had complete surgical excisions. The probability of a complete excision was influenced by Gynecologic Oncology Group (GOG) performance status (0-2 vs. 3, P = 0.05) and size of largest tumor deposit (< 10 cm vs. > 10 cm, P = 0.03). Eleven (30.1%) patients experienced morbidity and 1 (2.8%) died postoperatively. Of 27 symptomatic patients with at least 3 months of follow-up, 26 (96.2%) had resolution or improvement of their symptoms. Of 25 followed for at least 6 months postoperatively, 23 (92.0%) had a GOG performance status of 0 or 1. Survival was adversely influenced by the administration of salvage chemotherapy before surgery (P = 0.02), a preoperative GOG performance status of 3 (P = 0.01), and a brief disease free interval after completion of primary treatment (P = 0.01). The median survival was extended for patients completely resected before salvage chemotherapy or radiation, compared with those with macroscopic residual disease remaining (43 vs. 5 months, P = 0.03). CONCLUSIONS: Complete secondary cytoreductive surgery for recurrent epithelial ovarian cancer is technically feasible and has an acceptable operative complication rate. Survival is significantly improved for patients having complete resection. Subsequent relief of symptoms and performance status are excellent. PMID- 8635066 TI - Soluble interleukin-2 receptor alpha is elevated in sera of patients with benign ovarian neoplasms and epithelial ovarian cancer. AB - BACKGROUND: Previous studies have established that soluble interleukin-2 receptor alpha (sIL-2R alpha) levels are elevated in ascites and sera from individuals with advanced ovarian cancer (International Federation of Gynecology and Obstetrics [FIGO] Stage III/IV). This study was undertaken to evaluate sIL-2R alpha levels in individuals with benign ovarian neoplasms and early stage ovarian cancer (FIGO Stage I/II). Comparison with CA 125 levels was performed to assess screening potential. METHODS: Sera from 92 healthy individuals, 61 with benign adnexal masses, 12 patients with FIGO Stage I/II ovarian cancers, and 27 patients with FIGO Stage III/IV ovarian cancers were assayed for sIL-2R alpha by enzyme linked immunosorbent assay and CA 125 by radioimmunoassay. RESULTS: The mean serum sIL-2R alpha levels for benign pelvic masses, and Stage I/II and Stage III/IV epithelial ovarian cancer were 1507 +/- 82, 1631 +/- 274, and 2596 +/- 384 U/ml, respectively. The difference between mean serum sIL-2R alpha levels in individuals with benign adnexal masses and Stage III/IV epithelial ovarian cancer was statistically significant (P < 0.05). In addition, of the four individuals with FIGO Stage I/II ovarian cancer who had CA125 levels below 35 U/ml, the accepted upper limit of normal, three patients had elevated serum sIL-2R alpha levels. Eleven of 12 patients (92%) with potentially curable Stage I/II disease had elevated serum levels of either sIL-2R alpha or CA125 and 8 of 12 (67%) had elevations of both sIL-2R alpha and CA125. Sensitivity and specificity of a combination of CA 125 and soluble IL-2R alpha were 88.5% and 27.1%, respectively. CONCLUSION: Soluble interleukin-2 receptor alpha levels do not appear to differentiate between benign adnexal lesions and early malignancy; however, measurement of sIL-2R alpha levels in combination with CA125 warrants further evaluation to determine if together they will identify individuals with Stages I and II ovarian cancer. PMID- 8635067 TI - Surgery with adjuvant irradiation in patients with pathologic stage C adenocarcinoma of the prostate. AB - BACKGROUND: In recent years, the routine use of prostate-specific antigen (PSA) to detect cancer of the prostate (CaP) early has renewed the controversy regarding radiotherapy versus radical prostatectomy as the superior definitive treatment. Radiotherapy alone has been reported to result in a high incidence of local recurrence, whereas on the other hand surgical treatment has resulted in a high incidence of microscopic residual tumor. The purpose of this study was to review our treatment results with radical prostatectomy followed by planned courses of postoperative irradiation in patients with pathologic Stage (PS) C disease. METHODS: From 1972 to 1989, 95 patients with CaP with PS C tumors were treated with radical prostatectomy and bilateral pelvic lymphadenectomy. Pathologic stage distribution was: C1 in 26 (27%), C2 in 37 (39%), and C3 in 32 (34%) patients. The median follow-up was 6 years. All 95 study patients received postoperative pelvic irradiation as the only adjuvant treatment. Radiotherapy treated volume included the prostatic fossa and its immediate vicinity. The RT dose ranged from 33 Gy to 61.8 Gy (median, 45 Gy). RESULTS: The overall 5- and 10 year actuarial survival rates were 94% and 73%, respectively, with the 5 and 10 year disease specific survival of 98% and 91%, respectively. Clinical and/or prostate specific antigen recurrence was 31% at 5 years and 44% at 10 years. Prostate specific antigen elevation without clinical evidence of recurrent disease was recorded in 26 (27%) patients. Seminal vesicle involvement (C3) and high Gleason's score (8-10) were the most important factors predicting recurrence. Of the 95 patients treated, 2 had pelvic recurrence alone and 1 had local and distant metastatic disease. Radiotherapy was well tolerated with no clinically important morbidity. CONCLUSION: Based on this experience, moderate dose adjuvant radiotherapy after radical prostatectomy in patients with PS C CaP is recommended. PMID- 8635068 TI - A phase II study of subcutaneous recombinant human interleukin-4 in metastatic renal cell carcinoma. AB - BACKGROUND: A study was conducted to assess the response rate for and toxicity of recombinant human interleukin-4 (IL-4) administered subcutaneously to outpatients with metastatic renal cell cancer. METHODS: Human recombinant IL-4 provided by Schering-Plough Research Corporation was administered subcutaneously to 19 patients at a dose of 1 microgram/kg three times per week. Eligibility included Cancer and Leukemia Group B performance status of 2 or better, adequate hematologic (leukocyte count < or = 2500/microliters platelets < or = 75,000/microliters), renal (creatinine < or = 2.0 mg/dl), and hepatic (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and alkaline phosphatase < or = 3 times the upper limit of normal) function. Exclusion criteria included prior immunotherapy, clinically significant diabetes, pulmonary disease, and history of congestive heart failure or an ejection fraction of less than 40%. Soluble CD23 was measured in prospectively collected serum via an enzyme-linked immunosorbent assay technique. RESULTS: There was one minor response among 18 evaluable patients and median survival was only 35 weeks. Toxicities included fever, fatigue, myalgias, arthralgias, nausea, and anorexia. One patient each experienced a 14% asymptomatic decrease in the cardiac ejection fraction, a gastrointestinal bleed, and oral angioedema. Ten patients noted increased pain in tumor bearing areas, especially in areas of bony disease. Two patients with pre-existing vertebral disease experienced symptoms of cord compression. In 12 patients for whom complete data were available there was a mean increase in soluble CD23 of 2.6 ng/ml (P = 0.002) 1-2 weeks after therapy initiation. CONCLUSIONS: At this modest dose and schedule, IL-4 was tolerated in most patients. There was minimal biologic and clinical activity. Further development of IL-4 as a therapeutic agent in metastatic renal cell cancer at this dose and schedule is not supported by this study. PMID- 8635069 TI - The increasing incidence of malignant gliomas and primary central nervous system lymphoma in the elderly. AB - BACKGROUND: Studies indicate that the incidence of primary malignant brain tumors in the elderly is increasing, but this may reflect increased case ascertainment due to the introduction of computed tomography (CT) scanning. In addition, tumor histology was not available in these studies. This study was conducted to determine whether the incidence of primary malignant brain tumors in the elderly in Florida is increasing and to verify the histology of these increases. METHODS: Using the number of primary malignant brain tumors reported to the Florida Cancer Data System (FCDS) when CT scanning was available, incidence rates per 100,000 people were calculated. Incidence density ratios (IDRs) were calculated for 1986 1989 relative to 1981-1984. RESULTS: Tumor incidence at ages 20-64 years increased from 5.7 in 1981-1984 to 5.9 in 1986-1989, with an IDR of 1.05 (not significant). The incidence in those aged 65 years or older rose from 14.8 to 18.3, with an IDR of 1.23 (P < 0.001). The increase was 15% in those aged 65-69, 16% in those 70-74 years, 30% in those 75-79 years, 36% in those 80-84 years, and 254% in those 85 years or older. Indicence density ratios in those aged 65 years or older were 0.92 (not significant) for astrocytoma, 2.7 (p < 0.001) for anaplastic astrocytoma, 1.32 (p < 0.001) for glioblastoma and 3.56 (p < 0.001) for lymphoma. In those aged 65+ the incidence of all cancers rose 7.6% (not significant), and the incidence of pancreatic cancer (another neoplasm that requires CT scanning for diagnosis) rose 0.34% (not significant). CONCLUSIONS: The incidence of primary brain tumors in elderly Floridians has increased. This increase is independent of increased case ascertainment associated with the introduction of CT scanning and independent of a general increase of all cancers. The rise in brain tumor incidence is observed in anaplastic astrocytoma, glioblastoma, and lymphoma but not astrocytoma. This study confirms the increase is histologically specific and not due to increased case ascertainment. Further investigation into the etiology of this increase is warranted. PMID- 8635070 TI - Oncoproteins and tumor progression in papillary thyroid carcinoma: presence of epidermal growth factor receptor, c-erbB-2 protein, estrogen receptor related protein, p21-ras protein, and proliferation indicators in relation to tumor recurrences and patient survival. AB - BACKGROUND: The prognostic relevance of activated oncogenes and oncoproteins has not been well studied in papillary thyroid cancer, which is slow-growing, with only regional spread in most cases. Therefore, the influence of some protein markers and tumor cell proliferation on disease progress in a series of patients with surgically treated papillary thyroid carcinoma was studied. METHODS: One hundred twenty-seven patients with papillary thyroid carcinoma larger than 10 mm in greatest dimension were studied retrospectively, and the majority were treated with total or near-total thyroidectomy. Immunohistochemical and flow cytometric analyses of paraffin embedded tumor material were performed and the results were related to time to recurrence and thyroid cancer deaths using the univariate product-limit survival analysis and the multivariate Cox' regression method. RESULTS: Immunohistochemical expression of c-erbB-2 protein, estrogen receptor related protein (p29), which may be regarded as an indicator of hormone-dependent growth, S-phase, G2M-phase, sex, age, histologic grade, and primary tumor extent were all of significant prognostic importance in univariate analyses of patient survival. In the final Cox' model, however, only male sex (P = 0.017), older age, (P < 0.00005) and high grade histologic features (P = 0.006) were associated independently with decreased survival. Among females, decreased expression of c erbB-2 protein was independently related to decreased patient survival (P = 0.019). In multivariate analysis of time to recurrence, lymph node status (P = 0.0001), epidermal growth factor (EGF) receptor expression (P = 0.013) and estrogen receptor-related protein (P = 0.007) were independent risk indicators, and S-phase fraction (P = 0.074) showed a borderline significance. CONCLUSION: In this study, sex, age, and histologic grade were independent indicators of deaths from thyroid cancer. Lymph node metastases, EGF-receptor expression, and decreased estrogen receptor-related protein staining persisted as prognostic variables in the final multivariate analysis of recurrence free survival. Alterations in these oncogenes thus seem to play some role in disease progression in papillary thyroid carcinoma, although conventional variables are still important as prognostic indicators. PMID- 8635072 TI - Primary hypothyroidism as a consequence of 131-I-metaiodobenzylguanidine treatment for children with neuroblastoma. AB - BACKGROUND: 131-I-metaiodobenzylguanidine is a radioiodinated compound selectively concentrated by cells of neuroectodermal origin, including neuroblastoma cells, for this reason it may represent a promising treatment modality for neuroblastoma in childhood. Although a potential side effect of 131 I-MIBG administration is thyroid dysfunction, relatively few data are reported about this issue. METHODS: A series of 14 long term surviving patients with neuroblastoma who had been treated with 131-I-MIBG courses ranging from 2.5 to 5.5 gigabecquerels after surgical and conventional pharmacologic therapy is reported. RESULTS: Twelve patients developed primary hypothyroidism that was clinically overt in 8 patients and compensated in 4 patients within 6-12 months of completion of 131-I-MIBG administration. Only in two patients was thyroid function spared. Significant correlations between the cumulative dose of 131-I MIBG and the degree of thyroid failure were not found. CONCLUSIONS: Primary hypothyroidism appears to be a common side effect in children with neuroblastoma treated with 131-I-MIBG. This finding suggests that methods to preserve thyroid function other than oral administration of iodide should be sought. PMID- 8635071 TI - Accelerated fractionation radiotherapy and concomitant chemotherapy in patients with stage IV inoperable head and neck cancer. AB - BACKGROUND: Stage IV inoperable head and neck cancer has a 2-year mortality rate of greater than 70% when treated with conventional radiotherapy. A Phase II study was undertaken to evaluate the effects of concomitant chemotherapy and accelerated, interrupted, twice-a-day radiotherapy on tumor response, locoregional control, survival, and morbidity. METHODS: Thirty-four patients with Stage IV inoperable squamous cell carcinoma of the head and neck and a minimum follow-up of 36 months were evaluated. Concomitant chemoradiotherapy was administered during weeks 1, 3, and 5 (with planned breaks during weeks 2 and 4), consisting of cisplatin 60 mg/m2 on day 1, continuous 5-day infusion of 5 fluorouracil, 750 mg/m2 per day, and radiotherapy, 2 Gy twice a day, more than 6 hours apart, followed by 3 days of radiation therapy alone (final "boost") in week 6, for a total dose of 70 Gy and treatment duration of 5 1/2 weeks (38 days). RESULTS: Twenty-seven patients achieved a clinical complete response (82%). Actuarial locoregional control at 3 years was 73% and the actuarial 3-year survival probability, including all deaths, was 38%. All locoregional recurrences were manifested within 12 months. Of the 20 deaths, 12 were tumor related (locoregional and/or metastatic), 3 were treatment related, and 5 were due to other causes. Acute toxicity consisted of grade 3 mucositis and dysphagia and grade 2-3 leukopenia, not requiring treatment interruption or cessation. CONCLUSION: Concomitant accelerated radiation therapy and chemotherapy is a feasible treatment approach in this prognostically poor patient population, yielding dramatic tumor responses and impressive locoregional control at the cost of somewhat increased acute toxicity. Although serious late complications have not been observed, caution should be exercised in view of the relatively short follow up. PMID- 8635073 TI - Treatment of uveal melanoma metastatic to the liver: a review of the M. D. Anderson Cancer Center experience and prognostic factors. AB - BACKGROUND: Liver metastasis develops in approximately two-thirds of patients with recurrent uveal melanoma. Despite therapy, the median survival of those with liver metastasis is 5 to 7 months. The recognition of a grave prognosis associated with liver metastasis has led to evaluation of new modalities of therapy, including the use of regional therapies such as intrahepatic arterial chemotherapy and either embolization or chemoembolization of hepatic metastases. In this study, the results of an institutional experience over the past 2 decades are reviewed and prognostic factors that affect survival from the time the liver metastasis is diagnosed are assessed. METHODS: In this study of 201 patients with uveal melanoma involving the liver who were treated at M. D. Anderson Cancer Center between 1968 and 1991, the authors restrospectively reviewed the cases and compared the results of systemic therapies, hepatic intra-arterial chemotherapies, and chemoembolization of liver metastases. Cox's multivariate analysis and stepwise logistic regression were then computed to determine significant prognostic variables. RESULTS: The systemic therapies produced a response rate of less than 1%. Chemoembolization was the most effective treatment, inducing responses in 36% of patients. Survival curves were calculated using the life-table method of Kaplan and Meier. Patient- and tumor-related characteristics were examined and their relation to on survival from the time of diagnosis of liver metastasis was determined. Levels of serum alkaline phosphatase, total bilirubin, and lactic dehydrogenase plus response to treatment showed a strong relation to survival. In contrast, univariate analysis showed that patient age and gender, metastasis free interval, presence of extrahepatic metastasis, and type of therapy for liver metastasis did not influence survival. Multivariate stepwise regression analysis identified serum alkaline phosphatase and metastasis free interval as the main independent prognostic factors for survival after liver metastasis diagnosis. CONCLUSIONS: Of the three modalities of therapy used for choroidal melanoma metastatic to the liver, only chemoembolization using cisplatin-based regimens produced a meaningful response rate. Information from this analysis can be used to predict the outcome of patients with uveal melanoma metastatic to the liver. Patients with metastatic ocular melanoma confined to the liver should be treated with chemoembolization and should not be included in chemotherapy trials designed for cutaneous melanoma. PMID- 8635074 TI - The National Cancer Data Base report on pancreatic cancer. AB - BACKGROUND: The National Cancer Data Base (NCDB) of the Commission on Cancer gathers data on time trends in stage of disease, treatment patterns, and survival for selected cancers. The most current (1991) data for pancreatic cancer are described here. The NCDB data contain important reference information regarding patient and hospital demographics associated with the diagnosis and treatment of pancreatic cancer. METHODS: Three Calls for Data have yielded a total of 17,490 cases for 1985, 1986, and 1991, from 937 hospital cancer registries across the United States. The data for 1991 represent 32% of all pancreas cancer cases for U.S. RESULTS: A higher incidence of more advanced disease was reported for the youngest group of patients, Hispanics, African Americans, Asians, males and patients seen at smaller hospitals. For all patients combined, only 14.2% were reported to have had a pancreatectomy. Older patients, patients from lower income zip codes, and African Americans were somewhat less likely to have received a pancreatectomy. Cancer of the body of the pancreas was the anatomic subsite for which patients with pancreatic cancer were least likely to receive a pancreatectomy. Patients seen at hospitals with larger case-loads and at teaching hospitals were more frequently reported as having had a pancreatectomy. Between 1985-1986 and 1991, there was a trend from treatment with surgery only or radiation only toward more frequent use of combined chemoradiation without surgery. There was less radiation reported as received by patients at hospitals with fewer than 150 annual cancer caseloads compared with hospitals with larger caseload. For patients with resectable tumors, a moderate survival advantage was reported compared with nonresectable tumors: 1-year, 48% versus 23%; 2-year, 24% versus 9%; 3-year, 17% versus 6% respectively. CONCLUSIONS: The NCDB provides a powerful tool for examining practice patterns and outcome of cancer care in the U.S. The present report, covering one-third of all patients treated in the U.S. in 1991, confirms the advanced disease status of patients with pancreatic cancer. Minority groups present with more advanced disease and are less likely to undergo resection, currently the only potentially curative therapy. Resectability rates appear to be higher in large caseload hospitals. PMID- 8635075 TI - Hepatocellular carcinoma during pregnancy and its comparison with other pregnancy associated malignancies. PMID- 8635076 TI - Benefit of mammography screening in women ages 40-49 years: current evidence from randomized controlled trials. PMID- 8635077 TI - Detection of residual prostate cancer after external radiotherapy. PMID- 8635078 TI - Surgical management of prostate cancer. PMID- 8635079 TI - Second primary tumors in patients with head and neck squamous cell carcinoma. PMID- 8635080 TI - Survival of infants with malignant astrocytomas: a report from the Childrens Cancer Group. PMID- 8635081 TI - Etoposide, ifosfamide, and cisplatin therapy for refractory childhood solid tumors. PMID- 8635082 TI - Developing a prognostic index for ductal carcinoma in situ of the breast. Are we there yet? PMID- 8635083 TI - Understanding the relationship between relative and absolute risk. AB - BACKGROUND: Relative risks are the most common statistics used to quantify the risk of mortal or morbid outcomes associated with different patient groups and therapeutic interventions. However, absolute risks are of greater value of both patient and physician in making clinical decisions. METHODS: The relationship between relative and absolute risks is explained using graphical aids. A program to estimate absolute risks from relative risks is available on the internet (see ftp://ftp.vanderbilt.edu/pub/biostat/absrisk+ ++.txt). This program uses a competing hazards model of morbidity and mortality to derive these estimates. RESULTS: When a patient's absolute risk is low, it can be approximated by multiplying her relative risk by the absolute risk in the reference population. This approximation fails for higher absolute risks. The relationship between relative and absolute risk can vary dramatically for different diseases. This is illustrated by breast cancer morbidity and cardiovascular mortality in American women. The accuracy of absolute risk estimates will be affected by the accuracy of relative risk estimates, by the appropriateness of the reference groups used to calculate relative risks, by the stability of cross-sectional, age-specific morbidity and mortality rates over time, by the influence of individual risk factors on multiple causes of mortality, and by the extent to which relative risks may vary over time. CONCLUSIONS: Valid absolute risk estimates are valuable when making treatment decisions. They can often be obtained over time intervals of 10 to 20 years when the corresponding relative risk estimates have been accurately determined. PMID- 8635084 TI - Morphologic alterations in esophageal squamous cell carcinoma after preoperative high dose rate intraluminal brachytherapy. AB - BACKGROUND: Total esophagectomy specimens from 4 patients given preoperative high dose rate intraluminal brachytherapy (HDRILBT) of 20 Gray (GY) in 2 fractions of 10 Gy each week were reviewed for radiation changes. METHODS: In all patients, preoperative biopsy specimens showed moderate to poorly differentiated squamous cell carcinoma with minimal to negligible keratin production. The esophagectomy specimens were sampled at the resection margins, the edge of irradiated length, 1 cm from the proximal and distal edge of visible tumor, the center of the tumor, and the lymph nodes. RESULTS: Radiation change in the form of fibrosis was limited to the submucosa at the resection margins, the circular muscle layer at the edge of irradiated length, and full thickness at 1 cm from the edge of the visible tumor and the center of the tumor. Surface epithelium did not show any changes at the resection margins but did show basal cell hyperplasia at the edge of the irradiated length and ulceration at 1 cm from the edge of the visible tumor and the center of the tumor. Endarteritis obliterans was seen only 1 cm from the edge of the visible tumor and the center of the tumor. Necrosis, intense keratin formation, and giant cell reaction were observed at the center of the tumor. When compared with the preradiotherapy biopsies, the amount of keratin in the postradiotherapy specimens was extensive. HDRILBT may cause induction of the keratin gene in the irradiated cells to stimulate differentiation toward better differentiated cells. CONCLUSIONS: HDRILBT may cause the keratin gene in the irradiated cells to induce differentiation toward better differentiated cells. Preoperative high dose rate intraluminal brachytherapy may have a role in improving the prognosis of patients with early esophageal cancer treated with a combination of radiotherapy and surgery. PMID- 8635085 TI - Prognostic value of preoperative immunosuppressive acidic protein in patients with gastric carcinoma. Findings from three independent clinical trials. Tumor Marker Committee for the Study Group of Immunochemotherapy with PSK for Gastric Cancer. AB - BACKGROUND: Immunosuppressive acidic protein (IAP) has been reported to have close correlation with the impairment of host immune response. To evaluate the significance of IAP in clinical studies, the prognostic value of preoperative IAP was investigated in clinical trials of patients with gastric carcinoma after curative resection. METHODS: An appropriate IAP threshold value of 580 micrograms/mL was determined using Cox's proportional hazards model. Five-year survival rates were estimated for high and low IAP groups in three different clinical studies. Meta-analysis was performed based on individual patient data, and summarized hazard ratios were estimated using a stratified proportional hazards model. RESULTS: Meta-analysis of the three clinical trials demonstrated that patients with preoperative IAP levels above the threshold had significantly poorer cancer related survival (P = 0.0039) and absolute survival (P = 0.0023), even after adjustment for the major prognostic factors. CONCLUSIONS: Gastric carcinoma patients with an IAP value above the threshold level of 580 micrograms/mL have a higher risk of cancer death and absolute death than patients with an IAP value below the threshold value. PMID- 8635086 TI - Colorectal carcinoma metastases to the liver. Does primary tumor location affect its lobar distribution? AB - BACKGROUND: There is considerable evidence that blood returning from different abdominal organs does not mix completely but maintains streamline flow in the portal vein. This study tested the hypothesis that the location of primary colorectal carcinoma affects the intrahepatic distribution of liver metastases according to streamline flow in the portal vein. METHODS: Eighty-five patients with histologically verified liver metastases from colorectal carcinoma underwent potentially curative hepatectomy. Primary tumor location was the right-sided colon in 18 patients and the left-sided colon in 67. The liver was divided into two lobes by Cantlie's line. RESULTS: A total of 195 metastatic deposits were resected: 135 in the right lobe and 60 in the left. In the right-sided colon carcinoma group, 29 deposits were in the right lobe and 3 in the left. In the left-sided colon carcinoma group, 106 deposits were in the right lobe and 57 in the left. The pattern of lobar distribution was significantly different in the two groups (P = 0.003). CONCLUSIONS: Right-sided colon cancers selectively involve the right lobe, while left-sided tumors involve the entire liver, considering the ratio of weights of the right to left lobe is 2:1. This difference suggests that primary tumor location affects the pattern of lobar distribution of colorectal carcinoma liver metastases according to streamline flow in the portal vein. PMID- 8635087 TI - Hepatocellular carcinoma in the United States. Prognostic features, treatment outcome, and survival. AB - BACKGROUND: The purpose of this study was to investigate prognostic factors at presentation and the survival of North American patients with hepatocellular carcinoma (HCC). METHODS: A retrospective analysis of medical records was performed for 314 patients identified through the Tumor Registry as having been evaluated for hepatocellular carcinoma at the Deaconess Hospital, Boston, Massachusetts, from 1986 through 1995. Clinical characteristics were noted, including age, sex, TNM staging, serum biochemistries, serum alpha-fetoprotein (AFP), patency of portal vasculature, cirrhosis, history of alcohol abuse, hepatitis-B or C positivity, hemochromatosis, treatment received, and ultimate survival from the date of diagnosis. RESULTS: Overall median survival was 10 months. The presence of cirrhosis, a history of alcohol abuse, low albumin, high bilirubin, abnormal AFP, and portal vein obstruction (PVO) were each associated with significantly shorter survival, as was advanced stage. Only albumin, AFP, and PVO were independent risk factors by multiple regression analysis. Patients undergoing surgery had the longest median survival (45 months), followed by those receiving chemoembolization (14 months). Those patients who were untreated or received systemic chemotherapy alone had significantly shorter survivals (2-4 months). CONCLUSIONS: Despite the difference in the underlying etiology of HCC in this population compared with Asian patients, poor prognostic indicators are similar. In this large series of patients at a single Northeastern hospital, analysis of presenting clinical characteristics was found to offer useful prognostic information. PMID- 8635088 TI - Clinical and pathologic features of hepatocellular carcinoma in young and older Italian patients. AB - BACKGROUND: It is not known whether putative etiologic factors and clinical and pathological features of hepatocellular carcinoma (HCC) differ between young adult and older white patients. METHODS: We examined the characteristics of 498 consecutive patients with HCC age < 50 years (Group 1: 54 patients) and age > or = 50 (Group 2: 444 patients), an age beyond which the tumor occurrence rate briskly increases. RESULTS: Demographic characteristics, alcohol and coffee intake, and cigarette smoking did not differ between the two groups. Group 1 had a greater prevalence of the hepatitis B surface antigen (HBsAg) carriers (P = 0.006), while the prevalence of either past hepatitis B virus infection (P = 0.008) or antivirus C antibodies (P = 0.016) was higher in Group 2. The lack of both hepatitis B and C virus serologic markers was more common in Group 1 (P = 0.018). In these patients, HCC was less frequently superimposed on cirrhosis (P = 0.002) and was more advanced at the time of diagnosis. In fact, despite a better histologic differentiation grade (P = 0.019), monofocal (solitary and massive) tumors were larger (P = 0.012), small lesions (< or = 5 cm) less frequent (P = 0.028), and either diffuse (P < 0.001) or massive (P = 0.011) types more common. An elevation of serum alpha-fetoprotein was less frequent in group 1 (P = 0.016), but this difference disappeared when the "diagnostic" cut-off of 400 ng/mL was considered. Albeit the prevalence of presenting symptoms did not significantly differ between the two groups, the clinical stage was more advanced in young patients (P = 0.004). The 9-year cumulative rate of survival was similar in the 2 groups. CONCLUSIONS: An early exposure to the virus and/or an accelerated hepatocarcinogenesis in HBsAg carriers can be inferred. Moreover, in the period of life at low risk for hepatoma: (1) the impact of nonalcoholic chemical carcinogenesis seems to be greater; (2) the tumor occurrence is less dependent on cirrhosis development; (3) although histologically better differentiated, the neoplasm is more advanced at the time of diagnosis; and (4) the long term survival is similar to that of the patients age 50 years or older. PMID- 8635089 TI - Immunocytochemical detection of p53 protein from pancreatic duct brushings in patients with pancreatic carcinoma. AB - BACKGROUND: It is often difficult to distinguish pancreatic carcinoma preoperatively from chronic pancreatitis. Therefore, we have developed a new method of detecting p53 immunoreactivity in cytologic material obtained by endoscopic retrograde pancreatic duct brushing (ERPDB). METHODS: Twenty-eight patients with prominent strictures of the main pancreatic duct demonstrated by pancreatography including 20 ductal cell carcinoma and 8 chronic pancreatitis were studied. The ability to distinguish between these two groups preoperatively by conventional cytologic examination was compared with p53 immunocytochemistry using ERPDB: RESULTS: The sensitivity, specificity, and overall accuracy of conventional cytologic examination in distinguishing ductal cell carcinoma from chronic pancreatitis were 60%, 100%, and 71% respectively. In comparison, the sensitivity, specificity, and overall accuracy of p53 immunocytochemistry in distinguishing were 90%, 100%, and 93%, respectively. The sensitivity of p53 staining of specimens from patients with carcinoma of the body or tail of the pancreas (90%) was the same for those with tumors of the head of the pancreas (90%). CONCLUSIONS: These results suggest that p53 immunocytochemistry using ERPDB in conjunction with conventional cytologic examination can help differentiate ductal cell carcinoma from chronic pancreatitis preoperatively. PMID- 8635090 TI - An aggressive therapeutic approach to carcinoma of the body and tail of the pancreas. AB - BACKGROUND: Prognosis of patients with adenocarcinoma of the pancreatic body and tail is extremely poor. Anatomically, this part of the pancreas is thin, and cancerous invasion to the retropancreatic structures occurs easily. The majority of patients have residual tumor in the retroperitoneal tissues after conventional distal pancreatectomy. METHODS: Between 1962 and 1979, 10 patients with carcinoma of the pancreatic body underwent simple distal pancreatectomy. Between 1980 and 1990, 22 patients including 7 with distant metastasis underwent a more aggressive approach intended to achieve longer survival: distal pancreatectomy with extended dissection of the lymph nodes and adjacent structures, especially into the retropancreatic space. After 1984, intraoperative radiation (IORT) by electron beam and chemotherapy by hepatic infusion plus systemic injection of mitomycin C (MMC) were added for 7 patients without distant metastasis. RESULTS: The 10 patients who underwent pancreatectomy between 1962 and 1979 all died within 20 months after their operations. Conversely, the patients treated with an aggressive approach between 1980 and 1990 survived longer; the 5-year survival rate for 15 patients without distant metastasis was 29%, though the 7 patients with distant metastasis died within 10 months of their operations. There were 4 long term survivors (> or = 5 years); 3 of whom received IORT and chemotherapy with MMC. Invasion to the retropancreatic soft tissues was present in 95% of the resected specimens from the 22 patients. However, invasion to the surgical margin at the posterior surface of the resected specimen was present in only 36% after extended resection of the retropancreatic structures. CONCLUSIONS: Survival improved for this disease after distal pancreatectomy with extended dissection, especially of the retropancreatic structures, adjuvant IORT, and chemotherapy had been performed. PMID- 8635091 TI - nm23-H1 protein immunoreactivity in laryngeal carcinoma. AB - BACKGROUND: The nm23-H1 gene encodes a 17-kilodalton cytoplasmic and nuclear protein that has recently been shown to be reduced in a number of human carcinomas including breast, colorectal, lung, gallbladder, and biliary tract carcinomas. This study examines the immunohistochemical staining characteristics of the nm23-H1 protein in human laryngeal carcinomas and nonneoplastic laryngeal polyps, and attempts to determine if there is any relationship between reduction of nm23-H1 protein immunoreactivity and prognosis of patients with laryngeal carcinoma. METHODS: Routine streptavidin-biotin immunohistochemistry with a polyclonal antibody was employed to study the expression of the nm23-H1 protein in laryngeal squamous cell carcinoma (SCC) (N = 22) and nonneoplastic polyps (N = 8). The carcinomas were classified as well differentiated (N = 2), moderately differentiated (N =15), and poorly differentiated (N = 5). Tissues from metastatic tumor deposits in lymph nodes (N = 5) were also studied. A semiquantitative immunostaining index was derived from the intensity and extent of staining of the cells. RESULTS: All laryngeal polyps showed intense immunostaining for the nm23-H1 gene product in the squamous epithelium. However, reduced immunoreactivity was found in nearly half of the SCC cases (N = 10; 46%), with the least staining intensity found in tumor metastases in lymph nodes (N = 4; 80%), and were associated with a shorter median survival of 14.3 months. In contrast, tumors that demonstrated moderate to strong nm23-H1 protein immunostaining were associated with a longer median survival period of 20.4 months. CONCLUSIONS: There is reduced expression of the nm23-H1 gene in human laryngeal SCC compared with nonneoplastic laryngeal polyps. Reduction in the intensity and extent of nm23-H1 protein immunostaining appears to correspond to reduced duration of patients survival. PMID- 8635092 TI - Consolidation biochemotherapy for patients with advanced nonsmall cell lung carcinoma responding to induction PVM (cisplatin, vinblastine, mitomycin-C) regimen. A phase II study. AB - BACKGROUND: The authors investigated a consolidation biochemotherapy program with subcutaneous recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN alpha) biologic response modifiers (BRM) in patients with advanced nonsmall cell lung carcinoma (NSCLC) with responsive or stable disease to induction chemotherapy. METHODS: Patients with proven, advanced, previously untreated NSCLC were entered into the study. Induction chemotherapy consisted of cisplatin, 120 mg/m2 intravenously (i.v.), on Day 1; vinblastine, 6 mg/m2 i.v., on Day 1; and mitomycin-C, 6 mg/m2 i.v., on Day 1 (PVM), every 3 weeks. Subsequently, patients with complete response (CR), partial response (PR), and stable disease (SD) received consolidation biochemotherapy with subcutaneous rIL-2, 3 MU/m2 twice/day, and rIFN alpha, 3 MU once/day, 5 days a week, starting 2 weeks after the second PVM course. After 3 and 6 weeks of BRM treatment, patients had a 14 day rest period to intercalate consolidating PVM courses. RESULTS: Seventy-seven patients were enrolled in the trial. After 2 PVM induction courses, 16 patients progressed and went off the study, whereas 61 patients were eligible for consolidation biochemotherapy. Among the 61 patients, 9 were not treated with BRM for several reasons, whereas 52 patients began biochemotherapy and were evaluable for toxicity. Furthermore, a few days after starting BRM, 9 patients discontinued therapy due to side effects; the remaining 43 patients received adequate treatment and were fully evaluable. In the 52 evaluable patients, the following BRM related toxicities were observed: World Health Organization (WHO) grade 2-3 fever in 85% of patients, asthenia in 71%, anorexia in 63%, and flu-like syndrome in 18.5%. PVM-related vomiting was present in 19% of patients. WHO Grade 3-4 myelosuppression, from both BRM and PVM (overlapping toxicity), was anemia in 16% of patients, leukopenia in 12%, and thrombocytopenia in 19%. There were three toxic deaths: two due to BRM-induced hypotension and one from pneumonia. In the 43 fully evaluable patients (23 PR and 20 SD after induction chemotherapy), after a median of 6 weeks of biochemotherapy (range, 3-16 weeks), we observed 5 of 20 patients achieving PR from SD, and 6 of 23 with confirmed PR. In these 11 patients, the median duration of response was 21 weeks (range, 7-80 weeks). Overall response improvement was 11.6% in the 43 patients and 6.4% in the 77 total enrolled patients. Median survival was 41 weeks (range, 15-173 weeks) in the 43 patients and 38 weeks (range, 1.4-173 weeks) in the 77 patients. CONCLUSIONS: In this study, biochemotherapy, when administered by this dose and schedule, did not offer substantial benefit although it caused significant toxicity. PMID- 8635093 TI - Cell cycle analysis of 932 flow cytometric DNA histograms of fresh frozen breast carcinoma material. Correlations between flow cytometric, clinical, and pathologic variables. MMMCP Collaborative Group. Multicenter Morphometric Mammary Carcinoma Project Collaborative Group. AB - BACKGROUND: Confusing data have been presented for breast cancer patients on correlations between DNA ploidy and the percentage of S-phase cells and other prognostic variables. The aim of this study was to compare DNA ploidy classification and cell cycle variables with clinical, classic, and quantitative pathologic variables and clinical variables in a large group of patients. METHODS: DNA ploidy and cell cycle variables were extracted from MultiCycle (Phoenix Flow Systems, San Diego, CA) interpreted flow cytometric DNA histograms of fresh frozen material from 932 breast cancer patients and compared with clinical (age, hormonal status), classic pathology (lymph node status, tumor size and type), and quantitative pathologic variables (steroid receptor status, mitotic activity index [MAI], mean nuclear area [MNA]). RESULTS: The DNA ploidy correlated significantly with MAI, MNA steroid receptor status, and tumor type. No significant correlations were found with tumor size, lymph node status, age, and hormonal status. The first DNA index correlated significantly with MAI, MNA, and steroid receptor status. The percentage of S-phase cells significantly correlated with MAI, MNA, steroid receptor status, and lymph mode status. CONCLUSIONS: DNA index and DNA ploidy, as markers of genetic instability, correlated well with differentiation and proliferation markers and less well with lymph node status and tumor size as markers of metastatic potential and duration of disease. The percentage of S-phase cells was not independent of the percentage of differentiation markers and did not correlate strongly with mitotic activity. This indicates that the percentage of S-phase cells and the mitotic activity partially reflect different proliferative properties. PMID- 8635094 TI - A prognostic index for ductal carcinoma in situ of the breast. AB - BACKGROUND: There is controversy and confusion regarding therapy for patients with ductal carcinoma in situ (DCIS) of the breast. The Van Nuys Prognostic Index (VNPI) was developed to aid in the complex treatment selection process. METHODS: The VNPI combines three significant predictors of local recurrence: tumor size, margin width, and pathologic classification. Scores of 1 (best) to 3 (worst) were assigned for each of the 3 predictors and then totaled to give an overall VNPI score ranging from 3 to 9. Three hundred thirty-three patients with pure DCIS treated with breast preservation (195 by excision only and 138 by excision plus radiation therapy) were studied with detection of local recurrence as the end point. RESULTS: There was no statistical difference in the 8 year local recurrence free survival in patients with VNPI scores of 3 or 4, regardless of whether or not radiation therapy was used (100% vs. 97%; P = not significant). Patients with VNPI scores of 5, 6, or 7 received a statistically significant 17% local recurrence free survival benefit when treated with radiation therapy (85% vs. 68%; P = 0.017). Patients with scores of 8 or 9, although showing the greatest relative benefit from radiation therapy, experienced local recurrence rates in excess of 60% at 8 years. CONCLUSIONS: DCIS patients with VNPI scores of 3 or 4 can be considered for treatment with excision only. Patients with intermediate scores (5, 6, or 7) show a 17% decrease in local recurrence rates with radiation therapy. Patients with VNPI scores of 8 or 9 exhibit extremely high local recurrence rates, regardless of irradiation, and should be considered for mastectomy. PMID- 8635095 TI - Human papillomavirus DNA in uterine cervix squamous cell carcinoma and adenocarcinoma detected by polymerase chain reaction. AB - BACKGROUND: Substantial clinical, epidemiologic, and experimental evidence has reinforced the role of high risk human papillomavirus (HPV) types in the development of cervical carcinoma. The authors investigated HPV in the uterine cervix squamous cell carcinomas and adenocarcinomas of Finnish patients. METHODS: Specimens from 352 patients with uterine cervix squamous cell carcinomas and 108 with adenocarcinoma were examined for HPV DNA by polymerase chain reaction. The authors used consensus primers located in the L1 region, as well as HPV16, 18, and 33 type-specific primers located in the E6 region. RESULTS: HPV DNA was detected in 324 of 352 squamous cell carcinomas (92%), and 81 of 108 adenocarcinomas (75%). Two-hundred seventy-four of 352 squamous cell carcinomas (78%) and 18 of 108 adenocarcinomas (17%) contained HPV16 DNA, whereas 55 of 352 squamous cell carcinomas (16%) and 60 of 108 adenocarcinomas (56%) contained HPV18 DNA. Eight squamous cell carcinomas and 4 adenocarcinomas were positive for HPV33. Twenty-eight squamous cell carcinomas and 5 adenocarcinomas were positive for either HPV16 and HPV18 or HPV16 and HPV33. Unclassified HPV DNA was detected in 17 squamous cell carcinomas and 4 adenocarcinomas. Twenty-eight squamous cell carcinomas and 9 adenocarcinomas, which were positive for E6 DNA using type specific primers, were negative for the L1 gene. All 460 cervical specimens were tested twice with identical results. CONCLUSIONS: HPV DNA was highly prevalent in both uterine cervix squamous cell carcinoma and adenocarcinoma. HPV16 was detected more often in squamous cell carcinoma and HPV18 was detected more often in adenocarcinoma. Both consensus structural L1 gene-derived primers and type specific viral E6 oncogene-derived primers were necessary to detect HPV DNA in cervical carcinoma. PMID- 8635096 TI - Analysis of response to radiation therapy of patients with cervical adenocarcinoma compared with squamous cell carcinoma. MIB-1 and PC10 labeling indices. AB - BACKGROUND: The MIB-1 monoclonal antibody is a marker of cycling cells and the PC10 monoclonal antibody is a marker of proliferating cell nuclear antigen in paraffin sections. This study was conducted to elucidate the difference in response to radiotherapy (RT) between cervical adenocarcinomas and squamous cell carcinomas, focusing on cell proliferation. METHODS: A total of 196 biopsy specimens taken from the cervical carcinomas of 14 consecutive patients with adenocarcinoma and 62 patients with squamous cell carcinoma before and after RT at doses of 9 and 27 Grays (Gy) were investigated for MIB-1 and PC10 immunoreactivities. RESULTS: In adenocarcinomas, the mean MIB-1 labeling indices before and after RT at 9 and 27 Gy were 28%, 21%, and 26%, respectively, whereas the mean PC10 labeling indices were 15%, 13%, and 14%, respectively. In squamous cell carcinomas, the mean MIB-1 labeling indices before and after RT at 9 and 27 Gy were 38%, 53%, and 26%, respectively, and the mean PC10 labeling indices were 23%, 23%, and 11%, respectively. CONCLUSIONS: Cervical adenocarcinomas have a lower cycling cell population and their indices show no change during RT. Squamous cell carcinomas have a higher cycling cell population and show a transient increase of the MIB-1 cycling cell population at 9 Gy of RT. These findings suggest a difference in response to RT between adenocarcinomas and squamous cell carcinomas. PMID- 8635097 TI - The role of radioimmunoscintigraphy and computed tomography scan prior to reassessment laparotomy of patients with ovarian carcinoma. A preliminary report. AB - BACKGROUND: Accurate evaluation of patients with ovarian carcinoma who have completed primary therapy often requires surgical exploration. Radioimmunoscintigraphy (RIS) represents an evolving technique that may allow noninvasive detection and localization of persistent or recurrent disease in these patients. METHODS: Our prospective, blinded study enrolled patients with normal CA 125 levels and no clinical evidence of disease after primary cytoreductive surgery and cytotoxic chemotherapy for ovarian carcinoma. Each patient underwent RIS using Indium-satumomab pendetide (labeled antibody B72.3 to the tumor-associated antigen TAG-72) and abdominal/pelvic computed tomography (CT) prior to reassessment laparotomy. RESULTS: Twenty patients were enrolled from January 1994 to January 1995. Two patients with negative RIS scans refused reassessment laparotomy and were without evidence of disease > 15 months from the study. Twelve of the remaining 18 patients (66.7%) had histologically proven disease at reassessment laparotomy. RIS images indicated the presence of disease in all 12 patients, whereas CT scans detected disease in only 2 patients. In three of five patients, biopsy-proven microscopic disease (no gross disease at the time of laparotomy) was found only in specimens obtained by RIS-directed biopsies. RIS was superior to CT in sensitivity (100% vs. 16.7%), accuracy (72% vs. 33%), and negative predictive value (100% vs. 28.6%) (P < 0.005). CONCLUSIONS: Routine use of CT is of limited value in the assessment of ovarian carcinoma patients with negative physical examinations and normal CA 125 levels. With its high level of sensitivity and negative predictive value, RIS may play a role in the detection of persistent disease in this population and aid in the classification of patients into three distinct groups: those with gross residual disease, small volume or microscopic disease, and no disease. Separation of this heterogenous group without surgery may help guide subsequent consolidation therapy. However, attaining a high level accuracy with RIS, depends on optimizing the method of image acquisition, the timing of scans, and the reconstruction of data. PMID- 8635098 TI - The Performance Status Scale for Head and Neck Cancer Patients and the Functional Assessment of Cancer Therapy-Head and Neck Scale. A study of utility and validity. AB - BACKGROUND: The goal of this investigation was to examine the relationship between, and application of, two disease specific quality of life (QL) measures currently being employed for head and neck cancer patients: the Functional Assessment of Cancer Therapy-Head and Neck Scale (FACT-H&N) and the Performance Status Scale for Head and Neck Cancer Patients (PSS-HN). METHODS: The FACT-H&N and PSS-HN were administered to 151 head and neck cancer patients with a range of disease sites, treatment status (on vs. off treatment), and treatment modalities (surgery, radiation, and chemotherapy). RESULTS: FACT-H&N subscale and total scores and PSS-HN subscale scores proved sensitive to patients groups (showed significant and clinically meaningful differences) on the basis of treatment status (on vs. off treatment) and global performance status (Karnofsky scores). The pattern of correlations between FACT-H&N and PSS-HN subscales supported the scales' construct (convergent vs. divergent) validity. The strongest and most significant associations were observed between PSS-HN Normalcy of Diet and Eating in Public, and the head and neck subscale (HNS) of FACT-H&N, both of which were designed to measure the unique problems of head and neck cancer patients. More modest associations were observed between subscales measuring physical and functional areas of performance, social functioning, and emotional well-being. CONCLUSIONS: The FACT-HNS was found to be reliable and valid when applied to head and neck cancer patients. It clearly adds information to that collected by the parent (core) instrument. The PSS-HN also provides unique information, independent of that provided by the Karnofsky or the FACT-H&N. This study supported the multidimensional nature of QL for head and neck cancer patients, and thus the importance of assessing disease specific concerns in addition to general health status when assessing functional and QL outcome. PMID- 8635099 TI - Ifosfamide, etoposide, cytarabine, and methotrexate as salvage chemotherapy in relapsed or refractory aggressive non-Hodgkin's lymphoma. AB - BACKGROUND: Patients with relapsed or resistant non-Hodgkin's lymphoma (NHL) have a poor prognosis and are rarely cured with usual salvage chemotherapy. Intensive treatment with the support of peripheral blood stem cells (PBSC) may be an effective therapy for these patients. We used a combination of ifosfamide, etoposide, cytarabine, and methotrexate (IVAM) with the intention both to reduce tumor burden and collect PBSC prior to transplantation. METHODS: Thirty-one patients (17 with relapsed NHL and 14 with refractory NHL) were treated with 2 courses of chemotherapy: IVAM regimen (ifosfamide, 1500 mg/m2 daily for 5 days plus mesna; etoposide, 150 mg/m2 daily for 3 days; cytarabine, 100 mg/m2 daily for 3 days; and methotrexate, 3 g/m2 on Day 5, with leucovorin rescue). Twenty three patients had an intermediate grade and 8 patients had a high grade lymphoma. RESULTS: After IVAM therapy, 19 patients (61%) achieved complete response, 8 patients (26%) achieved partial response and 4 patients (13%) failed to respond. The major toxicity of IVAM was myelosuppression, but there were no toxic deaths. PBSC harvest could be performed in 29 patients (94%) with a median granulocyte-macrophage colony-forming unit count of 55 x 10(4)/kg (range, 2-391 x 10(4)/kg). Three patients could not undergo transplantation because of disease progression. One patient received a syngeneic transplant, 25 patients received PBSC transplantation, and 2 patients received a bone marrow transplant. In an intent-to-treat analysis, the overall survival rate at 4 years was 37% for the whole group (95% confidence interval: 22-55). CONCLUSIONS: We conclude that IVAM is an effective salvage chemotherapy for refractory or relapsed NHL and permits PBSC collection in most of these patients. PMID- 8635100 TI - Phase I study of escalating doses of mitoxantrone and paclitaxel with granulocyte macrophage colony stimulating factor support. AB - BACKGROUND: Both paclitaxel and mitoxantrone demonstrate significant antineoplastic activity in breast cancer patients. Colony stimulating factor support allows significant dose escalation of each of these drugs when administered as a single agent. METHODS: We performed a Phase I study employing escalating doses of paclitaxel and mitoxantrone with granulocyte-macrophage colony stimulating factor (GM-CSF) support. Initially the paclitaxel dose was fixed at 175 mg/m2 and an attempt was made to escalate mitoxantrone from the starting dose of 14 mg/m2. Subsequently, the dose of mitoxantrone was fixed at 14 mg/m2 and the dose of paclitaxel was increased. Treatments were given every three weeks. RESULTS: In neither case could we safely escalate beyond a combination of paclitaxel 175 mg/m2 and mitoxantrone 14 mg/m2 which is, therefore, the recommended Phase II dose. The dose limiting toxicity was neutropenia. No unexpected toxicities were observed, although two patients were removed from the study because of chest pain possibly related to GM-CSF. There were no complete or partial remissions. CONCLUSIONS: We conclude that GM-CSF does not allow significant dose escalation of this combination of agents. PMID- 8635101 TI - Severe myelotoxicity of oral etoposide in heavily pretreated patients with non Hodgkin's lymphoma or chronic lymphatic leukemia. AB - BACKGROUND: Promising results have been reported for patients with non-Hodgkin's lymphoma (NHL) receiving chronic oral etoposide. Due to the small number of patients reported, information regarding side effects is limited, and therefore warrants further evaluation. METHODS: Twenty eligible patients with NHL and chronic lymphatic leukemia (CLL), resistant to or relapsed after previous protocols of polychemotherapy were treated with oral etoposide at a dosage of 50 mg/m2/day for 21 days in a 28-day cycle. Response and toxicity were evaluated according to standard criteria. RESULTS: Total response was noted in 13 patients, complete response in 2 patients, and partial response in 11 patients. Two patients had stable disease and five patients had progression of disease during treatment. Seventy-five percent of patients experienced neutropenia below 1500/microL. Half acquired infection and required hospitalization. Fifty-five percent required blood transfusions. All patients needed course shortening and dosage reduction. CONCLUSIONS: Chronic daily administration of oral etoposide is effective in patients with NHL and CLL. In heavily pretreated patients, myelotoxicity is severe. Therefore, modification of the schedule plan is mandatory in this group of patients. PMID- 8635102 TI - The genetic attributable risk of breast and ovarian cancer. AB - BACKGROUND: The age-specific proportion of breast and ovarian cancer in the general population that is likely to be due to a breast/ovarian cancer susceptibility gene(s) is estimated. In addition, the age-specific penetrance of ovarian cancer for women predicted to be carriers of a susceptibility gene is calculated using population-based data. METHODS: Data are from the Cancer and Steroid Hormone Study, a population-based, case-control study conducted by the Centers for Disease Control, which includes 4730 breast cancer cases aged 20 to 54 years. Information regarding the occurrence of breast and ovarian cancer was collected for mothers and sisters of the cases during an in-home interview. The probability of being a breast cancer susceptibility gene carrier was calculated for each of the breast cancer cases using information on the family history of breast cancer. The calculated risk of ovarian cancer in the first-degree relatives of breast cancer cases with a high probability of being a gene carrier is compared with that seen in first-degree relatives of breast cancer cases with a low probability of being a gene carrier and used to calculate the proportion of ovarian cancer cases that are likely to be due to a breast/ovarian susceptibility gene(s) as well as the age-specific risk of developing ovarian cancer for gene carriers. RESULTS: Approximately 10% of ovarian cancer cases and 7% of breast cancer cases in the general population are estimated to be carriers of a breast/ovarian cancer susceptibility gene; these women are found primarily in families characterized by multiple cases of the early onset of breast cancer. The proportion of breast cancer cases predicted to be attributable to the gene decreases markedly with age; approximately 33% of cases age 20-29 years compared with approximately 2% of cases age 70-79 years. The proportion of ovarian cancer cases predicted to be due to the susceptibility gene ranges from 14% among patients diagnosed in their 30s to 7% among those diagnosed in their 50s. Carriers are predicted to have at least 15 times the age-specific risk of ovarian cancer of noncarriers. Among women predicted to carry the gene, the cumulative risk of developing ovarian cancer by the age of 59 years is approximately 10%. CONCLUSIONS: The estimates provided may prove helpful to clinicians until such time as large-scale population-based screening for breast and ovarian cancer susceptibility genes is possible. PMID- 8635103 TI - Coexistence of renal cell carcinoma and malignant lymphoma. A causal relationship or coincidental occurrence? AB - BACKGROUND: Secondary malignant neoplasms are increasingly being observed in cancer populations and a considerable amount of data have accumulated in the literature. Among the secondary malignant neoplasms that occur with a higher incidence in cancer patients are lymphomas and renal cell carcinoma (RCC), as well as melanoma, lung/bronchus carcinoma. METHODS: The authors analyzed the patient population at the Memorial Sloan-Kettering Cancer Center in New York City between 1985 and 1995 for coexisting carcinomas, and identified 15 patients who had both RCC and malignant lymphoma among a total of 1262 patients with RCC and 1660 patients with malignant lymphoma. The occurrence and time of diagnosis of both malignant neoplasms and their clinical features, types, and stages, as well as short term follow-up, results, are presented. RESULTS: The data show a greater than coincidental coexistence of RCC and malignant lymphoma (P < 0.01). In addition, there was a significant increase in the number of patients with both melanoma and RCC (P < 0.01), as well as melanoma and malignant lymphoma (P < 0.01). No significant increase was found in cases of coexisting RCC or malignant lymphoma with either lung/bronchus carcinoma or colorectal carcinoma (P > 0.05). CONCLUSIONS: Causes of this increased coexistence may include a genetic predisposition to cancer, similar immune mechanisms associated with these neoplasms, closer scrutiny of this group of patients, or a combination of these factors. Studies are underway to elucidate a common genetic component in these patients. PMID- 8635104 TI - Primary tracheal non-Hodgkin's lymphoma. A case report and review of the literature. AB - BACKGROUND: Primary tracheobronchial non-Hodgkin's lymphoma (NHL) is an uncommon occurrence. The authors report a patient who presented with primary tracheal NHL, the sixth such patient described in the literature. METHODS: Using a MEDLINE search, 41 additional patients presenting with symptomatic primary or secondary tracheobronchial NHL were identified. The characteristics, management, and outcome of these patients are described. RESULTS: Patients with NHL of the upper respiratory tract present with dyspnea, wheezing, and cough, and frequently are misdiagnosed as having asthma. The majority of patients have additional sites of intrathoracic disease with tracheobronchial involvement occurring in the setting of advanced or relapsed NHL. Low grade histology is seen most commonly in patients with primary tracheal NHL. Several patients demonstrate the typical histologic features of mucosa-associated lymphoid tissue. Surgery, chemotherapy, and radiation therapy have been used alone or in combination for treatment. The outcome of these patients does not appear different from that observed in patients with lymphomas of similar histology and stage that do not involve the tracheobronchial tree. CONCLUSIONS: Thoracic surgeons, pulmonologists, and oncologists should recognize that NHL can rarely be confined to the trachea or bronchi. NHL should be considered in the differential diagnosis of airway obstruction, because it represents a highly treatable malignancy. PMID- 8635105 TI - Epstein-Barr virus infection in the neoplastic and nonneoplastic cells of lymphoid malignancies. AB - BACKGROUND: The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied. METHODS: Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein-Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases. RESULTS: Although EBER-1 positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1, EBV genomic DNA was detected in 8 of the 39 examined cases. CONCLUSIONS: T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes. PMID- 8635106 TI - Participation in a women's breast cancer risk counseling trial. Who participates? Who declines? High Risk Breast Cancer Consortium. AB - BACKGROUND: Little is known about what factors influence women to participate its trials designed for women at high risk for breast cancer. Yet the recruitment phase is of utmost importance in determining whether the trial can achieve its goals. The purpose of this study was to examine predictors of participation in a risk counseling trial for first-degree relatives of breast cancer patients. METHODS: Subjects were identified by their relatives who had received a diagnosis of breast cancer at one of six institutions. Letters of introduction were sent to potentially eligible, unaffected relatives. They were informed about the risk counseling study and given the opportunity to decline. Letters were sent to 1392 first-degree relatives; of these, 1149 women were contacted for an interview and 14% refused. Of the 987 respondents, 47% accepted and 53% declined to participate. Bivariate and multivariate statistics were used to examine predictors of participation. RESULTS: In the total sample, there were five significant multivariable predictors of participation: level of education, recent clinical breast examination (CBE), objective and perceived breast cancer risk, and any current use of alcohol. Participation was moderated by time since the relative's diagnosis. There was a statistically significant relationship to education such that women with a higher level of education were significantly more likely to participate if they were approached within 2 months of their relative's diagnosis of breast cancer. CONCLUSIONS: Like many other kinds of trials, participants in this risk counseling trial were significantly more likely to have more than a high school education and more likely to be nonsmokers than the nonparticipants. They also were significantly more likely to drink alcohol (dichotomous measure), have had a recent CBE, and have higher subjective and objective breast cancer risks. Different types of recruitment strategies may be needed depending on the characteristics of the people being recruited and the timing of the invitation. PMID- 8635107 TI - 125I plaque therapy for uveal melanoma. Analysis of the impact of time and dose factors on local control. AB - BACKGROUND: 125I episcleral plaque therapy has gained wide acceptance for the treatment of uveal melanoma because of its potential to preserve vision, salvage the globe, and provide good local control. A rigorous analysis of the optimum radiation dose, dose rate, and overall treatment time has not been reported with this technique. METHODS: One hundred fifty patients with uveal melanoma treated with 125I plaques between 1982 and 1990 and included in the uveal melanoma study (UMS) database of the Wills Eye Hospital were analyzed. Mean patient age was 60.7 years (range: 17.7-84.6 years). Initial mean tumor size was 9.7 x 8.5 x 3.7 mm with a range of 4.5 to 21.5 mm in basal dimension and 1.2 to 11.8 mm in height. Mean dose to the tumor apex was 94.77 gray (Gy) (29.5-141 Gy). Mean dose rate to the tumor apex was 92.9 cGy/hr (10-292 cGy/hr); the mean dose to the base was 359 Gy (181-692 Gy); the mean dose rate to the base was 348 cGy/hr (112-893 cGy/hr); and mean duration of treatment was 124.7 hours (range: 28-333 hours) RESULTS: With a median follow-up of 68 months, there have been 33 local failures. Mean time to local failure was 19 months (range: 6-78 months). Actuarial local control is 81% at 5 years. Multivariate analysis demonstrates significant correlation of local failure with larger tumor dimension (P = 0.0046), close proximity to the optic disc (P = 0.0029), lower radiation dose to the tumor apex (P = 0.03), lower radiation dose rate to the tumor apex and base (P = 0.01 and 0.03), and longer overall treatment time (P = < 0.0001). CONCLUSIONS: This retrospective analysis reinforces the importance of dose rate, minimum tumor dose, overall treatment time, maximum tumor basal dimension, and proximity to the optic nerve in the treatment of uveal melanoma. PMID- 8635108 TI - Outpatient interstitial thermoradiotherapy. AB - BACKGROUND: Hyperthermia enhances the cytocidal effect of ionizing radiation. Several pilot studies demonstrated that the combination of interstitial hyperthermia and interstitial radiotherapy (interstitial thermoradiotherapy) is safe and effective. However, these studies mainly utilized low dose rate brachytherapy, and therefore, required hospitalization. With the availability of median or high dose rate brachytherapy devices, we piloted a study to evaluate the feasibility, toxicity and efficacy of interstitial thermoradiotherapy performed in an outpatient setting. METHODS: Between 1989 and 1993, 27 patients with a diagnosis of carcinoma of the head and neck region (n = 23), carcinoma of the breast (n = 3), or malignant melanoma (n = 1) received 1 or 2 sessions of interstitial thermoradiotherapy. Median patient age was 66 years (range: 37-83 years). Treatment consisted of 60 minutes of 915 MHz microwave interstitial hyperthermia, followed by iridium-192 seed implants, either by Micro-Selectron HDR (10-12 Gray [Gy] in 8.5-21 minutes) or high activity (5-8 mCi per seed) seeds (10-15 Gy in 2-4 hours). In addition to interstitial temperature measurements, a real-time thermal camera was used to monitor the surface temperature spatial distribution. power supply and/or position of interstitial microwave applicators was adjusted when appropriate. All but one patient also received external beam irradiation prior to implants. RESULTS: Patients tolerated treatments well although 16 (59%) of them required analgesics during hyperthermia sessions. Skin blisters or ulcerations occurred in only 6 (22%), and all but 2 healed. Complete response occurred in 24 patients (89%), partial in 3 (11%). With a median follow up of 16 months (range: 3-43 months), the 2-year actuarial local control rate was 74%. CONCLUSIONS: The results of this study indicate that outpatient interstitial thermoradiotherapy is convenient, safe, and efficacious for treating human neoplasms. PMID- 8635109 TI - Radiotherapy and neoadjuvant chemotherapy for cervical carcinoma. A randomized multicenter study of sequential cisplatin and 5-fluorouracil and radiotherapy in advanced cervical carcinoma stage 3B and 4A. AB - BACKGROUND: The locoregional failure rate remains high in advanced cervical carcinoma. Chemotherapy (CT) was added to radiotherapy (RT) in order to increase disease control and to improve 5-year survival. METHODS: CT + RT included cisplatin administered 100mg/m2, d.1 plus 5-fluorouracil 1000 mg/m2 D.1 to 5, ci (120 hrs), q every 3rd week for 3 cycles, followed by RT. RT included external beam irradiation 64.8 Gy in 1.8 Gy fractions, five days a week, by 4-field box technique. The median follow-up was 46 months. Ninety-four patients were evaluable for survival, 47 in the CT + RT group and 47 in the RT group. Ninety two patients were evaluable for response. Known prognostic factors were equally distributed between the two groups. RESULTS: Of the 43 patients evaluable before RT, 31 (72%) achieved a partial or complete response after CT alone. After RT, 52 patients attained a complete response, 25 in the CT + RT group and 27 in the RT group. Sixty-three patients developed distant metastases or local relapse, 30 in the CT + RT group and 33 in the RT group. In the CT + RT group 6 of the 9 patients with metastases also had local progression at relapse, in the RT group, 7 of 17 patients. The survival rates for the two groups are not statistically different. Thirty-seven patients are alive, 29 have no evidence of disease. Fifty seven have died, 29 in the CT + RT group and 28 in the RT group. Fifty-four deaths were related to cancer, and 3 to therapy. CONCLUSIONS: Sequential CT and RT did not improve the survival, local control, or metastasis rate compared with RT alone. PMID- 8635111 TI - Prostate specific antigen findings and biopsy results following interactive ultrasound guided transperineal brachytherapy for early stage prostate carcinoma. AB - BACKGROUND: Interactive, transrectal, ultrasound-guided transperineal implantation is a new technique for performing permanent brachytherapy implants of the prostate. Prostate specific antigen (PSA) findings, biopsy results, and morbidity are examined to demonstrate its efficacy and safety in treating early stage prostate carcinoma. METHODS: Ninety-seven patients underwent permanent implants for classifications T1 to T2 adenocarcinoma of the prostate gland with a median follow-up of 18 months (range: 6-51 months). Seventy-nine patients had negative laparoscopic pelvic lymph node dissections prior to implantation. Patients with positive lymph nodes were not implanted. The radioactive isotope used was I-125 in 71 patients and Pd-103 in 26 patients. RESULTS: PSA failure was defined as two consecutive increases in PSA above the nadir level. The actuarial freedom from PSA failure (FFPF) at 2 years was 76% for the entire group. Stage significantly affected FFPF. Patients classified as T1b to T2a (35) had a FFPF of 91% at 2 years compared with 68.5% for patients classified as T2b to T2c (62) (P = 0.04). The pre-treatment PSA also significantly affected FFPF. Patients with PSA values of < or = 10 ng/mL (44) had a FFPF of 83% at 2 years. A similar rate of 82% was found in patients with PSA values of 10.1 to 20 ng/mL (29). Patients with PSA values > 20 ng/mL (24) had a significantly poorer FFPF at 2 years of 58% (P = 0.02). The PSA values of patients free from a PSA failure (82) ranged from 0.1 to 12.9 ng/mL with a median of 0.8 ng/mL. Transrectal prostate biopsies were performed 18 to 36 months posttreatment in 39 patients. Negative biopsies were found in 74% (29/39) of cases. The procedure was associated with an actuarial preservation of erectile function rate and sexual potency at 2 years of 96% and 79%, respectively. There were no cases of urinary incontinence or radiation cystitis. Associated morbidity included urinary retention requiring catheterization in 4% of the patients, outlet obstruction requiring a transurethral resection of the prostate in 2% and Grade 2 rectal complications in 1%. CONCLUSIONS: Interactive, ultrasound-guided transperineal brachytherapy results in a low PSA failure rate, high negative biopsy rate, and is associated with low morbidity and preservation of erectile function. PMID- 8635110 TI - Radiation therapy tolerance limits. For one or for all?--Janeway Lecture. AB - BACKGROUND: The optimal use of radiation therapy for cancer treatment is hampered by the application of tolerance limits of normal tissues derived empirically from population averages. Such limits do not reflect the considerable differences from patient to patient in susceptibility to late radiation sequelae. Assays that accurately predict normal tissue tolerance in individual patients would permit real application of the concept of treatment to tolerance thereby increasing the probability of an uncomplicated cure for the population as a whole. METHODS: A summary of laboratory research is presented to test the hypothesis that the cellular radiosensitivity of normal skin fibroblasts can predict the severity of late connective tissue damage that develops following radiotherapy. The pathogenesis of radiation reactions and the possible role of radiation induced cellular senescence in the development of clinical late effects are briefly reviewed. RESULTS: Although the pathogenesis of radiation injury is highly complex, several clinical studies have demonstrated a significant correlation between fibroblast radiosensitivity and the severity of late sequelae from treatment. However, the precision and reproducibility of fibroblast cell survival assays are inadequate for routine clinical use. Newer assays incorporating insights into the effects of radiation on cellular senescence and cytokine production are being developed. Such assays may, in the future, be complemented or replaced by molecular and/or cytogenetic probes to derive robust estimates of individual tolerance. CONCLUSIONS: The principle of prediction of tolerance to radiotherapy has been established. Although current assays lack the precision required for clinical use, the goal of individualized treatment to tolerance ultimately should be achieved. PMID- 8635112 TI - Patterns of disease failure after trimodality therapy of nonsmall cell lung carcinoma pathologic stage IIIA (N2). Analysis of Cancer and Leukemia Group B Protocol 8935. AB - BACKGROUND: The impact of sequential trimodality therapy on the pattern of first site disease failure in pathologic Stage IIIA (N2) nonsmall cell lung carcinoma (NSCLC) was analyzed. METHODS: Seventy-four eligible patients with histologically documented Stage IIIA (N2) NSCLC underwent sequential trimodality therapy on Cancer and Leukemia Group B (CALGB) Protocol 8935. Treatment consisted of 2 cycles of induction cisplatin at 100 mg/m2 intravenously (i.v.) (Days 1 and 29) and vinblastine at 5 mg/m2 i.v. weekly for 5 weeks followed by surgery. Surgery included a thoracotomy with resection of the primary tumor and hilar lymph nodes and a mediastinal lymph node dissection. Patients with resected disease then received an additional a cycles of cisplatin at 100 mg/m2 i.v. and vinblastine at 5 mg/m2 i.v. biweekly for 2 total of 4 doses followed by consolidative thoracic irradiation. Patients with completely resected disease received 54 Gray (Gy) whereas those with incompletely resected disease received 59.4 Gy at 1.8 Gy/fraction (fx) once a day. Patients with unresectable disease underwent thoracic radiation therapy (TRT) treatments only to 59.4 Gy at 1.8 Gy/fx without any additional chemotherapy. Disease recurrence was determined by clinical, radiographic, or histologic criteria. Pattern of disease failure was identified by site of involvement at first recurrence as indicated by the CALGB Respiratory Follow-Up Form. RESULTS: Sixty-three of the 74 patients completed the induction chemotherapy as planned. Forty-six of the 63 patients underwent resection of disease whereas the remaining 17 were unresectable. Thirty-three of the 46 resected patients completed the entire adjuvant postoperative chemoradiation treatment as planned. Ten of 17 patients with unresectable disease completed postsurgical TRT. At a median follow-up interval of 27 months (range, 4-43), the 3-year overall survival and failure-free survival were 23% and 18%, respectively, for all 74 eligible patients. Overall, disease failure has occurred in 52 (70%) of the 74 eligible patients: local only: 13 (25%); distant only: 16 (31%); and both local and distant: 23 (44%), (P = not significant [NS]). Ten patients progressed during induction chemotherapy: local only: six patients; and both local and distant failure: four patients. Twenty-eight of 46 resected patients recurred: local only: 1 (4%); both local and distant failure: 11 (39%); and distant only: 16 (57%); (P < 0.001). Disease progression occurred in 14 of 17 patients with unresectable disease: local only: 6; both local and distant sites: 8. Among the 52 total patients experiencing disease relapse, isolated or combined local failure occurred commonly among patients during induction chemotherapy (n = 10, [28%]), in those with unresectable disease (n = 14, [39%]), or in those with resected disease (n = 12, [33%]), (P = NS). However, isolated or combined distant failure was more likely to occur among patients with resected disease (n = 27, [69%]) than either during induction chemotherapy (n = 4, [10%]) or in those with unresected disease (n = 8, [21%]), (P < 0.05). Among patients who relapsed, brain metastases occurred in 13 of 52 (25%) patients overall and in 12 of 28 (43%) patients with resected disease. CONCLUSIONS: Overall, disease failure was just as likely to occur in local, distant, or combined sites on CALGB Protocol 8935 using sequential trimodality therapy in the treatment of pathologic Stage IIIA (N2) NSCLC: Isolated or combined local failure occurred commonly during sequential tri modality therapy whereas isolated or combined distant relapse was prevalent among patients with resected disease. In addition, isolated local failure was rare among patients with resected disease. The pattern of disease failure on CALGB Protocol 8935 reflects the biology of locoregional NSCLC as much as the therapeutic impact of trimodality therapy. PMID- 8635113 TI - Angiosarcoma. A report of 67 patients and a review of the literature. AB - BACKGROUND: Angiosarcomas (AS) are rare, aggressive tumors. Optimal treatment has not been well defined. The authors undertook a retrospective review of patients seen at their institution with the intent of identifying prognostic factors and optimal treatment strategies. METHODS: Between 1955 and 1990, 67 patients with AS were seen at the University of California, at Los Angeles Medical Center. Follow up ranged from 1 to 173 months with a median of 30 months. RESULTS: The overall prognosis was poor. The actuarial 2- and 5-year disease free survivals (DFS) were 44% and 24%, respectively. Of 52 recurrences after primary treatment, 81% (42 of 52) had a component of local failure. Twenty-eight patients had developed distant metastases at last follow-up. Of patients who received surgery (S) and radiation therapy (RT), with or without chemotherapy (CT), 5-year actuarial DFS was 43%, compared with 17% for patients who underwent S +/- CT as initial treatment (P = 0.03). Only 9% of patients (1 of 11) treated with RT +/- CT were rendered free of disease. CONCLUSIONS: Patients with AS usually present with high grade histology, and with multifocal disease. There is a propensity for both local recurrence and distant metastases. Our results and a review of the literature, suggest that S plus RT offers the best chance for long term control of this aggressive tumor. The role of CT remains undefined. PMID- 8635114 TI - Hyperbaric oxygen therapy for the treatment of radiation-induced sequelae in children. The University of Pennsylvania experience. AB - BACKGROUND: The role of hyperbaric oxygen (HBO) therapy in the treatment of radiation-related sequelae in adults is well known. In contrast, its role in the management of radiation-related sequelae in children has not been well studied. In an effort to define its value better, the authors reviewed the University of Pennsylvania experience and hereby report the results of their analysis. METHODS: Between 1989 and 1994, ten patients who underwent radiation therapy for cancer as children were referred for HBO therapy. Six patients underwent HBO therapy as a prophylactic measure prior to maxillofacial procedures; dental extractions and/or root canals (four patients), bilateral coronoidectomies for mandibular ankylosis (one patient), and wound dehiscence (one patient). Therapeutic HBO was administered to four other patients; one patient for vasculitis resulting in acute seventh cranial nerve palsy and the other three after sequestrectomy for osteoradionecrosis (mastoid bone, temporal bone, and sacrum, respectively). Osteoradionecrosis was diagnosed both radiologically and histologically after exclusion of tumor recurrence. The number of treatments ranged between 9-40 "dives" (median, 30 dives). Treatments were given once daily at 2 atmosphere absolutes for 2 hours each. Adjunctive therapy in the form of debridement, antibiotics, and placement of tympanotomy tubes was administered to two patients. Ages at HBO treatment ranged from 3.5 to 26 years (median, 14 years). Six patients were male and four were female. The most commonly irradiated site was the head and neck region (eight patients; brain stem gliomas [one], posterior fossa primitive neuroectodermal tumor [one], rhabdomyosarcomas [three], nasopharyngeal cancer [one], carcinoma of the parotid gland [one], and Hodgkin's disease [one]). The remaining two patients received radiation therapy for pelvic tumors [Ewings's sarcoma and rhabdomyosarcoma). Radiation doses ranged between 4000 and 6660 centigray (cGy) (median, 5500 cGy). The interval between the end of radiation therapy and HBO treatment ranged between 2 months and 11 years (median, 15 years). The median follow-up interval after HBO therapy was 2.5 years (range, 2 months-4 years). RESULTS: Except for two patients who had initial anxiety, nausea, and vomiting, the HBO treatments were well tolerated. In all but one patient, the outcome was excellent. In the six patients who had prophylactic HBO, all continued to demonstrate complete healing of their orthodontal scars at last follow-up. In the four patients who received HBO as a therapeutic modality, all 4 had documented disappearance of signs and symptoms of radionecrosis and two patients demonstrated new bone growth on follow-up computed tomography scan. One patient with vasculitis and seventh cranial nerve palsy had transient improvement of hearing; however, subsequent audiograms returned to baseline. CONCLUSIONS: The use of hyperbaric oxygen for children with radiation-induced bone and soft tissue complications is safe and results in few significant adverse effects. It is a potentially valuable tool both in the prevention and treatment of radiation related complications. PMID- 8635115 TI - Randomized trials of radiotherapy alone versus combined chemotherapy and radiotherapy in stages IIIa and IIIb nonsmall cell lung cancer: a meta-analysis. PMID- 8635116 TI - Stress and the development of breast cancer: a persistent and popular link despite contrary evidence. PMID- 8635117 TI - Geriatric oncology. PMID- 8635118 TI - Tumor chemosensitivity and chemoresistance assays. AB - BACKGROUND: Chemosensitivity and chemoresistance assays have been used to select potentially more effective chemotherapy regimens and to avoid the toxicity of potentially ineffective drugs. METHODS: Literature review was conducted. RESULTS: The relevant outcome measure for chemosensitivity assays is improved patient survival. However, the relevant outcome measure for chemoresistance assays is more controversial. Advocates propose that the avoidance of the toxicity of potentially ineffective drugs is the relevant outcome, whereas others believe that the latter is an intermediate outcome and that improved patient survival is the appropriate outcome for both chemoresistance and chemosensitivity assays. There have been no clinical trials showing that either of these assays is associated with improved patient survival. CONCLUSIONS: In terms of improved patient survival, the clinical role of chemosensitivity and chemoresistance assays is unproven. The use of the assays will vary depending on the type and stage of the tumor involved, the proposed role of chemotherapy, and the role of the empiric judgement of the physician. PMID- 8635119 TI - Alternative and complementary cancer therapies. PMID- 8635120 TI - Prognostic value of the plasminogen activation system in patients with gastric carcinoma. AB - BACKGROUND: Patients with gastric cancer have a poor prognosis and can be cured by surgery only if the cancer is detected in an early stage. Extended surgery, down staging with chemotherapy before operation, and new postoperative treatments are recent approaches to increase survival rates. Categorizing patients' prognoses as good or poor by pathophysiologic markers, however, may be of great help in selecting therapies for these patients. For example, plasminogen activation (PA) parameters, that play an important role in tumor invasion and metastasis, have prognostic value for several human malignancies. METHODS: We evaluated the relation between several PA parameters in tissue with standard clinicopathologic parameters and with the overall survival of 50 consecutive patients with gastric carcinoma. RESULTS: Univariate analysis showed that a low tissue-type plasminogen activator (t-PA) activity in normal mucosa and in carcinomas and a high antigen level of inhibitor type-1 (PAI-1), and, to a lesser extent, of urokinase-type plasminogen activator (u-PA) receptor, in carcinomas are associated with a poor overall survival of the patients. In contrast, of the 14 clinicopathological parameters only the number of eosinophils in the tumors was associated with survival. Multivariate analysis revealed that the t-PA and PAI-1 levels are independently associated with survival. CONCLUSIONS: Plasminogen activation parameters in both normal and carcinomatous tissue of the stomach of patients with gastric carcinoma are of particular clinical interest because of their prognostic impact on overall survival. PMID- 8635121 TI - Helicobacter pylori infection in patients with gastric carcinoma in biopsy and surgical resection specimens. AB - BACKGROUND: The discrepancy between the high seropositivity for Helicobacter pylori (H. pylori) and the low diagnostic yield of H. pylori organism in gastric biopsies of patients with gastric carcinoma has yet to be clarified. The present study attempted to clarify this controversial point by performing a comparative evaluation between the detection rate of H. pylori in biopsy and in surgical specimens. METHODS: The presence of H. pylori in biopsy samples from 50 patients with gastric carcinoma and 50 age-matched controls was evaluated histologically. Six histologic sections were obtained from gastric noncancerous areas and the presence of H. pylori was evaluated in those H. pylori negative patients who underwent gastrectomy. RESULTS: H. pylori was positive in 35 of 50 controls (70%). In biopsy samples, H. pylori was detected in 29 of 37 patients (78.4%) with early gastric carcinoma, 7 of 13 (53.8%) with advanced carcinoma, 16 of 23 (69.6%) with intestinal type of gastric carcinoma, and 20 of 27 (74.1%) with diffuse type of carcinoma. Studies carried out in gastrectomy specimens increased the diagnostic yield of H. pylori to 33 (89.2%), 12 (92.3%), 19 (82.6%), and 26 (96.3%) in patients with early, advanced intestinal, and diffuse types of gastric carcinoma, respectively. Overall, H. pylori was positive in 36 biopsy specimens (72%) and 45 gastrectomy specimens (90%). Namely, the detection of H. pylori infection was significantly higher in patients with gastric carcinoma using gastrectomy specimens than in patients with gastric carcinoma using biopsy specimens only (P < 0.05). CONCLUSIONS: These results indicate that the actual prevalence of H. pylori in patients with gastric carcinoma is considerably higher than that previously reported. PMID- 8635123 TI - Serum transforming growth factor alpha level as a marker of hepatocellular carcinoma complicating cirrhosis. AB - BACKGROUND: Transforming growth factor alpha (TGF alpha) is alleged to play a role in malignant progression as well as normal cell growth in an autocrine manner and its serum levels have been reported to increase during this progression. Most hepatocellular carcinomas (HCC) develop in cirrhotic livers in which hepatocyte necrosis and regeneration prevail. The significance of serum TGF alpha levels in the diagnosis of HCC complicating cirrhosis should be clarified. METHODS: One hundred twenty-four patients with cirrhosis were studied, 80 with HCC (HCC) patients and 44 without (LC) patients. There was no difference in clinical features between the two groups. One hundred eighty-two healthy adults were also studied as controls. Serum TGF alpha levels were measured by enzyme linked immunosorbent diffusion assay (ELISA). RESULTS: Serum TGF alpha levels were significantly higher in HCC patients than in healthy adults or LC patients (mean +/- SD: 45 +/- 40 vs. 21 +/- 15 or 25 +/- 19 pg/ml, respectively). In LC patients, serum TGF alpha levels were significantly correlated with serum albumin and total bilirubin levels (r = -0.44 and 0.32, respectively). When the cutoff level was defined as 25 pg/ml from receiver operating characteristic curve, sensitivity and specificity for the diagnosis of HCC in the presence of cirrhosis were 69% and 66%, respectively. Serum TGF alpha levels were decreased after successful treatment for HCC in 60% of the HCC patients. Serum TGF alpha levels showed no correlation with serum alpha-fetoprotein levels; the levels were greater than 25 pg/ml in 67% of the HCC patients whose serum alpha-fetoprotein levels were within 20 ng/ml. CONCLUSION: Serum TGF alpha levels may provide useful information for the diagnosis of HCC developing in the presence of cirrhosis. PMID- 8635122 TI - Postoperative hepatitis status as a significant risk factor for recurrence in cirrhotic patients with small hepatocellular carcinoma. AB - BACKGROUND: Recurrence of small hepatocellular carcinoma (HCC) is common. Recent studies have suggested that the status of the underlying liver parenchyma is a significant risk factor for recurrence of HCC. METHODS: The postoperative values of transaminase were examined every 6 months after surgery in 57 patients with a surgically resected solitary small HCC measuring up to 3 cm in greatest dimension. Based on the patterns of the transaminase values, the patients were divided into two groups. Group 1 (n = 20) had a high transaminase level; the values of postoperative transaminase were always more than 100 IU/L. Group II (n = 37) had a low transaminase level; the values of postoperative transaminase were sometimes lower than 100 IU/L. RESULTS: The cumulative carcinoma-free survival rates in Groups I and II were 91% and 80%, respectively, at 1 year, 64.5% and 5.5%, respectively, at 3 years, and 48.2% and 0%, respectively, at 5 years after surgery. The disease free survival rates in Group I were significantly lower than those in Group II (P = 0.0007), although no significant differences in histologic risk factors for recurrence or in clinical backgrounds were observed. With regard to the recurrence pattern, solitary recurrence was more frequently observed in Group I (P = 0.02), compared with the patients in Group II. A histologic comparison between the primary and recurrent tumor in patients who underwent re resection for solitary recurrence demonstrated the possible multicentric occurrence of HCC in 2 of 8 patients (25%) in Group I. CONCLUSIONS: This study suggests that the hepatitis status of the remnant liver plays an important role in the recurrence rates and patterns of small HCC after hepatectomy. PMID- 8635124 TI - Sequential treatment of recurrent mesenteric desmoid tumor. AB - BACKGROUND: The optimal management of inoperable desmoid tumors is still unclear. We report a 26 year-old female patient with familial adenomatous polyposis suffering from a recurrent inoperable intraabdominal desmoid tumor and its sequential treatment. METHODS: Treatment strategies included low-dose tamoxifen (30 mg orally per day), high-dose tamoxifen (90 mg orally per day), and a subsequent combination of goserelin acetate (3.6 mg subcutaneously once every four weeks) plus low-dose tamoxifen, medroxyprogesterone acetate (1000 mg orally per day) and interferon gamma (3 Mio IU subcutaneously 3 times a week). RESULTS: The combination of goserelin acetate and low-dose tamoxifen resulted in a decrease in tumour size and a complete relief of symptoms for 17 months. Thereafter the tumor progressed and again growth was stopped with interferon gamma therapy for another 6 months. All other treatment modalities had no effect. CONCLUSIONS: This study demonstrates long-term regression of a desmoid tumor with combined endocrine therapy using goserelin acetate plus tamoxifen. Tumor progression after 17 months was again stopped by a combination of interferon gamma and goserelin acetate. PMID- 8635125 TI - Analysis of the prognostic significance of cytosolic determination of CA 125 tumor-associated antigen, carcinoembryonic antigen and squamous cell carcinoma antigen in patients with nonsmall cell lung carcinoma. AB - BACKGROUND: The use of new prognostic factors as guidelines for the indication of treatment ancillary to surgery has been advocated for patients with nonsmall cell lung carcinoma (NSCLC). This article is an analysis of the prognostic information derived from determination of tumor-tissue cytosolic concentration of CA 125 tumor-associated antigen (CA 125), carcinoembryonic antigen (CEA), and squamous cell carcinoma antigen (SCC) in patients with NSCLC. METHODS: Tumor samples were obtained from 97 patients who successfully underwent surgery for NSCLC with curative intent. CA 125 and CEA were determined by enzyme immunoassay. SCC was determined by radioimmunoassay. The relationship between the tumor-tissue marker level and survival or likelihood of disease free survival was analyzed. RESULTS: Thirty-month survival post-treatment in patients registering CA 125 levels less than 26 U/mg was 68%, versus 30% among patients with levels of CA 125 greater tha 26 U/mg (P < 0.005). For SCC, these values were 57% and 39%, respectively (P = 0.07). No significant difference was seen for CEA (60% versus 44%; P = 0.3). Patients whose tumors had CA 125 levels lower than cutoff recorded a disease free survival rate of 61% versus 29% (P < 0.001); with SCC, likelihood of remaining free of tumor recurrence was 55% versus 34% (P < 0.05). Again, no significant difference was seen for CEA (49% versus 45%; P = 0.5). For CA 125 and SCC, the relationship between marker level and outcome held only with the most favorable histologic types. For CA 125 however, this relationship also held for Stage I tumors (P < 0.005). CONCLUSIONS: Ascertainment of concentrations of CA 125 and SCC in tumor tissue is useful parameter in the determination of postoperative prognosis for patients with NSCLC. PMID- 8635126 TI - Whole blood harvested after granulocyte-colony stimulating factor (Neupogen) mobilization, and reinfused unprocessed after high-dose melphalan treatment, accelerates hematopoietic recovery in patients with multiple myeloma. AB - BACKGROUND: High-dose melphalan (HDM) is now a standard treatment for multiple myeloma (MM). Stem cell transplants are fast evolving as an alternative to bone marrow transplants because they are less traumatic and easier to perform. A study was undertaken to test if whole blood harvested after mobilizing peripheral blood stem cells with granulocyte-colony stimulating factor (G-CSF) reinfused as such (without stem cells being processed or concentrated through a cell separator) for rescue after HDM (dose 140-180 mg/m2) in MM accelerates hematopoietic recovery. METHODS: Eight consecutive patients with MM were given HDM after receiving 4-5 courses of infusional vincristine, doxorubicin, and dexamethasone (VAD) chemotherapy. Approximately 6 weeks after the last course of chemotherapy, at the start of the procedure, G-CSF (Neupogen) was given at a dose of 10 microgram/kg subcutaneously daily for 4 days to mobilize stem cells. All of the patients had brisk leukocytosis (18.4-64.6 x 10(9)/L). On day 0 (fifth day after G-CSF), 1 L of blood was removed by phlebotomy and kept at room temperature for 24 hours. This was followed by a rapid intravenous (i.v.) bolus of melphalan at a dose of 140 mg/m2 in the first three patients, 155 mg/m2 in the 4th, 170 mg/m2 in the 5th, and 180 mg/m2 in the 6th, 7th, and 8th patients, applies to all patients along with hydration and diuresis. Twenty-four hours later, I L of blood previously removed was transfused back to the patient without any processing through the cell separator. The numbers of mononuclear cells reinfused was between 0.76 and 3.2 x 10(8)/kg (mean: 1.82 x 10(8)/kg). The number of CD 34+ cells infused in 4 patients ranged from 1.9 to 2.8 x 10(6)/kg (mean: 2.35 x 10(6)/kg). G-CSF was restarted on day 2 at a dose of 5 microgram/kg and given daily until the granulocyte count was 0.5 x 10(9)/L or more for 3 consecutive days. Antibiotics were given whenever a patient developed pyrexia. Platelets were transfused if below 20 x 10(9)/L or for incidences of overt bleeding. RESULTS: Granulocyte counts touched 0 between Days 6 and 10, lasting for an average of 3.4 days. Duration of granulocyte count below 0.5 x 10(9)/L was 6.5 days (range: 5 9), and these rose to 1.0 or more x 10(9)/L by Day 13.7 (range: 13-16). The number of platelet transfusions given per patient was 2.5 (range: 0-4). Intravenous antibiotics were used for 9 days (range: 4-12), and patients were discharged on Day 19 (range: 16-22). Follow-up ranged from 45 to 380 days. CONCLUSIONS: Hematopoietic recovery was rapid in all patients, including those who received doses of melphalan higher than 140 mg/m2. This resulted in less need for platelet transfusions or antibiotics, and hospitalization was short. Our results are historically similar to conventional peripheral blood stem cell transplants but much better than autologous bone marrow transplants, indicating that whole blood after G-CSF provided sufficient progenitor cels for early engraftment. The procedure is less labor intensive insofar as it does not require separation and concentration of stem cells. It has the potential of becoming an alternative to autologous marrow and peripheral blood stem cell transplantation in patients with MM. PMID- 8635127 TI - Combined overexpression of urokinase, urokinase receptor, and plasminogen activator inhibitor-1 is associated with breast cancer progression: an immunohistochemical comparison of normal, benign, and malignant breast tissues. AB - BACKGROUND: A strong positive correlation exists between the breast cancer tissue content of either urokinase-plasminogen activator (uPA) or plasminogen activator, inhibitor type I (PAI-1), quantified in the tissue extracts by immunoassays, and the survival of patients with breast cancer. Furthermore, several studies assign to the urokinase-type plasminogen activator receptor (uPAR) a pivotal role in triggering the proteolytic activity of the urokinase pathway involved in tumor stroma degradation, tumor spread and metastasis. However, the pattern of distribution of uPAR in normal and cancerous human tissue and the pattern of coexpression of activators and inhibitors that occurs in breast cancer tissues is not completely known. METHODS: The immunohistochemical localization of uPAR, uPA, tPA) and PAI-1 was evaluated by using the avidin-biotin immunoperoxidase technique and affinity-purified monoclonal antibodies from American Diagnostica Inc. Studies were performed in formalin fixed, paraffin-embedded tissue prepared from 23 surgically excised non-neoplastic breast tissues and 18 ductal breast carcinomas. RESULTS: While the expression of uPAR protein represents a constant feature of invasive ductal breast cancer, it was also observed in most of the breast tissue samples, including the normal breast tissues. The staining for uPAR was mainly localized on normal or tumoral epithelial cells, even if the co expression of uPAR in stromal cells was frequently observed in adjacent slides. A semiquantitative analysis of immunohistochemical results showed that uPAR and PAI 1 were overexpressed in invasive breast cancer in comparison with normal and benign breast tissues. In addition, uPA was higher in both invasive breast carcinomas and benign breast lesions with respect to normal breast tissues. CONCLUSIONS: We showed that overexpression of uPAR, uPA, and its main inhibitor, PAI-1, is a constant feature of invasive ductal breast carcinomas. However, the expression of the above fibrinolytic reactants is not specific for breast cancer since positive staining for these molecules was frequently observed in benign breast lesions as well as in normal breast tissues. The combined increased expression of uPA and its cellular receptor, uPAR on the surface of tumor epithelial cells may account for the activation of the proteolytic system which occurs in breast cancer. PMID- 8635128 TI - Self-reported stress and risk of breast cancer. AB - BACKGROUND: Many women attribute the development of their breast cancer to psychosocial factors such as stress and depression. Yet investigations of the relationship between breast cancer and stressful life events have had inconsistent outcomes, due in part to studies with small sample sizes and reliance on hospital-based populations. METHODS: As part of a population-based, case-control study of breast cancer etiology, we evaluated the association between stressful life events and the risk of breast cancer among 258 breast cancer patients and 614 randomly selected population-based controls. Information on 11 stressful life events was collected in telephone interviews with women aged 50-79 who were participating in the ongoing study. RESULTS: Breast cancer patients and controls experienced the same number of stressful life events in the five years prior to diagnosis or an equivalent reference date (controls), averaging 2.4 and 2.6 events, respectively. After adjustment for known breast cancer risk factors, there was no association between weighted stressful life event scores and the risk of breast cancer (odds ratio [OR] = 0.90 per unit increase; 95% confidence interval [CI], 0.78-1.05). Only one life event, death of a close friend, was significantly more often reported by controls (OR = 0.72; 95% CI, 0.52-1.00). Other life events were inconsistently and nonsignificantly associated with breast cancer risk. CONCLUSIONS. The results of this retrospective study do not suggest any important associations between stressful life events and breast cancer risk. PMID- 8635130 TI - Quantitative analysis of vascular endothelial growth factor in primary breast cancer. AB - BACKGROUND: Recent clinical studies have demonstrated that tumor angiogenesis is a potent prognostic indicator for breast cancer patients. The quantitation of endothelial growth factors is thought to be useful to assess angiogenic phenotype in the tumor. Among the many new endothelial growth factors, vascular endothelial growth factor (VEGF) is known to be particularly responsible for promoting the neovascularization in human breast cancer. METHODS: Intratumoral protein levels of VEGF were measured by enzymatic immunoassay in 135 primary breast cancer tissues. The VEGF levels were compared with the microvessel density evaluated by immunostaining the endothelial antigen and also were compared with intratumoral protein levels of other endothelial growth factors, including basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). RESULTS: Intratumoral VEGF concentrations varied from 3.3 pg/mg protein to 2032 pg/mg protein (average 148 pg/mg protein). An immunocytochemical analysis using anti-VEGF antibody confirmed that VEGF was located mainly in the cytoplasm of the tumor cells. The VEGF concentrations were significantly higher in vascularly rich tumors than in vascularly poor tumors. No significant association was found between VEGF concentrations and the two other endothelial growth factor concentrations. CONCLUSIONS: The quantitation of intratumoral VFGF levels seems to be useful for assessing the activity of tumor angiogenesis. PMID- 8635129 TI - A prospective study of conservative surgery alone in the treatment of selected patients with stage I breast cancer. AB - BACKGROUND: Randomized clinical trials have clearly demonstrated that the use of radiation therapy (RT) following breast-conserving surgery (CS) substantially reduces the risk of local recurrence. However, the low rate of local recurrence after CS and RT for patients without known risk factors, and the recent increase in the detection of smaller cancers due to mammographic screening have led to the speculation that a subgroup of patients who have a low risk of local recurrence without RT might be identified. In 1986, we initiated a one-arm, prospective clinical trial of CS alone for treatment of highly selected breast cancer patients without known risk factors for local recurrence. METHODS: The study had a sequential design with a planned accrual of 90 patients. Criteria for entry into the trial were: a unicentric, clinical TI infiltrating ductal, mucinous or tubular carcinoma without an extensive intraductal component or lymphatic vessel invasion; a wide excision with a pathologically-documented negative margin of at least 1 cm; and histologically negative axillary lymph nodes. No adjuvant RT or systemic therapy was administered. Seventy-six per cent of the lesions were detected by mammography alone. The median gross pathologic tumor size was 0.9 cm. The median patient age was 67 years. RESULTS: Eighty-seven patients were enrolled in the trial before it closed prematurely in 1992 because the predefined stopping boundary was crossed (i.e., the sixth local recurrence was observed). At that time, the average annual local recurrence rate was 4.2%. With a median follow-up of 56 months, there are now 14 patients (16%) with local recurrence as their site of first failure (average annual local recurrence rate: 3.6%). Four patients without local recurrence developed distant metastases. Three patients have died, one of metastatic breast cancer and two of unrelated causes. CONCLUSIONS: Even in a highly selected group of patients with early-stage breast cancer, there is a substantial risk of early local recurrence for those treated with wide excision alone. PMID- 8635131 TI - Altered expression of transforming growth factor-beta 1 in cervical neoplasia as an early biomarker in carcinogenesis of the uterine cervix. AB - BACKGROUND: Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cell growth, but can also stimulate stromal cell growth. Loss of responsiveness to TGF-beta 1 or loss of TGF-beta 1 itself may be important in the progression of cervical intraepithelial neoplasia (CIN) to invasive cervical carcinoma. METHODS. To examine the expression of TGF-beta in early stages of malignant transformation of the uterine cervix, paraffin embedded tissue samples from 11 patients with normal cervical epithelium, 15 with CIN I III, 12 with microinvasive, and 18 with invasive squamous cell carcinoma were examined using an immunohistochemical technique. Tissues were immunostained with polyclonal antibodies that react with intracellular and extracellular forms of TGF-beta 1. RESULTS: Percent positive staining for the intracellular form of TGF beta 1 was 100% for normal epithelium, 73.3% for CIN, and 44.1% for invasive carcinomas, (P = 0.002). Percent positive staining for the extracellular form of TGF-beta 1 was 63.6% for stroma underlying normal epithelium, 60% for stroma associated with CIN, and 94.1% for stroma surrounding invasive cancer (P = 0.007). CONCLUSIONS: Decreased expression of intracellular TGF-beta 1 in neoplastic epithelium and increased expression of extracellular TGF-beta 1 in stroma associated with invasive cervical carcinoma suggest that an early event in the neoplastic transformation of cervical epithelia] cells may involve the loss of TGF-beta 1. Tumor progression may be indirectly promoted by TGF-beta 1 secreted into or produced by supporting stromal elements. PMID- 8635133 TI - A risk model for ovarian carcinoma patients using CA 125: time to normalization renders second-look laparotomy redundant. AB - BACKGROUND: We evaluated the incorporation of CA 125 normalization times into a prognostic model based on pretreatment variables in patients with ovarian carcinoma to determine if they could render second-look laparotomy (SLL) redundant. METHODS: A total of 54 consecutive patients with ovarian carcinoma who underwent SLL between 1985 and 1990 were included in this analysis. At diagnosis, all of the patients had abnormal CA-125 serum levels, which fell to within the normal range during chemotherapy. Cox's model was used to select pretreatment variables relevant for prognosis. The influence of the time to normalization of CA 125 (< or = vs. > 1 months) and the capability of SLL results to modify prognostic prediction, were also evaluated. RESULTS: The size of the residual tumor at the beginning of therapy, and Eastern Cooperative Oncology Group (ECOG) performance status (PS) were independently predictive of survival. The time to normalization of CA 125 serum levels (analyzed either as -a continuous or as a two-category variable) also had an independent prognostic role when included in the model. When we examined the inclusion of both CA 125 parameter and SLL into the model together, we found that only CA 125 continued to have an independent prognostic relevance. On the basis of the two pretreatment parameters (PS and tumor size) and of this response parameter (time to normalization of CA 125 values) we selected six subgroups of patients having different outcomes (log rank test of equality over-strata < 0.001). Patients with good prognostic pretreatment variables, and those with intermediate prognosis at the beginning of therapy who showed a quick normalization of CA 125, had an 80% 5-year survival, compared with 16% 5-year survival in the remaining patients. (P < 0.0001). CONCLUSIONS: Our data suggest that the survival of patients with advanced ovarian carcinoma could be accurately predicted by considering some pretreatment variables and time to CA 125 normalization together, without performing SLL. Our risk model, however, needs to be validated by larger prospective trials, to draw any definitive conclusions about the abandonment of surgically defined response. PMID- 8635132 TI - Pathologic models to predict outcome for women with endometrial adenocarcinoma: the importance of the distinction between surgical stage and clinical stage--a Gynecologic Oncology Group study. AB - BACKGROUND: Numerous pathologic factors have been identified as important in predicting outcome for women with endometrial adenocarcinoma. However, most patients have a mixture of good and bad factors. For these women, the prognosis is uncertain, and it is often unclear whether postoperative therapy is indicated. METHODS: Using univariate and multivariate analysis, we investigated the pathologic factors commonly reported to be of prognostic significance, using data from 819 patients with clinical Stages I and II endometrial adenocarcinoma from a Gynecologic Oncology Group study. Since the clinical stage frequently underestimated the surgical stage, models that designate the relative risk associated with each of the variables were created for both clinical and surgical Stage I and 11 patients. RESULTS: We confirmed the importance of age, depth of myometrial invasion, and to a lesser degree, histologic grade, and cell type, as independent prognostic variables. CONCLUSIONS: The relative risk of death can be determined using a simple multiplicative calculation, and the absolute risk can be estimated by inspection of the accompanying figures. These data can be used to provide patients with prognostic information and to help determine the need for postoperative adjuvant therapy. PMID- 8635134 TI - Epithelial ovarian tumors in the reproductive age group: age is not an independent prognostic factor. AB - BACKGROUND: While ovarian carcinoma is rare in the reproductive age group, these younger patients are known to fare better than the older patients. To determine whether age is an independent prognostic factor, as well as to investigate the clinicopathologic profile and survival rate of young women with ovarian carcinoma, a retrospective analysis in a series of patients aged 40 years or younger was performed. METHODS: We collected data on 74 patients with borderline or invasive ovarian carcinoma treated at the Department of Obstetrics and Gynecology at the University of Florence between 1969 and 1994. The median follow up was 72 months (range, 11-288 months). To assess the clinicopathologic profile and survival differences according to age, the series was subdivided into "very young" (30 years or younger) and "young" (31-40 years) groups of 34 and 40 patients, respectively. Survival rates (Kaplan-Meier method) were compared by the log rank test. A multivariate analysis (Cox proportional hazards) was used to determine the independent effect of each variable on survival. RESULTS: The overall 5-year and 10-year survival rates were 58.2% and 46.1%, respectively. Several prognostic factors were found significant by univariate analysis, including stage (P < 0.001), grade (P < 0.001), residual disease (P < 0.001), histologic type (P < 0.05), and age (< or = 30 years vs. 31-40 years; P = 0.009). Five year survival rates for the patients age 30 years and younger and patients age 31-40 years were 71.3% and 47.1%, respectively. In the former group, low malignant potential tumors and well differentiated carcinomas were significantly more frequent (68.8% vs. 37.5%; P = 0.01). In the multivariate analysis, only stage (I vs. >I; P = 0.004), grade (0-1 vs. 2-3; P = 0.03) and residual disease (P = 0.02) were found to be significant independent prognostic factors, whereas age (< or = 30 years vs. 31-40 years) yielded no independent information (P = 0.36). CONCLUSIONS: Epithelial ovarian cancer patients age 30 years or younger have a more favorable prognosis because of a higher rate of early stage, low grade tumors. Patients aged 31-40 years also show a more favorable disease profile and clinical outcome, although to a lesser extent, than the average ovarian cancer population. Age does not emerge as an independent prognostic factor for ovarian carcinoma in women younger than 40 years. PMID- 8635135 TI - The proportion of free to total prostate specific antigen: a method of detecting prostate carcinoma. AB - BACKGROUND: Prostate specific antigen (PSA) is the most useful marker for prostate carcinoma (CaP). However, the sensitivity and specificity for PSA are not sufficient for the diagnosis of organ-confined prostate carcinoma. Recent studies have revealed that anti-PSA antibody identifies both PSA complexed to alpha-1-antichymotrypsin and free PSA, whereas anti-gamma-seminoprotein antibody recognizes free PSA exclusively. To enhance the ability of PSA to detect CaP in patients with total PSA levels of 10 ng/mL or lower, we developed the ratio of gamma-seminoprotein and PSA (free/total PSA index). METHODS: We measured free/total PSA indices for 285 patients who had serum PSA levels of 10 ng/mL or lower and who were diagnosed pathologically. RESULTS: Of the 285 patients, 228 had no prostate carcinoma (NC) and 57 had CaP. The mean total PSA level for CaP (5.137 +/- 2.483 ng/mL; mean +/- standard deviation) was significantly greater (P < 0.0001) than that for NC (3.251 +/- 2.129). The mean free/total PSA index for CaP (0.774 +/- 0.468 was significantly lower (P < 0.0001) than that for NC (1.563 +/- 0.938). The sensitivity for the free/total PSA index was similar to that for total PSA (78.9% vs. 75.4%). However, the specificity, positive predictive value, and overall accuracy for the free/total PSA index (75.9%, 45%, and 76.5%, respectively) increased by 15-20% compared with those for total PSA (56.6%, 30.3%, and 60.4%, respectively). CONCLUSIONS: The free/total PSA index improved the specificity of PSA without impairing the sensitivity in detecting CaP among patients with serum PSA levels of 10 ng/mL or lower. PMID- 8635136 TI - Phase II study of the oral cyclophosphamide and oral etoposide combination in hormone-refractory prostate carcinoma patients. AB - BACKGROUND: Hormonotherapy temporarily controls symptoms in 80% of patients with metastatic prostate carcinoma. Once progression occurs, no consensus exists on further therapy. Oral etoposide (VP-16) has shown clinical efficacy in advanced small cell lung carcinoma, breast cancer, germ cell tumors, and lymphomas, A synergistic effect between etoposide and alkylating agents such as estramustine was recently reported. We began a prospective Phase II study of an oral combination of cyclophosphamide (CPM) and VP-16 in patients with hormone refractory [correction of refactory] prostate carcinoma (HRPC). METHODS: Patients were orally treated with CPM (100 mg/day) and VP-16 (50 mg/day) for 14 days every 28 days. Therapy continued until there was evidence of disease progression. RESULTS: From November, 1992, to February, 1995, 20 patients with HRPC were entered into the study. Patients were eligible if they had an ECOG performance status (PS) of 0 to 2. All of the patients presented with bone metastasis, and 70% presented with bone pain. Seventy-five percent had failed at least two hormonal manipulations. The mean duration of treatment was 5 months (range 2-12). Performance status improved in 26% of the patients, and bone pain was relieved in 71%. An objective response was defined as a decrease of 50% or more in the prostate-specific antigen (PSA) level. One patient demonstrated a complete response, and six patients had partial responses assessed by PSA plasma levels (objective response rate: 35%). The mean duration of response was 8 +/- 6 months (range: 2-24). Median survival was 11 months. Toxicities were minimal. CONCLUSIONS: The combination of oral CPM and VP-16 may be an active and well tolerated regimen for patients with HRPC. PMID- 8635137 TI - Phase II trial of subcutaneously administered granulocyte-macrophage colony stimulating factor in patients with metastatic renal cell carcinoma. AB - BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is involved in the differentiation and proliferation of various hematopoietic precursors. It also has been reported to enhance the antitumor activity of various mature effector cells. Previous reports have noted preclinical antitumor activity in a murine model utilizing genetically engineered tumor cells and instances of tumor regression in patients with solid tumors receiving GM-CSF. In the present study, a Phase II trial of human recombinant GM CSF (GM-CSFrh) in patients with metastatic renal cell carcinoma (RCC) was conducted to investigate further the potential antitumor activity of this cytokine. METHODS: Twenty-six eligible patients with metastatic RCC received 3 microgram/kg of GM-CSFrh subcutaneously for 14 days, with cycles repeated every 28 days. RESULTS: Two of 26 patients (8%; 95% confidence interval 1-25%) demonstrated partial tumor responses during GM-CSFrh therapy. Both individuals who responded had received prior therapy. A median of three cycles per patient were administered, and toxicity was mild. CONCLUSIONS: GM-CSFrh may mediate tumor regression in patients with metastatic RCC; however, the level of activity in previously treated patients is low. PMID- 8635138 TI - The correlation between the loss of chromosome 14q with histologic tumor grade, pathologic stage, and outcome of patients with nonpapillary renal cell carcinoma. AB - BACKGROUND: Conventional pathologic classifications of human renal cell carcinoma (RCC) give little insight into oncogenesis and little assistance in predicting the clinical behavior of this disease. Identification of specific genetic alterations involved in the development of RCC using fluorescence in situ hybridization (FISH) however, may help provide foundations for classification, prognosis, and clinical management of the patients. METHODS: Archival, paraffin embedded tissue sections from 30 human non-papillary RCCs were examined with a dual color FISH technique for loss of chromosomes 3p and 14q. Telomeric DNA probes from 3p or 14q and an internal ploidy control probe, centromeric probe of chromosome 2, were applied directly to the tumor sections. The correlations between loss of 3p or 14q, and tumor ploidy, with tumor grade, pathologic stage, and patient outcome were assessed. RESULTS: Ninety percent of the patients (27) lost chromosome 3p, and 36.7% of the patients (11) had chromosome 14q deletions. The loss of 3p in the samples tested was unrelated to patient age, gender, outcome, tumor stage, or histologic grade. However, the deletion of 14q was significantly correlated with higher stage (P = 0.01), histologic grade (P = 0.01), and patient outcome (P < 10(-4)). CONCLUSION: The close correlation of 14q loss with higher stage, higher histologic grade, and poorer outcome for patients with nonpapillary RCC indicates that it may be a promising prognostic marker. PMID- 8635139 TI - Necrosis as a prognostic factor in glioblastoma multiforme. AB - BACKGROUND: Many pathologists require the presence of tumor necrosis within an astrocytic neoplasm to establish the diagnosis of glioblastoma multiforme (GM). Two new grading systems for astrocytic neoplasms allow a tumor to be diagnosed at the highest level of anaplasia without requiring the presence of tumor necrosis. METHODS: To determine whether GMs without necrosis had biologic behavior most compatible with a diagnosis of GM or of anaplastic astrocytoma (AA), we examined the survival of 299 patients whose tumors were diagnosed as GM because they contained endothelial proliferation and who were treated according to prospective clinical protocols. Multivariate proportional-hazards survival analysis was used to assess the importance of tumor necrosis after adjustment for other prognostic factors. RESULTS: Of 275 patients with GMs containing endothelial proliferation, 88% had tumor necrosis. Absence of necrosis was associated with younger age and with less extensive surgical resection. The absence of necrosis predicted longer survival in univariate analysis (P = 0.02) and after adjustment for age, Karnofsky performance score, and extent of resection in a multivariate analysis (P = 0.04). However, the magnitude of the survival difference was not clinically important: patients without tumor necrosis had a median survival of 12.5 months, and those with tumor necrosis had a median survival of 10.9 months. Kaplan-Meier survival rates two years after diagnosis were 13.0% for patients with necrosis and 27.1% for patients without necrosis; the difference in 2-year survival was not statistically significant (difference in survival rates = 14.1%, 95% confidence interval -2.2% to 30.4%). CONCLUSIONS: The survival of patients with astrocytic neoplasms containing endothelial proliferation and no necrosis conforms best to the pattern expected for patients with GM rather than AA. This supports the classification of these tumors as GM in the revised World Health Organization grading system and as grade 4 astrocytomas in the St. Anne-Mayo grading system. PMID- 8635140 TI - The sequential changes in DNA synthesis, glucose utilization, protein synthesis, and peripheral benzodiazepine receptor density in C6 brain tumors after chemotherapy to predict the response of tumors to chemotherapy. AB - BACKGROUND: Monitoring therapy in patients with brain tumors is very difficult and unreliable. It has been shown that there is no good correlation between tumor sensitivity measured in vitro and in situ tumor response to therapies. METHODS: Sequential changes in tumor size, number of DNA synthesizing cells (labelling index [LI]), glucose utilization (LCGU), protein synthesis (LCPS), and peripheral benzodiazepine receptor (PBR) density were examined after chemotherapy for seven days. This was done using antibromodeoxyuridine immunohistochemical stain and multiple tracer quantitative autoradiography in a C6 rat brain with an implanted glioma. On Day 10 after inoculation, the rats were divided into 5 experimental groups: (1) a nontreatment group (control Group 1); (2) a group received 5% dextrose intraarterial (IA) administration (control Group 2); (3) a group received 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) intravenous (i.v.) administration (Group 3) (5% dextrose was solvent); (4) a group received BCNU IA administration (Group 4) (5% dextrose was solvent); and (5) a group received sarcosinamide chloroethyl nitrosourea (SarCNU) IA administration (Group 5) (solvent as for the BCNU group). RESULTS: Three treatments showed a significant decrease (P < 0.003) in tumor growth. The most effective treatment was BCNU IA and SarCNU IA was moderately effective. BCNU i.v. showed no effect on tumor growth when compared with the two control groups. The change in the peak LI correlated well with the peak LCGU. These parameters decreased markedly and significantly in both Group 4 and Group 5 from Day 1 after treatment. The rates of the decrease in these biologic factors also correlated well with a decrease in the tumor growth. The LCPS did not correlate with a decrease in the LI or LCGU. The dissociation constant (Kd) and densities of the receptors PBR (B max) did not change significantly in any of the treatment groups during the observation period. CONCLUSIONS: From the results presented, we concluded that changes in the LI and LCGU represent the most reliable parameters with which to predict the response or sensitivity of this glial tumor to the treatments applied. These data suggest that if changes in peak LCGU were measured in tumors using positron emission tomography, they might be instrumental in providing in vivo information about the sensitivity of a tumor to a given treatment without the need for repeated tumor biopsy. PMID- 8635141 TI - Prognostic significance of proliferative and apoptotic fractions in low grade follicle center cell-derived non-Hodgkin's lymphomas. AB - BACKGROUND: The biologic parameters, DNA ploidy and proliferative activity, have been suggested as prognostic factors in non-Hodgkin's lymphoma (NHL). However, reports on the prognostic importance of these factors in follicle center cell derived (FCC) centroblastic/centrocytic (CB/CC) NHL patients with long follow-up are scarce. METHODS: Apoptotic fractions were quantified in 60 patients with CB/CC NHL by in situ labeling of DNA strand breaks in nuclei [TdT-mediated dUTP/dATP in situ 3'OH--end labeling (TUNEL)]. The findings were related to S phase and MIB-1 counts, DNA ploidy, and clinical outcome. RESULTS: In CB/CC NHL, the percentages of proliferating and apoptotic cells were lower than in reactive germinal centers (GC; P < 0.05; mean, 0.188 vs 3.263% and 19.05 vs. 69.4% for TUNEL and MIB-1 positive cells in CB/CC and GC, respectively). Significantly higher percentages of MIB-1 and TUNEL positive cells were observed in patients with complete remission when compared with the partial remission / no response group (P < 0.01). The size of proliferative and apoptotic fractions did not correlate with the overall survival of the patients. However, follicular and diffuse growth pattern, elevated serum lactic dehydrogenase, advanced stage, and age indicated a lower probability of 5- and 10-year survival. CONCLUSIONS: The investigation of proliferative and apoptotic fractions in FCC lymphomas may help to define groups of patients to who would benefit from aggressive, high dose therapy protocols and patients to whom less aggressive strategies can be applied safely. PMID- 8635142 TI - Carpal tunnel syndrome associated with interleukin-2 therapy. AB - BACKGROUND: Interleukin (IL-2) has been extensively used in our institution in the treatment of cancer and has protean neurologic side effects. Carpal tunnel syndrome developing in patients receiving IL-2 appears to have a good prognosis and may spare the patient unneeded investigation. METHODS: A retrospective evaluation was undertaken for all patients using our institution's Patient Studies database. The patients were examined and their charts reviewed. RESULTS: We found eight patients with renal cell carcinoma who developed carpal tunnel syndrome (CTS) during treatment with IL-2, fluorouracil (5-FU), and alpha interferon (alpha-IFN). The symptoms were bilateral in five patients and all patients improved with cessation of therapy. Three patients had recurrent symptoms with subsequent courses of therapy. Symptoms occurred during or shortly after IL-2 infusion and resolved after therapy was completed with conservative management. The number of courses given did not seem to correlate with development of symptoms. Neurophysiologic studies demonstrated conduction velocity slowing without evidence of acute denervation. CONCLUSIONS: IL-2 can produce focal entrapment of the median nerve at the wrist, which reverses with drug withdrawal. IL-2 mediates the inflammatory response and can cause interstitial edema that likely causes CTS to develop in predisposed patients undergoing treatment. PMID- 8635143 TI - Cisplatin and ifosfamide with either vinblastine or etoposide as salvage therapy for refractory or relapsing germ cell tumor patients: the Institut Gustave Roussy experience. AB - BACKGROUND: Approximately 30% of patients with metastatic germ cell tumors require salvage chemotherapy for recurrent or refractory disease after first-line treatment. The optimal salvage chemotherapy regimen remains to be determined. METHODS: Fifty-four patients with metastatic germ cell tumors who failed to be cured with first-line therapy, were treated with a salvage VIP/VeIP regimen including cisplatin (20 mg/m2/d dl to d5), ifosfamide (1.2 gm/m2/d dl to d5), and either etoposide (75 mg/m2/d dl to d5) or vinblastine (0.11 mg/kg/d dl and d2) for 5 consecutive days every 3 weeks. RESULTS: A complete remission was observed in 24 patients (44%) at completion of VIP/VeIP chemotherapy. In 17 patients (31%), complete remission was reached with chemotherapy alone, whereas four (7%) were rendered tumor-free by resection of the residual inactive tumor. Three patients (6%) became tumor-free by resection of the residual carcinoma. Ten other patients (19%) achieved a partial response, with normalization of serum tumor markers. Eleven of those thirty-four patients additionally received high-dose chemotherapy with hematopoietic stem cell support as consolidation treatment. Twenty patients (37%) were judged to be treatment failures because of either incomplete response (3 patients) or progression of disease (17). Myelotoxicity was severe, but no toxicity deaths were noted. After a median follow-up of 30 months, 23 patients (43%) are alive, 16 of whom (30%) are without evidence of progression of disease. Among patients who received high-dose chemotherapy, the relapse-free survival was 63% compared with 35% for patients who did not receive this consolidation treatment. CONCLUSIONS: Currently available salvage chemotherapy with ifosfamide and cisplatin is predicted to cure approximately 30% of the patients who have failed first-line treatment. Whether high-dose chemotherapy with hematopoietic stem cell support after salvage VIP/VeIP could improve these modest results remains to be confirmed in a randomized study. PMID- 8635144 TI - Aggressive T/natural killer cell lymphoma presenting as testicular tumor. AB - BACKGROUND: Nonnasal CD56+ T/natural killer (NK) cell lymphomas with morphologic and immunogenetic features similar to those of the distinctive nasal T/NK cell lymphoma are uncommon and have been characterized only recently. They show predominantly extranodal presentation, high stage disease, a highly aggressive course, strong association with Epstein-Barr virus (EBV), and lack of T cell receptor gene rearrangement. Only one previously reported case had a testicular presentation, although the testis is not uncommonly involved during the course of disease in both nasal and nonnasal T/NK cell lymphomas. METHODS: Three patients with T/NK cell lymphoma who presented initially with a testicular mass are reported. RESULTS: The three patients underwent orchidectomy for testicular tumor. Histologically, the testes showed diffuse dense infiltration of medium sized or large lymphoma cells. Antiocentric growth and necrosis were prominent in two cases. The lymphoma had the following immunophenotype: CD2+ CD3 epsilon+ CD56+ compatible with T/NK cell lymphoma; two lacked staining with Leu4 (CD3), and one had weak staining. With immunohistochemical preparations, it was noted that the rete testis stained consistently for CD56, and the Leydig cells and Sertoli cells showed patchy staining. The neoplastic cells harbored EBV, as demonstrated by in-situ hybridization. Additional sites of disease were detected at the time of the diagnosis in one patient (nose) or appeared soon afterwards in all three patients (skin or gastrointestinal tract). All three patients died within 5 months. CONCLUSIONS: This study confirms that testicular CD56+ T/NK cell lymphoma tends to disseminate early, pursues an aggressive course, and is strongly associated with EBV. CD56 recognizes the neural cell adhesion molecule (NCAM), which exhibits homophilic binding properties. The expression of CD56 in the normal testicular constituents can perhaps explain the tendency for T/NK cell lymphoma to localize in this organ. PMID- 8635145 TI - Histopathologic risk factors in retinoblastoma: a retrospective study of 172 patients treated in a single institution. AB - BACKGROUND: Microscopic extrascleral involvement and involvement of optic nerve resection line are accepted risk factors for orbital and/or metastatic disease from retinoblastoma. Conversely, choroidal and retrolaminar optic nerve involvement are questionable risk factors. The aim of this retrospective study was to define the histopathologic risk factors for orbital and/or metastatic disease in patients treated by first-line enucleation. METHODS: Histopathologic review of 172 evaluable patients treated at Institut Curie between 1977 and 1990 determined the degree of choroidal (minimal or massive), scleral (intra- or extrascleral), optic nerve (prelaminar, retrolaminar with or without resection line involvement), and anterior chamber invasion. The degree of differentiation was also analyzed. The log rank test was used for univariate analysis and the Cox regression model was used for multivariate analysis. RESULTS. Eighty-seven percent of the 172 patients were disease free at 3 years. Twenty-three patients developed retinoblastoma recurrence. The disease-free survival was significantly different among the five subgroups of choroidal or scleral invasion (P = 3 x 10( 3). The differences among the four subgroups of optic nerve invasion were also significant (P = 10(-4)). Classical factors were confirmed in our series (extrascleral involvement and involvement of optic nerve resection line). Multivariate analysis of the 149 patients without these classical risk factors revealed two factors that increase the risk of orbital and/or metastatic disease: massive choroidal invasion and postlaminar optic nerve involvement. CONCLUSIONS: In our experience, retrolaminar optic nerve involvement, with free resection line, and massive choroidal invasion significantly increase the risk for orbital and/or metastatic disease. PMID- 8635147 TI - Azelaic acid decreases the fibrinolytic potential of cultured human melanoma cells in vitro. AB - Azelaic acid (AZA) has been used successfully in the treatment of lentigo maligna melanoma. Since it is generally accepted that the fibrinolytic potential of tumour cells is related to their malignant phenotype, it was the aim of this study to investigate the effect of AZA on the fibrinolytic potential of three different human melanoma cell lines (Bowes, GUBSB and MJZJ). Melanoma cells were incubated with AZA in doses ranging from 10(-2) M to 4 x 10(-2) M for 5, 8 and 24 h. The expression of tissue-type plasminogen activator (t-PA), urokinase-type PA (u-PA) and PA inhibitor-1 (PAI-1) in such treated cells was investigated by specific ELISAs on the protein level and by Northern blotting on the mRNA level. AZA caused a time and dose dependent decrease in the fibrinolytic potential of all three cell lines investigated by decreasing t-PA antigen in Bowes, by decreasing u-PA antigen in GUBSB and by increasing PAI-1 antigen in MJZJ cells, respectively. There was no significant difference between the viability of cells in control cultures and those treated with AZA. The effect of AZA on specific mRNA for t-PA in Bowes cells, u-PA in GUBSB and PAI-1 in MJZJ was consistent with its effect on the secretion of these fibrinolytic proteins by the respective cells. The results show that AZA decreases the fibrinolytic potential of the three human melanoma cell lines in vitro. This decrease may be operative in the mechanism by which AZA has been shown to affect malignant melanoma in vivo. PMID- 8635146 TI - Sequential detection of tumor cells in the peripheral blood and bone marrow of patients with stage IV neuroblastoma by the reverse transcription-polymerase chain reaction for tyrosine hydroxylase mRNA. AB - BACKGROUND: The aim of this study was to evaluate the changes of tumor cell contamination in bone marrow (BM) and peripheral blood (PB) during the clinical course of patients with advanced neuroblastoma by detecting tyrosine hydroxylase (TH) mRNA to clarify the appropriate source and time for harvesting hematopoietic stem cells for transplantation. METHODS: A total of 15 patients with Stage IV neuroblastoma were studied. All 15 patients had peripheral blood stem cell (PBSC) samples and BM samples examined for TH mRNA by using the reverse transcription polymerase chain reaction (RT-PCR) at the time of harvest. Nine of the 15 patients, also had BM and PB samples examined sequentially. RESULTS: Comparing the 45 paired samples concurrently drawn, 16 of 28 BM samples (57.1%) and 4 of 28 PB samples (14.2%) obtained during complete remission (CR) were positive for TH mRNA (P < 0.01), whereas 17 of 17 BM samples (100%) and 14 of 17 PB samples (82.3%) obtained before CR was achieved were positive (not significant). The incidence of TH mRNA positivity was significantly lower in the samples obtained during CR than those obtained before CR was achieved (P < 0.0001 for PB samples, P < 0.01 for BM samples). At the time of PBSC harvesting, the incidence of TH mRNA positivity was lower in PBSC samples (3 of 15, 20%) than in BM samples obtained concurrently (10 of 15, 66.7%; P < 0.03). CONCLUSIONS: These findings show that there is a substantial risk of tumor cell contamination in harvested PBSCs, although its incidence was lower than that in BM samples. We recommend that PBSCs would be better harvested during remission and should be examined for tumor contamination before use as a stem cell source. PMID- 8635148 TI - Repeated immunotherapy using intratumoural injection with recombinant interleukin 2 and tumour-infiltrating lymphocytes inhibits growth of breast cancer and induces apoptosis of tumour cells. AB - This study tested the effect of repeated intratumoural injection with recombinant interleukin-2 (rIL-2) and tumour-infiltrating lymphocytes (TILs) on inhibition of growth of breast cancer and on induction of apoptosis of tumour cells. The tumour cell line LDLX43 was used to induce breast cancer in Wistar rats. Group I (10 rats) was the control. Group II (12 rats) received repeated intratumoural injection with rIL-2 and TILs. rIL-2 at the dose of 5 x 10(5) IU/day was given for 7 days, and 1 x 10(7) TILs were injected on the second day of each rIL-2 therapy, for a treatment session. Overall, two treatment sessions of immunotherapy with rIL-2 and TILs were given in all treated animals. Rapid increased tumour volume was found in the control group. In the treated group the total response rate was 42%, of which 25% tumours showed partial regression and 17% tumours reached complete remission where infiltration of plenty of T lymphocytes was detected, indicating that T cell-mediated antitumour immunity is primarily responsible for tumour rejection. Further investigation showed the repeated immunotherapy using intratumoural injection with rIL-2 and TILs could induce the development of apoptosis of breast cancer cells. PMID- 8635149 TI - Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis and DMBA-DNA adduct formation by curcumin. AB - Curcumin, a constituent of the traditional Indian spice and medicine turmeric, was evaluated for its capacity to inhibit the mammary tumor initiating activity of 7,12-dimethylbenz[a]anthracene (DMBA) and the in vivo formation of mammary DMBA-DNA adducts in the female rat. Administration (i.p.) of curcumin at 100 mg/kg and 200 mg/kg doses was associated with a significant decrease in the number of palpable mammary tumors and mammary adenocarcinomas. The in vivo formation of mammary DMBA-DNA adducts also was depressed for animals administered curcumin doses from 50 mg/kg to 200 mg/kg. There was, however, no significant enhancement of liver glutathione-S-transferase activity following curcumin administration. Therefore, curcumin when administered i.p. can act as an effective chemopreventative agent towards DMBA-induced rat mammary tumorigenesis and mammary adduct formation. PMID- 8635150 TI - Identification and characterization of zinc finger encoding genes from the tumoral exocrine pancreatic cell line AR42J. AB - Zinc finger transcription factors are DNA-binding proteins that are known to determine the identity of cells by regulating cell-specific gene expression. In addition, mutations in some members of this family have been found to be associated with several neoplasias, including Wilms' tumor and leukemias. Because the mechanisms that regulate normal, as well as neoplastic, pancreatic cell differentiation are poorly understood, we are searching for pancreas-enriched transcription factors that may determine the identity of pancreatic cells. Towards this end, we have used the polymerase chain reaction and degenerate primers against the highly conserved (Cys2-His2) zinc finger domain to amplify novel transcription factor encoding cDNAs from the well-characterized pancreatic acinar cell line AR42J. Using this approach, we have identified 17 different zinc finger encoding cDNAs (AZF-1 to -17). Sequence analysis shows that all of these clones encode for different zinc finger peptides which share the consensus DNA binding motif with the Drosophila transcription factor kruppel. As a first step in the characterization of these genes, the purified PCR products were used to determine their spatial pattern of expression by northern blot analysis. Using these techniques, we find that numerous zinc finger encoding genes are expressed in AR42J acinar cells as well as in normal adult rat pancreas and suggest that they may play a role as transcription factors in exocrine pancreatic cells. PMID- 8635151 TI - Dietary phytohaemagglutinin slows down the proliferation of a mouse plasmacytoma (MPC-11) tumour in Balb/c mice. AB - Ten days after subcutaneous injection of MPC-11 cells, plasmacytoma tumours which developed in female Balb/c mice fed on a diet containing the kidney bean lectin phytohaemagglutinin (PHA) at a concentration of 7.0 mg g-1 diet, weighed only about 38% of those fed a lactalbumin (La) control diet. The reduction in growth caused by the lectin appeared to occur in a dose-dependent manner but the values did not reach significance before PHA was at a concentration of 7.0 mg g-1 diet. Pre-feeding with the lectin caused a further 50% reduction in tumour weight. In contrast to the reduction in tumour size the inclusion of PHA in the diet elevated the mean dry weight of the small intestine in a dose-dependent manner, values reaching significance at 3.5 mg g-1 diet. The results showed that gut hyperplasia was able to occur even in the presence of the developing tumour. A lypolytic effect of PHA occurred at high concentration. The observations suggest that PHA itself does not have a direct effect on the tumour cells, but an inter relationship between gut hyperplasia and decreased tumour growth is indicated. PMID- 8635152 TI - Lack of inhibitory effects of beta-carotene, vitamin C, vitamin E and selenium on development of ductular adenocarcinomas in exocrine pancreas of hamsters. AB - The effects of vitamins E and E, beta-carotene and selenium on development of N nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic tumours in hamsters were investigated. Dietary supplementation of vitamin C, alone as well as in combination with beta-carotene resulted in consistently lower numbers of advanced ductular lesions. The differences with the controls, however, did not reach the level of statistical significance. Beta-Carotene alone demonstrated no inhibitory effect on the development of (pre)neoplastic lesions in the pancreas. Vitamin E or Se, either alone or in combination, had no effect on the development of advanced ductular lesions in BOP-treated hamsters. PMID- 8635153 TI - Low frequency of p53 gene mutation and protein expression in mucinous colorectal carcinomas. AB - Immunohistochemical data indicate that the frequency of p53 protein overexpression is consistently lower in the mucinous than in the non-mucinous carcinomas of the breast, ovary, pancreas and colon. This peculiar immunohistochemical behavior of the mucinous phenotype could be due to the effect of large amounts of mucus on the staining or to an actual mutation frequency difference between mucinous and non-mucinous carcinomas. This question was investigated on a group of mucinous colorectal carcinomas. DNA was extracted from paraffin sections of 16 human mucinous colorectal carcinomas and the mutation frequency was determined by sequencing of p53 exons amplified in PCR. The expression of p53 protein was determined with the avidin-biotin complex peroxidase staining procedure and CM-1 antiserum. Twenty-five percent of the tumors, exhibited p53 protein overexpression and in 31% a mutation was detected. Concordance between the two techniques was found in 69% of tumors. Overexpression without mutation was observed in 12% and mutation without overexpression in 19%. G:C --> A:T transitions represented the most frequent lesion (80%), as previously observed in non-mucinous colorectal carcinomas. These data indicate that the mutation pattern in the p53 gene is similar in mucinous and non-mucinous colorectal carcinomas. The low frequency of p53 overexpression in the mucinous phenotype is not due to a mucus effect on the staining but is related to the low mutation frequency of p53 gene. These results lead to the hypothesis that in contrast to the nonmucinous tumors the development of the majority of colonic carcinomas with the mucinous phenotype may be independent from p53 mutations. PMID- 8635154 TI - L-myc, GST M1 genetic polymorphism and hepatocellular carcinoma risk among chronic hepatitis B carriers. AB - In order to assess associations between the genetic polymorphism of L-myc and glutathione S-transferase M1 (GST M1) and the risk of hepatocellular carcinoma (HCC), a total of 46 surgically treated HCC patients who were seropositive in hepatitis B surface antigen (HBsAg) and 88 HBs-Ag positive controls were recruited for this study. L-myc and GST M1 genetic polymorphism was examined using a polymerase chain reaction-based restriction fragment length polymorphism assay on DNA extracted from liver and peripheral blood samples. There was no significant difference in GST M1 genotypes between HCC patients and matched controls. A gene dosage trend of association with HCC risk was observed for L-myc genotype. The dose-response relationship remained statistically significant in the multiple logistic regression analysis. PMID- 8635156 TI - Ascorbic acid (vitamin C) improves the antineoplastic activity of doxorubicin, cisplatin, and paclitaxel in human breast carcinoma cells in vitro. AB - Utilizing a microplate ATP bioluminescence assay, two human breast carcinoma cell lines, MCF-7 and MDA-MB-231, were tested against doxorubicin (DOX), cisplatin (DDP), and paclitaxel (Tx) alone and in combination with ascorbic acid (Vit C). In both cell lines, Vit C exhibited cytotoxic activity at high concentrations (i.e. 10(2)-10(3) microM). Both cell lines also were resistant to DOX. MCF-7 was found to be DDP-resistant, MDA-MB-231 was moderately sensitive to DDP. Both cell lines were strongly sensitive to Tx. Vit C both at non-cytotoxic (1 microM) and moderately cytotoxic concentrations (10(2) microM) improved the cytotoxicity of DOX, DDP, and Tx significantly. Combination effects between Vit C and DDP or Tx were partly synergistic and partly additive or subadditive whereas a consistent synergism was found between Vit C and DOX. The mechanisms by which Vit C potentiates the cytostatics studied are yet unclear and should be evaluated further. PMID- 8635155 TI - Primary Ewing's sarcoma of the maxilla, a rare and curable localization: report of two new cases, successfully treated by radiotherapy and systemic chemotherapy. AB - Primary maxillary localization of Ewing's sarcoma is unusual. Involvement of facial bones is characterized by clinical and radiological features distinct from those commonly observed in other sites. Because of the above peculiarities a delay in diagnosis and thus in starting treatment is very probable in such cases. We report here two new cases of Ewing's sarcoma localized to facial bones, successfully treated by local high dosage radiotherapy and systemic chemotherapy. Our experience suggests that, especially for particular sites not suitable to radical surgery, radiation therapy can represent an effective tool to achieve local control of the tumor. PMID- 8635157 TI - Malignant tumours of the gastrointestinal tract in an area with an asbestos cement plant. AB - Data on persons who died of cancer of the gastrointestinal tract in a Croatian coastal area with an asbestos-cement plant were analysed for the period 1970 1990. By poll method applied to the families of deceased subjects, additional data on occupation, lifestyle, educational level, length of resistance and cancer mortality among relatives were collected. The investigation showed that in the study area, but also in certain narrower locations within it (subarea settlements), some of the tumours studied occurred at higher rates than expected. Although not conclusive, these findings may indicate a role of environmental exposure to asbestos, particularly in the occurrence of peritoneal mesothelioma. PMID- 8635158 TI - Modulation of matrix metalloprotease-2 and invasion in human glioma cells by alpha 3 beta 1 integrin. AB - We have investigated the effect of integrin antibodies to a well-characterized alpha 5 beta 1 (fibronectin receptor) and to a multi-specific alpha 3 beta 1 (laminin, collagen, and fibronectin receptor), on the expression of matrix metalloproteases and the invasion ability of two human glioblastoma cell lines, SNB19 and U251. Cell adhesion assays indicated that both cell lines adhere to fibronectin, type IV collagen and laminin. Adhesion of cells to fibronectin was inhibited by a RGD peptide. Cells treated with anti-alpha 3 beta 1 or anti-alpha 5 beta 1 antibodies expressed increased levels of MMP-2. An in vitro matrigel assay also showed that the alpha 3 beta 1 antibody-treated cells had greater invasive ability than the controls. Immunofluorescence data showed that glioma cells treated with either anti-alpha 3 beta 1 or anti-alpha 5 beta 1 antibodies expressed diminished alpha 3 beta-1 and alpha 5 beta 1 integrins relative to the controls. The data show that treatment of cells with alpha 3 beta 1 antibody diminishes the integrin expression on the cell surface and increases the MMP-2 activity and invasiveness. PMID- 8635159 TI - Induction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activation in rat lung microsomes by chronic ethanol consumption and repeated running exercise. AB - The effects of both chronic ethanol consumption and repeated exercise on metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl) 1-butanone (NNK) by rat lung microsomes were investigated. Keto aldehyde production was significantly enhanced 52% and 98%, respectively, by the ethanol and exercise, as was keto alcohol production (72% and 76%). 4-(Methylnitrosamino) 1-(3-pyridyl)-1-butanol (NNAL) production was significantly enhanced only by the exercise (+32%). The combined treatment enhanced the keto alcohol production by 39%, indicating non-additive effects on cytochrome P450 (CYP) isozymes by the different treatments. Immunoblot and metabolic studies revealed an increased activity and content of CYP1A2 and CYP2B induced by the ethanol and exercise treatments as well as an induction of CYP2E1 proteins by the ethanol and combined treatments. These results indicate that both ethanol consumption and running exercise enhance NNK activation by increasing the expression of the CYP enzymes responsible for NNK activation. PMID- 8635160 TI - Are retroviruses involved in the aetiology of human breast cancer? AB - To further investigate the possibility for retroviral involvement in the etiology of human breast cancer we processed peripheral blood monocytes and malignant breast tissue biopsies from 10 patients with breast cancer (infiltrating ductal carcinoma or infiltrating lobular carcinoma; ages 40-80 years) and 20 normal healthy women (with no evidence or family history of breast cancer. 10 age matched controls and 10 women age 22-27 years) for the assay of the retroviral enzyme, reverse transcriptase, using an ELISA and for election microscopy examination for the detection of retroviral-like particles. Reverse transcriptase activity was detected in 5 out of 10 samples of monocyte culture medium and in 1 out of 10 of malignant tissue biopsies from the patients with breast cancer. In contrast, reverse transcriptase was not detected in the culture medium of the monocytes from any of the control subjects. Electron microscopy did not reveal the presence of any retroviral-like particles in any sample of monocyte culture medium or in any of the malignant or normal breast tissue biopsies. Despite evidence for the presence of reverse transcriptase in a subsample of the monocyte culture medium and breast tissue biopsies from the cohort of breast cancer patients who participated in this study, the role of retroviruses in human breast cancer remains unclear. PMID- 8635161 TI - Infrequent somatic alteration of p16/MTS1 in human primary superficial bladder cancers. AB - Although superficial bladder cancer, usually presenting as low grade transitional cell carcinomas, are easily resected by transurethral intervention, their frequent recurrence and progression of satage or grade of the recurrent tumors in some cases is a major problem in urology. Deletion of chromosome 9, bands 9p21-22 in bladder cancers including the lowest grade and stage, suggest potential location of candidate tumor suppressor genes. Recently, p16/MTS1 was isolated from 9p21-22 as a multiple tumor suppressor gene, which regulates the cyclin dependent kinase 4 in the G1/S phase of the cell cycle. In the present study, somatic alterations of p16/MTS1 were examined concentrating on histologically defined superficial bladder carcinomas by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) technique using paraffin embedded materials. Infrequent alterations of p16/MTS1 in superficial bladder cancers, one deletion and one silent mutation in 15 cases, were detected. The results suggest that p16/MTS1 mutation is not involved in the development of superficial urinary bladder carcinomas. PMID- 8635162 TI - Evaluation of some antioxidant enzymes in lung carcinoma tissue. AB - This investigation was effected to determine the levels of the two antioxidant enzymes, superoxide dismutase (SOD) (EC 1.15.1.1) and catalase (CAT) (EC 1.11.1.6) in lung cancerous tissues and to compare with normal lung tissue in order to evaluate the antioxidant status in lung cancer. Fifteen lung carcinoma tissue samples and the normal counterparts from the same cases were homogenized and the cytosols obtained by ultracentrifugation (100,000 x g). SOD was assayed using a modification of the indirect nitroblue tetrazolium assay method, while CAT was measured by a spectrophotometric method. The data obtained are as follows: 1.42 +/- 0.24 U/mg protein (means +/- SEM) of SOD in lung cancer and 3.13 +/- 0.51 U/mg protein in normal lung tissue and 33.53 +/- 6.09 U/mg protein of CAT in lung cancer and 71.33 +/- 14.38 in normal lung tissue. The differences were found to be significant at the level of P < 0.01 for both enzymes. These low levels of the antioxidant enzymes in lung cancerous tissues can lead to elevated levels of reactive oxygen metabolites, resulting in damage to the key subcellular structures such as DNA, cell membranes, and other vital cellular components. PMID- 8635163 TI - Cancer prevention and early detection among Egyptians. AB - Although cancer is frequently not perceived as a significant health problem in developing countries, cancer death in developing countries is higher than in developed countries. Therefore, cancer prevention and early detection in developing countries is becoming a universal challenge to health care providers. The purpose of this exploratory study was to identify knowledge, attitudes, and behaviors related to cancer prevention and early detection among Egyptians. Data were collected from 99 people, and the design was descriptive and correlational, using a self-administered survey. Results showed that the Egyptians surveyed had some knowledge about warning signs of cancer as well as prevention and early detection strategies. In addition, results indicated a significant difference between males and females in warning signs of cancer as well as prevention and early detection methods. Participants were less likely to believe their lifestyle behaviors affect their chances of developing cancer. Current practices undertaken included dietary behaviors, abstinence from smoking, check-up for minor suspicious symptoms, and annual complete physical examinations. Barriers to undertaking practices included factors related to lack of preventive resources, lack of information about preventive strategies, family financial concerns, and specific attitudes toward cancer. The study findings indicate the need for providing people with information to help them make decisions regarding undertaking cancer prevention and early detection practices. PMID- 8635164 TI - Uncertainty, symptom distress, and information needs after surgery for cancer of the colon. AB - The purpose of this study was to determine the relationships between uncertainty, symptom distress, and discharge information needs in individuals after a colon resection for cancer. The theoretical framework for the study was derived from Lazarus and Folkman's stress, appraisal, and coping model, and Mishel's theory of uncertainty in illness. Uncertainty was measured by the Mishel Uncertainty Illness Scale (MUIS); symptom distress of pain, fatigue, constipation, diarrhea and loss of appetite by visual analogue scales; and discharge information needs by the Patient Learning Need Scale (PLNS). Forty individuals with a first diagnosis of cancer were interviewed after surgical resection of colon cancer. The study results indicated that they had moderate levels of uncertainty, low levels of symptom distress, and a moderate number of discharge information needs. Information related to treatment, complications, and activities of living were identified as highly important. An increase in uncertainty was significantly associated with an increase in discharge information needs. Increased attention to information needs at discharge may decrease an individual's level of uncertainty and facilitate the transition from hospital to home. PMID- 8635165 TI - Needs of caregivers of clinic and hospice cancer patients. AB - This prospective correlational study compared the self-identified needs of 55 caregivers of clinic (n = 25) and hospice (n = 30) cancer patients. Patients identified their family caregivers who filled out the Home Caregiver Need Survey (HCNS) and a demographic data form. The HCNS measures the importance and satisfaction of needs on two seven-point Likert-type scales. Descriptive statistics and t tests were used to analyze the data. Caregivers of both clinic and hospice cancer patients ranked the needs items in the Information and Spiritual categories as most important. Variation in types of information occurred between the groups. Clinic caregivers were less satisfied with how well their needs in the Information category were met. Differences between the groups in the importance scores were significant at the 0.001 level for the needs in the Patient Care and Personal Care categories. Differences in the satisfaction scores between the groups were significant at the 0.001 level for needs in the Household and Patient Care categories. The HCNS is helpful for assessing and screening caregiver needs and identifying differences in needs between groups. Health care providers in clinics and hospices must individualize teaching to meet the specific needs of caregivers, particularly those needs related to information. PMID- 8635166 TI - Multinational approaches to cervical cancer screening: a review. AB - Worldwide, cervical cancer incidence is 75% higher than the projected ideal set by the World Health Organization. Despite an effective screen for in situ stage cancer, many women do not adhere to regular screening schedules. These delays raise mortality rates, since curative therapies are available only for the early stages of disease. Seeking to improve cancer control among women, investigators have developed a variety of methods to improve adherence rates. This article presents these methods as well as the cost-effectiveness of each when evaluative data are available. The article suggests that nurses may draw upon current knowledge to design and test the effectiveness of cancer control models in the catchment areas served by their institutions and agencies. PMID- 8635167 TI - Hematopoietic growth factors. PMID- 8635169 TI - Pain assessment: a pilot study in an Israeli bone marrow transplant unit. AB - The incidence and quality of pain experienced by nine patients during the first phase of the bone marrow transplant (BMT) was assessed in an Israeli BMT Unit. Reports of 215 separate episodes of pain were collected, which included the quality, intensity, and different locations of pain reported by the patient and medical staff. It was found that patients experienced on an average five anatomically identifiable pains daily for a period of > 11 days. In addition, although nurses recorded these episodes of pain more frequently than physicians did (47 versus 33%), there was still a discrepancy between the nurses' and the patients' reporting. This pilot study highlights the need for a larger study to be conducted in the area. PMID- 8635168 TI - Development and validation of a patient needs assessment tool (PNAT) for oncology clinicians. AB - The Patient Needs Assessment Tool (PNAT) is an interviewer-rated scale that may be completed through a simple structured interview and screens cancer patients for potential problems in physical and psychosocial functioning. The instrument provides separate scores for physical, psychological, and social status, and can potentially clarify the types of interventions needed to address specific areas of dysfunction. Reliability and validity was tested in two studies that used prescreened patient videotapes and other materials to assess the performance of the PNAT in groups of oncology nurses, physicians, and social workers. The data demonstrate that subscale scores for the physical, psychological, and social dimensions have good inter-rater reliability and internal consistency (intraclass correlation coefficients of 0.71-0.97). Criterion and construct validity was suggested through high correlations of each subscale with the evaluation of expert raters (correlation coefficients of 0.85-0.95) and with scores on validated patient-rated instruments appropriate to the functional area. These analyses suggest that the PNAT is a valid scale for the assessment of a range of functional disturbances in the cancer population. PMID- 8635170 TI - Cancer detection: how effective is public education? AB - The American Cancer Society has been educating the public about cancer detection methods since 1922. Originally, only two warning signs were published; however, for more than 40 years, there have been seven cancer warning signs. In an attempt to evaluate the public knowledge of cancer detection and prevention, this pilot study examined the attitudes, knowledge and behaviors of 172 laypersons. The instrument used consisted of four sections and was designed by the investigator and the graduate nursing research class. The first section contained 30 questions about the individual, health practices, and risk status in a forced-choice format. Ability to identify the seven cancer warning signals was the second section. Attitudes toward Cancer Detection methods were evaluated in a semantic differential format as the third section. The list section contained 24 Likert formatted statements of beliefs about the importance of cancer detection. Before data analysis, a Cronbach's alpha was obtained on each scale and ranged from 0.8031 to 0.8897. Eighty nine (52%) of the respondents were women and 83 (48%) were men. The sample was 85% white, 11% African-American, and 4% other ethnic groups. Ninety-four percent of the population had some form of health insurance. Gender was not significantly related to scores on the Attitudes toward Cancer Detection or the Beliefs about Cancer Scale. Race was significantly related to scores on the Attitudes toward Cancer Detection Scale. Nineteen percent of the sample could not identify any of the cancer warning signs. The median number of warning signs correctly identified as warning signals was three. Thirty-two items were incorrectly listed as warning signs. Survival of cancer is linked with early detection. The inability to influence changes in knowledge and practices over the past 50 years is examined. Implications for nurses and teaching related to cancer warning signs are explored. PMID- 8635172 TI - Management of cerebral vasospasm in the 1990s. PMID- 8635171 TI - Neuroendocrine regulation of sodium and volume following subarachnoid hemorrhage. PMID- 8635173 TI - The use of adjunctive therapy to alter the pathophysiology of bacterial meningitis. AB - The inflammation in the subarachnoid space (SAS) that develops in the course of bacterial meningitis may have a role in eradicating the infection, but also, ultimately, is the cause of the neurological sequelae associated with this infection. The presence of an inflammatory exudate in the SAS leads to alterations in the blood-brain barrier, altered cerebrospinal fluid dynamics, cerebral edema and increased intracranial pressure, and loss of cerebral auto regulation. Our increasing understanding of the pathophysiology of meningeal inflammation has led to therapeutic interventions to limit the degree of meningeal inflammation and neurologic sequelae. The inflammatory cascade that leads to alterations in central nervous system physiology will be reviewed as well as the experimental evidence and clinical trials demonstrating the efficacy of adjunctive therapy in reducing meningeal inflammation and decreasing the incidence and severity of neurological sequelae in bacterial meningitis. PMID- 8635174 TI - The stress protein response and its potential relationship to prolonged seizure activity. AB - Recent investigations have shown that neuronal excitation can lead to a variety of changes in intracellular signaling that ultimately result in altered gene expression. In particular, the excessive neuronal activation seen with seizures leads to the induction of certain "immediate-early" genes that are transcription factors. These transcription factors presumably facilitate the subsequent expression of various "intermediate-late" or effector genes. Given the growing numbers of genes that appear to be modulated by seizures, it appears that seizures initiate a tremendous cascade of changes in gene expression over both the short and the long term. In this article, I discuss two main classes of gene products that are modulated by seizure activity: heat shock proteins and calcium binding proteins. By using both in vivo an in vitro systems, we and others are exploring the conditions that lead to altered expression of genes and the potential significance of such changes in expression. Although the functional meaning of the altered gene expression remains unknown, it seems likely that some of these changes will ultimately be related to certain components of neuronal vulnerability and epileptogenesis. PMID- 8635175 TI - Core temperature control in the management of CNS lesions. AB - Renewed interest in the regulation of core temperature as a means of treating central nervous system disease has been fueled by a wealth of literature describing the physiology of lowered core temperature. Hypothermia is now recognized for its anesthetic and cerebroprotective effects and is being applied in an increasing number of clinical paradigms. PMID- 8635176 TI - Folate deficiency, anticonvulsant drugs, and psychiatric morbidity. AB - The folic acid (FOA) level was determined in serum and erythrocytes in 100 epileptic patients and 100 control patients using a luminescence assay. A lowered FOA concentration in serum, erythrocytes, or both was observed in 15% of the epileptic patients and in 2% of the control group. In the epileptic patients, the FOA in the serum and in the erythrocytes was significantly lower than that in the control group. Patients receiving carbamazepine monotherapy had a significantly lower FOA level in the erythrocytes than did patients receiving phenytoin monotherapy. The FOA level showed a negative correlation to the duration of epilepsy. None of the patients with lowered FOA had a normal mental status. The course of the supplementation treatment with 5 mg folinic acid (or FOA) of four patients with FOA deficiency could be monitored psychopathometrically. All four patients showed an improvement in their well-being and the majority of measured variables of the cognitive performance. PMID- 8635178 TI - Therapy of fungal meningitis. AB - There has been an increase in recent years in the number of reported cases of meningitis and brain abscesses caused by fungi. This increase is due to the availability of better diagnostic techniques for fungal infections and the ever increasing population of immunocompromised hosts (1,2). The patients most susceptible to invasive fungal infections include those with hematologic malignancies; those receiving hyperalimentation, corticosteroids, or cytotoxic drugs; transplant recipients; injection drug abusers; and those with the acquired immunodeficiency syndrome (AIDS). Although many fungi infect only immunologically impaired patients, some will infect normal hosts as well. The successful treatment of central nervous system (CNS) fungal infections is highly dependent on the underlying immune status of the host, as well as on the prompt initiation of appropriate antifungal therapy. However, the diagnosis of these infections may be difficult, and proper therapy often delayed. Furthermore, information on treatment regimens ranges from extensive, as in the case of cryptococcal meningitis, to scanty or nonexistent in the case of rare, opportunistic fungi. For > 3 decades, the standard antifungal agent for the treatment of CNS fungal infections has been amphotericin B. However, the effectiveness of amphotericin B is often eliminated by poor CNS penetration, fungal resistance, and toxicity (3). Because of the problems associated with use of amphotericin B, newer azole antifungal agents have been developed, some of which are efficacious in the therapy of fungal meningitis. We give an overview of the antifungal agents currently available for clinical use and their utility in the treatment of fungal meningitis. PMID- 8635177 TI - Diethylpropion pharmacotherapeutic adjuvant therapy for inpatient treatment of cocaine dependence: a test of the cocaine-agonist hypothesis. AB - Fifty cocaine-dependent patients completed a 2-week double-blind, double-dummy, parallel-group comparison of four dosage levels of diethylpropion and placebo. This clinical trial was designed to evaluate both diethylpropion's ability to attenuate cocaine cue-induced craving and its potential for development as a medication with cocaine-agonist properties. The results indicated that diethylpropion was not superior to placebo and confirmed earlier reports that craving for cocaine diminishes over the course of an inpatient hospitalization. Moreover, the results showed that the cocaine cue-induced craving paradigm employed is effective in stimulating craving for cocaine. Medications that are effective in attenuating this type of "conditioned" craving may have relevance to the breaking of the cycle of relapse and long-term treatment of cocaine dependence. Diethylpropion may not be an appropriate candidate for future medication development because of its lack of obvious therapeutic efficacy and the emergence of a significant number of side effects. However, a cocaine-agonist medication strategy may be appropriate for a subgroup of cocaine-dependent patients with coexisting attention deficit-hyperactivity disorder. PMID- 8635179 TI - Tardive syndromes and other drug-induced movement disorders. PMID- 8635180 TI - The effects of flumazenil in neuropsychiatric disorders. AB - Flumazenil is a benzodiazepine receptor antagonist. It is currently used mainly in the anaesthetic and emergency rooms to reverse the effect of exogenous benzodiazepines. Its use in a variety of experimental animal models and in human neuropsychiatric disorders continues to generate a wealth of information on the possible role of the benzodiazepine-GABA(A) receptor complex in their pathogenesis. In addition, labelled with carbon-11, flumazenil has proved to be one of the most successful positron emission tomography ligands stimulating research on the role of the benzodiazepine receptor in these disorders. This review focuses on the current state of play of flumazenil as a therapeutic or investigative agent in neuropsychiatry, citing also the relevant animal models. PMID- 8635182 TI - The phencyclidine-glutamate model of schizophrenia. AB - For the past 20 years, it has been widely assumed that schizophrenia results from chronic dopamine (DA) hyperactivity. However large amounts of evidence exist that call into question this assumption. After examining the brains of schizophrenic patients, studies failed to find evidence of elevated levels of DA, alterations in DA-producing or degrading enzymes or both, or increased DA-receptor concentrations or affinity; thus, there are no direct observations linking psychosis to increases in DA activity. Therefore, it seems that mechanisms unrelated to altered dopaminergic functioning may be involved in the underlying pathology of schizophrenia. The anesthetic drug phencyclidine (PCP) is capable of inducing psychosis-like states through nondopaminergic mechanisms. PCP acts as a glutamate antagonist; glutamatergic abnormalities have been detected in the brains of schizophrenics. This evidence suggest that glutamate hypofunction may be involved in the pathology of psychosis. Additionally, a functional link exists between glutamate and DA neural systems. Based on these facts, as well as an extensive review of the literature, it is concluded that dysfunctional glutamatergic pathways are involved in psychotic pathology. PMID- 8635183 TI - L-deprenyl as an adjunct to low-dose bromocriptine in early Parkinson's disease: a short-term, double-blind, and prospective follow-up study. AB - The therapeutic efficacy of L-deprenyl (10 mg daily) as an adjunct to low-dose bromocriptine monotherapy (up to 25 mg daily) in patients with early Parkinson's disease (PD) was evaluated in a double-blind placebo-controlled short-term study (11 patients) and subsequently in a long-term prospective open follow-up (21 patients) until L-dopa was required, over a 4-year period. The combined regimen of bromocriptine plus L-deprenyl produced a mildly significant improvement, as shown by the majority of clinical rating scales used after 6 weeks of sustained treatment (as compared to bromocriptine alone and bromocriptine plus placebo). In the prospective long-term study, a stabilization of the clinical status was observed until 12 months of sustained treatment, whereas after that, a gradual worsening of the scores on all motor rating scales occurred. However, at 24 months, fewer than one third of the patients had required L-dopa, a proportion comparatively smaller than that reported in the literature with bromocriptine alone. This finding could be related to the persistence of initial symptomatic effect of L-deprenyl, but a slowing action on the course of the disease process exerted by the monoamine oxidase typeB (MAO-B) inhibitor cannot be ruled out. PMID- 8635181 TI - The drug-drug interaction effects of haloperidol on plasma carbamazepine levels. AB - The metabolic interaction between carbamazepine (CBZ) and haloperidol (HP) was studied in Japanese schizophrenic patients treated with HP but not with CBZ and with both CBZ and HP. The serum CBZ concentrations in patients treated without HP were significantly decreased (p < 0.05), on average approximately 40%, as compared to those in patients treated with both CBZ and HP, whereas the serum HP concentrations in patients treated with both HP and CBZ were significantly decreased (p < 0.05), as compared to those in patients treated with HP but not with CBZ. The effect of HP, which prevents the serum CBZ level from decreasing, was shown in this study. PMID- 8635184 TI - Acute administration of levodopa-benserazide and tolcapone, a COMT inhibitor, Parkinson's disease. AB - Tolcapone, a catechol-O-methyltransferase inhibitor, can interfere with the metabolism of levodopa and dopamine and could prolong the motor effect induced by levodopa in parkinsonian patients. To test this hypothesis, we studied the motor effect induced by three acute administrations of a dose of levodopa-benserazide (Madopar) with either 200 mg or 400 mg of tolcapone or placebo, in a double-blind latin-square design. The duration of the on-phase could be compared in 10 parkinsonian patients suffering from square-shaped motor effect. In comparison to placebo, 200 mg and 400 mg of tolcapone significantly increased the mean duration of the on-phase by 61.7 min ( +/- 19.4 SEM) and by 72.2 min ( +/- 18.5), respectively. This clinical effect is suggested to be related mainly to the increase in levodopa area under the curve and half-life induced by tolcapone. The intensity in dyskinesias was increased by 400 mg of tolcapone. Tolcapone appears to be well tolerated and could be helpful as an adjuvant treatment to levodopa in parkinsonian patients with motor fluctuations. PMID- 8635186 TI - Trauma, axonal injury, and amyotrophic lateral sclerosis: a clinical correlate of a neuropharmacologic model. AB - Axonal injury induces cell death in selectively vulnerable motor neurons of immature animals. This extensively studied animal model of trauma-induced motor neuron death is being used to develop the theoretical basis for the therapeutic use of neurotrophic factors to "rescue" dying neurons in amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. Antecedent trauma has been implicated as a precipitating factor for amyotrophic lateral sclerosis in susceptible individuals. The animal model of trauma-induced motor neuron death provides support for the concept that motor neurons in individuals susceptible to amyotrophic lateral sclerosis may be selectively vulnerable to trauma producing axonal injury. The case histories of six young adult men who developed amyotrophic lateral sclerosis after trauma with axonal injury are presented. PMID- 8635185 TI - Tremorlytic activity of budipine: a quantitative study with long-term tremor recordings. AB - The tremorlytic activity of the novel antiparkinson agent budipine was quantified in an open trial. Eleven patients with Parkinson's disease (PD) were treated with individual doses of budipine added to stable conventional antiparkinsonian medication. Tremor activity was measured using long-term electromyogram (EMG) recordings. Tremor intensity was reduced by 25%, tremor occurrence by 34%, and conventional "Unified Parkinson's Disease Rating Scale" (UPDRS) scores improved by 20% with this medication. There were two dropouts because of side effects. One dropout appeared not to be related to budipine. Apart from those, the drug was well tolerated by all patients. We conclude that budipine is an effective and well-tolerated tremorlytic drug and that the method of long-term EMG recording is suitable for tremor quantification in clinical studies. PMID- 8635187 TI - Retroperitoneal fibrosis in a patient with Parkinson's disease treated with pergolide. AB - We describe a 68-year-old patient with Parkinson's disease who developed retroperitoneal fibrosis during pergolide treatment. Because pergolide is an ergot derivative, it could be related to the development of this complication. PMID- 8635188 TI - Vigabatrin and multifocal myoclonus in adults with partial seizures. AB - We report the appearance of multifocal myoclonus in two adult patients treated with vigabatrin as an add-on drug for complex partial seizures. The myoclonus subsided after dose reduction or discontinuation of the drug. There were no electroencephalogram correlates during the myoclonic jerks. This phenomenon may represent a apparently dose-related rare adverse drug event, similar to that seen occasionally with other anticonvulsants. PMID- 8635189 TI - Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells. AB - To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26 8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotype-matched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host. PMID- 8635190 TI - Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients. AB - A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti ganglioside GD2 antibody mAb ch14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch14.18 at dose levels from 10 mg/m2 to 200 mg/m2. The plasma clearance of ch14.18 was biphasic. Following the first course of treatment t1/2,alpha was 3.4 +/- 3.1 h and t1/2,beta 66.6 +/- 27.4 h in 9/10 children. The t1/2,beta values were significantly less than those of 181 +/- 73 h previously reported in adult melanoma patients (P < or = 0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch14.18 in children and adults. After a second course of treatment, administered to 5/10 children, t1/2,beta decreased significantly from 72.9 +/- 19.8 h to 31.7 +/- 18.4 h (P = 0.015). We therefore conclude that the elimination kinetics of mAbs ch14.18 in children and adults are different, and furthermore that repeated administration of mAb ch14.18 to children with neuroblastoma leads to accelerated antibody clearance. PMID- 8635191 TI - Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2. AB - In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor gamma II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+ T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon gamma, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL. PMID- 8635192 TI - Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid. AB - A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23 alpha. These components had RF values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbent assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway. PMID- 8635194 TI - Low-dose-melphalan-induced up-regulation of type-1 cytokine expression in the s.c. tumor nodule of MOPC-315 tumor bearers and the role of interferon gamma in the therapeutic outcome. AB - We have previously shown the importance of endogenous tumor necrosis factor (TNF) production for the curative effectiveness of low-dose melphalan (L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor. In the current study we demonstrate that low-dose melphalan is actually associated with enhanced expression of mRNA for TNF alpha in the s.c. tumor nodule. Moreover, the expression of mRNA for interferon gamma (IFN gamma) and interleukin-12 (IL-12; p40) is also elevated at the tumor site. However, while elevation in the expression of mRNA for TNF alpha and IFN gamma is evident within 24 h after the chemotherapy, elevation in the expression of mRNA for IL-12(p40) is first evident 72 h after the chemotherapy. Moreover, neutralizing anti-IFN gamma mAb, like neutralizing anti-TNF mAb but not neutralizing anti-IL-12 mAb, reduced the curative effectiveness of low-dose melphalan for MOPC-315 tumor bearers. Studies into the mechanism through which IFN gamma mediates its antitumor effect in low dose-melphalan-treated MOPC-315 tumor-bearing mice revealed that MOPC-315 tumor cells, which are not sensitive to the direct antitumor effects of TNF, display some sensitivity to the antiproliferative activity of high concentrations of IFN gamma. However, unlike TNF alpha, IFN gamma is unable to promote the generation of anti-MOPC-315 cytotoxic T lymphocyte activity and, in fact, exerts an inhibitory activity on CTL generation. Taken together, our studies illustrate that low-dose melphalan therapy of MOPC-315 tumor bearers is associated with the rapid elevation in the expression of mRNA for IFN gamma and TNF, two cytokines which are important for the curative effectiveness of low-dose melphalan, and which mediate their antitumor effect, in part, through distinct mechanisms. PMID- 8635193 TI - Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor beta by treatment with bleomycin. AB - We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-beta (TGF beta) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was significantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGF beta antibody. TGF beta activities were found in KDH-8-tumor-tissue conditioned medium without acid treatment and were found in tumor-cell conditioned medium after acid treatment; TGF beta mRNA and TGF beta protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor derived TGF beta might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor derived TGF beta and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significant reduction of TGF beta activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGF beta activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGF beta. PMID- 8635196 TI - Protection against metastasis by immunization with an allogeneic lymphocyte antigen. AB - Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa 2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi syngeneic NFSA tumor was examined. First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness. PMID- 8635195 TI - Antitumor and antimetastatic effects of dacarbazine combined with cyclophosphamide and interleukin-2 in Lewis lung carcinoma (3LL). AB - The antitumor and antimetastatic activity of dacarbazine (DTIC) alone or in combination with cyclophosphamide (CY) was tested in C57BL/6 mice bearing Lewis lung carcinoma (3LL). Treatment with both agents significantly reduced tumor growth and the number of metastases. These effects were associated with marked changes of the biochemical and immunological properties of drug-treated 3LL cells, i.e. (a) reduction of alpha 6 integrin expression, (b) increased susceptibility to natural immunity in vivo, as measured in terms of rapid clearance from mouse lungs of prelabeled 3LL cells injected i.v. and (c) increased immunogenicity, as assessed by T-cell-mediated immune responses (i.e. graft rejection by intact syngeneic mice, and frequency of specific CTL precursors recognizing DTIC/CY-treated cancer cells). The immunotherapeutic advantage afforded by increased immunosensitivity and immunogenicity of 3LL cells exposed to DTIC + CY appears to be markedly reduced in vivo by the profound immunodepressive effects of these drugs. Within this context, addition of interleukin-2 was found to increase the antitumor and antimetastatic activity of this chemotherapeutic regimen. The present study shows, for the first time in a solid tumor model, that a biological response modifier increases the antitumor efficacy of drugs that are able to affect the immunological properties of cancer cells. PMID- 8635198 TI - Biotechnological developments in New Zealand. AB - Biotechnology has a long history in New Zealand, and there have been some innovations that are unique to the country. This article describes some developments that illustrate this point and gives some examples of current research projects that typify the strengths and interests of the biotechnology community. Examples are taken from the food industry, agriculture, forestry, production of industrial chemicals, and the treatment of hazardous chemicals. PMID- 8635197 TI - Prophylactic intervention in radiation-leukemia-virus-induced murine lymphoma by the biological response modifier polysaccharide K. AB - Polysaccharide K (PSK) is a biological response modifier used for adjuvant immunotherapy of malignant diseases. We studied the potential applicability of PSK for preventing tumor progression using an experimental model of murine lymphoma. Mice inoculated with the radiation leukemia virus (RadLV) develop thymic lymphomas after a latency of 3-6 months. However, 2 weeks after virus inoculation, prelymphoma cells can already be detected in the thymus. We found that PSK treatment induced hyperresponsiveness to concanavalin A and heightened production of interleukin-2 (IL-2) and IL-4 in spleen cells of both control and prelymphoma mice. The response was transient and was accompanied with a dominant usage of T cells expressing V beta 8, but other T cell subsets were also stimulated by PSK. T lymphoma cells expressing V beta 8.2 underwent apoptosis when incubated with PSK. Treatment of RadLV-inoculated mice with PSK delayed the onset of overt lymphoma (and mortality) but could not protect the mice from the disease. Combined treatment with PSK and a RadLV-specific immunotoxin prevented synergistically the progression of the prelymphoma cells to frank lymphoma. The results suggest that PSK contains a superantigen-like component that selectively activates V beta 8+ T cells. Its administration prelymphoma mice interfered with the process of lymphoma progression. PMID- 8635199 TI - Luminescence-based systems for detection of bacteria in the environment. AB - The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase. Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process. In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples. PMID- 8635201 TI - Aggregation of a subpopulation of vimentin filaments in cultured human skin fibroblasts derived from patients with giant axonal neuropathy. AB - Giant axonal neuropathy (GAN) is a generalized disorder of intermediate filament networks which results in the formation of an ovoid aggregate in a large variety of cell types. We investigated the cytoskeletal organization of cultured skin fibroblasts derived from three GAN patients by indirect immunofluorescence, confocal, and electron microscopy. Whereas the organization of microfilaments seemed normal, the microtubule network appeared disorganized and tangled. The organization of the intermediate filament network, composed of vimentin, was probed with three antibodies directed against different epitopes: two vimentin specific antibodies, a monoclonal antibody (mAb V9) and a polyclonal antibody, and a serum specific for all type III IFPs (PI serum). These experiments showed that 20% of cultured skin fibroblasts from GAN patients have a vimentin aggregate composed of densely packed filaments which coexists with a well-organized vimentin network. After depolymerization of microtubules with nocodazole, all fibroblasts from GAN patients contained a vimentin aggregate which seemed to arise from a subpopulation of vimentin filaments normally integrated in the vimentin network. Such aggregates were never observed in any condition in control fibroblasts. Moreover, the ultrastructural analysis of GAN cells revealed the presence of swollen mitochondria. We suggest that GAN may be due to a defect in a factor which stabilizes cytoplasmic intermediate filament networks, and we speculate on its identification and properties. PMID- 8635200 TI - Actin-based motility of isolated axoplasmic organelles. AB - We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity. PMID- 8635202 TI - Mammalian myosin I alpha is concentrated near the plasma membrane in nerve growth cones. AB - To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin I alpha (MMI alpha) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMI alpha has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMI alpha staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMI alpha is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMI alpha label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMI alpha and the plasma membrane. MMI alpha label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMI alpha label. These data suggest a complex relationship between MMI alpha, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMI alpha is also associated with tubulovesicular structures. This suggests that at this location MMI alpha may either act as an organelle motor or is passively transported to the plasma membrane on vesicles. PMID- 8635203 TI - F-actin forms transient perinuclear shells at the mitosis-interphase transition. AB - Intermediate filaments and microtubules are known to be involved in establishing and maintaining nuclear shape. F-actin may also be involved in determining nuclear shape, since we have found it associated with reforming nuclei very briefly after cell division. We stained cells from vertebrate tissue cultures (3T3 and NRK-49F) and epidermal cells from an insect with rhodamine-phalloidin and Hoechst #33342 to localize F-actin in relation to the nucleus. We found that F-actin forms shells only around nuclei during reorganization in late mitosis and early interphase. We suggest that perinuclear F-actin shells may be generally present in eukaryotes, but that they are easily missed because of their delicacy and transience. PMID- 8635204 TI - Cytoarchitecture and cell growth control. AB - Appropriate cell-to-substrate adhesion together with SGF stimulation is necessary to initiate and continue cell cycle progression of growth arrested cells. Adhesion-dependent signaling events, which likely occur through integrin receptors specifically organized with cytoskeletal components within focal contacts, can induce expression of specific genes and stimulate quiescent cells into the growth cycle. The mechanisms as to how: (1) cell-to-substrate adhesion complexes are formed and maintained, (2) adhesion-dependent signal transduction events interface with SGF initiated signalling events, (3) adhesion influences expression of growth-state regulated genes, and (4) an appropriate cytoarchitectural environment may coordinate these events to regulate cellular growth are unclear. While it is apparent that defining these mechanisms would be critical to understanding the basic events which control cell growth, many of the mechanisms are just beginning to be addressed and understood. PMID- 8635205 TI - Ability of paralyzed flagella mutants of Chlamydomonas to move. AB - Chlamydomonas mutants missing the central pair or radial spokes are paralyzed despite the fact that they have the full wild-type complement of functional dynein ATPases. We show here that these mutants can move under conditions of low ATP concentration, a combination of ATP and ADP, and a combination of ATP and ribose-modified ATP analogs. These conditions suggest an inhibitory role of ATP and that this inhibition can be relieved by ADP or analogs. The function of the central-pair/radial spoke complex may be to release this ATP inhibition in a controlled manner. PMID- 8635206 TI - Electron microscopic evidence of impaired intramembrane particles and instability of the cytoskeletal network in band 4.2 deficiency in human red cells. AB - To obtain direct evidence of impaired intramembrane particles (IMPs) and a deranged cytoskeletal network in situ in human red cells of band 4.2 deficiency, electron microscopic studies were performed utilizing the freeze fracture method for IMPs and the quick-freeze deep-etching method for the cytoskeletal network. Three patients with three different previously identified mutations of the band 4.2 gene, i.e., band 4.2 Komatsu (homozygous; codon 175 GAT --> TAT), band 4.2 Nippon (homozygous; codon 142 GCT --> ACT), and band 4.2 Shiga (compound heterozygous; codon 317 CGC --> TGC and codon 142 GCT --> ACT), were selected for this study. The decrease in the number of IMPs with increase in their size was most marked in band 4.2 Komatsu, which was clinically most severe with no band 4.2 protein. In this regard, in band 4.2 Nippon, which showed moderate severity in clinical hematology with a nearly missing band 4.2 protein, increased sizing was less marked. The abnormalities in IMPs were the least in band 4.2 Shiga, which demonstrated compensated hemolysis with band 4.2 protein in a trace amount. The extent of the impairment of IMPs may be reflected by the total absence or the presence of band 4.2 protein even in a trace amount and/or by the specific site(s) of the mutation of the band 4.2 gene. Derangement of the cytoskeletal network was also observed in these three patients. It was most abnormal in band 4.2 Komatsu, and less so in band 4.2 Nippon and in band 4.2 Shiga. These results clearly indicate that 1) band 4.2 plays an important role not only in its binding to band 3 but also to the skeletal network (mostly to spectrins) vertically, and 2) its deficiency produces critical abnormality in maintenance of the structural and functional integrity of the integral proteins (such as band 3), as well as the cytoskeletal network. PMID- 8635207 TI - Transgenic manipulation of myocardial G protein-coupled receptors and receptor kinases. PMID- 8635208 TI - Adverse effects of chronic endogenous sympathetic drive induced by cardiac GS alpha overexpression. AB - To study the physiological effect of the overexpression of myocardial Gsalpha (protein levels increased by approximately threefold in transgenic mice), we examined the responsiveness to sympathomimetic amines by echocardiography (9 MHz) in five transgenic mice and five control mice (both 10.3 +/- 0.2 months old). Myocardial contractility in transgenic mice, as assessed by left ventricular (LV) fractional shortening (LVFS) and LV ejection fraction (LVEF) was not different from that of control mice at baseline (LVFS, 40 +/- 3% versus 36 +/- 2%; LVEF, 78 +/- 3% versus 74 +/- 3%). LVFS and LVEF values in transgenic mice during isoproterenol (ISO, 0.02 micrograms/kg per minute) infusion were higher than the values in control mice (LVFS, 68 +/- 4% versus 48 +/- 3%; LVEF, 96 +/- 1% versus 86 +/- 3%; P < .05). Norepinephrine (NE, 0.2 micrograms/kg per minute) infusion also increased LVFS and LVEF in transgenic mice more than in control mice (LVFS, 59 +/- 4% versus 47 +/- 3%; LVEF, 93 +/- 2% versus 85 +/- 3%; P < .05). Heart rates of transgenic mice were higher than those of control mice during ISO and NE infusion. In three transgenic mice with heart rates held constant, LV dP/dt rose by 33 +/- 2% with ISO (0.02 micrograms/kg per minute) and by only 13 +/- 2% in three wild-type control mice (P < .01). NE (0.1 micrograms/kg per minute) also induced a greater effect on LV dP/dt in the three transgenic mice with heart rates held constant compared with three wild-type control mice (65 +/ 8% versus 28 +/- 4%, P < .05). Pathological and histological analyses of older transgenic mouse hearts (16.0 +/- 0.8 months old) revealed hypertrophy, degeneration, atrophy of cells, and replacement fibrosis reflected by significant increases in collagen volume in the subendocardium (5.2 +/- 1.4% versus 1.2 +/- 0.3%, P < .05) and in the cross-sectional area of myocytes (298 +/- 29 versus 187 +/- 12 micron2, P < .05) compared with control mouse hearts. These results suggest that Gsalpha overexpression enhances the efficacy of the beta-adrenergic receptor-Gs adenylyl cyclase signaling pathway. This in turn leads to augmented inotropic and chronotropic responses to endogenous sympathetic stimulation. This action over the life of the animal results in myocardial damage characterized by cellular degeneration, necrosis, and replacement fibrosis, with the remaining cells undergoing compensatory hypertrophy. As a model, this transgenic mouse offers new insights into the mechanisms of cardiomyopathy and heart failure and provides a new tool for their study. PMID- 8635209 TI - Extracellular ATP inhibits adrenergic agonist-induced hypertrophy of neonatal cardiac myocytes. AB - We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophy-inducing agents, increases expression of the immediate early genes c-fos and junB in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [14C]phenylalanine (14C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of 14C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P2 purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal alpha-actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca2+-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy. PMID- 8635210 TI - Aging does not affect the activation of the myocyte insulin-like growth factor-1 autocrine system after infarction and ventricular failure in Fischer 344 rats. AB - To determine whether the attenuation in the growth capacity of myocytes in the overloaded aging heart is associated with an impairment in the activation of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in the stressed cells, large myocardial infarcts were produced in Fischer 344 rats at 4 and 16 months of age, and the animals were killed 6 hours, 3 days, and 7 days later. After the documentation of cardiac failure, the unaffected myocytes were enzymatically dissociated, and the expression of IGF-1 and IGF-1R was measured at these three time points after surgery. The level of expression of IGF-1R mRNA increased at 3 days and remained elevated at 7 days in both age groups. In addition, an increase in IGF-1R protein in these cells was found, with no apparent difference with age. This phenomenon was coupled with an upregulation of IGF-1 mRNA of comparable magnitude in the younger and older animals. In contrast, the increases in the dimensional properties of myocytes were delayed and of smaller magnitude in the older infarcted rats. Moreover, the expression of atrial natriuretic factor, used as a molecular marker of myocyte cellular hypertrophy, was greater at 3 days in 4-month-old rats and at 7 days in 16-month-old rats. Thus, aging may affect the hypertrophic response of myocytes after infarction but has no impact on the ability of the cells to enhance the expression of IGF-1 and IGF-1R, which may sustain only in part the growth reserve mechanisms of the pathological heart. PMID- 8635211 TI - Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro. AB - Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies. PMID- 8635212 TI - Thrombin receptor actions in neonatal rat ventricular myocytes. AB - Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor-derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12 myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels. PMID- 8635213 TI - Mechanism of hydrogen peroxide and hydroxyl free radical-induced intracellular acidification in cultured rat cardiac myoblasts. AB - After a transient ischemic attack of the cardiac vascular system, reactive oxygen derived free radicals, including the superoxide (O2-.) and hydroxyl (.OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiota acidification (approximately 0.24 pH unit) is induced by the addition of 100 micromol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of .OH, and not O2-. or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHi regulators (the Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co transporter), (2) a rise in intracellular Ca2+ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHi decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia. PMID- 8635214 TI - Transient ischemia reduces norepinephrine release during sustained ischemia. Neural preconditioning in isolated rat heart. AB - Endogenous catecholamine release may play a role in ischemic preconditioning either as a trigger or as a target within the process of myocardial preconditioning. Therefore, we investigated the effect of transient ischemia (TI) on norepinephrine release during sustained ischemia in isolated rat hearts. TI was induced by multiple cycles of global ischemia followed by reperfusion with a duration of 5 minutes each, comparable to ischemic preconditioning protocols. After TI, norepinephrine release was evoked by either sustained global ischemia, anoxia, cyanide intoxication, tyramine, or electrical stimulation. During TI, no washout of norepinephrine was observed, and tissue concentrations of norepinephrine were not changed. TI, however, reduced norepinephrine overflow after 20 minutes of sustained ischemia from 239 +/- 26 pmol/g (control) to 79+/-8 pmol/g (67% reduction, P <.01 ). A similar reduction of ischemia-induced norepinephrine release from 192 +/- 22 pmol/g (control) to 90 +/- 15 pmol/g was observed when hearts underwent transient anoxia without glucose (P < .05). When reperfusion between TI and sustained ischemia was prolonged from 5 to 90 minutes, the inhibitory effect of TI on norepinephrine release was gradually lost. Susceptibility to TI was a unique feature of norepinephrine release induced by sustained ischemia, since release of norepinephrine evoked by anoxia, cyanide intoxication, tyramine, or electrical stimulation remained unaffected by TI. We propose a protective effect of TI on neural tissue, which may reduce norepinephrine-induced damage during prolonged myocardial ischemia. PMID- 8635215 TI - Evidence for the presence of a proteinase-activated receptor distinct from the thrombin receptor in vascular endothelial cells. AB - The thrombin receptor was the first cloned G protein-coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in Xenopus laevis oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F2alpha (PGF2alpha)-contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2-derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF2alpha-contracted porcine coronary arteries. SFLLRN or SLIGRL (30 micronmol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor NG-monomethyl-L-arginine (1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2-mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature. PMID- 8635216 TI - Regulated expression of the ets-1 transcription factor in vascular smooth muscle cells in vivo and in vitro. AB - Ets-1 regulates the transcription of several genes encoding extracellular matrix proteins (ie, osteopontin and tenascin) as well as enzymes involved in degradation and remodeling of the extracellular matrix (ie, stromelysin and urokinase plasminogen activator). In the present study, we investigated the regulation of c-ets-1 in cultured rat vascular smooth muscle cells as well as in the arterial wall after balloon injury in vivo. Serum-starved smooth muscle cells exposed to serum for various time points express a major c-ets-1 mRNA transcript of 5.3 kb and minor bands of 4.0 and 2.5 kb with a peak at 2 hours after stimulation. These effects were concentration dependent. Western blotting revealed an increase in 55- and 40-kD immunoreactive ets-1 proteins in cells treated with serum for 2 hours, and binding to an oligonucleotide containing the ets-1 consensus cis-acting motif was demonstrated by electrophoretic mobility shift assay. Ets-1 mRNA abundance was induced with a peak at 2 hours after stimulation with platelet-derived growth factor-BB and with angiotensin II. There was a distinct increase of ets-1 immunoreactivity in the inner layer of the media 2 hours after balloon catheter injury of rat arteries, which declined after 6 hours and returned to the basal level 1 day after vessel wall damage. Arterial c ets-1 mRNA content was induced with an identical time course. These findings suggest that c-ets-1 may be of importance in the mitogenic signaling pathway of smooth muscle cells grown in culture. In addition, ets-1 may play a role in the activation of smooth muscle cells in vivo after mechanical injury of the vessel wall. Because the ets-1 transcription factor activates the gene expression of a number of mRNA species involved in matrix deposition and degradation, these data are compatible with a role for ets-1 in vascular remodeling and/or cell migration. PMID- 8635217 TI - Regulation of beta1-integrin function in cultured human vascular smooth muscle cells. AB - Avidity modulation and function of beta1-integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the beta1 subunit of integrin heterodimers and induces a high avidity state. The adhesion of SMCs to extracellular matrix proteins, but not to poly-L-lysine, was enhanced by pretreatment with mAb 8A2. A qualitative alteration of beta1 integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the beta1 subunit. Binding of mAb 15/7 was enhanced by mAb 8A2 in a dose-dependent manner. Arg-Gly-Asp peptide and soluble fibronectin also enhanced expression of the 15/7 epitope, suggesting that the 15/7 epitope is closely related to the ligand-occupied state of beta1 integrin. Platelet-derived growth factor (PDGF)-AA and -BB increased SMC adhesion to type I collagen but did not augment mAb 15/7 binding, suggesting that PDGFs increase binding avidity by a postreceptor mechanism. In addition, mAb 8A2 inhibited PDGF BB-induced SMC migration through Matrigel-coated filters. These results suggest that avidity modulation of beta1 integrin may play an important role in the function of SMCs. PMID- 8635218 TI - Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells. Role of the vascular natriuretic peptide system in vascular growth inhibition. AB - Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-beta (TGF-beta) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF beta abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta. Furthermore, the conditioned medium from ECs stimulated by TGF-beta was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs. PMID- 8635219 TI - Specific accumulation of lipoprotein(a) in balloon-injured rabbit aorta in vivo. AB - To explore whether lipoprotein(a), Lp(a), may accumulate preferentially to LDL in the arterial wall at sites of injury, cholesterol-fed rabbits were injected intravenously with radiolabeled Lp(a) and/or LDL 3.1 +/- 0.1 days (mean +/- SEM, n = 30) after a balloon injury of the thoracic aorta. After 5 to 10 minutes' exposure to labeled lipoproteins, more labeled LDL than labeled Lp(a) was recovered in the intima-inner media of the balloon-injured segment (n = 9; paired t test, P < .0001); however, the amount of tightly bound labeled lipoprotein was similar for the two lipoprotein fractions. In the second set of experiments, 131I Lp(a) (or 131I-LDL) was injected 26 hours before and 125I-Lp(a) (or 125I-LDL) 3 hours before the aorta was removed. Permeability and fractional loss of labeled Lp(a) (n = 8) versus LDL (n = 7) in the balloon-injured aortic intima-inner media were: permeability, 0.46 +/- 0.10 microL/cm2 per hour versus 1.41 +/- 0.32 microL/cm2 per hour (nonpaired t test, P < .0001); and fractional loss, 0.12 +/- 0.02 h-1 versus 0.44 +/- 0.05 h-1 (nonpaired t test, P = .0001), respectively. Finally, after 23 hours' exposure to labeled lipoproteins, the total accumulation and the amount of tightly bound labeled Lp(a) in the balloon-injured intima-inner media were, respectively, 174% (n = 6; ANOVA, P = .03) and 256% ANOVA, P = .005) of the values for labeled LDL. For labeled Lp(a) in the balloon-injured compared with the normal aortic intima-inner media, the recovery after 5 to 10 minutes, the permeability, and the accumulation after 23 hours were all increased, whereas the fractional loss was unchanged. These data suggest that the accumulation of Lp(a) is much larger in injured vessels than in normal vessels. Moreover, the data support the idea of a specific accumulation of Lp(a) compared with LDL in injured vessels. PMID- 8635220 TI - Adenosine-induced vasoconstriction in vivo. Role of the mast cell and A3 adenosine receptor. AB - Adenosine, a vasodilator metabolite, is often produced in tissues where the demand for oxygen exceeds the supply. We have recently demonstrated in isolated cannulated arterioles that adenosine and its metabolite, inosine, can also cause vasoconstriction by stimulation of mast cells. Secondary release of histamine and thromboxane is responsible for the inosine-induced constriction in vivo. In the present study, we explored the vasomotor effects of adenosine in vivo and investigated the role of the A3 adenosine receptor in mediating vasoconstriction. In vivo, local application of adenosine (10-6 to 10-4 mol/L) to arterioles consistently caused dose-dependent vasodilation. A fraction of arterioles, however, exhibited a biphasic response, with constriction following dilation. This, too, was dose dependent; 37% of arterioles constricted by 12.7 +/- 4.3% of the initial diameter in response to 10-4 mol/L adenosine. In the presence of 8-(p sulfophenyl)theophylline (8-SPT), an antagonist of A1 and A2 adenosine receptors, dilation in response to the same dose of adenosine was reduced, and constriction was enhanced; 85% of the tested arterioles constricted by -44.3 +/- 6.0% of the initial diameter. The A3 adenosine receptor has been shown to facilitate mediator release from mast cells, and its role was also examined. N6-(3-Iodo-4 aminobenzyl)adenosine (I-ABA), an agonist of A1 and A3 adenosine receptors, produced dose-dependent vasoconstriction. 1,3-Dipropyl-8-(4 acrylate)phenylxanthine (BW-A1433), an antagonist of A1, A2, and A3 receptors, significantly reduced the vasoconstrictor response to adenosine, which was unmasked during treatment with 8-SPT. In addition, both adenosine and I-ABA stimulated mast cell uptake of ruthenium red, indicating degranulation. The I-ABA induced constriction was abolished by combined histamine and thromboxane receptor antagonists. We conclude that adenosine can cause vasoconstriction in vivo, which is often masked by A2 receptor-mediated vasodilation. Mast cells are stimulated in the course of the response, and the A3 adenosine receptor is involved in mediating constriction. PMID- 8635221 TI - Removal of Arg141 from the alpha chain of human hemoglobin by carboxypeptidases N and M. AB - Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (alpha-Arg141) of the alpha chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of alpha-Arg141 accelerated the dissociation rate of the tetramer deoxy-des-alpha-Arg141 hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-alpha-Arg141 hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-alpha-Arg141 hemoglobin had a higher oxygen affinity (P50, 5.51 mm Hg; control, 19.94 mm Hg [P50 is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P50, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des alpha-Arg141 hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the alpha-Arg141 residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity. PMID- 8635223 TI - Enhancement of the L-type Ca2+ current by mechanical stimulation in single rabbit cardiac myocytes. AB - Anion conductance is known to be activated by mechanical stimulation, such as osmotic cell swelling or cell inflation via the patch pipette, of canine or rabbit cardiac myocytes. The effects of mechanical stimulation on time-dependent currents, however, remain unsettled. Using the whole-cell voltage-clamp method, we have found that mechanical stimuli enhance the L-type Ca2+ current (ICa,L) in rabbit cardiac myocytes. At every membrane potential, ICa,L was reversibly increased by osmotic cell swelling and by cell inflation caused by applying a positive pressure of 10 to 15 cm H2O via the patch pipette. ICa,L was increased during cell inflation by 37 +/- 21% (mean +/- SD, n = 17) in atrial cells and by 37 +/ -8% (n = 7) in sinoatrial node cells in solution containing 2 mmol/L Ca2+. The current-voltage relationship, the inactivation time constant, the steady state inactivation curve, and the conductance properties of ICa,L were all virtually unaffected by mechanical stimulation except for the open probability, which appears to increase. The increase in ICa,L was not dependent on protein kinase A, since an inhibitor peptide of cAMP-dependent protein kinase failed to prevent the increase in ICa,L during mechanical stimuli (n=5). The increase in ICa,L caused by cell inflation was unaffected by the chelation of intracellular Ca2+ by the addition of 10 mmol/L EGTA or 10 mmol/L BAPTA to the pipette solution, suggesting that the effect was not mediated by changes in intracellular Ca2+. Thus, mechanical stimulation due to cell swelling or inflation may itself directly increase ICa,L in rabbit cardiac myocytes. PMID- 8635222 TI - Inhibitory effects of TA-993, a new 1,5-benzothiazepine derivative, on platelet aggregation. AB - TA-993, an l-cis 4',8-dimethyl derivative of the Ca2+ antagonist diltiazem, and some of its metabolites inhibited platelet aggregation induced by collagen, ADP, epinephrine, platelet activating factor, arachidonic acid, and U-46619 in human platelets in vitro. Among the metabolites, MB3 was the most potent (IC50, <1 micromol/L; several hundred times more potent than the parent compound). The d isomer of MB3 was >100 times less potent than the l isomer. Unlike acetylsalicylic acid (ASA), TA-993 inhibited both primary and secondary phases of ADP-induced platelet aggregation and also exhibited a disaggregating effect on human platelet aggregates. The inhibitory effect of TA-993 was enhanced when used in combination with ASA. In ex vivo studies involving rats, TA-993 (approximately 0.3 to 100 mg/kg PO) dose-dependently inhibited collagen-induced platelet aggregation (ED50, 3 mg/kg PO). In the whole-blood platelet aggregation system in rats, orally administered TA-993 was also inhibitory in single (3 to 30 mg/kg) or repeated daily (10 mg/kg per day for 10 days) dosage. Orally administered TA-993 dose-dependently inhibited ADP-induced platelet aggregation ex vivo in dogs (0.3 to 10 mg/kg), significantly protected mice against collagen + epinephrine-induced thromboembolic death (10 mg/kg), and inhibited thrombus formation in an arteriovenous shunt in rats (30 mg/kg). The Ca2+-antagonistic action of TA-993 was very weak in depolarized canine basilar arteries: the potency was approximately 1/10 that of diltiazem (d-cis) and d-TA-993. These results suggest that antiplatelet action is more characteristic of the l-cis than the d-cis 1,5 benzothiazepine structure and that TA-993 may become a clinically useful antiplatelet agent of this structure series. PMID- 8635224 TI - Reentrant wave fronts in Wiggers' stage II ventricular fibrillation. Characteristics and mechanisms of termination and spontaneous regeneration. AB - The mechanisms of Wiggers' stage II ventricular fibrillation (VF) are poorly understood. Using computerized mapping techniques, we studied the patterns of activation during Wiggers' stage II VF in 13 open-chest dogs. In 7 of the 13 dogs, the right ventricular Purkinje fibers and adjacent subendocardial myocytes were ablated with Lugol solution. VF was induced electrically, and 3 to 5 seconds of data were obtained beginning approximately 2.5 seconds after the onset of VF. Dynamic displays of the activation patterns and isochronal maps revealed the presence of reentrant wave fronts in 17 of 33 runs of VF in ablated ventricles and in 12 of 45 runs of VF in intact ventricles. The incidence of reentry was not different between the subendocardium-ablated group versus the nonablated group (1.7 +/- 1.6 versus 1.2 +/- 1.6 rotations per episode of VF, P = .19). There were no differences in the core size (25 +/- 19 versus 29 +/- 18 mm2), life span (3.4 +/- 1.1 versus 3.2 +/- 1.2 rotations), or cycle length (111 +/- 12 versus 107 +/- 8 ms) in ablated ventricles versus intact ventricles, respectively. The core was unstable as it meandered within the mapped area displacing the entire reentrant wave front. In all episodes, the reentrant wave fronts were spontaneously initiated by an interaction between two propagating wave fronts roughly perpendicular to each other. The second wave front met the tail of the first wave front 69 +/- 11 ms (range, 40 to 90 ms) after its latest activation, indicating that the interaction occurred during a vulnerable period. The reentrant wave fronts terminated spontaneously (n = 7), as the result of interference by an invading wave front (n = 19 or meandered off the mapped region (n = 3). We conclude the following: (1) Reentrant activities with short life spans and meandering cores are present during Wiggers' stage II VF in dogs. (2) New reentrant wave fronts are generated when one wave front interacts with another wave front during its vulnerable period. (3) The reentrant wave fronts terminate spontaneously or as the result of interference. (4) Chemical subendocardial ablation does not affect the incidence, life span, cycle length, or core size of the reentrant wave fronts. PMID- 8635225 TI - K+ currents in human coronary artery vascular smooth muscle cells. AB - K+ channels and their currents are important in vascular tone regulation and are potential therapeutic targets; however, K+ channels in human coronary artery vascular smooth muscle cells (VSMCs) have received little attention. We examined K+ currents in freshly isolated VSMCs from human coronary arteries (n=368 from 32 human hearts) with conventional patch-clamp or perforated-patch techniques with nystatin. We detected four different K+ currents: (1) the delayed rectifier K+ current, IK(dr); (2) the Ca2+-activated K+ current, IK(Ca); (3) the nonrectifying noninactivating outward ATP-dependent K+ current, IK(ATP); and (4) the spontaneous transient outward K+ current, IK(STOC). K+ channels underlying spontaneous transient outward currents probably represent a single clustered population of Ca2+-activated K+ channels functionally associated with Ca2+ release channels in the sarcoplasmic reticulum. Inwardly rectifying K+ currents were not observed. K+ currents were unevenly distributed in that they were not uniformly exhibited by all cells. The most prominent K+ currents were IK(Ca) (100%) and IK(dr) (46%). IK(STOC)s, which have not been previously described in humans, were present in 67% of VSMCs. IK(ATP) was small under physiological conditions; however, IK(ATP) increased markedly after cell stimulation with exogenous or endogenous coronary vasodilators. Thus, IK(ATP) may be particularly relevant in ischemia and could be of special importance as a therapeutic target. We conclude that human coronary VSMCs have unique K+ currents that differ sufficiently from those of other species, thus making the investigation of human material clinically relevant. The findings suggest potential avenues for further therapeutic research. PMID- 8635227 TI - Cellular basis for the negative dromotropic effect of adenosine on rabbit single atrioventricular nodal cells. AB - The effects of adenosine on action potentials, rate-dependent activation failure (the cellular basis for second-degree atrioventricular [AV] block), and the recovery of excitability in rabbit isolated single AV nodal cells were studied using the whole-cell patch-clamp technique. Adenosine (1 micromol/L) shortened the duration, depressed the amplitude, and reduced the rate of rise of the AV nodal cell action potential. Adenosine (10 micromol/L) caused a significant hyperpolarization (7 +/- 1 mV) of AV nodal cells. Adenosine increased the occurrence and the rate dependence of activation failure (Wenckebach periodicity) of AV nodal cells: this effect was concentration dependent and mediated by A1 adenosine receptors. The rate-dependent activation failure caused by adenosine was associated with a prolongation of the effective refractory period by 18 +/- 2 ms (P < .05), an increase in the duration of activation delay, and an elevation (from 0.22 +/- 0.04 to 0.30 +/- 0.03 nA, P < .05) of the threshold current amplitude required to activate AV nodal cells. The results suggest that the slowed recovery of excitability of AV nodal cells caused by adenosine forms the cellular basis for adenosine-induced second-degree AV block. Adenosine decreased ICa,L and activated IK,ADO of AV nodal cells. These actions of adenosine on ion currents may contribute to the effect of this nucleoside to depress excitability of AV nodal cells. The enhancement by adenosine of rate-dependent activation failure of AV nodal cells implies that the negative dromotropic effect of adenosine should be more pronounced during an episode of supraventricular tachycardia than during normal rhythm. PMID- 8635226 TI - Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. AB - Previous voltage-clamp studies have suggested that the delayed rectifier current (IK) is small or absent in the human ventricle and, when present, consists only of the rapid component (IKr); however, molecular studies suggest the presence of functionally important IK in the human heart, specific IKr blockers are known to delay ventricular repolarization and cause the long QT syndrome in humans, and we have shown that the expression of IK is strongly influenced by cell isolation techniques. The present experiments were designed to assess the expression of IK in myocytes obtained by arterial perfusion of right ventricular tissue from explanted human hearts. Of 35 cells from three hearts, 33 (94%) showed time dependent currents typical of IK. The envelope-of-tails test was not satisfied under control conditions but became satisfied in the presence of the benzenesulfonamide E-4031 (5 micromol/L). E-4031 suppressed a portion of IK in 32 of 33 cells, with properties of the drug-sensitive and -resistant components consistent with previous descriptions of IKr and the slow component (IKs), respectively. Action potential duration to 95% repolarization at 1 Hz was prolonged by E-4031 from 336+/-16 (mean +/- SEM) to 421 +/- 19ms (n = 5, P < .01), indicating a functional role for IK. Indapamide, a diuretic agent previously shown to inhibit IKs selectively, suppressed E-4031-resistant current. The presence of a third type of delayed rectifier, the ultrarapid delayed rectifier current (IKur), was evaluated with the use of depolarizing prepulses and low concentrations (50 micromol/L) of 4-aminopyridine. Although these techniques revealed clear IKur in five of five human atrial cells, no corresponding component was observed in any of five human ventricular myocytes. We conclude that a functionally significant IK, with components corresponding to IKr and IKs, is present in human ventricular cells, whereas IKur appears to be absent. These findings are important for understanding the molecular, physiological, and pharmacological determinants of human ventricular repolarization and arrhythmias. PMID- 8635228 TI - Ca2+-induced current oscillations in rabbit ventricular myocytes. AB - Transient currents are activated by spontaneous Ca2+ oscillations in rabbit ventricular myocytes. We investigated the ionic basis for these transient currents under conditions in which K+ currents would be expected to be blocked. Holding cells under voltage clamp at positive potentials leads to a rise in intracellular Ca2+ via reversal of the Na+-Ca2+ exchanger and subsequently to the initiation of spontaneous Ca2+ transients, presumably from a Ca2+-overloaded sarcoplasmic reticulum. The current transients associated with these Ca2+ transients reversed at about +10 to +15 mV under conditions of approximately symmetrical Cl-. In the absence of Cl-, this current was inward at all potentials examined over the range from -88 to +72 mV, consistent with a Na+-Ca2+ exchanger current. In the absence of Na+, the repetitive spontaneous Ca2+ transients could be initiated by a brief train of depolarizations to activate the inward Ca2+ current. Under such conditions, the current was found to reverse at -3 mV when the equilibrium potential of Cl- (ECl) was -2 mV, and the reversal potential shifted to -32 mV when internal Cl- was lowered, to make ECl -33 mV. Thus, in the absence of Na+, it appears that the current is exclusively a Ca2+-activated Cl- current. There is no evidence to indicate the presence of a Ca2+-activated cationic conductance. Further, our results demonstrate that the Ca2+-activated Cl conductance can carry inward current at potentials more negative to ECl in rabbit ventricular myocytes and is therefore likely to contribute to the arrhythmogenic delayed afterdepolarizations that occur in Ca2+-overloaded cells. PMID- 8635229 TI - Effect of membrane potential on the initiation of acetylcholine-induced Ca2+ transients in isolated guinea pig coronary myocytes. AB - The muscarinic stimulation of single voltage-clamped coronary arterial smooth muscle cells of the guinea pig was used to evaluate the effect of membrane potential on the inositol 1,4,5-tris-phosphate (IP3)-mediated changes of ionized [Ca2+] in the cytoplasm (Ca2+ transient) measured with indo 1. When applied at the membrane potential of -50 mV, 10 micromol/L acetylcholine (ACh) induced a [Ca2+]i increase after the mean latency of 2.6+/-0.9 s. The latency was reduced to 1.1 +/- 0.3 s when the same dose was applied at a holding potential of +50 mV. In paired experiments in the same cells, the latency of response at +50 mV was reduced by a factor of 2.2 +/- 0.3 compared with the response at -50 mV. Supramaximal [ACh] (100 micromol/L) induced Ca2+ transients with a 0.4 +/- 0.1-s latency, which was independent of membrane potential. When applied repetitively at -50 mV, ACh induced Ca2+ transients with a progressively reduced amplitude and slower rate of rise. Depolarization to +50 mV accelerated the rate of rise of the Ca2+ transient by a factor of 3.4 +/- 0.4 without affecting the amplitude. The modulation of the initiation of Ca2+ transient by a 100-mV depolarization can be explained by an approximately threefold increase in the rate of IP3 accumulation. PMID- 8635230 TI - Endothelin-dependent actions in cultured AT-1 cardiac myocytes. The role of the epsilon isoform of protein kinase C. AB - The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC) epsilon immunoreactivity from the soluble to the particulate cell fraction. The subcellular distributions of PKCalpha and PKCzeta (also expressed by AT-1 cells) are not influenced by endothelin. Using quantitative fluorescence microscopy with fura 2, we examined the effects of endothelin on intracellular calcium. In electrically driven myocytes, endothelin induces a rapid and transient increase in the amplitude of the calcium transient. This is blocked by both phorbol 12 myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKCepsilon plays a critical role in endothelin receptor-dependent increases in intracellular calcium. Endothelin also stimulates mitogen-activated protein kinase (MAPK). MAPK activation is markedly attenuated by pretreatment with PMA or pertussis toxin (PTX, to activate susceptible G protein alpha subunits); it is completely prevented by combined pretreatment with PMA and PTX. In contrast, it is not attenuated by chelation of intracellular calcium with BAPTA. These findings indicate that the pathway for endothelin receptor stimulation of MAPK involves PKCepsilon and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKCepsilon as a mediator of endothelin receptor-dependent increases in cytosolic calcium and MAPK activity in AT-1 cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKCepsilon and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes. PMID- 8635231 TI - Differential response to Na+ channel blockade, beta-adrenergic stimulation, and rapid pacing in a cellular model mimicking the SCN5A and HERG defects present in the long-QT syndrome. AB - The long-QT syndrome (LQTS) is a hereditary disorder characterized by an abnormally prolonged QT interval and by life-threatening arrhythmias. Recently, two of the genes responsible for LQTS have been identified: SCN5A, a voltage dependent Na+ channel on chromosome 3 (LQT3), and HERG, responsible for the rapid component of the delayed rectifier current (IKr), on chromosome 7 (LQT2). We developed an in vitro model to attempt reproduction of the expected alterations in LQT3 and LQT2 patients. Guinea pig ventricular myocytes were exposed to anthopleura toxin A (anthopleurin), an inhibitor of the inactivation of the Na+ current, and to dofetilide, a selective blocker of IKr. Both interventions significantly prolonged action potential duration (APD), by 54 +/- 13 and 62 +/- 16 ms, respectively. Cells pretreated with anthopleurin significantly shortened APD in response to mexiletine, isoproterenol, and rapid pacing (from 264 +/- 38 to 226 +/- 32 ms after mexiletine, P < .001). On the contrary, cells exposed to dofetilide did not shorten the APD after mexiletine and even prolonged it after initial exposure to isoproterenol (from 280 +/- 25 to 313 +/- 20 ms, P < .001); during rapid pacing, APD was shortened but less (38 +/- 9 versus 60 +/- 11 ms, P < .05) than in anthopleurin-treated cells. This study shows that a cellular model for LQTS, based on the recent advances in molecular genetics, can provide adequate "phenotypes" of prolonged repolarization amenable to the testing of interventions of potential clinical relevance. We found differential responses to Na+ channel blockade, to beta-adrenergic stimulation, and to rapid pacing according to specific pretreatment with either anthopleurin (to mimic LQT3) or dofetilide (to mimic LQT2). These different responses in myocytes bear striking similarities with the differential response to analogous interventions in LQTS patients with mutations on the SCN5A and HERG genes. PMID- 8635232 TI - Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. AB - Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion. PMID- 8635233 TI - Hepatocyte growth factor and its receptor are expressed in cardiac myocytes during early cardiogenesis. AB - In the mouse, the heart primordium arises when mesoderm is set aside during gastrulation, is induced by pharyngeal endoderm, migrates ventrally to the midline of the embryo, forms a tube, and begins beating. Little is known of the molecular mechanisms that mediate the determination, mitosis, differentiation, and migration that lead to the beating heart. Transcripts for hepatocyte growth factor/scatter factor (HGF) and its receptor are coexpressed transiently and dynamically in the premyocardium but not in other heart progenitor cells. Transcripts the HGF ligand and receptor are first detected before cardiac function and looping and persist through the first looping stage, when heart morphology begins to elaborate. HGF ligand and receptor mRNA are detectable after the putative heart transcription factor, Csx/Nkx2-5, and concomitantly with the heart structural gene, cardiac actin. HGF receptor mRNA is detected in the mesoderm of the headfold stage and persists in myocardial precursors of the ventricles and atria (but not in the outflow-tract smooth muscle cells) through the 14-somite stage at approximately 8.75 days after fertilization (day E8.75). At the headfold stage, between E7.5 and E8.0, HGF receptor mRNA was detected in myocardial cells before fusion at the ventral midline. HGF ligand and receptor mRNA transcripts are coexpressed in the embryo, except in the headfold state (when only the HGF receptor can be detected) and in the heart at the 14- to 18 somite stage (when only HGF ligand can be detected). The dynamic pattern of coexpression suggests an autoregulatory role for HGF and its receptor in early heart development. PMID- 8635234 TI - Upregulation of cardiac uptake 1 carrier in ischemic and nonischemic rat heart. AB - Neuronal uptake1 constitutes the main elimination process of cardiac norepinephrine under normoxic conditions. Uptake1 may be subject to changes during myocardial ischemia. We therefore studied the regulation of the uptake1 carrier in isolated perfused rat hearts, comparing ischemic and nonischemic conditions. Radioligand binding with [3H]mazindol was used to determine carrier densities and affinities, whereas cardiac clearance of[3H]norepinephrine served as a measure of the transport capacity of the uptake1 carrier. When exocytotic norepinephrine release was induced in nonischemic rat hearts by electrical field stimulations, we observed an increase in the cardiac density of uptake1 carriers (Bmax) to 210 +/- 5 fmol/mg protein (versus 134 +/- 3 fmol/mg in control hearts). Simultaneously, the cardiac clearance of [3H]norepinephrine increased to 41 +/- 4% versus 30 +/- 4% in control hearts. Both carrier density and norepinephrine clearance returned to baseline values within a period of 40 minutes after stimulation. Carrier affinities (Kd values) did not differ between the groups. Stop-flow ischemia induced a substantial overflow of norepinephrine by itself. Additionally, carrier density was increased to 144% after 40 minutes of stop-flow ischemia (P < .005 versus control hearts). When ischemia was followed by 20 minutes of reperfusion, the Bmax of the uptake1 carrier remained significantly elevated. With a further extension of the reperfusion period to 40 minutes, however, carrier density declined to baseline values. Kd values were not influenced by any of these interventions. Clearance of [3H]norepinephrine was suppressed (to 5 +/- 2%) in the first minutes of reperfusion, which may reflect the inverse transport direction of the norepinephrine carrier known to occur in ischemia. After 20 minutes of reperfusion, clearance increased to 39 +/- 5% (P < .005 versus control hearts) and then fell to 29 +/- 5% after 40 minutes of reperfusion (NS). These results demonstrate that after both electrical field stimulation and myocardial ischemia, the density of uptake1 carrier proteins temporarily increases, which may result in an increased transport capacity for norepinephrine. PMID- 8635235 TI - Effect of endothelin-1 on actomyosin ATPase activity. Implications for the efficiency of contraction. AB - Endothelin is a powerful inotropic peptide that increases isometric force in isolated papillary muscle and the extent of shortening in isolated single cardiac myocytes. Its mechanism of action has been variously attributed to increased Ca2+ activation, increased Ca2+ sensitivity of the contractile proteins, and increased intracellular pH, but the physiological function of the changes in cardiac performance remains obscure. In this study, the effects of endothelin-1 on both force development and the kinetics of contraction have been examined. Isometric force, actomyosin ATPase activity, and unloaded shortening velocity were measured. The effects were dose dependent. From 1 to 50 pmol/L endothelin-1 did not alter force development in isolated trabeculae with intact endothelial cells, but actomyosin ATPase activity was increased. Between 100 pmol/L and 10 nmol/L endothelin-1 raised isometric force, decreased actomyosin ATPase activity, and decreased unloaded shortening velocity. The reduction in ATPase activity was progressively enhanced as sarcomere length was increased from 1.9 t0 2.4 microns. These results indicate that the effects of endothelin-1 on the force of contraction and the rate of ATP hydrolysis are not tightly coupled and are changed in the opposite directions by endothelin-1 over most of its effective dose-range. This raises the possibility that endothelin-1 may increase the economy of contraction. A novel function of endothelin may be the modulation of the efficiency of contraction, particularly when increased preload raises the contractile work of the heart. PMID- 8635236 TI - Role of poly-ADP ribosyltransferase activation in the vascular contractile and energetic failure elicited by exogenous and endogenous nitric oxide and peroxynitrite. AB - Stimulation of vascular smooth muscle with bacterial lipopolysaccharide (LPS) and proinflammatory cytokines induces the expression of a distinct isoform of NO synthase (inducible NOS [iNOS]) contributing to the suppression of vascular contractility. We have obtained evidence of the involvement of an indirect pathway triggered by NO and its reaction product peroxynitrite (ONOO-) through the activation of the nuclear enzyme poly-ADP ribosyltransferase (PARS) in the pathogenesis of cellular energetic and contractile failure in vascular smooth muscle. Exposure of vascular smooth muscle cells caused DNA strand breaks, activation of PARS, depletion of NAD+, and inhibition of mitochondrial respiration. The NAD+ depletion and inhibition of mitochondrial respiration were reduced by pharmacological inhibition of PARS. Stimulation of vascular smooth muscle cells with LPS and interferon gamma (IFN-gamma) triggered the production of superoxide anion over 3 to 48 hours and NO and ONOO- over 24 to 48 hours and resulted in significant DNA strand breakage. The decrease in mitochondrial respiration in response to LPS and IFN-gamma stimulation was inhibited by the ONOO- scavenger uric acid (100 mumol/L) and by inhibitors of iNOS. The PARS inhibitors 3-aminobenzamide (1 mmol/L), nicotinamide (1 mmol/L), and PD 128763 (100 mumol/L) inhibited the reduction in cellular NAD+ and ATP and the suppression of mitochondrial respiration in response to LPS and IFN-gamma stimulation. Administration of 3-aminobenzamide also reduced PARS activation and vascular hyporeactivity of rat thoracic aortas exposed to ONOO- (300 mumol/L to 1.5 mmol/L) in vitro. 3-Aminobenzamide (10 mg/kg IP) preserved the ex vivo contractility of aortas obtained from endotoxic rats and improved survival in lethal murine endotoxic shock. These data suggest that PARS activation due to iNOS induction (1) is involved in the energetic depletion of vascular smooth muscle cells that express iNOS and (2) contributes to the pathogenesis of vascular energetic and contractile failure in endotoxic shock. Inhibition of PARS may be a novel concept of therapeutic potential in shock. PMID- 8635237 TI - Differential sensitivity of venular and arteriolar alpha-adrenergic receptor constriction to inhibition by hypoxia. Role of receptor subtype and coupling heterogeneity. AB - Reflex adrenergic constriction of the venous circulation is considerably less sensitive than the arterial circulation to local metabolic inhibition, but the basis for this difference remains unclear. The purpose of the present study was to determine whether alpha-adrenergic receptor (AR) constriction of venular smooth muscle is in fact protected against inhibition by hypoxia, per se, and to examine possible mechanisms for this protection. An intermediate level of alpha 1 AR (norepinephrine + rauwolscine) or alpha 2-AR (UK 14,304 + prazosin) tone was induced in rat cremaster skeletal muscle arterioles and venules (control lumen diameter, 134 and 194 micron respectively), and tissue bath PO2 was lowered from the control value (30 mm Hg). Arteriolar alpha 2-AR tone was inhibited by 29% at 5 mm Hg PO2 (P < .05), whereas arteriolar alpha 1-, venular alpha 1, and venular alpha 2-AR constrictions were unaffected. Like these findings obtained for in situ vessels with normal blood flow, alpha 1-AR tone induced in vascularly "isolated" venules and basal diameter were again unaffected by hypoxia, whereas alpha 2-AR tone was actually enhanced by 19% (P < .05). This constriction was prevented by indomethacin but not by endothelin or nitric oxide blockade; importantly, however, venular alpha 2- and alpha 1-AR tone still remained insensitive to inhibition by hypoxia. ATP-sensitive K+ (KATP) channels, which are known to participate in hypoxic inhibition of arteriolar smooth muscle, were examined for a role in this differential arteriolar versus venular sensitivity to hypoxia. Use of the KATP antagonists glibenclamide and U-37883A and the KATP channel opener cromakalim suggested that venular, unlike arteriolar, smooth muscle had no detectable basal or inducible KATP activity. Also, unlike arteriolar alpha 2-AR constriction, venular alpha 2-AR tone did not depend on KATP activity. Finally, venular alpha 2-AR tone was unaffected by nifedipine (0.06 to 3 mumol/L), whereas venular alpha 1-AR tone was inhibited by 50% (P < .05), findings opposite those found for arteriolar alpha 1 and alpha 2 tone. These data demonstrate that venular alpha 1- and alpha 2-AR constrictions are insensitive to inhibition by hypoxia and suggest that this may be due to a paucity of KATP channels on venular smooth muscle. In addition, venular alpha 1- but not alpha 2-ARs appear to couple to dihydropyridine-sensitive voltage operated Ca2+ channels. PMID- 8635238 TI - Differential regulation of L-arginine transport and nitric oxide production by vascular smooth muscle and endothelium. AB - Since NO production is dependent on the availability of L-arginie, we examined whether L-arginine transport and NO synthesis are coregulated by vascular smooth muscle cells and endothelial cells cultured from the same vessel wall source. L Arginine transport by both bovine aortic smooth muscle cells (BASMCs) and endothelial cells (BAECs) was primarily Na+ independent (approximately 70%) and was mediated by both a high- and low-affinity transport system. Treatment of BASMCs with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta) resulted in a significant increase in L-arginine transport (approximately 20%) and in the induction of NO release. Exposure of BASMCs to interferon gamma (IFN-gamma) or lipopolysaccharide (LPS) also stimulated NO release but did not affect L-arginine transport. In contrast, incubation of BAECs with TNF-alpha or LPS strikingly enhanced L-arginine uptake (2.5-fold), whereas IL-1 beta and IFN gamma had no effect. Treatment of BAECs with any of the inflammatory mediators did not stimulate NO production. These results demonstrate that L-arginine uptake and NO synthesis by these cells are differentially regulated. In BASMCs, the coinduction of L-arginine transport and NO formation may function to provide increased levels of substrate to the cell during activation of the NO synthase enzyme. In contrast, the selective stimulation of L-arginine uptake in BAECs indicates that L-arginine transport is dissociated from NO generation in these cells. PMID- 8635239 TI - In situ hybridization reveals extensive diversity of K+ channel mRNA in isolated ferret cardiac myocytes. AB - The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions. PMID- 8635240 TI - Alpha 1-adrenergic inhibition of the beta-adrenergically activated Cl- current in guinea pig ventricular myocytes. AB - alpha-Adrenergic receptor stimulation regulates the activity of a number of different cardiac ion channels, including those underlying one or more distinct Cl- conductances. The whole-cell patch-clamp technique was used in the present study to investigate the effects of alpha-adrenergic stimulation on the beta adrenergically regulated Cl- current in guinea pig ventricular myocytes. Neither alpha 1-adrenergic receptor stimulation with methoxamine (25 to 500 mumol/L) nor direct activation of endogenous protein kinase C (PKC) with phorbol 12,13 dibutyrate (PDBu, 100 nmol/L) evoked a Cl- current. On the contrary, the Cl- current activated by 30 nmol/L isoproterenol was inhibited by methoxamine, with an EC50 of 6.7 +/- 2.6 mumol/L, and this response was blocked by prazosin, an alpha 1-adrenergic receptor antagonist. Prazosin also decreased the EC50 for current activation by norepinephrine from 53 +/- 7.1 to 18 +/- 3.8 nmol/L, demonstrating that the ability of this endogenous neurotransmitter to activate the Cl- current through beta-adrenergic receptor stimulation is limited by its intrinsic ability to also activate alpha-adrenergic receptors. Methoxamine did not inhibit the Cl- current evoked by either direct activation of adenylate cyclase with forskolin or inhibition of phosphodiesterase activity with 3 isobutyl-1-methylxanthine, indicating that alpha-adrenergic stimulation inhibits beta-adrenergic responses at a point upstream of adenylate cyclase activation. Methoxamine also did not inhibit the Cl- current activated by histamine, suggesting that alpha-adrenergic stimulation specifically inhibits beta adrenergic receptor-mediated responses. The inhibitory effect of methoxamine was not mimicked by PDBu, and it persisted in the presence of bisindolylmaleimide, a selective PKC inhibitor. However, methoxamine inhibition of the isoproterenol activated Cl- current was sensitive to pertussis toxin. These results suggest that alpha-adrenergic receptor stimulation inhibits the beta-adrenergically activated Cl- current, demonstrating a novel mechanism by which alpha-adrenergic receptors may regulate ion channel activity in the heart. PMID- 8635241 TI - Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. AB - Previous results from our laboratory have suggested that opioid receptors are involved in ischemic preconditioning (PC) in rat heart. Furthermore, other investigators have suggested that mu- and delta-opioid receptors mediate analgesia and hypoxic cerebral vasodilatation via opening of ATP-sensitive K+ (KATP) channels. Thus, the purpose of the present study was to test the hypothesis that activation of opioid receptors mimics the cardioprotective effect of ischemic PC and that this effect is produced by activation of KATP channels in the rat heart. Anesthetized open-chest Wistar rats were subjected to six different protocols. All groups were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic PC was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Similarly, morphine-induced PC was elicited by three 5-minute drug infusions (100 micrograms/kg i.v. ) interspersed with 5-minute drug-free periods before the prolonged 30-minute occlusion. Infarct size (IS) as a percentage of the area at risk (AAR) was determined by triphenyltetrazolium staining. Ischemic PC and morphine infusions resulted in similar reductions in IS/AAR from 56 +/- 5% to 11 +/- 3% and 12 +/- 5%, respectively (P < .05). Administration of glibenclamide (0.3 mg/kg i.v.), a KATP channel antagonist, or naloxone (3 mg/kg i.v.), a nonselective opioid receptor antagonist, both blocked the cardioprotective effects of morphine. These results indicate that opioid receptor stimulation results in a reduction in infarct size similar to that produced by ischemic PC. The effect of morphine is most likely mediated via an opioid receptor-KATP channel-linked mechanism in the rat heart, since glibenclamide abolished its protection. PMID- 8635242 TI - Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding. AB - The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 channel expressed in mammalian cells at therapeutically relevant concentrations (EC50, 6.2 mumol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the internal mouth of the ionic pore and that binding is stabilized by hydrophobic interactions. We tested these hypotheses using site-directed mutagenesis of residues proposed to line the internal mouth of the channel or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 mumol/L, respectively), and preserved both the voltage-dependent open channel-block mechanism and the electrical binding distance (0.19 to 0.22). In contrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, at the external entrance of the pore, and at sites in the S4S5 linker. The approximately 10-fold increase in affinity with T505I and the reduction of the dissociation rate constant with the mutations that increased affinity are consistent with a hydrophobic stabilization of binding. Moreover, the T505 and V512 residues align on the same side of the putative alpha helical S6 segment. Taken together, these results localize the hydrophobic binding site for this antiarrhythmic drug in the internal mouth of this human K+ channel and provide molecular support for the open channel-block model and the role of S6 in contributing to the inner pore. PMID- 8635243 TI - Antiarrhythmic drug binding sites in cardiac K+ channels. PMID- 8635244 TI - Stress-activated protein kinases in cardiovascular disease. PMID- 8635245 TI - Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes. AB - An antisense oligodeoxynucleotide (ODN) approach was used to investigate whether mitogen-activated protein kinase (MAPK) is necessary for the hypertrophic response in cardiac myocytes. A phosphorothioate-protected 17-mer directed against the initiation of translation sites of the p42 and p44 MAPK isoform mRNAs was introduced into cultured cardiac myocytes by liposomal transfection. At an antisense ODN concentration of 0.2 mumol/L, p42 MAPK protein was reduced by 82% (immunoblot) after 48 hours, and p42 and p44 MAPK activities were reduced by 44% and 60%, respectively. The same concentration of anti-MAPK ODN inhibited development of the morphological features of hypertrophy (sarcomerogenesis, increased cell size) in myocytes exposed to phenylephrine. Phenylephrine-induced activation of the atrial natriuretic factor (ANF) promoter (measured by the activity of a transfected ANF promoter/luciferase reporter gene) and induction of ANF mRNA (measured by RNase protection assay) were also attenuated. We conclude that MAPK is important for the development of the hypertrophic phenotype in this model of hypertrophy. PMID- 8635246 TI - Ca(2+)-dependent mitogen-activated protein kinase activation in spontaneously hypertensive rat vascular smooth muscle defines a hypertensive signal transduction phenotype. AB - The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na(+)-H+ exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II-mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca2+ mobilization, since chelation of intracellular Ca2+ with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension. PMID- 8635247 TI - Novel adenovirus component system that transfects cultured cardiac cells with high efficiency. AB - Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a "component system" formed by preincubation of Ad5dl312 adenovirus, poly-L-lysine, and an expression plasmid (lacZ reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying beta-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions. LacZ-positive myocytes could be identified in intact myocytes with the fluorescent substrate C12-fluorescein di-beta-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase C delta. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells. PMID- 8635248 TI - Structure and characterization of the 5'-flanking region of the mouse smooth muscle myosin heavy chain (SM1/2) gene. AB - We have previously shown that smooth muscle myosin heavy chain isoforms (SMs), including SM1, SM2, and SMemb, are differentially expressed during vascular development, and in vascular lesions, such as atherosclerosis. The SM1/2 gene is expressed exclusively in smooth muscle cells and generates SM1 and SM2 mRNAs by alternative splicing. Whereas SM1 is constitutively expressed from early development, SM2 appears only after birth. In this study, we have isolated and characterized the 5'-flanking region of the mouse SM1/2 gene. Transient transfection assays using a series of promoter-luciferase chimeric constructs demonstrated that tandem elements of the CCTCCC sequence, located at -89 and -61 bp relative to the transcription start site, were essential for transcriptional activity of the SM1/2 gene in primary cultured rabbit aortic smooth muscle cells and smooth muscle cell lines derived from the rabbit aorta but not in non-smooth muscle cells. Gel mobility shift assays indicated that CCTCCC was a binding site for nuclear proteins prepared from smooth muscle cells. Double-stranded oligonucleotides containing either the CACC box or the Sp1 consensus sequence efficiently competed with the CCTCCC elements for binding the nuclear extracts. Site-specific mutations of CCTCCC elements resulted in a significant reduction of the promoter activity. Moreover, CCTCCC elements are evolutionary conserved between mouse and rabbit. In conclusion, the results of this study indicate an important role for the interaction of the CCTCCC sequence with Sp1 or related factors in activating transcription from the SM1/2 gene promoter. PMID- 8635249 TI - Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels from cardiac muscle. AB - Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca2+ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stablizes gating of the skeletal Ca(2+) release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca(2+)-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca(2+)-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca(2+)-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes. PMID- 8635251 TI - AHA leads new heart and stroke research coalition. PMID- 8635250 TI - Nitric oxide synthase (NOS3)-mediated cholinergic modulation of Ca2+ current in adult rabbit atrioventricular nodal cells. AB - We examined the role of endogenous NO in the autonomic regulation of atrioventricular (AV) nodal function by studying spontaneous action potentials (SAPs) and L-type Ca2+ current (ICa-L) in isolated single AV nodal cells from adult rabbit hearts. Both the perforated and the membrane-ruptured patch-clamp techniques in the whole-cell configuration were used under conditions known to alter NO production. Three NO donors, 3-morpholinosydnonimine (SIN-1, 0.1 mmol/L), S-nitroso-acetylcysteine (0.1 mmol/L), and sodium nitroprusside (0.1 mmol/L), suppressed the beta-adrenergic agonist isoproterenol (ISO, 1 mumol/L) stimulated increase in ICa-L. SIN-1 also decreased the frequency and amplitude of SAPs. In cells in which ICa-L had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 mumol/L), SIN-1 had no additive effect. CCh activated an acetylcholine-sensitive outward K+ current (IK(ACh)) in AV nodal cells, in addition to the ICa-L inhibition. Intracellular dialysis with the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh induced, but not SIN-1-induced, ICa.L attenuation. However, intracellular dialysis with methylene blue (20 mumol/L), which inhibits NO-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN 1 on ICa-L. In these cells, neither L-NMMA nor methylene blue affected the CCh activated IK(ACh). Direct application of cGMP (10 mumol/L) via internal dialysis significantly inhibited ISO-stimulated ICa-L. In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit, ICa-L but still activated IK(ACh). CCh-induced ICa-L attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1 methylxanthine (20 mumol/L). However, CCh still significantly suppressed ISO stimulated ICa-L after the cGMP-inhibited PDE isozyme (PDE3) had been selectively inhibited by milrinone (5 mumol/L). Immunohistochemical staining identified the presence of the endothelial constitutive NO synthase (ecNOS or NOS3) in both single AV nodal cells in vitro and in cryostat sections of AV nodal tissue in situ. These results demonstrate that endogenous NO is involved in the muscarinic cholinergic attenuation of ICa-L in AV nodal cell; the mechanism likely involves the cGMP-stimulated PDE. PMID- 8635252 TI - Cost of prevention. The case of lipid lowering. PMID- 8635253 TI - Insulin resistance and atherosclerosis. Common roots for two common diseases? PMID- 8635254 TI - Insulin resistance, its consequences, and coronary heart disease. Must we choose one culprit? PMID- 8635255 TI - Leukocyte adhesion to the coronary microvasculature during ischemia and reperfusion in an in vivo canine model. AB - BACKGROUND: Prompt reperfusion of ischemic myocardium or myocardium that is in the process of becoming infarcted is a cornerstone of current therapy for coronary artery disease. Paradoxically, experimental evidence suggests that cardiac damage may be caused by the reperfusion itself. Leukocyte attachment to the coronary vascular endothelium during reperfusion may be an initiating step in this detrimental process. Leukocyte adhesion to microvascular endothelium has never been demonstrated directly in a cardiac model of ischemia and reperfusion. METHODS AND RESULTS: Fluorescent videomicroscopy through a special "floating" objective that allows a series of lenses to move in unison with the beating dog heart was used on the left ventricular surface of open-chest dogs. Epicardial microvessels (25 to 130 microm), in focus throughout the cardiac cycle, were recorded after infusion of acridine orange (to fluorescently label leukocytes) during either 1 hour of ischemia followed by 2 hours of reperfusion, 3 hours of ischemia, or 3 hours of no ischemia. The amount of net fluorescence recorded along microvessel walls, which represented leukocyte accumulation, significantly increased in dogs during reperfusion (n = 8) compared with the same time period in the animals that were kept ischemic (n=5) (21.0 +/- 3.8 versus 10.9 +/- 4.5 gray scale; P = .0001). The rapid increase in fluorescence during reperfusion was also significantly different from values in the same group during the preceding period of ischemia (21.0 +/- 3.8 versus 5.1 +/- 2.1 gray scale; P =.0001), whereas no significant increase was seen over the same time periods in the animals that remained ischemic throughout the protocol. CONCLUSIONS: Reperfusion, compared with ischemia alone, promotes the rapid accumulation of leukocytes in the coronary microvasculature of dogs. PMID- 8635256 TI - Restricted usage of T-cell receptor Valpha-Vbeta genes in infiltrating cells in aortic tissue of patients with Takayasu's arteritis. AB - BACKGROUND: Infiltration by perforin-secreting killer lymphocytes, such as T cells and natural killer cells, has been shown to be involved in the pathogenesis of vascular cell damage in Takayasu's arteritis. METHODS AND RESULTS: To investigate the immunological mechanisms involved, especially the nature of T cell infiltration in Takayasu's arteritis as well as atherosclerosis, we analyzed the expression of T-cell receptor (TCR) Valpha and Vbeta genes in infiltrating cells in the aortic tissue of patients with Takayasu's arteritis and the atherosclerotic aortic aneurysm by polymerase chain reaction (PCR). We also analyzed the expression of cytokine genes by PCR. We found that the repertoires of TCR Valpha as well as Vbeta gene transcripts in Takayasu's arteritis were restricted. The infiltrating cells expressing Valpha2, Valpha16, Valpha17, Vbeta7, and Vbeta13.1 were found in 3 of 4 patients. In contrast, TCR Valpha Vbeta repertoires in atherosclerotic aortic aneurysm were polyclonal. There was no significant difference in the pattern of cytokine gene expression between the two diseases. CONCLUSIONS: The restricted usage of TCR Valpha as well as Vbeta genes by infiltrating T cells in Takayasu's arteritis may indicate that a specific antigen in the aortic tissue was targeted. Our findings provide the evidence that distinct immunological mechanisms are involved in the pathogenesis of Takayasu's arteritis and atherosclerotic aortic aneurysm. PMID- 8635258 TI - Cholesterol lowering and the use of healthcare resources. Results of the Scandinavian Simvastatin Survival Study. AB - BACKGROUND: Advances in the treatment of cardiovascular disease have increased costs; annual cardiovascular healthcare expenditure in the United States currently exceeds $100 billion. Physicians and third-party payers need to assess the economic impact of treatments that reduce cardiovascular morbidity and mortality. METHODS AND RESULTS: The Scandinavian Simvastatin Survival Study is a randomized, double-blind, placebo-controlled trial in which simvastatin reduced the risk of death by 30% (P=.0003) over the median follow-up period of 5.4 years in patients with previous myocardial infarction or stable angina pectoris as a result of a 42% reduction in the risk of coronary deaths (P=.00001). In the present report, data prospectively collected from hospital admissions were analyzed to evaluate the impact of simvastatin on healthcare resource use and perform a cost-minimization analysis. In the placebo group (n=2223), there were 1905 hospitalizations (average duration, 7.9 days) for acute cardiovascular events or coronary revascularization procedures among 937 patients, whereas in the simvastatin group (n=2221), there were 1403 such hospitalizations (average duration, 7.1 days) among 720 patients (all differences, P<.0001). The corresponding number of hospital days was 15089 and 9951 in the two groups, respectively (34% reduction,P<.0001). In the United States, the resulting reduction in hospitalization costs over the 5.4 years of the trial would be $3872 per patient, reducing the effective cost of simvastatin by 88% to $0.28 per day. CONCLUSIONS: In addition to reducing mortality and morbidity in coronary heart disease patients, simvastatin markedly reduces use of hospital services, thus offsetting most of its cost. PMID- 8635257 TI - Missense mutation in the pore region of HERG causes familial long QT syndrome. AB - BACKGROUND: Long QT syndrome (LQT) is an inherited cardiac disorder that results in syncope, seizures, and sudden death. In a family with LQT, we identified a novel mutation in human ether-a-go-go-related gene (HERG), a voltage-gated potassium channel. METHODS AND RESULTS: We used DNA sequence analysis, restriction enzyme digestion analysis, and allele-specific oligonucleotide hybridization to identify the HERG mutation. A single nucleotide substitution of thymidine to guanine (T1961G) changed the coding sense of HERG from isoleucine to arginine (Ile593Arg) in the channel pore region. The mutation was present in all affected family members; the mutation was not present in unaffected family members or in 100 normal, unrelated individuals. CONCLUSIONS: We conclude that the Ile593Arg missense mutation in HERG is the cause of LQT in this family because it segregates with disease, its presence was confirmed in three ways, and it is not found in normal individuals. The Ile593Arg mutation may result in a change in potassium selectivity and permeability leading to a loss of HERG function, thereby resulting in LQT. PMID- 8635259 TI - Utilization of the coronary balloon-expandable coil stent without anticoagulation or intravascular ultrasound. AB - BACKGROUND: The balloon-expandable coil stent has been proved effective in the management of acute and threatened closure after coronary balloon angioplasty and has been shown to reduce restenosis in patients with suboptimal results after coronary balloon angioplasty. Coronary artery stenting has been limited by the occurrence of stent thrombosis and comorbidity related to anticoagulation. This study was undertaken to determine whether anticoagulation may be removed from poststenting protocols, thus reducing comorbidity without increasing stent thrombosis. METHODS AND RESULTS: Between September 1994 and May 1995, 369 patients received balloon-expandable coil stents in native coronary arteries at our institution. Of these patients, 216 were selected for a protocol of aspirin and ticlopidine (for 1 month) without anticoagulation. Eligibility for this protocol followed satisfaction of certain procedural and angiographic criteria. These criteria included adequate coverage of intimal dissections, absence of residual filling defects, and normal (TIMI grade 3) flow in the stented vessel after high-pressure balloon inflations. Intravascular ultrasound was not used to guide stent deployment. The stenting procedure was planned in 37% of patients and unplanned in 63% of patients, including 25 (12%) for acute or threatened closure. During the 30-day follow-up period, stent thrombosis occurred in 2 patients (0.9%), there was 1 death (0.5%), and 2 patients (0.9%) underwent coronary bypass surgery. Vascular access-site complications occurred in 4 patients (1.9%), and bleeding that required blood transfusion occurred in 4 patients (1.9%). CONCLUSIONS: Patients who receive the coronary balloon-expandable coil stent with optimal angiographic results without intravascular ultrasound guidance can be managed safely with a combination of aspirin and ticlopidine without anticoagulation. PMID- 8635261 TI - Atherosclerotic arterial remodeling in the superficial femoral artery. Individual variation in local compensatory enlargement response. AB - BACKGROUND: In previous studies on atherosclerotic arterial remodeling, compensatory enlargement of the artery in response to plaque accumulation was inferred from pooled data based on one cross section per artery. We assessed local arterial remodeling individually by analyzing 45 artery segments at 0.5-cm intervals over a length of 10 to 15 cm. METHODS AND RESULTS: Twenty patients were studied by 30-MHz intravascular ultrasound (IVUS) before balloon angioplasty of the superficial femoral artery (370 cross sections), and 25 femoral artery segments were studied postmortem (551 cross sections). In each cross section, the area surrounded by the internal elastic lamina (IEL area) and the plaque area were measured. The IEL area was larger in the cross section with the largest plaque area than in the cross section with the smallest plaque area (32.5+/-13.0 and 32.0+/-11.5 mm2 versus 28.9+/-9.7 [P=NS] and 26.7+/-10.1 [P<.05] mm2 for IVUS and histology, respectively [mean+/-SD]). A significant positive correlation was found between plaque area and IEL area for the pooled data (r=.61 and r=.47 and slope=1.07 and 0.90 for IVUS and histology, respectively; both P<.001). In 12 of 20 and 16 of 25 individual arterial segments, however, no significant correlation was observed between plaque area and IEL area for IVUS and histology, respectively. A large variation was found in the correlation of the regression of plaque to IEL area (IVUS, r=-.40 to .89; histology, r=-.13 to .91) and slope (IVUS, -0.28 to 1.29; histology, -0.18 to 1.32). CONCLUSIONS: In the majority of atherosclerotic femoral arteries, significant compensatory enlargement could not be determined. It is inferred that arterial remodeling in response to plaque formation may vary among individuals. PMID- 8635260 TI - Insulin sensitivity and atherosclerosis. The Insulin Resistance Atherosclerosis Study (IRAS) Investigators. AB - BACKGROUND: Reduced insulin sensitivity has been proposed as an important risk factor in the development of atherosclerosis. However, insulin sensitivity is related to many other cardiovascular risk factors, including plasma insulin levels, and it is unclear whether an independent role of insulin sensitivity exists. Large epidemiological studies that measure insulin sensitivity directly have not been conducted. METHODS AND RESULTS: The Insulin Resistance Atherosclerosis Study (IRAS) evaluated insulin sensitivity (SI) by the frequently sampled intravenous glucose tolerance test with analysis by the minimal model of Bergman. IRAS measured intimal-medial thickness (IMT) of the carotid artery as an index of atherosclerosis by use of noninvasive B-mode ultrasonography. These measures, as well as factors that may potentially confound or mediate the relationship between insulin sensitivity and atherosclerosis, were available in relation to 398 black, 457 Hispanic, and 542 non-Hispanic white IRAS participants. There was a significant negative association between SI and the IMT of the carotid artery both in Hispanics and in non-Hispanic whites. This effect was reduced but not totally explained by adjustment for traditional cardiovascular disease risk factors, glucose tolerance, measures of adiposity, and fasting insulin levels. There was no association between SI and the IMT of the carotid artery in blacks. The association between SI and the IMT was stronger for the internal carotid artery than for the common carotid artery in all ethnic groups. CONCLUSIONS: Higher levels of insulin sensitivity are associated with less atherosclerosis in Hispanics and non-Hispanic whites but not in blacks. This effect is partially mediated by traditional cardiovascular risk factors. PMID- 8635262 TI - LDL-Apheresis Atherosclerosis Regression Study (LAARS). Effect of aggressive versus conventional lipid lowering treatment on coronary atherosclerosis. AB - BACKGROUND: Intensive lipid lowering may retard the progression of coronary atherosclerosis. LDL-apheresis has the potential to decrease LDL cholesterol to very low levels. To assess the effect of more aggressive lipid lowering with LDL apheresis, we set up a randomized study in men with hypercholesterolemia and severe coronary atherosclerosis. METHODS AND RESULTS: For 2 years, 42 men were treated with either biweekly LDL-apheresis plus medication or medication alone. In both groups a dose of simvistatin of 40 mg per day was administered. Baseline (mean+/-SD) LDL cholesterol was 7.8+/-1.9 mmol x L(-1) and 7.9+/-2.3 mmol x L(-1) in the apheresis and medication groups, respectively. The mean reduction in LDL cholesterol was 63% (to 3.0 mmol x L(-1)) and 47% (to 4.1 mmol x L(-1)), respectively. Primary quantitative coronary angiographic end points were changes in average mean segment diameter and minimal obstruction diameter. No differences between the apheresis and medication groups were found in mean segment diameter ( 0.01+/-0.16 mm versus 0.03+/-0.16 mm, respectively) or in minimal obstruction diameter (0.01+/-0.13 mm versus 0.01+/-0.11 mm, respectively), expressed as means per patient. On the basis of coronary segment, mean percent stenosis of all lesions showed a tendency to decrease; only in the apheresis group more minor lesions disappeared in comparison to the medication group. On bicycle exercise tests, the time to 0.1 mV ST-segment depression increased significantly by 39% and the maximum level of ST depression decreased significantly by 0.07 mV in the apheresis group versus no changes in the medication group. CONCLUSIONS: Two years of lipid lowering both with medication alone or LDL-apheresis with medication showed angiographic arrest of the progression of coronary artery disease. However, more aggressive treatment induced functional improvement, which may precede anatomic changes. PMID- 8635263 TI - Abnormalities in beat-to-beat dynamics of heart rate before the spontaneous onset of life-threatening ventricular tachyarrhythmias in patients with prior myocardial infarction. AB - BACKGROUND: Beat-to-beat analysis of RR intervals can reveal patterns of heart rate dynamics, which are not easily detected by summary measures of heart-rate variability. This study was designed to test the hypothesis that alterations in RR-interval dynamics occur before the spontaneous onset of ventricular tachyarrhythmias (VT). METHODS AND RESULTS: Ambulatory ECG recordings from 15 patients with prior myocardial infarction (MI) who had spontaneous episodes of sustained VT during the recording and VT inducible by programmed electrical stimulation (VT group) were analyzed by plotting each RR interval of a sinus beat as a function of the previous one (Poincare plot). Poincare plots were also generated for 30 post-MI patients who had no history of spontaneous VT events and no inducible VT (MI control subjects) and for 30 age-matched subjects without heart disease (normal control subjects). The MI control subjects and VT group were matched with respect to age and severity of underlying heart disease. All the healthy subjects and MI control subjects showed fan-shaped Poincare plots characterized by an increased next-interval difference for long RR intervals relative to short ones. All the VT patients had abnormal plots: 9 with a complex pattern, 3 ball-shaped, and 3 torpedo-shaped. Quantitative analysis of the Poincare plots showed the SD of the long-term RR-interval variability (SD2) to be smaller in all VT patients (52+/-14 ms; range, 31 to 75 ms) than in MI control subjects (110+/-24 ms; range, 78 to 179 ms, P<.001) or the normal control subjects (123+/-38 ms, P<.001), but the SD of the instantaneous beat-to-beat variability (SD1) did not differ between the groups. The complex plots were caused by periods of alternating sinus intervals, resulting in an increased SD1/SD2 ratio in the VT group. This ratio increased during the 1-hour preceding the onset of 27 spontaneous VT episodes (0.43+/-0.20) compared with the 24-hour average ratio (0.33+/-0.19) (P<.01). CONCLUSIONS: Reduced long-term RR-interval variability, associated with episodes of beta-to-beat sinus alternans, is a highly specific sign of a propensity for spontaneous onset of VT, suggesting that abnormal beat-to-beat heart-rate dynamics may reflect a transient electrical instability favoring the onset of VT in patients conditioned by structurally abnormal hearts. PMID- 8635264 TI - Cycle length dynamics and spatial stability at the onset of postinfarction monomorphic ventricular tachycardias induced in patients and canine preparations. AB - BACKGROUND: The aim of this study was to determine whether cycle length (CL) variations at the onset of monomorphic ventricular tachycardias follow distinctive patterns. METHODS AND RESULTS: We retrospectively analyzed 59 monomorphic ventricular tachycardias induced in 40 patients in whom intraoperative mapping was performed with 63 epicardial and 64 endocardial electrograms recorded simultaneously. Activation times and CL were determined at each electrode site over several beats (36+/-10 beats, mean+/-SD) starting with the first after programmed stimulation. In the majority of the tachycardias, CL variations were accounted for by fitting to an exponential function: CL=CLs+Ae b/tau, where CLs is the stable CL, b is beat number, tau is the time constant (in beat number), and A is the magnitude of CL relaxation. A decelerating trend (with reference to rate) (negative A) accounted for 21 tachycardias, an accelerating trend in rate (positive A) accounted for 12 tachycardias, and 4 others displayed a double dynamic behavior, with an initial acceleration followed by a decelerating trend in rate. Among the ventricular tachycardias that were not fitted to exponential models, 12 showed a constant trend and 10 others showed irregular CL fluctuations. The monomorphic character of the tachycardias was established by principal-component analysis, which also indicated that CL dynamics associated with the accelerating and decelerating trends may be related to shortening and prolongation of activation times, respectively, occurring in equal proportion at all recording sites. In canine preparations in which reentry circuits could be mapped with high resolution, CL showed an accelerating trend in rate when circus movement of excitation occurred around a transmural scar in muscle generating unipolar electrograms with relatively high -dV/dtmax, and a decelerating trend in rate occurred when functional reentry occurred in muscle generating unipolar electrograms with depressed -dV/dtmax. CONCLUSIONS: Beat-to beat CL variations may occur at the onset of sustained monomorphic ventricular tachycardia as a result of uniform acceleration or deceleration of activation times while the overall activation pattern remains constant. The associated initial trends in the rate of sustained monomorphic ventricular tachycardia follow typical patterns that might provide "signatures" corresponding to reentry substrates with distinctive functional properties. PMID- 8635265 TI - Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans. AB - BACKGROUND: Although local inhibition of the generation or actions of endothelin 1 has been shown to cause forearm vasodilatation, the systemic effects of endothelin receptor blockade in healthy humans are unknown. We therefore investigated the cardiovascular effects of a potent peptide endothelin ETA/B receptor antagonist, TAK-044, in healthy men. METHODS AND RESULTS: Two randomized, placebo-controlled, crossover studies were performed. In nine subjects, TAK-044 (10 to 1000 mg IV over a 15-minute period) caused sustained dose-dependent peripheral vasodilatation and hypotension. Four hours after infusion of the highest dose (1000 mg), there were decreases in mean arterial pressure of 18 mm Hg and total peripheral resistance of 665 AU and increases in heart rate of 8 bpm and cardiac index of 0.9 L x min(-1) x m(-2) compared with placebo. TAK-044 caused a rapid, dose-dependent increase in plasma immunoreactive endothelin (from 3.3 to 35.7 pg/mL within 30 minutes after 1000 mg). In a second study in eight subjects, intravenous administration of TAK-044 at doses of 30, 250, and 750 mg also caused peripheral vasodilatation, and all three doses abolished local forearm vasoconstriction to brachial artery infusion of endothelin-1. Brachial artery infusion of TAK-044 caused local forearm vasodilation. CONCLUSIONS: The endothelin ETA/B receptor antagonist TAK-044 decreases peripheral vascular resistance and, to a lesser extent, blood pressure; increases circulating endothelin concentrations; and blocks forearm vasoconstriction to exogenous endothelin-1. These results suggest that endogenous generation of endothelin-1 plays a fundamental physiological role in maintenance of peripheral vascular tone and blood pressure. The vasodilator properties of endothelin receptor antagonists may prove valuable therapeutically. PMID- 8635266 TI - Selective attenuation by N-0861 (N6-endonorboran-2-yl-9-methyladenine) of cardiac A1 adenosine receptor-mediated effects in humans. AB - BACKGROUND: To determine the adenosine receptor subtype selectivity of the novel antagonist N-0861, the A1 and A2 receptor-mediated cardiac effects of adenosine were investigated in 13 patients during continuous intravenous infusion and boluses of adenosine before and after intravenous infusion of N-0861. METHODS AND RESULTS: Measurements of the the atria-to-His (A-H) interval, chest pain severity, and coronary blood flow velocity were made before and after low-dose (69 microg x kg(-1) x min(-1)) intravenous infusion and bolus (2.5 mg) adenosine. Two doses of N-0861 were infused intravenously, and the adenosine protocol was repeated. N-0861 0.25 mg/kg abolished the negative dromotropic effect (A-H interval prolongation) and chest discomfort experienced during infusion of adenosine and attenuated discomfort observed during the boluses of adenosine; however, the increase in coronary blood flow velocity was not significantly affected. CONCLUSIONS: These actions of N-0861 support the concept that the negative dromotropic effect and anginalike pain caused by adenosine are A1 adenosine receptor-mediated, whereas the increase in coronary blood flow velocity is due to activation of A2 adenosine receptors. N-0861 appears to be an effective and selective A1 adenosine receptor antagonist in humans. PMID- 8635267 TI - Echocardiographic quantification of regional left ventricular wall motion with color kinesis. AB - BACKGROUND: Color kinesis is a new technology for the echocardiographic assessment of left ventricular wall motion based on acoustic quantification. This technique automatically detects endocardial motion in real time by using integrated backscatter data to identify pixel transitions from blood to tissue during systole on a frame-by-frame basis. In this study, we evaluated the feasibility and accuracy of quantitative segmental analysis of color kinesis images to provide objective evaluation of regional systolic endocardial motion. METHODS AND RESULTS: Two-dimensional echocardiograms were obtained in the short axis and apical four-chamber views in 20 normal subjects and 40 patients with regional wall motion abnormalities. End-systolic color overlays superimposed on the gray scale images were obtained with color kinesis to color encode left ventricular endocardial motion throughout systole on a frame-by-frame basis. These color-encoded images were divided into segments by use of custom software. In each segment, pixels of different colors were counted and displayed as stacked histograms reflecting the magnitude and timing of regional endocardial excursion. In normal subjects, histograms were found to be highly consistent and reproducible. The patterns of contraction obtained in normal subjects were used as a reference for the objective automated interpretation of regional wall motion abnormalities, defined as deviations from this pattern. The variability in the echocardiographic interpretation of wall motion between two experienced readers was similar to the diagnostic variability between the consensus of the two readers and the automated interpretation. CONCLUSIONS: Color kinesis is a promising new tool that may be used clinically to improve the qualitative and quantitative evaluation of spatial and temporal aspects of global and regional wall motion. In this initial study, segmental analysis of color kinesis images provided accurate, automated, and quantitative diagnosis of regional wall motion abnormalities. PMID- 8635268 TI - Retinoid receptor expression and all-trans retinoic acid-mediated growth inhibition in vascular smooth muscle cells. AB - BACKGROUND: Retinoids have been used in the successful treatment of a variety of human hyperproliferative diseases. Their role in smooth muscle cell (SMC) growth control, however, has not been clearly established. The present study was designed to assess the retinoid receptor mRNA expression profile in SMCs and to determine whether retinoids exert a growth-inhibitory effect in these cells. METHODS AND RESULTS: Five of the six retinoid receptors were expressed in both cultured SMCs and aorta as determined by Northern blotting or reverse transcriptase-polymerase chain reaction. Receptor activity was demonstrated in SMCs with the use of a reporter assay with a retinoid receptor DNA binding sequence linked to a chloramphenicol acetyltransferase reporter gene. DNA synthesis and cell proliferation assays were performed to show that all-trans retinoic acid (atRA) antagonized platelet-derived growth factor-BB and serum stimulated SMC growth. Growth inhibition was distal to early growth-signaling events because induction of c-fos, c-jun, and egr-1 mRNA was unaffected by atRA. However, with an activated protein-1-linked chloramphenicol acetyltransferase reporter, atRA was shown to inhibit the activity of activated protein-1-dependent transcription in a transient transfection assay. CONCLUSIONS: These results establish the presence of functional retinoid receptors in SMCs and document the growth-inhibitory action of atRA on these cells. Retinoid compounds, already in clinical use as antiproliferative agents for nonvascular indications, should be assessed further in in vivo models of intimal disease. PMID- 8635269 TI - Cardiomyocyte troponin T immunoreactivity is modified by cross-linking resulting from intracellular calcium overload. AB - BACKGROUND: During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity. METHODS AND RESULTS: Hearts from adult guinea pigs (n=32) were perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-reperfusion and Ca2+ overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A similar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. CONCLUSIONS: The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium-dependent activation of both calpain proteolysis and transglutaminase cross-linking. PMID- 8635270 TI - Aminoguanidine prevents the decreased myocardial compliance produced by streptozotocin-induced diabetes mellitus in rats. AB - BACKGROUND: A decreased cardiac compliance is a major feature of the cardiomyopathy of diabetes mellitus. Either an increase in the resistance afterload to the LV or an increase in collagen cross-linking induced by the formation of advanced glycosylation end products (AGEs) of collagen may be responsible for the stiff myocardium. To evaluate these hypotheses, we examined the effect of captopril, an afterload-reducing agent, and aminoguanidine, a nucleophilic hydrazine that prevents the accumulation of collagen AGEs, on left ventricular end-diastolic (LVED) compliance after 4 months of streptozotocin (0.26 mmol/kg)-induced diabetes mellitus in rats. METHODS AND RESULTS: Diabetes mellitus produced a decrease in LV chamber compliance as a result of an increased myocardial stiffness (slope of the linearized LVED stress-LVED strain relation [unitless]: diabetes mellitus, 47+/-4; control, 27+/-3; P<.001) and an increase in blood pressure as a result of an elevated vascular resistance. LV end-systolic elastance was unaltered by diabetes mellitus. The stiff myocardium was not associated with changes in the myocardial collagen volume fraction or total hydroxyproline concentration but was associated with an increased myocardial collagen fluorescence (fluorescence units/microg hydroxyproline) (diabetes mellitus, 11+/-1.1; control, 6.6+/-0.7; P<.01). Captopril therapy (0.22 mmol x kg(-1) x d(-1)), despite producing a decrease in blood pressure through alterations in vascular resistance, failed to decrease myocardial stiffness in rats with diabetes mellitus. Alternatively, administration of aminoguanidine (7.35 mmol x kg(-1) x d(-1)) prevented both the enhanced myocardial collagen fluorescence (7.1+/-1.2) and the increased slope of the linearized LVED stress LVED strain relation (29+/-2) but did not change markers of blood glucose control. CONCLUSIONS: These results demonstrate that diabetes mellitus can produce a stiff myocardium before the development of myocardial fibrosis. The stiff myocardium in the early stages of the development of the cardiomyopathy of diabetes mellitus is not a consequence of an increase in ventricular resistance afterload and in these circumstances is associated with the formation of collagen AGEs. PMID- 8635272 TI - Quantification of volumetric coronary blood flow with dual-energy digital subtraction angiography. AB - BACKGROUND: As a solution to the well-documented problems associated with visual interpretation of coronary arteriograms, more physiological methods of assessing coronary artery stenosis are being investigated. Volumetric coronary blood flow (BF) can be a valuable aid in the analysis of functional significance of arterial obstruction. METHODS AND RESULTS: The left anterior descending coronary artery (LAD) of 15 anesthetized pigs (40 to 50 kg) was dissected free from the epicardium in its proximal portion, and a transmit-time ultrasound flow probe of the appropriate size was applied. A vascular occluder was positioned distal to the flow probe for flow adjustments. Contrast injections (2 to 4 mL/s for 3 seconds) were made into the left main coronary artery during image acquisition with a motion-immune dual-energy digital subtraction angiography (DE DSA) system. Tissue-suppressed energy-subtracted images were used to generate time-density curves. BF measurements were made in the LAD vascular bed with use of the time density curve, with consideration that blood was momentarily replaced with contrast during the injection. In 19 comparisons, the mean BF, measured with the use of DE DSA, correlated extremely well with the mean ultrasound flow (DE DSA=0.90 ultrasound+3.10 mL/min, r=.96). Also, contrast injection increased the BF by an average of only 15% during the image-acquisition time interval. CONCLUSIONS: Accurate BF measurements can be made with motion-immune DE DSA. The BF measurements can be completed before the onset of significant changes in BF due to contrast injection. Furthermore, it is possible to make the BF measurements during routine coronary arteriography. PMID- 8635271 TI - Monoclonal antibody against tissue factor shortens tissue plasminogen activator lysis time and prevents reocclusion in a rabbit model of carotid artery thrombosis. AB - BACKGROUND: Tissue factor (TF)-dependent activation of the coagulation is important in the pathophysiology of intravascular thrombus formation. We tested the effects of a monoclonal antibody against TF (AP-1) on lysis time induced by tissue-type plasminogen activator (TPA) and on reocclusion rate in a rabbit model of carotid artery thrombosis. METHODS AND RESULTS: Intravascular thrombosis was obtained by placing an external constrictor around carotid arteries with endothelial injury. Carotid blood flow velocity ws measured continuously with a Doppler flow probe. Thirty minutes after thrombus formation, the rabbits received either AP-1 (0.15 mg/kg IV, n=8) or placebo (n=8). All rabbits also received TPA (80 microg/kg bolus plus 8 microg x kg(-1) x min(-1) infusion for up to 90 minutes or until reperfusion was achieved) and heparin (200 U/kg IV as a bolus). At reperfusion, TPA was discontinued, and the rabbits were followed for an additional 90 minutes. AP-1 shortened lysis time from 44+/-8 minutes (mean+/-SEM) in control rabbits to 26+/-7 minutes in AP-1 rabbits (P<.01). Reocclusion occurred in all control rabbits in 10+/-3 minutes, whereas it occurred in only two of eight AP-1 treated rabbits in 72 and 55 minutes (P<.01). No changes in prothrombin time and ex vivo platelet aggregation in response to various agonists were observed after AP-1 administration, indicating the absence of systemic effects by this antibody. CONCLUSIONS: TF exposure and activation of the extrinsic coagulation pathway play an important role in prolonging lysis time and mediating reocclusion after thrombolysis in this model. AP-1, a monoclonal antibody against TF, might be suitable as adjunctive therapy to TPA. PMID- 8635273 TI - Cardiovascular effects of estrogen and lipid-lowering therapies in postmenopausal women. PMID- 8635274 TI - In memoriam. Cesare Gianturco 1905-1995. PMID- 8635275 TI - Images in cardiovascular medicine. Percutaneous balloon angioplasty with adjunctive stent placement in the mesenteric vessels in a patient with Takayasu's arteritis. PMID- 8635276 TI - Adhesion molecules as targets for the treatment of autoimmune diseases. PMID- 8635277 TI - Characterization of anti-endothelial cell antibodies in the patients with systemic lupus erythematosus: a potential marker for disease activity. AB - To investigate the prevalence and possible role of anti-endothelial cell antibodies (AECA) in the pathogenesis of systemic lupus erythematosus (SLE), cell membrane antigen was prepared from cultured human umbilical vein endothelial cells and immunoblotting performed to detect AECA in SLE sera. IgG-AECA could be detected in 41 (86%) of 47 SLE patients. They were highly specific and failed to react with membrane antigens of human peripheral blood mononuclear cells or granulocytes. IgG-AECA reacted with endothelial membrane antigens which ranged from 15 to 200 kDa in molecular size. Further analysis of the antigens reacting with IgG-AECA revealed some interesting correlations between specific species of antibodies with certain clinical manifestations. Thus, patients having lupus nephritis, vasculitis, and hypocomplementemia had IgG-AECA against a 66-kDa membrane antigen; those with thrombocytopenia had IgG-AECA against a 55-kDa antigen; those with pleuritis had IgG-AECA against an 18-kDa antigen. These results indicate that IgG-AECA in the sera of SLE patients consist of heterogenous species. PMID- 8635278 TI - Complement-fixing islet cell antibodies in type-1 diabetes can trigger the assembly of the terminal complement complex on human islet cells and are potentially cytotoxic. AB - Forty-one sera of patients with IDDM (insulin-dependent diabetes mellitus) containing complement-fixing islet cell antibodies were analyzed for their ability to activate TCC (terminal complement complex). Eighteen sera were found to promote deposition of TCC on human islets of pancreatic cryostat sections with a nonhomogeneous pattern of distribution corresponding to that of insulin. Activation of TCC by IDDM serum and binding of this complex to islet cells was confirmed using purified islets. Flow cytometric analysis of islet cell treated with a TCC+ IDDM serum showed IgG binding to the cell surface. The same serum had a cytotoxic effect on islet cells in the presence of human C. These results obtained with a homologous system of C activation by IDDM sera suggest that TCC may contribute, at least in part, to the pancreatic beta cell damage. PMID- 8635280 TI - The relationship of adhesion molecules and leukocyte infiltration in chronic tubulointerstitial nephritis induced by puromycin aminonucleoside in Wistar rats. AB - We studied the expression of adhesion molecules on infiltrating leukocytes and tubular cells in chronic tubulointerstitial nephritis associated with puromycin aminonucleoside (PA) nephrosis. Rats received injections of PA (2 mg/100 g body wt) weekly for the first 3 weeks and every other week thereafter. Rats were killed at 0, 3, 5, 8, and 12 weeks after the start of injections. From the third to the fifth week, the initial infiltrating cells in interstitial tissue were mainly CD4+ T lymphocytes. At the fifth week, ICAM-1, CD44, and hyaluronate were expressed on infiltrating cells in interstitial tissue. At the eighth week, the number of infiltrating cells reached a peak and consisted of T lymphocytes (CD4, CD8) and macrophages (ED1, MHC class II, CD11b, and CD18). The severity of interstitial infiltration was correlated with the degree of proteinuria and with ICAM-1 expression. Our results suggest that CD4+ T lymphocytes may contribute to the production of initial tubular injury. Expression of ICAM-1 helps mononuclear cells migrate to the interstitium. In addition, expression of CD44 and hyaluronate may play important roles in the chronicity of tubulointerstitial nephritis. PMID- 8635279 TI - The mechanism of inhibitory effect of eicosapentaenoic acid on phagocytic activity and chemotaxis of human neutrophil granulocytes. AB - Free eicosapentaenoic acid (EPA) was found to inhibit dose dependently the chemiluminescence of human neutrophil granulocytes phagocytosing zymosan and their chemotaxis induced by C5a-containing zymosan-activated serum (ZAS) and platelet-activating factor. Rigidification of plasma membranes in the ZAS-treated cells could be observed by measuring the fluorescence anisotropy. The cells were labeled by 3-[p-(6-phenyl-1,3,5-hexatrienoil) phenyl] propionic acid, reporting plasma membrane for determination of membrane fluidity. In resting, nonstimulated neutrophils, EPA dose dependently increased the fluidity of plasma membrane. In zymosan-activated cells, however, after a short fluidization, the basic effect of EPA was a rigidification compared to very low fluorescence anisotropy values of activated control cells. This diminished fluidity, increased membrane stability of plasma membranes can be one of the reasons for the decreased functions (phagocytosis and chemotaxis) of human EPA-treated neutrophils. PMID- 8635281 TI - Investigation of activation markers demonstrates significant overexpression of the secretory component on salivary glands epithelial cells in Sjogren's syndrome. AB - Labial salivary glands biopsies (LSG) performed to support clinical anomalies suggestive of Sjogren's syndrome (SS) sometimes fail to confirm the diagnosis. Here we investigated whether epithelial activation markers could provide further information. Frozen cut sections of LSG from 40 patients, including 23 confirmed SS, were examined in immunofluorescence for the expression of HLA class II molecules, the protector of apoptosis bcl-2, the intercellular adhesion molecule 1 (ICAM-1) and secretory component (SC). Class II molecules were highly expressed on epithelial cells in SS patients (DR > DP > DQ). Bcl2 was expressed in infiltrating cells which were more numerous in the group of SS patients. ICAM-1 was present on endothelial and infiltrating cells of a few patients in both groups. Epithelial cells produced SC in 83% of SS patients samples vs four cases of non-SS patients (P = 0.0002). Investigation of the expression of SC on glandular epithelial cells could therefore be proposed as a marker of SS. PMID- 8635282 TI - Effect of IL-6 on alveolar fibroblast proliferation in interstitial lung diseases. AB - Alveolar macrophage-fibroblast interaction may be involved in the pathogenesis of interstitial lung diseases (ILD). Herein, we compared IL-6 secretion from alveolar macrophages (AM) and alveolar fibroblasts (AFb) recovered from patients with sarcoidosis (SA) and with diffuse interstitial fibrosis (DIF). Moreover, we evaluated the effect of IL-6 on the in vitro AFb proliferation in both diseases. AM and AFb from SA patients showed increased spontaneous secretion of IL-6 compared with cells from DIF subjects. Tumor necrosis factor-alpha (TNFalpha) and interleukin-1 (IL-1) enhanced IL-6 secretion and IL-6 mRNA transcription in AFb of SA patients. Addition of anti-IL-6 MoAbs increased AFb proliferation capacity in SA, but suppressed it in DIF. These results show that only SA AM and AFb secrete high levels of IL-6 which have suppressive effect on AFb proliferation. This may indicate a potential role of IL-6 in the fibrogenesis of ILD. PMID- 8635283 TI - Increased serum levels of IgA antibodies to hsp70 protein in patients with diabetes mellitus: their relationship with vascular complications. AB - We measured class-specific antibodies to the mycobacterial hsp70 protein in 67 patients with diabetes mellitus (27 type 1 and 40 type 2) with or without vascular complications. Using ELISA, the levels of IgG and IgM antibodies in the sera of diabetic patients did not significantly differ either from those of healthy control subjects or between both types of diabetes, regardless of gender, disease duration, HbA1 level, or type of vascular complication. In patients with type 2 diabetes, the mean serum IgA levels were significantly higher than those in their matched controls [274(71) mg/dl vs 208(88) mg/dl; P < 0.01]. In this group of patients, the IgA antibody titer was significantly correlated to the serum IgA level (r = 0.334; P < 0.01). Serological autoimmunity (IgG or IgM type) to hsp70 protein is common in both the normal and the diabetic population. The increased IgA levels and anti-hsp70 IgA titers in the sera of diabetics suggest a possible role of IgA in the pathogenesis of the vascular complications of diabetes mellitus. PMID- 8635285 TI - Clonally expanded CD8 T cells in patients with polymyalgia rheumatica and giant cell arteritis. AB - Giant cell arteritis (GCA) is a vasculitic entity which exclusively, affects individuals older than 50 years of age. Polymyalgia rheumatica (PMR) is a closely related condition which lacks clinically significant vasculitic lesions but shares with GCA the age dependence and the HLA association. To examine whether age-related changes in the T cell receptor repertoire represent a risk factor in these two diseases, we have analyzed the diversity of peripheral blood CD8+ T cells. Untreated PMR/GCA patients carried multiple clonally expanded CD8 populations. The frequency of clonal expansion was not different from age-matched healthy controls. Molecular analysis of the CD8+ clonotypes showed a restricted repertoire in the patients with a distinct Jbeta gene segment usage compared to normal controls. Jbeta2.7+ CD8+ clonotypes were exclusively found in patients. Further evidence for selective CD8 cell expansion came from the finding that multiple clonotypes in the same patient transcribed identical Jbeta segments despite diversity of the Vbeta element. Oligoclonality in the CD8 repertoire persisted despite successful control of the disease activity, suggesting that the CD8+ clonotypes are not an epiphenomenon of the inflammation. We propose that selected CD8+ cells are of functional importance in the pathogenesis of GCA and PMR through a Jbeta-specific mechanism. Age-related changes in the composition of the CD8 T cell receptor repertoire with the emergence of such clonotypes may predispose individuals to develop PMR/GCA. PMID- 8635284 TI - Genetic control of in vivo tumor necrosis factor production in mice. AB - We report on the genetic effect on in vivo production of tumor necrosis factor (TNF)-alpha induced by lipopolysaccharides (LPS) using various congenic mouse strains. B10.A, Bl0.A(3R), B10.AQR, B10.A(5R), and B10.S(7R) produced significantly high TNF-alpha compared with B10.BR, B10.S, C57BL/10, B10.A(2R), B10.A(4R), B10.G, B10.DA(80NS), and B10.RIII(71NS). This suggests that LPS induced TNF-alpha production is genetically controlled by H-2. Mice with the same alleles on K, A, E, or S loci produced various (high or low) levels of TNF-alpha, thus indicating that regulatory genes are located outside these loci. All strains with H-2Dd produced significantly high levels of TNF-alpha, but strains with other alleles in the H-2D locus produced low levels. Thus, TNF-alpha production appears to be genetically linked to H-2D itself or H-2D linked genes and the allele d is linked to a high responder gene. This was the case with the A background. C3H/HeN (H-2k), however, showed a high TNF-alpha production, suggesting the presence of another controlling gene outside H-2. In addition, high TNF-alpha productivity was transmitted into F1 mice (B10.A X B10.BR) in a dominant fashion. Both LPS-stimulated and unstimulated TNF-alpha mRNA expression in splenic macrophages were enhanced in high responder strains. Thus, we conclude that TNF-alpha production is closely related to genes within or linked to the H 2D locus as well as others outside H-2. PMID- 8635286 TI - Apoptosis of T cells in multicentric Castleman's disease. AB - Profound immunodeficiency in multicentric Castleman's disease has already been elucidated. In the present study, we investigated CD45RO and Fas antigen expression and apoptosis by T cells in three patients with this disease. T cell expression of CD45RO and Fas antigen was increased in two of the three patients, indicating in vivo lymphocyte activation. Apoptosis of T cells from the two patients with increased CD45RO and Fas antigen expression occurred after overnight culture in the presence of pokeweed mitogen. Occurrence of apoptosis was indicated by DNA laddering and nuclear chromatin condensation. Peripheral blood mononuclear cells from the two MCD patients revealed spontaneous apoptosis following 3 days of culture by quantitative assay using flow cytometry. These findings show that T cells are activated in some patients with multicentric Castleman's disease and suggest that this activation may promote apoptosis. PMID- 8635287 TI - Superantigen-mediated proliferation and cytotoxicity of T cells isolated from the inflammatory tissues and peripheral blood of arthritis patients. AB - Superantigens are thought to play a role in acute infections and in the pathogenesis of autoimmune diseases that are believed to have an infectious etiology. The effect of the superantigens staphylococcal enterotoxin A, staphylococcal enterotoxin B, and streptococcal M type 5 protein on T cells derived from inflammatory tissues and peripheral blood (PB) of arthritis patients was studied in seven rheumatoid arthritis (RA), two psoriatic arthritis, two reactive arthritis, and one ankylosing spondylitis patient. Superantigen-reactive T cells and T cell lines derived from the PB, synovial fluid (SF), and synovial membrane (SM) of all 12 arthritis tissues recognized the superantigens in an MHC unrestricted manner. Heterogeneities in proliferation and superantigen-directed T cell cytotoxicity were observed in E+ T cells and the T cell lines. Four SF-CD4+ mycobacteria heat-shock protein 65-kDa specific T cell clones generated from an RA patient could recognize and lyse each other when pulsed with staphylococcal enterotoxin A and used as targets. From another RA patient, four SF-CD4+ T cell clones that specifically recognize autoantigens were generated with human IgG fragments or collagen type II fragments. Heterogeneities of such superantigen mediated specific lysis were also demonstrated. The data presented by us suggest a model in which superantigens do not have to be involved in triggering the initial disease because autoreactive T cells elicited by antigen can, in the presence of superantigen, lyse cells that express MHC class II molecules, including activated T cells. PMID- 8635288 TI - Binding of peptides from the N-terminal region of alpha-gliadin to the celiac disease-associated HLA-DQ2 molecule assessed in biochemical and T cell assays. AB - Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong HLA association predominantly to the cis- or trans-encoded HLA-DQ(alpha1*0501, beta1*02) (i.e., DQ2) heterodimer. T cell recognition of gliadin peptides presented by DQ2 in the intestinal mucosa is central in the immunopathogenesis of CD. Here we describe a study where overlapping peptides from the N-terminal region of alpha-gliadin have been tested in biochemical assays for binding to affinity-purified DQ2 and DR3 (i.e., DR(alpha, beta1*0301)) molecules. The peptides were also tested for binding to DQ2 in a functional binding assay, where binding was measured as the capacity to inhibit the stimulation of a gliadin-specific, DQ2-restricted T lymphocyte clone RNnTalpha33. In both assay systems the overlapping gliadin peptides were found to bind with weak or intermediate affinity to DQ2. No or only very weak binding was found to DR3 in the biochemical binding assay. Overall, the results question the role of these peptides in the T-cell-mediated immunopathogenesis of CD. The in vitro assays described here provide new methods for the screening of potentially toxic peptides. PMID- 8635289 TI - Effect of influenza A virus infection on natural and adaptive cellular immunity. AB - Influenza A virus (FLU) is an important pathogen in humans. Although many features of the antiviral immune response have been elucidated in murine and human models of disease, little is known about the role of NK cells, which provide natural, innate immunity. The effects of experimental intranasal FLU (H1N1) inoculation on NK cells and other immune parameters were studied in 18 healthy, adult volunteers during the acute and convalescent phases of infection. Peripheral blood mononuclear cells (PBMNC) were assayed at baseline and on Postinoculation Days 1, 3, 4, 6, 7, 23, and 44. FLU infection and pathophysiologic upper airway responses were documented in all subjects, and there was no mortality. During both the acute (Days 1-3) and the convalescent (Days 23 and 44) stages of the FLU infection, significant increases in NK activity and decreases in the number of activated NK cells were observed. Reductions in the absolute number of T lymphocytes and in PBMNC proliferation to FLU virus antigen and mitogen were also observed. The current investigation extended those findings to include reductions in the number of CD4+ and CD8+ T lymphocytes and increases in the number of activated T lymphocytes. These results document that FLU infection was accompanied by enhancement of natural immunity and, as expected, suppression of most of the other measured parameters of cellular immunity. The normal response to FLU infection in humans may involve sequential modulation of the different components of the cellular immune system. PMID- 8635291 TI - Nonprecipitating anti-La(SS-B) autoantibodies in primary Sjogren's syndrome. AB - Anti-La(SS-B) precipitin-negative sera show a restricted epitope recognition and can be easily overlooked in routine laboratory testing. We have therefore determined the prevalence of nonprecipitating anti-La(SS-B) antibodies in patients with primary Sjogren's syndrome and studied their clinical and immunological associations. Clinical details were obtained from 68 patients with primary Sjogren's syndrome, and serum samples were examined by enzyme-linked immunosorbent assay using purified recombinant La, 60-kDa Ro, and 52-kDa Ro proteins and by counterimmunoelectrophoresis. Thirteen patients (19%) were identified with anti-La antibodies which were nonprecipitating. These patients had similar clinical findings to other groups of patients with Sjogren's syndrome, but had significantly lower rheumatoid factor and serum IgG levels than patients with anti-La precipitins. None of the patients with nonprecipitating anti-La antibodies had previously contained anti-La precipitins in their sera. Furthermore, they tended to have lower levels of antibodies directed against denatured 60-kDa Ro (but not 52-kDa Ro) compared with anti-La precipitin-positive patients. Patients with Sjogren's syndrome associated with nonprecipitating anti La antibodies represent a stable serological and clinical subset in which there appears to be limited diversification of the autoimmune response to the Ro60 and La proteins of the Ro/La ribonucleoprotein. PMID- 8635292 TI - Amniotic fluid embolism. AB - AFE, although rare, remains a significant cause of maternal mortality. Even with improvements in supportive care, the prognosis remains poor for mother and fetus. The guidelines presented may help the physician to recognize this condition when it occurs and give some direction for therapy. PMID- 8635290 TI - Role of cytokines, tyrosine kinase, and protein kinase C on production of superoxide and induction of scavenging enzymes in human leukocytes. AB - We investigated the effects of proximal modulators of cytokines, tyrosine kinase (TK), and protein kinase C (PKC) on reactive oxygen species (ROS) generation and the induction of scavenging enzymes, superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) of human neutrophils and lymphocytes, by using IL1-alpha, TNF-alpha, and IFN-gamma and neutralizing antibodies to these cytokines. Inhibitors of TK (ST638 and herbimycin) or PKC (H-7, calphostin, and staurosporine) were also used. The results revealed that both (O2)- generation stimulated by five different agents (opsonized zymosan, A23187, PAF, PMA, and fMLP) and the inductions of all three scavenging enzymes were potentiated by priming with TNF-alpha. In contrast, both (O2)- generation and enzyme induction were attenuated by priming with IL1-alpha, with the exception of PMA-stimulated (O2)- generation. IFN-gamma decreased (O2)- generation but increased scavenging enzyme induction. Antibodies to all three cytokines and all the TK and PKC inhibitors decreased (O2)- stimulated by most agents, but markedly enhanced (O2)- levels stimulated by PAF. Induction of all three enzymes was enhanced equally by low concentrations of each of the three anticytokine antibodies, while each of the TK or PKC inhibitors decreased induction of SOD and GSH-Px and increased catalase induction. These results suggest that both ROS generation and scavenging enzyme induction are controlled in complex ways by the actions of these three proximal mediators. This supports our hypothesis that disturbances in the regulation of early events of cell activation can lead to oxidative tissue injury. PMID- 8635293 TI - Pneumonia during pregnancy. PMID- 8635294 TI - Tuberculosis in pregnancy: interactions, diagnosis, and management. PMID- 8635295 TI - Acute respiratory failure in pregnancy. AB - Pregnancy increases the risk for respiratory failure from to numerous pulmonary diseases. Adult respiratory distress syndrome, aspiration, venous air embolism, asthma, thromboembolism, and heart disease are etiologies shared by non-pregnant women. However, their management is complicated by complex physiologic changes during pregnancy. Amniotic fluid embolism and tocolytic-induced pulmonary edema are unique to pregnancy and must be added to the list of causes of respiratory failure. Diagnostics and supportive care is difficult and must be directed with the mother and the fetus in mind. This dictates a thorough understanding of maternal physiology, and the safety of drug use during pregnancy. PMID- 8635296 TI - Vaginal ultrasound for the practicing clinician. Equipment selection. AB - This article has essentially been a list of secondary factors for selecting an ultrasound machine. When a clinician narrows his/her choices for a new machine by viewing the image quality and features, the secondary factors in this article also should be studied. By doing so, a number of traps will be avoided and complaints from staff will be decreased. All sonographers and staff should have input to machine selection because all may have an impact on office operations. By anticipating problems before they occur, the selection of a new ultrasound machine becomes a smooth, well defined operation. PMID- 8635297 TI - Scanning techniques in obstetrics and gynecology. AB - Using the appropriate scanning techniques and knowing the advantages and disadvantages of ultrasound equipment, the clinically useful pictures that enable better care of the obstetric and gynecologic patient can be generated. The extra advantage of applying the best possible scanning technique may enable the clinician or the imaging specialist to arrive at the correct diagnosis, despite problems created by anatomic circumstances, such as body habitus. The widespread use of ultrasound in general and transvaginal sonography in particular substantially has changed obstetric and gynecologic practice. High frequency transducer probes enable the creation of a crisp detailed picture that facilitates a more accurate and faster diagnostic procedure. Transvaginal sonography should be considered the first choice laboratory tool in the diagnosis of an increasing number of obstetric and gynecologic entities. PMID- 8635299 TI - The uterus and endometrium. AB - Transvaginal sonography is useful for the identification of physiologic and pathologic changes within the myometrium and endometrium. Endometrial ultrasonography is simple to learn because of the reliable identification of the uterus. It is useful for monitoring fertility treatment, evaluation of abnormal bleeding and monitoring patients in the menopause. PMID- 8635298 TI - Pulmonary pharmacology. AB - This article reviews the basic principles of pharmacodynamics and pharmacokinetics, with a special emphasis on the pharmacologic considerations that must be taken into account when treating the patient with respiratory disease who is also pregnant or nursing the neonate. A description of the four classes of therapeutic agents used for COPD is given with a discussion of the scientific evidence for their safety during pregnancy. The understanding of asthma suggests that bronchodilators relieve the symptoms, while antiinflammatories suppress the disease. Direct administration to the target tissue by inhalation of the bronchodilators (beta-adrenoreceptor agonists and anticholinergics) and immunosuppressors (corticosteroids and cromolyn) leads to low systemic levels of these drugs, which reduces fetal drug exposure. Oral administration of beta-adrenoreceptor agonists, corticosteroids, and theophylline may be necessary to obtain sufficient maternal lung function and ensure adequate oxygenation of the fetus. This must be carefully weighed against the potential fetal and maternal risks involved with increased systemic levels of these drugs. A brief description of classes of drugs used for upper respiratory diseases (antihistamines, alpha-adrenergic agonists, corticosteroids, antitussives, and expectorants) and their safety during pregnancy is also given. There is concern that most alpha-adrenergic agonists increase blood pressure at therapeutic doses needed to relieve nasal congestion. Therefore, for pregnant patients requiring decongestants, opinion favors administration of pseudoephedrine, which has the most favorable therapeutic index, to reduce potential cardiovascular adverse reactions in the fetus. Intranasal administration of the newer corticosteroids, which have limited absorption, is useful for suppression of allergic rhinitis, while minimizing the risk of adverse reactions. The purpose of this article has been to provide pharmacologic/toxicologic information about commonly used respiratory drugs. This will to enable the clinician to make an educated decision regarding the choice of therapy for respiratory disorders to ensure that fetal and maternal outcomes are optimal. PMID- 8635300 TI - Early pregnancy: normal and abnormal. PMID- 8635301 TI - Clinician's approach to ectopic pregnancy. PMID- 8635302 TI - "Can I get pregnant?": the basic infertility workup. PMID- 8635303 TI - The application of transvaginal ultrasound for ovulation induction and in vitro fertilization. PMID- 8635304 TI - Saline infusion sonohysterography. AB - Saline infusion sonography enhances endovaginal ultrasound examination of the uterine cavity. It is easily and rapidly performed at minimal cost. It is well tolerated by patients and virtually devoid of complications. It can prevent further invasive diagnostic procedures in some patients and optimize the preoperative triage process for those patients who will require therapeutic intervention. PMID- 8635305 TI - Current role of ultrasound in ovarian cancer screening. PMID- 8635306 TI - Normal cardiopulmonary physiology during pregnancy. PMID- 8635307 TI - Diagnostic imaging of the lung during pregnancy. PMID- 8635308 TI - Asthma in pregnancy. PMID- 8635309 TI - Pregnancy in patients with cystic fibrosis. AB - With increasing life span of patients with CF, more women with CF are becoming pregnant and others are seeking information about the risks involved during pregnancy and delivery. A striking limitation of the available information is the lack of large prospective studies of pregnant patients with CF matched for age and disease severity compared with their non-pregnant cohorts. A study investigating the effect of pregnancy on morbidity and mortality is being completed by the Cystic Fibrosis Foundation. We recommend that all women with CF be offered contraceptive measures and counseling on the maternal and fetal risks of pregnancy, including the genetic risks for the child. The issue of who will raise the child in the event of subsequent morbidity or maternal mortality should ideally be prospectively discussed. PMID- 8635310 TI - Deep venous thrombosis and pulmonary embolism. PMID- 8635311 TI - Just say no: how are visual searches terminated when there is no target present? AB - How should a visual search task be terminated when no target is found? Such searches could end after a serial search through all items, but blank trials in many tasks are terminated too quickly for that to be plausible. This paper proposes a solution based on Wolfe's (1994) Guided Search model. The probability that each item is a target is computed in parallel based on items' differences from each other and their similarity to the desired target. This probability is expressed as an activation. Activations are examined in decreasing order until the target is found or until an activation threshold is reached. This threshold is set adaptively by the observer--more conservative following misses, more liberal following successful trials. In addition, observers guess on some trials. The probability of a guess increases as trial duration increases. The model successfully explains blank trial performance. Specific predictions are tested by experiments. PMID- 8635312 TI - Infants' metaphysics: the case of numerical identity. AB - Adults conceptualize the world in terms of enduring physical objects. Sortal concepts provide conditions of individuation (establishing the boundaries of objects) and numerical identity (establishing whether an object is the same one as one encountered at some other time). In the adult conceptual system, there are two roughly hierarchical levels of object sortals. Most general is the sortal bounded physical object itself, for which spatiotemporal properties provide the criteria for individuation and identity. More specific sortals, such as dog or car, rely on additional types of properties to provide criteria for individuation and identity. We conjecture that young infants might represent only the general sortal, object, and construct more specific sortals later (the Object-first Hypothesis). This is closely related to Bower's (1974) conjecture that infants use spatiotemporal information to trace identity before they use property information. Five studies using the visual habituation paradigm were conducted to address the Object-first Hypothesis. In these studies, 10-month-old infants were able to use spatiotemporal information but failed to use property/kind information to set up representations of numerically distinct individuals, thus providing empirical evidence for the Object-first Hypothesis. Finally, infants succeed at object individuation in terms of more specific sortals by 12 months. The relation between success at our task and early noun comprehension is discussed. PMID- 8635313 TI - In quest of outcomes: the Larimer Project. AB - This article illustrates Larimer County Mental Health Center's response to the intensifying demand for mental healthcare organizations to develop treatment outcome evaluation systems. It describes the integration of the Target Complaints Assessment (TCA) procedure into the everyday clinical assessment and treatment processes of the organization. The article delineates the development of the procedure's practical application and its usefulness as a feedback mechanism to senior management and line clinicians. PMID- 8635314 TI - Case manager follow-up to failed appointments and subsequent service utilization. AB - Case manager responses to failed appointments were monitored for 83 seriously mentally ill persons in a rural community mental health center. Case manager actions taken were grouped into four categories of follow-up from most intensive to least intensive: home visit, phone call, letter, and no follow-up. On the whole, case managers most frequently did not follow-up missed appointments (56.7%), followed up by letters (21.3%), and telephone calls (18.7%), and home visits (3.3%). Analyses revealed that home visits were most intensive and all clients who were visited following failed appointments did not fail the subsequent appointment. Clients who received telephone calls or letters were about equally likely to fail the subsequent appointment, but were much more likely to attend the subsequent appointment than were clients who received no follow-up to the failed appointment. Interestingly, clients who failed appointments and received no follow-up were much more likely to need emergency services rather than a regular appointment as their next contact with the clinic. PMID- 8635315 TI - Service delivery using consumer staff in a mobile crisis assessment program. AB - Interest has developed in the use of mental health consumers as staff members in community programs for persons with serious mental illness. The present study investigates consumer service delivery in a mobile assessment program designed to assist homeless people with severe psychiatric disorders. Consumer and non consumer staff were generally comparable. Results suggest that consumer staff engaged in more street outreach and were less often dispatched for emergencies. There was a trend for consumer staff to be more likely to certify their clients for psychiatric hospitalization. In sum, consumer staff appear to provide a valuable contribution to this form of service delivery. PMID- 8635316 TI - Perspectives concerning consumers as case managers. AB - Consumer and non-consumer case managers participating in a randomized trial of consumer case management were interviewed regarding their perceptions of consumer and non-consumer case managers, their work and their clients. Consumer case managers were concerned about how they were accepted by other mental health professionals. The consumer team maintained less collateral contact with other professionals and more interpersonal contact with clients than the non-consumer team. Consumer case managers did not show any greater signs of stress, diminished self esteem, or burnout than non-consumer case managers. PMID- 8635317 TI - Consumers as community support providers: issues created by role innovation. AB - Using data from a CSP-funded research demonstration project designed to expand vocational services offered by case management teams serving people with serious mental illness, this paper examines the issues created by employing consumers as peer support specialists for the project. Roles and benefits of these positions are analyzed. Challenges experienced by specialists created by serving peers, the structure of the position, the mental health system and the community, and personal issues are analyzed using data from focus groups and the project's management information system. Implications for consumer role definition, supports for role effectiveness, and the structuring of these types of positions are discussed. PMID- 8635318 TI - What's in a name? Mental health? Behavioral health? PMID- 8635319 TI - The role of self-help programs in the rehabilitation of persons with severe mental illness and substance use disorders. AB - Substance abuse treatment programs in the United States frequently incorporate self-help approaches, but little is known about the use of self-help groups by individuals with dual disorders. This paper brings together several current studies on the role of self-help programs in treating substance use disorders among individuals with severe mental illness. These studies indicate that only a minority of individuals with dual disorders become closely linked to self-help. Psychiatric diagnosis and possibly social skills are correlates of participation. Dually disorders consumers often experience the use of 12-step philosophy and jargon by mental health professionals as alienating and unempathic. The authors propose suggestions for incorporating self-help approaches into the comprehensive community care of individuals with dual disorders. PMID- 8635320 TI - Electron microscopic data for the presence of post-meiotic gene expression in isolated ram sperm chromatin. AB - A whole mount electron microscopic technique facilitated a direct visualization of transcripts in ram spermatozoa in run-on experiments. The localization of transcripts in sperm chromatin by spreading enabled identification of regions where transcription complexes, presumably pre-mRNA species, were seen related to chromatin. In another series of experiments the localization of polymerase II was demonstrated using a specific antibody against the conservative tail domain of the polymerase II molecule, followed by protein A-gold visualization on spread chromatin and on thin sections from mature spermatozoa. Incorporation of bio-UTP in transcripts was visualized by streptavidin-gold on spreading and on thin sections. The data suggest that transcription occurs at the periphery of the mature spermatozoa. PMID- 8635321 TI - Pachytene karyotype analysis of the nematode Meloidogyne spartinae in relation to the genus Meloidogyne. AB - Seven synaptonemal complexes (SC) were present in the pachytene nuclei of Meloidogyne spartinae. The SC was bipartite and consisted of two lateral elements, while lacking a striated central element. This pattern was similar in all Meloidogyne species studied thus far with the exception of M. microtyla. Each SC was attached by only one end to the nuclear envelope while the other end remained unattached and was free in the nucleoplasm. A bouquet formation at pachytene was absent. An average of eight recombination nodules were present on the SC during all stages of pachytene, although the length of SC increased approximately 77%, from an average of 78 to 138 microm from early to mid-late pachytene. In M. spartinae, there was no apparent relationship between SC length and amount of recombination at pachytene. In addition, nuclear volume increased >60% during pachytene while nucleolar volume decreased by approximately 35%. Two nucleolar organizer regions (NOR) were present during mid-pachytene, whereas other stages had only one NOR. This is the first time that two nucleoli have been identified at pachytene in any nematode. They were both the same chromosome yet in reverse order relative to the telomere attached to the nuclear envelope, suggesting that each telomere was competent to interact with the nuclear envelope. PMID- 8635322 TI - Rates of decline in lung function among subjects who restart cigarette smoking. AB - Several studies have demonstrated that smokers who are able to break the habit generally experience reductions in respiratory symptoms and improvement in pulmonary function; however, far less attention has been given to smokers who are unsuccessful in their attempts at quitting. Recent reports have suggested that these subjects (restarters) may have steeper rates of decline in pulmonary function than subjects never attempting to quit smoking. In this study, we compared rates of decline in FEV1 between restarters and subjects who remained current, ex-smokers, or never-smokers throughout the observation period. The results showed that, in both sexes, subjects who attempt to quit the habit and then restart have significantly steeper rates of decline in their FEV1 than subjects who continue smoking uninterrupted. Female restarters also have significantly steeper rates of decline in FEV1 than ex-smokers. These effects were independent of the amount smoked and respiratory diseases. PMID- 8635323 TI - Decreased contents of surfactant proteins A and D in BAL fluids of healthy smokers. AB - Hydrophilic surfactant proteins, surfactant protein A (SP-A) and surfactant protein D (SP-D), have important roles in modulating the host defense functions in the peripheral airways. It has been reported that cigarette smoke may alter the component and function of pulmonary surfactant. In this study, we determined the contents of SP-A and SP-D in BAL fluids of healthy smokers and nonsmokers by enzyme-linked immunosorbent assay using monoclonal antibodies against each protein. The contents of SP-A and SP-D in BAL fluids were significantly (p<0.05) decreased in smokers compared to those in nonsmokers, although there was no significant difference of total phospholipid content between smokers and nonsmokers. These results suggest that the decreased levels of SP-A and SP-D in smokers may impair the host defense functions of surfactant in the peripheral airways and might have a crucial roles in the development of chronic obstructive lung disease. PMID- 8635324 TI - Pulmonary abnormalities and serum immunoglobulins in facsimile machine repair technicians exposed to butyl methacrylate fume. AB - Seven repair technicians (RT, site A) repeatedly exposed to facsimile machine fume developed recurring sore throat, fever, lymphadenopathy, chest tightness, dry cough, and dyspnea. The fume concentration was low (0.6 mg/m3 of breathing zone air) but it contained butyl methacrylate (BMA), a known skin sensitizer. Although chest radiographs were normal, three of the seven RT-A had lung crackles and spirometric abnormalities, and increased serum levels of immunoglobulins IgE or IgM. Symptoms and most other abnormalities improved when exposure to BMA was stopped. We later evaluated workers in two other sites (B and C). Six RT-B had daily contact with BMA fume (0.14 to 0.40 mg/m3 of air) at a field repair depot. Six administrative and six sales staff members (AS-B, SS-B) without regular fume exposure served as controls. All RT-B had elevated serum IgE levels (202+/-69 U/mL [SEM]; normal <41 U/mL). IgE and fume levels were positively correlated (r=0.83). four RT-B had lung crackles, but few symptoms and normal results of spirometry. The crackles cleared 8 weeks after substitution of a BMA-free paper, but IgE levels remained high (201+/-69). The nonexposed AS-B and SS-B had no crackles. Their IgE levels were normal (19+/-4 U/mL [SEM]; p<0.01). The crackles suggest BMA fume might have caused inflammation in terminal airways units. The significance of the IgE elevations is also uncertain since this class of antibodies is usually associated with asthma, not pneumonitis. In view of these uncertainties, BMA was eliminated from the facsimile transceiver process. Follow up of group C workers (n=32) found no symptoms, lung crackles, or abnormal results of spirometry. However, IgE concentrations were elevated in 15 and remained so for 21 months, perhaps because of continuing exposure to residual low levels of BMA. These findings suggest that BMA-bearing facsimile fume caused increased IgE levels in RT at sites A, B, and C, and might have resulted in permanent lung injury if such exposure had continued. PMID- 8635325 TI - Ventilator-associated pneumonia due to Pseudomonas aeruginosa. AB - OBJECTIVE: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa has been associated with higher case fatality rates than VAP caused by other bacterial etiologies. The causes of this excess mortality are unclear. DESIGN: Retrospective review of 38 consecutive ventilated patients with Pseudomonas pneumonia, documented by highly reliable methods. Charts of five additional patients were unavailable for review. SETTING: Medical ICUs of a university affiliated Veterans Affairs Medical Center and a university-affiliated municipal hospital. MEASUREMENTS: Prospectively collected hospital admission acute physiologic and chronic health examination (APACHE) II scores and cause of ICU admission. Retrospectively calculated organ failure and APACHE scores, VAP score. Clinical and microbiologic variables. Antibiotic treatment and outcome. Direct cause of death by standard definitions. RESULTS: Overall mortality was 69% (26/38), significantly higher than the APACHE II predicted mortality of 42.6% (p=0.037). At least 38% (10/26) of deaths were directly attributable to Pseudomonas VAP. Multivariate analysis of factors associated with death found infectious cause for ICU admission (odds ratio [OR]=8.67; 95% confidence interval [CI], 0.86 to 85.94) and number of organ dysfunctions on the day of diagnosis (OR=1.73, 95% CI, 1.02 to 2.92) were significant. Septic shock from Pseudomonas VAP, septic shock from subsequent infection, and multiple organ dysfunction syndrome were the most common immediate causes of death. Mortality increased linearly with increasing APACHE III score on the day of diagnosis. Of initial antibiotic regimens, 67% (26/36) were considered failures. Persistent pneumonia occurred in 35% of patients while recurrent pneumonia was unusual (1/38). CONCLUSIONS: Development of Pseudomonas pneumonia results in a mortality rate in excess of that due to the presenting illness. The attributable mortality determined by several means appears to approach 40%. The excess mortality appears to be related to the host defense response to the pneumonia rather than any characteristic of the pneumonia. Even standard antibiotic regimens fail frequently and do not prevent the excess mortality. Since at least 38% of deaths can be directly attributable to the Pseudomonas pneumonia, improvement in therapy is needed. PMID- 8635326 TI - Efficacy of subcutaneous silver-impregnated cuffs in preventing central venous catheter infections. AB - STUDY OBJECTIVES: To determine the efficacy of an attachable subcutaneous silver impregnated cuff in preventing local central venous catheter (CVC)-related infection and catheter-related sepsis in critically ill surgical patients. DESIGN: A prospective analysis of the use of an attachable subcutaneous silver impregnated cuff compared with a control group in two consecutive time periods. SETTING: Two surgical ICUs at the Queen's Medical Center at the University of Hawaii Surgical Residency Program, Honolulu. PATIENTS: All surgical ICU patients requiring insertion of central catheters. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULT: Two hundred thirty-five CVCs in 154 patients were prospectively evaluated. Silver-impregnated cuffs were used in the first 100 catheters, but none were used in the remaining 135 catheters. The incidence of catheter-related infection in both groups was 15% and 20%, respectively, not statistically significant. Catheter-related sepsis was 3% in both groups. CONCLUSIONS: The use of an attachable subcutaneous silver-impregnated cuff failed to decrease the incidence of CVC-related infection and sepsis. PMID- 8635327 TI - Epidemiology of sepsis and multiple organ dysfunction syndrome in children. AB - STUDY OBJECTIVES: To determine the cumulated incidence and the density of incidence of systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, and multiple organ dysfunction syndrome (MODS) in critically ill children; to distinguish patients with primary from those with secondary MODS. DESIGN: Prospective cohort study. SETTING: Pediatric ICU of a university hospital. PATIENTS: One thousand fifty-eight consecutive hospital admissions. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: SIRS occurred in 82% (n=869) of hospital admissions, 23% (n=245) had sepsis, 4% (n=46) had severe sepsis, 2% (n=25) had septic shock; 16% (n=168) had primary MODS and 2% (n=23) had secondary MODS; 6% (n=68) of the study population died. The pediatric risk of mortality (PRISM) scores on the first day of admission to pediatric ICU were as follows: 3.9 +/- 3.6 (no SIRS), 7.0 +/- 7.0 (SIRS), 9.5 +/- 8.3 (sepsis), 8.8 +/- 7.8 (severe sepsis), 21.8 +/- 15.8 (septic shock); differences among groups (p=0.0001), all orthogonal comparisons, were significant (p<0.05), except for patients with severe sepsis. The observed mortality for the whole study population was also different according to the underlying diagnostic category (p=0.0001; p<0.05 for patients with SIRS and those with septic shock, compared with all groups). Among, patients with MODS, the difference in mortality between groups did not reach significance (p=0.057). Children with secondary MODS had a longer duration of organ dysfunction (p<0.0001), a longer stay in pediatric ICU after MODS diagnosis (p<0.0001), and a higher risk of mortality (odds ratio, 6.5 [2.7 to 15.9], p<0.0001) than patients with primary MODS. CONCLUSIONS: SIRS and sepsis occur frequently in critically ill children. The presence of SIRS, sepsis, or septic shock is associated with a distinct risk of mortality among critically ill children admitted to the pediatric ICU; more data are needed concerning children with MODS. Secondary MODS is much less common than primary MODS, but it is associated with an increased morbidity and mortality; we speculate that distinct pathophysiologic mechanisms are involved in these two conditions. PMID- 8635328 TI - The effects of neuromuscular paralysis on systemic and splanchnic oxygen utilization in mechanically ventilated patients. AB - OBJECTIVE: To evaluate the effect of neuromuscular paralysis on systemic and splanchnic oxygen utilization in patients in respiratory failure during controlled mechanical ventilation. SETTING: A university-affiliated teaching hospital. INTERVENTION: Mechanically ventilated patients, who were undergoing hemodynamics monitoring and who had a gastric intramucosal pH (pHi) of less than 7.35, were studied. Prior to paralysis, the patients were sedated with lorazepam and morphine to standard end points, and the cardiac output and oxygenation were optimized. The patients were then paralyzed with doxacurium and the ventilator rate adjusted to keep the PaCO2 at baseline value. The hemodynamic and oxygenation profile and pHi were determined prior to paralysis and repeated 2 to 2.5 h later. RESULTS: Eight patients were studied; their mean age was 63 +/- 8 years and acute physiology and chronic health evaluation II score was 22 +/- 4. The mean fraction of inspired oxygen, positive end-expiratory pressure, and venous admixture ratio prior to the study was 0.7 +/- 0.14, 11.8 +/- 2.4 cm H2O, and 26 +/- 9%, respectively. Prior to paralysis, the mean set assist controlled ventilation rate was 15 +/- 2 breaths/min and the patient rate was 23 +/- 5 breaths/min. With neuromuscular paralysis, the cardiac index fell from 4.6 +/- 2.2 to 4.3 +/- 2.4 L/min/m2 (p=0.1), the oxygen delivery fell from 537 +/- 129 to 471 +/- 95 mL/min/m2 (p=0.03), and the oxygen consumption and extraction ratio fell from 200 +/ 77 to 149 +/- 35 mL/min/m2 (p=0.03) and 36 +/- 5 to 31 +/- 10, respectively (p=0.2). The pHi increased from 7.21 +/- 0.16 to 7.29 +/- 0.1 (p=0.02). CONCLUSION: In critically ill patients in respiratory failure, neuromuscular paralysis decreased whole body oxygen consumption and increased pHi. Presumably, by eliminating the work of breathing, there is a redistribution of blood flow from the respiratory muscles to the splanchnic and other nonvital vascular beds. PMID- 8635329 TI - Serial pleural fluid analysis in a new experimental model of empyema. AB - Prior attempts to create an animal model of empyema by direct inoculation of bacteria alone into the pleural space have been unsuccessful. The animals either died of overwhelming sepsis or cleared the infection from the pleural space without development of an empyema. We hypothesized that injection of bacteria with a nutrient agar into the pleural space would allow the bacteria to remain in the pleural space for an extended time period, permitting an empyema to develop. The bacterium Pasteurella multocida in brain heart infusion (BHI) agar was injected into the right hemithorax of 12 New Zealand white male rabbits. Our preliminary studies showed that the animals died in less than 7 days if they were not given parenteral antibiotics. For this reason, the rabbits were given penicillin, 200,000 U, IM, every 24 h starting 24 h after bacterial injection. Pleural fluid was sampled by thoracentesis at 12, 24, 48, 72, and 96 h after bacterial injection. Pleural fluid pH, glucose, lactate dehydrogenase (LDH), leukocyte count, and Gram's stain and culture (in one half of the animals) were obtained at each time point. Pleural biopsy specimens were obtained at autopsy after 96 h. The mean pleural fluid pH reached a nadir of 7.01 at 24 h and remained less than 7.1 throughout the experiment. The mean pleural fluid glucose level reached a nadir of 10 mg/dL at 24 h. The mean pleural fluid LDH peaked at 21,000 IU/L at 24 h and the mean pleural fluid leukocyte count peaked at 12 h with a value of 67,000 cells per cubic millimeter. Gram's stains revealed organisms and cultures were positive for growth in all animals at 12 and 24 h. Some animals had positive Gram's stains and growth on cultures up to 72 h after bacterial injection. At autopsy, all rabbits injected with bacteria had gross pus in the right pleural space and had developed a thick pleural peel. Microscopic specimens of the pleura revealed large numbers of leukocytes (primarily polymorphonuclear lymphocytes) with invasion of the adjacent lung and chest wall. In conclusion, this model more closely mimics the empyema that occurs in humans, relative to previous animal models. This model appears appropriate for additional randomized studies in which different methods for the treatment of empyema can be evaluated. PMID- 8635330 TI - Continuous fiberoptic arterial and venous blood gas monitoring in hemorrhagic shock. AB - STUDY OBJECTIVE: To compare the performance of continuous fiberoptic blood gas monitoring with standard, intermittent blood gas sampling in the measurement of arterial and central venous blood gases during marked hemodynamic changes. DESIGN: Prospective, consecutive, enrollment, experimental study. SETTING: Research laboratory at a university medical center. PARTICIPANTS: Seven anesthetized, mechanically ventilated pigs. INTERVENTIONS: Severe shock was induced by hemorrhage in pigs monitored by a pulmonary artery catheter, an arterial line, and two fiberoptic blood gas sensors: one intra-arterial, and the other inserted into the superior vena cava via right internal jugular vein cutdown. Fiberoptic blood gas monitor measurements were compared with standard intermittent blood gas sampling. MEASUREMENTS AND RESULTS: A total of 184 blood gas samples were compared in seven animals at baseline, during shock, and after resuscitation. The baseline mean (+/- 1 SD) cardiac output decreased from 4.0 +/- 0.9 to 1.2 +/- 0.6 L/min during shock and returned to baseline after retransfusion (3.9 +/- 1.3 L/min). The comparison of continuous fiberoptic blood gas monitoring with intermittent blood gas sampling showed a bias+/-precision of 0.035 +/- 0.047 for arterial pH, 0.021 +/- 0.031 for central venous pH, -4.09 +/- 2.96 mm Hg (-0.55 +/- 0.39 kPa) for arterial Pco2, -3.67 +/- 2.44 mm Hg (-0.49 +/ 0.3 3 kPa) for central venous Pco2, -5.79 +/- 9.64 mm Hg (-0.77 +/- 1.29 kPa) for arterial Po2, and -7.85 +/- 8.52 mm Hg (-1.05 +/- 1.14 kPa) for central venous Po2. CONCLUSIONS: Continuous fiberoptic blood gas monitoring agrees closely with standard intermittent blood gas sampling during severe hemodynamic shifts and has a comparable accuracy for both arterial and venous blood gas measurements. Changes in venous Pco2 have recently been shown to correlate with changes in global tissue perfusion (eg, changes in cardiac output). Such data, available immediately via continuous venous blood gas monitoring, may be useful for monitoring shock and the response to resuscitation. PMID- 8635331 TI - Goals of asthma management. A step-care approach. AB - The past 15 years have seen a rise in mortality and morbidity resulting from asthma, despite a concurrent rise in general knowledge about the disease. The step-care strategy recognized these changes in its approach to asthma management; however, this approach should be used only with attempts to control environmental allergens. Step-care therapy requires that patients be categorized by the severity of illness. Step-one therapy is used for mild, infrequent symptoms and involves treatment based primarily on inhaled bronchodilators. Step-two therapy is instituted in all asthmatics except the mildest cases; it involves treatment by inhaled corticosteroids, cromolyn, or nedocromil. Step-three treatment targets cases of severe asthma through the use of oral corticosteroids. In all phases of treatment, however, it should be remembered that patient education is of critical importance. Education improves patient compliance and is critical to the successful treatment of asthma. PMID- 8635332 TI - Pulmonary complications of bone marrow transplantation. PMID- 8635333 TI - Cross-shift changes in blood inflammatory markers occur in the absence of airway obstruction in workers exposed to grain dust. AB - Grain dust is well known to cause both acute and chronic respiratory disorders, and endotoxins are considered key components in this. Since endotoxins are known to elicit proinflammatory mediators, we investigated cytokine (tumor necrosis factor [TNF], interleukin-6, interleukin-8) release and a number of proinflammatory and anti-inflammatory proteins (soluble TNF receptors, lipopolysaccharide (LPS) binding protein, bactericidal permeability increasing protein (BPI), C-reactive protein) in plasma of workers exposed to grain dust. In two surveys during 1 week, lung function was measured daily before and after the shift, using flow-volume curves and/or forced oscillation measurements. On Monday and Friday, blood samples (30 mL) were drawn and cytokine release was determined by enzyme-linked immunosorbent assay in supernatant of isolated monocytes or whole blood culture, either unstimulated or on the ex vivo stimulation with 3 ng/mL or 1,000 ng/mL endotoxin. Individual exposures were determined from stationary dust measurements at every workplace combined with personal task analysis during all shifts. In both surveys, no cross-week change in lung function parameters was observed. In the first survey (average exposure: 20.2 mg/m3), monocyte spontaneous TNF release was increased sevenfold cross week (p<0.001) and was significantly related both to individual dust exposure (r=0.62) of that week and the increase in soluble TNF receptor 75 kD (r=0.85). In the second survey, where average exposure was much lower (3.67 mg/m3), impedance parameters indicated a significant improvement of airway function, and cross-week changes in inflammatory markers were minimal. Therefore, we conclude that inflammatory events can be used to monitor adverse respiratory effects of moderate grain dust exposure. PMID- 8635336 TI - Closure of a postpneumonectomy main bronchus fistula using video-assisted mediastinal surgery. AB - Disruption of a mainstem bronchus is a rare but dreaded complication of pneumonectomy. After an anatomic study on cadavers, we successfully used a video mediastinoscope to close a postpneumonectomy main bronchus fistula via a cervical incision. PMID- 8635335 TI - Management of pleural effusion of cirrhotic origin. AB - STUDY OBJECTIVE: To determine the indications and limitations of surgical videothoracoscopy for management of pleural effusion, an infrequent and often recurring complication of cirrhotic ascites whose pathogenesis involves direct passage of ascitic fluid into the pleural space through minute defects in the diaphragm. DESIGN/SETTING/PATIENTS/INTERVENTIONS: Eight cirrhotic patients with ascites and recurrent pleural effusion underwent surgical videothoracoscopy to localize and close any diaphragmatic defects and to achieve pleurodesis by application of talc. MEASUREMENTS AND RESULTS: Diaphragmatic defects were localized and closed in six patients; postoperative mean volume and duration of drainage were, respectively, 0.408 +/- 0.157 mL and 7.6 +/- 1.75 days. None of these six patients developed recurrent pleural effusion (follow-up, 7 to 36 months). In the 2 patients in whom no defect was found, drainage had to be maintained for 15 days and 18 days (drainage volumes, 3 and 4 L). At hospital discharge, both patients had a stable recurrent effusion occupying the lower third of the cavity. CONCLUSIONS: Utilization of videothoracoscopy appears particularly indicated for these fragile patients when medical therapy fails. The procedure's efficacy is immediate and durable once defects are identified and closed. If the technique proves unsuccessful, it does not hinder subsequent use of other methods. PMID- 8635334 TI - Nasal lavage cellularity, grain dust, and airflow obstruction. AB - To evaluate the clinical utility of nasal lavage (NL), we performed post-work shift NL on 172 grain workers and 78 postal worker control subjects. The grain worker group included a higher percentage of current smokers (25.7% vs 16.7%) and a lower percentage of former smokers (21.15% vs 35.9%) compared with the postal workers. The control subjects included more female workers and were slightly older than the grain workers. Compared with the postal workers, the grain workers were exposed to significantly greater concentrations of total dust (0.1 +/- 0.0 vs 6.8 +/- 1.4 mg/m3; mean +/- SEM) and total endotoxin (4.3 +/- 0.8 vs 2,372.4 +/- 653.8 endotoxin units/m3). NL from gain workers showed a higher concentration of total cells (55,000 +/- 14,000 vs 25,000 +/- 5,000 cells per milliliter; p=0.03), a higher concentration of squamous epithelial cells (17,029.0 +/- 4,177 .0 vs 7,103.7 +/- 1,479.8 cells per milliliter; p=0.03), and a higher concentration of neutrophils (40,058.0 +/- 12,803.2 vs 17,891.0 +/- 3,822.3 cells per milliliter; p=0.10) compared with postal workers. Importantly, these differences in NL cellularity between grain workers and postal workers were observed within the three strata of smokers. To further assess the importance of total cells, squamous epithelial cells, and neutrophils in the NL fluid of grain workers, we investigated the relationship between these cell concentrations and (1) measures of dust and endotoxin exposure during the work shift. (2) spirometric measures of airflow obtained immediately before the NL, and (3) work related respiratory symptoms. The concentration of total cells, the concentration of squamous epithelial cells, or the concentration of neutrophils in the NL was not associated with ambient levels of dust or endotoxin, with baseline or cross shift changes in lung function, or with work-related respiratory symptoms. These findings suggest that increased NL cellularity may be seen in workers exposed to high dust levels. However, the NL cellularity does not appear to be associated with ambient concentrations of dusts or endotoxins, with signs of airflow obstruction, or with work-related respiratory symptoms. PMID- 8635337 TI - Routine video-assisted thoracoscopy prior to thoracotomy. AB - We investigated the role of routine video-assisted thoracoscopy (VAT) prior to thoracotomy. From June 1993 to May 1995, we routinely performed VAT prior to all our elective thoracotomies in adults. Patients who planned to have video-assisted thoracic surgery (VATS), those who underwent emergency thoracotomy, and patients younger than 10 years old were excluded from this study. There were 63 patients (47 men and 16 women; age range, 16 to 84 years), of whom 39 (62%) had malignant disease and 24 (38%) had benign disease. In four cases, VAT could not be performed because of either pleural symphysis or inability to adequately collapse the upper lung. In six cases, thoracoscopic findings influenced subsequent management. Pleural metastases were found in two cases that led to abandonment of thoracotomy; in four cases, identification of chest wall involvement by a malignant or benign process led to proper planning of subsequent thoracotomy. There was no added morbidity from this procedure which took, on average, 6.2 min to complete (range, 3 to 17 min). There was no added cost for consumables. We concluded that: (1) routine VAT is a safe procedure; (2) it adds little to the overall cost or operating time; (3) it can provide useful information that could alter subsequent operative strategy. We recommend routine VAT prior to thoracotomy in patients with known or suspected intrathoracic malignancy and those suspected of having chest wall involvement on CT scans. PMID- 8635338 TI - Refractory hypoxemia in a morbidly obese 28-year-old woman. PMID- 8635339 TI - Late extrusion of pulmonary plombage. PMID- 8635340 TI - Left atrial myxoma and acute myocardial infarction. A dangerous duo in the thrombolytic agent era. AB - Systemic embolization is a common complication of left atrial myxoma; however, coronary embolism leading to acute myocardial infarction is rare. The use of echocardiography has increased the detection of intracardiac tumors when signs and symptoms are not evident. Echocardiography is the diagnostic procedure of choice in the initial evaluation of patients with suspected left atrial myxoma. PMID- 8635341 TI - Actinomyces odontolyticus thoracopulmonary infections. Two cases in lung and heart-lung transplant recipients and a review of the literature. AB - We present the first case of mediastinitis and the third case of pneumonia attributed to Actinomyces odontolyticus. The first patient presented 10 months after single-lung transplant with a subacute apical infiltrate in the native lung and responded to therapy with oral penicillin. The second patient developed pyogenic mediastinitis 25 days after a heart-lung transplant and required sternal debridement and intravenous penicillin. We also review the literature on thoracopulmonary infections due to A odontolyticus. PMID- 8635342 TI - Pulmonary embolism with duplicated inferior vena cava. AB - Congenital anomalies of the inferior vena cava (IVC) are common and usually of little clinical significance. We report the unusual cases of two patients with pulmonary embolism from thrombosis of part of a duplicated IVC. Both caval anomalies were easily identified with ultrasound, and the patients were successfully treated with anticoagulants. We encourage the reporting of similar cases since knowledge of the contribution of IVC duplication to thromboembolic disease is unknown. PMID- 8635343 TI - Pulmonary edema in a woman following fetal surgery. AB - Most cases of acute lung injury in pregnancy are attributed to hydrostatic pulmonary edema. In this report, however, we describe a 20-year-old pregnant woman who developed a unique case of increased permeability pulmonary edema following surgery for the repair of a fetal congenital diaphragmatic hernia. Two days after surgery, the patient developed acute respiratory failure and diffuse alveolar edema, requiring intubation and positive pressure ventilation for 5 days. The diagnosis of increased permeability pulmonary edema was confirmed by the ratio of pulmonary edema fluid to plasma protein (ratio=0.99). The patient received IV nitroglycerine for tocolysis. As a nitric oxide donor, the nitroglycerine may have combined with exogenous oxygen to form peroxynitrite, a known impediment to alveolar epithelial cell function. Many cases of pulmonary edema in pregnancy are diagnosed as hydrostatic based on clinical parameters, such as positive maternal fluid balance. In this case, these parameters would have been misleading. Measurement of the protein concentration in the pulmonary edema fluid allowed us to accurately determine that the patient had increased permeability pulmonary edema as the cause of her acute respiratory failure. Sampling of pulmonary fluid can differentiate the type of edema formation and in some cases help to identify mechanisms of acute lung injury. PMID- 8635345 TI - Transmission of invasive aspergillosis from a subclinically infected donor to three different organ transplant recipients. AB - OBJECTIVE: To describe a cluster of donor-transmitted cases of invasive aspergillosis. DESIGN: Case series of epidemiologically linked cases of invasive aspergillosis. SETTING: Two tertiary care centers with solid-organ transplant programs. PATIENTS: Two kidney recipients, one heart recipient, and the single donor. MEASUREMENTS: Routine clinical, microbiological, and pathologic investigation as dictated for patient care. Epidemiologic analysis to establish linkage among cases. RESULTS: Three allografts (two kidneys and a heart) from a single donor transmitted invasive aspergillosis to the recipients. Three weeks after transplantation, the two kidney recipients had fever and urine cultures positive for Aspergillus fumigatus. The infected kidneys had multiple Aspergillus abscesses and had to be removed to cure the patients. The heart recipient had a negative workup when a diagnosis of aspergillosis was made for the kidney recipients but presented three months later with aspergillus endocarditis with hematogenous spread to the eyes and to the skin. Treatment included eye surgery, aortic valve replacement, and antifungal therapy; control of infection ensued. The donor was intensely immunosuppressed (17 days post-liver transplantation with death from intracerebral bleeding) but had no clinical or autopsy evidence of aspergillosis. Donor tracheal secretions obtained at the time of organ harvest later grew A fumigatus. CONCLUSION: Expanded criteria for organ donation have to be balanced against infectious risk to organ recipients. A fumigatus can be transmitted from a subclinically infected donor to solid-organ transplant recipients. PMID- 8635344 TI - Traumatic aortic incompetence associated with transection of the thoracic aorta. AB - A case of blunt chest trauma resulting in transection of the descending thoracic aorta and disruption of the aortic valve is presented. Successful treatment required graft repair of the aortic injury followed by aortic valve replacement. PMID- 8635346 TI - Noninvasive diagnosis and treatment of a saddle pulmonary embolism. A case report in support of new trends in management of pulmonary embolism. AB - Transesophageal echocardiography and contrast-enhanced spiral CT of the chest helped to avoid a pulmonary angiography in an elderly patient with saddle pulmonary thromboembolism and allowed for direct evaluation of its resolution during treatment with subcutaneous low molecular weight heparin. PMID- 8635347 TI - Barotrauma vs volutrauma. PMID- 8635348 TI - Propofol for ICU sedation. PMID- 8635349 TI - Preflight evaluation. Patients and methods. PMID- 8635350 TI - Volume reduction surgery. How selective should we be? PMID- 8635351 TI - Treat patients who have nocturnal asthma with anti-inflammatory drugs first. PMID- 8635353 TI - Facilitation of percutaneous dilational tracheostomy by use of a perforated endotracheal tube exchanger. PMID- 8635352 TI - Consider anti-CMV therapy. PMID- 8635354 TI - Check out this CT! PMID- 8635355 TI - Not too conservative treatment of over anticoagulated patients. PMID- 8635356 TI - Posttransplant bronchiolitis obliterans syndrome. Where have we been and where are we going? PMID- 8635357 TI - Snoring in pregnancy. Disease or not? PMID- 8635358 TI - Hypertension and OSA. Silent bedpartners? PMID- 8635359 TI - Long-acting beta-agonists, tachyphylaxis, and corticosteroids. PMID- 8635360 TI - Is there a standard treatment for locally advanced non-small cell lung cancer? PMID- 8635361 TI - Lung reduction surgery. Where are we heading? PMID- 8635362 TI - Delayed development of obliterative bronchiolitis syndrome with OKT3 after unilateral lung transplantation. A plea for multicenter immunosuppressive trials. AB - There is no consensus regarding the optimal induction immunosuppression regimen after lung transplantation (LT). In addition to the potential benefit of a reduced incidence of early acute allograft rejection, cytolytic induction immunosuppression may impact on long-term allograft function. We retrospectively assessed our incidence of obliterative bronchiolitis syndrome (OBS) stages Ia and IIa in LT survivors given two different cytolytic induction immunosuppression regimens: (between March 1989 and October 1990) OKT3 (5 mg/d)x10 to 14 days (n=11) vs (between November 1990 and April 1993) Minnesota antilymphocyte globulin (MALG) (10 to 15 mg/kgdx5 to 7 days. Cyclosporine (CSA) (whole blood polyclonal assay=600 to 800 ng/mL), azathioprine (1 to 2 mg/kg/d), and maintenance prednisone (0.2 mg/kg/d) were similar. Surveillance spirometry was performed monthly, in accordance with accepted American Thoracic Society criteria. Fiberoptic bronchoscopy with transbronchial biopsies (TBBs) were performed for clinical indications. Surveillance TBBs were not performed during the era of this study. As defined by the ISHLT "Working Formulation for the Standardization of Nomenclature and for Clinical Staging of Chronic Dysfunction in Lung Allografts," latencies to development of OBS stages Ia and IIa were determined by Kaplan-Meir analysis. Stepwise regression (Cox proportional hazards model) was performed for the variables: cytolytic induction regimen, episodes cytomegalovirus (CMV) pneumonitis, episodes CMV infection, serologic CMV donor (+): recipient (-) mismatch, prior pregnancy, HLA (A,B,DR +/- DQ) mismatches, episodes greater than grade A1 acute cellular rejection (ACR). We found that the OKT3 cohort experienced longer latencies for OBS stages Ia and IIa. Latencies to OBS stages Ia for OKT3 ve MALG were 962 +/- 65 vs 354 +/- 85 days (X +/- SEM) respectively. Brookmeyer-Crowley 95% confidence intervals for median latencies were 744 to 1,180 vs 266 to 510 days for OKT3 vs MALG, respectively. The Cox model was significant only for the variable of the induction cytolytic immunosuppression regimen (p=0.0015). By physiologic criteria, a longer course of OKT3 appeared superior to the short-course MALG protocol in delaying chronic lung allograft dysfunction. These effects may be related either to inherent differences in the antilymphocyte preparations or, alternatively, the difference in duration of treatment between groups. Surveillance TBB and treatment of detected occult ACR may serve to negate the observed differences in latencies for OBS. PMID- 8635363 TI - Cytolytic therapy for the bronchiolitis obliterans syndrome complicating lung transplantation. AB - The bronchiolitis obliterans syndrome (BOS) is the major cause of late morbidity and mortality after lung transplant (LTx). Previous studies suggest cytolytic therapy may be effective for the BOS but this therapy has not been proved effective or safe. METHOD: A retrospective study of a predetermined treatment regimen to determine if the rate of fall in FEV1 can be reduced by corticosteroids and cytolytic therapy. Since August 1992, 10 to 65 long-term survivors of LTx (5 men, 5 women; mean age 36 +/- 10 years) developed BOS. All had previously had lymphocyte immune globulin, antithymocyte globulin (equine) (ATGAM sterile solution; Upjohn Pty Ltd; Sydney, Australia) induction therapy and corticosteroid avoidance for the first 7 to 10 days post-LTx. Therapy for the BOS was initiated with pulse methylprednisolone and ATGAM (aiming for an absolute CD3 count of < or - 100 cells per microliter for 5 days). ATGAM therapy was initiated at a mean 657 +/- 323 days post-LTx. Subsequent follow-up has been for 310 +/- 110 days (range, 163 to 530 days). RESULTS: Nine of ten patients had a response with tolerable side effects. Preintervention, there was a linear fall in FEV1 of 0.22 +/- 0.15% predicted FEV1 per day (mean +/- SD) (range, 0.06 to 0.56%) compared with a postintervention linear fall of 0.036 +/- 0.019% predicted per day (range, 0 to 0.13%) (paired t test; p<0.005). This effect is sustained over the follow-up period. CONCLUSION: The fall off in FEV1 that characterizes the BOS may be altered usefully by augmented immunotherapy. This effect can be rapid and sustained although it is neither completely arrested nor ever reversed. These data are preliminary but encourage a randomized control trial in the BOS. PMID- 8635364 TI - Selective assessment of single-lung graft function with 133Xe radiospirometry in acute rejection and infection. AB - In single-lung transplant recipients, the usefulness of spirometric indexes in detecting acute events involving the lung graft is limited due to the bias caused by the native lung. Selective functional monitoring is needed for the proper evaluation of complications after transplantation, but thus far, to our knowledge, no clinically feasible methods for selective graft-function assessment have been presented. In ten single-lung recipients, of whom six had a parenchymal lung disease and four had pulmonary hypertension, the relative ventilation (Vtx), perfusion (Qtx), and ventilation/perfusion ratio of the transplanted lung (V/Qtx) were determined with multidetector 133Xe radiospirometry. Additionally, the fractions of FEV1, FVC, and diffusing capacity for carbon monoxide (Dco) of the transplant (FEV1tx, FVCtx, Dcotx, respectively) were determined by using corresponding radiospirometric parameters for the calculation of their distribution between the lungs. The analysis included seven episodes of acute rejection and nine episodes of infection. The Qtx decreased during acute rejection but did not change during infection (p=0.001). Compared with the figures during infection, the V/Qtx increased during acute rejection significantly (p<0.05) in patients with underlying fibrosis or emphysema, but not in those with pulmonary hypertension. In detection of acute events, the sensitivity of the selective parameters, ie, FEV1tx (86%) and FVCtx (73%), was higher than that of the sum-function parameters, FEV1 (66%) and FVC (40%). Moreover, the sensitivity of Dcotx (80%) was higher than that of Dco (60%) in detecting acute rejection. The findings indicate that, in single-lung recipients with a parenchymal lung disease, the assessment of Qtx, V/Qtx, and Dcotx with a radioactive tracer can help to distinguish acute rejection from infection. The graft-selective parameters, ie, FEV1tx, FVCtx, and Dcotx, tended to be more sensitive than the corresponding sum-function parameters in detecting acute events, thus providing a more accurate functional profile of the single-lung graft. PMID- 8635365 TI - Self-reported snoring in pregnancy. Association with fetal outcome. AB - OBJECTIVES: To determine the incidence of self-reported snoring in pregnant compared with nonpregnant women. To compare indicators of fetal outcome in pregnant women with self-reported frequent snoring vs those without snoring. STUDY DESIGN: Prospective, nonrandomized screening and comparison between groups. PATIENTS: Three hundred fifty pregnant women and 110 age-matched nonpregnant women. METHODS: Survey evaluating self-reported snoring. For the pregnant women, infant birthweight, APGAR scores, and other indicators of fetal outcome were obtained by record review. RESULTS: Frequent snoring was reported in 14% of the pregnant women vs 4% of the nonpregnant women (Chi2=6.2; df=1; p<0.05). The pregnant women who reported frequent snoring did not have deliveries resulting in infants with evidence of an increase in compromised outcomes. CONCLUSIONS: Frequent snoring is reported more often in pregnant than in nonpregnant women. Snoring mothers do not appear to be at increased risk for delivering infants with fetal compromise as might be expected with the concomitant occurrence of obstructive sleep apnea. PMID- 8635366 TI - The role of sleep-disordered breathing in essential hypertension. AB - In recent years there have been numerous reports addressing the relationship between sleep-disordered breathing (SDB) and hypertension (HTN). This study investigated the relationship between SDB and BP after controlling for age, gross obesity, and notably, antihypertensive medications. Sixty-seven men and women between 30 and 60 years of age and between 0.90 to 1.5 times ideal body weight were studied. SDB was assessed over two nights of polysomnographic monitoring, and BP was measured over repeated visits to the hospital. The results indicate that respiratory disturbance index (RDI) independently predicts diastolic BP (DBP), accounting for 15% of the variance in DBP (p=0.02). In subjects with severe levels of SDB (RDI >30), RDI uniquely accounted for 36% of the variance in DBP (p=0.003). Interestingly, SDB was not independently related to systolic BP. The physiologic mechanisms responsible for these findings are currently being explored. PMID- 8635367 TI - Sleep fragmentation as a risk factor for hypertension in middle-aged nonapneic snorers. AB - Although a high prevalence of hypertension has been observed in snorers, whether there is a direct link between hypertension and snoring remains controversial. It has recently been demonstrated that an abnormal amount of breathing effort during snoring is responsible for sleep fragmentation even in the absence of sleep apnea syndrome criteria. We hypothesized that sleep fragmentation during snoring may be a direct risk factor for the development of hypertension. On the basis of polysomnographic data, 105 nonapneic patients between 40 and 65 years of age referred for snoring with social impairment were selected and categorized as snorers with (n=55) or without sleep fragmentation (n=50) based on whether the arousals index was 10 or greater or less than 10/h of sleep, respectively. Sleep distribution did not differ between the two groups, except for a longer duration of wake after sleep onset (58 +/- 43 min vs 42 +/- 38 min) and a shorter duration of slow-wave sleep in the group with sleep fragmentation (72 +/- 34 min vs 97 +/- 34 min). Although there were no statistically significant differences between the snorers with and without sleep disruption in terms of age (51.3 +/- 7.7 vs 48.6 +/- 6.0 years), body mass index (26.9 +/- 4.0 vs 27.2 +/- 5.5 kg/m2), sex ratio, respiratory indexes during sleep, daytime sleepiness, and daytime tiredness, prevalence of systemic hypertension was significantly higher in the sleep fragmented group (20/55 vs 7/50). This significant difference persisted (16/51 vs 6/49) when patients using antihypertensive drugs with possible effects on the CNS were excluded. Our data suggest that sleep fragmentation is common in patients who seek medical help for snoring with social impairment and may play a role in the development of hypertension. PMID- 8635368 TI - Upper airway resistance syndrome, nocturnal blood pressure monitoring, and borderline hypertension. AB - Upper airway resistance syndrome (UARS) is a sleep-disordered breathing syndrome characterized by complaints of daytime fatigue and/or sleepiness, increased upper airway resistance during sleep, frequent transient arousals, and no significant hypoxemia. Of a population of 110 subjects (58 men) diagnosed as having UARS, we investigated acute systolic and diastolic BP changes seen during sleep in two different samples. First, six patients from the original subject pool were found to have untreated chronic borderline high BP, and were subjected to 48 h of continuous ambulatory BP monitoring before treatment and another 48 h of BP monitoring 1 month after the start of nasal-continuous positive airway pressure (N-CPAP) treatment. Five of six subjects used their equipment on a regular basis and had their chronic borderline high BP completely controlled. No change in BP values was seen in the last subject, who discontinued N-CPAP after 3 days. A second protocol investigated seven normotensive subjects drawn from the initial subject pool. Continuous radial artery BP recording was performed during nocturnal sleep with simultaneous polygraphic recording of sleep/wake variables and respiration. BP changes were studied during periods of increased respiratory efforts and at the time of alpha EEG arousals. Increases in systolic and diastolic BP were noted during the breaths with the greatest inspiratory efforts without significant hypoxemia. A further increase in BP was noted in association with arousals. Three of these subjects also underwent echocardiography during sleep, which demonstrated a leftward shift of the interventricular septum with pulsus paradoxus in association with peak end-inspiratory esophageal pressure more negative than -35 cm H2O. Our study indicates that, in the absence of classic apneas, hypopneas, and repetitive significant drops in oxygen saturation (below 90%), repetitive increases in BP can occur as a result of increased airway resistance during sleep. It also shows that, in some patients with both UARS and borderline high BP, high BP can be controlled with treatment of UARS. We conclude that abnormal upper airway resistance during sleep, often associated with snoring, can play a role in the development of hypertension. PMID- 8635370 TI - Changes in cerebral oxygenation and hemodynamics during obstructive sleep apneas. AB - OBJECTIVE: To evaluate changes in cerebral tissue oxygenation and blood volume during obstructive sleep apnea (OSA). METHODS: We studied eight men with moderate to severe OSA by near-infrared spectroscopy (NIRS) simultaneously with polysomnography during nocturnal sleep (five patients) and daytime naps (three). RESULTS: In all patients, a consistent decrease of oxyhemoglobin (OxyHb) and increases of deoxyhemoglobin and total hemoglobin (TotalHb) in the regional cerebral tissue were observed during the episode of OSA at every sleep stage. Changes in each hemoglobin and apnea duration were significantly (p<0.01) more remarkable during rapid eye movement (REM) sleep than non-REM (NREM) sleep. Significant correlations of changes in OxyHb and TotalHb during the apneic episode with apnea duration were found during both NREM and REM sleep (p<0.01). CONCLUSIONS: Since TotalHb is used as an indicator of blood volume in the NIRS technique and the venous return is reported to increase during OSA, it is assumed that cerebral blood flow (CBF) increases during the episode of OSA. Because a decrease in OxyHb was observed and brain activity is reported to decrease during OSA, it is supposed that oxygen supply to the brain tissue decreases rather than oxygen consumption in the brain increases. The results of this study indicate that possibly increased CBF could not compensate for reduced arterial oxygen saturation and cerebral tissue hypoxia may occur during the episode of OSA. PMID- 8635369 TI - The effect of triazolam and flunitrazepam--two benzodiazepines with different half-lives--on breathing during sleep. AB - We performed a double-blind single-dose placebo/hypnotics crossover study randomized within groups to test the potential problems that a group of normal subjects, including subjects who snore, may face using hypnotic medications. Two benzodiazepine hypnotics--triazolam, 0.25 mg, and flunitrazepam, 2 mg tablets- were considered. Subjects were monitored with nocturnal polysomnography, including esophageal pressure (Pes) monitoring as a measure of respiratory efforts, and were given daytime performance tests. Results were analyzed for the total nocturnal sleep period and also by thirds of the night in consideration of the different half-lives of the studied drugs. Three specific respiratory variables were evaluated: mean breathing frequency for selected unit of time, "Delta Pes" (esophageal pressure at peak end-expiration minus Pes at peak end inspiration) expressed in cm H2O, and the ratio of Delta Pes/Delta TI (inspiratory time), taken as an index of respiratory drive calculated for each respiratory cycle. There was no significant increase in either the respiratory disturbance index or the oxygen desaturation index (number of drops in arterial oxygen saturation of 4% or more per hour of sleep, as measured by pulse oximetry). There was a significant increase in mean breathing frequency with flunitrazepam compared with placebo, as well as a significantly larger percentage of time during sleep with Delta Pes above 10 cm H2O (taken as a cutoff point for normal respiratory effort) with both triazolam and flunitrazepam compared with placebo. These respiratory changes, even if significant, were minor but may become a liability in association with specific abnormalities. PMID- 8635371 TI - Ventricular arrhythmias during sexual activity in patients with coronary artery disease. AB - STUDY OBJECTIVE: Information on appearance or increase of ventricular arrhythmias during intercourse in patients with coronary disease is still inadequate. This prospective study analyzes patients' rhythm disturbances on sexual activity and compares them with occurrences during daily activities and stress testing. PATIENTS: The study included 88 male outpatients with stable coronary disease; ages ranged from 36 to 66 years (mean, 52 years). INTERVENTIONS: Patients underwent ambulatory ECG monitoring, which included sexual activity, and a near maximal ergometric test. RESULTS: Arrhythmia was found during intercourse in 56% of patients, compared to 38% at exercise. Occurrence or exacerbation of ectopic activity was the dominant pattern in patients with arrhythmia at exercise testing (89%), but this exacerbation was found only in 11% of patients during intercourse. Complex ventricular arrhythmia during sex was detected in 12.5% of patients. CONCLUSIONS: Two main observations may be drawn from the study. First, rhythm disturbances were not exacerbated during intercourse in most patients. Second, if ventricular ectopic activity occurred on intercourse, it was most often simple and essentially similar to disturbances in daily activity. PMID- 8635372 TI - Relation between depressed cardiac response to exercise and autonomic nervous activity in mildly symptomatic patients with idiopathic dilated cardiomyopathy. AB - We investigated whether the depressed cardiac response to adrenergic stimulation is accompanied with impaired autonomic function in mildly symptomatic patients with idiopathic dilated cardiomyopathy (DCM). Twenty-seven patients with DCM (New York Heart Association class I or II) and 7 normal control subjects underwent exercise radionuclide ventriculography and 24-h ambulatory ECG. The following frequency components of heart rate variability were calculated: the areas under the low (low frequency component [LF], 0.04 to 0.15 Hz), high (high frequency component [HF], 0.15 to 0.40 Hz), and total frequency portions of the spectrum. HF and HF% (the ratio of HF to total power) were calculated as indexes of specific vagal influences, and LF% (the ratio of LF to total power) and the ratio of LF to HF were of sympathetic tone. The left ventricular ejection fraction (LVEF) increased by more than 5% in all normal control subjects during exercise, whereas 17 (63%) of patients failed to show more than a 5% increase in LVEF. The profile of the mean hourly HF% and LF/HF showed circadian variations in normal control subjects but not in patients. The HF and HF% during sleep were significantly lower and the LF/HF during sleep was higher in patients than in normal control subjects. In patients, the LVEF during exercise minus LVEF at rest was significantly correlated with HF, LF%, and LF/HF during sleep, and with the ratios of the mean values during early morning to the mean daytime values for those spectral indexes. Our results demonstrated that mildly symptomatic patients with DCM showed an attenuated cardiac response to exercise and altered autonomic function, and their close relationship, suggesting that autonomic nervous activity contributes to cardiac desensitization in DCM. PMID- 8635373 TI - Pulmonary function in patients receiving long-term low-dose methotrexate. AB - STUDY OBJECTIVE: Acute interstitial pneumonitis is the main pulmonary side effect during methotrexate (MTX) treatment for rheumatoid arthritis. The aim of the study was to determine the following: (1) the incidence of MTX-induced pneumonitis during low-dose long-term MTX treatment for chronic arthritis; (2) whether periodic pulmonary function tests were useful for detecting MTX pneumonitis before clinical symptoms; and (3) whether any subclinical abnormality of pulmonary function was present in asymptomatic patients receiving MTX treatment. DESIGN: Pulmonary function tests, including diffusing capacity for carbon monoxide (DCO) measurements, were performed in 124 patients receiving low dose MTX for rheumatologic diseases at the time of initiating treatment, and then at 3 months, 6 months, and at 6-month intervals thereafter. Mean duration of treatment was 23 months. RESULTS: MTX treatment was interrupted in six patients for acute onset of clinical symptoms; criteria for diagnosis of MTX pneumonitis were fullfilled in four cases (incidence: 3.2%); no risk factor could be identified. No significant decrease in pulmonary function parameters could be observed before the onset of clinical symptoms of MTX pneumonitis, and this adverse effect could not be predicted by periodic function tests. A statistically significant decrease was found in FVC (-2.2%, p=0.04), FEV1 (-5.0%, p<0.001), and diffusing capacity per alveolar volume, DCO/VA (-4.8%, p=0.03), but not DCO ( 1.3%, p>0.05), in the 118 other asymptomatic patients during MTX treatment. CONCLUSION: We found minor subclinical alterations in pulmonary function in asymptomatic patients receiving low-dose long-term MTX treatment, but periodic pulmonary function tests did not allow us to detect MTX-induced pneumonitis before clinical symptoms. Therefore, we recommend that these tests should not be systematically performed while patients are receiving treatment. PMID- 8635374 TI - A prospective study of pulmonary function in patients receiving mitomycin. AB - Mitomycin is a chemotherapeutic agent that is used to treat a variety of solid tumors. Pulmonary toxic reactions from this agent can be life threatening. We prospectively investigated the utility of pulmonary function tests (PFTs) in monitoring for the occurrence of pulmonary toxicity due to mitomycin. PFTs were obtained at baseline and after three cycles of mitomycin therapy. We analyzed the clinical course, radiologic studies, and PFT results in 133 patients with metastatic squamous cell carcinoma of the lung randomized to treatment with either mitomycin, vinblastine, and cisplatin or mitomycin alone as part of a prospective treatment protocol of the North Central Cancer Treatment Group (NCCTG). The diffusing capacity (DCO) was available in only 40 patients after the third cycle due to a high rate of progression and death from their underlying disease. After three cycles of chemotherapy, there was an average decline in the DCO of 14% (p<0.0001) and no changes were observed in expiratory flows. No differences were noted between treatment arms. A significant decline in the DCO (defined as a >20% change after correcting for hemoglobin) was noted in 11 of 40 patients (28%). This decline in the DCO was not associated with a worse prognosis (p=0.77). Seven patients (5%) developed severe pulmonary toxic reactions attributed to chemotherapy, including noncardiogenic pulmonary edema, interstitial pneumonitis, and pleural effusions. Corticosteroid therapy resulted in temporary subjective improvement in three patients. The Dco did not correlate with the development of pulmonary toxic reactions in these seven patients. In conclusion, (1) the incidence of clinically significant pulmonary toxic reactions from mitomycin is relatively low (5%), (2) mitomycin therapy resulted in a greater than 20% decline in the DCO in approximately one-fourth of patients receiving three cycles of chemotherapy, and (3) the use of serial PFTs in patients receiving mitomycin was not shown to be predictive of pulmonary toxicity. PMID- 8635375 TI - A comparative study of the clinical efficacy of nedocromil sodium and placebo. How does cromolyn sodium compare as an active control treatment? AB - Nedocromil sodium and cromolyn sodium are the only two currently available nonsteroid anti-inflammatory agents for treatment of asthma. Clinical differences between the two agents remain under continuous investigation with reports differentiating the two on the basis of atopy of the patient and reversibility of bronchoconstriction. This study investigated the efficacy of nedocromil sodium (4 mg, qid) for treatment of mild-to-moderate asthma in comparison to placebo using cromolyn sodium (2 mg, qid) as an active control treatment. Patients were primarily allergic asthmatics (with at least 15% reversibility) previously maintained on a regimen of regular bronchodilator therapy. During a 2-week run-in period, the patient's slow-release theophylline therapy was removed, and the patients were randomized to treatment after deterioration of asthma control (asthma symptom summary score of 3 for 7 of the 14 days). After 8 weeks of treatment, patients were returned to as occasion requires bronchodilator therapy, as per the 2-week baseline period. The results demonstrate that patients treated with nedocromil sodium showed statistically significant improvements during the primary time period (mean weeks 3 through 8) over placebo-treated patients as evidenced by all indexes of asthma symptoms, pulmonary function measures, and decreased bronchodilator reliance (p<0.05). Patients treated with cromolyn sodium demonstrated similar improvements over placebo-treated patients. Comparisons between nedocromil sodium and cromolyn sodium showed the two agents to be comparable in this group of primarily allergic patients with reversible disease. Between-group differences were noted for 3 of the 13 variables (nighttime asthma, FEV1, and forced expiratory flow rate between 25 % and 75% of the FVC) in favor of cromolyn sodium when the data were pooled during the primary time period. The number of patients missing 1 or more days from work/school/regular activity due to asthma was significantly fewer compared with placebo, and favoring nedocromil sodium over cromolyn sodium. No differences were observed among the three treatments for adverse events. This study demonstrated that in primarily allergic patients with reversible airways disease, nedocromil sodium and cromolyn sodium are both significantly more effective than placebo for treatment of mild-to moderate asthma. PMID- 8635376 TI - Inhaled corticosteroids do not prevent the development of tolerance to the bronchoprotective effect of salmeterol. AB - INTRODUCTION: Twice-daily inhaled salmeterol produces rapid reduction in its acute bronchoprotective effect against methacholine in patients with mild asthma. This investigation examined this effect in patients with moderate asthma who were using inhaled corticosteroids. SUBJECTS AND METHODS: Eight asthmatic volunteers who required inhaled corticosteroids for control of their symptoms and who were able to withhold treatment with beta 2-agonists for 4 weeks before and during the study participated in a double-blind, crossover, placebo-controlled study with two random-order treatment periods: inhaled salmeterol, 50 microg twice a day for seven doses, and placebo in similar fashion, with a 7-day or greater washout between these periods. Methacholine inhalation tests were done 1 h after doses 1, 3, 5, and 7, and then 24 h after the last dose of the study inhaler, 10 min post 200 microg salbutamol. RESULTS: Baseline FEV1 measurements before doses 3, 5, and 7 of salmeterol, ie, 12 h after salmeterol, were significantly higher than all other baseline values. Twenty-four hours after the last dose of salmeterol, the FEV1 was no different from that during the placebo period. The geometric mean methacholine concentration causing a 20% fall in FEV1 (PC20) following the third dose of salmeterol (6.8 mg/mL) was significantly lower than after the first dose of salmeterol (12.0 mg/mL; p=0.031), and this reduction of bronchoprotection persisted following doses 5 and 7. The methacholine PC20 10 min postsalbutamol measured after the salmeterol period was significantly lower than after placebo (5.6 vs 13.3 mg/mL; p<0.001). CONCLUSIONS: Tolerance to the acute bronchoprotective effect of salmeterol was significant after the first two doses and persisted after the seventh dose. Tolerance to the acute bronchoprotective effect of salbutamol was also significant after regular use of salmeterol for seven doses. These effects, in subjects using inhaled corticosteroids regularly, were similar to the those previously seen in patients with mild asthma using as required beta 2-agonists only, indicating that tolerance is not prevented by use of inhaled corticosteroids. PMID- 8635378 TI - Trends in compliance with bronchodilator inhaler use between follow-up visits in a clinical trial. AB - STUDY OBJECTIVE: To assess objectively measured, long-term trends in compliance with physician-prescribed metered-dose inhaler (MDI) use during a clinical trial. DESIGN: A prospective study. SETTING: The Lung Health Study, a 5-year clinical trial to determine the effect of special intervention with an intensive smoking cessation program and bronchodilator therapy in cigarette smokers 35 to 60 years of age with minimal to moderate airflow limitation due to COPD. PARTICIPANTS: Two hundred thirty-one participants who were issued an MDI with an attached Nebulizer Chronolog (NC) (Forefront Technologies Inc; Lakewood, Colo) which electronically records the date and time of each MDI actuation. One hundred two participants were not informed of the recording capabilities of the attached NC, while 129 participants were aware of the NC's monitoring function. INTERVENTION: Following an initial 12-week period of counseling, participants returned to the clinic every 4 months. MEASUREMENTS AND RESULTS: Analysis of the data from the NC collected over a period of 2 years indicates that compliance with the prescribed medication regimen was best immediately following each follow-up visit and gradually declined during the interval between follow-up visits. The level of compliance after each visit was lower for each successive follow-up. These trends could not be observed from self-report or weighting the medication canisters at follow-up visits. The participants who were informed of the NC's function and who were provided with detailed feedback about their inhaler use generally showed better compliance. PMID- 8635377 TI - Partial vs full beta-receptor agonism. A clinical study of inhaled albuterol and fenoterol. AB - STUDY OBJECTIVE: To compare the maximal extrapulmonary effects of the beta agonists albuterol and fenoterol in eight healthy volunteers. SUBJECTS AND METHODS: In this double-blind study, we have examined the maximum cardiac effects (electromechanical systole [QS2I]--a measure of inotropy, heart rate, BP) and metabolic effects (plasma K+ and cyclic adenosine monophosphate [cAMP]) of repeated inhalation of albuternol and fenoterol. In eight healthy volunteers, 400 microg of each drug was administered every 10 min until QS2I and plasma K+ had reached a plateau (+/- 0.1 mmo l/L for K+, and +/- 10 ms for QS2I). The maximum response (Emax) and the dose of albuterol required to produce 50% of the maximum response to fenoterol (ED50F) were calculated. RESULTS: The Emax for fenoterol was significantly greater than albuterol for plasma K+ (-1.4 vs -1.03 mmol/L; p<0.002), QS2I (-71.8 vs 57.5 ms; p=0.047), and cAMP (33.8 vs 18.1 nmol/L; p<0.002). The dose required to produce the ED50f was significantly greater for albuterol than for fenoterol with potency ratios of 1.75, 1.61, and 2.26 for plasma K+, QS2I, and cAMP, respectively. There were no significant differences between fenoterol and albuterol with respect to heart rate (Emax, 44.9 vs 32.5 beats/min; p=0.19; potency ratio, 1.98; p=0.052). CONCLUSIONS: These findings suggest that albuterol behaves as a partial agonist at beta-receptors when compared with fenoterol, and that when inhaled in doses currently recommended for severe asthma, albuterol will result in lesser maximum cardiac and metabolic effects than fenoterol. These findings are consistent with the hypothesis that the property of full receptor agonism may contribute to the increased risk of death associated with fenoterol. PMID- 8635379 TI - Inhalation of single vs multiple metered-dose bronchodilator actuations from reservoir devices. An in vitro study. AB - Differences in inhalation technique with reservoir or spacer devices may affect metered-dose inhaler (MDI) dose availability to a patient. PURPOSE: This study examined the effect of single vs multiple actuations of an MDI into reservoir devices on dose delivery of albuterol, with three clinically available reservoir brands. METHODS: An in vitro lung model simulated inspiration from the MDI reservoir system. Albuterol (Proventil; Schering) was delivered by MDI, with the Monaghan Aerochamber, the Diemolding Healthcare Division (DHD) aerosol cloud enhancer (ACE), and the Schering InspirEase, using standardized volumes and inspiratory flows of 30 L min(-1). The MDI was actuated into each brand of reservoir 1, 2, or 3 times in rapid succession, followed by a single inhalation. Aerosol dose at the reservoir mouthpiece was captured on a cotton filter, dissolved in ethanol, and measured with a spectrophotometer at 278 nm. RESULTS: For all three brands of reservoir, less accumulated dose of drug is delivered with multiple actuations than with multiple single actuations each followed by inhalation. The total dose in milligrams increased significantly with two multiple actuations compared with one actuation in the Aerochamber and ACE (p<0.01), but not in the InspirEase (p>0.05). The Aerochamber, ACE, and InspirEase delivered a mean total dose (SD) of 0.0264 mg (0.012), 0.0271 mg (0.007), and 0.0136 mg (0.006), respectively, with one actuation compared to 0.0485 mg (0.011), 0.0453 mg (0.013), and 0.0218 mg (0.009) with two multiple actuations. The increase in total dose with three multiple actuations was not significant compared to two actuations for any of the brands tested (p>0.05). Although total dose increased with multiple actuations, a decline in efficiency was seen with two and three multiple actuations, compared to single actuation. The dose delivered per actuation decreased for the Aerochamber, ACE, and InspirEase from 0.0264 mg (0.012), 0.0271 mg (0.007), and 0.0136 mg (0.006) with one actuation, to 0.0243 mg (0.006), 0.0226 mg (0.006), and 0.0109 mg (0.005), respectively, with two multiple actuations, for losses of 8.0%, 16.6%, and 19.9% in dose per actuation for each brand. A further decline in delivery per actuation to 0.0164 mg (0.001), 0.0184 mg (0.004), and 0.0097 mg (0.005) for the 3 brands, respectively, was found with 3 multiple actuations before inhalation. This was a loss of 37.9%, 32.1%, and 28.7% of the dose per single actuation in each brand. There was no significant difference between the Aerochamber and the ACE in dose availability with 1, 2, or 3 actuations, but both of these brands provided significantly more drug than the InspirEase. CONCLUSION: Maximal aerosol bronchodilator from an MDI reservoir was given by single actuations each followed by a breath. Two rapid actuations followed by a breath will give a significant accumulation of dose with some loss when compared to two single actuations each followed by inhalation. Three multiple actuations led to a loss of approximately one third of the drug dose obtainable with three single actuations each followed by inhalation, for all three brands. PMID- 8635380 TI - Effect of 30 mg of morphine alone or with promethazine or prochlorperazine on the exercise capacity of patients with COPD. AB - OBJECTIVE: We have shown that the administration of 0.8 mg/kg of morphine (M) to patients with COPD resulted in a 20% increase in the maximum oxygen consumption (Vo2max), but was associated with significant drowsiness and euphoria. The objective of the present study was to ascertain whether lower doses of M alone or in combination with prochlorperazine (PC) or promethazine (P) could elicit significant increases in exercise tolerance. DESIGN: The exercise capacity, psychological status, and reaction times were assessed before and 60 min after the patients received placebo (PLAC), 30 mg M orally, 30 mg M plus 10 mg PC (M PC), or 30 mg M plus 25 mg P (M-P) in a randomized double-blind crossover study. In a secondary study, nine patients were tested on three separate days before and after receiving PLAC, 25 mg P, or 30 mg M plus 25 mg P. PATIENTS: Seven COPD patients (FEV1=0.99 +/- 0.30 L, Vo2max=990 +/- 315 mL/min) who were ventilatory limited. SETTING: Veterans Affairs medical center. RESULTS: After the patients ingested M-P, the increase in the Vo2max (129.0 +/- 104 mL/min), the workload (10.0 +/- 6.5 W) and the maximum minute ventilation (4.0 +/- 3.9 L/min) were significantly greater (p<0.05) than after PLAC ingestion (-4.8 +/- 79 mL/min, 1.4 +/- 6.9 W, and -1.6 +/- 2.4 L/min, respectively). Changes after the ingestion of M, P, o r M-PC were intermediate. The M-PC combination adversely affected the patient's reported mental status (Bond visual analog scale) more than the M-P or M regimens. No regimen significantly affected the reaction time. CONCLUSIONS: We conclude that the administration of 30 mg of M plus 25 mg of P significantly improves the exercise tolerance of patients with COPD, without significantly impairing the mental capabilities of the subjects. The utility of this regimen over longer time periods needs to be evaluated. PMID- 8635381 TI - Evaluation of pulmonary lesions with FDG-PET. Comparison of findings in patients with and without a history of prior malignancy. AB - STUDY OBJECTIVE: The purpose of this study was to evaluate the accuracy of positron emission tomography (PET) using F-18 fluorodeoxyglucose (FDG) in differentiating benign from malignant pulmonary lesions both in patients with and without a history of prior malignancy. DESIGN: Forty-eight consecutive patients with pulmonary lesions suspicious for malignancy underwent FDG-PET scanning. Group 1 included 27 patients without and group 2 included 21 patients with a history of malignancy. Pathologic proof of diagnosis was obtained for 32 patients and 16 patients were followed up clinically and radiographically for at least 6 month. The standard uptake ratio (SUR) and the lesion to background (L/B) ratio were determined in 45 patients. SETTING: Vanderbilt University Medical Center. RESULTS: In group 1, the average SUR and L/B ratio for malignant lesions (n=14) were 8.9 +/- 4.9 and 20.6 +/- 14.2, respectively. For benign lesions (n=12), the average SUR was 3.3 +/- 3.2 and L/B ratio was 5.2 +/- 5.5. In group 2, the average SUR and L/B ratio for malignant lesions were not significantly different from group 1. Using either a SUR greater than 2.5 or L/B ratio greater than 5 as an cutoff level to differentiate benign and malignant lesions, the sensitivity and negative predictive value in both groups were 100%. There were five false positive studies in group 1 and one in group 2, including tuberculosis (n=2), a granulomatous lesion (n=1), an inflammatory lesion (n=1), a schwannoma (n=1), and a fibrous mesothelioma (n=1). The overall accuracy was 88%, 81% in group 1, and 95% in group 2. CONCLUSION: FDG-PET can identify malignant pulmonary lesions both in patients without and with a history of prior malignancy with a high sensitivity and negative predictive value for lesions greater than 1 cm (100% in this study). High FDG uptake by some inflammatory processes and benign tumors may cause false-positive results. Semiquantitative evaluation using SUR or L/B ratio provides similar accuracy. PMID- 8635382 TI - A clinicopathologic study of resected cases of adenosquamous carcinoma of the lung. AB - Adenosquamous carcinoma of the lung is an uncommon form of the lung cancer. Owing to the infrequent occurrence of this disease, no series reported to date (and to our knowledge) has been of adequate size for definitive statistical analysis. In this study, survival curves and background factors affecting prognosis in those with resected adenosquamous carcinoma of the lung were reviewed. In the period from 1973 to 1994, a total of 1,284 patients with primary lung cancer, including 44 cases (3.4%) of adenosquamous carcinoma, were surgically treated in our department. The cumulative 5-year postoperative survival rate, for all cases of adenosquamous carcinoma of the lung was 18.5%. When the survival rates were compared by histologic type, the outcomes of patients with adenosquamous carcinoma were statistically worse than for patients with squamous cell carcinoma and adenocarcinoma, owing to the highly aggressive pathologic stage of adenosquamous carcinoma. The background factors most closely associated with the survival rate in those with adenosquamous carcinoma, using Cox's proportional hazard model, were gender and the degree of nodal involvement. Five-year survival was obtained in seven patients as follows: T1N0M0 in one patient, T2N0M0 in three, T2N1M0 in two, and T3N0M0 in one. Of these seven patients, all had received complete resections, and five were N0 cases. Although our series is small, this study suggest that adenosquamous carcinoma of the lung is an aggressive tumor that grows rapidly. PMID- 8635384 TI - The role of induction (neoadjuvant) chemotherapy in stage IIIA NSCLC. AB - Induction (neoadjuvant) chemotherapy has become an accepted treatment for stage IIIA (T1-3N2M0) non-small cell lung cancer. In two recent randomized trials, neoadjuvant chemotherapy plus surgery gave an increase in median survival at least fivefold greater than surgery alone. The Spanish Lung Cancer Group trial of preoperative chemotherapy, in which the cisplatin dose was randomized to either 50 mg/m2 or 100 mg/m2 plus 3 g/m2 ifosfamide and 6 mg/m2 mitomycin, examines the effect of K-ras gene mutations on tumor response and survival. Patients whose tumors contain K-ras gene mutations are more likely to develop distant metastases and have lower median survival than patients without such mutations. Microsatellite instability seems to be a frequent mechanism of genetic aberrations. Knowledge about these genetic alterations could have prognostic importance and may identify the patients who should receive the most aggressive additional treatment. PMID- 8635383 TI - CYFRA 21-1 enzyme-linked immunosorbent assay. Evaluation as a tumor marker in non small cell lung cancer. AB - BACKGROUND: The CYFRA 21-1, a newly developed sandwich enzyme-linked immunosorbent assay (ELISA), was used to measure soluble cytokeratin 19 fragment in serum that is expressed in simple epithelium and its malignant counterpart. The present study was designed to investigate whether CYFRA 21-1 is a sensitive and specific tumor marker for non-small cell lung cancer. METHODS: CYFRA 21-1 assay, using two specific monoclonal antibodies (KS 19.1 and BM 19.21) for cytokeratin 19, was measured in 312 serum samples, including 164 lung cancer, 118 benign pulmonary disease, and 30 healthy individuals. The sensitivity of CYFRA 21 1 was also compared with two other markers, carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC), in 164 patients with lung cancer. RESULTS: The median value of healthy individuals was 1.3 ng/mL (95th percentile 1.8). In patients with benign pulmonary diseases, the median was 1.5 ng/mL (95th percentile 2.9). There is no significant difference between sexes, smoking habit, and the subgroups of benign pulmonary disease, such as tuberculosis, pneumonia, or COPD. Using the cutoff value of 3.3 ng/mL, defined at 95% specificity for benign lung disease, the sensitivities of CYFRA 21-1 for squamous cell carcinoma (n=74), adenocarcinoma (n=54), undifferentiated large cell carcinoma (n=11), and small cell lung cancer (n=25) were 62%, 39%, 36%, and 20%, respectively. Despite the cell types, the sensitivities of CYFRA 21-1 in non-small cell lung cancer (NSCLC, n=169) were 51% (CEA 42%, SCC 20%). The sensitivity of CEA was significantly higher in patients with adenocarcinoma (58%) than other markers; while in patients with squamous cell carcinoma, CYFRA 21-1 assay has the highest sensitivity. The median level of CYFRA 21-1 in squamous cell carcinoma is significantly higher than that of other cell types (Mann-Whitney test, p<0.001). The serum level and sensitivity of CYFRA 21-1 were well correlated with staging and tumor size in squamous cell carcinoma. The CYFRA 21-1 values were measured for monitoring progression of disease in 20 patients with squamous cell carcinoma. There is significant difference in paired observation of CYFRA 21-1 level in patients with progressive disease (Wilcoxon signed-rank test, p<0.05), but no difference was observed in patients with stabilized disease (p>0.1). CONCLUSION: For patients with NSCLC, especially in squamous cell carcinoma, CYFRA 21-1 is not only a sensitive and specific tumor marker, but also may be a useful adjunctive marker for disease monitoring. PMID- 8635385 TI - Chemotherapy for advanced NSCLC. Will meta-analysis provide the answer? AB - Meta-analyses of published data show a modest but significant survival or response rate benefit for chemotherapy plus supportive care over best supportive care alone in non-small cell lung cancer (NSCLC), but these findings may be biased in favor of positive results because they are literature based. A meta analysis of individual patient data from 11 published and unpublished randomized studies shows a significant benefit for cisplatin-based chemotherapy over supportive care, a small (not significant) benefit for vinca alkaloids and etoposide, and no benefit for alkylating agents. The benefit is independent of age, sex, stage, histologic type, or performance status. Single large trials would avoid the pitfalls of meta-analyses, but these have so far proved difficult to organize. Physicians need to be convinced that even a small increase in median survival in NSCLC as a result of chemotherapy may represent a considerable increase in life expectancy for individual patients. PMID- 8635386 TI - Quality of life as a new end point. AB - Quality of life (QOL) is a relatively new clinical end point that is particularly relevant to the typically palliative therapy for non-small cell lung cancer. Patients' assessments of their QOL are shown to differ from their physicians', emphasizing the subjective nature of QOL. A number of relevant instruments and assessment techniques are employed. Results from a study using the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 instrument before and during chemotherapy are presented. Some parameters improved while others did not, preventing a simple interpretation. There are arguments for compiling indexes of QOL while retaining measures for individual parameters and a desire for the consistent international use of an instrument such as the EORTC questionnaire. PMID- 8635387 TI - Quality of life and supportive care in the treatment of NSCLC. AB - The definition of quality of life (QOL) for individual patients with non-small cell lung cancer (NSCLC) is unclear and its evaluation difficult because of the heterogeneity of the patients involved. More research is needed to determine the best means of routinely expressing and comparing QOL assessments and implementing the results of these studies into daily patient care. The role of broad supportive care in optimizing patient comfort and functionality is also an important issue. Because it incorporates not only specific palliative treatment, but also nontumor-specific measures and medical intervention, supportive care is relevant to all patients with NSCLC, whether terminally ill or suffering treatment-related toxic reactions. More education, research, and financial support are needed to optimize QOL and supportive care of patients. PMID- 8635388 TI - Do radiosensitizers enhance the treatment of patients with NSCLC? The need for better models and alternative methods of treatment. AB - Radiotherapy as a treatment for non-small cell lung cancer (NSCLC) can potentially be optimized by the use of radiosensitizers, substances that enhance the effect of radiation on tumor tissue without an equal increase in the effect on normal tissue. Radiosensitizers may act by increasing the level of lethal damage caused by radiation or by causing a decrease in the repair of such lethal damage. While cell and animal models have been used in an attempt to establish the efficacy of radiosensitizers, trials in man have so far been inconclusive. The need to improve existing models and methods for combining modalities to best effect is clear. Radiotherapy/chemotherapy combinations are a logical alternative to radiosensitizers for managing NSCLC, and despite variability in the extent of local and metastatic control, evidence for improved survival exists. PMID- 8635389 TI - Biological treatment of NSCLC. The need for conclusive studies. AB - Despite extensive investigation, biological treatments for non-small cell lung cancer (NSCLC) remain largely undeveloped. The lack of satisfactory models has frequently led to inadequate phase II studies and to small and inconclusive phase III trials. Nonuniformity of trials has prevented clearer conclusions from being reached by meta-analysis. In general, immunotherapy has failed to fulfill expectations for clinical usefulness. The benefit with this approach, if any, seems to be marginal, but it is not clear whether this is a result of lack of activity or faulty clinical testing. The future of biological agents in cancer treatment lies in ongoing advances in molecular biology, for example in making tumors more immunogenic. Another avenue of further clinical research includes novel forms of therapy with monoclonal antibodies. Adequate models for testing and appropriate clinical trial settings could clarify the role of biological agents in NSCLC. PMID- 8635390 TI - Cancer genetics and cell and molecular biology. Is this the way forward? AB - Lung cancer, the most prevalent cancer in the western world, is predominantly caused by smoking and thus perceived as a "self-inflicted" disease. Nevertheless, only 20% of smokers develop lung cancer. This review examines the concept of high risk populations and screening. It looks at developments in the molecular epidemiology of the disease that shed new light on genetic changes that may predispose individuals to malignancy. Improvements in existing drug therapy are discussed as well as important new therapeutic developments, including antigrowth factors (antagonists G and D), antimetastatic agents (matrix metalloproteinase inhibitors), and natural products, arising from a greater understanding of signal transduction pathways and the process of cell metastasis. PMID- 8635391 TI - Oncogenes and antioncogenes in lung tumorigenesis. AB - The role of oncogenes and antioncogenes in lung tumorigenesis is discussed in this review, with particular emphasis on their prognostic significance. Mutations in the ras family of oncogenes, overexpression of the myc and neu families of oncogenes, and mutations of p53, the recessive tumor suppressor gene, occur with differing frequencies in small cell lung cancer and non-small cell lung cancer, and are usually associated with a poor prognosis. Loss of heterozygosity, notably on chromosomes 3p, 5q, 9p, 13q, and 17p, is a common feature in lung carcinomas and its importance is also discussed. PMID- 8635392 TI - Non Small Cell Lung Cancer: Planning for the Future. Workshop proceedings. St Paul de Vence, France, October 21-22, 1994. PMID- 8635393 TI - Chemotherapy for advanced disease. How to raise enthusiasm. AB - Over the last 5 years, the newer chemotherapeutic agents (cisplatin, vindesine, vinorelbine tartrate [Navelbine], taxoids, and gemcitabine) have given greater hope in the treatment of patients with non-small cell lung cancer. Despite this, it has proved very difficult to organize trials large enough to show significant differences. Reasons for this include negative physician attitudes, the high cost of entering patients in trials, lack of cooperation between research organizations, and the perception of a self-imposed disease. To overcome this, communication with lung physicians, surgeons, and patients as well as oncologists is needed, and they must be involved through an appropriate choice of journals for publishing results, interaction between organizations, an information network linking hospitals, general physicians, and patients, and briefing of local and national media experts. PMID- 8635394 TI - Efficient designs for testing new agents and regimens. AB - Current clinical chemotherapy trial procedures in non-small cell lung cancer (NSCLC) require large numbers of patients and last up to 10 years before providing results that are often disappointing. Different designs are available that offer the advantages of smaller numbers of patients, shorter duration, and earlier identification of ineffective or highly active agents and regimens. An alternative method is proposed, combining phase II screening, randomized phase II studies, and multicenter, comparative, randomized phase III trials. Issues under discussion included the combined study in phase II of different disease stages and the need for recruitment to larger trials. PMID- 8635395 TI - Aggressive vs nonaggressive therapy for metastatic NSCLC. AB - Clinicians tend to underestimate potential modest benefits of chemotherapy. They are often reluctant to refer patients for chemotherapy, perhaps because they expect the side effects to outweigh any perceived benefits. However, patients are much more ready to accept chemotherapy, even when the likely benefits are small. Quality of life, change in performance status, and relief of tumor-related symptoms are important additional parameters of treatment assessment. Taking account of these other factors will help clinicians balance quality and quantity of life in patients with metastatic non-small cell lung cancer. PMID- 8635396 TI - The role of neoadjuvant chemotherapy in NSCLC. AB - Neoadjuvant chemotherapy to reduce tumor size before surgery or radiologic treatment is now a feasible option in the treatment of non-small cell lung cancer (NSCLC). Patients in clinical stage IIIA N2 of the disease who were previously a poor prognostic subset when treated with surgery are now being treated with neoadjuvant chemotherapy, followed by surgery or radiotherapy, in an attempt to improve survival rates. Published trials of neoadjuvant chemotherapy report response rates of 50 to 80% and median survival of 18-27 months. Two small trials have reported striking benefits of neoadjuvant chemotherapy over surgery alone. Other larger ongoing trials should answer some of the questions raised by these two trials. PMID- 8635397 TI - Benefits of neoadjuvant chemotherapy in NSCLC. AB - There is a significant potential benefit for induction chemotherapy in the management of non-small cell lung cancer (NSCLC). The real extent of such benefit is difficult to assess on the basis of available data owing to the intrinsic limitations of phase II studies. In fact, in most studies on neoadjuvant chemotherapy, the aim of the treatment (local control vs systemic effect) is often unclear, the eligibility criteria poorly defined, and pretreatment staging inadequate. Consequently, the interpretation of the results of such studies has proved difficult. This article examines the critical role of thoracic surgeons in the selection, staging, and optimal treatment of patients enrolled in neoadjuvant studies, and also in monitoring the quality of data and providing adequate specimens for concurrent biologic research. Within the framework of controlled trials, with proper methods and minimum toxic reactions, induction chemotherapy may be offered in the future to a large number of patients and eventually combined with long-term adjuvant/chemopreventive strategies. PMID- 8635398 TI - [Could C- and D-network mobile phones endanger patients with cardiac pacemakers?]. PMID- 8635399 TI - [Could C- and D-network mobile phones endanger patients with pacemakers?]. AB - OBJECTIVE: To investigate prospectively the extent of potentially harmful interference of cardiac pacemakers by mobile phones in the C (analog) and D (digital) networks in use in Germany. PATIENTS AND METHODS: 104 patients (54 men, 50 women; mean age 75.8 [40-100] years) with 58 different implanted pacemaker models (43 one-chamber and 15 two-chamber systems) underwent uniform tests at various functional states with three different telephones (D1 portable 8 Watt, D1 Handy model 2 Watt, C Handy model 0.5 Watt). The distances between telephone aerial and pacemaker, as well as reception sensitivity and polarity of the pacemaker were varied. All tests were done during continuous ECG monitoring. RESULTS: 28 different pacemaker types (48.3%) in 43 patients (41.3%) showed interference in the form of pacemaker inhibition and switching to interference frequencies as well as triggering of pacemaker-mediated tachycardias in the DDD mode, as well as in the temperature-regulated frequency-adaptive function. D portables influenced pacemaker function more often and at greater distance than the D Handy model, which was little different from the c network hand phone. Reduction in pacemaker sensitivity as well as switching to bipolar reception only partly eliminated the interference. CONCLUSIONS: Patients with implanted pacemakers should if possible not use mobile phones in the C and D networks. Individual testing with suitable programming of pacemaker sensitivity and polarity can reduce the risk of interference. PMID- 8635400 TI - [Transvenous closure of persistent ductus arteriosus with an Ivalon plug]. AB - OBJECTIVE: To assess a new transvenous transcatheter method of closing a persistent ductus arteriosus, combining advantages of the Porstmann and Rashkind techniques. PATIENTS AND METHODS: Five patients (three men, two women, mean age 36.2 [19-56] years) underwent the procedure. The diameter of the duct was 3-6 mm. A compressed ivalon (poly-vinyl-alcohol) foam plug, introduced and held by a modified biopsy forceps, was placed into the duct via a percutaneously and transvenously placed catheter sheath. Small titanium legs attached to the plug at the aortic and pulmonary ends unfolded once the plug was correctly placed, ensuring safe fixation. RESULTS: Closure was achieved in all five patients and no shunt demonstrated immediately afterwards in four. In one patient a small shunt briefly persisted but was not longer present the day after. One patient had a fever of up to 39 degrees C for several weeks that required no treatment and was thought to have been a foreign body reaction. Follow-up examination after 5-19 months confirmed complete closure. CONCLUSION: The described method appears to be safe and superior to the Porstmann and Rashkind techniques, but the results must be tested on a larger number of patients with longer follow-up. PMID- 8635401 TI - [Successful multimodal therapy of a locally advanced non-small-cell bronchial cancer]. AB - HISTORY AND CLINICAL FINDINGS: A chest radiogram, performed on a 60-year-old man with unproductive cough for 3 months, showed a space-occupying lesion in the right upper lobe, and breath sounds were diminished in this area. He had been a heavy smoker. His general condition and nutritional state were good. INVESTIGATIONS: Computed tomography, skeletal scintigraphy, bronchoscopy with biopsy and mediastinoscopy established the diagnosis of a locally advanced non small-cell bronchial carcinoma (stage IIIB or T2N3M0). TREATMENT AND COURSE: Combined adjuvant treatment was begun in the hope of improving the median survival time of 8 months predicted for this tumour stage. After two cycles of a combined chemotherapy scheme (ifosfamide, carboplatin, etoposide) he received hyperfractionated-accelerated radiotherapy (total dose 45 Gy; 1.5 Gy twice daily) together with carboplatin and vindesine. This was followed by a right upper lobectomy with lymphadenectomy. Full remission was confirmed in both the resected specimen and the lymph nodes. The patients remains free of tumour 30 months after the diagnosis. CONCLUSION: Neoadjuvant treatment can significantly improve the prognosis of non-small-cell bronchial carcinoma in stage III. Such patients should therefore be treated according to the appropriate study protocol, if possible. PMID- 8635402 TI - [Diagnosis of hepatic encephalopathies]. PMID- 8635403 TI - [Current state of tracheal replacement]. PMID- 8635405 TI - [Fecal incontinence after rectal resection]. PMID- 8635404 TI - [Indications for chemotherapy in multiple myeloma]. PMID- 8635407 TI - [Early writing about smallpox vaccination]. PMID- 8635406 TI - [Spider naevi after discontinuation of oral contraceptives]. PMID- 8635409 TI - Disaster epidemiology. AB - Sound epidemiologic knowledge of the morbidity and mortality caused by disasters is essential when determining what relief supplies, equipment, and personnel are needed to respond effectively in emergency situations. All disasters are unique because each affected region of the world has different social, economic, and baseline health conditions. Some similarities exist, however, among the health effects of different types of disasters, that if recognized, can ensure that the limited health and medical resources of the affected community are well managed. PMID- 8635408 TI - Health care personnel in disaster response. Reversible roles or territorial imperatives? AB - Disasters frequently demand exceptional skills from medical responders. Providers work most efficiently and effectively, however, within the roles and hierarchical structures with which they are familiar. The goal of disaster medical response planners is to assign personnel to roles that are as familiar as possible and to simultaneously enhance flexibility of response to extraordinary circumstances. We have outlined the most common disaster medical response roles and the personnel types that fit most directly as a primary provider within each role. Medics excel in field operations and field care of patients, whereas the training of nurses and physicians makes them the most flexible all-around providers, if specially trained in field emergency care, and the sole providers of definitive care. None of the providers, by virtue of their basic training, is well equipped to manage the public health consequences of disasters, but nurses and physicians should be able to easily move into the role, given appropriate special training. Some of the special courses needed to make medics, nurses, and physicians capable of serving flexible roles already exist; others need to be developed or enhanced. PMID- 8635410 TI - Management of unique clinical entities in disaster medicine. AB - This article discusses the management of clinical problems encountered particularly in disasters. These include the principles of multiple-casualty triage, and field and hospital management of blast injury, crush syndrome, compartment syndrome, particulate inhalation, and traumatic asphyxiation. The indications for extraordinary measures, such as field amputation, are detailed. A brief review of the causes and epidemiology of these entities is provided, with emphasis on the clinical management in the disaster setting. PMID- 8635411 TI - Hazardous materials. Disaster medical planning and response. AB - Hazardous materials offer a variety of unique challenges to emergency personnel. These agents have immense economic impact, but when mishandled, they become notorious for turning contained accidents into disasters involving the entire community. During a hazmat accident, the victims often ignore the rules of the disaster plan by seeking out the nearest hospital for medical care, regardless of that institution's capabilities. Health care workers rushing to the aid of contaminated individuals, without taking appropriate precautions (i.e., donning PPE), potentially make themselves victims. Disaster preparedness requires planning, policy, and procedure development, hazard analysis, training, and the availability of personal protective equipment for all responding personnel. Presently, the level of hazmat preparedness varies greatly among different hospitals, EMS and fire services, and disaster response teams. These differences in hazmat preparedness can be linked to a variety of factors (lack of awareness, funding, and support) and controversies (types of PPE and level of training required) which have prevented the establishment of a national hazmat policy for most of these organizations. Despite these difficulties, emergency departments continue to be the primary provider of care to contaminated individuals. As a result, emergency physicians must work with their hospital to implement a hazmat decontamination program in order to appropriately care for these individuals. The appendix to this article presents a list of recommendations for hospital hazmat preparedness. It is modeled after existing CDC and OSHA guidelines. PMID- 8635413 TI - The federal response plan and disaster medical assistance teams in domestic disasters. AB - Through a variety of processes over the last 30 years, an organized federal plan has emerged for the response to domestic disasters. This plan incorporates several aspects of medical response into two areas: (1) health and medical and (2) urban search and rescue. This article discusses the development of the federal response plan with emphasis specifically on medicine. Highlighted are disaster medical assistance teams, urban search and rescue task forces, and roles and responsibilities of emergency physicians and other emergency health professionals in a federal disaster response. PMID- 8635412 TI - Radiation disasters and emergency department preparedness. AB - There are few radiation disasters that have occurred worldwide. These events, however, give some insight as to the potential for theoretical radiation disaster events of the future. Some of our current fears, such as terrorist uses of radiation, currently have no historical basis for planning guidance. Hospital facilities should assess the potential for radiation accidents and potential radiation disaster scenarios when in the process of disaster planning. Preparation and training will lead to confidence and improved emergency management of potentially chaotic events. PMID- 8635414 TI - Medical support for mass gatherings. AB - Fairs, concerts, parades, and rallies are some of the many events that cause large numbers of people to gather in one place. Whether the event lasts a day or a week, it is evident that the people attending may require organized medical care. Most of the medical needs are minor, but cardiac arrests and other serious medical problems, including trauma, must be dealt with by the medical team. Careful planning and integration of emergency physician efforts with local hospitals and the emergency medical services system allow for an optimal delivery of health care, from the routine incident to a mass-casualty event. PMID- 8635415 TI - Urban search and rescue. AB - Structural-collapse rescue is one of the most challenging and dangerous disaster response activities. Sophisticated, multidisciplinary search-and-rescue capabilities have evolved to address the difficulties in finding, reaching, and extricating deeply entombed survivors. An equally advanced medical team is required to provide health and medical support to rescuers and to optimize extrication and survival of the often critically injured victims. Adaptations in the usual approach to emergency medical services and emergency medical care are needed to evaluate and treat confined-space victims adequately. PMID- 8635416 TI - Infectious disease emergencies in disasters. AB - This article discusses the relationship between disasters and infections. Infections that are reviewed include those resulting from (1) a breakdown of the usual mechanisms of infection control, (2) the introduction or emergence of pathogens, and (3) the movement of populations into new areas. Components of infectious-disease surveillance and disaster teams are detailed. PMID- 8635417 TI - Operational medicine in disasters. AB - There are many similarities and differences between operational and disaster medicine. Over the past several years, there have been increasing requirements for the use of military and tactical law enforcement operational medicine in disaster, humanitarian assistance, and terrorist settings. Many of the TEMS techniques have a direct application to disaster medicine. PMID- 8635418 TI - Disaster planning, Part I. Overview of hospital and emergency department planning for internal and external disasters. AB - The definition and causes for internal and external disasters are discussed in this article. Features of a hospital disaster plan are outlined with special reference to the role of the emergency department. Examples of previous disasters involving hospitals are presented to demonstrate problems that disaster planners should anticipate. PMID- 8635419 TI - Disaster planning, Part II. Disaster problems, issues, and challenges identified in the research literature. AB - Problems, issues, and challenges to disaster planning and selected disaster planning principles are presented in this article. Methods of developing strategies to overcome resistance to disaster preparedness and measures to ensure that medical-response facilities will function are among the concepts discussed. Disaster-preparedness resources are provided. PMID- 8635420 TI - Decreased neuronal burst discharge near site of seizure onset in epileptic human temporal lobes. AB - We examined auto-correlation and interval distribution characteristics of neuronal discharge from patients with complex partial seizures. The objective was to compare the interictal firing patterns of neurons in mesial temporal structures ipsilateral to the site of seizure onset with firing patterns of neurons in homologous contralateral structures. Spontaneous interictal recordings of 258 single neurons were acquired from 23 patients. A "burst area" measure was derived from the neuronal auto-correlation to assess the likelihood of grouped action potential discharge (burst discharge). Large burst area measures indicate a tendency for single neuronal burst discharge, but do not disclose information about interspike intervals within bursts. Although several measures based on single neuronal interspike interval distributions showed no overall difference between hemispheres, burst area was significantly reduced in mesial temporal structures ipsilateral to the site of seizure onset. Possible mechanisms of decreased burst discharge in epileptogenic regions include selective loss of burst-discharging neurons and increased recurrent inhibition. PMID- 8635421 TI - Neuron-specific enolase is increased after single seizures during inpatient video/EEG monitoring. AB - Neuron-specific enolase (NSE) is a marker of brain injury after acute neurologic insults. We report changes in serum NSE (s-NSE) in 25 patients (15 with epilepsy and 10 patients with nonepileptic events) during continuous inpatient video/EEG monitoring. s-NSE was significantly increased as compared with baseline and normal controls after the first ictal event in the epileptic group, especially in patients with secondarily generalized tonic-clonic seizures (p = 0.01), but s-NSE was not increased in patients with nonepileptic events. These preliminary data indicate that s-NSE may be increased after complex partial seizures--and generalized tonic-clonic seizures (GTCS). PMID- 8635422 TI - Complex febrile seizures. AB - In the context of a prospective cohort study, we examined the associations between individual complex features of both first (n = 428) and recurrent (n = 240) febrile seizures and factors shown to predict outcome in children with febrile seizures. Thirty-five percent of first and 33% of recurrent febrile seizures had one or more complex features (focal onset, duration > or = 10 min, or multiple seizures during the illness episode). There were strong correlations between focality and prolonged duration for both first and recurrent febrile seizures. A low fever at the time of the seizure was marginally associated with prolonged duration. Most factors associated with either recurrent febrile seizures or subsequent unprovoked seizures were not associated with either the initial seizure being complex or the likelihood that a recurrence would be complex. However, the children with recurrent febrile seizures, complex features tended to repeat. This factor was statistically significant and particularly striking for prolonged duration. Genetic or other constitutional factors may explain why the prolonged feature recurs. Eleven (2.5%) children had three or four risk factors for recurrent febrile seizure that was prolonged. This very small group of children may be candidates for abortive therapy to be administered at the onset of a recurrent seizure. PMID- 8635423 TI - Interictal temporal hypoperfusion is related to early-onset temporal lobe epilepsy. AB - Previous studies of interictal regional cerebral blood flow (rCBF) in temporal lobe epilepsy have shown variable correlations with clinical measures. We used high spatial resolution hexamethyl propyleneamine oxime single photon emission computed tomography (HMPAO SPECT) in 80 consecutive patients with complex partial seizures (CPS), comparing results with those from a large series of normal subjects. Visual image analysis detected abnormalities of rCBF in 41 of 80 (51%; numeric analysis detected abnormalities in 38 of 80). Age at epilepsy onset was significantly younger in patients with temporal hypoperfusion (p = 0.002), and the frequency distribution of hypoperfusion versus age at epilepsy onset was reverse exponential. The results of numerical image analysis showed that degree of hypoperfusion did not vary with age at epilepsy onset. These data suggest a single insult operating early in life as a cause of temporal hypoperfusion, as has been shown for mesial temporal sclerosis (MTS). We could not demonstrate relationships with other clinical variables, including time since last seizure. PMID- 8635424 TI - Prevalence of epilepsy in the elderly: the Rotterdam Study. AB - We assessed the prevalence of epilepsy in an elderly population in The Netherlands. The study was conducted from 1991 to 1993 as part of the Rotterdam Study, a population-based door-to-door study of all elderly people living in Ommoord, a suburb of Rotterdam, and included 5,559 persons aged 55-95 years. All subjects were screened for epilepsy through direct questions regarding the existence of epilepsy and antiepileptic drug (AED) use, in addition to relevant questions from the World Health Organization (WHO) protocol for epidemiologic studies of neurologic diseases. Further evaluation of screen positives was made by a panel of 1 study physician and 4 epileptologists, who also classified all confirmed cases of epilepsy according to the classifications of the International League Against Epilepsy (ILAE). The overall prevalence of active epilepsy in our study population was 0.9% including special syndromes and 0.8% excluding special syndromes. The prevalence increased with age from 0.7% for those aged 55-64 years to 1.2% for those aged 85-94 years. The increase with age was detected among men and women both. Our study confirms other findings showing that the prevalence of active epilepsy increases with age in the elderly. The prevalence figures in our study were high as compared with those of other population-based studies. Epilepsy appears to be a major cause of morbidity in the elderly. PMID- 8635425 TI - The clinical course of epilepsy and its psychosocial correlates: findings from a U.K. Community study. AB - As part of a large community-based study, we retrospectively examined the clinical course of epilepsy in an unselected population of people who had a recent history of seizures or were receiving antiepileptic drugs (AEDs). Clinical information was collected from medial records, and information about psychosocial functioning was obtained by means of postal questionnaires sent to identified subjects. The response rate to the postal questionnaire was 71%. There were some deficiencies in the recording of clinical data, which is not unusual since data were taken from records held by primary physicians rather than from hospital clinics. Nevertheless, findings regarding the clinical course of epilepsy corresponded to those of earlier studies. Fifty-seven percent of the sample had had at least a 2-year seizure-free period and 46% of subjects were currently in a remission of at least 2-year duration. There was a clear relationship between current seizure frequency and levels of anxiety and depression, perceived impact of epilepsy, perceived stigma, and marital and employment status. The relationship of seizure frequency and other clinical variables to psychosocial function was explored by multivariate analysis techniques. The amount of variation in scores on the various measures of function accounted for by the clinical variables was small. The most important predictor was current seizure activity, which was the first variable to enter the regression analyses for six of the eight measures of psychosocial function considered. Age at epilepsy onset also emerged as a significant predictor for depression, stigma, and marital status. In individuals with epilepsy in remission, there was little evidence that psychosocial functioning was associated with length of remission, a finding which may in part reflect the nature of this study population. The results indicate that there are several more important predictors of psychopathology and social dysfunction in epilepsy and suggest several implications for treatment interventions. PMID- 8635426 TI - Partial epilepsy of long duration: changing semiology with age. AB - There are few data on changing patterns of localization-related epileptic syndromes with time and particularly on changes in seizure semiology as patients age. We retrospectively reviewed 53 patients aged > 60 years who had had partial epileptic seizures for a mean duration of 44 years. In 29 patients, seizures became progressively less elaborate and briefer with time. In 20 patients, seizures were unchanged. In four patients, seizures worsened, with the appearance of drop attacks, secondarily generalized seizures, increasing drug resistance, and mental deterioration. These latter 4 patients had cerebrovascular disease with multiple strokes. PMID- 8635428 TI - Cognitive consequences of two-thirds anterior temporal lobectomy on verbal memory in 144 patients: a three-month follow-up study. AB - Previous studies have shown that left temporal lobectomy for intractable epilepsy can lead to verbal memory deficits. However, patients with left temporal lobe epilepsy (LTLE) frequently have impaired verbal memory preoperatively. The present analysis of 144 patients who underwent temporal lobe resections for either left (n = 68) or right (n = 76) temporal lobe epilepsy (LTLE, RTLE) addressed the questions of (a) whether a left two-thirds anterior temporal lobectomy (ATL) increases deficits in these qualitative aspects of verbal memory already impaired preoperatively, and (b) whether other aspects of verbal memory are additionally affected. We also evaluated possible determinants of preoperative abilities and postoperative changes, using multiple regression analysis. Preoperatively, patients with LTLE differed from patients with RTLE only in poorer performance on measures of long-term consolidation/retrieval (delayed recall). This is related to hippocampal pathology and seizure severity. Only left temporal lobe resections resulted in significant deterioration in verbal learning and memory. Acquisition over learning trials and recognition deteriorated most markedly, whereas performance in long-term consolidation/retrieval showed only minor changes. Preoperative performance levels, chronological age, the extent of the en bloc resection, preoperative performance on figural memory, and preoperative seizure severity were valuable determinants of postoperative changes in acquisition and recognition. In contrast, changes in consolidation/retrieval related only to preoperative ability. Left two-thirds ATL leads to new impairment in addition to preexisting memory deficits. The finding that left temporal lobectomy affects verbal acquisition and recognition more than long-term consolidation/retrieval, including the different determinants of these changes, most likely reflects the differential effects of surgery on mesial temporal and neocortical temporal functions. PMID- 8635427 TI - Memory tests distinguish between patients with focal temporal and extratemporal lobe epilepsy. AB - We examined the ability of preoperative memory performance to distinguish between patients who had been diagnosed as having left (LTLE, n = 31), right (RTLE, n = 37), and extra-(ETLE, n = 17) temporal lobe focal epilepsy. All patients eventually underwent surgical resections. Analyses indicated that the ETLE group performed better than the RTLE group on nonverbal memory measures and better than the LTLE group on verbal memory measures. Discriminant function analyses indicated that use of a combination of measures that assess different aspects of memory were of significant value in distinguishing between patients with focal TLE and ETLE. This approach, as compared the use of single measures, improved classification rates of all three groups. The best single predictor of group membership, an index of verbal learning, yielded a 47% overall correct classification rate, with sensitivities ranging from 25 to 59%, and performed at worse than chance levels in classifying RTLE patients. A multivariate approach, which included an index of verbal and nonverbal learning, incidental nonverbal memory, and consolidation of organized and rote verbal material, yielded a 65% correct classification rate, with sensitivities ranging from 57 to 75%. This compares favorably with the other noninvasive techniques for lateralizing epileptogenic lesions. PMID- 8635429 TI - Assessment of drowsiness in epilepsy patients receiving chronic antiepileptic drug therapy. AB - Drowsiness is a common complaint among patients with epilepsy taking antiepileptic drugs (AEDs) and may be of particular importance because of the potential effects on cognitive abilities. We used a novel EEG-based measure (the Awake Maintenance Task, AMT) to determine objectively whether patients on chronic, stable AED therapy had impaired ability to maintain wakefulness. Thirty patients receiving AEDs [carbamazepine (CBZ), phenytoin (PHT), phenobarbital (PB), valproate (VPA)] were compared to 35 healthy controls, 12 seizure patients not taking AEDs, and 16 patients with multiple sclerosis. A structured EEG recording was conducted under controlled conditions, and subjects were tested to determine their ability to maintain wakefulness during a 6-min unstimulated trial. Testing also included Digit Symbol, auditory reaction time, and subjective measures of fatigue or sleepiness [Profile of Mood States (POMS), Stanford Sleepiness Scale (SSS)]. Patients receiving AEDs had a mean total drowsiness score of 101 s compared with < or = 12 s for each of the three control groups (P < 0.001). One third of the AED-treated patients had > 120 s of drowsiness, in contrast to only 1 of 63 controls (p < 0.001). Among patients receiving AEDs, objective EEG drowsiness did not correlate with AED levels or performance measures. Untreated seizure patients had significantly greater complaints of lack of vigor despite a near absence of objective drowsiness on the AMT. These results suggest that epilepsy patients receiving chronic AED therapy have impaired ability to maintain wakefulness. Patient self-reports of AED-related sleepiness may not accurately represent this problem. PMID- 8635430 TI - Amygdala kindling of forebrain seizures and the occurrence of brainstem seizures in genetically epilepsy-prone rats. AB - Forebrain seizures were kindled in rats by daily electrical stimulation of the amygdala. Genetically epilepsy-prone rats scoring 9 (GEPR-9s) on the seizure severity scale during audiogenic seizure (AGS) screening ("brainstem seizure experienced") required fewer stimulations to achieve fully kindled seizures (forelimb clonus with rearing and falling) than control rats. AGS-naive GEPR-9s required an intermediate number of stimulations, indicating a role for both genetic predisposition and previous acoustically evoked brainstem seizure experience. Other forebrain kindling indices such as afterdischarge threshold/duration and seizure latency/duration also involved genetic as well as phenotypic (previous seizure experience) factors. In most GEPR-9s in both groups, severe brainstem seizures occurred after forebrain stimulation. The occurrence of brainstem seizures had a random nature and was not related to the sequence of kindling-dependent forebrain seizure progression. The lack of a difference in the occurrence of brainstem seizures between seizure-experienced and AGS-naive GEPR 9s suggest that genetic predisposition is the major factor in forebrain seizure induced activation of brainstem seizure circuitry. This brainstem seizure activity appears to model pertinent aspects of secondary generalization observed in human partial seizures. PMID- 8635431 TI - Change in neurotrophins and their receptor mRNAs in the rat forebrain after status epilepticus induced by pilocarpine. AB - We studied the effects of status epilepticus (SE) induced by lithium chloride/pilocarpine treatment on gene expression of neurotrophins of the nerve growth factor (NGF) family and of their high-affinity receptors of the tyrosine protein kinase (trk) family in the forebrain. Using in situ hybridization (ISH), we demonstrated an early (3 h after treatment) increase in brain-derived neurotrophic factor (BDNF) and trkB mRNA expression in the dentate gyrus, amygdala, and piriform cortex, as well as widespread increases in the cerebral cortex. NGF mRNA, but not the mRNA of its receptor trkA, was increased in the dentate gyrus. In contrast, 12 h after treatment, neurotrophin-3 (NT-3) decreased, and its receptor trkC mRNA increased. There was no change in NT-4 mRNA levels. All changes were blocked by pretreatment with scopolamine, a muscarinic antagonist. The noncompetitive N-methyl-D-aspartate (NMDA) antagonist ketamine blocked NGF, BDNF, and trkB mRNA increases in the hippocampus and cerebral cortex, but not in the amygdala and piriform cortex. In contrast, ketamine did not affect NT-3 and trkC changes. These results provide a complete description of changes in mRNA levels of neurotrophins and their receptors in the forebrain after SE and supply additional data supporting the view that neurotrophin gene expression is related to abnormal neuronal activity. PMID- 8635432 TI - Weeping during psychogenic nonepileptic seizures. AB - Ictal weeping may help distinguish psychogenic nonepileptic seizures (PNES) from epileptic seizures. However, the prevalence of weeping during PNES or epileptic seizures is unknown. We reviewed videotapes of recorded events in 84 consecutive patients with PNES or epilepsy. Weeping was observed during 14% of PNES in 31% of patients with PNES. Ictal weeping was not observed in any epileptic seizures occurring in 48 patients. The differences were highly significant. One patient with epileptic seizures originating in the right temporal lobe wept soon after the end of a seizure. Depression was not more common in PNES patients with than without ictal weeping. Weeping during an apparent seizure strongly argues that the event is not epileptic. PMID- 8635433 TI - Emanuel Swedenborg. AB - How is it that the name of a brilliant 18th century scientist and philosopher, many of whose exceptional achievements were often advanced for his time, is almost never mentioned in the annals of science? And how did it happen that a man very deeply dedicated to the advancement of science experienced a vision that completely altered the course of his life? We suggest, based on his extensive self-analytical writings, that the source of his spiritual experiences was temporal lobe epilepsy (TLE) and that he is among the group of creative religious thinkers also suspected or known to have had epilepsy, from St. Paul and Mohammed to Dostoevsky, who have changed Western civilization. PMID- 8635434 TI - Dermal exposure to environmental contaminants in the Great Lakes. AB - This paper reviews the literature to determine the importance of the dermal route of exposure for swimmers and bathers using Great Lakes waters and summarizes the chemical water contaminants of concern in the Great Lakes along with relevant dermal absorption data. We detail in vivo and in vitro methods of quantifying the degree of dermal absorption and discuss a preference for infinite dose data as opposed to finite dose data. The basic mechanisms of the dermal absorption process, routes of chemical entry, and the environmental and physiological factors affecting this process are also reviewed, and we discuss the concepts of surface slick exposure to lipophilic compounds and the adsorption of contaminants to water sediment. After presenting mathematical constructs for calculating the degree of exposure, we present in vitro data concerning skin absorption of polyaromatic hydrocarbons adsorbed to Great Lakes water sediment to show that in a worst-case scenario exposure via the dermal route can be equally important to the oral route. We have concluded that prolonged exposure of the skin, especially under conditions that may enhance dermal absorption (e.g., sunburn) may result in toxicologically significant amounts of certain water contaminants being absorbed. It is recommended that swimming should be confined to public beaches, people should refrain from swimming if they are sunburned, and skin should be washed with soap as soon as possible following exposure. Future studies should be conducted to investigate the importance of the dermal exposure route to swimmers and bathers. PMID- 8635437 TI - Human health, the Great Lakes, and environmental pollution: a 1994 perspective. PMID- 8635435 TI - Approaches to the evaluation of chemical-induced immunotoxicity. AB - The immune system plays a crucial role in maintaining health; however, accumulating evidence indicates that this system can be the target for immunotoxic effects caused by a variety of chemicals including the environmental pollutants of polychlorinated biphenyls, chlorinated dibenzo-p-dioxins, pesticides, and heavy metals. Adverse chemical-induced immunomodulation, which is studied within the discipline of immunotoxicology, may be expressed either as immunosuppression/immunodepression or immunoenhancement. The former may be manifested either as decreased resistance to opportunistic viral, bacterial, fungal, and other infectious agents or increased susceptibility to cancer. Immunoenhancement on the other hand may either increase the risk of autoimmune reactions or result in allergic reactions. This paper attempts to integrate several aspects of the immune system that are relevant to the assessment of potentially immunotoxic chemicals. PMID- 8635436 TI - Immunotoxicity of heavy metals in relation to Great Lakes. AB - Heavy metals including mercury, lead, and cadmium are present throughout the ecosystem and are detectable in small amounts in the Great Lakes water and fish. The main route of exposure of humans to these metals is via the ingestion of contaminated food, especially fish. Extensive experimental investigations indicated that heavy metals alter a number of parameters of the host's immune system and lead to increased susceptibility to infections, autoimmune diseases, and allergic manifestations. The existing limited epidemiologic data and data derived from in vitro systems in which human peripheral blood leukocytes were used suggested that the human immune system may also be at increased risk following exposure to these metals. The magnitude of the risk that the presence of such metals in the Great Lakes may pose to the human immune system, and consequently to their health, is not known. In this review, the available data with respect to potential adverse effects of heavy metals on the immune system of humans and experimental animals are discussed, and additional data requirements are suggested. PMID- 8635438 TI - Immunotoxicity of PCBs (Aroclors) in relation to Great Lakes. AB - Polychlorinated biphenyls (PCBs) are among the most widespread environmental pollutants and a prominent contaminant of the Great Lakes basin. Due to their resistance to biodegradation and lipophilic properties, PCBs bioaccumulate in fish tissues and in fish-eating humans. PCBs are also known to cross the placenta and to be excreted into the mother's milk, thus predisposing the infant to potentially adverse health effects. For example, a higher incidence of bacterial infections was reported for breast-fed infants born to mothers who consumed large amounts of Great Lakes fish compared to the incidence in control infants whose mothers ingested low amounts of fish. While data regarding the PCB-induced immunotoxic effects in humans are scarce, data derived from the use of experimental animals, including nonhuman primates, indicate that the immune system is a potential target for the immunotoxic effects of PCBs. Such studies have used the commercially available PCB mixtures alone. However, PCBs have the potential of partially antagonizing the effects of other structurally related compounds including the highly toxic dioxins, which are also present in small amounts in the Great Lakes. Thus, to fully evaluate the magnitude of the immunotoxic risk PCBs pose to humans, consideration should be given to investigations in which the interactive effects of PCBs are combined with other contaminants present in the Great Lakes. PMID- 8635439 TI - Immunological effects of chlorinated dibenzo-p-dioxins. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar halogenated aromatic hydrocarbons cause a broad range of immunologic effects in experimental animals including decreased host resistance to infectious disease and suppressed humoral and cell-mediated immune responses. In the mouse, TCDD immunotoxicity has been shown to be an aryl hydrocarbon (Ah) receptor-dependent process. However, despite considerable research, the biochemical and molecular alterations that occur subsequent to Ah receptor activation that lead to altered immune reactivity remain to be elucidated. In addition to immune suppression, TCDD promotes inflammatory responses. This effect may result from an upregulation of the production of inflammatory cytokines such as interleukin-1 and tumor necrosis factor. Nonhuman primates exposed to TCDD show suppressed antibody responses and changes in lymphocyte subsets in the peripheral blood. The immunotoxic effects of TCDD in humans are poorly characterized, and few studies have examined the immune status of individuals with known, documented exposure to TCDD. It is important for laboratory research to focus on defining TCDD-sensitive immunologic biomarkers in animal models that can also be used in human subjects. Understanding the mechanisms that underlie species differences in TCDD immunotoxicity is also of critical importance for extrapolation of effects seen in laboratory animals to man. PMID- 8635440 TI - Pesticide-induced immunotoxicity: are Great Lakes residents at risk? AB - Several organophosphate and organochlorine compounds, including pesticides commonly found in the Great Lakes basin, have the potential to induce immunotoxicity. Because of biomagnification and accumulation in the food chain, Great Lakes residents may inadvertently be exposed to these compounds and thus face increased risk of immune dysfunction. In spite of the laboratory animal data and evidence from occupational exposures that suggest immunotoxicity, there is no definitive evidence as yet that environmental exposure to these xenobiotics poses a significant threat to the human immune system that is sufficient to predispose residents of the Great Lakes basin to increased disease. However, uncertainties with regard to exposure levels, predictability of tests, suitability of the animal models, and immune reserve cannot be ruled out when making risk assessment decisions such as this. PMID- 8635441 TI - The reproductive toxicology of Great Lakes contaminants. AB - The Great Lakes basin is characterized as a heavily populated and industrialized region in which a large number of environmental contaminants have been identified. Both the scientific community and the public have voiced concern that contaminants present in the Great Lakes may pose undue risk to human reproduction. Evidence from animal experiments, wildlife studies, and reports of occupational and accidental human exposures indicate that chemical contaminants can adversely affect reproduction. The purpose of this paper is to review the reproductive toxicity of some of the many contaminants known to be present in the Great Lakes. Since the number of chemicals present in the Great Lakes is far too great for each to be adequately reviewed here, discussion will be limited to those contaminants that have been identified in human serum, ovarian follicular fluid, and semen obtained from people residing in the Great Lakes region. It is concluded that a) the data at present is too limited to support the notion that reproduction, in the general population, has been impaired by exposure to chemicals present in the Great Lakes; b) the lack of data in some cases such as for hexachloroethane and 1,2,4-trichlobenzene does provide reason for concern and underscores the need for further research in this area; and c) the potential for a number of the compounds, including polychlorinated biphenyls (PCBs) and 1,1,1 trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), to disrupt endocrine functions suggests that additive or synergistic effects of these compounds may already be causing adverse effects on reproduction in sensitive individuals, which needs to be explored. PMID- 8635442 TI - Biomarkers for Great Lakes priority contaminants: halogenated aromatic hydrocarbons. AB - One of the major goals of the Great Lakes Action Plan is to actively accumulate and assess toxicological information on persistent toxic substances found in the Great Lakes basin. As part of Health Canada's commitment to this plan, a review of biomarkers for the environmental contaminants polychlorinated biphenyls (PCBs) and polychlorinated dibenzodioxins/dibenzofurans (PCDDs/PCDFs) was conducted. In general, while food consumption was identified as the major source of human exposure to both contaminant groups, certain commodities, such as fish, milk and dairy products, and meat, were found to predominate. Due to the ubiquitous nature of these environmental contaminants and their propensity to bioaccumulate, all humans will have detectable body burdens, which in certain cases can be positively associated with the consumption of particular foods (i.e., PCBs and freshwater fish from the Great Lakes). When dealing with environmental exposure only, relating specific effect biomarkers to contaminant exposure or tissue levels was difficult, due in part to the complex nature of the exposure and the nonspecific nature of the effect. For PCBs, the most likely biomarkers of effect included some form of alteration in lipid metabolism (serum triglyceride/cholesterol levels) and elevation of hepatic-related enzymes, aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT). Cross species extrapolation also indicates the potential for neurotoxicologic effects to occur in humans. For PCDDs/PCDFs, dermatologic lesions (chloracne) and indications of hepatic enzyme induction have been documented, but primarily due to occupational or high acute accidental exposures. Recent evidence suggests that neonates may represent a potential at-risk population due to relatively high exposure to PCDDs/PCDFs, as with PCBs, during breast feeding as compared to standard adult dietary intake. Future areas of potential benefit for biomarker development include immunologic and endocrine effects, primarily based on biologic plausibility from experimental animal research. PMID- 8635444 TI - Radionuclides in the Great Lakes basin. AB - The Great Lakes basin is of radiologic interest due to the large population within its boundaries that may be exposed to various sources of ionizing radiation. Specific radionuclides of interest in the basin arising from natural and artificial sources include 3H, 14C, 90Sr, 129I, 131I, 137Cs, 222Rn, 226Ra, 235U, 238U, 239Pu, and 241Am. The greatest contribution to total radiation exposure is the natural background radiation that provides an average dose of about 2.6 mSv/year to all basin residents. Global fallout from atmospheric nuclear weapons tests conducted before 1963 has resulted in the largest input of anthropogenic radioactivity into the lakes. Of increasing importance is the radionuclide input from the various components of the nuclear fuel cycle. Although the dose from these activities is currently very low, it is expected to increase if there is continued growth of the nuclear industry. In spite of strict regulations on design and operation of nuclear power facilities, the potential exists for a serious accident as a result of the large inventories of radionuclides contained in the reactor cores; however, these risks are several orders of magnitude less than the risks from other natural and man-made hazards. An area of major priority over the next few decades will be the management of the substantial amounts of radioactive waste generated by nuclear fuel cycle activities. Based on derived risk coefficients, the theoretical incidence of fatal and weighted nonfatal cancers and hereditary defects in the basin's population, attributable to 50 years of exposure to natural background radiation, is conservatively estimated to be of the order of 3.4 x 10(5) cases. The total number of attributable health effects to the year 2050 from fallout radionuclides in the Great Lakes basin is of the order of 5.0 x 10(3). In contrast, estimates of attributable health effects from 50 years of exposure to current nuclear fuel cycle effluent in the basin are of the order of 2 x 10(2). Although these are hypothetical risks, they show that the radiologic impact of man-made sources is very small compared to the effects of normal background radiation. PMID- 8635445 TI - Viewing distance variation and related ophthalmological changes in office activities with and without VDUs. AB - This experimental study was conducted on 14 volunteer female clerks to measure viewing distance variation and related ophthalmological changes in office activities with (session 1) and without (session 2) VDU. A video camera connected to an electronic elaborator was used to determine eye-screen distance of the VDU operators during the task. Before and after each of the two work sessions far refraction, far phorias, near point of accommodation (APP), and fusional convergence were measured. Blinking rate of each subject was measured by a transducer only during the "VDU session'. The objective measurements of the eye screen distance, whose range was between 48.42 and 65.33 cm, allowed a precise quantification of the occupational visual load during session 1. The accommodation was about 1.5-2D on average and the fusional convergence 10-13 delta on average. The symptomatology shows a number of disturbances in session 1 (71.4%) clearly higher than in session 2 (35.7%). The study of refraction before and after the task has shown a slight excess of accommodation compared to the theoretical value in both sessions, more marked in session 1. The APP shows no statistical significative variation. A tendency to exophoria was registered at the beginning and at the end of the trial and the fusional convergence clearly decreased, especially in the first session. PMID- 8635447 TI - An experimental evaluation of instantaneous self-assessment as a measure of workload. AB - Instantaneous self-assessment (ISA) is a technique that has been developed as a measure of workload to provide immediate subjective ratings of work demands during the performance of primary work tasks such as air traffic control. This paper reports a study that compared the results of ISA with those gathered from other established workload evaluation techniques; subjective ratings collected at the end of the task, mean heart rate and heart rate variability, and error in the primary task of tracking. ISA ratings were found to be correlated significantly with the post-task ratings of workload, heart rate variability, and task performance. Generally each of the techniques was sensitive to variations in task difficulty. However, performance on the primary tracking task was found to be poorer during periods when ISA responses were required, regardless of whether they were spoken or manual responses. This finding suggests that the usefulness of the technique is limited in comparison to less intrusive measures of workload. PMID- 8635446 TI - The effects of weight levels and gloves on the ability to discriminate weight difference. AB - In this study, six different types of gloves commonly used in industry (surgical, cotton, nylon, leather, rubber, and whizard knife hand gloves) were evaluated at four different weight levels (8.2, 3, 1.5 and 0.25 kg). Six males and six females voluntarily took part in this study. The dependent variable was the difference limen (or difference threshold) (abbreviated as DL(%)) of the discriminating weight difference (DWD). A nested-factorial design with the subject nested under gender was employed. The results indicate that the weight level had a negatively significant effect on the DL(%). The mean difference thresholds were 3.49, 5.39, 6.08, and 9.45 for the weight levels at 8.2, 3, 1.5, and 0.25 kg, respectively. The individual difference becomes obvious when the weight level is relatively light (0.25 kg). However, the effects of gender, glove, gender x weight, and weight x glove were found to be insignificant, and there was no significant correlation between the DL(%) and glove characteristics (thickness and weight). Subsequently, four additional weight levels (0.01, 0.5, 1 and 15 kg) were considered, and a psychophysical equation was derived: DL(%) = 34.33 x W(0.238) (R2 = 0.98), where W represents the weight level. These results support the power function. A minimum weight difference with 90% confidence of correct detection was suggested. PMID- 8635448 TI - Visual search performance with complete and incomplete visual lobe areas. AB - A simulation study of the effects of visual lobe shape and search strategy on search performance is reported. Using a homogeneous pattern search task that did not dictate eye fixation movements (as in reading), three basic categories of strategy and two lobe shapes were tested. Random strategies with controlled overlap of lobe areas, representing various degrees of memory, were found to produce better performance (faster search times with equal or better detection rates) than either a completely random strategy or systematic strategies with various degrees of controlled visual lobe area overlap. The controlled overlap random strategies were far better able to compensate for non-optimum interfixation distances and distortion of visual lobe shape than systematic strategies. PMID- 8635449 TI - Road traffic offending, stress, age, and accident history among male and female drivers. AB - This paper draws on data gathered during a larger study of driving behaviour to explore possible connections between stress, offending against the traffic laws, and accident rates, and gives particular attention to comparisons between male and female drivers. Data were gathered from 422 drivers by a postal questionnaire, and further measures of stress were obtained during semi structured interviews with a specially selected subset of 66 of them. Analyses of variance investigated links between offending rates, accident history, and up to seven measures of stress, taking age and gender into account. In the results accident history, although positively related to offending, played little part in links involving stress variables. The principal findings were that (a) stress, both on and off the road, was positively associated with offending among both male and female drivers, and (b) although females overall offended less than males, females experienced more stress than males whatever their level of offending. One possible interpretation of these results is suggested by the feminist literature, which could account for both the higher levels of stress and lower levels of offending found among women drivers. However, there is unlikely to be a clear-cut gender divide in offending-stress relationships, and some small groups of female drivers in the study behaved like male ones. It is suggested that people's driving reflects their lifestyles, and that women drivers' patterns of offending and stress will resemble those of male drivers to the extent that their lives and concerns are similar to men's. It is concluded that this position warrants further research. PMID- 8635443 TI - Neurotoxicity of lead, methylmercury, and PCBs in relation to the Great Lakes. AB - There is ample evidence identifying lead, methylmercury, and polychlorinated biphenyls (PCBs) as neurotoxic agents. A large body of data on the neurotoxicity of lead, based on both epidemiologic studies in children and animal models of developmental exposure, reveals that body burdens of lead typical of people in industrialized environments produce behavioral impairment. Methylmercury was identified as a neurotoxicant in both adults and the developing organism based on episodes of human poisoning: these effects have been replicated and extended in animals. High-dose PCB exposure was recognized as a developmental toxicant as a result of several episodes of contamination of cooking oil. The threshold for PCB neurotoxicity in humans is less clear, although research in animals suggests that relatively low-level exposure produces behavioral impairment and other toxic effects. Tissue levels in fish below which human health would not be adversely affected were estimated for methylmercury and PCBs based on calculated reference doses (RfDs) and estimated fish intake. Present levels in fish tissue in the Great Lakes exceed these levels for both neurotoxicants. Great Lakes fish and water do not pose a particular hazard for increased lead intake. However, the fact that the present human body burden is in a range at which functional deficits are probable suggests that efforts should be made to eliminate point sources of lead contamination in the Great Lakes basin. PMID- 8635450 TI - Surface electromyography as a tool to study the head rest comfort in cars. AB - In this study, two methods were presented in order to evaluate objectively the comfort of head rests in cars using surface electromyography (SEMG). First, the Amplitude Probability Density Function (APDF) was computed. Extremely low level intensity activities were measured. No significant differences were observed on SEMG global activity between experiments with and without a head rest. Second, instead of computing one APDF, several APDF were calculated over time. Analyses focused on the rate of evolution of APDF parameters. This was a time varying APDF (TAPDF). This last method allowed a better diagnosis than the first one. For drivers, significant decreases in SEMG activity were observed when using a head rest. For passengers, a contradictory increase in SEMG activity was observed during experiments with a head rest. This observation was explained by the seat back angle chosen by passengers. PMID- 8635451 TI - Reliability of myoelectric trapezius muscle activity in repetitive light work. AB - The aim of this study was to evaluate the reliability of selected electromyographic (EMG) parameters describing the muscle activity in repetitive light work. EMG from the trapezius muscle of twelve female workers were recorded during a 20-min work session, morning and afternoon; Monday, Wednesday, Friday and the following Monday. The estimated reliability coefficient was 0.59 for static muscle activity and 0.85 for short interruptions in muscle activity (EMG gaps) during work. The reliability coefficient was 0.94 for the EMG signal's average median frequency of test contractions associated with the work session. The median frequency declined significantly during the work session. No time trend was found during the day or the week for these parameters. Consequently, it is possible to get representative measurements of the mentioned EMG parameters during repetitive light work by a recording taken at any time of the day or week. PMID- 8635452 TI - Exploring the functional robustness of an enzyme by in vitro evolution. AB - The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations. In vitro protein evolution seeks to accelerate this process. RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies. Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement. The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness. Complete substitution of the segment still allowed fixation of mutations. By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection. PMID- 8635453 TI - Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells. AB - Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions. PMID- 8635454 TI - T cells from baxalpha transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA damage-induced cell death in the absence of p53. AB - Baxalpha was isolated due to its interaction with Bcl-2. Baxalpha overexpression in an interleukin (IL)-3 dependent cell line accelerates apoptosis upon removal of the cytokine. The ratio of Baxalpha to Bcl-2 appears to be crucial for the effect. To study the action of the bax gene product in vivo, we have generated transgenic mice overexpressing Baxalpha specifically in T cells. Such T cells show accelerated apoptosis in response to gamma-radiation, dexamethasone and etoposide. By crossing baxalpha mice with bcl-2 transgenics we show that the critical nature of the Baxalpha:Bcl-2 ratio holds in primary T cells and that it can be manipulated to elicit a strong response to previously resisted stimuli. p53 has a role in the regulation of apoptosis in response to DNA-damaging agents. p53 directly activates transcription of the bax gene. The presence of the baxalpha transgene accelerated apoptosis in thymocytes from both p53-l- and p53+l mice in response to dexamethasone. Thymocytes from p53-l- mice with the baxalpha transgene showed similar resistance to apoptosis by DNA-damaging agents as did p53-l- mice without the transgene. Baxalpha overexpression alone cannot restore the DNA damage apoptosis pathway, suggesting that p53 is required to induce or activate other factor(s) to reconstitute the response fully. PMID- 8635455 TI - The Mas20p and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: evidence for a single hetero-oligomeric receptor. AB - Protein import into yeast mitochondria is mediated by four integral outer membrane proteins which function as import receptors. These proteins (termed Mas20p, Mas22p, Mas37p and Mas70p) appear to exist as two subcomplexes: a Mas37p Mas70p heterodimer and a less well characterized Mas20p-Mas22p complex. The subcomplexes interact functionally during protein import, but it has remained uncertain whether they are in direct contact with each other in vivo. Here we show that Mas20p and Mas70p can be cross-linked in intact mitochondria, or co immunoprecipitated from digitonin-solubilized mitochondria. Furthermore, the cytosolic domains of these two proteins interact in the 'two-hybrid' system. Association of Mas20p and Mas70p is virtually abolished by a mutation in the single tetratricopeptide motif in Mas20p. This mutation specifically inhibits import of precursors that are first recognized by Mas37p-Mas70p and only then transferred to Mas20p-Mas22p. We conclude that the two receptor subcomplexes of the mitochondrial protein import receptor interact in vivo via their Mas20p and Mas70p subunits and that this interaction is functionally important. PMID- 8635456 TI - Linking microfilaments to intracellular membranes: the actin-binding and vesicle associated protein comitin exhibits a mannose-specific lectin activity. AB - Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. PMID- 8635457 TI - Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation. AB - Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton. PMID- 8635458 TI - Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. AB - The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc). PMID- 8635459 TI - Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. AB - A double mutation in the alpha-secretase site in the betaA4 region of mouse amyloid precursor protein (APP) reduced its secretion from COS cells, polarized MDCK cells and rat primary neurons. Expression of this mutant in the brain of mice, using the neuron-specific elements of the mouse Thy-1 gene promoter, resulted in transgenic mice that became progressively hyperactive, displayed seizures and died prematurely. In three different transgenic lines the severity of the phenotype was related directly to the expression levels of the transgene, estimated by both mRNA and protein levels. In addition, homozygous mice derived from each transgenic strain showed more severe symptoms which also occurred earlier in life than in heterozygotes. The observed symptoms were, however, not essentially different in the different lines. Increased aggressiveness, disturbed responses to kainic acid and N-methyl-D-aspartate, neophobia and deficiency in exploratory behavior were demonstrated in these mice. In the brain, the observed neuropathological changes included necrosis, apoptosis and astrogliosis in the hippocampus, cortex and other areas. The data demonstrate that incomplete or incorrect alpha-secretase processing of APP results in severe neurotoxicity and that this effect is expressed in a dominant manner. PMID- 8635460 TI - Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice. AB - Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses. PMID- 8635461 TI - Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. AB - Inactivating mutations in distinct G protein-coupled receptors (GPCRs) are currently being identified as the cause of a steadily growing number of human diseases. Based on previous studies showing that GPCRs are assembled from multiple independently stable folding units, we speculated that such mutant receptors might be functionally rescued by 'supplying' individual folding domains that are lacking or misfolded in the mutant receptors, by using a co-expression strategy. To test the feasibility of this approach, a series of nine mutant V2 vasopressin receptors known to be responsible for X-linked nephrogenic diabetes insipidus were used as model systems. These mutant receptors contained nonsense, frameshift, deletion or missense mutations in the third intracellular loop or the last two transmembrane helices. Studies with transfected COS-7 cells showed that none of these mutant receptors, in contrast to the wild-type V2 receptor, was able to bind detectable amounts of the radioligand, [3H]arginine vasopressin, or to activate the G(S)/adenylyl cyclase system. Moreover, immunological studies demonstrated that the mutant receptors were not trafficked properly to the cell surface. However, several of the nine mutant receptors regained considerable functional activity upon co-expression with a C-terminal V2 receptor peptide spanning the sequence where the various mutations occur. In many cases, the restoration of receptor activity by the co-expressed receptor peptide was accompanied by a significant increase in cell surface receptor density. These findings may lead to the design of novel strategies in the treatment of diseases caused by inactivating mutations in distinct GPCRs. PMID- 8635462 TI - Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. AB - The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. PMID- 8635463 TI - Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25. AB - The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast Schizosaccharomyces pombe. We have found that cdc25 is ubiquitinated and have cloned pub1, a gene which regulates this event. Pub1 contains a region highly homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. Disruption of pub1 elevates the level of cdc25 protein in vivo rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis which can be rescued by loss of cdc25 function. A ubiquitin-thioester adduct of pub1 was isolated from fission yeast and disruption of pub1 dramatically reduced ubiquitination of cdc25 in vivo. These results suggest that pub1 directly ubiquitinates cdc25 in vivo. PMID- 8635464 TI - Functional dissection of the mouse Hox-a5 gene. AB - The Hox genes are clustered in evolutionarily conserved complexes and encode DNA binding proteins that determine positional identity. Ubiquitous expression of fly or mammalian Hox proteins in Drosophila embryos provides an assay for gene function, since different Hox genes induce characteristic homeotic transformations. Drosophila Sex combs reduced (Scr) and its murine cognate Hox-a5 produce identical transformations in transgenic flies. To study the contributions of domains conserved between the two proteins, truncated versions of mouse Hox-a5 were assayed for their ability to activate transcription in cultured cells and to induce homeotic transformation and activate target gene expression in transgenic embryos. The homeodomain is essential for protein function and/or nuclear targeting; the N-terminal region contributes to transcription activity and transformation potential in the embryo, but plays no role in determining functional specificity. The YPWM motif is essential for biological specificity, although it does not contribute to transcriptional activation potential. It was recently shown that the Hox-a5 YPWM motif is necessary for in vitro interactions with the co-factor Pbx1. Our results suggest that this type of protein-protein interaction may be essential for the biological activities of Hox-a5 and Scr. PMID- 8635465 TI - Developmental timing and tissue specificity of heterochromatin-mediated silencing. AB - Heterochromatic position-effect variegation (PEV) describes the mosaic phenotype of a euchromatic gene placed next to heterochromatin. Heterochromatin-mediated silencing has been studied extensively in Drosophila, but the lack of a ubiquitous reporter gene detectable at any stage has prevented a direct developmental characterization of this phenomenon. Current models attribute variegation to the establishment of a heritable silent state in a subset of the cells and invoke differences in the timing of silencing to explain differences in the patch size of various mosaic patterns. In order to follow the course of heterochromatic silencing directly, we have generated Drosophila lines variegating for a lacZ reporter that can be induced in virtually all cells at any developmental stage. Our data indicate that silencing begins in embryogenesis and persists in both somatic and germline lineages. A heterogeneity in the extent of silencing is also revealed; silencing is suppressed in differentiated tissues but remains widespread in larval imaginal discs containing precursor cells for adult structures. Using eye development as an example, we propose that the mosaic phenotype is determined during differentiation by a variegated relaxation in heterochromatic silencing. Though unpredicted by prevailing models, this mechanism is evident in other analogous systems. PMID- 8635466 TI - The response regulator RssB controls stability of the sigma(S) subunit of RNA polymerase in Escherichia coli. AB - The rpoS-encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post-transcriptional growth phase-related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C-terminal output domain and is the first known response regulator involved in the control of protein turnover. PMID- 8635467 TI - Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics. AB - Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS. Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation. Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes. Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils. Despite their functional similarities, each molecule is capable of independent activities. Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro. We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression. PMID- 8635468 TI - Crystal structure of the RNA binding ribosomal protein L1 from Thermus thermophilus. AB - L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible. PMID- 8635470 TI - The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E. AB - The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule. PMID- 8635469 TI - Reading two bases twice: mammalian antizyme frameshifting in yeast. AB - Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30 fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal. PMID- 8635471 TI - Cytoplasmic retention and nuclear import of 5S ribosomal RNA containing RNPs. AB - Nuclear export of newly transcribed 5S ribosomal RNA in Xenopus oocytes occurs in the context of either a complex with the ribosomal protein L5 (5S RNP) or with the transcription factor IIIA (7S RNP). Here we examine nuclear import of 5S RNA, L5 and TFIIIA. The 5S RNP shuttles between nucleus and cytoplasm and only 5S RNA variants which can bind to L5 gain access to the nucleus. The 7S RNP is retained in the cytoplasm. Only TFIIIA which is not bound to 5S RNA is imported into the nucleus. As a novel mechanism for cytoplasmic retention, we propose that RNA binding masks a nuclear localization sequence in TFIIIA. In contrast to the nuclear import of L5, import of TFIIIA is sensitive towards the nuclear localization sequence (NLS) competitor p(lys)-BSA, suggesting that these two proteins make use of different import pathways. PMID- 8635472 TI - Three recognition events at the branch-site adenine. AB - An adenosine at the branch site, the nucleophile for the first transesterification step of splicing, is nearly invariant in mammalian pre-mRNA introns. The chemical groups on the adenine base were varied systematically and assayed for formation of early spliceosome complexes and execution of the first and second steps of splicing. Recognition of constituents of the adenine is critical in formation of a U2 snRNP-containing complex on a minimal branch-site oligonucleotide. Furthermore, the efficiencies of the first and second chemical steps have different dependencies on the functional groups of the adenine. In total, the chemical groups on the adenine base at the branch site are differentially recognized during at least three different processes in the splicing of pre-mRNA. Moreover, a protein, p14, interacts with the adenine in a base-specific fashion and may mediate early recognition of this base. PMID- 8635473 TI - RNA editing in bryophytes and a molecular phylogeny of land plants. AB - RNA editing has been observed to date in all groups of vascular plants, but not in bryophytes. Its occurrence was therefore assumed to correlate with the evolution of tracheophytes. To gain more insight into both the phylogeny of early land plants and the evolution of mitochondrial RNA editing we have investigated a number of vascular and non-vascular plant species. Contrary to the belief that editing is absent from bryophytes, here we report mitochondrial RNA editing in cox3 mRNA of the liverwort Pellia epiphylla, the mosses Tetraphis pellucida and Ceratodon purpureus and the hornwort Anthroceros crispulus. RNA editing in plants consequently predates the evolution of tracheophytes. Editing is also found in the eusporangiate ferns Ophioglossum petiolatum and Angiopteris palmiformis, the whisk fern Tmesipteris elongata and the gnetopsid Ephedra gerardiana, but was not detected in Gnetum gnemon.cox3 mRNA of the lycopsid Isoetes lacustris shows the highest frequency of RNA editing ever observed in a plant, with 39% of all cytidine residues converted to uridines. The frequency of RNA editing correlates with the genomic GC content rather than with the phylogenetic position of a species. Phylogenetic trees derived from the slowly evolving mitochondrial sequences find external support from the assessments of classical systematics. PMID- 8635475 TI - Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants. AB - Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop. PMID- 8635476 TI - Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases. AB - A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target. Individual target-recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C-terminal part of the variable region of multispecific C5-MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N-terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6-10 'linker' amino acids, they have, depending on the enzyme studied, towards their C terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other. PMID- 8635474 TI - The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography. AB - This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands. PMID- 8635477 TI - Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target. AB - In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5 MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities. PMID- 8635478 TI - Identification of ORD, a Drosophila protein essential for sister chromatid cohesion. AB - Attachment between the sister chromatids is required for proper chromosome segregation in meiosis and mitosis, but its molecular basis is not understood. Mutations in the Drosophila ord gene result in premature sister chromatid separation in meiosis, indicating that the product of this gene is necessary for sister chromatid cohesion. We isolated the ord gene and found that it encodes a novel 55 kDa protein. Some of the ord mutations exhibit unusual complementation properties, termed negative complementation, in which particular alleles poison the activity of another allele. Negative complementation predicts that protein protein interactions are critical for ORD function. The position and nature of these unusual ord mutations demonstrate that the C-terminal half of ORD is essential for sister chromatid cohesion and suggest that it mediates protein binding. PMID- 8635479 TI - Recombination by resolvase to analyse DNA communications by the SfiI restriction endonuclease. AB - The SfiI endonuclease differs from other type II restriction enzymes by cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The nature of this communication event between distant DNA sites was analysed on plasmids with recognition sites for SfiI interspersed with recombination sites for resolvase. These were converted by resolvase to catenanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions between SfiI sites on the same DNA therefore cannot follow the DNA contour and, instead, must stem from their physical proximity. In buffer lacking Mg2+, where SfiI is inactive while resolvase is active, the addition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging its sites and thus isolating the sites for resolvase into separate loops. The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+. PMID- 8635482 TI - The tetrapeptide AcSDKP, a physiological inhibitor of normal cell proliferation, reduces the S phase entry of continuous cell lines. AB - The tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP), a physiological negative regulator of cell proliferation, inhibits the progression of normal quiescent cell to the S phase of the cycle, while it is inactive in the proliferation of permanent cell lines and of freshly isolated leukemic cells. It protects normal hematopoietic stem cells and progenitors from the toxic effects of anticancer drugs. We studied the effects of AcSDKP on the S phase entry of mouse and chicken continuous cell lines MS-K, 3T3, MDCC-PA9, and MDCC-MSB1 lines when they are cultured under these defined conditions. They show that AcSDKP acts on cells previously partially synchronized by culture under conditions of low serum concentrations or serum starvation. Our results demonstrate that AcSDKP reduces the proliferation of these cell of continuous cell lines as it does on hepatocytes or hematopoietic cells in vivo or on freshly isolated cells in vitro, by blocking or retarding their entry into S phase from early G1. PMID- 8635480 TI - Tissue-specific posttranslational modification of the small heat shock protein HSP27 in Drosophila. AB - Drosophila sHSPs (small heat shock proteins) are expressed in the absence of stress in specific regions of the central nervous system and in gonads of young adults flies. In these two organs, the sHSPs show a cell-specific and developmental stage-specific pattern of expression suggesting distinct regulation and function(s) of each individual sHSP (R. Marin et al., Dev. Genet. 14, 69-77, 1993). Since mammalian HSP27 has been reported to be phosphorylated through a complex novel cascade implicating distinct kinases, we examined whether two of the sHSPs (HSP27 and HSP23) exist in different isoforms as a result of posttranslational modification in vivo. HSP27 and HSP23 were analyzed in various tissues in unstressed and heat-shocked flies. Four isoforms of HSP27 were found to be constitutively expressed in the nervous system and in testes and two in ovaries. The proportion of these isoforms relative to each other was specific to a given tissue. In the case of HSP23, two isoforms were expressed in the heads and in testes of unstressed flies. In ovaries, a low level of a single isoform of HSP23 was found. Heat shock caused an increase in the amount of preexisting HSP27 and HSP23 and the appearance of additional isoforms in ovaries. Susceptibility to phosphatase treatment indicated that isoforms of HSP27 were phosphoproteins. This was further supported by in vitro experiments in which Drosophila sHSPs were incubated with purified Chinese hamster HSP27 kinase. Only HSP27 was shown to be a substrate of this mammalian HSP27 kinase. The present data suggest that tissue- and HSP-specific posttranslational modification systems may modulate the function of these proteins in different cell types. Furthermore, the signal transduction pathways leading to phosphorylation of the sHSPs are conserved between mammals and Drosophila, and the sHSP kinase cascade may be developmentally regulated. PMID- 8635481 TI - RAR beta 2-mediated growth inhibition in HeLa cells. AB - Retinoic acid inhibits the growth of a variety of normal and transformed cells in vitro and in vivo. How retinoic acid inhibits cell growth is poorly understood but involves interactions between the ligand and a series of nuclear and cytoplasmic receptors. The nuclear receptors for retinoic acid are of two types, the RARs and the RXRs. Each can function as a ligand-inducible transcription enhancing factor. In previous studies, we have demonstrated that an isoform of one RAR, RAR beta 2, is transcriptionally up-regulated in senescent human dermal fibroblasts and senescent human mammary epithelial cells. Moreover, we have also shown that RAR beta 2 can inhibit oncogene-induced focus formation, in primary rat embryo fibroblasts, as effectively as the tumor suppressor gene p53. Here, we extend our studies of retinoid-regulated signal transduction pathways that inhibit cell proliferation by demonstrating that HeLa cells expressing an RAR beta 2 construct are growth inhibited by greater than 50% when compared to the parent cell lines. The RAR beta 2-expressing cell lines are inhibited further by the addition of exogenous all-trans-retinoic acid. Finally, soft agar assays show that the RAR beta 2-expressing cell lines also demonstrate an inhibition of growth in soft agar, when compared to the parent growth cell lines, and are inhibited further in the presence of added all-trans-retinoic acid. These data definitively show that RAR beta 2 can inhibit cell proliferation in an established tumor cell line and provide more strength to the notion that this isoform is an effective growth inhibitor in vitro and, most likely, in vivo. PMID- 8635484 TI - In vitro fibroplasia: matrix contraction, cell growth, and collagen production of fibroblasts cultured in fibrin gels. AB - Extracellular matrix (ECM) reorganization, cell growth, and collagen synthesis/deposition are key features of fibroplasia during tissue repair. An in vitro fibrin gel culture model system simulating fibroplasia of wound repair was characterized. In the model system, fibrin gels were stabilized on plastic culture plates as hemispheres. In this way, fibroblasts were able to reorganize fibrin fibrils, resulting in a measurable decrease in gel thickness with no change in gel diameter, thereby producing a matrix with tension relevant to that of a repairing tissue. Within the study period, human dermal fibroblasts exhibited dynamic activities in cell growth and in reorganization and remodeling of the fibrin matrix. In the first 2 days of culture, fibroblasts quickly reorganized the fibrin matrix to 10% of its original thickness. Fibroblast proliferation occurred at a much slower rate compared to monolayer cultures. Proliferation continued at the same rate throughout the study in contrast to monolayer cultures, which ceased proliferation at confluence. Collagen synthesis was detected as early as the second day in culture. Type I collagen was the major collagen synthesized by fibroblasts with small amounts of type V and type III collagen. Collagen from either monolayer or fibrin gel cultures appeared identical when analyzed by two-dimensional peptide mapping of their CNBr fragments. Although collagen was detected biochemically from Day 2, organized collagen fibrils were apparently only in the later stage of cultures in transmission electron micrographs. Also, at this time, fibrin fibrils were largely removed and the matrix was filled with collagen fibrils and other filamentous ECM. The growth factor TGF-beta stimulated both fibrin gel contraction and collagen synthesis by fibroblasts. Therefore, using the model system, we have demonstrated that fibroblasts can actively reorganize the fibrin matrix and subsequently remodel it into a collagen-containing scar-like tissue. The unique features of this model system allow for creative designs in studying the complex mechanisms underlying tissue repair. PMID- 8635483 TI - Effects of immortalization upon the induction of matrix metalloproteinases in rabbit synovial fibroblasts. AB - Induction of stromelysin and collagenase mRNAs in response to phorbol myristate acetate (PMA) and autocrine factors (CAF) was compared in primary cultures of lapine synovial fibroblasts and an immortalized line of these cells known as HIG 82. In both cell types, message induction was quicker for CAF than for PMA. Appearance of both stromelysin and collagenase mRNAs occurred earlier in HIG-82 cells and, unlike primary cells, HIG-82 cells partially resisted inhibition by cycloheximide. To determine whether differences in AP-1 activity could account for these observations, the induction of c-fos and c-jun mRNAs was studied in conjunction with gel shift assays for AP-1 binding. Both inducers increased the abundance of c-fos mRNA, although the response was weaker in HIG-82 cells. However, the increase in c-jun mRNA was more marked in HIG-82 cells; furthermore, this increase was sustained for over 6 h. Gel shift assays confirmed that in both types of cells PMA and CAF increased AP-1 binding activity. In primary cells, this activity was sensitive to cycloheximide, but in HIG-82 cells, there was only partial sensitivity to cycloheximide. The gel shift analyses and data from experiments using an AP-1-CAT reporter construct revealed, in many cultures, constitutive AP-1 activity in the absence of stromelysin and collagenase expression, suggesting that AP-1 alone is insufficient for matrix metalloproteinase induction. Antisense oligonucleotides to c-fos and c-jun strongly inhibited the induction of stromelysin mRNA in primary cells treated with PMA, but was only weakly active against message induction in HIG-82 cells. In neither primary cells nor HIG-82 cells did antisense oligonucleotides strongly inhibit stromelysin induction in response to CAF. These data suggest there may exist an AP-1-independent route to message induction or that factors other than c FOS and c-JUN may be used in certain circumstances. Western blot analyses detected no marked difference between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG 82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in the kinetics and cycloheximide sensitivity of MMP induction may reside in their abilities to modify posttranslationally the relevant transcription factors. PMID- 8635485 TI - A human transforming growth factor-beta type II receptor that contains an insertion in the extracellular domain. AB - In the signal transduction of transforming growth factor-beta (TGF-beta), the TGF beta type II receptor (betaR-II) binds TGF-beta, presents the ligand to type I receptor, and forms a heterodimeric receptor complex that functions as an active signaling receptor. We isolated cDNA clones encoding an isoform of human betaR-II from vascular endothelial cells by RT-PCR. The deduced structure of the isoform designated as betaR-IIb contained a 25-amino-acid residue inserted at the extracellular region. The insert exhibited 73% homology to a similar insertion found in a mouse betaR-II variant. BetaR-IIb mRNA was ubiquitously expressed in all human cell lines examined. BetaR-IIb as well as betaR-II stably expressed in betaR-II deficient DR26 cells acted as a functional receptor transducing signals from TGF-beta for growth inhibition and the transcriptional activation of the plasminogen activator inhibitor-1 promoter. These results indicated that the betaR-IIb expressed in mammalian cells functions indistinguishably from in betaR II. The potential function of betaR-IIb is discussed. PMID- 8635486 TI - Stimulatory effect of PDGF on HMG-CoA reductase activity and N-linked glycosylation contributes to increased expression of IGF-1 receptors in human fibroblasts. AB - In the present paper, we show that the prereplicative period in platelet-derived growth factor (PDGF)-stimulated human diploid fibroblasts (HDF) includes an early increase in 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity followed by an increased N-linked glycosylation. HMG-CoA reductase inhibitors abolished these events and also prevented progression of cells into S-phase, indicating that mevalonate (MVA)-dependent glycosylation might be required in PDGF-mediated cell growth. Furthermore, PDGF was demonstrated to increase the number of insulin-like growth factor 1 (IGF-1) binding sites in HDF. This event, which was dependent on MVA, took place late in the prereplicative period and was corrected to a significant increase in the expression of de novo synthesized IGF 1 receptor (IGF-1R) proteins at the cell membrane. The PDGF-induced IGF-1R expression was suppressed in the presence of tunicamycin, an inhibitor of N linked glycosylation. Taken together, our findings suggest an important role of MVA and N-linked glycosylation in PDGF-mediated growth activation of HDF. PMID- 8635487 TI - A reversible defect in alpha-beta T cell receptor assembly. AB - A human leukemia cell line, SUP-T13, can gain and lose TCR/CD3 expression at rates incompatible with spontaneous mutation. In this study, we determined (i) the generality of this phenomenon among other T cell lines, (ii) the specificity of this phenomenon to the TCR/CD3 complex, and (iii) the molecular mechanism of TCR/CD3 loss in the SUP-T13 cell line at a biochemical level. We show that two other T cell lines can undergo gain and loss of TCR/CD3 expression at similar rates. However, class I MHC molecules do not switch expression on and off, demonstrating that such switching is not an artifact of the analysis. To determine the mechanism for loss of surface TCR/CD3 expression, pulse-chase labeling and immunoprecipitation were performed on SUP-T13 TCR/CD3 negative cells. These analyses revealed that TCR alpha proteins are produced, but do not covalently associate with TCR beta-CD3 proteins in the negative cells. Thus, these variants represent a novel level of posttranslational regulation of TCR/CD3 expression, namely, the disulfide linkage of alpha and beta TCR chains. PMID- 8635488 TI - Type I collagen gel modulates extracellular matrix synthesis and deposition by tracheal epithelial cells. AB - Extracellular matrix (ECM) molecules, including those present in basement membranes, are known to play an important role in morphogenesis and differentiation. Primary rat tracheal epithelial (RTE) cells grown on permeable membranes in air-liquid interface (ALI) cultures differentiate into mucous and ciliated cells. We previously showed that RTE cell differentiation is accelerated and enhanced on type I collagen gel-coated membranes. The purpose of this study was to determine whether type I collagen gel matrix also regulates basement membrane formation by RTE cells and whether this correlates with differentiation. Therefore, we examined the synthesis and deposition of several ECM molecules by RTE cells maintained on uncoated and type I collagen gel-coated permeable membranes in ALI cultures. Fibronectin, thrombospondin-1, and alpha-1 type IV collagen gene expression were down-regulated in cultures maintained on type I collagen gel-coated membranes and in differentiated cultures. Fibronectin and laminin alpha, beta, and gamma protein levels also were decreased slightly in response to type I collagen gel and differentiation. Deposition of fibronectin and laminin were dependent on type I collagen gel substratum. While fibronectin and laminin deposition were evident underlying both undifferentiated and differentiated cultures on type I collagen gel, deposition of these proteins on uncoated membranes was not readily detectable in undifferentiated cultures and was limited to patches of fibronectin deposition beneath differentiated cultures. Evidence suggestive of a discontinuous basal lamina-like structure was most apparent underlying differentiated cultures on type I collagen gel. These date demonstrate that ECM RNA and protein expression by RTE cells in ALI cultures are down-regulated by type I collagen gel whereas differentiation and ECM deposition are accelerated and enhanced on type I collagen gel. PMID- 8635489 TI - Heat shock proteins increase resistance to apoptosis. AB - Heat shock treatment of cells increases their survival and resistance to apoptosis. The kinetics of development of this resistance correlates with the kinetics of synthesis of heat shock proteins (hsps). U937 and Wehi-s cells were cultured for 1 h at 42 degrees C, conditions which induced the synthesis of heat shock proteins 27, 70, and 90. The cells were subsequently permitted to recover for a 2-h period, prior to exposure to the apoptosis inducing agents actinomycin D (5 micrograms/ml), camptothecin (5 micrograms/ml), and etoposide (25 micrograms/ml). Apoptosis was determined by both DNA fragmentation and flow cytometric analysis. Heat-shocked cultures had a smaller number of apoptotic compared to control cultures when both were exposed to apoptosis inducing stimuli. Transfected Wehi-s cells constitutively overexpressing human hsp 70 or 27 were then examined for their resistance to apoptosis inducing by these drugs. Using the MTT assay, hsp 27 and 70 overexpressing cells exhibited an increased resistance to cell death when compared to the parental line. The parental line demonstrated features of apoptosis, that is, cell shrinkage and single- and double-strand DNA breaks. Taken together these results demonstrate that an increase in cellular levels of hsp 27 or 70, either by a mild heat shock treatment or by stable transfection, increases the resistance of U937 and Wehi-s cells to apoptotic cell death. PMID- 8635490 TI - A melanoma cell line sensitive to expression of a fusion protein binding the retinoblastoma protein family pocket domain. AB - An established melanoma cell line (MM96L) was transfected with selectable plasmid constructs encoding either whole SV40 large T antigen, or beta-galactosidase fusions with the retinoblastoma protein (Rb)-binding region of SV40 large T antigen and a nonbinding mutant derivative of it. Both of the beta-galactosidase fusions also encoded the large T nuclear targeting signal. Transcription of inserted genes was regulated through a Zn+2-inducible metallothionein IA promoter, which provides tight but not absolute control of expression. Only the wild-type large T segment fusion was functionally active in the binding of Rb protein. Stable lines derived from primary transfectants with the expression plasmid encoding the mutant large T segment fusion showed a normal FACS scan profile, a normal growth rate, and (upon induction) high levels of nuclear staining for beta-galactosidase. However, cells transfected with the wild-type (Rb-binding) large T segment fusion grew slowly, with surviving clones assuming a predominantly tetraploid karyotype and relatively much lower levels of beta galactosidase activity upon Zn+2 induction. The latter cells, but not those transfected with the corresponding non-Rb-binding fusion construct, also exhibited elevated cell death and apoptosis in response to the inducer Zn+2. These results implied that expression of an Rb-binding protein has deleterious effects on the melanoma cell line growth and may reflect a role for Rb of a related pocket protein in maintaining the differentiation state of these transformed cells. PMID- 8635491 TI - Role of protein kinase C in modulating epidermal growth factor- and phorbol ester induced mammary epithelial cell growth in vitro. AB - Normal mammary epithelial cells isolated from mid-pregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Chronic treatment with low doses (0.1-0.5 nM) of phorbol 12-myristate 13-acetate (PMA) had no mitogenic action when given alone, but significantly enhanced epidermal growth factor (EGF) induced growth. In contrast, similar treatment with high doses (10-100 nM) of PMA significantly stimulated mammary epithelial cell growth in the absence of EGF. Furthermore, growth of cells treated with high doses of PMA and EGF was similar to that observed in cells treated with PMA alone. In parallel experiments, treatment with similar doses of 4-alpha-phorbol 12-myristate 13 acetate, a phorbol ester which does not activate PKC, did not significantly alter mammary epithelial cell proliferation when given alone or in combination with EGF. Acute treatment with 10 ng/ml EGF or 20 nM PMA stimulated phospholipid-dependent PKC translocation from the cytosolic to the membrane fraction, and this effect was blocked by prior treatment for 7 days with 20 nM PMA. Western blot analysis showed that chronic treatment with 1-10 nM PMA for 6 days caused only slight decrease in relative PKC alpha levels in the cytosolic and membrane fractions, while similar treatment with 20-100 nM PMA caused a large down-regulation in total cellular phospholipid-dependent PKC alpha levels. Additional studies showed that treatment with 1-2 nM PMA caused an increase, whereas treatment with 5-100 nM PMA caused a dose-related decrease in EGF-dependent EGF-receptor (EGF-R) autophosphorylation, In summary, these findings suggest that submitogenic doses of PMA potentiate EGF-induced cell growth by enhancing EGF-R mitogenic signaling, whereas the mitogenic effects of high doses of PMA alone appear to be mediated through PKC- and EGF-independent mechanisms. PMID- 8635492 TI - The relationship between susceptibility to retinoic acid treatment and protein kinase C alpha expression in murine melanoma cell lines. AB - Retinoic acid (RA)-induced differentiation of B16 mouse melanoma cells is accompanied by a large increase in the amount of PKCalpha protein. Overexpression of PKCalpha in these cells results in a more differentiated phenotype. To determine if these findings had general applicability to murine melanomas, we investigated the relationship between sensitivity to RA and induction of PKCalpha in three different murine melanoma cell lines. RA inhibited the anchorage dependent growth of all three cell lines, with JB/MS being the most sensitive, S91 intermediate, and RPMI the least affected. RA also inhibited soft agar colony formation in JB/MS, but had little effect on RPMI. All cell lines expressed PKCalpha, but not beta or gamma. RA induced a large concentration-dependent increase in PKCalpha protein in JB/MS (6- to 10-fold), a smaller increase in S91 (2- to 3-fold), and very little induction of PKCalpha in RPMI. Previously we had observed that the amount of PKCalpha increased with the density of B16 cells in culture. We found that this density-dependent increase in PKCalpha occurred in three out of four melanoma cell lines examined. These results suggest that PKCalpha plays an important role in RA-induced murine melanoma cell differentiation. PMID- 8635493 TI - Gap junction distribution and connexin expression in human breast. AB - Expression of the gap junction proteins (connexins) in human breast epithelium was studied in vivo and in vitro. A panel of sequence-specific anti-peptide antibodies was used to examine four connexin (Cx) isoforms by indirect immunofluorescence labeling. Antibodies to Cx43 readily detected gap junctions between the basal cells in major ducts, but less so within lobular/alveolar structures. Cx26 immunoreactivity was less abundant in beast epithelium but was observed between the luminal cells in major ducts and to a lesser extent in lobular/alveolar structures. Ultrastructural studies of normal human breast showed gap junctions between basal cells in ducts and lobules, but not between luminal cells or between luminal and basal cells. Immunomagnetically separated luminal and basal cells were grown in vitro. Basal cells expressed Cx43 at cell cell attachment points whereas luminal cells showed only small amounts of immunolabeling with the Cx26 antibody which was generally not associated with the cell borders. Microinjection of Lucifer yellow into cultured luminal or basal cells indicated that basal cells have high levels of gap junctional communication, but dye transfer between luminal cells was difficult to detect by transfer of Lucifer yellow. Western blot analysis of purified luminal and basal cells indicated the presence of mainly Cx43 in both cell types. Polymerase chain reaction analysis of breast mRNA identified message for CX43 and to a lesser extent for Cx26; Cx32 was not detected in human breast, although it was present in mouse mammary gland. mRNA extracted from cloned cultures of human luminal and basal cells contained message for Cx43 and Cx26 in both cell types. PMID- 8635494 TI - Intracellular fate of endocytosed collagen in rat liver endothelial cells. AB - The fate of endocytosed collagen (COL) in sinusoidal liver endothelial cells was studied using COL labeled with FITC (F-COL) or iodine (125I-COL) or both (125I FCOL). In pulse-chase experiments in vitro, F-COL localized after 10 min along the limiting membrane of vesicles, taking the appearance of rings. After 20 min chase the probe appeared more concentrated in fewer but larger ring structures, and after 60 min the probe was observed mainly in the interior of smaller vesicles. Gel filtration of solubilized cultures after pulse-chase experiments using 125I-FCOL or 125I-COL revealed that degradation was initiated and largely completed in the small, filled vesicles, judged as a pre- or early lysosomal compartment. In the presence of monensin, or by incubation at 20 degrees C, the probe was arrested at the level of the larger ring structures, and degradation could not be observed. Lysosomal preloading by iv injection of TRITC-COL (T-COL) 24 h prior to pulse-chase experiments with culture cells using F-COL disclosed colocalization of the two dyes only after 8 h in perinuclear vesicles of a size larger than the early lysosomal vesicles observed after 60 min, suggesting a slow, unidirectional transport of F-COL to the T-COL labeled lysosomal compartment. Esterase reaction product was observed mainly in vesicles resembling the double-stained lysosomes. We conclude that (1) the early endosomes, (2) the vesicles appearing after 60 min are prelysosomes mediating degradation, and (3) the lysosomes accumulating in the probe after 8 h are responsible for final degradation and storage of residual stain. PMID- 8635496 TI - An apical tension-sensitive microfilament system in retinal pigment epithelial cells. AB - A tension-sensitive microfilament (MF) system in the plane of zonula adherens (ZA) junctions of retinal pigment epithelial (RPE) cells is described. Organ cultures and whole mounts of RPE from chick embryos were labeled for F-actin and examined with a confocal laser scanning microscope. The changes in the distribution of MFs during alternations in the direction of tensional stress, generated in the organ culture system or induced by mechanical wounding, were examined. In RPE cells throughout the organ culture, fine MF bundles, termed apical stress fibers (ASF), oriented parallel to the direction in which tension had been generated, were observed at the level of circumferential MF bundles (CMBs) associated with ZA junctions. Within 24 h after the direction of the tension was experimentally altered by 90 degrees, the orientation of ASF also changed, becoming aligned along the new lines of the tension. The ASF likewise react to changes in the direction of stress generated when RPE cells respond to wounding in organ culture. The presence of similar MFs in RPE cells in the posterior retina of 13-day chick embryos in situ may reflect mechanical stretching of the RPE cells that occurs during the development of the eye. The ASF probably limit the stretching of the CMBs of RPE cells in any one direction, thereby protecting the RPE cells against tensional stress. This system of MFs may be especially important in chick RPE cells, which lack desmosomes and keratin type intermediate filaments that normally have this function in other cell types. PMID- 8635495 TI - Ligand activation of overexpressed epidermal growth factor receptor results in colony dissociation and disturbed E-cadherin function in HSC-1 human cutaneous squamous carcinoma cells. AB - Various types of tumors show aberrant expression and overexpression of epidermal growth factor (EGF) receptor and the degree of receptor expression correlates with a malignant phenotype in many epithelial tumors. However, in vitro evidence supporting the advantageous role of receptor overexpression is deficient. In this study, we compared the effects of exogenous EGF on the cell colony morphology in monolayer and collagen gel culture between HSC-1 squamous carcinoma cells overexpressing EGF receptor and their revertant subline cells. These cells formed coherent cell colonies under routine culture conditions, but addition of EGF induced dissociation of cell colonies within 24 h in the parent HSC-1 cells, though not in the subline cells. Since the colony dissociation apparently involved loss of cell-cell adhesion, we also studied the effects of EGF on E cadherin expression and its function. Cell aggregation assays showed that EGF reduced E-cadherin function dose-dependently in the parent cells, but not in the subline cells. However, immunoblotting analysis and ELISA showed the absence of downregulation or degradation of E-cadherin. Instead, EGF tyrosine phosphorylated cadherin/catenin complex components including beta-catenin and increased the detergent solubility of E-cadherin in the parent cells. These results suggest that EGF modified the functional association between E-cadherin and actin filament through tyrosine phosphorylation of the cadherin/catenin complex and thereby made the adhesion molecule incompetent. Our results indicate that the ligand activation of overexpressed EGF receptor impairs E-cadherin-mediated cell cell adhesion and causes dissociation of the squamous carcinoma cell colonies, which facilitates tumor cell invasion in vivo. This might be relevant to the advantageous role of EGF receptor overexpression in malignant phenotype of epithelial tumor cells. PMID- 8635498 TI - Modifications of vimentin filament architecture and vimentin-nuclear interactions by cholesterol oxides in 73/73 endothelial cells. AB - Among the different targets of the cytodamaging effects of cholesterol oxides in endothelial cells, cytoskeleton is one of the most relevant, due to the large variety of biological events controlled by this subcellular structure. The modifications of the intermediate filament network caused by three cholesterol oxides (cholestane-3beta,5alpha,6beta-triol, CH, 7-keto-cholesterol, KC, and 25 OH-cholesterol, COH) was investigated in the endothelial cell line 73/73 using immunofluorescence and laser scanner confocal microscopy. All three cholesterol oxides promoted a redistribution of vimentin filaments that took place well before cell detachment and the occurrence of any detectable sign of cell death. CH-induced alterations were characterized by the polarization of vimentin to the edges of the cell and a concomitant destruction of its interaction with the nucleus. In KC-treated cells, vimentin filaments appeared cross-linked and formed a sort of circular network ring between the nucleus and the cell periphery. COH promoted the aggregation of vimentin filaments in thick and irregular bundles that delimited apparently empty regions. All these changes occurred independently of gross modifications in microtubule organization, which was generally retained except for the appearance of immunoreactive tubulin spots throughout the cytoplasm. These results indicate that the organization of the intermediate-size filament protein vimentin is markedly affected by cholesterol oxides. The different rearrangements caused by CH, KC, and COH may derive from different pathobiochemical processes triggered by these compounds. PMID- 8635499 TI - Adhesion mediated by fibronectin's alternatively spliced EDb (EIIIB) and its neighboring type III repeats. AB - Adhesion functions of cellular fibronectin's (FN) alternatively spliced EDb (EIIIB) domain, as well as its neighboring type III repeats III7 and III8, were investigated with several cultured murine and human cell types. Minigene constructs encoding various permutations of these repeats and expressed in bacteria were used as shown previously in function studies of EDa and its neighboring repeats (P.Xia and L. A. Culp Exp. Cell Res. 213, 253-265, 1994). When substrata of recombinant proteins were incubated with several fibroblastic or neuronal derivative cell lines, cell attachment responses varied widely in a cell-type-specific manner. Balb/c 3T3 cells were shown to adhere to recombinant protein substrata in dose-dependent and EDTA-sensitive manners. Responses also varied with which repeat combinations were being tested, from excellent (Balb/c 3T3, src-3T3), to intermediate (Platt cells), to poor (LZEJ, VA-13, and F11), with EDb plus-containing proteins generally giving better adhesion than EDb minus proteins. On select recombinant proteins, cells showed limited cytoplasmic spreading (3T3 and src-3T3) or neurite extension (Platt and F11), while other cell lines (VA-13 and LZEJ) did not show any morphological changes beyond attachment. Again, EDb plus-containing recombinants were more effective at inducing these morphological changes than the neighboring repeats. These results demonstrate that the EDb domain of cellular FNs and its neighboring type III homology repeats contain important adhesion-promoting sequences, which may be regulated by cells through alternative splicing of FN's primary transcript. PMID- 8635497 TI - Disruption of actin microfilament organization by cholesterol oxides in 73/73 endothelial cells. AB - Various cholesterol oxides generated during the oxidation of low-density lipoproteins have been reported to exert cytotoxic effects on cultured endothelial cells and to decrease their barrier function. The cytoskeleton, and in particular the actin microfilament meshwork, is one of the preferential targets in oxidative stress-and thiol-depleting agent-induced cell injury. The alterations occurring in the microfilament network were investigated using the endothelial cell line 73/73 treated with increasing concentrations (0.5-10 micrograms/ml) of cholestane-3 beta, 5 alpha, 6 beta-triol, CH, 5-cholesten-3beta ol-7one, KC, and 25-OH-cholesterol, COH, for up to 6 h. The distribution of microfilaments was visualized using immunofluorescence and laser scanner confocal microscopy. All cholesterol oxides caused a progressive disruption of actin microfilaments that was characterized by the disappearance of the stress fibers within the cell body and, in selected cells, by a complete marginalization and clustering of the filaments to one edge of the cell. In addition, COH promoted F actin fragmentation, as revealed by the presence of scattered fragments of F actin in various cell regions. The redistribution of actin microfilaments was associated with a similar redistribution of alpha-actinin, an actin-binding protein involved in bundle formation and in the anchorage of actin filaments to the adhesion plaques. Concomitantly, cholesterol oxides promoted a loss of vinculin, another actin-binding protein, from the focal adhesion plaques located under the cell body and their marginalization and thinning. These alterations preceded cell detachment and cell death by apoptosis as revealed by the subsequent leakage of cytosolic enzymes and nuclear fragmentation. These results suggest that cytoskeletal (microfilament) alterations caused by cholesterol oxides may be one of the cytopathological events involved in the detachment of endothelial cells from the inner vascular surface promoted by cholesterol oxides. PMID- 8635500 TI - Plasminogen activator inhibitor type-2 (PAI-2) in human keratinocytes regulates pericellular urokinase-type plasminogen activator. AB - Plasminogen activation is observed in the human epidermis during reepithelialization of epidermal defects and under certain pathological conditions. The activation reaction depends on keratinocyte-associated plasminogen activators (PAs), which convert the ubiquitous proenzyme plasminogen into the active trypsin-like serine proteinase plasmin. The PAs are controlled by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-2. In vitro and in vivo keratinocytes express both PAIs. In the current study, we have addressed the possible function of PAI-2 in regulating extracellular PA activity in cultured normal human epidermal keratinocytes (NHEK), the human keratinocyte cell line (HaCaT), and a Ha-ras transfected HaCaT variant (HaRas). PAI-2 was detected intracellularly in all three cell types. Whereas only the NHEK and the HaCaT cells secreted detectable levels of PAI-2 into the culture medium, all three cell types released urokinase-type PA (uPA) into the supernatants. When comparing HaCaT and HaRas cells, we found that the cell lines secreted comparable levels of uPA antigen, whereas the levels of uPA activity were low in the presence of PAI-2, indicating that PAI-2 serves to regulate uPA activity. This assumption was supported by the findings that PAI-2 formed complexes with secreted uPA and that uPA/PAI-2 complexes were present at the surface of the PAI 2-secreting HaCaT cells but not at the surface of PAI-2 nonsecreting HaRas cells. Finally, PAI-2 was found to counteract the uPA-dependent and plasmin-mediated detachment of cultured HaCaT cells. Taken together, our findings indicate that secreted PAI-2 serves to regulate the activity of extracellular uPA in keratinocytes. PMID- 8635501 TI - Ca2+-dependent and Ca2+-independent regulation of the thyroid epithelial junction complex by protein kinases. AB - The integrity of epithelial cell junctions is controlled by E-cadherin-mediated (Ca2+-dependent) cell-cell adhesion. In thyroid follicular cells the dissociation of junctions induced by transfer to low Ca2+ medium (Ca2+ switch) is prevented by thyrotropin acting via cyclic AMP/protein kinase A (cAMP/PKA) (Nilsson et al., Eur. J. Cell Biol. 56, 308-318, 1991). In MDCK kidney epithelial cells protein kinase inhibitors elicit a similar response which, however, is cadherin independent (Citi, J. Cell Biol. 117,169-178,1992; Citi et al., J. Cell Sci. 107, 683-692, 1994). As such inhibitors also may interfere with PKA, we examined in a single cell type, filter-cultured pig thyrocytes, the effects and possible interactions of the cAMP/PKA agonist forskolin (or thyrotropin) and the kinase inhibitor H-7 in Ca2+ switch experiments. We found that the epithelial barrier dysfunction, comprising loss of transepithelial resistance, increased transepithelial flux of [3H]inulin and redistribution of junction proteins (cadherin and ZO-1), which follows Ca2+ removal were inhibited by TSH, forskolin, and H-7. All agents were also able to induce recovery of resistance in low Ca2+. The maximal recovery effects of forskolin and H-7 were additive when given simultaneous with Ca2+ chelator. In contrast, forskolin-induced recovery initiated 10 min after Ca2+ removal was antagonized by H-7. The protection of junctions by forskolin in low Ca2+ was rapidly abolished by light trypsinization (0.001%), whereas the same concentration of trypsin had little or no effect on the corresponding action of H-7 or staurosporine, another potent kinase inhibitor. In H-7-treated cells kept in low Ca2+, trypsin caused redistribution of ZO-1 from the plasma membrane to the cytoplasm while the transepithelial resistance remained high. Taken together, the data indicate that TSH via cAMP/PKA and the protein kinase inhibitor H-7 reinforce the thyroid epithelial barrier under low Ca2+ conditions by distinct although interacting mechanisms. The high sensitivity to proteolysis in the absence of Ca2+ suggests that the cAMP regulated mechanism is cadherin-dependent. H-7 promotes or inhibits the cAMP/PKA mediated recovery of transepithelial resistance depending on the duration of the preceding low Ca2+ period. The trypsin-induced displacement of ZO-1 in H-7 treated cells in low Ca2+ suggests that the localization of ZO-1 to the tight junction is not necessary for the maintenance of junctional tightness. PMID- 8635503 TI - Retinoic acid-mediated decrease of G (alpha S) protein expression: involvement of G (alpha S) in the differentiation of HL-60 myeloid cells. AB - The amount of the heterotrimeric G protein subunit G (alpha S) decreases after the induction of human myeloblastic leukemia HL-60 cells to become granulocyte like cells in the presence of retinoic acid (RA). Compared to untreated control cells, HL-60 cells expressed decreased levels of G (alpha S) protein and mRNA levels after addition of RA to the cultures as shown by immunoblot and Northern blot analysis. The reduction of the G (alpha S) protein in HL-60 cells by antisense RNA expression was associated with (i) decreased cell doubling time; (ii) induction of a granulocyte-like phenotype; (iii) and expression of a surface marker characteristic of myeloid differentiation. Expression of a constitutively active mutant G (alpha S) (Q227L) in HL-60 cells blocked RA-induced differentiation. In contrast, treatment with forskolin, prostaglandin E2, or 8 bromo-cyclic AMP, which increase intracellular cyclic AMP (cAMP) levels, did not inhibit the RA-mediated differentiation process. No changes in cAMP levels occurred in response to RA. The present study provides insights into the involvement of G (alpha S) protein in growth regulation during differentiation of the human myeloid cell line HL-60. These data suggest that in HL-60 human myeloid cells RA-mediated decrease of G (alpha S) plays a critical role in the regulation of differentiation which is independent of intracellular cAMP. PMID- 8635502 TI - Specific binding of adenosine deaminase but not HIV-1 transactivator protein Tat to human CD26. AB - Adenosine deaminase (ADA) and the HIV-1 transactivator protein Tat have been reported to bind to human CD26, also known as dipeptidyl peptidase IV (DPP IV). In order to demonstrate the specificity of such binding under native conditions of CD26, i.e., when expressed on the cell surface, we established murine cell lines expressing transfected human CD26, either wild-type or mutated at its serine-630, which inactivates the DPP IV activity. This experimental system is advantageous since murine ADA does not bind human CD26, whereas human and bovine ADA bind. Consequently, murine cell clones expressing either the wild-type or mutated form of human CD26 were found to bind specifically bovine 125I-labeled ADA with a high affinity (KD = 12 +/- 2 nM and 11 +/- 4 nM, respectively). No specific binding of 125I-labeled ADA was observed to murine clones not expressing human CD26. The binding of 125I-labeled ADA to CD26 was further characterized by the use of monoclonal antibodies specific to human CD26. The results obtained were in accord with those reported previously using other experimental models. These observations indicated that the murine cells expressing human CD26 provide a highly suitable model to investigate the potential binding of HIV-1 Tat to CD26. In contrast to previously published results, however, we could not demonstrate a specific interaction between Tat and human CD26. The 125I-labeled ADA-specific binding to human CD26 was not affected by Tat, even at concentrations which induced cell death. Similarly, the binding of several monoclonal antibodies to human CD26 was not modified by the addition of Tat. More significantly, Tat binding to different murine cell clones (human CD26 negative or positive) was found not to be correlated with the expression of human CD26. Finally, the toxic effect of Tat on the growth of different murine cell clones was independent of human CD26 expression. Taken together, these observations further confirm the specific binding of ADA to human CD26 and point out that CD26 is not the target of HIV-1 Tat protein. PMID- 8635504 TI - Retardation of phenotypic transition of rabbit arterial smooth muscle cells in three-dimensional primary culture. AB - Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cells in vivo. We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC. PMID- 8635506 TI - Disintegrin interaction with alpha V beta 3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on beta 3 subunit. AB - The effect of seven disintegrins (albolabrin, barbourin, bitistatin, echistatin, eristostatin, flavoridin, and kistrin) and the neurotoxin analogue, mambin, on the adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and fibronectin has been studied. Adhesion to vitronectin was significantly inhibited by echistatin, kistrin, flavoridin, and mambin. Echistatin, flavoridin, and kistrin bound with high affinity to immobilized alpha V beta 3 in solid phase assay; other disintegrins bound at a much lower level. Echistatin and flavoridin had a modest inhibitory effect on HUVEC adhesion to fibronectin. HUVEC adhered to disintegrins with a high selectivity toward bitistatin, echistatin, flavoridin, kistrin, and mambin. Adhesion of HUVEC to fibronectin and vitronectin resulted in cell spreading, whereas cells adhering to immobilized echistatin remained globular and cells adhering to kistrin showed abnormal morphology. Echistatin and kistrin potently inhibited the binding of monoclonal antibody (Mab) 7E3, which recognizes the alpha V beta 3 complex, to HUVEC. Echistatin and kistrin also induced the binding to HUVEC of Mab 62, which recognizes the ligand-induced binding site (LIBS) epitope on the beta 3 subunit, enhancing HUVEC binding to immobilized Mab 62. Similar results with both antibodies were obtained in Chinese hamster ovary cells transfected with alpha V beta 3 genes. In conclusion, disintegrin interaction with HUVEC appears to be selectively mediated by alpha V beta 3 receptors, and it results in an expression of LIBS epitope that may play a role in the regulation of ligand-binding affinity and intracellular signaling. PMID- 8635505 TI - Functional interplay between interleukin-1 receptor and elastin binding protein regulates fibronectin production in coronary artery smooth muscle cells. AB - We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL) 1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL 1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production. PMID- 8635507 TI - Differential regulation of vitamin D receptors in clonal populations of a chronic myelogenous leukemia cell line. AB - RWLeu4 is a chronic myelogenous leukemia cell line that is sensitive to the antiproliferative and differentiation-inducing actions of 1alpha,25(OH)2-vitamin D3 (VD3). The JMRD3 cell line is a VD3-resistant variant of RWLeu4 that was selected by continuous passage of RWLeu4 in the presence of VD3. The isolation of a spontaneous VD3-resistant variant suggests that phenotypically different cells exist within the RWLeu4 cell population. Therefore, single-cell clones of RWLeu4 cells were isolated and characterized. Four clonal cell populations that fall into three groups differing in response to the antiproliferative and differentiation-inducing actions of VD3 were examined. Surprisingly, the extent of response of the clones to VD3 does not show a correlation with the basal level of the vitamin D receptor (VDR). RWLeu4-3 and RWLeu4-4 are the clones most sensitive to the antiproliferative actions of VD3 (ED50 approximately equal to 1 nM); however, RWLeu4-3 expresses basal levels of VDRs similar to those found in the parental cells and the RWLeu4-2 clone, while in RWLeu4-4, VD3 binding and VDR protein are below the limits of detection. Furthermore, RWLeu4-10 expresses the highest basal level of VDR protein but is relatively resistant to the antiproliferative actions of VD3 (ED50 > or = 30 nM). Like JMRD3, RWLeu4-10 is still capable of differentiating in response to VD3, as judged by the induction of biochemical processes and cell-surface antigen expression. Although VD3 treatment increases VDR protein levels and DNA-binding activity in all clones, altered DNA-protein complexes are detected in RWLeu4-4. Our results suggest that sensitivity to the antiproliferative and differentiation-inducing actions of VD3 is not dependent solely upon the level of VDR expressed, but may also require posttranslational modification of the VDR or complex interactions with other nuclear transcription factors. PMID- 8635508 TI - Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct matrix compartments. AB - We have characterized immunohistochemically and biochemically the collagens accumulating in two compartments of the matrix formed by mature bovine articular chondrocytes in alginate beads. At all times of the 28-day culture period, more than 90% of the collagen molecules were recovered from the rim of cell-associated matrix (CM) which encapsulates individual chondrocytes and chondrocyte clusters. Both the total amount and concentration of collagens in this matrix compartment rose progressively with time. The ratio of collagen/proteoglycan remained relatively constant with time and was always five to seven times higher in the CM than in the interterritorial matrix compartment further removed from the cells. In the CM, collagen types II, IX and XI were present on Day 28 in relative proportions (95/l/3) similar to those in adult cartilage. A higher proportion of newly synthesized collagen type XI than types II or IX molecules did not become incorporated into the pericellular rim of matrix but accumulated in the further removed matrix. Although collagen type I was synthesized in small amounts by flattened cells at the surface of the beads, it did not become incorporated as heterotrimers or homotrimers in the matrix. Mature pyridinium crosslinks, principally pyridinoline, were detected as early as Day 7 of culture but became much more abundant between Days 15 and 28, especially in the CM which contained at all times more than 90% of the crosslinks formed. The codistribution of collagen types II, IX and XI and mature collagen-specific crosslinks support the contention that mature chondrocytes cultured in alginate matrix surround themselves with a protective shell whose composition is very similar to that which encapsulated the cells in vivo. PMID- 8635509 TI - Inhibition of proliferation and of IL-2 production and utilization in lymphocytes by S-oxalylglutathione. AB - Previously we have shown that S-oxalins (monothiolesters of oxalic acid) are ubiquitous mammalian metabolites whose concentrations decrease when lymphocytes are stimulated to proliferate. The present study was undertaken to further examine the role of S-oxalins in the proliferation process. When added to lymphocytes stimulated with concanavalin A, the S-oxalin, S-oxalylglutathione (GS Ox), inhibited DNA synthesis by 50% when present at ca. 0.15 mM and virtually 100% at 0.5 mM. The inhibition was reversible. The presence of GS-Ox blocked IL-2 production, but addition of IL-2 did not permit DNA synthesis to proceed. GS-Ox also inhibited proliferation of an IL-2-dependent cell line, BT2. In primary lymphocytes GS-Ox reduced IL-2 receptor expression, but not in an IL-2-dependent blast cell line. Overall RNA synthesis and protein synthesis were not significantly altered by GS-Ox. Levels of the positive transcription factor, NF kappaB, were decreased after incubation of lymphocytes with GS-Ox, but the amount of a negative transcription factor, NREA, was largely unchanged. The results not only provide further evidence that S-oxalins are small-molecule cell proliferation inhibitors, they also clarify to some extent the specific steps in the activation response modulated by S-oxalins. PMID- 8635510 TI - Functional characterization of the spontaneously transformed human umbilical vein endothelial cell line ECV304: use in an in vitro model of angiogenesis. AB - To gain insight into the role of the endothelial cell during the pathophysiology of the angiogenic response, investigators have isolated micro- and macrovascular endothelial cells from a wide range of both animal and human vessels, including the umbilical vein. Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cords remain a readily available and popular source of endothelial cell. However, the isolation and culture of these cells have several disadvantages, including the risk of infection, exogenous growth factor requirement, and low proliferative capacity. The heterogeneity of endothelial cells from different vascular beds as well as the heterogeneity between HUVEC isolates from different cords can make the critical interpretation of results difficult. ECV304 is a unique spontaneously transformed human umbilical vein endothelial cell line. In this report, the novel use of ECV304 cells as an alternative to HUVECs in an in vitro model of angiogenesis using the reconstituted basement membrane extract (Matrigel) was investigated. ECV304 cells were characterized immunohistochemically and their angiogenic behavior on Matrigel was analyzed functionally by phase-contrast, electron, and time-lapse video microscopy. ECV304 cells had several practical advantages over HUVEC culture and in contrast to HUVECs, ECV304 cells exhibited an enhanced and highly reproducible capacity for in vitro angiogenesis. However, several differentiated functions were lost or reduced in the ECV304 cell line which also exhibited anomalous cytokeratin expression. ECV304 cells may provide novel insights into the mechanisms governing angiogenesis under both normal physiological and pathological conditions. PMID- 8635511 TI - Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. AB - Chloroquine improves drastically the transfection of cells upon exposure to plasmid DNA/glycosylated polylysine complexes. So far the mechanism of action of chloroquine is not well understood. In this paper, the effect of chloroquine was investigated by measuring the transfection efficiency of a human hepatocarcinoma (HepG2 cells) by pSV2LUC/lactosylated polylysine complexes involving their internalization via the galactose-specific membrane lectin of these cells. The luciferase activity in the transfected cells was maximal when the transfection was performed for 3 or 4 h in the presence of 100 microM chloroquine. The luciferase activity was also enhanced in the presence of primaquine, a chloroquine analogue, but was not increased when transfection was performed in the presence of ammonium chloride, methylamine, spermine, or monensin, compounds known to neutralize the pH of the endocytotic vesicle lumen as chloroquine does. Chloroquine enters cells and accumulates in vesicular compartments; the overall intracellular concentration increases to 9 mM, which means that in the vesicular compartment, the chloroquine concentration is still higher. At such high concentrations, chloroquine induces the dissociation of plasmid DNA/lactosylated polylysine complexes, as shown in acellular experiments. PMID- 8635512 TI - Age-dependent inhibition of neural crest migration by the notochord correlates with alterations in the S103L chondroitin sulfate proteoglycan. AB - In avian embryos, the notochord inhibits neural crest migration, resulting in the absence of neural crest cells from the perinotochordal space. Here, we test whether temporal changes in the ability of the notochord to inhibit neural crest migration correlate with alterations in the S1O3L chondroitin sulfate proteoglycan (CSPG). Because CSPGs are abundant in the perinotochordal space and the inhibitory effects of the notochord are chondroitinase sensitive both in vivo and in vitro, we examined the distribution and biochemical nature of a large CSPG whose core protein is recognized by the S103L antibody. The S103L CSPG is specific to the perinotochordal space during the course of neural crest migration and codistributes with the HNK-1 carbohydrate. Biochemical characterization reveals that the S103L CSPG bears the HNK-1 epitope and is the only HNK-l immunoreactive proteoglycan present around the notochord at these stages. Following neural crest migration, the S103L CSPG staining is maintained in the perinotochordal region and also is expressed later in cartilage. In 4-day-old embryos, however, the S103L CSPG undergoes a reduction of HNK-1 immunoreactivity. To examine the temporal nature of the notochord's inhibitory ability, we assayed the effects, on neural crest migration of grafting notochords from 2- to 5-day old donor quail embryos into 2-day-old host chick embryos. Donor notochords from 2- to 3-day-old embryos inhibit neural crest cell migration, whereas the degree of inhibition is reduced or absent when notochords are derived from > or = 4-day old donors. This suggests that older notochords lose their inhibitory ability. Interestingly, preincubation of younger notochords with the HNK-1 antibody blocks the inhibitory effect, suggesting that glycosylation of the perinotochordal matrix may be important. The time when the notochord loses its inhibitory ability as assessed by our in vivo grafting assay correlates with the biochemical and immunocytochemical changes in the notochordal S103L antigen. These data suggest that a species of S103L CSPG, which is expressed by the early notochord and bears the HNK-1 epitope, may be important for the inhibition of neural crest migration. PMID- 8635513 TI - NuMA: a bipartite nuclear location signal and other functional properties of the tail domain. AB - Nuclear Mitotic Apparatus protein (NuMA) is a 238-kDa protein of the nuclear matrix in interphase that relocates to the spindle poles in mitosis. The globular tail domain (residues 1701 to 2115) contains the nuclear targeting sequence, the site for binding to the mitotic spindle as well as a site responsible for nuclear reformation. To more precisely map these sites, we inserted full-length human NuMA and 16 derivatives with increasing truncations of the tail domain into the pCMV5 vector and induced transient expression. NuMA was found in the interphase nucleus of all transfected BHK cells expressing either full-length NuMA or NuMA mutant proteins ending at or after residue 2005. In contrast, mutants ending at or before residue 2003 remained in the cytoplasm. In the full-length NuMA molecule, point mutations at position 1988 or 1989 or a double mutation at residues 2004 and 2005 cause NuMA to accumulate in the cytoplasm of both BHK and HeLa cells. The combined results indicate a bipartite nuclear location signal involving the sequences RKR (1987-1989) and KK (2004-2005) which are separated by 14 amino acid residues. In 30% of BHK cells transfected by the full-length clone, cytoplasmic aggregates of NuMA that colocalize with the centrosomes were documented in addition to the nuclear staining. In cells with large aggregates the cytoplasmic microtubular profile was disturbed. Observation of micronuclei formation suggests that a region important for normal nuclear reformation lies in the C-terminal 130 residues. Finally, NuMA mutant proteins ending at or after residue 1800 bound to the spindle poles of mitotic cells, while NuMA proteins ending at or before residue 1750 did not. PMID- 8635514 TI - Ultrastructure, enzymatic, and transport properties of the PICM-19 bipotent liver cell line. AB - The pig epiblast-derived PICM-19 cell line was previously shown to spontaneously differentiate into liver-like cells and structures and to secrete serum proteins. A study was undertaken to further define the liver-like characteristics of the PICM-19 cell line. PICM-19 cells displayed in vitro ultrastructure, enzymatic, and transport characteristics similar to those of parenchymal hepatocytes and bile duct epithelium. The PICM-19 cells contained large oval nuclei, numerous oval to elongate mitochondria with flat cristae, extensive rough and smooth endoplasmic reticulum, Golgi complexes, lipid vacuoles, and glycogen granules. Biliary canaliculi with intraluminal projecting microvilli were delimited by the junctional apparatuses between adjacent PICM-19 cells. The PICM-19 cells rapidly transported fluorescein into their biliary canaliculi from the extracellular environment. PICM-19 cells that had differentiated into multicellular ductal structures had high gamma-glutamyltranspeptidase (GGT) activity at their apical surfaces as shown by histochemical staining. PICM-19 total GGT activity was at least 19 times higher than that found in porcine hepatocytes. Metyrapone induced cytochrome P-450 content of PICM-19 cells was at least one-fourth of that found in porcine hepatocytes. PICM-19 P-450 activity induced by 7-ethoxycoumarin was nearly equivalent to that of primary cultures of pig hepatocytes. The data support the proposal that differentiated PICM-19 cells resembled hepatocytes, or bile duct epithelium cells, and, therefore, the PICM-19 cell line behaved like early embryonic liver progenitor cells. PMID- 8635515 TI - Dominant negative mutant of retinoic acid receptor alpha inhibits retinoic acid induced P19 cell differentiation by binding to DNA. AB - Retinoic acid (RA) is a potent inducer of P19 cell differentiation. RA activity is thought to be mediated by nuclear RA receptors (RARs), transcription factors whose activity is dependent on RA. There are three RARs called alpha, beta, and gamma. We created truncated versions of the three RARs and compared their activities as inhibitors of RA-mediated gene transcription and of P19 cell differentiation. Only mutants of the RAR alpha were inhibitory in these assays. A mutant of RAR alpha carrying a 10-amino-acid insert was able to heterodimerize with RXRbeta or with the normal RAR alpha and the inhibitory activity of this mutant was dependent on an intact DNA binding domain. We conclude that dominant negative mutants of RAR alpha act by heterodimerizing with RXRs or RARs and binding to RA response elements on DNA, thereby preventing binding of the normal receptors to those sites. PMID- 8635517 TI - Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis. AB - Cell condensations are the initial structures in the formation of proper cartilage and skeletal patterning in the developing vertebrate limb. Chondrogenic differentiation is dependent upon the cell-cell and/or cell-matrix interactions which take place during the condensation process. Coincident with the onset of condensation is the expression by limb mesenchyme of specific cell surface binding sites for the extracellular matrix macromolecule hyaluronan. The association of hyaluronan with the cell surface can influence the behavior of cells, especially cell aggregation. In this study the possible involvement of hyaluronan as an extracellular linker molecule in the cell-cell adhesion event during mesenchymal condensation was investigated in the avian limb model. Hyaluronan hexasaccharides were used to prevent the multivalent interactions that occur between native hyaluronan macromolecules and the cell surface. Our studies show that hyaluronan is required for early adhesive cell-cell interactions of limb bud mesenchyme and that perturbations of hyaluronan-cell interactions with hyaluronan hexasaccharides result in a delay in the formation of condensations as well as a delay in chondrogenic differentiation of mesenchymal cells in micromass cultures. PMID- 8635516 TI - Myocardial infarction is coupled with activation of cyclins and cyclin-dependent kinases in myocytes. AB - To determine whether the molecular components implicated in the regulation of the cell cycle are activated in myocytes after infarction, the expression of cyclins E, A, and B and the levels of their associated kinase activity were measured at 1 and 7 days following surgery. The quantity of cdk2 and cdc2 and the level of their kinase activity were also determined. Myocardial infarction was characterized by an increase in cyclins E, A, and B and cdc2 proteins in the surviving myocytes at 1 and 7 days. Cyclin E, A, and B and cdk2 and cdc2 kinase activity also increased. The quantity of cyclins E and A and the level of cyclin E-associated kinase activity in myocytes after infarction were comparable with those measured in neonatal myocytes. Moreover, cdc2 protein and cdc2 kinase activity in myocytes reached levels after infarction which were similar to those in neonatal myocytes. Thus, myocytes react to myocardial infarction by activating cyclins and cyclin-dependent kinases which may be coupled with the regeneration of muscle mass and recovery of ventricular function. PMID- 8635518 TI - Functional analysis of eukaryotic 20S proteasome nuclear localization signal. AB - The 20S proteasome is widely viewed at as a cytoplasmic multicatalytic proteinase complex: immunocytochemical investigations, however, show that proteasomes are localized in the cytoplasm as well as in the nucleus within the same cell. Strong nuclear accumulation of proteasomes is observed in rapidly dividing cells such as in the early stages of Drosophila embryogenesis and in tumorigenic cells. In fact, dependent on the metabolic state of a certain tissue or cell type its cellular distribution appears differentially regulated. Several of the proteasomal alpha-type subunits carry putative nuclear localization signals which may or may not take part in the regulation of the intracellular distribution of 20S proteasomes. We have examined the functional role of the putative nuclear localization signal (NLS) -KKKQKK-in the Drosophila PROS-28.1 subunit by deletion mutagenesis and transfection experiments. Linkage of the putative PROS-28.1 NLS to BSA as reporter protein and in vitro import studies with permeabilized mouse NIH 3T3 cells show that this NLS is able to induce complete translocation of the reporter protein into the cell nucleus. For analysis of the NLS within the 28-kDa subunit, cDNA deletion constructs were cloned into a pSG5 expression vector and transiently transfected into mouse fibroblast cells. Whereas the deletion of the NLS alone resulted only in a slight impairment of subunit transport into the nucleus, removal of the C-terminal 96 amino acid residues abolished nuclear translocation completely. PMID- 8635519 TI - Formation of nuclear bodies in cells overexpressing the nuclear pore protein POM121. AB - POM121 is an integral membrane protein specifically localized in the pore membrane domain of the nuclear envelope. We have investigated the intracellular distribution of rat POM121 heterologously overexpressed in monkey COS cells by immunofluorescence and fluorescence digital imaging microscopy. At low levels of expression overexpressed POM121 was distributed in the nuclear envelope in a punctate fashion, partially overlapping with the distribution of nuclear pores. At high levels of expression, however, the overexpressed protein accumulated in intranuclear bodies. These bodies represent a novel subnuclear structure, displaying a defined cylindrical structure and a distinct localization at or adjacent to the inner nuclear membrane. The C-terminal portion of POM121, which contains a pentapeptide repeat domain common to a subfamily of related nucleoporins, was sufficient to mediate targeting to the nuclear envelope as well as formation of intranuclear bodies. PMID- 8635520 TI - Behavior of the cell cycle-associated proteins in an unusual G0-arrestable cancer cell line. AB - An adenocarcinoma cell line which has the ability to arrest in G0 phase under exhausted culture conditions was established. Using the cell line, we investigated the expression of cell cycle-associated proteins including cdc2, cdk2, cyclin A, cyclin Dl, and Rb during entry into or withdrawal from the cell cycle. MAP kinase expression was also investigated as one of the most downstream proteins of the signal transduction of growth factors. The cells in the quiescent state did not express cdc2. In contrast, cdk2 was expressed weakly, and cyclin A and cyclin D1 were strongly expressed in the quiescent cells. The expression of cdk2 and cyclin D1 in the quiescent cells was reduced after stimulation by renewal of the medium and then increased, accompanied by Rb phosphorylation and cdc2 expression around the G1/S transition. Cdc2 and the hyperphosphorylated form of Rb disappeared as the cells became quiescent. MAP kinase expression was unchanged throughout all the phases analyzed. The results indicate that down regulation of neither cdk2, cyclin A, cyclin D1, nor MAP kinase is necessary to arrest cells in G0, but that only Rb dephosphorylation and down-regulation of cdc2 are accompanied by an arrest of cell proliferation in G0 in the cell line. PMID- 8635521 TI - Fusion with activated mouse oocytes modulates the transcriptional activity of introduced somatic cell nuclei. AB - We have analyzed the transcriptional activity of somatic cell nuclei fused with artificially activated mouse oocytes. Two types of somatic cells have been used: transcriptionally silent thymocytes, obtained from the thymus of newborn mice, and transcriptionally active murine erythroleukemia cells (MEL) from in vitro culture. Cells were fused with activated oocytes, either less than 1 h, or 3 h post-ethanol-treatment. When the fusion occurred early after activation (1 h or less), the transferred somatic cell nuclei reacted by nuclear envelope breakdown (NEBD), which exposed their chromatin to the cytoplasmic environment of the oocytes, and the reconstituted nuclei underwent significant ultrastructural remodeling. No transcriptional activity was ever detected in these reconstituted nuclei during the subsequent 3-4 h of culture of the resulting hybrid cells. In the case of MEL, this means that transcriptional activity ceased as soon as they entered the cytoplasm. In contrast, somatic nuclei which entered the cytoplasm of activated oocytes 3 h postactivation did not undergo NEBD and their remodeling was less pronounced. In contrast to the first group, these nuclei were transcriptionally active during the following 3-4 h of culture. In both cases, the female pronucleus remained transcriptionally silent. PMID- 8635522 TI - Autocrine regulation of macrophage proliferation by tumor necrosis factor-alpha. AB - We have examined production of tumor necrosis factor-alpha (TNF-alpha) from mouse bone marrow-derived macrophages (BMM) after stimulation by lipopolysaccharide (LPS), macrophage colony-stimulating factor (M-CSF/CSF-1), or granulocyte macrophage colony-stimulating factor (GM-CSF). To control for effects of trace levels of LPS in culture media, we examined CSF-1 and GM-CSF-stimulated TNF-alpha production in relatively homogeneous populations of normal LPS-hyporesponsive C3H/HeJ BMM and a growth factor-dependent cell line (S1) of C3H/HeJ origin. We found that both TNF-alpha mRNA and protein are induced in these macrophage populations by CSF-1 stimulation alone. Stimulation with GM-CSF, however, appears to negatively regulate TNF-alpha protein levels. Most TNF-alpha detectable after CSF-1 or GM-CSF stimulation was cell associated. Only stimulation by LPS resulted in large amounts of released TNF-alpha detectable in culture supernatant. The hypothesis that endogenously produced TNF-alpha can function as an autocrine growth factor for CSF-1-stimulated proliferation was supported by the demonstration of a partial inhibition of BMM proliferation (p < 0.05) in the presence of a neutralizing anti-TNF-alpha antiserum. These results demonstrate that CSF-1 alone is capable of inducing expression of both TNF-alpha mRNA and protein, that GM-CSF may negatively regulate TNF-alpha protein production, and that endogenously produced TNF-alpha may provide additional growth signals for CSF-1 mediated BMM proliferation. PMID- 8635523 TI - Reversible differentiation of human monoblastic leukemia U937 cells by ML-9, an inhibitor of myosin light chain kinase. AB - Human monoblastic leukemia U937 cells are induced to differentiate into monocytes and macrophages by various agents. We have shown that 1-(5-chloronaphthalene-1 sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), an inhibitor of myosin light chain kinase, induces differentiation of monocytoid leukemia cell lines U937 and THP-1 but not of myeloblastic leukemic ML-1 cell or erythroleukemia K562 cells. In the present study, we further analyzed the effect of ML-9 in comparison with that of 1 alpha, 25-dihydroxyvitamin D3 (VD3) a typical inducer of monocytic differentiation. ML-9 induced nitroblue tetrazolium (NBT)-reducing activity of U937 cell more rapidly than VD3: This differentiation marker was induced significantly after incubation with ML-9 and VD3 for 4 hours and 1 day, respectively. ML-9 also induced alpha-naphthyl acetate esterase (ANAE) activity, another monocytic differentiation marker, more rapidly than VD3. The maximum levels of these markers induced by ML-9 were comparable to those induced by VD3, but after removal of ML-9 from the medium by washing the cells, the expressions of theses markers decreased within 4 hours and reached basal levels in 1 day, indicating that ML-9's induction of expression of differentiation-associated phenotypes was reversible. The growth inhibition of U937 cells by ML-9 was also reversible. Similar effects were observed in another line of human monoblastic cells, THP-1. ML-9 had little or no effect on the morphology of U937 cells but increased the expression of monocyte-macrophage lineage-associated surface antigen, CD14, to some extent. Irreversible terminal differentiation induced by VD3 is associated with down regulation of the expression of c-myc and upregulation of the expression of c-fos and c-jun, but ML-9 did not affect the expression of these oncogenes appreciably. ML-9-induced differentiation was also reversible when the cells were cultured with cultured with ML-9 plus an anti cancer drug such as 1-beta-D-arabino-furanosylcytosine or daunomycin. it became irreversible, however, upon simultaneous treatment with dexamethasone and transforming growth factor-beta 1 (TGF-beta 1), which did not induce differentiation of U937 cells but caused growth arrest of the cells in the G0/G1 phase of the cell cycle. These results suggest that ML-9 should be useful for studying the mechanisms of monocytic differentiation. PMID- 8635524 TI - Expression of adhesion molecules on cytoplasmic processes of human megakaryocytes. AB - Megakaryocytes generate cytoplasmic processes (CP) that penetrate endothelial cells in the bone marrow sinus, and these processes may release platelets into the circulation at their terminal stage. Adhesion between the CP and endothelial cells may be important during the extension of CP. We examined the expression of adhesion molecules of the integrin family (CDw49b, CDw49d, CDw49e, CDw49f, CD18, CD11a CD11c, and CD11b), the immunoglobulin superfamily (CD54, CD56, CD58, and CD31), the selectin family (ELAM-1, LECAM-1, and CD62), and CD44, CD41b, and CD42b on platelets, megakaryocytes, and megakaryocytes with CP. No specific adhesion molecules were observed on the megakaryocytes with CP. Three staining patterns of adhesion molecules-homogeneous, speckled, and accumulated-were observed on the megakaryocytes with CP, but not on those without CP. Platelet integrins (i.e., CD41a, CDw49b, CDw49e and CDw49f) and GPIb (CD42b) were strongly and homogeneously stained on the CP. GMP-140 CD62) was weakly stained, in a speckled pattern. CD31 (PECAM-1) was also weakly stained but accumulated selectively on the tip of the CP. ANTI-CD31 suppressed CP formation of megakaryocytes. We speculate that the homodimerization of CD31 expressed on the tips of CP and endothelial cells is important for the extension of the processes and for the migration of megakaryocytes. PMID- 8635525 TI - Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura. AB - Canine idiopathic thrombocytopenic purpura (ITP) is clinically analogous to chronic ITP in human beings. The objective of this study was to investigate the pathogenesis of canine ITP by determining whether immunoglobulins bound to the surface of platelets from dogs with ITP (platelet-bound immunoglobulins) were directed against host platelet antigen and whether platelet glycoproteins (GP) IIb and IIIa were target antigens in dogs with ITP. Thirty-two dogs with ITP were studied. Increased platelet-bound immunoglobulin concentrations were detected in 30 cases (94%), and increased concentrations of serum platelet-bindable immunoglobulins were detected in 11 cases (34%). Immunoglobulins eluted from the surface of platelets from dogs with ITP bound to homologous normal canine platelets in 11 of 19 cases (58%). Immunoglobulins against platelet membrane GP IIb and/or IIIa were detected in serum from four of 17 affected dogs. This study provides evidence that immunoglobulins bound to surface of platelets from some dogs with ITP are directed against host platelet antigens and that platelet target antigens are, in some cases, GP IIb and IIIa. This supports the hypothesis that canine ITP is an autoimmune disease, similar to the pathogenesis of chronic ITP in human beings. PMID- 8635526 TI - Role of p53 and RB on in vitro growth of normal umbilical cord blood cells. AB - Human umbilical cord blood (UCB) is rich in hematopoietic stem cells and progenitors and recently has been used in the clinic as an alternative source for graft and marrow repopulation. We tried to determine in vitro the roles of wild type (wt) p53 and wt RB tumor/growth suppressor genes in the regulation of proliferation and maturation of hematopoietic UCB cells. CD34+ cells, isolated from mononuclear cells of UCB, were cultured in semisolid medium under conditions that favor growth of hematopoietic cells. We studied the level of expression of p53 and RB mRNAs and proteins during cell culture by Northern blot and cytofluorometry analysis, respectively. Sense (S), antisense (AS), or scrambled (missense [MS]) p53 and RB oligodeoxynucleotides (ODNs) were used to study the behavior of these cells in the absence of expression of p53 and/or RB. Adequate doses of p53 or RB ODNs inducing maximal inhibitory effect were used to study the behavior of these cells in the absence of expression of p53 and/or RB. Adequate doses of p53 or RB ODNs inducing maximal inhibitory effect with minimal cellular toxicity were determined. Exposure of CD34+ cells to p53 or AS, RB AS, or both p53 and RB AS but not other ODNs (sense or missense) resulted in a significantly increased number of colony-forming units-granulocyte/macrophage (CFU-GM) induced by interleukin-3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor (GM-CSF). The number of erythroid colonies (CFU-E) and burst-forming units (BFU E) derived from CD34+ cells in the presence of erythropoietin (Epo) was not significantly increased, whereas the number of such colonies was markedly increased in the presence of IL-3 + EPO upon p53 AS and/or RB AS treatment with hypothesis that wt p53 and RB are proliferation suppressor genes that interfere with normal maturation of hematopoietic cells. PMID- 8635527 TI - In vivo protective effects of tetrapeptide AcSDKP, with or without granulocyte colony-stimulation factor, on murine progenitor cells after sublethal irradiation. AB - Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) demonstrated hemato-protective activity in mice after sublethal irradiation (7 GY). Bone marrow interleukin-3 (IL-3)-responsive colony-forming cells (CFC and high proliferative potential colony-forming cells (HPP-CFC) were significantly (p < 0.05) increased by day 10 after irradiation in mice receiving a continuous infusion of 1000 ng/day of AcSDKP compared to irradiated control mice. The maximum protective effect for bone marrow progenitors was achieved when AcSDKP was administered for 3 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days after AcSDKP infusion in irradiated mice, significantly increased numbers of IL-3 responsive CSF-only control mice. In addition, platelets were significantly (p < 0.05) increased in mice receiving AcSDKP and G-CSF on days 18 and 21 after irradiation compared with mice receiving G-CSF alone. We conclude that ACSDKP has a radioprotective effect in vivo for progenitor cells, and that time of initiation and duration of AcSDKP administration relative to irradiation are crucial for these effects. Further, AcSDKP has a significant additive protective effect not only for progenitor cells but also for platelets when given in combination with G-CSF. We suggest that these in vivo observations provide a basis on which to design optimal clinical hypothesis and protocols. PMID- 8635528 TI - Thrombocytopenia in the toothless (osteopetrotic) rat and its rescue by treatment with colony-stimulating factor-1. AB - Osteopetrosis in toothless (tl) rats is characterized by reductions in bone resorption, osteoclasts, and macrophages, resistance to cure by bone marrow transplantation, and skeletal improvement after treatment with colony-stimulating factor-1 (CSF-1). Reductions in skeletal osteocalcin tl rats, together with the recent demonstration of osteocalcin expression in platelets and its possible role in bone turnover, prompted us to examine whether this rat mutation is associated with altered platelet numbers. Our prediction of a thrombocytopenia was confirmed by examination of tl rats, in which a profound reduction (32%) in platelets was accompanied by a significant elevation (62%) in megakaryocytes (MKC) compared to normal littermates. Light and transmission electron microscopy confirmed increases in both number and size of MKC in mutants without morphologic abnormalities of circulating platelets. CSF-1 treatment (10(6) U/48 hours for 10 days) of mutants restored platelet numbers to those found normal littermates and increased osteoclasts and the frequency of MKC in numbers. Preliminary studies of another mutation the rat, osteopetrosis (op), revealed a similar reduction (33%) in platelets. These data demonstrate the coexistence of osteopetrosis and thrombocytopenia in two osteopetrotic rat mutations and an increase in osteoclasts and platelets in one mutation after CSF-1 treatment. Together, these data suggest a potential functional interaction of MKC and osteoclasts in bone turnover. PMID- 8635529 TI - Interactions between leukemia cells and bone marrow stromal cells: stroma supported growth vs. serum dependence and the roles of TGF-beta and M-CSF. AB - Leukemia cell lines that do not proliferate in the absence of serum grow well when cultured with stromal cells. To study this growth dependence on stroma, we selected the M1 myeloblast clone, since its stroma dependence is reminiscent of that exhibited by hematopoietic stem cells. Conditioned medium form a stromal cell line, prepared under serum-free conditions, contained an activity that induced the proliferation of M1 cells and was therefore designated M1 myeloid activity (MMA). Among the various cytokines tested for MMA-like activity, only transforming growth factor-beta (TGF-beta) and macrophage colony-stimulating factor (M-CSF) were found to affect M1 cell survival, and the two cytokines acted synergistically to induce M1 cell growth. Antibodies to both TGF-beta and M-CSF abolished most, but not all, of the MMA in the medium conditioned by stromal cells, indicating that additional factors contribute to MMA. A subclone of M1 cells, M1/M2, selected in medium conditioned by stroma, was found to respond to stromal stimulation but was unable to proliferate in fetal calf serum (FCS). Neutralization experiments indicated that M1/2 cell growth depended mainly on M CSF and also partially on TGF-beta. By contrast, the same neutralizing antibodies did not affect the ability of serum to support M1 cell growth. The molecules that promoted leukemia cell growth in serum seemed therefore to differ from those provided by stroma. This model system may offer novel information on the interactions of normal and leukemic hematopoietic cells with their stromal microenvironment. PMID- 8635531 TI - Stem cell factor, but not flt3 ligand, induces differentiation and activation of human mast cells. AB - We have examined the effect of human ligand for the flt3/flk2 tyrosine kinase receptor on the differentiation of human mast cells in suspension cultures. We also explored the effect of flt3 ligand (FL) on the human mast cell line HMC-1 and mRNA expression of flt3/flk2 on in vitro developed human mast cells and HMC 1. The growth of cord blood mononuclear cells in suspension cultures was increased when cells were cultured in the presence of FL compared with cells cultured in the presence of stem cell factor (SCF). When SCF and FL were combined, the total cell growth was increased further. Our data show that Fl by itself neither induced differentiation of mast cells nor acted in the SCF dependent differentiation of human cord blood-derived mast cells (CBMC). Furthermore, no effects of FL were found on the proliferation of HMC-1 cells or the induction of early-immediate response genes in HMC-1 cells. In addition, neither HMC-1 cells nor CBMC expressed mRNA for flt3. As has been shown before, SCF and FL have little biological effect on their own but synergize well with a number of other hematopoietic growth factors. This study shows that a major difference between FL and SCF is that only SCF affects the differentiation and activation of mast cells. PMID- 8635530 TI - Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34+ cells. AB - As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34+ cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34+ cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34+ cells into cycle were stem cell factor (SCF) plus one of the following: interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF). The maximum numbers of cells in S+G2M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34+ cells remained quiescent in the first G0/G1 phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34+CD38- subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-G pseudotyped virus containing the neomycin phospho-transferase gene (neo), we exposed CD34+ cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34+ cells and the CD34+CD38- subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT neoDNA was detectable in CD34+ cells that have been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34+ cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the 34+CD38- subset showed that RT-reoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34+ cells but not in CD34+CD38- cells. These results suggest during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34+CD38-cells in addition to the lack of mitotic activity. PMID- 8635533 TI - Effects of axonal injury on astrocyte proliferation and morphology in vitro: implications for astrogliosis. AB - Mechanisms inducing gliosis following injury in the central nervous sy stem are poorly understood. We evaluated the effect of axonal injury on astrocyte and Schwann cell proliferation and morphology in vitro. Purified rat dorsal root ganglion neurons grown on monolayers of rat neonatal cortical astrocytes (N ASneonatal cultures) or sciatic nerve-derived Schwann cells (N-SC cultures) were mechanically injured. Non-injured cultures served as controls. Cell proliferation near lesions was monitored by autoradiography 1,2,4, and 8 days postinjury. Axonal injury caused a significant transient increase in astrocyte proliferation immediately proximal and distal to the lesion. The lesion did not induce marked changes in the intensity of glial fibrillary acidic protein (GFAP) immunoreactivity. However, processes from GFAP-positive cells usually arranged in random fashion in noninjured cultures were aligned perpendicularly to the cut distal to lesions. Ultrastructural analysis in lesioned N-ASneonatal cultures indicated that proximal to the lesion filament-filled astrocytes were intermingled with axons. Distal to the lesion astrocyte processes formed layers, between which an increased amount of collagen-like material appeared with time postlesion. Axons distal to the lesion degenerated by 2 days, coinciding with the early disappearance of neurofilament immunoreactivity. In noninjured and proximally in injured N-SC cultures, Schwann cells extended processes, engulfing some axons. Distal to the lesion, Schwann cells appeared more rounded and neurites remained until 4 days postinjury. Media conditioned by injured or non injured N-ASneonatal cultures did not affect neuron-induced Schwann cell proliferation. These findings demonstrate that axonal injury and degeneration cause a transient increase in astrocyte proliferation and induce morphological changes in astrocytes consistent with the onset of gliosis. PMID- 8635532 TI - Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA. AB - The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies. PMID- 8635534 TI - Early effects of FGF-2 on glial cells in the MPTP-lesioned striatum. AB - Fibroblast growth factor-2 (FGF-2), locally administered in gelfoam to the striatum of mice treated with the neurotoxic drug 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), has restorative and neuroprotective effects on dopaminergic neurons and associated striatal transmitter systems. Most of the beneficial alterations are apparently indirect. FGF-2 must therefore act through a series of cellular and molecular intermediate steps, which have not been explored. We have previously shown that FGF-2 does not significantly affect the astroglial reaction at the time, when the neuroprotective effect of FGF-2 reaches a peak (Day 11). In this study we have investigated the effect of FGF-2 at earlier time points after MPTP treatment. We report now that as early as 6 h after administration of the gelfoam containing either FGF-2 or control protein, FGF-2 immunoreactivity disappears from astroglial nuclei, while appearing in small ramified GFAP- and S-100-negative cells, most likely microglia. At 18 h, numbers and staining intensities of GFAP-ir astroglial cells are greater in FGF-2 than in cytochrome C-treated animals. At this time FGF-2-ir reappears in astroglia nuclei of cytochrome C-treated animals, but remains undetectable in the striatum carrying the FGF-2-containing gelfoam. Ramified GFAP/S-100-negative presumed microglial cells are now intensely ir for FGF-2. Signs of an FGF-2 mediated astrogliotic reaction are very pronounced at 18 h and 2 days, but no longer at 11 days, when the astrogliosis reaction has become equally strong in FGF-2- and cytochrome C-treated striata. Our results suggest that administration of FGF-2 to the MPTP-lesioned striatum has early effects on astro- and presumed microglia cells, notably on the nuclear FGF-2-ir of astrocytes. These changes may be involved in mediating the neuroprotective effects of FGF-2 in the MPTP-model of Parkinsonism. PMID- 8635535 TI - Specific toxicity of tunicamycin in induction of programmed cell death of sympathetic neurons. AB - Tunicamycin belongs to a group of antibiotics which can cause severe a nd often fatal neurological malfunction in animals, commonly known as "annual ryegrass toxicity." At the cellular level, tunicamycin is a potent glycosylation inhibitor which is often used to elucidate the importance of glycosylation in protein functions. Earlier reports suggested that tunicamycin was able to interfere with the binding of nerve growth factor to its receptors. In this report, we showed that tunicamycin was able to kill sympathetic neurons in cultures. The mechanism of cell death was observed to be similar to that of "programmed cell death" in sympathetic neurons induced by nerve growth factor deprivation. Such tunicamycin induced cell death could be prevented by the protein synthesis inhibitor cycloheximide, which was known to prevent the programmed cell death in sympathetic neurons. These results demonstrated that, in addition to the proven CNS neurotoxicity in animals, tunicamycin causes programmed cell death in peripheral (sympathetic) neurons. PMID- 8635536 TI - Recovery of locomotion after spinal cord hemisection: an X-ray study of the cat hindlimb. AB - Hemisection of the spinal cord in adult cats is a suitable model to st udy the mechanisms underlying recovery of motor functions. The initial paresis of the hindlimb is followed by a considerable improvement of locomotor functions of the affected hindlimb. Kinematic analyses of treadmill locomotion were performed from 10 days to 8 months after complete hemisections (right side) of the spinal cord at the thoracolumbar level, using X-ray cinematography for precise measurements of the hindlimb joint angles. The footfall pattern and the electromyogram were recorded. Motor control of both proximal and distal hindlimb joints improved substantially during the 1st postoperative month. However, persistent locomotor deficits were still present several months after hemisection. They could be divided into three groups of symptoms: (1) The gait pattern was disturbed with regard to interlimb coordination. The stance-phase duration of the right hindlimb was shortened. (2) The flexor capacity of the affected hindlimb was reduced, resulting in a slow insufficient flexion of the hip, knee, and ankle during the swing phase. (3) The timing of the flexion-extension events was impaired. The onset of the E1-extension was delayed and the amplitude was reduced. Electromyographic patterns of muscle activity during locomotion of the lesioned side limb differed from the contralateral hindlimb, which served as a contro. The results indicate that in spite of a good short-term functional improvement there are long-term locomotor deficits present after spinal cord hemisection. PMID- 8635537 TI - Implantation of olfactory ensheathing cells in the adult rat brain following fimbria-fornix transection. AB - Ensheathing cells from fetal rat olfactory bulb were implanted into th e damaged adult rat brain to assess whether these cultured cells would survive in nonolfactory CNS areas and support the regrowth of nonolfactory axons. Cultures of primary ensheathing cells prelabeled with WGA-Au were embedded in a collagen matrix and implanted into a lesion cavity immediately following ablation of the fimbria-fornix in adult rats; the animals were sacrificed 4 weeks after surgery. Labeled ensheathing cells were observed only within the graft and not in the adjacent neural tissue. Immunostaining for p75 neurotrophin receptor revealed two characteristic morphologies of ensheathing cells within the graft; slender, spindle-shaped cells and larger, flattened cells. Although GFAP immunostaining revealed an intense glial reaction around the margin of the wound with host astrocytes sending cytoplasmic processes into the collagen matrices, no immunoreactive cells were found within the grafts. Histochemical detection of AChE revealed numerous reactive fibers confined to the regions of the grafts possessing ensheathing cells. Axons within the grafts were also immunoreactive for GAP-43. These initial experiments provide encouraging data concerning the survival of ensheathing cells for a minimum of 4 weeks following implantation into the adult rat brain and their ability to support the growth of new axons. PMID- 8635539 TI - Motor performance score: a new algorithm for accurate behavioral testing of spinal cord injury in rats. AB - To evaluate the usefulness of standard neurological tests in predictin g the neurological outcome after photochemically induced spinal cord lesions in rats, we inflicted injuries of different severity to adult female rats. The behavior of the rats was followed for 6 weeks and the results of the behavioral tests were correlated with morphological indicators of tissue destruction at the end of this period. We found many behavioral tests to be highly correlated with the loss of tissue, whereas some tests were inaccurate in correlating with degree of tissue destruction. Motor score, beam walk, and righting reflect were all highly correlated with the volume of the lesion as well as the depth of the lesion cavity at its epicenter. We propose a protocol for neurological evaluation of this type of spinal cord injury consisting of six individual tests, hierarchally organized such that injured rats can be divided into 11 groups ofn eurological deficit, scored from 10 to 0. This so-called motor performance score is fast and easy to perform and shows high correlation with the lesion volume, and is thus suitable for neurological evaluation of photochemically induced spnial cord injury. PMID- 8635538 TI - Haloperidol prevents ketamine- and phencyclidine-induced HSP70 protein expression but not microglial activation. AB - Noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, inclu ding ketamine and phencyclidine (PCP), produce abnormal intracellular vacuoles in posterior cingulate and retrosplenial cortical neurons in the rat. Ketamine also induces 70-kDa heat shock protein (HSP70) expression in pyramidal neurons in the posterior cingulate and retrosplenial cortex and, as shown by this study, activates microglia in the retrosplenial cortex of the rat. Whereas HSP70 protein expression was induced with ketamine doses of 40 mg/kg (ip) and higher, doses of 80 mg/kg and higher were required to activate microglia. HSP70-positive neurons were observed in 30- to 90-day-old rats but not in younger, 10- to 20- day old animals following ketamine (80 mg/kg, ip). Pretreatment with the antipsychotic drug haloperidol at doses of 1.0 mg/kg and above abolished all HSP70 immunostaining produced by ketamine (80 mg/kg). However, a single dose of haloperidol (5 mg/kg, im) did not decrease the number of microglia activated in retrosplenial cortex by ketamine (80-140 mg/kg). Similarly, PCP (10 and 50 mg/kg, ip)-induced microglial activation in the posterior cingulate and retrosplenial cortex of adult rats was not blocked by haloperidol (10 mg/kg, im, 1 h prior to PCP). These results suggest that ketamine and PCP injure neurons in the posterior cingulate and retrosplenial cortex of adults rats. Though haloperidol may afford some protection against this injury since it inhibits induction of HSP70 expression, the failure to prevent microglial activation suggests that single doses of haloperidol do not completely protect neurons from NMDA antagonist toxicity. PMID- 8635540 TI - Elevated potassium enhances glutamate vulnerability of dopaminergic neurons developing in mesencephalic cell cultures. AB - This study examines the effects of high K+ concentration on the growth and development of mesencephalic cells and their glutamate vulnerability. Mesencephalic cell cultures obtained from Wistar rat embryos on the 14th gestational day were maintained for 14 days in medium with either normal (4.2 mM) or elevated (24.2 mM) potassium concentration. There was no significant difference due to various K+ concentration in cell growth and survival up to day in vitro (DIV) 13-14. In order to test the glutamate (Glu) vulnerability, cultures were treated with 100 mu M Glu for 15 min in salt solution on the DIV 3,6,8 and 13. Glu-induced neuronal damage was estimated 24 h later by measuring the neuron-specific enolase (NSE) content in the culture medium and by counting the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Glu had no damaging effect on the cells on DIV 3, but became pronounced beyond DIV 6. Elevated potassium concentration 24.2 mM) in the culture medium during development significantly increased neuronal vulnerability to Glu treatment, indicated by a higher increase of NSE content in the medium and by a more pronounced Glu-induced decrease of the number of TH-IR cells. The Glu-induced decrease of the number of TH-IR cells and of NSE-IR cells let us conclude that dopaminergic neurons are more vulnerable to glutamate than other neurons from mesencephalic culture. PMID- 8635541 TI - Transplanted CG4 cells (an oligodendrocyte progenitor cell line) survive, migrate, and contribute to repair of areas of demyelination in X-irradiated and damaged spinal cord but not in normal spinal cord. AB - In this study, we have examined the behavior of a lac-Z-transfected O- 2A progenitor cell line, CG4, following transplantation into normal and X-irradiated adult rat spinal cord, and we have also addressed the issue of whether CG4 cells transplanted remotely from ethidium bromide-induced demyelinating lesions in both X-irradiated and nonirradiated spinal cord are able to contribute to their repair. Following transplantation into X-irradiated spinal cord, CG4 cells survive, divide, and migrate extensively. The migration occurs mainly within the parenchymal tissue of the cord without preference for white or gray matter. Moreover, CG4 cells migrating away from their point of introduction are able to enter areas of demyelination and remyelinate the demyelinated axons therein. In contrast, when CG4 cells are transplanted into nonirradiated spinal cord, their survival is limited to areas of damage created by the injection procedure. The CG4 cells do not survive in undamaged, nonirradiated spinal cord. When transplanted remotely from areas of demyelination they are unable to traverse intervening areas of normal white matter, although they may enter lesions if transplanted into their close vicinity. These results have important implications for the development of potential therapeutic strategies for the treatment of multifocal demyelinating disorders that are based on glial cell transplantation. PMID- 8635542 TI - Axonal growth into tubes implanted within lesions in the spinal cords of adult rats. AB - Cultured Schwann cells were implanted into the thoracic spinal cords o f adult rats inside thin tubes made of polycarbonate film coated with poly-L-lysine. Additional control tubes were implanted which did not contain cultured Schwann cells. Some of the control tubes were coated with poly-L-lysine and others were not. One week to 2 months later the animals were perfused with fixatives and the tubes were prepared for light or electron microscopy. Immunocytochemical studies of the Schwann cell tubes reveal that they contain axons. Most of these axons are grouped in fascicles that run longitudinally through the tubes. The distribution of these axons matches precisely the distribution of basal lamina within the tubes as displayed by immunolabeling with an antibody to laminin. Surprisingly, the same patterns of labeling are seen in the control tubes, although they contain fewer axons. Control tubes lacking poly-L-lysine contain the fewest. Electron microscopy verifies that the tubes, including control tubes, contain Schwann cells and axons of different diameters. Furthermore, the Schwann cells ensheathe and myelinate the axons. These results strengthen the hypothesis that Schwann cells can support axonal growth in the spinal cords of adult animals. They also demonstrate that these Schwann cells can be implanted or they can be derived from the host animal. This finding raises the possibility that therapies could be devised for bridging spinal cord lesions that are based on maximizing migration of endogenous host cells into the sites of lesions. PMID- 8635543 TI - Effect of REM sleep deprivation on sleep apneas in rats. AB - We studied the effects of 48 h of REM sleep deprivation on spontaneous and post sigh central apneas in Sprague-Dawley rates by simultaneously monitoring sleep by the EEG and respiration for 6 h. During the recovery sleep following REM deprivation a decrease in post-sigh apneas occurred in total sleep. There was no change in spontaneous apneas. The results suggest the existence of partially distinct mechanisms for the two types of apneas. PMID- 8635544 TI - Segregation of optic input in a three-eyed mammal. AB - Using a marsupial mouse, we have transplanted additional eye primordia to the midbrain at stages before host visual centers are normally innervated; transplant or host projections were later traced. In contrast to the normally continuous crossed retinocollicular input, both transplant and host projections were arranged as rostrocaudally aligned stripes or as discrete patches. We propose that competitive interactions between transplant and host axons have induced the discontinuous input. PMID- 8635545 TI - Basic behavior of migratory Schwann cells in peripheral nerve regeneration. AB - In axonal regeneration after a peripheral nerve injury, Schwann cells migrate from the two nerve ends and at last form a continuous tissue cable across the gap which guides the axons toward the bands of Bungner. However, the behavior of migratory Schwann cells and their possible role are obscure. Using a film model in which the proximal stump of a transected nerve in mice was sandwiched between two thin plastic films, we analyzed neural regeneration in the early phase up to the 6th day after axotomy. Regenerating neurites emerged from the nodes of Ranvier adjacent to the axotomized nerve stump within 3 h after axotomy and extended along the parent nerve onto the film. All of the regenerating neurites on the surface of the film consisted of naked axons for at least 2 days after axotomy. Thereafter, Schwann cells from the proximal nerve migrated along a network of the regenerating axons and then closely attached to the axons, ensheathing them. Some of the Schwann cells advanced ahead of the axonal growth cones and were distributed over regions in which axonal extension was not yet present. As calculated from the time course of regenerating neurites, the velocity of axonal regeneration showed two phases: an initial slow phase (77 mu m/day) up to the 2nd post-operative day followed by a faster phase (283 mu m/day). The first observation of Schwann cells coincided with the onset of the second phase. In addition, the length of regenerating axons on the surface of the film containing many Schwann cells was significantly greater than that on the surface where Schwann cells were not yet present. It meant that migratory Schwann cells stimulated axons to elongate for a longer distance. Furthermore, Schwann cells from a distal stump showed a stronger ability to accelerate the axonal outgrowth than these from a proximal stump. PMID- 8635546 TI - Labeling and identification of living donor cells in brain slices of recipient hemiparkinsonian model rats for physiological recordings: methods for physiological assessments of neural transplantation. AB - Physiological properties of grafted neurons, such as membrane and intr acellular properties, have not been reported. To fill this lack in knowledge, physiological recordings from the identified grafted cells are required. Fluorescent latex microspheres (FLM) are non-toxic and stable, and thus seem suitable for long-term labeling of donor cells. Therefore, we tested the feasibility of labeling with FLM to identify living donor cells in the recipients' brain slices. We also tested if physiological recordings from the identified cells are possible or not. Cell suspensions were prepared from the substantia nigra (SN) of Embryonic Days 15 or 16 rats with enzymatic and mechanical trituration. Cell suspensions were then incubated with 0.5% FLM for 30 min to 2 h. The longer cells were incubated, the more FLM were taken up. The FLM-labeled SN cell suspensions were injected in the striatum of the hemiparkinsonian model rats. Eight to 13 weeks later, 150-mu m thick coronal brain slices including the graft track were prepared from the recipients. Slices were kept in vitro for several hours. Grafted cells could be clearly identified in the slice preparations by the uptake of FLM under a fluorescence microscope. Voltage-dependent currents and intracellular Ca2+ transcients were successfully recorded from the identified grafted neurons. It is suggested that labeling and identification of living donor cells with FLM is feasible and thus can provide a powerful tool to study the mechanisms underlying graft-induced amelioration of neurological deficits in parkinsonism by enabling physiological assessments of grafted cells. PMID- 8635547 TI - Fibroblast growth factor receptor-1 in the lateral hypothalamic area regulates food intake. AB - Previous studies have shown that acidic and basic fibroblast growth fa ctor (aFGF and bFGF) and certain fragments of the aFGF N-terminal suppress food intake in rats due to their inhibitory actions on the glucose-sensitive neurons in the lateral hypothalamic area (LHA). The present study was planned to determine the role of FGF receptor-1 (FGFR-1), which was found in the LHA neurons of rats, on feeding regulation. The structure-activity relationship of aFGF fragments in feeding suppression was also investigated. An injection of anti-FGFR-1 antibody (250 and 350 ng) into the bilateral LHA significantly increased food intake. Synthesized aFGF fragments were infused into the III ventricle to elucidate the structure-activity relationship on the inhibition of feeding. Although aFGF-(1 29) did not affect food intake, [Ser16]aFGF-(1-29) (400 ng) and [Glu16]aFGF-(1 29) (400 NG), in which the cysteine residue at position 16 of aFGF(1-29) was replaced with structurally similar serine and glutamic acid, were observed to significantly inhibit food intake. These findings suggest that endogenous FGFR-1 in the LHA plays an important role in FGF-induced feeding suppression, while, in addition, the dissolving disulfide bond formation in aFGF fragments enhances their inhibitory effects on feeding. PMID- 8635548 TI - Early cytopathic features in rat ischemia model and reconstruction by neural graft. AB - Using a silver impregnation (argyrophil III) and immunohistochemistry, acute cytopathic features after cerebral ischemia were investigated. Additionally, functional recovery and interconnection between the host and graft was also explored after neural graft. Animals were embolized in unilateral middle cerebral artery for 1 h. Argyrophil III method demonstrated "collapsed" dark neurons in the striatum, cortex, reticular thalamus, amygdala, and hypothalamus on ischemic side. These neurons exhibited characteristic shrunken somata with corkscrew-like dendrites, suggesting changes in cytoskeletal protein. In the above mentioned areas, the loss of immunoreactivity for mu-calpain proenzyme and microtubule associated protein 2 was also detected. Neural graft into the ischemic striatum was made 2 weeks after the ischemia paradign. The grafted striatal cells were prepared from E15 fetuses to make cell suspension marked by rhodamine-labeled latex microspheres. Methamphetamine-evoked rotations were detected after ischemia. These motor alterations were reduced gradually but significantly at 8 weeks after the graft. Interconnecton between the host and grafted cells was then studied in a brain slice preparation after loading fura-2 AM. About 10% of grafted cells tested from rats that showed motor amelioration exhibited [Ca2+]i increase to the electrical stimulation applied to the neighboring host tissue. Data indicate that, in the very early stage after ischemia, cytoskeletal damages, especially on microtubules, started and this would lead to later infarct. The graft survived in the ischemic striatum having connections with the host, and this might be partly involved in the amelioration of motor function. PMID- 8635549 TI - Neural transplantation and trophic factors in Parkinson's disease: special reference to chromaffin cell grafting, NGF support from pretransected peripheral nerve, and encapsulated dopamine-secreting cell grafting. AB - Since adrenal medullary chromaffin cells produce catecholamines as wel l as several kinds of neurotrophic factors which affect dopamine neurons, the authors have investigated the methods to increase the survival of grafted chromaffin cells in parkinsonian model animals. Measurement of nerve growth factor (NGF) showed that NGF level at the distal stump of the pretransected peripheral nerve increased significantly, thus, we have applied cografting of chromaffin cells with this stump of the peripheral nerve to animal models of Parkinson's disease since chromaffin cell survival have been reported to be increased by supplementation of nerve growth factor (NGF) in vitro and in vivo. By this cografting approach, not only the chromaffin cell survival but also the host intrinsic dopaminergic system recovery were enhanced. This effect continued 2 years in our long-term study The effects of donor and host ages were investigated and the results showed that the effects were more prominent when young donors or hosts were used compared with aging donors or hosts. Although cografting of adrenal medulla with peripheral nerve was applied successfully in parkinsonian patients with favorable results, it may be difficult to apply this procedure in aged patients since this is autografting and adrenal medulla itself may be affected by the disease in aged patients. Polymer-encapsulated dopamine-secreting cells are another donor candidates and can be applied combined with stereotaxic thalamotomy or pallidotomy for the patients with Parkinson's disease in the near future. PMID- 8635550 TI - Adenosine and propentofylline inhibit the proliferation of cultured microglial cells. AB - Propentofylline is a xanthine derivative that has been known to protec t neurons against ischemia-induced damage. To assess its neuroprotective mechanisms, we examined the effect of propentofylline on microglial proliferation that is thought to play an important role in neuronal damage. We determined the proliferation of microglia cultured from neonatal rat brains by measuring [3H]thymidine update. Propentofylline inhibited microglial proliferation in a dose dependent manner; EC50 was about 3 mu M. Similar results were observed with 2-chloroadenosine (agonist for A1 and A2 adenosine receptors) and 2-chloro-N6 cyclopentyladenosine (A1 receptor agonist) but not with 2-p-(2 carboxyethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine hydrochloride (A2 receptor agonist). However, 8-cyclopentyl-1,3-dipropylxathine (A1 receptor antagonist) could not reverse the inhibitory effect of propentofylline. Our results suggest that the neuroprotection by propentofylline is, as least in part, due to the direct effect of the drug on microglia and that the drug inhibits the proliferation via a certain mechanism not directly mediated by adenosine receptors. PMID- 8635551 TI - Microtubule-associated protein 2 expressing COS7 cells are resistant to argyrophilia under oxygen- and glucose-free condition. AB - Argyrophil III silver impregnation is a very sensitive method to detec t the early damage to neurons following brain ischemia. The argyrophil III staining and microtubule-associated protein 2 (MAP2) immunocytochemistry were performed on PC12D cells and MAP2C cDNA-transfected COS7 cells to detect the changes cytoskeletal proteins (microtubules/MAP2). After exposing these cells to simulated ischemic condition (oxygen and/or glucose free), the correlation between the appearance of the argyrophilia and the disappearance of MAP2 was investigated. The PC12D cells expressed very low MAP2 and became argyrophilic very easily depending on the degree of the ischmeia-like insult, whereas MAP2C cDNA-transfected COS7 cells expressed a higher level of MAP2C and were resistant to argyrophilia, although the diameter of their immunoreactive processes became thinner. Thus, when MAP2C is expressed at a higher level, cells became resistant to argyrophilia, suggesting a correlation between the argyrophilia and the damage on microtubules and MAPs. PMID- 8635552 TI - Regulation of normal proliferation in the developing cerebrum potential actions of trophic factors. AB - We review here a computational model of neocortical histogenesis based upon experiments in the developing cerebral wall of the mouse. Though based upon experiments in mouse, commonalities of developmental history and structure of neocortex across mammalian species suggest that the principles which support this model will be generally applicable to neocortical evolution and development across species. In its scope the model spans the successive histogenetic events: cell proliferation, cell migration, and the positioning of cell somata in neocortical layers following migration. Neurons are produced in a pseudostratified epithelium (PVE) which lines the ventricular cavaties of the embryonic cerebrum. The parameters which determine the rate and total number of neurons produced in the PVE are (1) the size of the founder population, (2) the number of integer cell cycles executed by the founder population and its progeny in the course of the neuronogenetic interval, (3) the growth fraction, and (4) the fraction of cells which exits the cycle (Q fraction) with each integer cycle. There is a systematic relationship between the integer cycle of origin and the sequence of cell migration, position in the cortex, and the extent to which a set of postmigratory neurons will be diluted in the cortex by the combined effects of tissue growth and cell death. Variation across species in the number of integer cell cycles as a function of the rate of progression of Q may be expected to modulate profoundly the total numbers of neurons that are produced but not the relative proportions of neurons assigned to the major neocortical layers. PMID- 8635553 TI - Microglial ramification requires nondiffusible factors derived from astrocytes. AB - It is generally accepted that process-bearing microglial cells origina te from ameboid macrophage-like mesodermal cells. This transformation, often called ramification, accompanies down regulation of macrophage-like properties, but the mechanisms involved in ramification have not been clarified. We investigated factors to promote ramification in culture. Isolated ameboid microglial cells were seeded on living or paraformaldehyde-fixed astrocyte monolayers. About 80% of the cells ramified on the fixed astrocytes in astrocyte-conditioned medium as well as on the living astrocytes. In fresh culture medium, 50% of the cells on the fixed astrocytes ramified. On the other hand, ameboid cells rarely ramified on noncoated glass coverslips even in the conditioned medium. Ameboid cells cultured on extracellular matrices dervied from astrocytes ramified more than on those coated with plasma fibronectin or collagen type I. A synthetic peptide containing Arg-Gly-Asp sequence or a tyrosine kinase inhibitor genistein partially reversed the ramification induced on the fixed astrocyte monolayers. These results show that some nondiffusible factors derived from astrocytes are essential for microglial ramification. A part of the nondiffusible factors are present in the extracellular matrices, and the effects might be mediated by integrins. Some diffusible factors secreted by astrocytes seem to promote ramification, if the nondiffusible factors are present. The experiments using the fixed astrocyte monolayers may be useful to identify the diffusible factors responsibile for ramification. PMID- 8635554 TI - Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. AB - Epidermal Growth Factor (EGF)-responsive stem cells isolated from the developing central nervous system (CNS) can be expanded exponentially in culture while retaining the ability to differentiate into neurons and glia. As such, they represent a possible source of tissue for neural transplantation, providing they can survive and mature following grafting into the adult brain. In this study we have shown that purified rat stem cells generated from either the embryonic mesencephalon or the striatum can survive grafting into the striatum of rats with either ibotenic acid or nigrostriatal dopamine lesions. However, transplanted stem cells do not survive as a large mass typical of primary embryonic CNS tissue grafts, but in contrast form thin grafts containing only a small number of surviving cells. There was no extensive migration of transplanted stem cells labeled with either the lac-z gene or bromodeoxyuridine into the host region surrounding the graft, although a small number of labeled cells were seen in the ventral striatum some distance from the site of implantation. Some of these appeared to differentiate into dopamine neurons, particularly when the developing mesencephalon was used as the starting material for generating the stem cells. EGF-responsive stem cells could also be isolated from the mesencephalon of developing human embryos and expanded in culture, but only grew in large numbers when the gestational age of the embryo was greater than 11 weeks. Purified human CNS stem cells were also transplanted into immunosuppressed rats with nigrostriatal lesions and formed thin grafts similar to those seen when using rat stem cells. However, when primary cultures of human mesencephalon were grown with EGF for only 10 days and this mixture of stem cells and primary neural tissue was transplanted into the dopamine-depleted striatum, large well-formed grafts developed. These contained mostly small undifferentiated cells intermixed with a number of well-differentiated TH-positive neurons. These results show that purified populations of rat or human EGF-responsive CNS stem cells do not form large graft masses or migrate extensively into the surrounding host tissues when transplanted into the adult striatum. However, modifications of the growth conditions in vitro may lead to an improvement of their survival in vivo. PMID- 8635555 TI - The ganglioside GM1 enhances microtubule networks and changes the morphology of Neuro-2a cells in vitro by altering the distribution of MAP2. AB - The effect of ganglioside GM1 on components of the neuronal cytoskeleton was studied in Neuro-2a neuroblastoma cells using immunofluorescent, immunogold labeled, and Western-blot analysis. Exposure of cells to GM1 for 24 h resulted in an increased microtubular network and level of tubulin, a redistribution of MAP2 immunoreactivity from perikarya to distal neuritic processes, and an increased MAP2 gold label in the subplasmalemmal cytoplasm, neuritic spines, and growth cones. A similar change in the distribution of actin-positive fluorescent immunoreactivity was observed. In contrast to the redistribution of MAP2, immunolocalization of MAP5 and tau did not change following 24 h GM1 exposure. Our results suggest that gangliosides enhance neuritogenesis by selectively altering the distribution of MAP2 from perikaryon to neuritic spines. Furthermore, the enhanced presence of MAP2 in regions known to be rich in microfilaments following GM1 treatment suggests that an interaction of MAP2 with microfilaments may be necessary for early neurite formation. PMID- 8635556 TI - Expression of the lacZ reporter gene in the rat basal forebrain, hippocampus, and nigrostriatal pathway using a nonreplicating herpes simplex vector. AB - We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy. PMID- 8635557 TI - Pentobarbitone induces Fos in astrocytes: increased expression following picrotoxin and seizures. AB - Fos immunoreactivity was observed in astrocytes identified by immunoreactivity to glial fibrillary acidic protein, 1.5 h following intravenous pentobarbitone anesthesia (20-30 mg/kg). Fos-positive astrocytes were seen only in the hilus of the hippocampus. Pentobarbitone administered after picrotoxin but without seizures resulted in an increase in both the intensity of Fos labeling and areal distribution of Fos-positive hilar astrocytes. Pentobarbitone administered after single picrotoxin-induced seizures resulted in an increased number of Fos immunoreactive astrocytes in the hilus and other hippocampal regions and their presence in the upper layers of Fr1, Fr2, cingulate, visual, and perirhinal cortex. Our observations that Fos is expressed in astrocytes following pentobarbitone, provides support for the current view that glial cell processes can be activated by pentobarbitone stimulation of GABAa receptor-chloride channels. PMID- 8635559 TI - Intraparenchymal NGF injections in adult and aged rats induce long-lasting Trk tyrosine phosphorylation. AB - Neurotrophic factors, particularly the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and related molecules are proposed for the experimental treatment of neurode-generative disease. Earlier observations had suggested down-regulation of the neurotrophin receptor response with chronic stimulation. We therefore tested for effects of acute and chronic NGF treatment in vivo on the tyrosine phosphorylation response of Trk-type neurotrophin receptors in adult and aged rats. Rats were treated for 1 week with daily injections of NGF directly into the striatum. Surprisingly, this chronic neurotrophin treatment induced long-lasting tyrosine phosphorylation of Trk type receptors beyond the last injection. A similar result was obtained with 1 week of daily injections of BDNF into the hippocampus. Persistent TRK tyrosine phosphorylation was also observed after single neurotrophin injections. With 1 microgram of NGF injected, Trk-type receptors were maximally stimulated from immediately after the injection until 3 days after the treatment. Maintaining Trk tyrosine phosphorylation required maintained energy levels in the tissue. Incubation of microslices of brain tissue from NGF-injected animals in glucose free buffer completely abolished all Trk tyrosine phosphorylation signals. Recovery of tissue in presence of glucose restored the signals in microslices derived from NGF-injected animals, in absence of acute NGF treatment. This result, together with dose-response comparisons after 2-h and 2-day survival times suggest that Trk protein remains tyrosine phosphorylated due to trophic protein which is only slowly being cleared out of the tissue during several days after the injection. Experiments with aged rats indicated similar extent and duration of Trk receptor activation after NGF administration in young adult and in aged brain. PMID- 8635558 TI - Modulation of GABA transmission by diazoxide and cromakalim in the globus pallidus: implications for the treatment of Parkinson's disease. AB - An ATP-sensitive potassium channel (KATP) is known to modulate insulin release from pancreatic beta cells. It has been proposed that potassium channels related to KATP in the nervous system might similarly modulate neurotransmitter release. We have therefore investigated the effects of KATP opening agents on GABA release in the globus pallidus. Diazoxide and cromakalim decreased the K(+)-evoked release of [3H]GABA from pallidal slices. The maximum inhibition observed for diazoxide (59%) and cromakalim (66%) was achieved at a concentration of 100 microM. The effects of both cromakalim and diazoxide were significantly antagonized by the concurrent application of the sulfonylurea glibenclamide (100 microM). Intrapallidal injections of diazoxide in the reserpine-treated rat model of Parkinson's disease reduced akinesia in a dose-dependent manner. These data suggest that manipulation of neuronal potassium channels with pharmacological properties similar to KATP may prove useful in the treatment of Parkinson's disease. PMID- 8635560 TI - Embryonic neurons transplanted to regions of targeted photolytic cell death in adult mouse somatosensory cortex re-form specific callosal projections. AB - In the neocortex, the effectiveness of potential transplantation therapy for diseases involving neuronal loss may depend upon whether donor neurons can reestablish the precise long-distance projections that form the basis of sensory, motor, and cognitive function. During corticogenesis, the formation of these connections is affected by tropic factors, extracellular matrix, structural pathways, and developmental cell death. Previous studies demonstrated that embryonic neurons and multipotent neural precursors transplanted into neocortex or mice undergoing photolytically induced, synchronous, apoptotic neuronal degeneration selectively migrate into these regions, where they differentiate into pyramidal neurons and accept afferent synaptic input. The experiments presented here assess whether embryonic neurons transplanted into regions of somatosensory cortex undergoing targeted cell death differentiate further and develop long-distance axons and whether this outgrowth is target specific. Neocortical neurons from Gestational Day 17 mouse embryos were dissociated, prelabeled with fluorescent nanospheres and a lipophilic dye (DiI or PKH), and transplanted into adult mouse primary somatosensory cortex (S1) undergoing apoptotic degeneration of callosal projection neurons. Donor neurons selectively migrated into and differentiated within regions of targeted neuronal death in lamina II/III over a 2-week period, in agreement with our prior studies. To detect possible projections made by donor neurons 2, 4, 6, 8, or 10 weeks following transplantation, the retrogradely transported dye fluorogold (FG) was stereotaxically injected into contralateral S1, ipsilateral secondary somatosensory cortex (S2), or ipsilateral thalamus. Ten weeks following transplantation, 21 +/- 5% of the labeled donor neurons were labeled by FG injections into contralateral S1, demonstrating that donor neurons sent projections to the distant area, the original target of host neurons undergoing photolytically induced cell death. No donor neurons were labeled with FG injections into ipsilateral S2 or thalamus, nearby targets of other subpopulations of neurons in S1. These data indicate that in the adult neocortex: (1) transplanted immature neurons are capable of extending long-distance projections between hemispheres through the mature white matter of the corpus callosum and (2) these projections are formed with specificity to replace projections by neurons undergoing synchronous degeneration. These experiments provide an experimental system with which to test factors affecting such outgrowth and connectivity. Taken together, these results suggest that the reconstruction and repair of cortical circuitry responsible for sensory, motor, or cognitive function may be possible in the mature neocortex, if donor neurons or precursor cells are provided with the correct combination of local and distant signals within an appropriately permissive host environment. PMID- 8635561 TI - Delayed death of septal cholinergic neurons after excitotoxic ablation of hippocampal neurons during early postnatal development in the rat. AB - To investigate the role of neuron-target interactions in regulating the survival of developing septo-hippocampal cholinergic neurons, hippocampal neurons were excitotoxically ablated in early postnatal rats. Four weeks after hippocampal ablation, hippocampal levels of brain-derived neurotrophic factor and nerve growth factor (NGF) mRNA had fallen to 15% of control values, and ipsilateral septal levels of NGF protein had fallen to 45% of control values. Four weeks after hippocampal ablation, the number of immunoreactive septal cholinergic neurons had fallen to 30% of control values. The number of cholinergic neurons in the septum correlated significantly with the amount of hippocampal tissue present. Ultrastructural analysis of the septal region at 3 days after hippocampal ablation showed no evidence of excitotoxic damage, but at 7 or 10 days showed degenerative profiles compatible with the delayed cell death of large septal neurons. Two weeks of NGF administration, initiated at 4 weeks after hippocampal lesions, failed to increase the number of detectable cholinergic neurons in the septal region, suggesting that the loss of immunoreactive neurons seen at 4 weeks represented cell death rather than downregulation of cholinergic markers. These findings suggest that septal cholinergic neurons depend for survival during early postnatal development on interactions with hippocampal neurons and are compatible with the possibility that neurotrophins play a role these interactions. PMID- 8635562 TI - Astrocytes infected with replication-defective adenovirus containing a secreted form of CNTF or NT3 show enhanced support of neuronal populations in vitro. AB - Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations. PMID- 8635563 TI - beta-Amyloid increases enzyme activity and protein levels of glutamine synthetase in cultured astrocytes. AB - Previous studies have reported that beta-amyloid peptides induce properties of reactivity in cultured astrocytes. We report here that aggregated A beta peptides increase expression of the enzyme glutamine synthetase in cultured astrocytes, as assessed by enzyme assay, Western blot analysis, and immunocytochemistry. The enhanced enzyme levels occur gradually over a period of 4 days after A beta exposure and maintain peak values for at least several days thereafter. These data suggest that A beta-related reactive astrocytosis in Alzheimer's disease brain may benefit local neurons by enhancing glial capacity to regulate levels of the excitotoxin glutamate. PMID- 8635565 TI - Changes in open field behavior, spatial memory, and hippocampal parvalbumin immunoreactivity following enrichment in rats exposed to neonatal anoxia. AB - Behavioral and neurochemical changes following enriched housing were studied in Wistar rats neonatally exposed to anoxia (100% N2 for 25 min at approximately 30 h after birth) or to sham treatment. Neonatal anoxia provoked transient hyperactivity during the P25-P40 period, and spatial memory disturbances persisting into adult life. Enriched housing, from P21, at weaning, to P60, improved behavior in open field and spatial memory abilities in a water maze, reducing the deficits that followed neonatal anoxia. Changes in the expression of the calcium binding protein parvalbumin were present in the CA1, CA3, and DG regions of the hippocampus in both sham-treated and anoxic rats exposed to enrichment. The present findings give further support to the evidence of a positive effect of enriched housing on behavior and learning of normal and lesioned animals, which is sustained by modifications in the neuronal activity, and suggest that modifications in the environment can be useful to counteract the development of some neurological disturbances that follow neonatal insults, e.g., perinatal asphyxia. PMID- 8635564 TI - Loss and subsequent recovery of local cerebral glucose use in visual targets after controlled optic nerve crush in adult rats. AB - A mild crush of the adult rat optic nerve serves as a model to study the restoration of function after traumatic brain injury. It causes a progressive degeneration of retinal ganglion cells, but visually guided behavior is partially restored within 2-3 weeks. The purpose of this study was to determine to what extent local cerebral glucose use (LCGU) decreases and if it recovers in retinofugal targets following unilateral optic nerve crush. At intervals of 2, 9, and 22 days after crush, LCGU was monitored in rats in which the visual system was stimulated by a strobe-light and pattern. In the ipsilateral retinofugal targets there was only a minimal loss of LCGU use, but in the contralateral retinofugal targets, LCGU was reduced at Postlesion Day 2: to 50% in the superior colliculus (SC), to 60% in the lateral geniculate nucleus of the thalamus (LGN), and to 87% in the visual cortex. On Postoperative Days 9 and 22 we observed a partial restoration of LCGU in the contralateral SC and LGN to 68 and 79%, respectively. As recovery of visual performance is known to follow a similar time course, we conclude that restoration of metabolic activity in target structures may contribute to the restoration of vision after optic nerve crush. PMID- 8635566 TI - Influence of dexamethasone on neurotoxicity caused by oxygen and glucose deprivation in vitro. AB - There is conflicting evidence regarding the impact of glucocorticoid exposure on hypoxic ischemic brain injury. We examined the effects of timing, duration, and concentration of dexamethasone on neuronal injury following in vitro oxygen glucose deprivation (OGD). Dissociated embryonic rat basal forebrain cells were cultured and either preincubated with dexamethasone for 72 h or continuously exposed prior to, during, and after OGD. Injury was assessed by morphology rating and cholineacetyltransferase (ChAT) activity at Day 13 in vitro, 2 days after OGD. Preincubation with nanomolar concentrations of dexamethasone resulted in a dose-dependent exaggeration of injury. Combined glutamate receptor antagonist application negated this deleterious effect, suggesting that dexamethasone may increase glutamate release, decrease uptake, or upregulate glutamate receptor expression. Continuous application of a narrow concentration range of dexamethasone (100 nM and 1 microM) prior to, during, and after insult protected neurons. Dose, timing, and duration of glucocorticoid administration may each be critical variables influencing outcome of hypoxic ischemic brain insult. PMID- 8635567 TI - Generation of DOPA-producing astrocytes by retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model. AB - Astrocytes secreting high levels of L-3,4-dihydroxyphenylalanine (DOPA) have been generated by retrovirus-mediated transfer of the human tyrosine hydroxylase (TH) gene. Immature astrocytes obtained from prenatal rat brain were cocultured with TH virus producing psi-2 cells that had been pretreated with the mitosis inhibitor mitomycin-C. During the first week of coculture DOPA production gradually increased to reach a plateau after 7-9 days. At this time point virtually all cells were GFAP positive and over 80% of them expressed TH. DOPA production in the transduced astrocytes was largely independent of exogenous cofactor, and DOPA release into the medium was not influenced by addition of either KCl or tetrodotoxin or by removal of Ca2+ from the culture medium, indicating that the newly synthesized DOPA was constitutively released from the cells. Transplantation of the TH-transduced astrocytes to the striatum in unilaterally 6-hydroxydopamine lesioned rats reduced apomorphine-induced turning by about 50% at 2 weeks postgrafting. Microscopic analysis revealed that the transduced astrocytes survived very well after transplantation and that some of the grafted cells had migrated out, partly along blood vessels, into the surrounding striatum. TH expression was observed in cells with both the appearance of mature GFAP-positive astrocytes, as well as in more immature looking cells. However, only a few percent of all transplanted cells maintained significant expression of the transgene, as determined by TH immuno histochemistry. The results show that primary astrocytes may be highly useful as gene carriers for ex vivo gene therapy in the CNS. With future improvement in the gene transduction procedure for more efficient, sustained expression of the TH transgene in vivo, genetically engineered DOPA-producing astrocytes hold great promise as a tool to explore the potential of ex vivo gene therapy in Parkinson's disease. PMID- 8635568 TI - Increased perivascular norepinephrine following intracerebroventricular infusion of NGF into adult rats. AB - In the present study, we used high performance liquid chromatography coupled with electrochemical detection to examine perivascular catecholamines associated with the intradural segment of the internal carotid artery following a 2-week in vivo intracerebroventricular infusion of the neurotrophin nerve growth factor (NGF). Following administration of NGF, a significant increase (87.3%) in perivascular norepinephrine (NE; microgram/g) was observed when compared with vehicle-infused controls, suggesting that increased sympathetic neurotransmitter accompanies the NGF-induced sprouting response by sympathetic perivascular axons previously observed using electron microscopy (13, 15). The biochemical quantification of perivascular NE in the present study taken together with our previous morphological quantification of perivascular sprouts at the ultrastructural level reveal that the increase in NE is not proportional to the increase in the number of axons. Thus, when compared with controls, the relative amount of norepinephrine per axon apparently is reduced following NGF infusion. The apparent decrease in NE per axon following NGF infusion suggests that, during the 2-week infusion period, exogenous NGF did not stimulate the biosynthesis of perivascular NE beyond that necessary to accommodate the newly sprouted axons. These results extend our morphological findings and provide evidence for plasticity of neurotransmitter expression by adult sympathetic perivascular axons in vivo. In addition, we provide evidence that the increased perivascular catecholamine histofluorescence previously observed following NGF infusion results from an increase in the number of perivascular axons associated with the vessel rather than from an increase in the amount of NE within individual axons. PMID- 8635570 TI - The process of reinnervation in the dentate gyrus of adult rats: physiological events at the time of the lesion and during the early postlesion period. AB - Destruction of the entorhinal cortex (EC) triggers a number of cellular and molecular responses in the denervated hippocampus and dentate gyrus. The signals that trigger these changes are not known but could include physiological events that occur during the production of the injury or during the early postlesion period. Of particular interest is whether experimental lesions induce seizures and/or spreading depression (SD), both of which have been shown to dramatically alter neuronal and glial gene expression. In the present study, acute neurophysiological techniques were used to evaluate whether seizures or SD occur during the production of EC lesions. Chronic recording techniques were used to monitor electroencephalographic (EEG) activity during the first 24 h after the injury in order to evaluate the extent of postlesion seizures. One or more episodes of SD occurred in 9 of 13 animals during the production of electrolytic EC lesions. However, hippocampal seizures were not observed except for very brief episodes of seizure activity at the onset of an episode of SD. Chronic recordings of postlesion EEG activity revealed that spontaneous electrographic seizures occurred during the first 24 h postlesion in all animals. The spontaneous electrographic seizures were approximately 30 s in duration and were not accompanied by motor convulsions. The first seizures occurred within several hours after the lesion, and seizures continued to occur periodically (at an average frequency of 0.42 per hour) over the 24-h recording period. Seizures occurred on the side of the brain ipsilateral to the lesion in all animals and occurred on the side contralateral to the lesion in 3 of 5 animals. These results indicate that EC lesions produce physiological events that occur variably in different animals; these processes may account for some of the variability in the cellular responses to this "standardized" injury. PMID- 8635569 TI - Selective hippocampal lesions differentially affect the phenotypic fate of transplanted neuronal precursor cells. AB - RN33B cells, a CNS-derived neuronal precursor cell line, transplanted into normal adult rat hippocampus can survive and morphologically differentiate with their ultimate morphology dependent on the integration site. This study examined the differentiation and structural integration of RN33B cells transplanted into the lesioned adult hippocampus. Pyramidal neurons of the CA1-3 regions or granular neurons in the dentate gyrus were preferentially destroyed by unilateral intraventricular kainic-acid or intradentate colchicine injections, respectively. One week after the lesion, a suspension of undifferentiated beta-galactosidase (beta-gal)-labeled RN33B cells was stereotaxically transplanted into the lesioned or the contralateral hippocampus. After 5-7 weeks, sections of the recipient brains were analyzed by toluidine blue staining and immunohistochemistry for beta gal, GFAP, and OX-42. A reactive gliosis was observed on the lesioned side which persisted up to 7 weeks postlesion (the latest time point examined). RN33b cells survived in the lesioned hippocampus and assumed variable morphologies depending on the hippocampal layer into which they integrated. Only RN33B cells located in intact or partially damaged cell layers or in the unlesioned contralateral hippocampus differentiated with morphologies similar to those of endogenous neurons characteristic of those layers. Cells located in layers completely depleted of endogenous neurons assumed bipolar morphologies or sent out multiple processes with no structural polarity, unlike the neuronal morphologies characteristically seen in intact hippocampal cell layers. These data suggest that the presence of some endogenous neurons and partially conserved cytoarchitectural organization are essential for immortalized neuroepithelial precursor cells to differentiate into region-specific neuronal cell types. PMID- 8635571 TI - The role of postlesion seizures and spreading depression in the upregulation of glial fibrillary acidic protein mRNA after entorhinal cortex lesions. AB - Unilateral lesions of the entorhinal cortex have been shown to lead to dramatic increases in GFAP mRNA levels in denervated zones in the hippocampus and dentate gyrus and sometimes (but not always) in nondenervated zones in the contralateral hippocampus and dentate gyrus. The variable distribution of the increases in GFAP mRNA expression suggests that the events which trigger changes in GFAP mRNA levels occur to a variable extent in individual animals. The companion paper characterizes two candidate triggering events: spreading depression (SD) that occurs to a variable extent at the time of the lesion and recurrent seizures that occur during the early postlesion interval. The goal of the present study was to evaluate whether individual differences in the extent or spatial distribution of lesion-induced increases in GFAP mRNA are related to the occurrence of either SD or seizures. We quantified the increases in GFAP mRNA levels in individual animals that had been monitored physiologically to define the incidence of SD and postlesion seizures. The results revealed that the quantitative extent of the increases in GFAP mRNA in denervated zones and was not related to either SD or postlesion seizures. The increases in GFAP mRNA in nondenervated zones also were not related to episodes of spreading depression that occurred at the time of lesion production but were related to the spontaneous seizures that developed during the first 24 h postlesion after the animals had recovered from the surgical anesthesia. Taken together, these data indicate that physiological events that occur during the early postlesion interval can play an important role in determining the pattern and extent of altered cellular gene expression in response to an injury. PMID- 8635572 TI - Reductions in motoneuronal neurofilament synthesis by successive axotomies: a possible explanation for the conditioning lesion effect on axon regeneration. AB - Axons regenerate more rapidly after a test lesion if they received a conditioning lesion. Previous work suggests that the cell body reaction to injury is responsible for this conditioning lesion effect. Here we examined the effects of the second, test lesion on the expression of the major cytoskeletal proteins, tubulin, actin, and neurofilament proteins. Using 2D-SDS-PAGE to separate these cytoskeletal proteins synthesized in the facial nucleus, along with in situ hybridization and RNA blotting to measure corresponding mRNA levels, we found that previous conditioning had little effect on actin or tubulin responses to a test lesion, but resulted in further decrease in neurofilament synthesis. Immunocytochemistry and electron microscopy revealed a greater loss of neurofilaments from the proximal conditioned axons, and axonal shrinkage. We suggest that the reduction in neurofilaments in the proximal axons of conditioned neurons reduces interference with tubulin transport. This may allow more tubulin to be transported more rapidly into the growing axon, to support the faster elongation rate of conditioned axons following a test lesion. PMID- 8635573 TI - Furin/PACE/SPC1: a convertase involved in exocytic and endocytic processing of precursor proteins. AB - One of the most exciting breakthroughs of the 90's in the fields of biochemistry, cell biology and neuroendocrinology is the identification of a novel family of proteolytic enzymes called mammalian subtilisin-like convertases. This family is comprised so far of seven distinct endoproteases responsible for the proteolytic excision of biologically active polypeptides from inactive precursor proteins. Six years after the initial observation of a structural conservation between a characterized yeast enzyme (kexin) and a human gene product (furin), it is now well accepted that one of these convertases, furin, has the enzymatic capabilities to efficiently and correctly process a great variety of precursors. Furin's ability to cleave precursors within both the exocytic and endocytic pathways will require sustained efforts in order to delineate all of its physiological roles. PMID- 8635574 TI - A novel myb-related gene from Arabidopsis thaliana. AB - A novel myb-like gene (Atmyb5) has been isolated from a genomic library of Arabidopsis thaliana. The gene contains a single intron in the region coding for the Myb domains. The Myb domains are highly homologous to other animal and plant Myb proteins. Arabidopsis plants transgenic for a chimeric Atmyb5 promoter/GUS gene expressed the enzyme in a developmentally controlled and tissue specific manner. The GUS activity was detected in developing leaf trichomes, stipules, epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. PMID- 8635575 TI - Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor. AB - Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding. Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified. PMID- 8635577 TI - Cerebrovascular amyloidosis in squirrel monkeys and rhesus monkeys: apolipoprotein E genotype. AB - Some neuropathological changes characteristic of aging and Alzheimer's disease (AD) in humans are present also in senescent non-human primates. The human apoE4 allele is associated with an increased risk of developing late-onset familial and sporadic AD. We found that rhesus monkeys and three subspecies of squirrel monkeys are homozygous for apoE phenotype with arginine at positions 112 and 158 as in human apoE4. However, in both species threonine replaces arginine at position 61 of human apoE. It was previously shown that arginine 61 was critical in determining apoE4 lipoprotein distribution in humans. PMID- 8635576 TI - Purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri. AB - From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass. Image analysis of transmission electron micrographs revealed a barrel-shaped particle composed of two rings with 8-fold symmetry. Only one type of subunit could be detected. The corresponding gene has been cloned and sequenced. The deduced amino acid sequence shows high homology with the members of group II chaperonins. The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen. PMID- 8635578 TI - Expression of human inducible nitric oxide synthase in Escherichia coli. AB - We have expressed active full-length human inducible nitric oxide synthase (iNOS) in E. coli. Expression required co-expression with calmodulin, a particularly tight-binding cofactor. The extracts also required tetrahydrobiopterin to display activity. Specific activity of the purified recombinant iNOS was similar to iNOS purified from murine macrophages. This result indicates that no special processing events unique to eucaryotic cells are necessary for iNOS activity. PMID- 8635579 TI - Expression of rat chymotrypsinogen in yeast: a study on the structural and functional significance of the chymotrypsinogen propeptide. AB - The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non-specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen. PMID- 8635581 TI - Role of base-backbone and base-base interactions in alternating DNA conformations. AB - Sequence-specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar-phosphate backbone. The TT step is inflexible and B-like, as it has two methyl groups which interlock with each other and with the sugar-phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A-conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine-pyrimidine, pyrimidine-purine, purine-pyrimidine steps, respectively, but to less extent, depending on the number of non-A:T basepairs in the steps. PMID- 8635580 TI - Attempts to convert chymotrypsin to trypsin. AB - Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin-->trypsin specificity conversion. PMID- 8635583 TI - Tumor necrosis factor (TNF) stimulates the production of nerve growth factor in fibroblasts via the 55-kDa type 1 TNF receptor. AB - The role of the two types of tumor necrosis factor (TNF) receptors, TNF-R1 and TNF-R2, in mediating the capacity of TNF to stimulate nerve growth factor (NGF) production in fibroblasts has been investigated. Although Swiss 3T3 cells express both the TNF-R1 and TNF-R2, an agonistic anti-TNF R1 antibody, but not an agonistic anti-TNF-R2 antibody, increases the NGF mRNA level and stimulates the production of NGF protein in the cells. Treatment of the cells with a combination of anti-TNF-R2 antibody and interleukin-1 beta (IL-1 beta) does not increase the NGF production induced by IL-1 beta alone, although TNF's activity to stimulate NGF production is markedly enhanced by IL-1 beta. Furthermore, simultaneous addition of the both antibodies does not increase the NGF production above that observed with the anti-TNF-R1 antibody, indicating that TNF-R1 alone mediates the TNF's activity to stimulate NGF production in fibroblasts. PMID- 8635582 TI - Structure of protein kinase CK2: dimerization of the human beta-subunit. AB - Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. Using the two-hybrid system we could show that the alpha- or alpha'-subunits of CK2 can interact with the beta-subunits of CK2, but not with other alpha- or alpha'-subunits. By comparison, the beta-subunit of CK2 can interact with another beta-subunit. Important amino acids for successful dimerization of the beta-subunit were localized between amino acid residues 156 and 165. Furthermore, we identified residues between amino acid 170 and 180 which antagonize the dimerization. PMID- 8635585 TI - Identification of POMC processing products in single melanotrope cells by matrix assisted laser desorption/ionization mass spectrometry. AB - The use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in identifying proopiomelanocortin (POMC) processing products in melanotrope cells of the pituitary intermediate lobe of Xenopus laevis was explored. Mass spectra were obtained with such a high sensitivity of detection that the peptides could be identified in a single melanotrope cell. In addition to known POMC processing products of the Xenopus melanotrope cell, the presence of previously unidentified POMC-derived peptides was demonstrated. Together these POMC processing products accounted for the entire length of the POMC precursor. Furthermore, Xenopus possesses two genes for POMC and the sensitivity and accuracy of the MALDI-MS technique allowed identification of processing products of both the POMCA and POMCB gene. In addition, differences were obtained between the mass spectra of melanotrope cells from Xenopus laevis adapted to different conditions of background illumination. These results show that MALDI-MS is a valuable tool in the study of the expression of peptides in single (neuroendocrine) cells. PMID- 8635584 TI - The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant. AB - The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231. PMID- 8635586 TI - Glycosylation and stability of mature HIV envelope glycoprotein conformation under various conditions. AB - The role of the glycans of the mature human immunodeficiency virus (HIV) envelope (gp160) in its stability in various conditions was studied. gp160 conformation was monitored through its subsequent ability to bind [125I]CD4. Treatment of glycosylated (CHO+) gp160 with (i) sodium dodecyl sulfate (SDS) concentrations above 0.01% impaired subsequent CD4 binding while 0.3% SDS abolished it; (ii) beta-mercaptoethanol (MSH) concentrations above 0.01% impaired CD4 binding while 0.03% MSH abolished it; (iii) 2 M guanidine-HCl had no effect; (iv) temperatures between 50 degrees C and 80 degrees C altered CD4 binding while, above 80 degrees C, the binding was abolished; (v) CD4 binding was decreased by 50% by 2 freeze thaw cycles but was not further affected by subsequent (up to 15) cycles; (vi) gp160 incubation in serum or cell lysate had no effect on CD4 binding. Glycanase treated (CHO-) gp160 binding activity was only 3-fold lower than that of CHO+ gp160. Only 2 M guanidine-HCl and heating at 70 degrees C differentially affected the binding of CHO+ and CHO- gp160, the effects being larger for CHO- gp160. CHO- gp160 binding was impaired after incubation in either serum or cell lysate. Thus, glycans stabilize gp160 conformation in some environments. However, CHO- gp160 appears to be resistant to denaturation as compared to other glycoproteins reported in the literature. PMID- 8635588 TI - Opposite regulation of bilirubin and 4-nitrophenol UDP-glucuronosyltransferase mRNA levels by 3,3',5 triiodo-L-thyronine in rat liver. AB - The effects of 3,3',5 triiodo-L-thyronine (L-T3) on the constitutive levels of hepatic mRNA encoding two UDP-glucuronosyltransferase (UGT) isoforms implicated in the glucuronidation of planar phenolic substrates (UGT1*06) and bilirubin (UGT1*0) were investigated in rat liver. The amount of UGT mRNA was quantitated by reverse transcription and amplification methods (RT-PCR). Treatment with L-T3 significantly increased UGT1*06 and decreased UGT1*0 mRNA levels by 41% and 54%, respectively. The opposite situation was observed in thyroidectomised animals. A good relationship observed between UGT activity toward 4-nitrophenol and bilirubin and mRNA levels emphasizes the key role played by the thyroid hormone L T3 on UGT expression. PMID- 8635587 TI - Suppression of bcl-2 gene expression by sphingosine in the apoptosis of human leukemic HL-60 cells during phorbol ester-induced terminal differentiation. AB - Our recent studies have shown that intracellular levels of sphingosine, an endogenous PKC inhibitor, increase during apoptosis resulting from phorbol ester (PMA)-induced terminal differentiation of human myeloid leukemic HL-60 cells, and have suggested that sphingosine may function as an endogenous mediator of apoptosis in these cells [Ohta, et al. (1995) Cancer Res. 55, 691-697]. We report here that apoptosis induced by PMA, sphingosine, and N,N-dimethylsphingosine (DMS) was accompanied by a concomitant decrease of bcl-2 expression in both RNA and protein levels in HL-60 cells, while expression of bcl-XL and bax mRNA did not change, and neither sphingosine nor DMS induced differentiation of HL-60 cells. In contrast, in apoptotic cells induced by pharmaceutical PKC inhibitors H7 or staurosporine, expression of bcl-2 did not change nor did the intracellular sphingosine concentration. These results suggest that sphingosine may function as an endogenous mediator of apoptotic signaling in PMA-induced terminal differentiation of HL-60 cells through bcl-2 down-regulation, probably independent from PKC inhibition. PMID- 8635589 TI - Expression of the gene for the receptor of gonadotropin-releasing hormone in the rat mammary gland. AB - Recent findings have demonstrated that the GnRH gene is expressed in the mammary gland of pregnant and lactating rats but not of virgin rats. Indeed, significant concentrations of biologically active GnRH have been found in milk of human, cow, sheep and rat. We have, therefore, looked for expression of the GnRH receptor in the rat mammary gland. By reverse transcription (RT)-PCR amplification, we have demonstrated the presence of GnRH receptor mRNA in mammary gland samples derived from virgin, pregnant and lactating rats. The GnRH receptor transcript cloned from the mammary gland was sequenced and found to have an identical coding region to the one cloned from the pituitary gland. In addition, we have found that the mammary gland, as the pituitary gland, contains at least two transcripts having the same coding region but different 5' non-coding regions. Binding studies, however, could demonstrate only low-affinity binding sites. These results, therefore, suggest that the regulation of the GnRH receptor occurs posttranscriptionally rather than at the level of transcription. PMID- 8635591 TI - Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C terminal fragment containing both a copper and quino-cofactor (FEBS 15979). PMID- 8635590 TI - Specific characteristics of phosphofructokinase-microtubule interaction. AB - Muscle phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et al. (1993) J. Biol. Chem. 268, 10888-10894] and periodical cross linking of the tubules [Lehotzky et al. (1994) Biochem. Biophys. Res. Commun. 204, 585-591]. Microtubule polymers of 'tail-free' tubulin obtained by removal of the carboxy-termini with limited subtilisin digestion retain the binding domains for phosphofructokinase that cross-bridges microtubule 'bodies'. Microtubule associated proteins bound on tubulin 'tails' do not perturb the kinase binding. These data suggest that the tubulin carboxy-terminal domain is not involved in microtubule-phosphofructokinase interactions and phosphofructokinase and microtubule-associated proteins have distinct binding domains on microtubules. Of different isoforms of phosphofructokinase, occurring mainly in brain and tumor cells, the muscle isoform exhibits selective adsorption behaviour on microtubules. Phosphofructokinase M and C isoforms with different associative and allosteric properties may represent an auxiliary pathway to modulate energy production via glycolysis. PMID- 8635592 TI - Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger. AB - We report here the first demonstration that reversible metal exchange occurs between metallothionein (MT) and full-length estrogen receptor (ER). Specific binding of ER to estrogen response element is inhibited in the presence of 40 microM thionein and restored by 120 microM zinc. Moreover, ER in metal-depleted nuclear extracts exhibits reduced DNA binding which can be restored by 140 microM native MT. Hence, thionein inhibits DNA binding by abstracting zinc from functional ER while native MT is capable of restoring binding to metal-depleted extracts by donating metal to ER. This indicates MT may be an important physiological regulator of intracellular zinc and/or other metals. PMID- 8635593 TI - Intercellular transfer of shed tumor cell gangliosides. AB - Three distinct steps underlie immumosuppression by tumor gangliosides: (i) their shedding by the tumor cell, (ii) binding to target leukocytes in the tumor microenvironment, and (iii) action upon the target cell. While shedding is well documented, cell to cell transfer of shed gangliosides is not. To address this, we employed a dual chamber culture system. In this system, metabolically radiolabeled lymphoma cells shed gangliosides into the conditioned medium of the contralateral chamber, which contained normal fibroblasts as the target cell. The shed lymphoma cell gangliosides bound avidly to the target fibroblasts in a trypsin-resistant manner (1-2 x 10(6) and 7 x 10(6) molecules/fibroblast in 24 and 48 h). Significantly higher than binding rates of purified lymphoma gangliosides added exogenously, these binding rates in a system which models the in vivo microenvironment suggest that cell to cell ganglioside transfer is a highly efficient process. PMID- 8635595 TI - Interaction of native and partially folded conformations of alpha-lactalbumin with lipid bilayers: characterization of two membrane-bound states. AB - alpha-Lactalbumin (alphaLA) can adopt two different membrane-bound states depending on the physical properties of the lipid bilayer, namely adsorbed and inserted. The latter, but not the adsorbed state, is able to disrupt the permeability barrier of the bilayer. The structure of both states is strongly affected by the conformational properties of the alphaLA conformer considered: as protein flexibility increases the helical content of the membrane-bound conformation decreases, especially in the adsorbed form. Moreover, the adsorbed and the inserted states of those conformers containing 3 or 4 disulfides can interconvert in response to changes in the physical properties of the host membrane. PMID- 8635594 TI - Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction. AB - Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule. PMID- 8635597 TI - Amino-terminal deletions in the decorin core protein leads to the biosynthesis of proteoglycans with shorter glycosaminoglycan chains. AB - Analysis of the N-terminal sequence of decorin purified from connective tissues and comparison with the sequence deduced from the cDNA indicate that the nascent proteoglycan has a 14 amino acid residue N-terminal propeptide. Mammalian expression vectors encoding wild-type decorin and decorin with deletions in the propeptide were used to transform COS and CHO cells. Cells transformed with vectors encoding deletion variants of decorin synthesize proteoglycans with shorter galactosaminoglycan chains than cells transformed with wild-type decorin. This effect on the polysaccharide chain length may be due to a lower affinity between the core protein and the glycosyltransferases synthesizing the linkage region. Alternatively, the deletions may affect the intracellular transport of decorin. An antiserum prepared against the N-terminal propeptide immunoprecipitated decorin secreted by cultured cells, showing that decorin is exported with the N-terminal region intact. PMID- 8635596 TI - Molecular cloning and expression of human caldecrin. AB - Earlier we reported the primary structure of serum calcium-decreasing factor (caldecrin) from rat pancreas, a protein which is considered to be a member of the elastase family. In this report, we describe the isolation of the two homologous cDNA clones encoding caldecrin from human pancreas, the structures of which are identical except for one base and the corresponding amino acid residue. These human caldecrin isoforms are composed of a signal peptide of 16 amino acids, a propeptide of 13 amino acids, and a mature form of 239 amino acids. Both recombinant caldecrins showed the same chymotrypsin-type protease activity and hypocalcemic activity. The hypocalcemic activity of both remained intact even after treatment with PMSF to abolish their protease activity. These results suggest that human caldecrin possesses hypocalcemic activity that has no connection with its protease activity. PMID- 8635598 TI - Nonenzymatically evoked and cytochrome P450-dependent lipid peroxidation inhibits synthesis of phosphatidylethanolaminevia the ethanolamine base exchange reaction in rat liver microsomes. AB - In the present study the relationship between lipid peroxidation, changes in the redox state of membrane and phosphatidylethanolamine (PE) synthesis via base exchange reaction in rat liver microsomes was investigated. It was found that PE synthesis is enhanced in the presence of antioxidants, butylated hydroxytoluene (BHT), or unsaturated free fatty acids. Prooxidants, tert-butyl hydroxyperoxide (BHP), ferrous ions combined with ascorbate or NADPH (via cytochrome P450 dependent proteins), increased the amount of lipid peroxidation products in the membrane, and in consequence inhibited the reaction. The effect of BHP was fully reversed by reduced glutathione and dithiothreitol (DTT), whereas the effect of other compounds could be reversed only by BHT. In contrast, a reversal of the inhibitory effect of cadmium ions on base exchange activity was observed in the presence of DTT, but not BHT. Therefore, both the -SH/-S-S- ratio in the membrane, affected by BHP and cadmium ions, and the lipid hydroxyperoxides (rather than aldehydes), generated by ferrous ions and ascorbate or NADPH, are equally responsible for the inactivation of the ethanolamine base exchange enzyme in rat liver microsomes. This may suggest that the synthesis of PE via the base exchange reaction may be considered an element of the superfine cellular machinery involved in the repair of damage to unsaturated fatty acid chains of phospholipids caused by reactive oxygen species under oxidative stress. PMID- 8635599 TI - Calcium store depletion potentiates a phosphodiesterase inhibitor- and dibutyryl cGMP-evoked calcium influx in rat pituitary GH3 cells. AB - A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 microM - 1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 microM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre-incubation of cells with 1 microM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 microM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores. PMID- 8635600 TI - A human B-box-binding protein downregulated in adenovirus 5-transformed human cells. AB - Internal promoters of some genes transcribed by RNA polymerase III (e.g. tRNA genes, adenovirus VA1 RNA gene, human retroposons of the Alu family) contain a conserved sequence element, B-box, interacting with basal transcription factor TFIIIC2 which initiates assembly of the full transcription complex on the genes, and which represents the major determinant of the efficiency of their expression. In this study we have identified in human nuclear extracts a protein which interacts with VA1 B-box DNA and forms a high-affinity complex which is very stable after the addition of a large excess of competitor DNA. Unlike TFIIIC2, the B-box-binding activity of the B-box-binding protein is found to be decreased in adenovirus 5-transformed human cells. In these cells (line 293) increased transcription of VA1 and tRNA genes in vivo and in vitro was previously detected by other workers. Our results suggest that besides TFIIIC2, an additional B-box binding protein factor may be involved in the regulation of expression of the RNA polymerase III-transcribed genes. PMID- 8635601 TI - Enhancement of catalytic activities of serine proteases by tripeptides compounds. AB - The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis. PMID- 8635602 TI - Bacterial and plant-produced scFv proteins have similar antigen-binding properties. AB - A gene encoding a single-chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum-targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80-fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant-produced scFv fragments is reduced to a similar extent, implying that all the plant-produced scFv fragments are antigen binding. PMID- 8635604 TI - Actinic light density dependence of the O intermediate of the photocycle of bacteriorhodopsin. AB - The O intermediate of the photocycle of bacteriorhodopsin (BR) was studied by absorption kinetic measurements at different actinic light densities. With increasing exciting flash intensity, the relative yield of O slightly increases, while that of Mf strongly decreases at the expense of Ms. Kinetic calculations and the optical anisotropy of O show that O can be formed only from Mf although Mf and O have different light intensity dependences. In order to resolve the apparent contradiction, a phenomenologically new cooperative regulatory mechanism seems to be necessary. PMID- 8635603 TI - The third intracellular domain of the m3 muscarinic receptor determines coupling to calcium influx in transfected Chinese hamster ovary cells. AB - The m2 and m3 muscarinic acetylcholine receptors were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m2/m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was found that the third intracellular loop of m3 receptor plays a fundamental role in regulating Ca2+ influx predicted to occur through Ca2+ channels located in the plasma membrane in CHO cells. PMID- 8635605 TI - Substrate specificity of monomeric and dimeric alpha-sarcin. AB - The substrate specificity of monomeric and dimeric forms of alpha-sarcin was investigated by membrane blotting procedures. Dimeric alpha-sarcin fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, whereas monomeric alpha-sarcin catalyzes both substrates. Both monomeric and dimeric alpha-sarcin are effective ribonucleases that are displayed by in situ RNA impregnated gel electrophoresis. The same purine base specificity was detected for both dimeric and monomeric forms. alpha-Sarcin is also an effective deoxyribonuclease to supercoiled DNA. The action of alpha-sarcin as deoxyribonuclease and ribonuclease is inhibited by the presence of SDS (3.5 x 10( 6) M); the inhibition on ribonuclease, but not on deoxyribonuclease, is reversible if the proteins are renatured. PMID- 8635606 TI - Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function. AB - The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter-like back door near Trp84, a key- residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the omega loop, bearing Trp84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap-like back door. Both possibilities were examined by site-directed mutagenesis. The shutter-like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap-like back door was tested by de novo insertion of a disulfide bridge which tethered the omega loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the omega loop in the immediate vicinity of Trp84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible. PMID- 8635607 TI - Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins. AB - In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule. PMID- 8635608 TI - Purification and characterization of the cytoplasmic histone acetyltransferase B of maize embryos. AB - From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations. PMID- 8635609 TI - Proteolysis of thrombospondin during cathepsin-G-induced platelet aggregation: functional role of the 165-kDa carboxy-terminal fragment. AB - The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP 1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD. PMID- 8635610 TI - Fetal myosin heavy chain increases in human masseter muscle during aging. AB - Biochemical, immunohistochemical and molecular biological methods were used to detect fetal myosin heavy chain (MyHC) in the human masseter of elderly and young subjects. Samples from the elderly subjects contained larger amounts of fetal MyHC than those of young adults. Only a very small amount of embryonic MyHC could be detected in both age groups. Embryonic and fetal MyHCs were never detected in the control adult orofacial, limb and trunk muscles. Polymerase chain reaction (PCR) analysis revealed the presence of fetal mRNA sequences in elderly and young masseter muscles. We conclude that fetal MyHC is present in the human masseter throughout the life span and that there is an increase in the relative amount of this protein with age. PMID- 8635611 TI - Fertility after menopause: a case report. PMID- 8635612 TI - Low-dose gonadotrophin stimulation for luteal phase defects--does absence of LH help pregnancy rates? AB - Based on data suggesting that higher serum LH levels during the follicular phase may decrease subsequent pregnancy rates and increase spontaneous abortion rates, the study presented herein was designed to compare the pregnancy and abortion rates in patients treated with gonadotrophin preparations with and without LH content. Infertile patients with luteal phase defects related to releasing eggs prior to complete follicular maturation were randomized into two treatment arcs: ultra-low-dose (75IU) human menopausal gonadotrophin (hMG) versus pure FSH. However, they were given the right to refuse the recommended treatment and use the other one if they preferred. Pregnancy and spontaneous abortion rates were determined for first cycle of therapy. The pregnancy rates for hMG versus pure FSH was 22.7 percent and 20.3 percent, respectively. The spontaneous abortion rates were also similar (8 percent and 9.1 percent). There were no multiple births resulting from these 36 pregnancies. Ovarian hyperstimulation syndrome was not observed in any of the 164 stimulation cycles. These data demonstrate that the use of an ultra-low-dose gonadotrophin stimulation regimen is an effective method of correcting infertility related to luteal phase defects related to follicular maturation defects since the overall pregnancy rate per first cycle of treatment was 22 percent despite a minimum of 10 months of infertility duration. Furthermore, an ultra-low-dose gonadotrophin regimen is safe for treating luteal phase defects in that there was no ovarian hyperstimulation or multiple births demonstrated. These results also show no advantage of choosing a preparation devoid of LH, thus giving the patient the opportunity to purchase the least expensive medication that is available. PMID- 8635613 TI - Screening concerns. PMID- 8635614 TI - More on cancer screening. PMID- 8635615 TI - Patient evaluation and treatment planning for complete-denture therapy. AB - A checklist is presented for use in evaluating and planning the treatment of patients for complete-denture therapy. A thorough explanation of each item and classification included in the checklist list also is presented. Classifications from the classic prosthodontic literature are used wherever possible and their sources are referenced appropriately. PMID- 8635616 TI - Lingualized occlusion. An occlusion for all reasons. AB - This authors make practical recommendations for a wide variety of clinical situations, including various anteroposterior and buccolingual arch discrepancies. Many of the ideals of the anatomical and mechanical schools of thought are blended. In addition to being a valuable clinical adjunct, Lingualized Occlusion also has great application in undergraduate dental education. It is a simpler occlusal scheme to teach and learn. PMID- 8635617 TI - Denture base materials. AB - A variety of materials have been used in the fabrication of denture bases. The advantages and disadvantages of these materials are discussed. Special emphasis is placed on polymeric material. particularly poly (methyl methacrylate) (PMMA). The chemical basis for formation of PMMA is provided to facilitate understanding. PMID- 8635619 TI - Immediate complete dentures. AB - When dentists treat patients for immediate complete dentures, they assume responsibility not only for the clinical and laboratory techniques unique to immediate complete denture fabrication, but also the responsibility for informing their patients about their treatment options, instructing their patients in the care of their oral tissues and dentures, and for continuing maintenance of the immediate dentures. Immediate dentures can be a traumatic introduction to complete dentures if patients are not informed fully about the complexity of the clinical and laboratory procedures, the need for immediate denture maintenance, and the costs associated with immediate denture treatment. The importance of 8 to 12 months of continuing care for immediate denture patients must be explained to patients and the dangers of neglecting continuing care must be emphasized. PMID- 8635618 TI - Processing complete dentures. AB - When this procedure is followed carefully, a strip of 0.0005-inch shim stock meets resistance when an attempt is made to withdraw it from between the teeth in occlusion and the incisal pin and its table simultaneously. Therefore, it is possible to process complete dentures by using compression molding and external heat for polymerization. The procedure involves a precise step-by-step method with particular emphasis on (1) investing properly in dental stone, (2) packing only one denture at a time with only one mix of acrylic resin, (3) using minimum packing pressure with maximum flow time, (4) trial packing until no flash is evident, and (5) careful deflasking. PMID- 8635620 TI - Overdentures. Principles and procedures. AB - Although advances in methods and improved materials have contributed to better treatment results, caries and periodontal problems still remain significant threats to overdenture service life. The prevention of caries and recurrent periodontal disease thus becomes a most important aspect of postinsertion care. Undoubtedly, there are a number of successful overdenture methods. This article outlines two treatment sequences previously described by Brewer and Morrow that continue to be effective (Fig. 23). Common to most overdenture techniques is that an overdenture may be the only treatment that permits retention using the patient's last few retainable teeth. A conservative approach to root preservation still is a valid and practical measure in preventative dentistry. PMID- 8635621 TI - Preprosthetic surgery for the edentulous patients. AB - Preprosthetic surgery is a rapidly changing area of dentistry. A knowledge of the range, capabilities, and limitations of the commonly used surgical procedures is a must for anyone treating a patient who will receive a complete denture prosthesis. It cannot be overemphasized that the establishment of a clear treatment plan and close coordination of all parties involved in the reconstructive effort are essential to achieve the best overall result. PMID- 8635622 TI - Implants for the edentulous patient. AB - With the introduction of osseointegration, the use of dental implants to support and retain dental prostheses had become predictable and offers the patient and the dentist an alternative treatment option. This article describes the indications for implant prostheses, the factors to be considered in pretreatment patient evaluation, and the basic techniques in using implant prostheses in the mandible and the maxilla. PMID- 8635624 TI - Complete-denture therapy for the geriatric patient. AB - Denture therapy for the geriatric patient will be in high demand for the decades ahead. An older adult's medical, functional, and psychological status should be considered in each phase of complete-denture treatment. Patient satisfaction is dependent on how well the dentist has restored facial appearance and rehabilitated chewing efficiency. PMID- 8635623 TI - Complete dentures for the obturator patient. AB - Treatment of the edentulous obturator patient requires the same basic fundamentals as used in the treatment of the complete-denture patient. Problems and considerations unique to the development, delivery, and follow-up of the complete maxillary obturator prosthesis and mandibular denture in the patient with an acquired maxillary defect are discussed. Combining laboratory and clinical research in maxillofacial prosthodontics and plastic and reconstructive surgery continues to improve the rehabilitation of these patients. PMID- 8635625 TI - Predictable impression procedures for complete dentures. AB - This article outlines those techniques and materials that will optimize the clinician's ability to obtain predictable complete denture impressions using the selected pressure philosophy. Fabrication of a custom tray is crucial to the ultimate outcome of exemplary impressions. Alternative methods for border molding the custom tray are presented. PMID- 8635626 TI - Recording jaw relationships in edentulous patients. AB - The use of a facebow transfer is an important first step in making jaw relation records. Equally important is to make a careful study of the patient's existing dentures. This article suggests a technique that permits an accurate measurement of the patient's inter-ridge distance. A measurement can be made of the existing dentures, modified existing dentures, record bases, and trial dentures. Treatment "hints" are offered to enhance the use and application of the data collected during treatment. PMID- 8635627 TI - Complete-denture esthetics. AB - This review of complete denture esthetics addresses the process of tooth selection, tooth arrangement, and characterization of the denture bases. The guidelines discussed in this article are all gleaned from the classic prosthodontic literature. These principles, which were developed over the past century, coupled with state-of-the-art materials are artificial teeth enable contemporary dentists to fabricate complete dentures with a level of esthetics never before possible. PMID- 8635629 TI - Twins and other multiples: progress with a price. PMID- 8635628 TI - Complete denture occlusion. AB - Available research has not identified a superior tooth form or arrangement to fulfill the requirements of complete denture patients in the areas of comfort, function, and esthetics. Therefore, it appears logical to use the least complicated approach to fulfill these requirements. This approach begins with the development of a philosophy of occlusion and selection of a concept to satisfy the philosophy. A balanced articulation appears to be most appropriate because of tooth contacts observed during nonfunctional activities of patients. Occlusal schemes to fulfill these occlusal concepts are many and varied. In the opinion of the author, the lingualized articulation concept using tooth molds specifically designed for this concept seems to be the most logical and least complicated approach in occlusal rehabilitation of edentulous patients. PMID- 8635630 TI - Pulmonary hypertension, cardiac disease and pregnancy. AB - OBJECTIVE: To ascertain the significance of coexisting pulmonary hypertension in cardiac disease in pregnancy. METHODS: Over a 3-year period a group of pregnant women with cardiac disease was followed until 6 weeks postpartum. Twenty women with pulmonary hypertension were compared with 20 controls without pulmonary hypertension with particular reference to maternal and fetal outcome. Analysis of data was carried out using Fisher's exact test and Student's t-test. RESULTS: Except for Eisenmenger's syndrome, there were no differences in maternal morbidity and mortality between the two groups. There were more low birth weight babies but no significant differences in premature delivery rate, mode of delivery or perinatal mortality. CONCLUSION: Except for Eisenmenger's syndrome, coexisting pulmonary hypertension complicating cardiac disease in pregnancy generally has a favorable outcome for both mother and fetus. PMID- 8635631 TI - Screening for gestational diabetes mellitus in Korea. AB - OBJECTIVES: To examine the effect of clinical characteristics on the prevalence of gestational diabetes mellitus (GDM) and to find the most effective screening program for GDM in Korea. METHODS: Universal screening with a 50-g glucose load at 24-28 weeks' gestation, as recommended by the Third International Workshop Conference on Gestational Diabetes Mellitus, was carried out among 3581 consecutive Korean women. Women with a 1-h plasma glucose > or = 130 mg/dl underwent a 3-h 100-g oral glucose tolerance test. The women's clinical characteristics and risk factors for GDM were recorded at the time of the screening test. RESULTS: The overall prevalence of GDM in Korean women was 2.2 cases/100. Although only 1.3% of this population was obese, the prevalence of GDM was found to be significantly increased with increasing body mass index. When 135 mg/dl is used as a threshold, the number of women requiring a diagnostic test decreases to 19.5%, enabling identification of 98.8% of women with GDM. CONCLUSION: Universal screening using 135 mg/dl as a threshold and early screening of those with two or more risk factors represent the most effective paradigm for Korea. PMID- 8635632 TI - Lateral ventricular atrium: larger in male than female fetuses. AB - OBJECTIVE: To attempt to detect the presence of a gender difference in the size of the fetal lateral ventricular atrium. METHODS: The width of the lateral ventricular atrium was measured sonographically on 543 consecutive fetuses scanned at 17-40 weeks, with a normal structural survey and documented gender assignment. Lateral ventricular measurements of male and female fetuses were compared. RESULTS: The mean width (+/- S.D.) of the lateral ventricular atrium for the entire study population of 543 fetuses was 6.5 +/- 1.4 mm. The measurement was 6.7 +/- 1.3 in male and 6.3 +/- 1.4 in female fetuses (P < 0.001). The atrial measurements were > or = 8.5 mm in 10% of the male and 7% of the female fetuses (P > 0.05, NS). CONCLUSION: Male fetuses have slightly larger cerebral lateral ventricles than female fetuses. PMID- 8635633 TI - Familial predisposition to uterine leiomyomas. AB - OBJECTIVE: To study the clinical risk for gynecological disorders in first-degree relatives in families with uterine leiomyoma. METHOD: Ninety-seven families (215 female patients) were enrolled in this study; 97 patients and 118 of their near family relatives were examined. RESULTS: Leiomyoma was discovered in 24.7% of cases, 2.2 times more frequently (P < 0.001) among the first-degree female relatives in families with two or more verified leiomyoma cases. The rate of PCO disease was about 15% for both groups. CONCLUSION: These results confirm the concept of distinct predisposition to uterine leiomyoma and the PCO disease in first-degree relatives in families with leiomyoma accumulation. PMID- 8635635 TI - Synchronous primary carcinomas of the endometrium and ovary. AB - OBJECTIVES: Synchronous carcinomas of the endometrium and ovary may indicate either independently developing neoplasms or metastatic disease. The clinical implications and prognosis of these two categories are quite different. The objectives of this study were to identify and evaluate the empirical criteria and significant therapeutic implications. METHOD: The National Taiwan University Hospital Cancer Registry records and pathological reports from 1977 to 1994 were reviewed. Empirical criteria were used to identify synchronous primary cancers. RESULTS: A total of 322 patients had endometrial cancer and 421 patients had ovarian cancer in our Cancer Registry records. Eleven patients had simultaneous cancer involvement of both the endometrium and ovary. Six cases fulfilled the criteria of synchronous primary carcinomas of the endometrium and ovary. Of these, five were alive and free of disease for 35-144 months (median 94.2 months). The disease-free survival rates between patients with synchronous primary and metastatic cancers of different histologic types showed a statistically significant difference (P = 0.013). No statistical significance was noted for different histologic types (P > 0.5). CONCLUSIONS: The empirical criteria used here were useful in identifying synchronous primary cancers of the endometrium and ovary. The favorable clinical outcome may relate to early detection of early-stage disease and low-grade malignancy with an indolent growth rate. Surgical management with or without adjuvant therapy has a satisfactory outcome in our experience. PMID- 8635634 TI - Limited invasiveness to assess retroperitoneal spread in stage I-II ovarian carcinoma. AB - OBJECTIVE: To evaluate the incidence of retroperitoneal metastases, survival rate and site of recurrence in early ovarian tumors undergoing limited retroperitoneal surgery. METHOD: Three hundred seventy-three consecutive patients underwent assessment of the retroperitoneum consisting of intraoperative palpation with or without biopsies. RESULTS: Retroperitoneal metastases were detected in 10 stage-I tumors (3.2%) and in 10 stage-II tumors (16%). The risk was inversely related to tumor differentiation. Palpation revealed metastases in 10 cases. During follow up, none of the borderline tumors (1.9% of stage-I grade-1 node-negative, 2.7% of grade-2 and 7.0% of grade-3 tumors) recurred in the retroperitoneum. In stage II, two recurrences were observed in grade-2 tumors (11%) and one in grade 3 (4.5%). CONCLUSION: Limited retroperitoneal surgery enables satisfactory outcome in early ovarian cancers. Risk of retroperitoneal recurrence is minimal in grade 1 and non existent in borderline tumors. Less differentiated tumors have low risk but further investigation of the therapeutic role of lymphadenectomy is justified. PMID- 8635636 TI - Conservative management of ovarian cancer in a 15-year-old patient. AB - A 15-year-old patient with stage-Ia epithelial ovarian cancer was managed conservatively after simple excision of the tumor. The remaining ovary is screened at regular intervals by ultrasound. It is hoped that this policy is justified to allow future childbearing after which total pelvic clearance is proposed. PMID- 8635638 TI - Cavernous hemangioma of the uterus. PMID- 8635637 TI - Adolescent health: a Middle East and North African perspective. AB - Adolescence is a period of physical, psychological and social transition from childhood to adulthood. At present there are 95 million young people aged between 10 and 24 years in the Middle East and North Africa. Female education is increasing in these regions, but secondary school enrollment rates for girls lag behind those of their male counterparts. Average age at first marriage varies between 17.9 and 24.3 years, but teenage marriage is quite common. Total fertility rates are higher than the world average. Pregnancy at an early age often translates into a high fertility rate and high risks for women and their children. Female children's share of food, health care, work options and general care is often less than that of boys, because they are perceived to be less valuable than boys in some countries. PMID- 8635639 TI - ACOG technical bulletin. Domestic violence. Number 209--August 1995 (replaces no. 124, January 1989). American College of Obstetricians and Gynecologists. AB - Domestic violence is a common problem that affects the family unit in particular and society in general. It occurs in all segments of society and reflects the violence that is a part of the behavior of many. Physicians can learn to detect its presence among their patients and to offer ways in which the victims can find help. Such help may include counseling for the victims, the batterer, and their children or a constructive plan for the woman to exit the relationship and rebuild her life in safety. While the public has been very slow to understand the extent and seriousness of this problem, legislative initiatives have begun to address remedies for this potentially damaging and lethal situation. Physicians should attempt to identify individuals who are the victims of abuse and to help them understand the dynamics of their relationship and the dangers faced by them and their children. Information about available community, social, and legal resources; their legal rights; and a plan for dealing with the abusive partner should be made available to these women. The physician must remain caring and supportive of the patient as she works through these crises, even if she chooses to follow advice or courses of action other than leaving the relationship. PMID- 8635640 TI - ACOG technical bulletin. Health maintenance for perimenopausal women. Number 210- August 1995. American College of Obstetricians and Gynecologists. AB - Perimenopausal women are assuming increasing importance in the practice of obstetrics and gynecology. While continuing to have reproductive concerns, such as attaining or preventing pregnancy, these women are confronted with a decline in estrogen levels and the inherent protection estrogen provides against osteoporosis and cardiovascular disease. Preventive care, life style modification, and early diagnosis and intervention can play a valuable role in maintaining patients' overall health and quality of life. PMID- 8635641 TI - The role of cell adhesion molecules in the development of IDDM: implications for pathogenesis and therapy. AB - IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM. PMID- 8635643 TI - Expression of a specific subset of CD44 variant transcripts in NOD pancreatic islets. AB - Adhesion of lymphocytes to the endothelial venules inside the islets of Langerhans seems to initiate the infiltration of islets in NOD mice. An overexpression of the lymphocyte surface molecule CD44 in infiltrated NOD islets compared with peripheral blood lymphocytes was recently reported. The CD44 protein family includes a variety of molecules generated by alternative RNA splicing from 10 variant exons (v1-v10). By using reverse transcriptase polymerase chain reaction followed by Southern blotting and hybridization to exon specific cDNA probes, we investigated the expression of CD44 isoforms in highly purified islets of Langerhans from 4- and 10-week-old NOD mice. At least six CD44 isoforms were strongly overexpressed in NOD islets at 4 and 10 weeks when compared with age-matched BALB/c islets. Controls in different tissues indicate that these variants are specifically increased in the islets from the NOD strain. Islets from the NOD-scid/scid strain also expressed these variant exons. Splenocytes from BALB/c did not express CD44 isoforms, whereas splenocytes from 4 week-old NOD mice did express CD44 variants. Treatment with inflammatory mediators induced new isoforms; however, these transcripts have a different variant exon composition from that found in NOD mice islets. These results suggest that some isoforms are expressed very early in the development of insulitis by a component of the NOD islet itself and underscore a possible role of CD44 in islet infiltration. PMID- 8635642 TI - Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet beta-cells. AB - The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the beta-cells were identified by amplification of the mRNA for insulin. We found that in single beta-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of beta cells in the disaggregated islet cell preparation. These results indicate that normal beta-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet beta-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet beta-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types. PMID- 8635644 TI - The Gly40Ser mutation in the human glucagon receptor gene associated with NIDDM results in a receptor with reduced sensitivity to glucagon. AB - The pancreatic islet hormone, glucagon, stimulates hepatic glucose production and has also been shown to potentiate glucose-induced insulin secretion. Because glucagon is a key regulator of glucose homeostasis, its receptor, which mediates the actions of glucagon, was considered a candidate gene involved in the pathogenesis of NIDDM. We have previously reported that a single heterozygous missense mutation in exon 2 of the glucagon receptor gene, which changes a glycine to a serine (Gly40Ser), is associated with NIDDM in a French population. In the present study, the signaling properties of this mutant receptor were examined in baby hamster kidney cells and rat insulinoma cells (RIN-5AH) stably transfected with either the wild type or Gly40Ser mutant human glucagon receptor cDNAs. Competition assays using (125)I-labeled glucagon were performed, and in both cell types, the Gly40Ser mutant receptor was found to bind glucagon with an approximately threefold lower affinity compared with the wild type receptor. In both cell types, the production of cAMP in response to glucagon was decreased in cells expressing the mutant receptor compared with those expressing the wild type. Finally, glucagon-stimulated insulin secretion by RIN cells expressing the mutant receptor was decreased such that the dose-response curve was shifted to the right in comparison to that obtained with cells expressing the wild type receptor. These results indicate that this single-point mutation located in the extracellular region of the glucagon receptor decreases the sensitivity of target tissues to glucagon. PMID- 8635645 TI - Microalbuminuria in NIDDM is caused by increased excretion of unmodified albumin. AB - After an intravenous infusion Of L-arginine to inhibit tubular reabsorption of albumin, glomerular clearance, renal clearance, and tubular reabsorption of unmodified albumin (UMA) and glycated albumin (GA) were determined in 72 patients with NIDDM without (NIDDM-I; n = 47) or with microalbuminuria (NIDDM-II; n = 25) and in 24 healthy control subjects. Samples of serum albumin and dialyzed urine obtained 60 min before and during L-arginine infusion were applied to an affinity column to separate GA from UMA, and their albumin contents were assayed. The serum level of GA in NIDDM patients was higher than that in control subjects (P < 0.0001). Both UMA and GA were excreted in excess in NIDDM-II as compared with the other two groups (P < 0.0001), and UMA comprised 80% of total albumin excretion. In NIDDM-II, the glomerular clearance of UMA (2.5 +/- 0.16 > NIDDM-I [1.8 +/- 0.1] > control subjects [1.3 +/- 0.1 microliter/min], P < 0.001) and of GA (1.7 +/- 0.13 > NIDDM-I = control subjects [1.1 +/- 0.1 microliter/min], P < 0.001) were enhanced, as compared with the other two groups. Renal clearance of UMA (1.3 +/- 0.13 microliter/min) and GA (0.89 +/- 0.09 microliter/min) in NIDDM-II was greater than that in control subjects (0.27 +/- 0.03, 0.19 +/- 0.02 microliter/min) or in NIDDM-I (0.30 +/- 0.03, 0.11 +/- 0.01 microliter/min). Tubular reabsorption of UMA, as assessed by the difference between glomerular and renal clearances of albumin, in NIDDM-II (1.1 +/- 0.1 microliter/min) was less than in NIDDM-I (1.50 +/- 0.09 microliter/min), and that of GA in NIDDM-II was lower than that in the other two groups, despite exaggerated glomerular clearance of GA and UMA in NIDDM-II. In summary, microalbuminuria in NIDDM is caused by increased excretion of UMA resulting from the decompensated ability of tubular reabsorption, which is exceeded by increased glomerular clearance of UMA. PMID- 8635646 TI - Are insulin and proinsulin independent risk markers for premature coronary artery disease ? AB - Controversy persists about whether hyperinsulinemia and hyperproinsulinemia are independent risk markers for coronary atherosclerosis. A common limitation of most previous studies has been imprecise categorization of disease status in normal and coronary artery disease (CAD) groups. We assessed the relationship of pancreatic beta-cell secretory products and premature CAD in a case-control study of 134 nondiabetic subjects, aged < or = 55 years old, carefully defined for CAD status by catheterization and/or thallium stress studies. Case patients comprised 66 patients with premature CAD, and control subjects (non-CAD group) included 68 patients without CAD but with traditional CAD risk factors and chest pain and/or abnormal electrocardiograms but normal catheterization and/or thallium stress studies. In addition to the CAD and non-CAD group comparison, both groups were compared with a reference group of 27 mixed lean and obese control volunteers. All CAD and non-CAD patients had a 3-h 75-g oral glucose tolerance test with measurement of fasting and post-glucose load immunoreactive insulin (IRI), specific insulin (INS), proinsulin-like material (PI), and C-peptide. Increased fasting insulin and fasting proinsulin levels both were statistically significantly associated with higher odds of being in either the premature CAD and the non-CAD groups when compared with the reference group in a polychotomous logistic regression model (odds ratio of at least 1.20 for a 20% increase in each beta-cell secretory product in both comparisons, P < 0.05). However, increased pancreatic beta-cell secretory hormone levels did not show a statistically significant relative risk for being in the premature CAD group when compared with the non-CAD group. After adjustment for BMI, all statistically significant associations disappeared for IRI, INS, and PI when the odds favoring being in the CAD and non-CAD groups were compared versus the reference group. Furthermore, the odds of being in the premature CAD and non-CAD groups when compared with the reference group were not significantly associated to the ratio of PI to insulin and C-peptide. Thus, although there is a statistically significant association between the odds of having premature CAD with elevated insulin and proinsulin levels compared with the reference group, these findings are equally common in subjects with traditional CAD risk factors without detectable CAD. Furthermore, the association of higher insulin and proinsulin levels with the likelihood of a patient having or not having CAD disappears after adjustment for BMI, suggesting that insulin and proinsulin are not independent risk markers but are primarily dependent on obesity. PMID- 8635647 TI - Increased insulin resistance and insulin secretion in nondiabetic African Americans and Hispanics compared with non-Hispanic whites. The Insulin Resistance Atherosclerosis Study. AB - The etiology of NIDDM is still controversial, with both insulin resistance and decreased insulin secretion postulated as potential important factors. African Americans and Hispanics have a two- to threefold excess risk of developing NIDDM compared with non-Hispanic whites. Yet little is known concerning the prevalence of insulin resistance and secretion defects in minorities, especially in African Americans in population-based studies. Fasting and 2-h post-glucose load glucose and insulin levels, insulin-mediated glucose disposal (insulin sensitivity index) (S(I)), glucose effectiveness (S(G)), and first-phase insulin response (acute insulin response [AIR]) were determined in nondiabetic African-Americans (n= 288), Hispanics (n= 363), and non-Hispanic whites (n= 435) as part of the Insulin Resistance Atherosclerosis Study. Subjects received a standard 2-h oral glucose tolerance test on the first day and an insulin-modified frequently sampled intravenous glucose tolerance test on the second day. African-Americans and Hispanics were more obese than non-Hispanic whites. Both African-Americans and Hispanics had higher fasting and 2-h insulin concentrations and AIR but lower S(I) than non-Hispanic whites. No ethnic difference was observed in S(G). After further adjustments for obesity, body fat distribution, and behavioral factors, African-Americans continued to have higher fasting and 2-h insulin levels and AIR, but lower S(I) than non-Hispanic whites. In contrast, after adjustment for these covariates, no significant ethnic differences in S(I) or fasting insulin levels were observed between Hispanics and non-Hispanic whites. Hispanics continued to have higher 2-h insulin levels and AIRs than those in non-Hispanic whites. In this report, the association between S(I) and upper body adiposity (waist-to-hip, ratio) was similar in each ethnic group. Both nondiabetic African Americans and Hispanics have increased insulin resistance and higher AIR than nondiabetic non-Hispanic whites, suggesting that greater insulin resistance may be in large part responsible for the higher prevalence of NIDDM in these minority groups. However, in Hispanics. the greater insulin resistance may be due to greater adiposity and other behavioral factors. PMID- 8635648 TI - Cytokine gene expression in pancreatic islet-infiltrating leukocytes of BB rats: expression of Th1 cytokines correlates with beta-cell destructive insulitis and IDDM. AB - Cytokines produced by islet-infiltrating mononuclear leukocytes may be involved in islet beta-cell destruction and IDDM. To determine which cytokine(s) might be involved in islet beta-cell destruction, we used a reverse transcriptase polymerase chain reaction assay to compare levels of cytokine mRNA expression in mononuclear leukocytes freshly isolated from islets of four groups of BB rats aged 60-75 days: diabetes-prone (DP) rats, DP rats protected from diabetes by injection of complete Freund's adjuvant (CFA) at age 25 days, acutely diabetic rats, and diabetes-resistant (DR) rats. We found that islet mononuclear leukocyte levels of gamma-interferon (IFN-gamma) mRNA were significantly higher in DP and diabetic rats than in DR rats, whereas CFA-treated DP rats had similar IFN-gamma mRNA levels to DR rats. Also, interleukin (IL)-2 mRNA levels tended to be higher in islet leukocytes from DP and diabetic rats than from DR rats. Tumor necrosis factor-alpha, IL-4, and IL-10 mRNA levels were not significantly different in islet leukocytes from the four groups of rats. These findings suggest that production of T-helper 1 (Th1)-type cytokines, IFN-gamma and IL-2, by islet infiltrating cells in BB rats is associated with beta-cell destruction and IDDM development. PMID- 8635650 TI - LDL from patients with well-controlled IDDM is not more susceptible to in vitro oxidation. AB - Increased susceptibility of LDL to oxidation has been shown to be associated with the presence of coronary heart disease and may account for the accelerated vascular disease seen in diabetes. The response of LDL to in vitro oxidative stress has been proposed as a measure of the predisposition of LDL to the in vivo subendothelial oxidative stress. Increased susceptibility to oxidation has been demonstrated recently in diabetic patients with poorly controlled IDDM. Thus, we conducted studies to determine whether the increased susceptibility of LDL to oxidation was secondary to diabetes per se or to the level of glycemic control. Fifteen IDDM patients with good glycemic control and with no evidence of macrovascular disease or proteinuria were compared with healthy age-, sex-, race , and BMI-matched nondiabetic subjects. Fasting blood glucose levels averaged 12.1 +/- 1.1 (mean +/- SE) vs. 4.9 +/- 0.1 mmol/l in the diabetic versus the control groups, respectively. HbA1c levels averaged 7.7 +/- 0.5 vs. 4.4 +/- 0.2%, reflecting well-controlled diabetes (P < 0.0001). Total, LDL, VLDL, and HDL cholesterol, triglyceride, and lipoprotein(a) levels did not differ between the groups. The particle size, lipid composition, fatty acid content, antioxidant content, and glycation were similar for LDL isolated from both groups. A rapid LDL preparation technique was used to compare LDL susceptibility to oxidation under the following conditions: final LDL cholesterol concentration of 100 microg/ml, 5 micromol/l of CuCl2 at 25 degrees C. There was no difference in the susceptibility to in vitro oxidation of LDL isolated from IDDM patients compared with control subjects. There was no correlation of glycemic control with any of the parameters of the in vitro oxidation of LDL. LDL from patients with well controlled IDDM does not differ in composition or in susceptibility to in vitro oxidative stress compared with LDL from nondiabetic subjects. PMID- 8635649 TI - Associations of fasting blood glucose with cholesterol absorption and synthesis in nondiabetic middle-aged men. AB - NIDDM and the metabolic syndrome are characterized by a low serum, HDL cholesterol content and a high triglyceride level, whereas total and LDL cholesterol concentrations are not necessarily elevated. Variable results have been reported on cholesterol absorption, elimination, and synthesis in NIDDM, but no studies are available on subjects within the normal range of blood glucose. From serum samples collected in 1985 from 203 nondiabetic men aged 51-66 years, we examined lipids, cholesterol precursors (reflecting cholesterol synthesis), and plant sterols and cholestanol (reflecting cholesterol absorption) in relation to fasting blood glucose. The findings prompted us (in 1993) to further examine 11 men from the highest and lowest glucose thirds of 203 nondiabetic men by additional dietary, serum, and fecal analyses for absorption, elimination, and synthesis of cholesterol and insulin sensitivity. In 1985, blood glucose was significantly related to LDL apolipoprotein B (P = 0.05) but not to LDL cholesterol (P = 0.19). Significantly higher serum lathosterol and desmosterol-to cholesterol proportions and lower plant sterol and cholestanol proportions in the highest rather than the lowest glucose thirds suggested that the subjects with high normal blood glucose had decreased absorption and enhanced synthesis of cholesterol. In 1993, men with the lowest glucose versus those with the highest glucose had a lower waist-to-hip ratio, plasma HbA1c, fasting and postload insulin and glucose values, and a higher insulin sensitivity index. In agreement with the 1985 non-cholesterol sterol data, direct analyses of cholesterol metabolism showed further higher cholesterol absorption efficiency (P = 0.03) and serum plant sterol and cholestanol proportions (P < 0.001). Despite a slightly lower dietary cholesterol intake, cholesterol synthesis (P = 0.02) and serum lathosterol (P < 0.01) and desmosterol (P < 0.01) proportions were lowest in men with the lowest glucose third. We conclude that noncholesterol sterols in serum exhibits a long-lasting correlation with blood glucose level in a nondiabetic male population. Low intestinal absorption and high synthesis of cholesterol characterize men with high normal blood glucose. Differences in cholesterol metabolism could be due to underlying insulin effects associated with obesity like fat distribution and may thus imply novel aspects in the metabolic interrelation between insulin and cholesterol in humans. PMID- 8635651 TI - The insulin-induced increase of guanosine-3',5'-cyclic monophosphate in human platelets is mediated by nitric oxide. AB - To investigate whether the insulin-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP) in human platelets is mediated by nitric oxide or is influenced by the nitric oxide precursor L-arginine, we measured cGMP in platelet rich plasma obtained from healthy volunteers incubated for 3 min with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l) both with and without 1) a 20-min incubation with the nitric oxide-synthase inhibitor N(G)-monomethyl-L arginine (L-NMMA) (50, 70, 100, and 1,000 micromol/l; n = 5 for each dose) and 2) a 20-min incubation with the nitric oxide precursor L-arginine (300 micromol/l; n = 6). In a first set of experiments, insulin induced a dose-dependent cGMP increase, from 9.8 +/- 0.8 to 45.6 +/- 5.5 pmol/10(9) platelets (P = 0.0001); in the presence of 1 mmol/l L-NMMA, this increase was blunted, cGMP being 8.9 +/- 1.4 and 11.1 +/- 2.2 pmol/10(9) platelets at 0 and 1,920 pmol/l insulin, respectively (NS). In the experiments with 70 and 100 micromol/l L-NMMA, the insulin effect on cGMP was inhibited, whereas 50 micromol/l L-NMMA did not blunt this insulin effect. In another set of experiments carried out to investigate the effects of L-arginine, insulin induced a dose-dependent cGMP increase, from 23.6 +/- 6.9 to 59.0 +/- 12.0 pmol/10(9) platelets (P = 0.0001); with L-arginine, basal cGMP values increased to 35.5 +/- 6.6 pmol/10(9) platelets (P = 0.05), and insulin maintained its ability to enhance dose-dependently cGMP values, which rose to 76.8 +/- 19.4 pmol/10(9) platelets (P = 0.003). This study carried out in human platelets demonstrates that the cGMP increase induced by insulin, which accounts for the antiaggregating effect of the hormone, is mediated by nitric oxide. PMID- 8635652 TI - Strain-dependent differences in sensitivity of rat beta-cells to interleukin 1 beta in vitro and in vivo: association with islet nitric oxide synthesis. AB - The aim of this study was to investigate whether strain-dependent differences in beta-cell sensitivity to interleukin (IL) 1 beta exist in vitro and in vivo and if so, whether these differences correlate to variations in IL-1 beta-induced islet inducible nitric oxide synthase (iNOS) mRNA expression and nitrite production in vitro and islet iNOS protein content in vivo. Isolated islets of Langerhans in vitro from Wistar-Kyoto/Mollegarden (WK/Mol) rats were sensitive to the inhibitory effect of IL-1 beta on accumulated and acute insulin secretion, whereas islets from Brown Norway/Charles River (BN/CR) rats were resistant. Furthermore, IL-1 beta induced higher islet iNOS mRNA expression and nitric oxide production from WK/Mol islets compared with BN/CR islets. WK/Mol, WK/CR, BN/Mol, BN/CR, and Lewis-Scripps/Mol (LS/Mol) rats received one daily injection of recombinant human IL-1 beta (4.0 microg/kg) or vehicle for 5 days. All the strains investigated were susceptible to IL-1 beta-induced changes in body weight, food intake, temperature, and plasma glucagon and corticosterone. However, IL-1 beta induced hyperglycemia and impairment of beta-cell glucose responsiveness in WK/Mol and LS/Mol rats, but not in BN rats. Furthermore, IL-l beta-induced islet iNOS expression in vivo determined by immunostaining was greater in WK/Mol rats compared with WK/CR and BN/CR rats. No restriction fragment length polymorphisms, using 20 restriction enzymes, were identified in the iNOS gene in six rat strains including BioBreeding rats. In conclusion, the relative resistance of BN rat islets to IL-1 beta-induced inhibition of beta-cell function in vitro was associated with lower islet iNOS mRNA expression and nitrite production in this strain. Further, the resistance of BN rats to IL-1 beta-induced hyperglycemia was associated with a lower islet iNOS expression in vivo. PMID- 8635653 TI - Expression of transporter associated with antigen processing-1 in the endocrine cells of human pancreatic islets: effect of cytokines and evidence of hyperexpression in IDDM. AB - A possible role of transporter associated with antigen processing (TAP)-1 in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (beta and non-beta) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-gamma and IFN-alpha, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-alpha) beta-cells may express in their surface a high density of HLA class I-peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells. PMID- 8635654 TI - Isolation, characterization, and chromosomal mapping of the human insulin promoter factor 1 (IPF-1) gene. AB - Insulin promoter factor 1 (IPF-1) is a homeodomain-containing protein that is thought to be a key regulator of pancreatic islet development and insulin gene transcription in beta-cells. This report describes the isolation and characterization of the human IPF-1 gene. The coding region, which showed 83% nucleotide identity with the mouse IPF-1 gene, was encoded by two exons that extended over a 5-kb region of human genome. The deduced human IPF-1 protein contained 283 amino acids, 1 amino acid less than the mouse IPF-1 protein. The homeodomain region of IPF-1 was encoded by the second exon, and it was highly conserved among species. The human IPF-1 gene was mapped to chromosome 13q12(12.1) by fluorescent in situ hybridization (FISH) analysis. A simple sequence repeat polymorphism (ipf1CA2) was identified in the genomic clone. Polymerase chain reaction (PCR) amplification of this repeat region revealed two alleles (heterozygosity = 0.32). This simple sequence repeat polymorphism, and thus the IPF-1 gene, was incorporated into the human linkage map by genotyping reference Human Polymorphism Study Center (CEPH) pedigrees. Multipoint analysis with the CEPH genotype database placed the gene with equal likelihood between two marker intervals: D13S292-cdx3GA1 and cdx3GA1-D13S289 on chromosome 13, consistent with the results of FISH analysis. Two-point linkage analysis inferred that the most likely location for ipf1CA2 was at theta = 0 from cdx3GA1 locus. The exon-intron boundaries of the IPF-1 gene were sequenced, and primers were synthesized to search the homeodomain region for potential variants in patients with NIDDM. By single-strand conformational polymorphism analysis, no variants were found within this region in 61 Japanese patients, which could contribute to the pathogenesis of NIDDM. The isolation of the human IPF-1 gene, along with characterization of its genomic structure and chromosomal mapping, will now permit the assessment of the role of this gene in the pathogenesis of NIDDM in various populations. PMID- 8635655 TI - Cellular immune response to diverse islet cell antigens in IDDM. AB - In IDDM, T-cells are postulated to mediate the destruction of pancreatic beta cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease. PMID- 8635656 TI - Reduced myocardial 123I-metaiodobenzylguanidine uptake in newly diagnosed IDDM patients. AB - 123I-labeled metaiodobenzylguanidine (123I-MIBG) scintigraphy is a novel technique for the assessment of cardiac sympathetic dysinnervation. To evaluate defects of the cardiac autonomic nervous system at the onset of IDDM, this technique together with conventional electrocardiogram (ECG)-based cardiac reflex tests and measurement of the QT interval was applied to 22 newly diagnosed metabolically stabilized IDDM patients without myocardial perfusion abnormalities (99mTc-labeled methoxyisobutylisonitrile scintigraphy) and 9 matched control subjects. Seventeen diabetic patients (77%), but none of the control subjects, were observed to have a reduced global myocardial uptake of 123I-MIBG. In contrast, only two diabetic patients (9%) demonstrated an ECG-based cardiac autonomic neuropathy (two or more of five age-related cardiac reflex tests abnormal) (P < 0.001). In newly diagnosed IDDM patients, the uptake of 123I-MIBG was reduced more in the posterior myocardial region compared with the lateral and apical region (P < 0.01, P = 0.03). The septal myocardial region exhibited a smaller uptake than the lateral myocardial region (P = 0.02). The maximum/minimum 30:15 ratio correlated with the global, anterior, lateral, and septal myocardial uptake of 123I-MIBG (P < 0.05, P < 0.05, P < 0.01, P < 0.05). A correlation between global and regional myocardial 123I-MIBG uptake and HbA1c or QT interval was not observed. Newly diagnosed metabolically stabilized IDDM patients without myocardial perfusion defects show evidence of cardiac sympathetic dysinnervation, as indicated by a reduction of 123I-MIBG uptake, at a significant higher proportion than ECG-based cardiac autonomic neuropathy. Furthermore, they present with regional differences of myocardial 123I-MIBG uptake. PMID- 8635657 TI - VLDL triglyceride kinetics in Wistar fatty rats, an animal model of NIDDM: effects of dietary fructose alone or in combination with pioglitazone. AB - The effects of dietary fructose alone or in combination with a new oral agent, pioglitazone, on VLDL-triglyceride (TG) turnover were studied in genetically obese Wistar fatty rats characterized by hyperinsulinemia (7,488 +/- 954 pmol/l), hyperglycemia, (22.5 +/- 1.4 mmol/l), and hypertriglyceridemia (4.39 +/- 0.54 mmol/l). They had an increased hepatic TG production (16.2 +/- 0.1 micromol/min; lean rats, 5.4 +/- 0.3 micromol/min) as well as a longer half-life of VLDL-TG from lean donors (8.8 +/- 1.4 min, lean recipients; 2.3 +/- 0.9 min). In addition, in lean recipients, the half-life of VLDL-TG from fatty donors was longer than that from lean donors (4.80 +/- 0.56 vs. 3.14 +/- 0.23 min). Although feeding fructose into fatty rats did not change plasma glucose and insulin levels, it produced a twofold increase in TG levels (8.74 +/- 1.15 mmol/l). This was associated with a 1.7-fold increase in TG production to 27.5 +/- 1.2 micromol/min, while no significant change was found in the half-life of lean VLDL TG in fructose-fed fatty recipients (10.9 +/- 2.4 min) or in that of VLDL-TG from fructose-fed fatty donors in lean recipients (4.46 +/- 0.76 min). Daily administration of pioglitazone (3 mg/kg body weight) in fructose-fed fatty rats ameliorated glycemia and triglyceridemia to the level of lean rats (8.1 +/- 0.7 and 1.18 +/- 0.05 mmol/l, respectively) and insulinemia to a lesser extent (2,712 +/- 78 pmol/l). A fall in TG levels was associated with improvement of an impairment in the ability of fructose-fed fatty rats to remove lean VLDL-TG (half fife: 2.6 +/- 0.6 min). Pioglitazone, however, produced no change in TG production (25.9 +/- 2.7 micromol/min), the half-life of VLDL-TG from fructose fed fatty donors in lean recipients (4.17 +/- 0.38 min), or the activity of lipoprotein lipase and hepatic lipase in postheparin plasma. We conclude that in Wistar fatty rats 1) hypertriglyceridemia is attributed to TG overproduction and impaired TG catabolism, and the latter is due to changes in both VLDL, such that they are less able to be removed, and changes in the nature of Wistar fatty rats, such that they are less able to remove VLDL-TG; 2) fructose further increases hepatic TG production with a resultant deterioration in hypertriglyceridemia; 3) pioglitazone normalizes TG levels by altering the physiology of the Wistar fatty rats in a manner that increases their ability to remove VLDL-TG from the circulation. PMID- 8635658 TI - Genetic absence of gamma-interferon delays but does not prevent diabetes in NOD mice. AB - Cytokines, particularly interferons, may participate in the development of type I diabetes. This involvement could be from direct cytotoxic actions of the interferons on the pancreatic beta-cells or from an indirect influence on the number, activity, or type of inflammatory cells that invade the islets in type I diabetes. To examine directly the role of interferon (IFN)-gamma in a mouse model of type I diabetes, we have introduced an inactivating mutation in the IFN-gamma gene (ifg) into NOD mice. The genetic absence of IFN-gamma does not prevent either insulitis or diabetes in the NOD mice, but it does increase the time to onset. Although it might have been predicted that the absence of IFN-gamma in these mice would lead to an increase in expression of Th2 T-helper cell-related cytokines, we found instead a profound decrease in the expression of two of the characteristic Th2 cytokines, interleukin (IL)-4 and IL-10. We also demonstrate that the splenocytes taken from IFN-gamma-deficient diabetic mice are fully capable of transferring diabetes to naive recipients. PMID- 8635659 TI - Differential responsiveness to interferon-alpha in beta-cells and non-beta cells. AB - Interferon-alpha (IFN-alpha) is important in the innate immune defense, particularly in viral infections. IFN-alpha induces 2',5'A synthetase, the products of which, 2',5'-oligoadenine nucleotides, activate mRNA degrading enzymes. IFN-alpha is the first detectable cytokine in the insulitis lesion seen in recent-onset IDDM, and insulin promoter directed expression of IFN-alpha in transgenic mice leads to development of IDDM. Here, we demonstrate that IFN-alpha induces 2',5'A synthetase activity only in insulin-producing betaTC3 cells and in isolated single rat beta-cells but not in alphaTC3 cells or in isolated rat non beta-cells. The increased responsiveness of beta-cells but not non-beta-cells to IFN-alpha with the ensuing activation of the mRNA-degrading 2',5'A synthetase system suggests why only the beta-cells are destroyed in the diabetogenic process. PMID- 8635660 TI - Leptin concentrations in diabetic and nondiabetic Mexican-Americans. AB - Leptin, the product of the OB gene, is increased in obese individuals, suggesting resistance to its effect. We questioned whether subjects with NIDDM have an altered regulation of serum leptin levels. We used a radioimmunoassay to measure serum leptin levels in three groups from the San Antonio Heart Study: 1) 50 Mexican-Americans with NIDDM; 2) 50 nondiabetic Mexican-Americans matched by age and sex to the diabetic Mexican-Americans; and 3) 50 nondiabetic Mexican Americans matched by age, sex, and BMI to the diabetic Mexican-Americans. Leptin concentrations did not differ significantly by diabetic status. Leptin concentrations were significantly correlated with BMI in all groups (NIDDM women: r = 0.637; nondiabetic women: r = 0.772; NIDDM men: r = 0.849; and nondiabetic men: r = 0.686; all P < 0.001). Leptin levels were higher in women than in men regardless of diabetic status. We concluded that the leptin concentrations were not different in diabetic and nondiabetic subjects and that the association of leptin with obesity was similar in diabetic and nondiabetic subjects. PMID- 8635662 TI - Glucagon-like peptide I receptors in the subfornical organ and the area postrema are accessible to circulating glucagon-like peptide I. AB - The intestinal incretin hormone glucagon-like peptide I (GLP-I) inhibits gastric motility and secretion in normal, but not in vagotomized subjects, pointing to a centrally mediated effect. Therefore, our aim was to study the availability of rat brain GLP-I receptors to peripherally injected 125I-labeled GLP-I. The specificity of the binding was tested by co-injection of excess amounts of unlabeled GLP-I. Using light microscopical autoradiography of rat brain sections, we found specific 125I-GLP-I binding exclusively in the subfornical organ and the area postrema. This binding was abolished when an excess amount of unlabeled GLP I was co-injected with the labeled GLP-I. We conclude that cells in the subfornical organ and the area postrema could be responsive to blood-borne GLP-I. The observed binding of peripherally administered GLP-I to the subfornical organ and the area postrema, which both have close neuroanatomical connections with hypothalamic areas involved in water and appetite homeostasis, is consistent with the potential roles of circulating GLP-I in the central regulation of appetite and autonomic functions. PMID- 8635661 TI - Sequence variants in the sulfonylurea receptor (SUR) gene are associated with NIDDM in Caucasians. AB - NIDDM is a common heterogeneous disorder, the genetic basis of which has yet to be determined. The sulfonylurea receptor (SUR) gene, now known to encode an integral component of the pancreatic beta-cell ATP-sensitive potassium channel, IKATP, was investigated as a logical candidate for this disorder. The two nucleotide-binding fold (NBF) regions of SUR are known to be critical for normal glucose regulation of insulin secretion. Thus, single-strand conformational polymorphism analysis was used to find sequence changes in the two NBF regions of the SUR gene in 35 NIDDM patients. Eight variants were found; and three were evaluated in two Northern European white populations (Utah and the U.K.): 1) a missense mutation in exon 7 (S1370A) was found with equal frequency in patients (n = 223) and control subjects (n = 322); 2) an ACC-->ACT silent variant in exon 22 (T761T) was more common in patients than in control subjects (allele frequencies 0.07 vs. 0.02, P = 0.0008, odds ratio (OR) 3.01, 95% CI 1.54-5.87); and 3) an intronic t-->c change located at position -3 of the exon 24 splice acceptor site was also more common in patients than in control subjects (0.62 vs. 0.46, P < 0.0001, OR 1.91, 95% Cl 1.50-2.44). The combined genotypes of exon 22 C/T or T/T and intron 24 -3c/-3c occurred in 8.9% of patients and 0.5% of control subjects (P < 0.0001, OR 21.5, 95% CI 2.91-159.6). These results suggest that defects at the SUR locus may be a major contributor to the inherited basis of NIDDM in Northern European Caucasians. PMID- 8635664 TI - Endosulfine, endogenous ligand for the sulphonylurea receptor: isolation from porcine brain and partial structural determination of the alpha form. AB - Anti-diabetic sulphonylureas act via high affinity binding sites coupled to K-ATP channels. Endosulfine, an endogenous ligand for these binding sites, was shown to exist in two molecular forms, alpha and beta, in both the pancreas and the central nervous system. We describe here the isolation, and partial structural characterization of alpha endosulfine derived from porcine brains by means of a series of chromatography runs and gel electrophoresis. Porcine alpha endosulfine is a protein with a molecular mass of 13,196 daltons as determined by mass spectrometry and which is N-terminally blocked. Tryptic digestion followed by separation of the fragments by HPLC and automated Edman degradation yielded a total of 72 amino acids in four partial sequences. Comparison of these sequences with that present in the National Biomedical Research Foundation protein data bank indicated a 82% identity with a 112-amino acid protein with a molecular mass of 12,353 daltons called "cyclic AMP-regulated phosphoprotein-19', isolated from the bovine brain as a substrate for protein kinase A. PMID- 8635663 TI - Hypoglycaemic brain damage: effect of a dihydropyridine calcium channel antagonist in rats. AB - Hypoglycaemic brain damage consists of selective necrosis of cerebral neurons related to the extracellular release of excitatory amino acids. Neuronal excitatory amino acid receptors are activated and calcium channels are opened. The present investigation was designed to test the effectiveness of dihydropyridine blockade of voltage-sensitive calcium channels in hypoglycaemic brain damage. Sixty-four rats were given either high-dose nimodipine, consisting of an initial bolus of 300 micrograms/kg nimodipine administered at the stage of EEG slowing (blood glucose levels of 1.0-1.5 mmol/l), followed by continuous intravenous nimodipine infusion at 1.5 micrograms.kg-1.min-1, low-dose nimodipine, consisting of an initial bolus of 30 micrograms/kg at the time of EEG slowing, followed by 0.15 microgram.kg-1.min-1, an equal volume of vehicle solution, or 154 mmol/l NaCl. Animals receiving either low- or high-dose nimodipine had higher mortality, and increased brain damage compared with controls. Examination of the perfusion-fixed brains 1 week after recovery with glucose revealed that quantitated neuronal necrosis was worsened by nimodipine in the hippocampus, caudate nucleus and cerebral cortex. The present results in profound hypoglycaemia (accompanied by a flat EEG) contrast with the beneficial effect of nimodipine in brain ischaemia. PMID- 8635665 TI - Decreased skeletal muscle phosphotyrosine phosphatase (PTPase) activity towards insulin receptors in insulin-resistant Zucker rats measured by delayed Europium fluorescence. AB - In order to measure the phosphotyrosine phosphatase (PTPase) activity in small muscle biopsies, a sandwich-immunofluorescence assay was developed using the phosphorylated human insulin receptor as a substrate, a C-terminal insulin receptor antibody as catching antibody and Europium-labelled anti-phosphotyrosine as detecting antibody. Soluble and particulate muscle fractions were prepared from soleus muscle of obese, diabetic (fa/fa) Zucker rats and their lean littermates (Fa/-). In the soluble muscle fractions of the obese (fa/fa) rats PTPase activity was significantly reduced compared to control (Fa/-) rats (45.2 +/- 2.6% vs 61.3 +/- 4.7%, p < 0.02). This reduction was completely prevented by 24 days of metformin treatment which decreased plasma glucose and plasma insulin levels. In particulate muscle fractions, however, no difference in PTPase activity was found among any groups of rats examined. These results show that the alterations in soluble PTPase activity in the insulin-resistant, diabetic Zucker rat vary with the abnormality in glucose homeostasis. PMID- 8635666 TI - Renal fate of circulating advanced glycated end products (AGE): evidence for reabsorption and catabolism of AGE-peptides by renal proximal tubular cells. AB - The presence of excessive amounts of advanced glycation end products (AGE) in tissues or in the circulation may critically affect the progression of diabetic nephropathy. Circulating AGE levels, mainly in the form of small peptides, increase in diabetic patients or in patients with end-stage renal disease. This rise correlates with the severity of the nephropathy. However, so far little is known about the fate of AGE-proteins and AGE-peptides in renal tissue, and in order to elucidate this issue we undertook the present study. AGE-bovine serum albumin (AGE-BSA) and AGE-peptides were prepared, characterized by spectrophotometry, spectrofluorometry, chromatography and SDS-PAGE. AGE-peptides reacted in vitro with LDL producing biochemical and ultrastructural modifications. Using colloidal gold post-embedding immunoelectron microscopy with an anti-AGE antibody generated in our laboratory, we followed, in a short-term kinetic study, the cellular and sub-cellular localisation of circulating AGE products throughout the nephron. AGE-peptides or AGE-BSA were injected into otherwise normal rats and detected by protein A-gold immuno-cytochemistry after 15, 30 or 45 min of circulation. Most of the AGE-BSA was found in the lumen of capillary vessels and distributed along the endothelial side of the glomerular basement membrane. Presence on mesangial matrix was also apparent. AGE-peptides were easily filtered and actively reabsorbed by the proximal convoluted tubule. At 15 min, little labelling was found in the glomerular wall. Instead, the labelling was present in the urinary space and microvilli of epithelial cells. Early endosomes displayed intense labelling as well. At 45 min, late endosomes and lysosomes added to the pattern of labelling. The distal tubule epithelial cells were devoid of labelling for any of the intervals studied. AGE-peptides but not AGE-BSA could be detected in the urine of injected rats. These observations point to participation of the endo-lysosomal apparatus of the proximal convoluted tubule to the disposal of AGE-peptides, while giving an ultrastructural support for a key role of the kidney in AGE catabolism. PMID- 8635667 TI - Loss of regulation by sympathetic hepatic nerves of liver metabolism and haemodynamics in chronically streptozotocin-diabetic rats. AB - The consequences of autonomic diabetic neuropathy, a common complication of chronic diabetes mellitus, have been studied mainly with regard to heart and stomach function. Since the autonomic nervous system also regulates liver carbohydrate metabolism and haemodynamics via hepatic nerves, it was the purpose of this study to examine the function of hepatic nerves in chronically diabetic rats. Diabetes was induced by i.p. injection of streptozotocin. Rat livers were perfused via both portal vein and hepatic artery. Hepatic nerves were stimulated for 2 min using a platinum electrode placed around the portal vein and the hepatic artery; in an additional stimulation phase noradrenaline was infused into the portal vein. Stimulation of hepatic nerves as well as portal noradrenaline infusion increased hepatic glucose output and reduced flow in control and in acutely (48-h) diabetic animals, which still had almost normal glycogen content. In addition stimulation also caused an overflow of noradrenaline into the caval vein. However, nerve stimulation neither increased glucose output nor decreased flow in 4-month diabetic rats. In these rats noradrenaline overflow was nearly completely abolished and hepatic glycogen content was markedly depleted. Portal noradrenaline infusion in chronically diabetic rats reduced flow to a similar extent as in controls, yet the increase in glucose output was diminished. The lack of nerve stimulation-dependent glucose output, flow reduction and noradrenaline overflow is indicative of a profound loss of function of hepatic autonomic nerves in chronically diabetic rats. PMID- 8635668 TI - Renal enlargement and insulin-like growth factor-1 accumulation in the Wistar rat model of experimental diabetes is not prevented by angiotensin converting enzyme inhibition. AB - Experimental diabetes is associated with renal enlargement and glomerular hyperfiltration. Possible mechanisms for these changes could be the direct effects of growth factors such as insulin-like growth factor-1 and angiotensin II. We investigated whether treatment with trandolapril, an angiotensin converting enzyme inhibitor, prevented renal enlargement in streptozotocin diabetic rats. Seven groups of male Wistar rats were studied: C (control + placebo); CL (control + low-dose trandolapril, 0.01 mg.kg-1.day-1); CH (control + high-dose trandolapril, 0.5 mg.kg-1.day-1; DP (diabetic + placebo); DI (diabetic, insulin-treated); DL (diabetic + low-dose trandolapril); DH (diabetic + high-dose trandolapril) and DI (diabetic + insulin). From day 2 glucose concentrations and body weight were similar in the non-diabetic and diabetic animals treated with insulin. Diabetic animals treated with placebo and low-dose trandolapril weighed significantly less compared to the control group. The diabetic groups, not treated with insulin, showed marked hyperglycaemia throughout the study. Kidney weight was greater in the diabetic, non insulin-treated groups compared with the control and insulin-treated groups. After 24 h of diabetes, kidney insulin-like growth factor-1 content was significantly increased from baseline levels in groups DP, DL and DH but by 48 h these levels had returned to normal. Renal tissue angiotensin converting enzyme activity was similar in groups C and DI but significantly reduced in all trandolapril-treated animals. Despite inhibiting renal angiotensin converting enzyme activity renal enlargement with increased tissue insulin-like growth factor-1 still occurred. This suggests that neither angiotensin II nor glomerular hyperfiltration, with raised intraglomerular pressure, play a role in the initial renal enlargement seen in experimental diabetes. Renal accumulation of insulin-like growth factor-1 appears to be an important factor in early renal hypertrophy and its effects are not modulated by angiotensin converting enzyme or angiotensin II. PMID- 8635669 TI - Interactions between essential fatty acid, prostanoid, polyol pathway and nitric oxide mechanisms in the neurovascular deficit of diabetic rats. AB - Impaired omega-6 essential fatty acid metabolism and exaggerated polyol pathway flux contribute to the neurovascular abnormalities in streptozotocin-diabetic rats. The potential interactions between these mechanisms were examined by comparing the effects of threshold doses of aldose reductase inhibitors and evening primrose oil, alone and in combination, on neurovascular deficits. In addition, high-dose aldose reductase inhibitor and evening primrose oil treatment effects were challenged by co-treatment with the cyclo-oxygenase inhibitor, flurbiprofen, or the nitric oxide synthase inhibitor, NG-nitro-L-arginine. Eight weeks of diabetes caused an 18.9% reduction in sciatic motor conduction velocity (p < 0.001). This was only modestly ameliorated by a 0.1% dietary supplement of evening primrose oil or the aldose reductase inhibitors ZD5522 (0.25 mg.kg-1.day 1 and WAY121 509 (0.2 mg.kg-1.day-1 for the final 2 weeks. However, joint treatment with primrose oil and ZD5522 or WAY121 509 caused marked 71.5 and 82.4% corrections, respectively, of the conduction deficit. Sciatic nutritive blood flow was 43.1% reduced by diabetes (p < 0.001) and this was corrected by 67.8% with joint ZD5522 and primrose oil treatment (p < 0.001). High-dose WAY121 509 (10 mg. kg-1.day-1 and primrose oil (10% dietary supplement) prevented sciatic conduction velocity and nutritive blood flow deficits in 1-month diabetic rats (p < 0.001). However, these effects were abolished by flurbiprofen (5 mg.kg(-1).day 1 and NG-nitro-L-arginine (10 mg.kg-1.day-1) co-treatment (p < 0.001). Thus, the data provide evidence for synergistic interactions between polyol pathway/nitric oxide and essential fatty acid/cyclo-oxygenase systems in the control of neurovascular function in diabetic rats, from which a potential therapeutic advantage could be derived. PMID- 8635671 TI - Effect of adrenalectomy on the development of a pancreatic islet lesion in fa/fa rats. AB - Adrenalectomy prevents development of obesity and hyperinsulinaemia in obese (fa/fa) Zucker rats, thereby implicating the hypothalamo- pituitary-adrenal axis in the pathogenesis of obesity. In this study glucose-induced insulin secretion and glucokinase activity were investigated in isolated islets from adrenalectomized and control obese and lean female rats. Islets from control fa/fa rats were more sensitive to glucose with a half-maximal effective concentration (EC50) of 6.1 +/- 2.0 mmol. 1(-1) compared with 10.6 +/- 2.7 mmol. 1(-1) for adrenalectomized fa/fa rat islets. Adrenalectomy did not alter the islet sensitivity to glucose in the lean rats (EC50 of 9.4 +/- 1.5 mmol.1(-1) and 9.3 +/- 2.0 mmol. 1(-1) for adrenalectomized and control lean rats respectively). Mannoheptulose did not inhibit insulin secretion from control obese rats; however at concentrations of 1.0 mmol. 1(-1) or more it significantly inhibited glucose induced insulin secretion in adrenalectomized obese and lean, and control lean rat islets (P < 0.05). In adrenalectomized fa/fa islets the glucokinase Km was increased twofold compared with the control fa/fa rats (9.5 +/- 1.5 mmol. 1(-1) vs 5.0 +/- 1.5 mmol. 1(-1), respectively), but there was no significant change in glucokinase Km in the lean rat islets after adrenalectomy. Mannoheptulose (10 mmol.1(-1) caused a significant reduction in glucose phosphorylation in disrupted islets of adrenalectomized fa/fa and lean, and of control lean rats, but not of control fa/fa rats. These data demonstrate that development of abnormal regulation of glycolysis in pancreatic islet beta cells of fa/fa rats, as indicated by the insulin response to manno-heptulose and glucokinase activity, is dependent on an intact hypothalamo-pituitary-adrenal axis. PMID- 8635670 TI - Expression of the gene encoding glycogen phosphorylase is elevated in diabetic rat skeletal muscle and is regulated by insulin and cyclic AMP. AB - Glycogen phosphorylase regulates the breakdown of glycogen into glucose, but as previous studies have demonstrated, the control of glycogen metabolism becomes deregulated in diabetes mellitus. Messenger RNA levels encoding several different proteins are altered in skeletal muscle biopsies of patients with insulin dependent and non-insulin-dependent diabetes. The possible alteration of expression of the gene encoding the skeletal muscle isoform of glycogen phosphorylase during diabetes has not previously been investigated. We examined the effect of streptozotocin-induced diabetes and insulin treatment on glycogen phosphorylase mRNA in rat skeletal muscle; glycogen phosphorylase mRNA levels were elevated in diabetic rat muscle tissue, but were partially suppressed in diabetic rat muscle following insulin treatment. To distinguish between the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA levels, we employed differentiating rat L6 myoblasts in culture. Insulin stimulated the accumulation of glycogen phosphorylase mRNA as determined by Northern blot analysis. Moreover, insulin and dibutyryl cAMP stimulated expression of a transiently transfected chloramphenicol acetyl transferase reporter gene under the control of the muscle glycogen phosphorylase promoter in differentiating myotubes in culture, suggesting that the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA are at the level of transcription. These results suggest that insulin and epinephrine may participate in the induction of the glycogen phosphorylase gene during myogenesis; moreover, activation of this gene in muscle tissue may be a contributing factor in impaired glycogen storage during uncontrolled diabetes. PMID- 8635672 TI - C-peptide stimulates rat renal tubular Na+, K(+)-ATPase activity in synergism with neuropeptide Y. AB - This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na+, K(+)-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na+, K(+)-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na+ affinity of the enzyme and all subsequent studies were performed at non-saturating Na+ concentrations. C-peptide stimulation of Na+, K(+)-ATPase activity occurred in a concentration-dependent manner in the dose range 10(-8)-10(-6) mol/l. The presence of neuropeptide Y, 5 x 10(-9) mol/l, enhanced this effect and stimulation of Na+, K(+)-ATPase activity then occurred in the C-peptide dose range 10(-11)-10(-8) mol/l. C-peptide stimulation of Na+, K(+)-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca2(+)-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na+, K(+)-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca2(+)-dependent intracellular signalling pathways. PMID- 8635673 TI - Rapid formation of capillary endothelial cells in rat skeletal muscle after exposure to insulin. AB - Research has suggested a role for insulin delivery through capillaries in muscle in the regulation of insulin sensitivity. Therefore, the formation and turn-over of capillary endothelial cells in muscle were studied in relation to exposure to moderately elevated insulin concentrations with or without concomitant increase of corticosterone concentrations. Female rats were exposed to a moderate, physiological hyperinsulinaemia (approximately 450 pmol/l) for 24 h 48 h, 3 days, 7 days and 7 weeks. Propranolol was used to inhibit elevated adrenergic activity. In one insulin-exposed group, corticosterone secretion was controlled by adrenalectomy with substitution of corticosterone to maintain normal concentrations, while another group was left with adrenal corticosterone secretion intact. Rats were exposed to insulin with controlled, non-elevated corticosterone concentrations after adrenalectomy and corticosterone substitution; compared to controls, the number of mitoses in capillary endothelial cells in the soleus and extensor digitorum longus muscle were approximately doubled after 24 h, reaching a maximum, about fivefold higher than controls, after 3 days. After 7 weeks of insulin exposure there were no longer any significant differences between control and insulin-exposed rats. The number of capillaries per unit muscle surface area was moderately (10-15%) but significantly increased at 7 days (only the extensor digitorum longus muscle) and 7 weeks (the extensor digitorum longus and the soleus muscles). In rats exposed to insulin, with intact adrenals, endogenous corticosterone production resulted in concentrations about threefold higher than in rats adrenalectomized with subsequent corticosterone substitution. In these rats the increase in mitoses in capillary endothelium was totally abolished. The results of this study suggest that exposure to insulin in this rat model is followed by a dramatic short-term increase in the formation of new capillary endothelial cells in muscle. It is also suggested that this growth factor-like effect of insulin is abolished by corticosterone. It is suggested that insulin and corticosterone exert opposite effects on the capillary network in muscles, which might be important for the insulin supply to this tissue, and hence for regulation of insulin sensitivity. PMID- 8635674 TI - Effects of dietary sodium on blood pressure in IDDM patients with nephropathy. AB - The objectives of the study were to assess the effects of moderate sodium restriction on blood pressure in insulin-dependent diabetic (IDDM) patients with nephropathy and high normal or mildly hypertensive blood pressure (primary objective), and to document possible associated changes of exchangeable body sodium, body volumes, components of the renin-angiotensin-aldosterone system, atrial natriuretic peptide, and catecholamines (secondary objective). Sixteen patients with untreated systolic blood pressure > or = 140 < 160 mmHg and/or diastolic blood pressure > or = 85 < 100 mmHg were included in a double-blind, randomized, placebo-controlled trial. After a 4-week run-in period on their usual diet and a 2-week dietary training period to reduce sodium intake to about 90 mmol/day, eight patients received 100 mmol/day sodium supplement (group 2) and eight patients a matching placebo (group 1) for 4 weeks while continuing on the reduced-sodium diet. Patients were examined at weekly intervals. Main response variables were mean values of supine and sitting systolic and diastolic blood pressure as measured in the clinic and by the patients at home. The differences in blood pressure between the beginning and the end of the blinded 4-week study period were calculated and the differences in changes between the two patient groups were regarded as the main outcome parameters. During the blinded 4-week study period, average urinary sodium excretion was 92 +/- 33 (mean +/- SD) mmol/day in group 1 and 199 +/- 52 mmol/day in group 2 (p = 0.0002). The differences in blood pressure changes between the two patient groups were 3.9( 1.2 to 9) mmHg [mean (95% confidence intervals)] for systolic home blood pressure, 0.9(-3.7 to 5.5) mmHg for diastolic home blood pressure, 4.9(-3.3 to 13.1) mmHg for clinic systolic blood pressure and 5.3(1 to 9.7 mmHg, p = 0.02) for clinic diastolic blood pressure. Combining all patients, there were relevant associations between changes of urinary sodium excretion and blood volume (Spearman correlation coefficient r = 0.57), blood pressure and angiotensin II (diastolic: r = -0.7; systolic: r = -0.48), and exchangeable body sodium and renin activity (r = -0.5). In conclusion, in this study of IDDM patients with nephropathy and high normal or mildly hypertensive blood pressure, a difference in sodium intake of about 100 mmol/day for a period of 4 weeks led to a slight reduction of clinic diastolic blood pressure. Studies including larger numbers of patients with various stages of nephropathy and hypertension are needed to definitely clarify the effects of sodium restriction in IDDM. PMID- 8635675 TI - Chronic primary hyperinsulinaemia is associated with altered insulin receptor mRNA splicing in muscle of patients with insulinoma. AB - Alternative splicing of the 36-base pair exon 11 of the human insulin receptor gene results in the synthesis of two insulin receptor isoforms with distinct functional characteristics (the isoform containing exon 11 has lower insulin binding affinity and lower internalization rate). Altered expression of these insulin receptor isoforms has been previously demonstrated in skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM). However, this observation was not confirmed by other studies and is still a matter of controversy; furthermore, it is not known whether it represents a primary event or is secondary to hyperinsulinaemia and insulin resistance. In order to address this issue in patients with pure non-genetically determined hyperinsulinaemia, we examined the alternative splicing of insulin receptor mRNAs in skeletal muscle of eight patients with surgically confirmed insulinoma and insulin resistance and in eight healthy subjects, using the reverse transcriptase-polymerase chain reaction technique. The insulinoma patients displayed a significant increase in the expression of the insulin receptor isoform containing exon 11 (75.7 +/- 2.3%) when compared with normal subjects (57.9 +/- 1.5%); furthermore, this increase was positively correlated with plasma insulin concentration and negatively correlated with in vivo insulin sensitivity (glucose clamp). In conclusion, the increased expression of the insulin receptor isoform with lower insulin binding affinity in patients with primary non-genetically determined hyperinsulinaemia supports a role for insulin in the regulation of alternative splicing of insulin receptor pre-mRNA and suggests that in NIDDM an altered receptor isoform distribution might be secondary to the ambient hyperinsulinaemia rather than representing a primary defect. PMID- 8635676 TI - Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man. AB - The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU.kg-1. min-1) euglycaemia followed by: 1)240 min of graded hypoglycaemia (plasma glucose nadir 2.8 mmol/l) or 2) 240 min of euglycaemia. At 350-360 min a muscle biopsy was taken and indirect calorimetry was performed at 210-240 and 330 350 min. Hypoglycaemia was associated with markedly increased levels of adrenaline, growth hormone and glucagon and also with less hyperinsulinaemia. During hypoglycaemia the fractional velocity for glycogen synthase was markedly reduced; from 29.8 +/- 2.3 to 6.4 +/- 0.9%, p < 0.05. Total glucose disposal was decreased during hypoglycaemia (5.58 +/- 0.55 vs 11.01 +/- 0.75 mg.kg-1. min-1 (euglycaemia); p < 0.05); this was primarily due to a reduction of non-oxidative glucose disposal (2.43 +/- 0.41 vs 7.15 +/- 0.7 mg.kg-1 .min-1 (euglycaemia); p < 0.05), whereas oxidative glucose disposal was only suppressed to a minor degree. In conclusion hypoglycaemia virtually abolishes the effect of insulin on muscle glycogen synthase activity. This is in keeping with the finding of a marked reduction of non-oxidative glucose metabolism. PMID- 8635678 TI - Interleukin-1 beta and GTP-binding proteins. PMID- 8635677 TI - A stable peroxovanadium compound with insulin-like action in human fat cells. AB - Aqueous solutions of peroxovanadium (pV) compounds are potent insulin-mimics in various types of cell. Since chemical instability is a problem with these agents, we studied the insulin-like action in human fat cells of a stable pV complex, bpV(pic). It enhanced 14C-U-glucose uptake in a dose-dependent manner by approximately twofold which was slightly less than the effect of insulin (approximately threefold). The pV complex did not alter cell-surface insulin binding and submaximal concentrations did not influence cellular sensitivity to insulin action on glucose uptake. The bpV(pic) inhibited the lipolytic effect of isoprenaline to the same extent as insulin; however, when the cGMP-inhibitable low-K(m) phosphodiesterase (cGI-PDE) was blocked with the specific inhibitor OPC 3911, the antilipolytic effect of insulin, but not that of bpV(pic), was completely prevented. Moreover, when lipolysis was stimulated by the non hydrolysable cAMP analogue N6-monobutyryl cAMP, bpV(pic), in contrast to insulin, maintained an antilipolytic effect. These findings indicate that bpV(pic) exerts its antilipolytic effect not only through cGI-PDE activation, similar to the effect of insulin, but also by means of other mechanisms. The tyrosine kinase activity of insulin receptors from human placenta was not altered by the pV compound itself, whereas bpV(pic) clearly enhanced insulin-stimulated activity. In contrast, in situ tyrosine phosphorylation of the insulin receptor beta subunit as well as that of several other proteins was clearly increased in cells which were treated with bpV(pic), whereas vanadate only amplified insulin stimulated tyrosine phosphorylation. In conclusion, bpV(pic) exerts powerful insulin-like effects in human fat cells and may be a new and potentially useful agent in the management of insulin-resistant states. PMID- 8635680 TI - Molecular mimicry and the T-cell repertoire. PMID- 8635679 TI - Large-scale study of an A-to-G transition at position 3243 of the mitochondrial gene and IDDM in Japanese patients. PMID- 8635681 TI - Diabetes mellitus and Alzheimer's disease: glycation as a biochemical link. PMID- 8635682 TI - Glucagon receptor gene mutation in NIDDM. PMID- 8635683 TI - Treatment of patients with human insulin. PMID- 8635684 TI - Analysis of liver development, regeneration, and carcinogenesis by genetic marking studies. AB - The mechanism of generating new hepatocytes and bile ductule cells in the liver has been controversial. Oval cells are found in the periportal region under some circumstances and may represent multipotent stem cells. The role of stem cells in generating new liver cells in normal and pathological conditions is unclear, however. Genetic marking can be used to determine the ability of a particular cell to replicate and to migrate. Cells of known lineage are marked at an initial time point, and their developmental potential determined by the cluster size, position, and phenotype of marked cells at a later time point. Recently, genetic marking studies have demonstrated that the hepatocyte itself is the source of new hepatocytes in the normal postnatal liver and that daughter cells do not migrate. These studies have also demonstrated that the hepatocyte can replicate extensively when stimulated. Finally, genetic marking studies suggest that either hepatocytes or oval cells can develop into a hepatocellular carcinoma or cholangiocarcinoma if a sufficient number of genetic mutations accumulate. The implications of these results for hepatic gene therapy, treatment of liver insufficiency states, and liver cancer are discussed. Future genetic marking studies may help to address some remaining questions in liver biology. PMID- 8635685 TI - Structural flexibility and functional versatility of mammalian P450 enzymes. AB - P450 enzymes have evolved into a large superfamily that displays great diversity in substrate and product specificities by fixing the natural amino acid substitutions with high frequency. Site-directed mutagenesis has been used to correlate the substitutions with the diverse specificities in various P450s. As a result, the common residues that determine the specificities of various mammalian P450s have been identified and aligned to the corresponding residues in the substrate-heme pocket of the 3-dimensional structures of bacterial P450s. The substrate-heme pocket appears to be structurally variable so that only a minor substitution (Ala -> -> Val, for example) at the critical positions is enough to define the altered specificity. Thus, the structural variability of the P450s provides the inherent versatility in acquiring a novel activity. Recent mutational studies indicate that the side chain size is the major determining factor of specificity, outweighing other factors such as polarity. Further understanding of the paradoxical characteristics observed may provide us with the underlying principles that determine P450 activities, and may lead to the ability to predict P450 activities based on the types of key amino acid residues. PMID- 8635686 TI - Beta-carotene, carotenoids, and disease prevention in humans. AB - A growing body of literature exists regarding the effects of beta-carotene and other carotenoids on chronic diseases in humans. This article reviews and critically evaluates this literature and identifies areas for further research. This review is restricted to studies in humans, with a major emphasis on the most recent literature in the area of carotenoids and selected cancers. Effects of carotenoids on cardiovascular diseases, photosensitivity diseases, cataracts, and age-related macular degeneration are also discussed briefly. Numerous observational studies have found that people who ingest more carotenoids in their diets have a reduced risk of several chronic diseases. However, intervention trials of supplemental beta-carotene indicate that supplements are of little or no value in preventing cardiovascular disease and the major cancers occurring in well-nourished populations, and may actually increase, rather than reduce, lung cancer incidence in smokers. As a consequence of these findings, some of the ongoing trials of beta-carotene and disease prevention have been terminated or have dropped beta-carotene from their interventions. Researchers should now seek explanations for the apparently discordant findings of observational studies vs. intervention trials. The most pressing research issues include studies of interactions of carotenoids with themselves and with other phytochemicals and mechanistic studies of the actions of beta-carotene in lung carcinogenesis and cardiovascular disease. Paradoxically, the finding that lung carcinogenesis and cardiovascular disease can be enhanced by supplemental beta-carotene may ultimately lead to a clearer understanding of the role of diet in the etiology and prevention of these diseases. The conclusion that major public health benefits could be achieved by increasing consumption of carotenoid-rich fruits and vegetables still appears to stand; however, the pharmacological use of supplemental beta-carotene for the prevention of cardiovascular disease and lung cancer, particularly in smokers, can no longer be recommended. PMID- 8635687 TI - Allosteric enzymes as models for chemomechanical energy transducing assemblies. AB - In chemomechanical energy transducing assemblies such as muscle and ATP synthase, substrates and macromolecules are locked together as partners where energy available from (or required for) a chemical transformation is exchanged with protein conformational changes. Allosteric binding proteins and enzymes are also chemomechanical energy transducers, using binding energy to generate protein conformational changes, and transduce energy in amounts almost as large as those used to drive muscle contraction and the synthesis of ATP. The recently determined structure of the F1-ATPase reveals a direct correspondence between the types of conformational changes in this transducer and simpler allosteric binding proteins and enzymes. Therefore, we can examine the structural and energetic data available on allosteric proteins to understand the linkage between ligand binding and global conformational changes in more complex energy transducing assemblies. PMID- 8635688 TI - Antioxidant and redox regulation of gene transcription. AB - Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H202 may act as signal transduction messengers. To develop a better understanding of the exact mechanisms that underlie ROS-dependent disorders in biological systems, recent studies have investigated the regulation of gene expression by oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state. At least two well-defined transcription factors, nuclear factor (NF) kappa B and activator protein (AP) -1 have been identified to be regulated by the intracellular redox state. The regulation of gene expression by oxidants, antioxidants, and the redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications. Binding sites of the redox-regulated transcription factors NF-kappa B and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g., AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems, and the exogenous lipoate dihydrolipoate couple may therefore be considered to be effective regulators of redox-sensitive gene expression. The efficacy of different antioxidants to favorably influence the molecular mechanisms implicated in human disease should be a critical determinant of its selection for clinical studies. PMID- 8635689 TI - Immunobiology of transplantation. AB - Despite major advances resulting from the development of new immunosuppressive drugs in recent years, the field of clinical transplantation is currently limited by inadequate organ availability, chronic rejection, and complications of chronic, nonspecific immunosuppressive therapy. Because of the organ shortage, extensive research has recently focused on the potential to use donors from other species, i.e., xenotransplantation. The best way to avoid the problems of chronic rejection and complications of immunosuppressive therapy, and to overcome the considerable immunologic barriers to xenotransplantation, would be to induce a state of systemic tolerance to the donor in the recipient. Recent developments in the understanding of mechanisms of central and peripheral T cell tolerance have led to new strategies for inducing tolerance in experimental models, some of which are already being evaluated clinically. PMID- 8635690 TI - The cellular and molecular basis of gastric mucosal defense. AB - The association between colonization of the stomach by Helicobacter pylori and peptic ulcer disease has stimulated a renewal of interest in the factors that render the gastric mucosa resistant to injury induced by endogenous secretions and ingested toxins. Mucosal defense consists of a complex network of components that function in concert with one another. This network includes: 1) the extramucosal components such as acid, mucus, surface-active phospholipids, and bicarbonate; 2) the epithelium itself; 3) the microcirculation and sensory afferent neurons beneath the epithelium; 4) the mucosal immune system; and 5) the ability of the mucosa to undergo repair. In the past two decades, an enormous amount has been learned about the cellular and molecular basis of the various components of mucosal defense, including a better understanding of the chemical substances that coordinate mucosal responses to injury. In this paper, we review the factors that contribute to mucosal defense, the cellular and molecular mechanisms through which mucosal defense is modulated, and the chemical mediators that play key roles in this process. PMID- 8635691 TI - G protein-coupled receptors and signaling pathways regulating growth responses. AB - Hormones that interact with seven-transmembrane spanning receptors, generally considered to be involved in acute signaling functions, also induce longer term effects on gene expression and cell growth. These genetic and proliferative effects can be induced by activation of receptors that signal through heterotrimeric GTP-binding proteins (G-proteins) of the Gq family, pertussis toxin-sensitive Gi/Go proteins, Gs, or G12/G13. Numerous growth-promoting G protein-coupled receptors activate the low molecular weight G-protein Ras and stimulate mitogen-activated protein kinase. Recent data suggest that c-Jun NH2 terminal kinase is also activated, possibly through interaction with low molecular weight G-proteins of the Rho family. Because G protein-coupled receptors lack intrinsic tyrosine kinase activity, the mechanisms by which heterotrimeric G-proteins couple to these kinase cascades remain to be elucidated. By analogy to growth factor receptors, G protein-coupled receptors may access these kinase cascades through binding of adapter proteins or recruitment of cytosolic tyrosine kinases. It is likely that interactions between multiple signaling pathways are required for G protein-coupled receptors to propagate signals to the nucleus. PMID- 8635692 TI - The molecular defect in the renal sodium-phosphate transporter expression pathway of Gyro (Gy) mice is distinct from that of hypophosphatemic (Hyp) mice. AB - Two animal models of the human disorder hypophosphatemic vitamin D-resistant rickets exist, the Hyp and Gy mice. Affected mice and humans both manifest an X linked phenotype, and show decreased Na+/Pi transport activity in the renal proximal tubules, which is characterized by a decreased maximal velocity (Vmax). The defect in Hyp mice is most likely due to a decreased transcription rate of the renal Na+/Pi transporter gene. The current studies were designed to define the molecular defect in the Gy mice. Sodium-dependent uptake of phosphate (Pi) in renal BBMV showed uptake levels of 170.58 +/- 25 and 66.00 +/- 11 pmol x mg protein-1 x 6 s-1 in normal and Gy mice, respectively (n=3, P=0.0102). Glucose uptake levels in the BBMV were 1.94 +/- 0.87 and 1.91 +/- 0.35 pmol x mg protein 1 x 6 s-1 in normal and Gy mice, respectively (n=3). Northern blot analysis of kidney cortex in both mice revealed nearly equivalent message levels (normal/Gy=1.01 +/- 0.12, n=3). In situ hybridization localized the mRNA to the renal cortex in both mice and confirmed equal message levels. Western blot analysis of renal BBM proteins, using a polyclonal antiserum, showed one predominant band at 87 kDa in both mouse samples, with intensities being decreased in the Gy mice (normal/Gy=4.129 +/- 0.70, n=4, P< 0.04). Immunohistochemical analysis localized the protein to the apical membrane of proximal tubules in both mice. These results suggest that the molecular defect in the Gy mice is distinct from that in the Hyp mice, and furthermore, that the manifestation of the diseased phenotype in Gy mice is related to a different defect in the renal Na+/Pi transporter expression pathway. The molecular mechanism of the defect likely relates to protein processing, metabolic turnover rate, or translocation to the brush-border membrane. These results further suggest that two distinct X-linked factors modulate different steps in the expression pathway of the Na+/Pi transporter gene. PMID- 8635693 TI - Expression of erythropoietin by the human placenta. AB - Circulating levels of maternal erythropoietin (EPO) rise during gestation due to increased biosynthesis of the hormone. Our objective was to investigate the human placenta as a potential extrarenal site of EPO production. Using two monoclonal antibodies recognizing different antigenic determinants, we identified immunoreactive EPO associated with villous cytotrophoblast, endovascular and intravascular cytotrophoblast, cytotrophoblast cell columns, and syncytiotrophoblast of first and second-trimester placenta as well as syncytiotrophoblast and extravillous cytotrophoblast of normal third-trimester and preeclamptic placenta. In addition, cultured JAR (trophoblast-derived) choriocarcinoma cells, cytotrophoblasts isolated from term placenta, villous core cells, and possibly other nontrophoblast cells within the decidual basal plate expressed immunoreactive EPO. Using reverse transcription-polymerase chain reaction and EPO-specific primers, a 378 bp DNA product was amplified from placental tissues of various gestational ages, cytotrophoblasts isolated from term placenta, and JAR choriocarcinoma cells. The amplified product yielded restriction enzyme fragments of predicted sizes. On Southern analysis, hybridization was observed for two of these fragments in which the radiolabeled EPO cDNA probe did not overlap with the primer sequences. Finally, the JAR choriocarcinoma cells elaborated EPO into the culture medium as determined by enzyme-linked immunosorbant assay and expressed EPO mRNA as determined by Northern analysis, both of which were stimulated by hypoxia (15-20 torr). Taken together, these results suggest a new site of EPO expression: the trophoblast cell of the human placenta. PMID- 8635695 TI - Expression of inducible nitric oxide synthase in placenta of women with gestational diabetes. AB - Gestational diabetes is one of the most prevalent medical complications of pregnancy and causes increased fetal wastage. Investigation of placentas from diabetic mothers indicate chronic disturbances in intervillous, circulation, dilatation of capillaries, and a relatively immature villous structure. Abnormal levels of nitric oxide (NO) may contribute to maternal disorders such as the pathogenesis of diabetic vascular complications. In the normal placenta NO is generated only by endothelial NOS, which apparently serves to regulate vascular tone in the fetoplacental circulation. In contrast, studies have reported the absence of inducible nitric oxide synthase (iNOS) in human placentas under normal conditions. The aim of our study was to investigate whether iNOS is expressed in placentas from patients with gestational diabetes. Reverse transcription polymerase chain reaction and Western blot analysis demonstrated iNOS mRNA and protein expression in placental tissue only from patients with gestational diabetes. Immunohistochemistry localized iNOS staining to endothelial cells and trophoblasts. We conclude that iNOS can be expressed in human placenta. Its expression might play an important role in placental pathophysiology. PMID- 8635694 TI - Expression and release of LAG-3-encoded protein by human CD4+ T cells are associated with IFN-gamma production . AB - The lymphocyte activation gene (LAG) -3 is a member of the immunoglobulin super family that is selectively transcribed in human activated T and NK cells. In this work, the possibility that LAG-3 expression by human CD4+ T cells was preferentially related to one or another phenotype of cytokine secretion was investigated. Surface LAG-3 expression correlated with IFN-gamma, but not IL-4, production in antigen-stimulated T cells and it was up-regulated by IL-12. Most activated CD4+ T cell clones with established Th1 or Th0 profiles of cytokine secretion expressed LAG-3 on their surface, whereas the great majority of Th2 clones showed neither surface LAG-3 nor LAG-3 mRNA expression. After activation, the majority of CD4+ T cell clones also released soluble LAG-3-related peptides, and such a release correlated positively with the production of IFN-gamma and inversely with the production of IL-4. Thus, LAG-3 expression by activated CD4+ human T cells appear to be preferentially associated with the differentiation/activation pathway leading to the production of IFN-gamma. PMID- 8635696 TI - Interleukin-1beta, tumor necrosis factor-alpha, and LPS enhance calcium channel current in isolated vascular smooth muscle cells of rat tail artery. AB - Cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), are thought to be responsible for the compromised blood pressure regulation after systemic infection or other antigenic challenge. Because Ca homeostasis is critical for the maintenance of vascular tone, we hypothesized that cytokines may contribute to alterations in blood pressure by a mechanism involving the voltage-sensitive Ca channel in vascular smooth muscle (VSM) cells. Using nystatin-permeabilized patch techniques we examined the effects of IL-1 beta, TNF-alpha, and lipopolysaccharide (LPS) on the Ca channel of VSM cells isolated from rat tail artery. Both IL-1 beta (0.05--1 nM) and TNF-alpha (0.1--1 nM) increased, dose-dependently, the Ba2+ current carried in VSM Ca channels, whereas heat-denatured IL-1 beta was without significant effect on the channel. LPS (0.01--1.0 ng/ml) also increased the Ba2+ current with onset kinetics similar to the two cytokines. Prostaglandins were ruled out as an intermediary in VSM Ca channel modulation, as prostaglandin E2 had no effect and indomethacin (1 microM) failed to block TNF-alpha-induced Ca channel enhancement. The role of cyclic nucleotides in mediating TNF-alpha-induced changes in Ca channel activity was also assessed. Increasing intracellular cAMP via forskolin (1 microM) did not affect the response to TNF-alpha, but pretreatment with the membrane-permeant analog of cGMP, dibutyryl cGMP (100 microM), inhibited the response to TNF-alpha. These data demonstrate that IL-1 beta, TNF-alpha, and LPS have immediate effects on VSM cells via an interaction with the voltage-sensitive Ca channel, and these effects may he regulated by intracellular cGMP. Immunomodulation of Ca channels may represent an early signaling step in VSM cells mediating kinetically slower events, such as changes in gene transcription. PMID- 8635697 TI - RANTES and platelet-activating factor open Ca2+-activated K+ channels in eosinophils. AB - RANTES is a member of the low molecular cytokines and appears to be strictly a chemotactic and activating factor for eosinophils. We studied the effect of RANTES on the channel activity of the eosinophilic cell line (EoL-1 cells) using patch clamp techniques. Under cell-attached patch conditions, RANTES (20 ng/ml) activated channels in EoL-1 cells when applied to the recording pipette or to the bath. This channel was permeable to K+ with a unit conductance of 14 pS, and the reversal potential was close to the equilibrium potential for K+. The current voltage relationship for this K+ channel was almost linear, showing little or no rectification. The open time and closed time histograms could he fitted to a single exponential function with time constants of 6.4 and 2.7 ms, respectively. Extracellular application of the calcium ionophore A23187 (1 microM) under cell attached patch conditions or an increase in intracellular Ca2+ concentration under inside-out patch conditions increased K+ channel activity in a manner similar to that caused by RANTES, indicating that the RANTES-activated channel was the Ca2+ -activated K+ channel. Intracellular application of GTP gamma S (10 microM) opened the Ca2+V-activated K+ channels under inside-out patch conditions. Furthermore, RANTES failed to activate the K+ channels after the cells had been treated with pertussis toxin (100 ng/ml) for 2 h at 37 degrees C. These results suggest that RANTES opens the Ca2+ -activated K+ channels of EoL-1 cells through activation of G-proteins. PMID- 8635699 TI - The origins of intermediary metabolism at Columbia College of Physicians and Surgeons (P&S). PMID- 8635698 TI - Functional interaction of hexokinase with ATP requires participation by both small and large lobes of the enzyme: implications for other proteins using the actin fold as a nucleotide binding motif. AB - The actin fold is a structural motif involved in binding of ATP to an otherwise diverse family of proteins that includes hexokinase, actin, and the 70 kDa heat shock protein. Previous analyses have focused attention on a hydrophobic pocket within the large lobe of hexokinase (and analogous regions in other proteins possessing the actin fold motif) as the-site for binding of the adenine moiety. However, there is reason to believe that binding of the adenine moiety also involves a beta-sheet region located in the small lobe of hexokinase. It is postulated that functional interaction with ATP, required for catalysis, involves interactions with both regions. The expected structural consequences provide a basis for explaining the kinetic mechanism suggested for this enzyme, i.e., although both substrates can bind independently, the preferred kinetic pathway is not random but ordered, with glucose binding first. The structural features postulated to be involved in binding of ATP to hexokinase are also found in other proteins possessing the actin fold motif. Interaction of the nucleotide with the beta-sheet region, analogous to that found in the "small lobe" of hexokinase, may induce functionally important conformational changes in other proteins employing the actin fold as a nucleotide binding motif. PMID- 8635700 TI - Endosonography-guided cholangiopancreatography. AB - BACKGROUND: Detailed imaging of the common bile duct and main pancreatic duct is possible with endosonography. Utilizing a custom manufactured flexible needle, we have developed a technique of performing endosonography-guided cholangiopancreatography (EGCP). METHODS: Of 205 patients undergoing ERCP, complete ductography was not possible in 11 patients. Employing a linear scanning echoendoscope in conjunction with a 4 cm, 22 to 23 gauge aspiration needle, transduodenal cholangiography (n = 10) or transgastric pancreatography (n = 1) was attempted. RESULTS: Successful ductography was possible in 8 of 11 patients (EGCP success 73% vs ERCP 0%, p < .001, Fisher's exact test). In 5 patients, abnormalities identified on EGCP subsequently led to repeat ERCP with precut sphincterotomy. In all of these cases 100% agreement was found between EGCP and ERCP findings. One postprocedure case of pancreatitis occurred in a patient who underwent EGCP at the same setting as the failed ERCP. No early or late complications occurred in the patient group with EGCP performed at a separate setting. CONCLUSION: EGCP allows an alternative method for obtaining cholangiopancreatography in those patients in whom ERCP is unsuccessful. Further studies are necessary to define the safety and success rate of this new procedure. PMID- 8635701 TI - A prospective outcome study of patients with clot in an ulcer and the effect of irrigation. AB - BACKGROUND: The proper management of patients with clots in an ulcer base has not been clearly defined by prospective studies. Variable prevalence and rebleeding rates may be explained by differing degrees of vigor used to clear the clot, as removal may reveal other stigmata. We prospectively assessed the natural histories of patients with clots after vigorous irrigation, employing a management strategy of endoscopic therapy for patients with high-risk stigmata and observation of those with clots resistant to washing or low-risk findings. METHODS: Forty-six patients with upper gastrointestinal bleeding found to have a clot in an ulcer had irrigation with a 3.2 mm bipolar probe for up to 5 minutes. Stigmata after washing were recorded; endoscopic therapy was given for active bleeding that persisted > or = 5 min or for nonbleeding visible vessels. Patients were observed in the hospital for > or = 3 days. RESULTS: Findings revealed after irrigation were adherent clot, 26 (57%); clean base, 1 (2%); flat spot, 5 (11%); nonbleeding visible vessel, 7 (13%); oozing, 6 (13%); and spurting, 1 (2%). Two of the 26 (8%) with adherent clots after washing rebled; endoscopic therapy resulted in no further bleeding. One of the 14 (7%) with active bleeding or visible vessels treated with hemostatic therapy rebled; repeat endoscopic therapy resulted in no further bleeding. No deaths occurred. CONCLUSIONS: Irrigation appears to be useful in patients with upper gastrointestinal bleeding who have ulcers with clots. The endoscopic findings present after washing can be used to dictate the appropriate management at initial endoscopy. Application of hemostatic therapy in patients with active bleeding or nonbleeding visible vessels and observation of patients with other stigmata, including clots resistant to washing, resulted in an excellent outcome. PMID- 8635702 TI - Factors predicting failure of endoscopic injection therapy in bleeding duodenal ulcer. AB - BACKGROUND: The aim of this study was to assess the factors that may cause failure of endoscopic injection in patients bleeding from a duodenal ulcer. METHODS: One hundred twenty patients admitted for a bleeding duodenal ulcer with active arterial hemorrhage or a nonbleeding visible vessel were included. RESULTS: Endoscopic injection was not feasible in 14 of 120 (11.6%) patients because of inaccessibility or massive hemorrhage. The remaining 106 patients underwent endoscopic therapy by injection of adrenaline and polidocanol. The efficacy (achievement of definitive hemostasis) of endoscopy therapy was 83% (88 of 106). Univariate analysis showed that failure of endoscopic injection was related to age, presence of shock, ulcer size greater than 2 cm, and hemoglobin level. Multivariate analysis showed that ulcer size greater than 2 cm (p = 0.005) and the presence of shock (p = 0.03) were factors predictive of endoscopic treatment failure. Failure to achieve hemostasis (p < 0.001) and poor physical status measured by American Society of Anesthesiology classification (p = 0.02) were significantly related to mortality. CONCLUSIONS: Ulcer size and severity of hemorrhage are predictive of endoscopic injection failure in patients bleeding from high-risk duodenal ulcers. Survival is determined by clinical status and associated diseases. PMID- 8635703 TI - A prospective randomized trial of heater probe thermocoagulation versus injection therapy in peptic ulcer hemorrhage. AB - BACKGROUND: A prospective, randomized study was performed to compare the hemostatic effect of injection therapy and heater probe thermocoagulation in the treatment of peptic ulcer bleeding. METHODS: This study includes 104 patients with upper gastrointestinal bleeding in whom endoscopy revealed a gastric or duodenal ulcer with nonbleeding or bleeding vessel (n = 66), oozing hemorrhage (n = 21), or adherent red clot (n = 17). Patients with other stigmata or clean ulcers were excluded. Patients were randomly assigned during endoscopy to receive injection therapy (adrenaline and polidocanol) (n = 51) or heater probe thermocoagulation (10F probe, at setting of 30 J (n = 53). Therapy was considered successful if there was no further hemorrhage or only minor rebleeding that was controlled with a second endoscopic procedure. Patients with major rebleeding or failure of retreatment underwent emergency surgery. RESULTS: There were no significant differences in effectiveness between injection therapy and thermocoagulation in any of the assessed parameters: the percentage of patients with major recurrent hemorrhage (4% vs 6%) or minor rebleeding (16% vs 17%), need for emergency surgery (two patients from each group), transfusion requirement (0.45 +/- 0.9 units vs 0.51 +/- 1.1 units), the mean number of hospitalization days (7.1 +/- 4.2 vs 6.9 +/- 4.9), and mortality (one patient from each group died). CONCLUSION: Injection therapy and heater probe have similar efficacies in the treatment of bleeding peptic ulcers. PMID- 8635704 TI - Endoscopic biliary endoprosthesis for palliation of gallbladder carcinoma. AB - BACKGROUND: Carcinoma of the gallbladder is a major cause of malignant obstructive jaundice in India. It usually presents at an advanced stage and endoscopic palliation is the mainstay of treatment. We prospectively studied our results with endoscopic stenting in patients with carcinoma of the gallbladder. METHODS: Patients unfit for surgery were included in the study. Straight 10F plastic prostheses were placed endoscopically. Patients were assessed for procedure success, early and late complications, and stent patency. RESULTS: The success rate of stent placement was 84% (27 of 32). The five failures were caused by an inability to pass the guide wire across the stricture. Relief of pruritus and reduction in jaundice was seen in 25 of 27 (92%) patients. Double stents were placed in three patients. Four patients (11%) developed cholangitis in the first 30 days. Stent occlusion was detected in four patients after longer follow-up. The 30-day mortality was 5 of 27 (18%). There were no procedure-related deaths. CONCLUSION: Endoscopic endoprosthesis is a safe and relatively effective palliative measure for the majority of patients with unresectable carcinoma of the gallbladder. PMID- 8635705 TI - Why is colonoscopy more difficult in women? AB - BACKGROUND: In our experience colonoscopy in women is more difficult than in men. A retrospective review of 2194 colonoscopies performed by a single experienced endoscopist (CBW) showed that 31% of examinations in women were considered technically difficult compared with 16% in men. METHODS: To investigate a possible anatomic basis for this finding, normal barium enema series from 183 female and 162 male patients were identified. From these barium enemas, measurements of colonic length and mobility were independently taken by two physicians who were unaware of each patient's gender. RESULTS: Total colonic length was greater in women (median, 155 cm) compared to men (median, 145 cm), p = 0.005, despite women's smaller stature (p < 0.0001). Although there were no significant differences in rectum plus sigmoid, descending, or ascending plus cecum segmental lengths, women had longer transverse colons (female median length, 48 cm; male median length, 40 cm), p < 0.0001. There were no differences in mobility of the descending colon and transverse colon between the sexes, but the transverse colon reached the true pelvis more often in women (62%) than in men (26%), p < 0.001. CONCLUSIONS: Colonoscopy appears to be a technically more difficult procedure in women. The reason for this may be due in part to an inherently longer colon. PMID- 8635706 TI - Endoscopic sphincterotomy in patients with liver cirrhosis. AB - BACKGROUND: Nonsurgical alternatives in biliary diseases have not been studied in large series of patients with cirrhosis. Our aim was to determine the indications and results of endoscopic treatment in this subset of patients. METHODS: Fifty two patients (36 men, 16 women-Child-Pugh Class A, 18; B, 22; C, 12) aged 63 +/- 18 years underwent endoscopic sphincterotomy (ES) between 1988 and 1993. Antibiotic prophylaxis was routinely carried out and coagulopathy was corrected before ES when required. The data were collected retrospectively up to 30 days after ES. RESULTS: ES succeeded in 98% of the patients (12 cases of needle-knife papillotomy). Twenty-nine patients (55.7%) had choledocholithiasis, 18 had biliary strictures (12 malignant), and 5 had pancreatic or other biliary diseases. Five days after ES, morbidity was 13.5% and mortality was 7.7%. At 1 month, morbidity was 22.9% and mortality 12.5%. Only endoscopic procedures and Ineffective drainage were seen to be significant risk factors or morbidity. The results of the subgroup of cirrhotic patients with choledocholithiasis (n = 29) were compared with those of matched noncirrhotic patients (n = 58). The cirrhotic and noncirrhotic patients treated for choledocholithiasis showed similar results for stone clearance, morbidity, and mortality. CONCLUSION: ES is a safe and effective procedure for treating choledocholithiasis in cirrhotic patients. ES can therefore be considered as an alternative to surgery in Child class A and B patients and must be preferred for Child class C patients with life-threatening biliary complications. PMID- 8635707 TI - Gastrointestinal bleeding after hepatic transcatheter arterial embolization in patients with hepatocellular carcinoma. AB - BACKGROUND: Transcatheter arterial embolization is a popular palliative treatment for patients with hepatocellular carcinoma, but the incidence of post-treatment gastrointestinal bleeding is not well-defined. METHODS: We retrospectively analyzed 206 patients with hepatocellular carcinoma who received transcatheter arterial embolization and compared them with 193 patients with hepatocellular carcinoma who underwent angiography along. RESULTS: Twenty-three episodes (8.5%) of gastrointestinal bleeding occurred within 3 months of hepatic transcatheter arterial embolization following 269 procedures involving 206 patients with hepatocellular carcinoma. Eight episodes (3.0%) of esophageal variceal bleeding and 15 episodes (5.5%) of nonvariceal bleeding were found. The sites of the nonvariceal bleeding episodes were the stomach (n = 7), duodenum (n = 5), and colon (n = 3). When compared with other sources, bleeding from esophageal varices took place earlier, required intensive treatment, and led to a higher mortality. Among another 193 patients with hepatocellular carcinoma who received angiography only, 6 patients developed gastrointestinal bleeding within 3 months (3.1%), and all bled from esophageal varices. CONCLUSIONS: This study suggests that esophageal variceal bleeding may occur after both angiography and transcatheter arterial embolization. Nonvariceal bleeding episodes, which were usually milder than episodes of variceal bleeding, may be related to the embolization procedure itself. PMID- 8635709 TI - Combined endoscopic clipping and injection sclerotherapy for esophageal varices. PMID- 8635708 TI - CT-guided PEG in patients with unsuccessful endoscopic transillumination. PMID- 8635710 TI - Simple and effective endoscopic tamponade for duodenal bleeding. PMID- 8635711 TI - Laparoscopic examination of the bowel in trauma patients. PMID- 8635712 TI - Postradiotherapy benign biliary stricture: successful treatment by self expandable metallic stent. PMID- 8635713 TI - Trans-hepatic proximal migration of percutaneous-endoscopic biliary stent presenting as an abdominal wall abscess. PMID- 8635714 TI - Nitinol coil esophageal prosthesis: advantages of removable self-expanding metallic stents. PMID- 8635715 TI - Treatment of esophageal perforation with a covered expandable metal stent. PMID- 8635716 TI - Removal of an incompletely expanded ultraflex esophageal stent. PMID- 8635717 TI - An esophagopericardial fistula successfully treated with an expandable covered metal mesh stent. PMID- 8635718 TI - Successful endoscopic hemostasis of bleeding colonic diverticula with epinephrine injection. PMID- 8635719 TI - Fluoroscopy or no for esophageal dilation: the passing of an era. PMID- 8635720 TI - Endotherapy for gastric outlet obstruction. PMID- 8635721 TI - Consent for colonoscopy. PMID- 8635722 TI - Antibiotic prophylaxis of ERCP with ciprofloxacin. PMID- 8635723 TI - How many patients must we treat for Helicobacter pylori infection to prevent a recurrent duodenal ulcer hemorrhage? PMID- 8635724 TI - Biliary rendezvous or solo combined procedure for therapy of sump syndrome. PMID- 8635725 TI - High esophageal rings. PMID- 8635726 TI - Peroperative endoscopic sphincterotomy during laparoscopic cholecystectomy for choledocholithiasis. PMID- 8635727 TI - Endoscopic treatment of biliary leakage after laparoscopic cholecystectomy. PMID- 8635728 TI - Does fluoroscopic guidance for Maloney esophageal dilation impact on the clinical endpoint of therapy: relief of dysphagia and achievement of luminal patency. AB - BACKGROUND: Use of fluoroscopy for Maloney esophageal dilation is controversial. We designed this prospective, randomized, single-blinded study to determine whether fluoroscopic guidance has an impact on relief of dysphagia and achievement of luminal patency. METHODS: Patients with benign esophageal strictures were randomized to undergo Maloney dilation with or without fluoroscopic guidance. Strictures were dilated to size 48F. Dysphagia scores were obtained before and 1 week after dilation. RESULTS: Eighty-three patients underwent 100 dilation sessions with fluoroscopic guidance being used for 50 sessions (156 dilations) and blinded technique for 50 (161 dilations). A 12.5 mm barium pill passed after dilation following 62.0% of the fluoroscopic dilation sessions and 42.0% of the blinded dilations (p = 0.045). Dysphagia was improved in 93.0% of patients receiving fluoroscopic dilations and 69.0% of patients receiving blinded dilations (p = 0.006). The mean improvement in dysphagia score was -2.10 points for the fluoroscopic group versus -1.50 points for the blinded group (p = 0.057). Differences in these parameters between techniques were even greater in 12 patients re-randomized to both techniques at different sessions. CONCLUSIONS: The use of fluoroscopic guidance impacts favorably on the efficacy of Maloney dilation, resulting in greater relief of dysphagia and increased luminal patency compared to the blinded technique. Based on these results, use of fluoroscopy is recommended when Maloney esophageal dilation is performed. PMID- 8635729 TI - Through-the-scope balloon dilation for pyloric stenosis: long-term results. AB - BACKGROUND: Through-the-scope balloon dilation has been used for treatment of benign pyloric stenosis; however, long-term results are lacking in the literature. METHOD: A retrospective analysis using the Kaplan-Meier method. RESULTS: Between November 1986 and December 1993, 54 patients underwent through the-scope balloon dilations for pyloric stenosis. The mean age was 57.5 years. There were 5 (9.3%) initial treatment failures due to tight stenoses and perforations from dilation occurred in 4(7.4%) patients. Forty-five (83.3%) patients underwent successful dilation. Four patients developed rapid restenoses and were found to have malignant obstructions. Forty-one patients entered our study. Time at risk commenced on the date of initial dilation. The end point was defined at the time at which patients presented with recurrent obstruction or other ulcer complications. The median follow-up period was 39 months. The ulcer complication-free probability at 3 months, and at 1, 2, and 3 years was 79.1%, 73.4%, 69.3%, and 54.7%, respectively. In all, 21 (51.2%) patients required subsequent surgery: 18 for recurrent obstructions, 2 for interval perforations, and 1 for bleeding. CONCLUSION: While through-the-scope balloon dilation may palliate symptoms of obstruction, recurrent obstruction and other ulcer complications are common. It should be reserved only for patients at high risk for operative surgery. PMID- 8635730 TI - Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. AB - Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosynthesis of the polyketide immuno-suppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in 'late' modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster. PMID- 8635731 TI - Cloning of the Mycoplasma capricolum gene encoding peptide-chain release factor. AB - In Mycoplasma capricolum (Mc), a relative of Gram+ eubacteria with a high genomic A + T-content, the UGA codon is assigned to Trp instead of being a stop codon. We previously showed the lack of peptide-chain release factor (RF) activity in vitro responding to the UGA codon in this bacterium [Inagaki et al., Nucleic Acids Res. 21 (1993) 1335-1338]. To obtain more information on the translation termination mechanism of Mc, we isolated and sequenced the gene encoding RF. The deduced amino-acid sequence has no RF-2-specific + 1 frameshift site and shows 50 and 36% identity to Escherichia coli RF-1 and RF-2, respectively. We conclude that this gene encodes the putative RF-1 which would possess the conserved 'five-domain' structure of RF family found in various organisms. PMID- 8635732 TI - Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. AB - The ERG5 gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.15-kb SacI-EcoRI DNA fragment containing an open reading frame of 538 amino acids (aa). ERG5 contains a 10-aa motif consistent with its role as a cytochrome P-450 (CyP450) enzyme and is similar to a number of mammalian CyP450 enzymes. Gene disruption demonstrates that ERG5 is not essential for cell viability. PMID- 8635733 TI - Selectable cassettes for simplified construction of yeast gene disruption vectors. AB - Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of an KmR-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker. PMID- 8635734 TI - A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae. AB - We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2 related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa2CysXaa20CysXaa2Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation. PMID- 8635735 TI - Sequence of the Saccharomyces cerevisiae YTP1 gene encoding a deduced novel type III integral membrane protein with domains of sequence similarity to mitochondrial electron-transport enzymes. AB - The nucleotide sequence is reported for the Saccharomyces cerevisiae YTP1 (yeast putative transmembrane (TM) protein) gene, encoding a novel deduced protein of 459 amino acids (aa) in length (51 643 Da). The Ytp1 protein appears by computer analysis (hydropathy plots in conjunction with the combined predictions of several Internet on-line programs that deduce protein structure from primary sequence data) to be a type-III integral TM protein containing 10 or 11 TM spanning domains. Blocks of aa sequence similarity, predominantly to mitochondrial electron transport proteins, are consistent with the notion that Ytp1 is an integral TM protein and may reflect some aspect of its functional role. The C terminus of Ytp1 is both hydrophilic and highly negatively charged, with 11 of the last 33 aa corresponding to Glu or Asp. Although Northern blot analysis indicates that this gene is expressed, a disruption of YTP1 shows that it is not essential. YTP1 is located between SIN4 (TSF3) and KEX2 (SRB1) at position 205 (kb) on the chromosome XIV physical map. PMID- 8635736 TI - Differential expression of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe. AB - The rhp51+ gene encodes three transcripts of 1.9, 1.6 and 1.3 kb which have at least six polyadenylation sites. Primer-extension analysis revealed that two transcription start points (tsp) at - 166 and - 136 were responsible for the DNA damage inducibility of this gene. Northern blot analyses showed that the three transcripts were expressed differentially in response to a variety of DNA damage. During the mitotic cell cycle, only the largest transcript exhibited periodic expression, reaching the maximal level in front of the cdc22+ transcript which peaks at the G1/S boundary. Unexpectedly, the steady-state levels of the three transcripts were differentially regulated during the growth cycle. The largest and smallest transcripts accumulated in large quantity at the diauxic shift and during the entry into stationary phase, respectively. To localize the regions responsible for the differential expression of rhp51+, we constructed rhp51::ura4 and ura4::rhp51 hybrid genes, and analyzed their expression patterns in response to methyl methanesulfonate (MMS)-induced DNA damage. The results showed that the promoter region and 5' half of rhp51+ are sufficient to confer damage responsiveness while the 3' end of the gene alone can direct the formation of multiple, discrete 3' ends of the transcripts. From these results, we conclude that this novel one gene-multiple product system is possible through the cooperation of both the promoter and 3' terminal regions. PMID- 8635737 TI - A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene. AB - A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec. PMID- 8635738 TI - Identification of calmodulin-binding peptide consensus sequences from a phage displayed random peptide library. AB - The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OlambdaR, WRAAV or WRXXAAAL, where +, -, O, lambda and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025-23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides. PMID- 8635741 TI - New shuttle vectors for Actinobacillus actinomycetemcomitans and Escherichia coli. AB - A series of shuttle vectors for Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was developed. These vectors carry a multiple cloning site, the lacZalpha reporter gene, the replication origin for Aa derived from pVT736-1 and a replicon for Ec originated from pUC, P15A or pSC101. With these vectors, cloning and expression can be effectively performed in Aa and Ec. PMID- 8635739 TI - Sequence of the Bacillus stearothermophilus gene encoding aspartokinase II. AB - The complete nucleotide sequence of the gene encoding aspartokinase (Ask) II from thermophilic Bacillus stearothermophilus has been determined. Degenerate oligodeoxyribonucleotides primed the amplification of a 932-bp gene. This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction using, as template, self-ligating DNA fragments. The deduced amino-acid sequence is 68.7% identical with the sequence of the Bacillus sp. strain MGA3 Ask II. PMID- 8635740 TI - Sequences of the genes encoding the A, B and C subunits of the Haemophilus influenzae dimethylsulfoxide reductase complex. AB - The genes (dms) encoding the dimethylsulfoxide reductase protein complex have been cloned and sequenced from Haemophilus influenzae (Hi) type b (Hib) strain Eagan. The Hib dms genes are arranged as an operon whose genomic organization is similar to that of the Escherichia coli (Ec) dmsABC operon. The deduced Hib DmsA, and DmsB and DmsC amino-acid sequences are highly homologous to their Ec counterparts and nearly identical to the recently published sequences of the Hi type-d strain Rd Dms proteins. Hi dimethylsulfoxide reductase appears to be a new member of the superfamily of oxidoreductase enzymes. PMID- 8635742 TI - Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector. AB - A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the KmR gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5alpha and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 X 10(6) colonies per microgram of DNA. Plasmid pYGK exists at approx. 3-4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism. PMID- 8635743 TI - Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene. AB - We have cloned the genomic copy of the Ppras1 gene, a homologue of the ras proto oncogene, from the true slime mold Physarum polycephalum. Ppras1 contains five small introns, four of which have a high content of pyrimidines. The (dC) homopolymers present in introns 4 and 5 may be responsible for the observed recA independent deletion in Ppras1 upon amplification of the Ppras1-bearing plasmid by choramphenicol. Although Ppras1 exhibits amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Distyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently. PMID- 8635744 TI - Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region. AB - We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24 L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs. PMID- 8635745 TI - Characterization of the Haemophilus influenzae topA locus: DNA topoisomerase I is required for genetic competence. AB - A gene essential for the development of genetic competence in Haemophilus influenzae (Hi) was identified as a homolog of the Escherichia coli (Ec) topA gene, which encodes DNA topoisomerase I (TopI). The Hi topA locus was initially identified by mini-Tn10kan mutagenesis. Three independent insertion events within 500 bp of each other resulted in mutant strains that shared a similar phenotype. Each was deficient in competence-induced DNA binding, showed increased sensitivity to UV irradiation, and had an increased doubling time as compared to the wild-type (wt) strain. The nucleotide sequence of a 6.6-kb fragment containing the wt allele was determined. The sequence contained an open reading frame (ORF) of 868 amino acids (aa) that was interrupted by each of the mini Tn10kan mutations. The deduced aa sequence had a molecular mass of 98 155 Da, a pI of 8.59 and showed strong similarity to Ec TopI. Examination of the topoisomer distribution of a test plasmid in an Hi mutant carrying an insertion in this ORF showed an increase in the level of supercoiling, indicating that TopI is necessary to relax supercoiled DNA in Hi. Complementation studies and insertional inactivation of genes downstream from topA indicated that TopI and not some downstream gene product was essential for competence. Four other ORFs were identified and two of these had homology to known genes. ORF1, which was truncated at one end of the sequenced region, shared strong sequence similarity to the C-terminal end of Ec pyridine nucleotide transhydrogenase beta subunit. ORF4, which was also truncated, showed strong sequence similarity to the N terminal end of the Ec threonyl-tRNA synthetase. PMID- 8635746 TI - A new cysteine-rich protein-encoding gene family in Giardia duodenalis. AB - We have cloned a gene, CRP65, from genomic DNA of Giardia duodenalis (Gd) which contains four 228-bp tandem repeat units between a short (48bp) 5' and long (942 bp) 3' non-repeat region. CRP65 encodes a Cys-rich protein (CRP) with the typical transmembrane domain and CXXC amino acid (aa) motif of Gd CRP. Comparison of the nucleotide (nt) and deduced aa sequences of CRP65 and a gene we cloned previously. CRP136, indicates that the genes are highly homologous in the entire non-repeat regions, but not in the repeat regions. The repeat unit of CRP65 was found to be homologous to epidermal growth factor (EGF)-like domains from different proteins. Analysis of Gd genomic DNA showed that there are multiple copies of CRP65 and each copy varies in the number of repeat units, as well as in certain restriction sites in the units. In Gd strain WB-1B, a 2.0-kb transcript encoded by the gene was expressed, while in a metronidazole-resistant line (WB1B M3) induced from WB-1B, two longer transcripts (5.5 and 7 kb) were expressed. Based on our results, we suggest that there is a unique CRP family in the Gd genome, whose members, including CRP65 and CRP136, carry various repeat units within a highly conserved 'cassette'. CRP65 may be involved in EGF-like interactions with the host proteins. PMID- 8635747 TI - Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. AB - We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites. PMID- 8635748 TI - Positive selection vectors for allelic exchange. AB - We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46. These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi. In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences. In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae. PMID- 8635749 TI - BTag: a novel six-residue epitope tag for surveillance and purification of recombinant proteins. AB - Epitope tagging (Eta) is becoming an increasingly useful technique in molecular biology and biotechnology for the detection, characterisation and purification of recombinant proteins (re-proteins). Here we describe a novel Eta system composed of two different monoclonal antibodies (mAb; D11 and F10) and a 6-amino-acid Eta (Gln-Tyr-Pro-Ala-Leu-Thr or QYPALT). This Eta was derived from a highly conserved region of the major core protein, VP7, of bluetongue (BT) viruses, hence the name BTag. BTag is unique among current tagging systems in its lack of charge and the fact the tag sequence can be placed and detected in any region of a re-protein. Other useful features of BTag include its small size and its recognition by two different mAb. Using the BTag system, more than 30 re-proteins have been produced from a variety of host organisms, and the antigenicity of the tag sequence was maintained in all of the proteins tested to date. Our result demonstrated that BTag could be superior to other existing Eta systems for certain applications. PMID- 8635750 TI - A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. AB - A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (MalE) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme. PMID- 8635751 TI - A refined vector system for the in vitro construction of single-copy transcriptional or translational fusions to lacZ. AB - New single-copy vectors based on lambda phage have been developed for creating either transcriptional (operon) or translational (gene) fusions to the lacZ gene. The improvements of these vectors over the previous lambda TL61 vector include: (i) incorporation of a tetracycline-resistance-encoding gene (TcR) to permit direct selection of lysogens, (ii) low-background beta-galactosidase activity, (iii) the ability to accept DNA inserts up to 8 kb in size, and (iv) an expanded multiple cloning site (MCS). The new transcriptional fusion vector retains the RNase III processing site downstream from the MCS which ensures independent translation of lacZ. The set of three translational fusion vectors allow for convenient subcloning in any of the three translational reading frames. PMID- 8635752 TI - Overproduction and characterization of the StsI restriction endonuclease. AB - The StsI restriction endonuclease (R-StsI), a class-IIS restriction endonuclease, found in Streptococcus sanguis 54, is a heteroschizomer of R-FokI, which recognizes 5'-GGATG-3'. To overproduce R-StsI in Escherichia coli, the coding region of R-StsI was joined to the tac promoter of an expression vector, pKK223 3. By introduction of the plasmid into E. coli UT481 cells expressing the fokIM gene, R-StsI activity was overproduced, from which R-StsI was purified homogeneously. We compared the properties of R-StsI with those of R-FokI. The optimum reaction conditions for R-StsI were quite different fron those for R FokI. R-StsI is an acidic protein (pI 6.3). Anti-R-StsI serum did not cross-react with R-FokI, indicating three-dimensional structural dissimilarity. The domain structure of R-StsI was elucidated by digestion with trypsin. In the presence of substrate DNA, R-StsI was digested to yield 45-kDa N-terminal and 23-kDa C terminal fragments. The amino-acid sequences around the trypsin cleavage sites of R-StsI and R-FokI were quite homologous. PMID- 8635753 TI - The location of four fimbrin-encoding genes, agfA, fimA, sefA and sefD, on the Salmonella enteritidis and/or S. typhimurium XbaI-BlnI genomic restriction maps. AB - Four fimbrin-encoding genes, fimA (type-1 or SEF21 fimbriae), agfA (thin aggregative or SEF17 fimbriae), sefA (SEF14 fimbriae and sefD (SEF18 fimbriae) from Salmonella enteritidis (Se) 27655-3b were located onto the XbaI-BlnI genomic restriction maps of Salmonella typhimurium (St) LT2 and Se strains SSU7998 and 27655-3b. The XbaI or BlnI genomic fragments carrying these genes were identified by hybridization with labeled oligodeoxyribonucleotides or fimbrin-encoding genes. The fimbrin-encoding genes were not encoded by the virulence plasmids, but were located on chromosomal DNA fragments. The position of each gene on a given XbaI fragment was determined by hybridization of a series of XbaI-digested genomic DNA samples from previously characterized Tn10 mutants of Se and St with its respective probe. The fimA gene mapped near 13 centisomes (Cs) between purE884::Tn10 at 12.6 Cs (11.8 min) and apeE2::Tn10 at 12.8 Cs (12.3 min) beside the first XbaI site at 13.0 Cs in St or between purE884::Tn10 at 12.6 Cs and the XbaI site at 13.6 Cs in Se. The agfA gene mapped near 26 Cs between putA::Tn10 and pyrC691::Tn10 in St, but near 40 Cs between pncX::Tn10 and the XbaI site at 43.3 Cs in Se. This difference in map position was due to the location of agfA near one end of the 815-kb chromosomal fragment inverted between Se and St. The sefA and sefD genes mapped precisely at 97.6 Cs in Se, but were absent from the genome of St LT2. To verify the mapping procedures used herein, tctC was also mapped in both Salmonella serovars. As expected, tctC mapped near 60 Cs in both St and Se, thereby confirming previous studies. PMID- 8635754 TI - Isolation of Rhodobacter capsulatus transketolase: cloning and sequencing of its structural tktA gene. AB - Rhodobacter capsulatus transketolase (Tkt) protein has been isolated from strain B10 by heparin affinity chromatography. Oligodeoxyribonucleotides (oligo) constructed as based on the amino-acid sequences were used for polymerase chain reaction (PCR) amplification on total genomic DNA. Southern hybridization with the PCR product as a probe allowed the isolation of a 5-kb PstI DNA fragment containing the structural Tkt-encoding gene (tktA) which was cloned and sequenced. The deduced tktA product of 671 aa (72815 Da) shares 59% identity with Rhodobacter sphaeroides Tkt. PMID- 8635755 TI - A host-vector system for heterologous gene expression in Streptococcus gordonii. AB - We have developed a host-vector system for heterologous expression in Streptococcus gordonii (Sg) Challis (formerly Streptococcus sanguis), a commensal bacterium of the human oral cavity. The system is based on (i) integration of plasmid insertion vectors into the chromosome of specially engineered recipient hosts, and (ii) the use of the M6-protein-encoding gene (emm6) as a partner for construction of translational gene fusions. M6 is a streptococcal surface protein already proven useful as a fusion partner for the delivery of foreign antigens to the surface of Sg [Pozzi et al., Infect. Immun. 60 (1992) 1902-1907]. Insertion vectors carry a drug-resistance marker, different portions of emm6 and a multiple cloning site to allow construction of a variety of emm6-based fusions. Upon transformation of a recipient host with an insertion vector, 100% of transformants acquire both the drug-resistance marker and the capacity of displaying the M6 molecule on the cell surface. Chromosomal integration occurred at high frequency in recipient host GP1221. Transformation with 1 microgram of insertion vector DNA yielded 8.1 X 10(5) transformants per ml of competent cells. PMID- 8635756 TI - Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. AB - The three giant multifunctional polypeptides of the rapamycin (Rp)-producing polyketide synthase (RAPS1, RAPS2 and RAPS3) have recently been shown to contain 14 separate sets, or modules, of enzyme activities, each module catalysing a specific round of polyketide chain extension. Detailed sequence comparison between these protein modules has allowed further characterisation of aa that may be important in catalysis or specificity. The acyl-carrier protein (ACP), beta ketoacyl-ACP synthase (KS) and acyltransferase (AT) domains (the core domains) have an extremely high degree of mutual sequence homology. The KS domains in particular are almost perfect repeats over their entire length. Module 14 shows the least homology and is unique in possessing only core domains. The enoyl reductase (ER), beta-ketoacyl-ACP reductase (KR) and dehydratase (DH) domains are present even in certain modules where they are not apparently required. Four DH domains can be recognised as inactive by characteristic deletions in active site sequences, but for two others, and for KR and ER in module 3, the sequence is not distinguishable from that of active counterparts in other modules. The N terminus of RAPS1 contains a novel coenzyme A ligase (CL) domain that activates and attaches the shikimate-derived starter unit, and an ER activity that may modify the starter unit after attachment. The sequence comparison has revealed the surprisingly high sequence similarity between inter-domain 'linker' regions, and also a potential amphipathic helix at the N terminus of each multienzyme subunit which may promote dimerisation into active species. PMID- 8635757 TI - The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2). AB - A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network. PMID- 8635758 TI - Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli. AB - We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues. PMID- 8635759 TI - Acute overdose of zolpidem. AB - Zolpidem (Ambien) is an imidazopyridine hypnotic recently introduced in the USA. We report a case of a fatal overdose of Ambien. A 68-year-old female ingested at least 30 tablets of 10 mg Ambien (300 mg). She was found dead at home. Toxicological analyses revealed blood concentration of 4.1, 19.3 and 2.3 micrograms/ml of zolpidem, meprobamate and carisoprodol, respectively. PMID- 8635760 TI - Changes in DNA induced by toxic agents. AB - This is a preliminary report on significant alterations in the DNA profile caused by toxic substances which potentially has profound implications for the use of DNA techniques in identification. Acute DNA changes in the globus pallidus of the brain in man caused by carbon monoxide poisoning were detected by DNA profiling with probe 33.15. Chronic DNA changes in rabbits caused by methamphetamine were detected by DNA profiling with probe 33.15, AmpFLP on D1S80, TH01, CSF1PO and TPOX loci. Pre-intoxication bands appeared, disappeared or were discoloured after intoxication. With PCR-dot blot hybridization testing for HLADQ alpha, pre intoxication positive spots became negative after intoxication and pre intoxication negative spots became positive after intoxication. Intravenous injection of 10 mg/kg of methamphetamine every 2 days for 2 weeks was a large enough dose for inducement of genetic changes. In this investigation, clear changes in DNA due to intoxication were confirmed. PMID- 8635761 TI - The effect of storage at various temperatures on blood alcohol concentration. AB - There is a paucity of data available on the effect of storage on blood alcohol concentration (BAC) at elevated temperatures. Changes in serum alcohol concentration (SAC) and BAC were studied. Serum samples spiked with alcohol in the presence or absence of preservative were stored at 26.7 degrees, 32.2 degrees or 37.8 degrees C respectively. Serum alcohol concentrations were determined daily on days 1 through 14, and on days 21 and 35. Under these controlled conditions, no significant change in SAC was observed at the aforementioned temperatures. Whole blood samples submitted from outside agencies were initially analyzed (day 1), then stored for 35 days at different elevated temperatures before a second analysis. The average loss in BAC was 19.20 +/- 15.6, 9.95 +/- 5.7, and 15.60 +/- 6.9% when the samples were stored at 26.7, 32.2 and 37.8 degrees C, respectively. The alcohol loss from whole blood samples may be attributed to chemical oxidation rather than to elevated temperatures. It is, therefore, concluded that a whole blood sample obtained from a living individual and stored in a locker, glove compartment or other environment where the temperature is elevated, may lose 10-19% of its alcohol content over 35 days of storage. On the other hand, when a serum or plasma sample is exposed to the same environment, no significant change in SAC was observed. The utility of this information is significant to the forensic toxicologist. The results of this study suggest that a whole blood sample analyzed after exposure to elevated temperature may have had, originally, a higher BAC. PMID- 8635762 TI - Risk of dying after a free fall from height. AB - Falls from height are predominantly an urban phenomenon and represent an important form of blunt trauma. Disagreement predominates regarding the height at which death results. The aim of this study was to investigate the risk of dying after a free fall from height in relation to the distance fallen. Therefore, medical records of victims of a fall from height treated in 1989 at Viennese emergency units were analysed. In addition, post-mortem reports of deaths due to falls from height, examined in the same year at the Institute of Forensic Medicine in Vienna, were studied. For the purpose of an homogenous investigation sample in regard to physical condition, only people aged from 20 to 50 years were taken into account. A total of 11 females and 30 males suffered an accidental fall from buildings, seven men from scaffolding and two men from a tree. A total of 18 females and 18 males jumped from residential buildings. One woman and nine men intentionally fell from other buildings. All victims landed on concrete or pavement. Suicidal jumps occurred from significantly higher places than accidental falls. The results of this analysis suggest that death usually occurs when distance is more than five storeys. PMID- 8635763 TI - Case report. Delayed death after pressure on the neck: possible causal mechanisms and implications for mode of death in manual strangulation discussed. AB - Death from hypoxic cerebral damage 1 week after manual strangulation is described, with a discussion of the competing pathophysiological mechanisms responsible for the fatal outcome in this case, and in manual strangulation in general. PMID- 8635764 TI - Postmortem diffusion of ingested and aspirated paint thinner. AB - Post mortem diffusion of paint thinner (toluene/ethyl acetate/isobutanol 8:1:1 v/v) from gastric residue (25 ml or 100 ml) and airways contamination (25 ml) was assessed in a human cadaver model, with sampling after 24 h at room temperature. Four torso blood samples showed less toluene diffusion after gastric instillation (0.5-3.8 micrograms/ml) than after tracheal instillation (10.5-421 micrograms/ml). Isobutanol diffused more readily than toluene with four torso blood samples 1.8-256 micrograms/ml after gastric instillation and 26-576 micrograms/ml after tracheal instillation. Following 25 ml gastric instillation, toluene concentrations (microgram/ml or microgram/mg) were: pericardial fluid 0.7 4.0; bile 0.5-0.6; urine 0-0.6; brainstem 1.1; lung 0.4-4.4; liver 0-162; spleen 0.6-0.7; kidneys 0.4-0.6; peri-renal fat 0.3-30.3; psoas muscle 0.3-0.8; concentrations of toluene and isobutanol were markedly higher in the left lobe of the liver than the right. Ethyl acetate was mostly undetectable in tissue samples but variably present in five blood samples: 0-21.2 micrograms/ml following 25 ml or 100 ml gastric instillation and 0-198 micrograms/ml following 25 ml tracheal instillation. Ethyl acetate was always detectable in pericardial fluid but not always detectable in gastric contents. We conclude that post mortem diffusion of toluene from gastric residue or airways contamination is unlikely to compromise the analytical validity of femoral venous blood samples, brain, or liver from deep within the right lobe. Analysis of pericardial fluid and gastric contents allows identification of ethyl acetate and isobutanol thus implicating thinner solution. PMID- 8635766 TI - Anaesthetic-associated mortality and anaesthetic contributory death: the South African medicolegal approach. AB - The medicolegal approach to cases of anaesthetic-associated mortality and anaesthetic contributory death (ACD) occurring in Johannesburg, South Africa is presented. The approach adopted has been found to be reasonably workable in practice and places heavy emphasis on the surgical and anaesthetic-related reasons for perioperative deaths and, in particular, on their multifactorial aetiology. It is recognised that in view of the multifactorial nature of the anaesthetic environment certain errors in clinical anaesthetic management are neither precisely quantifiable nor assessable. It is further emphasized that perioperative quality control programs as well as Inquest Court proceedings should comprise input by a team of specialists and which include clinical anaesthesiologists, surgeons and forensic pathologists. PMID- 8635765 TI - Immunohistochemical study of fibronectin for postmortem diagnosis of early myocardial infarction. AB - The postmortem diagnosis of early myocardial infarction has been a puzzling problem in forensic practice. In the present study, an immunohistochemical study of fibronectin (FN) was performed for the first time on 34 autopsy hearts to determine early myocardial infarction with streptavidin/biotin/peroxidase technique. Five cases of definite myocardial infarction showed positive FN staining of cardiomyocytes; of 18 cases where early myocardial infarction was suspected, positive FN staining of cardiomyocytes was found in 15 cases, but no such staining was seen in 11 non-cardiac death controls. The results led to the conclusion that positive FN staining in cardiomyocytes is a reliable marker of acute myocardial infarction and could be used as a new, sensitive method for the postmortem diagnosis of early myocardial infarction. It is worth noting that all cases in this study were autopsied between 8 h and 4 days after death and 5 cases had been fixed in 10% formalin for over 10 years. FN immunohistochemistry still gave satisfactory results in those cases. It seemed that FN was not affected by postmortem autolysis and formalin-fixation and could be used in routine forensic practice, especially for retrospective analysis of cases. PMID- 8635767 TI - Statistical issues in comparing random DNA samples. PMID- 8635768 TI - Stressful events as a trigger of sudden death: a study of 43 medico-legal autopsy cases. AB - The reports relating emotional stress to sudden death are largely anecdotal. In addition to experimental and electrophysiological studies, an opportunity for a better understanding of possible stress-related sudden death (SSD) may be provided by medicolegal autopsies. The goal of our autopsy study was to analyze cardiovascular pathologic findings in cases of SSD and if possible identify mechanisms by which the stressful event (SE) could be the cause. Forty three cases were studied (29 males and 14 females). In all cases, the SE and the death were witnessed. The age range was 22 to 90 years in males (mean, 52) and 30 to 92 years in females (mean, 64). Death occurred in all cases without premonitory symptoms. In 20 cases, death occurred during the SE and in the other 23 cases occurred within 2 h of the event. SE included fear, 15 cases; altercation, 21 cases; sexual activity, 3 cases; police questioning or arrest, 4 cases. According to police reports, in 40 cases (90%), the victims had no previous clinical history of cardiovascular disease. At autopsy, the heart weight in males ranged from 255 to 1000 g with a mean of 517 g and in females the range was 250-700 g with a mean of 417 g. In only 3 cases, gross and microscopic examination of the heart was normal. In 2 of the remaining 40 cases the subjects died of subarachnoid hemorrhage. In 38 cases, a cardiac cause of death was found as follows: coronary heart disease, 27 cases; cardiomyopathy, 6 cases; aortic valvular stenosis, 2 cases and right ventricular dysplasia, 3 cases. A coronary artery thrombosis was found in 8 cases of sudden coronary death. Post myocardial infarction fibrosis was present in 25 cases (92%) of sudden coronary death. In conclusion, it appears from our autopsy study that SSD occurs primarily in those individuals with severe heart disease, especially coronary heart disease. PMID- 8635769 TI - Sudden infant death in a tropical environment: Singapore's experience. AB - A total of 206 cases of sudden infant deaths examined at the Institute of Science and Forensic Medicine, Singapore, over a 5 year period (1989-93) were identified to assess the pattern of sudden death in this age group, which was subdivided into the neonatal and post-neonatal periods. A total of 34% (70) of infant deaths occurred in neonatal life and the remaining 66% (136) in the post-neonatal period; 90% of the neonatal deaths were natural, of which over half were due to congenital heart disease and complications of prematurity. Unnatural deaths in this period were uncommon, there being only seven such deaths. In the post neonatal period, unnatural deaths constituted 25% of the total with trauma and aspiration heading the list. Natural deaths in the post-neonatal period are predominantly due to infections (34%) and a group of sudden natural deaths with minimal findings (31%). The latter group may arguably represent cases of Sudden Infant Death Syndrome (SIDS). The yearly incidence of this group in our study varied between 0.08 to 0.2 per 1000 live births, which is considerably lower than the incidence quoted for Western populations. The criteria for the classification and the impact of sudden infant deaths in Asian countries are discussed. PMID- 8635770 TI - Study of three AMPFLPs (D1S80, 3'ApoB and YNZ22) in the population of the north of Portugal. AB - Allele and genotype frequencies for D1S80, 3'ApoB and YNZ22 loci have been determined in a population sample of the North of Portugal using the polymerase chain reaction (PCR) amplification and nonradioactive detection. The distribution of genotypes in the three polymorphisms studied is in agreement with expected values according to the Hardy-Weinberg equilibrium. The combined chance of exclusion for the three systems is 0.96, and the combined power of discrimination is 0.99. PMID- 8635771 TI - Comparison of methaqualone excretion patterns using Abuscreen ONLINE and EMIT II immunoassays and GC/MS. AB - A study was performed to compare the ONLINE and EMIT II immunoassays with gas chromatographic/mass spectrometric (GC/MS) analysis of methaqualone metabolites on urine using samples obtained from a clinical study. Urine was collected over a 72 h period from six healthy adults (4 male, 2 female) after oral dosing with 200 mg methaqualone (MTQ). Each urine sample was analyzed by ONLINE and EMIT II. The samples were then analyzed by GC/MS, hydrolyzed with beta-glucuronidase and again analyzed by GC/MS. Both immunoassays showed greater than 600 ng/ml concentrations of drug in each sample by the second void and remained highly positive for the rest of the 72 h. Unhydrolyzed samples analyzed by GC/MS showed both low concentrations of MTQ as well as its five major hydroxylated metabolites. The hydrolyzed samples analyzed by GC/MS showed high concentrations of the hydroxylated metabolites with the 2'-hydroxy and 3'-hydroxy metabolites being present at the highest concentrations, the 4'-hydroxy metabolite at a lower amount and the 6-hydroxy and 2-hydroxy metabolites at the lowest concentrations. The GC/MS data coupled with the antibody cross-reactivity data indicate that the major species in clinical samples that cross-react in both immunoassays are the conjugated forms of the hydroxylated metabolites of MTQ. Therefore when confirming by GC/MS after an immunoassay screen it would be prudent to confirm for the major hydroxylated metabolites as glucuronides of MTQ instead of the parent drug. PMID- 8635772 TI - Validation of a frequency database for four STR loci for use in casework in the Strathclyde Police Forensic Science Laboratory. AB - Data for four STR loci have been collected from 400 samples taken from complainers and suspects encountered in casework at the Strathclyde Police Forensic Science Laboratory (SPFSL). This paper describes statistical testing which demonstrates that its use will provide operationally robust procedures. Comparisons made with data collected from other British samples confirmed no practical differences between the different frequency distributions. This work provides further confirmation of the reliability of the so-called "product rule' in estimating the frequency of multilocus genotypes in British forensic casework. PMID- 8635773 TI - Alcohol and drugs (medical and illicit) in fatal road accidents in a city of 300,000 inhabitants. AB - During a 1-year period all fatal road accidents in the police district of Aarhus, Denmark, were investigated regarding the presence of alcohol, medical drugs or narcotic substances, and the nature of the accident. Out of a total of 30 accidents 24 were investigated. Two thirds had been caused by the victim him- or herself. In one third of the accidents alcohol was present and considered an important contributory factor. Medical drugs and narcotic substances played a lesser role. PMID- 8635775 TI - Pediatric intensive care: concept and issues. PMID- 8635774 TI - Increased risk of suicide with exposure to pesticides in an intensive agricultural area. A 12-year retrospective study. AB - Several reports have suggested that exposure to agricultural pesticides (mainly chronic exposure to organophosphates) produces depression, and depression is a major risk factor for suicide. A retrospective epidemiological study of 251 suicide cases was undertaken to explore the possible relationship between the high suicide rates in an intensive agricultural area, and a specific group of population at risk, namely farmers with chronic exposure to pesticides, who are at risk to develop mood disorders (mainly depression). Our data show that the suicide rate in that area is significantly higher than the suicide rates from other geographic areas with very similar socioeconomic and demographic features. In addition, the mortality from suicide in this population (farmers) does differ significantly from that of the rest of the population. PMID- 8635776 TI - IAP policy on infant feeding. PMID- 8635778 TI - Antenatal period: an educational opportunity. AB - In this study the impact of an educational programme during antenatal period was evaluated. Pregnant women attending the antenatal clinic formed the study material. The first 100 mothers who were not given health education served as controls. The subsequent 201 cases constituted the study group and were given health education on certain aspects of maternal and child care. The control and study groups were well matched for age, parity, education, income and number of antenatal visits. The results indicated that the mothers in study group gained statistically significant knowledge regarding the purpose of antenatal care, hematinics and tetanus toxoid vaccination. The awareness regarding breast feeding and its advantages also increased significantly in the study group. The knowledge about individual vaccine especially measles and DPT was poor which increased significantly after the educational intervention in the study group. It is recommended that the antenatal period should be optimally utilized to impart health education on the various aspects of maternal and child health. PMID- 8635777 TI - Parenteral nutrition (PN) in the management of very low birth weight (VLBW) babies--a randomized controlled trial. AB - Eighty five very low birth weight (VLBW) babies with birthweight less than 1250 g were randomly assigned such that 43 received parenteral nutrition (PN) with amino acid based glucose electrolyte solution (Vamin) and lipid emulsion (Intralipid) in the first 16 days of life. The other 42 (control group) received conventional intravenous dextrose with or without electrolytes plus enteral milk regimen. Baseline clinical parameters and neonatal problems encountered in the two groups were similar. There was no significant difference in the mortality rate in the two groups (48.9% in PN group and 42.9% in control group: X2 = 0.3, p > 0.05). The commonest cause of mortality in both the groups was septicemia (16.3% and 26.1% in PN and control groups, respectively). Local complications, sepsis and fluid electrolyte disturbances were similar in the two groups. Azotemia (25.6%), hyperlipidemia (9.3%), metabolic acidosis (9.3%) and prolonged cholestasis (14%) were commoner in the PN group but were reversible with early recognition. Time taken to regain birthweight was also similar in the two groups (X2 = 14.2 and 15.2 days for PN and control groups, respectively). Thus, PN failed to improve the survival or early weight gain in the routine management of the VLBW babies in our unit. PMID- 8635779 TI - Immunoglobulin G and complement C3 levels in pregnancy induced hypertension. AB - The immunoglobulin G (IgG) and complement C3 (C3) were measured in the maternal as well cord blood sera of 30 cases of pregnancy induced hypertension (PIH) as well as 9 controls with normotensive pregnancy. A depression of IgG as well as C3 level was observed in the maternal as well as cord sera of the mothers with PIH. These findings suggest decreased immunological status of both mother and her offspring in PIH, irrespective of the gestation and intrauterine growth status. PMID- 8635780 TI - Lung function tests in normal Indian children. AB - The forced vital capacity, forced expiratory volume in one second, peak expiratory flow, mid-expiratory flow and maximum voluntary ventilation was measured in 632 healthy, normal children from Metropolitan city of Bombay using computerized spirometer. The children were between age range 6 years to 15 years and belong to high or middle and lower socio economic status. The pulmonary function data was separated by sex, and classified on the basis of height and age. The mean and standard deviation for was calculated for every such variable. The lung function variables show a linear positive correlation with height and age. Forced vital capacity and one second forced expiratory volume show a spurt after height 150 cm. Boys show higher values for lung function variables than girls except for mid expiratory flow rates where girls have higher values than boys over height 140 cm and age 9 yrs. Stepwise regression equation was calculated using height, age and weight as independent variables. Height explained the maximum variance in lung function parameters. Use of logarithmic equations for age, weight do not improve the degree of correlation. Hence, for clinical evaluation of child's lung function, height is the most significant independent parameter in comparison to age and weight. PMID- 8635781 TI - Effects of protein energy malnutrition on circulating thyroid hormones. AB - The effect of protein energy malnutrition (PEM) in the children on serum levels of total thyroxine (TT4), total triodothyronine (TT3) and thyrotropin (TSH) were evaluated. There were 107 children aged 2 to 60 months in the malnutrition group and 54 healthy age and sex matched controls. Serum TT4 and TT3 were all reduced in the malnutrition group. This decrease in TT3 was more significant (p < 0.01) in severe malnutrition than in mild PEM. Serum TSH levels in the malnutrition and control groups were similar. These results suggest that the children remained euthyroid and represent an adaptive response to protein energy malnutrition. PMID- 8635782 TI - Risk factors of nutritional blindness and determinants of a successful vitamin A prophylaxis programme. AB - Data on 7668 children (0-72 months) and their 4621 mothers and 81 Anganwadi Workers (AWWs) collected for the USAID Assisted ICDS Evaluation Surveys were analysed. The results indicated that the major risk factors of nutritional blindness were lack of nutrition and health knowledge among mothers; presence of iron deficiency anemia in the children; and history of the child having had measles in the past one year. Mother's health and nutrition, knowledge and maternal literacy status were the determinants of the success of a vitamin A prophylaxis programme. Factors that determined AWW's performance in vitamin A supplementation were her nutrition and health knowledge, her literacy status and the amount of supervisory assistance she received from Auxillary Nurse Midwife (ANM). PMID- 8635783 TI - Respiratory distress in newborn: treated with ventilation in a level II nursery. AB - Fifty consecutive neonates with respiratory distress persisting beyond 6 h of age were studied during a 18 month period (total deliveries 2000/y). Twenty two neonates were managed with oxygen hood with increasing oxygen concentration, 28 with continuous positive airway pressure (CPAP) ventilation using a nasal cannula. Of these babies on CPAP, 10 were shifted to intermittent positive pressure ventilation (IPPV) on a pressure limited, time cycled ventilator (Neovent, Vickers). Babies were monitored with continuous hemoglobin oxygen saturation (SaO2), hourly blood pressure and vital charting. Radial arterial blood gas analysis (ABG) was done when feasible and especially on clinical deterioration. Oxygen (FiO2 0.95) from an oxygen concentrator was used as a source of continuous supply of oxygen. Commonest cause of respiratory distress was hyaline membrane disease (18%), followed by wet lung syndromes (14%), meconium aspiration (12%), asphyxia (12%) and septicemia (8%). In 8 babies, a lung biopsy (postmortem) was done to confirm the diagnosis. Nineteen of the 50 babies with respiratory distress died, there was a survival of 50% on CPAP and 30% on IPPV. No case of oxygen toxicity or other major complications was encountered. Even with moderate resources, neonatal ventilation in a Level II nursery is a challenging task. Babies less than 1000g require aggressive measures which is not very economical in a special care baby unit (SCBU). PMID- 8635784 TI - Assessment of thyroid functions and its role in body growth in thalassemia major. AB - The present study was done to establish the role of thyroid gland in causing growth retardation in regularly transfused thalassemic children. Growth, skeletal maturation and thyroid functions were assessed in 25 patients of thalassemia major in the age range of 5-17 years (mean age 10.3 +/ 3.6 years). Thirteen patients were migrants from Pakistan and 12 were of Indian origin. Twenty-five age and sex matched children who were not anemic served as controls. Thalassemic children received multiple blood transfusions ranging from 36-350 units with a mean of 168.4 +/ 98.9 (+/ 1 SD). The mean pretransfusion hemoglobin was 8.7 +/ 1.6 g/dl. Twenty eight per cent patients were below the 5th percentile for height and another 24% between 5th and 10th percentiles. The height age retardation was more pronounced than bone age retardation. The mean serum total T3 and T4 levels were significantly lower (p < 0.001) and the mean serum TSH levels were significantly higher (p < 0.005) in patients with thalassemia major as compared to the controls. Eight patients had high TSH levels; of these 5 had compensated primary subclinical hypothyroidism (elevated TSH with normal T3 and T4) and 3 had uncompensated primary sub-clinical hypothyroidism (elevated TSH, low T4 and normal T3). Two patients had low T4 with normal T3 and TSH levels. Thyroid dysfunction was not related to age, sex, hemoglobin levels and country of origin but transfused iron load (units/kg, units/year) was significantly higher in patients with hypothyroid function compared to those with euthyroid function (p < 0.005). Height age, weight age and bone age retardations were more pronounced in patients with hypothyroid function; however, the difference was not statistically significant. It is concluded that hypothyroidism is unlikely to be the sole cause of growth retardation; however, it may have a potentiating or permissive role. The strong association of high transfused iron load and decreased thyroid function stresses the need for intensive chelation therapy. PMID- 8635785 TI - Subclinical vitamin A deficiency in infants and young children. PMID- 8635786 TI - Infantile myofibromatosis. PMID- 8635787 TI - Prevalence of intestinal parasites in children of the orphanage in Sivas, Turkey. PMID- 8635788 TI - Wolman's disease. PMID- 8635789 TI - Tuberous sclerosis with a brain tumor. PMID- 8635790 TI - Effect of myleran therapy in multitransfused thalassemic children. PMID- 8635791 TI - Mixed infection with Plasmodium-vivax and Salmonella typhi in an infant. PMID- 8635792 TI - Present status of hemoglobinopathies in India. PMID- 8635793 TI - Propranolol in supraventricular tachycardia. PMID- 8635794 TI - Changing trend in decision makers. PMID- 8635795 TI - Use of warm air dryers in neonatal intensive care units. PMID- 8635796 TI - ORS: controversies and perspectives. PMID- 8635798 TI - Typhoid vaccine. PMID- 8635799 TI - Randomized trial of SPf 66 P. falciparum vaccine in children. PMID- 8635797 TI - Bile ascites. PMID- 8635800 TI - IVIG, anti-D or oral prednisolone in acute ITP? PMID- 8635801 TI - Does the baby find the nipple by smell? PMID- 8635802 TI - Child labor in India: present status. PMID- 8635803 TI - Neuromuscular manifestations of diarrhea related hypokalemia. AB - Twenty-three children from 8-60 months (mean age 21.13 months) admitted with neuromuscular manifestations of diarrhea related hypokalemia were studied. Forty four per cent cases were suffering from diarrhea at the time of admission but in majority of cases (56%), the diarrheal episode had already terminated. Mild hypokalemia was seen in 17.4%, moderate in 43.5% and severe in 39.1%. Neck flop was the commonest (100%) neuromuscular manifestations followed by diminished bowel sounds (82.6%), truncal weakness (52.2%), weakness of limbs (52.2%), lethargy (43. 5%), abdominal distension (43.5%), respiratory involvement (4.3%) and phantom hernia (4.3%). Two cases (8.7%) had flaccid paralysis of both the lower limbs. Severe hypokalemia was more frequently observed in children below 24 months of age and those who had received i.v. fluids or salt sugar solution before reporting in the hospital. A significant correlation was noticed between severity of hypokalemia and frequency of stools (p < 0.05), degree of dehydration (p < 0.01), severity of nutrition (p < 0.01) and extent of neuromuscular involvement (p < 0.01). Our results highlights the importance of diarrhea related hypokalemia particularly in young malnourished children who are rehydrated with solutions inadequate in potassium. Early diagnosis and appropriate treatment can promptly reverse these manifestations within 48-72 hours. PMID- 8635804 TI - Maternal care receptivity and its relation to perinatal and neonatal mortality. A rural study. AB - A longitudinal study was conducted on 212 pregnant women from May 1987 to April 1988. Maternal Care Receptivity (MCR) "an innovative approach" was adopted for the assessment of maternal care services provided to pregnant mothers at their door steps. During follow-up, scores were allotted to each of the services rendered and antenatal status of pregnant women. Depending on the score--MCR was classified as high (11 to 8), moderate (7 to 4) or poor (3 to 0). Perinatal and neonatal deaths were recorded and an inverse relationship between MCR and perinatal and mortalities was observed (z = 5.46, p < 0.0001). Significantly, no perinatal or neonatal deaths occurred in women with high MCR. One of the most important cause of high PNMR and neonatal mortality rate in developing countries is poor MCR, i.e., under utilization of even the existing maternal health services. The main reasons for this under utilization appear to be poverty, illiteracy, ignorance and lack of faith in modern medicine. PMID- 8635805 TI - Biochemical abnormalities in neonatal seizures. AB - Early diagnosis and appropriate treatment of biochemical abnormalities accompanying neonatal seizures is important for effective seizure control and to avoid further brain damage. The present study was carried out on 35 neonates to determine the frequency of various biochemical abnormalities in neonatal seizures. Diagnostic evaluation included estimation of levels of serum calcium, phosphorus, magnesium, sodium, potassium, zinc, and blood glucose. Two-thirds of the neonates with seizures had biochemical disturbances in their sera. A variety of abnormalities occurred in asphyxiated infants, including hyponatremia, hypoglycemia, hypocalcemia, and hypomagnesemia. Primary metabolic disorders accounted for one-forth of the cases of neonatal seizures, the most common being hypoglycemia, hypoglycemia/hypocalcemia, and hypocalcemia/hyperphosphatemia. Inappropriate intrauterine growth, inadequate feeding, and feeding with cow's milk were the main risk factors for primary metabolic seizures. Hyponatremia was a frequent finding in seizures resulting from brain damage like birth asphyxia, meningitis, and intracranial hemorrhage. No infant had hypernatremia, hyperkalemia, hypokalemia, or low serum zinc. PMID- 8635807 TI - A clinical and cytogenetic study of Turner syndrome. AB - Forty five case of Turner syndrome diagnosed in the Genetics Clinic, between January 1986 and December 1993, were analyzed. The most commonly observed karyotype was 45, X (44.4%), followed by 45, X/46, XX mosaicism (24.4%). Less frequently demonstrated karyotypes were 45, X/46, X, i (Xq) mosaicism and 46, X, i (Xq) (13.3%). Mosaicism for chromosome was seen in 6.7% of patients. Patients with 45, X karyotype had short stature (85%), dysmorphic facies (60%), delayed appearance of secondary sexual characters (100%) and primary amennorhea (100%). Those with 45, X/46, XX mosaicism were less often dysmorphic and presented with either primary or secondary amenorrhea. Patients with 45, X karyotype were younger at diagnosis and had a significantly shorter mean adult height than those with 45, X/46, XX mosaicism. The phenotype in patients with other karyotypic abnormalities was similar to the 45, X group. Short stature and primary or secondary amenorrhea occurring together in a female strongly suggests the possibility of Turner syndrome, which should be confirmed by chromosomal analysis. PMID- 8635809 TI - Ultrasonic diagnosis of intracranial hemorrhage in high risk neonates. AB - One hundred and eleven high risk neonates were subjected to (cranial ultrasound (CR-USG) Caranial sonography was performed by 2D realtime scanner with 5 MHz transducer through anterior and posterior fontanelle and temporo-squamal suture. One quarter of these neonates developed intracranial hemorrhage (ICH) within 120 hours of birth. Of them 42.8% neonates recovered completely, 21.4% developed ventriculomegaly, 21.4% neonates expired, 10.4% developed pseudo-porencephalic cysts and 3.5% developed aqueductal block. It is concluded that CR-USG is a useful technique for detection and monitoring of complications of ICH and at least one screening sonogram is essential in the first week of life of all high risk neonates. PMID- 8635806 TI - Attitudes of future mothers regarding infant feeding. AB - Attitudes of 2500 urban college girls from Ludhiana city, aged 15 to 20 years were assessed towards infant feeding using a standardized Likert Scale (r = 0.82). The influence of subject opted in college and the presence of a child less than 3 years in the family on the attitudes was also studied. The overall attitudes of the whole group towards infant feeding were negative. Science students had better attitude scores on most of the themes tested (p < 0.05). The presence of a child aged less than 3 years in the family had no significant influence on the attitudes of girls towards infant feeding. Most of the girls were desirous of studying the concepts of infant feeding in their college curriculum. It is recommended that efforts should be made to incorporate these concepts in the formal educational curriculum so that girls grow up with positive attitude towards infant feeding. PMID- 8635808 TI - A comprehensive index for longitudinal monitoring of child health status. AB - The aim of this study was to develop a single comprehensive index of child mortality for longitudinal assessment of health status of children. The need for such a comprehensive index arose from conflicting trends in different child mortality indicators. The data for the study was taken from the Sample Registration System (SRS) reports of the Registrar General of India. SRS is known to provide reliable estimates of births and deaths at the State and the National level. The study included five child mortality indicators, namely, under five mortality rate (U5MR), infant mortality rate, neonatal mortality rate, perinatal mortality rate and still birth rate. These were available for fifteen states of India over the years 1972-1988. To develop this index we modified an earlier method based on factor analysis. Factor analysis of data on various indicators of child mortality revealed two factors which together explained 78% to 93% of the total variation in different years. The first factor was identified as representing mortality after birth and the second as before and during birth. The comprehensive index was obtained as a linear combination of these two factors. The resultant index thus fairly represented all five mortality indicators and provided a comprehensive and reasonably correct picture of child mortality. The lower the magnitude of this index, the better was the child health status. Trends in the index showed that the highest decline in the magnitude was in the state of Kerala followed by Punjab, Andhra Pradesh, Gujarat and Maharashtra in that order. This indicates steady improvement of the child health status over years in these states. In the State of Jammu and Kashmir, the index remained more or less constant over the years though the magnitude was low in the cross-sectional comparison with other states. Thus the comprehensive index developed by using factor analysis of the various mortality indicators can be used for the longitudinal monitoring of child health status in the states of India. PMID- 8635810 TI - Radiological findings in moyamoya disease. AB - The clinical, angiographic and computed tomographic features in eight children with Moyamoya disease were evaluated. The CT Scan findings were correlated with the angiographic features and the stage of the vascular disease. Stenosis/occlusion of the supraclinoid internal carotid artery (ICA) and the proximal parts of the anterior (ACA) and middle cerebral arteries (MCA) were commonest angiographic findings. The cervical ICA was narrow in four patients. Infarcts (100%), abnormal enhancement patterns (63%) and cerebral atrophy (88%) were the frequent CT scan findings. Although the CT scan findings did not correlate entirely with the angiographic and clinical findings, they were more frequently abnormal in later stages of the disease. The volume of Moyamoya increased progressively up to stage 3 of the disease only to decrease with further progression. The etiology of Moyamoya disease in Indian children is not clear. However, the clinical and radiological features are similar to that in the Japanese. PMID- 8635811 TI - Chronic pancreatitis in homocystinuria. PMID- 8635812 TI - Melnick Needles syndrome. PMID- 8635814 TI - Complicated anophthalmos. PMID- 8635813 TI - Neonatal herpes simplex virus-2 encephalitis. PMID- 8635815 TI - Hallervorden-Spatz disease. PMID- 8635816 TI - Oral acyclovir in treatment of suspected herpes simplex encephalitis. PMID- 8635817 TI - Successful management of breast abscess with ongoing breastfeeding. PMID- 8635819 TI - Macromastia in adolescent girls. PMID- 8635818 TI - Remediable recurrent meningitis. PMID- 8635820 TI - Breath holding spells in a very young infant. PMID- 8635821 TI - Histiocytosis X presenting as chronic discharging ears. PMID- 8635822 TI - Chronic gastric volvulus--an unusual complication of poliomyelitis. PMID- 8635823 TI - Why do hemophiliacs bleed? PMID- 8635824 TI - Pulse polio--should we be giving it? PMID- 8635825 TI - Surfactant therapy and nasal CPAP for newborns with RDS. PMID- 8635826 TI - Oral iron chelators. PMID- 8635828 TI - Effect of oral water soluble vitamin K on PIVKA-II levels in newborns. AB - Intramuscular administration of vitamin K for prophylaxis against hemorrhagic disease of the newborn has the disadvantage of increased cost, pain, anxiety to parents and risk of transmission of infection. Oral route is a better alternative. Oral absorption of vitamin K has been shown to be equally good using special oral preparations. However, this preparation is not available in India. A prospective study was carried out on 51 full term, healthy breastfed newborns to evaluate if the injectable water soluble preparation of vitamin K (menadione sodium bisulphite) could be as effective. Fourteen babies received 1 mg vitamin K intramuscularly, 24 received 2 mg vitamin K orally while 13 controls did not receive vitamin K at birth. PIVKA-II levels were measured in cord blood and at 72 78 hours of age in all babies as a marker of vitamin K deficiency. The overall PIVKA-II prevalence in cord blood was 64.7%. At 72-78 hours, PIVKA-II was present in 50% of babies in IM group, 58.3% of babies in oral group and in 76.9% of babies in 'no vitamin K' group (p > 0.05). The PIVKA-II levels decreased or did not change at 72-78 hours in 91.6% of babies in oral group versus 92.8% of babies in IM group (p > 0.05). On the other hand, PIVKA-II levels increased in 30.7% of babies who did not receive vitamin K as against in 7.8% of babies receiving vitamin K in either form (p < 0.05). Hence, vitamin K prophylaxis is required for all newborns at birth and injectable vitamin K (menadione sodium bisulphite) given orally to term healthy babies is effective in preventing vitamin K deficiency state. PMID- 8635827 TI - Efficacy and safety of oral iron chelating agent deferiprone in beta-thalassemia and hemoglobin E-beta thalassemia. AB - OBJECTIVES: To assess efficacy and safety of oral iron chelating agent deferiprone (DFP) in patients with beta thalassemia and hemoglobin E-beta thalassemia. DESIGN: Non-randomized study. SETTING: Hematology Out-Patient Department. SUBJECTS: Forty-one patients of beta thalassemia and hemoglobin E beta thalassemia. INTERVENTIONS: DFP was given to 20 patients, 10 patients of beta thalassemia and 10 with hemoglobin E-beta thalassemia; the rest were taken as controls. RESULTS: A significant fall in serum ferritin was observed in the study group along with rise in urinary iron excretion (p < 0.05). Adverse effects of DFP were nausea and vomiting (30%), significant arthropathy requiring stopping of the drug (30%), and reversible neutropenia in one patient. All these complications could be managed easily with medical supervision and no death or permanent disability was seen. CONCLUSIONS: DFP is an effective and fairly well tolerated oral iron chelating agent. The side effects that occur can be tackled easily if monitored properly. PMID- 8635829 TI - Intellectual functions in childhood malignant disorders. AB - Our study was designed to compare the intellectual functions in 35 children with lymphoreticular malignancies (ALL or NHL) who had received CNS directed therapy (Group A), with those in 21 patients with solid tumors (Group B). Intellectual assessment was done using the Malin's modification for Indian children of the Wechsler's intelligence scale. Using 5 verbal and performance subscales each, the verbal IQ, performance IQ and full scale IQ were derived. The mean VIQ, PIQ and FIQ were comparable in the two groups with the differences not being statistically significant. However, the dispersion of IQ scores was greater in Group A with a larger number of subjects having scores of < 80. Similarly, the scores obtained in the arithmetic, digit-span, picture completion and block design subscales were lower in Group A. Mean IQ scores were significantly lower in children over the age of 10 years at diagnosis. Sex duration since diagnosis, disease free survival and treatment variables did not affect IQ scores. In conclusion, a cross-sectional evaluation of intellectual functions has revealed only minimal differences in children treated with chemotherapy and CNS directed therapy (ITMTX and RT) in comparison to those treated with chemotherapy alone. PMID- 8635830 TI - Impact of nutritional supplements on hematological profile of pregnant women. AB - Eighty one pregnant women (20 +/- 2 weeks of gestation) were assessed for their nutritional status. They were divided into three groups; Group I women (n = 30) were given 200 mg ferrous sulphate (FeSO4) tablet per day for 15 weeks, Group II women (n = 25) were given FeSO4 tablets along with 2,000,000 IU of vitamin A as single dose in beginning of study; and Group III women (n = 26) were not given any supplements and served as controls. To study impact of supplements on hematological profile of pregnant women, Hb, PCV, RBC, TS%, TIBC and serum levels of iron, zinc, copper, manganese and vitamin A were assessed at 20 +/- 2 weeks and 36 +/- 2 weeks of gestation by standard techniques. Pregnant women in Group I (Fe suppl) and group II (Fe + vitamin A suppl) had significantly (p < 0.01) higher Hb, PCV, RBC, TS% and serum iron levels than the controls. Group II had significantly (p < 0.05) higher values of these indices as compared to Group I. Levels of serum zinc, copper, manganese were not affected by supplements. Iron supplements improved the hematological profile of pregnant mothers but Fe + vitamin A supplements were more beneficial. PMID- 8635831 TI - An outbreak of multidrug resistant. Salmonella typhimurium in a nursery. AB - A nursery epidemic caused by multidrug resistant Salmonella typhimurium is reported. In total, 21 infants developed symptomatic illness; of these, 17 had septicemia (7 blood culture positive) and 4 had diarrhea alone. Asymptomatic carrier state was identified in 13 infants. Male sex and birth asphyxia increased the risk for symptomatic illness. Fever, lethargy, and diarrhea were the most common clinical features. Amongst the septicemic infants there was no difference in clinical profile whether the blood culture was positive or negative for S. typhimurium. In the symptomatic group, S. typhimurium was isolated from feces in 19 cases and from blood in 7 cases. In both symptomatic and asymptomatic infants, all isolates of S. typhimurium, whether obtained from feces and/or from blood, were resistant to ampicillin, chloramphenicol, and trimethoprim, and a significant number (almost one-fifth) of them also showed resistance to third generation cephalosporins. More than 90% of isolates were sensitive to aminoglycosides and ciprofloxacin. On a combination of third generation cephalosporin (cefotaxime or ceftriaxone) and amikacin, 17 (81%) infants recovered, 2 succumbed to their illness, and 2 failed to improve and required ciprofloxacin. The origin of epidemic was traced to a carrier staff nurse working in nursery. PMID- 8635832 TI - Cerebrospinal fluid adenosine deaminase activity and C-reactive protein in tuberculous and partially treated bacterial meningitis. AB - Adenosine deaminase (ADA) activity measurement and C-reactive protein (C-RP) detection were done in CSF of 27 tuberculous meningitis (TBM) and 8 patients of partially treated bacterial meningitis, apart from routine biochemical tests. Both the groups had comparable CSF cell count, protein and sugar concentrations. The mean CSF ADA activity was significantly raised in TBM as compared to partially treated bacterial meningitis patients (p < 0.05). A cut-off ADA level < or = 5 IU/L and C-RP positively were used for differentiation of partially treated bacterial from TBM cases. Based on this, the sensitivity and specificity of ADA and C-RP were 62.5%, 88.9% and 75%, 100%, respectively. Since both the tests are simple and take lesser time to perform, they can be used as rapid diagnostic tests to remove diagnostic dilemma between the two diseases. PMID- 8635834 TI - Ondansetron. PMID- 8635833 TI - Airway foreign bodies in children. PMID- 8635835 TI - Initiation of breastfeeding in cesarean section mothers: antenatal advise versus postnatal assistance. PMID- 8635836 TI - Use of milk based commercial weaning foods amongst scheduled caste communities in Haryana. PMID- 8635837 TI - Maternal beliefs regarding diet during common childhood illnesses. PMID- 8635838 TI - The disadvantaged girl child in Bihar: study of health care practices and selected nutritional indices. PMID- 8635839 TI - Proptosis as a manifestation of cysticercosis. PMID- 8635840 TI - Nightmare due to ciprofloxacin in young patients. PMID- 8635841 TI - Malignant acanthosis nigricans in adrenal carcinoma. PMID- 8635842 TI - Weill-Marchesani syndrome. PMID- 8635843 TI - Large arachnoid cyst. PMID- 8635844 TI - Aberrant pancreatic tissue causing gastric ulcer and pyloric obstruction. PMID- 8635845 TI - Knowledge of Anganwadi workers about breastfeeding. PMID- 8635846 TI - Breast feeding practices. PMID- 8635847 TI - Alternatives to INH chemoprophylaxis. PMID- 8635848 TI - Transmission of Salmonella. PMID- 8635850 TI - Pulse immunization against poliomyelitis in India. PMID- 8635849 TI - Pulse polio immunization. PMID- 8635851 TI - Chemotherapy for tuberculosis. American Thoracic Society. PMID- 8635852 TI - Chemotherapy for tuberculosis. PMID- 8635853 TI - Tumour development, histology and grade of breast cancers: prognosis and progression. AB - Using 1,973 breast tumours from women aged 40-69 participating in the Swedish two county trial of mammographic screening for breast cancer, we examined the effect of histological type on prognosis and sojourn time (the duration of the preclinical screen-detectable phase) by age. The hypothesis of dedifferentiation, according to which a cancer of mixed malignancy grade drifts towards grade 3 as the more poorly differentiated part of the tumour grows faster than the well differentiated part, was also assessed. Ductal carcinoma in situ, invasive ductal carcinoma of grade 1, mucinous carcinoma and tubular carcinoma were all associated with good survival. Ductal carcinoma of grade 3 was associated with poor survival. Ductal carcinoma of grade 2, lobular and medullary carcinoma were associated with intermediate survival. These patterns were much the same in women aged 40-49 as in women aged 50-69. In women aged 40-49, sojourn time was estimated at about 2 years regardless of histological type. For women aged 50-69, there was a marked association of sojourn time with histological type, the shortest sojourn time being observed for lobular (2 years) and medullary (1.2 years) carcinoma, and the longest for ductal carcinoma grade 1 (7.7 years) and tubular carcinoma (7.1 years). There was strong evidence of a potential to dedifferentiation. A mover-stayer mixture of Markov chain models estimated that, in women aged 40-54, 91% of ductal tumours have the potential to dedifferentiate and, in women aged 55-69, 38% of ductal tumours have such a potential. PMID- 8635854 TI - Marked increases in cathepsin B and L activities distinguish papillary carcinoma of the thyroid from normal thyroid or thyroid with non-neoplastic disease. AB - Cathepsin B (CB) and cathepsin L (CL) are cysteine endopeptidases involved in the processing of thyroglobulin (Tg) in the normal thyroid. As thyroglobulin expression is frequently altered in thyroid carcinomas, we have analyzed 42 human thyroid tissues from 40 patients to study the effect of malignant transformation an the expression of these endopeptidases. Our samples included 18 cases of papillary carcinoma (of which 10 also had matched adjacent normal thyroid tissue), 6 cases of normal thyroid from autopsy patients, 1 case of follicular carcinoma, 2 cases of medullary carcinoma, 2 cases of follicular adenoma, 3 cases of Hashimoto's thyroiditis (HT) and 10 samples from 8 patients with multi-nodular goiter (MNG). Enzyme-specific activities were increased 15-fold for CB and 9-fold for CL in papillary carcinoma compared with normal adjacent thyroid tissue or normal thyroid from autopsies. CB mRNA content was also markedly increased in papillary carcinoma compared with normal thyroid, primarily due to elevated levels of the 2.2-kb CB mRNA transcript. In thyroids with nonneoplastic diseases, including MNG and HT, there was no significant increase in either CB or CL enzyme activities nor CB mRNA levels compared with normal thyroids from non-cancer cases. Immunohistochemical studies on papillary carcinomas revealed increased CB staining in papillary carcinoma cells, with prominent staining close to the basement membranes of many of the neoplastic cells. Our observations suggest that CB and CL enzyme activities are potentially useful new biochemical markers for distinguishing papillary carcinoma of the thyroid from non-neoplastic thyroid disease. PMID- 8635855 TI - Frequency and distribution of herpesvirus-like DNA sequences (KSHV) in different stages of classic Kaposi's sarcoma and in normal tissues from an Italian population. AB - The frequency and distribution of herpesvirus-like DNA sequences (KSHV) were investigated by PCR in the pathologic skin lesions of a series of 22 HIV-negative elderly patients with classic Kaposi's sarcoma (KS) from Italy, one of the few regions of the world where classic KS is prevalent. Viral sequences were clearly identifiable in 15 cases, in particular in 2 of 5 patch, in 3 of 6 plaque and in 10 of 11 nodular lesions. Our findings confirm the association of these herpesvirus-like DNA sequences with KS in unrelated populations, providing evidence of the putative KS-associated agent in all different histologic lesions of the disease, mainly in the nodular stage. The search for other herpesviruses by PCR showed that Epstein-Barr virus (EBV) sequences were present in 7 of 22 pathologic skin lesions. In 4 cases, both EBV and KSHV were present. On the contrary, all 22 classic KS specimens were negative for human herpesvirus-6 sequences. Two of 3 patch and the 1 nodular lesions from AIDS-related KS patients examined were positive for KSHV but negative for both EBV and HHV-6 sequences. Furthermore, we evaluated the prevalence of KSHV sequences in the normal population of the same geographical area. Thirteen peripheral blood mononuclear cell samples, 9 salivary gland tissues and 6 saliva samples from healthy subjects were invariably found negative for KSHV, using the same PCR technique. Of interest, 2 of 11 hyperplastic tonsils harboured these herpesvirus-like sequences, suggesting that, like other herpesviruses, the KS- associated agent may be harboured in a proportion of normal individuals and tonsils may represent at least one of the possible reservoirs of this putative lymphotropic gamma herpesvirus in vivo. PMID- 8635856 TI - Anthropometric and reproductive factors and the risk of pancreatic cancer: a case control study in Shanghai, China. AB - To examine the possible role of body size and reproductive factors in pancreatic cancer, data were analyzed from a population-based case-control study conducted in Shanghai, China. Cases (n = 451) were permanent residents of Shanghai, 30-74 years of age, newly diagnosed with pancreatic cancer between October 1, 1990, and June 30, 1993. Deceased cases (19%) were excluded from the study. Controls (n = 1,552) were randomly selected from permanent Shanghai residents and frequency matched to cases by gender and age. Information on body size and reproductive and other possible risk factors was collected through personal interviews. After adjustment for age, income, smoking and other confounders, a positive dose response relation between body mass index and risk of pancreatic cancer was observed in both sexes. Among women, the risk of pancreatic cancer was significantly associated with number of pregnancies and live births. Compared with 0-2 pregnancies or live births, the odds ratio (OR) for 8 or more pregnancies was 1.90, while that for 5 or more births was 1.88. A modest elevation in risk, independent of parity, was associated with early age at first birth. Risk increased over 40% among women with a first birth at or before age 19 years relative to those at age 26 years or older. Ever use of oral contraceptives was associated with excess risk, though based on small numbers of users. Our findings suggest that, in Shanghai, obesity, gravidity, parity and perhaps use of oral contraceptives are associated with moderate increases in risk of pancreatic cancer, indicating that hormonal determinants deserve further investigation. PMID- 8635857 TI - Oestrogen-related cancer risk in mothers of testicular-cancer patients. AB - The belief that oestrogens are involved in the pathogenesis both of testicular cancer in young men and of cancers of the endometrium and female breast has become widespread. In a search for possible hormonal links between these cancers, we investigated the cancer pattern in a cohort of women who had given birth to sons who developed testicular cancer. Particular focus, was given to oestrogen related cancers. The present retrospective population-based cohort study is based on data from the Danish Cancer Registry. Mothers of 2,204 testicular-cancer patients were followed for the occurrence of cancer over a total of 70,063 person years. The ratio of observed cancers in the cohort over the expected numbers based on cancer incidence in the underlying female population served as measure of the relative risk (RR). The RR of developing breast cancer among mothers of testicular-cancer patients was 0.8 (95% confidence interval 0.6-1.1), the relative risk of endometrial cancer 0.6 (0.3-1.0) and of ovarian cancer 1.0 (0.6 1.6). Mothers of testicular-cancer patients are not at increased risk of developing oestrogen-related cancers. PMID- 8635858 TI - Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas. AB - We have investigated the localization of urokinase-type plasminogen activator (u PA), type-1 plasminogen-activator inhibitor (PAI-1), u-PA receptor (u-PAR) and alpha(2)-macroglobulin- receptor/low-density-lipoprotein-receptor-related protein (alpha(2)MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of 133 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph-node metastases were stained with monoclonal antibodies. Formalin-fixed sections of 15 primary tumors and 2 lymph-node metastases were stained with polyclonal antibodies. In primary tumors, u-PA and PAI-1 immunoreactivities were intense in macrophages and mast cells, and moderate in benign and malignant epithelial cells as well as in myofibroblasts and endothelial cells. A sub-group of poorly differentiated tumors showed particularly strong staining of stromal fibroblasts. u-PA immunoreactivity was also present in lymphocytes. alpha(2)MR/LRP and u-PAR immunoreactivities were intense in macrophages, but apart from these cells, alpha(2)MR/LRP was found only in fibroblasts, and u-PAR only in tumor cells located peripherally in tumor-cell clusters and glands and some myofibroblasts in the adjacent stroma. Lymph-node metastases showed staining for u-PA and PAI-1 both of cancer cells and of stromal fibroblasts, also staining for u-PA of lymphocytes. Similarly to some of the poorly differentiated primary tumors, approximately half of the metastases showed very strong staining of stromal fibroblasts, and extracts of these metastases had higher u-PA and PAI-1 levels, as determined by ELISA, than extracts of metastases without this staining pattern. alpha(2)MR/LRP was present only in fibroblasts and u-PAR only in some tumor cells. The presence of u-PA, PAI-1, alpha(2)MR/LRP and u-PAR was controlled biochemically by immunoblotting analyses, ligand-blotting analyses, and direct and reverse zymography. The spatial distribution and the variation in concentration of the various components of the plasminogen-activation system point to a complex, multifunctional role for the 4 proteins in and/or during the development and spread of breast cancer. PMID- 8635859 TI - Novel HPV types present in oral papillomatous lesions from patients with HIV infection. AB - Patients infected with the human immunodeficiency virus (HIV) often develop multiple papillomatous lesions of the oral cavity. In the present study, a total of 67 biopsies from benign oral lesions were analyzed for the presence of human papillomavirus (HPV) DNA using Southern-blot hybridization in combination with a polymerase chain reaction designed to detect all known HPV types, as well as unidentified types. These samples, collected at random from a high-risk population, were subsequently divided into 57 biopsies originating from patients with confirmed HIV infection and 10 biopsies from patients with unknown HIV status. Each sample was amplified with 7 different combinations of degenerate primers. All amplified products were sequenced. HPV DNA sequences were detected in 67% (45/67) of the samples. HPV 7 (19%) and HPV 32 (28%) were the predominant HPV types. HPV 32 was present in 2/4 fibromas tested. Two new HPV types, HPV 72 and HPV 73, were identified in oral warts with atypia. The complete genomes of these viruses were cloned and sequenced. Other HPV types detected were HPV 2a, HPV 6b, HPV 13, HPV 16, HPV 18, HPV 55, HPV 59 and HPV 69. PMID- 8635860 TI - Altered expression of CD44 isoforms in squamous-cell carcinomas and cell lines derived from them. AB - CD44 is a transmembrane glycoprotein that binds hyaluronan, extracellular matrix proteins and growth factors. Multiple isoforms of CD44 are generated by alternative splicing of 10 separate exons (V1-V10). Expression of the variable exons has been correlated with tumour progression and metastasis in a range of cell types. However, multiple CD44 isoforms are expressed by normal stratified squamous epithelia, such as the epidermis and the lining of the oral cavity. The purpose of our study was to examine CD44 expression in squamous-cell carcinomas (SCC). By immunofluorescence we found reduced expression of one or more of the variant exons in a series of 13 oral SCC, with loss being most common in poorly differentiated tumours. Of the exons we examined, V3 was lost most frequently, but otherwise there was no consistent pattern as to which exons (V4/5, 6, 8) were missing. We also studied CD44 expression in a range of SCC lines, using Western blotting and semi-quantitative RT-PCR. All lines showed reduced expression of the terminal differentiation marker involucrin. Two lines showed selective loss of the largest forms of CD44 and one failed to express any of the variant exons. These cell lines, therefore, provide a useful experimental model with which to study the biological significance of exon loss in SCC. PMID- 8635861 TI - Prevalence of mucosotropic human papillomaviruses in squamous-cell carcinoma of the head and neck. AB - The prevalence of mucosotropic human papillomavirus (HPV) DNA in 63 squamous-cell carcinomas (SCC) from different anatomic sites in the head and neck was determined by general primer-mediated polymerase chain reaction (GP-PCR). HPV DNA was detected in 20.6% of SCC. Additional type-specific PCR for HPV 6, 11, 16, 18, 31 and 33 demonstrated the presence of HPV 16 alone in these carcinomas. HPV 16 was also detected in normal epithelium from the resection margins of the majority of HPV-positive SCC. HPV status did not correlate with tumour site, whether primary or recurrent, TNM stage, metastases, degree of differentiation, smoking or alcohol history, fate or survival. PMID- 8635862 TI - Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo. AB - Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen positive tumors and select for antigen-loss variants in vivo. PMID- 8635864 TI - Characterization of a murine lymphoma cell line by 31P-NMR spectroscopy: in vivo monitoring of the local anti-tumor effects of systemic immune cell transfer. AB - The intradermal ESb-MP murine T-cell lymphoma in syngeneic DBA/2 mice has been used as a model for adoptive immunotherapy (ADI). Cultured ESb-MP cells were characterized in suspension by 31P-NMR spectroscopy (MRS) at 11.7 T, and solid primary tumors were examined by 31P-MRS in vivo at 7.0 Tesla using surface-coil techniques. Growing tumors contained relatively high levels of phosphomonoesters (PME, predominantly phosphoethanolamine), nucleotides (NTP) and Pi, low levels of phosphodiesters (PDE) and no phosphocreatine. Mean tissue pH was found to be 6.7 6.9. The spectra of ESb-MP cells cultured in RPMI medium (containing choline but no ethanolamine) also showed low PDE and no phosphocreatine at an intracellular pH of 7.4; however, only a trace amount of phosphoethanolamine was detected and significant levels of nucleoside mono- and diphosphates were observed. The complete ADI treatment protocol involved low-dose irradiation (5 Gy) followed by i.v. transfer of immune spleen cells from allogeneic B10.D2 donors and resulted in 100% remission (responders); no treatment or incomplete ADI (irradiation or immune cell transfer alone) resulted in no remissions (nonresponders). In vivo MRS could best discriminate between responders and non-responders on the basis of tissue pH, which increased in responders to 7.0 by day 5-6 after complete ADI. Following therapy, the sum of PME + Pi (both absolute and as a percent of total phosphates) decreased significantly only for responders but only after a visible decrease in tumor volume was apparent. PMID- 8635863 TI - Pharmacokinetics, biodistribution and biological effects of intravenously administered bispecific monoclonal antibody OC/TR F(ab')2 in ovarian carcinoma patients. AB - The bispecific monoclonal antibody (biMAb) OC/TR combines the anti-ovarian-cancer reactivity of the MOv18 monoclonal antibody (MAb) with the reactivity of an anti CD3 MAb. Pre-clinical studies have indicated that this biMAb is able to redirect the cytolytic activity of T cells towards tumour cells, resulting in efficient tumour-cell lysis. To assess the clinical potential of systemic biMAb-based cancer therapy we initiated a study in ovarian-cancer patients. Five patients suspected of ovarian cancer received 123I-OC/TR F(ab')2 i.v. Unexpectedly, the first patient developed side effects (grade III-IV toxicity) starting 30 min after infusion (p.i.) of 1 mg of OC/TR F(ab')2. After approval of the Ethical Committee, the study was continued at lower dose levels (0.1 mg; 0.2 mg). However, at the 0.2-mg dose level similar side effects were observed. FACS analysis indicated that all peripheral T cells were coated with biMAb immediately following the infusion. The cytokines tumour necrosis factor-alpha, interferon gamma and interleukin-2 showed maximum serum concentrations 2 h p.i. Tumour uptake ranged from 0.8 to 1.9% ID/kg, resulting in tumour/background ratios of 3 to 8. Our results suggest that at higher antibody dose levels OC/TR F(ab')2 causes T-cell activation with acute release of cytokines. Only low doses of biMAb can be administered safely. Despite the interaction with T cells, OC/TR F(ab')2 preferentially localizes in tumours following i.v. administration, thus offering therapeutic perspectives. PMID- 8635865 TI - Camptothecin post-treatments inhibit the biochemical events linked to the tumor promoting component of carcinogenesis in mouse epidermis in vivo. AB - 20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10-14 hr after TPA or 4-6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3-3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16-64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10 hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion. PMID- 8635866 TI - Resistance to the chemosensitizer verapamil in a multi-drug-resistant (MDR) human multiple myeloma cell line. AB - Inhibitors of P-glycoprotein (P-gp) or chemosensitizers, such as verapamil, are used to reverse multi-drug resistance (MDR) in cancer patients. Clinical studies in patients with myeloma have shown that some patients with P-gp-positive cancer cells respond to the chemosensitizing effect of verapamil. However, this response is short-lived and tumor cells ultimately become resistant to chemosensitizers. To study mechanisms of resistance to chemosensitizers, a human myeloma cell line, 8226/MDR10V, was selected from a P-gp-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are consistently more resistant to MDR drugs than parent cells, Dox40. Chemosensitizers, including verapamil and cyclosporin A, were less effective in reversing resistance in MDR10V compared with Dox40 cells. Verapamil and cyclosporin A were only partially effective in blocking P-gp drug efflux in MDR10V compared to Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V cells express less P-gp in the plasma membrane than do its parent cells, Dox40. [3H]Azidopine photoaffinity labeling of P-gp and its binding competition with unlabeled verapamil showed similar affinity for P-gp between Dox40 and MDR10V cell lines. Non-P-gp-mediated mechanisms of drug resistance, including over-expression of MRP and alterations in topoisomerase II, were not different for MDR10V cells compared with Dox40 cells. PMID- 8635867 TI - Leukemia-inhibitory factor stimulates breast, kidney and prostate cancer cell proliferation by paracrine and autocrine pathways. AB - Leukemia-inhibitory factor (LIF) is an inflammatory cytokine with pleiotropic activities. LIF was originally described as a differentiation factor of a murine leukemia cell line and was subsequently found to possess a broad spectrum of biological functions. Although LIF has been extensively studied in the hematopoietic system, little is known about its effects in solid tumors. We investigated the role of LIF in breast, kidney and prostate cancers. Using a clonogenic assay, we found that LIF significantly stimulated proliferation of 2 estrogen receptor-positive breast cancer cell lines (MCF-7 and T47-D) in a dose dependent fashion at concentrations ranging from 10 to 200 ng/ml. This effect was observed both in the presence of FCS and under serum- and estrogen-free culture conditions, suggesting that the effect of LIF is direct and does not depend on estrogen or any other cytokine. Neither line produced LIF protein, as assessed by ELISA. In contrast, the estrogen receptor-negative breast cancer line MDA MB-231 produced LIF but did not respond to either LIF or its neutralizing antibodies. Similarly, increasing concentrations of LIF did not affect the growth of primary kidney (A-498), metastatic kidney (ACHN) and prostate (DU 145) cancer cell lines. These lines produce LIF, however, and antibodies to LIF significantly suppressed their proliferation, suggesting that they were maximally stimulated by the endogenously produced cytokine. Taken together, our data suggest that LIF acts as either a paracrine or an autocrine growth factor for breast, kidney and prostate cancers. PMID- 8635868 TI - Verapamil suppresses the emergence of P-glycoprotein-mediated multi-drug resistance. AB - Selection protocols were designed to determine whether non-cytotoxic chemomodifiers can influence the evolution of the drug-resistant phenotype. To this end, the human multiple myeloma cell line RPMI 8226 (8226/S) was selected with either doxorubicin, verapamil or doxorubicin plus verapamil. Using this approach low-level multi-drug-resistant (MDR) cell lines were obtained when 8226/S was selected with doxorubicin only or doxorubicin plus verapamil but not with verapamil only. The MDR phenotypes obtained were mechanistically distinct. In doxorubicin only-selected cells (8226/dox4), drug resistance was mediated by over-expression of the MDR1 gene and its cognate protein P-glycoprotein. In contrast, the drug resistance seen in the doxorubicin plus verapamil-selected cells was mediated through decreases in topoisomerase II protein levels and catalytic activity and not by P-glycoprotein over-expression. Cells selected with verapamil alone did not become resistant to any of the drugs tested. None of the 3 selected cell lines showed any changes in MRP gene expression when compared with 8226/S. Our results indicate that the inclusion of verapamil during drug selection with doxorubicin influences the drug-resistant phenotype by preventing the selection of MDR1/P-glycoprotein-positive cells. PMID- 8635869 TI - Potent anti-tumor effects of an anti-CD24 ricin A-chain immunotoxin in vitro and in a disseminated human Burkitt's lymphoma model in SCID mice. AB - A new anti-CD24 immunotoxin (IT), SWA11.dgA, was constructed by coupling the MAb SWA11 via the bivalent linker SMPT to deglycosylated ricin A-chain (dgA). The effects of SWA11.dgA were evaluated in vitro against the B-precursor leukemia cell line REH, the non-B-non-T acute lymphoblastic leukemia cell line NALM-6 and the Burkitt's lymphoma cell lines BL-2 and BL-38. Binding of SWA11 to the CD24 antigen was assessed by flow cytometry demonstrating high affinity of the MAb for all cell lines tested. SWA11.dgA inhibited the protein synthesis of BL-38, NALM 6, REH and BL-2 cells by 50% at concentrations (IC50) of 4.0 x 10(-11) M, 6.0 x 10(-11) M, 8.0 x 10(-11) M and 3.0 x 10(-9) M, respectively. SWA11.dgA was subsequently used for the treatment of disseminated human BL-38 Burkitt's lymphoma in a newly developed SCID mouse model. The mean survival time (MST) of BL-38-bearing SCID mice was extended from 23 days in untreated controls to more than 230 days when 6 microg SWA11.dgA was applied intraperitoneally one day after tumor challenge. All of the animals achieved continuous complete remissions. SCID mice treated with SWA11.dgA 4 days after tumor cell challenge or a reduced dose of SWA11.dgA (67%) also had a significantly extended MST (45.0 and 51.4 days, respectively, as compared to 22.7 and 23.1 days in the controls). We conclude that SWA11.dgA might be of potential use for the treatment of lymphoma in man. PMID- 8635870 TI - Ly-6C+ natural T (NT) cells mediate immune surveillance against NK-sensitive and NK-resistant transplantable tumours in certain strains of mice. AB - We have previously shown that anti-Ly-6C monoclonal antibody (MAb) 2B6-F2 identifies a subset of (CBA +/- C57BL/6)F1 splenic NK-1.1+ natural T (NT) cells which kill the NK-sensitive YAC-1 target in vitro. Furthermore, these Ly-6C+ cells are responsible for 40-50% of in vitro YAC-1 killing in all mouse strains tested. In BALB/c and DBA/2 mice, these cells killed not only YAC-1 but also the NK-resistant WEHI-164 M1/16 target via a receptor that recognises a shared determinant on these targets in vitro. In the present study, the anti-tumour role of Ly-6C+ cells against the NK-sensitive B16 melanoma and NK-resistant tumours WEHI-7 T lymphoma and WEHI-164/1C fibrosarcoma was studied in vitro and in vivo. In vitro, B16, WEHI-7 and WEHI-164/1C tumour cell lines were highly sensitive to Ly-6C+ cell killing. In vivo, these same tumours showed significantly increased growth when transplanted s.c. into syngeneic mice treated with 2B6-F2 (0.05 < or = p < 0.0005), and this was most marked in the first 15 days following tumour appearance, when tumours were <15 mm in diameter. Our results show that Ly-6C+ cells play a role in controlling the growth of transplantable NK-sensitive B16 melanoma, and in BALB/c mice, at least, the repertoire of susceptible tumours is extended to include NK-resistant WEHI-7 and WEHI-164/1C. We conclude that Ly-6C+ NT cells play a role in immunosurveillance against NK-sensitive as well as NK resistant tumours in certain strains of mice. PMID- 8635871 TI - Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line. AB - CRIPTO is a member of the epidermal growth factor (EGF) gene family originally isolated from undifferentiated human NTERA2 clone D1 (NT2D1) multipotent embryonal carcinoma cells. Retinoic acid (RA) treatment of NT2D1 cells leads to a neuronal differentiation program and to concomitant loss of CRIPTO mRNA expression. To assess the role of CRIPTO in the control of NT2D1 cell growth or differentiation, these cells were treated with 3 anti-sense oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. A dose-dependent inhibition of monolayer and soft agar growth was observed with each of these CRIPTO anti sense oligodeoxynucleotides but not with a control oligodeoxynucleotide of random sequence or with the 3 corresponding CRIPTO sense oligodeoxynucleotides. In addition, NT2D1 cells were transfected with a recombinant expression vector containing a 918-bp coding fragment of the human CRIPTO cDNA in the 3' to 5' orientation. NT2D1 CRIPTO anti-sense transfectants exhibited a significantly reduced endogenous CRIPTO mRNA and protein, a 4- to 5-fold decrease in growth rate in monolayer and a 50-70% reduction in cloning efficiency in soft agar as compared with NT2D1 parental cells or with NT2D1 cells transfected with a plasmid containing the neomycin-resistance gene alone (NT2D1 neo cells). Finally, we examined the expression of immunophenotypic markers that are modulated during the differentiation of NT2D1 cells following RA treatment. The globoseries stage specific embryonic antigen-3 recognized by the monoclonal antibody (MAb) SSEA-3 was expressed in 60% of undifferentiated parental NT2D1 or NT2D1 neo cells and in only 20% of NT2D1 CRIPTO anti-sense transfectants, whereas it was down-regulated in all cell lines following RA treatment. A neuroectodermal antigen recognized by the A2B5 MAb, which was not expressed in parental NT2D1, in NT2D1 neo or in CRIPTO anti-sense NT2D1 cells, was induced by RA treatment in all cell lines. Taken together, our results show that inhibition of endogenous CRIPTO expression in human embryonal carcinoma cells interferes with both transformation and differentiation. PMID- 8635872 TI - A novel anti-Thy-1 (CD90) monoclonal antibody induces apoptosis in mouse malignant T-lymphoma cells in spite of inducing bcl-2 expression. AB - Mouse malignant T-lymphoma CS-21 cells can survive and proliferate in vitro when co-cultured with CA-12 stromal cells isolated from lymph nodes, but CS-21 cells undergo apoptotic cell death with DNA fragmentation when cultured alone. We immunized rats with CS-21 cells and raised monoclonal antibodies (MAbs) that recognized Thy-1 (CD90) or CD45 protein. The majority of these MAbs were able to inhibit the adhesion and apoptosis of CS-21 cells. When anti-Thy-1 MAbs were examined for their recognition site on Thy-1 glycoprotein, one of them, MCS-34, was found to recognize both Thy-1.1 and Thy-1.2. In addition, MCS-34, just like the anti-Thy-1 MAb G7, recognized the Thy-1A epitope. G7 was known to induce apoptosis in some T-cell hybridomas and in thymocytes. In CS-21 cells, however, G7 could not induce apoptosis, but MCS-34 could. Interestingly, MCS-34 enhanced the expression of bcl-2 protein, in spite of its ability to induce apoptosis. Upon examining the apoptosis-inducing mechanisms of MCS-34, we found that it promoted a sustained increase in cytoplasmic-free calcium in CS-21 cells. Calcium ionophore A23187 was also found to induce apoptosis in a dose-dependent manner. These results indicate that a sustained increase in cytoplasmic-free calcium by MCS-34 induces apoptosis in CS-21 cells in spite of bcl-2 protein expression. PMID- 8635873 TI - A comparative study of cytokine gene transcripts in normal and malignant breast tissue and primary cell cultures derived from the same tissue samples. AB - Using a differential centrifugation method followed by culture in selective medium, we have successfully isolated and maintained individual epithelial and stromal cells from normal (n = 10) and malignant (n = 6) human breast tissue and characterised their phenotype by immunocytochemistry. Further, we have studied expression of the cytokine genes IL-1beta, IL-6, IL-8 and TNF-beta in each cell fraction by RT-PCR and have compared these results with cytokine gene expression in tissue extracts from which primary cultures were derived. In breast tumours, there was near complete absence of IL-1beta in both whole tissue and cell fractions, and in normals it was present in only 3/10 tissue preparations, with increased expression in stromal (6/10) and epithelial (5/10) cell samples. IL-6 was constitutively expressed in all tumour-derived breast tissue samples but down regulated in tumour cell cultures, with the opposite result in normal breast. Near identical levels of IL-8 expression were found throughout each preparation, irrespective of tissue origin. TNF-beta was expressed in all normal tissue samples, in 9/10 epithelial preparations but in only 6/10 stromal preparations. In tumours, TNF-beta was associated predominantly with whole tissue or stromal samples, with reduced expression in epithelial preparations. Our data confirm that primary cultures of normal and malignant human breast tissue can be successfully separated into epithelial and mesenchymal cell populations and their phenotype can be maintained in culture for up to 30 days. However, this cellular separation does alter the cytokine profiles; therefore, experimental findings with isolated cells should be treated with a caveat. PMID- 8635875 TI - In situ demonstration of renal-cell-carcinoma-specific T-cell clones. AB - Using mixed lymphocyte tumor-cell culture (MLTC) in a selected renal-cell carcinoma, we derived a tumor-specific T-cell line in which Vbeta14+ and Vbeta19+ T cells represented 70% of the whole T-cell population. Selected Vbeta19+ T cells were CD8+ and exhibited a HLA-restricted specific cytotoxicity against tumor cells. Independently, 2 CTL clones were obtained by direct cloning of tumor infiltrating lymphocytes, VIIIC2 CTL expressing a Vbeta19 and VIIB10 CTL a Vbeta13 T-cell-receptor transcript. VIIB10 lysed autologous tumor cells, normal kidney cells and EBV-transformed B cells. In contrast, VIIIC2 lysed tumor cells exclusively, demonstrating that the antigen structure recognized is tumor specific. In addition, we used a PCR-based method to search for the presence of these CTL in situ. TCR beta chain of VIIIC2 and VIIB10 CTL were sequenced and primers complementary to their N regions were synthesized. VIIIC2 CTL constituted up to 60% of Vbeta19 transcripts in MLTC T-cell lines derived from tumor infiltrating lymphocytes, 23% in tumor and 26% in a tumor-draining lymph node, while VIIB10 was not detected. Thus, VIIIC2 CTL was successfully derived from lymphocytes infiltrating a renal-cell carcinoma by direct cloning as well as by MLTC, probably because it was highly expanded in vivo within the tumor composing almost 2% of the TIL. PMID- 8635874 TI - Melanosomal and lysosomal alterations in murine melanocytes following transfection with the v-rasHa oncogene. AB - Melanomas exhibiting mutated ras genes are frequently invasive and amelanotic. Transfecting melanocytes with ras oncogenes causes transformation and a loss of visible pigmentation. We analyzed murine melanocytes rendered amelanotic by transfection with the v-rasHa oncogene. Consistent with previous reports, tyrosinase and tyrosinase-related protein-1 (TRP-1) were not expressed by transformed cells. In addition, lack of expression of TRP-2 and the product of the silver locus was documented. Levels of melanosomal matrix antigens, the pink eyed dilution locus protein and lysosome-associated membrane protein-1 were markedly reduced. Residual matrix antigens were localized by immunofluorescence to large vacuoles distributed peri-nuclearly in transfected cells. Electron microscopy demonstrated the absence of typical melanosomes and the presence of large vacuolar structures, also in a peri-nuclear distribution. Although levels of lysosomal hydrolases, such as beta-glucuronidase and cathepsin D, were diminished, marked elevations were observed in the expression of cathepsins B and L, 2 thiol proteases implicated in the acquisition of invasiveness. Our data demonstrate that transfection of melanocytes with v-rasHa is sufficient to disrupt the biogenesis of melanosomes and to up-regulate thiol protease synthesis, providing insights into the amelanotic and invasive nature of melanomas exhibiting mutations in ras genes. PMID- 8635876 TI - Isolation of genes differentially expressed in human primary myoblasts and embryonal rhabdomyosarcoma. AB - Using a subtractive hybridization method, we have cloned 48 cDNAs which are expressed in human primary myoblasts but down-regulated in the embryonal rhabdomyosarcoma (RMS) cell line RD. Twenty-nine sequences could be identified as coding for previously known gene products, while 19 encode unknown proteins. Twelve clones coding for known proteins that were highly down-regulated in the RD cells were chosen for further analysis on Northern blots containing additional normal and RMS cells. The expression pattern of TGF-beta-induced gene product-3 (beta(ig)H3), inhibitory G-protein alpha sub-unit (G(alpha)i2), osteoblast specific factor-2 (OSF-2), 22-kDa smooth-muscle protein (SM22), clone A3351 (homologous to mouse talin), testican, thrombospondin-1 and thrombospondin-2 suggests involvement of these proteins in the genesis of the neoplastic phenotype. Among the clones with unknown sequence, several are identical or homologous to expressed sequence tags or known cDNAs, such as integrins or laminin. These results suggest that several isolated clones might have an important role in the determination or maintenance of the normal phenotype, and thus their loss is possibly involved in the progression of malignancy. PMID- 8635878 TI - Introduction to symposium on recent research on Haemonchus contortus. PMID- 8635877 TI - Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation. AB - To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo. To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3 dimensional growth under well-defined conditions. The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-transfected clone of each (Rat1-T1 and MR1). Spheroid volume growth curves and [3H]thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to monolayer cultures. Studies on Rat1 and M1 aggregates showed that the potential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture. pO2 measurements confirmed that neither cell quiescence nor cell death in the pseudo-normal cell aggregate types is due to an oxygen deficiency. In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures. Cell-line-specific attributes in 3D culture that were not specifically related to ras transfection of the cells included histological structure, development of necrosis and thickness of viable cell rim. However, growth behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformation. PMID- 8635879 TI - The pharmacology of FMRFamide-related neuropeptides in nematodes: new opportunities for rational anthelmintic discovery? AB - The chemotherapeutic control of helminth parasites is compromised by the limited number of classes of anthelmintic drugs. Discovery of novel anthelmintics is impeded by the lack of novel screening technologies that overcome the difficulties inherent in screens based on whole organism toxicity. The development and implementation of mechanism-based screens for new anthelmintics offers great promise for the revitalization of antiparasitic drug discovery. However, mechanism-based screens must be based on a thorough understanding of the proteins or processes that offer the best chance for selective chemotherapeutic intervention. Basic research on the characterization of nematode FMRFamide related peptides (FaRPs) has revealed that these peptides are ubiquitously distributed in helminths. Chemical identification of a number of nematode FaRPs has been achieved, and these peptides have potent and profound effects on the nematode neuromuscular system. Physiological processes mediated by nematode FaRPs (and other helminth neuropeptides) offer potential targets for the discovery of novel anthelmintics. PMID- 8635880 TI - Progress on vaccination against Haemonchus contortus. AB - Control of Haemonchus contortus at present is largely by the use of anthelmintics, assisted in some regions by management programs. Widespread development of resistance, particularly in South Africa and Australia, and concerns associated with the manufacture and use of chemicals have led to increasing interest in vaccination as an alternative means of control. Vaccination strategies basically fall into 2 categories, 'hidden' antigens (usually derived from the gastrointestinal tract of the adult parasite), or 'natural' antigens (those exposed to the immune system of the host during the course of infection, usually derived from the infective larval stage). Particularly promising results have been obtained using the hidden gut antigen H11, or H110D, and more recently with another hidden antigen, H-gal-GP. The use of a natural antigen vaccine, however, would provide advantages such as boosting of the immune response by field challenge. This article will review recent developments in both types of vaccines against H. contortus and consider the advantages and disadvantages of the 2 approaches. PMID- 8635881 TI - Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii. AB - The protozoan parasite Toxoplasma gondii causes severe disease in animals and humans. In AIDS patients, for example, the encephalitis it produces is a major cause of death. Part of the very successful strategy adopted by the parasite centers on its ability to differentiate from the actively growing tachyzoite form to a chronic, almost latent state called the bradyzoite. The molecular signals and precise triggers involved in this differentiation process are not known. Drugs for treating toxoplasmosis are not capable of clearing the infection apparently because of their inability to eradicate the bradyzoites. Recently, as part of our efforts to understand the mode of action of a promising new drug, atovaquone, we have generated and analysed a mutant that is resistant to this drug. Surprisingly, we found that this mutant is predisposed to spontaneously differentiate from the tachyzoite to bradyzoite form in vitro (Tomavo & Boothroyd, submitted). Given that atovaquone is believed to act on the parasite mitochondria, we were interested to explore the relationship between mitochondrial function and differentiation. We find that atovaquone and a number of other drugs targeted to mitochondria will cause wild type parasites to differentiate from tachyzoites to bradyzoites suggesting some sort of adaptive response to a decrease in mitochondrial activities. The fact that atovaquone resistant mutants are hypersensitive to clindamycin, a drug believed to work on the putative plastid of these parasites, suggests a model for how the mitochondrion and plastid interact and how they may be tied into the process and state of differentiation. This model is presented and discussed. PMID- 8635882 TI - A ubiquitous intracellular parasite: the cellular biology of Toxoplasma gondii. AB - Toxoplasma gondii shares many features with other apicomplexan parasites but is unusual in its extremely broad host and tissue specificity. The parasite exhibits typical 'zoite' morphology, its highly polar structure being dictated by the complex cytoskeleton. Molecules on the surface of the zoite are prime candidates for interaction with the host cell and in vitro assays have implicated 2 of the 5 tachyzoite surface molecules in invasion: SAG1 as a ligand mediating host cell invasion, and SAG2 in enabling reorientation prior to invasion. The functional roles of other molecules, secreted from internal organelles during invasion and intracellular development, are also becoming clear through immuno-EM and biochemical studies, and from sequence data. Molecules from the rhoptries including the penetration enhancing factor ROP1 are secreted at the point of invasion and are integral to the newly formed parasitophorous vacuole membrane. Release of the dense granule molecules GRA 1-6, appears to be calcium regulated and occurs within 10 min of invasion leading to formation of the tubular membranous network and stabilization of the vacuole. The interaction between Toxoplasma and the host cell is stage specific. The tachyzoite divides rapidly and synchronously forming rosettes and causing host cell lysis, while the bradyzoite exhibits slow asynchronous division secreting a granular matrix and becoming enclosed within a cyst wall. This altered phenotype is a reflection of changes in gene expression. Bradyzoite specific molecules are found internally, on the parasite surface, and in the cyst matrix while important tachyzoite proteins such as SAG1 and SAG2 are downregulated. Differentiation between the 2 stages is reversible and is influenced by immunomodulatory agents. However a strong genetic element is involved and it is notable that virulent strains show a very low frequency of cyst production. PMID- 8635883 TI - Current research on Sarcocystis species of domestic animals. AB - The genus Sarcocystis is composed of about 130 species of heteroxenous cyst forming coccidia with differences in life cycle and pathogenicity. Pathogenic Sarcocystis spp. can cause disease in their intermediate hosts, in particular in ruminants. Research on Sarcocystis infections has been impeded by several facets of the parasites. Intermediate as well as definitive hosts can be parasitized by several different species with similarities in biology and morphology. Antigen preparations derived from pathogenic Sarcocystis spp. are highly cross-reactive with antibodies directed against non-pathogenic species. As a consequence, none of the currently available immunological tests is species-specific and can differentiate between pathogenic and non-pathogenic Sarcocystis spp. Over the last decade, new techniques in immunology, protein chemistry and molecular biology have facilitated more advanced studies on the molecular composition and molecular biology of Sarcocystis spp. in various laboratories. The development of species-specific monoclonal antibodies and analyses of the molecular composition of some life-cycle stages of Sarcocystis spp. of cattle and sheep showed that species-specific proteins and antigens exist in these species, although they are not highly abundant. In addition, comparisons of rRNA genes of different Sarcocystis spp. identified unique sequences in the rRNA of pathogenic Sarcocystis spp. that are suitable targets for species-specific identification. Thus, tools have become available that facilitate the development of methods for species-specific identification and differentiation of Sarcocystis spp. as well as the identification and study of molecules that are associated with pathogenicity of some of these parasites. PMID- 8635884 TI - Cytokines and immunological control of Eimeria spp. AB - Protozoan parasites belonging to the genus Eimeria cause considerable losses in livestock production in which stocking densities are high or environments restricted. The ability of hosts to mount immunological responses which limit parasite reproduction vary according to the particular species of Eimeria. Typically though, immune responses restrict parasite reproduction during primary infection and limit, if not prevent, subsequent infections. Although mechanisms of immunity are unknown, host immune responses have been exploited in the development of a method to control coccidiosis-immunisation with attenuated strains of Eimeria. Limitations of this control method, predominantly the cost of producing the attenuated parasites, necessitates identification of protective immune responses to facilitate selection of antigens for use in non-living vaccines. As in immune responses to many other parasitic infections of the gastrointestinal tract, the role of antibodies is at best minor, whereas T-cells are crucial. Numerous studies have shown that the intestinal mucosal T-cell population is dynamic; the number and phenotype of T-cells changes in response to Eimeria-infection. Specific changes in the intestinal T-cell population have not, however, been correlated with limitation of parasite reproduction. Experiments involving adoptive transfer of T-cell sub-populations and in vivo depletion of specific T-cells have shown that CD4+ T-cells and to a lesser extent CD8+ T-cells are important in immune responses which limit primary infection. In contrast, CD8+ T-cells are more important in subsequent infections with CD4+ T-cells having a lesser role. The effects of T-cells on Eimeria are partially mediated by the cytokines they release. Most attention has concentrated on interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNF-alpha) because these cytokines have been shown to limit other protozoan infections. IFN-gamma is produced in Eimeria infected hosts but evidence that it is present at the site of infection is limited. Intestinal levels of IFN-gamma increase earlier in response to primary Eimeria-infection in mice which are relatively resistant, than in mice which are relatively susceptible. Neutralisation of endogenously produced IFN-gamma has shown that this cytokine limits oocyst production in either primary or secondary infections depending on the species of Eimeria. Production of TNF-alpha is also increased in infected hosts. In comparison with relatively susceptible mice, TNF alpha is produced earlier and to a greater extent in the intestines of relatively resistant mice. Unexpectedly, injections of TNF-alpha into infected mice increased oocyst production. It remains to be determined whether the effects of endogenous TNF-alpha are the same as those of exogenous TNF-alpha. Mechanisms by which IFN-gamma and TNF-alpha modulate parasite reproduction have not been identified. A number of lines of experimentation have suggested that it is unlikely that IFN-gamma limits parasite reproduction through induction of the synthesis of reactive oxygen or reactive nitrogen intermediates, since both of these reactive intermediates have the capacity to exacerbate Eimeria-infection. PMID- 8635885 TI - Resistance and the control of sheep ectoparasites. AB - For about 100 years Australian woolgrowers have used a variety of chemicals to control blowflies, lice and other ectoparasites of sheep. While the chemicals have changed considerably the application technology has not changed very much at all. Chemicals registered for use on sheep have paralleled the evolution of synthetic insecticides with the unfortunate consequence of the development of resistance in the Australian sheep blowfly, Lucilia cuprina, following closely behind. Organochlorine (dieldrin) resistance peaked at about 70% in 1958 when unacceptable residues in meat and wool forced their withdrawal. Organophosphate (OP) resistance appeared in 1965. With no alternative insecticide classes until 1979, OP resistance reached near fixation levels by the early 1970s and has remained unchanged. OP resistance has reduced the protection period from over 16 weeks to about 6 weeks. Moreover, resistance has decreased the effectiveness of many flystrike dressings to unacceptably low levels. OPs are still very effective against sheep body lice, Bovicola ovis but control is hampered by inadequate application via plunge or shower dipping. Synthetic pyrethroid (SP) pour-on products were released in 1981 but resistance developed by 1985 and many woolgrowers were unable to eradicate lice with pour-on products. Highest Resistance Factors at this time were only about 26 x but this was sufficient to prevent pour-ons working efficiently. By 1991 a population from Hartley in NSW was found to be 642 x resistant to cypermethrin with side-resistance conferred to the other SPs. SP resistance was partially suppresible by piperonyl butoxide but field trials suggested that the resulting improvement in efficacy was not sufficient to be commercially attractive. OPs remain very effective if applied correctly and the release of ivermectin and 2 benzoylphenyl urea products significantly improves the prospects for resistance management. However the increasing environmental concern about the persistence of chemical residues in wool has stimulated interest in biological control of sheep lice by Bacillus thuringiensis. PMID- 8635887 TI - Elder abuse. AB - Though we live in a violent society, hurting the elderly and children--those who are most defenseless--seems especially deplorable. This month's Iowa Medicine examines the problem of elder abuse. The following articles by Iowa physicians discuss how to recognize it and what to do about it. PMID- 8635886 TI - Costs of major parasites to the Australian livestock industries. AB - A cost-benefit model is developed to estimate the costs of major parasites to Australian livestock industries and to evaluate the benefits from improving parasite management. The model disaggregates the Australian livestock industries into agro-climatic regions and various stock classes to estimate the total treatment and production loss costs of major parasites. Experimental trials and a computer simulation model are used to estimate the productivity of livestock under different treatment regimes. Using the model, the economic costs inflicted by cattle ticks, sheep gastrointestinal worms, sheep lice and sheep blowflies are discussed and the farm profitability resulting from improved sheep roundworm management is assessed. PMID- 8635889 TI - Elder abuse--intentional or unintentional? PMID- 8635888 TI - Elder abuse reporting and ethical dilemmas. PMID- 8635890 TI - Community resources. PMID- 8635891 TI - Concerns about the system. PMID- 8635892 TI - Diagnosing and managing elder abuse. PMID- 8635893 TI - A new year prayer: renew family values. PMID- 8635894 TI - Informationally challenged. PMID- 8635895 TI - Prostate cancer treatment article incomplete. PMID- 8635896 TI - Out of the classroom. AB - Educational focus for medical students at the University of Iowa is shifting from the classroom to the bedside and the clinic. With the advent of the new curriculum, students will get earlier exposure to a medical practice and spend more time with community physicians. PMID- 8635897 TI - UI cancer research links laboratory, clinic. PMID- 8635898 TI - Interest in electronic distribution of health physics. PMID- 8635899 TI - The linear no-threshold model: is it still valid for the prediction of dose effects and risks from low level radiation exposure? Proceedings of a conference to honor Victor Bond in his 75th year. November 1994. PMID- 8635900 TI - Accolade. Victor P. Bond. PMID- 8635901 TI - The present system of quantities and units for radiation protection. AB - The International Commission on Radiation Units and Measurements has over the last decade developed operational quantities, the ambient, directional and personal dose equivalent, suitable for the measurement of radiation fields in a variety of circumstances. Experience with the use of these quantities to represent the dose limitation quantities defined by the International Commission on Radiological Protection in 1977 has been an important part of recent radiation protection metrology. The definition by International Commission on Radiological Protection in 1991 of new limitation quantities, the equivalent dose and the effective dose has necessitated a redirection of this work. The metrology field has made good progress, however. It has found that for photons, at least above 50 keV, the effective dose can be measured by the ambient dose equivalent about as well as the former effective dose equivalent. Unfortunately, for neutrons the existing and already quite severe complications have been made somewhat worse by the new quantities although not any worse in the important region between 0.1 and 1 MeV. Neutron measurements over a broad energy range are the subject of extensive evaluation and some new suggestions as the metrology field wrestles with these problems. Values of wR constitute an important part of the International Commission on Radiological Protection recommendations. A brief history of the development of higher relative biological effectiveness values for fission neutrons and alpha particles leading to the selection of 20 for wR in each case, is provided. PMID- 8635902 TI - Stem cell responses after radiation exposure: A key to the evaluation and prediction of its effects. AB - A biomathematical model of granulocytopoiesis is described and used to analyze the blood granulocyte changes seen in the blood of dogs and humans after continuous and after acute external radiation exposure. This allows to relate the cell change pattern seen to the extent of stem cell damage in the hematopoietic bone marrow distributed as semi-autonomous units throughout the skeletal bones. The model is described briefly and consists of 8 cellular and 2 regulatory compartments and is described by 37 differential equations. With the help of this model, it can be shown that the chronic radiation exposure of dogs at a rate of between 0.003 and 0.12 Gy per day results in a system failure with subsequent death of the animal, if the stem cell pool decreases below 2.5% of its normal content. In human beings exposed to a single radiation exposure (as seen in radiation accidents) the simulation of the granulocyte pattern results in the finding that a reduction of the stem cell pool to 5-10% of normal is compatible with the assumption of its "reversible" damage (to be treated by conventional replacement therapy including cytokines), whereas the reduction of blood granulocytes to levels of less than 200-300 per mm3 on day 5-6 after exposure indicates that no stem cells remain from which a spontaneous regeneration could occur and hence would require a substitution therapy by stem cell transplantation. In order to test the approach, the same model was used to correlate the changing granulocyte pattern seen after autologous blood stem cell transfusion in patients treated with a supralethal radiochemo conditioning regimen. The results indicate a proportionality of progenitor cells in the transfusate with the calculated stem cell number of the modeling exercise. It is proposed to use the pattern of granulocyte changes in the blood as a principal indicator to predict the outcome of a radiation exposure and to select appropriate therapeutic strategies. PMID- 8635903 TI - Radioepidemiology of the A-bomb survivors. AB - Estimation of the risk of cancer and other health effects following exposure to the atomic bombing of Hiroshima and Nagasaki remains largely empirical, and the models used to adduce risk incorporate few, if any, of the advances in molecular biology of the past decade or so. These facts compromise the estimation of risk where the epidemiologic data are weakest, namely, at low doses and dose rates. Although the risk estimates may be sufficient for regulatory purposes, without a better understanding of the molecular and cellular events ionizing radiation initiates or promotes, it seems unlikely that the estimates will be as intellectually satisfying as they might be. Nor will the situation improve further without attention to the identification and estimation of the effects of those host and environmental factors that enhance or diminish risk of cancer or the effects on the developing brain. PMID- 8635904 TI - Neutron induced recoil protons of restricted energy and range and biological effectiveness. AB - Low energy neutrons (<2 MeV), those of principal concern in radiation protection, principally initiate recoil protons in biological tissues. The recoil protons from monoenergetic neutrons form rectangular distributions with energy. Monoenergetic neutrons of different energies (<2 MeV) will then produce overlapping recoil proton spectra. By overlapping the effects of individual deposition events, determined microdosimetrically for cell nuclear dimensions, from such neutron beams the biological effectiveness of recoil protons within defined energy and range bounds can be determined. Here chromosomal aberrations per cell have been quantified following irradiation of Vicia faba cells with monoenergetic neutrons of 230, 320, 430, and 1,910 keV. Aberration frequencies from cells from part of the cell cycle, thereby limiting nuclear dimensions, were linearly related to dose and to the frequency of proton recoils per nucleus. The 320 keV neutrons were the most biologically effective per unit absorbed dose and 430 keV neutrons most effective per recoil proton, with 21% of recoils inducing aberrations. After extraction of effectiveness per proton recoil within each energy and range bounds (0-230, 230-320, 320-430, and 430-1,910 keV), it was concluded that recoil protons with energies of about 200-300 keV, traveling 2.5-4 microm and depositing energy at about 80 keV micrometer(-1), are more efficient at aberration induction than those recoil protons of lesser range though near equivalent LET and those of greater range through lesser LET. This approach allows for assessment of the biological effectiveness of individual energy deposition events from low energy neutrons, the lowest dose a cell can receive, and provides an alternative to considerations of relative biological effectiveness. PMID- 8635905 TI - Role of low-level ionizing radiation in multi-step carcinogenic process. AB - In view of our current understanding of experimental in vitro and in vivo studies, as well as of the epidemiological data, carcinogenesis is the result of many endogenous and exogenous factors. No single factor "causes" cancer. A number of extant theories of carcinogenesis and of ionizing radiation's role in the process have been reviewed. An integration of the stem cell theory, the theory of "oncogeny as partially blocked ontogeny," the initiation/promotion/progression model of carcinogenesis, the oncogene/tumor suppressor gene theory, and mutation/epigenetic theories of carcinogenesis was attempted by linking all of them with the process of intercellular communication. This integration was done by examining how extra-, intra- and inter-cellular communication might he affected by the current known facts of the types of radiation-induced biological effects, such as gene and chromosomal mutations, cell killing, including apoptosis and epigenetic alterations of gene expression. Finally, an examination of the possible role of low-level radiation in the multi-step carcinogenetic process, which might have given rise to the excess cancers attributable to radiation exposure in the survivors of the atomic bombs, was attempted. PMID- 8635906 TI - Effects of low doses of radiation. AB - This is a brief review of what is known from experimental studies about the effects of low doses of radiation, and approaches that might improve risk estimates are discussed. The dose-response relationships for cancer induction by radiation vary markedly between tissues. The evidence suggests that 1) the induction of the initial events is dependent on the cell type because the size and/or the number of targets and how the cells handle the initial lesions differs between cell types; and 2) there are marked differences among tissues how initial lesions are expressed and proceed to overt cancer. The recent findings about adaptive responses are discussed in the context of what they contribute to our understanding about the response to irradiation. Lastly, the possibility of extending the approach of determining "The probability of causation," which Vic Bond played such an important role in establishing, is raised. PMID- 8635907 TI - Radiation physics and radiobiology. AB - Three quite general rules link radiation physics to radiobiology. They concern the dependence on linear energy transfer of relative biological effectiveness and of the cross section for cell killing, as well as the dependence of relative biological effectiveness on absorbed dose. These rules are accounted for in compound dual radiation action according to which damage in the nanometer domain depends linearly on dose with no dose rate dependence and on relative biological effectiveness that is limited to low values because of saturation. Energy concentration in the micrometer domain can cause large relative biological effectiveness in processes in which pairs of DNA lesions interact with quadratic dose dependence and dose rate dependence for low linear energy transfer radiations. Damage at both the nanometer and the micrometer level can cause observed effects and their relative contributions determine the maximum relative biological effectiveness at very low doses. PMID- 8635908 TI - Radiobiological challenges posed by microdosimetry. AB - The inapplicability of the concept of dose to cellular and subcellular structures is the paradox that has driven the development of radiation biophysics. It remains a central problem which necessitates the use of microdosimetry in radiobiology, radiation protection, and the clinical applications of ionizing radiation. The trend towards microdosimetric measurements in radiation protection practice is contrasted to the recent adoption of radiation protection quantities by ICRP that are not related to microdosimetric parameters, and it is argued that uncertainties in the numerical magnitude of radiation risks must not motivate ambiguity of definitions. In radiobiology there has been a variety of uses of microdosimetric data but an insufficient assessment of their differences and similarities, often compounded by widely different terminology. The use of microdosimetry in radiobiology is, at present, limited by the detail and the reproducibility of biological information, not by lack of microdosimetric data. New techniques of molecular biology are now beginning to permit analyses of the spatial distribution of DNA lesions that can be correlated to the spatial fluctuations of energy deposition by different ionizing radiations. PMID- 8635909 TI - Carcinogenic risk coefficients at environmental levels of radon exposures: a microdosimetric approach. AB - We report a microdosimetric-based evaluation of the effects of domestic exposure to radon. The risk coefficients obtained here are based on the microdosimetry of radon progeny alpha particles, on a function q(y) for in vivo radiogenic neoplasia, and on scaling A-bomb results (epidemiology + microdosimetry) to radon exposure. We do not use miner data, nor do we invoke such notions as quality factors, dose equivalent or equivalent dose. With basal cells as targets our estimated risk coefficients are in good agreement with the miner data, and thus a quality factor of about 20 (as suggested by ICRP 60) is not unreasonable. However, if we take as targets the secretory cells our risk coefficients are twice as large as those reported by BEIR-IV. The main uncertainty in these estimates remains the dosimetric model. PMID- 8635911 TI - Relative biological effectiveness of ionizing radiations determined in tissue (RBE) fails in assessing comparative relative effectiveness in the tissue cells. AB - The value of the RBE of a test radiation is conventionally determined against a known standard radiation for a chosen response of a selected biological tissue and is expressed as the ratio of tissue absorbed doses at equal effect, or as ratio of magnitudes of the effect at equal absorbed dose. If such an effect is observable as a consequence of responses of individual elements of this tissue, namely the cells, such as induction of cancer that arises from a single cell, the relative biological effectiveness should be expressed as the ratio of the incidences of the effects at equal mean absorbed dose to the cells rather than at equal absorbed dose to tissue. This cell based relative biological effectiveness is here termed the relative local efficiency. Since tissue absorbed dose is a product of the number of energy deposition events in cells of that tissue (N(H)) and the mean absorbed dose to these cells in the exposed tissue (z(1)), per tissue mass equal tissue absorbed doses from different radiation qualities have different values of N(H) and z(1) As a result, for pink mutations in Tradescantia cells, the relative biological effectiveness of 0.43 MeV neutrons is 48 but the relative local efficiency in fact is 2.8. PMID- 8635910 TI - Microdosimetric-based risk factors for radiation received in space activities during a trip to Mars. AB - A system for evaluating quality factors, Q, based on the microdosimetric distribution of the radiation field of interest has been set up; it makes use of a specific quality function (SQF) to obtain--given microdosimetric spectra- values for Q. The advantages of a system based on lineal energy are well recognized. Furthermore, recent studies have shown that spectra in 1-microm diameter tissue-equivalent spherical volumes reproduce correctly (in the sense of this formalism) measured RBE values, and thus a proportional counter would be usable as a practical instrument for radiation protection. All specific quality functions, q(y), available to date have been calculated from in vitro cellular data. To extend this approach to radiations of interest in space activities we have recently obtained a new function q(y) for in vivo radiogenic neoplasia using data on the Harderian gland of the mouse. These data were obtained for charged particles and energies relevant to space exposures. Furthermore, we introduce a new procedure that allows one to obtain--here with the use of microdosimetric distributions for the Hiroshima-Nagasaki radiation fields--risk factors scaled from the A-bomb survivorship results. We apply these concepts to particles and energies representing the galactic spectrum. We estimate that for a trip to Mars (450 d) the excess lifetime cancer mortality due to galactic cosmic ray (GCR) radiation is 0.037. This is about 50% lower than the risk coefficient obtained with the aid of standard (LET-based) quality factors. PMID- 8635912 TI - Various approaches to damage assessment. AB - There are two main approaches used to assess the damage to human health from exposure to low-level ionizing radiation. The first is a realistic best-estimate approach. The second is performed in support of the development of radiation standards to protect workers and the public, and tends to overestimate risk. This paper reviews these approaches to damage assessments as they have been applied to the development of radiation protection standards and current estimates of risk. Technical issues affected by these two different approaches include use of the linear hypothesis, use of relative and absolute risk projection models, dose-rate effectiveness factor, appropriateness of data sets, and the transfer of risks between populations. The prudent approach may be justified for radiation protection purposes, but scientific estimates of risk should reflect the state-of the-science and include estimates of uncertainty. PMID- 8635913 TI - The use of cell-oriented factors and the hit size effectiveness function in radiation protection. AB - It has long been argued that ionizing radiation can be considered to interact with matter in discrete, randomly occurring energy transferring events ("hits") and that the resulting microscopically nonuniform pattern of energy deposition strongly influences the biological effect of a given exposure. Microdosimetric measurements combined with cellular biological response data in the form of a "hit size effectiveness function" (HSEF) suggest a possible cell-oriented alternative method of correlating exposure with effect at low levels of any radiation or mixture of radiations. The instrumentation required, the validity of the approach, and its practical usefulness in radiation protection are examined, and its application to space radiation exposure is proposed as a test case. PMID- 8635914 TI - Current misinterpretations of the linear no-threshold hypothesis. AB - Contrary to the "linear no-threshold hypothesis," which implies that "any amount, however small" of radiation energy is a serious cancer threat, it is shown here that only relatively quite large amounts of such energy can pose such a threat to a person or population. Key to doing this is to make a sharp distinction between the actual amount of the radiation agent imparted energy, epsilon, which must be expressed in units of joules, and the average concentration or density of energy, epsilon/m (i.e., absorbed dose), which is expressed in units of Gy. With any cellular system, e.g., in tissue culture, one can easily adjust the numbers of cells used at each dose point so that a clearly significant number of radiation induced quantal responses (e.g., mutations, chromosome aberrations, malignant transformations, cell death), in the absorbed dose range of about 0.7 to 3 or more Gy, can be observed. However, if the number of cells is held constant as the absorbed dose is progressively reduced, a point is reached at which no significant excess is observable. This situation is frequently "remedied" by including more cells at that point, which, of course, can increase the number of malignant transformations sufficiently to render the excess statistically valid. However, because both axes are expressed in relative terms, the data point, despite having gained statistical significance, remains at the same location on the graph. This gives the false impression that no more of the agent energy was added or needed to achieve significance. However, if both coordinates are put in absolute terms, i.e., the actual number of quantal responses vs. imparted energy, and the same exercise of "improving the statistics" at low exposures is attempted, it then becomes evident that any point thus rendered significant must be relocated at a substantially higher energy point on the graph. This demonstrates unequivocally the fallacy in the proof of the "linear hypothesis" which is based on agent concentration response curves and not agent amount. It shows that the smaller the agent concentration (absorbed dose; epsilon/m), the larger the amount of radiation energy that must be added to the system in order to demonstrate a radiation-induced response. This suggests a minimum average energy requirement for production of a radiation-attributable cancer. It Ls concluded that the "linear hypothesis" should be abandoned as the cornerstone of radiation protection and practice. PMID- 8635915 TI - The use of the aquarium pump for the radon/thoron progeny monitor. PMID- 8635916 TI - Robley D. Evans. PMID- 8635917 TI - Failure to publish research: a form of scientific misconduct? PMID- 8635918 TI - Patterns of cognitive recovery in sudden cardiac arrest survivors: the pilot study. AB - OBJECTIVE: To determine the prevalence, type, severity, and natural evolution of cognitive impairments in survivors of sudden cardiac arrest over time and to assess the relation of selected clinical and psychologic variables to those outcomes. DESIGN: Longitudinal with repeated measures. Twenty-five consecutive patients underwent extensive neuropsychologic testing during hospitalization within 3 weeks of their initial cardiac arrest. Of these, 17 completed additional testing at 6 to 9 weeks, 12 to 15 weeks, and 22 to 25 weeks after the event. SETTING: Cardiac electrophysiologic services at a university teaching hospital, a community hospital, and home. OUTCOME VARIABLES: Orientation, attention, concentration, immediate recall, early retention, delayed recall, reasoning, motor speed, and motor regularity were measured. RESULTS: During hospitalization, 72% of the patients had mild to severe impairments in one or more cognitive areas. Memory, particularly delayed recall, was the most common deficit. At 6 months after the arrest event, 29% (5 of 17) of the patients continued to be impaired, and all had deficits in delayed recall. Depression was significantly related to deficits in attention and delayed recall at 6 months only. Time to postarrest awakening was the most reliable predictor of long-term cognitive functioning in this patient sample. CONCLUSION: A significant minority of sudden death survivors incur long-term cognitive impairments, particularly in delayed recall or short-term memory. The occurrence of long-term cognitive deficits in these patients can be estimated from the duration of unconsciousness after resuscitation (time-to-awakening). PMID- 8635919 TI - Infection precautions with temporary pacing leads: a descriptive study. AB - OBJECTIVE: To describe infection precautions used by nurses when caring for patients with temporary epicardial and transvenous pacemakers. DESIGN: Descriptive, nation-wide survey. SETTING: All U.S. hospitals performing cardiac surgery. RESPONDENTS: Nurse managers and clinical specialists from cardiac and thoracic intensive care, coronary care, and telemetry units at all U.S. hospitals performing cardiac surgery. INTERVENTION: The Pacemaker Electrode Care and Safety Survey. RESULTS: Responses came from 43% (388) of the 895 hospitals. Most respondents indicated that they wore gloves when handling electrodes (approximately 96%); usually the gloves were nonsterile (73%). A sterile procedure was often used to perform site care (37% for epicardial and 65% for transvenous). The most common agent used for site cleansing was povidone-iodine. Concern has been expressed in the literature about use of this agent. Gauze was the most common type of dressing (used by 60% for epicardial dressings and 31% for transvenous). CONCLUSION: There is diversity in clinical practice. Further study is recommended to establish the safety, efficacy, and cost-effectiveness of identified infection precautions. PMID- 8635920 TI - Patients' and nurses' knowledge of cardiac-related symptoms and cardiac misconceptions. AB - OBJECTIVE: To compare knowledge and reported incidence of cardiac-related symptoms among patients with nurses' knowledge and estimated incidence of symptoms in this patient group. DESIGN: Retrospective collection of patient data by means of postal questionnaire and postal survey of hospital nurses. SETTING: Five non-randomly selected hospitals in northeast England. SUBJECTS: One hundred seventy-five male patients with a first uncomplicated myocardial infarction (MI), cared for by 168 registered nurses working in coronary care or medical wards. OUTCOME MEASURES: Reported and estimated occurrence of 11 common cardiac symptoms in the 3 weeks after MI and 15-item knowledge and 10-item misconception scales about cause of and recovery from MI. RESULTS: The percentage of nurses who correctly estimated the incidence of symptoms was low, 25% of nurses did not make any correct estimates, and the mean number of correct estimates was not associated with nurses' experience or qualifications. The overall mean score for the knowledge scale was 9.6 (SD 1.9) for nurses, which was significantly higher (t=7.5, p<0.001) than that for patients (mean 7.9, SD 2.3); the nurses' score was not significantly associated with experience or place of work. For the misconception scale the mean score for nurses was 6.8 (SD 1.5), which was significantly higher (t=6.85, p<0.001) than the mean score for patients (5.5; SD 2.6); nurses working in specialized cardiac wards and more experienced nurses had significantly higher scores. CONCLUSIONS: Nurses should be provided with sound knowledge on cardiac symptoms and risks so that they can educate patients accordingly and, in particular, can correct misconceptions about the condition, prognosis, and appropriate lifestyle changes. Advanced-training personnel should recognize the need to enhance nurses' skills in patient education and rehabilitation; the importance of these skills also should be recognized in first level training. PMID- 8635921 TI - An introduction to oxygen free radicals. AB - Oxygen free radicals are byproducts from the fundamental metabolic activities within the body. Normally, radicals are neutralized by enzymatic activity or natural antioxidants. Thus the generation of free radicals poses no problem so long as the balance between oxygen radical production and eradication remains in balance. There are multiple medical conditions, such as myocardial infarction, carcinogenesis, and neurologic trauma, to name a few, that may be aggravated by the presence of oxygen free radicals. This article will present an overview of oxygen free radicals: their normal formation and control and how they might further injure tissue in particular diseases. The implications for health care professionals are highlighted. PMID- 8635922 TI - Functional status instruments: outcome measure in the evaluation of patients with chronic obstructive pulmonary disease. AB - The purpose of this article is to review the instruments developed to measure functional status in patients with chronic obstructive pulmonary disease. Because the ability to carry out day-to-day activities is of primary importance to patients with chronic obstructive pulmonary disease, it is necessary for clinicians to understand which instruments provide the best measures of patient activity levels. Furthermore, as a critical outcome in managed care services, pulmonary critical pathways, and patient disability, the measurement of functional status in clinical practice assumes greater relevance. Functional status instruments in this review will refer to questionnaires measuring the day to-day activities of patients. Questionnaires reviewed will include those that provide measures of general health status with activity-specific items, as well as questionnaires specifically designed to evaluate patients with pulmonary disease. The psychometric strengths, reliability and validity, and clinical utility of the instruments will be presented. PMID- 8635923 TI - Artificial airways: a survey of cuff management practices. AB - OBJECTIVE: To determine current endotracheal and tracheostomy tube cuff management practices in adult and pediatric populations, and to compare current adult cuff management practice with those reported in use in 1984 and 1987. DESIGN: Descriptive survey. SETTING: Sixty-four acute care hospitals in the northeastern United States. SAMPLE: Responders represented 93 critical care units: 59 adult and 34 pediatric units. MEASUREMENTS: Subjects completed a survey questionnaire. RESULTS: Forty-one percent reported cuffs were routinely deflated, with most (88%) reporting cuff deflation every 8 to 12 hours or daily. In the pediatric population, minimal occlusive volume was the most frequent technique (29%); whereas in the adult population, both minimal occlusive volume technique and minimal leak technique were used more frequently (36%). Most (93%) cuff pressures were measured every 8 to 12 hours or daily with a recommended maximum range of 20 to 30 mm Hg. Cuff deflation and cuff inflation were performed more often by the nursing staff (36%). Cuff pressures were performed more often by respiratory staff (71%). There were no statistically significant differences in the cuff management practices between the adult and pediatric populations. In comparing the results for adults to the data of 1984 and 1987, most cuff management practices changed from every 8 hours or less to every 8 to 12 hours or daily, and the nursing responsibility for these techniques increased (22%). CONCLUSION: Most responders do not routinely deflate cuffs. Cuff management practices are performed less frequently, and nursing responsibility for these techniques has increased. PMID- 8635924 TI - Desaturation events during oral feedings with and without a nasogastric tube in very low birth weight infants. AB - OBJECTIVE: To examine the desaturation events with the presence and absence of a nasogastric tube during an entire oral feeding in 20 very low birth weight (VLBW) infants. DESIGN: Prospective, quasi-experimental, random assignment. SETTING: Midwestern, university-affiliated, tertiary neonatal medical center. PATIENTS: Twenty VLBW infants without severe neurologic problems or physical anomalies. On the day of the study, postnatal days were 17 to 82 days (49+/-18.91). OUTCOME MEASURES: Desaturation events. INTERVENTION: These infants were observed during oral feedings, once with a nasogastric tube and once without, at 9 am and 3 pm feedings within 1 day, decided in random order. RESULTS: Fifteen infants experienced 166 desaturation events (<90%), 83 desaturation events without the nasogastric tube, and 83 events with the nasogastric tube present. Infants for whom desaturation events developed has a longer transition period from tube feedings to oral feedings (p<0.05), and started feedings with lower oxygen saturation (p<0.05). Nearly all desaturation events (97%) occurred with breathing pauses (11.32+/-6.67 seconds), a change in heart rate, and an increase in end tidal CO2. The presence of a nasogastric tube increased the duration of desaturation by an average of 8 seconds (p<0.05). CONCLUSION: Infants' oxygen saturation needs to be monitored with feedings, and feedings may need to be started with a baseline lowest saturation of 95% or higher, monitored with breathing and heart rate to prevent desaturation. PMID- 8635925 TI - Intentional drug overdose: predictors of clinical course in the intensive care unit. AB - OBJECTIVE: To examine the characteristics of patients admitted to the Medical Intensive Care Unit (MICU) after intentional drug overdose. DESIGN: Retrospective chart review, descriptive. SETTING: Midwestern teaching hospital. SUBJECTS: Convenience sample of 43 patients admitted to the MICU after intentional drug overdose. OUTCOME MEASURES: Survival and disposition of those patients who survived and were discharged from the MICU. RESULTS: Ninety-five percent of the patients survived and were discharged from the MICU; Acute Physiology and Chronic Health Evaluation II scores ranged from 1 to 29 (mean, 8); Glascow Coma Scale scores ranged from 3 to 15 (mean 12.5); five patients were intubated and two patients had serious electrocardiograph changes requiring pharmacologic intervention for dysrhythmia. CONCLUSIONS: Neurologic findings were the best indicators of serious complications after drug overdose. THerefore, patients with a Glascow Coma Scale score of more than six, and who are not intubated, may not need admission to an intensive care unit. PMID- 8635926 TI - Radiation oncology: historical development in Germany. AB - In the first period after the discovery of x-rays by Roentgen until World War II, German radiotherapy made major contributions to the development of the specialty. During the Nazi regime and World War II, German doctors were separated from international developments, and after the war the technical standard was poor. It took German radiotherapy a relatively long time to return to international standards again. Because of the difficulties caused by the war, mistakes were made in placing too much emphasis on technical, physical, and biological questions in the publications of the 1960s and 1970s, while in other countries clinical research was promoted. An exception is the work of K. Musshoff (47, 48) from Freiburg. His treatment results in Hodgkin's disease were known and acknowledged internationally. Because of a variety of reasons, it was and is difficult to perform prospective randomized trials in Germany even today. In addition, the turn to the English language in medicine made it difficult for publications written in German to be popular outside of Germany. Therefore, today German journals publish abstracts in English, too. In the meantime, the clinical and technical standard as well as scientific work in German radiotherapy are comparable again to the international level, especially relative to the United States, Great Britain, France, and the Scandinavian countries. PMID- 8635927 TI - The history and evolution of radiotherapy and radiation oncology in Austria. AB - Austria has a longstanding and eventful history in the field of radiotherapy and radiation oncology. The founder of radiotherapy, Leopold Freund, began his well documented first therapeutic irradiation on November 24, 1896, in Vienna. He also wrote the first textbook of radiotherapy in 1903. Further outstanding Viennese pioneers in the field of radiotherapy, radiobiology, radiation physics, and diagnostic radiology include Gottwald Schwarz, Robert Kienbock, and Guido Holzknecht. Because many of the leading Austrian radiologists had to emigrate in 1938, irreparable damage occurred at that time for the medical speciality of radiology. After World War II, the recovery in the field of radiotherapy and radiation oncology started in Austria in the early sixties. Eleven radiotherapy centers have been established since that time, and an independent society for radio-oncology, radiobiology, and medical radiophysics was founded in 1984. Finally, in March 1994, radiotherapy-radio-oncology became a separate clinical specialty. PMID- 8635928 TI - One century of radiotherapy in France 1896-1996. PMID- 8635929 TI - Young patients with prostate cancer have an outcome justifying their treatment with external beam radiation. AB - PURPOSE: The majority of young patients with early stage prostate cancer in the United States are treated with radical prostatectomy. To determine whether this preference for surgical care is justified, we analyzed by patient age the survival without biochemical evidence of disease (bNED) of men with clinically organ-confined prostate cancer treated with external beam irradiation. METHODS AND MATERIALS: One hundred and sixty-nine men with clinical stages T1-2 adenocarcinoma of the prostate received external beam radiation therapy alone at Fox Chase Cancer Center. All patients had serum prostate-specific antigen (PSA) values less than 10 ng/ml prior to initiation of treatment. Out of 169 patients, 167 had unstaged regional nodes (NX) and all had no evidence for distant metastasis (M0). The median age was 69 years. Criteria for bNED survival were posttreatment serum PSA < or = 1.5 ng/ml and not rising on two consecutive values. The median follow-up is 35 months. RESULTS: The actuarial 5-year bNED survival of all 169 patients was 85%. The bNED survival of patients less than 65 was not significantly different than that of patients 65 and older (89 vs. 84%, respectively). Patient age, American Joint Committee on Cancer (AJCC) stage, palpation stage, Gleason score, and dose to the center of the prostate were not found to be significant predictors of bNED survival on multivariate analysis. CONCLUSION: Our results using strict biochemical endpoints are comparable to reported series of similarly staged men treated with prostatectomy. In addition, the patient age of less than 65 is not a prognostic factor for worse outcome after radiation therapy. Young patients with clinically organ-confined prostate cancer who are fully informed of their treatment options can be appropriately accepted for external beam treatment. PMID- 8635930 TI - Lateral rectal shielding reduces late rectal morbidity following high dose three dimensional conformal radiation therapy for clinically localized prostate cancer: further evidence for a significant dose effect. AB - PURPOSE: Using conventional treatment methods for the treatment of clinically localized prostate cancer central axis doses must be limited to 65-70 Gray (Gy) to prevent significant damage to nearby normal tissues. A fundamental hypothesis of three-dimensional conformal radiation therapy (3DCRT) is that, by defining the target organ(s) accurately in three dimensions, it is possible to deliver higher doses to the target without a significant increase in normal tissue complications. This study examines whether this hypothesis holds true and whether a simple modification of treatment technique can reduce the incidence of late rectal morbidity in patients with prostate cancer treated with 3DCRT to minimum planning target volume (PTV) doses of 71-75 Gy. METHODS AND MATERIALS: The 257 patients with clinically localized prostate cancer who completed 3DCRT by December 31, 1993 and received a minimum PTV dose of 71-75 Gy are included in this report. The median follow-up time was 22 months (range: 4-67 months); 98% of patients had follow-up of longer than 12 months. The calculated dose at the center of the prostate was < 74 Gy in 19 patients, 74-76 Gy in 206 patients, and > 76 Gy in 32 patients. Late rectal morbidity was graded according to the Late Effects Normal Tissue (LENT) scoring system. Eighty-eight consecutive patients were treated with a rectal block added to the lateral fields. In these patients the posterior margin from the prostate to the block edge was reduced from the standard 15 to 5 mm for the final 10 Gy, which reduced the dose to portions of the anterior rectal wall by approximately 4-5 Gy. Estimates of rates for rectal morbidity were determined by Kaplan-Meier actuarial analysis. Differences in morbidity percentages were evaluated by the Pearson chi-square test. RESULTS: Grade 2-3 rectal morbidity developed in 46 out of 257 patients (18%) and in the majority of cases consisted of rectal bleeding. No patient has developed Grade 4 or 5 rectal morbidity. The actuarial rate of Grade 2-3 morbidity is 23% at 24 months and the median time to the development of Grade 2-3 complications is 15 months. A statistically significant dose effect is evident. The incidence of Grade 2-3 rectal morbidity increased as the dose at the center of the prostate increased (p = 0.05). In patients receiving minimum PTV doses of < or = 76 Gy the use of a rectal block significantly reduced the incidence of Grade 2-3 toxicity; 6 out of 88 (7%) with a block vs. 30 out of 137 (22%) without a block, (p = 0.003). CONCLUSION: The incidence of late rectal morbidity with 3DCRT to minimum PTV doses of 71-75 Gy is acceptable and to date no Grade 4-5 rectal morbidities have been observed. In our experience, higher doses to the center of the prostate are associated with an increased likelihood of developing Grade 2-3 rectal morbidity but treatment techniques that reduce the total dose to the anterior rectal wall have reduced the incidence of late rectal morbidity. If clinical studies indicate improved tumor control with minimum PTV doses above 71 Gy, then dose escalation above 76 Gy to the center of the prostate should be pursued cautiously with treatment techniques that limit the total dose to the anterior rectal wall. PMID- 8635931 TI - Conformal mixed neutron and photon irradiation in localized and locally advanced prostate cancer: preliminary estimates of the therapeutic ratio. AB - PURPOSE: To determine the incidence of chronic toxicity and the probability of biochemical and histologic complete response among patients with nonmetastatic prostate cancer, treated with three dimensional (3D) conformal mixed neutron and photon irradiation. METHODS AND MATERIALS: Between November 1991 and December 1994, 151 patients with prostate cancer were entered in three prospective dose finding studies of conformal mixed neutron and photon irradiation. Patients with low stage, low to intermediate grade prostate cancer (T1-2NXM0, Gleason Score < or = 7) received 38 Photon Gy (PhGy) plus 9 (51 patients) or 10 (53 patients) Neutron Gy (NGy) to the prostate and seminal vesicles. Forty-seven patients with locally advanced prostate cancer (T3-4 N0-1 M0 and/or Gleason Score > or = 8) received 15 NGy + 18 PhGy to the prostate and seminal vesicles and 9 NGy + 18 PhGy to the pelvic lymph nodes. RESULTS: The median follow-up was 16 months (range: 3-30 months). There was no Grade 3-5 GI or GU toxicity recorded. At 20 months, the actuarial rates of Grade 2 GI morbidity were 6 and 29% for the 9-10 and 15 NGy protocols, respectively (p = 0.07). At 20 months, the incidences of Grade 2 GU morbidity were 4 and 16%, respectively (p = 0.08). Stiffness in flexing or abducting the hips was seen in 20 and 42% of patients receiving 9-10 and 15 NGy, respectively (p = 0.01). Potency was maintained in 65% of all patients. Among patients with an initial PSA < or = 10, 100% had a 12-month PSA < 2 and 78% < 1 ng/ml. Negative postradiation biopsies were seen in 30% of patients 6 months, 79% at 12 months, and 84% of patients at 18 months. CONCLUSION: The use of conformal mixed neutron and photon irradiation has been well tolerated with no severe bladder or rectal complications observed. However, because of the enhanced toxicity seen with 15 NGy, the current maximum dose levels of neutron irradiation have been limited to 11 NGy. PMID- 8635932 TI - Sexual potency following interactive ultrasound-guided brachytherapy for prostate cancer. AB - PURPOSE: The effect of a therapeutic modality on sexual potency is often an important consideration for patients choosing a treatment for prostate cancer. We prospectively assessed patients' penile erectile function before and following interactive ultrasound-guided transperineal permanent radioactive seed implantation to determine its effect on sexual function. METHODS AND MATERIALS: Eighty-nine patients underwent permanent radioactive seed implantation from June 1990 to April 1994 for localized prostate cancer (T1-T2) and were followed for a median of 15 months (1.5-52 months). 125I seeds were implanted in 73 patients with a combined Gleason grade of 2-6, and 103Pd seeds were implanted in 16 patients with higher grade lesions. The sexual potency of these patients was assessed prior to, at 3 and 6 months, and every 6 months after implantation. Erectile function was graded using a numerical score of 0 to 3 (0 = impotent (no erections), 1 = ability to have erections but insufficient for vaginal penetration, 2 = erectile function sufficient for vaginal penetration but suboptimal, 3 = normal erectile function). The pretreatment potency scores were as follows: 0 in 24 patients, 1 in 6 patients, 2 in 22 patients, and 3 in 37 patients. RESULTS: The actuarial impotency rates (score = 0) following implantation for those patients possessing some degree of erectile function prior to implantation (65 patients) were 2.5% at 1 year and 6% at 2 years. The actuarial decrease in sexual function rates (a drop in score of at least one point) were 29% at 1 year and 39% at 2 years. Only two patients became impotent following treatment and this occurred at 1 year and 16 months. The time period for a decrease in erectile function to occur ranged from 1.8 months to 32.7 months, with a median of 6.8 months. Patients with higher grade tumors showed a greater decrease in potency score compared to patients with lower grade tumors. CONCLUSION: Interactive ultrasound-guided transperineal brachytherapy for the treatment of localized prostate cancer is associated with preservation of erectile function in the vast majority of patients, although a minor decrease in potency is not uncommon. PMID- 8635933 TI - Prostate cancer volume adds significantly to prostate-specific antigen in the prediction of early biochemical failure after external beam radiation therapy. AB - PURPOSE: A new clinical pretreatment quantity that closely approximates the true prostate cancer volume is defined. METHODS AND MATERIALS: The cancer-specific prostate-specific antigen (PSA), PSA density, prostate cancer volume (VCa), and the volume fraction of the gland involved with carcinoma (VCafx) were calculated for 227 prostate cancer patients managed definitively with external beam radiation therapy. 1. PSA density = PSA/ultrasound prostate gland volume. 2. Cancer-specific PSA = PSA - [PSA from benign epithelial tissue] 3. VCa = Cancer specific PSA/[PSA in serum per cm3 of cancer] 4. VCafx = VCa/ultrasound prostate gland volume A Cox multiple regression analysis was used to test whether any of these clinical pretreatment parameters added significantly to PSA in predicting early postradiation PSA failure. RESULTS: The prostate cancer volume (p = 0.039) and the volume fraction of the gland involved by carcinoma (p = 0.035) significantly added to the PSA in predicting postradiation PSA failure. Conversely, the PSA density and the cancer-specific PSA did not add significantly (p > 0.05) to PSA in predicting postradiation PSA failure. The 20-month actuarial PSA failure-free rates for patients with calculated tumor volumes of < or = 0.5 cm3, 0.5-4.0 cm3, and > 4.0 cm3 were 92, 80, and 47%, respectively (p = 0.00004). CONCLUSION: The volume of prostate cancer (VCa) and the resulting volume fraction of cancer both added significantly to PSA in their ability to predict for early postradiation PSA failure. These new parameters may be used to select patients in prospective randomized trials that examine the efficacy of combining radiation and androgen ablative therapy in patients with clinically localized disease, who are at high risk for early postradiation PSA failure. PMID- 8635934 TI - Apoptosis and downstaging after preoperative radiotherapy for muscle-invasive bladder cancer. AB - PURPOSE: To determine the relationship between pretreatment apoptosis levels and clinical-to-pathologic downstaging resulting from preoperative radiotherapy. METHODS AND MATERIALS: Between 1960-1983, 338 patients were dispositioned to receive preoperative radiotherapy 4-6 weeks prior to radical cystectomy for muscle-invasive transitional cell carcinoma of the bladder. Of these, adequate hematoxylin and eosin stained tissue sections for morphologic analysis of apoptosis were available in 158 patients. These patients were treated to a median dose of 50 Gy at 2 Gy per fraction. Median follow-up was 90 months. The apoptotic index (AI) was calculated from the ratio of the number of apoptotic cells divided by the total counted and multiplied by 100. A minimum of 500 cells were counted from each patient. RESULTS: The average AI for the whole group (n = 158) was 2.0 +/- 1.3 (+/- SD), with a median of 1.8. The association of AI to clinical stage was significant with AI averages of 1.8 for Stage T2 (n = 56), 1.9 for T3a (n = 51), and 2.4 for T3b (p = 0.038, Kendall Correlation). The relationship of AI to radiotherapy response also was significant with an average of 2.2 for those who were downstaged (n = 103), 1.9 for those in whom the stage remained unchanged (n = 20), and 1.7 for those who were upstaged (n = 35, p = 0.054, Kendall Correlation). The other significant correlations with AI were for the factors, grade, mitotic index, number of tumors, and gender. The AI was then categorized into three groups ( < or = 1, > 1, and < or = 3, and > 3) to examine the prognostic significance of this parameter. The distributions of patients by clinical stage, grade, mitotic index, number of tumors, radiotherapy response, and hemoglobin level were significantly associated with AI using this grouping. When the analysis of the distribution of patients by radiation response and AI was segregated by stage, a significant correlation was observed only for those with Stage T3b disease (p = 0.006); 93% of T3b patients with an AI > 3 were downstaged, while in 7% the stage remained unchanged and none were upstaged. The relationship of AI to 5-year actuarial patient outcome was investigated using several end points and although no significant correlations were observed, a trend was seen for improved survival when AI was > 3 (71% vs. 41%, p = 0.09) for Stage T3b patients. CONCLUSION: The AI correlated most strongly with radiotherapy response for patients with clinical stage T3b disease, the one subgroup of patients wherein preoperative radiotherapy is likely to be of the most benefit. Further investigation of pretreatment apoptosis levels as a marker of anticancer response is needed, especially for patients treated with chemotherapy and radiotherapy with the goal of bladder preservation. PMID- 8635935 TI - Once weekly irradiation for carcinoma of the bladder. AB - PURPOSE: A dose-searching study was carried out treating selected elderly patients or patients with poor performance with bladder cancer with once weekly fractionation to determine an effective dose per fraction, and to evaluate acute and late effects resulting from this schedule. METHODS AND MATERIALS: Seventy patients with invasive transitional cell carcinoma of the bladder were entered in the study. The dose used was 36-39 Gy in six fractions over 35 days in 27 patients (Group 1). The remaining 43 patients were treated with 34.5 Gy in six fractions over 39 days (Group 2). RESULTS: Six patients developed Grade 1-2 European Organization for Research on Treatment of Cancer (EORTC) bowel reaction. Three patients in Group 1 developed Grade 3 late bowel reaction and a fourth patient developed Grade 4 reaction requiring colostomy. However, only one patient in Group 2 developed Grade 3 reaction. The difference between the two groups was statistically significant (chi 2 = 3.794, p = 0.05). CONCLUSIONS: The acute and late reaction as well as the 5-year free survival for patients in Group 2 compare favorably with daily treatment. We conclude that 34.5 Gy given over 39 days is a safe and effective treatment for selected patients with bladder cancer. PMID- 8635936 TI - Omission of the pelvic irradiation in stage I testicular seminoma: a study of postorchiectomy paraaortic radiotherapy. AB - PURPOSE: To review the survival, cure rate, and pattern of relapse or progression of patients with histologically confirmed Stage I testicular seminoma who underwent orchiectomy and radiation therapy to paraaortic lymphatics only. The pelvic ipsilateral lymph nodes were not irradiated. METHODS AND MATERIALS: Between 1978 and 1992, 150 patients with Stages I or II testicular seminoma received treatment at the Department of Radiation Oncology of the University of Wuerzburg. The distribution by stage was Stage I, 117 patients of which 93 were pT1 N0 M0 and 24 were pT2 N0 M0. Four patients were staged as Stage II (pT3 N0 M0), and in 29 patients the T Stage was not specified. Eighty-six patients from the 117 Stage I (pT1-pT2, N0 M0 according to the TNM classification) seminoma received postorchiectomy irradiation, and are analyzed for outcome in this article. The distribution of the Stage I patients by pT Stage was 71 pT1 and 15 pT2 patients. All these 86 patients had their paraaortic nodes (the biological target volume extending from top of L1 to the bottom of L5) irradiated with four field technique. Tumor dose was specified at normalization point along the central axis. The median tumor dose was 30 Gy given in 1.8-2.0 Gy fractions. Elective irradiation to the ipsilateral hemipelvis (iliac nodes) was totally abandoned. RESULTS: The 10-year disease-free survival and overall survival were 95.3 and 100%. No recurrence in the irradiated field was noted. Four patients (4.7%) experienced relapse of disease outside the treated volume. The most common site of solitary failure was the ipsilateral hemipelvis (one iliacal and one inguinal). One patient developed metastatic disease to the lung. One patient developed a mediastinal recurrence with superior vena cava syndrome and was successfully salvaged by mediastinal irradiation and chemotherapy. CONCLUSIONS: Recommendation for the future management of Stage I seminoma include: reduced biological target volume to the paraaortal lymph nodes (from lumbar vertebra L1 to L5). Complete elimination of irradiation to the pelvic nodes is warranted. Radiation dose should not exceed 30 Gy. PMID- 8635937 TI - Olsalazine is contraindicated during pelvic radiation therapy: results of a double-blind, randomized clinical trial. AB - PURPOSE: A randomized clinical trial from Great Britain suggested a possible beneficial effect of acetylsalicylate in the prevention of radiation-induced bowel toxicity. Olsalazine is an orally administered drug designed to deliver 5 aminosalicylate to the large bowel with minimal systemic absorption. A randomized clinical trial was undertaken to assess the effectiveness of olsalazine in preventing acute diarrhea in patients receiving pelvic radiation therapy. METHODS AND MATERIALS: Patients receiving pelvic radiation therapy were randomized, in double-blind fashion, to olsalazine 250 mg, two capsules twice daily, or an identical appearing placebo, two capsules twice daily. Patients were then evaluated weekly during radiation therapy for the primary study endpoint, diarrhea, as well as rectal bleeding, abdominal cramping, and tenesmus. RESULTS: The study was closed early, after entry of 58 evaluable patients, when a preliminary analysis showed excessive diarrhea in patients randomized to olsalazine. The incidence and severity of diarrhea were worse in patients randomized to olsalazine (p = 0.0036). Sixty percent of the patients randomized to olsalazine experienced Grade 3 or 4 diarrhea compared to only 14% randomized to placebo. There was also a trend toward higher incidence and greater severity of abdominal cramping in patients who were randomized to olsalazine (p = 0.084). CONCLUSION: Administration of olsalazine during pelvic radiation therapy resulted in an increased incidence and severity of diarrhea. Olsalazine is contraindicated in patients receiving pelvic radiation therapy. PMID- 8635939 TI - Effect of adenosine 3':5'-cyclic monophosphate and inhibition of protein kinase A on heat sensitivity in H35 hepatoma cells. AB - PURPOSE: To investigate the role of the cyclic adenosine 3':5'-monophosphate (AMP) signal transductions pathway in heat-induced cell death and the development of thermotolerance. METHODS AND MATERIALS: Reuber H35 rat hepatoma cells were heated after preincubation with various compounds known to modulate the cyclic AMP signal transduction pathway. Cell survival was determined by colony-forming ability. RESULTS: Preincubation of H35 cells with forskolin, a stimulator of adenylate cyclase, in combination with IBMX (3-isobutyl-1-methylxanthine), an inhibitor of cyclic AMP phosphodiesterase, results in thermosensitization. Similar results are obtained with various cyclic AMP analogs. Maximum thermosensitization occurs with 0.5 mM dibutyryl cyclic AMP (DBcAMP) after a preincubation period of 5 h and heating in the presence of the drug. The same relative degree of thermosensitization is found with 8-Cl-cAMP, but at a 10-fold lower concentration. Thermosensitization by DBcAMP is prevented by H89, a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Without additional cyclic AMP-inducing factors, H89 induces thermoprotection. None of the drug treatments are cytotoxic at 37 degrees C. DBcAMP does not affect the development of heat-induced thermotolerance but it reduces its expression to an extent similarly found in the observed thermosensitization in nonthermotolerant cells. CONCLUSION: The results strongly indicate that the cyclic AMP signal transduction pathway is involved in the process of heat-induced cell death. DBcAMP reduces the expression of thermotolerance, but does not affect its induction. PMID- 8635941 TI - A method of incorporating organ motion uncertainties into three-dimensional conformal treatment plans. AB - PURPOSE: We describe a method of incorporating organ motion into three dimensional (3D) conformal treatment plans, which predicts the effect of organ motion on the calculated dose to both the clinical target volume (CTV) and nontarget organs. METHODS AND MATERIALS: The method is based on measurements of organ motion by means of multiple computed tomography (CT) scans from a group of "reference" patients, in which the data consist of previously drawn contours of the target and nontarget organs. A computer program records the differences in contour position and shape that occur between scans in the reference data, and according to those differences adjusts the contours and dose calculation points of a "study" patient currently being planned, thus simulating organ motion. Dose volume histograms (DVHs) are accumulated, and the process is repeated over the set of reference patient scans, resulting in a set of treatment plans that are ranked according to a dose-based endpoint. Two plans are selected corresponding to specified lower and upper confidence limits in the endpoint, and the DVHs from these plans are displayed for comparison with the DVHs from the nominal plan in the absence of motion. RESULTS: As an example of the method's use, it is applied to a 6-field conformal treatment plan for prostate cancer. Confidence limit DVHs of the CTV and rectal wall (in which the plans were ranked by probabilities for tumor control and normal tissue complication, respectively) are presented and compared to those from the nominal plan. CONCLUSION: The method provides a means of estimating the uncertainty in dose delivered by a treatment plan when organ motion is present. It is generally applicable to any treatment site for which data in the form of multiple CT scans are available, and can be extended to include other treatment uncertainties such as variation in patient positioning. PMID- 8635940 TI - High-precision prostate cancer irradiation by clinical application of an offline patient setup verification procedure, using portal imaging. AB - PURPOSE: To investigate in three institutions, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis [AvL]), Dr. Daniel den Hoed Cancer Center (DDHC), and Dr, Bernard Verbeeten Institute (BVI), how much the patient setup accuracy for irradiation of prostate cancer can be improved by an offline setup verification and correction procedure, using portal imaging. METHODS AND MATERIALS: The verification procedure consisted of two stages. During the first stage, setup deviations were measured during a number (Nmax) of consecutive initial treatment sessions. The length of the average three dimensional (3D) setup deviation vector was compared with an action level for corrections, which shrunk with the number of setup measurements. After a correction was applied, Nmax measurements had to be performed again. Each institution chose different values for the initial action level (6, 9, and 10 mm) and Nmax (2 and 4). The choice of these parameters was based on a simulation of the procedure, using as input preestimated values of random and systematic deviations in each institution. During the second stage of the procedure, with weekly setup measurements, the AvL used a different criterion ("outlier detection") for corrective actions than the DDHC and the BVI ("sliding average"). After each correction the first stage of the procedure was restarted. The procedure was tested for 151 patients (62 in AvL, 47 in DDHC, and 42 in BVI) treated for prostate carcinoma. Treatment techniques and portal image acquisition and analysis were different in each institution. RESULTS: The actual distributions of random and systematic deviations without corrections were estimated by eliminating the effect of the corrections. The percentage of mean (systematic) 3D deviations larger than 5 mm was 26% for the AvL and the DDHC, and 36% for the BVI. The setup accuracy after application of the procedure was considerably improved (percentage of mean 3D deviations larger than 5 mm was 1.6% in the AvL and 0% in the DDHC and BVI), in agreement with the results of the simulation. The number of corrections (about 0.7 on the average per patient) was not larger than predicted. CONCLUSION: The verification procedure appeared to be feasible in the three institutions and enabled a significant reduction of mean 3D setup deviations. The computer simulation of the procedure proved to be a useful tool, because it enabled an accurate prediction of the setup accuracy and the required number of corrections. PMID- 8635938 TI - Successful treatment of radiation-induced fibrosis using Cu/Zn-SOD and Mn-SOD: an experimental study. AB - PURPOSE: To establish how far liposomal copper/zinc superoxide dismutase (Cu/Zn SOD) and manganese superoxide dismutase (Mn-SOD), respectively, reduce radiation induced fibrosis (RIF), using a well-characterized pig model of RIF permitting the design of a controlled laboratory experiment. METHODS AND MATERIALS: In this model of acute localized gamma irradiation simulating accidental overexposure in humans, three groups of five large white pigs were irradiated using a collimated 192Ir source to deliver a single dose of 160 Gy onto the skin surface (100%) of the outer side of the thigh. A well-defined block of subcutaneous fibrosis involving skin and skeletal muscle developed 6 months after irradiation. One experimental group of five pigs was then injected i.m. with 10 mg/10 kg b.wt. of Cu/Zn-SOD, twice a week for 3 weeks, and another experimental group of five was injected with 10 mg/10 kg b.wt. of Mn-SOD, three times a week for 3 weeks. Five irradiated control pigs were injected with physiological serum. Animals were assessed for changes in the density of the palpated fibrotic block and in the dimensions of the projected cutaneous surface. Block depth was determined by ultrasound. Physical and sonographic findings were confirmed by autopsy 12-14 weeks after completing SOD injections. The density, length, width, and depth of the fibrotic block, and the areas and volume of its projected cutaneous surface were compared before treatment, 1, 3, and 6 weeks thereafter, and at autopsy, 12 14 weeks after treatment ended. RESULTS: The experimental animals exhibited no change in behavior and no abnormal clinical or anatomic signs. Whether they were given Cu/Zn- or Mn-SOD, significant and roughly equivalent softening and shrinking of the fibrotic block were noted in all treated animals between the first week after treatment ended and autopsy, when mean regression was 45% for length and width, 30% for depth, and 70% for area and volume. Histologic examination showed completely normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissue in experimental animals and the 50% decrease in the linear dimensions of the scar were comparable to the results obtained in previous clinical studies and highly significant compared to the clinical and autopsy results for the control animals. CONCLUSIONS: Our results are striking and comparable to the results obtained in our previous clinical study after liposomal Cu/Zn-SOD treatment. To our knowledge, this is the first time that two agents have been shown to reverse the radiation-induced fibrotic process in experimental animals and to permit the regeneration of normal tissue in a zone of well-established postirradiation fibrosis. PMID- 8635942 TI - Concurrent cisplatin, etoposide, and radiotherapy for unresectable stage III nonsmall cell lung cancer: a phase II study. AB - PURPOSE: Prognosis of unresectable Stage III nonsmall cell lung cancer (NSCLC) treated with thoracic radiotherapy alone has been disappointing. In recent years, several Phase III trials have demonstrated encouraging results with induction chemotherapy, but with poor long-term local control. Concurrent cisplatin alone during the radiation therapy course has resulted in improved local control, but without efficacy on occult metastatic disease. Intensification of chemotherapy during radiation has the potential of improving both local control and metastasis free survival. This Phase II study was undertaken to determine the feasibility, toxicity, response rate, local control, and survival of concurrent chemotherapy with cisplatin-etoposide and radiotherapy in unresectable Stage IIIA and IIIB nonsmall cell lung cancer. METHODS AND MATERIALS: Between February 1992 and April 1993, 50 patients with either medically or technically inoperable Stage III NSCLC were treated with concurrent chemoradiotherapy. Thoracic radiotherapy was administered to a total dose of 60 Gy. Concurrent chemotherapy consisted of cisplatin 20 mg/m2/day plus etoposide 50 mg/m2/day, from day 1 through day 5, every 4 weeks for four cycles. Medically operable patients were evaluated for surgical resection after 45 Gy and two cycles of concurrent chemotherapy. All patients received an esophagitis preventive regimen. RESULTS: Response rate was 84%, including 68% complete response. With a minimum follow-up of 23 months, overall survival was 70% at 1 year, 39.7% at 2 years, and 34.7% at 3 years. Median survival was 18 months. Age, performance status, histologic type and grade, and stage and tumor size, did not influence survival, with the exception of contralateral nodal involvement (p = 0.0055). Patients achieving a complete response (n = 34) had a 2-year survival of 58.4% compared to 0% for nonresponders (p < 0.0001). Patients who could benefit from surgery (n = 9) had a 2-year survival of 77.8% compared to 31.2% for nonoperated patients (p < 0.013). Seventeen patients (34%) are currently alive and free of disease. Actuarial local control was 63.4% at 1 year, and 58.5% at 2 and 3 years, respectively. Major hematologic toxicity occurred in 24% of the patients. CONCLUSIONS: Concomitant chemoradiotherapy with cisplatin and etoposide at this dose level is a well tolerated outpatient regimen, which resulted in a high local control rate, and an encouraging survival at 1, 2, and 3 years. A direct comparison of this treatment schedule to induction chemotherapy followed by radiotherapy, or concurrent chemoradiation therapy using cisplatin alone, appears warranted. PMID- 8635943 TI - Feasibility study of the treatment of primary unresectable carcinoma of the pancreas with 103Pd brachytherapy. AB - PURPOSE: The purpose of this study was to assess the feasibility of 103Pd brachytherapy in the management of primary unresectable carcinoma of the pancreas. METHODS AND MATERIALS: Between August 1988 and January 1992, 11 patients with biopsy-proven primary unresectable adenocarcinoma of the pancreas were treated with 103Pd brachytherapy during laparotomy. The median age was 66 (range 57-70). The most common presenting symptoms were weight loss (eight patients), pain (six patients), and nausea/vomiting (four patients). Less common symptoms were jaundice (two patients), early satiety (two patients), and ascites (one patient). All patients underwent laparotomy and surgical staging. Eight patients had T3N0M0 disease, two patients had T3N1M0 disease, and one patient had T3N1M1 disease. The surgical procedure performed was biliary bypass in six patients, biopsy only in four patients, and gastric bypass in one patient. The average tumor dimension was 4.0 cm. The median activity, matched peripheral dose (MPD) and implanted volumes were 95.3 mCi, 124.4 Gy, and 33 cm3, respectively. The median initial dose rate was 0.21 Gy per hour. Five patients received postoperative external beam radiation therapy (median 45 Gy) and seven patients received chemotherapy postoperatively. The median follow-up was 7 months (range 1 19). RESULTS: The median survival for the entire group of patients was 6.9 months. Ten of 11 patients have died, with 1 patient presently alive and receiving chemotherapy for metastatic disease to the liver, but without local progression radiographically. Five of 11 patients (45%) were locally controlled, defined as either a complete response or freedom from progression at the site of the implant as evaluated by computed tomography scan. In the other six patients, the median time to local progression was 6.9 months. Five patients developed distant metastases (four liver, one subcutaneous nodule). Two patients failed in regional sites (one omentum, one paraaortic lymph node). Four of 11 patients (36%) developed acute postoperative complications that included one gastric outlet obstruction, one duodenal perforation, and two with sepsis. One of 11 patients (9%) developed a late complication of radiation enteritis 5 months after implantation. The median survival for patients experiencing complications was 1.7 months as compared to 8.4 months for the patients who did not develop a complication (p = 0.10). Pain relief was obtained in five out of six (83%) of the patients presenting with pain for a median duration of 24 weeks. Local control did not appear to be related to the MPD, dose rate, implanted volume, treatment with external beam irradiation, or the use of chemotherapy. Patients were more likely to develop a complication if the MPD was greater than 115 Gy (four out of six patients) as compared to those whose MPD was less than 115 Gy (one out of five patients) (p = 0.12). CONCLUSIONS: Because there was no improvement in median survival over conventional modalities, and the complication rate was high; we do not recommend 103Pd brachytherapy as a component of the treatment of unresectable adenocarcinoma of the pancreas. PMID- 8635944 TI - Entire hemithorax irradiation following complete resection in patients with stage II-III invasive thymoma. AB - PURPOSE: To evaluate the feasibility and efficacy of prophylactic entire hemithorax irradiation (EH) in addition to mediastinal irradiation (MRT) following a complete resection in Stage II-III invasive thymoma. METHODS AND MATERIALS: Forty-three patients with invasive thymoma treated with surgery and radiation therapy between 1978 and 1993 were analyzed retrospectively. All 43 patients underwent a complete surgical resection and were judged to have Masaoka's Stage II-III invasive thymoma. Of these, 23 patients received EH and MRT (EH-MRT) and the remaining 20 received MRT. Of the 23 patients with EH-MRT, 11 were Stage II and 12 Stage III. Of the 20 with MRT, 11 were Stage II and 9 Stage III. In most cases, EH was 15 Gy per 15 fractions over 3 weeks (without lung compensation calculation). In both the EH-MRT and MRT group, the total radiation doses to the mediastinum were similar with a median of 40 Gy. The median follow-up time after surgery was 63 months and no patients were lost to follow-up. RESULTS: Only one of the 23 patients with EH-MRT relapsed. On the other hand, eight of the 20 with MRT relapsed, six of whom died of disease. The pleura was the most common site of failure. At 5 years, the relapse-free rate was 100% for those receiving EH-MRT and 66% for those with MRT (p = 0.03); the overall survival rate was 96% for those with EH-MRT, and 74% for those with MRT (p: not significant). The only significant treatment-related complication was radiation pneumonitis requiring treatment, in one patient who received MRT and three who received EH-MRT, including one death of a 72-year-old man and one 68 year-old woman with severe lung fibrosis. CONCLUSION: Except for elderly patients, EH-MRT following a macroscopically complete resection appears to be safe and feasible, and can reduce intrathoracic relapses. PMID- 8635945 TI - Pelvic fractures following irradiation for endometrial carcinoma. AB - PURPOSE: To investigate the incidence and etiologic factors of pelvic fractures following radiation therapy for endometrial carcinoma. METHODS AND MATERIALS: Tumor registry and radiation oncology records of patients treated for endometrial carcinoma at The Toledo Hospital between April 1989, and December 1992, were reviewed. Patients identified as having pelvic fractures without the presence of metastatic disease underwent total body mineral density measurement with dual x ray densitometry. RESULTS: Two of 75 patients (2.7%) were found to have pelvic fractures an average of 29 months from the completion of postoperative irradiation. One patient, who received preoperative irradiation, was also identified as having developed a fracture of the pelvis and was included in the analysis. All patients were treated prone with 10-15 MV photons in four fields daily. All three fracture patients received 45 Gy external beam radiation therapy. The two postoperative patients each received a single vaginal brachytherapy application delivering 20 Gy to 0.5 cm deep to the vaginal mucosa with a vaginal cylinder containing 30 mgRaeq 137Cs. The preoperative patient received a single brachytherapy application with tandem and colpostats delivering 20 Gy to point A. Only one of the three fracture patients had the entire pubis included in the field of external beam treatment. One patient was taking nonsteroidal anti-inflammatory medication, one patient thyroid hormone replacement, and one patient was taking both types of medication. CONCLUSION: The etiology of pelvic fractures after irradiation is multifactorial. A complete medication history should be obtained, and care should be exercised in positioning the radiation fields to avoid inclusion of the entire pubis prior to the initiation of the radiation treatment. PMID- 8635946 TI - Endosonographic and color Doppler flow imaging alterations observed within irradiate rectal cancer. AB - PURPOSE: To correlate the endosonographic and color Doppler flow imaging alterations observed in irradiated rectal cancers with the pathologic features of radiation response, and to evaluate the potential impact of altered blood flow on the integrity of the surgical anastomosis. METHODS AND MATERIALS: Endosonography with color and pulsed wave Doppler was performed on 20 rectal cancer masses before and after high dose preoperative radiation (XRT). Pre- and post-XRT observations included comparing alterations in tumor size, sonographic echotexture, color Doppler flow, and pulsatility indices. Comparisons were made with pathologic findings in the irradiated specimens and with the incidence of anastomotic failure. RESULTS: Compared to pre-XRT observations, irradiated rectal cancers decreased in size and became either mixed in echogenicity with less apparent color Doppler flow (16 of 20) or unchanged in color Doppler flow and echotexture (4 of 20). Those with less flow (16 of 20) were imaged later (mean = 90.2 +/- 12.1 days) than those without change in color Doppler flow (mean = 21.7 +/- 2.7 days). Pathologically, the group of four without change in color Doppler signal had features of acute inflammation which were not observed in 16 of 20 imaged later. Based on pulsatility index measurements, both high and low resistance vessels were detected and confirmed by immunohistochemical staining, and features of postradiation obliterative vasculitis were observed. Only one primary anastomosis in 14 patients with decreased flow failed. CONCLUSIONS: The sonographic and color Doppler flow imaging alterations observed within irradiated rectal cancer correlated with changes of postradiation obliterative vasculitis. The apparent diminished local blood flow within high and low resistance vessels post-XRT did not result in an increased incidence of anastomotic failures. PMID- 8635947 TI - Analysis of localization errors in the definition of the mantle field using a beam's eye view treatment-planning system. AB - PURPOSE: Reports of the treatment of Hodgkin's disease (HD) with radiotherapy using the mantle field technique have demonstrated that coverage of disease by the field blocks significantly compromises outcome. It is our hypothesis that the availability of computerized tomography images reduces the incidence of localization error, and that the use of beam's eye view treatment planning techniques may further improve localization. The purpose of this report is to assess the possible contribution of a three-dimensional treatment-planning system to tumor localization and mantle block drawing in patients with HD. MATERIALS AND METHODS: We evaluated the localization error rate of four experienced radiation oncologists as they drew the lung blocks for the mantle field. The analysis included 16 patients treated with mantle fields in our department between 1989 and 1991. In each case our computerized three-dimensional treatment planning system was used to generate a beam's eye view display of tumor volumes. Simulation radiographs for all 16 patients were overlaid with acetate film, and lung blocks were drawn by clinicians using only the simulation radiographs for reference. The process was repeated with the thoracic CT scans available for reference. The mantle block contours for each trial were then superimposed upon the beam's eye view plots of tumor volumes. The beam's eye view plot was our benchmark for the evaluation of errors of tumor localization. Localization errors were defined as touching or overlap of the shielding blocks onto tumors. RESULTS: There was a high degree (p < 0.0003) of consistency in scoring across all pairing of clinicians and the results from all four were polled for the analysis. The overall error rate using the simulation radiographs alone was 18%. The rate was significantly lower (13%) when the CT images were available (p = 0.038). The axillary region had the highest localization error rate (41.7% with CT available and 27.1% with CT available) and the superior mediastinum had the lowest error rate (10.7% without CT, 8.5% with CT). Compared with a system such as beam's eye view, which could reduce the localization error rate to zero, the error rate with CT scans available is still significant [95% confidence interval (CI = 10 17.1%)]. Localization errors were more likely with increasing tumor size when CT scans were not available (p = 0.029). A similar trend was not seen when CT scans were available (p = 0.2). In a multivariate analysis, the use of CT scans predicted for reduced localization error rate (p = 0.03). Tumors in the axilla and inferior mediastinum had a greater relative risk than those in the superior mediastinum (p = 0.0001) CONCLUSION: The availability of CT imaging offers an advantage in the outlining of the mantle field in the treatment of Hodgkin's disease. When the error rate is evaluated using a beam's eye view treatment planning system, a significant proportion of tumors may be overlapped by the outlined mantle blocks even when CT images are available for reference. The use of beam's eye view treatment planning in mantle field definition, especially for tumors in the axillary region, may reduce the incidence of geographic misses. PMID- 8635948 TI - Monte Carlo-based treatment planning for boron neutron capture therapy using custom designed models automatically generated from CT data. AB - PURPOSE: A Monte Carlo-based treatment planning code for boron neutron capture therapy (BNCT), called NCTPLAN, has been developed in support of the New England Medical Center-Massachusetts Institute of Technology program in BNCT. This code has been used to plan BNCT irradiations in an ongoing peripheral melanoma BNCT protocol. The concept and design of the code is described and illustrative applications are presented. METHODS AND MATERIALS: NCTPLAN uses thin-slice Computed Tomography (CT) image data to automatically create a heterogeneous multimaterial model of the relevant body part, which is then used as input to a Monte Carlo simulation code, MCNP, to derive distributions within the model. Results are displayed as isocontours superimposed on precisely corresponding CT images of the body part. Currently the computational slowness of the dose calculations precludes efficient treatment planning per se, but does provide the radiation oncologist with a preview of the doses that will be delivered to tumors and to various normal tissues, and permits neutron irradiation times in Megawatt minutes (MW-min) to be calculated for specific dose prescriptions. The validation of the NCTPLAN results by experimental mixed-field dosimetry is presented. A typical application involving a cranial parallel-opposed epithermal neutron beam irradiation of a human subject with a glioblastoma multiforme is illustrated showing relative biological effectiveness-isodose (RBE) distributions in normal CNS structures and in brain tumors. Parametric curves for the MITR-II M67 epithermal neutron beam, showing the gain factors (gain factor = minimum tumor dose/maximum normal brain dose) for various combinations of boron concentrations in tumor and in normal brain, are presented. RESULTS: The NCTPLAN code provides good computational agreement with experimental measurements for all dose components along the neutron beam central axis in a head phantom. For the M67 epithermal beam the gain factor for 1, boronophenylalanine for a small midline brain tumor under typical distribution assumptions is 1.4-1.8 x . Implementation of the code under clinical conditions is demonstrated. CONCLUSION: The NCTPLAN code has been shown to be well suited to treatment-planning applications in BNCT. Comparison of computationally derived dose distributions in a phantom compared with experimental measurements demonstrates good agreement. Automatic superposition of isodose contours with corresponding CT image data provides the ability to evaluate BNCT doses to tumor and to normal structures. Calculation of gain factors suggests that for the M67 epithermal neutron beam, more advantage is gained from increasing boron concentrations in tumor than from increasing the boron tumor-to-normal brain ratio. PMID- 8635949 TI - Collimator-related radiation dose for different cobalt machines and linear accelerators. AB - PURPOSE: In previous publications (12, 13) measurements are described of the dose outside the primary beam (the peripheral dose (PD)) for 60Co gamma radiation to 25 MV photons. Comparison with data published by other investigators for different treatment machines, showed good agreement. This can only be explained when the contribution to the PD of radiation leaking through and scattering from the collimator does not differ considerably between treatment machines from different manufactures, and it is the purpose of this article to investigate whether this assumption is valid. METHODS AND MATERIALS: A request was sent out to all radiotherapy departments in The Netherlands and one in Belgium to measure the dose outside the primary beam for as many machines as possible. The following geometry was given: field sizes of 10 x 10 cm2 and 20 x 20 cm2 at distances of 30 and 50 cm, for collimator angles 0 degrees and 90 degrees at the standard source surface distance. This, therefore, resulted in a dataset of eight measurements per photon energy. RESULTS: Data were collected for four cobalt machines and 37 linear accelerators, from seven different manufacturers. All together 56 datasets were collected for 12 different photon energies. Although the variation of the leakage radiation dose is small, there can be differences of about 50% in the collimator scatter dose between collimator angles of 0 degrees and 90 degrees, depending on the collimator design or on the design of the flattening filter. For dual energy machines with a large gap between the low and the high energy, the values for the high energy are higher by about 40%. Old cobalt machines show higher leakage radiation dose than modern ones. CONCLUSION: Although there is no large variation in leakage radiation dose between different makes of accelerators, some show higher collimator scatter dose than others. The magnitude of the leakage radiation dose is well within regulatory limits. As the PD not only consists of a contribution from collimator-related radiation, but also of patient scatter, the differences are negligible when estimating the peripheral dose for an individual patient. PMID- 8635950 TI - Radiotherapy vs. surgery for prostate cancer: an age old question. PMID- 8635951 TI - Translational research in bladder cancer. PMID- 8635952 TI - Para-aortic radiotherapy for stage I seminoma: a new standard. PMID- 8635953 TI - New wine in an old bottle? Dose escalation under dose-volume constraints: a model of conformal therapy of the prostate. PMID- 8635954 TI - Correction for the use of the SOMA LENT tables. PMID- 8635955 TI - Low-dose splenic irradiation in human immunodeficiency virus-related immune thrombocytopenia. PMID- 8635956 TI - Progress toward a thermal dosimetry system? PMID- 8635957 TI - Cracking walnuts with sledge hammers--an Australian perspective on Griep et al. IJROBP 32(5):1347-1350; 1995. PMID- 8635958 TI - More opinions on effects of reforming veterinary education. PMID- 8635959 TI - Recommendation for disclosure of financial interests. PMID- 8635960 TI - Need training for application of insecticides. PMID- 8635961 TI - Encomium on James Herriot. PMID- 8635962 TI - Control charts and the value of statistical inference for food animal practitioners. PMID- 8635963 TI - What is your diagnosis? Cranial displacement of the small intestine and substantial abdominal distention caused by a large intra-abdominal lipoma. PMID- 8635964 TI - Sepsis of the ulnaris lateralis bursa and elbow joint in a horse. PMID- 8635965 TI - Vaccination of dogs and cats in veterinary teaching hospitals in North America. PMID- 8635966 TI - Factors affecting treatment decisions and satisfaction of owners of cats with cancer. AB - Cancer in cats is being diagnosed with increasing frequency. Euthanasia or an active intervention such as chemotherapy, radiation therapy, or surgery are treatment choices for the owner at diagnosis of the cat's disease. In this study, 2 interviews with cat owners, one soon after diagnosis of cancer in the cat and one 6 months later, were used to identify owner characteristics associated with a decision of euthanasia or intervention, to identify factors associated with an owner's satisfaction with euthanasia or intervention, and to evaluate inappropriate expectations of the owners who selected an intervention. The study included 89 owners from 3 referral hospitals. In logistic regression analysis, significant factors were not found that affected the owner's decision to euthanatize the cat versus intervene. Satisfaction with the decision to euthanatize the cat was associated with the ability of the cat to groom itself, eat, and play at the first interview. Among owners who selected an intervention, 4 combinations of factors were associated with being satisfied. The first combination was clinic of origin (CLIN), having a live cat at the 6-month follow up interview (LIVE), and understanding the number of return visits required for the intervention. The second was CLIN, LIVE, and type and frequency of adverse effects from the intervention at the 6-month interview. The third was CLIN, LIVE, and feeling guilty at the 6-month interview. The fourth was CLIN, LIVE, and whether the cat had a good or excellent quality of life at the first interview. Thirty percent (21/69) of the owners tended to overestimate their cats' life expectancy. Owners also felt they had reasonably accurate estimations of adverse effects of treatment and number of return visits, but underestimated the costs required for an intervention. For owners who elect an intervention, a reminder from the veterinarian that emotional upheavals may develop even after the decision has been made is important. To provide optimal patient care and client education, veterinarians must find a middle ground between being knowledgeable, practical, and informed, and being compassionate and approachable. PMID- 8635967 TI - Gender redistribution in the veterinary medical profession. PMID- 8635968 TI - Use of the holmium yttrium aluminum garnet laser for percutaneous thoracolumbar intervertebral disk ablation in dogs. AB - OBJECTIVE-To evaluate a laser ablation-technique for treatment of thoracolumbar intervertebral disk disease in dogs. DESIGN-Prospective case series. ANIMALS-33 dogs with signs of recurrent back pain associated with intervertebral disk disease after the initial episode had been managed conservatively for a minimum of 2 weeks. PROCEDURE-Spinal needles were placed percutaneously through the annulus fibroses to permit delivery of an optical fiber into the nucleus pulposes of thoracolumbar intervertebral disks T10-11 through L3-4. Fluoroscopy was used to guide needle placement. Holmium yttrium aluminum garnet laser energy then was used to ablate the contents of each selected intervertebral disk. Intervals from time of treatment to time of assessment ranged from 3 to 114 weeks. RESULTS-All dogs recovered without complication. Results of follow-up radiography and histologic evaluation indicated that percutaneous holmium yttrium aluminum garnet laser ablation reduces the volume of nucleus pulposus in treated disks. CLINICAL IMPLICATIONS-Used as a clinical treatment and prophylactically, this minimally invasive procedure should prevent further extrusion of partially herniated disks and should reduce the chances of subsequent herniation of disks at other treatment sites. PMID- 8635969 TI - One-year follow-up evaluation of magnetic resonance imaging of the brain in dogs with pituitary-dependent hyperadrenocorticism. AB - OBJECTIVE: To evaluate magnetic resonance imaging (MRI) brain scans of dogs with pituitary-dependent hyperadrenocorticism (PDH) and no signs of CNS dysfunction 1 year after diagnosis and initial MRI. DESIGN: Prospective study of surviving dogs from a previous study. ANIMALS: 13 dogs underwent MRI of the brain at the time that PDH was diagnosed and prior to treatment. At that time, none of the dogs had clinical signs suggestive of an intracranial mass. Approximately 1 year after diagnosis and MRI, the brain was again evaluated by MRI. RESULTS: On the initial MRI scan, 5 of the 13 dogs had normal findings, and 8 had evidence of a mass (tumor) in the area of the pituitary gland. Of the 5 dogs that had no visible pituitary mass on the initial MRI scan, 3 had a normal MRI brain scan 1 year later. Of the 5 dogs that had no visible pituitary mass on initial MRI scan, 2 had a visible pituitary mass at 1 year. The 8 dogs that had a visible mass on the initial MRI brain scan had easily identified pituitary masses on the second MRI scan. Of these 8 dogs, 4 had no apparent change in pituitary mass size, and 4 had obvious increase in vertical height of the pituitary mass. Of the 4 dogs, 2 developed signs of neurologic dysfunction within 1 year after diagnosis of PDH, presumably attributable to that mass. Of the 13 dogs, 12 were treated with mitotane soon after completion of the initial MRI scan. Sensitivity to mitotane and initial pituitary mass size or growth were not correlated. Of the 13 dogs evaluated initially and 1 year after diagnosis, 10 had pituitary masses identified on MRI brain scans. CLINICAL IMPLICATIONS: The incidence of visible pituitary masses among dogs with PDH at the time of or within a year of diagnosis was > 75%. In 2 dogs, signs of CNS dysfunction developed within 1 year of PDH diagnosis when pituitary masses were > or = 10 mm. PMID- 8635970 TI - Nephron sparing by partial median nephrectomy for treatment of renal hemangioma in a dog. AB - A 6-year-old neutered male Golden Retriever was admitted for evaluation of intermittent hematuria of 2 months' duration. A 3-cm heterogeneous mass causing distortion of the caudomedial aspect of the left kidney was detected via ultrasonography. Histologic examination of a renal tissue sample obtained by ultrasound-guided biopsy revealed a telangiectatic vascular plexus of unknown origin. Low glomerular filtration rate was identified by a modified exogenous creatinine clearance test. Excretory urography revealed a filling defect in the medial aspect of the caudal pole of the kidney, near the hilus. Because total renal function was low, a decision was made to perform nephron-sparing surgery involving resection of centrally located renal parenchymal and pelvic tissue by en bloc resection in the median plane, instead of radical nephrectomy. After surgery, the hematuria resolved and further decrease in renal function was not evident. Nephron-sparing surgery is a viable option for dogs with compromised renal function when there is concern that radical nephrectomy may precipitate uremia. PMID- 8635971 TI - Clinical, hematologic, and biochemical features of a syndrome in Bernese mountain dogs characterized by hepatocerebellar degeneration. AB - Seven related Bernese Mountain Dogs developed a syndrome Characterized by progressive cerebellar and hepatic disease. Clinically, stiffness in the hind limbs, mild incoordination, and a slight head tremor were first noticeable when pups were 4 to 6 weeks old. The condition progressed, causing pups to assume a wide-based stance. Other signs included head bobbing, spontaneous nystagmus, and, finally, paresis. Hematologic findings included leukocytosis with a left shift; normocytic, normochromic anemia; hypoproteinemia, low serum creatinine, and urea nitrogen concentrations; excessive fasting plasma ammonia concentration; and an increase in concentration of serum bile acids. Portal venography performed on 1 dog revealed a small liver and extensive extrahepatic varicosities. Necropsy revealed cerebellar hypoplasia, nodular liver, extensive abdominal varicosities, and ascites. Histologically, degeneration and depletion of Purkinje's cells and vacuolation, degeneration, and nodular regeneration of hepatic tissues were evident. Preliminary analysis of the pedigree was suggestive of an autosomal recessive pattern of inheritance. PMID- 8635972 TI - Allogeneic marrow transplantation in a cat with acute myeloid leukemia. AB - A listless, anorectic 2-year-old cat was found to have a normocytic, nonregenerative anemia. A diagnosis of acute myeloid leukemia type M6Er was made. Because the cat was young, had negative FeLV and feline immunodeficiency virus test results, had a sibling that could be used as a bone marrow donor, had received only 1 transfusion, and was in good health other than being anemic, allogeneic bone marrow transplantation (BMT) was attempted using marrow from the cat's sibling. The most life-threatening complication following BMT was hemorrhage caused by severe thrombocytopenia. Complete hematopoietic engraftment was evident 36 days after BMT. The cat then was discharged to its owner. In the first year, the cat continued to improve with the exception of an intractable dermatophytosis, which resolved eventually. The cat continues to do well 4 years after BMT. To our knowledge, this represents the first report of successful allogeneic BMT for a cat with acute myeloid leukemia. PMID- 8635973 TI - Clinical and pathologic findings in ferrets with lymphoma: 60 cases (1982-1994). AB - OBJECTIVE: To examine clinical and pathologic findings in 60 ferrets with lymphoma. DESIGN: Retrospective case series. ANIMALS: 60 ferrets in which the diagnosis of lymphoma had been confirmed by means of histologic examination of biopsy or necropsy specimens. PROCEDURE: Information including age, sex, coat color, history, clinical signs, clinicopathologic abnormalities, treatment, outcome, and results of histologic examination of biopsy and necropsy specimens were retrieved from medical records of ferrets with spontaneous lymphoma examined between 1982 and 1994 at the Massachusetts institute of Technology or private veterinary practices in 10 states. Classification of lymphoma was assigned according to the National Cancer Institute's working formulation for non Hodgkin's lymphomas. Chi 2 Trend analysis was used to determine whether age was associated with history, clinical signs, hematologic abnormalities, stage, histologic grade, or outcome. RESULTS: Acute onset, mediastinal mass, lymphocytosis, and multicentric distribution were linked with younger ferrets, and lymphopenia and survival longer than 2 months after diagnosis was associated with older ferrets. Twenty percent of ferrets in this study had cohabitated with another ferret with lymphoma. Chemotherapeutic efficacy was not evaluated. CLINICAL IMPLICATIONS: Clinical and pathologic features linked with age should be considered when evaluating diagnostic and therapeutic options for ferrets with lymphoma. PMID- 8635974 TI - Evaluation of vaccination of horses as a strategy to control equine monocytic ehrlichiosis. AB - OBJECTIVE: To determine whether preferentially vaccinated horses were at risk for exposure to Ehrlichia risticii, whether horses with equine monocytic ehrlichiosis (EME) were likely to have been nonvaccinated, and whether clinical severity and financial costs associated with care and treatment of EME were less for vaccinated horses with EME than for nonvaccinated horses with EME. DESIGN: Cross sectional and case-control studies. PROCEDURE: Information on usage of E risticii bacterins to control EME was collected for 2,587 horses located on 511 farms throughout New York. Each horse was tested for serum antibodies directed against E risticii. Data on efficacy of vaccination to reduce the prevalence and clinical severity of EME and monetary losses associated with EME were collected from 68 horses with EME and 132 clinically normal horses. RESULTS: A correlation was not detected between the county seropositive proportion and the proportion of horses vaccinated against EME. Among horses diagnosed for EME, median date of diagnosis was not delayed for vaccinated horses, compared with that for nonvaccinated horses. Mean cost per case was not significantly different for nonvaccinated horses, compared with that for vaccinated horses ($ 1,082 and $ 1,001, respectively). Vaccination was not associated with a reduction in prevalence or in severity of EME-related clinical signs. CLINICAL IMPLICATIONS: Administering killed E risticii bacterin once a year to control EME in New York appears to have limited success. Among horses in which EME was diagnosed, severity of illness and financial costs attributable to EME were indistinguishable for vaccinated and nonvaccinated horses. PMID- 8635975 TI - Benefit-cost analysis of vaccination of horses as a strategy to control equine monocytic ehrlichiosis. AB - OBJECTIVE: To determine whether horses in New York should be vaccinated against equine monocytic ehrlichiosis (EME). DESIGN: Decision-tree analyses of data from a cross-sectional study and a case-control study. SAMPLE POPULATION: Horses in New York. PROCEDURE: Annual expected monetary loss per horse attributable to EME was calculated for vaccinated and nonvaccinated horses in New York. Because risk of being seropositive was dependent on county in which the horse was located, farm elevation, and use of each horse, decision-tree analyses were stratified by these factors. RESULTS: Annual expected monetary loss per horse attributable to EME for horses vaccinated by veterinarians ranged from $ 21 to $ 21.83/horse/y; for horses vaccinated by owners ranged from $ 10 to $ 10.83/horse/y; and for nonvaccinated horses ranged from $ 0 to $ 4.03/horse/y. Assuming 78% of vaccinated horses were protected and mean losses associated with EME included costs for horses that died, annual incidence density at which expected monetary loss for vaccinated horses was equal to that for nonvaccinated horses was 12 cases/1,000 horses/y and 25 cases/1,000 horses/y for horses vaccinated by owners or by veterinarians, respectively. CLINICAL IMPLICATIONS: Annual vaccination minimizes monetary losses attributable to EME only when the annual incidence density exceeds 12 to 25 cases/1,000 horses/y. In New York, expected monetary losses are minimized when horses are not vaccinated because of the low annual incidence density in most regions. PMID- 8635976 TI - Prognosis for return to racing after recovery from infectious pleuropneumonia in thoroughbred racehorses: 70 cases (1984-1989). AB - OBJECTIVE: To determine the percentage of Thoroughbred racehorses that would be capable of racing performance after recovery from infectious pleuropneumonia. DESIGN: Retrospective case series. ANIMALS: 70 Thoroughbred horses that had recovered from pleuropneumonia. Only horses < or = 5 years old and horses > 5 years old known to be in race training at the time of illness were included in the study. RESULTS: Forty-three of the 70 (61%) horses raced after recovery, and 24 of the 43 (56%) won at least 1 race. Horses that required placement of an indwelling thoracic drain apparently did not have a worse prognosis than did horses that did not require placement of a drain. The prognosis for racing for horses that developed pleuropneumonia-associated complications (pulmonary abscess, cranial thoracic mass, bronchopleural fistula) was worse than the prognosis for horses that did not develop these complications. Duration of hospitalization was not considered indicative of outcome. CLINICAL IMPLICATIONS: In Thoroughbreds, the prognosis for return to racing after recovery from uncomplicated pleuropneumonia appears to be good. PMID- 8635977 TI - Job analysis of specialists in veterinary surgery. PMID- 8635978 TI - BSE emergency meeting inspires confidence over US safeguards. PMID- 8635979 TI - Foodborne illness prevention before slaughter? Yes! PMID- 8635980 TI - Veterinarians join bird rescue effort after oil spill. PMID- 8635981 TI - Opposing view on elimination of brucellosis reservoir. PMID- 8635982 TI - Opposition to trap-sterilize-release programs for feral cats. PMID- 8635984 TI - What is your diagnosis? Tarsal bone slab fracture in a horse. PMID- 8635983 TI - Clarification on foal with cyanoses and intense murmur. PMID- 8635985 TI - What is your neurologic diagnosis? Spinal abnormalities and syringomyelia of the lumbar spinal cord. PMID- 8635986 TI - The human side of computer technology. PMID- 8635987 TI - Some employment issues affecting veterinarians--hiring at will. PMID- 8635988 TI - Food safety symposium: responding to the changing epidemiologic characteristics of foodborne diseases. PMID- 8635989 TI - Prosthodontic appliance for repair of an oronasal fistula in a cat. AB - A 4.5-month-old Himalayan cat was evaluated because of a cleft secondary palate. Multiple surgical procedures failed to provide soft-tissue coverage of the defect. A 3-cm silastic nasal septal button was trimmed and placed in the oronasal fistula. This prosthodontic appliance provided obturation of the defect without the need for removal or cleaning. Food accumulation between the appliance and oral mucosa was minimal, avoiding the need for appliance manipulation. The owner reported clinical signs related to oronasal fistula or appliance complications were not evident 12 months after placement. PMID- 8635990 TI - Evaluation of dogs and cats with tumors of the ear canal: 145 cases (1978-1992). AB - OBJECTIVE: To characterize the frequency, clinical signs, biologic behavior, and response to treatment of tumors of the ear canal in dogs and cats. DESIGN: Retrospective analysis of medical records. ANIMALS: Medical records of 81 dogs (48 malignant tumors, 33 benign tumors) and 64 cats (56 malignant tumors, 8 benign tumors). PROCEDURE: Data were analyzed for cats and dogs with malignant tumors, and risk factors were analyzed for their potential impact on survival time. RESULTS: Malignant tumor types most commonly reported included ceruminous gland adenocarcinoma, squamous cell carcinoma, and carcinoma of undetermined origin. Median survival time of dogs with malignant aural tumors was > 58 months, whereas that of cats was 11.7 months. A poor prognosis was indicated by extensive tumor involvement (dogs) and by neurologic signs at time of diagnosis, diagnosis of squamous cell carcinoma or carcinoma of undetermined origin, and invasion into lymphatics or blood vessels (cats). CLINICAL IMPLICATIONS: Malignant tumors of the ear canal in dogs and cats have a propensity for local invasion, but tend not to metastasize. Squamous cell carcinoma and carcinoma of undetermined origin were the most locally aggressive tumors. Malignant tumors of the ear canal are best managed by aggressive surgical excision. Radiotherapy may be useful when tumors cannot be completely removed. PMID- 8635991 TI - Clinical and clinicopathologic findings in dogs with acute respiratory distress syndrome: 19 cases (1985-1993). AB - OBJECTIVE: To describe clinical and clinicopathologic findings from dogs with histologic pulmonary lesions consistent with human adult respiratory distress syndrome and to identify potential factors. DESIGN: Retrospective study. ANIMALS: 19 dogs with acute respiratory distress. PROCEDURE: Medical records of dogs were reviewed. Signalment, physical examination and clinicopathologic findings at admission, and thoracic radiographic and necropsy findings were recorded. RESULTS: The most common clinical sign was dyspnea. Respiratory rate ranged from 36 to 140 breaths/min and abnormal breathing patterns were detected. Crackles were auscultated in 7 dogs. Severe diffuse interstitial and alveolar infiltrates were observed on thoracic radiography in 9 dogs shortly after arrival and developed later in 4 dogs. Four dogs were leukopenic and neutropenic. Disseminated intravascular coagulation was diagnosed in 2 dogs, and hypoalbuminemia was found in 8 dogs. Respiratory status deteriorated rapidly in all dogs, and 10 dogs were mechanically ventilated. Death was attributed solely to respiratory failure in 8 dogs. In the other 11 dogs, severe lesions in nonpulmonary organs, sepsis, or both may have contributed to death. The most common associated conditions that may have contributed to acute respiratory failure were microbial pneumonia, sepsis, aspiration pneumonia, and shock, with more than 1 factor found in 11 of 19 dogs. CLINICAL IMPLICATIONS: The index of suspicion for acute respiratory distress syndrome should be high in dogs with bilateral pulmonary infiltrates and acute respiratory distress that rapidly progresses to failure. PMID- 8635992 TI - Respiratory function and treatment in dogs with acute respiratory distress syndrome: 19 cases (1985-1993). AB - OBJECTIVE: To characterize respiratory function and treatment in dogs with findings compatible with those of human adult respiratory distress syndrome (ARDS) and to evaluate the application in dogs of clinical for diagnosis of ARDS. DESIGN: Retrospective review of medical records. ANIMALS: 19 dogs with acute respiratory distress syndrome. RESULTS: Arterial blood pH was 7.30 +/- 0.59 (mean +/- SD). Nine dogs had metabolic acidosis. In nonmechanically ventilated dogs, PaCO2 was 15.0 to 54.9 mm of Hg. Respiratory acidosis developed in 2 mechanically ventilated dogs. Hypoxemia was observed in 4 of 5 dogs breathing room air. In 4 mechanically ventilated dogs, oxygenation was inadequate, despite use of > 60% inspired O2 and positive end-expiratory pressure in 3 dogs. Alveolar-to-arterial oxygen tension gradients were widened in 4 dogs breathing room air and in 6 dogs ventilated with 100% O2. Ten dogs were mechanically ventilated; ventilatory rate was 18 to 60 breaths/min. Tidal volume was 12 +/- 3.8 ml/kg of body weight in 4 dogs, minute ventilation > 400 ml/kg/min in 2 dogs, and peak airway pressures > 25 cm of H2O in 6 dogs. Positive end-expiratory pressure was used in 8 dogs. Pneumothorax was detected in 5 ventilated dogs. Human clinical criteria for diagnosis of ARDS were fulfilled in 7 dogs. Fluid treatment consisted of IV crystalloids and synthetic colloids. Drugs most often administered were antibiotics and loop diuretics. CLINICAL IMPLICATIONS: Human clinical criteria for identification of ARDS may be helpful in diagnosis of acute respiratory distress syndrome in dogs. PMID- 8635994 TI - Mummified fetus in a mare. AB - A 12-year-old Arabian mare with a history of repeated early embryonic losses gave birth to a mummified fetus. The fetus was not the result of a pregnancy with twins. The mare had been given a progestogen throughout gestation and expelled the mummified fetus at about 325 days of gestation, 2 weeks after progestogen treatment was discontinued. We estimate that the size of the fetus was consistent with a fetal age of 5 months. The mare and mummified fetus illustrated that progestogen administration after 100 days of gestation can promote retention of a nonviable fetus. When the fetoplacental unit is incapable of producing progestogens in adequate amounts for pregnancy maintenance at that stage of gestation, then it is also unlikely to provide sufficient oxygen and nutrients to meet the needs of the growing fetus. Monitoring fetal viability would enable practitioners to prevent prolonged retention of a nonviable fetus. PMID- 8635993 TI - Infection of the intertubercular bursa in horses: four cases (1978-1991). AB - OBJECTIVE: To determine the clinical outcome of horses treated for infection of the intertubuercular bursa (infectious bicipital bursitis). DESIGN: Retrospective analysis of case records. ANIMALS: Four horses referred for treatment of infectious bicipital bursitis. PROCEDURE: Medical records of horses that were severely lame on admission were reviewed. RESULTS: In 3 horses, palpation over the bicipital bursa as well as flexion and extension of the scapulohumeral joint were resented. Ultrasonography performed in 1 horse revealed that the bicipital bursa was large and that excessive amounts of fluid containing hyperechoic material were evident within the bicipital bursa. Two horses were treated by the administration of antimicrobial and nonsteroidal antiinflammatory drugs. Both remained lame and failed to resume their former activity. Two horses additionally were treated surgically by means of a partial synovectomy. Both resumed their former activity although a subtle lameness remained in 1 horse. CLINICAL IMPLICATIONS: Partial synovectomy may be useful in the treatment of horses with infectious bicipital bursitis. PMID- 8635995 TI - Neospora species infection in a herd of dairy cattle. AB - OBJECTIVE: To investigate the route of transmission of Neospora sp in a herd of dairy cattle in which sporadic abortions had been observed since the establishment of the farm in 1980. DESIGN: Serum samples were screened for antibodies to Neospora sp, and records from an artificial insemination program were analyzed. ANIMALS: 58 female cattle. PROCEDURE: An ELISA was used to screen serum samples of antibodies to Neospora sp. Fertility, calf mortality, and relationships between specific cattle were investigated. Statistical analysis was performed on the fertility data. RESULTS: Antibodies were detected in 17 of 58 (29%) tested cattle. All seropositive cattle were descendants of 2 cows purchased in 1980. Cattle that were descendants of those 2 cows were compared with their herdmates, but significant differences were not detected in the number of inseminations per confirmed pregnancy or in the number of cattle that required more than 1 insemination/ pregnancy. Since 1980, there were 323 confirmed pregnancies in the herd, and calf mortality (prenatal and perinatal mortality) was 24 of 323 (7%). CLINICAL IMPLICATIONS: Congenital transmission of Neospora organisms together with the apparent lack of horizontal transmission observed in the herd reported here indicated that Neospora sp has the ability to be transmitted from dam to offspring for several generations. This mode of transmission would explain the maintenance of infection in a population of cattle despite the lack of a definitive host for the parasite. PMID- 8635996 TI - Prevalence of lesions associated with subclinical laminitis in first-lactation cows from herds with high milk production. AB - OBJECTIVE: To determine prevalence of lesions associated with subclinical laminitis in first-lactation Holstein cows during early lactation and pregnant Holstein heifers during late gestation in herds with high milk production. DESIGN: Cross-sectional study. ANIMALS: 203 cattle in 13 herbs. PROCEDURE: Cattle were placed in lateral recumbency to allow visual examination and photography of their hooves. Claws on a forelimb and hind limb were examined on all cattle. Observable categories of lesions considered to be associated with subclinical laminitis in our study included yellow waxy discoloration of the sole, hemorrhage of the sole, separation of the white line, and erosion of the heel. RESULTS: Lesions in at least 1 of the categories were found in all herds. Lesions in all categories were found in 11 of 13 herds. Among claws, hemorrhage of the sole was observed most frequently in the lateral claw of the hoof of the hind limb. When days in milk was treated as a covariate, significant (P < 0.01) differences were detected in the prevalence of lesions between herds. CLINICAL IMPLICATIONS: Because the prevalence of lesions differed significantly among herds, it is logical to believe that causative factors and corrective measures also may have differed among herds. PMID- 8635997 TI - Coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping. AB - OBJECTIVE: To monitor the prevailing viral respiratory tract infections in cattle after transportation to feedlots. ANIMALS: 100 cattle with signs of respiratory tract disease on arrival at 2 feedlots. PROCEDURES: Nasal swab samples were obtained from each animal and were used for inoculation of defined cell culture systems that detected bovine viruses known to cause respiratory tract infections, as well as viruses previously not recognized as respiratory pathogens for cattle. RESULTS: Bovine respiratory coronaviruses were isolated from 38 of the 100 cattle, including 6 of 50 cattle from California, 22 of 31 cattle from Oklahoma, 6 of 11 cattle from Texas, and 4 of 8 cattle of unknown origin. Parainfluenza 3 viruses also were isolated from 5 California cattle, but other bovine viruses were not detected. CLINICAL IMPLICATIONS: The high rate of coronavirus isolations from feedlot cattle with signs of respiratory tract disease implied wide distribution and high susceptibility among cattle to this infection, which had not been detected by use of viral isolation systems in previous etiologic evaluations of feedlot cattle affected with bovine respiratory disease complex. PMID- 8635998 TI - c-Cbl is inducibly tyrosine-phosphorylated by epidermal growth factor stimulation in fibroblasts, and constitutively tyrosine-phosphorylated and associated with v Src in v-src-transformed fibroblasts. AB - The c-cbl gene was cloned as the cellular homolog of the v-cbl oncogene that is the transforming component of a murine tumorigenic retrovirus, CAS NS-1, though the biological roles of c-Cbl remain to be elucidated. We have previously reported that c-Cbl is implicated in the signal transduction triggered by granulocyte-macrophage colony-stimulating factor or erythropoietin in hematopoietic cells. Here, we observed tyrosine phosphorylation of C-cbl in cells expressing epidermal growth factor receptor depending on EGF stimulation and in v src transformed cells. Furthermore, c-Cbl was revealed to associate with v-Src in vivo. By means of binding experiments using glutathione S-transferase fusion proteins, we have found that the SH2 and SH3 domains of many proteins bind to c Cbl. These findings strongly suggest that c-Cbl is implicated in a wide variety of signal transduction pathways, including those of EGF receptor and Src protein, as well as in the signaling pathways of hematopoietic cells. PMID- 8636000 TI - Effects of nine active ingredients in Chinese herbal medicine sho-saiko-to on 2 (2-furyl)-3-(5-nitro-2-furyl)acrylamide mutagenicity. AB - The antimutagenic effects of nine active compounds in the Chinese herbal medicine "sho-saiko-to" on mutagenesis induced by a direct-acting mutagen, 2-(2-furyl)-3 (5-nitro-2-furyl)acrylamide (AF-2) were investigated in Salmonella typhimurium, strain TA100. The active compounds examined were classified into two major groups, saponins and flavonoids, the former comprising glycyrrhizin, saikosaponins a, c, and d, and ginsenosides Rb1 and Rg1, and the latter, baicalin, baicalein and wogonin. Saikosaponin a and ginsenoside Rb1 were found to reduce the mutagenicity of AF-2 significantly when applied post-AF-2-treatment in the Salmonella mutagenicity assay. Ginsenoside Rb1 also decreased the mutagenic activity of AF-2 in a simultaneous treatment protocol. The results indicate that saikosaponin a and ginsenoside Rb1 may enhance DNA repair, and ginsenoside Rb1 may also have the ability to inactivate the mutagenic activity of AF-2 directly. On the other hand, saikosaponin d and baicalin showed a slight enhancing effect. None of the compounds, except baicalein, showed any toxic effect on the test strain. These findings may be useful for the development of chemopreventive agents. PMID- 8635999 TI - A novel somatic point mutation of the RET Proto-oncogene in tumor tissues of small cell lung cancer patients. AB - We examined whether the novel point mutation from GCC (Ala) to GAC (Asp) at codon 664 in exon 11 of RET proto-oncogene, which we had found in two small cell lung carcinoma (SCLC) cell lines, existed in genomic DNA of tumor tissues of the two SCLC patients from whom these SCLC cell lines were derived. Sequence analysis revealed that point mutation identical to that of the SCLC cell lines was present in amplified alleles of single-strand conformational variants in genomic DNA of the tumor tissues, whereas it was not detected in genomic DNA of non-tumor tissues of the patients. These results indicate that this mutation had initially occurred in the SCLC patients and was of somatic origin. PMID- 8636002 TI - Absence of ras mutations and low incidence of p53 mutations in renal cell carcinomas induced by ferric nitrilotriacetate. AB - Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N-ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3' half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-att transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA. PMID- 8636001 TI - Prevention by methionine of enhancement of hepatocarcinogenesis by coadministration of a choline-deficient L-amino acid-defined diet and ethionine in rats. AB - The effects of methionine on hepatocarcinogenesis induced by coadministration of a choline-deficient L-amino acid-defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline-supplemented L-amino acid-defined (CSAA)++ diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c-myc and c Ha-ras expression, as well as generation of 8-hydroxyguanine (8-OHGua) and 2 thiobarbituric acid-reacting substances (TBARS), by coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8, or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c-myc and c-Ha-ras, 8 OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocks these actions. These results indicate that addition of methionine prevents the induction of c-myc and c-Ha-ras expression, 8 OHGua formation and TBARS generation, as well as hepatocellular lesions, by coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents. PMID- 8636004 TI - Induction of programmed death/apoptosis androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal. AB - Shionogi Carcinoma 115 (SC 115) cells are a cloned cell line derived from androgen-dependent mouse mammary tumor. They can grow in serum-free culture if a physiological level of androgen is present in the medium, but can not proliferate in culture without testosterone. In the present study, the mechanism of cell death in SC 115 cells after androgen withdrawal was examined. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, androgen withdrawal induces programmed cell death (apoptosis) of SC 115 cells in serum-free culture. Northern blot analysis was used to identify a series of genes whose expression per cell is enhanced during the recruitment of cells from a nonproliferative (i.e. G0) state into G1 (i.e.,cyclins D1 and C), from G1 into the S phase of the cell cycle (i.e., cdk2), and during the programmed cell death pathway (i.e. testosterone repressed prostatic message-2 (TRPM-2), transforming growth factor-beta1 (TGF-beta1) and glucose regulated 78 kilodalton protein (GRP-78). Expression of TRPM-2, TGF-beta1, GRP-78, and calmodulin genes increases, but that of cyclins C and D1, and cdk2 genes decreases during programmed cell death of SC 115 cells. These results demonstrate that androgen-dependent SC 115 cells undergo programmed cell death induced by androgen withdrawal, and that this death does not require proliferation or progression into G1 of the proliferative cell cycle. SC 115 cells should be a good model for investigating programmed death of hormone-dependent cancer. PMID- 8636003 TI - Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate. AB - An iron chelate, ferric nitrilotriacetate (Fe-NTA, induces renal proximal tubular damage, a consequence of iron-catalyzed free radical reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Previous studies have identified, within 24 h after administration of Fe-NTA, lipid peroxidation products, aldehyde-modified proteins and a variety of modified DNA bases such as 8-hydroxyguanine that may be mutagenic in vivo. In the present study, pathological features of the RCCs were studied, and, in an effort to correlate them with carcinogen-specific molecular events in Fe-NTA-induced carcinogenesis, the H-, K- and N-ras oncogenes and the p53 tumor suppressor gene were investigated for the presence of mutations. Fe-NTA-induced RCCs showed similarity to human RCCs in that they are often invasive, metastatic and fatal. None (0 of 12) of the tumors had mutation in codons 12, 13 and 61 of the H-, K- and N-ras genes by direct sequencing. Only one (1 of 12) tumor with high grade histology revealed a CGC-to-CTC (Arg to Leu) transversion in codon 246 of the p53 gene by the use of single strand conformation polymorphism (SSCP) analysis and direct sequencing. High expression of mutant p53 protein was confirmed by Western blotting and immunohistochemistry. Study of three peritoneal mesotheliomas induced by Fe-NTA revealed no mutation in ras and p53 genes. These results suggest that the ras and p53 genes are not the major targets of mutation in Fe NTA-induced carcinogenesis of kidney and mesothelium. Instead, p53 mutation may work for potentiation of malignant character in Fe-NTA-induced renal carcinogenesis. PMID- 8636005 TI - Expression of CD44 variant exons 8-10 in gastric cancer. AB - The expression of CD44 variant containing variant exons 8-10 product (CD44v8-10) was studied by western blot analysis and immunohistochemistry in gastric cancers using a monoclonal antibody, 44-1V. On western blots, a single band of 130 kD was recognized in stomach cancer cell lines. CD44v8-10 expression, with reactivity localized in the cell membrane, was found in 65 (33.5%) of the 194 advanced gastric cancers. There was no correlation between CD44v8-10 immunoreactivity and serosal, lymphatic, or lymph node invasion. However, there was significant correlation with CD44v8-10 immunoreactivity and venous invasion. CD44v8-10 positive cancers were more frequently associated with hematogenous metastasis than those which were immunonegative. There was an inverse association between CD44v8-10 immunoreactivity and peritoneal dissemination, especially in diffuse type adenocarcinomas. These observations indicate that CD44v8-10 may play a role in the metastasis of gastric cancer. PMID- 8636006 TI - Establishment of a human cell line secreting neuron-specific enolase from a primitive neuroectodermal tumor of the retroperitoneal cavity. AB - Primitive neuroectodermal tumor (PNET) is one of the small round cell malignancies of presumed neural crest origin for which an effective treatment has not yet been established. In the present study, a human cell line, designated KU 9, was established from a 27-year-old male patient with PNET of the retroperitoneal cavity and has been successfully maintained in nude mice and in culture. On histological examination, the primary tumor was composed of poorly differentiated small round cells arranged in clusters showing a variety of mitotic changes, and contained Homer-Wright rosettes. The histopathological appearance of the KU-9 xenografts was similar to that of the primary tumor. Electron microscopy revealed neurosecretory granules and cytoplasmic processes in the xenograft. No significant amplification of N-myc gene was observed in the KU 9 cells. The KU-9 cells showed chromosome numbers ranging from 56 to 61 with consistent structural abnormalities being add(2)(q31), +add(11)(p11.2), +add(13)(p11.1), and +del(22)(q12). Cultured KU-9 cells grew exponentially with a doubling time of about 50 h and a time-dependent increase in medium levels of neuron-specific enolase (NSE) was noted. Serum levels of NSE in KU-9 tumor bearing nude mice were significantly elevated and a linear relationship between the serum NSE levels and the tumor NSE content or tumor volume was observed, suggesting that serum levels of NSE may reflect the PNET tumor burden and tumor extent. These results indicate that the KU-9 cell line provides a reproducible model system which could be useful in gaining some insight into the histogenesis and oncogenesis of PNET and in establishing an effective treatment for PNET. PMID- 8636007 TI - N-acetyltransferase activity in the urine in Japanese subjects: comparison in healthy persons and bladder cancer patients. AB - The activity of urinary N-acetylamino-transferase was determined by high performance liquid chromatographic assay of acetylisoniazid and isoniazid after administration of isoniazid to healthy Japanese male and bladder cancer patients in Japan. The healthy subjects were 47 college students and 44 company employees ranging from 18 to 64 years old (mean +/- SC = 34.5 +/- 13.7). The bladder cancer group consisted of 58 male and 13 female patients, ranging from 28 to 82 years old (mean +/- SD = 60.8 +/- 11.6), who were being treated at several hospitals. The slow phenotype, defined as an acetylation ratio (acetylisoniazid/isoniazid) of less than 2.0, was observed in 13 (14.3%) of the 91 healthy subjects, and in 20 (28.2%) of the 71 bladder cancer patients; the difference between the two groups is significant (p < 0.05). A histogram of the acetylation ratio values showed an overall leftward shift of the patient group, indicating low values of acetylation ratio in this group as a whole (p < 0.01). PMID- 8636008 TI - A quantative in vivo method of analyzing human tumor-induced angiogenesis in mice using agarose microencapsulation and hemoglobin enzyme-linked immunosorbent assay. AB - This study was conducted to develop a quantitative assay system for use in the in vivo evaluation in mice of angiogenesis induced by human tumor cells. The human epidermoid carcinoma cells, A431 cells, were cultured on microcarriers. Microcarrier-attached A431 cells (A431-MC) were microencapsulated with agarose hydrogel to isolate them from the immune system of the C57BL/6 mice after subcutaneous dorsal midline implantation. The agarose hydrogel-microencapsulated A431 cells (Aga-A431 cells; diameter=300 micron) survived for at least 10 days in vitro, and the proliferation profile of the Aga-A431 cells was indistinguishable from that of non-microencapsulated A431 cells. The Aga-A431 cells were subcutaneously injected into mice with an 18-gauge needle. Ten days later, few vessels had formed at the site implanted with cell-free agarose beads, whereas notable angiogenesis was observed at the site implanted with Aga-A431 cells. The degree of angiogenesis was evaluated by measurement of the hemoglobin content in the implanted site using a mouse hemoglobin (mHb) enzyme-linked immunosorbent assay (ELISA) system. This mHb-ELISA system has the advantages of great simplicity and reproducibility. The measured mHb-content of new blood vessels at the site implanted with agarose beads was in good agreement with the amount of angiogenesis observed under a stereoscopic microscope. This assay system enabled us to evaluate the angiogenesis induced by xenogeneic cells, such as human tumor cells. Thus, our novel method may be useful for the study of the angiogenic potential of various human tumor cells and in research on the anti-angiogenic properties of various agents. PMID- 8636009 TI - Dual pathways of tubular morphogenesis of vascular endothelial cells by human glioma cells: vascular endothelial growth factor/basic fibroblast growth factor and interleukin-8. AB - In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fibroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-beta mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-alpha mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8. PMID- 8636010 TI - Prospective evaluation of the feasibility of cisplatin-based chemotherapy for elderly lung cancer patients with normal organ functions. AB - A study was conducted to examine the feasibility of cisplatin-based chemotherapy in elderly patients (> or = 75 years old) with advanced non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). Thirty-four patients were enrolled between September 1993 and December 1994. Patients with normal organ function and good performance status (PS) received cisplatin-based chemotherapy (cisplatin 80 mg/m2 on day 1 and vindesine 3 mg/m2 on days 2 and 8 for NSCLC, or cisplatin 80 mg/m2 on day 1 and etoposide 100 mg/m2 on days 2 to 4 for SCLC). Ten patients (29%) were eligible for this study, 7 with NSCLC and 3 with SCLC. Reasons for exclusion were ischemic heart disease in 14, poor PS (> or = 2) in 11, reduced creatinine clearance (Cer) in 10, abnormal electrocardiogram without ischemia in 9 and noncompliance with the protocol in 2 patients. Eight patients had two or more reasons. Nine of the 10 eligible patients were able to tolerate two or more courses of chemotherapy. All 3 patients with SCLC responded (1 complete response and 2 partial response), but only 1 of the patients with NSCLC achieved partial response. Toxicity was evaluated according to Japan Clinical Oncology Group criteria. All but one patient experienced grade 4 neutropenia, and 6 patients had infectious episodes requiring antibiotics. Grade 3 anemia and thrombocytopenia were observed in 1 and 2 patients, respectively. Non hematological toxicities were mild. Only 10 of 34 patients (29%) satisfied our eligibility criteria and they experienced severe myelotoxicity. We conclude that chemotherapy should be given carefully to elderly patients even if they appear to have normal organ function. PMID- 8636011 TI - Phase I study of paclitaxel by three-hour infusion: hypotension just after infusion is one of the major dose-limiting toxicities. AB - The primary objectives of this study were to determine the maximum tolerated dose (MTD) of paclitaxel administered by 3-h infusion to patients with solid tumors, and to characterize the pharmacokinetics of a 3-h infusion in comparison with those of a 24-h infusion. Twenty-seven patients each received one of six levels of paclitaxel, 105, 135, 180, 210, 240 and 270 mg/m2, with premedication. Two patients given 240 mg/m2 and one patient given 270 mg/m2 unexpectedly had grade 3/4 hypotension just after finishing the paclitaxel infusion. Peripheral neuropathy was also dose-limiting at 270 mg/m2. Although granulocytopenia was significantly less severe than with a 24-h infusion, more than half of the patients experienced grade 4 toxicity at doses of 240 or 270 mg/m2. Severe hypersensitivity reactions (HSRs) were not observed. Pharmacokinetic studies using high performance liquid chromatography demonstrated proportionally greater increases in the peak plasma concentration and area under the curve, and decreases in clearance and volume of distribution with increasing dose, suggesting non-linear pharmacokinetics of paclitaxel when given by 3-h infusion. The MTD of paclitaxel given as a 3-h infusion was determined to be 240 mg/m2 with dose-limiting toxicities of granulocytopenia, peripheral neuropathy and hypotension. Hypotension just after infusion, induced by 3-h infusion of paclitaxel, is a new observation which has not been reported previously. The recommended dose for phase II study is 210 mg/m2. Although hypotension was observed as an unexpected toxic effect, paclitaxel could be administered safely over 3 h with premedication and proper monitoring, resulting in reduced myelotoxicity and with no increase in the incidence of HSRs as compared with a 24 h infusion. PMID- 8636012 TI - Sensitivity and specificity of mass screening for gastric cancer using the measurment of serum pepsinogens. AB - The aim of this study was to estimate the validity of mass screening for gastric cancer using serum pepsinogens (PG test). The study subjects were 4876 workers aged from 40 to 61 years old. Sera were obtained at the time of the health checkup and serum pepsinogen levels (PG I and PG II) were measured at the same time. PG I < 50 ng/ml and PG I/PG II ratio < 3.0 were adopted as the criteria for a positive result for the PG test. PG test-positive subjects were examined, in principle, by endoscopy. Furthermore, all subjects were followed up for a year to check for occurrence of gastric cancer. Among the total subjects, 911 (18.7%) were positive for the PG test and 650 (71.4%) among the positive subjects underwent further examinations, which revealed 11 cases of gastric cancer. Seven gastric cancer cases were diagnosed during the follow-up period within one year after the PG test. When the results of one years's follow-up from the time of screening were defined as the gold standard, the sensitivity and specificity of the PG test were estimated at 66.7% and 81.5%, respectively. The authors conclude that the validity of the PG test as a mass screening method may be comparable to that of X-ray screening, if optimum criteria of a positive test are selected. PMID- 8636013 TI - Therapy and imaging of pancreatic carcinoma xenografts with radioiodine-labeled chimeric monoclonal antibody A10 and its Fab fragment. AB - Recombinant mouse/human chimeric monoclonal antibody A10 (ch-A10) and its Fab fragment (ch-Fab) react with carcinoembryonic antigen on various gastrointestinal carcinomas. We performed biodistribution studies with 125I-labeled ch-A10 and ch Fab in an antigen-positive human pancreatic carcinoma (BxPC-3) xenograft model. We also evaluated the anti-tumor effect of 131I-labeled ch-A10, and studied the detection of BxPC-3 xenografts with 123I-labeled ch-Fab in whole body scintigraphy. In comparative biodistribution studies, the tumor uptake of 125I labeled ch-A10 was significantly greater than that of 125I-labeled ch-Fab 24 h post-injection. However, the tumor-to-blood ratio was 46.8 for ch-Fab at 24 h post-injection, while it was only 1.4 for ch-A10. Microautoradiography studies showed that ch-Fab penetrated more uniformly into the tumor nodules than did ch A10. In mice given a therapeutic dose of 131I-labeled ch-A10, a significant inhibition of tumor growth was seen, while control 131-I-labeled human IgG did not affect tumor growth. Leukocyte toxicity was observed within 3 weeks after injection of 131I-labeled ch-A10, but leukocyte counts recovered to normal levels at 8 weeks post-injection. In whole-body scintigraphy, clear and rapid tumor imaging was obtained with 200 microCi of 123I-labeled ch-Fab 24 h post-injection. These results suggest that radioiodine-labeled chimeric A10 antibodies could potentially be useful candidates for radioimmunotherapy and radioimmunodetection of pancreatic carcinomas. PMID- 8636014 TI - Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases. PMID- 8636015 TI - Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa. AB - The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine. The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase. The wild-type uORF conferred Arg-specific regulation on the reporter gene in N. crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N. crassa strains expressing arg-2-lacZ fusion genes. All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels. Both sequence-dependent and sequence-independent effects of uORFs were observed. Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF. Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition. Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated. Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation. These studies provide direct biochemical evidence for arg-2 uORF function in translational control. PMID- 8636016 TI - Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides. AB - In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370 25376, 1995). This study also revealed an efficient exchange of citrate and D lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose. PMID- 8636017 TI - Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation. AB - Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation. PMID- 8636018 TI - In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2. AB - The PocR protein of Salmonella typhimurium LT2 was overexpressed and used to demonstrate in vitro that it specifically binds to the cobalamin biosynthetic operon (cob) promoter region. Evidence is presented to show that PocR DNA-binding activity in vitro is regulated by the effector molecule 1,2-propanediol. Deletion analysis of the cob promoter (Pcob) suggested that two regions upstream of the promoter are needed for optimal activation of Pcob by PocR in vivo. DNase I footprinting experiments demonstrated that PocR binds to two sites within Pcob. The transcription initiation site of cob mRNA in response to 1,2-propanediol was identified and shown to be different from the one reported for transcription initiation under anoxic conditions in the absence of 1,2-propanediol. PMID- 8636019 TI - Analysis of the relationship between the decrease in pH and accumulation of 3 phosphoglyceric acid in developing forespores of Bacillus species. AB - Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes. PMID- 8636020 TI - Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides. AB - The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion. PMID- 8636021 TI - Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli. AB - Transcriptional activation of the promoters of the mar/soxRS regulons by the sequence-related but independently inducible MarA and SoxS proteins renders Escherichia coli resistant to a broad spectrum of antibiotics and superoxide generators. Here, the effects of MarA and SoxS on transcription of the marRAB promoter itself were assayed in vitro by using a minimal transcription system and in vivo by assaying beta-galactosidase synthesized from marR::lacZ fusions. Purified MarA and MalE-SoxS proteins stimulated mar transcription about 6- and 15 fold, respectively, when the RNA polymerase/DNA ratio was 1. Purified MarA bound as a monomer to a 16-bp "marbox" located 69 to 54 nucleotides upstream of a putative RNA initiation site. Deletion of the marbox reduced MarA-mar binding 100 fold, abolished the stimulatory effects of MarA and SoxS on transcription in vitro, and reduced marR::lacZ synthesis about 4-fold in vivo. Deletion of upstream DNA adjoining the marbox reduced MarA binding efficiency 30-fold and transcriptional activation 2- to 3-fold, providing evidence for an accessory marbox. Although MarA and the mar operon repressor, MarR, bound to independent sites, they competed for promoter DNA in band shift experiments. Assays of marR::lacZ transcriptional fusions in marRAB deletion or soxRS deletion strains showed that the superoxide generator paraquat stimulates mar transcription via soxRS and that salicylate stimulates mar transcription both by antagonizing MarR and by a MarR-independent mechanism. Thus, transcription of the marRAB operon is autorepressed by MarR and autoactivated by MarA at a site that also can be activated by SoxS. PMID- 8636022 TI - NADP+ -dependent malic enzyme of Rhizobium meliloti. AB - The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme. The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP+]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP+ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE cellulose chromatography column. We found that NADP+ -dependent malic enzyme activity was not required for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-fixing) bacteroids was only 20% of the level detected in free-living cells. Much of that residual bacteroid activity appeared to be due to utilization of NADP+ by DME. The functions of DME and the NADP+ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants. PMID- 8636024 TI - Production of actinorhodin-related "blue pigments" by Streptomyces coelicolor A3(2). AB - The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants. PMID- 8636023 TI - Induction of bacteriocin production in Lactobacillus sake by a secreted peptide. AB - Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal. PMID- 8636025 TI - Characterization of the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae. AB - The addition of glucose to maltose-fermenting Saccharomyces cerevisiae cells causes a rapid and irreversible loss of the ability to transport maltose, resulting both from the repression of transcription of the maltose permease gene and from the inactivation of maltose permease. The latter is referred to as glucose-induced inactivation or catabolite inactivation. We describe an analysis of this process in a maltose-fermenting strain expressing a hemagglutinin (HA) tagged allele of MAL61, encoding maltose permease. The transfer of maltose induced cells expressing the Mal61/HA protein to rich medium containing glucose produces a decrease in maltose transport rates which is paralleled by a decrease in Mal61/HA maltose permease protein levels. In nitrogen starvation medium, glucose produces a biphasic inactivation, i.e., an initial, rapid loss in transport activity (inhibition) followed by a slower decrease in transport activity, which correlates with a decrease in the amount of maltose permease protein (proteolysis). The inactivation in both rich and nitrogen-starved media results from a decrease in Vmax with no apparent change in Km. Using strains carrying mutations in END3, REN1(VPS2), PEP4, and PRE1 PRE2, we demonstrate that the proteolysis of Mal61/HAp is dependent on endocytosis and vacuolar proteolysis and is independent of the proteosome. Moreover, we show that the Mal61/HA maltose permease is present in differentially phosphorylated forms. PMID- 8636027 TI - Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans. AB - The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans. PMID- 8636026 TI - Characterization of transmembrane domains 6, 7, and 8 of MalF by mutational analysis. AB - Oligonucleotide mutagenesis was used to isolate mutations in membrane-spanning segments 6, 7, and 8 of MalF. MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli. The current structural model predicts eight transmembrane domains for MalF. Membrane spanning segments 6, 7, and 8 of MalF flank or are part of the EAA-X3-G-X9-I-X-LP consensus region present in the cytoplasmic membrane subunits of the bacterial ABC transporter superfamily members. Mutations with two novel phenotypes with respect to substrate specificity of the maltose transport system were isolated. One mutant grew on minimal maltose media but not on media containing either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative. The other class of mutations led to a maltose minus but maltoheptaose plus phenotype. Nine of the isolated mutations leading to changes in substrate specificity were tightly clustered on one face of the postulated transmembrane helix 6. A similar clustering of mutations was detected in transmembrane domain 7. The majority of mutations in membrane-spanning segment 7 led to a protease sensitive or a conditional phenotype with respect to MalF function or both. Mutations in transmembrane domain 8 appeared to be more randomly distributed. The majority of mutations in membrane-spanning segment 8 caused a Mal+ Dex- phenotype. Six Mal+ suppressor mutations isolated to two mutations in transmembrane domain 7 changed amino acid residues in membrane-spanning segment 6 or 8. PMID- 8636028 TI - Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences. AB - The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion. PMID- 8636029 TI - Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome. AB - The 5' end of the cipC gene, coding for the N-terminal part of CipC, the scaffolding protein of Clostridium cellulolyticum ATCC 35319, was cloned and sequenced. It encodes a 586-amino-acid peptide, including several domains: a cellulose-binding domain, a hydrophilic domain, and two hydrophobic domains (cohesin domains). Sequence alignments showed that the N terminus of CipC and CbpA of C. cellulovorans ATCC 35296 have the same organization. The mini-CipC polypeptide, containing a cellulose-binding domain, hydrophilic domain 1, and cohesin domain 1, was overexpressed in Escherichia coli and purified. The interaction between endoglucanase CelA, with (CelA2) and without (CelA3) the characteristic clostridial C-terminal domain called the duplicated-segment or dockerin domain, and the mini-CipC polypeptide was monitored by two different methods: the interaction Western blotting (immunoblotting) method and binding assays with biotin-labeled protein. Among the various forms of CelA (CelA2, CelA3, and an intermediary form containing only part of the duplicated segment), only CelA2 was found to interact with cohesin domain 1 of CipC. The apparent equilibrium dissociation constant of the CelA2-mini-CipC complex was 7 x 10(-9)M, which indicates that there exists a high affinity between these two proteins. PMID- 8636030 TI - Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element. AB - We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids. PMID- 8636032 TI - Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall. AB - The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed. PMID- 8636031 TI - Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: effect of Fur and PvdS on promoter activity. AB - The pvdA gene, encoding the enzyme L-ornithine N5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site. RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mapping experiments revealed two 5'termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation. The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites. A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigmaE -like factor PvdS were identified 5' of the T1 start site. The minimum DNA region required for iron regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA. We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Fur and PvdS in the iron regulated expression of pvdA. Two fur mutants of P. aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression. Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Fur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes. PMID- 8636033 TI - Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6 phosphate amidotransferase of Candida albicans. AB - Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4 methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change. PMID- 8636034 TI - The proton/electron ration of the menaquinone-dependent electron transport from dihydrogen to tetrachloroethene in "Dehalobacter restrictus". AB - In the anaerobic respiration chain of "Dehalobacter restrictus," dihydrogen functioned as the electron donor and tetrachloroethene (PCE) functioned as the electron acceptor. The hydrogenase faced the periplasm, and the PCE reductase faced the cytoplasmic side of the membrane. Both activities were associated with the cytoplasmic membrane. UV spectroscopy showed that membrane-bound menaquinone (MQ) was reduced by oxidation of H2 and reoxidized by reduction of PCE, indicating that MQ functions as an electron mediator. Fast proton liberation (t1/2 = 6 +/- 2 s) during electron transport from H2 to PCE and to trichloroethene (TCE) after addition of either PCE or TCE to H2-saturated cells resulted in an extrapolated H+/e- ratio of 1.25 +/- 0.2. This ratio indicated that besides the formation of protons upon oxidation of H2, vectorial translocation of protons from the inside to the outside could also occur. Proton liberation was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP), 2-n heptyl-4-hydroxyquinoline N-oxide (HOQNO), and CuCl2. Fast proton liberation with an H+/e- ratio of 0.65 +/- 0.1 was obtained after addition of the MQ analog 2,3 dimethyl-1,4-naphthoquinone (DMN) as an oxidant pulse. This acidification was also inhibited by CCCP, HOQNO, and CuCl2. Oxidation of reduced DMN by PCE was not associated with fast acidification. The results with DMN indicate that the consumption and release of protons associated with redox reactions of MQ during electron transfer from H2 to PCE both occurred at the cytoplasmic side of the membrane. The PCE reductase was photoreversibly inactivated by 1-iodopropane, indicating that a corrinoid was involved in the PCE reduction. PMID- 8636035 TI - Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes. AB - Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes. PMID- 8636036 TI - A complex four-gene operon containing essential cell division gene pbpB in Bacillus subtilis. AB - We have cloned and sequenced the promoter-proximal region of the Bacillus subtilis operon containing the pbpB gene, encoding essential penicillin-binding protein PBP2B. The first two genes in the operon, designated yllB and yllC, are significantly similar to genes of unknown function similarly positioned upstream of pbpB in Escherichia coli. Both B. subtilis genes are shown to be nonessential. The third B. subtilis gene, yllD, is essential, as is the correspondingly positioned ftsL gene of E. coli. The predicted product of yllD is similar to FtsL in size and distribution of charged residues but is not significantly related in primary amino acid sequence. The major promoter for the cluster lies upstream of the first gene, yllB, but at least one minor promoter lies within the yllC gene. The operon is transcribed throughout growth at a low level. PMID- 8636037 TI - Characterization of cis-acting mutations in the first attenuator region of the Bacillus subtilis pyr operon that are defective in pyrimidine-mediated regulation of expression. AB - A transcriptional attenuation mechanism for the regulation of pyr operon expression in Bacillus subtilis in which the PyrR regulatory protein binds pyr mRNA at three sites with similar sequences to cause transcription termination in response to elevated pyrimidine nucleotide pools has been proposed (R. J. Turner, Y. Lu, and R. L. Switzer, J. Bacteriol. 176:3708-3722, 1994). Twenty-seven mutants with cis-acting defects in the repression by pyrimidines of beta galactosidase expression of a pyr-lacZ fusion-integrant were isolated as blue colonies on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) agar plates containing uracil and uridine after UV irradiation or treatment with mutagens or following mutD mutagenesis. These mutants showed normal repression of the chromosomal pyr operon by exogenous pyrimidines. Sequence analysis revealed 12 unique sites of mutation, which occurred in the conserved putative PyrR binding sequence (10 of the 12) or in the stem of the transcriptional terminator structure. These mutants strongly support the proposed model for regulation of the pyr operon. PMID- 8636038 TI - Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth. AB - Growth of Pseudomonas putida (pWWO) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma 70-dependent promoter called Ps2. The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma54-dependent promoter located upstream from the xylS open reading frame, now called Ps1. In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gene through the effect of the two promoters working in tandem: the newly found sigma 70-dependent promoter, whose expression is XylR and toluene independent, and the sigma 54-dependent promoter, whose expression is dependent on XylR activated by its effectors. This expression pathway of the xylS gene explains why sigma 54-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway. PMID- 8636039 TI - Role of the recJ gene product in UV-induced illegitimate recombination at the hotspot. AB - Illegitimate recombination between a prophage and adjacent bacterial DNA is the first step in the formation of specialized transducing phage. Such recombination is rare, but it is greatly enhanced by UV irradiation. We studied the mechanism of UV-induced illegitimate recombination by examining the effect of rec mutations on the frequency of lambda bio transducing phage and found that an Escherichia coli recJ mutation reduces it by 3- to 10-fold. In addition, the recombination hotspot, which accounts for approximately 60% of lambda bio transducing phages in wild-type bacteria, was not detected in the recJ mutant. Introduction of a RecJ overexpression plasmid into the recJ mutant recovered the recombination at the hotspot. These results indicate that the RecJ protein preferentially stimulates illegitimate recombination at the hotspot. Both the hotspot and the non- hotspot sites have short regions of homology, but only the hotspot sites contain common direct-repeat sequences. We propose a model based on the 5'-3' exonuclease activity of RecJ to explain the involvement of this protein in illegitimate recombination at the hotspot. PMID- 8636041 TI - Properties of a Bacillus subtilis polynucleotide phosphorylase deletion strain. AB - The pnpA gene of Bacillus subtilis, which codes for polynucleotide phosphorylase (PNPase), has been cloned and employed in the construction of pnpA deletion mutants. Growth defects of both B. subtilis and Escherichia coli PNPase-deficient strains were complemented with the cloned pnpA gene. RNA decay characteristics of the B. subtilis pnpA mutant were studied, including the in vivo decay of bulk mRNA and the in vitro decay of either poly(A) or total cellular RNA. The results showed that mRNA decay in the pnpA mutant is accomplished despite the absence of the major, Pi-dependent RNA decay activity of PNPase. In vitro experiments suggested that a previously identified, Mn2+ -dependent hydrolytic activity was important for decay in the pnpA mutant. In addition to a cold-sensitive-growth phenotype, the pnpA deletion mutant was found to be sensitive to growth in the presence of tetracycline, and this was due to an increased intracellular accumulation of the drug. The pnpA deletion strain also exhibited multiseptate, filamentous growth. It is hypothesized that defective processing of specific RNAs in the pnpA mutant results in these phenotypes. PMID- 8636040 TI - Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product. AB - Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly. PMID- 8636042 TI - A hairpin structure upstream of the terminator hairpin required for ribosomal protein L4-mediated attenuation control of the S10 operon of Escherichia coli. AB - Ribosomal protein L4 of Escherichia coli regulates transcription of the 11-gene S1O operon by promoting premature termination of transcription (attenuation) at a specific site within the 172-base untranslated leader. We have analyzed the roles of various domains of the leader RNA in this transcription control. Our results indicate that the first 60 bases of the leader, forming the three proximal hairpin structures, are not essential for in vivo L4-mediated attenuation control. However, a deletion removing the fourth hairpin, which is immediately upstream of the terminator hairpin, eliminates L4's effect on transcription. Base changes disrupting complementarity in the 6-bp stem of this hairpin also abolish L4 control, but compensatory base changes that restore complementarity also restore L4's effect. In vitro transcription studies confirm that this hairpin structure is necessary for L4's role in stimulating transcription termination by RNA polymerase. PMID- 8636043 TI - Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells. AB - The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single stranded DNA encoded by bacterial retrons. PMID- 8636044 TI - Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1. AB - An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases. PMID- 8636045 TI - Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. AB - Evolutionary relationships among strictly anaerobic dissimilatory Fe(III) reducing bacteria obtained from a diversity of sedimentary environments were examined by phylogenetic analysis of 16S rRNA gene sequences. Members of the genera Geobacter, Desulfuromonas, Pelobacter, and Desulfuromusa formed a monophyletic group within the delta subdivision of the class Proteobacteria. On the basis of their common ancestry and the shared ability to reduce Fe(III) and/or S0, we propose that this group be considered a single family, Geobacteraceae. Bootstrap analysis, characteristic nucleotides, and higher-order secondary structures support the division of Geobacteraceae into two subgroups, designated the Geobacter and Desulfuromonas clusters. The genus Desulfuromusa and Pelobacter acidigallici make up a distinct branch within the Desulfuromonas cluster. Several members of the family Geobacteraceae, none of which reduce sulfate, were found to contain the target sequences of probes that have been previously used to define the distribution of sulfate-reducing bacteria and sulfate-reducing bacterium-like microorganisms. The recent isolations of Fe(III) reducing microorganisms distributed throughout the domain Bacteria suggest that development of 16S rRNA probes that would specifically target all Fe(III) reducers may not be feasible. However, all of the evidence suggests that if a 16S rRNA sequence falls within the family Geobacteraceae, then the organism has the capacity for Fe(III) reduction. The suggestion, based on geological evidence, that Fe(III) reduction was the first globally significant process for oxidizing organic matter back to carbon dioxide is consistent with the finding that acetate oxidizing Fe(III) reducers are phylogenetically diverse. PMID- 8636047 TI - Role of three chitin synthase genes in the growth of Candida albicans. AB - The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants. PMID- 8636046 TI - Cloning and characterization of motY, a gene coding for a component of the sodium driven flagellar motor in Vibrio alginolyticus. AB - The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motY) encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus. Nucleotide sequence analysis revealed that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is very similar (94.5% identity) to the recently described MotY of V. parahaemolyticus. Their C-terminal domains were similar to the C-terminal domains of many peptidoglycan-interacting proteins, e.g., Escherichia coli MotB and OmpA, suggesting that MotY may interact with peptidoglycan for anchoring the motor. By using the lac promoter-repressor system, motY expression was controlled in V. alginolyticus cells. Swimming ability increased with increasing concentrations of the inducer isopropyl-beta-D thiogalactopyranoside, and the swimming fraction increased after induction. These results are consistent with the notion that MotY is a component of the force generating unit. V. alginolyticus motY complemented the motY mutation of V. parahaemolyticus. However, motY appeared to lack a region corresponding to the proposed motY promoter of V. parahaemolyticus. Instead, sequences similar to the sigma54 consensus were found in the upstream regions of both species. We propose that they are transcribed from the sigma54 -specific promoters. PMID- 8636048 TI - The insE open reading frame of IS1 is not required for formation of cointegrates. AB - The role of the insE open reading frame in transposition of IS1 was reexamined by using an insE nonsense mutation that does not alter the amino acid sequence of InsA inhibitor or InsAB transposase. The mutant was active in all strains tested, showing that insE is not essential for formation of cointegrates. PMID- 8636049 TI - ATP-dependent H+ -pump activity in inverted vesicles of Methanosarcina mazei Go1 and characterization of membrane ATPase. AB - ATP-dependent H+ -pump activity was found in inverted vesicles of Methanosarcina mazei Go1 by using acridine orange as a fluorescent probe. The H+ -pump activity specifically required both Mg and sulfite ions, but azide, an inhibitor of F0F1 ATPase, did not inhibit the activity. The membranes prepared from M. mazei also had an Mg-ATPase activity, and at least the presence of vacuolar-type ATPase was detected. PMID- 8636050 TI - Purification and characterization of catabolic mannopine cyclase encoded by the Agrobacterium tumefaciens Ti plasmid pTi15955. AB - Catabolic mannopine (MOP) cyclase encoded by certain Agrobacterium Ti and Ri plasmids lactonizes MOP to agropine (AGR). The enzyme, purified to homogeneity from a recombinant clone, has a molecular mass of 45 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme catalyzed the lactonization of MOP to AGR without the need for any cofactors. The enzyme also converted AGR to MOP with the lactonizing activity being predominant over the reverse reaction. MOP cyclase is specific for imine conjugates of D-hexose and L-glutamine and was not inhibited by sugars or amino acids. The enzyme lactonized deoxyfructosyl glutamine, a natural intermediate of MOP synthesis and catabolism, to a product indistinguishable from chrysopine, a newly discovered crown gall opine. The enzyme also lactonized N-l (1,2-dideoxy-D-mannityl)-L-glutamine, indicating that a hydroxyl group at carbon atom 2 of the sugar moiety is not required for the enzymatic reaction. PMID- 8636051 TI - Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm. AB - The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1. PMID- 8636052 TI - Analysis of the spacer DNA between the cyclic AMP receptor protein binding site and the lac promoter. AB - The role of the spacer region DNA between the cyclic AMP receptor protein (CRP) site and the RNA polymerase in the lac promoter was examined. We wanted to determine whether the wild-type DNA sequence of this region was an absolute requirement for CRP activation of lac transcription. The sequence of a 9-bp stretch of the spacer, from -41 to -49 relative to the start of transcription, was randomized, and the effect of randomization on lac expression was investigated in vitro and in vivo. We found that the spacer contains no specific sequence determinants for CRP activation of lac transcription; fewer than 1% of the mutants displayed greater than a 50% decrease in CRP activation of lac transcription. PMID- 8636053 TI - Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. AB - Formate is the major source of C1 units in many species of the genus Clostridium. In this study we have cloned and characterized the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. The genetic and transcriptional organizations of the genes and the high level of homology exhibited by the respective gene products to their Escherichia coli counterparts indicate strong evolutionary conservation of these enzymes. PMID- 8636054 TI - Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K 12. AB - We isolated 26 suppressor mutations that allowed growth of a delta pdxH::omega null mutant in the absence of pyridoxal. Each suppressor mapped to pdxJ, and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide. This bypass suppression suggests that PdxJ may participate in formation of the pyridine ring of pyridoxine 5'-phosphate. PMID- 8636055 TI - Autogenous regulation of the Bacillus subtilis glnRA operon. AB - Purified Bacillus subtilis GlnR was shown to bind with high affinity to a specific region that overlaps with the glnRA promoter site. The GlnR binding site includes four copies of a repeated sequence that may be the recognition site for the protein. GlnR inhibited transcription from the glnRA promoter in vitro. PMID- 8636056 TI - Quinine specifically inhibits the proteolipid subunit of the F0F1 H+ -ATPase of Streptococcus pneumoniae. AB - Streptococcus pneumoniae is uniquely sensitive to quinine and its derivatives, but only those alkaloids having antimalarial properties, i.e., those in the erythro configuration, also possess antipneumococcal activity. Quinine and related compounds inhibit the pneumococcal H+ -ATPase. Quinine- and optochin resistant pneumococci showed mutations that change amino acid residues located in one of the two transmembrane alpha-helices of the c subunit of the F0F1, H+ ATPase. PMID- 8636057 TI - Lactobacillus bulgaricus asparagine synthetase and asparaginyl-tRNA synthetase: coregulation by transcription antitermination? AB - Genes encoding the ammonia-dependent asparagine synthetase (asnA) and asparaginyl tRNA synthetase (asnS) have been cloned from Lactobacillus bulgaricus ATCC 11842. The nucleotide sequence suggests that asnA and asnS are organized as one operon and regulated by the tRNA-directed transcription antitermination mechanism (T. M. Henkin, Mol. Microbiol. 13:381-387, 1994). PMID- 8636058 TI - Stabilization of a HemA-LacZ hybrid protein against proteolysis during carbon starvation in atp mutants of Salmonella typhimurium. AB - Transposon insertions that stabilize the beta-galactosidase activity of a HemA LacZ hybrid protein following carbon starvation were mapped to the atp operon of Salmonella typhimurium. This effect is similar to that seen with nuo mutants defective in the energy-conserving type I NADH dehydrogenase. Insertions in several other genes, including such highly pleiotropic mutants as rpoS, polA, and hfq, were isolated with the same phenotypic screen, but they do not affect the beta-galactosidase activity of HemA-LacZ. All of these mutants act indirectly to alter the colony color of many different fusion strains on indicator plates. PMID- 8636059 TI - Two transcription factors, Gln3p and Nil1p, use the same GATAAG sites to activate the expression of GAP1 of Saccharomyces cerevisiae. AB - We present an analysis of the DNA region located upstream of GAP1, the structural gene for the general amino acid permease, which contains the sites required for activation of transcription of this gene in response to the nitrogen source of the growth medium. This gene is not expressed in media containing glutamine, and its transcription is activated in response to Gln3p in cells using glutamate as the source of nitrogen and by Nil1p in cells using urea as the source of nitrogen. We show that full response to both activators requires the presence of two GATAAG sites, as well as the presence of auxiliary sites located in the interval between 602 and 453 bp from the translational start site. The fact that both Gln3p and Nil1p utilize GATAAG sites to activate transcription is reflected in the high homology of the zinc finger regions of the two proteins. PMID- 8636060 TI - The tyrT locus of Escherichia coli B. AB - The tyrT (tRNA(1TYr)) locus of Escherichia coli B differs structurally from that of K-12 strains by the absence of 2 of 3.14 terminal repeat sequences. PMID- 8636061 TI - Noradrenergic and serotonergic abnormalities in depression: stress-induced dysfunction? AB - Noradrenergic and serotonergic abnormalities have long been suspected in patients with major depression. Traditional tricyclic antidepressants block the reuptake of these neurotransmitters. In recent years, understanding of the complex receptor systems mediating serotonin (5-HT) and norepinephrine (NE) effects has increased substantially, and drugs selectively targeting these receptors are being developed. Presented here is a theory which suggests that stress mediated through the hypothalamic-pituitary-adrenal axis produces the disturbances seen in monoamine functioning. Hypersecretion of cortisol as well as the presence of cortisol receptors in the brain is suggested as the pathway for monoamine change. PMID- 8636062 TI - The pharmacologic profile of mirtazapine. AB - Mirtazapine (Org 3770) is a new antidepressant with prominent alpha 2-adrenergic auto- and heteroreceptor antagonistic properties and no effect on monoamine reuptake. Mirtazapine increases noradrenergic and serotonergic transmission, as measured by on-line microdialysis and by enhancement of noradrenergic locus ceruleus and serotonergic raphe nucleus cell firing. Mirtazapine has a low affinity for 5-HT1A receptors but shows 5-HT1A-agonistic-like effects in a conditioned taste aversion test and by causing lower lip retraction in rats. Mirtazapine therefore causes enhancement of 5-HT1-mediated transmission. Other studies show that both 5-HT2 and 5-HT3 receptors are specifically blocked. The enhancement of both noradrenergic and serotonergic transmission probably underlies the therapeutic activity of mirtazapine. Blockade of 5-HT2 and 5-HT3 receptors possibly prevents side effects associated with nonselective 5-HT activation and may also contribute to the anxiolytic and sleep-improving properties of mirtazapine. PMID- 8636064 TI - Adrenoceptors and serotonin receptor function: relevance to antidepressant mechanisms of action. AB - Hypotheses of antidepressant action which argue that even norepinephrine (NE) uptake inhibitors ultimately work through potentiation of serotonergic function are critically reviewed. Preclinical electrophysiologic data can be interpreted as evidence for enhanced serotonin (5-HT) throughput as a common mechanism of action of all antidepressants. Biochemical data in rats (e.g., microdialysis) and humans (e.g., opposite effects of ECT on 5-HIAA in cerebrospinal fluid), however, suggest that more is involved than simply enhanced 5-HT function when NE uptake inhibition is combined with 5-HT uptake inhibition. The case, however, for noradrenergic effects on 5-HT function is quite strong either with regard to stimulation of alpha 1 receptors on 5-HT cell bodies or alpha 2 heteroreceptors on 5-HT nerve endings. Even the reported ability of pindolol to potentiate the antidepressant effects of 5-HT uptake inhibitors may prove to involve noradrenergic effects and not simply antagonism of 5-HT1A receptors as currently hypothesized. The biochemically specific drugs needed to directly test these concepts are not yet available. On the other hand, compounds which combine noradrenergic and serotonergic effects (e.g., alpha 2 antagonism and 5-HT2 antagonism) that go beyond those of the classic uptake inhibitors are emerging as agents to test the clinical potential of selective manipulation of these interacting neurotransmitter systems. PMID- 8636063 TI - New approaches to the treatment of depression. AB - This review considers the various modes of action and possible advantages of the different types of second generation antidepressants. The biochemical changes that may be causally related to depression and the mechanisms whereby these changes may be attenuated by antidepressant treatment are briefly considered. Whereas the monoamine theory of depression and the receptor adaptation hypothesis of antidepressant action have been influential in helping to unify the mechanisms of action of antidepressants with the possible pathologic basis of depression, the introduction of novel antidepressants and the development of treatment strategies suggestive of a rapid onset of antidepressant response may initiate a revision of these concepts. The review ends with a consideration of the nonaminergic changes that may be of fundamental importance not only in the pathologic basis of depression but also to the means whereby antidepressants bring about their clinical effects. PMID- 8636065 TI - How do antidepressants affect serotonin receptors? The role of serotonin receptors in the therapeutic and side effect profile of the SSRIs. AB - The serotonin selective reuptake inhibitors (SSRIs) have proved to be an important development in the treatment of depression because of both their greater practical ease of use and their selective primary action on a single binding site. Their efficacy in depressive illness of a range of severities, but also in obsessive-compulsive disorder and panic, is largely established. The onset of action of the SSRIs may be delayed by autoinhibition at the cell bodies of raphe neurons. This suggests that blockade of autoreceptors may speed the onset of antidepressant action by SSRIs. The main unwanted effects of the SSRIs occur in relation to gastrointestinal, sleep, and orgasmic disturbances. However, the total side effect burden is less than that associated with the older, less selective compounds, and there are clear hypotheses for how the side effects that do occur may be mediated and hence how they may be reduced by either adjunctive treatment or design of new compounds. PMID- 8636066 TI - Differential effects of two hydrocephalus/MASA syndrome-related mutations on the homophilic binding and neuritogenic activities of the cell adhesion molecule L1. AB - The cell adhesion molecule L1 plays an important role in neural development. We have previously demonstrated that the second immunoglobulin-like domain (Ig2) of L1 contains both homophilic binding and neuritogenic activities (Zhao, X., and Siu, C.-H. (1995) J. Biol. Chem. 270, 29413-29421). Recently, two mutations (R184Q and H210Q) within the Ig2 region of the human L1 gene have been shown to be responsible for X-linked hydrocephalus and the related MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome. Glutathione S-transferase-Ig2 fusion proteins containing these mutations were used to evaluate their effects on L1. The homophilic binding activity of fusion proteins and their ability to promote neurite outgrowth from retinal cells were examined. The R184Q mutation led to a complete loss of both homophilic binding and neuritogenic activities, while the H210Q mutation resulted only in a partial loss. These results provide, for the first time, direct demonstration of the deleterious effects of hydrocephalus/MASA mutations on two intrinsic properties of L1. PMID- 8636067 TI - Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes. AB - P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx. PMID- 8636068 TI - Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. AB - Integrins are heterodimeric (alpha, beta) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains of both the alpha and beta subunits promote constitutive signaling by the integrin alphaIIbbeta3. By generating charge reversal mutations, we show these "activating" mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. Thus, the modulation of specific interactions between the alpha and beta subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently, somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival. PMID- 8636069 TI - The histone folds in transcription factor TFIID. AB - The transcription factor TFIID is a multimeric protein complex containing the TATA box-binding polypeptide (TBP) and TBP-associated factors. We have previously reported that the N-terminal regions of dTAFII62 and dTAFII42 have sequence similarities with histones H4 and H3. Here, we demonstrate that the histone homologous regions of dTAFII62 and dTAFII42 form a heteromeric complex both in vitro and in a yeast two-hybrid system. Neither dTAFII62 nor dTAFII42 forms a homomeric complex, in agreement with a nucleosomal histone character. Moreover, circular dichroism measurements show that the heteromeric complex is dominated by alpha-helical secondary structure. These results strongly suggest the existence of a histone-like surface on TFIID. PMID- 8636070 TI - A novel CCAAT-binding protein necessary for adhesion-dependent cyclin A transcription at the G1/S boundary is sequestered by a retinoblastoma-like protein in G0. AB - Loss of adhesion leads to cell cycle arrest at the G1/S boundary in normal, adhesion-dependent, mesenchymal cells. This arrest is accompanied by the inability to produce cyclin A. Using deletional and mutational analysis of the cyclin A promoter, we have identified a CCAAT element that mediates the adhesion dependent transcriptional activation of cyclin A in late G1 phase of the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric protein complex, which we have named CBP/cycA, to this CCAAT element was detectable in growing but not in G0-arrested or nonadherent normal rat kidney fibroblasts. During G0 CBP/cycA appears to be present but sequestered by a retinoblastoma family member. These results suggest that expression of cyclin A, which controls cell cycle progression by adhesion at the G1/S boundary, is regulated by CBP/cycA and the phosphorylation status of the retinoblastoma protein or a retinoblastoma-related protein. PMID- 8636071 TI - Developmental changes in serum UDP-GlcA:chondroitin glucuronyltransferase activity. AB - Bovine, rat, and chicken UDP-GlcA:chondroitin glucuronyltransferase activities in sera during prenatal and postnatal development were systematically measured with polymeric chondroitin as an exogenous acceptor and with UDP-[14C]GlcA as a donor. The results indicated that the activity changed markedly with development in all species examined. Specifically, the activity was the highest at the middle prenatal stage in the bovine and chicken sera and at the late prenatal stage in the rat serum, and it decreased sharply thereafter in all three species. Although the origin of the serum enzyme has not yet been determined, these changes may reflect developmentally regulated biosynthesis of chondroitin sulfate and also suggest that the glucuronyltransferase could be a regulatory enzyme controlling the expression of chondroitin sulfate. PMID- 8636072 TI - Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. AB - p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting. PMID- 8636073 TI - The HIV-1 nef protein interferes with phosphatidylinositol 3-kinase activation 1. AB - nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions. PMID- 8636074 TI - Sequence requirements for mitochondrial import of yeast cytochrome c. AB - Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states. PMID- 8636075 TI - Ca2+-calmodulin binds to the carboxyl-terminal domain of dystrophin. AB - The unique COOH-terminal domain of dystrophin (mouse dystrophin protein sequences 3266-3678) was expressed as a chimeric fusion protein (with the maltose-binding protein), and its binding to calmodulin was assessed. This fusion protein, called DysS9, bound to calmodulin-Sepharose, bound biotinylated calmodulin, caused characteristic changes in the fluorescence emission spectrum of dansyl calmodulin, and had an apparent affinity for dansyl-calmodulin of 54 nM. Binding in each case was Ca2+-dependent. The maltose-binding protein does not bind calmodulin, and thus binding resides in the dystrophin-derived sequences. Deletion mutation experiments further localize the high affinity calmodulin binding to mouse dystrophin protein sequences 3293-3349, and this domain contains regions with chemical characteristics found in the calmodulin-binding sequences in other proteins. The COOH-terminal domain provides sites of attachment of dystrophin to membrane proteins, and calmodulin binding may modulate these interactions. PMID- 8636076 TI - The leucine-responsive regulatory protein (Lrp) from Escherichia coli. Stoichiometry and minimal requirements for binding to DNA. AB - Lrp (Leucine-responsive regulatory protein) regulates the expression of a number of operons in Escherichia coli. A recent study of DNA sequences recognized by Lrp established the consensus as a 15-bp sequence, YAGHAWATTWTDCTR (Y = C/T, H = "not G," W = A/T, D ="not C," R = A/G) (Cui, Y., Wang, Q., Stormo, G. D., and Calvo, J. M. (1995) J. Bacteriol. 177, 4872-4880). Here we report the stoichiometry of Lrp binding (an Lrp dimer binds to a single binding site) and studies that define the minimal length of DNA required for binding. A double-stranded 15 mer having a sequence that closely matches the consensus does not show measurable binding to Lrp. One or two base pairs of DNA flanking each end are not sufficient for binding, but constructs having 3-5 additional base pairs (21 mer) show relatively strong binding. Single-stranded flanking DNA also contributes to strong binding. The extent of the contribution to binding is dependent upon whether the single strand is on the left or right of the double-stranded region and whether the polarity of the single-stranded DNA is 5' to 3' or 3' to 5'. PMID- 8636077 TI - Oxygen and one reducing equivalent are both required for the conversion of alpha hydroxyhemin to verdoheme in heme oxygenase. AB - Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-heme oxygenase complex, which is the first intermediate in the catalytic reaction. The active site structure of the complex was examined by optical absorption, EPR, and resonance Raman spectroscopies. In the ferric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structure of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and consequently the prosthetic group assumes a porphyrin structure. The alpha hydroxyheme group undergoes a redox-linked conversion between a keto and an enol form. The ferric alpha-hydroxyheme reacts with molecular oxygen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. Reaction of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxygen also yields the verdoheme-enzyme complex. We conclude that the catalytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxygenase requires molecular oxygen and one reducing equivalent. PMID- 8636078 TI - Fetal lung fibroblasts selectively down-regulate proteoglycan synthesis in response to elevated oxygen. AB - Cell proliferation is in part regulated by extracellular matrix. Therefore, it is possible that elevated O2 may indirectly affect lung fibroblast growth via modulation of extracellular matrix. In the present study, we investigated the effect of elevated O2 on the synthesis of glycosaminoglycans (GAGs) and proteoglycans (PGs) by fetal lung fibroblasts. A 48-h exposure to >/=50% O2 reduced the incorporation of [3H]glucosamine and 35SO4 into GAGs by fetal lung fibroblasts. The relative proportion of the individual GAG molecules was not altered by elevated O2. Fibroblasts exposed to 50% O2 secreted less [35S]proteoglycans into the medium than controls. Specifically, the synthesis of the small soluble PG, biglycan, was decreased by exposure to 50% O2. Fetal lung fibroblasts did not synthesize the small chondroitin/dermatan sulfate PG, decorin. Elevated O2 concentrations also reduced the synthesis of membrane- and matrix-associated PGs. Furthermore, exposure of fetal lung fibroblasts to >/=50% O2 resulted in a decreased mRNA expression for biglycan and versican core protein sequences. In contrast, elevated O2 increased the message for type I collagen and fibronectin without affecting that of beta-actin. The inhibitory effect of elevated O2 on biglycan mRNA and protein expression was overcome by incubating the cells in 3% O2 after the 48-h exposure to 50% O2. The latter treatment also reversed the increased mRNA expression of type I collagen associated with elevated O2 but not that of fibronectin. These results demonstrate that fetal lung fibroblasts, in response to elevated oxygen concentrations, selectively down regulate their GAG and PG synthesis and that this O2 effect is reversible. PMID- 8636079 TI - Physical and functional association of cortactin with Syk in human leukemic cell line K562. AB - Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80 kDa protein at an early stage of megakaryocytic differentiation and that this 80 kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment. PMID- 8636080 TI - Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site. AB - Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of beta-spectrin required for association with alpha spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10 fold. N- and C-terminal truncations of one or more motifs from beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha spectrin nucleation site recombinant peptide, alpha18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nM, while each additional motif added only 0.85 nM to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised. PMID- 8636081 TI - Stimulation of luteinizing hormone beta gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. AB - The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, alphaT3-1, where it is believed to enhance expression of the common gonadotropin alpha-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone beta-subunit (LH beta) gene promoter revealed the presence of a consensus GSE at -127 to -119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both alphaT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHbeta-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHbeta promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHbeta-luciferase reporter construct. When cotransfected with -209/+5 of the LHbeta promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHbeta-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHbeta promoter. These data suggest that SF-1 may participate in the expression of the LHbeta gene by the gonadotrope. PMID- 8636082 TI - Ultrafast glycerophospholipid-selective transbilayer motion mediated by a protein in the endoplasmic reticulum membrane. AB - A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due to biosynthetic processes between the two leaflets of the endoplasmic reticulum membranes within a time scale of seconds. PMID- 8636083 TI - Differential effects of the protein kinase C activator phorbol 12-myristate 13 acetate on calcium responses and secretion in adherent and suspended RBL-2H3 mucosal mast cells. AB - Adhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme. PMID- 8636084 TI - Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts. AB - Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2 fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts. PMID- 8636085 TI - Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. AB - The developmentally arrested third stage infective larva of hookworms resumes development upon entry into the definitive host. This transition to parasitism can be modeled in vitro by stimulating infective larvae with a low molecular weight ultrafiltrate of host serum together with methylated glutathione analogues. When stimulated to resume development in vitro, activated larvae of the hookworm Ancylostoma caninum released a 42-kDa protein, termed Ancylostoma secreted protein (ASP). ASP was the major protein released by activated hookworm larvae. Degenerate oligonucleotide primers, based on a partial internal amino acid sequence of the protein, were used together with flanking vector sequence primers to amplify a fragment from a third stage larval cDNA library by polymerase chain reaction. The fragment was used as a probe to isolate a longer clone from the larval cDNA library. The full-length ASP cDNA was found to encode a 424-amino acid protein with homology to the antigen 5/antigen 3 family of proteins from hymenopteran venoms and a family of cysteine-rich secretory proteins. ASP was expressed in bacterial cells, and a polyclonal antiserum against purified recombinant ASP was produced. The antiserum, which was demonstrated to be specific for ASP, was used as a probe to measure the kinetics of ASP release by hookworm larvae. ASP is released within 30 min of stimulation, with the majority released by 4 h. Low levels of ASP were released continuously following activation, but only if the stimuli were present in the incubation medium. The compound 4,7-phenanthroline, previously shown to inhibit larval activation, also inhibited release of ASP. The specific, rapid release of ASP by activated infective larvae suggests that this molecule occupies a critical and central role in the transition from the external environment to parasitism. PMID- 8636086 TI - Structural basis of galactose recognition by C-type animal lectins. AB - The asialoglycoprotein receptors and many other C-type (Ca2+-dependent) animal lectins specifically recognize galactose- or N-acetylgalactosamine-terminated oligosaccharides. Analogous binding specificity can be engineered into the homologous rat mannose-binding protein A by changing three amino acids and inserting a glycine-rich loop (Iobst, S. T., and Drickamer, K. (1994) J. Biol. Chem. 269, 15512-15519). Crystal structures of this mutant complexed with beta methyl galactoside and N-acetylgalactosamine (GalNAc) reveal that as with wild type mannose-binding proteins, the 3- and 4-OH groups of the sugar directly coordinate Ca2+ and form hydrogen bonds with amino acids that also serve as Ca2+ ligands. The different stereochemistry of the 3- and 4-OH groups in mannose and galactose, combined with a fixed Ca2+ coordination geometry, leads to different pyranose ring locations in the two cases. The glycine-rich loop provides selectivity against mannose by holding a critical tryptophan in a position optimal for packing with the apolar face of galactose but incompatible with mannose binding. The 2-acetamido substituent of GalNAc is in the vicinity of amino acid positions identified by site-directed mutagenesis (Iobst, S. T., and Drickamer, K. (1996) J. Biol. Chem. 271, 6686-6693) as being important for the formation of a GalNAc-selective binding site. PMID- 8636087 TI - Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors. AB - Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands. The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity. Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive. Binding of a series of N-acyl derivatives of galactosamine suggests that the 2 substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative. Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N acetylgalactosamine over galactose. Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N acetylgalactosamine has been identified and probed by site-directed mutagenesis. The results of these studies define a binding pocket for the 2-substituent of N acetylgalactosamine in the hepatic asialoglycoprotein receptor. PMID- 8636088 TI - The occurrence of novel 9-O-sulfated N-glycolylneuraminic acid-capped alpha2-->5 Oglycolyl-linked oligo/polyNeu5Gc chains in sea urchin egg cell surface glycoprotein. Identification of a new chain termination signal for polysialyltransferase. AB - We report the isolation and structural characterization of an oligo/polysialic acid-containing glycopeptide fraction (designated ESP-Sia) prepared from the egg cell surface complex of the sea urchin, Hemicentrotus pulcherrimus, by exhaustive pronase treatment. The carbohydrate chains isolated from ESP-Sia were shown to consist of O-linked oligo/polysialic acid-containing glycan units and N-linked carbohydrate chains. The present studies have revealed that the O-linked oligo/polysialic acid-containing glycan chains derived from the ESP-Sia were similar to those present in egg jelly coat polysialylated glycoprotein in being composed of tandem repeats of N-glycolylneuraminic acid (Neu5Gc) glycosidically linked in a novel fashion through the glycolyl group, (-->5-OglycolylNeu5Gcalpha2 ->)n. However, they differ from the egg jelly coat in two key respects. First, the average degree of polymerization of the oligo/polysialic acid chains of ESP Sia is only 3; a value far lower than that found in the jelly coat glycoprotein (average degree of polymerization was about 20). Second, ESP-Sia is uniquely characterized by the presence of 9-O-sulfated N-glycolylneuraminic acid (Neu5Gc9HSO3) residues at the nonreducing termini of the (-->5 OglycolylNeu5Gcalpha 2-->)n chains. The terminal sialyl residues in the Neu5Gc9HSO3 alpha2-->(-->5-OglycolylNeu5Gcalpha2-->)n chains were totally resistant to exosialidases. The discovery of Neu5Gc9HSO3 as the nonreducing terminal residue of oligo/poly(-->5-OglycolylNeu5Gcalpha 2-->) group is especially noteworthy in that Neu5Gc9HSO3 appears to be of limited distribution among glycoconjugates. Following the earlier discovery of oligo/polysialic acid chains capped with KDN, i.e. KDN alpha2-->(-->8Neu5Gcalpha2-->)n, found in rainbow trout egg polysialoglycoproteins, it now appears that the sulfated Neu5Gc can serve a similar capping function. PMID- 8636089 TI - Rat hepatocytes transport water mainly via a non-channel-mediated pathway. AB - During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23 degrees C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 x 10(-4) cm/s. In studies done at 10 degrees C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23 degrees C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10 degrees C, the osmotic water permeability coefficient increased by 198% (p < 0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p < 0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of water across hepatocyte membranes occurs mainly by diffusion via the lipid bilayer (not by permeation through water channels as in cholangiocytes). PMID- 8636090 TI - Growth hormone (GH) and a GH antagonist promote GH receptor dimerization and internalization. AB - It has previously been shown that a human growth hormone (hGH) analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C. , Wagner, T. E., and Kopchick, J. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T. E., and Kopchick, J. J. (1991) Endocrinology 129, 1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner, T. E., and Kopchick, J. J. (1994) J. Biol. Chem. 269, 15892-15897). In this study, we report the ability of hGH and hGH-G120R to be internalized by GH receptor expressing cells. Additionally, results of chemical cross-linking experiments revealed that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH-binding protein (bp). The molecular mass of the complex was determined to be approximately 280 kDa which is consistent with multiple receptors interacting with the ligand. The predominant radiolabeled band detected was a complex of approximately 140 kDa which probably represents one GH molecule bound to one GH receptor. The cross-linked complexes were not detected in the presence of excess unlabeled hGH or hGH-G120R and were not observed in cells which do not express detectable levels of GH receptors. Also, GH induced tyrosine phosphorylation of a complex of proteins of approximately 95 kDa in these cells whereas hGH-G120R did not. Thus, we have separated the hGH or hGH G120R/GHR binding and internalization capabilities from the ability to stimulate tyrosine phosphorylation of intracellular proteins. PMID- 8636091 TI - Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf. AB - Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant. PMID- 8636092 TI - Functional lecithin:cholesterol acyltransferase deficiency and high density lipoprotein deficiency in transgenic mice overexpressing human apolipoprotein A II. AB - The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/ 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism. PMID- 8636093 TI - Gene structure and cDNA sequence identify the beaded filament protein CP49 as a highly divergent type I intermediate filament protein. AB - The fiber cell of the vertebrate ocular lens assembles a cytoskeletal structure, the beaded filament, which contains two proteins unique to the fiber cell: CP49 (phakinin) and CP115/CP95 (filensin). We report here the complete primary sequence and gene structure for human CP49. These data show that CP49 is a member of the intermediate filament family, but highly unusual in several regards. 1) CP49 primary sequence does not permit unambiguous assignment to any existing class of intermediate filament protein, but exhibits a gene structure that is identical to the Type I cytokeratins. 2) CP49 essentially lacks one of the three major domains that characterize all intermediate filament proteins, the carboxyl terminal tail domain. 3) CP49 shows substitutions at 3 of 4 residues in the otherwise highly conserved intermediate filament protein motif LNDR. Notably, this divergence includes an Arg to Cys substitution that has only been observed in the mutant human cytokeratin K14, a mutation shown to cause the skin blistering seen in the genetic disorder Dowling-Meara epidermolysis bullosa simplex. PMID- 8636094 TI - Glutaredoxin-3 from Escherichia coli. Amino acid sequence, 1H AND 15N NMR assignments, and structural analysis. AB - The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. In addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys65) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific 1H and 15N nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, 3JHNalpha coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low Kcat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grx1 display large functional differences in in vitro protein disulfide oxido-reduction reactions. PMID- 8636095 TI - Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. AB - After accumulation of a Ca2+ load, the addition of uncoupler to respiring rat liver mitochondria is followed by opening of the permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A. The channel's voltage threshold is profoundly affected under conditions of oxidative stress, with a shift to more negative values that may cause MTP opening at physiological membrane potentials. In this paper we further clarify the mechanisms by which oxidative agents affect the apparent voltage dependence of the MTP. We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by monobromobimane, a thiol selective reagent. (ii) A second site coincides with the oxidation-reduction sensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by arsenite or phenylarsine oxide is matched by increased MTP open probability under conditions where the PN pool is kept in the fully reduced state; at variance from the first, this site can be blocked by both N-ethylmaleimide and monobromobimane and is probably in equilibrium with the glutathione pool. Based on these findings, we reassess the mechanisms by which many oxidative agents affect the MTP and resolve conflicting reports on the relative role of PN and glutathione oxidation in the permeability transition within the framework of MTP (dys)regulation at two separate sites. PMID- 8636096 TI - Two separate signal transducer and activator of transcription proteins regulate transcription of the serine proteinase inhibitor-3 gene in hepatic cells. AB - The serine proteinase inhibitor (SPI-3) gene expression is transcriptionally regulated by interleukin (IL)-6 and glucocorticoids in hepatic cells. To identify the transcription factors involved in regulation of the SPI-3 promoter chloramphenicol acetyltransferase constructs we overexpressed Signal Transducer and Activator of Transcription (STAT) proteins (STAT1, STAT3, STAT5B, and STAT6) and CAAT enhancer-binding protein beta. Specific signaling pathways were activated by cointroduced receptors for growth hormone, IL-3, IL-4, or chimeric receptors containing the cytoplasmic domain of gp130. STAT3 and STAT5B induced transcription via the SPI-3 promoter. The STAT5B response was substantially enhanced by truncation of the 5'-flanking region from -1021 to -148. The responsiveness to STAT3 and STAT5B required the STAT binding element at -132 to 124. This element was sufficient to confer regulation onto a heterologous promoter gene construct. In contrast, overexpression of CAAT enhancer-binding protein beta reduced the transcriptional activity of the SPI-3 promoter, presumably by interfering with STAT protein binding to the promoter element. The SPI-3 gene is the first example of an acute phase gene that is responsive to both STAT3 and STAT5B. PMID- 8636097 TI - Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. AB - Receptor-stimulated arachidonic acid (AA) mobilization in P388D1 macrophages consists of a transient phase in which AA accumulates in the cell and a sustained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A2 (sPLA2) is the enzyme responsible for most of the AA released to the incubation medium. By using selective inhibitors for each of the PLA2s present in P388D1 macrophages, we demonstrate herein that the cytosolic group IV PLA2 (cPLA2) mediates accumulation of cell-associated AA during the early steps of P388D1 cell activation. The contribution of both cPLA2 and sPLA2 to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase A2s (PLA2s). Furthermore, the results suggest the possibility that a functionally active cPLA2 may be necessary for sPLA2 to act. cPLA2 action precedes that of sPLA2, and overcoming cPLA2 inhibition by artificially increasing intracellular free AA levels restores extracellular AA release. Although this suggests cross-talk between cPLA2 and sPLA2, selective inhibition of one other PLA2 present in these cells, namely the Ca2+-independent PLA2, does not block, but instead enhances receptor-coupled AA release. These data indicate that Ca2+-independent PLA2 does not mediate AA mobilization in P388D1 macrophages. Collectively, the results of this work suggest that each of the PLA2s present in P388D1 macrophages serves a distinct role in cell activation and signal transduction. PMID- 8636098 TI - Effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-activated Ca2+ signaling in mouse lacrimal acinar cells. AB - In mouse lacrimal acinar cells, microinjection of the metabolically stable analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate ((2,4,5)IP3), stimulated both intracellular Ca2+ mobilization and Ca2+ entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate ((1,3,4,5)IP4), the inositol 1,4,5 trisphosphate-3-kinase product, was ineffective at mobilizing intracellular Ca2+ or activating Ca2+ entry. In lacrimal cells previously microinjected with submaximal levels of (2,4,5)IP3, the subsequent microinjection of low to moderate concentrations of (1,3,4,5)IP4 did not result in additional release of intracellular Ca2+, nor did it potentiate the Ca2+ entry phase attributable to (2,4,5)IP3. However, as previously demonstrated (Bird, G. S. J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165), additional injections of (2,4,5)IP3 induced further mobilization of intracellular Ca2+ and increased the elevated and sustained Ca2+ entry phase. Introduction of high concentrations of (1,3,4,5)IP4 appeared to inhibit or block the (2,4,5)IP3-induced Ca2+ entry phase. These results were consistent with the observed effect of (1,3,4,5)IP4 in permeabilized lacrimal cells, where (1,3,4,5)IP4 did not release cellular 45Ca2+ but at high concentrations inhibited the ability of submaximal concentrations of (2,4,5)IP3 to release 45Ca2+. Likewise, injection of a high concentration of (1,3,4,5)IP4 prior to injection of (2,4,5)IP3 blocked both release and influx of Ca2+. The inhibitory action of (1,3,4,5)IP4 on Ca2+ signaling observed in intact cells occurred at concentrations that might be obtained in agonist-stimulated cells. However, in permeabilized cells, (1,3,4,5)IP4 inhibited Ca2+ mobilization at concentrations exceeding those likely to occur in agonist-stimulated cells. These results suggest that physiologically relevant levels of (1,3,4,5)IP4 in the cell cytoplasm do not release Ca2+, nor do they potentiate inositol trisphosphate induced Ca2+ entry across the plasma membrane. Rather, the possibility is raised that (1,3,4,5)IP4 or one of its metabolites could function as a negative feedback on Ca2+ mobilization by inhibiting inositol 1,4,5-trisphosphate-induced Ca2+ release. PMID- 8636099 TI - The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence. AB - The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the "very rapid" EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6 methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay. PMID- 8636100 TI - Comparative studies on the substrate specificity of avian myeloblastosis virus proteinase and lentiviral proteinases. AB - The retroviral proteinase (PR) seems to play crucial roles in the viral life cycle, therefore it is an attractive target for chemotherapy. Previously we studied the specificity of human immunodeficiency virus (HIV) type 1 and type 2 as well as equine infectious anemia virus PRs using oligopeptide substrates. Here a similar approach is used to characterize the specificity of avian myeloblastosis virus (AMV) PR and to compare it with those of the previously characterized lentiviral PRs. All peptides representing naturally occurring Gag and Gag-Pol cleavage sites were substrates of the AMV PR. Only half of these peptides were substrates of HIV-1 PR. The Km values for AMV PR were in a micromolar range previously found for the lentiviral PRs; however, the kcat values were in a 10 30-fold lower range. A series of peptides containing single amino acid substitutions in a sequence representing a naturally occurring HIV cleavage site was used to characterize the seven substrate binding subsites of the AMV PR. The largest differences were found at the P4 and P2 positions of the substrate. Detailed analysis of the results by molecular modeling and comparison with previously reported data revealed the common characteristics of the specificity of the retroviral PRs as well as its strong dependence on the sequence context of the substrate. PMID- 8636101 TI - Cloning, sequencing, and expression in escherichia coli of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes. AB - OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli. The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxlT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxlT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxlT are retained on its expression in E. coli. PMID- 8636102 TI - Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. AB - The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H Ras binds with high affinity (KD = 20 nM) to Raf-RBD and with low affinity (KD = 1 microM) to RGF-RBD, Rap1A shows the opposite behavior. The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of guanine nucleotide, which is the basis for the quantitative measurements in this work. Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-guanine nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras. PMID- 8636103 TI - Wild-type Escherichia coli cells regulate the membrane lipid composition in a "window" between gel and non-lamellar structures. AB - Escherichia coli strain K12 was grown at 17, 27, and 37 degrees C. The acyl chain composition of the membrane lipids varied with the growth temperature; the fraction of cis-vaccenoyl chains decreased, and the fraction of palmitoyl chains increased, when the growth temperature was increased. However, the polar head group composition did not change significantly. The equilibria between lamellar and reversed non-lamellar phases of lipids extracted from the inner membrane (IM), and from both the membranes (IOM), were studied with NMR and x-ray diffraction. At temperatures above the growth temperature the lipid extracts formed a reversed hexagonal phase, or a bicontinuous cubic phase, depending on the degree of hydration of the lipids. It was observed that: 1) at equal elevations above the growth temperature, IM lipid extracts, as well as IOM lipid extracts, have a nearly equal ability to form non-lamellar phases; 2) IM extracts have a stronger tendency than IOM extracts to form non-lamellar phases; 3) non lamellar phases are formed under conditions that are relatively close to the physiological ones; the membrane lipid monolayers are thus "frustrated"; and 4) as a consequence of the change of the acyl chain structures, the temperature for the lamellar gel to liquid crystalline phase transition is changed simultaneously, and in the same direction, as the temperature for the lamellar to non-lamellar phase transition. With a too large fraction of saturated acyl chains the membrane lipids enter a gel state, and with a too large fraction of unsaturated acyl chains the lipids transform to non-lamellar phases. It is thus concluded that the regulation of the acyl chain composition in wild-type cells of E. coli is necessary for the organism to be able to grow in a "window" between a lamellar gel phase and reversed non-lamellar phases. PMID- 8636104 TI - Murine laminin B1 gene regulation during the retinoic acid- and dibutyryl cyclic AMP-induced differentiation of embryonic F9 teratocarcinoma stem cells. AB - Retinoic acid (RA) and cyclic AMP analogs cause the differentiation of F9 embryonic teratocarcinoma stem cells into parietal endoderm, an epithelial cell of the early mouse embryo. Laminin B1 is induced in this differentiation process, but is not transcriptionally activated until 24-48 h after RA addition and is not maximally induced until approximately 72 h. Cyclic AMP analogs enhance this transcriptional activation. Although several DNase I hypersensitive sites (DHSS) were observed in the LAMB1 5 -flanking DNA, one of the sites, DHSS2, was detected only after 72 h of RA treatment. Transient transfections have demonstrated that the DHSS2 region functions as a "late-acting RA-inducible enhancer," and motifs in this enhancer contain the homeobox protein-binding site TTATTAACA. Greater binding is observed at these sites by electrophoretic mobility shift assay when cells are cultured with RA and cyclic AMP analogs versus RA alone, and no binding is seen in extracts from RA-treated F9 RAR gamma-/- cells which lack RAR gamma mRNA and protein. Laminin B1 mRNA is not induced by RA in the RAR gamma-/- cells (Boylan, J. F., Lohnes, D., Taneja, R., Chambon, P., and Gudas, L. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9601-9605). Our data show that these DNA regulatory elements contribute to the transcriptional activation of the LAMB1 gene during the later stages of the differentiation process. PMID- 8636105 TI - Purification and characterization of the vnf-encoded apodinitrogenase from Azotobacter vinelandii. AB - The vnf-encoded apodinitrogenase (apodinitrogenase 2) has been purified from Azotobacter vinelandii strain CA117.30 (DeltanifKDB), and is an alpha2beta2delta2 hexamer. Apodinitrogenase 2 can be activated in vitro by the addition of the iron vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C2H2, H+, and N2 reduction. Under certain conditions, the alpha2beta2delta2 hexamer dissociates to yield the free delta subunit (the VNFG protein) and a form of apodinitrogenase 2 that exhibits no C2H2, H+, or N2 reduction activities in the in vitro FeV-co activation assay; however, these activities can be restored upon addition of VNFG to the FeV-co activation assay system. No other vnf-, nif-, or non-nif-encoded proteins were able to replace the function of VNFG in the in vitro processing of alpha2beta2 apodinitrogenase 2 (in the presence of FeV-co) to a form capable of substrate reduction. Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with unique properties, most notably the inability to reduce N2 and insensitivity to CO inhibition of C2H2 reduction. PMID- 8636106 TI - The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681. AB - The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase. PMID- 8636107 TI - Structural organization of the major autolysin from Streptococcus pneumoniae. AB - LytA amidase is the best known bacterial autolysin. It breaks down the N acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function. PMID- 8636108 TI - Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger. AB - Aggregation of physiologically produced soluble amyloid beta protein (Abeta) to insoluble, neurotoxic fibrils is a crucial step in the pathogenesis of Alzheimer's disease. Aggregation studies with synthetic Abeta1-40 peptide by the thioflavin T fluorescence assay and electron microscopy and cytotoxicity assays using rat pheochromocytoma PC12 cells showed that an antibiotic, rifampicin, and its derivatives, which possess a naphthohydroquinone or naphthoquinone structure, inhibited Abeta1-40 aggregation and neurotoxicity in a concentration-dependent manner. Hydroquinone, p-benzoquinone, and 1,4dihydroxynaphthalene, which represent partial structures of the aromatic chromophore of rifampicin derivatives, also inhibited A beta1 40 aggregation and neurotoxicity at comparable molar concentrations to rifampicin. Electron spin resonance spectrometric analysis revealed that the inhibitory activities of those agents correlated with their radical-scavenging ability on hydroxyl free radical, which was shown to be generated in cell-free incubation of Abeta1-40 peptide. These results suggest that at least one mechanism of rifampicin-mediated inhibition of A beta aggregation and neurotoxicity involves scavenging of free radicals and that rifampicin and/or appropriate hydroxyl radical scavengers may have therapeutic potential for Alzheimer's disease. PMID- 8636109 TI - Laminin interactions important for basement membrane assembly are promoted by zinc and implicate laminin zinc finger-like sequences. AB - Laminin is an abundant basement membrane (BM) glycoprotein which regulates specific cellular functions and participates in the assembly and maintenance of the BM superstructure. The assembly of BM is believed to involve the independent polymerization of collagen type IV and laminin, as well as high affinity interactions between laminin, entactin/nidogen, perlecan, and collagen type IV. We report here that Zn2+ can influence laminin binding activity, in vitro. Laminin contains 42 cysteine-rich repeats of which 12 contained nested zinc finger consensus sequences. Recently, the entactin binding site was mapped to one of these zinc finger-containing repeats on the laminin gamma chain (Mayer, U., Nischt, R., Poschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., and Timpl, R. (1993) EMBO J. 12, 1879-1885). Based on these observations, the effect of a series of essential ions (Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, and Zn2+) on laminin binding activity was evaluated. Zn2+ was found to be the most effective at enhancing laminin-entactin and laminin-collagen type IV binding. Laminin-bound Zn2+ was detected by flame atomic absorption spectroscopy at a maximum of 8 mol/mol of laminin. Furthermore, Ca2+-dependent laminin polymerization was unaffected by Zn2+, an observation consistent with the lack of zinc finger containing repeats in the terminal globular domains required for polymerization. We conclude that Zn2+-laminin complexes may generate high affinity binding sites which contribute to BM cross-linking important for its assembly and homeostasis. Zinc is likely a cofactor for 2 kinds of cross-linking interactions; one involving direct binding between laminin and collagen type IV and the other a ternary complex of laminin-entactin-collagen type IV. PMID- 8636110 TI - Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor. AB - We have used the technique of adenovirus-mediated gene transfer to study the in vivo function of the very low density lipoprotein receptor (VLDLR) in low density lipoprotein receptor (LDLR) knockout mice. We generated a replication-defective adenovirus (AdmVLDLR) containing mouse VLDLR cDNA driven by a cytomegalovirus promoter. Transduction of cultured Hepa (mouse hepatoma) cells and LDLR-deficient CHO-ldlA7 cells in vitro by the virus led to high-level expression of immunoreactive VLDLR proteins with molecular sizes of 143 kDa and 161 kDa. Digestion of the cell extract with the enzymes neuraminidase, N-glycanase, and O glycanase resulted in the stepwise lowering of the apparent size of the 161-kDa species toward the 143-kDa species. LDLR (-/-) mice fed a 0.2% cholesterol diet were treated with a single intravenous injection of 3 x 10(9) plaque-forming units of AdmVLDLR. Control LDLR (-/-) mice received either phosphate-buffered saline or AdLacZ, a similar adenovirus containing the LacZ cDNA instead of mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice indicates that in the AdmVLDL animals, total cholesterol is reduced by approximately 50% at days 4 and 9 and returned toward control values on day 21. In these animals, there was also a approximately 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment. By FPLC analysis, the major reduction in plasma cholesterol in the AdmVLDLR animals was accounted for by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. Plasma VLDL, IDL/LDL, and HDL were isolated from the three groups of animals by ultracentrifugal flotation. In the AdmVLDLR animals, there was substantial loss (approximately 65%) of protein and cholesterol mainly in the IDL/LDL fraction on days 4 and 9. Nondenaturing gradient gel electrophoresis indicates a preferential loss of the IDL peak although the LDL peak was also reduced. When 125I-IDL was administered intravenously into animals on day 4, the AdmVLDLR animals cleared the 125I-IDL at a rate 5-10 times higher than the AdLacZ animals. We conclude that adenovirus-mediated transfer of the VLDLR gene induces high-level hepatic expression of the VLDLR and results in a reversal of the hypercholesterolemia in 0.2% cholesterol diet-fed LDLR (-/-, mice. The VLDLR overexpression appears to greatly enhance the ability of these animals to clear IDL, resulting in a marked lowering of the plasma IDL/LDL. Further testing of the use of the VLDLR gene as a therapeutic gene for the treatment of hypercholesterolemia is warranted. PMID- 8636111 TI - The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants. AB - We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure. PMID- 8636113 TI - Functional organization of saposin C. Definition of the neurotrophic and acid beta-glucosidase activation regions. AB - Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid beta-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid beta-glucosidase activation properties to the carboxyl- and NH2-terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid beta-glucosidase. PMID- 8636112 TI - Mutations in the second largest subunit of RNA polymerase II cause 6-azauracil sensitivity in yeast and increased transcriptional arrest in vitro. AB - Yeast RNA polymerase II enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongation-related defects. Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6-azauracil. RNA polymerase II purified from three different 6-azauracil-sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro. The extent of this defect did not correlate with sensitivity to growth in the presence of 6-azauracil. The most severe effect resulted from mutation rpb2 10 (P1018S), which occurs in region H, a domain highly conserved between prokaryotic and eukaryotic RNA polymerases that is associated with nucleotide binding. The average elongation rate of this mutant enzyme is also slower than wild type. We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpb2 10's arrest prone phenotype. This mutant enzyme can respond to SII for transcriptional read through and carry out SII-activated nascent RNA cleavage. PMID- 8636114 TI - Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1. AB - Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing -13 kilobase pairs to -320 base pairs (bp) of the 5 flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between -320 and 180 bp from the start of transcription by transfection analysis. Five DNase protected regions, indicating multiple protein-DNA interactions within the -320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from -197 to -158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The -197 to -158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription. PMID- 8636115 TI - Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae alpha-glucosidase using 5-fluoro glycosyl fluorides. AB - Yeast alpha-glucosidase is a member of a sequence-related family of alpha glycosidases (Family 13) that includes important digestive alpha-amylases and alpha-glucosidases. These enzymes catalyze the hydrolysis of alpha-linked oligosaccharides by a two-step mechanism involving a glycosyl-enzyme intermediate. This intermediate can be trapped by use of 5-fluoro-alpha-D glucosyl fluoride or 5-fluoro-beta-L-idosyl fluoride, members of a new class of mechanism-based glycosidase inactivators. Both of these trapped 5-fluoro glycosyl enzyme intermediates are catalytically competent, turning over when freed of excess inactivator and releasing free enzyme. Two glycosylated peptides in proteolytic digests of these trapped glycosyl enzyme intermediates were identified by use of neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Further tandem mass spectrometric analysis in daughter ion scan mode allowed identification of Asp-214 as the catalytic nucleophile in yeast alpha-glucosidase, and this identification was confirmed by aminolysis of the labeled peptide and high resolution mass spectrometry. This residue is one of three active site carboxylates that are completely conserved in this family, thus confirming the role of Asp-214 and the equivalent residues in other family members as the catalytic nucleophile. The other two conserved carboxylates are likely involved in acid/base catalysis. PMID- 8636116 TI - Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit. AB - Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit. SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface. We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions. By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit. The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM. The data are analyzed and discussed in the context of several possible models for complex formation. In particular, the sedimentation data are not consistent with a model involving cooperative binding. The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C. With glycosylated SCF1-165, the Kd is considerably higher. PMID- 8636117 TI - Inactivation of calmodulin-dependent protein kinase IV by autophosphorylation of serine 332 within the putative calmodulin-binding domain. AB - When brain calmodulin-dependent protein kinase IV is incubated with calmodulin dependent protein kinase IV kinase under the phosphorylation conditions in the presence of Ca2+/calmodulin, rapid initial incorporation of 1 mol of phosphate into 1 mol of the enzyme by the action of the kinase kinase occurs, resulting in marked activation of the enzyme, and the subsequent incorporation of more than 3 mol of phosphate by autophosphorylation occurs, resulting in no significant change in the activity (Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and Fujisawa, H. (1995) J. Biochem. (Tokyo) 117, 686-690). After the maximal phosphorylation, the continued incubation in the presence of excess EGTA resulted in additional autophosphorylation of the enzyme, leading to a complete loss of the Ca2+/calmodulin-dependent activity, while causing no significant change in the Ca2+/calmodulin-independent activity. The amino acid sequence analysis revealed that the autophosphorylation after removal of Ca2+ occurred on Ser332, Ser333, Ser337, and Ser341. Analysis by site-directed mutagenesis clearly showed that the autophosphorylation site responsible for the inactivation is Ser332. Thus, calmodulin-dependent protein kinase IV activated by the kinase kinase may lose its Ca2+/calmodulin-dependent activity by autophosphorylation on Ser332 located within the putative calmodulin-binding domain in the absence of Ca2+. PMID- 8636118 TI - Ion coupling stoichiometry for the norepinephrine transporter in membrane vesicles from stably transfected cells. AB - We prepared membrane vesicles from stable LLC-PK1 cells expressing serotonin (5 HT) gamma-aminobutyric acid (GABA) and norepinephrine (NE) transporters (SERT, GAT-1, and NET). These vesicles accumulate transport substrates when the appropriate transmembrane ion gradients are imposed. For NET, accumulation of [3H]dopamine (DA) was stimulated by imposition of Na+ and Cl- gradients (out > in) and of a K+ gradient (in > out). The presence of Na+ or Cl-, even in the absence of a gradient, stimulated DA accumulation by NET, but K+ had little or no effect in the absence of a K+ gradient. Stimulation by a K+ gradient was markedly enhanced by increasing the K+ permeability with valinomycin, suggesting that net positive charge is transported together with DA. Cationic DA is likely to be the major substrate for NET, since varying pH did not affect Km. We estimated the Na+:DA stoichiometry by measuring the effect of the transmembrane Na+ gradient on peak DA accumulation. The results suggest a 1:1 cotransport of Na+ with DA. Taken together, the results suggest that NET catalyzes cotransport of one cationic substrate molecule with one Na+ ion, and one Cl- ion, and that K+ does not participate directly in the transport process. PMID- 8636119 TI - Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons. AB - At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT 3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations. PMID- 8636120 TI - UDP-glucose deficiency in a mutant cell line protects against glucosyltransferase toxins from Clostridium difficile and Clostridium sordellii. AB - We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile. We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/10(6) cells) was lower than in the wild type (3.7 nmol/10(6) cells); and (iii) sensitivity to toxin B was restored upon microinjection of UDP-glucose. Using the mutant as indicator cell we also found that the related Clostridium sordellii lethal toxin is a glucosyltransferase which requires UDP-glucose as a cofactor. Like toxin B it glucosylated 21-23-kDa proteins in cell lysates, but Rho was not a substrate for lethal toxin. PMID- 8636121 TI - MAUB is a new mucin antigen associated with bladder cancer. AB - The M344 tumor-associated antigen, expressed in 70% of superficial bladder tumors, is a sialylated carbohydrate present on a high molecular mass thiol reducible secreted mucin, which we named MAUB for mucin antigen of the urinary bladder. Herein we studied the relationship between MAUB and other known mucins in the MGH-U3 bladder cancer line where MAUB expression is modulated by culture conditions. Northern blots, immunoradiometric assays, and Western blots showed that only MUC1 and MUC2 are expressed in this MAUB-positive cell line. MUC1 differs from MAUB by its molecular mass and its non-oligomeric nature, while MUC2 has similar molecular mass and response to culture conditions. However, in double determinant immunoradiometric assays, MAUB and MUC2 did not cross-react. Moreover, confocal microscopy showed different subcellular localization of the two antigens. Treatment of MGH-U3 cells with MUC2 antisense oligodeoxynucleotides resulted in decreased expression of MUC2 and increased expression of MAUB, ruling out the possibility that monoclonal antibody M344 recognizes a different glycosylated form of MUC2. In addition, we identified a tumor specimen expressing MAUB but no MUC2 antigen or mRNA. Together, these results suggest that there is expression of at least three mucins in MGH-U3 cells and that MAUB is a cancer associated mucin distinct from those identified so far. PMID- 8636122 TI - Structural features of the central cannabinoid CB1 receptor involved in the binding of the specific CB1 antagonist SR 141716A. AB - The antagonist SR 141716A has a high specificity for the central CB1 cannabinoid receptor and negligeable affinity for the peripheral CB2 receptor, making it an excellent tool for probing receptor structure-activity relationships. From binding experiments with mutated CB1 and with chimeric CB1/CB2 receptors we have begun to identify the domains of CB1 implicated in the recognition of SR 141716A. Receptors were transiently expressed in COS-3 cells, and their binding characteristics were studied with SR 141716A and with CP 55,940, an agonist recognized equally well by the two receptors. The region delineated by the fourth and fifth transmembrane helices of CB1 proved to be crucial for high affinity binding of SR 141716A. The CB1 and CB2 second extracellular loops, e2, were exchanged, modifications that had no effect on SR 141716A binding in the CB1 variant but that eliminated CP 55,940 binding in both mutants. The replacement of the conserved cysteine residues in e2 of CB2 by serine also eliminated CP 55,940 binding, but replacement of those in CB1 resulted in the sequestration of the mutated receptors in the cell cytoplasm. The e2 domain thus plays some role in CP 55,940 binding but none in SR 141716A recognition, binding of the latter clearly implicating residues in the adjoining transmembrane helices. PMID- 8636123 TI - Activation of Gsalpha by the epidermal growth factor receptor involves phosphorylation. AB - Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving Gsalpha and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B. (1990) J. Biol. Chem. 265, 21317 21322; Nair, B. G., and Patel, T. B. (1993) Biochem. Pharmacol. 46, 1239-1245). Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates Gsalpha and activates this protein. Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha. As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation. However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha. In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha. Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase. PMID- 8636125 TI - Distinct functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells. AB - Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells. PMID- 8636124 TI - p150TSP, a conserved nuclear phosphoprotein that contains multiple tetratricopeptide repeats and binds specifically to SH2 domains. AB - Src homology 2 (SH2) domains are structural modules that function in the assembly of multicomponent signaling complexes by binding to specific phosphopeptides. The tetratricopeptide repeat (TPR) is a distinct structural motif that has been suggested to mediate protein-protein interactions. Among SH2-binding phosphoproteins purified from the mouse B cell lymphoma A20, a 150-kDa species was identified and the corresponding complementary DNA (cDNA) was molecularly cloned. This protein encoded by this cDNA, which we have termed p150TSP (for TPR containing, SH2-binding phosphoprotein), is located predominantly in the nucleus and is highly conserved in evolution. The gene encoding p150TSP (Tsp) was mapped to chromosome 7 of the mouse with gene order: centromere-Tyr-Wnt11-Tsp-Zp2. The amino-terminal two-thirds of p150TSP consist almost entirely of tandemly arranged TPR units, which mediate specific, homotypic protein interactions in transfected cells. The carboxyl-terminal third of p150TSP, which is serine- and glutamic acid rich, is essential for SH2 binding; this interaction is dependent on serine/threonine phosphorylation but independent of tyrosine phosphorylation. The sequence and binding properties of p150TSP suggest that it may mediate interactions between TPR-containing and SH2-containing proteins. PMID- 8636127 TI - Human DNA topoisomerase I-mediated cleavages stimulated by ultraviolet light induced DNA damage. AB - DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA. PMID- 8636126 TI - Expression of a cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT I). M-CPT I is the predominant CPT I isoform expressed in both white (epididymal) and brown adipocytes. AB - We set out to determine if the cDNA encoding a carnitine palmitoyltransferase (CPT)-like protein recently isolated from rat brown adipose tissue (BAT) by Yamazaki et al. (Yamazaki, N., Shinohara, Y., Shima, A., and Terada, H. (1995) FEBS Lett. 363, 41-45) actually encodes the muscle isoform of mitochondrial CPT I (M-CPT I). To this end, a cDNA essentially identical to the original BAT clone was isolated from a rat heart library. When expressed in COS cells, the novel cDNA and our previously described cDNA for rat liver CPT I (L-CPT I) gave rise to products with the same kinetic characteristics (sensitivity to malonyl-CoA and Km for carnitine) as CPT I in skeletal muscle and liver mitochondria, respectively. When labeled with [3H]etomoxir, recombinant L-CPT I and putative M-CPT I, although having approximately the same predicated masses (88.2 kDa), migrated differently on SDS gels, as did CPT I from liver and muscle mitochondria. The same was true for the products of in vitro transcription and translation of the L CPT I and putative M-CPT I cDNAs. We conclude that the BAT cDNA does in fact encode M-CPT I. Northern blots using L- and M-CPT I cDNA probes revealed the presence of L-CPT I mRNA in liver and heart and its absence from skeletal muscle and BAT. M-CPT I mRNA, which was absent from liver, was readily detected in skeletal muscle and was particularly strong in heart and BAT. Whereas the signal for L-CPT I was more abundant than that for M-CPT I in RNA isolated from whole epididymal fat pad, this was reversed in purified adipocytes from this source. These findings, coupled with the kinetic properties and migration profiles on SDS gels of CPT I in brown and white adipocytes, indicate that the muscle form of the enzyme is the dominant, if not exclusive, species in both cell types. PMID- 8636129 TI - Interaction between the insulin receptor and its downstream effectors. Use of individually expressed receptor domains for structure/function analysis. AB - A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) beta subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1 331) of IR were expressed in bacteria as (His)6 fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro5-Pro65) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions. PMID- 8636128 TI - Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes. AB - The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9 cells infected with recombinant virus expressing cPLA2 exhibited a time-dependent release of arachidonic acid in response to the calcium ionophore A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic acid also increased the level of phosphorylation of cPLA2. Okadaic acid, but not A23187, induced a gel shift of cPLA2 and increased the level of phosphorylation of Ser-727 by 4.5-fold, whereas the level of phosphorylation of the other sites increased by 60% or less in response to both agonists. To determine whether the same sites on cPLA2 were phosphorylated in mammalian cells, human monocytes were studied. Okadaic acid stimulation of monocytes induced a gel shift of cPLA2, increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA2 on serine residues. Comparison of two-dimensional peptide maps of tryptic digests of 32P-labeled recombinant cPLA2 and human monocyte cPLA2 demonstrated that the same peptides on cPLA2 were phosphorylated in mammalian cells as in insect cells. These results show that the Sf9 baculovirus expression system is useful for investigation of the phosphorylation sites on cPLA2. The results also suggest that phosphorylation of the cPLA2 by protein kinases other than mitogen-activated protein kinase may be important for the regulation of arachidonic acid release. PMID- 8636130 TI - The platelet cytoskeleton stabilizes the interaction between alphaIIbbeta3 and its ligand and induces selective movements of ligand-occupied integrin. AB - Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand. PMID- 8636131 TI - Accelerated evolution in inhibitor domains of porcine elafin family members. AB - Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones were obtained that can be distinguished by a combination of restriction enzymes. Sequence analysis of these clones revealed the presence of three homologous members whose genes, all consisting of three exons and two introns, are almost identical except the exon 2 sequences encoding the inhibitor domain called "WAP motif"; the intron sequences are related to each other with sequence similarities of 93-98%, whereas the exon 2 sequences exhibited only 60-77% similarities among the three members. The extreme divergence in the exon 2 sequences compared to the highly conserved intron sequences may be generated by accelerated mutations confined in a short stretch of the genes following recent duplication events of a single ancestral gene. An RNase protection assay indicated that the messages of the elafin family members are abundantly expressed in the trachea and intestine, suggesting that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms. PMID- 8636132 TI - Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor. Identification with an L-selectin alkaline phosphatase reporter. AB - Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-alpha. PMID- 8636133 TI - N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. AB - The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane. PMID- 8636134 TI - Mutants of eukaryotic initiation factor eIF-4E with altered mRNA cap binding specificity reprogram mRNA selection by ribosomes in Saccharomyces cerevisiae. AB - Recognition of the 5'-end of eukaryotic mRNA by the ribosomal 43 S preinitiation complex involves the eukaryotic translation initiation factor eIF-4E (eIF 4alpha). Deletion mutants of the eIF-4E gene of Saccharomyces cerevisiae (CDC33) encoded proteins with reduced affinity for the 5'-cap. One of these mutant proteins lacked any detectable binding to a cap analogue binding column, yet was still able to support cell growth. More than 17% of the total eIF-4E amino acid sequence could be removed without fully inactivating this factor. At least 30 of the N-terminal amino acids are not essential for function. The minimal functional eIF-4E protein segment therefore comprises at most 176 amino acids. The translation and growth defects of the deletion mutants could be at least partially compensated by increases in eIF-4E synthesis, possibly due to a mass action effect on mRNA binding. Electroporation of yeast spheroplasts with in vitro synthesized mRNA allowed us to characterize the ability of eIF-4E mutant strains to distinguish between capped and uncapped mRNAs in vivo. Our data show that the cap specificity of eIF-4E determines to what extent the translational apparatus differentiates between capped and uncapped mRNAs and indicate the minimum relative mRNA (cap) binding activity of eIF-4E required for yeast cell viability. PMID- 8636135 TI - A mutation in which alanine 128 Is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli. AB - Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase. A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, D., and Cox, G. B. (1985) J. Bacteriol. 162, 420-426). To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132. The properties of mutants in this series are consistent with the region from Val124 to Gly131 forming an alpha-helix. Two of the mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation. This suggested that Val124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions. This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b-subunits. Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer. PMID- 8636136 TI - Prostate-specific antigen expression is regulated by an upstream enhancer. AB - Prostate cancer can be detected using assays for blood-borne prostate-specific antigen (PSA), which is the clinically most useful diagnostic marker of malignant disease. This paper characterizes the 5 -flanking prostate-specific enhancer which controls expression of the human PSA gene This enhancer, located between 5824 and -3738, is androgen-responsive and requires a promoter for activity. Inductions of 12-100-fold activity occur at 1 nM concentrations of the testosterone analog R1881. The enhancer demonstrated tissue specificity as judged by transfections of several human cell lines. Electrophoretic mobility shift assays comparing nuclear extracts from breast cancer cells MCF-7, and prostate cancer cells LNCaP, showed three regions of prostate-specific binding. These three regions are -4168 to -4797 (region I), -4710 to 4479 (region II), and -4168 to -3801 (region III). Region III contained a putative androgen response element at -4136 that markedly affected activity if mutated. These data suggest that prostate-specific gene expression may involve interaction of prostate-specific proteins or protein complexes with the enhancer in addition to binding of the androgen receptor to androgen response elements. PMID- 8636137 TI - A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain. AB - Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe. Calcium dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S labeled calmodulin. Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids. The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins. Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle. By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein. Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch. The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex. The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells. Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin. The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily. The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants. PMID- 8636138 TI - Charge distribution of flanking amino acids influences O-glycan acquisition in vivo. AB - The elements that regulate O-glycosylation are poorly understood. We have developed a novel in vivo system to analyze the role of flanking sequence on the modification of a single well characterized O-glycosylation site derived from human von Willebrand factor (PHMAQVTVGPGL). A secreted chimeric reporter protein, containing the human von Willebrand factor sequence, an antibody recognition epitope, and a heart muscle kinase site, was engineered and expressed in COS7 and MCF-7 cells. Glycosylated and non-glycosylated forms of the immunoprecipitated reporter were resolved electrophoretically and their relative amounts quantitated. Using mutational analysis we find that the glycosylation apparatus of COS7 cells can accommodate a broad range of changes in the flanking sequence without compromising glycosylation, but that the distribution of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to the glycosylation site being particularly sensitive. A combination of acidic residues at positions -1 and +3 almost completely eliminates glycosylation of the reporter in both COS7 and MCF-7 cells. The overall density of charged amino acids is less important since substitution of acidic residues at position -2, +1, and +2 had no effect in the level of glycosylation observed. PMID- 8636139 TI - Mechanical strain induces pp60src activation and translocation to cytoskeleton in fetal rat lung cells. AB - We have previously shown that mechanical strain-induced fetal rat lung cell proliferation is transduced via the phospholipase C-gamma-protein kinase C pathway. In the present study, we found that protein-tyrosine kinase activity of fetal lung cells increased after a short period of strain, which was accompanied by tyrosine phosphorylation of proteins of approximately 110-130 kDa. Several components of this complex were identified as pp60srcsubstrates. Strain increased pp60src activity in the cytoskeletal fraction, which coincided with a shift in subcellular distribution of pp60src from the Triton-soluble to the cytoskeletal fraction. Strain-induced pp60src translocation did not appear to be mediated via the focal adhesion kinase-paxillin pathway. In contrast, strain increased the association between pp60src and the actin filament-associated protein of 110 kDa. Preincubation of cells with herbimycin A, a tyrosine kinase inhibitor, abolished strain-induced phospholipase C-gamma1 tyrosine phosphorylation and its coimmunoprecipitation with pp60src. It also inhibited strain-induced DNA synthesis. These results suggest that activation of pp60src is an upstream event of the phospholipase C-gamma-protein kinase C pathway that may represent an important mechanism by which mechanical perturbations are converted to biological reactions in fetal lung cells. PMID- 8636140 TI - The plasmid RK2 initiation protein binds to the origin of replication as a monomer. AB - The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication. Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the lambda cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo. Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin. PMID- 8636141 TI - Functional LCK Is required for optimal CD28-mediated activation of the TEC family tyrosine kinase EMT/ITK. AB - Activation of CD28 on T lymphocytes initiates a cascade of intracellular events, which in concert with activation of the T cell receptor, culminates in production of cytokines and a functional immune response. One of the earliest biochemical changes observed following stimulation of CD28 is tyrosine phosphorylation. We have demonstrated that both the LCK and the EMT/ITK/TSK (EMT) intracellular tyrosine kinases are activated following cross-linking of CD28. Utilizing somatic cell mutants lacking LCK, we demonstrate that functional LCK is required for CD28 induced activation of EMT as evidenced by increased tyrosine phosphorylation and kinase activity. In support of a role for LCK in EMT activation, reconstitution of a LCK-negative Jurkat T cell line by transfection with normal LCK recreates CD28-mediated EMT activation. Furthermore, co-transfection of LCK and EMT into COS-7 cells showed that EMT becomes phosphorylated in the presence of LCK. In addition, increases in EMT association with CD28 were eliminated in a LCK negative Jurkat cell line, but were restored following transfection of wild type LCK. The data are most compatible with a model in which LCK, either directly or indirectly, initiates EMT activation and association with CD28 following ligation of CD28. PMID- 8636142 TI - Selective interaction of voltage-gated K+ channel beta-subunits with alpha subunits. AB - To begin to study the molecular bases that determine the selective interaction of the beta-subunits of voltage-gated K+ channels with alpha-subunits observed in situ, we have expressed these polypeptides in transfected mammalian cells. Analysis of the specificity of alpha/bet a-subunit interaction indicates that both the Kvbeta1 and Kvbeta2 beta-subunits display robust and selective interaction with the five members of the Shaker-related (Kv1) alpha-subunit subfamily tested. The interaction of these beta-subunits with Kv1 alpha-subunits does not require the beta-subunit N-terminal domains. Thus, the previously observed failure of N-terminal mutants of Kv beta1 to modulate inactivation kinetics of Kv1 family members is not simply due to a lack of subunit interaction. Interaction of these beta-subunits with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a member of the Shal-related subfamily was found to interact with beta-subunits; however, this interaction had biochemical characteristics distinct from the beta-subunit interaction with Kv1 family members. In all cases, Kvbeta1 and Kvbeta2 exhibited indistinguishable alpha-subunit selectivity. These studies point to a selective interaction between K+ channel alpha- and beta-subunits mediated through conserved domains in the respective subunits. PMID- 8636143 TI - A single high affinity binding site for histone H1 in a nucleosome containing the Xenopus borealis 5 S ribosomal RNA gene. AB - We have reconstituted nucleosomes containing the Xenopus borealis 5 S rRNA gene, a single histone octamer, and 1 or 2 molecules of histone H1. We determine that the 1st molecule of histone H1 to associate with the 5 S nucleosome binds with high affinity (KD approximately 2 nM), and the 2nd molecule of H1 binds with a reduced affinity (KD approximately 10 nM). This latter binding is comparable with the association of histone H1 with naked DNA. Neither molecule of histone H1 alters the helical periodicity of DNA in the nucleosome as revealed by hydroxyl radical cleavage. We conclude that although multiple molecules of histone H1 can associate with nucleosomal DNA, there is only a single high affinity binding site for histone H1 within the 5 S nucleosome. PMID- 8636144 TI - Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis. AB - Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5 sequence. In contrast to the strict cell type specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression. PMID- 8636145 TI - Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase. AB - The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1 transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1 transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3* transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2). PMID- 8636146 TI - Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase. AB - Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role. PMID- 8636147 TI - The sequence of the dictyostelium myo J heavy chain gene predicts a novel, dimeric, unconventional myosin with a heavy chain molecular mass of 258 kDa. AB - The complete sequence of the Dictyostelium myo J heavy chain gene has been determined from overlapping genomic clones. The gene spans approximately 7400 base pairs, is split by two small introns, and encodes a 2241-residue, 258-kDa heavy chain polypeptide that that is composed of an N-terminal 944-residue myosin head domain, a central 863-residue domain that is predicted to form an alpha helical coiled-coil containing six hinges, and a C-terminal 434-residue globular domain. The head domain is notable in that it contains a approximately 30 residue insert near the nucleotide binding pocket, and five potential calmodulin/myosin light chain binding sites at the head/tail junction. The existence within the Myo J tail domain of both an extensive coiled-coil structure and a large globular domain suggests that this myosin is dimeric and incapable of self-assembly into filaments. While these properties, as well as the overall predicted structure of the Myo J protein, are reminiscent of class V myosins, the sequence of the 434 residue globular tail piece of Myo J shows no similarity to that of either yeast or vertebrate myosins V. Consistent with this, phylogenetic analyses based on myosin head sequence comparisons do not classify Myo J as a type V myosin. These and other sequence comparisons indicate that Myo J and two as-yet-unclassified unconventional myosins from Arabidopsis represent members of the newest class within the myosin superfamily (class XI). Northern blots analyses suggest that Myo J may function predominantly in vegetative Dictyostelium cells. Finally, Southern blot analyses suggest that Dictyostelium possesses another myosin that is very closely related to Myo J. PMID- 8636148 TI - SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I. AB - Transcription of the low density lipoprotein (LDL) receptor gene is regulated by intracellular cholesterol concentration, hormones, and growth factors. We studied the mechanisms by which insulin and estradiol stimulate promoter activity of the LDL receptor gene. Hormonal effects were analyzed in HepG2 cells after transient transfection with promotor reporter gene constructs. Successive 5' deletions of the LDL receptor promoter fragment from -537 to +88 revealed the sterol regulatory element 1 (SRE-1) between -65 and -56 as an insulin- and estradiol sensitive cis-element. If the SRE-1 is point mutated at position -59 (C to G), which abolishes the binding of the SRE binding proteins (SREBP-1 and SREBP-2), no insulin or estradiol stimulatory effect on reporter gene expression was observed, indicating a role of SRE binding proteins in this regulatory mechanism. The concentration of the 125-kDa membrane-integrated SREBP-1 precursor protein in LDL repressed HepG2 cells is not altered by hormone treatment. Concentrations of SREBP-1 mRNA and precursor protein are reduced significantly by high and stable expression of an SREBP-1 antisense cDNA fragment in HepG2 cells (SREBP1(-) cells). Transfection of SREBP1(-) cells with promoter construct phLDL4 (-105 to +88) reduces induction of reporter gene activity by insulin and insulin-like growth factor-I to 35 and 17%, respectively, compared with HepG2 cells. The stimulatory effect of estradiol remains unchanged, and the inductions by pravastatin are enlarged. We conclude that different regulatory effects converge at SRE-1, but that SREBP-1 is selectively involved in the signal transduction pathway of insulin and insulin-like growth factor-I leading to LDL receptor gene activation. PMID- 8636149 TI - Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action. AB - Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 - > Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, anti-phosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of 32P-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of approximately 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by anti-phosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 - > Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS 1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action. PMID- 8636150 TI - Differential ability to form the G protein betagamma complex among members of the beta and gamma subunit families. AB - We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two hybrid system. We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins. PMID- 8636151 TI - A blood group-related polymorphism of CD44 abolishes a hyaluronan-binding consensus sequence without preventing hyaluronan binding. AB - CD44 is a widely expressed integral membrane protein that acts as a receptor for hyaluronan (HA) and is proposed to be important to cell-extracellular matrix interaction. The Indian (In) blood group antigens reside on CD44, and most individuals express the Inb antigen. Homozygosity for the Ina allele occurs as a rare event and is associated with production of alloantibody to the common Inb antigen after transfusion or pregnancy. The present study demonstrates that a single point mutation (G252 --> C) causes an Arg46 --> Pro substitution, which is responsible for the Inb/Ina polymorphism. Additional mutations were found in In(a+b-) cDNA but were not necessary to the antigenic phenotype as determined in site-directed mutagenesis studies. In studies using CD44 chimeric constructs, Arg46 has previously been shown to be crucial for maintenance of HA-binding ability to a CD44 peptide. However, the present study demonstrates that the Arg46 --> Pro substitution does not reduce HA binding to the intact CD44 protein, which contains two proposed extracellular HA-binding motifs. Down-regulation of HA binding to In(a+b-) CD44 by anti-CD44 monoclonal antibody (mAb) ligands, however, was weakened, although all mAbs tested bound In(a+b-) and In(a-b+) CD44 equally well. Competitive inhibition studies using human anti-Inb also showed that some mAbs that inhibit HA binding to CD44 may do so by interacting with a domain separate from, but affecting the structure of, the Inb epitope. PMID- 8636152 TI - G-protein palmitoyltransferase activity is enriched in plasma membranes. AB - Heterotrimeric G proteins are covalently modified by lipids. Myristoylation of G protein alpha subunits and prenylation of gamma subunits are stable modifications. In contrast, palmitoylation of alpha subunits is dynamic and thus has the potential for regulating protein function. Indeed, receptor activation of Gs increases palmitate turnover on the alpha subunit, presumably by stimulating deacylation. The enzymes that catalyze reversible palmitoylation of G-protein alpha subunits have not been characterized. Here we report the identification of a palmitoyl-CoA:protein S-palmitoyltransferase activity that acylates G-protein alpha subunits in vitro. Palmitoyltransferase activity is membrane-associated and requires detergent for solubilization. The preferred G-protein substrate for the enzyme activity is the alpha subunit in the context of the heterotrimer. Both myristoylated and nonmyristoylated G-protein alpha subunits are recognized as substrates. The palmitoyltransferase activity demonstrates a modest preference for palmitoyl-CoA over other fatty acyl-CoA substrates. Palmitoyltransferase activity is high in plasma membrane and present at low or undetectable levels in Golgi, endoplasmic reticulum, and mitochondria of rat liver. The subcellular localization of this enzyme activity is consistent with a role for regulated cycles of acylation and deacylation accompanying activation of G-protein signal transduction pathways. PMID- 8636153 TI - Identification and characterization of the cytoplasmic antiproteinase (CAP) in human platelets. Evidence for the interaction of CAP with endogenous platelet proteins. AB - To define the presence and potential role of platelet-associated protease inhibitors, we initiated a study designed to characterize the platelet components that are responsible for the formation of two SDS-stable complexes of approximately 58 and 70 kDa initially observed following the incubation of 125I thrombin and human platelets. We demonstrate that thermal-mediated unfolding of the 58-kDa complex between 125I-thrombin and a nonsecreted platelet protein leads to an apparent molecular mass of 70 kDa. This platelet component is functionally and immunologically indistinguishable from the cytoplasmic antiproteinase (CAP), also known as placental thrombin inhibitor, a recently cloned member of the ovalbumin family of intracellular serpins (serine proteinase inhibitors). CAP specific mRNA and antigen were detected in human platelets, suggesting that CAP synthesis occurs concurrent with platelet development. Utilizing quantitative immunoblotting, CAP antigen was estimated at 1.014 +/- 0.181 microg/10(9) nonstimulated platelets. After platelet activation with the calcium ionophore A23187, CAP antigen was detected in released microparticles at approximately 0. 195 +/- 0.031 microg/10(9) platelets and a fraction of platelet CAP was proteolytically modified. We provide evidence that these lower molecular mass species arise by cleavage of CAP at or near the reactive site loop. Most importantly, molecular sieving chromatography indicates the presence of an approximately 68-kDa SDS-labile complex between cleaved CAP and a cellular component in A23187-stimulated platelets, suggesting a physiological target of this intracellular serpin and a potential role for this inhibitor in regulating proteolytic activity that may be formed during platelet activation. PMID- 8636154 TI - Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells. AB - Iron regulatory proteins (IRPs) are RNA-binding proteins that post transcriptionally regulate synthesis of iron uptake (transferrin receptor) and storage (ferritin) proteins. Our previous work demonstrating that IRP1 is phosphorylated by protein kinase C supported the hypothesis that factors in addition to iron modulate IRP function. We have investigated changes in activity and expression of both IRP1 and IRP2 during phorbol 12-myristate 13-acetate (PMA) induced differentiation of HL-60 cells. In contrast to IRP1, IRP2 was highly phosphorylated in untreated cells. PMA stimulated phosphorylation of IRP1 and IRP2 by at least 2-3-fold without affecting incorporation of [35S]methionine into the proteins. IRP1 and IRP2 isolated from PMA-treated cells displayed different phosphopeptides. Phosphorylation of IRPs was associated with a 2-fold increase in high affinity RNA binding activity without altering KD, and this was accompanied by a 50% increase in transferrin receptor mRNA abundance. PMA acted on a latent pool of binding activity that is present in a nonaconitase oxidized form and is largely composed of a stable but inactive species of IRP2. Desferal and hemin modulated iron-responsive element binding activity in HL-60 cells without affecting the phosphorylation state of IRP1. Hemin appeared to reduce the abundance of phosphorylated IRP2. Thus, multiple factors affect the function of both IRPs and indicate that extracellular agents may program changes in cellular iron metabolism by altering the phosphorylation state of these regulatory RNA binding proteins. PMID- 8636155 TI - Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct. AB - Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion. We analyzed this dual incision reaction at a single 1,3 intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate. Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited. The nicks were mapped by restriction fragment end labeling and primer extension analysis. Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe. Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7. All of these components were required in order to observe any cleavage. PMID- 8636156 TI - Differential effects of changes in the length of a signal/anchor domain on membrane insertion, subunit assembly, and intracellular transport of a type II integral membrane protein. AB - The length requirement for a functional uncleaved signal/anchor (S/A) domain of the paramyxovirus hemagglutinin-neuraminidase (HN) type II glycoprotein was analyzed. HN mutants with progressive NH2-terminal S/A deletions or insertions were expressed in HeLa cells, and the membrane targeting, folding, tetramer assembly, and intracellular transport of the proteins were examined. Changing the length of the S/A by two residues resulted in HN mutants that displayed aberrant endoplasmic reticulum (ER) membrane targeting or translocation. This phenotype did not simply reflect upper or lower limitations on the size of a functional S/A, because normal signaling was restored by further alterations involving three or four residues. Likewise, ER-to-Golgi transport of mutants containing deletions of one or two S/A residues was delayed (approximately 30% of WT) or blocked, but transport was restored for a mutant with a total of three deleted residues. HN mutants with S/A insertions of three or four Leu residues differed from wild-type HN by having heterogeneous Golgi-specific carbohydrate modifications. Differences in ER-to-Golgi transport of the mutants did not strictly correlate with defects in either native folding of the ectodomain or the assembly of two dimers into a tetramer. Together, these data suggest that efficient entry into and exit from the ER are sensitive to changes in the HN S/A that may reflect alterations to a structural requirement along one side of an alpha-helix. PMID- 8636157 TI - Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice. AB - The involvement of pancreatic cholesterol esterase (bile salt-stimulated lipase) in cholesterol absorption through the intestine has been controversial. We have addressed this issue by using homologous recombination in embryonic stem cells to produce mice lacking a functional cholesterol esterase gene. Cholesterol esterase knockout mice and their wild type counterparts were fed a bolus dose of [3H]cholesterol and a trace amount of [beta-14C]sitosterol by gavage. The ratio of the two radiolabels excreted in the feces over a 24-h period was found to be similar in the control and cholesterol esterase-null mice. Similar results were observed when the radiolabeled sterols were supplied in an emulsion with phospholipid and triolein or in lipid vesicles with phosphatidylcholine. Cholesterol absorption results were similar between the control and cholesterol esterase-null mice regardless of whether the animals were fed a low fat diet or a high fat/high cholesterol diet. The rate of [3H]cholesterol appearance in the serum of the gene-targeted mice paralleled that observed in control animals. In contrast to these results, when experiments were performed with [3H]cholesteryl oleate instead of [3H]cholesterol, a higher amount of the 3H radiolabel was found excreted in feces and dramatically less of the radiolabel was detected in the serum of the cholesterol esterase-null mice in comparison with that detected in control animals. Serum cholesterol levels were not significantly different between control and cholesterol esterase-null mice fed either control or an atherogenic diet. These results indicate that cholesterol esterase is responsible for mediating intestinal absorption of cholesteryl esters but does not play a primary role in free cholesterol absorption. PMID- 8636159 TI - GroEL binds to and unfolds rhodanese posttranslationally. AB - The Escherichia coli chaperone GroEL is a member of a class of molecular chaperones that possesses a stacked double ring structure containing seven subunits per ring, with approximately 60-kDa subunits. It has been suggested that newly synthesized proteins may interact with a eukaryotic homolog of GroEL co translationally, thereby sequestering the unfolded protein from other proteins in the cell. To test whether it is essential for GroEL to form a stable interaction with a nascent polypeptide co-translationally, we translated the well studied GroEL substrate rhodanese in bacterial and wheat germ translation extracts. We found that rhodanese formed stable complexes with GroEL solely posttranslationally. Upon binding to GroEL, the protease resistant N-terminal domain of rhodanese unfolds. This interaction with GroEL leads to productive folding of the full-length rhodanese. We conclude that GroEL is able to assist in the folding of newly synthesized proteins following release from the ribosome and that GroEL can unfold a trapped protein folding intermediate of rhodanese. PMID- 8636158 TI - Cloning and characterization of a novel transcriptional repressor of the nicotinic acetylcholine receptor delta-subunit gene. AB - We have identified a negative cis-acting regulatory element in the nicotinic acetylcholine receptor delta-subunit gene's promoter. This element resides within a previously identified 47-base pair activity-dependent enhancer. Proteins that bind this region of DNA were cloned from a lambdagt11 innervated muscle expression library. Two cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box family of transcription factors. MY1/1a RNAs are expressed at relatively high levels in heart, skeletal muscle, testis, glia, and specific regions of the central nervous system. MY1/1a are nuclear proteins that bind specifically to the coding strand of the 47-base pair enhancer and suppress delta promoter activity in a sequence-specific manner. These results suggest a novel mechanism of repression by MY1/1a, which may contribute to the low level expression of the delta-subunit gene in innervated muscle. Finally, the gene encoding MY1/1a, Yb2, maps to the mid-distal region of mouse chromosome 6. PMID- 8636160 TI - Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. AB - Tuberculosis continues to be a major disease threatening millions of lives worldwide. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. We have expressed and purified the 16-kDa antigen, an immunodominant antigen with serodiagnostic value, which has been previously cloned and shown to share low sequence homology with the alpha-crystallin-related small heat shock protein family. Sedimentation equilibrium analytical ultracentrifugation and dynamic light scattering demonstrate the formation of a specific oligomer, 149 +/- 8 kDa, consisting of approximately nine monomers. In 4 M urea, a smaller oligomer of 47 +/- 6 kDa (or trimer) is produced. Analysis by electron cryomicroscopy reveals a triangular shaped oligomeric structure arising from the presence of three subparticles or globules. Taken together, the data suggest an antigen complex structure of a trimer of trimers. This antigen, independent of ATP addition, effectively suppresses the thermal aggregation of citrate synthase at 40 degrees C, indicating that it can function as a molecular chaperone in vitro. A complex between the antigen and heat-denatured citrate synthase can be detected and isolated using high performance liquid chromatography. We propose to rename the 16-kDa antigen Hsp16.3 to be consistent with other members of the small heat shock protein family. PMID- 8636161 TI - Biochemical characterization of ezrin-actin interaction. AB - The highly related actin isoforms are thought to have different functions. We recently demonstrated a polarized distribution of actin isoforms in gastric parietal cells and association of gastric ezrin with the cytoplasmic beta-actin isoform (Yao, X., Chaponnier, C., Gabbiani, G., and Forte, J. G. (1995) Mol. Biol. Cell. 6, 541-557). Here we used ultrastructural immunocytochemistry to verify that beta-actin is located within canalicular microvilli and the apical cortex of parietal cells, similar to the localization reported for ezrin. Furthermore, we tested whether ezrin binds preferentially to cytoplasmic beta actin compared with the skeletal muscle alpha-actin isoform. Purified cytoplasmic beta-actin (from erythrocytes) and skeletal alpha-actin were assembled with gastric ezrin. Co-sedimentation experiments showed that gastric ezrin selectively co-pelleted with the beta-actin isoform and only very poorly with alpha-actin. Binding of erythrocytic beta-actin to ezrin is saturable with a molar ratio of approximately 1:10 (ezrin:actin) and a dissociation constant approximately 4.6 x 10(-8) M. In addition, ezrin promoted pyrene-labeled actin assembly, with predominant effects on filament elongation and a distinct preference for beta actin compared with alpha-actin. Given these isoform-selective associations, we speculate that actin isoforms might segregate into different functional domains and exert specificity by interacting with isoform-orientated binding proteins. PMID- 8636162 TI - Biochemical heterogeneity and phosphorylation of coatomer subunits. AB - The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae, and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the beta-COP and delta-COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products but rather a significant degree of protein phosphorylation. The observed pI of beta-COP was sensitive to alkaline phosphatase digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both beta-COP and delta-COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane recruitment, or the specificity of coatomer organelle interaction. PMID- 8636163 TI - Structural characterization of a novel neuropeptide from the central nervous system of the leech Erpobdella octoculata. The leech osmoregulator factor. AB - Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family. PMID- 8636164 TI - Is polyethylene still the best prosthetic bearing surface? PMID- 8636165 TI - Porosity reduction in bone cement at the cement-stem interface. AB - The fatigue failure of bone cement, leading to loosening of the stem, is likely to be one mode of failure of cemented total hip replacements. There is strong evidence that cracks in the cement are initiated at voids which act as stress risers, particularly at the cement-stem interface. The preferential formation of voids at this site results from shrinkage during polymerisation and the initiation of this process at the warmer cement-bone interface, which causes bone cement to shrink away from the stem. A reversal of the direction of polymerisation would shrink the cement on to the stem and reduce or eliminate the formation of voids at this interface. We have investigated this by implanting hip prostheses, at room temperature or preheated to 44 degrees C, into human cadaver femora kept at 37 degrees C. Two types of bone cement were either hand-mixed or vacuum-mixed before implantation. We found that the area of porosity at the cement-stem interface was dramatically reduced by preheating the stem and that the preheating temperature of 44 degrees C determined by computer analysis of transient heat transfer was the minimum required to induce initial polymerisation at the cement-stem interface. Temperature measurements taken during these experiments in vitro showed that preheating of the stem caused a negligible increase in the temperature of the bone. Reduction of porosity at the cement-stem interface could significantly increase the life of hip arthroplasties. PMID- 8636166 TI - Orthopaedic bone drills-can they be improved? Temperature changes near the drilling face. AB - We studied the various drill bits available for engineering purposes, and compared them with standard orthopaedic drill bits, using continuous temperature recording at 0.5 mm, 1.0 mm and 1.5 mm from the edge of a 2.5 mm hole as it was drilled in fresh cadaver human tibia. We found that some commercially available drill bits performed better than their orthopaedic equivalents, producing significantly less thermal injury to the surrounding bone and halving the force required for cortical penetration. Our work suggests that the optimal bit for orthopaedic purposes should have a split point and a quick helix. Theoretical knowledge of cutting technology predicts that the addition of a parabolic flute will further reduce thermal damage. Further work is being done on other drill sizes used in orthopaedic practice and on new custom-designed bits. PMID- 8636167 TI - Biochemical properties of cortical allograft bone using a new method of bone strength measurement. A comparison of fresh, fresh-frozen and irradiated bone. AB - There have been conflicting reports on the effects of gamma irradiation on the material properties of cortical allograft bone. To investigate changes which result from the method of preparation, test samples must be produced with similar mechanical properties to minimise variations other than those resulting from treatment. We describe a new method for the comparative measurement of bone strength using standard bone samples. We used 233 samples from six cadavers to study the effects of irradiation at a standard dose (28 kGy) alone and combined with deep freezing. We also investigated the effects of varying the dose from 6.8 to 60 kGy (n = 132). None of the treatments had any effect on the elastic behaviour of the samples, but there was a reduction in strength to 64% of control values (p < 0.01) after irradiation with 28 kGy. There was also a dose-dependent reduction in strength and in the ability of the samples to absorb work before failure. We suggest that irradiation may cause an alteration in the bone matrix of allograft bone, but provided it is used in situations in which loading is within its elastic region, then failure should not occur. PMID- 8636168 TI - Foreign-body reaction and the course of osteolysis after polyglycolide implants for fracture fixation: experimental study in sheep. AB - Foreign-body reaction to polyglycolide (PGA) implants has been described in man. Many animal experiments have verified the mechanical properties of fixation devices made from PGA, but a significant foreign-body reaction has not been described. We studied the effect of PGA rods in 12 sheep with standardised osteochondral fractures of the medial femoral condyle fixed with uncoloured, self reinforced PGA rods (Biofix). Radiographs were taken at intervals ranging from two weeks to two years, and the sheep were killed at intervals ranging from six to 24 months. All knees were examined histologically. Eleven of the 12 fractures healed radiologically and histologically. Moderate to severe osteolysis was seen at four to six weeks with maximum changes at 12 weeks in ten animals. Six knees showed fistula-like connections between the implant site and the joint space. Three developed synovitis, one with inflammatory changes involving the whole cartilage and one with destruction of the medial condyle. Although in our study osteochondral fractures fixed with PGA rods healed reliably, there were frequent, significant foreign-body reactions. Caution is needed when considering the use of PGA fixation devices in vulnerable regions such as the knee. PMID- 8636170 TI - Stress fracture of the pubic ramus in female recruits. AB - During a four-month period we observed 12 stress fractures of the inferior pubic ramus in 11 military recruits undergoing basic training. Eleven of these were in women. This high number was considered to be caused by the introduction of mixed training of male and female recruits; this forces women to increase their stride length when marching. The presenting symptom was chronic groin pain which failed to settle with rest, and the clinical diagnoses were confirmed by radiographs in all but one patient who was diagnosed by 99mTc bone scanning. Since the required stride length has been reduced there have been no new cases of stress fracture of the pelvis. PMID- 8636169 TI - Transforming growth factor-beta 1 stimulates bone ongrowth to weight-loaded tricalcium phosphate coated implants: an experimental study in dogs. AB - Bone growth into cementless prosthetic components is compromised by osteoporosis, by any gap between the implant and the bone, by micromotion, and after the revision of failed prostheses. Recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) has recently been shown to be a potent stimulator of bone healing and bone formation in various models in vivo. We have investigated the potential of rhTGF-beta 1, adsorbed on to weight-loaded tricalcium phosphate (TCP) coated implants, to enhance bone ongrowth and mechanical fixation. We inserted cylindrical grit-blasted titanium alloy implants bilaterally into the weight-bearing part of the medial femoral condyles of ten skeletally mature dogs. The implants were mounted on special devices which ensured stable weight-loading during each gait cycle. All implants were initially surrounded by a 0.75 mm gap and were coated with TCP ceramic. Each animal received two implants, one with 0.3 microgram rhTGF-beta 1 adsorbed on the ceramic surface and the other without growth factor. Histological analysis showed that bone ongrowth was significantly increased from 22 +/- 5.6% bone-implant contact in the control group to 36 +/- 2.9% in the rhTGF-beta 1 stimulated group, an increase of 59%. The volume of bone in the gap was increased by 16% in rhTGF-beta1-stimulated TCP-coated implants, but this difference was not significant. Mechanical push-out tests showed no difference in fixation of the implant between the two groups. Our study suggests that rhTGF-beta 1 adsorbed on TCP-ceramic-coated implants can enhance bone ongrowth. PMID- 8636171 TI - Two-way compression along the shaft and the neck of the femur with the Medoff sliding plate: one-year follow-up of 108 intertrochanteric fractures. AB - The Medoff sliding plate (MSP) is a new device used to treat intertrochanteric and subtrochanteric fractures. There are three options for sliding; either along the shaft or the neck of the femur, or a combination of both. In a prospective series of 108 consecutive displaced intertrochanteric fractures we used combined dynamic compression. The patients were followed clinically and radiologically for one year. All fractures healed during the follow-up period. The only postoperative technical failure was one lag-screw penetration. Combined compression of the MSP gives increased dynamic capacity which reduces the risk of complications. The low rate of technical failure in our series compares favourably with that of the sliding hip screw or the Gamma nail but randomised trials comparing the MSP with other hip screw systems are necessary to find the true role of the MSP with its various sliding modes. PMID- 8636172 TI - Unipolar or bipolar prosthesis for displaced intracapsular hip fracture in octogenarians: a randomised prospective study. AB - We performed a randomised prospective trial to compare a cemented unipolar prosthesis (Thompson) with a cemented bipolar prosthesis (Monk) in the treatment of displaced intracapsular fractures of the hip in patients over 80 years of age. Patients with a mental test score of less than 5/13 were excluded but the mortality was still about 30% at one year in both groups. We therefore feel that subjective criteria such as the level of pain and the return to the preinjury state are of paramount importance. Two years after operation there was no statistical difference between the rate of complications in the two groups. After adjusting for confounding factors such as differences in the level of function before injury between the groups, the degree of return to the preinjury state was significantly greater (p = 0.04) when using the unipolar prosthesis, which is one quarter of the price of the bipolar. We cannot therefore justify the use of an expensive bipolar prosthesis in patients over 80 years of age. PMID- 8636173 TI - Postoperative treatment of internally fixed ankle fractures: a prospective randomised study. AB - In a prospective, randomised trial of 81 patients with fractures of the ankle of AO types A, B and C we compared two regimes of postoperative management after internal fixation. The patients were mobilised either non-weight-bearing with crutches or weight-bearing in a below-knee walking plaster. We found a temporary benefit in subjective evaluation only (65 v 50 points, Mann-Whitney test, cft, p=0.02) for those with a below-knee walking plaster. There were no significant differences between the groups in the loaded dorsal range of movement (25 degrees v 23 degrees, Mann-Whitney test, cft, p = 0.16) or in the overall clinical result. Both treatments were considered to be satisfactory and their choice depends on the ability to mobilise non-weight-bearing, wound healing, the type of work and personal preference. PMID- 8636174 TI - Pathological fractures of the proximal femur with impending shaft fractures treated by THR and cemented intramedullary nailing: a report of nine cases. AB - We have used total hip replacement combined with cemented intramedullary nailing to treat a selected group of nine patients with pathological fractures of the proximal femur and impending fractures of the shaft due to metastases. One patient died from cardiopulmonary failure on the third postoperative day, but the others were able to walk within the first week after operation. Complications included one recurrent dislocation of the THR and one fracture of an osteolytic lesion of the femoral shaft during nail insertion. Both were managed successfully. The hybrid osteosynthesis which we describe is an alternative to the use of tumour or long-stem prostheses; it has the advantage of preserving bone stock and muscle attachments. PMID- 8636176 TI - Long-term results of conservative treatment for acromioclavicular dislocation. AB - We have reviewed 30 patients who had been treated conservatively for acromioclavicular dislocation between 1979 and 1982 at an average of 12.5 years after the injury. All except one had a good outcome as did five others contacted by telephone. In all patients reviewed the acromioclavicular joint remained subluxed or dislocated. With conservative treatment a good long-term outcome can be expected without restoration of the anatomical configuration of the joint. PMID- 8636175 TI - Redisplaced unstable fractures of the distal radius: a prospective randomised comparison of four methods of treatment. AB - We performed a prospective, randomised trial on 120 patients with redisplaced fractures of the distal radius comparing four methods of treatment. The four treatment groups, each containing 30 patients, were remanipulation and plaster, open reduction and bone grafting, and closed external fixation with and without mobilisation of the wrist at three weeks. The radiological results showed improvement in angulation of the distal radius for the open reduction and bone grafting group. Functional results at six weeks, three and six months and at one year, however, showed no difference between any of the four groups. The main influence on final outcome was carpal malalignment which had a statistically significant negative effect on function. PMID- 8636177 TI - Dynamic radio-ulnar convergence after the Darrach procedure. AB - We reviewed 23 patients who had had 25 Darrach procedures for traumatic or post traumatic disorders of the wrist at a mean follow-up of 75.5 months (36 to 121). The mean age at the time of operation was 61.1 years (34 to 82). All patients were reviewed in person. Assessment included a history, a questionnaire on patient satisfaction and a detailed physical examination. Standardised radiographs of both wrists were taken with the patient's hands in a resting position and during maximal grip. Convergence of the distal ulnar stump towards the distal radius during maximal grip (dynamic radio-ulnar convergence) was seen in 14 wrists including five with actual contact (dynamic radio-ulnar impingement), but this produced symptoms in only two cases. The presence of dynamic radio-ulnar convergence did not correlate with grip strength, pinch strength, range of movement or wrist score, but was associated with increased length of excision of the distal ulna. Nineteen of the 23 patients were satisfied with the procedure. Dynamic radio-ulnar convergence is common after the Darrach procedure, but is rarely symptomatic; resection of the distal ulna remains a reliable procedure in the older patient with pain and loss of movement. Excision of the lower end of the ulna should be restricted to the least required to restore full rotation. PMID- 8636178 TI - Late management of post-traumatic palmar carpal subluxation: a case report. AB - Only a few cases of palmar carpal subluxation associated with an ulnovolar fragment from the distal radius have been reported previously. We report a case which was treated operatively six months after the injury. PMID- 8636179 TI - Early migration predicts late aseptic failure of hip sockets. AB - We report a prospective, stratified study of 60 PCA-cups and 60 RM-polyethylene cups which have been followed for a median time of 90 months, with annual radiography. The radiological migration of cups was measured by the computer assisted EBRA method. A number of threshold migration rates from 1 mm in the first year to 1 mm in five years have been assessed and related to clinically determined revision rates. A total of 28 cups showed a total migration of 1 mm or more within the first two years; 13 of these cups have required revision and been exchanged. The survival curves of cups which had previously shown early migration were considerably different from those without early migration. For cups with a migration of less than 1 mm within the first two years the mean survival at 96 months was 0.96 +/- 0.02; for migrating cups, it was 0.63 +/- 0.11 (log-rank test, p=0.0001; chi-square value=39.4). Early migration is a good predictor for late loosening of hip sockets. PMID- 8636180 TI - Postoperative deep-vein thrombosis in Asian patients is not a rarity: a prospective study of 88 patients with no prophylaxis. AB - Postoperative deep-vein thrombosis (DVT) is believed to be rare in Asians. We studied 88 consecutive patients in Malaysia who had operations for fracture of the proximal femur or for total hip or knee replacement. No patient had prophylaxis against DVT; bilateral ascending venography was performed between six and ten days after operation. A total of 55 patients (62.5%) showed venographic evidence of DVT. The prevalence was greatest after total knee replacement (76.5%), less after total hip replacement (64.3%) and smallest in the fracture group (50%). One patient developed symptomatic pulmonary embolism. In contrast to other reports from Asia, we found an incidence of postoperative DVT which is similar to that reported in Western populations. This suggests that the present practice of withholding routine prophylaxis against thromboembolism in Asian patients undergoing high-risk orthopaedic procedures should be reconsidered. PMID- 8636181 TI - Serum C-reactive protein levels after total hip and knee arthroplasty. AB - Any operation induces an elevation in the level of serum C-reactive protein (CRP). After hip and knee arthroplasty the maximal values are seen on the second and third postoperative days, after which the CRP decreases rapidly. There is no difference between patients with cemented or uncemented prostheses. Major postoperative complications may cause a further increase in CRP levels at one and two weeks. PMID- 8636182 TI - Fibrinolytic inhibition with tranexamic acid reduces blood loss and blood transfusion after knee arthroplasty: a prospective, randomised, double-blind study of 86 patients. AB - We investigated the effect of a fibrinolytic inhibitor, tranexamic acid, on blood loss and blood transfusion in knee arthroplasty by a randomised, double-blind study of 86 patients. A dose of 10 mg/kg body-weight of either tranexamic acid or placebo was given intravenously shortly before the release of the tourniquet, and repeated three hours later. The mean total blood loss was 730 +/- 280 ml in the tranexamic acid group as against 1410 +/- 480 ml in the placebo group (p < 0.001). Both the number of patients receiving blood transfusion and the number of blood units transfused were reduced to one-third in the treated group, and mean postoperative Hb concentrations were significantly higher after prophylaxis. The number of thromboembolic complications was the same in both groups. Tranexamic acid should be given prophylactically in order to be effective. PMID- 8636183 TI - Survivorship analysis of the Kinematic Stabilizer total knee replacement: a 10- to 14-year follow-up. AB - The Kinematic Stabilizer is a posterior-cruciate-substituting design of total knee replacement. We have reviewed 109 primary total knee replacements in 95 patients at a mean follow-up time of 12.7 years (10 to 14). We used survival analysis with failure defined as revision of the implant. This gave a cumulative survival rate of 95% (95% CI +/- 5%) at ten years and (87% +/- 10%) at 13 years. These results from an independent centre confirm the value of an established design of cemented total knee replacement and question the wisdom of the introduction of modifications and new designs without properly controlled trials. PMID- 8636184 TI - Non-operative management of anterior cruciate ligament injuries in the general population. AB - The operative treatment of lesions of the anterior cruciate ligament (ACL) in athletes has been widely advocated and performed. We have investigated the outcome of non-operative management in a lower-demand, general population. We reviewed a consecutive group of 228 patients, which excluded professional and high-level athletes, for two to 12 years after an ACL lesion had been diagnosed by arthroscopy. There was a low incidence of secondary ACL and meniscal surgery, 5.4% and 3.5% respectively, and all these procedures were performed during the first three years after the ACL injury. We studied a subgroup of 109 patients with follow-up of at least five years (mean 8.5 years) and evaluated them using the IKDC score. The general outcome was reasonably satisfactory, with 23% in grade A, 50% in grade B, 21% in grade C and only 6.4% in grade D. We found no statistically significant prognostic effect within this group as regards age, activity levels, or the incidence of associated lesions. PMID- 8636185 TI - Magnetic resonance imaging of patellar tendonitis. AB - The radiological and MRI appearances of 24 knees with patellar tendonitis resistant to conservative therapy were analysed to identify the characteristic MRI appearance and to determine if the patellar morphology was abnormal. A significant thickening of the tendon was found in all cases; this was a more reliable diagnostic feature than a high signal within the superior posterior and central aspect of the tendon at its proximal attachment. The site of the lesion shown by MRI is more compatible with impingement of the inferior pole of the patella against the patellar tendon than a stress overload of the tendon. There were no significant differences in the length of the patella, inferior pole or length of the articular surface when the patellar morphology was compared with that of a matched control group. PMID- 8636186 TI - Arteriovenous fistula after fibular osteotomy leading to recurrent haemarthroses in a total knee replacement. AB - We present a patient with an arteriovenous fistula of the peroneal artery acquired after a left dome tibial osteotomy with midshaft fibular osteotomy. He had subsequently had a total knee replacement on that side. The arteriovenous malformation was only diagnosed when he represented with symptoms and signs of venous hypertension with sterile recurrent haemarthroses in the left knee. Percutaneous obliteration of the fistula, by a combination of coil embolisation and balloon occlusion, cured the symptoms. PMID- 8636187 TI - Syme's amputation revisited: a review of 46 cases. AB - We assessed 46 Syme's amputees attending our prosthetic clinics in terms of the clinical and radiological condition of their stumps, their level of function and problems with the prosthesis. Twenty-five were compared with a matching group of 25 transtibial amputees in regard to activity, function and prosthetic behaviour. Function was similar in the two groups, but Syme's amputees had a higher incidence of prosthetic failure. Overall, Syme's amputees were pleased with their prostheses and their function. Childhood amputations were associated with fewer long-term problems in terms of function and stump problems. Syme's amputation is indicated for congenital foot deformities, fibular hemimelia and severe injury to the foot as long as the heel pad remains viable. PMID- 8636188 TI - Extensile posterior approach to the ankle. AB - Surgical exposure of the ankle is usually obtained by an anterior approach, especially for replacement arthroplasty. The transfibular approach has been popular for arthrodesis. We describe a new posterior approach to the ankle and posterior subtalar joint based on an extra-articular vertical calcaneal osteotomy behind the subtalar joint. The posterior flap so formed is hinged medially and offers wide exposure of the back of the ankle and posterior subtalar joint. This hinge allows safe and stable reduction of the osteotomised calcaneum, and the plane of dissection follows an 'internervous plane' behind the fibula. We have had good results after using this incision in 12 patients with osteoarthritis or rheumatoid arthritis and there have been no difficulties with wound healing. PMID- 8636189 TI - Simultaneous open reduction and Salter innominate osteotomy for developmental dysplasia of the hip. AB - We have studied retrospectively 37 hips in 36 children at an average of 91 months after simultaneous open reduction and Salter innominate osteotomy for developmental hip dysplasia. At the latest review 97.3% were clinically and 83.8% radiologically good or excellent. In three hips (8%) there were signs of avascular necrosis, but only one had been symptomatic. There were no cases of recurrent posterior displacement. PMID- 8636190 TI - The prognosis for walking in osteogenesis imperfecta. AB - We report a postal survey of 59 families of children with osteogenesis imperfecta. From the 51 replies we collected data on developmental milestones and walking ability and related them to the Sillence and the Shapiro classifications of osteogenesis imperfecta. Twenty-four of the patients had been treated by intramedullary rodding. Both classifications helped to predict eventual walking ability. We found that independent sitting by the age of ten months was a predictor for the use of walking as the main means of mobility with 76% attaining this. Of the patients who did not achieve sitting by ten months, walking became the main means of mobility in only 18%. The developmental pattern of mobility was similar in the rodded and non-rodded patients. PMID- 8636191 TI - Acquired thumb flexion contracture in children: congenital trigger thumb. AB - We examined prospectively 4719 newborn infants to determine the congenital incidence of trigger thumb. No cases were found. Fifteen other children aged from 15 to 51 months had surgery for this condition. The anomaly had not been seen at birth and all thumbs presented with a flexion contracture without triggering. The condition is usually seen after birth as a flexion contracture of the interphalangeal joint. The term 'congenital' is a misnomer because patients acquire the deformity after birth. The term 'trigger' is inaccurate as most thumbs show a fixed-flexion contracture without triggering. We suggest that rather than 'congenital trigger thumb' a more appropriate description of this disorder is 'acquired thumb flexion contracture in children'. If the contracture persists after one year of age, treatment by dividing the A-1 pulley is simple and effective. PMID- 8636192 TI - Ultrasound assessment and conservative management of inversion injuries of the ankle in children: plaster of Paris versus Tubigrip. AB - We studied 45 children who presented with an inversion injury of the ankle. The clinical signs suggested injury to the distal growth plate of the fibula, but the plain radiographs appeared normal. Ultrasound examination of the joint in 40 patients showed a subperiosteal haematoma consistent with a growth-plate injury in 23 (57.5%). Children who had been treated with a tubular bandage and crutches by random selection had a mean time to return of normal activity of 14.22 days compared with 21.60 days for those treated with a plaster-of-Paris cast (t=3.60, p=0.0032; d=7.38, 95% CI 3.0 to 11.8). We conclude that children with inversion ankle injuries who have clinical signs of injury to the distal fibular growth plate but a normal radiological appearance, should be treated with a tubular bandage and crutches. PMID- 8636193 TI - Engelmann's disease: a 45-year follow-up. AB - We report a 45-year follow-up of a patient with Engelmann's disease previously described in 1950, showing progression of the disease with unique involvement of the femoral capital epiphyses. The case is compared with others to add some information about the later stages of a disease which is not fully understood. PMID- 8636194 TI - A hereditable combination of congenital anomalies. AB - A kindred of seven affected individuals in three generations is described with autosomal dominant inheritance of bilateral five-fingered hands, pedal polydactyly with syndactyly and agenesis of the tibia and of the lower end of the radius. PMID- 8636196 TI - Smith-Petersen vitallium mould arthroplasty: a 45-year follow-up. PMID- 8636195 TI - Drop attacks and instability of the degenerate cervical spine. PMID- 8636197 TI - The vascular supply to bone in distraction osteoneogenesis: an experimental study. PMID- 8636198 TI - Compartment syndrome in tibial shaft fracture missed because of a local nerve block. PMID- 8636199 TI - Chromosomal aberrations in musculoskeletal tumours: clinical importance. PMID- 8636200 TI - Which primary total hip replacement? PMID- 8636201 TI - Which primary total hip replacement? PMID- 8636202 TI - Which primary total hip replacement? PMID- 8636203 TI - Knee pain after tibial nailing. PMID- 8636204 TI - Knee pain after tibial nailing. PMID- 8636205 TI - Roles of molecular chaperones in protein degradation. PMID- 8636206 TI - XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. AB - The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging. PMID- 8636207 TI - Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes. AB - In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis-Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed. PMID- 8636208 TI - Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. AB - To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for post-translational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation "chase" incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radio labeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of p alpha F was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation. PMID- 8636209 TI - Role of CD3 gamma in T cell receptor assembly. AB - The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression. PMID- 8636210 TI - Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin. AB - Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel. PMID- 8636212 TI - Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium. AB - Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co sedimented with actin filaments, suggesting that the sequestering activity of d cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement. PMID- 8636211 TI - Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide. AB - Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2 terminal, 16-amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal. PMID- 8636213 TI - Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level. AB - Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2 terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system. PMID- 8636214 TI - PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. AB - Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility. PMID- 8636215 TI - Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex. AB - The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51 nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes. PMID- 8636216 TI - Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16. AB - Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well-characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site. PMID- 8636218 TI - Mitotic mechanisms in Alzheimer's disease? AB - The mechanism(s) leading to widespread hyper-phosphorylation of proteins in Alzheimer's disease (AD) are unknown. We have characterized seven new monoclonal antibodies recognizing independent phospho-epitopes in the paired helical filament proteins (PHF) found in AD brain. These antibodies show pronounced immunoreactivity with cultured human neuroblastoma cells that are in the M phase of cell division, but have no discernible reactivity with interphase cells. Immunoreactivity with these antibodies does not localize to the microtubule spindles or chromosomes in M phase, but is confined to the surrounding cytoplasm. Similar staining in M phase is observed with cultured cells of various tissue types and species. Cells arrested in M phase with the microtubule depolymerizing agent, nocodazole, show marked increases in immunoreactivity with the antibodies by immunofluorescence staining, ELISA, and immunoblotting. In neuroblastoma cells, the appearance of the TG/MC phospho-epitopes coincides with activation of mitotic protein kinases, but not with the activity of the neuronal specific cyclin-dependent kinase, cdk5. These data suggest that the TG/MC epitopes are conserved mitotic phospho-epitopes produced as a result of increased mitotic kinase activity. To investigate this possibility in AD, we examined the staining of human brain tissue with MPM-2, a marker antibody for mitotic phospho-epitopes. It was found that MPM-2 reacts strongly with neurofibrillary tangles, neuritic processes, and neurons in AD but has no staining in normal human brain. Our data suggest that accumulation of phospho-epitopes in AD may result from activation of mitotic posttranslational mechanisms which do not normally operate in mature neurons of brain. PMID- 8636219 TI - TrkA receptor ectodomain cleavage generates a tyrosine-phosphorylated cell associated fragment. AB - The extracellular domain of several membrane-anchored proteins can be released as a soluble fragment by the action of a cell surface endoproteolytic system. This cleavage results in the generation of a soluble and a cell-bound fragment. In the case of proteins with signaling capability, such as tyrosine kinase receptors, the cleavage process may have an effect on the kinase activity of the cell-bound receptor fragment. By using several cell lines that express the TrkA neurotrophin receptor, we show that this receptor tyrosine kinase is cleaved by a proteolytic system that mimics the one that acts at the cell surface. TrkA cleavage is regulated by protein kinase C and several receptor agonists (including the TrkA ligand NGF), occurs at the ectodomain in a membrane-proximal region, and is independent of lysosomal function. TrkA cleavage results in the generation of a cell-associated fragment that is phosphorylated on tyrosine residues. Tyrosine phosphorylation of this fragment is not detected in TrkA mutants devoid of kinase activity, suggesting that phosphorylation requires an intact TrkA kinase domain, and is not due to activation of an intermediate intracellular tyrosine kinase. The increased phosphotyrosine content of the cell-bound fragment may thus reflect higher catalytic activity of the truncated fragment. We postulate that cleavage of receptor tyrosine kinases by this naturally occurring cellular mechanism may represent an additional mean for the regulation of receptor activity. PMID- 8636217 TI - Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae. AB - The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth. A search for additional S. cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression. The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S. cerevisiae and other organisms. Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells. Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells. In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally. In this background, deletion of CDC10 had no detectable effect on sporulation. In the other genetic background tested, the consequences of the two deletions were reversed. Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores. Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced. Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation. PMID- 8636220 TI - CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia. AB - Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces. PMID- 8636221 TI - Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. AB - We characterized the role of the E-cadherin adhesion system in the formation of epithelial tight junctions using the calcium switch model. In MDCK cells cultured in low (micromolar) calcium levels, the tight junctional protein Zonula Occludens 1 (ZO-1) is distributed intracellularly in granular clusters, the larger of which codistribute with E-cadherin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to the cell surface, where it localized in regions rich in E-cadherin. Immunoprecipitation with ZO-1 antibodies of extracts from cells kept in low calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the association of ZO 1 with alpha-, beta-, and gamma-catenins. E-cadherin was not detected in the ZO-1 immunoprecipitates but it was found in beta-catenin immunoprecipitates that excluded ZO-1, suggesting that the binding of ZO-1 to catenins may weaken the interaction of these proteins with E-cadherin. Immunofluorescence and immunoelectron microscopy confirmed a close association of beta-catenin and ZO-1 at 0 and 2 h after Ca2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E cadherin and formed a distinct, separate apical ring. The ZO-1-catenin complex was not detected in fully polarized monolayers. MDCK cells permanently transformed with Moloney sarcoma virus, which expresses low levels of E-cadherin, displayed clusters of cytoplasmic ZO-1 granules and very little of this protein at the cell surface. Upon transfection with E-cadherin into Moloney sarcoma virus MDCK cells, ZO-1 redistributed to E-cadherin-rich lateral plasma membrane but later failed to segregate into mature tight junctions. Our experiments suggest that catenins participate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic transformation may block the formation of tight junctions, either by decreasing the levels of E-cadherin or by preventing a late event: the segregation of tight junction from the zonula adherens. PMID- 8636222 TI - Visualization of CD2 interaction with LFA-3 and determination of the two dimensional dissociation constant for adhesion receptors in a contact area. AB - Many adhesion receptors have high three-dimensional dissociation constants (Kd) for counter-receptors compared to the KdS of receptors for soluble extracellular ligands such as cytokines and hormones. Interaction of the T lymphocyte adhesion receptor CD2 with its counter-receptor, LFA-3, has a high solution-phase Kd (16 microM at 37 degrees C), yet the CD2/LFA-3 interaction serves as an effective adhesion mechanism. We have studied the interaction of CD2 with LFA-3 in the contact area between Jurkat T lymphoblasts and planar phospholipid bilayers containing purified, fluorescently labeled LFA-3. Redistribution and lateral mobility of LFA-3 were measured in contact areas as functions of the initial LFA 3 surface density and of time after contact of the cells with the bilayers. LFA-3 accumulated at sites of contact with a half-time of approximately 15 min, consistent with the previously determined kinetics of adhesion strengthening. The two-dimensional Kd for the CD2/LFA-3 interaction was 21 molecules/microns 2, which is lower than the surface densities of CD2 on T cells and LFA-3 on most target or stimulator cells. Thus, formation of CD2/LFA-3 complexes should be highly favored in physiological interactions. Comparison of the two-dimensional (membrane-bound) and three-dimensional (solution-phase) KdS suggest that cell cell contact favors CD2/LFA-3 interaction to a greater extent than that predicted by the three-dimensional Kd and the intermembrane distance at the site of contact. LFA-3 molecules in the contact site were capable of lateral diffusion in the plane of the phospholipid bilayer and did not appear to be irreversibly trapped in the contact area, consistent with a rapid off-rate. These data provide insights into the function of low affinity interactions in adhesion. PMID- 8636224 TI - Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content. AB - Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains. PMID- 8636223 TI - Human neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3. AB - Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell-substrate interaction. PMID- 8636225 TI - RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus. AB - Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have different defects; Q69L (glutamine 69 changed to leucine) is defective in GTP hydrolysis while T24N (threonine 24 changed to asparagine) is defective in binding GTP. Both protein and snRNP import are sensitive either to the presence of the two mutant Ran proteins, which act as dominant negative inhibitors of nuclear import, or to incubation with nonhydrolyzable GTP analogues. This demonstrates that there is a requirement for a GTPase activity for the import of U snRNPs, as well as proteins, into the nucleus. The dominant negative effects of the two mutant Ran proteins indicate that the pathways of protein and snRNP import share at lease one common component. PMID- 8636226 TI - Native 3D structure of eukaryotic 80s ribosome: morphological homology with E. coli 70S ribosome. AB - A three-dimensional reconstruction of the eukaryotic 80S monosome from a frozen hydrated electron microscopic preparation reveals the native structure of this macromolecular complex. The new structure, at 38A resolution, shows a marked resemblance to the structure determined for the E. coli 70S ribosome (Frank, J., A. Verschoor, Y. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, and Srivastava. 1996b. In press; Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava ., A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, and R. Agrawal. 1995. Nature (Lond.).376:441-444.) limited to a comparable resolution, but with a number of eukaryotic elaborations superimposed. Although considerably greater size and intricacy of the features is seen in the morphology of the large subunit (60S vs 50S), the most striking differences are in the small subunit morphology (40S vs 30S): the extended beak and crest features of the head, the back lobes, and the feet. However, the structure underlying these extra features appears to be remarkably similar in form to the 30S portion of the 70S structure. The intersubunit space also appears to be strongly conserved, as might be expected from the degree of functional conservation of the ribosome among kingdoms (Eukarya, Eubacteria, and Archaea). The internal organization of the 80S structure appears as an armature or core of high-density material for each subunit, with the two cores linked by a single bridge between the platform region of the 40S subunit and the region below the presumed peptidyltransferase center of the 60S subunit. This may be equated with a close contact of the 18S and 28S rRNAs in the translational domain centered on the upper subunit:subunit interface. PMID- 8636227 TI - A v-SNARE implicated in intra-Golgi transport. AB - We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles. PMID- 8636229 TI - Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. AB - VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole. PMID- 8636228 TI - An endoplasmic reticulum storage disease causing congenital goiter with hypothyroidism. AB - In humans, deficient thyroglobulin (Tg, the thyroid prohormone) is an important cause of congenital hypothyroid goiter; further, homozygous mice expressing two cog/cog alleles (linked to the Tg locus) exhibit the same phenotype. Tg mutations might affect multiple different steps in thyroid hormone synthesis; however, the microscopic and biochemical phenotype tends to involve enlargement of the thyroid ER and accumulation of protein bands of M(r) < 100. To explore further the cell biology of this autosomal recessive illness, we have examined the folding and intracellular transport of newly synthesized Tg in cog/cog thyroid tissue. We find that mutant mice synthesize a full-length Tg, which appears to undergo normal N-linked glycosylation and glucose trimming. Nevertheless, in the mutant, Tg is deficient in the folding that leads to homodimerization, and there is a deficiency in the quantity of intracellular Tg transported to the distal portion of the secretory pathway. Indeed, we find that the underlying disorder in cog/cog mice is a thyroid ER storage disease, in which a temperature-sensitive Tg folding defect, in conjunction with normal ER quality control mechanisms, leads to defective Tg export. In relation to quality control, we find that the physiological response in this illness includes the specific induction of five molecular chaperones in the thyroid ER. Based on the pattern of chaperone binding, different potential roles for individual chaperones are suggested in glycoprotein folding, retention, and degradation in this ER storage disease. PMID- 8636230 TI - Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells. AB - Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles. PMID- 8636231 TI - Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. AB - In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a Michaelis-Menten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion. PMID- 8636232 TI - Three-dimensional structure of the Z band in a normal mammalian skeletal muscle. AB - The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three-dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z-filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band. PMID- 8636233 TI - Cell cycle regulation of dynein association with membranes modulates microtubule based organelle transport. AB - Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes. PMID- 8636234 TI - DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation. AB - We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin-related proteins. Immunoblots using an antibody raised against a non-conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone. PMID- 8636235 TI - A purified Drosophila septin complex forms filaments and exhibits GTPase activity. AB - Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells. PMID- 8636236 TI - Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. AB - The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis. PMID- 8636237 TI - Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo. AB - The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum. PMID- 8636238 TI - Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites. AB - The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane. PMID- 8636239 TI - Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension. AB - DM-GRASP is an immunoglobulin superfamily cell adhesion molecule that is expressed in both the developing nervous and immune system. Specific populations of neurons respond to DM-GRASP substrates appears to require homophilic interactions between DM-GRASP molecules. We were interested in determining whether DM-GRASP interacts heterophilically with other ligands as well. We have found that eleven proteins from embryonic chick brain membranes consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion molecules were not detected in the eluate. The DM-GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreased neurite extension on laminin, suggesting that the antibody is not toxic or generally inhibiting motility. We present two possible models for the DM-GRASP NgCAM association and a hypothesis for neural cell adhesion function that features the dimerization of cell adhesion molecules. PMID- 8636240 TI - Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. AB - The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells. PMID- 8636241 TI - Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism. AB - The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo. PMID- 8636244 TI - Clinical review 77: evaluation of secondary amenorrhea. PMID- 8636243 TI - Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2. AB - Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met. PMID- 8636242 TI - An antiglycolipid antibody inhibits Madin-Darby canine kidney cell adhesion to laminin and interferes with basolateral polarization and tight junction formation. AB - Epithelial cells polarize not only in response to cell-cell contacts, but also to contacts with a substratum composed of extracellular matrix molecules. To probe the role of specific matrix constituents in epithelial cell polarization, we investigated the effects of an adhesion-blocking mAb, 12B12, on initial polarization of MDCK cells. The 12B12 antibody, raised against whole MDCK cells, blocks adhesion to laminin by 65% but has no effect on adhesion of cells to collagen type I. Taking advantage of this antibody's function-blocking activity, as well as the fact that MDCK cells secrete laminin, the role of endogenous laminin in polarization was examined by plating cells on collagen-coated substrata in the presence of the antibody. Under these conditions, cell spreading was reduced 1.5h after plating, and cells were flatter and had fewer microvilli after 24 h. Even though lateral cell membranes were closely apposed, transepithelial resistance in the presence of the antibody was significantly reduced relative to controls. When the polarization of specific apical and basolateral markers was examined both biochemically and immunocytochemically in the presence of the antibody, we observed that the apical marker polarized at normal rates while basolateral markers did not. Surprisingly, the 12B12 antibody was not directed against any known cell adhesion protein but reacted specifically with Forssman antigen, a glycosphingolipid. These results suggest that glycolipids may play a significant role in cell adhesion via laminin and in epithelial cell polarization. PMID- 8636245 TI - Acromegaly: what constitutes optimal therapy? PMID- 8636246 TI - Cytokine antagonists: new ideas for the management of Graves' ophthalmopathy. PMID- 8636247 TI - Interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptor inhibit IL-1 induced glycosaminoglycan production in cultured human orbital fibroblasts from patients with Graves' ophthalmopathy. AB - An accumulation of glycosaminoglycans (GAG) is a feature characteristic of orbital connective tissues from patients with Graves' ophthalmopathy (GO) that leads directly to the clinical expressions of the disease. Interleukin-1 (IL-1), produced by macrophages and fibroblasts within the diseased orbit, stimulates GAG synthesis by orbital fibroblasts. We designed the current study to determine whether particular agents might block this effect and thus be useful in the treatment of GO. Orbital fibroblast cultures were grown to confluence and incubated for 48 h with IL-1 (1-10 U/mL) alone or IL-1 (10 U/mL) in combination with IL-1 receptor antagonist (IL-1ra; 1-40 ng/mL) or soluble IL-1 receptor (sIL 1R; 0.25-10 micrograms/mL). Cells were labeled with [3H]glucosamine and processed for GAG quantitation. The addition of IL-1 alone stimulated GAG synthesis by 73 176% (mean, 104%; P < 0.05). Significant inhibition of IL-1-stimulated GAG synthesis was observed after treatment of normal fibroblasts with IL-1ra at a concentration of 5 ng/mL (12.5-fold molar excess; mean, 33%; P < 0.05); essentially complete inhibition was achieved at 40 ng/mL (100-fold molar excess; mean, 86%; P < 0.05). Significant inhibition of GAG synthesis by sIL-1R was observed at a concentration of 0.5 microgram/mL (720-fold molar excess; mean, 79%; P < 0.05), and inhibition was essentially complete at 1 microgram/mL (1440 fold molar excess; mean, 89%; P < 0.05). IL-1ra and sIL-1R are potent inhibitors of IL-1-induced GAG production by cultured human orbital fibroblasts. Our results suggest that these two compounds, shown in early trials to be safe when administered parenterally, may be useful in the prevention or treatment of GO. PMID- 8636248 TI - Clinical case seminar: Cushing's syndrome in an elderly woman with large thyroid and pituitary masses. PMID- 8636249 TI - Prismatic cases: 17,20-desmolase (17,20-lyase) deficiency. PMID- 8636250 TI - Beyond the somatopause: growth hormone deficiency in adults over the age of 60 years. AB - GH secretion declines by 14% decade of adult life, leading to the suggestion that people over the age of 60 yr are functionally GH deficient. If this is the case, one might not be able to detect a difference in GH secretion between the elderly with documented hypothalamic-pituitary disease and an age-matched control group. We studied GH secretion in 24 patients with hypothalamic-pituitary disease and 24 controls matched for body mass index and age using 24-h GH profiles, arginine stimulation tests, and serum insulin-like growth factor I (IGF-I) levels. The median (range) area under the curve of the GH profile [< 9.6 (< 9.6-20) vs. 18.5 (10.7-74.4) micrograms/L.24 h; P < 0.0001], the median stimulated peak GH response to arginine [< 0.4 (< 0.4-7.7) vs. 8.0 (1.6-37.0) micrograms/L; P < 0.0001], and the median serum IGF-I concentration [102 (< 14-162) vs. 147 (65 255) ng/mL; P = 0.0002] were significantly lower in the patients than in the controls. Fifteen patients showed no evidence of spontaneous or stimulated GH secretion, whereas all controls had evidence of both. The area under the GH curve in the 33 subjects with demonstrable GH secretion correlated significantly with the peak GH response to arginine (r = 0.71; P < 0.0001), but not with serum IGF-I concentration. This study suggests that organic GH deficiency in the elderly is distinct from the decline in GH secretion associated with the aging process. These patients may benefit from GH replacement therapy. PMID- 8636251 TI - Incidence of subacute thyroiditis recurrences after a prolonged latency: 24-year survey. AB - Subacute thyroiditis, which is considered to be a viral disease, rarely recurs after a complete recovery. We evaluated data on 3,344 patients with subacute thyroiditis who were seen at Ito Hospital between 1970 and 1993. Subacute thyroiditis recurred in 48 of 3,344 patients 14.5 +/- 4.5 yr after the first episode. Five patients experienced a third episode 7.6 +/- 2.4 yr after the second. The mean age of the patients at the first, second, and third episode was 38.4 +/- 6.3, 53.1 +/- 8.9, and 57.8 +/- 10.1 yr old, respectively. The mean incidence of a recurrence was 2.3 +/- 0.9% per year. The erythrocyte sedimentation rate and the duration of treatment were each significantly decreased at the second episode as compared with the first. Thus, recurrences of subacute thyroiditis do occur at least in 2% of patients and exhibited relatively mild clinical manifestations. PMID- 8636252 TI - X-linked adrenoleukodystrophy is a frequent cause of idiopathic Addison's disease in young adult male patients. AB - X-Linked adrenoleukodystrophy (ALD) is a genetic disease associated with demyelination of the central nervous system, adrenal insufficiency, and accumulation of very long chain fatty acids in tissue and body fluids. ALD is due to mutation of a gene located in Xq28 that encodes a peroxisomal transporter protein of unknown function. The most common phenotype of ALD is the cerebral form (45%) that develops in boys between 5-12 yr. Adrenomyeloneuropathy (AMN) involves the spinal cord and peripheral nerves in young adults (35%). Adrenal insufficiency (Addison's disease) is frequently associated with AMN or cerebral ALD and may remain the only clinical expression of ALD (8% of cases). The prevalence of ALD among adults with Addison's disease remains unknown. To evaluate this prevalence, we performed biochemical analysis of very long chain fatty acids in 14 male patients (age ranging from 12-45 yr at diagnosis) previously diagnosed as having primary idiopathic adrenocortical insufficiency. In 5 of 14 patients (35%), elevated plasma concentrations of very long chain fatty acids were detected. None of these patients had adrenocortical antibodies. By electrophysiological tests and magnetic resonance imaging it was determined that two patients had cerebral ALD, one had adrenomyeloneuropathy with cerebral involvement, and two had preclinical AMN. Our data support the hypothesis that ALD is a frequent cause of idiopathic Addison's disease in children and adults. PMID- 8636253 TI - Venous angiography is needed to interpret inferior petrosal sinus and cavernous sinus sampling data for lateralizing adrenocorticotropin-secreting adenomas. AB - Bilateral simultaneous venous sampling of ACTH from the inferior petrosal sinus is a reliable test for diagnosing Cushing's disease, but is not reliable for lateralizing ACTH-secreting pituitary adenomas. We reviewed 23 consecutive patients with Cushing's disease who underwent venous angiography of the cavernous and inferior petrosal sinuses followed by bilateral simultaneous venous sampling of ACTH in the inferior petrosal and cavernous sinuses. Venous drainage was bilaterally symmetric in 14 patients (61%) and asymmetric in 9 (39%). The most common asymmetric pattern (6 patients) was for blood from both cavernous sinuses to drain into the right inferior petrosal sinus, with no significant drainage into the left. Cavernous sinus sampling in 21 patients correctly lateralized the tumor in 12 cases of symmetric venous drainage, but in only 3 cases of asymmetric drainage. Inferior petrosal sinus sampling in all 23 patients correctly lateralized the tumor in 12 cases of symmetric drainage, but in only four cases of asymmetric drainage. Overall, venous sampling correctly lateralized 70% of the tumors. Incorrect lateralization in cases of asymmetric venous drainage is probably attributable to shunting of blood toward the side of dominant venous drainage. Our findings illustrate the need for venography in all patients undergoing venous sampling of ACTH because an understanding of the venous drainage patterns is essential to correctly interpret venous sampling data and warn physicians that the lateralization data may be incorrect or unreliable. PMID- 8636254 TI - Spontaneous cortisol and growth hormone secretion interactions in patients with nonclassic 21-hydroxylase deficiency (NCCAH) and control children. AB - Both exogenous and endogenous hypercortisolism result in reduced GH secretion and decreased somatic growth. However, little is known about the relation between endogenous cortisol and GH secretion under physiological or slightly disturbed conditions. To examine this, we measured and evaluated the pulsatility and circadian rhythmicity, and we cross-correlated the secretory patterns of cortisol and GH in six prepubertal patients with nonclassic 21-hydroxylase deficiency (NCCAH) and seven age-matched short-normal children. Cortisol and GH were secreted in a pulsatile fashion in both the NCCAH and control groups. The nocturnal peak cortisol increment and time-integrated area were lower in the NCCAH patients than in controls, whereas there was no difference in the total 24 h cortisol secretion between the two groups. The nocturnal increase of GH in NCCAH children, on the other hand, was associated with a significant augmentation of the pulse amplitude, whereas in control children there was an elevation of the baseline component. The cross-correlation analysis of the 24-h raw data showed that cortisol and GH were negatively correlated at brief lag times of 0-30 min, and positively correlated at long lag times of 12-12.5 h in both groups, with cortisol leading GH. The negative correlation might reflect either the negative effect of glucocorticoids on the adrenergic system, which stimulates GH secretion through GH-releasing hormone (GHRH) elevations and somatostatin (SRIH) decreases, or the absence of an inhibitory effect of CRH on SRIH. The positive correlation may reflect the positive effect of glucocorticoids on the GH gene. In conclusion, NCCAH children have a mild nocturnal cortisol deficiency compared with control children, as previously reported, and a distinct circadian pattern of pulsatile GH secretion. The hypothalamic-pituitary-adrenal (HPA) axis exerts both negative and positive influences on GH secretion, with mild disturbances in cortisol biosynthesis associated with slight alterations of GH secretion. PMID- 8636256 TI - Insulin receptor mediates inhibitory effect of insulin, but not of insulin-like growth factor (IGF)-I, on IGF binding protein 1 (IGFBP-1) production in human granulosa cells. AB - Insulin-like growth factor binding proteins (IGFBPs) may participate in regulating ovarian function by modifying effects of insulin-like growth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperinsulinemic insulin resistant states, such as polycystic ovary syndrome. We examined regulation of IGFBP-1 production in human granulosa cells by insulin and IGF-I. The cells were obtained during in vitro fertilization, plated in McCoy-5A tissue culture medium supplemented with 10% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% humidity, 5% CO2 for 48 h. After additional 24 h incubation without fetal calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 micrograms/mL monoclonal anti insulin receptor antibody IR-47-9. After 48 h incubation with insulin or IGF-I, the medium was collected and IGFBP-1 and progesterone concentrations were measured, using kits from Diagnostic Systems Laboratories, Webster, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) cells, without consistent stimulatory effect of either insulin or IGF-I. Control cells produced 7.0 +/- 1.7 ng/mL of IGFBP-1. Incubation with 1 or 10 ng/mL of insulin resulted in culture medium IGFBP-1 concentrations of 7.1 +/- 1.3 ng/mL and 5.4 +/- 0.7 ng/mL, respectively (P = NS). Incubation with 100 ng/mL of insulin reduced IGFBP-1 culture medium concentration to 1.6 +/- 0.3 ng/mL (P < 0.01, compared with controls). 1, 10, and 100 ng/mL of IGF-I inhibited IGFBP-1 concentrations in the conditioned culture medium to 1.3 +/- 0.3 ng/mL, 0.4 +/- 0.1 ng/mL and 0.3 +/- 0.1 ng/mL, respectively (P < 0.01, compared with controls). Preincubation with antiinsulin receptor antibody IR-47-9 alleviated inhibitory effect of insulin, but not of IGF-I on IGFBP-1 production. After preincubation with IR-47-9, IGFBP-1 culture medium concentrations were 5.9 +/- 0.8 ng/mL, 4.9 +/- 1.2 ng/mL, and 4.8 +/- 1.3 ng/mL for 1, 10, and 100 ng/mL of insulin, respectively. The latter number was significantly higher than IGFBP-1 concentration in the medium collected from cells incubated with 100 ng/mL of insulin without IR-47-9 (1.6 +/- 0.3 ng/mL, P < 0.01) and not significantly different from the control cells. For cells preincubated with IR-47-9 and then incubated with 1, 10, or 100 ng/mL of IGF-I, the IGFBP-1 conditioned culture medium concentrations were 1.7 +/- 0.1 ng/mL, 0.5 +/- 0.2 ng/mL, and 0.3 +/- 0.1 ng/mL, respectively. None of these were significantly different from the IGFBP-1 concentrations in the medium collected from cells incubated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFBP-1 production by cultured human granulosa cells; 2) IGF-I is a more potent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect on IGFBP-1 production via insulin receptor, while IGF-I appears to exert its effect via another receptor. PMID- 8636255 TI - Increased levels of methylglyoxal-metabolizing enzymes in mononuclear and polymorphonuclear cells from insulin-dependent diabetic patients with diabetic complications: aldose reductase, glyoxalase I, and glyoxalase II--a clinical research center study. AB - Levels of aldose reductase, glyoxalase I, and glyoxalase II in mononuclear and polymorphonuclear cells from insulin-dependent diabetes mellitus (IDDM) patients with long term diabetic complications were compared to levels in IDDM patients without complications and to those in nondiabetic controls. Cells were isolated from 22 asymptomatic long term IDDM patients, 22 symptomatic IDDM patients, and 16 controls, using a double gradient centrifugation procedure. Aldose reductase was determined by Western blots using polyclonal antiserum to human aldose reductase purified from skeletal muscle. Glyoxalase I and glyoxalase II were determined spectrophotometrically. Aldose reductase in mononuclear cells from symptomatic IDDM patients is significantly elevated compared to that in asymptomatic IDDM patients (mean +/- SEM, 0.96 +/- 0.20 vs. 0.46 +/- 0.08 microgram/mg protein; P < 0.02). Aldose reductase was not detected in polymorphonuclear cells. Glyoxalase I in mononuclear and polymorphonuclear cells from symptomatic IDDM patients is significantly elevated compared to that in controls [mean for mononuclear cells, 0.46 +/- 0.03 vs. 0.37 +/- 0.03 mumol/min.mg (P < 0.05); mean for polymorphonuclear cells, 0.16 +/- 0.01 vs. 0.10 +/- 0.01 mumol/min.mg (P < 0.002)]. Glyoxalase II is significantly elevated only in polymorphonuclear cells from symptomatic IDDM patients compared to controls (mean, 0.13 +/- 0.01 vs. 0.063 +/- 0.016 mumol/min.mg; P < 0.005). Glutathione peroxidase and glutathione S-transferase were not significantly different in these populations. Aldose reductase, glyoxalase I, and glyoxalase II are involved in the metabolism of methylglyoxal, suggesting that methylglyoxal may play a role in the etiology of diabetic complications. PMID- 8636257 TI - Impaired processing of proopiomelanocortin in corticotroph macroadenomas. AB - The regulation and secretion of the ACTH precursors POMC and pro-ACTH were assessed directly using a 2-site immunoradiometric assay in six patients with pituitary macroadenomas (> or = 1.2 cm in diameter) and 27 patients with Cushing's disease due to a microadenoma. ACTH precursor levels were elevated in patients with macroadenomas (150-3690 pmol/L; normal range, < 5-40 pmol/L) and significantly higher than those in microadenoma patients (median, 29 pmol/L; range, 9-104 pmol/L; P < 0.001). Patients with macroadenomas also had increased ACTH precursor/ACTH ratios (15-181:1) compared with microadenoma patients (median, 5:1, range, 0.7-18.5:1; P < 0.001). ACTH precursors were unresponsive to high dose dexamethasone in patients with macroadenomas, whereas ACTH and cortisol responses varied. After CRH administration, ACTH precursors were unchanged, whereas cortisol increased significantly, suggesting the release of biologically active ACTH. This study clearly demonstrates reduced processing of POMC to ACTH in large pituitary tumors, a characteristic usually associated with tumors causing the ectopic ACTH syndrome, and provides evidence for differential regulation of ACTH precursors and ACTH by glucocorticoid and CRH. Variation in the clinical symptoms of patients with corticotroph macroadenomas may be attributable to differences in biological potency between the ACTH precursors and ACTH. PMID- 8636258 TI - Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus. AB - Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed. PMID- 8636259 TI - Nocturnal blood pressure elevation is related to adrenomedullary hyperactivity, but not to hyperinsulinemia, in nonobese normoalbuminuric type 1 diabetes. AB - We tested the hypothesis that insulin is an independent risk factor for elevated blood pressure. As our model we selected type 1 diabetes with peripheral circulatory hyperinsulinemia induced by sc insulin treatment. In 15 nonobese normoalbuminuric patients with type 1 diabetes (23.7 +/- 0.8 yr old) and in 15 healthy controls matched for age, sex, and body weight, ambulatory blood pressure was recorded over 24 h. The areas under the curve of free insulin (605 +/- 135 vs. 275 +/- 35 pmol/L.h; P = 0.03) and basal plasma epinephrine concentrations were higher (170 +/- 10 vs. 130 +/- 10 pmol/L; P = 0.02), and the basal aldosterone level was lower (220 +/- 40 vs. 410 +/- 50 pmol/L; P = 0.009) in the patients. The nocturnal decline in systolic blood pressure was less pronounced (13 +/- 1 vs. 19 +/- 2 mm Hg; P = 0.007) in the patients. Multivariate adjustment (r2 = 0.75; P = 0.0002) showed an effect of basal plasma epinephrine and norepinephrine levels and body mass index on the mean nocturnal systolic blood pressure, but showed no effect of age, sex, hemoglobin A1c, aldosterone, or, in particular, insulin. We found a blunted nocturnal fall in blood pressure in nonobese, normoalbuminuric type 1 diabetic patients. These patients showed increased adrenomedullary activity, and this predominantly contributed to the blood pressure alterations. We also found hyperinsulinemia in these patients, but, after controlling for covariates, blood pressure was independent of the insulin level. PMID- 8636260 TI - Cytokine production in the bone marrow microenvironment: failure to demonstrate estrogen regulation in early postmenopausal women. AB - Recent studies in rodents and in human in vitro systems suggest that the action of estrogen on bone is modulated by various bone-resorbing cytokines produced by osteoblasts, bone marrow cells, or both, but results among studies have been conflicting. Thus, we studied 20 untreated women (controls) and 20 women who had received estrogen replacement therapy since the time of menopause; both groups were 5 +/- 1 yr (mean +/- SE) postmenopausal. From bone marrow aspirates, we obtained marrow plasma to evaluate the production of cytokines by marrow and trabecular bone cells in vivo and marrow mononuclear cells to assess in vitro the constitutive and lipopolysaccharide-stimulated production of cytokines and also to test the in vitro effects of 17 beta-estradiol (10(-8) mol/L). Interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-1 receptor antagonist, and IL-6-soluble receptor were measured by specific enzyme-linked immunosorbent assay. No statistically significant difference between the two groups in the level of any of these cytokines was detected in the bone marrow plasma or the conditioned medium in vitro. Furthermore, in vitro treatment with 17 beta-estradiol did not affect basal or lipopolysaccharide-stimulated cytokine production. Thus, our data suggest that for normal post-menopausal women, mediation of the effects of estrogen by a single cytokine is unlikely. Either multiple cytokines are involved and small changes in these are difficult to detect in clinical investigative studies, or other mechanisms are operative. PMID- 8636261 TI - Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects. AB - In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals. PMID- 8636262 TI - Alterations in pulsatile luteinizing hormone and follicle-stimulating hormone secretion in idiopathic oligoasthenospermic men: assessment by deconvolution analysis--a clinical research center study. AB - To investigate the nature of neuroendocrine disturbances of the hypothalamo pituitary-gonadal axis in idiopathic male infertility, we studied 14 infertile men with oligoasthenozoospermia (OLIGO) and 15 age-, body mass index-, and community-matched euspermic controls by blood withdrawal at 10-min intervals for 12 h to encompass basal (8-h) and exogenous GnRH-stimulated (4-h) pulsatile release of LH and FSH (by immunoradiometric assay) as well as testosterone (by RIA). Deconvolution analysis was used to estimate endogenous LH and FSH half lives, secretory burst frequency, amplitude, duration, and mass. OLIGO men exhibited normal serum concentrations of total, free, and percent dialyzable testosterone and estradiol, but distinct dynamic alterations within the LH and FSH axes; namely (P < 0.05), 1) a prolonged half-life of LH (OLIGO, 95 +/- 19 min; control, 80 +/- 9.3 min) and a reduced half-life of FSH (OLIGO, 260 +/- 79 min; control, 320 +/- 93 min); 2) a low LH, but normal FSH, secretory burst frequency (OLIGO, 12 +/- 3.4; control, 15 +/- 3.0 LH pulses/day); 3) a decreased serum testosterone peak frequency (OLIGO, 16 +/- 4.3; control, 21 +/- 3.2 peaks/day); and 4) an amplified mass of LH (1.1- to 1.3-fold higher in OLIGO) and FSH (2.4- to 2.7-fold higher in OLIGO) secreted per burst basally as well as after GnRH injection. These disturbances were readily distinguishable from the neuroendocrine dysregulation described in other states of male hypogonadotropism (e.g. uremia, fasting, and aging). PMID- 8636263 TI - New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism. AB - Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for the mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients' genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. PMID- 8636264 TI - Insulin, insulin-like growth factor-binding protein-1, and sex hormone-binding globulin in patients with Turner's syndrome: course over age in untreated patients and effect of therapy with growth hormone alone and in combination with oxandrolone. AB - We have studied the course over age of fasting insulin, sex hormone-binding globulin (SHBG), and insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) in untreated children with Turner's syndrome (TS) and measured the course of these parameters during therapy with GH alone and in combination with oxandrolone. Forty patients with TS, aged 3.7-16.4 yr, were investigated before any therapy. Fasting insulin levels increased significantly with chronological age, whereas SHBG and IGFBP-1 decreased with chronological age, and serum concentrations of these parameters were in the normal range. SHBG and IGFBP-1 were not coregulated by insulin in TS as previously reported under physiological conditions; IGFBP-1 was inversely correlated with insulin, but SHBG was not, and neither parameter was correlated with the other. Twenty-eight patients were further investigated 3, 6, 9, and 12 months after the start of GH monotherapy (12 18 IU/m2-week) and 3, 6, 9, and 12 months after the addition of oxandrolone (0.0625 mg/kg.day; n = 16). There was a significant increase in insulin levels during GH monotherapy and a further increase during combination therapy, with peak levels 3 months after the start of GH and combination therapy, respectively. Both SHBG and IGFBP-1 levels decreased significantly during GH monotherapy, with a further dramatic decrease after the addition of oxandrolone. Levels of free testosterone were unaffected by both treatment regimens. IGFBP-1 was inversely correlated with insulin concentrations at all time points after the start of therapy. SHBG was inversely correlated with IGF-I concentrations, but showed no relation to insulin concentrations during GH monotherapy. In conclusion, there were no abnormalities in serum concentrations of insulin, SHBG, and IGFBP-1 in untreated patients that could help to explain the retarded growth in patients with TS. All effects of combined GH and oxandrolone therapy on endocrine parameters such as insulin, SHBG, IGFBP-1 and IGF-I mimic the endocrine pattern normally observed during the pubertal growth spurt. Our data confirm the importance of insulin in the regulation of IGFBP-1, but do not point to a coregulation of IGFBP-1 and SHBG by insulin in patients with TS. PMID- 8636265 TI - Clinical performances of galactosyl hydroxylysine, pyridinoline, and deoxypyridinoline in postmenopausal osteoporosis. AB - We have previously shown that galactosyl hydroxylysine (GHYL), pyridinoline (PYD), and deoxypyridinoline (DPD) have a better accuracy and discriminate power than hydroxyproline in distinguishing postmenopausal osteoporotic women from premenopausal controls. In this study, we evaluated the clinical performances of GHYL, PYD, and DPD, alone or in combination, in distinguishing postmenopausal osteoporotic women (OPBD, n = 26) from age-matched controls (CBD, n = 19). The diagnosis of osteoporosis was based upon the bone density (BD) of the lumbar spine measured by quantitative computed tomography (CBD: BD > 108 mg/cm3; OPBD: BD < 70 mg/cm3). Urinary excretion of GHYL, PYD, and DPD were measured by HPLC, and all data were expressed as the molar ratio with the creatinine excretion (GHYL/CR, PYD/CR, and DPD/CR). The clinical performances were tested by: Z score analysis (Z), Receiver Operated Characteristic curve analysis (%Acc) and logistic regression analysis of the posterior probabilities for prediction from a logistic model (LOGIST). GHYL/CR, PYD/CR, and DPD/CR were significantly increased in OPBD compared with CBD. The clinical performances were similar for the three assays, with slightly better performances for GHYL/CR (GHYL/CR: Z = 3.14, %Acc = 70 +/- 8, LOGIST P = 0.01; PYD/CR: Z = 2.19, %Acc = 67 +/- 8, LOGIST P = 0.051; DPD/CR: Z = 2.13, %Acc = 65 +/- 8, LOGIST P = 0.06). None of the possible combinations of the three assays yielded better clinical performances than GHYL/CR alone. In conclusion, this study further confirms the validity of GHYL, PYD, and DPD as markers of bone resorption. PMID- 8636266 TI - Functional characteristics of three new germline mutations of the thyrotropin receptor gene causing autosomal dominant toxic thyroid hyperplasia. AB - We report three unrelated families in which hyperthyroidism associated with thyroid hyperplasia was transmitted in an autosomal dominant fashion, in the absence of signs of autoimmunity. Exon 10 of the TSH receptor gene was directly sequenced after PCR amplification from DNA of peripheral leukocytes. In one family, a C to A transversion resulted in an S505R substitution in the third transmembrane segment; in the second, an A to T transversion caused a N650Y substitution in the sixth transmembrane segment; and in the third family, an A to G transition resulted in an N670S substitution in the seventh transmembrane segment. When expressed by transfection in COS-7 cells, each mutated receptor displayed an increase in constitutive stimulation of cAMP production; no effect on basal accumulation of inositol phosphates (IP) could be detected. In binding studies, cells transfected with wild-type or mutated receptors showed similar levels of expression, with the mutated receptors displaying similar or slightly increased affinity for bovine TSH (bTSH) binding. Cells transfected with S505R and N650Y mutants showed a similar cAMP maximal TSH-stimulated accumulation over the cells transfected with the wild type, whereas N670S transfectants showed a blunted response with an increase in EC50. A higher IP response to 100 mU/mL bTSH over that obtained with the wild-type receptor was obtained in cells transfected with N650Y; in contrast, cells transfected with S505R showed a blunted IP production (50% less), and the N670S mutant completely lost the ability to stimulate IP accumulation in response to bTSH. The differential effects of individual mutations on stimulation by bTSH of cAMP or IP accumulation suggest that individual mutant receptors may achieve different active conformations with selective abilities to couple to Gs alpha and to Gq alpha. PMID- 8636267 TI - Insulin resistance in growth hormone-deficient adults: defects in glucose utilization and glycogen synthase activity. AB - Fourteen GH-deficient (GHD) adults were compared with 12 age-, sex-, and body mass index-matched control subjects using a baseline tritiated glucose equilibration period and euglycemic-hyperinsulinemic (approximately 55 mU/L) clamp in conjunction with paired muscle biopsies for measurement of glycogen synthase fractional velocity (FV0.1). Despite similar basal rates of total glucose disposal (Rd), there was a 64% reduction in the insulin-stimulated rise (delta) in Rd in the GHD adults compared to that in controls [16.6 +/- 2.8 vs. 44.7 +/- 6.0 mumol/kg fat free mass (FFM)/min; P < 0.001], which was mainly due to a decreased glucose storage (GS) rate (delta GS, 12.6 +/- 2.9 vs. 39.5 +/- 7.5 mumol/kg FFM/min; P < 0.01). Furthermore, the insulin sensitivity indexes of Rd (0.39 +/- 0.07 vs. 0.85 +/- 0.11; P < 0.05) and GS (0.25 +/- 0.07 vs. 0.72 +/- 0.13 mumol/kg FFM/min per mU/L; P < 0.02) were reduced in GHD adults compared to the control values. The insulin sensitivity of the glycolytic pathway was also reduced by approximately 50% in GHD adults (P = 0.07 vs. controls). Insulin stimulated FV0.1 was decreased in GHD adults (0.31 +/- 0.02 vs. 0.47 +/- 0.03; P < 0.005) despite similar basal FV0.1. Using multiple and stepwise regression analysis, duration of GH deficiency, fasting triglycerides and fasting insulin accounted for 67% of the variance in the insulin sensitivity index of Rd. In conclusion, the severe insulin resistance in GHD adults is mainly due to the inhibition of the GS pathway and glycogen synthase activity in peripheral tissues, which is related to the duration of GH deficiency, fasting triglycerides, and fasting insulin. PMID- 8636268 TI - The corpus luteum insufficiency: a multifactorial disease. AB - The pulsatile release pattern of LH during the entire menstrual cycle is well defined; however, the response of corpora lutea to these LH pulses in patients suffering from corpus luteum insufficiencies (CLI) is largely unknown. Patients suffering from CLI were selected from infertile patients on the basis of low progesterone (P < 25 nmol/L) in a blood sample withdrawn during a monitoring cycle. During the next cycle, nine blood samples were collected during the follicular and luteal phase and follicular development was assessed by vaginal sonography. Of 109 patients who had a CLI in the monitoring cycle, 55 had a CLI again, and 38 women agreed to undergo assessment of pulsatile hormone secretion. These women again had P < 25 nmol/L at days 6 and 7 of the luteal phase and blood samples were withdrawn through antecubital vein catheters from 0900-1700 h at 10 min intervals on days 7, 8, or 9 following ovulation. From 38 patients with such defined CLI, 16 (42%) had no LH episode and significantly lower basal LH levels in comparison with 14 control subjects. Thirteen (34%) of the patients had normal appearing LH episodes despite too low P and E2 concentrations, but their CL did not react to the LH episodes. The remaining 9 patients (24%) had normal LH episodes; their CL reacted to these episodes, but their basal P levels were too low. In all blood samples LH was not only determined using an immunoassay but also by the mouse Leydig cell testosterone production bioassay. It could be established that no CLI exists, which is due to the release of bioinactive LH. It is anticipated that the differentiation of three different types of CLI, one of hypothalamic and two of ovarian origin, may allow the development of differential diagnostic and therapeutic tools in the future. PMID- 8636270 TI - Dissociation between serum interleukin-6 rise and other parameters of disease activity in subacute thyroiditis during treatment with corticosteroid. AB - Increased serum interleukin-6 (IL-6) concentrations have recently been reported in patients with subacute thyroiditis, possibly because of cytokine release from damaged thyroid cells. To investigate the changes in serum IL-6 concentrations in subacute thyroiditis during treatment with corticosteroid, serum IL-6 concentrations were determined by an enzyme-linked immunosorbent assay method in five patients with subacute thyroiditis. Serum IL-6 concentrations were increased moderately, and simultaneously, serum levels of T4, thyroglobulin, and C-reactive protein and erythrocyte sedimentation rate were increased markedly. The treatment with prednisolone rapidly and progressively decreased serum levels of thyroglobulin, T4, and C-reactive protein and the erythrocyte sedimentation rate. In contrast, serum IL-6 concentrations increased markedly 7 days after the treatment with prednisolone in all five patients and two of five patients showed further increases in serum IL-6 concentration on the 17th day. The rise in serum IL-6 levels in untreated patients with subacute thyroiditis in this study is compatible with previous reports. The rise in serum IL-6 levels after treatment with corticosteroid in subacute thyroiditis may reflect the dissociation between the persistent release of IL-6 from the damaged thyroid cells, immediate inhibition of secondary inflammatory reactions by corticosteroid, and the release of thyroglobulin and T4 from performed colloid stores in follicular lumen destroyed by subacute thyroiditis. PMID- 8636269 TI - Mullerian inhibiting substance in humans: normal levels from infancy to adulthood. AB - Mullerian-inhibiting substance (MIS) is a gonadal hormone synthesized by Sertoli cells of the testis and granulosa cells of the ovary. To facilitate the use of MIS for the evaluation of intersex disorders and as a tumor marker in women with MIS-expressing ovarian tumors, we measured MIS in 600 serum samples from males and females. These data show that mean MIS values for males rise rapidly during the first year of life and are highest during late infancy, then gradually decline until puberty. In contrast, MIS values in females are lowest at birth and exhibit a minimal increase throughout the prepubertal years. Whereas MIS is uniformly measurable in all prepubertal boys studied, it is undetectable in most prepubertal female subjects. These data reveal an easily discernible sexually dimorphic pattern of expression and confirm that MIS can be used as a testis specific marker during infancy and early childhood. MIS values that are above the upper limits for females are discriminatory for the presence of testicular tissue or ovarian tumor, and those below the lower limits for males are consistent with dysgenetic or absent testes or the presence of ovarian tissue. These data will enable normal and abnormal levels of MIS to be differentiated with higher precision and will facilitate the use of MIS in the management of gonadal disorders. PMID- 8636271 TI - Gene screening of thyroxine-binding globulin (TBG) deficiencies in the Japanese: only two mutations account for TBG deficiencies in the Japanese. AB - T4-binding globulin (TBG) is the principal transport protein for thyroid hormone in the circulation. Twelve mutations in the human TBG gene have been reported, and the inheritance of those variant TBGs was shown to be X-chromosome linked. We previously reported a nucleotide deletion at the codon 352 (TBG-CDJ) and a nucleotide substitution at the codon 362 (TBG-PDJ) of the TBG gene in a Japanese male manifesting TBG complete deficiency and partial TBG deficiency, respectively. In this communication we investigate the prevalence of both mutations among 50 unrelated Japanese subjects manifesting complete or partial TBG deficiency from various areas of the Japanese Archipelago. Mutant alleles were identified by amplification of their genomic DNAs by PCR with allele specific primers. In addition, the presence of a polymorphic mutation in codon 283 (TBG-Poly) in these variants was investigated. All male subjects manifesting complete TBG deficiency (n = 30) and all female subjects manifesting partial TBG deficiency (n = 4) were demonstrated to be hemizygotes and heterozygotes for the mutation of TBG-CDJ, respectively. All male subjects manifesting partial TBG deficiency (n = 16) were shown to have the mutation of TBG-PDJ as hemizygotes. TBG-Poly was consistently absent from these variants. We conclude that only TBG CDJ and TBG-PDJ may account for complete and partial TBG deficiencies in the Japanese. PMID- 8636272 TI - The frequency of macroprolactinemia in pregnant women and the heterogeneity of its etiologies. AB - We surveyed pregnant women for macroprolactinemia and examined the heterogeneity of its etiologies. Serum samples obtained from 105 pregnant women (29.7 +/- 4.4 yr) during the third trimester were treated with polyethylene glycol, and 3 women (2.9%) were found to have a significantly high proportion of precipitated prolactin (PRL). Gel filtration studies revealed that big-big PRL (molecular weight greater than 100,000) was predominant (63.6, 74.0, 43.5% vs. 0.3 +/- 0.2% in normal pregnant women). The common clinical features of the 3 women included idiopathic hyperprolactinemia without any clinical symptoms such as amenorrhea and galactorrhea before pregnancy and very high levels of PRL (1680, 793, and 790 micrograms/L vs. 315 +/- 112 micrograms/L in normal pregnant women) during pregnancy. However, the nature of big-big PRL was different. Two of the 3 women possessed anti-PRL autoantibody (125I-PRL binding to the serum: 17.6%, 18.6% vs. 6.9 +/- 1.6% in normal pregnant women), but the other one did not have it. A significantly high proportion of PRL was absorbed to a concanavalin A column (41.1% vs. 4.0 +/- 2.1% in normal pregnant women), repetitive freezing and thawing of isolated big-big PRL resulted in a partial conversion to big and little PRL, and reduction of the isolated big-big PRL with 2-mercaptoethanol almost completely converted big-big PRL to little PRL in this woman. These findings suggest that this woman had a heterogeneous complex of covalently and noncovalently bound form of PRL with increased glycosylation. We conclude that macroprolactinemia is present with considerable frequency in pregnant women and that different etiologies are involved to form big-big PRL in macroprolactinemia. PMID- 8636273 TI - Androgen production by monkey luteal cell subpopulations at different stages of the menstrual cycle. AB - Androgens produced by the primate corpus luteum (CL) serve as precursors for estrogen synthesis; moreover, detection of androgen receptors in luteal tissue suggests a regulatory role within the CL. To determine the cellular source(s) and agonist regulation of androgen production during the lifespan of the primate CL, luteal tissues were collected from rhesus monkeys in the early (days 3-5 post-LH surge), mid (days 7-8), mid-late (days 11-12), and late (days 14-15) luteal phase of the menstrual cycle. Collagenase-dispersed cells (i.e., mixed cells) were analyzed by flow cytometry based on light scatter properties and sorted into populations of small (< or = 15 microns) and large (> 20 microns) luteal cells. Cells (n = 4 animals/stage) were incubated in Ham's F-10 and 0.1% BSA for 3 h at 37 C with or without hCG (100 ng/mL), PGE2 (14 mumol/L), or dibutyryl cAMP (dbcAMP; 5 mmol/L), and androstenedione (A4) and testosterone were measured. Basal A4 production by large cells was markedly higher (P < 0.05) than that by small cells (e.g. mid-late luteal phase, 821 +/- 188 vs. 69 +/- 25 pg/mL.5 x 10(4) cells/3 h; mean +/- SEM), whereas that by mixed cells was intermediate (317 +/- 205 pg/mL). In the early luteal phase, hCG stimulated A4 synthesis by mixed (1.6-fold; P < 0.05) and large (3.1-fold; P < 0.05) luteal cells, but not by small cells (1.3-fold). By the mid-late luteal phase, hCG did not increase A4 production by any cell type, although hCG responsiveness returned to large cells (2.0-fold increase; P < 0.05) by the late luteal phase. PGE2 responsiveness by cell types was similar to that of hCG, except large cell responsiveness did not return in the late luteal phase. In all cell types, dbcAMP stimulated the largest increase in A4 levels; in the mid-late luteal phase, small and large cells responded to dbcAMP with 8.2- and 3.0-fold increases (P < 0.05) in A4 production, respectively. When luteal cells were incubated with the steroidogenic substrates, 17 alpha-hydroxyprogesterone or 17 alpha-hydroxypregnenolone (1 mumol/L), large cells produced much more (P < 0.05) A4, testosterone, estrone, and estradiol than small cells. Both substrates elicited similar patterns of androgen production, with A4 synthesis predominant in all luteal cell types. Thus, cell subpopulations in the primate CL can be distinguished by their ability to produce androgen and estrogen. Changes in agonist-responsive androgen production may influence the local steroid milieu and function of the CL during the menstrual cycle. PMID- 8636274 TI - Genetic and environmental correlations among hormone levels and measures of body fat accumulation and topography. AB - In this study we partition the phenotypic correlations between body fat measures and serum levels of hormones with known, or suspected, lipolytic effects into their genetic and environmental components. Using variance decomposition techniques, we are able to estimate the pleiotropic effects of genes and/or shared environmental factors that give rise to the phenotypic correlations previously reported between these traits. We used data from a large sample of randomly ascertained Mexican-American families living in San Antonio, TX. Data were available for 582 individuals in 26 pedigrees. Levels of sex hormone-binding globulin, dehydroepiandrosterone sulfate, insulin, insulin-like growth factor I, total T4, and total T3 were assayed. The measures of body fat accumulation and topography included body mass index, subscapular/triceps ratio, and relative fat patterning index. The results of this analysis demonstrate that significant phenotypic correlations among these traits can arise from three underlying conditions: 1) entirely from shared genetic effects (pleiotropy), 2) entirely from shared random environmental effects, or 3) a combination of both effects. However, we also show that it is possible for significant genetic and environmental correlations to interact in such a way as to produce a phenotypic correlation that itself would not be considered significant. PMID- 8636275 TI - Pituitary-ovarian responses to leuprolide acetate testing in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. AB - To assess whether patients with congenital adrenal hyperplasia (CAH) due to 21 hydroxylase deficiency exhibit a steroidogenic response to GnRH agonist consistent with functional ovarian hyperandrogenism (FOH) and elucidate the relationship between adrenal and ovarian hyperandrogenism, the LH, FSH, estradiol, 17-hydroxyprogesterone (17-OHP), androstenedione, total testosterone, dehydroepiandrosterone, and 17-hydroxypregnenolone responses to a sc dose of leuprolide acetate (500 micrograms) were evaluated in 10 patients with classic CAH (mean age, 18.4 +/- 0.95 yr), 7 of whom had oligomenorrhea, pretreated with dexamethasone (2 mg/day for 5 days, including the day of the test). The results were compared with those obtained in 11 patients with FOH (mean age, 18.7 +/- 0.46 yr) and 17 normal women (mean age, 19.68 +/- 0.59 yr) not pretreated with dexamethasone. Leuprolide acetate stimulation caused a significant augmentation of plasma E2, 17-OHP, androstenedione, testosterone, and 17-hydroxypregnenolone concentrations in all CAH patients. However, in only 6 (60%) of them, all with oligomenorrhea, was the 17-OHP response (posttest minus pretest value) similar to that of FOH patients and significantly higher than that in controls. In this subset of CAH patients, LH plasma levels after stimulation were significantly higher than those of CAH subjects with 17-OHP responses in the normal range, controls, and FOH patients, whereas FSH levels were similar to those of controls. In this latter group, plasma FSH concentrations after stimulation were significantly higher than those in FOH. In conclusion, the results of the present study indicate that LH-dependent functional ovarian hyperandrogenism is frequent in patients with classic CAH. As ovarian hyperandrogenism might be partially responsible for the menstrual irregularities that are common complications in such patients, all classic CAH patients with oligomenorrhea should undergo short term stimulation with GnRH agonists to ascertain the presence of ovarian hyperandrogenism and receive appropriate treatment. PMID- 8636276 TI - Differences in bone and vitamin D metabolism between primary hyperparathyroidism and malignancy-associated hypercalcemia. AB - Bone and vitamin D metabolism are examined in patients with primary hyperparathyroidism (1 degree HPT), humoral hypercalcemia of malignancy (HHM), and local osteolytic hypercalcemia (LOH) with normal renal function. Among the bone resorption markers, T scores of total deoxypyridinoline (Dpyd) were highest in HHM and were significantly higher than those in 1 degree HPT. Among the formation markers, T scores of osteocalcin (OC) were highest in 1 degree HPT but were negative in HHM. The elevation in total Dpyd was associated with an increase in OC in 1 degree HPT, and the ratios of total Dpyd/OC were similar to those in controls. In contrast, many patients with HHM and LOH exhibited elevated total Dpyd and suppressed OC with increased total Dpyd/OC ratios, but the ratios varied widely. Serum 1,25-dihydroxyvitamin D [1,25(OH)2D] was elevated in 1 degrees HPT but was suppressed in HHM and LOH at any serum Ca levels. These results demonstrate that increased bone resorption is associated with enhanced bone formation in 1 degrees HPT but are uncoupled in many of the HHM and LOH patients, and that total Dpyd/OC ratio can be a useful index to estimate the coupling state of bone. It is suggested that the reduction in serum 1,25(OH)2D cannot be explained by an elevation in serum Ca in HHM and LOH, and that the differences in bone and vitamin D metabolism in HHM and LOH from those in 1 degree HPT may be caused by a common mechanism such as the secretion of some cytokines from tumors. PMID- 8636277 TI - Secretion of insulin-like growth factors and their binding proteins by human normal and hyperplastic prostatic cells in primary culture. AB - Benign prostatic hyperplasia (BPH) is the most common benign proliferative disorder of unknown etiology found in men. Because insulin-like growth factors (IGFs) with their binding proteins (IGFBPs) are involved in the control of cellular proliferation, differentiation, and metabolism, we compared their secretion by prostatic epithelial and stromal cells in primary culture from the four different zones of normal prostate and from hyperplastic tissue to assess their contributions to the hyperplastic development. IGF-I could not be detected in the conditioned medium from either epithelial or stromal cells from normal and BPH tissues. IGF-II concentrations were the same in the conditioned medium from the epithelial cells of the different zones of the normal prostate and that of BPH cells. IGF-II concentrations secreted in stromal cell culture medium, however, were higher in the periurethral zone than in the peripheral and central zones. Moreover, in the periurethral zone, stromal cells secreted higher concentrations of IGF-II than did epithelial cells. Also, BPH stromal cells secreted more IGF-II than did BPH epithelial cells. IGFBP-3, IGFBP-2, and IGFBP-4 were all secreted by both epithelial and stromal cells. In contrast, IGFBP-5 was only produced by stromal cells of the periurethral zone of the normal prostate and BPH tissue. IGFBP-3 was predominantly secreted by normal stromal cells of the transitional zone. We observed that BPH stromal cells presented the same pattern of IGF-II and IGFBP production as normal stromal cells of the periurethral zone. These data support the hypothesis that the periurethral zone is the main region of the prostate implicated in the development of BPH. They also suggest that the variability in both IGF-II secretion and the secreted forms of IGFBPs, depending on anatomical location within the organ, may be important for the autocrine regulation of normal and hyperplastic prostate growth. PMID- 8636278 TI - Proteolysis of insulin-like growth factor-binding protein-3 in the male reproductive tract. AB - Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is produced by prostate epithelial and stromal cells and either enhances or inhibits the effects of IGF on prostate epithelial cells. The levels of this protein in the male reproductive tract may be determined in part by proteases, including prostate specific antigen (PSA), produced by the prostate epithelium. In this study we examined the proteolytic activity of human seminal fluid on IGFBP-3. Seminal fluid and prostate massage fluid (PF) were examined for IGFBP-3 or its fragments by use of an IGFBP-3 RIA that detects intact IGFBP-3 as well as fragments, a two site immunoradiometric assay (IRMA) that detects intact IGFBP-3 and the larger fragments, Western ligand blots (WLB), and immunoblots (WIB). In seminal fluid, IGFBP-3 was readily detectable by RIA, but was detected in only 50% of the samples assayed by IRMA. No detectable IGFBP-3 was observed by WLB with [125I]IGF I as the ligand, but with IGF-II as the ligand, IGFBP-3 fragments at 16-17 kDa were noted. On WIB, the 16-kDa fragment of IGFBP-3 was most abundant, with a smaller amount of the 29-kDa fragment, but no intact IGFBP-3. These results indicated that most of the IGFBP-3 detected in seminal fluid was in small (< or = 16-kDa) fragments. When three or more seminal fluid samples collected 1 month apart were available from the same individual, the coefficient of variation was 10.0 +/- 1.26% (+/- SE) for IGFBP-3 by RIA vs. 73.3 +/- 11.2% for sperm counts in the same samples. In a group of 42 PF samples, the IGFBP-3 levels measured by either RIA or IRMA were approximately 3-fold higher than those in seminal fluid. Intact IGFBP-3 was detected by both WLB and WIB. There was a significant inverse correlation between PSA and IGFBP-3, measured by IRMA, in PF (r = -0.526; P < or = 0.004). Finally, in the PF of African-American men, PSA was significantly lower, and IGFBP-3 determined by IRMA was significantly higher compared to those in Caucasian men. PMID- 8636280 TI - Immunoradiometric assay for follistatin: serum immunoreactive follistatin levels in normal adults and pregnant women. AB - A sensitive and specific immunoradiometric assay for follistatin was developed using antifollistatin mouse monoclonal and rabbit polyclonal antibodies. The sensitivity of the assay was 0.5 micrograms/L, and cross-reactivities with recombinant human activin A and bovine inhibin were less than 0.1%. The intra- and interassay coefficients of variation were less than 10%, and the recovery rate was about 90% in human serum. The addition of activin A to the same sample resulted in a minimal influence on follistatin recovery, indicating that this assay system can measure the total level of activin-bound and unbound follistatin. Gel filtration analysis of human serum showed that the majority of immunoreactivity was eluted in a larger molecular size position than that of free follistatin, suggesting that the large part of follistatin is bound to other proteins, presumably activins, in serum. Using this assay, immunoreactive follistatin levels in various biological fluids and human sera were examined. The dose-response curves of porcine follicular and amniotic fluids were parallel to the standard curve, and porcine follicular fluid contained extremely high follistatin immunoreactivity (5.6 mg/L). The serum follistatin level in normal human volunteers was 13.3 +/- 4.7 micrograms/L (mean +/- SD; n = 60), with a tendency to increase gradually with age. On the other hand, the serum follistatin level was remarkably elevated in pregnant women (62.7 +/- 35.3 micrograms/L; n = 57), with a positive correlation with weeks of pregnancy. These data indicated that circulating immunoreactive follistatin is detectable in human serum, and the levels vary with physiological conditions such as aging and pregnancy. PMID- 8636279 TI - Changes in thyroid hormone metabolism in exertional heat stroke with or without acute renal failure. AB - The effects of exertional heat stroke (ExHS), with or without acute renal failure (ARF), on thyroid hormone metabolism were investigated. Eighteen ExHS patients were recruited and divided into two groups based on the presence or absence of ARF. Eleven age-matched healthy subjects served as a control group. Serum values of T3, T4, TSH, free T4 (FT4), rT3, and sulfated T3 (T3S) were measured in these groups during the acute and recovery stages of ExHS. Serum T3, T4, and FT4 levels were reduced, with reciprocal increases in rT3 and T3S levels as the severity of ExHS increased. The following mean levels of thyroid hormones were found (controls vs. ExHS without ARF vs. with ARF): T3, 1514 vs. 1164 vs. 393 pmol/L (P < 0.05 each); T4, 97 vs. 79 vs. 49 nmol/L (P = NS and P < 0.05, respectively); FT4, 20.5 vs. 19.5 vs. 19.0 pmol/L (P = NS each); rT3, 371 vs. 617 vs. 805 pmol/L (P < 0.05 and P = NS, respectively); and T3S, 30.1 vs. 34.2 vs. 71.1 pmol/L (P = NS and P < 0.05, respectively). The serum TSH levels were not significantly different among the three groups. Significantly negative correlations were found between serum creatinine and T3 (r = -0.75; P < 0.001) and T4 levels (r = -0.65; P < 0.001), whereas no relationship was noted between serum creatinine and rT3 values (r = 0.11; P < 0.05). In contrast, a correlation was observed between serum glutamic pyruvic transaminase and rT3 (r = 0.45; P < 0.01). Thyroid function tests returned to normal after patients recovered. In conclusion, our results show that patients suffering from ExHS, with or without ARF, displayed altered serum thyroid function in proportion to the severity of their condition. No significant changes in serum levels of rT3 were observed between the two groups, whereas a positive relationship was observed between serum rT3 and serum glutamic pyruvic transaminase values, suggesting that the changes in serum rT3 levels were more dependent on extrarenal illness than on renal disease per se. The moderate increase in serum T3S levels found in patients suffering from both ExHS and ARF may represent a decrease in tissue 5'-monodeiodinase activity as found in other nonthyroidal illnesses. A return of serum thyroid function tests to normal values after recovery from ExHS suggests that the low T3 state may play a protective role to prevent undesirable catabolic effects. Replacement therapy is thus not recommended. PMID- 8636281 TI - Improved final height in girls with Turner's syndrome treated with growth hormone and oxandrolone. AB - The spontaneous growth process in Turner's syndrome is characterized by a progressive decline in height velocity during childhood and no pubertal growth spurt. Therefore, therapy aimed at improving height during childhood as well as increasing final height is desirable for most girls with Turner's syndrome. Forty five girls with Turner's syndrome, 9-16 yr of age (mean age, 12.2 yr), were allocated to three study groups. Group 1 (n = 13) was initially treated with oxandrolone alone; after 1 yr of treatment, GH without (group 1a; n = 6) or with (group 1b; n = 7) ethinyl estradiol was added. Group 2 (n = 17) was treated with GH plus oxandrolone. Group 3 (n = 15) was treated with GH, oxandrolone, and ethinyl estradiol. The dosage were: GH, 0.1 IU/kg.day; oxandrolone, 0.05 mg/kg.day; and ethinyl estradiol, 100 ng/kg.day. A height of 150 cm or more was achieved in 61%, 75%, and 60% of the girls in groups 1, 2, and 3, respectively. The most impressive increase in height was seen in group 2. In this group the mean final height was 154.2 cm (SD = 6.6), which is equivalent to a mean net gain of 8.5 cm (SD = 4.6) over the projected final height. In group 3, in which ethinyl estradiol was included from the start of therapy, the initially good height velocity decelerated after 1-2 yr of treatment. Their mean final height was 151.1 (SD = 4.6) cm, equivalent to a mean net gain of 3.0 cm (SD = 3.8). A similar growth-decelerating effect of ethinyl estradiol was seen in group 1b. We conclude that in girls with Turner's syndrome who are older than 9 yr of age, treatment with GH in combination with oxandrolone results in significant growth acceleration, imitating that in normal puberty, leading to a more favorable height during childhood. This mode of treatment also results in a significantly increased final height, permitting a great number of the girls to attain a final height of more than 150 cm. However, early addition of estrogen decelerates the height velocity and reduces the gain in height. PMID- 8636282 TI - Relationship of antibodies to thyrotropin receptors and to thyroid ultrasonographic volume in euthyroid and hypothyroid patients with autoimmune thyroiditis. AB - It has been reported in hypothyroid patients with Hashimoto's thyroiditis and in patients with primary myxoedema that antibodies (Ab) to the TSH-receptor (R), which inhibit the thyroid gland, decrease both thyroid hormonogenesis and cell growth in vitro. We investigated, in 169 newly diagnosed patients with euthyroid (n = 83) or hypothyroid (n = 86) autoimmune thyroiditis, the relationship between thyroid autoimmunity expression and thyroid ultrasonographic volume or thyroid hormonal status. In patients positive for TSH-receptor (R) antibodies (Ab), negative correlations (P < 0.01) were found between TSH-R Ab levels and free T4 (FT4) values in a euthyroid (r = -0.63), as well as in a hypothyroid (r = -0.54) state, and between TSH-R Ab and total thyroid volume (TTV) values in an euthyroid (r = -0.62) as well as in a hypothyroid (r = -0.53) state. In contrast, no positive correlations were found between TSH levels and FT4 values or TTV levels in patients positive as well as negative for TSH-R Ab in an euthyroid or in a hypothyroid state. These data demonstrate in vivo, that, in contrast to TSH, TSH R Abs are related to both thyroid hormonal production and volume in euthyroid or hypothyroid patients with autoimmune thyroiditis. PMID- 8636283 TI - High bone density in hyperandrogenic women: effect of gonadotropin-releasing hormone agonist alone or in conjunction with estrogen-progestin replacement. AB - We studied 20 hirsute patients with high levels of serum testosterone (T), calculated free T, androstenedione, and dehydroepiandrosterone sulfate and 19 age matched nonhirsute normoandrogenic control women. The bone mineral density (BMD) in the lumbar spine, femoral neck, and trochanter major region in hirsute patients was higher than that in the controls. BMD in the lumbar spine and proximal femur correlated positively with the body mass index and with serum T and free T in hyperandrogenic women and the whole study group, but not with serum androstenedione or dehydroepiandrosterone sulfate levels. The hirsute women were treated with a GnRH agonist (goserelin, 3.6-mg implant) for 9 months. After the first 3 months of treatment, half of the patients were randomized to receive estrogen-progestin replacement therapy (HRT), and the other half served as controls. After the first 3 months of trial, BMD was unaffected, and the urinary output of collagen pyridinoline, deoxypyridinoline cross-links, and hydroxyproline (all markers of bone resorption) were increased, but serum markers, the carboxy-terminal telopeptide of type I collagen (marker of bone resorption) and that of bone-specific alkaline phosphatase (marker of bone formation) did not change. After 9 months of goserelin treatment, the lumbar spine had lost 5.4% of its BMD (P < 0.01), but regained bone density 6 months after cessation of treatment. Addition of HRT protected the spine and trochanter major against bone loss. The changes in serum telopeptide and urinary output of pyridinoline and deoxypyridinoline after 3 months of treatment (from prestudy levels) correlated with the decrease in BMD in the femoral neck at 9 months. In conclusion, our data show that patients with ovarian androgen excess 1) have high BMD, 2) lose bone during 9 months of treatment with GnRH agonist, 3) show a decrease in bone density preceded by biochemical alterations in bone metabolism at least 6 months earlier, and 4) can have their bone loss prevented by add-back HRT. PMID- 8636284 TI - Cortisol secretory patterns in Cushing's disease and response to cyproheptadine treatment. AB - To investigate whether cortisol secretory patterns are associated with a response to cyproheptadine treatment in Cushing's disease, we studied two patients with a hyperpulsatile pattern and one patient with a hypopulsatile pattern before and during chronic cyproheptadine therapy (24 mg daily). In the two patients with a hyperpulsatile cortisol secretory pattern, pituitary magnetic resonance imaging with gadolinium did not reveal a pituitary adenoma, whereas in the patient with a hypopulsatile cortisol secretory pattern, a microadenoma was identified. Plasma cortisol levels were measured every 30 min for 24 h. In the two patients with a hyperpulsatile cortisol secretory pattern, chronic treatment with cyproheptadine resulted in sustained clinical and biochemical improvement and normalization of the median of absolute and relative increments in cortisol spikes. In the patient with a hypopulsatile cortisol secretory pattern, only a reduction of cortisol spikes was noticed during treatment. These results suggest that patients with Cushing's disease who are characterized by a hyperpulsatile cortisol secretory pattern and in whom no pituitary lesion can be identified by magnetic resonance imaging, cyproheptadine treatment may be useful. PMID- 8636285 TI - Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF R correlates with tumor aggressiveness. AB - Epidermal growth factor (EGF) has been localized in several human neoplasms and has been shown to have a significant correlation to prognosis. We investigated the potential role of the EGF receptor (EGF-R) system in pituitary tumorigenesis by examining the expression of EGF and EGF-R in the different types of human pituitary adenomas. EGF was identified by immunohistochemistry in all cell types of the nontumorous adenohypophysis and in all types of morphologically characterized functional (n = 9) and nonfunctional (n = 17) adenomas. To confirm local EGF synthesis, ribonucleic acid (RNA) from human pituitary adenomas was reverse transcribed and PCR amplified. Transcript signals of the expected size were identified, with marked variation in 41 of 48 adenomas. To assess possible secretion in vitro, EGF was measured in pituitary tumor culture medium. No measurable quantities of EGF were present in conditioned culture medium from all 35 adenomas examined, consistent with the rapid uptake of EGF by unoccupied functional EGF-R sites. Using a specific monoclonal antibody that recognizes the extracellular ligand-binding domain of the human EGF-R, we found EGF-R in cells in the normal pituitary and in some functional and nonfunctional adenomas with extremely variable intensity. By RT-PCR, EGF-R messenger RNA (mRNA) expression was also identified, with marked variation in all 48 adenomas examined and in the nontumorous pituitary. The highest degrees of EGF-R mRNA expression were present in somatotroph adenomas and the aggressive silent subtype 3 adenomas. Tumors from patients with recurrent acromegaly demonstrated significantly higher levels of EGF-R mRNA than those from patients with nonrecurrent disease. In conclusion, EGF and EGF-R are expressed in a variable manner in all types of human pituitary adenomas. The overexpression of EGF-R in recurrent somatotroph adenomas and aggressive silent subtype 3 adenomas suggests a selective mechanism for the EGF/EGF-R family in the growth of aggressive pituitary tumors. PMID- 8636286 TI - Bone mass and subtle abnormalities in ovulatory function in healthy women. AB - Women with occasional anovulatory or short luteal phase menstrual cycles have been reported to lose bone mineral density (BMD) at a greatly accelerated rate compared to women without such abnormalities. To investigate this association, we performed a longitudinal study of BMD in a group of healthy premenopausal women enrolled in a comprehensive study of ovulatory function. Subjects had collected daily urine samples that were analyzed for estrone and progesterone metabolites by enzyme-linked immunoassay. The 53 participants collected urine for an average of 4.1 cycles. Computer algorithms identified 7 (13.2%) women with luteal phase abnormalities (> 1 anovulatory cycle or cycle with luteal phase length < or = 10 days) and 17 (32.1%) women with other menstrual abnormalities. Areal BMD (grams per cm2) was measured at the lumbar spine, hip, and whole body using dual energy x-ray absorptiometry; BMD was measured 2-3 times over an average observation period of 17.5 months. At baseline, women with luteal abnormalities had mean BMD similar to those of the 29 women with no abnormal cycles: lumbar spine, 1.06 vs. 1.09 g/cm2; total hip, 0.95 vs. 0.94 g/cm2; whole body, 1.15 vs. 1.11 g/cm2 (P > 0.10; adjusted for age and weight at baseline, parity, physical activity level, and calcium intake). When compared at follow-up to women with no abnormal cycles, women with luteal abnormalities tended to gain BMD at the spine and hip (P > 0.10). On whole body measurement, women with luteal abnormalities tended to lose BMD compared to women with no abnormal cycles (-1.1%/yr vs. 0%/yr; P = 0.08); however, the magnitude of loss was not unusual for women in this age range and was within the coefficient of variation for replicate measurements. Neither mean luteal phase length, percent time in luteal phase, nor average daily excretion of progesterone metabolites was associated with baseline BMD or percent annual change in BMD at any measurement site. Thus, we did not confirm a relationship between luteal abnormalities and accelerated bone loss in this population of healthy premenopausal women. PMID- 8636288 TI - Effectiveness of octreotide in controlling fasting hypergastrinemia and related enterochromaffin-like cell growth. AB - The effects of long term (6-month), high (500-micrograms), once a day administration of octreotide on enterochromaffin-like (ECL) cell proliferation were evaluated in eight patients with hypergastrinemic atrophic gastritis at risk for the development of gastric carcinoids. Fasting gastrin levels were determined during treatment and up to 6 months after the end of treatment. Chromogranin A, hCG alpha, and somatostatin-immunostained cells were morphometrically evaluated in biopsy specimens of corpus mucosa taken before and after treatment. The results showed that gastrin levels significantly decreased from 950 to 238 ng/L ( 74.9%; P < 0.01) at the end of treatment, a decrease that persisted 6 months after the end of treatment (450 ng/L; P < 0.05). The volume density of CgA cells (mostly ECL cells) decreased from 3.7% to 2.1% of the epithelial component (-43%; P < 0.014), that of hCG alpha-storing ECL cells decreased by 85% (P < 0.0007), and that of somatostatin-stained cells decreased by 74% (P < 0.04). No clinically significant side-effects were found. It is concluded that octreotide treatment as used in the present study is safe and effective in reducing hypergastrinemia and associated ECL cell changes in patients with atrophic gastritis. The decrease in D cells is consistent with the occurrence of somatostatin receptors and related autocrine regulation in these cells. PMID- 8636287 TI - Biological and immunological characterization of inhibin forms in human plasma. AB - A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B. PMID- 8636289 TI - Evidence for a hypothalamic-pituitary versus adrenal cortical effect of glycemic control on counterregulatory hormone responses to hypoglycemia in insulin dependent diabetes mellitus. AB - The epinephrine and cortisol responses to hypoglycemia are reduced in insulin dependent diabetes mellitus (IDDM) patients in strict glycemic control. However, it is not known whether these abnormalities are mediated at a central (hypothalamic-pituitary) or peripheral (adrenal) level. To examine this question, we measured counterregulatory hormone secretion during a 3-h hypoglycemic hyperinsulinemic clamp (12 pmol/kg.min) that lowered glucose from 5.0 to 2.2 mmol/L in steps of 0.55 mmol/L every 30 min in 13 well controlled IDDM subjects (hemoglobin A1, 7.8 +/- 0.2%), 14 poorly controlled IDDM subjects (hemoglobin A1, 12.3 +/- 1.5%), and 20 healthy volunteers. Basal levels of ACTH, cortisol, and epinephrine were similar in all 3 groups before hypoglycemia. At the nadir glucose level (2.2 mmol/L), ACTH, cortisol, and epinephrine levels were significantly lower in well controlled IDDM compared to healthy controls, and the glucose levels required for significant secretion of ACTH, cortisol, and epinephrine also were lower in well controlled IDDM compared to those in both poorly controlled IDDM and healthy volunteers (P < 0.05). During hypoglycemia, ACTH levels were significantly correlated with cortisol levels (r = 0.43; P < 0.05). Because adrenomedullary epinephrine synthesis is partially dependent on adequate adrenocortical function, we also determined whether the blunted epinephrine response might result from the reduced cortisol secretion. Eleven of the control subjects underwent a second identical insulin clamp study during which metyrapone was administered to produce adrenal cortical blockade. Despite higher basal ACTH levels after metyrapone and sustained elevations in ACTH during hypoglycemia, the cortisol response was abolished during metyrapone treatment, indicating effective blockade. However, epinephrine responses did not differ during hypoglycemia with or without metyrapone treatment. We conclude that 1) ACTH, cortisol, and epinephrine responses during hypoglycemia are reduced in IDDM patients in strict glycemic control; 2) the lower cortisol response is correlated with reduced ACTH levels; and 3) in healthy subjects, the cortisol response to hypoglycemia is abolished by adrenocortical blockade with metyrapone, whereas the epinephrine response to hypoglycemia remains intact. These data suggest that central adaptations in hypothalamic-pituitary responses to hypoglycemia rather than alterations in adrenal gland function per se underlie the reduced counterregulatory responses seen in IDDM subjects in strict glycemic control. PMID- 8636290 TI - Fasting as a metabolic stress paradigm selectively amplifies cortisol secretory burst mass and delays the time of maximal nyctohemeral cortisol concentrations in healthy men. AB - Serum cortisol concentrations are increased in fasted or malnourished human subjects. The dynamic mechanisms underlying this adaptive response have been investigated in eight normal men by analyzing serum cortisol concentrations measured in blood obtained at 5-min intervals over 24 h on a control (fed) day and on the fifth day of a fast (water only) assigned in randomized order. A multiple parameter deconvolution method was used to simultaneously resolve endogenous cortisol secretion and half-life. Five days of fasting induced a 1.8 fold increase in the 24-h endogenous cortisol production rate (fed, 2504 +/- 308; fasted, 4528 +/- 488 nmol/L distribution volume; P < 0.006). This enhanced cortisol production rate was accounted for by a 1.6-fold increase in the mass of cortisol secreted per burst (fed, 115 +/- 12.1; fasted, 183 +/- 17.3 nmol/L; P < 0.02). Cortisol secretory event amplitudes (maximal rates of cortisol release attained within a burst) increased in seven of eight men, and mean secretory burst durations remained unchanged by fasting. Moreover, the number of computer resolved cortisol secretory bursts per 24 h (fed, 22 +/- 1.4; fasted, 25 +/- 2.0; P = NS) and the interburst interval (fed, 65 +/- 4.0; fasted, 57 +/- 4.4 min) did not change significantly during a 5-day fast. The calculated half-life of endogenous cortisol was not significantly altered by fasting (fed, 108 +/- 9.7; fasted, 129 +/- 11 min). There was no significant change in the nyctohemeral pattern of varying adrenocortical secretory burst frequency in response to fasting. However, the mean (mesor) mass of glucocorticoid secreted per burst over 24 h rose significantly in response to fasting. In addition, by cosinor analysis, maximal serum cortisol concentrations occurred (95% confidence intervals) between 0930-1334 h in the fed state and between 1116-1612 h in the fasted state (P < 0.04). Fasting augmented the mesor (average value about which the diurnal rhythm oscillates; P < 0.0008 compared with fed state) and the amplitude (P < 0.04) of the 24-h serum cortisol concentration profile. Linear regression analysis disclosed a significant inverse relationship between mean serum cortisol and GH concentrations in fasted men (r = -0.76; P < 0.02). In conclusion, the present data indicate that starvation-induced enhancement of cortisol secretion in young healthy men is mediated by an increased glucocorticoid secretory burst mass, rather than changes in secretory burst frequency or duration or in cortisol half life. In addition, fasting modifies the diurnal secretory pattern of cortisol by delaying maximal serum concentrations to the early afternoon. The inverse relationship between serum cortisol and GH responses to fasting suggests differential regulation of the corticotropic and somatotropic axis by the metabolic stress of fasting and/or feedback interactions between these two axes when they are both activated. PMID- 8636292 TI - Insulin resistance in type I diabetes mellitus: a major role for reduced glucose extraction. AB - We determined whether insulin resistance in Type I diabetes is caused by a defect in glucose extraction or blood flow and whether it is the rate of glucose metabolism rather than insulin that increases blood flow in these patients. To make this determination, 9 Type I diabetic patients (age 33 +/- 3 yr, body mass index 24 +/- 1 kg/m2, HbA1c 8.3 +/- 0.1%) and 10 matched normal subjects were first studied under normoglycemic hyperinsulinemic conditions. The diabetic patients were then restudied under similar conditions, but now whole body glucose uptake was normalized by glucose mass-action (glucose 8.7 +/- 0.6 mmol/L). During normoglycemia, rates of whole body (46 +/- 2 vs. 66 +/- 3 mumol/kg.min, P < 0.001) and forearm (47 +/- 9 vs. 78 +/- 7 mumol/kg forearm.min, P < 0.05) glucose uptake were decreased in the diabetic patients, because of a 32% decrease in the glucose AV-difference (1.5 +/- 0.2 vs. 2.2 +/- 0.2 mmol/L, P < 0.05). Forearm blood flow was similar in the diabetic patients (3.6 +/- 0.7 mL/dl.min) and normal subjects (3.7 +/- 0.3 mL/dL.min). During matched rates of whole body glucose uptake (68 +/- 1 vs. 66 +/- 3 mumol/kg.min, normoglycemic study in controls vs. hyperglycemic study in the diabetic patients), the glucose AV difference across the forearm was 64% higher than during normoglycemia (2.4 +/- 0.3 vs. 1.5 +/- 0.2 mmol/L, P < 0.05). Forearm blood flow (3.6 +/- 0.4 mL/dL.min) under conditions of matched glucose flux was similar to that during the normoglycemic study. We conclude that a defect in glucose extraction rather than blood flow characterizes insulin resistance in uncomplicated Type I diabetes. The signal for the flow increase is insulin and not the rate of glucose metabolism. PMID- 8636291 TI - Binding of antithyrotropin receptor autoantibodies in Graves' disease serum to nascent, in vitro translated thyrotropin receptor: ability to map epitopes recognized by antibodies. AB - The binding of Graves' disease autoantibodies and xenogeneic antibodies to the human TSH receptor (TSH-R) has been studied using receptor preparations generated in an in vitro transcription and translation reaction. The complementary DNAs encoding for the full-length (764 amino acids) and the extracellular region of TSH-R (amino acids 20-414, lacking the signal sequence) were used to generate the translated receptor antigen. Stable [35S]methionine-labeled nascent protein for full-length and extracellular regions of TSH-R of approximate size 87 and 50 kDa, respectively, together with other smaller proteins were generated. The 87- and 50 kDa translated receptor proteins react by immunoprecipitation analysis with monoclonal antibodies, polyclonal antisera, and unfractionated Graves' disease serum containing autoantibody to TSH-R. The translated products of the extracellular TSH-R were examined in detail. Using three well characterized murine monoclonal antibodies whose epitopes encompass the amino-, central, and carboxyl-terminals of the extracellular region of the receptor led to immunochemical identification of the smaller translated products to derive from internal methionine start sites of TSH-R. These smaller, N-terminal-truncated translated proteins were also recognized by polyclonal antisera generated against recombinant TSH-R, thus allowing epitope mapping of some antibodies. A large proportion of Graves' disease autoantibodies (> 70%) bind to the translated extracellular region of TSH-R. This indicates that the majority of pathogenic anti-TSH-R autoantibody binds the nascent translated extracellular region of TSH R, which is not influenced by the lack of glycosylation of the receptor. PMID- 8636293 TI - The involvement of the opioid system in human obesity: a study in normal weight relatives of obese people. AB - The involvement of the opioid system in human obesity has been demonstrated, but whether the abnormalities in the endorphinergic system play a primary role in overfeeding and weight gain or represent a simple biochemical feature is still unclear. The objectives of this study were to investigate the effects of both physiological and pharmacological plasma beta-endorphin levels on some metabolic and hormonal parameters in a normal weight, but prone to obesity, young population consisting of first degree relatives of obese subjects and in body mass index-, sex, and age- matched control subjects without a family history of obesity. Each subject underwent a 1-h infusion of synthetic human beta-endorphin at a constant rate of 4.5 ng/kg.min (low rate), then after a 1-week interval, at a rate of 500 micrograms/h (high rate). Under basal conditions, there was no significant difference in plasma glucose and pancreatic hormones (insulin, C peptide, and glucagon) between the two groups, except for plasma beta-endorphin levels, which were significantly (P < 0.01) higher in relatives of obese individuals. The low rate of beta-endorphin infusion induced physiological elevations of plasma opioid levels in both groups; no significant change in plasma glucose and pancreatic products in control subjects; and a significant (at least P < 0.05) rise in plasma insulin, C peptide, and glucagon concentrations in relatives of the obese. The high rate of beta-endorphin infusion produced pharmacological elevations of opioid plasma levels in both groups; significant (at least P < 0.05) increments in plasma glucose and glucagon levels and no appreciable modification of plasma insulin and C peptide levels in the control group; and a significant (at least P < 0.05) positive response of plasma glucose, insulin, C peptide, and glucagon levels in relatives of obese subjects. These findings suggest that 1) opioid peptides at least in part play a primary, rather than secondary, role in some metabolic events of obesity; and 2) both physiological and pharmacological plasma levels of beta-endorphin are able to provoke marked islet hormone release in the early phase of human obesity. PMID- 8636295 TI - Sleep and the sleep electroencephalogram across the menstrual cycle in young healthy women. AB - Cyclic changes in hormones, body temperature, and metabolic rate characterize the menstrual cycle. To investigate whether these changes are associated with changes in sleep and the sleep electroencephalogram (EEG), a total of 138 sleep episodes from 9 women with no premenstrual syndrome symptoms were recorded every second night throughout one ovulatory menstrual cycle and analyzed in relation to menstrual phase. Ovulation and menstrual cycle stage were confirmed by measurements of temperature, urinary LH, and midluteal plasma levels of estrogen and progesterone. No significant variation across the menstrual cycle was observed for subjective ratings of sleep quality and mood as well as for objective measures of total sleep time, sleep efficiency, sleep latency, rapid eye movement sleep latency, and slow wave sleep. In nonrapid eye movement sleep, EEG power density in the 14.25-15.0 hertz band, which corresponds to the upper frequency range of the sleep spindles, exhibited a large variation across the menstrual cycle, with a maximum in the luteal phase. The data show that in healthy young women, sleep spindle frequency activity varies in parallel with core body temperature, whereas homeostatic sleep regulatory mechanisms, as indexed by the time course of EEG slow wave activity are not substantially affected by the menstrual cycle. PMID- 8636294 TI - Deletion of Asn281 in the alpha-subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization. AB - We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (< 10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn281 in the alpha-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients' cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn281 of the IR alpha-subunit plays a critical role in the inhibitory constraint exerted by the extracellular alpha-subunit over the intracellular kinase activity. PMID- 8636296 TI - Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve. AB - Day 3 serum FSH is an indirect assessment of ovarian reserve and has been shown to be prognostic of outcome in ovulation induction and assisted reproductive technology programs. The precise physiologic basis for day 3 serum FSH screening is unknown. We tested the hypothesis that dimeric inhibin is differentially secreted from luteinized granulosa cells collected from women in preparation for in vitro fertilization with low vs. high day 3 serum FSH levels. This prospective study consisted of luteinized granulosa cells harvested from 7 women with low day 3 serum FSH levels (< or = 6 IU/L) and from 8 women with high FSH levels (> or = 10 IU/L) in preparation for in vitro fertilization-embryo transfer. Following retrieval, cells were isolated and then pooled within each individual patient and plated at 50,000 cells per well. Media was removed at 24 and 48 h and analyzed for dimeric and total inhibin concentrations by immunoassay as well as estradiol and progesterone concentrations by RIA. Dimeric inhibin was secreted at 2-fold higher concentrations in the low FSH group 43.2 pg/ml (30.8-60.6) (geometric mean and 95% confidence interval) compared with the high FSH group, 21.0 pg/ml (15.0 29.6), P < 0.004. Total inhibin was secreted at 1.8-fold higher concentrations in the low FSH group, 1148.2 pg/ml (931.1-1415.8) compared with the high FSH group, 639.7 pg/ml (428.5-955.0), P < 0.013. No significant differences in either estradiol or progesterone concentrations were noted. These data suggest that day 3 serum FSH is an indirect bioassay of dimeric inhibin production at the granulosa cell level. Thus, these data provide a potential physiological basis for day 3 serum FSH screening in ovulation induction and ART programs. PMID- 8636297 TI - Different patterns of steroid secretion in patients with adrenal incidentaloma. AB - We previously found that a remarkable number of patients with adrenal incidentaloma display partially autonomous cortisol secretion. The amount of hypercortisolism is insufficient to give clinical expression, but enough to inhibit, in some cases, normal adrenal tissue. Other researchers found with high frequency a partial deficiency of 21-hydroxylase. The aim of the present study was to make a combined evaluation of these aspects of adrenal steroidogenesis. Twenty patients (6 men and 14 women, aged 25-74 yr; median, 59 yr) with incidentally discovered adrenal masses were studied. All had an adrenal adenoma histologically proven or diagnosed on the basis of size (< or = 4.0 cm in all but 1) and computed tomography picture (hypodense homogeneous mass with well defined margins). The following parameters were used to evaluate the ACTH-cortisol axis: overnight 1-mg dexamethasone suppression (4 nonsuppressors), ovine CRH stimulation (blunted ACTH-cortisol response in 2 cases), circadian serum cortisol rhythm (blunted night/day ratio in 4 and increased 24-h mean in 1), and 24-h urinary free cortisol excretion (always within the normal range). Three patients had 2 concomitant alterations, and 5 had a single abnormality. Partial deficiency of 21-hydroxylase was assumed in 6 patients who showed an exaggerated 17 hydroxyprogesterone response to ACTH, with a peak value of more than 10 ng/mL (> 30 nmol/L), according to New's nomogram. No abnormalities of the ACTH-cortisol axis were found in these patients, with the exception of low amplitude cortisol rhythm in 1 case. Therefore, 2 distinct patterns, dysregulated and partially autonomous cortisol secretion, on the one hand, and reduced 21-hydroxylase activity, on the other, can be found in a high number of patients bearing an adrenal incidentaloma. They appear mutually exclusive, and the differentiation by endocrine testing is quite clear. Serum dehydroepiandrosterone sulfate was below the third percentile in 13 of 20 patients and could represent a specific marker of cortical adenomas. This finding was evenly distributed among patients with subclinical hypercortisolism or partial enzymatic defect; therefore, low serum dehydroepiandrosterone sulfate is not readily attributable to suppressed ACTH secretion, which actually occurs in only some patients with subclinical hypercortisolism. PMID- 8636299 TI - Changes in bone mass as determined by ultrasound and biochemical markers of bone turnover during pregnancy and puerperium: a longitudinal study. AB - In a longitudinal study, we analyzed the speed of sound (SOS) and broadband ultrasound attenuation (BUA) of the os calcis as an index of bone mineral density (BMD) to define the effects of pregnancy and lactation on bone metabolism. We used an ultrasound bone densitometer and measured 6 biochemical markers of bone turnover in 18 healthy women throughout pregnancy and puerperium. The measurement of SOS and BUA by such an ultrasound device was clinically advantageous; not only is it radiation-free technology, but it also correlates highly with BMD measured by conventional X-ray bone densitometry. While a significant decrease in SOS was found in the 3rd trimester of pregnancy as compared with the early stage of pregnancy, there was no difference in both SOS and BUA between the breast-feeding women and the principally formula-feeding women during a 6-month period of puerperium. The analysis of biochemical markers revealed that both bone formation and bone resorption were elevated in the 3rd trimester of pregnancy as well as during puerperium, and that the breast-feeding women had significantly higher bone metabolism than the principally formula-feeding women. These results indicate that bone mass decreases as bone turnover itself is enhanced during pregnancy, while lactation does not substantially affect bone mass during at least 6 months of puerperium, although bone turnover is active. PMID- 8636298 TI - The biological activity of the corticotropin-releasing hormone receptor-adenylate cyclase complex in human myometrium is reduced at the end of pregnancy. AB - We recently suggested that placentally derived CRH might influence human parturition via specific receptor mechanisms. We identified a human myometrial CRH receptor that changes to a high affinity state in the later stages of pregnancy and becomes coupled to the adenylate cyclase system. The purpose of this study was to investigate the functional capacity of this receptor in myometrial tissue obtained from women being delivered electively by cesarian section at term (38-40 weeks gestation) and preterm (30-35 weeks gestation) before the onset of labor. Myometrial membrane suspensions were prepared by differential centrifugation, and the production of cAMP after stimulation with various test substances was measured by RIA. In preterm myometrium, both human CRH and cholera toxin stimulated cAMP production. This effect was significantly reduced in term myometrium. The adenylate cyclase was functionally active in term myometrium, as demonstrated by the use of forskolin. Furthermore, pertussis toxin pretreatment of term myometrial membranes did not increase the response to CRH. These results suggest that in human pregnant myometrium at term, there is a modification in the coupling mechanisms between CRH receptors and the catalytic component of adenylate cyclase, resulting in a reduction of CRH-stimulated cAMP production. PMID- 8636300 TI - Combined administration of levonorgestrel and testosterone induces more rapid and effective suppression of spermatogenesis than testosterone alone: a promising male contraceptive approach. AB - Studies using high dose testosterone (T) administration in normal men as a male contraceptive have resulted in azoospermia rates of only 50-70%. Previous studies of T and progestogen combinations have shown comparable rates of azoospermia, but have been uncontrolled or used T in doses less than that associated with maximal suppression of sperm production. We conducted a randomized, placebo-controlled, single blind trial comparing 6 months of T enanthate administration (100 mg, im, weekly) with the same dose of T enanthate in conjunction with the progestogen levonorgestrel (LNG; 500 micrograms, orally, daily) in 36 normal men, aged 20-42 yr (n = 18 in each group). The primary end points were induction of azoospermia or severe oligospermia (< 3 million sperm/mL). The combination of T plus LNG was much more effective in suppressing sperm production than T alone. Sixty-seven percent of the T plus LNG group (12 of 18) and 33% of the T alone group (6 of 18) achieved azoospermia by 6 months (P = 0.06). Severe oligospermia or azoospermia developed in 94% of the T plus LNG (17 of 18) group compared to 61% of the T alone group (11 of 18; P < 0.05). T plus LNG also suppressed sperm production more rapidly than T alone. Time to azoospermia was 9.9 +/- 1.0 vs. 15.3 +/- 1.9 weeks in the T plus LNG and T alone groups, respectively (mean +/- SEM; P < 0.05). Serum high density lipoprotein cholesterol decreased 21.7 +/- 3.6% in men given T plus LNG (P < 0.05), compared to only a 1.8 +/- 3.8% decrease in men in the T alone group. Average weight gain was 5.3 +/- 0.8 kg in the T plus LNG group and 2.3 +/- 0.9 kg in the T alone group (P < 0.05). Acne and increase in hemoglobin were similar in the two groups. We conclude that combination hormonal therapy with T plus a progestogen might offer a reversible male contraceptive approach with a more rapid onset of action and more reliable induction of both azoospermia and severe oligospermia than T alone. PMID- 8636301 TI - Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro. AB - We have investigated the regulatory role of nitric oxide (NO) in corticotropin releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy. PMID- 8636302 TI - Testosterone treatment alters melatonin concentrations in male patients with gonadotropin-releasing hormone deficiency. AB - Recently, we demonstrated that melatonin secretion is increased in untreated male patients with GnRH deficiency. As testosterone (T) can be aromatized to estradiol (E2), and both T and E2 increase during T enanthate treatment, we were interested in determining whether T treatment (when T and E2 levels were well matched with pubertal control values) has an effect on melatonin levels in these patients. We measured nocturnal serum melatonin levels during the administration of 250 mg testosterone enantale/month for 4 months in 12 male patients with idiopathic hypogonadotropic hypogonadism (IGD; n = 6) and delayed puberty (DP; n = 6). Serum samples for melatonin and LH determinations were obtained every 15 min from 1900 0700 h in a controlled light-dark environment. The results of melatonin profiles were compared with the pretreatment values in each group and with values obtained in six normal pubertal male controls. After 4 months of testosterone treatment, all patients attained normal serum testosterone (19.5 +/- 3.7 in IGD vs. 20.8 +/- 4.1 nmol/L in DP) and E2 levels (83 +/- 12 in IGD vs. 84 +/- 9 pmol/L in DP). Serum LH levels were suppressed in all patients during T treatment (0.12 +/- 0.1 in IGD vs. 0.12 +/- 0.2 IU/L in DP). Before T treatment, patient melatonin levels were greater than those in age-matched pubertal controls. Melatonin levels were equal in patients and controls when T and E2 levels were well matched. Mean (+/- SD) dark-time melatonin levels decreased from 286 +/- 23 to 157 +/- 36 pmol/L in IGD and from 217 +/- 32 to 133 +/- 47 pmol/L in DP (vs. 183 +/- 64 pmol/L in controls). The integrated melatonin values decreased to normal (from 184 +/- 16 to 102 +/- 21 in IGD and from 142 +/- 19 to 90 +/- 26 pmol/min.L x 10(3) in DP vs. 119 +/- 61 pmol/min.L x 10(3) in controls). The intraindividual variations in melatonin levels ranged from 7.2-14.5%. These data indicate that male patients with GnRH deficiency have increased nocturnal melatonin secretion. T treatment decreased melatonin secretion to normal levels. The results suggest that in GnRH deficient male patients, sex steroids, rather than LH, modulate pineal melatonin in a reverse fashion. PMID- 8636303 TI - Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development. AB - Recent investigations have suggested that human GH (hGH) and its receptor may have specific functions during human fetal life. To improve our understanding of the mechanisms of hGH action during gestation, we characterized the ontogenic appearance of hGH receptor messenger ribonucleic acid (mRNA) in multiple human fetal and postnatal tissues. Using RT-PCR assays, followed by Southern hybridization to confirm the specificity of the amplified fragments, we scanned the entire coding region of the hGH receptor mRNA. Transcription of the hGH receptor gene was observed in all fetal tissues studied (liver, kidney, skin, muscle, lung, adrenal, spleen, intestine, central nervous system, pancreas, and placental villi) from the earliest stage that could be examined [7-14.8 weeks fetal age (FA)]. Furthermore, we identified only 2 isoforms of the hGH receptor mRNA-coding region: exon 3 can be retained or deleted. Surprisingly, we found individual-specific, not tissue-specific, expression patterns of these two transcripts when we examined multiple tissues (n = 2-6) from 15 individuals (11.5 33 weeks FA); this individual-specific pattern of expression is maintained in cultured dermal fibroblasts for at least 12 generations (n = 2; 16 and 20 weeks FA). In addition, a cross-sectional study of 78 individuals (9 weeks FA to 43 yr postnatal age) showed that the exon 3-deleted transcript is predominantly expressed in tissues from fetuses of 9-20 weeks FA (P < 0.002). Finally, we showed that the absence of exon 3 from the mRNA is not due to genomic deletion of exon 3 by amplifying exon 3 from genomic DNA of 3 fetuses (13.3-19 weeks FA) expressing only the exon 3-deleted mRNA transcript. We conclude that 1) transcription of the hGH receptor gene occurs in multiple tissues as early as the first trimester of human fetal life; 2) the exon 3-retaining and -deleted transcripts are the only two isoforms of the hGH receptor mRNA-coding region during gestation; and 3) the pattern of expression of these transcripts is individual specific and may be developmentally regulated. PMID- 8636304 TI - Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas. AB - Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas. PMID- 8636306 TI - The effects of hypothyroidism and replacement therapy on cholesteryl ester transfer. AB - To characterize further the impact of thyroid dysfunction on the transport of cholesterol in plasma, we studied plasma lipids and cholesteryl ester transfer (CET) in 10 hypothyroid women before and 3 months after thyroid replacement therapy. CET, estimated as the net mass transfer of CE from HDL to the apolipoprotein B-containing lipoproteins (very low density and low density lipoproteins) was significantly decreased at 4 h (P < 0.05) and 6 h (P < 0.001) when the patients were hypothyroid (T4, 2.01 +/- 1.4; TSH, 55.5 +/- 39.9 microIU/mL) and increased to normal levels after hormone replacement and restoration of eumetabolism. Plasma lipid levels in the hypothyroid state closely resembled those in a female reference group, although total plasma cholesterol fell significantly [pretreatment, 218 +/- 36 vs. posttreatment, 192 +/- 49 (P < 0.025); control, 218 +/- 28 mg/dL (mean +/- SD)] after treatment. Concentrations of cholesteryl ester transfer protein (CETP) were unchanged (pretreatment, 2.35 +/- 0.83 vs. posttreatment, 2.30 +/- 1.19 mg/dL). The results of recombination studies using different lipoprotein fractions suggest that decreases in CET during hypothyroidism may be secondary to acceptor lipoprotein (low density and very low density lipoprotein) changes in the hypothyroid state and not to changes in the concentration of CETP itself. PMID- 8636305 TI - Hypothalamo-pituitary-adrenal function in human immunodeficiency virus-infected men. AB - We prospectively studied adrenal function in 51 human immunodeficiency virus positive male patients, including heterosexuals, homosexuals, and iv drug users, classified according to 1987 CDC criteria as belonging to stages II/III or IVC. Basal serum concentrations of cortisol (F), progesterone (P4) and 17 alpha hydroxyprogesterone (17 alpha-OHP4) were determined during the two stages. In stage IVC patients, the circadian rhythms of ACTH and F were assessed, and ovine CRH (oCRH) and immediate cosyntropin-stimulating tests were evaluated. Serum concentrations of hormones were analyzed in relationship to the absolute CD4 cell count in all subjects. The mean serum F concentration in stage IVC patients, the mean P4 concentration in stage II/III and IVC patients, and the mean 17 alpha OHP4 level in stage II/III patients were significantly increased compared to control values (P < 0.0001, P < 0.0001, and P < 0.002, respectively). The mean serum F concentration in stage IVC patients was significantly increased compared to that in stage II/III patients (P < 0.004), and the mean serum 17 alpha-OHP4 concentration in stage II/III patients was significantly increased compared to that in stage IVC patients (P < 0.02). In the 22 stage IVC patients, the circadian rhythms of ACTH and F were normal in all but 7 for ACTH and 5 for F, whereas oCRH test results indicated that 14 of them had reduced or blunted responses. By contrast, cosyntropin stimulation results were normal. CD4 cell counts were significantly negatively correlated with the serum F concentration (P < 0.02). In conclusion, during human immunodeficiency virus infection, the serum F concentration was negatively correlated with CD4 cell counts. Cosyntropin test results were normal, but 63% of the stage IVC men had abnormal responses to oCRH. PMID- 8636307 TI - The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation. AB - Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation. PMID- 8636308 TI - Human adrenal cells express tumor necrosis factor-alpha messenger ribonucleic acid: evidence for paracrine control of adrenal function. AB - Tumor necrosis factor (TNF) is gaining increasing importance in clinical medicine. It plays a role in the interaction of the immune system with the hypothalamic-pituitary-adrenal axis. In the present study various morphological methods, including immunohistochemistry, electron microscopy, and in situ hybridization were applied to characterize the localization and distribution of TNF in the human adrenal gland. Double immunostaining revealed an astonishing degree of intermingling of steroid-producing cells and chromaffin cells. Macrophages could be found in all regions of the adrenal gland, but particularly in the transition zone of cortex and medulla. The steroid-producing cells of the inner zone of the cortex express major histocompatibility complex class II molecules. On the ultrastructural level, immune cells, steroid cells, and catecholamine-producing cells were found in direct contact. The combination of immunohistochemistry and in situ hybridization was optimally suited to define the exact cellular source of TNF in the human adrenal. TNF is produced in macrophages, but above all in 17 alpha-hydroxylase-positive cells (steroid producing cells) in the zona reticularis and medulla. No signal was found in chromaffin cells. TNF may induce major histocompatibility complex class II in human adrenal gland in a paracrine or autocrine manner. It is concluded that TNF may have an important role in normal human adrenal physiology. PMID- 8636309 TI - Evidence for atrophy and apoptosis in the prostates of men given finasteride. AB - Finasteride, a 5 alpha-reductase inhibitor, decreases prostate size and improves symptoms in men with benign prostatic hyperplasia. However, little is known about prostate histopathology in men taking finasteride. To determine the mechanism by which finasteride reduces prostate size, tissue was collected at the time of prostatectomy from men taking either no medication (n = 10) or 5 mg finasteride daily for 6-18 days (n = 6; group 1), 23-73 days (n = 5; group 2), or 3 months to 4 yr (n = 5; group 3). To assess whether finasteride causes epithelial atrophy, morphometric measurement of epithelial cell and duct width was used. The mean epithelial cell width in control prostates (mean +/- SEM, 21 +/- 0.7 microns) decreased with duration of treatment to 19 +/- 1 microns in group 1, 15 +/- 2 microns in group 2, and 8 +/- 0.3 microns in group 3. Mean duct width decreased from 135 +/- 6 microns in the control prostates to 128 +/- 10 microns in group 1, 103 +/- 3 microns in group 2, and 63 +/- 6 microns in group 3. To assess whether prostate cell death was occurring, sections were in situ end labeled for DNA breaks and immunostained for tissue transglutaminase (tTG), a marker of apoptosis (programmed cell death). The percentage of epithelial cells staining for DNA breaks was 0.4 +/- 0.2 in control prostates, 2.8 +/- 0.9 in group 1, 1.7 +/- 0.5 in group 2, and 0.7 +/- 0.3 microns in group 3. Anti-tTG staining of epithelial cells was graded on a scale of 0-4. In control prostates, 3 +/- 1% of the ducts were grade 3 or 4 (> 50% of epithelial cells staining). In finasteride-treated prostates, 2 +/- 2% of the prostates in group 1, 13 +/- 4% of the prostates in group 2, and 0.5 +/- 0.5% of the prostates in group 3 were grade 3-4. These results indicate that a progressive decrease in epithelial cell size and function occurs during the first several months in the prostates of men treated with finasteride. The staining for DNA breaks and the tTG staining also indicate that an increased rate of apoptosis is occurring transiently in these prostates. We conclude that finasteride causes prostate involution through a combination of atrophy and cell death. PMID- 8636311 TI - Cell turnover in normal cycling human ovary. AB - We investigated cell proliferation and apoptosis in 37 normal cycling human ovaries to determine cell turnover in the various stages of follicular and luteal development. We examined cell proliferation by immunostaining for both Ki67 and proliferative cell nuclear antigen (PCNA) and by silver staining of nucleolar organizer regions (AgNORs). Apoptosis was examined by 3'-hydroxy nick-end labeling and by immunostaining of an apoptosis-related antigen, Ley. The labeling indexes of Ki67, PCNA, and AgNORs were significantly increased in antral follicles. However, there were no significant differences in the labeling index of Ki67, PCNA, and AgNORs between dominant and nondominant follicles, including nonovulated follicles in the luteal phase. These results indicate that the transformation of granulosa cells from quiescence to active growth is important in early folliculogenesis. Immunoreactivity for Ki67 and PCNA were observed predominantly in the functioning corpus luteum, but not in the degenerating corpus luteum, indicating proliferation only during the luteal phase. Immunoreactivity for Ley and nick end-labeling reactive cells were not observed in the follicular and luteal phases, except for scattered cells in the degenerating corpus luteum. This may be because of the relatively long process of human follicular growth and atresia. PMID- 8636310 TI - Androgen and estrogen receptors are present in primary cultures of human synovial macrophages. AB - Macrophages, as antigen-processing and -presenting cells to T lymphocytes, play a key role in the immune system and are suspected to be target cells of the sex hormone-related dimorphism in the immune response peculiar to rheumatoid arthritis (RA) pathology. In the present study, the use of specific monoclonal antibodies revealed immunostaining for androgen and estrogen receptors in primary cultures of macrophages obtained from synovial tissues of patients affected by RA and controls without RA disease. Soluble and nuclear type I (high affinity, low capacity) and type II (lower affinity, greater capacity) sites of androgen or estrogen binding were detected in primary cultures of RA macrophages using radioligand binding assay. Higher levels of type I and type II estrogen receptor compared to those of androgen receptor were found, particularly in the soluble fraction; however, contrary to what was observed in whole synovial tissues, higher steroid receptor concentrations were found in the soluble than in the nuclear fraction of RA synovial macrophages. Binding affinities and receptor contents of cultured synovial macrophages were comparable to those previously reported in other well established sex hormone-responsive cells and tissues. Further, specific messenger ribonucleic acids for sex hormone receptors, encoding for a sequence of the DNA-binding domain of the receptor proteins were revealed by RT-PCR. PMID- 8636312 TI - Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum. AB - In the nonhuman primate and human corpora lutea, gap junctions have been identified by means of electron microscopy. Gap junctions are formed by connexons, which consist of a multigene family of tissue-specific connexins. In the ovarian follicle, the gap junction protein connexin-43 is present and hormonally regulated. However, there is little evidence indicating the type of connexin present in the corpus luteum. Therefore, the aim of this study was to demonstrate the presence of gap junctions by electron microscopy and the presence of connexin-43 and messenger ribonucleic acid (mRNA) for this protein. Using immunocytochemical procedures, we have shown the presence of connexin-43 in baboon and human midluteal phase corpora lutea and in the atretic corpora lutea of the baboon. The intensity of immunoreactivity was lower in atretic corpora lutea than in the midluteal phase corpora lutea. Western analysis indicates the presence of two bands at 43-45 kDa, and that the levels of connexin-43 protein are abundant in the midluteal phase. The two bands suggest the presence of the protein in a phosphorylated or a nonphosphorylated form. Ribonuclease protection assay suggests that the mRNA levels of connexin-43 remain constant throughout the luteal phase. mRNA for connexin-43 was not detectable in atretic corpora lutea. Thus, connexin-43 is one of the connexin family of proteins forming the connexon of gap junctions in the baboon and human corpus luteum. The expression of the protein may be hormonally regulated by locally produced factors, such as estradiol and progesterone. We suggest that gap junctional communication between the cells of the primate and human corpus luteum may be important in hormone synthesis and secretion and may be involved in the process of luteolysis through luteal cell apoptosis. PMID- 8636313 TI - Usefulness of 123I-metaiodobenzylguanidine (MIBG) scintiscan in the diagnosis of juxta-adrenal schwannoma. AB - The adrenal scintiscan with 123I-metaiodobenzylguanidine (MIBG), a reliable morphofunctional technique to evaluate catecholamine turnover in adrenal tumors, can be a useful method to investigate adrenal incidentalomas with arterial hypertension. A male patient, 44 yr old with diabetes, unstable arterial hypertension, and sudden paroxysms of tachycardia is described. The presence of a disomogeneous right juxta-adrenal neoplasm with calcifications was evidenced with ultrasound tomography and confirmed by computerized tomography (CT) scan. Adrenal 123I-MIBG scintiscan revealed a unilateral uptake at level of the right juxta adrenal region, sized similarly to the neoplasm previously evidence by CT scan. Histological findings of the surgically removed neoplasm were consistent with an ancient schwannoma. Apart from pheochromocytomas, the MIBG uptake is commonly reported in neuroblastomas. In neuroblastoma, a bidirectional process of transdifferentiation has been previously reported in vitro between two coexistent cells: cells with specific uptake system for norepinephrine, with 123I-MIBG uptake capability, and cells oriented toward schwann/melanocytic line. The evidence of in vivo MIBG uptake in our schwannoma may be caused by the same possible phenotypic interconversion of above mentioned cell types. In conclusion, the presence of adrenal tumors with MIBG uptake capability, apart from pheochromocytomas, neuroblastomas, ganglioneuroblastomas, and ganglioneuromas, must be considered in the diagnosis of adrenal tumors. PMID- 8636314 TI - Lack of an independent association between the human leukocyte antigen allele DQA1*0501 and Graves' disease. AB - The association between the human leukocyte antigen (HLA) serotype DR3 and Graves' disease (GD) in Caucasian populations is well known. However, an even stronger association has been reported recently, especially in the male population, between the closely linked HLA allele DQA1*0501 and GD. We postulated that the reported association between DQA1*0501 and GD may be a result of the linkage of this allele with DR3 and may not represent an independent association. Accordingly, we screened a population of North American Caucasians (n = 218), including patients with GD (n = 101, 32 males, 69 females) and individuals with documented normal thyroid function (n = 117, 51 males, 66 females), for the presence of the DQA1*0501 allele and those alleles corresponding to the DR3 serotype (DRB1*03). Screening was accomplished using sequence specific PCR. A significant association was documented in the total study population between DR3 positivity and GD (P = 0.0002), but not between DQA1*0501 positivity and GD (P = 0.06). After gender stratification, significant associations were found only in the female population (DR3, P = 0.0004; DQA1*0501, P = 0.012) and not in the male population (DR3, P = 1.0; DQA1*0501, P = 1.0). Additionally, in those DR3 negative female subjects (n = 100), there was no independent association between DQA1*0501 positivity (n = 26) and GD (p = 0.82). P-values were corrected, where appropriate, for gender stratification and/or the number of HLA alleles tested. In conclusion, our results demonstrate a lack of independent association between the presence of the HLA allele DQA1*0501 and GD. We suggest that the apparent association between this allele and GD in the female population may be the result of its' close linkage to DR3. PMID- 8636315 TI - Are the differences between estradiol and other estrogens, naturally occurring or synthetic, merely semantical? PMID- 8636316 TI - High levels of corticotropin-releasing factor (CRF) are inversely correlated with low levels of maternal CRF-binding protein in pregnant women with pregnancy induced hypertension. AB - Corticotropin-releasing factor-binding protein (CRF-BP) is suggested to play a role in modulating the activity of the hypothalamus-pituitary-adrenal axis during pregnancy, counteracting the actions of circulating or locally acting CRF. The aim of the present study was to evaluate whether maternal levels of CRF-BP and CRF are modified in pregnant women with a high risk of developing pregnancy induced hypertension (n = 21). A group of nine patients developed the disease between 25-35 weeks gestation, and sequential blood samples were taken every 5 weeks throughout the pregnancy. As a control group, healthy pregnant women were studied (n = 9) using the same protocol; a group of women with pregnancy-induced hypertension (n = 5) was studied starting from the time of diagnosis. In a subgroup of patients (n = 10), CRF-BP and CRF levels were studied after 5 weeks of antihypertensive treatment. Levels of CRF-BP were determined using a specific RIA, whereas CRF was evaluated by a two-site immunoradiometric assay. In patients at risk, circulating levels of CRF-BP followed the same pattern as that in healthy controls, showing a significant decrease at term (36-40 weeks; P < 0.05). A significant and progressive increase in plasma CRF levels was observed in both groups of pregnant women; the highest values were found at term (P < 0.01). In the nine patients who developed pregnancy-induced hypertension, maternal levels of CRF-BP at the onset of signs and symptoms were lower than control values, and CRF levels were significantly higher at the onset of the disease (P < 0.01). Similarly, in these hypertensive patients studied at the time of hospitalization, CRF-BP levels were lower whereas those of CRF were higher than levels in healthy patients (P < 0.01). No effect of antihypertensive therapy on either CRF-BP or CRF levels was observed. The present study shows an inverse correlation between reduced plasma CRF-BP levels and increased CRF levels in the maternal circulation of patients with pregnancy-induced hypertension, and indicates that these hormonal changes do not occur before the onset of disease, suggesting that the measurement of these polypeptides in maternal plasma does not predict the development of hypertension. PMID- 8636317 TI - Evidence for a clinically important adverse effect of fiber-enriched diet on the bioavailability of levothyroxine in adult hypothyroid patients. AB - To evaluate the effect of dietary fiber supplements on levothyroxine (T4) bioavailability in hypothyroid patients, dietary fiber-containing supplementation was withheld from patients requiring disproportionately high doses of T4, in whom a dietary history revealed ingestion of a dietary fiber supplement. The dose of T4 was maintained at a constant level. Serum thyrotropin (TSH) was assessed before and after removal of the dietary fiber supplements. T4 requirements, reflected by either decreased serum TSH or by decreased T4 dose, was observed in conjunction with decreased dietary fiber intake compared with T4 requirement during increased dietary fiber intake. In vitro experiments carried out to determine the mechanism of interaction between dietary fiber and T4 revealed dose dependent, nonspecific adsorption of levothyroxine by wheat bran. These results indicate a decrease in T4 bioavailability by dietary fiber through a mechanism involving nonspecific adsorption of T4 to dietary fibers. Increased intake of dietary fiber may account for the need for larger than expected doses of T4 in some hypothyroid patients. PMID- 8636318 TI - Reduced insulin-like growth factor binding protein-1 (IGFBP-1) levels correlate with increased cardiovascular risk in non-insulin dependent diabetes mellitus (NIDDM). AB - IGF-I and -II levels are altered in patients with atherogenic lipid profiles and may contribute to the development of macrovascular disease in NIDDM. We examined cardiovascular risk factors, IGF-I, IGF-II and IGFBP-1 in 74 NIDDM patients analysed as a whole group and according to treatment type. IGF-I was not significantly associated with cardiovascular risk factors but IGF-II levels correlated positively with total and LDL cholesterol most markedly in the diet treated group (0.72, p < 0.01 and 0.76, p < 0.01 respectively). In the whole group reduced IGFBP-1 levels were significantly associated with factors known to increase cardiovascular risk: i.e. low HDL cholesterol (0.31, p < 0.01) and elevated blood pressure (-0.35, p < 0.01), BMI (-0.37, p < 0.01), insulin (-0.29, p < 0.01) and proinsulin (-0.24, p < 0.01). In the treatment groups IGFBP-1 was lower in patients on diet alone (n = 11, 42.6 +/- 11.6 mu g/l) and sulphonylurea +/- insulin (n = 39, 53.2 +/- 7.6 mu g/l) relative to insulin treatment (n = 24, 103.0 +/- 19, 7 mu g/l, p < 0.05). The lower levels of IGFBP-1 were not due to a significant change in phosphorylation status from the highly phosphorylated circulating form since lesser and non-phosphorylated variants were undetectable in 53/74 patients. Multiple regression analysis revealed the best predictors of IGFBP-1 were BMI and MAP (R2 = 0.2. p < 0.001) and for blood pressure, IGFBP-1 and age (R2 = 0.47, p < 0.001). These findings indicate that in NIDDM patients low IGFBP-1 levels are associated with multiple factors predisposing to atherogenesis. PMID- 8636319 TI - Lack of effect of a gonadotropin-releasing hormone agonist in a patient with prostate cancer and a gonadotroph adenoma. PMID- 8636320 TI - Clinical review 79: Angiotensin II: an insulin-sensitizing vasoactive hormone? PMID- 8636321 TI - Cortisol as mineralocorticoid. PMID- 8636322 TI - Calcium-sensing receptor mutations: insights into a structurally and functionally novel receptor. PMID- 8636323 TI - Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. AB - The predominant variety of familial benign hypocalciuric hypercalcemia (FBHH) is FBHH(3q), which is associated with presumed inactivating mutations of the cell surface calcium receptor (CaR) gene on chromosome 3q13.3-q21. We sought mutations of the CaR gene in FBHH by direct sequencing of PCR-amplified genomic DNA from 14 affected families: 8 mapped to 3q13, 1 mapped to chromosome 19p, and 5 unmapped. We sequenced the entire coding region of the gene (exons 2-7) in one or two affected members of each family and found six point mutations that altered one amino acid, cosegregated with hypercalcemia, and were absent in more than 100 unaffected persons. Four mutations were unique (S53P, D215G, S657Y, and P748R), and two had been reported previously (P55L and R185Q). Of four mutant CaR proteins expressed in Xenopus oocytes, three were deficient in extracellular Ca2+ induced signaling. No CaR mutations were found in eight families, including the one mapped to chromosome 19p. Three benign polymorphisms occurred in the COOH terminal region of the CaR protein in 10%, 15%, and 30% of more than 100 unaffected persons. Thus, FBHH-causing CaR mutations were clustered in the NH2 terminal extracellular and membrane-spanning regions of the receptor protein. We suggest that these are important functional domains, probably for calcium binding and signal transduction, respectively. Finally, mutations in regulatory or intronic regions of the CaR gene may also underlie many cases of FBHH. PMID- 8636324 TI - Measurement of circulating inhibin levels: revisiting the inhibin hypothesis. PMID- 8636325 TI - Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. AB - Inhibin is a glycoprotein hormone that is defined on the basis of inhibition of pituitary FSH production, However, previous data have not shown any correlation between RIA measurements of inhibin and FSH in men. New enzyme-linked immunosorbent assays, specific for inhibin A, inhibin B, and inhibin pro-alphaC related immunoreactivity, were applied to the measurement of inhibin in 32 healthy men. Further measurements of inhibin B and pro-alphaC-RI were carried out on groups of men exhibiting a wide range of FSH concentrations, including semen donors, infertile men, and men with elevated FSH concentrations. Inhibin A was undetectable (<2 pg/mL) in all men studied. The healthy men studied all had measurable concentrations of inhibin B (135.6 pg/mL; confidence interval, 108.4 169.4) and pro-alphaC-RI (426.3 pg/mL; confidence interval, 378.4-480.2). A close negative correlation was found between the inhibin B and FSH concentrations in the semen donors (r = -0.69; P < 0.001), the infertile men (r = -0.81; P < 0.001), and the men with elevated FSH concentrations (r = -0.54; P < 0.01), but not in a group of healthy volunteers (r = -0.08; P = NS). No correlation was observed between concentrations of pro-alphaC-RI and FSH in any of the groups studied. These results strongly suggest that the physiologically important form of inhibin in men is inhibin B, which has a critical effect on FSH release. Inhibin B may offer a clinically useful serum marker of testicular function. PMID- 8636326 TI - Spontaneous remission of primary hyperparathyroidism from parathyroid apoplexy. PMID- 8636327 TI - The resistant ovary syndrome in a patient with galactosemia: a clue to the natural history of ovarian failure. PMID- 8636328 TI - Seven deadly sins in confronting endemic iodine deficiency, and how to avoid them. AB - Iodine deficiency is a problem for almost all countries of the world. Goiter is its most obvious consequence, but others do more damage, particularly effects on the developing brain. In 1990, most countries and international agencies pledged the virtual elimination of iodine deficiency by the year 2000. The technology for the assessment and implementation is sufficient to attain this goal, but translating its potential into success requires careful planning. This article reviews seven major errors that frequently occur in iodine supplementation programs and offers suggestions for their avoidance. They are 1) unreliable assessment of iodine deficiency: the best indicators are urinary iodine concentration, thyroid size (preferably by ultrasound), blood spot thyroglobulin levels, and neonatal TSH determinations; the best group for surveys is schoolchildren; 2) poor iodine supplementation plan: iodized salt is the preferred supplement; its effective application frequently requires extensive changes in salt production and marketing, and poor handling of these changes will endanger the iodization program; other measures include iodized oil, iodized water, and iodine drops; all are occasionally useful, but the long range solution should generally be iodized salt; 3) exclusion of relevant stake-holders: the program should include not only health authorities but other arms of the government as well (education, commerce, agriculture, and standards), the salt industry, health professionals, and the iodine-deficient community itself; 4) inadequate education: an understanding of the effects of iodine deficiency and the means for its correction is essential at all levels, from government to affected population; 5) insufficient monitoring: the best instruments are urinary iodine levels, iodized salt use, and thyroid size, measured in representative groups at regular intervals with public reporting of results; 6) inattention to cost: the expense of iodization must be recognized and apportioned fairly; and 7) nonsustainability: for permanent success, an iodization program must be fair to all relevant parties and accompanied by a regular system of appropriate monitoring. Only with careful avoidance of these seven "deadly sins" can the goal of sustainable elimination of iodine deficiency be achieved. PMID- 8636330 TI - Different growth hormone (GH) response to GH-releasing peptide and GH-releasing hormone in hyperthyroidism. AB - Altered GH responses to several pharmacological stimuli, including GHRH, have been found in hyperthyroidism. The mechanisms underlying these disturbances have not been fully elucidated. GH-releasing peptide-6 (GHRP-6) is a synthetic hexapeptide that specifically stimulates GH release both in vitro and in vivo. The mechanism of action of GHRP-6 is unknown, but it probably acts by inhibiting the effects of somatostatin on GH release. The aim of this study was to evaluate the effects of GHRP-6 on GH secretion in patients with hyperthyroidism (n = 9) and in control subjects (n = 9). Each subject received GHRP-6 (1 microg/kg, iv), GHRH (100 microg, iv), and GHRP-6 plus GHRH on 3 separate days. GH peak values (mean +/- SE; micrograms per L) were significantly lower in hyperthyroid patients compared to those in control subjects after GHRH alone (9.0 +/- 1.3 vs. 27.0 +/- 5.2) and GHRP-6 plus GHRH (22.5 +/- 3.5 vs. 83.7 +/- 15.2); a lack of the normal synergistic effect of the association of both peptides was observed in thyrotoxicosis. However, a similar GH response was seen in both groups after isolated GHRP-6 injection (31.9 +/- 5.7 vs. 23.2 +/- 3.9). In summary, we have shown that hyperthyroid patients have a normal GH response to GHRP-6 together with a blunted GH responsiveness to GHRH. Our data suggest that thyroid hormones modulate GH release induced by these two peptides in a differential way. PMID- 8636329 TI - Putative melatonin receptors in benign human prostate tissue. AB - Melatonin, secreted by the pineal gland at night, inhibits pubertal development of rats and presumably men. In addition, it may directly suppress prostate growth in the adult rat. To investigate the possibility for a causal relationship between the age-related decline in melatonin production and increase in prevalence of benign prostate hypertrophy (BPH) in man, the presence of melatonin binding sites in human BPH tissue was examined. In vitro autoradiography indicated specific 125I-labeled melatonin (125I-melatonin) binding in the prostate, localized to the glandular epithelium. Separation and subcellular fractionation indicated that these sites were associated with the microsomal fraction of the epithelial cells. Kinetic and equilibrium 125I-melatonin binding experiments revealed that the binding was time dependent and reversible, with an apparent half saturation at 140 pmol/L. Competition experiments indicated high and low affinity melatonin binding sites; binding was inhibited by melatonin (IC50 1 nmol/L and 1 micromol/L, respectively) and partially by the putative melatonin antagonist, N-(2,4 dinitrophenyl)-5-methoxytryptamine (ML-23; IC50 0.1 nmol/L). Serotonin and 6-hydroxymelatonin were less potent, whereas up to 0.1 mmol/Lol/L of 5-methoxytryptamine, 6-methoxymelatonin, and tryptamine caused only a partial reduction in specific binding. The guanine nucleotide analogs, guanosine 5'-O-[3-thiotriphosphate] and guanosine 5'-O-[2-thio-diphosphate, inhibited specific 125I-melatonin binding, whereas 5'-guanylyl imidodiphosphate was less potent. The results indicate putative melatonin receptors in the human prostate epithelium. PMID- 8636331 TI - Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy. AB - Genes on the long arm of Y (Yq), particularly within interval 6, are believed to play a critical role in human spermatogenesis. Cytogenetically detectable deletions of this region are associated with azoospermia in men, but are relatively uncommon. It has been hypothesized that microdeletions of Yq may account for a significant proportion of men with infertility. The objective of this study was to validate a sequence-tagged site (STS)-mapping strategy for the detection of Yq microdeletions and to use this method to determine the proportion of men with idiopathic azoospermia or severe oligozoospermia who carry microdeletions in Yq. STS mapping of a sufficiently large sample of infertile men should also help further localize the putative gene(s) involved in the pathogenesis of male infertility. Genomic DNA was extracted from peripheral leukocytes of 16 normal fertile men, 7 normal fertile women, 60 infertile men (50 of whom had azoospermia and 10 of whom had severe oligozoospermia with no other recognizable cause of infertility), and 15 patients with the X-linked disorder, ichthyosis. PCR primers were synthesized for 26 STSs that span Yq interval 6. None of the 16 normal men of known fertility had microdeletions. Seven normal fertile women failed to amplify any of the 26 STSs, providing evidence of their Y specificity. No microdeletions were detected in any of the 15 patients with ichthyosis. Of the 60 infertile men typed with 26 STSs, 11 (18%; 10 azoospermic and 1 oligozoospermic) failed to amplify 1 or more STS. Interestingly, 4 of the 11 patients had microdeletions in a region that is outside the Yq region from which the DAZ (deleted in azoospermia gene region) gene was cloned. In an additional 3 patients, microdeletions were present both inside and outside the DAZ region. In 3 subjects, the microdeletions were verified by Southern analysis using labeled PCR products corresponding to the deleted STSs as probes. These data suggest a high prevalence (18%) of Yq microdeletions in men with idiopathic azoospermia/severe oligospermia. The physical locations of these microdeletions provide further support for the concept that a gene(s) on Yq deletion interval 6 plays an important role in spermatogenesis. The presence of deletions that do not overlap with the DAZ region suggests that genes other than the DAZ gene may also be implicated in the pathogenesis of some subsets of male infertility. PMID- 8636332 TI - Treatment of central precocious puberty: comparison of urinary gonadotropin excretion and gonadotropin-releasing hormone (GnRH) stimulation tests in monitoring GnRH analog therapy. AB - GnRH analogs (GnRH-a) have proven to be efficacious and have become the standard treatment for central precocious puberty (CPP). To confirm the diagnosis of CPP and to monitor the adequacy of hvpothalamic-pituitary-gonadal (HPG) axis suppression, GnRH stimulation testing has been essential. To determine whether 24 h urinary gonadotropin excretion could adequately assess HPG axis suppression, we compared the results of simultaneous GnRH stimulation tests and 24-h urinary gonadotropin determinations in 18 girls with CPP who were receiving GnRH-a therapy (leuprolide acetate, Depot-Lupron, TAP Pharmaceuticals). HPG axis suppression was defined as the absence of significant LH and FSH responses to GnRH stimulation. Simultaneous GnRH stimulation tests and urinary gonadotropin determinations had a concordance rate of 68% (42 of 62). The sensitivity and specificity of urinary LH determinations to detect inadequate HPG suppression were 75% and 64%, respectively. For urinary FSH determinations, the sensitivity and specificity were 90% and 28%, respectively. Hence, single timed urine collections lacked the sensitivity and specificity to assess HPG axis suppression and, thus, cannot replace GnRH stimulation tests for monitoring the adequacy of the GnRH-a dose. PMID- 8636333 TI - Progesterone induces Ca++-dependent 3',5'-cyclic adenosine monophosphate increase in human sperm. AB - Progesterone (P) has been reported to modulate numerous sperm functions through the binding of P to plasma membrane. One of the effects is an increase in sperm hyperactivation, which is known to be cAMP-dependent. To evaluate the effect of P on cAMP levels, human spermatozoa were incubated 2 h with increasing P concentrations. P significantly induced cAMP increase in a dose-dependent manner, reaching a 3-fold increase at 100 micromol/L (P < 0.01). During the study of the kinetics of P effect, two cAMP peaks were observed: one occurring after a 30-min incubation, with a 1.5-fold increase (P < 0.05), and the second one after a 120 min incubation, with a 2.5-fold increase (P < 0.01). These effects of P on cAMP levels correlated with significant rises in the percentage of hyperactivated spermatozoa, occurring at the same times as those of cAMP. To evaluate the Ca++ dependence of these P effects, the experiments were performed in the presence of and in the absence of Ca++ in the incubation medium. The effects of P at the 30th min and the 120th min were completely abolished in the absence of Ca++. Moreover, calcium ionophore A23187, after a 30-min incubation, induced an increase in cAMP levels identical to that obtained with P. The effect of P was partially reproduced by gamma-amino-butiric acid (GABA) and inhibited by GABA antagonist picrotoxin. It was also inhibited by tyrosine kinase inhibitor genistein but not by RU486. Based on these findings, we conclude that P induces Ca++-dependent cAMP increase in human sperm, that this effect is likely caused by the influx of Ca++ (previously reported), and that the effect partially involves GABA(A)-like receptors. PMID- 8636334 TI - 5 alpha-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro. AB - 5 alpha-reductase (5 alpha R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5 alpha-reductase type 1 (5 alpha R1) and type 2 (5 alpha R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5 alpha-reductase In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5 alpha R2. Finasteride and SKF105,657 were potent inhibitors of 5 alpha-reductase activity in BPH tissue and COS cells expressing 5 alpha R2, but weak inhibitors in the cancer lines and in COS cells expressing 5 alpha R1. In contrast, UK117,026 was a more potent inhibitor of 5 alpha-reductase activity in the prostate cancer cell lines and in COS cells expressing 5 alpha R1. These data indicate that human prostate cancer cell lines express 5 alpha-reductase activity similar to that in COS cells transfected with 5 alpha R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation in which case it will be important to select appropriate inhibitors in the clinic. PMID- 8636336 TI - Monitoring of growth hormone replacement therapy in adults, based on measurement of serum markers. AB - The optimal dose for GH replacement therapy in GH-deficient (GHD) adults is not known, nor is there a consensus as to which method is the most appropriate for the monitoring of treatment. To establish a general guideline for GH replacement therapy in adults, we evaluated the relationship between the administered GH dose and the achieved serum levels of three GH-dependent serum markers. Serum levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) were measured in 46 GHD men participating in a 1-yr, double blind, and placebo-controlled dose-response study. The doses of recombinant human GH ranged from 0.33-3.0 IU/m(2)-day. During GH treatment, dose reduction was necessary because of side-effects in 18 of 46 patients, i.e. in 18% of the patients receiving a maintenance dose of 1 IU/M(2)-day, in 35% of the patients receiving a dose of 2 IU/m(2)-day, and in 67% of the patients receiving a dose of 3 IU/M(2)-day. In the untreated state, serum levels of all three markers were below the normal range in 90% of the patients. The rise in serum marker concentrations during the first month of treatment was dose dependent. Significant increases in IGF-I, IGFBP-3, and ALS levels were observed with a dose as low as 0.33 IU/M(2)-day. The minimal GH dose required for normalization of the serum IGF-I concentration was 0.66 IU/M(2)-day, and it was 1.0 IU/M(2)-day for ALS and IGFBP-3. In patients receiving 2.0 IU/M(2)-day, the mean serum IGF-I concentration rose to an abnormally high level, whereas at this dose, the mean IGFBP-3 and ALS levels were not different from normal. The lower sensitivity of IGFBP-3 and ALS to GH doses in the high range was also apparent during long term treatment. The number of patients who developed IGFBP-3 or ALS levels that exceeded the upper normal limit was substantially smaller than the number of patients with elevated IGF-I concentrations (2, 8, and 19 of 46 patients, respectively). In conclusion, serum IGF-I appears to be the preferred biochemical marker for the detection of GH excess in adults receiving GH replacement therapy, because it is more sensitive than IGFBP-3 and ALS to GH doses in the high range. If normalization of the serum IGF-I concentration is taken as the criterion for optimal GH replacement therapy, the predicted optimal GH dose for GHD men 20 - 40 yr old is 1.4 IU/M(2)-day, and the 95% confidence interval is 1.2-1.6 IU/M(2) day. PMID- 8636335 TI - An activating mutation of the follicle-stimulating hormone receptor autonomously sustains spermatogenesis in a hypophysectomized man. AB - As both gonadotropins, LH and FSH, are required for normal spermatogenesis, patients with pituitary insufficiency need hCG plus human menopausal gonadotropin therapy to induce spermatogenesis and establish fertility. In a patient hypophysectomized because of a pituitary tumor, who, despite undetectable serum gonadotropin levels, had normal testis volume and semen parameters and fathered three children under testosterone substitution alone, we hypothesized an activating mutation of the FSH receptor. Exon 10 of the FSH receptor gene was amplified from genomic DNA by PCR, screened by single stranded conformation polymorphism gel electrophoresis, and sequenced. We identified a heterozygous A- >G base change at nucleotide position 1700, leading to an Asp-->Gly transition in codon 567 in the third intracytoplasmatic loop. COS-7 cells transiently transfected with the mutated receptor displayed a 1.5-fold increase in basal cAMP production compared to wild-type receptor, indicating that this mutation leads to ligand-independent constitutive activation of the FSH receptor. We conclude that this activating mutation of the FSH receptor, the first ever described, autonomously sustains spermatogenesis in the absence of gonadotropins. PMID- 8636337 TI - Overnight excretion of urinary catecholamines and metabolites in the detection of pheochromocytoma. AB - The detection and diagnosis of pheochromocytoma are highly dependent on the biochemical confirmation of excessive catecholamine release by the tumor. As the reliability of baseline plasma catecholamines in the detection of pheochromocytoma is questionable, assessment of the excretion rates of catecholamines or metabolites in 24-h urine collections remains the mainstay of initial biochemical investigation. However, diagnostic difficulties can arise from incomplete collection of 24-h specimens or equivocal increases in catecholamines due to stress. To investigate the diagnostic validity of shorter collection times for the biochemical detection of this tumor, we measured the excretion of catecholamines and metabolites after sleep, a period associated with decreased sympathetic activity. Overnight catecholamines, metanephrines, and 4 hydroxy-3-methoxymandelic acid (HMMA) levels were measured in 16 patients with histologically confirmed pheochromocytomas, 166 patients with hypertension, and 24 normotensive subjects. All measurements were performed by high performance liquid chromatography with electrochemical detection. Overnight excretion of norepinephrine in the tumor group (range, 86-1552 nmol/mmol creatinine) was significantly different (P <0.001) from that in the nontumor group (14-63 nmol/mmol creatinine). Autonomous secretion of norepinephrine was evident in all urine collections, including a patient with a predominantly epinephrine-secreting tumor. Overnight normetanephrine levels displayed a similar excretion pattern (P < 0.001), whereas overnight epinephrine and metanephrine levels were normal in 10 of the 16 patients with pheochromocytoma. In contrast, HMMA excretion in overnight urine collections was highly variable, with only 6 of the 16 patients in the tumor group having consistently elevated excretion. In the other 10 patients, overnight HMMA excretion showed a high intravariability. The measurement of catecholamines and total metanephrines after sleep is a viable approach for the exclusion of pheochromocytoma, as overnight urine collections completely differentiated patients with pheochromocytoma from hypertensive patients. Compared to 24-h results, overnight urinary norepinephrine levels provided a better diagnostic sensitivity and specificity (100% sensitivity and 98% specificity compared with 88% and 82%). Sleep urine samples simplify the collection protocol while avoiding the effects of stress and exercise. PMID- 8636338 TI - Effect of age on the intestinal absorption of vitamin D3-palmitate and nonesterified vitamin D2 in the term human infant. AB - This study was undertaken to investigate the utility of vitamin D3-palmitate as a nutritional supplement and thus define the intestinal absorption profile of vitamin D2 and vitamin D3 liberated after its cleavage from vitamin D3-palmitate in the human infant at various postnatal ages. The subjects for study consisted of 48 normal infants that were simultaneously administered 0.07 and 0.08 micromol/kg BW vitamin D as vitamin D3-palmitate and nonesterified vitamin D2 respectively, by orogastric tube. Blood samples were obtained before and 6, 12, and 24 h postadministration and analyzed simultaneously for vitamins D2 and D3. For data analysis, the infants were divided into two groups based on postnatal age: group 1, 1 day of age; and group 2, more than 10 days of age. Data were analyzed using the integrated peak area under the absorption curve for each subject. All subjects demonstrated the ability to absorb vitamin D after oral administration, although postnatal age as well as vitamin form had a profound effect on the absorption of vitamin D2 and vitamin D3 liberated from vitamin D3 palmitate. Nonesterified vitamin D2 is well absorbed both in very young and older infants, although absorption efficiency increases with age, perhaps due to increased bile acid secretion. Liberation of vitamin D3 from vitamin D3-palmitate was shown to increase, perhaps due to gastrointestinal tract maturation, beyond 10 days of age, probably coinciding with the secretion of intestinal esterases. Our data indicate that both forms of the orally administered vitamin approach equivalency in their abilities to elevate circulating vitamin D levels in the human infant at a postnatal age of approximately 89 days. Thus, vitamin D3 palmitate would appear not to be dietarily equivalent to free vitamin D as a nutritional source of vitamin D in the human neonate. PMID- 8636339 TI - Recombinant synthesis of insulin-like growth factor-binding protein-4 (IGFBP-4): Development, validation, and application of a radioimmunoassay for IGFBP-4 in human serum and other biological fluids. AB - Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione derivatized resin. Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging. PMID- 8636340 TI - Dose-dependent inhibition of growth hormone (GH)-releasing hormone-induced GH release by corticotropin-releasing hormone in prepubertal children. AB - Previous studies have shown that CRH is capable of inhibiting GH release in response to GHRH in adult subjects, and this effect appeared to be sex dependent and more pronounced in women than in men. To assess whether CRH has an inhibitory action on GH release in children also, the effects of graded doses of CRH on the GHRH-induced GH secretion were studied in three groups of prepubertal children. All subjects underwent a GHRH test (1 microgram/kg), followed, on separate occasions, by the combined administration of GHRH (1 microgram/kg) and CRH (1 microgram/kg, group A, n = 6; 1.5 microgram/kg, group B, n = 6; 2 microgram/kg, group C, n = 7). GH concentrations in response to the single GHRH injection were comparable in the three groups. The combined administration of GHRH and CRH resulted in serum GH concentrations similar to those obtained in the same subjects in response to GHRH alone when 1 and 1.5 microgram/kg CRH were given. In contrast, the administration of 2 microgram/kg CRH together with GHRH led to an increase in GH concentrations significantly lower than those after the GHRH injection alone (GH area under the curve, 1022.18 +/- 106.26 vs. 3109.16 +/- 794.29 microgram/Lx24 h; P < 0.05). No differences in the GH response to GHRH alone or to GHRH plus CRH were detected between male and female subjects. The results of the present study indicate that CRH is capable of inhibiting GHRH induced GH release in children. Moreover, the inhibitory effect by CRH appears to be dose dependent and not sex related. PMID- 8636341 TI - Measurement of dimeric inhibin B throughout the human menstrual cycle. AB - This report describes the development of a specific and sensitive assay for inhibin B and its application to the measurement of inhibin B concentrations in plasma during the human menstrual cycle. A monoclonal antibody raised against a synthetic peptide from the betaB-subunit was combined with an antibody to an inhibin alpha-subunit sequence in a double antibody enzyme-linked immunosorbent assay format. The validated assay had a limit of detection of 10 pg/mL and 0.5% cross-reactivity with inhibin A. Using this immunoassay, we found that the plasma concentration of inhibin B rose rapidly in the early follicular phase to a peak of 85.2 +/- 9.6 pg/mL on the day after the intercycle FSH rise, then fell progressively during the remainder of the follicular phase. Two days after the midcycle LH peak, there was a short lived peak in the inhibin B concentration (133.6 +/- 31.2 pg/mL), which then fell to a low concentration (<20 pg/mL) for the remainder of the luteal phase. In contrast, the inhibin A concentration was low in the early follicular phase, rose at ovulation, and was maximal during the midluteal phase. The concentration of inhibin B in individual follicular fluid samples was 20- to 200-fold higher than the concentration of inhibin A and was highest in follicular fluid samples from the early follicular phase. Inhibin B appears to be the predominant form of inhibin in the preovulatory follicle. The different patterns of circulating inhibin B and inhibin A concentrations observed during the human menstrual cycle suggest that these forms may have different physiological roles. PMID- 8636342 TI - Adrenocorticotropin causes vasodilatation in the human fetal-placental circulation. AB - During human pregnancy, ACTH is produced by both the placenta and fetal pituitary. ACTH has been shown to cause vasodilatation in the adrenal cortex in vitro. In this context we have investigated the vasoactive effects of ACTH in the human fetal-placental circulation. Single lobules of term human placentas were bilaterally perfused in vitro with Krebs solution (maternal and fetal, 5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3), and changes in fetal placental arterial perfusion pressure (FAP) were measured. ACTH (40-4000 pmol/L; n = 5) caused a dose dependent reduction of both KC1 and PGF2alpha-induced increases in FAP in the fetal placental circulation. The reductions were of a similar magnitude in the presence of either constrictor agent. ACTH was 187.4 (95% confidence limits, 162.7-215.9) times more potent than prostacyclin (PGI2; 1.2-1180 nmol/L; n = 6), which is a known vasodilator of the fetal-placental circulation. The threshold concentrations for ACTH and PGI2 were 40 pmol/L and 1.2 nmol/L, respectively. ACTH-induced reductions in PGF2alpha-induced increases in FAP in the fetal placental circulation were not inhibited by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (100 micromol/L; n = 5), the cyclooxygenase inhibitor indomethacin (3 micromol/L; n = 5), or a guanylate cyclase inhibitor LY83583(1 micromol/L; n = 5). The inhibitory effect of ACTH was attenuated by the antagonist, ACTH-(7-38) (240 pmol/L; n = 4), and a polyclonal ACTH antiserum (1:1000 dilution; n = 4). We have demonstrated that ACTH causes a reduction in fetal placental vascular resistance in the human fetal-placental circulation in vitro. The mechanism by which it exerts these effects has not been defined, but neither nitric oxide nor PG-mediated pathways appear to be involved. PMID- 8636343 TI - Atrial natriuretic peptide and plasma renin levels in assessment of mineralocorticoid replacement in Addison's disease. AB - Assessment of mineralocorticoid replacement therapy in Addison's disease relies on clinical features and laboratory measurements, including plasma renin and potassium. Previous studies have questioned the value of measuring the plasma renin concentration (PRC), particularly in the setting of fludrocortisone overreplacement. The aim of this study was to evaluate the usefulness of plasma atrial natriuretic peptide (ANP) measurements as a marker of sodium and volume status in Addison's disease. Fourteen patients with Addison's disease receiving their usual glucocorticoid doses were placed on various doses of fludrocortisone (FC; 0 mg, 0.05 mg, 0.1 mg and 0.2 mg) in random order for four 2-week periods. At the end of each period, blood pressure and clinical symptoms were assessed, and blood was drawn for measurement of PRC and ANP levels. PRC was significantly elevated in patients receiving placebo (54.2 +/- 57.9 ng/mL x h) compared with PRC in those receiving baseline FC (24.7 +/- 42.4 ng/mL x h), 0.1 mg FC (15.2 +/- 25.9 ng/mL x h), and 0.2 mg FC (5.5 +/- 5.7 ng/mL x h). ANP levels were measured by either an extraction method (ANP(ext)) or directly from plasma (ANP(dir)). ANP(dir) was significantly elevated at 0.2 mg FC (87.1 +/- 20.1 pg/mL) compared with baseline (63.3 +/- 8.1 pg/mL), placebo (56.1 +/- 5.5 pg/mL), 0.05 mg FC (60.5 +/- 16.0 pg/mL), and 0.1 mg FC (65.4 +/- 13.7 pg/mL) values. ANP(ext) was elevated in patients receiving 0.2 mg FC (42.7 +/- 41.8 pg/mL) compared with that in patients receiving placebo (7.9 +/- 5.4 pg/mL), 0.05 mg FC (16.2 +/- 11.2 pg/mL), or 0.1 mg FC (19.7 +/- 11.1 pg/mL). Our data suggest that PRC is of value in determining mineralocorticoid underreplacement, whereas ANP is a more sensitive index of FC overreplacement. ANP levels may, therefore, be complementary to PRC in adjustment of mineralocorticoid doses in the upper dose range, where clinical symptoms and signs appear to be of little value. PMID- 8636344 TI - Calorigenic effects of growth hormone: the role of thyroid hormones. AB - GH administration increases energy expenditure, independent of changes in lean body mass, in healthy, obese, and GH-deficient subjects. This may be causally linked to the well known GH-induced increase in peripheral T4 to T3 generation, but experimental data are sparse. In this study we have addressed whether 1) the calorigenic effects of GH administration could be reproduced by oral supplementation of T3 in a dose selected to mimic the GH-induced increase in peripheral T3 levels; and 2) combined GH and T3 administration have a synergistic effect on resting energy expenditure (REE). Eight normal male subjects (aged 21 27 yr; body mass index, 21.11-27.17 kg/m2) were randomly studied during four 10 day treatment periods with 1) daily sc placebo injections and placebo tablets, 2) daily sc GH injections (0.1 IU/kg x day) and placebo tablets, 3) daily T3 administration (40 microg on even dates, 20 microg on uneven dates) plus placebo injections, and 4) daily GH injections plus T3 administration. GH administration increased both free T3 (FT3) levels [mean +/- SE, 6.2 +/- 0.3 (control) vs. 7.3 +/- 0.5 (GH) pmol/L; P < 0.05] and REE [mean +/- SE, 1959 +/- 67 (control) vs. 2164 +/- 55 (GH) Cal/24 h; P < 0.01]. T3 administration yielded comparable levels of FT3 (7.7 +/- 0.5 pmol/L; T3 vs. GH, P = 0.37), but did not increase REE (2015 +/- 48 Cal/24 h; T3 vs. control, P = 0.23). Combined GH and T3 administration increased REE to a level higher than that seen with T3 alone (2279 +/- 68 Cal/24 h; T3 vs. GH plus T3, P < 0.01). Significant increments in serum levels of insulin-like growth factor I and insulin were recorded with GH administration, but not with T3 alone. Resting heart rate increased to a similar degree after GH administration and T3 supplementation, respectively. Tympanic temperature remained unaltered in all four studies. The results suggest that the calorigenic effect of GH is not mediated solely through increased conversion of T4 to T3. PMID- 8636345 TI - Novel mutations in the estrogen receptor messenger RNA in human breast cancers. AB - One mechanism that has been suggested to play a role in the progression of human breast cancer from hormone dependence to independence is the expression or altered expression of mutant and/or variant forms of estrogen receptor (ER). Two major types of variant ER messenger (m)RNA have been identified in human breast biopsy samples so far: truncated transcripts and exon deleted transcripts. In this study we provide data indicating the existence of a novel type of abnormal ER mRNA. These transcripts were identified as larger than wild-type ER mRNA RT PCR products in 9.4% of 212 human breast tumors analyzed. The data suggest nucleotide insertions are present in ER mRNA of some breast tumors. Cloning and sequencing of the larger RT-PCR products showed three different types: a complete duplication of exon 6 occurring in 7.5% of tumors; a complete duplication of both exons 3 and 4 occurring in 1 tumor; and a 69 nucleotide insertion between exons 5 and 6 occurring in 3 tumors. Open reading frame analysis suggested that exon 6 duplicated transcripts encoded a 51.4 kDa ER-like protein truncated just after exon 6 sequences; the exon 3 and 4 duplicated transcript encoded a 83.3 kDa protein containing duplication of ER amino acid residues encoded by exons 3 and 4; the 69 nucleotide insertion was inframe, adding 23 novel amino acid residues between residues 412 and 413 of the normal ER protein to produce a 68.8 kDa protein. It is unknown if these novel ER-like mRNAs are stably translated in vivo. Any resulting protein would be structurally altered, however, possibly resulting in altered function. PMID- 8636346 TI - Expression of endothelin-1 gene and protein in human granulosa cells. AB - Previous studies in animal models indicated an autocrine/paracrine action of endothelin-1 (ET-1) in the ovary. We now report evidence on the presence of ET-1 in human ovary during reproductive life. Immunohistochemical and in situ hybridization studies demonstrated a positive signal into cytoplasm of granulosa cells (GC) of follicles at different growth stages. The concentration of ET-1 like immunoreactivity (ET-1-LI) was also measured by a specific RIA in human follicular fluid (FF). FF samples were obtained from women in an in vitro fertilization program undergoing gonadotropin stimulation (group A; n = 24) or no treatment (group B; n = 7). The mean (+/-SD) ET-1-LI FF level in group A (4.85 +/ 2.06 pg/mL) was significantly higher than that in group B (1.29 +/- 0.43 pg/mL; P < 0.01), whereas the corresponding mean plasma levels were not significantly different and were not correlated to respective FF values. Our results indicate for the first time the presence of ET-1 and its messenger ribonucleic acid in the GC of the human ovary. The higher ET-1-LI levels found in the FF from women undergoing gonadotropin treatment suggest a modulation by gonadotropins and/or ovarian steroids of ET-1 production by GC. PMID- 8636347 TI - A two-site monoclonal antibody immunoradiometric assay for human follistatin: secretion by a human ovarian teratocarcinoma-derived cell line (PA-1). AB - The follistatin/activin/inhibin system increasingly appears to have important growth and differentiating effects in a variety of cell types, including cancer. We have developed a two-site immunoradiometric assay for measurement of human follistatin using two monoclonal antibodies against recombinant human follistatin. This cloned protein donor assay is sensitive (0.5 ng/mL), specific for free human follistatin, and precise (<5% within assay coefficient of variation). Using this assay, native human follistatin could be measured in human pituitary extracts, follicular fluid, and granulosa-luteal cell-conditioned medium. To identify and characterize human follistatin secreted by ovarian cancer cells, we screened five human ovarian carcinoma cell lines currently available from the American Type Culture Collection (Rockville, MD). One of these, a cell line derived from a teratocarcinoma (designated PA-1, American Type Culture Collection, CRL1572), secreted large (3 microg/10(6) cells per 24 h) quantities of immunoreactive follistatin constituitively. Increasing volumes of conditioned medium from these cultured cells generated response curves parallel to those of recombinant human follistatin 288 reference protein, human follicular fluid, or culture medium from human granulosa-luteal cells. Secretion of follistatin by PA 1 cells was time and cell-number dependent with 297.9 +/- 15.2, 654 +/- 29.8, and 940 +/- 49.1 ng follistatin secreted over 24 h by 1 x 10(5), 2 x 10(5), and 3 x 10(5) cells, respectively. Western and ligand blot analysis revealed that the immunoreactive follistatin secreted by PA-1 cells and isolated by sulfate cellufine chromatography was identical to the molecular weight variants (32,000 and 35,000 Mr) of recombinant human follistatin 288. PA-1 cell-conditioned medium suppressed basal secretion of FSH by cultured rat anterior pituitary cells in a dose-dependent fashion. This follistatin bioactivity was completely removed by adsorption with either solid-phase monoclonal antifollistatin or a dextran sulfate chromatography gel. Because activin suppressed the proliferation of PA-1 cells, secretion of bioactive follistatin may represent an autocrine mechanism opposing activin to maintain the rapid growth rate of PA-1 cells. These observations demonstrate that the ovarian teratocarcinoma cell line, PA-1, secretes considerable amounts of human follistatin that is biologically active, capable of binding human activin, and antigenically similar to recombinant human follistatin 288. The monoclonal antibodies and two-site assay reported herein should be useful in assessing the regulation of follistatin secretion and as a diagnostic tool, especially if follistatin measurements prove to be a marker for some ovarian cancers. PMID- 8636349 TI - Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. AB - Human adrenomedullin (hAM), a potent vasodilatory peptide originally identified in pheochromocytoma, has been shown to be present in various human tissues and circulate in human plasma. We measured plasma concentrations of immunoreactive hAM in patients with sepsis who had been admitted to intensive care unit (ICU). Plasma hAM concentrations in 12 septic patients upon entering the ICU were extremely elevated (107 +/- 139 fmol/ml: mean +/- SD) compared to those of 16 age matched normal subjects (7.9 +/- 3 fmol/mL). Among 10 patients with normal renal function, plasma hAM levels either decreased or increased during the hospital course; the former group survived and the latter group succumbed. Two patients with acute renal failure had markedly elevated plasma hAM levels during the early course, which declined rapidly during the recovery course. High performance liquid chromatography of plasma extracts from one patient with acute renal failure revealed a single major component of immunoreactive hAM coeluting with authentic hAM (1-52) during acute and recovery phase. Plasma hAM concentration showed positive correlations with heart rate, right atrial pressure, and serum creatinine concentration, but not with other hemodynamic variables. These data suggest that a marked increase in circulating hAM in sepsis may be caused by its decreased clearance and/or its enhanced synthesis by multiple organ dysfunction, and that increased endogenous hAM may be involved in the mechanism of cardiovascular abnormalities associated with sepsis. PMID- 8636348 TI - Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency. AB - The hereditary syndrome of unresponsiveness to ACTH is a rare autosomal recessive disorder characterized by low levels of serum cortisol and high levels of plasma ACTH. There is no cortisol response to exogenous ACTH. Recent cloning of the human ACTH receptor gene has enabled us to study this gene in patients with glucocorticoid deficiency. By using the PCR to amplify the coding sequence of the ACTH receptor gene, we identified three mutations in two unrelated patients. One mutation present in homozygous form converted the negatively charged Asp107, located in the third transmembrane domain, to an uncharged Asn residue. The second patient was a compound heterozygote: the paternal allele contained a one nucleotide insertion leading to a stop codon within the third extracellular loop, and the maternal allele contained a point mutation converting Cys251 to Phe, also in the third extracellular loop. Normal and mutant ACTH receptor genes were expressed in the M3 cell line, and intracellular cAMP production in response to ACTH was measured. For the mutant receptors, no response to physiological ACTH concentrations was detected, suggesting an impaired binding of ACTH to the receptors and/or an altered coupling to the adenylate cyclase effector. PMID- 8636350 TI - Role of progesterone and nonsteroidal ovarian factors in regulating gonadotropin releasing hormone self-priming in vitro. AB - We investigated the effects of gonadotropin surge-attenuating factor (GnSAF), inhibin, and follistatin on GnRH self-priming and its augmentation by progesterone. Two GnRH challenges, 60 min apart, were administered to rat pituitary monolayers after 90-min exposure to medium alone (control), progesterone, GnSAF, inhibin, or follistatin. Inhibin-stripped follicular fluid from superovulated women was used as a source of GnSAF bioactivity. Under control conditions, the greater response to the second GnRH challenge (peak 2, 9.2 +/- 2.1; peak 1, 4.4 +/- 0.9 ng LH/mL; P < 0.01) demonstrated GnRH self-priming. None of the treatments significantly altered the first LH peak. Progesterone markedly increased GnRH self-priming (peak 2, 12.6 +/- 2.5 ng LH/mL; P < 0.01). However, GnSAF and RU486 significantly reduced GnRH self-priming (peak 2, 4.6 +/- 0.9 and 5.6 +/- 1.6 ng LH/mL, respectively; P < 0.01). The augmentation of self-priming induced by progesterone was completely abolished by coincubation with either GnSAF or RU486 (peak 2, 7.5 +/- 1.6 and 4.3 +/- 0.9 ng LH/mL, respectively; P < 0.01). Neither inhibin nor follistatin had any effect on GnRH self-priming or its augmentation by progesterone. The actions of RU486 in the presence and absence of progesterone demonstrate a nonprogestagenic effect of RU486 on the gonadotropes. In conclusion, the suppression of GnRH self-priming, with or without progesterone augmentation, supports the hypothesis that GnSAF acts by maintaining the pituitary in an unprimed state of reduced responsiveness to GnRH. PMID- 8636351 TI - Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. AB - This report concerns the evaluation of various estrogens, estrone (El), estradiol (E2), and estrone sulfate (E1S), as well as E1S-sulfatase and aromatase activities in pre- and postmenopausal women with breast cancer. The levels (in picomoles per g; mean +/- SEM) of the various estrogens in the breast tissue from premenopausal patients (n = 11) are: El, 1.4 +/- 0.5; E2, 1.2 +/- 0.6; and E1S, 1.2 +/- 0.3. In postmenopausal patients (n = 23), the values are, respectively, 1.0 +/- 0.4, 1.4 +/- 0.7, and 3.3 +/- 1.9. These concentrations of estrogens in the tumors of postmenopausal patients are significantly higher than those found in plasma. The activity of E1S-sulfatase in both pre- and postmenopausal patients was 50-200 times higher than that of aromatase. E1S-sulfatase and aromatase activities are significantly higher in post-menopausal than in cycling patients. It is concluded that despite the low levels of circulating estrogens in postmenopausal patients, the tissue concentrations of these steroids are several fold higher than those in plasma, suggesting tumor accumulation of these estrogens. The physiopathology and clinical significance of these high levels of the various estrogens (E1, E2, and E1S) as well as sulfatase and aromatase activities in postmenopausal patients with breast cancer is yet to be explored. PMID- 8636352 TI - The influence of polydipsia on water excretion in hyponatremic, polydipsic, schizophrenic patients. AB - To determine whether polydipsia is responsible for the altered water excretion in the subset of polydipsic schizophrenic patients who develop hyponatremia, the regulation of antidiuretic function was assessed in polydipsic schizophrenic patients with hyponatremia (n = 5), polydipsic schizophrenic patients without hyponatremia (n = 5), nonpolydipsic schizophrenic patients (n = 6), and normal controls (n = 8). The severity and duration of polyuria were similar in the two polydipsic groups. After oral water loading, maximal free water clearance was similar across all four groups. Free water clearance diminished, however, at lower plasma osmolalities in the hyponatremic polydipsics (P < 0.02) and at higher plasma osmolalities in the normonatremic polydipsics (P < 0.05) relative to that in the nonpolydipsic schizophrenics and normal subjects. The increase in plasma vasopressin after osmotic stimulation with hypertonic saline was slightly, but significantly (P < 0.02), blunted in both polydipsic groups. Hyponatremia occurs in some polydipsic schizophrenics because the relationship between free water clearance to plasma osmolality/sodium is shifted to the left. Polydipsia per se is not responsible for this still unexplained shift. PMID- 8636353 TI - Measurement of circulating inhibin forms during the establishment of pregnancy. AB - This study investigated the forms of inhibin released into the circulation 1) in very early pregnancy, 2) after stimulation of the corpus luteum by exogenous hCG, and 3) in abnormal and failing human pregnancy. Samples were assayed by enzyme linked immunosorbent assays for inhibin A, inhibin B, and inhibin pro-alphaC related immunoreactivity (pro-alphaC-RI). The concentration of inhibin A rose steadily during the conception luteal phase to an initial peak 12 days after ovulation (104 +/- 23 pg/mL), then rose rapidly to a further peak 43 days after ovulation 424 +/- 6 pg/mL). The concentration of pro-alphaC-RI exhibited a much larger peak on day 15 after ovulation (1423 +/- 361 pg/mL), but fell thereafter. The concentration of inhibin B was low after ovulation and subsequently barely detectable in pregnancy. hCG treatment resulted in a significant rise in the concentrations of inhibin A and pro-alphaC-RI, but had no effect on the inhibin B concentration. The pro-alphaC-RI concentration was a better indicator of continuing pregnancy viability than either hCG or inhibin A. Early trophoblast secretes proportionately more bioactive inhibin than the corpus luteum. The corpus luteum and trophoblast do not secrete inhibin B into the circulation. These data support the concept of different physiological roles for different inhibin forms. PMID- 8636354 TI - Recombinant human growth hormone improves growth in children receiving glucocorticoid treatment after liver transplantation. AB - Linear growth is often impaired after successful liver transplantation. The cause is multifactorial; poor graft function and long term glucocorticoid treatment are the main factors responsible. The efficacy and safety of recombinant human GH (rhGH) treatment were assessed in eight growth-retarded children (five boys and three girls) with liver transplants. Immunosuppression comprised azathioprine, cyclosporin, and methylprednisolone. rhGH was administered in a dose of 1 IU/kg x week, given by daily sc injections. The median age at the start of treatment was 9.7 yr (range, 5.9-14.9 yr). All but one of the patients remained prepubertal during treatment. The median growth rate increased from 3.2 to 7.l cm/yr (P = 0.025) and height SD score increased from -3.9 to -3.1 (P = 0.036) during the first year of rhGH treatment. Serum insulin-like growth factor I and insulin-like growth factor-binding protein-3 levels increased significantly during treatment. Graft function was normal in all except one patient, and no rejections or other serious side-effects were documented. In conclusion, rhGH treatment is effective in short, non-GH-deficient, liver-transplanted children receiving long term glucocorticoid treatment. Due to potential risk of allograft rejection, close monitoring of liver function and immunosuppression is required. PMID- 8636355 TI - Gender differences in the temporal organization of proclactin (PRL) secretion: evidence for a sleep-independent circadian rhythm of circulating PRL levels- a clinical research center study. AB - Although a nocturnal rise in PRL secretion is well known, it has long been presumed to be evoked by sleep. To determine whether PRL secretion was driven by a sleep-independent circadian rhythm, we studied 12 men and 10 women using a constant routine protocol. Under the constant routine conditions of continuous semirecumbent wakefulness in constant indoor room light with hourly meals distributed throughout the day and night, a persistent circadian rhythm of PRL secretion was present in men and in women at the follicular and luteal phases of the menstrual cycle. Furthermore, the amplitude of this rhythm in women was significantly greater than that in men. The present data demonstrate the presence of a robust sleep-independent endogenous circadian rhythm of PRL secretion in humans. We hypothesize that this endogenous component of the circadian rhythm of PRL secretion together with those of body temperature, urine production, and cortisol, TSH, and melatonin secretion are driven by the central circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus. PMID- 8636356 TI - Antibodies to glutamic acid decarboxylase and insulin-dependent diabetes in patients with autoimmune polyendocrine syndrome type I. AB - To evaluate the association of autoimmunity to glutamic acid decarboxylase (GAD) with insulin-dependent diabetes mellitus (IDDM) and IDDM-associated human leukocyte antigen (HLA) types, we studied a unique group of 47 patients with autoimmune polyendocrine syndrome type 1, a recessive disease not associated with HLA. GAD65 antibodies (GAD65-Ab), GAD67-Ab, islet cell antibodies, and HLA-DQA1, DQB1, and -DRB1 were analyzed in relation to IDDM or a decreased insulin secretory capacity. GAD65-Ab were found in six of the eight diabetic patients 0.9 8.0 yr before the onset of IDDM and in 16 (41%) nondiabetic patients during a follow-up of 2.4-19.5 yr. Eleven (28%) nondiabetic patients had GAD67-Ab and islet cell antibodies. Fasting C peptide (mean +/- SD, 0.5 +/- 0.24 vs. 1.03 +/- 0.49 nmol/L; P = 0.003) and first phase insulin response (75.6 +/- 37.9 vs. 166.4 +/- 112.7 mU/L; P = 0.019) were lower in patients with than in those without GAD65-Ab. No HLA genotype predominated in the IDDM patients or GAD65-Ab-positive nondiabetic patients, but the IDDM high risk genotypes were decreased in frequency among the patients with GAD65-Ab. In conclusion, nondiabetic autoimmune polyendocrine syndrome type 1 patients frequently have GAD65-Ab together with a decreased insulin secretory capacity, suggesting subclinical islet cell inflammation not invariably progressing to diabetes. This is not associated with HLA haplotypes conferring susceptibility to or protection from IDDM. PMID- 8636357 TI - Characterization of reproductive hormonal dynamics in the perimenopause. AB - Medical therapy for women in the perimenopausal period is controversial, in part due to varying degrees of ovarian hormone secretion characteristic of this time of life. To extend our understanding of the reproductive endocrine milieu of perimenopausal women, we studied 6 cycling women, aged 47 yr and older, for 6 months with daily collections of first morning voided urine. Five additional older reproductive aged (43-47 yr old) women were studied with daily urine and serum sampling for a single menstrual cycle; their urinary hormone data were combined with the former group for menstrual cycle comparisons. Urine was assayed for LH, FSH, estrone conjugates, and pregnanediol glucuronide and normalized for creatinine (Cr). Eleven midreproductive aged (19-38 yr old) normally cycling women, 5 women with well defined premature ovarian failure, and 5 women aged 54 yr and older who were at least 1 yr postmenopausal were used for comparison. Perimenopausal women had shorter follicular phases (11 +/- 2 days vs. 14 +/- 1 days; P = 0.031) and, hence, shorter menstrual cycles than midreproductive aged controls. FSH excretion in perimenopausal women was greater than that in younger women (range of means, 4-32 vs 3-7 IU/g Cr; P = 0.0005). LH secretion was overall greater than that in younger normal subjects (range of means, 1.4-6.8 vs. 1.1-4.2 IU/g Cr; P < 0.026). Overall mean estrone conjugate excretion was greater in the perimenopausal women compared to that in the younger women [76.9 ng/mg Cr (range, 13.1-135) vs. 40.7 ng/mg Cr (range, 22.8-60.3); P = 0.023] and was similarly elevated in both follicular and luteal phases. Luteal phase pregnanediol excretion was diminished in the perimenopausal women compared to that in younger normal subjects (range for integrated pregnanediol, 1.0-8.4 vs. 1.6-12.7 microg/mg Cr/luteal phase; P = 0.015). Compared to postmenopausal women, perimenopausal women had more overall estrone excretion (2.5-6.2 ng/mg Cr in postmenopausal women; P = 0.02) and lower mean FSH (range of means for postmenopause, 24-85 IU/g Cr; P = 0.017) and LH (range for postmenopause, 4.3 14.8 IU/g Cr; P = 0.041). Compared to women with premature menopause, perimenopausal women again had lower FSH (range of means for premature menopause, 36-82 IU/g Cr; P = 0.0022), lower LH (range of means for premature menopause, 5.5 23.8 IU/g Cr; P = 0.0092), borderline higher mean estrone conjugates (range of means for premature menopause, 4-44 ng/mg Cr; P = 0.064), and far longer periods of ovarian activity (one to two cycles in prematurely menopausal women vs. three to six cycles in perimenopausal women). We conclude that altered ovarian function in the perimenopause can be observed as early as age 43 yr and include hyperestrogenism, hypergonadotropism, and decreased luteal phase progesterone excretion. These hormonal alterations may well be responsible for the increased gynecological morbidity that characterizes this period of life. PMID- 8636358 TI - Progesterone stimulates the induction of human endometrial CD56+ lymphocytes in an in vitro culture system. AB - An unusual population of large granular lymphocytes (LGL) with the surface phenotype CD56(bright+)/CD16-/CD3- increases in the human endometrium during the late secretory phase and early pregnancy. To identify the factor(s) that induces CD56+ LGL in the human endometrium, we isolated endometrial leukocytes from nonpregnant human endometrium and investigated changes in CD56+ cells during culture with various factors. Isolated endometrial leukocyte-rich fraction and peripheral blood mononuclear cells were cultured for 6 days in the presence of progesterone, estradiol, PRL, or hCG, then nonadherent cells were collected and examined immunocytochemically. Endometrial leukocyte-rich fractions were composed of leukocytes and endometrial stromal cells, and 53.2 +/- 5.8% of them expressed CD45 antigen before culture. Therefore, leukocytes and endometrial stromal cells were cocultured in these endometrial leukocyte-rich fraction cultures. The percentage of CD56+ cells in endometrial leukocyte-rich fractions cultured with progesterone was significantly higher than that in fractions without progesterone. On the other hand, estradiol, PRL, and hCG did not significantly induce CD56+ cells in endometrial leukocyte-rich fractions. There was no significant difference in the percentage of CD56+ cells between peripheral blood mononuclear cell cultures with and without progesterone. These findings suggest that progesterone is an important factor for the in situ proliferation or differentiation of CD56+ LGL in human endometrium. PMID- 8636359 TI - Comparison of the ability of bread versus bread plus meat to treat and prevent subsequent hypoglycemia in patients with insulin-dependent diabetes mellitus. AB - We sought to determine whether treatment of hypoglycemia with a snack containing both protein and carbohydrate results in more prolonged protection against subsequent hypoglycemia than ingestion of carbohydrate alone. We studied six insulin-dependent diabetic subjects on two occasions. On both occasions subjects received a variable overnight insulin infusion to achieve euglycemia followed by a constant insulin infusion (approximately 0.5 mU x kg(-1) x min(-1)) designed to produce hypoglycemia. When glucose reached 50 mg/dL, subjects were fed a snack consisting of either bread (approximately 85 kcal) or bread plus meat (approximately 205 kcal). Both contained 15 g of carbohydrate. The insulin infusion was continued for the next 3 h or until glucose again fell to 50 mg/dL. Although bread plus meat resulted in a more marked rise (P < 0.05) in glucagon than did bread alone, neither the post treatment peak glucose concentration (73 +/- 4 vs. 70 +/- 6 mg/dL) nor the subsequent rate of fall of glucose (0.42 +/- 0.10 us. -0.35 +/- 0.07 mg/dL/min) differed. The present study shows that the rate of redevelopment of hypoglycemia does not differ after eating bread or bread plus meat. Therefore treatment of hypoglycemia with a protein-enriched snack merely adds calories rather prolonged protection against subsequent hypoglycemia. PMID- 8636360 TI - Age effects of follicle-stimulating hormone and pulsatile luteinizing hormone secretion across the menstrual cycle of premenopausal women. AB - To characterize the differential aging response in gonadotropin secretion that occurs before menopause, we assessed pulsatile LH and serial FSH, estradiol (E2), and progesterone (P) concentrations in aging women across the menstrual cycle. We conducted 96 daytime studies during the follicular, midluteal, and late luteal phases of the same menstrual cycle in 32 volunteers, aged 40-50 yr (n = 16) and 19-39 yr (n = 16). Mean cycle length was shorter in the older women (26 +/- 0.4 vs. 27.6 +/- 0.6 days; P = 0.02), but mean plasma E2 and P values were similar in the two age groups. Mean plasma FSH was higher in the older group on all 3 study days. For LH, an age difference was observed during the late luteal phase, when mean plasma LH and pulse amplitude were higher in women over 40 yr of age (mean LH, 6.4 +/- 0.7 vs. 3.0 +/- 0.5 IU/L (P = 0.002); mean amplitude, 4.0 +/- 0.5 vs. 2.8 +/- 0.2 IU/L (P = 0.03)]. Pulse frequency was higher in the older group, but not different from that in younger women on all study days. When the subjects aged 35-39 yr were analyzed as a third age group (n = 8), age effects for mean LH persisted, and pulse frequency was higher in the group over 40 yr of age vs. women under age 35 yr (n = 8) in both the follicular phase (7.1 +/- 0.4 us. 5.6 +/- 0.8; P = 0.03) and late luteal phase (5.8 +/- 0.7 vs. 4.4 +/- 0.3; P = 0.03). Although highly variable, individual patterns of gonadotropin secretion in the women over age 40 yr included a sustained elevation in the FSH/LH ratio as well as a failure to demonstrate slow frequency, high amplitude LH pulses in the midluteal phase. In conclusion, 1) the age-related increase in FSH concentrations in ovulatory women, although more pronounced, is associated with phase-dependent enhancement of pulsatile LH secretion; 2) the higher LH concentrations are brought about by changes in both pulse frequency and amplitude; and 3) these age effects preempt overt reductions in cyclic E2 or P concentrations. PMID- 8636361 TI - Serum growth hormone-binding protein in obesity: effect of a short-term, very low calorie diet and diet-induced weight loss. AB - GH-binding protein (GHBP) is increased in obesity. It is not known whether the increase in GHBP is reversible with weight loss or modulated by acute changes in nutritional intake. To address these questions, we measured GHBP in 18 obese subjects [body mass index (BMI), 40.9 +/- 1.1 kg/m2 (mean +/-SEM)] before and after an average weight loss of 30.3 +/- 4.6 kg and in 18 age- and sex matched normal subjects (BMI, 23.0 +/- 0.4 kg/m2) and studied the effects of a very low calorie diet over 4 days in 5 normal subjects and a subgroup of obese subjects before (n = 6) and after (n = 5) weight loss. GHBP was elevated in the obese subjects compared to levels in age- and sex-matched normal controls (1.48 +/- 0.1 vs. 0.53 +/- 0.1 nmol/L; P < 0.0001). GHBP was positively correlated to BMI and waist circumference (r = 0.71; P < 0.00001 and r = 0.73; P < 0.00001, respectively). In addition, GHBP was positively correlated to insulin as well as proinsulin levels (r = 0.60; P < 0.001 and r = 0.55; P < 0.001, respectively). After diet-induced massive weight loss, GHBP levels were restored to normal in obese subjects (BMI, 27.8 +/- 1.4 kg/m2). Multiple stepwise regression analysis revealed that changes in waist circumference and abdominal sagittal diameter during weight loss were the major determinants of and accounted for 54% of the fall in GHBP levels. Neither insulin nor proinsulin was an independent predictor. No changes were observed in GHBP in normal, obese, or reduced weight obese subjects after 4 days of a very low calorie diet, although mean insulin levels fell significantly in the normal subgroup as well as in the obese subgroup studied after weight loss. In summary, GHBP levels are elevated in obesity, are restored to normal by massive weight loss, and are unaffected by short term hypocaloric feeding. We conclude that GHBP may be regulated by the same or closely related factors that regulate fat mass and abdominal fat mass in particular, but not by insulin or acute changes in nutrition. PMID- 8636362 TI - Factors influencing the development of melatonin rhythmicity in humans. AB - The emergence of melatonin rhythmicity was studied in 163 infants between 46-55 weeks postconception by monitoring the excretion of the urinary melatonin metabolite 6-sulfatoxymelatonin (aMT.6S). From this population, we examined the effects of gender, season, multiple birth, home birth, previous sudden infant death syndrome in the family, premature labor, spontaneous rupture of membranes, preeclampsia, intrauterine growth restriction, and nursery lighting on pineal rhythmicity. As previously reported, rhythmic excretion of aMT.6S appeared between 49-55 weeks postconception (9-15 weeks of age) in singleton babies born at term in the hospital. Full-term infants who had a sibling die of sudden infant death syndrome had a pattern of melatonin rhythm development no different from that of the control full-term infants. In contrast, full-term infants born at home and full-term twins born in the hospital had significantly lower aMT.6S excretion than hospital-born singleton infants at the same ages despite similar body weights (e.g. at 52 weeks postconception; 1.8 +/- 0.4, 1.1 +/- 0.3, and 3.6 +/ -0.5 nmol/day, respectively). In full-term infants, there was no difference in the development of melatonin rhythmicity between the sexes, with season or method of delivery (vaginal vs. caesarean). The premature infants were divided into 5 groups (babies born after premature labor, premature rupture of membranes, preeclampsia, intrauterine growth restriction, and fetal distress). All premature infants had a delay in the appearance of aMT.6S rhythms in the urine in relation to chronological age. When the infants were compared on the basis of weeks since conception, those infants born after spontaneous premature labor excreted amounts of aMT.6S no different from those of full-term singleton infants during the period of study. In contrast, the premature rupture of membranes, preeclampsia, and fetal distressed infants excreted 50% less aMT.6S, and intrauterine growth restricted infants excreted 67% less at the same postconceptional ages. These differences were due to reduced nocturnal excretion of the metabolite. In an attempt to accelerate the development of melatonin rhythmicity, premature labor and premature rupture of membranes infants were randomly assigned to be totally deprived of light (using phototherapy eye shields) or partially deprived of light by moving them to a dimly lit room each night for the last 3-8 weeks of their stay in the hospital nursery. Babies born after premature labor produced normal amounts of aMT.6S between 46-52 weeks postconception, and this pattern was not affected by the nocturnal light deprivation. Infants born after premature rupture of membranes and totally deprived of light at night had aMT.6S excretion rhythms at 52 weeks postconception no different from those of full-term hospital-born infants or premature labor infants, whereas those in infants placed in dim light were similar to those in untreated premature rupture of membranes infants. These results suggest that premature birth alone is not the sole cause of altered rhythm development; other factors, such as preeclampsia, growth restriction, and nursery lighting, play an important role. The consequences of the delayed appearance of melatonin in infants are not known, but deserve further study. PMID- 8636363 TI - Expression of beta 1 integrins in human endometrial stromal and decidual cells. AB - The present study was undertaken to investigate the expression of beta1 integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the beta 1, alpha 1, alpha 2, and alpha 5 subunits of the beta1 integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of beta1 integrins in vitro. The immunohistochemical distribution of beta1 integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and mesenchymal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of the beta 1, alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of beta 1 integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of beta 1, integrins expression. Immunohistochemistry confirmed the beta 1 integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. Autoradiography of immunoprecipitate subjects to SDS-PAGE revealed three major polypeptides with molecular weights of 130 kDa (beta 1 subunit), 165 kDa (alpha 2 subunit), and 210 kDa (alpha 1 subunit) under reducing conditions. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed beta1 integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, beta 1 integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation. PMID- 8636364 TI - Entrainment of ultradian oscillations in the secretion of insulin and glucagon to the nonrapid eye movement/rapid eye movement sleep rhythm in humans. AB - The cause of ultradian oscillations in the secretion of glucagon and insulin with a period length between 70-140 min has been atttributed to feedback mechanisms of glucose and insulin. Influences of the central nervous system on these ultradian glucagon and insulin oscillations remained to be elucidated. In the present study on one occasion, concentrations of glucose, glucagon, insulin, and GH were determined at 15-min intervals from 2100-0700 h in 16 healthy subjects while they were infused with saline solution. On another occasion, concentrations of these hormones during nocturnal sleep were determined in 10 of these subjects while they were constantly infused with glucose (4.5 mg/kg x min). The order of the treatments (placebo vs. glucose) was balanced across subjects, and experiments were performed in a double blind manner. Significant glucagon and insulin peaks were determined by the peak detection algorithm Cluster. Sleep was recorded somnopolygraphically. During the infusion of saline solution, glucagon concentrations showed spontaneous oscillations, with a mean periodicity of 107.9 +/- 13.2 min. During the constant infusion of glucose, oscillations of similar periodicity (110.1 +/- 10.3 min) were observed for insulin. The phases of glucagon and insulin secretory activity on the respective nights were entrained to the nonrapid eye movement (non-REM)/REM sleep cycle. Significant increases in the concentrations of glucagon (chi 5.23; P < 0.02) and insulin (chi= 7.32; P < 0.01) generally fell into epochs of non-REM sleep, with a preference for the beginning of the epochs, whereas decreasing concentrations of these hormones coincided significantly with epochs of REM sleep (P < 0.05). The time spent in the different sleep stages was not altered during glucose infusion. In conclusion, ultradian oscillations of insulin and glucagon concentrations are modulated by central nervous system mechanisms entraining secretory pulses of the alpha- and beta-cells of the endocrine pancreas to the non-REM sleep epochs of the non-REM/REM sleep cycle. PMID- 8636365 TI - Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas. AB - Forty-four thyroid autonomously hyperfunctioning adenomas were analyzed to assess the frequency of mutations occurring in the TSH receptor (TSHR). PCR-amplified fragments encompassing the entire exon 10 of the TSHR gene were obtained from the genomic DNA extracted from the tumors and their adjacent normal tissues and were examined by direct nucleotide sequencing. Point mutations were found in 9 of the 44 adenomas examined (20%). One mutation occurred in codon 619 (Asp to Gly), four in codon 623 (three were Ala to Ser, one Ala to Val substitution), two in codon 632 (both Thr to Ile), and two in codon 633 (Asp to Tyr or His). All the alterations were located in a part of the gene coding for an area including the third intracellular loop and the sixth transmembrane domain of the TSH receptor. All mutations were somatic and heterozygotic, and none was simultaneous with alterations of ras or gsp oncogenes. Thus, our data show that in our series of 44 hyperfunctioning thyroid adenomas, a somatic mutation of the TSHR, responsible for the constitutive activation of the cAMP pathway, occurs in 20% of the tumors. PMID- 8636366 TI - Altered insulin receptor messenger ribonucleic acid splicing in liver is associated with deterioration of glucose tolerance in the spontaneously obese and diabetic rhesus monkey: analysis of controversy between monkey and human studies. AB - There are two insulin receptor (IR) isoforms (designated type A and type B), derived from alternative splicing of exon 11 of the IR gene. Recently, we reported (Huang Z., Bodkin N.L., Ortmeyer H.K., Hansen B.C., Shuldiner A. R., 1994, J Clin Invest, 94:1289-1296) that an increase in the exon 11- (i.e. lacking exon 11) (type A) IR messenger RNA (mRNA) variant in muscle is associated with hyperinsulinemia, an early risk factor for noninsulin-dependent diabetes mellitus (NIDDM), in the spontaneously obese, diabetic rhesus monkey. To explore further the role of IR mRNA splicing in insulin resistance of NIDDM, we studied liver, another target organ that is resistant to insulin action in NIDDM. The relative amounts of the two IR mRNA-splicing variants in liver were quantitated by RT-PCR in normal, prediabetic, and diabetic (NIDDM) monkeys. The percentage of the exon 11- mRNA variant in liver (n = 24) was significantly correlated with fasting plasma glucose (r = 0.55, P < 0.01) and intravenous glucose disappearance rate (r = -0.45, P < 0.05). The exon 11- mRNA variant was increased significantly from 29.8 +/- 1.6% in monkeys with normal fasting glucose to 39.2 +/- 2.9% in monkeys with elevated fasting glucose (P < 0.01). These studies provide the first direct evidence in vivo that the relative expression of the two IR mRNA-splicing variants is altered in liver and suggest that increased expression of the exon 11 IR isoform may contribute to hepatic insulin resistance and NIDDM or may compensate for some yet unidentified defect. PMID- 8636367 TI - The control of ovulation in mothers of dizygotic twins. AB - Dizygotic twinning is familial, suggesting that there may be an inherited abnormality of the control of ovulation that predisposes to double ovulation and, therefore, dizygotic twins. The present study examines 17 mothers of dizygotic twins (MODZT) and 8 control mothers of singletons by daily blood sampling throughout an entire menstrual cycle. Blood samples were assayed for LH, FSH, estradiol, progesterone, and inhibin. The process of follicular development was followed by transvaginal ultrasound. The pituitary LH response to iv GnRH was also assessed. Three of the 16 MODZT double ovulated during the study compared to none of the 8 control mothers (P < 0.05). The number of small follicles (<6 mm) declined significantly in control women at midcycle, but not in MODZT. There was no significant difference in serum FSH, LH, estradiol, or inhibin levels between the 2 groups at any stage of the menstrual cycle. During the follicular phase, serum progesterone levels were significantly higher in MODZT. The response to GnRH stimulation was not different between MODZT and controls. In conclusion, this study demonstrates an increased tendency to double ovulate in MODZT that may be due to a reduced rate of atresia in advanced follicles. Furthermore, the elevated progesterone levels in MODZT during the follicular phase suggest altered intrafollicular steroidogenesis that is independent of gonadotropins. PMID- 8636368 TI - Transient congenital hypothyroidism and hyperthyrotropinemia: normal thyroid function and physical development at the ages of 6-14 years. AB - Since the introduction of screening programs for congenital hypothyroidism, transient disturbances of thyroid function, especially transient congenital hypothyroidism and hyperthyrotropinemia, mostly due to iodine deficiency or contamination have been observed with variable frequencies. This study was carried out to reevaluate the thyroid function and physical development of 61 schoolchildren with transient congenital hypothyroidism or transient congenital hyperthyrotropinemia. Abnormalities were observed in 3 children. Thyroid function and growth were normal in all children, except 2 with moderately elevated TSH levels at the age of 7.7 and 10 yr in the presence of normal thyroid hormone levels. In 1 child, the TSH elevation was due to an ectopic hemithyroidea; in the other child, an unknown familial cause was suggested. In 1 girl (aged 12 yr), a euthyroid goiter caused by autoimmune thyroiditis was detected. We conclude from our investigation that frequent monitoring of thyroid function in children with transient congenital hypothyroidism or transient congenital hyperthyrotropinemia is not necessary during childhood if, postnatally, thyroid function recovered spontaneously. However, the growth and development of children with neonatal thyroid dysfunction should be followed, and if abnormalities occur, thyroid function tests are essential. PMID- 8636369 TI - Improvement of insulin sensitivity by metformin treatment does not lower blood pressure of nonobese insulin-resistant hypertensive patients with normal glucose tolerance. AB - Nine hypertensive patients with body mass indexes between 24-27 kg/m2 and normal glucose tolerance with at least a postchallenge plasma insulin level greater than 360 pmol/L were recruited for a double blind, cross-over study with metformin (850 mg, twice daily) and placebo. Each treatment lasted 1 month. Before and after each treatment, hormone and substrate concentrations were determined, blood pressure was monitored over 24 h, and insulin sensitivity was measured by a euglycemic (4.7 mmol/L) hyperinsulinemic (450 pmol/L) clamp study. Renal cation excretion and erythrocyte membrane cation heteroexchange were measured. Metformin, compared to placebo, did not affect body weight (70 +/- 7 vs. 70 +/- 7 kg), fasting plasma glucose (4.8 +/- 0.1 vs. 4.8 +/- 0.1 mmol/L), total cholesterol (5.38+/0.33 vs. 5.48 +/- 0.38 mmol/L), or triglycerides (1.73 +/- 0.72 vs. 1.91 0.89 mmol/L). Nevertheless, after metformin treatment, the plasma high density lipoprotein cholesterol concentration increased (1.42 +/- 0.18 vs. 1.34 0.16 mmol/L), and the plasma insulin level dropped (62 +/- 10 vs. 88+/- 12 pmol/L; both P < 0.05). Insulin-mediated glucose disposal was higher after metformin treatment (26.1 +/- 2.4 vs. 19.3 +/- 2.3 micromol/min x kg; P < 0.01), whereas hepatic glucose production was completely suppressed. These positive metformin-induced metabolic effects were not associated with a significant change in mean daily blood pressure levels (141 +/- 6/89 +/- 3 vs. 142 +/- 7/90 +/- 3 mm Hg). Compared to placebo, metformin increased the excretion of sodium, potassium, and lithium by enhancing their glomerular filtration rate. Na+/Li+ countertransport was not affected by metformin. However, the apparent affinity for H+ of Na+/H+ exchange was increased, and the Hill coefficient was decreased. In conclusion, 1 month of metformin administration to patients with essential hypertension and normal glucose tolerance 1) reduces the basal plasma insulin concentration, 2) improves whole body insulin-mediated glucose utilization, and 3) improves plasma high density lipoprotein cholesterol levels. Despite these positive effects, metformin did not reduce arterial blood pressure. PMID- 8636370 TI - The individual responsiveness to growth hormone (GH) treatment in GH-deficient adults is dependent on the level of GH-binding protein, body mass index, age, and gender. AB - The aim of the present trial was to study the individual responsiveness to GH treatment in terms of body composition and to search for possible predictors of the response in GH-deficient adults. Sixty-eight patients (44 men and 24 women) with a mean age of 44.3 (1.2) yr and verified GH deficiency participated in a 2 phase treatment trial with an initial randomized, double blind, placebo controlled, 6-month period, followed by an open treatment period, thereby ensuring all patients 12 months of GH treatment. Recombinant human GH was administered sc daily at bedtime, with a target dose of 12 micrograms/kg x day. GHBP was measured by ligand-mediated immunofunctional assay, and serum insulin like growth factor I (IGF-I) was determined by RIA after acid-ethanol extraction, using a truncated IGF-I analog as the radioligand. Lean body mass (LBM) and body fat (BF) were determined by dual energy x-ray absorptiometry, and total body water (TBW) was determined by bioelectrical impedance. During the placebo control period, serum IGF-I,LBM, and TBW increased (P < 0.001), whereas BF decreased (P < 0.001) and serum GHBP was unchanged in the group treated with GH compared with the patients treated with placebo. After 12 months of GH treatment, the individual changes in BF ranged from -12.5 to 4.3 kg and from -4.5 to 10.1 kg in LBM. Age (P < 0.05) and baseline GHBP level (P < 0.01) were inversely correlated with the increase in LBM. The GH-induced increment in IGF-I and TBW was greater in men than in women (P < 0.01), whereas the decreases in BF were similar in men and women. This trial demonstrates the variability in responsiveness to GH administration in GH-deficient adults. The best response to GH was obtained in younger patients with low GHBP levels. Furthermore, men responded better than women. PMID- 8636371 TI - Nandrolone, a 19-nortestosterone, enhances insulin-independent glucose uptake in normal men. AB - The effect of exogenous androgens on glucose metabolism is controversial. This study was designed to clarify the impact of testosterone enanthate (TE), an aromatizable androgen, and nandrolone decanoate (ND), a nonaromatizable androgen, on glucose disposal. Eleven healthy men were enrolled in a randomized, double blind cross-over study. All subjects completed two treatment cycles consisting of two weekly injections of placebo followed by six weekly injections of either TE (300 mg/week) or ND (300 mg/week). Treatment periods were separated by a 4-week washout. A tolbutamide-modified, frequently sampled, iv glucose tolerance test was used to assess insulin-dependent and insulin-independent glucose disposal. Data were analyzed using Bergman's minimal model. Parameters examined included acute insulin response to glucose, fasting insulin level, glucose disappearance constant, insulin sensitivity index, glucose effectiveness at basal insulin (SG), and glucose effectiveness at zero insulin (GEZI). Neither androgen adversely affected glucose disposal. To the contrary, treatment with ND actually improved noninsulin-mediated glucose disposal as expressed by SG and GEZI. In ND-treated men, SG (x 10(-2) min(-1)) rose from 2.4 +/- 0.2 at the end of the placebo period to 3.7 +/- 0.6 after treatment (P < 0.05), whereas GEZI (x 10(-2) min(-1)) increased from 1.8 +/- 0.2 to 3.1 +/- 0.6 (P < 0.01). We conclude that the treatment of normal men with supraphysiological doses of either TE or ND does not adversely affect glucose metabolism. Treatment with a nonaromatizable androgen, such as ND, actually improves glucose metabolism by enhancing noninsulin-mediated glucose disposal. PMID- 8636372 TI - Volumetric bone mineral density in normal subjects, aged 5-27 years. AB - Concerns have been raised regarding the validity of using areal bone mineral density (aBMD) as a substitute for the true volumetric bone mineral density (vBMD) in the pediatric population. We studied 209 normal subjects (109 males), aged 5-27 yr, to examine the influence of age, gender and growth on vBMD. The femoral neck, midthird of the femoral shaft, and the four lumbar vertebral bodies (L1-L4) were studied. Using data on bone width and height obtained by dual energy x-ray absorptiometry, bone volume was calculated with the assumption that all three sites are cylinders. In contrast to aBMD, vBMD of the femoral neck bore no relationship to age or weight in both sexes, but was significantly related to height in females (r2 = 0.07; P = 0.01). Similarly, vBMD of the femoral shaft (vFBMD) did not change with age or height in either sex. In females, a significant inverse relationship was seen between vFBMD and weight (r2 = 0.14; P = 0.001). Male subjects had higher vFBMD than females (mean +/- SD, 0.73 +/- 0.11 vs. 0.70 +/- 0.12; P = 0.047), but no sex difference was seen in vBMD of the femoral neck. Conversely, vBMD of L1-L4 remained age and growth dependent, although the strength of the relationship was weaker than that for aBMD (data not shown). In conclusion, the vBMD of the femoral neck and shaft is independent of age and is less dependent on growth variables in children and young adults than is aBMD. These observations offer a different perspective from our previous concepts of aBMD. PMID- 8636373 TI - A highly sensitive growth hormone (GH) enzyme-linked immunosorbent assay uncovers increased contribution of a tonic mode of GH secretion in adults with organic GH deficiency. AB - The limited sensitivity of conventional GH assays has impeded a better understanding of the pathophysiology of GH secretion. In normal subjects, interpulse GH levels often fall below assay sensitivity, making it unclear whether secretion stops or is maintained at a tonic level below assay detectability. In patients with severe organic GH deficiency (GHD), GH levels are mostly undetectable. Using an ultrasensitive GH enzyme-linked immunosorbent assay to measure 24-h integrated GH concentrations, we recently provided evidence that these patients secrete low, but measurable, amounts of GH. In this report, we apply the same assay to characterize and compare 24-h GH profiles obtained by 20 min sampling in 10 subjects with organic GHD and 10 normal subjects matched for age, sex, and body mass index. With deconvolution analysis, which provides estimates of GH secretion and half-life, our aim was to determine 1) whether normal GH secretion is exclusively pulsatile, 2) how GH is secreted in subjects with organic GHD, and 3) the attributes of GH secretion that determine circulating insulin-like growth factor I (IGF-I) levels. All samples, including nadirs, from GHD subjects were well within the assay detection limit (1 ng/L). Peak 24-h GH levels in GHD subjects were lower and did not overlap those in the normal subjects. Nadir GH concentrations were significantly lower in GHD subjects (14 +/- 5 vs. 43 +/- 9 ng/L; P = 0.008), but the range overlapped that of normal subjects. Endogenous GH half-life did not differ significantly between the two groups. Normal subjects secreted GH in a mixed pulsatile and tonic mode, with pulsatile secretion accounting for 93 +/- 2% of the total production. Total daily GH production in GHD was approximately 5% of the production in matched normal subjects. This difference resulted from a greater reduction in the pulsatile (by 96%) than in the tonic (by 47%) component, so that the fractional daily contribution by tonic GH release in GHD subjects was markedly greater. There was a significant relationship between pulsatile GH secretion and serum IGF-I levels for the two groups combined. In summary, 1) peak, but not nadir, GH levels were completely segregated between GHD and normal subjects; and 2) although normal subjects secrete GH in a tonic and pulsatile mode, both modes are reduced in organic GHD, with a proportionately greater reduction in pulsatile secretion. We conclude that 1) nadir GH levels are not sufficiently discriminatory to be useful for the diagnosis of GH deficiency; 2) normal GH profiles arise from a mixed pattern of tonic and pulsatile secretion, whereas reduced GH secretion in organic GHD arises primarily from a marked diminution in the amount of pulsatile GH release; and 3) pulsatile GH release is a significant regulator of the IGF-I level in normal and GHD subjects. PMID- 8636374 TI - Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. AB - Most parathyroid adenomas and some pathological parathyroid glands from patients with primary parathyroid hyperplasia or severe uremic secondary/tertiary hyperparathyroidism show an elevated set-point [the extracellular Ca2+ concentration (Ca2+o) half-maximally inhibiting PTH secretion]. In the present study, we investigated whether expression of the Ca2+o-sensing receptor protein recently cloned from bovine parathyroid, a key component in Ca2+o-regulated PTH release, is altered in primary and uremic hyperparathyroidism. Using immunohistochemistry with specific antireceptor antibodies, we compared immunoreactivity of the receptor protein in 14 adenomas, biopsies of 24 normal glands from this same group of patients, and 8 hyperplastic parathyroid glands from 2 individuals with uremic hyperparathyroidism. The results show a substantial reduction in the intensity of immunostaining for the receptor protein that averaged nearly 60% for both adenomas and hyperplastic glands, as quantitated by image analysis. Although normal glands from normocalcemic controls were not available, the intensity of receptor staining in normal glands from patients with adenomas was comparable to that in normal bovine, rat, and mouse parathyroid glands. There was considerable variation in staining intensity among different pathological parathyroid glands, even in those from the same patient with secondary hyperparathyroidism. In addition, both adenomas and hyperplastic glands had, in some cases, isolated chief cells and groups of cells, sometimes around the periphery of an abnormal gland, with receptor staining equivalent to that of normal parathyroid cells, whereas the bulk of the cells in the same gland showed a marked decrease in staining. Thus, there is a variable, but substantial, reduction in the immunoreactivity of the Ca2+o-sensing receptor protein in both parathyroid adenomas and uremic hyperparathyroidism, as assessed by immunohistochemistry, that probably results from reduced expression of the receptor protein and may contribute to the increase in the set-point often observed in these patients. PMID- 8636375 TI - Imprinting and expression of insulin-like growth factor-II and H19 in normal breast tissue and breast tumor. AB - Insulin-like growth factor (IGF)-II is a mitogenic peptide that has been reported to play an important role in the formation and growth of a variety of tumors. In most tissues, the IGF-II gene (IGF2) is parentally imprinted, with only the paternal allele being expressed. IGF2 messenger RNA (mRNA) and protein are overexpressed in some benign and malignant tumors. H19, a tumor suppressor gene located directly downstream from IGF2, is also genomically imprinted, but the paternal allele is silenced. It has been suggested that alterations in the imprinting of these two genes, which are located at chromosome 11p15.5, may lead to a malignant diathesis. We examined 18 fresh-frozen (FF) breast tumors with their adjacent normal breast tissue and 14 sets of paraffin-embedded formalin fixed tissues for IGF2 and H19 gene expression and imprinting. IGF2 mRNA and H19 RNA could be quantitated in 15 of the 18 FF tumors. Although three of these tumors showed a > or = 2-fold increase in IGF2 expression when compared with the normal control tissues, the average abundance of IGF2 mRNA in 8 of 15 FF samples was < 50% that observed in the normal tissue. The expression of H19 RNA in these tumors was increased by > or = 2-fold in 5 tumors, but decreased by < or = 50% in 6 tumors when compared with normal adjacent tissue. By examining the ApaI and CA repeat polymorphisms in the IGF2 gene, we found that the imprinting of IGF2 was maintained in all but 2 of the 17 informative subjects. H19 imprinting was maintained in all 18 informative fresh-frozen and paraffin-embedded formalin fixed samples. Our data suggest that alterations in IGF2 and H19 gene expression and loss of imprinting do not occur reliably in breast cancer. PMID- 8636376 TI - Characterization of monoclonal antibodies specific for the human growth hormone 22K and 20K isoforms. AB - We have derived and characterized a set of monoclonal antibodies (mAb) specific for the different human GH (hGH) isoforms. The binding characteristics of each antibody to the hGH isoforms (22K and 20K) were analyzed in direct and competitive immunoassays as well as by Western blot. We studied the effects of these mAb on the biological activity of hGH and showed that they specifically block their respective activities. Using these mAb, we developed several immunoassays that have been applied for the quantitation of the different hGH isoforms in body fluids. Therefore, these mAb may help to unravel the biological function of these variants. PMID- 8636378 TI - Localization of insulinomas by selective intraarterial calcium injection. AB - This study examines the role of selective intraarterial calcium injection and hepatic venous sampling in the localization of insulinomas. Seven patients were studied. In all cases, ultrasound and computerized tomography scans were either negative or equivocal. Calcium gluconate was injected directly into the arteries supplying the pancreas after standard selective angiography. Insulin levels were measured in samples taken from the right hepatic vein before and 30, 60, 90, 120, and 180 s after each injection. Two doses were used, 0.025 milliequivalents Ca/kg (1 mg/kg) for the first two subjects and 0.00625 milliequivalents Ca/kg (0.25 mg/kg) for the remaining five subjects. Serum insulin levels rose at least 2 fold, the proposed diagnostic rise, from basal in six subjects; one test was negative. Of the six positive studies, a diagnostic rise was seen only in one artery in five cases. One patient did not undergo surgery. In the remaining five patients, surgery confirmed the position and histology of the tumor. The one patient with a diagnostic rise in more than one artery, however, had residual disease after surgery. The seventh subject referred specifically for localization had a negative calcium stimulation study and a subsequent diagnosis of intermittent sulfonylurea abuse was made after a positive screen. The present study shows that preoperatively, selective intraarterial calcium injection with hepatic venous sampling is a powerful technique for the localization of insulinomas. Smaller doses of calcium than previously reported can be used and may reduce the risk of hypoglycemia during the procedure. PMID- 8636377 TI - Somatic mutations of the ret protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence. AB - Germline point mutations in exons 10, 11, and 16 of the ret protooncogene have been identified as causative in multiple endocrine neoplasia type 2 and in familial medullary thyroid carcinoma (MTC). Somatic point mutations of the same gene, exclusively associated with codon 918 of exon 16, have also been reported in few cases of sporadic medullary thyroid carcinoma. We analyzed the blood and tumor DNA of 19 patients with sporadic MTC and 6 patients with primary parathyroid adenoma for point mutations at exons 10, 11, and 16 of the ret protooncogene by restriction analysis of the PCR-amplified product and by sequence analysis of exons 10 and 11. A Cys634-->Tyr mutation was found in both the tumoral and blood DNA of one patient, indicating that he was affected by an hereditary form of MTC, erroneously considered sporadic. In the other 18 patients with MTC, somatic point mutations of ret were found in 8 cases (44.4%). In 5 cases the mutation affected exon 16 (Met918-->Thr), and in 3 cases it affected exon 11 (Cys634-->Arg in 1 and Cys634-->Trp in 2); these 3 mutations were confirmed by sequence analysis. The remaining 10 patients had no mutation in exon 10 by either restriction analysis or sequence analysis. Clinical data showed that 75% of the patients whose tumor carried ret mutation had tumor recurrence and/or increased serum calcitonin concentrations during the postsurgical follow-up period as opposed to 10% of the patients without mutations (P < 0.02, by chi2 analysis). No ret mutation was found in the tumoral DNA from parathyroid adenomas. Our findings indicate that the somatic ret point mutation frequently found in sporadic MTC may affect not only exon 16 but also exon 11 and is associated with less favorable clinical outcome. PMID- 8636379 TI - Remission of acromegaly caused by pituitary carcinoma after surgical excision of growth hormone-secreting metastasis detected by 111-indium pentetreotide scan. AB - GH-secreting carcinomas of the pituitary are extremely rare. We describe a 37-yr old woman with refractory acromegaly 15 yr after transphenoidal surgery and radiotherapy, with no evidence of a recurrent pituitary mass. Scanning with 111 indium pentetreotide revealed an area of intense activity in the left neck. A 3.5 x 2.5-cm mass was excised from the neck after demonstrating an arterio-venous GH gradient of 7:1. GH levels (50 ng/mL) dropped to 0.8 ng/mL 3 h after surgery and remained normal. GH gene expression was demonstrated in the metastasis by Northern and Western blot analyses and by positive immunocytochemistry and immunoelectron microscopy. In vitro cultured cells responded to GHRH and TRH by increasing GH levels (P < 0.01). Medium GH was identical to authentic pituitary GH, as demonstrated by high pressure liquid chromatography. RT-PCR of hypothalamic hormone receptor messenger RNA in the mass revealed somatostatin receptor subtypes 2, 3, and 5 and GHRH, TRH, and dopamine receptor expression. No GH gene amplification, rearrangement, or gsp mutation was found. RB gene deletion and H-ras mutations, previously reported in PRL- and ACTH-secreting carcinomas, were not detected. In conclusion, clinical and molecular features of a GH secreting pituitary carcinoma are presented. This metastatic lesion synthesized GH and expressed functional hypothalamic hormone receptors. PMID- 8636380 TI - Hyperproinsulinemia in a three-generation Caucasian family due to mutant proinsulin (Arg65-His) not associated with imparied glucose tolerance: the contribution of mutant proinsulin to insulin bioactivity. AB - Familial hyperproinsulinemia is a genetic abnormality characterized by an increased proportion of proinsulin immunoreactivity in the circulation due to mutations affecting the posttranslational processing of proinsulin. In affected Japanese families, this has been associated with noninsulin-dependent diabetes mellitus or impaired glucose tolerance. A three-generation Caucasian family with hyperproinsulinemia was identified through unexplained hyperinsulinemia in a normal volunteer participating in a metabolic study. High pressure liquid chromatography analysis of fasting plasma revealed a major peak eluting close to the position of proinsulin. Direct sequencing of the proinsulin gene exon 3 showed a heterozygous point mutation (CGT-->CAT) resulting in the substitution of Arg-->His in position 65 (corresponding to the AC cleavage site) in the index case, his mother, and his maternal grandmother. Using specific enzyme-linked immunosorbent assay methods to quantify insulin and proinsulin (including its conversion intermediates), the impact of this mutation on B cell secretion and glucose tolerance was studied. All affected subjects had normal oral glucose tolerance. In the basal state and after oral glucose administration, their proinsulin responses were immense, but intact insulin responses were slightly reduced. However, when calculating insulin bioactivity by assuming 9% activity for mutant Arg65-->His proinsulin, responses in affected subjects were comparable to those in normal subjects. In conclusion, our data demonstrate hyperproinsulinemia in a three-generation Caucasian family due to heterozygous mutant Arg65-->His proinsulin. This was not associated with impaired glucose tolerance. These results suggest that this mutation in the heterozygous state per se does not affect glucose tolerance and that the biological activity of mutant proinsulin contributes to glucose homeostasis in this family. The association of the same mutation with impaired glucose tolerance or diabetes in previous studies may be the result of selection bias or associated conditions (e.g. the genetic background of the kindreds examined). PMID- 8636381 TI - Localization of the expression of complement component 3 in the human endometrium by in situ hybridization. AB - C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736 bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation. PMID- 8636382 TI - Short-term diabetic ketosis alters n-6 polyunsaturated fatty acid content in plasma phospholipids. AB - At present, no information is available about the possibility that acute loss of metabolic control might be able to modify fatty acid composition in IDDM patients. Therefore, the aim of this study was to determine whether a short-term period of ketosis has any effect on the composition of fatty acid of plasma phospholipids in a group of insulin-dependent diabetes mellitus (IDDM) patients. Eleven IDDM patients and nine healthy volunteers were studied. Patients were studied over a 2-day period; each 2-day period consisted of initial baseline measurements followed by a day of hyperglycemia and mild ketosis, which was promptly alleviated by insulin infusion. No significant difference in baseline percent fatty acid composition was observed between normal controls and IDDM patients. In IDDM patients, ketosis induced a significant decrease in percent arachidonic acid (20:4 n-6) content, with a significant parallel decline in n-6 total polyunsaturated fatty acids. A significant inverse correlation between the prevailing plasma glucose and the relative content of arachidonic acid in plasma phospholipids was observed (r = -0.35; P = 0.0488). A highly significant inverse correlation was observed between the change from baseline condition to ketosis of the ratio C20:4/C20:3, the product/precursor ratio for the reaction catalyzed by delta5-desaturase, and the values of hemoglobin A1c,(r = -0.855; P = 0.0015). In conclusion, short term diabetic ketosis is associated with a significant decrease in n-6 polyunsaturated fatty acid content in plasma phospholipids, especially arachidonic acid. This decrease in arachidonic acid appears to be related to the degree of metabolic derangement, whereas the influence of short-term diabetic ketosis on the ratio of C20:4 to C20:3 seems to be due to the degree of long-term metabolic control. PMID- 8636383 TI - Arm span as measurement of response to growth hormone (GH) treatment in a group of children with meningomyelocele and GH deficiency. AB - Children with meningomyelocele (MMC) frequently have impaired linear growth. A number have associated structural brain defects with resultant GH deficiency (GHD). Reproducible measurements of height or length in MMC patients are often hampered by lower limb contractures, spasticity, and scoliosis. Arm span has been proposed as a more reproducible measure of linear growth. Five MMC children documented to have GHD were treated with recombinant human GH (hGH) for 1-3 yr. Their height, arm span, and growth velocity were compared with 32 children with idiopathic GHD treated similarly with hGH. These measures are compared with normal children by being expressed as standard deviation scores. The results of this study indicate that arm span measurements in GHD MMC patients are almost identical to height measurements in idiopathic GHD patients both before and during hGH therapy. The physical condition of children with MMC makes reproducible longitudinal height measurements difficult. Routine determinations of arm span measurements for children with MMC will assist in recognizing growth failure as well as monitoring treatment results. PMID- 8636384 TI - Codon 972 polymorphism in the insulin receptor substrate-1 gene, obesity, and risk of noninsulin-dependent diabetes mellitus. AB - Because of the role of insulin receptor substrate-1 in insulin action, the insulin receptor substrate-1 gene is a candidate gene for noninsulin-dependent diabetes mellitus (NIDDM). Modest associations between NIDDM and a GGG-->AGG single base substitution (corresponding to a glycine-->arginine amino acid substitution) in codon 972 of the gene have been found, but none reached statistical significance. To examine further how large a proportion of NIDDM cases could be caused by the mutation, we performed a stratified analysis combining the results from the 6 earlier studies and those from our panel of 192 unrelated NIDDM subjects and 104 healthy controls. In addition, we looked for a possibility that the codon 972 mutation plays a role only in the presence of certain conditions. Genomic DNA samples obtained from NIDDM cases and healthy controls were genotyped using a PCR-restriction fragment length polymorphism protocol modified for genomic DNA. The GGG-->AGG substitution was found in 5.7% of the diabetic subjects (11 of 192) and 6.9% of the controls (7 of 104). The difference between groups was not statistically significant, and it was not different from the results of other studies. The Mantel-Haenszel summary odds ratio across all studies was 1.49 (P < 0.05; 95% confidence intervals, 1.01-2.2). This summary odds ratio is consistent with a small proportion of NIDDM cases (approximately 3%) being caused by the mutation. Exploratory subgroup analyses on our panel suggested a clustering of NIDDM, the codon 972 mutation, and overweight, raising the hypothesis that the mutation may predispose to NIDDM only in the presence of excess body weight. PMID- 8636385 TI - Familial Albright's hereditary osteodystrophy with hypoparathyroidism: normal structural Gs alpha gene. AB - Albright's hereditary osteodystrophy (AHO) is a characteristic skeletal phenotype, including short stature, obesity, round face, and brachydactyly. AHO appears in patients with pseudohypoparathyroidism (PHP) who have resistance to PTH and in their eumetabolic family members who have pseudopseudohypoparathyroidism (PPHP). The differential diagnosis of AHO in families without PHP includes brachydactyly E, whose existence as a distinct entity has been questioned. We studied a patient with familial AHO who presented with hypocalcemia. To our surprise, PTH levels were low, and the response to PTH administration was normal. This is the first case of familial AHO with hypoparathyroidism. The proband's family included 22 affected subjects spanning 3 generations, who had variable degrees of AHO manifestations, with an autosomal dominant inheritance trait. The metacarpophalangeal pattern profile was typical of that of PHP-PPHP. As deficient activity and inactivating mutations of Gs alpha were described in PHP as well as in PPHP, we measured the biological activity of Gs in family members, which was normal. To exclude subtle abnormalities in the Gs alpha gene, we sequenced the entire coding region of Gs alpha in the propositus, which was normal. We conclude that hypocalcemia should be adequately evaluated even in the presence of familial AHO, and that familial AHO can occur with a normal coding structural Gs alpha gene. Identification of the molecular defect in familial AHO without PHP will shed light on the pathogenesis of AHO in general. PMID- 8636386 TI - Serum sex hormone-binding globulin and osteocalcin in systemic nonthyroidal illness associated with low thyroid hormone concentrations. AB - To test the hypothesis that patients with systemic nonthyroidal illness (NTI) and impaired thyroid hormone metabolism most commonly present with the low T3 or low T4 syndrome are in an euthyroid status at the tissue level, we determined serum sex hormone-binding globulin (SHBG) and osteocalcin (OC) as parameters for thyroid hormone availability to liver and bone. Serum SHBG and OC concentrations were measured in 61 severely ill patients with decreased serum levels of either T3 alone (n = 47) or both T3 and free T4 (n = 14). None of the patients had primary thyroid disorder indicated by regular thyroid sonomorphology and normal basal TSH concentrations. Data were compared with values obtained from age- and sex-matched controls without impairment of thyroid hormone physiology. The respective results from the study groups and from control subjects were as follows: low T3 SHBG, 49 +/- 30 nmol/L; low T4 SHBG, 35 +/- 14 nmol/L; control SHBG, 45 +/- 28 nmol/L; low T3 OC, 0.87 +/- 0.75 nmol/L; low T4 OC, 0.75 +/- 0.71 nmol/L; control OC, 0.98 +/- 0.87 nmol/L. SHBG and OC do not differ significantly between NTI patients with low T3 or low T4 syndrome and controls, and no significant relationship was found between thyroid hormone parameters and SHBG or OC, respectively. We conclude that the term euthyroid sick syndrome applied for the condition of systemic NTI associated with reduced circulating thyroid hormone concentrations appears to be appropriate at the level of hepatocytes and osteoblasts. Data are in partial contradiction to the results of previous studies using alternative serum parameters for assessment of peripheral thyroid hormone action (e.g. angiotensin-converting enzyme). Due to the tissue specificity of the biochemical serum markers, conflicting results may be explained by organ-specific differences in local thyroid hormone exposure. In case of doubt, SHBG and OC determinations may help specify peripheral thyrometabolic status in NTI. PMID- 8636387 TI - The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma. AB - Thyroid epithelial cells have been shown to have a high APP expression and to produce large amounts of its metabolic derivatives, namely secreted APPs and a potentially amyloidogenic 41-kDa C-terminal fragment. It was the aim of the present study to analyze how APP production and metabolism were regulated in human thyroid cells. The effects of three cytokines, interferon gamma (IFN gamma), interleukin 1beta (IL-1 beta) and transforming growth factor (TGF) beta, were investigated. Cell extracts and supernatants were studied by immunoblotting using specific N- and C-terminal APP antibodies. Quantification was performed by densitometric scanning. We demonstrate that IFN gamma has a strong suppressive effect on the production and metabolism of APP. From a concentration of 30 U/ml upwards it reduces the cellular APP content, decreases the amounts of secreted APPs and inhibits the generation of the 41-kDa amyloidogenic APP fragment. In contrast, IL-1 beta has a stimulatory influence on the generation of the amyloidogenic 41-kDa APP metabolite, but does not affect the cellular holoprotein or APPs. TGFbeta has no significant effect on APP. Our results demonstrate that cytokines can regulate APP production and metabolism in thyroid cells. IFN gamma is the first naturally occurring agent described to inhibit the generation of amyloidogenic APP fragments. It may be of relevance in preventing amyloid deposition during inflammatory processes in the thyroid gland, but may exert a similar protective effect in other non-neuronal and neuronal tissues. PMID- 8636388 TI - In memoriam: a tribute to the work of Alexander Psychoyos. PMID- 8636389 TI - Glucagon treatment in NICTH. PMID- 8636390 TI - Pathogenesis of myxedematous endemic cretinism. PMID- 8636391 TI - Vitamin D receptor gene and osteoporosis. PMID- 8636392 TI - Would the real role(s) for secretory PLA2s please stand up. PMID- 8636393 TI - Molecules that protect: the defense of neurons and other cells. PMID- 8636394 TI - O=O NO: it's CO. PMID- 8636395 TI - Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone. AB - Post-heparin hepatic lipase activity is reduced in chronic renal failure (CRF). This could be due to reduced synthesis, decreased activity, and/or impaired secretion of the enzyme. Further, the factor(s) responsible for such derangements are not elucidated. We examined hepatic lipase metabolism in normal, 6-wk-old CRF rats, CRF-PTX (parathyroidectomized) rats, and CRF and normal rats treated with verapamil (CRF-V, normal-V) using liver homogenate, hepatic cell culture for 8 h, and in vitro liver perfusion. The Vmax of hepatic lipase in liver homogenate was significantly (P < 0.01) reduced and the Km was significantly (P < 0.01) increased in CRF rats, but the values were normal in CRF-PTX, CRF-V, and normal-V rats. Culture of hepatic cells for 8 h was associated with an increase in hepatic lipase activity but the increment in CRF rats was significantly (P < 0.01) lower than that of normal, CRF-PTX, CRF-V, and normal-V rats. Both parathyroid hormone (PTH)-(1-84) and 1-34 inhibited the production of hepatic lipase in cultured cells from normal, CRF-PTX, CRF-V, and normal-V rats. The expression of the mRNA of the hepatic lipase was significantly reduced in CRF animals with the ratio between it and that of house keeping gene G3DPH being 15 +/-3% compared to 40 +/- 1.3% in normal, 44+/-2.9% CRF-PTX, 44 +/- 5.4% in CRF-V, and 39 +/- 3.9% in normal-V rats. Infusion of heparin to the in vitro hepatic perfusion system increased the activity of hepatic lipase in the effluent in all groups of rat except in CRF animals. Infusion of PTH-(1-34) in dose of 10(-6) M into the liver perfusion system inhibited the increase in post-heparin hepatic lipase activity. The data show that in CRF (a) the mRNA of hepatic lipase is downregulated, and hepatic lipase production, activity and release are impaired, (b) that this is due to the state of secondary hyperparathyroidism of CRF since both acute and chronic excess of PTH were associated with these abnormalities, (c) and that prevention of excess PTH by PTX of CRF rats or blocking the effect of PTH by treatment with verapamil corrected the derangement in hepatic lipase metabolism. PMID- 8636396 TI - Chymase in exocytosed rat mast cell granules effectively proteolyzes apolipoprotein AI-containing lipoproteins, so reducing the cholesterol efflux inducing ability of serum and aortic intimal fluid. AB - Degranulated mast cells are present in human fatty streaks. Chymase in granules released from degranulated rat serosal mast cells, i.e., in granule remnants, proteolyzes human high density lipoprotein3 (HDL3), and so reduces its ability to induce cholesterol efflux from macrophage foam cells in vitro. In this study we found that remnant chymase, by proteolyzing human serum and human aortic intimal fluid, prevents these two physiologic fluids from effectively inducing cholesterol efflux from cultured macrophage foam cells. Inhibition was strongest when remnants were added to apolipoprotein AI (apoAI)-containing lipoproteins; the remnants had no effect on the weaker efflux produced by apoAI-deficient serum. Western blot analysis showed that granule remnants degrade apoAI in serum and in internal fluid. When released from remnants, chymase lost its ability to proteolyze HDL3 in the presence of serum. Thus, remnant chymase (but not isolated chymase) was able to resist the natural protease inhibitors present in serum and in intimal fluid. The results imply participation of exocytosed mast cell granules in foam cell formation in atherogenesis. PMID- 8636397 TI - Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat. AB - At birth water is rapidly reabsorbed from the distal lung in preparation for alveolar gas exchange. To investigate a potential role for the AQP1 water channel in development, lung membranes from fetal and perinatal rats were analyzed by immunoblot. First expression of AQP1 was noted in fetal rat lung at E19 (19th day of the 21-day gestation). The level of AQP1 increased fivefold from the last gestational day to the first postnatal day and persisted at high levels into adulthood. Maternal corticosteroids increased expression of AQP1 in fetal lung, an effect also seen in adult rats. AQP1 mRNA increased in rat pups treated with corticosteroids, suggesting at least partial regulation at the level of transcription. Immunohistochemical analyses with anti-AQP1 demonstrated the protein in peribronchial vessels and visceral pleura at E21 with increased postnatal expression. AQP1 was not expressed in airway epithelium, and only occasional alveolar pneumocytes were labeled. Immunoelectron microscopy revealed AQP1 on both apical and basolateral membranes of endothelial cells. The ontogeny and corticosteroid induction of AQP1 in rat lung coincide with major physiological alterations in lung development; however, the distribution of AQP1 predicts the existence of other water channels in the alveolar epithelium. PMID- 8636398 TI - Neutral endopeptidase 24.11 in neutrophils modulates protective effects of natriuretic peptides against neutrophils-induced endothelial cytotoxity. AB - This study was performed to determine effects of atrial and brain natriuretic peptides (ANP, BNP) on neutrophils-induced endothelial injury which is known to play a role in the pathophysiology of ischemia/reperfusion myocardial injury and to examine whether the effects of ANP and BNP on neutrophils are modulated by neutral endopeptidase 24.11 (NEP) in neutrophils themselves. The incubation of human neutrophils with ANP and BNP inhibited the neutrophils-induced detachment of cultured human endothelial cells (HEC). The inhibitory effect of ANP and BNP was associated with the suppressions of the neutrophils adhesiveness to HEC, CD18 expression on the neutrophils and elastase release from the neutrophils. Coincubation with UK73967 or phosphoramidon, inhibitors of NEP, potentiated all of the effects of ANP and BNP on the neutrophil functions, and the NEP inhibitors protected degradation of ANP and BNP by the neutrophils. NEP enzymatic activity in the particulate fractions and immunoreactive NEP expression were found to increase in the neutrophils from patients with early phase of acute myocardial infarction (AMI) by 5.2- and by 4.2-fold of the neutrophils from patients with late phase of AMI, respectively. In an in vivo canine model of myocardial ischemia/reperfusion, the intravenous administration of UK73967 suppressed the neutrophil adherence to endothelium and the neutrophil accumulation in the ischemic/reperfused myocardium. The results indicate that ANP and BNP, which are known to increase in AMI, modulate the neutrophil functions and exert protective effects against the neutrophils-induced endothelial cytotoxity. But the effects are suppressed due to their degradation by the neutrophil own NEP. Thus, neutrophil NEP, which also increases in AMI, may play a role in the pathophysiology of neutrophils-mediated ischemia/reperfusion endothelial and myocardial injury. PMID- 8636399 TI - Increased ferritin gene expression in atherosclerotic lesions. AB - To identify genes potentially implicated in atherogenesis, a cDNA library was constructed from human atherosclerotic aorta and differentially screened with 32P labeled-cDNAs prepared from human normal and atherosclerotic aortas. Two cDNA clones exhibiting higher hybridization to the 32P-labeled cDNAs from atherosclerotic vessels were isolated and identified to be genes encoding L ferritin and H-ferritin, respectively. Northern blot analysis confirmed that the expression of both ferritin genes was notably higher in human and rabbit atherosclerotic aortas than in their normal counterparts. A time-course study illustrated that both L- and H-ferritin mRNAs were markedly increased in aortas of rabbits after feeding with a high cholesterol diet for 6 wk, which was also the time period after which the formation of lesions became evident. In situ hybridization revealed that both L- and H-ferritin mRNAs were induced in endothelial cells and macrophages of human early lesions. The signals were also detected in the smooth muscle cells of advanced lesions. Immunostaining further identified the presence of ferritin protein in atherosclerotic lesions. On the other hand, Prussian blue stain revealed the presence of iron deposits in advanced lesions but not in early human or rabbit lesions. Further experiments with cultured human monocytic THP-1 cells and aortic smooth muscle cells demonstrated that ferritin mRNAs were subjected to up-regulation by treatment with IL-1 or TNF, while TGF, PDGF, and oxidized LDL did not affect the expression of either ferritin gene in both cell lines. Collectively, these results clearly demonstrate that ferritin genes are susceptible to induction in the course of plaque formation. PMID- 8636400 TI - Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma. AB - Fibrin is deposited on the endothelial cell surface in the vasculature of murine methylcholanthrene A-induced sarcomas after injection of tumor necrosis factor (TNF). Capillary endothelial cells of the tumor vascular bed become positive for tissue factor after TNF injection, based on immunocytochemistry and in situ hybridization. Intravascular clot formation was not dependent on tissue factor derived from tumor cells, since in vessels of tumors not expressing tissue factor, TNF also induced fibrin/fibrinogen deposition. However, the time course of fibrin/fibrinogen deposition after TNF differed in tumors expressing no, little, or greater amounts of tissue factor. Fibrin/fibrinogen deposition was more rapid in tumors in which the neoplastic cells expressed tissue factor than in tumors not expressing tissue factor. In the tumors not expressing tissue factor, activation of coagulation was dependent on TNF-induced synthesis of tissue factor by host cells, i.e., endothelium or monocytes/macrophages. Intravenous somatic gene transfer with tissue factor cDNA in the antisense orientation (but not sense or vector alone) reduced intravascular fibrin/fibrinogen deposition and restored blood flow to the tumor, showing that de novo tissue factor expression is central in TNF-induced activation of the coagulation mechanism. PMID- 8636401 TI - Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. AB - Development of the mammary gland during puberty, pregnancy, and lactation is controlled by steroid and peptide hormones and growth factors. To determine the role of the insulin-like growth factors (IGFs) in this process we developed a transgenic model using the whey acidic protein (WAP) gene to direct expression of rat IGF-I and human IGF binding protein-3 (IGFBP-3) to mammary tissue during late pregnancy and throughout lactation. High levels of expression of transgenic IGF-I and IGFBP-3 were seen in lobular-alveolar cells by in situ hybridization. There was no obvious effect on mammary development during pregnancy and lactation; indeed, mothers were capable of nursing their pups normally and the only structural difference seen in the mammary glands at peak lactation was an overall smaller size of the alveoli. We also evaluated the role of IGF-I and IGFBP-3 in the remodeling of mammary tissue during involution. Compared with control animals, the process of involution was modified in both transgenic lines. The degree of apoptotic cells was lower in the WAP-IGF-I and WAP-BP-3 expressing mice. In addition, there was a more quiescent pattern of involution with residual lobular secretary ability and a muted host inflammatory reaction with fewer lumenal microcalcifications. These results demonstrate that IGF-I and IGFBP-3 may modulate the involutionary process of the lactating mammary gland. PMID- 8636402 TI - Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate. AB - Group II PLA2 has been implicated in inflammatory processes in both man and other animals and has been shown to be involved in inflammatory conditions, such as arthritis and sepsis. Transgenic mice expressing the human group II PLA2 gene have been generated using a 6.2-kb genomic fragment. These mice express the group II PLA2 gene abundantly in liver, lung, kidney, and skin, and have serum PLA2 activity levels approximately eightfold higher than nontransgenic littermates. The group II PLA2 transgenic mice reported here exhibit epidermal and adnexal hyperplasia, hyperkeratosis, and almost total alopecia. The chronic epidermal hyperplasia and hyperkeratosis seen in these mice is similar to that seen in a variety of dermatopathies, including psoriasis. However, unlike what is seen with these dermatopathies, no significant inflammatory-cell influx was observed in the skin of these animals, or in any other tissue examined. These mice provide an important tool for examining group II PLA2 expression, and for determining the role of group II PLA2 in normal and disease physiology. They serve as an in vivo model for identifying inhibitors of group II PLA2 activity and gene expression. PMID- 8636403 TI - Glutathione depletion impairs transcriptional activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells. AB - When primary cultures of guinea pig gastric mucosal cells were exposed to heat (43 degree C), ethanol, hydrogen peroxide (H2O2), or diamide, heat shock proteins (HSP90, HSP70, HSP60, and HSC73) were rapidly synthesized. The extent of each HSP induction varied with the type of stress. Ethanol, H2O2, and diamide increased the syntheses of several other undefined proteins besides the HSPs. However, none of these proteins were induced by exposure to heat or the reagents, when intracellular glutathione was depleted to <10% of the control level by pretreatment with DL-buthionine-[S,R]-sulfoximine. Gel mobility shift assay using a synthetic oligonucleotide coding HSP70 heat shock element showed that glutathione depletion inhibited the heat- and the reagent-initiated activation of the heat shock factor 1 (HSF1) and did not promote the expression of HSP70 mRNA. Immunoblot analysis with antiserum against HSF1 demonstrated that the steady state level of HSF1 was not changed in glutathione-depleted cells, but glutathione depletion inhibited the nuclear translocation of HSF1 after exposure to heat stress. These results suggest that intracellular glutathione may support early and important biochemical events in the acquisition by gastric mucosal cells of an adaptive response to irritants. PMID- 8636404 TI - The double edged sword of the immune response: mutational analysis of a murine anti-pneumococcal, anti-DNA antibody. AB - Anti-double-stranded (ds) DNA antibodies are not only an important diagnostic marker for SLE, but also play an important role in tissue injury. Microbial antigen may be a stimulus for the production of these antibodies. We isolated 99D.7E, an IgG2b monoclonal antibody from a nonautoimmune BALB/c mouse that is cross-reactive with both dsDNA and phosphorylcholine, the dominant hapten on the pneumococcal cell wall. While partially protective against a bacterial challenge, 99D.7E is also pathogenic to the kidney. To identify those molecular motifs that confer on anti-PC antibodies the potential for autoreactivity, we created a panel of 99D.7E mutants with single amino acid substitutions in the heavy chain, and examined the changes in antigen binding and renal deposition. Our results support the hypothesis that charge and affinity for dsDNA are not adequate predictors of the pathogenicity of anti-DNA antibodies. Differential renal damage from anti dsDNA antibodies may be due to differences in fine specificity, rather than differential affinity for dsDNA. Importantly, high affinity IgG antibodies cross reactive with bacterial and self antigen exist and can display pathogenic potential, suggesting that defects in peripheral regulation of B cells, activated by foreign antigen but cross-reactive with self antigen, might lead to autoimmune disorders. PMID- 8636405 TI - Nitric oxide production and perivascular nitration in brain after carbon monoxide poisoning in the rat. AB - Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning. PMID- 8636407 TI - A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease. AB - The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children. PMID- 8636406 TI - Redox control of resistance to cis-diamminedichloroplatinum (II) (CDDP): protective effect of human thioredoxin against CDDP-induced cytotoxicity. AB - Thioredoxin is a small ubiquitous protein with multiple biological functions, including cellular defense mechanisms against oxidative stress. In the present study, we investigated the role of human thioredoxin (hTRX) in the acquisition of cellular resistance to cis-diamminedichloroplatinum (II) (CDDP). The expression and activity of hTRX in Jurkat T cells was dose-dependently enhanced by exposure to CDDP, as determined by immunoblot analysis and insulin reducing assay. Furthermore, chloramphenicol acetyltransferase analysis using the hTRX promoter reporter gene construct revealed that treatment of Jurkat cells with CDDP caused transcriptional activation of the hTRX gene, which might be mediated through increased generation of intracellular reactive oxygen intermediates. To examine the biological significance of hTRX induction, we established hTRX-overexpressing derivatives of L929 fibrosarcoma cells by stable transfection with the hTRX cDNA. The clones, which constitutively expressed the exogenous hTRX, displayed increased resistance to CDDP-induced cytotoxicity, compared with the control clones. After exposure to CDDP, the control cells showed a significant increase in the intracellular accumulation of peroxides, whereas the hTRX-transfected cells did not. Taken together, these results suggest that overexpressed hTRX is responsible for the development of cellular resistance to CDDP, possibly by scavenging intracellular toxic oxidants generated by this anticancer agent. PMID- 8636408 TI - Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension. AB - Since dopamine produced by the kidney is an intrarenal regulator of sodium transport, an abnormality of the dopaminergic system may be important in the pathogenesis of hypertension. In the spontaneously hypertensive rat (SHR), in spite of normal renal production of dopamine and receptor density, there is defective transduction of the D1 receptor signal in renal proximal tubules, resulting in decreased inhibition of sodium transport (Na+/H+ exchanger [NHE] and Na+/K+ATPase activity) by dopamine. To determine if impaired D1 receptor regulation of NHE in proximal tubules is related to hypertension, studies were performed in a F2 generation from female Wistar Kyoto (WKY) and male SHR crosses. A D1 agonist, SKF 81297, inhibited (37.6 +/- 4.7%) NHE activity in brush border membranes of normotensive F2s (systolic blood pressure < 140 mm Hg, n = 7) but not in hypertensive F2s (n = 21). Furthermore, a D1 agonist, SKF 38393, when infused into the renal artery, dose dependently increased sodium excretion in normotensive F2s (n = 3) without altering renal blood flow but was inactive in hypertensive F2s (n = 21). Since the major D1 receptor gene expressed in renal proximal tubules is the D1A subtype, we determined the importance of this gene in the control of blood pressure in mice lacking functional D1A receptors. Systolic blood pressure was greater in homozygous (n = 6) and heterozygous (n = 5) mice compared to normal sex matched litter mate controls (n = 12); moreover, the mice lacking one or both D1A alleles developed diastolic hypertension. The cosegregation with hypertension of an impaired D1 receptor regulation of renal sodium transport and the development of elevated systolic and diastolic pressure in mice lacking one or both D1A alleles suggest a causal relationship of the D1A receptor gene with hypertension. PMID- 8636409 TI - Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy. AB - Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility. PMID- 8636411 TI - Use of maltose hydrolysis measurements to characterize the interaction between the aqueous diffusion barrier and the epithelium in the rat jejunum. AB - Rates of intestinal absorption and surface hydrolysis are determined by the interaction of two barriers: poorly stirred fluid adjacent to the mucosa, and the epithelial cell. These two barriers commonly are modeled as a fixed, flat layer of epithelium covered by a fixed thickness of unstirred fluid. To more accurately simulate these barriers in a villous mucosa, maltase activity (measured in vitro) was distributed over an anatomically correct model of rat jejunal villi. We then determined what interaction of the aqueous and epithelial barriers best predicted in vivo maltose hydrolysis rates measured over a broad range of infusate concentrations. Hydrolysis was accurately predicted by a model in which unstirred fluid extended from 20 microm over the villous tips throughout the intervillous space. In this model, the depth of diffusion into the intervillous space is inversely proportional to the efficiency of epithelial handling of the solute. As a result, both the aqueous barrier and the functional surface area are variables rather than constants. Some implications of our findings (relative to the conventional model) include: higher predicted Vmax, efficient handling of low concentrations of a solute at the villous tips while high concentrations must penetrate thick aqueous barriers, and sensitive regulation of transport rates via ease of access to the intervillous space. PMID- 8636410 TI - A femtomolar-acting neuroprotective peptide. AB - A novel 14-amino acid peptide, with stress-protein-like sequences, exhibiting neuroprotection at unprecedented concentrations, is revealed. This peptide prevented neuronal cell death associated with the envelope protein (GP 120) from HIV, with excitotoxicity (N-methyl d-aspartate), with the beta amyloid peptide (putative cytotoxin in Alzheimer's disease), and with tetrodotoxin (electrical blockade). The peptide was designed to contain a sequence derived from a new neuroprotective protein secreted by astroglial cells in the presence of vasoactive intestinal peptide. The neurotrophic protein was isolated by sequential chromatographic methods combining ion exchange, size separation, and hydrophobic interaction. The protein (mol mass, 14 kD and pI, 8.3 +/- 0.25) was named activity-dependent neurotrophic factor, as it protected neurons from death associated with electrical blockade. Peptide sequencing led to the synthesis of the novel 14-amino acid peptide that was homologous, but not identical, to an intracellular stress protein, heat shock protein 60. Neutralizing antiserum to heat shock protein 60 produced neuronal cell death that could be prevented by cotreatment with the novel protein, suggesting the existence of extracellular stress-like proteins with neuroprotective properties. These studies identify a potent neuroprotective glial protein and an active peptide that provide a basis for developing treatments of currently intractable neurodegenerative diseases. PMID- 8636412 TI - Activation of heat shock protein (hsp)70 and proto-oncogene expression by alpha1 adrenergic agonist in rat aorta with age. AB - Induction of heat shock proteins (hsp) most likely is a homeostatic mechanism in response to metabolic and environmental insults. We have investigated signal transduction mechanisms involved in alpha1, adrenergic receptor stimulation of hsp7O gene expression in isolated aortas with age. We found that alpha1 adrenergic agonists directly induced hsp70 mRNA in rat aorta in vitro; the alpha1, selective antagonist prazosin blocked this effect whereas chloroethylclonidine, an antagonist which has some selectivity for alpha1B receptors, was ineffective. This response was insensitive to pertussis toxin and was partially blocked by the protein kinase C inhibitor H7. Removal of extracellular calcium attenuated induction of hsp70 mRNA but not the induction of c-fos or c-myc. The induction of hsp70 mRNA by either norepinephrine or by phorbol dibutyrate was blunted in aortas from old (24-27 mo) rats whereas c-fos responses were not diminished in the older vessels. The hsp70 response to elevated temperature (42 degrees C) was not changed with age. Activation of hsp70 expression most likely involves a pertussis toxin insensitive G protein which activates protein kinase C, and requires extracellular calcium. With age, hsp70 gene expression induced by stimulation of alpha1 adrenergic receptors is markedly attenuated, which could modify responses to stress or vascular injury with aging. PMID- 8636413 TI - T cell subsets, epitope mapping, and HLA-restriction in patients with allergic bronchopulmonary aspergillosis. AB - Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease characterized by Aspergillus fumigatus (Af) colonization, IgE and IgG anti-Af antibodies, pulmonary infiltrates, bronchiectasis, and pulmonary fibrosis. Little is known regarding T cell responses and their role in the pathogenesis of ABPA. To examine T cell reactivity to Af antigens, T cell clones (TCC) specific to the Asp f 1 antigen, an 18-kD protein of Af, were established from the peripheral blood of three ABPA patients. The majority of TCC isolated from ABPA patients, and specific for the Asp f 1 allergen of Af, are IL-4 producing CD4+ cells of the Th2 phenotype. Further analysis in this study revealed that the majority of TCC reacted to mainly two epitopes of Asp f 1, while the remaining TCC reacted to three additional "minor" epitopes. Blocking studies using monoclonal antibodies specific for class II HLA-D region gene products showed that most TCC, 19/21, were restricted by HLA-DR molecules, and the remaining two clones by HLA-DP molecules. The use of a panel of HLA-matched and mismatched EBV-transformed B cells as antigen presenting cells revealed that the HLA-DR restriction was mediated exclusively by either the HLA-DR2 or HLA-DR5 alleles. Genotyping of DRB1 gene products showed that class II presentation for most clones was not restricted to a single allele, representing DRB1 gene products of either HLA-DR2 or DR5. These studies offer insight into the cellular and molecular determinants which contribute to the immunopathophysiology of ABPA. PMID- 8636414 TI - Ca2+ channel activation by platelet-derived growth factor-induced tyrosine phosphorylation and Ras guanine triphosphate-binding proteins in rat glomerular mesangial cells. AB - We investigated the signaling pathways mediating 1-pS Ca2+ channel activation by PDGF in cultured rat mesangial cells. In cell-attached patches, intrapipette PDGF BB (PDGF B chain homodimer isoform) (50 ng/ml) dramatically stimulates channel activity (P < 0.003, n = 6). Tyrosine kinase inhibition (100 microM genistein or 10 microM tryphostin 9) abolished PDGF-induced channel activation (P < 0.02, n = 6). In excised patches, the effect of tyrosine kinase inhibition could be reversed by 200 microM GTPgammaS (P < 0.02, n = 4). In contrast, 200 microM GDPbetaS inhibited PDGF-induced channel activity (P < 0.04, n = 6). Pertussis toxin (250 ng/ml) had no effect on PDGF-induced channel activity (P = 0.45, n = 6). When excised patches were exposed to anti-Ras antibody (5 microg/ml), PDGF induced channel activity was abolished (P < 0.002, n = 11). Western immunoblots revealed that PDGF-BB binding stimulates the formation of a membrane-bound complex consisting of growth factor receptor-binding protein 2, son of sevenless, and the PDGF-beta receptor. Complex formation was abolished by genistein. In mesangial cells, the intrinsic tyrosine kinase activity of the PDGF-beta receptor stimulates the formation of a membrane-bound growth factor receptor-binding protein 2/son of sevenless/PDGF-beta receptor complex and activation of the pertussis toxin-insensitive GTP-binding protein, p21-Ras, which leads to the opening of 1-pS Ca2+ channels. PMID- 8636415 TI - The relationship between the fibrinogen D domain self-association/cross-linking site (gammaXL) and the fibrinogen Dusart abnormality (Aalpha R554C-albumin): clues to thrombophilia in the "Dusart syndrome". AB - Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gammaXL"). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 > I-9 > 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alphaC" region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alphaC" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome." PMID- 8636416 TI - Effects of a change in the pattern of insulin delivery on carbohydrate tolerance in diabetic and nondiabetic humans in the presence of differing degrees of insulin resistance. AB - While it is well established that people with non-insulin dependent diabetes mellitus have defects in both insulin secretion and action, the relative contribution of each to glucose intolerance is not known. Therefore, nondiabetic (lean and obese) and non-insulin dependent diabetes mellitus subjects were studied on two occasions. On each occasion, insulin secretion was inhibited with somatostatin and glucose was infused in a pattern and amount that mimicked the systemic delivery rate normally observed after ingestion of 50 g of glucose. Insulin also was infused so as to mimic postprandial insulin profiles observed in separate groups of diabetic and nondiabetic subjects after food ingestion. Glucose turnover was measured using the isotope dilution method. A delayed pattern of insulin delivery (i.e., a "diabetic" insulin profile) led to higher (P < 0.05) glucose concentrations in all groups; however, the effects were transient, resulting in only a modest increase in the integrated glycemic responses. An isolated defect in insulin action had little effect on peak glucose concentration; however, it prolonged the duration of hyperglycemia, leading to a 2.5-4.2-fold increase (P < 0.05) in the integrated glycemic response. A combined defect in the pattern of insulin secretion and action was additive rather than synergistic. Both defects caused hyperglycemia by altering suppression of endogenous glucose release and stimulation of glucose disposal. Whereas obese diabetic and nondiabetic subjects had comparable defects in glucose clearance, non-insulin dependent diabetes mellitus subjects also had defects in hepatic insulin action. Thus, abnormalities in the pattern of insulin secretion and action alone or in combination impair glucose tolerance. An isolated defect in insulin action has a more pronounced and prolonged effect than does an isolated change in the pattern of insulin secretion. Hepatic and extrahepatic insulin resistance results in marked and sustained hyperglycemia. PMID- 8636417 TI - Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice. AB - This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM 1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1. PMID- 8636418 TI - Cardiac secretion of adrenomedullin in human heart failure. AB - Adrenomedullin (ADM) is a newly discovered endogenous vasorelaxing and natriuretic peptide. Recently, we have reported that plasma ADM is increased in severe congestive heart failure (CHF) in humans and that increased immunohistochemical staining is observed in the failing human ventricular myocardium. The present study was designed to test the hypothesis that the failing human ventricle secretes ADM and that circulating ADM progressively increases with the severity of clinical CHF. Plasma ADM was significantly increased in human CHF (39.8 +/- 3.6 pg/ml, P < 0.001 vs. normal) as compared with normal subjects (14.4 +/- 2.7 pg/ml). Plasma ADM was increased in mild CHF (NYHA class II, 30.1 +/- 3.4 pg/ml, P < 0.01 vs. normal), moderate CHF (NYHA class III, 31.5 +/- 3.0 pg/ml, P < 0.01 vs. normal), and severe CHF (NYHA class IV, 66.1 +/- 9.4 pg/ml, P < 0.001 vs. normal). In 13 patients with CHF in whom plasma samples were obtained from aorta (AO), coronary sinus (CS) and anterior interventricular vein (AIV), there was a significant step-up in plasma ADM between AO and AIV (50.6 +/- 9.3 pg/ml and 62.1 +/- 11.1 pg/ml, respectively, P < 0.01) and between AO and CS (50.6 +/- 9.3 pg/ml and 58.6 +/- 11.4 pg/ml, respectively, P < 0.05). The current study demonstrates that the failing human heart secretes ADM in human CHF suggesting contribution to the increase in plasma ADM, and indicates for the first time an additional endocrine system of cardiac origin which is activated in human CHF and may function in cardiorenal regulation. PMID- 8636419 TI - In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia. AB - Gene products of all three distinct nitric oxide synthases are present in the mammalian kidney. This mosaic topography of nitric oxide synthase (NOS) isoforms probably reflects distinct functional role played by each enzyme. While nitric oxide (NO) is cytotoxic to isolated renal tubules, inhibition of NO production in vivo invariably results in the aggravation of renal dysfunction in various models of acute renal failure. We reasoned that the existing ambiguity on the role of nitric oxide in acute renal failure is in part due to the lack of selective NOS inhibitors. Phosphorothioated derivatives of antisense oligodeoxynucleotides targeting a conserved sequence within the open reading frame of the cDNA encoding the inducible NOS (iNOS) were designed to produce a selective knock-down of this enzyme. In vivo use of these antisense constructs attenuated acute renal failure in rats subjected to renal ischemia. This effect was due, at least in part, to the rescue of tubular epithelium from lethal injury. Application of antisense constructs did not affect endothelial NOS, as evidenced by a spared NO release after the infusion of bradykinin during in vivo monitoring with an NO-selective microelectrode. In conclusion, the data provide direct evidence for the cytotoxic effects of NO produced via iNOS in the course of ischemic acute renal failure, and offer a novel method to selectively prevent the induction of this enzyme. PMID- 8636420 TI - Farnesyl analogues inhibit vasoconstriction in animal and human arteries. AB - Recent studies have suggested that nonsterol, mevalonate-derived metabolites are implicated in the control of vascular tone and blood pressure. Because of the metabolic importance of farnesyl pyrophosphate, a 15-carbon (C15) intermediate of the cholesterol pathway, the vasoactive properties of the farnesyl motif were investigated. Two farnesyl analogues were used: farnesol, the natural dephosphorylated form of farnesyl pyrophosphate, and N-acetyl-S-trans,trans farnesyl-L-cysteine (AFC), a synthetic mimic of the carboxyl terminus of farnesylated proteins. Both compounds inhibited NE-induced vasoconstriction in rat aortic rings at micromolar concentration. Their action was rapid, dose dependent, and reversible. Shorter (C10) and longer (C20) isoprenols as well as N acetyl-S-geranyl-L-cysteine (C10) did not inhibit the response to NE. In contrast, N-acetyl-S-geranylgeranyl-L-cysteine (C20), exhibited vasoactive properties similar to AFC. It was further demonstrated that AFC and farnesol inhibited KCl and NaF-induced contractions, suggesting a complex action on Ca2+ channels and G protein-dependent pathways. Finally, the effect of farnesol and AFC on the NE response was reproduced in human resistance arteries. In conclusion, mevalonate-derived farnesyl analogues are potent inhibitors of vasoconstriction. The study suggests that farnesyl cellular availability is an important determinant of vascular tone in animals and humans, and provides a basis for exploring farnesyl metabolism in humans with compromised vascular function as well as for using farnesyl analogues as regulators of arterial tone in vivo. PMID- 8636422 TI - Sickle cell disease: a step closer to the dream. PMID- 8636423 TI - Do anti-ganglioside antibodies cause human peripheral neuropathies? PMID- 8636421 TI - Preconditioning rabbit cardiomyocytes: role of pH, vacuolar proton ATPase, and apoptosis. AB - Ischemic preconditioning signals through protein kinase C (PKC) to protect against myocardial infarction. This protection is characterized by diminished intracellular acidification. Acidification is also a feature of apoptosis, and several agents act to prevent apoptosis by preventing acidification through activation of ion channels and pumps to promote cytoplasmic alkalinization. We characterized metabolic inhibition, recovery, and preconditioning through a PKC dependent pathway in cardiomyocytes isolated from adult rabbit hearts. Preconditioning reduced loss of viability assessed by morphology and reduced DNA nicking. Blockade of the vacuolar proton ATPase (VPATPase) prevented the effect of preconditioning to reduce metabolic inhibition-induced acidosis, loss of viability, and DNA nicking. The beneficial effect of Na+/H+ exchange inhibition, which is thought to be effective through reduced intracellular Na+ and Ca++, was also abrogated by VPATPase blockade, suggesting that acidification even in the absence of Na+/H+ exchange may lead to cell death. We conclude that a target of PKC in mediating preconditioning is activation of the VPATPase with resultant attenuation of intracellular acidification during metabolic inhibition. Inhibition of the "death protease," interleukin-1-beta converting enzyme or related enzymes, also protected against the injury that followed metabolic inhibition. This observation, coupled with the detection of DNA nicking in cells subjected to metabolic inhibition, suggests that apoptotic cell death may be preventable in this model of ischemia/reperfusion injury. PMID- 8636424 TI - Animal models of human disease for gene therapy. PMID- 8636425 TI - Hypoxic encephalopathy after near-drowning studied by quantitative 1H-magnetic resonance spectroscopy. AB - Early prediction of outcome after global hypoxia of the brain requires accurate determination of the nature and extent of neurological injury and is cardinal for patient management. Cerebral metabolites of gray and white matter were determined sequentially after near-drowning using quantitative 1H nuclear magnetic resonance spectroscopy (MRS) in 16 children. Significant metabolite abnormalities were demonstrated in all patients compared with their age-matched normal controls. Severity of brain damage was quantified from metabolite concentrations and ratios. Loss of N-acetylaspartate, a putative neuronal marker, from gray matter preceded that observed in white matter and was more severe. Total creatine decreased, while lactate and glutamine/glutamate concentrations increased. Changes progressed with time after injury. A spectroscopic prognosis index distinguished between good outcome (n = 5) and poor outcome (n = 11) with one false negative (bad outcome after borderline MRS result) and no false positive results (100% specificity). The distinction was made with 90% sensitivity early (after 48 h) and became 100% later (by days 3 and 4). This compared with 50-75% specificity and 70-100% sensitivity based upon single clinical criteria. MRS performed sequentially in occipital gray matter provides useful objective information which can significantly enhance the ability to establish prognosis after near-drowning. PMID- 8636427 TI - Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat. AB - The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium. PMID- 8636426 TI - A somatically mutated human antiganglioside IgM antibody that induces experimental neuropathy in mice is encoded by the variable region heavy chain gene, V1-18. AB - IgM paraproteins associated with autoimmune peripheral neuropathy and anti-Pr cold agglutinins react with sialic acid epitopes present on disialylated gangliosides including GD1b, GT1b, GQ1b, and GD3. A causal relationship between the paraprotein and the neuropathy has never been proven experimentally. From peripheral blood B cells of an affected patient, we have cloned a human hybridoma secreting an antidisialosyl IgM mAb, termed Ha1, that shows identical structural and functional characteristics to its serum counterpart. Variable region analysis shows Ha1 is encoded by the same VH1 family heavy chain gene, V1-18, as the only other known anti-Pr antibody sequence and is somatically mutated, suggesting that it [correction of is] arose in vivo in response to antigenic stimulation. In the rodent peripheral nervous system, Ha1 immunolocalizes to dorsal root ganglia, motor nerve terminals, muscle spindles, myelinated axons, and nodes of Ranvier. After intraperitoneal injection of affinity-purified antibody into mice for 10 d, electrophysiological recordings from the phrenic nerve-hemidiaphragm preparation demonstrated impairment of nerve excitability and a reduction in quantal release of neurotransmitter. These data unequivocally establish that an antidisialosyl antibody can exert pathophysiological effects on the peripheral nervous system and strongly support the view that the antibody contributes to the associated human disease. PMID- 8636428 TI - Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist. AB - Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 fibroblasts. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis. PMID- 8636429 TI - Modulation of very low density lipoprotein production and clearance contributes to age- and gender- dependent hyperlipoproteinemia in apolipoprotein E3-Leiden transgenic mice. AB - Apolipoprotein E3-Leiden (APOE*3-Leiden) transgenic mice have been studied to identify factors modulating chylomicron and VLDL remnant lipoprotein metabolism. Transient elevated levels of VLDL/LDL-sized lipoproteins occurred in these mice with maximal levels during the period of rapid growth (optimum at 45 d of age). After about 100 d of age, serum cholesterol and triglyceride levels stabilized to slightly elevated levels as compared to control mice. The expression of the APOE*3-Leiden transgene was not age-dependent. In young mice the in vivo hepatic production of VLDL-triglycerides was 50% increased as compared to older mice. This is sustained by in vivo VLDL-apo B turnover studies showing increased (75%) VLDL-apo B secretion rates in young mice, whereas the VLDL-apo B clearance rate appeared not to be age dependent. On a high fat/cholesterol diet, females displayed significantly higher cholesterol levels than males (10 versus 7.0 mmol/liter, respectively). Serum levels of VLDL/LDL sized lipoproteins increased upon administration of estrogens, whereas administration of testosterone gave the opposite result. As compared to male mice, in female mice the hepatic VLDL triglyceride production rate was significantly elevated. Injection of estrogen in males also resulted in increased VLDL-triglyceride production, although not statistically significant. In vivo VLDL-apo B turnover experiments showed that the VLDL secretion rate tended to be higher in females. Although, the fractional catabolic rate of VLDL-apo B is not different between males and females, administration of estrogens in males resulted in a decreased clearance rate of VLDL, whereas administration of testosterone in females resulted in an increased clearance rate of VLDL. The latter presumably due to an inhibiting effect of testosterone on the expression of the APOE*3-Leiden transgene. We conclude that hyperlipidemia in APOE*3-Leiden transgenic mice is strongly affected by age via its effect on hepatic VLDL production rate, whereas gender influences hyperlipidemia by modulating both hepatic VLDL production and clearance rate. PMID- 8636430 TI - Altered interaction of Cis-dichlorodiammineplatinum(II)--modified alpha 2 macroglobulin (alpha 2M) with the low density lipoprotein receptor-related protein/alpha 2M receptor but not the alpha 2M signaling receptor. AB - Receptor-recognized forms of alpha 2-macroglobulin (alpha 2M*) bind to two macrophage receptors: an endocytic receptor, the low density lipoprotein receptor related protein/alpha 2M receptor (LRP/alpha 2MR), and a G protein-coupled receptor, the alpha 2M signaling receptor (alpha 2MSR). Binding of alpha 2M* to LRP/alpha 2MR but not alpha 2MSR is inhibited by receptor-associated protein. We now present binding characteristics of alpha 2MSR (kD approximately 50 pm; 1,530 sites/cell) using Scatchard analysis. We also demonstrate that chemical modification of alpha 2M* with cis-dichlorodiammineplatinum (cis-DDP) does not significantly alter binding to either receptor or signaling characteristics as compared with unmodified alpha 2M*. However, internalization by LRP/alpha 2MR is greatly affected. Cis-DDP-modified alpha 2M* (cis-DDP-alpha 2M*) and alpha 2M* show comparable internalization during a single round of endocytosis; however, cis-DDP modification of alpha 2M* results in a > or = 82% reduction in internalization involving receptor recycling and multiple rounds of endocytosis. Results from pH 5.0 dissociation and receptor recycling experiments suggest that the mechanism of decreased internalization of cis-DDP-alpha 2M* involves poor dissociation from the receptor in endosomes and a decrease in available surface receptors over the time of exposure to the ligand. PMID- 8636431 TI - Suppression of cell-mediated and humoral immune responses by an interleukin-2 immunoglobulin fusion protein in mice. AB - Interleukin-2 (IL-2) plays a pivotal role in the cellular and humoral immune responses directed against foreign antigens. We characterized the in vitro and in vivo properties of a chimeric protein consisting of mouse IL-2 fused to the mouse IgG2b Fc domains. This fusion protein binds to IL-2 and Fc receptors and supports IL-2-dependent cell proliferation but does not mediate lysis of IL-2 receptor positive cells in the presence of murine complement in vitro. However, in vivo the IL2-IgG2b fusion protein suppresses both cellular and humoral immune responses after immunization with sheep erythrocytes. Surprisingly, delayed hypersensitivity is inhibited despite a dramatic increase of splenic CD3+ and NK1.1+ lymphocytes, indicating that altered homing of IL2-IgG2b-activated lymphocytes rather than cytolysis prevents these cells from accumulating in areas of inflammation. Although in vitro the IL2-IgG2b fusion protein does not alter proliferation of B cells in response to mitogenic stimulation, IgM production in response to sheep erythrocytes is profoundly inhibited in mice treated with the IL2-IgG2b fusion protein. Since no side effects are observed, the IL2-IgG2b fusion protein may expand the therapeutic repertoire of reagents used for the treatment of allograft rejection and autoimmune diseases. PMID- 8636432 TI - Basolateral localization and export activity of the human multidrug resistance associated protein in polarized pig kidney cells. AB - The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl) glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance. PMID- 8636433 TI - Clonal T cell expansion induced by interleukin 2 therapy in blood and tumors. AB - In a phase I clinical trial on the effects of preoperative adjuvant IL-2 therapy given to patients undergoing hepatic resection of colorectal adenocarcinoma metastases, we monitored the putative induction of T cell clonal expansion in both tissues and blood. The presence of T cell clonotypes was analyzed with a PCR based method that determines V-D-J junction size patterns in T cell receptor (TCR) V beta subfamilies in samples before and after a 5-d IL-2 infusion. This high resolution method analyzing CDR3 sizes of TCR transcripts was used in conjunction with FACS analysis of the corresponding T cell subpopulations with TCR V beta-specific mAb. At time of surgery (day 8 after starting IL-2), we found in the three patients analyzed with V beta-C beta primers multiple dominant T cell clonotypes in the tumor and peritumoral tissues which had probably expanded as a result of therapy. In three control patients not treated with IL-2, multiple oligoclonal patterns were not observed with this set of primers. In the fourth control patient a unique V beta 21-C beta CDR3 pattern which corresponds to two dominant clonotypes was found in the tumor. The same dominant clonotypes identified in the tumor after IL-2 were also detectable in the blood and comparison of the profiles obtained before and after IL-2 therapy indicates that they were induced by IL-2. The relative expansion of the corresponding T cell subpopulations was maintained for varying periods of time after surgery (4-7 d and almost 2 yr in one case). Together, these results indicate that IL-2 induces marked expansion of several T cell clones. Systemic IL-2 administration may represent, either alone or as a vaccine adjuvant, an appropriate way of boosting antigen-specific immune responses. PMID- 8636434 TI - Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. AB - Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy. PMID- 8636435 TI - Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control and non insulin-dependent diabetes mellitus subjects and regulation by glucose and insulin. AB - We examined the activity of the rate-limiting enzyme for hexosamine biosynthesis, glutamine:fructose-6-phosphate amidotransferase (GFA) in human skeletal muscle cultures (HSMC), from 17 nondiabetic control and 13 subjects with non-insulin dependent diabetes. GFA activity was assayed from HSMC treated with low (5 mM) or high (20 mM) glucose and low (22 pM) or high (30 microM) concentrations of insulin. In control subjects GFA activity decreased with increasing glucose disposal rate (r = -0.68, P < 0.025). In contrast, a positive correlation existed between GFA and glucose disposal in the diabetics (r = 0.86, P < 0.005). Increased GFA activity was also correlated with body mass index in controls but not diabetics. GFA activity was significantly stimulated by high glucose (22%), high insulin (43%), and their combination (61%). GFA activity and its regulation by glucose and insulin were not significantly different in diabetic HSMC. We conclude that glucose and insulin regulate GFA activity in skeletal muscle. More importantly, our results are consistent with a regulatory role for the hexosamine pathway in human glucose homeostasis. This relationship between hexosamine biosynthesis and the regulation of glucose metabolism is altered in non-insulin dependent diabetes. PMID- 8636437 TI - A circulating, biologically inactive thyrotropin caused by a mutation in the beta subunit gene. AB - Mutation of a critical carboxy-terminal cysteine residue (C105V) in the thyrotropin-beta (TSH-beta) subunit gene was found in two related families with central hypothyroidism. Affected patients had low thyroid hormone levels and radioactive iodine uptake in the thyroid gland associated with measurable serum TSH. Thyrotropin-releasing hormone-stimulated TSH secretion did not increase thyroid hormone production in these patients as compared to their unaffected siblings, suggesting that the mutant TSH was biologically inactive in vivo. Recombinant TSH harboring this mutation was confirmed to be biologically inactive in an in vitro bioassay. Based on crystallographic structure of chorionic gonadotropin, a disulfide bond between C19 and C105 in the TSH-beta subunit is predicted to form the "buckle" of a "seat belt" that surrounds the common alpha subunit and maintains the conformation and bioactivity of the hormone. This natural mutation of the TSH-beta subunit confirms the importance of the seat belt in the family of pituitary and placental glycoprotein hormones. PMID- 8636436 TI - Dissociation of glomerular hypertrophy, cell proliferation, and glomerulosclerosis in mouse strains heterozygous for a mutation (Os) which induces a 50% reduction in nephron number. AB - We reported that the Os mutation in ROP mice induced a 50% reduction in nephron number, glomerular hypertrophy, and severe glomerulosclerosis. We examined two mouse strains with the Os mutation, ROP Os/+ and C57 Os/+ mice, to determine whether the genetic background influenced the development of glomerulosclerosis. Nephron number was decreased by 50% in both ROP Os/+ and C57 Os/+ mice, and a glomerular volume and labeling index were two- to threefold increased in both. Whereas glomerulosclerosis was severe in ROP Os/+ mice, it was absent or minimal in C57 Os/+ mice. ROP Os/+ glomeruli had two- to threefold more type IV collagen, laminin, and tenascin than C57 Os/+ by immunofluorescence microscopy. Glomerular alpha 1IV collagen and tenascin mRNA levels were increased (2.8- and 1.7-fold) in ROP Os/+ and in C57 Os/+ (1.7- and 1.4-fold) mice. Both ROP Os/+ and C57 Os/+ mice had a slight increase (1.5- and 1.7-fold) in 72-kD collagenase mRNA levels. Whereas laminin B1 mRNA levels were twofold higher in ROP +/+ than in C57 +/+ mice, there was no further change in the presence of the Os mutation. Thus, the response to the Os mutation depended on the mouse strain, since severe glomerulosclerosis occurred only in ROP Os/+ mice, even though cell proliferation and glomerular hypertrophy also were present in C57 Os/+ mice. PMID- 8636438 TI - A mutation in the lipoprotein lipase gene is the molecular basis of chylomicronemia in a colony of domestic cats. AB - Members of a domestic cat colony with chylomicronemia share many phenotypic features with human lipoprotein lipase (LPL) deficiency. Biochemical analysis reveals that these cats do have defective LPL catalytic activity and have a clinical phenotype very similar to human LPL deficiency. To determine the molecular basis underlying this biochemical phenotype, we have cloned the normal and affected cat LPL cDNAs and shown that the affected cat has a nucleotide change resulting in a substitution of arginine for glycine at residue 412 in exon 8. In vitro mutagenesis and expression studies, in addition to segregation analysis, have shown that this DNA change is the cause of LPL deficiency in this cat colony. Reduced body mass, growth rates, and increased stillbirth rates are observed in cats homozygous for this mutation. These findings show that this LPL deficient cat can serve as an animal model of human LPL deficiency and will be useful for in vivo investigation of the relationship between triglyceride rich lipoproteins and atherogenic risk and for the assessment of new approaches for treatment of LPL deficiency, including gene therapy. PMID- 8636439 TI - Biochemical and functional characterization of xenoreactive natural antibodies in hu-PBL-SCID mice. AB - An in vivo model system to understand the mechanism of xenograft rejection was established using human peripheral blood leukocyte-reconstituted SCID (hu-PBL SCID) mice. Human xenoreactive natural antibodies (XNA), of IgM and IgG subtypes, capable of binding to pig aortic endothelial cells (PAEC) were detected in the sera of hu-PBL-SCID by ELISA and flowcytometric methods. Western blot analysis of PAEC lysates showed that IgM and IgG XNA from hu-PBL-SCID recognized xenoantigens with similar molecular mass as those recognized by XNA from normal human serum (NHS). This result demonstrated that hu-PBL-SCID contained XNA representing the same repertoire as that of the NHS. XNA from NHS and hu-PBL-SCID were also able to induce intracellular Ca2+ signals in cultured PAEC several fold above the basal level. This result revealed their functional similarity and demonstrated for the first time that XNA in the absence of C can activate PAEC, which may lead to the pathology of xenograft rejection. In vivo, PAEC transplanted under the kidney capsule of hu-PBL-SCID mice showed deposition of human IgM and mouse C. In summary, the present study demonstrates that hu-PBL-SCID can serve as a useful model to characterize innate immunity against xenograft. PMID- 8636440 TI - H19, a marker of developmental transition, is reexpressed in human atherosclerotic plaques and is regulated by the insulin family of growth factors in cultured rabbit smooth muscle cells. AB - H19 is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in Wilms' tumorigenesis. In this report, we have performed in situ hybridization analysis of H19 expression during normal rabbit development and in human atherosclerotic plaques. We have also used cultured smooth muscle cells to identify H19 regulatory factors. Our data indicate that H19 expression in the developing skeletal and smooth muscles correlated with specific differentiation events in these tissues. Expression of H19 in the skeletal muscle correlated with nonproliferative, actin-positive muscle cells. In the prenatal blood vessel, H19 expression was both temporally and spatially regulated with initial loss of expression in the inner smooth muscle layers adjacent to the lumen. We also identified H19-positive cells within the adult atherosclerotic lesion and we suggest that these cells may recapitulate earlier developmental events. These results, along with the identification of the insulin family of growth factors as potent regulatory molecules for H19 expression, provide additional clues toward understanding the physiological regulation and function of H19. PMID- 8636441 TI - Angiotensin converting enzyme inhibition modulates endogenous endothelin in chronic canine thoracic inferior vena caval constriction. AB - Endothelin (ET) is a potent vasoconstrictor peptide which is elevated in plasma in congestive heart failure. Recent studies suggest an important role for angiotensin II (AII) in the activation of ET in cultured cardiomyocytes. Chronic thoracic inferior vena caval constriction (TIVCC) is a model of reduced cardiac output that mimics the neurohumoral activation observed in congestive heart failure. We hypothesized that activation of the renin-angiotensin system in TIVCC plays a role in the activation of ET and that the elevation of endogenous ET contributes to the systemic and renal vasoconstriction that characterizes this model of venous congestion. We studied conscious dogs after 7 d of TIVCC in the presence or absence of chronic angiotensin converting enzyme inhibition with enalapril. TIVCC resulted in marked activation of plasma AII and ET in plasma, right atrium, lung, and renal medulla which was further localized to cardiomyocytes, pulmonary, and renal epithelial cells. Chronic angiotensin converting enzyme inhibition abolished the increases in plasma AII and ET during TIVCC. Acute endothelin A receptor blockade with FR-139317 resulted in significant decreases in mean arterial pressure and systemic vascular resistance in TIVCC. We conclude that activation of the renin-angiotensin system contributes to the activation of circulating and local ET in TIVCC and that this activation plays an important role in the regulation of arterial pressure and systemic vascular resistance in this model of congestive failure. PMID- 8636442 TI - Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3. AB - The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5 lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5 lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5 lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3. PMID- 8636443 TI - Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen activated protein kinase by alpha 1-adrenergic receptors in Madin-Darby canine kidney cells. AB - We have characterized the mechanism whereby a G protein-coupled receptor, the alpha 1-adrenergic receptor, promotes cellular AA release via the activation of phospholipase A2 (PLA2) in Madin-Darby canine kidney (MDCK-D1) cells. Stimulation of cells with the receptor agonist epinephrine or with the protein kinase C (PKC) activator PMA increased AA release in intact cells and the activity of PLA2 in subsequently prepared cell lysates. The effects of epinephrine were mediated by alpha 1-adrenergic receptors since they were blocked by the alpha 1-adrenergic antagonist prazosin. Epinephrine- and PMA-promoted AA release and activation of the PLA2 were inhibited by AACOCF3, an inhibitor of the 85-kD cPLA2. The 85-kD cPLA2 could be immunoprecipitated from the cell lysate using a specific anti cPLA2 serum. Enhanced cPLA2 activity in cells treated with epinephrine or PMA could be recovered in such immunoprecipitates, thus directly demonstrating that alpha 1-adrenergic receptors activate the 85-kD cPLA2. Activation of cPLA2 in cell lysates by PMA or epinephrine could be reversed by treatment of lysates with exogenous phosphatase. In addition, both PMA and epinephrine induced a molecular weight shift, consistent with phosphorylation, as well as an increase in activity of mitogen-activated protein (MAP) kinase. The time course of epinephrine promoted activation of MAP kinase preceded that of the accumulation of released AA and correlated with the time course of cPLA2 activation. Down-regulation of PKC by overnight incubation of cells with PMA or inhibition of PKC with the PKC inhibitor sphingosine blocked the stimulation of MAP kinase by epinephrine and, correspondingly, epinephrine-promoted AA release was inhibited under these conditions. Similarly, blockade of MAP kinase stimulation by the MAP kinase cascade inhibitor PD098059 inhibited epinephrine-promoted AA release. The sensitivity to Ca2+ was similar, although the maximal activity of cPLA2 was enhanced by treatment of cells with epinephrine or PMA. The data thus demonstrate that in MDCK-D1 cells alpha 1-adrenergic receptors regulate AA release through phosphorylation-dependent activation of the 85-kD cPLA2 by MAP kinase subsequent to activation of PKC. This may represent a general mechanism by which G protein coupled receptors stimulate AA release and formation of products of AA metabolism. PMID- 8636444 TI - The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome. AB - Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome. PMID- 8636446 TI - Molecular determinants of acute inflammatory responses to biomaterials. AB - The frequent inflammatory responses to implanted medical devices are puzzling in view of the inert and nontoxic nature of most biomaterials. Because implant surfaces spontaneously adsorb host proteins, this proteinaceous film is probably important in the subsequent attraction of phagocytes. In fact, earlier we found that acute inflammatory responses to experimental polyethylene terephthalate implants in mice require the precedent adsorption of one particular host protein, fibrinogen. The present investigations were aimed at defining the molecular determinants of fibrinogen-mediated acute inflammatory responses to implanted biomaterials. We find: (a) plasmin degradation of purified fibrinogen into defined domains reveals that the proinflammatory activity resides within the D fragment, which contains neither the fibrin cross-linking sites nor RGD sequences; (b) the major (and, perhaps, exclusive) proinflammatory sequence appears to be fibrinogen gamma 190-202, previously shown to interact with CD11b/CD18 (Mac-1). The chemically synthesized peptide, cross-linked to albumin (which itself does not promote inflammatory responses), mimics the proinflammatory effect of adsorbed native fibrinogen; and (c) this sequence probably promotes inflammatory responses through interactions with Mac-1 because phagocyte accumulation on experimental implants is almost completely abrogated by administration of recombinant neutrophil inhibitory factor (which blocks CD11b fibrin(ogen) interaction). We conclude that improved knowledge of such surface protein-phagocyte interactions may permit the future development of more biocompatible implantable materials. PMID- 8636445 TI - NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF- I: possible role of nitric oxide in muscle protein synthesis. AB - In human skeletal muscle, insulin-like growth factor-I (IGF-I) exerts both growth hormone-like (increase in protein synthesis) and insulin-like (decrease in protein degradation and increase in glucose uptake) actions and augments forearm blood flow two- to threefold. This study was designed to address whether (a) the increase in blood flow due to IGF-I could be blocked by an inhibitor of nitric oxide synthase; and (b) the metabolic actions of IGF-I were altered by use of a nitric oxide synthase inhibitor. Forearm blood flow, glucose, lactate, oxygen, nitrite, and phenylalanine balances and phenylalanine kinetics were studied in a total of 17 healthy, adult volunteers after an overnight fast in two different protocols. In protocol 1, after basal samples IGF-I was infused alone for 4 h with samples repeated during the last 30 min. After the 4-h sample period, NG monomethyl-L-arginine (L-NMMA) was infused into the brachial artery for 2 h to bring flow back to baseline and repeat samples were taken (6 h). In response to IGF-I alone, forearm blood flow rose from 3.8 +/- 1.0 (bas) to 7.9 +/- l.9 (4 h) ml/min/100 ml (P < 0.01) and was reduced back to baseline by L-NMMA at 6 h (P < 0.01). In protocol 1, IGF-I alone increased forearm nitrite release at 4 h (P < 0.03), which was reduced back to baseline by L-NMMA at 6 h (P < 0.05). Despite the reduction in flow with L-NMMA, IGF+L-NMMA yielded increases in glucose uptake (P < 0.005), lactate release (P < 0.04), oxygen uptake (P < 0.01), and a positive shift in phenylalanine balance (P < 0.01) due to both an increase in muscle protein synthesis (P < 0.02) and a decrease in protein degradation (P < 0.03). In protocol 2, L-NMMA was coinfused with IGF-I for 6 h, with the dose titrated to keep blood flow +/- 25% of baseline. Coinfusion of L-NMMA restrained blood flow to baseline and also yielded the same, significant metabolic effects, except that no significant increase in muscle protein synthesis was detected. These observations suggest: (a) that IGF-I increases blood flow through a nitric oxide dependent mechanism; (b) that total blood flow does not affect the insulin-like response of muscle to IGF-I; and (c) that nitric oxide may be required for the protein synthetic (growth hormone-like) response of muscle to IGF-I. PMID- 8636447 TI - Identification of clonally expanded T cells in rheumatoid arthritis using a sequence enrichment nuclease assay. AB - Identification of expanded clones engaged in immune and autoimmune responses is still imperfect, since they are often diluted by irrelevant cells expressing diverse specificities. To efficiently characterize T cell receptors expressed by clonally expanded lymphocytes in rheumatoid arthritis (RA) and other inflammatory conditions, we developed an assay system, termed sequence enrichment nuclease assay (SENA). Key elements of SENA are the efficiency of heat-denatured DNA strand reassociation, which increases exponentially with concentration, and the elimination of unhybridized sequences by single-strand-specific DNase. T cell clonal expansions were identified primarily in synovial fluids, but also in peripheral blood of RA patients. Synovial fluids had more prominent expansions in the CD8 than the CD4 subset, whereas clonal expansions in the CD4 subset predominated among peripheral blood lymphocytes. Dominant clones exhibited diverse sequences with no clear conservation of junctional motifs, although the same amino acid sequence was identified in two patients. In most instances, dominant clones in the blood were discordant to those in the corresponding synovial fluid, suggesting local stimulation or preferential sequestration of T cells displaying particular specifities. PMID- 8636448 TI - Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects. AB - We studied 24-h profiles of circulating leptin levels using a sensitive and specific RIA in lean controls and obese subjects with or without non-insulin dependent diabetes mellitus (NIDDM) during normal routine activity. Serum leptin levels were significantly higher in obese (41.7 +/- 9.0 ng/ml; n = 11) and obese NIDDM (30.8 +/- 6.7; n = 9) subjects compared with those in lean controls (12.0 +/- 4.4, n = 6). In all the three groups, serum leptin levels were highest between midnight and early morning hours and lowest around noon to midafternoon. The nocturnal rise in leptin levels was significant when data were analyzed by ANOVA (lean: F = 3.17, P < 0.0001, n = 4; obese: F = 2.02, P < 0.005, n = 11; and obese NIDDM: F = 4.9, P < 0.0001, n = 5). The average circadian amplitude between acrophase and nadir was 75.6% in lean, 51.7%, in obese and 60.7% in obese NIDDM groups, respectively. No significant correlations (P > 0.05) were observed between circulating levels of leptin and either insulin or glucose levels in any of the 20 subjects studied for 24-h profiles. The nocturnal rise in leptin observed in the present study resembles those reported for prolactin, thyroid stimulating hormone, and free fatty acids. We speculate that the nocturnal rise in leptin could have an effect in suppressing appetite during the night while sleeping. PMID- 8636450 TI - Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs. AB - The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects, 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel, interfered with healing by PDL and bone-derived cells in the submembranous space. PMID- 8636449 TI - Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans. AB - Allelic variants of Fc gamma R confer distinct phagocytic capacities providing a mechanism for heritable susceptibility to immune complex disease. Human Fc gamma RIIa has two codominantly expressed alleles, R131 and H131, which differ substantially in their ability to ligate human IgG2. The Fc gamma RIIa-H131 is the only human Fc gamma R which recognizes IgG2 efficiently and optimal IgG2 handling occurs only in the homozygous state. Therefore, since immune complex clearance is essential in SLE, we hypothesized that Fc gamma RIIA genes are important disease susceptibility factors for SLE, particularly lupus nephritis. In a two-stage cross-sectional study, we compared the distribution of Fc gamma RIIA alleles in African Americans with SLE to that in African American non-SLE controls. A pilot study of 43 SLE patients and 39 controls demonstrated a skewed distribution of Fc gamma RIIA alleles, with only 9% of SLE patients homozygous for Fc gamma RIIa-H131 compared with 36% of controls (odds ratio, 0.18; 95% CI, 0.05-0.69, P = 0.009). This was confirmed with a multicenter study of 214 SLE patients and 100 non-SLE controls. The altered distribution of Fc gamma RIIA alleles was most striking in lupus nephritis. Trend analysis of the genotype distribution showed a highly significant decrease in Fc gamma RIIA-H131 as the likelihood for lupus nephritis increased (P = 0.0004) consistent with a protective effect of the Fc gamma RIIA-H131 gene. The skewing in the distribution of Fc gamma RIIA alleles identifies this gene as a risk factor with pathophysiologic importance for the SLE diathesis in African Americans. PMID- 8636451 TI - Root cementum appearance in healthy monkeys and periodontitis-prone patients after different etching modalities. AB - The purpose of the present study was to compare cementum surfaces after etching at neutral or low pH in both healthy monkey teeth and periodontitis-affected human teeth. 16 monkey teeth and 16 human periodontitis-affected teeth were used. Etching with phosphoric and citric acids as well as EDTA was performed on the following surfaces: healthy monkey cementum, human cementum surfaces coronal and apical to the level of periodontal breakdown as well as exposed human dentin surfaces. Results indicate a profoundly higher capacity of EDTA to selectively expose collagen fibers in both healthy cementum surfaces and periodontitis affected dentin surfaces compared to agents operating at low pH which seemed to erode the surfaces to varying degrees. Variable results were seen on cementum surfaces which had been exposed to the environment of the periodontal pocket or the oral cavity. In view of this, it would seem preferable to mechanically remove the superficial layer of "diseased" cementum prior to the etching procedure. In conventional periodontal surgery, etching may be of limited value. However, in regenerative procedures, exposure of an intact collagenous matrix provides a matrix for retention of implants of biologically active substances such as growth factors, in addition to serving as a biocompatible surface for periodontal ligament cell colonization. PMID- 8636452 TI - The efficacy of an anti-gingivitis chewing gum. AB - Chlorhexidine is a well-established agent used for the control of supragingival plaque but is not without disadvantages, such as tooth staining, which limits its clinical applications to short-term use. This clinical trial studied the clinical effectiveness and stain-forming potential of chlorhexidine in a chewing gum base. Subjects (151) were screened for baseline plaque and gingival indices before receiving a dental prophylaxis and randomized into 3 treatment groups: group 1 chewed 2 pieces of chlorhexidine diacetate gum for 10 min 2x a day (total daily chlorhexidine = 20 mg), group 2 chewed 2 pieces of placebo gum for 10 min 2x a day and group 3 rinsed with 10 ml of 0.2% chlorhexidine gluconate mouthwash for 1 min 2x per day (total daily chlorhexidine = 40 mg). Plaque, gingivitis and stain evaluations were made at 4 and 8 weeks. Plaque and bleeding scores were significantly lower at 4 and 8 weeks in the chlorhexidine gum group compared to the placebo gum group and similar at 8 weeks to the rinse group. Stain intensity at week 8 was significantly less for the chlorhexidine gum than rinse. The staining measured by extent was also less with the chlorhexidine gum than the rinse, but the difference was not significant at week 4. At week 8, stain extent was significantly lower in the chlorhexidine gum group than chlorhexidine rinse. In conclusion, the results of this study demonstrate that this chlorhexidine chewing gum used with normal tooth cleaning provides similar adjunctive benefits to oral hygiene and gingival health as a 0.2% chlorhexidine rinse. PMID- 8636453 TI - Topical metronidazole application compared with subgingival scaling. A clinical and microbiological study on recall patients. AB - The aim of this study was to compare the topical application of a metronidazole 25% dental gel with subgingival scaling. 30 patients from the recall program participated in this open randomised study with split-mouth design. Pocket probing depths (PPD) and bleeding on probing (BOP) were measured before and 2, 12 and 24 weeks after the end of the treatment period. In addition, subgingival plaque samples were taken from all mesial sites and analysed with dark-field microscopy. All patients had at least 1 tooth in each quadrant with a PPD of 5 mm or more that should bleeding on probing, when entering the study. The treatment consisted of 2 applications of the dental gel in 2 randomly selected quadrants (on days 0 and 7) as well as simultaneous subgingival scaling of the remaining quadrants. Oral hygiene instruction was given on day 21. The average PPD and the average frequency of BOP were calculated for all sites with an initial PPD of 5 mm or more and continued at each examination, using the same sites. The statistical analyses showed that both treatments were effective in reducing PPD and BOP over the 6-month period. At the end of the follow-up period, the mean reduction in PPD was 1.3 mm after gel treatment and 1.5 mm after subgingival scaling. BOP was reduced by 35% and 42%, respectively. No significant differences between the 2 treatments were detected. Dark-field microscopy showed a shift toward a more healthy microflora for both treatment modalities; this persisted throughout the 6-month period. Application of a 25% metronidazole dental gel on recall patients seems to be as effective on the investigated clinical and microbiological parameters as subgingival scaling. PMID- 8636454 TI - Oral fluid elastase as an indicator of periodontal health. AB - The study was designed to find out whether oral elastase activity could be used as a simple biochemical indicator of periodontal health. Both stimulated whole saliva and water rinse samples were collected from subjects with different degrees of adult periodontitis, gingivitis or healthy periodontium. In both sample types, elastase was mostly bound to insoluble fraction and preferred valine containing synthetic substrate, similar to neutrophil elastase. The elastase measurement required very little manipulation or time and its reproducibility was found to be good. The elastase levels were found to be negligible in edentulous subjects and usually very low in subjects with healthy periodontium. In about 85% of periodontitis cases having at least 1 deep periodontal pocket ( > or = 6 mm), clearly elevated elastases levels were detected in both the saliva and r rinse samples. In advanced periodontitis cases, the colour reaction took place in 0.5 to 2 h. In localized periodontitis cases, 2 to 18-h incubations were required for positive reaction. There was a good correlation between the elastase activity and the number of deep periodontal pockets and the average community periodontal index of the subjects. Elastase activity was not a good indicator of gingivitis. About 45% of gingivitis cases were positive with the elastase test, and the enzyme values were not significantly increased in experimental gingivitis. In a longitudinal study on advanced periodontitis cases, elastase levels dropped dramatically as a result of clinically successful therapy, close to the values of healthy subjects. The oral elastase test could serve as a valuable adjunct in periodontal screening and assessment of treatment efficacy. PMID- 8636455 TI - Increased release of free oxygen radicals from peripheral neutrophils in adult periodontitis after Fc delta-receptor stimulation. AB - The release of free oxygen radicals and degranulation was studied in neutrophils from 14 patients with adult periodontitis and 14 age- and sex-matched healthy controls. The neutrophils were activated by Fc gamma-receptor stimulation, using Staphylococcus aureus opsonized with gamma globulin. Release of oxygen radicals was measured as luminol-enhanced chemiluminescence. Degranulation was assessed as release of elastase, measured with a specific substrate and as release of lactoferrin measured with ELISA. The neutrophils from the patients showed a significantly higher chemiluminescence and a slightly higher release of elastase, whereas the release of lactoferrin was the same in both groups. In contrast, the ratio between the 2 degranulation products, elastase and lactoferrin, was significantly higher in the group with periodontitis. A flow cytometric analysis of the membrane expression of the adhesion molecules CD 11a, CD 11b, CD 15, CD 16, CD 35 and Mel 14 showed no differences in the median immunofluorescence between the 2 groups. This study showed a more than 2-fold higher release of free oxygen radicals from Fc-gamma-receptor stimulated neutrophils compared with healthy controls, which indicates a specific neutrophil-associated host response in adult periodontitis. PMID- 8636456 TI - Effect of toothbrush wear on plaque control. AB - The purpose of this study was to investigate the effect of progressive toothbrush wear on plaque control. At baseline (week 0), each of 20 subjects was given a new toothbrush which they used for the 9-week period of the study. At weeks 0, 3 and 6, all plaque was professionally removed. The amount of plaque which accumulated in each of the 3 successive 3-week experimental periods was assessed at weeks 3, 6 and 9. Toothbrush wear was evaluated by measuring the increase in the brushing surface area of toothbrushes at weeks 3, 6 and 9 as compared with week 0. The brushing surface area was measured by computer analysis of tracings of the brushing surface outlines obtained from standardized photographs. Despite progressive toothbrush wear, the amount of plaque which accumulated in each successive 3-week period decreased. The decrease in plaque scores between weeks 3 and 6 and between weeks 3 and 9 were found to be highly significant (p < 0.001). Toothbrush wear varied widely amongst the subjects. When plaque scores were evaluated for the 10 subjects with highest toothbrush wear, and the 10 with lowest wear, no significant differences were found between the 2 subgroups. Under the experimental conditions of this study, progressive toothbrush wear did not lead to a decrease in plaque control. The improvement in plaque scores may have been due to motivational effects resulting from study participation and anticipation of oral examinations. It was concluded that the wear status of a toothbrush may not be critical in ensuring optimal plaque control. PMID- 8636457 TI - Root surface etching at neutral pH promotes periodontal healing. AB - The purpose of the present investigation was to examine whether an etching agent operating at neutral pH (EDTA) can enhance healing compared to a low pH etching agent (citric acid) in an animal model. Maxillary molars and premolars, in total 32 teeth, in 4 monkeys were divided between test (EDTA or citric acid treatment) and matched control groups. Periodontal surgery on both palatal and buccal roots using the dehiscence model was performed with or without root surface etching. Healing results were evaluated histomorphometrically after 8 weeks. The statistically significant differences between EDTA treated surfaces (n=15) and control surfaces (n=11) were approximately 10% less failure (gingival recession and periodontal pocket), 10 to 15% more total histological attachment (long epithelial junction, connective tissue and reparative cementum), approximately 20% less long epithelial junction and approximately 20% more connective tissue in roots etched with EDTA. The statistically significant differences between citric acid-treated surfaces (n=14) and control surfaces (n=11) were approximately 10% more connective tissue and 15% less long epithelial junction in the citric acid etched roots. Thus, etching with EDTA appeared to improve healing, avoiding the superficial necrotizing effect on exposed periodontal tissues by citric acid documented in previous studies. Although etching at present is not routinely applied in conventional periodontal therapy, future potential applications of etching at neutral pH may include exposure of the collagenous matrix of dentin for retention of biologically active substances, such as growth factors. Such treatment may be argued to produce a biocompatible surface more conducive to periodontal membrane cell colonization after removal of root-surface- associated smear without compromising the vitality of the surrounding periodontium. PMID- 8636458 TI - Examiner agreement on periodontal indices during dental surveys of elders. AB - Indices used to evaluate periodontal health have been widely accepted in epidemiological studies, yet their reliability cannot be guaranteed. The aim of this study was to evaluate the reliability of periodontal indices applied on elders. 19 elderly subjects, 73-years-old on average, were examined at a 1st appointment by 2 independent examiners. They were re-examined 2 weeks later during a 2nd session. The examinations were performed in a dental chair with good illumination. Periodontal health was evaluated using the community periodontal index of treatment need, and tooth mobility was evaluated using 2 different indices. Inter and intra-examiner agreements were evaluated using kappa statistics. Taken as an overall measurement, the CPITN was a reliable assessment of periodontal treatment need in elders. Disagreement occurred mainly on the evaluation of bleeding and shallow pockets. The detection of fairly mobile teeth was reliable; however, the performance of the more sensitive scale was deceptive. it seems that, in the case of tooth mobility, a choice has to be made between sensitivity or reproducibility. It can be concluded that examiners should be trained carefully since the reliability of the CPITN and tooth mobility evaluation were good but close to a critical level for which an agreement is classified as poor. PMID- 8636459 TI - The effects of antimicrobial mouthrinses on de novo plaque formation at sites with healthy and inflamed gingivae. AB - The objective of the present investigation was to evaluate to what extent mouthrinses containing triclosan and chlorhexidine may modify the amount of de novo plaque that forms on tooth surfaces adjacent to healthy and inflamed gingival units. 10 volunteers were recruited. On day 0, gingival crevicular fluid (GCF) was obtained at predetermined sites and gingivitis (GI) was assessed. A careful oral prophylaxis was given to each of the volunteers who subsequently abstained from all mechanical plaque control measures for the following 18 days. During the first 4 days (rinse phase I), they rinsed with either 0.12% chlorhexidine, 0.06% triclosan or placebo solution. Clinical examinations (GCF, GI) were repeated and the amount of plaque formed determined on days 4, 7 and 14. On day 14, the participants received a new professional tooth cleaning after which rinse phase II was initiated. During this 2nd phase, the participants rinsed for 4 days with the same mouthwash preparation and in the same manner as during rinse phase I. The examinations were repeated on day 18. Each participant received a comprehensive oral prophylaxisis and was instructed to perform meticulous mechanical plaque control during the following 4 weeks. A 2nd experimental period was then initiated. A total of 3 experimental periods were repeated until all subjects had rinsed with the 3 different mouthwash preparations. The results demonstrated (i) that significantly more plaque formed at sites with gingivitis than at surfaces adjacent to healthy gingival units and (ii) pre-existing gingivitis significantly increased the amount of de novo plaque that formed in subjects who rinsed with mouthwash preparations containing chlorhexidine and triclosan. PMID- 8636461 TI - Metamorphosis of spinal-projecting neurons in the brain of the sea lamprey during transformation of the larva to adult: normal anatomy and response to axotomy. AB - The spinal projecting system of the sea lamprey (Petromyzon marinus) has been used extensively in studies of axonal regeneration in both larvae and adults. However, little is known about the changes that are undergone by this system during metamorphosis. In order to determine the developmental changes in the size of the descending spinal projection and in the morphology of its neurons, larval, transforming, and adult lamprey brains were labeled by retrograde transport of horseradish peroxidase (HRP) injected into the spinal cord at 25% of body length. Examination of brain wholemount preparations revealed that the total number of labeled neurons doubled during metamorphosis. Most of this increase could be explained by elongation of reticulospinal axons from the rostralmost segments of the spinal cord to locations caudal to the injection site. There were no additions or deletions of either identified reticulospinal neurons or of reticulospinal nuclear groups between the larval and the adult stages. The proportions of Muller and Mauthner cells that were labeled reached a maximum of 93% during the early stages of metamorphosis. Axons of these neurons are known to project almost the entire length of the cord, even in larvae. Therefore, the efficiency of retrograde transport appears to be greater during metamorphosis than during larval or adult stages. While changes in efficiency of retrograde transport could account for some of the apparent increase in reticulospinal neuron numbers between larvae and animals undergoing metamorphosis, this could not contribute to the further increase in the apparent size of the reticulospinal system in the adult, since efficiency of retrograde labeling in these animals was lower than that at earlier stages. With retrograde labeling, a significant increase was seen in the profusion of dendritic arborization of some Muller and Mauthner cells during the early stages of metamorphosis. This correlated with an increase in the incidence of extreme axonal die-back, as indicated by the presence of retraction bulbs within the brainstem. However, intracellular injection of Neurobiotin in untransected animals showed similar degrees of dendritic arborization at all examined stages of development. Therefore, the dendritic profusion did not reflect developmental changes in neuronal morphology but rather reflected an increased sensitivity to axotomy during metamorphosis. We conclude that, during the transformation of the lamprey from the large larval to the adult form, there is little change in either the size or the dendritic morphology of the identified giant reticulospinal neurons. With respect to the smaller reticulospinal neurons, the distance of projection of many of their axons increases during metamorphosis, but there is very little increase in the number of reticulospinal neurons. PMID- 8636460 TI - Heterogeneity of astrocytes in human optic nerve head. AB - Previous studies demonstrated regional differences in the synthesis of extracellular matrix by astrocytes during optic nerve head (ONH) maturation and in glaucomatous optic neuropathy, suggesting heterogeneity of astrocytes. To characterize different types of glial cells in human fetal and adult ONH, we used a variety of neural cell markers such as HNK-1/N-CAM, A2B5, galactocerebroside (GalC), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP). Cryostat or paraffin sections were prepared from fetal (16-25 weeks) and mature (8 months to 75 years old) ONH and processed for standard single/double immunocytochemistry. Two subpopulations of type 7 astrocytes were present in the mature prelaminar and laminar regions. Glial cells expressing only GFAP were identified as type 1A astrocytes at the edges of the cribriform plates. Cells forming the glial columns and lining the cribriform plates expressed both GFAP and HNK-1/N-CAM and were identified as type 1B astrocytes. In the myelinated nerve, type 1A astrocytes form the glial limiting membrane. Cells labeled with GFAP and A2B5 were identified as type 2 astrocytes, and GFAP-negative cells labeled with GalC, MBP, and HNK-1/N-CAM were identified as oligodendrocytes. In fetal ONH, all glial cells expressed HNK-1/N-CAM. In older fetal ONH, some glial cells also expressed GFAP. No type 2 astrocytes or oligodendrocytes were present in the fetal ONH. In conclusion, at least two subpopulations of type 1 astrocytes exist in human ONH: Type 1A astrocytes may serve as structural support for axons; type 1B astrocytes, which retain the developmental neural marker HNK-1/N-CAM, may have a more complex function by interfacing between blood vessels and other connective tissue surfaces. These findings demonstrate the heterogeneity of astrocytes in the human ONH and suggest differential regional responses to changes in their microenvironment. PMID- 8636462 TI - Synaptic inputs to single neurons in the lateral geniculate nuclei of normal and monocularly deprived squirrel monkeys. AB - Neurons in the dorsal lateral geniculate nucleus (dLGN) of normal and monocularly lid-sutured squirrel monkeys were recorded electrophysiologically, and some were injected intracellularly with horseradish peroxidase (HRP) to examine and compare their synaptic inputs. Limited tests of the receptive field properties did not show any differences between the normal, nondeprived, or deprived neurons. Sixteen injected neurons were examined at the light microscopic level with most of these located in the P-laminae (n = 14). Ten of these were either from normal monkeys (n = 9) or received input from the nondeprived eye of a monocularly deprived monkey (n = 1). The remaining six neurons received input from the deprived eye. The dendritic trees of deprived neurons did not differ from those of normal or nondeprived neurons. Three normal and five deprived neurons from the P-laminae were examined at the electron microscopic level. Afferent distributions were not significantly different between normal and deprived neurons. Retinal, cortical, and gamma aminobutyric (GABA)ergic afferents accounted for nearly all inputs (avg., 42%, 23%, and 32%, respectively) and selectively contacted proximal, distal, or all parts of the dendrites. Overall, synaptic densities (synapses per length of dendrite) were high proximally and decreased with distance from the soma. However, the synaptic densities onto deprived neurons were higher at all distances compared to those onto normal neurons. Furthermore, HRP-filled deprived neurons received an average of 25 synapses onto their somata compared with only an average of 7 somal synapses on the HRP-filled normal neurons. Most of the increase in the number of synapses onto the deprived neurons was from GABAergic type profiles. This abnormality of the deprived neurons of the dLGN could be the underlying cause of their lesser responses compared with normal or nondeprived dLGN neurons. It could also be the initial stage that causes blindness in monocularly lid-sutured primates. PMID- 8636463 TI - Centrifugal projections upon the retina: an anterograde tracing study in the pigeon (Columba livia). AB - Previous work has shown that the avian retina receives two types of centrifugal fibers from the brain. These types can be distinguished based on the size and the morphology of their terminal endings and have been termed convergent and divergent. The centrifugal fibers arise from the isthmooptic nucleus (ION) and the surrounding ectopic cell region (ECR). We used injections of anterograde tracers either to the ION/ECR or to the ECR only to determine the morphology, depth of termination, and regional distribution of the centrifugal fibers arising from each. We found that the ECR gives rise only to the divergent type of the centrifugal fiber, whereas the ION gives rise mainly to the convergent type but may also send some fibers of the divergent type. Most of the fibers project contralaterally, although a few from the ECR project ipsilaterally. The terminals of either type are not uniformly distributed throughout the retina; instead, they are found mainly in the inferior, midtemporal, to nasal portion of the retina and appear to avoid the fovea and most of the red field. By comparison, the ION receives a major projection from portions of the tectum that receive input from the fovea and the red field in a type of neural loop. The neural loop does not project to the same point (homotopic), but projects from the red field to the inferior retina (heterotopic), as was recently proposed by Holden (1990; Vis. Neurosci. 4:493-497). The distribution of centrifugal axons corresponds to displaced ganglion cells that selectively innervate the nuclei of the accessory optic system (AOS), including the nucleus of the basal optic root (dorsal, ventral, and lateral) and the nucleus lentiformis mesencephali, pars magnocellularis. We suggest that the centrifugal axons act by increasing the gain on the AOS, thereby enhancing retinal stabilization of gaze with improved accuracy of pecking of small objects. PMID- 8636464 TI - Amygdala efferents form inhibitory-type synapses with a subpopulation of catecholaminergic neurons in the rat Nucleus tractus solitarius. AB - The central nucleus of the amygdala (CNA) integrates visceral responses to stress partially through efferent projections to portions of the medial nuclei of the solitary tracts (mNTS) containing catecholaminergic neurons. To determine anatomical sites for CNA modulation of these neurons, immunoperoxidase detection of anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) was combined with immunogold-silver labeling of the catecholamine-synthesizing enzyme, tyrosine hydroxylase, in adult rat mNTS. From 350 anterogradely labeled terminals identified within the intermediate mNTS, 30% formed symmetric, inhibitory-type synapses and the remainder lacked recognized junctions as seen within a single plane of section. Of the terminals forming symmetric synapses, 16% were presynaptic to tyrosine hydroxylase immunoreactive dendrites and the remainder to unlabeled dendrites. The level of tyrosine hydroxylase immunoreactivity as assessed by density of gold-silver particles was significantly lower in dendrites receiving synaptic input from CNA efferents as compared with dendrites of the same sizes (2.0 microns 2 in mean area) which received synapses from unlabeled terminals or lacked recognizable synaptic inputs. When separately examined without regard to afferent input, the medium- and larger-sized dendrites having mean cross-sectional areas of 1-3 microns 2 also contained significantly less tyrosine hydroxylase immunoreactivity than small (< 1 micron 2) dendrites. These results suggest that CNA efferents to the mNTS inhibit non-catecholamine-containing neurons and a subpopulation of catecholaminergic neurons distinguished by their low levels of tyrosine hydroxylase. The findings also indicate that small, presumably more distal, dendrites in the intermediate mNTS may synthesize and/or release catecholamines. PMID- 8636465 TI - Migration of A7 immortalized astrocytic cells grafted into the adult rat striatum. AB - The A7 cell line is an SV40 large T antigen-immortalized astrocyte cell line produced from the neonatal rat optic nerve. Previous studies have demonstrated that A7 cells provide a favorable environment for the survival and growth of cultured neurons and can also stimulate axonal growth after grafting into the rat striatum. The current study was designed to investigate whether A7 cells grafted into adult rat striatum can migrate away from the implantation site. A7 cells were labelled in culture by incorporation of bromodeoxyuridine (BrdU) or by expression of an alkaline phosphatase transgene. The labelled cells were then transplanted into the left striatum of normal adult rats by introducing a blunt end 22 gauge needle through a trephine hole. The rats were euthanized at periods of up to 30 days after grafting. The A7 cells did not appear to alter the cytoarchitecture of the surrounding brain parenchyma. Labelled A7 cells were observed in both gray and white matter areas, and many were located in areas free of damage due to the implantation procedure. The migration of the BrdU-labelled A7 cells with respect to the implantation needle track was determined on coronal sections. The radial migration distance from the needle tract was similarly determined on horizontal sections. A7 cells migrated progressively longer distances with increasing survival time of the animals: The largest migration distance (1,125 +/- 52 microns) occurred at 30 days after grafting with an estimated migration rate of 31 microns per day. There was no significant directional polarity in the migration of these cells within the striatum. Some of the labelled A7 nuclear profiles were associated with blood vessels, some appeared to be associated with fiber bundles within the striatum, and some were found within the gray matter without apparent association with any anatomical structure. These results demonstrate that A7 immortalized astrocytic cells migrate away from a single implantation site following grafting into the adult rat striatum to populate a large area of the striatum. PMID- 8636466 TI - Neuropeptide changes following excitotoxic lesion of the insular cortex in rats. AB - Following middle cerebral artery occlusion in Wistar rats, the immunoreactivity of neuropeptide Y increased ipsilaterally in the insular cortex and basolateral nucleus of the amygdala. In addition, the immunoreactivity of leucine-enkephalin, dynorphin, and neurotensin increased in the ipsilateral central nucleus of the amygdala. The amygdalar neurochemical changes are likely the result of damage to the insular cortex, although other cortical areas were also affected by the ischemia. To investigate whether damage to the insular cortex is essential in eliciting these changes, a localized lesion of the right or left insular cortex was produced by microinjection of D,L-homocysteic acid. Control animals received injections of vehicle into the right or left insular cortex or D,L-homocysteic acid into the right primary somatosensory cortex. Neurochemical changes were examined immunohistochemically with the peroxidase-antiperoxidase reaction 5 days after the injection. The immunoreactivity of neuropeptide Y increased locally after excitotoxic damage to the insular cortex or primary somatosensory cortex. The amygdalar neurochemical changes, including neuropeptide Y increase in the basolateral nucleus and leucine-enkephalin, dynorphin, and neurotensin increase in the central nucleus, were seen only when the ipsilateral insular cortex was lesioned. These neurochemical changes were similar to those seen 5 days after middle cerebral artery occlusion. Our findings indicate that damage to the insular cortex is essential in eliciting the neurochemical changes in the ipsilateral amygdala. In addition, the change in neuropeptide Y in the cortex appears to be a local reaction occurring irrespective of location of the lesion and glutamate receptor activation may be involved. PMID- 8636467 TI - Postnatal development of periodontal ruffini endings in rat incisors: an immunoelectron microscopic study using protein gene product 9.5 (PGP 9.5) antibody. AB - Postnatal development of Ruffini endings was ultrastructurally investigated in the upper incisors of the rat from 1 day to 60 days after birth by means of protein gene product 9.5 (PGP 9.5) immunocytochemistry. The immunostaining with PGP 9.5 antibody clearly demonstrated chronological alterations of the distribution and ultrastructure of the Ruffini endings during postnatal development. At 1 day after birth, the PGP 9.5-positive nerve terminals contained a few mitochondria and vesicles immunonegative for PGP 9.5. Dendritic terminals appeared at 4 days after birth, with a small number of expanded or bulbous portions. These expanded portions possessed morphological features similar to those of the growth cone: several mitochondria and various kinds of vesicles. Typical Ruffini endings with dendritic ramification and expanded portions appeared 7-11 days after birth. At this stage, parts of the axon terminals extended through the slits of Schwann cell covering and formed finger-like projections called axonal spines. These Ruffini endings increased dramatically in number after 24-26 days and were identical in density and morphology to those seen in adult rats. After the commencement of the occlusion between the incisors, the number of large mitochondria increased, in contrast to the decrease of the vesicles in the axon terminals. Moreover, the axonal spines increased both in number and in length. Thus, the periodontal nerve endings showed stage-specific morphological features intimately related in timing to tooth eruption and occlusion. Functional stimuli possibly contribute to the final differentiation and maturation of the periodontal Ruffini endings. PMID- 8636468 TI - Laminar patterns of expression of GABA-A receptor subunit mRNAs in monkey sensory motor cortex. AB - Radioactive complementary RNA probes, made from monkey-specific cDNAs specific for the alpha 1, alpha 2, alpha 4, alpha 5, beta 1, beta 2, and gamma 2 subunits of the gamma-aminobutyric acid A (GABAA) receptor were used for in situ hybridization histochemistry of the primary motor, somatosensory, and anterior parietal areas of the cerebral cortex in macaque monkeys. mRNAs for the alpha 1, beta 2, and gamma 2 subunit polypeptides, which form receptors with the full range of classical properties, are expressed at much higher levels in all areas and show laminar- and sublaminar-specific concentrations. alpha 2, alpha 4, alpha 5, and beta 1 subunit transcripts are expressed at much lower levels but also display individual, laminar-specific concentrations; alpha 5 expression, in particular, is highly expressed in layer IV in the somatosensory and parietal areas and in a layer IV-like band in the motor cortex. In layers in which expression of a particular transcript is high, all neurons may express the gene, but in layers in which expression is moderate, it is possible to detect differences in the degree of labeling of individual neurons for a particular mRNA, and some neurons may not express certain subunit transcripts in detectable amounts. These findings indicate the variability in expression of different GABAA receptor subunits in the cerebral cortex. Laminar differences may indicate the assembly of functional receptors from different arrangements of available subunits in different classes of cells. PMID- 8636469 TI - Developmental expression of glycine immunoreactivity and its colocalization with GABA in the embryonic chick lumbosacral spinal cord. AB - The development of immunoreactivity for the putative inhibitory amino acid neurotransmitter glycine was investigated in the embryonic and posthatched chick lumbosacral spinal cord by using postembedding immunocytochemical methods. Glycine immunoreactive perikarya were first observed at embryonic day 8 (E8) both in the dorsal and ventral gray matters. The number of immunostained neurons sharply increased by E10 and was gradually augmented further at later developmental stages. The general pattern of glycine immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterward. Most of the immunostained neurons were located in the presumptive deep dorsal horn (laminae IV-VI) and lamina VII, although glycine immunoreactive neurons were scattered throughout the entire extent of the spinal gray matter. By using some of our previously obtained and published data concerning the development of gamma-aminobutyric acid (GABA)-ergic neurons in the embryonic chick lumbosacral spinal cord, we have compared the numbers, sizes, and distribution of glycine- and GABA-immunoreactive spinal neurons at various developmental stages and found the following marked differences in the developmental characteristics of these two populations of putative inhibitory interneurons. (i) GABA immunoreactivity was expressed very early (E4), whereas immunoreactivity for glycine appeared relatively late (E8) in embryonic development. (ii) In the ventral horn, GABA immunoreactivity declined, whereas immunoreactivity for glycine gradually increased from E8 onward in such a manner that the sum of glycinergic and GABAergic perikarya remained constant during the second half of embryonic development. (iii) Glycinergic and GABAergic neurons showed different distribution patterns in the spinal gray matter throughout the entire course of embryogenesis as well as in the posthatched animal. When investigating the colocalization of glycine and GABA immunoreactivities, perikarya immunostained for both amino acids were revealed at all developmental stages from E8 onward, and the proportions of glycine- and GABA-immunoreactive neurons that were also immunostained for the other amino acid were remarkably constant during development. The characteristic features of the development of the investigated putative inhibitory spinal interneurons are discussed and correlated with previous neuroanatomical and physiological studies. PMID- 8636470 TI - Representation of the face and intraoral structures in area 3b of the squirrel monkey (Saimiri sciureus) somatosensory cortex, with special reference to the ipsilateral representation. AB - Studies of the representation of the trigeminal nerve in the thalamus and cerebral cortex of mammals have revealed representations of both contra- and ipsilateral intraoral structures. However, the relative extent of both representations is subject to considerable species variation. The present study employed microelectrode mapping and anatomical tracing to investigate the location and extent of the ipsilateral representation in area 3b of the somatosensory cortex of squirrel monkeys. A small region, approximately 2 mm2, was found to be responsive to stimulation of ipsilateral intraoral structures. This region was located on the anteromedial border of area 3b, surrounded by the representation of the contralateral roof of the mouth. This region corresponded to areas of intense anterograde labeling following injections placed in the ventromedial portion of the ventral posterior medial nucleus of the thalamus at the only sites where neural responses could be elicited by stimulation of ipsilateral intraoral structures. The amount of thalamus and cortex given over to the ipsilateral representation in the squirrel monkey is small compared with that of the macaque monkey. This difference may be related to the lack of cheek pouches in the squirrel monkey, and therefore a different strategy for eating. The representation of the contralateral lower lip in area 3b was split by the representation of the contralateral upper lip. This split representation is in agreement with previous studies of the trigeminal representation in area 3b of the macaque monkey and may be a general feature of the representation of the trigeminal nerve in area 3b of primate cerebral cortex. PMID- 8636471 TI - Children growing up in smoke. PMID- 8636472 TI - Nursing-bottle caries: the importance of a development perspective. AB - Early diagnosis of nursing-bottle caries is so difficult because its first stage is difficult to detect, and occurs at an age the child does not visit a dentist regularly. Also, the first stage does not appear alarming. If for the parent serious complaints from the child arise, irreversible defects already have developed. The developmental stages of nursing-bottle caries are clearly linked to the eruption pattern of the primary teeth. The maxillary primary incisors are affected first, followed by the maxillary first molars. In all stages, a stabilized condition, arrested caries, can occur, if circumstances change. A correct diagnosis of the etiology of caries in toddlers and preschoolers is mandatory as a basis for introducing proper preventive measures. Further study is mandatory to clarify the cause and effect of the disease-pattern. PMID- 8636473 TI - The delivery of dental and medical services in Canada. PMID- 8636474 TI - Two-year results with box-only resin composite restorations. AB - One of the restorative alternatives in the treatment of proximal carious lesions is the box-only approach. This paper describes baseline and two-year results with 68 box-only resin composite restorations in a clinical study. The restorations were made using a glass ionomer cement as a dentin replacement and a bevelled outline. In terms of treatment time (32 minutes) and postoperative sensitivity (4 cases), the box-only restorations show favorable properties in comparison to conventional Class II resin composite restorations. At the two-year assessment no failures were observed, with fair results regarding clinical characteristics. It is concluded that, although applied to a limited number of teeth, the results indicate that the box-only approach with resin composites may make a contribution to the tooth-tissue-saving, restorative techniques. PMID- 8636476 TI - Modifications of the palatal crib habit-breaker appliance to prevent palatal soft tissue embedment. AB - The authors recommended the use of an acrylic palatal button and .040 to .045 inch palatal wires as modifications to the palatal crib appliance. By enhancing the strength of the wire, and providing anterior support, these modifications can reduce the likelihood of the crib becoming embedded in the palatal soft tissues. PMID- 8636477 TI - Abnormalities of the maxillary incisors in children with cleft lip and palate. AB - Dental anomalies of the maxillary anterior teeth were studied in seventy-seven children affected by unilateral and bilateral clefts of the lip and alveolar process, with or without involvement of the palate. As for the permanent lateral incisor in the cleft area, our results show that its congenital absence is the most frequent abnormality followed by anomalies in size and shape and supernumerary teeth. Enamel hypoplasia was found to affect the permanent central incisor on the cleft side more frequently. Early recognition of tooth abnormalities during the primary dentition phase for an interceptive treatment of potentially severe problems was emphasized. PMID- 8636478 TI - Planning for the children of your current pediatric dental patients. AB - Projections by the Bureau of the Census indicate that there will be an additional 8.1 million youngsters less than 15 years of age living in this country by the year 2020. Estimates are developed for each state and region of the needed numbers of pediatric dentists to maintain current practitioner levels. The availability of needed practitioners is considered in terms of the number of graduates from pediatric dentistry training programs. PMID- 8636475 TI - Dental caries and its determinants in 2-to-5-year-old children. PMID- 8636479 TI - Correction and update: interest in pediatric dentistry. AB - Correcting and updating the data on senior dental student plans for pediatric dentistry training indicates that there is a continuing and increasing interest in pediatric dentistry specialty training. Except for 1993, compared to the earlier period, this increase was at a slower rate, however, than that reported previously in the Journal. PMID- 8636481 TI - Corrosive properties of fluoride-containing odontologic gels against titanium. AB - OBJECTIVES: The purpose of this study is to determine, on a quantitative basis, the kinetics of ionization of pure and alloyed titanium in various electrolytic media including commercial dental gels containing fluoride ions (Fluogel). METHODS: The experimental method is a linear sweep voltammetric analysis, making use of a rotating disc electrode. Several successive analyses of different titanium samples are carried out, particularly as a function of the nature of the electrolytic solutions. The current corrosion I(cor) is measured by the determination of linear polarization resistance Rp. RESULTS: Rp and I(cor) measurements by the voltammetric and analysis confirm the excellent resistance of titanium in physiological solutions; they also show that a fluoride-containing dental gel, which is a fluoridated and acidic preparation, is significantly harmful with respect to the corrosion process of titanium. CONCLUSION: The results of this study must draw our attention towards the use of prophylactic gels in which pH is acidic. Indeed titanium suffers an important corrosion process in fluoridated acidic media. Recommendations for the prescription and for the optimized composition of the dental gels could be derived. PMID- 8636480 TI - Water absorption of (RTV) silicone denture soft lining material. AB - OBJECTIVES: The reason for high water absorption by room temperature vulcanizing (RTV) silicone denture soft lining materials has not been demonstrated previously. An experimental room-temperature vulcanizing (RTV) silicone soft lining material which had been shown to have good mechanical properties had high water absorption at equilibrium whilst losing only small amounts of soluble material after desorption. The volume change in the material was also high. A variety of formulations of the experimental material was devised in order to determine the cause of the high water sorption values. METHODS: Water sorption values were determined using standard experimental techniques. Sections of the polymerized materials were examined by scanning electron microscopy for microporosity. RESULTS: Neither removal of residual cross-linker nor changing the method of polymerization reduced the water absorption. Scanning electron microscopy did not demonstrate a porous structure of the polymerized material. A correlation was seen between filler content and water absorption. A formulation without filler showed a greatly reduced water absorption and volume change. CONCLUSION: It was concluded that the filler was directly responsible for the water absorption of the RTV material. PMID- 8636482 TI - The ball mill as a means of investigating the mechanical failure of dental materials. AB - OBJECTIVE: The main purpose of this paper is to present a new method of predicting clinical performance using mechanical loading in a ball mill. METHODS: A series of four experiments (two involving a hybrid composite and one each on orthodontic brackets and bands) is described in which the ball mill was used to subject specimens to mechanical fatigue. RESULTS: A reproducibility study using composite beam specimens showed no significant difference between the Mean Survival Time (MST) in all the three experimental runs (P = 0.42). When subjected to thermal cycling, the MST of the cycled group was 155.0 min compared to 247.0 min for the control group (P < 0.01). The MST of untreated and sandblasted brackets was 7.9 h and 14 h respectively (P < 0.01). There is also a significant difference (P < 0.001) in the MST of sandblasted bands when compared to the untreated bands. CONCLUSIONS: The ball mill proved to be a convenient and reproducible means of producing mechanical fatigue and may be useful in predicting the clinical performance of dental materials. PMID- 8636483 TI - Diffusion of monomers from bonding resin-resin composite combinations through dentine in vitro. AB - OBJECTIVES: Previous work has demonstrated diffusion of the monomer triethylene glycol dimethacrylate (TEGDMA) from resin composite through dentine in vitro. The objective of the present work was to examine monomer diffusion from bonding resins and resin composites used in combination. METHODS: Occlusal cavities were prepared in tooth crowns and restored with bonding resin-resin composite combinations. Aqueous samples from the 'pulpal chamber' of each tooth were removed at timed intervals for analysis by reversed phase-high performance liquid chromatography and mass spectrometry. RESULTS: Bonding resins contributed to monomer diffusion. A TEDGMA-containing bonding resin used in combination with a TEGDMA-containing resin composite hastened and increased TEGDMA diffusion through dentine. A 2-hydroxyethyl methacrylate (HEMA)-containing bonding resin used in combination with a TEGDMA-containing resin composite reduced TEGDMA diffusion only slightly compared with the resin composite alone and added substantial diffusion of HEMA. CONCLUSION: The bonding resins tested contributed to monomer passage to the pulp space and did not prevent movement of monomer from resin composites to the pulp. PMID- 8636484 TI - Composite inlay/luting resin bond strength--surface treatment effects. AB - PURPOSE: To investigate the effect of surface treatments on the bond strength between two resin' composite inlay materials and their luting materials. MATERIALS AND METHODS: A notched disc specimen design was used to evaluate the torque to fracture: test specimens consisting of two half notched disc specimens bonded together, controls consisting of complete resin composite material specimens. RESULTS: Both surface treatment and bond resin application had a significant influence on interfacial bond strength--the torque required to initiate failure being expressed as a percentage of the value required to fracture the control specimens. CONCLUSION: The results allow conclusions to be drawn regarding the attainment of optimal bonding between the resin composite inlays and luting materials investigated. PMID- 8636485 TI - The efficacy of practical participation in undergraduate dental education. AB - OBJECTIVE: This paper investigates the efficacy of practical participation as a vehicle for conveying knowledge of the subject of dental occlusion. METHODS: A fourth-year class of students was divided into two groups: one had benefited from a series of practicals focusing on the re-organized approach to occlusion, and the other had attended routine clinical sessions in conservative dentistry. Both had attended the same course of four lectures. Their knowledge was tested by means of a short answer question paper. RESULTS: The group attending the focused practicals out-performed the control group at the 0.01 level of significance. CONCLUSION: Practical participation would appear to enhance students' understanding of the subject of dental occlusion. PMID- 8636486 TI - Wear: mechanisms, manifestations and measurement. Report of a workshop. AB - OBJECTIVE: The purpose of this workshop was to bring together a tribologist (T.A.S.), a clinician (L.H.M) and a dental materials scientist (R.W.V) to discuss the fundamental mechanisms of wear and how these relate to the manifestations and measurement of wear in dentistry. Eighty delegates contributed to a valuable discussion led by the workshop chairman (C.H.L.). KEY POINTS: Wear is the net result of a number of fundamental processes: abrasion, adhesive effects, fatigue and corrosive effects which act in different combinations on the various classes of materials. In ceramics, sliding compression and surface corrosion greatly increase wear, whereas the ability to deform either plastically or elastically influences the wear of polymers. The filler particle distribution is a major variable influencing the wear of composites. In the mouth, wear can be considered in terms of its site, timing and mechanism. The latter may involve direct contact between surfaces or result from the action of slurries; both of which are affected by surface corrosion (erosion). Although wear can be categorized at the chairside, its precise measurement involves the use of replica models and surface contouring. Laboratory simulation is useful to study fundamental wear mechanisms; but it is not able to predict clinical wear. CONCLUSIONS: The management of clinical wear requires a proper understanding of the underlying mechanisms. This can only be achieved through close co-operation between all the disciplines which seek to understand and manage wear. The attendance of so many delegates at this workshop indicates the willingness to participate in this process. PMID- 8636487 TI - Chronic alcoholism: an important condition in the dentist-patient relationship. AB - OBJECTIVES: This paper aims to give the dental practitioner an insight into the impact of chronic alcoholism on sufferers. METHODS: A review of the psychiatric and dental literature covering the aetiology, prevalence and complications is presented. CONCLUSIONS: Chronic alcoholism has a number of serious medical and social problems which may affect the provision of dental treatment. It is important for the dental practitioner to be aware of the impact of this condition on patients as he or she may be in an ideal position to stop the alcoholic before a medical consultation has been sought. PMID- 8636488 TI - In-vivo evaluation of a feldspathic ceramic system: 2-year results. AB - OBJECTIVES: The aim of this clinical study was to evaluate feldspathic ceramic inlays by clinical criteria and quantitative margin analysis, and to compare clinical performance with the quantitative margin analysis results. METHODS: Fifty fibre-reinforced feldspathic ceramic inlays were placed in box-shaped, Class II preparations with all margins located in enamel. A low-viscosity type dual-curing resin luting material was used for luting. The inlays were evaluated clinically according to modified US Public Health Service criteria after 2 days, 1 year, and 2 years. In addition, quantitative margin analysis was performed under a scanning electron microscope using an image analysing system. RESULTS: Clinically the inlays performed very well after 2 years, showing no changes in colour, no recurrent caries and no marginal discolouration. Because of the wear of the composite resin luting material, margins were perceptible with an explorer in 54% of the cases after 2 years. Quantitative margin analysis showed significantly (P < or = 0.05) more marginal gaps at the composite/ceramic interface compared with the enamel/composite interface at each evaluation. There was a significant increase in marginal gaps at both interfaces during the first year, whereas marginal gaps at the composite/ceramic interface did not increase significantly during the second year. Quantitative margin analysis showed that both time and interface had a significant influence on marginal gaps. CONCLUSION: Quantitative margin analysis should be included in clinical long-term trials on this type of restoration to recognize possible deficiencies in ceramic, composite resin luting material, and the luting interfaces. PMID- 8636489 TI - Industry-university collaborations: a necessity for the future. AB - OBJECTIVES: The aim of this discussion document is to consider the potential benefits of collaborations between universities and industry. Universities and industry now exist in a harsh and competitive economic climate. Indeed, their very survival depends increasingly on their ability and willingness to adapt to an unprecedented pace of change. There are a number of compelling reasons for universities and industry to undertake collaborative research. The costs of research have escalated. Universities, of necessity, are increasingly seeking financial support from industry as conventional sources of funding have been systematically reduced. Companies can no longer be self sufficient in terms of the science and technology needed for success. The strength of universities is their science base, and industry recognizes that the rapid exploitation of scientific advances is the key to its survival. Both organizations can benefit from collaborative research programmes. Universities are able to make additional staff appointments which add to their intellectual resource and reputation. In addition the purchase of equipment enhances their infrastructure. CONCLUSIONS: Industry has the opportunity to recognize the potential relevance and value of funded research and must harness and commercially exploit new knowledge as rapidly as possible. Ultimately such collaboration will give rise to new approaches to the prevention, diagnosis and treatment of dental diseases with benefits to all concerned. PMID- 8636490 TI - Proximal wear rates by tooth position of resin composite restorations. AB - OBJECTIVE: The purpose of this study was to evaluate restorations, previously placed in clinical trials, for the rate of wear of the proximal surfaces, by tooth position. MATERIALS AND METHODS: Seventy resin composite restorations were evaluated in bicuspids and molars. Fifty restorations were direct placement restorations and 20 were indirect placement restorations. Thirty-five bicuspids and 35 molars were evaluated for proximal wear rate. All restorations were opposing unrestored enamel proximal surfaces. Ten enamel to enamel proximal surfaces were chosen as controls. At baseline, contacts were judged to be closed visually and with thin, unwaxed dental floss. Impressions were taken at baseline and after 6, 12 and 24 months for the fabrication of models. Measurements for proximal wear, as determined by a loss of space between indexed transfer copings, were made under a zoom stereomicroscope by two independent evaluators. A parametric ANOVA, Student-Newman-Keuls, and log curve fit were applied to the data. RESULTS: When all data were pooled regardless of material, there were no significant differences (P < 0.05) in wear values for 1st and 2nd bicuspids and 1st and 2nd molars. Wear rates showed a high correlation (log curve fit R2 > 0.950) regardless of actual amount of wear. CONCLUSIONS: Regardless of composites examined, there were no differences in the amount of proximal wear with regard to tooth position in the arch. PMID- 8636492 TI - Clinical significance of dental root canal microflora. AB - OBJECTIVES: Previous work by this group has shown that a significant association exists between pain and the presence of either Prevotella or Peptostreptococcus spp. in dental root canals. The aim of this study was to examine a more extensive series of canals microbiologically, to determine whether any other particular endodontic symptoms or clinical signs showed specific associations with individual bacterial species. METHODS: Seventy root canals were examined microbiologically and clinical data collected to investigate in detail such associations. RESULTS: Of the canals studied, 37 were associated with pain, 49 with tenderness to percussion, 23 with swelling, six with purulent exudate and 57 presented with wet root canals. Anaerobes were isolated from 70.3% of painful canals and from 29.7% of pain-free canals. Significant associations were found between (a) pain and either Prevotella spp. or peptostreptococci, both with P < 0.01; (b) tenderness to percussion and Prevotella spp. (P < 0.01) or anaerobes (P < 0.05); (c) swelling and Eubacterium spp. (P < 0.01), or with Prevotella spp. or Pstr. micros, both with P < 0.05; (d) purulent exudate and any one of F. necrophorum (P < 0.01), Prev. loescheii, Streptoccoccus constellatus or Bacteroides spp. (each P < 0.05); (e) wet canal and facultative anaerobes (P < 0.01), and any one of the genera of Eubacterium, Peptostreptococcus, Prevotella or Propionibacterium (each P < 0.05). CONCLUSION: It was concluded that several different endodontic clinical signs and symptoms are significantly associated with specific bacterial species. PMID- 8636491 TI - Effect of replacement of dental amalgam on oral lichenoid reactions. AB - OBJECTIVES: The objectives of this study were to investigate (i) healing of oral lichenoid reactions (OLR) following the selective replacement of restorations of dental amalgam, (ii) whether there were differences in healing between contact lesions (CL) and oral lichen planus (OLP), and (iii) whether there was a difference in healing potential when different materials were selected as a substitute for dental amalgam. METHODS: Patients included in the study presented with OLR confined to areas of the oral mucosa in close contact with amalgam restorations (CL; n = 142) or with OLR which involved other parts of the oral mucosa as well (OLP; n = 19). After examination, restorations of dental amalgam which were in contact with OLR in both patient groups were replaced. The effect of replacement was evaluated at a follow-up after 6-12 months. RESULTS: In the CL group, the lesions showed a considerable improvement or had totally disappeared in 95% of the patients after replacement of the restorations of dental amalgam (n = 474). This effect was paralleled by a disappearance of symptoms, in contrast to patients with persisting CL (5%) who did not report any significant improvement. The healing response was not found to correlate with age, gender, smoking habits, subjective dryness of the mouth or current medication. However, the healing effect in patients who received gold crowns was superior compared to that of patients treated with metal-ceramic crowns (MC; P < 0.05). In the OLP group (n = 19), 63% of the patients with amalgam-associated erosive and atrophic lesions showed an improvement following selective replacement. OLP lesions in sites not in contact with amalgams were not affected. Most of the patients (53%) with OLP reported symptoms also after replacement. CONCLUSION: From these data it can be concluded that the vast majority of CL resolve following selective replacement of restorations of dental amalgam, provided that a correct clinical diagnosis is established. It is also noteworthy that MC crowns did not facilitate healing of CL to the same extent as gold crowns. PMID- 8636493 TI - Morphological characterization of tube-like structures in hypersensitive human radicular dentine. AB - OBJECTIVES: The objective of this work was to determine if there were differences in the distribution of tube-like structures within the tubules of biopsies from hypersensitive versus non-sensitive human cervical radicular dentine. METHOD: Ten pairs of dentine were made from the exposed sensitive and non-sensitive radicular dentine of nine adult patients complaining of dentine sensitivity. The biopsies were made with a small hollow diamond bur. The specimens were submitted to HCl collagenase treatment to partially digest the surface to permit visualization of subsurface structures. Half of the specimens were embedded in resin to facilitate longitudinal sectioning, and the other half were processed with embedding for SEM and EDS observation. RESULTS: Biopsies from hypersensitive regions exhibited hollow, tube-like structures within the lumina of dentinal tubules in 75.8 +/- 9.4%) ( mean +/- S.D.) of the tubules. In biopsies of non-sensitive areas of the same teeth, these tube-like structures were only seen in 20.4 +/- 8.5% of the tubules. In specimens that were embedded prior to treatment with HCl-collagenase, resin tags penetrated many of the tubules from hypersensitive dentine but few of the tubules from non-sensitive dentine. CONCLUSION: It is speculated that tube like structures found in hypersensitive dentine play a role in maintaining the patency of the tubules which may prevent physicochemical processes from spontaneously occluding the tubules, thereby maintaining dentine sensitivity. PMID- 8636495 TI - Anorexia and bulimia nervosa (the eating disorders): conditions of interest to the dental practitioner. AB - OBJECTIVES: This paper aims to give the dental practitioner an insight into the impact of the eating disorders on sufferers. METHODS: A review of the psychiatric literature on the diagnosis, aetiology and treatment of these conditions is given. CONCLUSIONS: The dental practitioner is in an ideal position to identify the warning signs of these conditions before they have attracted medical attention. PMID- 8636494 TI - Rings and watches: should they be removed prior to operative dental procedures? AB - OBJECTIVES: There is no clear evidence to support the recommendation that rings and watches should be removed prior to operative dental procedures. The aim of this study was to measure and identify the bacteria isolated from the skin under rings and watches worn by a group of dental surgeons and to compare the results with a group of non-clinical staff. METHODS: Forty volunteers participated in the study; of these 20 were dental surgeons and 20 were non-clinical staff. Four skin sites were sampled for each volunteer; the skin directly under the ring and on the same finger of the other hand and the skin under the watch face and wrist of the control hand. Bacteria on the swabs were dispersed and inoculated onto plates, which were incubated aerobically for 24 h at 37 degrees C. RESULTS: In both groups of volunteers there was a significantly greater number of bacteria isolated from under rings and watches compared with control sites. Few qualitative differences were found between the microflora found on the skin under rings and watches in the two volunteer groups. CONCLUSIONS: The bacterial flora isolated from volunteers do not commonly cause oral infections but could pose a threat to the immunocompromised patient, particularly in the event of gloves becoming torn or perforated. Effective hand disinfection is difficult to achieve if ring and watches are not removed; they should therefore be removed prior to hand disinfection and donning of gloves. PMID- 8636496 TI - Adaptation of a resin composite in vivo. AB - OBJECTIVE: The adaptation of experimental dentine bonding systems has been evaluated in vivo. METHODS: A cylindrical cavity which extended into dentine was prepared in the facial surface of vital teeth that were to be extracted for orthodontic reasons. The preparations were restored with a commercial light activated resin composite that was mediated by experimental dentine bonding systems. The restored teeth were extracted as soon as possible following polymerization of the composite. The marginal and internal adaptation of the resin composite restoration was observed under a light microscope in both horizontal and longitudinal sections of the teeth immediately after extraction. RESULTS: Of the experimental dentine bonding systems investigated, those including a primer of glyceryl methacrylate showed complete adaptation whereas those with primers of 2-HEMA showed a contraction gap in three out of 10 specimens. PMID- 8636497 TI - Microbiological study of selected risk areas in dental technology laboratories. AB - OBJECTIVES: To investigate the microbiological status of certain risk areas in the dental technology laboratory, namely pumice slurry, impression agar and curing water baths. METHODS: Samples were inoculated onto selective and non selective media. Resultant fungal and bacterial colonies were counted and identified to genus or species level. RESULTS: Pumice slurry freshly made up using disinfectant was free from contamination, but colony counts approached 10(9) g-1 after 3 days' use. Impression agar was free from contamination, probably because of its method of preparation and maintenance, which appears to achieve sterility. Staphylococci were most commonly isolated from curing water baths (and from the air). Candida spp. and other yeasts were common in pumice slurry. CONCLUSIONS: Personnel and managers should be aware of the potential cross-contamination hazards posed by the presence of a range of opportunist pathogens in dental technology laboratories. PMID- 8636498 TI - An in vitro comparative study of a reciprocating scaler for root surface debridement. AB - OBJECTIVES: A number of instruments are available for debriding periodontally involved root surfaces. The aim of this SEM study was to assess and compare the efficiency of the recently marketed EVA scaling system with that of ultrasonic (Cavitron-Dentsply) and hand instruments (Ash Dental Products). METHODS: Forty five periodontally involved extracted teeth were divided into three equal groups for instrumentation. The teeth were mounted onto typodonts, set in phantom heads and instrumented to tactile smoothness and visual cleanliness. Replica specimens of the debrided roots were prepared for the SEM and views were recorded on video film. The efficiency of instrumentation was assessed using the Remaining Calculus Index (RCI) and the Roughness and Loss of Tooth Substance Index (RLTSI). Five original specimens from each group were prepared for light microscopy to confirm histologically the appearances seen under the SEM. RESULTS: Observations indicated that there were no statistically significant differences between the different instruments but the EVA system took significantly longer than either the ultrasonic (P < 0.0001) or hand instruments (P < 0.025) to achieve visual cleanliness. The results supported the view that no instrument removes all extraneous deposits and all were capable of cavity roughness and minor damage to root cementum. Only the hand and ultrasonic instruments left scratches after instrumentation, although the greater part of all the surfaces was surprisingly smooth and calculus free after root surface debridement. CONCLUSIONS: Recently marketed EVA inserts are comparable to ultrasonic and hand instruments for root planing, with respect to calculus removal and quality of the remaining root surface. PMID- 8636499 TI - Effect of mechanical load cycling on the marginal integrity of adhesive Class I resin composite restorations. AB - OBJECTIVES: The purpose of this study was to determine the effect of cycle loading on the sealing capacity of different adhesive systems in Class I restorations in vitro. METHOD: (1) Clearfil Liner Bond/Clearfil Photo Posterior, (2) Scotchbond Multipurpose/Z100, (3) Syntae/Heliomolar, and (4) Gluma 2000/Pekalux were employed to restore Class I cavities in extracted human premolars and molars. For each test group, half of the specimens were subjected to mechanical loading at 125 N for 5000 cycles in a dye solution. After sectioning, dye penetration was assessed. RESULTS: Without loading, no specimen showed leakage, while loading did affect the sealing capacity of groups 3 and 4. Groups 1 and 2 showed no significant increase in leakage after the loading procedure. CONCLUSION: The results indicate that mechanical cycle loading can adversely affect the interface of adhesive Class I resin composite restorations unless hybridizing adhesive systems are used. PMID- 8636500 TI - Caries inhibition by a resin-modified and a conventional glass ionomer cement, in vitro. AB - OBJECTIVES: The aim of this study was to compare inhibition, in vitro, of a resin modified and a conventional glass ionomer cement. METHODS: Standardized Class V cavities were prepared in extracted human molar teeth and restored with Fuji II LC (n = 14) or Chemfil II (n = 14) according to manufacturers' guidelines. The teeth were submerged in an acid gel (lactic acid, gelatin, thymol, pH 4.5) for 14 days and resin embedded. Photomicrographs of ground sections were examined for enamel surface lesions, dentine surface lesions, wall lesions and evidence of caries inhibition. RESULTS: There was no significant difference between the depths of enamel or dentine surface lesions between the two materials. No wall lesions were found. CONCLUSIONS: Fuji II LC proved more acid resistant than Chemfil II and both materials inhibited caries in vitro. PMID- 8636501 TI - Preparation of void-free casts from vinyl polysiloxane impressions. AB - PURPOSE: To investigate two techniques to produce void-free casts from vinyl polysiloxane impressions. MATERIALS AND METHODS: Thirty casts prepared using a conventional technique were compared with 30 casts prepared using a technique involving syringing of stone in terms of numbers of surface voids. RESULTS: Significantly fewer surface voids were observed in the casts prepared using syringing technique. CONCLUSION: The syringing technique investigated is considered to have advantages over the conventional technique for the production of casts from vinyl polysiloxane impressions. PMID- 8636502 TI - Sprue design in removable partial denture casting. AB - OBJECTIVES: Correct sprue design is a major factor in the reduction of defects in castings of cobalt-chromium alloy. The purpose of this study was to investigate the sprue arrangements decided by a group of dental technicians, for a series of proposed removable partial denture castings, in order to determine if there was any consistency between them which would suggest the application of criteria in their design. METHODS: Information was gathered by postal inquiry with recipients asked to indicate their sprue designs on diagrams of patterns for proposed cobalt chromium partial denture castings. RESULTS: Replies were received for 260 proposed castings from dental schools, technical colleges and commercial laboratories which gave an overall response rate of 52%. Results displayed a wide range of responses in relation to sprue number and dimension, attachment site and the intended direction of metal flow. CONCLUSION: It was concluded that the group of technicians studied arbitrarily chose sprue arrangements for the castings proposed rather than following definite design criteria that seek to minimize potential problems during casting procedures. PMID- 8636503 TI - Budget savvy dental administrators of the '90s. PMID- 8636506 TI - Effect on undergraduates--the candidate of the future. PMID- 8636505 TI - Predental advisors--an unrecognized asset of the profession. PMID- 8636504 TI - The candidate of the future. PMID- 8636507 TI - The application process influences the applicant pool--we can send the applicants a message. PMID- 8636509 TI - Effecting change--principles for the process. PMID- 8636508 TI - It's time to launch a counter-cultural movement! PMID- 8636510 TI - The IOM study and minority issues. PMID- 8636511 TI - Curriculum change. PMID- 8636512 TI - Faculty development. PMID- 8636513 TI - Admissions and student concerns. PMID- 8636514 TI - Mercury release during autoclave sterilization of amalgam. AB - Natural teeth are an invaluable teaching tool for preclinical instruction in operative dentistry and endodontic techniques. Cavity preparation in teeth containing amalgam restorations is a realistic simulation of an often experienced clinical situation. As various pathogens are contained in saliva, teeth must be disinfected before use by students. The purpose of this study is to indirectly evaluate whether mercury vapor is released from amalgam restorations in such teeth during steam autoclave sterilization. Mercury vapor detection, sample mass changes and x-ray fluorescence data were collected from experimental steam autoclave sterilization of amalgam samples sealed in autoclave bags. All of the data showed evidence of mercury vapor generation coincident to steam autoclave sterilization. Mercury vapor levels within the room where amalgam was exposed to steam autoclave sterilization reached levels that constitute an unnecessary health risk to dental personnel. The volume of amalgam tested simulated that contained in 175 amalgam restored teeth. Initial venting of the autoclave chamber produced mercury vapor concentrations significantly in excess of OSHA vapor concentration ceiling levels. Thus, the use of a steam autoclave for sterilization of amalgam containing teeth for use in preclinical laboratory exercises may be harmful to personnel involved. PMID- 8636515 TI - A cross-cultural comparison of perceived sources of stress in the dental school environment. AB - This study compared the perceived sources of stress in the dental school environment between an American and a South East Asian (Singapore) dental school across their entire curricula (D1-D4 years). The dental environmental stress (DES) questionnaire was administered to 137 (98 percent) Singapore dental students and results were compared to a previous study conducted at an American school. Significant differences were found for 12 questionnaire items across the four classes for the American cohort as compared to 13 in the Singapore cohort. While the greatest stressor for Singapore students was completing graduation requirements, American students were most stressed by examinations and grades. The overall perception of stress by Singapore students was higher than for their American counterparts. The primary concern for both cohorts was, however, related to the different areas of their academic courses. PMID- 8636516 TI - Using "literature" to evaluate new drugs. PMID- 8636517 TI - Wasted efforts and dyssynchrony: is the patient-ventilator battle back? PMID- 8636518 TI - Respiratory response and inspiratory effort during pressure support ventilation in COPD patients. AB - OBJECTIVE: Pressure Support Ventilation (PSV) is now widely used in the process of weaning patients from mechanical ventilation. The aim of this study was to evaluate the effects of various levels of PS on respiratory pattern and diaphragmatic efforts in patients affected by chronic obstructive pulmonary disease (COPD). SETTING: Intermediate intensive care unit. PATIENTS: We studied ten patients undergoing PSV and recovering from an episode of acute respiratory failure due to exacerbation of COPD. METHODS: Three levels of PSV were studied, starting from the lowest (PSb) one at which it was possible to obtain an adequate Vt with a pH > or = 7.32 and an SaO2 > 93%. Then, PS was set at 5 cmH2O above (PSb + 5) and below (PSb-5) this starting level. Ventilatory pattern, transdiaphragmatic pressure (Pdi), the pressure-time product of the diaphragm (PTPdi), the integrated EMG of the diaphragm, static PEEP (PEEPi, stat), dynamic PEEP (PEEPi, dyn), and the static compliance and resistance of the total respiratory system were recorded. RESULTS: Minute ventilation did not significantly change with variations in the level of PS, while Vt significantly increased with PS (PS-5 = 6.3 +/- 0.5 ml/kg vs. PSb = 10.1 +/- 0.9 [p < 0.01] and vs. PS + 5 = 11.7 +/- 0.6 [p < 0.01]), producing a reduction in respiratory frequency with longer expiratory time. The best values of blood gases were obtained at PSb, while at PSb-5, PaCO2 markedly increased. During PSb and PSb + 5 and to a lesser extent during PSb-5, most of the patients made several inspiratory efforts that were not efficient enough to trigger the ventilator to inspire; thus, the PTPdi "wasted" during these inefficient efforts was increased, especially during PS + 5. The application of an external PEEP (PEEPe) of 75% of the static intrinsic PEEP during PSb caused a significant reduction in the occurrence of these inefficient efforts (p < 0.05). Minute ventilation remained constant, but Vt decreased, together with Te, leaving the blood gases unaltered. The PTPdi per breath and the dynamic PEEPi were also significantly reduced (by 59% and 31% of control, respectively, p < 0.001) with the application of PEEPe. CONCLUSION: We conclude that in COPD patients, different levels of PSV may induce different respiratory patterns and gas exchange. PS levels capable of obtaining a satisfactory equilibrium in blood gases may result in ineffective respiratory efforts if external PEEP is not applied. The addition of PEEPe, not exceeding dynamic intrinsic PEEP, may also reduce the metabolic work of the diaphragm without altering gas exchange. PMID- 8636519 TI - Effects of breathing patterns on mechanically ventilated patients with chronic obstructive pulmonary disease and dynamic hyperinflation. AB - OBJECTIVE: To examine the circulatory and respiratory effects of breathing pattern in patients with chronic obstructive pulmonary disease (COPD) and dynamic hyperinflation (DH) during controlled mechanical ventilation. DESIGN: Prospective, controlled, randomized, non-blinded study. SETTING: Respiratory intensive care unit of a university hospital. PATIENTS: Nine patients with acute respiratory failure and DH due to acute exacerbations of COPD. INTERVENTIONS: Keeping tidal volume and total breath duration (TTOT) constant, patients were ventilated at six different values of expiratory time (TE). TE changes were randomly induced by alterations of constant inspiratory flow (VI) and/or end inspiratory pause (EIP). Patients were studied at three levels of VI(0.93 +/- 0.08, 0.72 +/- 0.06 and 0.55 +/- 0.04 l/s, mean +/- SE), with and without EIP (10% of TTOT). MEASUREMENTS AND RESULTS: Lung volumes, airflows, airways pressures, oxygenation indices and dead space were measured. Alveolar pressure and airway resistance (Rmin), as well as the additional resistance (delta R) due to viscoelastic pressure dissipation and time-constant inequalities, were estimated by rapid airway occlusion during inflation. In seven out of nine patients, right-heart catheterization was performed and hemodynamic parameters were obtained at each value of TE. A significant decrease of intrinsic positive end-expiratory pressure (PEEPi), end-inspiratory static and mean (mPaw) airway pressures, end-expiratory lung volume above passive FRC (Vtrap), delta R and venous admixture and a significant increase of peak airway pressure, Rmin, stroke volume index and mixed venous PO2 (PvO2) were observed when VI increased. At each VI, the addition of EIP significantly decreased iso-volume expiratory flows and PvO2 and increased Vtrap and mPaw. CONCLUSIONS: We conclude that in mechanically ventilated patients with COPD, the pattern of lung inflation and TE alteration have a significant impact on respiratory system mechanics, gas exchange and hemodynamics. Addition of EIP in patients with COPD may be detrimental. PMID- 8636520 TI - Effect of low-level PEEP on inspiratory work of breathing in intubated patients, both with healthy lungs and with COPD. AB - OBJECTIVE: Evaluation of low-level PEEP (5 cm H2O) and the two different CPAP trigger modes in the Bennett 7200a ventilator (demand-valve and flow-by trigger modes) on inspiratory work of breathing (Wi) during the weaning phase. DESIGN: Prospective controlled study. SETTING: The intensive care unit of a university hospital. PATIENTS: Six intubated patients with normal lung function (NL), ventilated because of non-pulmonary trauma or post-operative stay in the ICU, and six patients recovering from acute respiratory failure due to exacerbation of chronic obstructive pulmonary disease (COPD), breathing either FB-CPAP or DV-CPAP with the Bennett 7200a ventilator. INTERVENTIONS: The patients studied were breathing with zero end-expiratory pressure (ZEEP), as well as CPAP of 5 cm H2O (PEEP), with the following respiratory modes: the demand-valve trigger mode, pressure support of 5 cm H2O, and the flow-by trigger mode (base flow of 20 l/min and flow trigger of 2 l/min). Furthermore, Wi during T-piece breathing was evaluated. MEASUREMENTS AND RESULTS: Wi was determined using a modified Campbell's diagram. Total inspiratory work (Wi), work against flow-resistive resistance (W(ires)), work against elastic resistance (Wiel), work imposed by the ventilator system (W(imp)), dynamic intrinsic positive end-expiratory pressure (PEEPidyn), airway pressure decrease during beginning inspiration (P(aw)) and spirometric parameters were measured. In the NL group, only minor, clinically irrelevant changes in the measured variables were detected. In the COPD group, in contrast, PEEP reduced Wi and its components W(ires) and Wiel significantly compared to the corresponding ZEEP settings. This was due mainly to a significant decrease in PEEPidyn when external PEEP was applied. Flow-by imposed less Wi on the COPD patients during PEEP than did demand-valve CPAP. Differences in W(imp) between the flow-by and demand-valve trigger models were significant for both groups. However, in relation to Wi these differences were small. CONCLUSION: We conclude that the application of low-level external PEEP benefits COPD patients because it reduces inspiratory work, mainly by lowering the inspiratory threshold represented by PEEPidyn. Differences between the trigger modes of the ventilator used in this study were small and can be compensated for by the application of a small amount of pressure support. PMID- 8636521 TI - Inspiratory effort and measurement of dynamic intrinsic PEEP in COPD patients: effects of ventilator triggering systems. AB - OBJECTIVE: To investigate effects of ventilator triggering systems (pressure and flow triggering: PT and FT) on measurement of dynamic intrinsic PEEP (PEEPidyn) and patient-ventilator interaction in patients with chronic obstructive pulmonary disease during weaning from mechanical ventilation. DESIGN: Prospective study. SETTING: Medical/surgical intensive care unit of an academic hospital. PATIENTS AND PARTICIPANTS: 6 COPD patients with acute respiratory failure ready to wean. MEASUREMENTS: We measured flow, airway opening, esophageal and gastric pressures. Minute ventilation, breathing pattern and pressure time product (PTP) of the respiratory muscles and of the diaphragm were obtained during spontaneous ventilation through a mechanical ventilator (Puritan-Bennett 7200ae). Two triggering systems, namely PT and FT, were evaluated. RESULTS: The inspiratory muscles effort necessary to overcome the triggering system overestimated PEEPidyn measurement of an amount equal to 49 +/- 2 and 58 +/- 3% during respectively pressure and flow triggering. FT increased tidal volume and minute ventilation and decrease PTP/b and PTP/min of the respiratory muscles and diaphragm. CONCLUSIONS: To correctly measure PEEPidyn, the inspiratory effort produced to overcome PEEPi and to trigger the ventilator must be discriminated. Application of flow triggering requires less effort to initiate inspiration and provide a positive end-expiratory pressure level that is able to unload the respiratory muscles by reducing PEEPi. With flow triggering higher minute ventilation are obtained in COPD patients during the weaning phase. PMID- 8636522 TI - Respiratory effects of tracheal gas insufflation in spontaneously breathing COPD patients. AB - OBJECTIVE: To evaluate the effect of tracheal gas insufflation (TGI) in spontaneously breathing, intubated patients with chronic obstructive pulmonary disease (COPD) undergoing weaning from the mechanical ventilation. DESIGN: A prospective study in humans. SETTING: Polyvalent intensive care unit (14-bed ICU) in a 700-bed general university hospital. PATIENTS: Twelve patients with chronic obstructive pulmonary disease (COPD) who required intubation and mechanical ventilation were studied. All patients met standard criteria for weaning from mechanical ventilation. Seven patients (group 1) had been transorally intubated during episodes of acute respiratory failure. Five patients, all men (group 2), had previously undergone tracheostomy and had a transtracheal tube in place. INTERVENTIONS: Intratracheal, humidified, O2-mixture insufflation (TGI) was given via a catheter placed in distal or proximal position. Gas delivered through the intratracheal catheter was blended to match the fractional of inspired gas through the endotracheal tube. Continuous flows of 3 and 6 l/min in randomized order were used in each catheter position. Prior to data collection at each stage, an equilibration period of at least 30 min was observed, and thereafter blood gases were analyzed every 5 min. A new steady state was assumed to have been established when values of both PaCO2 and V CO2 changed by less than 5% between adjacent measurements. The last values of blood gases were taken as representative. The new steady state was confirmed within 35-50 min. Baseline measurements with zero Vcath were made at the beginning and end of the experiment. RESULTS: This study shows that VT, MV, PaCO2, and VD/VT are reduced in a flow-dependent manner when gas is delivered through an oral-tracheal tube (group 1). The distal catheter position was more effective than the proximal one. In contrast, when gas was delivered through tracheostomy (group 2), TGI was ineffective in the proximal position and less effective than in group 1 in distal position. CONCLUSION: Under the experimental conditions, tracheal gas insufflation decreased dead space, increased alveolar ventilation and possibly reduced work of breathing. From the preliminary data reported here, we believe that TGI may help patients experiencing difficulty during weaning. PMID- 8636523 TI - Comparison of the effects of pressure support ventilation delivered by three different ventilators during weaning from mechanical ventilation. AB - OBJECTIVE: To compare the effects of pressure support ventilation (PSV) delivered at the same level by three different ventilators on patients' work of breathing (WOB), breathing pattern and gas exchange. DESIGN: Prospective, self-controlled clinical study. SETTING: Intensive care unit of a tertiary university hospital. PATIENTS: Nine intubated adult patients during weaning from mechanical ventilation. INTERVENTIONS: Patients were randomly connected to one of three ventilators: the Siemens Servo 900 C (SC), the Ohmeda CPU 1 (CPU), and the Engstrom Erica (EE) during both zero cmH2O PSV and 15 cmH2O PSV. MEASUREMENTS AND RESULTS: During zero PSV, there was no significant difference in terms of WOB, VT, VE, or auto-PEEP among the three ventilators, although there was a trend towards higher levels of WOB with EE. During 15 cmH2O PSV, WOB was significantly less with SC than with EE or CPU (0.47 +/- 0.48 J/l for SC, 1.0 +/- 0.48 for EE and 0.78 +/- 0.51 for CPU1, p = 0.003). WOB was 64% less than at zero PSV with SC but only 38% less with EE. This was associated with a different pressurization shape, as assessed by the interior surface of Paw-VT loops (1.23 +/- 0.09 J/l for SC, 0.9 +/- 0.02 for EE, and 0.79 +/- 0.18 for CPU; p < 0.001). At 15 cmH2O PSV, auto-PEEP was significantly lower with SC than with EE (1.7 +/- 2.1 cmH2O for SC, 4.7 +/- 3.6 for EE, and 2.8 +/- 0.3 for CPU; p = 0.04). External expiratory resistances, in cmH2O/l/s, were significantly higher with EE than with CPU or SC (12.9 +/- 3.2 EE, 7.5 +/- 2.4 CPU, 5.9 +/- 0.5 SC; p < 0.001). CONCLUSION: During PSV, the different working principles of different mechanical ventilators profoundly affect patient's WOB. Among the various factors, velocity of pressurization of PSV may play a role in its efficacy in unloading the respiratory muscles. PMID- 8636524 TI - Topical upper airway anaesthesia with lidocaine increases airway resistance by impairing glottic function. AB - OBJECTIVE: To assess if two different forms of upper airway topical anaesthesia induce similar changes in airway flow resistance (Rrs). DESIGN: Serial measurements of Rrs before and after topical anaesthesia with acqueous or paste lidocaine. SETTING: Lung function test laboratory. PARTICIPANTS: 9 normal men with documented normal lung function tests. INTERVENTIONS: 2 different session of topical upper airway anaesthesia with 100 mg of liquid 5% lidocaine and 100 mg of 2% lidocaine paste, respectively. MEASUREMENTS AND RESULTS: Rrs was measured by the random noise forced oscillation technique. Fiberoptic upper airway examination was performed in two subjects. Rrs increased on average by 81% after lidocaine spray and by 68% after lidocaine paste (p < 0.005, respectively) with no difference in the magnitude of Rrs increase between the two modes of anaesthesia studied. This increase lasted 13 +/- 3 min (spray) and 12 +/- 3 min (paste), respectively (p = ns). Fiberoptic examination of the two most responders showed inspiratory laryngeal collapse. CONCLUSIONS: Topical upper airway anaesthesia transiently increases Rrs with no specific effects regarding the drug presentation. Laryngeal dysfunction may be one mechanisms involved in Rrs increase following upper airway topical anaesthesia. Such findings may explain some poor respiratory tolerance reported during endoscopy. PMID- 8636525 TI - Improved bronchial cleansing in intensive care patients with a new double-lumen catheter. AB - OBJECTIVES: A new double lumen catheter with a small channel for application of rinsing solution in deeper parts of the endobronchial tree was developed and its efficiency was tested in two trials. DESIGN: Comparison of the new catheter in 2 controlled studies with the traditional way of suctioning and with conventional endotracheal lavage in a randomized block design. SETTING: Intensive care unit of a university hospital. PATIENTS: In the first study, endobronchial cleansing with the new catheter was compared to the traditional way of suctioning in 12 long time ventilated patients. In the second study, 28 ventilated patients received either conventional lavage or lavage with the new catheter. INTERVENTIONS: In the first trial the bronchial system of each patient was suctioned 25 times with a conventional technique or cleansed by using the new catheter. In the second study, patients alternatively received conventional lavage 88 times or lavage 88 times with the new catheter. MEASUREMENTS AND RESULTS: Drained secretions averaged 0.84 +/- 0.28 ml using conventional cleaning as compared to 11.02 +/- 0.84 ml with the new catheter. This was accompanied by a significant (p < 0.001) increase in PaO2 of 24.20 +/- 7.90 mmHg after 10 min compared to nearly unchanged PaO2 after normal suctioning. In the second study, suctioned volume was 2.48 +/- 0.21 ml using conventional endotracheal lavage and 10.55 +/- 0.47 ml using the new catheter. Use of the double-lumen catheter induced a significant increase in PaO2 by 30.90 +/- 3.90 mmHg within 10 min. The changes in PaO2 correlated with the drained volume. CONCLUSION: Both studies show that suctioning with the new double lumen catheter allows drainage of a larger volume of secretions and results in a greater improvement of oxygenation. PMID- 8636526 TI - The impact of HIV testing on blood utilization in the intensive care unit in patients with gastrointestinal bleeding. AB - OBJECTIVE: To determine whether the AIDS epidemic has influenced physician use of blood products in intensive care unit management of gastrointestinal hemorrhage. METHODS: Retrospective chart review of 148 patients with gastrointestinal hemorrhage admitted to the intensive care unit. Forty-eight patients were admitted before the onset of HIV testing of the blood supply (group 1) and 100 were admitted after HIV testing was begun (group 2). RESULTS: Of the 148 patients, 18 (eight in group 1, ten in group 2) were not transfused and had higher median hemoglobin levels on admission and higher median hemoglobin nadirs during hospitalization than patients who were transfused. Transfused patients in group 2 did not have significantly lower median hemoglobin levels on admission [7.9(4.2-12.5) g/dl] than transfused patients in group 1 [9.3 (4.1-13.5) g/dl] (p = 0.058). Patients in group 2 had significantly lower median hemoglobin concentrations prior to the first transfusion event [7.4 (4.2-10.3) g/dl] than those in group 1 [8.5 (4.2-12.1) g/dl] (p = 0.016). There were no significant differences between the two groups in terms of the total number of units of packed red blood cells, fresh frozen plasma or platelets transfused. Neither was any significant difference in mortality observed, with 11 patients (22.9%) dying in group 1 and 23 patients (23.0%) dying in group 2. The cause of death in 13 of the 34 patients was related to cardiovascular and hemodynamic complications of gastrointestinal bleeding. There was no significant difference in mean age (group 1: 60.5 years, group 2: 59.4 years) or mean hemoglobin nadir (group 1: 7.0 g/dl, group 2: 7.1 g/dl) among those who died in the two groups. CONCLUSIONS: These data indicate that physicians are transfusing patients at lower hemoglobin levels than they did before the beginning of HIV testing. However, there has been no decrease in the total median amount of blood products transfused since that time. This change in practice may be due to increased concern about HIV transmission through blood products and suggests the need for greater awareness of existing transfusion guidelines. PMID- 8636527 TI - Systemic lithium reabsorption from lithium-chloride-coated heat and moisture exchangers. AB - OBJECTIVE: To evaluate possible reabsorption and systemic effects of lithium released by lithium-chloride-coated heat and moisture exchangers (HMEs) during prolonged mechanical ventilation. DESIGN: Prospective study, including all patients mechanically ventilated for 5-30 days. SETTING: A 7 bed general traumatological ICU in a University Hospital. PATIENTS: 27 consecutive ICU patients, admitted following trauma, neurosurgery and respiratory insufficiency, mechanically ventilated for at least 5 days, with a lithium coated hygroscopic HME in the circuit. MEASUREMENTS AND RESULTS: Serum lithium levels were measured daily, with a standard laboratory spectrophotometric method, from admission to discharge from the ICU, and showed a reabsorption of lithium in all the patients; in the adults, levels were 5 to 15 times lower than therapeutic range, while in a child therapeutic and even toxic levels were reached. CONCLUSIONS: LiCl coat enhances HMEs' performance greatly, but reabsorption and systemic action must be considered. In adults, serum lithium levels were lower than the therapeutic range, but lithium is effective at low concentrations and it has a narrow therapeutic range; moreover, toxicity can be observed within this range too. In children, the risk of toxicity is much greater. When lithium coated HMEs are used, the risk/benefit ratio between good performance and systemic reabsorption must be evaluated carefully. PMID- 8636528 TI - Inhaled cigarette smoke selectively reverses human hypoxic vasoconstriction. AB - The acute effects of the inhaled gas phase of cigarette smoke on pulmonary (PAP) and systemic (SAP) arterial pressures and on plasma arterial cGMP content were compared with those of inhaling 10, 20 and 80 ppm nitric oxide (NO) in one healthy adult volunteer spontaneously breathing a hypoxic gas mixture. Hypoxia (FIO2 0.12) induced a sustained, stable pulmonary vasoconstriction. Inhaled NO induced a dose-dependent fall in PAP; plasma cGMP rose from 39.4 (hypoxia) to 164 pmol/ml (hypoxia plus 80 ppm NO). Exposure to cigarette smoke induced a rapid, consistent and reversible fall in PAP; plasma cGMP rose from 45.5 (hypoxia) to 138 pmol/ml (hypoxia plus cigarette smoke). Neither NO nor cigarette smoke inhalation induced any change in SAP. These data suggest that exposure to cigarette smoke is able selectively to reverse acute hypoxic vasoconstriction in humans without causing systemic vasodilation, an effect likely mediated through the NO-cGMP pathway. PMID- 8636529 TI - The delivery of aerosolized steroids from MDIs with nozzle extensions: quantitative laboratory evaluation of a method to improve aerosol delivery to intubated patients. AB - OBJECTIVE: Pulmonary deposition of aerosolized drug from a metered dose inhaler (MDI) is low with intubated patients. In the laboratory, extension of the MDI nozzle to the endotracheal tube tip has been shown to increase the delivered dose of albuterol. The objectives of this study were to determine the dose of aerosolized steroid (beclomethasone and triamcinolone) delivered through a MDI nozzle extension, the effect of nozzle extension length and number of actuations on the delivered dose, and particle size delivered through the nozzle extension. DESIGN: A 19-G catheter was used as the MDI nozzle extension. The nozzle extension was attached to a 60-ml syringe via the Luer-Lok connection, and the distal end was directed through a hole drilled into a 15-ml capped tube. The MDI was placed into the syringe and actuated by pressing the syringe plunger. Drug delivered through the nozzle extension into the tube was dissolved in methanol (beclomethasone) or ethanol (triamcinolone). Nozzle extension lengths of 10 cm, 20 cm and 30 cm were studied. For each nozzle extension length, delivery was assessed using one, two, three and five actuations of each drug. Drug remaining in the nozzle extension was recovered by rinsing with the appropriate solvent. Aerosol particle size leaving the nozzle extension was determined using a seven stage cascade impactor. Beclomethasone and triamcinolone concentrations were determined by spectrophotometry at 239 nm. SETTING: Respiratory care laboratory of a university teaching hospital. RESULTS: For the pooled results, 70.2 +/- 14.1% of the dose was delivered through the nozzle extension, with no difference between beclomethasone and triamcinolone (p = 0.838). The proportion of drug delivered through the 10-cm extension (76.7 +/- 8.4%) was greater than that from the 20-cm (66.1 +/- 16.5%) and 30-cm (67.7 +/- 13.9%) extensions (p = 0.001). Less drug was delivered through the extension with one actuation (54.1 +/- 17.7%) than with two (71.2 +/- 7.7%), three (77.2 +/- 5.5%), or five actuations (78.2 +/ 4.3%) (p < 0.001). There was a decrease in MMAD with increasing nozzle extension length (3.14 +/- 0.61 microns for 10 cm, 2.97 +/- 0.28 microns for 20 cm, 2.37 +/ 0.27 microns for 30 cm; p = 0.005). CONCLUSIONS: A high proportion of aerosolized steroid was delivered with a MDI actuated through a nozzle extension. The proportion delivered through the nozzle extension was significantly less with longer nozzle extensions and with fewer actuations, but this may not be clinically important. Although particle sizes were smaller from longer nozzle extensions, all were within the respirable range. These results suggest that steroids can be delivered efficiently using a MDI nozzle extension. PMID- 8636530 TI - The economics and cost-effectiveness of critical care medicine. PMID- 8636531 TI - The Italian SEPSIS study: preliminary results on the incidence and evolution of SIRS, sepsis, severe sepsis and septic shock. AB - This prospective, multicenter, epidemiological study was carried out in 99 Italian ICUs, distributed throughout the country, from April 1993 to March 1994. In the study, we applied the new ACCP/SCCM classification system for sepsis (SIRS, sepsis, severe sepsis and septic shock) and determined the prevalence, incidence, evolution and outcome of these categories in critically ill patients. The preliminary analysis of 1101 patients showed that on admission SIRS accounted for about half of the diagnoses (52%) with sepsis, severe sepsis and septic shock accounting for 4.5%, 2.1% and 3% of patients, respectively. Patients with severe sepsis or septic shock more frequently had high SAPS scores than patients without sepsis. Mortality rates were similar in patients with SIRS (26.5%) and without SIRS or infection (24%), but rose to 36% in patients with sepsis, to 52% in those with severe sepsis and to 81.8% in those with septic shock. Sepsis, severe sepsis and septic shock were more common in patients with medical diagnoses, and neither severe sepsis nor septic shock was observed in trauma patients. With respect to evolution, the incidence of septic shock was progressively higher in patients admitted with more severe "sepsis-related" diagnoses, while only a trivial difference in rates of incidence was observed between SIRS patients and those admitted without SIRS or any septic disorder (nil). The breakdown of the various ACCP/SCCM "sepsis-related" diagnoses at any time during the study was: SIRS in 58% of the population, sepsis in 16.3%, severe sepsis in 5.5% and septic shock in 6.1%. It seems reasonable to expect from the final evaluation of our study answers to the questions raised by the ACCP/SCCM Consensus Conference about the correlations between "sepsis-related" diagnosis, severity score, organ dysfunction score and outcome. PMID- 8636532 TI - Is sepsis a mediator-inhibitor mismatch? PMID- 8636533 TI - Time course of cytokine levels in sepsis. AB - In severe sepsis, a network of proinflammatory cytokines (TNF, IL-1 beta, IL-6, IL-8) is activated and blood levels of these cytokines are elevated, albeit inconsistently and with large individual variations. In addition, elevated blood levels of anti-inflammatory cytokines (IL-10), as well as of soluble cytokine receptors (sTNF-RI and II, IL-1ra), have been found. They seem to have a regulatory function in the host response. Levels of TNF and IL-6 are usually highest at the time of admission, whereas the time course of IL-1 beta levels (when detectable) can vary considerably. Limited data on IL-8 levels suggest that they may remain elevated for longer periods. Elevated levels of sTNFR and IL-1ra may also persist for a prolonged period of time. The pathogenetic significance of these observations is still unclear, but persistingly high levels of proinflammatory cytokines may be associated with organ failure and mortality. PMID- 8636535 TI - New therapeutic approaches in sepsis: a critical review. PMID- 8636534 TI - Coagulation inhibitor substitution during sepsis. AB - This review presents the rationale for and main results of coagulation inhibitor substitution during experimental and human sepsis. Activation of the contact system induces activation of the classical complement pathway with generation of anaphylatoxins, of the kinins pathway and of fibrinolysis. Physiologic inhibition depends on the C1-inhibitor (C1-Inh.). Septic patients exhibit a relative deficiency of biologically active C1-Inh. Substitution with concentrations of C1 Inh has been safely performed and preliminary results are consistent with a possible beneficial effect on hypotension and vasopressor requirement in septic shock. The extrinsic pathway is the main initial coagulation process involved in sepsis-induced DIC. Endothelial and monocyte generation of tissue factor (TF) is activated by bacterial products and endotoxin. Activation of TF is counteracted by a specific tissue factor pathway inhibitor (TFPI). The potential for TFPI substitution to inhibit the activation of the coagulation cascade in sepsis requires further study. Thrombin generation is inhibited by antithrombin III (AT III) and the protein C-protein S system. During sepsis, AT III is consumed and degraded by elastase. Animal studies have shown that DIC and death were prevented by high doses of AT III concentrates. Although a significant reduction in the duration of biological symptoms of DIC has been reported in most human studies, the usefulness of AT III substitution in human sepsis is still debated. None of the studies was able to document a statistically significant reduction in mortality. Protein C is activated by thrombomodulin and, with its cofactor protein S, inhibits factors Va and VIIIa. The free level of protein S depends on the level of the C4b binding protein (C4bBP), an acute-phase complement regulatory protein. During sepsis, protein C activity is significantly reduced, either by acute consumption or by thrombomodulin down-regulation, and increased levels of plasma C4bBP inhibit protein S. Infusion of activated protein C and protein S substitution both protect animals from the lethal effects of bacteria. Combining these different coagulation inhibitors should be carefully studied before its use in septic patients is recommended. PMID- 8636536 TI - Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease. AB - OBJECTIVES: The objectives of this study were to test prospectively for an association between Chlamydia and atherosclerosis by comparing the incidence of the pathogen found within atherosclerotic plaques in patients undergoing directional coronary atherectomy with a variety of control specimens and comparing the clinical features between the groups. BACKGROUND: Previous work has suggested an association between Chlamydia pneumoniae infection and coronary atherosclerosis, based on the demonstration of increased serologic titers and the detection of bacteria within atherosclerotic tissue, but this association has not yet been regarded as established. METHODS: Coronary specimens from 90 symptomatic patients undergoing coronary atherectomy were tested for the presence of Chlamydia species using direct immunofluorescence. Control specimens from 24 subjects without atherosclerosis (12 normal coronary specimens and 12 coronary specimens from cardiac transplant recipients with subsequent transplant-induced coronary disease) were also examined. RESULTS: Coronary atherectomy specimens were definitely positive in 66 (73%) and equivocally positive in 5 (6%), resulting in 79% of specimens showing evidence for the presence of Chlamydia species within the atherosclerotic tissue. In contrast, only 1 (4%) of 24 nonatherosclerotic coronary specimens showed any evidence of Chlamydia. The statistical significance of this difference is a p value < 0.001. Transmission electron microscopy was used to confirm the presence of appropriate organisms in three of five positive specimens. No clinical factors except the presence of a primary nonrestenotic lesion (odds ratio 3.0, p = 0.057) predicted the presence of Chlamydia. CONCLUSIONS: This high incidence of Chlamydia only in coronary arteries diseased by atherosclerosis suggests an etiologic role for Chlamydia infection in the development of coronary atherosclerosis that should be further studied. PMID- 8636537 TI - Angiographically silent atherosclerosis detected by intravascular ultrasound in patients with familial hypercholesterolemia and familial combined hyperlipidemia: correlation with high density lipoproteins. AB - OBJECTIVES: This study sought to evaluate the extent of atherosclerosis in coronary and iliac arteries in patients with heterozygous familial hypercholesterolemia or familial combined hyperlipidemia, using intravascular ultrasound imaging. BACKGROUND: Intravascular ultrasound imaging provides cross sectional tomographic views of the vessel wall and allows quantitative assessment of atherosclerosis. METHODS: Forty-eight nonsmoking, asymptomatic patients with heterozygous familial hypercholesterolemia or familial combined hyperlipidemia underwent intravascular ultrasound imaging of the left anterior descending coronary, left main coronary and common iliac arteries. Angiography showed only minimal or no narrowing in these vessels. Intravascular ultrasound images obtained during catheter pullback underwent morphometric analysis. Plaque burden was expressed as the mean and maximal intimal index (ratio of plaque area and area within the internal elastic lamina) and as the percent of vessel surface covered by plaque. RESULTS: Intravascular ultrasound detected plaque more frequently than angiography in the left anterior descending (80% vs. 29%, respectively), left main (44% vs. 16%) and iliac arteries (33% vs. 27%). Plaque burden was higher in the left anterior descending (mean intimal index [+/- SD] 0.25 +/- 0.16) than in the left main (0.11 +/- 0.16, p < 0.001) and iliac arteries (0.02 +/- 0.04, p < 0.001). Angiography detected lumen narrowing only in coronary arteries with a maximal intimal index > or = 0.42 (left anterior descending artery) and > or = 0.43 (left main artery). The area within the internal elastic lamina increased with plaque area in the left anterior descending (r = 0.82, p < 0.001) and left main arteries (r = 0.53, p < 0.001). By stepwise multiple regression analysis, the strongest predictor for plaque burden in the left anterior descending artery was the level of high density lipoprotein (HDL) cholesterol and total/HDL cholesterol ratio for the left main artery. CONCLUSIONS: In patients with heterozygous familial hypercholesterolemia and familial combined hyperlipidemia, extensive coronary plaque is present despite minimal or no angiographic changes. Compensatory vessel enlargement and diffuse involvement with eccentric plaque may account for the lack of angiographic changes. Levels of HDL cholesterol and total/HDL cholesterol ratio are far more powerful predictors of coronary plaque burden than are low density lipoprotein cholesterol levels in these patients with early, asymptomatic disease. PMID- 8636538 TI - Contribution of inadequate compensatory enlargement to development of human coronary artery stenosis: an in vivo intravascular ultrasound study. AB - OBJECTIVES: This intravascular ultrasound study sought to examine to what extent native coronary artery stenosis is accompanied by vessel wall thickening or inadequate compensatory enlargement (relative vessel constriction), or both. BACKGROUND: In human femoral arteries, inadequate compensatory enlargement is reported to be a paradoxic mechanism for the development of severe arterial lumen narrowing. However, it is unclear in human coronary arteries whether inadequate compensatory enlargement contributes to the development of critical arterial stenosis. METHODS: Thirty-five primary coronary artery lesions from 30 patients (19 men, 11 women; mean [+/- SD] age 65 +/- 13 years) were imaged by intravascular ultrasound. The vessel cross-sectional area and lumen area were measured, and the wall area (vessel cross-sectional area minus lumen area) was calculated at the lesion site and at the proximal and distal reference sites. We defined compensatory enlargement to be present when the vessel cross-sectional area at the lesion site was larger than that at the proximal reference site, inadequate compensatory enlargement when the vessel cross-sectional area at the lesion site was smaller than that at the distal reference site and intermediate remodeling when the vessel cross-sectional area at the lesion site was intermediate between the two reference sites. RESULTS: Compensatory enlargement was observed in 19 (54%) of 35 lesions, inadequate compensatory enlargement in 9 (26%) of 35 and intermediate remodeling in 7 (20%) of 35. In the inadequate compensatory enlargement group, reduction of the vessel cross-sectional area contributed to 39% of lumen reduction. CONCLUSIONS: Compensatory enlargement commonly (54%) occurs at stenotic coronary lesions. However inadequate compensatory enlargement results in a substantial amount (39%) of the lumen area reduction in 26% of primary coronary artery lesions. PMID- 8636539 TI - Myocyte degeneration and cell death in hibernating human myocardium. AB - OBJECTIVES: The aim of this study was to analyze the morphologic characteristics of myocyte degeneration leading to replacement fibrosis in hibernating myocardium by use of electron microscopy and immunohistochemical techniques. BACKGROUND: Data on the ultrastructure and the cytoskeleton of cardiomyocytes in myocardial hibernation are scarce. Incomplete or delayed functional recovery might be due to variable degree of cardiomyocyte degeneration in hibernating myocardium. METHODS: In 24 patients, regional wall motion abnormalities were analyzed by use of the centerline method before and 6 +/- 1 months after coronary artery bypass surgery. Preoperative technetium-99m sestamibi uptake was measured by single-photon emission computed tomography for assessing regional perfusion. Fluorine-18 fluorodeoxyglucose uptake was measured by positron emission tomography to assess glucose metabolism. Transmural biopsy specimens were taken during coronary artery bypass surgery from the center of the hypocontractile area of the anterior wall. RESULTS: The myocytes showed varying signs of mild-to-severe degenerative changes and an increased degree of fibrosis. Immunohistochemical analysis demonstrated disruption of the cytoskeletal proteins titin and alpha-actinin. Electron microscopy of the cell organelles and immunohistochemical analysis of the cytoskeleton showed a similarity in the degree of degenerative alterations. Group 1 (n = 11) represented patients with only minor structural alterations, whereas group 2 (n = 13) showed severe morphologic degenerative changes. Wall motion abnormalities showed postoperative improvements, and nuclear imaging revealed a perfusion-metabolism mismatch without significant differences between the groups. CONCLUSIONS: Long-term hypoperfusion causes different degrees of morphologic alterations leading to degeneration. Preoperative analysis of regional contractility and perfusion-metabolism imaging does not distinguish the severity of morphologic alterations nor the functional outcome after revascularization. The insufficient act of self-preservation in hibernating myocardium may lead to a progressive structural degeneration with an incomplete and delayed recovery of function after restoration of blood flow. PMID- 8636540 TI - Persistent transient myocardial ischemia despite beta-adrenergic blockade predicts a higher risk of adverse cardiac events in patients with coronary artery disease. AB - OBJECTIVES: We evaluated the prevalence and prognostic significance of transient myocardial ischemia despite beta-adrenergic blockade in patients with coronary artery disease. BACKGROUND: Persistence of transient ischemia despite therapy may correspond to a subset of high risk patients with coronary disease. The impact of beta-blocker withdrawal in these patients remains unknown. METHODS: Patients (n = 313) with documented coronary artery disease and beta-blocker therapy, with (group I, n = 84) or without (group II, n = 229) transient ischemia on ambulatory electrocardiographic monitoring, were followed up during 21 +/- 9 months for cardiac events (death, myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass surgery and worsening angina). Occurrence of events was compared by log-rank test. RESULTS: The number of coronary stenoses did not differ significantly between groups I and II. Beta-blocker therapy was discontinued more frequently during follow-up in group II (25% vs. 14% in group I, p = 0.04). Cumulative percentage of death or myocardial infarction, or both, tended to be higher in group I a 30 months (17% vs. 5% in group II, p = 0.09). Coronary angioplasty and bypass surgery were significantly more frequent in group I (p = 0.01 and 0.0008, respectively). Transient ischemia was associated with a higher cumulative probability of adverse events (p = 0.004). The number of coronary stenoses, presence of transient ischemia and beta-blocker withdrawal were the only significant prognostic factors of cardiac events in the Cox model. In group I patients, the relative hazard of cardiac events was increased threefold when beta-blocker therapy was interrupted. CONCLUSIONS: These data suggest that 1) the occurrence of transient ischemia despite beta-blocker therapy identifies a subset of high risk patients with coronary artery disease, and 2) the interruption of beta-blocker therapy increases the risk of adverse cardiac events. PMID- 8636541 TI - Comparison of rest thallium-201 imaging and rest technetium-99m sestamibi imaging for assessment of myocardial viability in patients with coronary artery disease and severe left ventricular dysfunction. AB - OBJECTIVES: We prospectively compared myocardial uptake of thallium-201 (201Tl) at rest with rest technetium-99m (99mTc) sestamibi uptake in the same patients, using quantitative singlephoton emission computed tomography (SPECT). BACKGROUND: Because of only slightly delayed redistribution, 99mTc-sestamibi uptake at rest may be less than 201Tl uptake, thereby underestimating the extent of viability. METHODS: Twenty patients (2.25 stenoses per patient) with a mean left ventricular ejection fraction of 33 +/- 2% underwent early and 3-h delayed rest 201Tl SPECT, rest 99mTc-sestamibi SPECT and two-dimensional echocardiography. RESULTS: The 280 scan segments were classified as either a normal, mild reduction in viability, defined as delayed 201Tl uptake < or = 75% and > or = 5%, or a severe reduction in viability, defined as delayed 201Tl uptake < 50%. Mild and severe defects were further classified as fixed or having rest 201Tl redistribution. Comparisons by patients were made using repeated measures analysis of variance and Dunnett's multiple comparisons test to compare 99mTc-sestamibi with initial rest 201Tl and delayed 201Tl uptake. Twenty patients had at least one mild fixed defect (95 total segments). The average percent uptake in these defects for initial 201Tl, delayed 201Tl and 99mTc-sestamibi was 62.5 +/- 2.7%, 63.1 +/- 7.1% and 67.3 +/- 9.7%, respectively (p = NS). Twelve patients (27 segments) had mild redistribution defects on serial rest 201Tl imaging. The average percent uptake was 61.6 +/- 5.2% for initial 201Tl, 67.0 +/- 9.1% for delayed 201Tl and 67.7 +/- 12.4% for 99mTc-sestamibi defects. Technetium-99m sestamibi uptake was not significantly different than that for delayed 201Tl but was significantly greater than initial 201Tl uptake. Seventeen patients (52 segments) had severe fixed 201Tl defects. The average percent uptake was 38.9 +/- 7.3% for initial 201Tl, 38.3 +/- 12.2% for delayed 201Tl and 42.7 +/- 14.2% for 99mTc-sestamibi defects in these patients (p = NS). Ten patients (19 segments) had severe redistribution defects on rest 201Tl imaging. The average percent uptake was 37.0 +/- 8.5% for initial 201Tl, 42.9 +/- 8.6% for delayed 201Tl and 44.5 +/- 11.3% for 99mTc sestamibi defects. As was seen for mild 201Tl redistribution defects, 99mTc sestamibi uptake was significantly higher than initial 201Tl uptake, but not significantly different than delayed 201Tl uptake in these severe defects. CONCLUSIONS: Technetium-99m sestamibi uptake after injection at rest is comparable to 201Tl uptake after injection at rest in patients with severe coronary artery disease and left ventricular dysfunction, suggesting comparable worth for viability assessment. PMID- 8636542 TI - Radionuclide detection of myocardial ischemia and myocardial viability: is the glass half empty of half full? PMID- 8636543 TI - Comparison of dobutamine echocardiography and positron emission tomography in patients with chronic ischemic left ventricular dysfunction. AB - OBJECTIVES: The aim of this study was to correlate dobutamine-induced contractile reserve as detected by echocardiography with findings on positron emission tomography in patients with chronic ischemic left ventricular dysfunction. BACKGROUND: Contractile reserve induced by low dose dobutamine infusion has been proposed as a marker of myocardial viability. METHODS: Sixty patients with stable coronary artery disease and left ventricular dysfunction (mean ejection fraction [+/- SD] 29 +/- 10%) underwent transthoracic echocardiography with dobutamine infusion (up to 10 micrograms/kg body weight per min) and positron emission tomography with nitrogen-13 ammonia and fluorine-18 (F-18) fluorodeoxyglucose as a perfusion and a metabolic tracer, respectively. Regional wall motion, perfusion and metabolism were analyzed semiquantitatively by using a 16-segment model. Segments with F-18 fluorodeoxyglucose uptake > 50% were considered viable on positron emission tomography. RESULTS: After dobutamine infusion, hemodynamic variables changed significantly, and myocardial ischemia was evident in 17 patients. All 60 patients had dysfunctional myocardium considered viable on positron emission tomography (8 +/- 4 segments/patient), whereas 52 patients had dysfunctional myocardium with contractile enhancement by dobutamine echocardiography (4 +/- 2 segments/patient, p = 0.01). The extent of dysfunctional myocardium with contractile reserve appeared to correlate less closely with the total extent of viable dysfunctional myocardium identified by positron emission tomography than with the number of such segments associated with a pattern of perfusion-metabolism mismatch. CONCLUSIONS: In patients with chronic ischemic left ventricular dysfunction, echocardiography can be used to identify enhancement in the contractile function of viable dysfunctional myocardium after infusion of low dose dobutamine. In this study, the presence and extent of such enhancement were relatively less than the values obtained from positron emission tomography. PMID- 8636544 TI - Response of dysfunctional myocardium to dobutamine. "The eyes see what the mind knows!". PMID- 8636545 TI - Identification of severe and extensive coronary artery disease by automatic measurement of transient ischemic dilation of the left ventricle in dual-isotope myocardial perfusion SPECT. AB - OBJECTIVES: This study sought to assess whether a transient ischemic dilation ratio, determined from automatically derived stress and rest left ventricular volumes during stress technetium-99m (Tc-99m) sestamibi/rest thallium-201 dual isotope myocardial perfusion single-photon emission computed tomography (SPECT), is useful for the identification of patients with severe and extensive coronary artery disease. BACKGROUND: Transient ischemic dilation of the left ventricle on stress/redistribution thallium-201 scintigraphy has been shown to be a clinically useful marker of severe and extensive coronary artery disease. However, in practice, its assessment is highly subjective. This study automatically assessed the transient ischemic dilation ratio on the basis of a previously described algorithm to estimate three-dimensional ventricular boundaries. METHODS: Normal limits for the transient ischemic dilation ratio were developed using data from 54 patients with a low likelihood (< 5%) of coronary artery disease, and criteria for abnormality were developed based on data from 97 who under-went catheterization, of whom 34 had severe and extensive coronary artery disease, defined as > or = 90% stenosis in the proximal left anterior descending coronary artery or in two or more coronary arteries, and 63 had no coronary artery disease (15 patients) or mild to moderate coronary artery disease (48 patients). The criteria were then tested in a validation cohort of 77 additional patients who underwent catheterization, of whom 36 had severe and extensive coronary artery disease. The quantitative results of the dilation ratio were compared with the visual results of the dilation ratio and perfusion defect analysis. RESULTS: For normal limits, receiver operating characteristic curve analysis showed that abnormal transient ischemic dilation ratio values corresponded to left ventricular endocardial volume ratios > 1.22 (mean +/- 2 SD). Transient ischemic dilation assessment using these criteria for abnormality showed high sensitivity (24 [71%] of 34) and very high specificity (60 [95%] of 63) for severe and extensive coronary artery disease. When the analysis was applied to the prospective catheterization group, similar sensitivity and specificity for severe and extensive coronary artery disease were observed (77% and 92%, respectively). Significant agreement (p = 0.0001) was found between the degree of transient ischemic dilation and the Tc-99m sestamibi defect extent, the latter assessed by semiquantitative visual analysis (summed stress score). CONCLUSIONS: The automatic measurement of transient ischemic dilation in dual-isotope myocardial perfusion SPECT is a clinically useful marker that is sensitive and highly specific for detection of severe and extensive coronary artery disease. PMID- 8636546 TI - Cardioesophageal reflex: a mechanism for "linked angina" in patients with angiographically proven coronary artery disease. AB - OBJECTIVES: The purpose of this study was to investigate the presence of a cardioesophageal reflex in patients with coronary artery disease that may explain the mechanism of "linked angina." BACKGROUND: It has been previously shown that esophageal acid stimulation can reduce coronary blood flow in patients with syndrome X, suggesting the presence of a cardioesophageal reflex in humans. METHODS: We studied the effect of esophageal acid stimulation on coronary blood flow in 14 patients with angiographically documented significant coronary artery disease and in 18 heart transplant recipients. Hydrochloric acid (0.1 mol/liter) and 0.9% saline solution were infused in random, double-blind manner (60 ml over 5 min) through a fine-bore tube positioned in the patient's distal esophagus, and coronary blood flow measurements were obtained after each infusion by use of a 3.6F intracoronary Doppler catheter positioned in the proximal left anterior descending coronary artery. RESULTS: Coronary blood flow was reduced significantly by esophageal acid stimulation in the coronary artery disease group (before acid 70.4 +/- 14.3 ml/min, after acid stimulation 46.4 +/- 19.1 ml/min [mean +/- SD], p < 0.01). However, there was no significant difference in coronary blood flow during saline infusion (73.5 +/- 15.3 vs. 72.5 +/- 14 ml/min). Coronary blood flow in the heart transplant group was not affected by acid or saline infusion. CONCLUSIONS: Esophageal acid stimulation can cause animal attacks and significantly reduce coronary blood flow in patients with coronary artery disease. The lack of any significant effect in heart transplant recipients with heart denervation suggests a neural reflex. PMID- 8636547 TI - Patterns and behavior of transient myocardial ischemia in stable coronary disease are the same in both men and women: a comparative study. AB - OBJECTIVES: This study sought to compare the circadian variations in transient ischemic activity, mean heart rate and ischemic threshold between women and men with coronary artery disease. BACKGROUND: There is a circadian variation in ischemic activity, onset of myocardial infarction and sudden cardiac death in patients with coronary artery disease, but studies assessing ischemia have incorporated predominantly male subjects. METHODS: Thirty-one women and 45 men underwent at least 48 h of ambulatory ST segment monitoring. RESULTS: There was a similar and significant circadian variation in ischemic activity in both women and men (p < 0.0001 and p < 0.0001, respectively), with a trough at night, a surge in the morning and a peak between 1 and 2 PM, corresponding to a similar circadian variation in mean hourly heart rate (p < 0.0001) that was not different between men and women (p = 0.28, power to detect a shift 99.9%). Mean heart rate at onset of ischemia (ischemic threshold) had similar variability in women and men (p = 0.96), and harmonic regression analysis confirmed a significant circadian variation (p < 0.0001), with a trough at night and a peak during activity hours. Heart rate increased significantly in the 5 min before ischemia throughout the 24 h (p < 0.0001), with no gender differences in the pattern of preonset to onset heart rate changes over time (p = 0.52); the smallest differences were recorded in the middle of the night. The majority of ischemic episodes (80%) had a heart rate increase > 5 beats/min in the 5 min before ischemia, but there were no gender differences. CONCLUSIONS: Women with coronary artery disease have a pattern of ischemic activity and underlying pathophysiologic mechanisms very similar to men. The importance of increase in myocardial oxygen demand in the genesis of ischemia in both men and women is reflected by similar magnitude of heart rate increases before ischemia. The lower ischemic threshold during the nocturnal hours, when blood pressure is also lower, is consistent with a circadian variation in underlying coronary vascular tone. PMID- 8636548 TI - Functional, angiographic and intracoronary Doppler flow characteristics in symptomatic patients with myocardial bridging: effect of short-term intravenous beta-blocker medication. AB - OBJECTIVES: We sought to define the effects of short-term beta-adrenergic blocking medication on intracoronary flow characteristics, clinical symptoms and angiographic diameter changes in patients with severe myocardial bridging of the left anterior descending coronary artery. BACKGROUND: Controversy exists regarding the pathophysiology, clinical relevance and optimal therapy in symptomatic patients with myocardial bridges because antianginal drugs have not been systematically tested. METHODS: In 15 symptomatic patients with myocardial bridging of the left anterior descending coronary artery, maximal lumen diameter reductions were evaluated by quantitative coronary angiography. There were no angiographic signs of coronary artery disease. Coronary blood flow velocities (using a 0.014-in. [0.035 cm] Doppler guide wire) were measured at rest, during atrial pacing and during intravenous administration of a short-acting beta blocker (esmolol, 50 to 500 micrograms/kg body weight per min) with continuous atrial pacing. RESULTS: The maximal angiographic systolic lumen diameter reduction within the myocardial bridges was 83 +/- 9% at rest, with a persistent diastolic diameter reduction of 41 +/- 11% (mean +/- SD). Short-term intravenous beta-blocker therapy decreased the diameter reduction during both systole (from 83 +/- 9% to 62 +/- 11%) and diastole (from 41 +/- 11% to 30 +/- 9%, both p < 0.001). The average diastolic peak flow velocity was higher within the myocardial bridges (33 +/- 13 cm/s) than the proximal (26 +/- 13 cm/s) and distal bridges (17 +/- 4 cm/s, both p < 0.001). During tachypacing, average diastolic peak flow velocity increased within the bridged segments to 63 +/- 21 cm/s versus 29 +/- 12 cm/s in the proximal and 20 +/- 4 cm/s in the distal bridges (both p < 0.001). Beta-receptor blockade produced a return to baseline values (average diastolic peak flow velocity within bridge 35 +/- 16 cm/s, p < 0.001). ST segment changes and symptoms were abolished with beta-blocker administration. CONCLUSIONS: In patients with myocardial bridges, administration of short-acting beta-blockers during atrial pacing alleviates anginal symptoms and signs of ischemia. This effect was mediated by a reduction of vascular compression and maximal flow velocities within the bridged coronary artery segment. PMID- 8636549 TI - Time from symptom onset to treatment and outcomes after thrombolytic therapy. GUSTO-1 Investigators. AB - OBJECTIVES: This study sought to examine the relations among patient characteristics, time to thrombolysis and outcomes in the international GUSTO-I trial. BACKGROUND: Studies have shown better left ventricular function and decreased infarct size as well as increased survival with earlier thrombolysis, but the relative benefits of various thrombolytic agents with earlier administration are uncertain. METHODS: We evaluated the relations of baseline characteristics to three prospectively defined time variables: symptom onset to treatment, symptom onset to hospital arrival (presentation delay) and hospital arrival to treatment (treatment delay). We also examined the relations of delays to clinical outcomes and to the relative 30-day mortality benefit with accelerated tissue-type plasminogen activator (t-PA) versus streptokinase. RESULTS: Female, elderly, diabetic and hypertensive patients had longer delays at all stages. Previous infarction or bypass surgery was an additional risk factor for treatment delay. Early thrombolysis was associated with lower overall mortality rate (< 2 h, 5.5%; > 4 h, 9.0%), but no additional relative benefit resulted from earlier treatment with accelerated t-PA versus streptokinase (p = 0.38). Longer presentation and treatment delays were both associated with increased mortality rate (presentation delay < 1 h, 5.6% and > 4 h, 8.6%; treatment delay < 1 h, 5.4%, and > 90 min, 8.1%). As time to treatment increased, the incidence of recurrent ischemia or reinfarction decreased, but the rates of shock, heart failure and stroke increased. CONCLUSIONS: Earlier treatment resulted in better outcomes, regardless of thrombolytic strategy. Elderly, female and diabetic patients were treated later, adding to their already substantial risk. PMID- 8636550 TI - Plasma brain natriuretic peptide concentrations predict survival after acute myocardial infarction. AB - OBJECTIVES: This study sought to examine whether plasma brain natriuretic peptide levels can predict prognosis after myocardial infarction. BACKGROUND: It has been suggested that concentrations of plasma brain natriuretic peptide reflect left ventricular function. Although the prognosis after myocardial infarction depends on residual left ventricular function, it is not known whether plasma levels of brain natriuretic peptide after the onset of myocardial infarction can be used to predict long-term outcome. METHODS: Plasma brain natriuretic peptide and atrial natriuretic peptide levels as well as invasive hemodynamic variables were measured in 70 patients with acute myocardial infarction (53 men, 17 women; mean age 65 years). Measurements were obtained on admission (mean 6 h after onset) and on day 2 after onset. Mean follow-up period was 18 months. RESULTS: Plasma brain natriuretic peptide levels measured on admission and day 2 correlated significantly with hemodynamic variables, which are influenced by left ventricular function. However, plasma atrial natriuretic peptide levels correlated with none of the hemodynamic variables measured on admission; and of those measured on day 2, plasma atrial natriuretic peptide levels correlated only with left atrial filling pressure. During the follow-up period (mean 18 +/- 7 months), 11 patients died of cardiac causes. By Kaplan-Meier analysis, it was found that patients with plasma brain natriuretic peptide levels higher than the median level, both on admission and on day 2, had significantly higher mortality rates than those with the submedian level (on admission, p < 0.01; on day 2, p < 0.05). However, only the plasma atrial natriuretic peptide level obtained immediately after admission was significantly related to survival (p < 0.01). By Cox proportional hazards model analysis of the noninvasive variables, it was found that plasma brain natriuretic peptide concentration was more closely related to survival after myocardial infarction (p = 0.0001). CONCLUSIONS: Increased plasma brain natriuretic peptide concentrations in the early or subacute phase of myocardial infarction are a powerful noninvasive indicator of poor prognosis, possibly reflecting residual left ventricular function after myocardial infarction. PMID- 8636551 TI - Influence of cigarette smoking on rate of reopening of the infarct-related coronary artery after myocardial infarction: a multivariate analysis. AB - OBJECTIVES: This study sought to determine whether the reopening of the infarct related vessel is related to clinical characteristics or cardiovascular risk factors, or both. BACKGROUND: In acute myocardial infarction, thrombolytic therapy reduces mortality by restoring the patency of the infarct-related vessel. However, despite the use of thrombolytic agents, the infarct-related vessel remains occluded in up to 40% of patients. METHODS: We studied 295 consecutive patients with an acute myocardial infarction who underwent coronary angiography within 15 days (mean [+/- SD] 6.7 +/- 3.2 days) of the onset of symptoms. Infarct related artery patency was defined by Thrombolysis in Myocardial Infarction trial flow grade > or = 2. Four cardiovascular risk factors--smoking, hypertension, hypercholesterolemia and diabetes mellitus--and eight different variables-age, gender, in-hospital death, history of previous myocardial infarction, location of current myocardial infarction, use of thrombolytic agents, time interval between onset of symptoms, thrombolytic therapy and coronary angiography--were recorded in all patients. RESULTS: Thrombolysis in current smokers and anterior infard location on admission were the three independent factors highly correlated with the patency of the infarct-related vessel (odds ratios 3.2, 3.0 and 1.9, respectively). In smokers, thrombolytic therapy was associated with a higher reopening rate of the infard vessel, from 35% to 77% (p < 0.001). Nonsmokers did not benefit from thrombolytic therapy, regardless of infarct location. CONCLUSIONS: These observational data, if replicated, suggest that in patients with acute myocardial infarction, thrombolytic therapy may be most effective in current smokers, whereas nonsmokers and ex-smokers may require other management strategies, such as emergency percutaneous transluminal coronary angioplasty. PMID- 8636553 TI - Intravascular ultrasound predictors of restenosis after percutaneous transcatheter coronary revascularization. AB - OBJECTIVES: This study sought to evaluate preintervention and postintervention intravascular ultrasound studies for potential predictors of angiographic restenosis and to use ultrasound predictors of restenosis to enhance our understanding of the pathophysiology of the restenosis disease process. BACKGROUND: Restenosis remains the major limitation of percutaneous transcatheter coronary revascularization. Although its mechanisms remain incompletely understood, numerous studies have identified some of the clinical, anatomic and procedural risk factors for restenosis. Intravascular ultrasound imaging of target lesions before and after catheter-based treatment consistently demonstrates more target lesion calcium, more extensive reference segment atherosclerosis, smaller final lumen dimensions, significant residual plaque burden and a greater degree of tissue trauma than is evident by angiography. METHODS: Intravascular ultrasound studies were performed in 360 nonstented native coronary artery lesions (final diameter stenosis 18 +/- 11%) in 351 patients for whom follow-up angiographic data were available 6.4 +/- 3.6 months later. Hospital charts were reviewed, and qualitative and quantitative coronary angiographic and intravascular ultrasound analyses were performed by independent core laboratories. Four dependent angiographic end points were tested: restenosis as a binary definition (> or = 50% diameter stenosis at follow-up) was the primary end point; follow-up diameter stenosis, late lumen loss and follow-up minimal lumen diameter were the secondary end points. RESULTS: Reference vessel size, the preintervention quantitative coronary angiographic assessment of lesion severity and the postintervention intravascular ultrasound cross-sectional measurements predicted the late angiographic results. In particular, the intravascular ultrasound postintervention cross-sectional narrowing (plaque plus media cross-sectional area divided by external elastic membrane cross-sectional area) predicted the primary end point (restenosis) and two of the three secondary end points (follow-up diameter stenosis and late lumen loss) and was therefore the most consistent predictor of restenosis. CONCLUSIONS: Intravascular ultrasound variables are more powerful and consistent predictors of angiographic restenosis than currently accepted clinical or angiographic risk factors. PMID- 8636552 TI - Clinical outcome 10 years after attempted percutaneous transluminal coronary angioplasty in 856 patients. AB - OBJECTIVES: This study reports the 10-year outcome of 856 consecutive patients who underwent attempted coronary angioplasty at the Thoraxcenter during the years 1980 to 1985. BACKGROUND: Coronary balloon angioplasty was first performed in 1977, and this procedure was introduced into clinical practice at the Thoraxcenter in 1980. Although advances have been made, extending our knowledge of the long-term outcome in terms of survival and major cardiac events remains of interest and a valuable guide in the treatment of patients with coronary artery disease. METHODS: Details of survival, cardiac events, symptoms and medication were retrospectively obtained from the Dutch civil registry, medical records or by letter or telephone or from the patient's physician and entered into a dedicated data base. Patient survival curves were constructed, and factors influencing survival and cardiac events were identified. RESULTS: The procedural clinical success rate was 82%. Follow-up information was obtained in 837 patients (97.8%). Six hundred forty-one patients (77%) were alive, of whom 334 (53%) were symptom free, and 254 (40%) were taking no antianginal medication. The overall 5- and 10-year survival rates were 90% (95% confidence interval [CI] 87.6% to 92.4%) and 78% (95% CI 75.0% to 81.0%), respectively, and the respective freedom from significant cardiac events (death, myocardial infarction, coronary artery bypass surgery and repeat angioplasty) was 57% (95% CI 53.4% to 60.6%) and 36% (95% CI 32.4% to 39.6%). Factors that were found to adversely influence 10-year survival were age > or = 60 years (> or = 60 years [67%], 50 to 59 years [82%], < 50 years [88%]), multivessel disease (multivessel disease [69%], single-vessel disease [82%]), impaired left ventricular function (ejection fraction < 50% [57%], > or = 50% [80%]) and a history of previous myocardial infarction (previous myocardial infarction [72%], no previous infarction [83%]). These factors were also found to be independent predictors of death during the follow-up period by a multivariate stepwise logistic regression analysis. Other factors tested, with no influence on survival, were gender, procedural success and stability of angina at the time of intervention. CONCLUSIONS: The long-term prognosis of patients after coronary angioplasty is good, particularly in those <60 years old with single-vessel disease and normal left ventricular function. The majority of patients are likely to experience a further cardiac event in the 10 years after their first angioplasty procedure. PMID- 8636554 TI - Relation between preexistent coronary collateral circulation and the incidence of restenosis after successful primary coronary angioplasty for acute myocardial infarction. AB - OBJECTIVES: The purpose of this study was to test the hypothesis that the incidence of restenosis after primary percutaneous transluminal coronary angioplasty for acute myocardial infarction is largely influenced by the preexistent coronary collateral circulation to the infarct-related coronary artery. BACKGROUND: The occurrence of restenosis after coronary angioplasty is the most serious limitation of this procedure. However, prediction of restenosis is difficult. Severe preexistent stenosis of the infarct-related coronary artery causing the development of collateral circulation may result in a high frequency of restenosis. METHODS: The study group consisted of 152 consecutive patients undergoing primary coronary angioplasty within 12 h after the onset of a first acute myocardial infarction. Of this group, 124 patients were angiographically followed up during the convalescent period of infarction and were classified into two groups according to the extent of preexistent collateral circulation to the infarct-related coronary artery. RESULTS: Restenosis occurred in 26 (38%) of 69 patients with poor or no collateral circulation (group A) in contrast to 35 (64%) of 55 patients with good angiographic collateral circulation (group B, p < 0.005). The frequency of preinfarction angina was significantly lower (p < 0.05) in group A (26% [18 of 69]) than in group B (44% [24 of 55]). CONCLUSIONS: These findings indicate that the presence of well developed collateral circulation to the infarct-related coronary artery predicts a higher frequency of restenosis after primary coronary angioplasty. The difference in restenosis rates observed between the patients with and without good collateral circulation probably reflects the impact of underlying severity of stenosis on the long-term outcome after coronary angioplasty. PMID- 8636555 TI - Serial doppler echocardiographic evaluation of bioprosthetic valves in the tricuspid position. AB - OBJECTIVES: This study sought to evaluate bioprosthetic valve dysfunction in the tricuspid position by serial Doppler echocardiography. BACKGROUND: Few reports on the long-term results of tricuspid valve replacement with bioprosthetic valves are evaluated by serial Doppler echocardiography. METHODS: Between September 1979 and December 1993, 95 patients underwent tricuspid valve replacement with bioprosthetic valves at our facility. Sixty patients who underwent serial Doppler echocardiographic examination at intervals of at least 2 years after operation were included in the final analysis. These patients were followed up from 1.5 to 13.0 years (mean 5.8 +/- 2.5). RESULTS: The actuarial rates of freedom from bioprosthetic valve stenosis and regurgitation at 10 years were 46% and 51%, respectively. The prevalence of bioprosthetic valve stenosis and regurgitation increased progressively in a linear manner beginning 1 or 2 years after tricuspid valve replacement. Right heart failure developed during follow-up in 20 of the 25 patients with bioprosthetic valve dysfunction. CONCLUSIONS: The long-term durability of bioprosthetic valves in the tricuspid position was substantially lower in our study than that reported in previous studies. Tricuspid bioprosthetic valve dysfunction increased progressively in a linear manner beginning 1 to 2 years after tricuspid valve replacement. PMID- 8636556 TI - Failure of adjusted doses of subcutaneous heparin to prevent thromboembolic phenomena in pregnant patients with mechanical cardiac valve prostheses. AB - OBJECTIVES: This report describes our experience with the use of an anticoagulant regimen of adjusted doses of subcutaneous heparin during pregnancy in women with cardiac valve prostheses. BACKGROUND: Gravid patients with prosthetic heart valves require long-term anticoagulant therapy. To avoid the increased incidence of fetal morbidity and mortality associated with the use of coumarin agents in such patients during pregnancy, anticoagulation with subcutaneous heparin has been suggested. Controversy exists concerning the appropriate treatment of these patients. METHODS: Forty pregnancies in 37 women with prosthetic heart valves were prospectively followed up. Subcutaneous heparin was administered from the 6th until the end of the 12th week and in the last 2 weeks of gestation. Heparin was given every 8 h in the first 36 cases and every 6 h in the last 4 cases, and the dose adjusted to maintain the activated partial thromboplastin time at 1.5 to 2.5 times the control level. Acenocoumarol was used at other times. RESULTS: The incidence rate of spontaneous abortions was 37.5%; there was one neonatal death (2.5%) due to cerebral hemorrhage. No signs of coumarin-induced embryopathy were found in any of the 16 live-born infants studied by the geneticist. One mother died of gastrointestinal bleeding while receiving oral anticoagulant agents. There were two cases of fatal massive thrombosis of a mitral tilting-disk prosthesis during heparin therapy. The study was interrupted after the last of these two cases. CONCLUSIONS: The regimen of adjusted doses of subcutaneous heparin used in this study is not effective to prevent thrombosis of mechanical valve prostheses during pregnancy. The use of heparin from the 6th to the 12th week of gestation does not decrease the high incidence of fetal wastage associated with anticoagulant therapy. Coumarin agents provide adequate protection against thromboembolism during pregnancy in patients with mechanical valve prostheses. PMID- 8636557 TI - Anticoagulation in pregnant women with prosthetic heart valves: a double jeopardy. PMID- 8636559 TI - Distribution of atrial electrogram types during atrial fibrillation: effect of rapid atrial pacing and intercaval junction ablation. AB - OBJECTIVES: This study examined the anatomic distribution types and possible determinant of atrial electrogram types during atrial fibrillation. BACKGROUND: Different types of atrial electrograms during atrial fibrillation have been observed and classified, but their anatomic distribution patterns, determinants and potential usefulness in guiding future catheter ablation are unknown. METHODS: Two animal models of atrial fibrillation were used: the sterile pericarditis model (n = 10) and the rapid atrial pacing model (400 beats/min for 6 weeks, n = 6). The atrial electrogram of atrial fibrillation and the atrial effective refractory period were obtained from multiple sites of the right and left atrium. In addition, decremental rapid atrial stimulation was applied to the site of shortest and longest atrial effective refractory periods until atrial fibrillation induction in a subgroup of nine dogs. Ablation of the intercaval junction was performed using the radiofrequency catheter technique in dogs with atrial fibrillation duration > 1 min. RESULTS: In both models, organized atrial electrograms (type I) were predominantly observed at the left atrial sites and the right atrial appendage, whereas disorganized atrial electrograms (type III) were mainly observed at the right posterolateral atrium. The distribution of the atrial electrogram types closely followed that of the atrial effective refractory period, with the shortest atrial effective refractory period corresponding to organized atrial electrograms (type I) and the longest atrial effective refractory period corresponding to disorganized atrial electrograms (type III). The correlation of atrial electrogram type with the atrial effective refractory period was further demonstrated by the effect of rapid atrial stimulation. When rapid atrial stimulation was applied to the site with the shortest atrial effective refractory period, disorganized atrial electrograms were observed at sites with the longest atrial effective refractory period, whereas 1:1 atrial capture was still present at the stimulation site. Ablation of the intercaval junction made atrial fibrillation noninducible or tended to shorten the atrial fibrillation duration (from 26.4 +/- 24.2 to 8.8 +/- 22.6 min in the pericarditis group, p = 0.02, and from 33.7 +/- 29.2 to 12.1 +/- 23.8 min in the rapid pacing group, p = 0.09) but did not change the atrial electrogram types during atrial fibrillation. CONCLUSIONS: Various types of atrial electrograms are present at different locations during atrial fibrillation. The atrial electrogram characteristics of atrial fibrillation at a specific location are related to the atrial effective refractory period, with short effective refractory periods associated with organized atrial electrograms and long effective refractory periods associated with disorganized electrograms. PMID- 8636558 TI - Effectiveness and safety of diltiazem or lisinopril in treatment of hypertension after heart transplantation. Results of a prospective, randomized multicenter trail. AB - OBJECTIVES: The purpose of this study was to determine the effectiveness and safety of diltiazem or lisinopril for treatment of hypertension after heart transplantation. BACKGROUND: Systemic hypertension is common after heart transplantation, and to date there are no randomized, prospective multicenter treatment trials. METHODS: Members of the Cardiac Transplant Research Database Group developed and implemented a prospective, randomized multicenter trial of the effectiveness and safety of diltiazem or lisinopril in the treatment of hypertension in cyclosporine-treated patients after heart transplantation. RESULTS: One hundred sixteen patients with hypertension (blood pressure > or = 140/90 mm Hg) after heart transplantation were randomized for > or = 3 months of treatment. Of 55 diltiazem-treated patients, 21 (38%) were responders (diastolic blood pressure < 90 mm Hg), 23 (42%) were nonresponders (diastolic blood pressure > or = 90 mm Hg), and 11 (20%) were withdrawn from the study. Of 61 lisinopril treated patients, 28 (46%) were responders, 22 (36%) were nonresponders, and 11 (18%) were withdrawn. There was no difference in baseline characteristics or percent responders between the two groups. Systolic pressure decreased from 157 +/- 2.3 to 130 +/- 2.0 mm Hg (mean +/- 1 SEM) in the diltiazem-treated responders and from 153 +/- 2.1 to 127 +/- 2.7 mm Hg in the lisinopril-treated responders (p < 0.0001). Diastolic pressure decreased from 100 +/- 0.9 to 85 +/- 1.6 mm Hg in the diltiazem-treated responders and from 100 +/- 1.0 to 84 +/- 2.0 mm Hg in the lisinopril-treated responders (p < 0.0001). There were a total of 35 reported adverse events, 22 of which led to withdrawal of the patient from the study. All drug-related side effects were considered minor and resolved with discontinuation of the drug. CONCLUSIONS: These results indicate that both diltiazem and lisinopril are safe for treatment of hypertension after heart transplantation, although titrated monotherapy with either drug controlled the condition in < 50% of patients. PMID- 8636560 TI - Regional left atrial stasis during atrial fibrillation and flutter: determinants and relation to stroke. AB - OBJECTIVES: This study sought to 1) determine the location of left atrial stasis during atrial arrhythmia; 2) define the degree of stasis associated with significant risk of stroke; and 3) identify clinical or transthoracic echocardiographic data useful for predicting left atrial stasis. BACKGROUND: Prior studies suggest that stroke during atrial arrhythmia is related to stasis in either the body of the left atrium or the appendage. Recent data indicate that appendage stasis is associated with appendage thrombus formation, but stroke during atrial arrhythmia occurs frequently in the absence of appendage stasis. METHODS: Blood flow velocity was measured in multiple sites in the body of the left atrium and in the appendage by transesophageal pulsed wave Doppler echocardiography in 89 patients with atrial fibrillation or flutter. Regional velocities were related to the frequency of probable embolic stroke and to clinical and transthoracic echocardiographic variables. RESULTS: The lowest velocity region was either the posterior left atrium or the appendage. Stroke frequency increased progressively and steeply with velocity < 15 cm/s in either region; this cutoff value had an 87% sensitivity and 40% specificity for stroke. Factors related to stasis were low left atrial ejection fraction, mitral regurgitation < 3+, fibrillation (vs. type I flutter), left ventricular dilation and mitral valve area < 2.0 cm2. CONCLUSIONS: Posterior left atrial stasis appears to be as important as appendage stasis for the risk of stroke, which increases steeply with lower blood flow velocity in either region. Patients likely to have severe stasis during atrial arrhythmia are those with left ventricular dilation and low atrial ejection fraction accompanying left atrial dilation. Direct measurement of atrial velocity by transesophageal echocardiography appears to be useful for the identification of patients at risk for stroke during atrial arrhythmia. PMID- 8636562 TI - Fetal tachycardia: mechanisms and predictors of hydrops fetalis. AB - OBJECTIVES: This study had three objectives: 1) to determine the electrophysiologic mechanisms of fetal supraventricular tachycardia at presentation and postnatally; 2) to identify the clinical and electrophysiologic predictors of hydrops fetalis; and 3) to describe the medium-term follow-up (1 to 7 years) of patients with fetal supraventricular tachycardia. BACKGROUND: Fetal supraventricular tachycardia causes significant fetal and neonatal morbidity and mortality. Prenatal analysis and postnatal confirmation of fetal supraventricular tachycardia mechanisms have been limited. METHODS: Supraventricular tachycardia mechanisms were evaluated by prenatal Doppler/M-mode echocardiography, immediate neonatal surface electrocardiography and postnatal transesophageal electrophysiologic procedures in 30 consecutive patients presenting with fetal supraventricular tachycardia (17 managed prenatally, 13 first managed postnatally). RESULTS: The fetal supraventricular tachycardia mechanism was 1:1 atrioventricular conduction in 22 patients and supraventricular tachycardia with atrioventricular block (atrial flutter) in 8. At the postnatal transesophageal electrophysiologic procedure, tachycardia was induced in 27 of 30 patients; atrioventricular reentrant tachycardia in 25 (93%) of 27 and intraatrial reentrant tachycardia in only 2 (7%) of 27. Hydrops was present in 12 of 30 fetuses. Sustained supraventricular tachycardia (> 12 h) and lower gestation at presentation correlated with hydrops (p < 0.02, p < 0.05), but mechanism of tachycardia and heart rate did not. Gestational age at delivery was significantly greater in those who received intrauterine management (39 +/- 1.3 vs. 37 +/- 2.9 weeks, p = 0.04) despite earlier presentation (32.6 vs. 37.1 weeks). Cesarean section deliveries were reduced in the same group (3 of 17 vs. 11 of 13, p = 0.0006). CONCLUSIONS: Atrioventricular reentrant tachycardia was the predominant mechanism of supraventricular tachycardia in the fetus. There was a high association of supraventricular tachycardia with atrioventricular block in utero and accessory atrioventricular connections. Outcome at 1 to 7 years was excellent regardless of severity of illness at clinical presentation. PMID- 8636561 TI - Abnormal reflex venous function in patients with neuromediated syncope. AB - OBJECTIVES: We sought to compare the forearm reflex venous response to mental arithmetic stress in patients with neuromediated syncope and in normal subjects. BACKGROUND: Patients with neuromediated syncope have a paradoxic arterial vasodilation in response to stressors that usually provoke vasoconstriction. Given the postulated role of diminished preload in provoking the reflex responses resulting in syncope, we hypothesized that mental stress might provoke paradoxic reflex venodilation in patients with neuromediated syncope. METHODS: Twelve normal subjects (mean age [+/-SD] 47 +/- 9 years) and 27 patients with neuromediated syncope (mean age 42 +/- 13 years) were studied before and during a mental arithmetic stress test. Forearm venous pressure-volume relations were determined by using radionuclide plethysmography. RESULTS: During mental arithmetic stress, heart rate and systolic and diastolic blood pressure increased significantly and similarly both in normal subjects and in patients with neuromediated syncope. The heart rate and blood pressure changes were qualitatively similar in both groups. However, with mental arithmetic stress, forearm venoconstriction of 13 +/- 2% (mean +/- SEM) was noted in normal subjects (p < 0.001) but not in patients with neuromediated syncope (mean 2%, p = NS). This group response of patients with neuromediated syncope did not result from a lack of individual responses but occurred because these patients had a wide range of responses. The normal physiologic and methodologic variability of the method was +/- 4%. Thirteen of the 27 patients with neuromediated syncope had forearm venoconstriction of 14.5 +/- 6.8% during mental arithmetic stress, whereas 7 had paradoxic forearm venodilation of 14.6 +/- 8.8%, and 7 were considered nonresponders (-1.3 +/- 3.4%). Thus, 14 (52%) of the 27 patients with syncope did not have normal vasoconstriction in response to mental stress. CONCLUSIONS: Patients with neuromediated syncope have an abnormal range of forearm venomotor responses to mental arithmetic stress. Reflex control of the veins may play an important role in the pathogenesis of neuromediated syncope. PMID- 8636563 TI - Tetralogy of Fallot with diminutive pulmonary arteries: preoperative pulmonary valve dilation and transcatheter rehabilitation of pulmonary arteries. AB - OBJECTIVES: This study sought to determine the results of a novel transcatheter management approach in tetralogy of Fallot with diminutive pulmonary arteries. BACKGROUND: Tetralogy of Fallot with diminutive pulmonary arteries and severe pulmonary stenosis is rare and resembles tetralogy of Fallot with pulmonary atresia: There is a high incidence of aortopulmonary collateral channels, arborization abnormalities, stenoses and need for multiple operations. Because a combined catheter-surgery approach facilitates repair in these patients, such an approach may benefit those with diminutive pulmonary arteries and pulmonary stenosis. METHODS: Clinical, catheterization and surgical data were studied retrospectively for 10 such patients undergoing preoperative pulmonary valve balloon dilation, among other transcatheter interventions, from January 1989 to January 1995. RESULTS: Initially, the Nakata index ranged from 20 to 98 mm2/m2 (mean 67 +/- 28 mm2/m2). The pulmonary valve was first balloon dilated (mean balloon/annulus 1.5 +/- 0.3), and the mean initial valve annulus Z score (-4.0 +/ 1) increased to -33 +/- 1.1 (p < 0.01) Other interventions included branch pulmonary artery balloon dilation (7 patients, 23 vessels) and coil embolization of aortopulmonary collateral channels (8 patients, 31 collateral channels). At preoperative follow-up catheterization, the mean pulmonary annulus Z score was 3.1 +/- 0.7, and the Nakata index increased to 143 +/- 84 mm2/m2 (p < 0.03). All patients underwent complete surgical repair successfully. At a mean follow-up period of 2.6 +/- 2 years, right ventricular pressure was < 70% systemic in all patients and < 50% systemic in seven. CONCLUSIONS: In patients with tetralogy of Fallot, severe pulmonary stenosis and diminutive pulmonary arteries, initial pulmonary valve balloon dilation increases the annulus Z score and anterograde pulmonary blood flow and facilities simultaneous coiling of aortopulmonary collateral channels and access for branch pulmonary artery dilation, all of which results in pulmonary artery growth, simplifying surgical management. PMID- 8636564 TI - Emergency echocardiography telemedicine: an efficient method to provide 24-hour consultative echocardiography. AB - OBJECTIVES: This study sought to assess the clinical utility of interpreting emergency echocardiograms after regular working hours through a telemedicine connection to on-call cardiologists. BACKGROUND: Physician interpretation of emergency echocardiograms is often delayed during weekends, evenings or night hours. This delay places undue responsibility on less qualified personnel to interpret echocardiograms of vital importance. METHODS: Digital quad-screen cine loop format was transmitted over standard telephone lines. Clinical data and conventional and telemedicine interpretations were collected prospectively for 187 emergent or semiemergent tele-echocardiograms after regular working hours. RESULTS: Indications for the echocardiogram included assessment of left ventricular function, ischemia, pericardial effusion, valvular disease, heart donor status and arrhythmia. Three off-site echocardiographers received the standard echocardiogram and spectral, gray-scale and color flow Doppler images in cineloop format using a laptop computer. Laptop interpretation showed 19 technically limited studied, 153 abnormal studies and 54% with wall motion abnormalities. Overall mean agreement rate between telemedicine laptop interpretation and conventional workstation interpretation performed in blinded manner for serious disorders with classic echocardiographic findings (pulmonary hypertension, left ventricular thrombus, aortic dissection, severe valvular insufficiency and large pericardial effusion) was 99.0% (95% confidence interval [CI] 96% to 99%). For serious wall motion abnormalities, the agreement rate was 96.3% (95% CI 92% to 99%). The following mean times elapsed after completion of the echocardiogram: to laptop fax report, 2.14 (range 10 min to 8 h); to dictation of videotape, 11.74 h (p < 0.001); to transcription of videotape diction, 56.6 h (p < 0.0001). CONCLUSIONS: After-hours emergency echocardiography telemedicine using a laptop computer is more rapid than scheduled conventional interpretation from a videotape workstation, yet diagnostic accuracy is comparable. PMID- 8636565 TI - A practical guide to assessment of ventricular diastolic function using Doppler echocardiography. AB - Doppler assessment of diastolic function has become a standard part of routine echocardiographic examination and imparts information relevant to a patient's functional class, management and prognosis. This review describes the Doppler patterns of diastolic function relative to physical signs and physiology. A continuum of doppler patterns of diastolic function exists, including normal diastolic function, impaired relaxation, pseudonormal filling, restriction, constriction and tamponade. These patterns evolve from one to another in a single individual, with changes in disease evolution, treatment and loading conditions. New applications of continuous wave Doppler, color Doppler M-mode and Doppler tissue imaging are refining our understanding of diastolic function. PMID- 8636566 TI - Validation of three-dimensional echocardiography for quantifying the extent of dyssynergy in canine acute myocardial infarction: comparison with two-dimensional echocardiography. AB - OBJECTIVES: This study was designed to compare the accuracy of three- and two dimensional echocardiography for quantifying the extent of abnormal wall motion in experimental acute myocardial infarction, as correlated with the pathologic determination of infarct size. BACKGROUND: Two-dimensional echocardiographic estimations of the fraction of myocardium showing abnormal wall motion are often used as an index of infarct size even though they rely on image plane positioning and geometric assumptions that may not be valid. Three-dimensional echocardiographic reconstruction of the endocardial surface eliminates the need for these assumptions and may improve echocardiographic estimates of infarct size. METHODS: Coronary ligation was performed in 14 open chest dogs, and echocardiographic imaging of the ventricle was performed 6 h later. Three dimensional echocardiography used seven or eight spatially registered short-axis images to measure percent of endocardial surface and mass showing abnormal wall motion. Three two-dimensional echocardiographic methods using multiple, nonpatially registered images were evaluated. One method used seven or eight-axis slices and a summation of discs algorithm for computing surface area. The second method used the same images and a conical model for the left ventricle. The third used basal, middle and apical short-axis plus apical four- and two-chamber views comparing summed endocardial lengths showing abnormal wall motion with the total of the endocardial dimensions, expressed as percent. The percent of left ventricular mass and surface area infarcted was determined by staining with triphenyltetrazolium chloride. RESULTS: Three-dimensional echocardiographic measurements of endocardial surface and correlated more closely with infarct mass (r = 0.94, SEE +/- 3.6%) than did the two-dimensional method using the summation of discs algorithm (r = 0.85, SEE +/- 6.6%), he summation of conical sections algorithm (r = 0.82, SEE +/- 5.4%) or the method using summed endocardial lengths (r = 0.79, SEE +/- 7.4%). Limits of agreement analysis comparing mass showing abnormal wall motion with anatomic infarct mass surface area showing abnormal wall motion with anatomic infarct surface area showed the smallest limits for three-dimensional echocardiography. CONCLUSIONS: Three-dimensional echocardiography is a more accurate means of noninvasively estimating myocardial infarct size in this canine model than two-dimensional echocardiography. PMID- 8636567 TI - Hypomagnesemia: characterization of a model of sudden cardiac death. AB - OBJECTIVES: We sought to compare the incidence of sudden death in rats treated with magnesium-deficient and control diets and to address the electrophysiologic characteristics associated with these end points. BACKGROUND: Although magnesium deficiency is associated with an increased incidence of sudden cardiac death in patients, there has been no clear cause and effect relation because of a number of covariables, including diuretic use, hypokalemia, digitalis use and left ventricular dysfunction. METHODS: Hypomagnesemic rats and their paired control rats underwent in vivo electrophysiologic studies and measurements of the total calcium and magnesium content of their cardiac ventricles RESULTS: Serum magnesium levels were 0.5 +/- 0.3 mEq/liter (mean +/- SD) in hypomagnesemic animals and 1.2 +/- 0.9 mEq/liter in control animals. A modest but significant prolongation of the repolarization time was seen at the apical epicardial site (83 +/- 8 ms in hypomagnesemic rats vs. 68 +/- 13 ms in control rats, p < 0.05), but not at the other sites studied. Bradyarrhythmias and tachyarrhythmias were observed in 82% of the hypomagnesemic rats during the in vivo electrophysiologic studies, compared with 0% in the control group. During these studies, sudden, unexpected asystolic deaths were observed in 4 of 11 hypomagnesemic rats and 0 of 8 control rats. Polymorphic nonsustained ventricular tachycardia was provoked by rapid pacing in 5 to 11 hypomagnesemic rats and 0 of 8 control rats. Three of six hypomagnesemic rats exposed to auditory stimuli developed seizures, followed immediately by sudden deaths-two due to asystole and one due to ventricular fibrillation-although no end points occurred in the control animals. CONCLUSIONS: In this model, magnesium deficiency results in sudden cardiac death. The presence of startle induction of sudden death preceded by seizures suggests that sudden cardiac death results from a neurologic trigger. PMID- 8636568 TI - Preconditioning does not prevent postischemic dysfunction in aging heart. AB - OBJECTIVES: This study was performed to investigate the effect of single or multiple brief periods of ischemia and the administration of exogenous norepinephrine before a more prolonged ischemic period and after reperfusion in adult and senescent isolated and perfused rat hearts. BACKGROUND: The mortality rate for coronary artery disease is greater in the elderly. Ischemic preconditioning has been proposed as an endogenous form of protection against ischemia-reperfusion injury. However, the role of preconditioning in aging heart is unknown. METHODS: We compared the protective effect of preconditioning transient ischemic and norepinephrine stimuli against 20 min of global normothermic ischemia and 40 min of reperfusion in isolated perfused hearts of adult (6 months old) and senescent (24 months old) rats. Norepinephrine release in coronary effluent was determined by high performance liquid chromatography. RESULTS: Final recovery of percent developed pressure was improved after single preconditioning transient ischemic and norepinephrine stimuli in adult hearts (87.7 +/- 9% and 82.3 +/- 8.7%) versus unconditioned control hearts (50.6 +/- 4.8%, p < 0.01 [mean +/-SD]). The effect of preconditioning on developed pressure recovery was not present in senescent hearts after transient ischemic stimulus (39.8 +/- 4.9% vs. 41.6 +/- 5.8%, p = NS) but was present after norepinephrine stimulus (74.3 +/- 10.5, p < 0.01). Norepinephrine release significantly increased after preconditioning transient ischemic stimulus in adult but not in senescent hearts (p < 0.01 vs. adult). Transient ischemic- and norepinephrine induced preconditioning was blocked by alpha-adrenergic receptor antagonists in both adult and senescent hearts. Multiple transient ischemic stimuli were able to reduce postischemic dysfunction in adult but not in senescent hearts. CONCLUSIONS: Preconditioning transient ischemic stimulus significantly reduces postischemic dysfunction in adult but not in senescent hearts, whereas exogenous norepinephrine is able to mimic preconditioning in both adult and senescent hearts. Ischemic preconditioning induces an increase in norepinephrine release in adult but not in senescent hearts. Preconditioning induced by transient ischemic stimulus and norepinephrine was abolished by alpha-adrenergic receptor blockade in both adult and senescent hearts. Thus, our data demonstrate that preconditioning is absent in aging heart and is probably related to the reduction of norepinephrine release and alpha-adrenergic receptor stimulation in response to ischemic preconditioning. PMID- 8636569 TI - Effect of prolonged inotropic stimulation on ventricular remodeling during healing after myocardial infarction in the dog: mechanistic insights. AB - OBJECTIVES: We hypothesized that positive inotropic stimulation during healing after myocardial infarction might increase contractile pull on the infarct segment, increase expansion and promote ventricular dilation. BACKGROUND: The effect of prolonged inotropic stimulation on left ventricular remodeling during healing after myocardial infarction has not been studied. METHODS: The effects of 6 weeks of inotropic stimulation on in vivo changes in left ventricular topography, function and mass (serial two-dimensional echocardiograms), hemodynamic variables, postmortem topography (planimetry) and collagen (hydroxyproline content) were studied in 36 chronically instrumented dogs randomized, 2 days after small anterior infarction, to digoxin (0.125 mg daily) and no digoxin (control group). RESULTS: Heart rate and arterial and left atrial pressures were similar in the two groups, but the first derivative of left ventricular pressure (peak dP/dt), systolic thickening of the noninfarct wall and systolic thinning of the infarct wall were higher in the digoxin group during the 6 weeks. At 6 weeks, infarct scar size and collagen content were similar in both groups, but the digoxin group had more infarct expansion and thinning. Between 2 days and 6 weeks, the digoxin group showed more in vivo diastolic infarct expansion, thinning and bulging; more aneurysm but less global dilation and increase in mass; and no change in ejection fraction. The effects of inotropic stimulation on remodeling were more marked in infarcts with 100% than 85% transmurality. CONCLUSIONS: Prolonged inotropic stimulation with digoxin during healing after small anterior infarction increases infarct bulging without decreasing infarct collagen content and preserves global ventricular size, mass and systolic function. PMID- 8636570 TI - Neuropeptide Y modulation of sympathetic activity in myocardial infarction. AB - OBJECTIVES: We examined the possible effect of neuropeptide Y in modulating central sympathetic activity after myocardial infarction in rats. BACKGROUND: Previous studies have shown the coexistence of neuropeptide Y and norepinephrine in the brain and a possible functional interaction between the two. Neuropeptide Y inhibits the release of norepinephrine at the presynaptic level and can be considered to act as a neuromodulator. METHODS: Two groups of rats were examined in this study-an experimental group, defined as those rats undergoing left coronary artery ligation, and a sham group without coronary artery ligation, serving as the control group. The animal in both groups underwent microdialysis in the paraventricular nucleus at 2, 4 and 8 weeks after operation. Microdialysis samples were collected with and without injecting neuropeptide Y in the paraventricular nucleus. The concentration of norepinephrine was determined by injecting purified microdialysate samples during high performance liquid chromatography. To explore the receptor's possible role, autoradiographic localization of neuropeptide Y receptors in the paraventricular nucleus was also carried out in the experimental and sham groups. RESULTS: The concentration of norepinephrine measured in the samples was decreased by 50% with neuropeptide Y in 2- and 4-week old rats after infarction, but by only 20% (p < 0.05) in 8-week old rats after infraction. The diminished inhibitory effects of neuropeptide Y on norepinephrine release was associated with increased sympathetic activity, as reflected by plasma norepinephrine; 8-week old rats after infarction had almost a 100% (p < 0.05) increase in their plasma norepinephrine level compared with the sham group. Autoradiography revealed a significant decrease in density of neuropeptide Y receptors in the paraventricular nucleus in 8-week old rats after infarction (p < 0.05). CONCLUSIONS: The data presented in this report suggest that the reduction of the inhibitory activation of neuropeptide Y on sympathetic release may contribute to elevated norepinephrine levels after myocardial infarction. PMID- 8636572 TI - President's page: the new CME. PMID- 8636571 TI - Role of nitric oxide in regulation of coronary blood flow during myocardial ischemia in dogs. AB - OBJECTIVES: This study was undertaken to examine whether nitric oxide released in ischemic myocardium decreases the coronary vascular resistance and attenuates the severity of contractile and metabolic dysfunction. BACKGROUND: Endothelium derived relaxing factor, recently identified as nitric oxide, is a potent relaxant of coronary smooth muscle. METHODS: The left anterior descending coronary artery was perfused through an extracorporeal bypass tube placed in the carotid artery in 56 open chest dogs. After hemodynamic stabilization, we occluded this bypass tube to decrease coronary blood flow to one third of the control flow. Thereafter, we maintained a constant coronary perfusion pressure (40.9 +/- 3.1 mm Hg). RESULTS: Under ischemic conditions, the coronary arteriovenous differences in nitrate and nitrite (end products of nitric oxide) increased (from 3.5 +/- 0.4 [mean +/- SEM] to 12.9 +/- 2.1 mumol/liter, p < 0.01). NG-Monomethyl L-arginine (3 micrograms/kg body weight per min, intracoronary) decreased the coronary arteriovenous differences in nitrate and nitrite (5.0 +/- 0.9 mumol/liter, p < 0.05) and coronary blood flow (from 29.8 +/ 0.5 to 18.1 +/- 1.1 ml/100 g per min, p < 0.001). Fractional shortening (from 3.7 +/- 1.0 to -1.3 +/- 0.7%, p < 0.001) and lactate extraction ratio (from -44.0 +/- 4.1 to -59.2 +/- 4.9%, p < 0.005) of the perfused area also decreased. These values were restored by the concomitant administration of L-arginine. Blood flow to the endomyocardium was decreased relative to the epimyocardium. A reduction in coronary blood flow and worsening of myocardial contractile and metabolic functions due to the administration of NG-monomethyl L-arginine during ischemia were observed in denervated hearts. A reduction in coronary blood flow in ischemic myocardium was observed with the administration of NW-nitro-L-arginine methyl ester as well, although neither NW-nitro-L-arginine methyl ester nor NG monomethyl L-arginine changed coronary blood flow and myocardial contractile and metabolic functions in the nonischemic myocardium. The cyclic guanosine monophosphate content of epicardial coronary artery increased due to myocardial ischemia; this increase was attenuated with NG-monomethyl L-arginine treatment. CONCLUSIONS: We conclude that endogenous nitric oxide predominantly decreases the coronary vascular resistance of ischemic endomyocardium, thereby improving myocardial contractility and metabolic function. PMID- 8636573 TI - Identification of patent foramen ovale permitting paradoxic embolism. PMID- 8636574 TI - Transesophageal echocardiography: additional diagnostic and therapeutic role in critically Ill patients? PMID- 8636575 TI - Q wave and non-Q wave myocardial infarction after thrombolysis. PMID- 8636576 TI - Immunizations in long-term care facilities: policies and practice. AB - PURPOSE: The present study was conducted to assess the policies and practices of nursing homes in regard to immunizations for residents and employees. METHODS: A 20-item, cross-sectional survey was mailed to all 445 Minnesota nursing homes from May through July 1993. Questions asked were about facility characteristics and their policies, practices, and attitudes regarding immunizations for residents and employees. Second and third mailings were sent to nonresponding institutions. RESULTS: The median size of 399 (90%) responding institutions was 88 beds and 110 employees. The median number of residents on December 1, 1992, was 84. Although 99% of the facilities had an influenza vaccination program, only 69.3% had written policies. The mean influenza vaccination rate for residents was 84%, with higher rates in institutions having written policies, facility-wide standing orders for vaccine administration, and in institutions that did not require written consent for vaccination. One-third of the facilities had written policies for pneumococcal vaccination, and 16.3% had policies for tetanus/diphtheria. Twelve-month immunization rates for these vaccines were 11.9% and 2.9%, respectively. Most facilities had an influenza vaccination program for employees with a mean vaccination rate of 33%. These rates were higher if the facility offered vaccine onsite, free of charge, and provided special inservice education to employees. Employee 12-month vaccination rates for hepatitis B and tetanus/diphtheria were 23.2% and 1.7%, respectively. Few of the institutions included evaluation of immunization activities in their quality assurance programs. CONCLUSION: Many long-term care facilities have inadequate policies and practices for ensuring their residents and employees immunity to important vaccine preventable diseases. These institutions, as caretakers for a vulnerable population, should develop and implement organized programs to immunize their residents and employees. By doing so, they will be able to take advantage of important opportunities to prevent illness and protect the health of their residents and employees. PMID- 8636577 TI - Chair design affects how older adults rise from a chair. AB - OBJECTIVE: To determine how modifications of key chair design aspects, such as seat height, posterior seat tilt, backrest recline, seat compressibility, and armrest placement, affect how older adults rise from a chair and the seating comfort they experience. DESIGN: Cross-sectional comparison. SETTING: Congregate housing facility and university laboratory. SUBJECTS: Two groups of volunteers, Old (n = 29, mean age 84) and Young (n = 21, mean age 23). MEASUREMENTS: Analysis of time to rise, body motion (determined by use of digitized videotaping), and self-reported difficulty when subjects rose from a variety of controlled chair settings thought to represent important chair design specifications encountered by older adults. Subjects also reported their comfort while being seated in these settings. RESULTS: Lowered seat height, increased posterior seat tilt and backrest recline, and perhaps increased seat compressiblity cause increased time to rise, increased body motion, and increased self-reported ratings of rise difficulty in both Young and Old groups. Under the most challenging conditions, the effect appears to be stronger in the Old than in the Young: a few Old were unable to rise, and the Old took disproportionately longer to rise and used disproportionately greater neck motion (P generally < 0.001) compared with the Young. Arm rest placement did not alter rise performance or ratings significantly. The conditions in which rise difficulty increases or decreases do not correspond exactly to conditions in which comfort increases or decreases. Some aspects that increase rise difficulty, such as tilt/recline and seat compressiblity, may also increase comfort. CONCLUSIONS: Aspects of chair design such as lowered seat height, increased posterior seat tilt, increased back recline, and increased compressibility interfere with chair egress in older adults. While decreasing ease of egress, however, these same factors may increase seating comfort. Furniture designers and manufacturers must find a balance between degree of sitting comfort, ease of egress and the degree to which the seating device facilitates functional independence, particularly to meet the needs of disable older adults. PMID- 8636578 TI - Characteristics of rural homebound older adults: a community-based study. AB - OBJECTIVE: To determine the frequency and characteristics of homebound older adults in a rural community. DESIGN: An epidemiological survey of an age stratified random community sample. SETTING: The rural mid-Monongahela Valley in Southwestern Pennsylvania. PARTICIPANTS: A total of 878 noninstitutionalized persons aged 68 years and older, fluent in English, and with at least grade 6 education. MEASUREMENTS: The frequency with which subjects left their homes, the Mini-Mental State Examination (MMSE) score, and additional information on demographics, self-reported health problems, health services utilization, IADLs, depression, and social support were measured. RESULTS: 10.3% of the sample was classified as homebound. In univariate analyses, being homebound was found to be associated significantly (P < .001) with being older, female, and widowed and with MMSE and IADL impairment, with more depressive symptoms and worse social supports, fair to poor self-rated general health, weight loss, and histories of stroke, angina, arthritis of the spine, and falls. In a multiple regression model, variables associated independently with homebound status were gender (odds ratio = 9.4, 95% confidence interval = 3.6 - 24.9), weight loss (OR = 3.7, CI = 1.7 - 8.2), IADL impairment (OR = 2.6, CI = 2.1 - 3.1), and depressive symptoms (OR = 2.1, CI = 1.3 - 3.2). Being homebound was also associated with recent acute hospitalization and use of home health and social services. CONCLUSIONS: These data provide evidence that homebound older adults have a disproportionate share of morbidity and disability and suggest a sociodemographic and clinical profile to help identify those older people at risk of being or becoming homebound. They also point to the need for home-based health services for the older adults, particularly in medically underserved communities such as rural areas. PMID- 8636580 TI - Factors affecting sample selection in a randomized trial of balance enhancement: the FICSIT Study. AB - OBJECTIVE: To determine demographic, functional, and health-related factors that may have influenced the selection of older adults for a randomized trial of balance enhancement. DESIGN: Comparison of participants with nonparticipants at various stages of the recruiting process. SETTING: Northeastern suburban community. PARTICIPANTS: Registered voters aged 75 and older (n = 7191). MEASUREMENTS: Demographic, health-related, functional, balance, gait, and falling characteristics. RESULTS: The overall participation rate in the randomized trial was 1.5%. Compared with nonparticipants, participants were significantly more likely to be male, married, living with others, living in a house, highly educated, healthy, and physically active. CONCLUSION: Recruiting older subjects by mail to studies of rigorous interventions can produce significant selection biases that may limit the population to which results can be generalized. PMID- 8636579 TI - Markers of failure to thrive among older hip fracture patients. AB - OBJECTIVES: To determine whether there is a group of recent hip fracture patients who exhibit the signs of failure to thrive and to identify potential precursors to their decline in physical functioning. DESIGN: Prospective (nonintervention) study of hip fracture recovery; patients were assessed in the hospital and at 2, 6, 12, 18, and 24 months post-fracture. SETTING: Hip fracture patients admitted to one of eight Baltimore area hospitals from the community with a new fracture of the proximal femur between January 1, 1990, and June 15, 1991. PARTICIPANTS: Patients were 65 years of age and older and lived in the community before the fracture. A total of 804 patients were eligible for the study; the present study analyses were restricted to the 252 patients who survived 1 year and had a self report assessment at 6 and 12 months post-fracture. MEASUREMENTS: A questionnaire administered during hospitalization assessed pre-fracture functional and health status and current affective and cognitive status. In-home interviews post fracture ascertained dependence and difficulty with physical and instrumental activities of daily living. Abstraction of the medical records provided information about comorbidities, surgical procedure, and hospital length of stay. RESULTS: Patients who declined in ability to walk from 6 to 12 months post fracture had greater use of health resources (more hospitalizations) and poorer physical functioning up to 2 years post-fracture. Impaired function in physical activities of daily living at 6 months, high glucose, calcium, and CO2 at admission, and low BUN and creatinine at admission were more prevalent among decliners than among non-decliners. CONCLUSIONS: Findings indicate that certain older hip fracture patients begin to exhibit signs and symptoms of failure to thrive. About 10% of patients who survived at least 1 year after fracture could not retain their recovery level of functioning after 6 months and began to decline further. High glucose and CO2 and low BUN and creatinine on hospital admission were associated with later functional decline among hip fracture patients, but their clinical significance is uncertain. PMID- 8636581 TI - Intraindividual reproducibility of postprandial and orthostatic blood pressure changes in older nursing-home patients: relationship with chronic use of cardiovascular medications. AB - OBJECTIVES: Although postprandial and orthostatic hypotension are commonly observed in nursing home residents, their reproducibility, relationship to each other, and association with chronic use of cardiovascular medications are poorly understood. DESIGN: We examined blood pressure (BP) and heart rate (HR) before and after postural change, and before and after a 419-kcal meal in 22 nursing home residents (mean age 89 +/- 5 (SD) years), each on two occasions, to determine reproducibility changes. These studies were repeated in 17 residents, with and without previous administration of cardiovascular medications, in random order. SETTING: Hebrew Rehabilitation Center for the Aged, an academic long-term care facility. RESULTS: Systolic BP declined an average (+/- SE) of 16 +/- 4 mm Hg and 12 +/- 4 mm Hg during the first and second meal studies, respectively. Mean intra-class correlation of postprandial systolic BP values during the two studies was 0.88 (95% CI 0.85-0.97). Systolic BP increased significantly during the first posture test to a maximum of 8 +/- 6 mm Hg at 6 minutes. There was no significant difference over time in postural systolic BP between the two tests. Repeated postural studies showed a mean intra-class correlation of 0.72 (95% CI 0.62-0.92) for changes in systolic BP. Cardiovascular medications had no additional effect on postprandial or orthostatic BP and HR changes. During the first studies, 10 subjects had postprandial hypotension, and three subjects had orthostatic hypotension, but only two of 22 subjects had both. CONCLUSIONS: Patterns of systolic BP response to meals or postural change are reproducible. BP responses to meals and postural change seem to be unaffected by potentially hypotensive medications in chronic users. Postprandial hypotension is distinct from orthostatic hypotension, occurring more commonly than orthostatic hypotension and infrequently together in the same patients. PMID- 8636582 TI - Breast cancer in women 70 years of age or older. AB - OBJECTIVES: To investigate the impact of age as a prognostic factor in older patients with breast cancer and to discuss the role of surgery in this category of patients. DESIGN: A retrospective study. SETTING: A tertiary care university teaching hospital. PARTICIPANTS: One hundred ninety patients aged 70 years or older (mean age: 75 years) were treated for breast cancer from 1967 through 1991. These patients were compared with 190 younger patients (mean age: 52 years) and matched on the basis of T and N categories (TNM staging system) and surgical procedures. MEASUREMENTS: Disease-free survival, breast cancer-specific survival. RESULTS: The 10-year actuarial breast cancer-specific survival was 66% for older patients and 56% for younger patients (P = .224). The 10-year actuarial disease free survival was 54% for older patients and 45% for younger patients (P = .136). Univariate and multivariate survival analysis revealed that tumor size and nodal stage were significant prognostic factors for both older and younger patients. CONCLUSION: Treatment with curative intent, similar to that adopted in younger patients, is appropriate for women over the age of 70 with breast cancer. PMID- 8636583 TI - The epidemiology, clinical characteristics, and natural history of older nursing home residents with a diagnosis of Parkinson's disease. AB - OBJECTIVE: To determine the epidemiology, clinical characteristics and natural history of nursing home residents with a diagnosis of Parkinson's disease (PD). DESIGN: A cohort study with 18-month follow-up utilizing resident assessments from the National HealthCorp 1991-1992 dataset. SETTING: Seventy-one National HealthCorp nursing homes. PARTICIPANTS: A total of 5020 nursing home residents older than age 55 were studied. Residents with primary and secondary diagnoses of PD were identified from the population using the International Classification of Diseases, Ninth Revision, Clinical Modification code 332.0. MEASUREMENTS: Baseline demographic and clinical characteristics were compared for residents with and those without Parkinson's disease. Outcome measures over the course of 18 months included death and functional status. RESULTS: The prevalence of a diagnosis of PD was 6.8%. Significant factors associated independently with PD included: younger age (79 +/- 7 vs 81 +/- 9 years; P < .001), male sex (32% vs 23%; P < .001), severe dependence in activities of daily living (OR = 1.26; 95% CI 1.08-1.46), impared body control (OR = 1.38; 95% CI 1.03-1.68), symptoms of depression (OR = 1.29; 95% CI 1.02-1.64), and the number of daily medications (OR = 1.23; 95% CI 1.08-1.44). Residents with a diagnosis of PD had a faster rate of functional decline over 18 months (P < .001) but did not have a higher mortality rate than residents without PD. CONCLUSIONS: Parkinson's disease is a relatively common diagnosis among nursing home residents and is associated with increased functional disability. There are several potentially modifiable conditions associated with PD that may offer an opportunity to design specific interventions and health services to improve the quality of life and slow functional decline in this frail population. PMID- 8636584 TI - Attitudes of older adults' on being told the diagnosis of Alzheimer's disease. AB - OBJECTIVE: Controversy exists as to whether Alzheimer's disease (AD) patients should be told their diagnosis, yet no research has been done examining older patients' attitudes on this topic. This study examines patient's attitudes toward this topic. DESIGN: A prospective, community-based study. Participants read vignettes of two patients, one with AD and one with terminal cancer, and then answered questions regarding their attitudes toward these illnesses. SETTING: A community-based retirement community in Charlottesville, Virginia. PARTICIPANTS: One hundred fifty-six community-dwelling older persons (mean age 79.7 +/- 6.9 years). MEASUREMENTS: A structured questionnaire disclosed demographic data (age, sex, race, religion, marital status), personal experience with cancer and AD, and opinions about being told the diagnosis of these diseases. RESULTS: Most participants (n = 124, 79.5%) responded that they would prefer to know if they had AD, but the number was significantly fewer (Fischer exact test, P < .001) than those who would want to know if they had terminal cancer (n = 143, 91.7%) Interestingly, significantly fewer married subjects would want their spouse to know if the spouse had either illness. Only 65.7% (n = 69) of subjects would want their spouse to know if the spouse had AD (Fisher exact test, P = .008), whereas for cancer, 80.2% (n = 77) would want their spouse to know if the spouse had cancer (Fisher exact test P < .001). No demographic variables distinguished subjects who did from those who did not want to know the diagnosis for themselves or their spouses for either AD or cancer. Among the reasons some subjects gave for wanting to know of the diagnosis of AD was being able to consider suicide. CONCLUSION: Although these results may support disclosure of diagnosis for most patients with AD, clinical and ethical issues remain in individual cases. PMID- 8636585 TI - Apparent idiopathic hyponatremia in an ambulatory geriatric population. AB - OBJECTIVES: The purpose of this descriptive analysis is to demonstrate that among older patients with hyponatremia, there is a subset with apparent hyponatremia of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) type, which appears associated with the aging process. A retrospective chart review was done to identify patients with hyponatremia and to classify them into non-SIADH, SIADH, and idiopathic categories. DESIGN: The participants were the ambulatory population of The Mount Sinai Hospital's outpatient geriatric clinic. Pertinent data generated during the antecedent 24 months were collected from outpatient charts and included sets of laboratory values (consisting of one sodium, glucose, BUN and creatinine level), age, sex, race, clinical history, present diagnosis, medications, and prescribed diets for each patient in the total population. RESULTS: Patients demonstrating hyponatremia with serum sodium levels of less than 135 mEq/L were identified from the total population and were further divided into SIADH and non-SIADH etiologies. The SIADH patients were then subdivided into known etiology SIADH and apparent idiopathic SIADH. In an ambulatory geriatric population of 405 subjects, 46 had hyponatremia, with SIADH the apparent cause in 27. Of the SIADH population, seven patients were considered to have idiopathic SIADH. An apparent predilection for development of this syndrome was observed among non-black patients and the old old. CONCLUSIONS: The results support the hypothesis that aging is a risk factor for the development of SIADH-like hyponatremia in a subset of older patients who do not have an apparent underlying etiology. Thus, aging may be an independent etiology for the development of hyponatremia. PMID- 8636586 TI - Dementia and guns. PMID- 8636587 TI - The effects of antihypertensive treatment on cognitive function: results from the HOPE study. AB - OBJECTIVES: Hypertension is associated with impaired cognition, but it is unclear whether this impairment is reversible. We sought to evaluate the effect of blood pressure reduction on cognition. DESIGN: A randomized, double-blind trial. SETTING: A single center, with assessments in subjects' domiciles. PARTICIPANTS: Community-screened subjects more than 69 years of age who had median diastolic pressures > 99 mm Hg and systolic pressures > 159 mm Hg or diastolic > 85 mm Hg and systolic > 179 mm Hg with Mini-Mental State Examination scores of 20 to 28. Subjects had not previously received antihypertensive treatment. INTERVENTION: Captopril 12.5 mg twice daily or bendrofluazide 2.5 mg daily for 24 weeks, preceded by a 2-week placebo phase. MEASUREMENTS: Cognition was evaluated by a psychometric test battery comprising Immediate and Delayed Logical Memory, Paired Associates recall, Raven's Progressive Matrices, Halstead Reitan Trail Making A, and the Anomalous Sentences Repetition Test. RESULTS: Eighty-one subjects (28 male, 53 female) were treated (41 captopril, 40 bendrofluazide). At entry, mean age was 76.1 years (range 70-84), mean blood pressure was 191 (160-230) mm Hg systolic, 101 (88-110) mm Hg diastolic, and mean MMSE score 26.1. A total of 69 subjects completed the trial. The 25th, 50th, and 75th percentiles of the difference between pretreatment and Week 24 blood pressures wer 15 mm Hg, 35 mm Hg, and 50 mm Hg (systolic) and 5 mm Hg, 10 mm Hg, and 19 mm Hg (diastolic). There were no significant differences in any psychometric test between captopril and bendrofluazide. The 19 subjects in the quartile that lowered their diastolic blood pressure most ( > or = 19 mm Hg) had improved scores on Anomalous Sentences (P = .012) and Paired Associates (P = .044) compared to the 19 subjects in the least blood pressure responsive quartile (fall < or = 5 mm Hg)s. CONCLUSION: The treatment of hypertension is not hazardous to cognitive function in older people with pre-existing cognitive impairment. Long-term adequate blood pressure control may reverse cognitive impairment associated with pre-existing hypertension. PMID- 8636588 TI - An intervention on discharge polypharmacy. AB - OBJECTIVE: To determine if providing a way to cancel pre-admission prescriptions would reduce the number of active drug prescriptions (RXs) at discharge. DESIGN: A randomized non-blinded clinical trial. SETTING: Inpatient acute medical service of a university affiliated Veterans Administration medical center. PARTICIPANTS: Twelve medicine ward teams were randomized to control and intervention groups. Patients controlled had been discharged from these teams during 12 weeks and were receiving outpatient medications from this facility at hospital admission; control = 180, intervention = 168. INTERVENTION: At discharge, intervention teams used a computer-generated drug list to cancel or renew previous outpatient RXs or to prescribe new medications. Control teams could not cancel outpatient drugs and wrote all medications on individual prescriptions. MEASUREMENTS: The difference between admission and discharge RXs. RESULTS: There were no significant differences in patients' age, sex, race, Charlson Index (CI), or LOS between patient groups at discharge. The intervention group had fewer RXs on admission (5.4 vs 6.2, P < .05) and at discharge was not significantly different (2.9 vs 2.9, P = .87) from the control group. CONCLUSIONS: Providing a method for canceling pre-admission medications did not reduce the number of RXs at discharge. Further research is needed to evaluate the appropriateness of the large increase in RXs from admission to discharge for patients in acute hospital settings. PMID- 8636589 TI - Pyuria among chronically incontinent but otherwise asymptomatic nursing home residents. AB - OBJECTIVE: To determine the prevalence of pyuria and its relationship to bacteriuria in a representative sample of chronically incontinent nursing home residents. DESIGN: Prospective, descriptive case series. SETTING: Six nursing homes. PARTICIPANTS: Two hundred fourteen chronically incontinent, but otherwise asymptomatic, nursing home residents who were enrolled in a clinical intervention trial for urinary incontinence. MEASURES: Two hundred fourteen urine specimens were collected by a validated clean catch technique. Each specimen underwent dipstick testing for leukocyte esterase, microscopic urinalysis to determine the number of white blood cells per high power field of centrifuged urine, and quantitative urine culture using standard laboratory techniques. RESULTS: The overall prevalence of pyuria was 45%, as defined by > 10 white blood cells per high field of spun urine, and the overall prevalence of bacteriuria was 43%, as defined by the growth of > 100,000 colony forming units of a urinary pathogen. Fifty-nine percent of the specimens with bacteriuria and 34% of the specimens without bacteriuria had pyuria. Of specimens with pyuria, 56% had bacteriuria, and of specimens without pyuria, 31% had bacteriuria. When any colon change on the leukocyte esterase pad was considered positive, this finding had a sensitivity of 83% and a specificity of 52% for pyuria on microscopic urinalysis. CONCLUSIONS: Pyuria is common among chronically incontinent nursing home residents, both in the presence and absence of bacteriuria. Clinicians must therefore be cautious in interpreting the presence of pyuria to indicate symptomatic infection in this population. Using pyuria to determine the need for antimicrobial treatment could result in unnecessary expense and morbidity as well as contribute to the development of resistant organisms in nursing homes. Research is needed to define pyuria better, to determine its prevalence and relationship to bacteriuria among nursing home residents with symptoms of acute urinary tract infection, and to elucidate the etiology and significance of sterile pyuria in this population. PMID- 8636590 TI - Effects of prompted voiding on fecal continence among nursing home residents. AB - OBJECTIVE: To determine the effects of prompted voiding on fecal continence in nursing home residents. DESIGN: Prospective, uncontrolled trial of prompted voiding for urinary incontinence. PARTICIPANTS: One hundred sixty-five nursing home residents who completed a 9 to 10-week trial. MEASUREMENTS: Trained research aides performed physical checks for urinary and fecal incontinence hourly from 8 AM to 6 PM for 3 days (total of 33 checks) at baseline, for the last 3 days of a 1-week trial of prompted voiding, and after 9 to 10 weeks of prompted voiding. RESULTS: After 9 to 10 weeks of prompted voiding, there was no significant change in the frequency of incontinent bowel movements per resident (1.1 [95% CI.83, 1.4] to .87 [95% CI.67, 1.1]; P = 0.140). There was a significant increase in the number of continent bowel movements per resident (.17 [95% CI.10, .24] to .62 [95% CI.45, .80]; P = .000). This increase occurred in residents whose urinary incontinence responded well to prompted voiding as well as those whose urinary incontinence did no respond. The percentage of bowel movements that were continent also increased significantly from 18% (95% CI 8,29) at baseline to 45% (95% CI 32,57) after 9 to 10 weeks of intervention (P = .000). In addition to these findings, we noted a marked increase in the total frequency of bowel movements after the first week of prompted voiding. This may have resulted from the relief of fecal impactions caused by the increased toileting, mobility, and fluid intake that occurred with prompted voiding. CONCLUSION: Prompted voiding did not change the frequency of incontinent bowel movements significantly in this sample of nursing home residents. However, the number of continent bowel movements and the percentage of bowel movements that were continent did increase. Our data must be interpreted cautiously because our study was designed primarily as an intervention for urinary, not fecal, incontinence and the design was neither blinded nor controlled. Trials of systematic toileting schedules specifically directed at fecal incontinence, with attention to fecal impaction, diet, fluid intake and laxative use, should be conducted. PMID- 8636591 TI - Shoe characteristics and balance in older women. AB - OBJECTIVE: To determine whether shoe characteristics affect balance in older women. DESIGN: Randomized order, cross-over, controlled comparison. SETTING: A retirement village. PARTICIPANTS: Thirty women aged 60 to 89 years (mean 78.7 SD = 8.5). Twenty-five subjects were hostel residents, and five were living independently in the community. OUTCOME MEASURES: Postural sway, maximal balance range, and co-ordinated stability. MAIN RESULTS: The subjects underwent assessments of static balance (body sway) and dynamic balance (maximal balance range and co-ordinated stability) under four conditions: (1) barefoot, (2) in standard low-heeled shoes (walking shoes), (3) in standard high-heeled shoes, and (4) in their own shoes. Manova analysis revealed a significant overall shoe condition effect -- subjects performed best in bare feet or low-heeled shoes and worst in high-heeled shoes. There were no significant differences between subjects with and without foot abnormalities in any of the balance measures or test conditions. CONCLUSION: These findings suggest that bare feet and walking shoes maximize balance, whereas high-heeled shoes constitute a needless balance hazard for older women. PMID- 8636592 TI - Gait disorders in older adults. PMID- 8636593 TI - Electrocardiogram in the ambulatory clinic in older patients with cardiac disease: an assessment of the contribution to management. AB - OBJECTIVE: To assess the value of the routine electrocardiogram (EKG) in the management of older (> 65 years) patients with cardiac disease during return visits to the ambulatory clinic. DESIGN: Retrospective chart analysis. SUBJECTS: Seventy-one patients older than 65 years of age, with cardiac diagnoses, presenting for follow-up visits. METHODS: Chart notes from 254 encounters with 71 patients in a cardiology clinic were analyzed. Excluded were new patients as well as patients followed in special arrhythmia clinics for pacemakers or significant arrhythmias. All patients underwent an EKG during each clinic visit, irrespective of their clinical status, according to the clinic's protocol. The frequency and nature of therapeutic decisions made in the clinic, the contribution of the EKG to the decision-making process, the appropriateness of the EKG, and the physician response to the EKG were assessed. MAIN RESULTS: Therapeutic decisions, the most common of which (28%) was medication changes, were made in 78 (31%) patients. The routine EKG was considered inappropriate in 60%. Unexpected diagnostic information not obtained from history or examination was not present in any encounter. EKG findings were not addressed by physicians in 22%. CONCLUSIONS: Most older patients seen in the cardiology clinic for return visits with stable symptoms do not benefit from EKGs. Unexpected diagnostic information from the EKG leading to major therapeutic decisions is rare in older people with stable symptoms. PMID- 8636594 TI - Geriatric oral health and its impact on eating. AB - OBJECTIVE: Adequate food and fluid intake and nutritional health are requisites for sustaining life. The oral-pharyngeal region has evolved multiple, highly regulated processes to ensure that the intake, chewing, and swallowing of foods and beverages is maintained. The objective of this paper is to identify the independent and collective roles of oral health on eating in older people. DESIGN: Research reports from peer-reviewed scientific journals. Hypothesis driven research that objectively examined taste, smell, dental and oral mucosal health, dental prostheses, chewing, and swallowing in the context of aging. DATA EXTRACTION AND SYNTHESIS: Data results were extracted independently by multiple observers. A qualitative synthesis of data results from independent studies was made in order to form conclusions regarding the role of oral health on eating in older people. CONCLUSIONS: Many oral functions remain intact in healthy older adults. However, significant alterations arise from oral and systemic diseases and their treatments, and these may have a profound effect on eating, drinking, and the nutritional status of older individuals. The care of older persons with smell, taste, dental/alveolar, oral mucosal, chewing, and swallowing problems requires a multidisciplinary team of health care providers. Recognition of the interrelationship between oral, pharyngeal, and systemic physiological processes will help practitioners identify the etiology of these disorders and implement appropriate therapy. PMID- 8636595 TI - Failure to thrive in older people. PMID- 8636596 TI - Grandfather's gun: when should we intervene? PMID- 8636597 TI - Establishing links between the AGS and the BGS. PMID- 8636598 TI - Hearing impairment. PMID- 8636599 TI - Links with the British Geriatrics Society. PMID- 8636600 TI - The risk of postoperative deconditioning in older adults. PMID- 8636601 TI - Physicians, ethics, advance directives, and long-term care. PMID- 8636602 TI - No increased risk of Taxol toxicity in older patients. PMID- 8636603 TI - The neuroleptic malignant syndrome: often an unrecognized geriatric problem. PMID- 8636604 TI - Prolongation of ECT-induced seizures with theophylline. PMID- 8636605 TI - Case control study of agricultural injuries to older farmers in central Wisconsin. PMID- 8636606 TI - Septic arthritis and bilateral endogenous endophthalmitis associated with percutaneous transluminal coronary angioplasty. PMID- 8636607 TI - Multivariant analysis on the long-term results of hemiarthroplasty after subcapital fracture of the femur. PMID- 8636608 TI - Provision of nonacute, ambulatory geriatric services in a sparsely populated region of Norway. PMID- 8636609 TI - [Treatment of intraductal carcinomas. 91 cases treated at Oscar-Lambret Center]. AB - During a period of 10 years, 91 ductal carcinomas in situ (DCIS) were operated. After a study of clinical and therapeutics characteristics, the evolution of the DCIS was studied. Eleven recurrences were seen in a period of 41 months. The actuarial risk of recurrence was 11% at 5 years and 17% at 8 years. About half of the recurrences were invasive and occurred 10 times out of 11 in the initial area or nearby. Among several factors studied, the association of radiotherapy with conservative surgery was the only one which reduced significantly the risk of recurrences. PMID- 8636610 TI - [Diagnosis of local breast cancer recurrences by magnetic resonance imaging]. AB - The aim of this study is to determine the diagnostic value of magnetic resonance imaging in the diagnosis of local recurrence of breast cancer. From 1991 to 1994, 61 women were studied prospectively using magnetic resonance imaging. All examinations were made on a 1.5 Testa machine with T1 weighted images and after gadolium-dota injection and dynamic images (T1 weighted sequences every 47 seconds during injection of a gadolinium-dota bolus). All the pre-injection, images in the dynamic series were subtracted from the images after injection. A surgical biopsy was obtained in 39 patients yielding a diagnosis of local recurrence (n = 28) or a benign lesion (n = 11). Among the 28 local recurrences, pathology examination reported invasive cancer in 22 and intra-ductal carcinoma in 6. In 22 patients with normal magnetic resonance imaging, follow-up examinations were performed every 6 months. There were no local recurrences within a delay of 6 to 36 months. Twenty-six of the 28 patients with a local recurrence, cystosteatonecrosis and surgery scar tissue less than 6 months old showed contrast uptake 1 min and 34 s after gadolinium injection during the dynamic sequence. This product uptake yielded nodular images within the invasive carcinomas and linear images in the intraductal cancers. In all cases, it is easier to visualize this contrast uptake in subtraction images. In conclusion, magnetic resonance imaging is a simple reliable method for the diagnosis of local recurrence of breast cancer. PMID- 8636611 TI - [Stage I and II endometrial cancer: should lymphadenectomy still be done?]. AB - Surgical treatment for endometrial carcinomas stage I and II is radical hysterectomy. The role of lymphadenectomy (pelvic and paraaortic) is under discussion. From a retrospective study (multivariate analysis of 320 patients treated by radiosurgical association) and a review of the literature, the authors limit the indications of lymphadenectomy to stage I grade 1 or 2 tumours and without deep tumours invasion into the myometrium (in that case only 10% of pelvic nodes will be involved). Stage II patients or stage I with grade 3 and/or deep tumour invasion into the myometrium do not require lymphadenectomy as post operative pelvic external beam irradiation will be performed in all cases. Para aortic lymphadenectomy is not useful as it increases morbidity and the adjuvant treatment in case of lymph node involvement does not improve the survival rate. PMID- 8636612 TI - [Total laparoscopic hysterectomy. Operative technique, results and indications]. AB - Total hysterectomy was performed via laparoscopy alone in 50 patients. In all cases, the operation was carried out using conventional, re-usable instruments (grasping forceps, laparoscopic scissors, bipolar coagulation). The mean duration of the operation was 163 min (range: 110-270 min). The mean drop in haemoglobin was 1.97 g/100 ml (range: 0-4 g/100 ml) and the average length of hospital stay was 3.9 days (range: 2-13 days). In one case (2%), we converted to laparotomy because a lateral myoma made it impossible to achieve haemostasis of the uterine pedicule under suitably safe conditions. No serious peri or post-operative complications were encountered. No second surgery was necessary and no transfusion was required. These results confirm that total laparoscopic hysterectomy via laparoscopy is a safe, feasable and reproductible technique. Future work will establish the exact place and methods for laparoscopic surgery for hysterectomy. Laparoscopic surgery is only indicated when vaginal hysterectomy is contraindicated or impossible. Laparoscopic hysterectomy constitutes an alternative to laparotomy rather than the vaginal hysterectomy. The combination of an immobile uterus and poor vaginal accessibility is the prime indication for total hysterectomy via laparotomy. PMID- 8636613 TI - [Spontaneous pregnancies in couples after failed or successful in vitro fertilization]. AB - OBJECTIVE: To determinate the true incidence of treatment-independent pregnancy in an in vitro fertilization programme. To establish and to compare the characteristics of couples with and without spontaneous pregnancy. To analyze the outcome of pregnancies. TYPE OF STUDY: Retrospective. SETTING: In Vitro Fertilization Unit, Conception's Hospital, Marseille, France. SUBJECTS: 594 couples having attempted one or more IVF procedures. The study concerned low fertility couples (484) ruling our true sterile couples (110). RESULTS: Spontaneous pregnancies occurred in 54 couples (11.2%). The characteristics of the two populations were not statistically different, except a shorter duration of infertility (p < 0.05) in spontaneous pregnancies. The rate of ectopic pregnancies in the spontaneous pregnancies was statistically higher than that observed in IVP pregnancies (12% versus 6.5%). The fertility rare of these couples was very low (0.38%). CONCLUSION: Spontaneous pregnancies in vitro fertilization programmes are not rare. The evaluation of the results of the in vitro fertilization requires taking the possible spontaneous pregnancies into account. PMID- 8636614 TI - [Association of Morris' syndrome with squamous cell carcinoma of the vulva]. AB - We described a case of squamous cell carcinoma of the vulva in a patient with Morris' syndrome. In these patients, there is an increased risk of gonadal neoplasms. On the contrary, neoplasms originating from the vulvo-vaginal tract are very rare. Neoplasms arising from the neovagina have been reported. Therefore, a continued periodic surveillance of the external genitalia and the vagina is indicated. PMID- 8636615 TI - [Peritoneal and tubal Schistosoma haematobium bilharziasis. Two case reports]. AB - It is now recognized that eggs of Schistosoma haematobium are most commonly found in the bladder because this parasitic infestation usually affects the urinary tract. However, numerous papers have been published on the occurrence of schistosomiasis of the female genital tract. The frequency of distribution of schistosomal disease of the genital tract has not been estimated because only cases with overt disease are usually considered. The involvement of Fallopian tubes is not rare in endemic areas and may predispose to ectopic pregnancy and infertility. In this paper, we report two cases of Schistosomiasis in black African women due to Schistosoma haematobium localized in the peritoneum and the Fallopian tubes. Our first patient had primary infertility associated with bilateral hydrosalpinx and peritoneal inflammatory reaction. Our second patient had an ectopic pregnancy associated with chronic salpingitis. In the both cases, histological examination showed the presence of schistosomal eggs in the peritoneum and the Fallopian tubes. These cases suggest that bilharziasis should now be considered as a urogenital disease and not only as an urinary tract disease. PMID- 8636616 TI - [Rh typing by PCR on amniotic cells]. PMID- 8636618 TI - [Prenatal diagnosis of a small intestinal volvulus]. AB - Ninety-five percent of the neonatal cases of bowel atresia result from jejuno ileal obstructions. Frequency is estimated from 1/3,000 to 1/5,000. We observed a case of small bowel volvulus secondary to jejunal atresia diagnosed at 35 weeks gestation. The ultrasound examination was performed due to decreased perception of active fetal movements. Fetal extraction was successful before perforation and meconial peritonitis. PMID- 8636617 TI - [Prenatal diagnosis of fetal varicella in the second trimester of pregnancy]. AB - The first case of prenatal diagnosis of congenital varicella by amniotic fluid viral culture and PCR is reported. Chickenpox is a benign disease in children, but it can lead to severe complications in the adult, especially in the pregnant woman. Five percent of women in childbearing age are not immunised, and the incidence of gestational chickenpox is between 1 and 7 per 10,000. The consequences of this primary infection during pregnancy can be severe for the mother, because of the risk of serious varicella pneumonia, and for the fetus. The fetal infection depends on the gestational age at which the maternal infection occurs. The 2% evaluated risk of fetopathy is maximal between the 7th and 20th week of amenorrhoea. The reported congenital abnormalities are essentially cutaneous, neurological, ophthalmological and musculo-squeletal lesions. A prenatal diagnosis can be suggested: the revelation of defects by ultrasound scan confirms the fetal affection, and can justify pregnancy termination; on the other hand, amniocentesis and cordocentesis are not totally safe, and cannot always assert the fetal contamination or its level of affection. From the therapeutical point of view, prevention with polyvalent gamma-globulin is prescribed to non-immunised pregnant women who have been in contact with the virus. On the opposite, in case of contracted chickenpox, the treatment of the mother with an association of polyvalent gamma-globulin and acyclovir is still controversial since, although probably effective, it may not be safe for the fetus. The solution may reside in the vaccination, soon available, of non immunised women in childbearing age. PMID- 8636619 TI - [Comparative value of transverse abdominal diameter and fetal abdominal perimeter. 3844 biometric examinations]. AB - OBJECTIVE: Assess charts of abdominal size as they are used in a routine ultrasound screening, on a non selected population with 5 operators, to compare the interest of transverse abdominal diameter (TAD) with abdominal circumference (AC). METHOD: Retrospective study, in the department of Antenatal Diagnosis of the Centre Medico-Chirurgical et Obstetrical between September 1991 and August 1994. MAIN OUTCOME MEASURES: Abdominal biometry and gestational age to characterize the prenatal trophicity. Neonatal weight and gestational age at birth to characterize neonatal trophicity. RESULTS: Between 32 and 36 weeks, the TAD charts detected only one SGA (small for gestational age) out of 10. However, the AC sensitivity was 54.5% with a specificity of 94%. In the same period, the TAD charts suspect LGA (large for gestational age) for one exam out of two. The charts of AC have about the same performance to detect LGA and SGA. CONCLUSION: For a routine ultrasound screening between 32 and 36 weeks gestational age, the AC charts have to be preferred to TAD charts. PMID- 8636620 TI - [Amniotic adhesions. A case report]. AB - Amniotic adhesions occur in a wide variety of foetal malformations and can involve the limbs, the cranio-caudal region and the trunk. They usually occur after premature rupture of the aminos membranes. We report a case of amniotic adhesions diagnosed late at 37 weeks gestation. PMID- 8636621 TI - [Effect of smoking on the fetus and the child during pregnancy]. AB - Cigarette smoking remains a frequent problem during pregnancy. Nicotine has deleterious effects on the foetus. The most common problem remains "small for date" babies. However, this is a minor problem compared with two major toxic consequences: intellectual impairment and increased rate of infantile cancer. Many studies have shown diminished intellectual capacity, behavioural problems and an increase of sudden infant death syndrome. The cancerigenic and genotoxic effects of smoking are well documented with recent studies showing the genotoxicity of amniotic fluid in smoking pregnant women and lymphocyte chromosome mutation in newborns. The frequency of cancers, particularly leukaemia, lymphoma and cerebral tumours are increased in children born to women who smoke during pregnancy. It is therefore excessively important to help pregnant women and their doctors become aware of the toxic effects of active or passive smoking in pregnancy and encourage the patients and their friends and relations to stop smoking as soon as pregnancy is diagnosed or if it's possible before the pregnancy. PMID- 8636622 TI - [Consumption coagulopathy during pregnancy: a case of afibrinogenemia during the second trimester]. AB - We report a case of a membrane disruption at 19 weeks 3 days of gestation complicated by a consumption coagulopathy with clotting tests suggesting primary fibrinogenolysis rather than a disseminated intravascular coagulation usually seen in such cases. A review of the literature revealed 5 cases of coagulopathy during the second trimester of pregnancy among which only two were suspected to be associated with primary fibrinogenolysis. Management of this coagulopathy is discussed. PMID- 8636623 TI - [Tuberculosis and pregnancy]. AB - The authors report a case of pulmonary tuberculosis during pregnancy. They point out that the pregnancy does not change the natural course of the disease, congenital tuberculosis is rare but with a high rate of mortality, and the three most important antituberculosis (isoniazid, ethambutol and rifampicin) are safety during pregnancy. PMID- 8636624 TI - [Complication after an exploratory hysteroscopy: one more case]. PMID- 8636625 TI - Principles of opioid pharmacotherapy: practical implications of basic mechanisms. AB - Opioids form the cornerstone of the pharmacologic armamentarium for the treatment of pain. Despite their long history of use, much confusion and misperception still surrounds their use. This short review will focus on pharmacodynamic and physiologic considerations in the clinical use of oral and parenteral opioids. PMID- 8636626 TI - Long-term opioid therapy: assessment of consequences and risks. AB - Medical practice with respect to the use of opioids for the treatment of pain has been heavily influenced by societal perceptions of problems of addiction and by laws governing the use of opioids. To effectively use opioids for the treatment of chronic pain, physicians must recognize pervasive, but clinically often irrelevant societal and legal influences on prescribing, while at the same time respecting opioid properties which may lead to unwanted consequences in the course of opioid therapy. This paper reviews history and legislation that has shaped medical prescribing of opioids, examines the potential risks and consequences of long-term opioid therapy and assesses three models of long-term opioid therapy. PMID- 8636627 TI - Government regulatory influences on opioid prescribing and their impact on the treatment of pain of nonmalignant origin. AB - Interpretation of regulations establishing standards for prescribing opioids by government regulatory boards and drug-enforcement agencies is more restrictive for treatment of nonmalignant pain than for malignant pain. Authority to regulate opioids is provided by health practice acts enacted by state governments, and controlled substances acts, enacted by both state and federal governments. The methods used by boards/agencies to determine standards of practice for opioid use result in interpreting the language in these regulations based on myths, prejudices, and misinformation about opioids, and the unexamined belief that mere exposure of patients to these drugs causes psychological dependence (addiction) on them to all patients in all instances. Interpretation is also strongly influenced by a failure of regulatory and enforcement bodies to recognize their coequal obligation of making opioids readily available to those who need them for legitimate medical purposes, while simultaneously policing their diversion to illegitimate uses. Emphasis on the police function of preventing diversion is paramount. Disciplining practitioners using standards based on myths, prejudices, etc., reinforces physicians' fears of prescribing opioids for nonmalignant pain. Patients with nonmalignant pain who are not relieved if opioids are not provided will continue to suffer until regulatory boards/drug enforcement agencies define the standards of practice for opioid use for nonmalignant pain in clear and unequivocal terms. It is unlikely these standards will be developed until there is a consensus among pain specialists about opioid use for nonmalignant pain because boards/agencies have no consistent, reliable source of expert information. Pain specialists should initiate efforts to develop this consensus. PMID- 8636628 TI - A visual analogue thermometer for measuring pain intensity. AB - A new instrument for measuring pain intensity--the visual analogue thermometer (VAT)--was developed to overcome limitations and disadvantages of the conventional visual analogue scale (VAS). Two studies were performed to assess the validity and utility of the VAT as compared to conventional pain instruments whose psychometric qualities are scientifically recognized. The first study was carried out with a group of 65 chronic pain patients who provided pain intensity ratings using the VAT, a standard VAS, and the McGill Pain Questionnaire. A second set of measures was obtained from a group of 243 adult healthy volunteers who quantified the intensity of a set of descriptive pain terms with the VAT, a numerical scale (NUM), and a VAS. The results of both studies support the concurrent validity of the VAT as a pain measure. When assessing changes in pain levels, the VAT was able to distinguish between different pain intensities, confirming the construct validity of the instrument. No major difference emerged in the relative sensitivity of the VAT compared to the standard VAS, both scales yielding comparable pain estimates. In contrast, the NUM scale tended to produce higher pain ratings. Regardless of the pain scale used, the results showed unequal differences between descriptive pain terms that are commonly considered equidistant on an ordinal scale. No major problem was noticed in subjects' understanding or using either the VAT, VAS, or NUM scales. When questioned about pain scale preference, a substantial number of participants preferred the VAT to the standard VAS as a means of rating pain intensity. In view of the results obtained in the present studies, it is concluded that the VAT is a valid accurate, and clinically useful tool for measuring pain. Its design makes it suitable and effective for clinical use and as an outcome measure in clinical trials. PMID- 8636629 TI - Pediatric pain practices: a national survey of health professionals. AB - The purpose of this study was to examine how health-care providers in U.S. teaching hospitals assess and manage children's pain. A 59-item questionnaire was sent to institutions with pediatric residency programs listed in the 1992 National Residency Matching Program. Two hundred and twenty-seven questionnaires were sent and 113 were returned. Two-thirds were from nurses, one-third from physicians. Sixty percent of the respondents stated that they had standards of care or protocols for pain in their institutions, but only one-quarter reported that the standards were followed 80% or more of the time. Use of formal pain assessment tools was reported by 73% of the sample. Respondents reported that the effectiveness of pain assessment and management was lower for infants and younger children. Only 35% of the sample indicated it was "likely" or "very likely" that parents would be involved in planning prior to a painful event. Several obstacles to adequate pain management were identified by the respondents: knowledge deficit, attitudes, and resources. PMID- 8636631 TI - Time for reflection. PMID- 8636630 TI - Oral methadone for managing chronic nonmalignant pain. AB - Five patients with chronic nonmalignant pain were transferred from other analgesics to oral methadone. The method of transferring to oral methadone is described and issues of cost effectiveness, convenience, improved analgesia, and adverse effects are discussed. Oral methadone may provide a cost-effective alternative to other opioid analgesics and may provide improved pain control in certain patients, even when compared with other opioids administered parenterally. PMID- 8636633 TI - Craniofacial growth in bilateral cleft lip and palate patients following secondary premaxillary setback. AB - Data of an experimental group of bilateral cleft lip and palate patients (BCLP) who had undergone premaxillary setback at a mean age of 10.2 years were compared with a control group of standard cephalometric values for the white population, and with cephalometric data of BCLP patients from the Oslo Cleft Lip and Palate Archive who did not have premaxillary setback. Cephalometric lateral skull radiographs were taken at a mean age of 16.6 years when most facial growth is completed. Overall, the most marked difference between the two cleft samples was a slightly more concave profile in the experimental BCLP group, mainly due to clockwise rotation of the maxillary plane. Other differences were a longer face and a larger mandible in the experimental group. PMID- 8636632 TI - Hypertelorism: interorbital growth, measurements, and pathogenetic considerations. AB - Normal pre- and post-natal changes in the interorbital distance are described. Causes of illusory hypertelorism include flat nasal bridge, epicanthic folds, exotropia, widely-spaced eyebrows, narrow palpebral fissures, and dystopia canthorum. Measurements of hypertelorism may involve soft tissues or bone, and a number of indices have also been proposed. Various types of measurements are evaluated and recommendations suggested. Possible pathogenetic mechanisms for hypertelorism include: early ossification of the lesser wings of the sphenoid; failure in nasal capsule development allowing the primitive brain vesicle to protrude into the space normally occupied by the capsule resulting in morphokinetic arrest in the position of the eyes; and disturbances of the cranial base in Apert syndrome. Associations with increased interorbital distance are also discussed: orofacial clefting, nonprotruding lipomas of the corpus callosum, calcification of the falx cerebri, duplication of the crista galli, wrinkling of the nose, and tissue tags of the nose. Finally, experimental models of hypertelorism in animals are discussed. PMID- 8636634 TI - Titanium miniplate fixation for osteotomies in facial fibrous dysplasia--a histologic study of the screw/bone interface. AB - In four patients who had osteotomies of the jaws affected by fibrous dysplasia (FD), screws embedded in bone blocks were removed at a re-entry operation 20 months postoperatively. Morphometric measurement of the bone density and calculation of the bone contact percentage were performed. Both normal and dysplastic bone were found to have some direct bone contact with the titanium screws. Although the bone contact percentage was higher in the normal bone when compared with FD, statistics failed to show any significant difference (P < 0.05). The dysplastic bone healed well around the titanium screws without inflammatory reaction and direct dysplastic bone/screw contact was noted. Longer screws should be used in facial FD in order to compensate for the reduced bone contact percentage. PMID- 8636635 TI - Repeated mandibular lengthening in Treacher Collins syndrome: a case report. AB - A patient with mandibular hypoplasia associated with Treacher Collins syndrome was treated by bilateral distraction osteogenesis. Since less than optimal length was provided by the first distraction, a second corticotomy was performed in the newly formed bone 6 months after the first distraction. Thus bone gained by distraction osteogenesis was subjected to distraction once again. New bone formation occurred after the second lengthening. This case illustrated that distraction osteogenesis may be applied to the mandible at the site of previous distraction. PMID- 8636636 TI - Maxillofacial and associated injuries in severely traumatized patients: implications of a regional survey. AB - The aim of this paper was to study the incidence and causes of facial injuries occurring in conjunction with major trauma, and to examine the role of the maxillofacial surgeon in the management of severely injured patients. A prospective study was undertaken of 1088 patients seen in 16 hospitals over a 1 year period. A total of 161 (15%) patients sustained facial injuries. Of these, 33% died at the scene of the incident and 21% died in hospital. There was poor resuscitation in 32% of patients, and a total of 32 injuries were missed in 19 patients. The involvement of the maxillofacial surgeon in the management of severely injured patients is examined. Our findings emphasize the need for early referral to the maxillofacial surgeon. It is concluded that maxillofacial surgery should be an on-site speciality, closely associated with the neurosurgical centre. PMID- 8636637 TI - Imaging of unerupted and displaced teeth by cross-sectional CT scans. AB - Thirty-six impacted and displaced teeth in 29 patients were examined radiographically by using orthopantomography (OPG) and computed tomography (CT). CT scans were obtained by using both a conventional program and a dental CT software program. The radiographic images obtained were compared for visualization of the internal position, inclination of unerupted teeth, and diagnosis of contact and doubtful or clearly visible resorption of adjacent roots. Both CT methods were superior to OPG. As far as the internal position and inclination of the impacted teeth, direct contact, and clearly visible resorption are concerned, the conventional CT program yielded the same information as the dental CT program. However, the exact proportional cross-sectional views obtained by dental CT yielded significantly (P < 0.01) better information about doubtful root resorption in adjacent teeth than did conventional CT. PMID- 8636638 TI - Factors influencing the patterns of invasion of the mandible by oral squamous cell carcinoma. AB - The pattern of tumour invasion of the mandible depends on the extent of invasion. Both the width (P = 0.02) and depth (P = 0.01) in patients with an invasive or infiltrative pattern of disease were greater than in tumours showing the less aggressive erosive pattern in which the tumour mass is separated from the resorbing bone by a connective-tissue layer. Evidence in this study suggests that the erosive pattern develops through a mixed pattern to the invasive pattern of disease as the tumour progresses through the bone. The invasive pattern of disease was evident at a much shallower depth in the molar region of the mandible (mean 9 mm), with a decreased ratio of alveolar to basal bone, than in the premolar and parasymphyseal region (mean 25 mm) (P = 0.02). The hypothesis to explain this phenomenon is that the more superficial alveolar bone responds by resorbing in advance of the tumour, but the basal bone is unable to respond in the same way and becomes widely infiltrated. The attached mucosa with its firm collagen attachment to bone is proposed as the main route of tumour entry into the mandible in both dentate and edentulous mandibles. PMID- 8636639 TI - The implication of DNA content and S-phase fraction in oral carcinomas with and without metastasis. AB - Flow cytometry DNA analysis was performed on fresh tissue samples of 90 primary lesions and 32 metastatic lymph nodes of squamous cell carcinomas of the oral cavity and related regions to elucidate the characteristics of tumors with metastatic potency. The incidence of aneuploidy for carcinomas with metastasis was 67%, which was significantly higher than the 44% of carcinomas without metastasis, and aneuploid carcinomas (39%) had a higher tendency to metastasis than diploid carcinomas (20%). The incidence of aneuploidy and metastasis was related to the T classification, the degree of differentiation, and the histologic grade of malignancy. The incidence of aneuploidy and mean DNA index of metastatic lesions were 31% and 1.12, respectively, and the values were significantly lower than the 67% and 1.30 of the corresponding primary lesions. The results indicate that the chance of evolution of metastatic cell lines is higher in aneuploid carcinomas than diploid carcinomas, possible because the former is more heterogeneous than the latter, but most of the cell lines causing lymph-node metastasis are diploid cell lines. Metastatic lesions had a lower S phase fraction than primary lesions, indicating that a high S-phase fraction does not always reflect the presence of metastatic cell lines. PMID- 8636641 TI - Palatal surgery without denudation of bone favours dentoalveolar development in dogs. AB - The present study compared the dentoalveolar development in beagle dogs after palatal repair according to the partially split flap technique and the von Langenbeck method. It was concluded that palatal surgery according to the partially split flap technique resulted in significantly wider transverse distances of the maxillary dental arch than after the von Langenbeck procedure and that its final outcome closely resembled that of the control group. PMID- 8636640 TI - Oral submucous fibrosis: a new concept in surgical management. Report of 100 cases. AB - Oral submucous fibrosis (OSF) is a collagen disorder commonly seen in the Indian subcontinent. A series of 100 patients is presented. All lesions were biopsied. The condition was staged into four categories. Very early and early cases were treated by local injection of triamicinolone acetonide, while advanced cases were treated by surgical intervention. A new surgical technique of a palatal island flap based on the greater palatine artery in combination with temporalis myotomoy and bilateral coronoidectomy was used in 35 cases. A follow-up ranging from 6 months to 31/2 year showed good results. PMID- 8636643 TI - Chemokines as inhibitors of hematopoietic progenitors. PMID- 8636642 TI - Effect of particulate porous hydroxyapatite on osteoinduction of demineralized bone autografts in experimental reconstruction of the rat mandible. AB - In an experimental model in the rat, a nonhealing bone defect was created in the left ascending mandibular ramus to test the effect of particulate porous hydroxyapatite (HA) on osteoinduction of demineralized bone autografts. The bone fragment removed was demineralized in HCl and used as an autograft for mandibular reconstruction. Granules of HA were added to the lingual and vestibular surfaces of the graft. The effect of this material was evaluated by determining the number of mesenchymal cells induced in the biomaterial and the central and peripheral zones of the bone graft, at 2-and 6-week intervals. The results show that the sites containing HA showed inhibition of osteoinduction by the bone matrix. In all groups, a proliferative gradient from the peripheral zone toward the center of the bone was observed. Similarly, the HA experienced a greater cellular increase in the regions in contact with the demineralized bone matrix. PMID- 8636644 TI - Regulation and selectivity of leukocyte emigration. PMID- 8636645 TI - Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile. AB - Crystallization-inhibiting proteins can explain longer nucleation times associated with bile from gallstone-free subjects as compared with bile from patients with cholesterol gallstones. We partially characterized and examined the crystallization inhibitory potency of a newly purified 15 kd human biliary protein. Gallbladder bile was passed through an anti-apolipoprotein A-I (apo A-I) immunoaffinity column to extract lipid-associated proteins. The bound fraction was separated by 30 kd ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) was performed under nonreducing and reducing conditions. Cholesterol crystallization activity was tested in a photometric cholesterol crystal growth assay. Isoelectric focusing was performed by using a standard gel. The purified 15 kd protein was subjected to N-terminal amino acid sequencing. Although the whole apo A-I-bound fraction contained a variety of proteins and lipids, its 30 kd filtrate yielded a nearly pure 15 kd protein with only minor contamination from apo A-1. Amino acid sequencing showed that the protein was unique. Enzymatic deglycosylation revealed no evidence for glycosylation. At a protein concentration of 10 micrograms/ml, crystallization time was delayed as compared with control and apo A-I, and final crystal mass was reduced to 75% of control. Its isoelectric point was 6.1 without isoforms. Under nonreducing conditions, the protein formed a 30 kd dimer and a 60 kd tetramer. We conclude that this protein is a novel potent biliary crystallization inhibitor protein. PMID- 8636646 TI - Carboxyterminal peptides with the dimeric form of PF4 retain the inhibitory effect on the growth of human megakaryoblastic cell lines. AB - We have previously shown that platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) inhibit the growth of the human erythroleukemia cell line (HEL). We further studied the effect of PF4, beta-TG, and various related peptides on human leukemic lineages to determine the specificity and the relationship between the inhibitory activity and the molecular structure of PF4. The results showed that PF4 and beta-TG had an inhibitory activity on the megakaryocytic growth. Furthermore, peptides corresponding to the 1-24 and 13-24 residues but not to the 16-24 residue of the PF4 C-terminal region, the 21-29 and 20-28 C-terminal region of beta-TG and IL-8, inhibited only the megakaryocytic cell growth. Interestingly, when Gln and Asn located at positions 15 and 24, respectively, of the PF4 C-terminal region were replaced by Glu and Asp (C13-24DE), an increase in the inhibitory activity was observed. Moreover, the 13-24 monomeric form (13-24M) and modified form (13-24A), where a cysteine in C-terminal position 19 was substituted by arginine, were no longer active. These results suggest that the inhibitory activity of PF4 and its related peptides might be localized in their 13-24 C-terminal region and that a dimeric structure seems to be necessary to exert inhibitory activity. PMID- 8636648 TI - Direct and indirect tests of pore size and charge selectivity in nephrotic syndrome. AB - We studied direct and indirect methods of measuring membrane charge by detecting fixed anionic sites with polyethylenimine (PEI) on the glomerular basement membrane (GBM) and Alcian blue on red blood cell (RBC) membrane (ABRBC), respectively, in 40 children with nephrotic syndrome (NS). Size selectivity of the GBM was measured indirectly by fine analysis of urinary proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 22 of these children. Correlation between ABRBC and PEI was strongest (r = 0.79; p = 0.0037) in 11 children with steroid-responsive NS (SRNS), moderate (r = 0.31) in 10 children with focal glomerulosclerosis (FGS), and absent in 14 children with hepatitis B antigen membranous nephropathy (MGN) and 5 with mesangioproliferative glomerulonephritis (MPGN). ABRBC and PEI were reduced in the group as a whole as compared with their controls (ABRBC: 44.53 +/- 9.81 vs 71.54 +/- 12.14, p < 0.05; PEI: 16.31 +/- 4.34 vs 33.3 +/- 1.09, p < 0.005). This reduction was greater in SRNS (26.35 +/- 7.15 p = 0.004) but was also detected in the remainder of the group taken together (52.31 +/- 26.07, p < 0.001). Excretion of glomerular proteins was restricted by size (< or = 80 kd) in SRNS but unrestricted (< or = 80 kd plus > 80 kd) in FGS, MGN, and MPGN. The main cause of proteinuria is likely to be depletion of negative charge on the GBM in SRNS, and distortion of capillary pore size in MGN and MPGN, with probable overlap of these mechanisms in each disease, especially in FGS. Basement membrane injury appears widespread in SRNS but confined to the kidney in MGN and MPGN. PMID- 8636647 TI - Nicotine prolongs neutrophil survival by suppressing apoptosis. AB - Neutrophil accumulation in the lung is implicated in the pathogenesis of pulmonary emphysema and chronic bronchitis associated with cigarette smoking. To determine whether nicotine contributes to this accumulation through the prolongation of neutrophil survival, we examined the survival rates of isolated neutrophils cultured with or without nicotine. We found that nicotine prolonged neutrophil survival in a dose-dependent fashion, with a maximum effect at 10(-6) mol/L. The survival rate at 72 hours was 35.6% +/- 1.2% in medium with 10(-6) mol/L nicotine, compared with 15.5% +/- 0.5% in control medium (mean +/- SEM; p < 0.01), as determined by trypan blue dye exclusion. This prolongation was brought about by suppression of apoptosis, as evidenced by both transmission electron and fluorescence microscopy, and was associated with the preservation of neutrophil functions such as chemotaxis and O2- generation. The prolongation of survival caused by nicotine was abrogated by the addition of Pro-Lys-Arg-NH2, a competitive inhibitor of the specific binding of nicotine to noncholinergic receptors on neutrophils. However, the prolongation of survival caused by nicotine was not suppressed in the presence of K-252b, an inhibitor of protein kinase C. These findings suggest that nicotine prolongs neutrophil survival through noncholinergic nicotine receptors and new protein synthesis, without activation of protein kinase C. PMID- 8636649 TI - Accumulation of bisphosphonates in the aorta and some other tissues of healthy and atherosclerotic rabbits. AB - Clodronate, etidronate, and pamidronate are highly hydrophilic bisphosphonates used for the treatment of bone resorption and hypercalcemia. They also inhibit the development of experimental atherosclerosis without influencing serum cholesterol level. We studied the distribution and the accumulation of the carbon 14-labeled bisphosphonates in the aorta and some other tissues of healthy rabbits and in rabbits with diet-induced atherosclerosis. After intravenous injection, clodronate and pamidronate disappeared from circulation more slowly in atherosclerotic than in healthy rabbits, and the drug concentrations in the peripheral tissues were generally lower in atherosclerotic than in healthy animals. At 24 hours after dosing in healthy rabbits, the mean aorta to plasma ratios of clodronate, etidronate, and pamidronate were, respectively, 2.4 to 2.8, 2.4 to 4.0, and 8.6 to 10. The corresponding ratios in atherosclerotic rabbits were, respectively, 13 to 22, 1.5 to 2.2, and 13 to 24. Seven days after the injection the mean clodronate concentration in the aortas of healthy rabbits was 0.5% to 0.9% of the dose given per tissue weight, and the concentration in those of atherosclerotic animals was 3.8% to 5.2% of the dose given per tissue weight. The results indicate that hydrophilic bisphosphonates, known to inhibit the atherogenesis, concentrate markedly in the aortas of healthy and atherosclerotic rabbits. PMID- 8636650 TI - Relationship between skeletal muscle intracellular ionized magnesium and measurements of blood magnesium. AB - The current laboratory approach to assessing magnesium status is based on determining the concentration of total Mg ((Mg)) in serum or plasma. This strategy is problematic in that the amount of Mg in blood is less than 1% of total body Mg and does not accurately reflect (Mg) in other tissues. Furthermore, the (Mg) of blood does not distinguish biologically active, ionized Mg from the bound fraction. The goal of this study was to determine intracellular ionized Mg ((Mg++)i) of skeletal muscle in vivo and to compare results with the (Mg) of blood constituents. (Mg++)i was determined in resting skeletal muscle by using phosphorus 31 magnetic resonance (31P-MR) spectroscopy. (Mg) was measured in serum (S(Mg)), serum ultrafiltrate (UF(Mg)), mononuclear blood cells (MBC(Mg)), and red blood cells (RBC(Mg)) by using atomic absorption spectroscopy or a colorimetric assay. In a sample of 60 healthy adult subjects, skeletal muscle (Mg++)i = 557 +/- 97 mumol/L (mean +/- SD); S(Mg) = 0.78 +/- 0.09 mmol/L; UF(Mg) = 0.60 +/- 0.12 mmol/L; MBC(Mg) = 13.8 +/- 2.3 mmol/L; and, RBC(Mg) = 1.92 +/- 0.33 mmol/L. A significant negative correlation was found between (Mg++)i and S(Mg) (r = -0.43, p < 0.05). S(Mg) was significantly lower (p < 0.05) and (Mg++)i significantly higher (p < 0.05) in women than in men, but neither was related to age. These findings provide new insight into the relationship between blood Mg measures and (Mg++)i of the largest soft tissue mass of the human body. PMID- 8636651 TI - Renal expression of a transforming growth factor-alpha transgene accelerates the progression of inherited, slowly progressive polycystic kidney disease in the mouse. AB - Polycystic kidney disease (PKD) is a prevalent inherited disease in human beings. The pathogenesis of PKD is as yet unclear. The epidermal growth factor family of proteins has been implicated in PKD based largely on in vitro data. To determine whether these growth factors contribute to the progression of inherited PKD in vivo, we crossed mice with a transgene for human transforming growth factor-alpha (TGF-alpha, a member of the epidermal growth factor (EGF) family) and mice with the pcy gene (which causes a slowly progressive form of PKD very similar to human autosomal dominant PKD). Renal expression of the TGF-alpha transgene in cystic mice (homozygous for the pcy gene) accelerated the development of PKD as shown by an increased kidney weight as a percent of body weight and an increased volume density of renal cysts at 8.5 weeks of age. However, renal expression of the TGF alpha transgene did not appear to precociously initiate cyst development (at 6.5 weeks), nor did it cause an increase in the final degree of renal enlargement (at 29 weeks). Thus TGF-alpha accelerated the enlargement of cysts once initiated. At 8.5 weeks of age, renal expression of the TGF-alpha mRNA correlated positively with the amount of renal enlargement. At all time points studied, cystic kidneys exhibited increased expression of c-myc mRNA as compared with phenotypic normal kidneys, consistent with PKD being a hyperplastic disease of renal tubules. However, the renal expression of c-myc in 8.5 week cystic kidneys, with or without the transgene, did not correlate with the degree of renal enlargement. The results of this study suggest that EGF-like proteins may accelerate the progression of inherited renal cystic disease. However, the final degree of cystic change is dictated by the primary disease process rather than by the continued presence of growth factor. PMID- 8636654 TI - Sensitivity of conventional methods for bilirubin measurements. PMID- 8636652 TI - Oral glutamine to prevent chemotherapy induced stomatitis: a pilot study. AB - Mucositis is a common toxicity of cancer chemotherapy. Glutamine appears to be the major energy source for intestinal epithelium, and animal studies have suggested that dietary supplementation with glutamine may protect the gut from both radiation and chemotherapy. Patients experiencing stomatitis after a course of chemotherapy were offered the opportunity to enter the current study if no clinical parameters precluded receiving the same chemotherapy doses during the next course of treatment. Patients received the same chemotherapy regimen as during the previous treatment but in addition received a suspension of L glutamine, 4 gm swish and swallow twice a day, from day 1 of chemotherapy for 28 days or for 4 days past the resolution of any post-chemotherapy mucositis. Twelve patients receiving doxorubicin, 1 receiving etoposide, and 1 receiving ifosfamide, etoposide, and carboplatinum were entered into the study. The maximum grade (CALGB criteria) of mucositis decreased in 12 of 14 patients with glutamine supplementation (median score 2A vs 0.5, p < 0.001). Similarly, after glutamine supplementation, the total number of days of mucositis was decreased in 13 of 14 patients (2.7 +/- 0.8 (mean +/- SEM) vs 9.9 +/- 1.1, p > or = 0.001). Thirteen of the 14 patients felt that the mucositis was less severe with the addition of glutamine. No change in the nadir neutrophil count was noted with glutamine, and no toxicity of glutamine was observed. We conclude that oral supplementation with glutamine can significantly decrease the severity of chemotherapy-induced stomatitis, an important cause of morbidity in the treatment of patients with cancer. Glutamine supplementation in patients receiving therapy for cancer warrants further study. PMID- 8636653 TI - An autoantibody to C1-inhibitor recognizes the reactive center of the inhibitor. AB - An autoantibody to C1-inhibitor produced a clinical disorder resembling that of patients with hereditary angioneurotic edema. The antibody could not interact with C1-inhibitor after exposure to synthetic peptides representing the primary structure of the reactive center region of the protein. Therefore the antibody recognized this domain of the inhibitor, and it probably impaired the function of C1-inhibitor by altering its conformational properties. PMID- 8636655 TI - Progressive massive fibrosis in a zinc miner. AB - The diagnosis of progressive massive fibrosis (PMF) is traditionally associated with exposure to coal dust. Since the legislation of better control of mine dust, the incidence of PMF has declined significantly. Clinicians who are not regularly involved in the medical care of coal workers may have limited knowledge of this incapacitating problem. Physicians are reminded that PMF is a complex process that can occur after exposure to mixed respirable particles other than coal dust. PMID- 8636656 TI - The polymerase chain reaction: a revolutionary new procedure for the laboratory diagnosis of infectious disease. AB - The polymerase chain reaction (PCR) is a revolutionary new means of amplifying, ie, replicating, selected DNA sequences in vitro. This procedure is rapid, requiring approximately 5 hours for completion, and exquisitely sensitive. Studies have shown that as few as one microorganism can be detected. Thus, it has the potential for revolutionizing the diagnosis of infections. Because of its expense, its immediate role will probably be restricted to infections where the causative organism cannot be cultured or is difficult to detect by conventional means. As further progress occurs, however, this technique may well become a major new tool for diagnosing infections. PMID- 8636657 TI - Noncompetition agreements in Kentucky health care. PMID- 8636658 TI - Use of laparoscopy in the treatment of acute and chronic right lower quadrant pain. PMID- 8636659 TI - The patient's last advocate. PMID- 8636660 TI - Drug discrimination under a concurrent schedule. AB - Three pigeons were trained to discriminate a 5.0 mg/kg dose of pentobarbital from saline under a two-key concurrent schedule with responding on the key associated with the presession injection, under both stimulus conditions, producing four times as many reinforcers as responding on the other key. This concurrent schedule resulted in approximately 70% responding to the higher reinforcement key under the pentobarbital stimulus and approximately 30% responding to that key under the saline stimulus. During testing, then, the pigeons were able to dose dependently emit higher (>70%) or lower (<30%) values than were established under the control conditions. Dose-response curves were determined for pentobarbital (twice), methamphetamine, phencyclidine, chlordiazepoxide, and the combination of pentobarbital and the barbiturate antagonist bemegride. The results obtained with pentobarbital and chlordiazepoxide showed that, as the dose increased, pentobarbital-appropriate responding also increased. Methamphetamine produced relatively flat dose--response curves, whereas phencyclidine administration produced inconsistent effects on responding. The combination of the training dose of pentobarbital with increasing doses of bemegride produced a decrease in pentobarbital-appropriate responding. The results also showed that the dose response curves for pentobarbital and chlordiazepoxide, instead of being all or none, were graded functions of the drug dose. PMID- 8636661 TI - Food and water intake as functions of resource consumption costs in a closed economy. AB - In two experiments, rats living in a closed economy were offered continuous, concurrent access to four resources: food, water, a nest, and a running wheel. Costs of consuming food and water were imposed with bar-press requirements, and the price of either one or both resources was raised. As the consumption cost increased, less was consumed in each bout of resource use. Bout frequency increased, but not sufficiently to compensate for the fall in bout size, and total intake fell. Food and water tended to be complementary resources, in that as intake of one fell with its price, intake of the other also decreased. This interaction was accounted for by the defense of the ratio of body water to lean body mass. As amount consumed decreased, increases in feed efficiency (weight gain per unit of food ingested) and the use of stored calories compensated for the reduced energy intake. There was evidence of competition between feeding and drinking at the higher costs: When both commodities were expensive, the decline in the intake of each one was greater than when only one commodity was expensive. Although the time spent nesting, running, and in unmonitored activity was adjusted when feeding or drinking took more of the rat's day, there was no particular activity that was sacrificed. PMID- 8636662 TI - Commitment using punishment. AB - Experimental parameters were adjusted so that pigeons' pairwise choices among three alternatives reflected the following order of preference: (a) a smaller sooner reinforcer, (b) a larger-later reinforcer, and (c) the smaller-sooner reinforcer followed by a punishment (consisting of an extended blackout period). After this order of preference was established, the pigeons were exposed to a two link, concurrent-chain-like choice procedure. One terminal link consisted of a choice between the smaller-sooner and the larger-later reinforcer; the other terminal link was identical to the first except that the smaller-sooner reinforcer was followed by blackout punishment. The pigeons' preference (in their initial-link choice) for the terminal link with the punished smaller-sooner alternative increased as the delay between the initial and terminal links increased. By choosing this terminal link, the pigeons are said to have "committed" themselves to obtaining the larger-later reinforcer. However, unlike prior studies of commitment (e.g., Rachlin & Green, 1972), it was still possible after making the commitment for the pigeons to choose the smaller-sooner reinforcer and undergo the punishment. The pigeons did in fact occasionally make this highly deleterious choice. PMID- 8636663 TI - Speed analyses of stimulus equivalence. AB - The functional substitutability of stimuli in equivalence classes was examined through analyses of the speed of college students' accurate responding. After training subjects to respond to 18 conditional relations, subjects' accuracy and speed of accurate responding were compared across trial types (baseline, symmetry, transitivity, and combined transitivity and symmetry) and nodal distance (one- through five-node transitive and combined transitive and symmetric relations). Differences in accuracy across nodal distance and trial type were significant only on the first tests of equivalence, whereas differences in speed were significant even after extended testing. Response speed was inversely related to the number of nodes on which the tested relations were based. Significant differences in response speed were also found across trial types, except between transitivity and combined trials. To determine the generality of these comparisons, three groups of subjects were included: An instructed group was given an instruction that specified the interchangeability of stimuli related through training; a queried group was queried about the basis for test-trial responding: and a standard group was neither instructed nor queried. There were no significant differences among groups. These results suggest the use of response speed and response accuracy to measure the strength of matching relations. PMID- 8636665 TI - The effects of knowledge availability and knowledge accessibility on coherence and elaborative inferencing in children from six to fifteen years of age. AB - Two experiments are presented in which a novel knowledge base was acquired by 6- to 15-year-old children prior to hearing a multiepisode story, and where inferences from the story drew only on that knowledge base. Making knowledge equally available to all children did not attenuate age-related differences in either coherence or elaborative inferencing. Easily accessible knowledge was generally twice as likely to be used to make inferences during text comprehension as was knowledge that took longer to retrieve, though knowledge accessibility was more important for coherence inferencing in younger than in older children. Children made more coherence than elaborative inferences in the context of text comprehension, even though elaborative inferencing was more frequent in a simpler processing situation. Within the context of an available knowledge base, the results provide evidence for the importance of knowledge accessibility in children's inferencing, and for the changing developmental relevance of knowledge accessibility for coherence and elaborative inferencing. PMID- 8636666 TI - Learning-disabled readers' working memory as a function of processing demands. AB - The purpose of this study was to investigate whether limitations in the enhancement of learning-disabled readers' working memory performance are attributable to process or storage functions. For Experiment 1, performance of reading-disabled, chronological age-matched, and reading level-matched children was compared on verbal and visual-spatial working memory measures under initial (no probes or cues), gain (cues that bring performance to an asymptotic level), and maintenance conditions (asymptotic conditions without cues). The results indicated that (a) learning-disabled readers' working memory performance was comparable on visual-spatial measures, but inferior to CA-matched children on verbal working memory measures; (b) learning-disabled readers' performance was superior to reading-matched counterparts across working memory conditions; and (c) performance differences remained between learning-disabled and CA-matched children or gain and maintenance conditions, even when initial and processing efficiency (probe) scores were partialed out in the analyses. Experiment 2 included the same conditions as Experiment 1, except that verbal short-term memory scores were also partialed out in the analysis. The results indicated that learning-disabled readers are inferior on both verbal and visual-spatial working memory measures when compared to CA-matched children on high demand conditions (maintenance). Two findings that emerged across experiments were (a) intercorrelations among diverse WM measures increased on demanding conditions and (b) verbal WM was not directly related to reading skill. In sum, the results support the notion that learning-disabled readers' poor working memory performance on demanding conditions reflect constraints in a central executive storage system. PMID- 8636664 TI - Children's sensitivity to syllables, onsets, rimes, and phonemes. AB - It has been argued that children's performance on phonological awareness tasks varies with the linguistic level that is tapped by the task. For example, tasks that involve syllables are thought to be easier than tasks that involve lower level linguistic units, and tasks that tap the level of onsets are thought to be easier than tasks that require access to single phonemes. In previous research, however, the linguistic status of a unit has often been confounded with its size. Five experiments were carried out in an attempt to disentangle these variables and so to provide a better test of the linguistic status hypothesis. In the first study, preschoolers and kindergartners more readily judged that two stimuli shared a beginning sound when that sound was an onset on its own than when it was part of a cluster onset. In two additional experiments, there was an advantage for syllables over rimes in kindergarten and first-grade children when the shared units occurred in the middle syllables of trisyllabic stimuli. The superiority for syllables was largely masked in two other studies in which the stimuli that shared a unit rhymed. This latter result suggests that children's familiarity with rhyme can override the syllable advantage. Overall, the results support the linguistic status hypothesis by indicating that effects of linguistic level on phonological sensitivity cannot always be reduced to effects of unit size. PMID- 8636667 TI - Predicting acute maxillary sinusitis. PMID- 8636668 TI - Cholesterol-lowering agent in men without CHD. PMID- 8636669 TI - Conservative treatment of prostate cancer. PMID- 8636670 TI - Pap smears after hysterectomy. PMID- 8636671 TI - Epidural analgesia in labor. PMID- 8636673 TI - Implementation and termination of a computerized medical information system. PMID- 8636672 TI - Impact of medical student teaching. PMID- 8636674 TI - Failure of hematocrit to detect iron deficiency in infants. AB - BACKGROUND: Infants of low-income families have three times the risk of iron deficiency as those of families above the poverty level. Psychomotor deficits have been associated with iron deficiency once it produces anemia. High-risk infants are usually screened for iron deficiency between 9 and 12 months of age with a hematocrit measurement. This type of screening may miss iron-deficient infants who are not yet anemic. METHODS: In the well-child clinics for low-income families in Houston, Texas, a hematocrit (Hct) < or = 33% is the standard screening criterion for iron deficiency. Three hundred twenty-one infants between the ages of 9 and 18 months had capillary blood drawn for Hct testing. Serum ferritin levels were simultaneously measured. RESULTS: Six (1.9%) of the 321 infants were anemic, but none because of iron deficiency. Fifty-one infants (15.9%) were iron deficient (ferritin <10 micrograms/L), none of whom were anemic. Hematocrit and ferritin levels did not correlate statistically. CONCLUSIONS: The Hct is not an adequate screening test for iron deficiency in this population of infants. Although this population is usually considered high risk, iron deficiency was mild. Selective screening of high-risk infants in this population may be appropriate, but a more sensitive screening test is required. Further studies are needed to determine the prevalences of iron deficiency in this and other high-risk populations. PMID- 8636675 TI - Impact of medical student teaching on family physicians' use of time. AB - BACKGROUND: The purpose of this study was to determine how much and in what ways family physicians' time at work is affected by the presence of a medical student in the practice. METHODS: The study included work sampling of 22 non-academic family physicians, each observed during 1 day with and 1 day without a medical student, and 12 academic family physicians, of whom nine were observed for 8 half days and three for 2 or 4 half-days of clinical practice. Observations were made on average every 4 minutes at preselected random times during the workday. RESULTS: When a student was present at the practice, the amount of time private physicians actually spent working increased by 52 minutes per day, and their patient-care productivity decreased from 3.9 to 3.3 patients per hour. There was no significant change in time spent at work for academic physicians. With a student present, the physicians in private practice spent 27 fewer minutes per day in patient-care activities, whereas academic physicians spent 47.5 fewer minutes per day in these activities. Private and academic physicians spent 71 and 63 minutes per day, respectively, in student-centered activities. There were few differences between physician groups in how this direct teaching time was used. CONCLUSIONS: When a student is in the practice, private family physicians shift substantial amounts of work time from patient-centered to student-centered activities. They also use their personal time for teaching activities and experience a decrease in patient-care productivity of 0.6 patients per hour. PMID- 8636676 TI - 'See and treat' electrosurgical loop excision of the cervical transformation zone. AB - BACKGROUND: "See and treat" electrosurgical loop excision of the cervical transformation zone (ELECTZ) is an excisional surgical procedure that enables simultaneous histologic diagnosis and treatment of premalignant cervical disease, thus eliminating the need for a preliminary cervical biopsy and an additional patient visit. Indications for the procedure include an abnormal cervical Papanicolaou (Pap) smear and a colposcopic impression of cervical intraepithelial neoplasia (CIN). The purpose of this study was to assess the "see and treat" ELECTZ procedure performed by family physicians. METHODS: Women who were scheduled for colposcopic evaluation because of an abnormal cervical cytology report were enrolled from the practices of three family physician colposcopists located at three sites. The "see and treat" ELECTZ procedure was performed on patients with both abnormal Pap smear results and abnormal colposcopic findings. Procedural complications were documented. Subjects were evaluated at follow-up examinations during the first postoperative year to determine therapeutic cure. RESULTS: "See and treat" ELECTZ was performed on 48 women. The histologic results from "see and treat" ELECTZ were normal for 36.1% of subjects. When subjects with a low-grade lesion on Pap smear were considered, 40.7% had normal loop histologic findings. Of women with a preoperative colposcopic impression of low-grade lesion, 54.2% had normal histologic results, and 12% of women with a high-grade colposcopic impression had normal histologic results (P<.001). When the colposcopic impression was reported as high-grade disease, 82% of loop specimens were reported as CIN 2 or 3. CONCLUSIONS: Selective use of "see and treat" ELECTZ may be appropriate only when practiced by experienced colposcopists who are able to reliably differentiate low-grade from high-grade disease by means of colposcopy, and if cytologic and colposcopic findings unequivocally indicate high grade cervical disease. PMID- 8636677 TI - Adhering to inpatient geriatric consultation recommendations. AB - BACKGROUND: The purpose of this study was to evaluate the rate of and factors associated with attending physicians' adherence to geriatric consultation recommendations in an urban community hospital. METHODS: A retrospective review was performed of the charts of 47 patients referred for inpatient geriatric consultation over the previous 1 1/2-year period. Study variables included patient and attending physician demographics, length of stay in hospital before geriatric consultation, status of patient on discharge, level of expertise of consultant, number of diagnoses per patient, and types and number of recommendations per patient made by consultant and acted upon by attending physicians. RESULTS: The recommendations made included medical (23.4%), medication (28.6%), laboratory (15.8%), radiological (2.6%), nutritional (11.7%), psychosocial (7.7%), skin care (1.6%), rehabilitative (6.4%), and other (2.2%). The percentage of total recommendations acted upon was 55.5%. By multivariate analysis, decreasing length of time prior to consultation was statistically associated with referring physician adherence to consultation recommendations (P=.03). Slightly more than 40% of the variability in adherence was explained by this single variable. CONCLUSIONS: Inpatient geriatric consultations are aimed at providing a comprehensive assessment for attending physicians. Recommendations are acted upon more than 50% of the time. Physician adherence to recommendations does not appear to be dependent on patient or physician demographic variables, but to some extent, adherence is associated with less time in the hospital prior to consultation. This is a relatively new concept in hospital medicine. PMID- 8636678 TI - Norplant prescribing in family practice. AB - BACKGROUND: This study describes the Norplant prescribing experience, training, and attitudes of South Carolina family practice and general practice (FP/GP) physicians. METHODS: A survey was mailed to all FP/GP physicians licensed in South Carolina, with two follow-up mailings to nonrespondents. RESULTS: Responses were received from 520 physicians, representing 43% of all licensed FP/GP physicians and 73% of all board-certified FP physicians. Norplant had been inserted by 39% of the respondents, and fewer than 10% reported encountering complications during the insertion process. Norplant removal was reported by 135 physicians, averaging 4.4 removals per year. Complications during removal were reported by 52% of physicians, with the most common problem being difficulty finding the capsules. Some degree of training in Norplant insertion was reported by 82% of physicians; 69% reported having had some training, formal or informal, in removal; but only 57% reported having received formal training either during residency or at a workshop. Training reduced insertion time but did not affect removal time or the number of complications encountered. Formal training was more likely to prepare physicians to successfully manage procedural complications. CONCLUSIONS: FP/GP physicians are important providers of Norplant. Improved training is needed to ensure that insertion is performed properly and to disseminate effective removal techniques. PMID- 8636680 TI - Patient support networks: something for everyone. AB - With the emphasis of American health care shifting toward patient rights and education, the resources available through patient support networks are a valuable supplement to physician services. Support networks are groups organized around specific conditions or disorders, their diagnosis, treatment, and prevention. As with other forms of patient education, support networks enable patients to become more informed about their disease and to play a more active role in their health care. Following a summary of patient-physician-support network relationships and the benefits thereof, a list of patient support resources is provided. PMID- 8636679 TI - Nosocomial infection in the community hospital: severe infection due to Serratia species. AB - BACKGROUND: Serratia bacteremia is an uncommon illness in hospitalized patients. The aim of this study was to determine how frequently this disease occurs nosocomially and to discover the most common portals of entry and the underlying disorders. METHODS: Fifty-six cases of Serratia bacteremia documented by blood culture (17 cases over a 4-year period in a community hospital in Gainesville, Florida, and 39 cases over a 3-year period in three community hospitals in Dayton, Ohio) were reviewed. Comparison was made with 60 control cases of general bacteremia from three Dayton hospitals. RESULTS: Of the 56 study cases of Serratia bacteremia, 45 (80.4%) were classified as nosocomial, compared with 13 (21.7%) of the controls. Twenty-seven (48.2%) of the 56 Serratia cases occurred in intensive care units. The cases were evenly distributed over the two study periods, and no outbreaks on specific units were noted. The most common portals of entry for Serratia organisms were, in descending order, lung, genitourinary tract, unknown, intravenous line, gastrointestinal tract, and skin. The most common underlying disorder for Serratia bacteremia was malignancy, followed by renal failure (acute or chronic) and diabetes mellitus. Most of the Serratia organisms tested were sensitive to carbenicillin, trimethoprim/sulfamethoxazole, ceftizoxime, ceftriaxone, ceftazidime, cefotetan, aztreonam, ticarcillin/clavulanate, and ciprofloxacin. The organisms were largely resistant to ampicillin, tetracycline, cefazolin, cephalothin, and cefuroxime. Twenty-five percent of the patients with Serratia bacteremia died, compared with 13.6 of the bacteremic controls. CONCLUSION: Serratia bacteremia is often acquired nosocomially. The mortality rate among the study population was surprisingly low for this opportunistic bacteremia, but was higher (though not significantly so) than that of the controls. PMID- 8636681 TI - Appetite suppressants as adjuncts in the treatment of obesity. AB - Obesity is a common and challenging problem that often leads to other medical problems, including type II diabetes, hyperlipidemia, hypertension, coronary artery disease, and degenerative joint disease. Weight loss, which is central to the dietary treatments for obesity, is often of limited success. Recent studies have documented the safety and efficacy of certain appetite suppressants for assisting in long-term weight loss and maintenance of weight loss. Since appetite suppressants, alone or in combination, have been documented to be safe and effective adjuncts for treating obesity and complicated obesity, physicians should consider using these agents in the pharmacotherapy for obese patients. PMID- 8636682 TI - Premature rupture of membranes in the second trimester. AB - Spontaneous rupture of membranes during the second trimester presents difficult medical and ethical questions for the patient and physician. Such pregnancies are at high risk for preterm birth, chorioamnionitis, and neonatal complications. Treatment can range from expectant management to pregnancy termination. This case presentation describes a patient with premature rupture of membranes at 21 weeks' gestation who gave birth at 35 weeks. PMID- 8636684 TI - Preparticipation physical examination. PMID- 8636683 TI - The changing interface of primary and specialty care. PMID- 8636686 TI - Outpatient circumcisions. PMID- 8636685 TI - Outpatient circumcisions. PMID- 8636687 TI - Outpatient circumcisions. PMID- 8636688 TI - Antibiotic treatment of asthma. PMID- 8636689 TI - Tobacco advertising in pharmacies. PMID- 8636690 TI - Elevated ESR in stroke. PMID- 8636691 TI - Recognition of iritis. PMID- 8636692 TI - Oviductal protein produces fluorescence staining of the perivitelline space in mouse oocytes. AB - Mouse oocytes were previously observed to undergo structural changes involving the perivitelline space (PVS) within the oviduct following ovulation, as visualized by staining with fluorochrome-protein conjugates. In the present study, this phenomenon was investigated in detail to determine the role of the oviduct and oocyte. Mouse ovarian oocytes matured in vitro were further incubated in medium or within explanted oviducts in vitro for varying periods of time and then stained with fluorescein isothiocyanate (FITC)-casein. Twenty percent of oocytes incubated within explanted oviducts for 3 hr showed distinct fluorescence staining of the PVS, whereas after 20 hr incubation, most (89%) oocytes were similarly stained. In contrast, no ovarian oocytes was stained when incubated in medium alone. Puromycin treatment during incubation of oocytes within explanted oviducts produced a dose-dependent decrease in the percentage of oocyte exhibiting PVS staining after FITC-casein exposure. FITC-casein staining of the PVS also occurred in all oocytes following incubation of in vitro-matured oocytes with oviductal tissue extract. In contrast, no oocytes incubated with serum exhibited fluorescence staining. Additionally, the PVS of oocytes failed to stain after incubation with either 0.001% of trypsin- or heat-treated oviductal homogenate. When zona pellucida (ZP) ghosts, devoid of ooplasm, were incubated within explanted oviducts, their PVS was stained brightly following FITC-casein treatment. From these results, it is concluded that proteinaceous material(s) secreted by the mouse oviduct is responsible for the fluorescence staining of the PVS of mouse oocytes and of ghost ZP. The ooplasm does not appear to play any role in altering the properties of the PVS staining. PMID- 8636693 TI - Kinetics of sperm penetration and fertilization in vitro in hamster follicular and oviductal ova. AB - This study compared kinetics of in vitro sperm penetration and fertilization of in vivo matured follicular vs. oviductal hamster ova. Monospermic fertilization in cumulus-intact (C+) and cumulus-free (C-) follicular ova was highest at sperm concentrations of approximately 50 and 1.0 x 10(4) sperm/ml, respectively. The cumulus layer was a barrier to sperm penetration, but penetration of C-follicular ova was still lower than with C-oviductal ova. At 1 h of sperm:egg coincubation, 21% of C- follicular ova was penetrated vs. 77% of C- oviductal ova, while by 4 h these values were 71% and 98%, respectively. In contrast, activation of zona-free follicular and oviductal ova was not different (83-99%). However, at 2 h male pronuclear formation was lower in follicular ova (9%) than in oviductal ova (26%, P < 0.05). To test the hypothesis that lower and slower penetration with follicular ova compared with oviductal ova is due to lack of oviductal exposure, effects of hamster periovulatory oviductal fluid on penetration and fertilization kinetics of C- follicular ova were examined. Adding oviductal fluid did not improve penetration of follicular ova to levels equivalent to oviductal ova, but 3 h preincubation of follicular ova with oviductal fluid improved both zona penetration and male pronucleus formation to levels equivalent to those in oviductal ova. We conclude that kinetics of sperm penetration and fertilization in follicular ova are retarded, that these deficiencies derive from lack of ovum modulation by oviductal secretions, and that prolonged oviductal exposure of ova is essential for achieving full competence. PMID- 8636694 TI - Hepatitis E virus (87A strain) propagated in A549 cells. AB - A strain of hepatitis E virus (HEV), the 87A strain isolated in 2BS cells from the feces of a patient with hepatitis E, has been reported previously. In this study, the 87A strain was propagated in A549 cells, and the marked cytopathic effect (CPE) appeared in the infected monolayer cells. The size of this virus is about 30 nm in diameter. Furthermore, HEV-RNA from the supernatants of the virus of different passages was detected by polymerase chain reaction (PCR) amplification using ET1.1 HEV primers. A band of HEV for 239 bp from PCR products was revealed by electrophoresis. PCR products of the fourth passage were sequenced. These results show that the 87A virus replicates in the A549 cell line. PMID- 8636695 TI - Existing variations on the gene structure of hepatitis E virus strains from some regions of China. AB - The isolation and identification of the 87A strain of hepatitis E virus (HEV) by means of cell culture have been described previously. This paper reports the nucleotide sequence of a portion of this HEV strain. The RNA extracted from the supernatants of the different passages of the 87A strain cultured in the A549 cell line was reverse-transcribed (RT) to cDNA, and then the polymerase chain reaction (PCR) amplification was carried out using the primers of HEV ET1.1 region. The PCR products from 1) the supernatant of the infected cells at the fourth passage, 2) the virus concentrated by polyethylene glycol (PEG) precipitation at the tenth passage, and 3) the virus purified by a sucrose gradient at the tenth passage were sequenced. In addition, three other PCR products obtained from sera of acute hepatitis E patients in Beijing (B-9) and Guangzhou (G-9 and G-20) were also sequenced. The nucleotide sequences of the above four strains of HEV (located in the genome from positions 4545-4754) were compared to those of some reported HEV strains. The nucleotide sequences of the B 9 strain and the 87A strain were similar to the Burmese strain and may belong to the same branch of HEV. The nucleotide sequences of the G-9 strain and the G-20 strain were a novel and unique branch. The Chinese HEV strains are multiplex and variable in gene structure. PMID- 8636697 TI - Human herpesvirus-6 DNA in the saliva of paediatric oncology patients and controls. AB - Children with malignancy are immunosuppressed and susceptible to serious infections with herpesviruses. The majority of children on chemotherapy for malignancy are seropositive for human herpesvirus-6 (HHV-6), and although HHV-6 has been demonstrated to be a pathogen in severely immunocompromised patients, whether this is the case for paediatric oncology patients is unknown. HHV-6 is secreted in saliva and in this study samples were examined prospectively for HHV 6 DNA in healthy children and those with malignancy. In a nested polymerase chain reaction (PCR), a 287 bp outer fragment and 163 inner fragment of HHV-6 DNA were amplified. The resulting amplimer contained a Hind III restriction site present only in "B" type HHV-6 and this was used to identify the type of HHV-6 amplified. In saliva from healthy control children, 74% (28/38) of samples were HHV-6 DNA positive in either the supernate, pellet or both. In the patients, 58% (45/77) of all samples were HHV-6 DNA-positive. When sequential samples from twelve patients were examined the children appeared to fall into two groups: those who were frequently HHV-6 DNA-positive (60% of samples or more) and those who were rarely HHV-6 DNA-positive (33% of samples or less) (P < 0.0001). The only apparent difference between these two groups was that the less frequently HHV-6-positive group was more often febrile and unwell with neutropaenia. Hind III digestion demonstrated all the positive samples to be "B" type HHV-6. Possible explanations for this difference in HHV-6 secretion between the patient groups are discussed. PMID- 8636696 TI - Study of Norwalk-related viruses in Mexican children. AB - Two-hundred Mexican children monitored from birth to 2 years of age in a cohort study of diarrhea were tested for Norwalk virus (NV) and Norwalk-related virus infection. Blood was collected quarterly and tested by an enzyme immunoassay (EIA) using the recombinant NV (rNV) particles as antigen. Stool was collected weekly and tested by an EIA using hyperimmune anti-sera from animals immunized with rNV and a reverse transcription-polymerase chain reaction (RT-PCR) with primers in the RNA polymerase region of NV. A high prevalence of serum antibody to NV (85% at age 2 years) was found by the antibody EIA. In 54 stool specimens selected from children who developed a high titer of serum antibody to rNV, none was positive for NV by the antigen EIA, but 6 yielded products by the RT-PCR. One stool specimen (MX virus) yielded a 3.3 kb RT-PCR product from the 3' end of the viral genome. The MX virus cDNA has a genomic organization like other caliciviruses. Sequence comparison showed that MX virus shares 80% nucleic acid and 91% amino acid sequence identity with Snow Mountain agent (SMA), but only 62% and 60% identity, respectively, with NV in the RNA polymerase region, suggesting that MX virus is a SMA-like virus. PMID- 8636698 TI - Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1. AB - Before completion of polarization, Madin-Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca2+ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV-1 in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. PMID- 8636699 TI - Use of polymerase chain reaction and quantitative antibody tests in children born to human immunodeficiency virus-1-infected mothers. AB - The diagnosis of human immunodeficiency virus (HIV) infection in children born to HIV-infected mothers is complicated by the presence of passively acquired maternal antibodies, and exclusion of infection in these infants remains problematic. The use of genome detection by polymerase chain reaction (PCR) amplification and the quantification of anti-HIV-1 antibodies were examined as methods for early diagnosis. Blood samples were taken from 84 non-breast-fed infants of HIV-infected mothers in five Italian and Spanish centres, a subgroup of children enrolled in the European Collaborative Study (ECS) for whom clinical and immunological information has been documented from birth. Whole blood was added to glycigel cryopreservative, stored, and tested in the United Kingdom by a nested PCR method. Antibody to HIV-1 was detected and quantified by titration using a gelatin particle agglutination test. PCR sensitivity and specificity were assessed. Twenty-one of the 84 children tested were infected. The estimated PCR sensitivity ranged from 0% (95% CI 0-26%) on day 1, 57% (19-85) on day 7, to 63% (33-92) on day 30. The negative predictive value of PCR ranged from 85% (83-88) on day 0 to 98% (94-100) at 3 months of age. On average, the level of maternal antibody halved every 33 days (31-36.5) in uninfected children. Between 6 and 9 months of age, increases in antibody titres in infected children were not more informative than absolute levels. These findings suggest that antibody measurement may supplement genomic diagnosis and that this collection method provides an alternative to the use of dried blood spots. PMID- 8636700 TI - Selection of a precore mutant after vertical transmission of different hepatitis B virus variants is correlated with fulminant hepatitis in infants. AB - The incidence of perinatal transmission of hepatitis B virus (HBV) depends on the HBeAg/anti-HBe status of the mother. While children of HBeAg-positive mothers have a 90% probability of acquiring a chronic hepatitis B virus carrier state, babies of anti-HBe-positive mothers are more likely to develop fulminant hepatitis within the first 3 to 4 months of life. There is evidence that precore (pre-C) mutations of the HBV can be associated with fulminant hepatitis. The pre C region was therefore examined in sera from nine infants with fulminant hepatitis after vertical transmission, one HBeAg-positive and seven anti-HBe positive mothers by polymerase chain reaction (PCR) and direct sequence analysis. In five mother/infant pairs the virus populations were characterized in addition by analysing clones of the amplified products. All mothers were infected with two or four variants of HBV with mutations at different positions of the preC genome including position 1896, which results in a stop codon. While the precore stop codon was detected in a portion of the virus populations of the HBeAg-positive and of four anti-HBe-positive mothers the dominating viral strain was represented by the wild type virus in three. In contrast, the virus populations of all babies showed the 1896 precore variant as the prevalent virus strain during the phase of active disease. In the surviving baby only wild type sequences were detected after recovery. Subtype ayw was found in all mothers and infants and adw2 was present in three mothers and in the surviving child. The findings suggest that all mothers carried a wild type HBV population with a certain number of different HBV variants. After transmission of the mixed virus population a selection process was started in the baby. The association of subtype ayw with the precore mutations and with the fatal outcome of the hepatitis B might be the result of a directed selection of this variant with a particular advantage in the viral life cycle. PMID- 8636702 TI - Genetic and antigenic characterization of a serotype G6 human rotavirus isolated in Melbourne, Australia. AB - An unusual rotavirus strain, MG6, was isolated from a 16-month-old child admitted to hospital with acute gastroenteritis. The virus could not be serotyped (G typed) by enzyme immunoassay using standard reagents specific for common serotypes of human Group A rotaviruses. Nucleotide sequencing of cDNA derived from the gene encoding the outer capsid protein, VP7, and deduction of the VP7 amino acid sequence indicated that this strain belonged to serotype G6, a serotype normally associated with viruses causing disease in cattle. This was confirmed by polymerase chain reaction typing and enzyme immunoassay using a G6 specific monoclonal antibody. The VP4 genotype of MG6 was determined by hybridization of its VP4 cDNA to genomic RNA isolated from standard strains of defined P-types. This analysis, confirmed by deduced amino acid sequence analysis, classified MG6 into the novel genotype P13. MG6, therefore, is related to the previously described G6P13 human strain PA169, isolated in Italy. The emergence of strain MG6, the first human G6 rotavirus identified in Australia, provides further evidence of reassortment between human and animal rotaviruses. PMID- 8636701 TI - Immunohistochemical identification of varicella-zoster virus gene 63-encoded protein (IE63) and late (gE) protein on smears and cutaneous biopsies: implications for diagnostic use. AB - Early and specific recognition of varicella zoster virus (VZV) infection is of vital concern in immunocompromised patients. The aim of this study was to compare the diagnostic accuracy of histochemical and immunohistochemical identification of the VZV ORF63 encoded protein (IE63) and of the VZV late protein gE on smears and formalin-fixed paraffin-embedded skin sections taken from lesions clinically diagnosed as varicella (n = 15) and herpes zoster (n = 51). Microscopic examinations of Tzanck smears and skin sections yielded a diagnostic accuracy of Herpesviridae infections in 66.7% (10/15) and 92.3% (12/13) of varicella, and 74.4% (29/39) and 87.8% (43/49) of herpes zoster, respectively. Immunohistochemistry applied to varicella provided a type-specific virus diagnostic accuracy of 86.7% (13/15; IE63) and 100% (15/15; gE) on smears, and of 92.3% for both VZV proteins on skin sections. In herpes zoster, the diagnostic accuracy of immunohistochemistry reached 92.3% (36/39; IE63) and 94.9% (37/39; gE) on smears, and 91.7% (44/48; IE63) and 91.8% (45/49; gE) on skin sections. These findings indicate that the immunohistochemical detection of IE63 and gE on both smears and skin sections yields a higher specificity and sensitivity than standard microscopic assessments. PMID- 8636703 TI - Productive HIV-1 infection of human vascular endothelial cells requires cell proliferation and is stimulated by combined treatment with interleukin-1 beta plus tumor necrosis factor-alpha. AB - Vascular endothelial cells (EC) play a key role in viral tropism in vivo. Since conflicting reports have been published on the capability of HIV to infect EC in vitro, we analyzed some factors potentially capable of influencing the susceptibility of human umbilical vein endothelial cells (HUVEC) to HIV-1. Both primary cultures and differentiated immortalized HUVEC lines were used. HUVEC were negative for the expression of CD4, but weakly CD26- and galactosylceramide positive. Although binding of HIV to EC was substantial, the virus was apparently incapable of replicating in nonproliferating cultures. In resting cultures, the content of cell-associated HIV disappeared 4-6 days after infection without production of p24 and infectious progency. In contrast, infection of proliferating EC cultures led to the transient release of p24 and infectious virus (10(2.5)-10(3.5) SFU/ml) peaking 2-6 days postinfection. Antibody neutralization of cytokines that may be produced by EC (IL1, IL6, IL8, TNF, IFN beta) failed to modify virus adsorption and replication, whereas treatment with IL1-beta plus TNF-alpha stimulated both virus binding and virus release. As seen by gag polymerase chain reaction (PCR), the viral genome persisted up to 15 days in untreated EC cultures, but over 20 days in cultures exposed to IL1-beta plus TNF-alpha. This study shows that: (a) CD4-negative HUVEC are capable of binding substantial amounts of HIV-1; (b) binding is enhanced by proinflammatory cytokines; (c) the establishment of productive infection is favored by cell proliferation; and (d) exposure to IL1-beta plus TNF-alpha enhances virus replication. PMID- 8636704 TI - Pathogenesis of ocular cytomegalovirus infection in the immunocompromised host. AB - Balb/C nude and C.B-17 SCID mice were inoculated with salivary gland passaged cytomegalovirus (SG-MCMV) intraperitoneally. Dissemination of the virus in the systemic and ocular tissues was studied by the direct immunofluorescence test, and the virus growth in each tissue was titrated in mouse embryonic fibroblasts. The mode of viral spread was assessed by inhibiting macrophage function by silica and administering polyclonal murine anti-MCMV antibody in the circulation. The virus first reached the eyelid, conjunctiva, and cornea. Subsequently, it spread in the outer ocular muscles and chorioretinal layer. Ocular tissues were involved as part of a generalized infection. Abrogation of macrophage function by silica did not affect the outcome of the viral distribution. Administration of antibody prior to and 3 days after the viral infection prevented virus dissemination. Ocular CMV infection occurred initially at the anterior segment of the eye in an immunocompromised host. Free virus, not macrophage-bound virus, disseminated via the bloodstream. PMID- 8636705 TI - Establishment and characterization of a carrier cell culture producing high titres of polyoma JC virus. AB - This report concerns a carrier cell culture (designated JCI) infected persistently with JC virus (JCV). Immunostaining with an anti-JCV antiserum revealed that JCI was a carrier culture in which only a small fraction of the cells (approximately 1.5%) produced the virus. The JCV titre was increased strikingly by incubating confluent JCI cells for 4-6 days in medium containing a low concentration of fetal bovine serum (2%). Viral genomes cloned from the persistently infected JCI cells were heterogeneous with respect to size, but most clones had an alteration of the same regulatory region (designated CR-JCI). Transfection experiments with a chimeric JCV DNA (Mad-1/CR-JCI), in which the regulatory region was CR-JCI and the other region was derived from an infectious JCV (Mad-1) DNA, showed that CR-JCI was less efficient in inducing viral growth than the regulatory regions of IMR-32-adapted JCVs. The transfected cells could be readily subcultured, and they continued to produce JCV. It is concluded that a decrease in the activity of the JCV regulatory region is of importance for the maintenance of the carrier state of JCI cells. PMID- 8636706 TI - Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis. AB - Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis. PMID- 8636707 TI - Molecular characterisation of small round structured viruses associated with gastroenteritis in South Africa. AB - The application of the reverse transcriptase polymerase chain reaction (RT-PCR) has enabled several morphologically and physically similar small round structured viruses (SRSVs), including the prototype Norwalk virus (NV), to be classified within the Caliciviridae. This technique, using primers directed to the RNA dependent RNA polymerase region within the ORF1 of NV, was used to characterise SRSVs associated with epidemic gastroenteritis in adults and sporadic paediatric gastroenteritis in South Africa. Genomic variation was investigated by sequence analysis of the amplified 209bp cDNA region from six isolates and comparison with other characterised SRSVs including NV. Antigenic variation was investigated by the use of the recombinant enzyme immunoassay described recently for the detection of Snow Mountain agent-like antigen in stool specimens. Two distinct antigenic groups were evident with NV-like viruses associated with adult gastroenteritis, and Mexico viruslike viruses associated with paediatric gastroenteritis. Viral isolates from two of the outbreaks of adult gastroenteritis showed a high degree of nucleotide sequence identity with NV, i.e., 84% and 98%, respectively, whereas the paediatric isolates showed 92-95% sequence similarity with the Snow Mountain-like virus, MxV. These data show concordance between antigenic and genomic analyses. PMID- 8636708 TI - Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis. AB - A limitation to date of reverse transcriptase polymerase chain reactions (RT PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmental samples. PMID- 8636710 TI - Homotypic immune response to primary infection with rotavirus serotype G1. AB - Some aspects of rotavirus humoral immunity were assessed on the basis of distinguishing serotype-specific specificities (VP4/VP7) by using rotavirus reassortants, human and animal strains in neutralization assays in serum samples obtained during the acute phase, and 1, 6 and 12 months after primary natural infection. In this study, all the infecting virus strains were characterized as G type and some also as P type. Primary natural infection induces a significantly greater homotypic neutralization response than heterotypic response. In addition, there was no significant difference in the number of homotypic or heterotypic responses following reinfection. Transplacentally acquired homotypic antibodies were associated with protection against dehydration during rotavirus gastroenteritis. PMID- 8636709 TI - Seroepidemiology of hepatitis E virus in the Egyptian Nile Delta. AB - The seroendemicity of hepatitis E virus (HEV) in an entire village population located in the Egyptain Nile Delta is described. Serum specimens were obtained from 68% of the total population of 1,850 villagers. The lack of serum specimen was greatest in the youngest age group (< 5). Commercially available enzyme immunoassays (EIA) for antibody to hepatitis A virus (anti-HAV), to hepatitis B virus core antigen (anti-HBc), to second-generation hepatitis C virus (anti-HCV) core and nonstructural antigen, and to hepatitis E virus (HEV) were used. Only repeated reactive sera were coded as positive. Stool specimens were examined for Schistosoma mansoni by the Kato method and standard methods for the examination of the liver and spleen by ultrasonography were used. Unadjusted for nonrespone, the seroprevalence of anti-HEV was 17.2% (SE +/- 1.1). Anti-HEV seroprevalence increased by age and was not associated statistically with any of the other viral markers including HCV. Anti-HAV seroprevalence was consistently > 95%, even in the youngest age group (< 5). The overall sero-endemicity of HEV was higher than reported elsewhere and appears not to have been introduced into the village population recently. PMID- 8636712 TI - Virus-specific, antibody-secreting cells during upper respiratory infections. AB - The humoral immune response of 18 army recruits with febrile upper respiratory infection (URI) was studied by enumerating virus-specific, antibody-secreting cells in the peripheral blood. Diagnosis was based on viral antigen detection in nasopharyngeal specimens, virus isolation from throat swabs, or on antibody measurement from paired serum samples. At the time of the sample collection, three viruses, including adenovirus, influenza A, and influenza B, were found mainly to cause URIs among the recruits, and ELISPOT assay for enumeration of the specific antibody-secreting cells was selected for these viruses. Of the 36 patients with febrile URI studied, viral diagnosis was made in 18 cases, which included 11 patients with adenovirus infection, three with influenza A, and four with influenza B. The first blood sample was collected at the first signs of URI and the second and third samples at 2-week intervals. The adenovirus-positive patients developed a strong IgG class antibody-secreting cell response against the homologous virus, which peaked at the first sample and decreased steeply by the second and third samples. In the influenza A and B patients, the response was similar kinetically to that seen in adenovirus-positive patients. In those cases where also IgA and IgM class antibody-secreting cells were determined, the IgG response dominated. The ELISPOT method has potential also as a diagnostic tool for respiratory infections. PMID- 8636711 TI - Emergence of hepatitis B virus S gene mutant in a liver transplant recipient. AB - Immunological and genomic analysis of the "a" determinant was carried out in seven patients with concurrent HBsAg and anti-HBs, four of whom were immunized against hepatitis B virus at liver transplant, two with histologically characterized chronic hepatitis B virus infection, and one HBsAg healthy carrier. The immune reactivity of the HBsAg "a" determinant was evaluated by binding to specific monoclonal antibodies, and the corresponding genomic sequence was studied by differential hybridization in microtiter plates and nucleotide sequence analysis. A double mutation generating an amino acid change (glycine to lysine) at residue 145, able to impair recognition by monoclonal antibodies, was observed in the post-transplant serum from one patient. No significant alteration of the "a" determinant sequence or reactivity was detected in the other patients. Amino acid residue 145 appears therefore to be critical for the recognition by anti-HBs antibodies. A previously undescribed glycine to lysine substitution at this level interferes with the immune reactivity of the "a" determinant. PMID- 8636713 TI - Characterization of SA-11 rotavirus receptorial structures on human colon carcinoma cell line HT-29. AB - The involvement of different cell membrane components in the receptor structures for SA-11 rotavirus was investigated. As experimental model, the human enterocyte like HT-29 cell line, was used because of its closer resemblance to the in vivo viral cellular target as compared to other in vitro systems. Rotavirus was incubated with whole membranes or their separated protein and lipid fractions before infection. Either isolated cell membranes or lipid components were capable of binding to the virus and to prevent infection, whereas proteins did not show any inhibitory activity. Among lipids, the glycolipid fraction was shown to impede rotaviral antigen synthesis with a dose-dependent relationship, whereas phospholipids failed to prevent viral infection. To confirm these findings, membranes and target cells were subjected to different enzymatic treatments prior to infection. In addition, HT-29 cells were also incubated with different lectins before infection. The blocking activity of membranes was inhibited by treatment with ceramide glycanase, neuraminidase, and beta-galactosidase but not by treatment with proteases or heat (100 degrees C). Viral infection was prevented by preincubation of target cells with lectins specific for sialic acid and galactose or with ceramide glycanase, neuraminidase, and beta-galactosidase, whereas protease treatments were not active. The results of these experimental procedures indicate that glycolipids containing specific carbohydrate moieties, such as sialic acid and galactose, contribute to the SA-11 rotavirus receptor structure on HT-29 cells. PMID- 8636714 TI - Serological and salivary markers compared with biochemical markers for monitoring interferon treatment for hepatitis C virus infection. AB - Paired serum and saliva specimens were collected on a regular basis from 18 asymptomatic blood donors participating in a controlled clinical trial of interferon alpha 2a (IFN) treatment of chronic hepatitis C virus (HCV) infection. Nine patients were randomised to receive interferon and nine to observation only. Serum and salivary HCV RNA was detected by a "nested" polymerase chain reaction (PCR) assay. Complete follow-up data were available for 14 patients (7 treated and 7 untreated). Serum ALT levels declined to normal in five of the seven IFN treated patients by the twelfth week. Of these five, loss of hepatitis C viraemia was observed in three. Of the seven treated patients, the three responders had a lower viraemia level than the partial or nonresponders. Both nonresponders had infection with type 1 HCV, but the complete and partial responders were infected with types 2 or 3. HCV RNA was detected in the saliva of all seven observation patients during the follow-up period. HCV was also detected in the saliva of the two patients who did not respond to IFN treatment. No correlation was shown between the level of HCV RNA in serum and the presence of HCV RNA in saliva. A role for noninvasive salivary investigations in monitoring treatment is possible, but further refinement of the methodology is required. PMID- 8636715 TI - HCV infection in a rural population of the Central African Republic (CAR): evidence for three additional subtypes of genotype 4. AB - The prevalence of hepatitis C virus (HCV) antibodies, HCV infection, and genotypes was studied in a rural population of the Central African Republic. In five villages, blood samples were taken from all the inhabitants present during the survey, belonging to Pygmies (299) and to Bantu and Banda ethnic groups (247). Using a second-generation ELISA screening and confirmation by immunoblot assay for the detection of HCV antibodies, all the Pygmies were negative, whereas seven Bantus/Bandas, aged > 35 years and with no familial relationship, were positive, giving a prevalence of 2.8% in this ethnic group. Five samples were also PCR positive; all belonged to genotype 4, but with three new subtypes identified by phylogenic analysis. These results indicate the co-existence of different HCV subtypes and raise questions about the natural transmission of HCV in this secluded population. PMID- 8636716 TI - Large-scale screening for human parvovirus B19 DNA in clinical specimens by dot blot hybridization and polymerase chain reaction. AB - Large-scale screening for human parvovirus B19 (B19) DNA in serum samples was carried out by both dot blot hybridization and the polymerase chain reaction (PCR). Dot blot hybridization was undertaken with a digoxigenin-labeled DNA probe. Serum samples from four patients were pooled and tested by a dot blot hybridization assay. When a dot was positive, each of the four samples was tested separately to identify the positive sample. The PCR template was the DNA extracted from mixed serum samples from 10 patients. When B19 DNA was positive by PCR, each of the ten samples was tested separately. A total of 7,969 serum samples were tested by dot blot hybridization and 15 samples (11 patients) were positive for B19 DNA; 7,038 serum samples were tested by PCR and 71 samples (50 patients) were positive. Large-scale screening for B19 DNA by PCR suggested a broader spectrum of clinical manifestations associated with B19 infection. PMID- 8636717 TI - Persistent rubella infection after erroneous vaccination in an immunocompromised patient with acute lymphoblastic leukemia in remission. AB - A 16-year-old male patient with acute lymphoblastic leukemia in complete remission and on maintenance treatment with weekly oral methotrexate and daily oral 6-mercaptopurine for 3 months was immunized in error with the WI-RA 27/3-HDC live attenuated rubella vaccine. Increasing rubella HAI antibodies were noted from 3 to 7 months post-vaccination as well as high levels of IgM antibody up to 8 months in three different tests. High HAI antibody titers persisted for 12-18 months after vaccination. Persisting rubella virus was indicated by PCR detection of rubella-specific nucleic acid in whole blood, non-stimulated and stimulated mononuclear cells 8 months following vaccination. Further attempts to detect rubella virus RNA in two subsequent blood samples were negative. Since acute arthritis and arthralgia occurred in the second month (days 51-63) after vaccination, antileukemic chemotherapy had to be interrupted. Evidence of higher risk for chronic or relapsing rubella-associated arthropathy in immunologically compromised patients and the need to interrupt antileukemic chemotherapy should warrant immunoprophylaxis with polyvalent immune globulin in rubella-susceptible patients who are immunocompromised. PMID- 8636718 TI - Splice sites of human papillomavirus type 16 E6 gene or heterologous gene required for transformation by E7 and accumulation of E7 RNA. AB - Transformation of primary baby rat kidney cells by the human papillomavirus type 16 (HPV 16) E7 gene and efficient accumulation of E7 RNA have been shown by this laboratory to depend on the integrity of the nucleotide position (nt) 880 splice donor site. Here, the splice sites within the HPV 16 E6 open reading frame (ORF) and the sites of the SV40 splicing unit were examined for an ability to provide this requirement. Constructs containing the HPV 16 E6 sites and the SV40 splice site sequences were used for transformation and RNase protection assays. E6 splice sites supported a low level of transformation, in assays for complete HPV 16 early region constructs containing loss-of-function mutations of the nt 880 site. Using constructs with wild-type E6 or SV40 splice sites showed that both splice sites could substitute similarly for the requirement in cis of the nt 880 site for transformation. HPV 16 E6 mutated splice site and SV40 splice site in reverse, nonfunctional orientation relative to the promoter, were not transformation competent. The HPV 16 E7 RNA levels for the E6 splice site constructs correlated closely with the transformation frequency. The SV40 splice sites were required for E7 transcript accumulation. The results showed E6 splice site function and evidence for enhanced exon skipping from E6 splice donor site to acceptor sites 3' of the E7 ORF. This was shown with constructs containing loss-of-function mutations of the nt 880 site. These results confirmed the function of the splice sites by the transformation competent constructs and suggested lower transformation frequency than for wild type was due to skipping of the E7 exon. These patterns of transcripts may have a role in the regulation of gene expression during progression to malignancy. The combined results revealed that the general presence of a functional splice donor site was absolutely required for transformation by HPV 16 E7 and accumulation of E7 RNA. PMID- 8636719 TI - Hepatitis B virus strains in Thailand: genomic variants in chronic carriers. AB - Genetic heterogeneity of the hepatitis B virus (HBV) has been shown to influence the serological pattern and clinical picture in HBV infection. Thailand has a high transmission rate of HBV, but the molecular epidemiology of HBV strains circulating in this region was hitherto unknown. In this study, the HBV strains from 34 Thai HBsAg-positive patients were investigated. In a proportion of these samples, an antigenically important region of the S gene (n = 18), and the pre-S2 and precore genes (n = 15) were sequenced after PCR amplification. Four strains had in-frame deletions of an upstream region of the pre-S2 gene, with all deletions ending at the same nucleotide. In one of three anti-HBe positive strains without a translational stop at codon 28 of the precore gene, there was a one nucleotide insertion in the precore gene. This insertion would cause a frame shift and result in a nonsense protein being expressed, thus providing one explanation for the lack of HBeAg in this patient. Several rare or unique amino acid changes in the region between residues 120 and 161 of the S protein were found. Glycine 145 was changed to alanine in one strain, and this position showed an apparent mixture of glycine and arginine in another. In total, 10 strains displayed unexpected changes that were not related to the normal variability between subtypes or genetic subgroups. It is concluded that there is considerable heterogeneity in HBV strains in Thailand and that this could have clinical and epidemiological importance in a region with high HBV transmission rates. PMID- 8636720 TI - Polymerase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection. AB - The reliability of the polymerase chain reaction (PCR) for prenatal diagnosis of human cytomegalovirus (HCMV) infection was determined by retrospective testing of 35 amniotic fluids identified previously as positive or negative for HCMV by virus isolation. Amniocentesis was performed in 26 pregnant women with primary HCMV infection at 14-36 weeks gestation, 3-21 weeks after maternal infection. Blood samples were obtained from 20 fetuses for IgM determination and/or virus isolation. Amniotic fluid culture led to antenatal diagnosis of HCMV in 9 of the 13 infected fetuses (sensitivity 69.2%) with one case diagnosed at a second sampling. PCR was able to detect one additional infected fetus (10/13, sensitivity 76.9%). Nested PCR did not increase sensitivity of prenatal diagnosis. Three cases were not diagnosed by all the techniques employed. The specificity of virus isolation from and DNA detection by PCR in amniotic fluid was 100%. The negative predictive value for virus isolation from amniotic fluid was 76.5% and for DNA detection by PCR 81.2%, whereas the positive predictive value was 100% for both techniques. The results showed that neither approach can detect all cases of congenital HCMV infection prenatally, and that the time interval between maternal infection and sampling seems to be a major factor affecting the reliability of prenatal diagnosis. PMID- 8636721 TI - Molecular epidemiology of an outbreak of hepatitis A in Italy. AB - The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived from the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB. PMID- 8636722 TI - Parental history of premature coronary heart disease: an independent risk factor of myocardial infarction. AB - We assessed the role of parental history of premature coronary heart disease (CHD) in the risk of acute myocardial infarction (AMI) and the extent to which the risk associated with positive parental history was independent of other risk factors: smoking, high serum cholesterol, elevated blood pressure, diabetes and obesity, and of socioeconomic status. The study is a prospective 12-year follow up of 15,620 men and women aged from 30 to 59 years in eastern Finland. Parental history of premature CHD was defined as either fatal or nonfatal myocardial infarction or angina pectoris before the age of 60 years. The end point of the follow-up was either nonfatal AMI or coronary death. The risk ratio (RR) of AMI associated with positive family history of either parent was 1.61 in men and 1.85 in women. The risk decreased only slightly when an adjustment was made for other risk factors, and did not change at all when an adjustment was made for the indicators of socioeconomic status. The risk was slightly higher for early AMI (< 55 years) compared with later AMI (> or = 55 years), RR 1.71 versus 1.50, among men and markedly higher, RR 2.87 versus 1.49, among women. These results from this population with an exceptionally high risk of CHD support the hypothesis that positive family history is an independent risk factor of AMI. PMID- 8636723 TI - Problems in the health management of persons with spinal cord injury. AB - This study was conducted to clarify the features of complications attending spinal cord injury (SCI). A comparison was made of the prevalence of disease among patients with SCI (SCIP) with that in the general population in Japan (National Livelihood Basic Survey). For this purpose, a survey was conducted on 244 males at 8 Rosai Rehabilitation Centers (Workman's Accident Compensation Rehabilitation Workshops). The average age was 49.6 years. To eliminate age effects on this parameter, the prevalence rates were expressed as standardized outpatient morbidity ratios (SOMRs), with the value for the general population set at 100. The SOMR data for cystitis were particularly high (16,278, p < 0.01). The SOMRs for other diseases were also high: renal diseases, 2,642; disorders of the skin, 361; gastritis, 339; and hepatic disorders, 381 (p < 0.01). These disorders may be regarded as primary or secondary lesions associated with SCI. SCIP with diseases associated with aging, such as hypertension and diabetes mellitus, are on the increase in Japan. The SOMR for hypertension was 250 (p < 0.01), and for diabetes mellitus it was 323 (p < 0.01). PMID- 8636724 TI - Reliability and changes in validity of self-reported cardiovascular disease risk factors using dual response: the behavioral risk factor survey. AB - The authors previously studied the validity of self-reported cardiovascular disease (CVD) risk factors assessed by telephone surveys, and found the validity low, especially for self-reported hypertension and hypercholesterolemia. One way to improve validity is to combine repeated measurements (dual response) into a single measure. The authors explored this and the reliability of self-reported CVD data collected by the Behavioral Risk Factor Survey in three New York counties from January 1989 to May 1990. Nine hundred and eleven subjects were interviewed by telephone to collect CVD risk factor and health behavior information. Interviewees were offered physical examination and laboratory testing to verify self-reported CVD risk factors; 628 participated. Subjects were also reinterviewed to assess the test-retest reliability of the survey, and to study how validity of self-reported CVD data changes by dual response. Reliability coefficients for CVD risk factors, preventive health practices, and knowledge of risk factor levels ranged from 0.42 to 0.99. Minimal improvement in sensitivity of self-reported risk factors was found using dual response, and it did not improve specificity. Also, for prevalence of risk factors, dual response minimally improved self-reported rates compared to objective estimates. Combining self-reported measurements causes minimal changes in the validity of these variables. Physiological assessment for hypertension and hypercholesterolemia, or correction for misclassification, is needed for valid individual measurement and for community prevalence estimates from telephone surveys. Self-reported cigarette smoking, obesity, and diabetes mellitus have better validity, but physiological assessment or correction for misclassification may supplement these self-reported risk factors. PMID- 8636725 TI - Serum albumin and physical function as predictors of coronary heart disease mortality and incidence in older persons. AB - The role of traditional risk factors in predicting coronary heart disease (CHD) among men and women aged 65 years and over has been extensively debated, but the search for risk factors that are distinctive in the elderly is still ongoing. The relation of serum albumin levels and physical disability to risk of CHD morality and incidence was prospectively assessed in a cohort of 4116 men and women, aged 71 years and over, who were evaluated in 1987-1989 and followed for a mean of 4.0 years. Outcome events were based on death certificates and Medicare hospitalization records. Analyses were adjusted for major CHD risk factors. There were 275 CHD deaths (16.8/1000 person-years) among all participants and 503 incident (fatal and nonfatal) CHD events (39.4 per 1000 person-years) among participants free of prevalent CHD during the observation period. The relative risk (RR) of CHD morality for women with an albumin concentration < 38 g/liter was 2.5 times higher than for women with albumin > 43 g/liter (RR 2.5; 95% confidence interval [CI], 1.4-4.6). There was a significant and graded increase in CHD incidence with decreasing albumin concentration in women but not in men. The presence of physical disability doubled the risk of CHD mortality among both men and women, an increase in risk that was comparable to that imposed by a previous myocardial infarction and was independent of other coronary risk factors. Disability had a lesser impact on CHD incidence, which was significant only in women. Low albumin concentration (< 38 g/liter) identifies a group of women at higher risk of CHD mortality and incidence. Physical disability is an independent predictor of CHD mortality in both men and women and for CHD incidence only in women. PMID- 8636726 TI - Influence of air pollution on humoral immune response. AB - To learn more about the effects of ambient air pollution on the human immune system, immunological parameters-16 serum proteins and circulating immune complexes--were determined for more than 500 women from the polluted area of Cologne, Germany, and a control area, Borken. The geometric mean values for immunoglobulins, complement components, haptoglobin, alpha 1-acid glycoprotein, alpha 1-antitrypsin, ceruloplasmin, alpha 2-macroglobulin, prealbumin, and transferrin were statistically significantly higher in Cologne than in Borken. No difference were found for C-reactive protein, rheumatoid factors, and anti streptolysin O. For each of the parameters a logistic regression was fitted, thus controlling for the influence of a number of confounding factors. After controlling for possible confounders, the percentages of values above the norm for immunoglobulins, complement components, haptoglobin, and alpha-1-glycoprotein were statistically significantly higher in Cologne than in Broken. Important confounders included overweight, high blood pressure, acute cold, fever in the preceding week, and smoking. The biochemical mechanisms underlying the observed interarea differences in protein profiles are as yet unknown and should be the subject of further, nonepidemiological research. PMID- 8636727 TI - Limitations in upper-body strength associated with breast cancer: a comparison of black and white women. AB - We examined differences in reported upper-body limitations between black and white breast cancer cases and controls aged 40 to 84 years at 3 and 12 months after diagnosis in the Detroit metropolitan area (n = 954 cases and 1000 controls at 3 months; n = 879 cases and 909 controls at 12 months). At 3 months black cases were more likely than white cases to report limitations in upper-body strength (30.4 versus 19.8%). No difference was found between black and white controls (8.0 versus 9.4%). At 12 months, the proportion of white patients with upper-body limitation returned to the same level as white controls. Black patients with limitations, however, did not return to the same level as black controls. Stage of disease was strongly associated with upper-body limitations, especially for black women. Race and stage differences in upper-body limitation could not be explained by differences in breast cancer treatment, financial adequacy, education, marital status, or comorbidity. Recommendations are made for more comprehensive studies of rehabilitation. PMID- 8636728 TI - Respondent-specific information from the randomized response interview: compliance assessment. AB - In situations in which researchers ask potentially embarrassing questions, respondents may feel uncomfortable with revealing certain behavior. Consequently, response rates or accuracy may be low. The "randomized response interview" (RRI) was developed to enable researchers to better elicit responses to such questions. The technique has clear potential in estimating population proportions engaging in embarrassing behavior. It does not appear to have been recognized that one may also obtain more respondent-specific information from the application of the RRI. This article indicates that while still only probabilistic, respondent-specific information is obtainable from the RRI. PMID- 8636729 TI - The effect of experience of illness on health state valuations. AB - There is increasing interest in health status measurement and the relative weights that patients and the general public attach to different states of health and illness. One important question that has been raised is whether preferences differ according to the characteristics of the respondents, such as their experience of illness. The results presented in this article suggest that current health status has an important effect on the valuations attached to different health states, with those in poorer health generally giving higher valuations. Past experience of illness, on the other hand, appears to have a negligible effect on valuations. These findings pose real problems for policy makers. To the problem of whose values should count can be added the problem of when these values should count, since the results imply that different valuations may be given by the same respondent depending on how recent their experience of illness was. PMID- 8636730 TI - Developing a condition-specific measure of health for patients with dyspepsia and ulcer-related symptoms. AB - A patient-administered instrument for dyspepsia and symptoms suggestive of duodenal or gastric ulcer, based on the type of questions asked when taking a patient's history, was developed and tested using the following steps: literature reviews, devising the questions, testing the responses to the questions using factor analysis and internal consistency, assessing test-retest reliability, and validating the questionnaire by comparing patient responses to the SF-36 health survey questionnaire. The main sample consisted of 135 patients referred to an outpatient clinic with dyspepsia, and 152 patients in general practice who were not referred to a specialist. The final instrument produced a Cronbach's alpha of 0.72 and an intraclass correlation coefficient of 0.69. Patient scores on the dyspepsia questionnaire had small to moderate correlations with the SF-36 health survey, the largest correlation being with the SF-36 scale of pain. Patient scores were significantly related to general practitioner perceptions of symptom severity, family history of gastric ulcer disease, and whether the patient was referred. The questions asked in taking a clinical history from a patient with dyspepsia and other symptoms suggestive of ulcer disease can be used to construct a valid and reliable measure of the effect of dyspepsia on health. PMID- 8636731 TI - Acute myocardial infarction in Estonia: first results from the Tallinn AMI Register. AB - The acute myocardial infarction (AMI) register of Tallinn, the capital of Estonia, started in 1990. The register follows methodology recommended by the WHO MONICA Project for the registration of coronary events. By standardizing its procedures with the FINMONICA AMI register, the Tallinn AMI register aims at producing data comparable with those of the centers participating in the WHO MONICA Project. This article presents incidence, attack rates, and mortality rates of AMI in Estonia during the first year of registration in Estonians and non-Estonians (mostly Russians) of the study area. The total number of registered AMI events was 493 among men and 117 among women. The age-standardized mortality from AMI (per 100,000 population) was 249 (95% confidence interval, 201-297) in Estonian men and 234 (189-279) in non-Estonian men. In women the corresponding rates were 35 (20-50) and 39 (23-55), respectively. The incidence and attack rate of AMI were not different in Estonians and non-Estonians. The incidence of AMI seems to be relatively high in international comparison. The registration period of our study is thus far rather short, but it is the first investigation of the incidence of AMI in Estonia based on standardized data collection procedures. This study provides a basis for the development of surveillance of cardiovascular disease in Estonia. PMID- 8636732 TI - Effect of passive smoking on the development of respiratory symptoms in young adults: an 8-year longitudinal study. AB - The evidence of an association between passive smoking and occurrence of respiratory symptoms is relatively strong in children, whereas studies conducted in adult populations have provided inconsistent results. The objective of the present study was to examine the relations between exposure to environmental tobacco smoke (ETS) and development of respiratory symptoms in young adults during a study period of 8 years, with emphasis on the evaluation of potential dose-response pattern of the relations. The study population consisted of 117 "never smokers," who were 15 to 40 years of age at the time of initial examination, when they answered a standardized questionnaire on respiratory health, and who were reexamined 8 years later. ETS exposure at home and at work during the study period was recorded at the 8-year examination with a structured questionnaire. The symptoms studied as outcomes included wheezing, dyspnea, cough, and phlegm production. The relations between ETS exposure and development of respiratory symptoms were studied in multivariate logistic regression models controlling for age, gender, atopy, and the presence of other respiratory symptoms. Cumulative incidences of the respiratory symptoms, except of phlegm production, were consistently greater among subjects exposed to ETS compared with the reference group. A significant dose-related increase in the risk of developing dyspnea was observed in relation to ETS exposure, with an OR of 2.37 for an average exposure of 10 cigarettes/day (95% confidence interval, 1.25 4.51). The risk of developing other respiratory symptoms, apart from phlegm, was also related to ETS exposure, but these relations did not achieve statistical significance. The results provide evidence of adverse respiratory effects of ETS exposure in the home and office work environments in young adults. These findings emphasize the need for effective measures in the prevention of involuntary smoking during young adulthood. PMID- 8636733 TI - Factors predicting change in prescription and nonprescription drug use in a community-residing black and white elderly population. AB - The current study identifies characteristics that predict change in use of prescription and nonprescription drugs over a period of 3 years. A modified health care services use model was applied to information obtained from a probability-based sample of black (n = 1778) and white (n = 1446) community resident elderly, interviewed in 1986-1987 and 1989-1990. Analysis was by means of logistic and ordinary least-squares regression, with sample weights and design effects taken into account. The number of users and average number of prescription drugs used increased over the 3 years, and was best predicted by extent of prior drug use, older age, white race, poorer health, and number of health care visits. Conversely, nonprescription drug use declined significantly, and was best predicted by prior use, white race, and female gender. The reduced use of prescription drugs by blacks as compared to whites is of concern, suggesting that attention is needed to assure equitable access to prescription drugs. PMID- 8636734 TI - The law of halves. PMID- 8636735 TI - Attributable risks and genetic predisposition. PMID- 8636736 TI - Diagnostic certainty in pancreatic cancer. PMID- 8636737 TI - Error in statistics program. PMID- 8636738 TI - Factoring outcomes in ovarian cancer. PMID- 8636739 TI - Dexrazoxane for protection against cardiotoxic effects of anthracyclines in children. PMID- 8636740 TI - Anthracycline cardiotoxicity prevention by dexrazoxane: breakthrough of a barrier -sharpens antitumor profile and therapeutic index. PMID- 8636741 TI - nm23 in ovarian cancer: correlation with clinical outcome and other clinicopathologic and biochemical prognostic parameters. AB - PURPOSE: The aim of the study was to define the prognostic role of the metastasis suppressor gene, nm23, in 106 primary ovarian cancer patients. PATIENTS AND METHODS: Northern and Western blotting analysis of nm23-H1 and nm23-H2 expression were performed in a subset of ovarian tumors. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded specimens from 106 primary ovarian carcinomas by the antihuman nm23 monoclonal antibody. RESULTS: Northern and Western blotting analysis demonstrated a direct association between nm23-H1 and nm23-H2 levels. Moreover, an overall concordance of 86.7% between Northern blotting and immunohistochemical data was observed. Sixty-six specimens (68%) showed a positive nm23-H1 immunoreaction. The percentage of nm23-H1 positivity was higher in lymph node-negative (70%) than in lymph node-positive cases (44%) (P = .049). Moreover, the percentage of complete/partial responses to chemotherapy was higher in nm23-H1-positive (69%) than in nm23-H1-negative (44%) patients (P = .03). The percentage of epidermal growth factor receptor (EGFR) positive cases was lower in nm23-H1-positive (44%) than in nm23-H1-negative immunostained (72%) samples (P = .012). Lower ras/p21 levels (median, 1.77 absorbance units) were found in nm23-H1-positive than in nm23-H1-negative samples (median, 2.63 absorbance units) (P = .03). The 6-year progression-free survival (PFS) rate of nm23-H1-positive cases was 50% (95% confidence interval [CI], 33 to 67) versus 12% (95% CI, -2 to 26) for nm23-H1-negative patients (P = .0056). In multivariate analysis, only stage, ascites, and nm23-H1 content retained independent prognostic roles. CONCLUSION: The assessment of nm23 content may provide useful information for prognostic characterization of ovarian cancer patients. PMID- 8636742 TI - Intraperitoneal recombinant interferon gamma in ovarian cancer patients with residual disease at second-look laparotomy. AB - PURPOSE: The purpose of this study was to evaluate the efficacy and tolerance of recombinant human interferon gamma (rIFN-gamma) as second-line treatment in patients with persistent disease at second-look laparotomy. PATIENTS AND METHODS: One hundred eight patients with residual disease at second-look laparotomy were treated with rIFN-gamma (20 x 10(6) IU/m2) administered intraperitoneally (IP) twice a week for 3 to 4 months. In the absence of clinically assessable disease, response to rIFN-gamma was assessed with a third-look laparotomy. RESULTS: Of 98 assessable patients, 31 (32%) achieved a surgically documented response, including 23 patients (23%) with a complete response (CR). The age and size of residual tumor were significant prognostic factors for the response to rIFN gamma. A 41% CR rate was observed in 41 patients younger than 60 years and with residual tumor less than 2 cm. The probability of response was independent of previous response to first-line chemotherapy. The median duration of response was 20 months and the 3-year survival rate in responders was 62%. Response to rIFN gamma was the most significant prognostic factor for survival of patients with residual disease. Adverse events included fever, flu-like syndrome, neutropenia, and liver enzyme disturbances. No significant peritoneal fibrosis was noted. CONCLUSION: These results support the potential interest of IP rIFN-gamma as adjuvant treatment in ovarian cancer. Controlled prospective trials are required to determine its place in the therapeutic strategy of this malignancy. PMID- 8636744 TI - High-dose megestrol acetate in advanced or recurrent endometrial carcinoma: a Gynecologic Oncology Group Study. AB - PURPOSE: Progestins represent the most widely used form of endocrine therapy in advanced or recurrent endometrial carcinoma. Based on encouraging response rates in breast cancer with high-dose megestrol acetate (MA) 800 mg/d, this phase II trial assessed response rates in patients with endometrial carcinoma treated with high-dose MA. PATIENTS AND METHODS: Sixty-three patients with recurrent or advanced endometrial carcinoma were entered into this Gynecologic Oncology Group (GOG) study. Patients had either failed to respond to or were considered incurable with local therapy and had not received prior cytotoxic or hormonal therapy. MA 800 mg/d was administered orally in divided doses. Standard GOG toxicity criteria were used. RESULTS: Of 63 patients entered, 58 were assessable for toxicity and 54 for response. Of 13 responders (24%), six (11%) had a complete and seven (13%) a partial response. Four of the responses lasted greater than 18 months. Twelve patients (22%) had stable disease. The response rate of patients with grade 1 or 2 lesions (11 of 30, 37%) was significantly higher (P = .02) than that of patients with more poorly differentiated tumors (two of 24, 8%). There was no difference in response rates comparing advanced versus recurrent disease, cell type, including papillary serous lesions, site of disease, prior radiation, age, or weight. The median progression-free survival (PFS) and overall survival intervals were 2.5 and 7.6 months, respectively. Grade 3 weight gain (> 20%) was seen in three patients and grade 3/4 hyperglycemia in three. Three deaths secondary to cardiovascular events were possibly related to therapy; diabetes was also a contributing factor in all three cases. CONCLUSION: High-dose MA is active in endometrial carcinoma, but appears to have no advantage over lower-dose progestins. PMID- 8636743 TI - High-dose versus low-dose cisplatin in combination with cyclophosphamide and epidoxorubicin in suboptimal ovarian cancer: a randomized study of the Gruppo Oncologico Nord-Ovest. AB - PURPOSE: The aim of the study was to compare high-versus low-dose cisplatin in combination with cyclophosphamide and epidoxorubicin as primary chemotherapy for suboptimal stage III and IV ovarian cancer. PATIENTS AND METHODS: One hundred forty-five patients were randomized to receive six courses of cisplatin 50 or 100 mg/m2 plus epidoxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2. The two treatment arms were well balanced; all patients had greater than 2 cm and 37.2% had greater than 5 cm of residual disease; 29.6% had stage IV disease. RESULTS: Patients in the high-dose arm received a double dose-intensity and double total dose of cisplatin. The high-dose regimen induced significantly more episodes of leukopenia (47.8% v 32.8%, P = .05), thrombocytopenia (21.7% v 3.2%, P = .003), anemia (37.6% v 12.5%, P = .002), nephrotoxicity (six v one patient), and neurotoxicity (30.4% v 6.3%, P = .002). There were no significant differences in efficacy in terms of clinical response rate (high-dose 57.5% v low-dose 61.1%), pathologic complete response (CR) (9.6% v 18.1%), median survival times (29 v 24 months), and median progression-free survival (18 v 13 months). CONCLUSION: This study shows that doubling the dose-intensity and total dose of cisplatin in combination with epidoxorubicin and cyclophosphamide has significant toxic effects and does not improve clinical outcome in patients with suboptimal ovarian cancer. PMID- 8636745 TI - Randomized trial of the cardioprotective agent ICRF-187 in pediatric sarcoma patients treated with doxorubicin. AB - PURPOSE: We conducted an open-label, randomized trial to determine whether ICRF 187 would reduce doxorubicin-induced cardiotoxicity in pediatric sarcoma patients. METHODS: Thirty-eight patients were randomized to receive doxorubicin containing chemotherapy (given as an intravenous bolus) with or without ICRF-187. Resting left ventricular ejection fraction (LVEF) was monitored serially with multigated radionuclide angiography (MUGA) scan. The two groups were compared for incidence and degree of cardiotoxicity, response rates to four cycles of chemotherapy, event-free and overall survival, and incidence and severity of noncardiac toxicities. RESULTS: Eighteen ICRF-187-treated and 15 control patients were assessable for cardiac toxicity. ICRF-187-treated patients were less likely to develop subclinical cardiotoxicity (22% v 67%, P < .01), had a smaller decline in LVEF per 100 mg/m2 of doxorubicin (1.0 v 2.7 percentage points, P = .02), and received a higher median cumulative dose of doxorubicin (410 v 310 mg/m2, P < .05) than did control patients. Objective response rates were identical in the two groups, with no significant differences seen in event-free or overall survival. ICRF-187-treated patients had a significantly higher incidence of transient grade 1 serum transaminase elevations and a trend toward increased hematologic toxicity. CONCLUSION: ICRF-187 reduces the risk of developing short term subclinical cardiotoxicity in pediatric sarcoma patients who receive up to 410 mg/m2 of doxorubicin. Response rates to chemotherapy, event-free and overall survival, and noncardiac toxicities appear to be unaffected by the use of ICRF 187. Additional clinical trials with larger numbers of patients are needed to determine if the short-term cardioprotection afforded by ICRF-187 will reduce the incidence of late cardiac complications in long-term survivors of childhood cancer. PMID- 8636746 TI - Survival from locally invasive or widespread neuroblastoma without cytotoxic therapy. AB - PURPOSE: To test the hypothesis that cytotoxic therapy is not needed at diagnosis to assure the survival of most patients with non-stage 4 neuroblastoma. METHODS: Patients with non-stage 4 disease received no cytotoxic therapy in the absence of N-myc amplification. The International Neuroblastoma Staging System (INSS) was used. RESULTS: Of 84 consecutive patients with previously untreated, newly diagnosed neuroblastoma, 31 (37%) had non-stage 4 disease. All 31 patients initially received no cytotoxic therapy because none of them had N-myc amplification. Nine stage 1 patients are relapse-free. This report focuses on the 22 patients with locally invasive or distant disease: two stage 2A with gross residual tumor postsurgery, 11 stage 2B with ipsilateral or midline lymph node involvement, four stage 3, and five stage 4S. Eight of the 22 patients were older than 1 year. Postsurgery, 13 patients had visible residual disease, and two others had markedly increased urinary catecholamine levels for more than 1 year. Recurrent or enlarging tumors regressed spontaneously (n = 2) or were excised 5 to 39 months after diagnosis (n = 4). One of the latter had chromosome 1p deletions (common in poor-risk neuroblastoma) that were not detected in the patient's original tumor resected 23 months earlier--findings consistent with clonal evolution or multifocal disease. The patient received chemotherapy. All 22 patients are alive 24 to 98 months (median, 64) from diagnosis. CONCLUSION: Our results suggest that non-stage 4 patients without N-myc amplification can be spared cytotoxic therapy because (1) residual postsurgical or recurrent biologically favorable neuroblastoma rarely evolves into lethal stage 4 disease; and (2) neuroblastoma in lymph nodes has no prognostic significance. These findings are remarkable because no other cancer includes subtypes that are curable without therapy to ablate residual disease. PMID- 8636747 TI - High-dose melphalan and cyclophosphamide with autologous bone marrow rescue for recurrent/progressive malignant brain tumors in children: a pilot pediatric oncology group study. AB - PURPOSE: To determine the maximum-tolerated dose of cyclophosphamide (CTX) when administered sequentially with melphalan 60 mg/m2/d for 3 days, followed by autologous bone marrow rescue (ABMR), in children with recurrent or progressive malignant brain tumors, and to make preliminary observations on efficacy. PATIENTS AND METHODS: Nineteen patients between the ages of 2 and 21 years were enrolled and 18 were assessable for effects of therapy. CTX was administered to seven patients at 750 mg/m2/d for 4 days, to five patients at 975 mg/m2/d, to three patients at 1,200 mg/m2/d, and to three patients at 1,500 mg/m2/d. All patients received ABMR. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used in 15 patients. Toxicity, response to therapy, time to progression, and survival and monitored. RESULTS: The median time to a granulocyte count more than 500/dL was 19 days (range, 11 to 39), and for a platelet count more than 50,000/dL was 33 days (range, 16 to 60). Four heavily pretreated patients (22%) died of transplant-related complications. No dose-limiting, non-hematologic toxicities were defined for the study. Seven of 18 patients (39%) had a complete response (CR) or a partial response (PR). These included four patients with medulloblastoma (CR and three PRs), two with germinomas (two CRs), and one with ependymoma (one CR). The estimated 1-year survival rate was 39% (SE 12%). CONCLUSION: CTX, at a maximum total dose of 6,000 mg/m2, administered sequentially with melphalan and followed by ABMR was tolerable in children with recurrent brain tumors who had not been heavily pretreated. Responses were seen in patients with medulloblastoma and germinomas. Further trials in children with chemosensitive tumors, with minimal residual disease, are planned. PMID- 8636748 TI - Cytoreduction and prognosis in acute lymphoblastic leukemia--the importance of early marrow response: report from the Childrens Cancer Group. AB - PURPOSE: To quantify the residual marrow lymphoblast fraction that best defines patients at high risk for relapse, and the optimal time for assessment during remission induction. PATIENTS AND METHODS: The residual lymphoblast percentage was evaluated on day 7 (n = 220) and day 14 (n = 205) during a four- or five-drug induction in patients with poor prognostic factors. The rate of cytoreduction was related to event-free survival (EFS) and other factors. RESULTS: On the New York (NY) regimen, 68%, 14%, and 18%, and on the Berlin-Frankfurt-Munster (BFM) regimen, 56%, 15%, and 29% of patients had M1 (< 5% blasts), M2 (5-25%), or M3 (> 25%) responses on day 7 (P = .075). On day 14, the corresponding values were 87%, 6%, 7% on NY and 84%, 8%, 8% on BFM. For patients who achieved remission by day 28 and a day-7 marrow rating of M1, M2, or M3, the 6-year EFS rate was 78%, 61%, and 49% (P < .001). The day-14 ratings predicted for a 72%, 32%, or 40% EFS (P < .001). Patients with 5% to 10% blasts day 7 had three times as many events as those with less than 5% and had no better EFS than those with 11% to 25% blasts. Patients with a WBC count more than 200,000/microL at diagnosis and an M1 day 7 marrow had an EFS rate of 69%, while for those with M2 or M3, the EFS rate was 41%. Day-7 marrow had greater prognostic significance than the day-14 evaluation. For slow responders on day 7, the day-14 marrow provided additional information. EFS for patients who achieved M1 by day 14 was 65%. EFS decreased to 20% for those still M2 or M3 on day 14. Day-7 and -14 evaluations had significance for patients of all ages and WBC levels. CONCLUSION: Marrow aspiration on day 7 of therapy provided more useful information than that on day 14. However, day-14 marrow provided additional information for patients with a poor day-7 response. The rate of cytoreduction is a powerful, independent prognostic factor that can identify patients with a slow early response who are at risk for a short remission duration. PMID- 8636750 TI - Tumor angiogenesis correlates with metastatic disease, N-myc amplification, and poor outcome in human neuroblastoma. AB - PURPOSE: To determine if the clinical outcome of children with neuroblastoma (NB) is correlated with the degree of tumor neovascularization and to assess the relationship of stage, N-myc copy number, and histology to angiogenesis. MATERIALS AND METHODS: The vascularity of primary untreated NB from 50 patients diagnosed at a single institution between 1984 and 1994 was evaluated. An image processor was used to analyze the tumor tissue area for each histologic slide of tumor, and a vascular index (VI) was calculated, where VI = total number of vessels/mm2 of tissue area. Tumors were classified histologically according to the criteria of Shimada et al (J Natl Cancer Inst 73:405-416, 1984), and N-myc copy number was determined by Southern blot analysis. RESULTS: We found that higher VI (> 4.0) in NB strongly correlated with widely disseminated disease (P = .006) and poor survival (P < .0001). VI more than 4.0 was also statistically associated with N-myc amplification (P = .02) and unfavorable histology (P = .02). Univariate analysis demonstrated that disease stage, tumor histology, and N myc copy number were also predictive of outcome. Cox regression analysis showed that VI provided independent prognostic information. CONCLUSION: Our studies indicate that angiogenesis may play an important role in determining the biologic behavior of NB. Antiangiogenic therapy may prove to be effective in the treatment of children with highly vascular, widely disseminated NB. PMID- 8636749 TI - Etoposide achieves potentially cytotoxic concentrations in CSF of children with acute lymphoblastic leukemia. AB - PURPOSE: Although epipodophyllotoxins are commonly used in contemporary treatment regimens for acute lymphoblastic leukemia (ALL), their potential role in CNS directed therapy has received little attention. We prospectively studied 20 children during initial remission of ALL and 16 children at relapse to assess CSF penetration of etoposide. METHODS: Simultaneous plasma and CSF concentrations were assessed at a median of 2.8 hours (range, 0.4 to 5.3) after an intravenous (i.v.) or oral dose in 41 paired samples. RESULTS: Etoposide given at 300 mg/m2 i.v. to patients during first remission and at 50 or 25 mg/m2 orally to those in relapse resulted in median CSF levels of 0.175 mumol/L (range, .066 to 2.12), 0.011 mumol/L (range, .004 to .032), and 0.007 mumol/L (range, .003 to .014), respectively. The CSF etoposide concentration was > or = 10 nmol/L in 20 of 20, five of 10, and two of 11 courses following 300 mg/m2 i.v., 50 mg/m2 orally, and 25 mg/m2 orally, respectively, and was positively related to both the concurrent etoposide plasma concentration (R2 = .64) and to dose (R2 = .73). The median ratio of CSF to plasma concentration was 0.30% (range, 0.09% to 3.12%), which was not related to dose, plasma concentration, or time postdose at which samples were obtained, but was positively correlated with the CSF protein concentration (R2 = 0.43, P = .006). Both the absolute etoposide CSF concentrations (P = .008) and the ratio of CSF to plasma concentrations (P = .023) were higher among first remission patients who had CSF leukemic blasts at diagnosis compared with those without CSF blasts. CONCLUSION: Because etoposide concentrations as low as 10 nmol/L may be cytotoxic in vitro, prolonged daily oral low-dose (50 mg/m2) or conventional i.v. doses of etoposide may contribute to successful CNS-directed therapy in children with ALL. PMID- 8636751 TI - Attitudes of 47 mothers of pediatric oncology patients toward genetic testing for cancer predisposition. AB - PURPOSE: To assess attitudes toward testing for cancer susceptibility genes, we interviewed mothers of pediatric oncology patients about their cancer causation theories, interest in hypothetical predisposition testing for themselves and their healthy children, and anticipated impact of testing. PATIENTS AND METHODS: The subjects were 47 mothers of two or more living children, one of whom was 6 to 24 months postdiagnosis of cancer. Potential risks and benefits of hypothetical genetic predisposition testing for cancer susceptibility were described. A semistructured interview assessed the following: (1) recall of discussions with the pediatric oncologist about the possible role of heredity in causing the child's cancer; (2) mothers' personal theories of the etiology of their child's cancer; (3) family cancer history; (4) interest in genetic predisposition testing for themselves and unaffected (cancer-free) children; and (5) expected sequelae of testing. RESULTS: If genetic cancer predisposition tests were available, 51% of mothers would test themselves and 42% would test healthy children, even with no medical benefit. With established medical benefit, an additional 36% of mothers would seek testing for themselves and another 49% would test their healthy children. Interest in cancer predisposition testing among mothers extended far beyond those with significant family histories of cancer. Most mothers would consider minor children's wishes in the decision about testing and would tell children under age 18 their test results. CONCLUSION: As increasing numbers of cancer susceptibility genes are identified, parents of pediatric oncology patients may be receptive to opportunities to test themselves and their healthy children. Counseling will be important to aid in decisions about testing. Research is essential to evaluate the long-term impact of predisposition testing. PMID- 8636752 TI - Docetaxel in patients with metastatic breast cancer: a phase II study of the National Cancer Institute of Canada-Clinical Trials Group. AB - PURPOSE: The National Cancer Institute of Canada-Clinical Trials Group (NCIC-CTG) conducted a phase II study to assess the efficacy and toxicity of docetaxel as first-line chemotherapy in metastatic breast cancer (MBC). PATIENTS AND METHODS: Fifty-one patients with measurable MBC were studied. Three patients were ineligible and were excluded from analysis. The planned dose of docetaxel was 100 mg/m2 intravenously (i.v.) every 3 weeks. Prior adjuvant chemotherapy was allowed if at least 12 months had elapsed from completion of treatment to recurrence. RESULTS: The most severe toxicity was granulocytopenia. Ten patients (20.8%) were hospitalized for febrile neutropenia. The protocol was amended to a starting dose of 75 mg/m2 for the last 16 patients. Sixty percent of patients experienced hypersensitivity reactions (HSRs). After two protocol amendments, the use of a premedication regimen of oral dexamethasone and i.v. H1 and H2 blockers prevented significant HSRs. Edema developed in 62% of patients and was cumulative, was present in 50% who received greater than 400 mg/m2, and was not improved by premedication regimens. Following an independent radiology review, 22 partial remissions and four complete responses in 47 assessable patients were confirmed (response rate, 55%; 95% confidence interval [CI], 40% to 69%). The response rate for 15 assessable patients registered at 75 mg/m2 was 40% (95% CI, 16% to 67%); for 32 assessable patients registered at 100 mg/m2, the response rate was 63%, (95% CI, 43% to 78%). CONCLUSION: Docetaxel is an active agent in MBC. Its activity as a single agent is comparable to many combination chemotherapy regimens and is not affected by prior adjuvant chemotherapy. Studies are ongoing to improve its therapeutic index and to incorporate docetaxel in combination chemotherapy regimens. PMID- 8636753 TI - Antiatherogenic effects of adjuvant antiestrogens: a randomized trial comparing the effects of tamoxifen and toremifene on plasma lipid levels in postmenopausal women with node-positive breast cancer. AB - PURPOSE: To evaluate whether a novel antiestrogen, toremifene, has similar antiatherogenic effects as tamoxifen. PATIENTS AND METHODS: Forty-nine postmenopausal patients with node-positive breast cancer were randomized in a trial that compared the effects of tamoxifen and toremifene on serum lipoproteins. Tamoxifen was given at 20 mg and toremifene at 60 mg orally per day for 3 years. Serum concentrations of apolipoprotein (apo) A-I, A-II, and B, and lipoprotein(a) [Lp(a)], cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol were measured before and after 12 months of antiestrogen therapy. RESULTS: Both antiestrogens significantly reduced serum total and LDL cholesterol and apo B levels. However, the response of HDL cholesterol to treatments was clearly different between the groups. Toremifene increased the HDL level by 14%, whereas tamoxifen decreased it by 5% (P = .001). As a consequence, both cholesterol-to-HDL and LDL-to-HDL ratios decreased more in the toremifene than tamoxifen group (P = .008 and P = .03, respectively). Toremifene also increased the apo A-I level (P = .00007) and apo A I-to-A-II ratio (P = .018). Both tamoxifen and toremifene decreased the Lp(a) concentration significantly (change, 34% v 41%). CONCLUSION: These results provide positive evidence that toremifene has antiatherogenic properties with potency to improve all lipoproteins that are associated with increased coronary heart disease (CHD) risk. PMID- 8636754 TI - Effect of tamoxifen on endometrial proliferation. AB - PURPOSE: An increase in the incidence of endometrial cancer and a potential increase in related mortality has been associated with the administration of 20 mg tamoxifen, the dose adopted in breast cancer chemoprevention trials, thus urging studies on intermediate markers of risk. PATIENTS AND METHODS: Thirty three women who received 20 mg tamoxifen as adjuvant breast cancer treatment underwent endometrial biopsy. Samples were divided for histologic examination, including a quantitative analysis of stromal:epithelial ratio, and an assessment of DNA ploidy and proliferation by flow cytometry. Results were compared with 37 symptomatic subjects. RESULTS: All histograms were DNA diploid. Compared with controls, a significant increase in the risk of proliferation as measured by the hyperdiploid fraction was associated with tamoxifen duration (< or = 36 months: cumulative odds ratio = 16.5, 95% confidence interval, 1.85 to 146.5; > 36 months: cumulative odds ratio = 28.2, 95% confidence interval, 2.56 to 310.6, P for trend < .05). Tamoxifen-induced risk was significantly reduced by the extent of menopausal status. No cases of cancer or epithelial hyperplasia were observed in the tamoxifen group, whereas seven cases of epithelial hyperplasia without atypia were observed in the control group. The effect of tamoxifen on proliferation was associated with an increase in the stromal component. CONCLUSION: Tamoxifen at 20 mg/d exerts a time-dependent proliferative effect on the endometrium, particularly in premenopausal and early postmenopausal women. This effect appears to be mediated by the stromal component, which accounts for the discrepancy between flow cytometry and histology. Our study provides preliminary evidence that the DNA flow cytometric hyperdiploid fraction may be a useful tool for monitoring endometrial cell proliferation in women exposed to tamoxifen. PMID- 8636756 TI - Tumor control and morbidity following transperineal iodine 125 implantation for stage T1/T2 prostatic carcinoma. AB - PURPOSE: To quantify disease progression and morbidity following computer tomography (CT)-based transperineal iodine 125 prostate implantation. METHODS: Ninety-two patients with clinical stage T1 or T2, Gleason score 2 to 7/10, prostatic carcinoma had outpatient, CT-based transperineal 125I prostate implantation and were monitored for 1 to 7 years (median, 3). The prescribed minimum radiation dose was 140 to 160 Gy. Lymph node dissection and postimplantation prostatic biopsies were not routinely performed. RESULTS: In 46% of patients, radiation-related urinary symptoms were substantial enough at 1 month following implantation to require medication. Radiation-related urinary symptoms gradually resolved. Two years after implantation, 14% of patients had persistent urinary symptoms of Radiation Therapy Oncology Group (RTOG) > or = grade 2. Eight percent of patients underwent a transurethral resection of the prostate (TURP) within 2 years of implantation. Five patients developed radiation induced rectal ulcerations. Of 56 patients who were sexually potent preimplantation, 86% retained potency at 3 years. Twenty-five patients had biochemical disease progression. The overall actuarial freedom from biochemical failure rate at 4 years following implantation was 63%. In Cox proportional hazards multivariate analysis, the strongest predictor of failure was prostate specific antigen (PSA) level less than or greater than 10 ng/mL (P = .005), followed by Gleason score (2 to 4 v 5 to 7, P = .08) and stage (T1 v T2, P = .09). CONCLUSION: The 5-year biochemical freedom-from-progression rates following transperineal 125I implantation are comparable with those achieved with prostatectomy. The morbidity has decreased with increased physician experience. PMID- 8636755 TI - Adjuvant chemotherapy for high-risk clinical stage I nonseminomatous testicular germ cell cancer: long-term results of a prospective trial. AB - PURPOSE: To assess the impact of short-term adjuvant chemotherapy on relapse rates, treatment-related morbidity, and long-term toxicity in patients with clinical stage I nonseminomatous testicular germ cell tumor (NSGCT I) who carry a high risk of relapse, ie, who show blood-vessel invasion (VI) by the primary tumor. PATIENTS AND METHODS: From January 1985 to January 1995, 42 NSGCT I patients with VI were treated with two courses of cisplatin, etoposide, and bleomycin (PEB) after orchidectomy. Of these, 29 patients with a follow-up time of more than 2 years are the subject of this report. NSGCT I patients without VI were assigned to a surveillance program and served as controls for the assessment of long-term toxicity. RESULTS: During a median follow-up time of 79 months (range, 27 to 119), two patients relapsed. One developed fully differentiated mature teratoma; the other was a true chemotherapy failure and again developed embryonal carcinoma. Twenty-seven patients (93%) are alive without evidence of disease; one patient (3%) died of progressive testicular cancer and another of lung cancer. The two courses of PEB did not cause any severe acute adverse reactions. The assessment of late sequels of adjuvant chemotherapy based on clinical and laboratory evidence of cardiovascular and pulmonary disease, fertility, and secondary neoplasms, as well as on a psychosocial questionnaire, did not show any significant disadvantages versus the control group. CONCLUSION: Adjuvant chemotherapy with two courses of PEB is an effective and reasonable treatment option for patients with clinical stage I NSGCT who carry a high risk of relapse. No adverse late sequelae were detected within a median follow-up time of more than 6 years. PMID- 8636757 TI - Management of residual mass in advanced seminoma: results and recommendations from the Memorial Sloan-Kettering Cancer Center. AB - PURPOSE: Guidelines for management of postchemotherapy residual mass in patients with advanced seminoma remain controversial. We sought to characterize independent prognostic factor(s) for persistence of tumor to identify patients with a high risk of residual carcinoma. PATIENTS AND METHODS: One hundred four patients with advanced seminoma were assessed. All had achieved a complete response or partial response with normal markers to induction cisplatin-based chemotherapy and had radiographs available for review. Selected prechemotherapy and postchemotherapy characteristics were compared for patients who had either germ cell tumor histology at surgery or relapsed at the assessed site (defined as site failure) versus those who had only necrosis or fibrosis found at surgery and did not relapse at the assessed site (defined as site nonfailure). RESULTS: At a median follow-up time of 47 months (range, 5 to 153), 94 patients (90%) were designated as site nonfailures and 10 (10%) as site failures. Site failure correlated only with size of the residual mass (< 3 cm or normal v > or = 3 cm; P = .0006). Two of 74 patients (3%) with residual masses less than 3 cm were considered site failures, compared with eight of 30 (27%) with residual masses > or = 3 cm. CONCLUSION: Patients with advanced seminoma who have normal radiographs or residual masses less than 3 cm after chemotherapy can be observed without further intervention. The following three options exist for patients with a residual mass > or = 3 cm: observation, radiotherapy, or surgical intervention. We prefer the latter to define response, resect viable tumor when possible, and direct further treatment. PMID- 8636758 TI - Prognostic significance of the microvascular count in colorectal cancer. AB - PURPOSE: To investigate the potential correlations between a high microvascular count and the survival rate in colorectal cancer. MATERIALS AND METHODS: Three markers for endothelial cells--Ulex Europaeus Lectin (UEA), a polyclonal anti-von Willebrand factor (vWF) antibody, and a monoclonal anti-CD31 antibody (all from Dakopatts, Glostrup, Denmark)--were used for immunohistochemical detection of microvessels in whole-mount sections from 15 colorectal cancers. Areas with higher microvascular density were homogeneously distributed in the sections, regardless of the marker used. The anti-vWF antibody was subsequently used for quantification of microvessels in full-cross tumor biopsies collected from 212 consecutive surgical specimens. The correlations between the mean number of microvessels in areas with the highest microvascular density and tumor differentiation, tumor stage according to Dukes', and survival time were investigated. RESULTS: A significantly longer survival time was shown for patients who had tumors with a mean of more than 10 anti-vWF-positive microvessels, as compared with those who had < or = five. Tumors with a microvascular count between six and 10 microvessels behaved in-between. There was no correlation between the number of microvessels and tumor differentiation or Dukes' stage. CONCLUSION: The number of microvessels measurable in tumor biopsies seems to be a prognostic predictor independent of Dukes' stage in colorectal cancer. However, our results are opposite to the findings in other tumor types investigated so far; we found that a high microvascular count predicted a longer survival time, rather than a shorter one. Determination of the microvascular count can be of importance in therapy selection even before, or immediately after, surgery, ie, before Dukes' stage is known. PMID- 8636759 TI - Loss of heterozygosity of the RB gene is a poor prognostic factor in patients with osteosarcoma. AB - PURPOSE: The usual therapy of osteosarcoma is neoadjuvant chemotherapy, followed by surgery, then by postoperative chemotherapy. There is no prognostic factor to predict, at diagnosis, the histologic response and final outcome. Inactivation of the retinoblastoma-susceptibility gene RB is associated with the pathogenesis of several human cancers. In primary osteosarcomas, loss of heterozygosity (LOH) at the RB locus has been found in greater than 60% of cases. The aim of this study was to determine the potential early prognostic value of LOH of RB gene on the biopsy material at diagnosis. PATIENTS AND METHODS: Forty-seven patients with primary osteosarcoma, treated in four French institutions, were studied. LOH was studied by polymerase chain reaction (PCR) of an informative RB DNA polymorphism. RESULTS: Assessment of LOH at the RB gene could be completed on 34 heterozygous patients only. LOH was found in 24 cases (70%). The event-free survival (EFS) rate at 60 months is 100% for patients without LOH, 43% for all patients with RB LOH, and 65% for nonmetastatic patients with RB LOH. The difference in EFS is highly significant at P = .008 and P = .024, respectively. Histologic response after preoperative chemotherapy did not show significant correlation with LOH status. CONCLUSION: RB gene LOH appears to be an early predictive feature for osteosarcomas that indicates a potential unfavorable outcome. RB LOH study might shortly help to identify high-risk patients earlier. If this is verified, therapy could then be adapted earlier to the individual's real risk of relapse. PMID- 8636760 TI - Prognostic significance of a positive microscopic margin in high-risk extremity soft tissue sarcoma: implications for management. AB - PURPOSE: A positive microscopic margin (PMM) is a significant prognostic variable and leads to local recurrence (LR) in high-grade soft tissue sarcoma (STS) patients. Its effect on the rate of distant metastasis (DM) and tumor mortality (TM) remains controversial. PATIENTS AND METHODS: One hundred sixty-eight primary, high-risk (high-grade, deep, > or = 5 cm) extremity STS patients were identified from our data base, of which 42 had a PMM. Limb-sparing surgery (LSS) was the primary surgical therapy in 144 patients; 24 received amputation (AMP). Statistical analysis was by log-rank test and Cox model. Significance was defined as a P value less than .05. RESULTS: A PMM was a significant negative prognostic factor for both DM and TM (P = .002 and .002, respectively). However, those patients who received LSS with 28% PMMs showed no significant difference in the rate of DM or TM compared with patients who received AMP with only 8% PMMs (log rank, P = .057 and .28, respectively). A PMM was significantly associated with > or = 1,000 mL blood loss and more than 3 hours of operating time (P < .006 and .001, respectively). CONCLUSION: The strong statistical significance that relates a PMM to DM and TM in high-risk STS of the extremity is likely related to biologically aggressive tumors and LSS. Residual microscopic disease is not a guarantee of LR. The main problem in this group of patients is not LR, but DM and subsequent death. Therefore, to increase a disability with further surgery or amputate a patient's limb without clear evidence of LR in this group at high risk for distant recurrence is not recommended. PMID- 8636761 TI - Treatment of patients with melanoma of the extremity using hyperthermic isolated limb perfusion with melphalan, tumor necrosis factor, and interferon gamma: results of a tumor necrosis factor dose-escalation study. AB - PURPOSE: To evaluate response rates and systemic and regional toxicity of hyperthermic isolated limb perfusion (ILP) for treatment of in-transit metastases of extremity melanoma using escalating-dose tumor necrosis factor (TNF) in conjunction with melphalan and interferon gamma (IFN). PATIENTS AND METHODS: All patients received IFN 0.2 mg2 for 2 days followed by a 90-minute ILP with TNF and IFN (0.2 mg) given at time 0 and melphalan (10 mg/L limb volume) given at 30 minutes. Twenty-six patients were treated with 4 mg of TNF and 12 patients received 6 mg of TNF. All patients had assessable disease in the perfusion field and all but two patients were assessable for response at 1 month after treatment. RESULTS: Mean peak perfusate TNF levels in the 4-mg group were 4.8 micrograms/mL, compared with 7.4 micrograms/mL for the 6-mg group (P = .03). The complete response rate in the 4-mg TNF group was 76%, with an overall objective response rate of 92%, compared with 36% and 100% for the 6-mg group. Subgroup analyses showed that the lower complete response rate in the 6-mg TNF group was not explained by differences in disease burden or prior regional therapy. Systemic drug toxicity was short-lived, easily managed, and related to perfusate leak more than to TNF perfusate dose. Regional toxicity, particularly painful myopathy and neuropathy, was greater with the 6-mg dose level and was considered dose limiting. CONCLUSION: ILP with 4 mg TNF, IFN, and melphalan can lead to complete local responses in the majority of patients with extremity melanoma. Escalating the TNF dose to 6 mg did not increase the complete response rate and increased regional toxicity. PMID- 8636762 TI - The value of high-dose methotrexate-based neoadjuvant chemotherapy in malignant fibrous histiocytoma of bone. AB - PURPOSE: The value of high-dose methotrexate (HD-MTX)-based neoadjuvant chemotherapy was evaluated in patients with malignant fibrous histiocytoma (MFH) of bone. PATIENTS AND METHODS: Since 1977, MFH of bone was diagnosed in 17 patients (12 males and five females). Ten patients (59%), completed treatment with four courses of neoadjuvant chemotherapy as follows: HD-MTX, vincristine, doxorubicin, cyclophosphamide, bleomycin, and dactinomycin, or HD-MTX, 4(1) epidoxorubicin, and carboplatin followed by local tumor resection (n = 3), curettage-cryosurgery (n = 2), amputation (n = 2), or tumor resection endoprosthetic replacement or allograft (n = 3). After recovery from surgery, an additional six courses of polychemotherapy, including HD-MTX in nine patients, were administered. One patient changed to cisplatin- instead of HD-MTX-containing chemotherapy postoperatively. One additional patient received only adjuvant HD MTX-containing polychemotherapy. Neoadjuvant MTX-containing chemotherapy was contraindicated in five patients (29%) due to age, cardiac insufficiency, or mental disorder. In one patient, neoadjuvant chemotherapy was cancelled after one course due to renal failure. Treatment consisted of amputation (n = 2), one course of chemotherapy and amputation (n = 1), hyperthermic isolated limb perfusion (HILP; n = 1), intraarterial chemotherapy, radiotherapy, and endoprosthetic replacement (n = 1), and a combination of chemotherapy and radiation treatment (n = 1). RESULTS: Five of six patients who received no HD-MTX based neoadjuvant chemotherapy developed metastatic disease (83%); the median time to metastatic disease was 17 months (range, 3 to 44). In contrast, in 10 patients who completed treatment with HD-MTX-based neoadjuvant chemotherapy, with a mean follow-up time of 9.8 years (range, 2.3 to 15.7) and a median follow-up time of 10.8 years (range, 2.3 to 15.7) after diagnosis, no local recurrence or distant metastases were diagnosed (P < .005). CONCLUSION: Neoadjuvant HD-MTX containing chemotherapy in addition to surgery has dramatically improved the prognosis of patients with MFH of bone. PMID- 8636763 TI - Prognostic significance of abnormal p53 accumulation in primary, resected non small-cell lung cancers. AB - PURPOSE: This study was conducted to evaluate the prognostic significance of p53 abnormalities in primary, resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Methodologic validation of immunohistologic detection of p53 abnormalities in routine pathology sections was assessed using 31 lung cancer specimens for which p53 gene status was known from our previous molecular biologic studies. Applying the optimized cutoff value, we evaluated the prognostic significance of p53 abnormalities in an independent cohort of 208 NSCLC patients with complete follow-up data, whose resections were consecutively performed between January 1984 and December 1988. RESULTS: Immunohistologic detection of p53 abnormalities appeared to be reliable and showed approximately 90% concordance with the p53 gene status. Using the selected cutoff value of 10%, 46% of 208 NSCLCs showed p53 abnormalities. There was no relationship between p53 abnormalities and clinical outcome in the entire cohort, which represented all histologic subtypes of NSCLC (P = .58). Based on the reasoning that the influence of p53 abnormalities may have been obscured by distinct biologic roles depending on histologic subtypes, we also separately analyzed subsets of patients with adenocarcinomas (n = 100) and with squamous cell carcinomas (n = 88) and found that it may be a useful prognosticator only in adenocarcinoma patients (P = .04). CONCLUSION: p53 abnormalities are not a significant prognostic factor in primary, resected NSCLC when all histologic subtypes are combined, but may be a useful prognosticator for adenocarcinomas. Additional studies are warranted for further evaluation, specifically of adenocarcinomas. PMID- 8636764 TI - Phase II study of topotecan in patients with advanced non-small-cell lung cancer previously untreated with chemotherapy. AB - PURPOSE: This study was designed to assess the anti-tumor activity of topotecan (TPT) in patients with advanced non-small-cell lung cancer (NSCLC) previously untreated with chemotherapy. PATIENTS AND METHODS: Patients with stage IIIB or IV NSCLC with measurable disease in nonradiated fields were eligible. Other eligibility criteria were Zubrod performance status (PS) < or = 2 and adequate renal and liver function. TPT was administered at a dose of 1.5 mg/m2/d for 5 days over 30 minutes every 21 days. Of 48 registered patients, 40 were fully assessable. Nineteen patients had adenocarcinoma (AD), 14 squamous carcinoma (SCC), and seven poorly differentiated carcinoma. RESULTS: Six patients (15%) achieved a partial remission (PR) (durations: 8, 14, 18, 28, 56, and 61 weeks) and four patients a minor response; 10 patients had stable disease and 20 patients progressive disease. The PR rate was 36% (five of 14 patients) in patients with SCC versus 4% (one of 26 patients) in those with other histologies (P = .014). The overall median survival time was 38 weeks and 30% of patients were alive at 1 year. Grade 3 to 4 granulocytopenia and thrombocytopenia occurred after 76% and 10% of courses administered, respectively. No grade 3 to 4 nonhematologic toxicities were observed. Grade 1 or 2 nonhematologic toxicities consisted of nausea (46% and 5%), vomiting (31% and 7%), and fatigue (53% and 16%). CONCLUSION: TPT at the dose and schedule used has moderate antitumor activity in NSCLC; its activity is mostly limited to patients with SCC. TPT is well tolerated, with myelosuppression of short duration being the most common and limiting toxicity. PMID- 8636765 TI - Phase II trial of fludarabine monophosphate as first-line treatment in patients with advanced follicular lymphoma: a multicenter study by the Groupe d'Etude des Lymphomes de l'Adulte. AB - PURPOSE: Fludarabine monophosphate (FAMP) is a major drug in the treatment of chronic lymphocytic leukemia and showed efficacy in selected groups of patients with low-grade lymphomas, most of them pretreated. The aim of this trial was to assess the efficacy and the toxicity of FAMP in untreated patients with follicular lymphoma. PATIENTS AND METHODS: Fifty-four untreated patients with advanced follicular lymphoma were treated with intravenous (i.v.) fludarabine at a dose of 25 mg/m2/d during 5 days every 4 weeks, to a maximum of nine cycles. RESULTS: The toxicity of the drug was mild, mainly granulocytic. Granulocytopenia > or = 3 (World Health Organization [WHO]) was observed during 48 of 328 cycles (14.6%) and in 22 of 53 (41%) patients assessable for toxicity. Fludarabine had to be stopped prematurely because of toxicity in nine patients: marrow toxicity in five, peripheral neuropathy in two, and interstitial pneumonitis and hepatitis in one patient each. Among 49 patients assessable for response, the overall response rate was 65% and the complete response (CR) rate 37%. The median progression-free survival interval for all patients was 13.6 months. CONCLUSION: These results confirm that fludarabine is active when used as first-line treatment in patients with follicular lymphoma and has a low toxicity rate. It may be used as single treatment in elderly patients. Associations of fludarabine with other drugs active against follicular lymphoma need to be determined. PMID- 8636766 TI - Phase I/II trial of PIXY321 to enhance engraftment following autologous bone marrow transplantation for lymphoid malignancy. AB - PURPOSE: A phase I/II study of PIXY321 following high-dose therapy (HDT) and autologous bone marrow transplantation (ABMT) was conducted to evaluate the safety and clinical potential of this agent. PATIENTS AND METHODS: Fifty patients with Hodgkin's disease or non-Hodgkin's lymphoma (NHL) undergoing HDT and ABMT received PIXY321 post-ABMT in doses that ranged from 50 to 1,000 micrograms/m2/d either as intravenous (i.v.) or subcutaneous (SC) dosing until engraftment was reached. RESULTS: If all doses are considered together, the median time to reach an absolute neutrophil count (ANC) > or = 500/microL was 18 days and the median time to platelet transfusion independence was 21 days. At the estimated optimum dose of 750 micrograms/m2/d by SC injection once daily, the median time to reach an ANC > or = 500/microL was 15 days and the median time to platelet transfusion independence was 16 days. Historical control patients who received granulocyte macrophage colony-stimulating factor (GM-CSF) had a median time to an ANC > or = 500/microL of 19 days and a median time to platelet independence of 26 days. CONCLUSION: The administration of PIXY321 post-ABMT was generally well tolerated and resulted in prompt engraftment in the majority of patients who underwent HDT and ABMT for lymphoid malignancies. The optimum dose and route of administration of PIXY321 suggested by this trial was 750 micrograms/m2/d by once-daily SC injection. Compared with historical control patients who received 2-hour i.v. GM CSF, patients who received PIXY321 at 750 micrograms/m2/d by SC injection once daily had an improvement in the median days to neutrophil and platelet engraftment by 4 and 10 days, respectively. PMID- 8636767 TI - Vinblastine, bleomycin, and methotrexate chemotherapy plus extended-field radiotherapy in early, favorably presenting, clinically staged Hodgkin's patients: the Gruppo Italiano per lo Studio dei Linfomi Experience. AB - PURPOSE: To ascertain whether vinblastine, bleomycin, and methotrexate (VBM) (CT) combined with extended-field radiotherapy (EF RT) is effective enough to spare laparotomy in early, favorably presenting Hodgkin's disease (HD) patients. PATIENTS AND METHODS: Fifty patients with clinical stage IA or IIA HD with favorable histology and no bulky masses entered a prospective multicenter study started in January 1988. The median follow-up time was 38 months. RESULTS: All patients achieved a complete remission (CR). Five relapsed after 3 to 40 months and underwent successful salvage therapy. The actuarial remission rate was 0.89% at 3 years and 0.82% at 5 years. Two patients died in CR: one of severe pulmonary toxicity, the other of a second neoplasia (adenocarcinoma of the lung), 2 and 43 months after the end of therapy, respectively. The hematologic toxicity recorded during VBM CT was mild on the whole. Major toxicity was represented by pulmonary side effects and neurologic symptoms. Multiple regression analysis demonstrated that pulmonary toxicity was significantly related only to the amount of RT delivered to the mediastinum and not to the relative dose of bleomycin, to the dose-intensities of the three drugs in the regimen, or to patient age or sex. The same statistical technique showed that the only clinical factor related to grade of neurotoxicity was vinblastine dosage. CONCLUSION: VBM CT combined with EF RT is an effective treatment for early, clinically staged, favorable HD patients. However, the toxicity of this combination suggests that certain modifications should be evaluated. PMID- 8636768 TI - High-dose chemotherapy followed by autologous bone marrow transplantation versus dexamethasone, cisplatin, and cytarabine in aggressive non-Hodgkin's lymphoma with partial response to front-line chemotherapy: a prospective randomized italian multicenter study. AB - PURPOSE: To evaluate, in a prospective multicentric study, the efficacy of a conventional salvage chemotherapy (dexamethasone, cisplatin, and cytarabine [DHAP]) versus high-dose chemotherapy (carmustine, etoposide, cytarabine, and cyclophosphamide [BEAC]) followed by autologous bone marrow transplantation (ABMT) in patients with aggressive non-Hodgkin's lymphoma (NHL) in clinical partial response (PR) after two thirds of a conventional front-line therapy. PATIENTS AND METHODS: From August 1988 to August 1991, 286 patients with aggressive NHL were randomized in seven Italian institutions to receive fluorouracil, methotrexate, cytarabine, cyclophosphamide, doxorubicin, vincristine, and prednisone (F-MACHOP) or methotrexate with leucovorin, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (MACOP-B) as front-line therapy. Of the 286 patients enrolled onto the trial, 77 (27%) were considered in PR after two thirds of the front-line therapy, and 49 of 77 (64%) were randomized: 27 to receive DHAP chemotherapy and 22 to receive BEAC followed by ABMT. RESULTS: The response after second-line treatment was as follows: in the DHAP group, four patients (15%) achieved a complete remission (CR), 12 (44%) remained in stable PR, and 11 (41%) showed progressive disease; in the ABMT group, three patients (14%) obtained a CR, 18 (82%) obtained a stable PR, and one (4%) progressed, with an overall response (CR + stable PR) of 59% and 96% (P < .001) in the DHAP and ABMT groups, respectively. The overall survival was 59% versus 73% and the progression-free survival (PFS) was 52% versus 73% in the DHAP and ABMT groups, respectively (P, not significant). The toxicity was mild, particularly in the ABMT group, and no treatment-related deaths occurred in either group. CONCLUSION: Because of the small number of patients randomized, we were unable to determine whether ABMT or a standard salvage regimen (DHAP) is superior for PR patients. However, we confirmed that myeloablative treatment is a safe and well-tolerated procedure in this category of patients and this may enable us to evaluate its role as part of a front-line treatment in poor-risk NHL patients. PMID- 8636769 TI - No effect of 96-hour paclitaxel infusion in patients with relapsed non-Hodgkin's lymphoma refractory to a 3-hour infusion schedule. AB - PURPOSE: Preclinical data suggest that the efficacy of paclitaxel (Taxol; Bristol Myers Squibb Co, Princeton, NJ) is schedule-dependent. Schedule dependency is currently under investigation in ongoing randomized trials. PATIENTS AND METHODS: Twelve patients with relapsed non-Hodgkin's lymphoma (NHL) refractory to a 3-hour infusion of 200 mg/m2 Taxol were crossed over to receive a 96-hour infusion of 140 mg/m2 Taxol every 3 weeks in an outpatient setting. Premedication with corticosteroids and antihistamines was not used. Patients who did not achieve at least a partial remission (PR) after two courses or whose disease progressed after one course were removed from the study. RESULTS: All 12 patients were assessable for response. Eleven patients received at least two courses and one patient received one course of 96-hour Taxol infusion. None of the 12 patients crossed over to receive 96-hour Taxol infusion achieved a PR or complete response (CR). Eight patients (67%) developed progressive lymphoma, three (25%) had stable disease, and only one (8%) had a minor response. No major hypersensitivity reactions or life-threatening toxicities were observed. CONCLUSION: Ninety-six hour Taxol infusion does not produce significant responses in patients with NHL refractory to 3-hour Taxol infusion. Until the results of an ongoing multicenter trial comparing a 3- with a 96-hour infusion are published, the use of 96-hour Taxol infusion in NHL patients should be restricted to investigational programs. Because 48- to 72-hour infusions can produce higher plasma concentrations of Taxol than a 96-hour infusion, these schedules should be investigated to determine if they can induce better clinical responses. PMID- 8636770 TI - Treatment of primary cutaneous B-cell lymphomas of follicle center cell origin: a clinical follow-up study of 55 patients treated with radiotherapy or polychemotherapy. AB - PURPOSE: Primary cutaneous follicle center cell lymphomas (PCFCCL) are a distinct group of cutaneous B-cell lymphomas with a favorable prognosis after radiotherapy (RT) or polychemotherapy (PCT). In the literature, conflicting data exist regarding the efficacy and the relapse rate of both treatment modalities. In the present study, treatment results and follow-up data of a large group of PCFCCL are evaluated. PATIENTS AND METHODS: Fifty-five patients with a PCFCCL who presented with skin lesions on either the head (n = 12), the trunk (n = 35), or lower legs (n = 8), and who were initially treated with RT (40 cases) or PCT (15 cases) were studied. RESULTS: RT resulted in a complete remission in all 40 cases. Eight cases relapsed and three of these patients died as a result of their lymphoma. The estimated 5-year survival was 89%. Four of eight relapses and all three lymphoma-related deaths occurred in the group of patients presenting with tumor(s) on the lower legs. Treatment with cyclophosphamide, doxorubicin vincristine, and prednisone (CHOP) or cyclophosphomide, vincristine, and prednisone (COP) resulted in a complete remission in 14 of 15 cases. All four cases treated with COP relapsed, whereas only two of 11 patients treated with CHOP had a relapse. The estimated 5-year survival rate of the PCT group was 93%. CONCLUSION: Both RT and CHOP PCT are highly effective modes of treatment for PCFCCL. In localized PCFCCL, RT is the treatment of choice. In patients with multiple tumors involving anatomic nonrelated parts of the skin, CHOP rather than COP PCT is the preferred mode of treatment. PCFCCL on the lower legs, a subgroup that characteristically occur in elderly patients, have a higher relapse rate and a less favorable prognosis than PCFCCL presenting on the head or trunk. PMID- 8636771 TI - Preirradiation chemotherapy with cyclophosphamide, doxorubicin, vincristine, and dexamethasone for primary CNS lymphomas: initial report of radiation therapy oncology group protocol 88-06. AB - PURPOSE: This study was a prospective phase I/II trial performed by the Radiation Therapy Oncology Group (RTOG) to test the tolerance and efficacy of preirradiation cyclophosphamide, doxorubicin, vincristine, and dexamethasone (CHOD) chemotherapy followed by large-volume, high-dose brain radiation therapy (RT) for patients with primary CNS lymphoma (PCNSL). PATIENTS AND METHODS: Fifty four (52 assessable) human immunodeficiency virus (HIV)-negative patients with PCNSL were entered on study and received two (n = 20) or three (n = 32) cycles of CHOD (six patients with positive CSF cytology received intrathecal methotrexate in addition to CHOD). Whole-brain RT to 41.4 Gy and tumor boost to 18 Gy (total dose, 59.4 Gy) followed chemotherapy. RESULTS: As of July 1994, with a minimum potential follow-up time of 20 months, 12 of 52 assessable patients remain alive without evidence of progression. The median survival time for the entire group is 16.1 months, with a 2-year survival rate of 42%. By univariate analysis, patient age was found to be a significant prognostic factor with respect to survival (P = .005) in favor of age less than 60 years. Karnofsky performance status (KPS) was of borderline significance (P = .057). Survival for patients treated on RTOG 88 06 was compared with that of patients treated on RTOG 83-15, which tested RT alone. No difference in overall survival was found (P = .53). Grade 4 neutropenia developed in 29 of 51 patients during chemotherapy. There were two deaths during chemotherapy: one as a result of sepsis and one of a pulmonary embolus. The worst toxicity during RT was < or = grade 2 in 50 of 52 patients. CONCLUSION: Preirradiation CHOD chemotherapy does not significantly improve survival over RT alone for patients with PCNSL. Age remains a powerful prognostic factor independent of therapy and must be considered in testing alternative combined approaches. PMID- 8636772 TI - Leukemia following low-dose total body irradiation and chemotherapy for non Hodgkin's lymphoma. AB - PURPOSE: Low-dose total body irradiation (TBI) is used to treat non-Hodgkin's lymphoma (NHL) and several other malignancies. Large volumes of bone marrow and other tissue receive considerable exposure, but few studies have quantified late carcinogenic sequelae. PATIENTS AND METHODS: A cohort of 61 2-year survivors of NHL treated initially with low-dose TBI was monitored for second cancer occurrence. Data on primary and subsequent therapy were collected, and cumulative dose of radiation to active bone marrow (ABM) (median, 5.2 Gy) was reconstructed. RESULTS: Thirteen second primary cancers occurred. Four patients developed acute nonlymphocytic leukemia (ANLL), which represents a relative risk (RR) of 117 (95% confidence interval [CI], 31.5 to 300) compared with population rates. A fifth patient was diagnosed with myelodysplastic syndrome (MDS). All five patients with secondary hematologic malignancies subsequently received salvage treatment, with either alkylating agents alone (n = 1) or combined modality therapy (CMT) (n = 4). Overall, eight solid tumors were observed (RR = 2.0; 95% CI, 0.9 to 4.0). The 15-year cumulative risks of all second cancers and secondary ANLL were 37% and 17%, respectively. CONCLUSIONS: Despite the small number of subjects, a considerable risk of leukemia was observed among patients treated with low-dose TBI in combination with CMT including alkylating agents. Based on these results, approximately eight to nine excess ANLLs might be expected to occur among 100 NHL patients treated with low-dose TBI and salvage treatment and followed-up for 15 years. PMID- 8636773 TI - Bone marrow transplants from HLA-identical siblings in advanced Hodgkin's disease. AB - PURPOSE: To determine the outcome of HLA-identical sibling bone marrow transplants in advanced Hodgkin's disease. PATIENTS AND METHODS: We reviewed the data on 100 consecutive patients with Hodgkin's disease who received HLA identical sibling bone marrow transplants between April 1, 1982 and August 12, 1992, reported to the International Bone Marrow Transplant Registry (IBMTR). The median interval from diagnosis to transplant was 2.5 years (range, < 1 to 14). All had advanced disease. Eighty-nine of 100 patients were not in remission at the time of transplant. Fifty had pretransplant Karnofsky scores less than 90% and 27 had active infection in the week before transplant. Patients received a variety of conditioning regimens; 45 received total-body radiation. RESULTS: The 100-day probability of acute graft-versus-host disease (GVHD) was 35% (95% confidence interval [CI], 26% to 46%); the 3-year probability of chronic GVHD was 45% (95% CI, 31% to 59%). The 3-year probability of relapse was 65% (95% CI, 50% to 78%). The 3-year probability of survival was 21% (95% CI, 14% to 30%). The 3 year disease-free survival rate was 15% (95% CI, 9% to 24%). CONCLUSION: HLA identical sibling bone marrow transplants have a limited role in advanced Hodgkin's disease. PMID- 8636774 TI - Renal toxicity after allogeneic bone marrow transplantation: the combined effects of total-body irradiation and graft-versus-host disease. AB - PURPOSE: To evaluate retrospectively the cumulative risk probability and factors correlated with renal dysfunction after allogeneic bone marrow transplantation (BMT). PATIENTS AND METHODS: From October 1984 to July 1994, 84 patients with malignant hematopoietic diseases received allogeneic BMT after conditioning with high-dose chemotherapy and total-body irradiation (TBI). Seventy-nine patients with normal renal function before conditioning are included in this study. Conditioning included high-dose cyclophosphamide without (n = 46) or with (n = 33) other agents (daunorubicin, busulfan, cytarabine, and thiotepa) followed by TBI. The TBI dose prescribed to the center of the abdomen was 10 Gy for 24 patients, 12 Gy for 32, and 13.5 Gy for 23. In vitro T-cell depletion was undertaken in 48 cases. The post-BMT nephrotoxicity of aminoglycosides, vancomycin, amphotericin, and cyclosporine was assessed. Time to renal dysfunction was defined as the time to a persistent increase of serum creatinine (SCr) level greater than 110 mumol/L. The potential influence of sex, age, diagnosis, chimerism, and graft-versus-host disease (GvHD) on renal dysfunction was also assessed. RESULTS: The 18-month probability of renal dysfunction-free survival (RDFS) for the whole group was 77%. Only TBI dose and presence of GvHD were significantly correlated with renal dysfunction by multivariate analysis. The 18-month probabilities of RDFS were 95%, 74%, and 55% for the patients conditioned with 10, 12, and 13.5 Gy, respectively. The 18-month RDFS probabilities were 88% and 61% for patients without and with GvHD, respectively. Combining both variables, we have defined two risk categories: low-risk (ie, 10 Gy TBI with/without GvHD and 12 Gy TBI without GvHD) and high-risk (ie, 12 Gy TBI with GvHD and 13.5 Gy TBI with/without GvHD). The predicted 18-month RDFS rates were 93% and 52% for the low- and high-risk groups, respectively. CONCLUSION: Renal dysfunction after allogeneic BMT is strongly related to the delivered TBI dose (and dose per fraction) and to the presence of GvHD. Renal shielding should be recommended if a TBI dose greater than 12 Gy (fractionated twice daily over 3 days) is to be prescribed. Furthermore, in those cases with a high risk of developing GvHD (eg, unrelated allogeneic BMT, absence of T-cell depletion), these data suggest that kidney doses greater than 10 Gy should be avoided. PMID- 8636776 TI - Significance of cytogenetic findings for the clinical outcome in patients with T cell lymphoma of angioimmunoblastic lymphadenopathy type. AB - PURPOSE: The aim of this study was to evaluate the significance of cytogenetic findings for the clinical outcome of patients with Angioimmunoblastic Lymphadenopathy (AILD)-Type T-cell lymphoma. MATERIALS AND METHODS: In a retrospective analysis, the cytogenetic findings of 50 patients with AILD-type T cell lymphoma were correlated with the frequency of spontaneous and therapy induced remissions and with survival using the statistical methods of Kaplan and Meier and the model of Cox for multivariate analysis. Treatment was not uniform because the patients were treated in different hospitals during a period of 8 years and because a standard therapy has not yet been established. RESULTS: The following cytogenetic findings were associated with a significantly lower incidence of therapy-induced remissions and a significantly shorter survival duration: presence of aberrant metaphases in unstimulated cultures (P = .04 for both parameters); clones with an additional X chromosome (P = .0001 and P = .03, respectively); structural aberrations of the short arm of chromosome 1, preferentially involving 1p31-32 (P < .001 and P = .04, respectively); and complex aberrant clones with more than four aberrations (P = .0003 and P = .005, respectively). Multivariate analysis showed that these cytogenetic findings had a significant influence on survival, but therapy modalities did not. Only the presence of complex aberrant clones was an independent prognostic factor. Trisomy 3 had no effect on survival, but patients without trisomy 5 (P = .08) tended to live longer. CONCLUSION: This is the first study that seems to indicate that cytogenetic findings have prognostic significance in AILD-type T-cell lymphoma. These results must be proven in prospective studies of homogeneously treated patients. PMID- 8636775 TI - Survival benefit from high-dose therapy with autologous blood progenitor-cell transplantation in poor-prognosis non-Hodgkin's lymphoma. AB - PURPOSE: To compare standard and intensive treatment strategies for patients with high-grade non-Hodgkin's lymphoma (NHL) of poor prognosis, defined by the international prognostic index. PATIENTS AND METHODS: Thirty-four patients received standard chemotherapy with 11 weeks of doxorubicin, cyclophosphamide, vincristine, bleomycin, etoposide, prednisolone, and methotrexate (VAPEC-B), and 33 received intensive treatment with 7 weeks of VAPEC-B, three cycles of ifosfamide/cytarabine, then high-dose busulfan/cyclophosphamide followed by autologous blood progenitor-cell (BPC) transplantation. RESULTS: Twelve of 33 patients in the intensive group and 26 of 34 patients in the standard group have died. The median follow-up time for the surviving patients is 31 months and 68 months, respectively. At 2 years, the actuarial estimates of event-free survival (EFS) were 61% versus 35% (P = .01) and of overall survival, 64% versus 35% (P = .01). A significant reduction in the event rate (progression or death) was maintained after adjustment for age and the number of risk factors. The estimated risk of experiencing an event was 0.37 (95% confidence interval [CI], 0.16 to 0.84) in the intensive group compared with the standard group. CONCLUSION: Patients with poor prognostic features who received high-dose therapy and BPC rescue had a superior EFS. The survival differences observed in this study justify a formal comparison in a randomized study. PMID- 8636777 TI - Phase I study of weekly outpatient paclitaxel and concurrent cranial irradiation in adults with astrocytomas. AB - PURPOSE: Astrocytomas are extremely resistant to currently available treatments. Cranial irradiation is a mainstay of frontline therapy, but tumor recurrence is nearly universal. Paclitaxel has shown antitumor efficacy against astrocytoma cell lines, and is a potent radiosensitizer. For these reasons, we conducted a phase I study of weekly paclitaxel and concurrent cranial irradiation in patients with newly diagnosed astrocytomas. PATIENTS AND METHODS: Patients with astrocytomas were eligible for this study following initial surgery if they had a Karnofsky performance score (KPS) > or = 60%; normal hematologic, liver, and renal function; and could give informed consent. Beginning on day 1 of treatment, patients received paclitaxel by 3-hour infusion once weekly for 6 weeks, concurrent with standard cranial irradiation. Pharmacokinetic studies were performed on 10 patients. RESULTS: Sixty patients were enrolled; 56 were fully assessable. Forty-eight had glioblastomas (GBMs), 10 anaplastic astrocytomas (AAs), and two astrocytomas. Age ranged from 21 to 81 years (median, 55); KPS ranged from 60 to 100 (median, 70). The paclitaxel dose was escalated from 20 mg/m2 to 275 mg/m2. No clinically significant anemia or thrombocytopenia occurred. Only one patient (175 mg/m2) became neutropenic. Sensory neuropathy was dose-limiting. The maximum tolerated dose (MTD) was 250 mg/m2. Paclitaxel pharmacokinetic profiles in study patients were identical to those of previously reported patients with other solid tumors. CONCLUSION: The MTD of paclitaxel administered weekly for 6 weeks by 3-hour infusion is 250 mg/m2. Since patients with brain tumors often have preexisting neurologic deficits, we suggest 225 mg/m2 as the optimum dose for phase II trials in this group of patients. PMID- 8636778 TI - Phase I study of etoposide with SDZ PSC 833 as a modulator of multidrug resistance in patients with cancer. AB - PURPOSE: To determine the maximum-tolerated dose (MTD) and toxicity of PSC 833 infusion administered with etoposide for 5 days in patients with cancer, and to determine the effect of PSC 833 on etoposide pharmacokinetics. PATIENTS AND METHODS: Thirty-five patients were entered onto the study, one of whom was ineligible. Etoposide was delivered from day 1 as a 2-hour infusion over 5 consecutive days at a dose of 75 to 100 mg/m2/d. PSC 833 was administered from day 2 as a 2-hour loading dose and as a 5-day continuous infusion. Doses were escalated from 1 to 2 mg/kg (loading dose) and 1 to 15 mg/kg/d (continuous infusion). RESULTS: Thirty-four patients were treated with 53 cycles of PSC 833 and etoposide. Steady-state blood PSC 833 levels more than 1,000 ng/mL were achieved in all patients treated at PSC 833 doses > or = 6.6 mg/kg/d by continuous infusion. Myelosuppression was the most common toxicity. The major dose-related toxicity of PSC 833 was reversible hyperbilirubinemia, which occurred in 83% of cycles. The dose-limiting toxicity of PSC 833 was severe ataxia, which occurred in two of nine patients treated at 12 mg/kg/d and in both of the single patients treated at 13.5 and 15 mg/kg/d. PSC 833 concentrations more than 2,000 ng/mL resulted in an increase in etoposide area under the curve (AUC) of 89%, a decrease in etoposide clearance (Cl) of 45%, a decrease in volume of steady-state distribution (Vss) of 41%, and an insignificant increase in alpha half-life (t 1/2 alpha) and significant increase of beta half-life (t 1/2 beta) of 19% and 77%, respectively. CONCLUSION: PSC 833 can be administered in combination with etoposide with acceptable toxicity. The recommended continuous infusion dose of PSC 833 for this schedule is 10 mg/kg/d over 5 days. PSC 833 results in an increase in etoposide exposure and etoposide doses should be reduced in patients receiving PSC 833. PMID- 8636779 TI - Randomized placebo-controlled trial of granulocyte-macrophage colony-stimulating factor in patients with chemotherapy-related febrile neutropenia. AB - PURPOSE: To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) used in addition to standard inpatient antibiotic therapy shortens the period of hospitalization due to chemotherapy-induced neutropenic fever. PATIENTS AND METHODS: One hundred thirty-four patients with a hematologic (n = 47) or solid tumor (n = 87) who had severe neutropenia (< 0.5 x 10(9)/L) and fever (> 38.5 degrees C once or > 38 degrees C twice over a 12-hour observation period) were randomly assigned to receive GM-CSF 5 micrograms/kg/d (n = 65) or placebo (n = 69) in conjunction with broad-spectrum antibiotics for a minimum of 4 days and a maximum of 14 days. GM-CSF/placebo and antibiotics were stopped if the neutrophil count was greater than 1.0 x 10(9)/L and temperature less than 37.5 degrees C during 2 consecutive days, or for a leukocyte count > or = 10 x 10(9)/L, both followed by a 24-hour observation period (hospitalization period). RESULTS: Compared with placebo, GM-CSF enhanced neutrophil recovery. Median neutrophil counts at day 4 were 2.5 x 10(9)/L (range, 0 to 25) in the GM-CSF arm and 1.3 x 10(9)/L (range, 0 to 9) in the placebo arm (P < .001). No significant difference was observed with regard to median number of days with less than 1.0 x 10(9)/L neutrophils (4 v 4) or days of fever (3 v 3). The median number of days patients were hospitalized while on study was comparable in the GM-CSF and placebo groups at 6 (range, 3 to 14) versus 7 (range, 4 to 14), respectively, according to an intention-to-treat analysis (P = .27). Quality-of-life scores in 90 patients demonstrated significant differences in favor of the placebo group. Hospital costs were significantly higher for GM-CSF-treated patients if GM-CSF was included in the price (median costs, $4,140 [US] for GM-CSF v $590 for placebo; P < .05). CONCLUSION: These results indicate that GM-CSF does not affect the number of days for resolution of fever or the hospitalization period for this patient group, although a significant effect of GM-CSF was observed on neutrophil recovery. PMID- 8636780 TI - Comparative effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor after high-dose cyclophosphamide cancer therapy. AB - PURPOSE: We compared hematologic and clinical effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G CSF) after treatment with high-dose cyclophosphamide (HD-CTX, 7 g/m2), given as the first phase of a high-dose sequential chemotherapy program that includes a myeloablative therapy with mobilized progenitor cell autografting. PATIENTS AND METHODS: Forty-nine consecutive patients with non-Hodgkin's lymphoma, Hodgkin's disease, or poor-prognosis breast cancer received GM-CSF (n = 27) or G-CSF (n = 22) after HD-CTX in two consecutive, nonrandomized studies. Cytokines were administered in continuous intravenous (i.v.) infusion for 14 to 15 days at a median dose of 5.5 and 10 micrograms/kg/d, respectively, starting 24 hours after HD-CTX. RESULTS: Neutrophil recovery was faster with G-CSF administration (11.5 v 13.2 days; P = .01), whereas platelet counts recovered more rapidly with GM-CSF (13.7 v 16.6 days; P = .01). Prophylactic platelet transfusions were administered more frequently to patients treated with G-CSF than with GM-CSF (66% v 22% of the patients; P = .02). No clinically significant difference was observed between the two groups concerning days of absolute neutropenia or neutropenic fever. Both cytokines reduced the time to eligibility for subsequent chemotherapy administration compared with historical controls not given cytokine (14 to 16 v 20 days). Both cytokines increased circulation of hematopoietic progenitors. Most side effects were World Health Organization (WHO) median grade 1 to 2, were more frequent during GM-CSF than during G-CSF treatment, and were reversible by simple supportive measures and/or by dose reduction or suspension of the cytokine. Permanent suspension of cytokine administration was never required in either group. CONCLUSION: GM-CSF or G-CSF administration after HD-CTX reduces hematologic toxicity of high-dose chemotherapy and induces circulation of large amounts of hematopoietic progenitors suitable for autografting in cancer patients. PMID- 8636781 TI - Early lymphopenia after cytotoxic chemotherapy as a risk factor for febrile neutropenia. AB - PURPOSE: Febrile grade four (ie, < or = 500/microL) neutropenia (FN) is a frequent life-threatening complication of cancer chemotherapy. Although its incidence correlates to the dose of chemotherapy, FN may occur after almost any cytotoxic regimen. At present, there is no predictive method to identify patients who will experience FN. PATIENTS AND METHODS: Univariate and multivariate analyses of risk factors for FN were performed on a retrospective cohort of 112 consecutive patients treated with various chemotherapy regimens. Two independent risk factors were identified by the logistic regression and used to create a risk model for FN. The validity of the model was tested in three distinct groups of patients: two prospective groups of patients treated in two institutions (Centre Leon Berard [CLB] and Institut G. Roussy [IGR]) and the group of patients with intermediate- or high-grade non-Hodgkin's lymphoma (NHL) treated with the doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisone (ACVBP) regimen between 1988 and 1992 at CLB. RESULTS: Within the retrospective group, 23 of 47 (49%) patients with lymphocyte counts < or = 700/microL at day 5 after chemotherapy experienced FN compared with seven of 65 (11%) of other patients (P = .00002). The type of chemotherapy (high dose v others) was also significantly correlated to FN (48% v 11%, P = .0003). Age, performance status, the number of previous chemotherapy cycles, or polymorphonuclear leukocyte (PMN) counts, were not significantly correlated to the incidence of FN in univariate analyses. Two independent risk factors were identified in the logistic regression: day 5 lymphocyte counts (beta = 1.97 +/- 0.53) and the type of chemotherapy regimen (beta = 1.91 +/- 0.53). The calculated probability to experience FN in patients with none, one, and both of these risk factors was 4.3%, 24.0%, and 68.8%, respectively. The validity of this model was tested in the three groups of patients used as validation samples. The observed incidences of FN in the above defined risk subgroups were 3%, 19%, and 67%, respectively, within the CLB prospective series and 6%, 19%, and 75% within the IGR prospective series. In the ACVBP group, the incidence of FN was 33% and 72%, respectively, in patients from the intermediate- and high-risk groups. In the two prospective groups and in the ACVBP series, the observed numbers of FN in the different risk groups did not differ significantly from those calculated by the model (P = .89, P = .86, and P = .72 for these three groups, respectively). CONCLUSION: Day 5 lymphocyte counts < or = 700/microL and the type of chemotherapy regimen enable oncologists to define subgroups of patients treated with chemotherapy as those with a high intermediate, and low risk of FN. These criteria could be used to select subjects in whom prophylactic measures for FN, in particular hematopoietic growth factors, should be proposed. PMID- 8636783 TI - Inferior vena cava filters in cancer patients: indications and outcome. AB - PURPOSE: Our experience with inferior vena cava (IVC) filter placement to prevent pulmonary emboli (PE) in cancer patients with deep vein thromboses (DVT) was reviewed to identify indications, patient characteristics, complications, and long-term outcome. METHODS: Charts of 182 patients with cancer were retrospectively analyzed. All patients had received an IVC filter in our institution between January 1980 and April 1992. RESULTS: Of 182 patients, 103 were men and 79 were women. Median age was 59 years (range, 15 to 88). Eight patients (4%) had stage I disease, 22 patients (12%) stage II, 37 patients (20%) stage III, and 115 patients (63%) stage IV. A DVT was diagnosed in 97 patients (53%), a PE in 46 patients (25%), and a combination in 39 patients (21%). Indications for IVC filter placement were DVT or PE in the presence of contraindications to anticoagulation therapy (perioperative, n = 58; CNS metastases, n = 20; thrombocytopenia, n = 7; bleeding, n = 61; others, n = 24; total, N = 170) or anticoagulation failure (recurrent PE, n = 6; recurrent DVT; n = 6; total N = 12). Filter placement complications (n = 6, 3%) included malposition (n = 3), migration (n = 1), arrhythmia (n = 1), and wound infection (n = 1), but no deaths. After filter placement, four patients developed a recurrent PE, and 11 patients developed a recurrent DVT. No significant postthrombotic complications were observed. CONCLUSION: IVC filter placement patients with advanced cancer and thrombotic complications is safe, well tolerated, and can offer effective therapy/prophylaxis with a low incidence of treatment failure. PMID- 8636782 TI - Analysis of cumulative probabilities shows that the efficacy of 5HT3 antagonist prophylaxis is not maintained. AB - PURPOSE: Several investigators have reported that the efficacy of 5HT3 receptor antagonists is maintained over repeated cycles of chemotherapy. These investigators presented conditional probabilities of protection. Because conditional analyses by definition only include patients with protection in previous cycles, the results are flattered. PATIENTS AND METHODS: We applied a novel statistical approach to investigate whether the efficacy of the 5HT3 receptor antagonist ICS 205-930 (tropisetron) is maintained over repeated cycles of weekly high-dose cisplatin. Overall protection was determined based on cumulative probabilities with the Kaplan-Meier method. Complete protection was calculated with a three state model for transitional probabilities. Eighty-three patients were studied. RESULTS: Over six consecutive cycles, protection against both acute and delayed emesis decreased significantly. The initial complete and overall protection rates against acute emesis of 71% and 95%, respectively, decreased to 43% and 72% in the sixth cycle of chemotherapy. Similarly, the protection rates of 31% and 68% against delayed emesis decreased to 6% and 40%, respectively. CONCLUSION: We conclude that overall and complete long-term protection is more accurately measured by cumulative probabilities than with a method that is based on conditional probabilities. Our statistical approach shows that the efficacy of 5HT3 antagonists is not maintained. PMID- 8636784 TI - Extensive-disease small-cell lung cancer: the thrill of victory; the agony of defeat. PMID- 8636785 TI - Funding for clinical (patient-oriented) oncology research: current status and recommendations for improvement. American Society of Clinical Oncology. PMID- 8636786 TI - Outcomes of cancer treatment for technology assessment and cancer treatment guidelines. American Society of Clinical Oncology. AB - In 1993, the Health Services Research Committee of the American Society of Clinical Oncology (ASCO) charged an Outcomes Working Group with defining the outcomes of adult and pediatric cancer treatment to be used for technology assessment and development of cancer treatment guidelines. The Working Group defined by consensus outcomes for technology assessment and guideline development, focusing on cancer treatments. The Working Group considered a variety of perspectives on outcomes, including those of patients, physicians, clinical investigators, ASCO, and policy makers. Because ASCO's guidelines will define what constitutes the best treatment and not whether that treatment should be paid for, the Working Group gave higher priority to the clinical and clinical research perspectives than to the health policy perspective. Survival is the most important outcome of cancer treatment. An improvement in at least disease-free survival is a prerequisite for recommending adjuvant therapy. In the case of metastatic cancer, treatment can be recommended even without an improvement in survival, if it improves quality of life. Quality of life includes global quality of life, as well as its physical, psychologic, and social dimensions. To be an outcome of cancer treatment, quality-of-life measures must be sensitive to clinically meaningful changes produced by treatment; evaluations must control for placebo effects and determinants of quality of life not related to cancer or its treatment. Toxicity, both short and long term, is vitally important, with the latter being particularly critical in children. The value of cancer outcomes like tumor response (eg, complete or partial response) and biomarkers (eg, CA-125) for technology assessment and guideline development depends on their ability to predict patient outcomes (survival and quality of life) or to influence decisions about treatment. Complete response is an important outcome when it predicts survival. Progression is important because it signals the need to change or stop treatment. Cost-effectiveness is an especially important outcome to consider when the benefits of treatment are modest or the costs are high. Patient outcomes (eg, survival and quality of life) should receive higher priority than cancer outcomes (eg, response rate), but both types of outcomes are important in technology assessment and guideline development. Multiple outcomes should be considered because no single outcome adequately describes the results of cancer treatment. In general, there is no minimum benefit above which treatments are justified; rather, benefits should be balanced against toxicity and cost. PMID- 8636787 TI - Defining new standards for adjuvant therapy of testis cancer: a reply. PMID- 8636788 TI - Uniform criteria for childhood acute lymphoblastic leukemia risk classification. PMID- 8636789 TI - DNA repair and the risk of secondary leukemias. PMID- 8636790 TI - Bone marrow biopsy for staging Hodgkin's lymphoma: the value of bilateral or unilateral trephine biopsy. PMID- 8636791 TI - Osteosarcoma: relationship of response to preoperative chemotherapy and type of surgery to local recurrence. PMID- 8636793 TI - High-dose chemotherapy for metastatic breast cancer. PMID- 8636792 TI - Ethical questions on the testicular seminoma study. PMID- 8636794 TI - Dose-intensive metastatic breast cancer chemotherapy. PMID- 8636795 TI - High-dose chemotherapy for breast cancer. PMID- 8636796 TI - Risk stratification for high-dose chemotherapy in metastatic breast cancer. PMID- 8636797 TI - Cost-effectiveness: let's get "real". PMID- 8636798 TI - Tumor necrosis factor: is it a breakthrough? PMID- 8636799 TI - Megestrol acetate-induced adrenal suppression. PMID- 8636800 TI - The Journal of Neuroradiology. PMID- 8636801 TI - [The anatomy and the MRI anatomy of the interhemispheric cerebral commissures]. AB - Correlation of myelin-stained or cryotomic sections of human brain with inversion recovery MR images can display the cerebral commissures as white-matter tracts (in hypersignal on MRI), crossing the mid-line. MRI shows routinely in three orthogonal planes a) the corpus callosum stretched above the supra-tentorial ventricles, it's four portions (rostrum, genu, body and splenium) and connections with the Deep Grey Nuclei b) the fornix, intralimbic commissure joining anteriorly the mammillary bodies (through it's columns) to the alveus posteriorly and inferiorly (via it's two crura), arcing around the thalamus and lying over the hippocampus and the dentate gyrus as shown on the frontal sections c) the anterior commissure, white-matter tract connecting the two temporal lobes. In axial view, the anterior commissure has the shape of bicycle handlebars, coursing posteriorly, inferiorly and laterally behind the head of the caudate nucleus and passes into the lateral nucleus of the globus pallidus into the inferior and middle temporal gyri. Because the anterior commissure is easily recognisable in all planes, it's appears to be a important landmark for identification of the lateral and medial nuclei of the globus pallidus on axial and sagittal planes d) at least, the posterior commissure, anterior margin of the pineal region, closely related to the superior colliculi, acquire a major importance in the AC-PC line delineation becoming a reference landmark for stereotatical procedures. PMID- 8636802 TI - [Magnetic resonance imaging and meningiomas of the posterior cerebral fossa. 31 cases]. AB - To assess the value of MRI for meningioma of the posterior cerebral fossa, in correlation with surgical and pathological findings, we retrospectively reviewed 31 cases. The patients (24 females and 6 males ranging in age from 25 to 79 years) were preoperatively studies on a 1,5 T MR imager (GEMS Signa) between july 1989 and november 1993. The protocol included: 1. MR scan with axial sections in T2-weighted spin-echo sequence (3 mm thickness), T1-weighted spin-echo sequence before and after gadolinium injection (3-5 mm thickness), coronal and sagittal T1 weighted sections performed after injection. 2. Surgery reports. 3. Histopathological reports; the predominant histological subtype of each tumor was graded according to the classification scheme of Russel and Rubinstein. We focused on five items: 1. The site of the dural attachment of the meningioma. 2. Tumoral extensions (to the tentorium, to the jugular foramen, to the internal auditory canal). 3. The meningioma signal in T1- and T2-weighted sequence using the same visual scoring system for grading signal intensities as Elster and al. 4. Secondary features (necrosis, cysts, calcifications) within the tumor. 5. Interface between meningioma and encephalic structures. Meningiomas arose from the posterior surface of the petrous bone in 74% of the cases and from the clivus in 9.6%. Meningiomas were bulky at the time of diagnosis as since tumoral arrow overtook 2 cm in 64.5% of the cases. Surgical approach was guided by an anatomo radiologic classification based on the exact site of tumoral dural attachment. This determination relied on: 1. Osseous reaction noted in 58% of the cases (enostosic spur in 19%, localized osseous thickening in 16%). 2. The trigeminal nerve displacement by the tumor; in case of clival meningioma extended to the petrous apex, this nerve is displaced outside; otherwise, meningioma of the petrous bone extended to the clivus displaced the trigeminal nerve inside. 3. Radiate structure within tumor converging to vascular basal pole of the meningioma noted in 42% of the cases. Tentorial involvement remained a difficult diagnosis on MR images. It was affirmed when the tumor extended on the opposing surface of the tentorium and when focal hypersignal existed through the usual tentorial hyposignal on T2-weighted images and T1-weighted images after gadolinium. On the other hand, tentorial linear dural enhancement adjacent to the tumor was not a reliable sign (error in 15.8% of the predicted cases). The meningothelial (syncitial) type was noted in 67.7% of the cases. (ABSTRACT TRUNCATED AT 450 WORDS) PMID- 8636803 TI - [The evaluation of 3DFT time-of-flight MR-angiography versus angiography in the study of carotid atheromatous lesions with a review of the literature]. AB - In 37 patients suspected of having a stroke 71 carotid bifurcations were explored by MR-angiography and by digital angiography the reference technique. A 3D sequence was acquired with the time-of-flight technique, using a transmitter receiver cranial coil, followed by a strictly receiver Helmoltz coil on a 1 Tesla magnet. Two examiners evaluated the carotid bifurcations and measured the degree of stenosis in terms of diameters, according to the north american symptomatic carotid endarterectomy trial (NASCET). Five classes were established: class 1: normal; class 2: 1 to 29%; class 3: 30 to 69%; class 4: 70 to 99% and class 5: thrombosis. The results obtained in the determination of classes were identical with both coils: the coefficient of correlation with straight angiography were 0.973 with the cranial coil and 0.966 with the Helmoltz coil. Five stenoses were overestimated and classified as Class 3 instead of Class 2. The five stenoses greater than 70% (Class 4) showed a signal-void area at their level, due to severe dephasing induced by turbulences. Finally, there was a false-negative image of occlusion: the high-intensity signal of the thrombus was mistaken for one of flow. The data of our study were in accordance with the excellent results obtained by several authors in the literature, which makes it possible for us to propose this type of examination as a novel mean of investigating bifurcations of carotid arteries. Provided a strict technique is applied, and in addition to carotid bifurcation the Willis' circle and the cerebral parenchyma are explored, MR-angiography can complete the results of Doppler-echo. Standard arteriography could then be reserved to surgical patients and to those with discordant results of MR-arteriography and Doppler echo systems. PMID- 8636804 TI - [Dural fistula of the cavernous sinus. Clinical and angiographic aspects. Results of particulate intravascular treatment]. AB - Thirteen patients with dural fistula of the cavernous sinus were studied by angiography. Nine of them presented with ophthalmic symptoms (chemosis and oculomotor disorders caused by the fistula). In one patient the lesion was discovered by chance, and 2 other patients had consulted for a disabling tinnitus. Eight patients accepted to the treated by the endovascular route for embolization of the maxillary artery, using particles. Embolization was unilateral in 4 cases and bilateral in 4 other cases. All embolizations were followed by serial control angiography immediately performed. All subjects were seen again as out-patients at 3 month, and 5 of them accepted a control angiography. Three patients were then regarded as clinically and anatomically cured. Two patients with incomplete clinical and angiographic results had a second embolization which resulted in clinical and anatomical cure at a 4-month control examination. These 8 patients were re-examined clinically after one month of treatment and found to be symptomless. Only one complication (transient oedema of the face) was noted. Dural fistulae are lesions that are most probably acquired by alteration of the physiological dural arteriovenous shunts occurring soon after venous thrombosis. Their course is capricious, and they sometimes heal spontaneously. However, the cavernous sinus location with its repercussion on the eye usually requires treatment. This treatment is initially endovascular; surgery and multifascicular irradiation being reserved for failures. Particle embolization of maxillary arteries is a simple and efficient procedure which must be used initially. If it proves insufficient, embolization of other arterial feeders (but it is often more dangerous) or the venous route can be tried. PMID- 8636805 TI - [Dural fistula of the cavernous sinus. Treatment through an intravenous approach apropos of 4 cases]. AB - The authors report 4 cases of symptomatic dural fistula of the carotid cavernous sinus treated by transvenous coils. The objectives of dural fistula treatment are now clearly established and several methods are available. Abstention is the rule for asymptomatic forms, whereas symptomatic forms with ophthalmological complications may respond to manual compression of the jugular vein and the carotid artery, arterial embolization, radiotherapy or venous embolization. Arterial embolization results in complete cure in 72% to 78% of the cases, but it is limited by the meningeal supply of the internal carotid artery, with the microcatheter the transvenous route make it possible to treat the meningeal supply from the internal and external carotids without affecting the arterial system, but this technique is restricted by the anatomy of each patient. PMID- 8636806 TI - Extrapulmonary effects of antenatally administered steroids. PMID- 8636807 TI - Increased urinary nitrite excretion in children with minimal change nephrotic syndrome. AB - OBJECTIVE: We tested the hypothesis that nitric oxide synthesis by the kidney is increased in children with primary nephrotic syndrome. METHODS: We examined the urinary excretion of nitrite, a stable metabolite of nitric oxide, using the Griess reaction, in children with nephrotic syndrome. RESULTS: In comparison with healthy children, patients with minimal change nephrotic syndrome had increased urinary nitrite excretion regardless of whether the disease was in relapse or remission (p < 0.025). In contrast, urinary nitrite excretion was similar in control subjects and patients with focal segmental glomerulosclerosis or IgA nephropathy. CONCLUSION: These findings indicate that measurement of urinary nitrite excretion may be a useful test to help discriminate between minimal change nephrotic syndrome and focal segmental glomerulosclerosis in children with idiopathic nephrotic syndrome. PMID- 8636809 TI - Growth hormone treatment of children with neural tube defects: results from 6 months to 6 years. AB - OBJECTIVE: Patients with neural tube defects (myelomeningocele) have severe growth retardation, and treatment with recombinant human growth hormone (rHGH) for 6 months accelerates growth velocity. We examined patients treated for longer periods to determine whether accelerated growth persists, and whether patients demonstrated to be growth hormone deficient have a greater response to rHGH therapy. METHODS: We retrospectively evaluated the growth rate and length standard deviation score (SDS) of 22 patients in response to treatment with 0.3 mg/kg per week of rHGH for 7 to 72 months. Nine of 22 patients were growth hormone deficient (nocturnal and provocative growth hormone responses < 7 ng/ml). Treatment success was defined as an increase of length SDS of > 0.2 SD per year. RESULTS: Fourteen patients (64%) had treatment successes, and eight had treatment failures. Length SDS improved from a pretreatment value of -2.9 (+/- 1.2) to the most recent length SDS of -1.9 (+/- 1.4) (p < 0.001). The growth rate was significantly increased through year 4 of treatment. The annualized growth rate after 6 months of rHGH treatment was significantly different for the success and failure groups (11.0 +/- 2.6 cm/yr vs 5.1 +/- 3 cm/yr, p < 0.001). The annualized 6-month growth rate during treatment was related to the probability of treatment success. CONCLUSION: Treatment with rHGH significantly improves the growth rate and length SDS of children with neural tube defects. The 6-month annualized growth velocity during treatment was predictive of long-term treatment response. The effect on adult stature is unknown. PMID- 8636808 TI - Effects of recombinant human growth hormone on renal function in children with renal transplants. AB - OBJECTIVE: To provide accurate measurement of renal function during treatment with recombinant human growth hormone (rhGH). METHODS: We measured glomerular filtration rate and effective renal plasma flow by clearance of inulin and para aminohippuric acid before rhGH therapy, after 1 week, and then at 6-month intervals for up to 2 years of treatment in 16 children (mean (SD) age = 13.1 (2.2) years; glomerular filtration rate = 52 (27) ml/min per 1.73 m2). The mean (SD) time from transplantation was 6.5 (3.6) years. RESULTS: Linear growth velocity during rhGH therapy increased from 4.0 (1.8) to 8.8 (2.6) cm/yr (p < 0.0001). One child was withdrawn after 9 months because of abnormal glucose tolerance, and another child received a second renal transplant after 18 months. Glomerular filtration rate increased to 57 (29) ml/min per 1.73 m2 at 1 week (p = 0.004), remained improved at 6 months (63 (30); p = 0.013), but was not significantly better at 1 year (59 (33)). Effective renal plasma flow on day 1 was 237 (127) ml/min per 1.73 m2 and was unchanged on day 8 (244 (123)), at 6 months (271 (149)), and after 1 year (269 (157)). During the study there was no significant change in filtration fraction, blood pressure, or kidney volume, and excretion of microalbumin and N-acetylglucosaminidase was unaltered. There was one rejection episode per 14.8 patient-months in the year before treatment, 1 per 18.9 patient-months during the first year of treatment, and 1 per 13 patient months during the second year of rhGH therapy. CONCLUSION: Treatment with rhGH improves growth in children with renal transplants. Glomerular filtration rate was increased after 1 week and 6 months of rhGH therapy but returned to baseline values thereafter. The data indicate the need for long-term follow-up of children with renal transplants who are receiving rhGH. PMID- 8636810 TI - Effect of number of blood cultures and volume of blood on detection of bacteremia in children. AB - OBJECTIVE: To determine whether bacteremia can be detected more rapidly and completely by (1) obtaining two blood cultures instead of one and/or (2) collecting a larger volume of blood. STUDY DESIGN: Prospective comparison of different strategies in 300 patients undergoing blood culture for suspected bacteremia. Each patient had two samples of blood, A (2 ml) and B (9.5 ml), obtained sequentially from separate sites. The B sample was divided into three aliquots: B1 (2 ml), B2 (6 ml), and ISO (1.5 ml, quantitative culture). RESULTS: A pathogen was isolated from one or more blood cultures in 30 patients (10% of cases). When measured at 24 hours, the pathogen recovery rate for the B2 sample (72%) was higher than that for the individual small-volume samples (A = 37%, B1 = 33%; p < 0.01 for each comparison) and for the combination of the two small volume samples (A + B1 = 47%; p = 0.04). At final (7-day) reading the pathogen recovery rate for the B2 sample (83%) was higher than that for B1 (60%; p = 0.02) and similar to the recovery rate observed with the combination of the two small volume cultures (A + B1 = 73%; p = 0.55). CONCLUSIONS: Increasing the volume of blood inoculated into blood culture bottles improves the timely detection of bacteremia in pediatric patients and spares the patients the cost and pain of an additional venipuncture. PMID- 8636811 TI - Total and functional antibody response to a quadrivalent meningococcal polysaccharide vaccine among children. AB - OBJECTIVE: To determine total and functional serogroup C antibody response after vaccination with a quadrivalent meningococcal polysaccharide vaccine. DESIGN: Prospective, before and after intervention study. SUBJECTS: Study subjects were between the ages of 0.5 and 19.9 years, and were eligible for a community-wide public health immunization campaign against Neisseria meningitidis serogroup C. METHODS: Total and functional antibody response was measured by enzyme-linked immunosorbent assay and bactericidal assay, respectively. RESULTS: One month after vaccination, total capsular polysaccharide antibody significantly increased in all age groups; a significant rise in bactericidal antibody, that correlated with total capsular polysaccharide antibody, was seen in children 18 months of age and older. At 1 year bactericidal antibody titers were maintained but capsular polysaccharide antibody declined substantially in children younger than 5 years. CONCLUSION: Total capsular polysaccharide antibody concentration appears to be a useful surrogate measure of bactericidal antibody in children 18 months and older. Children who originally received the vaccine at less than 18 months of age should be considered for revaccination if there is a new or continuing risk of disease. Because of the differences in the total and bactericidal antibodies formed, vaccine efficacy trials are required to define which serologic measures are associated with protection. PMID- 8636812 TI - Small-bowel bacterial overgrowth in children with chronic diarrhea, abdominal pain, or both. AB - OBJECTIVE: To evaluate the frequency of small-bowel bacterial overgrowth (SBBO) as a cause of chronic digestive symptoms in a large cohort of children, using the glucose breath hydrogen test (BHT). DESIGN: Patients were 53 children (aged 2 months to 12 years) with chronic diarrhea, abdominal pain, or both. Diagnosis of SBBO was defined with a BHT by a change in H2 concentration of 10 ppm H2 or more in expired air after an oral glucose load. Patients with a positive BHT result were included in group 1 and treated with a combination of colistin and metronidazole for 10 days; a second BHT was performed 1 month later. Group 2 comprised patients with a negative BHT result. Group 3 (n = 15) was a control group of healthy subjects, and group 4 (n = 6) a comparison group of subjects with bacteriologically documented SBBO. RESULTS: Eighteen patients (34%) had a positive BHT result and 35 a negative result. The BHT results were comparable in groups 1 and 4 and in groups 2 and 3, respectively. Fasting H2 levels were higher in group 1 than in groups 2 (p < 0.001) and 3 (p < 0.01). In group 1, children were younger than in group 2 (1 +/- 1 year vs 3.9 +/- 3 years; p < 0.001) and diarrhea was frequent (83%), but 17% of patients had abdominal pain alone. Fetid stools (p < 0.01), mucus in stools (p < 0.01), and flatulence (p < 0.05) were more frequent in group 1 than in group 2. Antibiotic treatment of children in group 1 led to a rapid disappearance of symptoms and normalization of BHT results. CONCLUSION: SBBO appears to be a frequent cause of chronic digestive symptoms in children, especially before the age of 2 years. The BHT provides a simple and noninvasive method of detecting it. The recognition of SBBO in children leads to effective treatment. PMID- 8636813 TI - Reference intervals for serum granulocyte colony-stimulating factor levels in children. AB - OBJECTIVE: To determine age-adjusted reference intervals for children for serum granulocyte colony-stimulating factor (G-CSF) levels. DESIGN: We determined the serum G-CSF levels of 168 disease-free children who were term neonates to 15 years of age with a highly sensitive chemiluminescent enzyme immunoassay. RESULTS: The lowest values (5 ng/L) exceeded the detectable limit (1 ng/L) of this assay technique. The G-CSF levels were highest on the day of birth (mean +/- SD 147 +/- 146 ng/L); thereafter values decreased to 46 +/- 33 ng/L in the early neonatal period and to 23 +/- 10 ng/L in the late neonatal period. The G-CSF levels both on the day of birth and in the early neonatal period were significantly higher than in all older age groups. No significant differences were found among any of the age groups after 4 weeks of age. The G-CSF values were correlated with both blood leukocyte counts (r = 0.496, p < 0.0001) and neutrophil counts (r = 0.547, p < 0.0001). In children older than 4 weeks of age (n = 128), the 95% reference interval for G-CSF values was 5 to 42 ng/L. CONCLUSIONS: Our study in disease-free children revealed that change in serum G CSF levels are age dependent and are correlated with neutrophil counts. Determination of reference intervals for the neonatal period requires further study. PMID- 8636814 TI - Menetrier disease of childhood: role of cytomegalovirus and transforming growth factor alpha. AB - OBJECTIVE: Because the role of cytomegalovirus in Menetrier disease in children remains unclear and recent studies have implicated transforming growth factor alpha in the pathogenesis of this disease in adults, we investigated the possibilities that (1) cytomegalovirus is etiologic in Menetrier disease in children and (2) transforming growth factor alpha mediates its development. METHODS: The presence of a cytomegaloviral infection and the pattern of transforming growth factor alpha immunolocalization were determined in the gastric mucosa of four pediatric patients with Menetrier disease, in control subjects (children with normal gastric mucosa, gastritis, or prostaglandin E1 induced antral hyperplasia), and in adults with Menetrier disease. RESULTS: Evidence of a cytomegaloviral infection was present only in the four children with Menetrier disease. The pattern of transforming growth factor alpha immunostaining was identical in the specimens from pediatric and adult patients with Menetrier disease. This pattern was distinct from that found in the pediatric control specimens. CONCLUSIONS: These data strengthen the possibilities that cytomegalovirus is etiologic in children and that transforming growth factor alpha is involved in the pathogenesis of Menetrier disease in both children and adults. PMID- 8636816 TI - Natural history of Alstrom syndrome in early childhood: onset with dilated cardiomyopathy. AB - Alstrom syndrome is an autosomal recessive disorder characterized by cone-rod dystrophy, obesity, hearing impairment, and diabetes caused by insulin resistance. By reviewing the charts of eight patients followed for periods of 2 to 22 years, we established the natural history of this syndrome during childhood. Five patients, in four families, were seen between the ages of 3 weeks and 4 months with a dilated cardiomyopathy, a previously unrecognized feature of the syndrome. Photophobia and nystagmus were first documented in the eight patients between the ages of 5 months and 15 months. In all patients, electroretinography initially showed a severe cone impairment with mild (2/8) or no (6/8) rod involvement. Electroretinograms, obtained again at ages 9 to 22 years for four patients, revealed extinguished rod-and-cone responses. Obesity developed during childhood in seven patients, in at least three of them before age 2 years. Hearing impairment (5/8) and diabetes/glucose intolerance (4/8) were diagnosed at the end of the first decade or during the second decade. This constellation of features should facilitate early diagnosis of the syndrome. PMID- 8636815 TI - Pleuropulmonary blastoma: a marker for familial disease. AB - OBJECTIVE: To catalog and evaluate patterns of disease in families of children with pleuropulmonary blastoma (PPB). METHODS: Data have been collected since 1988 on 45 children with PPB and their families. All pathologic materials were centrally reviewed. Preliminary molecular genetic analyses were performed when possible. RESULTS: In 12 of 45 patients, an association was found between PPB and other dysplasias, neoplasias, or malignancies in the patients with or in their young relatives. The diseases found to be associated with PPB include other cases of PPB, pulmonary cysts, cystic nephromas, sarcomas, medulloblastomas, thyroid dysplasias and neoplasias, malignant germ cell tumors, Hodgkin disease, leukemia, and Langerhans cell histiocytosis. Abnormalities of the p53 tumor suppressor gene, Wilms tumor suppressor gene (WT1), and the putative second genetic locus for Wilms tumor (WT2) were not found in preliminary investigations. CONCLUSIONS: The occurrence of PPB appears to herald a constitutional and heritable predisposition to dysplastic or neoplastic disease in approximately 25% of cases. All patients with PPB and their families should be investigated carefully. Further research of this new family cancer syndrome may provide insight into the genetic basis of these diseases. PMID- 8636817 TI - Acute rectus muscle palsy in children as a result of orbital myositis. AB - OBJECTIVE: Acute rectus muscle palsy caused by orbital myositis is often confused with infectious orbital cellulitis. The purpose of this article is to clarify the former condition by explaining how it is manifested and treated. METHODS: The seven children less than age 11 years in this study were examined after having an acute onset of ocular pain. Physical examination indicated a loss of motility in the field of action of the involved extraocular rectus muscle. Computed tomographic examination confirmed the existence of orbital myositis. Treatment consisted of intravenous or oral administration of corticosteroids. RESULTS: In all the initial evaluations, computed tomography showed the isolated rectus muscle to be enlarged. In four of the patients in whom the lateral rectus was involved, esotropia was present in the primary position. In the fifth patient with lateral rectus involvement, esotropia was seen only in the field of gaze of the involved lateral rectus muscle. The patient with medial rectus involvement had exotropia, and the patient with superior rectus involvement had hypotropia on the involved side. Each had injection and chemosis of the conjunctiva and Tenon fascia only in the quadrant overlying the involved rectus muscle. CONCLUSIONS: Children with orbital myositis can have an acute rectus muscle palsy. The inflammation and strabismus are exquisitely sensitive to systemic corticosteroid therapy, which produces a rapid resolution of symptoms. If the steroid therapy is discontinued abruptly, the myositis can recur. PMID- 8636818 TI - Subarachnoid fluid collections: a cause of macrocrania in preterm infants. AB - We report the outcome of 12 very low birth weight infants with macrocrania caused by subarachnoid fluid collections. By the age of 15 to 18 months, head growth had stabilized along a curve above and parallel to the 95th percentile. No infant required neurosurgical intervention, nor was cerebral palsy or mental retardation diagnosed in any of the infants. PMID- 8636819 TI - Microbial dynamics of persistent purulent otitis media in children. AB - Repeated aspirations (for a period of 36 to 55 days) of the exudate through an open perforation were performed in seven children with acute otitis media that did not respond to antimicrobial therapy. Penicillin-resistant organisms were present in all but one of the first two aspirates. Failure to respond to antimicrobial therapy was associated with the emergence of resistant anaerobic and aerobic bacteria in the third and fourth cultures. The infection was cured in all instances after administration of antimicrobial agents effective against these bacteria. PMID- 8636820 TI - Persistence of fetal hemoglobin production after successful transplantation of cord blood stem cells in a patient with sickle cell anemia. AB - A girl with sickle cell anemia was treated with cord blood transplantation combined with hematopoietic growth factor. Cord blood cells were collected from a sister with an identical human leukocyte antigen complex who was a carrier of the sickle cell trait (hemoglobin AS). The patient had complete engraftment and no graft-versus-host disease. The persistence of a high level of fetal hemoglobin 6 months after engraftment was noted. PMID- 8636821 TI - Nonketotic hyperglycinemia: atypical clinical and biochemical manifestations. AB - A 16-year-old boy had intermittent chorea, delirium, and vertical gaze palsy precipitated by febrile illness. Nonketotic hyperglycinemia was confirmed by measurement of liver and lymphoblast glycine cleavage enzyme activity. Deficient but residual enzyme activity was demonstrated in both tissues, possibly accounting for the mild phenotype. Confirmation of an atypical variant of nonketotic hyperglycinemia with residual glycine cleavage enzyme activity has important implications for diagnosis and treatment. PMID- 8636822 TI - Recurrent necrotizing jejunitis in a child. AB - We report the case of a 5-year-old boy with segmental necrotizing jejunitis, present a review of the literature including a single previously described North American child, and give evidence to document disease recurrence. This uncommon disease must be differentiated from Crohn disease because the treatment and prognosis are different. PMID- 8636823 TI - Herpes simplex virus type 2 infection associated with urinary retention in the absence of genital lesions. AB - A 14-year-old boy had inguinal lymphadenitis and associated urinary retention; herpetic genital and anal lesions were absent. Lymph node biopsy established the presence of infection with herpes simplex virus type 2. We recommend that this infection be considered in the differential diagnosis of adolescent patients with urinary retention and constipation, even when the diagnostic clue provided by genital ulcers is absent. PMID- 8636824 TI - Circadian variations of urinary electrolyte concentrations in preterm and term infants. AB - OBJECTIVES: To determine whether a circadian variation of urinary excretion of calcium and phosphorus exists in preterm infants. STUDY DESIGN: We studied 70 newborn infants (median birth weight 1920 gm, range 660 to 3550 gm; median gestational age 34 weeks, range 25 to 42 weeks) at a median postmenstrual age of 36 weeks (range 32 to 42 weeks). Within a period of 24 hours, four urine specimens were collected during 6-hour periods. The concentrations of calcium, phosphorus, sodium, potassium, and creatinine were determined and creatinine quotients were calculated for each specimen. RESULTS: No clinically relevant circadian variation in urinary excretion for any of these minerals was found. CONCLUSION: If spot urine specimens are used to monitor calcium and phosphorus balance in preterm infants, the time of the day these are collected is not important. PMID- 8636825 TI - Adrenocortical function in the very low birth weight infant: improved testing sensitivity and association with neonatal outcome. AB - OBJECTIVE: To evaluate adrenocortical function in ill preterm infants and investigate potential relationships between plasma cortisol concentrations and major neonatal outcomes. STUDY DESIGN: Randomized trial of adrenocorticotropic hormone (1-24ACTH) stimulation testing, followed by a chart review. SETTING: Two level III neonatal intensive care units, Sacramento, Calif. PARTICIPANTS: Sixty seven very low birth weight infants, born at 32 weeks of gestation or earlier weighing 1500 gm or less, who had endotracheal intubation and indwelling arterial access. RESULTS: Most infants (76%) had baseline cortisol concentrations < 414 nmol/L (15.0 micrograms/dl), and of those, only 36% responded to stimulation with 1-24ACTH, 0.1 microgram/kg. Raising the 1-24ACTH dose to 0.2 microgram/kg resulted in a response rate of 67% (p = 0.09) but decreased the sensitivity of the test. An elevated mean 11-deoxycortisol/cortisol ratio indicated that decreased 11 beta-hydroxylase activity may limit cortisol production in some infants. Infants with baseline cortisol concentrations less than 414 nmol/L (15.0 micrograms/dl) were more likely to have chronic lung disease (p < 0.002) and less likely to have severe intraventricular hemorrhage (p < 0.02). Response to 1 24ACTH was not associated with a detectable difference in outcome. CONCLUSION: Many very low birth weight infants have low cortisol and ACTH concentrations and are unable to mount a cortisol response to physiologic doses (0.1 microgram/kg) of 1-24ACTH. These findings suggest that delayed maturation of adrenal response may result in physiologically inadequate cortisol concentrations in stressed very low birth weight infants. This delayed maturation may contribute to the development of chronic lung disease. PMID- 8636827 TI - Ultralente insulin treatment of transient neonatal diabetes mellitus. AB - In an infant with transient neonatal diabetes mellitus, control of the blood glucose concentration was attained with ultralente insulin treatment, without any episodes of hypoglycemia. We recommend subcutaneous injection of ultralente insulin, rather than lente or isophane (NPH) insulin, to avoid hypoglycemia during the treatment of transient neonatal diabetes mellitus. PMID- 8636828 TI - Pilot study to assess the effect of inhaled corticosteroids on lung function in patients with cystic fibrosis. AB - OBJECTIVE: To determine the effect of inhaled corticosteroids on lung function in patients with cystic fibrosis. STUDY DESIGN: We report a pilot study of 49 patients with cystic fibrosis and moderate to severe bronchial obstruction (forced expiratory volume in 1 second < or = 55% of the predicted value); 25 patients were given inhaled corticosteroids for 30 days (1500 micrograms of beclomethasone via spacer), and 24 patients had the same standard treatment but no inhaled corticosteroids. RESULTS: Forced vital capacity, forced expiratory volume in 1 second, and airway resistance showed significant improvement in both study groups, but thoracic gas volume and the diffusion capacity of the lung for carbon monoxide improved significantly only in the group given inhaled corticosteroids. When concomitant medications were taken into account, analysis of variance confirmed a significant effect of inhaled corticosteroids on the improvement of thoracic gas volume. CONCLUSION: Inhaled corticosteroids in combination with standard treatment can contribute to the improvement of lung function in patients with cystic fibrosis and moderate to severe bronchial obstruction. Our preliminary data seem encouraging enough to warrant a multicenter, long-term, blind control study. PMID- 8636826 TI - Prediction of neonatal hyperthyroidism in infants born to mothers with Graves disease. AB - OBJECTIVE: To determine whether determinations of thyrotropin-receptor antibody (TRAb) levels in newborn infants of women with Graves disease would predict which infants will have hyperthyroidism. METHODS: The TRAb levels, assayed in the sera of 14 infants born to 14 women with Graves disease, were measured sequentially in the infants with hyperthyroidism during the course of antithyroid medication therapy. RESULTS: Seven infants had TRAb values less than 0.15 and remained euthyroid. In seven infants whose initial TRAb values were more than 0.25 (range, 0.48 to 0.88), clinical and biochemical signs of hyperthyroidism developed. The infants were treated with antithyroid medication until day 57 to day 123 of life. Therapy was discontinued when the infants were free of symptoms and when serum thyroxine and triiodothyronine and free thyroxine levels remained normal during therapy with decreasing doses of antithyroid medication. When the medication was discontinued, TRAb values were less than 0.20. CONCLUSIONS: Infants born to mothers with Graves disease with initial TRAb values less than 0.15 remained euthyroid. The TRAb values greater than 0.25 were associated with the development of neonatal hyperthyroidism. During treatment of neonatal hyperthyroidism, TRAb values less than 0.20 may be helpful in deciding when to withdraw antithyroid medication. PMID- 8636829 TI - Macrophage activation syndrome in systemic juvenile rheumatoid arthritis successfully treated with cyclosporine. AB - A macrophage activation syndrome, possibly related to methotrexate toxicity, developed in a boy with systemic juvenile rheumatoid arthritis. Corticosteroid administration was ineffective, whereas a prompt response to cyclosporine was observed. Two months later, Pneumocystis carinii pneumonia developed. PMID- 8636830 TI - Cisapride-induced long QT interval. AB - A 2-month-old infant with gastroesophageal reflux was treated with cisapride. Bradycardia developed and an electrocardiogram revealed 2:1 atrioventricular conduction and a prolonged QT interval. After cessation of cisapride therapy, both the rhythm and the QT interval returned to normal. Prolonged QT interval during treatment with cisapride may occur in children as in adults. PMID- 8636831 TI - High-dose oral dexamethasone therapy for chronic childhood idiopathic thrombocytopenic purpura. AB - Because high-dose oral dexamethasone therapy has been reported to be effective for adults with idiopathic thrombocytopenic purpura, we assessed the short-term efficacy and toxicity of dexamethasone in seven children with chronic or refractory idiopathic thrombocytopenic purpura. Dexamethasone therapy was effective and well tolerated; further long-term studies are warranted. PMID- 8636832 TI - Treatment of atrichia pubis in adolescent girls with pituitary dwarfism. AB - Five girls with multiple pituitary insufficiency and atrichia pubis, aged 14 to 22 years, were instructed to apply 5% testosterone undecanoate cream on the pubic area twice daily. Pubic hair development became noticeable between days 15 and 20 of treatment; after 4 months the pubic hair density was at Tanner stage II to III. Testosterone blood levels did not increase. This treatment corrects an aesthetic problem, which is of great concern to adolescents with pituitary insufficiency. PMID- 8636833 TI - Successful medical treatment of severely decompensated Wilson disease. AB - Delayed response to medical treatment sometimes leads to unnecessary liver transplantation in patients with severely decompensated Wilson disease. We report the course of five patients (mean age 13.4 years, range 11 to 15 years) with severely decompensated Wilson disease who were successfully treated medically. Prothrombin time improved after a minimum of 1 month and returned to normal within 3 months to 1 year or more. PMID- 8636834 TI - Behavioral intervention to increase condom use among high-risk female adolescents. AB - OBJECTIVE: To determine whether condom use among high-risk female adolescents could be increased by a behavioral intervention, with the use of infection with Chlamydia trachomatis as a biomarker of condom practices. DESIGN: Prospective, randomized, controlled intervention. SETTING: Urban family planning and sexually transmitted disease clinics. PARTICIPANTS: Two hundred nine female adolescents, aged 15 through 19 years, who were treated for C. trachomatis genitourinary infection, were randomly assigned to standard (control) or experimental (behavioral intervention) groups. One hundred twelve subjects returned for follow up 5 to 7 months after enrollment and comprise the study subjects. MEASUREMENTS: Subjects completed a multiinstrument questionnaire measuring sexual behavior, condom practices, attitudes and beliefs, cognitive complexity, sociodemographics, and motivation at enrollment and follow-up. Endourethral and endocervical sites were sampled for C. trachomatis. RESULTS: Among the 112 subjects who returned for repeated examination, those who had received the experimental intervention reported increased use of condoms by their sexual partners for protection against sexually transmitted diseases (odds ratio = 2.4; p = 0.02) and for vaginal intercourse (odds ratio = 3.1; p = 0.005) at the 6-month follow-up. Multivariable logistic regression analysis controlling for condom use at enrollment demonstrated that the experimental intervention (odds ratio = 2.8; p = 0.03) and the higher cognitive complexity (odds ratio = 4.6; p = 0.02) independently contributed to greater condom use at follow-up. Despite greater use of condoms among the group who had received the intervention, use remained inconsistent and rates of reinfection with C. trachomatis were not significantly different (26% vs 17%; p = 0.3). CONCLUSION: Although a brief behavioral intervention among high risk female adolescents can increase condom use by their sexual partners, incident infection does not appear to be reduced, because condom use remained inconsistent. PMID- 8636835 TI - Reversibility of cerebral ventricular enlargement in anorexia nervosa, demonstrated by quantitative magnetic resonance imaging. AB - OBJECTIVE: To determine the reversibility of the loss of brain parenchyma and ventricular enlargement in patients with anorexia nervosa after refeeding. STUDY DESIGN: Quantitative magnetic resonance imaging was performed on three groups of subjects: (1) 12 female adolescents hospitalized with anorexia nervosa, (2) the same 12 patients after nutritional rehabilitation, a mean of 11.1 months later, and (3) 12 healthy age-matched control subjects. Sixty-four contiguous coronal magnetic resonance images, 3.1 mm thick, were obtained. With a computerized morphometry system, lateral and third ventricular volumes were measured by a single observer unaware of the status of the patient. RESULTS: On admission, patients were malnourished and had lost an average of 11.7 kg (body mass index, 14.3 +/- 2.0 kg/m2). After refeeding, they gained an average of 9.7 kg (body mass index, 17.9 +/- 1.5 kg/m2). Total ventricular volume decreased from 17.1 +/- 5.5 cm3 on admission to 12.4 +/- 3.0 cm3 after refeeding (p < 0.01) and returned to the normal range. The degree of enlargement of the third ventricle was greater than that of the lateral ventricles. There was a significant inverse relationship between body mass index and total ventricular volume (r = -0.63; p < 0.05). CONCLUSION: In patients with anorexia nervosa, cerebral ventricular enlargement correlates with the degree of malnutrition and is reversible with weight gain during long-term follow-up. PMID- 8636836 TI - Neuroimaging studies in children with developmental delay. PMID- 8636837 TI - Effect of calcium on blood pressure. PMID- 8636838 TI - Diazepam to reduce recurrences of febrile seizures. PMID- 8636839 TI - Extracellular matrix degradation by Haemonchus contortus. AB - To better understand the in vivo function of secreted cysteine proteases of Haemonchus contortus, the ability of live parasites to degrade connective tissue was investigated using [3H]proline-labeled extracellular matrix produced by smooth-muscle cells (R22). The matrix was composed of glycoprotein(s) (34%), elastin (49%), and collagen (15%) in an insoluble, multilayered, cross-linked structure. No degradation of the extracellular matrix by third-stage larvae (L3) (10,000/ml) occurred during 24-hr in vitro incubation. In contrast, fourth-stage larvae (L4) (1,000/ml) degraded 42% of the matrix, whereas adults (100/ml) degraded the entire matrix. The presence of Z-phe-ala-FMK (100 microM), a specific cysteine protease inhibitor, during incubation of adults, reduced matrix degradation to 30% without affecting parasite motility. Isolated adult excretory/secretory products (ESP) (0.1 mg protein/ml) degraded 64% of the total matrix; specific degradation consisted of 80.3% of the glycoprotein, 67.1% of the elastin, and 27.6% of the collagen matrix components. Degradation of the matrix by ESP was stimulated by dithiothreitol (2 mM) and inhibited by Z-phe-ala-FMK. Thus, the secretory cysteine proteases of H. contortus are active under physiological conditions and able to degrade the major components of connective tissue in an in vitro model system that simulates their structure in vivo. These data strengthen the proposed role of these enzymes in the breakdown of host tissue. PMID- 8636840 TI - Variation in the development of two isolates of Cryptocaryon irritans. AB - Two isolates of Cryptocaryon irritans obtained from Acanthopagrus australis from Moreton Bay (isolate C1) and Gymnocranius audleyi from Heron Island (isolate C2) were passaged on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C under identical laboratory conditions. There were significant differences between isolates in the diameter of trophonts and tomonts, the incubation period of tomonts, and the length of theronts. Trophonts of C1 were significantly larger on L. calcarifer than on M. novemaculeata and showed marked size variation with temperature, whereas trophonts of C2 developed equally well on both species and showed little size variation with temperature. Tomonts of C1 were significantly larger than those of C2 when grown on L. calcarifer, whereas on M. novemaculeata tomonts from C1 were significantly smaller than C2 tomonts. The incubation period of tomonts from C1 was significantly shorter than that for tomonts of C2, and theronts of C1 were significantly larger than theronts of C2 under all host/temperature conditions. The differences in the development of these isolates are of biological and epidemiological importance. This indicates that distinct intraspecific variants of C. irritans occur along the coast of southeast Queensland. PMID- 8636841 TI - On the distribution and abundance of eel parasites in Nova Scotia: influence of pH. AB - The geographic distribution of metazoan parasites of American eels (Anguilla rostrata) was determined from 28 sites in the Southern Upland and adjacent regions of Nova Scotia. Twelve parasite species were encountered. Component community diversity as measured by species richness, Shannon-Wiener index (H') and Hill's number (N1) decreased when pH < 5.4. Seven species (Azygia longa, Crepidostomum brevivitellum, Bothriocephalus claviceps, Proteocephalus macrocephalus, Paraquimperia tenerrima, Echinorhynchus salmonis, Ergasilus celestis) were found in both the Southern Upland and adjacent watersheds to the north. Three species (Pseudodactylogyrus anguillae, Diplostomum sp., Pomphorhynchus bulbocolli) were found only at sites beyond the Southern Upland, which flowed into the Bay of Fundy and had high pH values ( > 5.4). Two species (Daniconema anguillae, Echinorhynchus lateralis) were limited to sites within the Southern Upland. Digeneans were absent from rivers in the southwest with pH values < 4.7. Variation in occurrence of parasite species was observed among rivers within a single watershed and among years within individual sites. Nevertheless, the impact of acidity at the local level was observed regionally across the Southern Upland of Nova Scotia. These data support the hypothesis that parasite assemblages may be good indicators of environmental stress. PMID- 8636842 TI - Identification of a misleading trypanosomatid parasite from Gerbillus pyramidum and G. andersoni in a Leishmania major endemic area in north Sinai. AB - During an epidemiologic investigation of cutaneous leishmaniasis at a focus in north Sinai, Egypt, between June 1989 and December 1991, 897 desert rodents were trapped and examined to identify reservoir hosts for Leishmania major. Mixed forms of epimastigotes and promastigotes were isolated in Tanabe's medium from 4 Gerbillus pyramidum and 1 Gerbillus andersoni. The 2 forms were later grown and separated as distinct cultures in Schneider's medium. The isoenzyme profile of the gerbils' promastigotes was identical to Leishmania tropica but differed from those of L. major and the gerbils' epimastigotes. The protein pattern by sodium dodecyl sulfate poly acrylamide gel electrophoresis gave no conclusive results. The Hae III restriction endonuclease analysis of kinetoplast DNA of both morphological forms confirmed their similarity and distinguished them from L. tropica and L. major. The gerbils' promastigotes were 30% broader, with a smaller nucleus than those of L. tropica. Following several subcultures, epimastigotes were found to transform to promastigotes. These observations suggest that the 2 forms belong to the genus Trypanosoma. Further studies are in progress to classify this putative Trypanosoma species whose promastigote stages display isoenzyme patterns identical to L. tropica, and which can be misidentified microscopically as Leishmania promastigotes. PMID- 8636843 TI - Parasites of coral reef fish recruits, Epinephelus merra (Serranidae), in French Polynesia. AB - Honeycomb grouper recruits Epinephelus merra (Serranidae) from 2 recruitment events on Moorea Island, French Polynesia, were examined for internal parasites. Trypanorhynch blastocysts (Eucestoda: Trypanorhyncha) and encysted phyllobothriid metacestodes (Eucestoda: Phyllobothriidae) were found in recruits from both events. Fish from the second event were examined later as juveniles and were found to harbor apparently melanized parasites. Neither parasite was found in adults. Evidence suggests these patterns are not due to differential mortality among recruits but to the elimination of parasites by host immune responses. The data also suggest that pelagic fish larvae may represent a dispersal mechanism for parasites if transfer occurs before the death of the parasite. PMID- 8636844 TI - Distribution and abundance of two malarial parasites of the endemic Anolis lizard of Saba Island, Netherlands Antilles. AB - The ecology of 2 parasites, Plasmodium floridense and Plasmodium azurophilum, was studied in the endemic lizard, Anolis sabanus, on Saba island in the eastern Caribbean. Prevalence of the two parasite species was similar, but prevalence varied among sites. Lowest prevalence occurred at dry, windy sites or cool, wet mountain peaks (0-20%); the parasites were more common at most other locations on the island (40-80% infected; overall prevalence = 47%). High and low prevalence sites sometimes were only a few hundred meters apart. Prevalence was similar for males and females but increased with body size except for a decline in the largest ( = oldest) lizards. A surplus of mixed infections (P. floridense and P. azurophilum together in the same host) existed compared with chance proportions. Parasitemia generally was low; 70% of P. floridense infections were < or = 100 parasites/10,000 erythrocytes, and 70% of P. azurophilum infections were < or = 50/10,000. Parasitemia in solitary infections averaged more than twice that seen in mixed infections for both species. PMID- 8636845 TI - Seasonal population dynamics of Halipegus occidualis and Halipegus eccentricus (Digenea:Hemiuridae) in their amphibian host, Rana clamitans. AB - The seasonal population dynamics of Halipegus occidualis and Halipegus eccentricus (Hemiuridae) in their amphibian host, the green frog (Rana clamitans), were examined for 3 yr (weekly, April through October). Frogs were caught, marked, and examined for H. occidualis and H. eccentricus, both of which occur as immatures and adults in the buccal cavity. Frogs were then released and allowed to continue natural recruitment and loss of the parasites. It was thus possible to monitor individual infrapopulations over successive time periods. One hundred and forty-nine frogs were caught and released, with a total of 328 observations. Overall, the levels of both parasites among male and female frogs were similar, as were levels of infection among adult and juvenile hosts. There was, however, no correlation between the total number of H. occidualis and the body size (snout-vent length) of R. clamitans. In contrast, there was a significant correlation between the total number of H. eccentricus and frog size. Recruitment of both species began in May, peaked in June/July, and ended in July (H. eccentricus) or August (H. occidualis). In general, both the prevalence and relative density of H. occidualis was greater than that of H. eccentricus and may be related to space constraints in the buccal cavity of R. clamitans. Adults of both species were observed from April through October. Following increases in parasite recruitment, infrapopulation sizes declined in September 1992 and in August of 1993 and 1994. Examination of variance to mean ratios indicated that both species were overdispersed in the frogs. Large declines in the variance to mean ratios for H. occidualis after periods of greatest recruitment are most likely associated with the loss of larger infrapopulations, suggesting that there may be density-dependent regulation of infrapopulation size. By monitoring individual hosts using the mark-release-recapture protocol, dynamic changes in parasite infrapopulations were observed, e.g., there were losses of immature worms and rapid changes in infrapopulation sizes, observations that would not be made with typical host-parasite systems. PMID- 8636846 TI - The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases. AB - We determined the nucleotide (nt) sequence of the putative gene encoding acetyl coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns. The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species. Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs. This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family. PMID- 8636847 TI - Male reproductive success of Schistosoma mansoni-infected Biomphalaria glabrata snails. AB - Infection of Biomphalaria glabrata by Schistosoma mansoni results in a dramatic reduction in the snail's ability to produce eggs. We studied the ability of such parasitically castrated snails to fertilize the eggs of uninfected snails. Pigmented B. glabrata snails (13141 stock) were infected with S. mansoni miracidia and reared individually until they ceased laying eggs. These infected snails were then given the opportunity to mate with uninfected albino (NMRI) snails. Each of the infected snails was paired with a different albino partner each subsequent week. Sperm transfer by the infected snails was evident from the production of pigmented progeny by the uninfected albino snails. Infected snails successfully acted as males for up to 6 wk after parasitic castration had occurred. The duration of allosperm use by uninfected recipients was lengthy, regardless of the infection status of the pigmented sperm donor. PMID- 8636848 TI - A survey of the coccidian parasites of reptiles from islands of the Galapagos Archipelago: 1990-1994. AB - From 1990 through 1994, fecal samples were collected and examined for coccidian parasites from 26 giant land tortoises Geochelone nigra, from 715 lava lizards Tropidurus spp., from 139 land iguanas Conolophus subcristatus, and from 128 marine iguanas Amblyrhynchus cristatus, all of which inhabit various islands in the Galapagos Archipelago. None of the samples from A. cristatus or from C. subcristatus was infected with coccidia. Only 1 of 26 (4%) G. nigra was infected with a single Eimeria species that we describe here as new. A total of 262 of 715 (37%) individuals representing 3 species of Tropidurus discharged oocysts of 1-3 different coccidian species; these included 2 previously described species Eimeria tropidura and Isospora insularius, and an eimerian that we describe here as new. Additionally, 104 fecal samples from Tropidurus spp. were from 51 animals recaptured in either 2 or 3 yr; 21 had no infections in any year, 15 were infected at least once, 14 were infected in 2 yr, and only 1 was infected during 3 yr. No animal was recaptured and sampled during each of the 4 yr of this study. Of the 262 infected individuals, 30 (12%) had multiple coccidial infections at the time of collection (eimerian and isosporan, or 2 eimerians). Where determination of the sexes was possible in the lava lizards, there was no difference in prevalence rates between males (39%) and females (41%). Sporulated oocysts of the new eimerian from Tropidurus are ellipsoidal, 27.1 x 15.6 (25-31 x 14-18) microns, with a polar body, but without a micropyle or oocyst residuum; they contain ellipsoidal sporocysts, 11.8 x 6.7 (10-14 x 6-8) microns, without Stieda, sub-, or parastieda bodies, but with a sporocyst residuum. Sporulated oocysts of the new eimerian from G. nigra are ellipsoidal to ovoidal, 21.6 x 18.1 (18-25 x 16-20) microns, with a large polar body, but without a micropyle or oocyst residuum; they contain ellipsoidal sporocysts 10.7 x 7.0 (8-12 x 5-8) microns, with Stieda body but no sub- or parastieda bodies. Also present is a sporocyst residuum of medium to large granules randomly distributed among the sporocysts. PMID- 8636850 TI - The Nigerian I/CDC strain of Plasmodium ovale in chimpanzees. AB - The chimpanzee is the only animal host currently available that can support the development of the human malaria parasite Plasmodium ovale. Thirty-one infections with the Nigerian I/CDC strain were induced in splenectomized chimpanzees. Maximum parasite counts ranged from 1,240 to 127,224/microliters. Infections were transient and unpredictable. Anopheles stephensi, Anopheles gambiae, Anopheles freeborni, and Anopheles dirus mosquitoes were infected by feeding through parafilm membranes on heparinized blood containing gametocytes; each species supported development to sporozoites in the salivary glands. Mean oocyst counts per infected mosquito ranged from 1 to 85.1; 21.7% of infected lots of mosquitoes averaged > 20 oocysts per positive mosquito gut. One infection was induced via the bites of infected An. gambiae. The prepatent period was 16 days. PMID- 8636849 TI - Infectivity of low numbers of Toxoplasma gondii oocysts to pigs. AB - To define the infectiousness of the VEG strain of Toxoplasma gondii, 42 pigs were fed doses estimated at 10, 1, or < 1 mouse infective oocysts. They were killed 38 99 days after inoculation and 50 g of tissues from their tongue, heart, and brain were individually homogenized in acidic pepsin solution and bioassayed in mice. Pools of brain, heart, tongue, and skeletal muscle (total 500 g) were bioassayed in cats. Toxoplasma gondii was isolated by bioassays in mice and in cats from 13 of 14 pigs fed 10 oocysts, 13 of 14 pigs fed 1 oocyst, and 4 of 14 pigs fed "less than" 1 oocyst, indicating high infectivity of VEG strain of T. gondii to pigs. All infected pigs developed modified agglutination test antibodies (> 1:50). Control pigs (n = 6) remained seronegative (< 1:20) and T. gondii was not isolated from their tissues. Toxoplasma gondii was isolated from tongues of 27 (93%), brains of 21 (72%), and hearts of 13 (45%) of 29 experimentally infected pigs by bioassay in mice. The number of T. gondii-positive mice after inoculation of tongue, brain, and heart from infected pigs was 240 (80%), 84 (28%), and 36 (12%) of 300 mice inoculated with each organ, respectively. Thus, the VEG strain of T. gondii was localized more often and in higher numbers in the tongue than in the brain and the heart of pigs. The apparent muscle localization after infection with the low dose of the VEG strain of T. gondii agrees with other studies in livestock that suggest T. gondii is more neurotropic in mice than in livestock. PMID- 8636851 TI - Infestation of an introduced host, the European green crab, Carcinus maenas, by a symbiotic nemertean egg predator, Carcinonemertes epialti. AB - The recent introduction of the European green crab, Carcinus maenas, to the west coast of the U.S. has provided an opportunity for host transfer of the symbiotic nemertean egg predator, Carcinonemertes epialti, from its native shore crab host, Hemigrapsus oregonensis to the exotic C. maenas. Two surveys of C. maenas in Bodega Harbor, California, revealed that, in March 1995 prevalence of C. epialti on C. maenas was significantly lower than on H. oregonensis (11% versus 74%), but in November 1995 there was no significant difference between the 2 species (79% versus 98%). Only juvenile C. epialti were recovered from C. maenas in March 1995. However, in November 1995, ovigerous C. maenas were harboring actively feeding adult worms. Prevalence in both crab species significantly differed from March to November. Laboratory studies revealed that C. epialti fed and reproduced on eggs of C. maenas. The feeding rate of C. epialti on C. maenas eggs (2.5 eggs/trial) was not significantly different from that on H. oregonensis eggs (3.6 eggs/trial). Our findings suggest that this nemertean may have less host specificity than was previously thought. If C. epialti causes brood mortality of C. maenas in nature, it could potentially impact populations of this exotic crab. PMID- 8636852 TI - The inflammatory response promotes cutaneous metastasis in hamsters infected with Leishmania (Viannia) panamensis. AB - The influence of nonspecific and immunologically elicited inflammatory responses on the development of metastatic lesions was examined in the hamster model of Leishmania (Viannia) panamensis infection. Delayed type hypersensitivity (DTH) responses were induced using the contact sensitizing agent DNFB (2, 4-dinitro-1 fluorobenzene) and infection with L. panamensis followed by intradermal application of leishmanin. Nonspecific inflammatory response was achieved by the surgical excision of toes. The inductive and eliciting procedures were performed on the ears and fore and hind paws of the right side of experimental groups of hamsters that were inoculated in the snout with a highly metastatic strain of L. panamensis (MHOM/COL/84/1099). Skin metastases were detected by physical evaluation at 15-day intervals over a period of 7-8 mo. Suspected metastases were parasitologically confirmed by culture of tissue fluid aspirated from the lesion. The frequency of metastatic lesions was greater in hamsters subjected to inflammatory stimuli (14/38) than control animals (6/33; P = 0.035). Likewise, the frequency of metastases at the site of induction and elicitation of inflammation (18/22 lesions) in the experimental groups was greater than that observed at the same site in control animals (5/11 lesions; P = 0.017). These findings support a causal relationship between inflammatory response and the development of lesions in this model of secondary disease caused by L. panamensis. PMID- 8636853 TI - A new species of Phoreiobothrium (Cestoidea:Tetraphyllidea) from the great hammerhead shark Sphyrna mokarran and its implications for the evolution of the onchobothriid scolex. AB - Phoreiobothrium manirei n. sp. is described from the spiral intestines of 3 immature individuals of the great hammerhead shark Sphyrna mokarran collected off of the west coast of Florida. This cestode is unusual in that it possesses 4 muscular papillae on the anterior margin of the accessory sucker. Numerous small protrusions with central cilium-like projections were conspicuous throughout the posterior margin of each bothridium. This species possesses the unique combination of bothridia posteriorly subdivided into subloculi and hooks with extended bases and 2 rather than 3 prongs. In fact, the hooks are strikingly like those of species in the genera Dicranobothrium and Platybothrium. This combination of characters suggests that the bifid hook condition is plesiomorphic with respect to the trifid hook condition within the Onchobothriidae. These data further suggest that the triloculated bothridial condition is plesiomorphic relative to the biloculated condition. PMID- 8636855 TI - Emended description of Mazamastrongylus peruvianus (Nematoda:Trichostrongylidae), with comments on the relationships of the genera Mazamastrongylus and Spiculopteragia. AB - Resurrection of Mazamastrongylus as proposed by Jansen (1986) was validated and placement of Mazamastrongylus peruvianus in this genus was confirmed, based on characters of the synlophe, copulatory bursa, and spicules. The cervical synlophe consists of a strongly tapering pattern and prominent "hood ridges" at the level of the excretory pore; there are 40 ridges at the midbody of males. The bursa is of the 2-2-1 type, with rays 2 and 3 parallel and rays 4 and 5 of near equal length and only slightly divergent at the tips. The spoon-shaped dorsal process of the spicules, typical of M. peruvianus and other species of Mazamastrongylus is postulated as a synapomorphy for the genus. The genera Mazamastrongylus, Spiculopteragia, and Sarwaria are considered to be independent based on characters of the synlophe, spicules, and copulatory bursa. Host and geographic distributions for species of Mazamastrongylus appear to be consistent with a history of coevolution and colonization and relationships for M. peruvianus may parallel the pattern postulated for species of Nematodirus in cervids and camelids in the Neotropics. PMID- 8636854 TI - On the status of the genus Pinguicollum (Tetraphyllidea: Onchobothriidae) with a redescription of P. pinguicollum. AB - Material of the poorly known Pinguicollum pinguicollum from Raja spp. in California from the personal collections of Nathan Riser and Mike Moser was examined by light and scanning electron microscopy. The presence of unusual triloculated bothridia covered with a layer of tissue, but capable of protruding through an aperture in this tissue, was confirmed. The species is also unusual in its lack of a muscular pad and apical sucker on each bothridium and in its possession of bifid hooks in which the axial prongs are conspicuously shorter than the abaxial prongs. The scolex was interpreted to consist of a short bothridial region and an elongate cephalic peduncle containing 4 pairs of robust longitudinal muscle bundles. The segment morphology is described in detail and figured for the first time. The ovary is bilobed in cross section; the testes are arranged in multiple rows in cross section. The apex of the scolex was found to possess elongate, filiform microtriches. The distal surfaces of the protrusible regions of the bothridia were covered with short filiform microtriches in the anterior and both short filiform microtriches and spiniform microtriches on the posterior bothridial surfaces. The remainder of the scolex and cephalic peduncle were covered with densely packed, spiniform microtriches of varying sizes. Based on the unusual bothridia, robust cephalic peduncle, and lack of muscular pad and apical sucker on the bothridia, it is recommended that the genus be considered to be valid. As all attempts to locate the type material of the species failed, a neotype is designated to facilitate future study of the genus. PMID- 8636856 TI - Strelkovimermis acuticauda n. sp. and Strelkovimermis buccalis n. sp. (Nematoda:Mermithidae) from adult chironomids (Diptera:Chironomidae) from Lake Itasca, Minnesota. AB - Specimens of 2 mermithid species were obtained from adults of undetermined species of chironomids emerging from Lake Itasca (Clearwater County), Minnesota. Both species have the characteristics of species in the genus Strelkovimermis. The new species are distinguishable from one another and from the 8 currently accepted species in the genus by their small size, shape of the posterior end, length of the buccal funnel, length of the cephalic papillae, location of the mouth, and morphology of the spicule region and vagina. Strelkovimrmis acuticauda n. sp. is unique in possessing an acute posterior end, an auxiliary protractor muscle, and a relatively long vagina, whereas Strelkovimermis buccalis n. sp. is distinguishable from other Strelkovimermis species by its thick cuticle and exceptionally long buccal funnel. The significance of the tail muscles is emphasized as a taxonomic feature to be used in describing future species of Strelkovimermis. A key to the 10 species of Strelkovimermis is provided. PMID- 8636857 TI - A new species of Acanthobothrium van Beneden, 1849 (Eucestoda:Tetraphyllidea: Onchobothriidae) in Dasyatis longus Garman (Chondrichthyes:Myliobatiformes:Dasyatididae) from Chamela Bay, Jalisco, Mexico. AB - A new species of Acanthobothrium in Dasyatis longus from Chamela Bay, Jalisco, Mexico, is a member of a presumed clade of species diagnosed by being anapolytic or nearly so, having more than 100 testes per proglottis, with immature and mature proglottides wider than long to square, aspinose scolex, muscular bothridia fused to the scolex at their posterior ends, H- to V -shaped ovaries, relatively short symmetrical to asymmetrical ovarian arms that extend anteriorly to, or nearly to, the cirrus sac, and vitellaria arranged in fields rather than a single row of follicles. The new species most closely resembles Acanthobothrium terezae from the freshwater stingray Potamotrygon motoro in the following characters: bothridial hooks longer than 200 microns with inner hooks having bent asymmetrical prongs, an average of 130-140 testes per proglottis, and shallow genital atria located posterior to midline of proglottis. The new species differs from A. terezae by having outer hooks approximately the same size and shape as the inner hooks, inner hooks averaging 230 microns rather than 313 microns in total length, and cirrus sacs averaging 255 microns rather than 450 microns in length. The new species is unique among all described species of Acanthobothrium by having a cleft in the posterior margin of each apical bothridial pad. The apparent close relationship of the new species to one inhabiting a Neotropical freshwater stingray provides support for the hypothesized Pacific marine ancestry of Neotropical freshwater stingrays and raises the possibility that the Neotropical freshwater stingrays may not be monophyletic. PMID- 8636858 TI - Haemoproteus iwa n. sp. in great frigatebirds (Fregata minor [Gmelin]) from Hawaii: parasite morphology and prevalence. AB - We describe a new species of Haemoproteus Kruse, 1890 from great frigatebirds (Fregata minor [Gmelin]) captured on Tern Island-French Frigate Shoals and Laysan Island in Hawaii. Parasite prevalence on Laysan Island (35%) was not significantly different than that of Tern Island (36%). On Laysan, prevalence was highest in juveniles (52%), followed by adult males (29%) and adult females (19%). Prevalence on Tern was 36% both for adult females and juveniles, and 28% for adult males. Parasitemia was low (mean < 2 parasites/10, 000 red blood cell). Parasitized red cells had significantly greater areas than unparasitized cells. We named this parasite Haemoproteus iwa after the Hawaiian name for frigatebirds (iwa). This is the first documentation of a hemoparasite from tropical pelagic seabirds in Hawaii and the first description of an endemic hemoparasite in the archipelago. PMID- 8636859 TI - Infectivity of Hymenolepis diminuta for the jird, Meriones unguiculatus, and utility of this model for anthelmintic studies. AB - The jird (Meriones unguiculatus) has been shown to be a useful model host for the cestodes Taenia crassiceps and Echinococcus multilocularis. This report outlines a novel model in which hydrocortisone-treated jirds (0.02% in the feed) are infected with another cestode, Hymenolepis diminuta. Jirds were inoculated with 5 freshly harvested cysticercoids of H. diminuta prior to (day 0, -1, or -5) or after (day 1 or 5) switching to medicated feed; in some cases, jirds were never medicated. On days 7, 14, 21, or 28 postinoculation (PI), jirds were killed by CO2 inhalation and their small intestines were examined for tapeworms. Hymenolepis diminuta established, grew, and developed to the gravid adult state in jirds. They persisted longer in medicated (21 days) than in nonmedicated (7 days) animals, and generally higher levels of infection were obtained when jirds were inoculated immediately prior to switching to medicated feed. Treatment of infected jirds on day 4 or days 4, 5, and 6 PI with selected anthelmintics followed by necropsy on day 7 PI discriminated drugs with known activity against tapeworms from those with little or no activity. This rodent in vivo model should provide a useful adjunct for anthelmintic studies. PMID- 8636860 TI - Interaction of the human serine protease inhibitor alpha-1-antitrypsin with Cryptosporidium parvum. AB - The protozoan parasite Cryptosporidium parvum was studied for interaction with a human serine protease inhibitor (serpin), alpha-1-antitrypsin (AAT). A C. parvum homogenate (CPH) prepared from oocysts was incubated with purified human AAT and complexes formed between the serpin and CPH were detected using an enzyme-linked immunosorbent assay (ELISA). The optical density read at 450 nm of AAT:CPH reactivity was significantly increased (P < 0.001) relative to CPH in the absence of AAT treatment. Additionally, ELISA reactivity was blocked by incubating AAT with a cognate target enzyme, porcine pancreatic elastase (PPE), prior to treatment of the CPH. Incubation of a partially excysted sample of C. parvum with AAT (37 C x 60 min) demonstrated preferential fluorescence labeling of sporozoites by indirect immunofluorescence assay; AAT complexes were not detected on intact oocysts. Localization of AAT interactions with C. parvum sporozoites was visualized by transmission immunoelectron microscopy. Collectively, these data suggest that C. parvum sporozoites express a protease-like component that is recognized by human AAT. The ability to block ELISA reactivity with PPE suggests that the AAT interactions we detected are functionally similar to the serpin enzyme complex AAT forms with a protease target. PMID- 8636861 TI - Cypria reptans (Crustacea: Ostracoda) as an intermediate host of Neoechinorhynchus rutili (Acanthocephala: Eoacanthocephala) in Italy. AB - A total of 2,568 specimens of 3 species of ostracod, namely Herpetocypris sp., Ilyocypris sp., and Cypria reptans Brohnstein, 1928, was collected between November 1994 and April 1995 from the Ceresina Canal, a tributary of the River Brenta (northern Italy), and examined for larval helminths. Larvae of Neoechinorhynchus rutili Muller, 1780 were found in the hemocoel of C. reptans. The prevalence of infection ranged from 8.57 to 14.51%. Most larvae were in the cystacanth stage (a new geographical record for this cystacanth). Not more than 3 larvae were encountered in a single host. Among 48 aquatic alder fly larvae of Sialis lutaria collected over 6 mo, together with the crustacean, no infected larvae were encountered. Specimens of Gasterosteus aculeatus (Pisces: Gasterosteiformis) collected with the arthropods from the same site were found to be parasitized with adult N. rutili. Fish stomach content analysis showed ostracods to be a major food item of three-spined sticklebacks in the study area during much of the year. PMID- 8636863 TI - Synlophe of Batrachonema synaptospicula (Nematoda:Amphibiophilidae) collected from Malaysian frogs. AB - The synlophe of Batrachonema synaptospicula Yuen, 1965 collected from Rana limnocharis Boie, 1835 of peninsular Malaysia was found to be identical morphologically to that in the specimens from Rana narina Stejneger, 1901 of Okinawa, and R. limnocharis of Taiwan. In the midbody, 20-22 ridges are present, and the ridges increase gradually in size and are oriented from right to left in the dorsal and left ventral fields, whereas the right ventral ridges are small and almost perpendicular to the body wall. The orientation of ridges from right to left is considered to be a key characteristic of the genus Batrachonema. Because Amphibiophilus ranae Wang et al., 1978 and Amphibiophilus sp. from R. limnocharis of south China are regarded to be conspecific with B. synaptospicula, this nematode is surmised to be distributed widely in southeast and east Asia. PMID- 8636862 TI - Ticks parasitizing humans in Georgia and South Carolina. AB - From 1990 through 1995, 913 ticks removed from 460 human patients in Georgia or South Carolina were identified and recorded. The majority of these specimens (758, 83.0%) were lone star ticks, Amblyomma americanum. One hundred and four (11.4%) American dog ticks Dermacentor variabilis, 36 (3.9%) blacklegged ticks Ixodes scapularis, 9 (1.0%) Gulf coast ticks Amblyomma maculatum, and 6 (0.7%) brown dog ticks Rhipicephalus sanguineus were also recovered. All active stages (larvae, nymphs, and adults) of A. americanum were represented, whereas nymphs and adults of D. variabilis and I. scapularis and only adults of A. maculatum and R. sanguineus were recorded. Compared with data published for other regions in the U.S.A., A. americanum was a much more prevalent parasite of humans in the current survey. Only 1 (3%) of the I. scapularis collected was a nymph. Because these tick species are vectors of zoonotic pathogens or cause tick paralysis in humans, the data have epidemiological significance. PMID- 8636864 TI - Identification of the telomeres on Schistosoma mansoni chromosomes by FISH. AB - The telomeres (molecular termini) of chromosomes play an important role in maintaining the structural integrity of the chromosome. Using a deoxyoligomer that contains the core sequence TTAGGG, we identified by fluorescence in situ hybridization (FISH) the telomeres on all 8 pairs of Schistosoma mansoni mitotic metaphase chromosomes. Our results indicate that S. mansoni exhibits a telomere sequence typical of other species. PMID- 8636865 TI - Archaeoparasitology at a 17th century colonial site in Newfoundland. AB - Excavations at the 17th century site of Ferryland during the past 2 summers have revealed thousands of artifacts and the remains of several structures. Of particular interest here was the discovery of the remains of a privy and associated stable. Examination of privy contents revealed the presence of eggs of 4 parasites: Ascaris, Trichuris, Taenia, and Dicrocoelium. Their identification represents the first reported discovery of parasite remains in an archeological context in Canada. Due to possible contamination by domestic animal feces, it was not possible to determine with certainty if the eggs are of human origin. PMID- 8636866 TI - Systemic infection with an unidentified Toxoplasma-like protozoan in a neonatal Lichtenstein's hartebeest (Sigmoceros lichtensteinii). AB - An unidentified Toxoplasma gondii-like disseminated infection was found in tissues of a 2-day-old hartebeest. Protozoal tachyzoites were about 3.5 x 1.7 Microns, had electron-dense rhoptries, and divided by endodyogeny. The organism did not stain with polyclonol antibodies to T. gondii, Neospora caninum, or Sarcocystis cruzi. PMID- 8636867 TI - Prevalence and identity of Sarcocystis infections in armadillos (Dasypus novemcinctus). AB - Little is known about the prevalence or identity of Sarcocystis species infecting armadillos in North America. Sarcocysts were observed in the tongues of 23 (96%) of 24 armadillos collected between 1989 and 1994 from Texas, Oklahoma, Kansas, and Arkansas. The identity of the species present was determined in histological sections of tongue from armadillos. Sarcocystis dasypi was present in 21 (88%) and Sarcocystis diminuta was present in 5 (21%). Mixed infections with S. dasypi and S. diminuta were present in 3 (13%) armadillos. A single sarcocyst with ultrastructural features distinct from S. dasypi and S. diminuta was observed with transmission electron microscopy. PMID- 8636868 TI - Identification of a common filarial larva in Simulium damnosum s.l. (type D, Duke, 1967) as Onchocerca ramachandrini from the wart hog. AB - Filarial larvae resembling Type D (Duke, 1967), which are common in the Simulium damnosum s.i. vectors of human onchocerciasis ("riverblindness") in several parts of West Africa, were dissected from wild-caught flies in north Cameroon and examined morphologically. This was done in order to establish a possible synonymy with infective larvae (L3) of 2 recently discovered Onchocerca species of wart hogs (Onchocerca ramachandrini Bain, Wahl, and Renz, 1993 and Onchocerca sp. Wahl and Bain, 1995), which had been found to resemble Type D. After dissection of approximately 1,700 S. damnosum s.1., 13 Type D-like larvae were recovered from 12 infected flies. Their morphology corresponded to O. ramachandrini. PMID- 8636869 TI - Microwave applicator design for cardiac tissue ablations. AB - A variety of microwave applicators were designed, fabricated and tested for catheter applications: I-radiators, U-radiators, O-radiators, forward helical coil radiator, reverse helical coil, double coil radiator, loaded monopole radiator, leaky coaxial radiator and tee radiators. The comparative and relative radiation characteristics of these applicators were tested in a saline bath and tissues. Most radiators designed produced larger lesions than have been described previously. PMID- 8636870 TI - Effect of model fitting artifacts on the stepwise approach to identifying patterns of attachment loss. AB - The stepwise approach to the determination of periodontal attachment loss involves fitting linear, logarithmic, and exponential models to individual site data and concluding that the form of loss is consistent with the model that has the greatest r-value, provided that the model predicts loss in excess of a site specific threshold. Logarithmic and exponential fits are considered to define early and late bursts, respectively, while linear fit describes loss at a constant rate. In a recently published study, the stepwise approach was applied to 6,935 sites in patients with established periodontitis and, of 581 loss detections, 195 (33.6%) were linear, 224 (38.6%) logarithmic, and 162 (27.9%) exponential. However, curvilinear patterns may occur by chance and regression algorithms that can fit such curvature may have an advantage unrelated to the true mechanism(s) [correction of mchanism(s)] of periodontal destruction. To investigate the implications of this possibility, proportions of linear, logarithmic, and exponential fit were estimated by simulation under four different conditions. These conditions incorporated random or random plus linear change, but no nonlinear effects. The relative proportions of model fits described in the published study were approximated in all of these conditions. It would appear that the observed proportions are ubiquitous to the modelling approach itself, and do not constitute evidence of a causal non-linear biological mechanism. The stepwise approach may be useful for detecting change but relevance to causal processes seems problematic. PMID- 8636871 TI - Profiles of destructive periodontal disease in different populations. AB - In this study we evaluated the traditional view that the severity of periodontal disease varies between populations in that African and Asian populations are more severely affected than other populations. Our data on periodontal destruction in two random samples of a Kenyan and a Chinese adult population were recalculated to conform with the methods of examination and data presentation utilized in each of 6 other studies of attachment loss levels in different populations. The adult Kenyan and the adult Chinese group, who had very poor oral hygiene conditions and massive gingival inflammation, had attachment loss levels which were quite similar to those in a Japanese population (31), in a Norwegian population (27) and in a New Mexico group of adults (30). Attachment losses were similar in a population of young US adults (26) aged between 35 and 60 years relative to the corresponding Kenyan and Chinese groups while young US citizens had higher and elderly US citizens had lower mean attachment levels than either Kenyans and Chinese. Higher attachment loss levels beyond the age of 27 years were reported for a population of Sri Lankan Tamil tea workers (27) and across all ages in two South Pacific island populations (29). Overall, the analysis indicates that the periodontal attachment loss profiles may differ between populations, but that these differences do not conform with the traditional generalization that African and Asian populations suffer more severe periodontal breakdown than other populations. PMID- 8636872 TI - Biased T cell receptor V gene usage in tissues with periodontal disease. AB - In an attempt to characterize TCR V gene usage in human periodontally diseased tissue, V alpha 2, V beta 5.2-3, V beta 5.3, V beta 5.1, V beta 6.7, V beta 6.7, V beta 8 and V beta 12.1 expressions were examined. Serial cryostat sections obtained from 20 periodontitis and 9 gingivitis biopsies were then reacted with monoclonal antibodies directed to each repertoire. The technique was combined with a sensitive alkaline phosphatase-anti-alkaline phosphatase method. Peripheral blood was obtained from 10 periodontitis and 2 gingivitis patients. TCR repertoire was also quantified by flow cytofluorography with FITC-conjugated antibodies. Cells displaying binding of each antibody were counted. The proportions to CD3-positive cells were then calculated. The pattern of each TCR V gene product expression in inflamed gingiva exhibited individual variation, nevertheless, a consistent pattern emerged. The V beta 5 subfamily and V beta 6.7 were frequently used repertoires in gingiva, whereas the V alpha 2 and V beta 8 subfamily were underexpressed in most cases. Furthermore, the TCR V gene product expression in gingival tissue was biased compared with autologous peripheral blood. Three of 10 periodontitis subjects showed 1 or 2 strikingly overrepresented repertoire comparatively with autologous blood. In these 3 subjects V beta 6.7 was overexpressed in two cases and 5.2-3, V beta 8 and V beta 12.1 were overexpressed in one case. These results suggest that gingival T-cells are not randomly mobilized from peripheral blood and that local events influence the TCR repertoire at the level of T-cell recruitment or T-cell expansion. PMID- 8636873 TI - Comparison of randomly cloned and whole genomic DNA probes for the detection of Porphyromonas gingivalis and Bacteroides forsythus. AB - Whole genomic and randomly-cloned DNA probes for two fastidious periodontal pathogens, Porphyromonas gingivalis and Bacteroides forsythus were labeled with digoxigenin and detected by a colorimetric method. The specificity and sensitivity of the whole genomic and cloned probes were compared. The cloned probes were highly specific compared to the whole genomic probes. A significant degree of cross-reactivity with Bacteroides species, Capnocytophaga sp. and Prevotella sp. was observed with the whole genomic probes. The cloned probes were less sensitive than the whole genomic probes and required at least 10(6) target cells or a minimum of 10 ng of target DNA to be detected during hybridization. Although a ten-fold increase in sensitivity was obtained with the whole genomic probes, cross-hybridization to closely related species limits their reliability in identifying target bacteria in subgingival plaque samples. PMID- 8636874 TI - Immunohistochemical localization of very late activation integrins in healthy and diseased human gingiva. AB - The beta 1-integrins (VLA family) are cellular adhesion molecules (CAM) that play a major role in cell-cell and cell-matrix interactions. The expression pattern of CAM was studied in 5 clinically normal volunteers with healthy gingiva and in 18 patients with clinically different stages of periodontitis. In healthy human gingiva alpha 2, alpha 3 and alpha 6 integrin chains were found in a characteristic distribution, showing a broad continuous expression on the junctional and sulcular epithelium sites. The expression of these integrins was demonstrated primarily on the basal cell layers and in some cells of the stratum spinosum. Inflammatory stages of periodontitis revealed further upregulation of alpha 2, alpha 3 and alpha 6 integrins into the junctional and sulcular epithelial cells, which correlated with the stage of the periodontitis and the extent of the cellular infiltration. alpha 4 and alpha 6 were found to be the predominant beta 1 integrin chains on inflammatory cells. The amount of alpha 4 and alpha 6 positive infiltrative cells increased with the number of inflammatory cells. VCAM-1, the corresponding cell-cell ligand of VLA-4 (alpha 4) was present on the majority of subepithelial vessels in all stages of gingivitis and periodontitis. The alpha 5 subunit was expressed on both endothelium and gingival connective tissue cells. Samples from advanced periodontitis cases showed a higher number of alpha 5 positive mononuclear cells. In comparison to normal epidermis, human gingival epithelial cells express higher levels of integrins. This expression is further upregulated in advanced stages of periodontitis, indicating changes of the beta 1 integrin organization. PMID- 8636875 TI - Analysis in gingival crevicular fluid of two oligopeptides derived from human hemoglobin beta-chain. AB - HPLC on a reversed phase column, amino acid sequencing and mass spectrometry were used to determine the structure of two human gingival crevicular exudate oligopeptides (Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu and Leu-Val-Val Tyr-Pro-Trp-Thr-Gln-Arg-Phe) which were shown to have been derived from the beta chain of hemoglobin. These sequences may simply represent two degradation products of the beta-chain. However, their preservation in an exudate characterized by active peptidolysis may also prompt the question about their possible more specific role. PMID- 8636876 TI - Characterization of T lymphocyte clones derived from Porphyromonas gingivalis infected subjects. AB - Porphyromonas gingivalis plays a major role in the pathogenesis of periodontal disease, however some individuals with P. gingivalis infection do not experience periodontal breakdown. The aim of this study was to investigate the proliferative responses of two highly defined groups of subjects and to establish and characterize peripheral blood and gingival cell T cell lines and clones from subjects from these groups. The two groups were selected on the basis of P. gingivalis in their plaque and the presence of serum anti-P. gingivalis antibodies. Both groups therefore were seen to have P. gingivalis and to have responded to it. They however differed only in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. Dose responses of peripheral blood mononuclear cells extracted from the subjects showed a trend towards a lower response by the adult periodontitis group to P. gingivalis outer membrane (OM) antigens. Peripheral blood T cell lines and clones responsive to P. gingivalis OM were established from a high responding gingivitis subject and a low responding adult periodontitis subject. Gingival T cell lines and clones were also derived from cells extracted from the periodontal tissues of the same periodontitis subject. The majority of T cells in the peripheral blood T cell line from the gingivitis subject were CD4 while those from the adult periodontitis subject were CD8. The gingival T cell line was CD3+ve CD4-ve and CD8-ve. All lines and clones proliferated slowly to P. gingivalis OM but phytohaemagglutinin (PHA) induced an increase in DNA synthesis in those derived from the gingivitis subject with little to no effect on those established from the adult periodontitis subject. Furthermore, PHA inhibited the proliferative response of the CD8 clone derived from the adult periodontitis subject. Phenotypic analysis demonstrated that all the peripheral blood clones expressed the alpha beta TCR while the gingival T cell clones expressed the gamma-delta TCR. All clones had the memory/primed CD45RO+ve phenotype and at least 80% of cells in each clone were HLA-DR+ve. A lower percent of gingival cells expressed CD45RA than the CD4 peripheral blood clones and the two CD8 clones also had a decreased CD45RA expression. The gingival T cell clones also expressed a low percent CD25 as did the CD8 clone derived from the adult periodontitis subject. The results suggest that clones derived from the gingivitis and adult periodontitis subject may be functionally different. The presence of gamma-delta T cells in adult periodontitis remains to be confirmed and their function determined. PMID- 8636877 TI - Protein composition of whole and parotid saliva in healthy and periodontitis subjects. Determination of cystatins, albumin, amylase and IgA. AB - Cystatins are physiological inhibitors of cysteine proteinases and they are widely distributed in human tissues and body fluids including saliva. We previously reported an increased cystatin activity in whole saliva of gingivitis and periodontitis subjects. Based on this result we decided to investigate the type and origin of cystatins involved in this increased cystatin activity by collecting both whole and parotid saliva of 25 healthy and 30 periodontitis subjects. Saliva samples were quantified for cystatins S and C by enzyme-linked immunosorbent assay and cystatin activities were measured toward papain. Besides, three other salivary proteins were determined: the plasma protein albumin, the typical parotid derived amylase and the salivary immunoglobulin IgA. The present investigation shows that levels of total protein and cystatin activity as well as the levels of glandular derived proteins amylase and cystatin C were significantly higher in whole and parotid saliva of subjects with periodontitis than in healthy controls. Cystatin S, the major salivary cystatin, however was higher in the whole saliva of the healthy group. Whole saliva concentrations of albumin and IgA, originating from sources other than the glandular cells, were not different between healthy and periodontitis subjects and were also not correlated with the typical salivary gland proteins. In conclusion, this study provides additional evidence that the human salivary glands may respond to an inflammatory disease of the oral cavity, periodontitis, by enhanced synthesis of some acinar proteins. PMID- 8636878 TI - Loss of attachment in the marginal periodontium of the rat incisor under non inflammatory conditions. Expression of alkaline phosphatase activity. Experimental Oral Biology Group. AB - Alkaline phosphatase (ALP) has been suggested to play a role in acellular cementum formation and maintenance of periodontal attachment. In an attempt to determine whether changes in attachment level are associated with altered expression of ALP-activity in the periodontium we induced natural loss of attachment in rats by pinning the lower incisor to the jaw bone. Previous studies have shown that this procedure results in regressive changes in the marginal periodontium without any inflammatory response. Six months after blockage of eruption the attachment level on the experimental (right) side had shifted about 700 microm in the apical direction. On the control (left) side the apical termination of the junctional epithelium had remained stationary with respect to the alveolar crest. Our observations have shown that during the first few weeks of the experiment loss of attachment is accompanied by considerable reduction of ALP-activity in the supracrestal part of the periodontium. At later time intervals, however, no distinct relation was found between apical migration of junctional epithelium and loss of ALP-activity in the supracrestal region, indicating that the two phenomena are not directly related to each other. The domain of the ALP-positive fibroblasts in the supracrestal extension of the periodontal ligament decreased in size and was replaced by ALP-negative connective tissue cells probably coming from the outer gingival domain. Since at all time intervals a distinct demarcation could be observed between the ALP positive and ALP-negative areas, we interpret our data as indicating that ligament and gingival cells do not mix. PMID- 8636879 TI - Cell-bound and extracellular matrix-associated alkaline phosphatase activity in rat periodontal ligament. Experimental Oral Biology Group. AB - In previous studies it was noted that alkaline phosphatase (ALP) activity in periodontal ligament does not only seem to be related to cells but may also be associated with the extracellular matrix. In an attempt to clarify this we studied the distribution of the enzyme at the electron microscopic level. In addition, ALP-activity was assessed biochemically following extraction of the ligament with (i) agents dissolving the membrane or splitting the phosphatidylinositol anchor (Triton X-100 or phosphatidylinositol-phospholipase C, respectively), and (ii) a matrix-degrading enzyme cocktail (collagenase, hyaluronidase and elastase). Histochemical observations revealed (a) a heterogeneous distribution of ALP-activity, with highest activity adjacent to the alveolar bone and (b) two pools of activity; one bound to cells and one associated with the collagenous extracellular matrix. In line with this were the biochemical data indicating that approximately 10% of the enzyme activity was firmly bound to the extracellular matrix and 90% to plasma membranes. Isoelectric focusing did not reveal differences between the two fractions, both samples yielding a single broad band corresponding with an isoelectric point of about 4.4. PMID- 8636880 TI - Do facial expressions signal specific emotions? Judging emotion from the face in context. AB - Certain facial expressions have been theorized to be easily recognizable signals of specific emotions. If so, these expressions should override situationally based expectations used by a person in attributing an emotion to another. An alternative account is offered in which the face provides information relevant to emotion but does not signal a specific emotion. Therefore, in specified circumstances, situational rather than facial information was predicted to determine the judged emotion. This prediction was supported in 3 studies--indeed, in each of the 22 cases examined (e.g., a person in a frightening situation but displaying a reported "facial expression of anger" was judged as afraid). Situational information was especially influential when it suggested a nonbasic emotion (e.g., a person in a painful situation but displaying a "facial expression of fear" was judged as in pain). PMID- 8636881 TI - Effects of adult attachment and presence of romantic partners on physiological responses to stress. AB - The effects of presence vs. absence of a romantic partner on psychophysiological responses to a stressful laboratory situation were examined in a sample of 35 college women involved in serious dating relationships. Participants performed a standard psychological stress task both in the presence and in the absence of their romantic partners, with order counterbalanced across participants. Heart rate and blood pressure were measured in each of these conditions during both baseline and task performance periods. Avoidant (but not secure) and anxious (but not nonanxious) participants displayed heightened physiological responses across all conditions and periods if the partner-absent condition came first rather than second. Discussion focuses on 2 explanations for the findings in terms of (a) the anxiety-reducing function of attachment relationships and (b) the anxiety producing effect of separation in a stressful situation. PMID- 8636882 TI - Social constraints, intrusive thoughts, and depressive symptoms among bereaved mothers. AB - The study examined how social constraints on discussion of a traumatic experience can interfere with cognitive processing of and recovery from loss. Bereaved mothers were interviewed at 3 weeks (T1), 3 months (T2), and 18 months (T3) after their infants' death. Intrusive thoughts at T1, conceptualized as a marker of cognitive processing, were negatively associated with talking about infant's death at T2 and T3 among socially constrained mothers. The reverse associations were found among unconstrained mothers. Controlling for initial level of distress, there was a positive relation between T1 intrusive thoughts and depressive symptoms over time among socially constrained mothers. However, higher levels of T1 intrusive thoughts were associated with a decrease in T3 depressive symptoms among mothers with unconstrained social relationships. PMID- 8636883 TI - Close relationships and adjustment to a life crisis: the case of breast cancer. AB - When life crises occur, significant others are thought to help alleviate distress and resolve practical problems. Yet life crises may overwhelm significant others, eroding their ability to provide effective support. The accuracy of these contrasting accounts of relationship functioning was evaluated in a study of 102 breast cancer patients and their significant others, interviewed at 4 and 10 months after diagnosis. Results largely confirmed the negative account of relationship functioning. Although significant others provided support in response to patients' physical impairment, they withdrew support in response to patients' emotional distress. Moreover, support from significant others did not alleviate patients' distress or promote physical recovery. These results reveal limits to the effectiveness of close relationships in times of severe stress. PMID- 8636884 TI - Adult attachment security and symptoms of depression: the mediating roles of dysfunctional attitudes and low self-esteem. AB - Three studies investigated the relation between adult attachment security and symptoms of depression. Study l examined the overall magnitude of the association between adult attachment and depression, and Studies 2 and 3 tested whether this relation was mediated by dysfunctional attitudes and low self-esteem. Results from the three studies were consistent with a mediation model. This model suggests that insecure adult attachment styles are associated with dysfunctional attitudes, which in turn predispose to lower levels of self-esteem. Such depletions in self-esteem are directly associated with increases in depressive symptoms over time. Insecure attachment appears to lead to depressive symptoms in adulthood through its impact on self-worth contingencies and self-esteem. PMID- 8636885 TI - Development and validation of the State Hope Scale. AB - Defining hope as a cognitive set comprising agency (belief in one's capacity to initiate and sustain actions) and pathways (belief in one's capacity to generate routes) to reach goals, the Hope Scale was developed and validated previously as a dispositional self-report measure of hope (Snyder et al., 1991). The present 4 studies were designed to develop and validate a measure of state hope. The 6-item State Hope Scale is internally consistent and reflects the theorized agency and pathways components. The relationships of the State Hope Scale to other measures demonstrate concurrent and discriminant validity; moreover, the scale is responsive to events in the lives of people as evidenced by data gathered through both correlational and causal designs. The State Hope Scale offers a brief, internally consistent, and valid self-report measure of ongoing goal-directed thinking that may be useful to researchers and applied professionals. PMID- 8636886 TI - Postbereavement depressive mood and its prebereavement predictors in HIV+ and HIV gay men. AB - Prebereavement predictors of the course of postbereavement depressive mood were examined in 110 gay men who were their partner's caregiver until the partner's death of AIDS. In all, 37 HIV+ and 73 HIV- bereaved caregiving partners were assessed bimonthly throughout a 10-month period beginning 3 months before and ending 7 months after the partner's death. Throughout the 10 months, mean Centers for Epidemiology Scale-Depression (CES-D) scores on depressive mood were above the cutoff for being at risk for major depression. CES-D scores decreased for 63% bereaved caregivers over the 7 postbereavement months, and 37% showed little change from high CES-D scores or increasing CES-D scores. High prebereavement CES D scores and finding positive meaning in caregiving predicted diminishing depressive mood; HIV+ serostatus, longer relationships, hassles, and use of distancing and self-blame to cope predicted unrelieved depressive mood. PMID- 8636887 TI - IQ and ego-resiliency: conceptual and empirical connections and separateness. AB - The constructs of intelligence and ego-resiliency are discussed. The personality implications of "pure intelligence" and "pure ego-resilience" were identified. Intelligence (IQ) was indexed by the Wechsler Adult Intelligence Scale-Revised and ego-resiliency by an inventory scale. Residual scores measuring "pure intelligence" and "pure ego-resilience" were correlated with the items of the observer-based California Q-sort, used to describe participants. Persons relatively high on ego-resilience tend to be more competent and comfortable in the "fuzzier" interpersonal world; persons defined primary by raw IQ tend to be effective in the "clearer" world of structured work but tend also to be uneasy with affect and less able to realize satisfying human connections. Gender differences exist in the relations of ego-resilience and intelligence and in their adaptive relevance. PMID- 8636888 TI - Individual differences in repressive-defensiveness predict basal salivary cortisol levels. AB - Prior studies assessing the relation between negative affective traits and cortisol have yielded inconsistent results. Two studies assessed the relation between individual differences in repressive-defensiveness and basal salivary cortisol levels. Experiment 1 assessed midafternoon salivary cortisol levels in men classified as repressors, high-anxious, or low-anxious. In Experiment 2, more rigorous controls were applied as salivary cortisol levels in women and men were assessed at 3 times of day on 3 separate days. In both studies, as hypothesized, repressors and high-anxious participants demonstrated higher basal cortisol levels than low-anxious participants. These findings suggest that both heightened distress and the inhibition of distress may be independently linked to relative elevations in cortisol. Also discussed is the possible mediational role of individual differences in responsivity to, or mobilization for, uncertainty or change. PMID- 8636889 TI - Stereotypes of emotional expressiveness of northerners and southerners: a cross cultural test of Montesquieu's hypotheses. AB - Montesquieu argued that residents of warmer climates are more emotionally expressive than those living in cooler ones. More than 2,900 college students from 26 countries completed a brief questionnaire assessing the degree to which they considered Northerners and Southerners within their own countries to be emotionally expressive. In addition, individuals rated themselves on their own degree of expressiveness. In partial confirmation of Montesquieu's hypothesis, it was found that large within-country North-South stereotypes exist. Especially in Old World countries, Northerners are viewed as less emotionally expressive than Southerners. Regression and other analyses revealed that self-ratings of expressiveness were, in fact, related to being from the South and to warmer mean temperatures. Several possible explanations for these effects are discussed. PMID- 8636890 TI - Major life events and minor stressors: identifying mediational links in the stress process. AB - Whether the relationship between major life events and distress is mediated through minor stressors was examined in three stress groups: those who (a) experienced the death of a spouse, (b) divorced, or (c) were the parent of a child with asthma. Each of these major stress groups was compared with a control group. Path analyses conducted by aggregating the data cross major stress groups indicated that major life events exert both a direct influence on distress and an indirect influence through minor stressors. On the other hand, the nature of the mediational relation linking major life events with psychological distress through minor stressors was found to vary as a function of the major life stress situation under consideration. Methodological and theoretical implications for the study of stress processes are discussed. PMID- 8636891 TI - Effects of failure on subsequent performance: the importance of self-defining goals. AB - Extending R.A. Wicklund and P.M. Gollwitzer's (1982) self-completion theory, 2 experiments examined the role of self-defining goals in predicting performance effects of failure among students committed to professional goals such as becoming a physician (Experiment 1) or a computer scientist (Experiment 2). Results of Experiment 1 revealed that failure on a task characterized as being relevant to students' professional self-definition led to (a) enhanced performance on a subsequent task relevant to the same self-definition and (b) impaired performance on a subsequent task unrelated to the self-definition challenged through prior failure. Experiment 2 replicated these findings. In addition, performance effects due to self-definitional failure were annulled when participants experience intermittent social recognition for the aspired-to-self definition. PMID- 8636892 TI - Self-anchoring and differentiation processes in the minimal group setting. AB - In-group favoritism in the minimal group setting was hypothesized to be a function of 2 processes: a tendency to base in-group judgments on the self (self anchoring) and a tendency to assume 1 group to be opposite of the other (differentiation). In the first 3 experiments, in which the order of rating the self and target group was varied, was categorized and uncategorized participants were given trait information about 1 group and were asked to estimate the level of those traits in the other group. In-group judges tended to base group ratings on the self, whereas out-group and uncategorized judges inferred the 2 groups to be opposite of one another. Experiment 4 attempted to directly assess the direction of inference between self and in-group by giving feedback about self or in-group on unfamiliar dimensions and found that participants were more willing to generalize from self to in-group than from in-group to self. PMID- 8636893 TI - Overhelping. AB - Overhelping occurs when one attempts to spoil an observer's impression of a performer by explicitly helping the performer achieve a goal, thereby inviting the observer to attribute the performer's success to the help. The results of 4 experiments suggest (a) that people are most likely to overhelp when they believe that their interventions will be ineffective but will be considered effective by observers and (b) that when either of these beliefs is wrong, the strategy will backfire. The results point to an intervention principle that predicts how and when people may most effectively influence a performance so as to shape observer's inferences about the performer. PMID- 8636894 TI - Effect of perspective taking on the cognitive representation of persons: a merging of self and other. AB - Two experiments examined the possibility that perspective taking leads observers to create cognitive representation of others that substantially overlap with the observers' own self-representations. In Experiment 1 observers receiving role taking instructions were more likely to ascribe traits to a novel target that they (observers) had earlier indicated were self-descriptive. This pattern was most pronounced, however for positively valenced traits. In Experiment 2 some participants received role-taking instructions but were also given a distracting memory task. In the absence of this task, role taking again produced greater overlap--primarily for positive traits--between self- and target representations. In the presence of the memory task, the degree of self-target overlap was significantly reduced for all traits, regardless of valence. Possible explanations for these findings are discussed. PMID- 8636895 TI - Influence of prototypes on perceptions of prejudice. AB - Two studies examined the influence of cultural stereotypes and personal factors (one's race, gender) on perceptions of racial and gender discrimination. Overall, the data suggest that our perceptions of prejudice are strongly influenced by specific expectations regarding who are the prototypic perpetrators and victims of prejudice. More general expectations regarding out-group conflict or regarding only the characteristics of the perpetrator appear to have less of an impact on such perceptions. Additionally, women were found to be more likely than men to perceive sexism directed against men and racism directed at African Americans and Caucasians. Also, African Americans were more likely than Caucasians to perceive racist events against Whites and Blacks. The implications of these data are discussed. PMID- 8636896 TI - On being cool and collected: mood regulation in anticipation of social interaction. AB - This study examined the influence of anticipated social interaction on the regulation of moods. Study 1 induced happy and sad moods through exposure to music. All participants expected to perform a second, unrelated experimental task either by themselves of with another participant. Participants who expected to do the task alone subsequently selected positive and negative news stories equally, but those who expected to interact preferred stories containing material incongruent with their mood. Study 2 confirmed this outcome, but showed it was confined primarily to anticipation of interaction with partners who are expected to be in neutral or good moods themselves. In Study 3, participants whose mood was not manipulated reduced self-exposure to cheerful or depressing videos when they expected to interact with another. PMID- 8636897 TI - Internalization of biopsychosocial values by medical students: a test of self determination theory. AB - Two studies tested self-determination theory with 2nd-year medical students in an interviewing course. Study 1 revealed that (a) individuals with a more autonomous orientation on the General Causality Orientation Scale had higher psychosocial beliefs at the beginning of the course and reported more autonomous reasons for participating in the course, and (b) students who perceived their instructors as more autonomy-supportive became more autonomous in their learning during the 6 month course. Study 2, a 30-month longitudinal study, revealed that students who perceived their instructors as more autonomy-supportive became more autonomous in their learning, which in turn accounted for a significant increase in both perceived competence and psychosocial beliefs over the 20-week period of the course, more autonomy support when interviewing a simulated patient 6 months later, and stronger psychosocial beliefs 2 years later. PMID- 8636898 TI - Assessing individual differences in constructive versus destructive responses to anger across the lifespan. AB - Scenario-based, self-report measures were developed to assess how people characteristically experience and manage anger from middle childhood through adulthood. The Anger Response Inventories (ARIs) for children, adolescents, and adults each assess (a) anger arousal, (b) intentions, (c) cognitive and behavioral responses, and (d) Long-term consequences. Several independent studies provide support for the reliability and validity of the ARIs. Theoretically consistent patterns of correlations were observed with (a) global self-report measures of hostility, aggression, and anger-management strategies (adult version); (b) teacher reports of behavioral and emotional adjustment (child and adolescent versions); and (c) self- and family-member reports of behaviors on specific anger episodes (adolescent and adult versions). Findings from additional personality and developmental studies are summarized, further supporting construct validity. PMID- 8636899 TI - Relation of shame and guilt to constructive versus destructive responses to anger across the lifespan. AB - This study explored the relation of shame proneness and guilt proneness to constructive versus destructive responses to anger among 302 children (Grades 4 6), adolescents (Grades 7-11), 176 college students, and 194 adults. Across all ages, shame proneness was clearly related to maladaptive response to anger, including malevolent intentions; direct, indirect, and displaced aggression; self directed hostility; and negative long-term consequences. In contrast, guilt proneness was associated with constructive means of handling anger, including constructive intentions, corrective action and non-hostile discussion with the target of the anger, cognitive reappraisals of the target's role, and positive long-term consequences. Escapist-diffusing responses showed some interesting developmental trends. Among children, these dimensions were positively correlated with guilt and largely unrelated to shame; among older participants, the results were mixed. PMID- 8636901 TI - Perceiving interpersonal conflict and reacting to it: the case for agreeableness. AB - Two converging, multimethod studies probed the hypothesis that individual differences in Agreeableness are related to patterns of interpersonal conflict. In Study 1, participants (N = 263) evaluated the efficacy of 11 modes of conflict resolution within the context of 5 different interpersonal relationships. Across all relationships, high- and low-agreeable participants rated negotiation and disengagement tactics as better choices that power assertion tactics. However, low-agreeable participants rated power assertion as a better choice than did high agreeable participants. In Study 2, participants (N = 124) were assigned partners and were asked to resolve jointly 2 social conflict problems. Partners were videotaped, and observers coded behaviors. Participants also completed ratings of perceived conflict, partner perception, and liking of their partner. Agreeableness differences, sex of participant, and type of dyad partner were related to patterns of interpersonal conflict. Results were discussed in terms of personality and social influences during interpersonal conflict. PMID- 8636900 TI - Positive and negative global self-esteem: a substantively meaningful distinction or artifactors? AB - Global self-esteem based on M. Rosenberg's (1965) scale is typically treated as a unidimensional scale. However, factor analyses suggest separate factors associated with positively and negatively worded items, and there is an ongoing debate about the substantive meaningfulness of this distinction. Confirmatory factor analysis (CFA) was used to evaluate alternative 1- and 2-factor models and to test hypotheses about how the factors vary with reading ability and age. Responses based on the National Longitudinal Study of 1988 (S.J. Ingles et al., 1992) reflected a relatively unidimensional factor and method effects associated with negatively worded items. Such effects are common in rating scale responses, and this CFA approach may be useful in evaluating whether factors associated with positively and negatively worded items are substantively meaningful or artifactors. PMID- 8636902 TI - Dispositional and situational determinants of repression. AB - This research project posits a model of repression that incorporates both repressive personality and repressive social behavior. The 1st parameter of the model specifies the motivation for repressors' distancing of themselves from emotional events. Experiment 1 demonstrates that repressors are hypersensitive- in their cognitive attention--to both negative and positive emotional events. The 2nd parameter of the model specifies the conditions under which repressors distance themselves from emotional events. Experiments 2 and 3 demonstrate that repressors psychologically distance themselves when the situation threatens their self-evaluation and provides opportunity for them to attend to and process self relevant and non-self-relevant information. This 2-factor model extends the current conceptualization of repression in that it identifies motivation (dispositional emotional sensitivity) and context (situational threats to self evaluation and distraction availability) for repressors' distancing of themselves from negative and positive emotional events. PMID- 8636903 TI - Self-handicapping and intrinsic motivation: buffering intrinsic motivation from the threat of failure. AB - High and low self-handicappers (as measured by E. E. Jones & F. Rhodewalt's [1982] Self-Handicapping Scale) were asked to play a game of pinball (in a competitive or noncompetitive setting) after they had practices as much as they wanted on a related task (thus, not practicing could have served as a self handicap). High self-handicappers who did not practice much became more involved in the game and subsequently reported enjoying the game more than high self handicappers who practiced a lot. Furthermore, the effects on enjoyment were mediated by task involvement, suggesting that the protection afforded by self handicapping affects intrinsic motivation by allowing the individual to become absorbed in the activity instead of focusing on performance concerns. Individuals who self-handicap may be providing themselves with the "breathing room" they need to become absorbed in an activity and to experience the activity as enjoyable. PMID- 8636904 TI - The relationship of death anxiety with age and psychosocial maturity. AB - For this study, 194 respondents completed a biographical data sheet, the Templer (1970) Death Anxiety Scale and the Constantinople (1973) Inventory of Psychosocial Development to help assess the relationship among death anxiety, age, and psychosocial maturity. Findings showed that psychosocial maturity was a better predictor of death anxiety than age was. However, both variables were significantly negatively correlated with death anxiety, revealing that as psychosocial maturity and age increase, death anxiety decreases. PMID- 8636905 TI - Psychophysiological aspects of autistic disorders: overview. AB - The neurological, neurochemical, and neurotransmitter level differences as well as genetic influences associated with autism have been studied extensively in the last two decades. The varied findings from research offer hope for better understanding, effective treatment, and, perhaps, cure of this pervasive developmental disorder. PMID- 8636907 TI - Further contrasts between self-reflectiveness and internal state awareness factors of private self-consciousness. AB - Although widely used as a unitary measure of self-focused attention, the Private Self-Consciousness subscale (Fenigstein, Scheier, & Buss, 1975) contains two factors. In the present study, this subscale and its self-reflectiveness (SR) factor predicted greater shame, guilt, other-directedness, and social anxiety; but the internal state awareness (ISA) factor displayed relationships that were in the opposite direction. Contrasts between SR and ISA often became more obvious in partial correlations, when one factor was examined while controlling for the other. In relationships with personal and social identity, SR appeared to reflect public as much as private self-consciousness. These data support recent suggestions that it may be necessary to construct more adequate measures of private self-consciousness. PMID- 8636906 TI - His and her individualisms? Sex bias and individualism in psychologists' responses to case vignettes. AB - Individualism and sexism have been identified as important and problematic biases in psychotherapy. The extent to which psychologists use individualist values differentially in their responses to clinical case vignettes describing men and women was examined. Two samples of practicing psychologists (N=229) responded to 14 clinical vignettes by choosing initial hypotheses about the client that reflected either a utilitarian, instrumental (traditionally masculine) perspective or expressive (traditionally feminine) themes. Two sex-of-client manipulations were conducted, one of which resulted in a sex-of-client effect. A loglinear logit analysis of repeated measures conducted with the non-manipulated vignettes indicated a marked preference for the utilitarian form of individualism for males in response to their clinical difficulties, whereas the responses for female clients were more balanced on the utilitarian and expressive alternatives. There was little evidence of a sex-of-respondent effect. PMID- 8636908 TI - The independence of physical attractiveness and symptoms of depression in a female twin population. AB - The relationship between physical attractiveness and symptoms of depression was investigated in a general population simple of 1,100 female twins. Photographs were rated by 4 raters. Symptoms of depression were measured by the Depression sub-scale of the SCL-54, by a self-rating based on the DSM-III-R, and by an MD diagnosis based on a structured interview (SCID). No relationships between ratings of physical attractiveness and symptoms of depression were found. PMID- 8636909 TI - Examining the specificity of the orality-depression link: anal personality traits and depressive symptoms. PMID- 8636910 TI - Psychiatric disorders, inappropriate health service utilization and the role of consultation-liaison psychiatry. PMID- 8636911 TI - Two decades of alexithymia. PMID- 8636912 TI - Somatic complaints in the German population. AB - The Freiburg Complaint List (Freiburger Beschwerdenliste FBL-R) is a self-report questionnaire which consists of 80 somatic complaints. A survey was conducted in 1993 and a sample of N = 2070, representative for the German adult population of 16 years and above, was obtained. Factor analysis, cluster analysis and item analysis were employed to develop nine scales: General Condition, Tiredness, Cardiovascular, Gastrointestinal, Head-Throat, Tenseness, Emotional Reactivity, Pain, Sensory and Total Score. Regression analyses revealed substantial effects of gender, age group, and, to a lesser extent, social class on FBL-R scores. Furthermore, highly significant correlations were found between FBL-R scores and indicators of illness, health concern, and utilization of health services. The standardization provides normative data (stanine norms) that account for gender and age groups. This representative survey provides essential data, i.e. base rates of somatic complaints in the general population and subsamples. These base rates are relevant to the future development of criteria for diagnosis of somatization disorder. PMID- 8636913 TI - Coping strategies and defense mechanisms and their relevance for the recovery after discectomy. AB - Although several empirical studies have shown that psychological and social characteristics predict recovery after lumbar discectomy, the possible significance of the psychodynamic concept of defense mechanisms has been neglected. To investigate the predictive usefulness of defense mechanisms, coping strategies, and depression, 52 consecutive admissions were assessed before their operations and again six months later (n = 48). Using three outcome criteria, 8 patients (16, 7%) were classified as having poor operation outcomes. A stepwise discriminant analysis correctly classified 87.7% of these poor outcomes. The groups with poor and good outcome differed significantly in the prominence of two defense mechanisms: "rationalization" and "regression". PMID- 8636914 TI - The relationship between hassles, uplifts and irritable bowel syndrome: a preliminary study. AB - In a preliminary study, 30 sufferers of irritable bowel syndrome filled in daily symptom sheets and the combined Hassles and Uplifts questionnaire for five weeks. Initially, analyses were carried out for each symptom separately. Results showed that ratings on the hassles and symptoms questionnaires completed in the same week were more highly associated than ratings for hassles and symptoms in different weeks. No one symptom in any week was significantly associated with the following week's hassles. Similarly, hassles in any one week were not associated with the following week's symptoms. However, total symptoms were found to be significantly associated with hassles in the following weeks, whereas the association between hassles and total symptoms in the following weeks was not significant. The study suggests that there is a cumulative effect of symptoms such that an increase in the severity of the combined effect of symptoms is associated with an increase in severity of stress in the next week. Increased hassles do not appear to exacerbate symptoms. There was no evidence of an association between uplifts and IB symptoms. PMID- 8636915 TI - Alexithymia in primary health care patients. AB - Although many studies have been published about the relationship between alexithymia and different somatic diseases, little is known about the occurrence of alexithymia in primary health care patients. The aim of the present study was to shed light on this problem. The study forms part of a larger project dealing with psychiatric morbidity in primary health care patients. The original material consisted of 1,000 randomly selected adult patients in Turku in 1989-90. As part of a follow-up study (N = 748) three years later, alexithymia was measured using the Toronto Alexithymia Scale (TAS). Primary care patients seemed to have commonly alexithymic features: The mean of the TAS-score was 64.41 +/- 11.71 for male and 63.51 +/- 11.86 for female patients. The results indicated that alexithymia was associated with psychological distress, age, educational level, and socioeconomic status. The nature of alexithymia is discussed. PMID- 8636916 TI - Subjective complaints versus neuropsychological test performance after cardiopulmonary bypass. AB - The study by Newman et al. (Journal of Psychosomatic Research, 1989) compared subjective reports of cognition with assessed cognitive performance in patients one year after coronary artery bypass surgery. The current study reinvestigated this relation in a larger and more heterogeneous group--90 cardiac patients six months after cardiopulmonary bypass--using a more extensive checklist of subjective complaints and different neuropsychological tests. In agreement with previous research, the patients who reported complaints in specific cognitive areas were not found to have impaired cognitive functions as assessed with appropriate neuropsychological tests. The patients who reported deterioration in cognition after surgery were found to have higher levels of depression and state anxiety. These differences were significant for almost all evaluated cognitive functions. An alternative explanation of the relationship between mood and cognitive complaints based on personality traits, i.e., neuroticism, is offered. PMID- 8636917 TI - The acceptability of psychological treatment in patients with medically unexplained physical symptoms. AB - Patients with unexplained physical symptoms are considered to benefit from psychological treatment, but are believed to be reluctant to accept a referral to a psychiatrist or psychologist. As a part of a treatment study, we had the opportunity to examine to what extent somatising patients are willing to accept psychological treatment and how patients who are willing to accept it differ from those who are not. The study was introduced to the patient by the attending physician, and the treatment took place in the general medical outpatient clinic itself. Of 229 patients who had presented with unexplained physical symptoms to a general hospital medical outpatient clinic, 172 (75%) were interviewed at about three months after their initial visit to the clinic. Fourty-five (26%) patients appeared to have either improved or recovered from their presenting symptoms, and 26 (15%) were already receiving psychiatric or psychological treatment. Of 98 patients eligible for treatment, 79 (81%) were willing to participate. Compared with the patients who agreed to take part, the nonparticipants reported lower levels of physical symptoms and less functional impairment. In conclusion, most of the patients who might have benefitted from additional psychological help were willing to accept it. Somatising patients who rejected psychological treatment were those with the least serious problems. PMID- 8636918 TI - The effects of stress and coping upon the diagnostic intracavernous injection in men with erectile dysfunction. AB - The diagnostic intracavernous injection (ICI) can be considered to be a stressful situation because it may exacerbate worries about the 'sexual' function and because it is an invasive procedure. The individual's primary appraisal (harm or pain caused by the injection, performance anxiety, fear of success as well as fear of failure) and his secondary appraisal (general coping style as well as the (in)effectiveness of this function) could influence the ICI. Therefore, coping style and anxiety levels were assessed in men with erectile dysfunction. These psychometric characteristics were correlated with the subsequent ICI with prostaglandin E1 (PGE1): the individual's anxiety level does not, but the coping style does explain a significant part of the variation in ICI response. Indeed, avoidance and palliative coping have a negative impact on the penile response following ICI. The present study suggests that coping style should be included in further psychophysiological studies of the ICI response. PMID- 8636919 TI - Psychosocial factors in women with operable breast cancer. An association to estrogen receptor status? AB - The association between psychosocial parameters and estrogen receptor (ER) status was evaluated in 85 consecutive breast cancer patients with stage I and II disease. Patients were categorized into those with ER+ and ER- tumours. Life events, emotional control, neuroticism, social support, adjustments to illness, and psychological distress were measured the day before surgery. Group comparisons showed significant differences between the groups as to age, emotional control and distribution of the life event: "experienced another illness/accident/hospitalization during the last year". A multiple regression analysis indicated significant effect for variables "age" and "life event", explaining 19% of the variation in ER level. The variable "emotional control" was positively correlated to age (r = 0.369) and did not contribute in itself. We conclude that none of the factors were associated with ER status when age and personal health data were controlled for. Our study does not support ER status as a possible link between behavioural factors and disease course in breast cancer. PMID- 8636920 TI - Somatization: a transcultural study. AB - The primary aim of this study was to investigate the comparative rates of somatic complaints between Asian and Caucasian patients in a primary care setting and to characterize the factors associated with increased rates of somatization. One hundred and ninety-five individuals aged between 16 and 65 yr were interviewed with the Bradford Somatic Inventory (BSI) and the Hospital Anxiety and Depression Scale (HAD) respectively. The main finding was that the Asian patients reported significantly more somatic and depressive symptoms than the Caucasian patients. Ethnicity was the most important variable determining this result. PMID- 8636921 TI - Negative affectivity, somatic complaints, and symptoms of temporomandibular disorders. AB - This study examined the relationship between trait anxiety, subjective somatic symptoms, and pain associated with temporomandibular disorders (TMD) in a combined cross-sectional and prospective study. Compared with the initial measurements, a significant reduction in anxiety and pain was observed in the follow-up phase (two years after evaluation and therapy). No change in general somatic complaints was found. In both the initial phase and the follow-up phase, a consistent relationship between anxiety, somatic complaints, and TMD-related pain was demonstrated. The results are in agreement with a conceptualization of negative affectivity (e.g., anxiety, distress, tension) as a general dimension of somatopsychic distress. The best predictors of later TMD pain were general somatic complaints, followed by initial pain, and trait anxiety. Significant moderate correlations between anxiety, somatic complaints, and response to muscle palpation (feelings of tenderness) were also found. It is concluded that studies of TMD should incorporate measures of negative affectivity and general somatic complaints because these factors are important predictors of TMD pain, response to treatment, and chronicity. PMID- 8636922 TI - Personality disorder and sexual risk taking among homosexually active and heterosexually active men attending a genito-urinary medicine clinic. AB - 61 homosexually active men and 57 heterosexually active men attending a genito urinary medicine clinic were assessed for personality disorder and sexual risk taking. Of the homosexually active men, 23/61 (38%) were found to have personality disorders, as against 16/57 (28%) of the heterosexually active men. Multiple regression analysis indicated that antisocial personality disorder (p < 0.001) was the main predictor of sexual risk taking for the homosexually active clinic attenders. In the case of the heterosexually active men attending the clinic, sexual risk taking was predicted by cocaine use (p < 0.001) and antisocial personality disorder (p < 0.001). These results indicate a need to screen for personality disorders in genitourinary medicine clinics and at the time of pre-HIV test counseling. PMID- 8636923 TI - Psychosocial adjustment of thalassaemic children's siblings. AB - beta-Thalassaemia is a chronic disease causing serious symptoms to the patients and considerable burden to their families. The psychopathology and psychosocial adjustment of 71 siblings of thalassaemic patients and 71 matched control subjects were investigated. Siblings of thalassaemic patients were found to have significantly more psychiatric disorders than the control subjects (p < 0.05). The frequency of psychopathology among the patients' siblings, when compared to the controls, was significantly greater after the age of 10 years (p < 0.01), with no difference between the two groups before the age of 10. Siblings of thalassaemic patients also scored significantly lower on sports and nonsports activities and on social functioning in terms of number of friends and number of contacts with them. These findings show that siblings of thalassaemic patients are at risk for psychiatric symptomatology and for impaired psychosocial functioning. PMID- 8636924 TI - Cytokine regulation of HLA-G expression in human trophoblast cell lines. AB - HLA class I genes are differentially expressed among subpopulations of cells in first trimester human placentas. In this study, HLA class I protein was detected in extravillous cytotrophoblast cells by immunohistochemistry using the monoclonal antibody W6/32. In the same trophoblast subpopulation, class Ib proteins were identified with two monoclonal antibodies, 87G (anti-HLA-G) and 131 (anti-HLA-A/G) and class Ia protein was detected with the monoclonal antibody, 4E (anti-HLA-B/C). All of the antibodies also identified antigens on the human trophoblast-derived choriocarcinoma cell line, JEG-3. Therefore, the JEG-3 cells were used as a model system to study cytokine regulation of HLA-G in trophoblast cells. Northern blot hybridization studies showed that interferons (IFN-alpha, IFN-beta, IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) modestly enhanced steady state levels of HLA-G mRNA. Yet analysis of HLA-G protein by immunocytochemistry and flow cytometry failed to identify any changes in intracellular or membrane expression of HLA-G protein following cytokine treatment. Resistance to upregulation of HLA class I antigens was not a general feature of JEG-3 cells; IFNs enhanced expression of HLA-B/C as well as HLA class I light chain, beta 2-microglobulin. HLA null Jar choriocarcinoma cells did not contain HLA-G mRNA or antigen and exposure to cytokines had no effect on HLA-G. The results of this study are consistent with the postulate that trophoblast cell expression of HLA-G is stringently regulated and is controlled in part by post transcriptional mechanisms. PMID- 8636926 TI - Purification and characterization of a sperm antigen recognized by HSA-5 monoclonal antibody. AB - Among the monoclonal antibodies generated against acrosome-reacted human sperm, HSA-5 was shown to react with a sperm antigen localized predominantly to the equatorial region of the acrosome of human sperm and to the head and tail of mouse sperm. This antibody reacted with the methanol-fixed sperm, but not with fresh live sperm. When purified by immunoaffinity column, a major protein band with a molecular mass of approximately 100 kDa on SDS gel was isolated from fresh human sperm extract. The immunospecificity of isolated human sperm protein to this monoclonal antibody was verified by enzyme-linked immunosorbent assay and Western blot analysis. This antigen, designated as HSA-5, was susceptible to proteolytic degradation and revealed multiple immunoreactive bands in Western blot analyses of some preparations. Mouse sperm homogenates showed a similar polymorphic pattern to that of human samples. The tissue specificity of this antigen was examined immunohistochemically using various mouse and human tissues. HSA-5 did not cross-react with any other tissues except for sperm in adult testes and epididymis. This antibody also showed no binding activity to testicular tissue sections from mice of 13 and 21 days of age. The results of our study suggest that the sperm antigen recognized by HSA-5 monoclonal antibody is a differentiation antigen, which is expressed postmeiotically in testicular sperm but not in any somatic tissues. PMID- 8636925 TI - Interleukin-2 in human amniotic fluid during pregnancy and parturition: implications for prostaglandin E2 release by fetal membranes. AB - The potential role of interleukin 2 (IL-2) in human pregnancy was investigated by evaluating the following. (1) The presence and concentrations of IL-2 in amniotic fluid (AF) in 24 women at 16-18 weeks' gestation (Group 1) and in 27 women at term pregnancy, either before the onset of labor (Group 2, n = 10) or during spontaneous active labor (Group 3, n = 17). (2) The production of IL-2 by fetal membranes at term gestation (n = 7). (3) The release of prostaglandin E2 (PGE2) by the above tissues after stimulation with IL-2 (n = 10) or phytohemagglutinin (PHA) (n = 8). Immunoreactive IL-2 was detected only in AF samples obtained from women of Groups 1 and 3; the higher concentration was found in Group 1 samples; IL-2 was not detected in AF samples from women of Group 2. Tissues did not release IL-2. Both IL-2 and PHA exerted a significant stimulatory effect on PGE2 release by tissues. IL-2 stimulated PGE2 release by chorion tissue, but not by amnion tissue. The following conclusions can be drawn: (a) AF IL-2 might play a role in the maternal-fetal immune relationship during early pregnancy and, perhaps, during labor; (b) fetal membranes would not seem to represent a source of AF IL-2 in the absence of labor; (3) IL-2 might influence arachidonic acid metabolism through the cyclooxygenase pathway in the chorion tissue. PMID- 8636927 TI - Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody. AB - Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein containing 77 amino acid residues in the C-terminal region of MSAg-5. This 1 kb cDNA insert was engineered in a pGEX vector to express a recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified. PMID- 8636929 TI - Reduction of ovulation rate in the rat by administration of a neutrophil depleting monoclonal antibody. AB - To investigate the effect of systemic neutrophil depletion on ovulation rate, rats were synchronised with eCG and hCG, and concurrently were administered neutrophil-specific, cytotoxic RP-3 monoclonal antibody (mAb), or an irrelevant, class-matched mAb. Neutrophils in the peripheral blood and in the thecal-luteal area of corpora lutea were detected by immunohistochemical analysis with the neutrophil-specific mAb MCA149 and were found to be reduced in number by 70% and 38% respectively following RP-3 treatment compared to the control group. Ovulation rate, as assessed by counting the number of oocytes in the ampullary region of the oviduct 20 h after hCG administration, was found to be reduced by 27% in the neutrophil-depleted rats. This result provides further evidence that neutrophilic granulocytes play an active role in ovulation in the rat. PMID- 8636928 TI - Thymosin alpha-1 and FA-1 monoclonal antibody affect murine preimplantation embryo development by modulating protein phosphorylation. AB - The effects of thymosin alpha 1 (T alpha 1) and FA-1 monoclonal antibody (anti-FA 1 mAb) on murine preimplantation embryonic development were investigated by performing 2-cell embryo bioassay and by studying ova/embryos protein phosphorylation pattern (by 32P metabolic labeling and by in vitro kinase assay) and protein synthesis (by in vitro [35S]methionine labeling). T alpha 1 treatment (0.1, 0.5 and 5 ng/100 microliters) significantly increased blastulation rates (P < 0.01), blastocyst hatching rate (P < 0.0001), blastocyst diameter (P < 0.001) and number of cells per blastocyst (P < 0.0001) of the in vitro cultured 2-cell stage embryos. Anti-FA-1 mAb reduced blastulation rates (P < 0.001) primarily due to an arrest of development at morula stage. In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into 4 major protein bands of murine ova (M(r) 125, 90, 68 and 31 kDa, respectively), 7 protein bands of 2-cell (M(r) 90, 68 and 31; and 145, 52, 38 and 32 kDa, respectively), 10 protein bands of morula (M(r) 150, 110, 92, 82, 70, 54, 39, 34, 30 and 29 kDa, respectively), and 15 protein bands of blastocyst (150, 110, 92, 70, 68, 54, 39, 34 and 30; and 131, 105, 52, 44, 43 and 33 kDa, respectively) stage embryos. T alpha 1 treatment (0.1 0.5 ng/100 microliters) resulted in a general increase in 32P labeling in all proteins of 2-cell, morula and blastocyst stage embryos. Anti-FA-1 mAb completely blocked 32P labeling of various proteins of murine ova, 2-cell, morula and blastocyst stage embryos, whereas control mouse myeloma IgG did not affect phosphorylation of these proteins. In vitro kinase assay performed directly on various ova/embryos extracts revealed 6 phosphoproteins (M(r) 105, 82, 55, 38, 34 and 33 kDa, respectively) that were common to ova and 2-cell embryos, besides a 43 kDa protein detected only in the ova extract. Of these phosphoproteins, T alpha 1 treatment specifically enhanced whereas anti-FA-1 mAb inhibited autophosphorylation of a 55 kDa protein of 2-cell embryos.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 8636930 TI - From Rwanda to Wisconsin: the global relevance of diarrhoeal diseases. PMID- 8636931 TI - Clinical and molecular aspects of the pathogenesis of Staphylococcus aureus bone and joint infections. AB - Staphylococcus aureus is an important cause of bone and joint infections. In recent years, significant changes in the incidence of septic arthritis and osteomyelitis have occurred. Haematogenous osteomyelitis is now less common during childhood, but secondary spread of infection to bone or joint from a contiguous site in adults is increasing in incidence. Infection introduced at the time of surgery or arising by the haematogenous route is a significant complication of prosthetic joint implantation, and the effect of bone cement on local immune function may be important in this setting. ALthough S. epidermis is a more common cause of prosthetic joint infection, S. aureus is more difficult to treat. S. aureus produces a number of extracellular and cell-associated factors, but it is unclear what role these have as virulence factors in vivo. Furthermore, it is difficult in animal models to simulate transient bacteraemia followed by non-fulminating septic arthritis or osteomyelitis, as occurs in the patient. Surface factors which may be important in pathogenesis include the cell wall (activates complement and stimulates cytokine release), capsular polysaccharide (promotes adhesion to host cell surfaces), collagen receptors and fibronectin binding protein. Staphylococcal toxic shock syndrome toxin (TSST-1) and the enterotoxins are superantigens and have the potential to suppress plasma cell differentiation and antibody responsiveness. TSST-1-positive isolates have been shown to cause more severe joint infection in one animal model, but most other studies to date have focused on in-vitro rather than in-vivo effects. There is little evidence supporting a role for coagulase, lipase and the haemolysins in staphylococcal bone and joint infections. Despite the clinical importance of these infections, surprisingly little is known about pathogenesis at the cellular level. Future research should focus on the role of the host immune system in limiting spread of infection, and the expression of virulence factors in animal or other models incorporating isogenic mutant strains. PMID- 8636932 TI - IgA and IgG antibodies to distinct serotypes of Mycobacterium avium in HIV seropositivity and AIDS. AB - IgA and IgG antibodies to cytoplasmic and secreted antigens of serotypes 4 and 8 of Mycobacterium avium and the percentage of agalactosyl immunoglobulin (%Gal[0]) were measured by ELISA in groups of blood donors, HIV seronegative persons, HIV seropositive persons with CD4+ cell counts >300/mm3 and AIDS patients co-infected with M.avium. No differences were found between the control groups, but HIV seropositive persons were distinguished by their increased %Gal[0] (p<0.001) and increased IgA titre (p<0.05) to secreted antigens of both serotypes of M. avium. Patients going on to develop aviumosis differed from other HIV-positive individuals, having more IgA to secreted antigens of serotype 8 (p<0.03) and more IgG to secreted antigens of both serotypes (p<0.0001), IgA titres fell to both types of antigen from serotype 4 (p<0.01) and sonicate antigen of serotype 8 (p<0.001) and IgG fell to the secreted antigens of serotype 4 (p<0.03). On interpretation of these observations that antibody profiles to M. avium might be used to identify healthy persons at special risk of developing HIV seropositivity, and to identify persons with early AIDS who are likely to develop aviumosis. PMID- 8636933 TI - Serum antibody response to Staphylococcus aureus enterotoxins and TSST-1 in patients with septicaemia. AB - The prevalence of enterotoxins and toxic shock syndrome toxin (TSST-1) production in strains isolated from patients with Staphylococcus aureus septicaemia, and the serum antibody response in relation to toxin production in vitro of each isolate, were investigated. Among 63 strains of S. aureus isolated from the blood of patients with septicaemia, 51 from patients with superficial wounds and 49 from nasal carriers, 50-60% produced at least one of the enterotoxins A-D or TSST-1. The most frequent toxins produced were enterotoxins A and C and TSST-1. Among the 63 patients with staphylococcal septicaemia, 51 (81%) had a significant rise or a high antibody titre, or both, to at least one of the toxins. A positive serological response to toxin A was found in 78%, to enterotoxin B in 83%, to enterotoxin C in 80%, to enterotoxin D in 86% and to TSST-1 in 92% of the patients from whom the isolated strain produced the respective toxin. Antibodies against enterotoxins A, B, C and D and TSST-1 were also seen in 35%, 16%, 32%, 59% and 10%, respectively, in patients infected by strains that did not produce the specific toxin. Immunological cross-reactions between the toxins were demonstrated both in hyperimmune sera obtained from rabbits and in patients' sera, particularly between enterotoxins B and C. It is concluded that these potent toxins with superantigenic properties are produced in vivo during S. aureus septicaemia. No differences with regard to enterotoxin or TSST-1 production or antibody response were noted between patients with complicated versus uncomplicated septicaemia. PMID- 8636934 TI - Inter-centre comparison of pulsed-field gel electrophoresis for the typing of methicillin-resistant Staphylococcus aureus. AB - The results of pulsed-field gel electrophoresis (PFGE) of chromosomal DNA of the same 12 methicillin-resistant S. aureus (MRSA) strains of diverse geographical origin, performed in three different laboratories were compared; one laboratory used field-inversion gel electrophoresis (FIGE), one used contour clamped homogenous electrophoresis (CHEF) and one used both (all manufactured by BioRad Laboratories Inc., Hercules, CA, USA). No single method produced the maximum number of chromosomal fragments from all isolates. In only four instances were the same number of fragments identified by any two techniques. Although there were similar trends in strain identification the results showed many discrepancies even with a three-band difference rule to discriminate between strains. Plasmids in seven of the isolates produced a fragment, but this did not affect discrimination of the study isolates. There is a great need to standardise methodology and produce a standard set of strains to assist in this process. PMID- 8636935 TI - Comparison of rapid automated laser fluorescence analysis of DNA fingerprints with four other computer-assisted approaches for studying relationships between Acinetobacter baumannii isolates. AB - The relationships between isolates suggested by a novel DNA typing method (RAPD ALFA) that combines randomly amplified polymorphic DNA with automated on-line laser fluorescence analysis of DNA fragments were compared with those suggested by four other computer-assisted typing strategies (biotyping, antibiogram typing, pulsed-field gel analysis of chromosomal fingerprints and arbitrarily-primed DNA amplification with three different primers) for 25 isolates of Acinetobacter baumannii obtained from 12 different hospitals in four countries over a period of 12 years. The results obtained by cluster analysis with two different software packages confirmed that the relationships suggested by RAPD-ALFA were robust and essentially similar to those suggested by the other more laborious computer assisted typing methods. The technique of RAPD-ALFA appears to offer the possibility of routine on-line molecular identification and typing of isolates from particular hospital wards or units (e.g., intensive care units), and could, therefore, play a key role in the early recognition and prevention of outbreaks of infection. PMID- 8636936 TI - Assessment of clinical significance of positive blood cultures of relatively low virulence isolates. AB - In Omori Hospital, Toho University School of Medicine, relatively low-virulence blood isolates, including coagulase-negative staphylococci (CNS), enterococci and nonfermentative gram-negative rods other than Pseudomonas aeruginosa comprised c. 60% of total blood isolates. A retrospective study was conducted to assess their clinical significance by reviewing a total of 91 hospital charts. The physicians' assessments of these positive blood cultures as recorded in the charts were classified into four categories--sepsis, possible sepsis, contamination and no comment. The episodes classified as sepsis accounted for 5.0-19.6%. These episodes were also evaluated by a graded clinical significance score based on multiple factors, including number of positive cultures and clinical signs. The scores for the 91 episodes covered a wide range from 1 to 9, indicating that both contaminants and causative organisms may have been involved. The episodes judged as sepsis or possible sepsis tended to have higher scores. The scores for the episodes associated with enterococci were also higher than those involving CNS or non-fermentative gram-negative rods. The scores for episodes associated with intravenous hyperalimentation catheters were higher than those not associated with the catheters. PMID- 8636937 TI - Streptococcus agalactiae: a vaginal pathogen? AB - The significance of Streptococcus agalactiae as an aetiological agent in vaginitis was evaluated. A total of 6226 samples from women who presented with vaginal symptoms was examined. The presence of >10 leucocytes/high-power field (h.p.f.) was taken to be the criterion of active infection. S. agalactiae was isolated from 10.1% of these samples. The isolation rates of other common pathogens such as Candida spp., Gardnerella vaginalis and Trichomonas spp. were 54.1%, 27.2% and 4.2%, respectively, in the same group of patients. In contrast, the isolation rates of these micro-organisms in the group of patients who had no infection (<10 leucocytes/h.p.f.) were 4.2%, 38.3%, 33% and 0.5%, respectively. In the majority of samples from which S. agalactiae was isolated, it was the sole pathogen isolated (83%) and its presence was associated with an inflammatory response in 80% of patients. Furthermore, the relative risk of vaginal infection with S. agalactiae (2.38) in patients with purulent vaginal discharge was greater than that of Candida spp. infection (1.41) and lower than that of Trichomonas spp. infection (8.32). These data suggest that S. agalactiae in symptomatic women with microscopic evidence of inflammation should be considered a causative agent of vaginitis. PMID- 8636938 TI - Type characterisation and antibiotic susceptibility of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis in the United Kingdom and the Republic of Ireland. AB - The spread of Burkholderia cepacia among cystic fibrosis (CF) patients in the UK prompted an investigation into whether an epidemic strain was responsible. A total of 366 B. cepacia isolates from 178 CF patients in 17 centres was examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Associations were also sought between antibiotic resistance and strain type. More than 50 ribotype patterns were found but one, termed ribotype 1, was identified from 68 patients in eight centres. One centre had a single patient with this type while, in others, most or all patients harboured this organism. Small clusters of apparent cross-colonisation within centres were also evident for some other ribotypes. PFGE confirmed that ribotype 1 isolates were genetically similar. Ribotype 1 isolates were not markedly more resistant to antimicrobial agents than were other isolates, and the MICs of individual antibiotics were no more tightly clustered for ribotype 1 isolates than for others. Most isolates were resistant to ciprofloxacin, amikacin, gentamicin, tobramycin, carbenicillin, cefuroxime, cefotaxime, imipenem, biapenem, chloramphenicol, tetracycline, trimethoprim and sulphamethoxazole, but > or = 77% were susceptible to ceftazidime, piperacillin, piperacillin/ tazobactam and meropenem. We conclude that numerous strains of B. cepacia colonise CF patients in the UK and Ireland but that one epidemic strain has spread in at least eight centres. Isolates of this strain appear homogenous in total genomic profile but very variable in antibiotic susceptibility. PMID- 8636940 TI - Detection of the gene for toxic shock syndrome toxin 1 in Staphylococcus aureus by enzyme-labelled oligonucleotide probes. AB - A colony hybridisation method with enzyme-labelled oligonucleotide probes was developed to detect the gene for toxic shock syndrome toxin 1 (tst). For rapid identification, bacterial colonies were transferred from agar plates directly on to nylon membranes. These procedures took only 3 h. Results obtained by this test correlated well with those obtained by the reverse passive latex agglutination test. Thus, this method is convenient and reliable for the detection of tst in staphylococci, which could be useful for both research and clinical purposes. This method demonstrated that tst was more prevalent in methicillin-resistant Staphylococcus aureus (56%) than in methicillin-susceptible S. aureus (4%). PMID- 8636939 TI - Experimental endogenous septicaemia caused by Klebsiella pneumoniae and Escherichia coli in mice. AB - Multi-resistant Klebsiella pneumoniae have recently occurred in several nosocomial outbreaks of septicaemia. An animal model resembling the pathophysiology of these infections in man would be very useful. A new model of endogenous septicaemia caused by K. pneumoniae and Escherichia coli strains in mice has been established. The mortality rate of conventional ddY mice given cyclophosphamide (CY) or fluorouracil (5-FU), each 200 mg/kg intraperitoneally, every other day was 70 and 100%, respectively. Pseudomonas aeruginosa septicaemia was observed in all dead mice treated with CY, whereas Enterobacteriaceae, including K. pneumoniae, were isolated from 90% of mice given 5-FU. Specific pathogen-free mice, decontaminated with ampicillin and ceftazidime, were given multi-resistant K. pneumoniae CF504, CF514 or E. coli CF604, or CF614 carrying CAZ-1/TEM-5 plasmid by oral inoculation. Subsequent dosing with 5-FU induced lethal septicaemia caused by the inoculated strains in most of these mice, whereas CY did not regularly induce septicaemia. This model with 5-FU is considered to resemble closely the situation observed in man and to be beneficial for investigating pathophysiology and therapeutic strategies. PMID- 8636941 TI - A comparison of immunomagnetic separation, direct culture and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces. AB - Verocytotoxin-producing Escherichia coli O157 (O157 VTEC) has become well recognized as an important enteric pathogen. The number of organisms present in environmental and clinical samples may be low and efforts have been made to increase the sensitivity of O157 VTEC detection. Immunomagnetic seperation (IMS) has been shown to improve O157 VTEC detection in bovine faeces and food samples. A milkborne outbreak of O157 VTEC infection allowed us to compare the isolation rates from human faeces by IMS, direct faecal culture on sorbitol-MacConkey agar and a PCR test for verotoxin gene carriage. Of 142 faecal samples examined, 20 were positive on both direct culture and IMS and a further 13 on IMS alone. Therefore, IMS increased the detection rate of individual cases of O157 VTEC infection and also compared well with PCR. We recommend IMS for use in routine diagnostic laboratories where a more sensitive method than direct faecal culture is required for O157 VTEC isolation. PMID- 8636942 TI - A comparison of the Genie and western blot assays in confirmatory testing for HIV 1 antibody. AB - The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies. PMID- 8636943 TI - Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium avium complex. AB - The newly developed E test was compared with an extended 1% proportion dilution method for determining the susceptibility of Mycobacterium avium complex (MAC) strains to amikacin, streptomycin, fusidic acid, rifampicin, clarithromycin, ciprofloxacin, ofloxacin and fleroxacin. For all antibiotics tested except clarithromycin and ciprofloxacin, no more than one strain gave a different susceptibility result with the two methods. The discrepant results occurred near the chosen breakpoint concentration of clarithromycin and outside the concentration range of the E test for ciprofloxacin. For the minimum inhibitory concentration (MIC) values obtained within the range of antibiotic concentrations tested, there was good correlation between the two methods; the MICs differed by more than one two-fold dilution in no more than two strains per antibiotic. It is concluded that the E test is suitable for susceptibility testing of MAC. PMID- 8636944 TI - Multi-drug resistant typhoid: a global problem. PMID- 8636945 TI - Quinolones: structure-activity relationships and future predictions. AB - Development of the first clinically useful quinolone--nalidixic acid-- occurred in 1962, but the significant breakthrough with this class of agents occurred almost 20 years after the original discovery when the addition of a fluorine molecule at position C6 of the pharmacore created the 'fluoroquinolones'. It has been estimated that over 10000 analogues of nalidixic acid or the fluoroquinolones have now been synthesised. The benefits of some of these new compounds include: oral and parenteral dosing, a much broader spectrum of antibacterial activity, good tissue distribution, improved pharmacokinetic profiles, stability and a comparatively low incidence of adverse effects. This review considers the structure of the core fluoroquinolone molecule, some of the changes that feature on current class members under development, and the effects that these chemical modifications may have on the interaction of these compounds with man. PMID- 8636946 TI - Characterisation of strains of Salmonella serotype Livingstone by multiple typing. AB - Isolates of Salmonella serotype Livingstone (6,7:d:1,w) from man, water and various animals and animal products in Canada, England, France, Israel and Scotland were examined for ribotype, biotype and plasmid profile. Analysis by these methods indicated that an epidemic strain of Livingstone of ribotype 1/biotype 8/plasmid-type 6 was responsible for the major upsurge of Livingstone infection that occurred in man in Tayside (Scotland) between 1989 and 1991; that type was also isolated from spring water, animal feed and poultry. Livingstone isolates of ribotype 1/biotype 8 with plasmid profiles other than type 6 were also present in Scotland, England and France at that same time. Among representative Livingstone isolates from England, a strain of ribotype 2/biotype 1 was predominant in man and poultry products between 1988 and 1992, although strains of other ribotypes (1, 3 and 4) were also present. Strains of ribotype 3 of different biotypes were obtained from poultry and animal feed sources in Canada. A strain of ribotype 5/biotype 3 caused human infections in Israel between 1968 and 1992. Ribotyping, biotyping and plasmid profile analysis used together have helped to trace the sources and extent of spread of human infections caused by Salmonella Livingstone. PMID- 8636947 TI - Strain-specific variation in the dnaJ gene of mycobacteria. AB - The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was evaluated for species identification among mycobacteria by analysis of the dnaJ gene. Nine clinical isolates of Mycobacterium tuberculosis with different fingerprint patterns all gave the same distinct SSCP banding pattern and could be distinguished from other mycobacteria, such as M. avium. In contrast, considerable strain-specific dnaJ gene variations were observed amongst 42 clinical isolates of M. avium and 13 other atypical mycobacterial strains. Only 62% of the M. avium isolates hybridised to an M. avium-specific probe and only 14% could be identified correctly as M. avium by both probe and restriction fragment length polymorphism analysis. This finding was supported by direct sequence analysis. Variations were also observed in M. gordonae and M. scrofulaceum isolates. Computerised analysis of M. avium samples broadly identified three clusters. Results suggest that although the SSCP procedure may be useful for distinguishing M. tuberculosis from other mycobacteria, this technique applied to the dnaJ gene may not be suitable for strain identification. The results stress the importance of testing a large collection of clinical isolates before new molecular procedures are introduced into routine laboratories. PMID- 8636948 TI - The epidemiology of acinetobacter infections in Hong Kong. AB - A retrospective survey was conducted of the characteristics of acinetobacter infections in Hong Kong--seasonal and geographic distributions, frequency of isolation from various body sites, antimicrobial susceptibility and molecular epidemiology. Most (80%) isolates of Acinetobacter spp. belonged to DNA groups 2 (A. baumannii) or 13, as defined by growth at 44 degrees C. An increased isolation rate in summer was related to higher ambient temperatures. The notion that acinetobacters are opportunist nosocomial pathogens was supported by the body site- and ward-specific distributions, which were similar to those of Pseudomonas aeruginosa and in marked contrast to those of coagulase-negative staphylococci and Escherichia coli. Typing of Acinetobacter isolates by arbitrary primed polymerase chain reaction revealed extensive genotypic polymorphism, suggesting that numerous unrelated strains were circulating between patients. In view of the association with a high incidence of polymicrobial bacteraemia and multiresistance to antibiotics, a careful selection of appropriate antibiotics in combination is necessary for empirical therapy of infections caused by Acinetobacter spp. PMID- 8636949 TI - Antibody response in rabbits to serotype-specific determinants in lipopolysaccharides from Moraxella catarrhalis. AB - Antibodies against the serotype determinant epitopes of Moraxella catarrhalis lipopolysaccharides (LPS) were demonstrated in sera from rabbits immunised with whole bacterial cells. Purified LPS preparations from eight strains of M. catarrhalis were used as antigens in enzyme-linked immunosorbent assays (ELISA) and immunoblotting. The serotype specificity of the antibodies was shown by neutralisation with LPS and with purified polysaccharide obtained from LPS prepared from strains belonging to different serotypes. In immunoblots, antisera against the A and B LPS serotypes reacted only with LPS of its own type, confirming the presence of type-specific antibodies. A weak band was observed with type A LPS and antibody to type C, indicating cross-reactivity between the A and C serotypes. This cross-reaction can be explained on the basis of the known chemical structure of the LPS of the different serotypes. After heterologous absorption of sera, bands were obtained with homologous LPS antigen. These results suggest that the predominant antibody response in rabbits to LPS from M. catarrhalis is serotype-specific, unlike that previously observed in infected human patients. PMID- 8636951 TI - Adhesion of enteroaggregative Escherichia coli to formalin-fixed intestinal and ureteric epithelia from children. AB - The adhesion characteristics of enteroaggregative Escherichia coli (EAggEC) to the mucosal surfaces of formalin-fixed paediatric intestinal and ureteral tissue were studied. The technique offers a means of overcoming the problem of limited tissue access in childhood and a way of examining the initial steps of bacterial adhesion. Five EAggEC strains isolated from children with diarrhoea in the UK and a well characterised, prototype EAggEC strain (221) were examined. Five of the six EAggEC strains showed preferential adhesion to jejunal mucosa with limited adhesion to ileum and colon. Five of the six also adhered to ureteric tissue. EAggEC can adhere to proximal, as well as distal, regions of the gastrointestinal tract in children, a previously unrecognised characteristic. PMID- 8636950 TI - Potential of exocellular carbohydrate antigens of Staphylococcus epidermidis in the serodiagnosis of orthopaedic prosthetic infection. AB - The potential of exocellular carbohydrate antigens of Staphylococcus epidermidis as markers of infection in bone was investigated by immunoblotting and enzyme linked immunosorbent assay (ELISA). Exocellular antigens were prepared by gel filtration chromatography of concentrated brain heart infusion culture supernates. The antigenic material appeared as diffuse bands between 24 and 32 kDa on the immunoblots and was not susceptible to digestion with trypsin, indicating that the response in the patients was to non-protein (polysaccharide or teichoic acid, or both) exocellular material. Significant differences were detected between the immunoblot antigen profiles for serum IgG from patients with S. epidermidis bone infection and those with an uninfected prosthetic joint. Thirteen of 16 patients with S. epidermidis prosthetic joint infection showed an elevated serum IgG level by ELISA compared with controls with uninfected joints. However, the antigen was not specific for S. epidermidis bone infection; high levels of IgG were also detected in patients with other serious staphylococcal and streptococcal infections. The ELISA test may be valuable in distinguishing between staphylococcal infection of joints and aseptic loosening by excluding cases of infection. PMID- 8636952 TI - Degradation and utilisation of chondroitin sulphate by Streptococcus intermedius. AB - Streptococcus intermedius, part of the 'Streptococcus milleri group', has the ability to produce glycosaminoglycan depolymerising enzymes (hyaluronidase and chrondroitin sulphate depolymerase) which is unique amongst the viridans streptococci and may contribute to their virulence in brain and liver abscesses. The growth of S. intermedius strain UNS 35 was studied in basal medium supplemented with chondroitin sulphate A (CS-A, sulphated at position 4 of the N acetylgalactosamine moiety) or chondroitin sulphate C (CS-C, sulphated at position 6 of the N-acetylgalactosamine moiety) as the major carbohydrate source. CS-A but not CS-C supported the growth of S. intermedius. Extracellular degradation of CS-A resulted in the initial accumulation of 2-acetamido-2-deoxy-3 O-(beta-D-gluco-4-delta-enepyranosyluronic acid)-D-galactose (deltaUA GalNAc-0S), and low levels of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-delta-enepyranosyl uronic acid)-4-O-sulpho-D-galactose (deltaUA GalNAc-4S) in the medium with GalNAc 0S being subsequently utilised during bacterial growth. Metabolic end-products included formate and ethanol but not lactate, indicating that growth was probably carbon-limited. The CS-A contained 30% CS-C, which was also depolymerised resulting in the formation of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-delta enepyranosyluronic acid)-6-O-sulpho-D-galactose (deltaUA GalNAc-6S) in the culture supernate, but this unsaturated disaccharide was apparently not utilised during growth. The results indicate that S. intermedius produced CS-AC depolymerase, which was inducible and extracellular, and sulphatase activity. Experiments with authentic deltaUA GalNAc-4S and deltaUA GalNAc-6S demonstrated that deltaUA GalNAc4S rather than deltaUA GalNAc-6S was the preferred substrate for the sulphatase. Therefore, it is suggested that the CS-AC depolymerase of S. intermedius may play a role in the destruction of CS in host tissues, facilitating bacterial spread, and also in bacterial nutrition by the liberation of nutrients at the site of infection. PMID- 8636953 TI - Purification and properties of a novel glycosaminoglycan depolymerase from Streptococcus intermedius strain UNS 35. AB - A glycosaminoglycan (GAG) depolymerase that acts on chondroitin sulphate A (CS A), chondroitin sulphate C (CS-C) and hyaluronic acid (HA) was purified to apparent homogeneity from a culture of Streptococcus intermedius, strain UNS 35, grown in minimal medium supplemented with CS-A as the sole carbon source. The enzyme was purified by ammonium sulphate precipitation followed by serial chromatography on DEAE Trisacryl M, CM Trisacryl M and heparin-agarose. SDS-PAGE analysis of the purified enzyme yielded a single band with a mol.wt of c. 83000. The purified GAG depolymerase was unusual in its substrate specificity. The enzyme was initially regarded as a CS depolymerase because of its induction by CS A. However, the GAG depolymerase exhibited greatest activity against HA, whereas the degradation rates of CS-A and CS-C were c. 8% and 2%, respectively, of the rate with HA. On this basis the enzyme could be classified as a hyaluronidase rather than a CS depolymerase. The pH optimum was around neutrality and the enzyme was unusual in having a high pI of approximately 9.3. PMID- 8636954 TI - Rapid identification of Candida albicans, C. glabrata, C. parapsilosis and C. krusei by species-specific PCR of large subunit ribosomal DNA. AB - A rapid PCR-based method for the identification of four Candida species is described. Primers to conserved sequences in the V3 region of large subunit rDNA were used to amplify DNA from C. albicans, C. glabrata, C. parapsilosis and C. krusei. The sequences were aligned and areas of non-concordance were used to design four species-specific forward primers. In a blind study of 82 yeast strains, four PCRs based on these primers correctly identified nine C. albicans, 18 C. glabrata, 13 C. parapsilosis and 18 C. krusei strains after gel electrophoresis of amplified DNA. Furthermore, of 17 other Candida strains (10 species) and seven strains from other yeast genera (Saccharomyces, Cryptococcus and Trichosporon) only one false positive amplification product (with C. dubliniensis) was seen. These PCRs offer a rapid alternative to conventional techniques for the identification of C. albicans, C. glabrata, C. parapsilosis and C. krusei. PMID- 8636955 TI - Pneumococcal vaccine. PMID- 8636956 TI - Molecular genetic approaches to identification, epidemiology and taxonomy of non albicans Candida species. AB - The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HIV. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-alpha-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis. Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species specific probes. Synthetic oligonucleotide probes--which do not have to be species-specific and which can detect minor polymorphisms--have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomy--and the example of a recently discovered novel species, C. dubliniensis--is discussed. PMID- 8636957 TI - Effect of mucin and glucose on proteolytic and glycosidic activities of Streptococcus oralis. AB - The production of glycosidase and protease activities, which may play a role in the degradation of human glycoproteins, by Streptococcus oralis strains isolated from endocarditis, septicaemia or the oral cavity was investigated with a range of fluorogenic substrates. The pH optima of the proteases ranged from 6.0 to 9.3 and the pH optima for the glycosidases were lower (4.5-6.0), although the pH range over which both groups of enzymes acted was broad. Growth in a minimal medium supplemented with glucose resulted in repression of glycosidase activities and elevated proteolytic activity. Bacteria from cultures supplemented with porcine gastric mucin (PGM), a model glycoprotein, exhibited higher levels of glycosidase activity, while proteolytic activity was suppressed and glycoprotein derived monosaccharides were transported at significantly higher rates than those observed for cells grown in media with glucose. PGM-derived cells also exhibited high levels of N-acetylneuraminate pyruvate-lyase, the first intracellular enzyme in the pathway of sialic acid catabolism. Taken together, these data indicate that S. oralis strains produce a range of proteolytic and glycosidic enzymes that may play a role in the degradation of host-derived glycoproteins. PMID- 8636958 TI - The effects of inhibitors of vacuolar acidification on the release of Listeria monocytogenes from phagosomes of Caco-2 cells. AB - To evaluate the role of the acidic pH of phagosomes on the invasive ability and fate of Listeria monocytogenes within host cells, entry and replication of this gram-positive bacterium in a human enterocyte-like cell line (Caco-2) were investigated by a combination of biochemical and ultrastructural approaches. The effects of inhibitors of vacuolar acidification--the lipophilic weak base ammonium chloride, the carboxylic ionophore monensin and the vacuolar proton ATPase inhibitor bafilomycin A1--on the bacterial invasion pathway were analysed. These agents, which raise the intracellular vesicle acidic pH of living cells by different mechanisms, affected L. monocytogenes replication in Caco-2 cells. Bacteria internalised by bafilomycin-treated cells were unable to escape from phagosomes, as demonstrated by electronmicroscopy. The results provide evidence that low pH is required for efficient intracellular growth of L. monocytogenes. PMID- 8636960 TI - Biochemical characterisation, enteropathogenicity and antimicrobial resistance plasmids of clinical and environmental Aeromonas isolates. AB - One hundred and eight strains of Aeromonas from clinical and environmental samples were speciated. Seven species were identified, the most prevalent of which was A. hydrophila. Experimental studies in an animal model with 36 representative strains of different species revealed that all strains could cause significant fluid accumulation in rabbit ileal loops. Of 107 strains showing single or multiple antimicrobial resistance, the highest incidence of resistance was shown for beta-lactam antibiotics other than cefotaxime. Transferable resistance plasmids, encoding resistance to ampicillin, cephalexin, cefoxitin, erythromycin and furazolidone, either alone or in combination, were detected in 35 strains. A further proportion of strains could be cured of one or more resistance markers, including resistance to nalidixic acid, and this was accompanied by the loss of plasmid DNA. The plasmids ranged in size between 85.6 and > 1 50 kb. PMID- 8636961 TI - Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom. AB - Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties. PMID- 8636959 TI - Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease deficient mutant. AB - This study investigated how pH and the presence of urea affect the survival and growth of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was studied after incubation for 1 h in buffers at different pH values at 37 degrees C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH 3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain, which survived well. At pH 9, urea further reduced the viability of H. pylori and flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor flurofamide affected the viability of the urease-deficient mutant under the same conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH 5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9. The bactericidal activities of metronidazole and tetracycline were similar at the different pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and amoxycillin at pH 7 was of additive type. These results suggest that pH and growth conditions may be important in the antibacterial efficacy of different antibiotics in vivo and also provide a possible explanation for the potentiating effect of omeprazole with antibiotics in the treatment of H. pylori infections. PMID- 8636964 TI - Antigenicity of amino-acid sequences from Clostridium difficile toxin B. AB - Clostridium difficile toxins A and B cause antibiotic-associated colitis. Whereas antigenic determinants specifying neutralisation of toxin A have been partially elucidated, those of toxin B remain unknown. To define antigenic determinants of toxin B, synthetic peptides were prepared for five linear sequences selected by computer analysis for putative T and B epitopes. Peptides spanning the carboxy terminal region (aa 2155-2283) were also selected because this region contains repetitive units thought to bind the toxin to cell receptors. Multiple antigenic peptides were synthesised by linking four peptide copies to a core of four lysine residues (tetraMAP). Outbred mice were given four doses of each tetraMAP by intraperitoneal injection and specific immunoglobulins G and A were measured by enzyme-linked immunosorbent assay (ELISA) in serum, ascitic fluid and faeces. All 14 MAPs induced strong IgG responses against the homologous peptide; peptides representing aa 2155-2179 and 2246-2270 induced the strongest responses, of 592 and 493 ELISA units, respectively - although, to a lower extent, all 14 MAPs induced serum and faecal IgA responses against the homologous peptide. All MAPs induced IgG1 and IgG2b subclasses, documenting their capacity to elicit Th2 dependent mucosal immunity. IgG anti-MAPs were assayed for reaction with native toxins A and B; most anti-MAPs recognised the toxins only weakly or did not recognise them. Antibodies against peptide representing aa 2168-2192 recognised both native toxin B (19 ELISA units) and toxin A (2 ELISA units). None of the antibodies neutralised cytotoxicity of either toxin in cell culture. In contrast, four MAPs (aa 2080-2095, 2168-2192, 2220-2244 and 2233-2257) inhibited cytotoxicity when mixed with toxin B before addition to cells; inhibition was mediated by a direct interaction with toxin B. PMID- 8636963 TI - Development and evaluation of an ELISA to detect Escherichia coli K88 (F4) fimbrial antibody levels. AB - An enzyme-linked immunosorbent assay (ELISA) to determine IgG antibody levels against K88 (F4) fimbrial antigen from porcine enterotoxigenic Escherichia coli (ETEC) has been developed. The ELISA method was checked with serum samples obtained from rabbits and pigs, and the parameters affecting the method were also analysed. ELISA plates were optimally coated with K88 antigen 0.5 microgram/ml for testing rabbit antiserum or with 1.25 microgram/ml for testing pig serum. Optimal concentrations of H202 (0.5%) and orthophenylene-diamine (OPD) (0.125%) were chosen when a 10-min incubation period was used. The expression of antibody levels as enzyme-immunosorbent units (EIU) significantly decreased the variability of results between duplicate plates, when compared with the expression of results as direct OD values. ELISA-K88 applied to a field study with serum samples from 141 vaccinated and 52 unvaccinated sows was shown to be useful in differentiating between samples from vaccinated and unvaccinated animals. PMID- 8636962 TI - Analysis of the fim cluster of an avian O2 strain of Escherichia coli: serogroup specific sites within fimA and nucleotide sequence of fimI. AB - Escherichia coli MT78, an avian pathogenic strain of serogroup 02, produces a variant form of type 1 fimbriae with distinct antigenic properties and apparent mol. wt of the major subunit. The fim gene cluster of strain MT78 was cloned and its sequence was determined in a region spanning upstream of fimB to the beginning of fimD. Whereas most genes were well conserved relative to fim genes previously described, comparison of the fimA gene from strain MT78 with homologous sequences from other strains of E. coli and Klebsiella pneumoniae revealed that most differences were clustered in four well defined regions. A PCR assay, based upon these variable sequences, allowed amplification of a fragment of gene fimA which is specific for most 02 strains. In addition, the sequence of the previously uncharacterised gene fimI, which is located between genes fimA and fimC, was determined. PMID- 8636965 TI - Protective activity of a murine monoclonal antibody against acute and chronic experimental infection with type IV group B streptococcus. AB - A murine IgM monoclonal antibody (MAb H11) was developed against the type polysaccharide capsular antigen of group B streptococcus (GBS), serotype IV, after intraperitoneal immunisation of BALB/c mice with heat-killed bacteria. MAb H11 reacted in immunodiffusion with the purified polysaccharide in both its sialylated and desialylated form, giving a line of identity, and opsonised type IV GBS strains in an in vitro assay. When administered at the time of intraperitoneal lethal challenge with homologous GBS, or 4 h earlier, MAb H11 protected 90% of the mice. Protection was still observed when MAb H11 was given 4 h after the challenge. This MAb was strongly effective in preventing septic arthritis induced by type IV GBS. PMID- 8636966 TI - A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticus-A. baumannii complex. AB - Different PCR-based DNA fingerprinting techniques were evaluated for typing 26 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex. Seven isolates belonged to a previously defined outbreak while 19 isolates were unrelated epidemiologically. The PCR-based DNA fingerprinting techniques used were: (i) repetitive extragenic palindromic (REP) PCR; (ii) enterobacterial repetitive intergenic consensus (ERIC) PCR; (iii) randomly amplified polymorphic DNA with M13 forward primer; (iv) restriction analysis of the amplified 16S rRNA gene (ARDRA-16S); and (v) restriction analysis of an amplified region containing the 16S-23S rRNA spacer region and part of the 23S rRNA gene (ARDRA 23S + spacer). The discrimination index for the PCR-based DNA fingerprinting techniques was: 0.99 for REP; 0.94 for ERIC; 0.87 for M13; 0.60 for ARDRA-16S digested with Hpa II and <0.50 for ARDRA 23S + spacer. It was concluded that REP-PCR possessed high discriminatory power and reproducibility in comparison with the other PCR-based DNA fingerprinting techniques, and is a simple and rapid typing method for use in epidemiological studies of isolates belonging to the A. calcoaceticus-A. baumannii complex. PMID- 8636967 TI - Streptococcus pneumoniae in the nasal cavity of mice causes lower respiratory tract infection after airway obstruction. AB - A model of persistent colonisation in the nasal cavity of mice by Streptococcus pneumoniae has been established. S. pneumoniae NNP-4 was introduced to the lungs of CBA/J mice at a density of c. 10(4) cfu/lung by an aerosol method and a high dose of ampicillin was administered 1 h after infection. This antibiotic eliminated bacteria from the lungs and trachea, but did not affect the bacterial counts in the nasal cavity. In mice given ampicillin, the bacteria were recovered from the nasal cavity only more than 2 weeks after infection, but IgG antibody against the colonising organisms was produced in sera around day 8 after infection. Airway obstruction was induced by intratracheal injection of formalin 2% into mice. Organisms appeared in the lungs in greater numbers when formalin was injected before the antibody production than when the immunity was established. In the early stages of infection, 10(3)-10(4) cfu appeared in the lungs 6 h after the formalin injection and the bacterial counts increased to c. 10(6) cfu within 24 h. When ampicillin was administered again 1 h after formalin was given, no bacteria were recovered from lungs 6 h later. However, in some of the mice given ampicillin after formalin, bacteria appeared in the lungs on the next day and the bacterial counts increased thereafter. These results suggest that S. pneumoniae in the nasal cavity invade the lower respiratory tract and that these organisms can localise and proliferate in lungs in the event of damage to the airway. PMID- 8636968 TI - True identity of control Staphylococcus aureus strains and their performance in the tube coagulase test. AB - One hundred laboratories were asked to submit their control Staphylococcus aureus strains to determine the true identity of strains presumed to be S. aureus NCTC 6571, and also to evaluate the performance of those strains being used as controls in the tube coagulase test (TCT). Of the 60 who replied, 55 laboratories sent at least one strain labelled as S. aureus NCTC 6571 (total of 64 strains). Of these, 84% were identified as S. aureus, and were indistinguishable from a fresh type strain by a combination of phenotypic methods including biotyping, antibiotic susceptibility testing and phage typing. Six-to-ten strains (9-16%), depending on the degree of stringency, were not identifiable as S. aureus NCTC 6571. The time since last retrieval from storage ranged from daily to > or = 3 years, but there was no correlation between this duration and the likelihood of differing from S. aureus NCTC 6571. Forty-seven laboratories submitted 51 strains used as controls in the TCT; these included 31 strains labelled as S. aureus NCTC 6571, eight wild strains, three other NCTC strains and nine strains of uncertain origin. Generally, the S. aureus NCTC 6571 strains produced weaker clots than the remainder. None of the S. aureus NCTC 6571 strains was found to be inoculum dependent but four of the other control strains were. The study demonstrates that some laboratories must improve procedures for ensuring that control S. aureus strains retain their true identity, particularly by avoiding repeated subcultures. Laboratories are divided in their use of strong or weak (S. aureus NCTC 6571) positive controls for the TCT. S. aureus NCTC 6571 is a more stringent control for the TCT than other control strains presently being used and is, therefore, to be preferred. PMID- 8636969 TI - Crystal structure of an acidic phospholipase A2 from the venom of Agkistrodon halys pallas at 2.0 A resolution. AB - The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys pallas has been determined by molecular replacement at 2.0 A resolution to a crystallographic R-factor of 0.157. The overall structure of the molecule is very similar to those of other phospholipase A2 species of known structure. The catalytic site, the hydrophobic channel and the N-terminal region show greatest structural conservation. The Ca(2+)-binding region has a conformation that resembles closely that of bovine PLA2 rather than Crotalus atrox PLA2. Compared with other PLA2 species, the conformation of the C-terminal ridge shows significant difference due to the insertion of two residues. A unique aromatic patch appears on one face of the molecules, surrounded by two acidic residues, the relevant features of this structure and their possible biological implications are discussed. PMID- 8636970 TI - An in-the-groove view of DNA structures in complexes with proteins. AB - DNA structures bound to transcription factors are studied by using the crystal coordinates of complexes. Structural characteristics which are found at the sites where a protein secondary structure, an alpha-helix or a beta-sheet binds, can be understood in terms of fitting of the concave surface of the DNA major groove and the convex surface of the protein secondary structure; the former changes, becoming narrower or wider so that it fits the latter. An alpha-helix, independent of which groove it binds to, tends to produce a narrow major groove and a wide minor groove. When the major groove becomes narrower, the DNA helix axis bends around the major groove. Bending of DNA, which is overall structural change, is achieved by changes in local parameters (in particular, the roll parameter) so that an intermediate feature, the groove, fits the protein surface. PMID- 8636971 TI - The mitochondrial DNA of Allomyces macrogynus: the complete genomic sequence from an ancestral fungus. AB - We have determined the complete nucleotide sequence of the circular mitochondrial DNA (mtDNA) of the chytridiomycete fungus, Allomyces macrogynus (57,473 bp; A + T content 60.5%). The identified genes that are typical for most fungal mitochondria include those for the large (rnl) and small subunit (rns) ribosomal RNAs, a complete set of 25 tRNAs, three ATPase subunits (atp6, atp8 and atp9), apocytochrome b(cob), three subunits of the cytochrome oxidase complex (cox1, cox2 and cox3), and seven subunits of the NADH dehydrogenase complex (nad1, nad2, nad3, nad4, nad4L, nad5 and nad6). A total of 28 introns of both groups are found, some of which contain open reading frames (ORFs) coding for potential endonucleases (group I) or reverse-transcriptases (group II). All mitochondrial genes are transcribed from the same DNA strand, as is the case in many other eufungi. Particular features of the A. macrogynus mtDNA include: (1) the first documented case of a fungal mitochondrial ribosomal protein gene (rps3) that is clearly identified by similarity with bacterial homologues; (2) four unique ORFs; (3) the presence of an insert in the atp6 gene that may have been acquired by interspecific transfer; (4) more than 67 short, highly structured and conserved DNA elements inserted in intergenic spacers, introns, and variable regions of the rnl and rns genes: these elements are unusually G + C rich; (5) rRNA structures that resemble more closely those of eubacteria than their counterparts in other fungal mitochondria. The high degree of conservation of the A. macrogynus mitochondrial rRNA secondary structures, the existence of a mitochondrial rps3 gene (common to protist but unique in fungal mtDNAs), and phylogenetic relationships inferred from highly conserved protein genes, demonstrate consistently the ancestral character of this fungal mitochondrial genome. PMID- 8636972 TI - NMR studies of DNA three-way junctions containing two unpaired thymidine bases: the influence of the sequence at the junction on the stability of the stacking conformers. AB - DNA three-way junctions (TWJs) containing unpaired residues at the branch point can adopt a conformation in which one helix is stacked upon another, forming a coaxial, quasicontinuous double helix. As in four-way junctions (FWJs), two conformers with different stacking arrangements between the arms are possible. However, in both types of structures a markedly strong preference for one conformer has been observed. To investigate the basis for this preference, in particular the influence of the stacking proclivity of the base-pairs at the centre of the junction, two linear oligomers (36 nucleotides), TWJ1 and TWJ2, differing only in one base-pair (G.C versus C.G, respectively) at the branch point, were designed and chemically synthesized. Each one is expected to fold into a stable three-way junction, containing two unpaired thymidine bases at the junction region and two arms capped with a hairpin loop. The data obtained from 1H and 31P-NMR spectroscopy confirm that both oligomers are present as stable three-way junctions. In both TWJs two of the helical arms stack preferentially upon each other. However, the stacking arrangement is similar in both molecules. From this it is deduced that purine-purine stacking across the junction cannot be considered as a major factor that determines the preferred stacking arrangement. PMID- 8636973 TI - Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity. AB - The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and -170 degrees. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 A resolution for data collected at room temperature. The -170 degrees C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 A resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S1 through S4 sites, while density is apparent only in S1 and S2 for antipain. In the S1 site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P1 phenylalanyl side-chain of chymostatin. The P1 arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S1 site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approximation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseus proteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness. PMID- 8636974 TI - High-resolution crystal structure of magnesium (MgII)-iron (FeII) hybrid hemoglobin with liganded beta subunits. AB - The structures of deoxy (alpha(Mg(II))2 beta(Fe(II))2) and CO-liganded (alpha(Mg(II)2(Fe(II)-CO)2) forms of human hemoglobin were determined by X-ray crystallography to a resolution of 1.7 A and 1.9 A, respectively. The deoxy hybrid has virtually the same structure as that of the native deoxy HbA. Both the deoxy and CO-liganded hybrids assumed a T quaternary structure characteristic of native deoxy HbA. No significant structural difference was found between the alpha subunits of the CO-liganded hybrid and deoxy HbA, while in the beta subunit, significant tertiary structural changes were confined to the heme pocket. Baldwin showed by a comparison of COHbA and deoxy HbA that there is a 1.5 A shift of the beta subunit heme into its pocket. This shift was much reduced in alpha(Mg(II))2 beta(Fe(II)-CO)2. On the other hand, when the two structures are compared with superposition of hemes, the nearest neighbors of CO (Fe, E11 Val and E7 His) have shifted nearly to the same positions as those in COHbA. Thus the tertiary structure of the beta subunit of alpha(Mg(II))2 beta(Fe(II)-CO)2 is such that the CO molecule and the neighboring atoms assume nearly the same conformation as those of COHbA, while the block shift of these groups is impeded with respect to the structural invariant portions of the molecule. PMID- 8636975 TI - Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. AB - The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3 methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far. PMID- 8636976 TI - Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. AB - The Strep-tag is a selected nine-amino acid peptide (AWRHPQFGG) that displays intrinsic binding affinity towards streptavidin and has been used as an affinity tag for recombinant proteins. In order to elucidate the molecular mechanism underlying this type of artificial protein-peptide recognition, X-ray crystallographic analyses and binding measurements were carried out. The crystal structure of the complex between recombinant core streptavidin and the synthesized peptide was solved and refined at 1.7 A resolution (space group I4(1)22; unit cell dimensions a = b = 58.3 A, c = 176.9 A). The Strep-tag was bound at the same surface pocket where biotin, the natural ligand of streptavidin, gets complexed. The peptide backbone exhibited 3(10)-helical conformation, with eight of the residues involved in protein contacts. The C terminal Gly-Gly moiety of the Strep-tag participated in a salt bridge to Arg84 of streptavidin with its free carboxylate group. This finding explained why the use of the Strep-tag in fusions with recombinant proteins was restricted to their carboxyl end. Employing a synthetic peptide spot assay, the variant Strep-tag II was screened, which did not have this limitation. The isomorphous crystal structure of its complex with streptavidin revealed that a glutamate side-chain provided the salt bridge in this case, with an otherwise almost unchanged mode of binding. Affinity constants between the peptides and streptavidin were measured by isothermal titration calorimetry. A value of 2.7 x 10(4) M-1 was determined for the Strep-tag peptide, and slightly tighter binding was seen when the Strep tag was applied as part of a bacterially produced fusion protein. This affinity is significantly higher, compared with values previously reported for shorter streptavidin-binding peptides, and agrees well with the remarkable selectivity observed in recombinant protein purification applications. PMID- 8636978 TI - An allosteric theory for hemoglobin incorporating asymmetric states to test the putative molecular code for cooperativity. AB - The two-state (MWC) model for cooperative oxygen binding by tetrameric (alpha2beta2) hemoglobin based on concerted transitions between symmetric states (T and R) is extended to include a third, asymmetric state with one alphabeta dimer possessing high (R-like) oxygen affinity and the other alphabeta possessing low (T-like) oxygen affinity. The asymmetric state is assigned a stability that corresponds to the level reported by Ackers and colleagues in the studies on mixed valence hybrids that led to their proposed "molecular code for cooperativity in hemoglobin." However, this level of stability for the asymmetric intermediates significantly diminishes cooperativity in simulated oxygenation curves, to a degree (Hill n = 2.1) that is no longer compatible with the well established oxygenation properties of normal ferrous hemoglobin (Hill n approximately 3.0). Therefore, the cyanomet derivatives do not appear to be reliable analogues of intermediate oxygenation states. PMID- 8636977 TI - Reversible association of the equilibrium unfolding intermediate of lambda Cro repressor. AB - An extended differentiated scanning calorimetry study of the wild-type Cro repressor and of its V55C mutant has revealed a significant concentration dependence of the melting profiles, even though the two polypeptide chains forming the active repressor molecule are covalently bound within the mutant. An analysis of the temperature dependencies of the partial molar heat capacity suggests that in both cases equilibrium unfolding occurs via a highly-populated intermediate state corresponding to polypeptide tetramers. The results of thermodynamic analysis are confirmed by direct glutaraldehyde cross-linking experiments. Judging by heat effects and circular dichroism data, this intermediate state regains about 50% of the ordered structure and melts co operatively. PMID- 8636979 TI - Identification of a phage-coded DNA-binding protein that regulates transcription from late promoters in bacteriophage P4. AB - The genetic element P4 can propagate as a temperate phage or as a multicopy plasmid in its host Escherichia coli. Late in the lytic cycle and in the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P(LL) and P(sid), which control the transcription of the left and right operons, respectively. Both P4 late promoters are positively regulated by the product of the P4 delta gene, which is transcribed from P(sid). We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control. vis is the first gene downstream of P(LL) and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif. Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P(LL). By Northern hybridization analysis we showed that vis negatively regulates transcription from P(LL) and enhances transcription from P(sid). Thus, vis auto regulates its expression by repressing its own promoter and enhancing transcription of delta, which is required for P(LL) activation. The vis gene was fused with the glutathione S-transferase gene and the GST-Vis fusion protein was partially purified. By gel retardation assays and DNA footprinting we demonstrated that GST-Vis binds to a 32 bp long region immediately downstream of P(LL). We also showed, by gel retardation, that GST-Vis binds to the P sid region. A sequence present in both P(LL) and P(sid) regions may represent the Vis binding consensus sequence. The dual role of Vis on the control of P4 late transcription may be required for a regulated expression of the replication functions when P4 propagates in the plasmid state. PMID- 8636980 TI - RecA protein dynamics in the interior of RecA nucleoprotein filaments. AB - We characterize aspects of the conformation and dynamic state of RecA filaments when bound to dsDNA that are specifically linked to the presence of the second of the two bound DNA strands. Filaments bound to dsDNA exhibit a facile exchange between free and bound RecA monomers or oligomers in the filament interior that is not seen on ssDNA. The RecA mutant K72R, which binds but does not hydrolyze ATP, forms mixed filaments with wild type RecA protein under some conditions. In the presence of dATP, mixed filaments are formed on dsDNA or ssDNA in which the RecA K72R content approximately reflects the proportion of the K72R mutant in the total RecA protein present when the filament is formed. In the presence of ATP, mixed filaments are formed on dsDNA, but the mutant protein strongly inhibits the binding of wtRecA protein to single-stranded DNA. When RecA K72R is added to pre formed filaments containing only wild-type RecA protein on single-stranded DNA, little of the mutant protein exchanges into the filament. Exchange occurs readily, however, when the filament is bound to double-stranded DNA. The presence of a second DNA strand in RecA-dsDNA filaments produces as altered and more dynamic filament state relative to filaments formed on single-stranded DNA. The results point to a substantial alteration in filament state when synapsis occurs during RecA protein-mediated DNA strand exchange. PMID- 8636981 TI - Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase. AB - A specific tumour necrosis factor alpha ribozyme (TNF-alpha-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-alpha ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-alpha-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitro cleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse-chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitro non-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo. PMID- 8636982 TI - Posttranscriptional regulation of EcoP1I and EcoP15I restriction activity. AB - Efficient establishment of a DNA restriction-modification (R-M) system in a non modified cell requires a tight control of the potentially lethal activity of the restriction enzyme. The type III R-M systems EcoP1I and EcoP15I can be transferred to non-modified Escherichia coli cells by transfection, conjugation or transformation and become established without difficulty. Modification activity is expressed immediately after the R-M genes enter the cell, whereas the expression of restriction activity is delayed until complete protection of the cellular DNA is achieved by methylation. We have shown by Western blot analysis that the expression of the modification polypeptide subunit positively regulates the amount of restriction subunit present in the cell. The finding that ribosomal alterations affected the expression of restriction activity pointed to additional control at the translational level. The analysis of EcoP1I expression in E. coli strains mutated in either of the ribosomal proteins S12 (rpsL) or S4 (rpsD) suggests that the level of in vivo restriction activity can be modulated both by a decrease in the efficiency of translation and by varying ribosomal accuracy conditions. In addition, we have preliminary evidence from in vivo gene fusion studies that the res gene may code for more than one gene product. PMID- 8636983 TI - Activation of a yeast pseudo DNA methyltransferase by deletion of a single amino acid. AB - The biological methylation cytosine bases in DNA is central to such diverse phenomena as restriction and modification in bacteria, repeat induced point mutation (RIPing) in fungi and for programming gene expression patterns in vertebrates. Structural studies on HhaI DNA methyltransferase, together with the sequence comparisons of around 40 cytosine-specific DNA methyltransferases, have recently provided a molecular framework for understanding the mechanism of action of the related group of enzymes that catalyse this base modification. There are, however, a number of organisms, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster, which have no detectable DNA methylation. Here we report that the product of the pmt1 gene recently identified in S. pombe, which contains most of the primary structure elements of a typical cytosine-specific DNA methyltransferase, is catalytically inert owing to the insertion of a Ser residue between the Pro-Cys motif found at the active site of all such DNA methyltransferases. Following deletion of this Ser residue, catalytic activity is restored and, using a range of DNA binding experiments, it is shown that the enzyme recognises and methylates the sequence CC(A/T)GG, the same sequence that is modified by the product of the Escherichia coli dcm gene. The pmt gene of S. pombe therefore encodes a pseudo DNA methyltranferase, which we have called psiM.SpoI. PMID- 8636984 TI - Comparison of the structures of wild-type and a N313T mutant of Escherichia coli glyceraldehyde 3-phosphate dehydrogenases: implication for NAD binding and cooperativity. AB - The crystal structure of wild-type and N313T mutant glyceraldehyde 3-phosphate dehydrogenases from Escherichia coli was determined in the presence of NAD at 1.8 angstrom and 2.17 angstrom, respectively. The structure of the monomer and of the tetramer are similar to those observed for other GAPDHs. An exhaustive analysis of the hydrophobic clusters and the hydrogen bond networks explain the high degree of sequence conservation in GAPDHs. The structural effect of the N313T mutation is a change in the (phi,psi) angles of nearby residues Asn236 and Val237, while the structure around the mutated residue remains unchanged. A detailed comparison of the wild-type and N313T mutant E. coli GAPDH with the apo and holo forms of Bacillus stearothermophilus GAPDH is carried out in relation to the apo --> holo transition. An unbiased set of about 60 residues, whose C(alpha) atoms remain in the same relative position in the different forms of the tetramer, is defined as the tetramer "core" which acts as a fixed scaffold around which structural rearrangements occur during the apo --> holo transition. This core essentially includes beta-strands from the beta-sheets forming the O-P and Q R interfaces, in particular strand beta1 which bears catalytic residue His176. During the apo --> holo transition, dimer O-P rotates around the molecular P-axis by about +1 degrees, and dimer O-R by about -1 degrees. Further rotations of the NAD binding domain relative to the catalytic domain are discussed in relation to the molecular symmetry. The possible effect on NAD binding cooperativity of mutations around the tetramer core is exemplified by residue 252. The presence of a conserved hydrophilic patch embedded in the hydrophobic O-P interface is highlighted. A mechanism for substrate binding, different from those currently proposed, is described where the hydroxyl group of the substrate C(2) atom is hydrogen bonded to Cys149N. PMID- 8636985 TI - The crystal structure and ion channel activity of human annexin II, a peripheral membrane protein. AB - Annexin II binds in a calcium-dependent manner to acidic phospholipids and is a substrate of some protein kinases. An N-terminally shortened form of human annexin II was crystallized and its molecular structure determined. It is very similar to two previously described members of this protein family, annexin I and annexin V. The protein structure is nearly completely alpha-helical organized as four compact domains which consist of five alpha-helices each. The domains surround a hydrophilic pore. The calcium binding sites are located at the convex side of the structure as in annexin V. Recombinant and natural porcine annexin II are active as ion channel with characteristics similar to annexin V, while N terminally shortened annexin II and the heterotetramer (annexin II-p11)2 are inactive. Two cysteine residues, Cys133 and Cys262, form a disulphide bridge connecting domains II and III, adding further weight to the notion that ion channel activity does not require major structural rearrangements. PMID- 8636986 TI - The nuclear pore complex and lamina: three-dimensional structures and interactions determined by field emission in-lens scanning electron microscopy. AB - Three dimensional surface imaging was used to examine structural details of the nuclear pore complex. Subsurface structures were uncovered by detergent extraction, proteolysis, mechanical fracturing and combinations of these. Features observed in this way were mostly consistent with previous three dimensional structures with some novel observations. In addition to cytoplasmic and basket filaments attached to each coaxial ring, we have observed radiating filaments within the central channel. New details of basket organization are presented, showing that basket filaments branch and are woven together to form the basket ring. The "central transporter" is shown to be a regular, consistent structure revealed after removal of overlying internal filaments. The lumenal ring is visualised and we present evidence that the lamina may be attached to the spoke ring complex. Finally we show that there is evidence for a novel structure, the "star ring", sandwiched between the cytoplasmic ring and the membrane. PMID- 8636988 TI - Time-resolved fluorescence studies of the molten globule state of apomyoglobin. AB - We have investigated the structure of the molten globule state of horse heart apomyoglobin by energy transfer experiments. The tyrosine residue at position 146 was converted into 3-nitro-tyrosine and distances between this side-chain and the two tryptophanyl side-chains were obtained from the time-resolved tryptophanyl fluorescence decay curve. Since both Trp residues are located in the N-terminal A helix and the modified Tyr residue is located in the C-terminal H-helix, these measurements give information about this helix-helix distance. The energy transfer experiments provide direct evidence for a close contact between the A helix and the H-helix in the molten globule state. This gives a very strong indication of the presence of a single near-native hydrophobic cluster in this state, as previously proposed by other authors. The distance distribution suggests that the fluctuations in the compact states have correlation times shorter than 1 ns. The experiments also show larger fluctuations, both in the native state and in the molten globule state. In addition, the tryptophanyl fluorescence anisotropy decay curves have been measured. The results suggest loose tertiary contacts in the molten globule state, which is in good agreement with earlier studies. For the denatured state of apomyoglobin, both techniques indicate an extended random coil structure. PMID- 8636987 TI - Global changes in amide hydrogen exchange rates for a protein antigen in complex with three different antibodies. AB - The binding of anti-lysozyme monoclonal antibodies, D44.1 or D1.3, to their antigen reduces the rate of exchange for many amide hydrogens in lysozyme. The D44.1 antibody contacts a similar region of lysozyme to the HyHEL-5 antibody, while the D1.3 antibody binds to the side of lysozyme which is opposite to the HyHEL-5 and D44.1 epitopes. We compare the effects of binding these antibodies on amide hydrogen exchange rates in lysozyme. These comparisons suggest that there are regions of lysozyme that fluctuate in a coordinated manner such that the effects of binding can be propagated to regions that are distant from the epitope. The activation enthalpies for hydrogen exchange for 36 of the 126 amide hydrogens in lysozyme and for 25 of 126 lysozyme amide hydrogens in the lysozyme D1.3 complex are also reported. These data suggest that the reduction in amide hydrogen exchange rates upon antibody binding reflect changes in the dynamics of the antigen. These changes contribute to a reduction in the specific heat capacity upon binding. PMID- 8636989 TI - CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event. AB - The expansion and contraction of CTG and CGG trinucleotide repeat sequences have been associated with several heritable genetic diseases. We developed a system for investigating the expansion of triplet repeat sequences in Escherichia coli in order to elucidate molecular mechanisms. Analysis of expanded regions using the interrupting CTA triplet sequence as a location marker within the CTG tract revealed that the expansion of large CTG repeats is one event rather than an accumulation of multiple small expansions and that the expansions occur more frequently in the region distal from the replication origin. Also, we showed that a loss of interruptions increases the expansion frequency. Thus, the instability of large triplet repeats in hereditary diseases occurs by a mechanism different from the instability in microsatellite sequences caused by defects in mismatch repair systems for certain sporadic cancers and hereditary non-polyposis colorectal cancers. PMID- 8636990 TI - Deletion analysis of the che operon in the archaeon Halobacterium salinarium. AB - Halobacterium salinarium is a chemo- and phototactic archaeon whose signal transduction pathway includes the classical two-component system made up of CheA and CheY. Deletion analysis of the che operon in H. salinarium has been undertaken. Following the removal of the entire operon, the importance of each of the four individual members, cheY, cheB, cheA, and the novel member cheJ, was evaluated by their replacement in combinations of three. The mutant strains were investigated for their motility, their chemo- and phototactic signalling, and the rotational bias of their flagella. Loss of cheA, cheY or cheB led to the complete loss of chemo- and phototaxis, whereas the absence of cheJ caused a reduction in chemo- and phototactic ability. Reverse swimming and counterclockwise rotation of the flagella required the presence of cheA and CheY. The wild-type 50:50 distribution of forward and reverse swimming was observed in the strain lacking cheB, whereas this distribution was perturbed to 88:12 in the strain lacking cheJ. These results are compared with the corresponding deletion strains in Escherichia coli and provide new insights into the eu- and archeabacterial flagellar switch. PMID- 8636991 TI - Structural and functional characterisation of the voltage sensor in the ion channel human annexin V. AB - The ion channel properties of human annexin V, a calcium- and phospholipid binding protein of the annexin family, have been structurally and functionally investigated by analysing the mutant Glu112 -->Gly. Glu112 forms a salt bridge with Arg271 located in the interior of the hydrophilic pore of the molecule which is conserved within the annexin family. The crystal structures of the mutant and wild-type proteins are very similar and show only marginal conformational changes around the mutation site. Electron microscopic images show a conserved four domain structure upon membrane binding as in the wild-type annexin V. The channel properties of the mutant are drastically changed, as the mutant has lost the voltage-dependent channel gating and the selectivity for calcium ions over monovalent cations. These results strongly support the hypothesis that the central, hydrophilic pore is the ion-conducting pathway. PMID- 8636992 TI - Physical and functional analysis of the prokaryotic enhancer of the sigma 54 promoters of the TOL plasmid of Pseudomonas putida. AB - The physical and the functional organization of the upstream cis-acting sequence that controls at a distance the transcriptional activity of Pu and Ps, the two sigma 54-dependent promoters of the TOL (toluene/xylene biodegradation) operons of Pseudomonas putida, have been determined. DNase I and hydroxyl radical footprinting of the promoters with the purified and pre-activated enhancer binding protein XylR clearly indicated the presence of two distinct binding sites (proximal and distal) that were occupied independently and did not share an evident sequence similarity. However, alignment of the sequence on the basis of the cleavage protection patterns, along with those produced on Po, a third XylR responsive promoter of a phenol degradation operon, generated a consensus sequence 5'-TTGATCAATTGATCAA-3' having greater similarity to the proximal than to the distal boxes. To verify that this consensus was the sequence recognized by XylR, we footprinted in vitro a synthetic site, the results indicating that it was strongly bound by the activator with the predicted pattern of interactions. The mode of protection indicated that XylR recognized the sequence as a palindrome and not as a tandem repeat, interacting with it on one side of the DNA helix. In vivo experiments involving directed deletions through the entire 5' region of the Pu promoter confirmed that the proximal XylR-binding sequence suffices for promoter activity. In vivo data also suggested that XylR binding to the upstream sequences promoted the assembly of the oligomeric form of the activator that is competent for transcription initiation. PMID- 8636993 TI - In vitro activities of an N-terminal truncated form of XylR, a sigma 54-dependent transcriptional activator of Pseudomonas putida. AB - A truncated derivative of the XylR protein, which is able to constitutively activate the sigma 54-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to homogeneity and its various activities have been separately examined, in vitro. The truncated regulator XylR delta A was deleted of the signal reception N-terminal module present in wild type XylR, but retained its central activation domain and the DNA binding segment, located at its C terminus. XylR delta A bound to the region -120 to -190 bp upstream of the transcription initiation site of the Pu promoter, where previous analyses have located the XylR target site. XylR delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA containing the native upstream activation sequences of Pu. Both ATPase activity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPase activity but retained the nucleotide binding ability of the parental protein. XylR delta A was able to activate transcription in vitro with sigma 54-RNA polymerase alone, although its activity was enhanced up to 20-fold in the presence of the integration host factor protein. The requirements for activation of the Pu promoter in vitro are consistent with the view that DNA-facilitated oligomerization of the regulator for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma 54-dependent promoters. Furthermore, additional co-regulation elements seem to adjust promoter activity in vivo to the physiological status of the cells. PMID- 8636994 TI - Suppression of temperature-sensitive defects of polypeptide release factors RF-1 and RF-2 by mutations or by an excess of RF-3 in Escherichia coli. AB - The termination of protein synthesis in bacteria requires two codon-specific polypeptide-release factors, RF-1 and RF-2. A third factor, RF-3, stimulates the RF-1 and RF-2 activities in vitro. To clarify the in vivo role of RF-3 for the RF 2 dependent termination, we isolated and characterized suppressor mutations for the temperature-sensitive RF-2 mutation prfB286. One of the intergenic suppressor mutations, srb-1, acquired an up-promoter alteration in the RF-3 gene, which enhanced the RF-3 expression four- to fivefold. Consistently a threefold increase in the RF-3 level by a promoter-controlled expression plasmid suppressed prfB286. On the other hand, a temperature-sensitive mutation in RF-1, prfA1, was suppressed only slightly by the high-level expression of wild-type RF-3. The RF-3 mutations that suppress prfA1 were isolated and named sra. They were classified into four specific alleles; two each in the N and C-terminal regions. These altered RF-3 proteins restored the RF-1-dependent termination at UAG in prfA1 cells. Moreover, they enhanced the RF-2-dependent UGA termination in both wild type and prfB286 cells. The termination-stimulating activity of RF-3 was further additively increased by the double sra mutations, suggesting that they affected two distinct protein domains that modulate the termination reaction. Taking these and other results into consideration, RF-3 is likely to interact functionally and cooperatively with the release factors RF-1 and RF-2 in Escherichia coli. PMID- 8636995 TI - Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent. AB - The accessibility of the ribose groups in the phosphodiester chain of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, has been probed with an Fe(II)-EDTA reagent when the RNA is alone in solution, when it is in a complex with a tRNA precursor substrate, and when it is in the holoenzyme complex with its cofactor, C5 protein. The regions found to be protected under these various conditions, as well as those previously identified in other chemical probing experiments, have been mapped on a three-dimensional working model of M1 RNA and are generally compatible with the previously proposed placement of the substrate on the enzyme and with previous data and inferences regarding the interactions of C5 protein with M1 RNA. On the basis of the accessibilities of the C(4') atoms, refinements have been introduced in the model to accommodate the Fe(II)-EDTA protection data. The protein cofactor makes contact with several helical regions of the catalytic RNA on the opposite side of the surface to which substrates bind. PMID- 8636996 TI - Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene. AB - The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome. PMID- 8636997 TI - DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage. AB - DNA gyrase, the bacterial enzyme that supercoils DNA, is trapped on chromosomal DNA by the 4-quinolone compounds, as drug-gyrase complexes that contain DNA breaks. Examination of chromosomal DNA extracted from Escherichia coli indicated that bacteriostatic concentrations of oxolinic acid trap gyrase and block DNA synthesis without releasing broken DNA from gyrase-DNA complexes. Release, detected as free rotation of DNA in the presence of an intercalating dye, occurred only at high, bactericidal oxolinic acid concentrations. Release of DNA breaks and cell death were both blocked by chloramphenicol, an inhibitor of protein synthesis, suggesting that synthesis of additional protein activity is required to free the DNA ends. Ciprofloxacin, a more potent quinolone, released DNA breaks and killed cells even in the presence of chloramphenicol. It is proposed that this second, chloramphenicol-insensitive mode for release of DNA breaks and cell killing arises from dissociation of gyrase subunits. Ciprofloxacin also killed a gyrase (gyrA) mutant resistant to the prototype of quinolone, nalidixic acid, and created complexes on DNA detected by DNA fragmentation. This lethal effect of ciprofloxacin was eliminated by additional mutations mapping in parC, one of the two genes encoding topoisomerase IV. Thus, the fluoroquinolone compounds have two intracellular targets. In the absence of the gyrA mutation, the parC (CipR) allele did not by itself confer resistance to ciprofloxacin, indicating that gyrase is the major quinolone target in E. coli. These findings provide a molecular explanation for quinolone action in bacteria and a new way to study topoisomerase IV-chromosome interactions. PMID- 8636998 TI - Structural requirements for FokI-DNA interaction and oligodeoxyribonucleotide instructed cleavage. AB - The FokI restriction endonuclease recognizes the double-stranded (ds) 5'-GGATG-3' site and cuts at the 9th and 13th nucleotides downstream from the 5'-3' and 3'-5' strands, respectively. To elucidate the interaction between FokI and DNA, and the effect of Mg2+ on this interaction, we used FokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the FokI recognition site. Oligo- and dsDNA FokI interactions showed that for fully effective recognition, two or more base pairs were required outside the 5'-GGATG-3' site. When using FokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover, FokI cleavage, when followed by step-wise filling-in of FokI cohesive ends in the dsDNA, allowed FokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed with FokI (without Mg2+. Such a complex, when formed in the absence of Mg2+, did not accept the subsequently added Mg2+ for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg2+, the half-life of the complex FokI and dsDNA was 12 minutes at 22 degrees C. In the absence of Mg2+, such a complex, possessing a terminally located 5'-GGATG-3' site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5' GGATG-3' site. PMID- 8636999 TI - A mathematical analysis of in vitro molecular selection-amplification. AB - We construct a mathematical model for in vitro molecular selection with amplification. Using DNA-protein binding as the illustrative example, we obtain an expression for the probability that a randomly selected molecule from the final in vitro selection products is a molecule with the highest binding affinity. Experiments of this type have been reported for several examples of DNA binding proteins. Our study requires a model of the DNA-protein binding constant between DNA molecules and the target protein. The relationship between binding constants and selection probabilities is presented under simplifying but reasonable assumptions. From our analysis, we find that for successful in vitro selection experiments there should be a certain relationship between the number of polymerase chain reaction cycles and the concentration of free protein. The results obtained should be widely applicable to a variety of selection amplification procedures. PMID- 8637000 TI - Crystal structure of the pertussis toxin-ATP complex: a molecular sensor. AB - Pertussis toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. The protein is a hexamer containing a catalytic subunit (S1) that is tightly associated with a pentameric cell-binding component (B-oligomer). In vitro experiments have shown that ATP and a number of detergents and phospholipids assist in activating the holotoxin by destabilizing the interaction between S1 and the B-oligomer. Similar processes may play a role in the activation of pertussis toxin in vivo. In this paper we present the crystal structure of the pertussis toxin-ATP complex and discuss the structural basis for the ATP-induced activation. In addition, we propose a physiological role for the ATP effect in the process by which the toxin enters the cytoplasm of eukaryotic cells. The key features of this proposal are that ATP binding signals the arrival of the toxin in the endoplasmic reticulum and, at the same time, triggers dissociation of the holotoxin prior to membrane translocation. PMID- 8637001 TI - Structural studies of synthetic peptides dissected from the voltage-gated sodium channel. AB - Peptides representing transmembrane regions of the alpha-subunit of the voltage gated sodium channel were synthesised and their structures analysed, using 1H NMR and CD, in trifluoroethanol and in dodecylphosphocholine micelles. Sequence analysis suggests that the channel has six regions, S1 to S6, predicted to span the membrane in four homologous domains, designated, I, II, III and IV. Presented here are studies of representatives examples of possible single spanning segments (IS2, IS4, IVS4) and a double spanning segment, IS34, composed of segments IS3 and IS4. In addition, we investigated ISlink56, the putative linker region between segments IS5 and IS6. All of the peptides were found to have predominantly alpha-helical structures in both solvent systems. There was some evidence for bending of the longer helices but there was no discernible evidence for well-defined tertiary structure. PMID- 8637002 TI - Amino acid sequence determinants of beta-lactamase structure and activity. AB - TEM-1 beta-lactamase catalyzes the hydrolysis of beta-lactam antibiotics such as the penicillins and cephalosporins, thus providing for bacterial resistance to these compounds. To determine the amino acid residues critical for the structure and function of TEM-1 beta-lactamase, the codons for each of the 263 amino acid residues that constitute the mature form of the enzyme were randomized using a site-directed mutagenesis procedure. Functional random mutants were selected based on their ability to confer ampicillin resistance to Escherichia coli. The DNA sequence of several functional mutants was determined for each set of random mutants. It was found that 43 out of the 263 amino acid residues do not tolerate substitutions and therefore are critical for the structure and activity of the enzyme. In addition, a comparison of conserved residue positions among functional beta-lactamase mutants with conserved residues in the beta-lactamase gene family identified many positions which did not tolerate substitutions in the mutagenesis studies but are freely substituted among members of the gene family. This observation may be due to the accumulation of compensating mutations among members of the gene family. Finally, the sequence variability at residue positions among functional mutants was quantitated by calculating the effective number of substitutions at each position using information-theoretical entropy. These values were used to obtain a quantitative estimate of the correlation between the sequence variability at a position and the fractional accessible surface area of the residue. The correlation is found to be statistically significant in that buried residues tend to exhibit low variability and invariant residues tend to exhibit low solvent exposure. However, the correlation is weak because most residues are neither completely buried nor invariant. PMID- 8637003 TI - Intermediate states in protein folding. AB - The efficiency of protein folding suggests that this process proceeds through some intermediate states, raising the possibility of experimental observation of incompletely folded states of proteins. However, critical analysis of the observed partly folded stable states of proteins shows that they present either misfolded forms obtained under conditions inappropriate for folding, or partially unfolded states that retain folded the subpart of these molecules. This retained part, which unfolds last and folds first in a unfolding/refolding experiment, has a definite tertiary structure maintained by specific long-range interactions and can be isolated by fragmentation. Therefore, it can be regarded as a definite domain of the protein molecule. Provided the polypeptide chain of a small single domain protein does not become trapped in a misfolded form, its folding proceeds very rapidly, with all intermediates being transient and extremely unstable. PMID- 8637004 TI - Bacteriophage T4 host range is expanded by duplications of a small domain of the tail fiber adhesin. AB - The adsorption specificity of T4 is determined by the tip of the gene 37 tail fibers which bind to receptors on the bacterial surface. T4 infects only Escherichia coli and closely related Shigella species, but rare host range mutants can be isolated that infect Yersinia pseudotuberculosis I, an evolutionally distant bacterium. Some of these mutations result in amino acid residue substitutions in the C-terminal portion of gene 37, but others involve unequal exchanges between a series of sequence motifs (His boxes) in the same region. The duplication or mutational alteration of this segment apparently suffices for phage adsorption to a Yersinia receptor. It is suggested that recombination between the His box sequences can generate diversity in phage host range by shuffling receptor recognition domains. PMID- 8637005 TI - Inter-ring communication is disrupted in the GroEL mutant Arg13 --> Gly; Ala126 - > Val with known crystal structure. AB - The crystal structures of the chaperonin GroEL Arg13 --> Gly; Ala126 --> Val double mutant, without and in complex with ATP gamma S, have been determined at atomic resolution. Here, we show that the double mutation Arg13 --> Gly; Ala126 - > Val disrupts negative co-operativity between GroEL rings, with respect to ATP, but has little effect on the positive co-operativity within each ring. Our results help to explain why the double mutation facilitated the crystallization of GroEL and why breaking of dyad symmetry between rings is not observed in crystal structures of this mutant. Our results may also help to explain why the observed structural differences between the GroEL double mutant and its ATP gamma S-bound form are small. PMID- 8637006 TI - Transactivation of adenovirus E2-early promoter by E1A and E4 6/7 in the context of viral chromosome. AB - Transcription from adenovirus E2-early promoter is controlled by a unique array of four cis-acting elements which include an atypical TBP site, two E2F sites present in an inverted orientation relative to each other, and an ATF site. In virus-infected cells, this promoter is transactivated by E1A and the E4 6/7 proteins. In addition, it is also stimulated by the DNA-binding protein (DBP) in transient transfection assays. Here we describe a genetic analysis of the E2 transcriptional regulation in the context of the viral chromosome. By using genetically engineered mutant adenoviruses we have determined the interrelationship between the different cis-acting elements of the E2-early promoter during basal transcription, the extent to which E1A and E4 6/7 contribute to the E2 promoter activation and the E2 promoter elements that respond to these transactivators. We show that at eight hours following infection, E1A can transactivate the promoter about 21-fold whereas E4 6/7 can induce the promoter by only fivefold. DBP does not induce the promoter in the chromosomal context. Our mutational analysis suggests that the unique architecture of the E2-early promoter necessitates the concerted interaction of all three host transcription factors with their cognate recognition elements to form a stable and functional transcription complex. E1A mediated transactivation is dependent on this stable basal transcription complex and transactivation may involve simultaneous interaction of E1A with each of the three transcription factors present in the multicomponent basal transcription complex. The E4 6/7 protein can transactivate the E2-early promoter in the absence of ATF presumably by promoting the DNA binding capacity of transcription factor E2F and thereby stabilizing the basal transcription complex. We discuss some of the possible protein-protein interactions that may take place at the level of the multicomponent transcriptional complex at the E2-early promoter during transcriptional activation and the discrepancies that arise when a promoter is analyzed in infection versus transfection assays. PMID- 8637007 TI - Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage. AB - The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints. To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage. The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length. When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding. We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones. The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates. Cross reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently. The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides. PMID- 8637008 TI - Flexible regions of RNA structure facilitate co-operative Rev assembly on the Rev response element. AB - The oligomerisation of Rev on the Rev-response element (RRE) was studied using a series of model substrates. Only a monomer of Rev is able to bind efficiently to a high affinity site that is flanked by perfect duplex RNA. Addition of a bulge or a second stem structure adjacent to the high affinity site permits the co operative incorporation of a second Rev molecule to the RNA. Model RREs carrying bulges can bind Rev with a higher degree of co-operativity than the native structure. Oligomerisation was efficient when the bulge was moved to the opposite strand of the duplex, but was severely impaired when the distance between the bulge and the high affinity site was increased by more than 8 bp. Rev can oligomerise at either end of the RNA-protein complex formed at the high affinity site; when the duplex flanking a high affinity site is disrupted by a bulge or a stem, oligomerisation proceeds in the direction of the disruption regardless of the orientation of the high affinity site. The results are consistent with the "molecular rheostat" model for RRE function, which suggests that Rev binding to the RRE is highly distributive and provides a sensitive measurement of intracellular Rev concentrations. PMID- 8637009 TI - Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain. AB - We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals. PMID- 8637010 TI - Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8. AB - UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA. PMID- 8637011 TI - A model for the cooperative binding of eukaryotic regulatory proteins to nucleosomal target sites. AB - The mechanism by which gene regulatory proteins gain access to their DNA target sequences in chromatin is not known. We recently showed that nucleosomes are intrinsically dynamic, transiently exposing their DNA to allow sequence-specific protein binding even at buried sites. Here we show that this dynamic behaviour provides a mechanism for cooperativity (synergy) in the binding of two or more proteins to sites on a single nucleosome, even if those proteins do not interact directly with each other in any way. As a consequence of this cooperativity, two proteins binding to the same nucleosome facilitate each other's binding and also control the level of occupancy at each other's sites. This model, with no adjustable parameters, accounts quantitatively for recent reports of cooperative (synergistic) binding to nucleosomes in vitro. We assess the potential importance of this new cooperativity for gene regulation in vivo by comparing its magnitude to free energies of cooperative protein-protein direct contacts having known significance for gene regulation. Possible roles for nucleosome dynamics in eukaryotic gene regulation, and key remaining questions, are discussed. PMID- 8637013 TI - Effect of GroEL on the re-folding kinetics of alpha-lactalbumin. AB - The effect of GroEL on the re-folding kinetics of apo- and holo-alpha-lactalbumin from the acidic molten globule state has been investigated by stopped-flow fluorescence measurements. GroEL retards the re-folding of apo-alpha-lactalbumin by interacting with the molten globule state of the protein. The binding constant was estimated to be in the order of 10(5) M-1 by analyzing the kinetic data quantitatively and was found to be much weaker than the binding between GroEL and disulfide-bond reduced alpha-lactalbumin, whose binding constant is in the order of 10(7) M-1. Our present results, together with the previous results, suggest that the state recognized by GroEL is not unique and that the binding strength varies with the state of a target protein. The binding between GroEL and the molten globule state of apo-alpha-lactalbumin becomes stronger with an increasing salt concentration; the binding constant is increased tenfold (from 10(5) to 10(6) M-1) by an increase in salt concentration from 0.05 to 0.25 M. The study of the effect of GroEL on the re-folding kinetics of holo-alpha-lactalbumin, which is represented by a bi-phasic process, shows that the slow phase is affected by GroEL in the same manner as observed in the apo-alpha-lactalbumin re-folding but that the fast phase is not affected by GroEL at all. This indicates that the binding rate of GroEL is faster than the slow phase but slower than the fast phase of the re-folding, and the bi-molecular rate constant of GroEL binding to the molten globule state of alpha-lactalbumin was estimated to be in the order of 10(6) M-1S-1. PMID- 8637012 TI - Thermodynamic prediction of conserved secondary structure: application to the RRE element of HIV, the tRNA-like element of CMV and the mRNA of prion protein. AB - An algorithm for prediction of conserved secondary structure of single-stranded RNA is presented. For each RNA of a set of homologous RNAs optimal and suboptimal secondary structures are calculated and stored in a base-pair probability matrix. A multiple sequence alignment is performed for the set of RNAs. The resulting gaps are introduced into the individual probability matrices. These homologous probability matrices are summed to give a consensus probability matrix emphasizing the conserved secondary structure elements of the RNA set. Thus the algorithm combines the advantages of thermodynamic structure prediction by energy minimization with the information obtained from phylogenetic alignment of sequences. The algorithm is applied to three examples. The REV-responsive element of HIV, the structure of which is well known from the literature, was chosen to test the algorithm. The second example is the 3' terminal segment of genomic single-stranded RNAs of cucumber mosaic viruses; a structure similar to that of the related brome mosaic virus was expected and was confirmed. The third example is the prion-protein mRNA from different organisms; the structure of this mRNA is not known. By application of the algorithm highly conserved hairpins were found in the prion-protein mRNA. PMID- 8637014 TI - A discontinuous headful packaging model for packaging less than headful length DNA molecules by bacteriophage T4. AB - Bacteriophage T4 and other double-stranded DNA-containing bacteriophages package DNA by the classical headful packaging mechanism. In this mechanism, the packaging machinery cuts a DNA concatemer and packages a single unit length genome within the viral capsid. The length of the packaged DNA molecule is determined by the size of the viral capsid. Surprisingly, during large DNA cloning experiments, we observed that the in vitro phage T4 packaging system can package and transduce DNA molecules that are much smaller than the T4 headful size. We analyzed this phenomenon by using defined plasmid DNAs as substrates for in vitro packaging. The data showed that phage T4 can successfully package and transduce 4 to 29 kb plasmid DNA molecules. When two plasmid DNAs with different antibiotic markers were added to the packaging reaction mixture, transductants that are resistant to both the antibiotics were obtained, suggesting that both the plasmid DNAs are packaged within the same head. Analysis of the transducing particles by equilibrium CsCl density-gradient centrifugation showed that the particles have the same density as the wild-type phage. That the less than headful length molecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches. Finally, unit length plasmid DNA molecules of appropriate size were isolated from the in vitro packaged particles. Based on these data, we propose a discontinuous headful packaging model for packaging less than headful length molecules. In this model, the packaging machinery packages the first available less than headful length DNA molecule and generates a partially full head. The partially full head then reinitiates packaging on a second DNA molecule. This process continues until the head is filled with DNA. PMID- 8637016 TI - Structure and orientation of the mammalian antibacterial peptide cecropin P1 within phospholipid membranes. AB - Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly alpha-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospholipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cis positive transmembrane voltage difference. Taken together, the results further support a "carpet-like" mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration. PMID- 8637017 TI - A surface of minimum area metric for the structural comparison of proteins. AB - A new method for comparing protein structures, based on a minimal surface metric, is developed. A virtual polypeptide backbone is created by joining consecutive C alpha atoms in a protein structure. The minimal surface between the virtual backbones of two proteins (the Area Functional) is determined numerically using an iterative triangulation strategy. The first protein is then rotated and translated in space until the smallest minimal surface is obtained. Such a technique yields the optimal structural superposition between two protein segments. It requires no initial sequence alignment, is relatively insensitive to insertions and deletions, and obviates the need to select a gap penalty. The optimal minimal area can then be converted to the Area-C alpha distance, measured in angstroms, to determine the structural similarity. This technique has been applied to a large class of proteins and is able to detect not only small-scale differences between closely related proteins but also large-scale topological similarities between evolutionary unrelated proteins that lack any obvious sequence homology. To measure the similarity between structurally dissimilar proteins, an additional measure (the Fit Comparison) is developed. This is a scale-invariant measure of a structural similarity that is useful for determining topological similarities between dissimilar proteins with unrelated sequences. PMID- 8637015 TI - Human alpha-thrombin inhibition by the active site titrant N alpha-(N,N dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: a comparative kinetic and X-ray crystallographic study. AB - Kinetics for the hydrolysis of the chromogenic active site titrant N alpha-(N,N dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77 plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen).Dmc-azaLys acyl.enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 A resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin.Dmc azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin.Dmc-azaLys acyl.enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme "aryl-binding site". PMID- 8637018 TI - The search for cytotoxic synergy between anticancer agents: a case of Dorothy and the ruby slippers? PMID- 8637019 TI - Breast cancer care in old age: where do we go from here. PMID- 8637021 TI - Yeast's value rises as scientists map its 6,000 genes. PMID- 8637020 TI - Drug discovery shifts toward rapid, mass screening. PMID- 8637022 TI - Cancer researchers getting caught up in the net. PMID- 8637023 TI - Why patients enroll in clinical trials: physicians play a key role. PMID- 8637024 TI - New GAO report says insurance coverage growing for bone marrow transplants. PMID- 8637025 TI - Factors associated with surgical and radiation therapy for early stage breast cancer in older women. AB - BACKGROUND: In addition to demographic and health care-related characteristics, the age and physiologic status of women at the time of breast cancer diagnosis have been reported to influence receipt of standard treatments. Previous studies of the influence of age and comorbidity have not examined whether other patient-, region-, or health care-related characteristics altered the association of age and comorbidity with type of treatment received. PURPOSE: This study examined factors associated with receipt of breast-conserving surgery and radiation therapy, both of which are recommended treatments for breast cancer, among a cohort of 18,704 women aged 65 years or more who had breast cancer diagnosed during the period from 1985 through 1989. METHODS: A data file linking Medicare claims records to data from the Surveillance, Epidemiology, and End Results (SEER) Program of the U.S. National Cancer Institute was utilized. Logistic regression analysis was used to examine associations between patient, region, and hospital characteristics and the receipt of specific treatments. The likelihood test was used to assess the significance of observed associations (expressed as odds ratios [ORs]). Because of multiple comparisons, only those ORs with two sided P values <.01 were considered statistically significant. RESULTS: The frequency of breast-conserving surgery was highest (54%) among women aged 80 years or more, who had two or more comorbid conditions and stage I disease. However, in general, the receipt of radiation therapy among women undergoing breast-conserving surgery declined markedly with age, irrespective of comorbidity status and disease stage. Between the ages of 65-69 years and 80 years or older, radiation therapy declined from 77% to 24% among women with no comorbid conditions and from 50% to 12% among women with two or more comorbid conditions. In regression models that included hospital, region, and patient characteristics as variables, age and comorbidity remained independently associated with the receipt of radiation therapy (OR = 0.12 and 95% confidence interval [CI] = 0.10 0.14 for women aged 80 years or more compared with women 65-69 years of age and OR of 0.33 [95% CI = 0.24-0.46] for women with two or more comorbid conditions versus no comorbid conditions). CONCLUSIONS: After adjustment for multiple clinical and nonclinical factors influencing treatment, chronologic age remains an important independent factor associated with the receipt of radiation therapy after breast-conserving surgery among women aged 65 years or more who were diagnosed with early stage breast cancer. IMPLICATIONS: Future studies should determine whether these differences in treatment patterns among older women result in increased morbidity (e.g., from recurrence), shortened disease-free or overall survival, or decreased quality of life. PMID- 8637026 TI - Increase in testicular cancer incidence in six European countries: a birth cohort phenomenon. AB - BACKGROUND: For unknown reasons, the age-standardized incidence of testicular cancer has shown a rapid increase in virtually all countries (mostly Western) studied. For populations with a sufficiently long period of cancer registration, this development can be traced back to the first half of this century. PURPOSE: By evaluating data from six countries with long periods of cancer registration (Denmark, Norway, Sweden, the former German Democratic Republic [East Germany], Finland, and Poland), we sought to determine whether the increase in testicular cancer risk follows a birth cohort pattern and, if so, to quantify and compare any birth cohort effects. METHODS: A total of 30,908 incident cases of testicular cancer, diagnosed from 1945 through 1989 in men who were 20-84 years of age, were identified in population-based cancer registries in the six countries. In addition to performing simple trend analyses, we fitted several Poisson regression models (with the explanatory variables age, time period [calendar time], and birth cohort) to the data. Individual models were estimated by the maximum likelihood method. RESULTS: The age-standardized incidence of testicular cancer was found to vary among the six populations and, on the basis of total registration data, increased annually at rates ranging from 2.3% (in Sweden) to 5.2% (in East Germany). A comparison of several regression models indicated that birth cohort was a stronger determinant of testicular cancer risk than was calendar time for all six populations. Within each population, little variation in testicular cancer risk was observed for men born between 1880 and 1920; thereafter, the risk began to increase. Among men born in Denmark, Norway, and Sweden between 1930 and 1945 (the period encompassing the Second World War), the increasing trend in risk was interrupted (i.e., a leveling in risk occurred). After 1945, an uninterrupted increase in risk was observed for all six populations. With men born around 1905 as the reference group, the relative risk of testicular cancer for those born around 1965 varied from 3.9 (95% confidence interval [CI] = 2.7-5.6) in Sweden to 11.4 (95% CI = 8.3-15.5) in East Germany. CONCLUSIONS AND IMPLICATIONS: The increasing trend in testicular cancer risk observed for these six populations follows a birth cohort pattern. This distinct risk pattern provides a framework for the identification of specific etiologic factors. PMID- 8637027 TI - Cytotoxic effects of topotecan combined with various anticancer agents in human cancer cell lines. AB - BACKGROUND: Topotecan (TPT) is a topoisomerase I poison that exhibits antineoplastic activity. Analysis of the cytotoxic effects of combinations of TPT and other anticancer agents has been limited. PURPOSE: We assessed the cytotoxic effects produced by combinations of TPT and other antineoplastic agents in experiments involving multiple human cancer cell lines of diverse histologic origins. METHODS: The cytotoxic effects of various antimetabolites (fluorouracil, methotrexate, or cytarabine), antimicrotubule agents (vincristine or paclitaxel [Taxol]), DNA alkylating agents (melphalan, bis[chloroethyl]nitrosourea [BCNU], or 4-hydroperoxycyclophosphamide [4HC]), and a DNA-platinating agent (cisplatin), alone and in combination with TPT, were measured in clonogenic (i.e., colony forming) assays. HCT8 ileocecal adenocarcinoma, A549 non-small-cell lung carcinoma, NCI-H82ras(H) lung cancer, T98G glioblastoma, and MCF-7 breast cancer cell lines were used in these assays. The data were analyzed by the median effect method, primarily under the assumption that drug mechanisms of action were mutually nonexclusive (i.e., completely independent of one another). For each level of cytotoxicity (ranging from 5% to 95%), a drug combination index (CI) was calculated. A CI less than 1 indicated synergy (i.e., the effect of the combination was greater than that expected from the additive effects of the component agents), a CI equal to 1 indicated additivity, and a CI greater than 1 indicated antagonism (the effect of the combination was less than that expected from the additive effects of the component agents). RESULTS: When the mechanisms of drug action were assumed to be mutually nonexclusive, virtually all CIs for combinations of TPT and either antimetabolites or antimicrotubule agents revealed cytotoxic effects that were less than additive. The CIs calculated at low-to intermediate levels of cytotoxicity for combinations of TPT and the DNA alkylating agents melphalan, BCNU, and 4HC also showed drug effects that were less than additive; in most cases, however, nearly additive or even synergistic effects were observed with these same drug combinations at high levels of cytotoxicity (i.e., at > or = 90% inhibition of colony formation). Results obtained with combinations of TPT and cisplatin varied according to the cell line examined. With A549 cells, less than additive effects were seen at low-to intermediate levels of cytotoxicity, and more than additive effects were seen at high levels of cytotoxicity. With NCI-H82ras(H) cells, synergy was observed over most of the cytotoxicity range. CONCLUSIONS AND IMPLICATIONS: TPT cytotoxicity appears to be enhanced more by combination with certain DNA-damaging agents than by combination with antimetabolites or antimicrotubule agents. Interactions between TPT and other drugs can vary depending on the cell type examined. Further investigation is required to determine the basis of the observed effects and to determine whether these in vitro findings are predictive of results obtained in vivo. PMID- 8637028 TI - Hepatitis C virus infection and risk of hepatocellular carcinoma among Japanese: possible role of type 1b (II) infection. AB - BACKGROUND: Although hepatitis C virus (HCV) infection is recognized as an important risk factor for hepatocellular carcinoma HCC), the strength of this association has been inconsistent. In addition, the role of specific HCV genotypes in HCC progression has not yet been determined. PURPOSE: We conducted a case-control study to estimate the relative risk (RR) of HCC in relation to HCV infection among residents of the Fukuoka Prefecture, where HCC risk is among the highest in Japan, and to examine whether the risk differs according to HCV genotypes and/or HCV RNA titers. METHODS: Stored serum samples obtained from 91 patients with HCC and 410 healthy control subjects, who had been frequency matched to the patients with regard to sex and age, were tested for antibodies to HCV by use of second-generation immunoradiometric and immunoblot assays. The presence of serum HCV RNA and of specific HCV genotypes was determined by use of polymerase chain reaction-based assays, and HCV RNA titers were measured by use of a branched DNA assay. RESULTS: Antibodies to HCV were detected in 71 patients (78.0%) and in 30 control subjects (7.3%), of whom 57 patients and 25 control subjects had serum HCV RNA. One patient was positive for HCV RNA but not for antibodies to HCV. The sex- and age-adjusted RR of HCC among individuals positive for antibodies to HCV was estimated to be 53.7 (95% confidence interval [CI] = 27.1-106.2). Antibodies to HCV were much more prevalent among patients negative for serum, hepatitis B surface antigen (HBsAg) (69 of 72, 95.8 %) than among HBsAg-positive patients (two of 19, 10.5%); the RR increased to 339.6 (95% CI = 96.5-1195.8) in the separate analysis of HBsAg-negative subjects. The most frequent genotype among HCV RNA-positive subjects was type lb (also called type II) (found in 49 [86.0%] of 57 patients and in 15 [60.0%] of 25 control subjects); individuals with type 1b infection experienced a significantly elevated risk (RR = 3.8; 95% CI = 1.0-13.9) compared with the risk observed for individuals with type 2a (also called type III) infection. No statistically significant association between HCV RNA titers and HCC was evident. CONCLUSIONS: HCV infection, particularly type 1b infection, plays an important role in the development of HCC among the study population. We estimated that approximately 78% (95% CI = 69%-86%) of the HCCs that occur in this high-risk area are attributable to HCV infection, if we assume that the patients in this study were representative population samples. IMPLICATIONS: Further studies are needed to clarify potential risk factors, including specific HCV genotypes, for progression to HCC among HCV carriers. PMID- 8637029 TI - Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease. AB - BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT 29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene. PMID- 8637030 TI - DNA repair proficiency: potential susceptiblity factor for breast cancer. PMID- 8637031 TI - Reversal of relation between body mass and endogenous estrogen concentrations with menopausal status. PMID- 8637033 TI - Re: Differences in lung cancer risk between men and women: examination of the evidence. PMID- 8637032 TI - Re: Helicobacter pylori and atrophic gastritis: importance of the cagA status. PMID- 8637034 TI - Long-term freedom from disease progression with interferon alfa therapy in two patients with malignant hemangiopericytoma. PMID- 8637036 TI - Poloxamer-188 revisited: a potentially valuable immune modulator. PMID- 8637035 TI - Diethylstilbestrol for uncontrolled bleeding in cancer. PMID- 8637037 TI - Re: Consumption of black tea and cancer risk: a prospective cohort study. PMID- 8637038 TI - How much can we rely on the level of prostate-specific antigen as an end point for evaluation of clinical trials? A word of caution! PMID- 8637040 TI - Pancreatic cancer shows modest survival gain. PMID- 8637039 TI - Treasure hunt for human papillomaviruses in nonmelanoma skin cancers. PMID- 8637043 TI - Middle Eastern nations sign milestone pact to collaborate. PMID- 8637042 TI - Topotecan: after FDA and ASCO, what's next? PMID- 8637041 TI - Angiogenesis research enjoys growth spurt in the 1990s. PMID- 8637044 TI - PSA levels after radiotherapy: how low must they go? PMID- 8637045 TI - Suramin-induced decrease in prostate-specific antigen expression with no effect on tumor growth in the LNCaP model of human prostate cancer. AB - BACKGROUND: Suramin, a polysulfonated naphthylurea and a recognized antitrypanosomal agent, has shown some promise in phase II clinical trials in the management of hormone-refractory human prostate cancer. Reduction of serum prostate-specific antigen (PSA) levels has been proposed as an end point for evaluating the antitumor efficacy of treatments for hormone-refractory prostate cancer. PURPOSE: We examined the antitumor effect of suramin in an in vivo mouse model of hormone-refractory human prostate cancer to determine whether a decrease in PSA levels reflects a reduction in tumor growth (volume). The tumors were induced in castrated, athymic nude mice by use of the androgen-independent, tumorigenic human prostate cancer cell line C4-2, which is a subline of the androgen-dependent, parental nontumorigenic cell line LNCaP. We also evaluated the effects of suramin in vitro on cell growth and the expression of PSA messenger RNA (mRNA) in both LNCaP and C4-2 cells. METHODS: For the in vivo studies, 24 mice were given a subcutaneous injection of 5 x 10(6) C4-2 cells at each of four sites. Animals (n = 20) with tumor volumes greater than 1 mm3 or less than 5 mm3 were divided equally into two groups. Drug treatment was initiated in one group by administration of 1 mg suramin intraperitoneally, followed by 0.1 mg suramin at 10-day intervals to maintain constant serum levels. Tumor growth and PSA expression levels were monitored. For the in vitro studies, both LNCaP and C4-2 cells were exposed to 100-400 microgram/mL suramin, and cell growth was monitored by a quantitative crystal violet assay. PSA mRNA expression was assessed by northern blot analysis in cells treated with either 250 microgram/mL suramin, 400 ng/mL dihydrotestosterone (DHT) (positive control), or 0.5-75 microgram/mL hydrocortisone (to mimic the clinical use of hydrocortisone during suramin treatment to compensate for the loss of adrenocortical function). In some studies, the combined effect of DHT and suramin on PSA mRNA expression was also evaluated. A two-way analysis of variance was performed to evaluate the treatment differences, and P values were obtained from two-sided tests for statistical significance. RESULTS: In vivo, suramin did not significantly affect the growth of androgen-independent C4-2 tumors (relative to the growth of tumors in 5% glucose-treated control animals; P = .76). However, suramin significantly decreased the ratio of PSA level to tumor volume (ng/mL PSA per mm(3) of tumor) (P<.001). Mice developed bone metastases in both treatment arms. Suramin affected the in vitro growth of LNCaP cells but not of C4-2 cells. Suramin diminished PSA mRNA expression in both LNCaP and C4-2 cells grown in vitro. Hydrocortisone had no effect on PSA mRNA levels. CONCLUSIONS: Although suramin inhibited the growth of androgen-dependent LNCaP cells, it did not inhibit the growth of androgen independent C4-2 cells either in vitro or in vivo. Suramin significantly decreased PSA mRNA expression in both cell lines in vitro and depressed serum PSA levels in mice bearing androgen-independent C4-2 tumors. IMPLICATIONS: PSA level should be used with caution as an end point in clinical trials using suramin therapy for hormone-refractory prostate cancer. PMID- 8637047 TI - Representation of African-Americans, Hispanics, and whites in National Cancer Institute cancer treatment trials. AB - BACKGROUND: The National Cancer Institute (NCI)-sponsored clinical trials cooperative groups place more than 25 000 American patients in treatment trials every year. Equal access and proportional representation of all races/ethnicities is desired. PURPOSE: Our objectives were to evaluate the inclusion of African Americans, Hispanics, and non-Hispanic whites in NCI-sponsored treatment trials and to determine if there is proportional racial/ethnic representation. METHODS: During the period of January 1, 1991, through June 30, 1994, 99 495 cancer patients were enrolled in clinical trials and declared themselves as non-Hispanic black, non-Hispanic white, or Hispanic (of any race). In the analysis, participants in NCI treatment trials were subdivided into three age groups: birth to 19 years, 20-49 years, and 50 or more years. The racial/ethnic composition of each of these age groups was compared with the racial/ethnic makeup of the American population with cancer. Estimates of the number of incident cancer cases per year were made for each racial/ethnic group within each age group using data from the Surveillance, Epidemiology, and End Results (SEER) Program and the 1990 Census. The percentage of all cancer patients who were in each racial/ethnic group were compared with the population that entered clinical trials. Comparisons are also made separately for patients with leukemia and breast, colorectal, lung, and prostate cancers. RESULTS: Among patients 0-19 years old, 20-49 years old, and 50 years old or older there is relatively proportional representation of non Hispanic blacks, Hispanics, and non-Hispanic whites in trials. It is noted that more than 70% of cancer patients aged 0-19 years are estimated to enter cooperative group clinical trials compared with 4.0% of cancer patients aged 20 49 years and 1.5% of patients aged 50 years or older. CONCLUSIONS: Accrual of American cancer patients to NCI-sponsored treatment trials generally parallels the incident burden of disease among non-Hispanic African-Americans, Hispanics, and non-Hispanic whites. IMPLICATIONS: This study shows that the NCI clinical trials are, as a whole, racially/ethnically representative of the American population and suggests that there is equal access to NCI clinical trials. PMID- 8637046 TI - Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients. AB - BACKGROUND: Nonmelanoma carcinomas of the skin represent the most frequent cancers among the Caucasian population worldwide. They occur with high frequency in renal allograft recipient patients after prolonged immunosuppression. PURPOSE: We analyzed tumors obtained from both immunosuppressed and nonimmunosuppressed patients for human papillomavirus (HPV) DNA. METHODS: Twenty-nine specimens of nonmelanoma carcinomas of the skin were obtained from 19 renal allograft recipient patients; these included 20 specimens of squamous cell carcinoma (SCC) from 11 patients, five specimens of basal cell carcinoma (BCC) from four patients, and four specimens of carcinoma in situ (CIS) from four patients. Forty one specimens of nonmelanoma carcinomas of the skin were obtained from 32 nonimmunosuppressed patients; these included 26 SCC specimens from 19 patients, 11 BCC specimens from nine patients, and four keratoacanthoma (benign epithelial tumor) specimens from four patients. A polymerase chain reaction method involving use of degenerate oligonucleotide primers, in which the conserved region of the open reading frame of the HPV L1 (major capsid protein) gene is amplified, was used to amplify total cellular DNA purified from individual tumors. The DNA of each specimen was subjected to 16 different amplification reactions; different primer combinations were used in order to increase the sensitivity and specificity of HPV detection. Resulting products were probed with a radioactively labeled, degenerate oligonucleotide. HPV-specific DNA was either sequenced directly after elution from the gel or amplified with semi-nested, degenerate primers, after which the products were cloned and sequenced. Sequences were compared with all known papillomavirus sequences. RESULTS: Thirteen (65%) of the 20 SCC specimens and three of the five BCC specimens from immunosuppressed (renal allograft recipient) patients contained identifiable HPV-related sequences, among them 13 putative novel HPV genomes. In addition, all other malignant tumor specimens from this patient group revealed faint signals upon amplification and hybridization; the origin of these signals has not been identified in the present study. In nonimmunosuppressed patients, eight (31%) of 26 SCC specimens and four (36%) of 11 BCC specimens contained sequences of HPV types. Two putative novel HPV sequences could be identified in this group. Faint signals of yet undetermined origin were observed in eight of the SCC specimens and in two of the BCC specimens. Two of four keratoacanthoma specimens contained sequences of known HPV type. (Keratoacanthoma is a nonmalignant lesion for which the natural history has not been defined.) The spectrum of HPV types in both groups of patients differed substantially. CONCLUSIONS: These data point to the frequent presence of HPV sequences in SCCs and BCCs of the skin. The etiologic relationship of these infections to the respective malignant tumors remains to be evaluated. IMPLICATIONS: The presence of HPV DNA in a large percentage of specimens of nonmelanoma carcinomas of the skin from immunosuppressed patients, as well as from nonimmmunosuppressed patients, renders a papillomavirus infection as a possible factor in the etiology of this disease. PMID- 8637048 TI - Phase I and pharmacologic studies of topotecan in patients with impaired hepatic function. AB - BACKGROUND: Topotecan, a topoisomerase I inhibitor that has demonstrated anticancer activity toward leukemias and solid tumors in clinical trials, is eliminated via hepatic and renal routes. However, dosing guidelines for the administration of topotecan to patients with impaired hepatic function have not yet been established. PURPOSE: We compared the maximum tolerated doses (MTDs), the toxic effects, and the pharmacokinetics and pharmacodynamics of topotecan in patients who had refractory, malignant, solid tumors and who either had or lacked hepatic injury. The potential role of three substrate markers of liver function (indocyanin green [ICG]-- a marker of hepatic blood flow; lorazepam--a substrate marker of hepatic glucuronidation; and antipyrine--a substrate marker for cytochrome P450 activity) in optimizing topotecan doses for patients with liver injury was also evaluated. METHODS: Twenty-one cancer patients, 14 of whom had hepatic injury due to metastatic disease, biliary obstruction, or cirrhosis, were treated with intravenously delivered courses of topotecan consisting of 0.5, 1.0, or 1.5 mg/m2 of drug per day for 5 days. Most patients received more than one course of treatment, with new courses initiated at 3-week intervals. Patient responses (evaluated by tumor measurements) and treatment-induced toxic effects were assessed. Prior to the initiation of topotecan treatment, patients were given intravenous injections of ICG, lorazepam, and antipyrine to determine the plasma pharmacokinetics of these compounds. The pharmacokinetics of topotecan (both the lactone and the carboxylate forms) were determined by analysis of plasma and urine samples collected on the first day of the first course of drug treatment. Scatter plots of area under the plasma concentration versus time curves in relation to percent decreases in either absolute neutrophil count or platelet count were used to explore the pharmacodynamics of topotecan. The Student's t test and the Mann-Whitney U test were used to compare pharmacokinetic parameters between patients with and without abnormal hepatic function. Correlations were assessed using the Spearman's rank correlation coefficient (rs). Reported P values are based on two-tailed tests of significance. RESULTS: Patients with hepatic injury tolerated topotecan doses up to 1.5 mg/m2, i.e., the MTD of this drug established in previous studies. The nature and severity of treatment-induced toxic effects and the pharmacokinetics of topotecan were similar in patients with and without liver injury. No differences were observed in the urinary excretion of topotecan between the two patient groups. Clearances of total topotecan and its lactone species correlated only with clearance of ICG (rs = .64, P = .004; and rs = .68, P = .0017, respectively). The pharmacodynamic effects of topotecan were not altered by liver dysfunction. CONCLUSIONS AND IMPLICATIONS: Cancer patients with hepatic injury can be treated with topotecan at a starting dose of 1.5 mg/m2, given daily for 5 days and administered every 3 weeks. Topotecan dose modifications do not appear to be required for patients with hepatic dysfunction and normal renal function. PMID- 8637049 TI - Prevention of murine radiogenic thymic lymphomas by tumor necrosis factor or by marrow grafting. AB - BACKGROUND: Split-dose irradiation (1.75 Gy given weekly for 4 weeks) of C57BL/Ka mice induces the emergence of preleukemic cells (PLCs). These cells develop into leukemic cells after a latency period of 3-6 months. The survival and transformation of PLCs are dependent on radiation-induced alterations of the thymic epithelium and of resident lymphocyte (i.e., thymocyte) subpopulations in the thymus. PLCs can be eliminated, concomitantly with the restoration of the thymus, by grafting bone marrow cells immediately after the last irradiation. Our hypothesis was that any agent able to restore the thymus after leukemogenic irradiation would exert the same effects as a bone marrow graft. Tumor necrosis factor-alpha (TNF-alpha) is one such possible agent, since it has been shown to modulate some functions of the thymic epithelium and thymocyte subpopulations. PURPOSE: The goal of this study was to assess the ability of repeated intraperitoneal injections of TNF-alpha to functionally replace bone marrow transplantation in the restoration of normal intrathymic lymphopoiesis and in the prevention of thymic lymphomas in split-dose-irradiated mice. METHODS: We replaced the bone marrow graft with repeated injections of TNF-alpha (25 000 U/injection) in the split-dose-irradiated (4 x 1.75 Gy) C57BL/Ka mouse model. We analyzed the expression of the cell differentiation markers CD4 and CD8 on thymocytes by flow cytometry. We also studied the thymic environment by isolating thymic nurse cells, the bone marrow prothymocyte activity by analyzing thymic repopulation, and the evolution of PLCs by an in vivo transplantation assay. Local production of TNF-alpha after bone marrow grafting was examined by in situ hybridization. Injections of anti-TNF-alpha antibodies were given to split-dose irradiated mice to test the effect of neutralizing TNF-alpha in vivo. One-way analysis of variance and Newman-Keuls two-tailed tests were used to test statistical significance. RESULTS: Multiple injections of TNF-alpha into split dose-irradiated mice did not influence bone marrow prothymocyte activity but restored thymocyte subpopulations and thymic epithelium, induced the disappearance of PLCs, and prevented the development of lymphomas. Moreover, a bone marrow graft significantly stimulated intrathymic production of TNF-alpha messenger RNA (P<.01), and anti-TNF-alpha antibodies partially inhibited the antilymphomatous effects of bone marrow graft in split-dose-irradiated mice (P<.05). CONCLUSION: These data strongly suggest that TNF-alpha is a mediator that is involved in the mechanisms by which bone marrow transplantation functions to prevent thymic lymphomas in split-dose-irradiated mice. IMPLICATIONS: Cytokines might be used in some biological systems, particularly in the hemopoietic system, as a therapeutic agent for the secondary prevention of cancer. PMID- 8637050 TI - Second cancers after adjuvant tamoxifen therapy for breast cancer. PMID- 8637051 TI - Cancer therapy during Ramadan. PMID- 8637052 TI - Re: Reappraisal of hepatic arterial infusion in the treatment of nonresectable liver metastases from colorectal cancer. PMID- 8637053 TI - Re: Telomerase activity in human breast tumors. PMID- 8637054 TI - Re: Educational attainment and racial differences in cigarette smoking. PMID- 8637055 TI - Re: Multiple neoplasias: an oncologic reality. PMID- 8637056 TI - Toxicology of deoxynivalenol (vomitoxin). AB - Trichothecene mycotoxins are a group of structurally similar fungal metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a trichothecene, is prevalent worldwide in crops used for food and feed production, including in Canada and the United States. Although DON is one of the least acutely toxic trichothecenes, it should be treated as an important food safety issue because it is a very common contaminant of grain. This review focuses on the ability of DON to induce toxicologic and immunotoxic effects in a variety of cell systems and animal species. At the cellular level, the main toxic effect is inhibition of protein synthesis via binding to the ribosome. In animals, moderate to low ingestion of toxin can cause a number of as yet poorly defined effects associated with reduced performance and immune function. The main overt effect at low dietary concentrations appears to be a reduction in food consumption (anorexia), while higher doses induce vomiting (emesis). DON is known to alter brain neurochemicals. The serotoninergic system appears to play a role in mediation of the feeding behavior and emetic response. Animals fed low to moderate doses are able to recover from initial weight losses, while higher doses induce more long-term changes in feeding behavior. At low dosages of DON, hematological, clinical, and immunological changes are also transitory and decrease as compensatory/adaptation mechanisms are established. Swine are more sensitive to DON than mice, poultry, and ruminants, in part because of differences in metabolism of DON, with males being more sensitive than females. The capacity of DON to alter normal immune function has been of particular interest. There is extensive evidence that DON can be immunosuppressive or immunostimulatory, depending upon the dose and duration of exposure. While immunosuppression can be explained by the inhibition of translation, immunostimulation can be related to interference with normal regulatory mechanisms. In vivo, DON suppresses normal immune response to pathogens and simultaneously induces autoimmune-like effects which are similar to human immunoglobulin A (IgA) nephropathy. Other effects include superinduction of cytokine production by T helper cells (in vitro) and activation of macrophages and T cells to produce a proinflammatory cytokine wave that is analogous to that found in lipopolysaccharide-induced shock (in vivo). To what extent the elevation of cytokines contributes to metabolic effects such as decreased feed intake remains to be established. Although these effects have been largely characterized in the mouse, several investigations with DON suggest that immunotoxic effects are also likely in domestic animals. Further toxicology studies and an assessment of the potential of DON to be an etiologic agent in human disease are warranted. PMID- 8637057 TI - Neurotoxicity resulting from coexposure to pyridostigmine bromide, deet, and permethrin: implications of Gulf War chemical exposures. AB - Of the three-quarters of a million service personnel involved in the Persian Gulf War, approximately 30,000 have complained of neurological symptoms of unknown etiology. One contributing factor to the emergence of such symptoms may be the simultaneous exposure to multiple agents used to protect the health of service personnel, in particular, the anti-nerve agent pyridostigmine bromide (PB; 3 dimethylaminocarbonyloxy-N-methylpyridinium bromide), the insect repellent DEET (N,N-diethyl-m-toluamide), and the insecticide permethrin (3-(2,2-dichloro ethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methyl ester). This study investigated neurotoxicity produced in hens by individual or simultaneous exposure to these agents (5 d/wk for 2 months to 5 mg/kg/d PB in water, po; 500 mg/kg/d DEET, neat, sc; and 500 mg/kg/d permethrin in corn oil, sc). At these dosages, exposure to single compounds resulted in minimal toxicity. Combinations of two agents produced greater neurotoxicity than that caused by individual agents. Neurotoxicity was further enhanced following concurrent administration of all three agents. We hypothesize that competition for liver and plasma esterases by these compounds leads to their decreased breakdown and increased transport of the parent compound to nervous tissues. Thus, carbamylation of peripheral esterases by PB reduces the hydrolysis of DEET and permethrin and increases their availability to the nervous system. In effect, PB "pumps" more DEET and permethrin into the central nervous system. Consistent with this hypothesis, hens exposed to the combination of the three agents exhibited neuropathological lesions with several characteristics similar to those previously reported in studies of near-lethal doses of DEET and permethrin. If this hypothesis is correct, then blood and liver esterases play an important "buffering" role in protecting against neurotoxicity in the population at large. It also suggests that individuals with low plasma esterase activity may be predisposed to neurologic deficits produced by exposure to certain chemical mixtures. PMID- 8637058 TI - Sensitivity of platelet microtubules to disassembly by methylmercury. AB - With the aim of identifying a surrogate marker for the neurotoxic effects of methylmercury using a peripheral blood sample, the sensitivity of microtubules in circulating blood cells to depolymerization by methylmercury was compared. Methylmercuric chloride was added to samples of human venous blood or to isolated platelets and lymphocytes (human or rabbit) suspended in RPMI medium plus 10% fetal calf serum. After 1 h, microtubular networks were visualized by immunolabeling with antibody specific for tubulin. The percentage of platelets without visible, intact microtubules and the percentage of viable, unactivated lymphocytes without microtubules visibly radiating from the centriolar region through the cytoplasm were counted. A concentration-dependent loss of microtubules was observed in both cell types. Loss of microtubules was more easily quantitated and was observed at significantly lower concentrations in platelets compared to lymphocytes. The IC50 (concentration of methylmercuric chloride resulting in dissolution of microtubules in 50% of the cells) was 3.1 microM in platelets and 7.4 microM in lymphocytes in samples exposed in culture medium without erythrocytes. When methylmercury was added to whole blood for 1 h, the IC50 increased to 182 microM in platelets and >700 microM in lymphocytes, consistent with the known sequestration of methylmercury in erythrocytes. With longer durations of exposure, much lower concentrations of methylmercury were effective in both cell types. When rabbit lymphocytes and platelets were exposed to methylmercury under culture conditions, IC50s in platelets/lymphocytes were 2.5 microM/4.8 microM after 1 h of exposure, 0.77 microM/1.12 microM after 20 h, and 0.51 microM/0.63 microM after 70 h. The results of this study suggest that platelets may be more suitable than lymphocytes as a cell type in which to monitor in vivo effects of methylmercury on microtubules. PMID- 8637059 TI - Cadmium mobilization by nitrogen donor chelating agents. AB - The relative abilities of a series of acyclic polyamine chelating agents containing only nitrogen donors (N-donors) to induce the urinary excretion of cadmium has been examined in the rat. The compounds examined include triethylenetetramine dihydrochloride (TRIEN), tris(2-aminoethyl)amine trihydrochloride (TREN), tetraethylenepentamine pentahydrochloride (TETRAEN), and pentaethylenehexamine hexahydrochloride (PENTAEN). Sodium N-methyl-D-glucamine-N carbodithioate (NaG) was used as a positive control compound. The polyamines induced a significant increase in the urinary excretion of cadmium in rats that had been loaded with cadmium at least 4 d prior to the polyamine treatments. A comparison of these with similar data on macrocylic nitrogen donor systems, which form much more stable complexes with cadmium but are also ineffective in enhancing the excretion of cadmium from such aged deposits, suggests that the factors responsible for the relative inefficiency of these compounds may involve either a difficulty in penetrating cellular membranes or a slow rate of reaction with biologically bound cadmium. The occurrence of oliguria and anuria following the administration of the several of the polyamines indicates that their use is accompanied by significant renal damage in cadmium-exposed rats. PMID- 8637060 TI - Pulmonary toxicity of systemic terbium chloride in mice. AB - Terbium (Tb) is a rare earth metal that finds use in several emerging technologies. However, little is known about the biological effects of Tb. Thus, in this study the pulmonary toxicity of systemic Tb in mice was investigated. Mice were treated intravenously with a single dose of 20 or 200 mumol Tb/kg, as TbCly and killed at 3, 6, 12, 24, 48, or 72 h later. Administration of Tb at a dose of 200 mumol/kg increased pulmonary weight, lipid peroxidation, and protein content but decreased pulmonary glutathione content. Pulmonary gamma-glutamyl transpeptidase (gamma-GTP) activity was increased after Tb administration at a dose of 200 mumol/kg. Pulmonary alkaline phosphatase (ALP) activity was also increased after Tb administration at a dose of 200 mumol/kg. Investigation of the defense system against oxidative damage in the lung showed that superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were all decreased after Tb administration at the higher dose. The concentrations of Tb, Ca, and P in lung was increased by the dose of 200 mumol/kg. These results suggest that pulmonary lipid peroxidation may be an early and sensitive consequence of Tb exposure and that SOD, CAT, and GSH-Px might be considered as potential modulators of Tb-induced lipid peroxidation. The mechanisms involved in Tb-induced pulmonary lipid peroxidation deserve further study. PMID- 8637061 TI - Effect of soil loading on dermal absorption efficiency from contaminated soils. AB - The effect of soil loading on the dermal uptake of soil-borne contaminants was examined using an in vitro evaporation/penetration apparatus and abdominal skin from human cadavers. Dermal uptake of two 14C-labeled pesticides, lindane and 2,4 dichlorophenoxyacetic acid (2,4-D), was assessed at nominal soil loadings of 1, 5, and 10 mg/cm2. Sub-150-microns fractions of two soils with differing organic carbon contents were employed. Mean 24-h dermal absorption values ranged from 0.45 to 2.35% for lindane and from 0.18 to 1.64% for 2,4-D, depending upon soil load and type. Mean mass fluxes ranged from 8.8 to 32 pg/cm2/h for lindane and from 1.9 to 6.4 pg/cm2/h for 2,4-D. Results were aggregated as ratios of flux or percent absorption at 1 and 10 mg/cm2 to corresponding values obtained at 5 mg/cm2. Fluxes at 5 and 10 mg/cm2 did not differ significantly, but flux at 1 mg/cm2 was about one-half the value observed at the higher loadings. The most plausible explanation for this decrease in mass flux is incomplete (submonolayer) coverage of the skin. Evidence in the form of electron micrographs is presented in support of this conclusion. Relative percent absorption increased significantly with decreases in soil load from 10 to 5 and from 5 to 1 mg/cm2. This effect was inversely proportional to loading reduction in the former case, but was less than proportional due to the impact of contact area reduction (and, in the case of lindane, volatilization losses) in the latter. Percent dermal absorption data obtained in the laboratory require adjustment for differences in loading and coverage before application to assessment of exposure to contaminants in soils. Description of dermal absorption from soil in a manner comparable to that used to describe absorption from a liquid or vapor (i.e., using a driving force and a mass transfer coefficient) would reduce confusion on this point and is recommended. PMID- 8637062 TI - It is time we told the Emperor about his clothes. PMID- 8637064 TI - Failure of nonoperative treatment of a gunshot wound to the liver predicted by computed tomography. PMID- 8637063 TI - The length of hospital stay after an unnecessary laparotomy for trauma: a prospective study. AB - OBJECTIVE: To record length of hospital stay (LOS) for patients for whom unnecessary laparotomies for trauma (no repair, no drain) were performed. The influence of complications and associated injuries on the LOS would be studied. DESIGN: Prospective case series. MATERIALS AND METHODS: Data were recorded concurrently for consecutive patients on whom unnecessary laparotomies for trauma were performed at a trauma center. MEASUREMENTS AND MAIN RESULTS: The main and secondary outcome measures were LOS and the influence of complications or associated injuries on the LOS, respectively. From 1988 until 1991, unnecessary laparotomies for trauma were performed on 254 patients. The overall mean LOS was 8.1 days (median, 6 days; range, 1-80 days), whereas the overall mean LOS for 81 patients who had no associated injuries and on whom completely negative laparotomies were performed was 4.7 days (median, 5 days; range, 2-8 days). Complications occurred in 41.3% of the patients and increased the mean LOS from 5 days (no complication) to 9 days (complication) (p = 0.0002). Associated injuries occurred in 43.7% of the patients and increased the mean LOS from 5.3 days (no associated injury) to 11.7 days (associated injury) (p = 0.0001). CONCLUSIONS: Unnecessary laparotomies for trauma resulted in a significant LOS. The presence of a complication or an associated injury significantly prolonged the LOS. Current efforts to reduce the incidence of these unnecessary procedures and minimize the occurrence of complications are worthwhile. PMID- 8637065 TI - Intrafocal (Kapandji) pinning of unstable distal radius fractures: a preliminary report. AB - Seventeen consecutive patients (17 wrists) who underwent intrafocal pinning of unstable distal radius fractures, as described by Kapandji, were retrospectively reviewed at a mean of 42 weeks after surgery (range, 13-88 weeks). The patients were immobilized for 6 weeks postoperatively. All patients were assessed by a single physician and were asked a set of questions regarding subjective and functional status. All patients underwent physical examination of their upper extremities, and bilateral wrist PA and lateral x-rays were taken. There were 13 females and four males; the average age was 49 years. Ten dominant and seven nondominant extremities were involved. Average volar tilt on follow-up lateral wrist radiography was 7 degrees. This compares with -20 degrees at initial presentation, -12 degrees preoperatively, 6 degrees immediately postoperatively, and 10 degrees in the normal wrists. Radial shortening (average) was -2 mm at initial presentation, -1mm preoperatively, 1 mm postoperatively, 0 mm at follow up, and 1 mm in the normal wrists. Radial inclination was 17 degrees initially, 20 degrees preoperatively, 23 degrees immediately postoperatively, 23 degrees on follow-up, and 24 degrees in the normal wrists. There was a trend for patients with osteopenic bone to lose their postoperative reduction. However, this was not statistically significant. Patients older than 65 years of age had significantly inferior radiologic results. Loss of pronation and supination averaged 2 degrees (range 0-10 degrees) compared with the uninjured wrist. Loss of dorsiflexion averaged 6.5 degrees, and palmar flexion averaged 7.6 degrees. The patients' subjective complaints were minimal. Average pain on visual analog scale (VAS) was 0.44/10. Function measured 8.64/10 (VAS). Sixteen of the patients were happy with the surgery and the outcome of their wrists. Complications included extensor tendon rupture (one patient), pin migration requiring premature removal (one patient), and initial loss of reduction requiring reoperation (one patient). Intrafocal pinning of unstable distal radius fractures provides an effective means to stabilize these complex injuries. Early follow-up suggests that the patients have a satisfactory functional outcome. The complications in this series were preventable. Intrafocal pinning should be added to the surgical armamentarium in treating distal radius fractures. PMID- 8637066 TI - Closed reduction of coronal fractures of the capitellum. AB - Closed reductions of displaced coronal fractures of the capitellum were successfully obtained for nine patients. The method involves spontaneous reduction by allowing the elbow to fully extend under anesthesia and then gradually flexing the elbow while distracting the elbow joint. Distraction allows the radial head to capture the capitellar fragment in the joint rather than push it proximally. The elbow is immobilized at 90 degrees for 3 weeks. Gentle active motion is then started. Closed reduction is often or usually obtained, but open reduction and internal fixation are performed in those cases in which a closed reduction cannot be obtained. PMID- 8637067 TI - Computed tomographic scans of minimally displaced type II odontoid fractures. AB - OBJECTIVE: To determine the computed tomography (CT) appearance of minimally displaced type II odontoid fractures and the optimal protocols to evaluate these fractures by CT. MATERIALS AND METHODS: The CT scans of five patients with minimally displaced type II odontoid fractures and 71 patients without odontoid pathology were reviewed for signs of fracture. A phantom consisting of a cadaver specimen with a type II odontoid fracture was evaluated with several protocols on four CT scanners. The protocols differed in slice thickness and reconstruction algorithm. Helical scanning was also performed, and parasagittal and coronal reformations were created from each image set. MAIN RESULTS: Multiple cortical disruptions longer than 1 mm were demonstrated on the scans of all five patients with type II odontoid fractures. Only three of the 71 patients without odontoid fractures had multiple cortical disruptions, and none were longer than 1 mm. In the phantom study, thinner sections demonstrated cortical disruptions better than thicker sections. Similarly, images reconstructed with a bone reconstruction algorithm demonstrated the cortical disruptions better than images reconstructed with a soft-tissue reconstruction algorithm. Helical scans (1 mm thick) adequately demonstrated the reduced Type II odontoid fracture. Parasagittal and coronal reformations failed to demonstrate a fracture line through the base of the odontoid process on all image sets. CONCLUSIONS: Cortical disruptions greater than 1 mm and multiple cortical disruptions may be the only findings of odontoid fractures and should suggest the diagnosis. Evaluation of potential type II odontoid fractures is improved as CT section thickness is reduced (down to 1 mm) and is also improved by use of a bone reconstruction algorithm. The apparent absence of a fracture line through the base of the odontoid process on parasagittal or coronal reformations does not rule out the diagnosis. PMID- 8637069 TI - Serum lactate is not predicted by anion gap or base excess after trauma resuscitation. AB - OBJECTIVE: The inability to normalize lactate predicts death after trauma, but lactate may not be immediately available in every center. We postulated that, in a normal acid-base environment, lactate would correlate with the anion gap and the base excess of an arterial blood gas. METHODS: We studied 52 consecutive, invasively monitored patients with trauma admitted directly to the intensive care unit (ICU) from the emergency department or operating room in our level I center to determine whether base excess and anion gap could predict lactate. Lactate, base excess, and anion gap were recorded upon admission to the ICU and 8, 16, 24, 36, and 48 hours after admission. Correlation coefficients (r2) were calculated for the total patients, the 43 survivors, and the nine non-survivors. RESULTS: Serum lactate was significantly higher in nonsurvivors at 16 hours after post ICU admission (4.0 +/- 1.69 vs. 2.84 +/- 1.49, p < 0.05), and this trend persisted; the greatest difference was seen at 48 hours after admission (2.92 +/- 1.47 vs. 1.76 +/- 0.57, p < 0.001). There were no differences in base excess or anion gap between survivors and nonsurvivors. We found no consistent correlation between lactate versus anion gap, lactate versus base excess, or anion gap versus base excess. CONCLUSIONS: There is no correlation between lactate, base excess, and anion gap after initial resuscitation. Neither anion gap nor base excess was capable of predicting lactate; therefore, lactate must be directly measured. The lack of correlation of anion gap with base excess or lactate suggests the presence of unmeasured anions, an impairment in acid-base regulation after injury and resuscitation, or both. PMID- 8637068 TI - Causation, incidence, and costs of traumatic brain injury in the U.S. military medical system. AB - Hospital discharge records from military facilities and private facilities reimbursed by Civilian Health and Medical Program of the Uniformed Services for fiscal year 1992 were reviewed to identify head injury admissions. Incidence rates, case fatality rates, causes of head injuries, and direct cost for hospital admissions were computed in this well-defined population. For fiscal year 1992, there were 5,568 hospitalized cases of noncombat head injury in the military medical system. The age-adjusted head injury rates for ages 15-44 years are higher in active-duty individuals compared with other beneficiaries (1.6 times greater for men and 2.5 times greater for women). The total cost for hospitalization in this population was $43 million. Private facility rehabilitation accounted for 26% of all private facility costs but only 6% of head injury cases. Firearms and motor vehicle crashes caused the most severe injuries for cases admitted to military facilities. Motor vehicle crashes, falls, and fighting accounted for 80% of the total military facility cost for head injuries. Military active-duty individuals are at increased risk for noncombat head injury. Prevention of head injury in military settings should focus on motor vehicle crashes, fist fights (assault), and falls. PMID- 8637070 TI - A prospective analysis of transesophageal echocardiography in the diagnosis of traumatic disruption of the aorta. AB - OBJECTIVE: Recently, transesophageal echocardiography (TEE) has been proposed as the standard for the diagnosis of traumatic disruption of the aorta (TDA), replacing aortography. The purpose of this study was to evaluate the accuracy and practicality of TEE in the diagnosis of TDA. DESIGN: Prospective clinical trial. MATERIALS AND METHODS: Patients with blunt trauma admitted with a suspected diagnosis of TDA were evaluated with TEE and aortography. MEASUREMENTS AND MAIN RESULTS: Thirty-four patients were evaluated with TEE and aortography. TEE was unsuccessful in five patients (15%). Of the remaining 29 patients, TEE results were true-positive in four and true-negative in 20. TEE results were false positive in two patients, and three injuries were missed (two were proximal to the left subclavian artery, and one was a localized aortic disruption). Sensitivity and specificity of TEE were 57% and 91%, respectively, compared with aortography, for which sensitivity was 89% and specificity was 100%. CONCLUSION: Although the use of TEE in the diagnosis of TDA has several advantages, it is not more accurate than aortography. TEE should not replace aortography as the standard for the diagnosis of TDA. PMID- 8637071 TI - Diaspirin cross-linked hemoglobin is efficacious in gut resuscitation as measured by a GI tract optode. AB - The objective of this study was to compare the efficacy of diaspirin cross-linked hemoglobin (DCLHb) with that of standard resuscitative fluids in restoring intestinal mucosal oxygenation and villous architecture after hemorrhage. Male rats were bled to a base deficit of 5 +/- 2 nmol/l under propofol anesthesia and monitored for 90 minutes postresuscitation with DCLHb, blood, lactated Ringer's solution, albumin, or nothing (DNR) for mucosal oxygen tension (Pmo2) and physiologic and laboratory parameters. Small intestinal histologic specimens were obtained and scored independently by two investigators blinded to therapy on a scale of 0 (normal) to 4 (worst). All treatments restored Pmo2; only DCLHb did so without exceeding baseline values. For untreated rats (DNR), Pmo2 was not restored. Normal mucosal architecture was maintained only in DCLHb-treated rats. As Pmo2 increased, mucosal score improved. In a rat model of controlled hemorrhage, Pmo2 changes measured by an optode correlated with gut histological abnormalities. By these criteria, DCLHb is superior to crystalloid, colloid, and blood in gut resuscitation. PMID- 8637072 TI - Early resuscitation with low-volume PolyDCLHb is effective in the treatment of shock induced by penetrating vascular injury. AB - OBJECTIVE: To study the efficacy of an oxygen-carrying solution in early resuscitation of hemorrhagic shock induced by penetrating vascular injury. DESIGN: Experimental study with anesthetized rats. MATERIALS AND METHODS: Severe hemorrhagic shock was induced by a 25-gauge needle puncture to the infrarenal aorta. Forty animals were resuscitated 10 minutes after injury with either lactated Ringer's solution (LR; 60 mL/kg), 7.5% hypertonic saline (HTS; 5 mL/kg), or modified diaspirin cross-linked hemoglobin (PolyDCLHb; 5 or 20 mL/kg) or were not resuscitated (NR) and followed for 6 hours. RESULTS: Total blood loss was similar in all treatment groups. Mean arterial pressure was restored to baseline values, base deficit was corrected to base excess, and venous oxygen saturation improved with PolyDCLHb and more slowly with LR but persisted below baseline values with HTS and NR. The 6-hour mortality rates were zero of eight (low-dose PolyDCLHb), three of eight (high-dose PolyDCLHb), two of eight (LR), six of eight (HTS), and six of eight (NR). CONCLUSION: Early resuscitation with low-volume hemoglobin is effective in restoring tissue perfusion and improving survival in uncontrolled hemorrhagic shock. PMID- 8637073 TI - Arterial bleeding diagnosed by CT in hemodynamically stable victims of blunt trauma. AB - Although the presence of intra-abdominal blood is a common finding on abdominal computed tomography (CT) scans performed for trauma, acute intra-abdominal bleeding is rarely diagnosed by CT. A focal area of high-density contrast, as compared to the surrounding fluid and tissues, is the characteristic CT finding associated with acute intra-abdominal bleeding and should prompt immediate intervention. PMID- 8637074 TI - Role of repeat computed tomography after emergency endoscopic retrograde pancreatography in the diagnosis of traumatic injury to pancreatic ducts. AB - Endoscopic retrograde pancreatography (ERP) is performed on patients with pancreatic injury after abdominal trauma. To delineate pancreatic ductal injuries more accurately, we performed repeat computed tomography (CT) shortly after completion of ERP. We describe our experiences with six patients to demonstrate the feasibility and utility of this method. In our cases, the diagnosis of pancreatic ductal injury was made with certainty on the basis of the presence of extravasated contrast medium. This protocol is useful for reaffirmation of injuries noted on ERP, for diagnosis of injuries not noted on ERP, and for exclusion of injuries in patients with equivocal results of ERP. Moreover, the protocol is easy to implement because it involves only the transfer of the patient from the endoscopy to the CT suite. The technique can be used to clarify potentially confusing situations. PMID- 8637075 TI - Temporary intravenous bag silo closure in severe abdominal trauma. AB - Several temporary abdominal wall closure techniques have been described in the literature. We present our experience with an inexpensive and efficient method of temporary abdominal closure when bowel edema and distension preclude safe primary closure. Our technique is a variation of the silon (silo) closure used in the repair of gastroschisis and omphalocele, using a pre-gas-sterilized, soft 3-L plastic cystoscopy fluid irrigation bag cut to an oval shape and stapled or sutured to the skin edges of the wound. PMID- 8637076 TI - Flexible endoscopy for the diagnosis of esophageal trauma. AB - The role of flexible endoscopy in the diagnosis of esophageal trauma remains undefined. This study evaluates the use of immediate flexible fiberoptic esophagogastroduodenoscopy (EGD) as the primary diagnostic tool for detection of esophageal injury in trauma patients. Flexible EGD was performed on 31 patients for this purpose from August 1991 through January 1994. There were 28 males and 3 females with a mean age of 24.3 years (range, 16-54 years). Twenty-four of 31 patients (77%) were intubated at the time of the examination. Mechanism of injury was penetrating in 24 patients (20 gunshot wounds, four stab wounds) and blunt (motor vehicle crash) in seven patients. Penetrating injuries were located in the neck in 5 of 24 patients, in the chest in 15 of 24 patients, and in both the neck and chest in 4 of 24 patients. Upper gastrointestinal contrast studies were performed for 3 of 31 patients (10%), computed tomography was performed for eight patients (26%), bronchoscopy was performed for 13 patients (42%), angiography was performed for 17 patients (55%), and rigid esophagoscopy and laryngoscopy were each performed for one patient (3%). Evidence of esophageal trauma during EGD was seen in 5 of 31 patients. True-positive studies occurred for four patients, false positive results occurred for one patient, true-negative results occurred for 26 patients (as demonstrated by exploration in five and clinical follow-up in 21), and no false-negative examinations occurred. Sensitivity of flexible EGD was 100%, specificity was 96%, and accuracy was 97%. No complications occurred related to the performance of EGD. Flexible fiberoptic endoscopy seems to be a safe and effective method for both detection and exclusion of esophageal trauma. PMID- 8637077 TI - Conservative management of penetrating hypopharyngeal wounds. AB - OBJECTIVE: To demonstrate the applicability of nonoperative treatment to penetrating hypopharyngeal wounds. DESIGN: A prospective study. MATERIALS AND METHODS: An analysis of patients with penetrating hypopharyngeal wounds who were treated nonoperatively at the Hospital Universitario del Valle (Cali, Colombia) during 4 years (beginning January 1990) was performed. Patients older than 13 years with proven lesions produced by penetrating trauma were included. Patients with foreign-body-induced or iatrogenic lesions (orotracheal intubation, endoscopy) or immediate need for surgery (for associated lesions) were excluded. Nonoperative management consisted of nasogastric tube for feeding, suspension of oral intake, and parenteral antibiotics for 7 days. MEASUREMENTS AND MAIN RESULTS: Fourteen patients met the above criteria. Gunshot wounds (n = 11) were the most common trauma. An esophagogram/endoscopy demonstrated the lesions. The only complication in the group consisted of a cervical abscess, which presented in a patient during the first week of treatment. There were no complications such as fistula, leakage, and Horner's syndrome. CONCLUSIONS: Conservative management is a good and safe alternative when indicated in penetrating hypopharyngeal wounds. PMID- 8637079 TI - The failure of triage criteria to identify geriatric patients with trauma: results from the Florida Trauma Triage Study. AB - Since 1990, Florida has used a uniform set of eight triage criteria, known as the trauma scorecard, for triaging adult patients with trauma to state-approved trauma centers. If any one of the eight criteria are met, paramedics classify the patient as a "trauma alert" and transport to a state-approved trauma center. Widespread concern within the trauma care community that the scorecard was not providing an effective tool for adult trauma triage, particularly for older adults, was a motivating force for conducting an evaluation of the trauma scorecard's performance. Thus, the Florida Department of Health and Rehabilitative Services, Office of Emergency Medical Services initiated a research effort to assess the effectiveness of the state-adopted trauma triage criteria for adults, giving special attention to geriatric trauma. The results of the Florida Trauma Triage Study indicate that the eight triage criteria comprising the trauma scorecard produce unacceptable levels of undertriage in elderly patients (age 55 years or older) with life-threatening injuries. PMID- 8637078 TI - Reduction of circulating prostaglandin E2 level by antiserum against core lipopolysaccharide in a rabbit model of multiple organ failure. AB - OBJECTIVE: To determine the potential role of prostaglandin E2 (PGE2) in the development of multiple organ dysfunction or failure (MOF), the possible effects of antiserum directed against Re chemotype lipopolysaccharide (LPS, from Re mutant of Escherichia coli F515) on circulating PGE2 level and survival rate, and whether there is an elevation in the plasma LPS concentration that could account for the induction of arachidonic acid metabolite in a rabbit model of MOF caused by acute hypovolemic insult. DESIGN, MATERIALS, AND METHODS: An animal model of MOF in rabbits, engendered by feeding live Escherichia coli O111:B4 before hemorrhagic shock (35-40 mm Hg for 60 min), was used in the present study. Re-LPS antiserum was given intravenously in the treatment group at the onset of hemorrhage and 4 hours after resuscitation. The animals that received equal volumes of normal rabbit serum and antiserum served as the control group. MEASUREMENTS AND MAIN RESULTS: The circulating PGE2 level was not increased at the end of shock (p > 0.05), but it was found to be significantly elevated 24 hours after hemorrhage and resuscitation in both groups. However, Re-LPS antiserum administration markedly decreased peak PGE2 level (p < 0.05) and attenuated multiple organ damage caused by acute insult. Concomitantly, there were also lower LPS concentrations in the treatment group as compared with the control group (p < 0.05-0.01). The survival rate was significantly increased in antiserum-treated rabbits 96 hours postinjury (treatment vs. control: 58.0% vs. 11.1%, p < 0.01). CONCLUSIONS: The results suggest that an excessive generation and release of PGE2 may be involved in the pathogenesis of immunosuppression and MOF following hemorrhage and resuscitation. Re-LPS antiserum has an inhibitory effect on overproduction of circulating PGE2 and the ability to improve survival with MOF. Gut-derived endotoxemia, bacterial translocation, or both, could account, at least in part, for the PGE2 formation and release in animals response to acute hypovolemic insult. PMID- 8637081 TI - Lightning accident with eight victims: case report and brief review of the literature. AB - A case of a lightning accident with eight victims is presented. The patients presented with typical injuries, such as cardiocirculatory arrest, apnea, burns, lightning paralysis, and rupture of internal organs. Two of three patients with cardiocirculatory arrest were successfully resuscitated. A brief description of the spectrum of typical lightning injuries, as well as the recommended therapy concepts, are given. PMID- 8637080 TI - Alcohol as a risk factor for downhill skiing trauma. AB - OBJECTIVE: To assess the role of alcohol in downhill skiing injuries. DESIGN: Comparison of alcohol consumption habits and blood alcohol concentrations of injured skiers to those of randomly selected controls. MATERIALS AND METHODS: 121 injured skiers and 701 control subjects were interviewed and gave breath samples for the determination of blood alcohol concentration. MEASUREMENTS AND MAIN RESULTS: Neither mean blood alcohol concentration nor the number of subjects with an intoxicating level of alcohol in blood (> 0.5 g/L; 2.9% of control subjects and 3.3% of the injured patients) differed significantly between the groups. Also, the severity of the injury and the blood alcohol concentration seemed to be independent of each other; all of the most severe traumas occurred in subjects with no detectable alcohol in blood. CONCLUSIONS: Alcohol does not seem to be a major etiological factor in skiing-related injuries. PMID- 8637082 TI - Thoracic compartment syndrome. AB - Presented is a case of a thoracic gunshot wound resulting in descending thoracic aortic and biventricular cardiac injuries. Successful management of these wounds allowed the development of an unusual and previously undescribed complication of thoracic trauma--thoracic compartment syndrome. The clinical features, therapy, and potential sequelae of thoracic compartment syndrome are presented with a review of the literature. PMID- 8637083 TI - Posttraumatic Budd-Chiari syndrome treated with thrombolytic therapy and angioplasty. AB - Injury of the hepatic veins or suprahepatic inferior vena cava is a rare cause of Budd-Chiari syndrome. Treatment of this syndrome has primarily involved hepatic venous decompression with a variety of portosystemic shunts. We report a case of thrombosis of the inferior vena cava after blunt injury managed with interventional radiologic techniques. PMID- 8637084 TI - Nonoperative management of a splenic tear in a Jehovah's Witness with hemophilia. AB - Splenic laceration, the most common visceral lesion following blunt abdominal trauma, can be treated in a nonoperative fashion in only a select group of stable patients with minimal injury. We report a unique case of life-threatening splenic trauma in a Jehovah's Witness with hemophilia that was successfully managed without surgery. PMID- 8637085 TI - A case of rectal infarction after sigmoid colectomy for traumatic perforation in a patient with a major pelvic fracture. AB - Rectal ischemia and infarction are very uncommon. We report a case of rectal infarction complicating sigmoid colectomy for blunt trauma in a patient with a severe pelvic fracture. We advise that the superior rectal arteries must be preserved in severe pelvic fracture to prevent rectal ischemia. PMID- 8637086 TI - Conservative treatment of an injured hydronephrotic kidney: the role of percutaneous nephrostomy. AB - The authors report a child who presented with massive hematuria after blunt trauma to a hydronephrotic kidney (ureteropelvic junction stenosis). The insertion of a nephrostomy tube effectively decompressed the injured kidney and enabled an early reconstructive repair of the stenosed ureteropelvic junction. PMID- 8637087 TI - Extratemporal facial nerve paralysis after blunt trauma. AB - Presented are two patients in whom facial palsy developed after blunt trauma to the side of the face. Both patients made a complete and uneventful recovery. The etiology of a facial nerve lesion can be established through knowledge of the possible sites of injury to the facial nerve, good history taking and physical examination, together with appropriate tests. The prognosis of an injury secondary to blunt trauma is generally good. PMID- 8637088 TI - Salvage of a devascularized digit with free arterialized venous flap: a case report. AB - Significant soft tissue and vascular loss is always encountered in salvage of a devascularized digit that has sustained crush or avulsion injury. Ideal reconstruction should include repair of the vascular defect and provision of stable soft tissue coverage simultaneously in a single operation to facilitate early recovery and early rehabilitation. A venous flap is a composite cutaneous flap that contains a segment of vein traversing it. A free venous flap is used in a devascularized digit to cover the soft tissue defect and restore the digital circulation by perfusion of arterial blood through the flap's venous network (i.e., free arterialized venous flap). Satisfactory result, both for the flap and for the digit, is obtained. The free arterialized venous flap is a useful, up-to date option for such dual-purpose reconstruction. PMID- 8637089 TI - Bilateral popliteal artery injury from bumper crush injury. AB - Blunt trauma to the knee of sufficient force to result in knee dislocation or fracture is commonly associated with popliteal artery injury. The challenging problem of bilateral popliteal artery injury has been rarely reported. We describe a case of bilateral popliteal artery injury after bumper crush injury between two automobiles that illustrates a successful method of management. Expeditious revascularization with minimum ischemia time was obtained by using the posterior approach, rather than the conventional medial approach, allowing two surgical teams to work simultaneously. PMID- 8637091 TI - Introduction of a new tracheostomy exchange device after percutaneous tracheostomy in a patient with coagulopathy. AB - Presented is a case of a 22-year-old male bone marrow transplant recipient who developed severe methotrexate-induced oropharyngeal mucositis and respiratory insufficiency caused by sepsis. Coagulopathy and severe thrombocytopenia precluded surgical tracheostomy; however, dilatational percutaneous tracheostomy was performed uneventfully. The tracheostomy tube was later changed using a newly developed exchange device permitting intraprocedural oxygenation and stomal redilatation. We conclude that severe thrombocytopenia and coagulopathy are not an absolute contraindication for percutaneous tracheostomy and that the new tracheostomy exchange device optimizes airway management and safety during this procedure. PMID- 8637092 TI - Sudden severe barotrauma from self-inflating bag-valve devices. AB - Self-inflating bag-valve devices are commonly used for the ventilation of intubated patients, especially during resuscitation and transport. These devices are generally safe, but minor deviations in their recommended use can expose patients to airway pressures greater than 135 cm H2O. We present a patient in whom a sudden tension pneumothorax developed during ventilation with a bag-valve device. We believe that this complication resulted from high airway pressures generated in the bag-valve device. The ability of the device in question to cause barotrauma was confirmed by bench-top measurements of the peak airway pressures generated by minor deviations from proper use of the device. PMID- 8637090 TI - Traumatic blindness after airbag deployment: bilateral lenticular dislocation. AB - Airbags have been shown to save lives and reduce head, neck, and upper torso trauma associated with motor vehicle crashes. However, airbags have also been implicated as the source of facial and orbital injury. We report the case of a patient who suffered bilateral lenticular subluxation, anterior chamber hemorrhages, and minor facial lacerations as the result of airbag deployment. PMID- 8637093 TI - A serious vascular complication of internal fixation of a tibial plateau fracture: a cautionary tale from which several lessons can be learned. AB - A serious vascular complication of internal fixation of a tibial plateau fracture is described, along with the problems encountered in revascularisation of the leg. Several lessons can be learned from this case, which illustrates the importance of on-site vascular surgery services. PMID- 8637094 TI - Wound repair: from ritual practice to scientific discipline. PMID- 8637095 TI - Demographics of civilian cranial gunshot wounds. PMID- 8637096 TI - Abdominal aortic aneurysm repair in Veterans Affairs medical centers. AB - PURPOSE: This study was performed to define outcomes after abdominal aortic aneurysm (AAA) repair in Veterans Affairs (VA) medical centers during fiscal years 1991 through 1993. METHODS: With VA patient treatment file data, patients were selected from diagnosis-related groups 110 and 111 and were then classified in a patient management category. In the categories of repair of nonruptured and ruptured AAA, mortality and postoperative complication rates were defined for patients who underwent AAA repair in VA medical centers during the 3-year study period. RESULTS: Hospital mortality rates were 4.86% (166 of 3419) after repair of nonruptured AAA and 47.0% (126 of 268) after repair of ruptured AAA (p<0.001). Of 292 deaths after AAA repair, 136 (43.2%) followed repair of ruptured AAA, even though ruptured AAA comprised only 7.3% of total AAA surgical volume. AAA repairs were performed at 116 VA medical centers, with 31.8+/-23.1 (range, 1 to 140) procedures performed at each center. Although many lower-volume centers had excellent results, centers that performed >or=32 AAA repairs tended to have lower in-hospital mortality rates after repair of nonruptured AAA than those that performed or=45 minutes, neurologic complications occurred in 7 of 55 (13%) versus 7 of 18 (39%) (p=0.033). CONCLUSION: The period of risk during aortic cross-clamp time is reduced with the adjuncts of CSF drainage and DAP, which significantly lower the incidence of neurologic complications after repair of TAAA types I and II. PMID- 8637100 TI - The risk of ischemic spinal cord injury in patients undergoing graft replacement for thoracoabdominal aortic aneurysms. AB - PURPOSE: We developed a monitoring system to detect spinal cord ischemia during aortic cross-clamping (AXC). This system was used to prospectively determine in which patients ischemia occurs, in which patients reimplantation of intercostal arteries is unnecessary or mandatory, and when reperfusion of intercostal arteries (ICAs) is urgent. METHODS: Two hundred sixty patients underwent thoracoabdominal aortic aneurysm (TAA) repair with simple AXC. In 167 patients, two electrocatheters were placed before the onset of anaesthesia at level L1/L2 (stimulation) and level T5/T6 (recording) within the epidural space. During surgery, spinal cord function was monitored by recording spinal somatosensory evoked potentials (sSSEP). According to the extent of aortic replacement, most patients were expected to have a high risk of paraplegia. RESULTS: In group A (59 patients), sSSEP remained normal throughout surgery, and in 54 of these patients ICAs were not reattached outside the proximal aortic anastomosis. In the other five patients ICAs were reimplanted separately because of possible anatomic relation to spinal cord blood supply. No patient in group A had postoperative neurologic deficit. In group B (54 patients) sSSEP remained normal until 15 minutes after AXC but were impaired thereafter. Nineteen patients had early reimplantation of ICAs. Of the 19, three had paraparesis and two had paraplegia. Neurologic deficit developed in the patients without early reimplantation of ICAs. In four patients separate reimplantation of ICAs was performed late in the procedure because of incomplete sSSEP recovery. Subsequently, the sSSEP returned to normal and only one of the four patients had mild paraparesis. The total rate of neurologic deficits in this group was 13% (paraplegia, 3.5%; paraparesis, 9.5%). All 54 patients in group C showed rapid loss of sSSEP within 15 minutes of AXC. In 28 patients ICAs were reimplanted only within the proximal anastomosis. Twenty-one of these patients showed prompt signal recovery after blood-flow release into the reimplanted ICAs, and none had neurologic deficit. Seven patients had no or very late and incomplete sSSEP recovery. Of the seven, three had paraplegia and four had paraparesis. In 26 patients ICAs were reimplanted separately to the proximal anastomosis. This was done early during the procedure in 17 patients, of whom 13 had full recovery of sSSEP and normal neurologic status. Four patients had incomplete or no recurrence of sSSEP, followed by paraplegia in one and paraparesis in three. In nine patients ICAs were reimplanted after the aortic replacement had been completed because of sSSEP recovery was not satisfactory. In all patients in this subgroup sSSEP returned to normal. Six patients had a normal neurologic status and three had mild paraparesis. The total neurologic complication rate in group C was 26% (paraplegia, 7.5%; paraparesis, 18.5%). CONCLUSION: The risk of ischemic spinal cord injury during replacement for TAA can be assessed continuously by monitoring the sSSEP directly from the spinal cord. Patients without sSSEP changes during aortic reconstruction do not require ICA reattachment and will not have neurologic deficit. Patients who lose sSSEP after AXC are at risk for paraplegia. Patients with impairment or loss of sSSEP >15 minutes after AXC have some collateral vessels, and must have ICAs reimplanted only if sSSEP do not return within normal recovery time after blood-flow release into the proximal anastomosis. Loss of sSSEP within 15 minutes of AXC shows poor collateralization and mandates early restoration of spinal cord blood supply. If the surgeon can achieve the return of sSSEP to normal by subsequent separate reimplantation of ICAS, paraplegia will not occur and paraparesis will be rare and mild. Spinal cord monitoring is a valuable guide to detect whether the spinal cord is at risk and to take measures against par PMID- 8637101 TI - Aortic dissection: percutaneous management of ischemic complications with endovascular stents and balloon fenestration. AB - PURPOSE: The purpose of this study was to evaluate endovascular stenting (EVS) and balloon fenestration (BF) of intimal flaps for the management of lower extremity, renal, and visceral ischemia in acute or chronic aortic dissection. METHODS: Twenty-two patients (16 male, 6 female) with a median age of 53 years (range 35 to 77 years) underwent percutaneous treatment for peripheral ischemic complications of 12 type A (five acute, seven chronic) and 10 type B (nine acute, one chronic) aortic dissections. RESULTS: Ten patients had leg ischemia, 13 had renal ischemia, and 6 had visceral ischemia. Sixteen patients were treated with EVS including 11 with renal, 6 with lower extremity, 2 with superior mesenteric artery, and 2 with aortic stents. Three patients had BF of the intimal flap, and three had BF in combination with EVS. Revascularization with clinical success was achieved in all 22 patients. Two patients died 3 days and 13.4 months after the procedure was performed, respectively. Of the remaining 20 patients, 1 is lost to follow-up, and 19 have persistent relief of clinical symptoms. Mean follow-up time is 13.7 months (range 1.1 to 46.5 months). One case was complicated by guidewire-induced perinephric hematoma. CONCLUSION: EVS and BF provide a safe and effective percutaneous method for managing peripheral ischemic complications of aortic dissection. PMID- 8637102 TI - Prospective evaluation of new duplex criteria to identify 70% internal carotid artery stenosis. AB - PURPOSE: Large multicenter trials (North American Symptomatic Carotid Endarterectomy Trial, European Carotid Surgery Trial) have documented the benefits of carotid endarterectomy for treating symptomatic patients with >or=70% stenosis of the internal carotid artery. Although color-flow duplex scanning has become the preferred method for noninvasive assessment of internal carotid artery disease, no criteria have been generally accepted to identify this subset of patients. We previously reported a retrospective series to establish such criteria. This study details our results when these criteria were applied prospectively. METHODS: Carotid color-flow duplex scans were compared with arteriograms in 457 patients who underwent both studies. Criteria for >or=70% internal carotid artery stenosis were peak systolic velocity >130 cm/sec and end diastolic velocity >100 cm/sec. Internal carotid arteries with peak systolic velocity <40 cm/sec in which only a trickle of flow could be detected were classified as preocclusive lesions (95% to 99% stenosis). Arteriographic stenosis was determined by comparing the diameter of the internal carotid artery at the site of maximal stenosis to the diameter of the normal distal internal carotid artery. RESULTS: Internal carotid artery stenosis of >or=70% was detected with a sensitivity of 87%, specificity of 97% positive predictive value of 89%, negative predictive value of 96%, and overall accuracy of 95%. Eighty-seven percent of 70% to 99% stenoses were correctly identified. False-positive errors (n=10) were attributed to contralateral internal carotid artery occlusion or high-grade (>90%) stenosis (n=5) and to interpreter error (n=1); no explanation was apparent in the other four. Eleven of 12 false-negative examinations occurred in patients with 70% to 80% internal carotid artery stenosis. CONCLUSIONS: In our laboratories, prospective application of the above velocity criteria identified internal carotid artery stenosis of >or=70% with a reasonably high degree of accuracy. Errors occurred when stenoses were borderline and in patients with severe contralateral disease. With suitably modified velocity criteria, color flow duplex scanning remains the most reliable noninvasive method for identifying symptomatic patients who are candidates for carotid endarterectomy. PMID- 8637103 TI - Comparison of axillofemoral and aortofemoral bypass for aortoiliac occlusive disease. AB - PURPOSE: A comparison of aortofemoral bypass grafting (AOFBG) and axillofemoral bypass grafting (AXFBG) for occlusive disease performed by the same surgeons during a defined interval forms the basis for this report. METHODS: Data regarding all patients who underwent AOFBG of AXFBG for lower-extremity ischemia caused by aortoiliac occlusive disease were prospectively entered into a computerized vascular registry. The decision to perform AOFBG rather than AXFBG was based on assessment of surgical risk and the surgeon's preference. This report describes results for surgical morbidity, mortality, patency, limb salvage, and patient survival for procedures performed from January 1988 through December 1993. RESULTS: We performed 108 AXFBGs and 139 AOFBGs. AXFBG patients were older (mean age, 68 years compared with 58 years for AOFBG, p<0.001), more often had heart disease (84% compared with 38%, p<0.001), more often underwent surgery for limb-salvage indications (80% compared with 42%, p<0.001). No significant differences were found in operative mortality (AXFBG, 3.4%; AOFBG, <1.0%, p=NS), but major postoperative complications occurred more frequently after AOFBG (AXFBG, 9.2%; AOFBG, 19.4%; p<0.05). Follow-up ranged from 1 to 83 months (mean, 27 months). Five-year life-table primary patency, limb salvage, and survival rates were 74%, 89%, and 45% for AXFBG and 80%, 79%, and 72% for AOFBG, respectively. Although the patient survival rate was statistically lower with AXFBG, primary patency and limb salvage rates did not differ when compared with AOFBG. CONCLUSION: When reserved for high-risk patients with limited life expectancy, the patency and limb salvage results of AXFBG are equivalent to those of AOFBG. PMID- 8637104 TI - Results of bypass to the popliteal and tibial arteries with alternative sources of autogenous vein. AB - PURPOSE: The goal of an all-autogenous policy for infrainguinal arterial bypass requires that many bypasses be performed with alternative autogenous veins (AAV) because an adequate length of ipsilateral or contralateral greater saphenous vein (GSV) is not available. The durability and efficacy of infrainguinal vein bypasses constructed of venous conduits other than a single segment of greater saphenous vein (SSGSV) is, however, questioned. METHODS: AAV and GSV bypasses were reviewed from 1980 through 1994. Patients who required bypass to the popliteal or a tibial artery were compared for vascular surgical history and vascular disease risk factors and life-table survival. AAV and SSGSV procedures were compared for indications for surgery, morbidity and mortality rates, limb salvage rates in patients who underwent surgery for limb-salvage indications, subsequent need for revision, and life-table-assisted primary patency. RESULTS: Nine hundred nineteen autogenous vein bypasses were performed to the popliteal or a tibial artery--187 (20%) with AAVs, including whole or partial arm vein conduits in 144 grafts (77%). One hundred fourteen AAVs (61%) required vein splicing. The mortality rate was 2% for SSGSV bypasses and 1% for AAV bypasses. The morbidity rate was higher for GSV surgery as a result of increased wound complications (11% vs 5%; p=0.02). Sixty-seven percent of patients with AAV bypass extremities had undergone previous ipsilateral arterial surgery, compared with 20% of patients with SSGSV bypasses (p0.0005). AAV bypasses were more likely to be to a tibial artery (71% vs 45%; p<0.0001). Twelve percent of SSGSV and 15% of AAV popliteal bypasses required revision (p=NS). The 5-year assisted primary patencies were 82%, 77%, and 63%, with limb salvage rates of 91%, 86%, and 74% for ipsilateral SSGSV, contralateral SSGSV, and AAV femoropopliteal bypasses, respectively. Twelve percent of SSGSV and 30% of AAV tibial bypasses required revision (p=0.0001). The 5-year assisted primary patencies were 74%, 82%, and 72%, with limb salvage rates of 84%, 92% and 78% for ipsilateral SSGSV, contralateral SSGSV, and AAV femorotibial bypasses, respectively. CONCLUSION: AAV bypasses can provide overall results comparable with SSGSV bypasses. PMID- 8637105 TI - Vascular applications of telepresence surgery: initial feasibility studies in swine. AB - PURPOSE: Telepresence surgery is a novel technology that will allow procedures to be performed on a patient at locations that are physically remote from the operating surgeon. This new method provides the sensory illusion that the surgeon's hands are in direct contact with the patient. We studied the feasibility of the use of telepresence surgery to perform basic operations in vascular surgery, including tissue dissection, vessel manipulation, and suturing. METHODS: A prototype telepresence surgery system with bimanual force-reflective manipulators, interchangeable surgical instruments, and stereoscopic video input was used. Arteriotomies created ex vivo in segments of bovine aortae or in vivo in femoral arteries of anesthetized swine were closed with telepresence surgery or by conventional techniques. Time required, technical quality (patency, integrity of suture line), and subjective difficulty were compared for the two methods. RESULTS: All attempted procedures were successfully completed with telepresence surgery. Arteriotomy closures were completed in 192+/-24 sec with conventional techniques and 483+/-118 sec with telepresence surgery, but the precision attained with telepresence surgery was equal to that of conventional techniques. Telepresence surgery was described as intuitive and natural by the surgeons who used the system. CONCLUSIONS: Blood-vessel manipulation and suturing with telepresence surgery are feasible. Further instrument development (to increase degrees of freedom) is required to achieve operating times comparable to conventional open surgery, but the system has great potential to extend the expertise of vascular surgeons to locations where specialty care is currently unavailable. PMID- 8637106 TI - Modulation of monocyte adherence to endothelial cells by endothelin-1 involvement of Src (p60src) and JAK1-like kinases. AB - PURPOSE: The purpose of this study was to determine the transmembrane signaling pathway by which endothelin-1 (ET-1) enhances monocyte adherence to human umbilical vein endothelial cells (HUVECs) and to investigate the role of tyrosine kinases in this mechanism. METHODS: Adherence of purified human blood monocytes to HUVEC monolayers was assessed with radiolabeled monocytes. Tyrosine kinase activation was examined by immunoprecipitation and Western blotting. RESULTS: ET 1 potentiated monocyte adherence to HUVECs in a biphasic manner with peaks at 10( 10) mol/L and 10(-7) mol/L. A potent antagonist to ET B receptors, when used alone, had no effect. However, the antagonist, when combined with ET-1, significantly enhanced monocyte adherence to HUVECs. Incubation of ET-1 (10(-12) mol/L to 10(-7) mol/L) with HUVECs activated tyrosine kinases in a biphasic manner as identified by immunoblotting with PY20 antibody to tyrosine phosphorylated proteins. Phosphorylated proteins with Mr 60, 110, and 130 kDa were observed after ET-1 stimulation of HUVECs. Of interest, ET A or ET B receptor antagonists failed to antagonize the effect of ET-1. Rather, these receptor antagonists significantly augmented ET-1 induced tyrosine phosphorylation in HUVECs. Immunoprecipitation with antibodies to p60SRC and JAK1 kinases followed by immunoblotting with PY20 antibody suggested that ET-1 receptor response coupling in HUVECs involves the activation of p60SRC and JAK1 like kinases. CONCLUSIONS: These data suggest an association between activation of p60SRC and JAK1-like kinases and monocyte adherence in response to ET-1. ET-1 induced monocyte adherence is upregulated by ET B receptor antagonist, suggesting a negative feedback on cell adhesion through this receptor. PMID- 8637108 TI - Insulin-like growth factor-I binding in injury-induced intimal hyperplasia of rabbit aorta. AB - PURPOSE: The proliferation of arterial-wall smooth muscle cells is an important step in the formation of intimal hyperplasia. Insulin-like growth factor-I (IGF I) is a mitogen that exerts its effects through specific receptors located on the cell membrane. IGF-I has been found to promote the multiplication of vascular smooth muscle cells in culture. This study aimed to evaluate the status of IGF-I binding in injury-induced intimal hyperplasia in a rabbit model. METHODS: We used binding techniques to study IGF-I binding of control and hyperplastic aortas of adult White New Zealand rabbits. Hyperplasia was induced by balloon-catheter injury. At 2 weeks and 1, 2, 4, and 7 months after injury, segments of abdominal aortas were harvested from two control and six study rabbits, and 20-micrometer thick frozen sections were obtained. Hematoxylin and eosin-stained sections confirmed the presence of intimal hyperplasia in the hyperplastic aortas. Adjacent sections were incubated in a buffer solution containing 125I-IGF-I in the presence and absence of an excess of unlabeled IGF-I. Autoradiograms were then obtained by apposing the treated sections to autoradiography film, which was developed at 3 days and analyzed by comparison with the hematoxylin and eosin stained sections under light microscopy. A marked increase in IGF-I binding grain density was observed in the areas corresponding to the hyperplastic lesions. To characterize these binding sites, binding inhibition studies were performed and the dissociation constant (K d) and maximum binding capacity (B max) were obtained from Scatchard analysis. RESULTS: Six hyperplastic aortas for each time interval and a total of nine control aortas were evaluated. The K d of the hyperplastic aortas (1.5+/-0.2 nmol/L) was not significantly different from that of control aortas (1.3+/-0.2 nmol/L), which indicated similar high-affinity IGF-I binding sites in normal and hyperplastic arteries. The results of B max were 6.9+/-1.2, 8.5+/-2.1, 12.4+/-2.1, 20.4+/-5.9, 20.6+/-3.2, and 8.1+/-1.3 pmol/L for control, 2 weeks, 1 month, 2 months, 4 months, and 7 months, respectively. With analysis of variance (p<0.05), B max values at 1, 2, and 4 months were significantly higher than those of control aortas. B max values returned to levels not significantly different from those of control aortas at the 7-month interval. CONCLUSION: Increased IGF-I binding in the hyperplastic aortas suggests that IGF-I plays an important role in the proliferation of arterial wall cellular components during the hyperplastic process. PMID- 8637107 TI - Anti-CD 18 monoclonal antibody slows experimental aortic aneurysm expansion. AB - PURPOSE: Inflammation has been implicated as a contributing factor in the expansion of abdominal aortic aneurysms (AAA). To test this hypothesis, we examined the effects of a monoclonal antibody (MAB) to the leukocyte CD18 adhesion molecule on the expansion of experimental AAA. METHODS: Aneurysms were induced by perfusion of an isolated segment of the infrarenal aorta with elastase in 22 normotensive (WKY) and 17 genetically hypertensive (WKHT) rats. Animals of both strains were randomly allocated to control or MAB-treated groups (MAB, 5 microgram/100 gm body weight intraperitoneally, daily, beginning on the operative day for a total of four doses). The activity of the MAB against rat leukocytes had first been determined by in vitro immunofluorescence flow cytometry. Aortic size was directly measured initially and on day 14. At that time, a segment of aorta was stained with hematoxylin and eosin and mononuclear leukocytes and neutrophils were counted in each of 10 microscopic fields (400X). RESULTS: The initial aortic size in all animals was 1.11+/-0.15 mm. All groups developed aneurysms significantly larger than the initial aortic size (p<0.01). However, the MAB-treated animals had significantly smaller aneurysms than the untreated controls (mm): WKY: 3.63+/-1.26, WKY-MAB: 2.08+/-0.30, WKHT: 4.54+/-1.86, WKHT MAB: 2.37+/-0.40, p<0.0001. There also were significantly fewer monocytes in the MAB-treated normotensive rats: WKY:35.5+/-29.9, WKHT:40.6+/-28.8, WKY-MAB: 8.9+/ 8.5, WKHT-MAB: 32.3+/-25.7, p=0.03. Neutrophil counts did not differ significantly between the groups. CONCLUSIONS: Treatment with anti-CD18 monoclonal antibody slows the expansion of AAA in this experimental model. The associated inflammatory process at day 14, as indicated by monocyte infiltration, is reduced, but this effect may be opposed by the presence of hypertension. Further evaluation of the role of leukocytes and adhesion molecules in the expansion of AAA is warranted. PMID- 8637109 TI - Role of nitric oxide in the effect of blood flow on neointima formation. AB - PURPOSE: Neointima formation after arterial injury is inhibited by increased blood flow. The object of this study was to determine whether nitric oxide mediates the effect of increased blood flow on neointima formation. METHOD: Balloon catheter-denuded rat carotid arteries were exposed to increased blood flow or control blood flow by ligation of the contralateral carotid artery. Beginning 2 days before balloon denudation, rats were given either saline vehicle alone or the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl ester (L NAME) at a dose of 10 mg/kg/day or 2 mg/kg/day intraperitoneally. The normalized neointima area was measured 14 days after denudation. RESULTS: Blood flow was significantly increased by ligation of the contralateral carotid artery for all drug treatments (p<0.008). In rats given saline vehicle only, normalized neointima area was significantly reduced after increased blood flow compared with control blood flow (0.33+/-0.04 compared with 0.48+/-0.03; p=0.006). Systolic blood pressure was significantly elevated by treatment with high-dose L-NAME (p=0.002 compared with vehicle), but was not altered by low-dose L-NAME (p=NS compared with vehicle). Normalized neointima area was not significantly reduced after increased carotid blood flow for rats treated with either dose of L-NAME (p=NS). CONCLUSION: The inhibition of neointima formation by increased blood flow was abolished with hypertensive and nonhypertensive doses of the nitric oxide synthase inhibitor L-NAME, which suggests that the L-NAME effects are independent of systemic hemodynamic alterations. It is concluded that flow-induced inhibition of neointima formation is mediated in part by nitric oxide. PMID- 8637110 TI - Surgical results: a justification of the surgeon selection process for the ACAS trial. The ACAS Investigators. AB - PURPOSE: The selection of surgeons to participate in a prospective randomized trial comparing the efficacy of a surgical method with medical management is critically important because it will have a direct impact on the outcome of the study and the future use of the operation. We report the success of the method used for selecting surgeons who participated in the Asymptomatic Carotid Atherosclerosis Study (ACAS) by examining the surgical morbidity and mortality rates and the outcome of the study. METHODS: A Surgical Management Committee established criteria for auditing surgeons who wished to participate in the study. The parameters included a minimum performance of at least 12 carotid endarterectomies (CEA) per year and an audit of each surgeon's last 50 consecutive CEAs with required documentation of a combined neurologic morbidity and mortality rate of <3.0% for asymptomatic patients and <5.0% for all indications including symptomatic patients. RESULTS: As of February 1991, 164 surgeons from 48 medical centers applied for ACAS participation. One hundred seventeen were approved, and their aggregate experience of 5641 operations yielded a combined neurologic morbidity and mortality rate of 2.3% for asymptomatic and symptomatic patients combined. The morbidity and mortality rate for CEA on asymptomatic patients was 1.7%. These surgeons, plus those recruited after February 1991, became investigators in the ACAS trial and were responsible for the surgical care of 825 patients who were randomized to the surgical arm. Seven hundred twenty-four patients actually underwent CEA. One patient (0.14%) died and ten patients (1.38%) had strokes within the 30-day perioperative interval, for a combined stroke or death incidence of 1.52%. The 5-year stroke event rate in the surgical group (including perioperative morbidity and mortality rates) was 5.1%. compared with 11% of patients treated medically, yielding a relative risk reduction of 53% in favor of surgery (p=0.004). CONCLUSIONS: A method for selecting surgeons for participation in the ACAS trial was successful in providing low perioperative morbidity and mortality rates. This materially influenced the outcome of the study in favor of CEA. PMID- 8637111 TI - The merit of polytetrafluoroethylene extensions and interposition grafts to salvage failing infrainguinal vein bypasses. AB - PURPOSE: The purpose of this study was to evaluate the merit of polytetrafluoroethylene (PTFE) extensions and interpositions for the management of failing infrainguinal vein bypass grafts. METHODS: The treatment of 133 failing vein grafts in 125 patients over a 10-year period was retrospectively reviewed. Twenty-two graft-threatening lesions were detected in patients who did not have a usable autogenous vein conduit as determined by preoperative and intraoperative evaluations. A PTFE extension or interposition graft was used for the necessary reconstruction in all cases. RESULTS: Ten lesions were within the vein graft, 11 were proximal to the graft in the femoral or popliteal artery segments, and one was distal to the graft in the popliteal artery. The treatment of these lesions included 19 extensions and three mid graft interpositions. The vein graft lesions developed significantly sooner (mean 10.6+/-2.5 months) after the bypass (p<0.05) than the arterial lesions (mean 28.0+/-6.1 months). The 3 year cumulative secondary patency rate for these vein grafts treated with PTFE extensions or interpositions was 84%+/-8%. This was not significantly different from the 3-year cumulative secondary patency rate for vein grafts treated with vein extensions or interpositions at our institution over the same time period (82%+/-10%). The 3-year limb salvage rates were 95% and 89%, respectively. CONCLUSIONS: These results indicate that PTFE extensions and interpositions can be used successfully to maintain the patency of failing vein grafts and may serve to prolong limb salvage in patients without any usable autogenous vein. Early reintervention with a PTFE conduit in this difficult group of patients is appropriate to salvage a failing vein graft. PMID- 8637112 TI - Doxycycline inhibition of aneurysmal degeneration in an elastase-induced rat model of abdominal aortic aneurysm: preservation of aortic elastin associated with suppressed production of 92 kD gelatinase. AB - PURPOSE: Increased local production of matrix metalloproteinases (MMPs) is a potential mechanism underlying structural protein degradation in abdominal aortic aneurysms (AAA). With an elastase-induced rodent model of AAA, we determined whether pharmacologic treatment with an MMP-inhibiting tetracycline might limit the development of experimental AAA in vivo. METHODS: Forty-eight Wistar rats underwent a 2-hour perfusion of the abdominal aorta with 50 U porcine pancreatic elastase and were then treated with either subcutaneous doxycycline (25 mg/day; n=24) or saline solution vehicle (n=24). Aortic diameter was measured before and after elastase perfusion was performed and before the rats were killed at 0, 2, 7, or 14 days, and AAAs were defined as an increase in aortic diameter to at least twice that before perfusion. At death the aortic tissues were either perfusion-fixed for histologic evaluation or extracted for substrate zymographic evaluation. RESULTS: Aortic diameter was not different between groups at 0 or 2 days, but it was significantly less in animals treated with doxycycline at both 7 and 14 days (mean+/-SEM, p<0.01). After day 2 the incidence of AAA was reduced from 83% (10 of 12 rats treated with saline solution) to 8% (1 of 12 animals treated with doxycycline). By histologic assessment doxycycline prevented the structural deterioration of aortic elastin without decreasing the influx of inflammatory cells. Increased aortic wall production of 92 kD gelatinase observed in a saline solution-treated control group was markedly suppressed in animals treated with doxycycline. CONCLUSIONS: Treatment with an MMP-inhibiting tetracycline inhibits the development of experimental AAA in vivo. This inhibition may be due to selective blockade of elastolytic MMP expression in infiltrating inflammatory cells. Additional experiments, however, are necessitated to fully delineate this process. PMID- 8637114 TI - Durability of venous valve reconstruction techniques for "primary" and postthrombotic reflux. AB - PURPOSE: The durability of the variety of valve reconstruction techniques in "primary" reflux and postthrombotic reflux was studied. METHODS: A total of 423 valve repairs in 235 patients with a follow-up period ranging from 1 to 12 years were analyzed. End points for assessment consisted of ulcer recurrence and Doppler competence in serial duplex examination. Multivariate analysis with Cox proportional hazards model was used. RESULTS: Ulcer-free survival curves were similar for "primary" and postthrombotic reflux. No significant difference in ulcer recurrence was seen regardless of the technique used. Different results were obtained when valve competence instead of ulcer recurrence was used for assessment of durability. Reconstructions in "primary" reflux were more durable than those in postthrombotic reflux. Durability differences were also noted among different techniques. A cohort of posterior tibial repairs proved extraordinarily durable (0 failures in 23 repairs). CONCLUSION: Valve reconstruction in postthrombotic reflux can yield clinical results similar to those in "primary" reflux. Although any of the several described techniques can produce similar clinical results, Doppler competence suggests the following order for choice of procedures: (1) internal valvuloplasty, (2) prosthetic sleeve in situ, (3) external valvuloplasty, and (4) axillary vein transfer. PMID- 8637113 TI - Polytetrafluoroethylene bypasses to infrapopliteal arteries without cuffs or patches: a better option than amputation in patients without autologous vein. AB - PURPOSE: This study was undertaken to evaluate our results of polytetrafluoroethylene (PTFE) tibial and peroneal artery bypasses done for limb salvage. METHODS: Within a group of patients undergoing infrainguinal limb salvage bypasses at our institution between January 1986 and May 1995, 63 patients faced an immediate amputation, had no autologous vein on duplex examination and operative exploration, and had only a tibial or peroneal artery as an outflow vessel for bypass. Most of these patients (82%) had two or more prior ipsilateral infrainguinal bypasses. These 63 patients underwent 66 PTFE bypasses to a tibial or peroneal artery without a distal anastomotic vein cuff or an adjunctive arteriovenous fistula. Our results were then compared with those reported from infrapopliteal (crural) bypasses performed with alternate autologous vein sources or PTFE in conjunction with various recommended adjuncts. RESULTS: The 3- and 5-year cumulative primary graft patency rates for our PTFE infrapopliteal bypasses were 39%+/-7% and 28%+/-9%, respectively. Secondary graft patency rates were 55%+/-8% and 43%+/-10% at 3 and 5 years, respectively. Limb salvage rates were 71%+/-7% at 3 years and 66%+/-8% at 5 years. Two-year actuarial patient survival rate was only 67%+/-7%. CONCLUSIONS: These results indicate that a PTFE bypass to an infrapopliteal artery remains a worthwhile option in patients without usable autologous vein. The secondary patency and limb salvage rates were acceptable in this setting and were not significantly different from the best results reported with prosthetic tibial/peroneal bypasses with distal vein cuffs or patches (74% at 1 year; 58% at 3 years), arteriovenous fistulas (71% at 1 year) or composite arm vein grafts (39% and 29% at 3 and 5 years, respectively). PMID- 8637116 TI - Regarding "Surgical management of infrainguinal arterial prosthetic graft infections". PMID- 8637115 TI - The treatment of focal aortic arch branch lesions with Palmaz stents. AB - PURPOSE: The purpose of this study was to evaluate the safety and effectiveness of Palmaz stents as a less morbid alternative to traditional surgery for focal aortic arch branch lesions. METHODS: Twenty-two patients with symptoms and a mean age of 61.3 years were treated from July 1991 to May 1995 with 26 stents at the following locations: 8 innominate artery, 5 left common carotid artery, 1 right common carotid artery, and 12 left subclavian. Procedures were carried out in an operating room with patients receiving either local anesthetic in 12 cases or general anesthetic in 10. Surgical exposure of either the cervical common carotid or brachial artery allowed precautionary distal clamping before retrograde stent deployment to prevent atheroembolization. RESULTS: Initial success was possible in 92.3% (24 of 26) of cases. There were no strokes or deaths. During a mean follow-up period of 27 months, 22 of 26 (85%) vessels have remained patent and the patients symptom free. CONCLUSION: Focal aortic branch lesions can be effectively and safely treated with Palmaz stents. PMID- 8637117 TI - A randomized prospective clinical trial of the Taylor patch. PMID- 8637118 TI - Cardiac enzymes in the blood salvaged during abdominal aortic surgery. PMID- 8637120 TI - Special consultation on syringe laws addresses epidemics, airs controversy. PMID- 8637119 TI - Regarding "Lower extremity lymphedema caused by acquired immune deficiency syndrome-related Kaposi's sarcoma". PMID- 8637121 TI - Yeast genome sequencing raises human hopes. PMID- 8637122 TI - Making new drugs via combinatorial chemistry. PMID- 8637123 TI - Sustained deployments tax health of military. PMID- 8637124 TI - From the Centers for Disease Control and Prevention. AIDS associated with injecting-drug use--United States, 1995. PMID- 8637125 TI - From the Centers for Disease Control and Prevention. Tuberculosis morbidity- United States, 1995. PMID- 8637126 TI - From the Centers for Disease Control and Prevention. Tetrodotoxin poisoning associated with eating puffer fish transported from Japan--California, 1996. PMID- 8637127 TI - Medicine and the US embargo against Cuba. PMID- 8637128 TI - Medicine and the US embargo against Cuba. PMID- 8637129 TI - Medicine and the US embargo against Cuba. PMID- 8637130 TI - Sick building syndrome. PMID- 8637131 TI - Folic acid and prevention of birth defects. PMID- 8637132 TI - Folic acid and prevention of spina bifida. PMID- 8637133 TI - Peanut anaphylaxis from food cross-contamination. PMID- 8637134 TI - Continuing medical education: actually learning rather than simply listening. PMID- 8637135 TI - Outbreaks of diarrheal disease: a ship is a ship. PMID- 8637136 TI - Seroprevalence of antibody against poliovirus in inner-city preschool children. Implications for vaccination policy in the United States. AB - OBJECTIVES: To assess susceptibility to poliomyelitis in selected inner-city preschool children in the United States and to estimate the contribution of secondary spread of live attenuated oral poliovirus vaccine virus to type specific immunity. DESIGN: Cross-sectional seroprevalence study. METHODS: Serum neutralizing antibody levels against poliovirus types 1, 2, and 3 were analyzed according to vaccination status, age, and other sociodemographic variables. SETTING: Hospital and satellite clinics serving inner-city populations in Houston, Tex, and Detroit, Mich, 1990 to 1991. PARTICIPANTS: A total of 526 children aged 12 to 47 months seeking medical care were enrolled in the seroprevalence study; 144 children aged 12 to 35 months without a history of previous oral poliovirus vaccination were enrolled in the secondary spread study. RESULTS: Seropositive rates were similar in children in both cities, ranging from about 80% for types 1 and 3 in 12- to 23-month-old children to more than 90% in those aged 36 to 47 months. The most important predictor of seropositivity was the number of doses of oral poliovirus vaccine received (P < .01), with levels approximately 90% for all 3 serotypes among children who had received 3 or more doses. In children likely to have been unvaccinated, seropositive rates ranged from 9% to 18% for poliovirus types 1 and 3 and from 29% to 42% for type 2; secondary spread of vaccine virus appeared to have occurred among children who had previously received 1 dose or less but not those with 2 or more doses. CONCLUSIONS: Levels of immunity to poliovirus among inner-city preschoolers are high and may be predicted by the number of doses of oral poliovirus vaccine received. Secondary spread of the vaccine virus plays a modest role in increasing polio immunity in inner-city populations, especially against types 1 and 3. This role will decrease in importance if the recently attained high levels of immunization coverage in the United States are sustained and if the risk of importation of wild poliovirus continues to diminish. PMID- 8637137 TI - Morbidity and mortality in relation to cigarette smoking in Shanghai, China. A prospective male cohort study. AB - OBJECTIVE: To evaluate prospectively the health risk of cigarette smoking in middle-aged men in Shanghai, China. DESIGN: Prospective cohort study with annual follow-up. PARTICIPANTS: A total of 18 244 male residents of Shanghai, China, enrolled in the study during January 1, 1986, through September 30, 1989, and actively followed via annual visits. RESULTS: By September 30, 1993, 852 deaths and 554 incident cancer cases were identified during the follow-up period, which averaged 5.4 years per subject. The overall incidence rate for cancer was 568 per 100 000 man-years, with the 3 leading sites being lung (146/100 000), stomach (116/100 000), and liver (81/100 000). Forty-one percent of all deaths were from cancer. Stroke was the most frequent cause of death unrelated to cancer, with an age-adjusted rate 4.2 times higher than that of US white men (201/100 000 vs 48/100 000), followed by ischemic heart disease, with an age-adjusted rate one fifth that of US white men (69/100 000 vs 366/100 000). Compared with lifelong nonsmokers, the relative risks in heavy smokers (20 or more cigarettes per day) after adjustment for alcohol consumption were 2.2 for any incident cancer, 9.4 for incident lung cancer, 6.7 for head and neck cancer, and 1.8 for liver cancer. In terms of mortality, heavy smokers were at a 60% greater risk of death relative to lifelong nonsmokers; there was a 2.3-fold excess risk of death from cancer and 2-fold to 3-fold excess risk of death from heart disease. CONCLUSIONS: Cigarette smoking is an important predictor of risk of cancer and mortality in men in Shanghai. Among the study subjects, 36% of all cases of cancer and 21% of all deaths could be attributed to cigarette smoking. PMID- 8637138 TI - Mental stress--induced myocardial ischemia and cardiac events. AB - OBJECTIVE: To assess the clinical significance of mental stress-induced myocardial ischemia in patients with coronary artery disease (CAD). DESIGN AND SETTING: Cohort study in outpatients in a tertiary care teaching hospital assessed at baseline and followed up for up to 5 years. SUBJECTS: A total of 126 volunteer patients (112 men, 14 women; mean age, 59 years) with documented CAD and exercise-induced myocardial ischemia. OUTCOME MEASURES: Patients underwent baseline mental stress and exercise testing using radionuclide ventriculography and 48-hour Holter monitoring. Patients were subsequently contacted by mailed questionnaires or telephone to document cardiac events, including death, nonfatal myocardial infarction, and cardiac revascularization procedures. Logistic regression and Cox proportional hazards models were used to examine the prognostic value of the ischemic measures after adjusting for such potential confounding factors as age, baseline left ventricular ejection fraction (LVEF), and history of myocardial infarction. RESULTS: Twenty-eight patients (22%) experienced at least 1 cardiac event. Baseline mental stress-induced ischemia was associated with significantly higher rates of subsequent cardiac events (odds ratio, 2.8; 95% confidence interval [CI], 1.0-7.7; P < .05). The LVEF change during mental stress was significantly related to event-free survival (risk ratio [RR], 2.4; 95% CI, 1.12-5.14; P = .02), controlling for age, history of prior myocardial infarction, and baseline LVEF. This relationship remained significant after controlling for electrocardiogram (ECG)-defined ischemia during exercise (RR, 2.2; 95% CI, 1.01-4.81; P < .05). The RR for ECG-defined ischemia during exercise testing was 1.9 (95% CI, 0.95-3.96; P = .07) and the RR for ambulatory ECG ischemia was 0.75 (95% CI, 0.35-1.64; P = .47). CONCLUSIONS: The presence of mental stress-induced ischemia is associated with significantly higher rates of subsequent fatal and nonfatal cardiac events, independent of age, baseline LVEF, and previous myocardial infarction, and predicted events over and above exercise induced ischemia. These data suggest that the relationship between psychological stress and adverse cardiac events may be mediated by the occurrence of myocardial ischemia. PMID- 8637139 TI - Concurrent Lyme disease and babesiosis. Evidence for increased severity and duration of illness. AB - OBJECTIVE: To determine whether patients coinfected with Lyme disease and babesiosis in sites where both diseases are zoonotic experience a greater number of symptoms for a longer period of time than those with either infection alone. DESIGN: Community-based, yearly serosurvey and clinic-based cohort study. SETTING: Island community in Rhode Island and 2 Connecticut medical clinics from 1990 to 1994. STUDY PARTICIPANTS: Long-term residents of the island community and patients seeking treatment at the clinics. MAIN OUTCOME MEASURES: Seroreactivity to the agents of Lyme disease and babesiosis and number and duration of symptoms. RESULTS: Of 1156 serosurvey subjects, 97 (8.4%) were seroreactive against Lyme disease spirochete antigen, of whom 14 (14%) also were seroreactive against babesial antigen. Of 240 patients diagnosed with Lyme disease, 26 (11%) were coinfected with babesiosis. Coinfected patients experienced fatigue (P = .002), headache (P < .001), sweats (P < .001), chills (P = .03), anorexia (P = .04), emotional lability (P = .02), nausea (P = .004), conjunctivitis (P = .04), and splenomegaly (P = .01) more frequently than those with Lyme disease alone. Thirteen (50%) of 26 coinfected patients were symptomatic for 3 months or longer compared with 7 (4%) of the 184 patients with Lyme disease alone from whom follow up data were available (P < .001). Patients coinfected with Lyme disease experienced more symptoms and a more persistent episode of illness than did those (n = 10) experiencing babesial infection alone. Circulating spirochetal DNA was detected more than 3 times as often in coinfected patients as in those with Lyme disease alone (P = .06). CONCLUSIONS: Approximately 10% of patients with Lyme disease in southern New England are coinfected with babesiosis in sites where both diseases are zoonotic. The number of symptoms and duration of illness in patients with concurrent Lyme disease and babesiosis are greater than in patients with either infection alone. In areas where both Lyme disease and babesiosis have been reported, the possibility of concomitant babesial infection should be considered when moderate to severe Lyme disease has been diagnosed. PMID- 8637140 TI - The safety effects of child-resistant packaging for oral prescription drugs. Two decades of experience. AB - OBJECTIVE: To evaluate the effectiveness of child-resistant packaging in reducing the mortality rate for children younger than 5 years from the unintentional ingestion of oral prescription drugs. DESIGN: Annual mortality rates for children younger than 5 years associated with the unintentional ingestion of oral prescription drugs are constructed for 1964 through 1992. The effect of child resistant packaging on the child mortality rate during the postintervention period (1974 through 1992) is evaluated with a multivariate time series regression model. The analysis controls for changes in the consumption of oral prescription drugs over time and for long-term safety trends. SETTING: United States. SUBJECTS: Children younger than 5 years. MAIN OUTCOME MEASURE: Estimated reductions in the child mortality rate associated with the use of child-resistant packaging. RESULTS: After controlling for covariates, the use of child-resistant packaging was associated with an annual reduction in the oral prescription drug related mortality rate of 1.40 (95% confidence interval, 0.85-1.95) deaths per million children younger than 5 years. This suggests a reduction of about 460 child deaths from 1974, the year oral prescription drugs became subject to child resistant packaging requirements, through 1992-a mortality rate reduction of about 45% from levels projected without the child-resistant requirements. CONCLUSION: Child-resistant packaging reduces child mortality from the unintentional ingestion of oral prescription drugs. PMID- 8637141 TI - Can medical savings accounts for the nonelderly reduce health care costs? AB - OBJECTIVE: To understand how medical savings account (MSA) legislation for the nonelderly would affect health care costs. DESIGN: Economic policy evaluation based on the RAND Health Expenditures Simulation Model. SETTING: National probability sample of nonelderly noninstitutionalized households. PARTICIPANTS: Persons in 23 157 sampled households from the 1993 Current Population Survey. INTERVENTIONS: Medical savings account legislation would allow all Americans who are covered only by a catastrophic health care plan to set up a tax-exempt account that they can use to pay medical bills not covered by their health insurance. The interventions we evaluate differ in the deductibles of the catastrophic plan and in whether the employee or employer funds the MSA. MAIN OUTCOME MEASURES: Changes in national health expenditures and net societal benefits of health care. RESULTS: If all insured nonelderly Americans switched to MSAs, their health care expenditures would decline by between 0% and 13%, depending on how the MSAs are designed. However, not all nonelderly Americans would choose MSAs; taking into account selection patterns, health spending would change by + 1% to -2%. CONCLUSIONS: Medical savings account legislation would have little impact on health care costs of Americans with employer-provided insurance. However, depending on the size of the catastrophic limit, waste from the excessive use of generously insured care could be reduced, and MSAs would be attractive to both sick and healthy people. PMID- 8637143 TI - An 82-year-old woman with cataracts. PMID- 8637142 TI - Prevalence and incidence of adult pertussis in an urban population. AB - OBJECTIVES: To determine the prevalence of Bordetella pertussis infection among adults who have prolonged cough for 2 weeks or longer and to estimate the incidence of B pertussis infection in adults in a defined urban population. DESIGN: A prospective clinical study. SETTING: Kaiser Permanente, San Francisco (Calif) Medical Center. PARTICIPANTS: One hundred fifty-three referred and participating health plan members 18 years old or older with the complaint of cough persisting for 2 weeks or longer and 154 health plan members 18 years old or older with no cough for the past 3 months (controls) were enrolled. Medical records for an additional 100 patients randomly sampled from 676 patients 18 years old or older with an ambulatory diagnosis of cough (60 with prolonged cough) were also reviewed. MAIN OUTCOME MEASURES: Prevalence of adult pertussis as determined by enzyme-linked immunosorbent assay IgG antibody levels to pertussis toxin in individuals with prolonged cough for 2 weeks or longer and the incidence of adult pertussis in San Francisco Kaiser health plan members. RESULTS: The prevalence of adult pertussis was 12.4% of the participating referrals. The incidence of adult pertussis was estimated to be 176 cases per 100 000 person-years (95% confidence interval, 97 to 255 cases). CONCLUSIONS: Adult pertussis is a significantly greater public health threat than previously suspected. Booster doses of acellular pertussis vaccine after 7 years of age may be an effective approach to minimize transmission and infection. PMID- 8637144 TI - A 79-year-old musician with asymptomatic carotid artery disease, 1 year later. PMID- 8637146 TI - Tobacco--the growing epidemic in China. PMID- 8637145 TI - Polio eradication--how near? PMID- 8637147 TI - Blunt force violence in America--shades of gray or red. Ultimate/extreme fighting. PMID- 8637148 TI - The impact of managed care on patients' trust in medical care and their physicians. AB - Social trust in health care organizations and interpersonal trust in physicians may be mutually supportive, but they also diverge in important ways. The success of medical care depends most importantly on patients' trust that their physicians are competent, take appropriate responsibility and control, and give their patients' welfare the highest priority. Utilization review and structural arrangements in managed care potentially challenge trust in physicians by restricting choice, contradicting medical decisions and control, and restricting open communication with patients. Gatekeeping and incentives to limit care also raise serious trust issues. We argue that managed care plans rather than physicians should be required to disclose financial arrangements, that limits be placed on incentives that put physicians at financial risk, and that professional norms and public policies should encourage clear separation of interests of physicians from health plan organization and finance. PMID- 8637150 TI - Guns and violence. PMID- 8637149 TI - Franklin E. Zimring on law and firearms. PMID- 8637151 TI - Postal service tries to reverse violent image through employee assistance and team approach. PMID- 8637152 TI - Prediction of violence both art and science. PMID- 8637153 TI - Kids who kill: nature plus (lack of) nurture. PMID- 8637154 TI - Preparing for medical consequences of terrorism. PMID- 8637156 TI - Don't ignore patients' threats, psychiatrists told. PMID- 8637155 TI - Children and families need help after disasters. PMID- 8637157 TI - From the Centers for Disease Control and Prevention. Progress toward poliomyelitis eradication--India. PMID- 8637158 TI - The American public and the gun control debate. PMID- 8637159 TI - Firearms and fatalities. PMID- 8637160 TI - Firearms and fatalities. PMID- 8637161 TI - Firearms and fatalities. PMID- 8637162 TI - Clinical forensic medicine. PMID- 8637163 TI - Bone lead levels and delinquent behavior. PMID- 8637164 TI - Bone lead levels and delinquent behavior. PMID- 8637165 TI - Bone lead levels and delinquent behavior. PMID- 8637166 TI - Bone lead levels and delinquent behavior. PMID- 8637167 TI - Bone lead levels and delinquent behavior. PMID- 8637168 TI - Bone lead levels and delinquent behavior. PMID- 8637169 TI - School-associated violent deaths in the United States, 1992 to 1994. AB - OBJECTIVES: To conduct the first nationwide investigation of violent deaths associated with schools in the United States, to quantify the risk of school associated violent death, and to identify epidemiologic features of these deaths. DESIGN: Descriptive case series. SETTING: United States, July 1, 1992, through June 30, 1994. METHODS: School-associated violent deaths were identified by study collaborators and through 2 online news databases. Police reports, medical examiners' records, and interviews with police and school officials provided detailed information about each case. RESULTS: In a 2-year period, 105 school associated violent deaths were identified. The estimated incidence of school associated violent death was 0.09 per 100 000 student-years. Students in secondary schools, students of minority racial and ethnic backgrounds, and students in urban school districts had higher levels of risk. The deaths occurred in communities of all sizes in 25 different states. Homicide was the predominant cause of death (n = 85 [80.9%]), and firearms were responsible for a majority (n = 81 [77.1%]) of the deaths. Most victims were students (n = 76 [72.4%]). Both victims and offenders tended to be young (median ages, 16 and 17 years, respectively) and male (82.9% and 95.6%, respectively). Approximately equal numbers of deaths occurred inside school buildings (n = 31 [29.5%]), outdoors but on school property (n = 37 [35.2%]), and at off-campus locations while the victim was in transit to or from school (n = 37 [35.2%]). Equal numbers of deaths occurred during classes or other school activities (n = 46 [43.8%]) and before or after official school activities (n = 46 [43.8%]). CONCLUSIONS: School-associated violent deaths were more common than previously estimated. The epidemiologic features of these deaths were similar to those of homicides and suicides that occur elsewhere. A comprehensive approach that addresses violent injury and death among young people at school and elsewhere in the community is suggested. PMID- 8637170 TI - Hospitalizations for firearm-related injuries. A population-based study of 9562 patients. AB - OBJECTIVE: To determine the incidence, nature, demographics, severity, and hospital charges associated with inpatient treatment of firearm-related injuries. DESIGN: A retrospective, 1-year, population-based study of firearm-related hospitalizations based on the 1991 California Hospital Discharge Abstract Data Tapes. SETTING: California acute care hospitals that reported firearm-related discharges. PATIENTS: A total of 9562 patients discharged with firearm-related injuries. MAIN OUTCOME MEASURES: Per capita hospital discharge rates, according to age, race, and sex. RESULTS: A total of 9562 firearm-injured persons were discharged from California hospitals in 1991, representing a rate of 32 discharges per 100 000 population. Males aged 15 to 24 years accounted for 72% of the hospitalizations. For all causes of firearm-related injury, the highest age- and race-specific discharge rate was 439 per 100,000 for black persons aged 15 to 24 years. The highest county discharge rate was 55 per 100,000 for Los Angeles County. Statewide, there were 1.8 hospital discharges per firearm-related fatality (both in the hospital and in the community). Assaults accounted for 74% of cases. Among black males aged 15 to 24 years, assaults accounted for 598 discharges per 100 000 population. Hospital charges for 9193 patients exceeded $164 million; mean and median charges per patient discharged were $17,888 and $8535, respectively. Publicly financed health insurance programs sponsored 56% of patients; 25% had private insurance, and 19% were uninsured. Fifty-three percent of the discharges occurred at 13 of the 371 hospitals that discharged patients with firearm-related injuries. CONCLUSIONS: Firearm-related violence is a major cause of hospitalization of young urban black males and represents a significant cost to publicly financed health care. The impact on individual hospitals is highly disproportionate. While hospital discharge data can be used for population based surveillance of firearm-related trauma, there is need for improvement in local, state, and national surveillance of these injuries. PMID- 8637171 TI - Unintentional, nonfatal firearm-related injuries. A preventable public health burden. AB - OBJECTIVE: To describe the magnitude and characteristics of unintentional, nonfatal firearm-related injuries treated in US hospital emergency departments. DESIGN: Data were obtained from medical records for all firearm-related injury cases identified using the National Electronic Injury Surveillance System (NEISS) from June 1, 1992, through May 31, 1994. We report on cases classified as unintentional gunshot wounds. SETTING: NEISS comprises 91 hospitals that are a stratified probability sample of all hospitals in the United States and its territories that have at least 6 beds and provide 24-hour emergency service. MAIN OUTCOME MEASURES: Number of and population rates for unintentional, nonfatal firearm-related injuries. RESULTS: An estimated 34 485 (95% confidence interval [CI], 25 225-43 745) persons (6.7 per 100 000 population; 95% CI, 4.9-8.5) were treated for unintentional, nonfatal firearm-related injuries in US emergency departments during the 2-year study period. The majority of patients were male (87%) and aged 15 to 34 years (61%); 38% required hospitalization. Injuries were most often to an extremity (73%), were self-inflicted (70%), involved a handgun (57%), and resulted during common gun-related activities. CONCLUSIONS: Further development of effective interventions are needed to reduce the risk of injury from unintentional discharge of a firearm during routine gun-handling practices by those who own and use firearms. These injuries often occur during common gun related activities such as gun cleaning, loading/unloading, hunting, target shooting, and showing, handling, or carrying. Studies are needed to evaluate the efficacy of existing gun safety training courses and assess the potential role of various gun safety devices (eg, trigger locks and loading indicators) in future prevention strategies. PMID- 8637173 TI - The relationship between firearm design and firearm violence. Handguns in the 1990s. PMID- 8637172 TI - Population estimates of household firearm storage practices and firearm carrying in Oregon. AB - OBJECTIVES: To examine statewide data on exposure of adults and children to loaded and unlocked household firearms, and to estimate the prevalence of firearm carrying among adults in Oregon. DESIGN: Analyses of 1992 and 1993 telephone survey data from 6202 adults aged 18 years and older using the Oregon Behavioral Risk Factor Surveillance System. MAIN OUTCOME MEASURES: Number of adults and children exposed to household firearms always or sometimes stored loaded and unlocked. Adjusted odds ratios (ORs) for exposure to loaded and unlocked firearms and firearm carrying among adults by demographic and alcohol use patterns. RESULTS: Ten percent of adults (197 400 persons) lived in households with firearms that were always or sometimes stored loaded and unlocked. An estimated 6.2% of households with children had firearms that were loaded and unlocked, and about 40 000 children lived in these households. Overall, 4.4% of adults carried loaded firearms in the past month. Rural residence, male sex, and less than a college education were associated with living in a household with loaded and unlocked firearms and with firearm carrying. Drinking 5 or more alcoholic beverages on 1 or more occasions in the past month (OR, 1.7; 95% confidence interval, 1.3-2.3) or drinking 60 or more alcoholic beverages in the past month (OR, 1.8; 95% confidence interval, 1.2-2.7) were independently associated with living in households with loaded and unlocked firearms. CONCLUSIONS: Many adults and children are exposed to unsafely stored firearms in Oregon, and many adults carry loaded firearms. Improved public health surveillance of firearm storage and firearm carrying using standardized questions and definitions is needed at the national, state, and local levels. PMID- 8637174 TI - Gun acquisition and use by juvenile offenders. AB - OBJECTIVES: To learn how, when, where, and why juvenile offenders acquire guns. DESIGN: Following acquisition of informed consent, we conducted semistructured interviews between June and November 1995 with a convenience sample of 63 juvenile offenders aged 13 through 18 years, each of whom was incarcerated at a detention center in metropolitan Atlanta, Ga. SETTING: Five detention centers in metropolitan Atlanta. MAIN OUTCOME MEASURES: Frequency of handgun acquisition and use, age at and method of first handgun acquisition, feelings experienced when carrying guns, development of gun-carrying behavior, drug use, and gang membership. RESULTS: The mean age of respondents was 15.7 years. Forty-one male and 12 female respondents had owned a gun. Eighty-four percent of gun carriers acquired their first gun before the age of 15 years; more than half received their first gun passively, without any specific plan to do so. Adolescents who purposefully obtained their first handgun were more likely to become frequent or constant carriers. Forty percent felt safer and 40% said they felt more energized, excited, or powerful while carrying a gun. However, 34% reported increased anxiety about getting caught. Almost all stated that guns are readily available from a wide range of sources. CONCLUSION: Knowledge of the developmental patterns of gun carrying by delinquent adolescents could be useful in formulating effective strategies to reduce firearm violence. PMID- 8637175 TI - Effects of limiting handgun purchases on interstate transfer of firearms. AB - OBJECTIVE: To determine the effect of limiting handgun purchases to 1 per month on the illegal movement of firearms across state lines. DESIGN: Data from the Bureau of Alcohol, Tobacco, and Firearms firearms trace database were obtained for traces requested for firearms recovered in connection with criminal investigations. The analysis incorporates data on date and location of purchase for 14606 firearms purchased prior to (September 1989 through June 1993) and after (July 1993 through March 1995) enactment of a Virginia law limiting handgun purchases to 1 per month. MAIN OUTCOME MEASURES: Odds of tracing a firearm acquired prior to implementation of the law to Virginia vs another state in the Southeast compared with the odds for firearms acquired after the law took effect. RESULTS: For firearms recovered anywhere in the United States, 3201 (27%) of 11 876 acquired prior to the implementation of the law and 519 (19%) of 2730 purchased after the law was enacted were traced to Virginia (odds ratio [OR], 0.64; 95% confidence interval [CI], 0.58-0.71). For traces initiated in the northeast corridor (New York, New Jersey, Connecticut, Rhode Island, and Massachusetts), 1103 (34.8%) of 3169 of the firearms acquired before the 1-gun-a month law took effect and 142 (15.5%) of 919 firearms purchased after implementation were traced to Virginia (OR, 0.34; CI, 0.28-0.41). CONCLUSION: Gun control policies involving licensing, registration, and restricting the number of purchases represent efforts to limit the supply of guns available in the illegal market. This study provides evidence that restricting handgun purchases to 1 per month is an effective means of disrupting the illegal interstate transfer of firearms. PMID- 8637177 TI - Strategic thinking about gun markets and violence. PMID- 8637178 TI - A piece of my mind. Frozen in time. PMID- 8637176 TI - Nonfatal and fatal firearm injuries in a rural county. AB - OBJECTIVE: To measure the incidence of fatal and nonfatal firearm injuries in a rural setting. DESIGN: A prospective, population-based surveillance system using information from medical and legal records, newspapers, and members of the community. SETTING: A rural county in North Carolina. MAIN OUTCOME MEASURES: Incidence rates and case-fatality ratios (fatal:non-fatal). RESULTS: Of the 114 firearm injuries detected by the surveillance system from January 1, 1990, through December 31, 1991, nearly two thirds were nonfatal. The overall age adjusted annual incidence of firearm injuries was 66.4 per 100 000 population, and the incidence of nonfatal firearm injuries was 41.2 per 100 000 population. Subgroup annual incidence rates were greatest for African Americans. The overall case-fatality ratio was 1:1.8 (fatal:nonfatal). CONCLUSIONS: Incidence rates and case-fatality ratios for firearm injury in this rural setting were greater than expected, based on national estimates, perhaps because of greater proportions of rifle injuries and self-inflicted injuries. PMID- 8637180 TI - Neuropeptide-Y-ATP interactions at the vascular sympathetic neuroeffector junction. AB - Neuropeptide Y (NPY) and ATP are considered cotransmitters with norepinephrine (NE) in sympathetic neurons innervating some blood vessels, including those of the mesentery. A prominent action of NPY is to potentiate the postjunctional contractile effect of NE as well as that of other vasoactive agents. We wished to investigate whether NPY also potentiates the contractile effect of ATP and, if so, to determine which receptor subtype mediates such an effect. The effect of NPY, the NPY-Y1-selective agent Leu31Pro34 NPY, and the NPY-Y2-selective fragment NPY 14-36 on the increase in perfusion pressure produced by ATP was examined in rat perfused mesenteric arterial bed. Results demonstrated that both NPY and Leu31Pro34 NPY but not NPY 14-36 potentiated the increase in perfusion pressure produced by ATP. These results suggest that NPY acts on Y1 receptors to enhance the postjunctional response of ATP. The putative NPY antagonist PYX2, but not the putative antagonists benextramine or PYX1, attenuated the effect of NPY, indicating that PYX2 acts as an NPY antagonist in this system. A major action of NPY is to enhance the postjunctional response of both cotransmitters, ATP and NE at the vascular sympathetic neuroeffector junction in the mesenteric arterial bed, and this may be mediated by NPY-Y1 receptors. PMID- 8637179 TI - Potentiation of endothelium-dependent hyperpolarization to serotonin by dietary intake of NC 020, a defined fish oil, in the porcine coronary artery. AB - The membrane potential of vascular smooth muscle cells of the porcine coronary artery was measured to examine whether serotonin evokes endothelium-dependent hyperpolarization, and if it does, whether the electrical responses are modulated by the chronic dietary intake of NC 020, a defined fish oil. Serotonin induced transient, concentration-dependent hyperpolarizations of coronary arterial smooth muscle cells. The hyperpolarization was observed in tissues with, but not in those without endothelium. In coronary arteries obtained from pigs fed chronically with NC 020, serotonin induced significantly larger hyperpolarizations than those observed in control arteries. These results suggest that endothelium-dependent hyperpolarization may contribute to the endothelium dependent relaxation evoked by serotonin and to its potentiation by the dietary intake of fish oil (NC 020). PMID- 8637181 TI - Effects of losartan on contractile responses of conductance and resistance arteries from rats. AB - We investigated the selectivity of losartan as an angiotensin II (ANG II) antagonist in contractile experiments using segments of small mesenteric arteries and rings of aorta from rat. The concentration-effect curve of ANG II was not different in mesenteric arteries with an without endothelium. In both resistance and conductance vessels, it was shifted toward larger concentrations by losartan (3 nM) with similar apparent inhibition constant (KB) values: 4.1 +/- 1.8 nM (n = 6) in small mesenteric arteries and 1.9 nM (n = 6) in aorta. These values agree with the known affinity of losartan for AT1 receptors. At 1 microM, the AT2 selective ligand CGP 42112A had no effect on contractile responses induced by norepinephrine (NE), serotonin, or neuropeptide Y (NPY). However, it inhibited vasoconstriction elicited by prostaglandin F2 alpha (PGF2 alpha). This latter effect was also noted in the aorta. Similarly, losartan also competitively antagonized aortic contractile responses elicited by U 46619, a thromboxane A2 analogue (TXA2), with a pA2 value of 5.7. Two losartan analogues, DuP 532 and EXP 3174 (a metabolite of losartan), < or = 30 microM, did not antagonize U 46619, showing structural requirements for this antagonistic action of losartan. We conclude that in both rat resistance and conductance vessels, ANG II induces vasoconstriction through activation of AT1 receptors which are selectively blocked by losartan at nanomolar concentrations and that at micromolar concentrations, losartan may also block the vascular TXA2/PGF2 alpha (TP) receptor. PMID- 8637182 TI - Effect of doxorubicin on postrest contraction in isolated rat hearts. AB - Clinical use of doxorubicin is limited by its cardiotoxicity. In doxorubicin induced cardiomyopathy, vacuolization of the sarcoplasmic reticulum (SR) has been reported. We investigated whether doxorubicin had a direct action on the sarcoplasmic reticulum (SR) in isolated perfused rat hearts. The left and right atria were trimmed to maintain heart rate (HR) <200 beats/min. Postrest contractions, which are believed to be due primarily to Ca2+ release from the SR, were evoked with a programmable stimulator after variable rest intervals. The amplitude of the postrest contractions increased markedly as the rest interval increased. Doxorubicin (0.1 mM) significantly suppressed this potentiation of the postrest contractions. Furthermore, doxorubicin slowly induced aftercontractions and an increase in left ventricular diastolic pressure (LVDP), phenomena that are usually associated with ouabain intoxication. We conclude that doxorubicin induced cardiomyopathy may be due to SR dysfunction, leading to intracellular Ca2+ overload. PMID- 8637183 TI - Protective effect of K(ATP) openers in ischemic rat hearts treated with a potassium cardioplegic solution. AB - ATP-sensitive potassium channel (KATP) openers directly protect ischemic myocardium, which may make them useful for treating patients undergoing cardiopulmonary bypass, but whether high-potassium-containing cardioplegic solutions would inhibit their protective effects is not clear. We determined whether additional protection greater than that provided by cardioplegia could be found for KATP openers. We studied the effect of 10 microM cromakalim or BMS 180448 pretreatment (10 min before cardioplegia) on severity of ischemia in isolated rat hearts given normothermic or cold St. Thomas' cardioplegic solution (16 mM K+). After cardioplegic arrest, the hearts were subjected to 30-min (normothermic) or 150-min (hypothermic) global ischemia, each followed by 30-min reperfusion. The cardioplegic solutions significantly protected the hearts, as measured by increased time to onset of contracture, enhanced recovery of function, and reduced lactate dehydrogenase (LDH) release. Cromakalim and BMS 180448 both further significantly increased time to contracture in both normothermic and hypothermic arrested hearts; this was accompanied by enhanced recovery of reperfusion contractile function and reduced cumulative LDH release. This additional protective effect of the K ATP openers was abolished by glyburide. Because administration of the K ATP openers only with the cardioplegic solution (1 min before global ischemia) was not efficacious, >1-min pretreatment apparently is necessary. K ATP openers provide additional protection to that afforded by cold or normothermic potassium cardioplegia in rat heart, although the timing of treatment may be crucial. PMID- 8637184 TI - Ionotropic mechanisms involved in postsynaptic inhibition by the endothelins of ganglionic transmission in dog cardiac sympathetic ganglia. AB - We investigated the effects of endothelin-1 and endothelin-3 (ET-1, ET-3) on the ganglionic transmission of cardiac sympathetic ganglia in vivo by the direct administration of agents to the ganglia through the right subclavian artery while monitoring the heart rate (HR) as an indicator of the ganglionic function in spinal dogs. The positive chronotropic responses to dimethylphenylpiperazinium (DMPP) and McN-A-343 administered to the ganglia were similarly inhibited by ET-1 (0.05-0.2 microg) and ET-3 (0.5-2 microg), but ET-1 was approximately 10 times more potent than ET-3. The inhibition induced by ETs was antagonized by endothelin ETA receptor antagonist BQ-123 (20 microg). This inhibition was unaffected by pretreatment with indomethacin given intravenously (i.v.), ruling out the possible involvement of endogenous prostaglandins production. The voltage sensitive Ca2+ channel antagonist nifedipine had no effect on inhibition. However, the inhibition was antagonized by pretreatment with the low conductance Ca2+-activated potassium channel antagonists, such as apamin (20 microg intraarterially, i.a.), scyllatoxin (10 mug i.a.) and D-tubocurarine (0.6 mg i.a.). On the other hand, the voltage-sensitive K+ channel antagonist 4 aminopyridine (4-AP), ATP-dependent K+ channel antagonist, glibenclamide, and high-conductance Ca2+-activated K+ channel antagonists iberiotoxin and charybdotoxin failed to affect the inhibition by ETs. The results suggest that ETs inhibit the nicotinic and muscarinic ganglionic transmission through the ETA receptor-operated low-conductance Ca2+-activated potassium channel at postganglionic sites. PMID- 8637185 TI - Effects of genetic hyperinsulinaemia on vascular reactivity, blood pressure, and renal structure in the Zucker rat. AB - The association between insulin resistance, obesity, and hypertension is well recognised. We examined the hypothesis that hypertension in the obese Zucker rat is related to changes in vascular reactivity. Systolic blood pressure (SBP) in conscious Zucker rats was significantly greater in obese as compared with lean animals (157 +/- 9 and 117 +/- 8 mm Hg). Obese animals also had marked proteinuria and reduced urinary creatinine excretion in 24 h as compared with their lean counterparts. The reactivity of isolated aorta to phenylephrine (PE) and 5-hydroxy-tryptamine (5-HT) was modestly (twofold) increased in obese animals (EC50 13.8 nM as compared with 29.4 nM in lean animals and 0.19 nM as compared with 0.46 nM in lean animals, respectively). In the perfused mesenteric vascular bed, basal perfusion pressure was the same in both phenotypes, as was the pressor response to PE and depressor response to acetylcholine (ACh) and sodium nitroprusside (SNP). In the isolated aorta, from obese animals, insulin attenuated the contractile response to PE but markedly enhanced the vasoconstrictor potency of 5-HT. It had no significant effect on pressor or depressor responses in the perfused mesenteric bed. The data suggest that increased reactivity of central arteries to spasmogenic agents may be involved in the development of systolic hypertension in the hyperinsulinaemic Zucker rat. PMID- 8637186 TI - Somatostatin-induced contraction of human isolated saphenous vein involves sst2 receptor-mediated activation of L-type calcium channels. AB - A range of somatostatin (SRIF) analogues have been used to characterize the SRIF receptor-mediating contraction of the human saphenous vein. SRIF produced concentration-dependent contractions with an EC50 value of approximately 20 nM. The peptidase inhibitors phosphoramidon and amastatin did not alter the potency of SRIF. The sst2 receptor-selective peptide BIM-23027 was approximately three times more potent than SRIF in contracting the vein, whereas the sst5 receptor selective peptide L-362855 was approximately 50 times weaker. The sst3 receptor selective peptide BIM-23056 did not contract the saphenous vein. Contractions to SRIF were not antagonised by the putative SRIF receptor blocker cyclo(7 aminoheptanoyl-Phe-D Trp-Lys-Thr[Bzl]) (CPP), phentolamine, or indomethacin. Decreasing the external calcium concentration reduced the maximum contraction to SRIF in a concentration-dependent manner without altering the EC50 value. Nifedipine and verapamil also markedly reduced the SRIF-induced contraction. SRIF and several SRIF analogues caused contraction of the human saphenous vein by what appeared to be a direct effect on the smooth muscle. Their relative potencies suggest that their effects were mediated by a somatostatin receptor that is like the recombinant sst2 receptor. The receptor transduction mechanism appears to involve activation of L-type calcium channels and entry of extracellular calcium. PMID- 8637187 TI - Assessment of the cytotoxicity of the photosensitizing drug BPD verteporfin using human vascular smooth muscle cells in culture. AB - Photosensitizing drugs are selectively taken up by lipid-rich lesions such as atheromatous plaque which when exposed to light render the drugs cytotoxic. However, skin photosensitivity which persists for many weeks is a significant side effect. We investigated the cytotoxicity of a new photosensitizing drug, the benzoporphyrin derivative BPD verteporfin (Quadra Logic Technologies), which does not have this deleterious side effect. Vascular smooth muscle cells (VSMC) from normal human mammary and diseased human coronary arteries were grown in culture from explants and characterized with respect to their growth rates. The sensitivity to BPD with and without light was assessed by measuring viability after treatment. The lethal dose of drug for 50% viability loss (LD50) for BPD with light was approximately 12.5 ng/ml for mammary artery, with 52 +/- 8% cell survival (n = 6). The coronary artery VSMC from all patient sources, although differing significantly in growth rate, had a survival of 44 +/- 6% (n = 12) at the same concentration of BPD used for the mammary artery SMC (p = NS). Our results established the LD50 for BPD using human arterial sources of SMC and showed that the growth rates of the cells did not affect the cytotoxicity of the drug. PMID- 8637188 TI - Ambulatory blood pressure and left ventricular changes during antihypertensive treatment: perindopril versus isradipine. AB - Using digitized M-mode echocardiograms and 24-h ambulatory blood pressure (BP) monitoring, we compared the effects on left ventricle (LV) and BP of 6-month treatment with a calcium antagonist or an angiotensin-converting enzyme (ACE) inhibitor in 36 hypertensive patients with LV hypertrophy (group 1, 18 subjects treated with sustained-release isradipine; group 2, 18 subjects treated with perindopril). At the basal evaluation, the two groups had comparable BP and LV parameters. After treatment, both groups showed a similar and significant reduction in 24-h, day- and night-systolic and diastolic BP (SBP, DBP). The reduction in LV mass index was greater (p < 0.01) in group 2. In group 1, percentage of decrease of LV mass correlated significantly with percentage of decrease in 24-h and daytime BP; this was not true of group 2. Together with the reduction in LV hypertrophy, there was a significant increase of peak lengthening rate of LV diameter that was greater (p < 0.01) in group 1. Both drugs can reduce LV hypertrophy and improve diastolic function. The reduction of hypertrophy induced by perindopril appears to be partly independent of BP decrease and therefore partly related to a direct action of perindopril on the myocardium. PMID- 8637189 TI - Developmental differences in distribution of cyclic nucleotide phosphodiesterase isoforms in cardiomyocytes and the ventricular tissue from newborn and adult rats. AB - We characterized cyclic nucleotide phosphodiesterase (PDE) activities in neonatal rat cardiomyocytes and in whole ventricle from newborn and adult rats, to determine whether cyclic nucleotide hydrolytic activities might be influenced by the developmental stage of the animal or by cell isolation and culture procedures. Using anion-exchange high-performance liquid chromatography (HPLC), we obtained four soluble PDE activities from adult rat ventricle. Peak 1, a cyclic GMP-specific PDE was stimulated by Ca2+/calmodulin; peak 2 hydrolyzed cyclic AMP and cyclic GMP, the addition of cyclic GMP stimulating the hydrolysis of cyclic AMP. Peaks 3 and 4 selectively hydrolyzed cyclic AMP, peak 3 being very sensitive to inhibition by rolipram and peak 4 consisting of two different enzyme activities, one inhibited by cyclic GMP (peak 4b) and the other inhibited by rolipram (peak 4a). In cardiomyocytes and whole ventricle from newborn rats, the response of the two cyclic GMP-sensitive PDE isoforms (cyclic GMP-stimulated and cyclic GMP-inhibited PDE) to the effector cyclic GMP was markedly reduced as compared with that of the corresponding isoforms (peaks 2 and 4b) present in the heart ventricle of adult rats. The other peaks were analogous to peaks 1, 3, and 4a of the whole ventricle. The profile of PDE activities in nonmyocardial cells did not differ from that of myocytes. Therefore, the lack of cyclic GMP effects was not due to culture conditions. Differences in PDE activities observed between cardiomyocytes from newborn rats and ventricle from adult rats might be age dependent. PMID- 8637190 TI - Contractile responses to sumatriptan in isolated bovine pulmonary artery rings: relationship to tone and cyclic nucleotide levels. AB - We examined responses to the 5-hydroxytryptamine 1D (5-HT1D)-receptor agonist sumatriptan in bovine pulmonary artery rings (2-3 mm ID). The effects of agonist induced tone and agents that alter intracellular cyclic AMP [cyclic AMP]i or [cyclic GMP]i on responses to sumatriptan were investigated. At resting tension, responses to sumatriptan were slight or not evident. In the presence of tone induced by U46619, responses to sumatriptan (1 nM-30 mM) were greatly potentiated, as were responses to the alpha2-adrenoceptor agonist UK14304. Responses to the alpha 1-adrenoceptor agonist phenylephrine (PE) were potentiated only slightly. In the presence of U46619, addition of the adenylyl cyclase activator, forskolin (1 nM-0.1 microM or isoprenaline (ISO 1 microM) induced relaxations and increases in [cyclic AMP]i and resulted in further potentiation of the contractile response to sumatriptan. Addition of 0.1 microM sodium nitroprusside (SNP) inhibited sumatriptan-induced contractions. Whereas sumatriptan alone did not significantly affect [cyclic AMP]i, in the presence of U46619 it decreased [cyclic AMP]i. This effect of sumatriptan was further enhanced in the presence of forskolin. Sumatriptan increased [cyclic GMP]i. Using a nitric oxide (NO) synthase inhibitor and vessels denuded of endothelium, we showed that the increased [cyclic GMP]i in response to sumatriptan was endothelium-dependent and mediated by NO. This increase in [cyclic GMP]i was not observed in the presence of U46619. By measuring cyclic AMP and cyclic GMP phosphodiesterase (PDE) levels, we demonstrated that the point of "cross-talk" between cyclic nucleotides may not be at the level of total PDE activity. These results highlight the important role of [cyclic AMP], [cyclic GMP]i, and endothelium function in the control of 5-HT1D receptor-mediated vasoconstriction, which is dependent on a decrease in [cyclic AMP]i in the absence of an increase in [cyclic GMP]i. PMID- 8637191 TI - Terfenadine blocks time-dependent Ca2+, Na+, and K+ channels in guinea pig ventricular myocytes. AB - Terfenadine, which blocks delayed rectifier K+ channels (Ik), is structurally related to diphenylalkylamine L-type Ca2+ channel (ICa) blockers and has been reported to render Purkinje fibers inexcitable. We used standard whole-cell patch clamp techniques in isolated guinea pig ventricular myocytes to investigate the direct effect of terfenadine on ICa after discovering that the upstrokes of early afterdepolarizations in guinea pig myocytes were inhibited by the drug at concentrations > or = 10(-6)M. Some data analyzing the effect of terfenadine on time-dependent Na+ channels (INa) and IK also were obtained. All experiments were controlled for time of intracellular dialysis. Terfenadine (3 x 10(-6)M) reduced peak ICa (measured in either K+-containing or Cs+-substituted intracellular solutions from holding potentials of -40 mV) after 10 min exposure [peak at 0 mV in K+-deficient dialysis solution -4.2 +/- 2.3 pA/pF (mean +/- SD, n = 5) versus 13.02 +/- 4.33 pA/pF in control solution (n = 5), p < 0.01], and ICa was almost completely blocked after 15 min drug exposure. Ten minutes of exposure to terfenadine (3 x 10-6M) also caused near-complete blockade of peak INa when INa was measured at -40 mV after 300 ms conditioning pulses from a holding potential of -40 to potentials between -60 and -90 mV. The effect was much less pronounced when INa was measured from a holding potential of -90 mV. After exposure to terfenadine 3 x 10 (-6)M, IK density, measured as peak tail current at -40 mV after 300-ms depolarizations, was also reduced but not eliminated at membrane potentials between -20 and +60 mV. In contrast, exposure to terfenadine caused no significant change in the current-voltage relationship after 300-ms steps from 90 to +60 mV. Terfenadine had no effect on time constants of decay of IK or ICa. These results suggest that terfenadine blocks several time- and voltage-dependent channels, possibly by binding to a common protein structure, not related to ion selectivity, that is primarily associated with time-dependent activation of channel conductance. PMID- 8637192 TI - Myocardial release of endothelin (ET) and enhanced ET(A) receptor-mediated coronary vasoconstriction after coronary thrombosis and thrombolysis in pigs. AB - We investigated changes in vascular reactivity to endothelin (ET) and local release of ET-like immunoreactivity (ET-LI) induced by myocardial ischemia and reperfusion in a pig model of coronary thrombosis and thrombolysis and studied the possible mechanisms producing the changed vascular reactivity to ET-1. We induced coronary thrombosis by inserting a copper coil into the left anterior descending coronary artery (LAD) and achieved thrombolysis with tissue plasminogen activator (t-PA). Vascular reactivity to ET-1 in the nonischemic and ischemic/reperfused LAD diagonal branches was evaluated in vitro. ET-LI was analyzed in plasma from the great cardiac vein and aorta for estimation of local release. The vasoconstrictor response to ET-1 was enhanced twofold (p < 0.01) in the ischemic/reperfused arteries as compared with the nonischemic arteries. The vasoconstriction induced by the ETB receptor agonist [Ala 1,3,11,15] ET-1 or serotonin was not significantly affected by ischemia/reperfusion. The ETA receptor antagonist BQ-123 reversed the ET-1-induced vascular contraction to a similar degree in ischemic/reperfused and control arteries. The ET-1-induced vasoconstriction of control arteries was not affected by inhibition of nitric oxide (NO) synthase with NG-nitro-L-arginine (L-NNA) or cyclooxygenase with indomethacin. During reperfusion, the myocardial venoarterial plasma concentration difference of ET-LI and blood flow increased, resulting in an increased overflow of ET-LI. Our results demonstrate that coronary thrombosis and thrombolysis evokes enhanced local release of ET-LI during the reperfusion period and increases the vasoconstrictor effects of ET-1 through a mechanism related to ETA receptor activation but unrelated to altered endothelial function. These changes may play a role in the development of ischemic/reperfusion injury and no reflow phenomenon. PMID- 8637193 TI - Hemodynamic comparison of dopexamine and nitroprusside at high and low doses in patients with coronary artery disease and impaired left ventricular function. AB - Pure vasodilator drugs are currently the preferred agents for treatment of acute and chronic heart failure of all grades of severity. In contrast, the role of drugs that combine vasodilation with inotropic action remains highly controversial despite their several advantageous physiological actions and long therapeutic history in heart failure. We hypothesize that this uncertainty might be due first to subtle unfavorable hemodynamic effects not detectable by the relatively crude hemodynamic methods by which these agents are usually analyzed, particularly with regard to the quantification of afterload, and secondly to the narrow therapeutic range of these drugs, such that the dose administered is critical. We tested this hypothesis by comparing several refined hemodynamic measurements of dopexamine, an inotropic vasodilator, with a pure arteriovenous dilator (sodium nitroprusside, SNP) on left ventricular (LV) systolic and diastolic function, large arterial behavior, and coupling of the left ventricle to the arterial system at two dose levels in 35 patients with ischemic heart disease. The study protocol was a fixed order of 15-min infusions of saline, dopexamine 1 microg/kg/min, and dopexamine 3 + ++microg/kg/min, or saline, SNP 1 microg/kg/min, and SNP 3 microg/kg/min. Detailed hemodynamic observations were made at the end of each 15-min infusion period. Both drugs produced equivalent arterial vasodilation, as measured by the decrease in systemic vascular resistance index (SVRI), but dopexamine resulted in a significantly greater increase in cardiac index (CI). Myocardial contractility, assessed by several load-independent indexes, increased with dopexamine, as anticipated with an inotropic drug, but did not alter with SNP. Arterial compliance, a measure of the distensibility of large conduit arteries, was increased by SNP but not by dopexamine. Arterial wave reflection was increased by dopexamine, especially at high doses, but reduced by SNP. Increased arterial compliance and reduced wave reflection reduce LV afterload. SNP reduced preload, whereas dopexamine had no effect on this aspect of ventricular function. Vasodilator drugs and those which combine vasodilator and inotropy increase cardiac output (CO) and reduce SVR. Therapy with inotropic vasodilators has no effect on preload and does not reduce the dynamic components of ventricular afterload, although it does reduce its static components. These effects are dose dependent; there is less perturbation of afterload at lower doses. In contrast, vasodilator therapy reduces preload and both static and dynamic parts of afterload. PMID- 8637194 TI - Actions of 1,4-dihydropyridines in isolated mesenteric vascular beds. AB - Recent studies indicate that the vasorelaxation induced by nitrendipine may be mediated partially by increased release of nitric oxide (NO). To study this effect in more detail and to examine the vasodilating effect of other 1,4 dihydropyridines (1,4-DHP) with regard to a possible involvement of NO, we investigated the effects of nitrendipine, nifedipine, nisoldipine, and nimodipine on isolated mesenteric vascular bed an the influence of L-NG-nitroarginine (L NNA) on 1,4-DHP-induced vasorelaxation. Perfusion with these 1,4-DHP resulted in a concentration-dependent increase in global flow and vascular diameter in all vessel branches. Nifedipine exhibited a more pronounced effect on G4 vessels, whereas the actions of the other 1,4-DHP on the investigated vascular tree were more homogeneous. The dilating and flow increasing effects of nitrendipine and nifedipine could be significantly antagonized by treatment with LNNA. The vasodilating effects of nisoldipine and nimodipine could also be antagonized with L-NNA. We conclude that NO release plays an additional role in the relaxation of small resistance vessels by 1,4-DHP. PMID- 8637195 TI - Gender differences in atherosclerosis: possible role of nitric oxide. AB - The mechanism by which women in the reproductive age group are protected from developing coronary heart disease (CHD) as compared with men of similar age is not known. To elucidate whether there is a gender difference in the rate of atherosclerosis formation, we investigated the rate of development of atherosclerosis in both male and female rabbits fed an identical diet consisting of 2% cholesterol for 10 and 15 weeks. The extent of atherosclerosis was correlated with the amount of basal and stimulated release of nitric oxide (NO) from endothelium-intact aortic rings obtained from these animals. Under identical dietary conditions, the female rabbits fed a high cholesterol diet (HCD) for 10 weeks developed very little atherosclerosis (10% surface involvement) as compared with male rabbits (42% surface involvement). However, no significant gender differences in atherosclerosis were observed after 15 weeks of the HCD. The serum cholesterol, high and low density lipoprotein (HDL and LDL) cholesterol were similar in animals fed the HCD for 10 and 15 weeks. The basal release of NO from endothelium-intact aortic rings was significantly greater in control females as compared with males. The magnitude of endothelium-dependent relaxation of aortic rings obtained from both male and female rabbits fed the HCD were impaired to a similar extent, and this impairment correlated with the duration of hyperlipidemia but not with the extent of atherosclerosis. The arginine content of aortic rings were not different between males (257 +/- 52 nmol/g wet weight) and females (345 +/- 62 nmol/g wet weight) or between control and hyperlipidemic groups (males 312 +/- 69; females 301 +/- 65 nmol/g wet weight). Although the precise mechanism for the slower rate of development of atherosclerosis in the female rabbits as compared with males is not clear, the greater basal release of NO in females before they were fed a hyperlipidemic diet, as well as other factors, may be involved. The impairment of endothelium-dependent relaxation in hyperlipidemic animals is not due to a decrease in the availability of arginine, the substrate for NO. PMID- 8637196 TI - Investigation of the mechanism by which ketanserin prolongs the duration of the cardiac action potential. AB - Action potential duration (APD) lengthening is believed to underlie the cardiac arrhythmogenicity of ketanserin, a serotonin (5-HT)2A/2C receptor antagonist. We wished to determine (a) whether this activity involves blockade of 5-HT2A/2C receptors and (b) the precise mechanism of ketanserin-induced APD prolongation. APs were recorded in guinea pig isolated papillary muscles by conventional "floating" microelectrodes, and potassium currents in guinea pig isolated myocytes were recorded in the whole-cell configuration. Ketanserin (1-10 microM) increased APD (EC50 value for enhancing APD at 90% repolarization (APD90) 3.1 +/- 2.7 microM, n = 24), without affecting resting potential, maximum upstroke velocity (Vmax) or AP amplitude (APA). Pirenperone (10 microM), a ketanserin congener, similarly increased APD90 from 204 +/- 3 to 241 +/- 7 ms (p < 0.001, n = 6). No increase in APD was observed, however, with ritanserin or ICI 170809, even at high concentrations (10 microM, n = 6, respectively), two 5-HT2A/2C receptor antagonists chemically distinct from ketanserin, thereby excluding the involvement of 5-HT2A/2C receptors in mediating APD lengthening. That APD prolongation was mediated specifically by the benzolyl-piperidine moiety of ketanserin and pirenperone was confirmed by 1-propyl-4(4-fluorobenzoyl)piperidine (PFBP), which evoked APD lengthening effects remarkably similar to those produced by ketanserin and pirenperone (EC50 3.73 +/- 2.6 microM, n = 12). In isolated cardiomyocytes, ketanserin (1-32 microM) selectively and concentration dependently reduced the IKr component of the delayed outward current (IK) without affecting the inward rectifier current, IK1. Thus, ketanserin (32 microM) significantly reduced IK at a potential value of -20 mV from 813 +/- 65 to 569 +/ 55 pA (p < 0.001, n = 6), whereas at a potential value of -110 mV, IK1 was not significantly affected (730 +/- 103 vs. 603 +/- 143 pA, respectively; n=6). The results demonstrate that APD is prolonged by ketanserin and congeners but not be chemically different 5-HT2A/2C receptor antagonists. The benzoyl-piperidine moiety appears to mediate the APD-prolonging effects of ketanserin and pirenperone specifically. Furthermore, ketanserin-induced APD lengthening does not appear to involve 5-HT2A/2C receptors but is consecutive to direct blockade of myocardial potassium channels. PMID- 8637197 TI - Cardiovascular interactions characterizing the vasodilator components of nicorandil in conscious dogs. AB - We conducted cardiovascular interaction studies in conscious dogs to characterize the cyclic GMP and KATP channel opener (PCO)-dependent peripheral vasodilator components of nicorandil (NIC). Intravenously (i.v.) administered NIC (6-24 microg/kg/min for 6 h) reduced arterial blood pressure (BP) and increased heart rate (HR), and 15 mug/kg/min NIC was simulated by combining the cyclic GMP vasodilator nitroglycerin (NTG 1-5 microg/kg/min i.v.) with the PCO minoxidil (MNX 0.23 microg/kg i.v.). Tolerance to NTG-induced vasodilation was accelerated by oral isosorbide dinitrate (ISD 400 mg/2.5 days), and MNX was antagonized by the KATP blocker U-37883A (6 microg/kg/min i.v.). U-37883A attenuated NIC-induced hypotension with a mild tachycardia, whereas chronic ISD attenuated and delayed NIC-induced hypotension, with a pronounced tachycardia. Chronic ISD plus U-37883A completely blocked NIC-induced hypotension, although NIC-induced tachycardia persisted. These studies demonstrate that the cyclic GMP component of NIC induces a minimally tachycardiac acute vasodilation at lower drug concentrations resistant to K ATP blockade but cross-tolerant to ISD. Conversely, the PCO component of NIC induces a more tachycardiac vasodilation at higher concentrations free of ISD cross-tolerance but sensitive to KATP blockade. Therefore, low-dose NIC free of adjunctive nitrates should optimize its cyclic GMP vasodilator component for clinical indications. PMID- 8637198 TI - Effects of pulmonary hypertension on vasoconstrictor responses to endothelin-1 and sarafotoxin S6C and on inherent tone in rat pulmonary arteries. AB - Vasoconstrictor responses to endothelin-1 (ET-1) and the ETB receptor agonist sarafotoxin S6c (SXS6c) were investigated in the main pulmonary artery and pulmonary artery branch removed from rats previously exposed to 10% O2 [chronic hypoxic (CH) rats] or room air (control rats) for 2 weeks. The effects of nitric oxide synthase (NOS) inhibition with L-Nomega-nitroarginine methyl ester (L-NAME) (100 microM) on ET receptor-induced responses in these arteries were also investigated. In control rats, in rings of main pulmonary arteries and pulmonary artery branches. ET-1 induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were antagonized by the ETA receptor antagonist FR 139317 whereas SXS6c did not vasoconstrict. Chronic hypoxia had no effect on the sensitivity of the main pulmonary arteries to ET-1, whereas small vasoconstrictor responses to SCS6c were evident. ET-1 was more potent in the CH rat pulmonary artery branches than in controls. SXS6c also caused vasoconstriction with a maximum response 30% of that to ET-1 in both endothelium intact and endothelium-denuded vessels. L-NAME increased the sensitivity to ET-1 in the CH rat main pulmonary arteries and increased the responses to low concentrations of ET-1 in the control rat main pulmonary arteries but did not affect any ET-1 responses in any other vessels. It did disclose responses to SXS6c in control rat main pulmonary arteries. L-NAME itself increased vascular tone to a greater extent in CH rat pulmonary arteries than in controls. In preconstricted pulmonary arteries, however, relaxations to acetylcholine (ACh) were diminished in the CH rats as compared with their controls. All pulmonary artery branches, denuded of their vascular endothelium, relaxed to sodium nitroprusside (SNP) and therefore exhibited endogenous vascular tone. This effect was greatest in the pulmonary artery branches from the CH rats. The results suggest that rat large pulmonary artery responses to ET-1 are normally mediated by ETA receptors. Pulmonary hypertension can potentiate ETA receptor-mediated vasoconstriction and facilitate ETB receptor-mediated vasoconstriction. Endogenous NO may normally suppress ETA receptor-mediated responses in rat main pulmonary arteries. Rat pulmonary arteries exhibit endogenous tone, which is increased by exposure to chronic hypoxia. PMID- 8637200 TI - Acetazolamide-induced vasodilation in the carotid vascular bed in healthy volunteers. AB - Acetazolamide (ACTZ) vasodilating properties are used for the assessment of cerebral vasodilatory reserve not only in cerebral pathology investigation, but also in clinical pharmacology studies. However, the kinetics of these vasodilating properties are not clearly established; moreover, the cerebral selectivity of ACTZ-induced vasodilation has not been demonstrated. To address these issues, we performed an ACTZ test in 9 healthy volunteers and measured noninvasively the effects exerted by the drug simultaneously on common carotid artery blood flow (CCABF), middle cerebral artery mean blood flow (BF) velocity (MV) (transcranial Doppler technique), and facial cutaneous BF (CutBF). The time of the peak increase in MV was variable, occurring between 20 and 40 min after ACTZ, and brief. In addition, simultaneous increases (with variable mean peak times) occurred in flows in the CCA (+11% at 20 min) and in its intracranial (+25% at 30 min) and extracranial (+134% at 50 min) branches. The first finding suggests that the cerebral effects of ACTZ should be measured repeatedly to detect in each individual subject the time of MV peak increase; the transcranial Doppler is particularly suitable for such an approach. The second finding suggests that, especially in patients, extracranial actions of ACTZ must be taken into account for estimation of the cerebral vasodilatory reserve. PMID- 8637199 TI - Mechanical and electrophysiological effects of preconditioning in isolated ischemic/reperfused rat hearts. AB - Changes in action potential duration (APD) were studied during ischemic/reperfusion injury preceded or not by preconditioning in isolated rat hearts. Hearts were perfused on a Langendorff apparatus with Krebs-Henseleit carbonate buffer and submitted to 25-min global low-flow ischemia (coronary flow, 0.3 mol x min-1) followed by 30-min reperfusion. In hearts that had been preconditioned, two intermittent periods of total ischemia for 5 min each, separated by 5-min reflow, were performed before low-flow ischemia. At the end of the ischemic period, APs were significantly prolonged in nonpreconditioned hearts; this prolongation was abolished by preconditioning. Moreover, preconditioning increased the recovery of the contractile function. Therefore, ischemia can widen APD. The results also showed that in rats, preconditioning can be produced in a manner qualitatively similar to preconditioning in other species. Verapamil (3 x 10(-9) mol x min(-1)) or 4-aminopyridine (4-AP, 3 x 10( 6) mol x min(-1)) applied exclusively during low-flow ischemia significantly improved postischemic contractile function in nonpreconditioned hearts (25.9 +/- 4.4. and 37.9 +/- 2.4 vs. 12.9 +/- 5.3%, respectively) as well as in preconditioned hearts (61.8 +/- 4.2 and 55.5 +/- 4.7 vs. 36.0 +/- 1.4%, respectively). With verapamil, this protection was associated with a decrease in APD at 90% of repolarization in the nonpreconditioned hearts (APD90 32.2 +/- 0.1 vs. 71.1 +/- 6.7 ms at the end of ischemia). With 4-AP, this same protection was associated with an increase in APD in the preconditioned hearts (APD90 67.7 +/- 0.7 vs. 48.5 +/- 2.6 ms at the end of ischemia). Both agents given during a 25 min ischemic challenge improved myocardial recovery in nonpreconditioned and preconditioned hearts, despite discordant effects on the AP. Furthermore, the action of these agents was cumulative with the effect of preconditioning. PMID- 8637201 TI - Myth and reality in minimal access oncologic surgical management. PMID- 8637203 TI - Estrogen and progesterone receptor status determined by the Ventana ES 320 automated immunohistochemical stainer and the CAS 200 image analyzer in 236 early stage breast carcinomas: prognostic significance. AB - The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r=0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P=0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P=0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P=0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stroma-rich tumors, and early-stage lesions including intraductal carcinoma. PMID- 8637202 TI - Prognostic factors in angiosarcoma: a multivariate analysis of 55 cases. AB - Data for prognostic factors in angiosarcoma (AS) are limited, prompting a large scale study of AS with multivariate analysis. To analyze prognostic factors in angiosarcoma (AS), clinical and histologic findings in 55 patients collected from hospitals in Japan were reviewed. Prognostic factors were evaluated by univariate and multivariate Cox's proportional hazards models. The study involved 32 males and 23 females, ages 18-93 (median, 69) years. The primary sites of tumors included head and neck (32 cases), trunk (10), extremities (3), spleen (3), breast (3), and other (4). The overall 2-year survival rate was 21%. Univariate analysis of clinical factors including age, sex, size and depth of tumor, tumor related symptoms, interval between onset of symptoms and admission, surgical procedures, adjuvant chemotherapy, and adjuvant radiotherapy showed that age, tumor size, and mode of treatment were significant for survival. Histologic factors analyzed were mitotic counts, cellularity, cellular pleomorphism, extent of necrosis, vascular differentiation, and nonspecific diagnosis. Only mitotic counts were significant for prognosis. Multivariate analysis on these four factors revealed that tumor size, mode of treatment, and mitotic counts were independent prognostic factors. PMID- 8637204 TI - Analysis of variables affecting wound healing after musculoskeletal sarcoma resections. AB - Adjuvant treatment modalities, nutritional parameters, and allogenic blood transfusions are investigated as possible contributing variables which may adversely affect wound healing after tumor resections for musculoskeletal sarcomas. Statistical analysis determined that preoperative chemotherapy, depressed preoperative hematocrit, and allogenic blood transfusions (probably immunosuppression mediated) were the only significant factors affecting wound healing outcome. Suggestions to improve postoperative infection rates are discussed. PMID- 8637205 TI - Carcinoma of the ampulla of Vater: is radical lymphadenectomy beneficial to patients with nodal disease? AB - This study was undertaken to evaluate the effectiveness of radical lymphadenectomy in ampullary cancer with nodal disease. Thirty-five patients underwent the Whipple procedure with radical lymphadenectomy. The location and number of positive nodes was characterized. Eighteen patients (51%) had positive nodes. Patients without nodal disease (pN0 group) had an actuarial 5-year survival rate of 81%. Seven patients with metastasis confined to the pancreaticoduodenal nodes had a 5-year survival rate of 67%, which was comparable for the pN0 group (N.S.) and better than the 27% 5-year survival rate in patients with positive superior mesenteric nodes (P<0.05). Eleven patients with one to three positive nodes had a 5-year survival rate of 71%, which was also comparable to the pN0 group (N.S.) and better than the 0% 5-year survival rate in patients with four or more positive nodes (P<0.01). Radical lymphadenectomy is effective against a limited degree of nodal disease. PMID- 8637206 TI - Cisplatin-based chemotherapy after retroperitoneal lymph node dissection in patients with pathological stage II nonseminomatous germ cell tumors. AB - In order to assess the results of cisplatin-based chemotherapy after primary lymph node dissection in patients with pathological stage II nonseminomatous germ cell tumors of the testis, we retrospectively reviewed the long-term outcome of 44 patients who received adjuvant chemotherapy at Institut Gustave Roussy over a 7-year period. Two chemotherapy regimens were sequentially delivered. Twenty three patients were treated with vinblastine, cyclophosphamide, bleomycin, actinomycin D, and cisplatin (mVAB-6, four cycles), while 21 patients received a combination of etoposide and cisplatin (EP, four cycles). After a median follow up of 6 years, all patients remain free from progression. The long-term toxicity included retrograde ejaculation in eight patients and severe ototoxicity in two patients. We conclude that four cycles of cisplatin-based chemotherapy for pathological stage II testicular cancer resulted in a 100% cure rate with minimal toxicity. PMID- 8637207 TI - Role of surgery in the management of primary lymphoma of the gastrointestinal tract. AB - The role of surgery in the management of primary gastrointestinal lymphoma remains controversial. We retrospectively reviewed the management and outcome of 107 patients with the diagnosis of gastrointestinal lymphoma treated at the UCLA Medical Center during the period 1956-1990. Sixty-four patients underwent surgical exploration at the UCLA Medical Center; 35 of these underwent resection for cure. Sixteen of these 35 patients received no postoperative adjuvant therapy. Twenty-nine patients underwent palliative or "noncurative" resection. There were five postoperative deaths (mortality rate 8%). The overall morbidity rate was 48% There were 3 perforations in a total of 53 patients receiving multiagent chemotherapy. Five-year actuarial survival was as follows: 59% for curative resection alone, 51% for curative resection plus adjuvant therapy, and 28% for "noncurative" resection (P<0.05). Multivariate analysis revealed that stage of disease (P<0.01) and resection for cure (P<0.05) were independent predictors of survival. These results suggest that patients undergoing resection for cure have improved survival. The apparent low risk of perforation during chemotherapy, along with the considerable risk of morbidity and mortality associated with operation, suggests that a policy of debulking large tumors prior to chemotherapy is unwarranted. PMID- 8637208 TI - Insulin-like growth factor-II as a prognostic factor in pulmonary adenocarcinoma. AB - We stained resected specimens from 117 patients with pulmonary adenocarcinoma for insulin-like growth factor-II (IGF-II) by the avidin-biotin-peroxidase (ABC) method and evaluated the usefulness of IGF-II as a prognostic factor. The patients were classified into the IGF-II (+) groups showing staining of 1% or more cancer cells (60 patients) and the IGF-II (-) groups showing staining of <1% (57 patients). The 5-year survival rate was 22% in the IGF-II (+) group and 54% in the IGF-II (-) group (P<0.01). Our results suggest the usefulness of IGF-II stainability as a prognostic factor of pulmonary adenocarcinoma. PMID- 8637209 TI - Suppressive effect of iodine on DMBA-induced breast tumor growth in the rat. AB - Concerning the suppressive effect of inorganic iodine on the growth of 7,12 dimethyl-benz(a)anthracene (DMBA)-induced breast tumor in female Sprague-Dawley (SD) rats, we previously reported that although iodine itself had a suppressive effect on the tumor growth, its effect was not as strong as that of MPA (medroxy progesterone acetate). However, the combined medication of iodine at a low concentration + MPA showed a stronger effect than MPA alone. The purpose of the present study is to elucidate this mechanism of action by determining the uptake of the administered iodine into breast tumor tissue. Breast tumors were induced with DMBA in female SD rats, and these animals were treated with MPA + inorganic iodine at various concentrations for 4 weeks to determine tumor growth and tumor iodine content. In the comparison of tissue iodine content in growth-suppressive tumors with that in nonsuppressive tumors, the former showed a much higher iodine content. This suggests that direct uptake of inorganic iodine by breast tumors led to the suppression of tumor growth. PMID- 8637210 TI - Comparative analysis of imaging modalities in the preoperative assessment of nodal metastasis in esophageal cancer. AB - Preoperative radiological findings of nodal status in 74 patients, using endoscopic ultrasonography (EUS), computed tomography (CT), ultrasonography (US), and magnetic resonance imaging (MRI), were compared to histopathology reports following transthoracic total esophagectomy with radical lymphadenectomy (TTE), involving complete dissection of the mediastinal and abdominal nodes and lower cervical lymph nodes. Accuracy, sensitivity, and specificity of each radiological investigation were calculated for each anatomic group of nodes. Statistical analysis revealed that EUS is more accurate and significantly more sensitive (P<0.01) for lymph nodes along the right recurrent laryngeal nerve and those in the upper and mid-periesophageal, infracarinal locations. Paratracheal and lower paraesophageal nodes are assessed better using CT whereas MRI is better for mid paraesophageal and infra-aortic nodes. US is most accurate and sensitive for evaluation of cervical and abdominal nodes (P<0.01). PMID- 8637211 TI - Postoperative complications after pneumonectomy for treatment of lung cancer: multivariate analysis. AB - The charts of 62 patients with primary lung cancer who underwent a pneumonectomy at our department from 1979 through 1992 were reviewed for the evaluation of postoperative morbidity and mortality. The 30-day mortality was 3/62 or 4.8%. Postoperative complication occurred in 37 of 62 patients (60%). The most common complication was a supraventricular tachyarrythmia. A major complication, which was defined as one necessitating re-thoracotomy or one which caused death, occurred in 19 patients (31%). We analyzed 43 perioperative variables for their predictive value of postoperative morbidity and mortality. Univariate analysis indicated that an elevated serum LDH, low predicted forced vital capacity, low predicted forced expiratory volume in 1 sec (FEV1) were significantly associated with the occurrence of a major complication. A multivariate logistic regression model indicated that a high LDH level, a low predicted FEV1 and no extubation following surgery were associated independently with a postoperative major complication. Since only the complete removal of a tumor offers a chance for cure for the treatment of non-small cell lung cancer, it is sometimes necessary to perform a pneumonectomy for these high-risk patients. Patients identified as being at high risk of a major complication should be candidates for intensive preoperative evaluation and perioperative care. PMID- 8637212 TI - Expression of MRP and mdr1 in human gastrointestinal cancer cell lines: a correlation with resistance against doxorubicin. AB - The mRNA expression of mdr1 and MRP, each of which codes for a transport protein belonging to ATP-binding cassette superfamily and are reported to be responsible for multidrug resistance phenotype, were semi-quantified by RT-PCR in a panel of gastrointestinal cancer cell lines. Although the expression of MRP was predominant in esophageal cancer cell lines, expression of either or both of the genes was detected in all the cell lines tested. Expression of these two genes added together correlated significantly with chemosensitivity against doxorubicin, implicating that expression of both genes should be evaluated in the future analysis of multidrug resistance phenotype. The ID50 values for pirarubicin, although generally lower than the values for doxorubicin, correlated well with the latter, suggesting that the similar phenotype as that for doxorubicin might be responsible for drug resistance against this semisynthetic anthracycline glycoside. PMID- 8637213 TI - Pathologic fracture secondary to subungual melanoma. AB - Subungual melanoma is uncommon, and delays in diagnosis and misdiagnosis occur frequently. We describe a 61-year-old black male who presented with a non-healing area in his left thumb nailbed with many of the features of subungual melanoma. However, the patient also had a pathologic fracture of the distal phalanx, leading to some initial confusion about the diagnosis. Despite aggressive multimodality therapy, the disease rapidly progressed, resulting in the patient's death. Pathologic fracture due to subungual melanoma may indicate a particularly poor prognosis. PMID- 8637215 TI - Myelodysplastic syndromes in the elderly: the role of growth factors in management. AB - Myelodysplastic syndrome (MDS) comprises a group of heterogeneous clonal bone marrow disorders leading to peripheral cytopenia(s) and hypercellular marrow in the majority of the patients. The morphology of the cell lines is characterized by dysplastic features in some or all cell lines. The FAB classification has divided MDS in five subgroups, namely (1) RA (refractory anemia); (2) RARS (refractory anemia with ring sideroblasts); (3) CMML (chronic myelomonocytic leukemia); (4) RAEB (refractory anemia with excess blasts); and (5) RAEB-T (refractory anemia with excess blasts in transformation). Myelodysplastic syndrome remains primarily a disease of the elderly. With a reported median age of 74.4 years, patients have a chronic relentless course with complication of cytopenias, and a significant number of MDS patients, especially from the RAEB and RAEB-T categories, end up in acute myeloid leukemic transformation. Cytogenetic abnormalities are present in 40-58% of the cases and can provide not only help in diagnosis, but also understanding regarding the clinical course and prognostic aspect. Management of MDS is quite pragmatic and at this stage far from satisfactory. Various modalities have included use of differentiating agents, aggressive chemotherapy, bone marrow transplant and, more recently, significant interest has been generated in the use of hematopoietic growth factors. Differentiating agent trials have been unrewarding so far; chemotherapy trials have resulted in less benefit and more early toxic deaths, especially in the elderly MDS patients where the disease predominates. Bone marrow transplant appears suitable for some patients who are at a younger age. Salvation from this disease is being searched in the proper usage of hematopoietic growth factors and cytokines. There has been concern, however, that usage of growth factors has led to early and enhanced transformation of these patients to frank acute leukemic states. This concept appears to be somewhat refuted by newer controlled trials with GM-CSF and G-CSF, emphasizing that the acute leukemic transformation is the natural course of the disease and is not hastened by growth factor use. Preliminary studies are also suggesting that a combination of growth factors, especially G-CSF and erythropoietin as compared to chemotherapies, could be more beneficial in prolonging the survival of MDS patients who have progressed to the acute leukemic phase. More studies are needed for the understanding of the pathogenetic mechanism(s) in order to facilitate a more suitable and appropriate management strategy for MDS. PMID- 8637214 TI - Retroperitoneal cystic lymphangioma. AB - Two cases of retroperitoneal cystic lymphangioma (CL) are presented; the current literature on this rare, benign neoplasm of the lymphatic system is reviewed. This tumor consists of various numbers of cyst-like cavities filled with a serous, serosanguineous or chylous fluid. The histogenesis of CL is still uncertain. Most commonly CL occurs in the neck and in the axillary region, whereas it is rare in the retroperitoneum. Although retroperitoneal CL is a benign lesion, it may cause significant morbidity due to its large size, and its often invasive character with a strong tendency to secondary infection. The treatment of choice is surgical excision. PMID- 8637216 TI - Activity of Fgr protein-tyrosine kinase is reduced in neutrophils of patients with myelodysplastic syndromes and chronic myelogenous leukemia. AB - The Fgr protein-tyrosine kinase, p55(c-fgr), is specifically expressed and functions in cells of myelomonocytic lineages. We examined levels of expression and enzymatic activity of p55(c-fgr) peripheral blood neutrophils of patients with myelodysplastic syndromes (MDS) and chronic myelogenous leukemia (CML) by comparison with those of normal individuals. While neutrophils of eight normal subjects gave uniform results, the specific enzymatic activity of p55(c-fgr), a ratio of the total kinase activity versus the protein level was reduced in seven out of eight patients with MDS and all of five patients with CML. The specific kinase activity of p55(c-fgr) correlated significantly with the activity of neutrophil alkaline phosphatase (NAP) which has been considered to be a marker of neutrophil maturity (r=0.568, P<0.01). The reduced activity of this tyrosine kinase was considered to be a biological parameter for immaturity and to reflect dysfunction of neutrophils of patients with MDS and with CML. PMID- 8637217 TI - The function of src family tyrosine kinases in hematopoietic cells. PMID- 8637218 TI - Deletion of the acetylcholinesterase locus at 7q22 associated with myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). AB - The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) are located within regions subject to non-random chromosomal abnormalities in the myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Acetylcholinesterase is mapped to 7q22, within the critical deleted region presumed to contain a myeloid specific tumour suppressor gene. Butyrylcholinesterase is mapped to 3q26: abnormalities at this region are associated with sub-types of MDS and AML with thrombocytopenia, or with increased platelet counts. Both ACHE and BCHE have been implicated as playing a role in megakaryopoiesis and thrombopoiesis, and these genes have been observed to be co amplified in acute myeloid leukaemia. Recent findings suggest a more significant role for the ACHE gene in haemopoiesis by regulating multipotent stem cell proliferation, and apoptosis in cells undergoing erythroid and myeloid differentiation. This led us to investigate gene copy-number alterations at these genes in MDS and AML. Samples were screened by slot-blot hybridization, and if changes were observed, by Southern blotting. A total of 42 samples from 31 de novo AML patients, 10 samples from eight cases of post-MDS AML and 85 samples from 67 MDS patients were analysed with probes for ACHE, BCHE, c-MYC, MDR-1 and globin control. Changes in ACHE and/or BCHE were observed in 9/31 de novo AML patients, and in 7/67 MDS patients: 1/37 cases of refractory anaemia (RA), 1/10 cases of refractory anaemia with excess blasts (RAEB) and 5/20 chronic myelomonocytic leukaemia (CMML) patients. The amplification events observed generated copy numbers no greater than 10, showed normal restriction patterns and had no clear correlation with megakaryopoiesis or thrombopoiesis. Loss of signal at the ACHE locus was observed: haploid signal intensity was seen in seven samples: one RA with thrombocytopenia, three CMML, one AML-M5a (no karyotypic abnormalities of chromosome 7), one AML-M4 (monosomy 7), and one case of AML-M7 (karyotype unknown). Homozygous deletion was observed at relapse of an additional patient with AML-M4. These data reinforce the possibility that ACHE may play a role as a myeloid tumour suppressor gene. PMID- 8637219 TI - All-trans retinoic acid combined with interferon-alpha effectively inhibits granulocyte-macrophage colony formation in chronic myeloid leukemia. AB - We investigated the effect of all-trans retinoic acid (ATRA) alone and in combination with interferon-alpha (IFN-alpha) on the granulocyte-macrophage (GM) colony formation of peripheral blood progenitors isolated from patients with chronic myeloid leukemia (CML) (n = 12) or other myeloproliferative disorders (n = 10) as well as from healthy controls (n = 7). The ATRA or IFN-alpha alone inhibited slightly, but not significantly, the GM colony growth in CML. Granulocyte-macrophage colony formation decreased significantly (P<0.05) when ATRA and IFN-alpha were combined (114 +/- 96 versus 74 +/- 53 colonies/10(4) mononuclear cells). The combination did not have any inhibitory effect on the other MPDs. In healthy controls, ATRA or IFN-alpha alone or their combination stimulated GM colony growth, the increase being from 22 +/- 9 to 39 +/- 16 colonies for ATRA (P<0.05), up to 47 +/- 12 colonies for IFN-alpha (P<0.05) and up to 50 +/- 19 colonies for the combination (P<0.05). In conclusion, ATRA combined with IFN-alpha inhibits GM colony growth in CML. This combination may be worth testing clinically as a treatment of CML. PMID- 8637220 TI - Regulation of topoisomerase II expression during the VM-26 induced differentiation of IW32 murine erythroleukemia cells. AB - The effect of VM-26, a topoisomerase II targeting drug, on IW32 murine erythroleukemia cells was investigated. The VM-26 induced IW32 cells to differentiate at a non-toxic but cytostatic concentration (0.01 microgram/ml). More than 40% of the cells were induced to synthesize hemoglobin, and cells were arrested in G2/M phase of the cell cycle. Levels of beta-globin mRNA also increased significantly. Cells became committed to erythroid maturation after 16 h of continuous drug exposure. Replacement with fresh VM-26 after 48 h of drug treatment further increased the hemoglobin containing cells to greater than 80%. Unlike other drug induced erythroleukemia cell differentiation, c-myc mRNA expression was not affected by VM-26. Inhibition of topoisomerase II activity was observed during the first 12 h of VM-26 treatment; however, elevated enzyme activity was found thereafter. Northern blot analysis showed significant increase in the expression of topoisomerase IIalpha mRNA at 12 and 24 h after VM-26 addition. These findings indicate that VM-26 inhibited the activity of topoisomerase II and promoted the committed differentiation of IW32 cells along the erythroid pathway. In addition, a parallel increase in mRNA and activity levels of topoisomerase II in differentiated cells suggests that regulation of the enzyme expression occurred in the VM-26 induced erythroid maturation. PMID- 8637221 TI - Arylsulfatase B in Kurloff cells: increased activity of anionic isoforms in guinea pig acute lymphoblastic leukemia. AB - We examined the in vivo role of Kurloff cells (KC), guinea pig natural killer cells, during the development of L2C leukemia, by analysing changes in the arylsulfatases (Asases) in the lysosomal Kurloff body. The Kurloff body is rich in acid phosphatase, esterase and proteoglycans, as are large granular lymphocyte granules. Moreover, the Kurloff body contains lysosomal Asase B, with unusual anionic isoforms. Injection of L2C cells elicited a three-fold increase in KC Asase activity on day 6. The increase in KC Asase activity was correlated with the number of circulating L2C cells. The basic Asase form (pl 8) was lost, and a concomitant increase in anionic isoforms (pl 5-6) was observed on day 6. The role of the latter in cytolysis was investigated by examining their capacity to lyse L2C target cells. We conclude that Asase participates in cytolysis when lysis is mediated by the complete assembly of cytolytic proteins. Changing and increasing KC Asase activity during leukemia development may be a marker for activated KC in vivo. These findings suggest that the cytolytic activation of KC occurs during the preleukemic period. PMID- 8637222 TI - Serum TNF-alpha, gamma-INF, G-CSF and GM-CSF levels in neutropenic children with acute leukemia treated with short-course, high-dose methylprednisolone. AB - High-dose methylprednisolone (HDMP, 20-30 mg/kg/day po) treatment has been shown to increase the number of bone marrow and peripheral blood CD34 positive progenitors and serum granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in patients with ALL and AML. To investigate the effect of HDMP on some other hematopoietic regulatory cytokines, tumor necrosis factor-alpha (TNF alpha), gamma-interferon (gamma-INF), granulocyte colony-stimulating factor (G CSF) and interleukin-6 (IL-6) were studied by microplate ELISA technique in 15 chemotherapy-induced neutropenic episodes of 14 children with acute leukemia (eight with ALL and six with AML) in whom HDMP was given alone (30 mg/kg/day po) for 4 days. The absolute neutrophil counts increased significantly in all neutropenic episodes on the fourth day of HDMP treatment. The TNF-alpha was 93.5 +/- 161 pg/ml in ALL and 78.3 +/- 61.4 pg/ml in AML before treatment and 76.1 +/- 160 pg/ml in ALL and 19.1 +/- 39.8 pg/ml in AML after treatment. The gamma-INF was 204.1 +/- 210.3 pg/ml in ALL and 130.8 +/- 138.3 pg/ml in AML before treatment and 28.6 +/- 50.5 pg/ml in ALL and 23.3 +/- 20.4 pg/ml in AML after treatment (P<0.05). Serum G-CSF and GM-CSF levels increased in all episodes (100%). The GM-CSF levels increased from 12.2 +/- 10.9 pg/ml to 36 +/- 24.7 pg/ml after treatment in ALL (P<0.05) and from 13.3 +/- 4 pg/ml to 45 +/- 48.1 pg/ml in AML (P<0.05). Serum G-CSF levels increased from 13.3 +/- 11.7 pg/ml to 83.3 +/- 86.8 pg/ml after treatment in ALL (P<0.05) and from 6.6 +/- 12.1 pg/ml to 28.3 +/ 11.3 pg/ml in AML (P<0.05). However, IL-6 levels were undetectable in all patients before and after therapy. These preliminary data suggest that short course HDMP treatment could decrease serum TNF-alpha and gamma-INF and increase G CSF and GM-CSF levels. PMID- 8637223 TI - Interaction of reactive nitrogen and oxygen intermediates in HL60 and dimethylsulphoxide-differentiated HL60 cells. AB - The interaction of reactive nitrogen intermediates (RNI) with reactive oxygen intermediates (ROI) was inferred from the effect of added L-arginine on luminol dependent chemiluminescence (LCL) and cytochrome C reduction in HL60 cells, dimethylsulphoxide (DMSO)-differentiated HL60 cells and human neutrophils. Phorbol myristate acetate (PMA)-stimulated HL60 cells had no effect on LCL and a decreased rate of cytochrome C reduction in the presence of increasing concentrations of L-arginine. Inhibition of L-arginine-mediated cytochrome C reduction was relieved by L-N(G)-monomethyl arginine (L-NMMA), an inhibitor of nitric oxide synthesis, in a concentration-dependent manner. In contrast, DMSO differentiated cells and human neutrophils separated from blood showed decreased rates of LCL and cytochrome C reduction with increasing concentrations Of L arginine, which were relieved to some extent by L-NMMA in a dose-dependent manner. These results are consistent with a 40% increase in the production of nitrate following stimulation of DMSO-differentiated cells and human neutrophils by PMA compared with only a 6% rise in undifferentiated HL60 cells. Possible inhibition of NADPH oxidase has been suggested to explain the responses of LCL, cytochrome C reduction and nitrate production by nitric oxide in the presence of L-arginine. PMID- 8637224 TI - The location of MZF-1 at the telomere of human chromosome 19q makes it vulnerable to degeneration in aging cells. AB - A zinc-finger gene encoding a transcription factor that regulates hematopoiesis, MZF-1, is located at the extreme end of the q arm of human chromosome 19. Several lines of evidence indicate that MZF-1 lies less than 20 kb from the subtelomeric repeat region of 19q. Telomeres are known to degenerate as cells age; disruption of MZF-1 due to telomeric degeneration may play a role in the increased incidence of leukemia in the elderly. PMID- 8637225 TI - Chronic myelogenous leukemia: does interferon alpha prolong life? PMID- 8637226 TI - Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society. AB - Hemophagocytic lymphohistiocytosis (HLH) is a rare, often fatal, disease of early infancy. The diagnosis of HLH is frequently delayed or made at autopsy because no genetic or biologic marker has been identified. To improve the classification and treatment of HLH, the Histiocyte Society has established an 'International Registry for HLH'. Data collected included family history, clinical and laboratory features at the onset of illness, and treatment outcome. Stringent diagnostic criteria (ie fever, splenomegaly, cytopenia, hypertriglyceridemia, and/or hypofibrinogenemia, and hemophagocytosis without evidence of malignancy) were used for patient selection. One hundred and twenty-two patients (61 males, 61 females) were enrolled from 17 centers in 11 countries. The rate of parental consanguinity was 24%. A positive family history was reported in 49% of cases including two pairs of affected male twins. The median age at disease onset was 2.9 months, with no difference between familial and sporadic cases. Age at onset was similar in affected sibs from 10 of 14 families, but in four up to 3-year differences were observed. Hemophagocytosis was present at diagnosis in 75%. An associated infection (usually by common viral pathogens) was reported in 50 of the 122 (41%) cases, of which 25 had familial disease. Natural killer activity was impaired in 36 of 37 patients studied. Chromosome analysis was normal in all tested patients. A decreased frequency of HLA-B7 and B8 alleles and increased frequency of HLA-B21 and DQ3 were observed. The estimated 5-year survival (SE) was 21% (18.7) for all patients. It was 66% (37.8) for patients who received allogeneic bone marrow transplant and 10.1% (9.6) for patients treated with chemotherapy alone (P=0.0001). None of the previously proposed prognostic indicators (age, associated infection, cerebrospinal fluid pleocytosis, family history) correlated with treatment outcome. PMID- 8637227 TI - Acute myelogenous leukemia: a disorder of gene splicing? AB - The two common rearrangements t(8;21) and inv(16) are found in approximately 20% of all acute myelogenous leukemias. Both aberrations result in the formation of a fusion gene, involving subunits of the core binding transcription factor (CBF). In this manuscript we hypothesize that the alternative splicing of the fusion genes, leading to truncated CBF subunits, contributes to the pathogenesis of t(8;21) and inv(16) leukemias. PMID- 8637228 TI - PML/RAR alpha transcripts monitored by polymerase chain reaction in acute promyelocytic leukemia during complete remission, relapse and after bone marrow transplantation. AB - The translocation t(15;17)(q24;q21), unique to acute promyelocytic leukemia (APL), gives rise to PML/RAR alpha fusion transcripts detected by the sensitive reverse transcriptase-polymerase chain reaction (PCR) technique. PCR may help in the diagnosis and in monitoring minimal residual disease. Reversion of PCR to negative is obtained by chemotherapy (CT) alone or in combination with all-trans retinoic acid (ATRA). Here we show a serial PCR study of 10 APL cases. Five cases were studied at the time of diagnosis, and all were PCR positive for the rearranged transcripts (three bcr1 type, two bcr3 type). Seven cases in complete remission (CR) after one cycle of induction CT were persistently PCR negative, one case in CR after ATRA rescue was persistently PCR positive (bcr1 type), one patient (bcr3 type) relapsed 15 months after the PCR-negative CR and one patient died early. Seven patients underwent bone marrow transplantation (BMT) (five allogeneic, two autologous). One of them died early after take of the allogeneic BMT, the other six cases studied by serial PCR were persistently negative. At a median follow-up of 31 months (range 9-39), none of these six cases had relapsed. PCR data characterize the CR at the molecular level and evaluate the efficacy of different treatments, including BMT. The data may help to define a standardized schedule for PCR follow-up, and are also potentially useful to establish the time required before judging patients with persistently negative PCR to be cured. BMT as post-induction treatment in first CR is also discussed. PMID- 8637229 TI - Heterogeneity of hyperdiploid (51-67) childhood acute lymphoblastic leukemia. AB - We studied the fully banded chromosomes of 182 children with hyperdiploid (51-67) acute lymphoblastic leukemia (ALL) to better delineate the heterogeneity of this disease subtype. Forty-six percent of the cases had numerical changes exclusively, while the remainder had structural as well as numerical changes. Chromosome 21 was added most often (97% of cases), followed by chromosomes 6 (86%), X (81%), 14 (80%), 4 (76%), 18 (68%), 17 (68%), 10 (56%), 8 (34%) and 5 (26%). Chromosomal translocations, including the t(1;19)(q23;p13) and t(9;22)(q34;q11), were detected in only 20% of the cases, as compared with 50% in ALL in general. The most common structural alterations were duplication of the 1q arm and isochromosome of 17q, present in 25 (14%) and nine (5%) cases, respectively. The presence of absence of structural abnormalities in these cases did not influence event-free survival, as assessed in 168 patients enrolled in three successive protocols for children with newly diagnosed ALL. By contrast, patients with 51-55 chromosomes per leukemic cell (n=105) appeared to fare worse than the 56-67 subgroup (n=63) (5-year probability of event-free survival = 72 +/ 5% (s.e.) vs 86 +/- 5%; P=0.04 by the stratified log-rank test). The poorer prognosis of the 51-55 subgroup was partly due to the higher frequency of isochromosome of 17q; 6/7 patients with the isochromosome in this group have had an adverse event. Other unfavorable features within the hyperdiploid (51-55) ALL subgroup include a low prevalence of trisomies of chromosomes 4 and 10 and a higher proportion of patients with leukocyte counts greater than 50 X 10(9)/l when compared to hyperdiploid (56-67). Thus, ALL defined by 51-55 chromosomes appears to be a clinicobiologic entity quite distinct from cases with higher modal numbers. PMID- 8637230 TI - YAC clone H10 discriminates between 3q26.2 and 3q27 chromosome rearrangements in hematological disorders. AB - To discriminate with molecular-cytogenetic resolution between 3q26.2 breakpoint, associated to various myeloproliferative disorders, and 3q27 breakpoint, recurrent in several types of non-Hodgkin lymphoma, we tested the feasibility of using a yeast artificial chromosome, YAC clone H10, mapped on 3q26.3. Fluorescent in situ hybridization of the biotinylated polymerase chain reaction product of the YAC H10 was performed in three myeloproliferative diseases and one follicular non-Hodgkin lymphoma carrying different rearrangements of chromosome 3 involving region q26-q27. Our study shows that YAC H10 signal was telomeric to all three myeloid breakpoints, while it was centromeric in the lymphoid one thus showing that this probe can discriminate between these two subsets of chromosome 3 rearrangements. These results point out the opportunity of using additional YACs in the characterization of polymorphic chromosome alterations acquired in neoplastic cells. PMID- 8637231 TI - c-kit ligand stimulates tyrosine phosphorylation of a similar pattern of phosphotyrosyl proteins in primary primitive normal hematopoietic progenitors that are constitutively phosphorylated in comparable primitive progenitors in chronic phase chronic myelogenous leukemia. AB - Characteristic of Philadelphia (Ph)+ chronic myelogenous leukemia (CML) is the presence of the chimeric BCR/ABL (p210) protein possessing elevated protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Our previous studies demonstrated subtle differences in the growth, phenotypic and morphologic characteristics of the most primitive subpopulations of primary lin Ph+ chronic phase CML blasts and comparable primary lin- normal blasts. Recently, in comparing proteins phosphorylated on tyrosine in these cell populations, we reported a prominent 62 kDa phosphotyrosyl (P-tyr) protein constitutively present in primary primitive lin- CML chronic phase blasts which was virtually undetectable in primary primitive lin- normal blasts. In the present studies, we demonstrate that this P-tyr p62 from primary primitive lin- chronic phase CML blasts co-immunoprecipitates with ras-GAP. Furthermore, in addition to the p210 protein, we show in whole cell lysates the presence of other clearly consistent but less prominent P-tyr proteins with molecular weights of approximately 155, 140, 110, 55 and 45 kDa as well as more minor P-tyr proteins of approximately 190, 85, 52, 42 and 39 kDa constitutively present in primary primitive lin- chronic phase CML blasts. In analyzing proteins tyrosine phosphorylated in primary primitive lin- normal blasts in response to various hematopoietic growth factors, we found a striking similarity in the phosphorylation of four major (approximately 140, 110, 62 and 56 kDa) and three minor (approximately 51, 45 and 42 kDa) P-tyr proteins after stimulation with c-kit ligand and the P-tyr proteins constitutively phosphorylated in primary primitive lin- chronic phase CML blasts. Other growth factors tested (ie GM-CSF, G-CSF, IL-3, FLT3 ligand and EPO) were much less active or stimulated phosphorylation of other proteins. It is provocative that at least seven proteins rapidly and transiently phosphorylated on tyrosine in the c-kit ligand signal transduction pathway in lin- normal blasts may be constitutive substrates for the p210 activated tyrosine kinase in comparable lin- chronic phase CML blasts. In addition, it is intriguing that some of the biological effects on hematopoietic progenitors attributed to the c-kit ligand may be similar to some of the observed biological consequences of the p210 protein, including survival and expansion of a more mature stem cell population, probably at the time of lineage commitment rather than at the level of the earliest self-renewing stem cell. PMID- 8637232 TI - Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. AB - FLT3/FLK2 is a receptor tyrosine kinase (RTK) which is thought to play an important role in early stages of hematopoiesis. Monoclonal antibodies (mAbs) against the extracellular domain of human FLT3 were generated to study the cell surface expression of this class III RTK on normal bone marrow cells and on leukemic blasts from patients with acute leukemias. Functional analysis of five mAbs (SF1 series) revealed that all of them can mimic to variable extents the activity of the FLT3 ligand (FL) upon receptor activation and modulation, while only one mAb weakly inhibited ligand binding. Using flow cytometry, we detected surface expression of FLT3 on cell lines of the myeloid (4/8) and B lymphoid (7/10) lineages. On normal human bone marrow cells, the expression of FLT3 is restricted, in agreement with a presumed function of this receptor at the level of the stem cells and early committed progenitors. Expression of FLT3 was found on a fraction of CD34-positive and CD34-negative cells. Three-color analysis further revealed that most of the CD34 FLT3+ cells coexpress CD117 (KIT) at a high level. Finally, FLT3 is expressed on leukemic blasts of 18/22 acute myeloid leukemias (AML) and 3/5 acute lymphoid leukemias (ALL) of the B lineage, providing a possible application in diagnosis and therapy of these diseases. PMID- 8637233 TI - Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage. AB - We analyzed homozygous deletions and mutations of the CDKN2(p16(INK4A)/MTS1) gene, using polymerase chain reaction and Southern blot analysis, in 120 children with acute lymphoblastic leukemia (ALL). Homozygous deletion was found in 17 of 89 (19%) precursor B-ALL patients, in 11 of 24 (46%) T-ALL patients, and in 0 of 7 other phenotype ALL patients. After excluding 28 (23%) patients who showed a homozygous deletion of CDKN2, we found that three patients (3%) had mutation at exon 2 of CDKN2 using PCR-SSCP and sequencing strategy. One had a CGA to TGA nonsense mutation (Arg to stop) at codon 72, one had a 1-bp deletion at codon 117, and the third had a 2-bp deletion at codon 70, resulting in frameshifts in the two latter patients. All three of these patients were T phenotype ALL, and the incidence of mutation in the 24 T-ALL patients examined was 13%. In contrast, no mutation was detected in the remaining patients with precursor-B or other type ALL (0/96). Our results suggest that mutational inactivation of the CDKN2 gene may contribute to the leukemogenic growth, especially in some patients with T ALL. PMID- 8637235 TI - Effects of FLT3 ligand on human leukemia cells. I. Proliferative response of myeloid leukemia cells. AB - The growth of cells in vitro and in vivo is regulated by several environmental signals among which growth factors (cytokines) figure prominently. FLT3 is a novel cytokine receptor with intrinsic ligand-stimulated (FLT3 ligand, FL) tyrosine kinase activity. Here, using a specific anti-FLT3 monoclonal antibody (McAb) and flow cytometry we determined the expression pattern of the receptor protein in 55 human leukemia-lymphoma cell lines and in 20 primary samples from patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). FLT3 receptor surface expression was found predominantly in pre-B cell, myeloid and monocytic cell lines and in pre-B-ALL and AML cells, FL was overexpressed in baby hamster kidney cells producing a recombinant protein that was functional in receptor binding and signaling. Incubation with FL induced 3H-thymidine uptake measured proliferation in some myeloid cell lines and in 2/9 AML cases. The strongest proliferative response was seen in the two growth factor-dependent myeloid leukemia cell lines MUTZ-2 and OCI-AML-5. Long-term substitution of the commonly used cytokines with FL sustained the continuous proliferation of these two cell lines suggesting that also upon permanent activation FLT2 can function as a mitogenic signaling molecule. Despite the high density of FLT3 receptor expression on cultured and fresh pre-B-ALL cells, no proliferation could be stimulated in any of these specimens. Incubation with the anti-FLT3 McAb had agonistic proliferative effects in MUTZ-2 and OCI-AML-5; and anti-FL reagent blocked FL-stimulated proliferation. To summarize, we demonstrated that FL is effective in inducing proliferation of leukemic myeloid cells and that protein expression does not necessarily indicate an FL-responsive cell. While the present data clearly demonstrate that FL might play a proliferative role in leukemogenesis, further studies are needed to clarify whether the signals provided by FL:FLT3 interaction are confined to a proliferation-inducing function or whether maturational progression could also be elicited in certain cells. PMID- 8637234 TI - Homozygous deletions of cyclin-dependent kinase inhibitor genes, p16(INK4A) and p18, in childhood T cell lineage acute lymphoblastic leukemias. AB - p16(INK4A) and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase activities required for the overcoming of the G1 checkpoint in the eukaryotic cell division cycle. The frequent cytogenetic aberrations occurring in several human neoplasms at the level of their codifying genes along with their molecular function strongly suggest that they might be important tumor suppressor genes. We looked for homozygous deletions of p16(INK4A) and p18 genes in 21 cases of childhood T cell lineage acute lymphoblastic leukemia (ALL). Twenty of 21 patients (95%) had homozygous deletions of p16(INK4A) gene while three out of 21 (14%) showed p18 gene biallelic deletion. Loss of heterozygosity studies were performed in 18 of the T cell ALL investigated by means of two highly polymorphic 9p21 markers. The results obtained demonstrated that genetic deletions of different extension occur on the short arms of the 9 chromosome pair. Karyotypic analyses, performed in 13 cases, failed to demonstrate 9p alterations in 12 samples, (92%) thus demonstrating that p16(INK4A) gene homozygous deletions are not restricted to cases with cytogenetically detectable 9p aberrations. The high incidence of p16(INK4A) gene deletions in pediatric T cell lineage ALL suggests that this genetic alteration could represent an early and key event in the development of such a malignancy but it should not have any prognostic value. Conversely, the inactivation of p18 gene, observed in a lower but significant number of cases, could participate in the progression of acute leukemias towards a more aggressive disease. Finally, our results may suggest that p16(INK4A) protein plays a key role in the control of proliferation and/or differentiation of human T lymphocytes. PMID- 8637236 TI - Effects of FLT3 ligand on human leukemia cells. II. Agonistic and antagonistic effects of other cytokines. AB - We have previously shown that the growth factor FLT3 ligand (FL) is mitogenic for human primary and continuously cultured myeloid leukemia cells. Despite widespread expression of the receptor FLT3 among the leukemia cell lines from certain cell lineages, only two growth factor-dependent myeloid leukemia cell lines showed a significant proliferative response to FL. In the present study, we examined the proliferative effects of FL on a comprehensive set of growth factor dependent leukemia cell lines. A significant enhancement of cell growth by FL was seen in 10/12 myelomonocytic cell lines, while all cell lines with predominantly megakaryocytic and/or erythroid characteristics did not respond positively, despite the expression of the receptor. The cytokines interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) could independently enhance the FL-stimulated proliferation in a synergistic fashion. Transforming growth factor-(beta)1 (TGF-(beta)1), in a dose dependent fashion, partially inhibited the FL-promoted proliferation, but basic fibroblast growth factor (bFGF), on its own augmenting the response to FL, significantly abrogated the inhibitory effects of TGF-(beta)1. TGF-(beta)1 down regulated mRNA and protein expression of the FLT3 receptor. Taken together these data suggest that the effects of FL on the growth of normal and malignant hematopoietic cells can be positively and negatively modulated by other cytokines. PMID- 8637237 TI - Primary acute myeloid leukemia cells negative for c-kit receptor protein expression do not proliferate in vitro in response to colony-stimulating factors. AB - This study examined the effects of recombinant human stem cell factor (SCF) alone or in combination with colony-stimulated factors (CSFs) on colony formation of leukemic progenitors obtained from 29 acute myeloid leukemia (AML) patients. C kit receptor (c-kitR) protein expression was examined using an immunofluorescence method and was detected on more than 10% of leukemic cells in 20 of 29 cases (c kitR+). SCF alone could stimulate formation of colonies in 14 of 20 c-kitR+ cases. Granulocyte(G)-CSF, granulocyte-macrophage (GM)-CSF, and interleukin (IL) 3 alone stimulated colony growth in 18, 17 and 16 of c-kitR+ cases, respectively. In contrast, colony and cluster formations from eight of nine c-kitR- cases was not stimulated at all by SCF alone nor SCF in combination with CSF nor with any CSF alone. However, the in vitro culture behavior of fluorescence-activated cell sorter-sorted c-kitR- cells from c-kitR+ cases did not significantly differ from c-kitR+ cells. These results suggest that responses of leukemic progenitors to CSFs were variable but may be predicted by their c-kitR expressions, and that c kitR(-) cases may have a different disease entity from c-kitR(+) cases. PMID- 8637238 TI - Increased expression of c-Kit or its ligand Steel Factor is not a common feature of adult acute myeloid leukaemia. AB - Cell surface levels of the receptor tyrosine kinase P145(c-kit), the product of the c-kit proto-oncogens, in a panel of 80 primary adult acute myeloid leukaemia (AML) specimens collected at presentation were quantitated by immunofluorescence and flow cytometry, and compared with levels on CD34+ bone marrow cells from normal donors. Receptor levels on AML blast cells were extremely variable and were similar to, or less than, those on normal stem and progenitor cells. In general P145(c-kit) expression was higher on cells of immature phenotype (FAB M1 and M2). c-kit mRNA was quantitated by ribonuclease protection assay (RPA) and was shown to be correlated with cell surface protein expression (r=0.76; P<0.001). This indicates that ligand-mediated receptor internalisation or other mechanisms of increased protein turnover are not responsible for variations in the level of P145(c-kit) in AML specimens. Quantitative Southern blotting was used to examine c-kit gene copy number in 25 of these specimens and was found to be normal in all but one. Thus we have found little evidence of over-expression of c-kit in adult AML. mRNA for the c-kit ligand, Steel Factor (SLF) was also quantitated by RPA in these specimens. While SLF message was detectable (limit of detection approximately 10(4) copies per 10 microgram total RNA; equivalent to 1 copy per 100 cells) in 19% of cases, these specimens in general contained low levels of c-kit mRNA. Thus, an autocrine cycle involving c-kit and SLF does not appear to be a common feature of AML. PMID- 8637239 TI - Expression of the receptor MPL and proliferative effects of its ligand thrombopoietin on human leukemia cells. AB - Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets exerting its effects via the receptor MPL. We examined the expression of MPR on the cell surface of a panel of 43 myelomonocytic, erythroid and megakaryocytic leukemia cell lines and 21 primary acute myeloid leukemia (AML) cases by flow cytometry. With few exceptions MPL was found on all 32 erythroid/megakaryocytic cell lines and on all 11 growth factor dependent myelomonocytic cell lines, albeit at variable percentages and intensities per cell population (with a 10% cut-off level for positivity still 30/43 cell lines scored as MPL positive). The majority of the primary AML samples (including all seven M6/M7 cases) expressed the MPL protein regardless of the morphological and immunological subtype (13/21 cases had >10% MPL-positive cells). Recombinant TPO overexpressed in hamster cells induced a mitogenic response in seven cell lines (one growth factor-independent and six factor dependent lines) and in 3/21 AML specimens (two AML M2, one AML M7) as measured by 3H-thymidine incorporation. Expression of MPL clearly did not correlate with response to TPO. For further detailed studies of the interaction of TPO with other cytokines we used the AML M7-derived M-07e cells as an informative indicator cell line for which both murine and human TPO acted as a very potent mitogen in a dose-dependent fashion (3- to 11-fold proliferation increase relative to medium alone). This growth factor-dependent cell line which is normally cultured in conditioned medium containing several cytokines could be grown in long-term culture supplemented only with TPO. Co-incubation of M-07e with various cytokines and TPO showed additive proliferative effects for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM CSF) and synergistic responses for stem cell factor (SCF), interferon (IFN) alpha, and to a lesser extent for IFN-gamma and tumor necrosis factor (TNF) alpha. Erythropoietin (EPO), IL-1, IL-6, IL-11 and leukemia inhibitory factor (LIF), know as megakaryocytic maturation-inducing molecules, were not substantially effective, neither singly nor in combination with TPO, with regard to cell growth. Transforming growth factor (TGF)-beta1 antagonized the inductive effect of TPO on M-07e cell growth. Addition of TPO to cultures of megakaryocytic cell lines failed to significantly alter the ploidy distribution and the differentiation marker immunoprofile of the cells indicating a lack of maturation inducing effects in this model system. In summary, TPO represents an efficient in vitro potentiator of megakaryocytic leukemia proliferation of at least some primary cases or cell lines. While TPO seems to be the major physiological regulator of megakaryocytopoiesis, the present data suggest also some proliferative effects on certain leukemia cells, apparently on non-megakaryocytic leukemia cells as well, thus assigning to TPO a possible pathobiological role in leukemogenesis which would be of clinical relevance. Our data show that the response to TPO is not restricted to cells committed to the megakaryocytic differentiation pathway as we could demonstrate TPO-responsive megakaryocytic and non-megakaryocytic cell lines; thus, these cell lines represent powerful tools in such analyses. Consequently, this new cytokine needs to be properly examined so we can get a clear understanding of the clinical possibilities and dangers. PMID- 8637240 TI - The regulation of hematopoiesis in max 41 transgenic mice with sustained excess granulopoiesis. AB - max 41 transgenic mice consistently exhibit elevated numbers of mature granulocytes and monocytes in the peripheral blood and of immature and mature cells of these lineages in the marrow, spleen, lymph nodes and liver. The immature populations are not autonomous and exhibit a normal quantitative responsiveness to proliferative stimulation by the four colony-stimulating factors. The present studies examined three other candidate regulators of granulocyte formation and showed that max 41 cells exhibit normal quantitative responsiveness to stem cell factor, slightly enhanced responsiveness to IL-6 but reduced responsiveness to Flk-ligand. Serum levels of growth factors were not unusually elevated in max 41 mice before or after the injection of endotoxin nor were excessive levels of the four CSFs or IL-6 produced in cultures of max 41 organs. Responses to injected G-CSF were not unusually high in terms of fold elevations in max 41 mice. Levels of mRNA for various growth factors were not abnormal in max 41 marrow populations although, in crowded cultures, max 41 marrow cells exhibited a higher level of endogenously stimulated colony formation than control cells. max 41 cells also exhibited elevated responsiveness to stimulation by mixtures of growth factors, particularly those in organ conditioned media. The present observations suggest some possible mechanisms by which a max 41 mouse might achieve a sustained elevation of granulocyte and monocyte production but the data seem insufficient to provide a complete explanation and indicate persisting deficiencies in knowledge of how granulocyte and monocyte production is regulated. PMID- 8637242 TI - Multiple autoimmune manifestations in monoclonal gammopathy of undetermined significance and chronic lymphocytic leukemia. AB - In 18 cases of monoclonal gammopathy of undetermined significance, MGUS (monoclonal gammopathy of undetermined significance), admitted for diagnosed or suspected peripheral neuropathy, 11 patients showed other co-existing autoimmune manifestations. Two had POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-component, and skin symptoms), the others mainly endocrinopathy and polyclonal pseudolymphoma. There were 13 cases of sensorimotor neuropathy, two cases of neuritis, while neuropathy could not be confirmed in three cases. Compared with a retrospective review of autoimmunity in a randomly selected CLL (chronic lymphocytic leukemia) cohort of 115 patients, 13 out of 42 patients with clinical and/or laboratory features of autoimmunity showed co-expression of autoimmune signs, the dominating traits being Coombs positive AIHA (auto-immune hemolytic anemia), platelet autoantibodies, endocrinopathy mainly associated with the thyroid gland, serological and/or rheumatological symptoms, but only one case of sensorimotor neuropathy. Viewed from a current model of acquired autoimmunity it is perhaps not surprising that such autoimmunity is seen predominantly in patients with monoclonal gammopathy. Thus, a high concentration of cross-reacting polyreactive autoantibodies related to the M-component might be present in these patients. Furthermore, quantitative defects of the immunoglobulins including the hypogammaglobulinemia associated with M-components can presumably give rise to a defect of the anti-idiotypic network's regulation of natural autoantibodies and autoimmune manifestations in vivo. Such autoimmune manifestations, which are easily overlooked in CLL may call for additional treatment with immunosuppression and/or intravenous, polyclonal IgG. PMID- 8637241 TI - Antileukemic activity of recombinant humanized M195-gelonin immunotoxin in nude mice. AB - A leukemia-selective immunotoxin was constructed by linking recombinant gelonin (rGel), a single chain ribosome inhibitory protein, to recombinant humanized M195 antibody (HuM195), which recognizes the cell-surface protein designated CD33. CD33 is an antigen found on myeloid leukemia blasts as well as myeloid progenitor cells but it is not expressed in detectable amounts on the ultimate hematopoietic progenitor stem cell. Our previous studies indicated that a non-recombinant humanized immunotoxin displayed specific, potent toxicity towards CD33-positive cells but not to CD33-negative cells in vitro. In the current study, a recombinant humanized immunotoxin, HuM195-rGel, was evaluated in vivo in a nude mouse model of human myeloid leukemias. HuM195-rGel was found to target leukemia cells rapidly in vivo and was subsequently internalized into the cells. For trials in vivo, nude mice were injected (ip) with 10(7) log-phase HL60 human leukemia cells 10 days prior to the start of i.p. HuM195-rGel treatments. HuM195 rGel demonstrated significant tumor suppressive activity in this model. While all mice treated with either saline, rGel alone, or HuM195 plus unconjugated rGel (at 10 or 14 days after transplantation) had rapid tumor growth or early deaths, 50% of mice treated with HuM195-rGel failed to develop leukemic tumors for 5 months and the 50% had significantly retarded tumor growth after treatment with HuM195 rGel. Mice treated at later times (28 days after transplantation of leukemia cells) also showed delayed leukemia cell growth, but no cures. These data show that HuM195-rGel can target leukemia cells in vivo and can result in pronounced anti-leukemic effects. PMID- 8637244 TI - Engraftment of chronic prolymphocytic and T cell leukemia in SCID mice. AB - Engraftment of human acute leukemia cells in immunocompromised (SCID) mice has resulted in in vivo models for exploration of human tumor biology. Attempts at engraftment of chronic leukemia cells have been generally unsuccessful. We have engrafted cells from three human chronic leukemias in SCID mice. Cell populations were from two patients with chronic lymphocytic leukemia (CLL) and either increased proolymphocytes (CLL-Pro; patient 1), or prolymphocytic transformation (PLL; patient 2) and from a third patient with newly diagnosed T cell CLL. Both fresh and cryopreserved cells were used and were injected intravenously, intraperitoneally, or both, after conditioning with cyclophosphamide. In addition, cells derived from a mouse spleen engrafted with human leukemia were passaged into another mouse. The animals were observed daily for signs of disease or appearance of tumors and sacrificed when terminally ill. At intervals blood samples were obtained and analyzed for the presence of human cells or DNA. Human leukemic cells were demonstrated by polymerase chain reaction (PCR) analysis of the human DQalpha gene or positive staining for human leukocyte common antigen (LCA). The presence of Epstein-Barr virus (EBV)-positive cells was also investigated by PCR analysis. Disseminated tumors developed in most mice inoculated with cells from the first patient, and this was associated with shortened survival times. The methods of administration, use of fresh or frozen samples, or the size of the inoculum had no effect on the development of leukemia. Survival of the mouse receiving passaged cells was similar to mice inoculated with fresh cells. Extensive histologic, immunophenotypic, and DNA studies were performed on organs from mice engrafting with cells from patient 1. PCR analysis for EBV sequences was negative in the mice engrafting from all three cases. The successful engraftment of human CLL-Pro PLL and T cell CLL in SCID mice, and the reproducibility of this effect using frozen cells, will provide a model for exploration of disease biology and for investigations of new drugs or combinations that may be useful in the treatment of CLL. PMID- 8637243 TI - Primary adult T cell leukemia of bone: two patients with primary bone lesion showing monoclonal integration of HTLV-I proviral DNA. AB - Adult T cell leukemia (ATL), a neoplasm of mature helper T lymphocytes is etiologically associated with human T lymphotropic virus type-I (HTLV-I). ATL cells infiltrate various organs, the lung, skin, central nervous system, gastrointestinal tract, and bone, causing various clinical manifestations. Two unusual cases of ATL, in which lytic bone lesion was the primary site of ATL, are described. One patient had multiple lytic lesions in bones without any involvement of other organs, and the other patient had a bone lesion in the right radius, which disappeared after chemotherapy. In both cases, monoclonal integration of HTLV-I provirus was demonstrated in the genomic DNA from each bone lesion. Although their clinical courses and pathological findings were different, ATL in both patients began as a bone lesion, showing that primary lymphoma of bone can be manifested in ATL cases. PMID- 8637245 TI - Germline configuration of the p27(Kip1) gene in childhood acute lymphoblastic leukemia (ALL). PMID- 8637246 TI - Simultaneous presence of t(11;14) and a variant Burkitt's translocation in the terminal phase of a mantle cell lymphoma. AB - Little is known about the clinical significance of secondary chromosome aberrations in lymphomas with t(11;14)(q13;q32), the characteristic change of mantle cell lymphomas. Here we present a patient with mantle cell lymphoma, who showed a variant Burkitt's translocation t(2;8)(p12;q24) in addition to t(11;14) during the progression of the disease. An involvement of chromosome 8q24, the localization of the c-myc gene, has so far been described in only four patients, who seemed to have a fatal clinical course. Although no blastic transformation occurred in our patient, no remission could be induce by intensified treatment and survival was only 5 months. This case demonstrates that secondary chromosome aberrations can determine the clinical course of patients, even if morphologic and immunophenotypic findings fail to predict the poor outcome. PMID- 8637247 TI - Hepatitis C virus infection in subsets of neoplastic lymphoproliferations not associated with cryoglobulinemia. AB - Hepatitis C virus (HCV) is both hepatotropic and lymphotropic and a clear-cut association has been proposed between HCV infection and mixed cryoglobulinemia (MC), a benign lymphoproliferative disorder, which sometimes evolves into a frank malignant B cell non-Hodgkin's lymphoma (B-NHL). Moreover, in the presence of antibodies to HCV, as well as of HCV-specific genomes has been reported in the sera of over 37% patients with B-NHL, not associated with MC. Thus, we decided to perform both a serologic and a molecular study to give insights into a possible relationship between HCV infection and neoplastic lymphoproliferations. We used ELISA and RIBA tests to show that anti-HCV antibodies were present in the serum of 29 out of 69 unselected B-NHL patients (42%), while seropositivity in a healthy population was about 1%. The prevalence of anti-HCV antibodies was low in definite subsets of B lymphoid disorders, including multiple myeloma, Waldenstrom's macroglobulinemia and monoclonal gammopathies of undetermined significance. Then, using reverse transcriptase polymerase chain reaction, we detected HCV sequences directly in the pathologic lymph node biopsies in 13 out of 34 B-NHL cases, and in particular in six out of eight low-grade lymphomas of MALT type and in five out of eight centroblastic-centrocytic follicular lymphomas. In contrast, the peripheral blood samples from 10 B cell chronic lymphocytic leukemia patients resulted negative for the presence of HCV genomes. Similarly, viral sequences were absent in 10 T cell NHL, while only one out of the 14 Hodgkin's disease cases tested resulted positive. Finally, we used a PCR based assay to characterize the genotypes (I-IV) present in the positive lymphomatous tissues. The presence of both serologic and molecular markers of HCV infection in a high percentage of certain types of B-NHL, not associated with cryoglobulinemia, and its absence from other lymphoproliferative diseases extends the spectrum of HCV-associated lymphoproliferations arguing in favor of some role of this viral infection in the pathogenesis of the malignant proliferation of definite B lymphoid populations. PMID- 8637248 TI - Absence of p18 mutations or deletions in lymphoid malignancies. AB - p18 is a recently described cyclin-dependent kinase inhibitor (CDK-I) wih homology to p16 and p15. The latter two CDK-Is have been implicated as possible tumor suppressor genes in a wide variety of human tumors, including hematological malignancies. Because of p18's structural and functional homology to p16 and p15, we hypothesized that it may also function as a tumor suppressor gene in some lymphoid malignancies. To explore this possibility we examined 81 primary lymphoid tumors for deletion and mutation p18. The primary tumors included 40 T cell malignancies and 41 B cell malignancies. None of the lymphoid tumors studied possessed deletions of p18, including a group of lymphoblastic lymphomas which we previously reported to have deletions of p16 and p15. PCR-SSCP analysis of the p18 gene identified a single polymorphism of codon 114, but failed to demonstrate mutations in any of the lymphoid tumors. These results do not support a role for p18 in the pathogenesis of the lymphoid neoplasms studied. PMID- 8637249 TI - Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay. AB - Quantification of X-chromosome inactivation patterns (XCIPs) using PCR amplification of the human androgen receptor (HUMARA) locus is potentially valuable in a range of haematological disorders. Of 236 females screened, 203 (86%) were heterozygous. For quantitative XCIPs it was necessary to limit the number of PCR cycles to 20 to reduce preferential amplification of shorter alleles. The optimized PCR method was compared with Southern blotting results using either PGK, HPRT or M27beta in 51 haematologically normal females and blast cells from 27 patients with acute myeloid leukaemia (AML). Reproducible XCIP results were obtained in all 78 samples using digestion with Hpa II prior to amplification (median difference in duplicate values 3%, range 0-17%) and they correlated well with Southern blotting results, r=0.966. Greater variability was observed in the results using Hha I digestion (median difference 4%, range 0 48%). There were marked inconsistencies in repeated analyses of three AML samples and although the HUMARA-Hha I results correlated well overall with Southern blotting in the remaining 75 samples (r=0.922), in nine samples there were still discrepancies with > or = 20% difference between the two values. These results suggest that PCR analysis of the HUMARA locus in Hpa II-digested DNA is suitable for the quantification of XCIPs in haematological samples but results with Hha I should be treated with caution. PMID- 8637250 TI - RT-PCR in acute promyelocytic leukemia: second workshop of the European Retinoic Group, Paris, France, 17-18 December 1994. AB - Reverse transcriptase-polymerase chain reaction is a recent technique in the diagnosis and assessment of minimal residual disease of acute promyelocytic leukemia, by amplification, of the different PML-RARalpha transcripts resulting from the t(15;17) translocation. The main issues addressed by the Second Workshop on PML-RARalpha-RT-PCR which took place in Paris, France on 17-18 December 1994, were related to (1) defining the specific pitfalls of the PML-RARalpha-RT-PCR, and means to improve the sensitivity of the technique; (2) the validity of PCR results obtained in CR to provide information on the extent of the disease; (3) the frequency and prognostic value of the different PML-RARalpha transcripts. PMID- 8637252 TI - Emergence of novel transient clonal chromosomal bone marrow changes in remission of acute lymphoblastic leukemia. PMID- 8637251 TI - Infant leukaemia biology, aetiology and treatment. PMID- 8637253 TI - Differentiating atypical HCL from HCL-Japanese variant. PMID- 8637254 TI - The Pine Ridge-Mayo National Aeronautics and Space Administration telemedicine project: program activities and participant reactions. AB - OBJECTIVE: To determine the response of participants to the Pine Ridge-Mayo National Aeronautics and Space Administration telemedicine project. DESIGN: We describe a 3-month demonstration project of medical education and clinical consultations conducted by means of satellite transmission. Postparticipation questionnaires and a postproject survey were used to assess the success of the activity. MATERIAL AND METHODS: Patients and employees at the Pine Ridge Indian Health Service Hospital in southwestern South Dakota and employees at Mayo Clinic Rochester participated in a telemedicine project, after which they completed exit surveys and a postproject questionnaire to ascertain the acceptability of this mode of health care. RESULTS: Almost all Pine Ridge and Mayo Clinic participants viewed the project as beneficial. The educational sessions received favorable evaluations, and almost two-thirds of the patients who completed evaluations thought the consultation had contributed to their medical care. More than 90% of the respondents from Pine Ridge and more than 85% of the respondents from Mayo Clinic Rochester said that they would recommend participation in this project to others. More than 90% of respondents from Pine Ridge and 80% of Mayo respondents agreed with the statement that the project should continue. CONCLUSION: These data suggest that a program of clinical consultation services, professional education, and patient education available by telemedicine might be viewed as beneficial. PMID- 8637255 TI - Diagnosis of chronic venous disease of the lower extremities: the "CEAP" classification. AB - OBJECTIVE: To test a new classification of chronic venous disease (CVD)--based on clinical, etiologic, anatomic, and pathophysiologic data (the CEAP system)--in a series of patients by using objective tests to establish all diagnoses. MATERIAL AND METHODS: The CEAP classification was applied to 102 extremities in 70 consecutive patients with CVD. Diagnoses were based on objective testing with continuous-wave Doppler studies, duplex scanning, plethysmography, venous pressure, and phlebography, which were applied selectively (the more invasive methods were reserved for cases of greater severity). RESULTS: Use of this classification provided an organized categorization of the key elements of the venous abnormalities in each case and clarified the interrelationships among the clinical manifestations, cause of the process, and anatomic distribution of involvement. For example, in this series of 102 extremities, 79% had primary venous disease, 18% had secondary disease, and 3% had congenital abnormalities. Ulcers were found in 7% of extremities with primary CVD and 44% with secondary CVD. Of the cases with ulceration, 43% were due to primary incompetence and 57% to postthrombotic disease. Reflux was the pathophysiologic problem in 86% of the total series and in 80% of ulcer cases. Similar relationships can be delineated for cases with varicose veins, edema, or skin changes. Study of the specific facets of the CEAP classification provided precise information about the cause and the effect of venous abnormalities that could be compared with cases in other series. CONCLUSION: Use of the CEAP classification with diagnoses determined by objective testing accurately identifies categories of CVD. The objective date provide a clear description of the abnormalities in each case and may be used for analyses of meaningful relationships between categories of CVD. Adoption of this objective method of classifying CVD will facilitate interinstitutional studies. PMID- 8637256 TI - Genetic expression of endothelial nitric oxide synthase in human atrial myocardium. AB - OBJECTIVE: To investigate the expression of endothelial nitric oxide synthase (eNOS) in human cardiac tissues. DESIGN: We attempted to determine the genetic expression and localization of eNOS in normal human atrial tissue. MATERIAL AND METHODS: In normal human right atrial tissues from five donors during cardiac transplantation, eNOS expression and localization were assessed by using Northern blot analysis, in situ hybridization, and immunohistochemical staining. RESULTS: Northern blot analysis and in situ hybridization demonstrated that eNOS messenger RNA is present in cardiomyocytes. Positive immunohistochemical staining was observed in the cytoplasm of cardiomyocytes. CONCLUSION: These studies show for the first time the genetic expression and distribution of eNOS in human atrial myocardium and suggest that the eNOS mediated paracrine and autocrine pathway may participate in the control of myocardial function in humans. PMID- 8637257 TI - Video-assisted thoracic surgical procedures: the Mayo experience. AB - OBJECTIVE: To describe an initial 3-year experience with video-assisted thoracic surgical procedures (VATS) at Mayo Clinic Rochester. DESIGN: We review the cumulative data on 771 VATS performed between June 1, 1991, and May 31, 1994, and assess the applications for this technique. MATERIAL AND METHODS: The indications for VATS, our techniques used, and the associated mortality and morbidity are summarized. In addition, the frequency of conversion of VATS to open procedures and the reasons for choosing this strategy are discussed. RESULTS: The 771 study patients (401 male and 370 female patients) had a median age of 62 years (range, 7 to 96). For all VATS. we used one-lung general anesthesia, without carbon dioxide insufflation. Indications for performing VATS were a pulmonary nodule in 333 patients, pleural effusion in 208, pulmonary infiltrate in 117, pneumothorax in 51, mediastinal mass in 22, pleural mass in 17, air leak in 13, and other in 10. The procedure was a wedge excision in 352 patients, examination of the pleural cavity in 128, pleural biopsy in 86, talc pleurodesis in 85, wedge excision and mechanical pleurodesis in 46, decortication in 27, excision of a mediastinal mass in 12, sympathectomy in 4, and other in 16. The rate of conversion of VATS to thoracotomy was 33.1% and did not change throughout the period of the study. The most common reasons for conversion were to complete a resection of a malignant lesion or to remove a deep nodule. The overall operative mortality was 1.9%. Complications occurred in 43 patients (8.3%) who underwent VATS without conversion to an open procedure and included prolonged air leak in 14, respiratory failure in 8, pneumothorax in 6, and atrial fibrillation in 5. The median hospitalization was 5 days (range, 1 to 104). CONCLUSION: VATS is safe and useful for selected thoracic conditions. We favor conversion to thoracotomy when curative resection of a malignant lesion is intended. PMID- 8637258 TI - Daniel Nathans--geneticist and microbiologist wins Nobel prize. PMID- 8637259 TI - Effect of nebulized lidocaine on severe glucocorticoid-dependent asthma. AB - OBJECTIVE: To determine whether nebulized lidocaine is a useful therapy in patients with severe glucocorticoid-dependent asthma. DESIGN: We prospectively conducted an open study of the effects of administration of nebulized lidocaine four times daily in 20 patients with asthma who had side effects of exogenous hypercortisolism. MATERIAL AND METHODS: The 18 women and 2 men, who were 19 to 71 years of age, all had severe asthma that necessitated both topical and systemic administration of glucocorticoids to control symptoms of airflow obstruction. Treatment consisted of nebulized lidocaine, 40 to 160 mg four times daily. Initially, all topical and systemic glucocorticoid regimens were maintained; if peak flow rates remained stable and symptoms of asthma were well controlled, orally administered glucocorticoid regimens were slowly reduced. RESULTS: Thirteen patients were able to discontinue oral use of glucocorticoids entirely, despite prolonged glucocorticoid dependence (mean 6.6 years and median 3 years for the 20 patients); 4 achieved reduction in their daily glucocorticoid requirement while maintaining control of symptoms of asthma (duration of glucocorticoid dependence for responders, mean 6.2 years and median 3.2 years). Three patients had no apparent response, as determined by their continued severe asthma symptoms and inability to reduce oral glucocorticoid requirements. CONCLUSION: These results suggest that nebulized lidocaine is a useful therapy for chronic asthma, allowing reduction or elimination of oral glucocorticoid therapy. PMID- 8637260 TI - Electron beam computed tomography and coronary artery disease: scanning for coronary artery calcification. AB - OBJECTIVE: To review the association of coronary artery calcification with coronary atherosclerosis and its potential clinical application as detected on electron beam computed tomography (EBCT). DESIGN: A literature review of coronary artery calcification, coronary artery disease, and EBCT is presented, and clinical applications of EBCT are discussed. RESULTS: Recent studies have confirmed that arterial calcification is an active process intimately associated with atherosclerotic plaque evolution. Clinical investigations with use of EBCT have shown that a scan "negative" for coronary calcification is common in patients with normal or near-normal findings on coronary angiography, whereas patients with severe obstructive disease most commonly have "positive" scans- greater amounts of coronary artery calcium are associated with more severe luminal disease. Coronary artery calcium as evaluated on EBCT follows patterns that reflect the development of coronary atheromatous disease as a function of age and gender. Although histologic studies have confirmed that not all atherosclerotic segments have detectable calcification, the area of coronary artery calcification quantified on EBCT has a direct, positive relationship with the histopathologic coronary plaque area. CONCLUSION: The long-held notion of "degenerative" calcification of the coronary arteries with aging is incorrect. Although the incidence of coronary artery calcification increases with patient age, this relationship simply parallels the increased incidence of coronary atherosclerosis with advancing age. Data suggest that EBCT is a highly sensitive and specific test for coronary atherosclerosis and provide a basis for clinical applications when EBCT is viewed as a noninvasive method to estimate human coronary atherosclerotic involvement and "plaque burden." PMID- 8637261 TI - Protein-losing enteropathy after the Fontan operation: successful treatment by percutaneous fenestration of the atrial septum. AB - Protein-losing enteropathy (PLE) after the Fontan operation is a life-threatening complication that may be refractory to medical therapy. Herein we describe a percutaneous atrial fenestration that was performed in a 42-year-old man with a double-inlet left ventricle who had undergone a Fontan operation 9 years earlier. Severe PLE developed, and despite frequent infusions of protein, his albumin level was 1.8 g/dL. The diagnosis of PLE was confirmed by an alpha(1)-antitrypsin clearance of 425 mL in 24 hours (normal 27 or less). Percutaneous atrial fenestration resulted in dramatic clinical improvement and resolution of the PLE. At 5-month follow-up, the patient's albumin level was 4.2 g/dL, his alpha(1) antitrypsin clearance was normal, and he was free of ascites and edema. PMID- 8637262 TI - Squamous cell carcinoma in the ovary of a 14-year-old girl. AB - Squamous cell carcinoma (SCC), whether primary or metastatic, rarely involves the ovary. All previously reported cases have been in adults. Herein we describe a 14 year-old girl who underwent extensive neurologic and musculoskeletal assessment because of symptoms of generalized muscle weakness and pain. She was found to have SCC that involved the ovary, with widespread metastases. This case illustrates the importance of eliciting a thorough history and evaluating the patient's symptoms. A review of the differential diagnosis of ovarian SCC is included. To our knowledge, our patient is the youngest to be described in the literature with SCC in the ovary. PMID- 8637263 TI - Spinal shock. AB - The term "spinal shock" applies to all phenomena surrounding physiologic or anatomic transection of the spinal cord that results in temporary loss or depression of all or most spinal reflex activity below the level of the injury. Hypotension due to loss of sympathetic tone is a possible complication, depending on the level of the lesion. The mechanism of injury that causes spinal shock is usually traumatic in origin and occurs immediately, but spinal shock has been described with mechanisms of injury that progress over several hours. Spinal cord reflex arcs immediately above the level of injury may also be severely depressed on the basis of the Schiff-Sherrington phenomenon. The end of the spinal shock phase of spinal cord injury is signaled by the return of elicitable abnormal cutaneospinal or muscle spindle reflex arcs. Autonomic reflex arcs involving relay to secondary ganglionic neurons outside the spinal cord may be variably affected during spinal shock, and their return after spinal shock abates is variable. The returning spinal cord reflex arcs below the level of injury are irrevocably altered and are the substrate on which rehabilitation efforts are based. PMID- 8637265 TI - A polymerase chain reaction-based test for spinal and bulbar muscular atrophy. AB - A novel type of genetic alteration, trinucleotide repeat amplification, is responsible for spinal and bulbar muscular atrophy. PMID- 8637264 TI - Use and interpretation of rheumatologic tests: a guide for clinicians. AB - In recent years, several new autoantibody tests have been developed and are being used in the field of rheumatology, including the antineutrophil cytoplasmic antibody (ANCA) and myositis-specific antibodies such as anti-Jo1. Positive test results for ANCAs reveal one of two basic staining patterns: cytoplasmic (c-ANCA) or perinuclear (p-ANCA). The Jo1 antibody test is often helpful at the time of diagnosis of a new case of idiopathic inflammatory myopathy. Herein this article reviews the clinical utility of the new tests in conjunction with the established autoantibody tests including antinuclear antibodies and extractable nuclear antibodies. Both the antinuclear antibody and extractable nuclear antibody tests are helpful in diagnosing connective tissue diseases. Before the results of any of these tests can be interpreted, the physician must consider the sensitivity, specificity, and negative and positive predictive values. Positive results must be analyzed in the clinical context and in relationship to other autoantibody test results. PMID- 8637266 TI - 38-year-old woman with left flank pain. PMID- 8637267 TI - Symposium on epilepsy. Foreword. PMID- 8637268 TI - A clinical approach to the classification of seizures and epileptic syndromes. AB - Seizures and epilepsies are heterogeneous. Their classifications are essential for clinicians to achieve a common understanding of the disorders. The diagnosis, treatment, and prognosis of seizure disorders depend on the correct identification of the types of seizures and epilepsy. The two currently accepted classifications are the International Classification of Epileptic Seizures and the International Classification of Epilepsies and Epileptic Syndromes. Both are based on clinical and electrophysiologic data, and both maintain a basic dichotomy between partial (focal, localization-related) and generalized epileptic disorders. Partial seizures are further classified into those that are simple partial, complex partial, and partial with secondary generalization. Generalized seizures are classified predominantly on the basis of their motor manifestations. Epilepsies are divided into idiopathic, cryptogenic, and symptomatic types. The utility and the limitations of these two classifications are discussed. A simplified system that encompasses neuroradiologic advances is offered to enhance the clinical usefulness of classifying epileptic disorders. PMID- 8637269 TI - Primary care: specialists or generalists. AB - A critical appraisal of the literature on the cost and quality trade-offs of primary care provided by specialists in comparison with that provided by generalists is presented. PMID- 8637270 TI - Telemedicine: an application in search of users. PMID- 8637271 TI - Classification and reporting standards for venous disease. PMID- 8637272 TI - Atrial natriuretic peptide and blood pressure. PMID- 8637273 TI - Electroconvulsive therapy. PMID- 8637275 TI - [Supply of blood to heart surgery patients. Can transfusion habits be changed?]. AB - OBJECTIVE: In cardiac surgery, patients might receive unnecessary blood transfusions, due to the lack of a uniform criteria for blood replacement. We set out to evaluate the effect of a transfusion education program on the homologous blood requirements of subjects undergoing cardiac surgery. METHODS: In June of 1993 a transfusion education program based on international criteria was designed and put into effect in the intensive care unit. Since that date, 133 patients (group 1), on whom we attempted to evaluate the effects of the program, were included, comparing them with 150 control patients (group 2) from the previous year. Multiple variables were analyzed, in order to confirm that the factors that could affect the use of blood did not differ between the two groups. The impact of these variables and the program on the homologous blood expenditures was measured with uni and multivariant analyses. RESULTS: Blood requirements of the patients during the first day of the postoperative period were 0.9 +/- 0.1 U in group 1 versus 1.8 +/- 0.1 U in group 2 (p < 0.001) and the requirements during their entire stay in ICU were 1.8 +/- 0.1 versus 2.6 +/- 0.1 (p < 0.005). There were significant differences between both groups: Hemoglobin level at the admission in ICU, anoxia time, extracorporal circulation time, and blood losses in the first 24 hours of their stay in ICU. However, the analysis of covariance showed that the transfusion program exerted an independent influence, apart from other variables, over the decrease of blood transfusions. Morbidity and mortality were similar in both groups. CONCLUSIONS: Transfusion habits can be modified, permitting a lesser consumption of homologous blood through the adoption of education programs adapted to the diseases in which they will be applied. PMID- 8637274 TI - [Internal medicine in a group of 52 Spanish hospitals. Analysis of a case series and efficiency]. AB - BACKGROUND: To analyze the case-mix and efficiency in a group of Services of Internal Medicine (IM) from 52 Spanish hospitals with routinary clinical information system. METHODS: From a total amount of 566,726 discharges during 1992, we studied the 71,430 corresponding to IM, analyzing the Minimum Basic Data Set and the Diagnosis-Related Groups (DRG). The hospitals were classified as level A (< 250 beds), B (251-599) and C (600 or over). We analyzed: Major Diagnostic Category (MDC); DRG, raw average length of stay (ALOS); outliers, trimmed ALOS; mortality; case-mix index; performance index; ajusted ALOS (by standard performance and case-mix); associated complications; number of diagnoses per discharge. A Chi-square test was used for qualitative variables and a mean comparison test (independent data) for the quantitative ones. RESULTS: Among the Internal Medicine Departments, IM was the Service that cared most patients, even more in the small-size hospitals. The most frequent DRG were: chronic obstructive pulmonary disease (COPD) 6.5% (DRG 088); simple pneumonia & pleuresy 6.1% (DRGs 089-090); heart failure 5.9% (DRG 127); stroke 4.5% (DRG 014); and angor pectoris 2.4% (DRG 140). The ALOS was shorter in the A-level hospitals (10.1 days) vs. B level (11.6) and C-level (14.4). However, the trimmed ALOS was more homogeneous (A 8.8; B 9.9; C 11.7). The complexity indexes increased with the hospital level although the performance index also increased. CONCLUSIONS: The IM services provide care for a high percentage of hospital patients, especially in the small size centers. In these, less complex patients are seen, with decreased comorbidity, complications and mortality and they solve their patients with shorter ALOS than the IM services in the large-size hospitals. PMID- 8637277 TI - [Internal medicine at the end of the century]. PMID- 8637276 TI - [Risk factors in stable coronary disease. Relationship with ischemic threshold and prognostic implications]. AB - BACKGROUND: To assess the clinical evolution, in a 5-year follow-up, of the development of acute coronary syndromes, in patients with angina clinically stable, establishing its relationship with the ischemic threshold (IT) and the main modifiable cardiovascular risk factors. PATIENTS AND METHODS: 120 patients, 86 males (71%), with an age of 57 +/- 8 years, with stable angina. The presence of smoking, hypercholesterolemia, hypertension and diabetes mellitus was evaluated. We performed exercise testings (baseline and after vasodilator drugs) in the beginning, in order to characterize the IT (which was fixed in 72 patients and variable in 48). The later group underwent exercise testing each term during the first year of follow-up. Lesion at least of 70% in a main coronary vessel was required as inclusion criteria. The development of acute myocardial infarction (AMI), unstable angina pectoris and cardiac death was recorded. Cox's hazard function analysis and multivariant analysis were applied. RESULTS: 106 patients (88%) had one or more risk factors (40% hypertension, 43% hypercholesterolemia, 22% diabetes mellitus and 56% were smokers or ex-smokers). A significant association was shown between male gender and smoking and diabetes mellitus and female gender. 6 cardiac deaths, 8 AMI and 9 unstable angina were recorded. Within the 72 patients with fixed IT, 12.5% (9) suffered some acute syndrome. In the 48 with variable IT, in the 30 who continued in it, 20% (6) developed acute coronary pathology and in the 18 who modified their IT to fixed, the prevalence was 44.5% (8). Patients with modification of IT to fixed had a higher risk of acute coronary syndrome in the follow-up (p < 0.01) and the presence of hypertension and hypercholesterolemia allowed the prediction of modification of the IT. CONCLUSIONS: The prevalence of cardiovascular risk factors in the stable coronary artery disease is high (88%); male gender is associated smoking and female gender to diabetes mellitus. The presence of hypertension and hypercholesterolemia are associated to modification in the IT in patients with variable threshold, allowing the detection of a subgroup of high risk for the development of acute coronary pathology. PMID- 8637278 TI - [Pleural cryptococcosis in patients with human immunodeficiency virus infection]. AB - Four cases of pleural cryptococcosis as the form of onset of cryptococcosis in patients with human immunodeficiency virus (HIV) infection are reported. In two out of the four cases cryptococci were simultaneously isolated in other localizations (blood and meninges). In the two remaining patients the pleura was the only site of the disease, with serum determination of the cryptococci antigen being negative in one. The four patients evolved favourably, with three being exclusively treated with fluconazol. Pleuritis is an infrequent manifestation in cryptococcosis although it may be the form of onset and the only localization of the disease. Only 10 cases have been reported in patients with HIV infection. The present four cases represent 11% of the authors' series of cryptococcosis in AIDS patients. The diagnostic possibility of cryptococcosis should be considered in patients with human immunodeficiency virus infection presenting pleural effusion. PMID- 8637279 TI - [Evaluation of the therapeutic risk]. PMID- 8637280 TI - [Oral leukoplakia]. PMID- 8637281 TI - [The polyvalent internist: needs and opportunity]. PMID- 8637282 TI - [The primary care physician, the specialist and the internist? Keys to a crisis. Possible answers]. PMID- 8637283 TI - [Anaphylactic shock after ingestion of a laxative: usefulness of an allergologic study]. PMID- 8637284 TI - [The Oregon health care plan: beyond the experiment]. PMID- 8637285 TI - [Impact of hospitalization on drug consumption]. PMID- 8637286 TI - [Headache in an epidemic of multiple sclerosis: should other causes be excluded?]. PMID- 8637287 TI - [Thrombocytopenia and acute pancreatitis: the relationship with appearance of complications]. PMID- 8637288 TI - [p53 in Hodgkin's disease]. PMID- 8637290 TI - [Green-blue urine, an important sign of possible intoxication with a suicidal intent]. PMID- 8637289 TI - [Arthropathy associated with parvovirus B19 infection]. PMID- 8637291 TI - [Infection of sternal wound in heart surgery: analysis of 1000 operations]. AB - BACKGROUND: Sternal wound infection (SWI) is the most important complication in cardiac surgery. The aim of this study was to describe the frequency and clinical and microbiological features of this complication. METHODS: All the cases of SWI which were observed in the authors' hospital in the first 1,000 cardiac surgery operations performed with extracorporeal circulation were retrospectively reviewed. The cases were identified through the Infectious Diseases and Cardiac Surgery Department files and were classified according to the depth of the infection. During the study period neither the prophylaxis against infection nor the surgical techniques were modified. RESULTS: Forty-three patients (4.3%) presented SWI. Fourteen were superficial infections and 29 were deep infections of which 9 were classified as osteomyelitis and 20 as mediastinitis. A progressive decrease was observed in the proportion of SWI over time parallel to an increase in the number of operations performed. Staphylococcus aureus was the agent most frequently isolated (60.4%). Gram-positive aerobic cocci were found in 66.7% of the total number of isolations, being most frequent in the deep infections (83.3% of the isolations). The gram-negative aerobic bacilli were isolated more frequently in the superficial infections than in the deep infections (57.8% v.s. 16.7% of the isolations, respectively p < 0.01). In patients with SWI the predictive value of the positive blood cultures for the diagnosis of mediastinitis was 83.3%, with a sensitivity of 50% and specificity of 91.3%. Three patients with deep infection developed chronic complications and another three died (mortality by mediastinitis 15.0%). The mean postoperative stay was 52 days for the patients with deep infection and 39 days for those with superficial infection (p = NS). CONCLUSIONS: The percentage of surgical wound infection during the study period showed a trend to a decrease parallel with an increase in the number of operations. The gram-positive bacteria were responsible for most of the SWI. Although the depth of SWI is difficult to clinically predict, the presence of bacteremia suggests the existence of mediastinitis. Despite their lesser clinical importance, the superficial infections carry a long postoperative stay. PMID- 8637292 TI - [Relationship between blood levels of sex hormone binding globulin, testosterone levels in blood and saliva and body morphology in premenopausal women]. AB - BACKGROUND: The concentrations of the globulin binding of sexual hormones (GBSH) and serum and saliva testosterone were studied, as well as the free testosterone index (FTI), in a group of 75 premenopausic women of 42 years of age. These concentrations were correlated with the body mass index (BMI) and the relationship between the waist and hip perimeters (WHI) as an index of body fat distribution. MATERIAL AND METHODS: Anthropometric measurements were performed in all the women studied. The BMI was used to classify the women as obese or not obese, while the WHI was used to classify fat distribution as android or gynoid type. The hormone determinations were carried out by radioimmunoassay and the GBSH by fluorescence immunoassay. RESULTS: A significant positive correlation was observed between FTI and BMI (p = 0.024) and a negative correlation was seen between GBSH and WHI (p = 0.024). A significant decrease was found in the GBSH levels in obese women with respect to those who were not obese (p = 0.025) and between those with android fat distribution versus gynoid fat distribution (p = 0.032). CONCLUSIONS: The modifications observed in the concentrations of the globulin binding sexual hormone in obese premenopausic women and the absence of the same in the free testosterone indexes may be explained because of other steroids such as androstenodiol and dehydrotestosterone are elevated. The measurement of globulin binding sexual hormone concentrations may be an early marker of alteration in the androgenic status in obese women. PMID- 8637293 TI - [Treatment of hyperlipemia: opinions of primary care physicians of the La Rioja region]. AB - BACKGROUND: Primary health care physicians (PHCP) play a fundamental role in the detection and treatment of hyperlipemias in a general population. The aim of this study was to estimate the opinion of the PHCP in the Autonomic Community of La Rioja, Spain, regarding desirable lipemic values and treatment of hyperlipemias. METHODS: A survey with personal interview was carried out to all the PHCP in the Spanish National Health Care (INSALUD) in La Rioja in May and June 1994. RESULTS: Of the 177 PHCP included 97% completed the questionnaire. The main coronary risk factor for 46% of physicians was high blood pressure followed by serum cholesterol (30%). Fifty-six percent of the physicians believed that the desired populational cholesterolemia should be less than 200 mg/dl and this percentage rose if the physician worked in an urban environment (p < 0.01). Sixty-six percent considered 200 mg/dl as the desired triglyceridemia. For treatment and follow-up of hyperlipemia, 78% of physicians use cholesterol linked to low density lipoproteins (cLDL). This use of cLDL increased among those who were familiar with the recommendations of the Spanish Society of Arteriosclerosis (p < 0.002) or who worked in health care centers (p < 0.02). Regarding initiation of dietetic treatment most chose cholesterolemia between 200-250 mg/dl, a cLDL value between 150-185 mg/dl and triglyceridemia between 200-250 mg/dl. For the use of pharmacologic treatment these levels were 250-300 mg/dl, 150-185 mg/dl and 250 300 mg/dl, respectively. Ninety-nine percent of physicians indicated diet as the first therapeutic measure. In isolated hypercholesterolemia the resins were most used by the Family Medicine specialists (p < 0.002) and fibrates (p < 0.03) and statins (p < 0.02) the least used. The use of statins was lower in the physicians working in health care centers (p < 0.03). CONCLUSIONS: Knowledge of theshold values of serum cholesterol and triglicerides levels and the use of drugs among the primary health care physicians from La Rioja, Spain, are generally correct. The physicians with good knowledge of the National Consensus Guides on hyperlipemias had the most adequate opinion. PMID- 8637294 TI - [Interferences of human solidarity in intensive care]. PMID- 8637295 TI - [French words of misleading translation in medicine]. PMID- 8637296 TI - [Human babesiosis in a patient after splenectomy]. AB - A case of human babesiosis in a 34-year-old male patient with previous splenectomy is reported. This is the first description of babesiosis in a splenectomized patient in Spain. The clinical picture was mainly characterized by the presence of fever and hemolysis. The findings of a reactive hemophagocytic syndrome and lymph node enlargement, the description of which is exception in this entity are of note. The therapeutic aspects are reported emphasizing the good response to treatment with clindamycin and quinine sulphate which is unusual in the splenectomized cases reported in Europe. The diagnostic tods as well as the epidemiologic implications of the description of this zoonosis in Spain are discussed. PMID- 8637298 TI - [Murcia: the highest cardiovascular mortality in Spain?]. PMID- 8637297 TI - [Fever and abdominal pain in a 17-year-old patient]. PMID- 8637299 TI - [Importance of lactate dehydrogenase-C4 in the follow up of men after varicocelectomy]. PMID- 8637301 TI - Risk factors for the development of chronic obstructive pulmonary disease. AB - Cigarette smoking clearly has been shown to be the major environmental risk factor predisposing to the development of COPD. Occupational exposures to dust and fumes, air pollution, passive smoke exposure, childhood respiratory infections, and diet may also contribute. Airway hyperresponsiveness is a risk factor for the development of decline in FEV1, but its role in the development of COPD remains uncertain. Alpha1-antitrypsin deficiency is an important genetic risk factor for COPD in the small minority of COPD patients who inherit this deficiency. Other genetic factors are likely involved but have not yet been identified. Elucidation of additional genetic risk factors may provide useful insights into the pathogenesis of COPD. Potential interactions between the various environmental and genetic risk factors may be extremely important in determining the variable development of COPD. PMID- 8637300 TI - [Anaphylaxis caused by cefixime with tolerance to other beta lactams]. PMID- 8637303 TI - Diffusing capacity in the clinical assessment of chronic airflow limitation. AB - CAL remains an important cause of morbidity and mortality. The diffusing capacity has ranked high in the assessment of CAL because it represents the best pulmonary function test to assess the integrity of the pulmonary capillary bed. Unfortunately, numerous physiologic, pathologic, and technical factors affect the test, thus limiting its sensitivity and specificity. HRCT techniques offer the potential to assess the extent of emphysema more accurately, but the technique requires greater standardization and is more expensive and less noninvasive than DLcoSB testing. Although the CIBA symposium considered DLcoSB "essential" in the investigation of the CAL patient, 16 the use of conventional DLcoSB testing in the seated position at rest is not currently advised as a routine screening procedure. The test must be performed in a center with high degree of quality control, and the results can be of value only by integrating the result into a comprehensive clinical assessment. Within this context, conventional DLcoSB testing may provide limited information about the extent of emphysema because reductions in DLcoSB correlate with the extent of emphysema by HRCT. When DLcoSB is normal, it may point in the direction of considering asthma as the cause of the airflow limitation. It may also provide information about disease severity and prognosis in O2-dependent CAL patients. The test should be a part of the investigation of the patient with unexplained dyspnea. It remains controversial how emphysema correlates with the degree of impairment in CAL, and further work needs to be done to clarify this relationship. This requires a reexamination of current CT methods 110 and the relationship between DLcoSB, structural changes in the lung, and HRCT evidence of emphysema. Refinements in DLcoSB testing methods, such as the measurement of DLcoSB-3EQ are linked to rapidly responding CO analyzers and computer-driven software, which will potentially improve the accuracy and reproducibility of the test, particularly in the presence of airway obstruction and nonuniform distribution of ventilation. Such refinements, which offer the possibility that tests of diffusion could become more useful markers of disease, include measuring DLcoSB when the pulmonary capillary recruitment is near maximal (head-down position, exercise), enhancing the sensitivity of the test to alterations in the lung periphery, standardizing previous volume history, developing more precise corrections for Hb and COHb, and developing an index of diffusion nonuniformity. PMID- 8637302 TI - Early intervention in chronic obstructive pulmonary disease. A review of the Lung Health Study results. AB - The major findings of the LHS that have been reported thus far are that an effective smoking cessation program can be developed that can produce more than a 20% success rate in getting smokers to give up the habit permanently, and that by stopping smoking, individuals with early COPD benefit by having an initial improvement in lung function and a slowing of the annual loss of their FEV1. The use of a bronchodilator has a short-term effect in improving the FEV1, but it does not affect long-term changes in lung function. AHR is common in patients with mild-to-moderate COPD. The reward for a smoker to give up the habit is an initial gain in FEV1 and a subsequent close to normal annual rate of decline of this pulmonary function parameter. These results should provide a positive incentive for smokers to quit and thereby decrease the morbidity and mortality caused by the use of tobacco. PMID- 8637304 TI - Clinical exercise testing in chronic airflow limitation. AB - Exercise testing has become an essential tool in the management of patients with CAL. In addition to its ability to assess exercise limitation objectively, it has usefulness in detecting the presence or absence of associated disease processes, in assessing the response to therapies, in allowing assessment of the importance of psychological factors in exercise limitation, and in guiding prescription for exercise rehabilitation programs. Although much is known about the clinical usefulness of exercise testing in this disease, and much has been learned about how this disease functionally impairs the exercise capacity of the patient, additional study is necessary to appreciate fully the physiologic abnormalities demonstrated by patients with CAL during exercise. PMID- 8637305 TI - Current thoughts regarding treatment of chronic obstructive pulmonary disease. AB - The available evidence indicates that pulmonary rehabilitation benefits patients with symptomatic COPD. The effect of pulmonary rehabilitation programs on health care use is promising but requires further investigation. In contrast, aerobic lower extremity training is of benefit in several areas of importance to patients with COPD. These areas include exercise endurance, perception of dyspnea, quality of life, and self-efficacy. The exact role of upper extremity exercise training programs requires further studies but should be used in patients who develop symptoms with arm activities. Psychological support improves the awareness of the patient and increases his or her understanding of the disease, but when used alone it is of limited value. Pulmonary rehabilitation when coupled with smoking cessation, optimization of blood gases, and medications offers the best treatment option for patients with symptomatic airflow obstruction. PMID- 8637306 TI - Home oxygen therapy. AB - Both the efficacy and the indications for LTOT have been well defined. Most of the studies performed have focused on patients with hypoxemia caused by COPD, and the benefits observed are assumed to apply to all patients with correctable hypoxemia. For Medicare patients, oxygen is reimbursed under a prospective payment system with all delivery systems considered to be cost and therapeutically equal. Because there are, in fact, substantial clinical differences in the medical indications for individual oxygen delivery systems, it is imperative that the prescribing physician be prepared to order the therapy that is most appropriate for each patient. Most home oxygen therapy is now being ordered by primary care physicians, often functioning as gatekeepers in managed care organizations. Education of primary care physicians in this area is often inadequate, and decisions for therapy should not be delegated to the equipment suppliers. If the study of home oxygen therapy conducted by the Office of the Inspector General were repeated today, less misuse of home oxygen would probably be found because of more clearly defined indications and requirements for therapy, but it is likely that the study would find that the level of knowledge of the prescribing physician has not maintained pace with the advances in technology. Continuing education for primary care physicians in this area of respiratory care is essential for appropriate medical management now and in the future. PMID- 8637307 TI - Lung reduction surgery in chronic obstructive lung disease. AB - In the 1960s the promise of the Brantigan lung reduction surgery was shattered when it was shown that the improvement in airway conductance drifted back towards the preoperative value over a period of 12 to 18 months. Since then there has been a marked improvement in our understanding of emphysema, its pathology, and techniques for obtaining images of the lung. In addition, reliable automated cardiopulmonary and physiologic testing, advances in critical care medicine, and new pharmacologic agents have improved patient care. Surgical techniques now allow better control of air leaks and access to anatomic regions not previously accessible. The combination of all of the above makes lung reduction surgery worth re-examining as a palliative procedure for severely symptomatic patients. Clearly, it is not a panacea but can in some cases produce dramatic improvements in symptomatology and quality of life. This article presents the available data describing potential mechanisms of improvement and clinical outcomes following lung reduction surgery. It also outlines areas that need further work, such as patient selection and surgical techniques. PMID- 8637308 TI - Lung cancer and chronic obstructive pulmonary disease. AB - The application of current knowledge and technology could dramatically improve the survival rate in both lung cancer and COPD, even before physicians and other health workers are finally able to convince the population that both personal and environmental smoke must be eliminated to begin to reduce the premature morbidity and mortality from lung cancer, airflow obstruction, and other smoking-related diseases such as heart attack and stroke. PMID- 8637310 TI - [A new prescription form introduced to prevent falsifications]. PMID- 8637309 TI - Lung transplantation in chronic airflow limitation. AB - Lung transplantation is an option for appropriately selected patients with end stage chronic airflow limitation. The functional results have been excellent after single or bilateral lung transplantation, and the medium-term survival results have been good. Obliterative bronchiolitis, however, thought to be a manifestation of chronic rejection, occurs in approximately 40% of recipients, and it is the major cause of late morbidity and mortality in lung transplant recipients. PMID- 8637311 TI - [A French physician is at risk to be suspended because of a book about Mitterand's disease]. PMID- 8637312 TI - [The myocardial infarction group: secondary prevention in primary health care for the aged is needed]. PMID- 8637314 TI - [Unconventional medicine a topic for European cooperation]. PMID- 8637313 TI - [Medical tests in occupational environment are not uncontroversial. Consider the alternatives, and let the committee decide!]. PMID- 8637315 TI - [Unfortunate choice of words in the case of rectal cancer]. PMID- 8637316 TI - [Measure bone density, but follow up the result]. PMID- 8637317 TI - [Depressions among adolescents are not always correctly diagnosed]. PMID- 8637318 TI - [Health care indicators are needed]. PMID- 8637319 TI - [It is justified to question the diabetes registry]. PMID- 8637320 TI - [News about HIV. Combination therapy with new drugs gives hope]. PMID- 8637321 TI - [Ultrasound in anorectal diagnosis]. PMID- 8637322 TI - [Heroin is now the most popular drug among new addicts]. PMID- 8637323 TI - [Take neonatal jaundice seriously!]. PMID- 8637324 TI - [Angioedema following treatment with ACE inhibitors]. PMID- 8637325 TI - [Physician in the cyberspace. Internet is cheap and convenient for communication as well as training]. PMID- 8637327 TI - ["Concealed" decisions to restrain from cardiopulmonary resuscitation. Unified symbols and plain language should be required]. PMID- 8637326 TI - [The Tule-test is a good test of physicians' clinical competence. Low unemployment among those who passed the examination]. PMID- 8637328 TI - [Premature infants are followed up to the age of 5 years]. PMID- 8637329 TI - [Obese patients on the operating table. A laparoscopic technique is under way]. PMID- 8637330 TI - [Drugs are the most common cause of gynecomastia]. PMID- 8637331 TI - [Spektramox and liver damage]. PMID- 8637332 TI - [The psyche and the skin suffer most under the effect of Relifex]. PMID- 8637333 TI - The truth of torture. PMID- 8637334 TI - Diet, breast cancer, and case-control studies. PMID- 8637335 TI - Are we ready for factor V Leiden screening? PMID- 8637336 TI - Diseases that masquerade as motor neuron disease. PMID- 8637337 TI - Air ambulances: missions of mercy or flights of futility? PMID- 8637338 TI - HIV epidemic in India: opportunity to learn from the past. PMID- 8637339 TI - Intake of macronutrients and risk of breast cancer. AB - BACKGROUND: The association between risk of breast cancer and dietary fat and intakes of other energy sources remains controversial. The Italian population offers special opportunities to assess the influence of high intakes of unsaturated fat and starch and, because the population has low awareness of diet and cancer issues, there is less scope for recall bias. We have assessed the relations of various macronutrient intakes with risk of breast cancer. METHODS: In this case-control study, 2569 women with incident breast cancer (median age 55 years) and 2588 control women (median age 56 years) in hospital with acute, non neoplastic diseases, were interviewed in six different areas of Italy between 1991 and 1994. A validated food-frequency questionnaire was used. It included questions on 78 foods and recipes grouped into six sections, as well as specific questions on individual fat intake pattern. FINDINGS: The risk of breast cancer decreased with increasing total fat intake (trend p 0.01) whereas the risk increased with increasing intake of available carbohydrates (trend p = 0.002). The odds ratios for women in the highest compared with the lowest quintile of energy-adjusted intake were 0.81 for total fat and 1.30 for available carbohydrates. Starch was the chief contributor to the positive association with available carbohydrates. High intakes of polyunsaturated and unsaturated fatty acids (i.e., polyunsaturated fatty acids plus oleic acid) were associated with a decreased risk of breast cancer (odds ratios for highest vs lowest quintile 0.70 and 0.74, respectively). Conversely, the intakes of saturated fatty acids, protein, and fibre were not significantly associated with breast-cancer risk. INTERPRETATION: This case-controls study shows that unsaturated fatty acids protect against breast cancer, possibly because intake of these nutrients is closely correlated with a high intake of raw vegetables. The findings also suggest a possible risk in southern European populations, of reliance on a diet largely based on starch. PMID- 8637340 TI - Randomised, controlled trial of low-dose heparin for prevention of fatal pulmonary embolism in patients with infectious diseases. The Heparin Prophylaxis Study Group. AB - BACKGROUND: Fatal pulmonary embolism and other thromboembolic complications are common in hospital inpatients. However, there is little evidence on the routine use of pharmacological thromboprophylaxis in non-surgical patients. We assessed the efficacy and safety of low-dose heparin in the prevention of hospital acquired, clinically relevant, fatal pulmonary embolism in patients with infectious diseases. METHODS: Our study used the postrandomisation consent design. 19,751 consecutive patients, aged 55 years or older, admitted to departments of infectious diseases in six Swedish hospitals, were screened for inclusion in the randomised, controlled, unblinded, multicentre trial. Of the eligible patients, 5776 were assigned subcutaneous standard heparin (5000 IU every 12 h) until hospital discharge or for a maximum of 3 weeks; 5917 were assigned no prophylactic treatment (control group). We sought consent only from the heparin group. Follow-up was for 3 weeks after discharge from hospital or for a maximum of 60 days from randomisation. The primary endpoint was necropsy verified pulmonary embolism of predefined clinical relevance. FINDINGS: By intention-to-treat analysis mortality was similar in the heparin and control groups (5.3 vs 5.6%, p = 0.39) and the median time from admission to death was 16 days in both groups (IQR 8-31 vs 6-28 days). Necropsy-verified pulmonary embolism occurred in 15 heparin-treated and 16 control-group patients. There was a significant difference between heparin and control groups in median time from randomisation to fatal pulmonary embolism (28 [24-36] vs 12.5 [10-20] days, p = 0.007). This difference corresponds to the duration of heparin prophylaxis. Non fatal thromboembolic complications occurred in more of the control than of the heparin group (116 vs 70, p = 0.0012). INTERPRETATION: Our findings do not support the routine use of heparin prophylaxis for 3 weeks or less in large groups of non-surgical patients. Further studies are needed to investigate whether heparin prophylaxis of longer duration may prevent fatal pulmonary embolism. PMID- 8637341 TI - Which groups of patients benefit from helicopter evacuation? AB - BACKGROUND: The evacuation of emergency cases by air, usually by helicopter, is controversial because of the cost of the programme, the possibility of an accident (especially in an urban area), and unproven benefit. But such evacuations cannot be studied by a random intervention (eg, air versus ground ambulance). We used an expert-panel approach to estimate the health outcome for patients transferred by emergency helicopter compared with the potential outcome if they had gone by surface ambulance. METHODS: The helicopter programme is based at the University Hospital of Tromso in northern Norway. 370 case-reports of helicopter evacuation from rural areas were screened by anaesthetists for routine and case-specific data. Two expert panels assessed the cases for potential additional health benefit arising from the fact of helicopter evacuation. The panels used a modified Delphi technique to reach consensus in life-years gained. One panel met for cases aged under 15 and pregnant women, the other for older cases. FINDINGS: 240 of the 370 cases were male (65%); the age range for both sexes was 0-86 years. The most common diagnosis for the 55 cases aged under 15 was infection (49%); in older patients, cardiovascular disease dominated (50%). Trauma accounted for just under a fifth of cases in both groups. On average, the patients arrived 69 min (range 0-615) earlier in hospital than if they had gone by ground transport. For 283 cases, the initial screening by the anaesthetists indicated no additional benefit compared with that obtainable by ground-ambulance transport. The main reason was that no treatment was given during the flight or early on in hospital that could not have been given otherwise. 90 cases entered the expert panel system. Of these 90, 49 cases were judged to have received no additional benefit. This left 41 (11% of the total of 370 evacuated) who were judged to have benefited, gaining 290.6 life-years. 96% of the total number of life-years gained was achieved in nine patients, six of whom were aged below 7 (four were aged 0-7 months). The life-year-gain per adult patient with cardiovascular disease was 0.54. INTERPRETATION: We conclude that an emergency helicopter service can provide considerable health benefits for selected patients, at least in this rural setting. Given the costs and risks of such a service, the benefits for most patients are small. PMID- 8637342 TI - Identification of the enzyme responsible for oxidative halothane metabolism: implications for prevention of halothane hepatitis. AB - BACKGROUND: Fulminant hepatic necrosis ("halothane hepatitis") is an unusual and often fatal complication of halothane anaesthesia. It is mediated by immune sensitisation in susceptible individuals to trifluoroacetylated liver protein neoantigens, formed by oxidative halothane metabolism. The seminal event in halothane hepatitis is hepatic metabolism, yet the enzyme responsible for oxidative halothane metabolism and trifluoroacetylated neoantigen formation remains unidentified. This investigation tested the hypothesis that cytochrome P450 2E1 (CYP2E1) is responsible for human halothane metabolism in vivo. METHODS: 20 elective surgical patients received either disulfiram (500 mg orally, n = 10) or nothing (controls, n = 10) the night before surgery. Disulfiram, converted in vivo to an effective inhibitor of P450 2E1, was used as a metabolic probe for P450 2E1. All patients received standard halothane anaesthesia (1.0% end-tidal, 3 h). Blood halothane and plasma and urine trifluoroacetic acid, bromide, and fluoride concentrations were measured for up to 96 h postoperatively. FINDINGS: Total halothane dose, measured by cumulative end-tidal (3.8 SE 0.1 minimum alveolar concentration hours) and blood halothane concentrations, was similar in the two groups. Plasma concentrations and urinary excretion of trifluoroacetic acid and bromide, indicative of oxidative and total (oxidative and reductive) halothane metabolism, respectively, were significantly diminished in disulfiram treated patients. In control and disulfiram-treated patients cumulative 96 h postoperative trifluoroacetic acid excretion was 12,900 (SE 1700) and 2010 (440) mumol, respectively (p < 0.001) while that of bromide was 1720 (290) and 160 (70) mumol (p < 0.001). INTERPRETATION: The substantial attenuation of trifluoroacetic acid production by disulfiram after halothane anaesthesia suggests that P450 2E1 is a predominant enzyme responsible for human oxidative halothane metabolism. Inhibition of P450 2E1 by a single preoperative oral disulfiram dose greatly diminished production of the halothane metabolite responsible for the neoantigen formation that initiates halothane hepatitis. Single-dose disulfiram may provide effective prophylaxis against halothane hepatitis. PMID- 8637343 TI - Diet, acetylator phenotype, and risk of colorectal neoplasia. AB - BACKGROUND: Inherited or acquired differences in metabolic pathways that activate or inactivate dietary carcinogens may influence the risk of developing cancer. A polymorphism in N-acetyltransferase classified people into fast and slow acetylators. This enzyme catalyses the formation of mutagenic products from foodstuffs, especially cooked meat and fish. Some data suggest that fast acetylators are at higher risk of colorectal cancer. We have studied the adenoma and cancer risk in relation to meat intake and acetylator status. METHODS: In a case-control study, we compared 110 patients with colorectal cancer, 89 patients with colorectal adenomatous polyps, and 110 controls. Acetylator status was assessed by the rate of acetylation of sulphamethazine given orally. FINDINGS: The fast-acetylator phenotype was associated with odds ratios of 1.1 (95% Cl 0.6 2.1) and 1.8 (1.0-3.3) for adenoma and colorectal cancer, respectively. The highest risk occurred in the youngest tertile (< 64 years) of cases (2.5 [0.7 9.4] and 8.9 [2.6-30.4], respectively). There was no difference between the sexes. The risk of adenoma or cancer increased with increasing intake of meat in fast but not in slow acetylators: covariate-adjusted odds of disease over three levels of meat consumption were 2.1 (0.9-4.7) for adenoma, 1.7 (0.9-3.5) for cancer, and 1.9 (1.0-3.7) for all tumours. INTERPRETATION: Our findings indicate that acetylator status modulates the risk of colorectal neoplasia associated with meat intake. PMID- 8637344 TI - Resistance to activated protein C in an unselected population of patients with pulmonary embolism. AB - BACKGROUND: Resistance to activated protein C (APC) is the most frequent cause of inherited thrombophilia. This phenomenon has been reported in 10-50% of selected patients with venous thromboembolism, a variation that might result from different degrees of selection in different reports. METHODS: We measured the APC sensitivity ratio in 494 frozen blood samples from unselected consecutive outpatients suspected of pulmonary embolism and referred over a 30-month period to the emergency ward of the University Hospital of Geneva, the only public primary-tertiary care hospital in the region of Geneva (400,000 inhabitants). FINDINGS: Prevalence of resistance to APC was 5.5% (95% Cl 2.4-10.5%) (8/146) in patients with confirmed pulmonary embolism and 4.0% (2.2-6.7%) (14/348) in patients in whom the diagnosis could be ruled out (p = 0.66), giving an odds ratio of 1.36 (0.56-3.32). INTERPRETATION: The very different risks of venous thromboembolism in the presence of resistance to APC that have been reported in trials published so far are probably due to variable recruitment conditions. The lower prevalence observed in our totally unselected population of patients with pulmonary embolism may be more representative of the real risk with which clinicians will be confronted. Therefore, more data are needed from various populations of patients with venous thromboembolism to help decide which patients will benefit from screening for resistance to APC. PMID- 8637345 TI - A young man who liked lizards and lost his job. PMID- 8637346 TI - The war on cancer. AB - 25 years ago, then President Nixon "declared" War on Cancer. In this personal commentary, the war is reviewed. There have been obvious triumphs, for instance in cure of acute lymphocytic leukaemia and other childhood cancers, Hodgkin's disease, and testicular cancer. However, substantial advances in molecular oncology have yet to impinge on mortality statistics. Too many adults still die from common epithelial cancers. Failure to appreciate that local invasion and distant metastasis rather then cell proliferation itself are lethal, obsession with cure of advanced disease rather than prevention of early disease, and neglect of the need to arrest preneoplastic lesions may all have served to make victory elusive. PMID- 8637347 TI - The Ingelfinger rule, embargoes, and journal peer review--Part 1. AB - It is 27 years since Dr Franz Ingelfinger announced that a manuscript would be rejected by his journal, the New England Journal of Medicine, if it had been published elsewhere. Many other medical journals have since adopted this so called Ingelfinger rule. The restrictions resulting from the rule have generated enormous controversy in medical journalism, as shown by the first of the two-part article The Ingelfinger rule, embargoes, and journal peer review. Critics say that the rule restricts the free flow of information, whereas proponents claim that information from a paper released early may be inaccurate because the paper has not been subjected to peer review. Yet peer review itself has also come under scrutiny, with its many limitations rarely being openly discussed. PMID- 8637348 TI - The governance of clinical trials. PMID- 8637349 TI - Statins and coronary heart disease. PMID- 8637350 TI - A cofactor for HIV-1 entry into cells is identified. PMID- 8637351 TI - Glimmers of clinical relevance for Fas. PMID- 8637352 TI - Longstanding presence in Belgians of multiple non-B HIV-1 subtypes. PMID- 8637353 TI - Betrayal by the surgeons. PMID- 8637354 TI - Betrayal by the surgeons. PMID- 8637355 TI - Betrayal by the surgeons. PMID- 8637356 TI - Betrayal by the surgeons. PMID- 8637357 TI - A money-saving approach to antidote immunotherapy for digoxin toxicity. PMID- 8637358 TI - Limb defects and chorionic villus sampling. PMID- 8637359 TI - Limb defects and chorionic villus sampling. PMID- 8637360 TI - CD4 lymphopenia and risk of infection in immunogerontologically healthy elderly people. PMID- 8637361 TI - Blackwater fever after halofantrine. PMID- 8637363 TI - Roller-coaster headache due to spinal cerebrospinal fluid leak. PMID- 8637362 TI - Parasitism of HIV-infected patients by insect flagellates. PMID- 8637364 TI - International debt relief. PMID- 8637365 TI - World Bank. PMID- 8637366 TI - Promotion of Coversyl by Servier. PMID- 8637367 TI - Role of external medical volunteers after disasters. PMID- 8637368 TI - Signing up for authorship. PMID- 8637369 TI - Vancomycin resistance and avoparcin. PMID- 8637370 TI - Laboratory management of antibodies to blood-group antigens in pregnancy. PMID- 8637371 TI - Homocystinuria and transsexualism. PMID- 8637372 TI - Expression of CD44 variants in differential diagnosis of ulcerative colitis and Crohn's disease. PMID- 8637373 TI - Invasive pneumococcal disease in HIV-1 infected patients. PMID- 8637374 TI - Measurement of urinary iodine concentration. PMID- 8637376 TI - Body temperature and infection in acute stroke. PMID- 8637375 TI - DNR orders in acute stroke. PMID- 8637377 TI - Hantavirus pulmonary syndrome in Germany. PMID- 8637379 TI - Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders. PMID- 8637378 TI - Interferon beta and suicide in multiple sclerosis. PMID- 8637380 TI - Leprosy and community-based rehabilitation. PMID- 8637381 TI - Immunohistochemical study of cutaneous neuritis in positive lepromin reactions. AB - Sixty skin biopsies taken from positive tuberculoid and borderline-tuberculoid late lepromin reaction were studied using histological techniques. The distribution of mycobacterial antigen and nerves was demonstrated using immunochemical methods. A total of 557 nerve bundles was observed in 51 biopsies; 9 were devoid of nerves in the sections examined; 475 nerve bundles showed some relationship to the inflammatory infiltrate (85%); perineuritis being seen in 144 (30%) and endoneuritis in 5 (0.9%). Mycobacterial antigens inside the granuloma were detected in 59 of the 60 biopsies (98%). Only one specimen, showing a strong tuberculoid reaction, failed to show these antigens. On the contrary, mycobacterial antigen was absent in almost all nerves. Small deposits were detected in the perineurium of one nerve with perineuritis, and inside a Schwann cell of another, the latter belonging to a previously multibacillary patient. The neurotropic tendency of the granuloma does not seem to be stimulated by the presence of mycobacterial antigens inside the nerves, as normally these antigens do not penetrate them. The hypothesis of some antigenic fraction of the neural tissue which cross-reacts with Mycobacterium leprae antigens, thus eliciting a perineural or near-perineural inflammatory reaction is put forward, but needs further investigation. PMID- 8637382 TI - Extended studies on the viability of Mycobacterium leprae outside the human body. AB - Very little is known in leprosy regarding the transmission of the infection from the source to the susceptible host. One of the important factors which governs the transmission of the disease is the viability of Mycobacterium leprae outside the human body. In this study M. leprae obtained from untreated patients have been subjected to several adverse conditions. Their viability was verified by their multiplication in the footpads of normal mice. After drying in the shade the organisms were viable up to 5 months. On wet soil, they remained alive for 46 days. Kept in saline at room temperature, the organisms lived for 60 days. Surprisingly on exposure to direct sunlight for 3 hours a day the bacteria survived for 7 days. On refrigeration at 4 degrees C, the bacteria could be preserved for 60 days. On the other hand, keeping at -70 degrees C, the bacteria could be maintained in a living condition for only 28 days. On exposure to antiseptics like Savlon (R) and alcohol, the bacteria were rapidly killed. These results indicate the survival outside the human body of M. leprae under different environmental conditions in India where the disease is endemic. Transmission of infection by indirect contact and occurrence of new cases in the absences of any known source, are consistent with M. leprae being viable outside the human body for varying periods of time. The findings could also be pointers to understand the epidemiology of leprosy. PMID- 8637383 TI - Detection of IgA anti-PGL-I specific antigen to Mycobacterium leprae in mangabey monkeys inoculated with M. leprae. AB - Using sera from 4 pairs of mangabey monkeys inoculated with titrated doses of Mycobacterium leprae we demonstrated that IgA antibodies against M. leprae specific PGL-I antigen were present in 75% of inoculated monkey's sera. High IgA antibody was detected in 50% (3/6) of infected animals and all three developed lepromatous leprosy (LL). Antibody titers correlated with PGL-I antigen in serum. The highest IgA peak appeared late and corresponded to the beginning of treatment, and in two of them appeared shortly after or corresponded with neurological damage. Low IgA response was found in the other 3 monkeys (50%-3/6), two of which developed indeterminate leprosy (I) and the other one LL. Low IgA levels appeared late after IgG and IgM, and shortly after neurologic signs. Both I monkeys were negative for PGL-I in serum. The remaining 2 monkeys (25%-2/8) did not show an IgA response; one of them developed LL but the disease regressed to I. IgM seemed to correspond to the appearance of PGL-I in serum. The other animal did not develop clinical symptoms of leprosy, and PGL-I in serum was negative. Although there was no clear relation between the development of anti-PGL-I IgA and experimental leprosy, the finding of a high IgA response in some animals suggests that further studies are needed to evaluate the role of antigen-specific IgA in the disease process. PMID- 8637384 TI - Dapsone syndrome in a Filipino man. AB - A case of dapsone syndrome occurring in a Filipino man under treatment for multibacillary (MB) leprosy is described. The patient manifested progressive fever, erythroderma and jaundice 4 weeks after initiation of multidrug therapy (MDT) with rifampicin, clofazimine and dapsone. The clinical symptoms conformed well to the dapsone syndrome first described in the 1950s and this report proves that the syndrome does still exist. There was recovery after dapsone was omitted and therapy with systemic corticosteroids was started. In view of this potentially fatal hypersensitivity reaction, this case report emphasizes the need for caution when initiating MDT or dapsone therapy. It is also suggested that any patient on MDT or dapsone needs to be referred immediately to a dermatologist or internist if the patient develops a skin rash during the first 2 months of treatment. PMID- 8637385 TI - Leprosy of the eustachian tube (nasopharyngoscopic study). AB - The technique of nasopharynogoscopy affords an accurate assessment of the lesions at the orifices of the eustachian tube. It was performed in 30 patients suffering from lepromatous leprosy in order to determine the type, nature and site of the lesion. Involvement of the eustachian tube in leprosy may begin with a localized area of erythema progressing to granuloma formation or ulceration. Leprous lesion at the eustachian tube orifices was related with subsequent changes in the tympanogram pattern. Nasopharyngoscopy is also found to be of therapeutic value in removing the crust, discharge and granulations at the eustachian tube orifices. PMID- 8637386 TI - Leprosy in Croatia in the twentieth century. AB - Even today, leprosy is a relatively frequently occurring disease, especially in tropical regions of the world. From the eleventh to thirteenth century, leprosy pandemics affected Europe, including Croatia. Probably as a consequence of such history, one can still find endemic foci of leprosy in present-day Croatia. The aim of this study was to analyse all cases of leprosy registered in Croatia during the twentieth century; therefore, we studied thoroughly existing medical documentation and published reports on sporadic leprosy cases, and went on to collect the relevant data through on-site investigation in those parts of Croatia known as putative endemic foci of leprosy. In this way, we collected data concerning the number of leprosy cases, the probable sources of infection, and traced the possible paths of spread of the disease. During the twentieth century, 17 cases of leprosy were registered in Croatia. However, due to the loss of medical documentation concerning the cases from Metkovic, the total number was obviously slightly greater. Concerning the 17 analysed cases, 4 patients were most probably infected during their visits (as sailors or immigrant workers) to the Middle East, South America or Africa; 3 patients developed leprosy after prolonged close contact with previously infected family members, while the exact source of infection remains unsettled for the remaining 10. However, 2 of these patients originated from the area of Cazin in Bosnia and Herzegovina, which is known to be an endemic focus of leprosy. Furthermore, the remaining 8 came from the small area of the village of Blizna in the Croatian municipality of Trogir, and therefore it seems reasonable to conclude that Blizna represents the endemic focus of leprosy in Croatia. The last case of leprosy in Blizna was registered back in 1956. Nevertheless, it is clear that sporadic cases of leprosy can reappear in Croatia, originating either from this endemic focus of Blizna, or as an infected person returning to Croatia from abroad. So, we can conclude that, even today, Croatian medical doctors (and especially dermatovenereologists) should still be acquainted with the clinical diagnosis of leprosy and basic principles of its treatment. PMID- 8637387 TI - Interpretation of data on monolesion leprosy case vs total new case detection rate. PMID- 8637388 TI - Proportion of beneficiaries vs relapse in MDT programme. PMID- 8637389 TI - Problems due to migration of leprosy patients into urban areas. PMID- 8637390 TI - The risk of standardized regimens of corticosteroids for the treatment of leprosy reactions in the field. PMID- 8637391 TI - Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E cadherin. AB - Recent studies have shown that Cd2+ can damage the Ca(2+)-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd2+ with E-cadherin, a Ca(2+) dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd2+ might bind directly to the E cadherin molecule, we studied the binding of Cd2+ to E-CAD1, a recombinant, 145 residue polypeptide that corresponds to one of the extracellular Ca(2+)-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd2+ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd2+ binding site per E-CAD1 molecule. The apparent dissociation constant (KD) for the binding was about 20 microM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of CD2+ was greatly reduced when excess Ca2+ was included in the dialysis solution. These results suggest that Cd2+ can interact with the Ca2+ binding regions on the E CAD1 molecule, and they provide additional support for the hypothesis that E cadherin might be a molecular target for Cd2+ toxicity. PMID- 8637392 TI - Serotonin and the regulation of hypothalamic-pituitary-adrenal axis function. AB - That serotonin (5HT) is involved in regulating hypothalamic-pituitary- adrenal axis (HPA) function has long been recognized. A variety of drugs including precursors of 5HT such as 5HTP, drugs which release 5HT such as fenfluramine and drugs which act directly on 5HT receptors such as ipsapirone increase cortisol and ACTH concentrations. There is a general assumption that such stimulation occurs at a hypothalamic level. However, our increasing understanding of the complex interplay between 5HT and the HPA raises questions as to the validity of this simple model. An increasing volume of experimental research indicates that 5HT can act directly on the adrenal gland and possibly on the anterior pituitary as well. These findings have major implications for the interpretation of neuroendocrine studies of 5HT conducted in psychiatric conditions, such as depression. PMID- 8637393 TI - In vivo pharmacokinetics and in vitro production of cocaethylene in pregnant guinea pigs. AB - Simultaneous exposure to cocaine and ethanol results in the formation of cocaethylene, an active metabolite of cocaine. The concurrent abuse of both cocaine and ethanol is common during human pregnancy, but the kinetics of elimination and formation of this ethyl ester of cocaine have not been studied during pregnancy in any species. In the late gestation guinea pig (61 to 63 days), cocaethylene, at doses of 2 to 4 mg.kg-1, is rapidly eliminated with a half-life of 29 min and a total body clearance of 77 ml.min-1.kg-1. It is formed enzymatically by hepatic microsomal preparations from fetal, neonatal and maternal guinea pigs. The maximum rate of cocaethylene production (apparent Vmax) when either ethanol or cocaine are varied while the other substrate is held constant, increases with age, from the late fetal period (65 days gestation, term 70 days) to adulthood. However, the Michaelis-Menten constant (apparent KM) does not change with age. The rapid elimination of cocaethylene, coupled with the slow rate of formation (apparent Vmax of 140 pmol.min-1.mg microsomal protein-1) and the small amount of plasma analyzed most likely explains the inability to detect coacethylene in vivo after concomitant cocaine and ethanol administration. PMID- 8637394 TI - Interleukin-6 secreted from human myxoma reduces murine viral myocarditis. AB - The effect of interleukin-6 (IL-6) secreted by a human atrial myxoma in vitro was investigated in C3H female mice with acute viral myocarditis. A culture medium containing IL-6 (100 ng/ml) and IL-8 (250 ng/ml), was prepared; viral myocarditis was induced by exposure to the encephalomyocarditis virus. Mice were assigned to four groups: 1) intraperitoneal (i.p.) injection of supernatant with IL-6 and IL 8 (0.2 ml/mice) given simultaneously with virus, 500 pfu for 4 days (Group 1); 2) i.p. injection of supernatant with IL-6 starting on Day 4 for 4 days in the same manner (Group 2); 3) i.p. injection of culture medium simultaneously with the virus (Group 3); and 4) i.p. injection of PBS in the same manner (Group 4). Uninfected control mice were administered medium only (Group 5) or supernatant with IL-6 and IL-8 (Group 6) for 4 days without virus. The survival rate on Day 14 in Group 1 was 90% significantly (p < 0.01) prolonged. The ratio of heart weight-to-day weight in the Group 1 was significantly (p < 0.01) lower. Histopathological examination revealed that cardiac necrosis and cellular infiltration in Group 1 was reduced compared with Group 3. Moreover, the radio of spleen weight/body weight in Group 1 was significantly (p < 0.01) higher than that of Group 3 and of Group 4. To confirm the effect of IL-6 or IL-8, mice were treated with recombinant IL-6 to IL-8 simultaneously with virus for 4 days. IL-6 treated mice survived significantly compared with IL-8 treated mice and untreated mice. The viral titer on day 4 of IL-6 treated mice was significantly lower than IL-8 treated or untreated mice. Thus, IL-6 derived from human myxoma improved the survival of murime viral myocarditis and reduced myocardial necrosis when the myxoma-derived IL-6 was administered simultaneously with the virus, due to eliciting cellular immunity in the spleen. PMID- 8637395 TI - Expression of intercellular adhesion molecules 1 (ICAM-1) via an osmotic effect in human umbilical vein endothelial cells exposed to high glucose medium. AB - To clarify the etiology of accelerated atherosclerosis in patients with diabetes mellitus, we measured expression of intercellular adhesion molecule 1 (ICAM-1), vascular cellular adhesion molecule 1 (VCAM-1), and E-selection on the cell surface by enzyme-linked immunosorbent assay and ICAM-1 mRNA content in human umbilical vein endothelial cells exposed to 5.5 mM glucose (NG), 33 mM glucose (HG), or 27.5 mM mannitol plus 5.5 mM glucose (HM).1) Cell-surface ICAM-1 expression in HG and HM cells was maximally increased by 37% and 32% (P < 0.01), respectively. This effect was dependent on glucose concentration in the medium and was found as early as 24 h and maintained until 6 days after exposing cells of HG. However, neither VCAM-1 nor E-selection expression were affected by HG conditions. 2) Both HG and HM induced increased mRNA content between 6 and 12 h after the stimulation. 3) Adhesion of THP-1 cells to endothelial cells exposed to HG and HM was increased, when compared to NG conditions. These results indicate that osmotic effects can induce increased mRNA and cell-surface expression of ICAM-1 via an as yet unknown mechanism. PMID- 8637396 TI - Urinary output of endogenous monoamine oxidase inhibitory activity is related to everyday stress. AB - The MAO A and B inhibitory components of urinary tribulin were investigated in normal individuals (11 males and 24 females, mean age +/- SD; 27.1 +/- 4.5 years) in relation to everyday stress. Volunteers collected a urine sample at the same time (late evening) on five days over a single week. On each occasion subjects also completed a mood adjective checklist which measured perceived stress levels over the day in question. For each subject all daily measures were aggregated. Mean individual urinary MAO A and B inhibitory activity was found to be positively correlated with stress scores both before (r = 0.38, p < 0.05 and r = 0.37, p < 0.05 respectively, n = 35) and after (r = 0.35, p < 0.05 and r = 0.33, p < 0.05 respectively, n = 35) correction for the effects of urinary volume. These results suggest that in normal healthy individuals high endogenous MAO inhibitory activity in the urine is indicative of a relatively enduring state of everyday stress. PMID- 8637397 TI - Property of receptor for vasoactive intestinal contractor (VIC) expressed in Xenopus oocytes injected with mRNA from rat intestine. AB - Property of receptor for vasoactive intestinal contractor (VIC), a peptide related to the endothelin family, expressed in Xenopus oocytes by injecting mRNA obtained from the intestine of rat, were studied using the voltage-clamp method. Inward-current responses to VIC (1 nM-100 nM) were evoked in a concentration dependent manner in mRNA-injected oocytes. Non-injected and water-injected oocytes failed to respond to VIC. The reversal potential for the VIC response was around -20 mV and the depolarizing shift was approximately 18 mV, when the external concentration of Cl-was halved, in agreement with the Nernst equation. The response to VIC was suppressed either by the external application to BAPTA/AM (10 microM) or by pertussis toxin 0.5 microgram/ml). These results indicate that the receptor for VIC, functionally expressed in Xenopus oocytes injected with rat intestinal mRNA, is coupled to pertussis toxin-sensitive G-protein and its activation leads to mobilization of intracellular Ca2+. PMID- 8637398 TI - A sustained increase in beta-adrenoceptors during long-term therapy with metoprolol and bisoprolol in patients with heart failure from idiopathic dilated cardiomyopathy. AB - Effects of long-term therapy with beta 1-selective antagonists (metoprolol, bisoprolol) on beta-adrenoceptors in lymphocytes of patients with idiopathic dilated cardiomyopathy (DCM) were examined. There was a significant reduction in the number of lymphocyte beta-adrenoceptors in patients with DCM compared to that in healthy volunteers, as demonstrated by a selective decrease in maximum number of binding sites (Bmax) for (-)-[125I]iodocyanopindolol (CYP). A therapy with metoprolol and bisoprolol in these patients caused a marked increase in lymphocyte beta-adrenoceptor density. The significant increase was observed from 2 or 3 months after the start of therapy with these drugs, and it was maintained during the therapy for 24 months. The left ventricular ejection fraction in patients with DCM was improved by the long-term therapy with metoprolol and bisoprolol, and this effect seems to be correlated with an observed enhancement of lymphocyte beta-adrenoceptors in the time course. Also, the increase in lymphocyte beta-adrenoceptors appears to be correlated with a gradual amelioration in circulating catecholamine levels by the long-term therapy with beta-adrenoceptor antagonists in patients with DCM. Thus, the present study suggests that beta-adrenoceptors in lymphocytes of patients with DCM are up regulated by a long-term therapy with metoprolol and bisoprolol. PMID- 8637399 TI - Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect. AB - Globin digest (GD), prepared from globin by acidic protease treatment, suppressed the elevation of serum triglyceride level in not only total but also chylomicron fraction after oral administration of olive oil. By screening with this lowering activity, we concluded that Val-Val-Tyr-Pro (VVYP) would be most effective constituent having hypotriglyceridemic action in GD. The mode of their action was dose dependent and did not show species specificity. Neither the repression of peristaltic movement of intestine nor the delaying of gastric emptying was caused by intake of GD or VVYP, however, the excretion of administered lipid was much more than that of control. Furthermore, administration of GD caused more prominent activation of hepatic triglyceride lipase (HTGL) and the increase of hepatic free fatty acid (FFA) concentration in early phase after administration of fat. From these results, it could be elucidated that GD, and also VVYP, inhibited fat absorption from digestive tract and enhanced activity of HTGL, so that more rapid clearance of dietary hypertriglyceridemia was caused. PMID- 8637401 TI - Macrophage stimulation reduces the cholesterol levels of stressed and unstressed rats. AB - Male, Sprague-Dawley rats were either treated with zymosan, a nonspecific macrophage stimulator, or saline vehicle. Half of each group were then subjected to a stress procedure, the other half remained in their home cage. Results indicate that zymosan-treated animals had lower levels of total, low-density/very low-density, and high-density lipoprotein than vehicle controls. Stressed animals had higher levels of the cholesterol parameters than did home cage controls. Manipulation of macrophage levels may be a prophylactic manipulation to combat stress-induced increases in cholesterol. PMID- 8637400 TI - Binding of 125I-prothymosin alpha to lymphoblasts through the non-thymosin alpha 1 sequence. AB - The important immunological activities of Thymosin alpha 1 (T alpha 1), a peptide derived from the thymus, led to its use in combination therapies in cancer patients. Prothymosin alpha (ProT alpha) is a highly acidic polypeptide, first isolated as the putative precursor of T alpha 1. However ProT alpha is now known to be more immunoreactive than T alpha 1 in certain in vivo and in vitro assays. Recent results indicate that ProT alpha may be useful to design future therapeutic interventions in cancer patients if the mechanisms underlying these effects are puzzled out. With this in mind, we radiolabeled ProT alpha to obtain a high specific activity and a high biological activity for 125I-ProT alpha. Moreover, we also obtained autoantibodies exhibiting high titers and an unique specificity for anti-ProT alpha and anti-T alpha 1. With both tools we studied the presence of binding sites for ProT alpha on the surface of lymphoblast cells. We conclude that ProT alpha binds through the non-T alpha 1 sequence. PMID- 8637402 TI - Evaluation of melanotan-II, a superpotent cyclic melanotropic peptide in a pilot phase-I clinical study. AB - A pilot phase I study was conducted with a cyclic heptapeptide analog of alpha melanocyte stimulating hormone (alpha-MSH). The lactam-bridged molecule, called Melanotan-II (MT-II), has the structure Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10 alpha-MSH4-10-NH2 (MT-II) and has superpotent melanotropic activity in vitro. A single-blind, alternating day (saline or MT-II), placebo-controlled trial was conducted in 3 normal male volunteers at the starting dose of 0.01 mg/kg of MT II. Subcutaneous injections of MT-II or saline were given daily (Monday-Friday) for 2 consecutive weeks. Two subjects were escalated by 0.005 mg/kg increments to 0.03 mg/kg and one to 0.025 mg/kg. The 0.03 mg/kg dose produced Grade II somnolence and fatigue in one of two subjects (WHO standards). Mild nausea, not requiring antiemetic treatment, was reported at most MT-II dose levels. A stretching and yawning complex appeared to correlate with the onset of spontaneous, penile erections which were intermittently experienced for 1-5 hours after MT-II dosing, depending on the MT-II dose. Two subjects had increased pigmentation in the face, upper body and buttock, as measured by quantitative reflectance and by visual perception 1 week after MT-II dosing ended. These results demonstrate that MT-II has tanning activity in humans given only 5 low doses every other day by subcutaneous injection. The recommended single MT-II dose for future Phase I studies is 0.025 mg/kg/day. PMID- 8637404 TI - Preservation of intestine protein by peptide YY during total parenteral nutrition. AB - Maintaining rats on TPN for 7 days was associated with a 50% reduction in gut mass and protein content. Co-infusing PYY with total parenteral nutrition (TPN) resulted in significant savings in jejunal wet mass and elevated protein content of jejunum, ileum and colon as compared with rats maintained on TPN alone. No significant effects of PYY on plasma amino acid profile were noted. Although minor alterations in mucosal polyamines were observed in rats maintained on TPN, co-infusion of PYY had no significant effect on gut polyamine concentrations. These results suggest that PYY has trophic effects upon the gut during otherwise catabolic conditions. Therefore, co-infusion of PYY with TPN may suggest methods whereby loss of intestinal mucosa and atrophy-associated complications of TPN may be modulated. PMID- 8637403 TI - In vivo labeling of delta opioid receptors in mouse brain by [3H]benzylidenenaltrexone, a ligand selective for the delta 1 subtype. AB - (E)-7-Benzylidenenaltrexone (BNTX) is a selective ligand for the putative delta 1 (delta 1) opioid receptor. To explore the feasibility of labeling delta 1 sites in vivo; we determined the cerebral distribution of radioactivity after systemic administration of [3H]BNTX to CD1 mice. Uptake was highest in striatum and lowest in cerebellum throughout the 4 hr time course. Specific radioligand binding, approximated as the difference in radioactivity concentrations between striatum and cerebellum, peaked at 0.32 percent injected dose/g at 30 min and comprised a modest 23% of total striatal radioactivity. For seven brain regions, radioactivity concentrations correlated with delta site densities known from prior in vitro studies (rS = 0.79, p = 0.03), and also with the uptake of N1' ([11C]methyl)naltrindole in vivo (rS = 0.78, p = 0.04) in mice. Specific binding in striatum, olfactory tubercles and cortical regions was saturable by BNTX, and was inhibited stereoselectively by the optical isomers of naloxone. Naltrindole and naltriben (NTB), delta antagonists, blocked 65-99% of [3H]BNTX specific binding at a dosage of 5.0 mumol/kg. Similar doses of the mu antagonist cyprodime, or the kappa agonist U50,488H, did not inhibit binding. Adjusted for the four-fold greater brain penetration of NTB relative to BNTX, dose-response studies suggested that delta 1 selective BNTX (ED50 = 1.51 mumol/kg) was 50% more potent than delta 2 selective NTB (ED50 = 0.56 mumol/kg) in blocking specific [3H]BNTX binding in striatum. In CXBK mice, a strain with functional delta 1 but not delta 2 receptors in antinociceptive assays, radioligand uptake and distribution proved similar to that in CD1 mice. In sum, [3H]BNTX labels murine delta opioid receptors in vivo with a low extent of specific binding. The data is consistent with, but not conclusive for, selective labeling of the delta 1 subtype. PMID- 8637406 TI - Effect of dietary alpha-linolenic acid deficiency on habituation. AB - Three weeks before mating, two groups of SWISS OF1 mice were fed a diet that was similar but contained either peanut oil poor in alpha-linolenic acid [18:3(n-3)] (n-3 deficient = deficient mice = (n-3)-) or peanut + rapeseed oil rich in alpha linolenic acid (n-3 nondeficient = controls = (n-3)+). Pups, fed the same diet as their dams, aged 45 to 62 days were used for brain lipid analysis and for behavioral experiments, aimed at determining whether there is a relation between the dietary intake of alpha-linolenate and a simple form of learning: habituation. The behavior of mice was compared using four models: exploration recorded in a photocell actimeter, activity in an open-field, duration of immobility in the forced swimming test and number of escape attempts from a small closed space. Habituation was measured by testing the mice in the same situation after some time had elapsed since the first test. Exploration in the photocell actimeter was significantly reduced between day 1 and 4 in nondeficient mice, but, not in deficient mice. The number of square crossings in the open-field was significantly reduced on the second test neither in the control nor in the deficient mice. In the forced swimming test, the habituation (increase in duration of immobility) was significantly greater (255%) in nondeficient than in deficient mice (163%). In the escape attempt experiment, the habituation showed a trend to be greater in controls than in deficient mice (p = 0.061) and was significantly greater in females than in males (p = 0.028). These results suggest that a simple form of learning, habituation, occurs more slowly in mice fed a diet deficient in alpha-linolenic acid. PMID- 8637405 TI - Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes. AB - Two antitumor polysaccharide-protein complexes, PSPC and PSK from mushrooms, were compared for their modulating effect on cytokine and cytokine receptor gene expression. RNA samples were isolated from the splenocytes and peritoneal exudate cells of the untreated or treated mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the cytokine gene expression. Nine out of 17 cytokine mRNAs and 5 out of 6 cytokine receptor mRNAs were detected in the splenocytes and peritoneal exudate cells from both untreated and treated mice. However, IL-4 was only detected in the splenocytes while IL-7 and IL-1R(typeI) were only detected in the peritoneal exudate cells. Among the 9 cytokine genes, the expression level of M-CSF was up-regulated in splenocytes and peritoneal exudate cells of the mice by PSPC and PSK. The expression level of TNF-alpha was only up-regulated in the peritoneal exudate cells by PSK, but not by PSPC. PMID- 8637407 TI - Formation of tetrahydroharman (1-methyl-1,2,3,4-tetrahydro-beta-carboline) by Helicobacter pylori in the presence of ethanol and tryptamine. AB - Helicobacter pylori contains alcohol dehydrogenase which oxidizes ethanol to acetaldehyde. In the present study, H. pylori cytosol was incubated in a buffered media at pH 6.0 and 7.4 in the presence of ethanol and tryptamine. Under these conditions, tetrahydroharman (1-methyl-tetrahydro-beta-carboline) was produced as a condensation product of tryptamine and acetaldehyde. At pH 6.0, 20.60 +/- 5.00% of the added tryptamine was converted to tetrahydroharman, while 27.00 +/- 4.80% (mean +/-SD) was converted at pH 7.4. Similar reactions between acetaldehyde and other dietary amines seem likely. Such biogenic alkaloids, if formed in vivo, might contribute to the dysphoric effects of alcohol. PMID- 8637408 TI - Double-blind placebo-controlled study of velnacrine in Alzheimer's disease. AB - The present study assessed the safety and efficacy of the cholinesterase inhibitor, velnacrine, for treating the cognitive symptoms of Alzheimer's disease. Patients (N = 236) meeting NINCDS-ADRDA criteria for Alzheimer's disease entered a double-blind, placebo-controlled dose-ranging protocol (30, 75, 150, 225 mg/day each for one week) to identify velnacrine responders (> or = four point improvement on the cognitive subscale of the Alzheimer's Disease Assessment Scale [ADAScog]). After a two week drug washout, velnacrine responders were randomly assigned to their best velnacrine dose or placebo in a six week dose replication protocol employing the ADAScog and the Clinical Global Improvement scale as primary outcome measures. During dose-replication, intent-to-treat analysis revealed that velnacrine patients scored significantly better than placebo patients on the ADAScog after two (p < 0.004), four (p < 0.025) and six (p < 0.001) weeks of treatment. No significant treatment effect on Clinical Global Improvement scores was observed. The primary adverse event was an asymptomatic elevation of liver transaminases found among 28% of the 236 treated patients. Cholinergic side effects including diarrhea (14%), nausea (11%) and vomiting (5%) were observed and 8% of patients experienced skin rash. The present study identified a subgroup of Alzheimer's patients who demonstrated a significant, but modest, improvement during velnacrine treatment on structured cognitive testing. PMID- 8637409 TI - Role of ouabain-like compound in the regulation of plasma aldosterone concentration in rats. AB - A major biologically active Na,K-ATPase inhibitor in the mammalian circulation may be ouabain-like compound(OLC). We developed a population of immunized rats against ouabain to block the actions of circulating OLC. To investigate the roles of OLC in the regulation of aldosterone secretion and/or production, we measured plasma aldosterone concentration after a week of low salt diet. No significant changes in serum Na and K concentrations were observed in immune rats. The plasma aldosterone concentration was significantly decreased by 30% in 17 immune rats as compared with 11 control rats(control: 455 +/- 53, immune: 315 +/- 21 pg/mL, p < 0.05). These data indicate that chronic blockade of the circulating OLC significantly decreases plasma aldosterone concentration during salt depletion and suggest that endogenous OLC may play an important role in the regulation of aldosterone secretion and/or production. PMID- 8637411 TI - Effects of topical anandamides on intraocular pressure in normotensive rabbits. AB - A series of anandamide-type compounds were synthesized and studied for their effect on the intraocular pressure (IOP) of normotensive pigmented rabbits. Each test compound was dissolved in an aqueous 2-hydroxypropyl-beta-cyclodextrin solution and administered (31.25 - 62.5 micrograms) unilaterally to the eye. The most promising anandamides caused a statistically significant reduction of IOP in treated eyes, usually preceded by an initial transient elevation of IOP, compared to saline controls. In the contralateral untreated eyes, only a marginal or short hypotensive response was observed. Indomethacin pre-treatment (12.5 mg, s.c.) eliminated the IOP response to administered anandamides and arachidonic acid. PMID- 8637410 TI - Endothelins and endothelin receptor antagonists: binding to plasma proteins. AB - Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. PD-156707, L-749329, Ro 470203, and A-127722 are potent non-peptide ET receptor antagonists developed recently. When tested in human and rat plasma, both ET-1 and -3 and the four aforementioned antagonists exhibited a high degree (> 98%) of plasma protein binding. When ET-1 binding to the receptors was examined, 5% (v/v) of human plasma inhibited ET-1 binding to both ETA and ETB receptors by 80 - 90%. Similarly, 5% (w/v) of human serum albumin inhibited ET-1 binding by 82%, suggesting that the major protein component in plasma which interfered with ET-1 binding to the receptors was serum albumin. Competition studies show that, in the absence of human serum albumin, the IC50 values of PD-156707, L-749329, Ro-47 0203, and A-127722 were 0.37, 0.29, 5.7, and 0.22 nM, respectively. Addition of increasing doses of human serum albumin incrementally decreased the potency of the antagonists; in the presence of 5% of human serum albumin, the IC50 values increased to 62.8, 50.2, 122.7, and 6.72 nM for PD-156707, L-749329, Ro-47-0203, and A-127722, respectively. In conclusion, ET and ET receptor antagonists exhibit a high degree of binding to plasma proteins, especially serum albumin. Consequently, serum albumin inhibits ET binding to its receptors, and also decreases the potency of ET receptor antagonists. Our findings may explain the discrepancy observed for ET receptor antagonists between in vitro and in vivo potencies. PMID- 8637412 TI - Parathyroid hormone induces a rapid increase in the number of active osteoclasts by releasing histamine from mast cells. AB - Intraperitoneal injection (i.p.) of parathyroid hormone (PTH, 50 micrograms/kg) into young rats (7-day postnatal) induced, within one hour, an increase in the number of osteoclasts which showed well-developed clear zone. Histological observations showed that degranulation of mast cells adjacent to bone surface occurred within 15 min after the injection of PTH. Injection of histamine into rats pretreated with cimetidine, an H2 antagonist of histamine, also induced an increase in the number of active osteoclasts within one hour after the injection of histamine. Furthermore, pretreatment of mice with an H1 antagonist, pyrilamine, completely inhibited the rapid increase in the number of active osteoclasts by PTH. These results suggest that PTH may stimulate osteoclasts to the active form by releasing histamine from mast cells and by stimulating H1 receptors for histamine. PMID- 8637413 TI - Central and NO mediated mechanisms are involved in the inhibitory effects of CCK on the chicken cecorectal area. AB - In chickens CCK-8s induces defecation and causes an inhibition of rectal electrical activity (EA) and an increase in cecal motility. In contrast, CCK-4 inhibits the motility of both rectum and ceca. The cecorectal responses to CCK-8s and CCK-4, given intravenously (i.v.), were studied in conscious chickens prepared with electrodes for electromyography; the influence of atropine, phentolamine plus propranolol, hexamethonium and L-NAME on such responses was determined. Atropine and phentolamine plus propranolol did not cause any change in the response to CCK-8s or CCK-4 in the cecorectal area. Hexamethonium only induced a significant decrease in the number of defecations (ND) induced by CCK 8s. L-NAME slightly modified the decrease in rectal EA due to CCK-8s. The effects of intracerebroventricular (i.c.v.) administration of CCK-8s and CCK-4 were also studied. CCK-8s and CCK-4, given i.c.v., caused, in conscious chickens, a slight decrease in cecal EA, in the 15 minutes following administration. This effect was similar to that seen after i.v. administration of CCK-4. In conclusion, our results suggest that the inhibitory action of CCK on chicken rectum is mediated, at least in part, through nitric oxide release. In addition, nicotinic receptors mediate the increase in the ND caused by CCK-8s. Ganglionic, muscarinic, adrenergic and nitrergic blockade were not able to modify the excitatory cecal response to CCK-8s, which may indicate that the receptor mediating this effect is located on the cecal smooth muscle. Finally, the inhibitory action of i.v. CCK-4 on chicken cecum seems to be centrally mediated, as suggested by the fact that i.c.v. administration of either CCK-8s or CCK-4 induce a similar effect. PMID- 8637414 TI - Reduced cytochrome P450 and increased heme oxygenase in liver during rabbit aflatoxicosis. AB - The administration of aflatoxin B1 (AFB1) in New Zealand rabbit for 5 days at a daily oral dose of 0.05 or 0.1 mg/kg decreased microsomal hepatic cytochrome P450 whereas a dose-dependent increase in the reduced microsomal 420 nm absorption occurred. The nature of such an absorption was then investigated. Either in vitro incubation of control microsomal proteins with AFB1 up to 800 microM and NADPH, or primary rabbit hepatocyte cultures exposure to AFB1 up to 30 microM for 24 to 72 h, failed to produce any 420 nm absorbing species, suggesting that the 420 nm absorption observed in vivo was due to an hemoprotein increase. Chemical reductions of microsomal proteins from AFB1-treated rabbits confirmed this hypothesis. Enzyme activity determinations revealed an increase in both microsomal heme oxygenase and NADPH-cytochrome c reductase activities in AFB1 treated rabbits, suggesting that the 420 nm absorption observed in vivo was related to a particular increase in heme oxygenase. PMID- 8637415 TI - Increased kynurenic acid levels and decreased brain kynurenine aminotransferase I in patients with Down syndrome. AB - Excitatory amino acid (EAA) receptors are central to brain physiology and play important roles in learning and memory processes. Kynurenic acid (KYNA), a metabolite of tryptophan in the brain blocks all three classical ionotropic EAA receptors and also serves as an antagonist at the glycine site associated with the N-methyl-D-aspartate receptor (NMDA) complex. We measured the endogenous levels of KYNA and activities of KYNA synthesizing enzymes kynurenine aminotransferase I (KAT I) and kynurenine aminotransferase II (KAT II) in the frontal and temporal cortex of elderly Down syndrome (DS) patients (aged 46-69 years). Compared with control specimens (0.21 +/- 0.06 pmol/mg tissue), the measurement of KYNA content revealed a significant 3-fold increase in frontal cortex of DS patients (0.67 +/- 0.13 pmol/mg tissue; p < or = 0.01). In temporal cortex KYNA levels were increased by 151% (p < or = 0.05) of control (0.41 +/- 0.09 pmol/mg tissue) Using crude cell free homogenate KAT's activities were determined in the presence of the 1 mM 2-oxoacid as a co-substrate at their pH optima of 10.0 for KAT I and 7.4 for KAT II. KATs activities in the presence of 1 mM pyruvate were 2.79 +/- 0.52 and 4.55 +/- 1.98 pmol/mg protein/h for KAT I and 0.98 +/- 0.07 and 1.09 +/- 0.14 pmol/mg protein/h for KAT II in frontal cortex and temporal cortex, respectively. When compared with the brain samples of controls the activity of KAT I was reduced in frontal cortex (9.8 +/- 2.4%; p < or = 0.01) and temporal cortex (25.8 +/- 6.4 %) of DS patients, while KAT II levels were within the normal range. Measurement of the neuronal, cholinergic marker choline acetyltransferase (ChAT) in the frontal cortex, revealed a significant reduction (36.6 +/- 4.3% of control; p < or = 0.01) in DS. Our data demonstrate the involvement of KYNA-metabolism in the cellular mechanisms underlying altered cognitive function in patients with DS. Although the localisation of both, KAT I and KAT II is not stated yet the reduction of KAT I may suggest impairment of KYNA metabolism in neuronal and/or nonneuronal compartments. PMID- 8637416 TI - Epidermal growth factor receptor-dependent cytotoxicity for human squamous carcinoma cell lines of a conjugate composed of human EGF and RNase 1. AB - Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The EGF-RNase conjugate showed dose dependent cytotoxicity for EGF receptor-overexpressing A431 and TE-8 human squamous carcinoma cells with an IC50 of 2 x 10(-7)M and 10(-6)M, respectively, whereas the IC50 of RNase alone was almost 10(-4)M. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. The conjugate showed no detectable cytotoxicity against EGF receptor-deficient small cell lung cancer cells (H69). Addition of excess EGF in the medium protected A431 cells from the EGF-RNase conjugate cytotoxicity. The cytotoxicity of the EGF-RNase conjugate was positively correlated with the EGF receptor numbers of each cell line. The chimeric toxin composed of only human proteins might be a more useful anti-cancer agent with less immunogenicity than the conventional chimeric toxins. PMID- 8637417 TI - Autobiographical notes. Donald J. Jenden. PMID- 8637418 TI - Cholinergic and Related Mechanisms in Aging and Disease: a Tribute to Donald J. Jenden, M.D. Los Angeles, California, November 17-18, 1995. PMID- 8637419 TI - Chemical methods for the determination of acetylcholine. PMID- 8637420 TI - Regulatory mechanisms of choline production. AB - This paper gives a short account of the mechanisms regulating choline production. Although choline is absorbed from the GI tract and biosynthesized in the liver, its subsequent metabolism to choline esters and phospholipids seems to dominate in importance as to its regulatory role in maintaining a constant source of free choline extracellularly. PMID- 8637421 TI - In vivo 1H MRS choline: correlation with in vitro chemistry/histology. AB - We correlated the in vivo 1H Magnetic Resonance Spectroscopy (MRS) concentration of the choline peak (CHO) with in vitro chemical measures of choline-containing compounds and a histological grade of cellularity in 18 patients with neoplastic and infectious brain lesions. Gas-chromatography-mass-spectrometry (GCMS) was used to measure the concentrations of free choline (Cho), glycerophosphocholine (GPCho), phosphocholine (PCho) and phosphatidylcholine (PtdCho) from biopsies in the same area where MRS was performed. Cellular density, free Cho, PCho and GPCho were the strongest determinants of 1H MRS CHO while PtdCho was not. Just as the 1H MRS 2.0 peak reflects both the concentration of n-acetyl-l-aspartate and neuronal density, the 1H MRS 3.2 peak reflects the concentration of water-soluble choline-containing compounds and cellular density. PMID- 8637422 TI - Regulation of acetylcholine synthesis in the presence of hemicholinium mustard. AB - High affinity choline uptake (HACU) is a critical element in the synthetic pathway for acetylcholine (ACh), and is known to demonstrate activity-dependent regulation in vivo and in vitro. However, little is known about this important sodium-dependent transport protein at the biochemical level, and about the nature of its interaction with the ACh synthetic enzyme ChAT. Hemicholinium mustard (HCM), an irreversibly binding analog of hemicholinium-3 (HC3), was used to create a preparation with HACU that is completely inhibited in order to investigate the immediate source of Ch for ACh synthesis. Rat brain synaptosomes were pre-incubated with HCM and washed before transport incubations of increasing length (0-6 min) were carried out. The contribution of endogenous and extracellular (tracer) Ch to the ACh level was measured at each time point using a gas chromatography mass spectrometry (GCMS) system that allows quantitative measurement of endogenous (unlabelled; [2Ho]) Ch as well as tracer (deuterium labelled; [2H4]) Ch. The hypothesis was that if an endogenous intraterminal Ch pool can be used for ACh synthesis, an increase in unlabelled ACh across time would be observed. In neither HCM-treated nor control synaptosomes was an increase observed in intraterminal (pellet) unlabelled ACh. To test the effects of high tissue demand, in other experiments synaptosomes were depolarized with addition of 40 mM KCl to the buffer after HCM treatment; again, no significant increase in intraterminal unlabelled ACh was observed across time. These experiments demonstrate that endogenous unlabelled Ch does not contribute to ACh synthesis, even when HACU is inactivated, and under conditions of high demand. PMID- 8637423 TI - Lesions of the cholinergic nuclei in the rat basal forebrain: excitotoxins vs. an immunotoxin. AB - Infusion of the excitotoxins, ibotenic acid, quisqualic acid, or AMPA, into the medial septal nucleus, diagonal band, and the nucleus basalis magnocellularis of rats produced less cholinergic cell loss as assessed by choline acetyltransferase activity in the projection fields, cortex and hippocampus, than that obtainable by intraventricular administration of the immunotoxin, 192 IgG-saporin. All excitotoxins produced reductions in tissue levels of some monoamines, while no decreases were found for the immunotoxin. All toxins produced acquisitional impairment in the hidden platform water maze. This behavioral deficit was slightly greater for the excitotoxic-lesioned rats than for those given 192 IgG saporin at a dose which produced ChAT depletions similar to the most potent excitotoxin (AMPA). This supports the idea that some of the behavioral effects produced by excitotoxic lesions are due to the cholinergic basal forebrain lesion and some are due to noncholinergic damage. PMID- 8637424 TI - The AF64A model of cholinergic hypofunction: an update. AB - Based on numerous reports in the literature since 1980, one can now conclude that ethylcholine aziridinium (AF64A) is selective for the cholinergic system in vivo, and that the effect is both dose- and site-dependent. Thus, AF64A treatment, under the correct conditions of dose and time will result in selective reductions in levels of ACh, AChE, ChAT, HAChT, and K(+)- and ouabain-stimulated release of ACh. While other neurotransmitters may also be affected in brains of AF64A treated rats, the effect is only transient and is most probably secondary to the initial cholinergic deficit-induced by AF64A, reflecting an adaptive reaction of these neurotransmitter systems, which are normally integrated with cholinergic interconnections, to the cholinergic deficiency induced by AF64A. This paper provides a historical perspective for the development of AF64A as a selective cholinotoxin, and surveys its potential mechanisms of action at the neurochemical and molecular levels. Moreover, the availability of an animal model such as the AF64A-treated rat, in which the cholinergic system has been compromised selectively for an extended period of time, has allowed investigators to study a wide variety of questions that relate to factors controlling cholinergic function in vivo. Several key illustrations are presented at the end of this paper. PMID- 8637425 TI - Continuing the search for cholinergic factors in cognitive dysfunction. AB - The objective of the experiments reported here was to explore three hypotheses regarding cholinergic processes underlying the development of progressive degenerative dementia (PDD). One possibility involved the downregulation of muscarinic receptors (mAChR) with aging, thus reducing the capability of the cholinergic system to support normal memory and other cognitive functions. The results of downregulation to 10% of normal produced only a temporary effect, the system having the capability to repair the damage. A second hypothesis predicted that a chronic hypocholinergic state can produce structural changes that are reflected in persisting cognitive dysfunctions. Chronic administration of a false cholinergic transmitter in the diets of weanling rats mimicked such a state which, if maintained for a protracted period, produced many of the features of PDD in humans. When the diet was returned to normal, biochemical and physiological processes recovered fully. However, memory impairment continued. This suggested the possibility that the behavioral losses were mediated by persisting morphological changes in the CNS. The third hypothesis proposes that these changes may be due to cell loss resulting from impaired phospholipid metabolism. Changes in sphingomyelin, one of the two major Ch-containing lipids in cell membranes, could increase amounts of ceramide, an inducer of programmed cell death (apoptosis). Tests of this hypothesis are nearing completion. PMID- 8637426 TI - The interaction of 4-DAMP mustard with subtypes of the muscarinic receptor. AB - The compound 4-DAMP mustard (N-2-chloroethyl-4-piperidinyl diphenylacetate) is a 2-chloroethylamine derivative of the selective muscarinic antagonist 4-DAMP (N,N dimethyl-4-piperidinyl diphenylacetate). At neutral pH, 4-DAMP mustard cyclizes spontaneously into an oziridinium ion that binds covalently with muscarinic receptors. Analysis of the kinetics of receptor alkylation showed that the interaction of 4-DAMP mustard with M2 and M3 receptors was consistent with a model in which the aziridinium ion rapidly forms a reversible complex with the receptor which converts to a covalent complex at a relatively slower rate. The rate constant (k2) for alkylation of M2 and M3 receptors was approximately the same (k2 = 0.1 min-1); however, the affinity of the aziridinium ion for the M3 receptor (KD = 7.2 nM) was approximately 6.3-fold greater than that for the M2 receptor (KD = 43 nM). The results of competitive binding experiments on Chinese hamster ovary cells transfected with the M1 - M5 subtypes of the muscarinic receptor showed that the affinity of the aziridinium ion for the M1, M3, M4 and M5 subtypes was approximately the same and about 11-fold greater than that for the M2 receptor. 4-DAMP mustard is a useful tool for selectively inactivating all non-M2 muscarinic receptors, particularly when it is used in the presence of a reversible M2 selective antagonist to protect the M2 receptor from alkylation. The results of studies on isolated smooth muscle preparations that have had their M3 receptors alkylated with 4-DAMP mustard are consistent with the postulate that the M2 receptor can elicit contraction by inhibiting the relaxant effect of isoproterenol and forskolin on histamine induced contractions. PMID- 8637427 TI - Membrane delimited and intracellular soluble pathways in the somatostatin modulation of ACh release. AB - The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from acutely dissociated St 40 ciliary ganglion neurons. As in intact synapses, somal ACh release was blocked by 100 nM SOM or 100 microM dibutyril cGMP, and the SOM mediated inhibition could be reversed by 10 microM 1-NAME (a selective inhibitor of nitric oxide synthase, NOS) or 100 microM Rp-8p-CPT-cGMPs (a selective inhibitor of a cGMP protein dependent kinase, PKG). In whole cell recordings, SOM inhibition of Ca2+ current rapidly relaxes to control levels but is sustained in perforated patch recordings which decreases cell dialysis. Inhibition of NOS or PKG in perforated patch recordings, however caused SOM effects to become transient again. We hypothesize that PKG alters the characteristics of the membrane-delimited G protein inhibition of Ca2+ current. Therefore SOM receptors trigger a membrane-delimited signal transduction cascade that is modulated by soluble messengers, converging on voltage activated Ca2+ channels. When both pathways are active together, SOM causes a sustained inhibition of neuronal Ca2+ current leading to a decrease in transmitter release. PMID- 8637428 TI - Differentiation agents enhance cholinergic characteristics of LA-N-2 human neuroblastoma cells. AB - LA-N-2, a cell line derived from a human peripheral neuroblastoma, has a partially cholinergic phenotype and is a potential in vitro model of cholinergic neurons. The object of this study was to enhance the cholinergic phenotype of these cells with differentiation agents to improve the cell line's usefulness as a convenient model of cholinergic function. I treated cells in the presence of serum with 10 microM 5-azacytidine, 2.5 microM bromodeoxyuridine, 2 nM ciliary neurotrophic factor, 1 mM dibutyryl cAMP, 0.25 nM leukemia inhibitory factor and/or 3.8 nM nerve growth factor, N-2 supplement (without serum), or 10 microM retinoic acid for 9-14 days. Treated cells were loaded with [3H]choline for 30 min at 37 degrees and washed. The amounts of cellular and released (5 min, room temperature), labeled and unlabeled acetylcholine and choline were determined by HPLC. None of the differentiation agents induced Ca(2+)-dependent release of [3H]acetylcholine, but 5-azacytidine, dibutyryl cAMP, N-2, and retinoic acid increased Ca(2+)-independent release that was specific for acetylcholine. In addition, 5-azacytidine, bromodeoxyuridine, leukemia inhibitory factor, and N-2 substantially increased [3H]acetylcholine levels, and these increases correlated highly with increases in total acetylcholine levels. Overall, LA-N-2 cells should prove to be a good model for studying cholinergic function. PMID- 8637429 TI - Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue. AB - Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in cisternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N1-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [3H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [3H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [3H]NMN was specifically formed by a brain cytosolic enzyme. The production of [3H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance. PMID- 8637430 TI - The influence of aging on whole body choline release and clearance. AB - We have confirmed that hypoxia elicits a substantial rise in blood choline levels in young adult rats. An intravenous infusion of tracer quantities of [2H4]-Ch, serial measurements of blood [2H0]-Ch and [2H4]-Ch, and a simple pharmacokinetic model were used to assess the bidirectional flux of choline between the central pool and peripheral pools before, during and after a period of imposed hypoxia, in rats ranging from 56 to 780 days of age. The results indicate that the age dependence of the hypercholinemic response to hypoxia is predominantly due to an increase in the amount of choline released in response to hypoxia, and that changes in its clearance are relatively unimportant. PMID- 8637431 TI - Cholinergic control of cerebral blood flow in stroke, trauma and aging. AB - Enhancing the availability of endogenous acetylcholine by inhibition of cholinesterase with physostigmine, eptastigmine or soman at sub-toxic doses increases cerebral blood flow (CBF) and the response of this variable to changes in PaCO2. These effects are not correlated with metabolic activation, suggesting that the function of the cholinergic vasodilation is not merely to supply metabolic substrates. Since choline (Ch) can exchange between blood and the brain extracellular milieu the stage is set for possible feedback interactions between ACh synthesis and CBF. A negative feedback of CBF on ACh synthesis under conditions of a negative arteriovenous (A-V) difference for Ch across cerebral capillaries may contribute to stabilize GBF in ischemia. Eptastigmine and physostigmine significantly improve perfusion in experimental models of focal cerebral ischemia and traumatic brain injury respectively. During the short periods of time in which the A-V difference for Ch across the brain is positive, a positive feedback between cerebral free Ch and CBF may enhance the ability of the brain to recover Ch from the circulation for synthesis of membrane phospholipids. A loss of cholinergic cerebrovascular control may thus impair the survival of all cells within the CNS and contribute to the pathophysiology of dementia. Perhaps the view that the loss of cholinergic cells is the end point of Alzheimer's dementia could be modified to state that a cholinergic deficit may be the starting point of a decline in cerebral phospholipid turnover and cell membrane renewal that could lead to a generalized deterioration of cerebral function. PMID- 8637432 TI - Neurotrophins and Alzheimer's disease: beyond the cholinergic neurons. AB - Improvement of the cholinergic deficit in Alzheimer's disease (AD) by intracerebral application of nerve growth factor (NGF) serves as a paradigmatic example for a novel approach to the treatment of neurodegeneration. The first part of this paper presents and discusses experiments which were performed in our laboratory to study the NGF receptor response after intracerebral NGF treatment in vivo. We found that intraparenchymal NGF elicits prolonged tyrosine phosphorylation of Trk type NGF receptors. Our results indicate that intraparenchymal injections are preferable to intraventricular application for targeting specific neuronal populations with minimal side effects. Besides the cholinergic deficit, severely disturbed brain energy metabolism, particularly in cortical association areas, is another consistent feature of AD. Metabolic hypofunction is observed early in the disease progression and correlates with the cognitive impairment. Cell culture findings are presented which indicate that brain-derived neurotrophic factor (BDNF), and other neurotrophins with activity on the TrkB tyrosine kinase receptor, increase mRNA levels and biochemical activity of enzymes of the glycolytic pathway in brain cells. Treatment with these factors was also found to stimulate glucose utilization in rat embryonic cortex cells in primary cultures. Our observations suggest that selected neurotrophins should become useful not only for the treatment of the cholinergic deficit in AD, but also of the cortical metabolic hypofunction associated with this disease. PMID- 8637433 TI - Evidence for decline in intracellular calcium buffering in adrenergic nerves of aged rats. AB - Age-related alterations in neuronal intracellular calcium regulation and neurotransmitter release have been widely reported. We have investigated the impact of age on neurotransmitter release and intracellular calcium buffering in adrenergic nerve endings of the isolated rat tail artery and on intracellular calcium in acutely dissociated cells from the superior cervical ganglion. Advancing age, from 6 to 27 months, resulted in significantly increased stimulation-evoked norepinephrine release from the isolated rat tail artery, an effect which persisted when neuronal and extraneuronal uptake were blocked with cocaine and deoxycorticosterone and presynaptic alpha adrenergic receptors were blocked with idazoxan. Alterations in extracellular calcium had significant effects on stimulation-evoked norepinephrine release, but these were much more marked in old, compared to young, arteries. Blockade of mitochondrial calcium accumulation with dinitrophenol had no significant effect on stimulation-evoked norepinephrine release from 6-month-old arteries, but in 20-month-old arteries, treatment with dinitrophenol resulted in a substantial increase in stimulation evoked norepinephrine release. However, when extracellular calcium was increased to 5 mM in 6 month-old-arteries, then addition of dinitrophenol resulted in an increase in stimulation-evoked norepinephrine release. Measurement of intracellular calcium in acutely dissociated superior cervical ganglion cells using fura-2 revealed substantial age-related differences. Peak calcium transients in 20-month-old ganglion cells depolarized with 68 mM K+ were substantially higher than in 6-month-old cells. Together these findings support the hypothesis that in adrenergic nerves advancing age results in a disruption of intracellular calcium buffering leading to higher levels of intracellular calcium and increased transmitter release. PMID- 8637434 TI - Cysteine-string proteins: a cycle of acylation and deacylation? AB - We used tunicamycin, an inhibitor of protein fatty acylation, to examine the possibility that there is a cycle of acylation and deacylation of cysteine string proteins at nerve terminals. Using both physiological and immunoblot approaches, we obtained no evidence for a cycle of acylation and deacylation that affects these proteins. These data suggest that this lipid modification of cysteine string proteins is relatively more stable than that observed for other nerve ending proteins, like SNAP-25. PMID- 8637435 TI - Role of the stretch reflex in oxotremorine tremor. AB - Although the basic mechanism for parkinsonian tremor is known to be central, the case for the oscillator(s) of physiological tremor and shivering is still a matter of dispute. In this case, an important role has been proposed for muscle afferents. Oxotremorine tremor has in common with shivering its frequency range (10-28 Hz) and the co-contraction of flexors and extensors. On the other hand, in contradistinction, it can be blocked by atropine, as is the case with parkinsonian tremor. Thus it was of interest to analyze the role of muscle afferents in the production and maintenance of oxotremorine tremor in the acutely decorticated cat. This was studied with two experimental approaches. In one, either a front or hind limb of a cat was completely deafferented by dorsal rhizotomy and the tremor activity recorded electromyographycally in a pair of antagonistic muscles. In the other, the nerve and muscle electrical activity and force produced by the tremoring muscle (gastrocnemius) were recorded simultaneously. Tremor activity was induced by oxotremorine injection (200-750 micrograms/kg i.p.) given at different postoperative periods. The drug induced a regular tremor in the chronically deafferented animals (3 weeks to 5 months) but not in the acutely deafferented limb (3 hrs after dorsal rhizotomy). The tremor observed in the former group was very regular and had the same frequency range (10-28 Hz) for both normal and deafferentd sides. Section of all the muscle afferents of a given pool (agonist and its main synergist) did not have any effect on the regularity and frequency of the oscillation (tremor) of that pool. The role of other muscle and/or cutaneous afferents, although perhaps of some importance, remains unclear. A pure spinal mechanism to account for the regular oscillations (tremor) is proposed. PMID- 8637437 TI - Studies of working memory using 18FDG-positron emission tomography in normal controls and subjects with epilepsy. AB - We have studied three groups of subjects with a working memory paradigm, using 18FDG-PET. Controls show the greatest increase on uptake in dorsolateral prefrontal cortex, basal forebrain and angular gyrus. A group of subjects with focal frontal epilepsy did not show increases compared to a control task of attention. Primary generalized epilepsy subjects show the greatest changes in angular gyrus, dorsal temporal, medial frontal and parietal regions. Factor and regression analyses extend these observations and show reliance of both patient groups on the medial and inferior temporal lobe. We propose that the normal network of working memory is disrupted by these two forms of epilepsy and different networks are accessed. Declarative memory may be used as a compensatory system, which results in decreased performance. PMID- 8637436 TI - In vivo proton magnetic resonance spectroscopy of the normal aging human brain. AB - The effect of age on brain metabolite concentrations was evaluated using localized proton magnetic resonance spectroscopy. This technique allows in vivo measurements of N-acetyl compounds (NA), total creatine (CR), choline-containing compounds (CHO), myo-inositol (MI), glutamate and glutamine (GLX), as well as the percentage of cerebrospinal fluid (CSF) and the brain water content within the brain region studied. Frontal gray matter and frontal white matter brain regions were examined in 36 normal healthy volunteers (19-78 years of age). Using a rigorous absolute quantitation method, with an external reference and atrophy correction, we found relatively stable concentrations of NA, a neuronal marker. In contrast, CR, CHO, MI, and the percentage of CSF increased in the gray matter with age. However, the brain water content decreased significantly with age (r = 0.72; p < 0.0001). No significant age-related changes in metabolite concentrations, CSF or brain water content were observed in the white matter regions. These findings demonstrate that biochemical alterations are associated with aging in the frontal gray matter. There might be an increase in the brain density as indicated by increased metabolite concentrations and decreased brain water content with aging. PMID- 8637438 TI - The effect of low-glycemic carbohydrate on insulin and glucose response in vivo and in vitro in patients with coronary heart disease. AB - The insulin resistance syndrome has recently been implicated in the etiology of coronary heart disease, with a possible metabolic defect at the level of the adipocyte. We report the effects of a low- versus high-glycemic-index (LGI and HGI, respectively) diet on insulin and glucose response as assessed by oral glucose tolerance test (OGTT) and insulin-stimulated glucose uptake in isolated adipocytes in a group of 32 patients with advanced coronary heart disease. The area under the insulin curve following OGTT was significantly reduced after 4 weeks in the LGI group (P < .03), but not in the HGI group. Insulin-stimulated glucose uptake in isolated adipocytes harvested from a presternal fat biopsy was significantly greater following the LGI diet (P < .05). This study demonstrates that simple short-term dietary measures can improve insulin sensitivity in patients with coronary heart disease. PMID- 8637440 TI - Growth hormone normalizes low-density lipoprotein receptor gene expression in hypothyroid rats. AB - Hypothyroidism leads to a decreased activity of the low-density lipoprotein (LDL) receptor, which contributes to the hypercholesterolemia frequently seen during hypothyroidism. It is not known whether the decreased activity of the LDL receptor is directly due to the absence of thyroid hormone, or secondary to a deficiency of growth hormone (GH). Therefore, the effect of GH administration on LDL receptor activity was studied in hypothyroid rats. Following induction of hypothyroidism, the level of LDL receptor mRNA was significantly decreased in liver homogenates to 31 % +/- 6% of the control value. LDL binding to liver cell membranes and plasma membranes decreased during hypothyroidism to approximately 65% of the control value. The effect of hypothyroidism on the hepatic LDL receptor was reflected in a significantly increased half-life of (125)I-LDL of 29 hours in controls versus 48 hours in hypothyroid rats. Treatment of hypothyroid rats with human GH (hGH) resulted in normalization of both the amount of hepatic LDL receptor mRNA and LDL binding on liver cell membranes. The plasma half-life of human (125)I-labeled LDL decreased during GH substitution but did not normalize. GH treatment significantly reduced plasma LDL cholesterol levels by 36% (P < .05, n = 8), to levels that were still higher than in control animals. These data indicate that at least part of the decreased LDL receptor activity during hypothyroidism is secondary to GH deficiency. PMID- 8637441 TI - Very-low-density lipoprotein of uremic patients is a poor substrate for bovine lipoprotein lipase in vitro. AB - Very-low-density lipoprotein (VLDL) from 10 hemodialysis patients and 10 healthy controls was studied with respect to the substrate characteristics for bovine milk lipoprotein lipase (LPL). Compared with the control subjects, the hemodialysis patients had significantly higher serum triglyceride and apolipoprotein B-associated apolipoprotein CIII concentrations (1.03 +/- 0.31 v 1.98 +/- 0.86 mmol/L and 0.004 +/- 0.002 v 0.011 +/- 0.005 g/L, respectively), lower serum high-density lipoprotein (HDL) cholesterol and apolipoprotein AI concentrations (1.33 +/- 0.37 v 0.95 +/- 0.31 mmol/L and 1.29 +/- 0.25 v 1.09 +/- 0.23 g/L, respectively), and lower postheparin plasma LPL activity (82 +/- 24 v 35 +/- 14 milliU/milliL). There were also significant increases in the relative fat content and diameter of VLDL particles from patients versus controls. VLDL was labeled with a fluorescent phospholipid analog, DHPE, and the rate of the lipolytic reaction with purified bovine milk LPL was estimated from the increase in fluorescence intensity at 490 nm. There was no significant difference between initial reaction velocities in the study groups, but VLDL particles from hemodialysis patients were lipolyzed to a significantly lesser extent than those from healthy controls (mean increase in fluorescence intensity after completion of the reaction, 95 +/- 36 v 140 +/- 43 arbitrary units). These results are in accordance with the accumulation of remnant particles reported to occur in uremia despite only a moderately increased serum triglyceride concentration. PMID- 8637439 TI - Sitosterolemia: opposing effects of cholestyramine and lovastatin on plasma sterol levels in a homozygous girl and her heterozygous father. AB - Sitosterolemia is a genetic disorder characterized by sitosterol accumulation in plasma and clinically accelerated atherosclerosis. Under a condition of metabolic control with a 30% fat, low-sitosterol diet, we compared the effects of monotherapy and dual-drug treatment with lovastatin and cholestyramine on plasma sterol parameters and endogenous cholesterol synthesis in a homozygous sitosterolemic patient with concomitant heterozygous familial hypercholesterolemia (FH), her obligate heterozygous father, and hyperlipidemic control subjects. We found that for both the sitosterolemic homozygote and heterozygote, cholestyramine plus lovastatin dual therapy proved not to be superior to either drug treatment alone. In the homozygous patient, cholestyramine accounted for the decrease of plasma sterol (ie, lovastatin was ineffective), whereas in the heterozygote, lovastatin represented the margin of difference (ie, low-dose cholestyramine was relatively ineffective). Thus, the best treatment option for this homozygote child and her heterozygote father appears to be monotherapy with cholestyramine and lovastatin, respectively. Stimulation by bile acid malabsorption produced a dramatic decrease of plasma sterols in the homozygote, without increasing endogenous cholesterol synthesis, but this therapy was ineffective in the heterozygote. Decreasing endogenous cholesterol synthesis with lovastatin was effective in the heterozygote, but ineffective in the homozygote. In suspected sitosterolemia, a poor sterol response to lovastatin and a dramatic response to cholestyramine may differentiate homozygous from heterozygous and other familial forms of hyperlipidemia. PMID- 8637442 TI - The relationship between plasma insulin level, prostaglandin production by adipose tissue, and blood pressure in normal rats and rats with diabetes mellitus and diabetic ketoacidosis. AB - There is a correlation between circulating insulin levels and blood pressure over a wide range of insulin levels and in a variety of clinical conditions. Production of prostaglandin (PG)E(2) (PGE(2)) and prostacyclin (PGI(2)), two potent vasodilators, by adipose tissue is increased in severe insulin deficiency, eg, diabetic ketoacidosis (DKA), explaining the decreased peripheral vascular resistance in DKA. Conversely, decreased production of PGE(2) and PGI(2) may mediate the relationship between hyperinsulinemia and hypertension. Although insulin inhibits PG production in normal rat adipose tissue, PG production in adipose tissue from patients or experimental animals with nonketotic diabetes mellitus (DM) and DKA has not been studied. We examined the effect of plasma insulin levels on blood pressure and on adipose tissue PG production in rats with DM and DKA and normal rats. There was a significant relationship between plasma insulin level and blood pressure in rats with DM and normal controls (P < .021) and in rats with DKA and normal controls (P < .0001). There was an inverse linear correlation between plasma insulin levels and basal 6-keto-PGF(1 alpha) production by a mixture of adipocytes and endothelial cells from epididymal adipose tissue in rats with DKA and normal rats (P < .0252, R2 = .67). Rates of basal glycerol, PGE(2), and 6-keto-PGF(1alpha) production by a mixture of adipocytes and endothelial cells from epididymal adipose tissue were significantly higher in rats with DKA than in normal rats. These rates were also higher in rats with DM than in normal rats, but only glycerol values were statistically significant. In rats with DM, PGE(2) production induced by epinephrine 2 x 10(-5) mol/L (but not lower concentrations) was significantly greater than basal production (P < .05); production of 6-keto-PGF(1alpha) was not stimulated. In rats with DKA, 6-keto-PGF(1alpha) production induced by epinephrine 2 x 10(-5) mol/L (but not lower concentrations) was significantly greater than basal production (P < .05); production of PGE(2) was not stimulated. We conclude the following: (1) there is a close correlation between circulating insulin level and systemic blood pressure when rats with DM and DKA are compared with controls; (2) in insulin deficiency, PGI(2) and PGE(2) production are increased in adipose tissue versus normal tissue; and (3) the correlation between insulin level and blood pressure may be mediated by the inhibitory effect of insulin on vasodilative PG production by adipose tissue. PMID- 8637443 TI - Clustering of dyslipidemia, hyperuricemia, diabetes, and hypertension and its association with fasting insulin and central and overall obesity in a general population. Atherosclerosis Risk in Communities Study Investigators. AB - Clustering of elevated triglycerides, decreased high-density lipoprotein cholesterol (HDL-C), hyperuricemia, diabetes, and hypertension has been related to insulin resistance/high insulin levels and central and/or overall obesity. The extent to which these abnormalities cluster and whether hyperinsulinemia, central adiposity, and overall obesity each independently associate with this clustering were evaluated in 14,481 US whites and African-Americans 45 to 64 years of age. With the exception of hypertension, abnormalities rarely existed in isolated form. Clustering greatly exceeded chance association (P < .001). Although this clustering was greater in relative terms (ratio of observed to expected cluster frequency) in the lean and less centrally obese, it was greater in absolute terms (observed minus expected cluster frequency as a percent of total population) in the more centrally and more generally obese. The greatest excesses were found for clusters that included both hypertriglyceridemia and low HDL-C. Multiple logistic regression models showed strong and independent graded relationships of clusters with quintiles of fasting insulin (fifth quintile odds ratio, 10 to 54, P < .001) and to a lesser degree with quintiles of the waist to hip ratio (2.2 to 5.4, P < .001 for most) and of body mass index (1.6 to 4.5, P < .05 for most). In conclusion, all abnormalities cluster in excess of that predicted by chance, with clusters showing remarkable and graded independent associations with fasting hyperinsulinemia and to a lesser extent with central and overall obesity. Thus, a metabolic syndrome occurs in both lean and obese middle-aged US adults. PMID- 8637444 TI - Increased sodium influx and calcium uptake in erythrocytes in hyperthyroidism: role of abnormal membrane lipid levels. AB - The study was designed to examine the effects of thyroid hormones on red blood cell (RBC) membrane phospholipids and ion transport. We demonstrated that in untreated Graves' disease, an alteration in the phospholipid pattern is present at cellular levels, with a concomitant derangement in membrane permeability defined as (22)Na influx and (45)Ca uptake. Thionamide therapy replaced the normal membrane permeability, presumably as a consequence of restoring the normal phospholipid membrane composition. We conclude that thyroid hormones are able to induce a quick breakdown of a large number of membrane components such as membrane phospholipids. PMID- 8637445 TI - Effects of oral albuterol on serum lipids and carbohydrate metabolism in healthy men. AB - beta(2)-Selective adrenergic agonists are used in the management of bronchial asthma and preterm labor. Due to their ability to increase muscle strength and size in animal models, new applications for these agents are also being explored for neuromuscular disorders and in rehabilitation. However, the effects of long term beta(2)-agonist administration on lipoprotein and carbohydrate metabolism are incompletely understood. This investigation evaluated the effects of a beta(2)-agonist, albuterol, on serum lipids and carbohydrate homeostasis in eight healthy nonsmoking men aged 24 to 61 years. Collection of fasting blood samples was completed in duplicate on separate days at baseline, during 14 days of oral albuterol administration (Proventil Repetabs, 8 mg twice daily; Schering Pharmaceuticals, Kenilworth, NJ) and during a 7-day washout period. Carbohydrate homeostasis was evaluated using the minimal model technique at the end of the baseline and albuterol periods. Fasting glucose and insulin, intravenous glucose tolerance, acute insulin response to intravenous glucose (AIRg), insulin sensitivity (Si), and glucose effectiveness (Sg) were not significantly changed during albuterol administration. Significant alterations (P < or = .02) were observed in total cholesterol ([TC] -9.1% +/- 2.5%), low-density lipoprotein cholesterol ([LDL-C] -15.0% +/- 2.9%), and high-density lipoprotein cholesterol ([HDL-C] +10.4% +/- 3.2%) concentrations, as well as the TC/HDL-C (-17.4% +/- 2.6%) and LDL-C/HDL-C (-22.9% +/- 2.4%) ratios. During washout, TC and LDL-C returned to baseline levels, whereas HDL-C remained elevated by 5.8% +/- 2.4% (P < .05). Thus, albuterol administration was associated with favorable changes in the serum lipid profile without marked impairment of glucose tolerance or its physiologic determinants. PMID- 8637447 TI - The serum lathosterol to cholesterol ratio, an index of cholesterol synthesis, is not elevated in patients with glomerular proteinuria and is not associated with improvement of hyperlipidemia in response to antiproteinuric treatment. AB - The hypothesis that increased cholesterol synthesis provides a mechanism that contributes to nephrotic syndrome-associated hyperlipidemia is mainly based on experimental evidence. The serum level of the cholesterol precursor, lathosterol (expressed per millimole cholesterol), is a reliable marker of whole-body cholesterol synthesis in normocholesterolemia and primary hypercholesterolemia. Serum lathosterol and lipoprotein levels were measured in 11 moderately hyperlipidemic patients with nephrotic-range proteinuria and 22 matched controls. The proteinuric patients were evaluated before and during three antiproteinuric treatment periods with angiotensin-converting enzyme (ACE) inhibition therapy (n = 6) or a low-protein diet (n = 5) alone, in combination, and again as a single treatment. In untreated patients, serum total cholesterol, very-low-density (VLDL) and low-density (LDL) lipoprotein cholesterol, apolipoprotein B (apo B), and lipoprotein (a) [Lp(a)] levels were higher than in controls (P < .01 to P < .001), but the lathosterol to cholesterol ratio tended to be lower in patients (0.99 +/- 0.43 micromol/mmol) as compared with controls (1.29 +/- 0.41 micromol/mmol, P < .10). During combined antiproteinuric treatment, total and VLDL + LDL cholesterol, apo B, and Lp(a) decreased (P < .02 to P < .01), but remained higher than levels in controls. Yet the serum lathosterol to cholesterol ratio changed little and was even lower (P < .05) in treated patients than in controls. Serum total cholesterol (r = -.82, P < .01) and apo B (r = -.84, P < .01) were inversely correlated with serum albumin in untreated patients, whereas the serum lathosterol to cholesterol ratio was not (r = -.01, NS). In the patient group, multiple regression analysis showed that changes in the lathosterol to cholesterol ratio during the study were only related to changes in the dietary polyunsaturated to saturated fatty acids ratio (P:S) coinciding with the low protein diet (P < .01). In contrast, the decrease of VLDL + LDL cholesterol, apo B, and Lp(a) was independently related to reduction of proteinuria (P < .02 to P < .001), but not to changes in the lathosterol to cholesterol ratio. In conclusion, the present data, based on the serum lathosterol to cholesterol ratio, do not support the concept that increased cholesterol synthesis plays an important role in the maintenance of human nephrotic syndrome-associated hypercholesterolemia. Moreover, it appears unlikely that the decrease of apo B containing lipoproteins with antiproteinuric treatment is attributable to inhibition of cholesterogenesis. These findings warrant further documentation of cholesterol synthesis in human nephrotic syndrome by direct methods. PMID- 8637446 TI - The hypothalamus-pituitary-ovary and hypothalamus-pituitary-thyroid axes in spinal cord-injured women. AB - Sixteen women with spinal cord injury (SCI) underwent studies of the hypothalamus pituitary-ovary (HPO) and hypothalamus-pituitary-thyroid (HPT) axes with luteinizing hormone (LH) releasing hormone (LHRH) and thyrotropin (TSH) releasing hormone (TRH) stimulation tests during the early follicular phase. The mean interval from injury to participation in this study was 7.5 years (range, 1.5 to 13.1). All subjects were menstruating regularly. Five (35.7%) SCI subjects who were menstruating before injury had postinjury amenorrhea for 1 to 12 months, and the other nine (64.3%) SCI subjects had no interruption of menstruation after injury. Two SCI subjects whose injury occurred in preadolescence proceeded to menarche without any delay. The amount of menstrual flow was noted to be reduced in nine (64.3%) SCI subjects. Two and three SCI subjects had elevated follicle stimulating hormone (FSH) and prolactin (PRL) levels, respectively. LH responses to LHRH were significantly higher in the SCI group (P < .001). Ten (62.6%) SCI subjects had enhanced LH responses to LHRH. The mean TSH, PRL, and FSH responses to TRH and LHRH of the SCI group were not significantly different from those of age-matched controls. However, five (31.2%), four (25.0%), and five (31.2%) SCI subjects had enhanced TSH, PRL, and FSH responses to TRH and LHRH, respectively. Six (37.5%) SCI subjects had a delayed FSH response to LHRH. In total, 13 (81.2%) SCI subjects had at least one axis abnormality. These findings are consistent with the hypothesis that changes of central neurotransmitters may occur after SCI. PMID- 8637449 TI - Anterior pitutiary and pitutiary-dependent target organ function in men infected with the human immunodeficiency virus. AB - To evaluate pituitary and pituitary-dependent target organ function in men infected with the human immunodeficiency virus (HIV), 26 ambulatory HIV-positive men (13 with acquired immunodeficiency syndrome [AIDS]) and nine healthy control men were administered rapid sequential injections of thyrotropin (TSH)-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), ovine corticotropin (ACTH) releasing hormone (oCRH), and human growth hormone-(GH)-releasing hormone (hGHRH). Blood samples were collected before and for 90 minutes after the injections for immunoassay of pituitary hormones, cortisol, testosterone, and free thyroxine (fT(4)). Data were analyzed for each group of men considering basal, peak, and incremental responses to the releasing hormones, as well as the time course of response of each hormone. Mean basal serum GH concentrations were the same in all groups (control, AIDS, and non-AIDS HIV-positive), but stimulated GH levels were substantially higher at all time points in both groups of HIV positive subjects. Results for prolactin (PRL) were similar, although stimulated PRL levels were increased significantly only in the AIDS group. The mean basal serum TSH concentration and stimulated TSH levels at 60 and 90 minutes were significantly greater in the AIDS group than in the control group. Basal mean fT(4) concentration in the AIDS group was significantly less than in the control group. Mean basal and stimulated serum (total) testosterone concentrations in all groups were the same. However, basal serum luteinizing hormone (LH) concentrations in both groups of HIV-infected men were significantly greater than in controls; stimulated (peak) LH levels were not different from control levels. Basal and peak stimulated plasma ACTH concentrations were significantly increased in both HIV-infected groups. Basal serum cortisol levels were also greater, on average, in HIV-infected groups, although stimulated (peak) cortisol responses were not different. These results indicate that basal serum concentrations of TSH, LH, ACTH, and cortisol are modestly increased in men with AIDS, and that maximum levels of GH, PRL, TSH, and ACTH stimulated by the releasing hormones are also increased in this group. Measurements obtained in the non-AIDS HIV-infected men showed a pattern generally similar to that obtained in men with AIDS, but less marked. The basis for the increased pituitary activity is unknown; we speculate that it is due to modestly impaired target organ function and to increased hypothalamic stimulation. PMID- 8637450 TI - Relationships between glucose metabolism and thermogenesis with and without prior exercise in obese women with non-insulin-dependent diabetes mellitus. AB - The rate of insulin-stimulated glucose disposal is reduced in individuals with insulin resistance, and is associated with a blunted or absent increase in energy expenditure in response to a glucose load. The magnitude of the effect of glucose on energy expenditure (EGEE) may be a function of opposing changes in the rate of glucose disposal (Rd) and hepatic glucose production (HGP). In this study, six women with non-insulin-dependent diabetes mellitus (NIDDM) were studied on a metabolic ward in each of three conditions. On days 1 and 2, they did no exercise (NX) or else performed low-intensity exercise ([LO] 3,118 kJ [745 kcal]) at 50% maximal oxygen consumption [V0(2)max]) or high-intensity exercise ([HI] 3,114 kJ [744 kcal] at 75% V0(2)max). On day 3, infusion of 6,6(2)H-glucose in the basal state was immediately followed by infusion of glucose, 6,6(2)H-glucose, and insulin at fixed rates. Indirect calorimetry was performed during the last 30 minutes of each infusion. EGEE was not different among the three conditions (mean +/- SEM: NX -0.18 +/- 0.11, LO -0.08 +/- 0.05, and HI -0.08 +/- 0.07 kJ/min) and was inversely related to steady-state plasma glucose concentration, a direct measure of insulin resistance (r = -.89, P < .05). EGEE was positively correlated with glucose Rd (r = .94, P < .001) and negatively correlated with HGP (r = -.91, P < .05). The data indicate that the glucose effect on energy expenditure was slightly positive in the more insulin-sensitive individuals, but negative in the more insulin-resistant subjects. The EGEE appears to be determined by the relative balance between energy required to store glucose and energy saved by suppression of glucose production. PMID- 8637448 TI - Improvement of insulin sensitivity and dyslipidemia with a new alpha-glucosidase inhibitor, voglibose, in nondiabetic hyperinsulinemic subjects. AB - This study was undertaken to investigate the effect of voglibose, a new alpha glucosidase inhibitor, on glucose and lipid metabolism in nondiabetic hyperinsulinemic subjects. Sixteen nondiabetic subjects with hyperinsulinemia participated in the study. They were divided into two groups of eight subjects with normal (NGT) and impaired (IGT) glucose tolerance. A meal tolerance test and a 75-g oral glucose tolerance test (OGTT) were performed at the beginning (baseline phase) and end (treatment phase) of the 12-week treatment. Serum lipid levels were measured every 4 weeks throughout the treatment phase and follow-up phase (8 weeks). All patients received 1 0.2-mg tablet of voglibose before each test meal (3 tablets per day). We also measured insulin sensitivity using a steady-state plasma glucose (SSPG) method in eight normotensive hyperinsulinemic subjects and in eight age- and body mass index (BMI)-matched control subjects before and after the drug treatment. Voglibose significantly decreased the responses of plasma glucose and insulin on the meal tolerance test. The area under the curve for 2-hour insulin during the 75-g OGTT decreased after treatment, whereas that for 2-hour glucose did not change before and after treatment. SSPG was reduced after treatment, indicating improvement of insulin sensitivity. Moreover, treatment with voglibose resulted in a significant decline of triglyceride level and an elevation of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-1. These values returned to near-baseline levels after the drug was discontinued. Consequently, we conclude that this agent not only has a direct hypoglycemic effect through decreased absorption of carbohydrate, but also a hypoinsulinemic and hypolipidemic effect via improved insulin sensitivity. PMID- 8637451 TI - Glucose and lactate kinetics during a short exercise bout in pregnancy. AB - Pregnancy is considered diabetogenic. Although exercise has been advocated to assist in metabolic control of the nonpregnant diabetic individual, there is a paucity of data about the metabolic effects of exercise during pregnancy. To examine whether moderate exertion may be beneficial in the maintenance of maternal carbohydrate homeostasis, glucose and lactate kinetics were measured in the third trimester in five pregnant nondiabetic women (gestational age, 34.2 +/- 0.1 weeks [mean +/- SE]) by infusion of 45 microg x kg(-1) x min(-1) [6,6 2H2]glucose and 70 microg x kg(-1) x min(-1) [U-13C]lactate tracers. Subjects were observed at rest for determination of baseline steady-state kinetics over a 30-minute period, and then they exercised for 30 minutes at 60% maximum oxygen consumption (VO2max) and were evaluated for 30 minutes postexercise. Glucose and lactate kinetics and lactate oxidation were measured throughout the exercise protocol. This study was repeated postpartum in all individuals at least 6 weeks after delivery. Compared with the steady-state preinfusion period, plasma glucose concentration was not elevated during exercise in either group, nor was plasma lactate concentration significantly different in either group. Glucose kinetics did not change during exercise, but lactate kinetics increased in both groups. V02 and percent of lactate C contribution to CO2, an indication of lactate oxidation, increased proportionally in both groups during exercise. Metabolic perturbations, as measured by glucose and lactate kinetics, do not appear to be different during the third trimester of pregnancy during a relatively short bout of exercise compared with the nonpregnant state. PMID- 8637452 TI - Functional active receptors for insulin-like growth factors-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells. AB - Insulin-like growth factors I and II (IGF-I and IGF-II) are expressed at high levels in the endocrine pancreas during development and tissue regeneration. However, their effects at the endocrine pancreas are poorly understood. We searched for receptors of IGF-I and IGF-II and possible biological effects on clonal insulin-secreting (HIT), glucagon-secreting (INR1G9), and somatostatin secreting (RIN 1027 B2) cell lines. Our data showed that HIT cells and RIN 1027 B2 cells express specific type I and type 11 IGF receptors. INR1G9 cells possess type II IGF receptors and IGF-I binding sites with the same affinity for both IGF I and IGF-II. In HIT cells, insulin secretion was not influenced by either peptide. Proinsulin gene transcription was stimulated by IGF-II but not by IGF-I. IGF-I potently inhibited proglucagon gene transcription and glucagon secretion in INR1G9 cells, whereas IGF-II only inhibited glucagon release. In RIN 1027 B2 cells, IGF-I but not IGF-II increased somatostatin output, whereas both stimulated somatostatin gene expression. These data demonstrate the presence of classic type I and type II IGF receptors on insulin-, glucagon-, and somatostatin secreting cells. Both peptides may be important regulators of endocrine pancreatic function in terms of islet hormone release and gene expression. Therefore, both peptides may be involved in the regulation of intraislet cellular homeostasis. PMID- 8637453 TI - Metabolic response to radiation therapy in patients with cancer. AB - The effect of radiation therapy on substrate metabolism was evaluated in five patients with head and neck or lung cancer. Stable isotope tracer methodology was used to determine urea, amino acid, glucose, and lipid kinetics during postabsorptive conditions before initiation, near the midpoint (after receiving 2,672 +/- 36 rads), and at completion (after receiving 6,072 +/- 307 rad) of a 6- to 8-week course of radiation therapy. Nutritional status was maintained throughout the treatment period by providing supplemental enteral feedings as needed. Postabsorptive plasma insulin, catecholamine, and amino acid concentrations did not change during the course of treatment. Before radiation therapy was initiated, values for the plasma rate of appearance (Ra) of urea (3.35 +/- 0.33 micromol x kg(-1) x min(-1)), alpha-ketoisocaproate ([alpha-KIC] 2.16 +/- 0.19 micromol x kg(-1) x min(-1)), phenylalanine (0.59 +/- 0.052 micromol x kg(-1) x min(-1)), and glucose (10.56 +/- 1.31 micromol x kg(-1) x min(-1)) were in the normal range. However, glycerol and palmitate Ra values (3.11 +/- 0.30 and 2.01 +/- 0.33 micromol x kg(-1) x min(-1), respectively) were 25% higher than values observed previously in normal subjects. Substrate flux did not change during radiation therapy, and measurements obtained during the midpoint and at completion of treatment were similar to initial values. These results demonstrate that large doses of radiation therapy, administered over 6 to 8 weeks to the upper body, do not cause significant metabolic stress. PMID- 8637455 TI - Effect of insulin on urinary phosphate excretion in type II diabetes mellitus with or without renal insufficiency. AB - We investigated the effect of insulin on urinary excretion of phosphate in type II diabetes mellitus (DM) with respect to the absence or presence of renal insufficiency. A euglycemic-hyperinsulinemic clamp was performed in 37 type II DM patients. Subjects were divided into two groups: group A consisted of patients with serum creatinine levels less than 1.5 mg/dL (n = 22), and group B consisted of patients with serum creatinine levels of 1.5 mg/dL or greater (n = 15). Blood and urine samples were collected at the beginning and end of the clamp, and urinary excretion of phosphate was evaluated by calculating fractional excretion (FE-P). Tissue sensitivity to insulin in the whole body was expressed as the glucose infusion rate (M value) and that divided by steady-state plasma insulin levels (M/I ratio) during the last 30 minutes of the clamp. FE-P in group A patients significantly decreased during the clamp (from 9.46 +/- 0.67% before the clamp to 7.12 +/- 0.73% after the clamp, P < .004), whereas FE-P in group B patients did not change significantly during the clamp. The percent decrease of FE-P (decrease of FE-P during the clamp divided by FE-P before the clamp) in group A patients was significantly higher than in group B patients (22.5 +/- 7.0% and 2.5 +/- 5.1 %, respectively, P < .04). In all 37 patients, the percent decrease of FE-P was negatively correlated with blood urea nitrogen ([BUN] r = .36, P < .05), serum creatinine (r = -.34, P < .05), and serum beta2 microglobulin (r = -.44, P < .01) and positively correlated with creatinine clearance (r = .570, P < .004), but it was not correlated with the M value or M/I ratio. These results showed that the kidneys of diabetic patients with renal insufficiency are insulin-insensitive in terms of phosphate transport, and the insulin insensitivity is related to the glomerular filtration rate but not to systemic insulin insensitivity. The percent decrease of FE-P on clamp study could be useful for assessing the insulin insensitivity of the kidney, which probably occurs primarily in the renal tubules. PMID- 8637454 TI - Insulin increases distinct species of 1,2-diacylglycerol in isolated perfused rat heart. AB - Insulin and glucose increase the synthesis of 1,2-diacylglycerol (1,2-DAG), the physiological activator of protein kinase C (PKC) in a variety of tissues and cells. The effects of insulin and glucose on the abundance and fatty acid composition of 1,2-DAG were investigated in isolated perfused rat hearts with the use of capillary gas chromatography and 1,2-dipentadecanoin as an internal standard. A high concentration of insulin (25 mU/ mL) significantly increased cardiac contractility and reduced coronary flow. In addition, perfusion with 25 mU/mL insulin induced significant increases of 18.2% and 26.4% in 1,2-DAG mass after 5 and 30 minutes, respectively, in the presence of 8.6 mmol/L glucose, whereas there was no increase in 1,2-DAG with 2.5 mU/mL insulin. Analysis of the fatty acid composition of 1,2-DAG showed that only species containing specific fatty acids (16:0, 18:1, and 18:2) were increased in response to insulin. In contrast, an increase in glucose concentration in the perfusion medium from 3 to 17 mmol/L had no effect on the total mass or fatty acid composition of 1,2-DAG, cardiac contractility, or coronary flow. Addition of a high insulin concentration to the high-glucose medium increased the abundance of 1,2-DAG containing 16:0, 18:1, and 18:2 fatty acids, as well as cardiac contractility. It is concluded that the effect of insulin on cardiac contractility may be related to the associated increase in 1,2-DAG abundance. PMID- 8637457 TI - Making fatigue less tiresome. PMID- 8637458 TI - Beta-blockers in chronic heart failure: paradox proven? PMID- 8637456 TI - Effect of CL316,243, a highly specific beta3-adrenoceptor agonist, on sympathetic nervous system activity in mice. AB - To examine whether long-term administration of a beta3-adrenoceptor agonist influences sympathetic nervous system (SNS) activity, norepinephrine (NE) turnover, a reliable indicator of SNS activity, in the interscapular brown adipose tissue (IBAT), the heart, and the spleen, as well as urinary excretion of NE, were measured using mice treated with CL316,243 (CL), a highly specific beta3 adrenoceptor agonist, at a dose that stimulated thermogenesis and reduced body weight. CL significantly decreased NE turnover in the IBAT, heart, and spleen and decreased urinary excretion of NE without affecting food intake over 1 to 4 weeks of treatment. These findings show that long-term administration of the beta3 adrenoceptor agonist decreases SNS activity and urinary excretion of NE. PMID- 8637459 TI - Schizophrenia and the community. PMID- 8637460 TI - Fatigue in selected primary care settings: sociodemographic and psychiatric correlates. AB - OBJECTIVES: To determine the prevalence and sociodemographic and psychiatric correlates of prolonged fatigue syndromes among patients in primary care. DESIGN: Prospective questionnaire survey. PATIENTS AND SETTING: Adults over 18 years attending three general practices in metropolitan Sydney and one on the Central Coast, north of Sydney. RESULTS: Of 1593 patients, 25% had prolonged fatigue, while 37% had psychological disorder. Of the patients with fatigue, 70% had both fatigue and psychological disorder, while 30% had fatigue only. The factors associated with prolonged fatigue were concurrent psychological disorder, female gender, lower socioeconomic status and fewer total years of education. Patients with fatigue were more likely to have a current depressive disorder. CONCLUSIONS: Prolonged fatigue/neurasthenia syndromes are common in Australian primary care settings, and are commonly associated with current depressive disorders. Such syndromes, however, do not fit readily into current international psychiatric classification systems. PMID- 8637461 TI - Pattern of non-steroidal anti-inflammatory drug use in Australia 1990-1994. A report from the Drug Utilization Sub-Committee of the Pharmaceutical Benefits Advisory Committee. AB - OBJECTIVE: To determine the pattern of use of non-steroidal anti-inflammatory drugs (NSAIDs) in the Australian community, 1990-1994. DESIGN: Data from the national drug utilisation database were expressed in defined daily doses per 1000 population per day (DDDs/1000 population per day). Temporal trends were assessed and comparisons were made with NSAID use in other countries. Epidemiological data were used to estimate the likely impact of changing NSAID use on peptic ulcer hospitalisation rates. SETTING: Australian community (excluding hospitals). MAIN OUTCOME MEASURES: Estimated consumption of prescription NSAIDs, expressed in DDDs/1000 population per day. RESULTS: NSAID use in the Australian community fell from 50.1 DDDs/1000 population per day in 1990 to 34.6 DDDs/1000 population per day in 1994 (down 31%). From this reduced exposure we estimated that the number of admissions for NSAID-related upper gastrointestinal complications will have fallen by about 400 per year. Market research data for this period show a lower percentage use of NSAIDs for osteoarthritis and a decrease in the proportion of use in age groups over 50 years. CONCLUSIONS: The level of use of non-steroidal anti-inflammatory drugs in Australia has been high in comparison with other countries, but in recent years has fallen markedly. This fall occurred in conjunction with regulatory interventions, educational campaigns and increased concern in the medical and lay press regarding the risks associated with the use of NSAIDs. PMID- 8637462 TI - Tetanus immunity in an older Australian population. AB - OBJECTIVE: To evaluate the effectiveness in older Australians of the current tetanus vaccination program. DESIGN: A cross-sectional survey of tetanus immunity (enzyme immunoassay of serum samples) in an older population in New South Wales. Self-reported history of tetanus vaccination was compared with serologically measured immunity. PARTICIPANTS: 430 randomly selected adults, 49 years of age and older, from the Blue Mountains Eye Study population. RESULTS: Fifty-two per cent (95% confidence interval [CI], 47%-57%) of adults 49 years of age and older had protective levels of tetanus antitoxin ( > 0.15 IU/mL). There was a significant decline in the prevalence of immunity with increasing age (chi 2 for linear trend, P = 0.036), and women were less likely to be immune regardless of their age (Mantel-Haenszel weighted odds ratio, 0.65; CI, 0.43-0.92). Thirty-five per cent (95% CI, 31%-40%) of all participants reported that they had been vaccinated in the preceding 10 years. Although self-reported tetanus vaccination history was associated with tetanus immunity, it was neither sensitive nor specific as a test for immunity. CONCLUSIONS: About half the adults 49 years of age and older in the Blue Mountains area of New South Wales do not have protective levels of tetanus antitoxin because of inadequate vaccination coverage in this age group. Vaccination history is not a reliable indicator of tetanus immunity and a system is needed for accurate recording of adult vaccination. PMID- 8637463 TI - Iatrogenic and zoonotic Creutzfeldt-Jakob disease: the Australian perspective. AB - The transmissible brain diseases of humans and animals, the spongiform encephalopathies, continue to stimulate interest, and the announcement that exposure to "mad cow disease" (bovine spongiform encephalopathy [BSE]) is a possible explanation for more than 10 cases of a variant Creutzfeldt-Jakob disease in humans in the United Kingdom is a recent example. Cases of iatrogenic Creutzfeldt-Jakob disease (from previous use of human cadaveric tissues for pituitary hormone therapy and neurosurgical grafts) are still being identified, and the unique nosological status of this group of disorders-that they are both transmissible and inherited and that the only known component of their infectious agent is protein-alone makes these diseases remarkable. PMID- 8637464 TI - Neurosurgery and iatrogenic transmission of Creutzfeldt-Jakob disease. AB - The Neurosurgical Society of Australasia has not recommended that recipients of dura mater grafts be actively sought out and informed, but rather that neurosurgeons should attempt to discover for individual patients whether they have received dura mater grafts. PMID- 8637465 TI - Managing HIV. Common drug interactions in HIV medicine. PMID- 8637466 TI - Managing HIV. Antiretroviral therapy. AB - Initiating or changing antiretroviral therapy requires specialist knowledge of indications, side effects and drug interactions. This brief guide highlights the key points for monitoring antiretroviral therapy. PMID- 8637468 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.19 HIV and palliative care. AB - The goals of palliative care are the relief of pain and suffering in advanced disease and the support of the patient's carers. In HIV medicine, good palliative care often includes active treatment, and the palliative care team may work in support of the primary care doctor. PMID- 8637467 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.18 HIV and sexually transmitted diseases. AB - Sexually transmitted diseases (STDs) play a key role in the AIDS epidemic. The transmission of HIV is amplified by some STDs, and HIV has been found to alter the natural history and prevalence of others. The control of STDs is fundamental to effective prevention and management of HIV infection. PMID- 8637469 TI - Managing HIV. Part 5: Treating secondary outcomes. 5.20 HIV and autopsies. PMID- 8637470 TI - The significance of birthmarks. PMID- 8637471 TI - The risks and benefits of antithrombotic therapy in chronic atrial fibrillation. AB - As the risks of adverse effects of warfarin and aspirin in certain patients with chronic atrial fibrillation are high, intervention with these agents should be targeted towards those who will derive most benefit. Clearer guidelines are emerging to identify those patients and to standardise current prescribing practices. PMID- 8637472 TI - Assisted reproduction: a reassuring picture. AB - Many couples seek solutions to problems of infertility by using assisted reproduction techniques. Despite very different beginnings, children conceived after donor insemination, ovum donation, in-vitro fertilisation or gamete intrafallopian transfer show no adverse long term effects, either physically or psychosocially. PMID- 8637474 TI - Tampons and toxic shock syndrome. PMID- 8637473 TI - Tampons and toxic shock syndrome. PMID- 8637475 TI - Once-daily aminoglycoside dosing. PMID- 8637476 TI - Multiple endoscopies in a Sydney blood donor found positive for hepatitis B and C antibodies. PMID- 8637477 TI - Tetanus vaccination demands continued attention in adults. PMID- 8637478 TI - Unrecognised renal infarction. PMID- 8637479 TI - Benefits of a best friend. PMID- 8637480 TI - Familial male breast cancer. PMID- 8637481 TI - Clarithromycin and omeprazole for Helicobacter pylori. PMID- 8637483 TI - The prostatron: microwaves for benign prostatic hyperplasia. PMID- 8637482 TI - Azelaic acid-a new topical drug for acne. PMID- 8637484 TI - Diagnosis. PMID- 8637485 TI - Running on empty. PMID- 8637486 TI - Bone marrow transplantation. New strategies for treating malignant disease. AB - In the years since the world's first successful bone marrow transplant (BMT) was performed at the University of Minnesota in 1968, the field of bone marrow transplantation has evolved rapidly. Stem cells can be obtained from a variety of sources: bone marrow from a matched sibling or unrelated donor (allogeneic transplant), bone marrow or peripheral blood from the patient (autologous transplant), and umbilical cord blood that was collected and stored after delivery for later use by the infant, a matched sibling, or an unrelated patient. Bone marrow transplantation is a widely accepted and successful therapy for treating a variety of malignant and nonmalignant diseases. Continuing research should yield new methods to ensure successful engraftment and to prevent or reduce complications post-BMT. Many patients with cancer have been cured with BMT. However, much work remains to improve survival and to better manage complications. New approaches under investigation may expand the availability of transplantation and improve short- and long-term survival and quality of life. PMID- 8637488 TI - Break the gridlock in Washington. PMID- 8637487 TI - Genetic testing for familial cancer. A clinician's perspective. AB - Molecular genetics researchers have recently identified several genes (and mutations) that result in family cancers. This has prompted the development of molecular diagnostic tests to identify individuals at risk, making it possible to prevent certain types of cancer. However, detection and prevention of certain malignancies are impeded because few medical practitioners know of these tests, and because patients fear the emotional consequences of testing. To highlight some of these issues, we describe a patient who chose to be tested for the RET gene mutation. The mutation was identified, additional family members were tested, and several were found to be at risk. The RET gene mutation is 100% predictive for the ultimate development of medullary thyroid cancer. Testing and counseling provided this patient and her family with information not only about the thyroid cancer in their family, but also about multiple endocrine neoplasia, a syndrome resulting from mutation of this particular gene. Familial cancer clinics are being established to assist patients and physicians who face questions about familial diseases and the value of genetic testing. We include in this report information about the familial cancer clinics in Minnesota and list the services and testing opportunities available at these clinics. PMID- 8637489 TI - Wellstone and Ramstad debate health care issues -news-. PMID- 8637490 TI - Governors propose controversial Medicaid changes -news-. PMID- 8637491 TI - Mammography quality assurance. PMID- 8637493 TI - Cancer research and treatment. When money, morals, and medical science converge. PMID- 8637492 TI - Early detection of prostate cancer. Decreasing the mortality rate. AB - In conclusion, prostate cancer is a major menace to Western society. Since prostate cancer is asymptomatic in the early stages and no curative therapy exists for the advanced stages, our only hope for decreasing the mortality rate from prostate cancer is through early detection programs. Young men with life expectancies of 15 years or more should participate in early detection efforts. These men should be evaluated diligently, through the combined use of DRE and the newly described age-specific reference ranges. Radical prostatectomy, when performed on a man with a life expectancy of 30 to 35 years who has organ confined prostate cancer, is an effective treatment for the No. 1 cancer in men today. With limited health care dollars available in the future for the diagnosis and management of prostate cancer, physicians will be forced to become selective with their diagnostic and therapeutic efforts. It is much less expensive to treat a young man with early-stage, curable prostate cancer than to manage an elderly man terminally ill from advanced prostate cancer. Without question, young men will be the target of our early detection efforts and the ones who will benefit. PMID- 8637494 TI - Does screening with prostate/specific antigen improve outcomes? PMID- 8637495 TI - Mandates for unproven health care interventions. PMID- 8637496 TI - Building bridges to combat cancer. PMID- 8637497 TI - Prevention of perinatal group B streptococcal disease: a public health perspective. Centers for Disease Control and Prevention. AB - Group B streptococcus is a leading cause of serious neonatal infection. Most neonatal GBS infections can be prevented through the use of intrapartum antimicrobial prophylaxis in women who are at increased risk for transmitting the infection to their newborns. However, despite clinical trials that demonstrate the effectiveness of intrapartum antibiotic prophylaxis, prevention strategies have not been implemented widely or consistently, and the incidence of neonatal GBS disease has not declined. To promote a coordinated approach to prevention among obstetric- and pediatric-care practitioners and among supporting clinical microbiology laboratory personnel, CDC has developed prevention guidelines in conjunction with experts from relevant disciplines and with representatives of the American College of Obstetricians and Gynecologists, the American Academy of Pediatrics, and other professional organizations. This report provides the epidemiologic basis for prevention protocols, summarizes results of clinical trials demonstrating the efficacy of intrapartum antimicrobial agents, examines limitations of different approaches to prevention, and presents guidelines for the prevention of GBS disease. CDC recommends use of one of two prevention strategies. In the first strategy, intrapartum antibiotic prophylaxis is offered to women identified as GBS carriers through prenatal screening cultures collected at 35-37 weeks' gestation and to women who develop premature onset of labor or rupture of membranes at < 37 weeks' gestation. In the second strategy, intrapartum antibiotic prophylaxis is provided to women who develop one or more risk conditions at the time of labor or membrane rupture. Issues addressed by these prevention guidelines include the following: the appropriate clinical and laboratory methods required for prenatal screening programs designed to identify GBS carriers; risk conditions that indicate the need for intrapartum antibiotics; management of newborns whose mothers receive intrapartum antibiotic prophylaxis for GBS disease; and education of prenatal patients regarding GBS disease and the available prevention policy. These guidelines are intended for the following groups: a) providers of prenatal, obstetric, and pediatric care; b) supporting microbiology laboratories, hospital administrators, and managed-care organizations; c) childbirth educators; d) public health authorities; e) expectant parents; and f) advocacy groups for expectant parents. PMID- 8637498 TI - Guidelines for school health programs to promote lifelong healthy eating. Centers for Disease Control and Prevention. AB - Healthy eating patterns in childhood and adolescence promote optimal childhood health, growth, and intellectual development; prevent immediate health problems, such as iron deficiency anemia, obesity, eating disorders, and dental caries; and may prevent long-term health problems, such as coronary heart disease, cancer, and stroke. School health programs can help children and adolescents attain full educational potential and good health by providing them with the skills, social support, and environmental reinforcement they need to adopt long-term, healthy eating behaviors. This report summarizes strategies most likely to be effective in promoting healthy eating among school-age youths and provides nutrition education guidelines for a comprehensive school health program. These guidelines are based on a review of research, theory, and current practice, and they were developed by CDC in collaboration with experts from universities and from national, federal, and voluntary agencies. The guidelines include recommendations on seven aspects of a school-based program to promote healthy eating: school policy on nutrition, a sequential, coordinated curriculum, appropriate instruction for students, integration of school food service and nutrition education, staff training; family and community involvement, and program evaluation. PMID- 8637500 TI - The ultraviolet-sensitizing function of plasmid R391 interferes with a late step of postreplication repair in Escherichia coli. AB - The conjugative plasmid R391 increases the UV radiation sensitivity of wild-type, uvrA, and lexA cells of Escherichia coli, but not recA strains. To investigate the UV-sensitizing function of R391, we examined the effect of R391 on the repair of DNA daughter-strand gaps and on the UV radiation sensitivities of various repair and/or recombination-deficient mutants. The presence of R391 did not significantly inhibit the repair of DNA daughter-strand gaps in uvrB cells. The presence of R391 increased the UV radiation sensitivity of uvrA, uvrA recF, uvrB, uvrB recF, uvrB recB, and uvrB ssb-113 cells to UV irradiation, but did not significantly increase the UV radiation sensitivity of uvrA ruvA and uvrA ruvC strains. Based on these results, we propose that the UV-sensitizing activity of R391 acts by inhibiting or interfering with the ruvABC-mediated postsynapsis step of recombinational repair. PMID- 8637499 TI - Enhanced UV sensitivity of yeast cells induced by overexpression of Mg(2+) dependent protein phosphatase alpha (type 2C alpha). AB - The UV sensitivity of wild-type Saccharomyces cerevisiae cells was increased 2 fold when rat Mg(2+)-dependent protein phosphatase alpha (protein phosphatase type 2C alpha) was overexpressed in the cells. The overexpression of this enzyme rendered the rad 18 mutant (defective in postreplication repair) more UV sensitive than was observed in the wild-type cells. However, this increase in UV sensitivity disappeared when the host cells had a rad 1 mutation (defective in excision repair). These results suggest that the Mg(2+)-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system. PMID- 8637501 TI - Stability of microsatellites and minisatellites in Bloom syndrome, a human syndrome of genetic instability. AB - Bloom syndrome (BS) is a human cancer-prone genetic disorder essentially characterized by a generalized genetic instability including a high level of sister chromatid exchanges (SCEs). Although mutator and hyper-Rec phenotypes of BS cells present analogies with those of bacteria and yeast defective in DNA mismatch repair, we report that (CA)(n) microsatellite alterations are undetectable in BS cells. Thus, our results suggest that the origin of BS mutator phenotype is not a major defect in DNA mismatch repair, allowing us to eliminate an attractive hypothesis for the pleiotropy of BS. We previously suggested that at least some of the intra-allelic rearrangements occurring in minisatellites could result from unequal SCEs. Although SCEs are abnormally frequent in BS cells, the present study failed to show any significant variation of the mutation rates of the two hypermutable minisatellites we analyzed. Thus, our results show that, in spite of an overall genetic instability, alterations in structural motifs known to be predisposed to instability by different mechanisms are undetectable in BS cells. PMID- 8637502 TI - Differential expression of the apurinic / apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis. AB - The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA binding capability of Jun-Jun homodimers and Fos-Jun heterodimers by altering their redox state and to be involved in calcium mediated transcriptional repression of the parathyroid hormone gene. Previous studies examining the tissue specific distribution of the AP endonuclease (APE) transcript and protein by Northern analysis and enzymatic assays, respectively, have shown that this gene is expressed in all tissues at relatively similar levels. In the current study, adult and fetal rat tissue sections were examined for the expression of the APE transcript in specific subpopulations of cells and during development by in situ hybridization. In the adult brain, the APE transcript showed a widespread, but heterogeneous pattern of expression. Predominant levels of transcript were detected in the suprachiasmatic nuclei, the supraoptic and paraventricular nuclei, the hippocampus and the cerebellum. During fetal development, transcript was detected in all somatic sites examined with very high levels in the thymus, liver and developing brain. Examination of the adult testis indicated that the expression of the transcript varies with the stage of spermatogenesis with the highest levels being present over round spermatids. These results provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes and during development. PMID- 8637504 TI - The Escherichia coli DNA repair protein UvrA can re-associate with the UvrB: aflatoxin B1-DNA complex in vitro. AB - The UvrA and UvrB proteins form part of the UvrABc endonuclease, which is responsible for nucleotide excision repair in Escherichia coli. Using a mobility shift gel assay we have studied the binding of UvrA dimer, UvrB monomer and UvA(2)B trimer complexes with 40, 50 and 136 bp (32)P-end-labelled DNA fragments adducted with aflatoxin B(1). UvrA was shown to re-associate with adduct specific UvrB: DNA complexes, a phenomenon which could be reversed by the addition of 500 mM potassium chloride or anti-UvrA anti-sera. Re-association was shown to be UvrA concentration dependent. Re-association of UvrA(2)B to the UvrB:DNA complex was not seen. We have also shown that the UvrB:DNA complex, in the case of aflatoxin B(1), is extremely stable with a half-life excess of 400 min and that fragment termini are not a specific substrate for UvrA binding. PMID- 8637503 TI - A comparison of the genotoxic effects of carboplatin and cisplatin in Escherichia coli. AB - cis-Diammine(1,1,-cyclobutanedicarboxylato)platinum(II) (carboplatin) is a second generation platinum anticancer agent with antineoplastic properties like that of its parent compound, cis-diamminedichloroplatinum(II) (cisplatin) but with substantially less deleterious side effects in treated patients with cisplatin. We compared their genotoxic effects in Escherichia coli and found carboplatin to be less cytotoxic (measured as loss of colony forming ability) that cisplatin in that equitoxic doses required greater than 60 time more carboplatin. However, solutions of carboplatin containing chloride ion became more cytotoxic to E. coli after a 24 h incubation period than similar freshly made solutions. Two platinum conversion products which were neither present in freshly made solutions nor in solutions lacking chloride were resolved by thin-layer chromatography (TLC). One of the conversion products migrated like cisplatin and its occurrence in carboplatin solutions was associated with cisplatin-like properties, enhanced cytotoxicity and ability to induce the SOS responses in E. coli. The SOS-inducing abilities were determined by induction of a sulA::lacZ fusion. Likewise, adducts formed in end-labeled oligonucleotides treated with carboplatin appeared identical to those caused by cisplatin when carboplatin was preincubated in chloride-containing solutions but not by carboplatin in freshly made solutions. It is likely that responses evoked by carboplatin in biological systems are partly due to activation of carboplatin by its conversion of cisplatin. PMID- 8637505 TI - DNA single-strand scission in the pyloric mucosa of rat stomach induced by four glandular stomach carcinogens and three other chemicals. AB - Induction of DNA single-strand scission by four glandular stomach carcinogens and three other chemicals was studied in the pyloric mucosa of rat stomach after gastric intubation. DNA single-strand scission, as was measured by the alkaline elution method, was induced by four glandular stomach carcinogens; N-nitroso N methylurethane at doses of 1 and 9 mg/kg body wt, 4-nitroquinoline 1-oxide at 20 and 30 mg/kg body wt. N-ethyl-N'-nitro-N-nitrosoguanidine at 30 and 100 mg/kg body wt and N-propyl-N'-nitro-N-nitrosoguanidine at 30 and 100 mg/kg body wt. DNA single-strand scission was also induced dose-dependently by a direct acting mutagen, 1-nitrosoindole-3-acetonitrile at doses of 100, 500 and 800 mg/kg body wt. Omeprazole, a proton pump inhibitor, was equivocal in its effect in this assay at 30-500 mg/kg body wt: induction was statistically significant by Cochran Armitage binomial trend test. Loxtidine, an H2-receptor antagonist, did not induce DNA single-strand breaks in the pyloric mucosa at a dose of 400 mg/kg body wt. The present results together with previous information suggest that DNA single-strand scission is a good marker for tumor-initiating activity in rat stomach mucosa. PMID- 8637507 TI - Chromosomal aberrations in lymphocytes from car painters. AB - In the present paper we report the results of biological monitoring of a group of 25 car painters working in different automobile shops in Brasilia. There was a significantly higher frequency of aneuplodies and chromosome deletions in the peripheral lymphocytes of car painters than in control subjects. We also detected a significant correlation between the time worked as a car painter and the frequency of aneuploidy. Smoking habits do not represent a significant factor in terms of production of the various types of chromosome aberrations among car painters. These results permitted us to conclude that the individuals studied represent a risk group and should be medically followed up with judicious periodic examinations. PMID- 8637506 TI - Evaluation of the mutagenicity of the molluscicidal latex of Christ's Crown (Euphorbia milii var. hislopii) in mammalian cells in vitro and in vivo. AB - The latex of Christ's Crown (Euphorbia milii var. hislopii, syn. E. splendens var. hislopii) is a highly active plant molluscicide and could be used for snail control to reduce the prevalence of schistosomiasis in endemic areas. In the course of its toxicological evaluation, the mutagenicity of the latex of Euphorbia milii was tested in mammalian cells in vitro and in vivo. Latex was investigated for its capability of inducing gene mutations and chromosome aberrations in V79 cells in the absence and presence of S9-mix. Concentrations up to 800 micrograms/ml neither induced gene mutations at the HPRT locus nor chromosome aberrations. Latex had no effect on the frequencies of chromosome aberrations in the bone marrow of male and female rats at a dose of 1000 mg/kg. The results indicate that latex of E. milii is not mutagenic in mammalian cells in vitro and in vivo and its use as a molluscicide does not pose a mutagenic hazard for humans. PMID- 8637509 TI - DNA damage induced by seven N-nitroso compounds in primary cultures of human and rat kidney cells. AB - Seven N-nitroso compounds (NOC), known to induce kidney tumors in rats, were assayed for DNA-damaging activity in primary cultures of human and rat kidney cells. DNA fragmentation was measured by the alkaline elution technique. Positive responses were obtained in cells of both species with N-nitrosodimethylamine (32 mM), N-nitrosodiethylamine (32 mM), N-nitrosodi-n-propylamine (10 mM), N-ethyl-N hydroxyethylnitrosamine (18 mM), and streptozotocin (1 mM). N nitrosodiethanolamine and N-nitrosomorpholine were inactive at the highest concentration tested (32 mM). The responses of human kidney cells were qualitatively similar to those of rat kidney cells, but statistically significant differences between the two species in the DNA-damaging potencies were observed with N-ethyl-N-hydroxyethylnitrosamine and streptozotocin, both more genotoxic in rat cells. Taken as a whole, the results suggest on the one hand that the five active NOC might be carcinogenic for the kidney in humans, and on the other hand that the rat kidney cell/DNA damage assay is a valid model for predicting the genotoxic potential of NOC in human kidney cells. PMID- 8637510 TI - Detection of genotoxic activity in native hospital waste water by the umuC test. AB - The genotoxic potential of the waste water of a hospital was evaluated by the umuC test. Within 2 years over 800 native waste water samples were analysed. Genotoxic activity was found in 13% of the samples. The highest genotoxic activity occurred in the morning hours, but genotoxic samples were detected also during the day and at night. 96% of the genotoxic waste water samples revealed a genotoxic potential without growth inhibition of test bacteria monitored as OD600, in the same way as antineoplastic drugs like mitomycin C or cisplatin. 4% of the genotoxic waste water samples showed combined cytotoxic and genotoxic activities as seen in control experiments using glutaraldehyde containing disinfectants and certain antibiotics. PMID- 8637508 TI - Genetic and non-genetic biomarkers related to carcinogenesis in evaluating toxicological risk from Fenarimol. AB - A multibiomarker approach based on the study of toxicity mechanisms at both genetic and metabolic levels has been applied to Fenarimol. With regard to genotoxicity, particular attention was given to assays for chromosomal aberration and micronuclei; clastogenic potential was assessed in human peripheral blood lymphocytes in vitro, while the induction of micronuclei was studied in male CD1 mouse bone marrow polychromatic erythrocytes (PCE). Fenarimol did not induce any significant dose-related increase in micronucleated PCEs, up to 4-fold above the control level at a single dose of 75 mg/kg b.w., was observed 24 h after treatment. Using selective biochemical markers of effect Fenarimol was found to induce CYP 2B1 isoforms in liver, kidney and lung microsomes of Swiss Albino CD1 male and female mice, as shown by the significant increase in specific 2B1-probe pentoxyresorufin O-dealkylase activity. On the contrary, CYP 3A, probed by N demethylation of aminopyrine, were only induced in the liver. Results were corroborated by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 2B1 and 3A. Northern blotting analysis with CYP 2B1 and 3A cDNA biotinylated probes showed that the expression of such isoforms is regulated at mRNA level. Taken as a whole, these data indicate the possible (mutagenic) cotoxic/cocarcinogenic and promoting potential of this fungicide. PMID- 8637511 TI - Clastogenic activity of sodium fluoride to rat vertebral body-derived cells in culture. AB - The US National Toxicology Program has shown equivocal evidence of carcinogenic activity of sodium fluoride (NaF) in male F344/N rats based on the occurrence of five osteosarcomas in treated animals. In the study the osteosarcomas developed mainly in the rat vertebrae. To provide a possible mechanistic basis for the observed tumors, the genotoxic effects of NaF on the possible target organ of NaF carcinogenesis were examined. Rat vertebral body-derived (RVBd) cells were established from trabecular bone of vertebral bodies of a male F344/N rat 6 weeks of age and treated with NaF. RVBd cells in secondary culture exhibited a high level of alkaline phosphatase (ALP) activity when the cells at confluence were assayed by ALP staining. When the histochemical examination was performed on RVBd cell colonies, most of the colonies were stained positively for ALP. Confluent RVBd cells were responsive to 10(-8) M 1 alpha.25-dihydroxyvitamin D3 with a 7.7 fold increase in osteocalcin production over base line values. The von Kossa staining demonstrated that in the presence of 2 mM beta-glycerophosphate, RVBd cells that were allowed to grow past confluence for approximately 2 months formed mineralized nodules. When RVBd cells in tertiary culture were treated with NaF at 0.5-2.0 mM for 24-72 h, the growth and/or survival of the treated cells was reduced in a dose-dependent manner. Significant increases in the frequencies of chromosome aberrations were induced in a dose- and treatment time-dependent fashion when NaF was administered to RVBd cells at 0.5 and 1.0 mM for 24 and 48 h. The results indicate that NaF is genotoxic to rat vertebrae, providing a possible mechanism for the vertebrae, as a target organ of NaF carcinogenesis. PMID- 8637512 TI - The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. AB - BACKGROUND: Accurate estimates of the risk of transfusion-transmitted infectious disease are essential for monitoring the safety of the blood supply and evaluating the potential effect of new screening tests. We estimated the risk of transmitting the human Immuno-deficiency virus (HIV), the human T-cell lymphotropic virus (HTLV), the hepatitis C virus (HCV), and the hepatitis B virus (HBV) from screened blood units donated during the window period following a recent, undetected infection. METHODS: Using data on 586,507 persons who each donated blood more than once between 1991 and 1993 at five blood centers (for a total of 2,318,356 allogeneic blood donations), we calculated the incidence rates of seroconversion among those whose donations passed all the screening tests used. We adjusted these rates for the estimated duration of the infectious window period for each virus. We then estimated the further reductions in risk that would result from the use of new and more sensitive viral-antigen or nucleic acid screening tests. RESULTS: Among donors whose units passed all screening tests, the risks of giving blood during an infectious window period were estimated as follows: for HIV, 1 in 493,000 (95 percent confidence interval, 202,000 to 2,778,000); for HTLV, 1 in 641,000 (256,000 to 2,000,000); for HCV, 1 in 103,000 (28,000 to 288,000); and for HBV, 1 in 63,000 (31,000 to 147,000). HBV and HCV accounted for 88 percent of the aggregate risk of 1 in 34,000. New screening tests that shorten the window periods for the four viruses should reduce the risks by 27 to 72 percent. CONCLUSIONS: The risk of transmitting HIV, HTLV, HCV, or HBV infection by the transfusion of screened blood is very small, and new screening tests will reduce the risk even further. PMID- 8637513 TI - Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection. AB - BACKGROUND: For many people infected with the hepatitis C virus (HCV), the route of exposure, risk of transmission, and severity of associated liver disease are unknown. We studied these variables in people who donated blood voluntarily. METHODS: Blood donors who tested positive for HCV antibodies on enzyme immunoassay were classified according to whether the results of a confirmatory second-generation recombinant immunoblot assay (RIBA) for HCV were positive, negative, or indeterminate. The evaluations also included an assessment of risk factors, a physical examination, serial determinations of alanine aminotransferase levels and HCV serologic assays, a polymerase-chain-reaction assay for HCV RNA, testing of sexual contacts and family members, and liver biopsies in some participants who were HCV-positive by RIBA. RESULTS: A total of 481 donors were studied, among whom 248 were positive for HCV by RIBA, 102 had indeterminate results, and 131 were HCV-negative. In a logistic-regression analysis, significant risk factors for HCV infection among the HCV-positive participants were a history of blood transfusion in 66 (27 percent; P < 0.001 for the comparison with RIBA-negative donors), intranasal cocaine use in 169 (68 percent, P < 0.001), intravenous drug use in 103 (42 percent, P = 0.001), sexual promiscuity in 132 (53 percent, P = 0.002), and ear piercing among men (P < 0.05). Nine of 85 sexual partners of HCV-positive donors were anti-HCV-positive; 8 had used intravenous drugs or received transfusions. HCV RNA was found in 213 HCV-positive donors (86 percent), 3 who had indeterminate results by RIBA (2 of these 3 tested positive with a more specific, third-generation RIBA), and none who were HCV-negative. Of the HCV-positive donors, 69 percent had biochemical evidence of chronic liver disease; among 77 donors positive for HCV by RIBA who underwent liver biopsy, 5 had severe chronic hepatitis or cirrhosis, 66 had mild to-moderate chronic hepatitis, and 6 had no evidence of hepatitis. CONCLUSIONS: Among volunteer blood donors, prior blood transfusion, intranasal cocaine use, intravenous drug use, sexual promiscuity, and ear piercing in men are risk factors for HCV infection. The high frequency of intravenous drug use was unexpected, because these donors had denied such use when questioned directly at the time of their blood donations. PMID- 8637514 TI - Treatment of septic shock with the tumor necrosis factor receptor:Fc fusion protein. The Soluble TNF Receptor Sepsis Study Group. AB - BACKGROUND: A recombinant, soluble fusion protein that is a dimer of an extracellular portion of the human tumor necrosis factor (TNF) receptor and the Fc portion of IgG1 (TNFR:Fc) binds and neutralizes TNF-alpha and prevents death in animal models of bacteremia and endotoxemia. METHODS: To evaluate the safety and efficacy of TNFR:Fc in the treatment of septic shock, we conducted a randomized, double-blind, placebo-controlled, multicenter trial. A total of 141 patients were randomly assigned to receive either placebo or a single intravenous infusion of one of three doses of TNFR:Fc (0.15, 0.45, or 1.5 mg per kilogram of body weight). The primary end point was mortality from all causes at 28 days. RESULTS: There were 10 deaths among the 33 patients in the placebo group (30 percent mortality), 9 deaths among the 30 patients receiving the low dose of TNFR:Fc (30 percent mortality), 14 deaths among the 29 receiving the middle dose (48 percent mortality), and 26 deaths among the 49 receiving the high dose (53 percent mortality) (P = 0.02 for the dose-response relation). Baseline differences in the severity of illness did not account for the increased mortality in the groups receiving the higher doses of TNFR:Fc. CONCLUSIONS: In patients with septic shock, treatment with the TNFR:Fc fusion protein does not reduce mortality, and higher doses appear to be associated with increased mortality. PMID- 8637516 TI - Images in clinical medicine. Purpura fulminans in Neisseria meningitidis sepsis. PMID- 8637515 TI - Measurement of fractional flow reserve to assess the functional severity of coronary-artery stenoses. AB - BACKGROUND: The clinical significance of coronary-artery stenoses of moderate severity can be difficult to determine. Myocardial fractional flow reserve (FFR) is a new index of the functional severity of coronary stenoses that is calculated from pressure measurements made during coronary arteriography. We compared this index with the results of noninvasive tests commonly used to detect myocardial ischemia, to determine the usefulness of the index. METHODS: In 45 consecutive patients with moderate coronary stenosis and chest pain of uncertain origin, we performed bicycle exercise testing, thallium scintigraphy, stress echocardiography with dobutamine, and quantitative coronary arteriography and compared the results with measurements of FFR. RESULTS: In all 21 patients with an FFR of less than 0.75, reversible myocardial ischemia was demonstrated unequivocally on at least one noninvasive test. After coronary angioplasty or bypass surgery was performed, all the positive test results reverted to normal. In contrast, 21 of the 24 patients with an FFR of 0.75 or higher tested negative for reversible myocardial ischemia on all the noninvasive tests. No revascularization procedures were performed in these patients, and none were required during 14 months of follow-up. The sensitivity of FFR in the identification of reversible ischemia was 88 percent, the specificity 100 percent, the positive predictive value 100 percent, the negative predictive value 88 percent, and the accuracy 93 percent. CONCLUSIONS: In patients with coronary stenosis of moderate severity, FFR appears to be a useful index of the functional severity of the stenoses and the need for coronary revascularization. PMID- 8637517 TI - Persons found in their homes helpless or dead. AB - BACKGROUND: Health care providers and providers of emergency services are sometimes called to help with people who are found alone in their homes either helpless or dead. It is not known who is at risk for being found helpless or dead, what the mortality rates are among those found alive, or how frequently this situation occurs. METHODS: We conducted a population-based study of patients who were found in their homes either helpless or dead. Over 12 weeks, paramedics employed by the city of San Francisco identified 387 such events involving 367 persons. We obtained information on these patients from the emergency-medical services department or the hospitals to which they were taken and determined their outcomes. RESULTS: The median age of the persons found helpless or dead was 73 years; 51 percent were women. The frequency of such incidents increased sharply with age, from a rate of 3 per 1000 per year among those 60 to 64 years of age to 27 per 1000 per year among those 85 years of age or older. The highest rate was among men 85 years and older who were living alone (123 per 1000 per year). In 23 percent of the cases, the person was found dead; an additional 5 percent died in the hospital. Thus, total mortality was 28 percent. Of the patients found alive, 62 percent were admitted to the hospital. The average hospital stay was eight days, and 52 percent of those admitted required intensive care. Of the survivors, 62 percent were unable to return to living independently. The total mortality was 67 percent for patients who were estimated to have been helpless for more than 72 hours, as compared with 12 percent for those who had been helpless for less than 1 hour. CONCLUSIONS: For elderly people who live alone, becoming incapacitated and unable to get help is a common event, which usually marks the end of their ability to live independently. PMID- 8637518 TI - The tumor necrosis factor ligand and receptor families. PMID- 8637519 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 20-1996. A newborn triplet with episodes of respiratory distress and a pulmonary mass. PMID- 8637520 TI - Viral infections and the blood supply. PMID- 8637521 TI - Assessing the severity of coronary-artery stenoses. PMID- 8637522 TI - Home alone, and in danger. PMID- 8637523 TI - A proposal to restructure hospital care for dying patients. PMID- 8637524 TI - A reversible posterior leukoencephalopathy syndrome. PMID- 8637525 TI - A reversible posterior leukoencephalopathy syndrome. PMID- 8637526 TI - A reversible posterior leukoencephalopathy syndrome. PMID- 8637527 TI - A reversible posterior leukoencephalopathy syndrome. PMID- 8637528 TI - Medical response after an earthquake. PMID- 8637529 TI - Medical response after an earthquake. PMID- 8637530 TI - Acute spinal injury. PMID- 8637531 TI - Acute spinal injury. PMID- 8637532 TI - Tuberculous bronchiolitis. PMID- 8637533 TI - Tuberculous bronchiolitis. PMID- 8637534 TI - "Bouncing" creatinine levels. PMID- 8637535 TI - The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. AB - BACKGROUND: Athletes often take androgenic steroids in an attempt to increase their strength. The efficacy of these substances for this purpose is unsubstantiated, however. METHODS: We randomly assigned 43 normal men to one of four groups: placebo with no exercise; testosterone with no exercise; placebo plus exercise; and testosterone plus exercise. The men received injections of 600 mg of testosterone enanthate or placebo weekly for 10 weeks. The men in the exercise groups performed standardized weight-lifting exercises three times weekly. Before and after the treatment period, fat-free mass was determined by underwater weighing, muscle size was measured by magnetic resonance imaging, and the strength of the arms and legs was assessed by bench-press and squatting exercises, respectively. RESULTS: Among the men in the no-exercise groups, those given testosterone had greater increases than those given placebo in muscle size in their arms (mean [+/-SE] change in triceps area, 424 +/- 104 vs. -81 +/- 109 square millimeters; P < 0.05) and legs (change in quadriceps area, 607 +/- 123 vs. -131 +/- 111 square millimeters; P < 0.05) and greater increases in strength in the bench-press (9 +/- 4 vs. -1 +/- 1 kg, P < 0.05) and squatting exercises (16 +/- 4 vs. 3 +/- 1 kg, P < 0.05). The men assigned to testosterone and exercise had greater increases in fat-free mass (6.1 +/- 0.6 kg) and muscle size (triceps area, 501 +/- 104 square millimeters; quadriceps area, 1174 +/- 91 square millimeters) than those assigned to either no-exercise group, and greater increases in muscle strength (bench-press strength, 22 +/- 2 kg; squatting exercise capacity, 38 +/- 4 kg) than either no-exercise group. Neither mood nor behavior was altered in any group. CONCLUSIONS: Supraphysiologic doses of testosterone, especially when combined with strength training, increase fat-free mass and muscle size and strength in normal men. PMID- 8637536 TI - Trimethoprim-sulfamethoxazole (co-trimoxazole) for the prevention of relapses of Wegener's granulomatosis. Dutch Co-Trimoxazole Wegener Study Group. AB - BACKGROUND: Respiratory tract infections may trigger relapses in patients with Wegener's granulomatosis in remission. Uncontrolled data have suggested that treatment with trimethoprim-sulfamethoxazole (co-trimoxazole) may be beneficial. METHODS: We conducted a prospective, randomized, placebo-controlled study of the efficacy of co-trimoxazole (800 mg of sulfamethoxazole and 160 mg of trimethoprim) given twice daily for 24 months in preventing relapses in patients with Wegener's granulomatosis in remission during or after treatment with cyclophosphamide and prednisolone. Relapses and infections were assessed with predefined criteria based on clinical, laboratory, and histopathological findings. Patients were evaluated at least once every three months for signs of disease activity, compliance with the treatment regimen, side effects of the therapy, and evidence of infections. Titers of serum antineutrophil cytoplasmic antibodies were measured serially. RESULTS: Forty-one patients were assigned to receive co-trimoxazole, and 40 to receive placebo. In 8 of the 41 patients in the co-trimoxazole group (20 percent), the drug had to be stopped because of side effects. According to life-table analysis, 82 percent of the patients remained in remission at 24 months, as compared with 60 percent of the patients in the placebo group (relative risk of relapse, 0.40; 95 percent confidence interval, 0.17 to 0.98). There were fewer respiratory tract infections (P = 0.005) and non respiratory tract infections (P = 0.05) in the co-trimoxazole group than in the placebo group. There were no significant differences in antineutrophil cytoplasmic antibody titers at any time. Proportional-hazards regression analysis identified treatment with co-trimoxazole as an independent factor associated with prolonged disease-free survival and a positive antineutrophil cytoplasmic antibody test at the start of treatment as a risk factor for relapse. CONCLUSIONS: Treatment with co-trimoxazole reduces the incidence of relapses in patients with Wegener's granulomatosis in remission. PMID- 8637537 TI - Percutaneous balloon valvuloplasty for pulmonic stenosis in adolescents and adults. AB - BACKGROUND: Percutaneous balloon valvuloplasty has been the accepted first-line treatment for congenital pulmonic stenosis in children. Its efficacy in adolescents and adults is less well defined. METHODS: Between December 1985 and July 1995 we performed percutaneous pulmonic valvuloplasty with a single Inoue balloon catheter in 53 adolescent or adult patients 13 to 55 years of age (mean [+/- SD], 26 +/- 11). Follow-up studies were performed 0.2 to 9.8 years after the procedure (mean, 6.9 +/- 3.1) by Doppler echocardiography (in all the patients) and by cardiac catheterization and angiography (in nine patients). RESULTS: After balloon valvuloplasty, the systolic pressure gradient across the pulmonic valve decreased from 91 +/- 46 mm Hg to 38 +/- 32 mm Hg (P < 0.001), and the diameter of the pulmonic-valve orifice increased from 8.9 +/- 3.6 mm to 17.4 +/- 4.6 mm (P < 0.001). In the nine patients catheterized at follow-up, the systolic gradient decreased from 107 +/- 48 mm Hg before valvuloplasty to 50 +/- 29 mm Hg after valvuloplasty and to 30 +/- 16 mm Hg at follow-up (P < 0.001 for the comparison of the gradient before and after valvuloplasty; P < 0.001 for the comparison before valvuloplasty and at follow-up; and P < 0.05 for the comparison after valvuloplasty and at follow-up). In the same nine patients, the diameter of the pulmonic valve, as measured by right ventricular angiography, increased from 8.3 +/- 1.4 mm before valvuloplasty to 17.2 +/- 2.0 mm after valvuloplasty (P < 0.001) and to 18.4 +/- 1.4 mm at follow-up (P = 0.08). Incompetence of the pulmonic valve was noted in 7 of the 53 patients (13 percent) after balloon valvuloplasty, but it had disappeared at follow-up in all of them. CONCLUSIONS: Patients with congenital pulmonic stenosis who present in late adolescence or adult life can be treated with percutaneous balloon valvuloplasty with excellent short-term and long-term results that are similar to those in young children. PMID- 8637538 TI - Images in clinical medicine. Tropheryma whippelii. PMID- 8637539 TI - Surveillance and prevention of residential-fire injuries. AB - BACKGROUND: The majority of severe and fatal burn injuries result from residential fires. We studied the effectiveness of a smoke-alarm-giveaway program in the prevention of burn injuries in an area with a high rate of such injuries. METHODS: We collected data on burn injuries in Oklahoma City from September 1987 through April 1990. The target area for the intervention was an area of 24 square miles (62 km2) with the highest rate of injuries related to residential fires in the city. We distributed smoke alarms door to door in the target area and then surveyed alarm use and function in a sample of the homes that had received an alarm. We also calculated the rates of fire injury per 100,000 population and per 100 fires for both the target area and the rest of the city before and after the smoke-alarm giveaway. RESULTS: Before the intervention the rate of burn injuries per 100,000 population was 4.2 times higher in the target area than in the rest of Oklahoma City. An initial survey indicated that 11,881 of the 34,945 homes in the target area (34 percent) did not have smoke alarms. A total of 10,100 smoke alarms were distributed to 9291 homes; 45 percent were functioning four years later. The annualized fire-injury rates declined by 80 percent in the target area during the four years after the intervention (from 15.3 to 3.1 per 100,000 population), as compared with a small increase in the rest of the city (from 3.6 to 3.9 per 100,000 population). There was also a 74 percent decline in the target area in the injury rate per 100 fires (from 5.0 to 1.3; rate ratio, 0.3; 95 percent confidence interval, 0.1 to 0.6), as compared with a small increase in the rest of the city. CONCLUSIONS: A targeted intervention involving a smoke alarm-giveaway program can reduce the incidence of injuries from residential fires. PMID- 8637540 TI - Postherpetic neuralgia--pathogenesis, treatment, and prevention. PMID- 8637541 TI - Adhesion molecules--Part II: Blood vessels and blood cells. PMID- 8637542 TI - The illusion of certainty. PMID- 8637543 TI - The anabolic action of testosterone. PMID- 8637544 TI - Low-dose oral contraceptives and stroke. PMID- 8637545 TI - Empiricism and Wegener's granulomatosis. PMID- 8637546 TI - Managed care and mental health. PMID- 8637547 TI - Managed care and mental health. PMID- 8637548 TI - Managed care and mental health. PMID- 8637549 TI - Compensation for teaching medical students and house staff. PMID- 8637550 TI - Compensation for teaching medical students and house staff. PMID- 8637551 TI - Compensation for teaching medical students and house staff. PMID- 8637552 TI - Preinfarction angina. PMID- 8637553 TI - Preinfarction angina. PMID- 8637554 TI - Coronary-artery stenting. PMID- 8637555 TI - Massive infiltration of the skin by HIV-specific cytotoxic CD8+ T cells. PMID- 8637556 TI - Firearm injuries and deaths. PMID- 8637558 TI - A shot in the arm for clinical research. PMID- 8637559 TI - 1997 budget confirms US science faces a period of flat funding. PMID- 8637560 TI - Ministers seek legal shelter from Canada's HIV inquiry. PMID- 8637557 TI - Stroke in users of low-dose oral contraceptives. AB - BACKGROUND: Previous studies have linked the use of oral contraceptive agents to an increased risk of stroke, but those studies have been limited to oral contraceptives containing more estrogen than is now generally used. METHODS: In a population-based, case-control study, we identified fatal and nonfatal strokes in female members of the California Kaiser Permanente Medical Care Program and who were 15 through 44 years of age. Matched controls were randomly selected from female members who had not had strokes. Information about the use of oral contraceptives (essentially limited to low-estrogen preparations) was obtained in interviews. RESULTS: A total of 408 confirmed strokes occurred in a total of 1.1 million women during 3.6 million woman-years of observation. The incidence of stroke was thus 11.3 per 100,000 woman-years. On the basis of data from 295 women with stroke who were interviewed and their controls, the odds ratio for ischemic stroke among current users of oral contraceptives, as compared with former users and women who had never used such drugs, was 1.18 (95 percent confidence interval, 0.54 to 2.59) after adjustment for other risk factors for stroke. The adjusted odds ratio for hemorrhagic stroke was 1.14 (95 percent confidence interval, 0.60 to 2.16). With respect to the risk of hemorrhagic stroke, there was a positive interaction between the current use of oral contraceptives and smoking (odds ratio for women with both these factors, 3.64; 95 percent confidence interval, 0.95 to 13.87). CONCLUSIONS: Stroke is rare among women of childbearing age. Low-estrogen oral-contraceptive preparations do not appear to increase the risk of stroke. PMID- 8637561 TI - Panel calls for overhaul of AIDS research. PMID- 8637563 TI - A big step along the visual pathway. PMID- 8637564 TI - Reverse transcription. Foamy viruses bubble on. PMID- 8637562 TI - Bioterminology. PMID- 8637565 TI - Infrared astronomy. The real water-bearer. PMID- 8637566 TI - Blood. Taking the pressure off. PMID- 8637567 TI - Screening for genetic mutations. PMID- 8637568 TI - State-dependent life histories. AB - Life-history theory is concerned with strategic decisions over an organism's lifetime. Evidence is accumulating about the way in which these decisions depend on the organism's physiological state and other components such as external circumstances. Phenotypic plasticity may be interpreted as an organism's response to its state. The quality of offspring may depend on the state and behaviour of the mother. Recent theoretical advances allow these and other state-dependent effects to be modelled within the same framework. PMID- 8637569 TI - S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. AB - A dynamic cycle exists in which haemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated, and the NO group is released during arterial venous transit. The vasoactivity of S-nitrosohaemoglobin is promoted by the erythrocytic export of S-nitrosothiols. These findings highlight newly discovered allosteric and electronic properties of haemoglobin that appear to be involved in the control of blood pressure and which may facilitate efficient delivery of oxygen to tissues. The role of S-nitrosohaemoglobin in the transduction of NO related activities may have therapeutic applications. PMID- 8637570 TI - Infrared emission spectra of candidate interstellar aromatic molecules. AB - Interstellar dust is responsible, through surface reactions, for the creation of molecular hydrogen, the main component of the interstellar clouds in which new stars form. Intermediate between small, gas-phase molecules and dust are the polycyclic aromatic hydrocarbons (PAHs). Such molecules could account for 2-30% of the carbon in the Galaxy, and may provide nucleation sites for the formation of carbonaceous dust. Although PAHs have been proposed as the sources of the unidentified infrared emission bands that are observed in the spectra of a variety of interstellar sources, the emission characteristics of such molecules are still poorly understood. Here we report laboratory emission spectra of several representative PAHs, obtained in conditions approximating those of the interstellar medium, and measured over the entire spectral region spanned by the unidentified infrared bands. We find that neutral PAHs of small and moderate size can at best make only a minor contribution to these emission bands. Cations of these molecules, as well as much larger PAHs and their cations, remain viable candidates for the sources of these bands. PMID- 8637571 TI - A role for melanin-concentrating hormone in the central regulation of feeding behaviour. AB - The hypothalamus plays a central role in the integrated regulation of energy homeostasis and body weight, and a number of hypothalamic neuropeptides, such as neuropeptide Y (ref. 1), galanin, CRH (ref. 3) and GLP-1 (ref. 4), have been implicated in the mediation of these effects. To discover new hypothalmic peptides involved in the regulation of body weight, we used differential display polymerase chain reaction to identify messenger RNAs that are differentially expressed in the hypothalamus of ob/+ compared with ob/ob C57B1/6J mice. We show here that one mRNA that is overexpressed in the hypothalamus of ob/ob mice encodes the neuropeptide melanin-concentrating hormone (MCH). Fasting further increased expression of MCH mRNA in both normal and obese animals. Neurons containing MCH are located in the zona incerta and in the lateral hypothalamus. These areas are involved in regulation of ingestive behaviour, but the role of MCH in mammalian physiology is unknown. To determine whether MCH is involved in the regulation of feeding, we injected MCH into the lateral ventricles of rats and found that their food consumption increased. These findings suggest that MCH participates in the hypothalamic regulation of body weight. PMID- 8637572 TI - Learning to commit or avoid the base-rate error. AB - When predicting an event, people neglect overall frequencies (base rates) of various possibilities. We have previously shown that this base-rate occurs not only with word problems, but also in a procedure with repeated trials: a sample cue followed by two choice options, one of which the subject must choose, with feedback regarding the correctness of the choice. Perhaps this base-rate error depends on people's histories of matching physically similar items. In support of this suggestions, we begin by showing that the base-rate error is eliminated with physically unrelated items. We then show that when the relation between items is again arbitrary, but is a relation that subjects already know, the error reappears. Finally we show that teaching subjects new arbitrary relations reintroduces the error in later testing. These experiments demonstrate a fundamental base-rate error dependent on learned relationships without interference from re-existing associations between cues and options, predictions may be made more optimally. PMID- 8637573 TI - Orientation selectivity of thalamic input to simple cells of cat visual cortex. AB - More than 30 years after Hubel and Wiesel first described orientation selectivity in the mammalian visual cortex, the mechanism that gives rise to this property is still controversial. Hubel and Wiesel proposed a simple model for the origin of orientation tuning, in which the circularly symmetrical receptive fields of neurons in the lateral geniculate nucleus that excite a cortical simple cell are arranged in rows. Since this model was proposed, several experiments and neuronal simulations have suggested that the connectivity between the lateral geniculate nucleus and the cortex is not well organized in an orientation-specific fashion, and that orientation tuning arises instead from extensive interactions within the cortex. To test these models we have recorded visually evoked synaptic potentials in simple cells while cooling the cortex, which largely inactivates the cortical network, but leaves geniculate synaptic input functional. We report that the orientation tuning of these potentials is almost unaffected by cooling the cortex, in agreement with Hubel and Wiesel's original proposal. PMID- 8637574 TI - Functional recovery in parkinsonian monkeys treated with GDNF. AB - Parkinson's disease results from the progressive degeneration of dopamine neurons that innervate the striatum. In rodents, glial-cell-line-derived neurotrophic factor (GDNF) stimulates an increase in midbrain dopamine levels, protects dopamine neurons from some neurotoxins, and maintains injured dopamine neurons. Here we extend the rodent studies to an animal closer to the human in brain organization and function, by evaluating the effects of GDNF injected intracerebrally in rhesus monkeys that have had the symptomatology and pathophysiological features of Parkinson's disease induced by the neurotoxin 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The recipients of GDNF displayed significant improvements in three of the cardinal symptoms of parkinsonism: bradykinesia, rigidity and postural instability. GDNF administered every four weeks maintained functional recovery. On the lesioned side of GDNF treated animals, dopamine levels in the midbrain and globus pallidus were twice as high, and nigral dopamine neurons were, on average, 20% larger, with an increased fibre density. The results indicate that GDNF may be of benefit in the treatment of Parkinson's disease. PMID- 8637575 TI - Voltage-dependent modulation of N-type calcium channels by G-protein beta gamma subunits. AB - The most commonly used signal transduction pathway for receptor-mediated N-type Ca2+-channel modulation involves activation of a heterotrimeric G protein to produce voltage-dependent inhibition. Although it is widely assumed that Galpha mediates this effect, experiments to address this hypothesis directly are lacking. Here I show that transient overexpression of Gbetagamma in sympathetic neurons mimics and occludes the voltage-dependent Ca2+ channel modulation produced by noradrenaline (NA). Conversely, over-expression of Galpha produces minimal effects on basal Ca2+ channel behaviour but attenuates NA-mediated inhibition in a manner consistent with the buffering of Gbetagamma. These observations indicate that it is Gbetagamma, and Galpha, that mediates voltage dependent inhibition of N-type Ca2+ channels. The identification of Gbetagamma as the mediator of this pathway has broad implications as G-protein-coupled receptors, many of which are implicated in disease or are targets of therapeutic agents, couple to N-type Ca2+ channels and may modulate synaptic transmission by this mechanism. PMID- 8637576 TI - Modulation of Ca2+ channels by G-protein beta gamma subunits. AB - Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation. PMID- 8637577 TI - Inactivation of p27Kip1 by the viral E1A oncoprotein in TGFbeta-treated cells. AB - The adenovirus oncoprotein E1A and the simian virus SV40 large T antigen can both reverse the strong growth-inhibitory effect of transforming growth factor(TGF) beta on mink lung epithelial cells: exposure of TGF-beta causes these cells to arrest late in the G1 phase of the cell cycle (ref. 3). This arrest correlates with an increase in expression of the protein p15Ink4B (ref. 4), inactivation of the cyclin E/A-cdk2 complex by the inhibitory protein p27Kip1 (refs 5-7), and with the accumulation of unphosphorylated retinoblastoma protein. The rescue by E1A of cells from TGF-beta arrest is partly independent of its binding to retinoblastoma protein. Here we show that E1A directly affects the cyclin dependent kinase inhibitor p27Kip1 in TGF-beta-treated cells by binding to it and blocking its inhibitory effect, thereby restoring the activity of the cyclin-cdk2 kinase complex. In this way, E1A can overcome the effect of TGF-beta and modulate the cell cycle. To our knowledge, E1A provides the first example of a viral oncoprotein that can disable a cellular protein whose function is to inhibit the activity of cyclin-dependent kinases. PMID- 8637580 TI - NIH resists bill to promote research into Parkinson's. PMID- 8637579 TI - Kinetic trapping of oxygen in cell respiration. AB - Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase. In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre. It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak. This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre. Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping. Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2. This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration. PMID- 8637578 TI - Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. AB - DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription. PMID- 8637581 TI - Cancer genetics group drafts guidelines. PMID- 8637582 TI - Did UK 'dump' contaminated feed after ban? PMID- 8637583 TI - European vote raises bioethics stakes. PMID- 8637584 TI - Citation policy on sequences. PMID- 8637585 TI - Julius Marmur (1926-96) PMID- 8637586 TI - Immunology. An affinity for learning. PMID- 8637587 TI - Visual perception. An efficient code in V1? PMID- 8637588 TI - Growth factors. Mad connection to the nucleus. PMID- 8637589 TI - Preclinical test for prion diseases. PMID- 8637590 TI - Clutch size and malaria resistance. PMID- 8637591 TI - Skin grafts and cheetahs. PMID- 8637592 TI - Molecular chaperones in cellular protein folding. AB - The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains. PMID- 8637593 TI - The guinea-pig is not a rodent. AB - In 1991 Graur et al. raised the question of whether the guinea-pig, Cavia porcellus, is a rodent. They suggested that the guinea-pig and myomorph rodents diverged before the separation between myomorph rodents and a lineage leading to primates and artiodactyls. Several findings have since been reported, both for and against this phylogeny, thereby highlighting the issue of the validity of molecular analysis in mammalian phylogeny. Here we present findings based on the sequence of the complete mitochondrial genome of the guinea-pig, which strongly contradict rodent monophyly. The conclusions are based on cumulative evidence provided by orthologically inherited genes and the use of three different analytical methods, none of which joins the guinea-pig with myomorph rodents. In addition to the phylogenetic conclusions, we also draw attention to several factors that are important for the validity of phylogenetic analysis based on molecular data. PMID- 8637594 TI - Power laws governing epidemics in isolated populations. AB - Temporal changes in the incidence of measles virus infection within large urban communities in the developed world have been the focus of much discussion in the context of the identification and analysis of nonlinear and chaotic patterns in biological time series. In contrast, the measles records for small isolated island populations are highly irregular, because of frequent fade-outs of infection, and traditional analysis does not yield useful insight. Here we use measurements of the distribution of epidemic sizes and duration to show that regularities in the dynamics of such systems do become apparent. Specifically, these biological systems are characterized by well-defined power laws in a manner reminiscent of other nonlinear, spatially extended dynamical systems in the physical sciences. We further show that the observed power-law exponents are well described by a simple lattice-based model which reflects the social interaction between individual hosts. PMID- 8637595 TI - Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. AB - The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn 3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia. PMID- 8637596 TI - Emergence of simple-cell receptive field properties by learning a sparse code for natural images. AB - The receptive fields of simple cells in mammalian primary visual cortex can be characterized as being spatially localized, oriented and bandpass (selective to structure at different spatial scales), comparable to the basis functions of wavelet transforms. One approach to understanding such response properties of visual neurons has been to consider their relationship to the statistical structure of natural images in terms of efficient coding. Along these lines, a number of studies have attempted to train unsupervised learning algorithms on natural images in the hope of developing receptive fields with similar properties, but none has succeeded in producing a full set that spans the image space and contains all three of the above properties. Here we investigate the proposal that a coding strategy that maximizes sparseness is sufficient to account for these properties. We show that a learning algorithm that attempts to find sparse linear codes for natural scenes will develop a complete family of localized, oriented, bandpass receptive fields, similar to those found in the primary visual cortex. The resulting sparse image code provides a more efficient representation for later stages of processing because it possesses a higher degree of statistical independence among its outputs. PMID- 8637597 TI - Primary cortical representation of sounds by the coordination of action-potential timing. AB - Cortical population coding could in principle rely on either the mean rate of neuronal action potentials, or the relative timing of action potentials, or both. When a single sensory stimulus drives many neurons to fire at elevated rates, the spikes of these neurons become tightly synchronized, which could be involved in 'binding' together individual firing-rate feature representations into a unified object percept. Here we demonstrate that the relative timing of cortical action potentials can signal stimulus features themselves, a function even more basic than feature grouping. Populations of neurons in the primary auditory cortex can coordinate the relative timing of their action potentials such that spikes occur closer together in time during continuous stimuli. In this way cortical neurons can signal stimuli even when their firing rates do not change. Population coding based on relative spike timing can systemically signal stimulus features, it is topographically mapped, and it follows the stimulus time course even where mean firing rate does not. PMID- 8637598 TI - Absence of spectrally specific lateral inputs to midget ganglion cells in primate retina. AB - Visual information is conveyed to the brain by the retinal ganglion cells. Midget ganglion cells serve fine spatial vision by summing excitation from a receptive field 'centre', receiving input from a single cone in the central retina, with lateral inhibition from a receptive field 'surround', receiving input from many surrounding cones. Midget ganglion cells are also thought to serve colour opponent vision because the centre excitation is from a cone of one spectral type, while the surround inhibition is from cones of the other type. The two major cone types, middle(M)- and long-(L)wavelength sensitive, are equally numerous and randomly distributed in the primate central retina, so a spectrally homogeneous surround requires that the cells mediating lateral interactions (horizontal or amacrine cells) receive selective input from only one cone type. Horizontal cells cannot do this because they receive input indiscriminately from M and L cones. Here we report that the amacrine cells connected to midget ganglion cells are similarly indiscriminate. The absence of spectral specificity in the inhibitory wiring raises doubt about the involvement of midget ganglion cells in colour vision and suggest that colour opponency may instead be conveyed by a different type of ganglion cell. PMID- 8637599 TI - T-cell-receptor affinity and thymocyte positive selection. AB - Development of thymocytes involves two distinct outcomes resulting from superficially similar events. Recognition by thymocytes of major histocompatibility complex (MHC) proteins plus peptides leads to their rescue from apoptosis (positive selection), and recognition of antigenic peptide induces cell death (negative selection). Antigen analogues, and sometimes low concentrations of antigenic peptide, induce positive selection; such analogues are often antagonists of mature T-cell clones. Various models seek to explain how recognition of different peptide/MHC complexes leads to such different outcomes: quantitative models relate response to the affinity, avidity or kinetics of T cell-antigen receptor (TCR) binding, whereas qualitative models require conformational or spatial changes in the TCR or associated molecules to modulate signal transduction. We have used surface plasmon resonance to measure the kinetics of TCR interactions with positively and negatively selecting ligands to distinguish between these models, and find that affinity correlates to the outcome of selection. A 'window' of affinity resulting in positive selection extends over a 1-log range starting threefold below the affinity for negative selection. PMID- 8637601 TI - Intersubunit rotation in active F-ATPase. AB - The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP. PMID- 8637600 TI - A human Mad protein acting as a BMP-regulated transcriptional activator. AB - The TGF-beta/activin/BMP cytokine family signals through serine/threonine kinase receptors, but how the receptors transduce the signal is unknown. The Mad (Mothers against decapentaplegic) gene from Drosophila and the related Sma genes from Caenorhabditis elegans have been genetically implicated in signalling by members of the bone-morphogenetic-protein (BMP) subfamily. We have cloned Smad1, a human homologue of Mad and Sma. Microinjection of Smad1 messenger RNA into Xenopus embryo animal caps mimics the mesoderm-ventralizing effects of BMP4. Smad1 moves into the nucleus in response to BMP4. Smad1 has transcriptional activity when fused to a heterologous DNA-binding domain, and this activity is increased by BMP4 acting through BMP-receptor types I and II. The transactivating activity resides in the conserved carboxy-terminal domain of Smad1 and is disrupted by a nonsense mutation that corresponds to null mutations found in Mad and in the related gene DPC4, a candidate tumour-suppressor gene in human pancreatic cancer. Additionally, we show that DPC4 contains a transcriptional activation domain. The results suggests that the Smad proteins are a new class of transcription factors that mediate responses to the TGF-beta family. PMID- 8637603 TI - Human evolution: the study of Indian mitochondrial DNA. PMID- 8637602 TI - [Insulin producing cells as therapy in diabetes mellitus]. AB - Even with intensive insulin therapy it is impossible to reach physiological blood glucose levels in insulin-dependent diabetes mellitus. Because of the high costs and technical problems involved in islet cell transplantation broad applicability of this therapy seems uncertain. An alternative approach is the development of molecular-engineered insulin-producing clonal cell lines. The main interest is in rodent insulinoma cell lines and neuroendocrine AtT-20ins cells. This paper reviews the current knowledge about glucose-stimulated insulin secretion and the problems that have to be solved before these cells can be used for therapy in diabetes mellitus. PMID- 8637604 TI - From pattern recognition to sound localization: a by-product of growing larger during evolution. PMID- 8637605 TI - Oxygen concentration and the oxidation-reduction state of yeast: determination of free/bound NADH and flavins by time-resolved spectroscopy. PMID- 8637607 TI - The cell nucleus of cultured melanoma cells as a source of ultraweak photon emission. PMID- 8637606 TI - Functional compartmentalization in bacteria and archaea. A hypothetical interface between cytoplasmic membrane and cytoplasm. PMID- 8637608 TI - Serotonergic modulation of cholinergic function in the central nervous system: cognitive implications. AB - Accumulating evidence suggests that serotonin may modulate cholinergic function in several regions of the mammalian brain and that these serotonergic/cholinergic interactions influence cognition. The first part of this review is an overview of histological, electrophysiological and pharmacological (in vitro, in vivo) data indicating that, in several brain regions (e.g., hippocampus, cortex and striatum), there are neuroanatomical substrates for a serotonergic/cholinergic interaction, and that alterations in serotonergic activity may induce functional changes in cholinergic neurons. In the second part, the review focuses on experimental approaches showing or suggesting that central cholinergic and serotonergic mechanisms are cooperating in the regulation of cognitive functions. These arguments are based on lesion, intracerebral grafting and pharmacological techniques. It is concluded that not all mnesic perturbations induced by concurrent manipulations of the serotonergic and cholinergic systems can be attributed to a serotonergic modification of the cholinergic system. The cognitive faculties of an organism arise from interactions among several neurotransmitter systems within brain structures such as, for instance, the hippocampus or the cortex, but also from influences on memory of other general functions that may involve cerebral substrates different from those classically related to mnesic functions (e.g., attention, arousal, sensory accuracy, etc.). PMID- 8637609 TI - The neuroprotective effects of MK-801 on the induction of microgyria by freezing injury to the newborn rat neocortex. AB - Four-layered microgyria is associated with many developmental disorders, including mental retardation, epilepsy, and developmental dyslexia. Freezing lesions to the newborn rodent neocortex result in the formation of four-layered microgyria. Previous research had suggested this type of injury acts as an hypoxic/ischemic event to the developing cortical plate. The current study examines the effectiveness of the non-competitive N-methyl-D-aspartate receptor antagonist dizocilpine (MK-801) in protecting against freezing injury to the newborn rat cortical plate. Three groups of rats received freezing injury to the cortical plate on the first day of life (postnatal day 1). Two groups were treated with MK-801 (1 or 2 mg/kg) 0.5 h before the lesion and 6 and 14 h after, while one group received saline injections. A fourth group received MK-801 injections, but did not have a freezing lesion. The volume of neocortical abnormality was determined for all three groups in rats killed after postnatal day 7. Treatment with the higher dose of MK-801 (3 x 2 mg/kg) dramatically reduced the effects of freezing injury but also resulted in over 50% mortality in both lesioned and unlesioned groups. Animals in the lesioned group, however, had a decreased volume of abnormal cortex, and there were fewer animals with microsulci than in the untreated group. This is the first demonstration of a significant anatomical neuroprotective effect in newborns leading to a reduction of cortical malformation. PMID- 8637610 TI - Functional geometry of amino acid sensitive membrane of layer V neurons in the guinea-pig neocortex in vitro. AB - On guinea-pig neocortical slices the spatial organization of dendrites sensitive to excitatory amino acids was studied. Extracellular recording were obtained from the the soma of layer V neurons. Responses of 135 neurons to iontophoretically applied glutamate or aspartate have been analysed. An increased firing rate to somatic and most of dendritic applications were of short latency not exceeding 500 ms. Dendritic applications caused somatic responses with far longer latencies (up to 2-3 s) in 18% of cases. Latencies of responses to excitatory amino acids applied to several dendritic sites of the same neuron had similar values. The greatest reactions were obtained in response to excitatory amino acids imposed to the soma and proximal dendrites. At a distance of 100 microm beyond the soma in the basal region and region and further than 300 microm in the apical region excitatory amino acid applications produced two to three times less intensive somatic response. The area where dendritic activation gave rise to change in neuronal firing was confined to 350 and 800 microm for basal and apical dendrites, respectively. Topography of effective dendritic sites fell into the area corresponding to anatomically known outline of dendritic tree of pyramidal neurons. This fact implies that in our experiments we basically dealt with layer V pyramids. The results obtained suggest that local activation of distal dendrites may elicit spike generation in the soma. Different electrical properties of somatic and dendritic membranes are discussed. PMID- 8637611 TI - Phosphoinositide hydrolysis, G alpha q, phospholipase C, and protein kinase C in post mortem human brain: effects of post mortem interval, subject age, and Alzheimer's disease. AB - Influences of post mortem time interval, subject age and Alzheimer's disease were investigated on several components of the phosphoinositide second messenger system, including stimulation of [3H]phosphatidylinositol hydrolysis by GTP[S] and several receptor agonists and the levels of Galphaq, beta, delta and gamma subtypes of phospholipase C, and five protein kinase C isoforms, in membranes prepared from post mortem human prefrontal cortex. Most of these components were stable with post mortem delays in the range of 5-21 h, but decreases of Galphaq and the alpha and xi protein kinase C subtypes were detected. Within the subject age range of 19-100 years, G-protein- and agonist-induced [3H]phosphatidylinositol hydrolysis decreased, as did levels of Galphaq, but the levels of phospholipase C and protein kinase C subtypes were generally unchanged. In Alzheimer's disease, compared with age- and post mortem interval-matched controls, there was a decrease in [3H]phosphatidylinositol hydrolysis stimulated by G-proteins and by several receptor agonists, but the levels of Galphaq and most of the phospholipase C and protein kinase C isoforms were unaffected. The greatest deficits, which were >50%, occurred with GTP[S]- and carbachol-induced [3H]phosphatidylinositol hydrolysis, indicating that this G-protein function and the response to cholinergic stimulation are significantly impaired in Alzheimer's disease. In summary a comprehensive assessment of several components of the phosphoinositide second messenger system was made in post mortem human brain. Most elements were stable within the post mortem interval range of 5-21 h, lending validity to measurements using these tissues. Significant age-related reductions in several components were identified, indicating loss of responses with increasing age. Most importantly, severe reductions in responses to several stimuli were found in Alzheimer's disease brain, deficits in signal transduction which may contribute to impaired cognition and to the limited therapeutic responses to drugs, such as those used to activate cholinergic receptors coupled with the phosphoinositide system. PMID- 8637612 TI - GABAergic inhibition of granule cells and hilar neuronal synchrony following ischemia-induced hilar neuronal loss. AB - In the dentate gyrus, granule cells are ischemia-resistant, but at least five types of predominantly spiny hilar neurons are extremely vulnerable to ischemia. Many of the ischemia-sensitive subtypes of hilar neurons appear to be involved in: (i) the regulation of GABAergic inhibition in the dentate gyrus, and (ii) the generation of hilar neuronal synchrony. The present study examined functional consequences of ischemia-induced hilar neuronal loss on GABAergic inhibition of granule cells and hilar neuronal synchrony. Transient (15 min) forebrain ischemia was induced by a modification of the four-vessel-occlusion method producing a substantial hilar neuronal loss as demonstrated by the Gallyas silver stain method. Three months later, we have examined spontaneous and stimulus-evoked inhibitory postsynaptic currents mediated by both GABA(A) and GABA(B) receptors, and inhibitory bursts induced by 4-aminopyridine (50 microM) using whole-cell recordings in coronal brain slices maintained at 34-36 degree C in the presence of excitatory amino acid receptor blockers. Spontaneous dentate spikes reflecting hilar neuronal synchrony and synaptic responses evoked by perforant path stimulation were also recorded in vivo to assess synchrony and inhibition in the dentate gyrus. In spite of significant damage to several types of hilar neurons, there were no marked differences in the conductance, kinetics, and 4 aminopyridine-induced burst frequencies of synaptic GABA(A) and GABA(B) responses in granule cells. Furthermore, both paired-pulse inhibition and dentate spikes appeared to be normal in vivo. We conclude that there appears to be little impairment of GABAergic inhibition of granule cells or of hilar neuronal synchrony three months following a massive ischemic damage to spiny hilar neurons. PMID- 8637613 TI - The distributions of Purkinje cell perikaryon and nuclear volume in human and rat cerebellum with the nucleator method. AB - The Purkinje cells are among the largest cells in the central nervous system and are the output cells of the cerebellar cortex. They are, therefore, of special interest in cerebellar diseases. The estimation of total number and mean perikaryon and nuclear volume of Purkinje cells in five normal human and nine rat cerebella were obtained using unbiased methods based on stereological principles. The average total number of Purkinje cells was 30.5 x 10(6) (Coefficient of variation, CD = S.D./mean = 0.13) in humans and 0.61 x 10(6) (0.21) in rats. Thus the total number of Purkinje cells was 50 times higher in the human cerebellum compared with rats, while numerical density (number/mm3) was 13 times lower in humans (0.81 x 10(3)) compared with rats (10.1 x 10(3)). An unbiased stereological principle, the nucleator, was applied to estimate the volume of Purkinje cell perikarya and nuclei. In humans the average geometric mean volume of Purkinje cell perikaryon is 12,400 microm3 (interindividual coefficient of variation = 0.08), which is about three times larger than in rats, 4900 microm3 (CVi = 0.09). The intraindividual distributional variation (CVd) in perikaryon volume is much larger in humans compared to rats (CVd = 0.72 vs 0.32). One of the differences between the two species is the simple proportionality between perikaryon and nucleus size i humans, whereas larger Purkinje cells have relatively larger nuclei in the rat. PMID- 8637615 TI - Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain. AB - The large number of metabotropic glutamate receptor subtypes suggests diverse roles in brain function, although specific distribution patterns can give clues to subtype-specific functions [Hayashi Y. et al. (1993) Nature 366, 687-690; Nakajima Y. et al. (1993) J. biol. Chem. 268, 11868-11873; Nomura A. et al. (1994) Cell 77, 361-369; Ohishi H. et al. (1993), 1009-1018]. The metabotropic glutamate receptor mGluR7 is sensitive to the agonist L-2-amino-4 phosphonobutyric acid, a presynaptic inhibitor of neurotransmitter release. We examined the anatomic distribution of mGluR7 messenger RNA expression by in situ hybridization in the developing and adult rat central nervous systems. Our results demonstrate that mGluR7 messenger RNA is among the most widely distributed of metabotropic glutamate receptors in both the developing and adult rat nervous system and that mGluR7 messenger RNA is expressed in most neuronal groups known to respond to L-2-amino-4-phosphonobutyric acid, including mitral cells of the olfactory bulb, granule cells of the dentate gyrus and neurons of the entorhinal cortex and dorsal root ganglion. mGluR7 exhibits preferential expression in sensory afferent pathways and is highly represented in the periventricular zone of the hypothalamus, the latter implying a modulatory role for mGluR7 in neuroendocrine pathways. Most strikingly, the majority of neurons at all levels of olfactory circuitry are among the areas of highest mGluR7 messenger RNA content. The anatomic distribution of mGluyR7 messenger RNA suggests that mGluR7 activation may participate in the processing of hippocampal, sensory and olfactory information. PMID- 8637614 TI - Sex steroid hormones change the differential distribution of the isoforms of the D2 dopamine receptor messenger RNA in the rat brain. AB - The two isoforms of the rat dopamine D2 receptor are generated by alternative splicing of the pre-messenger RNA and differ in the length of their third cytoplasmic loop involved in coupling to G-proteins. As quantified by polymerase chain reaction, the long isoform D2L is predominant in the pituitary gland, the striatum and to a lesser extend in the olfactory tubercle, whereas the short isoform D2S is relatively more abundant in the hypothalamus and the substantia nigra. Changes in circulating sex hormone levels modulated the splicing without affecting the total amount of D2 receptor messenger RNA. Castration of male rats increased the ratio D2L/D2S in the pituitary, hypothalamus and substantia nigra, and decreased it in the olfactory tubercle. Testosterone substitution reversed the effect of castration in the pituitary and olfactory tubercle but not in the substantia nigra. In castrated rats, 17beta-estradiol had a similar effect to that of testosterone in the olfactory tubercle, indicating that testosterone may act after aromatization of estradiol. In the hypothalamus, 17beta-estradiol alone reversed the effect of castration. In the striatum, neither castration nor hormonal treatments modified the splicing of the D2 receptor mRNA. Treatment of animals with specific androgen and estrogen receptor blockers confirmed that steroids were acting through their specific intracellular receptors. These observations suggest a molecular mechanism, physiologically relevant, by which circulating sex hormones could modulate dopamine transmission in areas implicated in reproductive and parental behaviours. PMID- 8637616 TI - Immunohistochemical detection of thrombospondin in microglia in the developing rat brain. AB - The development of microglia involves the expression of a phenotype displaying phagocytic behaviour termed brain macrophage or amoeboid microglial cell. We have previously shown that rat brain macrophages purified in vitro secrete thrombospondin, an extracellular matrix protein, which acts on cultured neuronal cells by promoting neurite growth. In the present study, the expression of thrombospondin was investigated in tissue sections of the developing rat forebrain in relation to the distribution of microglia. These cells were identified using anti-macrophage antibodies and the isolectin B4 from Bandeiraea simplicifolia. Immunocytochemical detection of thrombospondin clearly outlined a cell population displaying the morphologies and distribution of brain macrophages, from the 17th day of embryonic life up to the end of the second postnatal week. These cells were most numerous in cortical and subcortical regions of developing fibre tracts such as the corpus callosum or the internal capsule. The localization of thrombospondin in brain macrophages was confirmed by double immunostaining using ED1 monoclonal anti-macrophage antibodies. Ramified microglial cells were also labelled transiently by anti-thrombospondin antibodies during early postnatal life. These results provide in situ evidence supporting the notion that microglial cells could favour axonal growth by producing thrombospondin during development. PMID- 8637617 TI - Evidence for a repetitive (burst) firing pattern in a sub-population of 5 hydroxytryptamine neurons in the dorsal and median raphe nuclei of the rat. AB - Previous electrophysiological studies have shown that spontaneously active mesencephalic 5-hydroxytryptaminergic neurons of anaesthetized or freely moving animals fire solitary spikes in a slow, regular pattern. In the present study, using extracellular single unit recordings from dorsal and median raphe neurons of the anaesthetized rat, an additional electrophysiological property of a sub population of presumed 5-hydroxytryptaminergic neurons was observed. These neurons, during their otherwise regular firing pattern, repeatedly fired two (or occasionally three or even four) spikes where only one was expected. Spikes in this burst-like repetitive firing mode (spikes in doublets or triplets) occurred in a short time interval (range: 2.4-11.5 ms), and with a diminishing spike amplitude. Cross-correlation analysis of spikes in doublets revealed a very high interdependency between them. The proportion of spikes in doublets to solitary spikes showed great variation between different neurons, ranging from 5 to 95% of the total spikes displayed. However, for each neuron the proportion of spikes in doublets to solitary spikes, and the time interval between the spikes in doublets, remained constant during control recordings. All these features are characteristic of single neurons firing in a repetitive firing pattern rather than simultaneous recordings of two separate 5-hydroxytryptaminergic neurons. Repetitive firing neurons were recorded with a similar frequency in both chloral hydrate and Saffan anaesthetized rats, and were detected using both glass and metal electrodes. Furthermore, neurons with a repetitive firing pattern were inhibited by intravenous administration of a selective 5-hydroxytryptamine1A receptor agonist and a 5-hydroxytryptamine reuptake inhibitor, thus displaying responses typical of 5-hydroxytryptaminergic neurons. Repetitive firing neurons occurred in both the dorsal and median raphe nuclei, although they were much more frequent in the dorsal raphe nucleus (91 of 332 neurons). The occurrence of repetitive firing neurons in the midbrain raphe nuclei is a newly described phenomenon which may indicate unique properties of a sub-population of 5 hydroxytryptaminergic neurons. In functional terms, it could modify both axonal and dendritic 5-hydroxytryptamine release, and provide an additional option for neuronal information signalling. PMID- 8637618 TI - The association of thirst, sodium appetite and vasopressin release with c-fos expression in the forebrain of the rat after intracerebroventricular injection of angiotensin II, angiotensin-(1-7) or carbachol. AB - The effect intracerebroventricular injections of angiotensin II (0.1 nm), angiotensin-(1-7) (1 or 100 nm) and carbachol (500 ng) on c-fos expression was examined in the forebrain of Lister hooded rats. Intense staining of the c-Fos protein was found in the median preoptic nucleus, organum vasculosum of the lamina terminalis, subfornical organ, paraventricular nucleus and supraoptic nucleus after angiotensin II and carbachol Angiotensin II caused significantly more c-fos expression in the ventral median preoptic nucleus and organum vasculosum of the lamina terminalis than carbachol, whereas in the paraventricular and supraoptic nuclei this was reversed, with carbachol having a greater effect on c-fos expression in these areas. Angiotensin-(1-7), however, only induced c-Fos protein in the organum vasculosum of the lamina terminalis and median preoptic nucleus with the number and the intensity of staining of the nuclei significantly less in both areas than after angiotensin II or carbachol. Separate groups of Lister rats were given i.c.v. injections of the same substances at the same doses, but excluding the lower dose of angiotensin-(1-7), and the intakes of water and 1.8% NaCl over 60 min were measured. Angiotensin II stimulated intakes of both water and NaCl. The effect on water intake was almost immediate (<1 min), whereas NaCl intake did not usually start until at least 5 min after injection. Over 60 min, water (12.4 +/- 1.0 ml) and NaCl (4.2 +/- 0.9 ml) intakes were significantly greater than water (1.1 +/- 0.2 ml) and NaCl (0.6 +/- 0.5 ml) intakes of the controls. Carbachol caused less drinking than angiotensin II, the water intake over 60 min being significantly less (4.8 +/- 0.7 ml) and the latency of response greater (>5 min). Carbachol, unlike angiotensin II, had little effect on NaCl intake (0.7 +/- 0.4 ml). Angiotensin-(1 7) had no effect on water (1.1 +/- 0.3 ml) or NaCl (0.3 +/- 0.3 ml) intakes. The plasma levels of vasopressin were measured after i.c.v. injection of the same three substances in the same doses, again excluding the lower dose of angiotensin (1-7), in further groups of rats. Angiotensin II and carbachol caused an approximate five-fold increase in plasma vasopressin levels compared to cerebrospinal fluid-injected rats, but angiotensin-(1-7) had no effect on vasopressin release. Therefore, three compounds with widely differing effects on thirst, sodium appetite and vasopressin release induce distinctive patterns of c fos protein expression in the forebrain. By combining experimental approaches in this way it is possible to determine areas of the brain which are involved in certain behavioural and endocrine responses. PMID- 8637619 TI - Effects of pretreatment with corticotropin-releasing factor on the electrophysiological responsivity of the locus coeruleus to subsequent corticotropin-releasing factor challenge. AB - Both acute central administration of exogenous, and stress-induced release of endogenous corticotropin-releasing factor result in electrophysiological activation of the noradrenergic neurons constituting the locus coeruleus. The present experiments were designed to examine whether single (1) or repeated (8) intracerebroventricular pretreatment with exogenous corticotropin-releasing factor would alter locus coeruleus electrophysiological responsivity to subsequent corticotropin-releasing factor challenge in rats. A single corticotropin-releasing factor (3 microg) pretreatment significantly attenuated challenge-induced locus coeruleus activation 24 and 72, but no 96 h later, while a single vehicle pretreatment had no significant effect on the response to subsequent challenge at any pretreatment-to-test interval. Repeated pretreatment with either corticotropin-releasing factor or vehicle completely attenuated locus coeruleus response to challenge 24 h after the final pretreatment. Seventy-two hours after the last vehicle pretreatment, challenge resulted in a significant increase in locus coeruleus activity, though the response was less than in naive controls. Challenge continued to produce no effect on locus coeruleus activity in repeated corticotropin-releasing factor-pretreated rats at this (72 h) time point. One week (168 h) after the cessation of repeated pretreatment, challenge resulted in a significant increase in locus coeruleus activity which was equal to that of naive controls in vehicle-pretreated rats, but reduced by comparison to controls in corticotropin-releasing factor-pretreated rats. Basal discharge rates of locus coeruleus neurons 24 h after the last repeated corticotropin-releasing factor pretreatment were significantly less than in naive controls. Thus, the failure of challenge to increase neuronal activity in these rats was not due to a "ceiling" effect caused by elevated tonic discharge rate. Repeated vehicle pretreatment produced a functional change similar to that produced by exogenous corticotropin-releasing factor administration. One hypothesis is that repeated vehicle pretreatment was stressful and caused the repeated release of endogenous corticotropin-releasing factor. This hypothesis was tested by determining whether locus coeruleus neurons remained responsive to challenge following repeated administration of a corticotropin-releasing factor antagonist. Thus, the effect if repeated pretreatment with the antagonist, [D-Phe, Nle, Calpha MeLeu]CRF was also examined. Challenge resulted ina significant increase in discharge rate 24 h after the final antagonist pretreatment, providing support for the hypothesis. Additionally, in rats repeatedly pretreated with vehicle, carbachol challenge induced an increase in locus coeruleus activity equal to that induced in naive controls. These results indicate that prior exposure to corticotropin-releasing factor, or the repeated mild stress of vehicle infusions, reduces locus coeruleus responsiveness to corticotropin-releasing factor, and reveal that the relationship between these two neurotransmitter systems is modifiable. This altered relationship may contribute to stress-related affective disorders in which both systems have been implicated. PMID- 8637620 TI - alpha 2-Adrenoreceptor-mediated inhibition of acetylcholine-induced noradrenaline release from rat sympathetic neurons: an action at voltage-gated Ca2+ channels. AB - [3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in 3H outflow which was half-maximal at about 300 microM and within 5 s. The overflow induced by 10 s exposure to 300 micro A acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 microM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow irrespective of the stimulus. The selective alpha2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximately 25% maximal inhibition) than electrically induced overflow ( > or = 45% maximal inhibition). These inhibitory effects were antagonized by the alpha2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 microM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 microM). UK 14,304 had no effect when 3H overflow was evoked by acetylcholine in the presence of 300 microM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of teh patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 microM) inhibited voltage dependent Ca2+ currents just as did UK 14,304 (10 microM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 microM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. alpha2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and effects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha2-adrenoceptor. PMID- 8637622 TI - Tachykininergic tone in the spinal cord of the rabbit: dependence on nociceptive input arising from invasive surgery. AB - In the decerebrated and spinalized rabbit, reflexes evoked in the gastrocnemius medialis muscle nerve by electrical stimulation of teh sural nerve are suppressed after blockade of NK-tachykinin receptors. This observation suggests that endogenous tachykinins tonically enhance transmission between sural nerve afferents and gastrocnemius motoneurons. In the present study we have investigated some possible sources of this tachykininergic tone. Electrical stimulation of the sural nerve at 1 Hz, as used in our previous investigation, leads to increased gastrocnemius reflex responses with successive stimuli. We examined reflexes evoked by pairs of sural stimuli separated by intervals of 10 1000 ms, and found that responses to the second stimuli of such pairs were significantly enhanced at intervals from 50 to 500 ms. Treatment with the NK receptor antagonist CP-99,994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2 phenylpiperidine; 1 mg/kg, i.v.] reduced gastrocnemius reflex responses per se, but did not alter the facilitatory effects of pairing sural stimuli. Subsequent treatment with the glutamate N-methyl-D-aspartate receptor blocker dizocilpine (0.5 mg/kg, i.v.) further reduced reflex responses and abolished paired-pulse facilitation. In a second set of experiments, rabbits were prepared so that reflexes could be studied with minimal surgical preparation of the hindlimb. Reflex responses recorded in this way were enhanced by treatment with CP-99,994 (up to 1 mg/kg, i.v.). Subsequent administration of the opioid receptor antagonist naloxone (1-10 microg/kg, i.v.) increased reflexes, as seen previously in surgically-prepared animals. These data show that tachkininergic modulation of spinal reflexes in the rabbit results from the nociceptive input arising from surgical preparation of the leg. In contrast, tonic opioidergic inhibition of reflexes is not substantially dependent on such input. PMID- 8637621 TI - Prior exposure to cocaine diminishes behavioral and biochemical responses to aversive conditioning: reversal by glycine/N-methyl-D-aspartate antagonist co treatment. AB - Animals will respond with stress-like behavioral and biochemical changes when exposed to a neutral stimulus that had previously been paired with a stressful stimulus. This phenomenon is generally known as aversive conditioning or conditioned fear. We tested the effect of prior exposure to cocaine on rats subjected to an aversive conditioning paradigm. Rats were given repeated doses of cocaine to develop a reverse tolerance or sensitization to the locomotor stimulant properties of cocaine. We blocked this sensitization to cocaine in one cocaine-exposed group by co-administering an antagonist of the strychinine insensitive glycine site of the N-methyl-D-aspartate receptor complex, R-(+)-HA 966, which prevented the development of locomotor sensitization to cocaine. After about three weeks, we examined the effect of cocaine sensitization and the prevention of sensitization by R-(+)-HA-966 on aversive conditioning. Rats were exposed to 10 tones (neutral stimuli) paired with footshock (stressful stimuli) over 30 min for the conditioning session. The following day, rts were returned to the cages, received 10 tones only over 30 min and were killed. No drugs were given to any rat before either session and control rats received the tones without footshock in both sessions. Prior exposure to cocaine caused an attenuation of the behavioral effects of aversive conditioning, namely the amount of time spent immobilized and the number of fecal boli expelled. Additionally, the elevated metabolic activity of dopamine in the medial prefrontal cortex, nucleus accumbens and ventral tegmental area associated with aversive conditioning was diminished in rats pre-exposed to cocaine. The behavioral and biochemical effects of pre-exposure to cocaine were reversed in rats that receive R-(+)-HA-966 co-treatment with the five day cocaine sensitization regimen. These data suggest that prior behavioral sensitization to cocaine diminishes the stressful effect of conditioned fear and that these effects are reversed when sensitization is prevented with R-(+)-HA-966. PMID- 8637623 TI - Expression of c-fos protein in the nucleus tractus solitarius in response to physiological activation of carotid baroreceptors. AB - This study has utilized unilateral physiological pressure stimulation f a vascularly isolated carotid sinus combined with c-fos immunohistochemistry to locate neurons of the nucleus tractus solitarius which are activated by carotid baroreceptors in the anesthetized, vagotomized dog. Carotid baroreceptor stimulation primarily activated neurons in the ipsilateral commissural and medial subnuclei of the caudal nucleus tractus solitarius. In the intermediate and rostral nucleus tractus solitarius, carotid baroreceptor stimulation activated neurons in the dorsal and medial subnuclei. Results from this study also suggested that different subgroups of nucleus tractus solitarius neurons may be activated by baroreceptors with different pressure thresholds. The use of c-fos immunohistochemistry in this study has enabled the definition of populations of dorsal medullary neurons in the carotid baroreflex pathway. The results also suggest a different projection of carotid baroreceptors with different pressure thresholds. PMID- 8637624 TI - [3H]Nisoxetine binding sites in the cat brain: an autoradiographic study. AB - The binding of [3H]nisoxetine, a selective inhibitor of the high-affinity noradrenaline uptake sites, was studied on frontal frozen sections of the cat brain. The highest densities in autoradiographic signal were observed in the nucleus locus coeruleus and its ascending pathways, in the area postrema and in the dorsal part of the inferior olive, the pontine nuclei, the raphe nuclei, the colliculi, the periventricular and lateral areas of the hypothalamus, the suprachiasmatic nucleus, the nucleus accumbens and the olfactory bulb. A moderately high concentration of binding sites was observed in the hippocampal formation, especially in the molecular layer of Ammon's horn, in the superficial layers of the entorhinal cortex and in the indusium griseum. Binding sites were visualized in all the subdivisions of the neocortex. The highest density of binding was generally detected in the outer edge of the superficial layer I. In some cortical areas, especially in the visual cortex, labeling with a prevalent laminar distribution in the superficial layers I-III and in the deep layers V-VI was clearly observed. Moderate to low densities of binding sites were seen in most other areas of the brain except in the white matter, the caudate nucleus and putamen, which were devoid of labeling. Overall these findings indicate a good correlation between the distribution of [3H]nisoxetine binding sites and the noradrenergic systems. Furthermore, data suggest that in several areas, high affinity noradrenaline reuptake mechanisms could play an important role in local interactions between the noradrenergic system and the other monoaminergic systems. PMID- 8637625 TI - Decrease of quantal size and quantal content during tetanic stimulation detected by focal recording. AB - End-plate potentials and miniature end-plate potentials were recorded focally (i.e., over a limited area of the end-plate with several or possibly only one active zone) in a cutaneous pectoris from neuromuscular junction during a prolonged (1-6 min) tetanic (20-100 Hz) nerve stimulation. End-plate potential amplitudes decreased and became more variable with prolonged stimulation. Synaptic depression thus occurs even when synaptic output is low, if release is evoked from only a few active zones, suggesting that there is little if any vesicular replenishment between the active zones. The probability density function of the end-plate potential amplitudes has been obtained using the Parzen estimate with a Gaussian weighting function, to reduce the number of end-plate potentials needed for the same accuracy. Quantal size of the end-plate potentials was estimated from the slope of the best fitted line to the prominent and apparently equidistant peaks of probability density functions or from the spectrogram of the probability density function of end-plate potentials. Quantal contents were initially (+/- S.D.):5.7 +/- 2.9, ranged from 2 to 12, and in all cases examined (n = 11) decreased with prolonged tetanic stimulation. The rates of the decrease of end-plate potentials amplitudes (and quantal contents) from different segments of the same nerve terminal were often different, even when they were initially comparable. This suggests that some active zones or some areas of the end-plates become depleted much faster than others. Quantal sizes of the nerve evoked and the spontaneously released quanta were generally similar at low frequencies of stimulation (0.5-2 Hz). Both decreased with high frequency stimulation, but the decrease of the quantal sizes of nerve evoked quanta was usually more pronounced. At different loci of the same end-plate the contribution of lower quantal size to the synaptic depression varied widely (from < 5% to > 80%). In conclusion lower quantal size can contribute significantly to synaptic depression. At uneven decrease of quantal sizes over the whole nerve terminal helps to explain both aspects of synaptic depression (lower synaptic efficacy and greater variability of quantal responses. PMID- 8637626 TI - Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe. AB - A non-invasive microspectrofluorimetric technique was used to investigate experimentally induced changes in cell water volume in single N1E-115 murine neuroblastoma cells, using calcein, a derivative of fluorescein, as a marker of the intracellular water compartment. The osmotic behavior of N1E-115 cells exposed to media of various osmolalities was studied. Exposure to hyperosmotic (up to +28%) or hyposmotic (up to -17%) solutions produced reversible decreases and increases in cell water volume, respectively, which agreed with near osmometric behavior. Increases in [Ca2+]i produced by exposing the cells to the ionophore ionomycin (1 microM) in isosmotic medium, resulted in a gradual decrease in cell water volume. Cells shrank to 40 +/- 7% (n = 7) below their initial water volume at an initial rate of -1.2 +/- 0.2%/min. It is concluded that N1E-115 cells are endowed with Ca2+-sensitive mechanisms for volume control, which can produce cell shrinkage when activated under isosmotic conditions. Because the technique used for measuring cell water volume changes is new, we describe it in detail. It is based on the principle that relative cell water volume in single cells can be measured by introducing an impermeant probe into cells and measuring its changes in concentration. If the intracellular content of the probe is constant, changes in its concentration reflect changes in cell water volume. Calcein was used as the probe because its fluorescence intensity is directly proportional to its concentration and independent of changes in the concentration of native intracellular ions within the physiological range. Because calcein is two to three times more fluorescent that other fluorophores such as 2,7,-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein or Fura-2, and it is used at its peak excitation and emission wavelengths, it has a better signal to noise ratio and baseline stability than the other dyes. Calcein can also be esterified allowing for cell loading and because of the possibility of reducing the intensity of the excitation light, measurements can be performed producing minimal photodynamic damage. The technique allows for measurements of cell water volume changes of < 5% and it can be applied to single cells which can be grown or affixed to a rigid substratum, e.g., a coverslip. PMID- 8637627 TI - Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye. AB - Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38 like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye. PMID- 8637628 TI - Growth cones and axon trajectories of the earliest descending serotonergic pathway of Xenopus. PMID- 8637629 TI - Reduction in the number of spinal motor neurons in neurotrophin-3-deficient mice. PMID- 8637630 TI - Lectins modulate calcium channels in chick sympathetic ganglia. PMID- 8637632 TI - Sensitivity to cross-like figures in the cat striate neurons. AB - The responses and orientation tuning in 48 out of 62 (77.4%) neurons of the cat striate cortex (area 17) significantly, but with different sign, changed at stimulation by specific cross-like figure flashing in receptive field as compared with single light bar of preferred orientation. Neurons of the first group (19 units from 62, 30.6%) were found to increase the responses by 3.3 times if stimulated by a certain cross-like figure, specific for each cell configuration and orientation. Under the same conditions, neurons of the second group (29 or 46.8% revealed a three-fold decrease of responses and all tuning characteristics worsened. Among them 8% of total number of cells showed bimodal or double orientation tuning when stimulated by some configurations of crosses due to an angle specific inhibition. Dependence of the revealed effects on excitatory convergence from neurons with different orientation tuning, on inhibitory influences from end-stop and side-zones of receptive field, as well as possible functional implication of the first group neurons for an angle and line-crossing detection are discussed. PMID- 8637631 TI - Time course of the neuroprotective effect of transplantation on quinolinic acid induced lesions of the striatum. AB - Injection of quinolinic acid in the rat striatum mimics neurochemical changes observed in Huntington's disease. We previously demonstrated that intrastriatal transplantation of fetal striatum or gelfoam protects against toxicity induced by a subsequent intrastriatal injection of quinolinic acid performed one week later. Herein, we examined whether fetal striatum or sham transplantation provides protection against quinolinic acid that lasts up to four weeks. Intrastriatal quinolinic acid injection produces neuronal loss and gliosis in Nissl staining, loss of cytochrome oxidase histochemical staining, decrease in autoradiographic binding of [3H]SCH 23390-labeled dopamine D1 and [3H]CGS 21680-labeled adenosine A2 receptors, and increase in autoradiographic binding of [3H]PK 11195-labeled peripheral benzodiazepine binding sites. None of these changes was observed in rats transplanted with fetal striatum one, two or four weeks before quinolinic acid injection. In animals transplanted with fetal striatal tissue, Nissl staining showed healthy grafts located in normal appearing striata. Although sham transplantation performed one week before quinolinic acid injection also protected against histological, histochemical and binding changes, sham transplantation performed two or four weeks before quinolinic acid injection was less effective in attenuating quinolinic acid-induced striatal toxicity. Thus, sham transplantation provides transient protection against quinolinic acid induced striatal toxicity, whereas implantation of tissue such as fetal striatum seems to be required for long-lasting protection. Our study suggests that intracerebral transplantation may also act through other mechanisms than restoration of deficient neurotransmitters or damaged pathways, a finding which may have significant clinical implications in assessing the potential benefit of this approach for the treatment of neurodegenerative disorders such as Huntington's disease. PMID- 8637633 TI - Substance P in the ventral pallidum: projection from the ventral striatum, and electrophysiological and behavioral consequences of pallidal substance P. AB - The ventral pallidum of the basal forebrain contains a high concentration of substance P and receives a massive projection from the nucleus accumbens. The present study was designed to determine whether the accumbens serves as a source for substance P-containing fibers in the ventral pallidum and characterize the function of this tachykinin peptide within the ventral pallidum. By combining in situ hybridization for messenger RNA of the substance P prohormone, beta preprotachykinin, with Fluoro-Gold retrograde labeling from iontophoretic deposits in the ventral pallidum, a population of substance P-containing neurons was demonstrated in the shell and core components of the nucleus accumbens and the ventromedial striatum. The function of substance P within the ventral pallidum was characterized at the level of the single neuron, and the behaving animal. Electrophysiological assessment revealed that approximately 40% of the 97 ventral pallidal neurons tested were readily excited by microiontophoretic applications of substance P or a metabolically stable agonist analog, DiMeC7 [(pGlu5, MePhe8, MeGly9)-substance P5-11]. Response characteristics were distinguished from glutamate-induced excitations by a slower onset and longer duration of action. Recording sites of tachykinin-sensitive neurons were demonstrated to be located throughout the ventral pallidum and within high densities of fibers exhibiting substance P-like immunoreactivity. When behaving rats received microinjections of DiMeC7 into this same region, the animals displayed an increase in motor activity, with a response threshold of 0.1nmol per hemisphere. These results verify the existence of a substantial substance P containing projection from the nucleus accumbens to the ventral pallidum. The projection likely serves to excite ventral pallidal neurons for these neurons readily increased firing following local exposure to tachykinins. Furthermore, an increase in motor behavior appears to be a consequence of this neuronal response. PMID- 8637634 TI - Distribution of limbic system-associated membrane protein immunoreactivity in primate basal ganglia. AB - The limbic system-associated membrane protein is a 64,000-68,000 mol.wt molecule known to be preferentially expressed by neurons in limbic structures of rats and cats. The present immunohistochemical study describes the distribution of this protein in the basal ganglia of Macaca fascicularis. The ventral striatum of the cynomolgus monkey displays a very intense immunostaining, whereas the dorsal striatum is much more weakly stained, except for some small zones scattered in the caudate nucleus and, to a lesser extent, in the putamen. These protein-rich zones are in register with striosomes, as visualized on adjacent sections immunostained for calbindin. At pallidal levels, immunostaining for the protein is observed only in the subcommissural regions, at the ventromedial tip of the internal pallidum, and in the caudoventral portion of the external pallidum. At nigral levels, the immunostaining is highly heterogeneous with a marked decreasing rostrocaudal gradient. The staining is most intense in nigral regions that receive striatal inputs and are enriched with calbindin. Nigral sectors populated by dopaminergic neurons, as visualized on adjacent sections immunostained for tyrosine hydroxylase, are largely devoid of immunoreactivity. In contrast, the immunostaining is uniformly intense in the ventral tegmental area. This study provides the first neuroanatomical evidence for teh existence of the limbic system-associated membrane protein in primate brain. It reveals that this glycoprotein is distributed in a highly heterogeneous manner in primate basal ganglia, where it preferentially labels regions that are anatomically and functionally linked to the limbic system. PMID- 8637635 TI - Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: comparison of stressors. AB - Repeated restraint stress induces an atrophy of apical dendrites of CA3c pyramidal neurons in the hippocampus, but the relationship between stress and adrenocortical activation has not been thoroughly investigated. In order to better understand the relationship between neural and non-neural indices of the severity of stress, we investigated the temporal relationship between CA3c dendritic atrophy and indices of adrenal steroid stress responsiveness. For this purpose, we used two different stress regimens: repeated restraint stress (6 h/day) and a chronic multiple stress paradigm (shaking, restraint and swimming, each day), differing in the degree of adrenal activation produced over 14 and 21 days. Atrophy of dendrites of CA3c neurons was found after 21 days of stress, but not after 14 days, and was of a similar magnitude for both stressors. However, non-neural measures differed between the two stress paradigms: (i) chronic restraint stress caused a significant habituation by day 21 in the corticosterone response to acute restraint, whereas chronic multiple stress exposure was not accompanied by habituation of the corticosterone response to restraint; (ii) chronic restraint stress caused neither adrenal hypertrophy nor thymus atrophy, but did reduce the rate of body weight gain throughout the 21 days, whereas chronic multiple stress caused a transient adrenal hypertrophy (on day 14), delayed suppression of thymus weight (on day 21) and transient reduction of body weight gain (on days 7 and 14, but not on day 21). Thus the non-neural indices of response to stress--although complex in their time course--suggest that the multiple stress regimen is a somewhat more potent chronic stressor for corticosterone and adrenal responses. Yet both stress regimens produced the same degree of apical dendritic atrophy in CA3c pyramidal neurons. These results are consistent with a model in which adrenocortical secretion plays a permissive role in enabling another agent, namely, excitatory amino acids, to produce the final effect. PMID- 8637637 TI - Development of the raphe-hippocampal projection in vitro. AB - In this study we examined whether the serotonergic raphe-hippocampal projection preserves its characteristic target selectivity for GABAergic interneurons when developing in vitro, in organotypic cultures. Hippocampal slices from one- to three-day-old rats were co-cultured with slices derived from the raphe nuclei of the same animals. After several weeks of in vitro incubation, a large number of raphe fibres--visualized by immunostaining for serotonin--were found to innervate the hippocampal tissue. In our random sample of over 250 serially sectioned boutons--52 of which were completely reconstructed from serial sections--only two were found to form conventional synapses in the electron microscope, and contacted dendritic spines. These results demonstrate that raphe-hippocampal serotonergic afferent are unable to form synaptic contacts with their normal targets in vitro, if explanted one to three days postnatally. Neurons in the afferent and/or target area may have passed a critical age when selective synaptic contacts can be formed, or unknown chemical or electrical signals may be missing under these conditions, which should serve to guide subcortical afferents to their synaptic target elements. PMID- 8637636 TI - Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. AB - Repeated restraint stress of rats for 21 days causes atrophy of apical dendrites of hippocampal CA3c pyramidal neurons. This effect is mimicked by daily corticosterone treatment for 21 days and is prevented y the anti-epileptic drug, phenytoin, known to interfere with excitatory amino acid release and action. The present study was designed to investigate the involvement of endogenous corticosterone secretion and excitatory amino acid receptors in the stress induced hippocampal dendritic atrophy. Treatment of chronically stressed rats with the steroid synthesis blocker cyanoketone prevented stress-induced dendritic atrophy. Cyanoketone-treated animals showed an impaired corticosterone secretion in response to the stressor, while basal levels were maintained. Besides the involvement of endogenous corticosterone secretion, N-methyl-D-aspartate receptors also play a role, since the competitive receptor antagonist, CGP 43487, blocked stress-induced dendritic atrophy. In contrast, NBQX, a competitive inhibitor of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, was ineffective at a dose that blocks ischemic damage. These results indicate that the reversible atrophy induced by 21 days of daily restraint stress requires corticosterone secretion and that excitatory mechanisms involving N methyl-D-aspartate receptors play a major role in driving the atrophy. PMID- 8637638 TI - Nasopharyngeal radium treatment of veterans. PMID- 8637639 TI - Post-traumatic stress disorder among World War II mustard gas test participants. AB - Open-minded and structured interviews were conducted to assess post-traumatic stress disorder (PTSD) and other psychosocial outcomes among 24 men who had participated in the military's mustard gas testing program during World War II. Most men had volunteered (92%) and had participated in chamber tests (96%). During the tests, few (22%) understood the danger involved. The majority (67%) were ordered to refrain from discussing their participation with anyone. Most men (83%) experienced physical symptoms subsequent to the test. At present, the men were less psychologically and physically healthy than expected for men of similar age. The current prevalence of PTSD due to the mustard gas was 17%. The current prevalence of subdiagnostic mustard-gas-related PTSD was 25%. Lifetime estimates for full and subdiagnostic PTSD was 17 and 33%, respectively. The only mustard gas experience that predicted lifetime full or subdiagnostic PTSD was the number of exposures to the gas. PMID- 8637640 TI - The peacetime trauma experience of U.S. Army surgeons: another call for collaborative training in civilian trauma centers. AB - OBJECTIVE: To document the ongoing trauma experience of U.S. Army surgeons during peacetime clinical practice. METHODS: Surveys were mailed to the trauma directors of military surgical services worldwide. Questions focused on numbers of operative cases and availability of trauma resources. Laparotomy, thoracotomy, and vascular repair for trauma were considered index cases and simple means were computed based on numbers of cases per general surgeon. RESULTS: Seventy percent of worldwide Army surgical services responded to the survey. In 1992, on the average, a general surgeon in the Army performed 1.3 trauma laparotomies, 0.3 thoracotomies, and 0.3 vascular repairs for trauma. Only 6 of 28 surgical services had an organized "trauma team," 3 held regular trauma conferences, and 1 service kept a registry and could calculate an average injury severity score. CONCLUSIONS: Our data support perceptions concerning lack of an ongoing trauma experience for military surgeons and reinforces the need for collaborative education and training in busy civilian trauma centers. A regional approach is suggested as a viable solution. PMID- 8637642 TI - The impact of sexual abuse on job attrition in military recruits. AB - OBJECTIVE: To determine whether recruits entering the U.S. Air Force with a history of sexual abuse had a higher attrition rate from basic military training (BMT). METHODS: Retrospective, case-control study involving 28,918 recruits entering BMT from October 1, 1991, to September 30, 1992. Self-report questionnaires were given to all recruits on the second day of BMT. We compared recruits revealing a history of sexual abuse to all other recruits at the end of BMT and at the end of the next fiscal year. RESULTS: We found that victims of sexual abuse had a higher attrition rate from BMT than non-victims (10.6 versus 4.1%, p < 0.0001). Four and one-tenth percent of all recruits (1,289) reported a history of sexual abuse, and fewer male than female recruits reported a history of sexual abuse (1.5 versus 15.1%, p < 0.0001). However, after BMT there were no differences in any job performance indicators between victims and non-victims. CONCLUSION: We conclude that recruits with a history of sexual abuse had a higher attrition rate from BMT than those without a history of abuse; however, those recruits who did complete BMT were as successful as those who did not report a similar history of abuse. PMID- 8637641 TI - Tincture of benzoin: clinical and microbiological implications of reusable containers. AB - At our institution, tincture of benzoin solution is commonly used as a topical adhesive agent. As a cost-saving practice, multiple-dose bottles are routinely used in the operating rooms and the clinic on multiple patients. Although clinically pathogenic organisms are known to be capable of survival in both benzoin and its isopropyl alcohol solvent, no prior controlled studies have investigated the potential for tincture of benzoin solution to support the growth of specific pathogens under clinically relevant conditions. In this study, multiple aerobic, anaerobic, and spore-forming bacteria were exposed to tincture of benzoin solution, as well as Candida albicans and Mycobacterium fortuitum. Bacillus cereus was the only index organism demonstrating a clear ability to survive a 15 minute incubation in tincture of benzoin, although 24 hours of exposure to tincture of benzoin resulted in no subsequent viable cultures of this organism after 72 hours of incubation. Thus although certain bacilli might, under ideal circumstances, remain viable and infectious within multiple-dose bottles of tincture of benzoin, the risk of causing iatrogenic infection appears to be rather minimal. Still, the use of multiple-dose dispensers of topical agents, particularly in surgical patients, should be carefully scrutinized for their clinical risk-to-economic benefit ratio. PMID- 8637643 TI - Prevalence of sand fly fever, West Nile, Crimean-Congo hemorrhagic fever, and leptospirosis antibodies in Pakistani military personnel. AB - To determine the prevalence of antibodies to viral diseases known or suspected to be present in Pakistan, we studied 570 sera from three groups of adults; two of the groups were involved in outbreaks of hepatitis, and the third included men admitted to a hospital for evaluation of febrile illnesses. Immunoglobulin G antileptospiral antibody was found in 1 to 6% of the subjects, with the highest rate in enlisted military personnel hospitalized for febrile illness. One man in the group with febrile illness had significantly elevated immunoglobulin M antileptospiral antibody titers. However, in a group of recruits experiencing suspected non-A, non-B hepatitis, 19 (11%) of 173 had a 4-fold rise in immunoglobulin M antibody to leptospirosis. Antibody to sand fly fever viruses was found in 27 to 70%. Antibody to West Nile virus was present in 33 to 41% of subjects. Antibody reactive with Japanese encephalitis virus was present in 25%, but plaque reduction neutralization tests suggested this to be cross-reaction with West Nile virus. All 212 specimens tested for antibody to Crimean-Congo hemorrhagic fever and Hantaan viruses were negative. This study indicates that diseases known to be prevalent in other areas of southwest Asia and the Middle East are also prevalent in northern Pakistan and may impact on those traveling or working in this area. PMID- 8637644 TI - Development of a Comprehensive Surgical Information System at Madigan Army Medical Center. AB - The Operative Registry (DA Form 4108) has been the information source for surgical data supporting quality assurance and utilization review efforts at Madigan Army Medical Center. Recently, Madigan's requirements for data and reporting changed. Like other government medical facilities, Madigan began pervasive quality-improvement efforts. This resulted in new ideas to measure hospital performance. Consequently, requirements for surgical data required to support quality and resource management reporting, utilization review, residency review reporting, research and credentialing changed. This article details Madigan's approach to addressing these requirements via development of a comprehensive computing solution. It discusses Madigan's fragmented data environment before system development, and gives the reader perspective on the decision-making process that led to system development rather than purchasing a commercial product. Finally, the article describes how a strong partnership between staff and developers was key to providing a solution that exceeded established goals. PMID- 8637646 TI - The evolving pattern of war-related injuries from the Afghanistan conflict. AB - This is a retrospective study of 922 victims of the Afghanistan war admitted to Mekkah Mukarramah Surgical Hospital, a 120-bed hospital, from April 1987 to April 1989. The range and extent of war-related injuries were reviewed. The lower limbs were the most common site of injuries. The ratio of critical-area wounds (trunk, head, and neck) to extremity wounds was 0.17, which reflects the lack of early emergency medical care for the wounded. This ratio can be used to monitor and evaluate the efficiency of the evacuation plans in a war situation. PMID- 8637645 TI - Stress, suicide, and military service during Operation Uphold Democracy. AB - The initial weeks of Operation Uphold Democracy were marked by a high rate of major axis I disorders among soldiers presenting for treatment at the 528th Combat Stress Center. The mission to Haiti was also marked by three suicides during the first 30 days. The author explores psychodynamic sources of stress that may have contributed to the high level of psychiatric acuity observed during that first month. Then, distinctions are made between psychiatric conditions that present in combat and those that occur on peacekeeping missions. PMID- 8637647 TI - Tobacco use habits of naval personnel during Desert Storm. AB - This study examined availability and usage of tobacco products, and their potential impact on the oral health of naval personnel deployed to Desert Storm. Of 4,200 surveys mailed to a randomly selected sample, 45.6% were returned (N = 1,915). The respondents included 55.9% who reported a present or former smoking habit, 34.1% who identified themselves as current smokers (SM), and 23.8% who were smokeless tobacco (ST) users. Tobacco products were easily and inexpensively accessible through ship stores, exchange, or military support organizations (USO). While in the Persian Gulf, 7.0% started SM and 9.3% started ST, resulting in an overall 4.7 and 6.1% increase in SM and ST, respectively. Of those who were already tobacco users, 29.2% reported more SM use and 19.0% used ST more often. Stress (35.1%) and boredom (21.4%) were the most frequently cited reasons to start or increase use. Although 30.5% of respondents reported military personnel have encouraged them to quit, 77.2% reported that anti-smoking efforts have been unsuccessful in influencing them to quit. Since the tobacco usage rate is higher in the military than in the civilian sector, greater emphasis on preventive efforts in warranted to promote health and wellness. PMID- 8637650 TI - Adrenocortical neoplasm disguised as a post-traumatic perinephric hematoma. AB - Trauma and cancer are entities that are encountered frequently by surgeons and occasionally may exist simultaneously in certain patients. Illustrated here is the case of a man with a perinephric mass, discovered during evaluation for blunt abdominal trauma, which was originally thought to be a perinephric hematoma and later proved to be an adrenocortical carcinoma. PMID- 8637648 TI - Baseline (laser) eye examination program. AB - Eye injury is the primary hazard associated with laser use. A baseline laser eye examination program allows for pre-injury examination and preventive early diagnosis of eye problems (glaucoma). Using portable equipment and a simplified exam format, and staffed by corpsmen and eye care providers, a rapid and accurate large-scale program that is cost efficient can be instituted. A single location for ophthalmic information coupled with recent refraction data will enhance readiness for deployment. For service members and their providers, a new and well run eye care program will foster a strong and positive bond with the medical department. PMID- 8637651 TI - Hernia through an iliac bone graft defect. PMID- 8637649 TI - Vascular injury secondary to gunshot wound: wanna be, gonna be, never was. AB - This case report is unusual not because of the history of gunshot wound but because the patient lied about the injury. The patient stated that he was wounded in combat while serving in Vietnam. When records were verified, it was discovered that the patient was not a veteran and, therefore, could not have been injured in combat. The fact that this patient claimed to be a combat veteran but never served in the military brings to mind the words from the well-known Airborne cadence "Wanna Be, Gonna Be." However, in this case the patient never was. PMID- 8637652 TI - Pseudomonas aeruginosa pneumonia/sepsis as an initial opportunistic infection in an HIV patient. PMID- 8637653 TI - Uterine packing in the combined management of obstetrical hemorrhage. AB - Uterine packing to control obstetrical hemorrhage has been generally discouraged over the past several decades. Intractable uterine hemorrhage postpartum or following an abortion is an extremely vexing management problem for the physician and continues to be a leading cause of maternal mortality. Uterine packing should be considered as a presurgical management tool after lacerations of the lower genital tract, uterine rupture, or retained products have been ruled out and when conventional therapy fails to control uterine hemorrhage. We describe two obstetrical patients with intractable uterine hemorrhage who were managed with uterine packing in combination with other methods of therapy. Causes of obstetrical hemorrhage and techniques of packing the uterus are discussed. PMID- 8637654 TI - Interview with professor Uwe Reinhardt of Princeton University. Interview by Steve Adubato. PMID- 8637655 TI - A physician's first option for quality. AB - John Adessa is unhappy with the direction managed care has taken in this country, and he is doing something about it. Adessa, the chief executive officer of First Option Health Plan, dislikes the burden of risk that falls on physicians through capitation arrangements, and he does not believe that patients should be severely restricted in their access to physicians. PMID- 8637656 TI - HCFA's Medicare mortality statistics. PMID- 8637658 TI - Acute acalculous cholecystitis secondary to Candida albicans. PMID- 8637657 TI - Limiting transesophageal echocardiography in valve endocarditis. PMID- 8637659 TI - Nutrition and behavior. PMID- 8637660 TI - Managed cancer care. A blueprint for discussion. PMID- 8637661 TI - New violence institute extends UMDNJ tradition of service to New Jerseyans. PMID- 8637662 TI - Physicians and nurse practitioners: can they work together? PMID- 8637663 TI - Funding for Medicaid. PMID- 8637664 TI - Raising money for charity care in New Jersey. PMID- 8637665 TI - Immunohistochemical analysis of the endocrine cells in the pancreatic islets of cattle. AB - The distribution of endocrine cells and the areas of islets in the bovine pancreas were investigated by immunohistochemical methods. The islets in the A region, which consisted of the left lobe, the ventral portion of the body, and the ventral and distal portions of the right lobe, contained a central core of insulin-containing (B-) cells surrounded by glucagon containing (A-) cells, a few somatostatin-containing (D-) cells and sporadic pancreatic polypeptide-containing (PP-) cells. The islets in the B-region, which consisted of the uncinate process, and the dorsal and proximal portions of the body and the right lobe, contained a central mass of B-cells, and peripheral cells which were predominantly PP-cells with a few D-cells but no A-cells. The areas of islets in the B-region were small as compared with those in the A-region. From these findings it is suggested that A- and PP-cells have a complementary relationship to one another. In view of this hypothesis, two types of islet, A-cell-rich and PP-cell-rich islets, were identified. The A-region contained A-cell-rich islets derived from the dorsal pancreatic primordium, while the B-region contained PP-cell-rich islets that originated from the ventral primordium. In the bovine pancreas, the areas containing PP-cell-rich islets are greater than those in previously examined species. PMID- 8637666 TI - The relationship between the palatal form and the maxillary sinus in orang-utan. AB - The relationship between the palatal form and the maxillary sinus was studied in 40 skulls of male and female Bornean orangutans (Pongo satyrus borneensis) ontogenetically. Univariate analyses of the measurements of the palate and the maxillary sinus showed that the sexual dimorphism appeared earlier in palatal length than in the maxillary sinus volume. Bivariate statistical analyses, including correlation and linear regression analyses, revealed close relationships between the characteristics of the palate and the maxillary sinus for both the growth period and the adult status. Partial correlation coefficients showed, however, that these relations were highly influenced by the skull size. Except significant correlations between the maxillary sinus width and the palatal volume in the adult status, all partial correlation coefficients were clearly below the Pearson's correlation coefficients. In contrast to the situation in humans, this study suggests that under physiological conditions the influence of the palatal form on the maxillary sinus size in the orang-utan is very small. Alterations of the craniofacial morphology during the evolution of the hominids, such as facial reduction and decrease of the maxillary sinus size, are considered important factors which led to changes of the relations between the palatal form and the maxillary sinus. PMID- 8637667 TI - Developmental-anatomical observations on cleavage line patterns of the skin in Chinese miniature pigs. AB - In twelve Chinese miniature pigs (CMP), of both sexes and ranging from newborn to 5 months of age, the postnatal developmental features of the cleavage lines of the skin of the whole body were examined by the stab-wound method, using a metal probe with a sharp conical point. 1). The CL patterns of the skin in the 5-month old CMP were largely similar to those of adult Landrace-strain pigs (Wakuri et al., 1993). 2). At each monthly stage, the CL of the skin of the inter-ocular area, medial ocular angle, umbilicus, preputial orifice and anus presented a convergent arrangement. An annular pattern was found in the skin around the eye, the base of the ear, and the vulva. 3). Some alterations in CL patterns were seen during the general and regional growth of CMP, as shown in the skin of the lateral side of the trunk, the prepuce and the scrotum. PMID- 8637668 TI - Studies on the reduction indices of the deciduous teeth of Chinese living in Taiwan. AB - The reduction index of deciduous teeth was investigated statistically. Materials used were plaster casts of the deciduous dentition taken from Chinese children living in Taiwan. The reduction indices showed no sexual differences. In the maxillary teeth, reduction indices of the bucco-lingual diameters (BL) had the largest values, followed by those of the mesio-distal diameters (MD), and those of the crown area (AREA) which were the least. In contrast to the maxillary teeth, in the mandibular teeth, the reduction indices of MD and those of BL had nearly the same values although the reduction indices of MD were slightly larger than those of BL in females (p < 0.05). With respect to the difference between the maxilla and the mandible, in deciduous incisors the reduction index of MD in the mandible was larger than that in the maxilla (p < 0.01). While in BL the reverse relation was noted. In the deciduous molars the reduction indices of BL and AREA in the maxilla were larger than those in the mandible (p < 0.01). The reduction indices showed no significant difference among the Mongoloid populations sampled. This result may be explained by the primitiveness of deciduous teeth. PMID- 8637669 TI - A facial growth analysis based on FEM employing three dimensional surface measurement by a rapid laser device. AB - Although there have been several studies of finite element method (FEM) analysis on two dimensional (2D) facial growth with cephalometric X-rays, there has been little FEM analysis on three dimensional (3D) facial growth of long term observation. Therefore the objective of our study is to use FEM model by 3D surface measurement of rapid laser device from human dried skull and to analyze the changes of facial growth based on FEM by the volume and the direction of strain in each stage. Samples were taken from each human dried skull for 5 stages; about 0, 3, 6, 9, 12 and 18 years of age, a total of 6 normal human dried skulls. (No abnormal skeletal growth patterns were selected and age was supposed by tooth development and eruption.) After measuring each human dried skull by 3D rapid device, we selected the clearest 16 anatomical reference points from about 70,000 points on face image to form accurate FEM shells. The study utilized Cosmos/M (SRAC) for FEM analysis, on a PC(NEC). From the strain analyses, it was revealed that (1) The vale of growth strain from 0 to 3 years of age and from 3 to 6 years of age gradually increased from condylar area toward mental area and the most vale of growth strain was showed at mental area. The vale of growth strain of corpus area was bigger than other areas. (2) As a whole the vale of growth strain of other areas except mental, corpus and nasal area were almost equal. The results indicate especially the growth change of mandible were predominantly showed in the early stages, and the direction of growth strain changed backward and above from mental area to condylar area. PMID- 8637670 TI - The non-effect of parathyroidectomy in the aquatic limbless newt (Apoda, Amphibia). AB - In the aquatic limbless newt, Typhlonectes compressicauda (Apoda, Amphibia), serum Ca levels of parathyroidectomized newts were no lower than for the control newts at 1 week after the operation. In this species, the parathyroid glands may not be functional in raising the serum Ca levels. PMID- 8637671 TI - [Aging as the final stage of differentiation: an approach to an aperiodic state?]. PMID- 8637672 TI - [The action of tiazofurin on the development of sea urchin embryos and larvae]. AB - The effects of the antitumor drug tiazofurin on development of sea urchins Sphaerechinus granularis, Paracentrotus lividus, Strongylocentrotus intermedius, and Arbacia lixula were studied. When 0.01-200 microM tiazofurin (TAF) was introduced in the incubation medium (artificial sea water) just after fertilization or at the midblastula stage, the development proceeded quite normally until the beginning of gastrulation. But later TAF blocked gastrulation and induced formation of mobile ball-shaped larvae with normal pigment cells but devoid of the nervous system, skeletal spicules and digestive tract. The threshold TAF concentrations varied from 0.05 microM (S. granularis) to 2-5 microM (all other species). When TAF was introduced during gastrulation and just after gastrulation, the larvae had defective nervous system and skeleton and suppressed expression of gangliosides. The nonhydrolyzable analog of GTP, GTP gamma-S (5-20 microM), introduced in artificial sea water no later than at the midblastula stage prevented all above mentioned developmental defects. PMID- 8637673 TI - Pediatric keratoplasty. PMID- 8637675 TI - Surgical alignment in infants for congenital esotropia. PMID- 8637674 TI - Formula revealed. PMID- 8637676 TI - Cotton-wool spots and giant cell arteritis. PMID- 8637677 TI - Preliminary procedure assessment: innovation, information, and knowledge. PMID- 8637678 TI - Incidence and progression of nuclear opacities in the Longitudinal Study of Cataract. AB - PURPOSE: To estimate incidence and progression rates of nuclear opacities in the Longitudinal Study of Cataract, an epidemiologic study of the natural history of all types of lens opacities. METHODS: The Lens Opacities Classification System III was used to assess longitudinal changes between baseline and follow-up lens photographs for the 764 Longitudinal Study of Cataract participants. Baseline data, collected until December 1988 as part of a case-control study, included color slit, retroillumination, and Scheimpflug photographs. The same data were collected by the longitudinal Study of Cataract at four subsequent visits at yearly intervals. RESULTS: Among patients free of nuclear opacities at baseline, the incidence of new opacities was 6% after 2 years and 8% after 5 years of follow-up. The progression of pre-existing nuclear opacities was much higher. After 2 years, nuclear opacities had progressed in more than one third of the patients with pre-existing opacities; after 5 years, almost half had progressed. Older age was significantly related to higher incidence of new nuclear opacities, but not to progression of pre-existing opacities. Patients with other opacity types had higher nuclear incidence and progression rates. CONCLUSIONS: In this clinic-based, older-patient population, new nuclear opacities developed in less than one tenth of the patients after 5 years of follow-up. In contrast, almost one half of the patients with pre-existing opacities had worsened after 5 years. These estimated rates can be used to plan intervention or other studies of nuclear changes in similar populations. PMID- 8637679 TI - Results of a prospective evaluation of three methods of management of pediatric cataracts. AB - BACKGROUND: Although a variety of approaches to manage cataracts in children have been studied, no consensus exists on the optimum approach. The authors, therefore, conducted a prospective, nonrandomized, consecutive study to evaluate three most commonly adopted methods of management of pediatric cataracts. METHODS: Lensectomy anterior vitrectomy (LAV), extracapsular cataract extraction with intraocular lens implantation (ECCE + IOL) and ECCE, primary posterior capsulotomy, anterior vitrectomy with IOL (ECCE + PPC + AV + IOL) were the surgical procedures performed. Aphakia in the LAV group was corrected with spectacles or contact lenses. Intraoperative and postoperative results were analyzed. Discrete variables among the three groups were compared using chi square test. RESULTS: One hundred ninety-two eyes were included in the study. There was no statistically significant difference in the intraoperative complications in the three groups. During a mean follow-up period of 11.3 months, postoperative obscuration of the visual axis was seen in 43.7% of eyes in the ECCE + IOL group and in 3.65% of eyes in the ECCE + PC + AV + IOL (p < 0.001). Two of the seven patients in the LAV group in whom contact lenses were prescribed developed corneal infiltrates. Severe postoperative anterior uveitis occurred in 15.9% and 13.8% of eyes in the ECCE + PPC + AV + IOL and ECCE + IOL groups, respectively. None of the eyes that underwent LAV developed this complication (P < 0.001). There was no statistically significant difference in the incidence of retinal detachment, endophthalmitis, or glaucoma in the three groups. CONCLUSION: Of the three approaches, ECCE + PPC + AV + IOL was conducive to at least short term maintenance of a clear visual axis, provided optimum refractive correction, and was not associated with increased risk of short-term complications. Continued follow-up of these eyes is necessary to conclude on the long term results of this technique. PMID- 8637680 TI - Ipsilateral hypertropia after cataract surgery. AB - BACKGROUND: Reports of acquired strabismus caused by injection of local anesthetics during cataract surgery have increased recently. The authors proposed a mechanism to explain the occurrence of strabismus with apparent overactive muscles after cataract surgery. METHODS: The authors studied 19 patients in whom strabismus developed after cataract surgery. Prism and cover test in the diagnostic positions of gaze and forced-duction testing were used to identify the affected muscles. RESULTS: The deviation was greater in the field of action of the presumed tight muscle in 16 of 19 patients. An ipsilateral hypertropia with superior rectus muscle overaction subsequently developed in two patients with an initial hypotropia. An overaction of the ipsilateral lateral rectus muscle causing an exotropia developed in one patient with initially limited abduction. CONCLUSIONS: Myotoxicity from direct injection of local anesthetics into an extraocular muscle probably causes transient paresis followed by segmental contracture of the involved muscle. Mild contractures result in strabismus with a motility pattern of an overactive muscle. Larger amounts of contracture lead to restrictive strabismus. The risk of strabismus may be decreased by administering the local anesthetic into sub-Tenon space using a blunt-tipped cannula when performing cataract surgery. PMID- 8637681 TI - Hyperopia correction by noncontact holmium:YAG laser thermal keratoplasty. Clinical study with two-year follow-up. AB - BACKGROUND: Thermal keratoplasty to correct hyperopia has been attempted with nonlaser and laser devices. Problems have included long-term regression and irregular induced astigmatism. The present clinical study was performed to investigate the safety, efficacy, and stability of a noncontact mode of holmium: YAG laser energy delivery and a modified laser thermal keratoplasty treatment procedure for correction of low hyperopia. METHODS: Seventeen patients underwent noncontact holmium: YAG laser thermal keratoplasty in their nondominant eyes for correction of hyperopia of up to 3.00 diopters. Treatment parameters included simultaneous delivery of eight holmium: YAG laser spots in a symmetrical octagonal array with a centerline diameter of 6mm, 10 pulses of laser light at 5 Hz pulse repetition frequency, and pulse energies of 159 to 199mJ. Follow-up was 2 years in 15 of 17 patients. RESULTS: In the 15 eyes examined at 2 years after surgery, mean uncorrected distance Snellen visual acuity improved from 20/125-1 to 20/50-2. The mean change in spherical equivalent of subjective manifest refraction was -0.79 diopter. Eleven of these 15 eyes (73%) had a mean refractive correction of -1.1 diopters (range, -0.38 to -2.63 diopters); regression between 14 days and 2 years was 0.2 diopter. Four eyes (27%) had no persistent refractive correction (within +/- 0.25 diopter). Mean induced refractive astigmatism was 0.18 diopter. None of the eyes lost two or more lines of spectacle-corrected distance vision. The amount of refractive correction at 2 years after surgery was correlated to the treatment pulse energy and the volume of the opacified corneal tissue observed immediately after treatment. CONCLUSIONS: This technique of noncontact laser thermal keratoplasty produced safe, effective, and persistent corrections of low hyperopia in the majority of treated eyes. PMID- 8637682 TI - One-year follow-up results of photorefractive keratectomy for low, moderate, and high primary astigmatism. AB - OBJECTIVE: To study the efficacy of excimer laser photorefractive keratectomy (PRK) for high, moderate, and low degrees of primary myopic astigmatism. PATIENTS AND METHODS: Ninety-two eyes of 54 patients with different degrees of compound myopic astigmatism underwent PRK. The eyes were divided by degree of refractive astigmatism into three groups-high (-2.75 to -5.0 diopters [D]), moderate (-1.25 to -2.50 D), and low (< or = 1.0 D). Refraction, corneal topography, slit-lamp findings, and visual acuity with and without correction were assessed. RESULTS: At 12 months, the mean reduction from the preoperative refractive cylinder was 80.7% in the high astigmatism group, 68.4% in the moderate astigmatism group, and 47.6% in the low astigmatism group. The post-treatment residual cylinder axis remained stable in 23 (38.3%) of 60 eyes and deviated in 37 (61.7%) of 60 eyes. The maximal deviation of the residual cylinder axis was 15 degrees. Of the 89.2% of eyes with low cylinder, 81.8% had moderate cylinder, and 85% of the eyes with high cylinder achieved a final uncorrected visual acuity between 20/20 and 20/35 at 12 months. CONCLUSIONS: A statistically significant reduction in the refractive cylinder was found in the high, moderate, and low astigmatism groups. The difference between the mean reduction of the high and moderate cylinders compared with the mean reduction of the low cylinders also was found to be statistically significant. The laser used in this study is an efficient tool for correcting high and moderate astigmatism. However, regarding low astigmatism, it was found to be less effective. PMID- 8637683 TI - Management of postoperative Acremonium endophthalmitis. AB - PURPOSE: Four patients presented after cataract surgery with delayed-onset endophthalmitis caused by Acremonium kiliense with in vitro sensitivity to amphotericin B. In all patients, ocular infection was recalcitrant to single-dose intravitreous amphotericin B injection. The authors reviewed the management of endophthalmitis caused by A. kiliense and presented treatment recommendations. METHODS: The authors retrospectively evaluated a cluster of four patients with delayed-onset postoperative endophthalmitis after phacoemulsification with posterior chamber intraocular lens implantation. All patients underwent vitreous sampling, intravitreous injection of amphotericin B, and systemic administration of fluconazole. Pars plana vitrectomy was performed in all patients for management of either primary (1 eye) or persistent infection (3 eyes). Two patients with persistent infection also underwent surgical explanation of their posterior chamber intraocular lens. RESULTS: Worsening infection developed in three of three eyes that underwent vitreous aspiration with intravitreous injection 5 micrograms amphotericin B. These patients subsequently responded to vitrectomy followed by additional intravitreous amphotericin B injection. One eye underwent primary vitrectomy and intravitreous injection of 7.5 micrograms amphotericin B. Although treatment of the initial infection was successful, fungal keratitis developed in this patient 3 months after presentation. Visual outcome was variable, ranging from visual acuity of 20/25 to no light perception with follow-up of 2 years. Epidemiologic investigation suggested a common environmental source for the A. kiliense organisms. CONCLUSIONS: Single-dose administration of intravitreous amphotericin B was inadequate treatment for fungal endophthalmitis caused by A. kiliense. Vitrectomy with repeated intravitreous administration of amphotericin B may be necessary to eradicate intraocular function caused by this organism. PMID- 8637684 TI - Aspergillus endophthalmitis. An unrecognized endemic disease in orthotopic liver transplantation. AB - PURPOSE: The authors discovered an unusually high incidence of Aspergillus endophthalmitis in an autopsy series of orthotopic liver transplantation recipients. This study was conducted to discern the frequency, topographic distribution, and potential significance of the infections. METHODS: Autopsy reports from liver transplant patients were reviewed. All patients with Aspergillus endophthalmitis were studied by gross and histologic examination. Histologic sections were stained with Grocott-Gomori methenamine-silver nitrate and periodic acid-Schiff stains. Some Grocott-Gomori methenamine-silver nitrate stained sections were counterstained with hematoxylin-eosin. The distribution of ocular infections in the eye was determined for each patient. The organs infected were determined at autopsy. RESULTS: The authors found seven patients with Aspergillus endophthalmitis. Six of these seven patients were from a group of 85 (7.1%) orthotopic liver transplantation recipients. Fourteen (16.5%) orthotopic liver transplantation recipients had invasive pulmonary aspergillosis and ten (11.8%) had disseminated disease. The eyes were the second most common site of infection. Two patients had ocular involvement as the only nonpulmonary site of infection. Aspergillus endophthalmitis was diagnosed in only one patient before death. Infection was located posterior to the equator in all patients; three patients were anterior to the equator as well. The retina (5/7), vitreous (5/7), and choroid (3/7) were common sites of infection. CONCLUSIONS: This is the first report of Aspergillus endophthalmitis associated with orthotopic liver transplantation recipients. Patients with orthotopic liver transplants are unusually susceptible to invasive aspergillosis and Aspergillus endophthalmitis. Aspergillus infection is frequently bilateral, begins posteriorly in the retina or choroid, and has vitreous involvement. Recognition of this entity is important because many patients die of disseminated Aspergillus infection that may be detected early with bedside funduscopic examination. PMID- 8637685 TI - Systemic drug toxicity trends in immunosuppressive therapy of immune and inflammatory ocular disease. AB - PURPOSE: To compare the relative toxicities of six systemic immunosuppressive drugs and systemic corticosteroids used to treat patients with severe ocular inflammatory disease and to identify factors influencing their occurrence. METHODS: The authors reviewed the clinical records of 602 patients with ocular inflammatory disease treated with immunosuppressive drug therapy and/or systemic corticosteroids for adverse systemic effects while undergoing therapy. Proportional hazards regression analysis was performed to identify demographic and clinical factors that influence the occurrence of drug toxicity in these patients. RESULTS: Immunosuppressive drug treatment was more likely to result in discontinuation of therapy because of toxic side effects than was corticosteroid treatment. However, unlike many of the side effects of corticosteroid treatment, the side effects of immunosuppressive therapy were reversible with reduction in dosage or discontinuation of the drug. Gastrointestinal symptoms and hematologic abnormalities accounted for the majority of reported side effects of the immunosuppressive medications. Neuro-psychiatric and endocrine side effects were common in patients taking prednisone. In 17 patients treated with prednisone, pathologic fractures developed, which involved the hips and the spine. Female sex and age older than 60 years also were identified as factors associated with intolerance to drug therapy in the authors' study population. Race and type of systemic ocular disease were not significant factors influencing tolerance to drug therapy. CONCLUSION: These findings suggest that when properly administered and monitored for adverse effects, most immunosuppressive agents used in the current study have similar risk profiles with relatively few serious therapeutic mishaps and largely reversible side effects. In contrast, corticosteroids can result in permanent disabilities as a result of long-term treatment. PMID- 8637686 TI - Infectious mononucleosis presenting with dacryoadenitis. AB - BACKGROUND: A case of severe, bilateral, acute dacryoadenitis in a rarely reported association with infectious mononucleosis is described. METHODS: A 16 year-old girl had acute, bilateral, lacrimal gland enlargement demonstrated with computed tomographic scanning of the orbits. Clinical findings and laboratory investigations confirmed the diagnosis of infectious mononucleosis. Treatment with systemic steroids was initiated. RESULTS: There was rapid resolution of the clinical findings with systemic steroids. CONCLUSION: Dacryoadenitis is an uncommon presentation of infectious mononucleosis and may overshadow the other manifestations of this disease. The diagnosis of infectious mononucleosis should be considered in patients with acute dacryoadenitis. Systemic steroids play an important role in rapidly resolving the dacryoadenitis. PMID- 8637687 TI - Prominent proptosis in childhood thyroid eye disease. AB - BACKGROUND: Orbital signs and symptoms occur in approximately one half of children with Graves disease, but the symptoms are usually minor and limited to the eyelids. Prominent proptosis is uncommon in children with this disorder. METHODS: Review of eight children with prominent proptosis associated with thyroid eye disease. Four patients were treated at the Children's Hospital of Philadelphia, the other four at the Columbia Presbyterian Medical Center. RESULTS: At initial presentation, children ranged in age from 3 to 16 years. There were five girls and three boys. Seven of eight children had hyperthyroidism at ophthalmic presentation. Four patients had restrictive myopathy, and all of the seven patients who underwent neuroimaging had extraocular muscle enlargement. Five patients were treated with lubrication. Two underwent orbital fat decompression. One patient had thyroid eye disease and myasthenia gravis. CONCLUSIONS: Proptosis in childhood thyroid eye disease usually is associated with a hyperthyroid state. The proptosis may be dramatic, but corneal exposure and restrictive myopathy are seen in only some of the patients. Neuroimaging shows enlarged extraocular muscles. Most children with this complication can be treated conservatively with topical lubrication, but orbital fat decompression may be considered in patients with more advanced conditions. PMID- 8637688 TI - "One-stitch" canalicular repair. A simplified approach for repair of canalicular laceration. AB - BACKGROUND: It has been widely believed that direct microsurgical re-anastomosis of the canalicular epithelium is necessary for satisfactory repair of canalicular lacerations. However, because repair is carried out in conjunction with placement of an indwelling silicone stent, this stent should keep the canalicular edges adequately approximate without the need for suturing. The authors report their results in repairing canalicular lacerations using a single, fine, horizontal, mattress suture to re-approximate the overlying pericanalicular orbicularis muscle and eliminate direct microsurgical re-anastomosis of the canalicular epithelium. METHODS: The authors retrospectively reviewed the charts of 67 patients who underwent repair of lacerated canaliculi with one-stitch re approximation of the overlying orbicularis muscle in conjunction with bicanalicular silicone tube intubation. Stents were left in place for 3 months postoperatively and then removed. Probing across the lacerated portion of the canaliculus was carried out at the time of stent removal to ensure patency. Dye disappearance testing with 2% fluorescein and irrigation through the canaliculus then was performed 6 weeks to 3 months after stent removal. RESULTS: Of the 67 patients, 59 were followed to stent removal. Probing with a 00 probe showed canalicular patency in all 59 patients. Irrigation resulted in reflux in two patients, indicating unrelated nasolacrimal duct obstruction. Of these 59 patients, 45 complied with scheduled follow-up 6 weeks to 3 months after stent removal. Dye disappearance testing using 2% fluorescein demonstrated delay in lacrimal outflow in 6 of the 45 patients. Only two patients had symptomatic epiphora, and in both patients there was an underlying nasolacrimal duct obstruction confirmed by irrigation. CONCLUSIONS: Simple re-approximation of the lacerated overlying soft tissue combined with bicanalicular silicone intubation proved highly successful in managing canalicular lacerations. Probing through the lacerated canaliculus demonstrated patency in 100% of the 59 patients followed to stent removal. Only 4% of patients had symptomatic epiphora postoperatively, and 13% demonstrated some delay in outflow with dye disappearance testing. This compares very favorably with previous reported series in which lacerated canaliculi were microsurgically re-anastomosed. PMID- 8637689 TI - Effect of light on the prevalence of simple anisocoria. AB - PURPOSE: Because simple anisocoria is believed to decrease in bright light, the authors determined the prevalence of simple anisocoria under different lighting conditions. METHODS: The authors measured the pupil size of 104 healthy subjects with infrared videography at four clinically accessible light levels: darkness; darkness with a hand-held light shining from below; room light; and room light with the hand light shining from below. RESULTS: Of the 104 subjects, 40 (38%) were men and 64 (62%) were women. The ages ranged from 12 to 71 years (mean, 36.3 +/- 12.5 years). The mean decrease in pupillary diameter from darkness to the brightest condition was 1.89 mm. Based on the traditional definition of a pupillary diameter difference of 0.4 mm or greater, the prevalence of simple anisocoria decreased from 18% in darkness to 8% in room light with the hand-held light shining from below. The prevalence of anisocoria varied considerably when other definitions were used. Repeated measures analysis of variance showed that pupillary area difference decreased with brighter conditions (P = 0.026). However, the ratio of the pupillary areas did not change with brighter conditions (P = 0.666). CONCLUSIONS: The prevalence of simple anisocoria decreases with brighter conditions based on pupillary diameter difference. However, this decrease is not apparent when anisocoria is expressed as pupillary area ratio. Those clinicians who measure pupils will find that simple anisocoria decreases in bright light. However, with gross observation where perception of an anisocoria may be related more to the ratio of the pupillary areas, simple anisocoria may not seem to change much with brighter conditions. PMID- 8637690 TI - Rapid growth of an optic nerve ganglioglioma in a patient with neurofibromatosis 1. AB - BACKGROUND: Optic nerve gangliogliomas are extremely rate tumors of the central nervous system composed of elements of glial and neuronal origin. The clinical and biologic behavior of gangliogliomas depends on the glial component. Pilocytic gangliogliomas generally have a low growth rate and good ultimate prognosis. No definitive relation has yet been established between gangliogliomas and neurofibromatosis type 1. METHODS: The authors describe the clinical, histologic, and immunohistochemical features of an optic nerve ganglioglioma with several atypical findings. A review of the literature is provided. RESULTS: An optic nerve glioma was diagnosed in a 16-year-old patient who had signs of neurofibromatosis type 1; the atypical clinical course involved a rapid progression of symptoms with a significant increase in tumor size. The tumor was removed surgically and found to be a ganglioglioma composed of pilocytic glial cells (immunoperoxidase-positive for glial fibrillary acid protein) and neurons (immunoperoxidase-positive for neuron specific enolase, synaptophysin, and neurofilament). A focal astrocytic area showed increased cellularity, several mitotic figures, and an elevated labeling index with Ki-67 immunoperoxidase staining. CONCLUSION: Optic nerve gangliogliomas are rare tumors that cannot be distinguished clinically from pilocytic astrocytomas. Although these tumors usually grow slowly, careful follow-up is advised. The atypical histologic features are considered to be a manifestation of rapid local growth rather than a harbinger of malignant behavior. The authors' findings suggest that gangliogliomas may be included in the diagnostic criteria for neurofibromatosis type 1. PMID- 8637691 TI - The use of bioerodible polymers and daunorubicin in glaucoma filtration surgery. AB - PURPOSE: Glaucoma filtering surgery usually fails from postoperative fibroblast proliferation, collagen deposition, and subsequent sclerostomy or bleb scarring. Daunorubicin inhibits fibroblast proliferation in vivo and in vitro. The authors studied the effect of a sustained subconjunctival release of daunorubicin by way of a bioerodible polymer on the success of glaucoma filtration surgery in a rabbit model. METHODS: Daunorubicin was incorporated into the copolymer by compression molding. The resultant implant measured 3.0 mm in diameter, 1.0 mm in thickness, and 8.0 mg in weight. A posterior lip sclerectomy was performed in both eyes of 23 rabbits by the same surgeon. One eye was randomized to receive the disc with daunorubicin, whereas the fellow eye received a blank disk as a control. The appropriate polymer disk then was placed on the scleral surface immediately posterior to the sclerostomy site and the conjunctiva was closed. Intraocular pressures and slit-lamp biomicroscopy were recorded preoperatively, then every other day after surgery for 31 days. Intraocular pressure, bleb survival, and complications were evaluated. RESULTS: The decrease in intraocular pressure from baseline was significantly greater in the daunorubicin eyes than in the control eyes during postoperative days 5 through 25. Bleb survival was significantly longer in the daunorubicin eyes than in the control eyes (bleb failure in 91% of control eyes versus 22% of treatment eyes by day 13). There were no statistically significant differences between the daunorubicin-treated and control eyes regarding corneal clouding, lens clarity, cataract formation, or conjunctival injection. However, conjunctival erosions occurred in four daunorubicin-treated eyes. Histopathologic examination was performed 2 weeks after surgery on the eyes of three randomly selected rabbits. CONCLUSION: Daunorubicin in a bioerodible delivery system may potentially be a useful adjunct to glaucoma filtering surgery and requires further evaluation. PMID- 8637692 TI - Clinical phenotype of juvenile-onset primary open-angle glaucoma linked to chromosome 1q. AB - PURPOSE: Recent reports have suggested that a gene responsible for juvenile-onset primary open-angle glaucoma exists on the long arm of chromosome 1 (1q). This report describes a previously unpublished family (UM:JG3) in which juvenile-onset glaucoma is segregating in an autosomal dominant manner. The clinical features in this family were compared with those seen in other pedigrees with this condition. Linkage analysis was performed to evaluate whether a glaucoma-causing gene in UM:JG3 is linked to genetic markers on chromosome 1q. METHODS: Affected family members, their siblings, children, and spouses were examined to identify the presence of glaucoma. Linkage studies were performed using short tandem repeat polymorphisms from chromosome 1q. Results of these studies were compared with those found for other families in which juvenile-onset primary open-angle glaucoma is linked genetically to the same chromosome 1q region. RESULTS: The UM:JG3 family includes 22 affected individuals over five generations, including 12 still living. The average age at diagnosis for living affected individuals was 26 years. An association between myopia and glaucoma was observed in this family, but the glaucoma was not associated with iris processes or other structural anomalies. The clinical course of disease and response to treatment were similar to other families with this disease. The disease phenotype in this family is linked to markers on chromosome 1q with a maximum lod score of 3.52 at a recombination fraction of 0.00 for marker D1S433. Haplotype analysis suggests the gene responsible for glaucoma in this family is located in an 8-cM region between markers D1S445 and D1S218. CONCLUSIONS: The glaucoma in UM:JG3 is linked to markers on chromosome 1q, with a candidate interval smaller than that in previous reports. In individuals with juvenile-onset open-angle glaucoma linked to chromosome 1q, the phenotype can range from mild ocular hypertension to blindness, resulting from marked elevations in intraocular pressure, with age at diagnosis ranging from 6 to 62 years. However, most affected individuals display a characteristic phenotype that includes onset in the first three decades of life, unusually high intraocular pressures, and the need for surgical therapy to prevent loss of vision. Whether differences in expression among families is due to allelic heterogeneity remains to be determined. PMID- 8637693 TI - Goldmann applanation tonometry using sterile disposable silicone tonometer shields. AB - PURPOSE: Placement of a sterile single-use cover over the tonometer tip may be a convenient and safe alternative method to repeated chemical disinfection. This study was undertaken to evaluate the accuracy and performance of Goldmann applanation tonometry using a sterile disposable silicone tonometer shield over the biprism tip. METHODS: The same investigator measured intraocular pressure in 120 eyes with and without the shield. The eyes were tested randomly first with either the uncovered or the covered tonometer to control for the possible effects of repeated tonometry influencing measurement differences. Readings were recorded independently in a masked fashion. RESULTS: The intraocular pressure measurements evaluated with the silicone shield caused an average overestimation of the true intraocular pressure of 1.9 +/- 1.9 mmHg (P = 0.0001). Covered and uncovered readings correlated well (r = 0.91, P = 0.0001). The sensitivity and specificity of the covered tonometry in detection intraocular pressures of 21 mmHg or higher (as measured without the shield) were 96.3% and 68.8%, respectively. Minor distortions of the normal-appearing fluorescein bands were seen in 5% of the tonometries, and the shield had to be replaced because of bubbles or wrinkles on the applanating surface in 3.3% of the measurements, which means that care must be taken when fitting the shield. CONCLUSIONS: Although covered and uncovered tonometry readings correlated well, an overall trend for a slightly increased measured intraocular pressure as a result of using the silicone shield over the Goldmann tonometer tip was found. The outstanding sensitivity of this procedure in detecting increased intraocular pressure suggests that it can be useful in screening for ocular hypertension and glaucoma. PMID- 8637694 TI - Study of lens autofluorescence by fluorophotometry in pregnant patients with diabetes. AB - PURPOSE: Lens autofluorescence originates from an accumulation of fluorescent substances such as the tryptophan-derived residues and glycosylated protein aggregations, which are associated with the process of cataractogenesis and lens aging. The purpose of this investigation is to determine whether pregnancy alters the typical constituents of the lens autofluorescence in patients with diabetes and, if so, to what degree this may occur. METHODS: Lens autofluorescence was studied with fluorophotometry in 127 eyes of 72 individuals: 23 control subjects, 6 healthy pregnant women, 21 patients with diabetes, and 44 pregnant patients with diabetes. RESULTS: The autofluorescence values were 311 +/- 130 ng/ml, 253 +/- 40 ng/ml, 378 +/- 110 ng/ml, and 562 +/- 164 Eq ng/ml (Eq ng/ml = Ng/ml equivalent fluorescein) in the four groups, respectively. The difference between the nonpregnant and pregnant patients with diabetes was significant (P < 0.001). CONCLUSION: These results suggest that there is an important deterioration in the metabolic state of the lens during gestation in patients with diabetes, as is the case for diabetic retinopathy. PMID- 8637695 TI - Risk factors for ciliochoroidal effusion after panretinal photocoagulation. AB - PURPOSE: To determine the incidence, duration, and risk factors for ciliochoroidal effusion after panretinal photocoagulation. METHODS: Thirty-nine consecutive patients with diabetic retinopathy underwent ultrasound biomicroscopy of both eyes to image the ciliochoroidal space immediately before and 1 day after unilateral argon-green panretinal photocoagulation. Imaging was repeated on days 3, 7, and 14 in patients in whom ciliochoroidal effusion developed. RESULTS: Low lying ciliochoroidal effusions were imaged in 23 (59%) of 39 eyes. Of 23 eyes, effusions resolved in 6 (26%), 12 (52%), and 5 (22%) eyes by 3, 7, and 14 days respectively. The number of laser applications (P = 0.02), shorter axial length (P = 0.01), and percentage of retinal surface area treated (P = 0.02) were associated with systemic hypertension, location of treatment, previous panretinal photocoagulation of cataract surgery, retinal surface area treated, and mean blood pressure before photocoagulation were not associated with effusion. All fellow, untreated eyes remained effusion-free. CONCLUSION: Ciliochoroidal effusion develops commonly after panretinal photocoagulation. Limiting the number of laser applications and the percentage of retinal surface area treated reduces the likelihood of this complication. Eyes with shorter axial lengths are at higher risk PMID- 8637696 TI - Breakdown of the blood-aqueous barrier after argon laser panretinal photocoagulation for proliferative diabetic retinopathy. AB - BACKGROUND: Breakdown of the blood-aqueous barrier (BAB) after panretinal photocoagulation (PRP) was measured with a laser flare photometer over a study period of 8 weeks. METHODS: Twenty-five eyes of 25 patients who had no previous photocoagulation and required such treatment for proliferative diabetic retinopathy (PDR) were included in the trial. They received 2000 burns (0.1 second exposure, 200 mu m spot) via a panfunduscope and 500 burns (0.1-second exposure, 500-mu m spot) with a Goldmann lens. Power levels were adjusted to produce a mild blanching of the retina. Only an argon green laser (514 nm) was used. Laser photometry was performed on both eyes at 3, 24, 48, 72, 96, and 168 hours and 8 weeks after laser treatment. RESULTS: Including all of the eyes treated, there was a significant increase in flare value of 3, 24, and 48 hours compared with baseline (Student's t test) but not at 72, 96, and 168 hours or at 8 weeks. Peak values occurred at 24 hours. When blue and brown irides were analyzed separately, there was a significant increase in flare for blue irides compared with baseline levels at 3 and 24 hours, whereas for brown irides the increased flare was sustained at 3, 24, 48, 72, and 96 hours (Student's paired t test). In addition, when the increase in flare value from baseline was compared between blue and brown irides (pooled Student's test), there was a sustained increase at 24, 48, 72, and 96 hours for brown irides compared with blue. Clinically significant uveitis, posterior synechiae, or peripheral anterior synechiae did not develop in any of the patients. CONCLUSIONS: Breakdown of the BAB may occur after PRP, particularly in more heavily pigmented irides. The time course of this suggests that the phenomenon is related directly to laser effects in the anterior segment, although other factors may contribute. PMID- 8637697 TI - The effect of solution temperature on the pain of peribulbar anesthesia. AB - BACKGROUND: Peribulbar anesthesia is the preferred technique of local anesthesia of the majority of cataract surgeons. Local anesthetic injections at other sites in the body have been shown to be less painful if the solution is warmed to body temperature before injection. To determine whether this is of advantage with peribulbar anesthesia, the authors performed a prospective, randomized, single, blind trial comparing local anesthetic injections that have been warmed to room temperature. METHODS: Forty consecutive patients undergoing routine cataract surgery were randomized into two groups of 20 patients and received local anesthetic at 20 degrees C or 37 degrees C. The peribulbar injection contained a solution of 5 ml 2% Lignocaine, 5 ml 0.5% bupivicaine (Marcaine), and 1550 IU hyaluronidase (HYlase) in a 10-ml syringe on a 25-mm, 25-gauge needle. Seven milliliters of the final solution was injected transcutaneously at the junction of the lateral and medial thirds of the lower lid. The patients graded the pain of the injection using a visual analogue scale. RESULTS: The pain sensation of local anesthesia is less when the solution is warmed to body temperature compared with room temperature (P = 0.026, using an unpaired Student's t test). CONCLUSION: Warming the local anesthetic used in peribulbar anesthesia to body temperature before injection reduces this iatrogenic pain significantly. PMID- 8637698 TI - Single-dose azithromycin in the treatment of trachoma. A randomized, controlled study. AB - PURPOSE: To compare the safety and efficacy of single oral-dose azithromycin with a 7-week topical tetracycline ointment course in the treatment of active trachoma. METHODS: A total of 64 patients with active trachoma were selected randomly to receive azithromycin (20 mg/kg) in a single dose or topical tetracycline eye ointment for 6 weeks. Clinical assessments were made before and at 4, 8, 12, and 24 weeks after treatment. Conjunctival scrapings were obtained before and after 24 weeks after treatment and fixed for Giemsa and direct immunofluorescence staining. RESULTS: Trachoma resolved in 17 (63.3%) patients who received azithromycin compared with 19 (65.4%) who were treated with tetracycline ointment. There were no significant differences in treatment effect or baseline characteristics between the treatment groups. Both treatments were well tolerated, and no adverse events were noted. CONCLUSION: Single-dose azithromycin is as effective as a 6-week course of topical tetracycline ointment in the treatment of active trachoma. The findings may help establish high compliance in treating trachoma and could contribute to the control of trachoma worldwide. PMID- 8637699 TI - The effects of California Proposition 187 on ophthalmology clinic utilization at an inner-city urban hospital. AB - PURPOSE: To determine the effect on ophthalmology clinic utilization at a major public inner-city hospital of California Proposition 187 and the debate surrounding its passage. Proposition 187 was a statewide referendum passed by 63% of the electorate in the November 1994 election that would restrict social services to undocumented immigrants and require providers to report them to immigration authorities. METHODS: The ophthalmology clinic volume at the Los Angeles County/ University of Southern California Medical Center was analyzed from October 1 to December 31, 1993 and 1994. RESULTS: New walk-in patients significantly decreased (P < 0.001) for a 2-month period around the election, but returned to baseline levels in December 1994. The new patient cancellation and no show rate was not affected. No change in return patient behavior was noted for general and specialty clinics. CONCLUSIONS: Proposition 187 may have caused a statistically significant decrease in new walk-ins to the ophthalmology clinics during a 2-month period surround the November 1994 election, but it had no measurable effect on other indicators of utilization. In addition, utilization rates returned to baseline after the implementation of Proposition 187 was stayed by the judicial system, and concern that providers would be required to report undocumented immigrants to authorities was alleviated. PMID- 8637700 TI - Automated lamellar keratoplasty. American Academy of Ophthalmology. AB - The Academy finds in its preliminary review of keratomileusis in situ and ALK reason for more rigorous evaluation of the safety, refractive accuracy, stability, and optical quality of these microkeratome-based procedures. To understand these issues more thoroughly, the ophthalmic community needs continued access to peer-review literature of sufficient quality to answer the questions posed earlier in this preliminary assessment. PMID- 8637701 TI - The TC21 oncoprotein interacts with the Ral guanosine nucleotide dissociation factor. AB - TC21 is a highly oncogenic member of the Ras superfamily of small GTP binding proteins. We have used the yeast two hybrid system to identify proteins that interact with an oncogenic form of the TC21 protein. cDNA clones encoding the carboxy-terminal region of the RalGDS protein were isolated from human B-cell and HeLa cDNA libraries. RalGDS is an exchange factor that stimulates GDP dissociation from Ral, another member of the Ras superfamily of proteins. The interaction between RalGDS to TC21 is direct and appears to be mediated by the effector domain of TC21 and the carboxy-terminal region of RalGDS. Moreover, RalGDS only binds to TC21 in its active, GTP-loaded configuration. These results suggest that RalGDS might be an effector molecule for TC21 and may participate in cross-talking between Ral and TC21 signalling pathways. PMID- 8637702 TI - Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. AB - Reversible growth arrest has been characterised for enhanced expression of a set of genes called gas (growth arrest specific). gas6 product (Gas6) is a secreted protein that was identified as the ligand for the tyrosine kinase receptor Axl. Here we report that Gas6 is able to induce cell cycle division entry in serum starved NIH3T3 cells. This mitogenic activity of Gas6 strictly correlates with its ability to interact with NIH3T3 endogenous Axl receptor since it can be abolished by soluble Axl extracellular domain and activates both Axl intrinsic kinase activity and the downstream MAPK pathway. Moreover when ectopic Axl overexpression is performed by microinjection in serum starved NIH3T3 cells, addition of a non mitogenic level of Gas6 induces selective entry into S phase in Axl overexpressing cells. Interestingly, Axl overexpression per se is not able to induce S phase entry. Finally we present evidences indicating that Gas6 is able to protect serum starved NIH3T3 cells from cell death by apoptosis as induced by complete growth factor depletion. The reported survival activity seems to be independent of Gas6 mitogenic activity, thus implicating a double and separable activity for Gas6 during growth arrest. PMID- 8637703 TI - Identification of tyrosine residues that are essential for transforming activity of the ret proto-oncogene with MEN2A or MEN2B mutation. AB - The c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A or 2B mutation can transform NIH3T3 cells with high efficiencies as a consequence of its constitutive activation. The MEN2A mutation induces ligand-independent homodimerization of the Ret protein on the cell surface while the MEN2B mutation appears to alter the catalytic activity without dimerization. In the present study, we investigated the role of tyrosine residues present in the kinase domain for the transforming activity of the mutant Ret proteins. Substitution of phenylalanine for tyrosine 905 (Y905F) that corresponds to tyrosine 416 of the Src protein abolished the transforming activity of Ret with the MEN2A mutation (MEN2A-Ret) but not with the MEN2B mutation (MEN2B-Ret). On the other hand, the transforming activity of MEN2B-Ret but not MEN2A-Ret significantly decreased by changing tyrosine 864 or 952 to phenylalanine. In addition, double mutations of these tyrosines (Y864/952F) completely abolished the activity of MEN2B-Ret. The Y905F and Y864/952F mutations resulted in severe impairment of the kinase activity of MEN2A-Ret and MEN2B-Ret, respectively. These results thus indicated that tyrosine residues essential for the transforming activity are different between MEN2A-Ret and MEN2B-Ret. PMID- 8637704 TI - Fusion of the EWS and CHOP genes in myxoid liposarcoma. AB - The translocation t(12;16)(q13;p11), which cytogenetically characterizes myxoid liposarcomas (MLS), results in a fusion of the CHOP gene in 12q13 and the FUS gene in 16p11, creating a chimeric FUS/CHOP gene. We have identified two cases of MLS with translocations giving rise to recombination between 12q13 and 22q12. The result was a fusion of the N-terminal part of the EWS gene in 22q12, involved in a number of mesenchymal tumor types, with the CHOP gene and the creation of an EWS/CHOP chimeric gene. The presence of the EWS/CHOP chimeric gene in MLS shows that (i) the N-terminal part of FUS may be replaced by the N-terminal part of EWS in a CHOP fusion oncoprotein (ii) the two N-terminal parts, when fused to certain transcription factors, have a common or very similar oncogenic potential and (iii) the tumorigenic process in MLS and the morphogenetically distinctly different EWS-associated tumor types may be related. PMID- 8637705 TI - SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals. AB - The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation. PMID- 8637706 TI - Ras-GTP levels are elevated in human NF1 peripheral nerve tumors. AB - Neurofibromin, the gene product of NF1, is a Ras GTPase Activating Protein. The absence of neurofibromin leads to increased levels of Ras-GTP, which contributes to the proliferation of NF1 neurogenic sarcoma cell lines. Whether this pathogenic mechanism is applicable to benign and malignant peripheral nerve tumours from NF1 and non NF1 patients is not known, due to lack of a tissue based assay. We have adapted a colorimetric enzymatic assay for determining levels of Ras bound guanine nucleotides in tissues. Ras-GTP levels were increased in NF1 neurogenic sarcomas (15 times) and benign NF1 neurofibromas (four times), compared to non NF1 schwannomas. Neurofibromin was not expressed in NF1 sarcomas, in support of its important negative Ras regulatory role in the pathogenesis of NF1 peripheral nerve tumors. PMID- 8637707 TI - HMGI-C rearrangements as the molecular basis for the majority of pulmonary chondroid hamartomas: a survey of 30 tumors. AB - Pulmonary chondroid hamartomas (PCH) are benign tumors of the lung characterized by a more or less high degree of mesenchymal metaplasia. In our series we investigated 30 PCH by a combination of cytogenetic and molecular methods. 18 tumors (60%) had cytogenetically detectable aberrations involving either 12q14-15 or 6p21 with a clear predominance of chromosomal abnormalities involving 12q14-15 (15 tumors). As in subgroups of pleomorphic adenomas of the salivary glands, leiomyomas of the uterus, and lipomas with 12q14-15 abnormalities the HMGI-C gene is frequently rearranged we tested PCH with either 12q14-15 abnormalities or normal karyotype by FISH and 3' RACE experiments for rearrangements of HMGI-C. Rearrangements were found in all cases with chromosomal 12q14-15 abnormalities and further six cases with an apparently normal karyotype. By the combination of cytogenetics with molecular techniques the percentage of cases with intragenic rearrangements of HMGI-C or rearrangements of its immediate surrounding was thus increased to 70% (21/30 cases). Considering all types of aberrations within this series 80% (24/30) of all PCH were aberrant. This is the first report on a combined molecular and cytogenetic analysis of a large series of pulmonary chondroid hamartomas indicating that rearrangements of HMGI-C, a member of the high mobility group protein gene family, are the leading molecular events in the genesis of PCH. PMID- 8637708 TI - Antisense RNA to the putative tumor suppressor gene BRCA1 transforms mouse fibroblasts. AB - Recently, BRCA1, a familial breast and ovarian cancer susceptible gene has been cloned and shown to be either lost or mutated in families with breast and ovarian cancers. BRCA1 has been postulated to encode a tumor suppressor, a protein that acts as a negative regulator of tumor growth. We have characterized the BRCA1 gene products by Western blot and immunoprecipitation analysis in mouse and tumor cells. Multiple BRCA1 polypeptides of approximately 225, 185, 160, 145, 100, 52 and 38 kD were identified in these cells. BRCA1 proteins were found to be localized mainly in the nucleus of normal Rat1 cells and human breast cancer cells. In order to understand the role of BRCA1 in cell transformation, we have established a stable NIH3T3 cell line expressing BRCA1 antisense RNA. The inhibition of expression of endogenous BRCA1 protein was detected in NIH3T3 transfectants by Western blot analysis. The antisense BRCA1 expressing NIH3T3 cells showed accelerated growth rate, anchorage independent growth and tumorigenicity in nude mice unlike the parental and sense transfectants. These results provide the first direct biological evidence for the possible function of BRCA1 as a tumor suppressor gene. PMID- 8637710 TI - Wilms' tumor 1 splice variants have opposite effects on the tumorigenicity of adenovirus-transformed baby-rat kidney cells. AB - The Wilms' Tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides which differ in the presence or absence of two stretches of amino acids: one of 17 residues (17aa) just N-terminal of the four zinc-fingers and of three residues (K-T-S) between zinc finger 3 and 4. In this study, four human cDNA constructs encoding the Wilms' tumor 1 splice variants were stably transfected into adenovirus-transformed baby rat kidney (Ad-BRK) cells. The in vivo produced WT1 proteins that lacked the KTS residues were found to bind efficiently to both the Egr-1 consensus sequence and the recently described WTE DNA sequence, as determined by electrophoretic mobility shift assays. Our studies show distinct effects of the different WT1 isoforms. Expression of the WT1 (-/+) protein, lacking the 17aa insert, strongly suppressed the tumorigenic phenotype of the Ad-BRK cells. Intriguingly, expression of the WT1 (-/-) protein, lacking both inserts, increased the tumor growth rate. In contrast to the growth in vivo, the growth rate of the transfectants in tissue culture is not influenced by any of the WT1 isoforms. However, the suppression of tumorigenicity appears to be correlated with a reduced ability of the cells to grow in serum-free medium. PMID- 8637709 TI - Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase. AB - The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32. PMID- 8637711 TI - IL-6-inducible complexes on an IL-6 response element of the junB promoter contain Stat3 and 36 kDa CRE-like site binding protein(s). AB - The junB gene is one of immediate-early genes whose expression are regulated by a variety of extracellular stimuli and play important roles in cellular responses to the given stimuli. Interleukin-6 (IL-6) activates the junB promoter through an IL-6 response element, JRE-IL6, that is composed of two cooperative DNA motifs, a low affinity Stat-binding site overlapping with an Ets-binding site (JEBS) and a cAMP responsive element (CRE)-like site. This element is a target for the Jak Stat signal transduction pathway. We showed that IL-6 induced novel complexes on JRE-IL6, termed JRE-IL6-BC1 and 2, which contained Stat3 but migrated more slowly than the complexes containing homo- or heterodimer of Stat3 and Stat1 in gel shift assays. These slow-migrating JRE-IL6-BCs appeared to contain CRE-like site binding proteins besides Stat3, since the formation of JRE-IL6-BCs required both the JEBS and CRE-like site of JRE-IL6 and oligonucleotides containing the CRE like site or somatostatin CRE efficiently competed with JRE-IL6 for making JRE IL6-BCs. The formation of the complexes correlated well with the responsiveness of JRE-IL6 to IL-6 signals. U.v.-cross linking study revealed that JRE-IL6 bound a 90 kDa protein, corresponding to Stat3, and a 36 kDa protein, most likely a CRE like site binding protein(s). Furthermore, we showed that the IL-6/interferon gamma (IFN gamma) response element in the IRF-1 promoter (IR/IRF-1), which contains a Stat-binding site and an adjacent CRE-like site, also makes IL-6 induced binding complexes similar to JRE-IL6-BCs. PMID- 8637712 TI - The induction of ret by Wnt-1 in PC12 cells is atypically dependent on continual Wnt-1 expression. AB - Wild type PC12 pheochromocytoma cells that had been infected with a Wnt-1 carrying virus and thus express Wnt-1 (PC12/Wnt-1) are known to acquire the same flat cell phenotype as that of spontaneously occurring PC12 flat cell variants except that the latter do not presently express Wnt-1. Flat cell variants of PC12 cells exhibit markedly altered morphology and gene expression. In order to assess the possibility that the spontaneously occurring flat cell variants could have been induced in wild type PC12 cells by previous transient expression of the cell's endogenous Wnt-1, we have isolated PC12/Wnt-1 cells expressing little or no Wnt-1. In spite of absent Wnt-1 expression, they retained their flat cell morphology, glutamate/aspartate transporter activity, increased neu mRNA levels and lack of both norepinephrine transporter activity and nerve growth factor induced differentiation. Thus, Wnt-1 expression is not required to maintain the flat cell phenotype. However, we identified one gene, ret, whose mRNA level in PC12 was not only increased by Wnt-1 expression, but whose increased mRNA level was also dependent on continual Wnt-1 expression. This finding suggests that the induction of ret by Wnt-1 can be used to elucidate the Wnt-induced signalling pathway in mammalian cells. PMID- 8637713 TI - Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF induced maturation of the murine pluripotent myeloid cell line 32Dcl3. AB - Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL 3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival. PMID- 8637714 TI - Sensitization of HER-2/neu-overexpressing non-small cell lung cancer cells to chemotherapeutic drugs by tyrosine kinase inhibitor emodin. AB - Overexpression of the HER-2/neu proto-oncogene which encodes tyrosine kinase receptor p185neu, has been observed frequently in many human cancers, including non-small cell lung cancer (NSCLC), and is correlated with poor patient survival in these cancers. In addition, HER-2/neu overexpression in NSCLC is known to induce chemoresistance. Recently, we demonstrated that emodin, a tyrosine kinase inhibitor, suppresses HER-2/neu tyrosine kinase activity in HER-2/neu overexpressing breast cancer cells and preferentially represses proliferation of these cells. The work described here was carried out to examine (1) whether the tyrosine kinase activity of p185neu is required for resistance to chemotherapeutic drugs of HER-2/neu-overexpressing NSCLC cells and (2) whether the tyrosine kinase inhibitor emodin can sensitize these cells to chemotherapeutic drugs. We found that emodin decreased tyrosine phosphorylation of HER-2/neu and preferentially suppressed proliferation of HER-2/neu overexpressing NSCLC cells. Furthermore, the combination of emodin with cisplatin, doxorubicin or etoposide (VP16) synergistically inhibited the proliferation of HER-2/neu-overexpressing lung cancer cells, whereas low doses of emodin, cisplatin, doxorubicin, or VP16 alone had only minimal antiproliferative effects on these cells. These results indicate that tyrosine kinase activity is required for the chemoresistant phenotype of HER-2/neu-overexpressing NSCLC cells and that tyrosine kinase inhibitors such as emodin can sensitize these cells to chemotherapeutic drugs. The results may have important implications in chemotherapy for HER-2/neu-overexpressing cancers. PMID- 8637715 TI - Overexpression of both RAR and RXR restores AP-1 repression in ovarian adenocarcinoma cells resistant to retinoic acid-dependent growth inhibition. AB - Retinoids including retinoic acid (RA) have been demonstrated to be effective growth inhibitors of a number of human cancer cell lines including ovarian adenocarcinoma cells. To begin to determine the mechanism of action by which RA inhibits the growth of ovarian carcinoma cells, we have examined AP-1 activity in two representative cell lines: CaOV-3 a RA-sensitive cell line and SK-OV-3 a RA resistant cell line. AP-1 activity was found to be inhibited by 50% upon RA treatment of the RA-sensitive cells while there was no change in AP-1 activity following RA treatment of the RA-resistant cells. Maximal inhibition of AP-1 activity could be achieved in the RA-resistant SK-OV-3 cells by overexpression of any one of the three retinoic acid receptor (RAR) subtypes in conjunction with retinoid X receptor (RXR) alpha. This inhibition of AP-1 activity was nearly comparable to that of the RA-sensitive cells. A similar change in AP-1 complex formation in vitro has also been observed. These results suggest that one mechanism by which RA inhibits growth of RA-sensitive ovarian carcinoma cells is by repressing AP-1 activity. Moreover, in the RA-resistant cells the RAR/RXR signalling pathway leading to inhibition of AP-1 activity is impaired however overexpression of one of the RAR subtypes along with RXR alpha is sufficient to restore this pathway. PMID- 8637716 TI - The human IL-11 receptor requires gp130 for signalling: demonstration by molecular cloning of the receptor. AB - We describe the molecular cloning of a cDNA for the alpha chain of the human IL 11 receptor (IL-11R alpha) and demonstrate the requirement of either the human or mouse gp130 molecule for signalling. cDNA clones encoding IL-11R alpha were isolated from a bone marrow cDNA library using a fragment from the murine IL-11R alpha as a probe. The human receptor was predicted to consist of 422 amino acids and was found to share 84% identity with the murine protein. In the extra cellular region it exhibited a single hemopoietin domain with conserved cysteine residues and WSTWS motif. The transmembrane region was followed by a short cytoplasmic tail which did not contain a tyrosine kinase domain. Interaction of the human IL-11R alpha with murine gp130 was demonstrated: expression of the human IL-11R alpha in murine M1 cells which constitutively express murine gp130 (and murine LIF receptor), resulted in the generation of specific high-affinity binding sites for IL-11 (Kd = 250 pM). In addition, expression of the human IL 11R alpha in these cells permitted the induction of macrophage differentiation in response to IL-11. These results suggested that the human IL-11R alpha chain was able to form a functional receptor complex in association with murine gp130. The requirement of gp130 for signalling was confirmed by expression of the human IL 11R alpha in Ba/F3 cells. BaF3 cells that expressed the human IL-11R alpha alone showed binding of radiolabelled IL-11 but no proliferative response. Introduction of human gp130 into these cells resulted in high-affinity IL-11 binding sites and IL-11 dependent cellular proliferation. Thus these results demonstrated the absolute requirement of gp130 for signalling. PMID- 8637717 TI - Characterization of p21Cip1/Waf1 peptide domains required for cyclin E/Cdk2 and PCNA interaction. AB - The cyclin-dependent kinase inhibitor p21Cip1/Waf1 is responsible for the p53 dependent growth arrest of cells in G1 phase following DNA damage. In the present study we investigated regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. To perform these studies we synthesized a series of overlapping peptides spanning the entire p21 sequence and used them in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40, antagonized p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding was, however, lost in a similar peptide lacking amino acids 15-20, or in a peptide in which cysteine-18 was substituted for a serine. These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonized p21 activity, but this peptide did not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides we tested inhibited cyclin E/Cdk2. We also found that a peptide spanning amino acids 21-45 bound recombinant p21 in ELISA assays, and additional studies revealed a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139 164 was found to bind PCNA in a filter binding assay and this peptide suppressed recombinant p21-PCNA interaction. Conformational analysis revealed that peptides spanning amino acids 21-45 and 139-164 tended towards an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, our results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. Our results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21. Continued exploration of these domains could prove useful in assessing p21-mimetic strategies for cancer treatment. PMID- 8637718 TI - Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1. AB - The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf 1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf 1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14 3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf 1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1. PMID- 8637719 TI - Overexpression of Mxi1 inhibits the induction of the human ornithine decarboxylase gene by the Myc/Max protein complex. AB - We have previously shown that the Myc/Max protein complex plays a role in the growth-associated expression of the human ornithine decarboxylase gene. Mxi1 and Mad, novel Max-associated proteins have been identified and shown to form heterodimers with Max which bind efficiently to the Myc/Max consensus recognition sequence, CACGTG, in vitro. However, formation of Max/Mxi1 or Max/Mad heterodimers results in a reduction in Myc/Max dependent transcriptional activation of reporter plasmid constructs containing the consensus element. In light of the evidence that ODC is transcriptionally regulated in vitro and in vivo by the Myc/Max protein complex and the potential role of Mxi1 and Mad as antagonists of Myc transactivation activity, we set out to determine if one of these Max associated proteins, Mxi1, could affect the regulation of ODC expression by Myc/Max and if this regulation was correlated to growth status. Our results show that overexpression of Mxi1 does in fact inhibit ODC gene expression in a dose-dependent manner both in vivo and in vitro. In addition, evidence is presented which shows that levels of Mxi1 are up-regulated during long term quiescence and down-regulated following growth stimulation by serum. These results suggest that alterations in the levels of Max-associated proteins such as Mxi1 can modulate critical levels of functional Myc/Max protein complexes. This can alter transcriptional transactivation of Myc-regulated targets and as a consequence affect levels of genes essential for initiation and/or maintenance of growth. PMID- 8637720 TI - Analysis of chimeric Gag-Arg/Abl molecules indicates a distinct negative regulatory role for the Arg C-terminal domain. AB - Arg and c-Abl represent the mammalian member of the Abelson family of nonreceptor protein tyrosine kinases. The two proteins are composed of SH2, SH3, kinase and C terminal domains. To examine Arg structure-function relationships we analysed a Gag-Arg fusion protein, analogous to the oncogenic Gag-Abl fusion protein of Abelson Murine Leukaemia Virus and found that in contrast to Gag-Abl, it lacked transforming activity. Three observations indicated that the difference in the transforming activity was mediated by the distinct Arg and Abl C-terminal domains. (1) The analysis of chimeric Gag-Arg/Abl molecules revealed that the Arg C-terminal domain completely abrogated Gag-Abl transforming activity and that the Abl C-terminus conferred transforming activity to Gag-Arg. Substitutions of SH2 and kinase domains did not affect activity. (2) Alterations in the Arg C-terminus were observed in spontaneous foci that developed in transfections of two nontransforming chimera. (3) An engineered Gag-Arg molecule containing a truncation of almost the entire C-terminal domain, including three SH3 domain binding sites, was oncogenic, whereas a slightly smaller truncation that deleted two of three SH3 domain-binding sites, lacked transforming activity. These observations indicate that the C-terminal domain regulates Arg biological activity in a manner distinct from c-Abl and suggest that this effect may be mediated in part by SH3 domain-binding sites. PMID- 8637721 TI - Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK. AB - JNK/SAPKs are identified as new members of the MAPK family; they phosphorylate c Jun protein in response to several cellular stimuli including ultraviolet irradiation, TNF and osmotic shock. We have identified a protein kinase, MUK, as an activator of the JNK-pathway, whose kinase domain shows significant homology to MAPKKK-related proteins such as c-Raf and MEKK. The over-expression of MUK or MEK kinase (MEKK) in NIH3T3 or COS1 cells results in the activation of JNK1 and the accumulation of a hyper-phosphorylated form of c-Jun. While MEKK also activates the ERK pathway, MUK is a rather selective activator of the JNK pathway. On the other hand, c-Raf activates the JNK pathway only slightly despite its remarkable ability to activate the ERK pathway. Even though we originally identified MUK as a MAPKKK-related protein kinase, a greater similarity to mixed lineage kinase (MLK) is found not only in the catalytic domain but also in the 'leucine-zipper'-like motifs located at the C-terminal side of the catalytic domain. The structural divergence between MUK and MEKK reveals the multiplicity of signaling pathways that activate JNK/SAPKs. PMID- 8637722 TI - Gene-specific DNA repair and steady state transcription of the MDR1 gene in human tumor cell lines. AB - We have explored the relationship between DNA repair and transcription in vivo. A gene-specific repair assay has been employed to study removal of ultraviolet light-induced cyclobutane pyrimidine dimers in the MDR1 gene at different levels of MDR1 mRNA expression. The parental human adenocarcinoma cell line, KB-3-1, has very low levels of MDR1 mRNA expression, but its multidrug resistant derivatives KB-8-5 and KB-C1 have 42-fold and 3800-fold increases in MDR1 mRNA expression, respectively. In the KB-3-1 cell line that has a low level of MDR1 mRNA expression, we find a low level of MDR1 gene-specific repair and inefficient repair of the transcribed strand of the gene. In the KB-8-5 cell line that has a modest increase in MDR1 mRNA expression, we find only a minor increase in dimer repair in the MDR1 gene. Here, the repair in the transcribed strand is not significantly higher than that in the KB-3-1 cell line. However, in the KB-C1 derivative, where there is a 3800-fold increase in the level of MDR1 mRNA expression, we find a substantial increase in the level of dimer repair in the MDR1 gene. In addition, the MDR1 transcribed strand repair is markedly more efficient than the repair in the nontranscribed strand. Our data suggest that the rate of transcription in the MDR1 gene must be substantially increased before there is any measurable effect on DNA repair. Repair in the housekeeping gene, dihydrofolate reductase (DHFR), was similar in all three tumor cell lines. Repair in its transcribed strand was markedly lower than previously reported in normal human fibroblasts. We suspect that these human HeLa-derived tumor cell lines have deficient gene-specific DNA repair. This may be an important aspect of their malignant phenotype. PMID- 8637723 TI - A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction. AB - A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120- >glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression. PMID- 8637724 TI - Allelic losses on chromosome 4 suggest the existence of a candidate tumor suppressor gene region of about 0.6 cM in gamma-radiation-induced mouse primary thymic lymphomas. AB - Mouse chromosome 4 was investigated to assess the involvement of tumor suppressor genes in primary thymic lymphomas induced by gamma-irradiation. PCR analysis using microsatellite DNA polymorphic markers was performed in F1 animals generated from a cross between the strains C57BL/6J and RF/J. Microsatellite markers were selected with focus on chromosome 4 around the region containing the interferon alpha gene cluster (D4Mit17, D4Wsm1, D4Mit9, and D4Mit205 and a more distal region (D4Mit12, D4Mit54, and D4Mit13). Allelic losses were detected in 21/47 (44.7%) gamma-radiation-induced thymic lymphomas. Analysis of markers located on six other chromosomes as well as on the proximal region of chromosome 4 illustrates the specificity of the occurrences of LOH appearing on the former markers. This analysis clearly suggests the existence of a candidate tumor suppressor gene region on mouse chromosome 4 of about 0.6 cM between the markers D4Wsm1 and D4Mit9 (TLSR1, Thymic Lymphoma Suppressor Region 1). In addition, another more distal region centered at the marker D4Mit54 could be also exist (TLSR2). In most tumors, allelic losses on these chromosome regions involved the paternal RF/J allele (19 of 20). PMID- 8637725 TI - BCR gene recombines with genomically distinct sites on band 11Q13 in complex BCR ABL translocations of chronic myeloid leukemia. AB - We have analysed a cloned 11q13/3'BCR junction fragment, one recombination product of a complex t(9;11;22) translocation in a patient with chronic myeloid leukemia. 3'M-Bcr recombined with chromosome band 11q13 at a specific point between two Alu elements lying in opposite orientation. We present new molecular data comparing the genomic location of the 11q13 breakpoint in our patient with that of one other recently reported to lie within the GSTP1 gene. This is the first time that specific breakpoint sites within a chromosomal region highly involved in complex Ph translocations have been relatively mapped. These early results argue against a precise site in 11q13 with which M-Bcr preferentially recombines and favour instead a larger recombination-prone domain. Both of the 11q13 breakpoint regions show Alu repeat elements in close proximity to the site of recombination. PMID- 8637726 TI - Absence of germline p53 mutations in familial lymphoma. AB - p53, a tumor suppressor gene, is frequently mutated in sporadic human cancer, and inherited mutations in p53 predispose to the early onset of cancer. p53 mutations occur frequently in sporadic lymphoma, and, in mice deficient for p53, lymphoma is the most common type of malignancy. Families with an increased incidence of lymphoma have been described, suggesting an inherited predisposition to lymphoma in these circumstances. To determine whether the predisposition to lymphoma in these families results from germline mutations in p53, we analysed exons 4-11 of the p53 gene in 35 individuals from 19 lymphoma-prone kindreds. We found no germline p53 mutations in any of the individuals tested. However, p53 expression assessed by immunohistochemistry, which suggests mutation, was observed in 35% of the tumor samples from the familial Hodgkin's disease cases and in 13% of the familial non-Hodgkin's lymphoma cases. These results suggest that p53 mutations do not play a critical role in heritable susceptibility to lymphoma. p53 may act by different, non-mutation related mechanisms in this setting, or be involved in late events in the pathogenesis of these tumors. PMID- 8637727 TI - Induction of murine O6-alkylguanine-DNA-alkyltransferase in response to ionising radiation is p53 gene dose dependent. AB - Expression of both the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) and the p53 tumour suppressor protein are inducible by a number of DNA damaging agents. It is probable that DNA strand breaks are the common inducing signals. This similarity, and the function of p53 as a transcription factor lead us to reason that p53 might be involved in ATase inducibility. We now report that the induction of ATase activity in mouse tissues following gamma-radiation is p53 gene dose dependent. While the extent and kinetics of induction in p53 wildtype mice are consistent with previous reports (a 2-3-fold peak increase at 36 h), no induction is observed in p53 null animals. Importantly the heterozygous mice show an intermediate response but the same kinetics. The basal levels of expression in all tissues examined are unaffected by p53 status. These data represent the first report of a discrete DNA repair function being p53 regulated in vivo and their potential clinical implications are discussed. PMID- 8637728 TI - Genetic alterations on chromosomes 3 and 9 of esophageal cancer tissues from China. AB - In previous studies, we had demonstrated that allelic losses in esophageal cancer (EC) tissues are frequently involved in chromosomes 3 and 9 and that EC patients and their blood relatives have low capacity to repair damaged DNA and showed genetic instability. To better define the deleted chromosomal loci and understand the genetic instability in EC tissues, we selected 12 microsatellite markers (D3S1232, D3S1238, D3S1289, D3S1480, D3S647, D3S966, D3S1317, D3S659, D9S156, D9S171, D9S176 and GSN) to examine 36 paired EC tissues for loss of heterozygosity (LOH) and microsatellite instability (MIN) on chromosomes 3 and 9. The frequent LOH was found at D9S156(9p21), D3S647(3p23) and D3S1480(3p14.2), implying the possible existence of tumor suppressor genes near the deleted loci. Higher LOH incidence at D9S156 (9/18) and D3S1480 (8/19) was observed in EC tissues from Beijing, a low EC area. More frequent LOH at D3S647 (6/14) was found in EC tissues from Yangquan, a high EC area. This geographic difference of LOH occurrence was indicative of genetic heterogeneity in the etiology of EC. 24 of 36 (66.7%) EC tissues showed MIN at one or more chromosomal loci. The putative EC suppressor genes on chromosomes 3 and 9 and the molecular basis of the genetic instability associated with EC remain to be elucidated. PMID- 8637730 TI - Opening remarks at the conference on air pollution impacts on body organs and systems. PMID- 8637729 TI - Otology through the ages. AB - Recounted are some, not all, of the most significant contributions to otology. The history of otology has, for the most part, followed the history of medicine: Little was known of otology by the ancient physicians, but their work must be reviewed to appreciate the progress that has been made. Most of the otology produced during the Middle Ages was limited to those structures that were readily accessible; otologic surgery was confined to trauma and removal of foreign bodies from the external auditory canals. Examination of the ear began after studies of the ear by Italian anatomists during the seventeenth century. It was not until the end of the eighteenth century that magnification was used so that the minute anatomic details could be observed. The clinical specialty of otology started in France (1850s), emerged as a scientific specialty in England, and received explosive progress from the German-speaking countries at the end of the nineteenth century. Otology has a remarkable background. PMID- 8637731 TI - The National Association of Physicians for the Environment: an introduction. PMID- 8637732 TI - Effects of air pollution on the upper aerodigestive tract. AB - The main route of contamination of the human body with airborne pollutants is through the upper air and food passages. Because of the delicate balance of the mucous membranes and special sensory organs of these passages with respect to mucociliary activity, local and recruited immune responses, rapid uptake of chemicals, and carcinogenic potential, the ingestion or inhalation of pollutants in the air can be harmful to these internal body barriers. The particular target organs for air pollution effects on the upper aerodigestive tract include the mucosa, olfactory epithelium, auditory receptor cells, glottic epithelium, and adjacent neural and muscular tissues. Hearing loss caused by noise exposure may be aggravated by the concomitant inhalation of solvents. The strongest evidence for the carcinogenic effect of occupational inhalants in the nasal cavity and paranasal sinuses is seen with exposure to hardwood dust, tobacco smoke, furniture making, and leather tanning. With the exception of tobacco smoke, which produces squamous cell carcinomas, the majority of the occupationally related cancers are adenocarcinomas, usually of the intestinal variety. Tobacco smoke, passive or active, may lead to end-artery obliteration at the level of the otic end organ, causing a progressive sensorineural hearing loss. Further environmental research in the upper aerodigestive tract should aim at developing biologic markers to determine early, premalignant tissue changes; identifying the effects of chronic, low-dose toxic exposure on mucous membranes and neurosensory organs; providing field-tested tools for the standardized screening of large at risk populations. PMID- 8637733 TI - Blood and air pollution: state of knowledge and research needs. AB - The ready access to blood (plasma and formed cellular elements) makes it unusually susceptible to the deleterious effects of pollutants whose origins may be in the air. The red blood cells' hemoglobin may be rendered useless for oxygen transport by combination with carbon monoxide or conversion to methemoglobin or sulfhemoglobin. Lead and arsine can damage the erythrocytes' membranes, resulting in anemia. Metabolites of benzene and other volatile polycyclic hydrocarbons are implicated in the causation of leukemias. The extensive use of pesticides and herbicides may be associated with the development of Hodgkin's disease, non Hodgkin's lymphoma, and aplastic anemia. The carcinogenic risks from ionizing radiation, especially for leukemia, are well known. More information is needed concerning the epidemiology of environmental factors responsible for damage to blood. Enhanced knowledge about the molecular biology of toxins' effects on the hematopoietic system and improved detection and prevention technologies are needed to answer environmentally related health questions. PMID- 8637734 TI - Cardiovascular effects of environmental chemicals. AB - This article presents recent data on several environmental toxins: lead, carbon disulfide, asbestos, arsenic, ozone, cadmium, vinyl chloride, fluorocarbons, freon, and pesticides. These environmental toxins produce both hypertension and cardiac arrhythmias in most studies, and they are not necessarily related to primary lung disease and secondary heart disease. The possible mechanisms that could cause the cardiovascular diseases include (1) damage to the endothelial barrier in the vascular system, (2) activation of leukocytes and platelets, (3) initiation of plaque formation, (4) stimulation of the inflammatory response, (5) kidney-related hypertension, and (6) direct damage to cardiac and blood vessel tissue. Recommendations are that more animal, human cultured cell, and epidemiologic studies should be conducted on the environmental toxins identified in this article. PMID- 8637735 TI - Lung (agricultural/rural). AB - Industrialization of farming, animal raising, and forestry has added chemical and mechanical hazards that need to be recognized and prevented. Lung disease among farmworkers can result from a wide variety of hazardous exposures, which include organic dusts, allergens, chemicals, toxic gases, and infectious agents. In addition to nonspecific symptoms of mucous membrane irritation, farmworkers can experience occupational asthma or bronchitis, organic dust toxic syndrome, hypersensitivity pneumonitis, silo filler's disease (toxic hemorrhagic pulmonary edema), and neuromuscular respiratory failure. At risk are farmworkers and those involved in the processing, stocking, transportation, handling, and inspection of unprocessed agricultural, animal, and forestry products; veterinarians; gardeners; game, river, and forest keepers; persons involved in building, supplying, or servicing farm operations; and residents of rural communities. Worker education on the risks of environmental exposures, adherence to safety regulations, and increased knowledge of the cause and prevention of environmental diseases will reduce their prevalence and their adverse human and animal health and socioeconomic effects. PMID- 8637736 TI - Skin effects of air pollution. AB - The skin is a target organ for pollution and also allows the penetration of exogenous agents into the body. About 700,000 new cases of skin cancer were diagnosed in 1993, and 9100 people died of cancer; 76% of the deaths were due to melanoma. Skin cancers are most closely associated with exposure to UVB (290 to 320 nm) irradiation. For every 1% decrease in ozone there is a 2% increase in UVB irradiance, and therefore a 2% increase in skin cancer is predicted. Therefore the atmospheric pollution by ozone-depleting chemicals is a major concern to dermatologists. In addition to being a target organ and site of neoplasms and contact allergens, the skin is the site of significant absorption of environmental pollutants. In the case of chloroform, the percutaneous absorption is equivalent to the respiratory uptake, emphasizing how important it is to recognize skin absorption in toxicologic exposures. PMID- 8637737 TI - Bone: target and source of environmental pollutant exposure. AB - The skeleton can play a unique role in modulating and responding to environmental air pollutants. Bone and its metabolic activities may be immediate targets for particular agents, leading to skeletal disease. However, bone is also an important storage site in the body. Chemical pollutants may be actively absorbed into bone mineral and released later during the normal process of bone remodeling. Critical periods when bone remodeling is enhanced, such as pregnancy, lactation, menopause, immobilization, and exposure to microgravity, may be important to recognize because of the potential for enhanced release of previously stored chemical agents. Lead has been identified as a "criteria air pollutant" by the 1970 Clean Air Act because of its ubiquity in the environment and its effect on a multiplicity of health outcomes. It is used in this article as an example of a substance that can have both a direct effect on bone and a latent effect on other organ systems through release from bone long after the initial exposure. PMID- 8637738 TI - Legacy of lead exposure: consequences for the central nervous system. AB - The central nervous system appears to be the primary target organ for lead. Children in particular remain at risk for the central nervous system effects of lead, not only in many American cities but also in numerous developing countries where the use of leaded gasoline continues. Those same children, moreover, may sustain many of the risk factors, such as low socioeconomic status and calcium and iron insufficiencies, known to exacerbate the manifestations of lead exposure, including its central nervous system effects. Physiologic conditions associated with bone resorption, including pregnancy, lactation, and aging can also potentiate the central nervous system effects of lead and enhance exposure of adults. Questions regarding safe and efficacious diagnosis and treatment of elevated lead burden, particularly as they relate to central nervous system-based effects of lead have recently been raised. Clearly, greater education of and awareness in the medical community are needed for recognition of problems accruing from lead exposure. PMID- 8637739 TI - Psychiatric aspects of air pollution. AB - Psychological and toxic effects of air pollution can lead to psychiatric symptoms, including anxiety and changes in mood, cognition, and behavior. Increased levels of some air pollutants are accompanied by an increase in psychiatric admissions and emergency calls and, in some studies, by changes in behavior and a reduction in psychological well-being. Numerous toxic pollutants interfere with the development and adult functioning of the nervous system. Manifestations are often insidious or delayed, but they can provide a more sensitive indicator of toxic effects than cancer rates or mortality data. Other medical effects of air pollution, such as asthma, can indirectly affect psychological health. The sick building syndrome and multiple chemical sensitivity are conditions with toxicologic and psychiatric aspects. Psychosocial stress can cause symptoms similar to those of organic mental disorders. Reactions to stress depend on cultural, individual, and situational variables. We must understand these factors to be able to alleviate and prevent the consequences of environmental trauma. Expanded research is recommended in three main areas: (1) how people perceive and cope with environmental health risks, (2) the effects of air pollution on behavior and neuropsychological functioning, and (3) neurotoxicologic evaluation of air pollutants with both behavioral and in vitro studies. PMID- 8637740 TI - Airborne pollutants and the immune system. AB - The effects of airborne pollutants on the immune system have been most widely studied in the respiratory tract. Entry may occur as a volatile gas (ozone, benzene), as liquid droplets (sulfuric acid, nitrogen dioxide), or as particulate matter (diesel exhaust, aromatic hydrocarbons). The subsequent interaction with the immune system may result in local and systemic responses, and studies have shown examples of disease occurring from both overactive immune responses and immunosuppression. For the most part, airborne pollutants (small molecular weight chemicals) have to be coupled with other substances (proteins or conjugates) before they can be recognized by the immune system and exert their effects. Fortunately, this encounter rarely causes immunologically mediated human disorders. The following briefly reviews some of the disorders that may occur. Immunologically nonspecific inflammation of the lung can occur after inhalation of ozone in anyone given sufficient dose and time of exposure. Immunologically specific cell-mediated (T lymphocyte) reactions appear to predominate in chronic beryllium disease, which results in a granulomatous form of lung disease. Beryllium alone does not appear to be antigenic but requires chemical linkage with a larger molecule. Mercury-induced autoimmune disease (immune system attacks self-antigens) affecting kidneys and lungs has been demonstrated in animal models (changes similar to those seen in people with Goodpasture's syndrome). Immunosuppression can be demonstrated after exposure to polycyclic aromatic hydrocarbons (2,3,7,8-tetrachlorodibenzo-p-dioxin). Hypersensitivity (or allergic) reactions can occur after exposure to toluene diisocyanate (occupational asthma). In summary, airborne pollutants may cause a wide spectrum of immunologically mediated disorders. There is clearly an underlying genetic basis for the susceptibility to immunologic disease resulting from exposure to pollutants, but knowledge in this area is rudimentary at present. Studies have been impeded by lack of appropriate in vitro models, as well as difficulties in identifying the biologically active substance. PMID- 8637741 TI - Air pollution and its effect on the upper respiratory tract and on allergic rhinosinusitis. AB - The nose is the first organ system encountered by inhaled air and its associated pollutants. Pollutants are deposited during inspiration through the nose. They accumulate on mucus and are absorbed in the nasal mucosa, resulting in a number of deleterious effects on the body. Irritation of the nose and sinus from these pollutants, resulting from direct contact with the nasal mucosa, leads to inflammation, edema, swelling, and blocked sinuses. The result is acute and chronic sinusitis. Absorption of these chemicals into the body produces systemic effects. Their effect on the immune system, although subtle, leads to dramatic changes in the allergic diathesis. The T suppressor cell is the most sensitive cell of the immune system and the first to be affected by exposure to chemical pollutants. Diminution of the suppressor activity and the relative increase in helper activity in turn lead to increased immunoglobulin production and the manifestation of allergy symptoms. The underlying biochemical reaction is caused by the effects of pollutants on the T suppressor cell. Patients with existing allergies become brittle and difficult to treat with the exacerbation of the allergic diathesis. Removal of these chemical pollutants from the body as quickly as possible is essential for effective treatment of this problem. Dietary antioxidants help reduce the oxidizing effect of the pollutants and act as conjugators to remove the pollutants from the body. PMID- 8637742 TI - Effect of air pollution in asthma and respiratory allergy. AB - Epidemiologic and controlled exposure studies of human volunteers have shown that exposure to a variety of pollutants induces asthma exacerbations. Interestingly, in the case of ozone, recent evidence suggests that this pollutant acts to enhance the effect of inhaled allergen in persons with asthma. These and other data also suggest that pollutants may influence lung function in persons with asthma by increasing airway inflammation. The interaction of pollutants and inhaled allergens and the effect of pollutant exposure on baseline airway inflammation may be a key mechanism of pollutant-induced exacerbation of asthma. Further study of this interaction, as well as interactions of multiple pollutants, will be crucial for rational development of intervention and regulatory strategies. PMID- 8637743 TI - Environment and the genitourinary tract. AB - A variety of environmental factors have been associated with conditions that affect the genitourinary system. Chronic lead exposure and halogenated hydrocarbons have been found to affect sperm production and fertility. Heavy metals such as mercury and cadmium have been associated with renal tubular toxicity and kidney failure. Aromatic amines, used in a number of industrial processes and most commonly found in the dye industry, have been associated with development of bladder cancer. The same substances that are metabolites in cigarette smoke have been found to explain the strong association between bladder cancer and cigarette smoking. Chlorination and fluoridation have recently been associated with bladder cancer. In each of these associations, host factors may contribute to the toxicity of these substances either by detoxifying them or by converting them to more active agents. Education and regulation are needed to decrease the environmental risk they create. PMID- 8637745 TI - Effects of air pollution on African-American and other minority populations. PMID- 8637744 TI - Air pollution in the United States. PMID- 8637746 TI - Hispanic environmental health: ambient and indoor air pollution. AB - Air pollution has serious deleterious effects on health and is a public health matter of the utmost importance. The National Coalition of Hispanic Health and Human Services Organizations (COSSMHO) believes that reducing exposure to air pollution is a priority issue for the Hispanic community because 80% of Hispanics (compared with 65% of non-Hispanic blacks and 57% of non-Hispanic whites) live in nonattainment areas--areas that fail to meet the Environmental Protection Agency ambient air quality standard. Although Hispanics in general live as long as or longer than non-Hispanic whites, what morbidity data are available reveal that the quality of that life is severely impaired by a variety of chronic conditions, such as asthma. This makes environmental health a pressing matter for Hispanic communities, particularly in the area of air pollution. Action items are included. PMID- 8637747 TI - Air pollution hazards to children. PMID- 8637748 TI - Impact of air pollution on women's health. Society for the Advancement of Women's Health Research. AB - Despite the growing interest in exploring environmental impacts on the health of men and women, more attention needs to focus on assessing the effect of air pollution on women's health. Current research efforts have observed possible correlations between exposures to environmental toxins and the development of disease and illness, including cancer, reproductive dysfunction, and immunologic and neurologic impairment. However, this research has not gone far enough for us to understand and explain the harmful effect of these toxins on women's health and the unique way women respond to toxic exposure. To address these gaps in knowledge, the Society for the Advancement of Women's Health Research recommends the following: an aggressive research commitment to identify and understand the unique way environmental toxins in the air interact in women; a commitment to research to understand the impact of chemical exposures in the workplace on the health of women; and an interagency review to evaluate the federal risk assessment policy and its impact on women's health. PMID- 8637749 TI - Importance of radon as a threat to public health. AB - Since the discovery of x rays, the public has shown increasing concern about exposure to radiation. In the mid-1980s, with the dissemination of information about the ubiquitous nature of radon, this concern about radiation exposure has taken on a new perspective. As the general public realizes that exposure to radiation is an unavoidable part of life, questions arise as to how much exposure is acceptable when weighed against the costs of reducing the exposure. Because limited resources are available to protect the public's health and the environment, these resources need to be used wisely. The cost-effectiveness of the various options to lessen the potential adverse health effects from radon must be considered. PMID- 8637750 TI - Aerospace air pollution issues. AB - Practitioners of aerospace medicine are mindful of the environmental effects, particularly air pollution, caused by aviation and spaceflight operations. To an aerospace medicine specialist, the environment includes not only the air, water, and soil of the earth, but also the cabin milieu of aircraft and space vehicles where crews must work, sleep, and in some cases, live. Consequently, this article will address the following areas of concern: cabin air quality of aircraft, cabin air quality of space vehicles, noise, air pollution, and aerial spraying. PMID- 8637751 TI - Direct and indirect exposure to air pollution. AB - Hazardous substances that originally are discharged as air pollutants may find their pathway to human exposure through multiple routes, including ingestion and dermal contact, as well as direct inhalation. The mechanisms for modeling and understanding the fate of air pollutants through atmospheric transport, deposition into water and soil, bioaccumulation, and ultimate uptake to receptor organs and systems in the human body are complex. Pollution prevention programs can be better engineered, pollution priorities can be identified, and greater environmental public health gains (attributable to pollution prevention) can be achieved by evaluating the multiple pathways to human exposure and through improved dosage calculations. A single contaminant source often may represent only a fraction of a total body pollutant burden. Further research is needed on source culpability and attributable risk, long-range transport of air pollutants, human dose contributions by various pathways, better techniques for health risk assessment, and an identification of human behavior patterns that affect exposure and dose. PMID- 8637752 TI - Air and water pollution are intertwined. PMID- 8637753 TI - International medicine and air pollution. AB - Environmental pollution worldwide has been increasing rapidly as a result of massive increases in industrial production, especially since 1950. Although the United States has begun to address the problem, many nations have not been able to, such as those formerly part of the Communist bloc and many emerging nations. The International Society of Doctors for the Environment was established to promote the information flow about environmental impacts on health. The National Association of Physicians for the Environment is a member of that organization. PMID- 8637754 TI - Effects on the natural world: animals and plants. PMID- 8637755 TI - Future of environmental pollution. AB - In recent years, the concept of pollution prevention has overtaken end-of-pipe controls as the paradigm of choice for effecting environmental protection. Through cooperative mechanisms, significant progress is being made to reduce or eliminate upstream processes and practices that can lead to downstream pollution. These efforts, coupled with productive interactions with the public, are making an impact. The Science Advisory Board released a report in January that described the "next wave" of pollution prevention. Specifically, the report described approaches for anticipating environmental problems of tomorrow so that preventive actions can be taken today. The potential of air pollution problems figures prominently in the Board's vision of the future. PMID- 8637756 TI - Treatment of Bloom syndrome patients: guidelines and report of a case. PMID- 8637757 TI - Neurofibromatosis type I involving the external auditory canal. PMID- 8637759 TI - Caustic and thermal epiglottitis in the adult. AB - The presence of dysphagia, drooling, and stridor in an adult subsequent to thermal or caustic injury should alert the treating physician to the possibility of injury to the supraglottic structures with resultant epiglottitis. These adults possess many of the features seen in acute infectious epiglottitis and should be handled with the same consideration for potential upper airway obstruction. Epiglottic injuries of this type should be suspected in adults with mental disorders or communication difficulties. PMID- 8637758 TI - Posttraumatic macroglossia complicated by hyaluronidase injection. AB - A patient with posttraumatic macroglossia has been presented. A literature review reveals that this is a rare sequela of tongue injury and is usually of acute onset. The delayed symptom of swelling in this patient and the exacerbation of swelling in association with hyaluronidase suggest a causal relationship between the two events. Macroglossia has not been reported in association with hyaluronidase injection, and it is recommended that the drug not be used to treat swelling after tongue injury. PMID- 8637760 TI - Parathyroid lipoadenoma: case report and review of the literature. PMID- 8637761 TI - Palatal myoclonus: treatment with Clostridium botulinum toxin injection. PMID- 8637762 TI - Hydroxyapatite deposition disease: an uncommon cause of acute odynophagia. PMID- 8637763 TI - Role of Haller's cell in headache and sinus disease: a case report. AB - Although anatomic variations in the development of the nose and paranasal sinuses such as the Haller's cell do not themselves represent a disease state, in many cases they are responsible for the patient's symptoms. Particularly in the absence of extensive associated mucosal changes, these conditions may be easily overlooked unless specifically sought. Haller's cells may cause recurrent or chronic sinusitis and persistent sinugenic headache, without significant findings on physical examination including nasal endoscopy. The presence of Haller's cells on coronal CT in a patient with corresponding symptoms deserves consideration as the potential cause of the symptoms. When medical therapy is ineffective, such cases respond well to surgical therapy through the functional endoscopic approach. PMID- 8637764 TI - Intraoperative fire with electrocautery. PMID- 8637765 TI - Alcaligenes xylosoxidans subsp xylosoxidans in children with chronic otorrhea. AB - Because other known pathogens are frequently isolated with AXX, its clinical significance may be overlooked. AXX should not be considered a colonizer or contaminant, particularly in the presence of clinical signs and symptoms of infection. For a pediatric patient with AXX in the ear fluid, the choice of an antibiotic regimen should be based on in vitro activity of both AXX and other concurrent pathogens. Although the sensitivity of AXX may be highly variable, a number of antibiotics appear to be clinically useful. PMID- 8637766 TI - Ethmoidal cemento-ossifying fibroma: the transglabellar/subcranial approach. PMID- 8637768 TI - Bile duct stones recurring around metal clips. PMID- 8637767 TI - Hemangiopericytoma of the oral cavity. PMID- 8637769 TI - Do dietary antibodies still play a role in the diagnosis and follow-up of coeliac disease? A comparison among different serological tests. AB - OBJECTIVE: Comparison between the usefulness of immunological markers and intestinal biopsy in the diagnosis and follow-up of coeliac disease. MATERIALS AND METHODS: Serum antibodies to gliadin, several dietary proteins and endomysium were appraised in 27 patients with biopsy proven coeliac disease, both while untreated and 6-8 months after gluten withdrawal, when an intestinal biopsy was repeated. Forty-six healthy volunteers entered the study as controls. Antibodies to gliadin and dietary proteins were assessed by ELISA, antibodies to endomysium by indirect immunofluorescence using monkey oesophagus as antigen. RESULTS: Mean antibody levels to dietary proteins were significantly higher in untreated patients as compared to controls. Their titers decreased after gluten withdrawal, but a significant difference was found, except for casein, for the IgA class only. However, because of their unlinear and unpredictable behaviour, they showed a poor reliability. Antigliadin antibodies showed higher diagnostic accuracy, although they also produced false-positive and false-negative results. Anti endomysium antibodies, albeit the more expensive, proved the more reliable, due to their 100% specificity. CONCLUSION: To date, anti-endomysium antibodies are the most reliable marker for coeliac disease: a positivity warrants an intestinal biopsy. The actual role of antibodies to gliadin, cheaper than endomysium, is during follow-up when many determinations are needed. Antibodies to dietary proteins, useful in the pre-endomysium era, only have a historical role. PMID- 8637770 TI - Surgery of the incidentally discovered mass of the adrenal gland (incidentaloma). AB - The authors present their experience about surgery in adrenal gland incidentaloma, during a period of 12 years (1982-1993), at the VI Division of General Surgery, University of Turin. In 17 patients, of 70 who underwent adrenalectomy, the adrenal neoplasm was located thanks to a diagnostic investigation (ECI or CT) executed for other reasons. In ten cases was diagnosed adenoma, in five cases carcinoma, in two cases cysts. In the diagnostic approach to adrenal incidentaloma we did not search for possible hormonal activity, since the patients had been previously selected from specialized endocrinological centres. At present, biological markers not being certain or absolute radiological significance so as to detect benignant from malignant forms. In accordance with international Literature the size of the neoplasm is the discriminant element for adrenalectomy. We have removed the incidentaloma in all cases, be it clinically or subclinically functional and the silent forms > 4 cm as we have observed a statistically significant difference (p < 0.0002) between the benign and malignant lesions, particularly those measuring more than 4 cm in diameter. We suggest a screening with CT scan every three months in lesions < 4 cm, silent and those > 4 cm in patients over sixty years with a morphological aspect of the bening form. PMID- 8637771 TI - Amikacin administration in open heart surgery. AB - Amikacin is usually administered to the patients during open heart surgery immediately after cardiopulmonary bypass (CPB) because the serum levels of this drug may decrease due to the volume overload and hemodilution caused by intravenously administered fluids (1.2-2 liters prime solution). We investigated whether this application of amikacin is necessary immediately after CPB. A total of ten patients from Dokuz Eylul University Hospital, Department of Thoracic and Cardiovascular Surgery, who were scheduled for open heart surgery were studied. Serum sodium, potassium, blood urea nitrogen and creatinine values of the patients were found to be within normal limits before the operation. Amikacin was administered to patients just before open heart surgery and 2 ml blood samples were collected from all patients 15 minutes after the drug administration, just before cardiopulmonary by-pass (CPB), at the 30th and 60th minutes of the CPB, after CPB and after the cessation of operation, 4 hours after drug administration. A significant decrease was observed between the values of 10 patients by ANOVA (p < 0.05). Serum amikacin values were found to be 9.80 +/- 0.96 micrograms/ml at the end of the operation, 4 hours after the drug administration. Since serum amikacin levels do not fall below the trough concentrations at the end of the fourth hour we conclude that it is unnecessary to repeat the dose at the end of the operation. PMID- 8637772 TI - Searching for a marker for Alzheimer's disease-Apo-E. AB - The authors present their study of 10 patients affected by Alzheimer's disease and 10 patients with multinfarctual dementia, who are already part of a CNR study, senile dementia project, longitudinal study. The method adopted was that of DNA crossbreeding and amplification, so as to have a precise individualisation of alleles APo-E, Apo E-3, Apo E-4 and of their various phenotype combinations. These isoforms are evaluated and compared with the disease studied, in order to help to identify a timely-marker for senile dementia. On the results obtained we hypothesised an association between Apo E-4 and the disease of about 40% for Alzheimer's disease, which leads us to increase our number of cases, regarding associations between demential diseases either Alzheimer or vascular and the presence of Apo E-4. PMID- 8637774 TI - Neuroimmunomodulation and psychoneuroendocrinology: recent findings in adults and aged. AB - In recent years the relationships among immune, endocrine and nervous systems have been extensively studied, and grouped in a new research field: psychoneuro immunoendocrinology. Since ancient times its has been known that, in humans, mood as well as environmental influences could affect health. In the late '70s, only, evidence of bi-directional pathways has been achieved, first in animal models and, later on, in humans. We reviewed current knowledge on neuroimmunomodulation, concerning the influence of stress and psychological status on immunity as well as neuroendocrine modulation by the immune system, reporting some data obtained from our studies. Particularly, having detected a relevant impairment concerning most of the parameters studied, we emphasized the effects of depressive disorders on immune function in the elderly. PMID- 8637773 TI - Endocrine-immunological homeostasis: the interrelationship between the immune system and sex steroids involves the hypothalamo-pituitary-gonadal axis. AB - The growing body of evidence, both experimental and clinical supports the concept of multiplicity of endocrine-immunological interactions. Such interrelationship should be viewed as bidirectional: the immune system like other homeostatic mechanisms is subject to regulation by endocrine factors, while vice versa the immune system by itself affects the function of the neuroendocrine system. The gonadal steroids influence immunity acting at two levels: at the level of primary lymphoid tissues or mature immunocompetent cells, and at the level of the hypothalamo-pituitary axis, influencing the release of hormones which by itself also have immunoregulatory properties. Studies indicate that antiestrogens also affect immune response in both agonistic and antagonistic mode of action, especially analyzed in patients with endocrine-responsive tumors related by hormone therapy. PMID- 8637776 TI - Successful surgical removal of partially expanded Palmaz stent from external iliac artery. AB - Insertion of intravascular stents into various arteries including coronary arteries has become an integral part of vascular interventions, particularly in the treatment of aortoiliac arteriosclerotic disease. The incidence of vascular complications remains relatively high despite the better stent design and inserting techniques. We report a case where successful removal of partially expanded Palmaz stent from external iliac artery was needed due to a rare complication: a balloon catheter burst resulting in an insufficient stent expansion in an improper position. PMID- 8637775 TI - Food intolerance and allergy. AB - Various food products can trigger disagreeable symptoms in certain people which are generally described as food intolerances. Given the complexity of the subject and the fact that a great deal remains to be learned about the mechanisms responsible for these reactions, the paper attempts to clarify the whole question. Food intolerances have been classified in various ways by different authors. The present paper considers only the three major groups: those due to enzyme deficiencies, those due to an allergic mechanism and those caused by histamine-release or histamine-releasing food products. PMID- 8637777 TI - Anti-Jkb delayed hemolytic transfusion reaction after coronary bypass surgery. AB - Delayed hemolytic transfusion reaction occurred in a 74-year-old woman after coronary bypass. Antibodies were not detected during preoperative screening but did appear late after exposure to Jkb-positive red blood cells, probably as an anamnestic response to previous exposure during childbirth or remote transfusion. The incidence, pathophysiology, clinical presentation, diagnosis, and management of this syndrome are discussed. PMID- 8637778 TI - A physical mechanism in the treatment of neurologic disorders with externally applied pico Tesla magnetic fields. AB - The clinical studies describing the treatment of some neurological disorders with an externally applied pico Tesla (10R Tesla, or 10(-8) gauss) magnetic field are considered from a physical view point. An equation relating the intrinsic or "rest" energy of a charged particle of mass with its energy of interaction in an externally applied magnetic field B is presented. The equation is proposed to represent an initial basic physical interaction as a part of a more complex biological mechanism to explain the therapeutic effects of externally applied magnetic fields in these and other neurologic disorders. PMID- 8637779 TI - Recent epidemiology of group A streptococcal infections in North America and abroad: an overview. PMID- 8637780 TI - Acute pharyngitis: etiology and diagnosis. PMID- 8637781 TI - Evaluation of penicillins, cephalosporins, and macrolides for therapy of streptococcal pharyngitis. AB - OBJECTIVE: To review recent clinical experience with treatment of acute streptococcal pharyngitis with penicillins, cephalosporins, and macrolide antibiotics. METHODS: Literature review and analysis. RESULTS AND CONCLUSIONS: Oral penicillin V administered two to three times daily for 10 days is the treatment of choice for acute streptococcal pharyngitis and is the oral standard against which other treatments should be measured. A single intramuscular dose of benzathine penicillin also remains highly effective. Recent studies evaluating alternative oral agents given for less than 10 days or in once-daily regimens have yielded promising results. Studies should make efforts to exclude chronic streptococcal carriers with intercurrent viral pharyngitis because their inclusion in treatment trials substantially confounds the data. As issues of health care costs assume increasing importance, the cost of newer antimicrobial agents will deter their usage for acute streptococcal pharyngitis. PMID- 8637782 TI - A review of the rationale and advantages of various mixtures of benzathine penicillin G. AB - Intramuscular benzathine (BZ) penicillin G has been accepted as the gold standard for treatment of patients with streptococcal pharyngitis since it was first introduced in 1952. Unfortunately, it has been associated with pain and tenderness at the site of injection. Efforts to lessen this by combining it with varied quantities of procaine (PC) penicillin G have been successful, decreasing the incidence of significant local reactions to 5% to 10%, little more than that seen with PC penicillin alone. A preparation containing 600,000 U BZ penicillin G and 600,000 U PC penicillin in 2 mL has been marketed since the mid-1950s as CR Bicillin 600/600 but the content of BZ penicillin G has been considered adequate only for children who weigh < 60 pounds. This prompted the evaluation of a preparation containing 900,000 U BZ penicillin G plus 300,000 U PC penicillin G in a 2-mL injection. Of 400 children with streptococcal pharyngitis 100 each received this preparation, 600,000 U BZ penicillin G alone in 1 mL, 1.2 million U BZ penicillin G alone in 2 mL or 600,000 U BZ penicillin G plus 600,000 U PC penicillin G in 2 mL. Clinical response was equal in all four groups; all patients were well in 36 to 48 hours. The two preparations containing PC penicillin G had significantly less severe local reactions and throat cultures were negative in all by 48 hours; it remained positive in some patients who received BZ penicillin G alone after 72 hours. The cure rate in patients receiving the 900, 000/300,000 combination of BZ and PC penicillin G was equal to that in patients who received 1.2 million U BZ penicillin G with these added advantages. This combination offers optimal parenteral treatment for streptococcal pharyngitis in all children who weigh < 140 pounds and it has been marketed for this purpose since 1976. PMID- 8637784 TI - Antibiotic resistance: relationship to persistence of group A streptococci in the upper respiratory tract. AB - Despite the use of penicillin for more than 40 years in treating GABHS infections, there has been no significant change in the in vitro susceptibility of GABHS to penicillin. Reported failures to eradicate GABHS from the upper respiratory tracts of patients with pharyngitis and the apparent resurgence of serious Group A streptococcal infections and their sequelae probably are not related to the emergence of penicillin resistance. Although erythromycin resistance in GABHS had been a major problem in Japan and continues to be a major problem in Finland, it has not been a problem in this country. The susceptibility of GABHS to the newer macrolide antibiotics appears to be similar to that of erythromycin. Comprehensive, community-wide programs to continuously monitor for erythromycin resistance in GABHS would be difficult to justify. However, because little is known about how erythromycin resistance in GABHS is acquired or spread, it would be reasonable to periodically monitor isolates of GABHS for erythromycin resistance. A substantial proportion of GABHS are currently resistant to tetracyclines and these agents are inappropriate for treating GABHS infections. Although little recent information is available about the susceptibility of GABHS to sulfonamides, these agents have been shown to be ineffective in eradicating GABHS from the upper respiratory tract regardless of the in vitro sensitivities. GABHS have not been shown to be resistant to any of the commonly used oral cephalosporins; however, there is a great deal of variability among these agents in their activity against GABHS. Clindamycin resistance in GABHS has remained unusual. This agent is an alternative for treating GABHS infections due to macrolide-resistant strains in patients who cannot be treated with beta-lactam antibiotics. There is no reason, based on the in vitro susceptibilities of GABHS, to change the current recommendations for treating GABHS infections with penicillin and for using erythromycin for patients who are allergic to penicillin. PMID- 8637783 TI - Epidemiology and control of acute respiratory diseases with emphasis on group A beta-hemolytic streptococcus: a decade of U.S. Army experience. AB - OBJECTIVE: To summarize the experiences of the U.S. Army regarding prevention and control, and frequencies, rates, trends, and determinants of febrile acute respiratory diseases (ARDs), particularly Group A beta-hemolytic streptococcus (GABHS). METHODOLOGY: Since 1966, the U.S. Army has conducted routine surveillance of ARDs among basic trainees. Since 1985, all trainees with fever and respiratory tract symptoms have been cultured for GABHS: Field investigations were conducted when outbreaks of acute respiratory or GABHS-associated illnesses were detected. Mass plus tandem benzathine penicillin prophylaxis were used to interdict and control training center GABHS outbreaks. RESULTS: During the period 1985 to 1994, there were 65,184 hospitalizations for acute febrile respiratory illnesses among Army trainees. The crude hospitalization rate was 0.45 per 100 trainees per week. The rate consistently declined over the period. Incremental declines were temporally associated with increased use of adenovirus immunizations and broader use of benzathine penicillin prophylaxis. During the period, 10,789 of 59,818 (18%) pharyngeal cultures were positive for GABHS: GABHS outbreaks were associated with diverse clinical manifestations including streptococcal toxic shock, acute rheumatic fever, and pneumonia. The emergence of mucoid colony morphology in clinical isolates was a consistent indicator of circulating virulent strains with epidemic potential. Outbreak-associated M types were M1, M3, M5, and M18. In response to six GABHS outbreaks, mass plus tandem benzathine penicillin chemoprophylaxis produced rapid and sustained GABHS control. ARD and GABHS recovery rates were lowest when benzathine penicillin prophylaxis was widely used. CONCLUSIONS: ARD rates among Army trainees have consistently declined to unprecedented levels. GABHS has reemerged as an important threat to military trainees. Benzathine penicillin chemoprophylaxis is safe and effective for interdicting and preventing GABHS outbreaks in closed, healthy young adult populations. PMID- 8637785 TI - Adherence to physicians' instructions as a factor in managing streptococcal pharyngitis. AB - Adherence to physicians' instructions, including taking medications as prescribed, is essential for the proper treatment of streptococcal pharyngitis and the prevention of rheumatic fever. Nonadherence can be in many forms, including failure to have prescriptions filled, omission of doses, errors in dosing or administration time, and premature discontinuation of medication. Adherence is dependent on the physician, the patient, the illness, and the medication. Proper communication by the physician and prescribing inexpensive medications that can be taken once or twice daily are simple, yet important actions that improve adherence. PMID- 8637786 TI - Allergic reactions in rheumatic fever patients on long-term benzathine penicillin G: the role of skin testing for penicillin allergy. PMID- 8637788 TI - Are the currently recommended doses of benzathine penicillin G adequate for secondary prophylaxis of rheumatic fever? AB - OBJECTIVE: To review the literature on dose and regimens of intramuscular benzathine penicillin G (BPG) for secondary prophylaxis of recurrent rheumatic fever. SETTING: For over 40 years BPG has been the gold standard for secondary prophylaxis, usually as a dose of 1,200,000 U (900 mg). Although studies have suggested that BPG injections every 3 weeks are superior to injections every 4 weeks, implementation of an every 3 weeks regimen can be problematic with regards to both patient compliance (adherence) and an increased burden on health resources. FINDINGS: Some of the earliest studies of BPG suggested that larger doses resulted in prolongation of detectable penicillin levels. A recent study assessing plasma penicillin levels after BPG doses of 1,200,000 U, 1,800,000 U, and 2,400,000 U suggested there may be benefits in a BPG regimen every 4 weeks with doses higher than the standard 1,200,000 U. CONCLUSIONS: Further studies of higher dose BPG regimens seem justified. In addition, further work is needed on quality and storage options for different BPG preparations; location and method of BPG injections; the importance of weight differences between individuals; and ways of improving access to and compliance with BPG regimens. PMID- 8637787 TI - Three- versus four-week administration of benzathine penicillin G: effects on incidence of streptococcal infections and recurrences of rheumatic fever. AB - OBJECTIVE: To investigate the effects of 3-week versus 4-week administration of benzathine penicillin G (BPG) on the incidence of Group A streptococcal infections and the recurrences of rheumatic fever (RF). STUDY DESIGN: We started, in 1979, randomly allocating all patients with RF to a 3-week or 4-week BPG prophylaxis program. They were examined at the RF clinic, every 3 to 6 months, and at any time they did not feel well. During 1979 to 1989, throat cultures and sera for antistreptolysin O and streptozyme titers were obtained at each clinic visit. Chest radiographs, electrocardiogram, color Doppler echocardiograms, and acute phase reactants were obtained. SUBJECTS: Two hundred forty-nine patients fulfilled the revised Jones criteria and were followed until December 1991: 124 in the 3-week and 125 in the 4-week program. Their age, sex, weight, percentage with history of RF, severity of cardiac involvement, follow-up duration, and compliance to program were comparable. Eight hundred eighty throat cultures were collected in the 3-week program and 770 were collected in the 4-week program. Six hundred sixteen and 627 sera were determined in each program for antistreptolysin O, and 582 and 592 sera for streptozyme titers. RESULTS: True streptococcal infections occurred in both programs: 39 infections in the 3-week program, and 59 infections in the 4-week program (7.5 vs 12.7 per 100 patient-years). Four infections with no antibody response occurred in the 3-week program, and three such infections in the 4-week program. Nine RF recurrences occurred in 8 patients in the 3-week program, and 16 recurrences in 16 patients in the 4-week program. Prophylaxis failure occurred in 2 of 124 patients in the 3-week program, and in 10 of 125 patients in the 4-week program (0.25 vs 1.29 per 100 patient-years). The overall recurrences/infections rate in each program was comparable, 13.6% vs 15.5%, but the recurrences/ infections rate due to prophylaxis failure was higher in the 4-week program than in the 3-week program, 3.0% versus 9.7%. CONCLUSIONS: This 12-year prospective and controlled study documented that streptococcal infections and RF recurrences occurred more often in the 4-week program than in the 3-week program. The risk of prophylaxis failure was fivefold greater in the 4 week program than in the 3-week program. PMID- 8637790 TI - Rheumatic fever prevention in industrializing countries: problems and approaches. PMID- 8637789 TI - Rheumatic fever prophylaxis using benzathine penicillin G (BPG): two- week versus four-week regimens: comparison of two brands of BPG. AB - OBJECTIVE: This prospective study was aimed at answering two important questions: 1) Is a biweekly schedule of 1.2 million U intramuscular benzathine penicillin G (BPG) superior to a 4-week one in the prevention of upper respiratory Group A beta-hemolytic streptococcal (GABHS) infections and rheumatic fever (RF) recurrences? 2) Is there a difference in the bioavailability of BPG obtained from different manufacturers? METHODOLOGY: Three hundred sixty rheumatic patients aged 4 to 20 years were randomly assigned to either a biweekly (190 patients) or 4 week (160 patients) BPG prophylactic schedule and were followed-up monthly for 2 years by clinical examination, throat swab culture for GABHS and measurement of antistreptolysin O titer to detect GABHS infection and/or recurrences of RF (according to revised Jones' Criteria). Thereafter, 34 rheumatic subjects, aged 8 to 16 years were randomly assigned to receive a 4-week injection of 1.2 million U of either a locally manufactured BPG brand (22 patients) or an imported one (12 patients). Sera of all patients were tested for penicillin level by plate diffusion method on days 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 after the intramuscular injection of BPG. RESULTS: The GABHS infection rate was found to be 0.2% and 0.3% for patients on the biweekly and 4-week BPG schedules, respectively, with no significant differences between them. However, the RF recurrence rate/patient/year for the 4-week schedule patients (0.12) was double that for the biweekly schedule ones (0.06). Estimation of the bioavailability of the two different brands of BPG demonstrated a difference in their pharmacokinetics and a decrease in the serum penicillin concentration below the minimum inhibitory concentration 3 weeks after the injection of either brand. CONCLUSION: Although a biweekly schedule may not be superior in preventing upper respiratory GABHS infection, it may play a role in preventing the sequelae of such infections. The short duration of penicillinemia explains the superiority of the 2-week schedule in RF prophylais. The difference in the pharmacokinetics of penicillin brands might contribute to the high recurrence rate of RF reported in Egypt. PMID- 8637791 TI - A few facts on life. PMID- 8637793 TI - Development of the H & H Lactation Scale. AB - Psychometric properties of the H & H Lactation Scale, based on the Insufficient Milk Supply (IMS) conceptual framework, were examined in two separate studies. The two convenience samples consisted of 110 mothers of low-birth-weight (LBW) infants and 120 mothers of healthy term infants. Both groups planned to breastfeed and were actively breastfeeding or pumping to maintain a milk supply. Subscales identified by factor analysis measured three concepts: maternal confidence/commitment to breastfeeding, perceived infant breastfeeding satiety, and maternal-infant breastfeeding satisfaction. All subscales showed moderate to high internal consistency (alphas .75 to .98) as well as concurrent and predictive validity. The total scale and the three subscales were prospectively significantly related to level of breastfeeding 8 weeks after delivery with both groups of mothers. PMID- 8637792 TI - Effects of nonnutritive sucking on behavioral organization and feeding performance in preterm infants. AB - The purpose of this study was to examine the effects of nonnutritive sucking (NNS) on behavioral organization and feeding performance in preterm infants. Thirteen preterm infants were observed at four bottle-feedings, two of which involved treatment with prefeeding NNS. NNS had a positive effect on oxygen saturation and behavior state, as well as on the initiation and duration of the first nutritive suck burst. PMID- 8637794 TI - Children's preoperative coping and its effects on postoperative anxiety and return to normal activity. AB - A model was tested to assess children's preoperative coping with major orthopedic surgery and how coping is related to two different postoperative outcomes, anxiety and return to normal activities. Ninety children, ages 8 to 17, participated. Data were collected the day before surgery, the second postoperative day, and at 3-, 6-, and 9-month recovery periods. A respecified model was not significantly different from the data (p = .90), indicating a good fit. Children who were older, more anxious, and more internal in locus of control exhibited more vigilant coping. Avoidant coping was associated with less anxiety 2 days postoperatively, and vigilant coping was associated with return to normal activities over the course of recovery. PMID- 8637795 TI - Coming to terms: parents' response to a first cancer recurrence in their child. AB - The purpose of this study was to explore, using grounded theory, the process experienced by parents who are dealing with the first recurrence of cancer in their child. The sample of 33 guardians (27 mothers, 1 grandmother, and 5 fathers) was drawn from three pediatric oncology settings. Data were collected through interviews, observations, and medical record review. Thirteen parents were interviewed to validate first the evolving and, later, the complete study findings. Four interactive components emerged: regulating shock, situation monitoring, alternating realizations, and eyeing care-limiting decisions. The overall organizing construct induced from these components was labeled "coming to terms." This construct represents the parents' efforts to overcome shock and despair to make wise decisions about treatment while accepting that the outcome if beyond their control, and to help their child have the optimal chance for cure while preparing for the child's possible death. PMID- 8637796 TI - Respiratory muscle strength in older adults. AB - Gender-related effects and two measures of muscularity, handgrip strength and fat free mass (FFM), were examined to determine their relationship to respiratory muscle strength. Subjects were 101 healthy older adults. In 75 subjects, the magnitude of learning effect was examined over four weekly sessions. Maximal inspiratory pressure (PImax) was lower with increasing age in women, and maximal expiratory pressure (PEmax) was lower with increasing age in both genders. The PEmax correlated with handgrip strength and FFM in men only. Performance of PImax plateaued by the third visit in both men and women. Performance of PEmax plateaued by the third measure in women and was unchanged across four measurements for men. PMID- 8637797 TI - Stress mediation in caregivers of cognitively impaired adults: theoretical model testing. AB - The purpose of the study was to test a midrange model of caregiver stress mediation based on Modeling and Role-Modeling theory. Perceived support and self worth were examined to determine if these self-care resources had a mediating effect between stress and well-being in caregivers of cognitively impaired adults. The sample of 117 caregivers completed measures of basic need status, perceived support, self-worth, stress, and well-being. There were significant correlations among the study variables in the expected directions. Resources mediated the relationship between stress and well-being, providing tentative support for theoretically proposed linkages. PMID- 8637798 TI - British nurses at war 1914-1918: ancillary personnel and the battle for registration. AB - The purpose of this study was to examine the use of ancillary personnel to augment nursing staff, a recurring theme in the history of nursing. The focus was the use of untrained nursing personnel in Great Britain during the years 1914 1918. The findings demonstrate both the nature and extent of dilution within nursing and its effects in terms of social relationships and occupational structure. PMID- 8637799 TI - Quality of life in cardiac patient research: a meta-analysis. AB - This article reports a meta-analysis of 84 studies of quality of life (QOL) in cardiac patient populations published in the 5-year period 1987-1991. Selected methodologies and substantive characteristics of the studies are described. An overall effect size of .31 indicated a small but significant positive effect of pharmacologic, mechanical, surgical, nursing, or other treatment on QOL. No negative effect of treatment was found for any cardiovascular diagnostic category. Homogeneity analysis revealed eight potential moderators of the overall effect size: quality of study, gender of sample, time dimension, sampling method, intervention, marital status of subjects, quality-of-life dimension measured, and sample size. PMID- 8637800 TI - Use of a meta-analytic database management system. PMID- 8637801 TI - The rough and ready jackknife. PMID- 8637802 TI - Nursing, medicine, quackery, and health care reform, 1844-1994. PMID- 8637803 TI - The evolving nature of perinatal nursing. AB - Current and future health-care trends create many challenges for perinatal nurses. A new mind set is required for the profession not only to accommodate the paradigm shift but provide leadership in designing a new future for health care. Interestingly, it has been predicted that the movement toward achieving healthier communities will result in a decentralized, community-based, nonhierarchical system that will not be male dominated. Nurses will be called on to participate in the development and implementation of health-care programs rather than assume a passive, recipient role. On the brink of the 21st century, nursing is in a position to emerge as a leader in providing cost-effective, needs-based, health care services aimed at improving the health status of a community. PMID- 8637804 TI - Women's health. The role of advanced practice nurses in the 21st century. AB - Advanced practice nurses have the capability of providing a variety of services in women's health care. The role and functions of certified nurse midwives, nurse practitioners, clinical nurse specialists, and certified registered nurse anesthetists are discussed. Current issues and barriers to advanced practice nursing are presented and include role definitions and regulations, second licensure, educational level, prescriptive authority, third party reimbursement, admitting privileges, and malpractice reform. Future directions and recommendations are provided. PMID- 8637806 TI - Stress, immune function, and relationship to pregnancy outcome. AB - Pregnancy and the postpartal period are a time of immunosuppression. The normal immunosuppression that occurs during the puerperium may be aggravated by stress. Normal mechanisms of immunosuppression are discussed, and the research related to stress and childbearing is examined in this article. PMID- 8637805 TI - A review of problems of universal access to prenatal care. AB - Despite the preponderance of evidence that points to the advantages of prenatal care, the number of women who receive adequate prenatal care has remained at a plateau or actually decreased since 1980. Over the past decades, many demographic and structural barriers to receiving prenatal care have been identified; financial obstacles have been cited as the major barrier. The assumption has generally been made that if financial barriers to prenatal care were removed, problems with access to prenatal care would be solved. Recent appreciation of the significance of nonfinancial barriers to prenatal care has resulted in recognition that even if all financial barriers were removed, there would still be access problems. PMID- 8637807 TI - Use of ultrasonography in the home care of high-risk childbearing women. AB - Antenatally, as more women are being treated in the home setting rather than the hospital setting, diagnostic modalities such as ultrasonographic imaging need to be available for use by the home care provider. In the home setting, the care provider and treatment modalities are brought to the patient, whereas patients managed in the hospital setting must attend the provider site to receive the same services. Without the availability of diagnostic ultrasonography in the home setting, pregnant women who are treated in the home rather than the hospital are required to incur an added financial and physical burden associated with attendance at care. Financially, nonreimbursed out-of-pocket costs incurred by the patient may include costs of transportation, costs of child care, and loss of wages. Physically, patients are prevented from maintaining recommended bed rest and dietary treatment regimens. Ultrasonographic evaluations can be performed safely in the home setting by nurses who have received added educational preparation and ultrasonographic imaging experience, thereby providing the essential care often necessary for women who experience pregnancies complicated by high-risk conditions. PMID- 8637808 TI - Bed rest and high-risk pregnancy. Differentiating the effects of diagnosis, setting, and treatment. AB - Current research on high-risk pregnancy frequently has confounded the effects of diagnosis, setting, and treatment. Studies of pregnancy bed rest have demonstrated a beginning attempt to differentiate the influences of confounding variables and, similar to high-risk studies, have found that depression, anxiety, other disturbing emotions, and separation from family are common side effects. It is likely that some of the effects previously attributed to high-risk pregnancy may be either caused or heightened by activity restriction. This article identifies the major issues to be considered when studying high-risk pregnant women. Furthermore, it suggests that, if activity restriction must be prescribed, the adverse effects of treatment upon both the women and her fetus should be considered in the decision making process. PMID- 8637809 TI - A model of home care for high-risk childbearing families. Women with diabetes in pregnancy. AB - With a national trend toward decreased hospitalizations for high-risk childbearing women, home care and follow-up services have gained increased importance. The Quality-Cost Model of Nurse Specialist Transitional Follow Up Care is presented as a method to provide home care for high-risk pregnant women, specifically those whose pregnancies are complicated by diabetes mellitus. The model offers opportunities for intensive education, assessment, intervention, and support throughout the pregnancy and postpartum period. In addition, this model can potentially decrease the burden of care on the woman and her family by providing a comprehensive program of home care, follow up, support, and education. PMID- 8637811 TI - Family response to the low birth weight infant. AB - Families of VLBW and LBW preterm infants are usually happy to have a new baby in the family, although they are anxious, depressed, and concerned about their infant's future health and well-being. During the first year of the infant's life, families of LBW and VLBW infants face many new challenges. They have infants with unique health-care needs who are often temperamentally difficult and who benefit enormously from excellent parenting. Preterm LBW and VLBW infants are often born to families who are poor and to mothers who are young. Families of VLBW and LBW preterm infants experience decreased employment possibilities at a time when they also have increased expenses. Families of LBW and VLBW preterm infants undergo a vulnerable period during which they may benefit greatly from receiving care from nurses who understand their needs. PMID- 8637810 TI - Controversial issues surrounding early postpartum discharge. AB - Throughout the world, early postpartum discharge programs are emerging as one strategy for reducing health care costs and, in some areas, relieving the shortage of hospital beds. This article summarizes the research findings to date regarding programs of early postpartum discharge. Additionally, findings from recently completed work on a program of early discharge for high-risk childbearing families is discussed. PMID- 8637812 TI - State of the science. Breastfeeding for mothers and low birth weight infants. AB - There is support in the research literature for encouraging mothers of preterm and LBW to breastfeed their infants, although the numerous barriers to their successful breastfeeding have been well documented. The emerging work addressing short- and long-term health benefits for preterm infants provide's scientific rationale for allocation of resources, that is, equipment and personnel, to assist these mothers in attaining their breastfeeding goals. The literature also has linked selected, individualized interventions to breastfeeding outcomes for this population. Examples of this linkage include milk expression recommendations for maximizing milk yield, techniques for in-hospital breastfeeding, and models for providing breastfeeding services in the NICU. Clinical priorities should focus on strategies for implementing the available research into practice and for using the research to establish standards of care for mothers who breastfeed preterm and LBW infants. Research priorities should focus on practice models for providing breastfeeding services that demonstrate improved breastfeeding outcomes and cost effectiveness for this vulnerable population. PMID- 8637813 TI - Integrating family-centered developmental assessment and intervention into routine care in the neonatal intensive care unit. AB - Incorporating developmental intervention into routine practices requires time available during caregiving for continued infant assessment and intervention and requires time after and between caregiving for relief of infant distress. In an era of health-care reform, reorganization, and restructuring, this added caregiver time appears unavailable. However, nurses need to work together in meeting the developmental challenges of the NICU. Further research needs to be done to validate which interventions are appropriate for which babies and with what medical procedures. In addition, collaboration and sharing of responsibilities and resources with all care providers needs to be investigated. Moreover, research needs to be done that acknowledges that the environment of the NICU is also a world in which many adults work and live a large portion of their daily life. This environment must be supportive of their social needs as well as the needs of the high-risk infant. There are many pieces to the puzzle of providing developmentally supportive caregiving in the NICU. The number of different issues increases the complexity of changing the standard of care: Infant, family, environment. Each has its own challenges. However, with sensitivity, a collaborative approach, and a sincere effort to change, neonatal health-care professionals can integrate developmental practices into the NICU. PMID- 8637815 TI - Advanced ventilation in the neonate. AB - Advanced ventilation therapies for the sick neonate, such as nitric oxide and liquid ventilation, may be effective in treating the sick neonate. Both techniques are discussed in terms of their current merit for treatment and what impact they may have on nurses caring for the sick neonate. PMID- 8637814 TI - Implementing a research-based kangaroo care program in the NICU. AB - Kangaroo care or skin-to-skin holding of preterm infants requires consistent implementation for best outcomes with infants and families. Successful implementation of a project of this type demands an organized approach. This article describes how a standard of care was developed using research findings and then implemented with a step-by-step approach in the neonatal intensive care unit. PMID- 8637817 TI - Molluscum contagiosum. PMID- 8637818 TI - Almost extinct diseases: measles, mumps, rubella, and pertussis. PMID- 8637819 TI - Enhancing patient compliance in pediatrics. PMID- 8637816 TI - Chronic asthma: an update. PMID- 8637820 TI - Malabsorption in childhood. PMID- 8637822 TI - Lipoprotein disorders. PMID- 8637821 TI - Consultation with the specialist. Growth hormone. PMID- 8637823 TI - Nephrogenic diabetes insipidus. PMID- 8637824 TI - Treatment of type II diabetes: what options have been added to traditional methods? AB - Clinical goals in patients with non-insulin-dependent (type II) diabetes are to control glucose levels and prevent microvascular complications (eye, kidney, and nerve damage) while improving risk factors associated with cardiovascular disease (obesity, smoking, hyperlipidemia, hypertension, and hyperinsulinemia or insulin resistance). A wide array of medications and approaches is available to treat type II diabetes. Still, establishing an effective treatment regimen can be difficult, because patients have varying degrees of insulin secretory defects and insulin resistance and different conditions that must be factored in. Therefore, an individualized plan centered on self-management is the key to successful therapy in type II diabetes. PMID- 8637825 TI - Insulin pump therapy: acceptable alternative to injection therapy. AB - Initiation of insulin therapy is an exhilarating although anxiety-provoking experience for diabetic patients. It is, in a metaphysical sense, a time of rebirth. Patients are no longer victims of their diabetes but truly masters of it. No statement better illustrates how basic this change is than that of a patient who says, "What a difference it has made in my life to eat a meal because I want to, not because I have to." This article examines a means of insulin delivery that is growing in acceptance--insulin pump therapy. PMID- 8637826 TI - Surveillance for complications of diabetes: don't wait for symptoms before intervening. AB - Surveillance and treatment of diabetes-related complications should be part of routine care of all patients with diabetes. The natural history and screening recommendations for diabetic retinopathy, nephropathy, and neuropathy must be understood, since even advanced disease can be asymptomatic. Most adults require yearly ophthalmologic evaluations and determinations of albuminuria. Regular foot examinations by the patient and physician are required, with special attention to identifying patients with increased susceptibility to neuropathic ulcer and lower extremity amputation (ie, the "high-risk foot"). Cardiac autonomic neuropathy has become easier to diagnose, and its presence has several implications. Measurement of lipid levels and glycosylated hemoglobin and assessment of nutritional health should also be included in evaluation. Although understanding and prevention of diabetes complications are improving, the impact of end-organ damage remains a major problem. Early diagnosis and treatment often improve outcome and should dramatically decrease the burden of diabetes in our society. PMID- 8637827 TI - Organ transplantation: two baseball greats challenge us to give the gift of life. PMID- 8637828 TI - Gestational diabetes: ensuring a successful outcome. AB - When gestational diabetes is diagnosed, the physician has an opportunity to help the patient deliver a healthy baby by encouraging optimal glycemic control. In this article, the authors describe risk factors, methods of diagnosis, and a plan of management that includes a nutritional program based on ADA guidelines, use of insulin if needed, home monitoring of glucose levels, and appropriate exercise. PMID- 8637829 TI - Diagnostic imaging: its role in evaluating chest, abdominal, and musculoskeletal infections. AB - Imaging studies can provide essential information when infection is suspected. Chest radiography remains the best radiographic screening tool for intrathoracic infections. Computed tomographic (CT) scanning is preferred for evaluation of solid-organ abscesses, peritoneal abscesses, and associated malignant tumors or adenopathy. Ultrasonography is used primarily in evaluation of the gallbladder, biliary system and, increasingly, the appendix. Magnetic resonance imaging is highly sensitive in detecting osteomyelitis. PMID- 8637830 TI - Neonatal jaundice: when to treat, when to watch and wait. AB - What clinical circumstances call for aggressive treatment of jaundice in newborns? Is cessation of breast-feeding necessary to help bring down the high bilirubin concentration? When should phototherapy be started--and stopped? The authors address these and other questions in this thorough guide to differential diagnosis and management of neonatal jaundice. PMID- 8637831 TI - Getting the lead out: when is treatment necessary? AB - Lead poisoning is a continuing health concern, especially in children. Screening mandates by states, in addition to evidence that lead is toxic at lower levels than previously thought, make it increasingly important for physicians to have an understanding of this problem. Careful history taking focused on potential lead exposure from environmental, occupational, and recreational sources aids in detection and treatment. Avoidance of further lead exposure is the chief intervention in all patients at risk. Chelation therapy should be considered when blood lead levels reach 25 mg/dL in children and 51 mg/dL in adults. PMID- 8637832 TI - Nosocomial pneumonia: trying to make sense of the literature. AB - Nosocomial pneumonia is hard to diagnose with any certainty. Widely accepted concepts regarding the infection are based on data that are far from conclusive. Published recommendations for diagnosis and treatment often reflect a concentrated effort to consolidate these data. According to the authors of this article, initial empirical treatment may be defined by dividing patients into specific host groups. However, this approach should always be supplemented by earnest attempts at identifying the cause with microbial cultures. PMID- 8637833 TI - Nongenital warts: when is treatment warranted? AB - Patients often request laser therapy or other dramatic measures for treatment of nongenital warts. Because some popular interventions may be painful and costly and lead to scarring, it is wise to make sure a procedure's merits outweigh its side effects before commencing. PMID- 8637834 TI - Colorectal cancer screening: present strategies and future prospects. AB - Considerable controversy remains as to what represents the most effective and cost-effective approach to screening for colorectal cancer. The American Cancer Society (ACS), whose guidelines are the most widely used in the United States, recommends annual fecal occult blood testing and flexible sigmoidoscopy every 3 to 5 years beginning at age 50 in asymptomatic, average-risk individuals. However, the high rates of false-positive and false-negative results associated with the fecal occult blood testing techniques currently available continue to represent a concern. There is sufficient information to encourage patients and physicians to comply with ACS recommendations for colorectal cancer screening. However, reducing the mortality and morbidity of colorectal cancer will require advances in screening methodology as well as new methods of educating both the public and physicians about the importance of screening. Future screening strategies may include improved fecal occult blood testing, use of colonoscopy (as either a one-time or a periodic examination), and molecular genetic testing performed on feces or blood. Advances in understanding of genetic alterations in colorectal cancer will undoubtedly improve our ability to target aggressive screening strategies and implement preventive measures. PMID- 8637835 TI - Mobility and balance in the elderly: a guide to bedside assessment. AB - Elderly patients often have impaired balance and loss of mobility. Regular assessment of mobility status is essential to ensure that appropriate support is provided and to monitor recovery. Functions that should be assessed include mobility in bed, ability to rise to a sitting position, ability to transfer from bed to chair and in and out of a bathtub, balance, ambulation, and gait. Evaluation can be performed quickly at the patient's bedside. For some patients, stretching and strengthening exercises may help restore function. PMID- 8637836 TI - Healing the healer. PMID- 8637837 TI - Hypertensive crises in patients with glaucoma. PMID- 8637838 TI - Resource in addiction psychiatry. PMID- 8637839 TI - Assessment of fever in HIV-infected patients. AB - Fever is common among HIV-infected patients. Because it may signal a serious, potentially fatal underlying infection, a thorough investigation is merited. Where does evaluation begin and end? Drs Gleckman and Czachor discuss a diagnostic approach for primary care physicians that emphasizes rapid identification of the source of fever. PMID- 8637840 TI - [Biochemical response of recombinant Hansenula polymorpha strains to oversynthesis of homologous dioxyacetone kinase and bacterial beta galactosidase]. AB - Changes in the activities of key enzymes responsible for utilization of methanol by recombinant strains of methylotrophic yeasts H. polymorpha R22-2B and H. polymorpha LAC-56 grown in a chemostat are described. The strain R22-2B displaying a high activity of dioxyacetone kinase had also a high activity of formaldehyde dehydrogenase, which increased the rate of dissimilation of formaldehyde. There was a decrease in ATP concentration in the strain LAC-56 oversynthesizing beta-galactosidase from Escherichia coli; this effect decreased the rate of assimilation of formaldehyde. PMID- 8637841 TI - [Scientific principles of complex ecologically-safe technology of mustard gas destruction]. AB - The principles of complex, ecologically-safe technology for the destruction of battle gas mustard were worked out. This technology was based on the reaction alkaline detoxication of mustard; the major component of reaction mixture obtained after detoxication was thiodiglycol. Thorough thiodiglycol mineralization was achieved by electrochemical treatment. Electrolysis products were biologically utilized in biosorber. PMID- 8637842 TI - [Biosensor models based on potentiometric and amperometric transducers for use in medicine, biotechnology, and environmental monitoring (review)]. AB - Various types of potentiometric and amperometric biosensors are characterized: microbial sensors with Gluconobacter oxydans cells with potentiometric (pH sensitive field-effect transistor) and amperometric (Clark-type) electrodes for determining glucose; a potentiometric enzymatic electrode with butyrylcholinesterase, which is used in the biosensor designed to detect pesticides; immunosensors with pH-sensitive field-effect transistors which detect the herbicide 2, 4-D; a biosensor for human immunoglobulin G; biosensors with anaerobic bacteria Clostridium thermocellum; chemical and enzymatic sensors containing a photosensitive membrane for determining ammonium ions and urea; and amperometric microbial sensors prepared with Pseudomonas cells for determining naphthalene, biphenyl, and polychlorinated benzoates. Practical applications of the developed models of biosensors to medicine, biotechnology, and environmental monitoring are discussed. PMID- 8637843 TI - Structural domains of P450-containing monooxygenase systems. AB - All known P450-containing monooxygenase systems share common structural and functional domain architecture. Apart from P450 itself, these systems can comprise several fundamentally different protein components or domains, all of which are shared by other multicomponent/multidomain enzyme systems with various functions: FAD flavoprotein or domain, FMN domain, Fe2S2 ferredoxin, Fe3S4 ferredoxin, and cytochrome b5. Either FMN domain, ferredoxins or cytochrome b5 serve as the electron transport intermediate between the FAD domain and P450. The molecular evolution of both P450-containing systems and of each particular component does not follow phylogeny in general. Gene fusion and horizontal gene transfer events can lead to the appearance of novel redox chains in the same manner that artificial chimeric proteins can be constructed by humans. Recent studies using genetic and protein engineering techniques to investigate the separate domains and their interaction are described. PMID- 8637844 TI - 1.85 A structure of anti-fluorescein 4-4-20 Fab. AB - The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4 4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1. PMID- 8637847 TI - Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling. AB - D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophilic eubacterium, Thermotoga maritima, is remarkably heat stable (Tm = 109 degrees C). In this work, we have applied homology modelling to predict the 3-D structure of Th.maritima GAPDH to reveal the structural basis of thermostability. Three known GAPDH structures were used as reference proteins. First, the rough model of one subunit was constructed using the identified structurally conserved and variable regions of the reference proteins. The holoenzyme was assembled from four subunits and the NAD molecules. The structure was refined by energy minimization and molecular dynamics simulated annealing. No errors were detected in the refined model using the 3-D profile method. The model was compared with the structure of Bacillus stearothermophilus GAPDH to identify structural details underlying the increased thermostability. In all, 12 extra ion pairs per subunit were found at the protein surface. This seems to be the most important factor responsible for thermostability. Differences in the non-specific interactions, including hydration effects, were also found. Minor changes were detected in the secondary structure. The model predicts that a slight increase in alpha-helical propensities and helix-dipole interactions also contribute to increased stability, but to a lesser degree. PMID- 8637845 TI - The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T. AB - A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113. Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R). The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 A resolution. Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain. The R-factors were reduced to 0.18 for both mutants. Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T. In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules. The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability. Denaturation by heating may be interpreted from these structural results. PMID- 8637846 TI - A simple protein folding algorithm using a binary code and secondary structure constraints. AB - We describe an algorithm to predict tertiary structures of small proteins. In contrast to most current folding algorithms, it uses very few energy parameters. Given the secondary structural elements in the sequence--alpha-helices and beta strands--the algorithm searches the remaining conformational space of a simplified real-space representation of chains to find a minimum energy of an exceedingly simple potential function. The potential is based only on a single type of favorable interaction between hydrophobic residues, an unfavorable excluded volume term of spatial overlaps and, for sheet proteins, an interstrand hydrogen bond interaction. Where appropriate, the known disulfide bonds are constrained by a square-law potential. Conformations are searched by a genetic algorithm. The model predicts reasonably well the known tertiary folds of seven out of the 10 small proteins we consider. We draw two conclusions. First, for the proteins we tested, this exceedingly simple potential function is no worse than others having hundreds of energy parameters in finding the right general tertiary structures. Second, despite its simplicity, the potential function is not the weak link in this algorithm. Differences between our predicted structures and the correct targets can be ascribed to shortcomings in our search strategy. This potential function may be useful for testing other conformational search strategies. PMID- 8637848 TI - Evolutionary dynamics of enzymes. AB - This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity. PMID- 8637849 TI - Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type. AB - Under anaerobic conditions, cytochromes P450 can reductively dehalogenate heavily halogenated hydrocarbons, such as one- and two-carbon organic solvents. This catalytic capacity has drawn attention to the potential use of engineered forms of P450s in the remediation of contaminated deep subsurface ecosystems. Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign, IL) and S.G. Sligar (personal communication) have observed recently that an active-site variant of cytochrome P450cam (F87W) dechlorinates pentachloroethane approximately three times faster than the wild-type enzyme. Molecular dynamics simulations have revealed that the mutant enzyme binding pocket remains smaller, and that pentachloroethane assumes configurations closer to the heme-Fe in the F87W mutant twice as often as in the wild-type enzyme. This result is consistent with a collisional model of dehalogenation, which agrees with experimental observations [Li and Wackett (1993) Biochemistry, 32, 9355-9361] that solutions containing wild-type P450cam dehalogenate pentachloroethane 100 times faster than those containing free heme. The simulations suggest that it is unlikely that Trp87 significantly stabilizes the developing negative charge on the substrate during carbon-halogen bond reduction. The design of improved microbial enzymes that incorporate both steric and electronic effects continues for use in remediating halogenated contaminants in situ. PMID- 8637850 TI - Functionally essential, invariant glutamate near the C-terminus of strand beta 5 in various (alpha/beta)8-barrel enzymes as a possible indicator of their evolutionary relatedness. AB - Twelve different (alpha/beta)8-barrel enzymes belonging to three structurally distinct families were found to contain, near the C-terminus of their strand beta 5, a conserved invariant glutamic acid residue that plays an important functional role in each of these enzymes. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif owing to their mutual evolutionary relatedness. For this purpose, the sequence region around the well conserved fifth beta-strand of alpha amylase containing catalytic glutamate (Glu230, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The isolated sequence stretches of the 12 (alpha/beta)8-barrels are discussed from both the sequence structural and the evolutionary point of view, the invariant glutamate residue being proposed to be a joining feature of the studied group of enzymes remaining from their ancestral (alpha/beta)8-barrel. PMID- 8637851 TI - Enhanced dead-end elimination in the search for the global minimum energy conformation of a collection of protein side chains. AB - Although the conformational states of protein side chains can be described using a library of rotamers, the determination of the global minimum energy conformation (GMEC) of a large collection of side chains, given fixed backbone coordinates, represents a challenging combinatorial problem with important applications in the field of homology modelling. Recently, we have developed a theoretical framework, called the dead-end elimination method, which allows us to identify efficiently rotamers that cannot be members of the GMEC. Such dead ending rotamers can be iteratively removed from the system under study thereby tracking down the size of the combinatorial problem. Here we present new developments to the dead-end elimination method that allow us to handle larger proteins and more extensive rotamer libraries. These developments encompass (i) a procedure to determine weight factors in the generalized dead-end elimination theorem thereby enhancing the elimination of dead-ending rotamers and (ii) a novel strategy, mainly based on logical arguments derived from the logic pairs theorem, to use dead-ending rotamer pairs in the efficient elimination of single rotamers. These developments are illustrated for proteins of various sizes and the flow of the current method is discussed in detail. The effectiveness of dead end elimination is increased by two orders of magnitude as compared with previous work. In addition, it now becomes feasible to use extremely detailed libraries. We also provide an appendix in which the validity of the generalized dead-end criterion is shown. Finally, perspectives for further applications which may now become within reach are discussed. PMID- 8637853 TI - Prediction of the antigenic sites of the cystic fibrosis transmembrane conductance regulator protein by molecular modelling. AB - Antibodies are powerful tools for studying the in situ localization and physiology of proteins. The prediction of epitopes by molecular modelling has been used successfully for the papilloma virus, and valuable antibodies have been raised [Muller et al. (1990) J. Gen. Virol., 71, 2709-2717]. We have improved the modelling approach to allow us to predict epitopes from the primary sequences of the cystic fibrosis transmembrane conductance regulator. The procedure involves searching for fragments of primary sequences likely to make amphipathic secondary structures, which are hydrophilic enough to be at the surface of the folded protein and thus accessible to antibodies. Amphipathic helices were predicted using the methods of Berzofsky, Eisenberg and Jahnig. Their hydrophobic hydrophilic interface was calculated and drawn, and used to predict the orientation of the helices at the surface of the native protein. Amino acids involved in turns were selected using the algorithm of Eisenberg. Tertiary structures were calculated using 'FOLDING', a software developed by R. Brasseur for the prediction of small protein structures [Brasseur (1995) J. Mol. Graphics, in press]. We selected sequences that folded as turns with at least five protruding polar residues. One important property of antibodies is selectivity. To optimize the selectivity of the raised antibodies, each sequence was screened for similarity (FASTA) to the protein sequence from several databanks. Ubiquitous sequences were discarded. This approach led to the identification of 13 potential epitopes in the cystic fibrosis transmembrane conductance regulator: seven helices and six loops. PMID- 8637852 TI - Common molecular scaffold for two unrelated RGD molecules. AB - The sequence arginine-glycine-aspartic acid (RGD) is important for recognition of cell adhesion proteins by cell surface receptors (integrins). This tripeptide sequence is present in a number of proteins including fibronectin, vitronectin, von Willebrand factor and fibrinogen. Specific and selective binding of the RGD sequence by different receptors suggests that the conformational orientation of the tripeptide is critical for stereochemical recognition. The crystal structures of two proteins that contain the RGD signal were determined: (i) the cell-binding type III module of fibronectin (FNIII10) and (ii) an anti-receptor antibody fragment (OPG2) that is a functional RGD ligand mimic with an RYD recognition site in the variable (VH) domain. Both of these modules are folded into beta barrels with two layers of antiparallel beta-sheets enclosing a hydrophobic core. Since these molecules each contain the RGD (RYD) sequence, there is a unique opportunity for direct structural comparison. The comparison has defined a common molecular scaffold in these two unrelated molecules. Within this framework, the RGD (RYD) sites are located in structurally related loops in the two modules, i.e. at one end of the scaffold in a long loop connecting the last two strands in one of the beta-sheets. This shared scaffold is used for the stereochemical presentation of the RGD site for receptor recognition. PMID- 8637854 TI - Redesigning the active site of Geotrichum candidum lipase. AB - Attempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et al., 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains approximately 10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties. PMID- 8637855 TI - Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography. AB - The bacterial production of recombinant rat calpain II has been improved greatly by the use of two compatible plasmids for the two subunits. The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements into pACYC177. This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these two vectors. A high level of activity was also obtained from a bicistronic construct containing both subunit cDNAs under the control of one T7 promoter. The addition of a C-terminal His-tag to the large subunit simplified purification without affecting subunit association or enzyme activity. The active site cysteine 105 was mutated to alanine, causing complete loss of activity. The yield of purified C105A-calpain II (80 + 21 kDa) dimer following three column chromatography steps was 10 mg/l of cell culture. This provides a purified calpain, stable to autolysis and oxidation, which is likely to facilitate crystallization in both the presence and absence of calcium. PMID- 8637858 TI - A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry. AB - Inflammatory infiltrates in tissue-specific autoimmune disease comprise a collection of T cells with specificity for an antigen in the target organ. These specific cells recruit a population of nonspecific T cells and macrophages. The rare tissue-specific T cells in the infiltrate have the capacity to regulate both the influx and the efflux of cells from the tissue. Administration of an altered peptide ligand for the specific T cell which triggers autoimmunity can lead to the regression of the entire inflammatory ensemble in a few hours. Interleukin 4 is a critical cytokine involved in the regression of the inflammatory infiltrate. PMID- 8637857 TI - The anatomy of T-cell activation and tolerance. AB - The mammalian immune system must specifically recognize and eliminate foreign invaders but refrain from damaging the host. This task is accomplished in part by the production of a large number of T lymphocytes, each bearing a different antigen receptor to match the enormous variety of antigens present in the microbial world. However, because antigen receptor diversity is generated by a random mechanism, the immune system must tolerate the function of T lymphocytes that by chance express a self-reactive antigen receptor. Therefore, during early development, T cells that are specific for antigens expressed in the thymus are physically deleted. The population of T cells that leaves the thymus and seeds the secondary lymphoid organs contains helpful cells that are specific for antigens from microbes but also potentially dangerous T cells that are specific for innocuous extrathymic self antigens. The outcome of an encounter by a peripheral T cell with these two types of antigens is to a great extent determined by the inability of naive T cells to enter nonlymphoid tissues or to be productively activated in the absence of inflammation. PMID- 8637856 TI - The molecular biology of apoptosis. AB - All multicellular organisms have mechanisms for killing their own cells, and use physiological cell death for defence, development, homeostasis, and aging. Apoptosis is a morphologically recognizable form of cell death that is implemented by a mechanism that has been conserved throughout evolution from nematode to man. Thus homologs of the genes that implement cell death in nematodes also do so in mammals, but in mammals the process is considerably more complex, involving multiple isoforms of the components of the cell death machinery. In some circumstances this allows independent regulation of pathways that converge upon a common end point. A molecular understanding of this mechanism may allow design of therapies that either enhance or block cell death at will. PMID- 8637859 TI - Viruses, cytokines, antigens, and autoimmunity. AB - To explain the pathogenesis of autoimmunity, we hypothesize that following an infection the immune response spreads to tissue-specific autoantigens in genetically predisposed individuals eventually determining progression to disease. Molecular mimicry between viral and self antigens could, in some instances, initiate autoimmunity. Local elicitation of inflammatory cytokines following infection probably plays a pivotal role in determining loss of functional tolerance to self autoantigens and the destructive activation of autoreactive cells. We also describe the potential role of interleukin 10, a powerful B-cell activator, in increasing the efficiency of epitope recognition, that could well be crucial to the progression toward disease. PMID- 8637860 TI - Checkpoints in the progression of autoimmune disease: lessons from diabetes models. AB - In the last few years, data from experiments employing transgenic models of autoimmune disease have strengthened a particular concept of autoimmunity: disease results not so much from cracks in tolerance induction systems, leading to the generation of anti-self repertoire, as from the breakdown of secondary systems that keep these cells in check. T cells with anti-self specificities are readily found in disease-free individuals but ignore target tissues. This is also the case in some transgenic models, in spite of overwhelming numbers of autoreactive cells. In other instances, local infiltration and inflammation result, but they are well tolerated for long periods of time and do not terminally destroy target tissue. We review the possible molecular and cellular mechanisms that underlie these situations, with a particular emphasis on the destruction of pancreatic beta cells in transgenic models of insulin-dependent disease. PMID- 8637862 TI - gamma/delta and other unconventional T lymphocytes: what do they see and what do they do? AB - T lymphocytes recognize specific ligands by clonally distributed T-cell receptors (TCR). In humans and most animals, the vast majority of T cells express a TCR composed of an alpha chain and a beta chain, whereas a minor T-cell population is characterized by the TCR gamma/delta. Almost all of our knowledge about T cells stems from alpha/beta T cells and only now are we beginning to understand gamma/delta T cells. In contrast to conventional alpha/beta T cells, which are specific for antigenic peptides presented by gene products of the major histocompatibility complex, gamma/delta T cells directly recognize proteins and even nonproteinacious phospholigands. These findings reveal that gamma/delta T cells and alpha/beta T cells recognize antigen in a fundamentally different way and hence mitigate the dogma of exclusive peptide-major histocompatibility complex recognition by T cells. A role for gamma/delta T cells in antimicrobial immunity has been firmly established. Although some gamma/delta T cells perform effector functions, regulation of the professional and the nonprofessional immune system seems to be of at least equal importance. The prominent residence of gamma/delta T cells in epithelial tissues and the rapid mobilization of gamma/delta T cells in response to infection are consistent with such regulatory activities under physiological and pathologic conditions. Thus, although gamma/delta T cells are a minor fraction of all T cells, they are not just uninfluential kin of alpha/beta T cells but have their unique raison d'etre. PMID- 8637863 TI - A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins. AB - The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery. PMID- 8637864 TI - The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head. AB - A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6 ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin. PMID- 8637865 TI - Temperature effects in hydrophobic interaction chromatography. AB - The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters. PMID- 8637866 TI - Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells. AB - A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src-associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection, Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication. PMID- 8637867 TI - Low-calcium-induced enhancement of chemical synaptic transmission from photoreceptors to horizontal cells in the vertebrate retina. AB - According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Our study shows that the enhancement of synaptic transmission between photoreceptors and horizontal cells of the vertebrate retina induced by low-calcium media is caused by an increase of calcium influx into presynaptic terminals. This paradoxical effect is accounted for by modifications of surface potential on the photoreceptor membrane. Since lowering extracellular calcium concentration may likewise enhance calcium influx into other nerve cells, other experimental observations of "calcium-independent" release may be reaccommodated within the framework of the classical calcium hypothesis without invoking unconventional processes. PMID- 8637861 TI - Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires. AB - Immunological self-tolerance is ensured by eliminating or inhibiting self reactive lymphocyte clones, creating physical or functional holes in the B- and T lymphocyte antigen receptor repertoires. The nature and size of these gaps in our immune defenses must be balanced against the necessity of mounting rapid immune responses to an everchanging array of foreign pathogens. To achieve this balance, only a fraction of particularly hazardous self-reactive clones appears to be physically eliminated from the repertoire in a manner that fully prevents their recruitment into an antimicrobial immune response. Many self-reactive cells are retained with a variety of conditional and potentially flexible restraints: (i) their ability to be triggered by antigen is diminished by mechanisms that tune down signaling by their antigen receptors, (ii) their ability to carry out inflammatory effector functions can be inhibited, and (iii) their capacity to migrate and persist is constrained. This balance between tolerance and immunity can be shifted, altering susceptibility to autoimmune disease and to infection by genetic or environmental differences either in the way antigens are presented, in the tuning molecules that adjust triggering set points for lymphocyte responses to antigen, or in the effector molecules that eliminate, retain, or expand particular clones. PMID- 8637868 TI - Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment. AB - A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA 2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM. PMID- 8637869 TI - beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger. AB - Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP) induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK 8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK 8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease. PMID- 8637870 TI - COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex. AB - Postnatal development and adult function of the central nervous system are dependent on the capacity of neurons to effect long-term changes of specific properties in response to neural activity. This neuronal response has been demonstrated to be tightly correlated with the expression of a set of regulatory genes which include transcription factors as well as molecules that can directly modify cellular signaling. It is hypothesized that these proteins play a role in activity-dependent response. Previously, we described the expression and regulation in brain of an inducible form of prostaglandin synthase/cyclooxygenase, termed COX-2. COX-2 is a rate-limiting enzyme in prostanoid synthesis and its expression is rapidly regulated in developing and adult forebrain by physiological synaptic activity. Here we demonstrate that COX 2 immunoreactivity is selectively expressed in a subpopulation of excitatory neurons in neo-and allocortices, hippocampus, and amygdala and is compartmentalized to dendritic arborizations. Moreover, COX-2 immunoreactivity is present in dendritic spines, which are specialized structures involved in synaptic signaling. The developmental profile of COX-2 expression in dendrites follows well known histogenetic gradients and coincides with the critical period for activity-dependent synaptic remodeling. These results suggest that COX-2, and its diffusible prostanoid products, may play a role in postsynaptic signaling of excitatory neurons in cortex and associated structures. PMID- 8637871 TI - Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage. AB - Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2' deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8 tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer. PMID- 8637873 TI - Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition. AB - RAG1 protein is essential for the activation of V(D)J recombination in developing lymphocytes (V, variable; D, diversity; J, joining). However, it has not been determined whether its role involves substrate recognition and catalysis. A single amino acid substitution mutation in the RAG1 gene has now been identified that renders its activity sensitive to the sequence of the coding region abutting the heptamer site in the recombination signal sequence. These results strongly imply that RAG1 interacts directly with DNA. PMID- 8637872 TI - Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes. AB - Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage. PMID- 8637874 TI - Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. AB - Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart. PMID- 8637875 TI - Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface. AB - Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges. PMID- 8637876 TI - Baculovirus-mediated gene transfer into mammalian cells. AB - This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells. PMID- 8637878 TI - Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms. AB - Intermittent electrical footshock induces c-fos expression in parvocellular neurosecretory neurons expressing corticotropin-releasing factor and in other visceromotor cell types of the paraventricular hypothalamic nucleus (PVH). Since catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla make up the dominant loci of footshock-responsive cells that project to the PVH, these were evaluated as candidate afferent mediators of hypothalamic neuroendocrine responses. Rats bearing discrete unilateral transections of this projection system were exposed to a single 30-min footshock session and sacrificed 2 hr later. Despite depletion of the aminergic innervation on the ipsilateral side, shock-induced up-regulation of Fos protein and corticotropin releasing factor mRNA were comparable in strength and distribution in the PVH on both sides of the brain. This lesion did, however, result in a substantial reduction of Fos expression in medullary aminergic neurons on the ipsilateral side. These results contrast diametrically with those obtained in a systemic cytokine (interleukin 1) challenge paradigm, where similar cuts ablated the Fos response in the ipsilateral PVH but left intact the induction seen in the ipsilateral medulla. We conclude that (i) footshock-induced activation of medullary aminergic neurons is a secondary consequence of stress, mediated via a descending projection transected by our ablation, (ii) stress-induced activation of medullary aminergic neurons is not necessarily predictive of an involvement of these cell groups in driving hypothalamic visceromotor responses to a given stressor, and (iii) despite striking similarities in the complement of hypothalamic effector neurons and their afferents that may be activated by stresses of different types, distinct mechanisms may underlie adaptive hypothalamic responses in each. PMID- 8637877 TI - Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis. AB - beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta 2m-containing amyloid fibrils to the pathogenesis of DRA. PMID- 8637880 TI - Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants. AB - Neuropathogenicity of poliovirus can be attenuated by mutations in the internal ribosomal entry site (IRES) within the 5' nontranslated region of its genome. The Sabin vaccine strains used in prevention of poliomyelitis carry such mutations in their IRES elements. In addition, mutations within the structural and nonstructural proteins of Sabin strains may equally contribute to the attenuation phenotype. Despite their effectiveness as vaccines, the Sabin strains retain a neuropathogenic potential in animal models for poliomyelitis and, at a very low rate, they can cause poliomyelitis in vaccine recipients. The elimination of the neurocytopathic phenotype was achieved through the exchange of the entire poliovirus IRES with its counterpart from human rhinovirus type 2 without affecting growth properties in nonneuronal cells. The attenuating effect of the human rhinovirus type 2 IRES within the context of a poliovirus genome has been mapped to the 3' portion of this genetic element. PMID- 8637879 TI - Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs. AB - In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels. PMID- 8637881 TI - Immunolocalization of estrogen receptor protein in the mouse blastocyst during normal and delayed implantation. AB - We previously showed that estrogen receptor (ER) mRNA is present in preimplantation mouse embryos. The apparent synthesis of ER mRNA by the blastocyst at the time of implantation when estrogen is required was of special interest. A demonstration of the presence of ER protein would support the idea that estrogen can act directly on the embryo. The mouse embryo at the blastocyst stage is differentiated into two cell types, the trophectoderm and the inner cell mass. To determine whether ER mRNA is translated into ER protein and its cell specific distribution, immunocytochemical analyses were performed in mouse blastocysts. ER protein was detected in all cell types of the normal, dormant, or activated blastocyst. To trace the fate of ER in these cell types, immunocytochemistry was performed in implanting blastocysts and early egg cylinder stage embryos developed in culture. Again, ER was detected in all cells of the implanting blastocyst. At the early egg cylinder stage, continued expression of ER was observed in cells derived from the inner cell mass or the trophoblast. In trophoblast giant cells, ER was concentrated in small regions of the nucleus, possibly the nucleoli, which was similar to that observed in dormant and activated blastocysts. The embryonic expression of ER at such early stages in a broad array of cells suggests that ER may have a general role during early development. PMID- 8637882 TI - Mapping striate and extrastriate visual areas in human cerebral cortex. AB - Functional magnetic resonance imaging (fMRI) was used to identify and map the representation of the visual field in seven areas of human cerebral cortex and to identify at least two additional visually responsive regions. The cortical locations of neurons responding to stimulation along the vertical or horizontal visual field meridia were charted on three-dimensional models of the cortex and on unfolded maps of the cortical surface. These maps were used to identify the borders among areas that would be topographically homologous to areas V1, V2, V3, VP, and parts of V3A and V4 of the macaque monkey. Visually responsive areas homologous to the middle temporal/medial superior temporal area complex and unidentified parietal visual areas were also observed. The topography of the visual areas identified thus far is consistent with the organization in macaque monkeys. However, these and other findings suggest that human and simian cortical organization may begin to differ in extrastriate cortex at, or beyond, V3A and V4. PMID- 8637883 TI - p140/c-Abl that binds DNA is preferentially phosphorylated at tyrosine residues. AB - EP is a DNA element found in the enhancer and promoter regions of several cellular and viral genes. Previously, we have identified the DNA binding p140/c Abl protein that specifically recognizes this element. Here we show that phosphorylation is essential for the p140/c-Abl DNA binding activity and for the formation of DNA-protein complexes. Furthermore, by 32P labeling of cells and protein purification, we demonstrate that in vivo the EP-DNA-associated p140/c Abl is a tyrosine phosphoprotein. By employing two different c-Abl antibodies, we demonstrate the existence of two distinct c-Abl populations in cellular extracts. p140/c-Abl is quantitatively the minor population, is heavily phosphorylated at both serine and tyrosine residues, and is active in autophosphorylation reactions. PMID- 8637884 TI - Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells. AB - Agrobacterium genetically transforms plant cells by transferring a single stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import. PMID- 8637885 TI - Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix. AB - Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites. PMID- 8637887 TI - GSP-1 genes are linked to the grain hardness locus (Ha) on wheat chromosome 5D. AB - An important determinant of wheat grain quality is the hardness of the grain. The trait is controlled by a major locus, Ha, on the short arm of chromosome 5D. Purified starch granules from soft-grained wheats have associated with them 15 kDa polypeptides called grain softness proteins (GSPs) or "friabilins." Genes that encode one family of closely related GSP polypeptides - GSP-1 genes - were mapped using chromosome substitution lines to the group 5 chromosomes. An F2 population segregating for hard and soft alleles at the Ha locus on a near isogenic background was used in a single-seed study of the inheritance of grain softness and of GSP-1 alleles. Grain softness versus grain hardness was inherited in a 3:1 ratio. The presence versus absence of GSPs in single seed starch preparations was coinherited with grain softness versus hardness. This showed that grain softness is primarily determined by seed, and not by maternal, genotype. In addition, no recombination was detected in 44 F2 plants between GSP 1 restriction fragment length polymorphisms and Ha alleles. Differences between hard and soft wheat grains in membrane structure and lipid extractability have been described and, of the three characterized proteins that are part of the mixture of 15-kDa polypeptides called GSPs, at least two, and probably all three, are proteins that bind polar lipids. The data are interpreted to suggest that the Ha locus may encode one or more members of a large family of lipid-binding proteins. PMID- 8637886 TI - Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus. AB - We recorded in the CA1 region from hippocampal slices of prion protein (PrP) gene knockout mice to investigate whether the loss of the normal form of prion protein (PrPC) affects neuronal excitability as well as synaptic transmission in the central nervous system. No deficit in synaptic inhibition was found using field potential recordings because (i) responses induced by stimulation in stratum radiatum consisted of a single population spike in PrP gene knockout mice similar to that recorded from control mice and (ii) the plot of field excitatory postsynaptic potential slope versus the population spike amplitude showed no difference between the two groups of mice. Intracellular recordings also failed to detect any difference in cell excitability and the reversal potential for inhibitory postsynaptic potentials. Analysis of the kinetics of inhibitory postsynaptic current revealed no modification. Finally, we examined whether synaptic plasticity was altered and found no difference in long-term potentiation between control and PrP gene knockout mice. On the basis of our findings, we propose that the loss of the normal form of prion protein does not alter the physiology of the CA1 region of the hippocampus. PMID- 8637888 TI - Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice. AB - Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced. PMID- 8637889 TI - Spontaneous avoidance behavior in Drosophila null for calmodulin expression. AB - The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses. PMID- 8637891 TI - Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa. AB - The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair. PMID- 8637890 TI - A long-range regulatory element of Hoxc8 identified by using the pClasper vector. AB - Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene. PMID- 8637892 TI - Constitutive production of granulocyte/macrophage colony-stimulating factor by hypodense mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes. AB - We recently described the development in vitro of cells with granules characteristic of eosinophils and basophils (hybrid granulocytes) from normal human cord blood mononuclear cells cultured for 14 days with recombinant human (rh) interleukin (IL)-3, rhIL-5, and a soluble basement membrane, Matrigel. Hybrid granulocytes constitutively produced granulocyte/macrophage colony stimulating factor (GM-CSF) and rapidly developed into eosinophils after the exogenous cytokines and Matrigel were removed. To characterize the developmental progression of hybrid granulocytes, cells were maintained for an additional 14 days in medium containing rhIL-3, rhIL-5, and Matrigel. After 28 days, 73% +/- 1% (mean +/- SEM; n = 6) of the nonadherent cells were mononuclear eosinophils, 13% +/- 3% were eosinophils with two or more nuclear lobes, 13% +/- 4% were hybrid granulocytes, and 0.2% +/- 0.1% were basophils. More than 90% of the mononuclear eosinophils were hypodense as determined by centrifugation through metrizamide gradients. After an additional 5 days of culture in medium without exogenous cytokines, 65% +/- 3% (n = 5) of the 28-day cells excluded trypan blue. In contrast, 2% +/- 1% of freshly isolated peripheral blood eosinophils survived 5 days of culture without exogenous cytokines (n = 5). Fifty percent conditioned medium from in vitro derived 28-day mononuclear eosinophils and 14-day hybrid granulocytes maintained the survival of 60% +/- 7% and 77% +/- 7%, respectively, of freshly isolated peripheral blood eosinophils for 72 h, compared with 20% +/- 8% survival in medium alone (n = 3). The eosinophil viability-sustaining activity of 50% mononuclear eosinophil-conditioned medium was neutralized with a GM-CSF antibody. A total of 88% of the 28-day cells exhibited immunochemical staining for GM-CSF. Thus, during eosinophilopoiesis, both hybrid eosinophil/basophil intermediates and immature mononuclear eosinophils exhibit autocrine regulation of viability due to constitutive production of GM-CSF. PMID- 8637893 TI - Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. AB - The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage. PMID- 8637894 TI - Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. AB - The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L arginine (L-NMMA) administration. Immunohistochemistry with a specific anti nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice. PMID- 8637895 TI - The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation. AB - The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins. PMID- 8637896 TI - Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG. AB - Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light. PMID- 8637897 TI - High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin, an analog 500 1000 times more potent: structure-activity relationship of daunosamine-modified derivatives of doxorubicin. AB - A convenient, high yield conversion of doxorubicin to 3'-deamino-3'-(2'' pyrroline-1''-yl)doxorubicin is described. This daunosamine-modified analog of doxorubicin is 500-1000 times more active in vitro than doxorubicin. The conversion is effected by using a 30-fold excess of 4-iodobutyraldehyde in anhydrous dimethylformamide. The yield is higher than 85%. A homolog of this compound, 3'-deamino-3'-(1'',3''-tetrahydropyridine-1''-yl)doxorubicin, was also synthesized by using 5-iodovaleraldehyde. In this homolog, the daunosamine nitrogen is incorporated into a six- instead of a five-membered ring. This analog was 30-50 times less active than its counterpart with a five-membered ring. A similar structure-activity relationship was found when 3'-deamino-3'-(3'' pyrrolidone-1''-yl)doxorubicin (containing a five-membered ring) and 3'-deamino 3'-(3''-piperidone-1''-yl)doxorubicin (with a six-membered ring) were tested in vitro, the former being 5 times more potent than the latter. To further elucidate structure-activity relationships, 3'-deamino-3'-(pyrrolidine-1''-yl)doxorubicin, 3'-deamino-3'-(isoindoline-2''-yl)doxorubicin, 3'-deamino-3'-(2''-methyl-2'' pyrroline-1''-yl)doxorubicin, and 3'-deamino-3'-(3''-pyrroline-1''-yl)doxorubicin were also synthesized and tested. All the analogs were prepared by using reactive halogen compounds for incorporating the daunosamine nitrogen of doxorubicin into a five- or six-membered ring. These highly active antineoplastic agents can be used for incorporation into targeted cytotoxic analogs of luteinizing hormone releasing hormone intended for cancer therapy. PMID- 8637898 TI - Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells. AB - Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp 650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells. PMID- 8637899 TI - Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function. AB - The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3' terminal nucleotides, and extended antisense RNA containing 17 additional 3' terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5' untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA. PMID- 8637900 TI - Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor. AB - Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5 integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process. PMID- 8637901 TI - The response regulator SprE controls the stability of RpoS. AB - In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter. PMID- 8637902 TI - Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis. AB - Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed. PMID- 8637903 TI - CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. AB - We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40 bearing cells in active lesions (perivascular infiltrates). Most of these CD40 bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS. PMID- 8637904 TI - Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex. AB - The human immunodeficiency virus type 1 transactivator protein, Tat, stimulates transcriptional elongation from the viral long terminal repeat. To test whether Tat associates directly with activated transcription complexes, we have used the lac repressor protein (LacR) to "trap" elongating RNA polymerases. The arrested transcription complexes were purified by binding biotinylated templates to streptaviridin-coated magnetic beads. Transcription complexes were released from the magnetic beads following cleavage of the templates with restriction enzymes and were immunoblotted with antibodies to Tat, LacR and RNA polymerase II. The Tat protein copurified with RNA polymerase bound to wild-type templates but did not copurify with transcription complexes prepared by using templates carrying mutations in the transactivation response element (TAR) RNA. We conclude that Tat and cellular cofactors become attached to the transcription complex during its transit through TAR. PMID- 8637905 TI - Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA. AB - Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 ( 10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands. PMID- 8637906 TI - The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast. AB - Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences. PMID- 8637907 TI - Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach. AB - Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N terminal amino acid sequence analysis of 7 tryptic fragments of the S pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH like RTK family, binds more than one receptor. PMID- 8637908 TI - The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. AB - The experiments reported here were designed to test the hypothesis that the two electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental. PMID- 8637909 TI - Intracortical and corticothalamic coherency of fast spontaneous oscillations. AB - We report that fast (mainly 30- to 40-Hz) coherent electric field oscillations appear spontaneously during brain activation, as expressed by electroencephalogram (EEG) rhythms, and they outlast the stimulation of mesopontine cholinergic nuclei in acutely prepared cats. The fast oscillations also appear during the sleep-like EEG patterns of ketamine/xylazine anesthesia, but they are selectively suppressed during the prolonged phase of the slow (<1 Hz) sleep oscillation that is associated with hyperpolarization of cortical neurons. The fast (30- to 40-Hz) rhythms are synchronized intracortically within vertical columns, among closely located cortical foci, and through reciprocal corticothalamic networks. The fast oscillations do not reverse throughout the depth of the cortex. This aspect stands in contrast with the conventional depth profile of evoked potentials and slow sleep oscillations that display opposite polarity at the surface and midlayers. Current-source-density analyses reveal that the fast oscillations are associated with alternating microsinks and microsources across the cortex, while the evoked potentials and the slow oscillation display a massive current sink in midlayers, confined by two sources in superficial and deep layers. The synchronization of fast rhythms and their high amplitudes indicate that the term "EEG desynchronization," used to designate brain-aroused states, is incorrect and should be replaced with the original term, "EEG activation" [Moruzzi, G. & Magoun, H.W. (1949) Electroencephalogr. Clin. Neurophysiol. 1, 455-473]. PMID- 8637910 TI - Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism. AB - Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500 s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction. PMID- 8637911 TI - Molecular mechanism of transmembrane signaling by the aspartate receptor: a model. AB - The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer. PMID- 8637912 TI - Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11. AB - Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma. PMID- 8637914 TI - Active control of waves in a cochlear model with subpartitions. AB - Multiscale asymptotic methods developed previously to study macromechanical wave propagation in cochlear models are generalized here to include active control of a cochlear partition having three subpartitions, the basilar membrane, the reticular lamina, and the tectorial membrane. Activation of outer hair cells by stereocilia displacement and/or by lateral wall stretching result in a frequency dependent force acting between the reticular lamina and basilar membrane. Wavelength-dependent fluid loads are estimated by using the unsteady Stokes' equations, except in the narrow gap between the tectorial membrane and reticular lamina, where lubrication theory is appropriate. The local wavenumber and subpartition amplitude ratios are determined from the zeroth order equations of motion. A solvability relation for the first order equations of motion determines the subpartition amplitudes. The main findings are as follows: The reticular lamina and tectorial membrane move in unison with essentially no squeezing of the gap; an active force level consistent with measurements on isolated outer hair cells can provide a 35-dB amplification and sharpening of subpartition waveforms by delaying dissipation and allowing a greater structural resonance to occur before the wave is cut off; however, previously postulated activity mechanisms for single partition models cannot achieve sharp enough tuning in subpartitioned models. PMID- 8637913 TI - Progression of human breast cancers to the metastatic state is linked to hydroxyl radical-induced DNA damage. AB - Hydroxyl radical damage in metastatic tumor DNA was elucidated in women with breast cancer, and a comparison was made with nonmetastatic tumor DNA. The damage was identified by using statistical models of modified base and Fourier transform infrared spectral data. The modified base models revealed a greater than 2-fold increase in hydroxyl radical damage in the metastatic tumor DNA compared with the nonmetastatic tumor DNA. The metastatic tumor DNA also exhibited substantially greater base diversity than the nonmetastatic DNA, and a progression of radical induced base damage was found to be associated with the growth of metastatic tumors. A three-dimensional plot of principal components from factor analysis, derived from infrared spectral data, also showed that the metastatic tumor DNA was substantially more diverse than the tightly grouped nonmetastatic tumor DNA. These cohesive, independently derived findings suggest that the hydroxyl radical generates DNA phenotypes with various metastatic potentials that likely contribute to the diverse physiological properties and heterogeneity characteristic of metastatic cell populations. PMID- 8637915 TI - Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor. AB - Transcriptional repression is an important component of regulatory networks that govern gene expression. In this report, we have characterized the mechanisms by which the immediate early protein 2 (IE2 or IE86), a master transcriptional regulator of human cytomegalovirus, down-regulates its own expression. In vitro transcription and DNA binding experiments demonstrate that IE2 blocks specifically the association of RNA polymerase II with the preinitiation complex. Although, to our knowledge, this is the first report to describe a eukaryotic transcriptional repressor that selectively impedes RNA polymerase II recruitment, we present data that suggest that this type of repression might be widely used in the control of transcription by RNA polymerase II. PMID- 8637916 TI - Vascular endothelial growth factor B, a novel growth factor for endothelial cells. AB - We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle. PMID- 8637917 TI - Multiplex genotype determination at a large number of gene loci. AB - To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way. PMID- 8637918 TI - CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. AB - Dendritic cells are potent antigen-presenting cells that initiate primary immune responses. Although dendritic cells derive from bone marrow stem cells, the intermediate stages in their development remain unknown. In this study, plastic adherent blood monocytes (CD14+, CD1a-) cultured for 7 days with granulocyte monocyte colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha were shown to differentiate into CD1a+ CD83+ dendritic cells. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were the most potent stimulatory cells in allogeneic mixed leukocyte reactions. The identification of specific culture conditions that generate large numbers of dendritic cells from purified monocytes uncovers an important step in dendritic cell maturation that will allow the further characterization of their role in autoimmune diseases, graft rejection, and human immunodeficiency virus infection. PMID- 8637919 TI - Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. AB - Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice. PMID- 8637920 TI - The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins. AB - We have isolated an Arabidopsis thaliana gene that codes for a receptor related to antifungal pathogenesis-related (PR) proteins. The PR5K gene codes for a predicted 665-amino acid polypeptide that comprises an extracellular domain related to the PR5 proteins, a central transmembrane-spanning domain, and an intracellular protein-serine/threonine kinase. The extracellular domain of PR5K (PR5-like receptor kinase) is most highly related to acidic PR5 proteins that accumulate in the extracellular spaces of plants challenged with pathogenic microorganisms. The kinase domain of PR5K is related to a family of protein serine/threonine kinases that are involved in the expression of self incompatibility and disease resistance. PR5K transcripts accumulate at low levels in all tissues examined, although particularly high levels are present in roots and inflorescence stems. Treatments that induce authentic PR5 proteins had no effect on the level of PR5K transcripts, suggesting that the receptor forms part of a preexisting surveillance system. When the kinase domain of PR5K was expressed in Escherichia coli, the resulting polypeptide underwent autophosphorylation, consistent with its predicted enzyme activity. These results are consistent with PR5K encoding a functional receptor kinase. Moreover, the structural similarity between the extracellular domain of PR5K and the antimicrobial PR5- proteins suggests a possible interaction with common or related microbial targets. PMID- 8637921 TI - Nonlinear control of heart rate variability in human infants. AB - Nonlinear analyses of infant heart rhythms reveal a marked rise in the complexity of the electrocardiogram with maturation. We find that normal mature infants (gestation greater than or equal to 35 weeks) have complex and distinctly nonlinear heart rhythms (consistent with recent reports for healthy adults) but that such nonlinearity is lacking in preterm infants (gestation > or = to 27 weeks) where parasympathetic-sympathetic interaction and function are presumed to be less well developed. Our study further shows that infants with clinical brain death and those treated with atropine exhibit a similar lack of nonlinear feedback control. These three lines of evidence support the hypothesis championed by Goldberger et al. [Goldberger, A.L., Rigney, D.R. & West, B.J. (1990) Sci. Am. 262, 43-49] that autonomic nervous system control underlies the nonlinearity and possible chaos of normal heart rhythms. This report demonstrates the acquisition of nonlinear heart rate dynamics and possible chaos in developing human infants and its loss in brain death and with the administration of atropine. It parallels earlier work documenting changes in the variability of heart rhythms in each of these cases and suggests that nonlinearity may provide additional power in characterizing physiological states. PMID- 8637923 TI - Excessive homoplasy in an evolutionarily constrained protein. AB - The evolution of monomorphic proteins among closely related species has not been examined in detail. To investigate this phenomenon, the glycerol-3-phosphate dehydrogenase (Gpdh) locus was sequence in a broad range of Drosophila species. Although purifying selection to remove amino acid variation is the dominant force in the evolution of Gpdh, some replacements have occurred. The sequences were compared in the context of the phylogeny of the genus, revealing a high proportion of amino acid parallelism and reversal (homoplasy) at four sites. The level of homoplasy is significantly greater than that seen in other proteins for which multiple sequences are available, showing that Gpdh is strongly constrained by both the number of amino acid differences and the types of changes allowed. These four sites evolve at a much higher rate than do the other variable positions in the protein, accounting for half of the interspecific amino acid replacements. However, unlike typical hypervariable sites, where multiple changes to several different amino acids are seen, evolutionary 'flip-flopping' between two amino acid states defines this new class of hypervariable site. PMID- 8637922 TI - Synaesthesia in phantom limbs induced with mirrors. AB - Although there is a vast clinical literature on phantom limbs, there have been no experimental studies on the effects of visual input on phantom sensations. We introduce an inexpensive new device--a 'virtual reality box'--to resurrect the phantom visually to study inter-sensory effects. A mirror is placed vertically on the table so that the mirror reflection of the patient's intact had is 'superimposed' on the felt position of the phantom. We used this procedure on ten patients and found the following results. 1. In six patients, when the normal hand was moved, so that the phantom was perceived to move in the mirror, it was also felt to move; i.e. kinesthetic sensations emerged in the phantom. In D.S. this effect occurred even though he had never experienced any movements in the phantom for ten years before we tested him. He found the return of sensations very enjoyable. 2. Repeated practice led to a permanent 'disappearance' of the phantom arm in patient D.S. and the hand became telescoped into the stump near the shoulder. 3. Using an optical trick, impossible postures--e.g. extreme hyperextension of the fingers--could be induced visually in the phantom. In one case this was felt as a transient 'painful tug' in the phantom. 4. Five patients experienced involuntary painful 'clenching spasms' in the phantom hand and in four of them the spasms were relieved when the mirror was used to facilitate 'opening' of the phantom hand; opening was not possible without the mirror. 5. In three patients, touching the normal hand evoked precisely localized touch sensations in the phantom. Interestingly, the referral was especially pronounced when the patients actually 'saw' their phantom being touched in the mirror. Indeed, in a fourth patient (R.L.) the referral occurred only if he saw his phantom being touched: a curious form of synaesthesia. These experiments lend themselves readily to imaging studies using PET and fMRI. Taken collectively, they suggest that there is a considerable amount of latent plasticity even in the adult human brain. For example, precisely organized new pathways, bridging the two cerebral hemispheres, can emerge in less than three weeks. Furthermore, there must be a great deal of back and forth interaction between vision and touch, so that the strictly modular, hierarchical model of the brain that is currently in vogue needs to be replaced with a more dynamic, interactive model, in which 're entrant' signalling plays the main role. PMID- 8637925 TI - Whooping cough epidemics in London, 1701-1812: infection dynamics, seasonal forcing and the effects of malnutrition. AB - Time series analysis of the London Bills of Mortality, 1701-1812, reveals that whooping cough appeared as a lethal endemic disease after 1700 with epidemics of progressively increasing amplitude after 1720. The interepidemic period changed from 5 years (1720-1750) to 3 years (1750-1785) before returning to 5 years during 1785-1812. The epidemiology of whooping cough can be described by the mathematics of linearized dynamic systems and the interepidemic interval is determined by population size and susceptibility. The latter was governed by fluctuating levels of malnutrition, which were directly associated with oscillations in the wheat prices. It is suggested that the epidemics were driven in 1720-1785 by fluctuating seasonal temperatures which interacted with oscillations in wheat prices to produce an oscillation in susceptibility, but after 1785 the dynamics escaped from the pattern predicted by mathematical theory and the epidemics were apparently driven only by the wheat prices which generated a regular oscillation in susceptibility. The results emphasize the importance of an adequate nutritive level in combating whooping cough in the Third World today where it remains a lethal disease in children because of immunodeficiency linked to fluctuating and severe malnutrition which is often a consequence of crop cycles. PMID- 8637926 TI - Cortical oscillations and the origin of express saccades. AB - The latencies of visually guided saccadic eye movement can form bimodal distributions. The 'express saccades' associated with the first mode of the distributions are thought to be generated via an anatomical pathway different from that for the second mode, which comprises regular saccades. The following previously published observations are the basis for a new alternative model of these effects: (i) visual stimuli can cause oscillations to appear in the electroencephalogram; (ii) visual stimuli can cause a negative shift in the electroencephalogram that lasts for several hundreds of milliseconds; and (iii) negativity in the electroencephalogram can be associated with reduced thresholds of cortical neurons to stimuli. In the new model both express and regular saccades are generated by the same anatomical structures. The differences in saccadic latency are produced by an oscillatory reduction of a threshold in the saccade-generating pathway that is transiently produced under certain stimulus paradigms. The model has implications regarding the functional significance of spontaneous and stimulus-induced oscillations in the central nervous system. PMID- 8637924 TI - Localization and functional analysis of human cortical area V5 using magneto encephalography. AB - Using a multi-channel SQUID-based neuromagnetometer, we have determined the location, temporal dynamics and functional response properties of the human homologue of the primate cortical area V5 (MT). We provide evidence that area V5 in humans is located near the occipito-temporal border in a minor sulcus immediately below the superior temporal sulcus. this area is selective for low spatial frequencies ( < or = 4.0 c/deg), responds to a wide range of temporal frequencies ( < or = 35 Hz) and shows response saturation for stimulus contrasts greater than 10%. In addition, we find that this area is not responsive to purely chromatic patterns but is responsive to motion-contrast stimuli. Our results are consistent with the hypothesis that area V5 in humans represents a stage of processing within the magnocellular pathway. We discuss our results in relation to the widespread belief that area V5 in humans is specifically concerned with motion perception. PMID- 8637927 TI - Development of cooperative territoriality in juvenile lions. AB - African lions, Panthera leo, engage in many cooperative activities including hunting, care of young, and group territoriality, but the contribution of juvenile lions to these activities has never been documented. Here we present experimental evidence that juvenile lionesses make a gradual transition to group territorial defence between weaning (8 months) and sexual maturity (42 months). When challenged by simulated intruders played from a loud-speaker, juvenile females (but not males) become progressively more likely to join the adult females in territorial defence with age, and their behaviour is affected by both the number of defending adults and the number of intruders. We interpret the ability of juveniles to assess relative numbers as an adaptation for assessing the risk of territorial conflict according to their own fighting ability, and the ability of their pride of successfully defend the territory. The difference between the sexes reflects the greater value of the natal territory to philopatric females. Adult females display a variety of strategies when defending the territory, including unconditional and conditional forms of cooperation. We show here that individuals display the rudiments of these strategies as juveniles. PMID- 8637928 TI - Molecular characterization of cyclic and obligate parthenogens in the aphid Rhopalosiphum padi (L.). AB - Holocyclic clones of the aphid Rhopalosiphum padi (L.) reproduce by cyclic parthenogenesis, whereas anholocyclic individuals are obligate parthenogens. Mitochondrial DNA and (mtDNA) and random amplified polymorphic DNA markers in R. padi as well as plasmid DNA markers of its bacterial endosymbiont, Buchnera aphidicola, were examined to determine the extent of genetic divergence between clones with these differing breeding systems. These analyses revealed that cyclically parthenogenetic lineages possessed differing mtDNA and plasmid haplotypes than most obligately asexual clones. The extent of sequence divergence between the maternally inherited molecules suggest a relatively ancient origin of asexuality. Our work also identified a random amplified polymorphic DNA marker linked to the life-cycle variation in R. padi. This marker not only permits the rapid diagnosis of breeding system but sets the stage for studies to identify the gene(s) controlling this variation in mode of reproduction. PMID- 8637929 TI - On the functional of neuromelanin. AB - The hypothesis is presented, based on the chemical structure of neuromelanin, that one of its functions in the catecholamine neurons in the brain is to protect the cell against toxic quinones (such as dopaminchrome and noradrenochrome, or their dihydroxy isomers) produced from the catecholamines dopamine and noradrenaline (and possibly adrenaline) during the course of prostaglandin synthesis by the enzyme prostaglandin H synthetase, or possibly by spontaneous oxidation. One aminochrome-adrenochrome-has been shown to be neurotoxic and to have psychotomimetic properties in humans. Depending on the site of production these compounds may be involved in the pathogenesis of Parkinson's disease or schizophrenia. PMID- 8637931 TI - Mobility of Australian flying-foxes, Pteropus spp. (Megachiroptera): evidence from genetic variation. AB - Black (Pteropus alecto) and grey-headed (Pteropus poliocephalus) flying-foxes inhabit large ranges in coastal north and eastern Australia. P. poliocephalus is endemic to region and is classified as vulnerable. The bats are known to migrate in response to flowering and fruiting of their food plants, but direct observation of movement patterns is difficult. Protein electrophoresis was used to investigate genetic subdivision among populations. High gene flow was inferred for both species with an estimated exchange of 15 (P. alecto) and 28 (P. poliocephalus) individuals between populations per generation. Wright's FST, an index of among population genetic variation, was low, 0.023 (P. alecto), 0.014 (P. poliocephalus), reflecting the homogenising action of movements across the species' ranges. An FST value of 0.028 has been reported for a third Australian species, the little red flying-fox, P. scapulatus. The FST values for Australian flying-foxes are closer to those found for birds than the typically higher values for mammals and we conclude that these flying-foxes are essentially panmictic and for management purposes should be treated as migratory species. PMID- 8637930 TI - Systemic administration of cholecystokinin (CCK) inhibits operant water intake in rats: implications for the CCK-satiety hypothesis. AB - The demonstration that intraperitoneal administration of the sulphated octapeptide of cholecystokinin (CCK-8S) inhibits food but not water intake in rats, has led to the hypothesis that endogenous peripheral CCK acts as a food specific satiety factor. As water-deprived rats given free access to water can satisfy their thirst fairly rapidly, it is conceivable that the apparent lack of effect of CCK on water intake reported previously may have been because the animals had satisfied their thirst before the full effects of the peptide had become apparent. To test this possibility, the effects of intraperitoneal (i.p.) administration of CCK-8S, at doses that had previously been shown to inhibit food intake, were investigated on water intake in rats trained to make operant responses for water reinforcements. Such a paradigm has the merit of slowing down the rate at which a water-deprived rat can quench its thirst, thus extending the period over which the effects of CCK-8S on water intake may be assessed. CCK-8S (2, 4 or 8 micrograms kg-1, i.p.) produced a dose-related suppression of operant water intake in 16 h water-deprived rats during the first 30 min after administration. Additional experiments indicated that, as with feeding, CCK-8S inhibits water intake by an action at peripheral CCKA receptors. These finding have important implications for the CCK-satiety hypothesis as they show that the effect of the peptide on ingestive behaviours in the rats is not specific for food intake and suggest that it is unlikely that CCK is a mediator of satiety. PMID- 8637932 TI - Location and line orientation as distinguishable primitives in spatial vision. AB - In geometry the same parameters define a straight line segment as its two endpoints, yet the human observer's perception of spatial relations can differ depending whether an actual line is shown or merely its terminators. Experiments are reported that demonstrate that the position and orientation senses can be largely decoupled. Because both point localization and line orientation each in its own way has a quality of immediacy, yet manifests high performance in the hyperacuity range, both must be regarded as a primitive each with its own neural processing mechanisms. This can give rise to perceptual dissonances (visual illusions) of which one example is analysed. PMID- 8637933 TI - Physical Therapy '96: scientific meeting and exposition. June 14-18, 1996. Abstracts. PMID- 8637936 TI - Cardiovascular risk-factor reduction in elderly patients with cardiac disease. AB - As a result of increased numbers of elderly persons in the United States, many of whom have coronary heart disease, the mean age of patients in coronary care programs following a myocardial infarction is now greater than age 65 years. One third of cardiac operations on adults are performed on elderly patients. Despite the fact that in the United States utilization of interventions such as coronary artery bypass grafting and angioplasty is on the rise in elderly patients, there appears to be some reluctance to manage risk factors in this group. The four major modifiable risk factors for the development and progression of coronary artery disease are hypertension, hypercholesterolemia, habitual smoking, and physical infectivity. The effectiveness of risk-factor modification for the secondary prevention of events of cardiac morbidity and mortality has been described for younger persons; however, less is known for elderly persons. The purpose of this review is to focus on these four major risk factors in elderly patients with cardiac disease by describing the effectiveness of modification of these factors, methods for doing so, and considerations for the future of such interventions. PMID- 8637935 TI - Physical activity in the prevention of cardiovascular disease. AB - Research during the past several decades confirms the health benefits of regular physical activity, including improved cardiovascular function and quality of life, with reduced risks of morbidity and mortality from cardiovascular heart disease (CHD). Health care professionals, including physical therapists, can assist the public by promoting physical activity and describing the type, quantity, and quality of activity that confers health benefits. Participation in vigorous physical activity for at least 30 minutes a day is recommended for cardiorespiratory fitness, but recent studies suggest that physical activity below this level can have benefits in reducing the risk for CHD. This article addresses the relationship between physical inactivity and the financial burdens on health care, the relationship between physical inactivity and the risks of developing CHD in different age groups, the health benefits associated with moderate levels of physical activity, and the amounts and types of physical activities that are needed to reduce the risk of CHD. PMID- 8637934 TI - The cardiopulmonary system and movement dysfunction. AB - Physical therapy primarily involves the identification and treatment of problems related to movement. Movement dysfunction usually is attributed to impairments of the neuromuscular and musculoskeletal systems. The cardiopulmonary system plays an important role in movement because of its function of transporting oxygen to skeletal muscle. Abnormalities of the cardiovascular and pulmonary systems can produce limitations in physical function. The purposes of this article are to describe the steps involved in the transfer of oxygen from atmospheric air to skeletal muscles and to provide examples of problems that can occur with each step of the process. Common signs and symptoms of potential problems involving the cardiovascular and pulmonary systems also will be discussed. PMID- 8637937 TI - Current trends in cardiovascular pharmacology. AB - To help resolve abnormalities in cardiovascular function, many different types of drugs have been developed. Only recently, however, has there been increased emphasis on determining flow these agents decrease morbidity and mortality. In some cases, standard drug therapy has been challenged somewhat by newer drugs or new applications of existing drugs that seem to provide better outcomes in terms of disease progression and survival. The purpose of this article is to provide an update on contemporary pharmacologic management of three common conditions: hypertension, congestive heart failure, and myocardial ischemia/infarction. Physical therapists should be aware of the rationale for using specific drugs in each condition and that these medications can have positive therapeutic effects and adverse side effects that can influence the patient's response to physical therapy. PMID- 8637938 TI - Cardiac Transplantation: a review. AB - Cardiac transplantation is now an accepted treatment for end-stage cardiac disease. To ameliorate the deconditioning that results from the preoperative disease state and to counteract or lessen the severity of the sequelae of postoperative immunosuppression, physical therapists are active participants in the rehabilitation of cardiac transplant recipients. Thus involvement requires a level of knowledge and understanding of the surgical procedures, pharmacology, and postoperative management with which the therapist may have,heretofore, been unexposed. This article reviews the development of cardiac transplantation and presents an overview of the current state of the art, with emphasis preoperative considerations, surgical and immediate postoperative care, and the effects of complications on selected exercise-related responses. PMID- 8637940 TI - The future of PT education. PMID- 8637939 TI - Heart failure. AB - Congestive heart failure (CHF) affects more than 2 million Americans and requires greater than 900,000 hospitalizations each year. The incidence of CHF is increasing because of the increasing age of the American population and the use of newer medications and technologies that have increased survival at the expense of increased morbidity. Physical therapists are treating many patients who have varying degrees of CHF. Recent research has demonstrated the importance of exercise conditioning in patients with CHF. This article will review the pathophysiology, compensatory mechanisms, and signs and symptoms of CHF as well as provide a discussion of physical therapy in the treatment of patients with CHF. PMID- 8637941 TI - Michael Shepherd (1923-1995). PMID- 8637942 TI - 'Under the influence' in British India: James Esdaile's Mesmeric Hospital in Calcutta, and its critics. AB - Mesmerism was for a period very popular in Victorian Britain. The special clinical approach developed by Dr J. Esdaile while on duty in British India is elaborated in detail. The controversy surrounding Esdaile's treatment of surgical, medical and psychiatric cases at the 'mesmeric hospital' at Calcutta is discussed, and the main arguments are set within their contemporary socio cultural context. Some of the arguments advanced for and against mesmerism contain concerns similar to those that have been raised during later decades in regard to hypnotism and hypnotherapy. PMID- 8637943 TI - The Royal Free interview for religious and spiritual beliefs: development and standardization. AB - We present the development and standardization of a measure of spiritual, religious and philosophical beliefs. An interview was constructed based on on going studies by the authors of the nature and strength of belief held by people hospitalized with an acute illness. The interview was tested with three standard populations--staff of a teaching hospital; attenders to an inner city general practice; and people with clearly defined, devout religious beliefs--in order to establish population norms, validity and reliability for each question. The interview performed well with satisfactory validity and high internal and test retest reliability. It is not presented, however, as a final product which will meet all needs in this complicated area of study. Rather, we have attempted to refine a measure of spiritual and religious belief that might apply to people with a range of personal and public faiths. It is clear that people are able to express these aspects of their lives in a way that can be measured with acceptable reliability and validity. We believe that this interview could, therefore, be applied in any medical, psychological or social setting in which a measure of belief is sought. PMID- 8637944 TI - Filters on the pathway to mental health care, I. Incident mental disorders. AB - This study investigates health and mental health services use by adults with and without newly incident mental disorders and uses prospectively gathered data from the Epidemiologic Catchment Area Program, a multi-site interview survey of adult household residents in the United States. Study subjects were 13,400 participants who completed interviews in the initial survey and also in a follow-up 1 year later, and who reported no contact with mental health services in either the specialized mental health sector or in the general medical sector in the 6 months prior to the initial survey. Case ascertainment was by means of a standardized interview method, the Diagnostic Interview Schedule. Separately, and before the mental health assessments were made, respondents were asked about their use of health and mental health services. 'Hierarchical' and 'filter' models of mental health services provide frameworks useful in understanding the possible effects of specific categories of psychiatric disturbances on use of health services. Only a minority of individuals with newly incident psychiatric disorder report discussion of mental health issues in a health care setting. Persons who developed a mental disorder during the follow-up interval were just as likely to consult a non-psychiatrist physician for their mental health problems as to consult a specialist in mental health, even accounting for other factors known to be associated with differential use of health care services. This large community study of incident psychiatric disorders strengthens prior evidence on the importance of the general medical sector in the care of individuals with psychiatric disturbances. PMID- 8637945 TI - Filters on the pathway to mental health care, II. Sociodemographic factors. AB - This study uses the prospectively gathered data of the Epidemiologic Catchment Area Program, a multi-site interview survey of mental disturbances among adult household residents in the United States, to compare health services use by individuals with different sociodemographic characteristics, accounting for the first-time occurrence of psychiatric disorder, over the course of a 1-year follow up interval. Case ascertainment was by means of a standardized interview method, the Diagnostic Interview Schedule. In the present investigation, 13,400 continuing participants in the household sample who reported no contact with mental health services in the 6 months prior to the initial interview were studied with regard to health services use and sociodemographic characteristics. Separately, and before the mental health assessments were made, respondents were asked about their use of health and mental health services. African-Americans were significantly less likely than whites to have consulted with a specialist in mental health (estimated relative odds, 0.22, 95% confidence interval 0.10 to 0.52), even accounting for coincident psychiatric disorder, gender, and other covariates known to be associated with differential use of health care services. Hispanics and other minorities were also less likely to have consulted a specialist in mental health (estimated relative odds, 0.37 and 0.26, respectively). This large community study extends previous work on mental health services and ethnicity. PMID- 8637946 TI - Remission and relapse in major depression: a two-year prospective follow-up study. AB - This paper reports the course with respect to remission and relapse of a cohort of predominantly in-patient RDC major depressive subjects, who were followed at 3 monthly intervals to remission and for up to 15 months thereafter. Remission was comparatively rapid with 70% of subjects remitting within 6 months. Only 6% failed to do so by 15 months. However, 40% relapsed over the subsequent 15 months, with all the relapses occurring in the first 10 months. Greater severity of the depression and longer duration of the illness predicted a longer time to remission. Greater initial severity of depression also predicted relapse. Subjects with a worse outcome had not received less adequate treatment than the remainder. Our results confirm the comparatively poor outcome subsequent to remission that has been reported in recent literature, in spite of the availability of modern methods of treatment. The clustering of relapses in the first 10 months gives some support to the distinction between relapse and later recurrence. PMID- 8637947 TI - Residual symptoms after partial remission: an important outcome in depression. AB - This paper draws attention to an important adverse outcome in depression, the occurrence of residual symptoms after partial remission. Among patients with definite major depression followed every 3 months to remission and thereafter, residual symptoms reaching 8 or more on the Hamilton Depression Scale 17-item total were present in 32% (19) of the 60 who remitted below major depression by 15 months. The pattern was of mild but typical depressive symptoms. Residual symptoms were more common in subjects with more severe initial illness, but were not related to any other predictors, including longer prior illness, dysthymia, or lower dose of drug treatment during the illness episode. There were weak associations with personality that might have been consequences of symptom presence. Residual symptoms were very strong predictors of subsequent early relapse, which occurred in 76% (13/17) of those with residual symptoms and 25% (10/40) of those without. PMID- 8637948 TI - Changes in rates of depressive symptoms in a Japanese working population: life table analysis from a 4-year follow-up study. AB - To investigate the effects of demographic variables on changes in rates of depressive symptoms in a non-clinical population, a 4-year follow-up study was conducted on a total of 6378 employees of a Japanese electrical company. Data were collected five times at 1-year intervals (T0-T4) using a questionnaire that included the Zung Self-rating Depression Scale (SDS). Of the non-depressed (i.e. having a SDS score of 47 or less) at baseline (N = 4857), 14% were found depressed at least once during T1-T4. Younger, never married, female and blue collar workers were significantly at greater risk for becoming depressed during the follow-up period (P < 0.05). Of the depressed at baseline (N = 505), 20% were depressed every time during T1-T4. Younger workers were significantly at greater risk for persistence of depressive symptoms during the follow-up period (P < 0.05). The results suggested that age is associated with both occurrence and persistence of depressive symptoms, while gender, marital status and occupation are associated only with the occurrence. PMID- 8637949 TI - The phenomenology and explanatory models of common mental disorder: a study in primary care in Harare, Zimbabwe. AB - In order to describe the explanatory models and the etic and emic phenomena of common mental disorder in Harare, Zimbabwe, 110 subjects were selected by general nurses in three clinics and by four traditional healers from their current clients. The subjects were interviewed using the Explanatory Model Interview and the Revised Clinical Interview Schedule. Mental disorder most commonly presented with somatic symptoms, but few patients denied that their mind or soul was the source of illness. Spiritual factors were frequently cited as causes of mental illness. Subjects who were selected by traditional healer, reported a greater duration of illness and were more likely to provide a spiritual explanation for their illness. The majority of subjects were classified as 'cases' by the etic criteria of the CISR. Most patients, however, showed a mixture of psychiatric symptoms that did not fall clearly into a single diagnostic group. Patients from a subgroup with a spiritual model of illness were less likely to conform to etic criteria of 'caseness' and they may represent a unique category of psychological distress in Zimbabwe. A wide variety of emic phenomena were elicited that have been incorporated in an indigenous measure of non-psychotic mental disorder. Kufungisisa, or thinking too much, seemed to be the Shona term closest to the Euro-American concept of neurotic illness. PMID- 8637950 TI - Proton magnetic resonance spectroscopy: an in vivo method of estimating hippocampal neuronal depletion in schizophrenia. AB - Diffuse loss of cortical volume and ventricular enlargement have been demonstrated in schizophrenia using imaging. In addition, histological studies have provided evidence that the number of neurons in the medial temporal lobe structures is reduced and that the cytoarchitecture is abnormal. In an attempt to correlate these histological findings with in vivo estimates of neuronal integrity we have studied the concentration of the neuronal marker N-acetyl aspartate (NAA) in the hippocampi of schizophrenics using in vivo Magnetic Resonance Spectroscopy (MRS). Compared with a group of healthy volunteers schizophrenics showed a 22% loss of NAA in the left hippocampus. Two other metabolites, choline and creatine showed bilateral reduction in schizophrenics and these achieved significance in the left hippocampus. These results indicate a significant depletion of NAA in schizophrenia and are in close agreement with the reported neuronal loss in the hippocampus detected histologically. We propose that in vivo MRS is a valid measure of integrity of neuronal populations in schizophrenia. PMID- 8637951 TI - Alzheimer's disease in the NAS-NRC Registry of aging twin veterans, IV. Performance characteristics of a two-stage telephone screening procedure for Alzheimer's dementia. AB - In the course of a large twin study of Alzheimer's disease we used a two-stage telephone screening procedure. The modified Telephone Interview for Cognitive Status (TICS-m) served as an initial screen for dementia in 12709 individuals. The telephone Dementia Questionnaire (DQ) was then asked of collateral informants for subjects with TICS-m scores below 28, as well as for samples of persons with higher TICS-m scores. Based upon DQ responses, individuals with cognitive impairment not attributable to focal causes underwent assessment for the clinical diagnosis of Alzheimer's disease ('Alzheimer's dementia'), as did their twins. Well-defined Alzheimer's dementia was apparent in 39 subjects. Employing a cut off of 27 or lower as indicative of cognitive impairment, the sensitivity of the TICS-m in the detection of Alzheimer's dementia was estimated at > 99% and specificity at 86%. Inclusion of the DQ increased the specificity at the 27/28 cut-point to 99%. The TICS-m score was associated with an area under the receiver operating characteristic (ROC) curve of 0.88 (95% confidence interval 0.81 to 0.94). The maximum number of cases of Alzheimer's dementia remaining undetected in the sample was estimated to be 34. PMID- 8637952 TI - Decline in working memory associated with HIV infection. HNRC Group. AB - HIV infection has been associated with decline in a number of cognitive functions that are components of 'working memory'. Thus, tests of working memory that require the interaction of these components may be particularly sensitive to cognitive dysfunction that arises from HIV infection. To assess this possibility, working memory was examined in 147 HIV-seropositive (HIV+) and 38 HIV seronegative (HIV-) males using the Reading Span Test and the Digit Span subtest from the Wechsler Memory Scale-Revised (WMS-R). Speed of information processing, a component of some working memory tasks, was assessed with a version of the Sternberg Memory Scanning task. Results indicated that symptomatic HIV+ subjects were impaired relative to HIV- control subjects on the Reading Span and Digit Span tests. Asymptomatic and mildly symptomatic HIV+ groups exhibited a trend toward impairment on these tests, and on the whole, a greater proportion of HIV+ subjects than HIV- subjects were impaired. The groups did not differ significantly in information processing speed. These results indicate that deficits in working memory are apparent in at least a subset of HIV-infected individuals. These deficits are most apparent in symptomatic HIV+ individuals, but the decline may begin during the asymptomatic phase of infection. PMID- 8637953 TI - Neuropsychological deficits in tests of executive function in asymptomatic and symptomatic HIV-1 seropositive men. AB - There has been much debate about the exact nature and time of onset of the cognitive impairments associated with infection by the human immunodeficiency virus type 1 (HIV-1). Studies to date have not reached consistent conclusions. The present study comprised 22 asymptomatic and 18 symptomatic HIV-1 seropositive men, whose only risk factor for contraction of the virus was sexual intercourse, and 18 seronegative controls matched for age and IQ. Subjects were given computerized neuropsychological tests from the CANTAB battery, which assessed visuospatial memory, attention and executive function. Both the asymptomatic and the symptomatic HIV-1 seropositive subjects showed a selective pattern of deficits relative to the controls. In addition, the seropositive subjects were subtly but significantly impaired on tests of executive function but unimpaired on certain tests of visual memory. This finding supports an hypothesis that frontostriatal dysfunction occurs in HIV-1 infected individuals prior even to the expression of clinical symptoms. PMID- 8637954 TI - Structural abnormalities of the brain in schizophrenia: sex differences in the Cantabria First Episode of Schizophrenia Study. AB - This paper examines structural brain abnormalities, as evaluated by the CT scan, in first episodes of schizophrenia and their association with sociodemographic, diagnostic and clinical variables. The investigation included all patients with a first episode of schizophrenia who, over a 2-year period, made contact with any of the public mental health services of the Autonomous Region of Cantabria in Northern Spain. Diagnostic and clinical characteristics were evaluated through the use of the Spanish version of the Present State Examination (PSE-9) and the Scales for the Assessment of Positive and Negative Symptoms (SANS and SAPS respectively). The study demonstrated the presence of structural brain abnormalities in this sample of first episode schizophrenics. These abnormalities were mainly expressed in the presence of larger VBR for schizophrenic patients than in the controls, these findings being more marked in women than in men. We failed to reveal, however, any evidence of an association of these brain abnormalities with diagnostic or clinical characteristics. PMID- 8637955 TI - Psychiatric diagnoses, sexual and physical victimization, and disability in patients with irritable bowel syndrome or inflammatory bowel disease. AB - We compared 71 patients with irritable bowel syndrome (IBS) and 40 patients with inflammatory bowel disease (IBD) using structured interviews for psychiatric, gastrointestinal and sexual/physical victimization histories, as well as self reported measures of personality, functional disability and dissociation. IBS patients had significantly higher lifetime prevalence rates of major depression, current panic disorder, and childhood sexual abuse. Despite the absence of organic pathology, IBS patients had significantly higher numbers of medically unexplained physical symptoms and disability ratings equal to, or greater than, those of patients with severe organic gastrointestinal disease. PMID- 8637956 TI - Procedural validity of the computerized version of the Composite International Diagnostic Interview (CIDI-Auto) in the anxiety disorders. AB - The procedural validity of the computerized version of the Composite International Diagnostic Interview (CIDI-Auto) was examined against the consensus diagnoses of two clinicians for six anxiety disorders (agoraphobia, panic disorder (+/- agoraphobia), social phobia, simple phobia, obsessive compulsive disorder (OCD), generalized anxiety disorder (GAD) and major depressive episode (MDE)). Clinicians had available to them all data obtained over a 2- to 10-month period. Subjects were 98 patients accepted for treatment at an Anxiety Disorders Clinic, thus, all subjects had at least one of the diagnoses being examined. While the CIDI-Auto detected 88.2% of the clinician diagnoses, it identified twice as many diagnoses as did the clinicians. The sensitivity of the CIDI-Auto was above 0.85 except for GAD, which had a sensitivity of 0.29. The specificity of the CIDI-Auto was lower (range: 0.47-0.99). The agreement between the CIDI Auto and the clinician diagnoses, as measured by intraclass kappas, ranged from poor (kappa = 0.02; GAD) to excellent (kappa = 0.81; OCD), with a fair level of agreement overall (kappa = 0.40). Canonical correlation analysis suggested that the discrepancies between the CIDI-Auto and clinicians were not due to different diagnostic distinctions being made. It is suggested that the CIDI-Auto may have a lower threshold for diagnosing anxiety disorders than do experienced clinicians. It is concluded that, in a sample where all subjects have at least one anxiety disorder diagnosis, the CIDI-Auto has acceptable validity. PMID- 8637957 TI - Development of a new scale for measuring acculturation: the Taiwan Aboriginal Acculturation Scale (TAAS). AB - As part of the Taiwan Aboriginal Study Project (TASP), a new acculturation scale (the Taiwan Aboriginal Acculturation Scale, or TAAS) has been developed among the aboriginal minorities of Austronesian origin in Taiwan. The design of the original 54 items was based on Milton Gordon's concept of assimilation in association with a careful consideration of cross-cultural validity. These items were administered to 144 subjects stratified by age and sex who were randomly sampled from four major Taiwanese aboriginal groups. Item analysis and factor analysis were applied to select an 18-item scale which has three subscales (factors): cultural assimilation, social assimilation, and social attitude. Results of validity and reliability studies of the TAAS were found to be acceptable. The development of TAAS demonstrates the applicability of the concept of acculturation as a process that involves changes both in attitude, and in behaviour, to non-western societies. PMID- 8637958 TI - The effect of prior stressful experience on coping with war trauma and captivity. AB - The study assessed the implications of childhood life events, Holocaust background, combat experiences, war captivity and negative post-captivity life events in the mental status of ex-POWs (164) and comparable controls (184), 18 years after the war. Findings confirm the association between stressful life events in the course of the life span and five outcomes: PTSD, intrusion and avoidance tendencies, psychiatric symptomatology, and impaired social functioning. Different life events have different effects. War captivity made the strongest contribution to all dependent variables. PMID- 8637959 TI - Education, gender and cognitive performance in a 62-year-old normal population: results from the Turva Project. AB - Four WAIS (Wechsler Adult Intelligence Scale) subtests, and tasks of memory and cognitive control were administered to a population sample of 143 men and 179 women. Subjects with a minor advantage in years of education out-performed those with only primary schooling. Gender-related effects were also remarkable. Whereas the general abilities were equal in the genders, women out-performed men on recall of word pairs and objects, on verbal cognitive control and on Digit Symbol. In contrast, men excelled on Trail Making A, and on Block Design. The findings emphasize the need for age norms by gender and education for cognitive tests. PMID- 8637961 TI - Motivated closing of the mind: "seizing" and "freezing". AB - A theoretical framework is outlined in which the key construct is the need for (nonspecific) cognitive closure. The need for closure is a desire for definite knowledge on some issue. It represents a dimension of stable individual differences as well as a situationally evocable state. The need for closure has widely ramifying consequences for social-cognitive phenomena at the intrapersonal, interpersonal, and group levels of analysis. Those consequences derive from 2 general tendencies, those of urgency and performance. The urgency tendency represents an individual's inclination to attain closure as soon as possible, and the permanence tendency represents an individual's inclination to maintain it for as long as possible. Empirical evidence for present theory attests to diverse need for closure effects on fundamental social psychological phenomena, including impression formation, stereotyping, attribution, persuasion, group decision making, and language use in intergroup contexts. PMID- 8637962 TI - Perceiving persons and groups. AB - This article analyzes the similarities and differences in forming impressions of individuals and in developing conceptions of groups. In both cases, the perceiver develops a mental conception of the target (individual or group) on the basis of available information and uses that information to make judgments about that person or group. However, a review of existing evidence reveals differences in the outcomes of impressions formed of individual and group targets, even when those impressions are based on the very same behavioral information. A model is proposed to account for these differences. The model emphasizes the role of differing expectancies of unity and coherence in individual and group targets, which in turn engage different mechanisms for processing information and making judgments. Implications of the model are discussed. PMID- 8637960 TI - Cognitive dissonance reduction as constraint satisfaction. AB - A constraint satisfaction neural network model (the consonance model) simulated data from the two major cognitive dissonance paradigms of insufficient justification and free choice. In several cases, the model fit the human data better than did cognitive dissonance theory. Superior fits were due to the inclusion of constraints that were not part of dissonance theory and to the increased precision inherent to this computational approach. Predictions generated by the model for a free choice between undesirable alternatives were confirmed in a new psychological experiment. The success of the consonance model underscores important, unforeseen similarities between what had been formerly regarded as the rather exotic process of dissonance reduction and a variety of other, more mundane psychological processes. Many of these processes can be understood as the progressive application of constraints supplied by beliefs and attitudes. PMID- 8637963 TI - Scintigraphic findings in schistosomiasis. AB - Schistosomiasis mansoni is a tropical parasitic disease caused by a blood fluke which inhabits the portal system of humans. Fifteen pediatric patients with the acute disease were evaluated with liver and spleen scintigraphy (LSS). Clinical history, physical examination, and serum chemistries failed to reveal any other underlying systemic disease. Liver and spleen scintigraphies were performed before therapy, 7 months and 9 years after therapy with oxamniquine. LSS initially showed hepatomegaly in 93% of the patients. In the first follow up study a reactive spleen was evident in 78% of the cases, with an unchanged hepatic image. Long term follow up revealed that from the initially enlarged livers, 93% became normal. However, 47% of the spleens were abnormal. The scintigraphic changes observed in the liver over the years were those expected for an acute infection. The findings in the spleen might indicate the persistence of an immunologic reaction with a continuous trigger, probably an antibody. These observations suggest that the LSS can be used in the evaluation and follow-up of these patients. PMID- 8637964 TI - The use of intraoperative autotransfusion as a second stage of autologous blood use in pediatric orthopaedic surgery. AB - The purpose of this study is to evaluate the use of Intraoperative Autotransfusion (IAT) as a second stage in the use of autologous blood to reduce the need of allogeneic transfusions. We reviewed the medical records of 41 pediatric patients who underwent spinal fusion procedures from September 1991, to August 1993. Our experience with IAT started in August 1992. The group of patients was divided into those who only received preoperative donation autologous blood (53.6%) and those who received preoperative donation autologous blood and IAT (46.4%). The use of preoperative autologous blood donation in combination with IAT has proven to be a safe method of operative blood loss replacement in pediatric age patients. Exclusive use of autologous blood can reduce or eliminate transfusion reactions and exposure to transfusion transmitted agents and helps to increase the blood reserve at blood banks. PMID- 8637965 TI - [Perforated appendicitis in children: evaluation of delayed diagnosis]. PMID- 8637966 TI - Dilated cardiomyopathy: a clinical review of patients evaluated at a tertiary care center in Puerto Rico. AB - OBJECTIVES: The purpose of this study was to determine the causes, epidemiologic baseline data and clinical characteristics of a group of patients referred to a tertiary care center in Puerto Rico with the diagnosis of dilated cardiomyopathy. METHODS: The medical records of 91 new patients with the diagnosis of dilated cardiomyopathy were analyzed. Data was recopilated regarding initial medical history and physical examination, basic blood chemistry, electrocardiograms, chest films and other cardiac studies including echocardiogram, radionuclear ventriculography, cardiac catheterization, contrast ventriculography and coronary angiography. After reviewing the records the etiology for dilated cardiomyopathy was identified in each patient following the World Health Organization Task Force criteria. RESULTS: Ischemic cardiomyopathy was identified as the most common cause of dilated cardiomyopathy, representing 37% of the reviewed patient population; an idiopathic etiology was found in 22% of the patients and valvular heart disease in 14%. DISCUSSION: A comparison with previously described series in the medical literature regarding this subject is presented, along with the most prominent clinical characteristics of the studied population. It is felt that the findings presented will require further study of the underlying causes and clinical course of this condition in a larger patient sample and a longer follow-up period. PMID- 8637967 TI - Shwachman-Diamond syndrome: the clinical imitator of cystic fibrosis. AB - In this case we report the autopsy findings of a four-month-old boy with the diagnosis of Shwachman-Diamond syndrome. The clinical and pathologic findings are discussed with emphasis on the differential diagnosis of cystic fibrosis and a brief review of current literature. PMID- 8637970 TI - The Office of the Student Ombudsman: its role in the medical sciences. PMID- 8637968 TI - Prevalence of cognitive and functional impairment in an elderly Puerto Rican population. AB - Data from the Gurabo census of the elderly, 1987-1988 (n = 1890) were analyzed to determine the prevalence rates for cognitive and functional impairment in that population. Besides socio-demographic questions, the census questionnaire included the Short Portable Mental Status Questionnaire to determine cognitive dysfunction, and the modified Katz Scale to detect functional impairment. The overall prevalence rates were 18.5% and 18.4% for cognitive and functional dysfunction respectively. After multiple logistic regression analysis, cognitive impairment was found to be associated with poor education (OR = 4.0, CI = 2.31 6.93), older age (OR = 2.67, C.I. = 2.00-3.58), functional decline (OR = 2.44, C.I. = 1.83-3.25), female sex (OR = 1.82, C.I. = 1.39-2.40) and low income (OR = 1.49, C.I. = 113-1.98). Functional impairment was found to be associated with cognitive dysfunction (OR = 2.45, C.I. = 1.84-3.27) and older age (OR = 2.08, C.I.-1.59 = 2,72). These findings suggest that a substantial proportion of the elderly in Puerto Rico may require assistance to deal with the consequences of these impairments. PMID- 8637969 TI - Functional disability and mental impairment as predictors of mortality in community-dwelling elderly Puerto Ricans. AB - This is a prospective study that explores the role of functional impairment as predictor of one-year mortality in the elderly of rural Puerto Rico. Between September, 1987 and June 1988, 1901 elderly were interviewed at their place of residence. Besides obtaining social and demographic information, assessment of functional status using a modified Katz scale and determination of mental status using the Short Portable Mental Status questionnaire were performed. During the one-year follow-up period 43 participants died. Multiple logistic regression analysis showed that the presence of functional (OR = 5.31) and mental impairment (OR = 2.22) were independent predictors of mortality when adjusted for age, education, gender and income. These findings are consistent with those of other studies, where the presence of functional and mental impairment, detected on admission to a hospital or while living in the community, were associated to a higher mortality. PMID- 8637971 TI - Inhibition of hematopoiesis by a plasma factor in a case of aplastic anemia associated with systemic lupus erythematosus. AB - Systemic Lupus Erythematosus (SLE) may be associated with inhibition of hematopoiesis mediated by antibodies, T-cells or both. A 41-year-old woman with a five-year history of SLE treated with prednisone was admitted to Cabrini Medical Center in New York. The patient complained of fever, chills, arthralgias, general malaise, weakness and dyspnea on exertion, and showed malar rash, pallor, and a systolic ejection murmur along the left sternal border. Admission work up included a CBC with evidence of moderate pancytopenia, a normal EKG, and a normal chest X-ray. The patient's anemia was symptomatic and required a transfusion of packed red blood cells (PRBC's). Bone marrow biopsy and aspiration revealed an aplastic marrow with few hypoplastic islands of hematopoietic elements. The patient was treated with plasmapheresis, achieving immediate progress towards recovery. Bone marrow culture studies (erythroid BFU-E, and myeloid CFU-GM) were done by incubating various titers of the patient's acute phase plasma with normal bone marrow cells. This was done to determine if the patient's plasma contained any hematopoietic inhibitory activity, as has been reported in other cases. Our experiments demonstrated marked inhibition of erymathropoiesis and myelopoiesis in vitro, when various titers of the patient's plasma were included in the culture media. Control plasma produced no inhibition. These studies support the hypothesis that a circulating antibody which inhibits hematopoiesis may be produced in SLE patients with aplastic anemia, and be responsible for it. PMID- 8637972 TI - Hearing loss. AB - Nearly 30 million Americans have hearing loss. Audiograms are used to determine if hearing loss is sensorineural (SNHL), conductive (CHL), or mixed (MHL). SNHL indicates dysfunction of the cochlea, cochlear nerve, or brain. The study of choice for adults with SNHL is gadolinium-enhanced magnetic resonance imaging, which is used to look for acoustic neuromas, abnormal labyrinth signal intensity or enhancement, and brain disease. In children with SNHL, computed tomography (CT) performed with bone algorithms demonstrates inner ear dysplasias. SNHL from trauma is best evaluated with CT studies. CHL indicates disease in the external auditory canal, tympanic membrane, or middle ear; CT with thin bone algorithms is the best study. Causes of CHL include external canal atresia and neoplasms, myringosclerosis, middle ear anomalies, effusion, cholesteatomas, and neoplasms. MHL is a combination of SNHL and CHL. Dysplasias such as otosclerosis and osteogenesis imperfecta, the most frequent diseases with radiologic findings, are best assessed on CT images. The combination of patient age and type of hearing loss determines the best imaging study. PMID- 8637973 TI - On measuring bone to predict osteoporotic fracture: moving beyond statistical inference. PMID- 8637974 TI - Retrospective reviews of breast cancer screening: what do we really learn from them? PMID- 8637975 TI - Thin-section CT, emphysema, air trapping, and airway obstruction. PMID- 8637976 TI - Comparing competing medical procedures: costs or charges--what should it matter? PMID- 8637977 TI - CT during arterial portography: comparison of injection into the splenic versus superior mesenteric artery. AB - PURPOSE: To determine whether the diagnostic quality of computed tomography (CT) during arterial portography (CTAP) performed via the splenic artery (SA) is better than that performed via the superior mesenteric artery (SMA). MATERIALS AND METHODS: The authors evaluated CTAP images obtained in 98 patients from 1991 to 1994; 47 examinations were performed via the SA and 51 were performed via the SMA. Images were reviewed, by consensus, by three radiologists blinded to catheter location. Hepatic enhancement was quantitatively assessed in 53 patients (31 in the SA group, 22 in the SMA group). RESULTS: The numbers of low attenuation non-tumor-related perfusion defects (19 in the SA group, 17 in the SMA group), high-attenuation non-tumor-related perfusion defects (six in the SA group, six in the SMA group), diffuse mottled perfusion abnormalities (six in the SA group, five in the SMA group), and portal venous flow defects (20 in the SA group, 20 in the SMA group) were similar in both groups (P > .05). Peak hepatic enhancement was similar in both groups (SMA group = 111 HU; SA group = 112 HU) (P > .05). CONCLUSION: There is no difference in quality between CTAP performed via the SA versus CTAP performed via the SMA. PMID- 8637978 TI - Acute mesenteric ischemia: diagnosis with contrast-enhanced CT. AB - PURPOSE: To evaluate the accuracy of dynamic, contrast material-enhanced computed tomography (CT) in the diagnosis of acute mesenteric ischemia. MATERIALS AND METHODS: Reviewers blinded to patient diagnoses retrospectively compared the CT scans in a study group with those in a control group. The study group comprised 39 consecutive patients (23 men, 16 women; aged 55-88 years) with surgically proved acute mesenteric ischemia. The control group comprised 24 patients (13 men, 11 women; aged 50-82 years) with suspected acute mesenteric ischemia that was disproved at surgery. RESULTS: For the diagnosis of acute mesenteric ischemia, each of the following findings had a specificity of more than 95% and a sensitivity of less than 30%: arterial or venous thrombosis, intramural gas, portal venous gas, focal lack of bowel-wall enhancement, and liver or splenic infarcts. When CT was used in the diagnosis of suspected acute mesenteric ischemia, the detection of at least one of these signs resulted in a sensitivity of 64% (25 of 39; confidence interval, 0.49, 0.79), a specificity of 92% (22 of 24; confidence interval, 0.81, 1.00), and an accuracy of 75% (47 of 63; confidence interval, 0.64, 0.86). CONCLUSION: Dynamic, contrast-enhanced CT is a valuable tool in the diagnosis of and determination of prognosis in acute mesenteric ischemia. PMID- 8637979 TI - Atherosclerotic renal artery stenosis: ostial or truncal? AB - PURPOSE: To evaluate a discrepancy between the location of renal artery stenoses on intraarterial digital subtraction angiographic (DSA) images and that on spiral computed tomographic (CT) angiograms. MATERIALS AND METHODS: The spiral CT angiograms and intraarterial DSA images of 40 consecutive patients with atherosclerotic renal artery stenoses were examined retrospectively. Stenoses were classified as truncal or ostial. The atherosclerotic changes in the abdominal aorta were graded. RESULTS: Fifty-eight stenoses were demonstrated. In 48 ostial stenoses, there was no discrepancy in the location of the stenoses on spiral CT angiograms and DSA images. In 10 patients, spiral CT angiography showed an ostial lesion, whereas DSA demonstrated an apparent truncal lesion. Most of these stenoses ("pseudotruncal" ostial stenoses) were in patients with severe aortic atherosclerotic disease. CONCLUSION: A renal artery stenosis at or within 10 mm of an atherosclerotic aorta at DSA may be diagnosed as an ostial stenosis. PMID- 8637980 TI - Flexible tantalum stents for the treatment of iliac artery lesions: long-term patency, complications, and risk factors. AB - PURPOSE: To evaluate the long-term success of tantalum stents implanted in iliac artery lesions and to determine potential predictive factors of early and late stent failure. MATERIALS AND METHODS: In 289 patients, flexible tantalum stents were implanted in iliac artery stenoses (n - 223) or occlusions (n - 66). Early and late stent failures were evaluated at 1-79 months (mean, 23 months). Four risk factors were evaluated: lesion type (occlusion vs stenosis), lesion location (common vs external iliac artery), lesion length (< 4 vs > 4 cm), and quality of runoff (good vs poor). RESULTS: The frequency of early stent thrombosis was significantly (P < .001) higher in occlusions (15.2%) versus stenoses (2.7%), in external (12.8%) versus common (1.1%) iliac arteries, in long (16.7%) versus short (0.5%) lesions, and in poor (14.0%) versus good (2.1%) runoff. At multivariate analysis, runoff and location were influencing factors. Primary patency rates at 3 and 5 years were 85% and 70%, respectively, for all stents. Three-year patency rates were significantly higher in short (88%) versus long (63%) lesions and in stenoses (92%) versus occlusions (63%). At multivariate analysis, lesion length was the only predictive factor for 3-year stent patency. CONCLUSION: Stent implantation offers valuable long-term treatment for atherosclerotic iliac artery disease. Success can be predicted on the basis of risk factors. PMID- 8637981 TI - Palliation of malignant esophageal strictures with self-expanding nitinol stents: drawbacks and complications. AB - PURPOSE: To evaluate the usefulness of self-expanding nitinol stents in the palliative treatment of malignant dysphagia. MATERIALS AND METHODS: Eighty self expanding nitinol stents were placed in 59 patients (43 men, 16 women; mean age, 55 years; age range, 23-75 years) with inoperable malignant stenosis due to squamous cell carcinoma of the esophagus (n = 36), adenocarcinoma (n = 19), invasion of the esophagus due to carcinoma of the lung (n = 2), and recurrent anastomotic carcinoma (n = 2). Dysphagia was graded on a scale of 0 to 3. Follow up esophagograms were obtained to evaluate stent patency. RESULTS: Stent placement was successful in all patients. The severity of dysphagia decreased at least one grade in all but one patient. Tumor ingrowth and overgrowth were seen in 21 (36%) patients 2 days to 7 months after stent placement and caused recurrent dysphagia. These 21 patients underwent balloon dilation and additional stent placement. A mediastinal fistula was seen in three patients (5%), ulceration in four (7%), stent torsion in three (5%), and incomplete expansion of the stent in two (2%). Repeat intervention was necessary in 51% of the patients. CONCLUSION: There is a substantial range of drawbacks and complications associated with the use of self-expanding nitinol stents for palliation of malignant esophageal strictures. A covering would be necessary to prevent tumor ingrowth. PMID- 8637982 TI - Detection and treatment of dysfunctional hemodialysis access grafts: effect of a surveillance program on graft patency and the incidence of thrombosis. AB - PURPOSE: To determine the value of a hemodialysis graft surveillance program in reducing the incidence of graft thrombosis and prolonging graft patency by means of early detection and percutaneous transluminal angioplasty (PTA) of graft related stenoses. MATERIALS AND METHODS: For 4-1/2 years, routine graft examination and measurement of several dialysis parameters were used to identify 106 cases of suspected graft dysfunction in 57 patients (56 men, one woman; aged 27-76 years). Graft-related stenoses detected with angiography were treated with PTA. RESULTS: Abnormal physical examination findings were the most common sole indication of graft dysfunction. Of the 106 cases referred for angiographic evaluation, 97 (92%) had at least one lesion. PTA was successful in 88 of 90 treated cases. The primary patency rates at 1 year were 16% for arteriovenous fistulas (AVFs) and 23% for polytetrafluoroethylene (PTFE) grafts. Early detection of stenoses by means of surveillance and repeated PTA enabled 1-year primary assisted patency rates of 67% for AVFs and 68% for PTFE grafts. The incidence of graft thrombosis fell from 48% in 1988 to 17% in 1994 (P < .001). CONCLUSION: The hemodialysis graft surveillance program resulted in a statistically significant reduction in the incidence of graft thrombosis. Although primary patency rates after PTA were low, repeated PTA of detected stenoses allowed good primary assisted patency rates. PMID- 8637983 TI - Transjugular intrahepatic portosystemic shunt procedure: efficacy of 10-mm versus 12-mm Wallstents. AB - PURPOSE: To compare results of transjugular intrahepatic portosystemic shunt (TIPS) placement with 10- and 12-mm Wallstents. MATERIALS AND METHODS: Forty-six TIPS procedures in 47 patients were retrospectively reviewed. Wallstents that were 10 mm in diameter were used in 23 patients, and those that were 12 mm in diameter were used in 23 patients. Immediate results were compared, which included initial portosystemic gradient and Doppler measurements of blood flow velocity through the shunt at 1 day. Long-term patency and velocities were also assessed. RESULTS: TIPS were successfully created in 46 of 47 patients (98%). In one patient in the 10-mm group, the portal vein could not be accessed. When compared with TIPS in the 10-mm group, TIPS placed in the 12-mm group required dilation to larger diameters (mean, 11.1 vs 9.2 mm; P < .0001) to achieve an identical target gradient of 10 mm Hg and exhibited lower 1-day velocities (mean, 1.3 m/sec vs 1.7 m/sec; P < .03). The 1-day occlusion rate was 17% (four of 23 patients) in the 12-mm group versus 0% in the 10-mm group (P < .02). Patient survival was statistically significantly less in the 12-mm group (P < .03). CONCLUSION: Twelve-millimeter Wallstents yield statistically significantly poorer short- and long-term results in TIPS procedures. This is most likely due to the decreased radial strength of the larger stent, which is 50% less than that of the 10-mm stent. PMID- 8637984 TI - Absence of coronary calcification on double-helical CT scans: predictor of angiographically normal coronary arteries in elderly women? AB - PURPOSE: To test the hypothesis that angiographically normal coronary arteries in elderly women are identifiable by the absence of coronary calcification on double helical computed tomographic (CT) scans. MATERIALS AND METHODS: Forty-eight consecutive women (age range, 60-76 years) underwent coronary angiography for chest pain evaluation, as well as double-helical CT. Thirty women (mean age, 65 years +/- 5) had coronary artery disease (CAD), defined as any angiographic disease, and 18 women (mean age, 66 years +/- 4) had angiographically normal coronary arteries. RESULTS: Women with angiographically normal coronary arteries had lower coronary calcification scores than those of patients with CAD: 5.7 +/- 11 versus 580 +/- 634, respectively (P = .0004). Seven women with angiographically normal coronary arteries demonstrated mild coronary calcification (score < 50). Of the 11 women without coronary calcification, none had CAD. Thus, the absence of coronary calcification on double-helical CT scans in elderly women was predictive of angiographically normal coronary arteries with 61% sensitivity, 100% specificity, and 85% accuracy. CONCLUSION: Double-helical CT is an accurate, noninvasive modality for diagnosing angiographically normal coronary arteries in elderly symptomatic women. PMID- 8637985 TI - Congenital heart disease in adults and adolescents: comparative value of transthoracic and transesophageal echocardiography and MR imaging. AB - PURPOSE: To compare the diagnostic value of transesophageal echocardiography and magnetic resonance (MR) imaging with that of transthoracic echocardiography in the evaluation of congenital heart disease in adults and adolescents. MATERIALS AND METHODS: Corresponding transesophageal echocardiographic and MR images obtained in 61 patients (aged 14-74 years; 28 female, 33 male) with congenital cardiac abnormalities were analyzed retrospectively. Results were compared with those of transthoracic echocardiography in all patients and were confirmed by using cardiac catheterization (n = 41) and/or surgery (n = 41). RESULTS: of the 344 abnormalities detected with at least one imaging technique, 259 were demonstrated with transthoracic echocardiography. Transesophageal echocardiography depicted the atrioventricular junction and atrial structures the best. MR imaging was the only technique to fully depict 31 extracardiac abnormalities. In complex cardiac defects, the combination of all three imaging techniques was best. CONCLUSION: Transesophageal echocardiography and MR imaging are beneficial complementary imaging techniques in adults or adolescents with congenital heart disease. PMID- 8637986 TI - Nonvisualization of the fetal gallbladder: frequency and prognostic importance. AB - PURPOSE: To assess the frequency of fetal gallbladder visualization through gestation and to determine the prognostic importance of nonvisualization. MATERIALS AND METHODS: Demonstration of the gallbladder was prospectively attempted in 578 consecutive second- and third-trimester obstetric ultrasound examinations. Data regarding gallbladder visualization were stratified into subgroups on the basis of estimated gestational age. Postnatal follow-up was performed in 80 fetuses with nonvisualization of the gallbladder. RESULTS: The gallbladder was seen on 477 of 578 (82.5%) fetal sonograms. The likelihood of gallbladder visualization increased with advancing gestational age, reaching a plateau of approximately 95% between 24 and 32 weeks. After 32 weeks, the frequency of visualization declined. Seventy-five of the 80 fetuses with nonvisualized gallbladders who underwent follow-up had normal outcomes. Except for one fetus with trisomy 21, all fetuses with abnormalities had relatively minor, non-life threatening problems that did not involve the gallbladder or biliary tract. CONCLUSION: Most fetuses with nonvisualization of the gallbladder have normal outcomes. The rate of nonvisualization of the fetal gallbladder is sufficiently high to undermine the utility of gallbladder visualization as a screen for fetal abnormality. PMID- 8637987 TI - MR imaging of physeal bars. AB - PURPOSE: To determine if three-dimensional (3D) rendered and projection images derived from magnetic resonance (MR) imaging data are advantageous for the preoperative assessment of physeal bars. MATERIALS AND METHODS: Fifteen patients with suspected physeal bars were examined with MR imaging. Gradient-echo sequences were used with both two-dimensional and 3D MR imaging techniques. The 3D rendered and projection images were then derived from the MR imaging data. Patients were categorized on the basis of the effect of MR images on the decision to perform physeal bar excision. RESULTS: MR findings had either a moderate (n = 7) or a major (n = 6) effect on the surgical treatment of 13 patients (87%). They had no effect in two patients (13%) and were misleading in none. CONCLUSION: MR imaging is an excellent modality for imaging physeal bars. Imaging data can be processed to yield both 3D rendered and projection physeal maps that are particularly useful in preoperative planning. PMID- 8637988 TI - Intussusception: US findings with pathologic correlation--the crescent-in doughnut sign. AB - PURPOSE: To determine the characteristic ultrasound (US) findings of intussusception and to explain its different components. MATERIALS AND METHODS: Three intussusceptions were surgically induced in pigs, and in vitro US scans were compared with the corresponding pathologic slices. US findings in 44 cases of pediatric intussusception confirmed by means of saline enema examination were analyzed. RESULTS: Axial images of intussusception showed a doughnut pattern. The hypoechoic external ring was formed by the everted returning limb of intussusceptum and, to a lesser degree, by the intussuscipiens. The doughnut's center varied according to the scan level. Scans obtained at the middle or at the base of the intussusception showed a characteristic hyperechoic crescent in all cases. This crescent was formed by the mesentery enclosing the entering limb of the intussusceptum, which the authors have termed the "crescent-in-doughnut sign". On scans obtained at the apex of the intussusception, the center was hypoechoic owing to the entering limb of the intussusceptum and the absence of the mesentery. CONCLUSION: The crescent-in-doughnut sign appears to be a characteristic feature of intussusception. PMID- 8637990 TI - Two-phase helical CT for pancreatic tumors: pancreatic versus hepatic phase enhancement of tumor, pancreas, and vascular structures. AB - PURPOSE: To quantitatively evaluate and validate a two-phase helical computed tomographic (CT) protocol for evaluation of pancreatic tumors. MATERIALS AND METHODS: Twenty-seven patients with pathologically proved pancreatic adenocarcinomas prospectively underwent two-phase CT examination with helical acquisition during the pancreatic phase (40-70 seconds after infusion of intravenous contrast material at 3 mL/sec) and the hepatic phase (70-100 seconds after infusion). Mean CT attenuation values of tumor, bordering pancreas, and all major peripancreatic vessels were obtained for both time intervals. RESULTS: Mean tumor-pancreas contrast was significantly greater during the pancreatic phase (67 HU +/- 19) than the hepatic phase (39 HU +/- 16) (P < .001) This was the result of both greater enhancement of normal pancreas and lower tumor enhancement during the pancreatic phase. Opacification of all vascular structures, including the portal vein, was also greater during the pancreatic phase (P < .001). CONCLUSION: Two-phase helical CT with pancreatic phase acquisition provides statistically significantly better pancreatic, arterial, and portal venous enhancement than that of hepatic phase imaging, with improved tumor-pancreas contrast. PMID- 8637989 TI - Inverted Meckel diverticulum: clinical, radiologic, and pathologic findings. AB - PURPOSE: To determine the clinical, radiologic, and pathologic findings of inverted Meckel diverticulum by retrospectively reviewing a large series of cases. MATERIALS AND METHODS: Among 84 cases of Meckel diverticulum, 18 (21%) were found at surgery to be inverted into the lumen of the bowel. Thirteen of these 18 (72%) cases were associated with small bowel intussusception and five (28%) were not. RESULTS: All 18 patients (median age at time of diagnosis, 32 years) were symptomatic, but the symptoms were subacute or chronic in 14 (78%). At barium examination in 15 cases, inverted diverticulum was depicted in 10 (67%) as a solitary, elongated, smoothly marginated, often club-shaped intraluminal mass in the distal ileum. At computed tomography (CT) in three cases, a central area of fat attenuation was surrounded by a thick collar of soft-tissue attenuation. At ultrasound (US) in two cases, a target-like mass contained a central area of increased echogenicity. At pathologic examination in all cases, the inverted sac contained mesenteric fat. CONCLUSION: Inverted Meckel diverticulum occurs more commonly than previously recognized and is associated with characteristic findings at barium examination, CT, and US. PMID- 8637991 TI - Small-cell carcinoma of the esophagus: radiographic findings. AB - PURPOSE: To determine the radiographic findings of small-cell carcinoma of the esophagus. MATERIALS AND METHODS: The authors retrospectively reviewed barium studies as well as medical and pathologic records for three cases of small-cell carcinoma of the esophagus contributed to the radiologic archives of the Armed Forces Institute of Pathology. RESULTS: Two patients presented with dysphagia and one with chest pain. In all three patients, barium studies revealed a smoothly marginated, sessile mass with a relatively flat central ulcer on the right postero-lateral wall of the midesophagus below the level of the carina. The masses all were 4-5 cm in diameter, and the ulcers were 2-3 cm in diameter. In all three patients, the results of endoscopy confirmed the presence of a sessile mass with central ulceration in the midesophagus. CONCLUSION: Small-cell carcinomas of the esophagus can have similar findings on barium studies. Although these findings are more likely to be caused by squamous-cell carcinoma, it is important to obtain endoscopic biopsy specimens, because preoperative histologic diagnosis of small-cell carcinoma can dramatically alter the management of these cases. PMID- 8637992 TI - Solid and papillary epithelial neoplasm of the pancreas: imaging-pathologic correlation on 56 cases. AB - PURPOSE: To evaluate the clinical, pathologic, and imaging findings of solid and papillary epithelial neoplasm (SPEN) of the pancreas and to correlate imaging and gross pathologic features. MATERIALS AND METHODS: A retrospective review was performed in 56 patients (53 female and three male patients aged 10-74 years [mean age at diagnosis, 25 years]) with pathologically proven SPEN of the pancreas. All patients underwent computed tomography (n = 49), ultrasonography (n = 31), or magnetic resonance (MR) imaging (n = 9). Tumor size, location, and imaging features were evaluated and correlated with gross pathologic and histologic features. RESULTS: Mean transverse diameter of these tumors was 9.0 cm (range, 2.5-17.0 cm). They were localized to the tail (n = 30), head (n = 18), and body (n = 8) of the pancreas. All tumors contained some degree of internal hemorrhage or cystic degeneration, and all were well encapsulated. Areas of hemorrhagic degeneration ranged from solid friable tumor to gelatinous or cystic cavities and therefore demonstrated variable imaging features. Calcification was noted in 16 patients. Fluid-debris levels were noted in 10 patients. CONCLUSION: Imaging studies of SPEN of the pancreas consistently demonstrate variable degrees of hemorrhagic degeneration. Calcification is common. Characteristic fluid-debris levels and signal intensities seen with MR imaging indicate blood products. In the appropriate clinical setting, these findings are useful in making a prospective diagnosis. PMID- 8637993 TI - Hepatic hemodynamic alterations after administration of oral CT contrast agents. AB - PURPOSE: To assess the effects of oral computed tomography (CT) contrast agents on hepatic hemodynamics with duplex Doppler ultrasound (US). MATERIALS AND METHODS: Thirty healthy subjects who fasted underwent duplex Doppler US of the hepatic artery and portal vein both before and after administration of oral CT contrast agents. Resistive indexes were determined for the hepatic arteries. Peak and mean velocity (centimeters per second) and mean diameter were calculated for the main portal vein. RESULTS: Statistically significant elevation of the resistive index (P < .01) of the hepatic arteries was observed after ingestion of oral CT contrast agents. Increase in the mean resistive index at 15 minutes was 12% in the proper hepatic arteries and 20% in the intrahepatic arteries. There was a postprandial increase in mean portal venous flow compared with baseline levels (1,110 mL/min +/- 210 vs 940 mL/min +/- 140) (P < .05). CONCLUSION: An elevation of hepatic artery resistance that is probably due to arterial vasoconstriction after ingestion of oral CT contrast agents can be observed with Doppler US. Hemodynamic changes in liver vasculature after ingestion of oral CT contrast agents may have as yet unknown implications for CT scanning of the liver. PMID- 8637994 TI - Effects of training and experience in interpretation of emergency body CT scans. AB - PURPOSE: To determine the effects of level of training and other factors on the rate of discrepant interpretation of emergency body computed tomographic (CT) scans by trainees and staff radiologists. MATERIALS AND METHODS: Five hundred ninety-eight consecutive emergency CT studies were prospectively interpreted by radiology residents or board-certified body imaging fellows over a 12-month period. Each interpretation was reviewed within 12 hours by an attending body CT radiologist. Major discrepancies between staff radiologists' and trainees' interpretations were defined and those with the potential to affect immediate patient therapy; minor discrepancies were defined ad those without such potential. The effects on discrepancy rates were examined for abnormal versus normal CT findings and trauma versus nontrauma cases. RESULTS: Major and minor discrepancy rates were 1.2% and 6.5%, respectively, between interpretations made by the trainee and the staff radiologist. Overall, fellows demonstrated statistically significantly lower discrepancy rates than did senior of junior residents (5.9%, 13.7%, and 13.3%, respectively). The discrepancy rate was higher when CT findings were abnormal than when they were normal (13.5% vs 2.6%). There were no differences between discrepancy rates for trauma and nontrauma cases. CONCLUSION: Experience appeared to decrease discrepancy rates. Trainees were more likely to miss findings than to read normal scans as abnormal. PMID- 8637995 TI - Free-hand technique with ordinary antisepsis in abdominal US-guided fine-needle punctures: three-year experience. AB - PURPOSE: To evaluate the adequacy of ordinary antisepsis in ultrasound (US) guided free-hand fine-needle puncture. MATERIALS AND METHODS: Diagnostic and therapeutic procedures (n = 573) were performed in 456 patients. No puncture attachments, sterile gloves, or drapes or covers were used. Before each procedure the transducer was cleaned with a solution of water and 70% alcohol. No needles were contaminated. Patients were monitored for 5 days to exclude sepsis. Subsequently, the patients underwent follow-up blood and laboratory testing, including testing for for hepatitis B and C markers and human immunodeficiency virus antibodies, every 3 months for 6 months. The operators underwent the same follow-up for the first 6 months and for an additional 6 months. RESULTS: No patient or operator presented with fever or sepsis or with negative viral or hepatitis markers that became positive during follow-up. CONCLUSION: Use of this free-hand US-guided technique with ordinary antisepsis is safe for patients and operators, and it allows savings in time and the cost of materials. PMID- 8637996 TI - Osteoporosis: association of recent fractures with quantitative US findings. AB - PURPOSE: To study the association of quantitative ultrasound (US) parameters and bone mineral density (BMD) in patients with and patients without recent fractures. MATERIALS AND METHODS: The authors studied 4,698 women (69 years or older) who had sustained 1,363 new fractures, including 106 hip fractures, during the 7 years prior to the study. Broadband ultrasound attenuation (BUA) and other velocity parameters were measured by means of quantitative US of the calcaneus. BMD was measured at the spine, hip, and calcaneus. RESULTS: The standardized age adjusted odds ratio for all fractures was 1.5 (95% confidence interval [CI] = 1.4, 1.7) for BUA and up to 1.6 (95% CI = 1.5, 1.7) for BMD. For hip fractures, the odds ratio was 1.9 (95% CI = 1.5, 2.4) for BUA and up to 2.6 (95% CI = 2.0,3.4) for BMD. Sensitivity and specificity with BUA, velocity parameters, and BMD were comparable. Results of multivariate analysis showed that both BUA and BMD were independently associated with fractures and that combined measurements improved sensitivity and specificity. CONCLUSION: Quantitative US parameters are strongly associated with risk of fracture and partly independent of BMD. This simple, low-cost, portable, and radiation-free approach may complement bone densitometry in assessing risk of osteoporotic fracture. PMID- 8637997 TI - Tarsometatarsal joint: anatomic details on MR images. AB - PURPOSE: To evaluate the diagnostic capabilities of magnetic resonance (MR) imaging in the tarsometatarsal ([TMT] Lisfranc) joint with close anatomic correlation. MATERIALS AND METHODS: Six normal cadaveric feet were imaged by using T1-weighted spin-echo (oblique axial) and three-dimensional spoiled gradient-recalled acquisition in the steady state ([SPGR] coronal, sagittal) sequences. Subsequently, gadolinium-enhanced arthrography was performed in three specimens followed by T1-weighted spin-echo and SPGR MR imaging. Specimens were sectioned in all three planes followed by correlation of the MR imaging results with gross anatomic findings. RESULTS: In all specimens, the oblique axial and, less effectively, the coronal and sagittal planes allowed visualization of the Lisfranc ligament. Intermetatarsal ligaments were seen almost exclusively on the coronal images, and TMT ligaments on the sagittal images. Bone alignment could be assessed on the oblique axial images. CONCLUSION: MR imaging reliably depicts the anatomy of the TMT joint including ligamentous and osseous structures. PMID- 8637998 TI - Acromial morphology: relation to sex, age, symmetry, and subacromial enthesophytes. AB - PURPOSE: To evaluate acromial shape in relation to age, sex, symmetry, and presence of subacromial enthesophytes. MATERIALS AND METHODS: Three hundred ninety-four cadaveric scapulas were reviewed. Specimens were categorized by sex and age (age range, 20-89 years). Acromial morphology was typed according to the Bigliani classification: type I, flat; type II, curved; and type III, hooked. The presence and degree of subacromial enthesopathy was recorded. Selective radiographic correlation was obtained. RESULTS: The relative percentages of acromial types I, II, and III were 22.8% (90 acromions), 68.5% (270 acromions), and 8.6% (34 acromions), respectively. There was a greater percentage of type III in men (10.2% [21 of 205] vs 6.9% [13 of 189] and type I in women (27.5% [52 of 189] vs 18.5% [38 of 205]). There was no relationship between acromial type and age (P = .667). Enthesophytes were most common in type III (20 [59%] of 34 acromions) versus type II (115 [42.6%] of 270 acromions) and type I (22 [24%] of 90 acromions). Acromial morphology was symmetric in 135 (70.7%) of 191 pairs of acromions and asymmetric in 56 pairs (29.3%). CONCLUSION: Acromial shape does not vary significantly with age. It does, however, differ between sexes. The relative percentages of the types differ from previously reported values. Acromial shape tends to be symmetric. A trend between acromial type and the presence of enthesophytes is observed. PMID- 8637999 TI - Neuroblastoma: positron emission tomography with 2-[fluorine-18]-fluoro-2-deoxy-D glucose compared with metaiodobenzylguanidine scintigraphy. AB - PURPOSE: To assess the uptake in neuroblastoma of 2-[fluorine-18] -fluoro-2-deoxy D-glucose (FDG) versus metaiodobenzylguanidine (MIBG). MATERIALS AND METHODS: Seventeen patients with known or suspected neuroblastoma underwent FDG positron emission tomography (PET) (20 scans) and MIBG scintigraphy. Tumor uptake of FDG was quantified on positive PET scans. RESULTS: Tumor uptake of FDG was detected in 16 of 17 patients (18 of 20 scans). Neuroblastomas and their metastases avidly concentrated FDG prior to chemotherapy or radiation therapy. Uptake after therapy was variable. Uptake of FDG was intense in one patient with neuroblastoma that failed to accumulate MIBG. In 13 of the 20 scans, however, MIBG was rated superior to FDG for delineation of tumor compared with background and normal organs. CONCLUSION: Most neuroblastomas accumulate FDG. The mechanism of MIBG uptake is more intense prior to therapy. Concentration of FDG is not dependent on type 1 catecholamine uptake. FDG PET helps define the distribution of neuroblastomas that fail to concentrate MIBG. PMID- 8638000 TI - Metastatic prostate cancer: initial findings of PET with 2-deoxy-2-[F-18]fluoro-D glucose. AB - PURPOSE: To evaluate the accuracy of positron emission tomography (PET) with 2 deoxy-2-[fluorine-18] fluoro-D-glucose (FDG) in the detection of osseous and soft tissue metastases of prostate cancer. MATERIALS AND METHODS: Thirty-four patients (mean age, 71 years) with biopsy-proved prostate cancer and known or suspected metastatic disease were examined. Blinded interpretation of the PET images was compared with bone scan, CT, and clinical follow-up findings. RESULTS: In 202 untreated osseous metastases in 22 patients, the sensitivity of FDG PET was 65% (131 of 202 metastases), with a positive predictive value of 98% (131 of 133 positive findings). The estimated standardized uptake value in metastases was 2.1 5.7. Soft-tissue metastases to the lymph nodes or liver were identified, but evaluation of pelvic lymph node metastases was severely limited because of bladder tracer activity. CONCLUSION: FDG PET can help identify osseous and soft tissue metastases of prostate cancer with a high positive predictive value but is less sensitive than bone scintigraphy in the identification of osseous metastases. PMID- 8638001 TI - Intrauterine adhesions: detection with transvaginal US. AB - PURPOSE: To evaluate the reliability of transvaginal ultrasonography (US) in the diagnosis of uterine adhesions. MATERIALS AND METHODS: Transvaginal US was performed before hysteroscopy as part of the routine diagnostic work-up in 77 women who had repeated spontaneous abortions. The sensitivity, specificity, and positive and negative predictive values were calculated for transvaginal US in the diagnosis of uterine adhesions. Hysteroscopic findings were considered the reference. RESULTS: Uterine adhesions were correctly identified with transvaginal US in 10 of 11 women in whom this finding was subsequently confirmed at hysteroscopy. The adhesions were minimal in 10 instances and moderate in one. The sensitivity of transvaginal US was 91%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 98.5%. CONCLUSION: Transvaginal US, which is a noninvasive and relatively inexpensive procedure, seems to be effective in screening for uterine adhesions in a population at risk. PMID- 8638002 TI - Occult primary tumors of the head and neck: detection with 2-[F-18] fluoro-2 deoxy-D-glucose SPECT. AB - PURPOSE: To evaluate 2-[fluorine-18]fluoro-2-deoxy-D-glucose (FDG) single photon emission computed tomography (SPECT) in the detection of clinically occult primary tumors of the upper aerodigestive tract. MATERIALS AND METHODS: Eighteen patients with histologically proved cervical nodal metastasis from squamous cell carcinoma (SCC) underwent FDG SPECT; 17 also underwent computed tomography (CT) and one underwent magnetic resonance (MR) imaging. All 18 patients underwent direct panendoscopy and biopsy. Biopsy and imaging results were correlated. RESULTS: Among 11 histologically proved primary tumors, FDG SPECT depicted nine tumors; CT depicted four lesions. Five patients had abnormal FDG uptake with negative biopsy results. Among five patients with normal SPECT findings, biopsy results were positive in two and negative in three. FDG SPECT at the initial reading had sensitivity of 81%, specificity of 38%, positive predictive value of 64%, and negative predictive value of 60% for detection of occult primary tumors. CONCLUSION: FDG SPECT guidance of endoscopic biopsies in patients with occult primary SCCs of the head and neck has the potential to yield a higher rate of positive biopsy results than that traditionally expected from "blind" endoscopic procedures with "random" or "speculative" biopsy. FDG SPECT and CT are likely to remain complementary studies for the detection of occult primary tumors of the head and neck. PMID- 8638003 TI - Sacral plexus: optimal imaging planes for MR assessment. AB - PURPOSE: To identify the optimal imaging planes for magnetic resonance (MR) evaluation of the sacral plexus (SP) and proximal sciatic nerve (SN). MATERIALS AND METHODS: The SPs of 10 health adult volunteers were prospectively studied with T1-weighted MR imaging with custom-built pelvic phased-array coils. The conspicuity of 12 anatomic characteristics (comprising the SP and their relationship to normal pelvic anatomy) on a series of coronal, axial, and oblique images was graded. Results were evaluated with the Kruskal-Wallis and Wilcoxon signed rank tests. RESULTS: At least two planes were necessary to assess the anatomy of the SP and SN. Analysis of average conspicuity scores showed that the direct coronal and direct axial planes were the best overall and were superior to other imaging planes in the demonstration of the L-4 and L-5 ventral rami, the lumbosacral trunk, the S-1 contribution to the SN, and the SN in the greater sciatic foramen. The sacral coronal plane was best for the visualization of the bony sacrum, sacral foramina, and proximal S-1 to S-4 nerve roots. The remaining imaging planes had limited utility. CONCLUSION: MR imaging with a combination of direct coronal and direct axial planes enables thorough evaluation of all components of the SP and proximal SN. PMID- 8638004 TI - MR relaxation times in human brain: measurement at 4 T. AB - PURPOSE: To determine the values for relaxation times in human brain for magnetic resonance (MR) imaging at 4 T. MATERIALS AND METHODS: T1 measurements were made with a progressive saturation sequence, an implementation of the Look-Locker sequence, and an inversion-recovery (IR) interleaved echo-planar imaging (IEPI) sequence. T2 measurements were made with a standard spin-echo (SE) sequence and an SE IEPI sequence. RESULTS: The T1 measurements yielded values of 1,724 msec +/ 51 for gray matter, 1,043 msec +/- 27 for white matter, and 4,550 msec +/- 800 msec for cerebrospinal fluid. The deep gray matter regions had T1 values of 1,458 +/- 38 (caudate nucleus) and 1,372 +/- 60 (putamen). The T2 measurements yielded results of 63 msec +/- 6.2 for gray matter and 49.8 msec +/- 2.2 for white matter. CONCLUSION: The T1 values measured at 4 T show a higher value than predicted from extrapolation at lower field strengths. The T2 measurements showed a slight decrease in values over those measured at lower-field strength. The gain in signal-to-noise ratio from the higher field strength may be substantially offset by these altered relaxation time values to a degree that is sequence dependent. PMID- 8638005 TI - MR imaging contrast in human brain tissue: assessment and optimization at 4 T. AB - PURPOSE: To evaluate multiple magnetic resonance (MR) imaging sequences for their ability to provide T1 and T2-weighted images at a field strength of 4 T, and to validate previously obtained relaxation time measurements. MATERIALS AND METHODS: Different spin-echo, inversion-recovery (IR), gradient-recalled acquisition in the steady state (GRASS), and magnetization transfer contrast-enhanced GRASS sequences were evaluated in a single section, each in at least four volunteers. Also, interleaved echo-planar imaging (IEPI) and interleaved gradient-recalled echo (IGRE) sequences were analyzed and compared with standard sequences. RESULTS: Predicted contrast behavior, according to MR relaxation time measurements, was found to agree well with that of the validation experiments. Fair T1 contrast can be achieved on MR images at 4 T, contrary to early predictions. Under other conditions, however, such as partially spin-density weighted parameters, lower contrast is observed at the high field strength when compared with conventional field strengths. CONCLUSION: The longer T1 values at higher field strengths have a substantial effect on image contrast. Depending on the sequence and parameters chosen, good image contrast can be realized at 4 T. IEPI and hybrid IR IGRE sequences are useful in shortening the prolonged examination times owing to the longer T1 values at higher field strengths. PMID- 8638006 TI - Hippocampal MR imaging morphometry by means of general pattern matching. AB - PURPOSE: To determine the repeatability and validity of a pattern-matching method for the segmentation and measurement of hippocampi on magnetic resonance (MR) images. MATERIALS AND METHODS: Comparable two-dimensional MR images obtained in 18 subjects (nine healthy control subjects [six men, three women; aged 24-54 years] and nine patients with schizophrenia [six men, three women; aged 22-61 years]) were twice segmented manually and twice segmented by using pattern matching with digital atlas transformation. The atlas transformation was accomplished in two steps: global followed by local matching. Global matching was performed with use of landmarks; local matching was performed with use of a viscous fluid model. RESULTS: The mean percentage of difference between two atlas based measurements was 1.33% +/- 1.23 (+/- standard deviation); that between two manual measurements was 4.67% +/- 4.71. The validity of the atlas transformation measurements was demonstrated by means of the high correlation (intraclass correlation coefficient = .96) with manual segmentation measurements. Schizophrenic hippocampal areas tended to be smaller; however, no differences in hippocampal shape were found between patients with schizophrenia and patients with control subjects. CONCLUSION: General pattern matching of a digital brain atlas to an individual MR image is a mathematically robust method of measurement that is reproducible and less variable than manual measurement. PMID- 8638007 TI - MR imaging of Creutzfeldt-Jakob disease. AB - PURPOSE: To describe the magnetic resonance (MR) imaging appearance of Creutzfeldt-Jakob disease (CJD). MATERIALS AND METHODS: MR images obtained in 29 patients who died of CJD (aged 53-77 years at death) were retrospectively reviewed by three neuroradiologists blinded to the diagnosis. RESULTS: Moderate to marked bilateral, symmetrically increased signal intensity was demonstrated in the putamen and caudate nucleus on T2- and proton-density-weighted MR images in 23 patients (79%). In six patients (21%), images showed no major signal intensity abnormalities. T1-weighted images revealed no signal intensity abnormalities and no contrast material enhancement. The degree of atrophy in the cortex and basal ganglia corresponded to the time between onset of symptoms and MR imaging. All patients with a disease duration of longer than 4 months had substantial volume loss. CONCLUSION: Although approximately 20% of the patients did not have MR imaging abnormalities, MR imaging did show signal intensity alterations due to gliosis and spongiform changes early in the course of CJD in the remaining 80%. The demonstration of bilateral areas of increased signal intensity that predominantly affected the caudate nuclei and the putamina on long-repetition time MR images in an elderly patient with rapidly progressive dementia represents a specific finding and clearly should suggest the diagnosis of CJD. PMID- 8638008 TI - T2 shortening in the motor cortex: effect of aging and cerebrovascular diseases. AB - PURPOSE: To evaluate the effect of aging and cerebrovascular diseases on T2 shortening in the motor cortex at magnetic resonance (MR) imaging. MATERIALS AND METHODS: High-field-strength (1.5-T) MR images of 298 neurologically normal patients (157 male patients, 141 female patients; age range, 2-86 years) and 107 patients with cerebrovascular diseases (66 men, 41 women; age range, 41-86 years) were evaluated retrospectively. On T2-weighted spin-echo images, the signal intensities of the motor, sensory, parietal, temporal, and prefrontal cortices were divided into three grades compared with the signal intensity of the frontal subcortical white matter. RESULTS: Decreased signal intensity (grade III) was not seen in any region in the neurologically normal patients younger than 60 years. After the age of 60 years, however, the signal intensity of the motor cortex decreased, and 43 (66%) of 65 neurologically normal patients reached grade III by age 80 years. In patients with cerebrovascular disease, grade III was seen in 12 (34%) of the 35 patients younger than 60 years (5th and 6th decades). CONCLUSION: T2 shortening of the motor cortex was seen frequently in the older neurologically normal individuals and in patients with cerebrovascular diseases. These findings are compatible with those of a previously reported histochemical study of normal iron deposition in the motor cortex. PMID- 8638009 TI - HIV-related metabolic abnormalities in the brain: depiction with proton MR spectroscopy with short echo times. AB - PURPOSE: To analyze brain metabolite changes in human immunodeficiency virus (HIV)-positive neurologically asymptomatic patients and patients with acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). MATERIALS AND METHODS: Twelve ADC patients, 11 HIV-positive asymptomatic patients, and 10 healthy control subjects underwent magnetic resonance (MR) imaging and proton MR spectroscopy with short echo times. Changes in N-acetyl aspartate (NAA), choline (Cho), myoinositol (MI), and creatine (Cr) were presented as ratios with Cr as the reference. RESULTS: Statistically significant (P < .05) differences were noted. In ADC patients, all with MR findings of brain atrophy and diffuse white matter disease, NA/Cr decreased (white matter, -31.8%; gray matter, -22.3%), MI/Cr increased (white matter, +42.5%; gray matter, +51.8%), and Cho/Cr increased (white matter, +20.6%; gray matter, +51.7%) compared with healthy control subjects. In HIV-positive asymptomatic patients, NA/Cr decreased slightly (white matter, -6.9%; gray matter, -5.1%), MI/Cr increased slightly (white matter, +13.7%; gray matter, +10.7%), and Cho/Cr did not change. CONCLUSION: ADC has a uniform pathologic metabolic pattern that affects NAA, MI, and Cho. Proton MR spectroscopy with short echo times helps detect early abnormalities in clinically asymptomatic patients. PMID- 8638010 TI - Screening interval breast cancers: mammographic features and prognosis factors. AB - PURPOSE: To review the mammographic features of screening interval breast cancers and to compare the tumor size, histologic grade, and lymph node involvement with those in screening-detected and unscreened symptomatic cancers. MATERIALS AND METHODS: Screening mammography was performed in 72,773 women aged 50-64 years. Ninety interval cancers were identified in 89 women. The mammographic and histopathologic features of these cancers were analyzed. RESULTS: At review of the screening mammograms, interval cancers were classified into four groups: 51 true-positive, 20 false-negative, seven mammographically occult, and 12 unclassified. The most common missed abnormality in the false-negative cases was architectural distortion. Interval cancers were larger, of higher grade, and more likely to have lymph node involvement than screening-detected tumors and were of similar size, histologic grade, and stage of lymph node involvement as symptomatic tumors. CONCLUSION: Prognosis in interval cancers is similar to that in symptomatic, unscreened tumors and statistically significantly worse than that in screening-detected cancers. PMID- 8638011 TI - Invasive breast cancer: mammographic measurement. AB - PURPOSE: To establish the accuracy of mammographic measurement of tumor size for invasive carcinoma. MATERIALS AND METHODS: For 85 invasive cancers, mammographic tumor size was determined as the largest dimension observed on any mammographic projection (craniocaudal, lateral, or mediolateral oblique). This was then compared with the largest tumor dimension in the gross specimen. RESULTS: The relationship between the mammographic size and the pathologic size was almost exactly 1:1, with low variability. Mammographic measurements were larger by an average of about 1 mm. Neither breast parenchymal pattern nor the presence of accompanying ductal carcinoma in situ affected accuracy. CONCLUSION: Mammography can allow tumor size to be measured accurately and can be used as an alternative when pathologic staging is not possible. PMID- 8638012 TI - Pulmonary emphysema: quantitative CT during expiration. AB - PURPOSE: To determine whether measurement of the relative area of lung with attenuation coefficients lower than a certain threshold on thin-section computed tomographic (CT) scans obtained during expiration is a valuable method of quantifying the extent of pulmonary emphysema. MATERIALS AND METHODS: Eighty-nine patients underwent CT (with 1-mm collimation) preoperatively during inspiration and expiration. Relative areas of lung with attenuation coefficients lower than various thresholds were calculated. These relative areas were compared with areas found macroscopically to have emphysema (59 patients [51 men, eight women; aged 40-77 years]) and with two microscopic indices (35 patients [29 men, six women; aged 42-77 years]) assessed on the resected specimens. RESULTS: The valid expiratory CT thresholds were found to be -820 and 910 HU for microscopic and macroscopic emphysema, respectively. However, results of stepwise multiple regression analyses showed that the inspiratory threshold of -950 HU was superior for both macroscopically and microscopically quantified emphysema. The correlation coefficients in expiratory CT were higher for the pulmonary volumes but similar for the diffusing capacity. CONCLUSION: Expiratory quantitative CT is not as accurate as inspiratory CT for quantifying pulmonary emphysema and probably reflects air trapping more than reduction in the alveolar wall surface. PMID- 8638013 TI - Bronchial wall thickness: appropriate window settings for thin-section CT and radiologic-anatomic correlation. AB - PURPOSE: To analyze the influence of computed tomographic (CT) window settings on bronchial wall thickness and to define appropriate window settings for its evaluation. MATERIALS AND METHODS: Three inflation-fixed lungs were scanned with a section thickness of 1.5 mm by using a high-spatial-frequency algorithm. Wall thickness in 10 bronchial specimens was measured with planimetry. Window centers were altered in a range of -200 to -900 HU and window widths in a range of 400 1,500 HU. Relative and absolute differences between CT and planimetric values were calculated. CT and planimetric measures were correlated. Inter- and intraobserver variabilities were determined. RESULTS: Window widths less than 1,000 HU resulted in a substantial overestimation of bronchial wall thickness, whereas widths greater than 1,400 HU resulted in an underestimation of bronchial wall thickness. There was no interaction between "width" and "center" regarding their influence on bronchial walls (F = 0.23; P = .99). Correlation between CT and planimetry was statistically significant (r = .85; P = .0001). Differences between the two observers were not statistically significant; results of the measurements of the two observers correlated well (r = .97; P = .001). CONCLUSION: Bronchial wall thickness on thin-section CT scans should be evaluated with window centers between -250 and -700 HU and with window widths greater than 1,000 HU. Other than window settings, notably window widths less than 1,000 HU, can lead to substantial artificial thickening of bronchial walls. PMID- 8638014 TI - Pulmonary nodules: effect on detection of spiral CT pitch. AB - PURPOSE: To compare spiral computed tomography (CT) performed at increased pitch with spiral CT performed at standard pitch in the detection of pulmonary nodules. MATERIALS AND METHODS: Spiral CT scanning of the thorax was performed with a pitch of 1.0 in 109 patients with pulmonary nodules due to metastases. The patients were also randomly assigned to undergo further scanning with a pitch of 1.2 (n = 34), 1.5 (n = 37), 2.0 (n = 38) at the same scanning session. The scan pairs were analysed for number, size, and distribution of nodules. RESULTS: A bias toward undercounting was noted on scans with a pitch of 1.5 and 2.0; however, this was not statistically significant. Correlation coefficients were r = .982, r = .977, and r = .989 for scans of pitch 1.2, 1.5, and 2.0, respectively. Disease in one patient would have been prospectively understaged from findings on a scan of pitch 2.0 because of poor conspicuity of a small solitary nodule. CONCLUSION: Findings from scans with increased pitch generally agree well with those from scans with standard pitch; however, there is a greater risk of understaging of disease in patients with solitary nodules as pitch increases. Pitch should be limited to no greater than 1.5 for initial staging of pulmonary metastatic disease. PMID- 8638015 TI - Effect of a computer-aided diagnosis scheme on radiologists' performance in detection of lung nodules on radiographs. AB - PURPOSE: To evaluate the effect of a computer-aided diagnosis (CAD) scheme on radiologists' performance in the detection of lung nodules, and to examine a new method of receiver operating characteristic (ROC) analysis. MATERIALS AND METHODS: One hundred twenty radiographs (60 normal and 60 abnormal with lung nodules of varying subtlety) were used. Sixteen radiologists (two thoracic, six general, and eight residents) participated in an observer study in which they read both conventional radiographs and digitized radiographs. The radiologists' performance was evaluated with ROC analysis with two different methods (independent testing and sequential testing) and a continuous rating scale. RESULTS: Az (area under the best fit binormal ROC curve when it is plotted in the unit square) values obtained from ROC analysis with and without CAD output were 0.940 and 0.894, respectively, in the independent test and 0.948 and 0.906, respectively, in the sequential test. Findings with both methods indicated that the CAD scheme statistically significantly improved diagnostic accuracy, particularly for radiologists with less experience (P < .001). Reading time was not increased when CAD was used. CONCLUSION: The CAD scheme can assist radiologists in the detection of lung nodules on chest radiographs. PMID- 8638016 TI - Bronchial dehiscence after lung transplantation: correlation of CT findings with clinical outcome. AB - PURPOSE: To determine whether computed tomography (CT) can help predict which patients will require surgical or bronchoscopic intervention during healing of bronchial anastomotic dehiscence after lung transplantation. MATERIALS AND METHODS: The authors followed up 25 bronchoscopically proved dehiscent anastomoses through healing in 19 patients who underwent lung transplantation. CT findings were correlated with bronchoscopic results and clinical outcome. RESULTS: A bronchial defect and extraluminal air were initially present at CT in all 25 dehiscent anastomoses. Of 12 bronchial defects less than or equal to 4 mm, only one required intervention during healing (P < .05). Of 12 bronchial defects greater than 4 mm, six required intervention during healing. Eight of nine dehiscences with a tiny or small amount of extraluminal air healed with conservative treatment. Of 16 dehiscences associated with a moderate to large amount of extraluminal air, nine were treated conservatively and six required therapeutic intervention. Three healing anastomoses required bronchial stent placement. One patient died in the perioperative period. CONCLUSION: In patients with small dehiscences ( < 4mm) and patients with a tiny or small amount of extraluminal air, the anastomosis tends to heal without sequela. When patients have larger amounts of extraluminal air or larger ( > 4mm) dehiscences at presentation, CT cannot help predict which patients will require intervention. PMID- 8638017 TI - Development of an MR simulator: experimental verification of geometric distortion and clinical application. AB - PURPOSE: To evaluate the geometric distortion on magnetic resonance (MR) images obtained with a permanent magnet system and determine the usefulness of MR imaging-assisted x-ray simulation in radiation therapy treatment planning (RTTP). MATERIALS AND METHODS: The authors measured the distortion on MR images of grid pattern phantoms. MR imaging-assisted x-ray simulation was performed with skin markers in 14 patients with bone tumors. Treatment planning had already been performed with a conventional system. RESULTS: On phantom images, most of the positional displacements within a 120-mm radius from the center of the static magnetic field were less than 2 mm; larger displacements were observed in the peripheral region of the images. MR imaging was useful in the RTTP of all patients. The original radiation field was modified after MR examination in six patients. CONCLUSION: The amount of image distortion within the practical area is acceptable for RTTP. MR imaging-assisted x-ray simulation is useful for patients with bone tumors and warrants further investigation. PMID- 8638018 TI - Role of intraluminal brachytherapy in extrahepatic bile duct and pancreatic cancers: is it just for palliation? AB - PURPOSE: To evaluate intraluminal brachytherapy (ILBT) in patients with extrahepatic bile duct or pancreatic cancers. MATERIALS AND METHODS: Thirty-one patients (aged 33-87 years) with unresectable extrahepatic bile duct (n = 18) or pancreatic (n = 13) cancer received ILBT exclusively or as part of a definitive treatment regimen. ILBT was performed with transhepatic percutaneous drainage in four patients and with endoscopic retrograde cholangiopancreatography in 27. Fourteen patients with no metastases, an Eastern Cooperative Oncology Group performance score of < or = 2, and good hematologic parameters received combined modality treatment: 30-Gy ILBT and 45-Gy external-beam radiation therapy with continuous infusion of fluorouracil. Seventeen patients underwent 50-Gy ILBT alone for palliation. RESULTS: No direct treatment-related acute toxic reactions were seen. Three patients had cholangitis early in the study. Three patients had late gastrointestinal bleeding. Jaundice was palliated in all patients (n = 29); pain, in 11 of 13 patients. The survival rate in patients with extrahepatic bile duct cancer was 62% (five of eight) at 2 years for combined modality treatment. No patient with pancreatic cancer lived for longer than 2 years. CONCLUSION: ILBT is an effective palliative treatment of unresectable extrahepatic bile duct and pancreatic cancers. Results suggest a possible "curative" role in specific clinical settings when properly integrated with other treatments. PMID- 8638019 TI - In vivo He-3 MR images of guinea pig lungs. AB - The authors imaged the lungs of live guinea pigs with hyperpolarized (HP) helium 3 as a magnetic resonance (MR) signal source. HP He-3 gas produced through spin exchange with rubidium metal vapor was delivered through an MR-compatible, small animal ventilator. Two- and three-dimensional lung images acquired with ventilation-gated, radial k-space sampling showed complete ventilation of both lungs. All images were of high quality, demonstrating that HP He-3 allows high signal-intensity MR imaging in living systems. PMID- 8638020 TI - Application of region-of-interest imaging techniques to neurointerventional radiology. AB - To reduce radiation exposure to patients and staff during neurointerventional procedures, region-of-interest (ROI) techniques were used with fluoroscopy, road mapping, and digital subtraction angiography in 19 patients. ROI filters, made of multiple layers of gadolinium, were attached to the collimators. Patient skin exposure was reduced by a factor of 3.3-10.0 across 85% of the field of view, and exposures were reduced to below thresholds for skin effects. PMID- 8638021 TI - Barium enema retention balloon: an additional note of caution. PMID- 8638022 TI - A perspective on k-space. PMID- 8638023 TI - Management of patients with life-threatening sustained ventricular tachyarrhythmias--the role of guided antiarrhythmic drug therapy. AB - Two recent studies have evaluated the utility of electrophysiologic (EP) testing in the treatment of patients with serious ventricular arrhythmias. The first study compared electrophysiologically guided antiarrhythmic drug therapy with nonguided beta-blocker therapy. Patients without inducible arrhythmias were assigned to oral metoprolol; patients with inducible arrhythmias were randomly assigned to receive either oral metoprolol or EP-guided drug therapy with propafenone, flecainide, disopyramide, sotalol, or amiodarone. Antiarrhythmic drugs were tested in a random order, but amiodarone was always tested last. A total of 170 patients were evaluated; 115 patients had inducible arrhythmias, and 61 patients were randomly assigned to serial drug testing, 54 to metoprolol without invasive testing, and the remainder who were noninducible to empiric metoprolol. The best outcome was observed in patients without inducible arrhythmias, all of whom received metoprolol. There was no difference in outcome between the two groups with inducible arrhythmias, either treated with metoprolol or with EP-guided serial antiarrhythmic drug testing. The second study evaluated survivors of out-of-hospital ventricular fibrillation (VF) without new myocardial infarction. Patients received assessment of left ventricular ejection fraction, Holter monitoring (HM), and EP testing. Only patients with inducible sustained ventricular arrhythmias or with sufficient ambulatory ventricular ectopy were included in the study. Therapy was randomized either to empiric amiodarone or conventional drug therapy guided by EP testing and/or HM. A total of 228 patients were treated, 113 with amiodarone and 115 with conventional antiarrhythmic drug therapy. The composite primary end points were total mortality, documented out-of hospital resuscitation from recurrent VF, or syncopal implantable cardioverter/defibrillator shock followed by return of consciousness. Patients treated with empiric amiodarone had a better outcome than did patients treated with guided conventional drug therapy. In those patients in whom an implantable cardioverter/defibrillator was used, patients treated with amiodarone had fewer total shocks and fewer syncopal shocks than did patients treated with conventional therapy. Patients with a history of out-of-hospital VF or sustained ventricular tachycardia without inducible ventricular arrhythmias at EP study have the best outcome. Empiric metoprolol is equivalent to conventional antiarrhythmic drug therapy guided by EP testing. Empiric amiodarone is superior to conventional antiarrhythmic drug therapy guided by HM and/or EP testing. PMID- 8638024 TI - Nonpharmacological therapy for malignant ventricular arrhythmias: implantable defibrillator trials. AB - Implantable cardioverter-defibrillators (ICDs) are an important nonpharmacological option in the treatment of malignant ventricular arrhythmias. Technological advances in current devices permit nonthoracotomy implantation with transvenous lead systems using biphasic shocks. Decreasing device size has resulted in pectoral implantation. Battery longevity is still short in comparison with that of pacemakers. Lead failure rates as well as pacing thresholds are significantly higher than those for cardiac pacing lead systems. Other complications of ICD systems include infection, perforation, and thrombosis. The long-term performance of nonthoracotomy lead systems for ICD devices has now been extensively studied. Sudden death recurrence rates for these systems are less than 2% in 3 years and less than 5% at 5 years. Clinical trials with both monophasic and biphasic systems show a high degree of prevention of sudden death. Comparison of ICD outcome with that of drug therapy in three large retrospective studies and two small prospective randomized trials favors improved survival and sudden death prevention with device therapy. However, these studies need corroboration from large prospective trials. Two large prospective trials, CIDS and the AVID study, are now in progress to address this issue. PMID- 8638025 TI - Implanted cardioverter-defibrillators are preferable to drugs as primary therapy in sustained ventricular tachyarrhythmias. AB - The choice of initial therapy for patients with malignant ventricular tachyarrhythmias is examined based on clinical efficacy, patient safety, and cost. Antiarrhythmic drug therapy can be administered using a guided or empiric approach. Guided type-1 antiarrhythmic drug therapy has been associated with high arrhythmia recurrence rates (> 40% at 1 year) and moderate sudden death rates (10% at 1 year). Sotalol is associated with lower arrhythmia recurrence rates (20% at 1 year) that increase to 50% at 4 years. Beta-blocking agents have a limited role as stand-alone therapy in this condition. Empiric amiodarone therapy has sudden death-free survival rates of 82% at 2 years but has significantly poorer results in patients with ejection fractions < or = 40%. In contrast, implantable cardioverter-defibrillator (ICD) therapy has reported sudden death recurrence rates of 1% to 2% per year, with a cumulative index of 10% at 5 years. Total survival rate of ICD recipients ranges from 85% to 92% at 2 years. In patients with good left ventricular function, it approaches 90% at 5 years, whereas it is between 50% to 60% in patients with severe left ventricular dysfunction. Data from device memory indicate an absolute reduction in mortality rates with ICD intervention. Comparison of drug and device therapy has been performed in retrospective and prospective studies. Improved survival with device therapy is noted, particularly in patients with ejection fractions < or = 35% to 40% in retrospective studies. The results of two small prospective randomized trials also show significant survival advantage as compared with those for type 1C drugs and a mixed group of antiarrhythmic drugs. An initial strategy of ICD therapy was shown to be superior in the Netherlands Cooperative Study. The 30-day perioperative mortality rate of ICD therapy of 0.8% contrasts favorably with a 13% mortality rate in the ESVEM trial with antiarrhythmic drugs and a 3.5% mortality rate in the CASCADE study. Economic analyses show that drug therapy and device therapy are both within the range of other current cardiovascular therapies. An improving economic profile for device therapy has been observed with nonthoracotomy and pectoral implantation and direct use of ICD therapy because primary therapy shortens hospital stay and reduces costs. Based on available data, ICD therapy is preferable as initial therapy in patients with malignant ventricular tachyarrhythmias. PMID- 8638026 TI - A perspective on the ESVEM trial current knowledge: sotalol should not be the first-line agent in the management of ventricular arrhythmias. AB - The Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) trial has recently shown the superiority of sotalol over class-1 agents in lowering the rate of recurrence of ventricular tachyarrhythmias. However, this study was not placebo-controlled, and amiodarone was not included as one of the antiarrhythmic drugs in the trial. Randomized comparative trials between sotalol and amiodarone are available, but the results are inconclusive mainly because of small sample sizes. Because of the specific pharmacokinetics of amiodarone, sotalol has become the first-line agent in the management of ventricular arrhythmias. Because this policy is based on expediency rather than follow-up data, the long-term efficacy, morbidity, and safety of sotalol should be compared with those of amiodarone as well as of nonpharmacological treatment modes for ventricular tachyarrhythmias, such as implantable cardioverter defibrillator therapy in prospective trials. Until these issues are resolved, it is incorrect to say that sotalol should be the first-line agent in the management of ventricular arrhythmias. PMID- 8638027 TI - A perspective on the ESVEM trial and current knowledge: catheter ablation for ventricular tachyarrhythmias. AB - Although an effective and potentially curative technique for treating idiopathic ventricular tachycardia, map-guided transcatheter radiofrequency ablation is far from optimal for ventricular tachyarrhythmias in patients with advanced ischemic or other types of organic heart disease. First, this technique can be applied only to a minority of patients with structural heart disease, who can tolerate relatively long episodes of induced ventricular tachycardia necessary for mapping and successful ablation. Second, the success rate is lower and recurrence higher in patients with organic heart disease. Finally, for patients who lose consciousness during tachycardia or who present with prehospital cardiac arrest, transcatheter radiofrequency ablation is inappropriate as definitive treatment. At best, it is palliative and may be used to suppress relatively slow, frequent, or incessant ventricular tachycardias but does not obviate the need for other therapies such as cardioverter-defibrillator implantation or antiarrhythmic drug therapy. PMID- 8638028 TI - Electrocardiographic predictors in the ESVEM trial: unsustained ventricular tachycardia, heart period variability, and the signal-averaged electrocardiogram. AB - Sudden death remains a major problem because the causes are uncontrolled and accurate predictors have not been identified. However, new forms of electrocardiographic (ECG) analyses may provide prognostic information. The Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) trial provides a unique perspective to this issue because baseline and follow-up data were prospectively acquired on a relatively large sample of patients who were homogeneous with respect to sustained ventricular tachyarrhythmias, frequent ectopic activity, and inducible sustained ventricular tachyarrhythmias. Although analysis of the large amount of ECG data collected is in progress, initial studies have provided information about unsustained ventricular tachycardia (VTu), heart period (R-R) variability, and the signal-averaged ECG. VTu has been reported to have prognostic implications in several disorders, but its clinical significance in patients with sustained ventricular tachyarrhythmias is unknown. The significance of VTu recorded in the baseline (antiarrhythmic drug-free) 48 hour ECG recording in ESVEM study patients was examined; no variable representing the presence of VTu, the frequency of VTu events, or the duration of the longest episode of VTu was a significant predictor of arrhythmia recurrence, arrhythmic death, or all-cause mortality, although a trend was present for worse all-cause mortality in patients with VTu. R-R variability provides powerful prognostic information after acute myocardial infarction (AMI) and in patients with chronic ischemic heart disease. In general, R-R variability decreases dramatically at the time of AMI and recovers somewhat during the year after infarction. Although most patients in the ESVEM trial had chronic ischemic heart disease, R-R variability, which has been determined in about three fourths of the patients, was much lower than that reported in patients 1 year after MI. Instead, the mean values were closer to the more depressed values observed shortly after MI. This suggests a greater degree of autonomic dysfunction in patients with sustained ventricular tachyarrhythmias, frequent ventricular ectopic activity, and low ejection fractions, as compared with that for patients with chronic ischemic heart disease in general. Signal-averaged ECGs have also been shown to predict arrhythmic events in patients with ischemic heart disease. In a subset of the ESVEM patients, antiarrhythmic drugs that block sodium channels were found to prolong the filtered, signal-averaged QRS duration, especially the late potential portion. This correlated with prolongation of the cycle length of induced ventricular tachycardia. Sotalol appeared to have a differential effect on the signal-averaged ECG; the signal-averaged QRS shortened slightly in patients in whom induction of VT was suppressed by sotalol, whereas it appeared to lengthen slightly in patients in whom VT remained inducible despite sotalol. This suggests that sotalol may affect conduction in diseased tissue in some patients, and that this may affect suppression of ventricular arrhythmia induction by programmed stimulation. PMID- 8638029 TI - Design and analysis of the ESVEM Trial. AB - Inadequate understanding of the design and statistical approach of a clinical trial and the failure to recognize subjective aspects of the analysis often result in misinterpretation of trial results. This is exacerbated by the push to shorten publications and the wish for a simple message that summarizes the outcome of the trial. The purpose of this review is to critically review the design and statistical analyses of the results, to evaluate the assumptions underlying the statistical tests, and to examine the results of exploratory analysis on the interpretation of major findings of the Electrophysiologic Study Versus Electrocardiographic Monitoring (ESVEM) trial. The trial was unusual because its primary objective was to compare testing methods instead of treatments. This necessitated using a subset of the original randomized groups for sensible analysis of the clinical question. Nevertheless, the two groups appeared to be well balanced. The absence of a difference in outcome could be verified by several analyses. In addition, confidence intervals were narrow, indicating the high precision and reliability of the findings. However, the comparison of antiarrhythmic drugs is problematic because the trial was not designed to address this issue. There were differences in the distribution of clinical characteristics between the groups who received different antiarrhythmic drugs. Nevertheless, using both univariate analyses and a variety of adjustments for important prognostic variables, treatment with sotalol appeared to be a significant predictor of reduced arrhythmia recurrence, and sotalol was consistently associated with a trend for nearly a 50% reduction in sudden death and all-cause mortality as compared with the other drugs administered in the trial. In conclusion, the ESVEM trial raises a number of interesting and instructive issues about clinical trial design and analysis. PMID- 8638030 TI - Epidemiology and control of tuberculosis in Israel. AB - OBJECTIVE: This review of the epidemiology and control of tuberculosis (TB) in Israel, covering the period from shortly before 1948 until the end of 1992 give a historical perspective to the TB situation and the various methods of control at different times. The effect of arrival in Israel of groups of immigrants from countries with a high prevalence of TB on the incidence in the host population is considered. DATA SOURCES: Data from unpublished reports and those published in Israel and abroad. Much data which was available form the Central TB Register of the Department of Epidemiology of the Ministry of Health was analyzed by the author during the time he was responsible for tuberculosis in Israel. Some of the data has been updated and presented here for the first time. RESULTS: With the mass immigration to Israel in the early 1950s, including many cases of active TB, there was a steep rise in the number of reported cases. Special services were developed. The TB rate dropped rapidly, and although there were later occasional peaks due to special immigrant groups, this did not appear to cause any serious increase in the TB incidence in the host population. CONCLUSIONS: TB control measures in Israel have passed through several phases and demonstrate the value of various methods which have to be changed according to the epidemiological situation. PMID- 8638031 TI - Definitions of attributable risk--revisited. AB - BACKGROUND: The concept of attributable risk is a popular approach to describe the disease risk associated with an exposure factor on the population level. The paper reviews this epidemiologic concept. METHODS: Definitions and interpretations of four measures of association term "attributable risk" in the epidemiologic literature are compared. By introducing the notation of exposure specific disease events, the intrinsic relationship between these definitions in unveiled from a new perspective. In addition, the terminologic confusion relating to this concept is discussed. RESULTS: It is shown that all four definitions focus on the exposure-specific disease incidence, but all in different subpopulations of the study population. Data of a German cohort study on the relationship between lipoprotein fractions and myocardial infarction further illustrate the different measures of attributable risk. DISCUSSION: It is pointed out that the evaluation of the public health impact of an exposure under study by means of attributable risk calculations should form an integral part of the epidemiologic analysis. PMID- 8638032 TI - Underutilization of influenza vaccine in Jerusalem. AB - BACKGROUND: Influenza is an annual winter disease which causes considerable morbidity in the general population and may be lethal in defined high-risk groups. A killed vaccine, recommended for the high-risk groups as well as for several occupational categories, is moderately protective against the disease, but vaccine sales suggest that use is low. METHODS AND RESULTS: In a hospital based convenience survey of 295 persons belonging to groups for whom influenza vaccine is recommended, 78 (26.4%) reported having received the vaccine in 1989, and 74 (25.1%) in 1990. All vaccine recipients belonged to the 244 subjects eligible because of advanced age and/or chronic illness, giving vaccination rates in this group of 32.0% in 1989 and 30.3% in 1990. None of the 51 hospital doctors and nurses interviewed had received vaccine. Among the 244 eligible because of age or illness, vaccine receipt in 1990 was related to older age and number of risk factors, but even in subjects with three separate indications for receipt of vaccine, the vaccination rate was only 48%. Among persons advised by anyone to have influenza vaccine, 62.7% received vaccine in at least one of the two years, compared to a rate of 16.4% among subjects never advised to be vaccinated. Vaccine recommendation by a medical professional increased the vaccination rate to 69.3%. CONCLUSIONS: Influenza vaccine uptake in vaccine eligibles in Jerusalem is low, about 30% in the sick and elderly and considerably lower in medical personnel. Since influenza vaccination can save lives, it would be worthwhile to convince the medical profession to enthusiastically recommend this vaccine to high risk individuals and to explore novel ways of increasing uptake in the high high risk groups such as persons in hospital or those residing in institutions. PMID- 8638033 TI - Failure of mass chemoprophylaxis to control hyperendemic meningococcal disease. PMID- 8638034 TI - Israel Society of Epidemiology 13th annual meeting. Jerusalem, Israel, 11 May 1995. Abstracts. PMID- 8638035 TI - Comments on Tengs et al., "Comparative study of the cost-effectiveness of life saving interventions". PMID- 8638036 TI - Developing a subject-derived terminology to describe perceptions of chemicals in foods. AB - Risk perception may be influenced by a number of factors, such as unfamiliarity, lack of control, perceived consequences, and hazards being seen as catastrophic and having risk for future generations. Risk perception researchers have typically used such investigator-selected characteristics to assess hazards. In the first study reported here, the repertory grid method was used to elicit the terminology that subjects (n = 30) use to distinguish between 30 different chemicals. The data were submitted to generalized Procrustes analysis. The first principal axis of the resulting consensus plot separated the chemicals ranging from "poisonous or toxic," "harmful or dangerous," and "sounds negative" at one end, to "positive effect on health," "often present in food nowadays," and "sounds positive" at the other end. The second principal axis ranged from "familiar with or knowledge of" and "chemical" to "natural." A second study (n = 226) was carried out to look at the general validity of the results of the repertory grid interviews using a fixed questionnaire. The data were submitted to principal components analysis and internal preference mapping. The first principal component ranged from "safe" and "healthy" at one end, to "poisonous" and "harmful" at the other end. The chemicals also separated in terms of "familiar," "chemical," and "natural." All three methods of data collection and analysis yield essentially similar results. PMID- 8638037 TI - A distributed parameter physiologically-based pharmacokinetic model for dermal and inhalation exposure to volatile organic compounds. AB - Estimates of dermal dose from exposures to toxic chemicals are typically derived using models that assume instantaneous establishment of steady-state dermal mass flux. However, dermal absorption theory indicates that this assumption is invalid for short-term exposures to volatile organic chemicals (VOCs). A generalized distributed parameter physiologically-based pharmacokinetic model (DP-PBPK), which describes unsteady state dermal mass flux via a partial differential equation (Fickian diffusion), has been developed for inhalation and dermal absorption of VOCs. In the present study, the DP-PBPK model has been parameterized for chloroform, and compared with two simpler PBPK models of chloroform. The latter are lumped parameter models, employing ordinary differential equations, that do not account for the dermal absorption time lag associated with the accumulation of permeant chemical in tissue represented by permeability coefficients. All three models were evaluated by comparing simulated post-exposure exhaled breath concentration profiles with measured concentrations following environmental chloroform exposures. The DP-PBPK model predicted a time lag in the exhaled breath concentration profile, consistent with the experimental data. The DP-PBPK model also predicted significant volatilization of chloroform, for a simulated dermal exposure scenario. The end-exposure dermal dose predicted by the DP-PBPK model is similar to that predicted by the EPA recommended method for short-term exposures, and is significantly greater than the end-exposure dose predicted by the lumped parameter models. However, the net dermal dose predicted by the DP-PBPK model is substantially less than that predicted by the EPA method, due to the post-exposure volatilization predicted by the DP-PBPK model. Moreover, the net dermal dose of chloroform predicted by all three models was nearly the same, even though the lumped parameter models did not predict substantial volatilization. PMID- 8638038 TI - Customer perceptions of agency risk communication. AB - A government agency commissioned a baseline study of how its customers view the agency's risk information. The 70% response rate to a mail survey allows analysis by subgroups representing customers' primary interests. Although this agency traditionally has been responsible for ensuring plant and animal health at the farm gate (or where imported), responses emphasized emerging customer concerns about the environment and human health. Customers think many risk communication activities are important, but that the agency is not especially effective in conducting those activities. Customers are moderately satisfied with much of the risk information they receive, although many have little contact from or interaction with the agency. Customers identified other sources they use, which suggest potentially effective channels for this agency's risk messages. The study provides a baseline for measuring change in the agency's risk communication effectiveness. It also can be a model when other organizations plan their own risk communication evaluations. PMID- 8638039 TI - Derivation of a target concentration of Pb in soil based on elevation of adult blood pressure. AB - The increase in systolic blood pressure in males appears to be the most sensitive adult endpoint appropriate for deriving a health risk-based target level of lead (Pb) in soil. Because the response of blood pressure to blood Pb concentration (PbB) has no apparent threshold, traditional approaches based on the application of a Reference Dose (RfD) are not applicable. An alternative approach is presented based on a model which predicts the population shift in systolic blood pressure from ingestion of Pb contaminated soil as a simultaneous function of exposure to Pb in soil, the baseline distribution of blood Pb concentration in the population and baseline distribution of systolic pressure in the population. This model is analyzed using Monte Carlo analysis to predict the population distribution of systolic pressure resulting from Pb exposure. Based on this analysis, it is predicted that for adult males 18-65 years old, exposure to 1000 ppm Pb in soil will result in an increase of approximately 1 mm Hg systolic pressure, an increase in the incidence of systolic hypertension (i.e., systolic pressure > 140 mm Hg) of approximately 1% and an increase in PbB of 1-3 micrograms/dl. Based on the proposition that these adverse effects can be considered de minimis, 1000 ppm Pb in soil is proposed as a target soil concentration for adult exposure. Available data do not appear to be adequate to predict the newborn PbB level which would result from exposure to this soil level during pregnancy. PMID- 8638040 TI - Ecological risks to fossorial vertebrates from volatile organic compounds in soil. AB - The past several years has seen an increased awareness of the need to conduct ecological risk assessments (ERAs) for hazardous waste sites. One technique used in ERAs involves estimating contaminant exposure to individual animals of selected species, which is then compared to a reference dose derived from the literature. Exposure estimates are conducted on those species which are representative of the different trophic levels found at the site. In many terrestrial systems, fossorial (burrowing) vertebrates are found in both lower and upper trophic levels. As part of the ERA conducted for Site 300, Lawrence Livermore National Laboratory's high-explosive test facility, contaminant exposures were estimated for fossorial and nonfossorial vertebrates spanning two trophic levels. The results of the evaluation revealed that a significant pathway by which fossorial vertebrates could be exposed to trichloroethylene in soil was through the inhalation of contaminated subsurface burrow air. This was the first time that the importance of this ecological exposure pathway has been recognized. The results of this analysis suggest that further research into the ecological significance of subsurface burrow air contaminated with volatile organic compounds is warranted. PMID- 8638041 TI - Risk assessment of nongenotoxic carcinogens based upon cell proliferation/death rates in rodents. AB - Increased cell proliferation increases the opportunity for transformations of normal cells to malignant cells via intermediate cells. Nongenotoxic cytotoxic carcinogens that increase cell proliferation rates to replace necrotic cells are likely to have a threshold dose for cytotoxicity below which necrosis and hence, carcinogenesis do not occur. Thus, low dose cancer risk estimates based upon nonthreshold, linear extrapolation are inappropriate for this situation. However, a threshold dose is questionable if a nongenotoxic carcinogen acts via a cell receptor. Also, a nongenotoxic carcinogen that increases the cell proliferation rate, via the cell division rate and/or cell removal rate by apoptosis, by augmenting an existing endogenous mechanism is not likely to have a threshold dose. Whether or not a threshold dose exists for nongenotoxic carcinogens, it is of interest to study the relationship between lifetime tumor incidence and the cell proliferation rate. The Moolgavkar-Venzon-Knudson biologically based stochastic two-stage clonal expansion model is used to describe a carcinogenic process. Because the variability in cell proliferation rates among animals often makes it impossible to detect changes of less than 20% in the rate, it is shown that small changes in the cell proliferation rate, that may be obscured by the background noise in rates, can produce large changes in the lifetime tumor incidence as calculated from the Moolgavkar-Venzon-Knudson model. That is, dose response curves for cell proliferation and tumor incidence do not necessarily mimic each other. This makes the use of no observed effect levels (NOELs) for cell proliferation rates often inadmissible for establishing acceptable daily intakes (ADIs) of nongenotoxic carcinogens. In those cases where low dose linearity is not likely, a potential alternative to a NOEL is a benchmark dose corresponding to a small increase in the cell proliferation rate, e.g., 1%, to which appropriate safety (uncertainty) factors can be applied to arrive at an ADI. PMID- 8638043 TI - [The cochlea and underwater diving]. AB - Internal ear barotrauma are dangerous for cochlea. Out of accidents, long-term scuba diving involves no deafness. A study carries on 272 skin divers of the French Navy shows that only presbyacusis and industrial noise exposure give audiometric damages. PMID- 8638042 TI - Uncertainty and variation in indirect exposure assessments: an analysis of exposure to tetrachlorodibenzo-p-dioxin from a beef consumption pathway. AB - Indirect exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other toxic materials released in incinerator emissions have been identified as a significant concern for human health. As a result, regulatory agencies and researchers have developed specific approaches for evaluating exposures from indirect pathways. This paper presents a quantitative assessment of the effect of uncertainty and variation in exposure parameters on the resulting estimates of TCDD dose rates received by individuals indirectly exposed to incinerator emissions through the consumption of home-grown beef. The assessment uses a nested Monte Carlo model that separately characterizes uncertainty and variation in dose rate estimates. Uncertainty resulting from limited data on the fate and transport of TCDD are evaluated, and variations in estimated dose rates in the exposed population that result from location-specific parameters and individuals' behaviors are characterized. The analysis indicates that lifetime average daily dose rates for individuals living within 10 km of a hypothetical incinerator range over three orders of magnitude. In contrast, the uncertainty in the dose rate distribution appears to vary by less than one order of magnitude, based on the sources of uncertainty included in this analysis. Current guidance for predicting exposures from indirect exposure pathways was found to overestimate the intakes for typical and high-end individuals. PMID- 8638044 TI - [Otologic manifestations presenting and predominating in necrotizing angiitis]. AB - The authors report on 5 observations of necrotising angiitis with a specifically otological onset, namely 4 Wegener's syndromes and one periarteritis nodosa. In all 5 cases, the initial symptomatology consisted of otological signs in a feverish context, producing pictures of serous or sero-purulent otitis. Its resistance to usual forms of therapy led in 3 cases to the installation of transtympanic aerators, and in 2 cases to the performance of an antro-attico mastoidectomy. One of the observations led to the discovery in the middle ear of a histological aspect specific to the disease, from which it can be considered that the otologic impairment could be a specific seat of the disease. The 5th observation is particular in that it was the otological signs which revealed each time a new progression of the disease, enabling the immediate implementation of an adapted treatment prior to the occurrence of the systemic signs. 3 observations proved the difficulty of diagnosis at the initial stage, when at times only the otological signs are present, together with the importance of its discovery prior to the occurrence of systemic lesions that are occasionally irreversible. Any isolated otological symptomatology in a feverish context, resisting usual therapies, should evoke the diagnosis, and lead to the performance of further duly adapted examinations (sedimentation rate, pulmonary X ray, proteinuria, hematuria). PMID- 8638045 TI - [Otologic signs, craniofacial pain and dental malocclusion]. AB - An occlusal problem can explain otologic symptoms and myofacial pains. A dental and an occlusal examination is necessary to find the true mechanism. If the occlusal examination is not immediately possible, the scheme of the biting retro incisive helps to the diagnosis, in motter to put in place a treatment. In this complete study with a backward regard of 79 patients, 62 have been treated with a total success. PMID- 8638046 TI - [Anatomo-radiologic diagnosis of mucoceles in the paranasal sinuses]. AB - The diagnosis of mucoceles of the facial sinuses is essentially based on CT scans. The authors define here the main features of these benign tumours, the evolution of which is sometimes formidable. PMID- 8638047 TI - [Current aspects of sinus barotrauma. The impact on treatment, prevention and fitness]. AB - Among the barotraumatism which affects divers and airplane pilots, sinus barotraumatism is one of the less known and studied, as the scarceness of series published can testify. The interest of this study stands in doing an up to date synthesis (recap) of the bibliography, and to understand the "physio-pathologic" mechanism after the exploitation of series of 22 observations. That work will allow a better knowledge of therapeutic on the one hand, and of the "prophylactic" on the other hand. PMID- 8638048 TI - [Distant metastases originating from tumors of the larynx and hypopharynx]. AB - Following a review of the world literature dealing with the incidence of remote metastases in tumours in the ENT field in general, and of the larynx and hypopharynx in particular, we present a study covering 927 patients suffering from cancer of the larynx and hypopharynx. The purpose of the research was to determine the incidence and position of metastases in this type of patient, and to determine their evolution. A total of 786 patients were followed over a minimum of 4 years, and 52 cases (6.7%) of metastases were recorded. They occur all the more frequently when the primary tumour is hypopharyngeal, of the low differentiated carcinoma type, or when there is a cervical ganglionic extension. PMID- 8638049 TI - Parapharyngeal masses. Report de 4 cases. AB - The reduced incidence of inflammatory pathology in the head and neck over the last 20 years has diminished the clinical importance of the potential fascial spaces in this region. However, the parapharyngeal space, because of its contents, is still important. Lesions in this region have variable presentations and their management often requires a multidisciplinary approach. The anatomy of this space is the key to the understanding of the pathophysiological effects from expanding lesions occurring within it. We report 4 unusual cases to illustrate the management policy and highlight the pitfalls of lesions in this area. PMID- 8638050 TI - Eosinophilic granuloma of the temporal bone. AB - Otological involvement in Histiocytosis X, although infrequent, may be present in any of the forms of this entity. The otologist must keep Histiocytosis X in mind in the differential diagnosis of cases presenting with post-aural swelling, non purulent otorrhea and absence of fever and pain in children under three-years old. Some aspects of clinical presentation and treatment are discussed. PMID- 8638051 TI - Oligosymptomatic glomus tympanicum and caroticium with two different histological pictures. AB - Though glomus tumors have been relatively frequently reported, there is very little material about their differential diagnostic problems. The authors present a case of glomus tympanicum and glomus caroticum with only otological symptoms, facial paralysis and traumatic arterio-venous shunts in the neck. These two tumors had few different patterns of glomus tissue. PMID- 8638052 TI - [Extracranial meningioma: apropos of a case developing in the nasal cavity and paranasal sinuses]. PMID- 8638053 TI - [Complicated acute sphenoiditis. Apropos of a case]. PMID- 8638054 TI - [Inverted papilloma. Apropos of a multicentric case]. AB - Inverted papilloma is a benign, neoplastic condition of the sinusonasal cavity, independent from other polypoid lesions of the same area. Our case, presented below is, we believe, unique in that it lends credit to the possible multicentric origin of the disease; which started in the right nasal fossa, followed by the left and then the right tympanic cavities in that order. PMID- 8638055 TI - [Florid oral papillomatosis: evaluation apropos of 2 cases]. AB - Oral florid papillomatosis is a rare, so-called pre-neoplastic non-obligatory complaint. The authors, on the basis of two cases, attempt to define the concept of malignity associated with this complaint and the resulting surgical principles. PMID- 8638056 TI - [treatment of an epistaxis using a bilateral nasolabial flap in Osler-Rendu disease]. AB - Severe and recurrent epistaxis in cases of Rendu-Osler disease (Hereditary hemorrhagic telangiectasia) are very difficult to treat. For one case the authors propose a bilateral nasolabial flap with inferior pedicle. They describe the technique and after 15 months of follow-up there is no recurrency. This treatment is used for particular cases: old people with short expectation of life, severe anaemia involving the vital prognosis. PMID- 8638057 TI - Craniofacial resection. An alternative incision and skull base repair. AB - Neoplasia of the superior group of paranasal sinuses (frontal, ethmoidal and sphenoidal) has an extremely poor prognosis. Resection, preceded or followed by radiotherapy offers a 5 year survival of less than 30% (1). Satisfactory surgical excision has been undermined by difficulties of access to the cribriform plate area, a common site for residual disease. Furthermore the practice of piecemeal removal invariably resulted in inadequate clearance. The craniofacial approach allows not only excellent access to the anterior skull base but also in bloc excision. The results of treating 7 patients with craniofacial resection for tumours in this area are presented. Particular reference is made to a horizontal forehead skin crease incision for intracranial access. A new technique using a combination of surgical, pericranium, partial thickness skin and fibrin glue to repair the skull base defect is described. PMID- 8638058 TI - [Reconstruction of the base of the skull after ethmoidectomy using a mixed approach. Apropos of a necroscopy case]. AB - Concerning a necropsic observation in an irradiated patient, the authors report on the current state of their technique for the reconstruction of the base of the skull, using the Galea flap technique and homologous, cryopreserved bone. PMID- 8638059 TI - [Stapedectomy and micro-stapedotomy in the treatment of otospongiosis. A comparative study]. AB - This is a comparative study of 622 patients undergoing either stapedectomy or stapedotomy for otosclerosis. 379 underwent stapedectomy, which in the majority of cases employed interposition of the posterior crus of the stapes into a venous graft placed over the newly created fenestra. The results of this technique are compared with 243 microwindow stapedotomies performed since 1976. For all patients a minimum of 5 years had elapsed between surgery and evaluation of outcome. In some instances, this time period was 20 years. One month post-surgery the closure of the air-bone gap was more frequently superior with stapedectomy, particularly in the low frequencies. The degree of an incidence of overclosure was comparable for both techniques. However, at 4,000 Hz, bone threshold levels deteriorated more usually following stapedectomy. The air-bone gap widened progressively over the proceeding years post-stapedectomy. This was in contrast to the effect seen during the same period after stapedotomy, namely a narrowing of the air and bone threshold levels. Additionally a progressive loss of bone thresholds was noted after the latter technique. The incidence of sudden total deafness, either immediate or delayed, was less than 1% for both procedures. Even after 15 years or more poststapedectomy, the air-bone gap (13 dB average) and bone thresholds remained stable. Revision surgery was necessary in 8% of stapedectomies and in 4% stapedotomies. However, the post-operative follow up period is much shorter for the latter. PMID- 8638060 TI - [An experimental and clinical study of a new intubation tube with a foam-filled cuff]. AB - An experimental and clinical study of an endotracheal tube with a foam-filled cuff has been carried out. The experimental study showed that, during inspiration, the cuff was insufflated through the "T piece" connecting the cuff in the inspiratory limb of the ventilator circuit, preventing inspiratory leak. The intracuff pressure was equal to airway pressure. During expiration the gas insufflated into cuff leaked out through the "T piece" and intracuff pressure rapidly returned to zero. When N2O in 50% O2 was used for one hour, intracuff pressure did not increase. Twenty patients intubated with a foam-filled cuff tube, for ENT surgery, have been studied. The mean intubating time was 151 min +/ 36 and two patients were intubed, respectively, 26 hours and 28 hours. No complications were noted, 24 hours after extubation, during laryngeal fibroscopic control. Only two patients had light edema of the vocal cords and three of them had a light inflammation of the subglottic mucosa, without sore throat. No tracheal ischaemic damage nor tracheal mucosal inflammation were observed. PMID- 8638061 TI - [Medical treatment of seromucous otitis]. AB - The authors list the main modalities for medical therapy proposed for sero-mucous otitis. Treatments such as antibiotherapy, corticotherapy and antihistamines have not proved to be effective, although they are useful at certain stages of the disease. The authors present a study of a mucolytic, carbocysteine, for the treatment of sero-mucous otitis. The study was performed using a double blind procedure against placebo in 44 children aged from 30 months to ten years. Monitoring included a clinical examination, impedancemetry, and audiometry. With carbocysteine, the auditive recovery was significantly faster on the side of the least affected ear. When transtympanic drainage had to be performed, the mucolytic effect in vivo on the fluidity of the secretions was directly noted. An improved ear evacuation was obtained. Tolerance was perfect in prolonged treatment with a high posology in young children. PMID- 8638062 TI - Forensic sleep medicine and nocturnal wandering. PMID- 8638063 TI - Complex motor behavior arising during the sleep period: forensic science implications. PMID- 8638064 TI - Sleep-related violence: a medical and forensic challenge. PMID- 8638065 TI - Sleep-related violence. AB - We hypothesized that sleep-related violent behavior associated with parasomnias occurs as the result of a diathesis and is precipitated by stressors and mediated by disturbed nonrapid eye movement (NREM) sleep physiology. Sixty-four consecutive adult patients (mean age 30 years) who were investigated for sleepwalking or sleep terrors were categorized according to clinical history into three groups: serious violence during sleep to other people or to property or self (n = 26); harmful, but not destructive behavior (n = 12); and nonviolent behavior (n = 26). Log linear analysis showed that a diathesis (childhood parasomnia and/or family history of parasomnia) and a stressor (psychologic distress, substance abuse and sleep schedule disorder) predicted the presence of sleepwalking or night terror. Serious violent acts were more likely to occur with males (p < 0.004) who showed sleep schedule disorder (p < 0.03). Both harmful and serious violent sleep behavior occurred with drug abuse (p < 0.009). In comparison to all other groups, those who were violent to other people were males who experienced more stressors (p < 0.02), drank excessive caffeinated beverages, abused drugs (p < 0.03) and showed less stage 4 sleep (p < 0.02) and less alpha (7.5-11 Hz) electroencephalogram NREM sleep (p < 0.02) on polysomnography. Being male and having < 2% stage 4 sleep provided 89% sensitivity, 80% specificity and 81% diagnostic accuracy for individuals who were violent to others. The forensic implications of these findings are discussed. PMID- 8638066 TI - Forensic sleep medicine: nocturnal wandering and violence. AB - Forty-one subjects between 12 and 63 years of age with a complaint of nocturnal wandering were reviewed retrospectively, and a prospective investigation of their compliance to treatment was performed. Twenty-nine of 41 subjects committed violence against themselves or others ("violent group"). Clinical investigation of their problem involved polysomnography, wake and sleep EEGs and ambulatory EEG recording in the home environment. The nocturnal wandering may have started from NREM sleep or REM sleep, and violence was observed in both of these sleep states. Arousal from sleep may have been triggered by sleep-disordered breathing or may have been related to temporal lobe abnormalities, and, in some cases, no abnormal polygraphic features were noted. Violence was always preceded by many instances of nocturnal wandering that had received little clinical attention. Temporal lobe abnormalities, a rare cause of nocturnal wandering, were present only in the "violent" group. This group also had a higher percentage of men than the "nonviolent" group. In both groups, the frequency of nocturnal wandering increased with an increase in daytime stressors. Pharmacological and psychiatric treatment approaches were beneficial in both groups. PMID- 8638067 TI - Epileptic nocturnal wanderings. AB - Video-polysomnographic monitoring in four patients complaining of stereotyped paroxysmal ambulation and other complex motor activities during sleep demonstrated ictal epileptic discharges at the onset of the sleepwalking episodes. In addition, all patients displayed minor motor episodes resembling short-lasting nocturnal paroxysmal dystonia (NPD) attacks and paroxysmal arousals (PAs). NPD with short-lasting attacks, PAs and episodic nocturnal wanderings represent the same epileptic syndrome with variable presentations. PMID- 8638068 TI - Prolonged confusion with nocturnal wandering arising from NREM and REM sleep: a case report. AB - A 51-year-old man with Machado-Joseph disease had a 3-year history of prolonged confusion following nightly nocturnal wandering. Polysomnography with videotape monitoring revealed 19- to 120-minute sleepwalking episodes emerging from non rapid eye movement (NREM) sleep and occasionally from rapid eye movement (REM) sleep, followed by 22-47 minutes of prolonged confusion and disorientation. The patient also had a periodic limb movement disorder and obstructive sleep apnea syndrome. Excessive daytime sleepiness was evident by results from the Epworth Sleepiness Scale and Multiple Sleep Latency Test. A sleep-deprived electroencephalogram (EEG) and a polysomnogram with an expanded EEG montage before and during these episodes revealed no epileptiform activity. A contrast enhanced brain magnetic resonance imaging (MRI) scan demonstrated findings consistent only with Machado-Joseph disease. The patient improved with a combination of temazepam and carbidopa-levodopa. PMID- 8638069 TI - A polysomnographically documented case of adult somnambulism with long-distance automobile driving and frequent nocturnal violence: parasomnia with continuing danger as a noninsane automatism? AB - A case of childhood-onset somnambulism is reported in which a 43-year-old man presented with repeated sleep-related injuries incurred during violent nocturnal activity, which included frenzied running, throwing punches and wielding knives. He had also driven an automobile a long distance during a presumed somnambulistic state. His wife had been repeatedly injured, and she felt that her life was threatened by his nocturnal violence 2-3 times yearly. Polysomnography (PSG) documented multiple episodes of complex and violent behaviors arising exclusively from stage 3/4 sleep, thus confirming the diagnosis of somnambulism. Other causes of sleep-related violence were excluded. The patient responded promptly to treatment with bedtime clonazepam, and benefit was maintained at 5-year follow up. Although this strictly clinical case did not have any legal repercussions, it does carry forensic implications, particularly when placed in the context of the published medical literature on PSG-documented parasomnias (somnambulism, rapid eye movement sleep behavior disorder) containing explicit examples of recurrent violence, at times life-threatening, directed toward the bed partner and others. Thus, a new medical-legal concept is proposed, consisting of "parasomnia with continuing danger" as a noninsane automatism. Treatment guidelines, within the context of forensic medicine, are presented. PMID- 8638070 TI - Night terrors in an adult precipitated by sleep apnea. AB - Parasomnias are generally described as disorders of arousal that arise out of stage 3 and 4 nonrapid eye movement (NREM) sleep without identifiable cause. We present a case of a 35-year-old man who during nasal continuous positive airway pressure (nCPAP) treatment for severe obstructive sleep apnea experienced an intense night terror triggered by a residual obstructive apnea during rebound deep sleep. The role of rebound deep sleep was thought to be essential in creating a state of sleep with a high arousal threshold hypothesized to be important for the occurrence of parasomnias. This case supports the clinical wisdom that identifiable sources of arousal can trigger parasomnias. PMID- 8638071 TI - Homicidal behavior and sleep apnea: a case report and medicolegal discussion. AB - This case report documents the use of sleep apnea as a criminal defense for a man who fatally shot his wife during his usual sleeping hours. The defendant, who had severe sleep apnea as determined by a clinical evaluation and a polysomnographic study, admitted to shooting his wife but claimed that he was asleep at the time. Two physicians testified for the defense that the sleep apnea was of sufficient severity that the defendant may have had a confusional arousal related to the sleep apnea in which he could have shot his wife accidentally. Another physician, testifying for the prosecution, found no evidence to support this defense after a review of the patient's history and polysomnographic records and a review of relevant literature which may have linked sleep apnea with sleep-related violence. In this case, there was substantial apparent motive for the murder, including a past history of spousal and child abuse and a note written by the victim around the time of the shooting describing her intention to take the children and leave the suspect. The jury rejected the sleep apnea defense, handing down a first-degree murder verdict. In the discussion, we briefly review medicolegal issues related to the case as well as prospective guidelines for the medicolegal assessment of future cases. PMID- 8638072 TI - Seizures and arousals from sleep--which comes first? AB - Epileptic seizures may be associated with arousals from sleep. The temporal sequence of seizures and arousals is often uncertain and it may be impossible to determine their relationship by surface electroencephalogram (EEG) recordings alone. We describe a 28-year-old right-handed man with medically refractory partial epilepsy in whom seizure onset appeared to follow arousal from stage 2 nonrapid eye movement sleep based on the surface EEG. Inspection of simultaneously recorded intracranial EEG, however, demonstrated that the seizure onset preceded the arousal. This study illustrates the limitations of surface EEG and the utility of intracranial electrode recordings in investigating the relationship between arousals and seizures. PMID- 8638073 TI - Review of regulations and guidelines for commercial and noncommercial drivers with sleep apnea and narcolepsy. AB - Studies show that persons with sleep disorders, such as sleep apnea and narcolepsy, have an increased incidence of automobile accidents. The goal of this study was to review any regulations or guidelines dealing with fitness to drive of persons with sleep disorders in all the 50 states and countries around the world. Several authorities in the United States and abroad in fact have produced guidelines or regulations stating that certain of these persons are not fit to drive. As of March 1994, only four states in the United States (Maryland, North Carolina, Oregon and Utah) had guidelines for narcolepsy, while two had guidelines for both narcolepsy and sleep apnea (California and Texas). In Maine, guidelines had been proposed for sleep apnea. In contrast, almost all Canadian provinces have guidelines for both sleep apnea and narcolepsy, as does the United Kingdom. There are, however, considerable variations in the nature of the regulations used in different states, Canadian provinces and countries. These variations are not based on scientific data. Currently the impact of these regulations on crash rates or on the practice of sleep medicine has not been assessed. PMID- 8638074 TI - Sleep deprivation in the rat: XX. Differences in wake and sleep temperatures during recovery. AB - We examined the relationship between wake and sleep peritoneal temperature (T(ip)) during recovery from short-term (five rats, 5 days of deprivation) and long-term (nine rats, 14-21 days) total sleep deprivation (TSD). Mammalian body temperature normally declines in the passage from wakefulness to sleep. Recovery from TSD featured reductions of the typical wake-sleep T(ip) differences. Previous studies from our laboratory have shown that chronic TSD in the rat produces a progressive rise in energy production and an initial rise in wake T(ip), followed by a later fall in T(ip) to below baseline that becomes more acute as death becomes imminent. During recovery from both short-term TSD (wherein pre-recovery wake T(ip) was still above baseline) and long-term TSD (wherein pre-recovery wake T(ip) had fallen to below baseline), wake T(ip) and energy production quickly returned towards baseline. On the first recovery day, both short- and long-term TSD rats showed mean non-rapid eye movement (NREM) and paradoxical sleep (PS) T(ip) values that were slightly, although not significantly, above mean wake T(ip). In short-term TSD rats, wake-NREM and wake PS T(ip) differences were reduced from baseline significantly (p < 0.0025) on the first recovery day and nonsignificantly on the remaining three recovery days. In long-term TSD rats, wake-NREM and wake-PS T(ip) differences were significantly (p < 0.001) reduced from baseline on the first four recovery day block. On the last four recovery day block, wake-sleep T(ip) differences tended to return toward baseline. Hypothalamic wake-sleep temperature differences in long-term TSD rats showed similar reductions during recovery. The reduction of wake-sleep temperature differences in recovery does not support either energy reduction or cooling functions for sleep. PMID- 8638076 TI - Bibliography of recent literature in sleep research. PMID- 8638075 TI - Homicidal somnambulism. PMID- 8638078 TI - [Schematic sections of the female pelvis]. AB - The anatomic complexity of the female pelvis is pointed out. The diagrammatic frontal and lateral sections through the female pelvis presented in this article are based on results of anatomic studies of cadavers of adults and fetuses and on pelvic ultrasonography and magnetic resonance imaging findings. The horizontal floor of the pelvis, the perineum, and the pelvic cavity are visible on both sections. Newly identified structures are the urogenital fossa, the pubosacral perineal cord, and the urethro-clitorido-vulvar erectile body. Special attention is devoted to the description of the ischiorectal fossa and its extensions and of the uro-genito-rectal mesentery. A study of the functional anatomy of the female pelvis would be feasible. PMID- 8638077 TI - [Tissue glue in endoscopic surgery]. AB - Tissue-glue stands out by virtue of its ability to ensure non-traumatic tissue apposition and hemostasis, leading to a reduction in operative time. This explains the use of this technique in a wide range of indications in gynecological endoscopy. These include ovarian reconstruction after the extraction of cysts, tubal anastomoses and even closure of iatrogenic perforations of the uterus. In these indications, the application of tissue-glue can be considered as the method of choice because of its excellent long term results. In contrast, its effect in the sealing of peritoneal or serosal defects with the aim of avoiding adhesions has not yet been clearly demonstrated. Analysis of a sufficient number of cases with long follow-up is essential before any final assessment can be made. Among 75 instances of endoscopic use of tissue glue up to now, we have never encountered any post-operative complications, nor during subsequent follow-up. In addition to its ability to stimulate tissue healing, tissue-glue offers a simple and non-traumatic alternative to the lengthy technique of endoscopic sutures, with a hemostatic action in parallel. PMID- 8638079 TI - [Vaginal environment. The vaginal climate and its variations]. AB - Variations in the vaginal environment that occur throughout the life span are described. The nature and cybernetics of factors involved in these variations are discussed. PMID- 8638080 TI - [Does the incidence of premature labor vary according to the season?]. AB - To investigate the epidemiology of premature delivery, in particular regarding potential seasonal influences, a retrospective medical-record study was conducted in 3345 women who delivered prematurely over a six-year period (1988-1993). The control group was composed of 53,162 women who carried their pregnancies to term. Each study subject had delivered at least one liveborn baby, prematurely or at full term. The premature delivery rate (ratio of premature over term deliveries) was highest in May through July and decreased significantly in January and February (p < 0.05). Premature delivery rates were similar in primiparas and multiparas (p > 0.05) and were not influenced by the gender of the fetus (p > 0.05). In conclusion, premature delivery rates vary across seasons, suggesting an influence of weather conditions. PMID- 8638081 TI - [Is the length of gestation a multiple of a fundamental cycle?]. AB - Pregnancy duration was accurately determined using early ultrasonography in 21 patients prior to spontaneous abortion and in two successive pregnancies in each of 57 women prior to spontaneous delivery. Pregnancy durations before abortion or delivery approximated multiples of 14 days (14.4 and 13.3 days, respectively). In addition, in a given woman, the duration of two successive pregnancies carried to term was often identical; in a smaller number of cases, a difference of about 12 days (calculated value, 11.8 days) or, rarely, 24 days, was found. These values may correspond to the same functional period. These observations suggest that the duration of pregnancy (mean, 266 days) may be a multiple of the duration of the luteal phase of the menstrual cycle (12 to 14 days). Interindividual variations in luteal phase duration (standard deviation, 0.5 to 1 day) may explain inter individual variations in pregnancy duration (standard deviation of about 15 days). The mean multiple may be 20. Thus, pregnancy duration may be determined both by fetal maturation and by a maternal time-counting mechanism. This hypothesis is discussed in the light of current data. PMID- 8638082 TI - [Recurrent vulvovaginal mycoses]. AB - The treatment of recurrent vulvovaginal fungal infections is difficult. Pathogenic mechanisms are discussed. Available pharmacologic treatments and their mechanisms of action are reviewed. Patients clearly prefer oral treatment. Ketoconazole is toxic. Only topical or nonabsorbed oral agents can be used during pregnancy. Agents absorbed via the digestive tract can be used in women who use effective contraceptive methods. Published data demonstrate that trifluconazole (which has not yet been granted a license in France) is potent and less toxic than ketoconazole, and that recurrences at discontinuation of this drug are less common. A few hypotheses for future research are presented. PMID- 8638083 TI - Improving employees' telephone skills. PMID- 8638084 TI - From wood to gold: the story of homemade dentures. PMID- 8638085 TI - A systematic approach to improved anterior esthetics: part 2. PMID- 8638086 TI - Porcelain veneers: single-die technique made simple. PMID- 8638087 TI - Significance of serum laminin and type IV collagen levels for metastasis in murine RCT sarcoma. AB - We investigated in vivo the correlation between pulmonary metastasis and serum concentrations of laminin and type IV collagen in high-metastatic RCT(+) and low metastatic RCT(-) clones established from poorly differentiated RCT sarcoma in C3H/He mice. The in vitro invasiveness of these cell clones through the extracellular matrix was also studied. In the in vitro invasion assay, high metastatic RCT(+) cells showed significantly more invasiveness than low metastatic RCT(-) cells. The different invasiveness of these cloned cells was associated with their different ability to attach to and degrade the matrix. In mice inoculated with RCT(+) cells, serum levels of laminin and type IV collagen rose with the increase in the number of pulmonary metastatic nodules. Serum levels of these extracellular matrix components started to rise when pulmonary metastatic nodules were macroscopically observed in the RCT(+) group. In mice bearing RCT(-) cells with lower metastatic ability, there were no significant increases in serum extracellular matrix levels. These findings suggested that serum concentrations of laminin and type IV collagen might be useful markers for the early detection of metastasis. PMID- 8638088 TI - Implications of acidic tumor microenvironment for neoplastic growth and cancer treatment: a computer analysis. AB - The acidic microenvironment found in most solid tumors appears to be a main regulator for the self-organized development of neoplastic growth and invasion. Induced by mitogenic stimulation and/or oncogenic alterations in their signal transduction network, tumor cells develop an improved capability for acid extrusion. This clamps intracellular pH to slightly alkaline values permissive for growth and proliferation and provides tumor cells with an enhanced resistance against acidic extracellular conditions. Since rapid tumor cell growth under hypoxic conditions is accompanied by glycolytic metabolism, and consequently, acid production, acid export is further enhanced. Local extracellular acidification is facilitated by microcirculatory inadequacy resulting in both reduced buffering capacity and functional heterogeneity inside the tumor. Significant microenvironmental pH gradients promote tumor cell invasion and inhibit the immune response. The scenario is checked by means of a dynamic computer model. The stochastic minimal model supports the hypothesis that acidification of the microenvironment by malignant cells cannot only be regarded as a supplementary side effect of tumor cell metabolism, but rather as a strategic principle to 'enslave' processes normally counteracting neoplastic growth and invasion. The results are discussed with respect to current concept for cancer treatment. PMID- 8638089 TI - B16F10 murine melanoma cells express interleukin-2 and a functional interleukin-2 receptor. AB - In this study, we analyzed interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression in murine B16F10 melanoma and studied the effect of recombinant IL-2 (rIL-2) on the proliferation of these cells. Flow cytometry analysis revealed the presence of the IL-2R alpha subunit in B16F10 melanoma, with a mean positivity rate of 30%. Using confocal microscopy, the expression of this chain could be visualized on the surface of B16F10 cells and in intracellular compartments when the cells were permeabilized with ethanol. In addition to the alpha subunit, the IL-2R beta subunit was also expressed in B16F10 cells as shown by reverse transcription and polymerase chain reaction analysis. The functionality of the IL 2R on B16F10 cells was shown by the fact that cell proliferation increased dose dependently with the addition of rIL-2 to the culture medium. We also detected expression of the IL-2 gene in B16F10 cells. In Northern blot assays, a typical band of 0.9 kb corresponding to IL-2 mRNA was observed, although supernatants from B16F10 cultures had no detectable IL-2 activity. Furthermore, the addition of neutralizing antibody (anti-IL-2) to cell cultures had no effect on cell proliferation. From these results, we concluded that an IL-2 signalling system is present in murine B16F10 melanoma cells and that IL-2 favors B16F10 cell proliferation, suggesting a role for this cytokine in the tumoral activity of these cells. PMID- 8638090 TI - Enhancing effects of epidermal growth factor on human squamous cell carcinoma motility and matrix degradation but not growth. AB - In order to ascertain the effects of epidermal growth factor (EGF) on human cancer invasion abilities, three cell lines of human oral squamous cell carcinoma were studied using a phagokinetic track assay and zymography. EGF (1-100 ng/ml) was found to inhibit the growth but enhance the random motility of all three cell lines in a concentration-dependent fashion. Exposure to EGF, dose-dependently, led to an increased production of urokinase-type plasminogen activator and M(r) 92 kD matrix metalloproteinase by the same cells. These results strongly suggest that EGF may promote human squamous cell carcinoma invasion and metastasis. PMID- 8638091 TI - Membrane-bound (MUC1) and secretory (MUC2, MUC3, and MUC4) mucin gene expression in human lung cancer. AB - Abnormalities of mucin-type glycoproteins have been described in lung cancers, but their molecular basis is unknown. In this study, mucin-core-peptide-specific antibodies and cDNA probes were used to determine the relative expression of mucin genes corresponding to one membrane-bound mucin (MUC1), two intestinal mucins (MUC2 and MUC3), and one tracheobronchial mucin (MUC4) in normal (nonneoplastic) lung, and in lung neoplasms. Normal lung tissues exhibited a distinct pattern of mucin gene expression, with high levels of MUC1 and MUC4 mRNA and low to absent levels of MUC2 and MUC3 mucin immunoreactivity and mRNA. In contrast, lung adenocarcinomas, especially well-differentiated cancers, exhibited increased MUC1, MUC3, and MUC4 mRNA levels. Lung squamous-cell, adenosquamous, and large-cell carcinomas were characterized by increased levels of MUC4 mucin only. We conclude that the expression of one membrane-bound and several secretory type mucins is independently regulated and markedly altered in lung neoplasms. The frequent occurrence of increased MUC4 transcripts in a variety of non-small cell lung cancers indicates the potential importance of this type of mucin in lung cancer biology. PMID- 8638092 TI - Bureaucrats save lives. PMID- 8638093 TI - Lioness leadership. PMID- 8638095 TI - AIDS office budget morass settled. PMID- 8638094 TI - Treatment of chronic Lyme disease. PMID- 8638096 TI - Klausner's unconventional 'field station' in Seattle. PMID- 8638097 TI - Donna Shalala: 'leaving footprints' at HHS. PMID- 8638099 TI - FDA reform starts down the track. PMID- 8638098 TI - Vaccine drought spurs NIAID plan to improve industry ties... PMID- 8638100 TI - Is hippocampal cell death a myth? PMID- 8638101 TI - An intriguing new lead on Huntington's disease. PMID- 8638103 TI - Investors sought for AIDS vaccine trials. PMID- 8638102 TI - T cell inactivation linked to Ras block. PMID- 8638104 TI - The whole lactose repressor. PMID- 8638105 TI - Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. AB - The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA. PMID- 8638106 TI - A distinct G1 step required to specify the Chinese hamster DHFR replication origin. AB - Nuclei isolated from Chinese hamster ovary (CHO) cells at various times during the G1 phase of the cell cycle were stimulated to enter S phase by incubation in Xenopus egg cytosol. Replication of DNA initiated within the dihydrofolate reductase (DHFR) origin locus in nuclei isolated late in G1, but at random sites in nuclei isolated early in G1. A discrete transition point occurred 3 to 4 hours after metaphase. Neither replication licensing nor nuclear assembly was sufficient for origin recognition. Thus, a distinct cell cycle-regulated event in the nucleus restricts the initiation of replication to specific sites downstream of the DHFR gene. PMID- 8638107 TI - Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. AB - T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12 myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy. PMID- 8638108 TI - Blocked Ras activation in anergic CD4+ T cells. AB - T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate binding protein p21ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation. This block in Ras activation may lead to defective transactivation at activator protein 1 sites in anergic cells and may enable T cells to shut down IL-2 production selectively during anergy. PMID- 8638109 TI - Antigen presentation and T cell development in H2-M-deficient mice. AB - HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells. PMID- 8638110 TI - Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase. AB - Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme. PMID- 8638111 TI - Somatic mutation of immunoglobulin V genes in vitro. AB - The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection. PMID- 8638112 TI - Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers. AB - In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow. PMID- 8638114 TI - Bidirectional control of quantal size by synaptic activity in the hippocampus. AB - Analysis of strontium-induced asynchronous release of quanta from stimulated synapses revealed that long-term potentiation and long-term depression in the CA1 region of the mammalian hippocampus are associated with an increase and a decrease, respectively, in quantal size. At a single set of synapses, the increase in quantal size seen with long-term potentiation was completely reversed by depotentiating stimuli. Long-term potentiation and depression are also associated with an increase and decrease, respectively, in the frequency of quantal events, consistent with an all-or-none regulation (up or down) of clusters of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a change in the release of transmitter, or both. PMID- 8638115 TI - Self-fertilization, linkage disequilibrium, and strain in Plasmodium falciparum. PMID- 8638113 TI - HIV-1 Langerhans' cell tropism associated with heterosexual transmission of HIV. AB - Heterosexual transmission by vaginal intercourse accounts for most transmission of human immunodeficiency virus-type 1 (HIV-1) in Africa and Asia but is less important in the HIV-1 epidemics of the United States and Western Europe. Epithelial Langerhans' cells (LCs) represent a possible source of initial cell contact for vaginal infection. Fifteen primary isolates of HIV-1 from U.S. homosexuals and 18 HIV-1 isolates from Thailand heterosexuals were evaluated for growth in LCs of U.S. origin. All the viruses from the Thai heterosexuals, which were subtype E, grew more efficiently in the LCs than any of the viruses from the U.S. homosexuals, which are subtype B. These results suggest that LC tropism is associated with the efficiency of heterosexual transmission of HIV. PMID- 8638116 TI - Faunal evidence and Sterkfontein Member 2 foot bones of early hominid. PMID- 8638117 TI - More Haemophilus and Mycoplasma genes. PMID- 8638118 TI - More Haemophilus and Mycoplasma genes. PMID- 8638119 TI - Structures of an MHC class II molecule with covalently bound single peptides. AB - The high-resolution x-ray crystal structures of the murine major histocompatibility complex (MHC) class II molecule, I-E(k), occupied by either of two antigenic peptides were determined. They reveal the structural basis for the I-E(k) peptide binding motif and suggest general principles for additional alleles. A buried cluster of acidic amino acids in the binding groove predicted to be conserved among all murine I-E and human DR MHC class II molecules suggests how pH may influence MHC binding or exchange of peptides. These structures also complement mutational studies on the importance of individual peptide residues to T cell receptor recognition. PMID- 8638120 TI - Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. AB - During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions. Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage. PMID- 8638121 TI - Enhanced degradation of EGF receptors by a sorting nexin, SNX1. AB - The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery. A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes. PMID- 8638122 TI - Determination of life-span in Caenorhabditis elegans by four clock genes. AB - The nematode worm Caenorhabditis elegans is a model system for the study of the genetic basis of aging. Maternal-effect mutations in four genes--clk-1, clk-2, clk-3, and gro-1--interact genetically to determine both the duration of development and life-span. Analysis of the phenotypes of these mutants suggests the existence of a general physiological clock in the worm. Mutations in certain genes involved in dauer formation (an alternative larval stage induced by adverse conditions in which development is arrested) can also extend life-span, but the life extension of Clock mutants appears to be independent of these genes. The daf 2(e1370) clk-1(e2519) worms, which carry life-span-extending mutations from two different pathways, live nearly five times as long as wild-type worms. PMID- 8638123 TI - The role of zinc in selective neuronal death after transient global cerebral ischemia. AB - Zinc is present in presynaptic nerve terminals throughout the mammalian central nervous system and likely serves as an endogenous signaling substance. However, excessive exposure to extracellular zinc can damage central neurons. After transient forebrain ischemia in rats, chelatable zinc accumulated specifically in degenerating neurons in the hippocampal hilus and CA1, as well as in the cerebral cortex, thalamus, striatum, and amygdala. This accumulation preceded neurodegeneration, which could be prevented by the intraventricular injection of a zinc chelating agent. The toxic influx of zinc may be a key mechanism underlying selective neuronal death after transient global ischemic insults. PMID- 8638124 TI - Increase in single L-type calcium channels in hippocampal neurons during aging. AB - Voltage-activated calcium (Ca2+) influx is increased in mammalian CA1 hippocampal neurons during aging. However, the molecular basis for this elevation is not known. The partially dissociated hippocampal ("zipper") slice preparation was used to analyze single Ca2+ channel activity in CA1 neurons of adult and aged rats. Total L-type Ca2+ channel activity in patches was found to increase with aging, primarily because of an increase in the density of functional channels. Learning in aged animals was inversely correlated with channel density. This increase in functional Ca2+ channels with aging could underlie the vulnerability of neurons to age-associated neurodegenerative conditions. PMID- 8638125 TI - Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein. AB - The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function. PMID- 8638126 TI - Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. AB - The adenomatous polyposis coli gene (APC) is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of ARMADILLO, a component of the WINGLESS signaling pathway in Drosophila development. Here it is shown that when beta-catenin is present in excess, APC binds to another component of the WINGLESS pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila ZESTE WHITE 3. APC was a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region was dependent on phosphorylation by GSK3 beta. PMID- 8638128 TI - DNA replication fork pause sites dependent on transcription. AB - Replication fork pause (RFP) sites transiently arresting replication fork movement were mapped to transfer RNA (tRNA) genes of Saccharomyces cerevisiae in vivo. RFP sites are polar, stalling replication forks only when they oppose the direction of tRNA transcription. Mutant tRNA genes defective in assembly of transcription initiation complexes and a temperature-sensitive RNA polymerase III mutant (rpc160-41) defective in initiation of transcription do not stall replication forks, suggesting that transcription is required for RFP activity. PMID- 8638129 TI - Misconduct: judgment called for. PMID- 8638130 TI - Gifted public servants. PMID- 8638127 TI - Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase. AB - The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo. PMID- 8638132 TI - A $1 billion 'tax' on R&D funds. PMID- 8638131 TI - Gifted public servants. PMID- 8638133 TI - UCSF, Stanford hospitals to merge. PMID- 8638134 TI - Varmus proposes to scrap the RAC. PMID- 8638135 TI - Patent law closes drug loophole. PMID- 8638136 TI - Unique protein database imperiled. PMID- 8638137 TI - After 9 years, a tangled case lurches toward a close. PMID- 8638138 TI - Worm genes imply a master clock. PMID- 8638139 TI - Flies unmask evolutionary warfare between the sexes. PMID- 8638140 TI - Chemokines take center stage in inflammatory ills. PMID- 8638141 TI - Another twist to MHC-peptide recognition. PMID- 8638142 TI - Regulating cell proliferation: as easy as APC. PMID- 8638143 TI - Spread of synaptic depression mediated by presynaptic cytoplasmic signaling. AB - Postsynaptic activity may modulate presynaptic functions by transsynaptic retrograde signals. At developing neuromuscular synapses in Xenopus nerve-muscle cultures, a brief increase in the cytosolic calcium ion (Ca2+) concentration in postsynaptic myocytes induced persistent depression of presynaptic transmitter secretion. This depression spread to distant synapses formed by the same neuron. Clearance of extracellular fluid did not prevent the spread of depression, and depression could not be induced by increasing the Ca2+ concentration in a nearby myocyte not in contact with the presynaptic neuron. Thus, the spread of depression is mediated by signaling in the presynaptic cytoplasm, rather than by a retrograde factor in the extracellular space. PMID- 8638144 TI - Methylene chloride. PMID- 8638145 TI - Canavan gene therapy protocol. PMID- 8638146 TI - Clotting dispute. PMID- 8638147 TI - Wrong hookworm. PMID- 8638148 TI - HERG sequence correction. PMID- 8638149 TI - Space biology. Surgery confounds mission review. PMID- 8638151 TI - Seeking life's bare (genetic) necessities. PMID- 8638152 TI - Studly sheep by non-Mendelian means. PMID- 8638150 TI - Rifkin's latest target: genetic testing. PMID- 8638153 TI - Synapse-making molecules revealed. PMID- 8638154 TI - Zebrafish embryology builds better model vertebrate. PMID- 8638155 TI - Viral counts count in HIV infection. PMID- 8638156 TI - Mammalian cytochrome c oxidase, a molecular monster subdued. PMID- 8638157 TI - The cerebellum: a neuronal learning machine? AB - Comparison of two seemingly quite different behaviors yields a surprisingly consistent picture of the role of the cerebellum in motor learning. Behavioral and physiological data about classical conditioning of the eyelid response and motor learning in the vestibulo-ocular reflex suggests that (i) plasticity is distributed between the cerebellar cortex and the deep cerebellar nuclei; (ii) the cerebellar cortex plays a special role in learning the timing of movement; and (iii) the cerebellar cortex guides learning in the deep nuclei, which may allow learning to be transferred from the cortex to the deep nuclei. Because many of the similarities in the data from the two systems typify general features of cerebellar organization, the cerebellar mechanisms of learning in these two systems may represent principles that apply to many motor systems. PMID- 8638158 TI - The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. AB - The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified. PMID- 8638159 TI - GS28, a 28-kilodalton Golgi SNARE that participates in ER-Golgi transport. AB - Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28 kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex. PMID- 8638160 TI - Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. AB - The relation between viremia and clinical outcome in individuals infected with human immunodeficiency virus-type 1 (HIV-1) has important implications for therapeutic research and clinical care. HIV-1 RNA in plasma was quantified with a branched-DNA signal amplification assay as a measure of viral load in a cohort of 180 seropositive men studied for more than 10 years. The risk of acquired immunodeficiency syndrome (AIDS) and death in study subjects, including those with normal numbers of CD4+ T cells, was directly related to plasma viral load at study entry. Plasma viral load was a better predictor of progression to AIDS and death than was the number of CD4+ T cells. PMID- 8638161 TI - Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. AB - The absence of CTLA-4 results in uncontrolled T cell proliferation. The T cell receptor-specific kinases FYN, LCK, and ZAP-70 as well as the RAS pathway were found to be activated in T cells of Ctla-4-/- mutant mice. In addition, CTLA-4 specifically associated with the tyrosine phosphatase SYP, an interaction mediated by the SRC homology 2 (SH2) domains of SYP and the phosphotyrosine sequence Tyr-Val-Lys-Met within the CTLA-4 cytoplasmic tail. The CTLA-4 associated SYP had phosphatase activity toward the RAS regulator p52SHC. Thus, the RAS pathway and T cell activation through the T cell receptor are regulated by CTLA-4-associated SYP. PMID- 8638162 TI - Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. AB - The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation. PMID- 8638163 TI - Entorhinal-hippocampal interactions revealed by real-time imaging. AB - The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to gamma-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus. PMID- 8638164 TI - TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction. AB - Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction. PMID- 8638165 TI - Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves. AB - Highly correlated neural activity in the form of spontaneous waves of action potentials is present in the developing retina weeks before vision. Optical imaging revealed that these waves consist of spatially restricted domains of activity that form a mosaic pattern over the entire retinal ganglion cell layer. Whole-cell recordings indicate that wave generation requires synaptic activation of neuronal nicotinic acetylcholine receptors on ganglion cells. The only cholinergic cells in these immature retinas are a uniformly distributed bistratified population of amacrine cells, as assessed by antibodies to choline acetyltransferase. The results indicate that the major source of synaptic input to retinal ganglion cells is a system of cholinergic amacrine cells, whose activity is required for wave propagation in the developing retina. PMID- 8638166 TI - Polyclonal origin of colonic adenomas in an XO/XY patient with FAP. AB - It is widely accepted that tumors are monoclonal in origin, arising from a mutation or series of mutations in a single cell and its descendants. The clonal origin of colonic adenomas and uninvolved intestinal mucosa from an XO/XY mosaic individual with familial adenomatous polyposis (FAP) was examined directly by in situ hybridization with Y chromosome probes. In this patient, the crypts of the small and large intestine were clonal, but at least 76 percent of the microadenomas were polyclonal in origin. PMID- 8638167 TI - X chromosome dosage compensation in Drosophila. PMID- 8638168 TI - Medical complications of glue sniffing. AB - Glue sniffing refers to the deliberate inhalation of volatile solvents, commonly found in adhesives, for the purpose of intoxication. The increasing prevalence of inhalant use suggests that many physicians will encounter a glue-sniffing patient at some time during their practice. Knowledge of the epidemiology, toxicology, and medical complications associated with glue sniffing is essential in obtaining an accurate history of substance abuse and in clinically managing these patients. This review of sources is intended to aid clinicians in the recognition of glue sniffing patients and in the diagnosis of acute and chronic medical complications associated with the abuse of glues, solvents, and related substances. Glue sniffing has been linked to sudden death and chronic damage to the heart, lungs, kidneys, liver, peripheral nerves, and brain. Inhalant abuse in general is associated with mortality and morbidity, including social, educational, and economic deprivation in adolescents and young adults. PMID- 8638169 TI - Diabetic eye disease: a primary care perspective. AB - Patients with diabetes are at risk for multiple visual complications, most notably diabetic retinopathy, but also glaucoma, cataracts, optic nerve disease, and strabismus. Diabetic retinopathy is the leading cause of blindness for the majority of Americans. Microvascular damage from diabetes leads to microaneurysms, hemorrhage, exudates, and cotton-wool spots. Further progression of disease leads to new vessel growth, or neovascularization. Growth of new blood vessels can cause severe hemorrhage, scarring, and permanent visual loss. Various randomized, prospective studies have clearly shown benefit from laser therapy at specific stages of progression of retinopathy. Proper referral and close follow up are paramount to the preservation of visual function. PMID- 8638170 TI - Prevalence of lead intoxication in urban, adult medical inpatients. AB - Medical inpatients in inner-city hospitals have both potentially lead-related disorders and potential lead exposure. To see whether there is substantial unexpected lead intoxication in this population, we did a cross-sectional study of 117 consecutively admitted patients to a general medical ward of an inner-city university hospital. The mean (SD) blood lead level was 6.7 (2.8) micrograms/dL, with a range of 0 to 37 micrograms/dL. Twenty-one patients (18%) had mildly elevated lead levels (10 to 19 micrograms/dL) and 2 patients (2%) had moderately elevated lead levels (20 to 44 micrograms/dL). Only 1 of 117 patients (0.9%, 95% CI: 0.0% to 2.6%) had lead intoxication (a lead level > or = 20 micrograms/dL or a lead level > or = 15 micrograms/dL and a free erythrocyte protoporphyrin level > 90 micrograms/dL of erythrocytes, with no alternative explanation for the laboratory abnormalities). We conclude that lead intoxication is uncommon in this population and that routine screening is not warranted. Although mild elevation of blood lead level is common, the clinical significance remains to be determined. PMID- 8638172 TI - Systemic inflammatory response syndrome caused by chronic salicylate intoxication. AB - Systemic inflammatory response syndrome (SIRS) is characterized by body temperature abnormalities, tachypnea or hyperventilation, tachycardia, and leukocytosis or leukopenia. Although it is typically associated with a serious infection and referred to as sepsis, SIRS can stem from noninfectious causes, as well. We report the cases of four patients with toxic serum levels of salicylate (33.5 to 67.6 mg/dL) and SIRS, and we discuss mechanisms responsible for SIRS. Our patients showed temperature disturbances (35.5 degrees C to 39.8 degrees C), noncardiogenic pulmonary edema, and mixed acid base disturbances. Other abnormalities included coagulopathy (disseminated intravascular coagulation), encephalopathy, and hypotension. All four patients recovered from SIRS, probably due to early recognition and treatment; only one patient did not survive the hospitalization. Chronic salicylate toxicity should be considered as a cause of SIRS in the absence of a source of infection, since survival appears to be dependent on prompt diagnosis and management. PMID- 8638171 TI - The air gun: toy or weapon? AB - Originally used in warfare, air guns are commonly used in target shooting, as toys, and as "beginner" guns for children. The projectile force of these weapons can rival that of many conventional handguns. Pneumatic weapons pose a serious threat to the pediatric population, and their potential for serious injury must be recognized. PMID- 8638173 TI - Physician reporting of and referral for patient complaints about sleep disorders. AB - The first goal of this Nebraska-based study was to determine physician reporting of patient complaints about sleep. The second goal was to determine the patterns of referral to sleep disorders laboratories by geographic location and specialty practice. A total of 299 Nebraska physicians (177 in urban areas and 122 in rural areas) completed a questionnaire concerning sleep complaints among patients. We found no statistically significant differences between urban and rural physicians in the percentage of patients reporting insomnia, excessive daytime sleepiness, snoring, or other sleep complaints. Psychiatrists reported on significantly more patients with insomnia and excessive daytime sleepiness than did other physicians; however, they tended to refer fewer patients to sleep disorders laboratories than did physicians practicing internal medicine. Urban physicians referred significantly more patients to sleep disorders laboratories than did rural physicians. We concluded that physician reporting of patient complaints about sleep is similar in urban and rural areas of Nebraska. However, physicians in rural areas tend to refer fewer patients to sleep disorders laboratories than do physicians in urban communities. PMID- 8638174 TI - Epiglottitis: a 9-year case review. AB - A retrospective chart review of all cases of suspected epiglottitis from 1985 to 1993 at Arkansas Children's Hospital identified 29 patients treated for epiglottitis during this 9-year period. Review of the immunization status of these patients showed 72% without Hib vaccination, 11% who had initiated the Hib series, and 17% with unknown immunization status. The incidence of epiglottitis declined over the duration of the study with 0.61/1,000 admissions in 1985, 0.27 in 1986, 1.42 in 1987, 0.9 in 1988, 0.39 in 1989, 0.0 in 1990, 0.35 in 1991, 0.0 in 1992, and 0.0 in 1993. Comparing the years before available conjugate vaccine- 1985 to 1988--with the years after conjugate vaccine--1989 to 1993--shows a significant change in the incidence of epiglottitis. With increasing populations of susceptible children receiving Hib immunization, Hib epiglottitis may become a vanishing entity. PMID- 8638175 TI - Visceral leishmaniasis in Pakistani children. AB - Visceral leishmaniasis is endemic in Pakistan. No studies of visceral leishmaniasis in childhood have been reported from Pakistan. We prospectively studied clinical and laboratory features in 58 Pakistani children with visceral leishmaniasis. Mean age of the children was 2.9 years. Fever, pallor, and abdominal distention were the most common clinical manifestations, and hematologic abnormalities were the most common laboratory findings. All children recovered after antimicrobial therapy. Pakistani children with visceral leishmaniasis tended to be younger than affected children from Africa and were less likely to have lymphadenopathy. PMID- 8638176 TI - Significance of chest trauma in children. AB - Chest trauma in children is a marker of injury severity and is associated with a high mortality rate. This retrospective study of 1,356 trauma patients from a private pediatric hospital over a 2.5-year period identified 82 patients with chest injuries and a mortality rate of 22%. Results of Injury Severity Score, Glasgow Coma Scale, and Revised Trauma Score all indicated that children with chest injuries sustained more severe injuries. The presence of any extrathoracic injury was associated with a higher mortality (29%) than chest injury alone (4.3%). The type of extrathoracic injury was important, with head and neck injuries resulting in the highest mortality. Specific chest injuries, such as rib fractures and pulmonary contusions, were not related to increased mortality unless there was an associated extrathoracic injury. Many reports have shown a high mortality associated with chest trauma. This study suggests that it is the associated extrathoracic injury, rather than the chest injury itself, that is the real cause of the high mortality. PMID- 8638177 TI - Abdominal wall reconstruction after temporary abdominal wall closure in trauma patients. AB - We retrospectively analyzed 36 patients requiring temporary abdominal wall closure on admission to a level I trauma center from 1988 to 1992. There were 10 deaths (28%) in the study population. Of the 26 survivors, 8 patients (31%) had primary fascial closure at initial hospitalization, whereas 18 patients (69%) required split-thickness skin grafting to visceral granulation tissue. Of these 18 patients, 13 have had ventral herniorrhaphy at subsequent admission. Eight of these patients had primary fascial closure, 4 required primary fascial approximation with prosthetic onlay reinforcement, and 1 required multiple operations including prosthetic reconstruction and eventual complex tissue transfer. Complications occurred in 3 patients (14%) and included two wound seromas, which were drained nonoperatively, and a wound infection necessitating removal of prosthetic material and subsequent reconstruction with complex tissue transfer. Follow-up reveals no recurrent hernia at 24 months. Abdominal wall reconstruction after temporary closure can be done safely and promptly, with good functional and esthetic results. PMID- 8638178 TI - Microbiologic assessment of the transabdominal ultrasound transducer head. AB - The objectives of this study were to determine (1) the rate of bacterial isolation from the abdomen of women having obstetric ultrasonography, (2) the rate of bacterial transmission to the transducer head, and (3) the eradication rate after routine wiping of the transducer head. A total of 191 obstetric patients participated in this study. At the start of each day, the transducer head and the coupling gel were cultured. Aerobic cultures were obtained from each patient's periumbilical and suprapubic areas before the transabdominal scan and from the transducer head before and after wiping off the gel with a dry cloth. Daily transducer head and gel cultures were negative. Of the abdominal skin cultures, 175 (92%) were positive; 35 (18%) were positive for serious organisms, and 140 (74%) were positive for organisms of low virulence. Sixty percent of the transducer head cultures from women with abdominal skin pathogens were positive before the gel was wiped off. None of the cultures from the transducer head were positive after removal of the gel. We conclude that many women carry potentially virulent pathogens on the abdominal skin and that transmission of these organisms to the transducer head commonly occurs. Physical removal of the gel from the transducer head effectively eradicates these microorganisms, minimizing patient to-patient transmission. PMID- 8638179 TI - Pulmonary fibrosis and lung cancer in the United States: analysis of the multiple cause of death mortality data, 1979 through 1991. AB - We determined the relationship between pulmonary fibrosis and lung cancer in the United States from 1979 through 1991 by analyzing death certificate reports compiled by the National Center for Health Statistics. Of the 26,866,600 people who died during the study period, 107,312 died with pulmonary fibrosis, 1,739,725 died with lung cancer, 2,040,634 died with chronic obstructive pulmonary disease, and 7,807 died with asbestosis. Lung cancer occurred less frequently among decedents with pulmonary fibrosis (4.81%) and more frequently among decedents with chronic obstructive pulmonary disease (10.06%) and decedents with asbestosis (26.60%) than among decedents in the general population (6.48%). We conclude that the prevalence of lung cancer among people who died with a diagnosis of pulmonary fibrosis is lower than the 10% to 40% prevalence that has been reported in case series of pulmonary fibrosis. PMID- 8638181 TI - Secondary arterial hypertension linked to Freon exposure. AB - Freons are generally considered to be minimally toxic. There are no reports in the literature of Freons causing secondary arterial hypertension. We report two cases of acute, massive Freon exposure that preceded secondary arterial hypertension. We hypothesize that the arterial hypertension was precipitated by renal proximal tubular damage, although several other mechanisms are possible. PMID- 8638182 TI - False aneurysm of the thyrocervical trunk. AB - False aneurysm of the thyrocervical trunk as a complication of central venous catheterization is rare. We report a case of this complication in a 63-year-old white man and review the history, diagnosis, pathophysiology, and treatment of these aneurysms. PMID- 8638180 TI - Primary squamous cell carcinoma of the breast. AB - Primary squamous cell carcinoma of the breast is a rare cancer. Published reports of prognosis are variable, and most studies are case reports of one or a few patients. We report an additional case of squamous cell carcinoma of the breast occurring in a 53-year-old black woman. In reviewing the reported cases of this tumor over the past 20 years, we compared features of this cancer with those of the more common breast adenocarcinoma with squamous metaplasia. When squamous cell carcinoma of the breast is encountered, a skin primary lesion and metastasis from a distant site should be excluded. Prognosis is determined largely by stage and does not differ significantly from the prognosis in breast adenocarcinoma with squamous metaplasia. PMID- 8638183 TI - Mycobacterium avium-intracellulare complex pneumonia in a non-HIV-infected individual: an increasingly recognized disease. AB - In the past, Mycobacterium avium complex (MAC) was considered a colonizing microbe in the immunocompetent host. Today it should be considered a potential pathogen. We present a case of MAC necrotizing pneumonia in a 27-year-old man who tested negatively for the human immunodeficiency virus, had no typical granulomas, and responded rapidly to antimicrobial therapy. PMID- 8638184 TI - Malignant mesenchymoma of the pleura. AB - Malignant mesenchymomas are uncommon tumors of soft tissues. Three such tumors involving the pleura have been reported in the literature. We report a case of malignant mesenchymoma of the pleura that had liposarcomatous, rhabdomyosarcomatous, chondrosarcomatous, and osteosarcomatous elements. PMID- 8638186 TI - Sudden cardiac death due to primary coronary sinus thrombosis. AB - Coronary sinus thrombosis has been reported only as a complication of cardiac transplantation and right heart catheterization in noninfected patients. We report a case of acute coronary sinus thrombosis occurring in the absence of these procedures and resulting in sudden cardiac death. PMID- 8638187 TI - Gastric intramural pseudocyst with associated gastric outlet obstruction: recognition and management. AB - Pancreatic pseudocyst is a well-recognized complication of pancreatitis. Most pseudocysts either resolve spontaneously or are amenable to internal drainage. Occasionally, the pseudocyst extends to distant areas within the abdominal cavity or may invade a nearby anatomic structure. Direct extension into the stomach is infrequently noted both radiographically and clinically. Recognition and management of this entity are described. PMID- 8638185 TI - Verapamil therapy for persistent antral spasms in a child. AB - Little is known about the use of calcium channel blockers in children with gastric motility disorders. We report the case of an 11-year-old child who was hospitalized after 3 weeks of persistent vomiting, abdominal pain, and a 12-lb weight loss. When his symptoms failed to improve with traditional treatment, verapamil therapy was begun. Within 24 hours, his symptoms totally resolved. PMID- 8638188 TI - Double-lumen esophagus: formation of a new lumen or septation of the existing one? AB - Presented here are two cases in which two esophageal lumens were identified at endoscopy. One patient had a history of antireflux surgery and both patients had received esophageal dilations. Both patients have done poorly with standard esophageal dilation and are not considered likely to gain from surgery. The mechanism of formation of the second lumen is not certain in Case 1, but in Case 2 a self-contained perforation appears to be the likely mechanism. There is no clear definition of "double-lumen esophagus" in the literature. The term is used at times interchangeably with esophagogastric fistula. We propose that the phrase "double-lumen esophagus" is a morphologic description and should be used only when the two lumens are of nearly equal diameter. The term "fistula" should be used whenever that is the likely mechanism. Hence, Case 1 of our report is the true double-lumen esophagus, while Case 2 is an esophagogastric fistula. Since patients do poorly with standard dilation, newer endoscopic modalities may have a role in the management of these rare but difficult cases. PMID- 8638190 TI - Placenta increta occurring in a blighted ovum. AB - We present a rare case of placenta increta, confirmed postoperatively by pathologic analysis, occurring before 20 weeks' gestation in a blighted ovum. Hysterectomy was necessary for control of hemorrhage. PMID- 8638189 TI - Paclitaxel: severe mucocutaneous toxicity in a patient with hyperbilirubinemia. AB - Paclitaxel is a novel anticancer drug that is being increasingly used to treat cancer of the breast and other organs. We describe a patient with metastatic breast cancer and liver dysfunction who had severe mucocutaneous toxicity after administration of a standard dose of paclitaxel. Another interesting finding in this patient was that the administration of paclitaxel led to a prompt resolution of parathyroid hormone-related protein (PTHrP)-mediated hypercalcemia, which had previously proven to be refractory to multiple conventional antihypercalcemic agents as well as anthracycline-containing chemotherapy combination. The need for definitive guidelines for paclitaxel administration in the setting of hepatic dysfunction and the potentially unique sensitivity of PTHrP--producing cells to paclitaxel are discussed. PMID- 8638191 TI - Changing clinical perspectives toward gastroesophageal reflux. PMID- 8638192 TI - Laryngeal mask airway: uses in anesthesiology. AB - The laryngeal mask airway (LMA), developed in 1983, is a new device to assist in the management of the pediatric and adult airway. In 1991, the Food and Drug Administration gave its approval for use of the LMA in the United States. The LMA is reusable and appears to provide cost-effective airway management in numerous situations. The LMA is simple to use, atraumatic to insert, and helpful in overcoming an obstructed airway. Its role in management of the difficult airway and the traumatic airway is still evolving. This review will introduce the LMA to the nonanesthesiologist and review for the anesthesiologist the origins of the LMA, its physical structure, the technical aspects of insertion, problems with aspiration, its role in the difficult airway, and experience with the pediatric population. PMID- 8638194 TI - The Dutch health care system: lessons for reform in the United States. AB - The Dutch health care system achieves universal access with high-quality outcomes at a cost below that in the United States. As US policy makers struggle with issues regarding access, quality, and cost, scrutiny of successful systems in other nations may be useful. Review of health care in the Netherlands and contrast with that in the United States offer lessons of potential use in US health care reform. PMID- 8638193 TI - Current medical therapy for inflammatory bowel disease. AB - Traditional medical therapy for inflammatory bowel disease (IBD) includes corticosteroids and sulfasalazine. In recent years, several mesalamine derivatives of sulfasalazine have become available. These allow delivery of increased dosages of active medication with minimal side effects. Newer steroid preparations, all investigational at this point, likely will offer efficacy similar to that of prednisone but with an improved side effect profile. Immunosuppressive agents, including 6-mercaptopurine, azathioprine, and likely also methotrexate, are beneficial in treating refractory IBD, particularly in patients with chronic steroid dependence. Cyclosporine has been shown to be remarkably effective in delaying colectomy for severe ulcerative colitis, but its long-term role remains uncertain. PMID- 8638196 TI - Decreasing length of hospital stay by early excision and grafting of burns. AB - All acutely burned patients admitted to one surgeon's practice during a 1-year period were considered for burn excision and grafting. A total of 222 patients were enrolled; 57 did not have surgery. In all, 130 patients having surgery within 24 hours after admission were compared with 48 patients having excision later than this. Sex, age, burn size, number of operative procedures, and number of deaths were not significantly different statistically. The proportion of acute readmissions was not significantly different. The patients in the early excision group had a significantly shorter hospital stay for the first admission and for total length of stay for acute care, since if the length of stay for the first acute admission was added to the duration of hospitalization at any second acute admission, the early excision group again had a significantly shorter total length of stay. It appears that early burn excision (defined as within 24 hours of admission in this series) results in a reduced length of hospital stay without adverse effects on clinical outcome. PMID- 8638195 TI - Felodipine as an alternative to more expensive calcium antagonists in mild to moderate hypertension. AB - We studied the therapeutic substitution of a less expensive but equally effective antihypertensive agent and assessed patient outcome. The medication of 39 patients with hypertension was changed from once-daily diltiazem hydrochloride (Cardizem CD) or nifedipine (Procardia XL) to felodipine (Plendil). Titration to a final dose was based on home and office blood pressure measurements assessed over subsequent follow-up clinic visits. Self-administered questionnaires measured different aspects of well-being and symptoms before and after the change in medication. Eighty percent of the cohort switched successfully to felodipine. Office systolic and diastolic pressures improved after the medication change (systolic: 150 mm Hg versus 144 mm Hg; diastolic: 92 mm Hg versus 87 mm Hg). No statistically significant differences were found among the 39 symptoms measured. A yearly savings potential for our institution was estimated to be $72,000. PMID- 8638197 TI - Variation in recommendations for breast and cervical cancer screening among primary care physicians in North Carolina, 1991. AB - Physicians play an important role in recommending and doing cancer screening for their patients. In 1991, the North Carolina Department of Environment, Health and Natural Resources and the Centers for Disease Control and Prevention conducted a survey to determine the counseling and referral practices of primary care physicians practicing in North Carolina. Physicians were asked to report the percentage of patients in various age groups that they recommended receive a clinical breast examination, referral for mammography screening, or a Pap test and at what intervals they recommended each study. They were also asked their perceived barriers to cervical cancer screening. Obstetrician-gynecologists recommended annual Pap smear screening more frequently than did physicians in other specialties for patients of all ages. For mammography, variation existed for recommended screening intervals for all specialties by patient age (35 to 39 years, 40 to 49 years, 50 to 64 years, and over age 65). Recommendations for mammography also differed significantly according to physicians' age and sex. The medical community and the public need a consistent and clear message to promote effective screening maneuvers. PMID- 8638198 TI - Radical hysterectomy for stage IB cervical cancer: recurrence interval as a predictor of survival. AB - Two hundred patients with stage IB carcinoma of the cervix were treated with radical hysterectomy. We evaluated the impact of recurrence interval on survival and determined its relationship to other prognostic factors. Thirty-one patients (15.5%) had recurrence, with a median follow-up of 2.8 years (range, 1 to 5 years). Multivariate analysis, using the Cox proportional hazard regression model, showed the impact of recurrence interval on survival and its relationship to other prognostic factors. Patients were 19 times more likely to die during follow-up if recurrence occurred shortly after the operation. However, the risk of death from recurrence decreased exponentially as recurrence interval increased, by a multiple of 0.93 m where m is recurrence interval in months. We conclude that in patients with stage IB carcinoma of the cervix treated initially with radical hysterectomy, the shorter the recurrence period after operation, the greater the likelihood the patient would die during 5-year follow-up. This information may help clinicians determine a patient's prognosis after confirmed recurrence. PMID- 8638200 TI - Interpretive and procedural skills of the internal medicine clerkship: performance and supervision. AB - This descriptive study prospectively examined the performance and supervision of interpretive and procedural skills during an internal medicine clerkship. Students (N = 150) documented having done 7 required and 12 elective skills. Preceptors of required skills were interns (44%), residents (29%), attending physicians (12%), and others (16%). The elective procedures and the percentage of students doing them were as follows: skin tests, 78%; nasogastric tube insertion, 57%; paracentesis, 44%; bone marrow sampling, 35%; lumbar puncture, 34%; thoracentesis, 34%; Papanicolaou smear, 29%; central line placement, 27%; cardioversion, 13%; bladder catheter insertion, 11%; arthrocentesis, 9%; and skin biopsy, 7%. Elective procedures per student ranged from 0 to 9 (mean = 4) and were done less often in the first clerkship group than later in the academic year. Preceptors of electives were interns (46%), residents (39%), and attending physicians (9%). House staff were more likely and faculty less likely to precept electives than required procedures. Students' exposures to these skills are unequal. Their preceptor are generally house staff. To prepare medical students for postgraduate training, technical skills should be specifically addressed in the curriculum. PMID- 8638199 TI - Frankel A paraplegia: a comparison of two spinal instrumentation systems. AB - Seventeen patients with unstable thoracolumbar fracture dislocations and Frankel A (complete) paraplegia had posterior spinal fusion and instrumentation--nine with Cotrel-Dubousset instrumentation and eight with Luque rod instrumentation (LRI). Both systems maintained fracture reduction, and fusion occurred in all cases within a mean of 42 months (range, 30 to 66 months). Operative time and associated blood loss was less in the LRI group. One patient with LRI had fracture of the sublaminar wires that did not lead to loss of reduction or nonunion. Both instrumentation systems provided long-term posterior spinal stabilization in these patients. PMID- 8638201 TI - Persistent cutaneous larva migrans due to Ancylostoma species. AB - Cutaneous larva migrans is considered to be a self-limited parasitic infection of about 2 to 8 weeks' duration, though it has been reported to persist for as long as 55 weeks. In this case, a healthy 47-year-old white man had multiple serpiginous lesions typical of cutaneous larva migrans for 18 months. A biopsy taken 2 months before presentation showed a parasite consistent with Ancylostoma species deep in a hair follicle. The patient initially responded to topical thiabendazole, but relapse occurred when therapy was discontinued. Oral thiabendazole cured the problem after 22 months of infestation. Cutaneous larva migrans may sometimes be long-standing, here almost 2 years, even in a healthy patient. Organisms may reside deep in the hair follicles. Topical thiabendazole may not penetrate to this depth, necessitating oral thiabendazole therapy. PMID- 8638202 TI - Agranulocytosis and near fatal sepsis due to 'Mexican aspirin' (dipyrone). AB - The use of "unconventional" or alternative medicine has been reported in up to one third of American households, yet only 28% report the use of such agents to their physician. We present here a case of near fatal sepsis and agranulocytosis. The agranulocytosis is attributed to the use of dipyrone (Dolo-Tiaminol), which the patient obtained in Mexico as a stronger form of generic "aspirin." The pyrazolone class of analgesics, of which dipyrone is a derivative, was introduced in the late 19th century and had a meteoric rise in use until an associated rise in fatal agranulocytosis was discovered. These agents were banned by the Food and Drug Administration (FDA) in 1977. Dipyrone is thought to induce agranulocytosis by inducing an antibody response. With the widespread use of alternative treatments, it is important for physicians to inquire as to the use of unprescribed drugs. Several resources are available to aid with the identification of foreign drugs. PMID- 8638204 TI - Insulin-resistant diabetes mellitus in a black woman with ataxia-telangiectasia. AB - A 24-year-old woman with ataxia-telangiectasia had traumatic arthritis, elevated serum transaminase values, polyuria, polydipsia, and a serum glucose level of 575 mg/dL. A relatively high daily dose of insulin (2.8 U/kg) was required to achieve near normoglycemia. The fasting insulin concentration was elevated. During an insulin-modified frequently sampled intravenous glucose tolerance test, the first phase of insulin release in response to the administration of glucose was blunted. The insulin sensitivity was similar to that found in individuals with non-insulin-dependent diabetes mellitus. Insulin receptor antibodies were not detected in the serum. We conclude that insulin resistance and islet beta-cell dysfunction are characteristics of diabetes mellitus in ataxia-telangiectasia. Contrary to a previous report, our findings do not support a cause-and-effect relationship between insulin receptor antibodies and insulin resistance in this disorder. PMID- 8638203 TI - Mosquito-transmitted malaria acquired in Texas. AB - Malaria was endemic in the United States before World War II. However, locally acquired malaria was thought to have been eradicated. Since the mid-1980s, cases of locally acquired malaria have been described. We report the case of a 62-year old man who came to the Houston Veterans Affairs Medical Center with fever, malaise, and weakness and was found to have Plasmodium vivax infection on peripheral blood smear. He had not left the country for 37 years and had no previous history of malaria. On specific questioning, he mentioned heavy exposure to mosquitoes. Thus, malaria was presumably transmitted locally by mosquitoes. Subsequently, two other cases of apparently locally acquired, mosquito transmitted malaria were identified in Houston. Symptoms, signs, and general laboratory test results do not typically suggest a specific diagnosis. Therefore, malaria should be considered in all patients with febrile illnesses, even those without a history of travel. PMID- 8638205 TI - Extranodal sinus histiocytosis with massive lymphadenopathy: isolated central nervous system involvement mimicking meningioma. AB - We present the fifth reported case of extranodal sinus histiocytosis with massive lymphadenopathy (SHML) isolated in the central nervous system. This case emphasizes the importance of recognizing the clinical and radiographic presentation of SHML and shows that immunohistochemical evaluation is required for definitive diagnosis. PMID- 8638206 TI - Persistent paralysis after prolonged use of atracurium in the absence of corticosteroids. AB - Neuromuscular blocking agents (NMBAs) are often used for patients requiring prolonged mechanical ventilation. Reports of persistent paralysis after the discontinuance of these drugs have most often involved aminosteroid-based NMBAs such as vecuronium bromide, especially when used in conjunction with corticosteroids. Atracurium besylate, a short-acting benzylisoquinolinium NMBA that is eliminated independently of renal or hepatic function, has also been associated with persistent paralysis, but only when used with corticosteroids. We report a case of atracurium-related paralysis persisting for approximately 50 hours in a patient who was not treated with corticosteroids. PMID- 8638207 TI - Tripe palms: a cutaneous paraneoplastic syndrome. AB - Tripe palms is a cutaneous paraneoplastic syndrome. We report a case of tripe palms in a 71-year-old man with non-small cell lung cancer. Approximately 90% of patients with tripe palms have an associated cancer, most commonly involving the lung or the stomach. Any patient with tripe palms must have a complete cancer workup, especially for lung and stomach cancer. PMID- 8638208 TI - Escherichia coli lobar pneumonia: fatal infection in a patient with mental retardation. AB - Lobar pneumonia due to Escherichia coli is rare. Most lobar pneumonias are caused by either Streptococcus pneumoniae or Klebsiella pneumoniae, and most E coli pneumonias are bronchopneumonias. We report an acute fulminant course of E coli lobar pneumonia in a 37-year-old patient who was profoundly retarded, institutionalized, and nonimmunosuppressed and who died within 2 days of developing initial symptoms. Antemortem blood and postmortem blood and lung specimens isolated pure cultures of E coli. The source of infection in E coli lobar pneumonia is not clear in this patient or in the few cases that have been reported. We postulate that nasopharyngeal colonization of E coli in those who are institutionalized with mental retardation may predispose these patients to E coli pneumonia. Our case illustrates features of pneumonias that are unique in the institutionalized, mentally retarded patient population (ie, the relatively high prevalence of nasopharyngeal colonization of E coli, a higher incidence of E coli pneumonia than in other institutionalized populations, the often fulminant course of the disease), as well as the need for early, aggressive treatment including antibiotics effective against gram-negative bacteria. PMID- 8638209 TI - Diagnosis and management of laryngeal trauma in sports. AB - The management of sports-related laryngeal trauma presents a diagnostic and therapeutic challenge. The application of basic treatment principles such as airway monitoring, thorough physical examination, and fiberoptic laryngeal examination will direct the management. Further radiologic examination or surgical intervention may be required to adequately assess and manage the injury. We review the diagnosis, treatment, and management in a case of laryngeal fracture that occurred during a National Hockey League game. PMID- 8638210 TI - Cranial migration of a lumboperitoneal shunt catheter. AB - We describe a patient with pseudotumor cerebri for which a lumboperitoneal (LP) shunt was placed. After a pseudomeningocele was noted at the lumbar incision site, an LP shunt revision was done, at which time migration of the catheter into the thecal sac was noted. Three months later, radiologic studies revealed cranial migration of the LP shunt into the posterior fossa. We believe no similar complication has been reported. PMID- 8638212 TI - Heri, hodie, cras... yesterday, today, and tomorrow. PMID- 8638211 TI - Intravenous pamidronate for hypercalcemia of primary hyperparathyroidism. AB - Intravenous pamidronate disodium has been used successfully in the treatment of malignancy-associated hypercalcemia and Paget's disease of bone. Although the definitive treatment of primary hyperparathyroidism (PHPT) is surgical, intravenous pamidronate has been used to treat hypercalcemia of PHPT when surgery was contraindicated. We report two cases of PHPT in which intravenous pamidronate effectively reduced hypercalcemia and significantly improved the clinical symptoms. The results of our study and the experience in the literature suggest that pamidronate is a safe and effective method of reducing serum calcium in patients with PHPT when definitive surgery is contraindicated or must be postponed. PMID- 8638213 TI - Malpractice claims. PMID- 8638214 TI - Interferon alfa and autoimmunity. PMID- 8638215 TI - One step closer to gene therapy for Parkinson's disease. PMID- 8638216 TI - Polyposis coli, craniofacial exostosis and astrocytoma: the concomitant occurrence of the Gardner's and Turcot syndromes. AB - BACKGROUND: Up to 60% of the patients with known adenomatous polyposis coli may present hyperostosis of the skull and facial bones, and/or a susceptibility to fibromas. This is known as the Gardner's syndrome, and is considered as an allelic variant of familial adenomatous polyposis (FAP). Also, although very rare, an adenomatous polyposis coli may occur with malignant tumors of the central nervous system, known as Turcot syndrome. If both syndromes are different phenotypic presentation of FAP, this would explain a simultaneous occurrence. METHOD: We report the history of a patient who showed clinical signs of the simultaneous occurrence of both Gardner's and Turcot syndromes. The syndromes are compared, and in view of the literature, a genetic explanation for the concomitant occurrence is discussed. RESULTS: Evidence obtained from the literature to consider Turcot syndrome as a phenotype of FAB is as follows: (1) The occurrence of Gardner's and Turcot syndromes in one family, but in different members; (2) The presence of congenital hypertrophic retinal pigmented epithelium (CHRPE), which correlates with the expression of polyps in FAP patients, in both syndromes; (3) Linkage of the Turcot phenotype to the adenomatous polyposis coli locus by genetic markers. Evidence obtained from this case report indicates that there is a manifestation of both syndromes in one patient together with a positive family history for FAP. CONCLUSION: This concomitant occurrence of both Gardner's and Turcot syndromes in one patient clinically supports genetic and ophthalmic investigation to consider Turcot syndrome (like Gardner's syndrome) as a phenotypic variant of FAP. Patients with FAP should be examined for the presence of Gardner's syndrome. In case a Gardner's syndrome is suspected, a computed tomography scan of the brain is recommended because of the possible existence of a simultaneous Turcot syndrome. PMID- 8638217 TI - Triple primary malignant neoplasms including a malignant brain tumor: report of two cases and review of the literature. AB - BACKGROUND: Two rare cases of triple primary malignant neoplasms (PMN), including malignant brain tumors, which were glioblastoma multiformes, are described. METHODS: The clinical characteristics and underlying genetic alterations in triple or more PMN, including malignant brain tumors are discussed with intensive review of the literature. RESULTS: The first patient, a 77-year-old male, suffered metachronously from tubular adenocarcinoma of the stomach, transitional cell carcinoma of the bladder, and glioblastoma in the brain. This glioblastoma had loss of heterozygosity in exons 7-8 in p53 gene. The second patient, a 68 year-old male, developed papillary adenocarcinoma of the lung, adenocarcinoma of the rectum, and glioblastoma in the brain during a period of 7 years. In 42 such cases described in the literature, age distribution demonstrated two characteristic peaks, one in the third decade and the other over 50 years of age. The younger group consisted mainly of Turcot's syndrome, and of a case of Li Fraumeni familial cancer syndrome. On the other hand, neither of these hereditary cancer syndromes were contained in the elder group. Regarding the site of PMN, colorectal cancers were associated most frequently with malignant brain tumors, followed by stomach cancers, and thyroid cancers. Malignant brain tumors, mostly glioblastoma multiforme, tend to occur as the last tumor of triple or more PMN. CONCLUSIONS: These results suggest that genetic background might play an important role in tumorigenesis of PMN in the younger group, whereas epigenetic factors would be more important in the older group. Characteristic organ association and factors influencing carcinogenesis, such as aging, environmental carcinogens, and underlying genetic alterations in these tumors are further discussed. PMID- 8638218 TI - Expression of BCL-2 gene product in embryonal tumors of the central nervous system. AB - BACKGROUND: The expression of bcl-2 is associated with suppression of programmed cell death and prolonged cell survival. Recently, immunoreactivity to the bcl-2 gene product has been reported not only in a variety of embryonal and adult nonhematopoietic tissues, but also in neuroblastoma. However, the study of bcl-2 expression has not been performed in brain tumors. METHODS: In this study, we examined the incidence and significance of bcl-2 expression in 25 cases of embryonal tumors of the central nervous system, including medulloblastoma, neuroblastoma, ependymoblastoma, and PNET (primitive neuroectodermal tumor), which has the possibility of neuronal differentiation. RESULTS: The results demonstrated that 13 tumors (52%) positive for bcl-2 belonged to early differentiated and neuronal types, and 7 negative tumors (28.0%) mostly belonged to undifferentiated type. CONCLUSIONS: These results have led us to speculate on the possibility that tumor cells begin expressing bcl-2 along with their neuronal differentiation from primitive cells. PMID- 8638219 TI - Moyamoya disease associated with persistent primitive trigeminal artery variant in identical twins. AB - Identical twin cases of moyamoya disease associated with persistent primitive trigeminal artery variant are presented. Both of the children suffered from the cerebrovascular occlusive disease called "moyamoya disease," but there existed a remarkable time lag in the manifestation of their first clinical symptoms. Coexistence of persistent primitive trigeminal artery variant and high occurrence of moyamoya disease in identical twins suggest some congenital factors in the development of this disease. However, the time lag of the first clinical manifestation between the twins suggests certain acquired factors may also play a role in the manifestation of this disease. PMID- 8638220 TI - Simultaneous occurrence of aneurysm and multiple meningioma in Klippel-Trenaunay patients: case report. AB - Klippel-Trenaunay syndrome is a rare neurocutaneous disorder in which skeletal hypertrophy, vascular nevi, and vessel anomalies coexist. Involvement of the cranial bone is rare, and intracranial anomalies associated with features of this syndrome are exceptional. We report a case of an adult Klippel-Trenaunay woman with a huge cranial hypertrophy harboring of the same time multiple meningiomas and a fusiform intracranial aneurysm. Although meningioma and intracranial aneurysms have been described in Klippel-Trenaunay patients, such an association has not been reported previously in patients with this syndrome. PMID- 8638221 TI - Large cerebral arteriovenous malformations: experience with 27 cases. AB - BACKGROUND: The management of large cerebral arteriovenous malformations is difficult. Surgical excision is often attempted after embolization. Interventional neuro-radiology is available in only a few centers. If large AVMs can be safely excised without pre-operative embolizaton, then these lesions can be treated at many neurosurgical centers. METHODS: Between January 1986 and June 1992, a total of 210 patients with cerebral AVMs were diagnosed by angiographic studies. Twenty-seven of them had large AVMs wider than 5 cm in the longest diameter. The case records of these patients were retrieved and studied. RESULTS: Two patients were treated with Bragg peak proton beam therapy. Twelve patients were treated conservatively with no improvement. Thirteen patients underwent surgery and total AVM excision was achieved in 11. None of the patients who underwent surgery had any pre- or intra-operative embolization. All the patients had no neurological deterioration post-operatively. CONCLUSIONS: Large cerebral AVMs can be treated by surgical excision alone with acceptable results. PMID- 8638222 TI - Dural arteriovenous fistulas of superior sagittal sinus: case report and review of literature. AB - A case report and review of the literature of 16 dural arteriovenous fistulas (DAVFs) involving the superior sagittal sinus region are presented. In our case, magnetic resonance angiography detected the DAVF with multiple arterial feeding vessels from both external carotid arteries. The patient was successfully treated endovascularly, with complete occlusion of arterial feeders and a total resolution of symptoms. PMID- 8638224 TI - Intracranial adenoid cystic carcinoma: case report and review of the literature. AB - BACKGROUND: Adenoid cystic carcinoma (cylindroma) is a relatively common head and neck tumor that is slow growing, but locally aggressive and thus prone to recurrence. It is of particular interest to neurosurgeons and neurologists because of its tendency to locally infiltrate neural structures and to spread perineurally. Intracranial involvement has been regarded as rare. METHODS: A case report of a patient with adenoid cystic carcinoma involving the Gasserian ganglion region is presented. The world literature on intracranial involvement of adenoid cystic carcinoma is reviewed. A discussion of the characteristics of this lesion is provided. RESULTS: Our literature review revealed 119 previously reported cases of adenoid cystic carcinoma with intracranial involvement. Our case represents only the tenth reported intracranial case with an unknown primary site. CONCLUSIONS: Although intracranial adenoid cystic carcinoma is regarded as rare, we have accumulated over 100 such reports. A wide variety of primary sites and intracranial sites have been described. PMID- 8638223 TI - Ventriculolumbar perfusion chemotherapy with methotrexate and cytosine arabinoside for meningeal carcinomatosis: a pilot study in 13 patients. AB - Thirteen patients with meningeal carcinomatosis were treated by ventriculolumbar perfusion using methotrexate (MTX) and cytosine arabinoside (Ara-C). MTX (10-30 mg) and Ara-C (40 mg) were infused at 8- to 12-hour intervals on six or nine occasions via an Ommaya reservoir placed in the lateral ventricle. Nine of thirteen patients had evaluable response (69% response rate with a mean survival of 8.8 months among responders) and ventriculolumbar perfusion therapy was effective in improving cerebral, cranial nerve, and spinal root signs and symptoms, especially sensorimotor disturbance in the lower limbs. Three of the six bedridden patients became ambulatory without assistance and two of the four patients who were walking with assistance became ambulatory without assistance. Urinary incontinence also markedly improved, except in one nonresponder. Lumbar cerebrospinal fluid parameters (cytological findings and tumor markers) also improved in association with the clinical improvement. Our pilot results were encouraging, especially the improvement of sensorimotor function in the lower limbs. However, the toxicity was unacceptable when compared with that of standard intrathecal chemotherapy. Thus, this therapy needs to be investigated further to establish the most appropriate drug doses and perfusate volume to reduce toxicity as well as determine its true efficacy in the treatment of meningeal carcinomatosis. PMID- 8638225 TI - Endoscopic treatment of septated chronic subdural hematoma. AB - BACKGROUND: Chronic subdural hematomas are well delineated collections of fluid (blood) between the dura mater and the arachnoid space. Two types of encapsulated chronic subdural hematoma can be distinguished: the nonseptated and the septated form. The nonseptated form can be treated easily using the burrhole-drainage method, whereas treatment of septated chronic subdural hematoma remains a therapeutical problem. The main problem is the division of the hematoma by neomembranes into compartments, which hinder the efflux of the hematoma fluid through one or two burrholes. METHODS: Since 1991 we have operated on 14 patients with septated subdural hematoma using flexible steerable endoscopes through a burrhole approach. The flexible endoscopes are fixed and guided with the help of the Marburg Neuroendoscopy Fixation and Guiding System. For resection of neomembranes small microscissors or microforceps were used. This technique avoids blunt rupture of the membranes which may cause bleeding. A closed drainage system is applied temporarily to guarantee the efflux of the remaining hematoma. RESULTS: From January 1991 to May 1994, 14 patients with the septated variant of chronic subdural hematoma were operated on using the endoscopic technique. After the neuroendoscopic intervention 12 patients had a sufficient or complete hematoma evacuation. One patient had to be operated on a second time, and there was one postoperative subdural infection. Long term follow up of all patients shows no recurrence of the subdural hematoma. CONCLUSION: Treatment of membraneous septated CSH using an endoscopic operative technique combined with the application of a closed drainage system is a minimally invasive method and a therapeutic alternative to the craniostomy-membranectomy technique. PMID- 8638226 TI - The fiberoptic intraparenchymal cerebral pressure monitor in 244 patients. AB - BACKGROUND: The fiberoptic intraparenchymal pressure monitor has been shown to provide reliable data in patients monitored for a short period of time. An analysis of a large population, including patients monitored for a prolonged period such as weeks, has never been performed. METHODS: We conducted a retrospective study in which we evaluated the fiberoptic intraparenchymal cerebral pressure monitor for complications and accuracy. RESULTS: The fiberoptic intraparenchymal cerebral pressure (ICP) monitor was used in 244 patients since 1988. Pathology included trauma in 180, intracerebral hemorrhage (ICH) in 19, aneurysmal subarachnoid hemorrhage in 16, arteriovenous malformations in 10, cerebral edema in 12, and tumor in 7. The mean length of monitoring was 7 days with 49 patients (20%) monitored 1-2 days, 105 (43%) monitored 3-6 days, 48 monitored for 7-10 days (20%), and 42 (17%) monitored from 10-24 days. Seventy four patients also had ventriculostomies and there was strong correlation with the ICP monitor (p < 0.001). Complications from insertion were 2 patients (0.8%) with ICH, both with hepatic dysfunction. In the group monitored greater than 6 days, 34 patients (38%) required at least one catheter change due to upward drift of the ICP. Fiberoptic breakage requiring replacement was documented in 41 patients (16%). An insertion-site infection occurred in one case monitored for 23 days. Two infected bone flaps occurred that had a monitor placed in the flap. There were no cases of bacterial meningitis or cerebral abscess. CONCLUSIONS: The monitor is easy, safe, and reliable to use with a very low rate of infection. Coagulopathic patients may best be monitored by an alternative method. PMID- 8638227 TI - Surgical treatment of a sacral nerve root cyst with intermittent claudication in an 85-year-old patient: case report. AB - The following presents a case of an 85-year-old woman with intermittent claudication of a sacral nerve root cyst. The cyst wall was incised and oversewn while preserving the surrounding nerve fibers by microscopic procedures. All symptoms were eliminated following the operation. The syndrome of intermittent claudication may possibly arise while walking from the transient influx of cerebrospinal fluid into a cyst in a relatively narrow sacral canal. The present case may indicate that a sacral nerve root cyst is curable even in aged patients through thorough evaluation and management. PMID- 8638228 TI - Dermoid cyst within an upper thoracic meningocele. AB - A case of dermoid cyst within an upper thoracic meningocele is reported. The incidental feature of this combination is extremely rare. As upper thoracic meningocele is often associated with other spinal and intracranial abnormalities, close observation and radiologic evaluation of the craniospinal axis are necessary to obtain an exact diagnosis and to achieve an appropriate treatment. PMID- 8638229 TI - Cerebrospinal fluid pseudocyst after anterior stabilization for cervical spine injury treated by ventricular drainage: case report. AB - The author describes postsurgical formation of a cerebrospinal fluid (CSF) pseudocyst after anterior decompression and stabilization for cervical spine injury. The CSF pseudocyst was caused by extensive lacerations to the spinal dura mater. A further contributory factor was a circulation blockage of CSF in the spinal canal, brought about by edema of the contused spinal cord. Ventricular drainage caused rapid collapse of the pseudocyst and its subsequent healing. PMID- 8638230 TI - Are steroids useful in the treatment of head-injured patients? PMID- 8638231 TI - Kirwan "non-stick" bipolar forceps. PMID- 8638233 TI - Perspectives on a neurosurgical practice. PMID- 8638232 TI - Ethics, AIDS, and the neurosurgeon. PMID- 8638234 TI - Unintended durotomy. PMID- 8638235 TI - Extruded cervical disc. PMID- 8638237 TI - Spinal giant intradural perimedullary arteriovenous fistula: clinical and neuroradiological study in one case with review of literature. AB - BACKGROUND: Giant intradural perimedullary arteriovenous fistula with massive spinal cord compression is rare. The therapeutic difficulties include whether endovascular embolization or direct surgical excision should be selected. We present a patient with the largest giant spinal intradural perimedullary arteriovenous fistula shown by magnetic resonance imaging so far reported, who was successfully treated by a combination of endovascular embolization and direct surgery. CASE DESCRIPTION: A 16-year-old girl presented with a giant intradural arteriovenous fistula (perimedullary Type II) at the C4-5 level, manifesting as progressive cervical myeloradiculopathy. The single-hole fistula was supplied by the anterior spinal artery and an ascending artery arising from both the costocervical and highest intercostal arteries with a rapid transit time, and drained superiorly to the foramen magnum, and inferiorly to the thoracic spinal canal, through a huge venous lake at the site of the arteriovenous connection. The patient was treated by transarterial embolization with platinum coils and silk, followed by surgical excision with excellent results at 12 months' follow up. CONCLUSIONS: We recommend that such a huge perimedullary arteriovenous fistula with a rapid transit time, and severe cord and root compression, should be treated with embolization followed by surgical excision. PMID- 8638236 TI - Surgical treatment of thoracic disc herniation: a reappraisal of Larson's lateral extracavitary approach. AB - Twenty patients with thoracic disc herniations underwent removal of the transverse process, articular facet, pedicle, and rib--a procedure described by Larson as the lateral extracavitary approach. Eleven patients presented with myelopathy: five mild, nine moderate, and four severe. Fifteen patients showed significant neurologic improvement after the operation and five patients none. Postoperative follow-up ranged from 1 to 8 years. The pros and cons of each of the surgical approaches to this type of lesion are considered with reference to the published data. PMID- 8638239 TI - Intramedullary epidermoid cyst in the brain stem: case report. AB - BACKGROUND: The incidence of intracranial epidermoid cysts is between 0.2% to 1.8% of all brain tumors. They usually occur in the cerebellopontine angle or parasellar region. Intramedullary epidermoid cyst is rare and only nine cases have been previously reported. METHODS: A 69-year-old woman had progressive left hemiparesis for 2 years. Computed tomography scanning and magnetic resonance imaging (MRI) revealed that there was a cystic tumor in the pons that was compressing the brain stem. A part of the tumor cyst wall and the content of the cyst were surgically removed by right subtemporal transtentorial approach. The histologic diagnosis was epidermoid cyst. After the surgery, the left hemiparesis was improved and there was no sign of the tumor growth by MRI. CONCLUSION: The outcome of the previous cases with radical tumor resection was poor compared with the present case in which only a part of the cyst wall and the content were removed. Therefore, radical removal of the whole tumor may not be necessary for intramedullary epidermoid cyst of the brain stem. PMID- 8638238 TI - Cine-mode magnetic resonance imaging of a thoracic intradural arachnoid cyst: case report. AB - We report the appearance of a thoracic intradural arachnoid cyst on cine-mode magnetic resonance imaging (MRI). Based on the operative findings, cine-mode MRI was more sensitive for identifying the intradural location of arachnoid cysts than was conventional MRI. The value of cine-mode MRI in diagnosing this rare entity is discussed. PMID- 8638240 TI - Indirect revascularization for moyamoya disease: is there a beneficial effect for adult patients? AB - BACKGROUND: It is generally accepted that excellent development of collateral circulation can be achieved through indirect nonanastomotic bypass procedures for pediatric patients with moyamoya disease. However, there are no definitive conclusions about the effect of indirect revascularization for adult patients. To clarify the value of indirect bypass surgery for adult moyamoya disease, we have analyzed their follow-up angiographic results in comparison with those of the pediatric patients. METHODS: Between 1989 and 1993, 23 patients underwent combined direct and indirect bypass surgery. They consisted of 16 adults (mean age, 35; range, 20-59) and seven children (mean age, 10; range, 3-16). The main symptoms were those due to cerebral ischemia in all but 1 of 23 patients. Preoperative cerebral blood flow studies showed all patients to have decreased vascular reserve (misery perfusion). Postoperative follow-up angiography was done in all patients at a median of 6 months after the surgery. RESULTS: All pediatric patients showed good or moderate development of collaterals through the indirect bypass. Among the adult group, seven patients aged 20 to 29 had angiographic results similar to those of the pediatric group . On the other hand, nine patients older than 30 had results contrary to those of pediatric patients: (1) the degree of indirect revascularization declined to moderate or poor grades (especially in patients older than 40) and (2) the degree of direct bypass filling improved to high or medium grades. CONCLUSIONS: The results suggest that advancing age apparently affects the development of collateral formation through the indirect bypass. Consequently, direct bypass is thought to be the main treatment option for patients older than 40. PMID- 8638241 TI - Omental transplantation using a superficial temporal artery previously used for encephaloduroarteriosynangiosis. AB - BACKGROUND: Childhood moyamoya disease is a chronically progressive cerebrovascular occlusive disease affecting the territories of the anterior, middle, and posterior cerebral arteries. Surgery used in treatment of moyamoya disease to vascularize the brain include direct and indirect anastomoses. METHODS: Intracranial omental transplantation (OMT) was performed using a branch of the superficial temporal artery (STA) that had been used previously for encephaloduroarteriosynangiosis (EDAS) in five children with moyamoya disease. All five children continued to have paraparetic transient ischemic attacks (TIAs), urinary incontinence, and/or progressive mental retardation even after EDAS and/or STA-middle cerebral artery (MCA) anastomosis and encephalomyosynangiosis (EMS) to the territory of the MCA. Previously performed EDAS gave insufficient collaterals to the territory of the MCA in four of the five patients and sufficient collaterals to the territory of the MCA in the remaining patient. OMT was performed after stripping of a branch of the STA used in EDAS that gave insufficient collaterals to the brain in the former four patients; and the latter patient was performed using a parietal branch of the STA distal to the distal burr hole drilled in the previous EDAS. RESULTS: OMT resulted in marked improvement in neurologic conditions in all five patients. Four of the five patients suffered no TIAs postoperatively, while the remaining patient still had TIAs but at a markedly decreased frequency. CONCLUSIONS: In summary, OMT using a branch of the STA used in previously performed EDAS is required for patients with moyamoya disease who continue to manifest paraparesis, urinary incontinence, and/or progressive mental retardation even after multiple EDAS. PMID- 8638243 TI - Traumatic laceration of the intracranial vertebral artery causing fatal subarachnoid hemorrhage: case report. AB - A 36-year-old man who had been drinking alcohol had a fatal subarachnoid hemorrhage immediately after suffering a moderate craniofacial injury. Autopsy revealed a 3-mm longitudinal laceration of the left intracranial vertebral artery proximal to the posterior inferior cerebellar artery. There was no finding of arterial dissection. We discuss the mechanisms of the traumatic laceration of the vertebral artery in relation to traumatic dissection of the vertebral artery. PMID- 8638242 TI - Very poor prognosis in cases with extravasation of the contrast medium during angiography. AB - BACKGROUND: The rebleeding of a ruptured intracranial aneurysm in the acute stage has been thought to indicate a very poor prognosis. In our experience, the outcome of patients with extravasation of the contrast medium is worse than that of patients with rerupture under circumstances other than angiography. We demonstrated the poorer outcome of the patients with extravasation, compared to that of the patients developing rerupture under circumstances other than angiography, and examined the factors that contributed to the poorer outcome of the patients with extravasation. METHODS: Among the 641 cases of ruptured intracranial aneurysm, we have handled over the past 10 years, 36 (5.6%) patients have rebled before surgery was performed. Thirteen patients rebled during angiography (Group I), and the remaining 23 patients rebled in other circumstances (Group II). We compared the outcome of both groups. RESULTS: The rebleeding occurred within 6 hours of the initial rupture in 29 (80%) patients. The outcome of Group I was significantly poorer than that of Group II (p < 0.05). In Group I, SD was achieved in only 1 patient and the remaining 12 patients died, while in Group II, 8 patients were in GR or MD, 2 were in SD, and the remaining 13 patients died. CONCLUSION: Because rebleeding during angiography most often occurs in the acute stage and because the outcome of patients with extravasation is very poor, we recommend that the performance of angiography be delayed at least between 3 and 6 hours after the initial rupture. PMID- 8638244 TI - The relationship between brain surface elastance and brain reexpansion after evacuation of chronic subdural hematoma. AB - BACKGROUND: Brain stiffness has been thought to be a factor affecting brain re expansion after the evacuation of chronic subdural hematoma. METHODS: As an index of brain stiffness, the pressure for compressing the brain using an ophthalmodynamometer was defined as brain-surface elastance. This elastance in 14 patients was measured and analyzed in relation to brain re-expansion. RESULTS: Patients with an enlarged subdural space on computed tomography 1 month after the operation had higher elastance. There was a correlation between high elastance and high age. CONCLUSIONS: Measuring the elastance after evacuation of the hematoma may help predict the persistence of a subdural space. PMID- 8638245 TI - Cerebral alveolar hydatidosis: case report. AB - A young male patient, who previously was presumed inoperable due to an existing hepatic mass with multiple cerebral metastases, was referred to us from an oncology clinic ith signs of transtentorial herniation. An operation was performed promptly; the right parietal mass, which was totally removed, proved to be an alveolar hydatid cyst. He refused the operation proposed for the hepatic lesion and was discharged. PMID- 8638246 TI - The First Interim meeting of the European Society for Paediatric Neurosurgery. March 19-24, 1995; Eliat, Israel. PMID- 8638247 TI - Octogenarian pearls. PMID- 8638248 TI - Unintended "incidental" durotomy. PMID- 8638249 TI - Approaches to MCA aneurysms. PMID- 8638250 TI - A teratologist to extinction? PMID- 8638251 TI - Clinical teratology counseling and consultation case report. PMID- 8638252 TI - Ethanol-induced teratogenesis: free radical damage as a possible mechanism. AB - To investigate the possibility of a free radical mechanism for ethanol-induced teratogenesis, gestational day 8 mouse embryos were exposed for 6 hr in whole embryo culture to a teratogenic dosage of ethanol alone (500 mg%) or in conjunction with an antioxidant, superoxide dismutase (SOD; 300 U/ml). For subsequent analysis, some embryos were examined at the end of this 6-hr period, while others were removed to control medium and cultured for an additional time period. Ethanol exposure resulted in increased superoxide anion generation and increased lipid peroxidation (as noted 6 hr after initial ethanol exposure) and in excessive cell death (as noted 12 hr after initial exposure) in the embryos. Following a total of 36 hr in culture, a high incidence of malformation, including failure of the anterior neural tube to close in 63% of the ethanol exposed embryos, was noted. The ethanol-induced superoxide anion generation, lipid peroxidation, excessive cell death, and dysmorphogenesis were diminished in embryos co-treated with SOD, suggesting that the teratogenicity of ethanol is mediated, at least in part, by free radical damage. PMID- 8638253 TI - Effects of chloroquine and its enantiomers on the development of rat embryos in vitro. AB - The effects of the antimalarial drug chloroquine (CQ) and its enantiomers [(+)-CQ and (-)-CQ] on 9 1/2-day post-implantation rat conceptuses were studied by the whole-embryo culture technique over a 48 hr period. At a concentration of 500 ng/ml of culture medium (0.97 microM), which falls within serum levels attained during long-term CQ therapy, the drugs produced varying degrees of growth retardation and dysmorphogenesis in the conceptuses. These effects were most severe with exposure to racemic CQ (100%) and also to equimolar concentrations of the two enantiomers (90%). Dysmorphogenesis was least with (+)-CQ (30%) and intermediate in severity in those exposed to (-)-CQ (32%). In the CQ and combined enantiomer groups, there was significant reduction in yolk sac diameter to 81% and 73%, the crown-rump length to 77% and 71%, the number of somites to 76% and 74%, and the embryonic protein content to 64% and 49%, respectively, of control values. In the (-)-enantiomer group the only parameter significantly affected was the somite number which was reduced to 83% of control. The growth parameters of (+)-CQ-treated embryos did not differ significantly from controls. The commonest morphological abnormalities observed in all treatment groups were those of axial rotation. Unfused and underdeveloped cranial neural tube, microophthalmia and abnormal otic primordium also occurred frequently, especially in the CQ and enantiomeric combination groups. The results suggest that the commercially available form of chloroquine, CQ, is embryotoxic in doses comparable to serum levels reached during long-term therapy with the drug. It also appears that although the individual enantiomers show minimal embryotoxicity at the dosage used, they potentiate each other's effects in the racemic mixture. PMID- 8638254 TI - Pantothenic acid decreases valproic acid-induced neural tube defects in mice (I). AB - The effect of the administration of pantothenic acid (PTA) on valproic acid (VPA) induced teratogenesis was examined in ICR mice. VPA (300, 400, and 500 mg/kg, s.c.) or PTA (3 x 10, 3 x 100, and 3 x 300 mg/kg, i.p.) was injected on day 8.5 of gestation (plug day = day 0.5). Exencephaly was induced dose dependently by single injections of VPA. Three administrations of PTA alone at any dose levels showed neither embryocidal nor teratogenic effects. In combined treatment experiments, PTA (3 x 300 mg/kg) was injected 1 hr before, immediately before, and 1 hr after VPA administration. PTA significantly reduced VPA-induced exencephaly, while none of the other external malformations such as open eyelid or skeletal malformations such as fused, absent, or bifurcated ribs and fused thoracic vertebrae and fused sternebrae were reduced. The results suggest that PTA reduces the incidence of neural tube defect induced by VPA in mice. PMID- 8638255 TI - Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis stage rat embryos: effect of varying duration of deficiency. AB - Zinc deficiency is teratogenic in all species in which it has been examined. Zinc is an essential component of enzymes involved in DNA synthesis and cell proliferation, and may play an as yet undetermined role in apoptosis. To further our understanding of the role of zinc in normal development, we examined cell death and cell cycle parameters in embryos of pregnant rats fed a zinc-deficient diet for 2 to 10 days (0.5 microgram zinc/g diet; zinc-adequate diet was 25 micrograms zinc/g). To elucidate sensitive periods of development and susceptible cell populations, dams were fed the zinc-deficient diet from gestation day 1, 3, 7, or 9 and killed on day 11. Embryos were examined for morphology and developmental stage. From each litter, 2-3 embryos were stained with Nile blue sulfate (NBS) to visualize cell death, 3 embryos were frozen for flow cytometric cell cycle analysis and cell counts, and selected embryos were preserved for histological examination. Dams fed the zinc-deficient diet for more than 3 days reduced their food intake through gestation day 8 but increased food intake on day 9. Maternal plasma zinc dropped to 10-25% of control levels in the zinc deficient groups. Zinc deficiency from gestation day 1 or 3 resulted in two categories of affected litters on day 11. One category had embryos which were morphologically normal but displayed extensive NBS staining in the visceral arches, neural tube, and somites. The second category had developmentally retarded or maldeveloped embryos which showed little NBS staining. Zinc deficiency from gestation day 7 produced cell death in the posterior dorsal midline in the area of premigratory neural crest cells, which was confirmed by histological examination. Zinc deficiency from gestation day 9 did not affect morphology or NBS staining. Percentages of cells in the G0/G1, S, and G2M phases of the cell cycle on gestation day 11, determined by flow cytometry, were similar to controls in all groups. This study shows that as few as 4 days of maternal zinc deficiency can produce excess embryonal cell death, and that neural crest cells may be particularly sensitive. PMID- 8638256 TI - Estimating intracellular pH in developing rodent embryos using a computer imaging technique: changes in embryonic pH and proliferation rates following maternal treatment with acetazolamide. AB - Using the transplacental distribution of the weak acid 5,5-dimethyloxazolidine 2,4-dione (DMO), a computer assisted imaging technique has been developed to permit the estimation of intracellular pH (pHi) in very specific areas of the developing rodent embryo. The study reported here demonstrates the heterogeneity of radiolabeled DMO distribution in the developing mouse forelimb. The pattern of pHi distribution shifts from one of high pHi values in the proximal core of the mesoderm on day 10 of gestation to one of higher pHi values in the mesoderm just underlying the ectoderm on day 11. Studies [Scott et al. (1990) Toxicol. Appl. Pharmacol. 103:238-254] in which DMO concentration was monitored following treatment with acetazolamide or acetazolamide plus amiloride were done in whole embryo homogenates or pooled limb samples which allow for the calculation of an average pHi but may not reflect the pHi in very specific locations of the limb. Two hours after acetazolamide administration, the pHi pattern was not significantly changed from control. Intracellular pH was raised above control levels but was not significant statistically except in the peripheral mesoderm in the ventral third of the forelimb. Fifteen hours after acetazolamide treatment, there was a significant decrease in pHi values with no change in pattern. However, treatment with acetazolamide plus amiloride for 15 hr produced a marked reduction of pHi values throughout the forelimb bud. Changes in bromodeoxyuridine labeling index (an indication of proliferative activity) following treatment with acetazolamide or acetazolamide plus amiloride are reported. The combination treatment reduced the labeling index by approximately 15% below that of control embryos in the limb region where absence of digit(s) will occur. However, we found no overall correlation of proliferative rate and pHi of limb bud mesoderm in treated embryos. Consequently, we were unable to causally associate reduced pHi with decreased proliferative rate. PMID- 8638258 TI - Teratogen update: congenital toxoplasmosis. PMID- 8638257 TI - Comparative disposition, receptor affinity, and teratogenic activity of sulfon arotinoids. AB - To investigate the relationship between sulfon arotinoid biotransformation and teratogenic activity, the potency of the ethyl (Ro 15-1570) and methyl (Ro 14 9706) arotinoid sulfones and their in vivo disposition in pregnant hamsters were studied. Administration of Ro 15-1570 was teratogenic, but Ro 14-9706 showed no such activity. Total absorbed doses of the ethyl and methyl sulfones (measured as maternal plasma AUC) were very similar. Total delivered dose of Ro 14-9706 to liver and lung was 120-160% that of Ro 15-157, and Ro 14-9706 was transferred in greater amounts to the embryo as well. Placenta AUC for parent sulfon arotinoids was 160-250% that in the embryo. Plasma analyses by HPLC suggested that the ethyl sulfone was oxidized and appeared in maternal plasma as the corresponding sulfinic (Ro 14-9572) and sulfonic (Ro 14-3899) acids, amounting to 10% and 16%, respectively, of the mean maternal ethyl sulfone Cmax value. The concentrations of sulfinic and sulfonic metabolites were always less than the analytical limit of detection in placenta and embryo after maternal ethyl sulfone intubation. Neither the sulfinic nor the sulfonic acid were ever detected in maternal circulation, placenta, or embryo after methyl sulfone intubation. Comparisons of their binding affinities found that neither the ethyl nor the methyl arotinoid sulfone could act as a ligand for cellular retinoic acid-binding protein (CRABP), nor could these compounds bind retinoid nuclear receptors (RAR). Transcriptional activation of RARs was weak and similar for both compounds. The sulfinic and sulfonic acid arotinoids bind and transactivate RARs, and bind CRABP with efficiencies similar to all-trans-retinoic acid. Furthermore, they are active in cultured limb bud chondrocytes. The results suggest that the methyl sulfone (in accord with its lack of activity in cultured limb bud chondrocytes) is of no toxicologic significance in hamster embryo--even after relatively high delivered dose. Teratogenicity of the ethyl sulfone (which shows marked inhibition of chondrogenesis in cultured limb bud) does not appear to depend on measurable concentrations of these sulfinic/sulfonic acid metabolites in the hamster embryo. PMID- 8638260 TI - [Day surgery]. PMID- 8638259 TI - [Physicians from all countries--unite! Growth and return of severe infections]. PMID- 8638261 TI - [Surgical treatment of colorectal cancer. Are we at a crossroad?]. PMID- 8638262 TI - [Chemotherapy in colorectal cancer. Recommendations of the Norwegian Gastrointestinal Cancer Group]. AB - The scientific documentation for fluorouracil-based chemotherapy in patients with colorectal cancer has increased during recent years, both for the adjuvant primary situation and in recurrent and metastatic disease. In the case of operable colorectal cancer (stage Dukes B and C), the patients should be included in the ongoing randomized trial on use of fluorouracil and levamisol. In advanced disease, fluorouracil combined with calcium folinate may have palliative effects in terms of relief of symptoms, improved quality of life, delay of onset of symptoms and probably also prolonged survival in 40-50% of the patients, without serious toxicity. In many cases, however, the patient should not be given chemotherapy. PMID- 8638263 TI - [Neurologic paraneoplastic syndromes and anti-neuronal antibodies]. AB - A neurologic paraneoplastic syndrome may be the first sign of an occult and treatable cancer. Some syndromes are associated with autoantibodies against neuronal antigens. Patients with cerebellar degeneration and ovarian or breast cancer have antibodies against 34 and 62 kilodalton (kDa) proteins in Purkinje cell cytoplasm: anti-Yo antibodies. Patients with encephalomyelitis or sensory neuronopathy and small cell lung cancer have antibodies against 35-40 kDa neuronal nuclear proteins: anti-Hu antibodies. Patients with opsoclonus-myoclonus and breast cancer have antibodies against 55 and 80 kDa neuronal nuclear proteins: anti-Ri antibodies. Patients with Lambert-Eaton myasthenic syndrome and small cell lung cancer have antibodies against voltage-gated calcium channels (anti-VGCC) in motor nerve terminals. The presence of anti-neuronal antibodies strongly indicates that a neurological syndrome is paraneoplastic, and often identify the site of an occult neoplasm. However, the absence of detectable antibodies does not rule out the presence of an underlying tumour. PMID- 8638264 TI - [Laryngomalacia--endoscopic microsurgical treatment]. AB - Laryngomalacia is caused by inspiratory collapse of the supraglottic structures in babies. This predominant cause of stridor in the first years of life has a graded occurrence from an insignificant sound phenomenon to insufficient alveolar ventilation with possible serious complications and development of dystrophy. The laryngomalacia must be sub-classified by direct laryngoscopy with a flexible endoscope under spontaneous respiration. Microsurgical techniques for performing supraglottoplasty have recently been introduced. The authors describe symptoms and results in four cases of laryngomalacia, in one of whom a supraglottoplasty was performed. The authors emphasise the importance of recognising laryngomalacia as a possible cause of delayed development in the first year of life. PMID- 8638265 TI - [Ictus emeticus. Vomiting as epileptic manifestation]. AB - Autonomic and visceral phenomena are well-known manifestations of epileptic seizures, but recurrent vomiting as ictal epileptic events are less known. Three patients with ictus emeticus, i.e. with nausea and vomiting as their main ictal symptoms, are described. Vomiting is a complex symptom preceded by several phenomena in the gastrointestinal tract and transmitted by the vagal nerve to the vomiting centre in the lateral reticular formation of the medulla oblongata. This autonomic centre in the brain stem is influenced by several cerebral structures. There is both experimental and clinical evidence to support the hypothesis that epileptic disturbances in the insula and the mesial temporal structures are responsible for ictus emeticus. PMID- 8638266 TI - [The extent of ambulatory surgery. A questionnaire study among hospital directors and senior physicians]. AB - Hospital directors and surgical chairmen at 34 Norwegian hospitals were interviewed by questionnaire about the extent of day surgery, and their attitudes towards changes in capacity for the future. They were asked about medical, economic and administrative aspects in relation to the extent of day surgery. 88% responded. They estimated the average extent of day surgery to 24% of all surgical procedures. The estimates varied considerably, and those of the hospital directors were significantly higher than the estimates made by senior surgeons at the same hospitals. 64% considered the extent of day surgery to be less than ideal and 52% had plans for more use of day surgery. The majority of the respondents felt that an increase in the extent of day surgery was warranted, in terms of all three aspects investigated. PMID- 8638267 TI - [Utilization of a unit of ambulatory surgery]. AB - The productivity in an operating theatre can be defined as surgical time divided by the total work hours of the operating room staff. This productivity factor is normally reduced in the case of operations that take only a short time to perform, since the time interval between operations is usually not reduced correspondingly. In the unit for outpatient surgery at Akershus Central Hospital, 4.6 operations were performed daily in 206 days in 1993, with a productivity factor of 42% and an operating theatre utilization of 66% (operating theatre time spent on specific patients/total operating theatre time). The mean interval from the time the surgeons finished one operation until they could start the next was 33 minutes (confidence interval 32-34), with a mean operating time of 44 minutes (42-46). The productivity seemed good compared with the central operating theatre suite at the same hospital, and with US standard. PMID- 8638268 TI - [Treatment of fixed, primarily non-resected rectal cancer. Recommendations from Norwegian Gastrointestinal Cancer Group]. AB - In most cases (70%), fixed rectal cancers are primarily non-resectable, and of the ones that are resected, local recurrence will occur in 50-70%. The same trend, but less pronounced, is also seen for partly fixed or tethered tumours. High-dose irradiation (45-60 Gy) has been reported to yield resectability rates up to 70%, with 17% local recurrences and a 5-year disease-free survival of up to 60-70%. Combined chemotherapy and sequential radiation therapy, hyperthermia or addition of intra-operative radiotherapy, have shown promising results, but no randomized studies have been published comparing the different treatment modalities. Surgery should be performed 4-6 weeks after preoperative treatment. Resectability can only be determined by exploratory laparotomy and serious attempts to remove the tumour-bearing segment by anatomic dissection. PMID- 8638269 TI - [The AIDS epidemic in Norway--where did it go?]. AB - The 1986 prognosis for the AIDS-epidemic in Norway has not been fulfilled. The epidemic has been far less extensive than anticipated. In 1986, little knowledge existed about the parameters that influence the spread of the human immunodeficiency virus (HIV): the prevalence of HIV, the HIV transmission rate, the duration of the infectious period and the extent of risk behaviour. The prognosis was therefore not reliable. The causes of the variability in HIV transmission and in the duration of the infectious period are still poorly understood and thus cannot be easily influenced. A reduction of risk behaviour among homosexual men has been observed. In the general population there has been a slight increase in use of condoms. The behavioural changes may express HIV preventive measures. However, the large discrepancy between the early HIV/AIDS prognosis and the observed epidemic in Norway cannot be explained by behavioural change alone. PMID- 8638270 TI - [Treatment with antiepileptics of mentally retarded patients. Experiences from the 1980s a challenge in the 1990s]. AB - Epilepsy is the most frequent additional handicap in mentally retarded persons. Brain injury and mental retardation may predispose to side effects of antiepileptic drugs (AEDs) on the central nervous system. 63 institutionalized mentally retarded patients were treated for epilepsy in the 1980s. AED treatment was carefully monitored, aiming at the lowest effective dose and an optimal balance between seizure control and adverse effects. In 15 patients, AEDs could be withdrawn. Drugs with less cognitive side effects, such as carbamazepine and valproate, were preferred to longer established drugs, such as phenobarbital and phenytoin. A pronounced decrease in the frequency of seizures was achieved during the study. Based on the calculated Defined Daily Doses, the prescription of AEDs was reduced by 18%. Phenobarbital constituted 39% of the AED consumption at the beginning and 7% at the end of the study. The corresponding figures for phenytoin were 20 and 16%. The fraction of carbamazepine increased from 31 to 44% and that of valproate from 6 to 32%. Less sedative side effects were reported. Several factors other than AEDs may have modified control of seizure in this long term study. These issues are discussed. After the reform of the system of care for the mentally retarded in Norway, it is a challenge to the health authorities to provide an adequate comprehensive epilepsy service to this group of patients. PMID- 8638271 TI - [Half-yearly evaluation of specialist residency]. AB - The author describes experience from regular half-yearly evaluations carried out since 1988 at the psychiatric department in Stavanger. The head of the department, the chief psychiatrist on the resident's ward, the psychotherapy instructor, the resident and the leader of the education committee take part in these 45 minute long meetings. A report is written after each evaluation. This is used as a basis for the candidate's certification from the department. All participants find the half-yearly evaluations rewarding. Weak and strong aspects of the candidate and her/his progress, the education programme and the quality of the instruction can be discussed. In the end a plan is prepared for the education during the next six months for the particular candidate. The persons in charge find half-yearly evaluations especially useful when a candidate is not found to be suited for this particular speciality. When several share this opinion it is also easier to get the candidates themselves to understand and accept the situation. We warmly recommend this arrangement for other specialities too. PMID- 8638272 TI - [Use of influenza vaccine in Norway]. AB - Following the 1993/94 influenza season a questionnaire study was carried out to evaluate the use of influenza vaccine among at-risk groups. 275,000 doses of influenza vaccine were provided free of charge by the National Institute of Public Health to about 500 municipal health care units. 104 of 150 randomly selected units responded to our questionnaire. A majority correctly estimated the vaccine efficacy to be 70-80%, and 90% of the persons who were vaccinated belonged to the defined target groups. Two of three respondents were in favour of the government's support. Influenza among the target groups was mainly regarded as an individual problem, but considered to be a community issue for the general public. The strain on public health budgets for treatment of the old and infirm during influenza epidemics is obviously not fully appreciated. PMID- 8638273 TI - [Prostate-specific membrane antigen]. PMID- 8638274 TI - [Acute psychosis--an unusual clinical symptom in primary hyperparathyroidism]. PMID- 8638275 TI - [Trends, environment or a mystery?]. PMID- 8638276 TI - [Trends, environment or a mystery?]. PMID- 8638277 TI - [Parkinsonism of Johan Sebastian Welhaven]. PMID- 8638279 TI - [Are new thinking and development of medicine blocked?]. PMID- 8638278 TI - [Are new thinking and development in medicine blocked?]. PMID- 8638280 TI - [Understanding the inside driving forces]. PMID- 8638281 TI - Immunotoxicity assessment of biotechnology products: a regulatory point of view. PMID- 8638282 TI - Side-effects of a monoclonal antibody, muromonab CD3/orthoclone OKT3: bibliographic review. AB - Orthoclone OKT3 is a very powerful immunosuppressive drug marketed by Laboratory Cilag and the first monoclonal murine antibody to become available for therapy in humans. It is indicated in acute allograft rejection treatment and its side effects are strongly linked with its mechanism of action, ORT/OKT3 is an Ig2a immunoglobulin produced by hybridoma technique that recognizes, binds and blocks the CD3 complex of the T-cell receptor. Two types of side-effects may occur that are either the consequences of overimmunosuppression or activation of immune system, since ORT/OKT3 behaves as a foreign antigen. This report is a bibliographic review of the suspected side-effects of this product. PMID- 8638283 TI - Immune-mediated side-effects of cytokines in humans. AB - A large body of clinical experience on the adverse consequences of cytokine administration has accumulated since the last decade. Side-effects reported after the therapeutic use of cytokines has provided evidence that activation of the immune response may sometimes have deleterious consequences. Several effects appeared as a direct consequence of the immune activation induced by cytokines, e.g. flu-like reactions, vascular leak syndrome. Cytokine-induced exacerbation of underlying diseases or immune dysregulation were other complications of growing concern. Interferon-alpha (IFN-alpha) treatment has now been clearly linked with the exacerbation or the occurrence of several types of autoantibodies or autoimmune diseases (thyroiditis, systemic lupus erythematosus, hematologic disorders, insulin-dependent diabetes mellitus) or diseases involving altered cell-mediated immune functions (inflammatory dermatologic diseases, nephritis, pneumonitis, colitis). By contrast immunological side-effects of IFN-beta and IFN gamma have been seldom reported. However, the extent of clinical experience with both of these cytokines is still very limited. Interleukin-2 (IL-2) has also been implicated in various conditions that may involve immunopathological processes (thyroid disorders, rheumatoid arthritis, dermatological diseases, interstitial nephritis). Growth factors have been more specifically linked with the development or the exacerbation of dermatological inflammatory diseases through neutrophils, monocytes/macrophages or eosinophils activation (e.g. cutaneous vasculitis and generalized cutaneous eruption, Sweet's syndrome, bullous eruption, psoriasis). Exacerbation of autoimmune thyroiditis was described with granulocyte-macrophage colony-stimulating factor (GM-CSF) only. The immunogenicity of cytokines is also of great relevance and the occurrence of antibodies binding IFN-alpha and IFN-beta, IL2 and GM-CSF have been reported. While the clinical significance of non-neutralizing antibodies is not clearly established, an absence of response or reversal of clinical efficacy has been described in patients developing neutralizing antibodies. Finally, several isolated reports have recently suggested that IFN-alpha treatment may be associated with several immunosuppressive effects while IL-2 is clinically associated with an increased incidence of infectious complications. PMID- 8638285 TI - Cytokine knockout mice: possible application in toxicological research. AB - Knockout mice obtained by homologous recombination technology may be valuable tools for in vivo investigations in toxico-pathogenesis. A short review of the phenotype of mice with distinct cytokine deletions, which occur either naturally or are obtained by homologous recombination, is given. The possible application in pharmacological and toxicological research is discussed with examples of endotoxic shock, hepatic, renal and haematological toxicity. Further applications include a wide variety of cutaneous, pulmonary, vascular, infectious and autoimmune pathology. PMID- 8638284 TI - Safety evaluation of biological and biotechnology-derived medicines. AB - Evaluating the 'safety' of drugs produced by biotechnology resembles the assessment of conventional 'new chemical entities,' but with certain major differences. The 'quality' of the product requires careful control because of concern about the carry-over of DNA, immunogenic proteins, endotoxin and process chemicals. Equally the potency and purity of the product must also be considered, as well as its identity. The toxicity testing of rDNA-derived proteins, monoclonal antibodies and vaccines, although increasingly being swept under the umbrella of conventional studies, should be empirically devised according to the nature and physiological effects of the substance, taking account of the responsiveness of suitable species for non-clinical testing, the potential immunogenicity of heterologous proteins and any effect the drug may have on physiological mechanisms and the immune status of the test animals. Conventional types of single and multidose and reproduction toxicity experiments can then be adapted to detect and investigate any hazard of the novel drug. Kinetics, metabolism and drug interactions should be explored and the regulatory demand for genotoxicity data satisfied. If appropriate, immunological actions, including auto-immunity, can be sought. 'Safety-in-use' should then be predictable with some confidence, because of the extent of the toxicological investigations and because activities that cannot be examined will have been delineated, e.g. lack of a responsive species or of a suitable laboratory procedure. PMID- 8638286 TI - Recent developments and perspectives of biotechnology-derived products. AB - Recent advances in molecular biology especially in the area of rDNA, gene transfer, polymerase chain reaction and hybridoma techniques have provided the necessary molecular tools for the development of a new class of biopharmaceuticals. These biopharmaceuticals include antisense drugs, carbohydrate-based macromolecules and agents that interfere with apoptosis pathways. Cytokines and other immunomodulators also represent an exciting class of new therapeutic entities. The safety evaluation, efficacy, manufacturing and quality control of these complex biopharmaceuticals represent a challenge to the pharmacologist and toxicologist. Finally, the regulatory issues associated with the new biopharmaceuticals need to be addressed to insure the safety of these evolving therapeutic substances. PMID- 8638287 TI - Determination of lymphocyte subsets and cytokine levels in cynomolgus monkeys. AB - Lymphocyte subset counts and cytokine assays are useful to investigate the interactions of pharmaceuticals, particularly new biotechnology products, with the immune system. As no specific reagents are available to label monkey lymphocytes or to assay monkey cytokines by ELISA, cross reactivities of a panel of monoclonal antibodies specific for human lymphocytes or cytokines were studied in the Cynomolgus monkey. The proportions of B, T, CD4+ and CD8+ cells were determined by flow cytometry using a whole blood technique with at least one monoclonal antibody for each subset. Background data were obtained for more than 300 samples. Monkey and human cultured white blood cells were stimulated with standard mitogens. PHA + LPS in humans and Con A + PWM in monkeys triggered the greatest proliferation. IL-1 beta IL-2, IL-6, IL-8, TNF-alpha, TNF-beta and IFN gamma, but not IL-1 alpha, were detected in the monkey using human reagents. In addition, the cytokine profile and the kinetics of cytokine production compared well in humans and Cynomolgus monkeys. PMID- 8638288 TI - Recombinant human interleukin-6: safety issues of a pleiotropic growth factor. AB - Interleukin-6 (IL-6) plays a pivotal role in hematopoiesis, immune reactions and acute phase responses. It has been demonstrated, however, that the abnormal expression and dysregulation of IL-6 homeostasis are involved in the pathogenesis of a range of diseases. It is the purpose of this article to review briefly some of the biological properties of IL-6 in order to discuss issues related to the safety evaluation of recombinant human IL-6 (rhIL-6). Knowledge of the biological properties is important for, (1) selection of a responsive species for toxicological studies and (2) proper interpretation of the side-effect profile. PMID- 8638289 TI - GABAC receptors. AB - gamma-Aminobutyric acid (GABA) is an important neurotransmitter that mediates inhibition in the vertebrate CNS. Until recently, two receptor subtypes were known: bicuculline-sensitive GABAA and baclofen-sensitive GABAB receptors. Several lines of evidence now indicate the existence of a third class of GABA receptor, which is distinct pharmacologically from GABAA and GABAB receptors and is found predominantly in the vertebrate retina. These novel GABAC receptors are Cl- pores. They are insensitive to drugs that modulate GABAA and GABAB receptors and are activated selectively by cis-4-aminoacrotonic acid. PMID- 8638290 TI - A united theory for the multiple forms of LTP? PMID- 8638291 TI - Gap junctions and diseases of the nervous system. PMID- 8638292 TI - Neural component placement. AB - A range of neuroanatomical results supports the idea that 'save wire' is an organizing principle of brain structure: that the theory of combinatorial optimization of networks applies to the anatomy of the nervous system. In particular, optimization of the placement of components operates at several hierarchical levels in the nervous system, from gross to microscopic anatomy, and from invertebrates to primates. That is, when anatomical positioning of interconnected neural components is treated like a problem of wire minimization in microchip layout, a hypothesis of 'best of all possible brains' is consistent with the observed siting of brains, ganglia, and even somata of individual neurons that minimizes the length of interconnections. In the case of the positioning of ganglia of Caenorhabditis elegans, optimization predictions of one in-a-million precision can be verified. PMID- 8638293 TI - Striatal interneurones: chemical, physiological and morphological characterization. AB - The neostriatum is the largest component of the basal ganglia, and the main recipient of afferents to the basal ganglia from the cerebral cortex and thalamus. Studies of the cellular organization of the neostriatum have focused upon the spiny projection neurones, which represent the vast majority of neurones, but the identity and functions of interneurones in this structure have remained enigmatic despite decades of study. Recently, the discovery of cytochemical markers that are specific for each of the major classes of striatal interneurones, and the combination of this with intracellular recording and staining, has revealed the identities of interneurones and some of their functional characteristics in a way that could not have been imagined by the classical morphologists. These methods also suggest some possible modes of action of interneurones in the neostriatal circuitry. PMID- 8638294 TI - Adenylate cyclases: critical foci in neuronal signaling. AB - Current findings show that adenylate cyclases comprise a heterogeneous multigene family, members of which are variously regulated by the alpha and beta gamma subunits of G proteins, by Ca2+ and by protein kinases. In the CNS, individual isoforms of adenylate cyclase are expressed discretely in select regions of the brain. At the subcellular level, adenylate cyclases can be concentrated into dendritic spines, thereby increasing their susceptibility to multiple regulatory influences. Altogether, such findings greatly expand knowledge of the potential role of this archetypical signaling system in the modulation of neuronal function. PMID- 8638295 TI - The nuclear hormone-receptor family in the brain: classics and orphans. AB - Nuclear hormone receptors comprise a superfamily of over 40 transcription factors. About half of them are classical receptors for lipophilic ligands such as steroids and vitamins. Almost all of these true receptors are present in the brain, where they transduce chemical signals from endocrine organs or signals of nutritional origin into cellular responses. The other members resemble the classical receptors in structure, but have no known ligands, and are hence called 'orphan receptors'. The issue of whether ligands for nuclear orphan receptors exist is controversial. Evidence is emerging that orphan receptors might be activated by signal transduction pathways or might be constitutive enhancers or repressors that interact with the classical receptors. Thus, nuclear orphan receptors are placed in strategic positions in the regulation of gene expression in the nervous system. PMID- 8638297 TI - [Surgical treatment of varices]. PMID- 8638298 TI - [Partial or total stripping of the great saphenous vein. 5-year recurrence frequency and 3-year frequency of neural complications after partial and total stripping of the great saphenous vein]. AB - One hundred and sixty-three patients with primary long saphenous vein varices were randomized to either classical (total) stripping of the long saphenous vein (n = 84) or partial stripping, i.e. only of the femoral part of the vein (n = 79). Permanent nerve lesions were evaluated clinically three years and recurrence of varicosities evaluated five years postoperatively. It was found that 24 patients (29%) who had total stripping performed had permanent lesions of the saphenous nerve, whereas only four of the patients (5%) who had partial stripping of the vein had lasting nerve lesions (p < 0.01). Ten percent of patients in both groups had recurrence of varicosities. The present - one and only - long-term, randomized study of different stripping procedures shows that stripping the long saphenous vein below the knee increases the permanent nervedamage six-fold without reducing long-term recurrency. Total stripping of the long saphenous vein should be abandoned as a routine in varicose vein surgery. PMID- 8638296 TI - Anesthetic actions within the spinal cord: contributions to the state of general anesthesia. AB - The behavioral state known as general anesthesia is the result of actions of general anesthetic agents at multiple sites within the neuraxis. The most common end point used to measure the presence of anesthesia is absence of movement following the presentation of a noxious stimulus. The actions of general anesthetics within the spinal cord have been shown to contribute significantly to the suppression of pain-evoked movements, an important component of clinical anesthesia. Studies in the spinal cord are likely to increase our understanding of the pharmacology by which general anesthetics alter the transmission of somatomotor information. It now appears that the pharmacology responsible for the production of anesthesia is agent- and site-selective, and not the result of a unitary mechanism of action. PMID- 8638299 TI - [BiPAP (Biphasic Positive Airway Pressure)--an apparatus for non-invasive respiratory support]. AB - Ventilatory support to patients suffering from respiratory insufficiency using a non-invasive technique has gained increasing popularity during the last few years. BiPAP (biphasic positive airway pressure) (Respiconics) offers inspiratory support and expiratory resistance to this group of patients both in the hospital and, in particular, in the home. The apparatus has proven to be effective as for instance a long term support device for patients suffering from neuromuscular diseases, sleep apnoeas and during the postoperative period. It works without pressurized air and is portable. PMID- 8638300 TI - [Invasive ventilation. Classification, technique and clinical experiences with BiPAP/APRV (Biphasic Positive Airway Pressure/Airway Pressure Release Ventilation)]. AB - BiPAP (bilevel or biphasic positive airway pressure) and APRV (airway pressure release ventilation) are new, and from a technical viewpoint closely related techniques recently introduced to the field of invasive ventilatory support. BiPAP/APRV can be described as a pressure controlled continuous high flow positive airway pressure system with a time-cycled change between a high inspiratory pressure level and a lower expiratory pressure level. Due to highly sensitive valves placed in the inspiratory and expiratory part of the system, unrestricted spontaneous breathing is possible at any moment of the mechanically supported ventilatory cycle. During invasive ventilation BiPAP offers potential advantages by allowing unrestricted spontaneous breathing thus reducing the need for sedation and facilitating weaning. APRV has primarily been investigated in conditions of moderate to severe acute lung injury and it seems that APRV is associated with less detrimental effects on the cardiopulmonary system compared to conventional ventilatory strategies. Apart from a review of the literature the article gives a classification and a technical description of the systems and focuses on the practical approach to BiPAP/APRV, e.g. the initiation and adjustment of respiratory support and the weaning from ventilatory support when applying these techniques. PMID- 8638301 TI - [Results of radiotherapy of ureteral obstruction in invasive bladder cancer]. AB - Retrospective evaluation of the records of 574 patients with muscle-invasive bladder cancer revealed 90 patients (16%) with ureteric obstruction; the obstruction was bilateral in 24%. The effect of radiotherapy was assessed in 55 patients with 68 obstructed kidneys. Six patients with eight obstructed kidneys required percutaneous nephrostomy or ureteric catheters in addition to radiotherapy. Drainage improved in only 20% of kidneys and the diverting catheter could be withdrawn permanently in only one (17%) of the diverted patients. The median survival was 11 months. Irradiation was followed by significant complications in 37 patients (67%). This raises doubts about the assumed beneficial effect of irradiation on ureteric obstruction due to muscle invasive bladder cancer. The short median survival of 11 months confirms that ureteric obstruction is a poor prognostic factor in muscle invasive bladder cancer. PMID- 8638302 TI - [Management of prolonged pregnancy in Denmark. A questionnaire study]. AB - In order to describe how post-term pregnancy was managed in Denmark, a questionnaire was sent to all 50 maternity departments with a 100% response rate. Thirty-nine departments managed post-term pregnancy; twenty-one departments conservatively (i.e.expectantly) and 18 departments with induction of labour. Cardiotocography in the surveillance of the foetus was routine in 35 departments; 21 departments monitored with ultrasound and 18 used human placental lactogen. Gestational age was confirmed by ultrasound before 20th week of gestation in nine of ten departments with > 2000 births per year, in four of 19 departments with 1000-2000 births per year, and in five of ten departments with < 1000 births per year. Ten departments which managed post-term pregnancy by induction did not routinely confirm gestational age by ultrasound. Danish obstetricians differ in their management of post term pregnancy. Cumulative data from prospective, randomized studies can be interpreted as showing an active policy of induction to be advantageous. A large multicentre study is difficult, but desirable in order out to discuss for and against induction or expectant management. PMID- 8638303 TI - [Emergency ambulances manned by physicians]. PMID- 8638304 TI - [Secondary pulmonary hypertension in a patient with severe chronic restrictive pulmonary insufficiency--normalized during treatment with non-invasive intranasal ventilation and oxygen]. AB - The present case report describes a young woman with severe restrictive lung failure, who developed pulmonary hypertension. She was treated successfully with nocturnal noninvasive nasal ventilation and oxygen, which removed symptoms and signs of cardiac incompensation. The improvement has persisted for five years. PMID- 8638305 TI - [Weaning from respirators via biphasic positive airway pressure]. AB - Weaning procedures during the course of intermittent positive pressure ventilation are often difficult. A case is presented in which biphasic positive airway pressure (BiPAP) seemed to be superior to other well known ventilator weaning regimens. The course of the weaning procedure is described. PMID- 8638306 TI - [Systemic lupus erythematosus during pregnancy]. AB - Systemic lupus erythematosus (SLE) is a connective tissue disease most commonly affecting women of a fertile age. It is recommended that pregnancy is planned during periods with remission or low activity of he disease because it seems that SLE is more likely to be exacerbated by pregnancy when the disease is active at the time of conception. Presence of circulating anti-phospholipid antibodies is associated with a poor prognostic outcome for the foetus and increases the risk of complications related to pregnancy. PMID- 8638307 TI - [Manganese, hepatic failure and discoloration of laundry]. PMID- 8638308 TI - [Surgery, anesthesia and lithium treatment]. PMID- 8638309 TI - [What is significant in the scientific curriculum vitae?]. PMID- 8638310 TI - [Fluticasone propionate--a new inhalation steroid for the treatment of bronchial asthma]. PMID- 8638311 TI - [Sequelae of Chlamydia trachomatis should be prevented]. PMID- 8638312 TI - [Chlamydia trachomatis]. AB - Almost 11,000 cases of Chlamydia trachomatis (CT) are diagnosed every year in Denmark. The most important epidemiologic factor is age under 25 years. The prevalence of CT among women applying for legal abortion is five to eight percent and in a recent study the prevalence of CT among second trimester pregnant women was found to be 2.9 percent. The consequences of CT-caused infections are many, including risk of infertility, ectopic pregnancies, perinatal transmission etc. The perinatal transmission rate is high in untreated cases of CT during pregnancy, but it can be prevented by treatment of the expecting woman with erythromycin. The Danish National Board of Health do recommend screening for CT before transcervical interventions, but there are no such recommendations concerning pregnancy health care. A recent Danish study as well as many other studies have shown that it is cost-effective to screen high risk women i.e. women younger than 25 years. An introduction of such screening of Danish pregnant women younger than 25 years should therefore be considered. PMID- 8638313 TI - [Chlamydia trachomatis in pregnant women in the county of Vestsjaelland. Prevalence, prevention of perinatal transmission and cost-effectiveness of screening]. AB - The prevalence of genital Chlamydia trachomatis (CT) in pregnant women and the perinatal transmission after treatment was investigated. An analysis of the cost effectiveness of introducing a screening program among women at risk of having CT was made. Out of 339 pregnant women 2.9% had cervical CT. CT-positive women were treated with erythromycin. CT-positive women were significantly younger than CT negative women and the odds ratio (OR) of having CT if nulliparous was 3.35. The CT-prevalence was 6.6% among women younger than 25 years and 1.6% among women 25 years or older (p = 0.0163). OR of having CT if younger than 25 years was 4.3. The young women were significantly younger at sexual debut. None of the children of women treated for CT during pregnancy developed neonatal CT-conjunctivitis. The screening of women younger than 25 years was considered to be cost-effective. It is concluded that women younger than 25 years are at risk of having CT. Treatment of CT-positive women with erythromycin during pregnancy seems to be effective in eradicating this microorganism and thus preventing perinatal transmission and neonatal CT-conjunctivitis. It therefore seems rational to screen all pregnant women under the age of 25 years for cervical CT, especially in high risk areas. PMID- 8638314 TI - [Prevalence of Chlamydia trachomatis among conscripts. A comparative study of urine samples and urethral swabs]. AB - The purpose of the investigation was 1) to determine the prevalence of C. trachomatis among young men and women enrolled in military service, 2) to compare enzyme-immunoassay (EIA) of a urethral swab with a sample of first voided morning urine, 3) to determine frequency of earlier venereal diseases (VD) and actual symptoms of urethritis and cervicitis. EIA positive specimens were confirmed by immunofluorescent microscopy. The investigation comprised 831 men and 80 women (17-26 years). The prevalence was 5.7% (95% confidence limits 4.2-7.4%) and 15% (8-25%) for men and women, respectively (p < 0.005, chi 2). The agreement between the results of the urethral swab and the urine sample was low. Sensitivity and positive predictive value for urethral swab was better than for urine. The handling of urine specimens was more laborious and confirmation more difficult. The percentage of actual symptoms among Chlamydia positive men was 6. The frequency of earlier VD was 7.5% and 10% among men and women, respectively. Treatment with tetracycline was effective, which makes control of eradication unnecessary. We conclude that genital chlamydial infection among young asymptomatic people is common and screening would be desirable. We still recommend urethral swabbing as the routine method until simpler and/or more reliable assays for urine specimens are developed. PMID- 8638315 TI - [Infections related to central venous catheters]. AB - Central venous catheters (CVC) are a major source of nosocomial bacteraemia. The majority of cases are due to Staphylococcus aureus and coagulase-negative staphylococci. Severe complications, in particular septic shock, develop in 30% of all CVC-associated septicaemia cases and are usually caused by S. aureus. Some patients develop metastatic suppurative complications. Risk factors include prolonged catheterization, localization of catheter to the groin, the number of times the system is entered, use for parenteral nutrition and for dialysis, immunosuppresion and loss of skin integrity especially in burns. Important measures to decrease the rate of CVC-related infections include aseptic technique during insertion, changing transparent semipermeable dressing at least every 48 hours and replacement of the entire delivery system every 72 h. Technological advances such as bonding antimicrobial agents to the intravascular device may reduce the risk of infection. Treatment of CVC-related infections usually requires catheter removal. PMID- 8638316 TI - [Percutaneous lumbar diskectomy]. AB - The results after percutaneous lumbar discectomy were evaluated in 52 patients with low back pain corresponding to the L5 or S1 root. All patients were examined with a median follow-up time of 14 weeks. The patients were divided in two groups. Group 1: patients with an obvious indication for surgery (29 patients) and group 2: patients with a relative indication for surgery (23 patients). The follow-up results for patients in group 1 were comparable with the results after conventional discectomy. The results concerning follow-up low back pain in groups 1 and 2 were good or excellent in 69% and 43% of the patients respectively. The corresponding results for sciatica were 69% and 65% respectively. It is concluded that percutaneous lumbar discectomy is a possible alternative to conventional discectomy in selected patients. PMID- 8638317 TI - [Survival after childhood cancer in Denmark 1943-1987. A population-based study]. AB - Survival from cancer in childhood and adolescence was studied in 8312 children aged 0-19 years notified to the Danish Cancer Registry during 1947-1987. During the first period (1943-1972), five-year survival rates from all malignant neoplasms increased from 23% (1943-1952) to 33% (1963-1972). The greatest improvement was seen during the period 1973-87 when five-year survival rates reached 64% (1983-1987). Between 1973-1977 and 1983-1987, five-year survival rates increased from 32% to 62% for leukaemia, from 40 to 70% for acute lymphoblastic leukaemia, from 35 to 54% for non-Hodgkin's lymphoma, from 50 to 66% for central nervous system neoplasms and from 25 to 49% for bone tumours. An improvement in five-year survival rates for Wilms' tumour was seen between 1960 (19%) and 1980 (81%). Up to 1972, the five-year survival rate from germ-cell neoplasms was approximately 40%; among patients diagnosed in 1973-1987, 76% survived for five years. Survival was similar for boys and girls during the early period, but was significantly higher for girls subsequently. A marked effect of age at diagnosis was seen in the early registration period where survival rates for the age group 0-9 years was substantially lower compared to the age group 10 19 years. This inequality persisted only for children less than two years of age in the later period. PMID- 8638318 TI - [Increasing waiting lists for cataract surgery in the county of Vejle]. AB - For several years the waiting list for cataract surgery has increased in the county of Vejle, Denmark. To reveal any change of indications for cataract surgery accounting for this increase, we analyzed referral notes and files in two samples of 86 and 72 patients examined preoperatively in 1991 and 1993. No significant difference was found between the groups regarding age, visual acuity, other eye diseases than cataract, diabetes, treatment with steroids, anisometropia or occupational disability due to poor vision. Thus no signs of changed indications for cataract surgery were found comparing the two samples. Median waiting time from referral to surgery had increased highly significantly from 1991 to 1993 (p << 0.01). The number of cataract operations per year was unchanged in the period. We propose that the increasing waiting time could be attributed to more people wanting cataract surgery, reflecting growing demands for good visual functioning and better expectations of the outcome after surgery. PMID- 8638319 TI - [Systemic mastocytosis with eosinophilia]. AB - A 67-year old female patient presented with marked eosinophilia in the peripheral blood. The patient had asymptomatic systemic mastocytosis which she had probably had for many years. At the time of diagnosis, the eosinophilia had already produced symptoms of cardiac insufficiency. The patient was treated with prednisone and hydroxyurea which reduced the eosinophil count. In spite of treatment cardiac symptoms progressed, the patient developed atrial flutter, and died in pulmonary oedema. The autopsy showed fibrotic changes in the heart as seen in the fibrotic end stage of Loeffler's endomyocardial disease. The possible reasons for eosinophilia in the patient are discussed. PMID- 8638320 TI - [Thrombolytic therapy in acute cerebral ischemia]. PMID- 8638321 TI - [Needle aspiration biopsy--statistics]. PMID- 8638322 TI - [False increase of triiodothyronine levels in radioimmunoanalysis]. PMID- 8638323 TI - [Malnutrition at hospitals]. PMID- 8638324 TI - [Nutrition therapy in 542 hospitalized patients]. AB - The purpose of the study was to evaluate the feasibility of nutritional therapy in a university hospital. Over a four-year period, 542 adult patients from 16 different departments were included in the study due to malnutrition and/or severe disease. Energy requirement including a surplus for weight gain was calculated by the factorial method and feeding was undertaken by means of food, liquid supplements, tube feeding or parenteral nutrition for an average period of 4.8 weeks. In patients with benign disease only, the average weight gain was as expected from energy balance but in patients with a benign stress-catabolic disease weight gain was only 40% of that expected. In malnourished patients with malignant disease, radiation- or chemotherapy could be carried out without further loss of body weight. During bone-marrow transplantation only a minor weight loss occurred. In conclusion, nutritional therapy is feasible in a clinical setting and the methods employed can identify groups of patients that require only nutritional support and other groups of patients that in addition require treatment of a stress-catabolic state. PMID- 8638325 TI - [Wernicke's encephalopathy]. AB - Wernicke's encephalopathy (WE) is a neuropsychiatric disorder caused by thiamine (vitamin B1)-deficiency. WE is most commonly seen among patients with alcohol abuse, and thiamine deficiency is here caused by several factors, among others inadequate diet, insufficient gastrointestinal absorption and enzymatic abnormalities. The syndrome, however, is also seen among non-alcoholic, undernourished patients, e.g. certain patients with cancer or AIDS. The diagnosis WE has traditionally been given when the triad of confusion, ataxia and ophthalmoplegia was present. However, it should be recognised, that these three symptoms are not always present at the same time, partly because the mental symptoms often dominate and cloud, possible ocular abnormalities and ataxia. The syndrome is, according to the author's opinion, still underdiagnosed. The treatment of WE, consisting of large doses of intravenous thiamine, is effective and safe, and therefore it is important to be aware of WE among risk-patients, especially among patients with alcohol abuse, and to institute treatment with intravenous thiamine at the slightest suspicion. PMID- 8638326 TI - [Idiopathic pulmonary hemosiderosis]. AB - Idiopathic pulmonary haemosiderosis (IPH) is a rare disease characterized by recurrent episodes of intrapulmonary bleeding, chronic iron deficiency anaemia and pulmonary fibrosis. IPH is a diagnosis made by exclusion of other causes. It occurs in both adults and children. Other conditions than IPH can cause pulmonary haemosiderosis. The etiology is unknown, but might be an immunological mechanism causing a defect in the basement membrane of the pulmonary capillary. IPH should be suspected in patients with recurrent episodes of coughing, haemoptysis, dyspnoea and anaemia. Chest X-ray shows pulmonary infiltrates during an acute attack. Examination of sputum or lung biopsy discloses large numbers of haemosiderin-laden pulmonary macrophages. The mortality-rate is high, but the prognosis is difficult to evaluate because many patients survive for a long time either with a course of recurrent attacks or with chronic symptoms, such as dyspnoea and persistent anaemia. Steroids may improve the condition of the patient during a bleeding episode. PMID- 8638327 TI - [Abnormal neurological findings at first admission in patients with schizophrenia or schizophreniform disorders. Results of computer tomography and measurement of regional cerebral blood flow]. AB - Forty-five patients with schizophrenia or schizophreniform disorder admitted to hospital for the first time had a neurological examination, including integrative sensory and complex motor acts, by a trained neurologist. The patients were studied by CT and regional cerebral blood flow as well. A control group of 24 healthy volunteers was included. The patients had significantly more neurological abnormalities (NA) than the healthy volunteers. Medication did not explain the discrepancy. The NA were associated with sulcal enlargement and smaller brains as visualized by CT but not with ventricular enlargement. There was no association between the regional flow values and NA. PMID- 8638328 TI - [Schistosomiasis at Marselisborg hospital 1981-1990]. AB - Schistosomiasis cases diagnosed and treated at Marselisborg Hospital, Denmark from 1.1.1981 to 31.12.1990 were reviewed. In all cases the infection was acquired in Africa. Among 41 patients 15 were Danes and 26 were immigrants, and a total of 57 episodes of schistosomiasis was recorded. Schistosoma mansoni was found in 27 patients, Schistosoma haematobium in 11 patients and in three cases the diagnosis was based on serology. Forty-eight percent of patients with S. mansoni had symptoms, compared to 82% of the patients with S. haematobium. The immigrants more often had symptoms than the Danes, possibly due to the occurrence of other infections. The treatment was in all cases praziquantel. Residual infection was observed in 24%. We recommend that subjects from endemic areas who may have been exposed to infection should be tested for schistosomiasis by examination of urine, stool, or snips of the rectal mucosa. Re-examination after treatment is recommended especially in cases of S. mansoni infection due to risk of residual infection. PMID- 8638329 TI - [Cervical cancer in Frederiksborg County 1990-1991]. AB - One hundred and eight patients from Frederiksborg County, Denmark with cervical cancer diagnosed from 1990 to 1993 were analysed concerning type of carcinoma, tumour stage and screening history. The following types of carcinoma were found: 87 (81%) squamous, 5 (5%) adenosquamous, 15 (14%) adenocarcinoma and one (1%) small cell carcinoma. All women aged 23-60 receive a written invitation to participate in the screening programme. Of the 57 patients who had never or only sporadically been screened 23 were outside the target population. Tumour stage was generally higher for the non-screened, i.e. only 57.9% stage I compared to 82.4% for the screened population. In 51 cases the following errors had occurred: seven sampling errors, 21 screening errors, 15 lack of follow-up of abnormal or inadequate smears, six inadequate cryotherapy and two interval cancers. PMID- 8638330 TI - [Supinator syndrome. Entrapment of the posterior interosseous nerve]. AB - Activity related pain on the lateral side of the elbow or proximal on the forearm may be caused by compression of the posterior interosseous nerve in the radial tunnel. A number of different specialties can be involved in this patient. Often there is no effect of conservative treatment. Several investigations show that the condition can be treated surgically with good to excellent results in the majority of patients. Often the patients suffer a number of concomitant overuse syndromes. The clinical characteristics and the surgical approach to the problem is described and illustrated by two case stories. PMID- 8638331 TI - [Idiopathic pulmonary hemosiderosis in a 16-year old female]. AB - Idiopathic pulmonary haemosiderosis (IPH) is a rare disease of unknown aetiology. The clinical and paraclinical findings consist of recurrent haemoptysis, pulmonary infiltrates and iron deficiency anaemia. Examination of sputum or bronchoalveolar lavage fluid obtained at fiberoptic bronchoscopy discloses large numbers of haemosiderin-laden alveolar macrophages. Pulmonary interstitial fibrosis may develop. As pulmonary haemosiderosis can be observed in association with several diseases, IPH is basically a diagnosis made by exclusion of other causes. The treatment consists of immunosuppression with steroids and cytotoxic drugs. PMID- 8638332 TI - [Epidermodysplasia verruciformis, human Papillomavirus infection, genital epithelial neoplasia and possible immunodeficiency]. AB - Papilloma virus infections are common in patients with immune defect/suppression. We describe a case of regional localised epidermodysplasia verruciformis in combination with genital intraepithelial neoplasia, refractory anaemia, and impaired immunity as demonstrated by lymphopenia with reduced B-cell/T-cell ratio, but normal T-helper/T-suppressor (CD4/CD8) ratio, and neutrophils with reduced spontaneous migration and chemotaxis. The regional localisation of the lesion and the changes in the cell mediated immunity are not classical for epidermodysplasia verruciformis. It is concluded that an increased tendency to papilloma virus infections and/or intraepithelial neoplasia should alert the clinician to the possibility of an immune defect. PMID- 8638333 TI - [Biotechnological clearing of secretions]. PMID- 8638334 TI - [Audiologic care]. PMID- 8638335 TI - [Leading or misleading]. PMID- 8638336 TI - [Doppler ultrasound: its use in obstetrics?]. PMID- 8638337 TI - [The use of Doppler-ultrasound in obstetrics]. AB - Several investigations have suggested an association between intrauterine growth retardation or an adverse neonatal outcome and increased vascular resistance in the fetal and maternal uteroplacental circulation assessed by Doppler-ultrasound. Meta-analysis of nine randomized controlled trials showed that the use of Doppler ultrasound in the surveillance of high-risk pregnancies reduced the perinatal mortality considerably. Taken together, there is evidence to suggest that Doppler ultrasound is a valuable tool for discriminating between normal small and "sick" small fetuses. Screening in unselected populations of low-risk pregnancies, however, seems to be without any benefit. PMID- 8638338 TI - [Sick sinus syndrome. Pacemaker with atrial versus ventricular stimulation]. AB - In patients with sick sinus syndrome, single chamber atrial pacing has been reported in retrospective studies to be associated with lower frequencies of atrial fibrillation, thromboembolism, heart failure, and mortality than ventricular pacing. We did a prospective randomised trial in 225 consecutive patients (142 women, 83 men; mean age 76 years) with the sick sinus syndrome, randomised to atrial (n = 110) or ventricular (n = 115) pacing and followed for up to five years (mean 40 [SD 18] months). During follow-up, the frequency of atrial fibrillation was higher in the ventricular group. Thromboembolic events (stroke or peripheral arterial embolus) occurred in 20 patients in the ventricular group and in six patients in the atrial group (p = 0.008). Twenty five patients died in the ventricular group compared with 21 in the atrial group (p = 0.74). The number of cases of heart failure did not differ between the two groups. Atrioventricular block occurred in two patients in the atrial group. It is concluded that patients with sick sinus syndrome should be treated with atrial pacing rather than ventricular pacing because atrial pacing is associated with lower frequencies of atrial fibrillation, thromboembolic complications, and a low risk of atrioventricular block. PMID- 8638339 TI - [Treatment with implantable cardioverter-defibrillators. Experiences from Skejby hospital during the period 1989-1994]. AB - The aim of the present study was to evaluate survival and therapy for ventricular tachyarrhythmia in patients treated with implantable cardioverter-defibrillator (ICD)-implantation at Skejby Hospital. Seventy-two patients, of which 54 were male, have received an ICD since 1989. Mean (range) age was 54 (16-74) years. Forty-nine (68%) had ischaemic heart disease. The patients were followed for a median (range) of 14 (1/2-50) months. Kaplan-Meyer plots are presented for total mortality, cardiac mortality, sudden cardiac mortality, appropriate therapy, and therapy for life-threatening tachyarrhythmia. After one, two and three years respectively, mortality was respectively 13, 27, and 32%, cardiac mortality was 5, 19, and 24%, sudden cardiac mortality was 3, 6, and 12%, cumulative incidence of appropriate therapy was 56, 66, and 90%, and cumulative incidence of therapy for life-threatening tachyarrhythmia was 19, 29 and 52%. It is concluded, that the majority of patients treated with an ICD developed ventricular tachyarrhythmia and had appropriate or lifesaving ICD-therapy during follow-up. PMID- 8638340 TI - [Involuntary diurnal urinary incontinence among children--is it curable?]. AB - Thirty-two children aged 5-13 years were treated with urotherapy for daywetting. After treatment and observation for three months 41% became dry, 38% were improved and 16% were unchanged. Two patients were lost to follow-up. Our conclusion is that urotherapy has a place in the treatment of daywetting in children that are well-motivated to cooperate. PMID- 8638341 TI - [Eight days of naproxen therapy can prevent heterotopic ossification after total hip alloplasty]. AB - The effect of one week of treatment with naproxen on the formation of heterotopic ossification after cemented total hip arthroplasty was studied in a prospective trial. Twenty-seven patients received 500 mg naproxen twice daily for seven days postoperatively. The medication was started on the morning of the operation. The results were compared to a control group of 23 patients from a previous study who had not received any kind of nonsteroidal anti-inflammatory drug. All radiographs were randomly mixed and patient identification was blinded. Three months after the operation 12/23 patients in the control group and 3/27 in the naproxen treated group had developed heterotopic ossification. One year after the operation 4/27 in the naproxen-treated group and 12/23 in the control group had heterotopic ossification (p < 0.05). Three patients in the control group and none in the naproxen-treated group developed severe ossification. It is concluded that naproxen given for one week can decrease the incidence of heterotopic ossification after total hip arthroplasty. PMID- 8638342 TI - [New cancers after squamous cell skin cancer]. AB - The subsequent cancer experience in 5,100 patients with squamous cell skin cancer (SCC) was compared with the cancer incidence in the Danish population. Ratios of observed to expected cancers served as the measure of the relative cancer risk (RR). Overall, SCC patients were at significantly increased cancer risk due to cancers of the respiratory organs (RR = 1.7); cancers of the lip, buccal cavity and pharynx (RR = 3.1); non-Hodgkin's lymphoma (RR = 2.3); leukaemia (RR = 2.5); malignant melanoma (RR = 2.6); and small intestine cancers in men (RR = 4.1). The risk of new cancers was higher in patients diagnosed with SCC before the age of 60 years than in those diagnosed with SCC after that age. A previously undocumented, significant excess of smoking-related cancers was observed after a diagnosis of SCC. Since a variety of other squamous cell cancers have been linked to smoking, the authors hypothesize that some general effect of smoking might act on all human squamous epithelia. PMID- 8638343 TI - [DNA-fingerprinting and tuberculosis]. AB - DNA-fingerprinting or RFLP (Restriction Fragment Length Polymorphism) is a molecular biological technique which allows differentiation between tuberculosis strains. The technique is based on the occurrence of repetitive elements in the mycobacterial genome and is a powerful tool in epidemiological studies. DNA fingerprinting is also of value in tracking the spread of infection in patients with tuberculosis. The latter is illustrated in two case stories. PMID- 8638344 TI - [Traumatic total rupture of the musculus pectoralis major tendon]. AB - Total or partial rupture of the major pectoral muscle is a rare lesion. Early treatment is recommended to regain full strength in the shoulder, but even without surgery 70 to 90% of original strength may be recovered within a year after trauma. We report a case in which a carpenter three months after total rupture of the major pectoral muscle tendon on the dominant side had spontaneously recovered sufficiently to perform his usual job. The diagnosis was visualized by MR imaging. The muscle strength was tested by use of a dynamometer. PMID- 8638345 TI - [A new ultrasensitive bioanalysis of estrogen]. PMID- 8638346 TI - [Hepatitis A vaccine dosing]. PMID- 8638347 TI - [Intracardiac injection]. PMID- 8638348 TI - The 1995 staging requirement for approved cancer programs. PMID- 8638349 TI - Radical prostatectomy: the perineal approach. PMID- 8638350 TI - Radical prostatectomy: the retropubic approach. PMID- 8638351 TI - Temperature-correlated histopathologic changes following microwave thermoablation of obstructive tissue in patients with benign prostatic hyperplasia. AB - OBJECTIVES: To determine the intraprostatic pathologic changes following accurately measured doses of transurethral microwave thermal energy in patients with benign prostatic hyperplasia. METHODS: Eight patients scheduled for prostate surgery were treated for approximately 1 hour without anesthesia using a newly designed microwave treatment catheter that allows a close impedance match to prostate tissue and concentrates thermal energy preferentially in the anterior and lateral prostate gland. Interstitial, urethral, and rectal temperatures were continuously measured using a novel stereotactic thermal mapping technique. Serial sections of prostate tissue harvested during subsequent surgery were evaluated pathologically with prostate mapping. RESULTS: Microwave treatment resulted in marked and continuous intraprostate temperature elevation, while urethral and rectal temperatures remained low. Peak intraprostate temperatures in individual patients reached as high as 80 degrees C. Mean temperature reached a maximum of 54 degrees C at a radial distance of approximately 0.5 cm from the urethra and remained 45 degrees C or higher up to a distance of 1.6 cm. The predominant pathologic findings were uniform hemorrhagic necrosis and tissue devitalization without significant inflammation. The mean distance from the urethra to the viable-necrotic tissue border was 1.6 +/- 0.2 cm (range, 0.5 to 2.5). At this border, no more than 1 mm in thickness, temperature averaged 45.7 +/- 0.6 degrees C, and there was a suggestion that pure stromal nodules were more resistant to thermal injury. CONCLUSIONS: Microwave treatment can destroy obstructive prostate tissue while maintaining innocuous urethral and rectal temperatures. Temperatures of 45 degrees C or higher for approximately 1 hour cause uniform thermoablation of prostate tissue. PMID- 8638354 TI - In situ transitional cell carcinoma involvement of prostatic urethra: bacillus Calmette-Guerin therapy without previous transurethral resection of the prostate. AB - OBJECTIVES: Bacillus Calmette-Guerin (BCG) has demonstrated both the efficacy in patients with carcinoma in situ (CIS) of the bladder and the penetration in the prostate (granulomas) after endovesical treatment. We treated patients with CIS of the prostatic urethra with bladder instillations of BCG without previous transurethral resection to assess the local therapeutic effect. METHODS: Eighteen patients with CIS of the prostatic urethra (15 multifocal CIS and 15 associated to superficial bladder carcinoma) were treated with endovesical instillations of 81 mg of BCG Connaught weekly for 6 weeks. RESULTS: After a mean follow-up of 31 months, 14 of 18 patients had complete response, 3 had progression (2 in the prostate and 1 in the bladder); diffuse CIS of the bladder persisted in 1 patient. Three patients required cystectomy and 1 underwent palliative transurethral resection. Fifteen patients are alive with no evidence of disease, 2 patients died due to progression of the disease, and 1 is alive with tumor. CONCLUSIONS: The presence of CIS in the prostatic urethra can be managed with endovesical BCG as initial treatment with fairly good success. There is no need for transurethral resection, even though a loop biopsy may have to be obtained at the first control examination to make sure there is no stromal invasion. PMID- 8638353 TI - A comparison of the W-stapled ileal reservoir with hand-sewn reservoirs for orthotopic bladder replacement. AB - OBJECTIVES: Confidence has increased in the use of an orthotopic reservoir to the urethra after a cystoprostatectomy for bladder cancer; however, many surgeons would welcome a method to simplify the operative procedure. The availability of absorbable staples on a GIA stapler allows study of the incorporation of stapling procedures into formation of a reservoir. Because of the success of hand-sewn W configured ileal reservoirs, we initiated a Phase II study to evaluate absorbable staples in formation of a W-configured reservoir. We now present an expanded contemporary series comparing a W-stapled ileal neobladder with hand-sewn ileal (Studer) or hand-sewn ileocolic (Le Bag) reservoirs. METHODS: Forty-five selected patients underwent orthotopic urinary diversion after cystoprostatectomy for bladder cancer (n = 42) or prostate cancer (n = 3) using one of the three methods of reservoir construction. There were 43 men and 2 women. The evaluation included a urodynamic evaluation and a questionnaire sent to patients inquiring about urinary function. RESULTS: Most patients did well with the W-stapled ileal reservoir but 6 of 19 evaluable patients had unsatisfactory reservoir characteristics. Three patients needed an augmentation cystoplasty and 3 had higher pressure, smaller volume reservoirs. Reservoir function appeared to be consistently more favorable in patients with either of the hand-sewn reservoirs using an ileal or ileocolic segment. CONCLUSIONS: Although the W-stapled ileal reservoir is safe and allows reservoir formation quickly, the inconsistencies of the results discourage its use in the particular configuration described. Failure of the reservoir to distend could be a function of reservoir design, areas of ischemia in the reservoir, or reaction to staple material. Absorbable staples on the GIA instrument may work satisfactorily for formation of an ileocolic reservoir for continent cutaneous diversion. However, the W-configured orthotopic reservoir as constructed using absorbable staples in this study is inferior to a hand-sewn ileal or ileocolic neobladder. PMID- 8638352 TI - Additional experience with continent urinary diversion using cutaneous ileocecocystoplasty. AB - OBJECTIVES: We have used the technique of ileocecocystoplasty with a cutaneous continent catheterizable limb to incorporate native bladder into the urinary reservoir to treat patients with functional and anatomic bladder disorders. This review was performed to evaluate the outcome of the first 8 patients who underwent this procedure. METHODS: A retrospective analysis was performed by chart review and telephone interview. Patients' preoperative and postoperative bladder status and management, satisfaction rating, and renal function are reported. RESULTS: Follow-up of 9 to 64 months (mean, 33.9) in our first 8 patients reveals excellent results in all, including 1 patient who prefers an indwelling catheter due to progressive neuromuscular disability. CONCLUSIONS: Cutaneous ileocecocystoplasty offers urologists and patients continent urinary diversion with minimal morbidity and is a much less extensive procedure than complete bladder replacement. PMID- 8638355 TI - Prostate zones in three-dimensional transrectal ultrasound. AB - OBJECTIVES: This study was undertaken to evaluate the efficacy of three dimensional transrectal ultrasound to identify and measure the prostate zones. METHODS: In an anatomic-sonographic study, eight specimens were investigated by means of three-dimensional transrectal ultrasound. Subsequently, the volumes of the prostate and the transition zone were measured by means of a special planimetric program; the images and results obtained were compared with anatomic sections and water displacement volume measurement. Then two groups of patients were investigated by means of three-dimensional transrectal ultrasound; the first group included 90 patients ranging in age from 55 to 85 years who presented with benign prostatic hyperplasia (BPH), which was verified by means of histologic examination. Three-dimensional transrectal ultrasound was performed prior to ultrasound-guided biopsies of the prostate. The second group comprised 10 patients aged between 17 and 30 years whose prostates were free of disease. RESULTS: Three-dimensional transrectal sonography is the first imaging technique that can simultaneously demonstrate relevant structures in three planes; apart from the sagittal and the horizontal (or axial) plane, the region of interest can be examined in the coronal plane as well. The structural differences of the prostatic zones in BPH and the juvenile gland can be clearly identified with the help of three-dimensional transrectal ultrasound. The coronal plane provides important additional information; the central zone and the enlarged transition zone can be identified best in this third plane. CONCLUSIONS: The prostate zones, their interrelations, and, in particular, the hyperplasia of the transition zone in BPH are clearly demonstrable. Furthermore, the size of the enlarged transition zone can be exactly measured. PMID- 8638356 TI - Significance of intermittency in men with lower urinary tract symptoms. AB - OBJECTIVES: To determine the relationship between the symptom of intermittency, objective evidence of intermittency on uroflow traces, benign prostatic enlargement, and bladder outlet obstruction (BOO) in men with lower urinary tract symptoms (LUTS). METHODS: The prevalence of the symptom of intermittency was determined from a symptom questionnaire in 165 men presenting with LUTS. Objective evidence of intermittency during voiding was assessed from uroflow traces, and prostate volume was measured by transrectal ultrasound. Combined pressure-flow studies were performed to determine the presence or absence of BOO. RESULTS: There was relatively poor agreement between the symptom of intermittency and objective evidence of its presence. Of the 35 patients who reported intermittency most or all of the time, 21 showed no objective evidence of intermittency on uroflowmetry. The symptom of intermittency was not significantly related to the presence of BOO (P = 0.42) and the group-specific urethral resistance factor (URA) did not differ significantly (P = 0.87) between those men who complained of intermittency and those who did not (median URA, 35 versus 32 cm H2O). However, objective evidence of intermittency on uroflow traces was significantly related to BOO (P = 0.016), and those patients with objective evidence of intermittency had higher URA values (median, 43 versus 32 cm H2O). Objective evidence of intermittency had a specificity of 98% and positive predictive value of 92% for the presence of BOO. Neither the symptom of intermittency nor objective evidence of its presence was significantly related to prostate size. CONCLUSIONS: Although the symptom of intermittency does not seem to be related to BOO or prostate size, objective evidence of intermittency on flow curve traces is specific for outflow obstruction, and as such its presence could potentially be of value in the assessment of men with lower urinary tract symptoms. PMID- 8638357 TI - A dose-response study of the effect of flutamide on benign prostatic hyperplasia: results of a multicenter study. AB - OBJECTIVES: The objective of this study was to evaluate efficacy, safety, and dose-response profiles of four dosing schemes of flutamide over 24 weeks. METHODS: Patients were randomized to receive one of the following five treatment regimens for a period of 24 weeks: placebo capsule, flutamide capsules 125 mg twice daily, 250 mg once daily, 250 mg twice daily, and 250 mg three times daily. Patients were then evaluated at baseline (0 weeks) and at 4, 6, 12, 18, and 24 weeks after the start of treatment, and 8 weeks after the end of treatment (32 weeks). Evaluation of efficacy was performed by noting changes in urine flow rate, residual urine volume, symptom score, prostate volume, and prostate specific antigen level. A total of 372 patients were enrolled into the study at 32 centers (14 centers in the United States and 18 international centers). RESULTS: Baseline peak urinary flow rate and percent change from baseline in maximum flow rate showed a dose-related increase at 4 and 6 weeks; this increase was significant in the 250 mg three times daily group. At later time points, no significant differences between the flutamide and placebo groups were observed, largely because of the decreasing number of evaluable patients. At 4 and 6 weeks, 25% of patients in the 250 mg three times daily group had more than 3 cc/s increase in uroflow compared to about 10% of placebo patients (P < 0.05). All flutamide-treated groups had a significant decrease in prostate volume from baseline to the last treatment visit compared to placebo and this reduction was dose related (in comparison to placebo: P < 0.05 for 125 mg twice daily and P < 0.001 for all other treatment arms). Median decrease for the flutamide-treated groups ranged from 6% to 23% at 12 weeks and from 14% to 29% at 24 weeks. All treatment groups showed a subsequent increase in prostate volume after treatment was stopped. Furthermore, there was a significant reduction in residual urine volume at 24 weeks only in the 250 mg three times daily group. It increased following cessation of therapy. Urinary symptoms at 6, 12, 18, and 24 weeks did not show any significant difference between placebo and any flutamide dose group. The most common adverse events were nipple and breast tenderness (42% to 52%), diarrhea (29% to 34%), and gynecomastia (14% to 19%). Each of these adverse events had a significantly higher incidence in all flutamide dose groups compared with placebo, but none appeared to occur in a dose-related fashion. Sixteen percent of patients in the placebo group and 25% to 39% of patients in flutamide groups were discontinued due to diarrhea (12% to 17%) or nipple and breast tenderness (4% to 8%). A total of 1% to 3% of patients in various treatment arms discontinued due to deranged liver enzymes (1% for placebo); and 1% to 4% due to impotence (1% for placebo). CONCLUSIONS: Flutamide reduced the prostate volume in a dose-related fashion and resulted in an increase in peak flow rate at 4 weeks (3% for 250 mg three times daily, P value < 0.05), but the early positive effects did not maintain statistical significance due to an increasing number of dropouts due to adverse events. Effect on postvoid residual volume was observed only at the highest dose and at 24 weeks (median reduction, 23 mL, P < 0.05). Despite volume reduction and early improvement in peak flow rate, there were no significant differences in urinary symptoms among the placebo and flutamide groups. Higher incidences of diarrhea, breast tenderness, and gynecomastia, however, were the main limiting factors in this study and until these problems are overcome, the role of flutamide in the management of benign prostatic hyperplasia remains investigational. PMID- 8638358 TI - Transurethral vaportrode electrovaporization of the prostate: physical principles, technique, and results. AB - OBJECTIVES: This is a study to evaluate technique, efficacy, and safety of a new electrosurgical instrument (Vaportrode) in management of benign prostatic hyperplasia (BPH). METHODS: Forty-two symptomatic BPH patients form the subject of this study. Preoperative and postoperative International Prostate Symptom Score (IPSS), peak flow rates (PFR), postvoid residual urine (PVR), operative details, and complications were recorded in each patient. Baseline, 1, 3, and 6 months follow-up data were used for analyses. RESULTS: Results reveal that transurethral electrovaporization of the prostate (TUEVP) is a reasonable procedure to treat symptomatic BPH, and at 6 months, this procedure results in 68% reduction in IPSS, 128% improvement in PFR, and 58% reduction in PVR. Even in patients who are in retention prior to TUEVP, at 6 months the IPSS is 7.7, PFR is 20.4 cc/s, and PVR reduces to 130 cc. The procedure is well tolerated without any significant short-term complications. CONCLUSIONS: The results of this modality in improving the symptoms and PFR in symptomatic BPH patients in the short term appear promising. Advantages over transurethral resection (TUR) of the prostate and laser prostatectomy include familiarity of the transurethral route, lack of need for high-cost laser equipment and fibers, excellent intraoperative hemostasis, lack of bleeding or fluid absorption, and ability to cause a predictable TUR-like prostate defect at the end of the procedure. Further studies involving larger numbers of patients and longer follow-up are warranted to assess further the utility of this procedure. PMID- 8638359 TI - Ejaculation increases the serum prostate-specific antigen concentration. AB - OBJECTIVES: To determine the effect of ejaculation on the serum prostate-specific antigen (PSA) concentration in men at risk for developing prostate cancer. METHODS: A prospective, community-based study was conducted in which 64 men, aged 49 to 79 years, underwent a serum PSA determination immediately before ejaculation (baseline) and at 1 hour, 6 hours, and 24 hours following ejaculation. The serum PSA also was measured 48 hours and 1 week after ejaculation if the concentration had not returned to the baseline value by the previous time interval. All subjects abstained from ejaculation for a minimum of 7 days prior to the study and until the PSA concentration returned to the baseline level. Absolute and relative change in serum PSA concentration, as well as the time to return to baseline PSA concentration following ejaculation, were assessed. RESULTS: The serum PSA concentration increased following ejaculation in 87% of the subjects. The mean baseline PSA was 1.8 ng/mL (median, 0.7 ng/mL). The mean absolute PSA change +/- standard deviation 1 hour, 6 hours, 24 hours, and 48 hours after ejaculation was 0.8 +/- 1.32 ng/mL, 0.3 +/- 0.66 ng/mL, 0.2 +/- 0.33 ng/mL, and 0.4 +/- 0.40 ng/mL, respectively. The mean relative PSA change +/- standard error 1 hour, 6 hours, 24 hours, and 48 hours after ejaculation was 41 +/- 4%, 9 +/- 1.5%, 8 +/- 1.3%, and 10 +/- 2.3%, respectively. The absolute and relative changes in PSA concentration noted 1 hour, 6 hours, and 24 hours after ejaculation were statistically significant (P = 0.0001). A strong correlation was observed between absolute change in PSA and baseline serum PSA, at each time interval (1 hour: r = 0.68, 6 hours: r = 0.77, 24 hours: r = 0.70; P < 0.0001) after ejaculation. Similarly, a significant correlation was noted between absolute change in PSA and patient age at each time interval (1 hour: r = 0.37, 6 hours: r = 0.38; P = 0.002, 24 hours: r = 0.55; P < 0.0001). Ninety-two percent of subjects returned to baseline by 24 hours (95% confidence interval (Cl) = 83% to 97%), whereas 97% of subjects returned to baseline by 48 hours (95% Cl = 89% to 99%). CONCLUSIONS: Ejaculation causes a significant increase in the serum PSA concentration in men between 49 and 79 years of age that may persist for up to 48 hours. This change appears to correlate with age and baseline PSA. It is recommended that men abstain from ejaculation for 48 hours prior to having a serum PSA determination. PMID- 8638360 TI - Using proportions of free to total prostate-specific antigen, age, and total prostate-specific antigen to predict the probability of prostate cancer. AB - OBJECTIVES: This study was undertaken to define the probability of prostate cancer as a function of the proportion of free to total prostate-specific antigen (FTPSA), total PSA, and age for those patients with total PSA levels between 2.5 and 20.0 ng/mL. METHODS: Prebiopsy serums were obtained from 428 untreated patients (165 malignant, 263 benign) who had undergone sextant six-core biopsy. Each patient had no prior history of prostate cancer and a prebiopsy total PSA value between 2.5 and 20.0 ng/mL. Total PSA levels were determined using the PA immunoassay performed on the TOSOH AIA-1200 automated immunoassay instrument. Free PSA levels were determined using a monoclonal-polyclonal antibody sandwich radioimmunoassay. RESULTS: In men with total PSA values between 2.5 and 20.0 ng/mL, the FTPSA significantly differentiated between patients with benign and malignant histologic states. Log linear modeling indicated distinct differences in the risk for cancer as a function of FTPSA, total PSA, and age. The highest probability for cancer was observed in men greater than 70 years of age who had a FTPSA less than 7% and total PSA more than 10.0 ng/mL. Conversely, the lowest probability for cancer was observed in patients less than 60 years of age who had a FTPSA more than 25% and a total PSA less than 4 ng/mL. CONCLUSIONS: The probability that prostate cancer will be found on biopsy has a marked gradient that is associated with age, total PSA, and FTPSA. The extreme ends of FTPSA of less than 7% and more than 25% are diagnostic for prostate cancer and benign prostatic disease, respectively. PMID- 8638361 TI - Clinical significance of the determination of noncomplexed prostate-specific antigen as a marker for prostate carcinoma. AB - OBJECTIVES: The aim of this study was to investigate whether a significant difference consists between patients with and without prostate carcinoma (CaP) regarding the ratio of prostate-specific antigen (PSA) in complex with major proteinase inhibitors to noncomplexed (free) PSA (FPSA). METHODS: We analyzed the sera of 29 patients with untreated CaP, 34 patients with benign prostatic hyperplasia (BPH), and 33 men with no symptoms of prostate disease for the amount of FPSA and total PSA (TPSA) with the Immulite chemiluminescent enzyme immunometric assay. RESULTS: FPSA was found only as a minor fraction in all sera tested. Calculation of the percentage of FPSA from TPSA revealed a significant difference between patients with CaP (median, 9.55%) versus patients with BPH (median, 17.07%; P = 0.00001) or versus healthy men (median, 16.11%; P = 0.0006). Considering only patients with PSA values less than 10 ng/mL, the difference between patients with CaP versus patients with BPH remained significant (P = 0.026). The specificity for differentiation between CaP and BPH at a sensitivity level of 89% for the combined evaluation of the proportion of FPSA and TPSA increased from 30% (TPSA considered alone) to 61%. The cutoff level for TPSA was determined at 4 ng/mL and for the proportion of FPSA at 21.1%. CONCLUSIONS: These data indicate that the differentiation between CaP and BPH can be considerably improved by measuring both FPSA and TPSA and calculating the ratio of the one to the other. PMID- 8638362 TI - Reference range of prostate-specific antigen after transurethral resection of the prostate. AB - OBJECTIVES: The aim of the present study was to investigate how transurethral resection of the prostate (TURP) affected the serum levels of prostate-specific antigen (PSA) and to establish reference ranges of PSA in patients who have undergone TURP. METHODS: PSA was determined preoperatively and 3 months postoperatively in 190 patients who underwent TURP because of benign prostatic hyperplasia (BPH). RESULTS: Mean PSA levels were reduced by 70%, from 6.0 to 1.9 ng/mL. Prostate volume was reduced by 58% from 63.3 to 26.5 cc, which is close to the reported normal volume in men without BPH. Ninety percent of the patients had a postoperative PSA value of less than 4 ng/mL and 98% less than 10 ng/mL. CONCLUSIONS: After a complete TURP with a benign histopathologic specimen, PSA should be expected to be within the normal reference range, that is, less than 4 ng/mL. PMID- 8638363 TI - Improved continence with tubularized bladder neck reconstruction following radical retropubic prostatectomy. AB - OBJECTIVES: Incontinence after radical retropubic prostatectomy remains a common problem. This study determines the efficacy of a tubularized bladder neck reconstruction for improving the time to continence and also the total rate of continence following radical retropubic prostatectomy. METHODS: Tubularized bladder neck reconstruction was performed in 29 patients by creating a trapezoidal-shaped anterior bladder flap and performing tubularization over a 30 F red rubber catheter prior to urethrovesical anastomosis. This flap measured approximately 5 cm at its base and 3 cm at its apex. Continence rates of these patients were compared to results of 30 randomly selected patients in whom a bladder flap was not used. RESULTS: Seven of 29 (24%) patients with tubularized bladder neck reconstruction were fully continent within 24 hours of catheter removal and 27 of 29 (93%) were fully continent by 3 months follow-up. An additional patient was continent at 6 months for a total continence rate of 28 of 29 (97%). These continence rates were significantly higher at all follow-up times than for patients in whom an anterior bladder flap was not used. One patient developed a bladder neck contracture and was successfully managed with visual urethrotomy. He is fully continent. CONCLUSIONS: Tubularized bladder neck reconstruction following radical prostatectomy may both increase the successful achievement of postoperative continence and decrease the time needed to achieve full continence without increasing morbidity. PMID- 8638364 TI - Incidence of inguinal hernias following radical retropubic prostatectomy. AB - OBJECTIVES: During the last decade, the number of patients undergoing radical retropubic prostatectomy (RRP) has substantially increased. Over this same time period, we have noted that a significant number of these patients have developed postoperative inguinal hernias. We sought to identify the incidence of postoperative inguinal hernias after RRP and compared this with the stated 5% incidence in the general adult male population. METHODS: Ninety-two consecutive RRPs performed by three surgeons (I.J.S., N.L.C., S.W.D.) were retrospectively reviewed. The operative reports for each patient who subsequently underwent inguinal herniorrhaphy were analyzed to determine whether the hernias were direct or indirect. RESULTS: The overall incidence of postoperative inguinal hernias was 12% (11 of 92). All hernias were found within approximately 6 months of the prostatectomy. Ninety-one percent (10 of 11) of these hernias were indirect, and only 9% (1 of 11) were direct. CONCLUSIONS: We believe this to be the first report in the English literature describing postoperative inguinal hernias following retropubic prostatectomy. A significantly higher incidence (12%) of inguinal hernias was noted in the postprostatectomy population compared with the general adult male population (5%). This finding suggests that inguinal hernias can be a consequence of RRP. Urologists should be cognizant of this possibility in order to screen all patients carefully prior to surgery for subtle weakness in the inguinal canal, as well as to inform patients properly of the possibility of developing an inguinal hernia after surgery. PMID- 8638365 TI - An analysis of the time course of postoperative prostate-specific antigen failure in patients with positive surgical margins: implications on the use of adjuvant therapy. AB - OBJECTIVES: The role of adjuvant therapy in the postprostatectomy setting for positive margin patients is an unresolved issue. The purpose of this study is to provide the rationale for patient selection in Phase III trials that test the impact of adjuvant therapy on survival in positive margin prostate cancer patients. METHODS: Early (12 months or less) and delayed (more than 12 months) postoperative prostate-specific antigen (PSA) failure have been correlated with distant and local failure, respectively, as the site of first failure. In this study, a Cox regression multivariate analysis was used to determine the significant independent clinical and pathologic predictors of early and delayed postoperative PSA failure in 143 margin-positive prostate cancer patients. RESULTS: Margin-positive patients with positive pelvic lymph nodes, seminal vesicle invasion, or prostatectomy Gleason sum 8 or higher were excluded. For the remaining patients, a prostatectomy Gleason sum of 7, preoperative PSA more than 20 ng/mL, and an endorectal coil magnetic resonance imaging (erMRI) scan showing extensive disease were significant independent predictors of early postoperative PSA failure. Conversely, a prostatectomy Gleason sum of 6 or less, preoperative PSA 20 ng/mL or less, and an erMRI showing limited disease predicted delayed PSA failure. CONCLUSIONS: Preliminary data suggest that the pattern of first failure can be predicted by the time course of rise in the postoperative PSA. The preliminary results of this study suggest that patient selection for clinical trials examining the efficacy of postoperative adjuvant therapy in the positive margin patient may be determined on the basis of the clinical and pathologic characteristics that predict early versus delayed postoperative PSA failure. PMID- 8638366 TI - Deoxyribonucleic acid ploidy status as no basis for pathologic stage prediction in clinically resectable prostate cancer. AB - OBJECTIVES: Assessment of deoxyribonucleic acid (DNA) ploidy status of a tumor as a means for predicting pathologic stages in prostate cancer. METHODS: DNA ploidy of each focus of a cancer in 70 radical prostatectomy specimens was determined by nuclear image analysis. DNA ploidy was compared with the pathologic features of tumors. RESULTS: One hundred three individual cancers in 70 patients were used for DNA ploidy analysis. Of these, 39 (38%) were diploid and 64 (62%), nondiploid. DNA ploidy was weakly correlated with increase in tumor volume (P = 0.049) but not tumor grade. The relationship between DNA content and extraprostatic spread was not statistically significant. Increase in tumor volume and grade promoted extraprostatic spread more than nondiploidy, as shown by multivariate analysis. CONCLUSIONS: The incidence of a nondiploid cell population in a tumor focus in prostate cancer is essentially the same, at least for Americans and Japanese. DNA ploidy of a tumor cannot serve as a means of pathologic stage prediction in clinically resectable prostate cancers, contrary to many previous studies. Thus, DNA content analysis would be of no use for deciding on the treatment strategy of prostate cancer. PMID- 8638367 TI - Treatment of subclinical intraurethral human papilloma virus infection with interferon alfa-2b. AB - OBJECTIVES: Anogenital human papilloma virus (HPV) infection represents a growing concern among physicians in the United States. An intraurethral reservoir of the virus has been suggested as a possible source for reinfection between sexual partners, and may contribute to the increase in the number of affected individuals. Treatment reports of intraurethral HPV infection with adequate follow-up have been lacking. Our goals in this study were to identify the patients with cytologic evidence of HPV intraurethral infection, and to attempt treatment with intraurethral instillations of interferon alfa-2b. METHODS: Eighty nine men with anogenital lesions or known exposure to HPV underwent cytologic examination using a urethral swab after all visible disease was adequately treated. Sixteen patients with positive cytology results were treated with weekly instillations of 25 million U of interferon alfa-2b solution for 6 weeks. Urethral cytology was monitored at 2 and 6 weeks post-treatment, as well as every 3 months thereafter up to a year. Those who had a recurrence during the study were retreated with a 6-week course using 50 million U per instillation. Patients were monitored for possible side effects. RESULTS: Seventeen (19%) of 89 patients who entered the study had urethral cytology positive for HPV infection with no evidence of visible disease. Seven (41%) of these 17 patients did not show external (meatal or skin) manifestations of the disease. Fourteen of 16 (88%) men who underwent the therapy were followed for an average of 11.8 months. Nine of those 14 (64%) remained disease free throughout the follow-up. Of the 5 who had a recurrence, 3 were successfully retreated, with a mean of 7.2 months of disease free follow-up after the second course. No adverse effects of the treatment were noted by blood testing, semen analysis, and patient report. CONCLUSIONS: The urethra is a significant HPV reservoir and should be investigated in patients exposed to the virus. Interferon is a potentially safe and effective treatment option for intraurethral HPV. PMID- 8638368 TI - Operative trauma to the genitourinary tract in the treatment of anorectal malformations: 15 years' experience. AB - OBJECTIVES: To determine the incidence of urologic trauma during surgical correction of anorectal malformations (ARM). METHODS: The records of 228 children with ARM were retrospectively reviewed. RESULTS: Operative trauma to the genitourinary tract was seen in 12 patients, all male. Of 133 patients with a high ARM, 92 underwent an abdominoperineal pull-through and 41 underwent a posterior sagittal anorectoplasty (PSARP); the incidence of urologic trauma was 12% and 0%, respectively. In the subgroup of male infants with a high ARM (99 patients), the incidence of genitourinary trauma was 11%; in those with a low anomaly, the incidence was 1 of 43 (2%). There were 3 urethral tears, 4 urethral strictures, 5 urethral diverticula, 2 vas deferens injuries, and 1 ureteric injury. Nine of the 12 patients do not have any long-term sequelae resulting from the injuries; however, 2 are now infertile and 1 has urinary incontinence. Four patients are additionally compromised because of an absent or nonfunctioning kidney on one side. CONCLUSIONS: In children with ARM, those undergoing a conventional pull-through repair are more likely to have a genitourinary injury than those undergoing the PSARP, and boys with high ARM are at greatest risk. PMID- 8638370 TI - Cost-effective endoscopic examination of the contralateral inguinal ring. AB - OBJECTIVES: Laparoscopic examination of the contralateral inguinal ring has recently been advocated to exclude contralateral hernia in young children. We report a modification using nondisposable cystoscopic equipment, which is quick and reliable. METHODS: Either an 8 or 10 F soft straight catheter or a cystoscope sheath is passed through the open hernia sac. Air is insufflated into the abdomen with a syringe. Once the pneumoperitoneum is completed, a 4-mm cystoscope lens (110 degrees) is used to inspect the contralateral ring. RESULTS: Twenty-four children between the ages of 6 weeks and 4 years (median, 6 months) underwent exploration for presumed unilateral inguinal hernia and laparoscopic examination of the contralateral inguinal ring. Thirteen patients (54%) had an open processus vaginalis and underwent contralateral inguinal herniorrhaphy. No false-positive or false-negative results were found, and there were no complications associated with the procedure. CONCLUSIONS: This method of laparoscopic examination of the contralateral inguinal ring using nondisposable cystoscopic equipment is rapid, safe, reliable, and cost effective in evaluating a contralateral patent processus vaginalis. Laparoscopic examination spares the need for formal surgical exploration in patients with a closed processus vaginalis. PMID- 8638369 TI - Femoral nerve neuropathy after the psoas hitch procedure. AB - We report the first 3 cases of femoral nerve neuropathy after a psoas hitch vesicopexy, a technique commonly used with ureteroneocystostomy. The condition in 2 patients resolved with conservative therapy, and the third patient required reoperation with removal of an offending suture. All 3 patients recovered completely with no residual neurologic deficit. Urologists who use the psoas hitch must be familiar with this potential complication to prevent its occurrence. PMID- 8638371 TI - A unique perineal herniation of large bladder diverticulum: successful surgical repair through posterior sagittal approach. AB - We report a case of symptomatic perineal herniation of a massive posterior bladder diverticulum following abdominoperineal resection in a 75-year-old man with Crohn's disease and renal failure. Presentation, evaluation, and management issues are discussed. PMID- 8638372 TI - Lichen nitidus. PMID- 8638373 TI - Neurilemoma of the kidney. AB - Neurilemomas are neoplasms that originate from Schwann cells of the central and peripheral nervous systems. Thirteen case reports of neurilemomas involving the kidney have been described in the English literature. We report the fifth case of a neurilemoma within the renal sinus and review the previous cases of renal neurilemoma. This neoplasm is usually found incidentally. Presenting signs are nonspecific and radiographic features are highly variable. These factors make a preoperative diagnosis extremely difficult. Accurate identification relies heavily on detailed pathologic evaluation. Wide local excision is the treatment of choice, since malignancy is found in up to 30% of cases and there is a tendency for local recurrence and distant metastasis. PMID- 8638374 TI - Special considerations in endopyelotomy in a horseshoe kidney. AB - We report a patient with ureteropelvic junction obstruction in a horseshoe kidney who was successfully managed with an antegrade endopyelotomy. Follow-up at 1 year confirmed a good result. Special considerations are necessary for the successful performance of an endopyelotomy in a horseshoe kidney. Recommendations will be made regarding percutaneous access and orientation of the incision as well as a review of the literature. PMID- 8638375 TI - Traumatic pyeloduodenal fistula: a case report and review of the literature. AB - A case of a traumatic pyeloduodenal fistula secondary to a gunshot wound is reported. After initial conservative management failed, the fistula tract was surgically excised without removal of the involved kidney. Previous treatments of pyeloduodenal fistulas secondary to gunshot wounds have involved nephrectomy. We present the first case of a pyeloduodenal fistula secondary to gunshot wound managed without nephrectomy along with a review of the literature regarding traumatic pyeloduodenal fistula. PMID- 8638377 TI - Bilateral primary non-Hodgkin's lymphoma of the testis. AB - We report the case of a 72-year-old man with bilateral testicular masses that, on histologic section, were found to be synchronous non-Hodgkin's lymphomas. Workup was negative for systemic disease, indicating the possibility of bilateral primary testicular lymphomas. We discuss the evaluation and treatment of this lesion and review the literature concerning this subject. PMID- 8638376 TI - Conservative management of rectocutaneous fistula following radical perineal prostatectomy. AB - Inadvertent rectal injury during radical perineal prostatectomy may sometimes lead to the development of a rectocutaneous fistula. This has traditionally been managed with diverting colostomy until closure of the fistula is assured. We report 3 cases of rectocutaneous fistula following radical perineal prostatectomy, which were managed in a more conservative fashion. This included appropriate wound care, antibiotics, bowel rest, parenteral hyperalimentation, and, in the presence of a concomitant urine leak in 1 case, optimal urinary diversion and bed rest. All fistulas healed without incident. PMID- 8638378 TI - Strangulated urethral prolapse. AB - Urethral prolapse is rarely diagnosed. We describe the clinical findings in an 86 year-old woman. The treatment chosen was the excision of the urethral prolapse over an indwelling Foley catheter and suturing the mucosa edges to the vaginal skin. Etiology, differential diagnosis, and treatment modalities will be discussed. PMID- 8638379 TI - Chemotherapy for invasive bladder cancer. PMID- 8638380 TI - Should we treat localized prostate cancer? An opinion. PMID- 8638381 TI - Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene. AB - We have developed a combined polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for the detection and differentiation of Cryptosporidium parvum, Cryptosporidium muris, and Cryptosporidium baileyi. The assay utilizes PCR to amplify an approximately 1750 basepair product of the Cryptosporidium 18S rRNA gene. Following double digestion with restriction endonucleases Dral and Vsp1, this PCR product yields DNA fragments that can be resolved electrophoretically into RFLP profiles that are distinct for C. parvum, C. muris, and C. baileyi. Previous PCR-restriction analyses could not simultaneously differentiate all three species. Future application of this technique could include predicting the disease-causing potential of oocyst contaminated environmental specimens and helping to determine the source of oocyst contamination. PMID- 8638382 TI - Cytochemical study of the germinal membrane of the Echinococcus granulosus cyst. AB - The present cytochemical study was undertaken to provide more information on the localization of enzymatic and glycoconjugates in the germinal membrane of the Echinococcus granulosus cyst. The distinctive distribution of binding sites for two lectins (peanut agglutinin and Dilochos biflorus agglutinin) in the germinal membrane are described. An investigation is made of the distribution and specific activity of adenosine triphosphatase, alkaline phosphatase and acid phosphatase. The results suggest that cells located in the deeper layer of the germinal membrane are intrinsic in the cellular differentiation process. The dissimilarities detected in both the enzymatic activity and the lectin-binding receptors could be associated with metacestode development or degeneration. PMID- 8638383 TI - Response of cattle treated with a fenbendazole slow release bolus to challenge from nematodes the following season. AB - Nematode infection of cattle treated in their first year at pasture with the fenbendazole slow release bolus ('Bolus group') was compared during the second year with that of untreated cattle. Ostertagia was the most prevalent parasite associated with Cooperia. Except for the Dictyocaulus spp. which caused clinical signs of bronchitis in the 'Bolus' group, the infection during the second year resulted in a moderate response of the cattle whatever the group. Rises in both pepsinogen and gastrin levels were correlated with the number of Ostertagia L3 on herbage. Damages in the abomasal mucosa were more frequent and severe in the 'Bolus' group where more inflammatory signs were observed in spite of a smaller number of worms. Nevertheless, the differences in total weight gains were not significant thanks to a compensatory effect during the second part of the grazing season in the 'Bolus' group. Hypotheses related to a minimum threshold of infection during the first year necessary to develop high enough protection during the second year are discussed. The pathological effects of gastrointestinal nematodes seem to vary more according to the inflammatory response than to the number of worms. PMID- 8638384 TI - Development of immunity to lungworm in vaccinated calves treated with an ivermectin sustained release bolus or an oxfendazole pulse release bolus at turnout. AB - The ivermectin sustained release bolus (IVSRB), when used at turnout as recommended, will provide season-long control of parasitic bronchitis, thus obviating the need for use of a lungworm vaccine. However, some concerns have been expressed that calves treated with an IVSRB will receive so little exposure to Dictyocaulus viviparus that it will compromise their immunity in subsequent grazing seasons, which would be of particular importance in dairy herds. Although there is evidence that IVSRB-treated calves can develop immunity to D. viviparus when exposed to pasture infection, it was considered worthwhile to evaluate the compatibility of the IVSRB and lungworm vaccination to allow veterinary surgeons the option of using these products concurrently when they have particular concerns about the long term immune status of replacement dairy heifers. Thirty two dairy replacement heifers were vaccinated with two doses of lungworm vaccine and, at turnout, half the calves received an IVSRB and the remainder an oxfendazole pulse release bolus (OPRB). At the end of the grazing season four replicate bolus treated pairs and four parasite-naive calves were challenged with 1000 D. viviparus infective larvae. At slaughter there was a 95% and 93% reduction in D. viviparus burdens in the IVSRB and OPRB treated calves respectively, compared with the unvaccinated, untreated controls. These results indicate that where it is considered necessary to use lungworm vaccination in addition to an IVSRB or an OPRB, the compatibility of these products with lungworm vaccine will allow development of a protective level of immunity to D. viviparus. PMID- 8638385 TI - Experimental trichinellosis in goats. AB - The susceptibility and distribution of Trichinella spiralis infection in goats were examined in ten autochthonous kids, 2 months old and about 10 kg body weight. The animals were divided into two groups: one experimental group with eight animals, infected with 10,000 T. spiralis 'T1' encysted larvae and a control group with two non-infected animals. All the animals of the experimental group infected by the parasite showed that Trichinella larvae have a special affinity for the tongue, masseters, diaphragm, flexor-extensor muscles, intercostal muscles and myocardium in decreasing order. The ELISA test carried out showed the first increments of optical density (OD) on Day 16 postinfection (p.i), peaking on Days 37-44 p.i. and remaining elevated from this day on, with a slight fall at the end of the experiment (Day 90 p.i.). No alterations were observed in the OD obtained in control animals throughout the experiment. The great muscular establishment of T. spiralis larvae and the sigmoidal evolution of antibody levels confirm the host character of the goat to the parasite. PMID- 8638386 TI - The development of protective immunity in canine scabies. AB - Seven of eight dogs that had been previously infested with Sarcoptes scabiei var. canis and then cured, expressed protective immunity when experimentally reinfested with scabies. All seven dogs that expressed resistance were spontaneously cleared of scabies by 64 days after they were experimentally reinfested. Five of the eight dogs were free of scabies by 24 days. The sequential changes in the inflammatory/immune cellular infiltrate in the scabietic lesions of each dog were determined during the sensitizing infestation, cure and the subsequent experimental reinfestation (challenge). During the initial infestation and in the subsequent challenge reinfestation, dogs developed mixed cellular infiltrates in their scabietic lesions that contained mononuclear cells, neutrophils, plasma cells and mast cells. Reinfestation induced more rapid increases in the densities of these cells than had occurred during the sensitizing infestation. Mononuclear and mast cells were the most numerous infiltrating cells during the sensitizing phase. During the challenge phase the most numerous infiltrating cells were mononuclear cells and neutrophils. The sensitizing and challenge infestations induced circulating scabies-specific antibody responses, but the response was more rapid during the reinfestation challenge. Both the cell-mediated response in the skin and the circulating antibody response waned in parallel with clearing of the mites following reinfestation. PMID- 8638387 TI - Evaluation of the monitoring of papular dermatitis lesions in slaughtered swine to assess sarcoptic mite infestation. AB - We investigated the association between the presence of papular dermatitis and sarcoptic mite infestation in pigs slaughtered in southern Minnesota. Following dehairing, a sample of 30 pigs from each of 50 herds was inspected for papular dermatitis lesions. Herds were selected after being categorized into one of five categories according to the prevalence and severity of dermatitis lesions. Herd infestation with sarcoptic mange was determined by recovery of mites from ear scrapings of slaughtered pigs and also by survey of producer opinion. Mite infestation was detected in 28 herds (56%) and 215 of 1500 pigs (14%). Considerable variability in prevalence of positive scrapings, ranging from one pig (3%) to 19 pigs (63%), was found among infested herds. Prevalence of mite infestation was positively associated with severity of papular dermatitis lesions in groups. For individual pigs, estimates of the specificity of localized lesions ranged from 0.70 to 0.90. Generalized lesions appear highly specific (> 0.98) for sarcoptic mite hypersensitivity. Generalized lesions occurred in 36.7% of pigs from herds confirmed to be infested, compared with 0.4% of pigs in herds confirmed free from mange. Our date indicate that monitoring of dermatitis lesions in slaughtered pigs might be a useful test for sarcoptic mange in the Midwest, USA. PMID- 8638388 TI - Functional bovine immunoglobulins in Boophilus microplus hemolymph. AB - The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. The knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle. PMID- 8638389 TI - Western blot analysis of tick antigens from a Rhipicephalus sanguineus unfed larval extract and identification of antigenic sites in tick sections using immunohistochemistry. A comparative study between resistant and susceptible host species. AB - Most parasite-host relationships are characterized by the development of resistance by the host, thus limiting the number of parasites. However, some cases are very unusual. In the relationship of the domestic dog with the brown dog-tick Rhipicephalus sanguineus this does not occur, whereas guinea pigs develop efficient resistance. Sera from domestic dogs, crab-eating foxes and guinea pigs collected before and after infestation with R. sanguineus ticks, and after immunization with a whole tick adult or larval homogenate, were used in Western blot analysis to compare and identify potential important antigens from a tick larval homogenate. The same sera were tested in an indirect immunohistochemistry assay in an attempt to compare relevant antigenic sites on histological tick sections. The immunoblotting displayed antigens recognized only by the guinea pigs, as well as several shared antigens between host species, depending on the king of immunization. Immunohistochemistry revealed probable antigenic sites on the cells and tissues of ticks, which varied depending on the kind of immunization (infestation or vaccination) and the animal species involved. PMID- 8638390 TI - A repetitive DNA sequence specific for Trypanosoma (Nannomonas) godfreyi. AB - The satellite DNA sequence of Trypanosoma (Nannomonas) godfreyi, a recently described parasite of Suidae, was determined. The sequence is 373bp in length, and contains two imperfect internal repeats of approximately 170bp. Like other trypanosome satellite DNAs, it has no extensive open reading frames and is probably non-coding. There is no significant homology with other major repetitive DNAs within subgenus Nannomonas. We have developed a PCR test that is specific for T. godfreyi and used it to identify the parasite in natural tsetse infections from Zimbabwe and Cote d'Ivoire. This test shows no cross reaction with non target trypanosomes, even within subgenus Nannomonas, and will be invaluable in studies of the prevalence and distribution of T. godfreyi. PMID- 8638391 TI - In vitro responsiveness of Babesia bovis to imidocarb dipropionate and the selection of a drug-adapted line. AB - A South African stock of Babesia bovis was successfully resuscitated from liquid nitrogen, and cultured in microaerophilous stationary phase. The in vitro susceptibility of the B. bovis stock to titrated concentrations of imidocarb dipropionate was observed and the 50% inhibitory concentration (IC) was determined (8.7 x 10(7) g ml-1). A drug-adapted line was developed by culture in the presence of sub-inhibitory concentrations of imidocarb dipropionate and it had an IC50 eight times higher than that of its original stock (6.6 x 10(-6) g ml 1). The drug-adapted line was cryopreserved and resuscitated from liquid nitrogen. Continuous culture of the non-drug adapted line through 15 subcultures did not change the IC50 (8.3 x 10(-7) g ml-1). PMID- 8638392 TI - Trypanocidal resistance in Trypanosoma evansi in vitro: effects of verapamil, cyproheptidine, desipramine and chlorpromazine alone and in combination with trypanocides. AB - A study was conducted in vitro to assess the ability of calcium antagonists to reverse trypanocidal resistance in Trypanosoma evansi. Susceptibility patterns of sensitive and resistant parasites were evaluated against calcium antagonists of several chemical classes (verapamil, cyproheptidine, desipramine and chlopromazine), alone and in combination with suramin, diminazene aceturate or melarsen oxide cyteamine. The putative resistance modulators were intrinsically antitrypanosomal, but were unable to reverse resistance to any of the trypanocides tested. It was thus concluded that resistance to these trypanocides in T. evansi may differ from drug resistance mechanisms occurring in cancer cells, malaria or in South American trypanosomosis, where calcium antagonists have successfully reversed resistance. PMID- 8638393 TI - Observations on the use of ELISA for detection of Babesia bigemina specific antibodies. AB - An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated to study the cause of the high level of background reactions which hinders the application of ELISA as a field diagnostic test for Babesia bigemina. Different blockers to improve the specificity of the ELISA were compared. THe use of soya milk (25%), gelatin (2.5%) and chicken serum (2%) did not significantly improve the specificity of the test. It was noted that the presence of fibrinogen contributed to the positive ELISA results more than the presence of B. bigemina specific antigen. This conclusion was confirmed by testing bovine fibrinogen as a host protein antigen in ELISA which strongly responded against B. bigemina positive control sera. It is suggested that application of ELISA for B. bigemina is still unreliable until a more purified Babesia-specific antigen or specific monoclonal antibodies are available. PMID- 8638394 TI - Distribution of potential intermediate hosts for Fasciola hepatica and Fascioloides magna in Montana, USA. AB - A collection of lymnaeid snails in Montana was made over a 3 year period, in conjunction with a state-wide survey of the distribution of liver flukes in Montana. Collection areas were selected based on reports of infected cattle, sheep or wildlife, and with the intent of covering all geographic regions of the state. Snails were found at all 97 of the locations chosen for collections, with lymnaeids collected at 71 of the locations. The 97 sites were located in 28 of Montana's 56 counties. Nine lymnaeid species were collected, five of which have been reported either as natural or experimental intermediate hosts for Fasciola hepatica or Fascioloides magna. The two snail species most widely distributed over the areas enzootic for the flukes were Lymnaea modicella and Lymanaea caperata. PMID- 8638395 TI - Immune responses of chronically infected adult cattle to Fasciola hepatica. AB - Eight adult cows, with an existing chronic Fasciola hepatica infection, were experimentally infected with 1300 metacercariae of F. hepatica, given as trickle infections, over two separate 10-day periods. Two fluke-naive heifers were similarly treated. Analysis of parasite-specific immunoglobulin isotypes IgM, IgG1, IgG2 and IgA showed IgG1 to be the dominant isotype in both chronically infected and previously naive animals. Lymphocyte proliferation assays demonstrated (a) an association between lymphocyte response and mature fluke burden in the chronically infected cattle and (b) no association between lymphocyte response and mature or immature fluke burden in naive heifers. There was no production of gamma-interferon (IFN gamma) by lymphocytes responding to adult fluke antigen. At post-mortem examination the burden of immature flukes in chronically infected and previously naive heifers was similar. This suggests that chronically infected animals may be tolerant to a moderate superinfection and that the prevailing immune mechanism in operation may be a non-protective response generated by the Th2 lymphocyte subset. PMID- 8638396 TI - Effects of praziquantel on experimental Schistosoma bovis infection in goats. AB - The effect of praziquantel against experimental Schistosoma bovis infection in West African Dwarf goats was investigated. Thirty goats were exposed to 2000 cercariae each and 15 of those received a praziquantel treatment (60 mg kg-1) 13 weeks post-infection. One day, 1 week and 4 weeks post-treatment representative goats from each group were killed and worms were recovered by perfusion. For comparison, parasite-free control animals were monitored, some of which were given praziquantel. Every second week during the study, faecal samples were collected. The cure rate was 100% 1 day, 99.4% 1 week and 95.7% 4 weeks post treatment. Tissue egg counts were significantly reduced (P < 0.001) 4 weeks post treatment in all parts of the intestines, but not in the liver. Faecal egg counts were reduced by 84.1% 1 week and by 98.3% 3 weeks after treatment, the reduction being highly significant both 1 week 3 weeks after treatment (P < 0.001). Overall strong correlations between the number of worm pairs, tissue egg counts and the final faecal egg count were observed, indicating that the faecal egg counts during infection and following treatment can be used as a guideline for the pathology associated with the infection. PMID- 8638397 TI - Use of genomic DNA probes for the diagnosis of acute sarcocystosis in experimentally infected cattle. AB - Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10. These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples. Of five calves in the experiment, four were each orally dosed with approximately 200,000 S. cruzi sporocysts; one calf served as non-infected control. Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma. Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with [alpha-32P]dATP. The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25-39 in the granulocyte fraction. Parasitemia (i.e. merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy, Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner. As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S.cruzi; however this hypothesis requires further study. PMID- 8638398 TI - Experimental ovine fasciolosis: antipyrine clearance as indicator of liver damage. AB - Antipyrine clearance was used to assess microsomal oxidative function in eight female Churra breed sheep at 20, 30, 40, 60, 80 and 100 days after infection by an oral administration of 150 metacercariae of Fasciola hepatica. Experimental infection was ascertained by an ELISA test and by faecal analysis. A significant increase in plasma glutamate dehydrogenase (GLDH) activity from 20 days post infection and in gamma-glutamyltransferase (GGT) activity from 40 days post infection was found. Both enzyme activities reached maximum levels in plasma of infected sheep at 80 days post-infection, progressively decreasing thereafter. A significant reduction in the total plasma clearance of antipyrine occurred from 60 to 100 days post-infection and a significant increase in mean residence time occurred by 80 days post-infection. The decrease of antipyrine metabolism coincided with the entrance of parasites in bile ducts and the highest liver damage caused by migrating juvenile flukes. PMID- 8638399 TI - When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. AB - Double-stranded RNA is a potent inducer of interferon, a modulator of the expression of a number of other genes involved in the response of cells to virus infection, an activator of the interferon-induced antiviral state, and may be involved in differentiation, induction of apoptosis, and control of oncogenic transformation. This review will attempt to summarize what is known about the cellular proteins that act to mediate the response of cells to double-stranded RNA and the viral and cellular macromolecules that may be able to modulate these responses. PMID- 8638400 TI - Homologous and heterologous complementation of HBV and WHV capsid and polymerase functions in RNA encapsidation. AB - Successful encapsidation of hepadnaviral pregenomic RNA requires the orchestrated interaction of the viral capsid and polymerase proteins with each other and with the RNA packaging substrate. The early steps of this process involve binding of the polymerase to the encapsidation signal, epsilon, and are already understood in some detail. However, the underlying macromolecular interactions resulting in the subsequent encapsidation of this polymerase-epsilon complex by capsid proteins are less clearly defined. To approach this issue we have examined the ability of two different hepadnaviruses to encapsidate each other's pregenomic RNA. H. Okamoto et al. ((1990) J. Gen. Virol. 71, 959-963) have previously demonstrated that WHV polymerase could encapsidate an HBV pregenome, but the origin of the capsid proteins (i.e., HBV- or WHV-derived) required for this reaction was not clear; some evidence suggested that heterologous capsid and polymerase proteins might not be capable of interaction. To clarify this, we analyzed encapsidated RNA isolated from cytoplasmic cores produced following transient transfection of HepG2 cells with different combinations of plasmids encoding HBV or WHV core and polymerase genes. We found that (i) the essential encapsidation signal of WHV is comprised of a short region including epsilon, as in HBV; (ii) HBV and WHV polymerases are each competent to recognize both HBV and WHV packaging signals; therefore the encapsidation signals are functionally interchangeable; and (iii) HBV capsids encapsidate a WHV polymerase-epsilon complex, and vice versa, although the efficiency of heterologous packaging is slightly lower than that of homologous encapsidation. Our results underscore the close relationship of these two mammalian hepadnaviruses. PMID- 8638401 TI - Importance of localized skin infection in tick-borne encephalitis virus transmission. AB - Arboviruses are transmitted to vertebrates by the "bite" of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels > or = 2.0 log10 ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission. PMID- 8638402 TI - Lack of dichotomy between virus load of peripheral blood and lymph nodes during long-term simian immunodeficiency virus infection of African green monkeys. AB - During the asymptomatic phase of human immunodeficiency virus 1 (HIV-1) infection the lymphatic tissues seem to function as a major reservoir of HIV. We have examined the viral load in peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) of 12 naturally and 4 experimentally long-term simian Immunodeficiency virus (SIV)-infected African green monkeys (AGM) to help explain the apathogenicity of the AGM isolates of SIV (SIVagm) in their natural host. The mean number of SIVagm producing cells determined by limiting dilution assay was found to be 1.7 +/- 2.2 and 2.1 +/- 3.3 per 10(5) PBMC or LNMC, respectively. Similarly, polymerase chain reaction analysis of serially diluted cells showed the mean provirus carrying cell number to be 2.8 +/- 3.7 per 10(5) PBMC and 4.0 +/- 5.5 per 10(5) LNMC. When normalized for CD4+ cells the provirus and infectious virus loads in the LNMC and PBMC were also similar. No trapping of virus particles could be detected by in situ hybridization or immunohistochemistry. The data demonstrate that in contrast to HIV-1-infected humans, the viral burden in the lymph nodes of long-term SIV(agm)-infected AGMs is comparable to that in the PBMC. PMID- 8638403 TI - Stabilization of vesicular stomatitis virus L polymerase protein by P protein binding: a small deletion in the C-terminal domain of L abrogates binding. AB - We showed previously that cells expressing the vesicular stomatitis virus (VSV) L polymerase gene via the vaccinia-T7 RNA polymerase system accumulated 2- to 5 fold more L protein when the P protein was coexpressed (Canter et al., 1993, Virology 194, 518-529). The results presented here provide an explanation for this phenomenon. Pulse-chase analysis revealed that L was unstable with a half life of 3 to 6 hr if expressed in the absence of P protein, but was stable for at least 16 hr when coexpressed with a 10- to 15-fold molar excess of P. The P protein, in contrast, was stable under both conditions. Stabilization correlated with formation of a P:L polymerase complex evidenced both by coimmunoprecipitation and by glycerol gradient sedimentation analyses. A mutant L protein, lacking amino acids 1638 to 1673, was not stabilized by coexpression and showed no binding to P protein. Its anomalous sedimentation, however, suggested misfolding and/or aggregation as the cause for the failure to bind P. Transcription reconstitution in vitro, using extracts from cells expressing excess of P over L protein, strongly depended on coexpression of the proteins for optimal activity. We propose that the coexpression dependence for polymerase reconstitution documented here for VSV, as well as that reported previously for the Sendai paramyxovirus, reflects the protective effect of P protein on L protein stability. PMID- 8638404 TI - Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin. AB - The genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated by Agrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV, Micrutalis malleifera (Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whitefly Bemisia tabaci Genn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus. PMID- 8638405 TI - Characterization of DNA-binding proteins and protein kinase activities in Chlorella virus CVK2. AB - Several capsid proteins were selectively released from the viral core of Chlorella virus CVK2 by treatment with 4M urea. Among the viral core proteins, seven species (Vp154, Vp73, Vp63, Vp52, Vp48, Vp42, and Vp25) were shown to have DNA-binding activities by Southwestern blot analysis. Except for Vp154 and Vp25, these DNA-binding proteins showed a specific affinity for the viral genomic DNA. The viral core also contained three proteins with protein kinase activity (Vp73, Vp60, and Vp37); Vp73 seemed to have both DNA-binding and protein kinase activities. Antisera raised against Vp73 were used to screen a lambda-CVK2 expression library for the gene encoding Vp73. Three different clones (Vp73-3, Vp73-29, and Vp73-42) were obtained and analyzed. ORFs found in these clones all contained characteristic proline-rich motifs. The Vp73-42 ORF showed a strong similarity with histone H1 of various organisms and the Vp73-29 ORF contained two regions with leucine-zipper motifs. All three genes were expressed late in infection. PMID- 8638406 TI - Natural variation in HIV-1 protease, Gag p7 and p6, and protease cleavage sites within gag/pol polyproteins: amino acid substitutions in the absence of protease inhibitors in mothers and children infected by human immunodeficiency virus type 1. AB - Reduced sensitivity of human immunodeficiency virus type 1 (HIV-1) to protease inhibitors is associated with multiple amino acid substitutions in the virus encoded protease. The combination of changes that contribute to drug resistance is dependent in part upon the amino acid residues comprising protease alleles prior to drug therapy. We analyzed within peripheral blood mononuclear cells from HIV-1-infected mothers and their children viral gag/pol regions, which included p7, transframe p6/p6*, and protease coding sequences, as well as six protease cleavage sites. Sixty protease alleles from 12 individuals differed by at least 3 to as many as 10 amino acids from proteases encoded by molecular clones of HIV-1, indicating that there is no prototype or consensus wild-type HIV-1 protease sequence. Protease variants with a proline at position 63, a substitution associated with resistance to protease inhibitors, appeared in the absence of antiprotease therapy in 7 patients and were transmitted by 2 mothers to their infants. Gag p7 p6 regions were significantly more variable than protease. The p6/p6* region contained length variants and amino acid repeats in both reading frames. Five protease cleavage sites (B, D', D, E, and F) contained highly conserved amino acid sequences in individuals infected by epidemiologically distinct viruses. In contrast, C cleavage sites, localized between Gag p2 and Gag p7, displayed considerable amino acid variability, were unique among groups of infected individuals, and appeared to be related to particular protease alleles. Genetic variability in vivo in protease, in cleavage sites, and in proteins upstream of protease provides the potential to modulate enzyme activity and susceptibility to protease inhibitors. PMID- 8638407 TI - Multiple virus genes involved in the nematode transmission of pea early browning virus. AB - Mutations were introduced into four genes encoded by RNA2 of pea early browning virus (PEBV) to determine their possible involvement in the transmission of this virus by nematodes. Deletion of 28 amino acids from the C-terminus of the coat protein abolished the formation of virus particles. Deletion of 15 amino acids at the C-terminus did not affect particle formation but did abolish nematode transmission. In contrast, deletion of 13 amino acids immediately preceding the 16 C-terminal residues did not affect particle formation and decreased rather than abolished nematode transmission. A deletion in the gene encoding a 29-kDa protein and a frameshift mutation in the gene encoding a 23-kDa protein both abolished transmission without affecting virus particle formation. Mutations in an ORF encoding a 9-kDa protein, which is located on the genome between the coat protein gene and 29K gene, gave conflicting results. Removal of the AUG initiation codon from the 9K ORF had no effect on transmission, whereas the introduction of a frameshift mutation, which would prematurely terminate expression of the putative 9-kDa protein, decreased the frequency of transmission. The results show that the coat protein and probably all three of the other RNA2-encoded proteins play a role in the transmission of PEBV by vector nematodes. PMID- 8638408 TI - Differential effects of intronic and exonic locations of the human immunodeficiency virus type 1 Rev-responsive element. AB - The influence of the location of the Rev-response element (RRE) on human immunodeficiency virus type 1 (HIV-1) protein and RNA expression in COS cells was assessed. The RRE was placed into nef where it would be present in all HIV-1 RNAs. At this location, Gag and Env proteins were produced and the unspliced gag/pol and partially spliced env/vpu RNAs were able to accumulate in the cytoplasm. The RRE was also relocated from its normal location in the env exon to the env intron. In this way, the RRE would be present in the nuclear env pre mRNA, but not in the spliced env mRNA. Gag, but not Env protein production was detected. Th presence of the RRE in the env pre-mRNA allowed the cytoplasmic accumulation of the spliced env mRNA, which lacked the RRE. However, this mRNA accumulated at a reduced level relative to that produced by constructs containing the RRE within the env mRNA. The cytoplasmic accumulation of this mRNA was dependent on the presence of Rev and the RRE. These results demonstrate that the location of the RRE can have differential effects on the fate of HIV-1 RNAs. PMID- 8638409 TI - Mutational analysis of the bacteriophage P4 capsid-size-determining gene. AB - Satellite phage P4 (11,624 bp) depends on the morphopoietic genes (capsid, tail) and lysis genes of its helper phage P2 (33.5 kb) for its lytic development. In the morphopoietic process, P4 redirects the assembly pathway of large, P2 size, capsids (diameter = 60 nm) to yield smaller, P4 size, capsids (diameter = 45 nm), 1/3 in volume of that of its helper. The P4-specified capsid size determination is dependent on the function of the 27-kDa gpSid. To study the capsid size determining function, we carried out a mutational analysis of the P4 sid gene. Use of a P4-derived genome of 29.1 kb (P461), which can be packaged only into large, P2 size, capsids allowed us to select P4 Sid- mutants. By DNA sequencing we characterized 25 P4 Sid- mutants, of which 10 contain base pair substitutions and 15 contain deletions. Both types of mutations are clustered in separate locations within the sid gene. Our results suggest that the Sid polypeptide contains three distinct functional domains. PMID- 8638410 TI - Bacteriophage P4 capsid-size determination and its relationship to P2 helper interference. AB - The sid (size determination) gene product of phage P4 is known to be involved in capsid-size determination. Moreover, the capsid-size determination function interferes with the lytic development of its helper P2, presumably because the helper genome is too large to be packaged into P4-size capsids. In order to study P4-specified helper interference, we cloned the sid gene for expression during phage infection. Even though gpSid restores the capsid-size determination function of a sid defective P4 mutant, we find that gpSid alone is not sufficient to establish full interference of helper P2 phage production. Complete helper interference requires some P4 function in addition to gpSid. Complementation tests show that none of the known P4 genes display this property. We propose that P4 encodes a yet-unidentified function that in concert with gpSid establishes full P2 helper interference at the level of capsid-size determination. PMID- 8638411 TI - Persistence of vesicular stomatitis virus New Jersey RNA in convalescent hamsters. AB - Persistence of vesicular stomatitis virus New Jersey (VSV-NJ) was studied in experimentally infected hamsters (Mesocricetus auratus). We used reverse transcription and nested polymerase chain reaction (RT-NPCR) to probe tissues of hamsters inoculated with VSV-NJ Hazelhurst. Viral genomic RNA was detected in the brain, cerebellum, spleen, liver, kidney, and lung 2 months after infection, but only in the central nervous system at 10 and 12 months. Viral messenger RNA was detected in the brain of one hamster at 2 months after infection. Replicative intermediate was detected in the spinal cord of one hamster at 12 months. These results suggest that VSV-RNA persists in animals for long periods following infection, disease, and convalescence. However, infectious virus was not recovered from tissues by conventional serial passages of tissue extracts in Vero cells or by cocultivation. PMID- 8638412 TI - Rod-like shape of vesicular stomatitis virus matrix protein. AB - The shape of purified matrix protein (M) of vesicular stomatitis virus was determined using biophysical techniques like analytical centrifugation, dynamic light scattering, and small-angle neutron scattering. The data obtained are consistent with a rod-like model for M protein with a length of about 100 +/- 10 A and a radius of 9 +/- 1 A. These dimensions are in agreement with the substructure of M protein aggregates and with the fine morphology of the axial channel material found inside the viral nucleocapsid coil. This morphological information was combined with CD measurements and secondary structure predictions on four vesiculovirus M proteins leading to a proposal for the structure of M protein. PMID- 8638413 TI - Novel NTP binding property of rice dwarf phytoreovirus minor core protein P5. AB - Rice dwarf phytoreovirus (RDV) mRNA synthesized from purified virion has a cap structure, m7GpppAm-, which suggests the presence of guanylyltransferase activity in the virion. We attempted to identify the enzyme involved in the cap formation by using a nucleoside triphosphate binding assay. Incubation of virion with [alpha-32P]GTP resulted in labeling of an 89-kDa protein that had not previously been identified in purified virus preparations. Interestingly this protein also covalently bound UTP and ATP, which is not a property of the known guanylytransferases. RDV particles catalyzed GTP-PPi, dGTP-PPi, ATP-PPi, and UTP PPi exchange reactions. In SDS-polyacrylamide gel electrophoresis, the 89-kDa protein comigrated with the S5-coded protein, P5, which had been expressed by a baculovirus vector. Moreover, the labeled 89-kDa protein was precipitated by an antiserum against this recombinant RDV P5. Careful reinvestigation of purified virus particles by SDS-polyacrylamide gel electrophoresis and Western blotting analyses showed that they contained a small amount of P5 (<0.5% of the total protein) within the core. These results may suggest that the minor core protein of RDV, which is coded by S5, is a candidate guanylyltransferase, although the biological significance of its ATP and UTP binding activities remains largely unknown. PMID- 8638414 TI - Murine gammaherpesvirus-68 encodes homologues of thymidine kinase and glycoprotein H: sequence, expression, and characterization of pyrimidine kinase activity. AB - We have sequenced a 4.5-kb fragment of DNA spanning the junction of the BamHI D and E fragments of murine gammaherpesvirus-68 (MHV-68). This sequence was found to code for two major open reading frames (orfs) of 1934 and 2192 bp which showed significant homology to the thymidine kinase (TK) and glycoprotein H (gH) sequences of other gammaherpesviruses. Upstream from the TK gene another orf was found which showed amino acid sequence homology to the HSV1 UL24 gene. Analysis of the 1934-bp orf revealed the presence of all six of the recognized sites that are conserved between herpesvirus TKs although, uniquely among sequenced herpesvirus TK enzymes, MHV-68 lacks the consensus nucleotide binding site GXXGXGK, the second glycine being replaced by alanine. The MHV-68 TK has a predicted M(r) of 68,443, while the gH is predicted to have a M(r) of 82,890. Northern blot analysis showed an early TK message of 2.6 kb and a late gH specific message of 2.5 kb. Both TK and gH probes detected a 4.3-kb late message, implying that this late message spans gH and TK. The TK coding sequence was expressed using an in vitro transcription translation system and was shown to encode functional TK activity. PMID- 8638415 TI - Vesicular stomatitis New Jersey virus RNA persists in cattle following convalescence. AB - To test the hypothesis that vesicular stomatitis New Jersey virus (VSV-NJ) persists in convalescent cattle, we used explant cultures and reverse transcription nested polymerase chain reactions to probe for viral genomic, replicative intermediate, and mRNA in two cows experimentally inoculated in the tongue 5 months earlier and three cows naturally infected 4-14 months previously. Virus was not isolated from any tissues of any animal. Sequences of the viral polymerase and nucleocapsid genes were consistently identified in the tongue and lymph nodes draining the tongue of both experimentally infected animals but not in the three naturally infected animals. Replicative intermediate but not messenger RNA sequences were detected. These results showed for the first time the long term persistence of VSV-NJ RNA in its bovine host. PMID- 8638416 TI - Restriction of HIV-1 (subtype B) replication at the entry step in rhesus macaque cells. AB - Human immunodeficiency virus type 1 (HIV-1) is restricted for replication in rhesus macaque cells and does not establish infection in this species. The block to productive infection of macaque peripheral blood mononuclear cells (PBMC) in culture was investigated. A chimeric virus SHIV containing HIV-1 tat, rev, and env and all other genes from a simian immunodeficiency virus clone pathogenic in macaques (i.e., SIVmec239) replicated efficiently in macaque PBMC. Thus, the attachment step, involving interaction of the HIV-1 env glycoprotein with the cell surface CD4 receptor, is not blocked. Analysis of uptake of HIV-1 particles in these cells revealed a small reduction in virion entry; however, viral DNA synthesis, measured by PCR amplification, was greatly reduced. Taken together, these results indicate that the block to HIV-1 (subtype B) replication in rhesus macaque cells involves release of the virion core into the cytoplasm and/or a step immediately prior to initiation of reverse transcription. Further studies are required to characterize this block through identification of species specific cellular proteins that interact with HIV-1 proteins in the early phase of viral replication. PMID- 8638417 TI - Recruitment during infectious mononucleosis of CD3+CD4+CD8+ virus-specific cytotoxic T cells which recognise Epstein-Barr virus lytic antigen BHRF1. AB - Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis (IM) which is a common sequel to primary EBV infection. Thereafter, the virus is maintained as a lifetime latent infection. Although the proteins expressed during the latent EBV infection provide a rich source of immunogenic epitopes, very little is known about cytotoxic T lymphocyte (CTL) control of primary EBV infection. The present report is based on an analysis of CTL clones derived from a patient suffering from acute IM. An intriguing feature of six CTL clones that displayed an HLA-restricted pattern of cell lysis was their initial coexpression of the T cell markers CD3, CD4, and CD8. Detailed analysis of one of these clones, which was restricted through the class II MHC antigen DR2, revealed reactivity with an epitope within the EBV lytic cycle early antigen, BHRF-1, which corresponds to the C-terminal region of the protein (AGLTLSLLVICSYLFISRG) (residues 171-189). There have been no previously published reports describing a CTL response during acute IM directed against an EBV lytic antigen. Interestingly, the coexpression of CD4 and CD8 by these CTLs during acute IM suggests that CD3+CD4+CD8+ cortical thymocytic precursor cells are recruited in order to overcome the EBV infection. PMID- 8638418 TI - Clean hand--dirty hand. PMID- 8638419 TI - Discovering life quality in coping with dementia. AB - Using qualitative techniques, data were obtained from 20 informants, comprising 10 caregiving dyads--that is, the caregiver and the spouse diagnosed with possible or probable Alzheimer's disease in the mild to moderate stages. Processes were identified in caregiving reactions and interactions during implementation of a cognitive remediation intervention designed to disrupt the trajectory of decline in dementia. Of interest was the impact of the implementation process on both the Alzheimer-diagnosed care recipient and the caregiver, as individuals and as a dyad. A four-phase process model of discovering life quality was delineated through analysis of log recordings, interviews and observational notes, and written evaluative responses of the families. The phases that emerged from the data address movement from an initial state of depleted interaction to a regaining of familial life quality. PMID- 8638420 TI - The moderating effect of age on self-care. AB - The study examined the moderating effect of age on the relations between symptom severity, self-care and others' care, patients' perception of the extent of problem solution, their satisfaction with the solution, and their perception of control over their health. Participating in the study were 121 female and 167 male chronically ill patients. Results indicated that age did play a major role in the relations between self-care and others' care and self-care outcomes. The young population sought maximum relief of symptoms with minimum effort from the self. The elderly, in contrast, could be said fo maximize control because what was important for them was not to depend on others but to be self-sufficient in solving their health problems. PMID- 8638421 TI - Family members' experiences living with members with depression. AB - Using interview data from 11 family members and grounded theory methods, this study describes family members' experiences in living with a member with depression. Findings suggest that this process can be described as family transformations. In the first stage of this process--acknowledging the strangers within--family members described observing the metamorphosis of the person and other family members, finding socially acceptable explanations, living two lives, searching for reasons and solutions, and hoping for what was. In the second stage -fighting the battle--family members alternated between the strategies of holding our ground (protective) and of moving forward (coercive) to counteract the metamorphosis, and the strategy of working the system to get help for their ill member. In the third and final stage, family members described gaining a new perspective and identified preserving oneself, refocusing on others, redesigning the relationship, and becoming hopeful as strategies used in this stage. PMID- 8638422 TI - The meaning of relocation among elderly religious sisters. AB - The meaning of involuntary, intra-institutional relocation (a move in primary living space) was constructed in this qualitative study through semistructured interviews with 12 elderly religious sisters. A thematic analysis of the data elicited from audiotape-recorded interviews was conducted. Overall, themes emerging from the sisters' discussion of the relocation included relocation as preparation for the future (to ensure their care in advancing years) and possibilities and consequences (resulting in improved living conditions together with sacrifice, uprooting, isolation, and distancing). Although relocation was seen as a largely positive event, it also had negative meaning for the subjects. PMID- 8638423 TI - Assuring quality in narrative analysis. AB - Many nurse-researchers using qualitative strategies have been concerned with assuring quality in their work. The early literature reveals that the concepts of validity and reliability, as understood from the positivist perspective, are somehow inappropriate and inadequate when applied to interpretive research. More recent literature suggests that because of the positivist and interpretive paradigms are epistemologically divergent, the transfer of quality criteria from one perspective to the other is not automatic or even reasonable. The purpose of this article, therefore, is to clarify what the terms quality, trustworthiness, credibility, authenticity, and goodness mean in qualitative research findings. The process of assuring quality, validation, in qualitative research will be discussed within the context of the interpretive method, narrative analysis. A brief review of quality in narrative analysis nursing research will also be presented. PMID- 8638424 TI - Patient education materials nurses use in community health. AB - This study examined the reading level of patient education materials commonly used by nurses in a local health department, a community health center, and a home health care agency, and it also examined the cultural and ethnically related information included in the content of the selected educational materials. Results showed that a ninth-grade reading level was required for these patient education materials. A commercially developed booklet on parenting skills for teenage parents required the lowest reading level (fifth grade). In contrast, graduate reading level was required for materials dealing with the patient's bill of rights. Culturally sensitive information was found in only 4 of the 47 materials. In the clinical settings in which these materials were used, more than 90 ethnic and cultural groups were served. The materials used to educate these groups failed to recognize cultural beliefs, values, languages, perceptions, and attitudes held by patients and families. PMID- 8638425 TI - Asking substantive theory questions of naturalistically derived data. PMID- 8638426 TI - Teaching baccalaureate nursing students to use research. PMID- 8638427 TI - [Morphology of basement membrane and associated matrix proteins in normal and pathological tissues]. AB - Basement membranes (BM) are specialized structures of the extracellular matrix. Their composition is of particular importance for the maintenance of normal morphological and functional properties of a multitude of organs and tissue systems and it is thus required for regular homeostasis of body function. Generally, they possess three main functions, i.e. participation in the maintenance of tissue structure, control of fluid and substrate exchange, and regulation of cell growth and differentiation. BMs are made up by various components which are in part specifically localized within the BM zone, or which represent ubiquitous matrix constituents with specific quantitative and/or qualitative differences in their localization. On the basis of a thorough immunohistochemical analysis of normal and diseased tissues, we provide here a concept of "functional morphology/pathomorphology" of the different BM components analyzed: 1.) The ubiquitous BM-constituent collagen IV primarily stabilizes the BM-zone and thus represents the "backbone" of the BM providing mechanical strength. Its loss leads to cystic tissue transformation as it is evidenced from the analysis of polycystic nephropathies. Thus, in other cystic tissue transformations a similar formal pathogenesis may be present. 2.) The specific localization of collagen VII as the main structural component of anchoring fibrils underlines the mechanical anchoring function of this collagenous protein. Defects in this protein lead to hereditary epidermolysis. The rapid re-occurrence of epidermal collagen VII during normal human wound healing indicates a quick reconstitution of the mechanical tensile strength of healing wounds. 3.) The BM specific heparan sulfate proteoglycan (HSPG, Perlecan) with its highly negative anionic charge can be assumed to exert filter control. This assumption is corroborated by the localizatory findings of a preferential deposition of HSPG in endothelial and particularly in glomerular BM. Similarly, the lack of HSPG in the BM of lymph capillaries can be regarded as the correlate for a free fluid influx into lymphatic capillaries. The relative reduction in HSPG-staining in the developing glomerular BM also explains the still immature filter function. Furthermore, the low content of HSPG in placental chorionic capillaries can be regarded as morphological correlate for the required free fluid exchange between maternal and fetal blood systems. In diabetic glomerulopathy, the loss of HSPG coincides with a reduced filter function providing further support for the function of the HSPG. In further analyses of diabetic glomerulopathy, we provide evidence for an extensive matrix dysregulation resulting in either the overexpression of certain BM-components (diffuse glomerulosclerosis) or microfibrillar collagen VI (nodular glomerulosclerosis) indicating changes in cell function and possibly also cellular "differentiation". The analysis of congenital nephropathies additionally indicates that also the HSPG side chains with their negative charges may be involved in certain diseases with filter impairment. 4.) Furthermore, HSPG serves as a binding site for growth factors, particularly for the basic fibroblast growth factor (bFGF). It is of particular interest that the localization of HSPG and bFGF is not completely identical indicating some tissue specific differences in the receptor-ligand interaction. The functional importance of the bFGF-HSPG-interaction is exemplified by arteriosclerotic intima lesions where in highly cellular lesions high amounts of bFGF and HSPG coincide and low levels of both appear in poorly cellular lesions. Similarly, the granulation tissue in wound healing contains large amounts of bFGF positive mesenchymal cells. 5.) The role of individual matrix components can be deduced from the normal human wound healing process, where epithelial cells migrate on a fibronectin matrix without complete BM. PMID- 8638429 TI - Variability of vitamin E content in pumpkin seeds (Cucurbita pepo L.). AB - Pumpkin (Cucurbita pepo L.) seed oil is a common salad oil which is produced in the southern parts of Austria, Slovenia and Hungary. It is dark green and has a high content of free fatty acids. Due to its colour, the oil cannot be used for cooking. The content of vitamin E, especially gamma-tocopherol, is very high. The oil content of the pumpkin seed is about 50%. The seed itself can be eaten. Therefore a pumpkin variety with high vitamin E content is desirable. The aim of this work was to find a variety of Cucurbita pepo which has a high oil yield and a high vitamin E content. A total of 100 breeding lines were tested for their tocopherol content. The tocopherols and tocotrienols are extracted with hexane and analysed by NP-HPLC/FLD with hexane/dioxan (96/4) as eluent, with fluorescence detection at 292/335 nm. The gamma-tocopherol content, which is about 5-10 times as much as that of alpha-tocopherol varies over a broad range (41-620 mg/kg dry pumpkin seeds). Beta- and delta-tocopherol are found at low levels. PMID- 8638428 TI - Mobilization and analytical availability of drug residues in food. AB - A complete mobilization of veterinary drug residues for quantitative analytical detection is often unsuccessful. For some drugs, one reason for this is the quite different binding forms between the drug and the food matrix, which for three groups of veterinary drugs may be described by sorption-isothermals. The influence of glutathione on sorption-isothermals and residue mobilization is discussed. Thus, better interpretations regarding the availability of residues are possible. PMID- 8638430 TI - Effects of freezing and canning of papaya slices on their carotenoid composition. AB - An HPLC study of the carotenoid composition of fresh, frozen and canned papaya fruit slices was done. There were no qualitative differences between the carotenoid patterns of fresh and frozen papaya fruit slices (cultivar Sunrise). The major carotenoids found in papaya extracts were lycopene and carotenol fatty acid esters of beta-cryptoxanthin and beta-cryptoxanthin-5, 6-epoxide. Other xanthophylls detected were beta-cryptoxanthin, trans-zeaxanthin and cryptoflavin. It was possible to determine the quantitative losses of carotenoids in papaya slices as a result of the freezing process and frozen storage, since samples of these fruits were available before processing. The pigment pattern of the canned product showed lycopene as being a major pigment. Thermal treatment induced the degradation of carotenol fatty acid esters of xanthophylls. The freezing and canning processing of papaya slices led to significant decreases in the total carotenoids quantified by HPLC, with frozen female slices and canned samples showing lower amounts of pigments. Hunter colour values of frozen slices were similar to those of fresh papaya fruit slices. PMID- 8638431 TI - [Dietary fiber and pectin fractions of Beta vulgaris var. conditiva]. AB - The alcohol-insoluble substance (AIS) from red beet (Beta vulgaris var. conditiva Alef.) (3.31% of the edible substance) was extracted sequentially with water, ammonium oxalate, 0.05 N HCl and 0.05 N NaOH. Accordingly 3.93 g, about 0.8 g, 2.96 g resp. 3.80 g galacturonan/100 g AIS were extracted with this procedure. These pectin extracts were purified as Cu2 +-salts and fractionated into a water soluble and a water-insoluble part. The composition of neutral monosaccharide units was estimated in the fractions. Gal, Ara and Glc dominated; Xyl, Rha and Man were also present but in smaller amounts. A higher GalA content was found in the soluble fractions (with the exception of the alkali extract). Pectins from red beet are middle- or low-esterified and partially acetylated. The composition of the AIS and of the residue after pectin extraction (RE) was determined (14.6 resp. 9.5% pectin; 10,6 resp. 17.6% protein; 58.7 resp. 64.9% total polysaccharides). In the AIS 23.3% soluble and 54.7% insoluble dietary fiber were estimated, whereas in RE 15.3 resp. 54.7% were found (enzymatic method). Following dietary fiber fractions were determined by the detergent method for both preparations: 39.0 resp. 52.7% NDF; 6.3 resp. 4.5% NDF filtrate; 23.6 resp. 41.8% ADF; 1.2 resp. 1.8% lignin. The water binding capacity decreased from 19.85 g water (AIS) to 11. 53 g water (RE) related to 1 g AUS. From these just 50% were found in the NDF fractions and about 13% in the ADF fractions. Alterations of the grown biological structures during pectin extraction and dietary fiber analysis (detergent method) were investigated by scanning electron microscopy. PMID- 8638432 TI - Thermospray-LC-MS analysis of various groups of polyphenols in tea. II: Chlorogenic acids, theaflavins and thearubigins. AB - An account is given of the application of thermospray LC-MS in the analysis of caffeoyl- and p-coumaroylquinic acids, theaflavins and thearubigins in black tea. All compounds, except for the thearubigens, could be detected as the pseudo molecular ion [M + H]+. In addition to [M + H]+, other species such as adducts with sodium, ammonium and potassium, as well as solvent clusters were observed. The formation of those adducts depended upon on the structure of the compound. A fragmentation of chlorogenic acids occurred at elevated temperatures yielding the constituents of the molecules (caffeic, p-coumaric and quinic acids). PMID- 8638433 TI - Corrosion of aluminium in soft drinks. AB - The corrosion of aluminium (Al) in several brands of soft drinks (cola- and citrate-based drinks) has been studied, using an electrochemical method, namely potentiodynamic polarization. The results show that the corrosion of Al in soft drinks is a very slow, time-dependent and complex process, strongly influenced by the passivation, complexation and adsorption processes. The corrosion of Al in these drinks occurs principally due to the presence of acids: citric acid in citrate-based drinks and orthophosphoric acid in cola-based drinks. The corrosion rate of Al rose with an increase in the acidity of soft drinks, i.e. with increase of the content of total acids. The corrosion rates are much higher in the cola-based drinks than those in citrate-based drinks, due to the facts that: (1) orthophosphoric acid is more corrosive to Al than is citric acid, (2) a quite different passive oxide layer (with different properties) is formed on Al, depending on whether the drink is cola or citrate based. The method of potentiodynamic polarization was shown as being very suitable for the study of corrosion of Al in soft drinks, especially if it is combined with some non electrochemical method, e.g. graphite furnace atomic absorption spectrometry (GFAAS). PMID- 8638435 TI - Behaviour of Yersinia enterocolitica and Aeromonas hydrophila in skim milk during fermentation by various lactobacilli. AB - In this study, behaviour of Yersinia enterocolitica and Aeromonas hydrophila in skim milk during fermentation by various Lactobacillus sp. were determined. pH values of the skim milk samples were also examined during fermentation. The amount of produced lactic acid and diacetyl/acetoin productions of the Lactobacillus sp. were estimated. Antimicrobial effects of the lactobacilli on Y. enterocolitica and A. hydrophila were also determined by an agar diffusion method. While Y. enterocolitica was not inhibited and grew during fermentation, A. hydrophila was inhibited, in part, and the growth was retarded. Results were supported by the agar diffusion method for Y. enterocolitica, whereas inhibition activity was not found for A. hydrophila. The highest lactic acid productions were estimated in L. bulgaricus (7.50 mg/ml) and L. acidophilus (5.63 mg/ml) and four out of six Lactobacillus sp. were found to be diacetyl/acetoin producers. PMID- 8638434 TI - Effect of microwave heating on the migration of dioctyladipate and acetyltributylcitrate plasticizers from food-grade PVC and PVDC/PVC films into olive oil and water. AB - Migration of dioctyladipate (DOA) and acetyltributylcitrate (ATBC) plasticizers from plasticized polyvinylchloride (PVC) and polyvinylidene chloride (PVDC)/PVC (Saran) films into both olive oil and distilled water during microwave heating has been studied. The plasticizer migrating into olive oil and water was determined using an indirect GC method after saponification of the ester-type plasticizer (DOA or ATBC) and subsequent collection of the alcohol component of the ester, namely: 2-ethyl-1-hexanol and 1-butanol, respectively. Migration was dependent on heating time, microwave power setting, the nature of the food simulant and the initial concentration of the plasticizer in the film. Migration of DOA into olive oil reached equilibrium after heating for 10 min at full power (604.6 mg DOA/l). Migration into distilled water was 74.1 mg/l after 8 min of microwave cooking at full power. The amount of ATBC migrating into olive oil reached equilibrium after heating for 10 min at full power (73.9 mg ATBC/l). Migration into distilled water was 4.1 mg/l after heating at full power for 8 min. Control samples containing olive oil gave DOA migration values which were significantly higher than the upper limit for global migration (60 mg/l) set by the European Community. It is proposed that PVC should not be used in direct contact with food in the microwave oven, while Saran may be used with caution in microwave heating and reheating applications, avoiding its direct contact with high fat foodstuffs. PMID- 8638437 TI - Enzyme and immunohistochemical studies on acute monocytic leukemia (FAB M5): proposal for a new immunohistochemical subclassification. AB - Using only morphological criteria as proposed by the French-American-British (FAB) Study Group, the subclassification of acute monocytic leukemia (FAB M5) into the categories M5a and M5b can be difficult. We therefore investigated 13 cases of well-established M5 leukemias. The results show that immunohistochemical techniques allow a better subdivision of the acute monocytic leukemias. The less mature types are characterized by a focal lysozyme and a negative CD68 reaction, whereas the more differentiated types express a diffusely positive lysozyme and also a positive CD68 phenotype: a staining pattern also found in the rare true histiocytic lymphomas. We regard these results as a useful addition to the FAB classification. PMID- 8638436 TI - Peptides inhibitory to endopeptidase and aminopeptidase from Lactococcus lactis ssp. lactis MG1363, released from bovine beta-casein by chymosin, trypsin or chymotrypsin. AB - Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm of Lactococcus lactis ssp. lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and alpha-chymotryptic hydrolysates of beta-casein (beta-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin released beta-CN f193-209, kinetic constant (Ki) of which for inhibition of PepO was 60 microM. This peptide also inhibited (Ki = 1700 microM) the 95-kDa aminopeptidase (PepN) from L. lactis ssp. lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one, beta-CN f69-97, was not degradable by PepO (Ki = 4.7 microM), while the other, beta-CN f141-163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide, beta-CN f69-84, which inhibited PepO with a Ki of 8.1 microM, was isolated from the alpha-chymotryptic hydrolysate. Peptides released from beta-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degraded beta CN f193-209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (beta-CN f193-209, f69-97, f69-84, f141-163) were readily degraded by thermolysin. PMID- 8638438 TI - Megakaryocyte-related interleukins in reactive thrombocytosis versus autonomous thrombocythemia. AB - The primary thrombocytosis (thrombocythemia) associated with myeloproliferative disorders is believed to be due to autonomous platelet production. Secondary or reactive thrombocytosis can be observed in a number of clinical circumstances, and may be related to persistent overproduction of some thrombocytopoietic factors acting on megakaryocytes. Several cytokines, including IL-6, IL-1 and IL 4 have been shown to act alone or in concert, to affect various cellular stages of megakaryocytopoiesis in humans. The aim of this study is to assess the serum concentrations of these cytokines in myeloproliferative disorders (MPD) with thrombocythemia and in rheumatoid arthritis (RA) with marked reactive thrombocytosis. Twenty-two patients (14 men, 8 women) with MPD and thrombocythemia (platelet counts > 500 x 10(9)/1; range 507-996 x 10(9)/1), 33 RA patients (28 women, 5 men) with marked thrombocytosis (platelet counts > 500 x 10(9)/1; range 500-745 x 10(9)/ 1), 27 RA patients (24 women, 3 men) with normal platelet counts (range 168-399 x 10(9)/1) and 15 healthy volunteers (8 women, 7 men) with normal platelet counts (range 161-385 x 10(9)/1) enrolled in the study. Serum IL-1 alpha, IL-1 beta, IL-4 and IL-6 concentrations were measured in these four groups. Of the 22 patients with MPD, 10 had chronic myelogenous leukemia, 5 had polycythemia vera, 6 had essential thrombocytosis and 1 had osteomyelofibrosis. Serum interleukin concentrations in patients with MPD and thrombocythemia were either suppressed or similar to those of normal subjects, whereas IL-6, IL-1 beta and IL-4 levels were increased in RA patients with reactive thrombocytosis. We conclude that thrombocythemia associated with MPD is an autonomous phenomenon, and is not regulated by cytokines which affect megakaryocytopoiesis. PMID- 8638439 TI - A quantitative ELISA for measuring red cell-bound immunoglobulins. AB - An ELISA was developed for quantitating red-cell-bound IgG and IgA and its feasibility assessed on 50 blood donations and 32 clinical specimens with raised cell-bound IgG. Test and quality control samples and immunoglobulin standards (in red-cell lysate buffer) were assayed together. Calibration curves were derived from the standards, test values being read off and related to cell count. The working range was around 5-70 ng/ml, the upper limit being indefinitely extendable by dilution with lysate buffer. Ranges of results (and medians) in molecules per red cell were < 26-240 (40) for IgG and < 29-94 ( < 29) for IgA in the donations with 240-62,700 (1,200) for IgG and < 29-4,500 (73) for IgA in the clinical specimens. The method which can be adapted for any cell-bound immunoprotein should be particularly valuable in investigating autoimmune haemolysis. PMID- 8638440 TI - Combination therapy with interferon alfa-2b and hydroxyurea during the accelerated phase of chronic myelogenous leukemia. AB - Inferferon alfa-2b (IFN) plays a major role in the current management of previously untreated patients with chronic myelogenous leukemia (CML) as well as patients with CML who have relapsed after bone marrow transplantation. Hydroxyurea (HU) is the best conventional drug for treatment of CML in the chronic phase. Ten patients, six men and four women, 40-70 years of age, were treated during the accelerated phase of CML with a combination of IFN and HU. Patients had received only HU during the chronic phase of the disease. All patients were positive for the Philadelphia chromosome and had an excess number of blasts in peripheral blood smears (more than 10%), as well as increased numbers of basophils and eosinophils but a low leukocyte level of alkaline phosphatase. Eight of them had splenomegaly. Five patients (50%) survived for 1-3 years, achieving complete hematological remission. Three patients had a partial hematological response and died within 1-2 years. Two patients with aggressive disease died within 3 months of the blastic crisis. It appears that combination therapy with IFN and HU might be a useful alternative for patients in the accelerated phase of CML who have failed to respond to HU alone. PMID- 8638441 TI - Leukemia with megakaryocytic differentiation following essential thrombocythemia and myelofibrosis. Case report and review of the literature. AB - Leukemias of megakaryocytic lineage are rare and heterogeneous clinical entities. The nomenclature published in the literature is confusing and perhaps inappropriate to designate these primary myeloproliferative disorders. We describe a patient with essential thrombocythemia who evolved through myelofibrosis and myeloid metaplasia to a final picture of leukemia with megakaryocytic differentiation in the peripheral blood. This case illustrates different aspects of a chronic myeloproliferative disorder where myelofibrosis and myeloid metaplasia are frequent but secondary events. We have reviewed the literature focusing on the role of clonal megakaryocytic proliferation in myelofibrosis and on the clinical characterization of leukemia with megakaryocytic phenotype. We also present our interpretation of the literature which indicates that a formal review of the nomenclature is urgently needed. PMID- 8638442 TI - Acute myelogenous leukemia presenting with bulky lymphadenopathy. Case report and literature review. AB - We report a patient with acute myeloid leukemia (AML) presenting with generalized lymphadenopathy, clinically stimulating aggressive non-Hodgkin's lymphoma. This patient presented with anemia and bulky lymphadenopathy in the oropharyngeal (Waldeyer's ring), submandibular, supraclavicular and inguinal nodal regions. Lymph node biopsy was initially suggestive of a T-cell lymphoblastic lymphoma, based on morphologic features together with positive immunohistochemical staining for CD7 and CD43 (Leu 22). Definitive diagnosis of AML was established when a more detailed immunophenotypic analysis showed expression of the myeloid markers CD13 and CD33, and by the demonstration of rare Auer rods and positive peroxidase staining in bone marrow blast cells. Although this is a rare presentation, AML must always be considered in the clinical and pathologic differential diagnosis of aggressive non-Hodgkin's lymphoma. PMID- 8638443 TI - Leptomeningeal seeding with acute hydrocephalus--unusual central nervous system presentation during chemotherapy in Ki-1-positive anaplastic large-cell lymphoma. AB - An unusual central nervous system (CNS) manifestation in a 18-year-old male with Ki-1- positive anaplastic large-cell lymphoma is presented. The diagnosis of Ki-1 lymphoma was first confirmed by the distinct pleomorphic morphology, expression of Ki-1 (CD30) antigen on neoplastic cells and the specific chromosome translocation, t(2;5)(p23;q35). Although young age is thought to be a good prognostic factor in this disease, the course in our patient was very aggressive. At presentation, there was already extensive extranodal involvement, with malignant cell found in the pleural cavity and bone marrow. In spite of rapid shrinkage of whole-body lymph nodes and a decrease in malignant pleural effusion soon after starting chemotherapy, headaches and vomiting ensued in the following days. A computerized tomography scan of the brain showed poor corticomedullary differentiation without definite mass lesions, and numerous malignant cells were found in the cerebrospinal fluid (CSF). Although intrathecal methotrexate was given, the patient died following the sudden onset of acute hydrocephalus. CNS involvement in Ki-1 anaplastic large-cell lymphoma is very rare and most cases present as focal mass lesions in the brain parenchyma. Leptomeningeal seeding of the lymphoma cells with acute hydrocephalus contributing directly to death has never been reported. The experience from this case suggests that CNS involvement may present in variable forms in Ki-1 lymphoma and may be an important cause of mortality in young patients, especially those in advanced stages of the disease. Early detection of CNS involvement by CSF investigation or even prophylactic CNS therapy may be mandatory in these patients. PMID- 8638444 TI - Chronic neutrophilic leukemia associated with monoclonal gammopathy of undetermined significance. AB - A 30-year-old man with chronic neutrophilic leukemia (CNL) in association with monoclonal gammopathy is presented. Physical examination on admission revealed moderate hepatosplenomegaly. Initial blood count showed neutrophilic leukocytosis (42.2 x 10(9)/1 with 90% mature neutrophils). Leukocyte alkaline phosphatase (LAP) score was elevated. Bone marrow aspiration showed myeloid hyperplasia without dysplastic features. Karyotypic and molecular analyses of bone marrow cells showed the absence of Philadelphia (Ph1) chromosome and bcr gene rearrangement. Because there was no underlying infection or neoplasm, he was diagnosed as having CNL associated with IgG kappa-type monoclonal gammopathy (IgG, 1,269 mg/dl). In addition to its association with monoclonal gammopathy of undetermined significance (MGUS), the present case was also characterized by spontaneous remission of CNL during the 12-year follow-up, accompanied by a gradual increase in serum IgG levels up to 3,000 mg/dl. As far as we know, there have been 19 cases of CNL associated with monoclonal gammopathy in the literature. The median survival of these cases was 5 years. Although there have been only 6 cases of CNL associated with MGUS, survival of these cases was particularly favorable. Taken together with the observation that leukocytosis and hepatosplenomegaly in the present case subsided without specific treatment, we speculate that myeloid proliferation in the present case may have been a leukemoid reaction to underlying monoclonal gammopathy. PMID- 8638446 TI - Myelodysplastic syndrome associated with hypotriploidy. AB - We describe a case of de novo myelodysplastic syndrome (MDS) with hypotriploidy, a condition in which polypoid chromosome abnormalities are rarely observed. A 64 year-old male was diagnosed with refractory anemia with ring sideroblasts (RARS). Cytogenetic analysis of bone marrow cells revealed 65, X,-Y,+2,+6,-7,+8,-9, 10,+11,-12, add (12) (p11.2), +14,-16,-18,-19,20,-21, 21,+3mar[11/22]/46,XY[11/22]. One month later, rapid transformation to refractory anemia with excess of blasts in transformation was observed in spite of a hematological improvement obtained by therapy with corticosteroids, anabolic steroids and pyridoxine. Two months after the diagnosis of RARS, he died of multiple organ failure. In our case, the hypotriploid chromosome abnormality may have represented a poor prognosis. PMID- 8638445 TI - Acute myelomonocytic leukaemia with 11q23 abnormality during multiple myeloma: is this related to anthracycline? AB - The clinical course of a 66-year-old man diagnosed with multiple myeloma is described. Chemotherapy including alkylating agents had no effect, and so he was treated with vincristine, doxorubicin, and dexamethasone. His bone tumor was treated with localized radiation after two courses of chemotherapy. After these treatments, monocytosis was found and dysplastic changes were noted in the bone marrow. A cytogenetic study revealed t(9;11)(p22;q23), an abnormality which had previously been absent. A diagnosis of myelodysplastic syndrome (MDS) was newly established, and transformation to acute non-lymphocytic leukaemia (ANLL) was observed 6 months later. The cumulative doses of melphalan, cyclophosphamide, doxorubicin, and radiation therapy were 432 mg, 4,200 mg, 120 mg, and 4,000 cGy, respectively. The cytogenetic abnormality suggested that this patient's MDS/ANLL was related to doxorubicin and not talk to alkylating agents, although the dose of doxorubicin administered was quite low. PMID- 8638447 TI - Cytogenetics and its prognostic value in myelodysplastic syndromes. AB - In this review we describe the cytogenetics of primary and secondary myelodysplastic syndromes, discuss their relationship with other hemopoietic malignancies, and also attempt to put the described chromosome abnormalities into a clinically more useful perspective by emphasizing the prognostic impact they carry. In addition, we discuss their use as clonal markers in determining the mode of action and efficiency of different therapeutic approaches. PMID- 8638448 TI - Established role of magnesium sulfate as prophylactic anticonvulsive agent in preeclampsia/eclampsia. PMID- 8638449 TI - Controversies in assessment of ovarian tumors with transvaginal color Doppler ultrasound. AB - BACKGROUND: The observations made in the late 1980s indicated that the transvaginal color Doppler ultrasound can be used in the detection of ovarian cancer. This has generated a stream of clinical trials. The conflicting results of numerous publications, however, have led to a major controversy. The aim of this communication is to review the literature and our own observations in order to provide more insight into the subject. RESULTS: The performance characteristics of the 32 previous transvaginal color Doppler ultrasound studies showed a considerable heterogeneity: the sensitivity was 25%-100%, the specificity was 46%-100%, the positive predictive value was 28.6%-100% and the negative predictive value was 63%-100%. The accuracy varied between 57% and 100%. An indisputable overlap was found between the ranges of pulsatility index (PI) and resistance index (RI) values from the benign and malignant adnexal tumors. The RI was <0.40 in 43% of benign tumors and in 25% of the normal vascular ovaries. CONCLUSION: The current cut-off levels for PI or RI values should not be used in clinical decision making due to their poor specificity. PMID- 8638450 TI - Endometrial destruction by hyperthermia--a possible treatment of menorrhagia. An experimental study. AB - BACKGROUND: Treatment of menorrhagia by heat-destruction of the endometrium, intended to be an alternative to hysterectomy, was investigated in an experimental study. METHOD: A specially designed catheter (CavatermTM) with a silicone balloon containing a self-regulating containing heating element is inserted into the uterus, filled with glycine to a pressure of around 180 mmHg and heated to about 75 degrees C. We investigated the treatment effect in vitro in five extirpated uteri (series A) and in vivo in three patients treated peroperatively just before hysterectomy, temperatures being monitored in the surrounding tissues (series B). In both series we monitored the following variables: heating-power, balloon-pressure, temperature of the heating element (around 85 degrees C) and the temperatures at five locations from top to bottom of the balloon surface. RESULTS: After 30 min in vitro treatment at 75 degrees C, the endometrium was partly destructed, and condensed. Histological examination showed smooth muscle cells to be destroyed to a depth of 2-5 mm close to the endometrium. With in vivo treatment for 30 min at a heating power of about 20 W (resulting in balloon surface temperatures of 58-65 degrees C), the increases in temperature of the surrounding tissue were too small to measure accurately (+/-1 degree C). Histological examination showed destruction of cells in the corpus uteri to a maximum depth of 8 mm. CONCLUSION: Findings in in vitro and in vivo experiments suggest that 30 min heating of the endometrium to 58-65 degrees C with an intrauterine silicone balloon filled with a liquid to a pressure of 180 mmHg exerts therapeutic effects on both endometrium and uterine cavity smooth muscle cells without damage to surrounding tissues. PMID- 8638451 TI - A prospective study of antibodies against parvovirus B19 in pregnancy. AB - BACKGROUND: Parvovirus infection during pregnancy has been reported to be associated with spontaneous abortion and fetal loss. OBJECTIVE: To show the incidence of antibodies against Parvovirus B19 early in pregnancy and sero reactivities during and after pregnancy. STUDY DESIGN: In a prospective study during a non-epidemic period, 457 women admitted to an antenatal care center were included. Serum samples were collected at 7-13, 21, and 33 weeks of gestation, and 7-9 weeks after delivery. METHOD: Samples were evaluated for parvovirus specific IgG and IgM against two different parvovirus antigens. RESULTS AND CONCLUSION: Parvovirus specific antibodies were present in 81% of the women in the first sample. Six women (six of 88 susceptible, 6.8%) seroconverted and 28 women (28/369, 7.6%) boosted their antibody response during or after pregnancy. All gave birth to healthy infants. One woman free of symptoms experienced an intrauterine fetal death at 37 weeks of gestation. She had no rise in B19 antibodies during pregnancy, but parvovirus DNA was found in maternal serum samples and in the placenta. PMID- 8638452 TI - Toxic hepatitis: a rare complication associated with the use of ritodrine during pregnancy. AB - OBJECTIVES: To evaluate the incidence of toxic hepatitis associated with the use of ritodrine in pregnancy. DESIGN: A prospective study. SETTING: A large teaching hospital in Barcelona, Spain. PATIENTS: One hundred and twenty-three consecutive pregnant women, 101 singleton and 22 multiple pregnancies, receiving intravenous ritodrine with normal transaminase levels prior to the initiation of therapy. MEASUREMENTS: Serum glutamic pyruvic transaminase (SGPT) and glutamic oxaloacetic transaminase (SGOT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (PA), direct and total bilirubin. RESULTS: In two (l.9%) of 101 singleton and two (9.1%) of 22 multiple pregnancies, elevation of SGOT and SGPT levels was detected. PA, GGT, and bilirubin were normal in all women studied. Other causes of hepatitis were ruled out, and no other signs of liver impairment were found in these women. Transaminase levels recovered rapidly in all cases after discontinuation of therapy. CONCLUSION: Elevation of liver enzymes is a rare complication of ritodrine. Discontinuation of therapy does not appear to be indicated as long as other signs of liver dysfunction are not present and tocolytic treatment is required. PMID- 8638453 TI - Effect of dehydroepiandrosterone sulfate on fetal middle cerebral artery flow velocity waveforms in term pregnancy. AB - OBJECTIVE: To investigate whether bolus injection of dehydroepiandrosterone sulfate (DHAS) is associated with changes in fetal middle cerebral artery flow velocity waveforms in term pregnancy. METHODS: Ten normal full-term pregnant women received the administration of a 200-mg intravenous dose of DHAS in 20 ml of 5% dextrose. Ten normal full term pregnant women received 20 ml 5% dextrose as controls. Color Doppler flow imaging and pulsed Doppler ultrasonographic assessments were made on fetuses in each group before and 10 min, 30 min, 60 min, 90 min. and 120 min after DHAS or dextrose administration. The pulsatility index (PI) values for the middle cerebral artery, and umbilical artery, and fetal heart rate were recorded. RESULTS: In the DHAS group, middle cerebral artery PI decreased from baseline by 24% (p<.05) after 10 min, and the mean reduction was 22% (p<.05) after 30 min. The PI returned to the baseline value 60 min later. In the control group, there was no change in middle cerebral artery PI. No change was found in umbilical artery PI or fetal heart rate in the control or DHAS group. CONCLUSION: DHAS induces a significant decrease in the fetal middle cerebral artery PI, which suggests a possible decrease in fetal cerebral vascular impedance in term pregnancy. PMID- 8638454 TI - The influence of glucose tolerance tests on subsequent carbohydrate metabolism in pregnancy. AB - BACKGROUND: This present study was designed to prospectively confirm a previous observational finding that a 75g glucose load exerts an influence on the results of a subsequent 50g glucose tolerance test performed after an interval of one week. METHODS: Pregnant Chinese patients were given both 50g 3-hour and 75g 2 hour oral glucose tolerance test (OGTT) in random order, 7 days apart, between 28 and 32 weeks of gestation. Patients were divided according to whether they received 50g test first (group 1) or 75g test first (group 2). Student's t-test for independent samples and for paired samples were used for between group comparisons and within patient comparisons respectively using SPSS for Windows software. RESULTS: Significant decrease in fasting glucose levels (p<0.0001) between paired 50g and 75g OGTT was found when 75g test was performed first. The mean difference in 2-hour values was also 35% lower. Similar results were also found when individual timed glucose levels were compared between groups. CONCLUSIONS: Differences in the blood glucose levels between OGTTs were confirmed to be related to which glucose load was given first. The 75g test exerts a significant effect on the fasting glucose and the response to a 50g glucose load up to one week later. These effects are consistent with improvement in glucose utilization. The differences observed will not significantly alter current clinical practice but may necessitate modification in the diagnostic crieria of mild abnormal glucose tolerance in pregnancy. We postulate that in some cases of mild glucose intolerance, the response to diet may not be due to caloric reduction but rather stimulation of a previously inadequate insulin response since a proportion of our local pregnant women consistently have calorice intakes significantly below the 1800 kcal per day. PMID- 8638456 TI - Smoking habits among pregnant women in a Norwegian county 1987-1994. AB - BACKGROUND: The aim of the study was to investigate changes in smoking habits among pregnant women in the city of Trondheim and its surroundings. We also wanted to register the effect of a national campaign against smoking in pregnancy introduced in December 1989. MATERIAL AND METHODS: From 1987-1994 midwives interviewed 21 348 women in a non-selected population during a routine ultrasound examination at 18 weeks of pregnancy. Smoking habits and cigarette consumption were recorded. RESULTS: The point prevalence smoking rate among pregnant women decreased from 34% in 1987 to 22% in 1994 (p<0.001). A stratified analysis indicated that the reduction was not confounded by changes in age and parity over time. The mean number of cigarettes per pregnant smoker decreased from 8.6 cigarettes per day to 7.0 cigarettes per day during the study period (p<0.001). In 1987, 49% of the pregnant smokers consumed > or = 10 cigarettes compared with 35% in 1994 (p<0.001). The national campaign may have reinforced the on-going decline in smoking rate, but no significant effect was observed. CONCLUSIONS: The study demonstrated a statistically significant reduction in smoking prevalence among pregnant women in the study area. A similar decrease was not observed among women in the general population. No significant effect of the national campaign against smoking during pregnancy was observed. PMID- 8638455 TI - Validity of self-reported smoking habits in pregnancy: a saliva cotinine analysis. AB - BACKGROUND: We examined the validity of self-reported cigarette smoking during the third trimester of pregnancy using saliva cotinine as a marker. METHODS: Eligible for the study were 109 pregnant women attending the outpatient Prenatal Service of the Luigi Mangiagalli Clinic (the largest maternity clinic in Milan) for routine prenatal visits during the third trimester of pregnancy on twenty days in 1994. Women self-reporting current smoking or quitting smoking in pregnancy were asked to provide a saliva sample. Cotinine concentration was analyzed and classified as follows: cotinine not detectable, not probable nicotine use or passive exposure; cotinine <10 ng/ml, not probable nicotine use/probable passive exposure; cotinine, > or = 10 ng/m, probable occasional or regular nicotine use. RESULTS: A total of 57 (52.3%) women were non-smokers at conception and were excluded from any subsequent analysis. Of the remaining 52 women, 25 self-reported quitting smoking in pregnancy and 27 were current smokers. Saliva cotinine levels were below 10 in 20 of the 25 subjects reporting quitting smoking in pregnancy. The five cases with cotinine > or = 10 reported a husband smoking more than 10 cigarettes per day. Among the 26 current smokers, seven had a cotinine level <10 ng/ml (four reported smoking fewer then five cigarettes per day and two reported smoking five or more per day); in 20 cases the cotinine value was > or = 10 ng/ml. CONCLUSIONS: These findings provide evidence of a satisfactory validity of self-reported smoking habits in pregnancy. PMID- 8638457 TI - Maternal risk factors for preterm birth and low birthweight in Cape Verde. AB - BACKGROUND: Fifteen years after the implementation of an antenatal risk screening program in Cape Verde, the first assessment of an association between maternal obstetric characteristics and preterm birth or low birthweight (LBW) infants was undertaken. METHODS: A cohort of 353 systematically selected antenatal clinic attenders in the county of Praia, Cape Verde, was studied prospectively during the period October 1991 through December 1992. The cohort was followed past the perinatal period and information was obtained according to a pretested structured questionnaire. In the analysis of preterm birth and LBW, multiple logistic regression was listed to estimate the relative risks of ll background variables. RESULTS: The prevalence of preterm birth (<37 gestational weeks) was 12%, and the prevalence of LBW infants was 8%. Low birthweight (<2500 grams) was significantly associated with low maternal age (< or = 19 years, RR=3.7); nulliparity (RR=5.2) and obstetric history of previous LBW infant (RR-6.5). The risk of preterm birth was significantly increased if the woman had an obstetric history of hypertension or convulsions (RR=2.6). CONCLUSIONS: In the setting studied, teenage women and women with previous pregnancy hypertension should be given selective attention in antenatal care to achieve improved pregnancy outcome. Primary prevention is needed to lower the prevalence of teenage pregnancies. PMID- 8638458 TI - Possible influence of expatriation on perinatal outcome. AB - BACKGROUND: In order to test the hypothesis of possible influence of environmental stress on the length of gestation the data on deliveries in the Maternity Unit, Zagreb University School of Medicine in three six months periods; May-October 1991 (active war in Croatia), May-October 1990 (pre-war period), and the same period in 1992 are analysed. METHODS: Deliveries of 7845 women from free areas of Croatia (non-displaced population) and deliveries from 712 women from occupied areas of Croatia, as well of 593 Croatian refugees from Bosnia and Herzegovina and Serbia (expatriated population) were compared. The duration of pregnancy, fetal weight, immediate neonatal condition, mode of delivery and perinatal outcome in non-displaced and expatriated population were compared using chi-square test in statistical analysis. RESULTS: During 1992 and 1991, there was a slight increase in total number of deliveries in comparison to 1990. The number of deliveries by displaced women more than doubled. The incidence of major pregnancy complications was almost the same for both groups in all three time periods. The two populations were comparable regarding their age, parity and previous obstetric history. Slight increase in preterm delivery rate (7.7% in l990, 8.7% in 1991 and 9.4% in 1992), and a subsequent slight decrease in birth weight was found in all women. There was no significant change in the proportion of growth-retarded newborns. Expatriated women both in 1990 and in 1991 delivered twice as often prematurely in comparison to non-displaced women (17.5% and l4.3% deliveries), respectively. Birth weight of their infants was significantly more often under 2500 grams. Slight increase in overall perinatal mortality was observed. Perinatal mortality in the experiated population was significantly higher than in the non-displaced population. Increase in perinatal mortality could be attributed exclusively to increase in prematurity rate. CONCLUSION: Our results support the concept of possible influence of stress, fear, exile and inadequate antenatal surveillance on the length of gestation. PMID- 8638459 TI - The hazards of reproduction in space. AB - NASA Medical Standards for space flight (JSC 11570) specifically disqualify any flight crew member who is pregnant. A variety of factors led to this recommendation including the effects of radiation exposure on fetal development. Additionally, other concerns such as toxic chemical exposure, microgravity, pregnancy accidents, plus alterations in breathing gas mixtures and decompression preclude pregnant flight crew members from missions in the foreseeable future. The future of space travel will require reproduction beyond the confines of earth's environment. This makes it all the more necessary to identify the potential hazards associated with reproduction in space. PMID- 8638460 TI - Prospective study of incidence and predisposing factors for clavicular fracture in the newborn. AB - OBJECTIVE: To determine the incidence of clavicular fracture, associated fetal and maternal risk factors and its connection with quality care control. SUBJECTS AND METHODS: A total of 3030 newborns delivered vaginally were evaluated for clavicular fractures by three separate physicians. The study group included all newborns with fractured clavicle. A control group consisted of 52 newborns delivered vaginally with no history of fractures. Maternal records were evaluated for possible predisposing factors. RESULTS: Forty-six (l.5%) newborns were found to have clavicular fractures. When compared to the control group, they were found to have a higher birthweight (3710+/-352gm vs 3235+/-405gm) an older maternal age (30.5+/-5 bs 27.7+/-6), a longer second stage of labor (34 min vs 23 min), higher rate of instrumental deliveries (13/46 vs 6/52) and shoulder dystocia (6/46 vs 1/52). Nearly 80% of newborns with clavicular fractures weighed less than 4000 gm. Multivariate analysis demonstrated two independent variables; birthweight over 3500 gm and maternal age >29. CONCLUSION: Clavicular fractures are associated with higher birthweight, older maternal age, longer second stage of labor, instrumental deliveries and shoulder dystocia. However, in most cases this injury cannot be predicted prior to delivery and thus cannot be an indicator of quality control. PMID- 8638461 TI - The effect of post-natal exercises to strengthen the pelvic floor muscles. AB - BACKGROUND: The purpose of the present study was to evaluate the effect of post natal pelvic floor muscle exercise. METHODS: A prospective comparison design comprising 66 matched pairs (n=132) of mothers, divided into a training group (TG) and a control group (CG) was used. The TG attended an eight week special pelvic floor muscle exercise course, training in groups led by a physiotherapist 45 minutes once per week. In addition they exercised at home at least three times per week. The CG followed the ordinary written postnatal instructions provided by the hospital. Pelvic floor muscle strength was measured at the 8th and the 16th week after delivery using a vaginal balloon catheter connected to a pressure transducer. Vaginal palpation and observation of inward movement of the balloon catheter during contraction, were used to control the ability to perform correct pelvic floor muscle contraction. In addition the women were interviewed about breast feeding, menstruation, general physical activity and pelvic floor muscle exercise during pregnancy, between birth and test l, and between test l and test 2. Wilcoxon-Signed-Ranks-Test for matched pairs was performed to compare change in pelvic floor muscle strength within and between groups. RESULTS: While a statistically significant change in pelvic floor muscle strength was found in both the TG and the CG, the improvement for the training group was significantly greater. CONCLUSION: was concluded that a specially devised pelvic floor muscle strength training programme can add significantly to physical recovery after childbirth. PMID- 8638462 TI - The effects of estradiol valerate plus medroxyprogesterone acetate and conjugated estrogens plus medrogestone on climacteric symptoms and metabolic variables in perimenopausal women. AB - BACKGROUND: Two sequential hormone replacement regimens, containing either estradiol valerate plus medroxyprogesterone acetate (E2V/MPA) or conjugated estrogens plus medrogestone (CE/MED), were compared with respect to effects on climacteric symptoms, lipid metabolism, and hemostasis. METHODS: In an open, multicenter study, 51 perimenopausal women were randomized to E2V/MPA and 50 to CE/MED. Assessment of climacteric complaints was performed at baseline and at months 1, 3, and 6. The effects on lipid and hemostatic variables were measured at baseline and at month 6. Quantitative data were analyzed using analysis of variance, the paired t-test or the chi2 Mantel-Hanszel test, where appropriate. RESILTS: Efficacy regarding treatment of climacteric symptoms was with E2V/MPA as good as with CE/MED, with a statistically significant reduction of most symptoms in both groups. After 6 months, total cholesterol and triglycerides had remained unchanged in both groups. High-density lipoprotein cholesterol showed no significant change with E2V/MPA, whereas an increase was noted in the CE/MED group (p<0.05). Low-density lipoprotein cholesterol was decreased with E2V/MPA (p<0.01) and was unchanged in the CE/MED group. Hemostatic parameters showed no significant changes after 6 months, with the exception of a decreased prothrombin time with E2V/MPA (p<0.05). Acceptability was excellent, expressed by the low incidence of treatment-related drop-outs in both groups. CONCLUSIONS: E2V/MPA is a one tablet per day sequential HRT regimen, which is as effective and acceptable as hormone replacement therapy with CE/MED regarding treatment of climacteric symptoms. Neither preparation had negative effects on lipid metabolism and hemostatic variables. PMID- 8638463 TI - Previous ectopic pregnancy should be considered a contraindication for microsurgery. AB - BACKGROUND: To estimate the risk of subsequent ectopic pregnancy (EP) after tubal surgery, given that the woman becomes pregnant, by means of a logistic model, a retrospective study was initiated. METHODS: During the period 1986-1990, 221 women with tubal infertility underwent microsurgery. Subsequent fertility was evaluated in 1991. Ninety women conceived, of whom 84 were included in the study (30 with EP and 54 with intra-uterine pregnancy as the only outcome). Clinical background factors of importance, surgical procedures used, scoring systems for tubal lesions, adnexal adhesions and risk of EP were analysed for possible correlation to subsequent EP. These factors were further used in a logistic model to estimate the risk of subsequent EP as only outcome. RESULTS: The risk of EP after microsurgery is minimum 15% without any risk factors. Previous EP and endometriosis could be identified as factors with prognostic power in the logistic model. One previous EP implies a 60% risk, whereas two previous EPs and endometriosis increase the risk to 95%. CONCLUSION: Patients with previous EP should generally not be considered for microsurgery owing to the high risk of recurrence and to the reduced chance of intra-uterine pregnancy. PMID- 8638464 TI - The cytological screening history of 469 patients with squamous cell carcinoma of the cervix uteri; does interval carcinoma exist? AB - BACKGROUND: To determine whether interval carcinomas occurred and to determine the level of screening-participation by women who developed a cervical carcinoma. METHODS: A retrospective study of the cytological history of 469 patients diagnosed between January 1980 and December 1989 with cervical squamous cell carcinoma in 12 hospitals in the western part of The Netherlands. Clinical data, and cervical smear histories in 3.5 years preceding the diagnoses were obtained. Cervical smears diagnosed as Pap I, II or IIIA were traced for review. RESULTS: 306 patients' data were completed. Two hundred and twenty-three patients (72.9%) had never been screened and 83 patients (27.1%) had had at least one smear, of which 39 were normal. The percentage of women over 54 was higher in patients who had never been screened (58%) than in those screened (46%). Women over 54 were in higher Figo stage. Interval carcinoma was proven in six of 306 (2%) patients. Of the normal smears 53% were false negative. CONCLUSION: The high number of non participants still forms the main reason for the failure of cervical cancer screening. Secondly, the assumed existence of frequent interval carcinoma could not be demonstrated. There are grounds for changing the age limits of the current Dutch screening program without changing the screening interval. PMID- 8638465 TI - Abdominal hysterectomy should not be considered as a primary method for uterine removal. A prospective randomised study of 100 patients referred to hysterectomy. AB - OBJECTIVE: The present study is a prospective randomized comparison of laparoscopically assisted vaginal hysterectomy (LH) with total abdominal hysterectomy (TAH). METHODS: 100 patients referred for uterine removal were included in the study, 46 undergoing LH and 54 TAH, at two teaching hospitals in Norway. RESULTS: In the LH group, the duration of surgery was longer while duration of hospitalization and time from operation to return to normal activity were shorter as compared to the TAH group. Postoperative pain, assessed by the need for analgesics, was less in the LH group. All these differences were statistically significant. There were two ureteral lesions in the LH group, and one center withdrew early from the study for this reason. CONCLUSIONS: In expert hands, LH as a primary method for uterine removal is superior to TAH. PMID- 8638466 TI - Disseminated implantation of peritoneal trophoblastic tissue secondary to laparoscopic removal of a tubal pregnancy. PMID- 8638467 TI - Omental herniation following laparoscopic sterilization. PMID- 8638468 TI - Acardius (acardius acephalus). PMID- 8638470 TI - [Homocysteinemia as a risk factor for cerebrovascular disorders. The role of age and homocysteine levels]. AB - PURPOSE: To evaluate importance of homocysteinemia as risk factor of thrombotic cerebrovascular disease, in terms of age and homocysteinemia levels. METHODS: A group of patients under 55 years old (n = 35, 21 males) that had suffered a stroke 3 months to 1 year before the study, defined by clinical criteria and the presence of cerebral infarction confirmed by tomography, without history or predisponents to embolic disease. The patients were matched with a group of controls without vascular pathology of a check-up program, in terms of age and sex. Patients and controls with history of alcoholism, signs or laboratory of renal or hepatic insufficiency or with history of recent ingestion of vitamins of the group B were excluded since these conditions could influence homocysteinemia levels. We measured to patients and controls the plasmatic basal homocysteinemia and homocysteinemia 6 hours after methionine overload of 0.1 g/Kg body weigh. We estimated case-control odds ratio of hyperhomocysteinemia globally and by age groups, and odd ratio of different levels of homocysteinemia. RESULTS AND CONCLUSIONS: Hyperhomocysteinemia case-control global odds ratio was 5.7, being higher in younger patients (8.8 below and 3.5 after the age of 45 years). Homocysteinemia as a risk factor of cerebrovascular disease presented as a continuous effect: low homocysteinemia was protective, and the higher the homocysteinemia, the higher the cerebrovascular risk proved to be. In these circumstances, heterozygozyty of cysthationine beta synthase deficiency, refered as the more important cause of hyperhomocysteinemia, cannot account for most of the cases of hyperhomocysteinemia. PMID- 8638469 TI - Hydrops fetalis and intrauterine deaths due to anti-M. PMID- 8638472 TI - [Cerebral toxoplasmosis in AIDS patients, CT and MRI images and differential diagnostic problems]. AB - Cranial computed-tomographies (CT) and magnetic resonance imaging (MRI) of 14 patients with AIDS and central nervous system toxoplasmosis (CNST) were reviewed. In spite of the low specificity of CT and MRI findings in CNS mass lesions of AIDS patients, there are some features which have been observed as more typical of CNST, namely: 94.9% of the lesions were round shaped and 94.5% had ring or nodular enhancement; 81.3% of the cases presented multiple lesions; 60.2% of the lesions were localized at the cerebral cortical or corticomedullary junction (100% of the cases showed at least 1 lesion in this localization); 34.6% of the lesions had less than 1cm in diameter.; on nonenhanced CT, 91.3% of the lesions were hypodense.; On T2-weighted MR images, 53.4% of the lesions had at least one hypointense zone on T2-weighted images. The existence of target-shaped lesions with hypointense centre on T2-weighted MR images (29.3% the observed lesions) is also suggestive of CNST, which, to our belief, had not been previously reported and will need confirmation with larger series. The visualization of iso/hyperdense lesions on nonenhanced CT or irregular shape lesions is uncommon in CNST. The finding of a solitary lesion, on CT or MR, it is not, by itself, a good criterion of differential diagnosis. PMID- 8638471 TI - [Expectations of control in the context of personality. More about the factorial structure of Levenson's IPC scale]. AB - The goal of this study was to review some of the psychometric properties concerning the IPC scale as well as to confirm the locus of control structure as proposed by Levenson. Having that in mind we reviewed some previous studies on this subject, and tried to work out what went wrong when reports couldn't make a clear cut between social and chance factors within the external dimension of the proposed construct. The sample was formed by a homogeneous group drawn from a population of university students. This allowed for comparisons with similar studies. The innovation here resides in the analytical approach proposed, which should account for the oblique factors determined by the two alleged existent clusters for the external locus of control. It did, thus corroborating the original invariants proposed. The study also reinforces the conviction previously noted that questions 3, 4, and 10 should be ignored or even suppressed, eventually leading ulterior studies to report through standard transformation of the raw scores. PMID- 8638473 TI - [Arterial hypertension in pregnancy]. PMID- 8638474 TI - [Meta analysis of diagnostic methods in urinary stress incontinence]. AB - The analysis of existing diagnostic methods of urinary stress incontinence suggest that it is almost always clinical. Urodynamic evaluation has a place in special situations, as atypical symptom; neurologic disease; recurrent USI after surgery or when it is necessary to confirm the clinical diagnose. PMID- 8638475 TI - [Idiopathic pulmonary hemosiderosis]. AB - We report a case of a 23 year-old man admitted to hospital with cough, fever and recurrent episodes of haemoptysis. Laboratory findings, which included a pulmonary biopsy, established the diagnosis of Idiophatic Pulmonary Hemosiderosis (IPH). Despite a number of morphologic, immunologic and ultrastructural studies, the etiology and pathogenesis of this disease remain indetermined. To diagnose an IPH all the other causes of pulmonary hemorrhage must be excluded. This is a rare disease, even more rare in adults. Based on these facts the authors publish this article, which includes the case report and a literature review. PMID- 8638476 TI - [Adult type respiratory distress syndrome (ARDS) induced by hydrocarbons]. AB - The authors present a case of a child aged 32 months, victim of accidental ingestion of an unidentified hydrocarbon product. Sixteen hours following hospitalization, the patient began polypnea with progressive hypoxemia, with the radiograph showing ARDS pattern. Mechanical ventilation was initiated on the 2nd day of admission. During hospitalization the patient developed pneumomediastinum and bilateral pneumothorax which justified active drainage. Inspite of the initial poor prognosis, the patient improved, with no symptoms after discharge. The authors discuss certain theoretical considerations regarding hydrocarbon intoxication. PMID- 8638477 TI - [Risk of radiologic examinations during pregnancy]. AB - Considering that there's a lack of information concerning the risks of radiation exposure in pregnancy, the author identifies such risks and estimates its magnitude. He underlines the fact that radiological examinations deliver lower doses in relation to those that can be proved to be malefic to the embryo, both in the somatic and genetic plan. PMID- 8638478 TI - [Cerebral metastases. Review of a hospital population]. AB - Cerebral Metastases are diagnosed in approximately 20-30% of patients with primary tumours. Because of the improvement of central nervous system imagining technology and of the newly effective therapeutic schemes, the incidence of this particular type of cerebral lesion tends to increase. In this retrospective study the authors review the patients admitted in the Neurology, Neurosurgery and Medicine departments and in the outpatient chemotherapy consultation, with a diagnosis of cerebral metastases. Sex and age, neurologic symptoms at presentation, neuroradiologic findings, primary tumour origin, treatment outcomes and prognosis were evaluated in this population. PMID- 8638479 TI - [Accreditation. Priority and challenge]. AB - This article aims at rousing the discussion of a subject in which Portugal does not have a definitive position and on which the doctors' contribution, although fundamental, is not yet defined. The concept and evolution of accreditation, its advantages and inconveniences, the method (standards, accreditation body and process) and experience of some countries are reviewed according to recent bibliography. PMID- 8638480 TI - [Hypertension associated with pregnancy. Epidemiologic study of 311 consecutive cases]. AB - The epidemiologic aspects of 311 consecutive cases of hypertension associated with pregnancy seen in the Department of Obstetrics and Gynecology, Hospital de Santa Maria/University of Lisbon Medical School between January 1st 1988 and December 31st 1992, are reviewed. Seventeen cases were multifetal pregnancies. Using the criteria proposed by the American College of Obstetricians and Gynecologists the cases were classified as follows: Mild preeclampsia, 64 cases (7 in twins); severe preeclampsia 50 cases (5 in twins); chronic hypertensive disease, 81 (1 in twins); chronic hypertension with superimposed preeclampsia, 16 (all singleton pregnancies); transient hypertension of pregnancy, 84 (4 in twins); unclassified hypertension, 16 cases of singleton pregnancies. No maternal deaths occurred. The most frequent maternal complications (eclampsia, HELLP syndrome, abruptio placentae and acute renal failure) were seen in preeclampsia (mild and severe forms). Only 2 significant maternal complications were observed in the cases of superimposed preeclampsia on chronic hypertensive disease. In the other groups maternal complications were seldom seen. Excepting in transient hypertension, perinatal morbidity and mortality were frequent in all groups, specially in severe preeclampsia and superimposed preeclampsia, when the delivery occurred before 34 weeks; after that time of pregnancy there were no neonatal deaths in any of the groups and intrauterine growth retardation and fetal distress were the most common fetal complications in all groups. In the whole, uncomplicated chronic hypertension and transient hypertension of pregnancy were the clinical situations in which maternal and perinatal complications were milder and less frequent. No perinatal problems were found in the group of unclassified hypertension. PMID- 8638481 TI - Fibronectin and cell adhesion: specificity of integrin-ligand interaction. PMID- 8638482 TI - Tyrosine hydroxylase. PMID- 8638484 TI - Eukaryotic dihydrofolate reductase. PMID- 8638483 TI - Exopolysaccharide alginate synthesis in Pseudomonas aeruginosa: enzymology and regulation of gene expression. PMID- 8638485 TI - Periorbital aesthetic surgery with the KTP laser. AB - The eyes are regarded as the windows to the soul. Many expressions of mood may be derived from the appearance of the eyes--mad, sad, bad, criminal, sweet, friendly, mystic. In addition, love and flirtatiousness, self-consciousness, pride, modesty, anger, youth, and age are shown in the expression of our eyes. The eyes and the periorbital region therefore challenge our surgical skill to improve the patient's overall well-being to be looked at each day in the mirror. The potassium titanyl phosphate (KTP) laser in many indications helps us to fulfill the patient's expectations concerning pain, oozing, bruising, swelling, outpatient surgery, and early return to work and normal social activities. With the cutting fiber device, an accurate removal of skin and fat or even tumors is possible in this region with practically no side effects. The frontal lift, eyebrow lift, direct or through coronal incision, as well as temporal lifting are easily accessible and carried out by this device. Glabellar frowns may also be removed endoscopically. Further, the KTP laser may be used for transconjunctival blepharoplasty. PMID- 8638486 TI - Transconjunctival blepharoplasty: further applications and adjuncts. AB - Recent enthusiasm for transconjunctival blepharoplasty (TCB) has encouraged us to extend the technique not only to patients who need removal of excess orbital fat, but also to those with fine wrinkles, excess skin, and redundant or ptotic orbicularis muscle. We describe our technique that combines the transconjunctival removal of fat with a trichloroacetic acid--Jessner's solution chemical peel, a phenol peel, a skin pinch excision, or lateral canthoplasty performed at the same time. The combined procedures require minimal operative time and there is less morbidity during recovery than with the standard external approach. The procedure has produced eyelids with a rejuvenated, natural, and unoperated on appearance. PMID- 8638487 TI - Aging of the upper lip: a new treatment technique. AB - The signs of aging of the upper lip, the pathogeny, and different treatments proposed up to now are discussed. A personal technique used since 1992 is presented. It is based on an earlier personal technique, described in 1970, which consisted of dissecting the skin from the orbicularis muscle and inserting a temporary (three weeks) silicone sheet. The present technique consists of a skin excision at the nasolabial junction, based on that described by Cardoso and Sperli in 1971. However, I dissect the skin of the vermilion border and insert a trapezoidal graft of the pretemporal areolar tissue, taken during rhytidectomy. The graft is fixed with Vicryl sutures at the level of the nasolabial folds. In a few patients this has been combined with a peel. The technique corrects both the superficial and the deep wrinkles caused by the retraction of the fibers that join dermis and orbicularis muscle. It achieves an eversion of the vermilion, enhancing its convexity and producing a fuller look, and shortens the lip, which adopts a concave, youthful appearance because of the improvement of the philtrum and of Cupid's arch. Our technique has been used in 15 patients who were satisfied with the results. PMID- 8638488 TI - Nasal tip repair with axial flap of nasal muscle. AB - The procedure described here consists of a myocutaneous axial flap with lateral pedicle. It uses the transverse part of the nasal muscle that goes forward in rotation and the lateral and superior portions of the flap in the V-Y. The procedure is useful for repairing losses of nasal tip and the surrounding area. It is a simple method that can be used to reconstruct the brim near the tip of the nose, yielding an aesthetically and functionally efficient nose. PMID- 8638489 TI - Musculoaponeurotic plication in abdominoplasty: how durable are its effects? AB - Abdominal myofascial plication has become an integral part of aesthetic abdominoplasty in an effort to narrow the waistline. Out of concern for the durability of this procedure, we performed a study using standard radiographs and clinical photographs to determine if there was long-term separation of the plication. Nine patients were entered into the study. In one patient immediate total separation of the plication occurred, and in another no separation occurred at all. The remaining patients' plication separated over only a 1-3-cm distance in the central part of the abdomen (original maximum width of plication was 12-16 cm). Aesthetic appearance was not compromised by this minor degree of separation. We conclude that the effects of musculoaponeurotic plication are durable. PMID- 8638490 TI - Salvaging the exposed mammary prosthesis. AB - The authors present a new approach to handling the problem of extrusion of mammary implants. The prosthesis is removed and reintroduced in the same operation. Five cases are discussed. PMID- 8638491 TI - "L" technique compared with others in mammaplasty reduction. AB - With more than 20 years' experience with using my L method in reduction mammaplasty, I realize that very little has changed in my philosophy. With the L technique one can achieve a perfect shape with a minimal scar and avoid a medial scar toward the sternum. I use only the lower access with upper vascular protection, abandoning the inferior vascular base of the flap. This procedure has new competition including techniques that yield a simple vertical scar or only a periareolar scar. However, up to now it is not possible, with any technique, to achieve a satisfactory shape for exigent patients. When I explain to potential patients the advantages and disadvantages of the different methods, in most cases they choose the L technique. PMID- 8638493 TI - Use of an anesthetic cream before infiltration with local anesthesia in aesthetic surgery. AB - The use of local anesthesia in aesthetic surgery must be provided to the patient based on its risk/benefit ratio, effective control of postoperative pain, and lower incidence of postoperative disorders. The use of a new anesthetic cream before infiltration with a local anesthetic may cause reduction or elimination of the pain provoked by the needle during the infiltration, increasing the compliance of local anesthesia. Here we describe the modalities and the advantages of using anesthetic cream. PMID- 8638492 TI - Suction-assisted lipectomy does not increase the risk of random flap necrosis in a randomized study in pigs. AB - Thirty-two dorsally based random flaps were raised in eight Yorkshire pigs (26-31 kg) after infiltration with 200 mL of dilute lidocaine (0.1%) with epinephrine (1:1,000,000) into each flap. Prior to elevation 16 of the flaps were treated with syringe-assisted liposuction, while the remaining flaps served as controls. One week postoperatively the viability of the flaps was assessed with computer assisted planimetry of the necrotic area and by measurement of capillary blood flow using radioactive microspheres. The mean area of necrosis was nearly identical in the flaps treated with liposuction and in the control flaps, and there was no statistically significant difference in blood flow between the groups. The results show that liposuction performed with the "super-wet" or "tumescent" technique can be done prior to elevation of flaps in pigs without increasing the risk of flap necrosis. PMID- 8638494 TI - Aesthetic aspects of reconstructive surgery of the lower lid. AB - Aesthetic repair of the lower eyelid should use palpebral skin and restrict the cutaneous scars to the orbital area. Except in young patients or those having previously undergone a blepharoplasty, it is usually possible to raise a 10- or 12-mm-wide flap from the upper eyelid. The use of such a flap lined with an alar chondromucosal graft is advocated in a one-stage procedure. This graft ensures a good functional result and the stability of the new eyelid because the cartilage is as high in its bulk as the lid. In spite of histologic differences, where the tarsus is not a cartilage and the inner lining of the ala nasi is not actually a mucosa, the alar chondromucosal graft is very much like the tarsoconjunctival complex. When the alar defect is accurately repaired, no deformation of the nose results. PMID- 8638495 TI - Side-to-side earlobe variations with respect to surface area and shape: a quantitative study. AB - Side-to-side earlobe variations, based on the size of the earlobe surface area and a classification into earlobe types, were quantitatively analyzed in 58 female students, ranging from 20 to 22 years old. Using a new earlobe measurement technique, a closed-circuit outline of each ear was directly traced onto a clear polyethylene sheet, after which the surface area of the earlobe portion of each ear was identified, measured by a digital planimeter, and analyzed. Results revealed that side-to-side differences in the size of the earlobe surface area existed in all subjects. In contrast, based on classifying the earlobe shape into three types (i.e., tapering, square, or pendulous), differences in the mean earlobe surface area value among the three types were not significant. However, when the classification of the earlobe shape was reduced to only two types (pendulous or nonpendulous), the mean earlobe surface area value between the two types was highly significant. These results thus suggest that for a quantitative analysis of earlobe variations, classification of the earlobe shape into two types rather than into three types is the better method. This method makes it easy to distinguish earlobe differences. Further, if the earlobe differences are noted before ear piercing, a balanced ear piercing effect can be achieved. PMID- 8638496 TI - The family physician's role in adolescent idiopathic scoliosis. PMID- 8638497 TI - Hypertension and heart disease: how much is enough? PMID- 8638498 TI - Behavioral management of patients with dementia. PMID- 8638499 TI - Immune response to Pityrosporum orbiculare and seborrheic dermatitis. PMID- 8638500 TI - Blood pressure readings in the elderly: seated or standing? PMID- 8638501 TI - Transvaginal ultrasonograph and endometrial thickness. PMID- 8638502 TI - AAFP's role in public education about antibiotic resistance. PMID- 8638503 TI - HIV, Ebola virus and public health measures. PMID- 8638504 TI - Use of quinine for leg cramps. PMID- 8638505 TI - Cost of vaginal delivery vs. repeated cesarean section. PMID- 8638506 TI - Management of hypertension, Part I. AB - Elevated systolic blood pressure increases the risk of a coronary heart disease event to as great or greater a degree as elevated diastolic blood pressure. The fifth Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure has incorporated systolic blood pressure into the new definitions of hypertension. Although the syndrome of white coat hypertension has been well defined, physicians should still rely on casual blood pressure readings taken in the office or clinic for treatment decisions. The levels of blood pressure used to determine risk or estimate prognosis have been based on office or clinic blood pressures. The JNC's suggestion that diuretics or beta blockers be the preferred initial therapy is based on evidence from long-term trials showing that these medications reduce morbidity and mortality not just for cerebrovascular, but also for cardiovascular diseases. The possibly adverse effects of diuretics or beta blockers on lipids and glucose metabolism have probably been overemphasized. PMID- 8638507 TI - Identification and assessment of the slowly growing child. AB - Reliable measurements taken by trained personnel using appropriate equipment are essential in assessment of the slowly growing child. Linear growth plotted on appropriate statistical charts and expected growth based on parental heights are indicators of inappropriately reduced linear growth. Children older than three years of age who grow less than 1.75 in (4.5 cm) per year should be evaluated. Family and personal medical history (including prenatal and birth information) are important in the identification of familial short stature, constitutional delay and other causes of proportionate growth disorders. Other conditions, including chromosomal disorder, systemic disease and endocrine dysfunction, may be discovered during physical examination or appropriate laboratory investigation. Disproportionate growth of the limbs and trunk suggests the presence of a bone or collagen disorder. Bone age data based on radiographs of the left hand may assist in predicting the child's final height. PMID- 8638508 TI - Lower extremity bursitis. AB - Bursitis is a common cause of lower extremity pain in patients presenting to primary care physicians. Several bursae in the lower extremity account for most of these injuries, including the ischiogluteal, greater trochanteric, pes anserine, medial collateral, prepatellar, popliteal and retrocalcaneal. Often the symptoms are mild, with the patient successfully self-treating through activity modification and other conservative measures. A systematic approach to the evaluation and treatment of patients with bursitis, including prevention, relative rest, ice, compression, elevation, anti-inflammatory medication and treatment modalities such as ultrasound and electrical stimulation, combined with a structured rehabilitation program, will greatly facilitate the healing process. PMID- 8638509 TI - Adolescent idiopathic scoliosis: an update. AB - Adolescent idiopathic scoliosis is defined as a lateral spinal curvature of greater than 10 degrees, for which no pathologic cause can be determined. The initial assessment of adolescents with scoliosis focuses on identification of any treatable underlying pathology. Adolescents with scoliosis typically are asymptomatic and have normal neurologic and physical examinations, with the exception of curvature of the spine. Treatment strategies are determined by the risk of progression. This risk depends on the extent of the curvature and anticipated future spinal growth. The extent of the curvature may be estimated by use of a scoliometer and verified by calculation of the Cobb angle on radiographic evaluation. Skeletal maturity may be estimated by several methods, including radiologic estimates of ossification by bone atlas or Risser sign. Treatment strategies include bracing and surgery. PMID- 8638510 TI - Initial management of auricular trauma. AB - The exposed and unprotected position of the auricle makes it susceptible to injuries. Because many of these injuries are initially managed in a primary care setting, family physicians should have an understanding of the management of auricular hematomas, lacerations, abrasions and thermal injuries. Auricular hematoma requires prompt drainage and pressure applied to the site for several days. Treatment of lacerations and abrasions includes meticulous cleaning and minimal debridement of viable tissue. Thermal injuries require recognition of the extent of injury. Prompt treatment of auricular injuries will help avoid aesthetic and functional complications. PMID- 8638511 TI - Azapirones: an alternative to benzodiazepines for anxiety. AB - The azapirones are a relatively new class of psychotherapeutic drugs with both anxiolytic and antidepressant properties and a favorable benefit-to-risk ratio. They represent a significant advance in psychotherapeutic drug development. Buspirone, the only azapirone currently in clinical use, is a partial serotonin agonist with low abuse potential, no sedative effects, no cognitive or psychomotor impairment properties and no significant withdrawal symptoms. It is well-tolerated by elderly patients. Clinical indications for which buspirone is particularly appropriate are chronic anxiety and mixed anxiety/depression states. Buspirone has demonstrated some efficacy in the treatment of a broad range of other serotonin-related disorders. PMID- 8638512 TI - AAFP position paper: fluoridation of public water supplies. AAFP Commission on Public Health. PMID- 8638513 TI - AAP tuberculin testing recommendations. PMID- 8638514 TI - Some statistical procedures for analytical method accuracy tests and estimation. AB - In 1974 the National Institute for Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration joined to complete exposure standards promulgated by federal regulations. In that effort NIOSH scientists developed an accuracy criterion (AC) and a statistical protocol for evaluating fulfillment of that AC by an analytical method. This article extends that foundation and proposes a new approach to accuracy analyses. It concentrates on the case of known bias, but attempts to bridge the procedures from that case to one in which the bias is estimated. The article emphasizes a general and flexible approach to the design and analysis of more informative and effective method accuracy studies. These empower the user/investigator to design and analyze studies to be most useful and informative for specific requirements. PMID- 8638515 TI - Corrections to the target and critical values for the National Institute for Occupational Safety and Health validation tests. AB - In 1974 the National Institute for Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration joined to complete exposure standards promulgated by federal regulations. In that effort NIOSH scientists developed an accuracy criterion (AC) and a statistical protocol for evaluating its fulfillment. That AC and those procedures have been widely used ever since. This article presents corrections to the target and critical coefficients of variation published as part of the statistical protocol. PMID- 8638516 TI - An isoinertial predictor for maximal acceptable lifting weights of Chinese male subjects. AB - The practicality of using restrained as well as standard isoinertial strength tests in predicting the maximum acceptable weight of lift (MAWL) of Chinese male subjects was examined using a restrained strength test, which allows the subject to pull on the load in front of the body and to apply force in a functional free posture. Prediction models were constructed and evaluated under task conditions of two lifting ranges, two box sizes, and three lifting frequencies. The following measurements were taken for each subject in each test: (1) the body posture and joint angles, and (2) electromyographic activities of the arm muscles (biceps brachii), back muscles (erector spinae), and leg muscles (rectus femoris). Results indicate that the restrained knuckle height isoinertial strength (RK) was related significantly to the MAWL for all tasks, and the correlation coefficients ranged from 0.42 to 0.69. A step-wise multiple regression analysis based on data from the 22 subjects showed that RK, chest circumference, and thigh length accounted for 90 to 94% of the variance. A validation effort on a different sample (N = 10) showed that the MAWLs can be predicted within an absolute mean (SD) error of 0.84 (3.30) kg. Percentages of muscle activation levels indicated that, other than strength from the erector spinae, the strongest contribution to the RK value was that of biceps brachii strength. Since the strength of the body's upper extremity was also recruited, the weak upper extremity strength of Chinese subjects was better reflected. Evidence for the existence of a close match between MAWL and RK values, and the conditions of the existence, suggest that RK is good predictor for the MAWL. PMID- 8638517 TI - Effects of 5 ppm hydrogen sulfide inhalation on biochemical properties of skeletal muscle in exercising men and women. AB - This study compared the acute effects of 5 ppm hydrogen sulfide (H2S) inhalation (50 % of its occupational exposure limit) on the biochemical properties of skeletal muscle in exercising men and women. Twenty-five healthy volunteers, 13 men and 12 women, completed two 30-minute submaximal tests at 50% of their predetermined maximal aerobic power (VO2max) while breathing 0 ppm (control) or 5 ppm H2S from a specially designed flow system in a single-blind manner. Immediately after exercise, biopsies were obtained from the vastus lateralis muscle under local anaesthesia. They were subsequently analyzed for concentrations of the following markers of anaerobic and aerobic metabolism: lactate (La), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome oxidase (CytOx). Repeated measures analysis of variance indicated that in men, the CS concentration decreased significantly (p = 0.006) as a result of H2S exposure. There was also a tendency for their La and LDH concentrations to increase and CytOx concentration to decrease in the presence of H2S, but these changes were not significant (p > 0.05). In women no significant changes were observed in any of these biochemical properties. These results suggest that (1) exposure to H2S at 50% of its OEL might inhibit aerobic metabolism during exercise in healthy men, thereby increasing their dependency on anaerobic metabolism; and (2) there could be a significant gender difference in the acute response to sub-OEL exposures of H2S. PMID- 8638518 TI - Experimental and numerical studies on the impact of work practices used to control exposures occurring in booth-type hoods. AB - The observation that the between-worker variance component of exposure is significant for those performing the same tasks suggests that work practices are an important determinant of exposure. Decisions to implement engineering controls may be less than optimal if these work practices are not carefully identified. This study examines the position of the worker with respect to an object and the airflow direction in a large booth-type hood, and its implications for control of exposure. Experiments are conducted in a wind-tunnel using a mannequin and tracer gas techniques to measure exposures in the various positions at different air velocities. Smoke-wire, flow-visualization techniques are employed to correlate the exposures with the airflow patterns. Numerical predictions of these flow patterns and exposures compare favorably with experimental data, despite limitations. Further work is underway to examine more realistic situations such as spray-painting applications. PMID- 8638519 TI - Adjusting the evaluation limit for broad-band whole-body vibration. AB - The vibration levels of 87 axes in 29 conventional vehicles were measured, recorded, and analyzed. The mean deviation of the overall frequency-weighted vibration levels (Lw) and the greatest single one-third octave band frequency weighted vibration levels (Lwi-max) in corresponding axes were statistically calculated. The results showed that the vehicle vibrations were broad-band vibrations. The mean deviation of Lw - Lwi-max is 6 dB. This article suggests a 6 dB value as an appropriate adjustment value for evaluating broad-band whole-body vibrations of vehicles when using the most sensitive limits given in International Organization for Standardization document 2631/1. PMID- 8638520 TI - Assessment of worker exposure to airborne molds in honeybee overwintering facilities. AB - Airborne fungi in honeybee overwintering and equipment cleaning facilities were enumerated and identified to determine worker exposure during cleaning and routine beekeeping operations. Testing was prompted by observations of extensive mold growth on dead bees and associated material and by results of a preliminary study at one Alberta beekeeping facility that showed very high numbers of mold colonies on air samples taken during worker activity. To evaluate whether high mold counts were indicative of a problem at a single site or were industry wide, approximately 120 air samples were collected with a Reuter centrifugal sampler inside 10 overwintering facilities before and during routine beekeeping activity during fall, winter, and spring periods. A set of 30 samples was collected from 15 sites used for annual equipment cleaning. This study showed that average spore counts per overwintering site ranged from 238 to 1442 colony-forming units (CFU)/m3 prior to disturbance by workers and from 2200 to 13,931 CFU/m3 while workers swept up dead bees. Levels of airborne molds recovered during annual cleaning of beekeeping equipment ranged from 300 to 54,700 CFU/m3 with an average of 16,083 CFU/m3. Potentially toxigenic, pathogenic, or allergenic molds were recovered at all sites. Since the data indicate that exposure to high levels of airborne molds is widespread throughout the industry, actions that might help minimize worker exposure are discussed. PMID- 8638521 TI - Effect of nitrates on myocardial remodeling after acute myocardial infarction. AB - Nitrates are effective for the therapy of acute coronary syndromes, including acute myocardial infarction. Their application in acute infarction has established that vasodilators are beneficial provided hypotension is avoided. Nitrates limit early ventricular remodeling in infarction. New dosing strategies and formulations that permit chronic use after infarction with less tolerance might limit late remodeling. Over the last decade, the demonstrated effectiveness of angiotensin-converting enzyme (ACE) inhibitors in limiting ventricular dilation postinfarction has generated controversy over the usefulness of nitrates for that indication. The uncertainty has been intensified by 2 large mortality trials that tested both agents as adjuncts to conventional therapy. These trials were not designed to test whether nitrates might limit remodeling. Mechanistic experimental and clinical studies that tested whether nitrates or ACE inhibitors could effectively limit ventricular remodeling showed that both improved remodeling endpoints. However, experimental studies raise some concern about the decrease in infarct collagen associated with ACE inhibition and emphasize the fact that final outcome represents a balance of effects. That nitrates do not decrease infarct collagen could be important. Nitrate-induced early recruitment of ventricular function after late reperfusion of acute infarction might also be important. In the mortality trials, >50% of patients received open-label nitrates as per indication. Thus, the trial results to date do not suggest that nitrates are ineffective for remodeling, but rather that ACE inhibitors can confer added benefit. There has been no large clinical trial to test the efficacy of nitrates for remodeling as there has been for ACE inhibitors. PMID- 8638522 TI - Nitric oxide and nitrovasodilators: similarities, differences, and interactions. AB - The endothelium functions as a semipermeable membrane separating the blood from the body and allowing the transport of macromolecules from the blood to the interstitial space. The endothelium secretes a number of diffusible substances. These include endothelium-derived relaxing factor (EDRF), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin, in addition to vasoconstrictors including endothelin, angiotensin, and endothelium-derived contracting factor. EDRF is now known to be nitric oxide, or a closely related molecule, which affects signaling by stimulation of soluble guanylate cyclase, causing increased intracellular levels of cyclic guanosine monophosphate (cGMP), in turn leading to relaxation of vascular smooth muscle as well as a variety of additional effects that include altered function of platelets and cardiac myocytes. Nitric oxide can be made available to cellular elements in two ways: by endogenous synthesis via one or more of the three nitric oxide synthases now known to exist in mammalian species; or by exogenous administration of pharmacologic sources of nitric oxide, usually as organic nitrate vasodilators that can be metabolically converted to biologically activated nitric oxide. This process appears to require free sulfydryl groups. The metabolic machinery necessary to convert organic nitrates to a biologically active form exists mainly in the vasculature and not in the myocardium. Numerous studies have demonstrated that the presence of coronary artery disease is associated with interruption of the endogenous production of nitric oxide. Under these circumstances, exogenous nitrates still produce coronary vasodilation as well as relaxation of vascular smooth muscle in the periphery. Other articles in this supplement will focus on the vascular effects of nitric oxide and nitrovasodilators; this article will conclude with a brief discussion of the role of the nitric oxide pathway in the control of cardiac autonomic responsiveness and the potential role of cytokines and the nitric oxide pathway to impair the ability of the myocardium to respond to catecholamines or other stimuli with a normal increase in contractile function. PMID- 8638523 TI - New insights into mechanisms underlying nitrate tolerance. AB - The hemodynamic and anti-ischemic efficacy of organic nitrates is rapidly blunted due to the development of nitrate tolerance. The mechanisms underlying this phenomenon remain poorly understood and likely involve several independent factors. More recent experimental observations suggest that tolerance may be the consequence of intrinsic abnormalities of the vasculature, including enhanced vascular superoxide and endothelin production. Superoxide anions degrade nitric oxide derived from nitroglycerin, whereas autocrine-produced endothelin within vascular smooth muscle sensitizes the vasculature to circulating neurohormones, such as catecholamines and angiotensin II, all of which may compromise the vasodilator potency of nitroglycerin. Interestingly, these vascular consequences of in vivo nitroglycerin treatment can be mimicked by incubating cultured endothelial and smooth muscle cells with angiotensin II. Further, nitrate tolerance and rebound following sudden cessation of prolonged nitroglycerin therapy can be prevented by concomitant treatment with high-dose angiotensin converting enzyme inhibition or angiotensin-I receptor blockade. These data strongly suggest that increased circulating levels of angiotensin II, which are encountered during in vivo nitroglycerin treatment, initiate cellular events that ultimately attenuate the nitroglycerin vasodilator effects during prolonged treatment periods. PMID- 8638524 TI - Beneficial actions of nitrates in cardiovascular disease. AB - Nitroglycerin and the long-acting nitrates have been used in cardiovascular medicine for >100 years. Nitrates are widely utilized for the various anginal syndromes and are also used in congestive heart failure and patients with left ventricular dysfunction. The potential mechanisms for relief of myocardial ischemia with nitrates are multiple. The nitrovasodilators are a related group of drugs that result in the formation of nitric oxide (NO) within vascular smooth muscle cells. NO stimulates the enzyme guanylate cyclase, which results in increases in cyclic guanosine monophosphate and vasodilation. In the presence of atherosclerosis, endothelial dysfunction is ubiquitous and associated with decreased NO availability, probably due to increased destruction of NO by free radical anions. Nitrovasodilators, including the nitrates, supply exogenous NO to the vascular wall and improve the vasodilator state. When nitrates are administered, endothelial-dependent stimuli cause relaxation rather than constriction in the setting of endothelial dysfunction. Nitrates also have antiplatelet effects, and recent evidence confirms that these drugs decrease platelet aggregation and thrombosis formation. This may play an important role in the therapy of acute unstable myocardial ischemia, including unstable angina and myocardial infarction. Nitrate hemodynamic effects have been long known. They are primarily modulated through a decrease in myocardial work that results from smaller cardiac chambers operating with lower systolic and diastolic pressures. These changes are caused by a redistribution of the circulating blood volume away from the heart to the venous capacitance system, with a fall in venous return to the heart. The afterload or arterial effects of nitrates are also useful in decreasing myocardial oxygen consumption. Considerable evidence confirms a variety of mechanisms whereby nitrates increase coronary blood flow, including epicardial coronary artery dilation, stenosis enlargement, enhanced collateral size and flow, improvement of endothelial dysfunction, and prevention or reversal of coronary artery vasoconstriction. These effects help increase nutrient coronary blood flow to zones of myocardial ischemia. Recent data with the nitroglycerin patch confirm that myocardial ischemia is decreased after nitrate administration. Nitroprusside, another nitrovasodilator, is a commonly used intravenous agent for lowering arterial pressure and left ventricular filling pressure. This drug is highly effective for the treatment of acute or severe hypertension and congestive heart failure. However, there are data suggesting that nitroprusside may be deleterious in the presence of acute myocardial ischemia, perhaps by shunting blood away from zones of jeopardized myocardial blood flow. Therefore, nitroprusside cannot be recommended to treat myocardial ischemia; intravenous nitroglycerin should be used in this context. PMID- 8638525 TI - Organic nitrates: new formulations and their clinical advantages. AB - The organic nitrates have been used for more than a century in the management of patients with myocardial ischemia. The most commonly used agents at this time include nitroglycerin, isosorbide dinitrate, and isosorbide-5-mononitrate. These agents all exert their therapeutic effects through biodegradation to nitric oxide, which stimulates guanylate cyclase in vascular smooth muscle cells with the production of cyclic guanosine monophosphate. The latter induces vasodilation by reducing the availability of ionized calcium to the contractile proteins. Tolerance to the organic nitrates occurs when the agents are administered in an attempt to provide therapeutic effects throughout 24 hours each day. There are probably several mechanisms responsible for nitrate tolerance, but there is no evidence at this time that concurrent medications will modify the development of tolerance. The only available method at this time is to give these agents intermittently to provide a period of washout. In so doing, it is possible to provide therapeutic nitrate effects for approximately 12 hours throughout each 24 hour period. PMID- 8638526 TI - Nitrates in the treatment of congestive heart failure. AB - Nitrates have been widely used for the treatment of patients with chronic congestive heart failure. Although the use of these drugs has not been evaluated by large-scale studies traditionally used for evaluation of new therapy, multiple studies over the years have demonstrated their favorable effects. Organic nitrates have been shown to have a beneficial effect on ischemia, hemodynamic profile, magnitude of mitral regurgitation, endothelial function, and cardiac remodeling. These drugs alone or in combination with hydralazine have improved exercise capacity, maximal oxygen consumption, cardiac function, and survival. The use of nitrates in patients with heart failure has been limited by reduced responsiveness (resistance) and early development of tolerance. Nitrate resistance is due to reduced vascular response and results in the need to use a larger dose of any nitrate preparation when used for the treatment of patients with heart failure compared to patients without heart failure. Recent information suggests that nitrate tolerance is caused by increased levels of superoxide at the vascular wall, which leads to reduced nitric oxide level and to increased sensitivity to vasoconstrictive mechanisms, such as endothelin and angiotensin II. Intermittent dosing of nitrates allowing a 12-hour nitrate-free interval is effective in preventing nitrate tolerance and is, therefore, recommended. Recent information suggests that augmentation of nitrate dose by the use of an escalating dose regimen and a concomitant use of hydralazine can prevent or overcome the effect of nitrate tolerance in patients with heart failure. PMID- 8638527 TI - The renin-angiotensin system in left ventricular remodeling. AB - Left ventricular remodeling is a dynamic process that occurs in reaction to an insult to the myocardium. The response to either loss of cells, as may occur following myocardial infarction, or to hemodynamic overload, as may occur in aortic stenosis, is an attempt to maintain cardiac output and normalize wall tension. This is accomplished through the activation of the renin-angiotensin system and hypertrophy of noninfarcted segments of the myocardium. in the case of moderate or large infarctions these mechanisms fail to normalize wall stress. The stimulus to further remodeling remains, viable myocytes hypertrophy (with greater increases in cell length than width), the mass-to-volume ratio increases, and an exponential increase in wall stress results. This increase in myocyte tension has been associated with premature myocyte cell death. Thus, a vicious cycle is established wherein overstretch of the myocardium while sustaining cardiac output leads to progressive myocyte loss and left ventricular dilation. The renin angiotensin system plays an integral role in this process. Its inhibition by angiotensin-converting enzyme (ACE) inhibitors both chronically and immediately after myocardial infarction has been shown to decrease left ventricular volumes and reduce mortality. Controversy exists regarding the mechanism through which ACE inhibitors exert their effects. ACE inhibitors reduce afterload/preload, circulating angiotensin II levels, and raise circulating levels of bradykinin. It is not yet clear which mechanism is responsible for the greatest impact on left ventricular dilation and mortality. inhibition of the renin-angiotensin system is clearly beneficial to cardiac performance as well as morbidity and mortality when myocardium is lost and heart failure ensues. Specific modes of action require further definition, including local and systemic effects. Possible benefits of angiotensin receptor blockade versus augmentation of bradykinin requires definition, setting the stage for further study, while the beneficial therapeutic use of these agents continues. PMID- 8638528 TI - PSA divergence. A new parameter for the accurate longitudinal assessment of prostatic disease. AB - Prostate-specific antigen (PSA) and the various parameters derived utilizing this marker have increased our ability to diagnose early prostatic disease; however, their accuracy in identifying the etiology of the disease remains somewhat limited. We propose a new PSA derivative, termed "PSA divergence" (PSADI), defined as the change in serum PSA over time (years) divided by the change in prostatic volume over time (years), to more accurately distinguish benign, premalignant, and malignant prostatic diseases. In this study, we evaluated 160 subjects with a PSA >4.0 ng/ml who were found by transrectal ultrasound-guided biopsy (TRUS) to have either benign prostatic hyperplasia or prostatic intraepithelial neoplasia. These men were followed at 6 or 12 months with serial PSA, digital rectal exam (DRE), and TRUS with rebiopsy. Data analysis demonstrated a statistically significant (p < 0.05) correlation between PSADI and each final pathologic outcome, suggesting that PSADI is useful in distinguishing among intraepithelial neoplasia and benign and malignant prostatic disease. PMID- 8638529 TI - Management of stage I-B, II-A, and II-B carcinoma of the cervix with high-dose rate brachytherapy: initial results of an institutional clinical trial. AB - In 1989, the University of Miami began a program incorporating high-dose-rate (HDR) brachytherapy into the definitive treatment of patients with invasive carcinoma of the cervix. Patients received an average total dose to point A of 5,511 cGy (range 4,280-6,360 cGy) in an average of 57 days (range 39-84 days). An analysis of the first 24 cases found 11 FIGO Stage I-B, four Stage II-A, and nine Stage II-B tumors. At the end of all radiation therapy, 19/24 patients' tumors (79.2%) had undergone a clinical complete response (CR). With median follow-up of 26 months (range 14-63 months), three have relapsed locally, two regionally, and six in extrapelvic sites. Almost two-thirds of all failures occurred in patients with tumors >4 cm, who also took more than 8 weeks to complete their treatment. Overall 2-year actuarial survival for the entire study group is approximately 74%. A univariate analysis determined that clinical stage (P = 0.02), overall treatment time (P = 0.03), tumor size (P = 0.05), and response at the end of therapy (P = 0.005) were significant prognostic factors. Multivariate analysis showed that tumor response to therapy was the most important prognosticator of outcome (P = 0.001). Besides five cases of apical vaginal stenosis, there have been no reported chronic complications in this cohort of patients. A prospectively randomized trial is recommended to compare the efficacy of HDR vs. low-dose-rate brachytherapy in cervical carcinoma. PMID- 8638530 TI - Treatment of metastatic adrenal cortical carcinoma with etoposide (VP-16) and cisplatin after failure with o,p'DDD. Clinical case reports. AB - A 42-year-old woman with liver metastasis from adrenal cortical carcinoma was treated with mitotane (o,p'DDD) for 11 months with a minimal response. Mitotane was stopped because of severe adverse effects. Thereafter a combination of cisplatin and etoposide was given every 3 weeks. A partial response was achieved after five chemotherapy courses. Following this chemotherapy, the patient had a surgical excision of the liver metastasis and entered into a complete remission for 17 months. Etoposide plus cisplatin may be an active combination for the treatment of adrenal cortical carcinoma. PMID- 8638532 TI - Management of early-stage Hodgkin's lymphoma. The radiation oncology experience at Northwestern University/Northwestern Memorial Hospital. AB - Early-stage Hodgkin's lymphoma patients treated with radiotherapy alone or combined modality therapy were retrospectively analyzed for survival, patterns of failure, salvage, and toxicity. Of 75 evaluable patients, 47 were given radiotherapy alone and 28 were given combination radiotherapy and chemotherapy. Of the patients studied, 26 were clinical stage I and 49 were clinical stage II, with nine patients upstaged at laparotomy. Minimum follow-up was 2 years, with a median of 81 months. Complete response rate was 95%. Relapse-free survival and overall survival were 89% and 96%, respectively, at 2 years; 78% and 86% at 5 years; and 76% and 82% at 10 years. Of 16 patients who relapsed (21%), 13/47 patients were treated with radiotherapy and 3/28 were treated with combined modality therapy. Salvage rates were higher in those treated with radiotherapy alone. There were 13 deaths: six from disease, two from treatment-related complications, and five from second primary malignancies. There was a higher incidence of second malignancies and deaths due to complication in patients treated with combined modality therapy. Radiotherapy alone or with chemotherapy is an effective modality in the treatment of Hodgkin's lymphoma. Treatment should be selected properly to optimize results and decrease complications. PMID- 8638533 TI - A pilot trial of 5-FU, leucovorin, and cisplatin with or without adriamycin in advanced cancer. AB - In a pilot trial, we treated patients with solid tumors with 5-FU (5 fluorouracil), leucovorin (LV), and cisplatin (FLP) with Adriamycin in selected patients (FLAP). 5-FU/LV were administered weekly for 6 weeks, with cisplatin at two dose levels (and Adriamycin in some patients) at weeks 1 and 4. Nine patients received FLP; 11 received FLAP.FLP was able to be administered with a cisplatin dose of 75 mg/m2; the maximum-tolerated dose of FLAP included a cisplatin dose of 60 mg/m2. Significant toxicities included granulocytopenia, thrombocytopenia, and diarrhea. Preliminary activity was demonstrated with FLAP in patients with adenocarcinomas of the stomach and gastroesophageal (GE) junction. Ongoing trials of FLAP are underway in patients with gastric and GE junction carcinomas in the neoadjuvant and advanced disease settings. PMID- 8638531 TI - Prophylactic filgrastim (G-CSF) during mitomycin-C, mitoxantrone, and methotrexate (MMM) treatment for metastatic breast cancer. A randomized study. AB - Patients with metastatic breast cancer were randomly assigned to receive as second-line chemotherapy either MMM (mitomycin 8 mg/m2 day 1; mitoxantrone 8 mg/m2 days 1 and 22; methotrexate 35 mg/m2 days 1 and 22) alone or in combination with filgrastim (5 micrograms/kg s.c. days 4-17, 24-37). The courses were repeated every 42 days for a maximum of six courses. Thirty-one patients are evaluable for safety and efficacy. The 16 patients in the filgrastim arm received a total of 42 cycles compared with 34 cycles in the 15 control patients. Tumor responses were few in both patient groups (one partial response in the filgrastim group and two partial responses in control group). Nevertheless, a difference in survival was seen (filgrastim median 10.7 months, control median 6.5 months; p = 0.02 log rank). The treatment was well tolerated. Doses were reduced six times in the filgrastim arm and eleven times in the control arm. Grade IV neutropenia was seen in four patients in the filgrastim arm and in twelve patients in the control arm. The observed survival benefit needs to be confirmed in a larger patient group. PMID- 8638534 TI - Phase I study of a carboplatin-cisplatin-etoposide combination chemotherapy in advanced non-small-cell lung cancer. AB - Cisplatin (CDDP)-containing chemotherapy has become the mainstay of clinical trials in unresectable non-small-cell lung cancer (NSCLC), but the role of chemotherapy in the routine management of NSCLC remains controversial. We conducted a phase I study with the combination carboplatin (CBDCA), CDDP, and etoposide (Etop) in unresectable NSCLC. CBDCA, at a starting dose of 80 mg/m2, on day 1, was combined with a fixed dose of CDDP (80 mg/m2, day 1) and Etop (80 mg/m2, days 1-3). Escalation was performed after four patients entered at each dose level. If no World Health Organization (WHO) grade 4 toxicity developed after the first cycle in more than half of the patients or WHO grade 3/4 toxicity in more than two thirds, the dose was escalated. The maximum tolerated dose was established at 300 mg/m2 for CBDCA. Thrombocytopenia and leukopenia were the dose limiting toxicities. No grade 3/4 nonhematologic toxicities were seen. The recommended dose of CBDCA to be combined with CDDP (80 mg/m2, day 1) and Etop (80 mg/m2, days 1-3) is 280 mg/m2. This trial suggests that our combination chemotherapy may be effective in patients with advanced NSCLC. A multicenter phase II study based on these findings is now under way. PMID- 8638535 TI - Phase I-II trial of recombinant interferon alpha-2b with cisplatin and 5 fluorouracil in recurrent and/or metastatic carcinoma of head and neck. AB - The aims of this study were to establish the feasibility and toxicity of the biochemical modulation of the cisplatin (CDDP)-5FU combination by interferon alpha-2b (INF), and to assess its therapeutic efficacy in recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). The mandatory eligibility criteria included histologically proven SCCHN; a performance status <2; adequate bone marrow, hepatic, renal, and cardiac functions; and measurable and/or evaluable disease. The protocol was CDDP, 100 mg/m2 i.v. day 1; 5-FU, 1,000 mg/m2 in a c.i.v. infusion over 96 h; and INF 3.10(6) U/day s.c., begun 2 h before cisplatinum for 5 consecutive days, repeated every 3 weeks. Twenty patients were included and received 76 cycles (median number cycles/patient = three). Eighteen patients were evaluable for activity with an overall response rate (RR) of 30% [2 complete responses (CR) + 4 partial responses (PR)], which was 55% (5/9) in previously untreated and 9% (1/11) in previously treated patients. Myelosuppression (50%), mucositis (40%), loss of electrolytes (15%), and asthenia (20%) were the most frequent severe toxic effects. Notwithstanding, the protocol was feasible and well tolerated in this overall population with a poor prognosis. Median duration of response was 8 months, and median survival for the overall population was 8.5 months. This schedule is the test arm of an ongoing international multicentric phase III trial versus standard CDDP-5FU in the same SCCHN population. PMID- 8638537 TI - Long-term cure of a non-small-cell lung cancer treated by monochemotherapy. AB - Cure after 8 years of a non-small-cell lung cancer (NSCLC), inoperable because of locoregional spread, is reported. The authors believe this case represents the longest survival after monochemotherapy reported to date and provides the opportunity to review studies of prognostic factors concerning long-term survival in NSCLC. PMID- 8638538 TI - Concurrent 5-fluorouracil, daily low-dose cisplatin, and radiotherapy in stage IIIB cervical cancer. A phase II prospective study. AB - Between June 1987 and May 1991, 30 patients with Stage IIIB cervical cancers were treated using synchronous radiotherapy, 5-fluorouracil (5-FU), and daily low-dose cisplatin. External radiotherapy (3,600-3,960 cGy) was given to the whole pelvis in 4 weeks. Two courses of intracavitary brachytherapy were given 2 weeks later. Parametrial boost was then given. Continuous infusion of 5-FU 750 mg/m2 was given for 5 days during the first and third week of pelvic irradiation. Cisplatin (6 mg/m2) was given 30 min before every irradiation in the second and fourth week. The complete response rate was 87%. The 3-year local control rate was 77%. The 3 year overall and disease-free survival rate was 66% and 56%, respectively. Distant metastases were the major causes of treatment failure. Toxicities were acceptable. Our preliminary results indicate that this synchronous combination treatment is feasible. Further follow-up is required to determine whether this regimen has a genuine favorable impact on survival and chronic toxicity. PMID- 8638539 TI - Fludarabine: a phase II trial in patients with previously treated low-grade lymphoma. AB - The aim of the study was to investigate the therapeutic activity of Fludarabine in patients with low-grade non-Hodgkin's lymphoma (LG-NHL) no longer responding to standard treatment. In this Phase II study patients were treated with Fludarabine 25 mg/m2 intravenously daily for 5 days repeated at 28-day intervals. Twenty-two patients with LG-NHL, no longer responding to standard treatment, were entered in the study. Among twenty-one evaluable patients, seven had a complete and six a partial response. The median time to treatment failure and survival time are 4.6 months and >28.0 months, respectively. The most important toxicity was hemogram suppression, which was usually manageable but occasionally severe. Fludarabine is not only an active agent with definite therapeutic value in patients with treatment-resistant LG-NHL, but effective and well tolerated in patients no longer responding to standard treatment. PMID- 8638536 TI - The role of neoadjuvant intraarterial infusion chemotherapy with cisplatin and bleomycin for locally advanced cervical cancer. AB - To clarify the effect of neoadjuvant intraarterial infusion chemotherapy on the cure rate in advanced cervical cancer with bulky tumor, a total of 50 patients were examined prospectively. The clinical stage according to the International Federation of Gynecology and Obstetrics (FIGO) classification included 23 IIb, 6 IIIa, and 21 IIIb. These patients were randomly divided into the neoadjuvant intraarterial infusion chemotherapy group and the control group. There were no significant differences in mean age, FIGO clinical stage, and tumor histology between groups. Twenty-five patients in the former group were given 25 mg/m2 of cisplatin and 15 mg/m2 of bleomycin via each internal iliac artery. If the results of the evaluation indicated that surgery was feasible, radical surgery was performed. The patients whose tumors were inoperable received radiation therapy consisting of external irradiation and intracavitary irradiation. Twenty five patients in the control group also underwent the same radiation therapy. The overall response rate was 80.0%. Eighteen of 20 responders underwent surgery. The 3-year survival rate was 85.7% for operated patients, 42.9% for patients receiving neoadjuvant intraarterial infusion chemotherapy followed by irradiation, and 49.5% for the control group. In the present study, neoadjuvant intraarterial infusion chemotherapy did not improve the prognosis of patients with advanced cervical cancer compared to radiation therapy alone, and only responders who underwent surgery obtained an advantage in survival. PMID- 8638540 TI - TPA, TATI, CEA, AFP, beta-HCG, PSA, SCC, and CA 19-9 for monitoring transitional cell carcinoma of the bladder. AB - A total of 76 patients with transitional cell carcinoma of the bladder were prospectively monitored with simultaneous serum value estimations of tumor polypeptide antigen (TPA), tumor-associated trypsin inhibitor (TATI), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (beta-HCG), prostatic specific antigen (PSA), squamous cell carcinoma antigen (SCC), and CA 19-9 in different stages and phases of their disease. In locally advanced disease positive values were noted for TATI in 22/28 patients (78.5%), for TPA in 17/28 (60.7%), for CA 19-9 in 10/28 (35.7%), for CEA 11/28 (39.2%), for beta-HCG in 3/28 (10.7%), for PSA in 6/28 (21.4%), for SCC in 6/28 (21.4%), and for AFP in 0/28. In metastatic disease elevated levels were observed for TATI in 43/48 patients (89.5%), for TPA in 41/48 (85.4%), for CA 19 9 in 19/48 (39.5%), for CEA in 20/48 (41.6%), for beta-HCG in 6/48 (12.5%), for PSA in 7/48 (14.5%), for SCC in 8/48 (16.6%), and for AFP in 1/48 (2.1%). In metastatic disease TATI and TPA values were significantly modified in patients with complete remission and TATI, TPA, and CA 19-9 in patients with partial remission and nonresponders. In T2-T4-N0M0 tumors, TPA, TATI, CA 19-9, and CEA were significantly increased in nonresponders. In patients with complete remission, a change in serum TATI, TPA, and CA 19-9 levels cannot be evidenced with the available numbers. The concurrent determination of TATI and TPA in T2 T4N0M0 tumors and TATI, TPA, and CA 19-9 in generalized disease could predict the response to chemotherapy. This study indicates that only the determination of TATI and TPA and in some degree the CA 19-9 is a potential tool for monitoring the efficacy of treatment. PMID- 8638541 TI - Treatment of stage III-IV non-small-cell lung carcinoma with vinorelbine in combination with ifosfamide plus MESNA: a study by the Southern Italy Oncology Group (GOIM). AB - Thirty-five patients affected by stage III-IV non-small-cell lung carcinomas were treated with ifosfamide 3 gr/m2 plus MESNA as uroprotector on day 1 and vinorelbine 25 mg/m2 i.v. bolus on day 1 and 8. This cycle was repeated every 21 days. Over a total of 35 evaluable patients, the overall response rate was 34% (95% CL 18-54%). One patient experienced a complete response with a duration of 7.2+ months, and 11 patients a partial response with a mean duration of 5.9+ months. Seven patients had no change and 16 improved. The overall survival was 7.6+ months. Over a total of 145 cycles, the most frequent toxicity was myelosuppression, but grade 3 leukopenia and grade 2 thrombocytopenia were seen only in 14% and 9% of cases, respectively. Only one patient suffered grade 4 leukopenia. Gastrointestinal toxicity was minimal; only five patients (14%) complained of grade 3 vomiting. This combination regimen can be safely given on an outpatient regimen, but it is relatively active in advanced non-small-cell lung cancer. However, it should be noted that >50% of the patients in this series had a performance status of <80 and >50% were older than 65 years. PMID- 8638542 TI - Microwave thermoradiotherapy for intraocular melanoma. AB - We evaluated the use of microwave hyperthermia as an adjuvant to ophthalmic plaque radiotherapy for patients with intraocular tumors. Forty-eight patients were offered combined microwave plaque thermoradiotherapy (TRT) as a primary treatment for their uveal melanomas. A dish-shaped microwave antenna was placed on the sclera beneath the tumor at the time of plaque brachytherapy. While temperatures were measured at the sclera, the tumor's apex was targeted to receive a minimum of 42 degrees C for a 45-min duration. Patients were also given full or reduced doses of plaque radiotherapy (125I or 103Pd). We reduced the minimum tumor radiation dose (apex dose) for 42 (88%) of our patients to <70 Gy (mean, 52.5 Gy). The 48 TRT patients were followed for < or =8 years (average, 45 months). To date, there has been one documented postoperative tumor enlargement, for a 97.2% local control rate. Two patients were lost to follow-up. Four eyes have been enucleated: two due to neovascular glaucoma, one to glaucoma secondary to intraocular inflammation, and one for progressive tumor enlargement. Although 13 patients have died, only four deaths were due to metastatic melanoma. Thirty five patients (73%) have maintained within two lines or had better than their preoperative visual acuity. Side effects attributable to heating included chorioretinal scar formation within and around the targeted zone and decreased intraocular pressures without hypotony. Our experience with this heat delivery system suggests that adjuvant microwave thermotherapy can be used with plaque radiotherapy for the treatment of uveal melanoma. PMID- 8638543 TI - Taxol is active in platinum-resistant endometrial adenocarcinoma. AB - Taxol, 170 mg/m2 by 3-h infusion every 3 weeks, was administered to seven patients with primarily advanced or recurrent endometrial cancer with progressive disease on platinum analogues. By standard response criteria, there were three clinical responders (43%; 95% confidence limits, 6-80%) and one disease stabilization. All three responses were clinical partial responses. The response duration ranged from 3 to 7 months. These preliminary results suggest that taxol is active in patients with platinum-resistant endometrial adenocarcinoma. PMID- 8638544 TI - Radiation therapy for pituitary adenomas. A retrospective study of the University of Louisville experience. AB - A retrospective analysis of treatment outcome was performed on patients treated with radiation for pituitary adenomas at the University of Louisville from January 1988 to December 1992. The study population included 27 patients. Twenty received radiation as a component of their initial treatment while seven received radiation as part of their treatment for recurrent disease. Nineteen patients were treated with post-operative radiation, and eight were treated with radiation alone. Follow-up interval ranged from 1 month to 109 months, with a median of 28 months. All three patients with stage I disease were controlled with radiation alone (1/3) or combined surgery and postoperative radiation (2/3), whereas six of eight stage II patients had disease control following surgery and postoperative radiation. Both patients with stage III adenomas treated with radiation alone had local control, whereas local control was achieved in six of seven with post operative radiation. Three of five patients with recurrent disease had local control with radiation alone, whereas both patients undergoing surgery and postoperative radiation had local control. This retrospective analysis supports previous findings that radiation therapy alone or combined with transphenoidal resection is effective in long-term control of pituitary adenomas. It further suggests that immediate radiation therapy may be superior to radiation for surgical or medical failures. PMID- 8638545 TI - Recombinant interferon ALFA-2A in combination with carboplatin, vinblastine, and bleomycin in the treatment of advanced malignant melanoma. AB - Thirty-four patients with advanced malignant melanoma were treated with recombinant alpha-interferon (IFN) and chemotherapy consisting of carboplatin, vinblastine, and bleomycin (CVB). CVB was given for four cycles and IFN for 1 year or until progression. Of the 34 analyzed patients, 17 (50%) achieved an objective response, including two complete (6%) and 15 partial responses (44%). Responses were noted in cutaneous, lymph node, and pulmonary sites, with a median time to disease progression of 5 months (range, 3-20 months). The median survival from onset of therapy was 8 months (range, 1-22 months) for the 34 patients. Ninety-four percent of the patients experienced flu-like symptoms and 82% fatigue or weakness. Leukopenia grade 3-4 was observed in four patients (12%). There were two toxicity-related deaths (6%); one from bleomycin-induced pneumonitis and one from neutropenic sepsis. It is concluded that the addition of IFN to CVB regimen, in this study, showed no apparent advantage on response rates, disease-free interval, or survival. The observed treatment-related mortality was unacceptably high. IFN administered as maintenance therapy following CVB conferred no survival benefit. PMID- 8638546 TI - Randomized trial of 5-fluorouracil and high-dose folinic acid with or without alpha-2B interferon in advanced colorectal cancer. AB - We evaluated the role of low-dose alpha-2b interferon, added to chemotherapy, for advanced colorectal cancer; we randomized patients, to either a combination chemotherapy of 5-fluorouracil (5-FU) and high-dose folinic acid (HDFA) or the same regimen plus interferon. Between January 1990 and March 1992, 100 untreated patients (PTS) with advanced colorectal cancer, 53 men and 47 women, with an ECOG performance status (PS) of < or = 3, were randomized to either HDFA 200 mg/m2 iv bolus and 5FU 370 mg/m2 in 15-min iv infusion days 1-5 every 4 weeks (arm A), or the same chemotherapy plus IFN 3 x 10(6) IU subcutaneously three times a week in chemotherapy intervals (arm B). A total of 97 PTS are evaluable for response, toxicity, and survival; 3 PTS are not evaluable in arm B for major protocol violations. PTS characteristics were well balanced in both arms for age (median, 64 years), disease-free survival, and disease site. ECOG PS was 0 in 28% of PTS in arm A and in 13% in arm B. Response rates were as follows: arm A, 40%; and arm B, 23%. Median time to failure was as follows: 10.2 months arm A versus 9 months arm B. Median survival was as follows: 13.3 months arm A versus 10.9 months arm B. Grade 3 haematological toxicity was 9% of PTS in both arms. Gastrointestinal toxicity was as follows: 17% arm A versus 22% arm B. The cost of drugs expressed per m2/month was $60 in arm A and $390 in arm B. The results show that IFN at the schedule and doses employed adds no benefit to the combination of 5FU/HDFA. PMID- 8638547 TI - Adenocarcinoma of the duodenum with liver metastases. Complete remission and long term survival with 5-fluorouracil chemotherapy--a case report. AB - Primary duodenal carcinoma is an uncommon malignancy, and experience with chemotherapeutic regimes in this condition is limited. We report a case of duodenal carcinoma with liver metastases in which complete remission was achieved with 5-fluorouracil chemotherapy. The patient remains well 30 months after completing treatment. PMID- 8638548 TI - High-dose folinic acid and fluorouracil with or without ifosfamide is an inactive combination in advanced pancreatic cancer. A randomized phase II study of the Italian Oncology Group for Clinical Research (G.O.I.R.C.). AB - The Italian Oncology Group for Clinical Research (GOIRC) randomized 55 naive patients with advanced pancreatic cancer (APC) between intravenous fluorouracil (5FU) 400 mg/m2, days 1-5 and folinic acid (FA) 200 mg/m2, days 1-5 alone, using Machover's schedule, or with FU, FA, and ifosfamide (IFO) 5 g/m2, day 1 and Mesna. In both arms, treatment was repeated every 28 days. Fifty-one patients were evaluable for response. The overall response rate was 6% (3 out of 51), 1 out of 29 (3%) complete response (CR) in the arm with FU plus FA, and 2 out of 22 (9%) partial responses (PR) in the arm with IFO. The duration of response rate was 39, 55, and 74 weeks, respectively. Median survival time was 21 weeks (range, 4-83 weeks) for 5FU/FA and 16 weeks (range, 3-106 weeks) for the FU/FA/IFO arm. Diarrhea, mucositis, and vomiting occurred in the majority of patients. One patient died due to toxicity. The combination of 5FU plus FA failed to demonstrate therapeutic activity in patients with APC and was associated with moderate to severe toxicity that could lower the quality of life of these patients. Ifosfamide did not potentiate the activity of this combination. Neither of these combinations should be considered for treatment of patients with APC. PMID- 8638549 TI - Physician involvement and assessment in the development of hospital oncology programs. AB - Hospitals and other healthcare facilities are focusing resources on the care of the cancer patient. Physicians should become involved in all aspects of the development of oncology programs. Physicians need to be involved from the initial program development, through the strategic planning to the formulation of the business plan. Strong physician leadership is needed to coordinate care provided by physicians and ancillary staff. Educational programs for patients, their families, and physicians need to be developed by the medical director, physicians, and ancillary staff. Continuing evaluation and assessment of the entire cancer program is essential to monitor the achievement of goals and objectives set forth in the strategic plan. PMID- 8638551 TI - Neuroendocrine primary small cell carcinoma of the breast. PMID- 8638550 TI - Phase I/II study of concomitant irradiation and carboplatin for locally advanced carcinoma of the uterine cervix: an interim report. AB - The outcome of women treated with either definitive irradiation alone or in combination with cisplatin-based chemotherapy for locally advanced (>IIb) squamous cell carcinoma of the cervix has been disappointing. To improve upon our reported results with irradiation alone, a trial using irradiation plus carboplatin chemotherapy was designed for these patients. Twenty-seven women with unresectable squamous cell carcinoma of the uterine cervix were referred to our institution between July 1991 and September 1994. Seven of these patients were enrolled in a phase I/II protocol combining concurrent irradiation and carboplatin chemotherapy. Megavoltage irradiation was used to deliver 45-50.4 Gy to the pelvis (and paraaortic chain when nodes were involved) through a multiple field technique followed by the application of Fletcher-Suit-Delclos tandem and ovoids to boost the point A dose to 85 Gy. Chemotherapy consisted of intravenous carboplatin (60 mg/m2) administered in conjunction with irradiation to a total dose of 300 mg/m2. The enrolled patients consisted of six women with stage IIIb disease and one with stage IIa with concomitant paraaortic adenopathy. All seven patients enrolled in the study completed the planned course of treatment and tolerated the treatment without severe acute morbidities. No dose modifications were required for the radiation therapy regimen. For one patient, a dose of carboplatin was withheld to allow recovery from thrombocytopenia. The overall response rate was 100% (four complete response, three partial response). The combination of concurrent irradiation (pelvic or pelvic + paraaortic fields) and carboplatin chemotherapy can be safely administered to patients with locally advanced squamous cell carcinoma of the cervix. The treatment is well tolerated and is associated with a high rate of response. Longer follow-up will be necessary to assess the durability of response. In the meantime, we have elected to escalate the dose of carboplatin (90 mg/m2) in the hope of increasing the rate of complete response without incurring unacceptable toxicity. PMID- 8638552 TI - Race, gender, and the response to stress: autoworkers' vulnerability to long-term unemployment. AB - A three-wave panel study of auto plant closings focused on the mental health effects of unemployment on blue-collar workers. This paper explores how the impact of long-term unemployment varies across race and gender. We also examine whether other demographic factors can themselves modify the impacts of race and gender. Dependent variables include two measures of distress and two drinking measures. Results showed that the effect of long-term unemployment on distress and drinking was more severe among less educated workers, and responses of blacks were especially sensitive to level of education. In addition, men showed a greater association of long-term unemployment with depression (and to some extent anxiety) than did women. Marriage affected the responses of men but not of women, and of whites but not of blacks. Explanatory variables--the worker's experiences of financial hardship, other negative life events, and lack of a confidant- largely accounted for male-female differences. We conclude by discussing theoretical implications of these effects and address the limitations of the traditional term "vulnerability" in describing them. PMID- 8638553 TI - A meta-analysis of bibliotherapy studies. AB - Used meta-analysis to examine the efficacy of bibliotherapy. Bibliotherapy treatments were compared to control groups and therapist-administered treatments. The mean estimated effect size (d) of the 70 samples analyzed was + 0.565. There was no significant differences between the effects of bibliotherapy and therapist administered treatments, as well as no significant erosion of effect sizes at follow-up. Bibliotherapy did appear more effective for certain problem types (assertion training, anxiety, and sexual dysfunction) than for others (weight loss, impulse control, and studying problems). Overall the amount of therapist contact during bibliotherapy did not seem to relate to effectiveness, but there was evidence that certain problem types (weight loss and anxiety reduction) responded better with increased therapist contact. Recommendations for future research were given, especially for more research on the commonly purchased books and moderator analyses by personality type and reading ability. PMID- 8638554 TI - "Taking another route": daily survival narratives from mothers who are homeless. AB - Homelessness among families has become a growing social problem for communities, yet little is known about the types of daily survival strategies such families employ. This paper presents results of a qualitative study of the coping narratives of 64 mothers living in temporary emergency shelters with their children. The women reported using a variety of coping responses to daily stressful events. These included the use of direct actions and more palliative strategies. Results suggest that stress and coping theory may be useful for understanding homelessness. Implications for program development and future research are discussed. PMID- 8638555 TI - Cultural orientation and psychological well-being in Chinese Americans. AB - Examined cultural orientation in the domains of language proficiency, cultural activity, and social relationship, and its relationship with psychological well being in a group of 143 Chinese Americans in San Francisco. Four indicators of psychological well-being were utilized, two assessing the experience of distress (depression level and negative affect) and two assessing positive well-being (positive affect and life satisfaction). Domain-specific cultural orientations were found to hold differential relationships with psychological well-being. For instance, bicultural activity orientation predicted the best psychological well being (regardless as to how it was assessed), but socially separatist individuals experienced less negative affect than assimilated and bicultural respondents. Findings were discussed in the context of the multicultural setting of San Francisco. It is suggested that future research retain separate assessments of domain-specific cultural orientation, examine the contribution of the community's ethnic/cultural composition to its members' cultural orientations (i.e., address the role of person--environment fit) and study their impact on psychological well being. PMID- 8638556 TI - Orthodontic and orthognathic surgical correction of Class III malocclusion. AB - This case was presented as part of the student case displays at the 1994 AAO meeting, sponsored by the College of Diplomates of the American Board of Orthodontics. It was selected to be submitted for publication in the American Journal of Orthodontics and Dentofacial Orthopedics by a CDABO committee. PMID- 8638557 TI - A longitudinal cephalometric study of the soft tissue profile of short- and long face syndromes from 7 to 17 years. AB - The longitudinal growth and development of the soft tissue drape for boys and girls with long and short vertical patterns was examined from age 7 to 17 years. The sample was taken from the Denver Growth Study and consisted of 32 subjects who were selected on the basis of their percentage of lower anterior vertical face height. All subjects were of northern European ancestry, and none had undergone orthodontic treatment. The sexual dimorphism was evident as anticipated for several soft tissue measurements. The boys showed continued growth through age 16 years in contrast to the girls who attained the adult size of the soft tissue integument around 14 years. A significant difference between vertical facial patterns was reported for all soft tissue variables with the exception of the soft tissue thickness at A point and the upper lip height. The boys and girls with long vertical patterns exhibited a thicker and longer soft tissue drape for the most variables when compared with those with short facial patterns. These soft tissue differences are believed to be compensatory mechanisms in long-face subjects, which may attempt to mask the vertical dysplasia, thereby producing a more normal facial profile. Individual growth assessments revealed that the perioral soft tissues follow a pattern similar to that of the mean group patterns. The subjects with long vertical facial patterns experienced their pubertal growth spurt earlier than the short-face subjects. This may have clinical implications in the timing of orthodontic intervention and treatment. PMID- 8638558 TI - Permanence of skeletal changes after function regulator (FR-2) treatment of patients with retrusive Class II malocclusions. AB - The purpose of this study was to examine the skeletal changes produced by the Frankel function regulator (FR-2) appliance during the treatment of patients with mandibular retrusive Class II malocclusions and to characterize the permanence of these changes in the years after treatment (5 years, on average). Data from the pretreatment, posttreatment, and long-term serial cephalograms of 14 patients who received FR-2 treatment were compared with data obtained from untreated controls and from published standards. Relative to controls, FR-2 therapy produced a statistically significant decrease in the ANB angle and an increase in the rate of mandibular growth. At the same time, no maxillary effect was noted. During the post-FR-2 period, the rate of mandibular growth showed no compensatory decline or "rebound." Instead, it was remarkably similar to that inferred from age-matched and sex-matched normative standards. The present study thus supports the conclusion that FR-2 therapy, in conjunction with a period of postfunctional fixed- or removable-appliance therapy designed to perfect the occlusion, can produce a statistically and perhaps clinically significant relative increase in mandibular length. PMID- 8638559 TI - Effects of reverse headgear treatment on sagittal correction in girls born with unilateral complete cleft lip and cleft palate--skeletal and dental changes. AB - Patients with cleft lip and cleft palate often develop maxillary retrognathism. This is due to the combined effects of the congenital deformity and surgical repairs. Early protraction of the maxilla with extraoral forces helps to achieve more balanced skeletal harmony and favorable occlusion for future growth to occur. The purpose of the present study is to investigate the proportion of the skeletal and dental changes contributing to the improvement in a group of Southern Chinese girls born with unilateral complete cleft lip and cleft palate treated by the reverse headgear. This study only focused on treating a homogenous sample group, i.e., only girls with unilateral complete cleft lip and cleft palate. This design was deliberate so as to avoid having boys and girls with various types of cleft all pooled together for analyses as seen in most of the previous reports. In addition, comparison was made with girls matched in having a similar deformity, presenting a similar skeletal structure and maturity status to reveal the genuine treatment effect. The 9.7 months of reverse headgear treatment improved the sagittal jaw relationship (p < 0.01) and overjet (p < 0.01), which was effected by about two-thirds skeletal and one-third dental changes. PMID- 8638560 TI - Nickel allergy in adolescents in relation to orthodontic treatment and piercing of ears. AB - The aim of this study was to investigate the frequency of nickel hypersensitivity in adolescents in relation to sex, onset, duration and type of orthodontic treatment, and the age at which ears were pierced. The subjects were 700 Finnish adolescents, from 14 to 18 years of age, of which 476 (68%) had a history of orthodontic treatment with metallic appliances. The study consisted of patch testing for a nickel allergy and a patient history obtained by a questionnaire and from patient record. The frequency of nickel sensitization in the whole group was 19%. Nickel allergy was significantly more often found in girls (30%) than in boys (3%) and in subjects with pierced ears (31%) than in those with no piercing of ears (2%). Orthodontic treatment did not seem to affect the prevalence of nickel sensitization. None of the girls who were treated with fixed orthodontic appliances before ear piercing showed hypersensitivity to nickel, whereas 35% of the girls who had experienced ear piercing before the onset of orthodontic treatment were sensitized to nickel. The results suggest that orthodontic treatment does not seem to increase the risk for nickel hypersensitivity. Rather, the data suggests that treatment with nickel-containing metallic orthodontic appliances before sensitization to nickel (ear piercing) may have reduced the frequency of nickel hypersensitivity. PMID- 8638561 TI - The role of the periodontal ligament in bone modeling: the initial development of a time-dependent finite element model. AB - Current remodeling theories, as applied to long bones, suggest that such processes are controlled by mechanical strains either within or on the bone surface. In this study, the stresses and strains within the periodontal ligament and surrounding bone, consequent to orthodontic loading of a tooth, were investigated by application of the finite element method. Previously, various authors have applied two and three dimensional instantaneous (essentially static) models to analyze the problems. The study reported in this article describes an initial time-dependent (continuous/dynamic) finite element model for tooth movement that uses newly developed software, the results being cross-referenced against historical data. These early results, from a two-dimensional mathematical model of a loaded canine tooth, suggest that the remodeling process may be controlled by the periodontal ligament rather than the bone. In the finite element model, bone was found to experience a low strain of 1 x 10(-5), whereas the periodontal ligament experienced a strain of 0.1 when the "tooth model" is loaded. Only this latter figure is above the threshold usually reported to be necessary to initiate the remodeling process. Further developments in this rapidly advancing area of biomechanical research should facilitate a greater increase in our knowledge of tissue stress and strain after loading. PMID- 8638562 TI - Craniofacial structure and obstructive sleep apnea syndrome--a qualitative analysis and meta-analysis of the literature. AB - The etiologic relevance of craniofacial structure to obstructive sleep apnea syndrome (OSAS) is controversial yet the premise of a causal association serves to justify many treatments. A qualitative and quantitative analysis of the literature was performed to examine the foundation for any relationship between craniofacial structure and OSAS. A MEDLINE search and investigation of the published and unpublished literature on diagnostic imaging and OSAS was toxonomically arranged. Each sample study was evaluated by using the following criteria: (a) appropriate control group, (b) "blinding" of evaluators, (c) reliability measured, (d) random assignment of treatment, and (e) "success" was defined adequately in efficacy studies. Morphologic variables were combined among studies and compared with controls drawn from either the same patient pool as the OSAS group, or matched for gender, age, and body mass index. Analysis revealed 32 review articles, 16 case reports, and 95 sample studies. Only seven sample studies drew a control group from the same patient pool, whereas five used matched controls. Only one of these studies satisfied all the qualitative criteria. Of the treatment efficacy studies, 10 defined outcome adequately. However, none of these met all the qualitative criteria. The most consistent, strong effect sizes with the highest potential diagnostic accuracies were for mandibular plane to hyoid, mandibular plane angle, and mandibular body length. Only mandibular body length demonstrated a clinically significant association with and diagnostic accuracy for OSAS. However, since this variable's controls were selected from the literature, possible explanations for a positive association include methodologic differences between studies, varying magnification factors, and morphologic differences. PMID- 8638563 TI - Comments on the phase I versus phase II treatment and point-counterpoint articles. PMID- 8638564 TI - One phase versus two phase orthodontic treatment. PMID- 8638565 TI - Comment on long-term stability. PMID- 8638566 TI - Skeletal and functional effects of treatment for unilateral posterior crossbite. AB - Unilateral posterior crossbite (UPXB) is a common malocclusion, frequently presenting a lower midline deviation, accompanied by Class II subdivision relationships in final closure and a very high prevalence of the reverse sequencing (RS) pattern of jaw movement. These features often persist even after the elimination of the crossbite. The purpose of the present study was to examine in detail the morphologic, skeletal, and functional effects of the treatment for this malocclusion category. The experimental group consisted of 24 children in the mixed dentition stage with UPXB who were treated with removable expansion plates and a control group of 10 age-matched children with normal occlusion. Longitudinal follow-up revealed a stable dental maxillary arch expansion of at least 1.5 mm but a complete elimination of crossbite in only 50% of the cases. The frequent persistence of Class II subdivision relations and lower midline deviation that were not due to functional mandibular shift was striking. The pretreatment posteroanterior (P-A) cephalograms indicated reduced facial and maxillary widths. After treatment, the achieved maxillary width increase was greater than expected with normal growth. Longitudinal assessment of the mandibular movement response revealed by the electrognathograph showed a high prevalence of RS, which was reduced after treatment. In conclusion, (1) a higher than expected prevalence of skeletal transverse aberrations at the maxillary and zygomatic levels were found in the UPXB group; (2) the removable expansion appliance induces transverse growth of the maxilla; and (3) an inherent pattern of jaw movement is characteristic to the UPXB and does not change significantly with orthodontic treatment. PMID- 8638567 TI - Comment on mandibular and facial asymmetries. PMID- 8638568 TI - Superimposition and structural analysis. AB - Superimposition on the lower border of the mandible has been severely criticized. It has allegedly been responsible for causing chaos and confusion in the literature. This report indicates that this is not true and that superimposition on the lower border of the mandible is valid. We have shown that changes resulting from superimposing on implants are a part of the sella-nasion superimposition. The implant changes are contained within the sella-nasion superimposition and are a part of it. The gonion angle grows approximately parallel to the mandibular plane. The lower border of the mandible is a part of the big picture all through the growing period, always containing the implant changes. The mandibular plane changes its inclination as the relation of vertical to horizontal growth changes. Examples are shown to illustrate the "give and take" between vertical and horizontal growth, and its effect on the facial complex. Objective evidence is shown that seems to indicate that the implant changes may have been counted and then counted again. This new analysis places a different interpretation on the implant studies. PMID- 8638569 TI - Superimposition and structural analysis. PMID- 8638570 TI - The Bolton ratio studied with the use of spreadsheets. PMID- 8638571 TI - Comment on bond strength of rebonded brackets. PMID- 8638572 TI - Comment on the transfer of patients' records. PMID- 8638573 TI - Principles of ethics and code of professional conduct. PMID- 8638575 TI - New findings in the degenerating tissues of the periodontal ligament during experimental tooth movement. AB - The degenerating tissues found in rat periodontal ligaments during tooth movement were examined morphologically, histochemically, and elementally, with decalcified and unfixed, undecalcified frozen sections. There were two types of degenerating tissues found in the compressed periodontal ligaments: One (type A tissue) was stained differently from collagen and the other (type B tissue) showed the same color as collagen. Type A tissue also showed the deposition of fibrin in Martius scalet blue and Weigert stain. The electron micrograph also showed the deposition of fibrin in type A tissue. No collagen fibers with typical bandings were seen in either tissue. The digestion experiment showed that type A tissue was digested by trypsin but not type B, whereas most of type B tissue was digested by collagenase but not type A. The backscattered electron image by scanning electron microscopy of type A tissue of the unfixed undecalcified frozen sections showed the presence of many small pieces. The elemental analysis of the pieces showed high peaks of phosphorous and calcium. These results indicate that collagen degradation, fibrin deposition, and calcification occurred in the degenerating tissues, especially in type A tissue during the experimental tooth movement. PMID- 8638576 TI - Distal movement of premolars to provide posterior abutments for missing molars. AB - In 24 patients with missing molar teeth in the upper and/or in the lower jaw, 32 premolars were distalized. The mean orthodontic distalizing distance was 9.4 mm (SD 2.6). After distalization all these teeth served as posterior abutments for fixed restorations. The investigation period ranged between 2.5 to 14.1 years, average 9.6 years (SD 3.2). The clinical examination criteria were sensitivity, mobility, probing depth, sulcus bleeding index; the radiologic criteria were root resorption (lateral and apical) marginal bone level and axial position. None of the 32 premolar abutments were lost during investigation period. All the teeth maintained their vitality. The measured probing depths and sulcus bleeding indices were low. Of the teeth tested 40.6% revealed localized lateral root resorption on the pressure side; the average postorthodontic depth of root resorption was 0.7 mm (SD 0.3), and the length 2.3 mm (SD 0.6). The follow-up examination revealed a partial repair of the lateral root lesions. The extent of apical root resorption amounted to 0.9 mm (SD 1.1). The marginal bone level showed a bone loss of 0.5 mm mesially and 0.2 mm distally. The findings confirm that the distalized premolar functioning as a posterior bridge abutment represents a prognostically favorable alternative to an implant. PMID- 8638574 TI - Combination of bionator and high-pull headgear therapy in a skeletal open bite case. PMID- 8638578 TI - Treatment and postretention changes in dental arch width dimensions--a long-term evaluation of influencing cofactors. AB - The aim of the present long-term follow-up study of orthodontically treated patients was to analyze postretention changes in arch width dimension and to isolate factors that may serve as predictors of long-term prognosis. Pretreatment, end-of-treatment, and postretention (at least 10 years) models of 226 cases with different malocclusions were used to measure intercanine and intermolar width, arch length, sum of the mesiodistal dimension of the incisors, irregularity index, crowding, molar and canine relationship, overjet, and overbite. To assess the influence of initial and end-of-treatment alignment, kind of treatment (extraction versus nonextraction) and the amount of expansion in postretention stability, the sample was divided into different subgroups. The findings indicate that postretention arch width relapse occurred more frequently in the upper intermolar (25.8%) and lower intercanine region (23.9%) than in the lower intermolar (19.0%) and upper intercanine (13.8%) region. Pretreatment and posttreatment alignment as well as the kind of treatment and the amount of expansion were found to be influencing factors. The study concludes by proposing a reassessment of the definition of stability. The influence of the pretreatment anomaly, kind of treatment, amount of expansion, and posttreatment alignment on long-term stability should be recognized. Patients should be apprised of treatment limitations before treatments. PMID- 8638579 TI - Distal movement of buccal segments with the "en masse" removable appliance: its value in treating patients with mild Class II, Division 1 malocclusion. Part II: the model measuring system and results. AB - The model of 25 children with mild Class II, Division 1 malocclusions who had their upper buccal segments moved distally with an en masse appliance were measured at the beginning of treatment and at the completion of buccal segment retraction. A reflex microscope, interfaced to a personal computer, was adapted for this purpose. A custom-made jig and linear stepping motor permitted the recording of both buccal and incisal measurements of the teeth in occlusion, as well as individual arch parameters. Software was designed to record and calculate the required measurements. The method error associated with this approach ranged from 0.01 to 0.5 mm. Results indicated that mean distal movement of the buccal segments approximated 6 mm, equivalent to a full cusp of buccal segment retraction. A small spontaneous reduction in overjet was seen. The upper arch showed spontaneous alignment and increases in width, length, and perimeter. In the lower jaw, transverse expansion was accompanied by a small increase in arch perimeter. Arch length, however, was slightly reduced. It was concluded that the system was an acceptable method of recording occlusal changes during orthodontic treatment, supplementing routine cephalometry. Furthermore, a full unit of buccal segment retraction could be expected, by using a removable appliance/headgear technique. PMID- 8638577 TI - Rapid maxillary expansion. A study of the long-term effects. AB - A study was made to determine whether skeletal alterations usually produced by rapid maxillary expansion may be compensated for in time by growth and/or comprehensive orthodontic treatment. In 30 patients, orthodontic treatment was started with rapid maxillary expansion, followed by fixed appliances, not combined with any other form of orthopedic device. Mean treatment time was 3.1 years. Nine measurements from the Ricketts analysis were studied, based on lateral cephalometric radiographs. Records were taken before orthodontic treatment and after completion of active therapy. A statistical analysis was made of the nine variables used, reflecting the vertical and anteroposterior skeletal proportions of the face, contrasting the changes before and after treatment. Of all the variables studied, the four that change with age according to the Ricketts analysis (mandibular plane angle, maxillary height, facial depth and facial convexity), yielded statistically significant differences after treatment, indicative of normal growth. The five remaining variables that remain constant with age according to the Ricketts analysis (facial axis, lower facial height, total facial height, palatal plane inclination and maxillary depth) showed no significant changes after treatment, also indicative of normal growth. PMID- 8638580 TI - Long-term stability of Class II, Division 1, nonextraction cervical face-bow therapy: II. Cephalometric analysis. AB - The long-term stability of Class II, Division 1 nonextraction therapy, using cervical face-bows with full fixed orthodontic appliances was evaluated for 42 randomly selected patients. Part 1, a study model analysis, was published in the March 1996 issue of the JOURNAL. Each patient was treated by the same practitioner, with the same techniques, and the treatment goals had been attained for all patients. Pretreatment records were taken at a mean age of 11.5 years; the posttreatment and postretention records were taken 3.0 and 11.6 years later, respectively. The results showed that the ANB angle decreased 2 degrees during treatment, most of which was due to the 1.6 degree decrease of the SNA angle. The mandibular plane angle was not changed significantly during treatment. Although upper incisor inclination was maintained during treatment, the lower incisor was proclined 2.3 degrees and the lower molar was tipped back 4 degrees. Of the 22 cephalometric measures evaluated, only four indicated relapse related with the treatment change. Three of the four measures pertain to lower incisor retroclination subsequent to excessive proclination. The ratio of treatment proclination of incisors to posttreatment retroclination is approximately 5:1. Similarly, for every 3 degrees of molar tip back, there was approximately 1 degree of relapse. It is concluded that nonextraction therapy for Class II malocclusion can be largely stable when the orthodontist ensures proper patient selection and compliance and attains treatment objectives. PMID- 8638581 TI - The decision to extract: part II. Analysis of clinicians' stated reasons for extraction. AB - In a recently reported study, the pretreatment records of each subject in a randomized clinical trial of 148 patients with Class I and Class II malocclusions presenting for orthodontic treatment were evaluated independently by five experienced clinicians (drawn from a panel of 14). The clinicians displayed a higher incidence of agreement with each other than had been expected with respect to the decision as to whether extraction was indicated in each specific case. To improve our understanding of how clinicians made their decisions on whether to extract or not, the records of a subset of 72 subjects randomly selected from the full sample of 148, have now been examined in greater detail. In 21 of these cases, all five clinicians decided to treat without extraction. Among the remaining 51 cases, there were 202 decisions to extract (31 unanimous decision cases and 20 split decision cases). The clinicians cited a total of 469 reasons to support these decisions. Crowding was cited as the first reason in 49% of decisions to extract, followed by incisor protrusion (14%), need for profile correction (8%), Class II severity (5%), and achievement of a stable result (5%). When all the reasons for extraction in each clinician's decision were considered as a group, crowding was cited in 73% of decisions, incisor protrusion in 35%, need for profile correction in 27%, Class II severity in 15% and posttreatment stability in 9%. Tooth size anomalies, midline deviations, reduced growth potential, severity of overjet, maintenance of existing profile, desire to close the bite, periodontal problems, and anticipation of poor cooperation accounted collectively for 12% of the first reasons and were mentioned in 54% of the decisions, implying that these considerations play a consequential, if secondary, role in the decision-making process. All other reasons taken together were mentioned in fewer than 20% of cases. In this sample at least, clinicians focused heavily on appearance-related factors that are qualitatively determinable by physical examination of the surface structures of the face and teeth. They appear to have made primary use of indicators available on study casts and facial photographs and relatively little use of information that is available only on cephalograms or that involves the application of specialized orthodontic theories. PMID- 8638582 TI - Effect of varying etching times on the bond strength of ceramic brackets. AB - Damage to the enamel surface when debonding orthodontic ceramic brackets has been a clinical concern. Ideally, bond failure at the bracket-adhesive interface should occur without damaging the enamel surface. The purpose of this study was to determine the shear bond strength and debonding failure modes of ceramic brackets with varying etching times. Sixty freshly extracted human premolars were pumiced and divided into six groups of 10 teeth. Each group was assigned an etching time interval of either 30, 20, 15, 10, 5, or 0 seconds with 37% phosphoric acid. Ceramic orthodontic brackets were bonded to each etched tooth by using the same orthodontic bonding system. The teeth were mounted in phenolic rings and stored in deionized water at 37 degrees C for 48 hours. A Zwick universal testing machine (Zwick GmbH and Co., Ulm, Germany) was used to determine shear bond strengths. The residual adhesive on the enamel surface was evaluated with the Adhesive Remnant Index. The results of the analysis of variance indicated that there were significant differences in bond strengths between the various etching times (p=0.0001). The Duncan multiple range test revealed that the 5-second and no etch group exhibited significantly lower bond strengths. The results of the Chi square test evaluating the residual adhesives on the enamel surface also revealed significant differences (p=0.0001). However, when the 5- and 0-second groups were dropped from the test, the Chi square test revealed no significant differences between the 30-, 20-, 15-, and 10-second groups (p=0.211). In conclusion, decreasing etching time between 30 and 10 seconds does not significantly affect either bond strength or the site of bond failure. PMID- 8638583 TI - Cephalometric assessment in obstructive sleep apnea. AB - It is reported that some specific craniofacial characteristics are associated with obstructive sleep apnea syndrome (OSAS). To test this finding, the present study developed and assessed the feasibility of a craniofacial index score (CIS) in differentiating patients with OSAS from habitual snorers. Anthropometric measurements and lateral head radiographs were obtained on 24 male and 4 female patients with OSAS who had physician-diagnosed OSAS (respiratory disturbance index (RDI) >20), and 25 male and 5 female habitual snorers (RDI <20). Thirteen cephalometric and four anthropometric measure- ments were used in a discriminant model to construct the CIS. The model was able to correctly classify 82.1% of the OSAS group and 86.7% of the snoring group. In addition, variables that were related to the soft tissues, hyoid bone to mandibular plane, Body Mass Index, and soft palate length had the highest predictive value. These findings indicate that a CIS constructed from cephalometric and anthropometric measurements can be used to identify subjects with and without OSAS. PMID- 8638584 TI - Surface preparation for orthodontic bonding to porcelain. AB - This study evaluated the effect of various porcelain surface treatments on the tensile strength of orthodontic brackets bonded to a feldspathic metal ceramic porcelain. The porcelain was fused to flat gold alloy tabs and divided into six groups that were subjected to sandblasting, silane application, intermediate resin, or etchants (9.6% hydrofluoric acid or 4% APF gels). Two brackets were bonded onto each porcelain/metal tab (n=60) with Bis-GMA resin (Concise, 3M Corp., St. Paul, Minn.) or 4-META resin (MCP-bond, Sun Medical Co. Ltd., Tokyo, Japan). The samples were stored in 37 degrees C water, thermocycled 1000 times from 5 degrees C to 55 degrees C and tested in tension. Alignment and uniform loading during testing were secured by engaging a hook in a circular ring soldered onto the bracket slot before bonding. Similar control brackets (n=12) were bonded with Concise to extracted caries-free mandibular incisors. Bond failure sites were classified according to a modified Adhesive Remnant Index (ARI) system. Silane application to the sandblasted porcelain surface significantly increased the bond strengths according to analysis of variance and Duncan's multiple range test. The quality of the bonds was further enhanced by the addition of the intermediate resin. Etching the porcelain with 9.6% hydrofluoric acid provided similar bond strengths, but the 4% APF gel was less effective. The MCP-bond was not significantly better than Concise in bond strength to sandblasted porcelain. Several difficulties associated with the clinical interpretation of laboratory data on bonding to dental porcelains are discussed, and clinical trials are necessary for final evidence of efficacy. PMID- 8638585 TI - Orthodontic brackets bonded to glazed and deglazed porcelain surfaces. AB - Edgewise orthodontic brackets were bonded with a light activated composite resin to 100 glazed and 100 deglazed feldspathic porcelain blocks for an in vitro comparative determination of shear/peel bond strengths. The independent variables studied were porcelain surface preparation and the time interval between activation of the composite resin and the debonding procedure. The initial shear/peel bond strength achieved on bonding to deglazed porcelain was statistically significantly greater than that on bonding to glazed porcelain (p<0.05), but after the 10-minute interval, the bond strength in the glazed porcelain sample was greater than in the deglazed sample. Many more porcelain fractures occurred on deglazed porcelain (71%) than on glazed porcelain (36%) during bond strength determination. Scanning electron microscopy evaluation suggests that deglazing does not substantially increase micromechanical adhesion of the composite resin. Bond strength adequate to withstand the application of orthodontic forces were achieved when bonding to glazed porcelain. This study indicates that deglazing may not be necessary for attachment of orthodontic brackets to porcelain surfaces. PMID- 8638586 TI - The physical properties and behavior of magnets used in the treatment of anterior open bite. AB - This investigation examined the magnetic flux and repulsive force of neodymium iron-boron (Nd-Fe-B) magnets used in repulsion in the treatment of anterior open bite. Three different grades of magnets each of five different dimensions were used. Magnetic flux was measured by a Hall probe and the repulsive force by an Instron mechanical testing machine. Results showed that the different grades of magnet displayed similar magnetic flux with a relationship between thickness and flux. There was a significant difference between the flux measured at the end as opposed to the center of the magnet. The force between two magnets and the flux above each magnet was directly related. Using magnets in groups did not produce higher forces than using single magnets of an equivalent size. It was concluded that Nd-Fe-B magnets can be used to give predictable repulsive forces in the mouth. PMID- 8638587 TI - Straight talk about extraction and nonextraction: a differential diagnostic decision. AB - At one stage or another, orthodontics is usually a space management procedure, particularly during the correction of a Class I or Class II malocclusion. Orthodontists use space that is available or create space to correct malocclusions. There are anterior, posterior, lateral, and vertical dimensions of the dentition and its supporting structures. If the muscular balance is normal, the clinician should try to respect these dimensions. The orthodontic clinician should not be an extractionist or a nonextractionist. Rather, the clinician should use differential diagnostic skills and artistic ability to arrive at the most appropriate treatment outcome for each patient. PMID- 8638588 TI - The Electronic Study Club and electronic media in orthodontics. PMID- 8638589 TI - Two phase treatment of a severe Class II, division I malocclusion. PMID- 8638591 TI - A pressure-distribution sensor (PDS) for evaluation of lip functions. AB - The purpose of this study was to develop and to test a pressure-distribution sensor (PDS) for evaluating lip functions. The PDS is fabricated as a disposable cartridge and is based on the principle of optical-pressure conversion used in tactile sensors of robot arms. Its advantages are in measuring sealing forces, contact area, and pressure-distribution patterns of the lips at maximum effort. We used the PDS to evaluate pressure-distribution patterns of long-face subjects with extremely large interlabial distances. The results suggest that the PDS is a useful device for evaluating lip sealing functions and their changes after orthodontic therapy or orthognathic surgery. PMID- 8638590 TI - Fluoride released from orthodontic bonding agents alters the enamel surface and inhibits enamel demineralization in vitro. AB - In this study, we examined two fluoride-releasing orthodontic agents in respect to fluoride release, enamel demineralization inhibition, as well as alterations observed on the enamel surface after their use. Fluoride release was found to occur in the highest concentrations within the first 24 hours for both materials examined. After this period, a dramatic decline in fluoride release was observed in both adhesives, and after 90 days no fluoride was detectable. Both fluoride releasing materials showed significant differences on enamel demineralization around the brackets when compared with a conventional adhesive, and the agent with the initial higher amounts of fluoride release showed the best results. Scanning electron microscopy of the enamel surface revealed particle depositions of microglobular form, after the examined adhesives were used. These particles most likely represent deposition of calcium fluoride, a salt with clearly cariostatic properties. The results of this in vitro study show that certain fluoride-releasing orthodontic bonding systems may provide an additional degree of safety against caries susceptibility in patients with fixed appliances for a limited period. PMID- 8638592 TI - Thermographic assessment of temporomandibular disorders symptomology during orthodontic treatment. AB - The relationship between orthodontic treatment and temporomandibular disorder (TMD) symptoms has been the focus of many subjective studies. Objective studies are now needed. Electronic thermography (ET) has shown promise as an objective tool for assessing temporomandibular disorders. Clinical TMD examinations and ET were performed on 21 control subjects, 18 subjects undergoing orthodontic treatment, and 20 subjects with TMD pain. Standardized blinded clinical examinations that used algometry were conducted. The ET was performed with an Agema 870 unit (Agema Infrared Systems, Secaucus, N.J.) under controlled conditions. Data were analyzed to determine the usefulness of ET as an objective measure of TMD symptoms. The ET alone identified the subjects with painful clicking TMD with a sensitivity of 87%. Subjects with no painful clicking (controls) were identified with a specificity of 86%. The ET findings also had a strong correlation with pain to muscle palpation. This study indicates that ET shows promise as an objective tool for selecting normal subjects from subjects with TMD symptoms. The ET could prove to be valuable in accessing the relationship between orthodontic treatment and TMD symptoms in future longitudinal studies. PMID- 8638594 TI - The effects of loops on the torsional stiffnesses of rectangular wires: an in vitro study. AB - Various loop designs have been investigated with respect to the increased torsional flexibility given to the wire by their inclusion. The torsional flexibility of the wire was shown to be enlarged from 26% to 63%, depending on loop design. Both variation in wire cross-section and loop geometry will influence torsional stiffness. Wires with small cross-sectional dimensions will show greater flexibility in torsion. Even small deviations from the stated cross sectional dimensions can influence the change in torsional stiffness caused by a specific loop design. Inclusion of a reverse-closing loop led to the greatest increase in flexibility, while a Bull loop changed torsional stiffness the least. When vertical flexibility is needed along with reduced torsional stiffness, the T loop is the loop of choice. PMID- 8638593 TI - Canine retraction with rare earth magnets: an investigation into the validity of the constant force hypothesis. AB - The objective of this study was to test the hypothesis that a prolonged constant force provides more effective tooth movement than an impulsive force of short duration. Six human subjects were selected, the main criterion being a need for extraction of their upper first premolars. Canine retraction on these subjects was executed on one side with the application of a force rapidly declining in magnitude, produced by a vertical loop, and on the other side with the application of a relatively constant force. This type of force was achieved by a similar vertical loop which was constantly activated by three parylene-coated neodymium-iron-boron (Nd2Fe14P) block magnets. The vertical loop on the control side was reactivated 6 weeks after the initial activation. No reactivation was necessary on the experimental side for the duration of the experiment. The rate of tooth movement on the two sides was compared over a period of 3 months, on the basis of maxillary impressions taken at frequent intervals during the course of the study. The canines retracted with a constant force moved statistically significantly more than the control canines (p < 0.05) during the experimental period. The average differences in the mean rates of tooth movement between the two sides were in the order of 2:1 in favor of the experimental side. There were no statistically significant differences in the changes of angulation (tipping) or rotation about the y axis between the two sides. The duration of force application seems to be a critical factor in regulating rate of tooth movement. Conversely, magnitude of the applied force did not appear to be of primary significance. PMID- 8638595 TI - Tooth surface and pulp chamber temperatures developed during electrothermal bonding. AB - The rationale of electrothermal bonding is based on the premise that when an electric current is passed across the beaks of tweezers holding a stainless steel orthodontic bracket, heat will be generated by virtue of the electrical resistance of the steel bracket. This study was carried out to evaluate the temperatures generated on the tooth surface at the bracket/tooth interface and within the pulp chamber during electrothermal bonding. Temperatures were recorded with 5 and 7.5 A current levels applied as a 1 second pulse with time intervals between pulses of 1, 2, 3, and 4 seconds. The data showed that after three pulses with a 5 A current, the temperature on the tooth surface ranged between 43.3 degrees C (4 second intervals) to 53.6 degrees C (1 second intervals). By using a 7.5 A current, the temperature ranged from 77.5 degrees C (4 second intervals) to 85.9 degrees C (1 second intervals). The pulp chamber temperatures were evaluated in vitro for a mandibular incisor, the maxillary central and lateral incisors, a canine, a premolar, and a molar. The pulp chamber temperature of a mandibular incisor responded most, whereas that of premolars and molars responded least to temperature changes on the labial surface. The increase in mandibular incisor pulp chamber temperature after three pulses was 2.1 degrees C for 5 A and 2.8 degrees C for 7.5 A current while for a premolar the increase ranged from 0.9 degree C to 1.6 degrees C. On the basis of current evidence the increase in pulp chamber temperatures during electrothermal bonding may be considered to be clinically safe. PMID- 8638596 TI - Orthodontics and temporomandibular joint internal derangement. AB - The purpose of this investigation is to compare the prevalence of internal derangement of the temporomandibular joints (TMJ) in asymptomatic volunteers versus symptomatic subjects using magnetic resonance imaging (MRI), with a detailed comparison to clinical signs and symptoms and with attention to a prior history of orthodontic treatment. Bilateral MRI scans were obtained of the TMJs in 76 asymptomatic volunteers and 102 symptomatic patients. A comparison was made to the clinical signs and symptoms, a history of orthodontic treatment, and to the MR findings. The MRI scans were reviewed using established criteria for disk displacement and the reviewers were blinded as to the clinical information. Our results show a prevalence of disk displacement in 25 of 76 (33%) volunteers and 79 of 102 (77%) patients with a statistically significant difference (p < 0.001). No statistical link was noted between a history of prior orthodontic treatment and internal derangement of the TMJ. PMID- 8638597 TI - Effects of buccal shields on the maxillary dentoalveolar structures and the midpalatal suture--histologic and biometric studies in rabbits. AB - The mechanism of the buccal shields with regard to regulating the transversal relationship of the maxillary dental arch is uncertain. The periosteal pull theory, growth at the midpalatal suture and changes in equilibrium have all been proposed as explanatory factors. The aim of the study was to investigate the transversal development of the dental arch and the osseous remodeling at the lateral surfaces of the maxillary alveolar process and in the midpalatal suture after stretching the bucca (cheek) with buccal shields in the vestibulum. Ten New Zealand white male rabbits 12 weeks old were used. The animals were divided into two groups (control and experimental). Initial and final impression had been taken from the two groups and plaster models were made using biometric analysis in all animals of each group. Transversal measurements were taken of the distances between the maxillary right and left teeth. Tetracycline hydrochloride and Alizarin complexone were used to label the mineralizing tissues. After the animals had been killed, the biopsies from the midpalatal suture, the alveolar wall, and the periosteum were studied under a microscope. The results from the biometric analysis showed maxillary dental arch expansion in the two groups that was significantly larger in the experimental group. Microscopically, the growth at the midpalatal suture was also significantly larger in the experimental group, while no significant differences were found at the maxillary alveolar bone in the two groups. The conclusions drawn from this study are that the vestibular buccal shields bring about an increased expansion of the maxillary dental arch and increased growth at the midpalatal suture. No increased bone deposition was found at the buccal dentoalveolar wall, inducing doubts as to the accuracy of the periosteal pull theory for widening of the dental arch. PMID- 8638598 TI - The effect of etch duration on the microstructure of molar enamel: an in vitro study. AB - This in vitro study evaluated the influence of varying etch time on the microstructure of molar buccal enamel. The buccal surfaces of four groups of extracted first molar teeth were etched for 15, 30, 45, and 60 seconds. The etch patterns obtained were photographed with a scanning electron microscope and rated with a 3-grade scale. The 15-second etch time failed to produce any optimal etch patterns. The best etch patterns were found in molars etched for 60 seconds. PMID- 8638599 TI - Frictional resistances in stainless steel bracket-wire combinations with effects of vertical deflections. AB - This research evaluated the effects of different bracket-wire combinations and second-order deflections on kinetic friction. Thirteen different brackets, six with 0.018 x 0.025 inch slots and seven with 0.022 x 0.028-inch slots were evaluated with six different sizes and shapes of stainless steel orthodontic wire, i.e., 0.016, 0.016 x 0.022, 0.017 x 0.025, 0.018, 0.018 x 0.025 and 0.019 x 0.026 inch for four second order deflections of 0.00, 0.25, 0.50, and 0.75 mm. The wires were ligated into the brackets with elastomeric modules. Bracket movement was implemented by means of an Instron universal testing instrument (RMO, Denver, Colo.), and frictional forces were measured by a tension load cell and recorded on an X-Y recorder (Hewlett-Packard, Anaheim, Calif.). Second-order deflection was created by a specially designed and machined testing apparatus that allowed two alternate pairs of the four total brackets to be offset in increments of 0.25 mm. The kinetic frictional force increased for every bracket wire combination tested as the second-order deflection increased. Friction also increased with an increase in wire size, whereas rectangular wires produced greater friction than round wires. Bracket designs that limited the force of ligation on the wire generated less friction at low second-order deflections (0.00 and 0.25 mm). PMID- 8638600 TI - An evaluation of root resorption incident to orthodontic intrusion. AB - A new radiographic method was developed for measuring changes in root length. With this technique, orthodontic intrusion was investigated as a potential cause of apical root resorption of maxillary incisors. The experimental group consisted of 17 patients with excessive overbite who were treated with a Burstone-type intrusion arch, which delivered a low level of force (about 15 gm per tooth). A control group was made up of 17 patients in full-arch fixed appliances who were randomly selected. After a period of approximately 4 months, the intrusion group had only slightly more root resorption than the controls, 0.6 mm versus 0.2 mm (statistically significant difference). Intrusion measured at the center of resistance of the central incisor averaged 1.9 mm. The amount of resorption was not correlated with the amount of intrusion. A weak correlation, r = 0.45, was found between resorption and movement of the apex (i.e., in addition to intrusion, there was often palatal root movement). Results of this study seem to indicate that intrusion with low forces can be effective in reducing overbite while causing only a negligible amount of apical root resorption. PMID- 8638602 TI - Ten rules for reading clinical research reports. AB - This was not a scientific assessment of the scientific quality of the papers published by The American Journal of Surgery. It was an informal audit of the adequacy of the data analysis in the clinical research reports appearing in the 1987-1988 issues. As one who has devoted more than three decades to helping a great variety of people make sense of scientific data, I found the overall quality of data analysis in these papers to be above average for the medical literature; and yet, I found many instances of errors so serious as to render invalid the conclusions of the authors. My 10 proposed rules for reading clinical research reports constitute only an interim solution to a very worrisome problem. The real solution must come from the producers of and the gatekeepers for the medical literature. PMID- 8638601 TI - The performance of bonded magnets used in the treatment of anterior open bite. AB - This investigation examined the magnetic flux and repulsive force of neodymium iron-boron (Nd-Fe-B) magnets based on Nd2Fe14B with acrylic coatings in different orientations. The flux was measured with a Hall probe and the force measured by electronic scales with the magnets mounted on a laboratory jack. Results show that there were no magnetic losses after embedding the magnets in acrylic bite blocks, although there were significant flux losses when the magnet blocks were directly heated. The alignment of the magnets over each other was of the utmost importance and significantly affected the repulsive force between the magnets. When the magnets were mounted in an articulator to simulate jaw movement, the force levels between the magnets was further reduced as a result of the effects of the rotation of the articulator. It is concluded that Nd-Fe-B type magnets embedded in acrylic can be used to give predictable repulsive forces in the mouth. The dramatic reduction in force levels when the magnets are not in optimal alignment, however, makes their effectiveness in aiding intrusion of teeth doubtful. PMID- 8638603 TI - Searching the internet. AB - Your first experiences using the Internet search and retrieval programs will be profitable if you understand the environment within which they were designed to work. When used properly, they represent the Internet's directory assistance that will aid you in information discovery. PMID- 8638604 TI - The interview and hiring process according to the Americans with Disabilities Act and the Equal Employment Opportunities Commission. Part I. PMID- 8638605 TI - Random suppression of T cells that bear specific T cell receptor V beta sequences in adult T cell leukemia/lymphoma (ATLL) patients at each clinical stage: carrier, smoldering, chronic, and acute. AB - Human T cell leukemia virus type I (HTLV-I) is associated with adult T cell leukemia/lymphoma (ATLL), which is well known as a T cell malignancy. In order to clarify whether HTLV-I plays a role as a virus-encoded superantigen in the neoplastic process, we examined the TCR V beta families in the peripheral blood at four different clinical stages: carrier, smoldering leukemia, chronic leukemia, and acute leukemia. An increased number of CD4 T cells was found in each of the four clinical stages. However, we found neither uniform specific losses nor uniform clonal expansion of particular TCR V beta gene families in any case from the four clinical stages. However, a suppression of the random TCR V beta families was found. Our data did not therefore directly suggest the existence of a common superantigen model of HTLV-I which induces an increase in CD4 T cells. The random suppression in the TCR V beta repertoire is most likely caused by the influence of HTLV-I neoplastic pathogenesis rather than by virus encoded superantigens. In the patients with acute leukemia, one or two families of the V beta repertoires were very strongly expressed, while in chronic leukemia, no such repertoire of strong expression was observed. The immunological reaction of the hosts might thus be different between the above described groups. PMID- 8638606 TI - Reduction of antithrombin III, protein C, and protein S levels and activated protein C resistance in polycythemia vera and essential thrombocythemia patients with thrombosis. AB - Patients with polycythemia vera (PV) or essential thrombocythemia (ET) show a high frequency of thrombosis. The reduction of hematocrit after phlebotomy and normalization of platelet counts do not completely eliminate thrombotic risk. Some preliminary studies reported a reduction in the concentration of natural anticoagulants (NA) in this group of patients. For this reason we evaluated protein S (PS) total antigen, antithrombin III (AT III), and protein C (PC) activity in 81 patients with chronic myeloproliferative disorders (33 with PV and 48 with ET). Data were compared with those obtained in 70 healthy sex- and age matched subjects. Fifty-seven percent of patients (46 out of 81) showed one or more thrombotic episodes at diagnosis or during follow-up. Interestingly, we found a NA deficit in 43.5% of patients with thrombosis versus only 5.7% in the group of patients without thrombosis. These results may suggest new interpretations about the pathogenesis of thrombosis in PV or ET patients. PMID- 8638608 TI - Flow cytometric analysis of T and Tn epitopes on chronic lymphocytic leukemia cells. AB - Immunophenotyping of peripheral blood lymphocytes from six patients with B-cell chronic lymphocytic leukemia (B-CLL) and five normal volunteers was done and their T and Tn epitopes analyzed using specific monoclonal antibodies and flow cytometry. Lymphocytes from all patients showed strong Tn expression as compared to normal control lymphocytes. By contrast, T antigen was not expressed, The TN expression may be a useful diagnostic and prognostic marker for B-CLL. PMID- 8638609 TI - Japanese beta zero-thalassemia: molecular characterization of a novel insertion causing a stop codon. AB - During a physical checkup, a 42-year-old Japanese man with liver dysfunction was diagnosed as having beta-thalassemia. Using molecular biological techniques including PCR, we investigated the chemical basis of the hematological disorder. We found that a frameshift attributable to the insertion of a thymidine into or following the TTT sequence of codon 42 transformed codon 43 (GAG) into a stop codon (TGA). We believe that this mutation has not been previously reported. PMID- 8638610 TI - Leukemogenic risk of hydroxyurea therapy in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. AB - In polycythemia vera (PV), treatment with chlorambucil and radioactive phosphorus (p32) increases the risk of leukemic transformation from 1% to 13-14%. This risk has been estimated to be 1-5.9% with hydroxyurea (HU) therapy. When compared with historical controls, the risk with use of HU does not appear to be statistically significant. The leukemogenic risk of HU therapy in essential thrombocytosis (ET) and in myelofibrosis with myeloid metaplasia (MMM) is unknown. HU remains the main myelotoxic agent in the treatment of PV, ET, and MMM. We studied 64 patients with these three disorders, seen at our institution during 1993-1995. The patients were studied for their clinical characteristics at diagnosis, therapies received, and development of myelodysplasia or acute leukemia (MDS/AL). Forty-two had PV, 15 ET, and 6 MMM, and 1 had an unclassified myeloproliferative disorder. Of the 42 patients with PV, 18 were treated with phlebotomy alone, 16 with HU alone, 2 with p32, 2 with multiple myelotoxic agents, and 2 with interferon-alpha (IFN-alpha). Two patients from the phlebotomy-treated group, one from the HU treated group, and 1 from the multiple myelotoxic agent-treated group developed MDS/AL. In the larger group, 11 received no treatment or aspirin alone, 18 were treated with phlebotomy alone, 25 with HU, 5 with multiple myelotoxic agents, 2 with p32, 2 with IFN-alpha, and 1 with melphalan. Study of the entire group of 64 patients showed that only one additional patient (total of 5 out of 64) developed MDS/AL. This patient had been treated with HU alone. Statistical analysis did not show any association between clinical characteristics at diagnosis, or HU therapy, and development of MDS/AL (P=0.5). Thus, our data provide no evidence suggestive of increased risk of transformation to MDS/AL with HU therapy in PV, ET, and MMM. Larger, prospective studies are needed to study this issue further. PMID- 8638607 TI - Epstein-Barr virus infection and associated products (LMP, EBNA2, vIL-10) in nodal non-Hodgkin's lymphoma of human immunodeficiency virus-negative Japanese. AB - Sixty cases of B-cell nodal non-Hodgkin's malignant lymphoma (B-ML), and 46 cases of T-cell nodal lymphoma (T-ML) were surveyed for Epstein-Barr virus (EBV) genomes, RNA, and associated proteins. We used a Southern blot analysis, polymerase chain reaction (PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the presence of EBV. We performed an immunohistochemical study on EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 (EBNA-2), latent membrane protein (LMP), and viral interleukin 10 (vIL-10). In addition, we also analyzed the terminal repetitive sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin's lymphomas were grouped into three types by number of EBV-infected cells: I) almost all lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed an EBV presence; and III) only a few cells showed such a presence, which was probably due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infected cells were found; 7 were type I, 1 was type II, and 17 were type III. In 27 of 46 T-MLs, EBV-infected cells were found; no cases were type I, 5 cases were type II, and 22 cases were type III. Seven B-MLs and 3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-2/LMP-positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was detected in almost all lymphoma cells; in T-MLs, however, LMP was found in only a small portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In summary, it was thus speculated that EBV infection was associated with the various states of lymphomagenesis. PMID- 8638611 TI - Monoblastic leukemia in an HIV-infected patient: absence of viral expression in RNA blasts. AB - A small number of patients seropositive for the human immunodeficiency virus (HIV) have been reported as developing acute non-lymphoblastic leukemia (ANLL). In the cases previously published, the authors never reported a study of the link joining HIV infection and leukemia. We describe here the case of a 41-year-old HIV positive patient who developed ANLL (FAB classification M5). Using molecular techniques, we looked for a direct link between these two co-existing diseases. We showed the absence of HIV expression in the malignant clone, suggesting that the association of ANLL and Acquired Immune Deficiency Syndrome is not a direct consequence of the myeloid precursors infection. Nevertheless a relationship may exist through a disorganization of the bone marrow micro-environment. PMID- 8638612 TI - T-cell receptor J beta 1/J beta 2 locus rearrangements in an HTLV-1-positive T cell lymphoma with complex chromosomal aberrations. AB - We report a case of human T-cell lymphotropic virus type 1 (HTLV-1)-infected adult T-cell lymphoma that has multiple chromosomal abnormalities, including the presence of an additional 7q22-36, which contains the locus of the T-cell receptor (TCR) beta chain gene. Specific TCR J beta 1/J beta 2 gene rearrangements were detected in both marrow and peripheral blood DNA, with evidence of further evolution of the transformed clonal population within the peripheral lymphocytes. To our knowledge, this is the first case in which gene rearrangements have been associated with additional TCR loci. Consequently, it is advised that every effort should be made to correlate chromosomal abnormalities with gene rearrangement by molecular methods. PMID- 8638615 TI - Inhibitors to factor VIII:C in nonhemophiliacs. PMID- 8638613 TI - Paraneoplastic pemphigus in a patient with non-Hodgkin's lymphoma. AB - Paraneoplastic pemphigus (PNP) is an autoimmune disorder occurring in the setting of an underlying neoplasm in which patients have polymorphous skin and mucous membrane lesions. We describe a patient with non-Hodgkin's lymphoma who developed bullous, ulcerating lesions in an area being treated with radiation therapy. The diagnosis of PNP was confirmed by indirect immunofluorescence of the patient's serum on rat bladder. The disorder was refractory to therapy, and ultimately the patient expired. PMID- 8638614 TI - Multiple myeloma presenting as plasmacytoma of the base of the skull. PMID- 8638616 TI - Recombinant interleukin-1 followed by immunosuppressive therapy for aplastic anemia. PMID- 8638617 TI - Abnormal chromatin clumping in polymorphonuclears from a patient with AIDS. PMID- 8638618 TI - Rearrangement of the bcl-6 gene in Hodgkin's disease, lymphocyte predominant type. PMID- 8638619 TI - Two cases of epidemic mucormycosis infection in patients with acute lymphoblastic leukemia. PMID- 8638620 TI - Translocation 21;22 may be involved in control of differentiation in erythroleukemia. PMID- 8638622 TI - Physiological neutrophilia is associated with elevated serum level of macrophage colony-stimulating factor (M-CSF) PMID- 8638621 TI - Hodgkin's lymphoma in a cyclist treated with growth hormone. PMID- 8638623 TI - Kung Fu phlebitis: an unusual presentation of Mondor's disease. PMID- 8638624 TI - Inherited deficiency of multiple vitamin K-dependent coagulation factors and coagulation inhibitors presenting as hemorrhagic diathesis, mental retardation, and growth retardation. PMID- 8638625 TI - Orthotopic liver transplantation for acute grade IV hepatic graft-versus-host disease following bone marrow transplantation. PMID- 8638626 TI - Spurious automated white cell count with Coulter STKS in the myelodysplastic syndromes suggests the presence of a red cell membrane defect. PMID- 8638627 TI - Cerebral and vein thrombosis, transient protein S deficiency, and anticardiolipin antibodies. PMID- 8638628 TI - Enzyme-linked immunosorbent assay detects a potential soluble form of the erythropoietin receptor in human plasma. AB - The erythropoietin receptor (EpoR) is a type I transmembrane protein that is a member of the family of hemopoietin receptors. Several members of this family have soluble receptor forms that are secreted by the cells rather than expressed on the cell surface. An alternatively spliced EpoR transcript has been described in human erythroid precursors that, if translated, would produce a truncated, soluble EpoR lacking the transmembrane domain. To determine if the human EpoR is expressed in a soluble form, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for the EpoR, and we analyzed human serum and plasma. Sheep were immunized with a fusion protein (EREx) consisting of glutathione-S-transferase (GST) and the human EpoR extracellular domain. The sheep antiserum was affinity purified on immobilized EREx, and then used in a two-stage antigen capture ELISA. The plasma from 20 normal subjects was studied with this assay. There was a wide variability in the levels of soluble EpoR in these subjects (range, <10-2,200 ng/ml). An average value of 550 +/- 735 ng/ml for soluble EpoR was obtained in these normals. Protein A adsorption of the test plasma prior to the assay had no effect on the values obtained. Assay of serum from the same normal subjects showed an average decrease of 88% in soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR levels compared to plasma. There was no correlation between hematocrit and soluble EpoR level. This assay may have utility in the further elucidation of erythropoietin physiology. PMID- 8638629 TI - Core decompression in avascular necrosis of the hip in sickle-cell disease. AB - Sickle-cell disease (SCD) is the most common cause of avascular necrosis (AVN) of the hip in childhood. It results in significant physical impairment and chronic pain, and often progresses to require hip replacement. Conservative therapy is ineffective. We evaluated whether core decompression can arrest progression of AVN. We performed 13 coring procedures in 10 patients with SCD and AVN. Patients ranged from age 9-21 years at diagnosis (mean, median age, 15 years); five hips were stage I, six hips were stage II, and two hips were stage III. Mean follow-up on these patients was 3.7 years. Efficacy of the procedure was evaluated by clinical improvement in pain, radiographic progression, and need for further surgery. All 5 stage I patients had substantial improvement in pain, and only one showed X-ray progression. Five of the 6 (83%) stage II patients had improvement in pain, and 2 patients progressed on X-ray. Both stage III patients progressed on X-ray, but one was clinically improved. None of the 10 patients has required further surgery. Our results demonstrate that in early AVN, core decompression was beneficial for almost all patients, even with progression on X-ray. Core decompression should be considered in the management of SCD patients with early AVN. PMID- 8638630 TI - First report of a B cell lymphoproliferative disorder arising in a patient treated with immune suppressants for severe aplastic anemia. AB - Aplastic anemia is a disorder characterized by pancytopenia and bone marrow hypocellularity. There is some evidence that aplastic anemia may be due to suppression of hematopoiesis by activated T-suppressor cells. Thus, immunosuppressive agents have been used as an alternative to bone marrow transplantation for treatment. We report on a unique case of a patient with aplastic anemia who was treated with a course of immunosuppression including cyclosporine (CSA), anti-thymocyte globulin (ATG), and prednisone. Five months after this treatment, the patient developed a B cell lymphoproliferative disorder which was successfully treated with radiation therapy. Prior reports of CSA associated lymphoproliferative disorders have appeared in the literature as potential side effects of immunosuppression following transplantation. This is the first report of a lymphoproliferative disorder associated with immunosuppressive treatment of aplastic anemia in a nontransplant setting. Thus, when presenting options for treatment of aplastic anemia, lymphoproliferative disorders should be included as a rare complication of immunosuppressive therapy. PMID- 8638631 TI - Negligible synergistic effect of beta2-glycoprotein I on the reactivity of antioxidized low-density lipoprotein antibody to oxidized low-density lipoprotein. AB - We conducted this study to investigate whether antioxidized low-density lipoprotein (a-oxLDL) is an antibody to cryptic and/or neo-antigen on beta2 glycoprotein I (GPI), which is introduced by binding to anionic phospholipid, similar to that of GPI-dependent anticardiolipin antibody (aCL) employing a-oxLDL ELISA. We found that no significant optical density differences existed among systemic lupus erythematosus patients, including cases with aCL and/or lupus anticoagulant positivity, before and after the addition of GPI. Our results suggest that a-oxLDL is not an antibody to denatured GPI, but rather to oxLDL. PMID- 8638633 TI - Pseudo-"acid retinoic syndrome" mimicked by severe influenza A infection. PMID- 8638632 TI - Reinstituting warfarin in patients who develop warfarin skin necrosis. AB - Skin necrosis is a rare but serious complication of oral anticoagulation with coumarin derivatives. Frequently, the necrosis can be extensive and may result in major morbidity and mortality. The majority of these patients require prolonged anticoagulation for life-threatening conditions such as deep various thrombosis and pulmonary embolism. Resuming oral anticoagulants in the face of skin necrosis is a difficult decision for both the patient and the physician. Because long-term heparin therapy is inconvenient and is associated with significant side effects, we reviewed the literature to find alternative treatment strategies. A Medline search was done, and all papers published in English since 1967 were reviewed. Of 58 cases with skin necrosis attributed to oral anticoagulants, oral anticoagulation was resumed in 7 patients with no resulting adverse effects. Warfarin is the most widely used coumarin derivative in the United States. Based on our review, we make recommendations for resuming warfarin in patients who have developed skin necrosis when the clinical condition absolutely requires prolonged anticoagulation. PMID- 8638634 TI - Heat exhaustion with thrombocytopenia and leukopenia in a car wash attendant. PMID- 8638635 TI - Factor XI deficiency in a Bedouin family. PMID- 8638636 TI - Hemorrhagic cystitis associated with BKV in patients with refractory acute lymphoblastic leukemia. PMID- 8638637 TI - Idiopathic myelofibrosis with unusually high erythroblastosis in the peripheral blood. PMID- 8638638 TI - Leukoerythroblastosis following the use of G-CSF. PMID- 8638639 TI - Possible cytokine mechanism of increased megakaryocytic proliferation in 5q syndrome. PMID- 8638640 TI - Iron granules in plasma cells: a particular morphologic aspect. PMID- 8638641 TI - Homozygous thalassemia and difficult endotracheal intubation. PMID- 8638642 TI - An unusual case of untreated chronic lymphocytic leukemia patient presenting with Rhodococcus equi bacteriemia. PMID- 8638643 TI - Osteoporosis in severe congenital neutropenia: inherent to the disease or a sequela of G-CSF treatment? PMID- 8638644 TI - Granulocyte colony-stimulating factor production by human bone marrow fibroblasts stimulated with interleukins. AB - Granulocyte colony-stimulating factor (G-CSF) is a cytokine that mediates the clonal proliferation and differentiation of progenitor cells into mature granulocytes. The kinetics of G-CSF production by human bone marrow fibroblasts (BMF) were investigated by quantitative immunoassays. The spontaneous production of G-CSF by BMF was below the detectable level. Interleukin-1 (IL-1) induced a dose-dependent production of G-CSF, and the production reached a plateau at 50 U/ml of IL-1. G-CSF production by BMF stimulated with IL-1 was cell concentration dependent. Detectable G-CSF was produced by 5 x 10(2) BMF in a 35 x 10-mm plastic dish. The optimal range was 1 x 10(4)-5 x 10(4) BMF. Production of newly synthesized G-CSF was detectable 6 hr after stimulation and continued for approximately 48 hr. A 6-hr pulse exposure to IL-1 was necessary to induce production of G-CSF, and after 48 hr, the adherent BMF could not be restimulated. IL-2, IL-3, IL-4, IL-5, and IL-6 were unable to induce G-CSF production. However, IL-4 promoted G-CSF production after stimulation with IL-1. These results provide useful data with regard to the mechanism of G-CSF production by human BMF. PMID- 8638645 TI - HyperCVAD for VAD-resistant multiple myeloma. AB - More effective and safer regimens are needed for patients who have advanced multiple myeloma resistant to or relapsing despite prior treatment with alkylating agents and VAD. We treated 58 such patients using the combination of twice daily cyclophosphamide (total dose 1.8 g/m2) and VAD (hyperCVAD). Treatment was given to outpatients followed by G-CSF at 5 microgram/kg/d until granulocyte recovery. Twenty-three patients responded (40%), with a median duration of granulocyte depression to less than 500/microliter of 4 days and a mortality rate of 2%. The median survival time for all patients was 15 months, and the median remission time of responding patients was 8 months. Patients who had low LDH, low B2M, or primary resistant disease lived significantly longer than patients without these features. The combination of fractionated cyclophosphamide and VAD provided an effective and safe rescue treatment for many patients who had advanced myeloma resistant to standard therapies. PMID- 8638646 TI - Lack of B cells precursors in marrow transplant recipients with chronic graft versus-host disease. AB - B cell reconstitution after bone marrow transplantation is slow in patients with chronic graft-vs.-host disease (cGVHD). Could this be secondary to decreased production of B cells in the bone marrow? We determined the relative amount of B cell precursors in the marrow of 26 patients at approximately 1 year after marrow transplant (10 patients with and 16 patients without clinical cGVHD) and 8 normal adult controls. In the controls (median), 3.1% of all marrow mononuclear cells were B cell precursors. The patients without cGVHD tended to have higher than normal percents of B cell precursors (median 6.5%; the difference from normal adults was not significant). In contrast, the patients with cGVHD had barely detectable B cell precursors (median 0.2%; the difference from normal adults was significant, P = 0.004). Therefore, delayed reconstitution of 8 cells in patients with cGVHD appears to be due at least in part to decreased B cell production by the marrow. PMID- 8638647 TI - Generation of antibodies to heparin-PF4 complexes without thrombocytopenia in patients treated with unfractionated or low-molecular-weight heparin. AB - The incidence of antibodies to heparin-PF4 complexes (H-PF4) has been evaluated in patients who were under heparin therapy for more than 7 days: 109 patients treated with unfractionated heparin (UH) and 100 patients with low-molecular weight heparin (LMWH). The presence of antibodies was identified in 17% of the former group and 8% of the latter. In both the UH and the LMWH groups, IgM antibodies were found in all but four patients who showed IgA antibodies. IgG isotypes were only detected in five patients and were consistently associated to either IgM or IgA antibodies. The follow-up of H-PF4 antibodies in 76 patients treated with UH from 1 to > or = 12 days showed a relationship between the incidence of antibodies and the duration of therapy. Despite the presence of anti H-PF4 antibodies there was no thrombocytopenia (<150 10(9)/L) in the patients. A significant drop of platelets requiring the discontinuation of heparin was observed, however, in three patients, but their platelet count consistently remained >150 10(9)/L. Our study demonstrates that the induction of antibodies to H-PF4 is a frequent phenomenon in patients treated with UH or with LMWH. The absence of thrombocytopenia and of clinical complications in these patients demonstrates that other conditions must be associated with H-PF4 antibodies for inducing type II HIT: optimal concentrations of heparin and PF4 in the blood circulation to allow the formation of macromolecular H-PF4 complexes, presence of activated platelets that present an increased binding of H-PF4 complexes, increased expression of FcgammaRIIA receptors, or presence of their H 131 phenotype. We conclude that the measurement of antibodies to H-PF4 complexes allows the detection of heparin-treated patients at risk of developing type II HIT. PMID- 8638648 TI - Cardiovascular function during rest and exercise in patients with sickle-cell anemia and coexisting alpha thalassemia-2. AB - Cardiac function was measured at rest and during exercise in 9 patients with sickle-cell anemia (SS) and coexisting homozygous alpha thalassemia-2 (alpha thal 2). Results were compared with 18 sickle cell patients with normal alpha globin genes, who were matched to the study group by age, gender, and size, and to published normal values. SS alpha thal-2 patients were less anemic: 9.9 +/- 1.0 vs 8.2 +/- 1.2 gm/dl for SS alone (P<.05). Left ventricular dimensions were normal in SS alpha thal-2 (4.9 +/- 0.7 cm), but increased in SS (5.4 +/- 0.7, cm P=.05) (normal range, 3.7-5.6 cm). Left ventricular wall thickness was, however, dramatically increased in the SS alpha thal-2 patients (free wall, 1.8 +/- 0.6 cm; septum, 1.6 +/- 0.4 cm), though SS controls had normal wall thickness (free wall, 1.0 +/- 0.2 cm; septum, 1.0 +/- 0.2 cm, P<.001) (normal range, 0.6-1.1 cm). At rest, Doppler indices of systolic function were not significantly different between sickle groups and normal values. SS alpha thal-2 patients did have abnormal diastolic filling at rest, as evidenced by a reduced ratio of early/late diastolic filling, 1.4 +/- 0.3 vs. 2.0 +/- 0.5 for SS controls (P<.01), and 1.8 +/- 0.4 for normals. An analysis of covariance suggested that this abnormality persisted after taking into account the previously demonstrated hypertrophy. During exercise, SS alpha thal-2 patients had higher heart rates and blood pressures than SS controls in spite of performing the same or less work. This resulted in a higher double product (an estimate of oxygen consumption) in SS alpha thal-2 patients (37,470 +/- 2,310 mm Hg-BPM) than in SS controls (33,310 +/ 1,490 mm Hg-BPM, P<.01). Work capacity, peak heart rate, and blood pressure were all abnormally decreased in both sickle-cell groups when compared to normal. Cardiac abnormalities noted at rest and during exercise in SS alpha thal-2 patients suggest a role of microvascular occlusion and a protective effect of decreased hemoglobin. PMID- 8638650 TI - Cellular and molecular physiology of volume-sensitive anion channels. AB - Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed. PMID- 8638649 TI - Effect of Ca2+ influx on intracellular free Ca2+ responses in antigen-stimulated RBL-2H3 cells. AB - We undertake a quantitative investigation of changes in intracellular free Ca2+ concentration ([Ca2+]i) in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells, which include contributions of both Ca2+ store release and Ca2+ influx from the medium. Following Keizer and De Young (J. Keizer and G. De Young. Biophys. J. 61: 649-660, 1992), we develop a highly constrained mathematical model for [Ca2+]i oscillations in RBL-2H3 cells, which includes activation of the inositol trisphosphate receptor (IP3R) by inositol 1,4,5-trisphospate, indirect Ca2+ activation of the IP3R via Ca2+ -dependent activity of phospholipase C gamma, slow inhibition of the IP3R by cytosolic Ca2+, refilling of Ca2+ stores by a Ca2+ -ATPase (SERCA)-type pump, and a simple representation of the dependence of plasma membrane (PM) fluxes on experimental conditions. Using this full (open cell) model, we simulate [Ca2+]i responses for protocols in which antigen concentration and external Ca2+ are manipulated and compare out calculations with experimental data. In protocol A, cells are stimulated in the presence of external Ca2+, in protocols B and C, cells are stimulated in the absence of external Ca2+, with external Ca2+ later reapplied in protocol C. We are able to reproduce quantitatively the important features of all three protocols, including the dose response of protocol B, the [Ca2+]i response to thapsigargin, and lag time results, and we provide qualitative explanations for the responses derived from our calculations. We also develop a simplified (closed cell) version of the model in which PM fluxes are neglected and total free Ca2+ concentration ([Ca2+]T) is a slowly varying parameter. This permits us to explain in a simple graphical fashion how PM fluxes may influence [Ca2+]i responses in RBH-2H3 cells through modulation of [Ca2+]T. PMID- 8638651 TI - Differential regulation of Na+-K+-ATPase gene expression by corticosteriods in vascular smooth muscle cells. AB - To determine whether gluco- and mineralocorticoids have specific actions on Na+ K+-ATPase gene expression in vascular tissue, we used Northern blot analysis to compare the effects of dexamethasone (Dex) and aldosterone (Aldo) on Na+-K+ ATPase alpha1 and beta1-subunit mRNA expression in cultured vascular smooth muscle cells from rat aortae. Dex at 10(-6)M increased alpha1 -mRNA level 2.5 fold at 24 h and beta1-mRNA level 9.9-fold at 12 h. Aldo at 10(-6)M increased alpha1-mRNA 2.7-fold at 48 h and beta1-mRNA level 10.9-fold at 6 h. The half maximal stimulation of both alpha1 and beta1-mRNA levels occurred at a concentration of 5-7 X 10(-9)M Dex, whereas it occurred at a concentration of 2-3 X 10(-9)M Aldo. The glucocorticoid receptor antagonist RU-38486 inhibited both Dex- and Aldo-mediated induction of beta1-mRNA. The mineralocorticoid receptor antagonist spironolactone inhibited Aldo-mediated induction of beta1-mRNA, whereas it had no effect on Dex-mediated induction of beta1-mRNA. Removal of Na+ from the extracellular medium (isosmotic replacement with choline) caused no effect on Dex-mediated induction beta1-mRNA, whereas it inhibited Aldo-mediated induction of beta1-mRNA. Addition of a specific inhibitor of the Na+/H+ exchange, ethylisopropylamiloride, had no effect on Dex-mediated induction of beta1-mRNA, whereas it resulted in a significant inhibition of Aldo-mediated induction of beta1-mRNA. We conclude that 1) both Dex and Aldo induce Na+-K+-ATPase alpha1- and beta1-mRNA expression in a time- and dose-dependent manner; 2) Dex-mediated induction of beta1-mRNA occurs only through glucocorticoid receptors, whereas Aldo-mediated induction of beta1-mRNA occurs through both gluco- and mineralocorticoid receptors; and 3) Dex-mediated induction of beta1-mRNA occurs through Na+-independent mechanisms, whereas Aldo-mediated induction of beta1 mRNA, at least in part, occurs through Na+-dependent mechanisms, including stimulation of the Na+/H+ exchange. PMID- 8638652 TI - Protein kinase C modulates natriuretic peptide receptors in astroglial cultures from rat brain. AB - We determined previously that astroglia cultured from newborn rat brain contain both guanylyl cyclase-coupled and atrial natriuretic peptide (ANP)-C natriuretic peptide receptors. Here, we investigated the effects of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) on these receptor subtypes in cultured astroglia to understand the intracellular processes involved in the modulation of natriuretic peptide receptors in these cells. PMA (10 nM to 1 microM; 15 min to 24 h) treatment elicited a time- and concentration-dependent decrease in the numbers of 125I-labeled ANP specific binding sites, which was inhibited by the PKC antagonist staurosporine (500 nM). Furthermore, PMA (100 nM, 2 or 24 h) treatment elicited a significant decrease in the specific binding of 125I-des-Cys-Cys-ANP, an ANP-C receptor selective ligand. PMA (10 nM to 1 microM; 30 min) treatment also significantly decreased ANP (100 nM)-stimulated guanosine 3', 5'-cyclic monophosphate levels in cultured astroglia, an effect unmodified by phosphodiesterase inhibition. These data indicate that PKC modulates both guanylyl cyclase-coupled and ANP-C natriuretic peptide receptors in cultured astroglia. PMID- 8638653 TI - Generation of nitric oxide and superoxide during reperfusion after focal cerebral ischemia in rats. AB - We investigated the levels of nitrosyl hemoglobin (HbNO) in rat jugular blood by electron spin resonance (ESR) spectroscopy during and after middle cerebral artery occlusion. The levels of plasma nitric oxide (NO) end products, nitrate plus nitrate, were compared with the levels of HbNO. Small amounts of HbNO were detected in sham-operated rats (n=4) and those subjected to 2 h of occlusion (n=4), whereas nitrite plus nitrate was increased only in the latter (P<0.01; vs.sham). Upon reperfusion after 2 h of occlusion both HbNO and nitrite plus nitrate clearly increased after 15 min (n=4) and 30 min (n=6) reperfusion (P<0.01; vs.occlusion). Administration of superoxide dismutase (5 mg/kg) significantly increased HbNO (P<0.05) but not plasma nitrate plus nitrate (n=5). The increase in HbNO suppressed by administration of NG-nitro-L-arginine methyl ester (20mg/kg; n=4,P<0.01), and this suppression could be reversed by L-arginine (200 mg/kg) (n=4). The present study clearly showed that the L-arginine-NO synthase pathway was activated during reperfusion after focal cerebral ischemia and indicated the involvement of a reaction between NO and superoxide during early reperfusion. PMID- 8638655 TI - Role of weight-bearing function on expression of myosin isoforms during regeneration of rat soleus muscles. AB - The expression of myosin isoforms was studied in regenerated rat soleus muscle during either normal or altered postural activity. Regeneration was induced following injury by venom from the Notechis scutatus scutatus snake. Immunohistochemical analysis showed that, in regenerating soleus muscle after 3 wk of hindlimb suspension, nearly all fibers reacted positively with the myosin heavy chain (MHC) antibody associated with fast-twitch muscle fibers (fast MHC). When 3 wk of recovery with normal weight-bearing activity followed hindlimb suspension, the regeneration soleus muscle exhibited a nearly homogeneous staining with the MHC antibody associated with the slow-twitch muscle fibers (slow MHC). These findings were in accordance with quantitative analysis of the electrophoretic separation of the native myosin isoforms. Immunohistochemical data showed that removal of weight bearing in the 21-day old regenerated soleus muscles resulted in an increase in fast MHC expression. Together, the results of the present study clearly demonstrate that the postural load is an important component in the induction of slow MHC in regenerating muscle and that the control of the expression of MHC in muscle comprising a homogeneous population of fibers deriving from satellite cells appears more homogeneous and more complete than in a nondegenerated one. PMID- 8638654 TI - Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling. AB - Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins. PMID- 8638656 TI - Xenopus oocytes express multiple receptors for LPA-like lipid mediators. AB - In Xenopus laevis oocytes, both lysophosphatidic acid (LPA) and a cyclic phosphate-containing analogue 1-acyl-sn-glycero-2,3-cyclic phosphate (cLPA) isolated from Physarum polycephalum activated oscillatory Cl- currents. cLPA elicited oscillatory currents only when applied extracellularly and, similarly to LPA, evoked homologous desensitization. cLPA applied to oocytes previously desensitized b y LPA failed to elicit a current, indicating that LPA completely desensitized the cLPA receptors. In contrast, when oocytes were desensitized by cLPA, LPA still evoked large currents. The lack of heterologous desensitization between cLPA and LPA indicates that the former acts on a distinct receptor subpopulation(s), which is also activated by LPA. The alkyl-ether analogue 1 hexadecyl-2-lyso-sn-glycero-3-phosphate (16:0-GP) and dioleoyl-phosphatidic acid (18:1-PA) showed heterologous desensitization patterns similar to that of LPA with regard to cLPA. Complete heterologous desensitization was obtained between LPA and 16:0-GP or 18:1-PA. These observations demonstrate the simultaneous expression of at least two different types of receptors for LPA-like lipid mediators on Xenopus oocytes and that these receptors show different pharmacological properties in their selectivity to cLPA. PMID- 8638657 TI - Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability. AB - Regulation of endothelial permeability is poorly understood. Previous studies have shown that endothelial cells contain phosphodiesterase (PDE) isoenzymes II IV and that simultaneous adenylate cyclase activation and/or PDE inhibition blocked endothelial hyperpermeability (J.Clin.Invest. 91: 1421-1428, 1993). We now focused on a possible role for guanosine 3',5'-cyclic monophosphate (cGMP) dependent mechanisms and studied H2O2-exposed porcine pulmonary artery endothelial cell monolayers. Pretreatment of cells with different nitric oxide (NO) donors or atrial natriuretic peptide (ANP) increased endothelial cGMP content severalfold and blocked H2O2-related effects on permeability; opposite results were obtained with a NO synthase inhibitor. Determination of cGMP degradation in nitroprusside-exposed endothelial cells identified PDE II as the major cGMP metabolizing pathway, whereas PDE III and IV contributed little or nothing. Inhibition of PDE II reduced H2O2-related endothelial hyperpermeability, an effect that could be enhanced synergistically by simultaneous guanylate cyclase activation. In summary, these studies indicate that cGMP-dependent mechanisms (NO donors, ANP, and dibutyryl-cGMP) blocked H2O2-related increases in endothelial permeability. The major cGMP degrading pathway in endothelial cells was PDE II, thereby substituting the missing PDE V in these cells. Simultaneous guanylate cyclase activation and/or PDE II inhibition may be a valuable approach to treat endothelial hyperpermeability. PMID- 8638658 TI - Sodium-bicarbonate cotransport in guinea pig ileal crypt cells. AB - Prior studies show that ileal HCO3- secretion is of crypt origin, possibly involving Na+-HCO3- cotransport. To test for the latter, we isolated crypt cells from guinea pig ileum and determined effects of medium HCO3-, Na+, K+, disulfonic stilbenes, and gramicidin on intracellular pH [pHi;2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein fluorescence], cell volume (electronic sizing), and Na+ efflux from 22Na+ -preloaded cells. Ileal crypt cells alkalinized when placed in sodium gluconate-HCO3- medium containing N-5-methyl-5-isobutyl amiloride (1 microM), bumetanide (10 microM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250 microM which blocks Cl-/HCO3- exchange but not Na+ dependent HCO3- uptake). Depolarization with either gramicidin (50 microM) or 50 mM K+ caused a further 4 acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-inhibitable increase in pHi. Gramicidin also caused SITS-inhibitable cell swelling. Both gramicidin effects were Na+ dependent: at 0 mM Na+, gramicidin acidified and did not alter cell volume; at 25 mM, gramicidin also acidified; at 90 and 140 mM, gramicidin alkalinized and induced cell swelling. HCO3- -dependent SITS inhibitable Na+ efflux from 22Na+ -preloaded cells was also seen. We conclude that ileal crypt cells engage in electrogenic Na+ -HCO3- symport. PMID- 8638659 TI - Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells. AB - The restitution of epithelial integrity is accomplished in part by cell migration. Studying this process, we have found that nitric oxide (NO) release migrating epithelial BSC-1 cells displayed a biphasic response to the inflicted wounds; an initial transient release of NO is followed by a delayed sustained elevation. Whereas the constitutive endothelial NO synthase (NOS) did not show any spatial or temporal changes associated with wounding, the inducible NOS became expressed 3 h after wounding and showed higher abundance at the edges of epithelial wounds. L-Arginine (L-Arg) or NO donor, S-nitroso-N-acetyl-DL penicillamine, exerted motogenic effect in epithelial and endothelial cells. Inhibition of NOS with NG-nitro-L-arginine methyl ester (L-NAME) or a selective knockout of inducible NOS with antisense oligodeoxynucleotides reduced the rate of spontaneous or epidermal growth factor (EGF)-induced BSC-1 cell migration. Migrating cells showed the polarized expression of NOS, suggesting a head-to-rear NO gradient. Several growth factors (EGF, insulin-like growth factor I, hepatocyte growth factor, and fibroblast growth factor) were motogenic for BSC-1 cells, but this effect was abrogated by pretreatment with L-NAME. We conclude that endogenous NO production is a prerequisite for BSC-1 cell migration. A vectorial NO release may be essential for the spatially and temporally coordinated reciprocal phenomena that occur at the leading and trailing edge of locomoting epithelial cells. Although the exact mode of NO action remains uncertain, it is conceivable that the production of NO serves as a cellular switch from the stationary to the locomoting epithelial phenotype. PMID- 8638660 TI - Role of glucosamine synthesis in the stimulation of TGF-alpha gene transcription by glucose and EGF. AB - Transforming growth factor-alpha (TGF-alpha) gene transcription is regulated by both epidermal growth factor (EGF) and glucose. Previous studies have suggested that the metabolism of glucose to glucosamine through the enzyme L-glutamine: D fructose-6-phosphate amidotransferase (GFAT) plays a critical role in the glucose signaling. In this paper, we compared the role of GFAT in the glucose and EGF signals. We found that, although EGF stimulates GFAT mRNA accumulation in MDA-MB 468 cells, this effect of EGF occurred several hours after TGF-alpha transcription increased. MDA-MB-468 cells also exhibited a TGF-alpha transcriptional response to low concentrations of glucose. The TGF-alpha response to glucose but not EGF could be inhibited by a blocker of GFAT activity. Blockade of GFAT was confirmed by using Western blotting with the RL2 antibody, which recognizes an epitope on proteins containing N-acetylglucosamine. Exposure of cells to glucose increased the RL2 signal on several polypeptides, but this change could be blocked by inhibition of GFAT. These results support the notion that glucose stimulation of TGF-alpha expression requires GFAT, but EGF stimulation does not. PMID- 8638662 TI - Modulation of cell membrane potential in cultured vascular endothelium. AB - The present study explores the role of different ionic conductances in the regulation of membrane potential under resting conditions and after bradykinin (BK) or thapsigargin (TG) stimulation of cultured bovine aortic endothelial cells. Under resting conditions, the cell membrane potential observed was -62+/- 5 mV. The main conductance under these conditions is an inwardly rectifying potassium (IRK) channel. Application of 50 nM BK induced a transient hyperpolarization to -87 +/- 4 mV followed by sustained depolarization to -35 +/- 5 mV. The transient hyperpolarization was eliminated by 1 microM noxiustoxin, a blocker of calcium-activated postassium channels (K(Ca)). the sustained depolarization induced by BK was prevented by incubating the cells with the calcium channel blocker lanthanum. TG evoked a similar response in membrane potential, with the exception that the onset of the hyperpolarization was slower compared with BK. The results presented here indicate that the cell resting potential is maintained at -62 +/- 2 mV by the IRK channel. BK or TG stimulation induces a transient hyperpolarization of approximately -20 mV produced by activation of a KCa. This hyperpolarization is followed by a sustained depolarization produced by activation of a calcium-selective channel sensitive to lanthanum. PMID- 8638661 TI - Nitric oxide: a modulator for the epidermal growth factor receptor expression in developing ovarian granulosa cells. AB - the present study was designed to assess the involvement of nitric oxide (NO) in the regulation of the epidermal growth factor (EGF) receptor during development of rat granulosa cells, which were prepared by puncturing ovaries of diethylstilbestrol-treated rats. The immature cells were cultured for 48 h with follicle-stimulating hormone (FSH) to be transformed into mature cells. A marked accumulation of guanosine 3',5' -cyclic monophosphate (cGMP) was observed during development. The accumulation of cGMP, but not of adenosine 3',5'-cyclic monophosphate (cAMP), was suppressed by specific inhibitors of NO synthase, L-NG monomethyl-L-arginine (L-NMMA) and L-N(G)-nitro-L-arginine, and a selective inhibitor of the inducible NO synthase, aminoguanidine. The L-NMMA-induced suppression was partially reversed by addition of L-arginine to cultures but not D-arginine, indicating that NO formation is inhibited by competing with analogues of L-arginine for NO synthase. Treatment with 2-(4-carboxyphenyl)-4,4,5,5, tetramethylimidazoline-1-oxyl-3-oxide , an antagonist of NO, caused suppression in the increase of EGF binding sites, whereas exposure of the cells to sodium nitroprusside (SNP), an NO donor, caused elevation in EGF binding sites, with increasing extra- and intracellular cGMP levels. Analysis of the EGF receptor by affinity labeling with 125I-labeled EGF revealed that the intensity of the cross linked receptor molecular with a molecular mass of 180 kDa was increased by exposure to SNP. The facilitatory effect of SNP on the EGF receptor was observed when the cAMP-dependent pathway was fully activated by FSH. However, the NO effect may be mediated by a cGMP-independent pathway, as 8-bromo-cGMP did not mimic the action of SNP. These results indicate that the L-arginine-NO system may contribute to the regulation of EGF receptor expression in developing granulosa cells stimulated by FSH. PMID- 8638663 TI - Coordinate modulation of glucocorticoid receptor and glutaminase gene expression in LLC-PK1-F+ cells. AB - The effect of glucocorticoid receptor on glutaminase gene expression and related glutamine metabolism was studied in proximal tubule-like LCC-PK1-F+ cells. These cells express functional glucocorticoid receptors as demonstrated by immunoreactivity with antiglucocorticoid receptor antibody, specific ligand binding, and a 14-fold increase in chloramphenicol acetyltransferase (CAT) reporter gene activity after exposure to dexamethasone (10(-6)M). Dexamethasone exposure for 18 h increased glutaminase mRNA and activity by 32 and 42%, respectively (both P< 0.05, paired t-test), associated with a small (9%) but significant increase in glutamine utilization (P<0.05). In an effort to elicit a greater response, endogenous glucocorticoid receptors were supplemented by transfecting cells with a plasmid, pMAMGR, expressing the rat glucocorticoid receptor gene. Transfected cells expressed a 39-fold increase in CAT activity with dexamethasone treatment, confirming a higher level of functional receptors, but glutaminase mRNA and activity were now decreased by 34 and 32%, respectively, associated with a 15% fall in glutamine utilization after 18-h exposure to dexamethasone. This biphasic response in glutaminase gene expression was mirrored by glucocorticoid receptor mRNA that increased 41% after dexamethasone in LLC PK(1)-F+ cells, but decreased 63% after transfection (both P<0.05). These findings are consonant with glucocorticoid receptor gene modulation of glutaminase gene expression and glutamine utilization. PMID- 8638664 TI - Production and localization of cGMP and PGE2 in nitroprusside-stimulated rat colonic ion transport. AB - Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase activity in other tissues, but stimulation of this enzyme has not previously been described for intestine. We report a 20-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) production by radioimmunoassay in colonic mucosal strips stimulated with SNP. SNP also caused a significant increase in prostaglandin (PG) E2 release but did not stimulate release of the prostanoids thromboxane B2 or 6 keto-PGF1alpha. Stimulation of isolated colonic crypts and the remaining subepithelial mucosa demonstrated that the latter was the major source of the increases in cGMP and PGE2. Immunostaining of colonic mucosa revealed minimal basal cGMP immunoreactivity but large increases in abundance, localizing to the subepithelium, after SNP treatment. Under basal conditions, there was diffuse immunostaining for constitutive NO synthase in both the epithelial and subepithelial compartments, which was corroborated with NADPH diaphorase staining. In conclusion, SNP was an NO donor stimulates production of cGMP and PGE2 from the subepithelium. NO may be an important mediator of colonic secretion and other processes predominantly via its direct effects on cells of the lamina propria. PMID- 8638665 TI - EGF modulation of Na+/H+ antiport in rat hepatocytes: different sensitivity in adult and fetal cells. AB - The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells. PMID- 8638666 TI - Regulation of colonic ion transport by GRP. I. GRP stimulates transepithelial K and Na secretion. AB - Regulation of electrolyte transport across porcine distal colon epithelium by gastrin-releasing peptide (GRP) was examined using mucosal sheets mounted in Ussing chambers. Serosal GRP produced a biphasic response consisting of a transient increase in short-circuit current (ISC) followed by a long-lasting decrease. Indomethacin and tetrodotoxin inhibited the ISC increase without affecting the secondary decrease. Addition of GRP to the mucosal solution produced a decrease in ISC similar to that observed with serosal treatment, but no transient increase in ISC was observed. GRP and bombesin (50% effective concentrations of 26 and 30 nM, respectively) were more effective than neuromedin B in decreasing the ISC, and the GRP receptor antagonist [D-Phe(6)]bombesin(6-13) O-methyl produced a sixfold dextral shift in the GRP concentration-response relationship. The GRP-stimulated decrease was reduced in the absence of Cl and by serosal bumetanide. Flux measurements showed that GRP increased Rb and Na secretion while having no effect on transepithelial Cl transport. Phosphoinositide turnover was increased by GRP, suggesting that the ion transport changes may be mediated by intracellular Ca concentration. The results of this study demonstrate that GRP stimulates K and Na secretion across the porcine distal colon epithelium and that these processes are dependent, in part, on a bumetanide-sensitive transport pathway located in the basolateral membrane. PMID- 8638667 TI - Regulation of colonic ion transport by GRP. II. GRP modulates the epithelial response to PGE2. AB - The purpose of this study was to examine the potential modulatory effects of gastrin-releasing peptide (GRP) on prostaglandin (PG) E2-stimulated electrolyte transport across the distal colon epithelium. In an earlier study, PGE2 was shown to reduce net Cl absorption without altering the serosal-to-mucosal unidirectional Cl flux in porcine distal colon (19). In the present study, tissues were pretreated with serosal or mucosal GRP and subsequently stimulated with PGE2. The resulting increase in short-circuit current (ISC) was 152% (serosal GRP) and 49% (mucosal GRP) greater than control PGE2 responses alone. Serosal, but not mucosal, GRP also enhanced the ISC response to vasoactive intestinal peptide. On the basis of flux measurements, the combined effects of serosal GRP and PGE2 resulted in the activation of a transcellular pathway for Cl secretion, which was not activated by either mediator alone. The time course of the PGE2 response was also affected by GRP. Serosal GRP shortened the time to maximum ISC by 35%, whereas mucosal peptide lengthened the time to maximum ISC by 68% These results suggest that GRP acts as a modulator of PG action on electrolyte transport in the distal colon. PMID- 8638668 TI - Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport. AB - The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis. PMID- 8638669 TI - BAY K 8644 depresses excitation-contraction coupling in cardiac muscle. AB - We determined the effect of the dihydropyridine L-type Ca channel agonist BAY K 8644 (BAY) on excitation-contraction (E-C) coupling in isolated ferret ventricular myocytes using whole cell voltage clamp. The sarcoplasmic reticulum (SR) Ca load during the test pulses, assessed by caffeine-induced contractures, was similar in the presence and absence of BAY, with extracellular Ca concentration lowered from 3 to 1 mM in BAY. The relationship between L-type Ca current (ICa) and contraction was assessed, with current and contractions measured during depolarizations from -40 to between -30 and +50 mV after a conditioning train (to ensure constant SR Ca load). BAY shifted the current contraction relationship downward, such that, for a given ICa and SR Ca load, the contraction elicited was much smaller in the presence of BAY. BAY also induced a characteristic negative shift in the the current-voltage relationship. We conclude that BAY decreases the efficacy of a given Ca current to induce SR Ca release during E-C coupling in ferret cardiac tissue (in contrast to the BAY induced increase of resting SR Ca release). This may reflect an alteration in the state of the SR Ca release channel due to BAY binding to dihydropyridine receptors. PMID- 8638670 TI - Cloning of GRK2 cDNA from S49 murine lymphoma cells. AB - Beta-adrenergic receptor kinase is a member of the G protein-linked receptor kinase (GRK1) family that elicits receptor desensitization. We have cloned GRK2 from S49 mouse lymphoma cells. The nucleotide sequences of rat GRK2 and GRK3 were aligned and conserved primers chosen for use in reverse transcription-polymerase chain reaction (RT-PCR) of S49 mRNA. Direct sequencing of the PCR fragment provided a rapid means to identify the expression of the GRK2 but not the GRK3 transcript in these cells. Unique expression of GRK2 in S49 cells was confirmed by Western blotting. Three additional pairs of primers were chosen from the rat GRK2 sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to ligate the four fragments of S49 cell GRK2 cDNA by using PCR proved unsuccessful, the intact cDNA was assembled by digesting the PCR products in the region of the overlaps and ligating them in a single step into pBlue-script SK(+). PMID- 8638671 TI - The effect of temperature on charge movement repriming in amphibian skeletal muscle fibers. AB - Cut twitch muscle fibers, mounted in a triple Vaseline-gap chamber, were used to study the effects of temperature on intramembranous charge movement and, in particular, on the repriming of charge 1 (the intramembranous charge that normally moves in the potential range between -100 and +40 mV). Changing the holding potential from -90 to 0 mV modified the voltage distribution of charge movement but not the maximum movable charge. Temperature changes between 16 and 5 degrees C did not modify the fiber linear capacitance, the maximum nonlinear intramembranous charge, or the voltage distribution of charge 1 and charge 2 (the intramembranous charge moving in the membrane potential range between approximately -4 and -160 mV). We used a pulse protocol designed to study the repriming time course of charge 1, with little contamination from charge 2. The time course of charge movement repriming at 15 degrees C is described by a double exponential with time constants of 4.2 and 25 s. Repriming kinetics were found to be highly temperature dependent, with two rate-limiting steps having Q10 (increase in rate of a process by raising temperature 10 degrees C) values of 1.7 and 7.1 above and below 11.5 degrees C, respectively. This is characteristic of processes with a high energy of activation and could be associated with a conformational change of the voltage sensor or with the interaction between the voltage sensor and the calcium release channel. PMID- 8638672 TI - Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1. AB - Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost. PMID- 8638673 TI - AlphaB-crystallin protects glial cells from hypertonic stress. AB - AlphaB-crystallin and the small stress protein, heat shock protein of 27 kDa (HSP27), share structural similarities and are coordinately induced by classical stress stimuli. We have recently observed that hypertonic stress produced by high NaCl concentrations selectively induces alphaB-crystallin in glial cells. To examine divergence of the functional properties of these two related proteins, we have constructed stable alphaB-crystallin-expressing glial cell lines from the U 251 MG glioma cells, which are normally deficient in alphaB-crystallin expression but constitutively express HSP27. These transfected cells lines are more resistant to acute hypertonic stress than the parental line from which they were derived. Moreover, the parental line acclimates to stepwise increases in hypertonicity and upregulates endogenous alphaB-crystallin in the process but not HSP27. The overexpression of HSP27 and alphaB-crystallin in NIH/3T3 fibroblasts, a cell line that normally expresses little alphaB-crystallin and no HSP27, does not result in increased survival. This suggests that alphaB-crystallin interacts with cell-type specific mechanisms to aid in protection from hypertonic stress. PMID- 8638674 TI - Xenobiotic transport differences in mouse mesangial cell clones expressing mdr1 and mdr3. AB - P-glycoprotein (PGP), which confers multidrug resistance to cancer cells, is expressed in mouse kidney proximal tubule and mesangium. We report on the expression of PGP and its xenobiotic transport function in mesangial cells. Studies were performed in a mouse mesangial cell line (TKGM) and two cell clones. Ribonuclease protection assay and Western blot analysis demonstrated that TKGM cells expressed mdr1 and mdr3, the isoforms responsible for multidrug resistance. TKGM-F12 cells coexpressed mdr1 and mdr3 whereas TKGM-G2 cells expressed only mdr1. The drug transport function, measured by rhodamine 123 (R-123) efflux, was smaller in TKGM-F12 than in TKGM-G2 cells. The PGP substrates adriamycin, cyclosporin A, vinblastine, and verapamil inhibited R-123 transport in TKGM and TKGM-G2 cells. In the cells studied, PGP conferred some resistance to adriamycin; concomitant exposure to adriamycin with another PGP substrate impaired cell growth. The differential expression of mdr1 and mdr3 in mouse mesangial cell clones, the ability of mdr1 PGP to transport R-123, and the impairment of PGP mediated transport in TKGM-F12 cells, coexpressing mdr1 and mdr3 products, are demonstrated. PGP may play a physiological role in mesangial cells. PMID- 8638675 TI - Response of LLC-PK1-F+ cells to metabolic acidosis. AB - The role of extracellular glutamate formation as opposed to cellular glutamate removal in regulating monolayer glutamate content in response to metabolic acidosis was studied in LLC-PK1-F+ cells. Exposure to metabolic acidosis (14 mM bicarbonate; pH 7.1) for 18 h resulted in 24% fall in monolayer glutamate content. Of this, approximately one-half could be attributed to enhanced glutamate removal via glutamate dehydrogenase, consistent with a rise in ammonium production. The remainder appears due to reduced extracellular glutamate formation as a consequence of diminished gamma-glutamyltranspeptidase (gamma-Gt) activity. Metabolic acidosis, but not respiratory acidosis, resulted in a 33% fall in gamma-Gt activity and a proportional fall in extracellular glutamate formation; glutamate transport into these cells was not rate limiting in acidosis. Overall glutamine utilization decreased 36%, reflecting the fall in gamma-Gt activity as well as a decrease in a pH-sensitive glutamine uptake, whereas glutamine transport coupled to the phosphate-dependent glutaminase flux increased. It is noteworthy that the increased ammonium produced in metabolic acidosis was preferentially secreted into the apical compartment; acid secretion, but not production, was similarly increased. Thus reduced cellular glutamate appears to coordinate activation of intracellular glutaminase to the apical membrane exchanger, consistent with the functioning kidney response to metabolic acidosis. PMID- 8638676 TI - Rapid kinetics of second messenger production in bitter taste. AB - The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5 trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste. PMID- 8638678 TI - Fractal analysis and imaging of the proximal nephron cell. AB - Cells of the S1 proximal renal tubule were examined to determine whether their peculiar shapes are a result of certain constructs of fractal mathematics. Morphometric measurements of the cell perimeter were made at several levels of cell height by measuring the intercellular boundaries that appear on electron micrographs of tubule cross sections. When the measurements were made over a range of scale lengths, the fractal dimension, D, of the cell perimeter was found to increase from 1.3 near the cell apex to 1.78 near the cell base. The length of scale was found to range between 8 and 0.4 micron and to represent the approximate dimensions of actual cell processes. Fractal patterns that conformed to the measured parameters were then constructed from a fractal generator composed of budlike formations that originated near the cell apex and that increased in number and decreased in size with cell depth according to a fractal scaling. It was found that the fractal rule of keeping a constant relative scale could be maintained between budding processes but, to obtain patterns that resemble biological structure, the processes must be positioned randomly on the cell periphery. It is shown that when the relative sizes of the buds decrease exponentially and their numbers increase geometrically, the perimeter can grow to the correct length without overlap. This suggests that patterns of the cell periphery corresponding to different levels of cell height obey a law of scale but occur randomly in a way that increases to high fractal dimension or near plane-filling values at the cell base. The fractal patterns that correspond to the measured fractal dimensions can be assembled into a three-dimensional model that closely resembles the known shape of the proximal tubule cell. PMID- 8638677 TI - NH4Cl-induced hypertrophy is mediated by weak base effects and is independent of cell cycle processes. AB - Renal hypertrophy occurs in a number of clinical conditions, some of which are associated with increases in ambient ammonia concentrations. NH4Cl induces hypertrophy in cultured renal epithelial cells. The present studies examined the mechanism of NH4Cl-induced hypertrophy in NRK-52E cells. Hypertrophy was also induced by methylammonium chloride, a related weak base, but not by tetramethylammonium chloride, a weak base analogue that can neither accept nor donate protons. Bafilo-mycin A1, an inhibitor of vacuolar proton pumps, also induced hypertrophy. Together, these studies suggest that NH4Cl-induced hypertrophy is mediated by its weak base property, allowing it to enter and alkalinize acid vesicular compartments. Additional studies demonstrated that NH4Cl-induced hypertrophy is not mediated by modulation of cell cycle processes. NH4Cl addition had no effect on the following: c-fos mRNA abundance, typically associated with entrance into the cell cycle; cyclin E protein abundance, which increases as cells progress through G1; or protein synthesis, which also increases during G1. In addition, inactivation of pRB by overexpression of human papilloma virus-16 carrying the E7 gene, which inhibits cell cycle-dependent hypertrophy, had no effect on the ability of NH4Cl to induce hypertrophy. Based on these data, we postulate that, in hypertrophic conditions associated with increased ammoniagenesis, hypertrophy is mediated by vesicular alkalinization and occurs independently of processes that govern progression through the cell cycle. PMID- 8638679 TI - Electrophysiological characterization of RACTK1 K+ channel in stable cell line. AB - RACTK1 is a pH-sensitive K+ channel cloned from rabbit renal collecting tubule cells. To characterize the function of this K+ channel in more detail, RACTK1 was transfected to an established cell line and the patch-clamp study was performed. cDNA of RACTK1 was inserted in the pMAM vector and transfected to Chinese hamster ovary cells. In one of 36 cell lines, the channel protein as expressed by the dexamethasone-induced mRNA and was detected by the specific antibody. The RACTK1 K+ channel with 80 pS was consistently observed. In inside-out patch, Ca2+ at concentrations higher than 500 nM activated the channel. Open probability was decreased by protein kinase A (from 45 to 4.2%, n+6) but not by protein kinase C. Whole cell currents of the transformed cells represented K+ conductance that was not blocked by an addition of charybdotoxin but by apamin. RACTK1 K+ channel has similar, though not identical, characteristics to the Ca2+ -activated K+ channel. RACTK1 might therefore encode a subunit of the intermediate conductance Ca2+ activated K+ channel observed in the apical membrane of the rabbit renal collecting duct. PMID- 8638680 TI - Effect of purinergic blockers on outward current in isolated smooth muscle cells of the sheep bladder. AB - Freshly dispersed cells from sheep urinary bladder were voltage clamped using the whole cell and inside-out patch-clamp technique. Cibacron and Basilen blue increased outward current in a dose-dependent manner with a half-maximal response at 10(-5)M. Suramin, in concentrations to 10(-3)M, had no such effect. The Cibacron blue response was abolished in Ca2+ -free physiological salt solution, suggesting that it was acting on a Ca2+ -dependent current. Similarly, the Cibacron blue-sensitive current was significantly attenuated by charybdotoxin. Cibacron blue did not modulate inward current nor were its effects modified by caffeine or heparin, suggesting that its effect on outward current was not secondary to an increase in intracellular Ca2+. Application of 10(-4)M Cibacron blue to the inside membrane of excised patches caused a rapid increase in open probability of a large conductance (300 pS) K+ channel. These results suggest that Cibacron blue is a potent activator of a Ca2+ -dependent outward current in bladder smooth muscle cells in addition to its action as a purinergic blocker. PMID- 8638681 TI - Diverse signaling pathways in the cellular actions of insulin. AB - Insulin is one of the most important regulators of glucose and lipid homeostasis. Many of its cellular actions are mediated by changes in protein phosphorylation. The consequences of these phosphorylation events extend from a series of different short-term metabolic actions to longer-term effects of the hormone on cellular growth and differentiation. Although the insulin receptor itself is a tyrosine kinase that is activated upon hormone binding, the ensuing changes in phosphorylation occur predominantly on serine and threonine residues. Moreover, insulin can simultaneously stimulate the phosphorylation of some proteins and the dephosphorylation of others. These paradoxical effects of insulin suggest that separate signal transduction pathways may emanate from the receptor itself to produce the pleiotropic actions of the hormone. PMID- 8638682 TI - Chronic ouabain infusion does not cause hypertension in sheep. AB - Ouabain is claimed to be a hormone of adrenal origin, capable of raising arterial pressure in rats. We infused ouabain in conscious sheep under carefully controlled circumstances to determine its effects on blood pressure, urine electrolytes, and vasoactive hormones. Eight healthy ewes were studied while taking a constant intake of dietary sodium and potassium. Ouabain infusion at 0.25 mg daily over 22 days reduced heart rate and arterial pressure and had no effect on pressor responsiveness to incremental intravenous infusions of angiotensin II. Ouabain induced minor, but statistically significant, decrements in urine volume, urinary sodium excretion, plasma renin and angiotensin II concentrations, and a rise in plasma aldosterone and cortisol. Plasma ouabain levels averaged 1.37 +/- 0.28 nmol/l during ouabain infusion. In conclusion, high dose chronic ouabain infusion in sheep did not elevate arterial pressure or alter pressor responsiveness to angiotensin II, was antidiuretic and antinatriuretic, and induced minor perturbations in circulating renin, angiotensin II, aldosterone, and cortisol. PMID- 8638683 TI - Forearm vasodilator mechanisms during mental stress: possible roles for epinephrine and ANP. AB - The contribution of epinephrine (Epi) to forearm vasodilator responses to mental stress was evaluated in 12 healthy men by comparing hemodynamic and plasma catecholamine responses to mental stress and to intravenous and intra-arterial infusions of epinephrine. Mental stress decreased forearm vascular resistance (FVR) by 45%, increased arterial Epi from 0.23 to 0.44 nmol/l in arterial plasma, and increased forearm norepinephrine overflow. Intra-arterial Epi infusion decreased FVR concentration dependently by up to 43%. Intravenous Epi infusion decreased diastolic arterial pressure and increased heart rate and systolic blood pressure dose dependently. FVR decreased by up to 39% at 4.60 nmol/l Epi in arterial plasma. The average Epi contribution to forearm vasodilation during mental stress was calculated to be between 9 and 30%, depending on if responses to stress were compared with intravenous or intra-arterial Epi infusion. Arterial atrial natriuretic peptide immunoreactivity increased by 23% during stress, supporting a vasodilator influence, whereas vasopressin immunoreactivity was unaffected. Thus secretion of Epi explains only part of the stress-induced forearm vasodilation. Intravenous infusion of Epi appears to activate sympathetic counterregulation. PMID- 8638684 TI - Metabolic responses of canine gracilis muscle during contraction with partial ischemia. AB - The metabolic effects of partial ischemia on canine skeletal muscle were examined during 20 min of isometric contraction. A reduction in blood flow of approximately 75% resulted in an approximate 40% reduction in contractile function. Muscle lactate accumulation and phosphocreatine (PCr) hydrolysis were greater during ischemia, indicating a greater reliance on anaerobic ATP regeneration. Pyruvate dehydrogenase transformation to its active form (PDCa) during contraction was not affected by ischemia, such that PDCa did not appear to be a determinant of skeletal muscle fatigue. Acetylcarnitine concentration was greater during ischemic contraction and inversely correlated with PCr concentration (r = -0.79, P<0.01). Furthermore, acetylcarnitine accumulation and PCr degradation correlated with the degree of skeletal muscle fatigue (r = 0.56, P<0.05 and r = 0.70, P<0.01, respectively). Thus the greater the acetyl group oxidation, the lesser the contribution from anaerobic ATP provision and, subsequently, the smaller the degree of muscle fatigue observed. The metabolic characteristics of this model of ischemic muscle contraction are indistinguishable from the normal metabolic responses observed with increasing contractile intensity. PMID- 8638685 TI - Androgens contribute to the stimulation of cancellous bone formation by ovarian hormones in female rats. AB - We investigated whether androgens, for which the ovaries are the major source in female rats, contribute to the stimulation of cancellous bone formation by ovarian hormones in female rats. Ovariectomized animals were administered 5alpha dihydrotestosterone (DHT; 10 and 100 microgram/kg) by daily subcutaneous injection for 13 days, after which histomorphometric analysis was performed at the proximal tibial metaphysis. To prevent ovariectomy from stimulating bone turnover, and hence complicating the interpretation of changes in bone formation, animals were also given the resorption inhibitor 3-amino-1-hydroxypropylidene-1 bisphosphonate. We found that ovariectomy markedly suppressed cancellous bone formation, which was partially prevented by DHT (100 microgram/kg). To further address whether androgens contribute to the stimulation of bone formation by ovarian hormones, we treated intact and ovariectomized female rats with the androgen antagonist flutamide (15 mg/kg/day) for 28 days. Whereas flutamide had no effect in ovariectomized rats, it significantly reduced cancellous bone formation in intact animals. We conclude that, in the female rat, androgens contribute under physiological conditions to the stimulation of cancellous bone formation by ovarian hormones. PMID- 8638686 TI - Enteral glutamate is almost completely metabolized in first pass by the gastrointestinal tract of infant pigs. AB - We studied the absorption of enteral glutamate and phenylalanine using isotopic tracer and arteriovenous difference techniques. Six piglets, implanted with portal, carotid, and gastric catheters and an ultrasonic portal flow probe received a 6-h intragastric infusion of [U-13C] glutamate and [2H] phenylalanine, with a high-protein diet offered one time each hour. Amino acid concentrations and the isotopic enrichments of all mass isotopomers of glutamate, glutamine, and phenylalanine were measured in portal and arterial blood over the last hour. There was significant (P<0.025) net absorption of the indispensable amino acids as well as arginine, proline, serine, and alanine. There was no portal uptake of glutamate, aspartate, and glycine, and arterial glutamine was removed by the portal drained viscera (P<0.05). At isotopic steady state, 72% of the [2H] phenylalanine but only 5% of the [U-13C] glutamate tracer appeared in the portal blood. We conclude that, in fed infant pigs, the gut metabolizes virtually all of the enteral glutamate during absorption. Therefore, glutamate and glutamine in the body as a whole must derive almost entirely from synthesis de novo. PMID- 8638687 TI - Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo. AB - Based on our quantum concept for mechanically adaptive bone formation, we hypothesized that a single bout of loading would increase bone formation at the endosteal surface in rat tibiae, with a maximal response 4-8 days after loading and a stimulus-response relationship for load magnitude. Bending loads were applied to right tibiae of rats at 31, 43, 53, or 65 N for a single bout of 36 or 360 cycles; bone formation was assessed 1-4, 5-8, or 9-12 days after loading. A single loading episode increased lamellar bone formation rate (BFR) in all groups (P<0.05) and was maximal 5-8 days after loading. A distinct dose-response relationship was not evident among all load magnitudes or for duration, but 65 N was significantly more osteogenic than loads of 31-53 N (P<0.05), consistent with a threshold response to loading. There was also evidence for a significant increase in BFR (P<0.05) and double-labeled surface (P<0.01) within 4 days of loading, suggesting that bone-lining cells were activated directly by the stimulus. Thus subtle changes in BFR may occur by modulating the activity of surface cells, but large modeling drifts and anabolic responses require recruitment and differentiation of osteoprogenitor cells near the bone surface. PMID- 8638688 TI - Differential effects of acute hypertriglyceridemia on insulin action and insulin receptor autophosphorylation. AB - Experimentally induced hypertriglyceridemia (HTG) and high plasma free fatty acid (FFA) levels impair in vivo insulin action. To determine if this is a consequence of impaired in vivo insulin receptor autophosphorylation and related to defective receptor signaling, hyperinsulinemic euglycemic clamps, indirect calorimetry, and skeletal muscle biopsies were performed in nine healthy subjects. In vivo insulin action was determined from the glucose infusion rate (GINF) and glucose oxidation (Glcox) during 40 and 120 mU/m2 /min clamps with (HTG clamp) and without (control clamp) a triglyceride emulsion infusion. The percentage of receptors autophosphorylated in vivo was determined by 125I-labeled insulin tracer binding in skeletal muscle immunoprecipitates of insulin receptors and phosphorylated receptors. Compared with the control clamps, plasma triglycerides and FFA increased four- and twofold, whereas GINF and Glcox decreased 15 and 35%, respectively, during the HTG clamps (all P<0.05). However, the percentages of receptors phosphorylated after the 40 and 120 mU/m2/min HTG clamps (9.2 +/- 1.5 and 21.1 +/- 2.6%, respectively) were similar to the control clamps (9.0 +/- 0.6 and 18.6 +/- 2.2%, respectively). These results indicate that, if impaired insulin signal transduction is a mechanism by which HTG and FFA impair insulin action, it occurs at a site downstream from insulin receptor autophosphorylation. PMID- 8638689 TI - IL-1 receptor antagonist attenuates sepsis-induced alterations in the IGF system and protein synthesis. AB - The purpose of the present investigation was to determine whether endogenously produced interleukin (IL)-1 mediates the changes in insulin-like growth factor (IGF) I and IGF binding proteins (IGFBP) induced by chronic abdominal sepsis in rats and to correlate the changes in the IGF system with the alternations in protein synthesis. A constant infusion of IL-1 receptor antagonist (IL-1ra) was begun after the induction of sepsis and was continued for 5 days. Sepsis decreased IGF-I levels in the blood, liver, and gastrocnemius muscle, increased the content in the kidney, and did not alter IGF-I levels in heart, jejunum, and spleen. IL-1ra attenuated the sepsis-induced decrease in plasma IGF-I and completely prevented the changes in IGF-I observed in liver, kidney, and the gastrocnemius. IGFBP-1 was increased in the blood, liver, and muscle of septic rats. IL-1ra prevented this increase in IGFBP-1 in blood and liver but not in muscle. The rate of in vivo protein synthesis was decreased in the gastrocnemius and kidney and unaltered in the heart, liver, jejunum, and spleen. A strong linear correlation existed between levels of IGF-I and the rate of protein synthesis determined simultaneously in the gastrocnemius. These results provide evidence for the role of IL-1 as an endogenous mediator of the sepsis-induced changes in IGF-I and IGFBP-1 and suggest that the accompanying changes in muscle protein synthesis are partially mediated via changes in IGF-I. PMID- 8638690 TI - Cocaine and exercise: temporal changes in plasma levels of catecholamines, lactate, glucose, and cocaine. AB - To determine the combined sympathoadrenal effects of cocaine and exercise in awake animals, rats were assigned to one of four treatment groups: saline-rest (SR), saline-exercise (SE), cocaine-rest (CR), and cocaine-exercise (CE). Venous blood samples from jugular catheters were obtained at -40, 0-4, 7, 10, 13, 16, 19, 26, and 36 min after intravenous injection of cocaine (5 mg/kg) or saline and the simultaneous onset of a 16-min treadmill run (26 m/min, 10% grade). CE increased plasma epinephrine (24.2 nM at 16 min), norepinephrine (28.0 nM at 10 min), and lactate (11.2 mM at 4 min) to levels 2-5 times greater than either treatment (SE and CR) alone (P<0.05) and 11-35 times higher that SR. Blood glucose values were significantly depressed in CE (-33% vs. SE) but increased in CR (+26% vs. SR). Plasma cocaine peaked < 2 min after injection in both CR and CE, and the peak was 69% higher in CE (P<0.05); however, the plasma elimination half-life (12-14 min) was not different. These results indicate that the combined effect of the two sympathoadrenal stimulants, exercise and cocaine, amplify the catecholamine responses to levels far greater than when each stimulant is used alone. PMID- 8638691 TI - Metabolic fate of an oral long-chain triglyceride load in humans. AB - To determine the steps involved in the metabolism of ingested triglycerides (TG), 10 healthy women were studied during 6 h after ingestion of 30 g olive oil labeled with [1,1,1-13C3] triolein. The appearance of 13C was followed in chylomicron-TG (CM-TG), nonesterified fatty acid (NEFA), very low-density lipoprotein (VLDL)-TG, and in expired gas. Indirect calorimetry was used to determine total lipid oxidation. After 90 min, labeling was higher in CM-TG than in NEFA or VLDL. At 180 min, a plateau of enrichment was obtained for CM-TG and NEFA, demonstrating the entry of exogenous lipids in the NEFA pool. After 300 min, a plateau was observed for VLDL-TG with levels of enrichment (0.38 +/- 0.04%) similar to those observed for NEFA (0.36 +/- 0.03%), suggesting a precursor-product relationship. Only 19 +/- 2% of the load was oxidized. From 300 to 360 min, 70% of total lipid oxidation was from exogenous TG. We conclude that, after ingestion of a lipid load, a cycle of fatty acids-TG occurs from CM to NEFA and from NEFA to VLDL. Furthermore, this lipid load has a sparing effect on endogenous lipid stores. PMID- 8638692 TI - Leucine and glucose kinetics during growth hormone treatment in intrauterine growth-retarded preterm infants. AB - The present study was performed with the objective to investigate the possible effects of recombinant human growth hormone (rhGH) supplementation on protein and glucose metabolism during the early postnatal period in seven intrauterine growth retarded (IUGR) preterm infants. Eight infants were studied as controls. The metabolic rate was measured by indirect calorimetry, and whole body protein and glucose kinetics were measured using constant-rate infusions of [1-13C] leucine and [U-13C]glucose during continuous adequate oral feeding. Energy expenditure was similar in both groups. In the rhGH-treated group of infants, leucine turnover (470 +/- 76 vs. 409 +/- micromol/ kg/day), leucine used for protein synthesis (402 +/- 72 vs. 337 +/- 36 micromol /kg/day), and leucine derived from protein breakdown (365 +/- 71 vs. 304 +/- 41 micromol/kg/day) were increased. However, net leucine balance was not increased (37 +/- 17 vs. 34 +/- 13 micromol/kg/day). The total rate of appearance of glucose was 10.1 +/- 1.2 vs. 10.0 +/- 1.3 mg/kg/min. We suggest that immediate postnatal treatment with rhGH is not effective in stimulating protein gain and has no effect on glucose kinetics in IUGR preterm infants. PMID- 8638693 TI - Increments in skeletal muscle GLUT-1 and GLUT-4 after endurance training in humans. AB - We investigated the time course of training-induced changes in the expression of GLUT-1 and GLUT-4 in human skeletal muscle. Seven healthy males trained for 2 h/day (approximately 60% pretraining VO2peak) for 31 days (31D). Muscle biopsies were obtained before training (PRE) and after 5 (5D) and 31 days (31D) of training. Training resulted in progressive increases in muscle GLUT-4 with increasing training duration (PRE<5D<31D; P<0.01). Muscle GLUT-1 content was also increased (P<0.05) after training; however, the increase was not observed until 31D (131%). Increases in muscle hexokinase (HK) activity were complete by 5D (P<0.01). Muscle malate dehydrogenase activity was not elevated after 5D of training but was increased (+35%; P<0.01) at 31D. Results from this study show that increases in both GLUT-4 and HK represent early training-induced adaptations to prolonged exercise training. As training progresses, further increases in GLUT 4, but not HK, occur in conjunction with an increase in muscle mitochondrial potential and GLUT-1. PMID- 8638694 TI - Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM. AB - The current study was undertaken to examine the impact that obesity and non insulin-dependent diabetes mellitus (NIDDM) have on the ability of glucose to stimulate its own uptake and oxidation in muscle. Euglycemic and hyperglycemic clamp experiments were performed with somatostatin infusions so that insulin could be replaced to basal levels or to physiological hyperinsulinemia. Arteriovenous leg balance methods were used to measure the pathways of leg muscle glucose uptake, oxidation, and storage. Percutaneous biopsies of the vastus lateralis muscle were taken to determine the pyruvate dehydrogenase complex or glycogen synthase activities. During basal insulin replacement, obese compared with lean nondiabetic subjects had higher values for glucose uptake, respiratory quotient, and glucose oxidation (all P<0.05) and a higher proportion of leg energy expenditure derived from glucose. Obese NIDD patients had a greater reliance on fat calories than lean diabetics during basal insulin replacement (P< 0.05). Hyperinsulinemia increased leg glucose metabolism (P<0.001) in all groups, but obese NIDD patients were significantly more insulin resistant. Hyperglycemia in NIDDM compensated for insulin resistance to the extent that rates of glucose metabolism were the same as those for nondiabetics studied at euglycemia. When nondiabetics were studied at hyperglycemia matched to the diabetics, the insulin resistance was still readily apparent. PMID- 8638695 TI - Interaction between nitric oxide and prostaglandin E2 pathways in pregnant rat uteri. AB - We examined the possible interrelationship between nitric oxide (NO) and cyclooxygenase (COX) products in the uterus during pregnancy and labor. Results indicate that 1) rat uteri during labor, at term, demonstrated a 69% decrease in nitrite production and a 217% increase in prostaglandin E2 (PGE2) production compared with day 18 of pregnancy; 2) interleukin-1 beta (IL-1 beta) induced a pronounced elevation of both nitrites and PGE2 in rat uteri; 3) diethylenetriamine/NO, a donor of NO, induced a significant increase of PGE2 production by the uterus in a dose-dependent manner; 4) NG-nitro-L-arginine methyl ester, an inhibitor of NO synthesis, markedly inhibited IL-1 beta-induced nitrite and PGE2 in rat uteri; this inhibitory action was reversed by coincubation with L-arginine; 5)exogenous PGE2 significantly inhibited IL-1 beta induced, but not constitutive, nitrite production; and 6) inhibition of endogenous PGE2 by indomethacin substantially increased IL-1 beta-induced nitrite production. Thus the interaction between NO and COX pathways might be important in the regulation of uterine contractility during pregnancy and labor. PMID- 8638696 TI - Effect of chronic hypoxia on myometrial responsiveness in the pregnant rat. AB - Mechanisms involving the timing of normal parturition are not well understood in most animal species. To gain a greater understanding of the mechanisms, we employed hypoxia to perturb the normal system of parturition. The present study was designed to investigate the effects of chronic hypoxia on myometrial contractility in the near-term pregnant rat. Rats were exposed to room air (control) or to continuous hypoxia (10.5% O2) either from experimental days 19 through 21 (2-day exposure) or from experimental days 15 through 21 (6-day exposure). On day 21, blood was collected for hormone assays, and the uterine horns were collected from each dam. One horn was snap-frozen in liquid nitrogen for oxytocin (OT) receptor analysis, and the other was used for in vitro assessment of myometrial contractile responses to cumulative doses of OT or arginine vasopressin (AVP). Hypoxic exposure resulted in approximately 60% reduction of the maximal myometrial contractile response to OT and a significant reduction in OT binding sites from 256.9 +/- 34.9 to 84.9 +/- 21.3 fmol/mg protein (P<0.01). In contrast, the contractile response to AVP was unaffected after exposure to chronic hypoxia (P> 0.05). Additionally, we observed no difference in the plasma concentrations of estrogen, progesterone, and corticosterone. We conclude that chronic hypoxia decreased the effectiveness of OT-specific contractile mechanisms, at least partially through a decrease in OT binding sites. PMID- 8638697 TI - Effect of aging on the metabolism of phosphorus and 1,25-dihydroxyvitamin D in healthy men. AB - We tested the hypothesis that aging alters physiological regulation of the serum concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] by inorganic phosphorus. In seven elderly men [age 71 +/- 1 (SE) yr] and 9 young men (29 +/- 2 yr), dietary phosphorus was first normal, then increased and decreased within its normal range. At each intake of phosphorus, serum concentrations of 1,25(OH)2D in the elderly did not differ from those in young men, but fasting and 24-h mean serum concentrations of phosphorus were lower in elderly men. With phosphorus restriction, in each group serum 1,25(OH)2D increased by 47%, and 24-h mean serum phosphorus decreased by 0.6 +/- 0.1 mg/dl. Serum concentrations of 1,25(OH)2D varied inversely with 24-h mean serum phosphorus (R= -0.92, P<0.0001). Thus, in healthy elderly men in whom glomerular filtration rate is normal or near normal, serum concentrations of 1,25(OH)2D increase when dietary phosphorus is restricted; the magnitude of response at steady state is unaffected by aging, but the time course of response is delayed. At any level of serum phosphorus, serum 1,25(OH)2D is lower than that in young men, as reflected by a lower intercept of regression of serum 1,25(OH)2D on 24-h mean phosphorus. PMID- 8638698 TI - Placental transport and fetal utilization of leucine in a model of fetal growth retardation. AB - Placental transport and fetal utilization of leucine were studied at 130 days of gestation in six control ewes and in seven ewes in which intrauterine growth retardation (IUGR) had been induced by exposure to heat stress. Leucine fluxes were measured during simultaneous intravenous infusion of L-[1-13C]leucine into the mother and L-[1-14C] leucine into the fetus. In the IUGR group, the following leucine fluxes, expressed as micromol/min/kg fetus, were reduced compared with control: net uterine uptake (3.44 vs. 8.56, P<0.01), uteroplacental utilization (0.0 vs. 4.7, P<0.01), fetal disposal rate (6.4 vs. 8.9, P<0.001), flux from placenta to fetus (5.0 vs. 7.1, P<0.01), direct transport from mother to fetus (1.6 vs. 3.4, P<0.01), flux from fetus to placenta (1.5 vs. 3.2, P<0.001), and oxidation of fetal leucine by fetus plus placenta (2.1 vs. 3.2, P<0.02). Uterine uptake, uteroplacental utilization, and direct transport were also significantly reduced per gram placenta. We conclude that maternal leucine flux into the IUGR placenta is markedly reduced. Most of the reduced flux is routed into fetal metabolism via a decrease in placental leucine utilization and a decrease in the leucine flux from fetus to placenta. PMID- 8638699 TI - Preservation of insulin secretory responses to P2 purinoceptor agonists in Zucker diabetic fatty rats. AB - The role of P2 purinoceptor agonists in regulatory insulin secretion in Zucker diabetic fatty (ZDF) rats was studied using the isolated perfused pancreas and intracellular Ca2+ concentration ([Ca2+]i) microfluorimetry. The relative potency of different purinoceptor agonists to stimulate the insulin secretory process was consistent with the conclusion that responses in [Ca2+]i and insulin secretion are mediated by the P2y subtype of purinoceptors. Additional studies using specific antagonists of the Ca2+ signaling pathway indicated that activation of P2y purinoceptor releases Ca2+ from intracellular stores and promotes Ca2+ entry through voltage-independent rather than voltage-dependent Ca2+ channels on the beta-cell membrane. Perfused pancreas and isolated islets from ZDF rats demonstrated markedly reduced or absent insulin secretion and [Ca2+]i responses to glucose and KCl. In contrast, responses to P2y purinoceptor agonists were normal, indicating that the secretion coupling pathway activated by these agonists is preserved in glucose-unresponsive islets from diabetic animals. These observations raise the possibility that the purinoceptor pathway may play an important role in regulating insulin secretion in hyperinsulinemic non-insulin dependent diabetes mellitus. PMID- 8638701 TI - Integrated mathematical model to assess beta-cell activity during the oral glucose test. AB - A model describing beta-cell secretion during an oral glucose tolerance test (OGTT) is introduced. The aim was to quantify beta-cell activity in different pathologies by analyzing peripheral concentration data of insulin, C-peptide, and islet amyloid polypeptide (IAPP). Insulin appearance in periphery is given by the fraction of C-peptide secretion, CPS(t), which accounts for liver degradation. A novelty of this study is the inclusion of IAPP delivery assumed proportional to CPS(t). Although IAPP fractional clearance is estimated in every subject, the clearances of insulin and C-peptide are assigned from a wide set of previous independent studies. Sensitivity analysis was performed to quantify the "error" in the estimated variables due to these assignments. All parameters relating to beta-cell secretion increased in the glucose-intolerant states [integrated CPS(t)=56 +/- 8 nmol/l in 180 min vs. 32 +/- 3 of controls, P<0.05; total IAPP delivery= 83 +/- 21 pmol/l in 180 min vs. 41 +/- 6, P<0.05]. Elevated plasma IAPP concentration of the patients was due to augmented secretion since IAPP clearance was found to be even slightly greater than in controls, (0.053 +/- 0.011 vs. 0.034 +/- 0.004 min-1) and markedly lower than that of insulin (0.14 +/- 0.02, P<0.01). In conclusion, the model introduced here allows the characterization of beta-cell secretory parameters during a simple test such as OGTT. PMID- 8638700 TI - Neuroendocrine and cardiovascular effects of serotonin: selective role of brain angiotensin on vasopressin. AB - Central serotonin (5-HT) and angiotensin (ANG II) stimulate arginine vasopressin (AVP), oxytocin (OT), and adrenocorticotropin (ACTH) secretion and increase blood pressure. Studies were conducted in conscious rats to determine whether neuroendocrine activation by 5-HT requires a brain angiotensinergic intermediate pathway. In the first study, ANG II formation was inhibited by the angiotensin converting enzyme inhibitor enalapril before injection of the 5-HT releaser/uptake inhibitor d-fenfluramine. Fenfluramine (2 mg/kg ip) stimulated AVP, OT, corticosterone, and prolactin (PRL) secretion (P<0.01). Enalapril (60 mg/l in drinking water for 4 days and 10 mg/kg ip 2 h before the rats were killed) inhibited only the AVP response (P<0.01) to d-fenfluramine. In the second study, the effect of intracerebroventricular injection of the 5-HT2A/2C antagonist LY-53857 (10 microgram), or the ANG II AT1 antagonist DuP-753 (10 microgram), on intracerebroventricular 5-HT (10 microgram)-stimulated AVP, OT, ACTH, PRL, renin secretion, mean arterial pressure (MAP) and heart rate (HR) was tested. LY-53857 inhibited the AVP, OT, and ACTH responses to 5-HT (P<0.01), whereas DuP-753 inhibited only the AVP response (P<0.01). Intraventricular injection of 5-HT increased MAP and decreased HR. The MAP response was not affected by LY-53857 or DuP-753, and at no time did MAP decline below starting levels. The decreased HR was inhibited by LY-53857 but not by DuP-753. These results demonstrate that 5-HT-induced AVP secretion is mediated selectively via brain angiotensinergic mechanisms by way of the AT1 receptor. PMID- 8638702 TI - The hot but not the cold minimal model allows precise assessment of insulin sensitivity in NIDDM subjects. AB - Assessment of insulin sensitivity in subjects with non-insulin-dependent diabetes mellitus (NIDDM) is of paramount importance but intrinsically difficult. The standard (hereafter cold) minimal model, in conjunction with an insulin-modified protocol, has been recently proposed, but the estimates of insulin sensitivity showed poor precision (Saad et al. Diabetes 43: 1114-1121, 1994). We propose the tracer (hereafter hot) minimal model as a highly reliable method to estimate insulin sensitivity (SI*) and fractional glucose clearance (SG*), reflecting glucose disposal only, in NIDDM subjects. A [6,6- 2H2] glucose-labeled insulin modified intravenous glucose tolerance test was performed in seven NIDDM subjects. In particular, SI* was 1.07 +/- 0.34 x10(-4)min(-1).microU-1.ml estimated with an average precision (mean coefficient of variation of 12%, range 4-22%), whereas the cold minimal model SI was 0.96 +/- 0.26 x 10(-4) min-1. microU-1.ml (mean coefficient of variation of 105%, range 3-353%). Another advantage of the hot indexes with respect to the cold indexes is their ability to reflect glucose and insulin effect on glucose disposal only, and not also on hepatic glucose production. Finally, we also studied by simulation the effect of glucose urinary loss on cold and hot minimal model indexes; only cold glucose effectiveness (SG) was significantly affected, resulting in a mean approximately 40% lower. The hot minimal model appears therefore more reliable than the cold model for assessing glucose tolerance in NIDDM subjects. In particular its ability to dissect disposal from production processes, coupled with the very good precision of the estimated metabolic indexes, supports the clinical use of this method in NIDDM subjects. PMID- 8638703 TI - Human skeletal muscle malonyl-CoA at rest and during prolonged submaximal exercise. AB - Previous literature has indicated that contraction-induced decreases in malonyl CoA are instrumental in the regulation of fatty acid oxidation during prolonged submaximal exercise. This study was designed to measure malonyl-CoA in human vastus lateralis muscle at rest and during submaximal exercise. Eight males and one female cycled for 70 min (10 min at 40% and 60 min at 65% maximal O2 uptake). Needle biopsies were obtained at rest and at 10 min, 20 min, and 70 min of exercise. Malonyl-CoA content in preexercise biopsy samples determined by high performance liquid chromatography (HPLC) was 1.53 +/- 0.18 micromol/kg dry mass (dm). Malonyl-CoA content did not change significantly during exercise (1.39 +/- 0.21 at 10 min, 1.46 +/- 0.14 at 20 min, and 1.22 +/- 0.15 micromol/kg dm at 70 min). In contrast, malonyl-CoA content determined by HPLC in perfused rat red gastrocnemius muscle decreased significantly during 20 min of stimulation at 0.7 Hz [3.44 +/- 0.54 to 1.64 +/- 0.23 nmol/g dm, (n=9)]. We conclude that human skeletal muscle malonyl-CoA content 1) is less than reported in rat skeletal muscle at rest, 2) does not decrease with prolonged submaximal exercise, and 3) is not predictive of increased fatty acid oxidation during exercise. PMID- 8638704 TI - Prostaglandin endoperoxide synthase: why two isoforms? AB - Prostaglandin endoperoxide synthase-1 [prostaglandin G/H synthase-1 (PGHS-1)] and PGHS-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. We refer to these isoforms as cyclooxygenase-1 (COX-1) and COX-2 in this review. This brief review focuses on recent developments in the study of these enzymes. Alterations in the expression levels of COX-2 result in distinct phenotypic changes in intestinal epithelial cells. Overexpression of COX 2 in intestinal epithelial cells results in increased adhesion to extracellular matrix proteins and inhibition of apoptosis. Disruption of the COX-2 gene in mice results in renal dysplasia, cardiac fibrosis, and defects in the ovary. Interestingly, disruption of the COX-1 gene results in distinct phenotypic changes different from those observed for COX-2. COX-1 null mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice. These two closely related enzymes must have distinct functions in the organisms, since lack of their expression causes distinct phenotypic changes for each respective isoform. PMID- 8638705 TI - Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha hydroxylase at the molecular level. AB - The present study examines the feedback control governing human cholesterol 7alpha-hydroxylase mRNA expression in the human hepatoblastoma cell line, Hep G2. Glycochenodeoxycholate (GCDC) and glycodeoxycholate, hydrophobic bile salts, decreased cholesterol 7alpha-hydroxylase mRNA levels and bile acid synthesis in a concentration-dependent (76 +/- 8%, P<0.001, and 48 +/- 3%, P<0.01, respectively) and time-dependent manner. Cholesterol 7alpha-hydroxylase mRNA levels were repressed with a half-maximal inhibitory concentration of <12.5 microM by GCDC and a half-life of 30 min by 100 microM of the bile acid. The addition of actinomycin D (10 microgram/ml) alone or in combination with GCDC (100 microM) led to similar concentration-and time-dependent suppression of cholesterol 7alpha hydroxylase mRNA. Glycocholate (100 microM), not internalized based on lack of uptake of a fluorescent cholate analogue, had no effect on cholesterol 7alpha hydroxylase mRNA or total bile acid synthesis. In cultures transfected with a rat cholesterol 7alpha-hydroxylase promoter construct, reporter gene activity was decreased (31%, P<0.01) by GCDC (100 microM). Hep G2 cells maintain the intracellular machinery to express and rapidly regulate human cholesterol 7alpha hydroxylase by hydrophobic bile acids. These data suggest that Hep G2 cells will support functional studies of the human cholesterol 7alpha-hydroxylase gene. PMID- 8638706 TI - Impaired intracellular signal transduction in gastric smooth muscle of diabetic BB/W rats. AB - The pathophysiological mechanisms responsible for diabetic gastroparesis remain unclear. Diabetes mellitus occurs spontaneously in 90% of a partially inbred colony of BB/W rats. This animal model resembles human insulin-dependent diabetes and is suitable for investigating the mechanism of diabetic gastroparesis. Diabetic BB/W rats were killed 6 mo after the onset of diabetes. Muscle contraction experiments and [3H]acetylcholine release studies were performed with muscle strips of the gastric body. Biochemical measurements of inositol trisphosphate (IP3) and protein kinase C (PKC) in gastric muscle were performed to characterize abnormalities of the intracellular signal transduction system in gastric myocytes. Circular muscle contractions in response to direct myogenic stimulants, carbachol (10(-7) - 10 (-3)M) or substance P (10(-7) - 10(-5)M), were significantly impaired in diabetic BB/W rats compared with controls. Similarly, muscle contractions in response to NaF (10 mM), a direct stimulant of G proteins, were also impaired in diabetic BB/W rats. In contrast, muscle contractions in response to KCl (25-75 mM) were similar between control and diabetic BB/W rats, indicating normal voltage-dependent Ca2+ entry in muscle strips obtained from diabetics BB/W rats. [3H]acetylcholine release from gastric myenteric plexus in response to electrical transmural stimulation remained intact in diabetic BB/W rats. In separate studies, we demonstrated that carbachol (10(-6) - 10(-4)M) induced IP3 responses were significantly reduced in diabetic rats compared with control. In addition, there was also impairment of translocation of PKC in diabetic BB/W rats. These observations indicate that myogenic impairment occurred in diabetic BB/W rats. This resulted from altered intracellular signal transduction involving abnormal IP3 production and PKC translocation. PMID- 8638707 TI - cGMP stimulates bile acid-independent bile formation and biliary bicarbonate excretion. AB - The effect of guanosine 3',5'-cyclic monophosphate (cGMP) on hepatic bile formation was studied in isolated perfused rat livers and rat hepatocytes. Studies in isolated perfused rat livers showed that infusion of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 3 micromol/min or 100 microM) 1) increased bile flow without affecting biliary excretion of simultaneously infused taurocholate, 2) increased biliary concentration and excretion of HCO3(-) but did not affect biliary excretion of glutathione, and 3) increased net perfusate H+ efflux without affecting hepatic O2 uptake. Studies in isolated rat hepatocytes showed that 1) 8-BrcGMP increased intracellular pH in the presence (but not in the absence) of extracellular HCO-3, and effect inhibited by 4,4' diisothiocyanostilbene-2,2'-disulfonic acid and Na+ replacement, 2) 8-BrcGMP did not affect taurocholate uptake and intracellular [Ca2+], and 3) bile acids, like ursodeoxycholate and cholate, did not increase cellular cGMP. Taken together, these results indicate that cGMP stimulates bile acid-independent bile formation, in part by stimulating biliary HCO3- excretion. cGMP may increase HCO3- excretion by stimulating sinusoidal Na+ - HCO3- cotransport, but not Na+/H+ exchange. cGMP, unlike adenosine 3',5'-cyclic monophosphate, may not regulate hepatic taurocholate transport, and bile acid-induced HCO3- rich choleresis may not be mediated via cGMP. PMID- 8638708 TI - Toxic dilatation of colon in a rat model of colitis is linked to an inducible form of nitric oxide synthase. AB - The contribution of nitric oxide (NO) to the altered colonic contractility of acute colitis was investigated in the 2,4,6-trinitroben-zenesulfonic acid model. NO synthase was measured in colonic tissue; the effects of NO synthase inhibition on colonic contractility were studied in vitro and in vivo. Inducible NO synthase was not detected in normal colons, whereas inflamed colons showed high activity. Acute inflammation was associated with enlarged colonic perimeter. NO synthase inhibitors or selective inhibitors of the inducible enzyme prevented colonic dilatation. In vitro, contractile responses to KCl were lower in muscle from colitic than control rats. After NO synthase inhibition, however, no difference was observed between colitic and control muscle contractility. In vivo, intracolonic pressure was lower in colitic than in control rats. Selective inhibition of inducible NO synthase increased intracolonic pressure in colitic but not in control rats. In conclusion, NO generation by inducible enzymes impairs smooth muscle contractility in colitis and may be involved in the pathogenesis of toxic dilatation of the colon. PMID- 8638709 TI - Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1. AB - The effect of hyperosmolarity on cloned Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 was studied in stably transfected PS120 fibroblasts. Na+/H+ exchanger activity was determined spectrofluorometrically in acidified cells that were exposed to isosmolar (300 mosmol/kg) or hyperosmolar (450 mosmol/kg) media, in which the only difference is the presence or absence of 150 mM mannitol. Hyperosmolar solution reversibly inhibited NHE2 and NHE3 with a delay of approximately 15 s. Hyperosmolarity significantly reduced their maximal reaction velocity compared with isosmolar medium but did not alter their Michaelis-Menten constant for intracellular H+. The Michaelis-Menten constant of the exchangers for extracellular Na+ in hyperosmolar medium was not different from that in isosmolar medium. Pretreatment of PS120/NHE3 cells with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the tyrosine kinase inhibitor genistein, and the serine/threonine protein phosphatase inhibitor okadaic acid did not affect the hyperosmolar inhibition of NHE3. Hyperosmolar inhibition of Na+/H+ exchanger activity was also observed in PS120 cells transfected with truncated NHE3 cDNAs (E3/585, E3/543, E3509, and E3/475) and NHE2 cDNA (E2/499). We conclude that 1) hyperosmolarity inhibits NHE2 and NHE3, in contrast to the stimulatory effect on the housekeeping isoform NHE1, 2) this inhibition is reversible, and 3) the COOH termini of NHE2 and NHE3 are not necessary for hyperosmolar inhibition of NHE2 and NHE3. PMID- 8638710 TI - Anaphylactic intestinal response to milk proteins during malnutrition in guinea pigs. AB - We investigated whether sensitization to cow's milk occurs during malnutrition and alters intestinal ion and macromolecular transport. Malnourished guinea pigs received a low-protein diet containing either 4% soy or 4% milk proteins, and well-nourished sensitized controls received 26% soy plus 4% milk proteins. To assess milk sensitization, we measured immunoglobulin (Ig) G and passive cutaneous anaphylactic (PCA) responses to beta-lactoglobulin (beta-Lg) and the intestinal anaphylaxis, reflected by the rise in short-circuit current (delta Isc) induced by beta-Lg in tissues mounted in Ussing chambers. To assess intestinal function, we measured ionic conductance and unidirectional fluxes of 14C-mannitol and -3H-horseradish peroxidase (HRP). In malnourished animals fed milk proteins, IgG, PCA, and delta Isc (beta-Lg) increased more than in well nourished animals. Ionic conductance and mannitol permeability rose in both malnourished groups. Malnourished animals fed milk proteins also displayed enhanced permeability to HRP. These data suggest that increased paracellular permeability is due to malnutrition per se, whereas increased macromolecular transport seems to require both malnutrition and sensitization. They indicate that intestinal anaphylaxis in response to milk proteins is persistent and even enhanced during experimental malnutrition. PMID- 8638711 TI - Electrophysiological effects of cholinergic agonists in surface epithelium of Necturus gastric antrum. AB - Intracellular microelectrode techniques were used to characterize voltage and conductance properties of the basolateral membrane of surface epithelial cells in in vitro Necturus antral mucosa. Flux studies confirmed that this tissue secretes HCO3- under resting conditions and during response to cholinergic stimulation. In studies using intracellular microelectrodes, exposure to cholinergic agonists such as acetylcholine, bethanechol, or carbachol elicited an initial hyperpolarization followed by depolarization of the basolateral cell membranes associated with up to fourfold increases in basolateral membrane conductance. Effects of acetylcholine were dose dependent (10(-6) - 10(-4) M) and prevented by pretreatment of tissues with the nonselective muscarinic receptor blocker atropine. Some variation in this response to cholinergic stimulation was observed and appeared to be related to the season (fall/winter/early spring vs. late spring/summer). Despite such variability, circuit analysis and ion substitution studies indicated that the carbachol-induced increases in basolateral conductance were due to increases in conductance to K+ and Cl- . These increases in basolateral transport processes may serve to stabilize cell ion composition and membrane electrical properties during cholinergic stimulation of mucus and HCO3- secretions. PMID- 8638712 TI - Gastrin stimulation of histamine synthesis in enterochromaffin-like cells from rabbit fundic mucosa. AB - This work aimed to investigate the molecular role of gastrin in histamine synthesis in isolated rabbit fundic mucosal cells enriched in enterochromaffin like (ECL) cells (37%). Gastrin stimulated histidine decarboxylase (HDC) activity by increasing the maximal velocity (Vmax) from 0.240 +/- 0.017 (basal value) to 0.332 +/- 0.012 pmol/mg protein/h and by decreasing the Michaelis-Menten constant value -Km; 73.90 +/- 2.2 vs. 93.42 +/- 4.32 microM (basal value)]. Pertussis toxin (PTX) (200 ng/ml) reduced the stimulation of HDC induced by 10 nM gastrin from 41.8 to 15.9%, whereas cholera toxin (CTX) (100 ng/ml) was without effect. Staurosporine and polymyxin B inhibited in a dose dependent manner the HDC activity stimulated by 10 nM gastrin. Phorbol 12-myristate 13-acetate (PMA; 100 nM) decreased Vmax (0.558 +/- 0.021 pmol/ mg protein/h) but did not change the Km. Furthermore, cycloheximide (0.1-10 microM) inhibited the gastrin-induced stimulation of HDC activity, whereas actinomycin D (up to 10 microM) was without effect. Finally, incubation of cells with gastrin (10 microM) left the expression of HDC mRNA unchanged. We concluded that gastrin, acting through "gastrin/CCK-B type" receptors coupled to PTX-sensitive G protein, exerts a short-term regulation of histamine synthesis in gastric ECL cells by increasing both the affinity of HDC for L-histidine and the number of active enzyme molecules. This last event, related to protein kinase C activation, could be due to a translational or posttranslational mechanism. PMID- 8638713 TI - Frequency of gastric pacesetter potential depends on volume and site of distension. AB - Little is known about the response of the frequency of gastric pacesetter potential (PP) to luminal distension. When volume distension occurs as a result of a meal, gastric emptying may play an important role, since the site of distension shifts as the meal is displaced from the stomach to the small bowel. In this study, using does equipped with duodenal fistulas and serosal electrodes on the antrum, we compared the frequency of gastric PP during the course of gastric emptying while isolating the volume distension to either the stomach or the small bowel. We found that 1) the frequency of gastric PP decreased linearly with greater initial meal volume when volume distension was isolated to either the stomach -P<0.05, analysis of variance (ANOVA)- or small bowel (P<0.01, ANOVA), and 2) the frequency of gastric PP decreased linearly with increased volume remaining in the stomach or increased volume entering the small intestine. We conclude that the frequency of gastric PP depends on the volume and site of distension. PMID- 8638714 TI - Comparison between activation of ornithine decarboxylase and histidine decarboxylase in rat stomach. AB - We compared the responses of rat stomach ornithine decarboxylase (ODC) and histidine decarboxylase (HDC) to food intake, oral treatment with antisecretagogues, NaHCO3, and hypertonic NaCl, antrectomy, intravenous infusion of gastrin-17, the selective cholecystokinin (CCK)-B/gastrin receptor antagonist L-365,260, and the somatostatin analogue RC-160. The serum gastrin concentration and oxyntic mucosal ODC and HDC activities were higher in freely fed rats than in fasted rats. Food intake in fasted rats raised the serum gastrin concentration and the ODC and HDC activities. Ranitidine, omeprazole, and NaHCO3 raised the serum gastrin concentration and activated ODC and HDC. Hypertonic NaCl raised the ODC activity 200-fold, whereas circulating gastrin and HDC activity were increased only moderately. Infusion of gastrin-17 activated HDC but not ODC. L 365,260 prevented the activation of HDC but not of ODC in response to food intake and treatment with omeprazole, NaHCO3, or hypertonic NaCl. Antrectomy prevented the food- and omeprazole-evoked rise in oxyntic mucosal HDC activity but not the rise in ODC activity. RC-160 suppressed HDC activity after food intake and treatment with omeprazole, NaHCO3, or NaCl. In contrast, RC-160 suppressed omeprazole- and NaHCO3-evoked ODC activation but not that evoked by food intake or NaCl. The results support the view that HDC in the oxyntic mucosa is activated by gastrin and suppressed by somatostatin. The induction of ODC is not mediated by gastrin; ODC activation appears to be related to acid inhibition per se or to mucosal maintenance and repair; somatostatin, or rather the lack of it, might contribute to the induction of ODC after acid blockade. The mechanism behind the activation of rat stomach ODC seems to differ depending on the type of stimulus. PMID- 8638715 TI - Measurements of the jejunal unstirred layer in normal subjects and patients with celiac disease. AB - Normal intestinal absorption of nutrients requires efficient luminal mixing to deliver solute to the brush border. Lacking such mixing, the buildup of thick unstirred layers over the mucosa markedly retards absorption of rapidly transported compounds. Using a technique based on the kinetics of maltose hydrolysis, we measured the unstirred layer thickness of the jejunum of normal subjects and patients with celiac disease, as well as that of the normal rat. The jejunum of humans and rats was perfused with varying maltose concentrations, and the apparent Michaelis constant (Km) and maximal velocity (Vmax) of maltose hydrolysis were determined from double-reciprocal plots. The true Km of intestinal maltase was determined on mucosal biopsies. Unstirred layer thickness was calculated from the in vivo Vmax and apparent Km and the in vitro Km of maltase. The average unstirred layer thickness of 11 celiac patients (170 micron) was seven times greater than that of 3 controls (25 micron). The unstirred layer of each celiac exceeded that of the controls. A variety of factors could account for the less efficient luminal stirring observed in celiacs. Although speculative, villous contractility could be an important stirring mechanism that would be absent in celiacs with villous atrophy. This speculation was supported by the finding of a relatively thick unstirred layer (mean: 106 micron) in rats, an animal that lacks villous contractility. Because any increase in unstirred layer slows transport of rapidly absorbed compounds, poor stirring appears to represent a previously unrecognized defect that could contribute to malabsorption in celiac disease and, perhaps, in other intestinal disorders. PMID- 8638717 TI - Rat intestinal and hepatic ferritin subunit expression during development and after dietary iron feeding. AB - Ferritin consists of 24 heavy (H) and light (L) subunits in varying proportions in different tissues and plays a significant role in iron metabolism. We studied rat ferritin subunit expression in the duodenum and liver during early life, when a cycle of iron depletion and repletion occurs. In both tissues, ferritin contents decreased to low levels from day 3 to day 12. The ferritin on day 3 had an H/L mRNA ration of 0.9 and an H/L subunit ratio of 0.6. The decrease of tissue ferritin levels, but not mRNA, on day 12 suggests translational repression consistent with iron depletion. In the duodenum, a twofold increase in both H and mRNA and subunit protein occurred on day 18. The subsequent increase of H mRNA was accompanied by a 50% decrease in L mRNA, resulting in the increase of H/L mRNA and subunit ratios to 7.9 and 9, respectively, by day 32. In contrast, liver H/L mRNA and subunit ratios were similar throughout development. The possibility that dietary iron regulates duodenal ferritin subunit expression was investigated. When day 12 rats were fed 6 ml of a milk formula containing 56 microgram/ml iron for 18 h, dietary iron increased the duodenal levels of L mRNA but not H mRNA. In contrast, hepatic H and L mRNA levels did not change. Dietary iron promoted greater increases in ferritin protein than mRNA in both tissues. Thus a shift from L-rich to H-rich ferritin isoforms occurs in the duodenum but not in the liver during neonatal development. This change is regulated at the pretranslational level and is independent of dietary iron. PMID- 8638716 TI - Evidence for VIP-induced increase in NO production in myenteric neurons of opossum internal anal sphincter. AB - A significant interaction between vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) has been reported in neurotransmission of the gastrointestinal tract, including the internal anal sphincter (IAS). The exact site of this NO release from the IAS in response to VIP is not known. Studies were carried out to determine the site of this VIP-induced NO release in opossum IAS. NO synthase (NOS) activity was quantitated by determining L--3H-citrulline production from L[3H]arginine in isolated myenteric ganglia and smooth muscle cells of the IAS. L [3H]citrulline production was determined before and after treatment with either the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), VIP, or peptide histidine isoleucine (PHI) in the absence and presence of the neurotoxin tetrodotoxin and the NOS inhibitor NG-nitro-L-arginine (L-NNA). Smooth muscle cells and ganglia were preloaded with L-[3H]arginine for 5 min and treated with VIP for 1 and 5 min. DMPP and VIP caused a significant increase in L [3H]citrulline formation in myenteric ganglia at both time periods, whereas in smooth muscle cells there was a moderate but significant increase in L [3H]citrulline production only at 5 min of VIP treatment. VIP-induced relaxation of isolated smooth muscle cells of the IAS was not affected by L-NNA. The increase in NOS activity of myenteric ganglia by DMPP and VIP was sensitive to neurotoxin and the NOS inhibitor. The data suggest that the increase in NO production in response to VIP in the IAS occurs mainly from the myenteric neurons, with some contribution from the smooth muscle cells. PMID- 8638718 TI - Progesterone and estrogen are potential mediators of gastric slow-wave dysrhythmias in nausea of pregnancy. AB - Women in pregnancy experience nausea, which correlates with gastric slow-wave rhythm disruption. Mediators of these dysrhythmias were explored. To quantitate slow-wave disruption, eight pregnant women with first-trimester nausea underwent electrogastrography after a 250-kcal meal. Results were compared with nonpregnant women with nausea during a prior pregnancy who received estradiol and/or progesterone to levels of the first trimester of pregnancy. Five pregnant women exhibited dysrhythmias, with increases in combined recording time in tachygastria plus bradygastria, as well as decreases in the percentage of electrogastrography signal power in the normal 3 cycle/min range (cpm), compared with nonpregnant women (P<0.05). Estradiol did not evoke dysrhythmias in nonpregnant women; however, progesterone induced increases in recording time in bradygastria plus tachygastria and increases in bradygastric signal power with corresponding decreases in signal power in the 3-cpm range (P<0.05). With estradiol and progesterone coadministration, an additive effect was observed at 3.3 +/- 0.8 h, with increased recording time in bradygastria alone and in bradygastria plus tachygastria with corresponding increases in bradygastric signal power and decreases in power in the 3-cpm range (P<0.05). In conclusion, women with nausea of pregnancy exhibit slow-wave rhythm disruption. Similar dysrhythmias are evoked in nonpregnant women by progesterone alone or in combination with estradiol in doses that reproduce levels in pregnancy. Thus gastric dysrhythmias in pregnancy may be due to a combination of elevated progesterone and estrogen levels. PMID- 8638719 TI - Rab11 is an apically located small GTP-binding protein in epithelial tissues. AB - Rab proteins are involved in many aspects of dynamic vesicle processing within eukaryotic cells. We have previously identified Rab11 in gastric parietal cell tubulovesicle membranes. We have produced a monoclonal antibody that is specific for Rab11. In all rabbit tissues examined, Rab11 immunoreactivity was highly enriched in epithelial cells. In the gastric fundus, parietal cells were stained in a pattern consistent with localization on tubulovesicles. Surface mucous cells of both the fundus and antrum demonstrated punctate subapical staining. Ileal and proximal colonic enterocyte labeling was observed deep to the brush borders. In the distal colon, staining was observed in the apical regions of mid-crypt cells. In skin and esophagus, punctate immunoreactivity was present in the medial layers of the squamous epithelia. Prominent Rab11 immunoreactivity was present in hepatocytes deep to the bile canaliculi. Punctate subapical staining was observed in pancreatic acinar cells. Apical staining was also observed in collecting duct cells and in the glandular cells of the prostate. These results indicate the Rab11 is expressed in apical vesicular populations in discrete epithelial cell populations. PMID- 8638720 TI - Facilitating effect of CCK on nicotinic neurotransmission in cat pancreatic ganglion. AB - Previous studies have demonstrated the presence of cholecystokinin (CCK)-like peptides in nerve terminals surrounding ganglion neurons of the cat pancreas. The present study was undertaken to determine the effect of cholecystokinin octapeptide (CCK-8) on ganglionic transmission. Recordings were made intracellularly in vitro from ganglion neurons in isolated pieces of the pancreas. Sulfated CCK-8 (S-CCK-8) and nonsulfated CCK-8 initiated or increased ongoing fast excitatory postsynaptic potential (fEPSP) activity, an effect antagonized by hexamethonium. Superfusion of S-CCK-8 in concentrations ranging from 10(-11) to 10(-8) M significantly augmented the amplitude of nerve-evoked subthreshold fEPSPs without a significant change in either membrane potential or membrane input resistance. S-CCK-8 (10(-8)M) also increased the quantal content and quantal size of nerve-evoked fEPSPs and increased the response to exogenously applied acetylcholine (ACh). Concentrations of S-CCK-8 higher than 10(-8)M caused depolarization and an increase in membrane input resistance, an effect unaltered by a low-Ca+, high-Mg2+ solution. It was concluded that S-CCK-8 potentiated nicotinic transmission by facilitating release of ACh from preganglionic nerve terminals and by increasing the postsynaptic membrane sensitivity to ACh. PMID- 8638721 TI - Pyruvate reduces anoxic injury and free radical formation in perfused rat hepatocytes. AB - The effects of 5 mM pyruvate on anoxic injury, superoxide (O2-.) and hydrogen peroxide (H2O2) generation, and lactate dehydrogenase (LDH) release during reoxygenation after 2.5 h anoxia were studied in perfused rat hepatocytes. When pyruvate was present during anoxia and reoxygenation, there was little anoxic injury, and the generation of free radicals and LDH release during reoxygenation were reduced 50-60%. When Pyruvate was added during reoxygenation, there was no decrease in O2-. or LDH release, although H2O2 formation was depressed. Free radical formation and anoxic/reperfusion injury were significantly reduced when pyruvate was added during the anoxic period only. Pyruvate reduced the deleterious effects of 10 microM antimycin A by preventing the increase in O2-. formation and LDH release evoked by the inhibitor. These results indicate that pyruvate protected hepatocytes against anoxic injury and that its protective action occurred principally during anoxia and not during reoxygenation. Pyruvate appeared to act at a mitochondrial site, since it reduced the deleterious effects of antimycin A. PMID- 8638722 TI - Lung alveolar epithelial cell migration in vitro: modulators and regulation processes. AB - After acute lung injury, altered bronchioloalveolar epithelia must be repaired quickly in order to restore lung function. During reepithelialization, type II cells initially appear to migrate and spread over a remodeled matrix; then a secondary proliferative phase occurs. It was hypothesized that 1) type II cells can develop locomotion in vitro that is modulated by growth factors, proinflammatory cytokines, and substrate adhesion molecules and 2) migration and proliferation of type II cells can occur as distinctive processes. Chemotaxis assays were elaborated using short term cultures of rat type II pneumocytes. Epidermal growth factor (EGF), transforming growth factor-alpha, laminin, fibronectin were found to be the main attractants for type II cells with respective increases of approximately 8.5-, 10.5-, 8-, and 7-fold in cell migration (P<0.05 vs. control). Laminin induced gradient-dependent and random cell migration. Addition of laminin with EGF had a synergistic effect in promoting cell migration (approximately 30-fold increase over control, P<0.05). Interferon-gamma and interleukin-6 inhibited EGF-induced type II cell migration, whereas tumor necrosis factor-alpha and interleukin-1beta acted as primers for type II cell migration (approximately 1.5-fold increase over control, P<0.05. Type II cells did not need to be in a proliferative phase in order to exhibit motility. New insights regarding the regulatory processes for type II cell migration are especially relevant in our understanding of early events occurring during epithelial repair after acute lung injury. PMID- 8638723 TI - Expression and distribution of surfactant proteins and lysozyme after prolonged hyperpnea. AB - We have induced prolonged hyperpnea in rats and examined the distribution of surfactant-associated proteins (SP-A and SP-B) and lysozyme in lamellar bodies (lb) and two alveolar fractions, one tubular myelin rich (alv-1) and the other tubular myelin poor (alv-2). We have also examined the expression of SP-A, SP-B, SP-C, and lysozyme mRNA in lung tissue and alveolar type II cells. Hyperpnea resulted in significant increases in lb SP-A, lysozyme, and phospholipid (PL) but no change in the protein-to-PH ratios, suggesting that lb stoichiometry is constant. The SP-A and SP-B-to-PL ratios were 33 and 18 times greater, respectively, in control alv-1 than in lb, suggesting that alv-1 is enriched with these proteins. In contrast, the lysozyme-to-PL ratio was similar in control alv 1 and lb. Hyperpnea did not alter the alv-1 SP-A or SP-B-to-PL ratios, suggesting some constant stoichiometry to their lipid association; however, the lysozyme-to PL ratio was reduced. Whereas hyperpnea significantly elevated the PL, SP-A, and lysozyme levels in alv-2, the SP-B level was unchanged. We suggest that surfactant-associated lysozyme is secreted with lb, the majority of SP-A is linked to lipid secretion but not necessarily with lb, and the majority of SP-B secretion is independent of PL secretion. Hyperpnea did not alter the mRNA expression of SP-A, SP-B, SP-C, or lysozyme in alveolar type II cells, but expression of SP-A and SP-B mRNA was significantly increased in lung tissue. PMID- 8638724 TI - Stimulation of surfactant lipid secretion from fetal type II pneumocytes by gastrin-releasing peptide. AB - Gastrin-releasing peptide (GRP) and bombesin apparently enhance the rate of secretion of surfactant lipids from cultured fetal rat type II pneumocytes. This effect, evident within 1h of addition of the peptide, is concentration-dependent, with a maximal response at 3.0 nM. When the effect of GRP was assessed in comparison with other known secretagogues, it was found that, whereas GRP and isoproterenol were additive in their effect, there was no response to GRP in the presence of saturating concentrations of A23187 or phorbol 12-myristate 13 acetate. This suggests that the secretory response to GRP is via activation of Ca2+/calmodulin-dependent protein kinase and/or protein kinase C and is independent of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. This conclusion is supported by the observation that the GRP-induced secretion is inhibited by calphostin C, an inhibitor of protein kinase C, but not by H-89, an inhibitor of cAMP-dependent protein kinase. The fact that GRP regulates surfactant secretion from type II pneumocytes suggests that it and/or related peptides may play a significant role in the physiological maturation of the lung. PMID- 8638725 TI - Sodium nitroprusside stimulates Ca2+ -activated K+ channels in porcine tracheal smooth muscle cells. AB - To directly investigate the possible role of large-conductance Ca2+ -activated K+ (KCa) channels in nitro-vasodilator-induced relaxation of airway smooth muscle, we used cell-attached patch-clamp techniques to test the effects of sodium nitroprusside (SNP) on KCa channels in freshly dispersed porcine tracheal smooth muscle cells. Channel open-state probability (nPo) increased approximately 13 fold with exposure to 10(-5) M SNP, and this was partially reversed by addition of the guanylate cyclase inhibitors methylene blue (3 X 10(-4) M) or LY-83583 (5 X 10(-5) M). Pretreatment with the guanosine 3',5' -cyclic monophosphate (cGMP) dependent protein kinase (G kinase) inhibitor Rp-8-(p-chlorophenylthio) cGMP phosphorothioate (2 X 10(-5) M) prevented activation of KCa channels by SNP. We also tested the ability of G kinase to directly activate KCa channels in inside out patches. G kinase (2.5 U/microliter) with ATP (0.5 mM) and cGMP (0.1 mM), but not ATP and cGMP alone, increased nPo approximately 23-fold. We conclude that SNP activates KCa channels in airway smooth muscle via guanylate cyclase and G kinase. Phosphorylation of the channel protein by G kinase may account for this response. Consequent membrane hyperpolarization and inhibition of Ca2+ entry through voltage-dependent channels may contribute to SNP-induced relaxation of airway smooth muscle. PMID- 8638727 TI - Parathyroid hormone-related protein, an autocrine regulatory factor in alveolar epithelial cells. AB - Alveolar epithelial cells in vivo, primary cultures of adult rat type II cells, and human A549 alveolar carcinoma cells express parathyroid hormone-related protein (PTHrP). Here we demonstrated that type II cells and A549 cells also express the PTHrP receptor and that they exhibit differentiation-related responses to the amino-terminal PTHrP fragment, PTHrP-(1-34). PTHrP receptor expression in A549 cells was shown by detection of a 0.3-kb reverse transcriptase polymerase chain reaction product formed by primers specific for PTHrP receptor. In situ hybridization studies localized the site of production of PTHrP and PTHrP receptor mRNA in rat lung cells with morphology and location typical of type II cells. Primary cultures of such type II cells also expressed PTHrP receptor mRNA. Incubation with PTHrP-(1-34) stimulated disaturated phosphatidylcholine (DSPC) synthesis in A549 cells and increased the release of newly synthesized DSPC by cultured type II cells and A549 cells. In addition, PTHrP-(1-34) increased the number of lamellar bodies per type II cell and increased their expression of alkaline phosphatase in a dose-dependent manner. Thus PTHrP-(1-34) promoted a differentiated type II cell phenotype. Since cultured type II cells, alveolar epithelial cells in vivo, and A549 cells express PTHrP and the PTHrP receptor, PTHrP-(1-34) may be an autocrine regulatory factor in type II cells and lung cancer cells. PMID- 8638728 TI - Nitroprusside inhibits serotonin-induced mitogenesis and tyrosine phosphorylation of smooth muscle cells. AB - Serotonin (5-hydroxytryptamine, 5-HT) produces hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells (SMC) in culture. The growth responses are associated with early elevations of c-myc and actin gene expressions and are blocked by agents that elevate cellular adenosine 3',5' cyclic monophosphate or inhibit 5-HT transport or tyrosine phosphorylation. A rapid enhancement of tyrosine phosphorylation of a 120-kDa protein (p120) is associated with the 5-HT-induced mitogenesis. In the present studies, sodium nitroprusside (SNP, 10-100 micromol/l), a NO-generating agent, dose dependently inhibited 5-HT-induced thymidine incorporation by SMC. Inhibition of the 5-HT stimulatory effect was also observed with isosorbide dinitrate and nitroglutathione, which are also NO donors. Incubation of cells with 8 bromoguanosine 3',5'-cyclic monophosphate (1 micromol/l) mimicked the antimitogenic action of SNP. The antiproliferative effect of SNP was inhibited by hemoglobin (50 micromol/l) and potentiated by superoxide dismutase (200 U/ml), supporting the role of NO in the process. Enhancement of tyrosine phosphorylation of p120 by 5-HT was prevented by preincubation with SNP or exogenously added guanosine 3',5'-cyclic monophosphate. The data indicate that 5-HT acts as a mitogen for SMC through a signal transduction pathway involving tyrosine phosphorylation. SNP likely prevents the 5-HT-induced mitogenesis of SMC through elevation of intracellular guanosine 3',5'-cyclic monophosphate and inhibition of tyrosine phosphorylation of p120. PMID- 8638726 TI - Interleukin 6-receptor expression on human bronchial epithelial cells: regulation by IL-1 and IL-6. AB - Airway epithelial cells have a potential to participate in regulation of local homeostasis by releasing active compounds including cytokines and growth factors. Several factors such as transforming growth factor-beta and endothelin have been shown to regulate airway epithelial cell functions through an autocrine mechanism. We studied the expression of the specific receptor for a multifunctional cytokine interleukin 6 (IL-6), which is expressed and released by airway epithelial cells. Specific binding assay demonstrated a single set of binding sites on human primary and transformed bronchial epithelial cells. Human interleukin-1alpha (IL-1alpha) increased maximal binding sites to IL-6. Northern blot analysis demonstrated that airway epithelial cells constitutively expressed mRNA for IL-6 receptor (IL-6R), and IL-1alpha and IL-6 itself upregulated IL-6R gene expression. Moreover, exogenously added human recombinant IL-6 had a stimulatory effect on IL-8 release from human bronchial epithelial cells. These results indicated that human bronchial epithelial cells expressed IL-6R, and IL-6 might be involved in the regulation of the epithelial functions via an autocrine as well as a paracrine mechanism. PMID- 8638729 TI - Effects of interleukin 5-induced pulmonary eosinophilia on airway reactivity in the guinea pig. AB - Administration of interleukin 5 (IL-5) to guinea pigs by tracheal injection was associated with increased recovery of eosinophils and neutrophils from bronchoalveolar lavage (BAL) fluid. The number of eosinophils recovered from BAL fluid increased in a dose-dependent manner from 9 +/- 2 X 10(3)/ml to a plateau of 143 +/- 29 X 10(3)/ml after the administration of recombinant human IL-5 (rhIL 5). Tracheal administration of recombinant guinea pig IL-5 (gpIL-5) also increased eosinophil recovery but was less potent than rhIL-5. Histological analysis confirmed the presence of inflammatory cells in the lung; there were higher grades of inflammation in airway than in parenchymal tissue after gpIL-5 administration. In addition, the histological grade of airway inflammation was greater 24 and 72 h after gpIL-5 administration than it was 6 days after administration. Airway hyperresponsiveness is reported to occur in guinea pigs exposed to rhIL-5 by intraperitoneal cellular production. It is surprising that airway infiltration with eosinophils induced by the topical application of IL-5 was not associated with hyperresponsiveness to substance P, histamine, or platelet-activating factor in intact animals or to methacholine in tracheally perfused lungs. Furthermore, the microvascular leakage induced by substance P was not altered by rhIL-5 administration. These findings indicate that the presence of eosinophils alone is not sufficient for the expression of airway hyperresponsiveness. Our ability to separate eosinophil recruitment and retention in the tissues from airway hyperresponsiveness indicates that these two processes are distinct and that the presence of eosinophils in lung tissue, by itself, is not sufficient to alter airway contractile responses. PMID- 8638730 TI - Serine proteinase inhibitors influence the stability of tropoelastin mRNA in neonatal rat lung fibroblast cultures. AB - Elastin, an elastic extracellular structural protein, is a polymer comprised of soluble tropoelastin (TE) monomers that are joined by covalent cross-links and become insoluble. In cultured vascular smooth muscle cells, the steady-state level of TE mRNA is influenced by soluble elastin moieties in the culture medium, either TE or its fragmentation products. We have hypothesized that an enzyme mediated proteolytic event may modulate the quantities of TE and its fragmentation products in the culture medium of mesenchymal cells, and thereby indirectly regulate the steady-state level of TE mRNA. Neonatal rat lung fibroblasts were cultured in the presence or absence of the serine proteinase inhibitor, aprotinin, and the quantities of soluble elastin and TE mRNA were analyzed. Exposures to aprotinin lasting up to 12 h increased the soluble elastin content of the culture medium. The increase in the soluble elastin content did not reflect an increase in TE mRNA, which diminished after exposures for 12 h or longer. The decrease in TE mRNA resulted from a decrease in its half-life, rather than a decrease in the rate of TE gene transcription. Aprotinin did not reduce TE mRNA in plasminogen-depleted cultures, but the effect of aprotinin was evident when purified plasminogen was added back to the cultures. Therefore, a serine proteinase, possibly plasmin, may participate in a feedback mechanism and modulate the quantity of TE in lung fibroblast cultures. This mechanism may help ensure that intracellular TE synthesis occurs in tandem with extracellular elastin deposition and cross-linking. PMID- 8638731 TI - Metalloporphyrin chloride ionophores: induction of increased anion permeability in lung epithelial cells. AB - 5,10,15,20-Tetraphenyl-21H,23H-porphine manganese (III) chloride [TPPMn(III)] is a positively charged lipophilic anion carrier that is widely used as a Cl- sensor. TPPMn(III) increased anion permeability of cultured mouse lung epithelial (MLE) cells as measured by short-circuit current (ISC) to a level similar to that induced by forskolin analogues. Anion permeability was also studied in cultured human lung epithelial (A549) cells by measurement of the rates of change of fluorescence of the anion sensitive fluorescent dye, 6-methoxy-N-(3 sulfopropyl)quinolinium (SPQ). In these studies, cells were incubated with SPQ in SO2-4- medium, washed free of extracellular SPQ, and then perfused with medium containing anions that are known to quench SPQ fluorescence. The effect of TPPMn(III) on anion transport was then determined either microscopically in single cell studies or using cell monolayers mounted in a front face fluorimeter. TPPMn(III) in the range from 1 to 100 micrograms/ml induced a dose-dependent increase in Br- transport. The half-maximal quenching effect was estimated to be approximate 5 micrograms/ml. TPPMn(III) increased the rates of fluorescence quench of anions by up to fourfold. TPPMn(III) was without effect on -Ca2+-i level in A549 cells as measured with fura 2-AM. This indicates that TPPMn(III) effects were not mediated through effects on Ca+2 -activated Cl- channels, or by compromise of energy metabolism or membrane integrity of the cells. This study suggests that TPPMn(III) and, by extension, other lipophilic Mn(III) or Co(III) derivatives wherein the selectivity of lipophilicity is altered, could increase the anion permeability of biological membranes, and suggests a new approach for treatment of diseases such as cystic fibrosis, where transport of Cl- is defective. PMID- 8638732 TI - Sensory denervation by neonatal capsaicin treatment exacerbates Mycoplasma pulmonis infection in rat airways. AB - Mycoplasma pulmonis infection in rats results in life-long disease, characterized by chronic inflammation of the airway mucosa with widespread accumulation of lymphoid tissue, mucous cell hyperplasia, and mucosal thickening. In addition, there is angiogenesis and increased sensitivity of mucosal blood vessels to substance P (SP), so tachykinins released from sensory nerve fibers cause an abnormally large amount of plasma leakage. We sought to learn whether the sensory nerves influence the severity of the chronic inflammatory response of M. pulmonis infection. Our strategy was to destroy the nerves by capsaicin pretreatment at birth, infect the rats with M. pulmonis at 8 wk of age, and then study the animals 6 wk later. We found that capsaicin pretreatment increased the severity of the infection, exaggerated the pathological changes in the tracheal mucosa, and increased the amount of SP-induced plasma leakage, as quantified with Monastral blue. The thickness of the tracheal mucosa in these infected rats was 80% greater than in their vehicle-pretreated counterparts and 200% greater than in the pathogen-free controls. The area density of Monastral blue-labeled blood vessels averaged 20% in the infected rats pretreated with capsaicin, which represented a 40-fold increase over the leakage in the pathogen-free group. By comparison, the amount of Monastral blue labeling was only 13% in rats pretreated with vehicle (P<0.05), which was a 22-fold increase over the corresponding pathogen-free group. The number of SP-immunoreactive nerve fibers was reduced both by neonatal capsaicin and by infection (87 and 63% reductions, respectively); but when the two conditions were combined, their effects were not additive (79% reduction), perhaps because of nerve regrowth. We conclude that destruction of sensory nerves increases the severity of infection- induced chronic inflammation in the airway mucosa, with exaggerated mucosal thickening, angiogenesis, plasma leakage, and nerve remodeling. PMID- 8638733 TI - Substance P (NK1) receptor immunoreactivity on endothelial cells of the rat tracheal mucosa. AB - Substance P released from sensory nerve fibers causes plasma leakage through an action on neurokinin-1 (NK1 or substance P) receptors. However, it is unknown whether the leakage results from a direct action of substance P on endothelial cells. We determined the distribution of NK1 receptors at sites of plasma leakage in the rat tracheal mucosa, using NK1 receptor-immunoreactive endosomes as markers of substance P-induced receptor internalization. We found that immunoreactive endosomes were located in the endothelial cells of venules and capillaries but not in those of arterioles. Five minutes after vagal stimulation for 1 min, the number of immunoreactive endosomes in endothelial cells was increased 5-fold in postcapillary venules (mean of 17.4 endosomes/100 micron2 compared with a baseline value of 3.4), 15-fold in collecting venules (12.1 compared with 0.8), and 4-fold in capillaries (2.5 compared with 0.7). No endosomes were found in arterioles under either condition. The number of immunoreactive endosomes in individual vessels corresponded to the amount of stimulus-induced plasma leakage. Both the receptor internalization and the plasma leakage were blocked by the selective NK1 receptor antagonist SR-140333 (100 microgram/kg iv). Although both substance P (5 microgram/kg iv) and platelet activating factor (5 microgram/kg iv) caused plasma leakage, only substance P induced receptor internalization. We conclude that substance P, released from sensory nerve fibers, causes plasma leakage through a direct action on endothelial cells of venules, and that this action is followed by the internalization of NK1 receptors into endosomes. PMID- 8638734 TI - PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells. AB - The effect of recombinant platelet-derived growth factor (PDGF) isoforms (PDGF AA, -BB and -AB) on mitogenesis of human cultured airway smooth muscle (ASM) was examined using the MTT-reduction assay and [3H]thymidine incorporation. Results were correlated with expression of PDGF receptor (PDGFR)-alpha and -beta subunits in the absence and presence of fetal calf serum (FCS). When FCS was absent PDGF AB and -BB were potent mitogens, whereas PDGF-AA was weakly mitogenic, evoking <20% of the maximum response induced by the B-chain isoforms. When FCS (2.5%) was present, all PDGF isoforms stimulated marked ASM proliferation with similar efficacy and potency. Cross-competition binding analysis in FCS-deprived cells revealed that ASM cells in culture express mainly PDGFR-beta. Preincubation with PDGF-AA or PDGFR-alpha neutralizing antiserum abolished PDGF-AA binding and decreased total receptor number by approximately 15%. The ratio of PDGFR-alpha to beta subunits was approximately 1:8, supported by intense immunofluorescence staining for PDGFR-beta and weak staining for PDGFR-alpha. In parallel studies, uptake of [3H]thymidine stimulated by PDGF-AA, but not PDGF-AB or -BB, was inhibited by PDGFR-alpha immobilization. Western immunoblot analysis confirmed expression of mature PDGFR-alpha and -beta subunits in ASM cells. FCS did not cause any detectable increase in PDGFR-alpha expression or in PDGF-AA binding. These data support a role for PDGFR-beta mediating ASM mitogenesis during FCS free conditions, but in the presence of FCS, both PDGFR-alpha and -beta subunits are linked to mitogenesis. The enhanced mitogenicity of PDGF-AA in the presence of FCS was independent of any detectable upregulation of PDGFR-alpha, suggesting that the inability of PDGF-AA to promote mitogenesis in the absence of FCS is not simply due to relative numbers of PDGFR-alpha and PDGFR-beta. PMID- 8638735 TI - In situ activation of mouse alveolar macrophages by aerosolized liposomal IFN gamma and muramyl tripeptide. AB - Interferon-gamma (IFN-gamma) was entrapped with an efficiency of 30-40% in muramyl tripeptide-containing liposomes by a freeze-thawing procedure. A microcytotoxicity assay was developed to measure the tumoricidal activity of mouse alveolar macrophages (AM) against tumoral target cells with a colorimetric viability test. Free IFN-gamma and liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) were found to be only slightly effective to activate in vitro AM, whereas encapsulation of both INF-gamma and MTP-PE within the same liposomes produced higher activation of AM. Aerosolized IFN-gamma and liposomal immunomodulators enhanced antitumor properties of AM recovered in mice 24 h postinhalation. Whereas free IFN-gamma also induced a substantial activation of peritoneal macrophages, liposomal encapsulation significantly reduced the systemic activity of inhaled immunomodulators. This approach provides a useful model for the compartmentalized organ-specific activation of AM in mice. PMID- 8638737 TI - Glutathione supplements protect preterm rabbits from oxidative lung injury. AB - The main objective of this study was to determine if glutathione (GSH) supplementation attenuated hyperoxic lung injury. Preterm (29 days) rabbits were delivered and exposed for 24 h to 1) room air, 2) room air and GSH, 3) 95% oxygen and GSH. GSH supplements (1 mM) were delivered in the nutritional support of 10% dextrose and saline through a peritoneal catheter. Animals assigned to oxygen had decreased lung volumes at 35 cmH2O, decreased lung compliance, increased edema, decreased cell viability, and decreased lung tissue and lavage-reduced/oxidized GSH levels, compared with control animals. Despite exposure to hyperoxia, animals supplemented with GSH were not different from room air controls with respect to lung mechanics, edema, cell viability, or tissue and lavage GSH. These studies suggest that GSH supplementation maintains normal lavage and lung tissue GSH levels in preterm animals exposed to hyperoxia and attenuates the changes in lung mechanics associated with oxygen-induced lung injury. PMID- 8638736 TI - Effect of cold preservation on pulmonary arterial smooth muscle cells. AB - The efficacy of preservation fluids on the cytoskeleton and contractile function of porcine pulmonary arterial smooth muscle (SM) cells during cooling and rewarming was evaluated, using EuroCollins solution (EC), University of Wisconsin solution (UW), Marshall's solution (MS), and tissue culture growth medium (GM). Functional studies included passive distensibility and contraction to prostaglandin F2a (PGF2a) in arterial rings and wrinkling of silicone membranes by cooled-rewarmed cultured SM cells. Immunofluorescence measurements were made of actin brightness in cooled arterial rings. Cultured SM monolayers were stained with antibodies to SM alpha-actin, SM myosin, and tubulin. In cooling, all solutions resulted in increased arterial distensibility, whereas EC and MS reduced cell wrinkling. With the use of all solutions, actin cables thinned, myosin filaments dissociated, and microtubules depolymerized. During rewarming, resistance to imposed tension increased in all arterial rings. After GM,ED, and MS preservation, contraction to PGF2a increased. Wrinkling increased and actin myosin cables shortened after GM and EC; after UW, wrinkling decreased and actin myosin cables thinned. No recovery occurred after MS. Thus the type of preservation solution influenced contractility during preservation and after rewarming. The absence of spontaneous contraction in cells cooled in UW may be advantageous. PMID- 8638738 TI - Compartmentalization of glucose utilization after intravenous vs. intratracheal challenge with LPS. AB - The rate of glucose utilization (Rg) of various tissues including lung and bronchoalveolar lavage (BAL) fluids cells was measured, using the 2-deoxyglucose technique in Sprague-Dawley rats 4 h after challenge with either 1 mg/kg intravenous or 0.3 mg/kg intratracheal lipopolysaccharide (LPS). After intravenous LPS, Rg increased in whole lung and nonrespiratory tissues, but was unaltered in BAL cells. After intratracheal LPS, the Rg of nonrespiratory tissues was unchanged, but the Rg of BAL cells increased from 3.7 +/- 0.3 to 71.5 +/- 16.0 nmol/min. This increase in the Rg of BAL cells was explained by a doubling of the macrophage specific Rg, by a 100-fold increase in polymorphonuclear leukocytes (PMN) number, and by a higher Rg in PMN than in macrophages. These results demonstrate that increased glucose utilization after intratracheal LPS is confined to the respiratory system and that intra-alveolar phagocytes participate in this increase. PMID- 8638739 TI - Identification of receptors binding fibronectin and laminin on fetal rat lung cells. AB - Fibronectin and laminin have been implicated in regulating lung morphogenesis. In the present study, the cell surface receptors involved in fetal lung cell binding to laminin and fibronectin were identified. Messages for alpha5- and beta1 integrin subunits were detected in both fetal lung epithelial cells and fibroblasts. The presence of alpha5 beta1 -integrin on both cell types was demonstrated by immunocytochemistry and confirmed by cell adhesion experiments with fibronectin and RGD-containing peptides. Epithelial cells adhered more readily to laminin than fibroblasts. The alpha4 beta1-integrin, and RGD independent fibronectin receptor, was weakly expressed on either cell type. Both cell types expressed alpha6-integrin subunit mRNA and stained immunopositive for the alpha6-subunit. Although either cell type expressed nonintegrin 67-kDa laminin-elastin receptor mRNA, no positive immunoreactivity for this laminin elastin binding protein was detected. None of these findings explain the enhanced attachment of distal fetal lung epithelial cells to laminin compared with fibroblasts. Previously, we have reported that epithelial cells were enriched in alpha3-integrin subunit mRNA and protein expression. Herein, we found that epithelial cell attachment to laminin was nearly completely inhibited by alpha3- but only partially by alpha6 -monoclonal antibodies. A peptide near the globular region at the long arm of the laminin A-chain, which contained the IKVAV sequence, and the laminin A-chain amino acid sequence representing the alpha3 beta1 -integrin binding site, inhibited the adherence of epithelial cells to laminin. Fetal lung epithelial cells attached to substrata coated with the alpha3 beta1-integrin binding site peptide and the peptide containing the IKVAV sequence. These data suggest that both fetal lung cell types bind to fibronectin via the fibronectin receptor, alpha5 beta1, and fetal lung epithelial cells interact with laminin via alpha3 beta1 and proteins that recognize the IKVAV containing sequence on the laminin A-chain. PMID- 8638740 TI - MgSO4 relaxes porcine airway smooth muscle by reducing Ca2+ entry. AB - Magnesium sulfate (MgSO4) is used clinically, but its mechanism of action is unknown. To determine whether MgSO4 relaxes airway smooth muscle and to investigate the pathways involved, we compared effects of MgSO4 in porcine tracheal and bronchial muscles contracted with either carbachol or KCl and measured the effects of MgSO4 on the concentration of intracellular free calcium ([Ca2+]i). Lungs were dissected after anesthesia and exsanguination. Tracheal strips and bronchial rings were suspended in tissue baths for measurement of isometric tension in the presence of different concentrations of MgSO4. In separate experiments, tracheal smooth muscle tension and [Ca2+]i were measured simultaneously, using the fluorescent dye fura 2. MgSO4 (1.2, 2.2, 9.2 mM) produced a concentration dependent rightward shift of contraction dose-response curves to KCl but not to carbachol. MgSO4 relaxed trachealis muscles precontracted with KCl or carbachol and simultaneously decreased [Ca2+]i. These findings indicate that MgSO4 directly relaxes airway smooth muscle and that the mechanism involves a decrease in [Ca2+]i. Because initiation and maintenance of contraction during KCl stimulation and maintenance of contraction during carbachol stimulation require Ca2+ entry through voltage-dependent calcium channels, MgSO4-induced relaxation may involve a decrease in Ca2+ entry via these channels. PMID- 8638742 TI - Relative and absolute reliability of the KT-2000 arthrometer for uninjured knees. Testing at 67, 89, 134, and 178 N and manual maximum forces. AB - We assessed the reliability of the KT-2000 knee arthrometer at 67, 89, 134, and 178 N and at manual maximum forces on 30 college students who were free from present or previous knee injuries. Two examiners tested all subjects on two occasions. Anterior laxity (P < 0.0001) and side-to-side difference (P < 0.05) significantly increased as force increased. There was a significant difference (P < 0.0001) between testers for anterior laxity but not for side-to-side difference. We used intraclass correlation coefficients to estimate relative reliability. Anterior laxity intraclass correlation coefficients (2,1) between testers ranged from 0.81 to 0.86 and within tester correlations ranged from 0.92 to 0.95. Intraclass correlation coefficients for between testers for side-to-side differences ranged from 0.38 to 0.58 and within tester correlations ranged from 0.53 to 0.64. Subject-to-subject variability needs to be taken into account when interpreting intraclass correlation coefficient values. Our absolute reliability estimates (95% confidence intervals) were small, indicating little variability. Our data demonstrate the KT-2000 arthrometer to be reliable. Researchers should present both relative and absolute reliability estimates, although we believe absolute estimates are of greater clinical value. Side-to-side differences are better discriminators than individual absolute values. We recommend that a < 3 mm side-to-side difference be used to indicate stable knees. PMID- 8638741 TI - Vitamin A deficiency enhances ozone-induced lung injury. AB - The present study determined the effects of vitamin A (vA) deficiency on the responses to ozone (O3) challenges in two inbred strains of mice that are differentially susceptible to O3-induced lung inflammation. Susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) dams at 2 wk gestation were fed test diets containing either 0 or 10 micrograms retinol/g diet. In mice that were maintained on vA-sufficient (vA+) diet, lung and liver tissue concentrations of vA and retinyl palmitate (RP) were significantly (P<0.05) lower in the B6 strain compared with C3, as measured by high-performance liquid chromatography techniques. vA and RP levels were significantly (P<0.05) reduced in lung and liver tissues of 8-wk old B6 and C3 mice that were maintained on a vA deficient (vA-) diet. vA+ and vA- mice of both strains were exposed to air or 0.3 ppm O3/72 h, and lung injury was assessed by differential cell count and total protein concentration in bronchoalveolar lavage (BAL) returns. O3 exposure caused significantly (P<0.05) greater increases in inflammatory cells and a total protein in BAL returns of vA+ B6 mice than vA+ C3 mice. vA deficiency significantly (P<0.05) enhanced O3-induced increases in polymorphonuclear leukocytes in C3 mice and epithelial cells loss in both strains. Compared with vA+ mice, lung permeability was also significantly (P<0.05) enhanced in vA- mice of both strains exposed to O3. vA replacement partially reversed the O3-induced lung injury that was enhanced by vA- diet. Results indicate that vA may have an important role in the pathogenesis of O3-induced lung injury in differentially susceptible inbred strains of mice. PMID- 8638744 TI - Stress fractures of the ribs in golfers. AB - During a collaborative review at three institutions, we documented 19 cases of stress fractures of the ribs in golfers. There were 13 men and 6 women with an average age of 39 years (range, 29 to 51). The 4th to 6th ribs were the most commonly injured. All fractures occurred along the posterolateral aspect of the ribs, and nine patients had fractures in more than one rib. Sixteen golfers sustained injury on the leading arm side of the trunk. Eighteen golfers were beginners, and the one experienced golfer had dramatically increased his practice time on the driving range before injury. Plain radiographs were usually diagnostic. However, bone scintigraphy was necessary to reach a diagnosis in three cases. A delay in diagnosis of 6 to 8 months occurred in two cases that were originally misdiagnosed as back strains. Stress fractures of the ribs in golfers may be more common than previously realized and may be incorrectly diagnosed as recalcitrant back strains. Based on the findings of other studies, we think fatigue of the serratus anterior is the mechanism of injury. We recommend strengthening the serratus anterior as rehabilitation after this injury and in a general conditioning program for golfers. PMID- 8638743 TI - The long-term biomechanical and viscoelastic performance of repairing anterior cruciate ligament after hemitransection injury in a goat model. AB - To test the healing of the partially torn anterior cruciate ligament, we transected the posterolateral bundle in 11 adult female goats and tested the ligaments at 12, 24, and 52 weeks and 3 years after surgery. As early as 12 weeks after surgery translucent fibrous tissue covered the wound. The differences in anteroposterior laxity between right and left knees measured at 45 degrees and 90 degrees of flexion were not significantly different at each period. Results of Instron testing of the posterolateral bundle revealed the normalized changes in load-relaxation and Young's modulus were not significantly different at each period, but the ultimate tensile strength and stiffness at 3 years were significantly higher than at 12 weeks (P < 0.05) Failure started at the repair site for the 12-week group, but at 24 and 52 weeks the failure occurred throughout the ligament. At 3 years, the specimens failed with bony avulsion, indicating the repaired tissue was not the weakest link of the bone-ligament-bone complex. This study shows that under favorable conditions, partial anterior cruciate ligament injuries are capable of repair. What is more important, the high ultimate tensile strength and stiffness of the 3-year repaired tissue indicate full structural repair of such an artificial transection injury may be possible. PMID- 8638745 TI - Fracture of the subtalar joint in springboard divers. A report of two cases. PMID- 8638746 TI - Open versus closed chain kinetic exercises after anterior cruciate ligament reconstruction. A prospective randomized study. PMID- 8638747 TI - Grip lock injuries to the forearm in male gymnasts. AB - We performed an epidemiologic survey to estimate the number of grip lock injuries occurring among male high school and college gymnasts. These injuries occur when dowel grips used by the gymnast become locked on the bar as the gymnast's momentum carries him through the skill being performed. We also questioned injured gymnasts to obtain details of their injuries. Thirty-eight high school coaches reported 17 injuries and 32 college coaches reported 21 injuries for a 10 year period; 36% of the coaches responding reported at least one such injury in their program. Of the 23 injured gymnasts who returned detailed questionnaires, 20 had sustained fractures and 9 required surgery. The distal forearm or wrist were the areas injured most often. Fourteen gymnasts had residual pain, seven had functional limitations, and eight had limited motion in the wrist. Fifteen of the 23 athletes were using a cubital (hyperpronated) grip at the time of injury and 19 were using dowel grips. Among the reasons cited for the injury, 18 gymnasts thought that their grips were either too large, worn, or stretched; 8 said the grips slid up their wrists, and 7 cited technical errors. PMID- 8638748 TI - The role of the popliteofibular ligament in stability of the human knee. A biomechanical study. AB - The popliteal tendon has a significant attachment to the fibula, the popliteofibular ligament. The role of this ligament in knee stability has not been determined. In this study we used selective cutting techniques to measure the static contribution of the popliteal tendon attachments to the tibia and the popliteofibular ligament for stability of the knee. Sectioning of all the posterolateral structures except the popliteal tendon attachments to the tibia or the popliteofibular ligament resulted in increased primary posterior translation, varus rotation, external rotation, and coupled external rotation. Although statistically significant, these increases were small. Sectioning of all the posterolateral structures resulted in larger increases in primary posterior translation, varus rotation, external rotation, and coupled external rotation. Our data indicate that the popliteal tendon attachments to the tibia and the popliteofibular ligament are important in resisting posterior translation and varus and external rotation. If an isolated injury to the posterolateral structures occurs, anatomic reconstruction of the major ligaments that restrain posterior translation and varus and external rotation may provide the best functional result. Reconstruction for isolated posterolateral instability should include anatomic attachment of the popliteal tendon to the tibia and the popliteofibular ligament. PMID- 8638749 TI - The biceps femoris muscle complex at the knee. Its anatomy and injury patterns associated with acute anterolateral-anteromedial rotatory instability. AB - We dissected 30 cadaveric knees to provide a detailed anatomic description of the biceps femoris muscle complex at the knee. The main components of the long head of the muscle are a reflected arm, a direct arm, an anterior arm, and a lateral and an anterior aponeurosis. The main components of the short head of the biceps femoris muscle are a proximal attachment to the long head's tendon, a capsular arm, a confluens of the biceps and the capsuloosseous layer of the iliotibial tract, a direct arm, an anterior arm, and a lateral aponeurosis. We examined 82 consecutive, acutely injured knees with clinical signs of anterolateral anteromedial rotatory instability for the incidence and anatomic location of injuries to the biceps femoris muscle. Injuries to components of that muscle were identified in 59 (72%) of these knees; 29 knees (35.4%) had multiple components injured. There were 3 injuries to the long head of the biceps femoris muscle (all in the reflected arm) and 89 to the short head. A statistically significant correlation (P = 0.01) was found between increased anterior translation with the knee at 25 degrees of flexion as demonstrated by the Lachman test and injury to the biceps-capsuloosseous iliotibial tract confluens. Additionally, adduction laxity at 30 degrees of flexion correlated with a Segond fracture (P = 0.04). These data establish, in part, the relationship of the biceps femoris complex injury to anterior translation instability. PMID- 8638752 TI - Full-thickness rotator cuff tears. A biomechanical comparison of suture versus bone anchor techniques. AB - We performed a biomechanical comparison of two rotator cuff repair techniques using fresh-frozen human cadavers. Nine pairs of cadaveric shoulders had standardized full-thickness tears made at the supraspinatus tendon insertion. One of each pair of the cadaveric shoulders was repaired by pulling the tendon into a bone trough in the humeral head using standard sutures. The remaining half of the pairs was repaired using anchor sutures. The repairs were tested using a servohydraulically operated material testing system. The anchor suture repair was significantly stronger than the standard suture technique irrespective of bone quality. Failure occurred predominantly through bone in the suture repairs and as a result of suture breakage in the anchor repairs. The anchors should be placed into the edge of the subchondral bone adjacent to the articular surface. The surgeon should direct the anchor so that the direction of the pull is approximately 90 degrees to the anchor, with the humerus at 30 degrees of abduction. PMID- 8638751 TI - Knee hyperextension gait abnormalities in unstable knees. Recognition and preoperative gait retraining. AB - Five patients with symptomatic knee hyperextension thrusting patterns due to posterolateral ligament complex injury underwent gait analysis before and after a gait retraining program. Patients were trained to avoid knee hyperextension by 1) walking with their knees slightly flexed throughout stance, 2) maintaining ankle dorsiflexion in early stance, and 3) maintaining an erect trunk-hip attitude during stance. Kinematic and kinetic measurements were obtained using automated gait analysis. Four of the five patients significantly reduced hyperextension at the knee and abnormal motion patterns at the hip and ankle. Patients showed increases in knee flexion throughout stance conversions of knee flexion-extension moments to more normal biphasic patterns with a 79% decrease in extension moments at terminal extension, and a 22% decrease in knee adduction moments. Posttraining values also showed a 30% decrease in the calculated medial tibiofemoral loads (P < 0.05). At the hip, there were significant decreases in abduction and adduction moments (36% and 18%, respectively, P < 0.01). Ankle plantar flexion motion decreased significantly by 42% (P < 0.01). Gait retraining can alter the biomechanics of hip, knee, and ankle function to approximately normal levels, and therefore is recommended before ligament reconstruction because abnormal knee motions, if resumed postoperatively, can stretch soft tissue reconstructions. PMID- 8638750 TI - Arthroscopic treatment of osteoarthritis of the knee: a prospective, randomized, placebo-controlled trial. Results of a pilot study. AB - The reasons why many patients seemingly benefit from arthroscopic treatment of osteoarthritis of the knee remain obscure. The purpose of this pilot study was to determine if a placebo effect might play a role in arthroscopic treatment of this condition. After giving full informed consent, including full knowledge of the possibility and nature of a placebo surgery, five subjects were randomized to a placebo arthroscopy group, three subjects were randomized to an arthroscopic lavage group, and two subjects were randomized to a standard arthroscopic debridement group. The physicians performing the postoperative assessment and the patients remained blinded as to treatment. Patients who received the placebo surgery reported decreased frequency, intensity, and duration of knee pain. They also thought that the procedure was worthwhile and would recommend it to family and friends. Thus, there may be a significant placebo effect for arthroscopic treatment of osteoarthritis of the knee. The small numbers in this preliminary study preclude a valid statistical analysis, and no conclusions can be drawn regarding the superiority of one treatment over another. A larger study is needed to evaluate fully the efficacy of an arthroscopic procedure for this condition and to decide if it is reasonable to expend health care resources for this treatment; the larger study should include a placebo control group. PMID- 8638753 TI - Postexercise muscle cramping associated with positive malignant hyperthermia contracture testing. PMID- 8638754 TI - Acute dislocation of the patella. A correlative pathoanatomic study. AB - The objective of our study was to elucidate the characteristic pathoanatomy associated with patellar dislocation and report the preliminary results of early surgical repair. Twenty-three patients with documented patellar dislocation had standard radiographs and a magnetic resonance imaging scan. Intraarticular lesions were evaluated and treated arthroscopically followed by an open exploration of the medial aspect of the knee in 16 patients. Twelve patients were observed for a minimum of 2 years after surgical repair (average, 34 months). Eleven patients returned for a follow-up examination. Magnetic resonance imaging revealed effusion (100%), tears of the femoral insertion of the medial patellofemoral ligament (87%), increased signal in the vastus medialis muscle (78%), and lateral femoral condyle (87%) and medial patellar (30%) bone bruises. Arthroscopic examination revealed osteochondral lesions involving the patella and the lateral femoral condyle in 68% of cases. Open surgical exploration revealed tears of the medial patellofemoral ligament off the femur in 15 of 16 patients (94%). After medial patellofemoral ligament repair, none of the patients experienced recurrent dislocation. Overall 58% of the results were considered to be good or excellent and 42% were fair. Fifty-eight percent of the group returned to their previous sport with no or minor limitations. PMID- 8638755 TI - Patellar taping in the treatment of patellofemoral pain. A prospective randomized study. AB - The purpose of this prospective study was to evaluate the efficacy of a patellar taping program in the conservative management of patellofemoral pain. Twenty-five patients with patellofemoral pain were randomized into two groups. One group underwent a standard physical therapy program for patellofemoral pain. The other group underwent the same physical therapy program, but use of a patellar taping technique was added to this program. Results of a subjective visual analog scale and changes in isokinetic strength and electromyographic activity of the quadriceps muscle were analyzed. Both the tape and no-tape groups experienced a statistically significant decrease in symptoms (P < 0.05), but no difference in improvement of patellofemoral pain was noted between the groups. Likewise, both groups demonstrated significant improvement in quadriceps muscle isokinetic strength (P < 0.05) and activity (P < 0.001), but no difference in improvement was noted between groups. The results of this study suggest no beneficial effect of adding a patellar taping program to a standard physical therapy program in the conservative treatment of patellofemoral pain. Larger prospective studies are warranted to support this opinion. PMID- 8638756 TI - Anterior cruciate ligament graft rotation. Reproduction of normal graft rotation. AB - The purpose of this study was to determine normal rotation of the anterior cruciate ligament and to provide a technique for reproduction of this rotation. Ten fresh-frozen knees were dissected of all soft tissue except for the anterior cruciate ligament. Specimens were secured in a vise in 60 degrees of flexion. Each tibia was allowed to spin freely on the femur, and rotation was recorded. Anterior cruciate ligament reconstructions, using bone-patellar tendon-bone grafts, were then performed on all specimens using four graft rotations. Each specimen was then tested to assess how the graft twist affected tibial rotation. The average tibial rotation of the normal anterior cruciate ligaments was 55 degrees internally. Previous descriptions of anterior cruciate ligament reconstructions have advocated medial or internal rotation of the graft to reproduce normal anatomic rotation of the anterior cruciate ligament. Our cadaveric dissections have demonstrated that the anterior cruciate ligament normally produces internal rotation of the tibia in relation to the femur. Reproduction of this anatomic rotation is accomplished with 90 degrees of lateral rotation of the tibial plug toward the fibula. PMID- 8638757 TI - Radiographic evaluation of native anterior cruciate ligament attachments and graft placement for reconstruction. A cadaveric study. AB - We examined seven cadaveric knees to determine the radiographic location of the native anterior cruciate ligament insertion sites as well as the location of tunnels used in anterior cruciate ligament reconstruction. Posteroanterior and lateral views at several flexion angles were taken with radiopaque markers around the insertions of the native anterior cruciate ligament and subsequent reconstruction tunnels. The femoral insertion was best seen on the 60 degrees notch view. On the lateral view, the femoral tunnel was easily seen as it crossed the roof of the intercondylar notch; however, because of the angle of the tunnel, the actual entrance into the knee may be well distal and anterior to this location. The tibial insertion and tunnel were easily seen at any flexion angle. The center of the insertion was 40% of the tibial diameter from the anterior margin. The lateral view in extension allowed determination of the tibial tunnel's location in relation to the intercondylar notch roof, but by itself did not allow accurate determination of the femoral tunnel's position. Notch and extension lateral radiographs together provided sufficient information for evaluation of anterior cruciate ligament graft position in a convenient, cost effective format. Neither view by itself provides enough information to evaluate the position of the graft. PMID- 8638759 TI - Paradoxical phenomena of the McMurray test. An arthroscopic investigation. AB - We evaluated 200 patients who had a positive McMurray test and found atypical McMurray test results in 24 patients (12%). These patients revealed pain or clicking or both either in the medial compartment of the knee when the leg was internally rotated or in the lateral compartment of the knee when the leg was externally rotated. The authors analyzed these paradoxical findings at arthroscopic examination to identify the relationship between the type of meniscal tear and the direction of leg rotation that elicited the catching and displacement of the torn meniscal portion during the McMurray test. Contrary to conventional McMurray test findings, three different types of meniscal tears were found on the side of the knee where pain or a clicking sound occurred. The three types were 1) anteriorly based posterior oblique tears with anterior displacement of the meniscus, 2) bucket-handle tears in the posterior half of the menisci, and 3) peripheral detachment of discoid menisci in the posterior half of the torn portions. PMID- 8638758 TI - The effect of ambient temperature on the shoe-surface interface release coefficient. AB - Previous studies of the shoe-surface interface correlated foot fixation with cleat length, configuration, and material composition as well as turf type and surface conditions. Our study examined the effect of temperature on the rotational torsion resistance of artificial turf football shoes. Five football shoe models, a flat-soled basketball-style turf shoe, a natural grass soccer style shoe, and three multistudded turf shoes, were studied on dry Astro Turf at five temperatures (range, 52 degrees F to 110 degrees F). An assay device, a prosthetic foot mounted on a loaded stainless steel shaft, was used to determine the force necessary to release a shoe from the turf's surface. We used a torque wrench to apply a rotational force so that each shoe was pivoted counterclockwise through an arc of 60 degrees. Our results indicated that release coefficients differ within and among the shoe models at various turf temperatures. We also found that an increase in turf temperature, in combination with cleat characteristics, affects shoe-surface interface friction and potentially places the athlete's knee and ankle at risk of injury. Based on an established risk criterion, which correlated shoe-surface interface combinations in the laboratory with documented clinical occurrences, only the flat-soled basketball-style turf shoe could be designated "safe" or "probably safe" at all five temperatures. PMID- 8638760 TI - Acute injuries in off-road bicycle racing. AB - A descriptive study was conducted to investigate injuries sustained at a major off-road bicycling race at Mammoth Mountain, California, July 6 to 10, 1994. A total of 4027 individual starts in five events during the race were reported. Overall, the total number of competitors in the 5 events was 3624, with some cyclists participating in multiple events. Injuries were considered significant if they occurred during competition and prevented the rider from completing the event. Sixteen cyclists had injuries that met these criteria for an overall injury rate of 0.40%. These 16 cyclists had 44 injuries. Abrasions were the most common injury, followed by contusions, lacerations, fractures, and concussions. The mean injury severity score was 3.0 (range, 1 to 5) with 81.2% of the injuries resulting from cyclists going downhill. Injuries were more severe when the riders were thrown from the bicycles (P = 0.03). We observed different mechanisms of injury in various events, suggesting that the risk factors for sustaining a traumatic injury may vary according to the type of competition involved. PMID- 8638761 TI - Factors associated with wrist pain in the young gymnast. AB - We conducted a cross-sectional survey of 52 nonelite gymnasts (32 girls, 20 boys; average age, 11.8 years) to assess their history of training and wrist pain within the last 6 months. An intensity index was created using the number of training hours per week and the athletes' skill levels. Wrist pain was prevalent in 38 (73%) of the gymnasts. Gymnasts with wrist pain were older (12.6 years versus 9.7 years; P = 0.0002), trained more hours per week (13.5 versus 7.7; P = 0.0002), trained at a higher skill level (P = 0.01), and began training at an older age (7.0 years versus 5.1 years; P = 0.006). Analysis of intensity versus age suggested that a threshold of training intensity may be important in the development of wrist pain. Logistical regression found these factors to be independently associated with wrist pain: intensity (P = 0.036), age > 10 years (P = 0.018), age < 14 years (P = 0.016), and the age of initiation of training (P = 0.020). This study demonstrates that wrist pain is a common problem among nonelite young gymnasts. Training intensity, relative to the age of the participant and the age when training was initiated, appears to be an important determinant of the development of wrist pain in this population. PMID- 8638762 TI - Pathogenesis of sports-related spondylolisthesis in adolescents. Radiographic and magnetic resonance imaging study. AB - We reviewed radiographs and magnetic resonance images of 77 young athletes with spondylolysis and spondylolisthesis (more than 5% vertebral slip) (slip group). The results were compared with similar studies in 88 patients with spondylolysis only (nonslip group). Endplate lesions were found in all patients in the slip group and in 60 (68%) of those in the nonslip group. Slippage between the osseous and cartilaginous endplates was identified in the T1-weighted sagittal magnetic resonance images and categorized according to the type of slippage: total slip of L-5 or S-1, partial slip of L-5 or S-1, or a combination of these (mixed type). In a study of 31 patients whose slippages progressed, no slippage was associated with the early stage of a pars interarticularis defect. Most vertebral slippages developed or progressed in the cartilaginous or apophyseal stage of the lumbar skeletal age. Wedging of the L-5 vertebral body and rounding of the sacrum progressed as the slippage developed; these did not occur in the nonslip group. These results indicate that the advanced stage of a pars interarticularis defect in an immature spine is a risk factor for spondylolisthesis. The deformities of the lumbosacral spine are thought to be the secondary changes caused by vertebral slippage. PMID- 8638763 TI - Magnetic resonance imaging of knee disorders. Clinical value and cost effectiveness in a sports medicine practice. AB - To prospectively evaluate the clinical value of magnetic resonance imaging of the knee in a referral sports medicine practice, we performed a three-part study. First, we asked 72 consecutive patients a series of clinically relevant questions regarding the ordering of their magnetic resonance imaging scans. Second, we asked the treating physicians at our center if the magnetic resonance imaging findings changed the diagnosis or treatment. Third, we compared the clinical evaluation with the findings on magnetic resonance imaging scans for 37 patients who had arthroscopic confirmation. From the physician's perspective, in only three cases would the results of the scan have changed the diagnosis. Information from the scans was judged to contribute to patient treatment in only 14 of 72 patients. Finally, comparison of clinical evaluation and magnetic resonance imaging findings with findings during arthroscopic procedures showed that clinical evaluation had a sensitivity and specificity of 100% for diagnosis of anterior cruciate ligament injuries, whereas magnetic resonance imaging was 95% sensitive and 88% specific. For isolated meniscal lesions, the clinical assessment had a sensitivity and specificity of 91% compared with 82% and 87%, respectively, for magnetic resonance imaging. For evaluation of articular surface damage, the predictive value of a positive test was 100% for clinical assessment and 33% for the magnetic resonance imaging. We conclude that magnetic resonance imaging is overused in the evaluation of knee disorders and not a cost-effective method for evaluating injuries when compared with a skilled examiner. Clinical assessment equals or surpasses the magnetic resonance imaging in accuracy. PMID- 8638765 TI - Fine structure of the afferent synapses in the paratympanic organ of the chicken, with special reference to the synaptic bodies. AB - The afferent synapses of the paratympanic organ in the chicken were studied by TEM. These synapses were formed by non-myelinated fibres which reached the basal part of the hair cells. The fibres contained a number of irregular mitochondria and a few pale vesicles. In the hair cells, near the presynaptic membrane, typical synaptic bodies formed by an electron-dense core surrounded by several small pale vesicles were present. The core was connected with the vesicles by numerous thin filaments, and at same time with the presynaptic membrane by some dense projections. Moreover, we have observed that the connections between the core and the adjacent vesicles also consisted of similar structures to the dense projections. We suggest that this device is involved in the movement of the vesicles towards the presynaptic membrane. Our hypothesis is in agreement with that formulated by some authors who believe that the electron-dense core of the synaptic bodies is able to channel the vesicles to the presynaptic membrane (conveyor-belt hypothesis). Moreover, our work showed that the synaptic bodies of the paratympanic organ in the chicken are variable in density and in shape. These morphological aspects might be linked to regression-reconstitution cycles of the SBs and to the functional level of the afferent synapses. PMID- 8638764 TI - In memoriam Janos Szentagothai 31. 10. 1912--8. 9. 1994. PMID- 8638766 TI - Variations in the branching of the internal pudendal artery in the ischioanal fossa. AB - The branches of the ischiorectal part of the internal pudendal artery include the inferior rectal artery and small branches to the obturator internus muscle. For our research we had 164 embalmed half pelves, the arteries of which has been injected by the Thiel method. After the dissection of the ischioanal fossa the arteries were documented. Four types of branching of the inferior rectal artery were found. I. (43%) One artery on each side. II. (31%) Two arteries on one side. III. (4%) Three arteries on one side. IV. (22%) Two arteries or more on each side. In the specimens examined we found one branch to the obturator internus muscle in 31%, two branches in 43% and three branches in 11%. In 15% of cases this branch was absent. In seven cases two branches formed an anastomosis parallel to the internal pudendal artery. PMID- 8638767 TI - [Architecture and fine structure of the terminal blood vessels in the marginal papillae of the tongue in newborn piglets]. AB - The vascular architecture of the marginal papillae of the tongue was examined in one to five day old piglets by means of light and transmission electron microscopy and scanning microscopy of vascular corrosion casts. The marginal papillae of the tongue exhibit a "rope ladder-like" principle of blood supply. Every papilla contains one ascending arteriole, which branches into single capillary loops each supplying a protrusion of the multiply branched base of the capillary body. The transition of the arteriole takes place on the tip of the papilla. Subsequently the capillary loops converge onto this venule. Precapillary sphincters are observed at the origin of the capillary loops. This type of vessel arrangement offers the opportunity to use the capillary system at maximum efficiency, thus allowing an erection of the papillae marginales during the process of suckling. PMID- 8638768 TI - Ultrastructural study of the dorsal lingual epithelium of the Asian snail-eating turtle, Malayemys subtrijuga. AB - The Asian snail-eating turtle, Malayemys subtrijuga, is classified phylogenetically as a member of the family Emydinae. Members of this family usually live in small rivers or ponds. However, this species is relatively well adapted to terrestrial life. We describe here the light, scanning electron and transmission electron microscopic appearance of the dorsal lingual epithelium of the snail-eating turtle and we compare the results to those obtained from other freshwater turtles in an attempt to clarify the relationship between the histological and ultrastructural differences in the lingual epithelium and the living circumstances of the turtles. The tongue is triangular with a rounded apex when viewed dorsally but it appears flattened when viewed laterally. Under the scanning electron microscope, no lingual papillae were visible on the dorsal surface of the tongue. Instead, plicae were seen all over the dorsal surface. On the surface of the epithelium of the outermost side, dome-shaped bulges, each of which was coincident with an individual cell, were compactly distributed. At higher magnification, scanning electron microscopy revealed numerous microvilli and microridges on the surface of these cells, and the thickening of cell-margins was clearly seen. Light microscopy revealed that the mucosal epithelium of the tongue was of the non-keratinized, stratified squamous type. Under the transmission electron microscope, the cells of the basal and deep intermediate layers of the epithelium appeared irregularly elliptical in shape. The nucleus was large and also irregularly elliptical, lying in the central region of each epithelial cell. The cytoplasm of these cells contained mitochondria, free ribosomes, rough endoplasmic reticulum and bundles of tonofibrils. Cell membranes formed processes around individual cells. Desmosomes were intercalated between the processes of adjacent cells. In the shallow intermediate layer, the cells were also elliptical, and the elliptical nucleus was located in the central area of each cell. A large part of the cytoplasm was occupied by electron-dense, discoid granules. Filamentous structures filled the spaces between these granules. Small numbers of free ribosomes, mitochondria and rough endoplasmic reticulum were scattered in the cytoplasm. Cell membranes still formed processes around cells. Desmosomes were intercalated between the processes of adjacent cells. The cells of the surface layer were still elliptical, as were their nuclei. Most of the cytoplasm was filled with electron-dense, discoid granules. Fine filamentous structures were dispersed between these granules. Cell membranes formed processes around cells which were coincident with microvilli and microridges. Intercalated desmosomes were also seen. In some cells, many of the electron-dense, discoid granules were secreted into the oral cavity. In conclusion, the histology of the lingual epithelium of the snail-eating turtle is very similar to that of the freshwater turtle, reflecting similarities in the gross morphology of the tongues of these species, in spite of the differences in their life styles. PMID- 8638769 TI - Development of pericyte-like cells during angiogenesis in quail chick chimeras as detected by combined Feulgen reaction and immunohistochemistry. AB - Perivascular fibroblasts have been proposed as possible precursor cells for microvascular pericytes. To investigate the development of pericytes during angiogenesis we examined interspecific grafts between chick and quail embryos. Limb buds of three-day old quail embryos were transferred to the chorioallantoic membrane (CAM) of ten to fourteen day-old chick embryos. Six days after grafting, the limb buds were explanted and histologically examined by combined Feulgen reaction and immunohistochemistry using an antibody to quail endothelial and hemopoietic cells (QH-1). Limb buds were found to be vascularized by a network of capillaries which were partially derived from sprouts of the chick CAM microvasculature. Numerous hybrid capillaries were detected, consisting of host endothelial cells (chick) and graft pericytes (quail). These results provide further support for the idea that microvascular pericytes can evolve from perivascular fibroblasts. PMID- 8638770 TI - C-cells of marmosets (Callithrix jacchus). AB - The localization and fine structure of C-cells of the thyroid glands from 31 marmosets (Callithrix jacchus) of different age groups (newborn to 11 years old) were investigated by electron microscopy and immunomorphology (APAAP, calcitonin). Calcitonin-positive cells were found concentrated in the middle third of the gland. Their localization in the follicular wall or in the interstitial space could not clearly be demonstrated by light microscopy. In adult animals, they were always located within the basal lamina, as revealed by electron microscopy. In newborn animals, their localization was difficult because of the dense cell packing. The fine structure of the C-cells with their 200 nm electron-dense granules corresponded to that of other species. Two cell types could be distinguished: (1) granule-rich cells in the storing phase and (2) cells with fewer granules and well-developed rough endoplasmic reticulum in the synthesis phase. The concentration of C-cells in the middle third of the marmoset thyroid gland renders this species especially suitable for morphological investigations and in vitro experiments. PMID- 8638771 TI - Unusual variants of the tributaries of the main pancreatic duct revealed by postmortem and endoscopic pancreatography. AB - Analysis of 97 human postmortem pancreatograms and 103 endoscopic pancreatograms revealed a total of 3 cases (1.5%) with isolated variants in the branches of the main pancreatic duct. In the first one, tortuous branches were present in the upper head-body region. The second case involved joint tributaries bridging over an unstenosed segment of the main pancreatic duct. In the third case, we found three branches from the uncinate process running down to the main duct. In all of these cases no pathological substrate was found, and they should be considered as anatomical variants observed during interpretation of a radiogram. PMID- 8638772 TI - The sensory innervation of the shoulder joint of the mouse. AB - The ultrastructure and location of sensory nerve endings in the shoulder-joint capsule, its tendinous reinforcements and in the periarticular connective and muscle tissue have been studied by means of light and electron microscopy in adult female white NMRI-F2 laboratory mice, aged 2.5-13 months. Most of the sensory nerve endings were detected in the fibrous layer of the joint capsule or in the inserting tendons. The identified lamellated corpuscles of the Pacini type are small and sometimes associated with Golgi tendon-organs. Large Vater-Pacini corpuscles were not detected. Ruffini corpuscles are found in small numbers only in the moderately dense connective tissue of the joint capsule. Golgi tendon organs were found mainly at the muscle-tendon junction of the muscles surrounding the joint. Muscle spindles have been identified mainly in periarticular muscles close to the muscle-tendon junctions. The number and distribution of the different types of mechanoreceptors investigated in the present study suggest that periarticular corpuscular sensory nerve endings play an important role in shoulder-joint control and mobility. The occurrence of small uniformly shaped lamellated corpuscles of the Pacini type in qualitatively different areas of surrounding tissue implies that they are susceptible to different kinds of mechanical stimuli. PMID- 8638773 TI - Two cases of the superficial dorsalis pedis artery observed in man. AB - A superficial dorsalis pedis artery was found in two (0.7%) of 296 Japanese feet. The sciatic and saphenous arteries were not present. This artery arose from the anterior tibial artery at the ankle, penetrated the inferior extensor retinaculum and ran forward over the deep fascia of the foot. The artery gave off a superficial dorsal metatarsal artery and continued into the deep plantar branch perforating the first dorsal interosseous muscle. In the second case, a thin deep dorsalis pedis artery anastomosed with the superficial dorsalis pedis artery to form an arterial circle surrounding the tendons of the extensor hallucis muscles. In man, the saphenous artery usually degenerates and therefore the superficial dorsalis pedis artery has not hitherto been recorded. The origin of this artery is considered to be a remnant of the distal part of the saphenous artery or an enlarged by-pass supplying the cutaneous nerves and subcutaneous tissue. PMID- 8638774 TI - Bony markers at the distal end of the radius for estimating handedness and radial length. AB - Measurements of the size of the bony markers at the distal end of the radius as well as the length of the radius in 61 left and 64 right dry radii were statistically analyzed. Since 90-95% of the general population is right-handed, as based on differences in the size of the right sided markers relative to the left, it is proposed that the greater distance between the dorsal tubercle and styloid process and the greater dorso-palmar diameter of the carpal articular surface opposite the dorsal tubercle are indicative of right-handedness. The length of the radius correlated with: the radio-ulnar transverse diameter at the distal end; the distance between the dorsal tubercle and the styloid process; the dorso-palmar diameter of the distal end opposite the dorsal tubercle; the dorso palmar diameter of the carpal articular surface opposite to the dorsal tubercle; the dorso-palmar diameter of the distal end opposite the medial margin of the groove for the extensor pollicis longus; the dorso-palmar diameter of the distal end opposite the floor of the groove for the extensor pollicis longus and, finally, the height of the dorsal tubercle in relation to the posterior margin of the carpal articular surface (P < 0.001). Regression equations of the length of the radius for these markers have been derived. PMID- 8638775 TI - Perspective from the editor-in-chief: anesthesia providers, patient outcomes, and costs. PMID- 8638776 TI - Perspective from a chairman of anesthesia in Minnesota: anesthesia providers, patient outcomes, and costs. PMID- 8638777 TI - Perspective from a chairman of anesthesia outside Minnesota: anesthesia providers, patient outcomes, and costs. PMID- 8638779 TI - Aprotinin prolongs activated and nonactivated whole blood clotting time and potentiates the effect of heparin in vitro. AB - This study was designed to evaluate the effect of aprotinin on activated versus nonactivated whole blood clotting time using two different on-site methods and to quantify these anticoagulant properties when compared to heparin in a controlled, in vitro environment. Blood specimens were obtained prior to heparin administration from 56 patients undergoing cardiac surgery. Specimens obtained from the first consecutive 20 patients were mixed with either normal saline (NS) or aprotinin (400 kallikrein inhibiting units (KIU)/mL), inserted into Hemochron tubes containing either NS or heparin (0.3 or 0.6 U/mL) and then used to measure celite-activated (celite ACT) and nonactivated whole blood clotting time (WBCT1) using four Hemochron instruments. Accordingly, specimens obtained from the second consecutive 20 patients were mixed with either NS or aprotinin, inserted into Automated Clot Timer cartridges containing either NS or heparin (0.06, 0.13, or 0.25 U/mL) and then used to measure kaolin-activated (kaolin ACT) or nonactivated whole blood clotting times (WBCT2) using four Automated Clot Timer instruments. Specimens obtained from the last 16 patients were mixed with either incrementally larger doses of aprotinin (0, 100, 200, 300, or 400 KIU/mL) or heparin (0, 0.12, 0.24, 0.36, 0.48, or 0.72 U/mL) and were then used for measurement of whole blood clotting time (WBCT2) using six Automated Clot Timer instruments. Aprotinin significantly prolonged activated or nonactivated whole blood clotting time and potentiated the prolongation of whole blood clotting time by heparin. The linear relationship between whole blood clotting time and either heparin concentration (WBCT2 = H x 357 + 280, mean adjusted r2 = 0.88) or aprotinin concentration (WBCT2 = A x 0.97 + 300, mean adjusted r2 = 0.94) was variable among patients. On average, 200 KIU/mL of aprotinin prolonged WBCT2 to the same extent as 0.69 +/- 0.28 U/mL of heparin using linear regression models within each patient. Aprotinin significantly prolongs activated or nonactivated whole blood clotting time measurements in a dose-dependent manner. Since prolongation of whole blood clotting time by heparin is potentiated by aprotinin in vitro, aprotinin's anticoagulant properties may in part account for the prolonged celite activated clotting time values observed in the presence of aprotinin. PMID- 8638778 TI - Continuous small-dose aprotinin controls fibrinolysis during orthotopic liver transplantation. AB - Large doses of aprotinin (1,000,000-2,000,000 kallikrein inhibitor units [KIU] initial dose and a 500,000 KIU/h infusion) have been used during orthotopic liver transplantation (OLT) to reduce the incidence and severity of fibrinolysis. This double-blinded study was designed to investigate whether a small-dose infusion of aprotinin (200,000 KIU/h) would control fibrinolysis. A controlled study was undertaken to compare small-dose aprotinin with a placebo in patients undergoing OLT with veno-venous bypass. Forty-four patients were randomized either to the aprotinin group (n = 21), which received an intravenous infusion of 200,000 KIU/h without an initial dose, or to a control group (n = 23), which received normal saline. Coagulation variables, thrombelastograms, and postoperative blood drainage were measured. Blood levels of fibrin degradation products (FDP) were significantly higher in the control group (95% > 20 micrograms/mL) at the end of surgery compared to the aprotinin group (53% > 20 micrograms/mL, P < 0.01). The transfusion of cryoprecipitate units was more in the control group versus the aprotinin (12.6 +/- 12.8 vs 5.7 +/- 7.5; P < 0.04), as was the number of fresh frozen plasma units (6.6 +/- 3.5 vs 3.6 +/- 6.1; P < 0.05). We conclude that an infusion of a small dose of aprotinin can safely control fibrinolysis during liver transplantation with a concomitant reduction in transfusion of blood products. PMID- 8638780 TI - Transesophageal echocardiography in myocardial revascularization: I. Accuracy of intraoperative real-time interpretation. AB - Transesophageal echocardiography (TEE) is increasingly used intraoperatively as a monitor of ventricular function and volume. Despite its increasing use, whether data from TEE monitoring can be interpreted accurately on-line in real-time is unknown. We studied the performance of five community-based, full-time cardiac anesthesiologists during 75 surgical procedures in which biplane TEE monitoring was used. Every 10 min intraoperatively, each anesthesiologist evaluated the video cine loop display of echocardiographic images to provide a real-time visual estimate of left ventricular ejection fraction area (EFA) and left ventricular filling at the level of the short axis and to assess regional wall-motion of the short axis and transgastric longitudinal views using a predefined scoring system. The same video images were analyzed quantitatively off-line by two blinded investigators. Intraoperative real-time estimates of EFA correlated moderately with off-line quantification (r = 0.8, P = 0.0001). Of the 662 cine loops analyzed by both off-line and real-time techniques, 386 (55%) were within +/-5% of each other, 495 (75%) were within +/-10% of each other, 561 (85%) were within +/-15% of each other, and 617 (93%) were within +/-20% of each other. The overall sensitivity and specificity of real-time echocardiographic ischemia detection were both 76%. However, there was individual variation among the five anesthesiologists. Recognition of normal and severe regional wall-motion abnormality, such as akinesis, had more concordance between real-time and off line analysis, 93% and 79%, respectively, than recognition of mild regional wall motion abnormalities. Anesthesiologists can estimate EFA in real-time to within +/-10% of off-line values in 75% of all cases. Real-time identification of normal regional function is more accurate than identification of abnormal function, i.e., there is variability in quantifying the severity of regional dysfunction. PMID- 8638781 TI - Transesophageal echocardiography in myocardial revascularization: II. Influence on intraoperative decision making. AB - This study was conducted to determine how transesophageal echocardiography (TEE) guides intraoperative decision making during myocardial revascularization. Although its usefulness in influencing clinical decision making during cardiac valvular surgery is well documented, the clinical utility of TEE in patients undergoing myocardial revascularization is less clear. We studied the performance of five community-based, full-time cardiac anesthesiologists during 75 surgical procedures. All patients were monitored with radial artery and pulmonary artery catheters as well as biplane TEE. Immediately after each clinical intervention, the anesthesiologist was asked to determine how real-time TEE influenced the therapy, which single monitor was most influential, and why each therapy was initiated. Of the 584 interventions, TEE was the single most important guiding factor in 98 instances (17%). Interventions involving fluid administration contributed to 277 of 584 (47%) of the total clinical decisions. TEE was the most important monitor influencing fluid administration in 82 of 277 instances (30%), versus the pulmonary artery catheter in 20 of 277 instances (7%). TEE was the single most important monitor in guiding other therapies as follows: antiischemic therapy, 8 of 38 = 21%; vasopressor or inotrope administration, 4 of 115 = 3%; vasodilator therapy, 1 of 38 = 3%; antiarrhythmic medications, 0 of 16 = 0%; and depth of anesthesia, 1 of 72 = 1%. In 2 of 75 patients (3%), critical surgical interventions were made solely on the basis of TEE. Also, TEE was found to act in concert with other monitors in 254 of 584 interventions (43%). TEE is often influential in guiding decision making in myocardial revascularization when incorporated as a routine monitor in the intraoperative setting. Information from TEE has been most commonly used to guide the management of fluid administration and institution of antiischemic therapy. In a small subset of patients, TEE appears to be useful in guiding critical surgical interventions. PMID- 8638782 TI - The relationship between cerebral blood flow and transcranial Doppler blood flow velocity during hypothermic cardiopulmonary bypass in adults. AB - A noninvasive, simple, and continuous method to assess cerebral perfusion during cardiopulmonary bypass (CPB) could help prevent cerebral ischemia. Transcranial Doppler sonography (TCD) allows a noninvasive, on-line measurement of blood flow velocity in cerebral arteries. The correlation of TCD-estimated and actual cerebral blood flow (CBF) has not been well studied during CPB. We determined the correlation of middle cerebral artery (MCA) mean velocity and CBF determined by the Kety-Schmidt method during nonbypass and two hypothermic bypass flow conditions. Sixteen patients undergoing hypothermic (27 degrees C) CPB for coronary artery bypass grafting and/or valve replacement surgery were enrolled in the study. We were able to determine MCA velocity in only 12 patients. We determined CBF and MCA velocity in each patient during four 15-min study periods: 1) prebypass after sternotomy before aortic cannulation; 2) hypothermic (27 degrees C) CPB with 1.2 L.min-1.m-2 pump flow; 3) hypothermic CPB with 2.4 L.min 1.m-2 pump flow, and 4) 30 min after weaning from CPB. There was no difference in the mean arterial pressure between the two CPB pump blood flows. The pooled change in MCA velocity and CBF as percentage of baseline (prebypass) for all patients and at all time points had a correlation of 0.33 (r). A decrease or increase in MCA velocity did not necessarily indicate a corresponding decrease or increase in CBF. This technology may be of limited usefulness during the circulatory condition of hypothermic, nonpulsatile CPB. PMID- 8638783 TI - The dose-dependent effects of halothane on right ventricular contraction pattern and regional inotropy in swine. AB - The right ventricle (RV) is comprised of two embryologically distinct units, the inflow and outflow tracts, which normally contract sequentially and differ in the magnitude of increased inotropy during sympathetic nervous stimulation. The present study examined the dose-response effects of halothane on the RV contraction pattern and regional contractility in seven open-chest pigs instrumented for measurement of inflow and outflow tract pressures and segment lengths. The RV contraction pattern was evaluated by comparing the phase of inflow and outflow tract shortening, and regional contractility was determined by calculation of preload recruitable stroke work (PRSW) slope. Using this methodology, an inflow-outflow tract contraction phase difference of -27 degrees (inflow tract shortened earlier) was evident at baseline, but was abolished by 1.0 and 1.5 minimum alveolar anesthetic concentration (MAC) halothane; PRSW slope of both the inflow and outflow tracts, however, demonstrated similar dose-related change. To determine whether alterations in cardiac sympathovagal balance played a role in the RV response to halothane, an additional four animals were studied after pretreatment with hexamethonium, propranolol, and atropine. In these animals, there was no difference in the regional contraction phase either at baseline or during halothane administration, and dose-related depression of PRSW by halothane was again similar in both regions. However, when halothane effects on regional PRSW in animals with autonomic blockade were compared to those of neurally intact animals, a 20% greater depression of outflow tract PRSW by 0.5 MAC halothane was evident. This study demonstrates that halothane abolishes the normal sequential pattern of RV contraction without exerting markedly variant negative inotropic effects within different regions of the RV, and provides evidence to suggest that alterations in cardiac sympathovagal balance may contribute to the effect of halothane on RV contraction dynamics. PMID- 8638785 TI - The effect of postoperative analgesia with continuous epidural bupivacaine after cesarean section on the amount of breast feeding and infant weight gain. AB - The purpose of this study is to determine the effect of postoperative analgesia on the amount of breast feeding and infant weight gain. Thirty parturients undergoing elective cesarean section under spinal anesthesia were randomly allocated to receive postoperative pain management with (S-E group, n = 15) or without epidural bupivacaine (S group, n = 15). Epidural analgesia was performed for 3 days with a continuous epidural infusion (0.7 mL/h) of 0.25% bupivacaine. Diclofenac was available on demand in all patients. The weight of milk fed by breast and the infant weight were measured for 11 days after cesarean section. In the S-E group, the visual analog pain score after surgery was significantly lower and both the weight of milk fed by breast and the infant weight during the study were significantly more than the respective values in the S group. The S group required a larger dose of diclofenac after the operation than did the S-E group. We suggest that satisfactory postoperative pain relief with continuous epidural bupivacaine for 3 days after cesarean section improved the amount of breast feeding and the gain of infant weight. PMID- 8638786 TI - The neuromuscular effects of mivacurium chloride during propofol anesthesia in children. AB - Previous studies examined the neuromuscular effects of mivacurium in doses up to, but not exceeding, 2.5 times 95% effective dose (ED95) in children. To determine whether larger doses offer clinical advantages, we compared the onset and duration of neuromuscular block, intubating conditions, and changes in plasma histamine concentration (PHC) after mivacurium (0.2, 0.3, or 0.4 mg/kg) with those after succinylcholine (2.0 mg/kg) during propofol/N2O anesthesia in 48 children aged 3-10 yr. The evoked electromyograph (EMG) of the adductor digiti minimi after supramaximal train-of-four (TOF) stimulation was recorded. When T1 was 10% of control, laryngoscopy and intubation were performed. PHC was measured immediately before and at 2 and 5 min after administration of the relaxant. Venous blood was sampled for determination of plasma cholinesterase activity. Axillary temperature was measured. Increasing the dose of mivacurium from 0.2 to 0.3 mg/kg accelerated the onset of block (time to 90% block, 1.6 +/- 0.2 vs 1.2 +/- 0.2 min) (P < 0.001), but did not significantly prolong recovery (time to 95% recovery, 16.0 +/- 3.8 vs 18.6 +/- 3.6 min). A further increase in dose to 0.4 mg/kg produced no significant decrement in onset time, but did prolong recovery (time to 95% recovery, 23.8 +/- 5.0 min) (P < 0.001). The duration of action of mivacurium 0.3 and 0.4 mg/kg correlated inversely with plasma cholinesterase activity. PHC increased significantly after mivacurium 0.3 and 0.4 mg/kg; however, mean arterial pressure did not change significantly. We conclude that mivacurium 0.3 mg/kg provides a relatively rapid onset and short duration of neuromuscular block in healthy children. Increasing the dose to 0.4 mg/kg does not significantly accelerate the onset of neuromuscular block. PMID- 8638784 TI - The hemodynamic and renal effects of sevoflurane and isoflurane in patients with coronary artery disease and chronic hypertension. Sevoflurane Ischemia Study Group. AB - In patients without significant cardiovascular disease, the hemodynamic effects of sevoflurane and isoflurane are similar; however, the hemodynamic effects of sevoflurane in patients with hypertension and ischemic heart disease are unknown. To examine the effects of sevoflurane in comparison to isoflurane in this high risk population, 214 patients scheduled for elective surgery were enrolled if they had evidence of ischemic heart disease or multiple risk factors for ischemic heart disease. Patients were randomly assigned to receive sevoflurane (n = 106) or isoflurane (n = 108) for anesthetic maintenance in conjunction with fentanyl and nitrous oxide in oxygen. Deviations in arterial blood pressure or heart rate of more than 20% from preinduction values that persisted after adjustment of the volatile anesthetic concentration were treated with intravenous phenylephrine, ephedrine, nitroglycerin, atropine, or esmolol as needed. Creatinine, blood urea nitrogen (BUN), and urine protein were measured before surgery, immediately after surgery, and 24 and 48 h postoperatively. For analysis, patients were divided into those with and those without the diagnosis of chronic hypertension. Heart rate and arterial blood pressure responses to sevoflurane and isoflurane were not different for the patients with or without chronic hypertension. Neither anesthetic was associated with a more frequent treatment for hemodynamic deviation. After surgery, creatinine and BUN decreased in both the sevoflurane and isoflurane groups without significant differences between groups. The incidence of post-operative proteinuria was similar in the sevoflurane and isoflurane groups. We conclude that hemodynamic stability in patients with hypertension and ischemic heart disease is similar with sevoflurane and isoflurane. No differences in renal function were observed between the sevoflurane and isoflurane groups. PMID- 8638788 TI - Dose-response study of intrathecal morphine versus intrathecal neostigmine, their combination, or placebo for postoperative analgesia in patients undergoing anterior and posterior vaginoplasty. AB - This study was designed to examine postoperative analgesia with intrathecal neostigmine in a randomized, blinded trial with morphine as the active control in patients undergoing anterior and posterior vaginoplasty. A secondary aim was to provide preliminary data on the interaction between these two drugs. The incidence of adverse effects was also assessed. Forty-eight patients were divided into eight groups (50 micrograms, 100 micrograms, and 200 micrograms morphine [M]; saline; 50 micrograms, 100 micrograms, and 200 micrograms neostigmine [N]; and 50 micrograms morphine + 50 micrograms neostigmine). Anesthesia was provided with a balanced technique. All patients stayed 24 h in the recovery room where adequacy of postoperative analgesia and side effects were assessed. Increasing doses of intrathecal morphine (50 micrograms, 100 micrograms, and 200 micrograms) and intrathecal neostigmine (50 micrograms, 100 micrograms, and 200 micrograms) showed a dose-dependent pattern of analgesia (P < 0.001). The M50 + N50 combination resulted in a better analgesic effect with fewer side effects than M50, N50, and control groups. These preliminary data suggest that spinal neostigmine produces analgesia for vaginoplasty surgery similar in duration to spinal morphine and that the combination of morphine and neostigmine may allow a reduction in the dose of each component for postoperative analgesia. PMID- 8638789 TI - Unilateral spinal anesthesia using low-flow injection through a 29-gauge Quincke needle. AB - Restriction of sympathetic denervation during spinal anesthesia may minimize hemodynamic alterations. Theoretically, the use of nonisobaric anesthetics may allow unilateral anesthesia and thus restrict sympathetic denervation to one side of the body. The present prospective study investigates the incidence of unilateral spinal anesthesia using hyperbaric bupivacaine 0.5% (1.4 mL, 1.6 mL, 1.8 mL, or 2.0 mL) injected via a 29-gauge Quincke needle with a pump-controlled injection flow of 1 mL/min. In 96 consecutive patients undergoing unilateral surgery of the lower extremities, spinal anesthesia was performed in the lateral decubitus position, which was maintained for 20 min postinjection. Increases in foot temperature of at least 0.5 degrees C were defined as sympathetic blockade. The incidence of unilateral block was not significantly influenced by the amount of bupivacaine. For all 96 patients, the incidence of unilateral sympathetic and complete motor block was 69% and 77%, respectively. Frequency of unilateral sensory block (assessed by pinprick and temperature discrimination) was significantly lower (28%). Strict unilateral spinal anesthesia was achieved in 24 cases (25%). Twenty minutes after injection of the local anesthetic, mean arterial blood pressure decreased significantly in patients with bilateral sympathetic blockade from 87 +/- 8 to 83 +/- 8 mm Hg (P < 0.01) but not in patients with unilateral sympathetic blockade (from 87 +/- 11 to 85 +/- 10 mm Hg). In conclusion, low-flow injection (1 mL/min) of hyperbaric bupivacaine 0.5% via a 29-gauge Quincke needle prevented bilateral sympathetic blockade in more than 69% of the patients. The data further suggest that loss of temperature discrimination alone is not a reliable estimation of sympathetic block. PMID- 8638787 TI - Intravenous ketorolac tromethamine worsens platelet function during knee arthroscopy under spinal anesthesia. AB - Ketorolac prolongs bleeding time and inhibits platelet aggregation and platelet thromboxane production in healthy, awake volunteers. However, platelet function was recently shown not to worsen after ketorolac was given during general anesthesia. The purpose of this study was to investigate platelet function changes during a standardized spinal anesthetic and surgery, as well as after a single intraoperative dose of intravenous (IV) ketorolac. The study comprised 30 ASA physical status I patients undergoing spinal anesthesia for knee arthroscopy. Subjects were randomized to receive either ketorolac 60 mg IV 15 min after skin incision or placebo IV. Platelet function testing consisted of an Ivy bleeding time, platelet aggregometry with adenosine diphosphate (ADP) and collagen, thromboelastography (TEG), and serum thromboxane B2 (TxB2) assays. Platelet function testing was performed: 1) 15 min prior to the performance of spinal anesthesia; 2) 10 min after surgical skin incision; and 3) 45 min after administration of study drug. The placebo group demonstrated no changes in any platelet function variable during spinal anesthesia and surgery relative to preoperative values. The ketorolac group, however, demonstrated a significant increase in bleeding time from postincision to poststudy drug data points (213 +/ 60s to 275 +/- 85s, mean +/- SD; P < 0.01). Further, platelet aggregometry to collagen was diminished in the ketorolac group from preoperative to poststudy drug data points (90.8% +/- 7.6% to 60.5% +/- 32.5%; P < 0.01). Platelet aggregometry with ADP, however, was unchanged in the ketorolac group. Platelet TxB2 production decreased dramatically in the ketorolac group from preoperative to poststudy drug data points (157.2 +/- 129.4 to 0.3 +/- 0.3 ng/mL; P < 0.01). Platelet function does not appear to be accentuated during spinal anesthesia as it is during general anesthesia. Unlike during general anesthesia, platelet function during spinal anesthesia is impaired, with respect to bleeding time and platelet aggregometry to collagen, by a single intraoperative dose of IV ketorolac. PMID- 8638790 TI - Oral clonidine premedication enhances the quality of postoperative analgesia by intrathecal morphine. AB - Since clonidine potentiates the analgesia by morphine, the current study was performed to investigate whether oral clonidine premedication would enhance the postoperative analgesia by intrathecal morphine. Twenty-six patients, aged 37-60 yr, schedule for abdominal total hysterectomy under spinal anesthesia, were studied. Patients were randomly allocated to one of two groups; the clonidine group (n = 13) received oral clonidine approximately 5 micrograms/kg, and the control group (n = 13) received no clonidine. All patients received hyperbaric tetracaine 12 mg dissolved in 10% dextrose and morphine 0.2 mg for spinal anesthesia. We measured duration of analgesia (time to the first request for supplemental analgesics) and motor block. We also recorded the total number of injections of supplemental analgesics, and intensity of postoperative visual analog pain scores, nausea, and pruritus for 48 h after intrathecal administration. Duration of analgesia in the clonidine group was longer than the control group (2017 +/- 263 vs 1190 +/- 199 min, mean +/- SEM; P < 0.05). Although there was no difference in the total number of injections of supplemental analgesics (1.1 +/- 0.4 and 2.2 +/- 0.3 in the clonidine and control groups, respectively), the number of patients not requiring supplemental analgesics during the entire study period was larger in the clonidine group than the control group (six patients versus one patient; P < 0.05). There were no differences at any observation point between groups in visual analog pain scores, or the incidence of nausea and pruritus. Oral clonidine preanesthetic medication enhances the postoperative analgesia of intrathecal morphine plus tetracaine without increasing the intensity of side effects from morphine. PMID- 8638791 TI - Preoperative airway assessment: predictive value of a multivariate risk index. AB - Using readily available and objective airway risk criteria, a multivariate model for stratifying risk of difficult endotracheal intubation was developed and its accuracy compared to currently applied clinical methods. We studied 10,507 consecutive patients who were prospectively assessed prior to general anesthesia with respect to mouth opening, thyromental distance, oropharyngeal (Mallampati) classification, neck movement, ability to prognath, body weight, and history of difficult tracheal intubation. After induction of anesthesia, the laryngeal view during rigid laryngoscopy was graded and the ability of experienced anesthesia personnel to ventilate via a mask was determined. Poor intubating conditions (laryngoscopy Grade IV) and inability to achieve adequate mask ventilation were identified in 107 (1%) and 8 (0.07%) cases, respectively. Logistic regression identified all seven criteria as independent predictors of difficulty with laryngoscopic visualization. A composite airway risk index (derived from nominalized odds ratios calculated from the multivariate model) as well a simplified (0 = low, 1 = medium, 2 = high) risk weighting exhibited higher positive predictive value for laryngoscopy Grade IV at scores with similar sensitivity to Mallampati class III, as well as higher sensitivity at scores with similar positive predictive value. Compared to Mallampati class I fewer false negative predictions were observed at a risk index value of 0. We conclude that improved risk stratification for difficulty with visualization during rigid laryngoscopy (Grade IV) can be obtained by use of a simplified preoperative multivariate airway risk index, with better accuracy compared to oropharyngeal (Mallampati) classification at both low- and high-risk levels. PMID- 8638792 TI - Attenuation of cardiovascular responses to tracheal extubation: verapamil versus diltiazem. AB - We studied the effect of intravenous injection of verapamil (0.05 mg/kg or 0.1 mg/kg) on cardiovascular changes during tracheal extubation and emergence from anesthesia and compared the efficacy of the drug with that of diltiazem (0.2 mg/kg). Eighty patients (ASA physical status I) who were to undergo elective gynecological surgery were randomly assigned to one of four groups (n = 20 each): saline (control), 0.05 mg/kg and 0.1 mg/kg verapamil, and 0.2 mg/kg diltiazem. These medications were given 2 min before tracheal extubation. Anesthesia was maintained with 0.5%-1.8% isoflurane and 60% nitrous oxide in oxygen. Muscle relaxation was achieved with vecuronium. Changes in heart rate (HR) and arterial blood pressure (AP) were measured during and after tracheal extubation. In the control group, the HR and systolic and diastolic AP increased significantly during tracheal extubation. Both calcium channel blockers attenuated the increases in these variables. The inhibitory effect was greatest with verapamil 0.1 mg/kg, while the alleviative effect of verapamil 0.05 mg/kg was inferior to that of diltiazem 0.2 mg/kg. These findings suggest that a bolus injection of verapamil 0.1 mg/kg given 2 min before tracheal extubation is a more effective prophylactic for attenuating the cardiovascular changes associated with extubation than is diltiazem 0.2 mg/kg. PMID- 8638793 TI - Anticholinesterase drugs stimulate phosphatidylinositol response in rat tracheal slices. AB - Some anticholinesterase (anti-ChE) drugs induce airway smooth muscle contraction. Whether anti-ChE drugs stimulate muscarinic receptors in airway smooth muscle as well as nicotinic receptors in neuromuscular junction is unknown. Since there is a direct relationship between phosphatidylinositol (PI) response and airway smooth muscle contraction induced by muscarinic agonists, we examined the effects of neostigmine, physostigmine, pyridostigmine, and edrophonium on PI response in the airway smooth muscle. The rat tracheal slices were incubated in Krebs Henseleit solution containing LiCl and [3H]myo-inositol in the presence of carbachol, anti-ChE, or none of them. [3H]inositol monophosphate (IP1), which is a degradation product of PI response, was counted with a liquid scintillation counter. Inositol monophosphate accumulation was stimulated by neostigmine, physostigmine, and pyridostigmine in a dose-dependent manner, but was not affected by edrophonium. These increases were completely inhibited by atropine. The results suggest that neostigmine, physostigmine, and pyridostigmine stimulate PI response in the airway smooth muscle, which would cause bronchoconstriction, while edrophonium does not affect PI response. PMID- 8638795 TI - Right ventricular function during revision total hip arthroplasty. AB - Total hip arthroplasty (THA) is associated with pulmonary embolization of cement bone marrow debris leading to cardiopulmonary complications including cardiac arrest. These complications are more prevalent during revision THA. This report assessed right ventricular function using a right ventricular ejection fraction pulmonary artery catheter (RVEF) and transesophageal echocardiography (TEE) in 18 patients undergoing revision THA. During femoral prosthesis insertion, all patients exhibited hemodynamic changes, but most of these were small and clinically insignificant. Four patients demonstrated a decrease in RVEF > or = 10% and an increase in mean pulmonary artery pressure > or = 10 mm Hg, requiring physician intervention. Two of these patients exhibited signs of pulmonary embolization postoperatively. All patients studied by TEE had detectable intracardiac emboli during femoral arthroplasty. The acute decreases in RVEF and increases in mean pulmonary artery pressures during hip arthroplasty, suggest a role for the embolization of bone marrow debris in the development of the "bone cement implantation syndrome." PMID- 8638794 TI - The role of the phrenic nerves in stress response in upper abdominal surgery. AB - Previous studies have failed to demonstrate a block of the endocrine response to upper abdominal surgery by thoracic epidural analgesia. To clarify the bases for this failure, we compared the effects of epidural analgesia of different dermatome levels up to C8-T2 or C3-4. The patients who received general anesthesia alone showed significant increases of adrenocorticotropic hormone (ACTH) and arginine vasopressin (AVP) immediately after skin incision. The patients with C8-T2 blocked developed significant increases in these hormones, not after the skin incision, but after the intraabdominal procedure. Of the eight patients with C3-4 block, six developed no such responses throughout the study period. The responses of oxytocin (OXT) and prolactin (PRL) were more susceptible to epidural analgesia and were blocked at the C8-T2 level. Growth hormone (GH) showed no correlation with surgical procedures and epidural block. These findings indicate that the nociceptive neural information during upper abdominal surgery is conveyed by the sensory fibers included in both the thoracic and lumbar spinal nerves that innervate the abdominal wall and the intraabdominal viscera, and by the phrenic nerves that innervate the diaphragm. The rationale for postulating the involvement of the phrenic nerves can be referred to the embryonal descent of the diaphragm from the C3-5 myotomes that serves as the upper wall of the abdominal cavity. PMID- 8638796 TI - In vitro diagnosis of malignant hyperthermia susceptibility with ryanodine induced contractures in human skeletal muscles. AB - The in vitro contracture test with ryanodine is a new method to distinguish malignant hyperthermia (MH) susceptible (MHS) from normal (MHN) patients. The purpose of our investigation was to determine whether smaller concentrations of ryanodine than those used previously may result in a better differentiation. We performed a ryanodine contracture test (RCT) using concentrations of 1 and 2 microM in muscle specimens of 41 MHS, 58 MHN, and 19 MH-equivocal (MHE) patients. Nine patients were excluded from the study due to neuromuscular diseases. All contracture levels (i.e., start of contractures, contractures of 0.2 g and 1.0 g) were attained significantly earlier in MHS than in MHN muscles at both concentrations of ryanodine. Using a ryanodine concentration of 2 microM, all contracture levels were reached significantly faster than with 1 microM. There was no overlap in the range of times between groups at all contracture levels with ryanodine 1 and 2 microM. The median threshold times for all MHE patients were always between those of MHS and MHN. Defining arbitrarily threshold times for MHS and MHN, an assignment of MHE patients to either MHS or MHN using 1 or 2 microM ryanodine was possible in most cases. Ryanodine administration at a concentration of 1 microM led to a better distinction of MHS from MHN patients than 2 microM. The RCT with ryanodine 1 microM should therefore be added to the current diagnostic methods. PMID- 8638797 TI - Alkaloid delta agonist BW373U86 increases hypoxic tolerance. AB - Activation of delta opioid receptors increases survival time during acute, lethal hypoxia in mice. delta Agonists therefore present a promising avenue for therapeutic application to reduce the morbidity and mortality associated with clinical hypoxia in settings such as drowning, head injury apnea, and complicated childbirths. However, most delta agonists now available are peptides, and may have limited clinical utility. In the present study, we evaluate the neuroprotective ability of an alkaloid delta agonist, BW373U86. Alkaloid compounds, due to increased stability and increased systemic distribution, may be more favorable for clinical use. We found that BW373U86, like the peptide delta agonist, DPDPE ([D-Pen2, D-Pen5]-enkephalin), increases survival time of mice during lethal hypoxia. The mechanism of neuroprotection induced by delta receptor activation appears to involve decreasing body temperature. Further, using selective opioid receptor antagonists, it appears that BW373U86 exerts these neuroprotective effects by acting at delta-opioid receptors. PMID- 8638799 TI - Human brain activity response to fentanyl imaged by positron emission tomography. AB - Positron emission tomography (PET) is a noninvasive imaging technique that can be used to observe drug actions on human brain in vivo. We used 15O-water PET scanning in six volunteers to examine the effects on regional cerebral activity as reflected by regional cerebral blood flow (rCBF) of a small intravenous bolus of fentanyl. rCBF was compared between scans obtained after fentanyl or a placebo using three separate statistical criteria including a pixel-by-pixel t statistic; significance was stringently defined at P values < 0.01. Anatomic locations of regional cerebral activity changes were verified by aligning rCBF PET scans with cranial magnetic resonance images using mathematical coregistration. Fentanyl administration was associated with significant increases in rCBF consistent with regional neuronal activation in both cingulate and orbitofrontal and medial prefrontal cortices, as well as caudate nuclei. These areas are responsive to nociceptive stimuli and are involved in avoidance learning, reward and addiction, visceromotor control, maintenance of attention, and pain-related affective behavior. Significant decreases were noted in both frontal and temporal areas and the cerebellum, a distribution far less extensive than that of opiate receptors in general. These data indicate that fentanyl's effects are highly localized and specifically affect cerebral regions associated with a range of pain-related behaviors. PMID- 8638798 TI - Inhibition of a fast inwardly rectifying potassium conductance by barbiturates. AB - Whole cell voltage clamp recordings were used to study the effects of two barbiturates, methohexital and pentobarbital, on inwardly rectifying K+ currents in the plasma membrane of a rat basophilic granulocyte cell line (RBL-1). Inwardly rectifying K+ currents are responsible for maintaining the resting membrane potential in a variety of cell types including skeletal and cardiac muscle, neurons, glia, blood cells, and endothelial cells. RBL-1 cells are unusual because the inward rectifier is the only apparent voltage-dependent current in these cells. Steps to command potentials between + 80 and -120 mV evoked only this strongly rectifying, rapidly developing current at membrane potentials more hyperpolarized than the reversal potential for K' ions. Extracellular Cs+ (10 mM) and Ba2+ (100 microM and 1 mM) blocked this current in a reversible and voltage-dependent manner. The voltage threshold for activation of the inwardly rectifying K+ current is dependent on the extracellular K+ concentration as predicted by the Nernst equation. Methohexital and pentobarbital reversibly inhibited the current in a concentration-dependent fashion with 50% inhibitory concentration (IC50) values of 145 microM and 218 microM respectively. The Hill slopes for both of these effects were approximately 1. The inhibition was not voltage dependent. These results indicate that fast inwardly rectifying K+ channels are potential molecular targets for barbiturates and could explain some of the diverse clinical effects of these drugs. PMID- 8638800 TI - Vecuronium-induced depression of phrenic nerve activity during hypoxia in the rabbit. AB - We investigated the effects of a close carotid injection of vecuronium on changes in phrenic nerve activity during different states of oxygenation. Experiments were performed on normothermic and mechanically ventilated adult New Zealand White rabbits with a tracheostomy under continuous thiopental infusion. Carotid arteries and carotid bodies were identified bilaterally followed by glomectomy on the contralateral side and positioning of a lingual artery catheter with its tip located in the carotid bifurcation on the ipsilateral side. From the dissected ipsilateral C-4 nerve root, changes in integrated phrenic nerve activity (IPA) were recorded after isocapnic step reductions from hyperoxia to normoxia (fraction of inspired oxygen [FIO2] 0.21, normoxic challenge) and from hyperoxia to moderate hypoxia (FIO2 0.15, hypoxic challenge) immediately after a close carotid bolus injection of either normal saline or 1 or 10 microgram of vecuronium. Arterial blood gases were analyzed during each ventilatory state. Phrenic nerve response to hypoxia expressed as chemosensitivity (Sch) was computed as the relative change in phrenic nerve activity per unit decrease in arterial oxygen saturation (Sao2), that is: Sch = (IPA challenge/IPA hyperoxia) - 1/Sao2 hyperoxia - Sao2 challenge. Hypoxic challenges were associated with reduced phrenic nerve response (Sch) after injection of 1 and 10 micrograms of vecuronium compared with normal saline. During normoxic challenges, no change occurred after 1 microgram, but reduced chemosensitivity was seen after 10 micrograms of vecuronium. We conclude that vecuronium depresses phrenic nerve activity during hypoxia. PMID- 8638801 TI - The effect of stabilization on the onset of neuromuscular block when assessed using accelerometry. AB - Accelerometry is increasingly being used for neuromuscular monitoring. We sought to determine whether this system is sensitive to the period of stabilization of muscle twitch prior to the administration of neuromuscular relaxant. We recruited 20 patients. No premedication was given, and anesthesia was induced with propofol and alfentanil and maintained by a propofol infusion. An accelerometer was attached to each wrist. One of the ulnar nerves was stimulated for 20 min and the other for 3 min using a train-of-four pattern at 15-s intervals. Ten patients then received vecuronium 0.1 mg/kg and a subsequent 10 received atracurium 0.5 mg/kg. The time to onset of maximum block was recorded. The data collected was subjected to a paired t-test with P < 0.05 taken as significant. The mean onset times for patients who received vecuronium was 148.5s for the arms stabilized for 3 min and 151.5s for the arms stabilized for 20 min, and in those who received atracurium it was 138.0s and 130.5s, respectively. We conclude that there is no significant difference in the onset of neuromuscular block with either vecuronium or atracurium after stabilization periods of 3 or 20 min when assessed by accelerometry. PMID- 8638802 TI - Isoflurane preserves adenosine triphosphate levels in anoxic isolated rat hepatocytes by stimulating glycolytic adenosine triphosphate formation. AB - The hypothesis that general anesthetics protect energy reserves by decreasing energy demand is widely accepted but poorly substantiated. Isoflurane at clinical doses preserved adenosine triphosphate (ATP) levels in anoxic isolated hepatocytes. Specific inhibitors were used to block mitochondrial and/or glycolytic ATP formation to ascertain whether pathways of energy supply or demand, or both, were involved in ATP preservation by isoflurane. Hepatocytes were isolated from fed adult male rats after perfusing livers with Krebs buffer containing collagenase. Cells were incubated in Krebs buffer for 0-30 min at 25 degrees C under N2/CO2 (95%/5%) +/- isoflurane 0.63 mM in liquid phase. Oligomycin, iodoacetate, or fasting were used to block mitochondrial and glycolytic ATP formation. Under anoxia alone, ATP levels declined more slowly in the presence than in the absence of isoflurane, confirming the ATP-protective effect of isoflurane reported previously. With oligomycin plus iodoacetate blocking all ATP formation, ATP decline (representing pure ATP consumption) was not slowed by isoflurane. Isoflurane's protective effect recurred when glycolytic ATP supply was restored by incubating with oligomycin only. The protective effect was accompanied by increased lactate accumulation, and both effects-ATP preservation and lactate formation-were similarly dependent on isoflurane concentration. We conclude that the protective effect of isoflurane on energy status in anoxic isolated hepatocytes was not associated with reduced ATP demand but with enhanced ATP supply via stimulation of glycolysis. PMID- 8638803 TI - Sevoflurane versus isoflurane for maintenance of anesthesia: are serum inorganic fluoride ion concentrations of concern? AB - Sevoflurane administration can result in increased serum inorganic fluoride ion concentrations, which have been associated with inhibition of renal concentrating ability. We measured serum fluoride levels, renal function, and recovery variables as a function of time in ASA grade I-III patients administered general anesthesia with isoflurane or sevoflurane for at least 1 h. Fifty patients were exposed to sevoflurane (< or = 2.4% inspired concentration) or isoflurane (< or = 1.9% inspired concentration) for maintenance of anesthesia as part of a multicenter trial. Blood was collected for determination of serum fluoride ion concentration, electrolytes, blood urea nitrogen, and creatinine at various time points pre- and postoperatively. Mean serum fluoride levels were significantly increased in sevoflurane versus isoflurane groups at all time points; the mean peak serum levels were 28.2 +/- 14 mumol/L at 1 h for sevoflurane and 5.08 +/- 4.35 mumol/L at 12 h for isoflurane. Sevoflurane-mediated increases in serum fluoride levels peaked at 1 h and, in general, decreased rapidly after discontinuation of the anesthesia. Three of 24 patients exposed to sevoflurane had one or more fluoride levels > 50 mumol/L. One of these patients had a serum inorganic fluoride ion level > 50 mumol/L at 12 h after sevoflurane, and an additional patient had fluoride levels > 33 mumol/L for up to 24 h after sevoflurane discontinuation. Those two patients also demonstrated an increase in serum blood urea nitrogen and creatinine at 24 h after sevoflurane administration compared with baseline. The elimination half-life of serum fluoride ion was 21.6 h. The results of this study suggest the possibility of sevoflurane induced nephrotoxicity. PMID- 8638804 TI - Anesthesia providers, patient outcomes, and costs. PMID- 8638805 TI - Telephone reporting of blood analysis results into the operating room. PMID- 8638806 TI - Fiberoptic-guided endotracheal intubation via the laryngeal mask airway in pediatric patients: a report of a series of cases. PMID- 8638807 TI - A modification of retrograde wire-guided, fiberoptic-assisted endotracheal intubation in a patient with ankylosing spondylitis. PMID- 8638808 TI - Single-lung ventilation in a patient with a freshly placed percutaneous tracheostomy. PMID- 8638809 TI - Failure of steroid supplementation to prevent operative hypotension in a patient receiving chronic steroid therapy. PMID- 8638810 TI - Meningitis after injection of intrathecal baclofen. PMID- 8638811 TI - Quantitative electroencephalography for detection of cerebral ischemia. PMID- 8638812 TI - TEE and ascending aortic atherosclerosis. PMID- 8638813 TI - Sodium nitroprusside metabolism in children. PMID- 8638814 TI - Simple technique to remove laryngeal mask airway "guide" after endotracheal intubation. PMID- 8638815 TI - Long-term postdural puncture auditory symptoms: effective relief after epidural blood patch. PMID- 8638816 TI - A simple and reliable method of subcutaneous tunneling of epidural catheters. PMID- 8638817 TI - Efficacy of epidural test doses? PMID- 8638818 TI - Procoagulant action of aprotinin. PMID- 8638819 TI - Factors affecting the production of carbon monoxide in inhalation anesthetic use. PMID- 8638820 TI - Reducing the pain on injection of propofol. PMID- 8638821 TI - Bronchoscopy and occlusion of tracheoesophageal fistula. PMID- 8638822 TI - Assessment of neuromuscular block: aspects of stimulation. PMID- 8638823 TI - Cerebral oxygenation during hypothermic cardiopulmonary bypass: clinical findings support mathematical model. PMID- 8638824 TI - Laryngeal mask airway for access to the upper gastrointestinal tract. PMID- 8638825 TI - New video system improves teaching of direct laryngoscopy. PMID- 8638826 TI - Alveolar oxygen partial pressure, alveolar carbon dioxide partial pressure, and the alveolar gas equation. PMID- 8638827 TI - Annual meeting of the Society of Neurosurgical Anesthesia and Critical Care. Atlanta, Georgia, October 20, 1995. PMID- 8638828 TI - Nosocomial infection in the critically ill: the lung as a target organ. PMID- 8638829 TI - Histopathologic and microbiologic aspects of ventilator-associated pneumonia. AB - BACKGROUND: The relationship between microbiology and histology in patients with ventilator-associated pneumonia has been sparsely described. METHODS: Twenty-five patients who died in the intensive care unit after their lungs had been mechanically ventilated for 72 h were studied. Twenty of the 25 died with clinical suspicion of pulmonary infection. A total of 375 immediate postmortem pulmonary biopsies were obtained after death and processed for quantitative microbiology and histology. Four evolutionary stages of pneumonia were defined: early, intermediate, advanced, and resolution. RESULTS: At least one specimen with histologic evidence of pneumonia was found in all but two patients (92%). Histologic pneumonia was a widespread and frequent process (46%) of biopsies examined) involving predominantly the lower lobes (55% of all biopsies with pneumonia) and showing different histopathologic stages of progression coexisting in the same lung lobes. Lung cultures were frequently polymicrobial (149 of 375, 40% of the pulmonary biopsy cultures, and 20 of 25, 80% of the cases) and not always yielding the same pathogen (19 microorganisms) when comparing one lung to the other. Histopathology and microbiologic biopsy cultures showed a weak relationship (28% and 49% of species had counts > or = 10(3) cfu/g in samples without pneumonia from patients with and without prior antibiotic treatment, respectively). Histopathologic evolutionary stages were not associated with any differences in quantitative culture results of pulmonary biopsies, independently of prior administration of antibiotics. Higher bacterial concentrations of biopsy cultures were associated with the absence of prior antibiotic treatment. CONCLUSIONS: Ventilator-associated pneumonia is a frequent diffuse and polymicrobial process showing different coexisting degrees of evolution and involving preferentially the lower lobes. Microbiology and histology can be dissociated even in the absence of prior antibiotic treatment. Lung histology appears more reliable than bacteriology as a diagnostic reference test. PMID- 8638830 TI - Cardiovascular events in the postanesthesia care unit: contribution of risk factors. AB - BACKGROUND: The purpose of this study was to determine the relationship of four postanesthesia care unit (PACU) cardiovascular events to long-term outcomes (unplanned critical care admission or mortality) and to evaluate the contribution of anesthetic management compared with other perioperative factors in predicting these events. METHODS: For patients admitted to the PACU after receiving general anesthesia (n = 18,380), the risk of long-term outcomes was examined for patients in the PACU with hypertension, tachycardia, bradycardia, or hypotension. Using logistic regression (P < 0.05), risk factors (grouped as patients, surgical, anesthetic, operating room observations, and other PACU observations) for each cardiovascular event were determined. For each factor grouping, the relative contributions to each cardiovascular event were compared using maximum likelihood chi-square analysis. RESULTS: Patients in the PACU with hypertension or tachycardia had more unplanned critical care admissions (2.6% and 4.0% vs. 0.2% for patients with no events) and greater mortality (1.9% and 2.3% vs. 0.3% and 0.4%) (P < 0.01). For PACU hypertension (rate 2.0%), age, smoking, renal disease, female gender, and angina were significant risk factors. For PACU tachycardia (0.9%), intraoperative tachycardia and dysrhythmia were the major contributors. Patient factors also increased the risk of bradycardia (2.5%); namely age, ASA physical status 1 or 2, and preoperative beta blocker therapy. For hypotension (2.2%), duration of surgery > 2 h, completion after 6 PM, and gynecologic intraabdominal procedures were significant risk factors. Compared to patient, surgical, intraoperative, or PACU observations, anesthetic factors studied (premedication, induction agent, ventilation, use of opioids) provided only a small contribution in predicting these events. CONCLUSIONS: Hypertension and tachycardia in the PACU, although infrequent, are associated with increased risk of unplanned critical care admission and mortality. Patient, surgical, intraoperative, or PACU observations contribute more to cardiovascular events in the PACU than do differences in anesthetic management identified in this study. PMID- 8638831 TI - Interaction of isoflurane and nitrous oxide combinations similar for median electroencephalographic frequency and clinical anesthesia. AB - BACKGROUND: The volatile anesthetic sparing effect of nitrous oxide in clinical studies is less than might be expected from the additivity of minimum alveolar concentration values. Other studies identify nonadditive interactions between isoflurane and nitrous oxide. The aim of this study was to quantify the interaction of isoflurane and nitrous oxide at a constant median electroencephalographic frequency. METHODS: Twenty-five patients were studied during laparotomies. Nitrous oxide was randomly administered in concentrations of 0, 20, 40, 60, and 75 vol%, to ten patients for each nitrous oxide concentration. Isoflurane vaporizer settings were chosen so that the median electroencephalographic frequency was held between 2 and 3 Hz. The relationship between nitrous oxide concentrations and required isoflurane concentrations was examined with the method of isoboles. RESULTS: Nitrous oxide linearly decreased the isoflurane requirement. Addition of every 10 vol% of nitrous oxide decreases the isoflurane requirement by approximately 0.04 vol%. The total anesthetic requirement of isoflurane and nitrous oxide, expressed in terms of previously reported minimum alveolar concentration values, increased significantly with increasing nitrous oxide concentrations. CONCLUSIONS: The interaction of isoflurane and nitrous oxide in the dose range 0-75 vol% on median electroencephalographic frequency is compatible with additivity. The potency of nitrous oxide as a substitute for isoflurane is less than on a minimum alveolar concentration basis. Maintaining median electroencephalographic frequency more appropriately reflects the clinical usage of isoflurane and nitrous oxide than does maintaining minimum alveolar concentration. PMID- 8638832 TI - Long-term angiotensin-converting enzyme inhibitor treatment attenuates adrenergic responsiveness without altering hemodynamic control in patients undergoing cardiac surgery. AB - BACKGROUND: The sympathoadrenal and the renin-angiotensin systems are involved in blood pressure regulation and are known to be markedly activated during cardiac surgery. Because unexpected hypotensive events have been reported repeatedly during anesthesia in patients chronically treated with angiotensin-converting enzyme (ACE) inhibitors, the authors questioned whether renin-angiotensin system blockade would alter the hemodynamic control through attenuation of the endocrine response to surgery and/or through attenuation of the pressor effects of exogenous catecholamines. METHODS: Patients with preserved left ventricular function undergoing mitral valve replacement or coronary revascularization were divided into two groups according to preoperative drug therapy: patients receiving ACE inhibitors for at least 3 months (ACEI) group, n = 22) and those receiving other cardiovascular drug therapy (control group, n = 19). Anesthesia was induced using fentanyl and midazolam. Systemic hemodynamic variables were recorded before surgery, after anesthesia induction, during sternotomy, after aortic cross-clamping, after aortic unclamping, as well as after separation from cardiopulmonary bypass (CPB) and during skin closure. Blood was sampled repeatedly up to 24 h after surgery for hormone analysis. To test adrenergic responsiveness, incremental doses of norepinephrine were infused intravenously during hypothermic CPB and after separation from CPB. From the dose-response curves, pressor (defined as mean arterial pressure changes), and vasoconstrictor (defined as systemic vascular resistance changes) effects were analyzed, and the slopes and the dose of norepinephrine required to increase mean arterial pressure by 20% were calculated (PD(20)). RESULTS: At no time did the systemic hemodynamics and the need for vasopressor support differ between the two treatment groups. However, for anesthesia induction, significantly less fentanyl and midazolam were given in the ACEI group. Although plasma renin activity was significantly greater in the ACEI group throughout the whole 24-h study period, plasma concentrations of angiotensin II did not differ between the two groups. Similar changes in catecholamines angiotensin II, and plasma renin activity were found in the two groups in response to surgery and CPB. The pressor and constrictor effects of norepinephrine infusion were attenuated markedly in the ACEI group: the dose-response curves were shifted to the right and the slopes were decreased at the two study periods; PD(20) was significantly greater during hypothermic CPB (0.08 micro/kg in the ACEI group vs. 0.03 micro/kg in the control group; P < 0.05) and after separation from CPB (0.52 micro/kg in the ACEI group vs. 0.1 micro/kg in the control group; P < 0.05). In both groups, PD(20) was significantly less during hypothermic CPB than in the period immediately after CPB. CONCLUSIONS: Long-term ACE inhibitor treatment in patients with preserved left ventricular function alters neither the endocrine response nor the hemodynamic stability during cardiac surgery. However, a significantly attenuated adrenergic responsiveness associated with incomplete blockade of the plasma renin angiotensin system supports the hypothesis that inhibition of angiotensin II generation and of bradykinin degradation within the vascular wall mediates some of the vasodilatory effects of ACE inhibitors. PMID- 8638834 TI - Laryngeal mask airway in pediatric practice: a prospective study of skill acquisition by anesthesia residents. AB - BACKGROUND: A prospective study was conducted to determine the rate of skill acquisition with the laryngeal mask airway in pediatric anesthesiology practice. The aim of the study was to provide information about the amount of supervised training required before satisfactory levels of skill were achieved. METHODS: Eight anesthesia residents in their third year of training with no prior experience using the laryngeal mask airway were observed using the device in 75 pediatric patients each (600 patients in total). Residents were given standardized guidelines for laryngeal mask airway usage in accordance with the manufacturer's recommendations and followed a predetermined protocol for anesthetic management. Induction was achieved with propofol followed by either a propofol infusion or isoflurane and either controlled or spontaneous ventilation as clinically indicated. Predefined major and minor problems were documented during the induction, maintenance, and recovery phases of anesthesia by a randomly selected supervising consultant trained in the study protocol and problem definitions. RESULTS: The total number of problems was 189 occurring in 121 children. Fifty-five children had one problem, sixty-four children had two problems, and two children had three problems. Of the problems, 77 were major and 112 were minor. The problem rate per patient for overall, major, and minor problems was 31.5%, 12.8%, and 18.7%, respectively. The problem rate comparing the first to last epochs of 15 uses decreased from 62% to 2% for overall problems, 23% to 2% for major problems, and 39 to 1% for minor problems. The residents with the most problems in the final epoch had problem rates of less than 10% after 60 uses. There was a significant decrease in the overall problem rate for induction, maintenance, and recovery (P < 0.05). The major problem rate decreased significantly for induction and maintenance (p < 0.05), but not for recovery. The minor problem rate decreased significantly for induction and recovery (P < 0.05). CONCLUSIONS: This study confirms that there is a rapid improvement in laryngeal mask airway skills when the standard recommended technique is employed and that a low problem rate can be achieved within 75 uses. Pediatric anesthesiologists with problem rates greater than 10% should determine if they are using the device suboptimally. PMID- 8638833 TI - Randomized, single-blinded trial of laparoscopic versus open appendectomy in children: effects on postoperative analgesia. AB - BACKGROUND: The benefit of laparoscopy to patients has been clearly established in adults undergoing cholecystectomy. Results are less clear for appendectomy. The current study was undertaken to compare the respective 3-day postoperative periods after laparoscopic and open appendectomy in children. METHODS: Sixty three children (aged 8-15 yr) scheduled for appendectomy were randomly assigned to two groups: open and laparoscopic. Postoperative evaluation included delay of postoperative recovery (walking and feeding), pain assessment by visual analog scale during the 3 subsequent days, amount of nalbuphine administered via a patient-controlled analgesia system during the first 48 h and responses by children, parents, and nurses on the overall quality of analgesia. RESULTS: There was no difference between groups for demographic data (particularly macroscopic aspect of appendix) analgesia, sedation, delay before eating and walking, incidence of urinary retention, nausea, vomiting. Operative time was long (P < or = to 0.05) in the laparoscopic group (54 +/- 17 min) than in the open group (39 +/- 18 min). Thirty five percent of the children had pain at the shoulder in the LAP group versus ten percent in the open group (P < or = 0.05). CONCLUSIONS: Laparoscopy did not improve analgesia and postoperative recovery after appendectomy in children. PMID- 8638835 TI - Pharmacokinetics and pharmacodynamics of remifentanil in volunteer subjects with severe liver disease. AB - BACKGROUND: Remifentanil, a new mu-opioid agonist with an extremely short duration of action, is metabolized by circulating and tissue esterases; therefore, its clearance should be relatively unaffected by changes in hepatic or renal function. This study was designed to determine whether severe hepatic disease affects the pharmacokinetics or pharmacodynamics of remifentanil. METHODS: Ten volunteers with chronic, stable, severe hepatic disease and awaiting liver transplantation and ten matched controls were enrolled. Each subject was given a 4-h infusion of remifentanil. The first five pairs received 0.0125 microgram x kg(-1) x min(-1) for 1 h followed by 0.025 microgram x kg(-1) x min( 1) for 3 h; the second five pairs received double these infusion rates. During and after the infusion, arterial blood was obtained for pharmacokinetic analyses, and the ventilatory response to a hypercarbic challenge was assessed. Simultaneous pharmacokinetic and pharmacodynamic analyses were performed. The pharmacokinetics were described using a one-compartment intravenous infusion model, and ventilatory depression was modelled using the inhibitory E(max) model. The pharmacokinetics of the metabolite GR90291 were determined using noncompartmental methods. RESULTS: There were no differences in any of the pharmacokinetic parameters for remifentanil or GR90291 between the two groups. The subjects with liver disease were more sensitive to the ventilatory depressant effects of remifentanil. The EC(50) values (the remifentanil concentrations determined from simultaneous pharmacokinetic/pharmacodynamic analyses to depress carbon dioxide-stimulated minute ventilation by 50%) in the control and hepatic disease groups were 2.52 ng/ml (95% confidence interval 2.07-2.97 ng/ml) and 1.56 ng/ml (95% confidence interval 1.37-1.76 ng/ml), respectively. CONCLUSIONS: The pharmacokinetics of remifentanil and GR90291 are unchanged in persons with severe, chronic liver disease. Such patients may be more sensitive to the ventilatory depressant effects of remifentanil, a finding of uncertain clinical significance, considering the extremely short duration of action of the drug. PMID- 8638837 TI - Pharmacokinetics and analgesic effect of ropivacaine during continuous epidural infusion for postoperative pain relief. AB - BACKGROUND: The pharmacokinetics and clinical efficacy of ropivacaine (2.5 mg/ml) during a 24-h continuous epidural infusion for postoperative pain relief in 20 patients scheduled for abdominal hysterectomy were characterized using an open label, increasing-dose design. METHODS: Through an epidural catheter inserted at T10-T12, a test dose of 7.5 mg ropivacaine was given 3 min before a bolus dose of 42.5 mg and immediately followed by a 24-h continuous epidural infusion with either 10 or 20 mg/h. Peripheral venous plasma samples were collected up to 48 h after infusion, and urinary excretion was followed up to the end of infusion. Postoperative pain at rest, on coughing, and at mobilization was assessed by means of a visual analog scale 2,4,6,8,12, and 24 h after the end of surgery. Sensory (pinprick) and motor block (modified Bromage scale) were assessed at the same intervals. RESULTS: The total plasma concentrations of ropivacaine increased markedly and consistently during the 24-h epidural infusion, in contrast to stable unbound concentrations. Both total and unbound plasma concentrations at the end of infusion were proportional to the total dose, although only the latter was proportional to the infusion rate. The total and unbound plasma clearance was independent of dose. Total mean clearance decreased on average by 21% (P < 0.001) during the last 12 h of epidural infusion, i.e., from 539 +/- 191 ml/min to 418 +/- 138 ml/min, indicating time-dependent kinetics. The unbound clearance also varied between estimates after 8 h of infusion and the end of treatment, i.e., a 5.3% decrease from 10.4 +/- 5.3 l/min to 9.5 +/- 3.9 l/min (P < 0.05). The unbound fraction of ropivacaine in plasma decreased during treatment, and this was related to the increase in alpha1-acid glycoprotein concentration. Pain was generally well controlled, and median visual analog scale scores during mobilization were less than 30 mm in patients receiving ropivacaine at 20 mg/h. CONCLUSIONS: The pharmacokinetics of ropivacaine were independent of dose, but total clearance decreased with time over 24 h. The consistent increase in total plasma concentration during the postoperative epidural infusion contrasted to much less variation in the unbound plasma concentrations of ropivacaine. PMID- 8638836 TI - Remifentanil versus alfentanil: comparative pharmacokinetics and pharmacodynamics in healthy adult male volunteers. AB - BACKGROUND: Remifentanil is an esterase-metabolized opioid with a rapid clearance. The aim of this study was to contrast the pharmacokinetics and pharmacodynamics of remifentanil and alfentanil in healthy, adult male volunteers. METHODS: Ten volunteers received infusions of remifentanil and alfentanil on separate study sessions using a randomized, open-label crossover design. Arterial blood samples were analyzed to determine drug blood concentrations. The electroencephalogram was employed as the measure of drug effect. The pharmacokinetics were characterized using a moment analysis, a nonlinear mixed effects model (NONMEM) population analysis, and context-sensitive half-time computer simulations. After processing the raw electroencephalogram to obtain the spectral edge parameter, the pharmacodynamics were characterized using an effect compartment, inhibitory maximum effect model. RESULTS: Pharmacokinetically, the two drugs are similar in terms of steady-state distribution volume (VD(SS)), but remifentanil's central clearance (CLc)) is substantially greater. The NONMEM analysis population pharmacokinetic parameters for remifentanil include a CLc of 2.9 l x min(-1), a VDss of 21.81, and a terminal half-life of 35.1 min. Corresponding NONMEM parameters for alfentanil are 0.36 l x min(-1), 34.11, and 94.5 min. Pharmacodynamically, the drugs are similar in terms of the time required for equilibration between blood and the effect-site concentrations, as evidenced by a T(12)k(e0) for remifentanil of 0.75 min [corrected] and 0.96 min for alfentanil. However, remifentanil is 19 times more potent than alfentanil, with an effective concentration for 50% maximal effect of 19.9 ng x ml(-1) versus 375.9 ng x ml(-1) for alfentanil. CONCLUSIONS: Compared to alfentanil, the high clearance of remifentanil, combined with its small steady-state distribution volume, results in a rapid decline in blood concentration after termination of an infusion. With the exception of remifentanil's nearly 20-times greater potency (30-times if alfentanil partitioning between whole blood and plasma is considered), the drugs are pharmacodynamically similar. PMID- 8638839 TI - Pharmacokinetics of dichloroacetate in patients undergoing liver transplantation. AB - BACKGROUND: Dichloroacetate (DCA) is an effective alternative to bicarbonate to treat lactic acidosis and stabilize acid-base homeostasis during liver transplantation. Although DCA presumably is metabolized by the liver, the impact of end-stage liver disease and liver transplantation on DCA distribution and elimination is unknown. Therefore, the pharmacokinetics of DCA were determined in patients with end-stage liver disease undergoing orthotopic liver transplantation. METHODS: Thirty-three patients undergoing liver transplantation were given DCA as two 40-mg/kg infusions over 60 min, the first after induction of anesthesia, the second 4 h later. Plasma DCA concentrations were determined by gas chromatography-mass spectroscopy. One- and two-compartment pharmacokinetic models were fitted to DCA concentrations versus time data using a mixed-effects population approach. Various models permitted changes in central compartment volume and/or plasma clearance to account for changes in hepatic mass and function and circulatory status during the paleohepatic, anhepatic, and neohepatic periods. RESULTS: The optimal model had two compartments. DCA clearance was 0.997, 0.0, and 1.69 ml x kg(-1) x min(-1) during the paleohepatic, anhepatic, and neohepatic periods, respectively (P < 0.05). Interindividual variability in central compartment volume differed during the paleohepatic and neohepatic periods. There was no apparent influence of blood or fluid requirements during surgery on DCA clearance or volume of distribution. CONCLUSIONS: Absence of DCA clearance during the anhepatic period indicates that DCA is metabolized exclusively by the liver. Differences in interindividual variability in central compartment volume during the paleohepatic and neohepatic periods possibly result from physiologic changes during surgery. Finally, the results indicate that the newly transplanted liver eliminates DCA better than the native liver. PMID- 8638838 TI - Stellate ganglion block is associated with increased tibial nerve muscle sympathetic activity in humans. AB - BACKGROUND: Left stellate ganglion block has been shown to increase heart rate and blood pressure, possible because of blockage of afferent vagal fibers from arterial baroreceptors in the aortic arch. Because efferent muscle sympathetic nerve activity (MSNA) is influenced by the arterial baroreflex, the hypothesis that left stellate ganglion block increases efferent MSNA recorded from the tibial nerve of humans was tested. METHODS: Twenty healthy male volunteers were sequentially assigned to one of three groups: stellate ganglion block (n = 10), in which 7 ml 1% mepivacaine was injected into the left stellate ganglion; placebo (n = 5), in which 7 ml of saline was injected into the left stellate ganglion; and intramuscular injection (n = 5), in which 7 ml mepivacaine was injected into the left deltoid muscle. Direct intraneural microneurographic recording with a tungsten microelectrode was used to record MSNA in the left tibial nerve. MSNA, heart rate, and blood pressure were recorded before and after injection in all groups. An additional five volunteers were studied with transthoracic echocardiography to examine the effect of stellate ganglion block on preload changes. RESULTS: Tibial nerve MSNA increased after mepivacaine injection to the left stellate ganglion but was unchanged after saline injection to the left stellate ganglion or mepivacaine injection into the deltoid muscle. Heart rate increased significantly after the left stellate ganglion block but did not change significantly after saline injection to the left stellate ganglion or after mepivacaine injection to the deltoid muscle. Systemic blood pressure did not change significantly in all groups. Left ventricular end-diastolic area and left ventricular end-diastolic circumference did not change after stellate ganglion block. CONCLUSIONS: Tibial nerve MSNA increased during left stellate ganglion block with mepivacaine. PMID- 8638840 TI - Predictors of pulse oximetry data failure. AB - BACKGROUND: Pulse oximeters have been reported to fail to record data in 1.12 2.50% of cases in which anesthesia records were handwritten. There is reason to believe that these may be underestimates. Computerized anesthesia records may provide insight into the true incidence of pulse oximetry data failures and factors that are associated with such failures. METHODS: The current study reviewed case files of 9,203 computerized anesthesia records. Pulse oximetry data failure was defined as the presence of at least one continuous gap in data > or = 10 min in duration in a case. A multivariate logistic regression model was used to identify predictors of pulse oximetry data failure, and a modified case control method was used to determine whether extremes of blood pressure and hypothermia during the procedure were associated with pulse oximetry data failure. RESULTS: The overall incidence of cases that had at least one continuous gap of > or = 10 min in pulse oximetry data was 9.18%. The independent preoperative predictors of pulse oximetry data failure were ASA physical status 3,4, or 5 and orthopedic, vascular, and cardiac surgery. Intraoperative hypothermia, hypotension, hypertension, and duration of procedure were also independent risk factors for pulse oximetry data failure. CONCLUSIONS: Pulse oximetry data failure rates based on review of computerized records were markedly greater than those previously reported. Physical status, type of surgery, and intraoperative variables were risk factors for pulse oximetry data failure. Regulations and expectations regarding pulse oximetry monitoring should reflect the limitations of the technology. PMID- 8638841 TI - Anesthetic potency of remifentanil in dogs. AB - BACKGROUND: Remifentanil is an opioid that is rapidly inactivated by esterases in blood and tissues. This study examined the anesthetic potency and efficacy of remifentanil in terms of its reduction of enflurane minimum alveolar concentration (MAC) in dogs. METHODS: Twenty-five dogs were anesthetized with enflurane. One group received incremental infusion rates of remifentanil from 0.055 to 5.5 micrograms x kg(-1). A second group received constant rate infusions of remifentanil of 1.0 micrograms x kg(-1) x min(-1) for 6-8 h. Enflurane MAC was measured before, hourly during remifentanil infusion, and at the end of the experiment after naloxone administration. A third group received alternating infusions of 0.5 and 1.0 micrograms x kg(-1) x min(-1) with MAC determinations made 30 min after each change in the infusion rate. Heart rate, mean arterial pressure, and remifentanil blood concentrations were measured during MAC determinations. RESULTS: Enflurane MAC was reduced up to a maximum of 63 +/- 10.4% (mean +/- SD) in a dose-dependent manner by remifentanil infusion. The dose producing a 50% reduction in the enflurane MAC was calculated as 0.72 micrograms x kg(-1)x min(-1) and the corresponding blood concentration was calculated as 9.2 ng/ml. Enflurane MAC reduction remained stable during continuous, constant rate infusions for periods of 6-8 h without any signs of tolerance. Recovery of enflurane MAC to baseline occurred in 30 min (earliest measurement) after stopping the remifentanil infusion. CONCLUSIONS: Remifentanil is equally efficacious and about half as potent as fentanyl, judging from the blood concentrations causing equivalent reductions in enflurane MAC in the dog. The characteristics of MAC reduction are similar to those of other opioids, including the ceiling effect. Recovery from remifentanil anesthesia is much more rapid than for any other opioid studied to date, especially after continuous infusions maintained for 6 or more h. PMID- 8638842 TI - Dexmedetomidine injection into the locus ceruleus produces antinociception. AB - BACKGROUND: Alpha(2)-Adrenergic agonists such as clonidine and dexmedetomidine are known to produce sedation and analgesia in humans. The sedative effect of these agents is thought to occur through supraspinal pathways, involving the locus ceruleus (LC) and its projections in rats. While the antinociceptive response to alpha(2) agonists, given intrathecally, is mediated predominantly in the spinal cord, other sites of action have not been systematically studied. The authors examined whether alpha(2)-adrenergic receptors in the LC mediate an antinociceptive effect. METHODS: For administration of different drugs into the LC, guide cannulas were placed with their tips in the LC in male Sprague-Dawley rats. Dexmedetomidine (3.5 micrograms/0.2 microliter) was microinjected into the LC through the cannula, or given systemically by intraperitoneal injecton (50 micrograms/kg). The antinociceptive effect of dexmedetomidine was measured using the tail-flick latency response. To determine the sites through which dexmedetomidine injection into the LC produces antinociception, the authors examined whether this response could be perturbed by the specific alpha(2) adrenergic antagonists atipamezole and L659,066 and pertussis toxin administered either into the LC or intrathecally before injection of dexmedetomidine systemically or directly into the LC. To eliminate the possibility that drug administered in one site (LC or intrathecal) could reach the other site, the dispositional characteristics of radiolabeled dexmedetomidine (LC) or atipamezole (intrathecal) were studied. RESULTS: Dexmedetomidine placed into the LC produces a dose-dependent increase in the tail-flick latency. This antinociceptive effect was blocked by pertussis toxin and by the alpha(2) antagonists atipamezole and L659,066 placed in the LC. Intrathecal administration of atipamezole and pertussis toxin also blocked the antinociceptive effect of dexmedetomidine placed in the LC. (3)H-dexmedetomidine introduced into the LC did not reach the spinal cord in pharmacologically active concentrations; also, intrathecally administered (3)H-atipamezole did not reach the LC in appreciable amounts. The systemic administration of dexmedetomidine produced an increase in tail-flick latency, and this effect was attenuated by the injection of atipamezole and L659,066 into the LC. CONCLUSIONS: Part of the mechanism by which dexmedetomidine produces an antinociceptive effect is by an action directly on the LC, demonstrated by these studies in which antinociception produced by injection of this drug into the LC can be blocked by specific alpha(2) antagonists injected into the LC. Furthermore, the action of dexmedetomidine in the LC in turn may result in an increase in activation of alpha(2) adrenoceptors in the spinal cord, because the antinociceptive effect of LC dexmedetomidine injection also can be blocked by intrathecal injection of antipamezole and pertussis toxin. PMID- 8638843 TI - Ventilation with constant versus decelerating inspiratory flow in experimentally induced acute respiratory failure. AB - BACKGROUND: Recognition of the potential for ventilator-associated lung injury has renewed the debate on the importance of the inspiratory flow pattern. The aim of this study was to determine whether a ventilatory pattern with decelerating inspiratory flow, with the major part of the tidal volume delivered early, would increase functional residual capacity at unchanged (or even reduced) inspiratory airway pressures and improve gas exchange at different positive end-expiratory pressure levels. METHODS: Surfactant depletion was induced by repeated bronchoalveolar lavage in 13 anesthetized piglets. Decelerating and constant inspiratory flow ventilation was applied at positive end-expiratory pressure levels of 22, 17, 13, 9, and 4 cm H(2)O. Tidal volume, inspiration-to-expiration ratio, and ventilatory frequency were kept constant. Airway pressures, gas exchange, functional residual capacity (using a wash-in/washout method with sulfurhexafluoride), central hemodynamics, and extravascular lung water (using the thermo-dye-indicator dilution technique) were measured. RESULTS: Decelerating inspiratory flow yielded a lower arterial carbon dioxide tension compared to constant flow, that is, it improved alveolar ventilation. There were no differences between the flow patterns regarding end-inspiratory occlusion airway pressure, end-inspiratory lung volume, static compliance, or arterial oxygen tension. No differences were seen in hemodynamics and oxygen delivery. CONCLUSIONS: The decelerating inspiratory flow pattern increased carbon dioxide elimination, without any reduction of inspiratory airway pressure or apparent improvement in arterial oxygen tension. It remains to be established whether these differences are sufficiently pronounced to justify therapeutic consideration. PMID- 8638844 TI - Location and characteristics of nitric oxide synthase in sheep spinal cord and its interaction with alpha(2)-adrenergic and cholinergic antinociception. AB - BACKGROUND: Nitric oxide synthase is located in the spinal cord dorsal horn and intermediolateral cell column, where it may modulate sensory and sympathetic neuronal activity. However, the biochemical characteristics of this enzyme have not been examined in these different areas in the spinal cord. Although alpha(2) adrenergic agonists, muscarinic agonists, and nitric oxide may interact in the spinal cord to produce antinociception, these interactions have not been characterized. METHODS: Sheep spinal cord tissue was homogenized ad centrifuged at high sped to separate soluble and membrane-bound fractions. Nitric oxide synthase activity was determined by conversion of [(14)C]-L-arginine to [(14)C]-L citrulline and its kinetic characteristics, dependency on cofactors, and sensitivity to inhibitors determined. Sheep spinal cord was stained for nicotinamide adenine dinucleotide phosphate diaphorase as a marker for nitric oxide synthase. Antinociception to a mechanical stimulus from intrathecal clonidine alone and with neostigmine was determined and the effects of L-arginine and n-methyl-L-arginine were determined. RESULTS: More than 85% of nitric oxide synthase activity was present in the soluble form and its kinetic, cofactor, and antagonist properties were similar to those of the neuronal isoform of nitric oxide synthase. Biochemical and histochemical studies localized nitric oxide synthase to the superficial dorsal horn and the intermediolateral cell column. Clonidine antinociception was enhanced by L-arginine and neostigmine, but not by D-arginine. Neostigmine's enhancement of clonidine antinociception was blocked by n-methyl-L-arginine. CONCLUSIONS: These results confirm those of previous studies demonstrating localization of nitric oxide synthase to superficial dorsal horn and intermediolateral cell column of mammalian spinal cord, and suggesting its identity as the neuronal isoform. Spinal alpha(2)-adrenergic agonist antinociception may be partly dependent on cholinergic and nitric oxide mechanisms. PMID- 8638845 TI - Inhibitory effects of ketamine and halothane on recombinant potassium channels from mammalian brain. AB - BACKGROUND: There is increasing evidence that direct interactions between volatile anesthetics and channel proteins may result in general anesthesia. Using voltage-clamp techniques, the authors examined the effect of two general anesthetics (ketamine and halothane) on a rat brain potassium channel of known amino acid sequence, and further assessed whether the inhibition of the channel is altered by a partial deletion of the C-terminal sequence of this channel. METHODS: Xenopus laevis oocytes were microinjected with either Kv2.1 or delta C318 (a mutated channel in which the last 318 amino acids of the C-terminus have been deleted) cRNA, and channel function in translated channels was observed before, during, and after exposure to graded concentrations of ketamine (25, 50, and 75 micrometers) and halothane (1%, 2%, and 4%). RESULTS: Ketamine and halothane reduced Kv2.1 and delta C318 peak current amplitude in a dose-dependent and reversible fashion. The inhibition of current was voltage dependent for halothane but not for ketamine. Halothane accelerated the time constant of current inactivation, whereas ketamine affected this parameter minimally in both channel types. Use dependence of ketamine and halothane action was observed in both Kv2.1 and the mutant channel, attributable to augmentation of C-type inactivation. CONCLUSIONS: Although both ketamine and halothane inhibit potassium currents through the Kv2.1 channel, their mechanisms of action at this potential target may be different. Deletion of the C-terminal sequence resulted in decreased sensitivity to both anesthetics. Although it is not clear whether anesthetics interact directly with the C-terminus, which is thought to reside intracellularly, this portion of the channel protein clearly influences the actions of both anesthetics. PMID- 8638846 TI - Demonstration of halothane-induced hepatic lipid peroxidation in rats by quantification of F2-isoprostanes. AB - BACKGROUND: Halothane can be reductively metabolized to free radical intermediates that may initiate lipid peroxidation. Hypoxia and phenobarbital pretreatment in Sprague-Dawley rats increases reductive metabolism of halothane. F(2)-isoprostanes, a novel measure of lipid peroxidation in vivo, were used to quantify halothane-induced lipid peroxidation in rats. METHODS: Rats were exposed to 1% halothane or 14% O(2) for 2 h. Pretreatments included phenobarbital, isoniazid, or vehicle. Rats also were exposed to halothane, enflurane, and desflurane at 21% O(2). Lipid peroxidation was assessed by mass spectrometric quantification of F(2)-isoprostanes. RESULTS: Exposure of phenobarbital pretreated rats to 1% halothane at 21% O(2) for 2 h caused liver and plasma F(2) isoprostane concentrations to increase fivefold compared to nonhalothane control rats. This halothane-induced increase was enhanced by 14% O(2), but hypoxia alone had no significant effect. Alanine aminotransferase activity at 24 h was significantly increased only in the 1% halothane/14% O(2) group. The effect of cytochrome P450 enzyme induction on halothane-induced F(2)-isoprostane production and liver injury was determined by comparing the effects of isoniazid and phenobarbital pretreatment with no pretreatment under hypoxic conditions. Halothane caused 4- and 11-fold increases in plasma and liver F(2)-isoprostanes, respectively, in non-pretreated rats, whereas isoniazid pretreatment had no effect. Phenobarbital pretreatment potentiated halothane-induced lipid peroxidation with 9- and 20-fold increases in plasma and liver F(2)-isoprostanes, respectively. Alanine aminotransferase activity was increased only in this group. At ambient oxygen concentrations, halothane but not enflurane or desflurane, caused F(2)-isoprostanes to increase. CONCLUSIONS: Specific halothane-induced lipid peroxidation was demonstrated in Sprague-Dawley rats using quantification of F(2)-isoprostanes and was increased by hypoxia and phenobarbital pretreatment, but not isoniazid pretreatment. PMID- 8638847 TI - Metabolism of glucose, glycogen, and high-energy phosphates during transient forebrain ischemia in diabetic rats: effect of insulin treatment. AB - BACKGROUND: Hyperglycemia associated with diabetes mellitus will exacerbate neurologic injury after global brain ischemia. Studies in a rat model of forebrain ischemia (bilateral carotid occlusion plus hypotension for 10 min) discovered that acute restoration of normoglycemia in diabetics, using an insulin infusion, resulted in a neurologic outcome that was similar to normoglycemic rats without diabetes. The current study evaluated cerebral glucose, glycogen, lactate, and high-energy phosphate concentrations to identify metabolic correlates that might account for an alteration in postischemic outcome. METHODS: Fifty-four pentobarbital-anesthetized Sprague-Dawley rats were assigned to three groups: chronically hyperglycemic diabetic rats (D; N = 18); insulin-treated, acutely normoglycemic diabetic rats (ID; N = 18); and nondiabetic rats (ND; N = 18). These groups were further divided into groups of six rats each that received either no ischemia, forebrain ischemia of 10 min duration without reperfusion, or ischemia plus 15 min of reperfusion. Brains were excised after in situ freezing, and metabolites were measured using enzymatic fluorometric techniques. RESULTS: Before ischemia, D rats had greater concentrations of brain glucose (12.18 +/- 2.67 micromol/g) than did either ID (5.10 +/- 1.33) or ND (3.20 +/- 0.27) rats (P < 0.05). Preischemic brain glycogen was similar in all groups. At the completion of ischemia, brain lactate concentrations in D were 86% greater than in ID and 61% greater than in ND (P < 0.05), reflecting a higher intraischemic consumption of glucose plus glycogen in D (P < 0.05). High-energy phosphate concentrations, as assessed by the energy charge of the adenylate pool, were better preserved in D (energy charge = 0.60 +/- 0.28) than in either ID (0.29 +/- 0.09) or ND (0.36 +/- 0.07; P < 0.05) rats. After 15 min of reperfusion, the energy charge returned to preischemic values (i.e., 0.91-0.92) in all groups. CONCLUSIONS: These studies demonstrated greater intraischemic carbohydrate consumption and lactate production in D than in ID or ND rats. Under these conditions, intraischemic-but not postischemic-energy status was better in D rats. Acute insulin therapy in ID rats resulted in a metabolic profile that was similar to that of ND rats. These results suggest that, in this model, primary energy failure during ischemia is not the origin of greater injury in hyperglycemic diabetics, nor is energy enhancement the origin of improved outcome after acute insulin treatment. PMID- 8638848 TI - Continuous intrathecal administration of shortlasting mu opioids remifentanil and alfentanil in the rat. AB - BACKGROUND: Lipid soluble mu opioids given intrathecally produce a potent, dose dependent analgesic response, which because of rapid clearance, is of short duration. Such agents delivered by continuous infusion can result in systemic accumulation and significant extraspinally mediated side effects. The effects of intrathecal infusions of two lipid-soluble mu opioids were investigated: remifentanil, an esterase metabolized agent with an inactive metabolite, and alfentanil. METHODS: Rats with chronic lumbar intrathecal catheters received intrathecal infusions (in flow rates of 1.0 microliters/min and 0.1 microliters/min) of remifentanil or alfentanil and were tested for hind paw thermal withdrawal latency, supraspinal side effects (sedation, block of pinna, and corneal responses) and motor impairment. Remifentanil was delivered either in a glycine formulation (R(g)) or in a saline vehicle (R(s)). Separate studies with the glycine vehicle also were undertaken. RESULTS: At an infusion rate of 0.1 microliters/min, remifentanil and alfentanil produced naloxone-reversible, dose dependent analgesia and supraspinal side effects with the intrathecal ED(50) (micrograms/min; 95% confidence interval) for analgesia: R(s) = 1.5 (1.2-1.8), R(g) = 1.2 (0.7-2.3); alfentanil = 1.5 (1.4-1.6) and for supraspinal side effects: R(s) = 1.7 (1.4-1.9); R(g) = 1.9 (1.6-2.4); alfentanil = 1.5 (1.4-1.7). There was no difference in potency or time until onset for analgesia at either delivery rate (12-20 min), whereas for supraspinal side effects, 1.0 microliters/min resulted in a faster onset for R(g). Recovery of normal thresholds after equianalgesic doses was faster in R(s) than alfentanil and for the supraspinal index faster in R(s) and R(g) groups. R(g), but not R(s), or alfentanil, produced a dose-dependent motor impairment after 90 min of intrathecal infusion at a flow rate of 0.1 microliters/min. Both glycine in R(g) and glycine (matching glycine dose) alone showed parallel time courses for motor impairment and similar intrathecal ED(50) (6.6 vs. 6.4 micrograms/min over 90 min) for this nonnaloxone reversible effect. Intrathecal bolus administration of the same total dose of glycine showed no significant motor effects. CONCLUSIONS: Remifentanil has a rapid onset like alfentanil but shows a faster recovery of action after intrathecal infusion. Despite its rapid clearance, remifentanil induces supraspinal side effects at analgesic effective doses. Moreover, in the current formulation, with glycine, a reversible motor impairment can occur after intrathecal delivery. PMID- 8638849 TI - Pseudocholinesterase-mediated hydrolysis is superior to neostigmine for reversal of mivacurium-induced paralysis in vitro. AB - BACKGROUND: The metabolic hydrolysis of mivacurium (and succinylcholine) is markedly impaired in the presence of hereditary or acquired defects of pseudocholinesterase. Clinical reports are conflicting as to the utility of anticholinesterases, in the reversal of mivacurium paralysis. In the current study, the role of exogenous cholinesterases and/or of anticholinesterase, neostigmine, in the reversal of deep mivacurium-induced paralysis, was studied. The rat phrenic-diaphragm preparation, in a fixed volume of Krebs solution, was chosen to eliminate the confounding effects on the dissipation of neuromuscular effects caused by hydrolysis, elimination, and redistribution of the drug. METHODS: In the phrenic-diaphragm preparation, mivacurium was administered to obtain >90% single twitch inhibition. Single twitch responses (0.1 Hz) were monitored for 60 min, after which the response to train-of-four stimulation was tested. The reversal of mivacurium by 0.5, 1.0, or 2.0 units/ml of (true) acetylcholinesterase, bovine pseudocholinesterase, or human plasma cholinesterase and by neostigmine, 0.1, 1.0, or 10.0 micrograms/ml tested. The efficacy of human plasma cholinesterase, 1 unit/ml in combination with each of the above neostigmine concentrations, also was examined. The reversal of succinylcholine induced paralysis by the acetylcholinesterase, bovine pseudocholinesterase, or human plasma cholinesterase (1 unit/ml) alone and in the presence of neostigmine (10.0 micrograms/ml) was additionally tested as a positive control. A train-of four ratio > 0.75 was considered adequate reversal. RESULTS: Acetylcholinesterase was a poor hydrolyzer of mivacurium, as bioassayed by reversal of paralysis. Bovine pseudocholinesterase in concentrations of 0.5 and 1.0 units/ml did not effectively reverse single twitch and train-of-four responses by 60 min, but bovine pseudocholinesterase (2 units/ml) and all concentrations of human plasma cholinesterase did. Neostigmine alone, tested at all concentrations, was an incomplete reversal drug. Clinical or therapeutic concentrations (0.1 and 1.0 micrograms/ml) of neostigmine did not, but pharmacologic concentrations (10 micrograms/ml) interfere with the efficacy of human plasma cholinesterase (1 unit/ml). Bovine pseudocholinesterase and human plasma cholinesterase equally reversed the effects of succinylcholine but acetylcholinesterase did not, whereas the addition of 10 micrograms/ml neostigmine to the enzymes inhibited the reversal of succinylcholine. CONCLUSIONS: Human plasma cholinesterase will reverse mivacurium more effectively than bovine pseudocholinesterase, but both will effectively reverse succinylcholine. Acetylcholinesterase has no effects on either relaxant. The anticholinesterase neostigmine was an incomplete reversal drug. Pharmacologic concentrations of anticholinesterases do, while clinical or therapeutic concentrations do not, completely inhibit the metabolic activity of pseudocholinesterases. PMID- 8638850 TI - Pontine cholinergic mechanisms modulate the cortical electroencephalographic spindles of halothane anesthesia. AB - BACKGROUND: Halothane anesthesia causes spindles in the electroencephalogram (EEG), but the cellular and molecular mechanisms generating these spindles remain incompletely understood. The current study tested the hypothesis that halothane induced EEG spindles are regulated, in part, by pontine cholinergic mechanisms. METHODS: Adult male cats were implanted with EEG electrodes and trained to sleep in the laboratory. Approximately 1 month after surgery, animals were anesthetized with halothane and a microdialysis probe was stereotaxically placed in the medial pontine reticular formation (mPRF). Simultaneous measurements were made of mPRF acetylcholine release and number of cortical EEG spindles during halothane anesthesia and subsequent wakefulness. In additional experiments, carbachol (88 mM) ws microinjected in the the mPRF before halothane anesthesia to determine whether enhanced cholinergic neurotransmission in the MPRF would block the ability of halothane to induce cortical EEG spindles. RESULTS: During wakefulness, mPRF acetylcholine release averaged 0.43 pmol/10 min of dialysis. Halothane at 1 minimum alveolar concentration decreased acetylcholine release (0.25 pmol/10 min) while significantly increasing the number of cortical EEG spindles. Cortical EEG spindles caused by 1 minimum alveolar concentration halothane were not significantly different in waveform, amplitude, or number from the EEG spindles of nonrapid eye movement sleep. Microinjection of carbachol into the mPRF before halothane administration caused a significant reduction in number of halothane-induced EEG spindles. CONCLUSIONS: Laterodorsal and pedunculopontine tegmental neurons, which provide cholinergic input to the mPRF, play a causal role in generating the EEG spindles of halothane anesthesia. PMID- 8638851 TI - Who said childbirth is natural? The medical mission of Grantly Dick Read. PMID- 8638852 TI - The 34th Rovenstine Lecture. 40 years behind the mask: safety revisited. PMID- 8638853 TI - General anesthesia in a child with a dynamic, vascular anterior mediastinal mass. PMID- 8638854 TI - Performance of local anesthetic and placebo splanchnic blocks via indwelling catheters to predict benefit from thoracoscopic splanchnicectomy in a patient with intractable pancreatic pain. PMID- 8638855 TI - Skin burn associated with pulse oximetry during perioperative photodynamic therapy. PMID- 8638856 TI - Practice guidelines for perioperative transesophageal echocardiography. A report by the American Society of Anesthesiologists and the Society of Cardiovascular Anesthesiologists Task Force on Transesophageal Echocardiography. PMID- 8638857 TI - The changing relationship of oral and maxillofacial surgery to anesthesia. PMID- 8638858 TI - Efficacy of a topical anesthetic on pain and unpleasantness during scaling of gingival pockets. AB - The efficacy of a topical anesthetic on pain and unpleasantness provoked by scaling of gingival pockets was investigated in 20 patients with mild chronic periodontitis. A eutectic mixture of local anesthetics (EMLA) and a placebo cream, both occluded by Orahesive Oral Bandages, were applied in a balanced, randomized, double-blind, split-mouth design, which enabled within-subject comparison of the anesthetic and the placebo in the upper and the lower jaw. Pretreatment interviews showed that approximately two-thirds of the patients considered gingival scaling to be associated with some degree of pain and unpleasantness. Pain intensity and unpleasantness were evaluated on 100-mm visual analog scales (VAS). Application of EMLA reduced both pain intensity and unpleasantness significantly compared to placebo cream. Median reductions in VAS pain intensity in the upper and lower jaw were 58.9% and 61.9%, and corresponding reductions in VAS unpleasantness were 31.9% and 25.6%, respectively. Generally, the patients accepted the anesthetic procedure well. The residual perception of pain and unpleasantness following topical anesthesia may be dependent on activation of nonanesthetized nociceptive fibers in the tooth pulp. However, the present study clearly demonstrates the efficacy of a topical anesthetic in a clinical situation, which may be recommended as a simple pharmacologic strategy to reduce pain and unpleasantness during scaling procedures. PMID- 8638859 TI - Topical anesthesia: differentiating the pharmacological and psychological contributions to efficacy. AB - Pain can be influenced by both pharmacologic and psychologic factors. The purpose of this study was to investigate the pharmacologic and psychologic factors. The purpose of this study was to investigate the pharmacologic and psychologic processes that may play a role when topical anesthesia is used to reduce the pain of dental injections. Subjects were assigned to one of two belief manipulation conditions: one-half of the subjects were led to believe they would receive a placebo, while the remaining subjects were told they would receive the active agent. In reality, all subjects received two separate injections at contralateral sites, one preceded by a placebo and the other by a 20% benzocaine gel. The order in which injections were given was associated with differences in pain report. Second injections were more painful than first injections. Whether injections were preceded by an active or placebo agent did not alter subjects' experienced pain. Likewise, the belief manipulation did not affect the pain report. However, subjects who believed they would receive the active agent anticipated significantly less pain than subjects who thought they would receive placebo. The widespread belief that topical anesthetics are effective at reducing injection pain may serve to reduce the anticipatory anxiety associated with an impending dental injection, thus making the injection experience less aversive. PMID- 8638861 TI - Dentists and anesthesia: commentary. PMID- 8638862 TI - Preinduction of anesthesia with rectal midazolam. PMID- 8638863 TI - Prospective study of vascular events and cerebral perfusional changes following transient ischemic attacks. AB - A prospective case-control study was carried out to clarify associations of cerebral transient ischemic attacks (TIAs) and other stroke risk factors with progression and exacerbation of cardiovascular and cerebrovascular disorders; 243 neurologically normal controls and 123 TIA patients without prior history of stroke were followed up for a mean interval of 4.4 years of TIA patients, 26 (21%) developed other events (excluding recurrent TIAs); 10 died of vascular causes (8.1%). Of controls, 44 (18%) developed events; 13 died of vascular causes (5.4%) and 3 from cancer. TIA patients were at 2.3 times greater risk than normal controls for stroke or death from vascular causes. They were predominantly male with significantly higher associations of risk factors for stroke, including hypertension, heart disease, diabetes mellitus, smoking, hyperlipidemia, alcohol consumption, and limited education. Controls developing vascular events compared with controls who did not were older, more frequently male, and with greater incidences of heart disease. TIA patients had lower rates of cerebral perfusion compared with controls that persisted throughout the study, with similar rates of decline related to aging among both groups. Among TIA patients, stroke risk factors were more prevalent than among controls. The longer their duration, the greater the incidence and the more rapid the rate of severe, often fatal cardiovascular complications. PMID- 8638864 TI - Comparison of lower limb arterial assessments using color-duplex ultrasound and ankle/brachial pressure index measurements. AB - The strength of agreement between two noninvasive methods of assessing lower limb arterial disease and their relationship to patient symptoms following exercise have been investigated. Color-duplex ultrasound (CDU) and ankle/brachial pressure index (ABPI) (before and afer exercise) measurements were obtained from 200 consecutive patients referred to a vascular investigations laboratory. From these patients, 290 limbs were available for study, comprising limbs without previous vascular surgery, from patients without diabetes and who could attempt a walking exercise test. The overall level of agreement between CDU and resting ABPI measurements was 83% (Kappa 0.66). The ABPI technique identified the more serious disease; a resting ABPI of less than 0.6 gave 100% agreement with CDU. With higher resting ABPIs the level of agreement became poorer: 83% (0.6 < or = ABPI <0.9) and 76% (normal ABPI > or = 0.9). The addition of postexercise ABPI measurements in determining significant arterial disease increased the strength of relationship between the two techniques by only 2% (85%, Kappa 0.69). The exercise test was generally limited by the most symptomatic limb in each patient, and the agreement between CDU and postexercise ABPI measurements in these limbs was higher at 93% (Kappa 0.81). In comparison, agreement for the least symptomatic group of limbs was found to be poor (69%, Kappa 0.37). Compared with symptoms after exercise, overall agreements with CDU and ABPI were both 67% (Kappa 0.27). The agreement was better (91%) when the resting ABPI was less than 0.6. The ABPI is biased toward the detection of more severe disease and is more consistent with CDU when the most symptomatic limbs are compared. The relationship between either test and symptoms after exercise is strong only for limbs with major disease. PMID- 8638865 TI - ACE inhibition with spirapril improves diastolic function at rest independent of vasodilation during treatment with spirapril in mild to moderate hypertension. AB - The effects of the ACE inhibitor spirapril and of hydrochlorothiazide on left ventricular diastolic function were studied. Thirteen patients with mild to moderate essential hypertension completed this randomized, double-blinded, placebo-controlled, crossover study. After a three-week run-in period the patients entered three periods lasting four weeks each, wherein they were treated with placebo, spirapril, or hydrochlorothiazide. Blood pressure, hemodynamic variables (stroke volume, heart rate, cardiac output, index of contractility, and systemic vascular resistance), echocardiography (left ventricular mass), and Doppler-derived atrial to early (A/E)-ratio velocity time integrals (VTI) were measured at the end of each of the four periods. Spirapril lowered the A/E-ratio VTIs (0.57, 0.12-1.00) (P < 0.02) as compared with both placebo (0.80, 0.50-2.67) and hydrochlorothiazide (0.83, 0.44-1.25), and the drug normalized the A/E-ratio VTI in those patients with elevated values. The hemodynamic variables, left ventricular mass, and end-systolic wall stress were unchanged during all three treatments. There were no significant changes in mean blood pressure during the treatment periods. These results indicate that spirapril lowers A/E ratio within four weeks in patients with mild to moderate essential hypertension. It thereby seems able to improve left ventricular diastolic function. The effect is not dependent upon changes in hemodynamic variables, blood pressure, left ventricular mass, or end-systolic wall stress. PMID- 8638867 TI - Quality of life in a group of patients with intermittent claudication. AB - In view of the increasing importance of chronic diseases in recent years, not only quantity but also quality of life has been taken into consideration. The aim of this study was to verify the quality of life in patients with peripheral obstructive arteriopathies of second degree according to the Leriche-Fontaine classification. Therefore, the authors administrated three different questionnaires to patients selected among those who came to the Angiology Ambulatory Care of the Medical Clinic of the University of Trieste. The questionnaires used were the following: McMaster Health Index Questionnaire (MHIQ), "Squibb" Quality of Life, General Health Index Questionnaire (GHIQ). The analysis of the results of the questionnaires shows the severe limitation of physical capacities of this kind of patients (overall mean functional limitation of 69% of the maximum value of 1, corresponding to an ideal state of well-being, with negative peaks of 50%). A negative tendency regarding social life and relationship is represented by different scores (from 52% to 66% of ideal maximum) from different items and questionnaires. Also the items concerning somatic disorders, sleeplessness, and states of anxiety gave results near the percentage of 70% and for this reason were indicative of a state of unwell-being. The data obtained from the items regarding the presence of a state of depression gave a result less predictable: a score of 87% with peaks over 90% could be indicative of a positive tendency. This last result, consistent with others of analogous studies in the literature, seems to indicate that peripheral arterial disease, even if physically restricting, does not have a strong impact on the psychological and emotional equilibrium of the patients. PMID- 8638866 TI - Secondary prevention of coronary heart disease in patients with extracoronary atherosclerosis: a need for accuracy of low density lipoprotein determination. AB - According to the new guidelines of the National Cholesterol Education Program (NCEP) for secondary prevention in adults with evidence of coronary heart disease or other clinical atherosclerotic disease, lipoprotein analysis is required and classification is based on low density lipoprotein (LDL) cholesterol. The aim of the present study was to analyze the reliability of calculated LDL cholesterol by the Friedewald formula compared with measured LDL cholesterol after separation by ultracentrifugation in 202 male patients with extracoronary atherosclerosis (100 patients with ischemic cerebrovascular disease and 102 patients with peripheral vascular disease) and in 117 health control subjects. Calculated LDL cholesterol coincided with measured LDL cholesterol, with less than 10% error, in 118 patients (58.4%) with extracoronary atherosclerosis and in 87 controls (74.4%). Calculated LDL cholesterol was overestimated, with an error of 10% or more compared with measured LDL cholesterol, in 34.6% of patients and 22.2% of controls, and underestimated in 6.9% and 3.4% respectively. Despite a good correlation between calculated and measured LDL cholesterol, the intraclass correlation coefficients demonstrate a poor concordance between calculated and measured LDL cholesterol, both in patients and controls. The authors underline the need for caution in assessing the reliability of calculated LDL cholesterol. PMID- 8638868 TI - Plasma viscosity increase with progression of peripheral arterial atherosclerotic disease. AB - Increased blood and plasma viscosity has been described in patients with coronary and peripheral arterial disease. However, the relation of viscosity to the extent of arterial wall deterioration--the most important determinant of clinical manifestation and prognosis of the disease--is not well known. Therefore, the authors studied plasma viscosity as one of the major determinants of blood viscosity in patients with different stages of arterial disease of lower limbs (according to Fontaine) and its relation to the presence of some risk factors of atherosclerosis. The study encompassed four groups of subjects: 19 healthy volunteers (group A), 18 patients with intermittent claudication up to 200 m (stage II; group B), 15 patients with critical ischemia of lower limbs (stage III and IV; group C), and 16 patients with recanalization procedures on peripheral arteries. Venous blood samples were collected from an antecubital vein without stasis for the determination of plasma viscosity (with a rotational capillary microviscometer, PAAR), fibrinogen, total cholesterol, alpha-2-macroglobulin, and glucose concentrations. In patients with recanalization procedure local plasma viscosity was also determined from blood samples taken from a vein on the dorsum of the foot. Plasma viscosity was most significantly elevated in the patients with critical ischemia (1.78 mPa.sec) and was significantly higher than in the claudicants (1.68 mPa.sec), and the claudicants also had significantly higher viscosity than the controls (1.58 mPa.sec). In patients in whom a recanalization procedure was performed, no differences in systemic and local plasma viscosity were detected, neither before nor after recanalization of the diseased artery. In all groups plasma viscosity was correlated with fibrinogen concentration (r=0.70, P < 0.01) and total cholesterol concentration (r=0.24, P < 0.05), but in group C (critical ischemia) plasma viscosity was most closely linked to the concentration of alpha-2-macroglobulin (r=0.78, P < 0.01). These results indicate that in patients with peripheral arterial disease plasma viscosity increases with the progression of the atherosclerotic process and is correlated with the clinical stages of the disease. PMID- 8638869 TI - Deep vein thrombosis as a predictor of cancer. AB - An association between cancer and venous thrombosis has long been recognized. In an attempt to find whether venous thrombosis can serve as a reliable predictor of subsequent cancer, 196 cases of confirmed venous thrombosis treated over a seven year period were reviewed. Of a total of 196 cases, 113 were found to suffer from primary type deep venous thrombosis (DVT), whereas 83 were allocated to secondary type DVT. Of 113 cases with primary DVT, 23 had suffered from recurrent episodes, whereas 90 had only a single episode. From the secondary DVT group of 83 patients, 14 suffered from recurrent episodes and 69 had a single episode. Of the patients in the primary DVT group, 2.65% (3/113) developed cancer subsequently, whereas none was detected among the group of 83 patients who had secondary DVT. In the group of 113 patients with primary DVT, the incidence was higher among those who had recurrent episodes (4.34%, 1/23) than among those who had a single episode (2.22%, 2/90). The overall incidence of cancer among 196 cases of DVT was found to be 1.53% (3/196). The findings do not support the routine screening of all DVT patients by expensive diagnostic modalities. These patients should rather be assessed by careful clinical examinations and baseline investigations. The detailed investigations should be resorted to if there is any suggestion of presence of occult malignancy by these simpler means. One should be more vigilant in case of patients suffering from primary type DVT, for the incidence in this group is significantly higher. PMID- 8638860 TI - Summary of the scientific literature for pain and anxiety control in dentistry. PMID- 8638871 TI - Cardiovascular effect of oral calcium supplementation: echocardiographic study in patients with essential hypertension. AB - Oral calcium (Ca) supplementation mildly reduces blood pressure. The authors studied the effects of Ca supplementation on the cardiovascular system in patients with mild to moderate essential hypertension. Twelve patients aged forty nine to seventy years (7 men and 5 women, mean age with 60.3 +/- 7.2 years) participated. The investigators orally administered Ca (1.0 g/day for one week) under hospitalization, adding to a dietary intake of Ca (0.6 g/day). Left ventricular function and systemic arterial compliance were evaluated by M-mode and pulsed Doppler echocardiographies before and after seven days of Ca supplementation. Left ventricular contractility and afterload were not changed. Preload indicated by end-diastolic volume was significantly decreased after Ca supplementation (109.6 +/- 8.5 vs 107.3 +/- 8.2 mL, P < 0.05). Myocardial relaxation evaluated by IIa-mitral valve opening time (87.7 +/- 6.7 vs 82.1 +/- 6.2 ms, P < 0.01) and maximum descending rate of the left ventricular posterior wall (10.6 +/- 1.0 vs 12.4 +/- 1.0 cm/s, P < 0.01), and atrioventricular net compliance assessed by the descending slope of rapid filling flow in the left ventricular inflow tract (2.63 +/- 0.24 vs 2.26 +/- 0.17 m/s2, P <0.05), as well as systemic arterial compliance (2.05 +/- 0.20 vs 2.73 +/- 0.26 mL/mmHg, P < 0.01) were significantly improved by Ca supplementation. Oral Ca supplementation improved the disturbed left ventricular diastolic function and systemic arterial compliance. PMID- 8638870 TI - Underestimation of the severity of pulmonary outflow tract obstruction in the first day of life--Doppler echocardiographic study. AB - To find out whether there is an underestimated severity of narrowing in obstructive lesions of the pulmonary outflow tract in the first day of life, Doppler measurement of the pulmonary outflow tract gradient and estimation of the pulmonary artery systolic pressure in 15 neonates with pulmonary outflow tract obstruction with a variety of associated lesions were studied in the first twenty four hours of life (mean thirteen hours, range six to twenty-four) and repeated at the age of twenty-six to seventy-two hours (mean forty-nine). The maximal instantaneous gradient across the pulmonary outflow tract obstruction (22.4 +/- 9.0 mmHg) in the first day of life was significantly lower than at the repeated study (61.0 +/- 19.4 mmHg, (P < 0.001). In 12 patients with patent ductus arteriosus, transductal gradient increased significantly at the repeated examination (19.6 +/- 9.3 vs 48.2 +/- 6.1 mmHg, P < 0.001). The pulmonary outflow tract gradient in these 12 patients was 22.3 +/- 8.4 vs 62.9 +/- 21.1 mmHg, (P < 0.001). The severity of the pulmonary outflow tract obstruction was underestimated in the first twenty-four hours of life. Low gradient across pulmonary outflow tract and low transductal gradient in the first day of life mirrored high pulmonary arterial systolic pressure. At the repeated study the increase in transductal gradient and the increase in the pulmonary outflow tract gradient more accurately represented the severity of pulmonary outflow tract obstruction owing to the decline in the systolic pulmonary arterial pressure. PMID- 8638872 TI - Jaundice induced by streptokinase. AB - Streptokinase is the mainstream therapy for acute myocardial infarction. A fifty seven-year-old man with acute MI was admitted to the intensive cardiac care unit and received streptokinase and heparin. At the time of admission, he was not receiving any drugs and denied any previous exposure to a hepatotoxic agent. Five hours later he developed a dramatic hypersensitivity reaction including high fever, pulmonary edema, cyanosis, and convulsions. Within twelve hours, his clinical state was stabilized. After forty-eight hours, he developed jaundice and transaminasemia, which subsided by the eighth day. Only a few reports of overt jaundice are associated with streptokinase. PMID- 8638873 TI - A serious complication of percutaneous mitral valvuloplasty: systemic embolism. How can we decrease it? Case history. AB - Systemic embolism is a potential and severe complication of percutaneous balloon mitral valvuloplasty (PBMV). The incidence of systemic embolism during PBMV has been reported to be less than 5% and only 0.6% with the Inoue technique. This is less than that reported in closed commissurotomy series and about the same as in open commissurotomy. In the authors' series of 50 cases, the incidence of systemic embolism was 2% (1 case). The patient had mitral restenosis (after closed commissurotomy) with mild to moderate valvular and subvalvular calcification, and cerebral embolism occurred during the procedure. To prevent systemic embolism, the authors' standard policy was to perform transesophageal echocardiography (TEE), computed tomography, and magnetic resonance imaging before the procedure and to give heparin during the procedure and two months preceding it in cases with atrial fibrillation or with a history of previous embolism and to limit manipulation of the catheter in the left atrium. The authors believe that a close scrutiny in the selection of patients, improved technology of the dilating system, good experience with PBMV, and adequate heparinization are also of great importance in the prevention of thromboembolic complications. PMID- 8638874 TI - Coronary artery spasm in the transplanted human heart. A case report. AB - This report describes a patient manifesting spontaneous and catheter-induced coronary artery spasm in a transplanted, denervated heart. This diagnosis should be considered in patients undergoing posttransplant coronary angiography. Intracoronary nitroglycerin should routinely be administered prior to coronary artery injections during posttransplant angiography. PMID- 8638876 TI - Coexistence of cerebral venous sinus and internal carotid artery thrombosis associated with exogenous sex hormones. A case report. AB - A forty-six year-old premenopausal woman developed headache, nausea and vomiting, left hemiparesis and seizure two days after parenteral use of progesterone and estradiol. Diabetes mellitus (DM) was found during admission. Computed tomography showed a hemorrhagic infarct in the right frontal lobe and increased density in the superior sagittal sinus (SSS). Left carotid angiography found occlusion of the left internal carotid artery (ICA). Right carotid angiograms failed to show the SSS and inferior sagittal sinus, suggestive of venous sinus thrombosis. Coexistence of the cerebral artery and the venous sinus occlusion has been described infrequently. In this case, the authors postulate that the use of estradiol and progesterone and the underlying DM increased vascular thrombogenicity, which provided a common denominator for thrombosis of both the ICA and the venous sinus. PMID- 8638875 TI - Postinfarction ventricular septal rupture following thrombolysis: long-term survival in the presence of normal coronary arteries. A case report. AB - A case of postinfarction ventricular septal rupture in a thrombolyzed patient is described. Coronary angiography revealed normal coronary arteries. The interrelations of long-term survival of postinfarction ventricular septal rupture, thrombolysis, and normal coronary arteries are discussed. PMID- 8638877 TI - Guide wire-induced coronary artery spasm during percutaneous transluminal coronary angioplasty. A case report. AB - Coronary artery spasm induced by a guide wire is a very rare complication of percutaneous transluminal coronary angioplasty (PTCA). The authors describe a patient who developed coronary vasoconstriction distal to the dilated lesion during PTCA, which was refractory to intracoronary nitroglycerin and intravenous calcium antagonist injection and balloon angioplasty but responded promptly and completely to withdrawal of the guide wire from the coronary artery, suggesting that the spasm had been induced by the guide wire. PMID- 8638878 TI - Recurrent myocardial infarction provoked by multiple giant coronary aneurysms. A case report. AB - A case of multiple coronary aneurysms, complicated with repeated myocardial infarction, is presented. The cine-coronary angiogram and autopsy disclosed marked dilatation of epicardial coronary arteries associated with multiple giant thrombosed aneurysms, which is rare in patients with atherosclerosis. PMID- 8638880 TI - The symbiosis between basic and applied research. AB - To illustrate the interdependence between the solution of practical problems and the search for fundamental mechanisms, their relationship within the contexts of the history of night myopia and the problem of nighttime traffic accidents is discussed. Night myopia, or nearsightedness at night, which has been a problem since the late 18th century, was shown to be the result of a recently discovered oculomotor mechanism, the intermediate resting position of accommodation. With this knowledge, the handicap of nearsightedness at night ( a major problem in nighttime viewing such as driving and military operations) is readily amenable to solution. Recent developments in our knowledge of the functional significance of the nervous system has led to an increased understanding of the cause and to amelioration of nighttime traffic accidents. These developments illustrate the symbiotic relationship between basic and applied research and the benefits to be gained by consideration of both objectives. PMID- 8638879 TI - Takayasu's arteritis associated with antiphospholipid antibodies. Report of two cases. AB - The authors describe 2 patients with Takayasu's arteritis in whom lupus anticoagulant was positive and the titer of anticardiolipin antibody was elevated. One patient developed diffusely stenotic and occlusive changes in the multiple larger arteries. Histology of the small-sized arteries in another patient showed occlusive vasculitis without thrombosis, in addition to the findings in large-sized arteries compatible with Takayasu's disease. These findings are uncommon in Takayasu's arteritis. These findings suggest that antiphospholipid antibodies may have contributed to the pathogenesis of the extensive vasculopathy and may have triggered vasculitis in these patients. PMID- 8638882 TI - [An unbiased study of the perception of smile and sadness by analysing the integration of emotions]. AB - This article describes a simple and easy-using clinical tool for investigating the visual perception of emotions. The method was validated with healthy persons. On the one hand, the results confirm and sharpen the data of the literature. On the other hand, they enable a precise quantification of the results. Nevertheless, only the perception of the smile and sadness has been studied. Therefore, the extension and validation of this tool to other fundamental emotions is requested. This will enable a simple and easy clinical investigation of troubles of the visual recognition of emotions. On the one hand, for many neurological pathologies: strokes, neurologic disease. On the other hand for psychiatric pathologies: Korsakoff's syndrome, schizophrenia, Alzheimer's disease, other dementias and as on. PMID- 8638881 TI - Professional psychology and deaf people. The emergence of a discipline. AB - This article depicts obstacles and opportunities that face students and consumers who are deaf and who interface with the profession of psychology. The rapid evolution of scholarship, specialized education and service programs, and related professional endeavors regarding psychology and deaf individuals is described. The emergence of a field of professional psychology and deaf people as a discipline in its own right is posited. Professional standards and ethics in this emerging discipline are discussed, especially those pertaining to fluency in American Sign Language (ASL) and to the accessibility of deaf people to the profession of psychology as well as to the services of the profession. The potential for the American Psychological Association to further or hinder this emerging discipline and the advancement of all psychologists with disabilities are considered. PMID- 8638883 TI - [Ethnic and psychophysiological analysis of brief reactive psychosis. Autobiographical data and the position of reactive psychosis among psychoses]. AB - The account of an acute schizophrenic episode personally experienced by the author is given here. It relates its rare early stages, the sudden outbreak of the psychotic state, the collapsing of the ego and the internal journey with its catastrophic and mystical thematic. The present positions on acute schizophrenic episodes are related. It is desirable that they should be considered independently from schizophrenics. PMID- 8638885 TI - [Modification of body image and the evolution of psychological dependence among drug addicts]. PMID- 8638884 TI - [The therapist facing the problem of the homeless. Ethnic and psychophysiological considerations]. AB - The problem of "severe poverty" is more socio-economic than psychiatric. Approximately three to four million people living in France receive a monthly minimum income allowance from the state. An ethno-psychiatric approach offers the possibility of situating homeless people within the context of their everyday living conditions and habits. If the scope of the problem goes beyond the realm of the psychiatric's intervention, he is nonetheless still involved, and we believe that this involvement is important. Setting up centers for homeless people run by multi-disciplinary teams would help to resolve concrete problems facing this population. In the end, however, it is not a question of managing the urgency of the situation, but of establishing a social policy for all citizens. PMID- 8638887 TI - [Clinical imaging and bodily invasive techniques. From fantasy to reality]. AB - Patients must cope with intrusive medical investigations, endoscopic exams, open methods in intensive care unit which all disturb body boundaries and body image. Reality and fantasies ares mixed. Psychodynamic way-out are discussed. PMID- 8638886 TI - [A contribution of brain imaging techniques to the diagnosis of psychiatric emergencies]. AB - This study was performed in the psychiatric emergency unit of Rene-Dubos General Hospital (Pontoise, France). Two cases of female patients with motor deficiencies suggesting a diagnosis of conversion disorder because of preceding psychological stressors and lability of symptoms, are presented. In both cases, the diagnosis was reconsidered after the use of RMI for multiple sclerosis in one case, HIV related encephalopathy in the other. Recent studies have shown that organic diseases with a psychiatric presentation represent 1,2 to 4% of patients examined in emergency setting. Criteria for the diagnosis of conversion disorders are discussed (they are classified as dissociative disorders in ICD-10 and as somatoform disorders in DMS-IV). A diagnosis of conversion disorder should not be made before a thorough clinical and neurological evaluation has been performed. PMID- 8638888 TI - [Compulsive clinical voyeurism: a special case of medical families]. PMID- 8638889 TI - ["I have something in the head". The evolution of the body image in children according ot the data of medical imagery in the course of a brain tumor]. PMID- 8638890 TI - [Clinical imaging in psychiatry]. AB - Brain imaging has made surprisingly remarkable progress since the early, and now historic days, of invasive radiology, which has now been replaced with a number of spectacularly precise techniques: structural (CT Scan, MRI) and functional (PET, SPECT) imaging, direct imaging during neurosurgery, EEG and its computer assisted derivatives, and transcerebral ultrasonography. We present five cases with two alleged autisms, a cerebral malaria, a panic disorder and to Parkinson disease with a depressive component. Using modern imaging methods the following respective diagnoses were arrived at: a left temporal cyst, a Sanfilippo mucopolysaccharidosis, a septum lucidum agenesis, a right temporal cyst, and a pituitary adenoma. These cases illustrate the scientific, emotional and philosophical impact, on physicians, and patients alike, of modern imaging technology. Neuroradiology, biochemistry and surgical imaging require a multi disciplinary approach and a perfect knowledge of psychiatric semeiology. In addition, they stimulate us to carefully reassess our sociocultural understanding to mental illness. PMID- 8638891 TI - Measuring patient benefit from otorhinolaryngological surgery and therapy. AB - The Glasgow Benefit Inventory (GBI) is a measure of patient benefit developed especially for otorhinolaryngological (ORL) interventions. Patient benefit is the change in health status resulting from health care intervention. The GBI was developed to be patient-oriented, to be maximally sensitive to ORL interventions, and to provide a common metric to compare benefit across different interventions. The GBI is an 18-item, postintervention questionnaire intended to be given to patients to fill in at home or in the outpatient clinic. In the first part of the paper, five different ORL interventions were retrospectively studied: middle ear surgery to improve hearing, provision of a cochlear implant, middle ear surgery to eradicate ear activity, rhinoplasty, and tonsillectomy. A criterion that was specific to the intervention was selected for each study, so that the patient outcome could be classified as above and below criterion. In all five interventions, the GBI was found to discriminate between above- and below criterion outcomes. The second part of the paper reports on the results and implications of a factor analysis of patient responses. The factor structure was robust across the study, and so led to the construction of subscales. These subscales yield a profile score that provides information on the different types of patient benefit resulting from ORL interventions. The GBI is sensitive to the different ORL interventions, yet is sufficiently general to enable comparison between each pair of interventions. It provides a profile score, which enables further breakdown of results. As it provides a patient-oriented common metric, it is anticipated that the GBI will assist audit, research, and health policy planning. PMID- 8638892 TI - Quality of life after acoustic neuroma surgery. AB - A questionnaire was sent to 134 patients who had undergone surgery for a unilateral acoustic neuroma between 1980 and 1993, to obtain data on the consequences on their quality of life, physical condition, social life, employment, and use of medical facilities. Distinctions were made between the translabyrinthine-transotic approach, the suboccipital approach, the tumor size, and the number of operations per patient. We found that the patients' reported state of health after surgery was poorer than that in a group of comparable nonoperated patients. Recuperation after an operation took many months and did not always result in full recovery. Surgery had various effects on preoperative symptoms such as hearing loss, tinnitus, vertigo, and facial nerve dysfunction: improvement, no change, or deterioration. Surgery had severe consequences on social life and occupation, but far less effect on income. Almost one third of the patients required postoperative home help, and a proportion were declared unfit to work. The surgical approach, tumor size, and reoperations had a definite influence on the study parameters. After suboccipital surgery, there were more reports of pain, more declarations of incapacity to work, poorer facial nerve function, and more frequent visits to the general practitioner. The translabyrinthine-transotic approach was associated with more severe pain and more complaints of postoperative vertigo. A greater proportion of the patients with larger tumors were declared unfit to work. The general state of health after suboccipital reoperations was better than after the initial operation; there was no reasonable explanation for this. Facial nerve function deteriorated after reoperation(s). PMID- 8638893 TI - DentaCT for evaluating mandibular and maxillary invasion in cancer of the oral cavity. AB - We used a retrospective analysis of charts, imaging studies, and histologic findings when available in 17 patients with cancer of the oral cavity to define the value of DentaCT or multiplanar reformation software in assessing bony involvement by malignant tumors of the oral cavity. From two academic tertiary referral medical centers, the 17 patients had a mean age of 63 years and suspected maxillary (2) or mandibular (15) invasion. All patients had conventional computed tomography scanning and multiplanar reformation scans with several additional imaging studies obtained. Two patients demonstrated no bony involvement by imaging and histologic studies. Twelve of the remaining 15 patients had surgically documented bony involvement also correctly detected by DentaCT. DentaCT was found to be a valuable tool in defining extent of bony invasion by tumor. Although not compared to other imaging modalities, our experience supports the use of DentaCT where available for this purpose. PMID- 8638894 TI - Subtotal carbon dioxide laser arytenoidectomy by endoscopic approach for treatment of bilateral cord immobility in adduction. AB - Subtotal carbon dioxide (CO2) laser arytenoidectomy for endoscopic treatment of bilateral immobility of the vocal folds in adduction is a variant of total arytenoidectomy. The principal modification involves preservation of a thin posterior shell providing good postoperative fixation of the arytenoid region. The risk of aspiration is thus averted and collapse of arytenoid mucosa into the larynx during inspiration is prevented. The risk of synechia with the posterior commissure is avoided. The CO2 laser is operated at a working distance of 400 mm with a continuous 7-W beam in superpulse mode. Operation time is thus reduced to approximately half an hour and the risk of postoperative edema is reduced. Tracheotomy is not necessary. Forty-one patients, including 16 men and 25 women, were treated by this technique between 1985 and 1994. Their mean age was 55 +/- 17 years, ranging from 11 to 83 years. Follow-up ranged from 1 month to 111 months (9 years 3 months), with a mean of 56 +/- 29 months (4 years 8 months). The mean peak forced expiratory flow-peak inspiratory flow ratio (normal = 1), which permits a measurement of respiratory quality, is improved from 3.7 +/- 1.4 preoperatively to 1.6 +/- 0.5 postoperatively (p<.001). Postoperative voice measurements show a mean vocal intensity of 61 +/- 3 dB hearing level, a mean maximum phonation time of 8 +/- 4 seconds, and a mean phonation quotient of 397 +/- 150 mL/s. As for vocal quality, 38% of the patients now have a near-normal voice according to our high-resolution frequency analysis, and all of the patients retained satisfactory voice quality. PMID- 8638895 TI - Value of electromyography in differential diagnosis of laryngeal joint injuries after intubation. AB - Laryngeal joint injury or arytenoid dislocation is not an uncommon complication resulting from intubation trauma, and is best evaluated by laryngeal electromyography (EMG) combined with laryngoscopic examinations. Two cases of cricoarytenoid joint injuries after intubation are reported along with laryngeal EMG findings. Early diagnosis of arytenoid dislocation is important for appropriate surgical management and better prognosis. However, the reported cases, because of delayed referrals, showed prolonged cricoarytenoid joint injuries associated with thyroarytenoid muscle denervation or myopathy, and resultant vocal fold immobility. The results of laryngeal EMG in cricoarytenoid joint injuries can be classified into three different patterns: 1) normal recruitment, 2) myopathy, and 3) denervation or reinnervation of the thyroarytenoid muscles. It is particularly valuable to sample different portions of the thyroarytenoid muscles with EMG in order to evaluate different patterns or pathologic changes of the muscles and nerve paralysis. PMID- 8638897 TI - Histopathologic changes of the tympanic membrane in acute and secretory otitis media. AB - The histopathologic changes observed in 40 normal and inflamed temporal bones of infants and children are reported. The tympanic membranes of patients with acute and secretory otitis media underwent considerable swelling compared to those of normal controls. Tympanic membranes with acute otitis media were thicker than those with secretory otitis media, but the difference was not statistically significant. The epithelial layer showed an increase in the number of cell layers. The lamina propria demonstrated the most significant changes of all layers with a marked swelling due to edema, engorged blood vessels, and inflammatory cell infiltration. Increased numbers of distended capillaries were present, predominantly in the subepithelial connective tissue layer, while infiltration of inflammatory cells was mainly in the submucosal connective tissue layer. By contrast, the involvement of the mucosal layer was modest and was composed of cuboidal cells, except for small islands of metaplastic mucosa with mucus production occasionally encountered in specimens with secretory otitis media. PMID- 8638896 TI - Morphologic and motility changes of nasal cilia in primary culture caused by Haemophilus influenzae. AB - We focused on the pathogenicity of otitis media with effusion (OME) with respect to the susceptibility of the upper respiratory tract mucosa to Haemophilus influenzae. Human nasal polyps in outgrowth culture were used to study H influenzae disturbance of the ciliary beat frequency (CBF) and the morphology of cilia. The CBF of control primary culture was 11.7 +/- 2.7 Hz. The CBF slowed down significantly, to 8.5 +/- 5.7 Hz, after incubation with the filtrate of 10(8) CFU/mL of H influenzae, and to 4.1 +/- 4.1 Hz with a suspension of 10(8) CFU/mL of H influenzae (p<.05). In the morphologic study, we classified the shapes of the cilia into five types: normal cilia, immotile cilia, swollen cilia, clumped cilia, and exfoliated ciliated cells. The abnormal shapes of cilia increased after incubation with the suspension or the filtrate of H influenzae. By scanning electron microscopy, we saw that aggregated bacteria tended to associate with cilia. Thus, the findings suggest that H influenzae disturbs the ciliary clearance of nasal cells and makes them more susceptible to infections. PMID- 8638898 TI - Pediatric sino-orbital desmoid fibromatosis. AB - Desmoid fibromatosis is a rare tumor of the head and neck. Only five cases involving the sino-orbital region have been previously reported in the literature. In this report we describe the sixth such case, involving a 2-year old boy, and review the literature. PMID- 8638899 TI - Facial canal dehiscence: a report of 1,465 stapes operations. AB - We retrospectively reviewed 1,465 stapes operations for facial canal dehiscences. The incidence of facial canal dehiscence was found to be 11.4%. A facial canal dehiscence with protrusion of the facial nerve is more common than a dehiscence without protrusion. In addition, 3 cases (0.2%) had a herniation of the facial nerve. Our data suggests that heredity could be one of the factors associated with a facial canal dehiscence. If an operated ear shows a dehiscent facial canal, there is a 29% possibility that the contralateral ear also has a dehiscence. Bilaterality of facial canal dehiscence is, therefore, exceptional. PMID- 8638900 TI - Reversibility of compound action potential during the acute phase after transitory local ischemia. AB - We investigated the reversibility of compound action potential (CAP) changes induced after transitory local ischemia induced by rose bengal photochemically induced thrombosis of the rat anterior inferior cerebellar artery (AICA). Cochlear blood flow (CBF) was measured with a laser-Doppler flowmeter positioned on the lateral bony wall of the basal turn of the cochlea, and the CAP to an 8 kHz half-wave of sinusoid sound at 100 dB sound pressure level was monitored. The irradiation was started 5 minutes before the rose bengal administration and continued through the thrombosis formation. Tissue-type plasminogen activator (t PA, 1 mg/kg) dissolved in saline was injected intravenously 2 minutes after complete photothrombotic blockade of the AICA in the rats presenting complete abolition of CAPs just after the vascular occlusions. Nineteen of the 51 rats presented complete abolition of the CAP just after the AICA occlusion, and the thrombosed AICA was successfully reperfused by t-PA administration in 11 of the 19 rats. The result showed that duration of ischemia was the more important determinant for the reversibility of CAPs during the acute phase (p = .00001), and the residual level of cochlear blood flow during ischemia was also an important factor (p = .066). It appeared that the critical time of ischemia for the complete recovery of CAPs was around 5 minutes, and that the critical limit for the irreversibility of CAPs within the acute phase was between 20 and 25 minutes. PMID- 8638901 TI - Effect of SC-41930, a potent selective leukotriene B4 receptor antagonist, in the guinea pig model of middle ear inflammation. AB - Arachidonic acid metabolites such as prostaglandins and leukotrienes have been shown to play an important role in the pathogenesis of otitis media (OM). Among these mediators, leukotriene B4 (LTB4) is one of the most potent inducers of inflammatory processes. SC-41930 has been shown to be a specific LTB4 receptor antagonist both in vitro and in vivo. In this study, anti-inflammatory effects of SC-41930 were investigated in a guinea pig model of OM induced by middle ear (ME) inoculation of killed Staphylococcus aureus. Outcome of treatment was determined by measurement of myeloperoxidase activity in the samples of ME mucosa, evaluation of temporal bone histopathology, and presence of ME fluids. Myeloperoxidase activity in the SC-41930-treated group was found to be significantly lower than that in the control group. Histopathology of temporal bones indicated decreased inflammation in the treated group as compared to the controls. In addition, ME fluids were absent in four out of six treated animals. These results demonstrate that SC-41930 can produce significant anti-inflammatory effects in this model of OM. PMID- 8638903 TI - Bazex's acrokeratosis paraneoplastica. PMID- 8638902 TI - Extramedullary laryngeal plasmacytoma. PMID- 8638904 TI - Don't confuse dental soft tissues with odontogenic tumors. AB - Surgical pathologists are cautioned against the misinterpretation of immature dental tissues (dental papillae and follicles) and dental pulp as odontogenic tumors, especially odontogenic myxomas and fibromas. The close histologic similarity of the immature tissues to tumors may require a clinical-radiologic correlation with the histopathologic specimen in order to distinguish the locally aggressive tumors from innocuous dental tissues. PMID- 8638905 TI - Morphologic patterns and molecular pathways of AIDS-related head and neck and other systemic lymphomas. AB - Head and neck manifestations of human immunodeficiency virus (HIV) infection include lymph nodal and extranodal localization of non-Hodgkin's lymphoma (NHL). A histopathologic characteristic common to head and neck NHLs and to all the other systemic lymphomas in HIV patients is the frequent occurrence of cases displaying pleomorphic features and some overlap between established histologic subtypes. This article highlights the difficulties in defining HIV-related NHLs correctly, and proposes a specific pathologic categorization of these disorders accounting for their pathogenic aspects as gathered from molecular data. PMID- 8638906 TI - [Prostatic biopsy: technic, value and complications]. PMID- 8638907 TI - [Cell cycle and carcinogenesis]. AB - This presentation allows us to understand the terminology used for oncogenesis by Molecular Biology investigators. Thanks to the minute description of the normal physiology of the cell cycle, its regulatory mechanisms and growth stimulating and inhibiting effects, we are able to understand the language used. We are now able to fill the gap in our knowledge and to establish the increasingly necessary relationship between routine practice and Molecular Biology. PMID- 8638908 TI - [Percutaneous alcoholization of simple serous cysts of the kidney]. AB - Benign renal cysts are usually asymptomatic: They require a minimally invasive treatment if they cause complaints such as flank pain or other compressive complications. During a 6 month period, 14 patients were treated for benign renal cyst using an ultrasound guided puncture. Presenting complaints were flank pain (12 cases), hypertension (1 case), polycythaemia (1 case). Only cyst of 5 centimeters diameter or more were assigned to this treatment. After needle puncture, the cyst was evacuated and then filled with sterile 95% alcohol. Clinical and anatomical results, after 6 months, were good in 9 cases, 2 cases showed good anatomical results with persistence of initial symptoms, and there were 3 failures corresponding to large cysts of 10 cm diameter or more. Percutaneous alcoholization of moderate benign cysts is a safe, simple and reliable method. PMID- 8638909 TI - [Pseudocystic ureteritis. Apropos of a case]. AB - The authors report one case of ureteritis cystica in a young adult. The diagnosis was made by intravenous urography and confirmed by histologic examination. The patient was treated surgically (nephro-ureterectomy). The etiopathogenic, clinical and therapeutic aspects are discussed with a review of literature. PMID- 8638911 TI - [BCG therapy. Its role in the treatment of tumors of the bladder]. PMID- 8638910 TI - [Endoscopic treatment of intermittent vesico-ureteral reflux in women. Apropos of 40 cases]. AB - To evaluate the clinical efficacy of endoscopic treatment of intermittent vesicoureteric reflux in adult females. METHODS: Forty women presenting with recurrent urinary tract infection related to intermittent vesicoureteric reflux, underwent subureteric Teflon injection according to the O'Donnell procedure (mean volume of Teflon 1 cc). Thirty patients required one injection, while nine patients needed two injections. Meatostomy or urethral dilatation were associated in 29 cases (72.5%). RESULTS: No complication occurred. Clinical results were evaluated using a questionnaire. The mean follow-up was 33.5 months (range 12 72), complete disappearance of infection was obtained in 31 out of 40 cases (77.5%). Recurrent urinary infection occurred in 9 cases. After complementary Teflon injection, urinary infection disappeared in 5 patients and finally a complete cure was obtained in 36 out of 40 cases (90%). CONCLUSION: Intermittent vesicoureteric reflux could be easily cured by an endoscopic procedure. This minimally invasive technique is simple and well adapted to this relatively benign disease. PMID- 8638912 TI - [Results of the treatment of stress urinary incontinence in women]. PMID- 8638913 TI - [Bladder dysfunction in children]. PMID- 8638915 TI - [Scrotal reconstruction after necrotizing cellulitis of the perineum and external genital organs. Apropos of 21 cases]. AB - The author report twenty one cases of tissue reconstruction defects secondary to necrotizing cellulitis of perineum and external genital organs, treated at the burns and plastic surgery unit of CHU Ibn Rochd of Casablanca, between 1992 and 1994. Depending on the extent of the defect, the techniques used were gracilis musculocutaneous flap in 8 cases, thigh fasciocutaneous flap in 7 cases, groin flap in 4 cases, simple skin sutures and skin grafts in one case, respectively. The morphological results were satisfactory. The mean hospital stay after reconstruction was 15 days. PMID- 8638914 TI - [Seminoma in a male pseudohermaphrodite. Apropos of a case]. PMID- 8638916 TI - Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase. AB - Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+) agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase. PMID- 8638917 TI - Expression of mRNAs for lysyl oxidase and type III procollagen in cultured fibroblasts from patients with the Menkes and occipital horn syndromes as determined by quantitative polymerase chain reaction. AB - The Menkes syndrome and the occipital horn syndrome are two X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a reduction in the activity of lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme that initiates the crosslinking of collagens and elastin. We report here that the amount of lysyl oxidase mRNA, as studied by Northern blotting, and the number of lysyl oxidase mRNA molecules per picogram of RNA, as determined by a quantitative PCR method, were decreased in three cultured skin fibroblast lines from patients with the Menkes syndrome and two from patients with the occipital horn syndrome compared with four control cell lines. The decreased lysyl oxidase activity found in these disorders thus appears to be a least in part due to a pretranslational mechanism. No decrease was found in the number of the beta-actin mRNA molecules in the Menkes cell lines, but rather a slight increase, whereas a decrease was found in these molecules in the occipital horn cell lines. An additional abnormality found in the Menkes cell lines was a significant increase in the number of mRNA molecules for type III procollagen in two of the three cell lines investigated. The present and previous data indicate that the Menkes syndrome may involve several abnormalities in the expression of genes for connective tissue proteins. PMID- 8638919 TI - Inhibition of RNA synthesis by bradykinin involves both the B1 and B2 receptor subtypes. AB - The efficacy of angiotensin converting enzyme inhibitors in the treatment of heart disease is due in part to the accumulation of bradykinin (BK). Since BK can exert its effect by influencing cell proliferation, we chose to study the effect of BK on the growth of A10 vascular smooth muscle cells. Ligand binding studies to determine which BK receptor subtypes are present on A10 cells showed that both B1 and B2 receptors were present in approximately equal numbers. Examination of RNA synthesis demonstrated that BK inhibits uridine incorporation in a dose dependent manner. This decrease in RNA synthesis was blocked by both B1 and B2 receptor antagonists, as well as by addition of indomethacin, a cyclooxygenase inhibitor. The latter result suggested that prostaglandins mediate the biological actions of BK. Consequently, we examined the direct effect of two prostaglandins, PGE2 and PGI2 (prostacyclin), on A10 cells. PGE2 caused a decrease in RNA synthesis, thus mimicking the effect of BK, while PGI2 did not. Therefore, the inhibition of RNA synthesis in A10 vascular smooth muscle cells by BK requires both B1 and B2 receptor subtypes and this action of BK is apparently mediated by de novo synthesis of prostaglandins. PMID- 8638918 TI - The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. AB - Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone. PMID- 8638920 TI - Reversible thermal denaturation of staphylococcal nuclease: a Fourier transformed infrared spectrum study. AB - The secondary structures of staphylococcal nuclease (SNase) have been assigned and semiquantitatively estimated from the deconvoluted Fourier transformed infrared (FTIR) spectrum. The changes in the secondary structures accompanying unfolding and refolding of SNase during reversible thermal denaturation up to 70 degrees C are followed by FTIR measurements. Only slight perturbation was observed up to 35 degrees C. The unfolding transition temperatures of beta structure and alpha-helix are almost the same at 48.0-48.5 degrees C. During refolding the formation of the beta-structure follows the same pathway but that of the alpha-helix does not, although it recovers its original content almost completely. The final thermally denatured state at high temperature (60-70 degrees C) contains nonrandom structures in the complicated interaction, energetically resembling many kinds of structures. The occurrence of local conformational change before the final cooperative transition to the unfolded state during thermal denaturation as judged by FTIR spectra indicates that the unfolding and refolding of SNase may not follow the typical two-state model. PMID- 8638921 TI - Involvement of calpain in integrin-mediated signal transduction. AB - An antibody specific to the calpain cleavage site in talin, a cytoskeletal protein, was produced. This antibody selectively recognizes the C-terminal 200 kDa fragment generated when talin is digested by calpain and does not react at all with intact talin or the N-terminal 47-kDa fragment. To assess the involvement of calpain in the integrin-mediated signaling pathway, the effect of limited proteolysis of talin by calpain on platelet activation and aggregation was analyzed using this antibody. It was revealed that thrombin-stimulated platelet aggregation accompanies the autolytic activation of mu-calpain and the accumulation of the mu-calpain-generated 200-kDa fragment of talin. These changes were blocked by RGDS peptide which inhibits the binding of fibrinogen, an adhesive ligand, to the major integrin in platelets, alpha IIb beta 3, while RGES peptide, which has no fibrinogen-binding-inhibitory activity, had no effect. Membrane-permeable calpain inhibitors calpeptin and E-64d inhibited platelet aggregation, mu-calpain activation, and the limited proteolysis of talin. These results strongly suggest that calpain is involved in the integrin-mediated signal transduction pathway. PMID- 8638923 TI - Functional high level expression of cytochrome P450 CYP2D6 using baculoviral expression systems. AB - Cytochrome P-450 CYP2D6 plays a central role in the metabolism of many widely used therapeutic drugs including beta-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and used to express CYP2D6 in Spodoptera frugiperda (Sf9) cells. High levels of recombinant protein have been produced using either polyhedrin or basic protein promoters (0.05-0.20 nmol/mg cell protein; 0.05-0.15 nmol/liter). The enzyme is catalytically active toward CYP2D6 substrates such as bufuralol and metoprolol. In order to optimize catalytic activity human reductase was coexpressed with CYP2D6 in Sf9 cells; reductase activity was in the region of 1000-1500 units per mg cell protein, while spectrally active CY2D6 was in the range 10-20 pmol/mg cell protein. The K(m) and K(cat) values for bufuralol metabolism were estimated as 4.7 microM and 12.23 min-1, respectively. The use of the conventional very late promoters such as the polyhedrin promoter generate a large proportion of inactive CYP2D6. The problem was to a degree circumvented using the "late" basic promoter which is active earlier in the baculovirus infection cycle. The yield of functional CYP2D6 was at least as high as with very late promoters, but the proportion of inactive protein was reduced. Bufuralol hydroxylase activity could be measured directly by HPLC analysis of cell culture media supplemented with bufuralol, and we have developed a plate assay system which provides a simple method for the analysis of drug metabolism reactions using Sf9 cells. Expression using baculovirus provides a valuable source of functional CYP2D6 for work aimed at elucidating the structure and function of the enzyme. PMID- 8638922 TI - Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens. AB - The collagen fibrils of some ligaments attach to bone by passing through a zone that consists of nonmineralized and mineralized fibrocartilages. Very little, however, is known about the cells, the biochemical composition, the extracellular matrix organization, and function of these fibrocartilages. In this study, the collagens present in the fibrocartilages of the bovine medical collateral and anterior cruciate ligaments femoral attachments to bone were isolated, characterized, and their distribution at these sites was assessed by laser confocal microscopy. Types II, IX, and XI collagens were identified after pepsin digestion of the tissues in addition to the types I and V collagens. Immunoblotting using specific polyclonal antibodies confirmed the presence of types II and IX collagens at these sites. Immunofluorescence using confocal microscopy showed that type II collagen was prominent in the nonmineralized area and to a lesser extent in the mineralized zone of the insertion. Type IX collagen showed similar distribution as type II collagen. Type II collagen isolated from the ligament-bone interface contained half hydroxypyridinium cross-linking residues when compared to type II collagen isolated from articular cartilage of the same animals. These data indicate that the fibrocartilaginous zones at the ligament-bone interface are cartilaginous in nature. The cartilage collagens may play a role of anchoring the ligament to bone or the cartilage-like tissue may participate in the modulation of the mechanical stresses which are known to exist at the soft tissue-hard tissue interface. PMID- 8638924 TI - Purification and analysis of an 80-kDa carcinoembryonic antigen-binding protein from Kupffer cells. AB - The receptor-mediated interaction of Kupffer cells with carcinoembryonic antigen (CEA) has led to the identification of an 80-kDa CEA-binding Kupffer cell protein. This study is aimed at the isolation and analyses of this protein from rat Kupffer cells. The binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis (PPAGE), and affinity chromatography using a CEA-Sepharose column. Fractions obtained from the gel filtration produced two major and few minor peaks with CEA-binding activity. Maximum reactivity was detected in the first major peak. The first major peak protein was partially precipitated following fractionation with 30% loss of activity in the precipitate. Fractions with CEA-binding activity were pooled and separated on the basis of molecular weight (MW) in PPAGE. The fractions between MW 70 and 90 kDa were pooled and affinity purified using CEA-Sepharose affinity chromatography. The purity of the 80-kDa protein was demonstrated by a single protein band on SDS-polyacrylamide gel. The protein was further identified by an anti-80-kDa binding protein antibody in Western blot analysis. The pI of the 80 kDa protein is 4.95. Amino acid analysis demonstrated no histidine; higher percentages of glutamine (13.3%), leucine (11.2%), asparagine and alanine (10.4%), and lysine (9.2%) were observed. Protein microsequencing revealed two unique sequences, one with 16 amino acids and the other with 11 amino acids. The 16-amino-acid sequence has less than 50% homology with a large sample of unrelated proteins, whereas the sequence containing 11 amino acids has 60-70% homology with the alpha chain of collagen from a variety of species but no significant homology with other known proteins, suggesting the presence of collagen-like domains in the 80-kDa receptor. PMID- 8638925 TI - Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress. AB - We have previously shown that incubation of the model protein glucose-6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc mesenteroides with 4 hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein. HNE-modified protein is resistant to proteolytic degradation and acts as an inhibitor of the multicatalytic proteinase. It was therefore important to establish the chemistry of the cross linking reaction. The formation of cross-linked Glu-6-PDH is associated with the nearly exclusive loss of lysine residues. For this reason the reaction of N acetyllysine with HNE has been investigated. The epsilon-amino group of lysine reacts with the double bond (C3) and the carbonyl (C1) functions of HNE via Michael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link. Chromatographic detection of this adduct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that this chemistry is responsible for the formation of cross-linked protein. Antibody to the reduced form of the 2:1 lysine-HNE adduct was prepared. The antibody was used to demonstrate that exposure of isolated liver mitochondria to oxidative stress led to the formation of intra- and intermolecular protein-HNE cross-links. The results of the present study indicate that modifications to protein by lipid peroxidation products may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein. PMID- 8638926 TI - Purification and molecular characterization of a novel b5-type cytochrome of the parasitic nematode, Ascaris suum. AB - A b5-type cytochrome was extracted from Ascaris suum muscle at pH 4.5 with 0.3% aluminum sulfate and purified by ammonium sulfate fractionation, ion-exchange chromatography on CM-500 Cellulofine, and gel filtration on Sephadex G-75. The hemoprotein displayed a typical absorption spectrum of cytochrome b with a midpoint redox potential of 78 mV. The N-terminal amino acid sequence was determined and revealed the N-terminus to be highly homologous to the heme binding domain of vertebrate cytochrome b5. Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encoding A. suum cytochrome b5 was isolated from the lambda ZAP II cDNA library. The entire nucleotide sequence of 563 bases comprised an open reading frame of 339 bases encoding a precursor protein of 112 amino acid residues. The purified cytochrome B5 was predicted to contain 82 amino acids with a molecular mass of 9141 Da, matching the 9140 Da obtained from electrospray ionization mass spectometry, and to lack of membrane-anchor domain at the C-terminus. In contrast, an N-terminal extension of 30 amino acids, characteristic of signal peptides, was apparent. Immunoblots revealed the presence of an A. suum cytochrome b5 of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochrome b5 possessing a presequence. PMID- 8638927 TI - Phospholipase A2 from Naja naja sputatrix venom is a muscarinic acetylcholine receptor inhibitor. AB - A variety of snake venoms was tested for the ability to inhibit the binding of antagonists to specific muscarinic acetylcholine receptors (mAChRs); the highest activity was found in the venom of Naja naja sputatrix. The active principle in this venom was isolated by column chromatography on Sephadex G-50, Sephadex G 150, and CM-Sephadex C-25. The final preparation was homogeneous as determined by polyacrylamide gel electrophoresis and HPLC; about sevenfold purification was achieved with a yield of 12%. The isolated active component, which was designated "muscarinic inhibitor," was found to displace various antagonists from rat synaptosomal membranes, which contain all subtypes of mAChRs. The m1 and m2 recombinant human receptors were also competitive with N. naja sputatrix muscarinic inhibitor. This antagonist-displacing action was dose dependent, but was independent of the reaction temperature. The isolated muscarinic inhibitor was determined to be a 13.6 kDa, monomeric, neutral protein and to have an N terminal amino acid sequence which is highly homologous with phospholipase A2 from the venoms of genus Naja. N. naja sputatrix muscarinic inhibitor could hydrolyze phosphatidylcholine in a dose- and temperature-dependent manner. This phospholipase A2 enzymatic activity was augmented by the addition of the calcium ion, while it was almost completely abolished by a competitive inhibitor of phospholipase A2 enzymes. However, the antagonist-displacing activity was only slightly affected by these agents. The treatment of the muscarinic inhibitor with p-bromophenacyl bromide, which selectively modifies the histidine residue in the catalytic site of a phospholipase A2 enzyme, caused completed elimination of both activities. These findings indicate that N. naja sputatrix muscarinic inhibitor is a protein with two distinct activities, phospholipase and antagonist displacement, the active centers of which may be in close physical proximity for both actions. This is the first finding that a phospholipase A2 is an inhibitor of the muscarinic receptor. PMID- 8638928 TI - Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases. AB - Quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases. The crystal structures of Escherichia coli quinone oxidoreductase (QOR) and Thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase. A structurally based comparison of these three enzymes confirms that they possess extensive overall structural homology despite low sequence identity. The most significant difference is the absence of the catalytic and structural zinc ions in QOR. A multiple structure-based sequence alignment has been constructed for the three enzymes and extended to include zeta-crystallin, an eye lens structural protein with quinone oxidoreductase activity and high sequence identity to E. coli quinone oxidoreductase. Residues which are important for catalysis have been altered and the functions and activities of the enzymes have diverged, illustrating a classic example of divergent evolution among a superfamily of enzymes. PMID- 8638929 TI - Distinct effects of phenobarbital and its N-methylated derivative on liver cytochrome P450 induction. AB - The relationship between barbiturate structures and their effects on induction of rat cytochrome P450 forms was studied in primary cultured hepatocytes. Treatment of hepatocytes cultured on matrigel with 1 mM barbital, N-methylbarbital, cyclobarbital, hexobarbital, phenobarbital (PB), or mephobarbital (N-methyl-PB) resulted in increased amounts of CYP2B1/2 and CYP2C6 forms. Microsomal CYP3A content was also enhanced by treatment with these barbiturates, except for barbital. Although no relationship was observed between the levels of CYP2B1/2 and CYP3A, ratios of CYP3A/CYP2B1 plus CYP2B2 contents were invariably higher with hepatocytes treated with N-methylated barbiturates than with the nonmethylated analogs. Consistent results were also observed in vivo in rats treated with PB and N-methyl-PB. These results indicate the difference in the structure requirement for induction of CYP2B and CYP3A. In addition, N-methyl-PB was found to suppress PB-mediated induction of CYP2B1. Hepatic levels of CYP2B1 mRNA and protein were increased by treatment with PB or N-methyl-PB alone, but decreased by cotreatment with 1 mM PB and N-methyl-PB. The suppression has been shown to occur at the transcriptional level of the CYP2B1 gene by using a chloramphenicol acetyltransferase reporter-CYP2B1 fused gene system. PMID- 8638930 TI - Simultaneous isolation of endogenous digoxin-like immunoreactive factor, ouabain like factor, and deglycosylated congeners from mammalian tissues. AB - DLIF (digoxin-like immunoreactive factor) and OLF (ouabain-like factor) are endogenous steroid-like ligands (approximately 781 and 595 Da, respectively) with molecular and structural properties similar to the plant-derived cardiac glycosides, digoxin and ouabain. We developed a purification method with a sufficiently wide range of extraction solubility to separate compounds with polarities spanning those of ouabain and digoxin. This technique provides a rapid, reliable, and efficient method for simultaneously isolating DLIF, OLF, and several naturally existing deglycosylated congeners, including three deglycosylated species of DLIF (DLIF-genin, DLIF-mono, and DLIF-bis) and one deglycosylated species of OLF (OLF-genin). Separation is achieved using acid extraction, C-18 reverse-phase HPLC chromatography, and signal detection using two antibodies, one specific for digoxin and one for ouabain. The average extraction efficiency is 400 pmol digoxin equivalent (range 300-500) and 42 pmol ouabain equivalent (range 37-50) per gram of adrenal cortex for DLIF and OLF, respectively. The relative molar immunoreactivity of DLIF is 10(3)-fold less than that of digoxin, whereas that of OLF is unity compared to ouabain, suggesting that OLF is structurally more similar to ouabain than DLIF is to digoxin. Of interest is the presence of a compound reacting with both digoxin and ouabain antibodies. This unique immunoreactive species is liekly to have structural similarity to both digoxin and ouabain and thus may represent a metabolic link between DLIF and OLF. PMID- 8638931 TI - Metabolic activation of the potent carcinogen dibenzo[a,h]anthracene by cDNA expressed human cytochromes P450. AB - The metabolic activation of the potent carcinogen dibenzo[a,h]anthracene (DB[a,h]A) was investigated with recombinant human cytochrome P450 enzymes 1A2, 2B6, 2C8, 2C9, 2E1, 3A3, 3A4, and 3A5 expressed in hepatoma G2 cells and with 14 different human liver microsomes. Three dihydrodiols, three phenols, and one diphenol were formed and separated by high-performance liquid chromatography and identified by UV absorption and mass spectra. Of all P450s tested, 1A2 and 2C9 were the most active and 2B6 was moderately active in the rate of total DB[a,h]A metabolism (2.5- to 12-fold greater activity than that for other P450s). The trans-3,4-dihydrodiol, generally recognized as a precursor of the ultimate carcinogenic 3,4-diol-1,2-epoxides, was produced most actively by 2C9, then 1A2 and 2B6. The values of enzymatic kinetics (K(m) and V(max)) indicated that 2C9 had the highest catalytic efficiency (V(max)/K(m) = 9.7) in the formation of 3,4 dihydrodiol, in contrast to 1A2 (5.9) and 2B6 (4.4). 1A2 had the highest activity toward production of the 1,2-dihydrodiol, which is considered to be a weakly carcinogenic metabolite. Although specific activities of human liver microsomes in overall metabolism of DB[a,h]A markedly differed between individuals, metabolic patterns were observed similar to that generated from 1A2. Since human 1A1, a predominant enzyme for metabolism of polycyclic aromatic hydrocarbons, is not significantly expressed in the liver, hepatic microsomal 2C9, 1A2, and 2B6 all probably contribute to the metabolic activation of DB[a,h]A. PMID- 8638932 TI - Nitric oxide diffusion in membranes determined by fluorescence quenching. AB - Quenching of pyrene derivative fluorescence by nitric oxide was used to evaluate the apparent diffusion coefficients of nitric oxide in artificial and biological membranes. The apparent second-order quenching constants of nitric oxide were obtained from Stern-Volmer plots using methyl- and undecylpyrene derivatives incorporated into liposomes and erythrocyte plasma membranes in order to assess the ability of nitric oxide to interact with molecules located at different positions in the membrane. Diffusion coefficients were estimated from the determined second-order quenching constants and compared to that of oxygen obtained under the same conditions. Oxygen and nitric oxide presented similar diffusional behavior in agreement with their similarity in structures, with the differences observed attributable to the higher lipophilicity of oxygen compared to nitric oxide. In solution, both showed the same quenching efficiency while in liposomes and erythrocyte ghosts oxygen diffusion was twice that of nitric oxide (k(O2)/k(NO) = 2). Nitric oxide diffusion coefficients determined at 20 degrees C ranged from 1.3 x 10(-5) cm2 s-1 in liposomes to 0.4 x 10(-5) cm2 s-1 in surface erythrocyte plasma membranes. Both nitric oxide and oxygen had larger quenching constants for the undecyl derivative compared to the methylpyrene compound incorporated into erythrocyte plasma membranes, indicating an increased solubility of both gases toward the center of the membrane. PMID- 8638933 TI - Effect of NADPH-associated keto-reducing domain on substrate entry into 6 hydroxymellein synthase, a multifunctional polyketide synthetic enzyme involved in phytoalexin biosynthesis in carrot. AB - 6-Hydroxymellein synthase is a polyketide biosynthetic enzyme induced in carrot cells which is organized as a homodimer composed of multifunctional subunits. The synthase liberates triacetic acid lactone, instead of 6-hydroxymellein, as a derailment product when the keto-reducing reaction at the triketide intermediate stage is interrupted. However, the efficiency of the triacetic acid lactone forming reactions is markedly lower than that of the normal reaction, and the kinetic analyses have revealed that the affinity of the enzyme protein for acetyl CoA is appreciably reduced in the abnormal reactions. It is assumed that the interaction of the NADPH-associated keto-reducing domain with a putative primary binding site(s) of the acyl-CoA in the enzyme structure affects the entry of the starter unit into the protein. The present finding should provide an example of the novel class of "subunit communication" of multimer enzymes. PMID- 8638934 TI - Anomeric specificity of the native and mutant forms of human beta-cell glucokinase. AB - The anomeric specificity of wild-type human beta-cell glucokinase and six of its mutant forms toward alpha- and beta-D-glucose was examined over 6-min incubation at 30 degrees C. When D-[U-14C]glucose at anomeric equilibrium was used as substrate, the wild-type form yielded a maximal velocity of 76 U/mg, a K(m) of 4 5 mM, and a Hill coefficient close to 1.2. The maximal velocity (2 to 89 U/mg) and K(m) (2.4 to 209.8 mM) of the mutant forms both covered a range of about two orders to magnitude. Wild-type glucokinase displayed a higher affinity for alpha D-glucose but greater maximal velocity with beta-D-glucose. A variance, however, in four mutant forms, the maximal velocity was higher with alpha- than beta-D glucose. These findings indicate that the higher insulinotropic efficiency of alpha- than beta-glucose cannot be ascribed to the intrinsic catalytic properties of human beta-cell glucokinase. They also suggest that the perturbation of the anomeric specificity of glucose-stimulated insulin release in type-2 diabetes could conceivably be attributable, on occasion and at least in part, to a mutation of the glucokinase gene. PMID- 8638935 TI - Oxidation of omega-oxo fatty acids by cytochrome P450BM-3 (CYP102). AB - Cytochrome P450 enzymes oxidize aldehydes either to the corresponding acid or, via a decarboxylation mechanism, to an olefin one carbon shorter than the parent substrate. To explore the factors that control partitioning between these two pathways, we have examined the cytochrome P450BM-3 (CYP102)-catalyzed oxidation of fatty acids with a terminal aldehyde group. P450BM-3 oxidizes 18 oxooctadecanoic, 16-oxohexadecanoic, 14-oxotetradecanoic, and 12-oxododecanoic acids exclusively to the corresponding alpha,omega-diacids. The rates of these oxidations decrease in the order C16 > C18 approximately = C14 > C12. No kinetic isotope effect is observed nor is the catalytic outcome altered when the aldehyde hydrogen is replaced by a deuterium in 16-oxohexadecanoic acid. The only product observed with 16-oxohexadecanoic acid is the diacid even when a 13,14-double bond or 15-methyl groups, substitutions that should stabilize the proposed radical intermediate generated by decarboxylation, are present. The oxidation of 16 oxohexadecanoic acid is not supported by H2O2. The results demonstrate that aldehyde oxidation by cytochrome P450BM-3 is insensitive to changes in substrate structure expected to stabilize the transition state for decarboxylation. Decarboxylation, in contrast to the oxidation of aldehydes to acids, depends on specific substrate-protein interactions and is enzyme-specific. PMID- 8638936 TI - Differential phosphorylation of human tau isoforms containing three repeats by several protein kinases. AB - The paired helical filaments (PHF) found in the brain of patients with Alzheimer disease (AD) are composed primarily of the microtubule-associated protein tau. Six isoforms of tau have been recognized and all are present in a hyperphosphorylated state in PHF. It is not known whether all tau isoforms serve equally well as substrates for various kinases. In this study we have compared the phosphorylation of human tau isoforms containing three microtubule-binding repeats and zero (tau 3), one (tau 3S), or two (tau 3L) N-terminal inserts. Four kinases (A-kinase, CK-1, CaM kinase II, GSK-3) were used for this purpose. With A kinase, CK-1, and CaM kinase II the extent of phosphorylation was tau 3L > tau 3S > tau 3. With GSK-3 it was tau 3L approximately = tau 3S > tau 3. Tau 3 was a poor substrate for either CaM kinase II or CK-1, 32P incorporation being only 5 and 11%, respectively, of that observed by these kinases when tau 3L was the substrate. After prephosphorylation of the three tau isoforms by A-kinase, a subsequent phosphorylation by GSK-3 was stimulated several fold over tau that was not prephosphorylated. Under these conditions the extent of 32P incorporation was tau 3L > tau 3S > tau 3. Both CK-1 and GSK-3 phosphorylated ser 396 more rapidly in tau 3L compared to tau 3 or tau 3S. Our results suggest that (1) the presence of N-terminal inserts in tau isoforms are important structural determinants that modulate the specificity of several kinases; (2) the different tau isoforms may be present at different states of phosphorylation in PHF. PMID- 8638938 TI - Stoichiometric conversion of all trans-beta-carotene to retinal by pig intestinal extract. AB - beta-Carotene and other provitamin A carotenoids are major dietary sources of vitamin A for humans and for many animals throughout the world. Two pathways for this oxidative reaction in mammals are: (1) central cleavage by beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) to yield two molecules of retinal per molecule of beta-carotene consumed and (2) eccentric (or random) cleavage via several beta apo carotenals to yield one molecule of retinal and various smaller fragments per molecule of beta-carotene oxidized. By the use of improved methods to minimize nonenzymatic reactions and to measure isomers of retinal, the mean experimentally measured molar ratio of retinal formed to beta-carotene consumed by pig intestinal preparations (800 and 10,000g supernatants) was 1.88 +/- 0.08, close to the theoretical value of 2.0 for central cleavage. beta-Apo carotenals, retinol, and retinoic acid were detected, if at all, in trace amounts during incubation. Thus, preparations of pig intestinal mucosa, which is considered a good physiologic model for human intestine, clearly convert beta-carotene to retinal, in large part if not solely, by central cleavage. Whether eccentric cleavage plays a greater role in vivo than it does in vitro is still uncertain. PMID- 8638937 TI - Destruction of cholera toxin receptor on HeLa cell membrane using microbial endoglycoceramidase. AB - The sensitivity of HeLa cells to cholera toxin decreased by Corynebacterium sp. endoglycoceramidase treatment. This endo-enzyme destroyed the cholera toxin receptor, ganglioside G(M1), on the cell surface membrane by liberating intact oligosaccharide from it, which was confirmed by the decrease of intracellular cAMP accumulation and the results of the analysis of released oligosaccharide with a combination of pyridylamination method and HPLC. Fluorescence microscopy using the immunofluorescence method revealed that the amount of cholera toxin attached to the cells decreased in endoglycoceramidase-treated cells. The enzyme acted on cellular glycosphingolipids without addition of any activator protein which is required by other similar enzymes. Corynebacterium endoglycoceramidase is a useful tool to elucidate the function of glycosphingolipids on the cell surface in situ. PMID- 8638939 TI - Catabolism and loss of proteoglycans from cultures of bovine collateral ligament. AB - This paper investigates the kinetics and mechanism of loss of the two major proteoglycan species from cultures of bovine collateral ligament. Following incubation of ligament with [35S]sulfate after 6 days in culture, the rate of loss of the predominant proteoglycan species, decorin, from the matrix was shown to be much slower (t1/2 approximately 18 days) than that of the large chondroitin sulfate proteoglycan (t1/2 approximately 1.4 days). Analysis of 35S-labeled proteoglycans released into the medium between Days 11 and 15 of the culture period on a column of Sepharose CL-4B revealed that these macromolecules constituted mainly decorin of similar hydrodynamic size to that present in the matrix. Furthermore, analysis of core proteins using gel electrophoresis followed by fluorography or immunodetection with LF-94, an antibody directed against the amino-terminal region of decorin, indicated that the core proteins of decorin released into the medium and those remaining in the matrix of ligament cultures had a similar molecular mass (approximately 49 kDa). Analysis of both the 35S labeled and endogenous macromolecules using 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides, revealed that three core proteins with molecular masses greater than approximately 200 kDa were present in the matrix. Four additional core proteins (range approximately 80-200 kDa) derived from the large proteoglycan were detected in the medium of ligament cultures. These findings indicate that, unlike decorin, the loss of the large chondroitin sulfate proteoglycan from the matrix of ligament cultures involved proteolytic cleavage of its core protein. No difference in the hydrodynamic size of the 35S-labeled glycosaminoglycan chains derived from either proteoglycan species remaining in the matrix or released into the medium of ligament cultures was observed. PMID- 8638940 TI - Glycosylation and phosphorylation of lysosomal glycosylasparaginase. AB - Glycosylasparaginase (EC 3.5.1.26) is a lysosomal amidase which hydrolyzes the bond between asparagine and the sugar moiety in N-linked glycoproteins. Deficiency of the enzyme results in aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation. Mature enzyme is formed by two proteolytic cleavage steps subsequent to removal of its signal peptide: (1) an activation cleavage in the ER of the initial single-chain 49-kDa polypeptide into a 27-kDa alpha- and 19-kDa beta-subunit; (2) a cleavage in lysosomes which removes 10 amino acids from the C-terminus of the alpha-subunit without affecting enzyme activity. Each subunit of glycosylasparaginase contains one N-linked oligosaccharide (N38, alpha-subunit; N308, beta-subunit). Both oligosaccharides were phosphorylated and releasable by Endo-H digestion, indicating they were of the high-mannose type. These glycosylation sequenons were mutagenized to determine the role of the oligosaccharide at each site in proper folding and transport of glycosylasparaginase. An N38D mutant underwent the lysosomal processing step, indicating that targeting to lysosomes can be via the phosphorylated beta-subunit oligosaccharide alone. Deletion of the beta-subunit oligosaccharide oat N308 by an aspartic acid substitution resulted in very little protein or enzyme activity in the transfected cells, reemphasizing that glycosylation of the beta-subunit site is important for efficient folding and/or targeting. A different mutation to eliminate the same N-glycosylation sequenon (T310A) yielded more protein and enzyme activity, and a double mutant N38D/T310A yielded the same results as the single beta-subunit substitution. Yield of enzyme for all mutants was increased in cells treated with brefeldin A. The N308 glycosylation site of the beta-subunit appears to be more important in maintaining normal transport and stability of human glycosylasparaginase. PMID- 8638941 TI - Stability and folding of precursor and mature tryptophan-substituted ribose binding protein of Escherichia coli. AB - A mutant ribose binding protein (RBP) of Escherichia coli was obtained by site directed mutagenesis, replacing Phe-187 in the wild-type RBP (WT-RBP) with a Trp residue, in order to compare its stability and folding behavior with those of the WT-RBP. The equilibrium unfolding properties and the folding kinetics of these proteins were monitored by fluorescence and circular dichroism (CD). For both WT RBP and the Trp-substituted RBP (Trp-RBP), the conformational stabilities of the precursor proteins and the mature proteins were the same, indicating that the signal peptide had no influence on the property of the mature domain. The Phe/Trp substitution in the mature domain, however, brought about a significant decrease in the conformational stability. The signal peptide had an appreciable retarding effect on the folding of the precursor Trp-RBP as was reported for the WT-RBP. Refolding kinetics of the WT-RBP and Trp-RBP showed a two-step reaction when monitored by fluorescence and by CD. PMID- 8638943 TI - Rate of translation of natural mRNAs in an optimized in vitro system. AB - We report results on in vitro translation of an mRNA coding for elongation factor TuB which was in vitro transcribed from the tufB gene from Escherichia coli. Translation occurs at a rate of about 10 codons per second, which is close to the in vivo rate. Protein elongation obeys Michaelis-Menten kinetics with respect to the concentrations of the elongation factors EF-Tu and EF-G in the translation system. The measured K(m) values for EF-Tu and EF-G are 10 and 0.25 microM, respectively. The obtained k(cat) and K(m) values were used to estimate the average k(cat)/K(m) of about 24 x 10(6) s-1 M-1 for the interaction of individual EF-Tu*GTP*aa-tRNA complexes with ribosomes. The estimated k(cat)/K(m) value for EF-G is 36 x 10(6) s-1 M-1. We have also studied translation with a "hyperaccurate" ribosome variant that is pseudodependent on streptomycin (SmP). We have found that SmP ribosomes translate the TuB mRNA significantly slower than wild-type ribosomes do. This is mainly due to a threefold lower k(cat)/K(m) for the interaction of EF-Tu*GTP*aa-tRNA complexes with SmP ribosomes. PMID- 8638942 TI - Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. AB - Nitric oxide (.NO) released by S-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective .NO concentration of 0.1 microM, while succinate- and NADH-cytochrome-c reductase activities were half-maximally inhibited at 0.3 microM .NO. Submitochondrial particles treated with .NO (either from GSNO or from a pure solution) showed increased O(-)(2) and H202 production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with .NO also showed increased H2O2 production. Cytochrome spectra and decreased enzymatic activities in the presence of .NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone-cytochrome b region of the respiratory chain, the latter leading to the increased O2- production. Electrochemical detection showed that the buildup of a .NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of .NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial O2- reacts with .NO to form peroxynitrate and, by removing .NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of .NO, inhibition of electron transfer, .NO-induced O2- production, and ONOO- formation participate in the regulatory control of mitochondrial oxygen uptake. PMID- 8638944 TI - Quantification of the carbon flow through the folate-dependent one-carbon pool using radiolabeled histidine: effect of altered thyroid and folate status. AB - Hyperthyroidism and folate deficiency are known to alter the concentration of hepatic folate pools and the activity of a number of enzymes related to one carbon metabolism. To evaluate the physiological significance of this relationship on a functional basis, we have developed and utilized an in vivo tracer kinetic technique to quantify the carbon flow through the one-carbon pool as a function of thyroid and folate status. Control, hyperthyroid, and folate restricted rats were continuously infused with L-[ring-2-14C]histidine to measure the oxidative flow of carbon from histidine through the one-carbon pool to CO2. As expected, the hepatic activities of a number of enzymes involved in the catabolism of histidine to CO2 were markedly decreased in hyperthyroid (histidase, 69%; urocanase, 30%; 10-formyltetrahydrofolate dehydrogenase, 65%) and folate-restricted (10-formyltetrahydrofolate dehydrogenase, 44%) rats. In addition, folate-restricted animals exhibited a 63% decrease in the hepatic concentration of total reduced folates. However, tracer kinetic analysis indicated an enhanced catabolism of histidine: the carbon flux from histidine to CO2 was increased approximately threefold in hyperthyroid rats and twofold in folate-restricted animals. Thus, in the case of hyperthyroidism and dietary folate restriction, changes in static measurements such as metabolite concentrations and enzyme activities do not reflect the dynamic tracer kinetic assessment of the carbon flux that is actually occurring in vivo. The kinetic data also demonstrate that the percentage of total entry into the 10 formyltetrahydrofolate pool originating from histidine catabolism was almost threefold greater compared to 5-methyltetrahydrofolate pool. PMID- 8638945 TI - Oxidant-inducible adapt 15 RNA is associated with growth arrest- and DNA damage inducible gadd153 and gadd45. AB - We have recently described a novel RNA, designated adapt15, which is strongly induced by a pretreatment concentration of hydrogen peroxide in hamster HA-1 fibroblasts under conditions where a protective "adaptive response" occurs. adapt15 may therefore be involved in protecting cells against the damaging effects of oxidative stress. Since two other known sequences, gadd45 and gadd153m were also found to be induced under our pretreatment conditions and are known to be growth arrest and DNA damage inducible, we decided to also assess the possible association of adapt15 with growth arrest and DNA damage. We found that, like gadd45 and gadd153, the levels of adapt15 RNA were low during proliferation, but high during density saturation- and low serum (G0)-growth arrests. Exposure of HA 1 cells to DNA-damaging agents revealed significant induction of adapt15 RNA by methylmeth- anesulfonate and cis-platinum but not X-irradiation. Near identical responses were also observed for gadd45 and gadd153 RNAs, suggesting coordinate regulation of adapt15, gadd45, and gadd153. All three RNAs were also increased relative to control following heat shock, a nongenotoxic treatment. Finally, the induction of adapt15 by hydrogen peroxide was strongly dependent upon calcium, a hallmark of gadd153 induction. The coordinate inductions of adapt15, gadd45, and gadd153 by multiple agents, and their induction by an adaptive- but not by a nonadaptive-response level of hydrogen peroxide, suggest these RNAs may act in concert to protect cells against the damaging effects of oxidative stress. PMID- 8638946 TI - Characterization of the human plasmalemmal carnitine transporter in cultured skin fibroblasts. AB - Carnitine is an essential cofactor for long-chain fatty acid oxidation. We characterized the human carnitine transporter in vitro in a cultured skin fibroblast model both at the previously established Km concentration of carnitine uptake in fibroblasts (5 mumol/liter) and at 0.05% Km (0.25 mumol/liter). A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake was demonstrated with increasing concentrations of nigericin, but no significant decrease was found with equimolar amounts of valinomycin. This would suggest that the Na+ gradient is integral to carnitine transport function. Interference of the Na+ (out-in) gradient by nigericin may be secondary to cytoplasmic acidification by this K+ proton ionophore. The rate of uptake was fully saturated at an extracellular Na+ concentration of 150 mmol/liter. Replacement of 150 mmol/liter extracellular Na+ with Li+ resulted in an 80 and a 50% reduction, and replacement with K+ and Rb+ ions resulted in a 100 and an 85 to 90% reduction in carnitine uptake, respectively, at carnitine concentrations of 0.25 and 5 mumol/liter, underlining the specific requirement for the Na+ ion. The effects of different site-specific respiratory chain toxins, namely, rotenone (complex I), antimycin A (complex III), and potassium cyanide (KCN) (complex IV) on carnitine uptake was also examined. A rapid exponential dose-dependent decrease in mean percentage of carnitine uptake with increasing concentrations of inhibitors was demonstrated. These data suggest either a metabolic energy requirement of the carnitine transporter or interference of the Na+ (out-in) gradient by a proton gradient (in out) secondary to the accumulation of intracellular H+ ions, due to the action of the respiratory chain toxins, further suggesting that the transporter is sensitive to and inhibited by intracellular H+ ions. The effects of several sulfhydryl-binding agents, namely 2,4-dinitrofluorobenzene, N-ethylmaleimide, and mersalyl acid, were examined, and a significant inhibition of carnitine uptake was demonstrated, suggesting that free sulfhydryl groups are also integral to the import function of the human fibroblast transporter. PMID- 8638947 TI - (i, i + 4) Ion pairs stabilize helical peptides derived from smooth muscle caldesmon. AB - The central region of smooth muscle caldesmon contains 10 repeats of a 13-amino acid residue motif that is rich in charged side chains. This region has been predicted to have a strong alpha-helical propensity, and the helical structure was thought to be stabilized by the interactions between oppositely charged side chains of residues at positions i and i + 4 (Wang, C.-L. A., Chalovich, J.M., Graceffa, P., Lu, R.C., Mabuchi, K, and Stafford, W.F., J.Biol. Chem. 266, 13958 13963, 1991). Two synthetic peptides corresponding to these repeats, each containing 25 and 60 amino acid residues, indeed assume an alpha-helical conformation that is stable over a wide range of salt concentration and pH, and exhibit a typical helix-coil transition upon heating. Most significantly, when the amino acid sequence of the 25-mer is randomized without losing the i-to-i + 4 ion pairs, the peptide maintains a helical content comparable to that of the wild type peptide, whereas another peptide variant with a sequence rearranged to eliminate all i-to-(i + 4) ion pairs has much less helicity, suggesting that specific interactions between residues with (i,i + 4) spacings are important determinants for the maintenance of secondary structure in this region of caldesmon. PMID- 8638948 TI - Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8 tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls. AB - Hepatic uroporphyria is a well-known effect of halo- genated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; IUPAC 77) when the heme precursor delta-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (approximately 80%) and heptacarboxylporphyrin (approximately 20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl (IUPAC 81) and 3,3',4,4',5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4'5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin 0-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this prophyria. Addition of 3,3',4,4'-TCB to TCDD treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model System for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists. PMID- 8638949 TI - Tyrosinase-catalyzed oxidation of 3,4-dihydroxyphenylglycine. AB - The oxidation chemistry of dopa in relation to melanin biosynthesis has been extensively studied. However, the oxidation of its lower homolog viz., 3,4 dihydroxyphenylglycine has not been described. Using 3,4-dimethoxybenzaldehyde as the starting material, the chemical synthesis of 3,4-dihydroxyphenylglycine was accomplished by Strecker's synthesis and demethylation reactions. Tyrosinase readily oxidized this unusual amino acid to the expected quinone. The glycyl-o benzoquinone thus formed was highly unstable like its higher analog, dopaquinone. However, unlike its counterpart, it failed to exhibit intramolecular cyclization reaction. Rather, glycyl-o-benzoquinone exhibited facile transformation(s) to ultimately generate 3,4-dihydroxybenzaldehyde as an isolatable product. A probable mechanism involving intermediary formation of unstable quinone methide and carbinolamine intermediates is proposed to account for the novel transformation of glycyl-o-benzoquinone to 3,4-dihydroxybenzaldehyde. PMID- 8638950 TI - Characterization of a large chondroitin sulfate proteoglycan present in bovine collateral ligament. AB - Bovine collateral ligament synthesized a 35S-labeled large proteoglycan species which eluted with a Kav of approximately 0.27 on Sepharose CL-2B and contained only chondroitin sulfate chains with a molecular mass of approximately 32 kDa. Fluorography of the 35S-labeled core proteins derived from the large ligament proteoglycan revealed a broad range of molecular masses above approximately 200 kDa, which was of comparable size to the four major endogenous core protein bands derived from this proteoglycan detected with 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides. The core proteins derived from the large ligament proteoglycan exhibited immunoreactivity of 12/21/1-C-6, an antibody specific for a peptide epitope common to both the G1 and G2 domains of aggrecan. Four major core protein bands with molecular masses greater than approximately 200 kDa derived from the large ligament proteoglycan, were detected using the antibodies raised against versican from bovine aorta or human fibroblasts. Compared with aggrecan, the 35S-labeled large ligament proteoglycan was distributed over a broader range of buoyant densities in an associative caesium chloride density gradient. This polydispersity may be indicative of differences in the degree of glycosylation as well as heterogeneity in the size of the large ligament proteoglycan core proteins. The 35S-labeled large ligament proteoglycan also demonstrated the ability to form complexes with an aggrecan aggregate preparation, the majority of which could not be dissociated by the presence of HA10-50. These findings indicate that the large chondrotin sulfate proteoglycan synthesized by bovine collateral ligament may be a versican like proteoglycan which exhibited the potential to form like protein-stabilized complexes. PMID- 8638951 TI - Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8 methoxypsoralen. AB - We report the formation of photocrosslink (diad-duct) between duplex poly(dA-dT) and 8-methoxypsoralen inferred on the basis of UV and fluorescence characteristics, Tm, nuclease S1 digestibility, and hydroxyapatite column chromatography. The photocrosslink was highly immunogenic inducing high titer antibodies in goat. The affinity constant of antigen-antibody interaction quantitated by precipitin titration was found to be 2.71 x 10(9) M-1, calculated by Langmuir isotherm plot. These antibodies were highly specific showing recognition of almost exclusively of nucleic acid-furocoumarin photoadducts and could be used as a probe to detect and quantitate DNA modified with furocoumarins in health and disease. Antibodies against poly(dA-dT)-furocoumarin photocrosslink might be a better probe because of repeated A-T sequences on the immunogen. PMID- 8638952 TI - A role for molecular oxygen in the formation of DNA damage during the reduction of the carcinogen chromium (VI) by glutathione. AB - Although well-established as carcinogens, the way in which chromium (VI) compounds exert their carcinogenic, mutagenic, and DNA-damaging potential remains obscure. It is clear that inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH). The present study is intended to clarify if Fenton mechanisms are likely to be important in the formation of DNA lesions by chromium(VI) in combination with GSH. In buffer solutions which were treated to remove Fenton-active metal ions as well as in those not further purified, chromate and GSH induced similar numbers of single strand breaks (SSB) in isolated PM2 DNA. Molecular oxygen was found to be essential for the formation of SSB, but chromium(V) species arising from chromate/GSH, unless activated by oxygen, appeared to be unreactive toward DNA. Upon addition of Mn(II) to solutions of chromium(VI) and GSH a diminution of Mn(II) ESR signals was observed, good evidence for the presence of chromium(IV) species. Using gas chromatography/mass spectrometry in selective ion-monitoring mode and high-performance liquid chromatography with electrochemical detection, we were able to show that Cr(VI)/GSH failed to induce base modifications typical of hydroxyl radical attack on DNA. Experimental conditions which readily induced SSB gave rise to the formation of chromium-DNA adducts, clearly demonstrating that the generation of these two DNA lesions is not mutually exclusive. We conclude that models which ascribe the induction of chromium-DNA adducts to chromium(V) and the generation of oxidative DNA damage including SSB to hydrogen peroxide are oversimplistic. It is not necessary to invoke a mechanism requiring the presence of added hydrogen peroxide to account for the ability of Cr(VI)/GSH to cause SSB. Our findings suggest that the combination of GSH, molecular oxygen, and chromium(VI) can damage DNA via non-Fenton pathways. PMID- 8638953 TI - Antibody binding to a peptide but not the whole protein by recognition of the C terminal carboxy group. AB - Antipeptide antibodies have become indispensable tools in modern biochemistry and molecular biology. Unfortunately, not all antipeptide antibodies react with their target proteins. The reasons why certain antipeptide antibodies fail to do so are not always clear, although it is commonly assumed that conformational difference between the peptide antigens and the corresponding sequences in proteins accounts for most failures. Here, we report detailed characterization of an antipeptide mAb which reacted avidly with the peptide antigen but did not react with the same sequence in a protein. ELISA analysis using analogs of the antigen peptide revealed that this mAb did not react with a C-terminus-extended analog of the antigen peptide and reacted poorly with a peptide amide analog of the antigen peptide. These results suggest that the mAb recognizes an epitope including the C terminal-free carboxyl group of the peptide. This analysis also revealed that the epitope recognized by this mAb was located in the C-terminal pentapeptide, RY IRS. Four amino acid side chains (R,I,R, and S) in this pentapeptide were shown by alanine-scanning to be critical for antibody recognition. Analysis of the polyclonal antisera raised against this peptide revealed that antibodies reacting with this unique carboxyl-containing epitope are most abundant. This unexpected finding has since been shown in several other cases in this laboratory, suggesting that generation of antibodies that recognize carboxyl-containing artificial epitopes may be rather common. we also found that the use of a peptide amide (instead of peptide acid) antigen did not prevent a similar problem; in this case, the C-terminal amide became part of the epitope. Based on these findings, we suggest a method for enhancing the probability of isolating protein reactive mAbs. PMID- 8638954 TI - A universal tag for recombinant proteins. AB - Incorporation of tags into recombinant proteins can facilitate their identification and purification. In addition, these tags can also be used to monitor the trafficking or localization of the recombinant proteins inside the cells. Several such tags have been developed. However, the lengths of these tags make it cumber-some to incorporate them into the desired proteins. Typically, one must subclone the desired cDNA into a plasmid containing the tag sequence at a suitable restriction site or ligate a synthetic oligonucleotide containing the tag sequence at a suitable restriction site in the cDNA of the desired protein. These manipulations can be avoided, if one uses a short peptide tag that can be incorporated by PCR. We show here that a short peptide tag, RYIRS can be easily incorporated at the C termini of recombinant proteins by PCR. We also showed that by using a mAb specific for this peptide sequence, the tagged proteins could be easily detected in Western blot analysis, immunofluorescence staining, and immunoprecipitation. The interactions between this tag sequence and the mAb have been well characterized. One can take advantage of this information and control the reactivities between the tagged proteins and the mAb by varying the lengths of the peptide tags. Furthermore, we showed that this tag can be used to monitor whether a recombinant protein is properly translated and terminated because the interactions between this tag sequence and the mAb requires that the tag be at the C-terminus of the protein. PMID- 8638955 TI - Photo-enhanced production of the spin adduct 5,5-dimethyl-1-pyrroline-N-oxide/.OH in aqueous menadione solutions. AB - The production of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/.OH in aqueous solutions of menadione and DMPO is enhanced by fluorescent room light. The formation of DMPO/.OH requires oxygen and water, is enhanced by superoxide dismutase, and occurs to a much smaller extent for benzoquinone than for menadione. This process is assigned as photo-initiated redox cycling of the menadione, which causes oxidation of DMPO to DMPO+. and reduction of oxygen to superoxide. DMPO+. reacts with water to produce DMPO/.OH. Although DMPO/.OOH was not observed in the menadione solutions, the possibility that some of the DMPO/.OH was produced by decomposition of DMPO/.OOH cannot be ruled out. There is no evidence for participation of hydroxyl radicals. Because benzosemiquinone is less readily oxidized than the semiquinone of menadione, redox cycling is less favorable for benzoquinone than for menadione and smaller quantities of DMPO/.OH are produced by photoexcitation of benzoquinone than of menadione. PMID- 8638956 TI - 1 alpha,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. AB - Several 1 alpha,25-(OH)2-vitamin D3 (1 alpha,25-(OH)2-D3) analogs have significant antiproliferative effects in vitro but do not elevate serum calcium in vivo. We tested whether the lack of a calcemic response of a vitamin D analog in vivo is due to its inability to stimulate intestinal calcium absorption by examining the effect of several such compounds on transepithelial calcium transport in the human colonic carcinoma cell line Caco-2. The relative stimulations of calcium transport by the four A-ring diastereomers of 1 alpha, 25 (OH)2-D3 (1 alpha,3 beta) and a 3 beta-bromoacetate analog (1 alpha,3 beta-BrAc) of the vitamin following 48-h treatment of cells at 10 nM were 1 alpha,3 beta (=100%), 1 alpha,3 alpha (+45.2%), 1 beta,3 beta (-15.6%), 1 beta,3 alpha (+6.5%), and 1 alpha,3 beta-BrAc (+50.6%). This was similar to the reported affinity of these compounds for the vitamin D receptor (VDR) and suggests that VDR binding predicts calcium transport. In contrast, three noncalcemic, sidechain or D-ring-modified analogs of vitamin D, 1 alpha,25-(OH)2-16-ene-D3, 1 alpha,25 (OH)2-16-ene-23-yne-D3, and 1 alpha,25,28-(OH)3-D2 (at 10 nM for 48 h), showed a different relationship between VDR affinity (150, 60, and 63% of 1 alpha, 25 (OH)2-D3, respectively) and calcium transport (74.1, 126, and 10%, respectively). Elevated calcium transport was accompanied by higher 24-hydroxylase and calbindin D9k mRNA levels. The data demonstrate that although some vitamin D compounds cannot stimulate calcium transport due to an inability to interact with the VDR (e.g., 1 beta isomers), other factors, e.g., differential cellular metabolism, may account for variations in biological response in vivo to various vitamin D analogs. PMID- 8638957 TI - Establishment of transgenic mice carrying human fetus-specific CYP3A7. AB - CYP3A7 is a cytochrome P450 isozyme expressed prenatally in humans. Six lines of mice transgenic for human CYP3A7 were established by microinjecting a CYP3A7 cDNA downstream of a mouse metallothionein-1 promoter gene into the male pronucleus of fertilized mouse oocytes. The inserted CYP3A7 transgene was expressed at a mRNA level in a variety of tissues including the liver, kidney, lung, spleen, testis, small intestine, thymus, brain, skin, and heart of adult mice. The protein expression of the transgene was also detected in the liver and testis of line M10 mice. A significantly higher level of total testosterone in the serum was found in line M10 male mice. In addition, this transgenic line exhibited weight increases in the liver, kidney, and uterus but a decrease in the testis (P < 0.01). The transcript of the integrated CYP3A7 gene possessed the ability to activate aflatoxin B1 in Ames test in which the his+ revertants of Salmonella typhimurium TA100 per plate were significantly higher (P < 0.01) when liver microsomes of line M10 transgenic mice were used. This result demonstrates that the CYP3A7 gene has been integrated into the mouse genome and translated into a catalytically active enzyme. These transgenic mice are expected to give useful information for studies on fetal toxicities in humans. PMID- 8638958 TI - Length-dependent modulation of myosin phosphorylation and contractile force in coronary arterial smooth muscle. AB - Phosphorylation of the 20,000 dalton myosin light chain (LC) is a central regulatory mechanism of smooth muscle contraction. Our previous findings on airway smooth muscle have led us to hypothesize that length-dependent and length independent modulation of myosin phosphorylation coexist in smooth muscle. In this study, we tested the general applicability of this hypothesis by investigating the length dependences of myosin phosphorylation and contractile force in bovine coronary arterial smooth muscle. Comparison of the time courses of myosin phosphorylation at optimal (Lo) and preshortened lengths indicated that the initial peak myosin phosphorylation induced by K(+)-depolarization, histamine, and endothelin were all length dependent. Additional experiments focusing on the length dependence of steady-state myosin phosphorylation revealed that the length dependence of K(+)-depolarization-induced steady-state myosin phosphorylation (0.13 mol P(i)/mol LC/Lo) was significant, but smaller than that found in airway smooth muscle. In contrast, the length dependence of endothelin induced steady-state myosin phosphorylation (0.03 mol P(i)/mol LC/Lo) was insignificant. The different length dependences of depolarization- and endothelin induced myosin phosphorylation were found to correlate with different length force relations. The length-force relationships in K+-depolarized and endothelin activated tissues remained different even when the force was normalized by the maximum value at Lo. Variable length-force relations have been reported but the biochemical basis is not understood. Results from this study suggest that length dependent modulation of myosin phosphorylation may be an important determinant of length-force relationship in smooth muscle. PMID- 8638959 TI - Evaluation of serum lipid concentrations among U.S. workers exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin alters lipid metabolism in animals; however, evidence for such an effect in humans is conflicting. This conflict was addressed using data from a cross-sectional medical study conducted between 1987 and 1988. The exposed participants had been employed at least 15 y earlier in the manufacture of 2,4,5-trichlorophenol or one of its derivatives at two chemical plants in the United States. A total of 281 workers and 260 unexposed referents participated. Workers had substantial exposure to 2,3,7,8-tetrachlorodibenzo-p dioxin, evidenced by a median serum 2,3,7,8-tetrachlorodibenzo-p-dioxin concentration of 406.6 femtograms/gram of serum (fg/g serum), compared with 36.9 fg/g serum among the referents. A slight association between triglyceride concentration and serum 2,3,7,8-tetrachlorodibenzo-p-dioxin concentration was found (p = .05). Over the range of observed 2,3,7,8-tetrachlorodibenzo-p-dioxin values (i.e., 37-19000 fg/g serum), triglyceride concentration increased only about 0.4 mmol/I. No association was found between an abnormally elevated triglyceride (i.e., > 2.82 mmol/I) concentration and serum 2,3,7,8 tetrachlorodibenzo-p-dioxin concentration. An association was also found between serum 2,3,7,8-tetrachlorodibenzo-p-dioxin concentration and an abnormal high density lipoprotein concentration (p = .09). in summary, there was evidence of an effect on lipid metabolism in a group of workers with high serum 2,3,7,8 tetrachlorodibenzo-p-dioxin concentrations. The influence of serum 2,3,7,8 tetrachlorodibenzo-p-dioxin on lipid concentrations, however, was small, compared with the influence of other factors. PMID- 8638960 TI - Effects of exposure to elemental mercury on the nervous system and the kidneys of workers producing natural gas. AB - Early signs of alterations in renal and neurological functions were studied in three groups of workers who were exposed to different levels of mercury that were below the current biological exposure index of 35 microg/g creatinine. There were no differences among the three study groups with respect to either motor nerve conduction velocity or tremor frequency spectra of physiological tremors. Also, no significant correlations were found between the results of the neurological tests and any of the present or historical biological monitoring data. In contrast, N-acetyl-beta-D-glucosaminidase was increased significantly in the group with the higher exposure, compared with either the lower-exposure or control groups. N-acetyl-beta-D-glucosaminidase was correlated strongly with mercury concentration in urine and was correlated weakly with historical biological monitoring data; however, there was no correlation with duration of exposure. These results suggest that after exposure to mercury at levels below the biological exposure index, a transient increase in N-acetyl-beta-D glucosaminidase can be observed, but is not an early indicator of developing renal dysfunction. PMID- 8638961 TI - Lead's legacy? Early and late mortality of 454 lead-poisoned children. AB - A series of 454 pediatric hospital patients who were diagnosed with lead poisoning between 1923 and 1966 were traced through 1991 to examine possible mortality effects. Numbers of observed deaths were compared with those expected, based on the rates of the U.S. population. Eighty-six deaths were observed (O/E = 1.7, 95% confidence interval (95% CI) = 1.4-2.2), of which 17 were attributed to lead poisoning. Mortality from all cardiovascular disease was elevated (O/E = 2.1, 95% CI = 1.3-3.2), and cerebrovascular deaths were particularly common among women (O/E = 5.5, 95% CI = 1.1-15.9). Among men, 2 deaths resulted from pancreatic cancer (O/E = 10.2, 95% CI = 1.1-36.2), and 2 deaths resulted from non Hodgkin's lymphoma (O/E = 13.0, 95% CI = 1.5-46.9). Chronic nephritis was not a significant cause of death. Despite limitations in the data, the pattern of mortality suggests that effects of lead poisoning in childhood may persist throughout life and may be experienced differently by men and women. PMID- 8638962 TI - Risk factors for high levels of lead in blood of schoolchildren in Mexico City. AB - Risk factors associated with blood lead levels exceeding 15 microg/dl were analyzed in this report. This relatively high lead level was selected because, at the time the study commenced, it was considered to be a "safe" level. A total of 1583 schoolchildren were studied. The students were from (a) two areas in Mexico City (Tlalnepantla and Xalostoc) that have had historically high concentrations of lead in air, and (b) three areas (Pedregal, Iztalpalapa, and Centro) with less impressive air lead levels. Parents were presented with a questionnaire that solicited information about lead risk factors. A bivariate analysis and a multilogistic analysis were conducted to identify associations and to identify the model that most accurately explains the variability of the sample. High blood lead concentrations were found in children who lived in Xalostoc and Tlalnepantla (16.1 and 17.0 microg/dl, respectively), and the lowest concentration (i.e., 10 microg/dl) was found in children from Iztapalapa. The strongest association was with area of residence, followed by education level of parents, cooking of meals in glazed pottery, and chewing or sucking of yellow or other colored pencils. A child's area of residence is the most significant risk factor that must be accounted for when any study of lead and blood lead concentrations is undertaken. Follow-up in similar populations should assist greatly in the evaluation of the impact of governmental actions on public health. PMID- 8638963 TI - Involuntary exposure to tobacco smoke in adolescents: urinary cotinine and environmental factors. AB - The relationship between environmental tobacco smoke exposure and urinary cotinine was studied in 434 14-y-old schoolchildren. To estimate the independent contribution of physiological and environmental variables to cotinine concentrations, we conducted a multiple regression analysis of log-transformed cotinine (R(2) = .21, p < .0001). Environmental tobacco smoke exposure was associated with sharing a household with members who smoked. The most profound associations were linked to (a) the smoking habits of the mother (beta = 5.135, p = .0397); (b) the combined smoking habits of the mother and other family members (beta = 8.201, p = .0020); and (c) the total number cigarettes smoked each day by family members in the household (beta = 0.217, p = .0008). Passive smoke exposure of adolescents is a preventable risk that could be reduced by improving ventilation and by increasing the living space available to each family member. Parents should avoid smoking at home in the presence of their children. PMID- 8638964 TI - Effects of age, socioeconomic status, and menstrual cycle on pulmonary response to ozone. AB - The purpose of this study was to investigate the effects of age, socioeconomic status, and menstrual cycle phase on the pulmonary response to ozone exposure. Three hundred seventy-two healthy white and black young adults, between the ages of 18 and 35 y, were exposed only once to 0.0, 0.12, 0.18, 0.24, 0.30, or 0.40 ppm ozone for 2.3 h. Prior to and after exposure, pulmonary function tests were obtained. Prior to exposure, each subject completed a personal and family-history questionnaire. The responses to this questionnaire were used to investigate age, socioeconomic status, and menstrual cycle phase effects on pulmonary responsiveness to ozone. We concluded that the ages of subjects, within the age range studied, had an effect on responsiveness (i.e., decrements in forced expiratory volume in 1 s decreased as the subjects' ages decreased). Socioeconomic status, as reflected by education of fathers, also appeared to affect forced expiratory volume in 1-s responsiveness to ozone, with the middle socioeconomic group being the most responsive. The phase of menstrual cycle did not have an impact on individual responsiveness to ozone. PMID- 8638965 TI - A portable inhalation system for personal exposure to ozone. AB - A low-cost portable inhalation system was developed for exposing an individual subject to 60-600 parts per billion of ozone in a 30-l clear-plastic head dome. The inhalation system had the following novel features: a canister vacuum cleaner that supplied room air without the need for precleaning or humidification; a 7% oxygen-in-nitrogen feed to a commercial ultraviolet ozonator that avoided an excess production of ozone; a compact inline mixer that assured homogeneous mixing of the 200-300 liters per minute room air supply with the 0.5-1.0 liters per minute of ozonated gas flow, positioning of gas inlet and exhaust hoses on the head dome that provided fresh gas delivery in the vicinity of the mouth; a quick-disconnect neck seal that allowed rapid donning of the head dome by the subject, and mounting of most system components on a small mobile cart. Temperature, humidity, and ozone and carbon dioxide concentrations were measured inside the dome while a subject exercised on a bicycle ergometer. An air flow of 200 liters per minute between rest and light exercise created a suitable microenvironment in the dome. During moderate and heavy exercise, however, a higher flow of 300 liters per minute should be used to suppress the build-up of carbon dioxide and humidity. PMID- 8638966 TI - Use of a 24-hour recall diary to assess exposure to environmental tobacco smoke. AB - Methods to assess exposure to environmental tobacco smoke need to be valid and relatively easy to use. We therefore explored the use of a 24-h environmental tobacco smoke exposure-recall diary by comparing data from the 24-h diary with questionnaire responses and levels of salivary cotinine--a biochemical marker of environmental tobacco smoke exposure. A total of 875 nonsmokers at five Rhode Island worksites participated in the study. Twenty-five percent of the participants lived with smokers, and 96% had regular exposure to environmental tobacco smoke at work. Individuals who lived with smokers reported more exposures in the 24-h diary, both outside of work and during work hours, compared with those who had no smokers in their household. The correlation between saliva cotinine concentrations and the exposures recorded in the diary was weak (r = .10). Brief instruments for assessment of environmental tobacco smoke should be viewed cautiously, and use of this 24-h recall diary is not recommended. PMID- 8638967 TI - Effect of sulfur dioxide on cytokine production of human alveolar macrophages in vitro. AB - Tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and transforming growth factor-beta are cytokines synthesized by alveolar macrophages. We investigated the effect of sulfur dioxide, a major air pollutant, on the production of these cytokines by alveolar macrophages. The cells were layered on a polycarbonate membrane and exposed for 30 min to 0.0, 1.0, 2.5, and 5.0 ppm sulfur dioxide at 37 degrees C and 100% air humidity. The cells were incubated for 24 h after exposure, thus allowing cytokine release. Cytotoxic effects of sulfur dioxide were evaluated by trypan blue exclusion. Cytokines were measured with enzyme-linked immunosorbent assays (i.e., tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) or by use of a specific bioassay (i.e., transforming growth factor-beta). The toxicity of sulfur dioxide for alveolar macrophages ranged from 3.1 % to 9.5 %. A 30-min exposure to sulfur dioxide induced a significant decrease in spontaneous and lipopolysaccharide-stimulated tumor necrosis factor-alpha (p < .001) and lipopolysaccharide-stimulated interleukin-1beta release (p < .05). The release of interleukin-6 and transforming growth factor-beta was not affected significantly by sulfur dioxide exposure. Our results demonstrated a functional impairment of alveolar macrophages after sulfur dioxide exposure (i.e., release of tumor necrosis factor alpha and interleukin-1beta). Neither spontaneous nor stimulated release of interleukin-6 and transforming growth factors were influenced by exposure to sulfur dioxide. PMID- 8638968 TI - Inorganic particles in bronchoalveolar lavage fluids from nonoccupationally exposed subjects. AB - This study comprised 30 patients who had not been exposed occupationally to dusts, but for whom a diagnosis of suspected pulmonary carcinoma had been made. Bronchoalveolar lavage fluids from these patients were analyzed by transmission electron microscopy and by energy-dispersive x-ray microanalysis in an effort to study the mineral particulate present in the alveolar region. Particles of silica, silicates, oxides, sulphates, and metal alloys were detected in various percentages in each subject. The smoking habits of two groups of patients that were defined by their bronchoalveolar lavage particulate concentrations (i.e., lower or higher than the median of the distribution) differed significantly. PMID- 8638969 TI - Dose-response relationship between urinary cadmium concentration and beta2 microglobulinuria using logistic regression analysis. AB - Logistic regression analysis was used to investigate the dose-response relationship for environmental cadmium exposure and to consider the effect age had on this association. The target population comprised 3178 inhabitants of Japan who were more than 50 y of age and who lived in a cadmium-polluted area and 1134 inhabitants who lived in nonpolluted areas of Japan. Logistic regression analysis was completed on the dose-response relationship between urinary cadmium concentration (i.e., an indicator of cadmium body burden) and beta2 microglobulinuria (i.e., an index of renal tubular dysfunction caused by exposure to cadmium). Both age and urinary cadmium concentration were associated significantly with beta2-microglobulinuria. Based on the relationship that was determined, we calculated, by age and sex, the values of urinary cadmium concentration that corresponded to the prevalence rates of beta2 microglobulinuria in the nonpolluted population. The resulting values were 1.6 3.0 micrograms/g creatinine for men and 2.3-4.6 micrograms/g creatinine for women. PMID- 8638970 TI - Lead, chromium, and cadmium exposure during abrasive blasting. AB - An evaluation of lead, cadmium, and chromium exposure was conducted during abrasive blasting of a steel bridge to remove paint in preparation for repainting. Airborne lead concentrations were measured at several locations inside the containment structure, as well as near the workers' breathing zones. Airborne cadmium and chromium were also measured in the containment area. Blood lead levels were monitored in each worker. Airborne lead and cadmium levels in containment exceeded the Occupational Safety and Health Administration's permissible exposure limits by factors of 219 and 3.1, respectively. The use of supplied air-blasting helmets will not effectively reduce workers' lead exposure to the permissible exposure limits when airborne levels are as high as were measured in this study. Studies are needed to evaluate additional engineering controls and alternative paint removal methods. Evaluation of workers' exposure to lead and other hazardous metals is needed for projects involving abrasive blasting. Medical surveillance for cadmium and lead may be necessary for similar projects. PMID- 8638971 TI - [Tuberculosis in the province of Mahajanga]. AB - Comparative study of compulsory declarations registered at the provincial level and at the central level between January 1st 1993 and 31st December 1994 showed a high prevalence of tuberculosis in the province of Mahajanga and an under declaration of cases at the provincial level was almost 25% with regard to data collected at the central level. Smear-positive pulmonary tuberculosis patients were the most frequent (86%), which showed respect towards the National Control Programme instructions. Extrapulmonary tuberculosis were under-estimated (less than 10%) because of the weak diagnosis means. Smear-positive pulmonary tuberculosis under treatment in the city of Mahajanga were cured globally at 80%. The difference between the centres diminished between 1992 and 1993, then it persisted requiring the improvement of tubercular management conditions within the province structures and most particularly within the city of Mahajanga structures which are at the present taking charge of more than 50% of the province tubercular. PMID- 8638972 TI - [Tuberculosis in the prison milieu at Antananarivo from 1990 to 1993]. AB - Within 42 months, from June 1990 to December 1993, 454 cases of tuberculosis have been recorded in the central remand home of Antananarivo. The tubercular prevalence observed was there eight times superior to that of the global population of Madagascar. Among the 360 pulmonary tuberculosis, only one did not have any bacteriological proof; among the 94 extrapulmonary tuberculosis, 37 have been proved by histology or bacteriology. Among the extrapulmonary tuberculosis, pleurisies were obviously preeminent (79/94). Association of tubercular localizations could be observed with 21% of the patients. New cases of smear positive pulmonary tuberculosis (PMT+) represented 81% of all the PMT+, recurrences were 9% and revivals 10%. Since February 1991, the 8 months short course regimen was the standard applied; before, the lack of stock did not allowed any standardization. The PMT+ new cases recovery rate increased from 42.5% in 1990 to 74% in 1993, whereas lethality decreased from 23% in 1990 to 8% in 1993. Patient dropouts were noted only with released or escaped individuals. Treatment failure rate was 4%. The diminution of cases despite the constancy of prisoners number and the carrying out of activities by the same health team make questionable the explanatory factors of the burst of tuberculosis-diseases in a prison milieu. Because of the importance of prison tubercular foci in terms of public health and the satisfactory results obtained, the Programme proposes to apply the model of partnership developed between the Tonga soa NGO and the prison administration to other prisons in Madagascar. PMID- 8638973 TI - [AIDS and tuberculosis: the situation in Madagascar]. AB - Because of the known epidemiological links between tuberculosis and HIV infection in developing countries, a systematic study of HIV infection prevalence among tuberculous patients has been conducted since 1989 in some centres of the capital and extended to other towns in 1992. HIV infection prevalence is still low (<200/100,000) with tuberculous patients. This result must incite to continue the surveillance of the ineluctable growth of HIV prevalence and to strengthen the tuberculosis Program in anticipation of subsequent problems. PMID- 8638974 TI - [Management of tuberculosis patients at the Antananarivo Military Hospital from 1989 to 1993]. AB - The experience of a 51 months continuous action of tuberculosis control in a pneumophysiology department of an important general hospital which works according to the principle of cost recovery, is reported. This centre, with an average of 345 annual cases, is the third in Madagascar. During the studied period (from September 1989 to December 1993), 1418 tubercular patients have been diagnosed, distributed into 57.7% of pulmonary tuberculosis and 42.3% of extrapulmonary tuberculosis. The number of extrapulmonary tuberculosis is obviously higher than in the rest of the country structure (16%); among them, pleurisies are distinctly prevailing (present in 29.6% of tubercular patients), other serositis take an important place, immediately after peripheric adenopathies (101 cases that is to say 7.1%); the high proportion of laryngitis shows the importance and oldness of bacilli infected pulmonary lesions. 13.7% of the patients have two or more tubercular localizations. Bacteriological proof has been done for 97.3% of the pulmonary tuberculosis and 7% of the extrapulmonary tuberculosis. A certitude proof has globally been acquired for 82.5% of the patients. 97.9% of the sick started a treatment. 7% of death were noted (95 cases), two thirds of them during the first month after diagnosis and two thirds due to pulmonary tuberculosis with positive microscopy. The average recovery rate within the studied period was 68.2% for all patients without distinction; 67.6% (456/674) for pulmonary tuberculosis with positive microscopy and 76% (265/349) for pulmonary tuberculosis with positive microscopy among civil servants and equivalent. It has been noted that private persons who pay their medical expenses showed a significantly less good compliance (60.9% of recovery rate) than civil servants whose medical expenses are entirely refunded. PMID- 8638975 TI - [Tuberculosis in children in Madagascar. 122 cases observed at the Soavinandriana Antananarivo Hospital Center]. AB - A group of 122 observations of pediatric tuberculosis has been studied. Nurslings represented 34% of the group, children under 8 years old 75% and only 10% were above 12. The sex ratio was 1,1. In a statistically significant way, tuberculous children were less often immunized by BCG than reference children not infected by tuberculosis. Contact has been traced back to close family in 42% of cases. Weight loss was significant at diagnosis time and after treatment the difference with the reference group disappeared. Extrapulmonary localizations were less frequent with children under 2, pulmonary and extrapulmonary localizations associations could be observed with 14% of children under 2 and with 31% of the whole children developing a proved tuberculosis. The importance of bronchial fibroscopy has been pointed out, for it allowed to detect 30% of abnormalities and to prove the diagnosis of tuberculous in 25% of cases. It is regrettable that the National Tuberculosis Control Programme did not prescribe chemoprophylaxis of contact children in its routine instructions, yet it is well known that child tuberculosis is rarely contagious and is not considered a priority by a programme. Finally, the authors reported that the number of pediatric tuberculosis managed in the country showed an obvious underestimation of the problem and they hope this work would lead to think more frequently of that diagnosis in the future. PMID- 8638976 TI - [Role of catholic centers in the control of tuberculosis]. AB - This study briefly reports the results of a survey conducted by the National Tuberculosis Control Programme (NTCP) to asses the Catholic Centres and particularly the leper colonies activities of tuberculosis control. Among the 34 Catholic Centres taking care of the lepers, 11 take part in the NTCP. Within less than 3 years, most of these centres have taken charge of twice more tuberculous patients: the number of cases increased from 540 to 1045. Leprosy prevalence is constantly decreasing, thus multibacillary leprosy cases declared by these centres decreased of 28% from 1992, to 1994. Centres which did not begin conversion would feel this necessity soon. Germs responsible for leprosy and for tuberculosis are "first cousins". Technical and operational approaches for the control of both affections are very much alike. Those considerations logically induce to propose the conversion of antileprosy centres for tuberculosis control. Other arguments are partially exposed in this work. The Central Division knows the existence of 28 Catholic Centres throughout the country, taking charge of tuberculous patients. In 1994, they put more than 1600 patients under treatment, thus 15% of the tubercular in Madagascar. Those Catholic Centres implementing tuberculosis control programme ought set up a "federation" as a privileged interlocutor for the NTCP and for the financial backers when allowing support. Responsibles of the Programme expect to convince Centres of the necessity of conversion and of the interest of tuberculosis control. PMID- 8638977 TI - [The National Tuberculosis Control Program in Madagascar]. AB - In 1991, the National Tuberculosis Control Program could start in Madagascar, thanks to the financial support of the French Cooperation. Within 3 years, this allocation of resources allowed the management, respecting the new standards, of 56% of the country's health structures and of more than 75% of the sick. The number of detected and treated patients increased of 80%. During the same period, the recovery rate increased from less than 35% to more than 65%. Those primary results were satisfactory in terms of working but they were not enough in epidemiological terms as the aims were still far: the detection rate of smear positive pulmonary tuberculosis was 40% whereas it ought be 60%, and their recovery rate was 65% whereas it ought to be over 80%. The geographic extension of the Program and its progress depend on a structural strengthening needing an obvious political will and on the intervention of financial partners cooperating with France and willing to set up a long lasting partnership. PMID- 8638978 TI - [Role of the Lutheran Non-Governmental Health Organization in tuberculosis control]. AB - Ever since its establishment in Madagascar, the Lutheran Church has been very active in medical field. In 1983, the creation of a medical non governmental organization called SALFA (Sampanasa Loteriana momba ny Fahasalamana) gave a new impulse. Since 1987, the SALFA has been seriously involved in the management of tuberculous patients. This document is a synthesis of actions conducted by SALFA whose experience in tuberculosis control is widely recognized by the Health Department and specially by the National Tuberculosis Control Program (NTCP). With an annual average of 1250 tuberculosis taken in charge, this NGO assumes 10% of the whole of the Program work. The diagnosis work is good (more than 85% Of PMT+) and therapeutic follow up of patients is excellent (more than 80% of the cured); those two elements of screening-treatment, basis of all tuberculosis control program incited us to describe this exemplary program. PMID- 8638979 TI - [National Laboratory for Mycobacterias. Evolution, missions and activities from 1991 to 1994]. AB - The setting up of a new nationwide tuberculosis control programme allowed the creation of a National Mycobacteria Reference Laboratory. This latter originated from the small bacteriology laboratory of the antitubercular dispensary of the Institut d'Hygiene Sociale (IHS) and its activities increased tenfold within three years. Extension of rooms, a more numerous staff and the acquisition of a modern equipment explained those results. However, to carry out the tasks of such a laboratory a rapid modification of structures and a new job distribution facilitated by a cooperation with the Institut Pasteur de Madagascar are necessary. PMID- 8638980 TI - [Evolution of the Mycobacteria Laboratory of the Pasteur Institute of Madagascar from 1991 to 1994]. AB - In 1991, the Laboratory of Mycobacteria was a small laboratory, part of the Clinical Biology Centre (CBC) of the Institut Pasteur de Madagascar: 656 pathological samples have been analysed for the account of the CBC and the National Control Programme activities. Within 4 years, the number of samples tested increased by more than threefold and the technical ability has evolved in an important way, specially for the identification and the antibiotic sensitivity testing. The scientific equipment have been modernized and the rooms surface increased by fourfold. In 1995, this laboratory was officially designated as the National Reference Laboratory for the culture, the identification and antibiogramme for the account of the National Control programme and for the private clinicians. It also participates to the tuberculosis research programmes of Institut Pasteur de Madagascar. It is associated to the Laboratory of Mycobacteria in the Institut d'Hygiene Sociale of Antananarivo which is the National Reference Laboratory for the bacilloscopy, the teaching and the supervision of the peripheral laboratories. PMID- 8638981 TI - [Genetic polymorphism of M. tuberculosis strains in Antananaviro]. AB - The genetic polymorphism of the mycobacteria of the tuberculosis complex in the city of Antananarivo was studied on 126 strains isolated from positive microscopy pulmonary tuberculosis patients. The genetic profiles established using the RFLP technic and the IS6110 marker yielded 83 clusters of 1 to 29 strains. There were 34 strains with a IS6110 unique band profile of which 29 had a band located at 1.4-1.5 kb. These strains could be differentiated using a second marker, the DR marker. 3 strains with an unique IS6110 band located at 1.8-1.9 kb were identified as M. bovis. In general, there was no evident epidemiological relationship between the patients presenting with identical profiles. In the prison of Antananarivo, the IS6110 typing of 36 strains yielded 28 clusters of 1 to 3 strains. Excepting 2 clusters showing an internal contamination, the absence of profiles specific to the jail suggests that the patients were probably contaminated before their entrance. This preliminary study shows that the RFLP profiles of M. tuberculosis, using the IS6110 and the DR markers, were polymorphic enough for using this method to study the transmission in Antananarivo. PMID- 8638982 TI - [Difficulties in the realization and implementation of a national survey on the Annual Risk of Tuberculosis Infection in Madagascar]. AB - The authors describe on the basis of their own experience, the difficulties that may arise during the achievement of a nationwide survey of the Annual Risk of Tubercular Infection and the analysis of the results. They stress the need of a close methodology at all stages of the survey, from conception to analysis, in order to get reliable and comparative results. PMID- 8638983 TI - [Tuberculosis and patient compliance: proposition for a study methodology of patient dropouts]. AB - Patient dropouts are one of the main concerns of tuberculosis programme in high prevalence countries. Perception of renunciation factors are generally weak or/and feeble from the part of agents working within programmes, while there are some a priori and confusions. This work proposes the setting up of a malagasy study, after the adequate definition of some hypothesis, then the writing of a survey form. The aim is to draw remedial behaviours and to make physicians and paramedical working close to tubercular patients, aware of the importance of a good mutual comprehension. PMID- 8638985 TI - [Extra-pulmonary tuberculosis in Antananarivo. Principal localizations and biological diagnosis]. AB - We describe the state of extrapulmonary tuberculosis in the capital of Antananarivo, a city of high endemicity for tuberculosis but very low endemicity for HIV infection. The Laboratory of Mycobacteria in the Institut Pasteur of Madagascar had examined from August 94 to April 95, 543 pathological samples issued from 295 patients clinically suspected of extrapulmonary tuberculosis (64% male and 36% female). The diagnosis of tuberculosis was confirmed for 47.7% of the patients (141/295), using either the culture technique or the histopathological method: 93% of them had an unique localization whereas 7% had a double localization. The most frequent form encountered was the pleural localization (77.8%), followed by the lymphadenopathic form (8,4%) and the abdominal form (6.9%). The confirmation rate on biopsies was 67% by histopathological method compared to 55% by the culture. On the fluid samples, the confirmation rate was 20.9% using the culture. The agreement between histology and culture was 70.3%. Of the 138 strains identified, 135 were M. tuberculosis, 1 M. bovis and 2 environmental mycobacteria. PMID- 8638984 TI - [Comparison of routine therapeutic protocols used in Madagascar for the treatment of smear-positive pulmonary tuberculosis (preliminary results)]. AB - A survey was undertaken in April 1993 to compare the respective benefits of 2 regimens containing either streptomycin (SHRZ) or ethambutol (EHRZ) in the first two months of treatment of smear-positive pulmonary tuberculosis in Madagascar. This operational research was justified by the risks related to the use of parenteral streptomycin in a country where single use material is rare and its purpose was to provide arguments for an eventual recommendation to replace this drug by oral ethambutol which is also less expensive. 907 patients were included. The compliance was not significantly different between the 2 groups, although it was traditionally assumed to be better with streptomycin. The frequency of side effects was significantly lower with EHRZ. Overall treatment failure rates were not significantly different, but all of 6 patients who were negative at 5 months and were again positive at 8 months had received EHRZ. This point obliged to be careful before concluding, because 24% of patients were lost for follow-up. A 2 years surveillance will be necessary to compare the frequency of recurrences. PMID- 8638986 TI - [660 cases of histologic extra-pulmonary tuberculosis at the Pasteur Institute in Madagascar]. AB - A 33 months retrospective study (from September 1992 to May 1995) of 8525 patients indicated that the incidence of histologic extrapulmonary tuberculosis (EPT) was 7,7%. The 8341 malagasy native patients presented an incidence of 7.8%. Male are more frequently infected than female. Most samples came from Antananarivo, with a prevalence reaching 12,5% while it was only 2,9% in the provinces. Most pathologic EPT were pleural tuberculosis (55,6%) and ganglial tuberculosis (34%). Peritoneal tuberculosis were much less frequent. According to literature data in Madagascar, the incidence of histologic EPT is obviously increasing. PMID- 8638987 TI - [Laryngeal tuberculosis in Antananarivo]. AB - From September 1989 to December 1992, 49 patients had been observed at the Centre Hospitalier de Soavinandriana and 31 cases at the prison infirmary of Antananarivo from April 1990 to December 1992. The 31 laryngeal tuberculosis observed in prison existed among 9.1% of tubercular patients, were associated to 10.9% of pulmonary tuberculosis and represented 24.2% of extrapulmonary tuberculosis. The 49 laryngeal tuberculosis of the CENHOSOA were present among 4.6% of tubercular patients, were associated to 7.9% of pulmonary tuberculosis and represented 8.4% of all the extrapulmonary tubercular localizations. Voice disorders, even if always present, rarely represented (<30%) a reason for tuberculosis discovery. Comparison with the whole of tuberculosis cases showed that laryngitis was not most frequent in retreatments and that there was no statistically significant difference in death risk. Laryngeal tuberculosis being mostly in the shadow of pulmonary tuberculosis, it develops in the same way under the same treatment, except when a very ancient laryngitis has given rise to irreversible fiber lesions. Recall of this very early known localization is made in a didactic perspective: in a developing country, all chronical voice disorders must induce to research of tuberculosis. PMID- 8638988 TI - [Pericardial tuberculosis in Madagascar. 23 cases]. AB - A 56 months retrospective study, from October 1990 to May 1995, at the Centre Hospitalier de Soavinandriana in Antananarivo pointed out 29 tuberculous pericarditis among the 97 pericardial effusions discovered by the echocardiography of 5600 patients. The sex-ratio was 0,81 and the mean age 38,6 years old (+/- 14,3). Hospitalization was justified by dyspnea (18 cases), thoracic pain (18 cases), lower limbs edema (6 cases) and ascitis (3 cases). Moreover, electrocardiography showed microvoltage in 18 cases and thoracic radiography showed one heart enlargement. Even if for 15 cases a pleural effusion was associated, only 2 patients had a pulmonary image suggestive of tuberculosis. Tuberculous pericarditis has been proved by the following examinations: pericardium puncture (21 cases), pericardium and pleural biopsy (respectively 11 and 13 cases), ganglionic biopsy and search of alcohol-acid-fast bacilli in sputum: 1 case. Histologic proof has been obtained 8 times out of 9 pericardial biopsies and 6 times out of 7 pleural biopsies. Bacteriological proof has been obtained 11 times by pathological samples cultivation: twice from fresh caseous material taken from the pericardium, once from 13 pleural fluids, 5 times from 6 pericardial biopsies, 3 times from 3 pleural biopsies. The patients have been put under antituberculous treatment associated with prednisone. 20 patients have been declared cured at the end of the treatment, 5 were dead and 4 were lost out of sight. Tuberculous pericarditis has become rare in developed countries but it is still challenging in Madagascar. In spite of the antituberculous treatment associated with corticoids, prognosis is severe (evolution towards pericardial constriction death. PMID- 8638989 TI - [Peritoneal tuberculosis in Madagascar, 55 cases at the Soavinandriana Hospital Center in Antananaviro]. AB - This retrospective study conducted in Antananarivo for a 42 months period, from September 1991 to March 1994, allowed to record 55 peritoneal tuberculosis, all of them in malagasy patients. The sex ratio was 0,83 and the mean age 36 years old. Ascites puncture was done each time there was an effusion (44 cases). For all cases. For all cases, the diagnosis based on laparoscopy allowed an investigation of the liver and the peritoneum, and 10 peritoneum biopsies could be done. The tubercular bacillus has been isolated in 2 ascites fluids out of 8 incubations, and in 5 biopsies of peritoneal granulation out of 8. For 8 cases, another tubercular localization has been discovered: 5 pleurisis, 2 evolutive tuberculosis and 1 pericardial effusion. All the patients have been put under antitubercular treatment and have regained health. Peritoneal tuberculosis has become rare in the West but is still a frequent pathology mostly in young women, in Africa and up in Madagascar. PMID- 8638990 TI - A guide to the understanding and use of tricyclic antidepressants in the overall management of fibromyalgia and other chronic pain syndromes. AB - The purpose of this review is to present relatively detailed information on the characteristics of tricyclic antidepressants, mainly amitriptyline hydrochloride and doxepin hydrochloride, for use as an integral part of the safe and effective management of fibromyalgia and, to a lesser extent, other chronic pain syndromes. Data sources include MEDLINE searches in English, relevant reference books and textbooks, my personal database and library, as well as personal clinical experience. I discuss these data with regard to the pharmacologic characteristics, mechanisms of action, adverse effects, and precautions involved with the use of tricyclic antidepressants. Additional information is given on drug selection and dosage titration. Much emphasis is placed on the fact that while tricyclic antidepressants play a major role in the management of fibromyalgia and other chronic pain syndromes, lifestyle alterations (eg. physical reconditioning and exercise), as well as behavior modification, are also vital to a successful outcome in management. PMID- 8638992 TI - Advance end-of-life treatment planning. A research review. AB - The year 1996 marks the fifth anniversary of the federal Patient Self Determination Act. The Patient Self-Determination Act required hospitals, nursing homes, and health plans to ask whether patients have advance directives and to incorporate them into the medical record. A "living will" is an advance directive by which a person tells caregivers the circumstances in which life-sustaining treatment is to be provided or forgone if the patient is unable to communicate. A "durable power of attorney for health care" enables one to designate a person to speak on his or her behalf if the author loses decision-making capacity. "Advance planning" is the process of reflection, discussion, and communication of treatment preferences for end-of-life care that precedes and may lead to an advance directive. PMID- 8638991 TI - Community-based outbreaks of tuberculosis. AB - Numerous recent reports have detailed outbreaks of tuberculosis in hospitals and other congregate settings. The characteristics of such settings, including high concentrations of infectious patients and immunocompromised hosts, the potential for sustained daily contact for weeks and often months, and improper precautions taken for protection, make them well suited for tuberculosis transmission. However, community-based outbreaks, which are the source of much public concern, have not been reviewed since 1964, when 109 community outbreaks were examined. Since few of the characteristics of institutional settings are present in the community, the lessons learned may not be applicable to community-based outbreaks. Furthermore, recent studies with analysis by restriction fragment length polymorphisms have documented unexpectedly high rates of primary disease in certain urban communities, suggesting that our understanding of community based transmission may be incomplete. We reviewed all reported community-based outbreaks of tuberculosis occurring in the last 30 years to assess the basis of our current understanding of community-based transmission. More than 70 outbreaks were identified, with schools being the most common site. In most, a delay in diagnosis, sustained contact with the index case, inadequate ventilation, or overcrowding was contributory. We conclude that community-based outbreaks of tuberculosis continue to occur and that well-established risks contribute to most outbreaks. Many outbreaks can be prevented or limited by attention to basic infection control principles. PMID- 8638993 TI - Cancer screening and informed patient discussions. Truth and consequences. AB - While screening for asymptomatic cancer has become one of the principal clinical activities of primary care physicians, patients are generally not involved directly in screening decisions. To help physicians better communicate the potential benefits and burdens of cancer screening, this article concisely presents information necessary for patients to make a reasoned decision as to whether to proceed with screening: the probability of developing cancer, the operating characteristics of available screening tests, the likelihood that screening will result in an improved outcome for the individual patient, and the potential burdens associated with screening. Screening tests for breast, colorectal, cervical, and prostate cancers are reviewed, including mammography, clinical breast examination, fecal occult blood testing, Papanicolaou smear, digital rectal examination, and prostate-specific antigen. Better communication about cancer screening will promote shared decision making--a central tenet of the physician-patient relationship. PMID- 8638994 TI - Duration of the survival benefit of zidovudine therapy in HIV infection. AB - BACKGROUND: Zidovudine therapy improves survival in advanced human immunodeficiency virus (HIV) infection and delays progression from earlier stages to advanced stage of HIV disease. The duration of the benefit of zidovudine therapy, however, may be limited. OBJECTIVE: To quantitate the duration of the survival benefit of zidovudine therapy in a heterogeneous patient population receiving care for HIV infection in an urban clinic. METHODS: We analyzed data from 393 HIV-infected patients with CD4+ cell counts of 0.5 x 10(9)/L (500 cells/microliter.) or less who first presented for care at The Johns Hopkins HIV Clinic, Baltimore, Md, from July 1989 through December 1993. Follow-up extended to a maximum of 3 years (median, 2 years). Survival probabilities in patients who received and who did not receive zidovudine therapy were analyzed by Kaplan-Meier methods and by multivariate Cox proportional hazards regression analysis adjusting for both time-dependent and fixed prognostic covariates. RESULTS: Adjusting for baseline differences in CD4+ cell count, clinical stage of HIV disease, and prophylaxis for Pneumocystis carinii pneumonia, Cox regression analysis showed a significant effect of zidovudine compared with no treatment on the risk of dying during the first year of therapy (relative hazard for death, 0.32; 95% confidence interval [CI], 0.18 to 0.59). However, analysis of the time dependent effect of zidovudine therapy showed that there was a diminishing relative hazard between treatment and no treatment of 0.75 (95% CI, 0.45 to 1.26) at 1 to 2 years of therapy and a relative hazard of 1.61 beyond 2 years (95% CI, 0.70 to 3.71). CONCLUSION: The survival advantage of zidovudine therapy is time dependent, lasting between 1 and 2 years in patients with CD4+ cell counts of 0.5 x 10(9)/L or less. Alternative antiretroviral treatment may be indicated at that time. PMID- 8638995 TI - Effectiveness of low-dose crystalline nicotinic acid in men with low high-density lipoprotein cholesterol levels. AB - BACKGROUND: Hypoalphalipoproteinemia (low serum concentration of high-density lipoprotein cholesterol [HDL-C]) is a common pattern of dyslipidemia associated with coronary heart disease. High doses of nicotinic acid effectively raise HDL-C levels in this condition, but they are commonly accompanied by side effects. The efficacy of low doses of nicotinic acid that may produce fewer side effects has not been adequately studied. OBJECTIVE: To determine the effects of low-dose nicotinic acid on HDL-C levels in patients with hypoalphalipoproteinemia. METHODS: Forty-four men with low HDL-C levels (< 1.03 mmol/L [< 40 mg/dL]) entered the study. Twenty-four patients otherwise had normal lipid levels, and 20 were moderately hypertriglyceridemic (range of plasma triglyceride levels, 2.82 to 5.64 mmol/L 250 to 500 mg/dL). The trial consisted of 3 phases; each phase lasted 8 weeks. The first phase was diet only (30% fat diet); in the second phase, crystalline nicotinic acid was added at 1.5 g/d; and in the third phase, the dose was increased to 3 g/d. RESULTS: Of the 44 patients who entered the study, 37 completed the low-dose phase (1.5 g/d); the remaining patients were withdrawn because of side effects to nicotinic acid. Four other patients who completed the low-dose phase were excluded from the higher dose phase because of side effects that developed when they were receiving the low dose. Ten other patients withdrew during the high-dose phase because of side effects. In both groups, responses to nicotinic acid therapy tended to be dose-dependent. For both groups, the higher dose generally produced a greater reduction in apolipoprotein B-containing lipoproteins and a greater rise in HDL-C levels. However, for both groups, the low dose of nicotinic acid gave an average 20% increase in HDL-C levels. CONCLUSIONS: A low dose (1.5 g/d) of crystalline nicotinic acid causes an average 20% increase in HDL-C levels and significantly lowers triglyceride levels in both normolipidemic and hyperlipidemic patients with low HDL-C levels. Although the changes induced by this dose are less than those that can be achieved by a higher dose, the lower dose is better tolerated. Nicotinic acid may be useful in combined drug therapy for secondary prevention of coronary heart disease, and if higher doses cannot be tolerated, use of a lower dose should still be useful for producing a moderate rise in HDL-C levels in patients with hypoalphalipoproteinemia. PMID- 8638996 TI - Comparison of elderly and younger patients with out-of-hospital chest pain. Clinical characteristics, acute myocardial infarction, therapy, and outcomes. AB - BACKGROUND: Acute myocardial infarction is associated with significantly higher mortality in elderly patients compared with younger patients. OBJECTIVES: To determine clinical differences in elderly and younger patients with acute myocardial infarction. To assess differences in therapies and outcomes between the age groups. METHODS: Over a 3.5-year period, 2482 consecutive adult emergency medical services patients with chest pain received prehospital electrocardiograms and were entered in the Milwaukee Prehospital Chest Pain Database in Milwaukee, Wis. Clinical characteristics that included cardiac history, description of chest pain, time of onset to presentation, and prevalence of acute myocardial infarction were obtained for all patients. Patients with acute infarction were further analyzed in reference to type of infarction (Q wave vs non-Q wave), therapeutic interventions, and mortality. Patients were stratified in 3 age groups: younger than 70 years (younger), 70 years or older (elderly), and 80 years or older (very elderly); differences were compared among the age groups. RESULTS: Even though more than 50% had a history of documented coronary artery disease, elderly patients with ischemic chest pain delayed more than 6 hours in seeking medical assistance after onset of pain. In elderly patients whose chest pain represented an acute myocardial infarction, hospital mortality was double that of younger patients. Thrombolytic therapy reduced hospital mortality by approximately 50% in both younger and elderly patients, although thrombolytic therapies were used in only 17% of the elderly patients compared with 50% of the younger patients (P < .001). Revascularization procedures were also beneficial in elderly patients as well as in younger patients, although this procedure, as with thrombolytic therapy, was less frequently used in elderly patients (48% vs 32%, P < .001). CONCLUSIONS: Effective methods for reducing time delays from onset of chest pain to accessing health care for elderly persons deserve investigation. Physicians should be aware of the benefits of thrombolytic and revascularization therapies in elderly patients with acute myocardial infarction. PMID- 8638997 TI - Prevalence of undiagnosed pernicious anemia in the elderly. AB - BACKGROUND: Existing information about the prevalence of pernicious anemia is largely based on older surveys that favored florid manifestations, tended to be retrospective analyses of previously diagnosed disease, and usually studied homogeneous European populations. The lack of current data in the United States has, among other things, hampered discussions of the proposal to increase folate intake by the general population. OBJECTIVE: To estimate the prevalence of undiagnosed and untreated pernicious anemia among the elderly. METHODS: A prospective survey of cobalamin levels and anti-intrinsic factor antibody was done in the elderly. Blood testing was done in 729 people aged 60 years or older and follow-up assessment with the Schilling test and other tests was offered when results were abnormal. RESULTS: Seventeen subjects were found to have pernicious anemia, usually with only minimal clinical manifestations of cobalamin deficiency. Although cobalamin deficiency had been suspected by the physicians of three subjects, they had been treated inadequately and still had evidence of deficiency. Excluding these three partially treated subjects from the analysis, 1.9% of the survey population had unrecognized and untreated pernicious anemia. The prevalence was 2.7% in women and 1.4% in men; 4.3% of the black women and 4.0% of the white women had pernicious anemia. CONCLUSIONS: Undiagnosed pernicious anemia is a common finding in the elderly, especially among black and white women. If these findings can be extrapolated, almost 800000 elderly people in the United States have undiagnosed and untreated pernicious anemia, and, thus, would be at possible risk for masked cobalamin deficiency if exposed to large amounts of folate. This number does not include those elderly with cobalamin deficiency caused by other disorders or the still unknown number of younger people with unrecognized pernicious anemia and other causes of deficiency. PMID- 8638998 TI - Somatized psychiatric disorder presenting as palpitations. AB - BACKGROUND: Psychiatric disorder is underdiagnosed in primary care practice, often because it is somatized and the patient reports only physical symptoms. Palpitations are among the symptoms that often are somatized. METHODS: We studied prospectively 125 consecutive medical outpatients referred for ambulatory electrocardiographic monitoring to evaluate a chief complaint of palpitations. They completed an in-person research interview at the time of monitoring and a telephone follow-up interview 3 months later. The referring physicians completed questionnaires about their patients before receiving the results of the monitoring and again 3 months later. RESULTS: Forty-three patients had clinically significant cardiac arrhythmias. Twenty-four (29%) of the remaining 82 patients had a current psychiatric disorder, and 20 of these patients (83%) had major depression or panic disorder. These patients were significantly younger and more disabled, somatized more, and had more hypochondriacal concerns about their health than did patients who had no psychiatric disorder. Their palpitations were more likely to last longer than 15 minutes, were accompanied by more ancillary symptoms, and were described as more intense. At 3-month follow-up, about 90% of the patients in both groups continued to experience palpitations. Symptoms of somatization, hypochondriacal concerns, and impairment of intermediate activities had improved in both groups, but remained higher in patients with psychiatric disorder than in patients without psychiatric disorder. During the follow-up interval, patients with psychiatric disorder had more emergency department visits. The physicians of patients with psychiatric disorder were more likely to ascribe the palpitations to anxiety or depression, and ordered fewer laboratory tests on them, but few patients who had not already been in psychiatric treatment were referred or started on psychotropic medication. CONCLUSIONS: Physicians are aware of a psychiatric component to the clinical presentation of palpitation, but this observation does not result in psychiatric treatment or referral in most cases. PMID- 8638999 TI - A 1-year trial of nasal mupirocin in the prevention of recurrent staphylococcal nasal colonization and skin infection. AB - BACKGROUND: The usefulness of nasal mupirocin in preventing recurrent staphylococcal nasal colonization and skin infection has been examined in immunodeficient patients and in healthy staphylococcal carriers but not in immunocompetent staphylococcal carriers who experience recurrent skin infections. We studied 34 such patients. METHODS: After an initial 5-day course of nasal mupirocin ointment for all patients, 17 patients continued to apply a 5-day course of nasal mupirocin every month for 1 year, and the other 17 patients applied a placebo ointment. Nasal cultures were obtained monthly, and all episodes of skin infection were recorded. RESULTS: The overall number of positive nasal cultures was 22 in the mupirocin group and 83 in the placebo group (P < .001), and the number of skin infections was 26 and 62, respectively (P < .002). Eight of the 17 mupirocin-treated patients but only 2 in the placebo group remained free of positive staphylococcal nasal cultures. One of the 10 patients who were free of colonization during the 12-month treatment period had skin infections, in contrast to all 24 of the patients with positive cultures (P < .01). Staphylococci resistant to mupirocin were observed in 1 patient. No adverse effects were reported. CONCLUSIONS: A monthly application of mupirocin ointment in staphylococcal carriers reduces the incidence of nasal colonization, which in turn lowers the risk of skin infection. PMID- 8639000 TI - Phenytoin toxicity associated with ticlopidine administration. PMID- 8639001 TI - Definition of screening status in case-control studies of the efficacy of endoscopy. PMID- 8639002 TI - Appendicitis due to Mycobacterium avium complex in a patient with AIDS. PMID- 8639003 TI - Ulcerative esophagitis during primary HIV infection. PMID- 8639004 TI - A low-dose perspective on the calcium antagonist controversy. PMID- 8639005 TI - Physician-assisted suicide. PMID- 8639006 TI - Testosterone treatment of men treated with glucocorticoids. PMID- 8639007 TI - Preventive efficacy of nutritional counseling. PMID- 8639008 TI - Hypomagnesemia and diabetes mellitus. A review of clinical implications. AB - Hypomagnesemia has long been known to be associated with diabetes mellitus. Mather et al confirmed the presence of hypomagnesemia in nearly 25% of their diabetic out-patients. Low serum magnesium level has been reported in children with insulin-dependent diabetes and through the entire spectrum of adult type I and type II diabetics regardless of the type of therapy. Hypomagnesemia has been correlated with both poor diabetic control and insulin resistance in nondiabetic elderly patients. PMID- 8639009 TI - Alcohol-related problems in older persons. Determinants, consequences, and screening. AB - Demographic trends reveal the elderly to be the fastest growing segment of the population. Physicians can therefore anticipate encountering increasing numbers of older patients with alcohol-related problems. These problems include liver disease, dementia, confusion (masquerading as dementia), peripheral neuropathy, insomnia, late-onset seizure disorder, poor nutrition, incontinence, diarrhea, myopathy, inadequate self-care, macrocytosis, depression, fractures, and adverse reactions to medications. Despite the prevalence of alcohol use in older people, their risks and problems are often unrecognized. We reviewed published literature on the determinants and consequences of alcohol-related problems in persons aged 65 years and older and the usefulness of available screening measures. Thirteen of 25 eligible studies on determinants and consequences met quality criteria and were reviewed. Nine additional studies on screening tests were also evaluated. Determinants include history of alcohol use and abuse, social isolation, and reduced mobility; consequences consist of risks of hip fracture from falls, neoplasms, and psychiatric illness. Currently accessible screening tests focus on high levels of alcoholic beverage use and abuse and dependence. They are not useful in screening for hazardous consumption that may result from relatively low levels of alcohol use alone or in combination with medications, medical illness, or preexisting diminished physical, emotional, or social function. Research is needed on the consequences of lower levels of alcohol consumption on the physical and psychosocial health of older individuals and on methods for distinguishing alcohol-related from age-related problems. Existing screening tests should be expanded or new screening methods developed in anticipation of a growing public health problem. PMID- 8639010 TI - Meta-analysis, clinical trials, and transferability of research results into practice. The case of cholesterol-lowering interventions in the secondary prevention of coronary heart disease. AB - OBJECTIVE: To evaluate, in the comprehensive scenario of "evidence-based" medicine, the transferability of the results of published randomized clinical trials and meta-analyses on cholesterol-lowering interventions to clinical practice. METHOD: Overview of randomized clinical trials on cholesterol-lowering interventions in the secondary prevention of coronary heart disease. RESULTS: The present overview on secondary prevention of coronary heart disease included 34 trials with cholesterol-lowering interventions in 24968 individuals. There was a 12.5% mortality in the group that was allocated active intervention and a 17.2% mortality in the control group (risk reduction, 13%; 95% confidence interval, 19% to -6%). Coronary and cardiovascular odds of deaths were significantly reduced. No clear association was found between noncoronary mortality and cholesterol-lowering interventions. Baseline total cholesterol levels had no clear influence on total mortality. Intermediate (10%-20%) and high ( > 20%) total cholesterol reductions were associated with similar reductions in the odds of death (-23% and -30%, respectively). No conclusion could be reached for patients who were less represented in the studies (ie, women and elderly persons). Patients with more complicated baseline clinical conditions (eg, congestive heart failure) had little nonsignificant benefit from cholesterol lowering interventions. CONCLUSIONS: The effect of cholesterol-lowering interventions at least in the secondary prevention of coronary heart disease can be considered as established, but the transferability of such results to real life patients remains the critical, unanswered question. PMID- 8639011 TI - Testosterone therapy in glucocorticoid-treated men. AB - BACKGROUND: Treatment with glucocorticoid drugs is a valuable therapy, but the use of these drugs is associated with major side effects, including osteoporosis, muscle wasting, and obesity. In men who take glucocorticoids, circulating testosterone concentrations are reduced, and this might contribute to the changes in bone and soft-tissue mass. OBJECTIVE: To asses the effect of testosterone replacement on these above-mentioned parameters in glucocorticoid-treated men. METHODS: Fifteen asthmatic men who were receiving long-term glucocorticoid treatment were randomly allocated to receive therapy with testosterone esters (30 mg of proprionate, 60 mg of phenylprionate, 60 mg of isocaproate, and 100 mg of decanoate [Sustanon]) (250-mg/mo intramuscular depot injection) or to act as control subjects during 12 months. After a washout period for those men who were receiving testosterone, the groups were then crossed over and studied for a further 12 months. Bone density and body composition were assessed by dual energy, x-ray absorptiometry. Paired or unpaired 2-tailed t tested were calculated. Unless otherwise stated, all values are given as mean +/- SEM. RESULTS: Bone density in the lumbar spine increased 5.0% +/- 1.4% (mean +/- SEM) (P = .005) during testosterone supplementation, but it did not change during the control period (between-groups difference, P = .05). These changes were accompanied by a decrease in the indexes of bone turnover. There was a gain in body fat mass (2.1 +/- 0.06 kg, P = .01) and a loss of lean body mass (1.4 +/- 0.5 kg, P = .02) during the control period, with both changes being reversed by testosterone treatment (P < .03). CONCLUSION: Testosterone treatment reverses the deleterious effects glucocorticoid drugs on skeletal and soft tissues in men. PMID- 8639012 TI - Correlates and consequences of diffuse atherosclerosis in men with coronary heart disease. Veterans Affairs High-Density Lipoprotein Intervention Trial Study Group. AB - BACKGROUND: Peripheral atherosclerosis is a strong and independent predictor of mortality even in patients with known coronary heart disease. However, the prevalence, correlates, and potential adverse effects on quality of life associated with combined coronary heart disease and clinically evident cerebrovascular or lower-extremity atherosclerosis are not known. Identification of patients with "diffuse atherosclerosis" may enhance treatment of modifiable risk factors and alter therapeutic strategies. METHODS: We conducted a cross sectional analysis of 2531 men younger than 73 years with coronary heart disease, low-density lipoprotein cholesterol levels of 3.62 mmol/L (140 mg/dL) or less, and high-density lipoprotein cholesterol level of 1.03 mmol/L (40 mg/dL) or less who were participating in Department of Veterans Affairs Cooperative Study 363 (the Veterans Affairs High-Density Lipo-protein Intervention Trial. Baseline demographic, medication, comorbidity, and atherosclerotic risk factor data were assessed by means of a standardized questionnaire. All plasma lipid levels were determined after a 12-hour fast by a central standardized lipid laboratory. Health status was determined by baseline reported symptoms, medical comorbidities, and the Psychological General Well-being Index. Clinically evident diffuse atherosclerosis was defined as a documented history of lower-extremity atherosclerosis or cerebrovascular disease. RESULTS: The mean age of all participants was 63.5 years. The mean plasma lipid values were as follows: total cholesterol, 4,52 mmol/L (174.6 mg/dL); high-density lipo-protein cholesterol, 0.81 mmol/L (31.5 mg/dL); low-density lipoprotein cholesterol, 2.88 mmol/L (111.2 mg/dL); and triglycerides, 1.81 mmol/L (160.6 mg/dL). Diffuse atherosclerosis was present in 525 (21%). Lower-extremity atherosclerosis was reported in 10%, while cerebrovascular disease was present in 13%. After controlling for other variables, the following factors were associated with the presence of diffuse atherosclerosis: increased age, being unmarried, being retired, having less than a high school education, increased alcohol use, hypertension, cigarette smoking, and diabetes. There was no association between lipid levels and the presence of diffuse atherosclerosis. After adjustment for age, race, and comorbidities, men with diffuse disease still had a reduced quality of life compared with men without diffuse atherosclerosis, as defined by having a greater number of clinical symptoms, lower psychological well-being scores, and more advanced or complicated coronary heart disease. CONCLUSIONS: Clinically evident diffuse atherosclerosis is common in men with coronary heart disease and low levels of high-density lipoprotein cholesterol. Because diffuse atherosclerosis is associated with a reduced quality of life and several modifiable risk factors, early detection and aggressive risk factor intervention appear justified. PMID- 8639013 TI - Prevalence of adrenal and extra-adrenal Conn syndrome in hypertensive patients. AB - BACKGROUND: Primary aldosteronism (PA) is caused by an adrenal aldosterone producing tumor (A-APT) or adrenal hyperplasia. An extra-adrenal APT (E-APT) as a cause of PA has been reported in 5 cases. Autopsy studies show a high incidence of ectopic adrenocortical tissue. We did a prospective study of the prevalence of A-APTs and E-APTs and the biochemical features of E-APTs in patients with PA. METHODS: Hypertensive patients (N = 3900) referred to our unit were screened for PA by measuring renin activity, urinary aldosterone-18-glucuronide, tetrahydroaldosterone, and 18-hydroxycorticosterone (18-OH-B). Primary aldosteronism was found in 257 cases. The differentiation between A-APTs and adrenal hyperplasia was based on the results of postural response of renin, plasma aldosterone, 18-OH-B, computed tomography, isotope scanning, or adrenal venous aldosterone. Ultrasound examination of the abdomen was used to screen for E-APT. RESULTS: The cause of PA was bilateral adrenal hyperplasia in 101 cases, unilateral adrenal hyperplasia in 2, an A-APT in 146, and an E-APT in 1. The site of aldosterone production was uncertain in 7 patients who had normal adrenal glands on computed tomography but refused to undergo isotopic scanning and adrenal venous catheterization. Ultrasound examination disclosed normal retroperitoneum in 4 of the 7 cases but could not rule out E-APT in 3 cases. The biochemical features of the patient with the E-APT were similar to classic A-APT, with low renin, high aldosterone, and high 18-OH-B values without appropriate response to posture or to short-term volume expansion. The excision of the E-APT in the right kidney resulted in normalization of blood pressure and renin, aldosterone, and 18-OH-B levels. CONCLUSION: Although E-APT is rare, it should be considered in the interests of specific therapy for PA because aldosterone secreting malignant ovarian tumors also have been reported. PMID- 8639014 TI - Management and prognosis of life-threatening bleeding during warfarin therapy. National Consortium of Anticoagulation Clinics. AB - BACKGROUND: The incidence of explicity defined life-threatening bleeding during warfarin sodium therapy is largely unknown, as are the prognosis for and treatment of patients who have such bleeding. In addition, the location of the source of the life-threatening bleeding and the risk factors associated with life threatening bleeding are not well-defined. OBJECTIVES: To determine the incidence of explicitly defined life-threatening bleeding during warfarin therapy, to identify the site of bleeding, to determine the risk factors for life-threatening bleeding, and to determine the risk of subsequent bleeding among patients receiving warfarin therapy. METHODS: A cross-sectional prevalence study was conducted and data were combined with those obtained during prospective observation of a dynamic cohort of patients followed up in 2 university affiliated and 3 Veterans Administration anticoagulation clinics. RESULTS: For this study, 1999 patients were followed up for 3865 patient-years; 32 patients (11 women, 21 men, mean age of 60 years) met criteria for life-threatening bleeding, an incidence of 0.83 events/100 patient-years (95% confidence interval, 0.54-1.12). The most common indication for warfarin was to prevent thromboembolism because the patient had a mechanical heart valve (17/32 patients, 53%). The gastrointestinal tract was the definite or likely site of bleeding in 21 (66%) of the 32 patients. The prothrombin time ratio was longer than 2.0 or the international normalized ratio was longer than 4.5 in 16 (55%) of the 29 patients in whom a coagulation time was measured. Fourteen (44%) of the 32 patients had a history of peptic ulcer disease or gastrointestinal bleeding. Warfarin was restarted in 26 (81%) of the 32 patients. Twenty-five of 26 patients were followed up for a median of 30 months (range, 5-143 months); 14 (56%) of the 25 patients had a subsequent bleeding event, with 8 (57%) of the 14 having 1 or more additional life-threatening bleeding events (1 fatal) after a median of 11.5 months (range, 0.5-22 months). CONCLUSIONS: We conclude that in this cohort: (1) the incidence of life-threatening bleeding was rare, (2) the gastrointestinal tract was the site of bleeding in two thirds of the patients who experienced life threatening bleeding, (3) most patients who experienced life-threatening bleeding had multiple risk factors for bleeding, including excessive anticoagulation, and (4) the risk of subsequent bleeding was extremely high among the patients who continued to receive warfarin therapy. PMID- 8639015 TI - Cholesterol-lowering intervention program. Effect of the step I diet in community office practices. AB - BACKGROUND: A randomized study was conducted to test the feasibility of cholesterol lowering in physician office practices using the National Cholesterol Education Program Adult Treatment Panel 1 guidelines. METHODS: Twenty-two physician practices in phase 1 and 23 in phase 2 were recruited from communities in Western Pennsylvania and West Virginia. These physicians treated a total of 450 adults in phase 1 (190 men and 260 women) and 480 adults in phase 2 (184 men and 296 women) with hypercholesterolemia. Three models (Usual Care [phase 1], Office Assisted [phase 2], and Nutrition Center [phase 2]) for implementing the National Cholesterol Education Program Adult Treatment Panel 1 guidelines were tested over an 18-month period. The baseline serum cholesterol levels were as follows: 6.51 mmol/L (252 mg/dL) in the Usual Care Model; 6.80 mmol/L (262 mg/dL) in the Office Assisted Model; and 6.96 mmol/L (269 mg/dL) in the Nutrition Center Model. RESULTS: In the patients who were not taking lipid-lowering medication, the mean cholesterol response was significantly different between the 3 models (P < .01). Serum cholesterol levels declined by 0.14 mmol/L (5.4 mg/dL) in the Usual Care Model; by 0.31 mmol/L (12 mg/dL) in the Office Assisted Model; and by 0.54 mmol/L (20.9 mg/dL) in the Nutrition Center Model. Two factors-length of time to follow-up measurement and change in weight-were independently related to cholesterol response across all models. African Americans demonstrated a significantly smaller response than whites in the Usual Care Model, while men demonstrated greater declines in serum cholesterol levels than women in the Office Assisted Model. Patient satisfaction was very favorable in both enhanced conditions; however, those treated in the the Nutrition Center Model were more satisfied (P < .05) with program components. CONCLUSIONS: The impact of nutrition intervention delivered through physician practices on serum cholesterol levels is less than clinically desirable, and new approaches with more aggressive therapy should be tested and implemented. PMID- 8639016 TI - Gender and ethnic differences in hospital-based procedure utilization in California. AB - OBJECTIVE: To examine several hospital-based procedures for systematic utilization differences between the genders and among ethnic groups (Asian, black, Latino, and white). METHODS: California hospital discharges in 1989 and 1990 were sampled by principal diagnosis. Odds ratios for treatment by demographic class were estimated for heart transplantation, kidney transplantation, extracorporeal shockwave lithotripsy, hip replacement, carotid endarterectomy, coronary artery bypass grafting, percutaneous transluminal coronary angioplasty, pacemaker implant, and automatic cardioverter-defibrillator implant. Logistic regression controlled for insurance status, age, diagnosis, and comorbidity count. RESULTS: The following results were statistically significant (P < .05). Males' odds of receiving most procedures exceeded those of females by 115% (odds ratio, 2.15) for coronary artery bypass grafting, 86% for heart transplantation, 38% for defibrillator implants, 34% for angioplasty, 28% for pacemaker implants, and 24% for hip replacement. Whites' odds of receiving several procedures exceeded those of blacks by 204% for kidney transplantation, 186% for defibrillator implant, 144% for coronary artery bypass grafting, 127% for endarterectomy, and 100% for angioplasty. Whites' odds of receiving some procedures also exceeded those of Latinos by 72% for angioplasty, 58% for kidney transplantation, and 49% for coronary artery bypass grafting. Whites' odds of receiving endarterectomy or angioplasty exceeded those of Asians by 108% and 30%, respectively. Asians had 113% higher odds than whites of receiving hip replacement. CONCLUSIONS: The array of utilization differences across 4 demographic comparisons and 9 hospital procedures suggests systematic trends in high-technology allocation. Generally, women received procedures less often than men and minorities less than whites. PMID- 8639017 TI - Marked improvement in recognition and completion of health care proxies. A randomized controlled trial of counseling by hospital patient representatives. AB - BACKGROUND: Advance directives provide a means for patients to retain influence on their medical care should decisional capacity be lost. Several studies have now demonstrated that advance directives that are completed in the ambulatory care setting are rarely available and recognized when patients are admitted to the acute care hospital. OBJECTIVE: To evaluate a generalizable model for improving recognition of previously completed advance directives and for promoting appointment of health care proxies in hospitalized patients. METHODS: Hospitalized elderly patients were randomly assigned to receive the intervention or usual care (n = 190). Intervention patients with capacity were counseled by hospital patient representatives about advance directives and encouraged to complete health care proxies. Patients with existing proxies had this information noted in their charts. For patients without capacity, counselors reviewed their charts for proxy documentation and if absent, contacted patients' next of kin and private physicians to determine proxy status. Usual care patients were not contacted by patient representatives. RESULTS: Forty-eight percent of intervention patients completed a new proxy or had a previously completed proxy identified compared with 6% of controls (P < .001). For patients with capacity, 22% of intervention patients had a previously appointed proxy agent identified compared with 6% of controls (P < .001). Thirty-six percent of intervention patients appointed a proxy decision maker compared with 0% of controls (P < .02). For patients without capacity, 31% of intervention patients had previously appointed proxies identified compared with 6% of controls (P < .001). CONCLUSIONS: Counseling by hospital patient representatives is an effective and generalizable means of improving recognition and execution of advance directives in the acute care hospital. PMID- 8639018 TI - Switch therapy in community-acquired pneumonia. PMID- 8639019 TI - Agranulocytosis associated with yohimbine use. PMID- 8639020 TI - N-acetylcysteine inhibits production of tumor necrosis factor alpha and interleukin-1 beta. PMID- 8639022 TI - Characterization of the lipopolysaccharides from Rhizobium meliloti strain 102F51 and its nonnodulating mutant WL113. AB - Lipopolysaccharides from the Rhizobium meliloti wild-type strain 102F51, which is effective in symbiosis with alfalfa, and from the nonnodulating mutant WL113, defective in root hair adhesion, derived thereof, were isolated and comparatively analyzed. Both preparations were composed of galactose, glucose, glucuronic acid, galacturonic acid, glucosamine, 3-deoxyheptulosaric acid, and 2-keto-3 deoxyoctonic acid as the major sugar constitutents. After a modified methylation analysis (consisting of the following consecutive steps: methylation, carboxyl reduction, remethylation, mild acid hydrolysis, reduction, and trideuterio methylation), all of the 3-deoxyheptulosaric and some of the 2-keto-3 deoxyoctonic acid residues were converted into their corresponding 3-deoxyalditol derivatives, which carried trideuteriomethyl groups at positions C-2, C-4, and C 6. Another part of the permethylated 3-deoxyoctitol was also found as 2,5,6- and 2,6,8-tri-O-trideuteriomethyl derivatives. NMR data obtained with the separated oligosaccharides and the results of methylation analysis indicated that the majority of 2-keto-3-deoxyoctonate was present in the fraction of permethylated disaccharide alditols, namely as 6-O-CD3-alpha Glc(1-->5)3-deoxyoctitol, 6-O-CD3 beta GlcNMeAcyl(1-->4)-3-deoxyoctitol, and as the permethylated trisaccharide alditol, alpha GalA(1-->3)-[6-O-CD3]-beta-Glc(1-->5)-[4-O-CD3]-3-deoxyoctitol. The presence of trideuteriomethyl groups at C-4 of both 3-deoxyalditols and at C 6 of the glucosaminyl or glycosyl residues indicated the linkage points of the released acid-labile ketosidic substituents, such as 3-deoxyheptulosarate and 2 keto-3-deoxyoctonate, in these oligosaccharides. The main differences between the preparations from the wild-type 102F51 and its mutant strain WL 113 were found in the higher content (in strain 102F51) of the following oligosaccharides: alpha glucuronosyl(1-->4)2-keto-3-deoxyoctonate and alpha-galacturonosyl-(1-->3)alpha glucosyl-(1-->5)2-keto-3-deoxyoc tonate and in the decreased content of beta glucosaminyl(1-->4)2-keto-3-deoxy-octonate. PMID- 8639021 TI - Sec-dependent preprotein translocation in bacteria. AB - Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multisubunit protein complex termed translocase, which consists of the integral membrane heterotrimer Sec YEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with Sec YEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. PMID- 8639023 TI - Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans. AB - Cytochrome cd1-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd1-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri. PMID- 8639024 TI - Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud). AB - A delta sud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The delta psr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity. PMID- 8639025 TI - Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit. AB - A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3'-terminal part encoding the C terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of 63Ni2+. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal. PMID- 8639026 TI - Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda. AB - Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional beta-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the beta-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the beta-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436 bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5 alpha(pETH3E) is very likely the beta-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5 alpha(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli. PMID- 8639027 TI - Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8. AB - Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. PMID- 8639028 TI - Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes. AB - Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A mutant that could grow on 2-bromoethanol with a growth rate of 0.034 h 1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2 chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2 bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2 chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8-4 microM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V(max) values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme of bromoacetaldehyde. PMID- 8639029 TI - Evidence that two genomic species of Rhizobium are associated with Medicago truncatula. AB - Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization. PMID- 8639030 TI - Violence, crime, and mental illness. How strong a link? PMID- 8639031 TI - Mental disorder and crime. Evidence from a Danish birth cohort. AB - BACKGROUND: Evidence has accumulated since the mid 1960s from a number of different countries indicating an association between mental disorder and crime and particularly between the major mental disorders and violence. Registries in Denmark were used to identify a birth cohort and to document all psychiatric admissions and all criminal proceedings of the 324401 members of this cohort up to the age of 43 years. METHODS: Persons who had been admitted to a psychiatric ward were assigned to a diagnostic category according to a hierarchy of principal discharge diagnoses. They were compared with persons never admitted to a psychiatric ward as to the prevalence, type, and frequency of criminal convictions. RESULTS: Women and men who had been hospitalized in psychiatric wards were more likely to have been convicted of a criminal offense than persons with no history of psychiatric hospitalization. The offenders who were hospitalized committed all types and, on average, as many offenses as did the never-hospitalized group of the same sex. CONCLUSIONS: These findings confirm those from 2 other post-World War II Scandinavian birth cohorts that have found an association between psychiatric hospitalization and criminal convictions. They also concur with findings that patients discharged from psychiatric wards are more likely than other persons living in the same community to commit crimes and with results from North America showing elevated rates of major mental disorders among incarcerated offenders. Generalization of these findings is limited to nations with similar criminal justice, mental health, and social welfare systems. PMID- 8639032 TI - Mental disorders and homicidal behavior in Finland. AB - BACKGROUND: Owing to the fact that Finnish police have been able to solve about 95% of all homicides during recent decades and because most homicide offenders are subjected to an intensive psychiatric evaluation, it was possible to examine data on 693 of 994 homicide offenders during an 8-year period. METHODS: The prevalences of mental disorders of the homicide offenders were used to calculate odds ratios (ORs) for the statistical increase in risk associated with specific mental disorders. RESULTS: The results indicate that schizophrenia increases the OR of homicidal violence by about 8-fold in men and 6.5-fold in women. Antisocial personality disorder increases the OR over 10-fold in men and over 50-fold in women. Affective disorders, anxiety disorders, dysthymia, and mental retardation did not elevate the OR to any significant extent (OR < 5.0). CONCLUSION: Homicidal behavior in a country with a relatively low crime rate appears to have a statistical association with some specific mental disorders classified according to DSM-III-R classifications. PMID- 8639033 TI - Prevalence of psychiatric disorders among incarcerated women. I. Pretrial jail detainees. AB - BACKGROUND: There are little epidemiologic data on psychiatric disorders of women in jails. Accurate data on female jail detainees are critical because of their increasing numbers and their unique treatment needs. METHODS: Using the Diagnostic Interview Schedule, independent interviewers assessed a randomly selected, stratified sample of 1272 female jail detainees awaiting trial in Chicago, Ill. We tabulated lifetime and 6-month prevalence rates of disorders by race or ethnicity (African American, non-Hispanic white, Hispanic), age, and education and compared the jail rates with general population rates for women in the Epidemiologic Catchment Area program. We also examined whether or not psychiatric disorder was associated with the severity of the detainee's current arrest charges. RESULTS: Over 80% of the sample met criteria for one or more lifetime psychiatric disorders; 70% were symptomatic within 6 months of the interview. The most common disorders were drug abuse or dependence, alcohol abuse or dependence, and post-traumatic stress disorder. Major depressive episode was the most prevalent major mental disorder. Rates were generally highest among non Hispanic whites and among older detainees. Rates for all disorders were significantly higher than general population rates, except for schizophrenia. Most detainees with psychiatric disorders were arrested for nonviolent crimes. CONCLUSION: These results suggest substantial psychiatric morbidity among female jail detainees. PMID- 8639034 TI - Prevalence of psychiatric disorders among incarcerated women. II. Convicted felons entering prison. AB - BACKGROUND: No unbiased estimates of the rates of psychiatric disorder among women prison inmates are available. Nonetheless, available data suggest that some psychiatric disorders are prevalent in this population. The objective of the study was to determine the rates, risk factors, and outcomes of specific psychiatric disorders among women prison inmates. METHODS: A virtual census of women felons (N = 805) entering prison in North Carolina was assessed using in person interviews. Assessments were conducted for 8 disorders, using the Composite International Diagnostic Interview as the primary assessment measure. For validation purposes, one quarter of the inmates were reassessed for 2 of these disorders, using structured clinical interviews. RESULTS: Inmates were found to have high rates of substance abuse and dependence and antisocial and border-line personality disorders compared with women in community epidemiologic studies. Rates among inmates were also somewhat elevated for mood disorders but not for anxiety disorders. The rate of reports of lifetime exposure to traumatic events was also high. Rates of disorder tended to be higher among white than among African American women. CONCLUSION: High rates of substance abuse, psychiatric disorder, and psychological distress associated with exposure to traumatic events suggest that women in prison have a need for treatment for substance abuse and other mental health problems. PMID- 8639035 TI - A prospective follow-up study of alcoholic violent offenders and fire setters. AB - BACKGROUND: This study investigated biochemical and family variables and predictors of recidivism among forensic psychiatric patients who had committed violent offenses or set fires. METHODS: One hundred fourteen male alcoholic violent offenders and fire setters were followed up for an average of 4.5 years after release from prison. At the beginning of their incarceration, the first half of the offenders were administered clinical diagnostic interviews, whereas the latter half received the Structured Clinical Interview for DSM-III (SCID) that was blind rated. A structured family history questionnaire was administered to all available first-degree relatives of offenders. The offenders also received lumbar punctures for monoamine metabolites, an oral glucose tolerance test, and a measurement of fasting plasma cholesterol level. At the end of the follow-up, the Finnish criminal registry was searched for recidivist crimes. RESULTS: Among all offenders, low cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) concentrations were associated with a family history positive for paternal alcoholism with violence. Low plasma cholesterol concentration was associated with a family history positive for paternal alcoholism without violence. The recidivists, who committed violent offenses or set fires during the follow-up period, had low CSF 5-HIAA and 3-methoxy-4 hydroxyphenylglycol (MHPG) concentrations compared with those in nonrecidivists. Early family environments of the recidivists, compared with those of the nonrecidivists, were characterized by common paternal absence from and presence of brothers at home. CONCLUSION: Among male alcoholic violent offenders and fire setters, low CSF 5-HIAA and HVA concentrations are strongly associated with a family history positive for paternal violence and alcoholism, while low fasting plasma cholesterol concentration is associated with a family history positive for paternal alcoholism. Recidivist violent offenders and fire setters are predicted by low CSF 5-HIAA and MHPG concentrations and a developmental history positive for early paternal absence from and presence of brothers in the family of origin. PMID- 8639036 TI - Impulsive aggression in personality disorder correlates with tritiated paroxetine binding in the platelet. AB - BACKGROUND: To examine the relationship between binding parameters of the platelet central serotonergic (5-HT) transporter and measures of aggression and impulsivity in adult human subjects. METHODS: Maximal number of platelet tritiated paroxetine binding sites (Bmax) and dissociation constant (Kd) values were measured in patients with personality disorder (n = 24) and healthy volunteers (n = 12). Measures of aggression and impulsivity included the total score and aggression subscale of the Life History of Aggression, the Motor Aggression factor and the assault subscale of the Buss-Durkee Hostility Inventory, and the total score and motor impulsivity subscale of the Barratt Impulsiveness Scale. RESULTS: The Bmax, but not Kd, values of platelet tritiated paroxetine binding was inversely correlated with the Life History of Aggression total score and aggression score and with the Buss-Durkee Hostility Inventory assault score in patients with personality disorder but not in healthy volunteer subjects. This relationship was independent of influences of factors related to depression, global function, or history of alcoholism or drug abuse. CONCLUSIONS: Reduced numbers of platelet 5-HT transporter sites may covary with life history of aggressive behavior in patients with personality disorder. This may represent another abnormality in 5-HT function in individuals with personality disorder and aggressive behavior. PMID- 8639037 TI - Excessive mortality in young free-ranging male nonhuman primates with low cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations. AB - BACKGROUND: The purpose of this study was to assess the impact of central serotonin turnover rate on survival to adulthood among nonhuman primates living in a large, free-ranging colony. METHODS: The rate of mortality was ascertained over a 4-year period after obtaining blood and cisternal cerebrospinal fluid (CSF) samples from 49 free-ranging, 2-year-old prepubertal male rhesus monkeys. Cerebrospinal fluid was assayed for 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, 3-methoxy-4-hydroxyphenylgycol, and homovanillic acid. Blood plasma was assayed for adrenocorticotropic hormone, cortisol, and testosterone. Following the sampling of body fluids, records of scars and wounds and aggressive encounters were used to rank the subjects from low to high in aggressiveness. Direct observations of aggressive behavior were collected from 27 of the subjects over a 3-month period. RESULTS: Four years later, 6 of the 49 subjects were known to be dead and an additional 5 had been missing for more than 2 years and were presumed dead. The CSF 5-HIAA concentrations were predictive of which subjects died, with 46% of the subjects with low CSF 5-HIAA concentrations dead or presumed dead. None of the subjects from the highest CSF 5-HIAA concentration quartile were dead or missing. Indeed, 91% of the dead subjects came from the 2 lowest quartiles of CSF 5-HIAA concentrations. Direct observations of aggressive behavior showed that dead or missing subjects had initiated escalated aggression, a measure of unrestrained aggression that has a high probability of trauma or injury, at a higher rate than subjects that were known to be alive. The cause of death could be ascertained for 6 of the 11 missing subjects. The 4 subjects that were known to die as a consequence of aggressive encounters came from the lowest quartile of CSF 5-HIAA concentrations and had been rated as more aggressive during their initial capture. Subjects captured more than once possessed lower CSF 5-HIAA concentrations, were rated as more aggressive, and were more likely to suffer early death than those captured only once. CONCLUSION: These findings suggest that low CSF 5-HIAA concentrations quantified early in life is a powerful biological predictor of future excessive aggression, risk taking, and premature death among nonhuman primate males. PMID- 8639038 TI - High rates of violence, crime, academic problems, and behavioral problems in males with both early neuromotor deficits and unstable family environments. AB - BACKGROUND: It is commonly assumed that individuals with both biological and psychosocial deficits are more likely to become criminal, but there is surprisingly little empirical support for this assumption. We test the hypothesis that a group with biosocial risk factors are more likely to develop behavioral and academic problems in adolescence and violent criminal offending in adult-hood compared with groups with only biological or only social risk factors. METHODS: Hypotheses were tested on a sample of 397 male subjects, using obstetric and early neuromotor measures collected in the first year of life; family, social, demographic, and behavioral measures at age 17 to 19 years; and criminal data at 20 to 22 years of age. RESULTS: Cluster analysis of the risk factors indicated a group with obstetric risk factors only, a group with poverty risk factors only, and a biosocial group with both early neuromotor deficits and unstable family environments. The biosocial group had more than double the adult violence, theft, and total crime rates of the other 2 groups and had significantly more behavioral and academic problems in adolescence. CONCLUSIONS: When early neuromotor deficits and negative family factors cluster together, individuals are particularly likely to become criminal and violent compared with those with only poverty or only obstetric risk factors. Because this biosocial group accounted for 70.2% of all crimes committed in the entire sample, early interventions that tackle these deficits might significantly reduce violence. PMID- 8639039 TI - The association of sexual assault and attempted suicide within the community. AB - BACKGROUND: Lifetime community rates of attempted suicide were compared between those who reported a history of sexual assault and a control group without such a history. METHODS: The 2918 respondents in the Duke University Epidemiological Catchment Area Study were placed into groups with reported sexual assault (n = 67) and those with no known history of such (n = 2851). Multivariate and bivariate procedures were used to examine the relation between sexual assault and attempted suicide. RESULTS: Subjects reporting a history of sexual assault were more likely to be female, younger, and to report higher rates of lifetime suicide attempt and post-traumatic stress symptoms; no differences were found in the number of chronic medical disorders, major depression, substance abuse or substance dependence, or panic attacks. Nine (14.9%) of the 67 index group subjects reported a suicide attempt, 4 of whom reported their first sexual assault as occurring before age 16 years. A sexual assault history was associated with increased prevalence of lifetime suicide attempt after controlling for sex, age, education, posttraumatic stress symptoms, and psychiatric disorder. Findings were similar in the female-only subsample (n = 1778). For women, the odds of attempting suicide was 3 to 4 times greater when the first reported sexual assault occurred prior to age 16 years compared with age 16 years or older. CONCLUSIONS: Sexual assault is associated with an increased lifetime rate of attempted suicide. In women, a history of sexual trauma before age 16 years is a particularly strong correlate of attempted suicide. PMID- 8639040 TI - Two Persian Gulf veterans with lymphadenopathy. PMID- 8639041 TI - False-positive antineutrophil cytoplasmic antibody in aspergillosis with oxalosis. PMID- 8639042 TI - Shadow cells in extracutaneous locations. PMID- 8639043 TI - Obtaining permission for postmortem examination. PMID- 8639044 TI - Mycobacterial testing in clinical laboratories that participate in the College of American Pathologists Mycobacteriology Surveys. Changes in practices based on responses to 1992, 1993, and 1995 questionnaires. AB - OBJECTIVE: To determine whether the trend of increasing use of rapid methods of mycobacterial testing among participants in the College of American Pathologists (CAP) Mycobacteriology E Proficiency Testing Survey noted between 1992 and 1993 continued through 1995, and to collect information concerning mycobacterial staining and culture protocols from laboratories that do limited mycobacterial testing. METHODS: The 1993 CAP E Survey questionnaire addressing mycobacterial laboratory practices, test volumes, and rate of recovery of drug-resistant Mycobacterium tuberculosis was included with the CAP 1995 E-A Survey. A shortened list of these same questions, excluding those addressing mycobacterial identification and susceptibility test methods, was added to the CAP 1995 E1-A Survey, to which laboratories that do limited mycobacterial testing subscribe. RESULTS: A total of 802 and 1490 participants in the E and E1 surveys, respectively, returned responses to the CAP by the cutoff date for data analysis. For E Survey participants who answered questions concerning test methods in the years being compared, the percentage who used rapid techniques increased significantly over the study period. More participants used the fluorochrome stain (58% in 1992, 62% in 1993, and 72% in 1995), BACTEC TB plus a solid medium for culture (36% in 1992, 42% in 1993, and 50% in 1995), DNA probes for identification of M tuberculosis (68% in 1993, 79% in 1995), and BACTEC TB for susceptibility testing (65% in 1993, 71% in 1995). The percentages of E1 Survey participants who used a fluorochrome stain for detection of acid-fast bacilli and both a liquid and a solid medium for mycobacterial culture were lower than the percentages of E Survey participants who used these methods. Among participants who responded in all years being compared, the percentage processing respiratory specimens at least 7 times per week increased from 26% in 1992 to 30% in 1993 and 43% in 1995 (P < .001), and the percentages reporting an identification of M tuberculosis within 21 days and susceptibility test results within 28 days increased significantly over the study period (29% in 1992, 40% in 1993, and 56% in 1995 for identification; 13% in 1992, 19% in 1993, and 30% in 1995 for susceptibility testing). Turnaround times for E Survey participants were significantly shorter than those for E1 Survey participants. The number of specimens tested per month appeared to remain relatively stable between 1993 and 1995; however, the number of new patients with tuberculosis and the number of known tuberculosis patients with positive cultures declined significantly. CONCLUSIONS: The recent emphasis placed on utilization of rapid methods of mycobacterial testing appears to have influenced laboratories that subscribe to the CAP E Survey. Significantly more of these laboratories were following the Centers for Disease Control and Prevention's recommendations in 1995 than in 1993 and 1992. However, many laboratories that provide only limited mycobacterial testing still have not adopted the more rapid techniques. Because tuberculosis remains a public health problem, the efforts directed at its control must not wane if the recent downward trend in incidence is to be maintained. PMID- 8639045 TI - Susceptibility testing of Mycobacterium avium complex in clinical laboratories. Results of a questionnaire and proficiency test performance by participants in the College of American Pathologists Mycobacteriology E Survey. AB - OBJECTIVES: To obtain information regarding the frequency and methodology of susceptibility testing of Mycobacterium avium complex (MAC) in clinical microbiology laboratories, and to assess interlaboratory reproducibility of MAC susceptibility testing. DESIGN: Questions addressing MAC susceptibility testing were added to the College of American Pathologists' 1994 Mycobacteriology E Proficiency Testing Survey, and participants were asked to complete the questionnaire. In addition, participants in the 1994 E Survey were asked to test susceptibility of a MAC isolate recovered from a proficiency testing specimen as an ungraded exercise if they offered such testing for patients. RESULTS: Of the 1003 participants enrolled in the 1994 Mycobacteriology E-A Survey, 806 responded to one or more supplemental questions. In regard to the demand for MAC susceptibility testing, 606 participants indicated that the test is requested by physicians in their institutions, and 188 said that they do the test routinely on at least one MAC isolate per patient. Eighty-two percent (630/765) of participants refer the test to an outside laboratory, most commonly a commercial reference laboratory or state health laboratory. Of the 70 participants who perform MAC susceptibility testing in-house and indicated the method on the questionnaire, 54 (77%) used a solid medium, whereas only 14 (20%) used BACTEC TB, which currently is the recommended method. The most frequently tested drugs were ethambutol, rifampin, isoniazid, and streptomycin; other commonly evaluated agents were ciprofloxacin, amikacin, and clarithromycin. Only eight participants modify the pH of the medium when testing a macrolide. In regard to reporting test results, 56% (45/80) report a qualitative result only, 35% (28/80) report a quantitative result with a qualitative interpretation, and 9% (7/80) report only a quantitative result. Participant performance on the MAC proficiency testing specimen showed lack of interlaboratory reproducibility; 80% or fewer participants reported the correct result for all drugs except amikacin, for which 92% (11/17) of laboratories responded correctly. CONCLUSIONS: Given the obvious interest in MAC susceptibility testing, standardized methodology that demonstrates interlaboratory reproducibility and, optimally, shows some correlation with clinical outcome is needed. Moreover, recommendations concerning indications for performing the test would be useful. PMID- 8639046 TI - Atypical squamous cells of undetermined significance. Current laboratory practices of participants in the College of American Pathologists Interlaboratory. Comparison Program in Cervicovaginal Cytology. AB - OBJECTIVE: To evaluate current laboratory practices and rates for atypical squamous cells of undetermined significance (ASCUS), a category of epithelial cell abnormality in the Bethesda System. DESIGN: Questionnaire surveys were mailed in December 1993 and March 1994. SETTING: Cytopathology laboratory participants in the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology (PAP). RESULTS: Most responding laboratories (82.5%) limited the use of "atypia" terminology to abnormalities of undetermined significance. Nearly half of the laboratories employed only the term ASCUS for squamous epithelial changes in this category. The median rate of ASCUS in 1993 was 2.8%, with 10% of laboratories reporting rates greater than 9.0%. The median squamous intraepithelial lesion rate was 2.0%, with a median ASCUS-squamous intraepithelial lesion ratio of 1.3. The majority of laboratories qualified a portion of ASCUS cases and issued recommendations for follow-up when appropriate. Fifty-six percent of laboratories surveyed included patients diagnosed with ASCUS in follow-up programs. Laboratories estimated that about 20% (median response) of patients with ASCUS smears had a squamous intraepithelial lesion or equivalent diagnosis made within a year's follow-up. CONCLUSIONS: The ASCUS category is used by the majority of laboratories as recommended by the Bethesda System, but reporting rates vary. The results of this survey and associated surveys provide laboratories with useful benchmark figures for interlaboratory comparison of ASCUS practices. PMID- 8639047 TI - Quality improvement of diagnostic microbiology through a peer-group proficiency assessment program. A 20-year experience in Ontario. The Microbiology Committee. AB - OBJECTIVE: To evaluate the microbiology laboratory performance in Ontario over a 20-year period of participation in a quality assessment program and to assess the impact of quality improvement strategies. DESIGN: Longitudinal review of isolation, identification, and antimicrobial susceptibility testing of bacteria from lyophilized, simulated patient samples. SETTING: Ontario medical laboratories, licensed by the Ministry of Health, have been subjected to mandatory testing by the Laboratory Proficiency Testing Program of the Ontario Medical Association since 1974. Survey reports, information bulletins, correspondence, on-site consultations, educational assistance tutorials, and teleconference education are used as quality improvement strategies. PARTICIPANTS AND INTERVENTIONS: Laboratories were subjected annually to 20 external quality assessment challenges. Performance was assessed against consensus reference values. Single survey and cumulative profiles were reviewed by a peer-group panel for acceptable or unacceptable performance. Specific interventions are used to improve collective and individual laboratory performance. RESULTS: The number of microbiology laboratories declined from 335 in 1974 to 190 in 1994. Twenty-one percent failed expected performance standards on initial review. One hundred forty-two on-site consultations and 61 educational assistance tutorials have been provided. Twenty-five laboratories were declared nonproficient. Since 1989, 50% of the laboratories have scored at or above 80% for isolation and identification, but 25% have scored at or below 50% on susceptibility testing, and 10% or fewer have scored at or above 80%. Poor susceptibility testing performance is due to inappropriate agent selection, not testing errors. CONCLUSIONS: The emphasis of the Laboratory Proficiency Testing Program is on quality improvement, not punishment. Performance has improved, but poor performers have the same characteristics as in 1974. Identification to species is common owing to the use of commercial systems. Automated susceptibility testing has increased to 45% of participants. PMID- 8639048 TI - Pseudomeningitis again. Association with cytocentrifuge funnel and Gram-stain reagent contamination. AB - OBJECTIVE: To report an "epidemic" of pseudomeningitis related to cytocentrifuge funnel and Gram-stain reagent contamination, and our evaluation and responses. DESIGN: Investigation was stimulated by the recognition of Gram-stained, smear positive, culture-negative cerebrospinal fluid (CSF) specimens. Cytofunnels, glass slides, Gram-staining reagents, and an automated Gram-staining apparatus were subjected to repeated staining and culture. Control stains and cultures using fetal bovine serum (simulated CSF) were performed for comparison. SETTING: The clinical microbiology laboratory of Parkland Memorial Hospital, a large acute care teaching hospital. SPECIMENS: Cerebrospinal fluid specimens were submitted to the clinical microbiology laboratory in the course of routine patient care. MAIN OUTCOME MEASURES: Gram's stains and cultures of test and control preparations. RESULTS: Most of the smear-positive, culture-negative, original CSF specimens contained Gram-positive bacilli or Gram-negative bacilli. Smears of cytofunnels revealed similar organisms, and cultures revealed Bacillus species. Cytofunnels from several lots were culture-positive. Glass slides were not contaminated. Of 25 CSF specimens stained during the initial week of investigation, 23 were negative by culture and two grew Cryptococcus neoformans (from acquired immunodeficiency syndrome patients). Control stains and cultures of simulated CSF were negative. Gram-stain reagents were frequently smear positive, and cultures repeatedly yielded Flavimonas oryzihabitans from the crystal violet well of an automated Gram-staining apparatus. These latter contaminants could not be eliminated consistently. INTERVENTIONS: No alternative sources of cytofunnels were found. The Gram-staining apparatus was cleaned and reagents changed frequently. Cytocentrifugation and use of automated Gram staining was discontinued for CSF and other normally sterile fluids. The laboratory staff was repeatedly educated about the problem. CONCLUSIONS: Contamination of cytocentrifuge funnels and an automated Gram-staining apparatus contributed to an "epidemic" of pseudomeningitis. The problem was corrected by education of the laboratory staff and by altered management of CSF and other sterile body fluid specimens. PMID- 8639049 TI - Changing patterns of autopsy findings among persons with acquired immunodeficiency syndrome in an inner-city population. A 12-year retrospective study. AB - OBJECTIVE: To determine patterns of autopsy findings among persons with acquired immunodeficiency syndrome (AIDS) during a 12-year period. DESIGN AND SETTING: All 168 autopsies performed on adult AIDS patients at the Bronx-Lebanon Hospital Center in New York City between 1982 and 1993 were analyzed. The Center is a 725 bed, acute-care facility in the south Bronx, which serves a population of approximately half a million people. High poverty levels, tuberculosis and AIDS incidence, and intravenous drug abuse are common in this population. Subjects were predominantly Hispanic and black. MAIN OUTCOME MEASURES: Changes in the frequency of single and multiple opportunistic infections in general and frequencies of specific infections during the 12-year period. RESULTS: Through 1986, 75% of AIDS autopsies demonstrated single infections, particularly Pneumocystis carinii pneumonia. Since 1987, 72% of autopsies demonstrated multiple infections related to P carinii pneumonia, mycobacteriosis, cytomegalovirus, and various fungi. During the last 3 years, the prevalence of mycobacterial infections was higher than in the previous 9 years combined. In contrast, P carinii pneumonia decreased from 52% in 1988 to 14% in 1993. CONCLUSIONS: We note the recent emergence of multiple infections by multiple organisms, a significant decrease in the prevalence of P carinii pneumonia, and a slight increase in mycobacterial tuberculosis. Awareness of these changing patterns of infection may be useful in treating persons with AIDS. PMID- 8639050 TI - Immunohistochemical markers of prolonged survival in small cell carcinoma of the lung. An immunohistochemical study. AB - OBJECTIVE: To investigate the association of a variety of cell surface and cytoplasmic antigens in small cell carcinoma of the lung with long-term survival (greater than 2 years). DESIGN: Using immunohistochemical analysis of small cell carcinomas, the tissue expression of corticotropin, bcl-2, p-glycoprotein, cathepsin B, cathepsin D, CD44, carcinoembryonic antigen, collagenase IV, Leu-7, neu oncoprotein, p53, S100, and synaptophysin was assessed. RESULTS: Compared with the control group of short-term survivors, tumors from prolonged survivors were unique in their relative absence of staining for cathepsin B (0/13 vs 3/13 [23%], P = .037), cathepsin D (5/13 [38%] vs 13/15 [87%], P = 0.006), carcinoembryonic antigen (5/13 [38%] vs 11/15 [73%], P = .047), and neu oncoprotein (5/13 [38%] vs 14/15 [93%], P = .0014). A variety of histologic characteristics were also compared, and none were shown to be associated with differences in survival in this study. CONCLUSIONS: Negative immunohistochemical staining for cathepsin B, cathepsin D, carcinoembryonic antigen, and neu oncoprotein is associated with prolonged survival in small cell carcinoma of the lung. Evaluation of these antigens should be considered in future attempts to stratify patients with small cell carcinoma of the lung for prognostic or therapeutic purposes, as this study is limited by the small size of the study group and the large number of clinical and pathologic variables. PMID- 8639051 TI - Relationship of standardized mitotic indices to other prognostic factors in breast cancer. AB - OBJECTIVE: To examine the relationship between mitotic index (MI), calculated from direct microscopic counts, and other prognostic features in breast cancer. DESIGN: Mitotic index was based on direct microscopic observations of mitotic figures in 10 consecutive microscopic fields, and the average cell number was determined by counts of population density in three of those fields. Tumor grade and type were established from tissue sections, whereas metastases were detected in lymph node biopsy, chest roentgenograms, and bone scan. Estrogen receptor (ER) and progesterone receptor (PgR) levels were determined by flow cytometry. RESULTS: The MI for 242 patients ranged from 0.2 to 37.6, with a mean of 5.8 mitoses per 1000 cells. More than 85% of the tumors with an MI below 1.0 were diploid and contained an S-phase fraction of 6.7% or less. In contrast, more than 75% of tumors with an MI above 5.0 were aneuploid with more than 6.7% of cells in S-phase. There was an inverse relationship between ER and PgR status and MI. Eighty percent of tumors with an MI less than 1.0 were both ER and PgR positive while only 25% of those with an MI above 10.0 were both ER and PgR positive. Receptor-positive tumors with high S-phase and MI values had ER and PgR levels below 100 fmol/mg. CONCLUSIONS: Lower MI values calculated from direct cell counts are correlated with negative node status, diploid DNA content, low S-phase fraction, and positive receptor status. Thus, there is a significant relationship between objective MI values and several other factors that predict the probability of breast tumor recurrence. PMID- 8639052 TI - Neuroendocrine differentiation in gastric adenocarcinomas. An immunohistochemical study. AB - BACKGROUND: The stomach contains a wide variety of neuroendocrine cells. Early studies with argyrophilic stains documented the presence of these cells in gastric adenocarcinomas. Immunohistochemical techniques for demonstrating hormones are more sensitive and specific and have been applied only sporadically to gastric adenocarcinomas. Thus, the true incidence of neuroendocrine cells in gastric adenocarcinomas is questionable. METHODS: Formalin-fixed, paraffin embedded archival tissue specimens from 48 gastric adenocarcinomas were immunostained with antibodies to chromogranin A, synaptophysin, serotonin, gastrin, and neuron-specific enolase. The percentage of cells staining positively was evaluated semiquantitatively. RESULTS: Among 48 gastric adenocarcinomas, 36 (75%) stained positively for chromogranin A, 33 (69%) stained for synaptophysin, 29 (60%) stained for neuron-specific enolase, 17 (36%) stained for gastrin, and 15 (31%) stained for serotonin. The distribution of positivity was highest for chromogranin A (7 cases positive in 26% to 75% of cells) and lowest for serotonin (14 out of 15 cases stained in fewer than 1% of the cells present). CONCLUSIONS: Immunohistochemical evaluation of neuroendocrine markers in gastric adenocarcinomas indicates that a high percentage of tumors contain widely scattered single cells with neuroendocrine differentiation. Most often, however, such cells constitute only a small percentage of the total number of tumor cells present. PMID- 8639053 TI - Sarcoma in association with bone infarcts. Report of five cases. AB - Sarcoma associated with bone infarct is rare, and only 41 well-documented cases have been published. We describe five additional patients, three women and two men, aged 39 to 57 years. The tumors involved the femur (three patients), tibia (one patient), and humerus (one patient). In three patients, the infarcts were idiopathic. Radiologic evidence of malignancy was found in all patients, and bone infarcts were suspected in four. Four of the patients had malignant fibrous histiocytoma and one an osteosarcoma. Histologically, bone infarcts were seen in all patients, but in three they were mostly replaced by tumor. Portions of intact infarcts were seen adjacent to the tumor, indicating that they had preceded the development of the sarcoma. No hypercellular or atypical reparative tissue was found in the infarcted bones or in three additional uncomplicated infarcts studied from the same patients. The pathogenesis of sarcoma arising in bone infarct is unknown. The prognosis is poor; four of our five patients died within 2 years. PMID- 8639054 TI - Expression of the fibroblast growth factor receptor-1 in human normal tissues and tumors determined by a new monoclonal antibody. AB - OBJECTIVE: To characterize the distribution of the receptor for the fibroblast growth factor in human normal tissues and tumors using a new monoclonal antibody. DESIGN: Monoclonal antibodies for a kinase-insert region of fibroblast growth factor receptor-1 (FGFR-1) were generated. We conducted an immunohistological analysis of FGFR-1 using a highly specific antibody we generated, and we examined the distribution of this receptor in normal human tissues and in tumors. RESULTS: Intense positivity of FGFR-1 was observed in astrocytes in the brain, smooth muscles in the uterus, cardiac myocytes, respiratory epithelium in the lung, tubular epithelium in the kidney, acinar cells in the pancreas, follicular cells in the thyroid, and ductal and lobular epithelium in the breast. We also observed FGFR-1 expression in the fibroblasts and the tissue microvasculature. In addition to some nonepithelial tumors, some epithelial tumors expressed FGFR-1, including pancreatic adenocarcinomas, thyroid papillary carcinomas, invasive ductal carcinomas of the breast, lung adenocarcinomas, renal cell carcinomas, and colonic adenocarcinomas. Although FGFR-1 was expressed in colonic adenocarcinomas, which have invasive potential, tubular adenomas, which are noninvasive, did not express FGFR-1. CONCLUSION: We have been able to define the distribution of FGFR-1 in human normal tissues and tumors. Especially in colonic tumors, FGFR-1 expression may lead adenoma cells to invade and grow in the surrounding tissue. PMID- 8639055 TI - Expression of bcl-2 in uveal malignant melanoma. AB - OBJECTIVE: To evaluate the expression of the bcl-2 proto-oncogene in uveal malignant melanomas. CASE MATERIAL: We studied 20 uveal malignant melanomas (19 choroidal and 1 ciliary body) by immunohistochemistry with the bcl-2 oncoprotein monoclonal antibody and the cell proliferation marker, MIB-1. RESULTS: Expression of bcl-2 was found in 100% of cases and was not correlated with the histologic subtype of melanoma or the MIB-1 proliferative index. Normal choroidal melanocytes were negative for bcl-2. CONCLUSION: Our results suggest that altered expression of bcl-2 is common in uveal melanomas and is not related to histologic grade. PMID- 8639056 TI - Yersinia enterocolitica bacteremia associated with red blood cell transfusion. AB - A chronically ill, 80-year-old man received over three fourths of a unit of CPDA 1 red blood cells contaminated with Yersinia enterocolitica serotype 0:3. He experienced rigors, high fever, elevated blood pressure, and increased pulse rate. He was treated with antibiotics and discharged in stable condition 6 days later. The blood donor, an Air Force recruit, was asymptomatic at the time of donation but recalled having had diarrhea 4 days earlier. In addition, several other flight members were reported to have had diarrhea at the same time. Serologic evidence suggested that the donor had had a recent infection with Y enterocolitica serotype 0:3. PMID- 8639057 TI - Carcinosarcoma arising in eccrine spiradenoma of the breast. Report of a case and review of the literature. AB - Although the vast majority of eccrine spiradenomas behave in a benign fashion, 23 cases of malignant transformation have been reported to date. We describe a unique example of malignant eccrine spiradenoma that arose in the right breast of a 68-year-old woman. The quiescent mass, which was present for approximately 50 years, experienced sudden enlargement with erythematous changes of the overlying skin and nipple discharge. Microscopically, the tumor showed the typical features of an eccrine spiradenoma with areas of adenocarcinoma, squamous cell carcinoma, and sarcoma. The sarcomatous component consisted of rhabdomyosarcoma and osteosarcoma. The immunoperoxidase staining revealed p53 protein expression only in the carcinomatous and sarcomatous components. This suggests that accumulation of p53 protein may be an important event in the malignant transformation of spiradenomas. Because of its location and biphasic nature, this malignant eccrine spiradenoma should be distinguished from metaplastic breast carcinoma. To our knowledge, this represents the first carcinosarcomatous transformation of eccrine spiradenoma in the breast. This case led us to conclude that breast tissue, which often undergoes apocrine metaplasia and gives rise to apocrine neoplasms, is also capable of originating benign and malignant tumors with eccrine sweat duct phenotype. PMID- 8639059 TI - Eclamptic seizures, magnesium, and semantics. PMID- 8639058 TI - Intrathoracic extramedullary hematopoietic tumor in hemoglobin C disease. AB - A case of intrathoracic extramedullary hematopoietic tumor associated with hemoglobin C disease is reported in a 27-year-old Bangladeshi man. The patient's initial complaints were generalized weakness, weight loss, and left upper abdominal pain. Hospital workup revealed a solitary intrathoracic mass in the posterior mediastinum, which was suspected to be a lymphoma based on fine-needle aspiration. The mass was excised and proved to be an extramedullary hematopoietic tumor. The problem of differential diagnosis is discussed. PMID- 8639060 TI - Herpesviruses in multiple sclerosis. PMID- 8639061 TI - Herpes simplex virus in postmortem multiple sclerosis brain tissue. AB - BACKGROUND: Herpes simplex virus (HSV) is a common neurotropic virus that is capable of long latencies. It can cause focal demyelination in animals. OBJECTIVE: To test for the presence of HSV-1 and -2 in postmortem brain samples from patients with multiple sclerosis (MS) and controls using polymerase chain reaction and Southern blot hybridization. METHODS: Dissected plaque tissue classified as active or inactive and unaffected white matter (WM) and gray matter (GM) from 37 cases of MS were screened for HSV using polymerase chain reaction and Southern blot hybridization. White matter and GM from 22 cases of Alzheimer's disease, 17 cases of Parkinson's disease, and 22 cases without neurologic disease served as controls. RESULTS: Forty-six percent (17/37) of the MS cases and 28% (17/61) of the control cases had samples that were positive for HSV (P = .11). Forty-one percent (9/22) of active plaques and 20% (6/30) of inactive plaques were positive for HSV. Twenty-four percent (9/37) and 14% (5/37) of MS cases and 23% (14/61) and 13% (8/61) of non-MS cases had HSV in WM and GM, respectively. No significant differences were found among all subgroups (P = .10). CONCLUSIONS: Herpes simplex virus was present in more MS cases than control cases and in more active plaques than inactive plaques. The presence of HSV in WM and GM in cases of MS as well as in control cases makes an etiologic association to the MS disease process uncertain, but cellular localization of HSV and its relationship to oligodendrocytes and latency may reveal such an association in future studies. PMID- 8639062 TI - Relationship of age, education, and occupation with dementia among a community based sample of African Americans. AB - OBJECTIVE: To explore the relationship between age, education, and occupation with dementia among African Americans. DESIGN: Community-based survey to identify subjects with and without evidence of cognitive impairment and subsequent diagnostic evaluation of a stratified sample of these subjects using formal diagnostic criteria for dementia. SETTING: Urban neighborhoods in Indianapolis, Ind. SUBJECTS: A random sample of 2212 African Americans aged 65 years and older residing in 29 contiguous census tracts. MEASUREMENTS: Subjects's scores on the Community Screening Instrument for Dementia (CSI-D), formal diagnostic clinical assessments for dementia, years of education, rural residence, primary occupation, self-reported disease, and alcohol and smoking history. Caseness was defined by four separate criteria: (1) cognitive impairment as defined by the subject's performance on the CSI-D cognitive scale; (2) cognitive impairment as defined by the total CSI-D score that included a relative's assessment of the subject's functional abilities; (3) dementia as defined by explicit diagnostic criteria; and (4) possible or probable Alzheimer's disease as defined by explicit diagnostic criteria. RESULTS: The mean age was 74 years (age range, 65 to 100 years), 65% of subjects were women, the mean education was 9.6 years (age range, 0 to 16 years), 98% of the subjects were literate, and 32% reported living in a rural area until age 19 years. Service, domestic, and production occupations accounted for 55.2% of the subjects' primary occupations with a mean of 25.8 years (range, 1 to 75 years) in the primary occupation. Years of education, rural residence to age 60 years, and primary occupation were highly correlated. Caseness defined by any of the four criteria was associated with functional impairment, but the frequency of impairment increased with increasing diagnostic specificity. Age, education, and rural residence to age 60 years were significantly independently associated with caseness for cognitive impairment, dementia, and Alzheimer's type dementia. White-collar occupation was independently associated only with caseness for cognitive impairment. History of stroke was associated with caseness for cognitive impairment and dementia but not Alzheimer's disease, while history of smoking was negatively correlated with Alzheimer's disease. CONCLUSIONS: Education was independently associated with cognitive impairment and dementia among a representative community-based sample of African Americans and the association remains significant across a variety of sensitivity analyses designed to control for measurement and confounding biases. The potential protective role of education against the development of dementia among African Americans deserves further evaluation. PMID- 8639063 TI - The Amyotrophic Lateral Sclerosis Functional Rating Scale. Assessment of activities of daily living in patients with amyotrophic lateral sclerosis. The ALS CNTF treatment study (ACTS) phase I-II Study Group. AB - OBJECTIVES: To test the utility of a new, easy to administer instrument for assessing activities of daily living in patients with amyotrophic lateral sclerosis (ALS), to validate its accuracy, and to assess its ability to record disease progression in patients with ALS against other functional scales, quantitative isometric muscle testing, and global assessment scales. DESIGN: Serial assessments of patients who presented to four ALS treatment centers in two multicenter studies. PATIENTS: Study 1 (cross-sectional) evaluated 75 consecutive patients who presented to four ALS treatment centers during a 2-month period. Study 2 (longitudinal) evaluated the progression of 53 patients who were enrolled in a multicenter, phase I-II clinical trial of recombinant human ciliary neurotrophic factor for treatment of ALS. OUTCOME MEASURES: The ALS Functional Rating Scale (ALSFRS) was compared with quantitative myometry and with other measures of daily function in patients with ALS both cross-sectionally and longitudinally. RESULTS: The first study of 75 patients evaluated the internal consistency, the test-retest reliability, and the construct validity of the ALSFRS. Internal consistency and test-retest reliability were high. Patient self rating of upper- and lower-extremity-dependent tasks were highly correlated with measures of upper- and lower-extremity strength, respectively. Thus, the ALSFRS has good construct validity. In the second study, ALSFRS scores declined in tandem with deterioration in motor and pulmonary function, indicating its sensitivity to change. CONCLUSIONS: The ALSFRS is a useful instrument for evaluation of functional status and functional change in patients with ALS. Its results are in close agreement with objective measures of muscle strength and pulmonary function. The ALSFRS may be used as a screening measure for entry into clinical trials, as a surrogate measure of function in situations in which muscle strength cannot be measured directly, or as an adjunct to myometry. PMID- 8639064 TI - Influence of cognitive reserve on neuropsychological functioning in asymptomatic human immunodeficiency virus-1 infection. AB - OBJECTIVE: To evaluate the influence of cognitive reserve or brain reserve capacity on neuropsychological performance in early human immunodeficiency virus (HIV)-1 infection. DESIGN: Cross-sectional group comparison study, based on neuropsychological performance, of HIV-1 seropositive and HIV-1 seronegative participants. SUBJECTS: Seventy-five medically asymptomatic HIV-1-seropositive homosexual or bisexual men and 50 HIV-1-seronegative homosexual or bisexual male controls. Subjects were grouped by HIV-1 status (seropositive vs seronegative) and by cognitive reserve scores (low reserve vs high reserve). MEASURES: Cognitive reserve scores were based on a combination of years of education, a measure of occupational attainment, and an estimate of premorbid intelligence. Performance on a battery of neuropsychological tests was summarized by empirically derived factor scores and clinical summary ratings. RESULTS: The HIV 1-seropositive subjects with low cognitive reserve scores exhibited significantly greater deficits on measures of attention and information processing speed, verbal learning and memory, executive functioning, and visuospatial performance than did the HIV-1-seropositive subjects with high cognitive reserve scores. In contrast, there were no significant group differences on these measures between both groups of HIV-1-seronegative subjects. CONCLUSIONS: Early neuropsychological impairments in HIV-1 infection are most evident in individuals with lower cognitive reserve. As has been found in other neurologic disorders, such as Alzheimer's disease, individuals with greater cognitive reserve may be less sensitive to the initial clinical effects of the underlying neuropathologic process. PMID- 8639065 TI - Cognitive performance and regional brain volume in human immunodeficiency virus type 1 infection. AB - BACKGROUND: Brain atrophy has been reported to occur in advancing human immunodeficiency virus (HIV) infection, particularly in patients with HIV-related dementia. Atrophy of the caudate region, as assessed by magnetic resonance imaging measures, has been reported to correlate with cognitive impairment in patients with HIV infection; however, differences in the severity of HIV-induced immunosuppression may have contributed to these findings. OBJECTIVE: To determine the relationship between regional brain volumes and cognitive performance in individuals with HIV infection. PATIENTS AND METHODS: We evaluated 11 patients with advanced HIV disease by using neuropsychologic tests and quantitative magnetic resonance imaging volume analysis. SETTING: University hospital, involving patients from a clinical trial. RESULTS: Caudate volume, expressed as a ratio of total intracranial volume, correlated with performance on the Trails A and Grooved Pegboard tests, but not with other tests of memory, motor speed, or mood (adjusted for age and education). Hippocampal volume did not correlate with any of the neuropsychologic tests. CONCLUSIONS: Caudate volume in patients with advanced HIV disease is associated with poor performance on neuropsychologic tests of complex motor and sequencing skills. Hippocampal volume does not appear to be related to impairment on neuropsychologic tests. These findings are independent of the degree of immunosuppression and the overall extent of brain atrophy; however, these results must be interpreted with some caution, given the limited sample size. PMID- 8639066 TI - Accuracy and reliability of periodic sharp wave complexes in Creutzfeldt-Jakob disease. AB - OBJECTIVE: To assess the sensitivity, specificity, and interobserver reliability of periodic sharp wave complexes in the electroencephalograms of patients with Creutzfeldt-Jakob disease. DESIGN: Sixty-eight electroencephalograms in 29 patients who had been suspected of having Creutzfeldt-Jakob disease were reanalyzed by an investigator who was unaware of the clinical data. The incidence of periodic sharp wave complexes in neuropathologically confirmed Creutzfeldt Jakob disease vs progressive dementia other than Creutzfeldt-Jakob disease was assessed. Blinded electroencephalogram analysis was performed by a second investigator. The interobserver reliability was assessed by the kappa value. SETTING: University hospital, base of the German National Creutzfeldt-Jakob Disease Surveillance Study. PATIENTS: Fifteen patients with neuropathologically confirmed Creutzfeldt-Jakob disease and 14 patients who had been suspected of having Creutzfeldt-Jakob disease because of rapidly progressive dementia but in whom other dementias were diagnosed by unblinded investigators based on clinical and electroencephalographic criteria. MAIN OUTCOME MEASURE: Sensitivity and specificity of periodic sharp wave complexes assessed by their incidence in Creutzfeldt-Jakob disease vs other dementias. Interobserver reliability of periodic sharp wave complexes was expressed by the kappa value. RESULTS: For periodic sharp wave complexes, blinded electroencephalographic analysis resulted in a sensitivity and a specificity of 67% and 86%, respectively. Interobserver reliability was excellent (kappa = 0.95). CONCLUSION: This blinded electroencephalographic study in Creutzfeldt-Jakob disease confirms the high diagnostic value of electroencephalography, as previously reported by open studies. PMID- 8639068 TI - The occurrence of depression in Parkinson's disease. A community-based study. AB - OBJECTIVE: To investigate the frequency of major depression (MD) and the severity of depressive symptoms among patients with Parkinson's disease (PD). DESIGN: The PD population was derived from a community-based prevalence study. Total case ascertainment and a high diagnostic accuracy of PD were attempted through a detailed community study and the use of a new clinical diagnostic classification. Major depression was diagnosed according to the criteria in the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition. The severity of depression in the prevalence population was scored with the Montgomery and Asberg Depression Rating Scale. The occurrence of depressive symptoms among patients with PD was compared with the occurrence among age-matched groups of patients with diabetes mellitus and of healthy elderly. In addition, the patients with PD and the control groups completed the Beck Depression Inventory. SETTING: Depression among patients with PD derived from a prevalence study in the county of Rogaland, Norway. PATIENTS: Two hundred forty-five patients with PD. Two age matched control groups (each including 100 patients); one group included patients with diabetes mellitus and the other, healthy elderly. RESULTS: Of the 245 patients with PD, 7.7% met the criteria for MD. Based on their Montgomery and Asberg Depression Rating score, 5.1% of the patients were moderately to severely depressed whereas another 45.5% had mild depressive symptoms. Among the patients who scored 20 or more on the Mini-Mental State Examination, 3.6% had MD compared with 25.6% of the patients with a score below 20. The frequency of patients with a Beck Depression Inventory score of 18 or more was higher in the PD group (24.1%) than among patients with diabetes mellitus (11%) and the healthy elderly controls (4%). CONCLUSION: This study suggests that the prevalence of MD in PD is lower than previously assumed, but a substantial proportion of patients with PD have less severe depressive symptoms. PMID- 8639067 TI - Regional gray and white matter metabolite differences in subjects with AD, with subcortical ischemic vascular dementia, and elderly controls with 1H magnetic resonance spectroscopic imaging. AB - OBJECTIVE: To use 1H magnetic resonance spectroscopic imaging to study differences in neuron density (N-acetylaspartate [NAA]), membrane phospholipid metabolites (choline [Cho]), and creatine-containing metabolites (creatine plus phosphocreatine [Cr]) in subjects with Alzheimer's disease (AD), with subcortical ischemic vascular dementia (SIVD), and elderly controls. DESIGN: Cross-sectional, between groups. SETTING: A Veterans Affairs medical center and university memory clinic. PARTICIPANTS: Forty elderly subjects with AD (n = 14), with SIVD (n = 8), and elderly controls (n = 18). MAIN OUTCOME MEASURES: We used 1H magnetic resonance spectroscopic imaging to acquire spectra from a 80 x 100 x 17-mm volume superior to the lateral ventricles. Spectra were analyzed from voxels in anterior, medial, and posterior gray and white matter using nuclear magnetic resonance-1 and the results were compared between groups using repeated measures analysis of variance (ANOVA), Tukey's test, and individual Student's t tests. RESULTS: Using ANOVA, significantly lower levels of NAA/Cho and NAA/Cr and significantly higher levels of Cho/Cr were observed across both gray and white matter voxels in subjects with AD. Using individual Student's t tests, a significantly lower level of NAA/Cho and a higher level of Cho/Cr were observed in the posterior gray matter in subjects with AD. Using ANOVA in subjects with SIVD, significantly lower gray and white matter NAA/Cr levels were observed. Using Tukey's test, the NAA/Cr level was significantly lower in frontal white matter voxels in subjects with SIVD compared with controls. CONCLUSIONS: Our findings in subjects with AD suggest neuron loss in gray matter, axon loss in white matter, and altered Cho metabolism in posterior brain regions. Our findings in subjects with SIVD are consistent with higher levels of creatine-containing metabolites and/or lower levels of NAA in frontal white matter. PMID- 8639069 TI - A double-blind study of subcutaneous dihydroergotamine vs subcutaneous sumatriptan in the treatment of acute migraine. AB - OBJECTIVE: To assess the efficacy and tolerability of subcutaneous dihydroergotamine mesylate (DHE-45) vs subcutaneous sumatriptan succinate (Imitrex) for the treatment of acute migraine with or without aura. DESIGN: Double-blind, randomized trial with parallel treatment arms. SETTING: Clinics and private neurology practices. SUBJECTS: Patients of either sex, with migraine with or without aura, between the ages of 18 and 65 years. INTERVENTIONS: Patients with moderate or severe head pain were randomized to receive either 1 mg of subcutaneous dihydroergotamine mesylate or 6 mg of subcutaneous sumatriptan succinate. Patients rated head pain, functional ability, nausea, and vomiting at baseline and at 0.5, 1, 2, 4, and 24 hours after the injection. Presence or absence of headache at 3 hours was calculated from collected data. If pain persisted after 2 hours, a second injection of the same study medication was allowed, and self-ratings were repeated 30 and 60 minutes later. Follow-up data were collected at 24 hours. MAIN OUTCOME MEASURES: Relief of head pain and recurrence of successfully treated headache. RESULTS: There were 295 evaluable patients. At 2 hours, 73.1% of the patients treated with dihydroergotamine and 85.3% of those treated with sumatriptan had relief (P = .002). There was no statistical difference in headache relief between the groups at 3 or 4 hours. Headache relief was achieved by 85.5% of those treated with dihydroergotamine and by 83.3% of those treated with sumatriptan by 4 hours. By 24 hours 89.7% of dihydroergotamine-treated patients and 76.7% of sumatriptan-treated patients had relief (P = .004). Headache recurred within 24 hours after treatment in 45% of the sumatriptan-treated patients and in 17.7% of the dihydroergotamine-treated patients (P < or = .001). CONCLUSIONS: Both sumatriptan and dihydroergotamine were effective in aborting migraine headaches. Headache recurrence was two and a half time as likely with sumatriptan as with dihydroergotamine. PMID- 8639070 TI - The effects of amantadine and pemoline on cognitive functioning in multiple sclerosis. AB - BACKGROUND: Amantadine hydrochloride and pemoline, both frequently used to treat the fatigue of multiple sclerosis (MS), may also improve attention and other cognitive functions in MS. To our knowledge, these agents have never been compared in a placebo-controlled trial of patients with MS. OBJECTIVE: To evaluate the effects of amantadine and pemoline on cognitive functioning in MS. METHODS: A total of 45 ambulatory patients with MS and severe fatigue were treated for 6 weeks with amantadine, pemoline, or placebo using a parallel group design. They underwent comprehensive neuropsychological testing to determine treatment effects on cognitive functioning. Primary outcome measures were tests of attention (Digit Span, Trail Making Test, and Symbol Digit Modalities Test), verbal memory (Selective Reminding Test), nonverbal memory (Benton Visual Retention Test), and motor speed (Finger Tapping Test). RESULTS: Fatigue did not significantly correlate with any of the neuropsychological outcome measures at baseline or after treatment. All three treatment groups improved on tests of attention (P < .003), verbal memory (P < .001), and motor speed (P < .002). There were no significant differences between amantadine, pemoline, and placebo. CONCLUSIONS: Cognitive functioning in MS is independent of fatigue. Neither amantadine nor pemoline enhances cognitive performance in MS compared with placebo. PMID- 8639071 TI - Cerebrospinal fluid concentrations of soluble amyloid beta-protein and apolipoprotein E in patients with Alzheimer's disease: correlations with amyloid load in the brain. AB - OBJECTIVE: To compare soluble amyloid beta-protein and apolipoprotein E levels in cerebrospinal fluid (CSF) and brain extracts from patients with definite Alzheimer's disease. SETTING: University medical center. PATIENTS: Nineteen patients with definite Alzheimer's disease. MAIN OUTCOME MEASURES: Soluble amyloid beta-protein and apolipoprotein E levels in CSF, in neutral and low-pH brain extracts, and in formic acid-treated sections of the frontal, temporal, and cerebellar cortices, measured using enzyme-linked immunosorbent assay. RESULTS: Soluble amyloid beta-protein and apolipoprotein E levels in CSF were significantly lower in patients with congophilic angiopathy than in those without angiopathy. The levels did not correlate with the number of amyloid plaques in the neocortex. There was, however, a tendency toward an inverse correlation between the amount of amyloid beta-protein in the frontal cortex extracts and the soluble amyloid beta-protein level in CSF. CONCLUSION: Soluble amyloid beta protein levels in CSF may reflect amyloid accumulation in brain blood vessels. PMID- 8639072 TI - Rippling muscles and myasthenia gravis with rippling muscles. AB - OBJECTIVE: To describe a patient with rippling muscles and myasthenia gravis. DESIGN: Clinical, laboratory, electrophysiologic, and muscle biopsy data are reported. SETTING: Medical office and hospital. PATIENT: We describe a patient with rippling muscles (as seen in rippling muscle disease) and myasthenia gravis (MG) with thymoma. There was no family history of rippling muscle disease in our patient. Diplopia and other symptoms of MG were initially overshadowed by the striking rippling phenomenon. The rippling resolved when the MG became florrid. INTERVENTIONS: The patient was treated with pyridostigmine, prednisone, and plasmaphereses before removal of a thymoma. MAIN OUTCOME AND RESULTS: His MG improved with treatment and the rippling has not recurred 4 months after thymectomy. CONCLUSIONS: Rippling muscle disease is rare and usually inherited. Our patient with rippling muscles (but no family history of rippling muscle disease) and MG suggests that rippling muscles may also be triggered by an autoimmune phenomenon. PMID- 8639073 TI - In vivo assessment of corneal endothelial function in diabetes mellitus. AB - OBJECTIVE: To evaluate in vivo corneal endothelial function in patients with non insulin-dependent diabetes mellitus with or without diabetic retinopathy. METHODS: In age-matched samples of 45 eyes of diabetic patients and 15 eyes of healthy control subjects, corneal endothelial function was evaluated by using pachometric measurements of the cornea following hydrophilic contact lens wear of low oxygen transmissibility. Corneal deswelling regression was quantitated to arrive at the percentage recovery per hour as an index of endothelial function. The status of retinopathy was determined in diabetic patients by using standard criteria. RESULTS: In patients with diabetes mellitus and diabetic retinopathy, the assessed mean (+/- SD) value for the percentage recovery per hour following contact lens-induced edema was 40.03% +/- 7.27%, which was significantly lower than in eyes without changes of diabetic retinopathy (54.31% +/- 6.88%). Also, diabetic patients without retinopathy had a significantly lower percentage recovery per hour than did healthy control subjects (65.27% +/- 12.02%). CONCLUSIONS: This study demonstrates significantly lower corneal endothelial function in patients with non-insulin-dependent diabetes mellitus and thus suggests a higher potential to decompensate following any deleterious stress. PMID- 8639074 TI - Anterior stromal punctures for bullous keratopathy. AB - OBJECTIVE: To evaluate the therapeutic effects of anterior stromal punctures (ASP) in patients with bullous keratopathy (BK). PATIENTS AND METHODS: Twenty seven patients awaiting penetrating keratoplasty with a diagnosis of BK were examined. They were seen before treatment with ASP and 1, 4, and 12 weeks after treatment. The examination included slit-lamp examination, photography of the cornea, ultrasonic pachymetry, central esthesiometry, and pneumotonometry. Subjective evaluations of pain, discomfort, and photophobia were also done using a visual scale model. Photographs were analyzed by computer-assisted planimetry and used to measure the corneal surface covered by bullae and microcysts. Pretreatment and posttreatment values (mean +/- SEM) were compared using the Student paired t test. RESULTS: At 3 months, a significant reduction in pain was noted. A decrease in the mean corneal surface covered by bullae (BKPreASP = 2733 +/- 553 microns2; BK3mo = 1006 +/- 356 microns2, P = .004) was observed. A decrease in the esthesiometry (E) measurement (EPreASP = 3.5 +/- 0.4 cm; E3mo = 1.3 +/- 0.3 cm, P < .001), an increase in corneal thickness ([CT] CTPreASP = 869 +/- 24 microns; CT3mo = 902 +/- 21 microns, P < .001), and a decrease in the number of quadrants through which iris (I) details could be seen (IPreASP = 1.7 +/- 0.3; I3mo = 1.2 +/- 0.3, P = .015) were also noted. These findings corroborate the clinical observation of increased subepithelial fibrosis following ASP. CONCLUSIONS: Anterior stromal punctures reduce bullae formation and alleviate pain in patients with BK, and they constitute a valuable alternative to penetrating keratoplasty should surgery be delayed or contraindicated. PMID- 8639076 TI - Plateau iris syndrome associated with multiple ciliary body cysts. Report of three cases. AB - OBJECTIVE: To describe plateau iris syndrome associated with multiple neuroepithelial cysts of the pars plicata. METHODS: Case reports of 3 patients with plateau iris syndrome who were found to have multiple bilateral ciliary body cysts on ultrasound biomicroscopic examination. RESULTS: Ultrasound biomicroscopy revealed classic features of plateau iris syndrome in each patient but also showed multiple neuroepithelial cysts of the ciliary body in each eye. CONCLUSION: Plateau iris syndrome may be associated with multiple ciliary body cysts. PMID- 8639075 TI - Reduced trabecular meshwork height in juvenile primary open-angle glaucoma. AB - OBJECTIVE: To compare trabecular meshwork height in a series of patients with juvenile primary open-angle glaucoma (JPOAG) with that in normal control patients. METHODS: Ultrasound biomicroscopy and A-scan biometry were performed on 16 eyes with JPOAG and 24 normal eyes. A radial, perpendicular image in the horizontal temporal meridian detailing the line of Schwalbe, scleral spur, and angle anatomy was obtained for each eye by a single examiner. Trabecular meshwork height was defined as the distance from the scleral spur to the Schwalbe line. RESULTS: Mean patient age (P = .85, t test), refractive error (P = .68), sex distribution (P = .26, Fisher exact test) and axial length (P = .39) were similar between the groups. Mean +/- SE trabecular meshwork heights were 0.36 +/- 0.03 mm (range, 0.19-0.53 mm) for JPOAG and 0.58 +/- 0.02 mm (range, 0.40-0.80 mm) for controls (P < .001). Eyes with greater axial length tended to have larger trabecular meshworks in both groups (P = .012, multivariate regression). A trabecular meshwork height-axial length ratio of 0.021 or less was associated with a significantly increased risk for JPOAG being present (odds ratio, 57; 95% confidence interval, 6.0-541). CONCLUSIONS: The trabecular meshwork is smaller in eyes with JPOAG compared with that in normal eyes. This finding suggests a structural abnormality that may underlie the reduced outflow. PMID- 8639077 TI - Echographic findings in benign reactive lymphoid hyperplasia of the choroid. AB - BACKGROUND: Benign reactive lymphoid hyperplasia is an uncommon uveal tumor. Misdiagnosis may lead to unnecessary enucleations. Clinical evaluation alone is usually not sufficient to diagnose this tumor. OBJECTIVE: To evaluate the utility of standardized echography in the recognition of this disease. METHODS: Retrospective review of the medical and echographic records of 6 patients with choroidal involvement of benign reactive lymphoid hyperplasia. RESULTS: Extraocular episcleral lymphoid nodules were present in 5 of the 6 patients and were usually seen as a late manifestation of the disease. Two patients had repeated echography after treatment of the tumor and showed marked regression of the episcleral nodules. CONCLUSIONS: A combination of clinical and echographic findings can assist in the accurate diagnosis of this rare uveal tumor. The presence of episcleral nodules on standardized echography supports the diagnosis. PMID- 8639078 TI - Five-year follow-up of fellow eyes of individuals with ocular histoplasmosis and unilateral extrafoveal or juxtafoveal choroidal neovascularization. Macular Photocoagulation Study Group. AB - OBJECTIVES: To document the incidence of choroidal neovascularization (CNV) in unaffected fellow eyes among individuals with ocular histoplasmosis and extrafoveal or juxtafoveal CNV in 1 eye; to determine whether the location and type of "histo spots" in the macula predict the site of future CNV development in second eyes; to describe changes over time in neovascular lesions present in fellow eyes at baseline; and to describe changes in visual acuity of fellow eyes with and without CNV at baseline. DESIGN, PATIENTS, AND SETTING: Five-year prospective follow-up study of fellow eyes of 516 patients enrolled in 2 randomized clinical trials of laser photocoagulation of extrafoveal and juxtafoveal CNV. Best-corrected visual acuity and reading vision were measured, and both maculas were photographed at baseline and at 6-month intervals. MAIN OUTCOME MEASURES: Cumulative incidence of CNV in fellow eyes free of neovascular maculopathy at the time of study enrollment, 5-year change in visual acuity of fellow eyes from baseline, and incidence of legal blindness (visual acuity < or = 20/200 in the better eye). RESULTS: Photographically documented CNV developed in 35 (9%) of 394 eyes initially free of neovascular maculopathy; nevertheless, good visual acuity was maintained in most newly affected eyes until the end of the 5 year follow-up period. Histo spots of any type in the macula at baseline tripled the risk for later development of CNV in comparison to eyes without histo spots in the macula. Although the type of histo spots present in the central macula at baseline did not predict future CNV development, in 32 of 35 second eyes in which CNV developed and in 7 of 9 fellow eyes in which a second area of CNV developed during follow-up, CNV was preceded by an "atypical" histo spot in the same location. Among 122 patients who had bilateral neovascular maculopathy initially, 100 were examined 5 years later; 8 (8%) were legally blind, compared with 3 (1%) of 339 patients examined who had unilateral CNV initially. At the 5-year examination, 355 (81%) of 439 patients examined had a visual acuity of 20/20 or better in at least 1 eye, including 74 (55%) of 134 patients who had bilateral neovascular maculopathy. CONCLUSIONS: Although the incidence of CNV in fellow eyes that initially were unaffected remained low throughout 5 years of follow-up, it persisted at a nearly constant rate. The risk of legal blindness was low, even for patients who had bilateral involvement. Perhaps most important, 81% of all patients followed up for 5 years retained a visual acuity of 20/20 in at least 1 eye, and 20% retained this visual acuity in both eyes. Retrospective review of photographs suggests that the ophthalmologist should pay special attention to areas of the central macula in which new "atypical" histo spots are observed, with the goal of treating CNV that may develop in the same area at a time when the benefits of laser treatment may be greatest. PMID- 8639079 TI - The long-term outcome of central serous chorioretinopathy. AB - OBJECTIVE: To assess the long-term outcome of central serous chorioretinopathy (CSR) among a group of patients who previously participated in a prospective argon laser photocoagulation study of CSR. DESIGN: Thirty-eight of 41 surviving patients with CSR participating in an earlier study were invited to participate in a follow-up study that included history taking, ophthalmoscopy, biomicroscopy, and fundus photography. RESULTS: Thirty-seven (38 eyes) of 38 surviving patients (97%) were available for follow-up between 11 and 15 years after participation in the earlier study. There were no clinically documented or historical recurrences of CSR among the six eyes previously treated by direct laser photocoagulation. There were 13 clinically documented recurrences and four historical recurrences among the 32 eyes not treated with direct laser photocoagulation. The difference in recurrences was statistically significant (P = .02). Pigment changes indistinguishable from age-related macular degeneration frequently occurred in eyes with CSR. The difference in the development of such pigment changes between eyes with CSR (33 of 38) and nonaffected fellow eyes (12 of 35) was significant (P = .001). CONCLUSIONS: The decreased rate of CSR recurrence after direct laser photocoagulation reported in an earlier study was sustained with follow-up beyond 10 years. Pigmentary changes in the fundus indistinguishable from those associated with age-related macular degeneration developed in eyes affected with CSR, probably as a consequence of the presence of subretinal fluid accompanying the CSR rather than from early age-related macular degeneration. PMID- 8639080 TI - Indocyanine green-guided laser photocoagulation of focal spots at the edge of plaques of choroidal neovascularization. AB - OBJECTIVE: To determine whether indocyanine green-guided laser photocoagulation of focal spots at the edge of plaques of neovascularization is beneficial for patients with untreatable occult choroidal neovascularization by fluorescein imaging. PATIENTS: A pilot series of 23 eyes were identified that had untreatable occult choroidal neovascularization secondary to age-related macular degeneration with focal spots at the edge of a plaque of neovascularization on the indocyanine green study. After informed consent was obtained, indocyanine green-guided laser photocoagulation was applied solely to the focal spot at the edge of the plaque. RESULTS: Anatomical success with resolution of the exudative findings was noted in 15 (79%) of 19 eyes at 6 months, 13 (68%) of 19 eyes at 12 months, and six (37.5%) of 16 eyes at 24 months. The median follow-up period was 18 months (range, 3 to 44 months). Visual acuity was stabilized or improved in nine (69%) of 13 successfully treated eyes at 1 year. Of the successfully treated cases at 1 year, nine (69%) of 13 had a final visual acuity of 20/100 or better. CONCLUSIONS: Our preliminary study of indocyanine green-guided laser photocoagulation of focal spots at the edge of plaques of neovascularization suggests that this technique may improve the visual prognosis of these patients with presently untreatable disease. A prospective, randomized, controlled clinical trial is warranted to evaluate this treatment approach. PMID- 8639081 TI - Vision in Leber congenital amaurosis. AB - OBJECTIVE: To determine if vision changed with age in infants and children with Leber congenital amaurosis. PATIENTS: Grating acuity and dark-adapted visual thresholds were tested in 36 patients with Leber congenital amaurosis. Longitudinal assessments were obtained for 24 patients and analyzed for significant changes over time. Visual acuity and threshold and the courses of visual acuity and threshold were examined for significant associations with hyperopia, fundus appearance, and complicated vs uncomplicated status. RESULTS: Measurable grating acuities ranged from 0.16 to 6 cycles per degree (median, 1.27 cycles per degree or about 20/500), and dark-adapted visual thresholds were elevated 1.0 to 5.6 log units (median, 2.33 log units). Eighteen patients never had demonstrable grating acuity, and 12 had no light perception. Among those with serial tests, visual acuity improved or remained stable in 10 patients and declined in 4. Dark-adapted visual thresholds were stable in those with improving or stable visual acuities but worsened in 5 patients, including the 4 whose visual acuity worsened. No significant associations of visual acuity, dark adapted visual threshold, the course of visual acuity, or the course of dark adapted visual threshold with hyperopia, fundus appearance, or complicated vs uncomplicated status were found. CONCLUSIONS: Visual capabilities varied widely. Vision was stable in the majority by longitudinal measures but increased in a few and deteriorated in others. Neither ocular characteristics nor complicated vs uncomplicated status predicted visual function. Thus, if vision and its course are to be known in a patient with Leber congenital amaurosis, it must be tested. PMID- 8639082 TI - Spontaneous visual improvement in orbital apex tumors. AB - OBJECTIVE: To describe patients with orbital apex masses involving the optic nerve who enjoyed spontaneous improvement in visual function without therapy. METHODS: A retrospective chart review of cases in an academic neuro-ophthalmology practice identified three cases of tumorous compression of the optic nerve in which there was spontaneous improvement in visual function. Each patient had undergone a full neuro-ophthalmologic evaluation, followed up with serial imaging. RESULTS: The first patient initially had a visual acuity of 2/200 and central visual field defects. Over a 4-year period, her visual acuity improved to 20/40 and her visual field expanded centrally. The second patient initially had a visual acuity of 20/40 and a cecocentral visual field defect. After a 4-year follow-up, her visual acuity had improved to 20/15 and her cecocentral defect had shrunk. The third patient had an initial visual acuity of 20/25, an afferent pupillary defect, and optic atrophy. Over a 2-year period she developed a paracentral scotoma and her visual acuity worsened to 20/30. In the next 2 years her visual acuity improved to 20/10 and her visual field normalized. CONCLUSIONS: In exceptional instances, visual dysfunction from tumorous compression of the optic nerve in the orbital apex may spontaneously improve. Clinicians who use the natural history of visual loss in differential diagnosis should be aware that spontaneous remission is compatible with the presence of tumor. PMID- 8639083 TI - Management of cavernous sinus-dural fistulas. Indications and techniques for primary embolization via the superior ophthalmic vein. AB - OBJECTIVE: To describe indications and surgical techniques for embolization of cavernous sinus-dural fistulas (CDF) by passing platinum coils through a cannulated superior ophthalmic vein based on our clinical experience. DESIGN: Retrospective clinical review. SETTING: University tertiary referral hospital and eye institute. PATIENTS: Over a 3-year period, 10 consecutive patients with CDF and progressive orbital congestion underwent transvenous embolization. All patients had a dilated superior ophthalmic vein. All 10 patients had indications for treatment of fistulas on the basis of progressive glaucoma refractory to medical management, venous stasis retinopathy with retinal ischemia, optic neuropathy, diplopia, exophthalmos with exposure keratopathy, cortical venous congestion with risk for intracranial hemorrhage, or a combination of these findings. INTERVENTION: Nine of the 10 patients underwent anterior orbitotomy via a lid-crease or sub-brow incision with cannulation of the ipsilateral superior ophthalmic vein and embolization of the cavernous sinus with platinum coils, following an unsuccessful transarterial embolization. One patient underwent a primary transvenous embolization. MAIN OUTCOME MEASURES: Successful closure of the fistula on angiography, return of baseline visual acuity, normalization of postoperative intraocular pressure, and cosmetically acceptable cutaneous scar. RESULTS: All 10 patients had prompt resolution of symptoms and halt of progressive visual loss following occlusion of the fistulas. Two patients had no flow in the anterior superior ophthalmic vein on angiography suggesting thrombosis, yet the superior ophthalmic vein was easily accessed in the anterior orbit, and transvenous embolization was successfully performed. In 2 additional patients with nondilated superior ophthalmic veins, we were unable to gain surgical access and in 1 case severe bleeding occurred during attempted access of the small vein. CONCLUSIONS: When performed by an experienced orbital surgeon and neuroradiology team, transvenous embolization of CDF via a dilated anterior superior ophthalmic vein is a technically straightforward, safe, and effective treatment for CDF and perhaps should be employed as primary therapy in cases with progressive orbital congestive symptoms. If the superior ophthalmic vein is not dilated or if it is located deep in the orbit, transorbital venous access may not be possible. PMID- 8639084 TI - Quantitative videographic analysis of blinking in normal subjects and patients with dry eye. AB - OBJECTIVE: To study patterns of eye blinking in normal subjects and patients with dry eye. METHODS: We developed an automated, noninvasive blink monitor that permits quantitative analysis of 6 parameters of blinking. We used this method under normal conditions and then examined the effects on the patterns of blinking in patients with dry eye; several steps in this method were designed to exacerbate or ameliorate ocular surface desiccation. RESULTS: The mean (+/-SD), maximum, and coefficient of variation of the interblinking time in normal subjects and patients with dry eye were 4.0 +/- 2.0 and 1.5 +/- 0.9 seconds, 8.9 +/- 4.0 and 4.2 +/- 2.4 seconds, and 55% +/- 21% and 65% +/- 24%, respectively. Those values for the blinking time were 0.20 +/- 0.04 and 0.27 +/- 0.16 seconds, 0.35 +/- 0.12 and 0.99 +/- 1.30 seconds, and 23% +/- 9% and 46% +/- 34%, respectively. The use of artificial tears or spectacles with moist panels and moist inserts tended to normalize the patterns of blinking in the patients with dry eye, whereas exposure to wind made them more abnormal. CONCLUSIONS: Our technique permitted a rigorous analysis of blinking that was previously unavailable. We have shown that local ocular surface conditions alone can significantly affect patterns of blinking. This method should be applicable to studying psychologic and any other factors that may influence blinking. PMID- 8639085 TI - Ultrastructural age-related changes on the posterior iris surface. A possible relationship to the pathogenesis of exfoliation. AB - OBJECTIVES: To verify the presence of age-related changes in the human iris and to determine whether such changes could be related to the pathogenesis of the exfoliation syndrome. DESIGN: Ultrastructural examination of the posterior surface of the normal iris in 9 enucleated eyes. PATIENTS OR OTHER PARTICIPANTS: Eyes of persons aged 1 day, 3 months, and 3, 9, 27, 52, 59, 59, and 65 years. MAIN OUTCOME MEASURE: The presence of aging changes in the iris. RESULTS: Aging changes included duplication of the basal lamina of posterior iris pigment epithelial cells, formation of atrophic invaginations in the posterior cell membranes containing interlacing basal lamina, formation (or deposition) of microfibrils 11 to 13 nm in diameter, with a banding periodicity of 12 to 16 nm, deposition of electron-dense material in relation to the basal lamina and/or microfibrils, and the presence of some fine granular material overlying the basal lamina. CONCLUSION: These changes have been consistently described before in association with exfoliation material, which suggests the possibility that exfoliation is an eventual aging process. PMID- 8639086 TI - Interferon alfa induces leukocyte capillary trapping in rat retinal microcirculation. AB - BACKGROUND: Interferon alfa has been suggested as a possible treatment for choroidal neovascularization. However, retinal complications following interferon therapy have been reported. OBJECTIVE: To evaluate the effects of interferon alfa on leukocyte dynamics in the rat retinal microcirculation. METHODS: Interferon alfa of different doses was intravenously administered in rats. Leukocyte dynamics were observed with acridine orange digital fluorography, which uses a nuclear fluorescent dye of acridine orange and scanning laser ophthalmoscopy. This technique allows visualization of leukocyte movements in the retinal microcirculation in vivo. RESULTS: After interferon alfa was administered, leukocytes adhered to vascular walls and became trapped in the retinal microcirculation. Leukocyte trapping was dose-dependent. CONCLUSIONS: Interferon alfa increased leukocyte adherence to vascular endothelium and subsequent leukocyte trapping in the retinal capillaries. Interferon alfa may activate leukocytes, and activated leukocytes may be involved in the pathogenesis of microinfarction associated with interferon-induced retinopathy. PMID- 8639087 TI - Hyalocytes synthesize and secrete inhibitors of retinal pigment epithelial cell proliferation in vitro. AB - BACKGROUND: Retinal pigment epithelial (RPE) cells that enter the vitreous in pathologic conditions, such as retinal detachment, may proliferate and contribute to the formation of epiretinal membranes. OBJECTIVE: To study whether hyalocytes, endogenous vitreous cells, play a role in modulating the proliferation of RPE cells. METHODS: Cell proliferation was measured by tritiated thymidine incorporation in density-arrested human RPE cells after incubation with media that had been conditioned by cultured bovine hyalocytes. Preliminary characterization of inhibitory activity in hyalocyte-conditioned medium was performed, including blocking experiments with a neutralizing antibody to transforming growth factor-beta 2 (TGF-beta) and proliferation assays that used MV-1-Lu mink lung epithelial cells. Northern blots were done to asses hyalocyte expression of TGF-beta messenger RNA. RESULTS: Hyalocyte-conditioned medium inhibited tritiated thymidine incorporation in RPE cells and MV-1-Lu mink lung epithelial cells in the presence or absence of serum or protease inhibitors. A portion of the inhibitory activity was neutralized by an antibody directed against TGF-beta. Northern blots of hyalocyte RNA demonstrated the presence of messenger RNA for TGF-beta 2. These data suggest that TGF-beta is responsible for a portion of the inhibitory activity secreted by hyalocytes. Additional inhibitory activity is attributable to one or more low-molecular-weight molecules distinct from TGF-beta. CONCLUSIONS: Hyalocyte-conditioned medium inhibits RPE cell proliferation in vitro through TGF-beta and at least one other molecule. Production of these factors by hyalocytes in vivo could provide a deterrent for epiretinal membrane formation that may be perturbed under pathologic conditions. PMID- 8639088 TI - Sorsby fundus dystrophy. A family with the Ser181Cys mutation of the tissue inhibitor of metalloproteinases 3. AB - Sorbsy fundus dystrophy (SFD) is an autosomal dominant disorder that is characterized by bilateral loss of central vision secondary to choroidal neovascularization and/or pigment epithelial atrophy in the macula, with onset of visual symptoms usually in the fourth or fifth decade. Drusenlike changes may occur, with impaired dark adaptation and abnormal electroretinographic results. PMID- 8639089 TI - Associations between siblings for esotropia and exotropia. AB - OBJECTIVE: To quantify familial aggregation of esotropia and exotropia in children examined in a large multicenter study. METHODS: Pregnant women and their children were examined in the Collaborative Perinatal Project of the National Institute of Neurological Disorders and Stroke, Bethesda, Md. Strabismus was evaluated in the children during follow-up examinations up to the age of 7 years. The second-order generalized estimating equations approach to logistic regression was used to estimate familial aggregation of esotropia and exotropia. RESULTS: For any pair of siblings, the odds for one sibling having esotropia more than doubled when the other sibling had esotropia. For exotropia, there were differences in sibling associations based on birth relationships. In particular, there was no statistically significant association between siblings from separate single births. On the other hand, for the pairs of siblings from multiple births (ie, twins, triplets, and quadruplets), the odds for exotropia in one sibling were increased by at least a factor of 17 when the other sibling from that birth also had exotropia. For both esotropia and exotropia, adjustment for previously identified risk factors only somewhat reduced the magnitudes of the observed associations. Limited data on zygosity showed a stronger association between monozygotic twins than between dizygotic twins. CONCLUSIONS: There is a significant familial component in the cause of strabismus. Furthermore, there are important contributions to this familial aggregation beyond those associated with known risk factors for strabismus. PMID- 8639090 TI - Topical beta-adrenergic antagonists and quinidine. A risky interaction. PMID- 8639091 TI - Indocyanine green angiography. Can it help preserve the vision of our patients? PMID- 8639092 TI - Techniques and surgical approach for transvenous embolization. PMID- 8639093 TI - Metastatic choroidal melanoma to the contralateral orbit 40 years after enucleation. AB - Metastatic uveal melanoma to the contralateral orbit is extremely rare, with only eight cases in the literature having been reported. We reviewed the clinical presentation, the histologic findings of the primary and secondary tumors, and the clinical outcome of these cases and studied an additional case of metastatic choroidal melanoma to the contralateral orbit that occurred 40 years after enucleation for the primary tumor. The long interval between recognition of the primary tumor and the appearance of clinically manifest metastatic disease in the contralateral orbit is the longest reported in the literature to date. The histologic features of the tumor cells of the enucleation specimen and those of the orbital metastasis were similar, consisting of spindle-cell type with minimal atypia. Combined use of immuno-phenotyping and electron microscopy substantiated the diagnosis of melanoma. PMID- 8639094 TI - Neonatal hepatoblastoma metastatic to the choroid and iris. AB - A newborn boy was diagnosed with hepatoblastoma after a systemic workup was prompted because of abnormal-appearing eyes. Radiological examination revealed mass lesions involving the liver, brain, eyes, and ribs. A liver biopsy was performed before death and results showed hepatoblastoma, an unusual solid tumor of childhood. At autopsy, hepatoblastoma was also found histopathologically in the irides and choroid of both eyes. No previous report of hepatoblastoma metastatic to the eye could be found in the medical literature. In addition, we believe this is the first report in a child of a solid tumor metastatic to the choroid and iris, and of a solid tumor metastatic to the iris that is confirmed histopathologically. PMID- 8639095 TI - Sixty-six magnet operations with successful extraction of particles of iron from the inferior of the eye in fifty-three cases. 1894. PMID- 8639097 TI - Endophthalmitis following trabeculectomy with releasable sutures. PMID- 8639096 TI - Pregnancy and corneal allograft rejection. PMID- 8639099 TI - Bilateral orbital involvement with massive allergic fungal sinusitis. PMID- 8639098 TI - Endophthalmitis associated with a releasable suture. PMID- 8639101 TI - Late visual loss secondary to filtering bleb exuberance. PMID- 8639100 TI - Epibulbar and ocular tuberculosis. PMID- 8639102 TI - Loss of vertical palpebral fissure height on downgaze in acquired blepharoptosis. PMID- 8639103 TI - The surgeon's dilemma. PMID- 8639104 TI - Posterior dislocation of plate haptic silicone lenses. PMID- 8639105 TI - Forceps-induced scratching of a foldable acrylic intraocular lens. PMID- 8639106 TI - A light pipe with a twist. PMID- 8639107 TI - Hypodontia of the permanent dentition. Case reports. AB - Hypodontia has been observed as one of the most common human dental developmental anomalies. It may be defined as agenesis of one or more teeth. The prevalence in the permanent lower central and lateral incisor region is low, ranging from 0.23 percent to 0.08 percent, respectively. This is compared with an overall incidence of hypodontia of 3.49 percent. However, significant racial variation occurs. Treatment options generally available are: no treatment, closure of spaces orthodontically, or prosthetic replacement. Aetiology, associated anomalies, and factors involved in treatment choice are discussed. A report of four cases of hypodontia of the permanent lower anterior teeth and their orthodontic management is presented. PMID- 8639108 TI - Cerebral abscess complicating dental treatment. Case report and review of the literature. AB - A case history and brief literature review of cerebral abscess related to dental therapy is presented. The 19-year-old male patient presented with a cerebral abscess caused by Actinobacillus actinomycetamcomitans. He was otherwise healthy, and had a recent history of periodontal surgery prior to the onset of symptoms. The patient was treated successfully with stereotactic aspiration and antibiotics. PMID- 8639109 TI - Temporomandibular disorders. Part 3. Surgical treatment. AB - Surgery of the temporomandibular joint has a small but nonetheless important role in the overall management of temporomandibular disorders. Appropriate case selection is the mandatory requirement for successful surgical intervention in order to achieve the desired outcome of treatment, such as relief of symptoms and improved function. In this, the third article in the series, a general overview of the current surgical treatment modalities for temporomandibular disorders will be presented. PMID- 8639110 TI - The stability of facial osteotomies. Part 5. Maxillary advancement with miniplate and screw fixation. AB - Surgical repositioning of the dento-skeletal components of the middle-third of the face, combined with appropriate orthodontic treatment, can be used to improve function and aesthetics. However, the attainment of three-dimensional stability following corrective jaw surgery continues to be a major problem in the postsurgical period. This paper examines the short-term (six weeks postoperative) and long-term (12 months postoperative) horizontal skeletal stability of Le Fort I maxillary advancement in 15 patients. The mean horizontal advancement of the maxilla was 8.76 +/- 0.99 mm. Six weeks later, a mean relapse of 0.22 +/- 0.19 mm was identified. The mean relapse at long-term follow-up was 0.61 +/- 0.26 mm (6.96%). These results indicate that rigid miniplate and screw fixation of Le Fort I osteotomy undertaken to correct horizontal mid-dentofacial deficiency is both statistically and surgically predictable and stable when reviewed up to twelve months after surgery. PMID- 8639111 TI - Cementation of vented crowns with low deformation. AB - Crown deformation during cementation was measured with a circumferential strain gauge. The effects of different seating forces (3, 10 and 25 N), three different cements (Phosphacap, Ketac-cem Applicap and Fuji-cap I) and the presence or absence of venting on deformation and seating discrepancy were investigated. Forty microns of cement space was provided for all experiments. Venting increased deformation and reduced seating discrepancy, whilst increased force increased deformation and decreased seating discrepancy. Ketac-cem Applicap allowed the lowest seating discrepancy with median crown deformation. It was possible, by using venting, low forces (10 N) and Ketac-cem Applicap, to achieve a low marginal discrepancy with low crown deformation. Venting also diminished the seating discrepancy to acceptable levels when higher forces (25 N) were used with Phosphacap. PMID- 8639112 TI - Oral conditions and their social impact among HIV dental patients. AB - This study aimed to assess oral health status and the social impact of oral conditions among dental patients with HIV infection in comparison with general dental patients receiving public-funded care in Adelaide, South Australia. DMFT and CPITN indices were recorded by one dentist at a clinic for HIV dental patients. The data were compared with information from an existing survey of general dental patients. Social impact was assessed using the Oral Health Impact Profile questionnaire and responses from HIV dental patients were compared with responses from a telephone interview survey of Adelaide residents. HIV patients were aged 21 to 49 years (median = 34), 90.7 percent were male and 29.6 percent had stage 4 HIV infection. Oral candida was present among 32.0 percent, hairy leukoplakia among 24.1 percent, HIV gingivitis among 18.5 percent, and HIV periodontitis among 33.3 percent. The DMFT index and its components did not differ significantly between HIV and general dental patients, while CPITN scores were lower among HIV patients (p = 0.01). However social impact among HIV patients was frequent: 64.6 percent reported toothache, 43.7 percent avoided foods, and 16.7 percent avoided going out because of dental problems. HIV patients reported significantly greater levels of social impact than the Adelaide sample (p < 0.01). Patients to this clinic frequently presented with severe and disabling oral conditions which were not adequately captured using standard clinical indices. PMID- 8639114 TI - Establishing malocclusion severity levels on the Dental Aesthetic Index (DAI) scale. AB - The Dental Aesthetic Index (DAI) is an orthodontic index based on socially defined aesthetic standards. It is useful in both epidemiological surveys to identify unmet need for orthodontic treatment and as a screening device to determine priority for subsidized orthodontic treatment. An earlier study established the score of 36 on the DAI scale to identify handicapping malocclusions. The purpose of the present study was to determine decision points on the DAI scale that identify malocclusion severity levels less severe than handicapping. Two sources of data were used: 1) The frequency distribution of DAI scores on a probability sample of 1306 study models representing the untreated occlusions found in half a million adolescents. 2) The percent distribution of US youths aged 12-17 by specified case severity reported in an assessment of the occlusion of youths by the National Center for Health Statistics (NCHS). The decision points separating specific case severities on the DAI scale were determined by relating the proportions of the NCHS population with specified case severities to the cumulative percentages of the frequency distribution of DAI scores on the 1306 models. The NCHS report found 45.8 percent of the sample to have normal or minor malocclusion with no need or slight need for treatment. DAI scores 25 and below corresponded to that proportion of the sample. The NCHS report found 25.2 percent of the sample to have definite malocclusion with treatment being elective. DAI scores between 26 and 30 corresponded to that proportion of the sample. The NCHS report found 13 percent of the population to have severe malocclusion with treatment highly desirable. Fifteen percent were included in this category. DAI scores 31 to 35 corresponded to that proportion of the sample. PMID- 8639113 TI - Fluoride content of infant formulae in Australia. AB - The prevalence of dental fluorosis in Australia and the United States of America has increased in both optimally fluoridated and non-fluoridated areas. This has been attributed to an increase in the fluoride level of food and beverages through processing with fluoridated water, inadvertent ingestion of fluoride toothpaste, and the inappropriate use of dietary supplements. A major source of fluoride in infancy is considered to be infant formula which has been implicated as a risk factor for fluorosis in a number of studies. In this study the fluoride content of the infant formulae commonly used in Australia was determined. The acid diffusible fluoride of each powdered formula was isolated by microdiffusion and measured using a fluoride ion-specific electrode. The fluoride content of milk-based formulae ranged from 0.23 to 3.71 micrograms F/g and for soy-based formulae from 1.08 to 2.86 micrograms F/g. When reconstituted, according to the manufacturer's directions, with water not containing fluoride, the formulae ranged in fluoride content from 0.031 to 0.532 ppm, with the average fluoride content 0.240 ppm. Using average infant body masses and suggested volumes of formula consumption for infants 1-12 months of age, possible fluoride ingestion per kg body mass was estimated. None of the formulae, if reconstituted using water containing up to 0.1 ppm F, should provide a daily fluoride intake above the suggested threshold for fluorosis of 0.1 mg F/kg body mass. However, if reconstituted with water containing 1.0 ppm F they should all provide a daily fluoride intake of above the suggested threshold for fluorosis with intakes up to 2-3 times the recommended upper 'optimal' limit of 0.07 mg/kg body mass. Under these conditions the water used to reconstitute the formulae would provide 65-97 percent of the fluoride ingested. These figures are likely to be overestimates due to the intake of nutrients from other sources reducing formulae consumption and also due to the lower bioavailability of fluoride from milk-based formulae. Further, it is generally believed that the maturation stage of enamel formation is the critical period for fluorosis development by chronic, above-threshold fluoride exposure. The maturation stage for the anterior permanent teeth, however, is after the first twelve months of life where fluoride intake from infant formula consumption per kg body mass is highest. The level of fluoride in the commonly used Australian formulae would suggest that infant formula consumption alone is unlikely to be a risk factor for dental fluorosis in a non fluoridated community, but could make a major contribution to an infant's daily fluoride intake. However, prolonged consumption (beyond 12 months of age) of infant formula reconstituted with optimally-fluoridated water could result in excessive amounts of fluoride being ingested during enamel development of the anterior permanent teeth and therefore may be a risk factor for fluorosis of these teeth. PMID- 8639115 TI - A comparison of patients attending general dental practices employing or not employing dental hygienists. AB - Previous studies have shown that dental practices employing dental hygienists provide a more periodontally oriented mix of dental services. Little is known about differences in the characteristics of patients who attend these practices, and whether these reflect the orientation of service delivery. In this study, a comparison was made between patients attending private general dental practices employing hygienists and patients attending practices without hygienists in Adelaide, South Australia. A questionnaire was mailed to 2391 patients, and valid responses were received from 632 patients attending 12 practices employing hygienists, and 1052 patients attending 22 practices not employing hygienists. Bivariate analysis of the responses comparing the two groups of patients was conducted using ANOVA for continuous variables and chi-square statistics for categorical variables. A multiple logistic regression model was developed to identify characteristics of patients attending practices with hygienists. Examination of significant (p < 0.05) findings revealed that patients attending practices with hygienists had a higher socio-economic status, had a more preventive pattern of use of services, had better knowledge of periodontally related topics and it was more likely that the dental profession was their main source of information of these topics. These findings indicate that patient profiles do reflect the orientation of service delivery within dental practices and raise the question of whether the dental practice alters the patient's knowledge and behaviour or whether certain patients selectively seek care from practices with hygienists. PMID- 8639116 TI - Oral health and personnel needs in the Pacific. AB - A regional review of oral health in the Pacific showed the major problems to be dental caries, periodontal diseases, poor dental health service management and lack of appropriate dental personnel. A strategy for training appropriate dentists to manage oral health services in the Pacific was suggested. Such a strategy must include training of ancillary and auxiliary dental health workers guided by dentists with clinical and managerial competencies. The training programme for dentists must be career-ladder, problem-based, and community oriented with competency-based learning of a spiral of tasks with increasing sophistication. The curriculum content must contain about 50 percent on public health and clinical aspects, respectively. PMID- 8639117 TI - Customer, client, patient? Dentistry in 1996. PMID- 8639118 TI - An unusual facial sinus. AB - An unusual presentation of a chronic suppurative granuloma on the alar base area originating from a maxillary lateral incisor is reported. Treatment involved adequate endodontic therapy with simultaneous apical surgery and excision of the granuloma and sinus tract. Awareness of the possible dental origin of facial sinuses, despite their unusual location, is emphasized. PMID- 8639120 TI - The use of acupuncture in dentistry. PMID- 8639119 TI - Methadone and oral hygiene. PMID- 8639121 TI - Endodontic cellulitis 'flare-up'. PMID- 8639122 TI - Endodontic cellulitis 'flare-up': clinical implications. PMID- 8639123 TI - Interocclusal appliance therapy as assessed by three-dimensional reconstructions of MRI scans of temporomandibular joints. PMID- 8639124 TI - Mouthguard protection in sports injury. Author's reply. PMID- 8639126 TI - Who manages breast cancer? PMID- 8639125 TI - Benign cementoblastoma. Case report. AB - Benign cementoblastoma is a rare benign odontogenic tumour of mesenchymal origin. A case of a 24-year-old man with benign cementoblastoma is presented. The lesion manifested as a round, radiopaque mass attached to the root of the left first molar of the mandible. The tooth was extracted and the lesion was removed. A diagnosis of benign cementoblastoma was made microscopically. There has been no evidence of recurrence during the three-year follow-up period. A review of the literature and a discussion of the histologic features of this case are included. PMID- 8639127 TI - Mammographically negative breast cancer at the Strathfield Breast Centre. AB - BACKGROUND: The current diagnostic modalities used to detect breast cancer are mammography, together with clinical examination, ultrasound and fine needle aspiration biopsy (FNAB). The accuracy rates for each modality varies and a combination of the modalities is recommended to detect cancer early. Some authors have suggested that mammography should be used primarily as a screening tool because of the false negative mammography results that have been reported in the past 10 years. The records of patients at the Strathfield Breast Centre were reviewed to determine the accuracy of other modalities. METHODS: The records of 371 breast cancer patients treated at the Strathfield Breast Centre in the 6 years form 1989 to 1994 were reviewed to determine the accuracy of mammography, ultrasound, clinical examination and fine needle aspiration biopsy. Of the 371 women with histopathologically diagnosed breast cancer, 349 had mammography. RESULTS: The accuracy rate of mammography in the present study was 91% with a false negative rate of 9%. It was found that there was no significant delay in treatment of breast cancer in mammogram-negative patients. CONCLUSIONS: Mammographically negative breast cancer was found to be more common in younger women, to be similar in size to mammogram-positive cancer, to occur in all histological types and grades and was usually invasive rather than noninvasive. The rate of lymph node involvement was similar to the mammogram-positive group. PMID- 8639128 TI - Paraffinomas of the breast: an oriental curiosity. AB - BACKGROUND: The injection of liquid paraffin wax was a form of breast augmentation practised in Hong Kong 30-40 years ago. Patients may present many years later with complications of this treatment. METHODS: The records of 43 patients diagnosed with paraffinomas of the breast at a teaching hospital in Hong Kong were reviewed. These patients had received paraffin injections 3-41 years (median 17) previously. RESULTS: Patients presented with hard masses in the breast 4-18 cm (median 17) diameter. Ulceration or infection occurred in 10 patients. Mammography revealed a honeycomb appearance in the affected breast. Treatment included biopsy only (7), excision of masses (3) and total mastectomy (30, bilateral in 27). Histology demonstrated hyalinized and densely sclerotic fibrous tissue with cystic spaces of various sizes. CONCLUSIONS: Awareness of this condition facilitates the differentiation from tuberculosis and carcinoma of the breast which often present late in this population of patients. Adequate treatment usually require total mastectomy. PMID- 8639129 TI - The prevalence of MEN-1 in Tasmania. AB - BACKGROUND: An extensive programme was undertaken to trace and screen four known families in Tasmania with multiple endocrine neoplasia type 1 (MEN-1). METHODS: Written and personal contact was made with family members over the age of 20 years recommending a review by family practitioners for the purpose of recording their medical history and collecting a blood sample. Those suspected of MEN-1 were referred to our Department for further investigation. RESULTS: In January 1993, the total number of individuals alive and known to be affected by MEN-1 was 107, giving a prevalence of MEN-1 disease in Tasmania of 23/100 000. The estimated prevalence of MEN-1 trait in Tasmania (including affected cases and those considered at 50% risk of possessing the trait) is 45/100 000. CONCLUSION: The prevalence of MEN-1 has never previously been determined accurately. The prevalence of MEN-1 in Tasmania is at the upper end of the possible range and would justify the allocation of resources for screening programmes equal to those available for the detection of several less prevalent genetic diseases. PMID- 8639130 TI - Factors affecting morbidity and mortality following surgical intervention in patients with intracranial meningioma. AB - BACKGROUND: Meningiomas usually grow slowly but they may cause recurrences despite surgical resection. The impact of clinical, neuroradiological and surgical characteristics on operative morbidity and mortality of patients operated on for intracranial meningioma was analysed. METHODS: A series of 450 patients operated on for intracranial meningiomas at the Department of Neurosurgery, Hacettepe University Hospital during the period 1964-1992 is reported. The surgical results were analysed with regard to intracranial site, extent of removal, histological type, and different time periods. Computed tomography (CT) and magnetic resonance imaging (MRI) facilitated the diagnosis and helped with the planning of treatment. RESULTS: Two hundred and ninety-two patients were examined with both CT and MRI. Overall mortality was 4% but showed a decline from 9% in the pre-CT era to 3% in the post-CT era and to 1% in the past 3 years. CONCLUSIONS: Operative mortality and recurrence rates are affected by the intracranial location of the tumour, histological type, and extent of tumour removal. Emphasis is also given to the importance of the introduction of the imaging techniques, and the microsurgical techniques with the Cavitron ultrasonic surgical aspirator (CUSA), laser, and/or bipolar coagulator which have further improved the operative mortality and recurrence rates. PMID- 8639131 TI - Intertrochanteric fractures of the femur: a randomized prospective comparison of the Gamma nail and the Ambi hip screw. AB - BACKGROUND: The Gamma nail has been introduced as an advance over the Ambi hip screw in intertrochanteric femoral fractures. Its efficacy in an Australasian setting has not been documented. METHODS: A prospective randomized study was used to compare the Ambi hip screw and the Gamma nail for the treatment of 69 patients over the age of 50 years with intertrochanteric femoral fractures. The groups were similar with respect to age, sex, prefracture mobility and abode, anaesthetic risk grade, CT measured bone density and fracture pattern. RESULTS: Those treated with the Gamma nail had a significantly longer image intensifier screening time (P < 0.05), greater blood loss (P < 0.01) and more operative complications. There was no difference in the length of hospital stay but the level of mobility recovered was significantly better in the Ambi group at 6 months follow up. Urine retention was the most frequent complication but did not correlate with the implant or method of anaesthesia. Two implants cut out, one in each group. Limb shortening was similar in both groups and was not affected by leaving the Gamma nail unlocked distally in unstable fractures. Thirteen patients died from pre-existing medical conditions. CONCLUSIONS: The Gamma nail proved technically more demanding with higher intra-operative complications and inferior return of mobility. PMID- 8639132 TI - Treatment of humeral shaft fractures with the Seidel intramedullary nail. AB - BACKGROUND: The use of intremedullary nails for the management of humeral shaft fractures has been controversial. Recently, the Seidel nail has become available. The purpose of this study was to review our initial experience with the Seidel nail. METHODS: A retrospective clinical and radiographic review of 25 consecutive patients treated with Seidel intramedullary humeral nail was performed. The nail was used for non-union in 10 patients, delayed union in four, acute fracture in eight and pathological fracture in three. Eighteen of the 19 survivors were clinically reviewed at an average of 15 months (range 8-15). Pain, function, satisfaction, shoulder power, range of motion and clinical outcome were graded using the UCLA shoulder score. RESULTS: Pain was present at the shoulder in four patients and at the fracture site in nine. Average shoulder abduction was 99 degrees and nine patients could not abduct the shoulder past 90 degrees. Sixty six percent of patients reviewed were graded as only fair or poor using the UCLA shoulder score. In three patients rotational control was not achieved with the distal locking device at the time of surgery. Complications included non-union in 10 patients and three intra-operative fractures. CONCLUSIONS: Non-union was more likely to occur if rotational control was not obtained, or if the patient had the nail inserted for a previous non-union. Use of the Seidel nail frequently leads to shoulder pain and dysfunction. The distal locking device is unreliable and predisposes to non-union. We do not recommend the continued use of the Seidel nail. PMID- 8639133 TI - Matchett Brown hemiarthroplasty for displaced subcapital fractures of the hip. AB - BACKGROUND: Matchett Brown hemiarthroplasty has been routinely performed at Middlemore Hospital in elderly patients following subcapital fracture of the hip. The outcome of patients undergoing Matchett Brown hemiarthroplasty was evaluated. METHODS: Matchett Brown hemiarthroplasties performed at Middlemore Hospital during 1987 were retrospectively reviewed. Medical records were reviewed and where possible patients were interviewed, examined and radiographs of their hip taken. RESULTS: The overall survival at follow up was 34%, with the greatest predictor of survival being whether the patient had been living alone prior to the accident. The majority of patients who survived the 4 year follow up had excellent mobility at the time of fracture. At follow up most patients had little or no pain from their hip, but three complained of constant pain. CONCLUSIONS: Hemiarthroplasty proved to be a satisfactory form of replacement in this group. If one were to select a patient for total hip replacement, rather than hemiarthroplasty, then age alone is not as important as other factors such as degree of mobility and independence of living. PMID- 8639134 TI - Biliary complications after liver transplant: the Victorian experience. AB - BACKGROUND: Biliary complications remain a continuing problem in liver transplantation. The goals of this study were to document the frequency of biliary complications following orthotopic liver transplantation in the Victorian programme, and to examine associations with suspected risk factors with reference to biliary stenosis. METHODS: Data were collected from 129 consecutive transplants in 123 patients (106 adults, 17 children) at the Austin Hospital, Melbourne during the period 1988-94. The 2 year actuarial survival was 88%. Biliary reconstruction was by end-to-end anastomosis in 89 patients and Roux-en-Y in 40. Complications were suspected on clinical, biochemical or microbiological evidence. Biliary stenoses were considered to be radiological evidence of duct narrowing. RESULTS: Biliary complications occurred in 19% and biliary stenosis in 8.5%. Of the stenoses, 1/35 occurred in the first 20 month period, 9/47 in the second and 1/47 in the third. There was a significant difference between the middle period and other periods (P<0.05, Chi-square test). This change may be related to incomplete flushing of bile from the donor liver. Recurrence of the original disease was suspected for one stenosis. The length of the donor bile duct from hilum to anastomosis, cold ischaemia time and total hepatic artery flow at transplant did not relate to stenosis. Cholangitis was not diagnosed in patients without strictures. Strictures were managed by dilatation (5/11) and by operative repair (6). CONCLUSIONS: Stenoses were not related to the length of the donor bile duct, cold ischaemia time or total hepatic artery flow. Meticulous adherence to the protocol for flushing out bile at the donor operation was associated with a significant reduction in frequency of biliary stenoses. PMID- 8639135 TI - Hepatic cystic disease in an adult polycystic kidney disease transplant population. AB - BACKGROUND: A study of the incidence of polycystic liver in a transplant population and an assessment of the impact of this disease on the group was undertaken. Clinical presentation, investigation and treatment of hepatic polycystic disease are explored. METHODS: The study examined the morbidity incurred by polycystic liver disease in patients, patient and graft survival, incidence of hepatic cysts and presentation, investigation and management of morbid hepatic cystic disease. One hundred and eleven patients were studied. RESULTS: It was found that hepatic cysts occurred in the majority of patients that underwent transplants for renal failure because of polycystic kidney disease at this institution. Symptomatic hepatic cystic disease was found to be primarily responsible for three deaths and affected between 15 and 20% of the group. Gall bladder disease was found to be associated with highly symptomatic hepatic cystic disease. CONCLUSION: Hepatic cystic disease is common in patients that have had kidney transplants because of polycystic kidney disease. Morbidity is likely to increase as patients survive for longer periods. Cholecystectomy should be considered in patients with significant hepatic cystic disease. PMID- 8639136 TI - Primary peritonitis in children. AB - BACKGROUND: Primary peritonitis is a rare cause of the acute abdomen in children. It is often found at laparotomy for suspected appendicitis, or diagnosed by paracentesis in a child with an associated medical disorder. METHODS: Our experience over the past 5 ++years with this condition at the Royal Children's Hospital is reviewed and compared with a larger series previously published from the same institution 25 years ago. RESULTS: There were 26 cases from 1989 to 1994; 22 (85%) were female and 6 (23%) of the patients had a major underlying medical disorder. CONCLUSIONS: The incidence of primary peritonitis has remained unchanged at this institution since 1970 (4.7 cases per year), but neonatal primary peritonitis is no longer seen. Primary peritonitis is more common in females in whom there is no clear aetiology, but is presumed to be related to the retrograde passage of organisms through the genital tract. By contrast, in males primary peritonitis is rare but may be related to underlying medical conditions such as alpha-1-antitrypsin deficiency and renal disease. PMID- 8639137 TI - Optimizing the hospital management of leg ulcers. AB - BACKGROUND: Leg ulcers are common and are often the cause of a long hospital admission. However, little information is available on the efficacy and efficiency of inpatient leg ulcer management. The inpatient management of leg ulceration was examined and areas for improvement were sought. METHODS: The management of patients admitted to a teaching hospital with a primary diagnosis of leg ulceration was examined, the costs estimated and areas for improvement identified. A retrospective analysis of 174 admissions to Heidelberg Repatriation Hospital between 1 January 1991 and 31 December 1992 was performed. RESULTS: Of 119 patients, 61 had ulcers due to arterial disease and 34 due to venous disease. Over 2 years, leg ulcers accounted for 5259 inpatient bed days, a mean of 44.2 days per patient. The estimated cost exceeded $2,750,000, averaging over $12,000 per admission. Thirty-three percent of patients had no recorded investigations into the cause of their ulcer and fewer than 50% had documented improvement at discharge. CONCLUSIONS: Leg ulcers are costly due to their extended treatment on an inpatient basis. Unfortunately, hospital admission does not guarantee optimal wound healing rates. A leg ulcer protocol is proposed to minimize inpatient stay and improve investigation and management in an outpatient or community setting. PMID- 8639138 TI - Cellulitis and the occult diabetic. AB - BACKGROUND: Occult diabetes may be an important factor in the development of cellulitis and cellulitis may act as a precipitant to the diagnosis of diabetes mellitus. The present study defines the prevalence of diabetes and glucose tolerance impairment in a group presenting with cellulitis to a teaching hospital. A description of the demographic and pathological presentation of the group is undertaken. METHODS: Five hundred consecutive admissions for cellulitis to a Sydney teaching hospital were analysed. The cases presented between 1985 and 1994. Precipitating factors, length of stay, site of infection, white cell count, degree of fever, blood sugar estimation, history of diabetes mellitus and microbiological diagnosis were recorded. RESULTS: Forty-nine patients had a prior diagnosis of diabetes mellitus. Twenty-one percent of patients (56/265) were noted to have abnormal glucose tolerance on routine testing. Thirty-seven per cent of the 1994 cohort (14/38) demonstrated abnormal glucose tolerance. The most common precipitant was trauma (137/500). Mean length of stay was 8.7 days. Microbiological diagnosis was made in 32% of cases. CONCLUSIONS: Cellulitis requiring admission to hospital is a significant problem in terms of cost and bed occupation. A presentation with cellulitis may be a clinical indicator of impaired glucose tolerance. All patients presenting with cellulitis should have a fasting blood sugar level determined as part of routine workup. PMID- 8639140 TI - A novel method of rectal occlusion during anterior resection of the rectum. AB - An inexpensive and effective method of rectal occlusion during low anterior resection using a commercially available cable tie is described. Its use facilitates cross stapling of the rectum distal to the tumour. PMID- 8639139 TI - A prospective trial of open versus laparoscopic appendicectomy. AB - BACKGROUND: An assessment of the value of laparoscopic appendicectomy was performed. METHODS: During 1993, all patients above the age of 13 years admitted with clinical appendicitis to Box Hill were assigned to an open or laparoscopic procedure depending on whether the surgeon on call was an 'open' or 'laparoscopic' surgeon for the purpose of the study. Ninety-two patients were entered in the study, of whom 57 were in the open group. The rate of histologically proven appendicitis was 73.9%. RESULTS: No significant difference between the groups was found in the use of narcotic analgesia, length of stay or incidence of wound infection. However, operating time was significantly longer in the laparoscopic group. CONCLUSIONS: Laparoscopic appendicectomy on an unselected group of patients does not confer many advantages but laparoscopy may be beneficial in certain subgroups. PMID- 8639141 TI - Divisions in surgery. PMID- 8639142 TI - Malignant sacrococcygeal germ cell tumour in an adult. AB - In adults, malignant sacrococcygeal germ cell tumour is a rare cause for a presacral tumour, with only 17 cases having been reported in the literature since 1907. We report the case of a 34 year old male who presented with a 6 month history of symptoms relating to a malignant presacral tumour which required en bloc excision including the lower sacrum and rectum. He died with lung and mediastinal metastasis 7 months following surgical excision and adjuvant chemotherapy using Cisplatin, Bleomycin and Etoposide. Prior to his death, he had a severe polyarthritis of his peripheral joints and evidence of hypertrophic osteo-arthropathy. The literature indicates that adults with these tumours have a poor prognosis, with only one reported long-term survivor. Surgical excision offers the only chance of cure, with the role of adjuvant therapy not having been defined because of the small numbers. PMID- 8639143 TI - Bilateral scapular fractures from electrical injury. PMID- 8639144 TI - Biomechanical explanation of a simultaneous closed rupture of both flexor tendons in the same digit. AB - Closed ruptures of both normal flexor tendons in the same finger are extremely rare, only nine cases having been reported in the literature. The authors describe the case of a patient who sustained a closed rupture of both flexor digitorum profundus and flexor digitorum superficialis of the ring finger, following a forced hyperextension injury. The present paper highlights the importance of the mechanism of injury and explains biomechanical basis. The patient was treated by a two-stage reconstruction of the flexor digitorum profundus and regained full flexion and extension of the finger. PMID- 8639145 TI - Spinal extradural angiolipoma. PMID- 8639146 TI - Assortative mating for relative weight: genetic implications. AB - Most work on the genetics of relative weight has not considered the role of assortative mating, i.e., mate selection based on similarity between mates. We investigated the extent to which engaged men and women in an archival longitudinal database were similar to each other in relative body weight prior to marriage and cohabitation. After controlling for age, a small but statistically significant mate correlation was found for relative weight (r=.13, p=.023), indicating some assortative mating. Furthermore, we examined whether mate similarity in relative weight prior to marriage predicts survival of the marriage. No significant effects were found. In sum, these results are consistent with those of other studies in suggesting that there is a small but significant intermate correlation for relative weight. However, they are unique in showing that these results cannot be explained on the basis of (a) cohabitation, (b) age similarity, or (c) selective survival of marriages between couples more similar in relative weight. The implications of these findings for heritability studies, linkage studies, and the estimation of shared environmental effects are discussed. PMID- 8639148 TI - Social contact, social attitudes, and twin similarity. AB - The nature of the relationship between social contact and attitude similarly between twins was investigated using longitudinal data from a sample of Australian twins. Earlier research has suggested that social attitudes are not explained solely by shared environment; rather there are both genetic and environmental components that explain variance in social attitudes. Using three types of analyses we investigated the magnitude of the relationship and the direction of causation between attitude similarity and social contact. Longitudinal analysis of within-pair variance by level of contact suggests that attitude similarity leads to contact among the females and that similarity is both genetically and environmentally based. Analyses using a crosslag regression model suggest that similarity causes contact among MZ females. Biometrical analyses indicate differences in direction of causation for males and females. Among females, both genetic and shared environmental parameter estimates could be equated across contact groups, suggesting little relationship between contact and similarity. Among males, findings of smaller estimated heritability in the high contact group suggest that similarity causes contact. However, an increased estimate of the contribution of shared environmental variance in the high-contact males could additionally suggest that contact leads to similarity. PMID- 8639149 TI - The independent prediction of general intelligence by elementary cognitive tasks: genetic and environmental influences. AB - Current theories of intelligence have, in some cases, begun to include elementary cognitive tasks. Behavioral genetic studies of intelligence have not taken these theories into account. The current study includes 135 MZ and 128 DZ twin pairs from the Western Reserve Twin Project. The 11 WISC-R subtests as well as 6 elementary cognitive tasks were employed. Using a Schmid-Leiman (1957) transformation, analyses indicate a four-group factor model, supported by a second-order general factor at both phenotypic and biometric levels. Results indicate that the general factor, group factors, and specific residuals are necessary when examining additive genetic variance. Common environmental variance can be collapsed into a single general factor. PMID- 8639147 TI - Assessing the statistical power to detect linkage in a sample of 51 bipolar affective disorder pedigrees. AB - We used computer simulation method to address the question of power in an initial collaborative sample of 51 bipolar affective disorder pedigrees. Simulations were performed for all possible combinations using (1) two levels of diagnostic stringency, (2) three transmission models, (3) locus heterogeneity, and (4) different assumed phenocopy rates. Some of the factors affect the power to detect linkage are (1) the specification of the correct genetic model, (2) the degree of locus heterogeneity, and (3) the frequency of phenocopies. The first two assertions were supported by our simulation results, but varying the rates of phenocopy did not substantially alter the power of the sample until a critical point. However, it is important to point out that these results are dependent on the genetic models under study and on the use of the "correct" model (i.e., the one used to simulate the data). If we assume a dominant mode of inheritance and locus homogeneity, the power to detect linkage is 97.5% at a theta of .01. However, the power declines dramatically, to 60.5% and 14.7%, if only 75 and 50% of the families are linked, respectively. Locus heterogeneity has a similar effect on the power of the sample to exclude linkage. The relative lack of power in our data, in the presence of significant locus heterogeneity, and for an intermediate mode of inheritance, underscores the need for multicenter collaboration. PMID- 8639151 TI - Using multivariate genetic modeling to detect pleiotropic quantitative trait loci. AB - Large numbers of sibling pairs or other relatives are needed to detect linkage between a quantitative trait locus (QTL) and a marker, especially if the variance of the QTL is low relative to the total phenotypic variance of the trait. One strategy to increase the power to detect linkage is to reduce the environmental variance in the trait under analysis. This approach was explored by carrying out a series of simulation studies in which multivariate observations were used to estimate individual genotypic values at a QTL, that pleiotropically affected more than one trait. Simulations for different QTL allele frequencies with a completely informative marker showed that the power to detect the QTL increased substantially when estimates of individual genotypic values at the QTL were used in the linkage analysis instead of phenotypic observations. An advantage of this approach is that, rather than employing phenotypic selection, individuals with extreme genotypes may selected when ascertaining a sample of extreme families. PMID- 8639150 TI - Type I and type II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred mouse strains. AB - Effective mapping strategies for quantitative trains must allow for the detection of the more important quantitative trait loci (QTLs) while minimizing false positives. Type I (false-positive) and Type II (false-negative) error rates were estimated from a computer simulation of QTL mapping in the BXD recombinant inbred (RI) set compromising 26 strains of mice, and comparisons made with theoretical predictions. The results are generally applicable to other RI sets when corrections are made for differing strain numbers and marker densities. Regardless of the number or magnitude of simulated QTLs contributing to the trait variance, the p value necessary to provide adequate protection against both Type I (alpha=.0001) and Type II (beta=.2) errors, a QTL would have to account for more than half of the between-strain (genetic) variance if the BXD or similar set was used alone. In contrast, a two-step mapping strategy was also considered, where RI strains are used as a preliminary screen for QTLs to be specifically tested (confirmed) in an F2 (or other) population. In this case, QTLs accounting for approximately 16% of the between-strain variance could be detected with an 80% probability in the BXD set when alpha = 0.2. To balance the competing goals of minimizing Type I and II errors, an economical strategy is to adopt a more stringent alpha initially for the RI screen, since this requires only a limited genome search in the F2 of the RI-implicated regions (approximately 10% of the F2 genome when p < .01 in the RIs). If confirmed QTLs do not account in the aggregate for a sufficient proportion of the genetic variance, then a more relaxed alpha value can be used in the RI screen to increase the statistical power. This flexibility in setting RI alpha values is appropriate only when adequate protection against Type I errors comes from the F2 (or other) confirmation test(s). PMID- 8639152 TI - Selective breeding for extremes in open-field activity of mice entails a differentiation of hippocampal mossy fibers. AB - The brains of mice selectively bred for differential locomotor activity in an open field (DeFries et al., Behav. Genet. 8:3-13, 1978) were analyzed for selection-dependent changes in the size of synaptic fields at the midseptotemporal level for the hippocampus. Timm-stained areas of all hippocampal fields from both left and right hippocampi were measured on five horizontal sections from the midseptotemporal level. The sample included 25 mice from two replicate lines, each one consisting of a high (HI); a low (LO), and a control line (CTL). The main selection effect was an enlargement of the intra infrapyramidal mossy fiber (IIP-MF) projection in both HI lines by about 70% compared to LO and CTL mice (p < .0001), while other mossy fiber fields did not show differences. These findings confirm that genetic variations of the IIP-MF projection influence hippocampal processes mediating exploratory activities. PMID- 8639154 TI - Models of spouse similarity: applications to fluid ability measured in twins and their spouses. AB - Genetic influences have consistently been reported to be the principal explanation for resemblance among relatives for intelligence, with shared environmental effects playing a much smaller role. However, crucial to understanding the nature of environmental influences are the mechanisms of assortative mating. Phenotypic assortment, albeit widely assumed or modeled in biometrical analyses, may be less important than other assortment processes, such as social homogamy. Consequently, effects of shared environment may play a greater role than prior studies have suggested. The goal of this study was to resolve environmental and genetic influences on fluid ability based on alternative models of assortment by examining the similarity of monozygotic (MZ) and dizygotic (DZ) twins and their spouses. Raven's Progressive Matrices scores were available from a population-based Swedish sample of 138 twin kinships. The effects of both social homogamy and phenotypic assortment were tested simultaneously in each of two alternate assortment models. A factor/delta path model represented social homogamy as a common factor and phenotypic assortment as a delta path, while a delta/delta path model represented both social homogamy and phenotypic assortment as delta paths. Overall, the factor/delta path model was found to be superior. Results suggested that social homogamy completely explained spouse similarity; phenotypic assortment was not significant. The results of these analyses suggest the presence of shared environmental effects among twins and their spouses, which would have been underestimated if only phenotypic modeled phenotypic assortment may have underestimated the effects of environment. PMID- 8639153 TI - Circadian rhythm and the per ACNGGN repeat in the mole rat, Spalax ehrenbergi. AB - Individual variability in circadian locomotor activity has recently discovered in the blind mole rat, Spalax ehrenbergi. An interesting association was found between different circadian types and two DNA fragments, 5.6 and 5.9 kb long, that contain the ACNGGN repeat sequence, homologous to a part of the period gene of Drosophila. Nine of 12 arrythmic animals showed the 5.6-kb band, while 13 of 17 circadian rhythmic animals had the 5.9-kb band. This repeat exists also in the brain RNA of the mole rat, apparently in higher quantities during the sleeping phase, suggesting that an unusual protein(s), composed of a poly-Thr-Gly segment, affects in circadian rhythm. PMID- 8639155 TI - Genetics of educational attainment in Australian twins: sex differences and secular changes. AB - The relative effects of genetic and environmental factors in producing individual differences in educational achievement are compared across women and men and over birth cohorts. In a large sample of Australian twin pairs, the heritability of self-reported educational attainment did not vary among women and men born before and after 1950. In a "psychometric" model of twin resemblance, based on separate self-reports in 1981 and 1989, genetic factors explained 57% of the stable variance in educational achievement, while environmental factors shared by twins accounted for 24% of the variance. Corrections for phenotypic assortative mating for educational level, however, suggested that estimated common-environmental effects could be entirely explained by the correlation between additive genetic values for mates. Taking this into account, heritability "true" educational attainment in Australia may be as high as 82% with the remaining variation being due to individual environments or experiences. Unlike previous studies in Scandinavian countries, results in Australia suggest that factors influencing educational success are comparable between women and men and for individuals born at different points during this century. PMID- 8639156 TI - New strategies for treating AIDS. AB - To date, the effective management of HIV-1 infection by anti-retroviral drugs has proved remarkably difficult to achieve. This is primarily due to the ease with which HIV-1 becomes resistant to drugs which initially may be very effective at blocking viral replication. In a recent issue of Science, two promising new AIDS treatments were reported. The first described the use of retroviral-type zinc finger structures found in the HIV-1 nucleocapsid protein as targets for antiretroviral drugs. THe second demonstrated the feasibility of the reverse transcriptase inhibitor (R)-9-(2-phosphonylmethoxypropyl) adenine as a postexposure prophylaxis in blocking HIV-1 infection. PMID- 8639158 TI - Do imprinted genes have few and small introns? AB - A gene is described as imprinted if its pattern of expression depends on whether it passed the previous generation in a male or female germ line. A recent paper reports that imprinted genes have fewer and smaller introns than a control set of genes. The differences are striking but their interpretation is unclear. The loss of introns after a gene becomes imprinted is not sufficient to explain why imprinted genes have fewer introns than average, because related unimprinted genes also have few introns. Similarly, small introns appear to be a property of chromosomal region rather than of imprinting status itself, because neighboring unimprinted genes also have small introns. PMID- 8639157 TI - Brain POU-er. AB - Developmental coordination is vital in the temporally coordinated appearance of cell types within the precise spatial architecture of the vertebrate brain and this, combined with the rich interplay between the developing brain and its target organs, is a biological problem of monumental complexity. An example is the genesis and subsequent integration of the neuroendocrine hypothalamus and the pituitary. Two recent papers use the developing hypothalamo-pituitary axis in order to gather a deeper understanding of these integrative mechanisms. In addition, they show that a sub-family of homeodomain factors, the POU-domain proteins, play a critical role in coordinating the respective ontogenies of the hypothalamus and the pituitary. PMID- 8639159 TI - Hierarchical guidance cues in the developing nervous system of C. elegans. AB - During embryogenesis, the basic axon scaffold of the nervous system is formed by special axons that pioneer pathways between groups of cells. To find their way, the pioneer growth cones detect specific cues in their extracellular environment. One of these guidance cues is netrin. Observations and experimental manipulations in vertebrates and nematodes have shown that netrin is a bifunctional guidance cue that can simultaneously attract and repel axons. During the formation of this basic axon scaffold in Caenorhabditis elegans, the netrin UNC-6 is expressed by neuroglia and pioneer neurons, providing hierarchical guidance cues throughout the animal. Each cue has a characteristic role depending on the cell type, its position and the developmental stage. These roles include activities as global, decussation and labeled-pathway cues. This hierarchical model of UNC-6 netrin mediated guidance suggests a method by which guidance cues can direct formation of basic axon scaffolds in developing nervous systems. PMID- 8639160 TI - Molecular mechanisms of anti-inflammatory action of glucocorticoids. AB - Glucocorticoid hormones are effective in controlling inflammation, but the mechanisms that confer this action are largely unknown. Recent advances in this field have shown that both positive and negative regulation of gene expression are necessary for this process. The genes whose activity are modulated in the anti-inflammatory process code for several cytokines, adhesion molecules and enzymes. Most of them do not carry a classical binding site for regulation by a glucocorticoid receptor, but have instead regulatory sequences for transcription factors such as AP-1 or NF-kappa B. This makes them unusual targets for glucocorticoid action and emphasizes the need for novel regulatory mechanisms. Recent studies describe an important contribution by protein-protein interactions, in which several domains of the receptor participate; these studies provide a better understanding of the action of the receptor and offer opportunities for the design of steroidal compounds that could function more effectively as anti-inflammatory drugs. PMID- 8639161 TI - Intracellular trafficking of lysosomal membrane proteins. AB - Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins. PMID- 8639162 TI - Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis: roles of thyroid hormone and its receptors. AB - Amphibian metamorphosis is a post-embryonic process that systematically transforms different tissues in a tadpole. Thyroid hormone plays a causative role in this complex process by inducing a cascade of gene regulation. While natural metamorphosis does not occur until endogenous thyroid hormone has been synthesized, tadpoles are competent to respond to exogenous thyroid hormone shortly after hatching. In addition, even though the metamorphic transitions of individual organs are all controlled by thyroid hormone, each occurs at distinct developmental stages. Recent molecular studies suggest that this competence of premetamorphic tadpoles to respond to the hormone and the developmental stage dependent regulation of tissue-specific transformations are determined in part by the levels of thyroid hormone receptors and the concentrations of cellular free thyroid hormone. In addition, at least two genes, encoding a cytosolic thyroid hormone binding protein and a 5-deiodinase, respectively, are likely to be critical players in regulating cellular free thyroid hormone concentrations. This review discusses how all of these molecular components coordinate to induce amphibian metamorphosis in a correct spatial and temporal manner. These studies provide us with general clues as to how and why tissues become competent to respond to hormonal signals. PMID- 8639163 TI - Dosage-dependent modification of position-effect variegation in Drosophila. AB - Many loci in Drosophila exhibit dosage effects on single phenotypes. In the case of modifiers of position-effect variegation, increases and decreases in dosage can have opposite effects on variegating phenotypes. This is seemingly paradoxical: if each locus encodes a limiting gene product sensitive to dosage decreases, then increasing the dosage of any one should have no effect, because the others should remain limiting. An earlier model put forward to resolve this paradox suggested that dosage-dependent modifiers encode protein subunits of a macromolecular complex that is sensitive to mass action equilibrium conditions. Because chemical equilibria are dynamic, however, such hypothetical complexes will be unstable to an extent that is inconsistent with the known properties of molecules that make up chromatin. An alternative model accounts for the dosage effects in terms of interactions between structural proteins that bind at multiple linked sites. These might include indirect interactions occurring between regulatory proteins and genes for structural proteins or their protein products. The large number of direct and inverse regulatory genes which are known to exist in Drosophila could account for the apparent genetic complexity that is seen for modifiers of position-effect variegation and for other systems of phenotypic modification. PMID- 8639164 TI - Meiotic recombination: a mechanism for tracking and eliminating mutations? AB - The function of meiotic recombination has remained controversial, despite recent inroads into mechanisms. Ideas concerning a possible role of recombination in the elimination or efficient incorporation of mutations have been backed by theoretical studies but have lacked empirical support. Recent investigations into the basis for local variations in recombination frequency in yeast have uncovered a strong association between recombination initiation sites and transcriptional regulatory sequences. Other recent studies indicate a strong correlation between transcription and mutation rates in yeast genes. Taken together, these data imply that distributions of recombination and mutation frequencies may be strongly correlated. This suggests that recombination may be targeted to genomic sites of high mutation frequency; such a 'mutation-tracking' function would clearly aid in the shuffling of mutations to break up unfavorable and create favorable allelic combinations. Moreover, recent insights into the mechanism of gene conversion in yeast reveal a very strong inherent bias in favor of alleles on the non initiating homolog. Combined with mutation tracking, these findings suggest a novel and general mechanism by which allelic gene conversion may act to eliminate mutations. PMID- 8639165 TI - Simple sequences and the expanding genome. AB - Recent analysis of the contribution of replication slippage to genome evolution shows that it has played a significant role in all species from eubacteria to humans. The overall level of repetition in genomes is related to genome size and to the degree of repetition that can be measured within individual ribosomal RNA genes, suggesting that the entire genome accepts simple sequences in a concerted manner when its size increases. Although coding sequences accept simple sequences much less readily than non-coding sequences, they accept some repeats, particularly (CAG)n, preferentially. This may have consequences for the evolution of the genes involved in trinucleotide expansion diseases and the transcriptional networks of which they may form a part. PMID- 8639166 TI - Genome analysis with gene expression microarrays. AB - Advances in biochemistry, chemistry and engineering have enabled the development of a new gene expression assay. This 'chip-based' approach utilizes microscopic arrays of cDNAs printed on glass as high-density hybridization targets. Fluorescent probe mixtures derived from total cellular messenger RNA (mRNA) hybridize to cognate elements on the array, allowing accurate measurement of the expression of the corresponding genes. Array densities of > 1,000 cDNAs per cm2 enable quantitative expression monitoring of a large number of genes in a single hybridization. A two-color fluorescence detection scheme allows rapid and simultaneous differential expression analysis of independent biological samples. Mass-produced microarrays provide a new tool for genome expression analysis that may revolutionize genetic dissection, drug discovery and human disease diagnostics. PMID- 8639167 TI - Guidelines for the management of rheumatoid arthritis. American College of Rheumatology Ad Hoc Committee on Clinical Guidelines. AB - RA is a chronic progressive polyarthritis (with varying systemic features) associated with substantial disability and economic losses. Successful treatment to limit joint damage and functional loss requires early diagnosis and timely initiation of disease-modifying agents. The goal of treatment is to arrest the disease and to achieve remission. Although remission rarely occurs, patients may still benefit from pharmacologic, nonpharmacologic, and if necessary, surgical interventions. Optimal longitudinal treatment requires comprehensive coordinated care and the expertise of a number of providers. Essential components of management include 1) establishment of the diagnosis of RA (versus other forms of polyarthritis), 2) systematic and regular evaluation of disease activity, 3) patient education/rehabilitation interventions, and initial treatment with NSAIDs, 4) use of DMARDs, 5) possible use of local or low-dose oral glucocorticoids, 6) minimization of the impact on the individual's function, 7) assessment of the adequacy of the treatment program, and 8) general health maintenance. PMID- 8639168 TI - Guidelines for monitoring drug therapy in rheumatoid arthritis. American College of Rheumatology Ad Hoc Committee on Clinical Guidelines. AB - Drugs used to treat RA may cause death, disability, and diseases, especially if the treatment continues in the setting of undetected toxicity. Prevention of toxicity may be enhanced by pretreatment assessment of individual risk factors for toxicity and by careful patient and physician education about safe use of the drug. Patients and their physicians must be alert to the signs and symptoms of toxicity that should prompt discontinuation of the drug and physician reassessment. Some drug toxicity may be discovered by appropriate laboratory monitoring before serious problems become clinically apparent. The 3 major drug categories for the treatment of RA are the NSAIDs, DMARDs, and glucocorticoids. Most NSAIDs have common GI and renal toxicity that may be averted by careful patient selection and administration of the drug. The individual DMARDs have specific toxicities for which monitoring protocols have been developed. The serious side effects of systemic glucocorticoids are largely related to dose and duration of treatment. The recommendations summarized in Table 1 are for basic monitoring in patients with uncomplicated RA. Additional monitoring may be appropriate for patients with comorbid disease, concurrent medication, or other risk factors. PMID- 8639169 TI - Cigarette smoking increases the risk of rheumatoid arthritis. Results from a nationwide study of disease-discordant twins. AB - OBJECTIVE: To test the hypothesis that cigarette smoking is associated with susceptibility to rheumatoid arthritis (RA) by comparing smoking history between twins with RA and their unaffected co-twins. METHODS: Interview questionnaires on smoking history were administered to 79 identical (monozygotic [MZ]) and 71 same sex nonidentical (dizygotic, [DZ]) twin pairs who were discordant for RA, recruited from the Arthritis and Rheumatism Council Twin Study. Results were expressed as odds ratios (OR) with 95% confidence intervals (95% CI). RESULTS: Although most twin pairs were concordant for smoking history, there was a strong association between ever smoking and RA in the MZ pairs (OR 12.0, 95% CI 1.78 513), with a similar trend observed in the DZ pairs (OR 2.5, 95% CI 0.92-7.87). CONCLUSION: The discordance in cigarette smoking history for individuals who are at presumed identical genetic risk for RA supports other data suggesting the role of smoking in disease susceptibility. PMID- 8639171 TI - Bone mineralization and bone mineral metabolism in children with juvenile rheumatoid arthritis. AB - OBJECTIVE: To identify mechanisms of the osteopenia associated with juvenile rheumatoid arthritis (JRA) by determining parameters of bone mineralization, and bone mineral content and density (BMC and BMD), in children with JRA. METHODS: BMC and BMD were measured by dual x-ray absorptiometry in 41 children with JRA and 62 healthy children. Serum samples were analyzed for concentrations of minerals, vitamin D, parathyroid hormone, osteocalcin, bone-specific alkaline phosphatase (BAP), procollagen I carboxy-terminal propeptide, and tartrate resistant acid phosphatase (TRAP), and urinary excretion of deoxypyridinoline crosslinks and calcium. RESULTS: BMD was decreased in all sites in JRA patients. BMD, corrected for age, height, weight, and bone area, was decreased at cortical bone sites (1/3 radius, upper and lower extremities, and whole body). Low concentrations of osteocalcin and BAP suggested reduced bone formation, and low TRAP levels suggested decreased resorption. Clinical scales of disease severity were negatively correlated with measures of bone mass. Laboratory markers of disease severity were highly correlated with decreases in markers of bone formation, but not with those of resorption. Although laboratory findings were similar for children with oligoarticular and polyarticular disease, differences in bone mass were greater in children with polyarticular disease. CONCLUSION: These data suggest an association between decreased bone mineralization in JRA and low bone formation that is related to disease severity. Efforts to stimulate bone formation, therefore, need to be considered clinically in prepubertal children with active JRA. PMID- 8639170 TI - Detection of Borrelia burgdorferi by DNA amplification in synovial tissue samples from patients with Lyme arthritis. AB - OBJECTIVE: To compare the detection rates of chromosomal flagellin gene from Borrelia burgdorferi in synovial tissue (ST) and synovial fluid (SF) using polymerase chain reaction (PCR) techniques. METHODS: B burgdorferi DNA was sought in SF and ST from 12 consecutive patients with Lyme arthritis and from 29 patients with noninfectious diseases (controls). RESULTS: No DNA amplification was observed in samples obtained from the 29 control patients, whereas B burgdorferi DNA was detected in all ST and/or SF samples from the 12 patients with Lyme arthritis. Results from 1 ST sample were not interpretable because of PCR inhibitors. Among the 11 remaining patients, 10 had positive ST samples, whereas only 4 had positive SF samples (P < 0.05). CONCLUSION: These data suggest that detection of chromosomal B burgdorferi DNA may be more efficient in ST than SF. PMID- 8639172 TI - Anti-endothelial cell IgG antibodies from patients with Wegener's granulomatosis bind to human endothelial cells in vitro and induce adhesion molecule expression and cytokine secretion. AB - OBJECTIVE: To elucidate the role of anti-endothelial cell antibodies (AECA) in vascular inflammation in patients with Wegener's granulomatosis (WG). METHODS: IgG fractions from 3 AECA-positive WG patients, IgG from 3 AECA-negative WG patients, and IgG from healthy donors were tested for their ability to: a) bind to endothelial cells and to display complement-dependent or antibody-dependent cellular cytotoxicity, b) modulate cell membrane expression of adhesion molecules, as evaluated by cytofluorometry and by immunoenzymatic assay, and c) induce the secretion of interleukin-1 beta (IL-1 beta), IL-6, IL-8, and monocyte chemotactic protein 1 (MCP-1). RESULTS: We found that AECA IgG from WG patients do not display any significant cytotoxicity but are able to up-regulate the expression of E-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and to induce the secretion of IL-1 beta, IL-6, IL-8, and MCP 1. CONCLUSION: AECA in patients with WG could play a potential pathogenetic role by activating endothelial cells, and thus facilitating leukocyte recruitment and adhesion to endothelial surfaces, rather than by displaying a cytotoxic activity. PMID- 8639173 TI - Mixed monoclonal cryoglobulinemia and monoclonal rheumatoid factor cross-reactive idiotypes as predictive factors for the development of lymphoma in primary Sjogren's syndrome. AB - OBJECTIVE: To prospectively investigate whether mixed monoclonal cryoglobulinemia (MMC) and monoclonal rheumatoid factor (mRF)-associated cross-reactive idiotypes (CRI) serve as predictive factors for the development of lymphoma in patients with primary Sjogren's syndrome (SS). METHODS: One hundred three consecutive patients with primary SS were evaluated from 1986 to 1991. In all patients, the amount of cryoglobulin was measured by ultraviolet absorption at 280 nm and 260 nm. The type of cryoglobulinemia was identified by agarose gel electrophoresis, combined with immunofixation. Sera from all patients were evaluated by enzyme linked immunosorbent assay, using the corresponding monoclonal or polyclonal antibodies, for the presence of immunoglobulins bearing the idiotypes 17109 (V kappa IIIb associated), G-6 (VH1 associated), and 3rd SS (a rabbit polyclonal antibody raised against the Fab fragment of an IgM kappa mRF from a patient with primary SS). Data analysis was performed by logistic regression. RESULTS: Eighteen of the patients with primary SS (17.4%) had MMC during the first evaluation. There was a statistically significant correlation between the presence of MMC and a higher prevalence of autoantibodies to Ro/SS-A and La/SS-B, as well as extraglandular manifestations. During a 5-year period, 7 patients developed lymphoma. Six of the 7 (86%) had MMC before the appearance of lymphoma, compared with 12 of 96 (12.4%) of the remainder (r = 0.421, P < 0.0009). Patients who developed lymphoma had higher amounts of cryoglobulin than those who did not (mean +/- SD 53.4 +/- 44.7 mg/dl versus 26.8 +/- 20.6 mg/dl). CRIs 17109 and G-6 were also correlated with lymphoma development (r = 0.321, P < 0.006 and r = 0.22, P < 0.03, respectively). For both CRIs, this correlation was dependent on the presence of MMC, since a stepwise multiple comparison analysis revealed that their individual significance was abolished when their correlation with lymphoma in association with MMC was assessed. CONCLUSION: The determination of MMC can be used as a laboratory predictive factor for lymphoma development in primary SS. CRIs 17109 and G-6 may also be used to predict lymphoma development, especially when the monoclonal component is absent. PMID- 8639174 TI - Increased expression of human thioredoxin/adult T cell leukemia-derived factor in Sjogren's syndrome. AB - OBJECTIVE: To determine the involvement of human thioredoxin/adult T cell leukemia-derived factor TRX/ADF) in Sjogren's syndrome (SS) and the correlation with Epstein-Barr virus (EBV). METHODS: Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and the presence of EBV, using 6 normal tissues and 23 surgical specimens. The kinetics of expression of TRX/ADF induced by EBV was examined in vitro with peripheral blood B cells from EBV-seronegative donors. RESULTS: Marked expression of TRX/ADF was found in the infiltrating B cells and the epithelial cells of salivary gland tissues from patients with SS (11 of 12 cases), but not in those from patients with other salivary gland inflammatory conditions (0 of 11 cases) or those of normal individuals (0 of 6 cases). In immunohistologic analyses, a striking topographic correlation between TRX/ADF and EBV was found. The coexistence of TRX/ADF messenger RNA and EBV DNA was detected by polymerase chain reaction (r = 0.75, P < 0.01). Peripheral blood B cells from EBV-seronegative donors showed de novo synthesis of TRX/ADF following in vitro infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRX/ADF was not detected in non-EBV-infected cells. Tumors in SCID mice reconstituted with mononuclear cells of salivary glands from SS patients, which were composed of human B cells carrying EBV DNA, were positive for TRX/ADF. CONCLUSION: These findings suggest that TRX/ADF expression closely reflects the intracellular event of EBV reactivation in SS. This is also the first report to show the ectopic in vivo expression of TRX/ADF in human autoimmune disease. PMID- 8639175 TI - Aberrant expression pattern of the SS-B/La antigen in the labial salivary glands of patients with Sjogren's syndrome. AB - OBJECTIVE: Salivary glands of patients with Sjogren's syndrome (SS) have been shown to be a site of anti-SS-B/La antibody production. The present study investigated differences in the localization of the SS-B/La antigen in labial salivary gland (LSG) tissue between SS and non-SS patients, which may explain the local antigen-driven anti-SS-B/La response. METHODS: Distribution of SS-B/La was studied immunohistologically in the LSG biopsy samples of 9 SS patients, 10 non SS patients, and in normal tissues obtained at autopsy within 2 hours after death, using a mouse monoclonal antibody directed to SS-B/La. In 3 SS and 3 non SS patients, LSGs were also studied with affinity-purified biotinylated human antibodies directed against SS-B/La. RESULTS: In the non-SS patients, SS-B/La was primarily observed in the nucleoli of acinic cells of the LSGs. Patients with either primary SS or secondary SS showed an accumulation of SS-B/La in the nucleoplasm of acinic cells. In the SS patients, SS-B/La was also detected in the cytoplasm as a diffuse or perinuclear staining. Sometimes, SS-B/La was found along the membrane of acinic cells as well. This aberrant nuclear and cytoplasmic distribution of SS-B/La in SS patients correlated well with abnormalities in the composition of the plasma cell population in the LSGs, but not with a lymphocytic focus score > 1. CONCLUSION: The accumulation and redistribution of SS-B/La in the LSGs may play an important role in the local antigen-driven anti-SS-B/La response in SS, and can also be used to improve the diagnostic possibilities of the LSG biopsy. PMID- 8639176 TI - Elevated serum level of soluble HLA class I antigens in patients with systemic lupus erythematosus. AB - OBJECTIVE: To examine the clinical significance of serum soluble HLA class I antigens (sHLA class I) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). METHODS: Serum levels of sHLA class I were measured by enzyme-linked immunosorbent assay, using a monoclonal antibody against monomorphic determinant of HLA class I (W6/32) and an enzyme-labeled polyclonal antibody to human beta 2-microglobulin. RESULTS: The serum sHLA class I concentration was 1.85 +/- 1.15 micrograms/ml (mean +/- SD) in 27 patients with SLE (P < 0.0001 versus normal controls, P = 0.0001 versus RA), 0.61 +/- 0.34 micrograms/ml in 16 patients with RA (P = 0.02 versus normal controls), and 0.41 +/= 0.20 micrograms/ml in normal controls. The HLA class I levels were significantly correlated with the SLE Disease Activity Index (r = 0.62, P = 0.0004) and with a reduction of CH50 levels (r = -0.60, P = 0.0007). A longitudinal analysis of patients with SLE indicated that serum sHLA class I levels fluctuated in conjunction with other disease activity markers. CONCLUSION: Serum sHLA class I may be useful as a disease activity marker of SLE. The mechanism of secretion and the physiologic role of sHLA class I require further study. PMID- 8639177 TI - Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra. AB - OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated with antibodies against murine TNF alpha and IL-1 alpha/beta at different time points of the disease. IL-1 receptor antagonist (IL-1Ra) was administered using Alzet osmotic minipumps. The effect of anticytokine treatment was monitored by visual scoring. Histology and cytokine reverse transcription polymerase chain reaction (RT-PCR) analyses were performed at the end of the treatment period. RESULTS: Anti-TNF alpha treatment showed efficacy shortly after onset of the disease, but had little effect on fully established CIA. Histologic analysis after early treatment revealed that anti-TNF alpha significantly reduced joint pathology, as determined by infiltration of inflammatory cells and cartilage damage. Anti-IL-1 alpha/beta treatment ameliorated both early and full-blown CIA. This clear suppression of established arthritis was confirmed by administration of high doses of IL-1Ra. Dose-response experiments showed that a continuous supply of 1 mg/day was needed for optimal suppression. Histologic analysis showed markedly reduced cartilage destruction both in the knee and the ankle joints. Autoradiography demonstrated full recovery of chondrocyte synthetic function of articular cartilage. In addition, we found that the IL-1 beta isoform plays a dominant role in established CIA. Profound suppression of CIA was observed with anti-IL-1 beta, although elimination of both IL-1 alpha and IL-1 beta still gave better protection. Analysis of messenger RNA with RT-PCR revealed that IL-1 beta was highly upregulated in synovium and cartilage at late stages of CIA, whereas anti-IL-1 beta treatment markedly reduced IL-1 beta message in the synovium. CONCLUSION: The present study identified different TNF alpha/IL-1 dependencies in various stages of CIA and revealed that blocking of TNF alpha does not necessarily eliminate IL-1. Continuous, high doses of IL-1Ra are needed to block CIA. PMID- 8639179 TI - Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo. AB - OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints. PMID- 8639178 TI - Cellular adhesion molecules in rat adjuvant arthritis. AB - OBJECTIVE: To examine adhesion molecule expression during the progression of inflammation in a rheumatoid arthritis model of adjuvant-induced arthritis (AIA) in rats. METHODS: Immunohistochemical analysis was used to determine the distribution of the following adhesion molecules: lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), Mac-1 and p150/95 (CD11bc/CD18), intercellular adhesion molecule 1 (ICAM-1), and CD44 in tissue sections from the ankle joints of rats with AIA. Control animals and those with AIA were killed at intervals over a 54-day period after injection with mineral oil and Mycobacterium butyricum, respectively. RESULTS: CD44 and LFA-1 were expressed on lymphocytes, macrophages, and synovial (ST) lining cells. CD44 expression on macrophages was found to be increased compared with control animals by day 18, and was significantly increased by day 41. CD44 expression on lymphocytes significantly increased earlier, on days 11-18. Increased LFA-1 expression on macrophages occurred late, on day 41. LFA-1 expression on lymphocytes was significantly increased on days 25, 47, and 54. ST lining cells exhibited two distinct periods of increased expression, one early, on days 11-25 and one later, on days 41-54. CD11b/c was expressed on macrophages and ST lining cells, showing a significant increase on AIA rat ST lining cells compared with control animals on day 4. No differences in ICAM-1 expression on endothelial cells between rats with AIA and controls were found on any of the days examined. CONCLUSION: CD44 expression is up-regulated on macrophages and lymphocytes during the early development of AIA, while LFA-1 expression is up-regulated later in the development of AIA. The up regulation of CD44 and LFA-1 at different times in the development of AIA suggests an important role for these adhesion molecules in establishing and sustaining an inflammatory response in the AIA joint. PMID- 8639180 TI - Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-10 and interleukin-4. AB - OBJECTIVE: Inflammation-induced articular cartilage degradation is a major problem in rheumatoid arthritis (RA). Type 1 T cell activity (characterized by interferon-gamma/interleukin-2 [IL-2] production), and consequently, the production of the proinflammatory cytokines IL-1 and tumor necrosis factor alpha (TNF alpha), have been reported to play a major role in cartilage damage. IL-10 and IL-4, both produced by type 2 T cells, are cytokines with the capacity to down-regulate proinflammatory responses. The present study was undertaken to investigate the way in which these cytokines affect activated mononuclear cells (MNC) of RA patients in relation to human articular cartilage degradation in vitro. METHODS: MNC from synovial fluid and peripheral blood of RA patients were stimulated with bacterial antigen and treated with IL-10 and/or IL-4. Bacterial antigen is known to activate type 1 T cells and to induce proinflammatory IL 1/TNF alpha-dependent cartilage damage. Cytokine production and effects of conditioned media, as well as effects of IL-10 and IL-4 on proteoglycan (PG) turnover (as a measure for cartilage damage), were determined. RESULTS: IL-10 and IL-4 inhibited proinflammatory cytokine production of stimulated RA MNC and completely reversed inhibition of cartilage PG synthesis induced by these stimulated RA MNC. IL-10 was more potent than IL-4 in this respect, and the combination of IL-10 and IL-4 had an additive effect. In addition, IL-10 directly stimulated cartilage PG synthesis. CONCLUSION: IL-10 reverses the cartilage degradation induced by antigen-stimulated MNC, and IL-4 has an additive effect on this process. Furthermore, IL-10 has a direct stimulatory effect on PG synthesis, and IL-4, as a growth factor for type 2 T cells, can reduce the ratio of type 1 to type 2 T cell activity. These results provide evidence in favor of the use of a combination of the two cytokines in the treatment of RA. PMID- 8639181 TI - Accelerated generation of CD14+ monocyte-lineage cells from the bone marrow of rheumatoid arthritis patients. AB - OBJECTIVE: To examine the capacity of bone marrow progenitor cells to generate CD14+ cells, in order to assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA). METHODS: CD14- cells purified from bone marrow specimens of 11 patients with active RA and 8 control patients (osteoarthritis or trauma) were cultured in the presence or absence of granulocyte-macrophage colony stimulating factor (GM-CSF; 100 pg/ml). After incubation for various lengths of time, the cells were analyzed by flow cytometry for expression of CD14 and HLA DR. RESULTS: The spontaneous generation of CD14+ cells from bone marrow CD14- progenitor cells was accelerated in RA patients compared with control patients. Moreover, the expression of HLA-DR on the bone marrow-derived CD14+ cells was also accelerated in RA patients compared with controls. GM-CSF significantly enhanced the generation of CD14+ cells, as well as the expression of HLA-DR, on CD14+ cells of control patients, but not those of RA patients. GM-CSF levels in the culture supernatants of bone marrow CD14- cells were not significantly different between RA patients and control patients (undetectable in most cases). CONCLUSION: These observations strongly support the hypothesis that the accelerated generation of CD14+ cells from bone marrow progenitor cells and the accelerated maturation of such CD14+ cells into HLA-DR+ cells play an important role in the pathogenesis of RA. Moreover, the data suggest a functional alteration of RA bone marrow CD14- cells in their responsiveness to GM-CSF. PMID- 8639182 TI - Enrichment of differentiated CD45RBdim,CD27- memory T cells in the peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis. AB - OBJECTIVE: To delineate in greater detail the phenotype of T cells that reside in the synovial tissue (ST) and synovial fluid (SF) of patients with rheumatoid arthritis (RA), in order to determine their precise differentiation status, and to determine whether the accumulation of these specific T cell subsets in these synovial compartments could be related to their capacity for transendothelial migration. METHODS: Lymphocytes from normal subjects or from the peripheral blood (PB), ST, and/or SF of RA patients were phenotypically analyzed by flow cytometry. Normal PB CD4+ T cells were also characterized using an in vitro assay of transendothelial migration. RESULTS: ST and SF were found to be enriched with memory (CD45RA-,CD45RO+,CD11abright,CD44bright and activated (CD69+) T cells. Moreover, ST and SF cells from RA patients were enriched in differentiated CD4+,CD45RBdim,CD27- T cells, a subset of mature memory T cells that develops after prolonged antigenic stimulation. In addition, PB of some RA patients contained an increased number of CD4+,CD45RBdim,CD27- T cells. The CD4+,CD11abright,CD44bright memory T cells, which included the CD45RBdim,CD27- more mature memory cells, exhibited an enhanced capacity for transendothelial migration that is likely to contribute to their enrichment in the rheumatoid synovium. CONCLUSION: RA patients manifest an increased number of mature memory T cells in the SF and ST, and some also have an increased number of these cells in PB that is likely to reflect chronic antigenic stimulation. The enrichment of these cells in the SF and ST reflects, in part, an enhanced capacity to migrate from the vascular space into inflamed tissue. PMID- 8639183 TI - An epitope on Ki antigen recognized by autoantibodies from lupus patients shows homology with the SV40 large T antigen nuclear localization signal. AB - OBJECTIVE: Epitopes on Ki antigen were analyzed using synthetic peptides, including KILT, a 16-mer peptide with an amino acid sequence homologous to the SV40 large T antigen nuclear localization signal (SV40 T NLS). METHODS: In addition to KILT, 4 synthetic peptides, all potential epitopes on Ki antigen according to computer analysis, were prepared and tested for reactivity with 49 anti-Ki-positive lupus sera by enzyme-linked immunosorbent assay. RESULTS: Eighteen sera reacted with KILT, but not with other peptides. The reaction of anit-Ki sera with KILT was specifically inhibited by recombinant Ki antigen. Eight of 49 anti-Ki sera reacted with a 7-mer synthetic peptide of SV40 T NLS, and the reaction was specifically inhibited by KILT. CONCLUSION: The 16-mer Ki peptide containing the sequence homologous to the SV40 T NLS is one of the antigenic epitopes recognized by anti-Ki antibodies in lupus sera. PMID- 8639184 TI - Detection of anticentromere antibodies using recombinant human CENP-A protein. AB - OBJECTIVE: To evaluate CENP-A reactivity with anticentromere antibodies (ACA) using recombinant protein (rCENP-A). METHODS: Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the ACA+ sera studied (n = 38), 95% were positive when tested against the rCENP-A in the ELISA system. Of the ACA- sera (n = 100), only 2% gave false-positive results in the assay. There was good correlation between the recombinant and bona fide antigens in assaying for ACA reactivity. CONCLUSION: CENP-A is a significant ACA target. The availability of the rCENP-A assay is a valuable adjunct to the previously described rCENP-B assay in analyses of the clinical significance of ACA. PMID- 8639185 TI - Strong association of dermatomyositis-specific Mi-2 autoantibodies with a tryptophan at position 9 of the HLA-DR beta chain. AB - OBJECTIVE: To characterize the clinical and immunogenetic features of patients with Mi-2 autoantibodies. METHODS: Eighteen adult white patients with Mi-2 antibodies were clinically characterized and compared with 41 Mi-2-negative dermatomyositis (DM) patients. HLA class I and class II typing for DRB alleles was done by microcytotoxicity assay and for DQA and DQB alleles by polymerase chain reaction-based oligotyping. RESULTS: Seventeen of the 18 Mi-2-positive patients had DM. Symptoms of scleroderma, lung involvement, and arthritis were less common in this group than in the Mi-2-negative DM patients; the V-sign rash and nailfold involvement were found more frequently. Mi-2 antibodies were strongly associated with HLA-DR7 (88% versus 24% in healthy controls), HLA DQA1*0201 (86% versus 23%), and DR7 "homozygosity" (31% versus 0%). A tryptophan residue at position 9 of the HLA-DR beta chain was present in all Mi-2-positive patients (100% versus 62%; homozygous in 81% versus 15%). CONCLUSION: Our results reemphasize the specificity of Mi-2 antibodies for DM, and extend previous reports that Mi-2 antibody production is associated with certain HLA class II antigens. We propose beta 9-Trp as a candidate epitope on the HLA-DR beta chain as a prerequisite for this type of autoimmune response. PMID- 8639187 TI - Prevalence of human parvovirus B19 infection in children with Henoch-Schonlein purpura. PMID- 8639186 TI - Chronic intestinal pseudoobstruction associated with autoantibodies against proliferating cell nuclear antigen. AB - Autoantibodies against proliferating cell nuclear antigen (PCNA) have been detected almost exclusively in sera from patients with systemic lupus erythematosus (SLE). Because of the extremely low prevalence of anti-PCNA, however, it has been difficult to establish its disease specificity and clinical associations. We present 2 cases in which patients with anti-PCNA developed severe gastrointestinal dysfunction that led to chronic intestinal pseudoobstruction. Neither patient had manifestations suggestive of SLE. Although sclerodermatous skin changes were minimal or absent in these patients, physical and radiographic findings indicated that systemic sclerosis was a likely cause of the gastrointestinal dysfunction. The similarity of the clinical features in these patients implies that the unusual detection of this unique autoantibody may not be a coincidence. PMID- 8639188 TI - Mycobacterium kansasii septic arthritis in a patient with acquired immunodeficiency syndrome. PMID- 8639189 TI - Tumor necrosis factor receptor p75 correlates better than p55 with disease activity in juvenile rheumatoid arthritis: comment on the article by Mangge et al. PMID- 8639190 TI - Serum pro-matrix metalloproteinase 3 in rheumatoid arthritis: a reflection of local or systemic inflammation? PMID- 8639192 TI - "Fast tracking" patients in an urban pediatric emergency department. AB - Modern health care reform emphasizes efficient resource and facility management and the need to develop strategies to direct patients with lower-acuity concerns away from the relatively cost-inefficient full-service pediatric emergency department (ED). This study examined a pediatric fast track system for triage accuracy and turnaround times. Egleston Children's Hospital is a regional, urban, tertiary-care academic center which is a major teaching affiliate of Emory University School of Medicine. The pediatric ED has an annual census of more than 30,000 patient encounters. During the 9-month period from December 1993 through August 1994, 2,243 lower-acuity patients were evaluated in the fast track section of the ED. Patients assigned to the fast track system maintained a quicker turnaround time than the aggregate of all patients seen in the ED (107 [95% CI 0, 245] minutes versus 149 [95% CI 0, 341] minutes, P < .01). Their total turnaround time was also less than that for patients with similar acuity levels seen during the hours that the fast track system was not in operation (120 [95% CI 0, 300 minutes], P < .01). Only 63 of the 2,243 (2.8%) patients assigned to fast track were found to have higher acuity levels than suspected at initial triage. In all cases they were appropriately cared for in the fast track area. The fast track system appears to be an effective method by which an urban pediatric ED can efficiently maintain patient flow in light of limited resources, space constraints, limited manpower, and an increasing census. PMID- 8639191 TI - A meta-analysis of blunt cardiac trauma: ending myocardial confusion. AB - The purpose of this study was to use a meta-analysis of the current literature to identify which patients with blunt cardiac trauma develop complications. All studies on myocardial contusion since 1967 were reviewed. Three separate meta analyses were performed: one with only prospective studies, one with only retrospective studies, and one with all studies combined. Data on electrocardiogram (ECG), creatine phosphokinase-MB isoenzyme (CPK-MB), radionuclide scans, and echocardiogram were analyzed using the Mantel-Haenszel procedure. Results of the three meta-analyses were similar. Abnormal ECG and abnormal CPK-MB were found to correlate directly with complications requiring treatment. Conversely, normal ECG and CPK-MB correlated with the lack of clinically significant complications. Radionuclide scans did not correlate with complications. The results for echocardiogram were not congruent between the prospective and retrospective studies. The data support the use of ECG and CPK-MB in the diagnosis of clinically significant myocardial contusion. Radionuclide scanning is not useful in the evaluation of patients with blunt cardiac trauma. Further studies need to define the role of echocardiography. PMID- 8639193 TI - Emergency department uses of physician assistants and nurse practitioners: a national survey. AB - A study was undertaken to determine the extent to which physician assistants (PAs) and/or nurse practitioners (NPs) are a source of health care delivery in emergency departments (EDs) in the United States. The National Hospital Ambulatory Medical Survey (NHAMCS) uses a multistage probability sample that examines patient visits within EDs. The sample included 437 hospitals with EDs. Visits were mostly from self-referred patients to EDs within nonfederal, short stay hospitals, or general hospitals. Analysis of NHAMCS data found that a PA and/or NP was seen for 3.5 million ED visits in 1992. Remarkably little difference in gender, reason for visit, diagnosis, and medication prescribed was found between PA/NP visits and visits to all providers. This was the first study that systematically identified the extent of PA/NP-delivered ED services in the United States and compared it with physician services. Overall, PAs and NPs were found to be significant sources of health care service for hospital EDs. They are involved in care for almost 4% of all ED visits nationally and manage a wider range of conditions than has been previously reported. When types of visits are analyzed, including reasons for ED care, diagnosis, and treatment, it appears that visits associated with care by ED-based PA/NPs are similar to all ED visits, including those attended by emergency medicine physicians. More studies are needed to better understand the role of PAs and/or NPs in various ED settings. Recruitment and use of PAs and NPs may be a cost-effective strategy for improved delivery of emergency services. PMID- 8639194 TI - Pattern of basic life support ambulance use in an urban pediatric population. AB - To evaluate the pattern of use of basic life support (BLS) ambulances in a pediatric population, emergency medical service (EMS) and pediatric emergency department (PED) records from an urban hospital PED for all children transported to PED by ambulance during a 1-month study period were retrospectively reviewed. Excluded were: (1) advanced life support transport, (2) transport from other medical facility, (3) patients with chronic medical disability without acute decompensation, and (4) patients in police custody. BLS transport was considered inappropriate if: (1) no intervention by BLS technicians, (2) minimal to no intervention in the PED, and (3) discharge without prescription medication. Of 376 ambulance transports evaluated, 238 (63%) met entry criteria, and 105 (44%) transports met criteria for being inappropriate. The mean charge for appropriate transport was $240.68, and for inappropriate, $237.12 (P = .2). The total charge for inappropriate transports was $26,523.20. Patients on federal assistance had a significantly higher rate of inappropriate transport (51%) compared with patients who had commercial insurance (30%) and those who self paid (42%). Trauma was the most common cause for transport, 48% of which was inappropriate. It was concluded that inappropriate BLS transport of pediatric patients is common. This use is costly and may disrupt the delivery of EMS care to the remainder of the community. Efforts aimed at public education and providing alternative means of transport may significantly reduce charges and improve the delivery of EMS care. PMID- 8639195 TI - C-reactive protein for the evaluation of acute abdominal pain. AB - The diagnostic value of C-reactive protein (CRP) was established in 143 patients with acute abdominal pain, 67 men and 76 women with mean age of 48 +/- 20 years old. Clinical and laboratory variables were collected after the patients' arrival at the emergency department. The attending clinicians did not consider the CRP value during the study period and did not use it for their management. When patients were grouped by final disposition, which was according to severity, only CRP and leukocyte count were identified as significant quantitative variables by multivariate analysis. CRP can detect the serious conditions, ie, in 79% of the hospitalized group, although specificity was 64%, the total accuracy was 73%. When elevated CRP was combined with leukocytosis, the diagnostic value was much improved, with specificity of 89% and positive predictive value of 88%. The sensitivity was improved to 90% when elevated CRP or leukocytosis was used. It is thus concluded that CRP is a helpful quantitative variable for disposition decision-making in patients with acute abdominal pain. PMID- 8639196 TI - Development of computer-assisted patient control for use in the hospital setting during mass casualty incidents. AB - Hospital resolution of mass casualty incidents can have difficulties involving "command and control" and information management, ineffective use of triage classes, and missed diagnostic procedures, leading to lower quality of care. A computer system has been developed to supply continuously updated group and patient data. The system uses barcoded identifiers to represent patients, injuries, facilities, and locations, in order to minimize errors and make exchange of data possible. The system communicates with the permanent hospital information system. This article reports the use of this technology during several experiments and real incidents. Computer registration based on bar codes, despite the greater number of items entered, still showed 25% fewer inaccuracies when compared with handwritten medical charts. Extensive training was shown to be unnecessary. Paramedical personnel judged the automated procedures to be an improvement during the admission of 143 evacuated patients. PMID- 8639197 TI - Intramuscular prochlorperazine versus metoclopramide as single-agent therapy for the treatment of acute migraine headache. AB - To compare the efficacy of intramuscular prochlorperazine and metoclopramide in the short-term treatment of migraine headache in the emergency department 86 eligible adult patients with moderate to severe migraine headache were evaluated prospectively at a university-affiliated community hospital. After randomization, each subject received a 2-mL intramuscular injection of sterile saline, prochlorperazine (10 mg), or metoclopramide (10 mg). No other analgesics were administered during the 60-minute study period; patient assessment of relief was followed using visual analog scales. Reduction in median headache scores was significantly better among those treated with prochlorperazine (67%) compared to metoclopramide (34%) or placebo (16%). Similarly, symptoms of nausea and vomiting were significantly relieved in the prochlorperazine group (chi 2 = 17.1, P < .001). However, rescue analgesic therapy was necessary in the majority of patients treated with prochlorperazine (16/28) and metoclopramide (23/29) after the 60-minute study period. Although intramuscular prochlorperazine appears to provides more effective relief than metoclopramide, these results do not recommend either drug as single-agent therapy for acute migraine headache. PMID- 8639198 TI - Alcohol, the elderly, and motor vehicle crashes. AB - To document the prevalence of alcohol or drug use among elderly drivers admitted to a Level 1 trauma center after motor vehicle crashes, charts from 180 drivers age 60 years or older who were admitted to an urban Level 1 trauma center after motor vehicle crashes were retrospectively reviewed. Overall, 14% of the patients had a positive blood alcohol screen; among men, 21% had a positive screen. Only 55% of the patients were discharged to home. Only one patient ( < 1%) had a toxicology screen positive for another drug abuse. Alcohol/drug abuse counselling was offered to only one patient. These results suggest a relatively of high prevalence of alcohol use in elderly drivers involved in motor vehicle crashes, particularly men. However, abuse of other drugs was uncommon. Physicians treating intoxicated drivers should consider referral for alcohol counselling. PMID- 8639200 TI - Obstructive sleep apnea presenting as acute delirium. AB - Obstructive sleep apnea syndrome (OSA) has not been previously reported as a cause of acute delirium. A patient who presented to the emergency department with acute delirium according to DSM-IIIR criteria was noted to have an abnormal respiratory pattern with periods of apnea associated with oxygen desaturation. The observation that the patient had episodes of apnea while sleeping led to the suspicion that this patient had OSA, and formal polysomnography confirmed the diagnosis. Other causes for acute delirium were ruled out. The delirium resolved after the OSA was treated. PMID- 8639199 TI - Pulse oximeter analysis of peripheral cyanosis distal to an AV fistula. AB - Arteriovenous (AV) fistulas are routinely constructed for hemodialysis in patients with renal failure. The case of a hemodialysis patient with cyanosis of the hand distal to an AV fistula is described. Pulse oximeter analysis found a saturation of 72% in the cyanotic hand with a normal saturation of 97% in the opposing hand. Evaluation of 11 additional patients with AV fistulas and no cyanosis or symptoms found no difference in oxygen saturation between extremities. Pulse oximetry was found to be useful for evaluating the oxygenation status distal to an AV fistula, and in asymptomatic patients with an AV fistula, the oxygenation status distal to the fistula should be no different from that of the opposing extremity. PMID- 8639201 TI - Enteric fistulization of a common iliac artery aneurysm: an unusual cause of gastrointestinal hemorrhage and shock. AB - A 78-year-old man with a history of recent unexplained lower gastrointestinal bleeding presented to the emergency department with the acute onset of abdominal pain, tenesmus, and shock. Computed tomography of the abdomen showed a fistula between a common iliac artery aneurysm and the small intestine. Laparotomy demonstrated a saccular aneurysm of the common and proximal internal iliac arteries with fistulous communication to the distal ileum. Aneurysmectomy, arteriorrhaphy, and segmental ileal resection with primary anastomosis were successfully performed. This case illustrates a rare complication of an uncommon aneurysm, emphasizing the need for emergency physicians to consider complicated vascular disease in the evaluation of a patient with abdominal pain and shock. PMID- 8639202 TI - Acute confusional migraine and trauma-triggered migraine. AB - Migraine in children can present as a state of confusion or agitation with or without a history of migraine. These events can arise spontaneously or can be triggered by mild head trauma. Transient blindness and hemiplegia may accompany the confusional state. We present two cases of children with histories of confusion and agitation, one with multiple episodes after mild head trauma. The symptomatology, differential diagnosis, theories on pathogenesis, and natural history of confusional migraine are discussed. PMID- 8639203 TI - Tracheobronchial ruptures in children. AB - Tracheobronchial ruptures in children after blunt trauma are rare, with an incidence at our clinic of 0.17% of juvenile thoracic injuries in the years 1985 to 1994. The symptoms are variable, and these injuries frequently occur in conjunction with other thoracic injuries, leading to delays in diagnosis. Diagnosis, conservative and operative treatment, and late complications are discussed by presenting four different cases of children with lesions of the tracheobronchial tree after blunt trauma. PMID- 8639204 TI - Primary cardiac chondrosarcoma. AB - A case of primary cardiac chondrosarcoma in a 41-year-old woman who presented with cardiac tamponade and cardiac intracavitary obstruction is described. The tumor originated from the right atrium and invaded the adjacent right ventricular wall and interatrial septum. Primary cardiac chondrosarcoma is extremely rare, and its clinical, computed tomographic, echocardiographic, and magnetic resonance imaging findings are described. PMID- 8639205 TI - Femoral neck fractures in the elderly patient: a preventable injury. AB - There is growing evidence that many fractures of the femoral neck in the elderly occur spontaneously because of stresses imposed on osteoporotic bone, rather than because of the trauma of the fall. A case of a spontaneous femoral neck fracture (Garden type IV) in an elderly woman with osteoporosis is presented. Early detection and medical management of osteoporosis complemented with assistive technology could have prevented this common injury. PMID- 8639206 TI - Traumatic atlanto-occipital dislocation: a potentially survivable injury. AB - Although survival with traumatic atlanto-occipital dislocation (AOD) is rare, there have been reports of victims who have sustained this injury with good neurological outcome. Plain lateral cervical spine radiography is the initial diagnostic procedure but may miss subtle dislocations. Several methods for the interpretation of the normal atlanto-occipital alignment have been devised and are discussed. Computed tomography (CT) and magnetic resonance imaging (MRI) are valuable studies in the diagnosis and management of AOD. Halo immobilization and posterior spinal fusion are the preferred modes of treatment. Vascular injury may contribute to the neurological deficits seen with AOD and is potentially reversible. Three cases are reported, two with survival of 1 day, and one long term survivor with poor neurological outcome because of associated cerebral trauma. PMID- 8639207 TI - Pulmonary barotrauma as the cause of pneumoretropharynx in pulmonary lymphangioleiomyomatosis. AB - A 55-year-old white woman with pulmonary lymphangioleiomyomatosis (LAM) presented to the emergency department with odynophagia and subplatysmal emphysema after a paroxysm of coughing. Lateral neck films showed subcutaneous emphysema and a retropharyngeal air stripe. Chest radiographs showed neither pneumothorax nor pneumomediastinum. Patients with LAM frequently develop pulmonary barotrauma and pneumothoracies. This patient, however, had undergone prior bilateral talc pleuradesis as treatment for recurrent pneumothoracies and, thus, could not manifest this complication of barotrauma. This case illustrates the uncommon occurrence of superior dissection of air after pulmonary barotrauma. PMID- 8639208 TI - Adenosine-induced atrial fibrillation. PMID- 8639209 TI - Accessing and using the Internet's World Wide Web for emergency physicians. AB - This report describes the use of the internet's World Wide Web (WWW) for emergency physicians. WWW is a graphic means of presenting information over the internet. Because this graphic presentation is derived from a point-and-click method of navigating through information, emergency physicians with little or no computer background should be able to benefit. There are currently many resources available to emergency physicians on the WWW that can be viewed at no charge. It is essentially a worldwide library of information that is growing at a rapid rate. An algorithm is presented to assist emergency physicians in deciding how to best access the WWW, depending on the physician's current resources, and some background information required to gain access to WWW is described. PMID- 8639210 TI - Emergency medicine physicians saving time with ultrasound. AB - In an attempt to provide comprehensive and timely patient care, emergency physicians have begun to use ultrasonography to perform and interpret goal oriented examinations. Reducing time to diagnosis can potentially have a major impact on the treatment of patients with ruptured ectopic pregnancy, leaking aortic aneurysm, and cardiac tamponade, who require time-sensitive surgical intervention. A review of three cases reveals how ultrasonography performed by emergency physicians can rapidly provide valuable diagnostic information and expedite patient care in three different clinical scenarios. PMID- 8639211 TI - Cases in electrocardiography. PMID- 8639212 TI - Uncommon complications of odontogenic infections. PMID- 8639213 TI - A new approach to chronic pain in the ED. AB - Patients presenting to emergency departments (EDs) for primary management of chronic or recurrent nonmalignant pain conditions and their physicians frequently report dissatisfaction, in part because of the impressions created by a small percentage of such patients that frequently visit EDs requesting opioids. Treating such patients with opioids is contrary to many published guidelines, but refusing them increases dissatisfaction. Narcotic registers serve to label patients who are suspected of seeking drugs, thus creating anxiety and often distrust in health care professionals treating them. The four Calgary adult EDs have developed a system that will attempt to remove labels associated with some of these patients, insure communication between patients, their family doctors, and ED staff, and facilitate optimal care of the patients' real problems, be they difficult home management of pain, drug dependence or addiction, or other social issues. Emphasis will be shifted to home management and the family doctor's office. If successful, the system will minimize ED visits by frequent attendees seeking medication for pain control, and should also decrease overall expenditure to the health care system. PMID- 8639215 TI - ED security. PMID- 8639214 TI - Safer subclavian vein puncture. PMID- 8639216 TI - A tool for the emergency medicine evaluation of psychiatric patients. PMID- 8639217 TI - Self-limiting pulmonary edema with alveolar hemorrhage during diving in cold water. PMID- 8639218 TI - Spontaneous rupture of the spleen. PMID- 8639219 TI - Bonus/incentive programs to increase physician productivity in academic emergency medicine. PMID- 8639220 TI - Injuries by emus. PMID- 8639221 TI - Historical, physical, and laboratory characteristics of female ED patients with positive chlamydia and gonorrhea cultures. PMID- 8639222 TI - Questions remain about shocking asystole. PMID- 8639223 TI - Physician assistants in control: ED mayhem. PMID- 8639224 TI - Bicarbonate buffering of local anesthetics. PMID- 8639225 TI - Lipoxygenases in plants--their role in development and stress response. AB - Lipoxygenases catalyze the hydroperoxidation of polyunsaturated fatty acids and thus the first step in the synthesis of fatty acid metabolites in plants. Products of the LOX pathway have multiple functions as growth regulators, antimicrobial compounds, flavours and odours as well as signal molecules. Based on the effects of LOX products or on the correlation of increases in LOX protein and the onset of specific processes, a physiological function for LOXs has been proposed for growth and development and for the plant response to pathogen infection and wound stress. PMID- 8639226 TI - Structure elucidation of azotobactin 87, isolated from Azotobacter vinelandii ATCC 12837. AB - Chromopeptide siderophores (azotobactin 87-I and -II) were isolated from an iron deficient culture medium of Azotobacter vinelandii ATCC 12837 (= DSM 87). Their structures were elucidated by chemical degradation studies and spectroscopic methods, especially 2D-NMR-techniques. Total assignments of 1H-, 13C-, and 15N resonances based on 2D-HOHAHA-, 1H/13C-HMQC-, 1H/13C-HMBC-, 1H/15N-HMQC/TOCSY-, and 1H/15N-HMBC-experiments are given as well as sequential information derived from 1H/1H-NOESY-, 1H/13C-HMBC- and 1H/15N-HMBC-experiments. Both Az 87-I and Az 87-II consist of a tetracyclic chromophore-- (1S)8,9-dihydroxy-4-oxo-2,3,4,5 tetrahydro-1H,10cH-3a,5,10b- triazaacephenantrylene-1-carboxylic acid--and a decapeptide chain linked with the N-terminus to the carboxy group of the chromophore containing also modified, non-proteinogenic amino acids. The sequence L-Ser-D-Ser-L-Hse-Gly-D-threo-OHAsp-Hse-Hse-Hse-D-N5OH-N5-R- Hbu-Orn-L-Hse was determined for Az 87-I, while Az 87-II contains a C-terminal L-Hse-lactone instead. Iron is chelated by the catecholic group of the chromophore, the beta hydroxy aspartic acid, and the hydroxamate function formed by N5-hydroxyornithine and R-beta-hydroxybutyric acid. PMID- 8639227 TI - Trehalose protects yeast pyrophosphatase against structural and functional damage induced by guanidinium chloride. AB - Trehalose is accumulated at very high concentrations in yeasts when this organism is submitted to a stress condition. This report approaches the question on the protective effect of trehalose and its degradation product, glucose, against structural and functional damage promoted by guanidinium on yeast cytosolic pyrophosphatase. Here it is shown that both, 1 M trehalose or 2 M glucose, are able to attenuate at almost the same extent the conformational changes promoted by guanidinium chloride on the pyrophosphatase structure. On the other hand while 1 M trehalose increases 3.8 times the Ki (from 0.15 to 0.57 M) for guanidinium chloride inhibition of pyrophosphatase activity, 2 M glucose did not even duplicate this parameter (from 0.15 to 0.25 M). These data support evidences for a functional reason for the accumulation by yeasts of trehalose, and not other compound, during stress conditions. PMID- 8639228 TI - Radioadapted chicken embryo cells: challenge specificity and alterations in higher-order DNA structure. AB - Radioadapted chicken embryo cells (X-irradiation in ovo with 10 cGy at the 14th day of development with priming periods of 24 h) were treated in vitro by challenge doses of 14 different DNA- and/or chromatin-interactive agents, including X-rays. A decrease in the cellular damage, as measured by scheduled DNA synthesis, was only observed with X-irradiation. Sedimentation of nucleoids as well as viscosity of alkaline lysates from ethidium bromide (0.35-400 micrograms/ml)-, novobiocin (125-1800 micrograms/ml)-, and hyperthermia (30 min at 43 degrees and 45 degrees)-treated cells suggest a higher tendency of radioadapted cells to undergo positive DNA supercoiling. When DNA from adapted and non-adapted chicken embryo cells was used as substrate, neither its digestion by DNase I nor the inhibition of DNase I activity by various DNA-interactive agents was changed in primed cells. From the previous investigations as well as from the present results it is concluded that an increase of tightening of protein-DNA interactions within the nuclear matrix is a molecular determinant of the elevated radiation resistance in radioadapted chicken embryo cells. PMID- 8639229 TI - Comparison of the action of an organophosphorus insecticide and its metabolite on chloride and sulfate transport in erythrocyte membrane. AB - The effect of the organophosphorus insecticide methylparathion and its main metabolite methylparaoxon on chloride and sulfate equilibrium exchange in pig erythrocytes was investigated using an isotope labelling technique. Efflux of both radioactive isotopes with time followed a single exponential. Methylparathion and methylparaoxon inhibited the chloride equilibrium exchange in erythrocyte ghosts in a dose- and time-dependent manner. There was no difference between effects evoked by these two compounds. Methylparathion and methylparaoxon inhibited sulfate efflux from resealed ghosts. The effect was also dose- and time dependent. Again, there was no difference between the action of both agents. Dixon analysis revealed a non-competitive character of the inhibition of the exchange of both anions with apparent Ki values 183 and 184 microM for methylparathion and methylparaoxon, respectively in the case of chloride transport; for sulfate exchange these values were 675 and 648 microM. It was suggested that structural similarity between the parent agent and its metabolite accounts for their identical effects. Methylparathion and methylparaoxon might inhibit the anion exchange indirectly by changing the fluidity of the erythrocyte membrane or directly by binding to the band 3 protein and evoking conformational changes that lead to the inhibition of the anion transport. The insecticides, due to their ability to phosphorylate, might also disturb some regulation processes in the band 3 protein and affect anion transport in this way. PMID- 8639230 TI - A cytotoxic principle of Tamarindus indica, di-n-butyl malate and the structure activity relationship of its analogues. AB - Bioassay-guided fractionation of the methanolic extract of Tamarindus indica fruits led to the isolation of L-(-)-di-n-butyl malate which exhibited a pronounced cytotoxic activity against sea urchin embryo cells. In order to study structure-activity relationships, close-structure relatives of di-n-butyl malate were synthesized using D-(+)- and L-(-)-malic acid as starting materials, and their cytotoxic activities were examined for the sea urchin embryo assay. L-(-) Di-n-pentyl malate was the most effective inhibitor to the development of the fertilized eggs. Significant inhibitory activity was not seen in the esters of D (-)-isomer. PMID- 8639231 TI - Interaction of penicillin G with the human erythrocyte membrane and models. AB - Penicillin G (PEN) is a widely used antibiotic whose mechanism of action is related to the interference with the synthesis of bacteria cell wall. In order to evaluate its perturbing effect upon human cell membranes PEN was made to interact with human erythrocytes, isolated resealed human erythrocyte membranes and molecular models. The latter were multibilayers of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) as well as DMPC large unilamellar vesicles. These studies were performed by scanning electron microscopy, fluorescence spectroscopy and X-ray diffraction methods. The observed results coincide in that PEN did not exert any significant effect upon the structures of the red cell membrane neither on its molecular models. This is in agreement with its reported lack of major toxicity and hematological reactions. PMID- 8639232 TI - Optimization of fast-fluorescence in situ hybridization with repetitive alpha satellite probes. AB - A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive DNA probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of alpha-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. As examples the systematic optimization for the two probes D12Z2 (major binding site on the centromere of chromosome 12) and D8Z2 (major binding site on the centromere of chromosome 8) are shown. The optimal hybridization conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 degrees C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maximum Smax of the corresponding FISH-spots. PMID- 8639234 TI - Electronic publishing. PMID- 8639233 TI - The relationship between abutment taper and resistance of cemented crowns to dynamic loading. AB - This study investigated the relationship between abutment total occlusal convergence angle (taper) and the resistance of cemented crowns subjected to dynamic loading. Crown and abutment analogs were placed using zinc-oxide-eugenol, zinc-phosphate, glass-ionomer, or resin composite cement. Total occlusal convergence angles of 2.5, 5, 10, 15, 20, 30, and 40 degrees were used. Dynamic stresses were applied to the luted components until the bond failed or the components reached 10(6) load cycles. The data were analyzed using the staircase technique. The relationship between convergence and resistance was approximately linear for all the cements tested. Crowns luted with resin composite cement were more resistant to dynamic lateral loading than those placed using glass-ionomer or zinc-phosphate cements. Crowns luted with zinc-oxide-eugenol cement presented the least resistance to cyclic lateral stresses. PMID- 8639236 TI - Tissue conditioners protected by a poly(methyl methacrylate) coating. AB - Interim soft denture liners or conditioners may be valuable therapeutic materials. The life of these liners varies, but it can be extended by the use of a poly(methyl methacrylate) coating material (Monopoly). This study evaluated the ability of Monopoly to prevent water absorption and plasticizer loss from an underlying tissue conditioner (Visco-gel). Disk specimens of Visco-gel coated with Monopoly were immersed in water that was sampled at 24 hours, 1 week, 2 weeks, and 1 month. Uncoated Visco-gel samples acted as controls. Water absorption was determined gravimetrically at each time interval and decanted water was subjected to separation by reversed phase high-performance liquid chromatography for component identification. All samples suffered significant initial weight loss followed by a trend toward weight gain in the uncoated control group, probably because of water absorption. The Monopoly coating appeared to reduce this effect. Plasticizer loss from the tissue conditioner was below quantifiable limits by high-performance liquid chromatography, but methyl methacrylate monomer loss from the Monopoly coating was demonstrated. Some clinical implications are discussed. PMID- 8639235 TI - Intermittent loading of teeth restored using prefabricated carbon fiber posts. AB - This in vivo study evaluated the fracture resistance of bovine teeth with prefabricated carbon fiber posts. Fourteen bovine teeth having similar lengths and dimensions were mounted in an acrylic resin block having a simulated periodontal ligament. The post space was prepared using two calibrated drills that provided an 8.5-mm post length. The prefabricated carbon fiber post was luted with a resin luting agent, and the core was made using the system's autopolymerizing resin core material. A crown was luted to each prepared tooth. Each test specimen was intermittently loaded (250 N) at an angulation of 45 degrees to the long axis of the tooth at a frequency of 2 loads per second. Four of the roots had an incomplete longitudinal fracture after loading. The results of this study were compared to a previous study by the authors that had been conducted under similar conditions with prefabricated parallel-sided posts (Para Post) and tapered, individually cast posts. The failure rates of the two types of posts from the previous study were significantly higher (Logrank test; P<.02) than those of the carbon fiber posts. PMID- 8639237 TI - Behavior of calcium phosphate coatings with different chemistries in bone. AB - Calcium phosphate ceramic coatings with a hydroxyapatite chemistry applied on the surface of dental implants eliminate the need for initial mechanical retention and decrease the time necessary for bonding the implants to the bone. Hydroxyapatite-coated implants retrieved from patients were found to be compatible and to have bonded strongly to the bone, but the coatings showed thinning because of partial or total loss of coating material. This study compared the behavior in bone of newly developed fluorapatite and heat-treated hydroxyapatite coatings, with the clinically used hydroxyapatite coatings used as controls in experimental studies in dogs. The biologic responses to fluorapatite and heat-treated hydroxyapatite coatings were the same as those to hydroxyapatite coatings, and bone condensation around all coatings was histologically evident. However, the coating thickness of the fluorapatite and heat-treated hydroxyapatite coatings remained stable with only minor changes during the observation period of 24 months. PMID- 8639238 TI - The effect of implant/abutment hexagonal misfit on screw joint stability. AB - A series of 10 incrementally larger, machined ASTM Grade 23 titanium non segmented (UCLA type) abutments was loaded off axis with 133 N and cycled at 1150 vertical strokes per minute and 28 counterclockwise rotations per minute to determine screw joint stability. Abutment internal hexagonals ranged from 0.1065 to 0.1110 inches. External hexagonal mean flat-to-flat width was 2.684 mm. Rotational misfit between international and external hexagonals ranged from 1.94 degrees for the smallest abutment to 14.87 degrees for the largest. Screw joint failure ranged from 134,000 to 9.3 million cycles. The tightest matrix/patrix hexagonal screw joint failed at a mean of 6.7 million cycles. This study indicated that there was a direct correlation between hexagonal misfit and screw joint loosening. A rotational misfit of under 2 degrees provided the most stable and predictable screw joint. PMID- 8639239 TI - Resin-bonded inlay retainer prostheses for posterior teeth. A 5-year clinical study. AB - This study evaluated posterior resin-bonded prostheses using inlays as retainers. Thirty-nine patients missing at least one premolar or first molar received 51 resin-bonded fixed partial dentures with high noble alloy inlay retainers and a metal ceramic pontic. Resin luting material bonding to the framework was secured by the Silicoating method (24 fixed partial dentures), lost sugar crystal method (13 fixed partial dentures), or tin plating (14 fixed partial dentures). Clinical examinations were performed 1 week, 1 month, 6 months, 1 year, 2 years, and 5 years after cementation. None of the fixed partial dentures with silicoating or sugar crystal impressions lost retention, whereas two of the tin-plated fixed partial dentures required replacement. Resin-bonded inlay-retained prostheses appear to be a favorable alternative to other types of fixed partial dentures. PMID- 8639240 TI - Bone strength of resilient lining materials to various denture base resins. AB - Adhesion failure of resilient lining materials to poly(methyl methacrylate) denture base material is a problem that is encountered clinically. This study compared the shear bond strength of some of the commercially available resilient lining materials to various denture base resins. Significant differences were observed in the shear strengths between the selected resilient liners and the denture base materials. Coe Super Soft specimens failed principally cohesively. Molloplast-B specimens always failed cohesively and Novus specimens always failed adhesively. Therefore, it was apparent that the bond strengths of Molloplast-B and Coe Super Soft specimens exceeded the cohesive strength. The bond strength of Novus was dependent on the denture base material and was greatest with Lucitone 199 and TS 1195. PMID- 8639241 TI - Factors correlated with caraniomandibular disorders in young and older adults. AB - This study was conducted to examine prevalence of craniomandibular disorders in young adults compared with that in older subjects. In addition, the relationship between various oral conditions and this dysfunction was studied. No difference was found in the incidence of craniomandibular disorders between the groups. Classification according to the Helkimo index showed significant difference in complaints of craniomandibular disorders symptoms between the groups. Most of the older patients did not complain of mild disorders. The results suggest that conditions such as the number of teeth, presence or absence of dentures, and the type of denture worn are not important factors in the pathogenesis of craniomandibular disorders. PMID- 8639242 TI - An in vitro study of the influence of occlusal scheme on the pressure distribution of complete denture supporting tissues. AB - This study compared lingualized occlusion and completely balanced occlusion using a simulation device. Sixteen pressure transducers were placed in the simulated residual ridge area supporting the test dentures. Lingualized occlusion was found to transfer stresses from the occluding side to the opposite, nonworking side to stabilize the mandibular denture. PMID- 8639243 TI - Working times of elastomeric impression materials determined by dimensional accuracy. AB - The working times of five poly(vinyl siloxane) impression materials were estimated by evaluating the dimensional accuracy of stone dies of impressions of a standard model made at successive time intervals. The stainless steel standard model was represented by two abutments having known distances between landmarks in three dimensions. Three dimensions in the x-, y-, and z-axes of the stone dies were measured with a traveling microscope. A time interval was rejected as being within the working time if the percentage change of the resultant dies, in any dimension, was statistically different from those measured from stone dies from previous time intervals. The absolute dimensions of those dies from the rejected time interval also must have exceeded all those from previous time intervals. Results showed that the working times estimated with this method generally were about 30 seconds longer than those recommended by the manufacturers. PMID- 8639244 TI - A study of discoloration of the gingiva by artificial crowns. AB - In this study, soft tissue discoloration including the marginal gingiva, gingival papilla, and attached gingiva were evaluated in subjects with artificial crowns and compared to the soft tissue of natural teeth using a spectrophotometer. Gingivae around artificial crowns had a lower value (L*) and chroma (C*ab) than the gingivae surrounding natural teeth. Hue (Hab o) shifts toward red-purple were frequently noted. There was generally a more noticeable color difference (E*ab) in the marginal gingiva and gingival papilla of teeth with crowns than was found in the attached tissue. PMID- 8639245 TI - Oligonucleotides induce apoptosis restricted to the t(14;18) DHL-4 cell line. AB - Most human follicular B-cell lymphomas are associated with t(14;18) chromosome translocation that joins the bcl-2 gene with the IgH locus. This hybrid gene causes upregulation of BCL-2 protein expression, endowing cells with survival advantage. Although early BCL-2 overexpression is definitely responsible for immortalization/transformation, its exact role in the overt transformation as well as in the maintenance of the tumor phenotype is not known. The capacity of oligodeoxynucleotides (ODN) to modulate gene expression specifically has been exploited to downregulate the overexpression of BCL-2 protein in the SU-DHL-4 human follicular B-cell lymphoma line by the use of sense ODN or antisense ODN or antisense ODN designed to encompass the unique nucleotide sequence in the fusion region of the hybrid transcript. The specific downregulation of the bcl-2 transcript and of the relevant BCL-2 protein in the treated cells activated programed cell death and inhibited growing cells. The antitumor activity was restricted to the DHL-4 cell line carrying the specific nucleotide sequence at the bcl-2/IgH joining region. Thus, DHL-4 lymphoma cells derived from the acute phase of human follicular B-cell lymphoma, although endowed with additional activated oncogenes, were growth inhibited by bcl-2 downregulation with additional activated oncogenes, were growth inhibited by bcl-2 downregulation in a genetically restricted fashion. The biological activity was exerted exclusively by ODNs synthesized in the sense orientation. The sense ODNs have been proposed to anneal the hybrid bcl-2/IgH antisense RNA as identified in this study. PMID- 8639246 TI - Design, synthesis, cytotoxic properties and preliminary DNA sequencing evaluation of CPI--N-methylpyrrole hybrids. Enhancing effect of a trans double bond linker and role of the terminal amide functionality on cytotoxic potency. AB - In an approach to the design and exploration of the properties of cyclopropylindole (CPI)-lexitropsin conjugates as potential anticancer agents. CPI--N-methylpyrroles of two separate classes have been synthesized and characterized. These comprise structures (i) in which the N-methylpyrrole moiety bears amide groups of different sizes and (ii) in which both flexible and rigid linkers are introduced between the CPI and N-methylpyrrole units. The extent and the relative rates of DNA cleavage following alkylation and thermal treatment by these CPI conjugates were determined by an agarose gel mobility shift assay. The DNA sequence preferences of the seven new agents were also determined in a preliminary study by high-resolution polyacrylamide gel electrophoresis and contrasted with that of CC-1065. The CPI--N-methylpyrrole agents avoid the major alkylation sites of CC-1065, but all alkylate the minor CC-1065 site of 5'AATA and exhibit a consensus sequence of 5'-N.A/T.A/T.A. The cytotoxicities of these compounds were determined against KB human tumor cells in vitro. Compound 6, bearing a 4-butyramide group in the N-methylpyrrole, is 100 times more potent that 7 which lacks an amide group, while 10 which bears a rigid trans double bond linker is 1000 times more potent than its flexible ethyl-linked counterpart. PMID- 8639247 TI - Interaction of geiparvarin and related compounds with purified microtubular protein. AB - Geiparvarin is an antiproliferative compound whose mechanism of action has not yet been identified. We have investigated the biochemical action on purified microtubular protein, as well as on tubulin, of geiparvarin and of some derivatives resulting from its conjugation with two synthetic oestrogens, diethylstilboestrol and meso-hexoestrol, in comparison with the antimicrotubular action of the reference oestrogens. Geiparvarin and derivatives did not inhibit colchicine binding to tubulin nor did they significantly influence GTP-induced polymerization. On the contrary, they effectively counteracted the stimulating effect of taxol on microtubule formation, both in the presence and in the absence of microtubule-associated proteins. A competitive inhibition mechanism at the taxol binding site of tubulin may thus be proposed to explain the antimicrotubular action of geiparvarin. PMID- 8639248 TI - Synthesis and biological evaluation of a series of hydroxybenzylphenylamine derivatives as inhibitors of EGF receptor-associated tyrosine kinase activity. AB - In order to obtain non-degradable and more potent protein-tyrosine kinase inhibitors, derived from the 5-(2,5-dihydroxybenzyl)-aminosalicylates already described, we have developed a new series of 5-(2,5-dihydroxybenzyl)phenylamines. The compounds, diversely substituted on the phenyl ring by alcohol, nitrile, ether, ketone, amide and thioamide groups, were tested for their ability to inhibit epidermal growth factor (EGF) receptor-associated tyrosine kinase activity in vitro. They inhibit the phosphorylation of the peptide substrate RR Src by the EGF receptor purified from ER 22 cells, with IC50 values in the range 0.02-0.45 microns. Several of these compounds inhibit EGF-dependent DNA synthesis in ER 22 cells with IC50 values of around 1 micron and furthermore their inhibition has been found to be specific for various protein kinases. PMID- 8639249 TI - QSAR of acridines, III. Structure-activity relationship for antitumour imidazoacridinones and intercorrelations between in vivo and in vitro tests. AB - A study on quantitative relationships between the biological activity and physicochemical properties of antitumour 5-alkylaminoimidazoacridinone derivatives was carried out. The activity was based on the results of several in vitro tests as well as experimental antileukaemic therapy. The capacity factor, log k', determined by the reverse-phase HPLC method, was a measure of lipophilic properties. UV and NMR spectra of the compounds were employed to describe electronic parameters. Values of steric descriptors were calculated as topological indexes. Results obtained by means of principal component analysis (PCA) allow us to group biological tests into two subsets: the lipophilicity dependent and lipophilicity-independent test groups. The highest intercorrelation, R = 0.92, was shown between the optimal dose, pOD, determined in leukaemia P388-bearing mice and cytotoxicity expressed as pEC50 in leukaemia cells. The equation describing this relationship could be applied to predict the therapeutic doses of imidazoacridinone derivatives which would be effective in experimental antileukaemic therapy. The quantitative structure-activity relationship (QSAR) study showed that lipophilic properties significantly influence cytotoxicity, pEC50, and antileukaemic potency, pOD, only in the case of 8-hydroxy analogues of imidazoacridinones, whereas the activity of the remaining derivatives is very low and does not depend on lipophilicity. Electronic resonance properties seem to influence this specific impact of lipophilicity on the biological activity of 8-hydroxy derivatives. Hence, it may be possible to improve the antitumour activity of 8-hydroxyimidazoacridinones by obtaining more hydrophilic derivatives, up to the optimal value of the lipophilic parameter. PMID- 8639250 TI - Endocrine stress reaction to surgery in brain-dead organ donors. AB - We studied the course of plasma levels of the stress markers adrenocorticotropic hormone (ACTH), cortisol, human growth hormone (h-GH), beta-endorphin, and prolactin during retrieval surgery in eleven brain-dead organ donors scheduled for multiple organ explantation. Donors were divided into two groups according to hemodynamic stability. Hormones demonstrated a great variability in plasma levels and in the pattern of reaction, revealing a different degree of remaining pituitary function. Beta-Endorphin was the only stress hormone that showed a response to surgical stimuli in six patients. Only three of them developed a concomitant rise in ACTH. Cortisol, prolactin, and h-GH plasma levels did not change during the observation period. In the three cases with a slight elevation in ACTH, no subsequent change in cortisol was detectable. Beta-Endorphin showed greater variability and a tendency to higher levels in the group presenting with a higher arterial pressure, which resulted in a significant difference (P < 0.005) when distributions were compared using the Mann-Whitney U-test. No correlation was found between hypotensive episodes and deficiencies of other stress hormones. We conclude that pituitary function varies considerably in brain dead organ donors without demonstrating a correlation to the onset of hypotension. Thus, we feel no need for a substitution treatment with any of the hormones investigated prior to organ explanation. PMID- 8639251 TI - Living related kidney donors over 60 years old. AB - The lack of available cadaveric organs for transplantation has resulted in an increased number of kidney transplants from living donors. During a period of 6 years, 149 kidney transplantations were performed from living related donors in our institute, 33.5% of whom were older than 60 years of age. In this study we examined the survival of patients and grafts as well as the graft function in 50 patients with transplants from donors over 60 years (mean age 65 years) as compared with those of 99 patients with transplants from donors younger than 60 years (mean age 47 years). There were no significant differences in the course of donor nephrectomy, postoperative complications, or remnant kidney function. However, delayed graft function occurred more frequently in recipients of transplants from older donors. Improvement in graft function was also slower in recipients of kidneys from older donors, with significant differences in serum creatinine levels observed during the first 12 months after transplantation. More frequent acute complications and more progressive chronic graft failure, irrespective of the causes, occurred during the 1st post-transplant year in recipients with grafts from older donors. Five-year patient survival (77% vs 92%) and kidney graft survival differed significantly for the same period with worse results for patients receiving grafts from older donors. It may be concluded that kidney grafts from donors older than 60 years -- and especially those older than 70 years -- may be used for living related kidney transplantation, but with precautions. PMID- 8639252 TI - The beneficial effects of oral nifedipine on cyclosporin-treated renal transplant recipients--a randomised prospective study. AB - The aim of this study was to test the hypothesis that nifedipine will improve graft survival in cyclosporin A (CyA)-treated renal transplant recipients. One hundred and forty-seven patients were randomised to one of three regimens. Group A received CyA, 7 mg/kg per day, and prednisolone; group B followed the same regimen as group A plus oral nifedipine and group C received CyA, 4 mg/kg per day, prednisolone and azathioprine. Calcium channel blockers were avoided in groups A and C. The crude 2-year (P = 0.0223) and 4-year (P = 0.0181) graft survival was significantly better in group B (86% and 81%, respectively) than in group A (75% and 63%, respectively). Delayed initial function was seen least frequently in group B (10.2%) compared to groups A (31%) and C (28%; P < 0.01). Group B also experienced fewer rejection episodes than groups A and C (P < 0.05). We conclude that the combination of oral nifedipine and CyA significantly improves initial graft function, rejection frequency and long term graft survival. PMID- 8639253 TI - Duct-to-duct biliary reconstruction following liver transplantation for primary sclerosing cholangitis. AB - The biliary complications in patients undergoing biliary reconstruction by duct to-duct (D-D) anastomosis or with a Roux-en-Y loop (RL) at the time of liver transplantation for primary sclerosing cholangitis (PSC, 16 D-D, 10RL) or primary biliary cirrhosis (PBC, 31 D-D, 1 RL) were reviewed and compared. Patients were followed up for a mean period of 32 months. Extrahepatic biliary strictures occurred in 18.7%, 10% and 9.7% of DD-PSC, RL-PSC and DD-PBC patients, respectively, leaks in 6.2%, 20% and 6.4% DD-PSC, RL-PSC and DD-PBC patients, respectively (P = NS). Four intrahepatic biliary abnormalities developed in the PSC group. Duct-to-duct anastomosis did not significantly increase the risk of stricture formation or bile leaks in PSC patients compared to PBC patients. We conclude that duct-to-duct biliary reconstruction following liver transplantation for PSC is satisfactory unless the distal common bile duct is strictured. PMID- 8639254 TI - Value of Doppler echocardiography in the detection of low-grade rejections after cardiac transplantation. AB - Modifications of the diastolic parameters pressure half-time (PHT) and isovolumic relaxation time (IVRT), recorded using cardiac Doppler echocardiography (CDE), were studied in 23 heart transplant recipients and compared to the results of 345 endomyocardial biopsies (EMB) performed on the same day. Two different protocols, analyzing respectively (1) a decrease of 20% or more in IVRT and/or PHT with respect to the mean and (2) a decrease of 20% or more in IVRT and/ or PHT with respect to its preceding value, were used to evaluate the efficiency of CDE in diagnosing mild and moderate rejections. When a mild rejection was detected by EMB, a statistically significant decrease was found in the average CDE parameter values of the patient population. However, these variations were weak and did not differ from the spontaneous variations observed in each patient in the absence of rejection. Thus, it is not surprising that the sensitivity of CDE in the detection of mild rejections was very low (45%) using the most sensitive protocol (variations of the parameters from their preceding value). We conclude that CDE alone does not seem to be sufficient to perform the noninvasive diagnosis of low grade rejections and must be complemented by other noninvasive methods. PMID- 8639255 TI - Infection-associated cellular activation accelerates chronic renal allograft rejection in rats. AB - The etiology of chronic rejection is unknown, although acute rejection, viral infection, and initial graft ischemia have been implicated. To test the effects of infections on the process of chronic rejection, we simulated bacterial infection by the administration of the endotoxin lipopolysaccharide (LPS), a potent activator of various cell types in an established rat model of chronic rejection. Lewis recipients of Fisher 344 kidneys were treated with a single dose of LPS or vehicle 8 weeks following transplantation and grafts were examined at various time points. In the chronically rejecting controls, leukocytic infiltration and the expression of cytokines peaked at 16 weeks. In LPS-treated hosts, leukocyte infiltration and cytokine expression peaked at 12 weeks. By 16 weeks, glomeruli in LPS-treated recipients had become far more sclerotic than those in controls, mimicking the changes observed in controls at 24 weeks. We conclude that infections may play an important role in the development of chronic rejection. PMID- 8639256 TI - Pulmonary hypertension: considerations in the liver transplant candidate. AB - Pulmonary hypertension is a potentially lethal complication of end-stage liver disease with a prevalence of 2%. In the setting of liver transplantation, the prevalence may be as high as 12%. Given the potential importance of this syndrome to the transplantation community, the purpose of this review is to summarize the current state of understanding of portopulmonary hypertension and to suggest potential management strategies for (1) liver transplant candidates with suspected pulmonary hypertension and (2) intraoperative pulmonary hypertension following liver allograft reperfusion. PMID- 8639257 TI - Serum HGF levels in acute renal rejection after living related renal transplantation. AB - Hepatocyte growth factor (HGF), a long sought-after hepatotrophic factor, has recently been shown to act as a renotrophic factor in regeneration of the kidney. We investigated serum HGF levels in 16 renal transplant patients. In patients with acute rejection, the serum HGF level was markedly increased (over 1 ng/ml), and its elevation was accompanied by an increase in serum creatinine and blood urea nitrogen (BUN). In contrast, serum HGF levels were continuously low in patients without rejection. We conclude that serum HGF may become a clinically useful marker for the assessment of acute renal rejection. PMID- 8639258 TI - Antibody removal and subsequent transplantation of a highly sensitised paediatric renal patient. AB - We report a successful renal transplant in a highly sensitised paediatric recipient following removal of HLA-specific antibodies by extracorporeal immunoadsorption. The immediate pretransplant cytotoxic titre against the donor was greater than 1:512; this was reduced to negativity by two immunoadsorption sessions prior to transplant surgery. We also describe the presence of unexpected non-HLA-specific antibody activities in this immunoadsorbed patient. PMID- 8639259 TI - Extrahepatic biliary stricture associated with cytomegalovirus in a liver transplant recipient. AB - We describe a patient who developed a stricture in the distal common bile duct 6 weeks after orthotopic liver transplantation. Histopathologic examination of the bile duct epithelium in the region of the stricture showed characteristic cytomegalovirus (CMV) inclusions. CMV was also identified in pulmonary alveoli and in the duodenum. Although CMV has been demonstrated in the biliary epithelium of AIDS patients with extrahepatic biliary strictures and biliary obstruction, this entity has not, to our knowledge, been described in liver transplant recipients. This report confirms that CMV infection should be included as a probable cause of extrahepatic biliary strictures and bile duct obstruction in liver transplant patients. PMID- 8639260 TI - Need for reduction of cyclosporin dosage in renal transplant patients with hypertriglyceridemia but not hypercholesterolemia. AB - Currently there is a paucity of data regarding the influence of high serum triglyceride levels on cyclosporin A (CyA) levels and dosing. We therefore undertook a retrospective study to determine the relationship of serum lipid levels to CyA levels and CyA dosages. Renal transplant patients at a 0.5-to-3 year post-transplant stage, with a stable CyA dosage, who were not on medications that affect CyA metabolism or renal function, were entered into the study. The CyA dosage was adjusted by clinicians to maintain whole blood. 12-h CyA trough levels between 200 and 250 ng/ml (monoclonal TDX method, which measures the parent compound). Forty-four patients qualified for the study. The data clearly indicated that high cholesterol levels (> 300 mg/dl and with normal triglyceride levels) did not influence the CyA levels or the dosages. Conversely, high triglyceride levels ( > 500 mg/dl) significantly reduced the amount of CyA required. A decreased clearance of CyA in the presence of hypertriglyceridemia led to high CyA levels in some patients. Reducing the CyA dosage to achieve levels between 200 and 250 ng/ml improved renal allograft function and decreased other side effects attributed to CyA toxicity. These studies indicate that high triglyceride levels, but not high cholesterol levels, increase CyA levels, which can lead to CyA toxicity. PMID- 8639261 TI - Tissue oxygen saturation of human hepatic grafts after reperfusion: paradoxical elevation in poor graft function. AB - The present study investigated the pathophysiology of primary nonfunction (PNF) of grafted livers with regard to hepatic tissue oxygenation. Hemoglobin oxygen saturation in hepatic tissue (H-So2) after reperfusion was determined using near infrared spectroscopy. Graft tissue oxygen consumption was also estimated according to Fick's principle. Six grafts with PNF were compared with 40 functioning grafts. One PNF graft with extremely low and heterogeneous H-So2 after reperfusion was found to contain multiple intrahepatic portal thrombi. However, five other PNF grafts showed no lower and, on the contrary, more homogeneous H-So2 at the end of the operation. As a whole, mean H-So2 was negatively correlated and the coefficient of variation (CV) of H-So2 was positively correlated with graft tissue oxygen consumption at the end of the operation; grafts whose H-So2 showed a secondary decrease had better initial function. In later relaparotomy, the H-So2 of the five PNF grafts was significantly higher and more homogeneous than that of the functioning grafts. These results suggest that H-So2 level reflects tissue oxygen consumption as well as oxygenation, and that the dissociation of both factors can occur in hepatic graft reperfusion. Not only low and heterogeneous H-So2 but also high and homogeneous H-So2, suggesting some shunt mechanism, can be signs of poor graft function. PMID- 8639262 TI - The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera. AB - Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others. PMID- 8639263 TI - Human drug-metabolizing enzyme polymorphisms: effects on risk of toxicity and cancer. AB - A growing number of human genetic polymorphisms in drug-metabolizing enzymes (DMEs) are being characterized. Some of these have been shown, quite convincingly, to be correlated with risk of toxicity or cancer, whereas others presently remain equivocal. There is good evidence that the correlation is stronger in populations exposed to a variety of environmental procarcinogens; perhaps 30% of DME substrates are able to be metabolically potentiated. Phase I DMEs, most of which represent cytochromes P450, metabolically activate procarcinogens to genotoxic electrophilic intermediates, and Phase II DMEs conjugate the intermediates to water-soluble derivatives, completing the detoxification cycle. It follows that genetic differences in the regulation, expression and activity of genes coding for Phase I and Phase II DMEs would be crucial factors in defining cancer susceptibility and the toxic or carcinogenic power of environmental chemicals. Not all Phase I and Phase II DMEs are implicated in detoxification; previous work from this and from other laboratories has identified candidate Phase I and Phase II genes in which certain alleles are more likely to be associated with cancer susceptibility. In some cases, the allelic frequencies vary dramatically between ethnic groups. In this review, our current knowledge about polymorphisms in the following genes are updated: the aromatic hydrocarbon receptor (AHR), the CYP1A1 structural gene (which encodes aryl hydrocarbon hydroxylase activity), the CYP1A2 structural gene (arylamine oxidations), the CYP2C19 gene (S-mephenytoin 4'-hydroxylase), the CYP2D6 gene (debrisoquine hydroxylase), the CYP2E1 gene (N,N-dimethylnitrosamine N demethylase), the null mutant for the GSTM1 gene (glutathione transferase mu), and the NAT2 gene (arylamine N-acetyltransferase). If unequivocal biomarkers of genetic susceptibility to cancer and toxicity can be developed successfully, then identification of individuals at increased risk would be very helpful in the fields of public health and preventive medicine. PMID- 8639265 TI - Spontaneous apoptosis in mouse F4N-S erythroleukemia cells induces a nonrandom fragmentation of DNA. AB - This study characterizes the fragmented DNA of mouse erythroleukemia (MEL) cells spontaneously entering apoptosis. Fragmented DNA was isolated by introducing a novel procedure that ensured a complete extraction of the characteristic oligonucleosomal ladder. As the results show, less than 10% of DNA of apoptotic cells is fragmented in this form. The main conclusion from experiments to characterize the nature of fragmented DNA is that spontaneous apoptosis induces a nonrandom cleavage of genomic DNA. The Southern analysis performed with total apoptotic DNA revealed that it is strongly enriched in interspersed repetitive sequences. In situ hybridizations with such DNA showed further than in interphase nuclei these sequences flock together and form clusters spread throughout the whole nuclear area whereas in mitotic chromosomes they are located predominantly at their pericentromeric/peritelomeric ends. Partial cloning and sequencing reinforces the notion that the apoptotic DNA is representative for a heterochromatinic portion of the mouse genome. Support for such an unexpected conclusion is coming also from experiments indicating that this DNA is heavily methylated and poorly transcribed. PMID- 8639264 TI - Cloning and sequence analysis of human breast epithelial antigen BA46 reveals an RGD cell adhesion sequence presented on an epidermal growth factor-like domain. AB - The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation. PMID- 8639267 TI - The Surf-6 gene of the mouse surfeit locus encodes a novel nucleolar protein. AB - The Surfeit locus contains the tightest cluster of mammalian genes so far described. The five Surfeit genes (Surf-1 to -5) that have been previously isolated and characterized do not share any DNA or amino acid sequence homology. These Surfeit genes appear to be housekeeping genes, with the Surf-3 gene encoding the 1.7a ribosomal protein and the Surf-4 gene encoding an integral membrane protein most likely associated with the endoplasmic reticulum. In this work, we have isolated the Surf-6 gene, a sixth member of the Surfeit locus. The Surf-6 gene contains four exons spanning a genomic region of 14 kb and specifies a mRNA of 2,571 bases. Surf-6 has features common to housekeeping genes because its transcript is present in every tissue tested, its 5' end is associated with a CpG-rich island, and its promoter does not contain a canonical TATA box. The Surf 6 long open reading frame encodes a novel highly basic polypeptide of 355 amino acids (28% Arg and Lys). By immunofluorescence and immunoblot analyses, the Surf 6 protein has been found to be located in the nucleolus and by immunocytochemical microscopy to be localized predominantly in the nucleolar granular component, a structure that is involved in ribosome maturation. These results indicate that the novel Surf-6 gene is involved in a nucleolar function. PMID- 8639266 TI - Isolation and characterization of BART-1: A novel balloon angioplasty responsive transcript in rat carotid arteries. AB - In previous studies, differential display analysis of balloon angioplasty-damaged rat carotid arteries identified a temporally expressed partial cDNA termed RC9. This message is undetectable in undamaged vessels, reaches maximal levels 3 days post-procedure, and reduces to half-maximal expression by 14 days post angioplasty. Using RC9 to screen a cDNA library, we now report the isolation and characterization of a full-length clone, termed BART-1. BART-1 is 98% homologous to RC9 and shows the same mRNA expression pattern as RC9 in rat carotid arteries subject to balloon angioplasty. Northern analysis of various rat tissues reveals tissue specificity and possible differential processing. Neither the nucleic acid nor amino acid sequences demonstrate similarity to previously reported expressed sequences. Predicted amino acid analysis reveals two strongly hydrophilic and one hydrophobic region, suggesting a type II integral membrane protein. The cDNA sequence hybridized to genomic DNA from a variety of species, suggesting evolutionary conservation. Thus, BART-1 mRNA appears to represent an inducible, tissue-specific transcript encoding a putative integral membrane protein transiently expressed in response to vascular trauma. PMID- 8639268 TI - Distinct mRNA-binding proteins interacting with short repeat sequences of the 3' UTR may be involved in the post-transcriptional regulation of the mouse catalase gene, Cas-1. AB - The 3' untranslated region (UTR) of the mouse catalase gene (Cas-1) is demonstrated to be an active site for specific protein interactions. We have identified two regions of the Cas-1 3' UTR mRNA that bind to distinct cytoplasmic proteins: one containing a (CA)31 repeat with UA octomer (RNA 5) and another with a (U)15 tract (RNA 6). RNA 5 interacts with one set of protein complexes (a, b, and c) whereas RNA 6 interacts with another (x, y, and z) in a sequence-specific manner. These RNA-protein complexes are development-, tissue-, and genotype specific. The proteins involved in the two sets of complexes are different. Further characterization of the proteins involved in these interactions has revealed the presence of a single protein of approximately 70 kD that binds RNA 5, and two proteins approximately 38 kD and approximately 47 kD that bind to RNA 6. The approximately 70-k D and approximately 38-kD proteins are also associated with the polysomal fractions and may play a role in the post-transcriptional regulation of Cas-1. Although the observed 3' UTR RNA-protein interactions are hypothesized to be important in post-transcriptional regulation of this gene in rodents, specific RNA sequences and their associated proteins identified in this report would now permit the elucidation of the mechanisms of their action at the molecular level. PMID- 8639269 TI - Characterization of the rat A2a adenosine receptor gene. AB - To understand the molecular basis for the regulation of rat A2a adenosine receptor (A2a-R) expression, we have characterized the rat A2a-R gene and defined its promoter regions. Through a combination of restriction mapping and sequence analysis, we have demonstrated that the rat A2a-R gene is composed of two exons interrupted by a 7.2-kb intron. Primer extension and RNase protection on RNA isolated from PC12 cells suggested that the A2a-R gene encoded two clusters of alternative transcripts. The most upstream transcription start site was designated as +1. The sequence of the proximal 1.5 kb of 5'-flanking region demonstrated no potential TATA box, CCAAT box, or initiator element in the appropriate location. Varying lengths of 5'-flanking regions were inserted into a transient expression vector (pGL2-basic), which contained bacterial luciferase as the reporter gene, to determine its promoter region(s) in PC12 cells, CHOP cells, and C6 cells. Consistent with two clusters of transcription start sites, two independent functional promoter regions (designated P1, -67/-1; and P2, +272/+304) for the rat A2a-R gene were identified. Although both promoters are in use in PC12 cells, only P2 is active in CHOP cells, indicating possible cell line specific usage of these two promoters. PMID- 8639271 TI - A non-isothermal bioreactor utilizing immobilized baker's-yeast cells: a study of the effect on invertase activity. AB - The behaviour of the enzyme invertase, located on the cell wall of baker's-yeast cells and entrapped in a gelatin membrane, was studied under isothermal and non isothermal conditions. The reaction rate linearly increased with the applied transmembrane temperature gradient, with reference either to the average temperature or to the temperature on the warm side of the catalytic membrane. These results were obtained both when the bioreactor was operated under conditions of closed volumes and when the substrate-containing solutions are recirculated. The mathematical relationships have been elaborated between the temperatures read in the working solutions and those on the two faces of the catalytic membrane. Since the temperature difference across the membrane is smaller than that indicated by the thermocouples, the observed effects are greater than expected. The potential advantages of the use of a non-isothermal bioreactor in processes of industrial interest are discussed. PMID- 8639270 TI - Molecular cloning of prohormone convertase 1 from the atrial gland of Aplysia. AB - We have screened an Aplysia atrial gland cDNA library using a prohormone convertase (PC)1 probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia PC1-related full-length 3.6-kb cDNA clone. The cDNA sequence (3,565 bp) encoded a putative preproendoprotease (APC1) of 703 amino acid residues that showed considerable sequence identity with other eukaryotic PC1s, and indicated a high degree of sequence identity with an Aplysia nervous system PC sequence (aPC1B). Northern blot analysis of atrial gland RNA identified two APC1 transcripts of 3.9 kb and 5.0 kb. APC1 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in atrial gland secretory cells and represents one of the first examples of PC1 expression in an exocrine tissue. PMID- 8639272 TI - Affinity purification and binding characteristics of Citrobacter freundii AmpR, the transcriptional regulator of the ampC beta-lactamase gene. AB - The transcriptional regulator of the Citrobacter freundii ampC beta-lactamase gene, AmpR, was purified as a single SDS/PAGE-gel band by using various techniques, including DNA-Sepharose 4B affinity chromatography. The purified AmpR consisted of a 32.5 kDa monomer that interacted with three operator sequences: two binding sequences, at positions -75 to -70 and -67 to -51 with respect to the transcriptional start site, were located in the LysR motif (-72 to -60), and the third sequence was at position -43 to -38. Equilibrium binding studies raise the possibility that the adjacent operator sequence could exert a positive influence on the ability of AmpR to bind to these sites. PMID- 8639273 TI - Development of an enzyme immunoassay for the measurement of human tumour necrosis factor-alpha (hTNF-alpha) using bispecific antibodies to hTNF-alpha and horseradish peroxidase. AB - The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF alpha in an experimental environment or in clinical trials. PMID- 8639274 TI - Behaviour of isolated rat and human red blood cells upon hypotonic-dialysis encapsulation of carbonic anhydrase and dextran. AB - Rat and human carrier red blood cells (RBCs) were prepared by hypotonic-dialysis encapsulation. The standard conditions used for encapsulation were 80 mOsm/kg for 60 min. The encapsulation behaviour of rat and human RBCs was studied using radiolabelled carbonic anhydrase and fluorescently labelled dextran. Both markers are incorporated to slightly greater extents by human than by rat RBCs by hypotonic treatment. Cell recovery of rat and human RBCs loaded with either carbonic anhydrase or fluorescent dextran accounted for 49% and 80% respectively. The cellular integrity of the loaded cells was revealed by the presence of fluorescence labelling in rat and human RBCs. Fluorescence studies showed an increase of size dispersion in loaded rat and human RBCs, giving cellular volume variations in both types of cells resulting from the encapsulation procedure. Two loaded cell populations were evident in both species, one with high fluorescence content and another with background staining. Apparently the proportion of high fluorescently labelled loaded cells was higher in the case of the human RBCs. A reduced level of fluorescence labelling was observed in rat and human RBC membranes, which indicates a process of adsorption of dextran to the membranes. PMID- 8639275 TI - Determination of residual pectin methylesterase activity in food products. AB - A method for the determination of a low level of pectin methylesterase activity from vegetable products is described. The method is based on an affinity chromatography technique that employs a resin-bound pectin methylesterase inhibitor, purified from kiwi fruit, which selectively binds the pectin methylesterase. The resin has the capacity to concentrate the enzyme, allowing measurement of enzyme activities too low to be determined by commonly employed techniques and commensurate with those found in pasteurized food products. The enzyme is eluted from the resin at alkaline pH (9.5) and assayed by a pH-stat method. Depulped orange juices containing different amounts of pectin methylesterase were prepared and used to determine enzyme recovery. The results show a recovery of 90% with a standard deviation of 6.8%. PMID- 8639276 TI - Status and transcriptional activity of a bovine-papillomavirus-I-based expression vector in a recombinant production cell lines. PMID- 8639277 TI - A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation. AB - A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5 bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine leukaemia-virus RT. The assay was found to be useful with various types of cell culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications. PMID- 8639278 TI - Applications of electrochemical immunosensors to environmental monitoring. AB - This paper discusses basic electrochemical immunoassay technology. Factors limiting the practical application of antibodies to analytical problems are also presented. It addresses the potential use of immunoassay methods based on electrochemical detection for the analysis of environmental samples. It provides examples for the detection and quantitation of environmental samples using conducting electroactive polymers (CEPs). CEP-based immunosensing systems are compared with conventional environmental immunoassay procedures. The advantages of using these types of sensors for rapid, sensitive, and cost-effective analysis of pesticides and toxic chemicals are analysed and discussed. CEP-based immunosensing technology might eventually be used for continuous monitoring of effluents such as waste streams to determine compliance with regulations. CEP based sensors are suitable for monitoring ground-water, waste stream effluents, agricultural run-offs and for monitoring the effectiveness of remediation, or for other situations where a real-time monitoring capability is desired. PMID- 8639279 TI - Development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine. AB - A simple immunosensor based on a conductivity method was developed for determination of methamphetamine (MA, a stimulant drug) in urine. Anti-MA antibody was immobilized onto the surface of a pair of platinum electrodes. The reaction of MA with the antibody causes a decrease in the conductivity of the anti-MA immobilized layer between the electrodes. A linear relationship was obtained between the conductivity and MA concentration in the range of 1-10 micrograms/ml. The method requires the sample to be rinsed with water on the electrodes after the immunoreaction. This detection system was applied to the determination of MA in urine and proved to be a useful and a simple detection technique of MA in forensic science in comparison with a gas chromatography-mass spectrometry method. PMID- 8639280 TI - Use of the USDT flow immunosensor for quantitation of benzoylecgonine in urine. AB - The flow immunosensor works on a principle somewhat distinct from other immunoassay systems in that it performs a displacement immunoassay. Antibody coated matrices are saturated with fluorescently labeled benzoylecgonine (BE), which is released and measured in the presence of BE-containing urine and measured downstream from the matrix. The same antibody matrix can be used for many samples. A flow immunosensor instrument, built by US Drug Testing, Inc., has recently received "Premarket Notification' (510(K)) from the Food and Drug Administration (FDA) to screen urine for the presence of the cocaine metabolite BE. The performance of the flow immunosensor for screening BE in urine was validated by comparison with results of a blind study using the Syva EMIT, the Abbott TDx and gas chromatography-mass spectroscopy (GC-MS). Potentially interfering drugs were also spiked into urine and evaluated using the flow immunosensor. While the FDA approval is for determining whether the BE concentration is above or below the 300 micrograms/l cutoff recommended by the National Institute of Drug Abuse, we have also shown that the flow immunosensor can be adapted to produce quantitative determinations of the amount of BE in the urine samples. The reliability of the quantitation was confirmed by testing 100 urine samples containing unknown amounts of BE using the flow immunosensor. GC-MS and the Abbott TDx system. Comparison of quantitative data obtained using the immunosensor and GC-MS showed a 97% correlation, compared with a much lower value for data from the TDx and GC-MS. PMID- 8639281 TI - Enzyme-epoxy membrane based glucose analyzing system and medical applications. AB - A semi-automatic glucose analyzing system has been developed. It consists of thin film glucose sensors and a flow injection system with a computerized data processing unit. The sensor is made of titanium and platinum, using the well established semiconductor process technology. Glucose oxidase is immobilized into the network of epoxy on the electrode surface together with ferrocene, which is used as an electron mediator for reducing the operation voltage of the sensor. The system can measure glucose from 50 to 4000 mg/dl with good linearity up to 400 mg/dl. This can cover the range needed in medical and biotechnological applications. The system can measure samples at a speed of more than 60 samples per hour and has a coefficient of variation of 3%. Selectivity tests were performed and the results showed that there was no interference from sucrose, lactose, maltose, ascorbic acid and uric acid. The sensors were then employed in detecting glucose in human blood plasma samples. The results show good correlation with the results from commercial machines. PMID- 8639282 TI - Carbon fibre micro-electrode and in vitro or in brain slices voltammetric measurement of ascorbate, catechol and indole oxidation signals: influence of temperature and physiological media. AB - Carbon fibre micro-electrodes have been used to determine the influence of temperature and physiological media on the oxidation potential value of three carboxylic acids of physiological interest such as ascorbate (AA), 3,4 dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIAA). Standard calibrations at room temperature (18-20 degrees C) in phosphate buffered saline (PBS, pH 7.4), in Krebs (pH 7.4) or in artificial cerebral spinal fluid (ACSF, pH 7.4) have been compared with calibrations performed at 37 degrees C under 95% oxygen, 5% carbon dioxide. Ex vivo experiments were then performed with the electrode inserted in the striatum of rat brain slices maintained in ACSF at 37 degrees C under 95% oxygen, 5% carbon dioxide. The results obtained from both in vitro and ex vivo experimentation indicate that the oxidation potential of peak 2 (DOPAC) is highly sensitive to changes in temperature and medium. Therefore the extrapolation from in vitro electrode calibrations performed in PBS at room temperature to ex vivo (brain slices) and possibly in vivo measurements of DOPAC oxidation should be reconsidered. PMID- 8639284 TI - A fractal analysis of the influence of non-specific binding on antigen-antibody binding kinetics for biosensor applications. AB - A fractal analysis of the influence of non-specific binding on the specific binding of antigen in solution to antibody immobilized on a biosensor surface is presented for first-, one and a half-, second, and other-order reactions occurring under external diffusion-limited conditions. Both single-step and dual step binding of antigen in solution to antibody immobilized on the surface is considered. For a first-order reaction, an increase in the fractal parameters, b, leads to a decrease in the amount of antigen in solution bound specifically to the antibody on the surface when non-specific binding is either absent or present. The presence of non-specific binding leads to a decrease in the amount of antigen bound to the antibody on the surface. For a one and a half- and for second-order reactions and when non-specific binding is either absent or present to a small degree (alpha = 0.01), an increase in the fractal parameter, b, leads to a decrease in the amount of antigen bound specifically to the antibody immobilized on the biosensor surface. However, for an alpha value of 0.1, the maximum rate and the amount of antigen bound specifically to the antibody immobilized on the biosensor surface is obtained for fractal parameter values of 0.2 and 0.4, and 0.4 for the one and a half- and for second-order reactions, respectively. Apparently, some amount of heterogeneity is helpful in obtaining the optimum amount and rate of antigen in solution bound specifically to the antibody on the surface for reaction orders higher than one. The applicability of the approach to real antibody surfaces is demonstrated. PMID- 8639283 TI - A 'mixed' self-assembled monolayer for an impedimetric immunosensor. AB - A synthetic peptide with the amino acid sequence 135-154 of the capsid protein VP1 of the foot-and-mouth-disease virus was modified with omega hydroxyundecanethiol and applied together with non-derivatised omega hydroxyundecanethiol for consecutive adsorption onto gold electrodes according to self-assembling procedures. The binding of a specific antibody to prepared recognition layers could be monitored by measurement of impedance or capacitance. In order to avoid non-specific effects, all measurements were performed in the presence of BSA. The complex between the antigenic peptide and the antibody was split by applying 6 M urea solution. The gold electrodes were mounted into an optimised flow-through system in order to perform capacitance-time measurements. The immobilised peptide can be recognised repeatedly by specific antibodies. PMID- 8639285 TI - In vivo and in vitro deactivation rates of PTFE-coupled glucose oxidase. AB - The deactivation of immobilized enzymes is a major lifetime limiting factor in several types of potentially implantable biosensors. The deactivation rate of covalently immobilized glucose oxidase was examined in vitro in mock physiologic environments and in the peritoneal cavity of mice. A first order deactivation model describes the observed exponential decay of the enzyme. Deactivation rate constants ranging from 0.198 to 1.3 per day were measured depending on experimental conditions. Enzymes immobilized on PTFE (Teflon) substrates in the peritoneal cavity of mice exhibited greater catalytic lifetimes than control samples kept in glucose solution in vitro. PMID- 8639286 TI - What's in a name? A lot. PMID- 8639287 TI - George E. Shambaugh, Jr, MD. A pioneer of American otomicrosurgery. PMID- 8639288 TI - Reverse gullibility and scientific evidence. AB - A 19th century image of the medical profession's attitude toward disease transmission is introduced through Ignaz Semmelweis' hypothesis: Infection can be caused by an external agent transmitted when physicians fail to sterilize their hands between patient examinations. Semmelweis' test of his hypothesis reduced the obstetrical death rate from 18% to almost 1%. However, he was degraded, defrocked, and driven to death by a profession whose emotions contradicted the evidence. Medical professionals like to believe they are not gullible, a trait defined as being easily duped. They rightly believe in their ability to avoid the error of accepting a result not supported by adequate evidence. They are not so free of the complementary error: refusing to accept a result that is supported by adequate evidence, which might be thought of as reverse gullibility. It is just as bad a logical error and just as serious a denial of the best medical care for our patients. Clearly Semmelweis, and later Louis Pasteur and others who were disbelieved, were correct. The profession was duped by its emotional adherence to current practice. To be truly professional, let us believe our evidence rather than our biases and not suffer from reverse gullibility. PMID- 8639289 TI - Magnetic resonance imaging determination of tracheal orientation in normal children. Practical implications. AB - OBJECTIVE: To determine normative anatomical data on the pediatric upper airway. DESIGN: Anatomical study of the trachea and surrounding structures in vivo using magnetic resonance imaging. Three-dimensional positions of the hyoid bone, sternum, skin, and trachea were determined. Tracheal angulation was measured from the vertical plane. SUBJECTS: One hundred children (53 males and 47 females) undergoing magnetic resonance imaging of the brain or chest. INTERVENTION: None. RESULTS: Ages ranged from 2 days to 20.4 years, with a median of 6.05 years. Gender differences were not significant. The mean distance from the hyoid bone to the sternal notch was 63.0 mm (SD, 22.04 mm). The skin to anterior tracheal wall distance averaged 9.34 mm (SD, 3.29 mm), and tracheal diameter averaged 8.48 mm (SD, 2.88 mm). The trachea was not found to be a linear structure. An anterior angulation change of 9.9 degrees (SD, 7.89 degrees) was detected. The point of inflection was found to lie below the sternal notch in children younger than 2 years and above the notch in older children. CONCLUSIONS: Normal anatomical data of the pediatric airway have been acquired using magnetic resonance imaging. Because the trachea exhibits a previously unknown anterior angulation, current design of tracheotomy tubes may not be ideal. Rigid tracheotomy tubes that do not conform to the anatomical structure of the trachea may be responsible for suprastomal collapse or innominate artery erosion. PMID- 8639290 TI - Injury prevention counseling opportunities in pediatric otolaryngology. AB - OBJECTIVE: To determine the prevalence of injury prevention counseling opportunities in children referred to a pediatric otolaryngologist. DESIGN: A caregiver questionnaire was administered during office registration. SETTING: Hospital-based pediatric otolaryngology practice in a metropolitan area. PATIENTS: Random sample of 300 caregiver questionnaires stratified by the age of the child. MAIN OUTCOME MEASURES: Prevention counseling opportunities, defined as ignorance of hottest water temperature, child exposure to passive smoke, missing smoke detectors on one or more floors in the home, or failure to use a seat belt or bicycle helmet. RESULTS: The hottest water temperature was unknown by 72% of the caregivers, smokers were present in 25% of the households, bicycle helmets were not used by 22% of the children, car seats or seat belts were not used by 11% of the children, and 10% of the homes did not have a working smoke alarm on each floor. Older children were significantly less likely to use a seat belt than were younger children. Although 98% of the caregivers had a regular pediatrician, 91% of the families still offered one or more counseling opportunities (95% confidence interval, 87% to 94%). CONCLUSIONS: Substantial opportunities exist for injury prevention counseling in pediatric otolaryngology. At least one opportunity for counseling is present for about 90% of the families, most often concerning the maximum safe hot water temperature. PMID- 8639291 TI - Laryngeal dyskinesia as a cause of stridor in infants. AB - OBJECTIVE: To describe 9 cases of stridor attributed to the failure of the vocal cord to abduct during inspiration. DESIGN: Case series. SETTING: Pediatric otolaryngology referral center. PATIENTS: Nine hospitalized infants, aged 1 to 13 months, presented over a 3-year period for exploration of inspiratory stridor that was attributed to a condition that we have termed laryngeal dyskinesia. RESULTS: A consistent clinical presentation was noted in all cases. The laryngeal dyskinesia occurred during calm breathing, crying, or sleep and was associated with gastroesophageal reflux in 8 cases (diagnosed clinically and/or with pH monitoring). In addition, 3 infants suffered from fainting spells associated with vagal hypertonia that was confirmed by 24-hour Holter monitoring. True paralysis of the abductor muscles was ruled out in all infants because of the presence of normal glottic motion during calm breathing or induction of anesthesia. The stridor resolved between the ages of 4 and 13 months in 7 of the patients. Improvement was progressive and had no clear relationship to treatment for gastroesophageal reflux. CONCLUSION: Laryngeal dyskinesia in infants seems to be a distinct clinical entity, frequently associated with gastroesophageal reflux. PMID- 8639292 TI - Refinements in pediatric microtia reconstruction. AB - Auricular construction for a child born with microtia is a rewarding endeavor that can provide the patient with a realistic pinna. However, not all reconstructive problems have been solved. For example, the donor sites for the rib harvest and for subsequent skin grafting are not without morbidity and scar formation. Furthermore, in some children, an unexpected shortage of rib can be encountered. Surgical techniques that address these problems are described. PMID- 8639293 TI - Risk factors for advanced-stage oral cavity cancer. AB - OBJECTIVE: To assess the association between risk factors for inadequate surveillance of oral cavity cancer and stage of disease (localized, T1, T2/N0 vs advanced, T1, T2/N1-3, T3, T4/N0 or N1-3). DESIGN: Convenience sample from a case series. SETTING: Otolaryngology clinic in a tertiary care hospital. PARTICIPANTS: Fifty-three patients with cancer of the oral cavity who were treated at The University of Iowa, Iowa City, from October 1990 through March 1994, participated in the study. Selection criteria included pathologic confirmation of squamous cell carcinoma (SCC) of the oral cavity, the capacity to retrieve data regarding tumor characteristics at initial presentation, and completion of a 30-item questionnaire by the patient. INTERVENTION: Administration of questionnaire identifying factors contributing to inadequate surveillance of cancer of the oral cavity. OUTCOME MEASUREMENTS: Advanced-stage cancer of the oral cavity was identified by the presence of large tumors (T3, T4) and cancer metastatic to the neck lymph nodes (N1, N2, N3). Comparison groups were built to determine the relationship between these two dependent variables and multiple independent variables. Descriptive statistics and tests of association were used to assess relationships. RESULTS: Two of the 53 patients performed self oral examinations specifically designed to screen for cancer prior to finding cancer of the oral cavity. Knowledge of the warning signs of cancer of the oral cavity was denied by 87%. The rate of cancer growth in the oral cavity was variable from first discovery by the patient to the time of tumor staging by otolaryngologists. The interval from discovery of the tumor to tumor staging (delay in diagnosis) was greatest for floor of the mouth cancers and shortest for those cancers located on the tongue. Thirty-seven percent of the patients younger than age 64 years were edentulous in contrast to 62% edentulism in patients older than 65 years. There was a significant, inverse relationship between time since last dental visit and late-stage disease. CONCLUSIONS: Patients with advanced-stage cancer of the oral cavity tended to be elderly, more often wore dentures, and seldom visited the dentist. Treatment of cancer of the oral cavity as localized disease, with an associated decrease in morbidity and mortality, is likely to result by targeting this population as one in need of more intense surveillance. PMID- 8639295 TI - Lower gingival carcinoma. Clinical and pathologic determinants of regional metastases. AB - OBJECTIVE: To determine which clinical and pathologic features are associated with regional metastases in patients with lower gingival squamous cell carcinoma. PATIENTS AND METHODS: The medical charts of 155 previously untreated patients seen between 1970 and 1990 were retrospectively analyzed. All patients underwent surgical resection of the primary tumor. In addition, 66 patients underwent elective neck dissection, while a therapeutic neck dissection was performed in 28. Sixty-one patients who had clinically N0 neck disease did not undergo treatment of the cervical lymphatics. RESULTS: T stage (P = .01), radiologic (P = .03) or histologic (P = .01) evidence of mandibular invasion, and decreased tumor differentiation (P = .004) significantly correlated with the presence or evolution of regional metastases. In addition, tumors involving the symphyseal region were associated with an increased incidence of nodal metastases, although the relationship did not achieve statistical significance (P = .08). Occult regional disease was found in 18% of patients who underwent elective neck dissection, and the presence of metastases was pathologically confirmed in 68% who underwent a therapeutic dissection. Six patients with clinically N0 neck disease did not undergo elective dissection and later developed regional metastases. In all patients, survival was adversely impacted by the presence or later development of regional metastases (P < .001). Two- and 5-year survival rates for patients with no cervical metastases were 0.91 and 0.85, respectively, while for those with cervical metastases, the survival at 2 and 5 years declined to 0.72 and 0.59. More importantly, the 2- and 5-year survivals of patients with clinically N0 necks who were found to have lymph node metastases histologically after neck dissection were 1.00 and 0.78. This contrasts with the 0.50 survival rate at 2 and 5 years for those who did not undergo elective dissection and later developed cervical metastases (P = .36). CONCLUSIONS: Patients with adverse clinical and pathologic features, even in the absence of demonstrable neck disease, are at risk for harboring regional metastases. Elective treatment of the cervical lymphatics should be considered for patients with primary tumors that overlie the mandibular symphysis, moderately or poorly differentiated tumors, or radiographic or histologic evidence of mandibular invasion. PMID- 8639294 TI - Correlation of tumor markers p53, bcl-2, CD34, CD44H, CD44v6, and Ki-67 with survival and metastasis in laryngeal squamous cell carcinoma. AB - BACKGROUND: Recent basic discoveries about the biological significance of nuclear and cell-surface marker proteins have opened new areas of research into head and neck cancer. However, the clinical significance of these markers is not yet understood. OBJECTIVE: To perform a historical prospective study of 70 patients with squamous cell carcinoma of the larynx who were treated at our institution between 1979 and 1989 to correlate tumor marker expression with survival and metastasis. DESIGN: Archival tissue was immunohistochemically stained for the p53 tumor suppressor gene product, the inhibitor of apoptosis (bcl-2), the stem cell marker CD34, the cell adhesion molecules CD44H and CD44v6, and a marker of cellular proliferation (Ki-67). The slides were examined using a light microscope and scored according to intensity and percentage of cells labeled. The patients were stratified by tumor stage, and survival and metastatic data were correlated with staining scores. RESULTS: For the stage IV group, increased expression of p53 and decreased expression of CD44H and CD44v6 correlated with a decreased survival (P = .03, P = .03, and P = .02, respectively), and decreased expression of CD44H correlated with an increase in metastasis (P = .01). For all stages, excluding metastatic cases, increased p53 expression was consistent with a shorter survival (P < .03), while increased CD44v6 expression was consistent with a longer survival (P < .02). CONCLUSIONS: The present study suggests that a loss of cell proliferation control implied by overexpression of p53 and loss of cell adhesion implied by decreased expression of CD44 may be determinants of survival in patients with carcinoma of the larynx. The tumor markers bcl-2 and Ki-67 were not prognostic discriminators in this limited series. This study also indicates that the stem cell marker CD34 is rarely expressed by laryngeal carcinoma cells. PMID- 8639297 TI - Regional metastases in patients with advanced laryngeal cancer. Department of Veterans Affairs Laryngeal Cancer Study Group. AB - OBJECTIVES: To determine patterns of regional metastases in patients with advanced squamous cell carcinoma of the larynx and the effect of these patterns on regional and distant tumor recurrence and disease-free and overall survival. METHODS: One hundred fifty-nine patients treated with conventional surgery and radiation in the Veterans Affairs Cooperative Study were studied. Ninety-three of the patients underwent modified or classic radical neck dissection. Median follow up was 5 years. Patient data collected included age, gender, alcohol consumption, tobacco use, and performance status. Tumor variables evaluated included TNM classification, tumor site, surface area, presence of ulceration and invasion, and histologic growth pattern. Histologically positive regional lymph nodes were examined for level, number, site, and extracapsular spread. Outcome variables included time and location of recurrence, distant metastases, and survival. RESULTS: Regional metastases were more common in supraglottic than glottic or subglottic tumors (P < .001) and in tumors with larger surface dimensions (P = .004). Cervical metastases were associated with decreased disease-free interval (P < .001) and survival (P = .001) and increased distant metastases (P < .001). Presence of 3 or more positive nodes predicted distant recurrence (P < .001) and decreased survival (P < .001, multivariate analysis). An analysis of lymph node involvement (levels I-V) indicated that level 1 and 5 involvement was a significant prognostic factor. Age, sex, race, and tobacco or alcohol use were not associated with number or extent of regional metastases. CONCLUSIONS: These findings confirm the prognostic importance of number and level of lymph nodes in advanced laryngeal cancer. The association of distant metastases with number and level of regional nodes indicates a use for these variables in considering adjuvant chemotherapy. PMID- 8639296 TI - Upper aerodigestive tract squamous cell carcinoma. The human immunodeficiency virus connection. AB - OBJECTIVE: To evaluate the incidence, distribution, and course of squamous cell carcinoma (SCC) of the upper aerodigestive tract in patients infected with the human immunodeficiency virus (HIV) and compare it to SCC in non-HIV-infected patients. DESIGN: Case-control study of all patients with SCC during a 9.5-year period from January 1985 through June 1994. SETTING: Two academic tertiary care centers in a metropolitan location. PARTICIPANTS: Five hundred thirty-nine patients (18 to 95 years old) with SCC of the upper aerodigestive tract. RESULTS: Infection with HIV was present in 4.5% of the patients with SCC of the upper aerodigestive tract. Patients infected with HIV were significantly younger than noninfected patients (P < or = < .001), accounting for 21.3% of those patients younger than 45 years (P < .001). No significant difference in tumor location was present between HIV-infected and noninfected patients; however, HIV-infected patients had larger tumors (P = .004) and a more advanced tumor stage (TNM classification) at presentation (P = .05). Tumor-related survival was significantly poorer in patients with HIV infection (P = .01), with 57% at 1 year and 32% at 2 years, compared with 74% and 59%, for non-HIV-infected patients. The detrimental effect of HIV infection on survival remained significant after adjusting for the confounding effects of age, tumor stage, and location of the tumor. All study patients with HIV infection had cancer risk factors such as tobacco and/or alcohol abuse. CONCLUSIONS: Infection with HIV possibly accelerates the development of SCC in patients with significant risk factors, presumably by impairing normal immune surveillance mechanisms. The decreased survival rates among these patients suggests that the SCC may be more aggressive or that other cofactors assume greater importance. A history of tobacco and/or alcohol abuse in patients with HIV infection warrants aggressive screening and early detection, to allow for early detection, which may help increase survival. PMID- 8639298 TI - Reconstruction of the laryngeal mucosa. A three-dimensional collagen gel matrix culture. AB - OBJECTIVE: To prepare an in vitro reconstruction of the porcine laryngeal mucosa, for use in cell biological investigations and as a model for the study of laryngeal disease. DESIGN: Using separately obtained epithelial cells and fibroblasts from porcine laryngeal mucosa, we reconstructed the laryngeal mucosa in a three-dimensional collagen gel matrix culture with air-liquid interface. SUBJECTS: Porcine larynges were from 6-month-old pigs obtained from a local abattoir. RESULTS: We successfully reconstructed the laryngeal mucosa in vitro, including epithelium and lamina propria. The epithelial cells showed five to eight cells in thickness and were well differentiated on the reconstructed lamina propria. The differentiation-specific cytokeratin was positive. CONCLUSIONS: To our knowledge, this is the first report of a reconstruction of the laryngeal mucosa in a three-dimensional collagen gel matrix culture. Fibroblasts and air liquid interface treatment exert a great influence on the proliferation and differentiation of the cultured epithelial cells. This culture system will help to provide an appropriate physiologic environment to study the differentiation and disease of the larynx. PMID- 8639299 TI - Preservation of the eye in the treatment of sinonasal malignant neoplasms with orbital involvement. A confirmation of the original treatise. AB - OBJECTIVE: To continue the retrospective analysis reported in 1988 that supported preservation of the eye in the treatment of sinonasal cancers when bony erosion of the orbit was noted on pretreatment radiographic analysis. DESIGN: Using the eye-sparing protocol previously reported, which included preoperative radiotherapy (with or without chemotherapy, depending on tumor size, involvement, or other characteristic) for malignant neoplasms of the superior nasal vault that had eroded the bony orbit, retrospective analysis of the results of therapy for the additional 33 patients treated between 1986 and 1993 was performed. These data were added to those from the original series to provide a total of 74 patients. Forty-one patients (55%) showed bony erosion at initial evaluation and 14 (19%) had periorbital involvement. During surgery, the periorbita was evaluated by frozen section control. If tumor was found, that region of periorbita was resected and, if necessary, replaced with fascia or split thickness graft. RESULTS: Five patients from the original series had orbital sacrifice. Of the remaining 36 patients, 4 (11%) had recurrent disease involving the orbit, but not at the primary site. Twenty (55%) of the 36 patients had no orbital complications, and only 1 (3%) had a permanent motility disturbance and 4 (13%) developed cataracts after radiotherapy. CONCLUSION: With the use of preoperative radiotherapy in resection of involved periorbita with frozen section control in tumors of the sinonasal vault that involve the bony orbit, the eye can be spared in most instances without compromising oncologic safety. PMID- 8639300 TI - Evaluation of prevertebral muscle invasion by squamous cell carcinoma. Can computed tomography replace open neck exploration? AB - OBJECTIVE: To compare computed tomography (CT) with open neck exploration in determining prevertebral invasion by squamous cell carcinoma of the oropharynx or hypopharynx. DESIGN: Retrospective analysis using the findings at open neck exploration and results of histopathologic studies as the criterion standards. SETTING: Tertiary care referral center. PATIENTS: Twenty-nine of 40 patients with advanced squamous cell carcinoma of the oropharynx or hypopharynx treated between January 1, 1986, and December 31, 1994, were selected for analysis based on CT findings of posterolateral extension of the primary tumor placing the prevertebral muscle (PVM) at risk. All study patients had no previous therapy and underwent neck exploration to determine resectability. RESULTS: Overall accuracy of CT in predicting PVM status was 55.2%. The sensitivity of preoperative CT for PVM invasion was 50%; the specificity was 61%. Using an estimate of 21% for the prevalence of PVM invasion, the predictive value of a positive CT scan was 0.254 and the predictive value of a negative CT scan was 0.821. Open neck exploration correctly predicted PVM status in all cases. CONCLUSIONS: Open neck exploration is superior to CT to evaluate possible PVM invasion by squamous cell carcinoma of the oropharynx or hypopharynx. The predictive value of a negative CT scan for PVM invasion is high, so it may be useful in treatment planning. Patients with advanced squamous cell carcinoma of the oropharynx or hypopharynx at risk for PVM invasion who are otherwise surgical candidates should be considered for open neck exploration to determine resectability most accurately. PMID- 8639301 TI - Wide polytef (Gore-Tex) implants in lip augmentation and nasolabial groove correction. AB - OBJECTIVE: To describe a new technique of polytef (Gore-Tex) implantation into the upper and lower lips and nasolabial grooves by using large implants as a method that achieves effective cosmetic improvement. SETTING: A private cosmetic surgery center. PARTICIPANTS: Thirty-three (female) patients who desired fuller lips and 62 patients (52 female and 10 male) who requested less prominent cheek lip grooves. MAIN OUTCOME MEASURE: Significant patient satisfaction after 12 to 54 months. RESULTS: Conspicuous aesthetic effect that related to both lip and nasolabial groove correction was documented. All patients but 4 (2 in each group) were pleased with the final outcome of the treatment. CONCLUSIONS: In the opinion of the authors, the threading technique of polytef implantation creates inconspicuous improvement-both in lip augmentation and nasolabial groove correction. Large polytef implants that were inserted through a tunneling technique produced consistently good results. Implants (lip augmentation: width, < or = 10 mm, and thickness, 4 mm; nasolabial groove correction: width, 8 mm, and thickness, < or = 8 mm) were found to be safe, simple, and effective. PMID- 8639303 TI - Pathologic quiz case 1. Bronchogenic cyst. PMID- 8639302 TI - Lateral mandibular reconstruction using soft-tissue free flaps and plates. AB - OBJECTIVE: To assess the outcome of patients who are undergoing reconstruction of segmental lateral mandibular defects by using soft-tissue free flaps combined with mandibular reconstruction plates. DESIGN: Retrospective case series of 15 patients who were undergoing primary reconstruction of mandibular segments posterior to the mental foramen, resulting from treatment of head and neck cancer. All patients received either preoperative or postoperative radiation therapy. SETTING: Academic tertiary care referral center. INTERVENTIONS: Fourteen patients had mandibular continuity restored by using the titanium hollow screw reconstruction plate system, and 1 patient received a stainless steel mandibular reconstruction plate. Associated soft-tissue defects were repaired by using radial forearm (n = 11), rectus abdominis (n = 2), scapular and parascapular (n = 1), or lateral arm (n = 1) free flaps. MAIN OUTCOME MEASURES: Early and delayed complications. RESULTS: All 15 microvascular free tissue transfers were successful. Early complications were minor and occurred in 5 (33%) of 15 patients. One patient in whom the titanium hollow screw reconstruction plate system had been used experienced a fracture at 15 months after reconstruction. Three patients experienced delayed external plate exposure between 7 and 15 months after primary oromandibular reconstruction. Patients who experienced delayed external plate exposure required secondary reconstruction with a vascularized bone-containing free flap. The overall rate of delayed reconstructive failure was 40% in patients who were followed up for a minimum of 1 year. CONCLUSIONS: For patients who are undergoing free flap reconstruction of lateral mandibulectomy defects, the technique that used soft-tissue free flaps combined with mandibular reconstruction plates has been abandoned in favor of using vascularized bone-containing free flaps or a combination of free flaps to achieve optimal long-term results. PMID- 8639304 TI - Pathologic quiz case 2. Ossifying fibroma of the premaxilla. PMID- 8639305 TI - Mechanism of microbial utilization of biphenyl sorbed to polyacrylic beads. AB - A microbial consortium mineralized biphenyl sorbed to polyacrylic beads faster than the slow rate at which much of the compound was desorbed. Pure cultures of bacteria isolated from the consortium mineralized biphenyl in solution but not the sorbed compound. However, combinations of two strains did degrade biphenyl. The consortium did not reduce the surface tension in media containing sorbed biphenyl or biphenyl in solution, and addition of synthetic and microbially produced surfactants to pure cultures did not result in utilization of sorbed biphenyl by isolates able to use the soluble molecule. Cells from the consortium that were attached to continuously washed beads degraded the substrate. We suggest that bacteria may act on sorbed compounds without the necessity of an initial desorption and that the mechanism may involve cells attached to the particles rather than the excretion of a surfactant. PMID- 8639306 TI - Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis. AB - A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10-hydroxystearic acid. PMID- 8639307 TI - Novel mechanisms of antiprogestin action. AB - Endocrine therapy used either prophylactically or therapeutically for the treatment of locally advanced or metastatic breast cancers offers many advantages to patients whose tumors contain functional estrogen (ER) and progesterone (PR) receptors. The range of treatments defined as endocrine include surgical ablation of endocrine glands, administration of pharmacologic doses of steroid hormones, chemical blockade of steroid hormone biosynthesis, and inhibition of endogenous steroid hormone action at the tumor with synthetic antagonists. The last of these approaches is the most widely used, making the antiestrogen tamoxifen the preferred first-line therapeutic agent for treatment of hormone-dependent metastatic breast cancer. The wide-spread use of tamoxifen reflects its efficacy and low toxicity, and the fact that it makes good physiological sense to block the local proliferative effects of estrogens directly at the breast. But are estrogens the only hormones with a proliferative impact on the breast and on breast cancers? This chapter focuses on evidence that progesterone also has proliferative actions in the breast; on preliminary data showing that progesterone antagonists may be new tools for the management of metastatic breast cancer; and on recent data suggesting that antiprogestin-occupied PR have novel mechanisms of action that bear on tissue specificity and development of hormone resistance. PMID- 8639308 TI - Monitoring palliative chemotherapy in advanced gastrointestinal cancer using serial tissue polypeptide specific antigen (TPS) measurements. AB - Tissue polypeptide antigen specific (TPS) was analysed in serum taken prior to chemotherapy in 90 patients with advanced gastrointestinal cancer and prior to every treatment course in 68 of these patients in order to explore whether serial tumour marker measurements can be of importance in monitoring patients treated with palliative chemotherapy. Elevated TPS levels were seen in 83/90 (92%) patients (48/52 colorectal, 9/9 pancreatic, 9/11 biliary, 17/18 gastric). Baseline TPS level correlated with performance status, tumour response and survival. Based upon the change in TPS levels after the first two courses in relation to baseline, a decrease by >50% had a high sensitivity for a favourable treatment outcome (partial remission and prolonged stationary disease (90%) or a subjective response (100%)), whereas the specificity was lower (72% and 73% respectively). A similar result was seen when the TPS levels were analysed at the time of the response evaluation after 2 months (sensitivity 91 and 95%, specificity 74 and 75% for an objective or subjective response respectively). In 7 out of 15 patients with an initially favourable outcome, an increase in TPS levels of >50% at two occasions was seen 8-20 weeks prior to clinical disease progression. In advanced gastrointestinal cancer serial TPS measurements can with high accuracy early identify patients who will not benefit from the treatment. On the other hand, a response must be confirmed using other methods in the presence of a decrease, since this was also seen in non-responding patients. PMID- 8639309 TI - Quality of life evaluation by the EORTC questionnaire technique in patients with generalized malignant melanoma on chemotherapy. AB - A longitudinal quality of life (QOL) study was performed on patients with advanced melanoma during chemotherapy. The purpose was to describe QOL in this palliative context and to compare the clinical outcome variables and patients' self-assessed QOL. QOL was assessed by the EORTC core questionnaire technique (QLQ-C36), a study-specific melanoma (MM module and the Hospital Anxiety and Depression (HAD) scale. The questionnaires displayed good psychometric qualities and the technique proved to be applicable in this longitudinal study of severely ill patients. Only six patients, out of 95 in total, complied with the full one year study. Drop-outs occurred early in the course of treatment, most of them due to progressive disease or death. Pretreatment, patients reported a low level of dysfunction and symptom burden but 9 weeks later they exhibited significant deterioration in all QOL measurements, with the exception of pain and emotional functioning. The mean duration of response was short and there was considerable observed treatment-related toxicity. However, no correlation was found between physician-rated clinical outcome variables and QOL measurements, except for neuropathy. Our results are in accordance with earlier data on the supplementary value of QOL measurements to define endpoints in clinical trials. PMID- 8639310 TI - Antiemetic efficacy of ondansetron and metoclopramide, both combined with corticosteroid, in malignant lymphoma patients receiving non-cisplatin chemotherapy. AB - The aim of the present study was to compare the antiemetic efficacy of ondansetron (OND) with metoclopramide (MCP), both combined with corticosteroid (CS) in patients with malignant lymphoma. A total of 109 patients with malignant lymphoma receiving their first series of non-cisplatin chemotherapy (CT) (CHOP or MOPP) were divided into prospective, randomized, open and parallel groups and analyzed at two hematological centres at university hospitals in Copenhagen, Denmark. The patients were randomized to receive one of the two following regimens; 1) OND 8 mg/methylprednisolone 80 mg i.v. before CT and OND 8 mg p.o. after 8 h and at bedtime. OND 8 mg tid days 2-3, and 8 mg tid prn days 4-5 and prednisolone 75-100 mg qds days 2-5 and 2) MCP 30 mg/metylprednisolone 80 mg i.v. before CT and MCP 20 mg p.r. after 4 and 8 h respectively. MCP 20 mg p.r. prn days 1-5 and prednisolone 75-100 mg qds days 2-5. In the acute phase OND/CS was superior to MCP/CS in the control of nausea and emesis, resulting in no emesis in 92% of the OND/CS treated group vs. 50% treated with MCP/CS (p < 0.001), and no nausea in 79% (OND/CS) vs. 42% (MCP/CS) (p < 0.001). The ultimate aim - neither nausea nor emesis - was reached in 77% (OND/CS) vs. 35% (MCP/CS) day 1 (p < 0.001). OND/CS is significantly better than MCP/CS in the control of delayed nausea, 81% (OND/CS) vs. 58% (MCP/CS) (p < 0.026). Both the OND/CS and MCP/CS regimens are highly effective in the control of delayed emesis, 94% (OND/CS) vs. 85% (MCP/CS) (p < 0.26). Adverse events were mild and experienced in 31% of the patients. In the OND/CS group 13% had constipation vs. 8% in the MCP/CS group. Nine percent treated with OND/CS had headaches compared to none treated with MCP/CS (p < 0.08). One extrapyramidal reaction was recorded in the MCP/CS group. In malignant lymphoma patients receiving moderately emetogenic CT, the combination of OND and CS was very effective and significantly better than low dose MCP and CS in the control of acute emesis, acute nausea and delayed nausea. PMID- 8639311 TI - Methyl-GAG, ifosfamide, methotrexate and etoposide (MIME) as salvage therapy for non-Hodgkin's lymphomas: a Swedish national prospective study. Swedish Lymphoma Study Group. AB - One hundred and two patients with recurrent or refractory non-Hodgkin's lymphoma (NHL) were treated with MIME (methyl-GAG, ifosfamide, methotrexate, etoposide) in accordance with a prospective protocol. Of 75 patients with high-grade malignant NHL (median age 57 years, range 21-79), 15 patients (20%) obtained a complete response (CR) and 27 patients (36%) a partial remission (PR), giving an overall response rate of 56%. The remissions were usually short when not consolidated with ABMT or radiotherapy. The probability of progression-free survival after 2 years was 13%, and the cause-specific survival was 23%. Of 27 patients with low grade NHL (median age 46 years, range 37-86), 7% had a CR and 37% a PR giving a response rate of 44%. The remissions were again usually short when not consolidated, and the probability of progression-free survival at two years was 11%, and the cause-specific survival 26%. The main toxicity was hematological with septicemia in 20% of the patients and other severe infections in 19%. Fifteen patients (11 high-grade NHL and 4 low-grade NHL) were consolidated with high-dose therapy followed by ABMT, of whom 6 are in continuous CR. We conclude that MIME can induce remissions in NHL patients, and that the remission rates are comparable with those of many other salvage regimens. The remissions are, however, generally of short duration and need consolidation. There was considerable toxicity therefore patients not suitable for ABMT preferably should be treated with less toxic salvage regimens. PMID- 8639312 TI - High-grade non-Hodgkin's lymphoma stage I. A retrospective study of treatment, outcome and prognostic factors in 213 patients. AB - In a retrospective study of 213 patients with high-grade non-Hodgkin's lymphomas clinical stage I, diagnosed 1985-1990, pretreatment prognostic variables and result of treatment were analysed. The median age of the patients was 67 years. Treatment consisted of radiotherapy in 61%, chemotherapy (10%) chemotherapy followed by radiotherapy (23%) and surgery alone (5%) of the patients. Complete response was achieved in 89% of the patients with estimated relapse-free survival at 5 years of 73%. Relative 5-year survival of all patients was 73%. After chemotherapy followed by radiotherapy the relapse rate was 15% compared with 29% after radiotherapy only. The 5-year relative survival differed between 58% and 74% in the treatment groups. Age, sex, nodal versus extranodal lymphoma, systemic symptoms, bulk of tumor and level of serum lactic dehydrogenase (s-LDH) were analysed as prognostic factors. In multivariate variate analysis, only age 65 years or older and elevated s-LDH were significant independent adverse prognostic factors. PMID- 8639313 TI - Cancer incidence for children born in a smelting community. AB - The Ronnskar smelter in Skelleftea, Sweden, produces significant environmental pollutants, such as lead, arsenic, copper, cadmium and sulphur dioxide. The purpose of the present study was to determine whether children born to women living near the smelter during pregnancy had an increased risk of childhood cancer. The study group consisted of children born between 1961 and 1990 in the municipality of Skelleftea and parish of Holmsund. Through linkage to the Swedish Cancer Registry cancer diagnoses in the study group were obtained and compared with the expected ones based on the national incidence rates. Thirteen cases of childhood cancer were identified among children born in the vicinity of the smelter against 6.7 expected (SIR 195, 95%CI 88-300). Among distant born the observed number of cases (n = 42) was similar to that expected (n = 41.8). PMID- 8639314 TI - Lung cancer in West Sweden 1976-1985. A study of trends and survival with special reference to surgical treatment. AB - We reviewed 3 285 consecutive cases of lung cancer diagnosed in West Sweden during the period 1976-1985. Data were collected from the regional cancer registry, the Swedish National Population Registry, and medical records. During the study period, the annual female/male ratio increased from 0.29 to 0.42. In females, there was an increase primarily in the incidence of tobacco-related morphologic tumour types (i.e. squamous and small cell lung cancers). In males, a moderate increase of adenocarcinomas was seen, although squamous cell cancer remained the most common tumour type. The overall 5-year survival rate was 8.3%. In 641 patients (20%) a surgical tumour resection was carried out. The 5-year survival rate following resection was 38%, and the probability of 10-year survival was estimated at 25%. In a multifactorial model including gender, age, histology, pTNM stage and extent of resection, pTNM stage and, to a lesser degree, age were statistically significant independent predictors of postoperative survival. The five-year survival was 57% in stage 1, 21-27% in stage II and IIIa, and 10% in stage IIIb. Of all resected patients, 4.2% died within two months after resection. In males, early postoperative mortality was predicted by preoperative bicycle ergometry. The prognosis in non-resected patients was poor, with only 2% surviving 5 years or longer. In conclusion, the results indicate that some progress has been made with regard to surgical management of lung cancer, but they also point to the fact that the vast majority of patients are not amenable to curative treatments, and that the overall survival in lung cancer has improved only marginally during the last decades. PMID- 8639315 TI - Variants of Kaposi's sarcoma in Southern Africa. A retrospective analysis (1980 1992). AB - All types of Kaposi's sarcoma (KS) are represented in the Southern African region. We present a retrospective analysis of patients with KS, treated and followed up at the Johannesburg General Hospital over a 12-year period (1980 1992). One hundred and nineteen patients with KS, divided into four groups according to their etiology (classical; endemic African; renal transplant recipients; epidemic AIDS-related) were analyzed. Choice of treatment (radiotherapy or chemotherapy) was individualized and based on clinical criteria, extent of disease and severity of symptoms. Kaposi's sarcoma showed a very high response rate to radiation therapy, regardless of variant, radiation modality or schedule. Chemotherapy was also effective in the more aggressive pattern of endemic African KS. Epidemic Kaposi's sarcoma showed the same poor outcome as demonstrated by its Western counterpart. We conclude that radiation therapy can provide excellent palliation with only minimal side-effects in all variants of KS seen in Southern Africa. PMID- 8639316 TI - Toxicity in patients with testicular seminoma treated with radiotherapy. Different dose levels and treatment fields. AB - The aim of this study was to evaluate the acute and late effects of irradiation in 56 patients with stage I and II testicular seminomas. A retrospective study of patients' records was performed paying attention to the acute and late toxicity of radiation in relation to treatment fields and radiation doses. Treatment groups were compared using the chi squared-test. Mild to moderate nausea and/or vomiting was seen in 66% of patients and occurred equally independent of the treatment volume or radiation dose. Increased bowel frequency was seen in 59% and was more common when a larger treatment volume was used. Skin reactions increased with increase in treatment volume and dose (p = 0.046). Severe late complications were recorded in two patients (myocardial damage-1/4 at risk, duodenal ulcer-1/56 at risk). These could not be attributed solely to the irradiation as other contributing factors might play a role. Overall the data suggest that the risk of major posttreatment morbidity is minimal for patients with testicular seminoma treated with postoperative radiotherapy. PMID- 8639317 TI - Different intravenous administration techniques for 5-fluorouracil. Pharmacokinetics and pharmacodynamic effects. AB - The pharmacokinetics after 20 min intravenous infusion or a 2 min bolus (push) injection of 5-fluorouracil (500 mg/m2) were studied in 14 colorectal cancer patients. Treatment effects and toxicity related to the administration technique of 5-fluorouracil were retrospectively analysed in 198 colorectal cancer patients. The AUC after bolus injection was 6158 +/- 874 micromol/l*min compared to 3355 +/- 428 micromol/l*min after short-time infusion of 5-fluorouracil (p < 0.01). The mean peak-level after bolus injection was 341 +/- 34 microM versus 161 +/- 17 microM after a short-time infusion (p < 0.01). Patients receiving bolus injections had significantly better treatment result (32% partial remission) than patients receiving infusion (10% partial remissions, p < 0.001). Toxic side effects were more frequently encountered after bolus injection but subjective improvement was also more frequently experienced by these patients. Bolus 5 fluorouracil push injection rather than a short-time infusion appears to be the more efficient administration technique. PMID- 8639318 TI - Nicotinamide pharmacokinetics in normal volunteers and patients undergoing palliative radiotherapy. AB - The influence of nicotinamide formulation on absorption characteristics and incidence of adverse side-effects has been studied in normal volunteers and in patients undergoing radiotherapy. Escalating single or repeated oral doses of nicotinamide were administered in tablet or liquid form under fasting or non fasting conditions. Drug absorption was slowed both by the presence of food in the stomach and by the administration of nicotinamide in tablet form compared with when it was dissolved in orange juice. Peak concentrations were generally slightly higher following the liquid preparation, but the incidence of adverse side-effects (chiefly nausea) was increased. A single dose of 9 g (88-97 mg/kg) nicotinamide in tablet form was well tolerated in two fasting normal volunteers, and in patients, doses of up to 133 mg/kg as tablets were tolerated twice/week for three weeks. Daily administration of 80 mg/kg nicotinamide was tolerated when given as tablets, but not in a liquid formulation. Neither the peak concentration nor the area under the concentration/time curve (AUC) of nicotinamide, nor the main metabolites of nicotinamide appeared to correlate with the incidence of toxicity. PMID- 8639319 TI - Primary mediastinal malignant germ cell tumour. Single institution experience in Chinese patients and correlation with specific alpha-fetoprotein bends. AB - Ten Chinese patients were reviewed, all with mediastinal germ cell tumours and treated in our centre during the past 8 years. Three patients with pure seminomas were given chemotherapy with or without radiotherapy. AB achieved complete remission with no relapse. Seven patients with non-seminomatous germ cell tumours (NSGCT) were given chemotherapy, with or without surgery. Two patients with rapid decay of alpha-fetoprotein (AFP) levels (half-life less than or equal to 7.2 days) during chemotherapy achieved complete remission with no relapse. Five patients with prolonged decay of AFP levels (half-life > 7.2 days) failed to achieve complete remission with initial chemotherapy and all but one patient died between 5 and 9 months later. One patient developed acute megakaryocytic leukaemia. Using isoelectric focusing, AFP bands specific to NSGCT were quantified, and comparison was made with the total AFP in five cases. In each case the change in NSGCT-specific AFP concentration in response to therapy closely paralleled that of total AFP. Estimation of NSGCT-specific AFP offers no apparent advantage in monitoring disease response or progression. PMID- 8639320 TI - Absence of misincorporation of pyrimidines in DNA after treatment with a combination of cisplatin (CIS-diammine-dichloro-platinum) amd 5-fluorouracil of mouse sarcoma cells. AB - The effects on incorporation into DNA of the deoxyribonucleotides dCTP and dTTP and the DNA synthesis rate after treatment with cisplatin (CDDP), 5-fluorouracil (5-FU) or a combination of CDDP and 5-FU were studied in ascites sarcoma (Bp8) growing in mice. Single administration of CDDP gave an early (1 h) transient increase in the DNA-synthesis followed by a decrease. 5-FU as single agent did increase the rate of DNA synthesis after 6 h with a maximum at 10 h. The combination of CDDP and 5-FU markedly increased the rate of DNA synthesis up to 6 h as compared to single drug treatment. Although the dCTP pool increased after combined treatment, while the dTTP pool was unchanged, no alterations in the proportions of dTTP and dCTP incorporated into DNA could be detected. Hence, misincorporation of pyrimidines is not the mechanism for the synergistic effect of the combination of CDDP and 5-FU. PMID- 8639321 TI - The RBE of fast neutrons for in vitro inactivation of human tumour cells determined by the ratio of mean inactivation doses. AB - In an effort to clarify the relationship between sensitivity of human tumour cells to low-LET and to fast neutron irradiation, 10 human tumour cell lines were exposed to cobalt gamma-rays and to 60 MeV (p -> Be+) neutron beam. The data were pooled with results of 31 human tumour cell lines previously published. The analysis of date using the linear-quadratic model indicated that not only alpha values increased after neutron irradiation, but so did beta values too, although to a lesser extent. The mean inactivation dose (MID) was derived for each cell line from the linear-quadratic parameters after low-LET and high-LET exposure. MID values following neutron irradiation were closely correlated to those after gamma-ray irradiation. In these 41 cell lines, the extreme values of RBE derived by the ratio of MID varied by a factor of 3 among the cell lines. RBE was positively correlated to photon MID, meaning that intrinsically radiation resistant tumour cells have a higher neutron RBE, on average. Similar findings were observed if alpha ratios were used instead of MID ratios. In addition, the RBE/dose variations were more marked in cells with the higher RBE. Taken together, these data suggest that, although considerable variations exist among human tumour cell lines, intrinsically radioresistant cells are relatively more sensitized when exposed to high LET beams than radioresponsive tumours. An 'intrinsic gain factor' may thus be expected in irradiating radiation resistant tumours with fast neutrons, in addition to the hypoxic or kinetic gain factors. Because the quadratic component is still present after neutron irradiation, we suggest using MID ratio as a reference RBE when comparing survival curves of cells exposed to radiations of different qualities. PMID- 8639323 TI - Improving awareness of the psychosocial needs of the patient -- a training course for experienced cancer doctors. PMID- 8639322 TI - Pediatric small cell variant of Ki-1 (CD30) + T-cell lymphoma with germ-line configuration of the T-cell receptor gene. PMID- 8639324 TI - A pilot study of interferon alpha and 5-fluorouracil in hepatocellular carcinoma. PMID- 8639325 TI - Insights into the molecular basis of thermal stability from the structure determination of Pyrococcus furiosus glutamate dehydrogenase. AB - The structure determination of the glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus has been completed at 2.2 A resolution. The structure has been compared with the glutamate dehydrogenases from the mesophiles Clostridium symbiosum, Escherichia coli and Neurospora crassa. This comparison has revealed that the hyperthermophilic enzyme contains a striking series of networks of ion-pairs which are formed by regions of the protein which contain a high density of charged residues. Such regions are not found in the mesophilic enzymes and the number and extent of ion-pair formation is much more limited. The ion-pair networks are clustered at both inter domain and inter subunit interfaces and may well represent a major stabilising feature associated with the adaptation of enzymes to extreme temperatures. PMID- 8639326 TI - Archaeal transcription factors and their role in transcription initiation. AB - Archaeal RNA polymerases show a weak ability in vitro to bind to promoter DNA and/or to initiate transcription with low activity independent of upstream regulatory DNA sequences. Active transcription in vitro and in vivo, however, depends strictly on a TATA box resembling the TATA box of eucaryal polII promoters. This TATA box is recognized by a polypeptide related to eucaryal TATA binding protein (TBP) that was formerly designated aTFB. Template competition studies showed that this archaeal TATA-binding protein (aTBP) is stably sequestered at the promoter by interaction with the second archaeal transcription factor, aTFA, which is related to eucaryal transcription factor IIB (TFIIB). The association of archaeal TFIIB (aTFIIB) with the aTBP-promoter complex leads to template commitment, indicating that aTFIIB recruits archaeal RNA polymerase to the preinitiation complex. These analyses suggest the following order for assembly of transcription factors on the archaeal promoter: aTBP, aTFIIB, RNA polymerase, and provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerase II and archaeal RNA polymerases. The sequence of the genes encoding aTBP and aTFIIB (TFB) showed all the characteristics conserved in their eucaryal counterparts. The degree of sequence similarity between archaeal and eucaryal transcription factors is between 27 to 35% for TFIIB and between 36 to 41% for TBP. The findings discussed here indicate that TBP and TFIIB perform analogous functions in Archaea and Eucarya and show that four essential components of archaeal and eucaryal transcriptional machineries. RNA polymerase, TATA box, TBP and TFIIB are homologous. PMID- 8639327 TI - On the origin of respiration: electron transport proteins from archaea to man. AB - All aerobic organisms use the exergonic reduction of molecular oxygen to water as primary source of metabolic energy. This reaction is catalyzed by membrane residing terminal heme/Cu-oxidases which belong to a superfamily of widely varying structural complexity between mitochondrial and bacterial members of this family. Over the last few years, considerable information from this and other laboratories accumulated also on archaeal respiratory chains and their terminal oxidases. In the following, the molecular and catalytic properties of the latter are discussed and compared to those from bacteria and eucarya under the aspect of their energy conserving capabilities and their phylogenetic relations. The Rieske iron-sulfur proteins being important functional constituents of energy transducing respiratory complexes are included in this study. A number of essential conclusions can be drawn. (1) Like bacteria, archaea can also contain split respiratory chains with parallel expression of separate terminal oxidases. (2) The functional core of all oxidases is the highly conserved topological motif of subunit I consisting of at least 12 membrane spanning helices with the 6 histidine residues of the heme/Cu-binding centers in identical locations. (3) Some archaeal oxidases are organized in unusual supercomplexes with other cytochromes and Rieske [2Fe2S] proteins. These complexes are likely to function as proton pumps, whereas on a structural basis several subunit I equivalents alone are postulated to be unable to pump protons. (4) The genes of two archaeal Rieske proteins have been cloned from Sulfolobus; phylogenetically they are forming a separate archaeal branch and suggest the existence of an evolutionary ancestor preceding the split into the three urkingdoms. (5) Archaeal oxidase complexes may combine features of electron transport systems occurring exclusively as separate respiratory complexes in bacteria and eucarya. (6) As far back as the deepest branches of the phylogentic tree, terminal oxidases reveal a degree of complexity comparable to that found in higher organisms. (7) Sequence analysis suggests a monophyletic origin of terminal oxidases with an early split into two types found in archaea as well as bacteria. This view implies an origin of terminal oxidases prior to oxygenic photosynthesis in contrast to the widely accepted inverse hypothesis. PMID- 8639328 TI - Histones and chromatin structure in hyperthermophilic Archaea. AB - HMf is a histone from the hyperthermophile Methanothermus fervidus. It is the archetype and most studied member of a family of archaeal histones that have primary sequences and three-dimensional structures in common with the eukaryal nucleosome core histones and that bind and compact DNA molecules into nucleosome like structures (NLS). HMf preparations are mixtures of two similar, small (approximately 7.5 kDa) polypeptides designated HMfA and HMfB that in vivo form both homodimers and heterodimers. HMfA synthesis predominates during exponential growth but the relative amount of HMfB increases as M. fervidus cells enter the stationary growth phase. Analyses of homogeneous preparations of recombinant (r) (HMfA)2 and (rHMfB)2 have demonstrated that these proteins have different DNA binding and compaction properties in vitro, consistent with different roles in vivo for the (HMfA)2, (HMfB)2 and HMfA. HmfB dimers, and for the NLS that they form, in regulating gene expression and in genome compaction and stability. PMID- 8639329 TI - Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: strategies of protein stabilization. AB - The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202, 715-728). Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima, it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds (delta delta Gstab approximately equal to 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents. PMID- 8639330 TI - Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea. AB - We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSVI of S. shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus types carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S. solfataricus. PMID- 8639331 TI - The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea. AB - Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5' type 1 DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri, the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type 1 DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type 1 DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure. PMID- 8639332 TI - General vectors for archaeal hyperthermophiles: strategies based on a mobile intron and a plasmid. AB - Although there are currently no cloning and expression vectors available for archaeal hyperthermophiles, small cryptic plasmids have been characterized for these organisms as well as viruses and introns capable of spreading between cells. Below, we review the recent progress in adapting these genetic elements as vectors for Pyrococcus furiosus and Sulfolobus acidocaldarius. An efficient and reliable transformation procedure is described for both organisms. The potential of the mobile intron from Desulfurococcus mobilis, inserted into the bacterial vector pUC18 to generate a new type of vector, was investigated in S. acidocaldarius. A polylinker was inserted upstream from the open reading frame encoding the homing enzyme I-DmoI. Both the polylinker and a 276 bp fragment of the tetracycline gene from pBR322 could be inserted into the intron-plasmid construct and spreading still occurred in the culture of S. acidocaldarius. Experiments are in progress to test the co-mobility of the alcohol dehydrogenase and beta-galactosidase genes from Sulfolobus species with the intron. A shuttle vector pCSV1 was also produced by fusing the pGT5 plasmid from Pyrococcus abyssi and the bacterial vector pUC19 which, on transformation, is stable in both organisms without selection. Growth inhibition studies indicate that both P. furiosus and S. acidocaldarius are sensitive to the antibiotics carbomycin, celesticetin, chloramphenicol and thiostrepton as well as butanol and butylic alcohol. Spontaneous mutants resistant to these drugs have been isolated carrying single site mutations in their 23S rRNA gene; they include mutants of S. acidocaldarius resistant to chloramphenicol, carbomycin and celesticetin with the mutation C2452U and thiostrepton-resistant mutants of P. furiosus carrying the mutation A1067G (both numbers corresponding to Escherichia coli 23S rRNA). These mutated genes are being developed as selective markers. Moreover, two beta galactosidase genes from P. furiosus have been cloned as possible phenotypic markers; one of these exhibits maximum activity at 95 degrees C with O nitrophenyl beta-D-galactopyranoside as substrate. PMID- 8639333 TI - Networking for action on sustainability and health. PMID- 8639334 TI - Why research findings are not used by commissions--and what can be done about it. PMID- 8639335 TI - Clinical and economic consequences of patients as producers. AB - NHS market 'reforms' and the world-wide drift toward managed competition in health services rest on a fundamental misunderstanding of the nature of health production through medical and nursing care. Optimally efficient health production depends on a general shift of patients from their traditional role as passive or adversarial consumers, to become producers of healthy jointly with their health professionals, in an essentially co-operative rather than competitive public service. PMID- 8639336 TI - A study on the epidemiology of hepatitis C infection among blood donors in Singapore. AB - BACKGROUND: Hepatitis C viral (HCV) infection has been identified as the main cause of post-transfusion hepatitis (PTH) since 1989. Despite this, little is known regarding the prevalence and mode of transmission of the disease. The purpose of this study was therefore to study the demographic factors associated with HCV infection among Singaporean blood donors. METHODS: In this study, the screening questionnaire records of HCV-positive donors were analysed. A total of 241 donors, tested positive for HCV between 7 December 1992 and 31 August 1994, were included. Demographic details studied included the age, sex, race, citizenship, occupation and number of previous donations. In additions, the associations of HCV infection with other screened diseases were analysed. RESULTS: The prevalence of HCV infection was found to be 0.370 per cent (241/65208) among the donors. Of these, 200 (0.389 per cent prevalence) were male and 41 (0.298 per cent prevalence) were female. The mean age was 34.2, SD = 9.4. The prevalence of the disease was found to increase with age. Significant differences were seen among the races (chinese versus Malay, 0.329 percent versus 0.513 percent, p < 0.05). There was also a significant association of HCV with Human Immunodeficiency Virus (HIV) infection among the donors (0.4 percent co infection versus 0.004 percent in the general donor population, p < 0.01). CONCLUSION: The incidence of HCV infections is relatively low among blood donors in Singapore. The differences in prevalence seen among the different groups studied suggest that the disease is community acquired and may be due to the lifestyle of the donors. PMID- 8639338 TI - Asthma deaths in Mersey region 1989-1990. AB - BACKGROUND: We examined the circumstances surrounding death from asthma in Mersey Region and the feasibility of conducting a regional confidential enquiry, as suggested by the British Thoracic Society in response to the proposal in the Green Paper the health of the nation that asthma should be a key area for action. METHOD: Sixty-three Mersey residents, aged 16-65 years, died in Mersey Region in 1989 and 1990 with ICD code 493. Diagnostic criteria for asthma were applied to each case. Anonymized case-note summaries were assessed by a chest physician (M.P.) for (1) severity of symptoms/disease, (2) risk of a respiratory death and (3)avoidable factors in the circumstances surrounding death. RESULTS: Some written information was available on every patients. In 43/63 (68 percent) cases asthma was the true cause of death; 41/43 had had asthma diagnosed in life. Mean age (47 years) and median age of onset of asthma (10 years) were similar to those found in previous studies, but the proportion of men (58 percent) was higher. Only 6 (14 percent) deaths occurred after more than a few hours in hospital; for the rest, hospital contact was either too late (19 percent) or did not occur (67 percent). Of 22/43 (51 percent) patients considered at high risk of a respiratory death, 15 had poorly controlled symptoms for at least a year before their deaths. Avoidable factors were present in 29/43 (67 percent) cases; most commonly lack of assessment, inadequate doses of steroids and over-reliance on bronchodilators. CONCLUSION: These findings resemble those of previous studies, showing that little has changed. Asthma deaths occur mostly outside hospital; thus to replace deaths, resources should be directed at the community to improve the recognition of asthma by patients, their families and the primary care team. A regional confidential enquiry is feasible and could be an effective mechanism for proving management. PMID- 8639337 TI - Practices and policies for influenza immunization in old people's homes in Nottingham (UK) during the 1992-1993 season: potential for improvement. AB - BACKGROUND: The elderly residents of nursing and old people's homes are at substantially increased risk from influenza and its complications. Annual vaccination is therefore strongly recommended by the UK Departments of Health. However, few data exist on the uptake of influenza vaccine in this setting; this was last estimated to be 45 percent in 1988-1989. A major epidemic of influenza A occurred in 1989-1990 which may have changed vaccination behaviours. METHODS: A structured interview survey was conducted in 49 old people's homes in Nottingham to determine practices and policies for influenza vaccine. The medical records of 151 randomly selected elderly residents were examined to determine vaccine uptake in the 1992-1993 season. RESULTS: Twenty-five homes (52.1 percent) claimed to operate a policy for influenza immunization but only 3 (12.0 percent) were written down and none set targets. Methods of vaccine promotion were generally weak. Accordingly, vaccine uptake was only 39.6% percent (95 percent confidence interval 33.2-46.0). CONCLUSIONS: In contrast to other high-risk groups living in the open community, no improvement in influenza vaccine uptake has occurred in nursing and old people's homes since the late 1980s. Future attempts to increase vaccine uptake in this setting should focus on developing joint policies between general practitioners and home staff to facilitate better co-ordinated programmes of vaccine promotion and administration. PMID- 8639339 TI - Coronary heart disease mortality trends in England and Wales, 1952-1991. AB - BACKGROUND: The 'programming' hypothesis for the causation of heart disease suggests that there are critical periods of development in early life during which a stimulus or insult has permanent effects on the subject's organ or tissue structure and physiology. Exposure to such effects might be expected to be linked to year of birth and produce cohort (generation) effects in heart disease death rates. Evidence is sought for such effects in data from England and Wales. METHODS: The age- and period-specific heart disease death rates for men and women aged 30-69 in England and Wales have declined recently in both men and women. The trends have two components. The first is described by a peak in rates during 1977 1981 that applies to both sexes and all age-groups. This is consistent with risk conferred by adult diet and lifestyle. The second component required to explain the trends also applies to both sexes and is described by a risk that is lower in each successive year of birth. This is consistent with risk linked to poor growth in early life. CONCLUSION: Poor early growth has become less common as maternal health, physique and nutritional status have improved through the century. The trends are therefore consistent with the'programming' hypothesis for the causation of coronary heart disease. PMID- 8639341 TI - Why did treatment rates for colorectal cancer in south east England fall between 1982 and 1988? The effect of case ascertainment and registration bias. AB - BACKGROUND: We had two aims in undertaking this study, as follows: (1) to describe regional and district trends in incidence and treatment for colorectal cancer in South East England from 1982 to 1988; (2) to examine the effect of registration practice and case ascertainment on district variations in incidence and treatment using data on death certificate only (DCO) registrations, mortality and stage. METHODS: We included all cases registered by the Thames cancer registry diagnosed with colon or rectal cancer between 1982 and 1988 and resident in 28 districts in the two South Thames regions. Indirect standardized incidence ratios were calculated for the districts and a alpha 2 test for trend was carried out. RESULTS: In the SE England regional analysis, between 1982 and 1988 there was a significant increase in the incidence of cases of colon and rectal cancer in the over-75s, but treatment rates remained unchanged. Treatment rates fell significantly in the under-65s although incidence rates remained unchanged. Age is a strong predictor of nontreatment. Between 1982 and 1988 the relative risk of not receiving treatment increased for all ages over 65 years. DCO registrations accounted for 22 percent and 15 percent of all colon and rectal cancer cases, respectively, between 1982 and 1988. The proportions rose (between 1982 and 1988) from 10 and 8 percent to 25 and 19 percent in colon and rectal cancer, respectively. DCO registration rates increased over time and in all age groups in South East England for both colon and rectal cancer between 1982 and 1988, but the largest increase was in the over-75s. Thirty-two per cent of colon and 25 per cent of rectal cases were unstaged. Although the proportion of unstaged cases remained constant over time, they were increasingly the result of DCO registrations. Errors in the registry staging data rendered those cases which were staged unusable. In the district analysis, there were significant variations in age-standardized incidence, treatment and DCO registration ratios across the 28 districts for men and women with colon and rectal cancer between 1982 and 1988. DCO registrations show a negative correlation with treatment for both colon and rectal cancer (p < 0.05) and with incidence for only rectal cancer. CONCLUSIONS: We report significant differences in age-standardized incidence and treatment ratios across 28 districts in South East England, some of which, can be accounted for by differences in registration practice. There is a complex relationship between DCO registrations and incidence and treatment for both colon and rectal cancer. DCO registrations are a good proxy for under-ascertainment of incidence in rectal cancer but not colon cancer, and are a good proxy for under ascertainment of treatment in both colon and rectal cancers. Information from the cancer registry can be used to examine registration and treatment rates across districts. However, if variations are to be adequately explained, meticulous data collection on stage and quality control are essential. PMID- 8639340 TI - Variations in the incidence, management and outcome of stroke in residents under the age of 75 in two health districts of southern England. AB - BACKGROUND: The aim of the study was to determine the incidence, outcome and health service resources consumed by stroke care in defined populations. METHODS: Patients under the age of 75 experiencing their first stroke between August 1989 and July 1991 were assessed at the onset, and at three and 12 months after their stroke. The settings were West Lambeth (WL) and Tunbridge Wells (TW) health authorities in southern England. The main outcome measures used were: age- and sex-specific incidence rates, hospital admission rates, length of stay and use of rehabilitation services. Functional disability was assessed using the Barthel scale. RESULTS: Four hundred and fifty-six strokes were registered. The annual incidence rates/1000 population aged under 75 years old [with 95% confidence interval (CI)] were 0.77 (0.67-0.87) in WL and 0.66 (0.58-0.75) in TW. The age- and sex-standardized incidence ratios were significantly higher in WL (126; 95% CI 110-144) than in TW (84%; 95% CI 74-95) (p < 0.001). There were independent associations of incidence with age group (p < 0.001), sex (p < 0.001) and ethnic group (p < 0.001). One year case-fatality was 36% (80/225). At one year, 11% (14) of surviving patients were moderately to severely disabled and 23% (28) mildly disabled. Seventy-one percent (326) of patients were admitted to hospital and the average health service cost per case was pound 3800 in WL and pound 2650 in TW, 93% of the cost being for in-patient care. CONCLUSION: The study has demonstrated a significantly increased incidence of stroke in an inner-city district compared with a district in rural southern England. It has also established ethnic group as a significant independent risk factor for stroke in the United Kingdom. The cost of care to the health services is considerable, and largely reflects nursing costs in hospital rather than effective treatment packages for stroke. PMID- 8639342 TI - Outcome following fractured neck of femur--variation in acute hospital care or case mix? AB - BACKGROUND: This study examined the quality of care given to patients admitted to hospital with a fractured neck of femur by assessing the link between outcome, case severity and resource use. Fractured neck of femur was chosen for this study as it is a common condition amongst elderly people which causes considerable morbidity and mortality, uses a high proportion of acute hospital resources and is a condition where virtually all new cases will come under the care of the hospital service. METHODS: Three hospitals which had different case fatality rates and costs were included in the study. These were an inner-city teaching hospital (Hospital 1), an inner-city associated teaching hospital (Hospital 2) and an associated teaching hospital in an urban location (Hospital 3). Patients were recruited for this study over a 12-month period. Details on case severity and basic demographic date were collected on admission, and information on the process of care was collected during the hospital stay. Four outcome measures were addressed: activities of daily living (ADL) before discharge and at three months post-fracture; mortality up to 12 months post-fracture; complications occurring after admission to hospital; and destination on discharge. RESULTS: A total of 492 patients were recruited into the study, with a male to female ratio of 1:4 and an age range of 60-101 years. Patients admitted to the three hospitals showed no difference with respect to the presence of co-morbidities, medication, pre-fracture ADL, mental state, age and sex. There were some differences observed in pre-fractured place of residence. Hospital 1 had the highest proportion of patients admitted from sheltered housing and other hospital. Hospital 2 the highest proportion from residential homes, and Hospital 3 the highest proportion admitted from their own homes. Hospital 3 discharged patients at an earlier stage of recovery in that a higher proportion were discharged with a poor ADL index. This hospital also had more orthopaedic complications but fewer medical complications; however, the outcome in terms of ADL at three months post-fracture and mortality at 12 months was similar in all three hospitals. The severity variables which predicted poor outcome were co-morbidities, impaired mental state, impaired ADL pre-fracture, increasing age and an extracapsular fracture. After controlling for severity variables, the resource variables had not further impact on mortality, either in hospital or within one year. An epidural anaesthetic was related to a poor ADL at three months and more orthopaedic complications but fewer medical complications. There was also a hospital effect in that Hospital 3, which performed the most epidurals, had the highest proportion of orthopaedic complications but the lowest proportion of medical complications. When the operating surgeon was a consultant, there were more orthopaedic complications, but this was not related to these patients having a worse case severity on admission. However, among the cases operated on by consultants, there were no hospital deaths. No other resource variables were related to ADL at three months, or orthopaedic or medical complications. CONCLUSIONS: The results show that a poor outcome following a fractured neck of femur was related to increased case severity at the time of fracture. The resource variables had very little impact on the outcome. PMID- 8639343 TI - Development of public health training in Hungary--an exercise in international co operation. AB - Following the fall of Communism, the countries of Central and Eastern Europe have been restructuring their health systems. Restructured medical education is vital to, and has been supported by, the European Union TEMPUS (Trans-European Mobility Scheme for University Studies) scheme. An example is a three-year project in Hungary to develop undergraduate and postgraduate medical education in public health. The project team (CNPHH) included representatives from all Hungarian medical schools and universities in Western Europe and Canada. The project involved assessing the needs of the Hungarian departments and developing a strategy involving transfer of skills in modern public health and educational methods, study visits and courses in Hungary and at Western universities, and capital funding. The project provides a model for others wishing to develop or restructure their education at national or local levels. This paper describes the methods used, outcomes achieved, and lessons learned. PMID- 8639344 TI - Hearing aids: value for money and health gain. AB - BACKGROUND: The aim of this study was to assess the ability of hearing aids to reduce the handicap associated with hearing impairment in adults. METHODS: Fifty adults aged over 60 who were supplied with an NHS hearing aid had their hearing handicap and communication function recorded at initial hearing aid assessment, and after three months of aid use. Hearing communication function was assessed by a key informant. RESULTS: Hearing aid use was associated with considerable score improvements for social and emotional function [20.5, 95 per cent confidence interval (Cl) 15.4-25.6] and communication function (22.9, 95 percent Cl 14.6 31.2). Younger people recorded the greatest reduction in handicap. Overall satisfaction with hearing aid performance was high. CONCLUSIONS: Hearing aids are effective in reducing hearing handicap in adults. They represent a good buy for purchasers seeking to achieve health gain for adults with hearing impairment. PMID- 8639345 TI - Audit of the support for breastfeeding mothers in Fife maternity hospitals using adapted 'Baby Friendly Hospital' materials. AB - BACKGROUND: The objective of this study was to assess the level of support given to breastfeeding mothers during their stay in maternity hospitals. The audit was carried out in maternity hospitals in Fife with the co-ordination of the Fife Joint Breastfeeding initiative. METHODS: The subjects consisted of ten maternity hospital staff (medical and midwifery), and 12 antenatal and 21 postnatal women. The design of the study consisted of an audit of hospital policies and practices in comparison with ten internationally recognized standards. This was carried out by adapting the external evaluation instruments from the WHO-UNICEF "Baby Friendly Hospital" materials. Methods relied not only on reported practices but also on direct observation and enquiry. RESULTS: Action was taken to address areas of practice which fell below the WHO-UNICEF standards: supplementary feeding of breastfed babies, particularly overnight, was reduced; discharge "bounty packs" advertising baby milk manufacturer products were discontinued; a hospital breastfeeding support group was established; the hypoglycaemia policy was revised; and the need for an orientation session on breastfeeding policies for medical staff was recognized. CONCLUSIONS: This audit approach using "Baby Friendly Hospital' materials has helped to define policy, measure performance against recognized standards, identify quality specifications for maternity service agreements and has improved hospital support for breastfeeding mothers. This approach is suitable for maternity hospitals whose breastfeeding rates make them ineligible for "Baby Friendly Hospital" accreditation, and has the potential to be extended to encompass wider "health-promoting hospital" issues such as promotion of infant car seats. PMID- 8639346 TI - Antibiotic prophylaxis for bacterial meningitis: overuse and uncertain efficacy. AB - BACKGROUND: There is little evidence supporting the efficacy of prophylactic antibiotics in preventing secondary cases of bacterial meningitis, and recent guidance extended the use of prophylactic antibiotics amongst children who attend pre-school groups. METHODS: We examined the volume of rifampicin prescribed, and that recommended to contacts of cases of meningococcal and Hib meningitis in Somerset over a three-year period using case note records of the Consultant for Communicable Disease Control (CCDC) and PACT data. RESULTS: There was evidence of excessive prescribing over and above that recommended by the CCDC. CONCLUSIONS: Excessive prescribing increases the chance of serious drug side effects and the development of antibiotic resistance. It is suggested that both meningitis contacts and information about early symptoms of meningitis, as well as an explanation of the rationale behind the prescribing of antibiotic prophylaxis to contacts. This may reduce the likelihood of unnecessary prescribing and subsequent complications. PMID- 8639348 TI - The Abrams report--communicable disease control; how do Health Districts measure up to the recommendations? AB - BACKGROUND: The aims of the study were to examine whether Health Districts in the North Western Region complied with the recommendations in the Abrams report regarding the control of communicable disease [incorporated into the Annex to Circular HSG(93)56], and to identify areas that need further attention. METHODS: The recommendations were extracted and arranged in questionnaire form. Further items were included dealing with the use of Epinet in communicating with the profession. A compliance score was derived from affirmative and qualified affirmative responses. RESULTS: Many recommendations were met by all or most Districts. Compliance was 90 percent or over for 58 percent of the questions where an assessment was appropriate. Of the 16 Districts in consortia, 75 percent did not have a consortium plan. Day-to-day plans were informal in 21 percent of Districts. In 63 percent of Districts the Family Health Services Authority (FHSA) was not involved to the extent that it should be. The Consultant in Communicable Disease Control (CCDC) had insufficient District Health Authority support in 42 percent of Districts and insufficient Local Authority support in 16 percent of Districts. In 58 percent of Districts there was lack of inclusion of matters relating to the control of infectious disease in contractual statements between purchaser and provider. There was a lack of audit in 47 percent of Districts. CONCLUSIONS: One plan or a compatible series of plans are required across each District. Informal day-to-day plans should be formalized. The FHSA should be fully involved in infectious disease control plans. Certain Districts require a Community Infection Control Nurse, accountable to the CCDC and/or administrative support to input and scan surveillance data. Contractual statements between purchaser and provider should include appropriate infection control requirements when this is not already the case. Communicable disease control audit should be a regular part of CCDC duties. PMID- 8639349 TI - The type and quality of randomized controlled trials (RCTs) published in UK public health journals. AB - BACKGROUND: Randomized controlled trials (RCTs) are increasingly being used to evaluate the effectiveness of health care interventions. Systematic reviews of RCTs form the basis of the Cochrane Collaboration, which aims to synthesize all RCTs concerned with the provision of health care. This paper reports the quantity and methodological quality of RCTs published in five UK public health journals, which were searched as part of a planned register of RCTs in public health. METHODS: Five journals were hand searched: The International Journal of Epidemiology, Health Trends, Journal of Public Health Medicine, Public Health and The Journal of Epidemiology and Community Health. All RCTs were identified and their methodological characteristics reviewed. RESULTS: Ninety-one trials were identified from the five journals. A wide variety of topics were covered, the most common being prevention strategies )46 percent of the trials). Although the actual number of reports of RCTs published increased over time, there was no detectable improvement in the quality of reports. The aspect of bias most well controlled was control of bias at entry (randomization), in 83 (91 per cent) of trials. However, even in these trials details about the process of randomization were poor. CONCLUSION: These trials will form the basis of a register of RCTs in public health. The diversity of topics covered illustrates the broad-based nature of public health. For this reason, many other RCTs relevant to the practice of public health are likely to be found in non-public health journals. PMID- 8639347 TI - How common is heart failure? Evidence from PACT (prescribing analysis and cost) data in Nottingham. AB - BACKGROUND: The aim of the study was to determine the prevalence of heart failure in Nottinghamshire by an analysis of prescriptions for loop diuretics. METHODS: An observational study was carried out in the Nottingham Health District, on the basis of Prescribing Analysis and Cost (PACT) data relating to nearly 400,000 kg of frusemide prescribed in the year 1991-1992 and general practitioner (GP) records on two groups of patients (total 903) prescribed frusemide. RESULTS: The total amount of frusemide prescribed on a daily basis in Nottinghamshire in 1,048,566 mg. The mean daily dose of frusemide per patient is 60 mg and the median dose 40 mg. There are between 13,107 and 26,214 patients taking frusemide in Nottinghamshire. Fifty-six per cent of patients prescribed loop diuretics by their GP fulfil diagnostic criteria for heart failure. CONCLUSION: The prevalence of heart failure in Nottinghamshire estimated from loop diuretic prescribing increases from 0.1 per cent in the age-group 30-39 years to 5.45 percent in patients aged over 90 years. PMID- 8639350 TI - Can general practice data be used for needs assessment and health care planning in an inner-London district? AB - BACKGROUND: The development of primary care led commissioning will increase the need for practice-based information on health and morbidity, and the NHS information strategy recommends that routinely collected health and morbidity information held on general practice computer systems should be use to inform local health needs assessment. The aim of this study was to evaluate the quality of information in six computerized practices. METHODS: A comparison was carried out of the recording of registration and social information, health risk factors, medication and record on consultations on the computer and in the manual records for a sample of patients, with an audit of morbidity coding by computer. A comparison was made of computerized disease registers with prescribing for diseases. RESULTS: Computer disease registers identified over 90 percent of diabetic patients on medication, 67 percent of asthmatics, 61 percent of epileptics, and 48 percent of patients with angina. Computer recording of problems was inconsistent; practices which recorded clinical information from every consultation did not have substantially more complete disease registers. Over 90 percent of encounters and prescriptions were computerized, but there was bias in recording consultation problems. Blood pressure, smoking, alcohol, weight and height were recorded for over 50 percent of patients aged 15-74, and computerized for 79 percent (291/370) for height, but only 56 percent (274/488) for the most recent blood pressure recorded. Limited social information was recorded about patients: 45 percent (410/915) had occupation or employment status and 35 percent (230/915) ethnic group; computerized for 26 percent and 18 percent, respectively. CONCLUSIONS: At present, the routine collection of information from practices would not provide reliable information for health care planning. Greater use of information in practices would improve data quality, and practice data could be used to address specific issues, if augmented by additional data, and for practice-based needs assessment. PMID- 8639351 TI - Quarterly Communicable Disease Review April to June 1995. From the PHLS Communicable Disease Surveillance Centre. PMID- 8639352 TI - The detection and management of hypertension. PMID- 8639353 TI - Safety and place of birth in Scotland. PMID- 8639354 TI - Does routine child health surveillance reach children most at risk of accidental injury? PMID- 8639355 TI - Whitening toothpastes: meeting needs or satisfying demands? PMID- 8639356 TI - Evidence based assessment? AB - There have been many calls recently for a more 'evidence based' approach to clinical dentistry. This article argues the need for a similar approach to be adopted by the private dental schemes when setting criteria for practice inspection. The author recognises the laudable attempt to revise the 'quality' of dental practice but questions whether the methods adopted to date are justifiable. PMID- 8639357 TI - A supernumerary paper. PMID- 8639360 TI - Artificial intelligence. PMID- 8639359 TI - Whitening toothpastes--do we need them? PMID- 8639361 TI - Localising maxillary canines. PMID- 8639362 TI - Tobacco advertising damages dental health. PMID- 8639363 TI - Inappropriate health care. PMID- 8639364 TI - School for dental therapists. PMID- 8639365 TI - Whose radiograph is it anyway? PMID- 8639366 TI - An unpleasant taste. PMID- 8639367 TI - Mortality and morbidity in general anaesthesia. PMID- 8639368 TI - Reversion of cyclosporine-induced overgrowth. PMID- 8639369 TI - Criteria for caries removal at the enamel-dentine junction: a clinical and microbiological study. AB - In the UK cavity preparation at the enamel-dentine junction (EDJ) is considered complete when all soft and/or stained tissue is removed. The aim of the present work was to link the clinical criteria of dentine consistency and dentine colour with the microbiological status of the tissue. Cavities were prepared in 564 teeth. Under rubber dam, access to the EDJ was gained and the sample sites selected (n = 847). Their consistency was noted as hard or soft and wet or dry when probed. The colour of the site was noted as stain-free or stained. Dentine was sampled using a round bur. After microbiological processing the total anaerobic count and counts of mutans streptococci and lactobacilli were expressed as log10 [CFU per sample]. Soft sites harboured significantly more bacteria than hard sites (P < 0.001). Soft and wet sites harboured significantly more bacteria than soft and dry sites (P < 0.001). Stained and hard sites harboured more bacteria than stain-free and hard sites (P < 0.05) but this difference was not considered to be clinically relevant. The relatively simple clinical criteria used here correlated significantly with the microbial findings. All soft dentine must be removed at the EDJ to ensure minimal infection of the underlying hard dentine. However, it may not be necessary to continue preparation until the EDJ is also stain-free. PMID- 8639370 TI - Efficacy of dental health advice as an aid to reducing cigarette smoking. AB - Ninety-eight cigarette smokers attending the Newcastle upon Tyne Dental Hospital, wishing to reduce their tobacco consumption, received smoking reduction advice in combination with dental health instruction and periodontal care. Those advised against smoking showed a greater reduction (P < 0.001) in reported cigarette consumption during treatment and follow-up, compared with 38 control subjects who received dental health instruction but no advice against smoking. Fifty percent of intervention subjects reported reducing to half or less than half of their initial cigarette consumption, compared with 24% for controls. Eighty percent of those advised against smoking reported some reduction, against 29% for controls. The reported smoking cessation rate in the intervention group was 13.3%, compared with 5.3% in the control subjects. The results indicate that advice against smoking combined with dental health instruction and periodontal care can be an effective aid to reducing tobacco consumption. PMID- 8639371 TI - The use of general anaesthesia for tooth extraction in young handicapped adults in France. AB - The anaesthetic management of handicapped young adults is often difficult. However, few examples were found in the literature that attempted to specifically measure the frequency of general anaesthesia in the dental treatment of this group of patients. This paper focuses on the management of mentally or physically handicapped young patients undergoing extractions at a specialist dental service in Southern France. A survey of 184 young patients treated during a 39-month period, either under general (34 cases) or local (150 cases) anaesthesia was carried out. A minimum one-year follow-up period was used for each patient. The main characteristics of the patients who received general or local anaesthesia were compared and the factors which indicated the choice of general rather than local anaesthesia were assessed. This study attempts to define precise selection criteria, including dental and non-dental factors. Limited past treatment history was found to be a strong indicator of the need for general anaesthesia. The results of this study suggest that non-dental background factors were also important. The need for general anaesthesia was markedly increased in the groups with severe behavioural disturbances or low levels of contact with the general dental practitioner. If provision of regular dental services for disabled young patients can significantly reduce the need for general anaesthesia, care will have to be taken in developing the relationships between the hospital and general dental practitioner. PMID- 8639372 TI - Demographics of HIV-infected persons attending a dental clinic. AB - The demographics of 147 HIV-infected persons attending a special care dental clinic in South West England are reported. The majority of attendants were homosexual/bisexual males, reflecting the UK epidemiology of HIV disease at the time of study. There was a substantial rise in patient numbers from 1988 onwards but patients often did not reveal their route of HIV acquisition or increasingly had acquired HIV disease via heterosexual routes. Patients were usually referred for routine dental treatment, not HIV-related oral disease. The HIV-infected patients generally attended the clinic irregularly, despite being offered many appointments. It is concluded that most patients with HIV disease attend clinics for routine dental care, yet many may be unable or unwilling to attend regularly. PMID- 8639373 TI - The role of occlusal auscultation in assessing dental occlusions. AB - Little attention is paid to the noises made when the teeth occlude, yet there is a wealth of information stored in these sounds. It is remarkable how quickly the ear can learn to discern the difference between a good occlusion and one which is disrupted, by using a stethoscope. With the addition of relatively inexpensive instrumentation, occlusal sounds may also be recorded for later reference, analysis, comparison or audit. PMID- 8639374 TI - Denture fixatives--an update for general dental practice. AB - While some clinicians accept that denture fixatives have a valuable function in certain situations and contribute significantly to denture retention and stability, others disapprove of their use, believing that there is no substitute for a properly constructed, accurately fitting denture. It is also believed that their inappropriate use can lead to problems such as impairment of the development of the neuromuscular system which is important for the retention and stability of dentures. However, because these fixatives are available over the counter, it is important that clinicians, whatever their opinion, can give patients informed advice. The aim of this paper, therefore, is to provide an update, advice and recommendations on the use of denture fixatives. PMID- 8639375 TI - Antibody catalysis of pericyclic reactions. AB - In an effort to increase our insight into the catalysis of pericyclic reactions we have initiated a detailed study of an antibody that catalyzes an oxy-Cope rearrangement. We have determined the stereochemistry of the antibody-catalyzed reaction, and experiments are in progress to determine the conformation of the substrate bound in the antibody combining site. The genes encoding the variable regions of this antibody have been cloned and sequenced, and we have made use of a bacterial expression system to produce this antibody as a Fab fragment in recombinant form, making it amenable to genetic manipulations such as site directed mutagenesis. The recombinant Fab fragment has been crystallized in the presence of its transition state analog, and we are now in the process of determining its active site structure. PMID- 8639376 TI - Antibody catalysis via strategic use of haptenic charge. AB - General acid-base catalysis contributes substantially to the efficacy of many enzymes. Similar effects can be exploited in antibody catalysis by taking advantage of charge complementarity between immunoglobulin and hapten (the template used to induce the antibody) to elicit functional groups in the combining site. This strategy has proved useful in the catalysis of a diverse set of chemical transformations, including elimination reactions. Provided that hapten design is optimized and the immune response is screened extensively, the efficiency of the resulting antibody catalysts can rival that of analogous natural enzymes. PMID- 8639378 TI - Cancer chemotherapy and biological response modifiers annual 16. Introduction. PMID- 8639377 TI - Nickel-catalyzed oxidations: from hydrocarbons to DNA. AB - Nickel(II) complexes of tetradentate ligands such as cyclam and salen are catalysts for olefin epoxidation using PhIO and NaOCl, respectively. In order to understand the lack of enantioselectivity observed with chiral cyclam and salen complexes, studies of DNA and RNA oxidation were carried out in which evidence for diffusible oxidants might be found. A variety of square-planar, tetradentate nickel(II) complexes were observed to mediate guanine-specific modification in the presence of KHSO5 or magnesium monoperphthalate. In particular, the cationic complex, [(2,12-dimethyl-3,7,11,17-tetraazabicyclo [11.3.1]heptadeca 1(17),2,11,13,15-pentaenato)nickel]2+, [NiCR]2+, has been studied as a probe of nucleic acid folding. The extent of guanine reaction depends upon the exposure of N7, a good transition metal binding site, thus implicating nickel-guanine binding during DNA oxidation. If this is the case, related systems should be able to confer enantioselectivity during the use of chiral nickel complexes and achiral substrates for oxidation. Mechanistic studies, including radical quenching and DNA enantioselectivity, are described and their mechanistic implications discussed. PMID- 8639379 TI - Antimetabolites. PMID- 8639380 TI - Multidrug resistance. PMID- 8639381 TI - New anticancer agents. PMID- 8639383 TI - Cytokines and immunological monitoring. PMID- 8639382 TI - Monoclonal antibody therapy of cancer. PMID- 8639384 TI - Biological response modifiers. PMID- 8639385 TI - Adoptive immunotherapy. PMID- 8639386 TI - Alkylating agents. PMID- 8639387 TI - Gene therapy of cancer. PMID- 8639388 TI - Hematopoietic growth factors in cancer chemotherapy. PMID- 8639389 TI - Reference databases of cytokine structure and function. PMID- 8639390 TI - Leukemias and myeloma. PMID- 8639391 TI - Lymphomas. PMID- 8639392 TI - Bleomycin. PMID- 8639393 TI - AIDS-associated malignancies. AB - Although currently diagnosed in about 40% of HIV-infected individuals at some point in their clinical course, AIDS-associated malignancies may increase in frequency as survival is prolonged and, particularly, as greater numbers of HIV infections occur in women. Although this review has been devoted to AIDS-defining cancers, there are suggestions from epidemiological studies that some of the more common cancers in the general population are beginning to be diagnosed at increased rates in HIV-infected people [141]. In addition, as children born with HIV infection show longer survival, increased cancer rates may be seen in this group as well. While recent advances have been made in understanding the pathogenesis of AIDS-associated malignancies, particularly KS, and modest therapeutic advances have been made, optimal therapy has yet to be defined for any of these neoplasms. PMID- 8639394 TI - Chemotherapy of head and neck cancer. PMID- 8639395 TI - Lung cancer. AB - In small cell lung cancer, combination chemotherapy including platinum compounds (cisplatin and carboplatin) and the podophyllotoxins (teniposide and etoposide) continues to be the backbone of therapy. Complete plus partial responses to combination chemotherapy occur in 80-90% of all patients. Median survival is at present 11-17 months depending on the initial tumour stage. Overall 5-year survival is approximately 5%. The duration of treatment has been shortened to 5-6 months. The results of the use of hematopoietic growth factors have hitherto been disappointing with respect to survival, while a definite effect on the degree and number of infections has been documented, when aggressive chemotherapy is administered. For epidermoid-, adenoid- and large cell carcinoma, results are emerging indicating that preoperative and preirradiatory chemotherapy in stage IIIa non-small cell lung cancer might have a substantial role in the overall management based on results from phase III trials. The studies are, however, rather modest in number and longer follow-up is necessary before recommendations for general use can be made. For patients with advanced non-small cell lung cancer (stage IIIb and IV), meta-analyses have yielded a small survival benefit (6 weeks) in favour of patients receiving chemotherapy vs. best supportive care. The treatment of this type of NSCLC should, however, still be considered to be experimental in order to define new innovative treatments. No improvement of the therapy of mesotheliomas has been reported. PMID- 8639396 TI - Mitomycins. PMID- 8639397 TI - Upper gastrointestinal tumors. PMID- 8639398 TI - Cancers of the large bowel and hepatobiliary tract. PMID- 8639399 TI - Endocrine tumors. PMID- 8639400 TI - Genitourinary malignancy. PMID- 8639401 TI - Gynecological malignancies. AB - A large study of tumors of low malignant potential confirmed the favorable survival in this group of patients compared to invasive epithelial ovarian tumors. Only 8% of patients died with recurrent disease after surgery. Patients with stage IA borderline tumors with mucinous histology tended to recur later and carried a poorer prognosis than patients with serous histology and similar stage. The group at highest risk for relapse were age greater than 70, stage II or III tumors, and histology other than serous. Long-term survival in this group was less than 75%. This high-risk group of patients should be targeted for innovative adjuvant treatment strategies. This year several well-designed studies with large sample sizes showed DNA ploidy to be an important new independent prognostic factor in stage I ovarian carcinoma. In patients with well-differentiated early stage ovarian cancer, DNA flow cytometric analysis may indicate subgroups with less favorable prognostic characteristics. This method of analysis may be beneficial in determining the need for additional treatments after surgery for early stage ovarian carcinoma. Recommendations for the definitive management of early stage ovarian cancer awaits completion of current GOG and European randomized prospective studies. Paclitaxel given in combination with platinum containing agents is an intense area of research for treatment of advanced stage disease. Early data from a prospective randomized trial of patients with advanced ovarian cancer showed a higher response rate and longer disease-free survival in patients treated with paclitaxel and cisplatin compared to a standard regimen of cyclophosphamide and cisplatin. The impact of this treatment on long-term survival awaits maturation of data. Preliminary results evaluating G-CSF in combination with paclitaxel and cisplatin for dose escalation was reported. Paclitaxel, 250 mg/m2, and cisplatin, 75 mg/m2, were the maximally tolerated doses, with peripheral neuropathy or myalgias the dose limiting toxicities. Further studies are now underway to test the effect of dose-response with escalation therapies and to determine the optimal dose and schedule for the management of patients with advanced ovarian cancer. IL-3 significantly ameliorated neutropenia but did not prevent cumulative platelet toxicity in a regimen utilizing high-dose carboplatin. This mild improvement in myelosuppression was obtained at the cost of significant toxicity. Nausea, vomiting, malaise, bone pain, headache, fever, chills and facial flushing were frequent. Intraperitoneal chemotherapy was tested as a means of consolidation treatment for patients after having a negative second-look laparotomy. These treatments were shown to be feasible; however, prospective randomized trials will be necessary to determine a benefit over operative therapy alone. Several studies addressed to problem of residual disease after primary surgery and adjuvant chemotherapy. A large phase II study conducted by the GOG confirmed the activity of salvage cisplatin-based intraperitoneal chemotherapy in patients with small volume residual ovarian cancer with favorable pretreatment characteristics. Whether intraperitoneal platinum-based therapy represents an advantage over systemic platinum therapy is being addressed in a prospective SWOG study. The use of six additional cycles of CAP for treatment of residual disease after primary treatment of surgery and adjuvant chemotherapy did not significantly improve complete pathological response and survival. Prolonged duration of chemotherapy above six cycles is not likely to impact treatment for residual disease. A regimen of high dose carboplatin was compared to whole abdominal radiotherapy for treatment of residual disease after initial chemotherapy. There was no difference in survival or disease-free survival between treatments.(ABSTRACT TRUNCATED) PMID- 8639403 TI - Breast cancer. PMID- 8639402 TI - Taxanes. PMID- 8639404 TI - Soft tissue and bone sarcomas. PMID- 8639405 TI - Brain tumors. PMID- 8639406 TI - Supportive care. PMID- 8639407 TI - DNA topoisomerase inhibitors. PMID- 8639408 TI - Cisplatin. PMID- 8639409 TI - Use of geographic information systems in epidemiology (GIS-Epi). PMID- 8639410 TI - Introduction of new vaccines in the National Vaccination Programs. PMID- 8639411 TI - 25 years without smallpox in the Americas. PMID- 8639412 TI - International workshop on population genetics and control of triatominae. Santo Domingo de los Colorados, Ecuador 24-28 September 1995. PMID- 8639413 TI - Annotation. The unhinged antithrombins. PMID- 8639414 TI - Stem cell factor and the regulation of dendritic cell production from CD34+ progenitors in bone marrow and cord blood. AB - Dendritic cells (DC) are essential for the presentation of antigen in primary immune responses and they develop from CD34+ cells in the bone marrow. Although both granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor (TNF) are known to stimulate the development of mature DC from their progenitor (CFU-DL), the function of stem cell factor (SCF) in this pathway remains to be determined. The interactions of SCF with GM-CSF, TNF, interleukin-3 (IL-3) and macrophage colony stimulating factor (M-CSF) in promoting CFU-DL development have now been studied in serum-free cultures of unfractionated as well as progenitor enriched cells from either bone marrow or cord blood. Although SCF alone is without effect on colony formation, it enhances both the numbers and size of DC colonies generated in vitro by GM-CSF and TNF. It acts directly on progenitors and in the presence of GM-CSF can also induce suboptimal DC growth even in the absence of TNF. SCF appears to recruit very early progenitors of a high proliferative potential with the capacity to differentiate into erythroid and myeloid as well as dendritic cell progeny. In combination with other cytokines it may therefore be useful for the ex vivo generation of large numbers of DC for clinical purposes. PMID- 8639415 TI - Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor beta1. AB - Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta1 (TGFbeta1). Development of MK from CD34+ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFbeta1. Inhibition of MK colony formation by PF4 was reversible because CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFbeta1-preincubated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF) binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34+ cells exhibited a higher potential in MK colony formation in the presence of 5 fluorouracil (5FU). These results demonstrate that both PF4 and TGFbeta1 inhibit MK development from CD34+ CB cells by different mechanisms, and suggest that PF4, unlike TGFbeta1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU resistant cell population. PMID- 8639416 TI - Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells. AB - Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 microM 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 microM ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units-erythroid (BFU-E), or colony forming units granulocyte/macrophage (CFU-GM). At the highest concentration tested, 100 microM BDCRB only moderately affected BFU-E or CFU-GM formation (31% and 47% inhibition, respectively). This concentration is 10-fold higher than that required to produce a 10000-fold reduction in virus titre. Evaluation of the 2-chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5'-deoxy analog (5'-dTCRB) which is more potent, and the 2-unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB > or = 5'-dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU-E and 86% for CFU-GM at 100 microM. These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure-activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action. PMID- 8639417 TI - Expression of rhombotin 2 in normal and leukaemic haemopoietic cells. AB - The rhombotin 2 gene was identified by its association with a recurrent chromosome translocation (11;14)(p13;q11), occurring in T-cell acute lymphoblastic leukaemias (ALLs). High levels of RBTN2 have been found in T-cell leukaemias carrying the translocation and in some T-cell ALLs that lack, by cytogenetic and molecular techniques, translocations involving 11p13. In normal murine tissues RBTN2 has been found to be widely expressed, with high levels present in brain and early B cells. Studies carried out in mice lacking RBTN2 have demonstrated the importance of this gene in erythropoiesis. We have investigated the expression of RBTN2 in human leukaemic cells, and in human and murine normal myeloid progenitor cells. RBTN2 is expressed in the leukaemic cells of patients with pre-B ALL, T-ALL and acute myeloblastic leukaemia (AML). By cytogenetic and molecular techniques no abnormalities were noted in 11p13. Using clonogenic assays and single cell PCR we found that RBTN2 is expressed strongly in the precursors of mixed erythrocyte/macrophage/mast, erythrocyte, megakaryocyte, neutrophil and macrophage colonies. In contrast, RBTN2 message was low to undetectable in the mature progeny. These findings indicate that RBTN2 is expressed in leukaemias of both the myeloid and lymphoid lineages. Further, these studies show that in normal myeloid and lymphoid cells the expression of RBTN2 is present in progenitor cells and is lost as the cells terminally differentiate. This latter finding further illustrates that the detection of a RNA in a population of cells should not be interpreted to mean that all of the cells in that population express the RNA. PMID- 8639418 TI - Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity. AB - The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vbeta gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the V beta gene usage were determined by comparing the TCR Vbeta gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro. Diverse Vbeta genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro. Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vbeta gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo, the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development. PMID- 8639419 TI - Leucocyte filterability: comparing diluted blood with purified cell suspensions. AB - Current rheological trends in filtration studies involve purification of cell subpopulations in order to study near-homogenous populations of cells, and this means that time-consuming and possibly cell-damaging procedures are used. Filtration of diluted blood offers the possibility of determining the properties of subpopulations of cells with minimal cell manipulation. The filtration properties of granulocytes and lymphocytes result in their being detected as one kinetic population, and therefore a combined granulocyte/lymphocyte pore transit time is calculated. There was no significant change seen in the combined granulocyte/lymphocyte transit time over the normal range of differential count. Filtration of diluted blood is a more reproducible way of measuring granulocyte and lymphocyte filterability, because purification of these cells gives rise to more scatter and larger ranges in the measured pore transit time, and significant day-to-day variation. A combined granulocyte/lymphocyte transit time is therefore an acceptable and reproducible way of assessing leucocyte rheology in diluted blood, when the differential count lies within the normal range. If changes in the granulocyte/lymphocyte transit time are detected, then cell purification may yield additional information on the subpopulations. Therefore, particularly in pathological samples, a combination of these approaches will yield more information than either test alone. PMID- 8639420 TI - Nerve growth factor enhances survival and cytotoxic activity of human eosinophils. AB - Phenotypic change of blood-type eosinophils to tissue-type eosinophils is induced by various cytokines. We examined the effect of nerve growth factor (NGF) as a candidate for a constitutive cytokine which is able to induce the phenotypic change of eosinophils. The viability of human peripheral blood eosinophils cultured for 4 d was increased from a mean of 26% to a mean of 50% (P<0.001) by the addition of NGF (50 ng/ml). Cytotoxic activity of eosinophils determined by larvicidal activity was increased 2-3-fold by the addition of NGF (50 ng/ml) (P<0.05). Furthermore, eosinophil chemotactic activity of NGF was demonstrated by the blind well chamber method. Since NGF is produced constitutively from various kinds of cells in local tissues, it is suggested that NGF might be a cytokine responsible for phenotypic change to tissue type eosinophils in conditions without immune stimuli. PMID- 8639421 TI - Thrombo-embolic disease after splenectomy for hereditary stomatocytosis. AB - Nine cases of hereditary stomatocytosis (HSt) are presented which show documented thrombotic complications after splenectomy. In three cases, patients became severely ill with pulmonary hypertension and a fourth developed portal hypertension. One unsplenectomized affected adult relative had suspected but unconfirmed thrombotic pathology; the six other affected unsplenectomized adults did not. Since splenectomy is of only limited therapeutic benefit in stomatocytosis, it should not be performed without careful consideration. A tendency to iron overload, even without hypertransfusion and irrespective of splenectomy, is evident in many of these patients. PMID- 8639422 TI - Comparison of the deoxyuridine suppression test with serum levels of methylmalonic acid and homocysteine in mild cobalamin deficiency. AB - Both the deoxyuridine suppression test (dUST) and the cobalamin-dependent metabolites, methylmalonic acid (MMA) and homocysteine, are valuable tools for identifying clinical cobalamin deficiency. Examination of these metabolic changes in mild or marginal deficiency can provide useful comparisons of diagnostic frequencies and sensitivities and help define the sequence of metabolic changes in early deficiency. These tests were therefore compared directly with each other in 50 patients with low cobalamin levels and few or no obvious signs of deficiency. Serum homocysteine (P=0.0003) and MMA levels (P=0.0004) correlated with dUST results. However, the dUST results were abnormal significantly more often (38/50 patients) when matched against levels of homocysteine (25 abnormal results of 50; P=0.007) or MMA (20/50; P=0.008). Abnormalities of one or both serum metabolite levels (30/50 patients) occurred almost as often as dUST abnormalities (P=0.059). Metabolite levels, even when originally 'normal', fell with cobalamin therapy in many cases. The results indicate that both the dUST and serum metabolite levels become abnormal before macrocytic anaemia develops in mild cobalamin deficiency. The dUST appears to be most frequently abnormal of the tests; metabolite levels appear to rise almost concurrently but they do not become diagnostically abnormal as soon. PMID- 8639423 TI - In vitro progenitor analysis in a Diamond Blackfan anaemia patient who responded once but not twice to interleukin-3 therapy, using short-term and long-term cultures and c-kit analysis. AB - Interleukin-3 (IL-3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL-3 (+EPO) generated well-haemoglobinized BFU-E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL-3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL-3 therapy, but not a second time following relapse, using short-term culture, long-term culture and c-kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long-term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c-kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL-3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested. PMID- 8639424 TI - Aplastic anaemia patients with clonal X-chromosome inactivation pattern in haemopoietic cells exhibit polyclonal TCRgamma and IgH gene rearrangements. AB - Previously, we reported that 13/18 (72%) female patients with aplastic anaemia (AA) exhibited a clonal X-chromosome inactivation (XCI) pattern in all haemopoietic lineages. To study the consequences of a clonal haemopoiesis for the randomness of immunoreceptor rearrangements in lymphocytes we determined clonality of T-cell receptor gamma (TCRgamma) and immunoglobulin heavy chain (IgH) gene rearrangements in purified cell fractions. Peripheral blood granulocytes, monocytes, and B and T lymphocytes from 18 female patients in remission from AA were studied by PCR for randomness of XCI and rearrangement at the IgH and TCRgamma locus. 13 patients were informative at the phosphoglycerate kinase-1 (PGK1) and monoamine oxidase A (MAOA) loci. Five of them displayed an clonal XCI pattern in all lineages studied and one patient had a clonal XCI in all lineages, except the T cells. In three cases skin biopsies were also available, exhibiting a polyclonal pattern in two of them, and a reversed skewed pattern in the third. Analysis of the rearrangement patterns at the immunoreceptor loci revealed a polyclonal ladder of bands, irrespective of XCI status in the lymphocyte populations. These results demonstrated that in AA a clonal XCI pattern of the lymphoid compartment is compatible with a polyclonal immunoreceptor rearrangement pattern. PMID- 8639425 TI - The release of nitric oxide and superoxide anion by neutrophils and mononuclear cells from patients with sickle cell anaemia. AB - The aim of this work was to investigate the release of nitric oxide and superoxide by neutrophils and mononuclear cells from patients with sickle cell anaemia. Nitric oxide release was assayed by the ability of leucocytes to inhibit thrombin-induced washed platelet aggregation. Superoxide release was assessed by a cytochrome c reduction assay. Neutrophils from sickle cell anaemia patients released nitric oxide in a similar manner to those from healthy controls, because inhibition of platelet aggregation by neutrophils from sickle cell anaemia or from healthy controls was blocked by the inhibitor of nitric oxide synthesis N(omega)-nitro-L-arginine methyl ester (300 microM), but not by N(omega)-nitro-D arginine methyl ester (300 microM) and was reversed by L-arginine (1 mM). Additionally, a similar number of neutrophils from sickle cell anaemia patients and from healthy controls was required to inhibit platelet aggregation. Mononuclear cells from sickle cell anaemia patients inhibited platelet aggregation only in the presence of superoxide dismutase (60 U ml(-1)). Phorbol 12-myristate 13-acetate (PMA, 30 nM)- or zymosan (100 particles/cell)-induced release of superoxide by mononuclear cells from sickle cell anaemia patients was significantly higher than that observed in mononuclear cells from healthy controls (P<0.001 and P<0.01 respectively, Mann-Whitney test). The levels of superoxide released by neutrophils from sickle cell anaemia patients were similar to those from healthy controls. We conclude that mononuclear cells from sickle cell anaemia patients release more superoxide than those from healthy controls, when stimulated with PMA or zymosan in vitro. Considering that superoxide inactivates nitric oxide, that nitric oxide is an important endogenous vasodilator, and that superoxide produces oxidant damage, this greater production of superoxide by mononuclear cells from sickle cell anaemia patients may represent an additional risk factor for the obstruction of the microcirculation and tissue damage in these patients. PMID- 8639426 TI - Long-term salvage treatment by cyclosporin in refractory autoimmune haematological disorders. AB - We report on long-term treatment (13-62 months) with cyclosporin A (CyA) in eight patients with autoimmune haematological disorders, resistant to all usual therapies. Three patients had an autoimmune haemolytic anaemia (AHA); four an idiopathic thrombocytopenic purpura (ITP), and one an Evans syndrome. All patients were responsive: six achieved complete remission and two partial remission. The side-effects were moderate and transient. The majority of surviving patients remain dependent on continued drug administration. The CyA appears to be recommendable as a salvage treatment in life-threatening, resistant autoimmune haematological diseases. PMID- 8639427 TI - Presenting features and prognosis in 72 patients with multiple myeloma who were younger than 40 years. AB - The purpose of this study was to analyse the presenting clinical and laboratory features and the outcome of 72 patients with multiple myeloma (MM) who were younger than 40 years. The records of all Mayo Clinic patients with MM younger than 40 years who were seen between 1 January 1956 and 31 December 1992 were reviewed. Survival was measured from the date when treatment was required to the date of last follow-up or death. The frequency of MM in patients younger than 40 and 30 years in 3278 Mayo Clinic patients was 2.2% and 0.3%, respectively. The main presenting clinical features were bone pain (66%), fatigue (26%), extramedullary plasmacytomas (19%) and bacterial infection (11%). Renal function impairment (creatinine level > or = 177 micromol/l) and hypercalcaemia (serum calcium value > or = 2.75 mmol/l) occurred in 29% and 30% of patients, respectively. Among the 57 patients evaluable for response the objective response rate was 54%. 14/35 patients treated with a single alkylating agent achieved an objective response, whereas 17/22 patients given combination chemotherapy had an objective response (P=0.013). However, this higher response rate did not result in a significantly longer survival. The median survival for the 72 patients was 54 months. Patients with good prognostic features (normal renal function or low beta 2-microglobulin level) had a median survival of 8 years. The actuarial survival at 5 and 10 years after initiation of therapy was 43% and 13%, respectively. In summary, survival in very young patients with myeloma is longer than that observed in series of patients of all ages, especially in those with good prognostic factors. PMID- 8639428 TI - Elevated circulating levels of TNFalpha and its p55 soluble receptor are associated with an adverse prognosis in lymphoma patients. AB - In 88 newly diagnosed lymphoma patients, tumour necrosis factor alpha (TNFalpha) and soluble TNF type I receptor (p55-R-TNF) were prospectively determined in plasma by immunoradiometric assay (IRMA) and ELISA methods respectively. These 88 patients included 19 with centrocyto-centroblastic lymphoma, 13 patients with other low-grade lymphoma, and 56 with high-grade lymphoma. Median TNFalpha plasma values were 20 pg/ml (range 5-380 pg/ml) in patients versus 7 pg/ml (range 4-9 pg/ml) in 20 healthy control subjects. Presence of TNFalpha level > or = 20 pg/ml was significantly associated with elevated LDH level (P<0.0001), serum beta2 microglobulin level > or = 3 mg/l (P<0.0001), haemoglobin < or = 12 g/dl (P=0.0001), Ann Arbor stage III or IV disease (P<0.005), and with bulky tumour (P=0.01). High level of TNFalpha was also associated with B symptoms (P<0.005), poor performance status (P<0.05), and serum albumin < or = 35 g/l (P<0.05). Levels of p55-R-TNF were also markedly elevated in these lymphoma patients (median of 3.5 ng/ml, range 0.8-18.8 ng/ml) versus 1.45 ng/ml in control subjects (range 1.1-2.3 ng/ml). Level of p55-R-TNF > or = 3.5 ng/ml was significantly associated with poor performance status (P<0.0001), B symptoms (P<0.0001), beta2 microglobulin levels > or = 3 mg/l (P<0.0001), serum albumin < or = 35 g/l (P=0.0001), C-reactive protein > 6 mg/l (P=0.0003), elevated (>20 pg/ml) IL-6 level (P<0.005), haemoglobin < or = 12 g/dl (P<0.005), and bulky tumour (P<0.001). In the whole group of 88 patients, both high TNFalpha and p55-R-TNF levels strongly predicted short progression-free survival (P<0.005 for both variables) and overall survival (P<0.001 and P<0.001 respectively). In multivariate analyses the elevation of p55-R-TNF retained a higher significance over the other variables and therefore improved the predictive value of the International Prognostic Index. This study suggests that elevated TNF gamma and p55-R-TNF levels have high correlation with other adverse prognostic factors in lymphoma patients and may predict a poor outcome. PMID- 8639429 TI - All-trans-retinoic acid increases adhesion to endothelium of the human promyelocytic leukaemia cell line NB4. AB - Pulmonary distress symptoms and thrombotic complications are side-effects of all trans-retinoic acid (ATRA) therapy for remission induction in acute promyelocytic leukaemia (APL). The ATRA-induced increase of leukaemic cell adhesive molecules may be responsible. To explore this we used a functional assay to study the effect of ATRA treatment on the adhesion of blast cells to cultured human endothelial cells (EC), endothelial cell matrix (ECM), and interleukin 1beta activated EC (IL1 + EC). NB4 cells, a maturation-inducible human promyelocytic leukaemia cell line, were treated with 1 microM ATRA or the vehicle (control), labelled with 51Cr and tested in the adhesion assay. ATRA increased NB4 adhesion to EC (P<0.01), ECM (P<0.001) and IL1 + EC (P=n.s.). An inhibition study with anti-EC adhesion receptors MoAbs indicated that anti-E-selectin, anti-VCAM-1 and anti-ICAM-1 effectively inhibited cell adhesion to IL1 + EC (18+/-7%, 45 +/-6.9% and 29+/-6% inhibition, respectively) and to unstimulated EC. Preincubation of ATRA-treated NB4 cells with MoAbs anti-VLA4 and anti-LFA1, the VCAM-1 and ICAM-1 counter-receptors respectively, resulted in a significant inhibition of adhesion. Cytofluorimetric analysis of the NB4 cell membrane molecules confirmed the increase under ATRA of VLA4, LFA1, MAC1 and ICAM-1. Therefore ATRA increases NB4 cell adhesion to the endothelium and the subendothelial matrix. These findings parallel the increment of NB4 surface adhesive molecules, among which VLA4 and LFA1 appear to play an important part. These mechanisms may contribute to the complications of ATRA therapy in APL. PMID- 8639430 TI - Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia. AB - Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA-5, VLA-4, LFA-1, ICAM-1, L-selectin and c-kit) on bone marrow CD34++ cells from 16 CML patients by three-colour flow cytometry. The mean percentage of cells expressing L-selectin in the CD34++CD38+(or)++ fraction from untreated CML patients was significantly lower, and that in the CD34++CD38- fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon-alpha (IFN-alpha), the mean percentage of the cells expressing L-selectin in the CD34++CD38- fraction from three patients with a low percentage of Ph1(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph1(+) cells. In addition, L-selectin expression rate was inversely correlated to the percentage of Ph1(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34++ fraction except LFA-1. These data suggest that decreased L-selectin expression in CML CD34++ cells reflects one of the features of malignant CML progenitors. PMID- 8639431 TI - Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl positive targets. AB - Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56+CD3- cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl+ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl+ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P=0.002 and P=0.029, respectively) in the BCR/abl- cultures than those in the six BCR/abl+ cultures. 5/6 BCR/abl- cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56+CD3+ cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33+) were more frequently detected in the BCR/abl+ than BCR/abl- A-NK cell cultures (P=0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl+ cells from these cultures. PMID- 8639432 TI - Donor leucocyte transfusions for relapse in myelodyplastic syndrome after allogeneic bone marrow transplantation. AB - A 31-year-old man with refractory anaemia of excess blasts, which had karyotypic abnormalities, received an allogeneic bone marrow transplant (BMT). At time of relapse, 3 months after BMT, he was treated with donor leucocyte transfusion (DLT). Grade III acute GVHD (graft-versus-host disease) occurred 35 d after DLT which was fully reversed with cyclosporin and prednisolone. His condition was complicated by a herpes zoster infection. 2 months after DLT, neutrophil and platelet count were increased and karyotypic abnormalities disappeared. This observation demonstrates that DLT is an effective treatment for relapse of myelodysplastic syndrome (MDS) after BMT. PMID- 8639433 TI - Progression from myelodysplastic syndrome to acute lymphoblastic leukaemia with Philadelphia chromosome and p190 BCR-ABL transcript. AB - We describe a patient with Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) who developed it 2.5 years after being diagnosed with myelodysplastic syndrome (MDS). The patient initially had refractory anaemia (RA), but progressed to refractory anaemia with excess blasts (RAEB) 2 years later, that terminated in ALL. An immunophenotypic analysis of the lymphoblasts revealed CD10 and CD19 positive cells. The karyotype was normal 46,XY in RA phase, 46,XY,20q-during the RAEB phase, and 46,XY,t(9;22)(q34;q11),20q-during the ALL phase. Furthermore, p190 BCR-ABL mRNA was detected in the ALL blasts. These findings indicate that this ALL arose from the MDS clone through multiple cytogenetic evolutions, the final event of which was the acquisition of p190 BCR ABL type Ph1. PMID- 8639434 TI - Differential regulation of GPI-linked molecules on leukaemic promyelocytes treated in vitro with all-trans retinoic acid. AB - It has been demonstrated that certain cell-surface proteins are anchored to the cell membrane by a unique structure known as the glycosylphosphatidylinositol (GPI) anchor whose absence has been reported on blood cells from patients with paroxysmal nocturnal haemoglobinuria. We have investigated the expression of CD16/Fc(tau)R-III and CD66b GPI linked molecules at the surface of blast cells from five acute promyelocytic leukaemia (APL) patients before and after in vitro stimulation with all-trans retinoic acid (ATRA). We observed that whereas CD66b antigen exhibited a strong ATRA-driven up-regulation in all cases studied, CD16 expression was unaffected by the treatment with the drug. PMID- 8639435 TI - Idiopathic myelofibrosis in children. AB - Childhood myelofibrosis (Mf) is rare with variable outcome reported in the literature. We present clinical and investigate details of three children who presented with idiopathic Mf in early childhood. Two of these children were identical twins and have been haematologically stable over the past 7 years since their diagnosis. The third patient underwent an allogeneic bone marrow transplant (BMT) procedure as her clinical status was deteriorating. She remains engrafted at 13 months post BMT. None of the children had hepatosplenomegaly although extramedullary haemopoiesis was demonstrated on 52Fe studies in two patients. Circulating progenitors in these patients were increased in the face of reduced marrow precursors. The aetiology of childhood Mf is unclear and its natural history seems different from the adult disease. Allogeneic BMT can be an option for definitive treatment. PMID- 8639436 TI - Soluble interleukin-6 receptor (sIL-6R), a new prognostic factor in multiple myeloma. AB - sIL-6R is a 55 kD soluble molecule mediating the interleukin-6 (IL-6) signal through the IL-6 receptor-associated transmembrane signal transducer, gp130. It has recently been suggested that sIL-6R serum levels may reflect disease severity in multiple myeloma (MM). We determined sIL-6R serum levels in 25 normal controls (NC) and in 80 MM patients at diagnosis and during the course of the disease. Measurements were done by ELISA. In NC, sIL-6R levels ranged from 14 to 40 ng/ml (median 28 ng/ml) whereas in MM patients the range was 10-200 ng/ml (median 38 ng/ml) (P<0.01). 61 patients entered remission and 19 were resistant. Median sIL 6R value at diagnosis was 36 ng/ml (10-120) in responding patients, and 82 ng/ml (20-200) in non-responding patients (P<0.001). During a follow-up from 12 to 89 months, sIL-6R values remained more or less stable in most patients. High sIL-6R levels correlated with poor survival. PMID- 8639437 TI - Acute myeloblastic leukaemia presenting with herpes simplex type-1 viraemia and pneumonia. AB - We report a patient with acute myeloblastic leukaemia who presented with a pneumonia and herpes simplex viraemia associated with primary herpes simplex virus-1 infection. The importance of detecting and treating viral infections in haematology patients is discussed. PMID- 8639438 TI - BCR rearrangement without juxtaposition of ABL in pre-T acute lymphoblastic leukaemia. AB - The incidence and clinical relevance of the Philadelphia (Ph) translocation t(9:22) (q34:q11) in T-lineage acute lymphoblastic leukaemia (ALL) are unknown. We describe a patient with pre-T-ALL and a clonal 22q-aberration detected by conventional cytogenetics, suggestive of a Ph translocation. However, fluorescence in situ hybridization (FISH) using BCR and ABL probes revealed a translocation with one breakpoint within the BCR gene on chromosome 22 without juxtaposition of ABL on chromosome 9. We discuss the diagnostic and possible pathogenetic implications of this Ph-like chromosomal aberration. PMID- 8639439 TI - Cyclosporine A alleviates severe anaemia associated with refractory large granular lymphocytic leukaemia and chronic natural killer cell lymphocytosis. AB - Large granular lymphocytic (LGL) leukaemia and chronic natural killer cell lymphocytosis (CNKL) are chronic indolent disorders often associated with neutropenia and constitutional symptoms. Severe anaemia occurs in about 20% of patients and is currently treated with corticosteroids followed by oral cyclophosphamide in non-responders. 30% of patients fail initial measures, and salvage therapy is inadequate. We describe three transfusion-dependent patients (two with T-LGL leukaemia, one with CNKL) refractory to corticosteroids, cyclophosphamide, and in one case fludarabine. Cyclosporine A (CSA) initiation resulted in prompt transfusion-independence and was well tolerated in all patients, making it an attractive alternative therapy for this disorder. PMID- 8639441 TI - Graft-versus-leukaemia activity can be predicted by natural cytotoxicity against leukaemia cells. AB - We have investigated graft-versus-leukaemia (GVL) effects after allogeneic bone marrow transplantation (BMT), using three murine leukaemia models, A20 (B lymphocytic), WEHI-3 (myelomonocytic) and PU5-1R (myeloid). Injection of leukaemia cells in a high number (10(6) cells) into syngeneic Balb/c mice (H-2d) invariably led to death with a median survival time of 22 d (A20), 18 d (WEHI-3) and 45 d (PU5-1R). A lower tumour load of A20 (5 x 10(5) cells) was used in some experiments resulting in a leukaemic death rate of 94%. Lethal total-body irradiation followed by syngeneic BMT prolonged survival (P<0.05) for animals bearing the leukaemia A20 and WEHI-3 but was unsuccessful for animals injected with cells from the monocytic leukaemia PU5-1R. Graft-versus-host (GVH) nonreactive marrow of (C57 x Balb/c)F1 mice (H2bxd) exerted a significant GVL effect with reduced relapse rate and improved survival in mice receiving the leukaemia cell line A20. In animals with low tumour load a significant reduction of the relapse rate from 82% following syngeneic BMT to 47% following allogeneic, GVH-nonreactive BMT could be achieved. Depletion of natural killer (NK) cells from the GVL-reactive semi-allogenic bone marrow graft enhances the relapse rate of the leukaemia A20 to 65%. In mice bearing the leukaemias WEHI-3 or PU5-1R allogeneic GVH-nonreactive BMT did not improve survival compared to syngeneic BMT. Transplantation of GVH-reactive bone marrow from DBA mice (MHC identical to Balb/c, minor difference) caused only a limited and insignificant reduction of relapse rate for animals with the leukaemia A20. These in vivo data are in close correlation with in vitro natural killer cell (NK) activity of the donor strains against the respective leukaemia targets. Depletion of NK cells from the GVL reactive (C57 x Balb/c)F1 bone marrow resulted in a significant loss of GVL activity. We conclude that NK cells are involved in graft-versus-leukaemia effects independent of graft-versus-host disease (GVHD). PMID- 8639440 TI - 2-Chlorodeoxyadenosine in the treatment of hairy cell leukaemia: differences in response in patients with and without abdominal lymphadenopathy. AB - We treated 26 patients with hairy cell leukaemia (HCL) with 2 chlorodeoxyadenosine, including nine with abdominal lymphadenopathy and of whom two had HCL-variant; 18 were previously treated. The overall response in 23 evaluable HCL patients was 100% with 87% complete remission (CR). The CR rate was 57% in patients with abdominal lymphadenopathy. Neither of the patients with HCL variant achieved CR. Of four patients who had become refractory to 2' deoxycoformycin, two achieved CR and one partial remission (PR). The lower response in HCL with abdominal lymphadenopathy supports the view that this represents a more resistant form of the disease. PMID- 8639442 TI - Isolation and characterization of cysteine proteinase in thrombotic thrombocytopenic purpura. AB - Thrombotic thrombocytopenic purpura (TTP) is an uncommon disorder characterized by thrombocytopenia, schistocytic haemolytic anaemia, fever, neurologic lesions, and renal failure. A platelet aggregating factor has been postulated to be responsible for this disorder, but its precise identity remains debated. Two different groups of investigators have provided evidence that the platelet aggregating factor is a cysteine proteinase. We have suggested that it was calpain, whereas others have suggested that it was calpain, whereas others have suggested that it was cathepsin L. To help resolve this issue, we have studied the platelet activating activity found in the acute serum samples from 10 different TTP patients as well as purified calpain and cathepsin L. The TTP activity was measured functionally (platelet serotonin release assay and casein lysis assay) and antigenically (immunodepletion using anti-calpain and anti cathepsin L antibodies and antigenic analysis using SDS PAGE). The TTP serum paralleled the activity of the purified calpain and had optimal pH activity of 7.5. The purified cathepsin L activity had optimal activity at low pH (5.5) and activity was no longer measurable at pH 7.5. Similarly, a specific cathepsin L inhibitor (Z-Phe-Phe-CHN2) had no effect on the activity of the TTP samples nor on purified calpain, but it did abolish the activity activity of purified cathepsin L. The platelet activating of the TTP samples was detectable in the microparticle pellet following ultracentrifugation of TTP serum, and could be immunodepleted using antibodies to calpain but not to cathepsin L. These studies indicate that the microparticle-associated platelet activating factor in TTP corresponds to calpain. PMID- 8639443 TI - Fusidic acid induced acute immunologic thrombocytopenia. AB - Fusidic acid is used in hospitals as second-line therapy for multidrug-resistant staphylococcal infections. We report the first fully documented case of fusidic acid induced thrombocytopenia, in a 48-year-old patient. The thrombocytopenia was abrupt and severe but resolved spontaneously 7 d after drug withdrawal. The thrombocytopenia transiently relapsed 6 d later, when fusidic acid was reintroduced. Haemorrhagic signs were observed, but no severe bleeding occurred. Platelet transfusions failed to increase the platelet count. We detected an IgG platelet antibody in the patient's serum, that specifically recognized platelet glycoprotein IIb/IIIa only in the presence of fusidic acid. Fusidic acid induced thrombocytopenia should be considered as a possible cause for the thrombocytopenia frequently seen in the intensive care setting. PMID- 8639445 TI - Comparison of lyophilized plasmas with fresh plasmas for calibration of thromboplastin reagents in oral anticoagulant control. AB - In a multicentre study of four thromboplastin reference materials (three from rabbit brain and one from bovine brain combined with adsorbed bovine plasma), three types of lyophilized plasmas were included. One was a pooled normal plasma, another was made by pooling plasmas of coumarin-treated patients, and the third was prepared by adsorption of vitamin K dependent clotting factors (artificially depleted). The purpose of this study was to investigate whether these lyophilized plasmas could be used for reliable calibration of the four thromboplastins. Calibration line slopes were calculated for the lyophilized plasmas and compared to the slopes based on fresh plasmas. When a rabbit thromboplastin was calibrated against a similar but not identical reagent (like-to-like comparison), the slopes for lyophilized plasmas and for fresh plasmas were the same. However, significant differences between lyophilized and fresh plasmas were observed in the calibration of unlike thromboplastins. The deviation of the slope with artificially depleted plasma was greater than that with lyophilized coumarin plasma. Indiscriminate use of these lyophilized plasmas for thromboplastin calibration should be avoided. PMID- 8639444 TI - Effects of desmopressin on circulating P-selectin. AB - Von Willebrand factor (VWF) and P-selectin share an identical intracellular storage compartment and transport to the cell surface. We induced degranulation of the Weibel-Palade bodies and measured circulating (c)P-selectin in plasma to test whether soluble P-selectin is present in the Weibel-Palade bodies, or if P selectin bound to the cell membrane of endothelial cells (EC) is rapidly proteolytically cleaved in vivo. A dose of 0.3 microgram/kg of desmopressin (DDAVP) or placebo was infused over 30 min to eight healthy men in a double-blind cross-over study. Plasma levels of cP- selectin and VWF-Ag were followed for 24 h. Despite a twofold increase in VWF-Ag levels immediately after a 2 h after infusion of DDAVP, no significant change in plasma concentrations of cP-selectin were observed. In summary, degranulation of the Weibel-Palade bodies is an unlikely source of cP-selectin. Thus cP-selectin in healthy subjects is conceivably attributable to other mechanisms, such as minor degrees of platelet activation or transcription induction of an alternatively spliced P-selectin. PMID- 8639446 TI - Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity. AB - The integrity of the gamma-carboxylic glutamic acid (GLA) residues of coagulation factor VII are thought to be essential for both the interaction of factor VII with its cell-surface lipoprotein receptor tissue factor and for the activated protein to manifest its serine protease activity. During the course of transiently expressing recombinant human factor VII in monkey COS cells it was noted that the factor VII synthesized in the absence of added vitamin K had <20% of expected procoagulant activity yet retained 65% of its binding activity to recombinant human tissue factor. Similar results were obtained when vitamin K was omitted from human 293 cell cultures permanently expressing recombinant factor VII. In contrast, both transient and permanent expression of factor VII in human 293 cell cultures containing physiological concentrations of vitamin K resulted in the synthesis of fully functional factor VII. Furthermore, factor VII in plasma samples from 24 patients undergoing warfarin therapy bound quantitatively to tissue factor whereas factor VII procoagulant activity averaged 65% of normal. Thus, data from both in vitro and in vivo situations indicated that factor VII molecules with suboptimal GLA content retained most of their ability to bind tissue factor but exhibited reduced procoagulant activity. PMID- 8639447 TI - Molecular characterization of haemophilia A in southern Chinese. AB - 41 unrelated southern Chinese haemophilia A patients were studied. The 5' promoter region, all 26 exons, their immediate 5' and 3' flanking splice junctions and the 3' untranslated region of the FVIII gene were amplified (including 16 different segments of exon 14) using GC-clamped primers. The GC clamped PCR products were screened by denaturing gradient gel electrophoresis (DGGE) and fragments showing an abnormal migration pattern were sequenced. 17 mutations were identified, of which four were splicing defects, involving the first 1-6 nucleotide (nt) in the intervening sequences (IVS), six missense mutations, three nonsense mutations and four frameshift mutations. 14 other patients carried the type 1 inversion, affecting the distal copy of the FSA gene at the telomere of the X chromosome and the same gene in intron 22 of the FVIII gene. All the mothers studied (12/14) were carriers of the inversion. Two of these patients with inversion also have a co-existing missense mutation. In most cases the clinical severity of the disease corresponds to the genotype. PMID- 8639448 TI - Transmission of symptomatic parvovirus B19 infection by clotting factor concentrate. AB - The risk of acquiring diseases from transfusion of blood and blood products is well recognized and the issue of parvovirus in haemophiliacs is not a new one. We report two patients with haemophilia acquiring iatrogenic parvovirus B19 infection, resulting in life-threatening sepsis in one, and immunocompetent adult. Over the last 10 years there has been great progress in manufacturing safer products with regard to enveloped viruses such as HIV, hepatitis B and C. A recent outbreak across Europe of hepatitis A in haemophiliacs treated with plasma derived factor VII concentrates has made haemophilic treaters concerned about the known (parvovirus B19 and hepatitis A) and the unknown non-lipid enveloped viruses that may be contained in the clotting factor concentrates, because these are resistant to the existing viral inactivating techniques. The possibility of HIV itself mutating into a non-lipid enveloped virus emphasizes the need to seek and use safer products. PMID- 8639449 TI - The incidence and cause of coagulopathies in an intensive care population. AB - We studied 235 patients admitted to an adult intensive care unit in order to determine the incidence and cause of coagulation disturbances. Clinical coagulopathy, defined as bleeding unexplained by local or surgical factors, was identified in 13.6% of patients. Laboratory evidence of coagulopathy was more common: a prothrombin time (PT) ratio > or = 1.5 was found in 66% of patients and a platelet count <100 x 10(9)/l in 38% of patients. Both factors were predictive of excessive bleeding and poor outcome. In a retrospective analysis of plasmas from 45 of the above patients who had PT ratios > or = 1.5 the most common cause was vitamin K deficiency (20%). PMID- 8639450 TI - Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet-reactive anti-HIV antibodies in thrombocytopenic narcotic addicts. AB - The levels of platelet-associated Igs (PAIgs) and plasma circulating antiplatelet antibodies were evaluated by a flow cytometric immunofluorescence assay (FCIFA), an enzyme-linked immunoassay (ELISA), and a platelet radioactive antiglobulin test (PRAT), in a group of 45 human immunodeficiency virus (HIV)-infected intravenous drug users (IVDUs), with or without thrombocytopenia (TCP). Direct tests demonstrated an increased amount of PAIgs in 40% of the patients, irrespective of their platelet count. These PAIgs were mainly of IgG class and could not be eluted with ether. Plasma IgG with antiplatelet activity was found in 70% of the thrombocytopenic individuals, whereas it was detected in only one patient without TCP. The relative frequencies of antibodies against the platelet glycoproteins (GPs) Ib/IX and IIb/IIIa were assessed in plasma from all patients by means of the monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). Plasmas from all non-thrombocytopenic patients were negative when tested by indirect MAIPA. In contrast, 10/23 plasma from thrombocytopenic patients reacted with either GP IIb/IIIa, GP Ib/IX, or both GPs. Finally, aiming to investigate whether HIV antibodies from these patients are reactive with normal platelets, we performed absorption-elution experiments, followed by evaluation of HIV antibodies in the indirect eluates by ELISA and Western blot. Interestingly, we detected anti-HIV antibodies that bind to normal platelet antigens in 50% of the ether eluates prepared from control platelets sensitized with plasma from patients with TCP, but in only 5% of eluates obtained from platelets sensitized with plasma from non-thrombocytopenic patients. The present study provides direct evidence that specific autoantibodies against platelet membrane GPs Ib/IX and IIb/IIIa are common in HIV positive thrombocytopenic individuals. The finding in these patients of HIV antibodies cross-reactive with normal platelets, suggests that mimicry of human antigens by HIV could play a key role in the pathophysiology of the HIV-related TCP. PMID- 8639451 TI - Molecular analysis of blood group Rh transcripts from a rGr variant. AB - The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the rGr phenotype. Nucleotide sequence analysis revealed that the rG allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid rG gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C. PMID- 8639452 TI - Large-cell variants of mantle cell lymphoma: cytologic characteristics and p53 anomalies may predict poor outcome. AB - Large-cell variants are uncommon in mantle cell lymphoma (MCL). Here we describe the pathologic and clinical findings in five patients with large-cell lymphoma related to MCL (L-MCL), and compare them to a group of classic small-cell MCL (s MCL) cases. Histologically, the MC origin of the large cells was evinced by their association with a small mantle cell component in the same tissue, or their distribution in a classic mantle zone pattern, or their development in a patient with previous s-MCL. The large cells were either pleomorphic mantle cells (case 1) or transformed blast-like cells (case 2-5). The median nuclear diameter, median nuclear area and proliferation index of L-MCLs and s-MCLs, were statistically different. Immunophenotypic characterization of four specimens of L MCL and 10 of s-MCLs with a large panel of antibodies showed the classic findings of MCL, i.e. the IgM+ D+/-, CD5+, CD10-, CD23- phenotype in all cases except two (one CD5- and one CD23+), and the association with a loose follicular dendritic cell network. Two of four L-MCLs and 5/10 s-MCLs demonstrated rearrangements of the bcl-1 gene by Southern blot or by polymerase chain reaction (PCR); 2/4 L-MCLs and 1/9 s-MCLs had p53 mutations on single-strand conformation polymorphism analysis; none of the 14 specimens showed rearrangement of bcl-2 by PCR or bcl-6 and c-myc by Southern blot. All patients with 'transformed' histology (versus 37% of all others) died of lymphoma; their survival (15-18 months; median 17) was much shorter than that of all the others (28-117+ months; median 43) (P=0.0035). All three patients with p53 anomalies, two of whom had tumours with transformed histology, died of their disease in a short time (15, 18 and 28 months). In contrast, the presence of bcl-1 rearrangements did not have prognostic implications. This study documents the existence of large-cell variants of MCL and the poor prognosis associated with the 'transformed' cytologic type and/or p53 abnormalities. PMID- 8639454 TI - High-dose dexamethasone in adult refractory idiopathic thrombocytopenic purpura. PMID- 8639455 TI - Endogenous cytokine levels after immunoselected CD34+ peripheral blood progenitor cell transplantation. PMID- 8639453 TI - Oral contraceptives enhance the risk of clinical manifestation of venous thrombosis at a young age in females homozygous for factor V Leiden. AB - In 29 patients (17 females) homozygous Arg 506 Gln mutation (FV Leiden) was identified. 25 had been investigated because of venous thromboembolism (VTE); four asymptomatic patients were found during family studies. The first VTE had occurred significantly earlier in females (median age [m] 26 years, range 17-49) than in males (m=38 years, range 21-82) (P=0.01). 12 females (80%) had taken oral contraceptives (OC, estrogen content 0.02-0.1 mg) for 6-150 months prior to thrombosis. Further triggering conditions in females were hormone replacement (n=1) and pregnancy (n=2). In 8/10 males the first VTE had occurred spontaneously -in two after surgery. The sites of VTE were deep vein thrombosis, pulmonary embolism, caval vein thrombosis and superficial thrombophlebitis. From our data we conclude that OC medication is the most important precipitating factor for VTE in females with homozygous FV Leiden. PMID- 8639456 TI - Mantle cell leukaemia? PMID- 8639457 TI - Platelet counts during pregnancy in essential thrombocythaemia treated with recombinant alpha-interferon. PMID- 8639458 TI - Accessory cell function may be retained in CD34-selected G-CSF mobilized peripheral blood mononuclear cells. PMID- 8639459 TI - Chemotaxis of G-CSF induced neutrophils from healthy volunteers. PMID- 8639460 TI - Anti-HCV seroreversion in HIV-negative haemophiliacs. PMID- 8639461 TI - Lymphoid cell resistance to glucocorticoids in HIV infection. AB - In humans infected with the HIV-1 virus there may be a disproportionate severity of signs and symptoms of illness compared to the fraction of CD4+ infected T lymphoid cells. In part, this may be due to altered intercellular signalling systems and intracellular signal transduction. Glucocorticoids are well known for their effects on the vitality and function of lymphoid cells. Patients with HIV infections often show elevated circulating levels of cortisol, suggesting some misfunction in the regulatory systems that maintain the levels of this critical hormone. At the cellular level, it is known that both acute HIV infection and glucocorticoids can cause apoptotic cell death in thymic lymphocytes. However, chronically HIV-infected cells appear to be resistant to glucocorticoid-evoked cell death. Glucocorticoid receptor-ligand binding studies on patients' cells have shown reduced affinity between the receptor binding sites and test steroids. In vitro, chronically HIV-infected cells of the lymphoid CEM line displayed resistance to glucocorticoid-induced apoptosis. These cells showed reduced numbers of binding sites with little alteration in apparent affinity between ligand and receptor. Thus it appears that there may often be malfunction of the normal glucocorticoid response in HIV-infected cells probably due to altered interactions between the glucocorticoid receptor and its hormone. Such alterations may have clinical consequences, including the possibility of a relatively longer life span of infected CD4+ T-lymphocytes, as well as systemic effects of chronically elevated cortisol level. PMID- 8639462 TI - Demonstration of vitamin D receptor expression in a human megakaryoblastic leukemia cell line: regulation of vitamin D receptor mRNA expression and responsiveness by forskolin. AB - We have shown earlier that 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] induces cell growth suppression and cell differentiation of a human megakaryoblastic leukemia cell line, HIMeg. However, the molecular mechanism of 1,25(OH)2 D3 action is still unknown. Prompted by this, we have searched here for the presence of 1,25(OH)2 D3 receptor (VDR) expression in HIMeg cells by reverse transcription polymerase chain reaction (RT-PCR). The amplified product showed an identical size to the product amplified from the control human VDR cDNA and hybridized specifically with the digoxigenin-labeled human VDR cDNA fragment. As expected, VDR mRNA is also expressed in HOS-8603, a human osteosarcoma cell line. These results represent the first reported evidence that VDR mRNA is expressed in megakaryoblastic cells. In addition, the regulation of VDR mRNA expression in HIMeg cells was studied by quantitative RT-PCR. It was found that [correction of the] VDR mRNA expression in HIMeg cells could be down-regulated rapidly by 1,25(OH)2 D3 (10 nM) in a time-dependent manner, reaching a maximal reduction to about 15% of control. However, VDR mRNA expression in HOS-8603 cells was not regulated by 1,25(OH)2 D3 at any time-point tested. Treatment of HIMeg cells with forskolin (1 microM), an activator of adenylate cyclase, caused an increase in VDR mRNA levels. Similarly, VDR mRNA expression in HOS-8603 cells was also up regulated by forskolin. Consistent with the functionality of the VDR in other target cells, we found that the up-regulation of VDR expression in HIMeg cells by forskolin was accompanied by an increased responsiveness of HIMeg cells to 1,25(OH)2 D3 even though forskolin alone had no effects. Exposure to 1,25(OH)2 D3 in combination with forskolin resulted in a much more significant inhibition of cell proliferation than to 1,25(OH)2 D3 alone. Similarly, forskolin could also augment the differentiation induced by 1,25(OH)2 D3 reflected by a more evident morphological change and a higher percentage of development of cells with multilobular nuclei. These alterations were accompanied by a loss of clonogenic capacity and a decrease in the number of cells in the S phase. These data establish that HIMeg cells express functional VDR, which served to mediate actions of its ligand on the proliferation and differentiation of these cells. PMID- 8639463 TI - Progestins and danazol effect on cell-to-cell adhesion, and E-cadherin and alpha- and beta-catenin mRNA expressions. AB - The first step of invasion and metastasis is the detachment of cancer cells in the primary tumor, which is mainly controlled by the function in the adherens junction, consisting of E-cadherin associated proteins (E-cadherin, alpha- and beta-catenins, vinculin, alpha-actinin, and actin). The cell-to-cell aggregation activity and the expressions of E-cadherin, and alpha- and beta-catenin mRNAs in Ishikawa cells of well-differentiated endometrial cancer were significantly suppressed by estrogen. These suppressions were reversed by progesterone, medroxyprogesterone acetate (MPA) and danazol. Proteins in the adherens junction appeared to be expressed intact and to be functional in Ishikawa cells. Persistent estrogen predominant milieu might contribute to the detachment of well differentiated endometrial cancer cells, leading to spreading of those cells, while progestins and danazol protect estrogen-induced spreading of those cells. PMID- 8639465 TI - Structural and functional analysis of N-terminal point mutants of the human estrogen receptor. AB - Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER. PMID- 8639464 TI - Retinoid regulation of human cathepsin D gene expression. AB - Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect. PMID- 8639466 TI - Androgens are not major down-regulators of androgen receptor levels during growth of the immature rat penis . AB - This study was undertaken to investigate the prevalent hypothesis that androgens are responsible for the organ-specific down-regulation of penile androgen receptors (ARs) and decline of penile growth in the rat during sexual maturation. Sexually immature male rats (21 days old) were castrated and treated for 3 days ("short-term"), with high doses of: (a) testosterone and the alpha-reductase inhibitor finasteride (T/F); (b) dihydrotestosterone (DHT); or (c) finasteride alone (F). Intact and castrate controls received vehicle only. PolyA + RNA was analysed by Northern blot hybridization and ARs were estimated in the penis and ventral prostates by (3-H)R-1881 binding in the cytosol. Short-term castration, with or without F, increased penile AR mRNA, whereas high doses of T/F and DHT reduced it considerably. Although penile cytosol AR concentration in the control castrates, with or without F, paralleled the AR mRNA rise, treatment with androgens left cytosol AR content per organ and AR concentration above those of the intact rat penis despite the drop in AR mRNA. A "long-term" treatment (10 days) on 19-day-old rats with either medium or high doses of T/F and DHT also failed to down-regulate penile cytosol ARs below the intact controls. Western blot analysis of penile cytosol AR levels confirmed these results. Block of pituitary FSH and LH release by a GnRH antagonist in castrates receiving T/F or DHT at high doses did not modify the response. In the case of intact rats, high doses of T/F or DHT actually increased penile cytosol AR content. No difference was observed between T/F and DHT effects. In contrast to what occurs during sexual maturation, the prostate ARs and growth rate responded to all treatments in a similar way to what was observed in the penis. Our results suggest that increases in serum T or DHT are not major factors in the physiological down regulation of ARs and androgen-dependent growth in the rat corpora cavernosa. PMID- 8639468 TI - An investigation of cut-points for primary breast cancer oestrogen and progesterone receptor assays. AB - Oestrogen and progesterone receptor (ER and PgR) assay values are frequently used in medical decision-making for breast cancer patients. We have proposed statistical standardization of receptor assay values to improve inter-laboratory comparability, and now report the use of standardized log units (SLU) to investigate the effects of ER and PgR cut-points on time to first recurrence outside the breast (DFS). Between 1980 and 1986, there were 678 primary breast cancer patients treated at the Henrietta Banting Breast Centre (HBBC). The effects of ER and PgR cut-points were examined with multivariate analyses considering the variables: age, tumour size, nodal status, weight and adjuvant treatment. We considered receptor assay cut-points ranging from - 1.0 to + 1.0 SLU (ER between 7 and 166 fmol/mg protein; PgR between 7 and 181 fmol/mg protein). PgR was included in the multivariate prognostic models more often than ER, although patients had a better prognosis with both larger ER and PgR values. There was no best cut-point for ER or PgR, and there was strong evidence that ER and PgR should be considered as continuous rather than dichotomous (negative, positive) variables. Patient prognosis should also be more comparable with SLU. PMID- 8639467 TI - Sulfhydryl groups are involved in the binding of agonists and antagonists to the human mineralocorticoid receptor. AB - To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4 degrees C inhibited the binding to hMR of both [3H]aldosterone and [3H]RU26752 (a synthetic aldosterone antagonist). At 4 degrees C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4 degrees C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20 degrees C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hmR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action. PMID- 8639470 TI - Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes. AB - In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7alpha-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the "acidic" and "neutral" pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7alpha-hydroxylase. Sterol 27 hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 microM; > 7 fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 microM), dibutyryl-cAMP (5O microM), nor squalestatin 1 (15O nM-1.0 microM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 microM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27 hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-beta-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7alpha-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [14C]cholesterol remained relatively high and consisted of beta-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids. PMID- 8639469 TI - Pharmacological profile of the tachykinin receptor involved in the stimulation of corticosteroid secretion in the frog Rana ridibunda. AB - It has recently been shown that the adrenal gland of the frog Rana ridibunda is densely innervated by a network of fibers containing two novel tachykinins, i.e. ranakinin (the counterpart of substance P) and [Leu3, Ile7]neurokinin A. Both ranakinin and [Leu3, Ile7]neurokinin A stimulate corticosteroid secretion from frog adrenal glands in vitro. In the present study, we have investigated the pharmacological profile of the receptors involved in the stimulatory action of ranakinin on perifused frog adrenal slices. The selective NK-1 receptor antagonists [D-Pro4, D-Trp7,9]substance P 4-11 and CP-96,345, did not affect the stimulatory action of ranakinin. The selective NK-1 agonist substance P 6-11 had no effect on corticosteroid secretion. The non-peptidic NK-1 receptor antagonist RP 67580 significantly reduced the stimulatory effect of ranakinin on corticosterone and aldosterone secretion by 57 and 55%, respectively. In addition, the dual NK-1/NK-2 receptor antagonist FK-224 significantly inhibited the effect of ranakinin on corticosterone (- 80%) and aldosterone secretion (- 95%). Finally, the amphiphilic analogue of substance P, [D-Pro2, D-Phe7, D Trp9]substance P, had no effect on corticosteroid secretion. These data suggest that in the frog adrenal gland the stimulatory action of ranakinin on steroid secretion is mediated by a novel type of receptor which differs substantially from the mammalian NK-1 receptor subtype. PMID- 8639471 TI - Testosterone metabolism in new squamous cell carcinoma cell line (RSS18) from 7,12-dimethylbenz[a]anthracene-induced submandibular gland of female rat. AB - We established a new squamous cell carcinoma cell line, designated RSS18, from a 7,12-dimethyl-benz[a]anthracene (DMBA)-induced submandibular gland of the female rat, and investigated a testosterone metabolism in the cells. During 6 h incubation of RSS18 cells with testosterone as a substrate, the cells produced a significant amount of 5alpha-dihydrotestosterone (DHT) and three kinds of minor metabolites, and their percentages metabolized against total metabolites were in descending order of DHT (89 %) > 5alpha-androstane-3alpha,17beta-diol (9.0 %) > 5alpha-androstanedione(1.6%) > 4-androstene-3,17-dione (0.69%). Therefore, testosterone in RSS18 cells was predominantly converted to DHT by 5alpha reductase. Growth of RSS18 cells was stimulated by DHT (10(-11)-10(-9) M) to around 170%. By reverse transcription-polymerase chain reaction, the androgen receptor mRNA was significantly detected in RSS18 cells. As a result of these findings, DHT production from testosterone and expression of androgen receptor mRNA, we concluded that RSS18 proliferation may be stimulated by DHT through 5alpha-reductase from testosterone. PMID- 8639472 TI - Biotransformation XXXIX. Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in Absidia coerulea culture. AB - The strain of Absidia coerulea was used to investigate the transformations of testosterone, androstenedione, progesterone and testosterone derivatives with additional Cl-C2 double bond and/or 17alpha-methyl group. All the examined substrates were transformed, mainly hydroxylated. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. The first three substrates (hormones) underwent hydroxylation at C-14, and additional hydroxylation at 7alpha was observed in progesterone. The presence of the double bond (C1-C2) in 1 dehydrotestosterone did not influence the position of hydroxylation, but the product with additional C14-C15 double bond (at the same site as hydroxylation) was formed. 17alpha-Methyltestosterone was hydroxylated at the 7alpha position, and also the dehydrogenated product (at the same site, with C6-C7 double bond) was obtained. The testosterone derivative with both C1-C2 double bond and 17alpha methyl group underwent hydroxylation at the 7alpha or 11beta position, and a little amount of 14alpha, 15alpha epoxide was formed. PMID- 8639473 TI - Gas chromatography/mass spectometry identification of long-term excreted metabolites of the anabolic steroid 4-chloro-1,2-dehydro-17alpha methyltestosterone in humans. AB - The misuse of anabolic steroids by athletes has been banned by sports organizations and is controlled by the analysis of urine samples obtained from athletes using gas chromatography/mass spectrometry (GC/MS). To extend the retrospectivity of the analytical methods, research is focused on long-term excreted metabolites. Preliminary results concerning the long-term detection of metabolites of the anabolic androgenic steroid 4-chloro-1,2-dehydro-17alpha methyltestosterone I are presented. A new metabolite 4-chloro-3alpha, 6 beta, 17beta-trihydroxy-17alpha-methyl-5beta-androst-l-en-16-one was isolated by high performance liquid chromatography (HPLC) from urine following a single oral administration of 40 mg of I and characterized. Metabolite II was excreted into urine with a maximum excretion rate at approximately 48 h after administration and could be detected by gas chromatography/high resolution mass spectrometry (GC/HRMS) for up to 14 days. Two further partly characterized metabolites III and IV were confirmed for more than 9 days. The same three metabolites, II-IV, in varying amounts were also detected in urine samples from athletes who administered I. PMID- 8639475 TI - Three-dimensional degradable porous polymer-ceramic matrices for use in bone repair. AB - A degradable polymer-ceramic matrix for use as a bone graft material is described. The fabrication method used produces 3-dimensional macroporous matrices which are structurally similar to cancellous bone in their porosity, mechanically similar to cancellous bone in compressive elastic modulus and chemically comparable to the mineral matrix of bone in that they contain hydroxyapatite (HA). A 50:50 copolymer of poly(lactide/glycolide) (PLAGA) reinforced by a particulate calcium phosphate ceramic, HA, was used to create a matrix composed of polymeric microspheres. The channels between these spheres were pores approximately 100 microns in diameter. Four polymer/ceramic ratios were used in matrix fabrication: 1:0, 1:1, 2.5:1, and 5:1. The mechanical behavior of the material was found to vary with ceramic content. Increased levels of HA resulted in increased compressive elastic moduli. Prior to polymer degradation, moduli ranged from a high of 1459 MPa (50% HA) to a low of 293 MPa (0% HA). Degradation studies over a 6-week period showed that 0 and 16.7% HA containing matrices lost up to 50% of their original weight, while the 28.6 and 50% IIA-containing matrices lost up to 20% of their original weight. Increased HA matrix content translated into decreased rates of matrix degradation. Environmental scanning electron microscopy (ESEM) confirmed that the polymer matrix contained pores that were interconnected during degradation. Viewed via ESEM, 10% HA containing matrices completely degraded by 6 weeks, while 50% HA matrices remained relatively stable. These studies indicate that the porous 3 dimensional polymer/ceramic matrix may potentially be useful as a synthetic material for bone repair. PMID- 8639474 TI - Preparation and characterization of alkylated poly(vinyl alcohol) hydrogels using alkyl halides. AB - A poly(vinyl alcohol) hydrogel coated onto polyethylene was partially alkylated by reaction with an alkylhalide (C4, C8, or C18) in the presence of a deprotonating agent (sodium ethoxide or potassium tert-butoxiDATE Surface coverage determined by X-ray photoelectron spectroscopy (XPS) was respectively approximately 34, 25, and approximately 8% for the C4, C8, and C18 modified surfaces. Statistically significant differences were observed in the fraction of C8 and C18 grafted alkyl groups as a function of depth (i.e. take-off angle) indicating the presence of a verticle composition gradient. All three surfaces showed maximal surface coverage of alkyl groups after 1 h reaction. At this reaction time, no further coverage was observed beyond a base/PVA ratio twenty times greater than the stoichiometric ratio. The advancing contact angle data exhibited an increase in hydrophobicity that correlated with the degree of coverage obtained by XPS: 90 +/- 1, 83 +/- 0.5, and 71 +/- 1 deg for C4, C8 and C18 alkylated PVA, and 55 +/- 2 deg for PVA respectively. Large contact angle hysteresis was observed on all three surfaces consistent with surface heterogeneity. PMID- 8639476 TI - Swelling/syneresis phenomena in gel-forming interpolymer complexes. AB - Grafted poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) copolymers were synthesized and their pH sensitivity investigated as a function of copolymer composition and PEG graft molecular weight. Interpolymer complexation occurred by hydrogen bonding between carboxylic groups on poly(methacrylic acid) (PMAA) and ether groups on poly(ethylene glycol) (PEG). This complexation was sensitive to the surrounding environment as complexes formed at pH levels low enough to insure substantial protonation of PMAA acid groups. At high pH, the acid groups became neutralized and did not form complexes. P(MAA-g-EG) membranes showed pH sensitivity due to complex formation and dissociation. Uncomplexed equilibrium swelling ratios were much higher than those of the complexed states and varied according to copolymer composition and PEG graft length. Mesh sizes in the two states were determined. Swelling under oscillatory pH conditions and constant ionic strength revealed the dynamic sensitivity of P(MAA-g-EG) membranes. Under changing pH conditions, network syneresis (complexation) occurred more rapidly than network expansion (decomplexation) because of the rates of diffusion of specific ions causing the responses. No distinct water fronts were observed. Instead, water transport was continuous through the gel. These gels show great promise for a number of biomedical applications where fast biomaterial response is necessary. PMID- 8639477 TI - Poly(1-vinyl-2-pyrrolidinone) hydrogels as vitreous substitutes: histopathological evaluation in the animal eye. AB - A homopolymer of 1-vinyl-2 pyrrolidinone and its copolymer with 2-hydroxyethyl methacrylate, both cross-linked with divinyl glycol, were produced as possible substitutes for the vitreous body of the eye. The hydrated polymers behaved like viscoelastic gels, displaying excellent physical and optical properties. The sterile gels (0.7-1.5 ml) were injected into the vitreous cavity of rabbits, which previously underwent gas-mediated vitrectomy. Clinically, the eyes were quiet, with the exception of transient opacities in the vitreous. After 4 weeks, the operated eyes were enucleated and subjected to histopathological analysis using light and transmission electron microscopy. The common feature in all sections was the invasion of inflammatory cells. Vacuoles containing granular material, assumed to be polymer, were seen in the intercellular spaces of the neural retina, in the retinal pigment epithelium cells, and in macrophages. These findings indicated the fragmentation and phagocytosis of synthetic gels. It appeared that the biodegradation of the internalized polymers did not proceed further, however, the fate of polymers and their usefulness as vitreous substitutes should be investigated through long-term experiments. PMID- 8639479 TI - Alkylation of cellulosic membranes results in reduced complement activation. AB - 4-Vinyl pyridine was grafted to the surface of the cellulosic membrane Cuprophan, and subsequently alkylated with both C10 and C16 aliphatic chains. Complement activation of heparinized human blood, corrected for anaphylatoxin adhesion, was measured by radioimmunoassay. The surface treatments both yielded substantial reductions in C5a activity, with a lessor reduction in C3a and C4a activity. Alkylation with 10 and 16 carbon chains resulted both in enhancements of albumin adsorption and stability. These enhancements as well as the reductions in complement activation were statistically indistinguishable between the two treatments. The reduction in complement activation was influenced more by adsorption of endogenous albumin and possibly by the vinyl pyridine graft, than the removal of surface active hydroxyl groups from Cuprophan. PMID- 8639478 TI - Amine effect on phenylboronic acid complex with glucose under physiological pH in aqueous solution. AB - A new "intelligent' polymer system was developed utilizing the binding and exchange of phenylboronic acid (PBA) with polyols and/or glucose. In this improved system, an amine component was incorporated into the polymer chain along with PBA, to enhance binding between PBA and glucose under physiological conditions. The PBA-based polymer was formed by free-radical copolymerization of 3-methacrylamidophenylboronic acid (MAPB) with comonomers, N,N dimethylaminopropylacrylamide (DMAPAA) and acrylamide (AAm) in the presence of N,N'-methylenebis(acrylamide) (Bis-AAm) as a cross-linker. The proportion of the amount of PBA groups complexed with glucose vs total amount of PBA groups was determined by the batch method. Compared to PBA copolymers synthesized without amine component, the proportion increased as a function of the amine content as well as the pH of the buffer. These results confirm that the interaction of neighboring amines (unprotonated) to PBA strengthens the binding with glucose, especially at pH 7.4 and above. This new PBA-amine copolymer is promising as a material useful for polyol separation, protection of polyols, and possibly, as an insulin delivery device. PMID- 8639480 TI - New biodegradable oligoesters for pharmaceutical application. AB - Tartaric acid, malic acid, and glyceric acid were copolycondensed with glycolic acid at various molar ratios in feed to quickly synthesize biodegradable oligoesters. They were likely to have a moderately cross-linked structure with relatively low molecular weights and hydrophilic groups on the chains. In addition to macroscopic gels which were insoluble in any solvents, we could obtain the oligoesters which were insoluble in water but soluble in N,N dimethylformamide. The degradation rate of the oligoesters was higher than that of lactic acid (LA) oligomers having molecular weights of a few thousands. On the contrary, their glass transition and flow temperatures were much higher than those of LA oligomers, indicating that their handling during the preparation of drug delivery dosage forms was much improved. The formulation of microspheres containing drugs from the oligoesters revealed that they were useful as biodegradable matrices having high degradation rates. PMID- 8639481 TI - The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin. AB - The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to +/- 80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin. PMID- 8639482 TI - Diffusion of mineral elements evaluated by PIXE at the bone-coral interface. AB - Substituting the tissue of human organs with biomaterials is problematic. However, its importance and relevance justify all the efforts made. An interdisciplinary approach is required. We report on our study of a product for bone substitution. Coral is a natural product, the interest of which we have already demonstrated in our previous work. Following sterilization, natural coral was implanted in sheep femurs. We regularly extracted the implants from the femurs to study the kinetics of elemental mineral transformation of the bone substitutes. For the first time ever, and thanks to the PIXE method (particles induced X-ray emission), we were able to measure the concentration of mineral elements at different time intervals after implantation over a whole cross section. We found a discontinuity of the mineral elements (Ca, P, Sr, Zn, Fe) at the interface between the implant and the receiver. This shows that the osseous attack is not global but, on the contrary, centripetal. Moreover, the fit of the concentration time course indicates that the kinetics of ossification are different for each atomic element and characterize a distinct biological phenomenon. Our analyses confirm the biocompatibility and the ossification of the implanted coral. PMID- 8639483 TI - From good substrates to good inhibitors: design of inhibitors for serine and thiol proteases. AB - Serine and thiol proteases react with peptide substrates to form an acyl-enzyme. We have synthesized inhibitors which are pseudo-substrates and react with the proteases to generate acyl-enzymes which hydrolize slowly. This is achieved by incorporating an electron-donating group near the carbonyl group of inhibitors I [Ac-Phe--C(O)NH--NH--C(O)X] and II [benzyl-O-C(O)-psiAla-Leu-ArgOMe]. The acyl enzymes derived from the reaction of I with papain and II with chymotrypsin hydrolyze with t1/2 of 12 and 1 h, respectively. The increased electron density on the carbonyl group of the inhibitor also reduces the rate of acyl-enzyme formation. Components were incorporated into the inhibitor which interact with the leaving group binding site (S' subsite) and which accelerate the rate of reaction of inhibitor with enzyme. For inhibitor I, X = NH(CH3), k(on) < 0.13 M( 1) s(-1) for the reaction papain, but if X = psiLeu(CH3)2,k(on) =10(5) M(-1) s( 1). Similar results were obtained with II and chymotrypsin. Concomitant with acyl enzyme formation, X is released and a slowly hydrolyzing acyl-enzyme remains. PMID- 8639484 TI - Temperature dependence of the reorganization energy for charge recombination in the reaction center from Rhodobacter sphaeroides. AB - The rate of charge recombination from the primary quinone to the bacteriochlorophyll dimer of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides has been investigated using time-resolved optical spectroscopy. Measurements were performed at temperatures from 293 to 10 K on reaction centers that have specific mutations that result in a range of 425 780 meV for the free energy difference of charge recombination compared to 520 meV for wild type [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Allen, J. P., & Williams, J.C. (1994) Proc.Natl.Acad.Sci.U.S.A. 91, 10265-10269]. In all cases, the rate increased as the temperature decreased, although the details of the dependence were different for each mutant. The observed dependence of the rate upon temperature is modeled as arising principally from a several hundred meV change in reorganization energy. The relationships among the rate, temperature, and free energy differences can be well fit by a Marcus surface using two modes centered near 150 and 1600 cm(-1)with a total reorganization energy that decreases from 930 to 650 meV as the temperature decreases from 293 to 10 K. In the inverted region, where the driving force is greater than the reorganization energy, the rate is found to be approximately independent of the free energy difference. This is modeled as due to the additional coupling of high frequency modes to the reaction. An alternative model is also considered in which a 140 meV increase in the reorganization energy is matched by a 140 meV increase in the free energy difference as the temperature decreases. The possible role of solvent dipoles in determining this temperature dependence of the reorganization energy and the implications for other electron transfer reactions are discussed. PMID- 8639486 TI - Cardiotoxin II segregates phosphatidylglycerol from mixtures with phosphatidylcholine: (31)P and (2)H NMR spectroscopic evidence. AB - The interaction of the cationic protein cardiotoxin II (CTX II) with mixtures of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was investigated using phosphorus ((31)P) and deuterium ((2)H) nuclear magnetic resonance (NMR) spectroscopy. Adding CTX II to 1:1 POPC/POPG mixtures produced a two-component (31)P NMR spectrum, in which the second component had a decreased chemical shift anisotropy. Simultaneously, the (2)H NMR quadrupolar splitting measured from both POPC-alpha-d(2) and POPC-beta-d(2) decreased. Thus, CTX II produces an altered macroscopic phase state of the lipid bilayers, and this obscures any effects on bilayer surface electrostatics observed by (2)H NMR. Using magic angle spinning (MAS) (31)P NMR spectroscopy, two isotropic resonances were resolved in the absence of CTX II and were assigned to POPG (0.47 ppm) and POPC (-.58 ppm). Adding CTX II produced two new isotropic resonances shifted approximately 0.5 ppm downfield. Quantifying the intensities of the various resonance lines revealed that the binding isotherms for different POPC/POPG mixtures shifted onto a universal curve when expressed as a function of the CTX II/POPG ratio. The results indicate that CTX II binds preferentially to POPG and is able to laterally segregate POPG from POPC. Fitting of the binding isotherms was achieved using a two-site model derived from statistical-thermodynamic considerations. One class of binding site is specific for POPG and the other is nonspecific, capable of binding both POPC and POPG. PMID- 8639485 TI - Molecular cloning and functional expression of a new human CC-chemokine receptor gene. AB - The cloning of several receptors activated by either CC or CXC chemokines and belonging to the G protein-coupled family of receptors has been reported recently. In the present work, we describe the cloning of a human gene, named ChemR13, encoding a new CC-chemokine receptor. The gene encodes a protein of 352 amino acids with a calculated molecular mass of 40 600 Da and displaying a single potential site for N-linked glycosylation. Using a set of overlapping lambda clones, the genomic organisation of the locus was investigated, demonstrating that the ChemR13 gene is physically linked, and in the same orientation, as the CC-CKR2 gene that encodes a receptor for the monocyte chemoattractant protein-1 (MCP-1). A distance of 17.5 kb separates the two coding regions, which share 75% identity in nucleic acid and amino acid sequences. Human ChemR13 was functionally expressed in a stably transfected CHO-K1 cell line. Physiological responses to chemokines were monitored using a microphysiometer. Macrophage inflammatory protein 1 alpha (MIP-1 alpha) was the most potent agonist. MIP-1 beta and RANTES were also active at physiological concentrations. The other CC-chemokines, MCP-1, MCP-2 and MCP-3, as well as CXC-chemokines (IL-8, GRO alpha) had no effect. ChemR13 receptor transcripts were detected by Northern blotting in the promyeloblastic cell line KG-1A, suggesting a potential role in the control of granulocytic lineage proliferation or differentiation. ChemR13 is thus a new member of the growing family of chemokine receptors that mediate the recruitment of cells involved in immune and inflammatory processes. Being the fifth functionally identified receptor in his class, this new CC-chemokine receptor (CC CKR) is tentatively designated CC-CKR5. PMID- 8639487 TI - Enforced interaction of one molecule of plastocyanin with two molecules of cytochrome c and an electron-transfer reaction involving the hydrophobic patch on the plastocyanin surface. AB - Laser flash photolysis is used to study the photoinduced electron-transfer reaction cyt(III)//pc(II) + 3Zncyt --> cyt(III)//pc(I) + Zincyt+ at pH 7.0 and 25 degrees. In the covalent (symbol//) complex cyt(III)//pc(II) the acidic patch in cupriplastocyanin is directly cross-linked to the basic patch in ferricytochrome c. The triplet state of zinc cytochrome c reduces the pc(II) moiety, not the cyt(III) moiety, of the covalent complex. The reaction is strictly bimolecular in the entire range of ionic strength studied, from 1.25 mM to 1.00 M. The two reactants interact only transiently, in a collisional complex, and do not form a persistent complex cyt(III)//pc(II)/Zncyt. Because noncovalent (symbol/) association of three separate protein molecules is far less probable than association of the covalent complex and another protein molecule, we conclude that, without the aid of covalent cross-links, one molecule of plastocyanin will not form a ternary complex with two molecules of cytochrome c, cyt/pc/cyt. Dependence of the rate constant on ionic strength is analyzed in terms of van Leeuwen theory of electrostatic interactions, which recognizes the importance of dipole moments of the proteins. This analysis shows that 3Zncyt reacts with the hydrophobic patch in the pc(II) moiety of the covalent complex cyt(III)//pc(II). At high ionic strength, at which electrostatic interactions are practically abolished, the blue copper site is reduced with approximately equal rates via the hydrophobic patch in the pc(II) moiety of the complex and via the acidic patch in free pc(II). This is evidence that the two distinct patches on the plastocyanin surface are comparable in their intrinsic "conductivity" for electrons coming to the copper site. Positively charged and electroneutral redox partners tend to react at the acidic patch (although not necessarily at the initial docking site in this broad patch) for electrostatic, not electronic, reasons. Earlier theorectical studies disagreed about the relative electronic conductivities of the two patches. This experimental study corroborates very recent theoretical studies that found the two patches to be comparable in the efficiency of electron transfer. PMID- 8639488 TI - Water-soluble, recombinant CuA-domain of the cytochrome ba3 subunit II from Thermus thermophilus. AB - Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm( 1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study. PMID- 8639489 TI - Mechanism of lipoxygenase inactivation by the linoleic acid analogue octadeca 9,12-diynoic acid. AB - During the irreversible inactivation of soybean Fe(III)-lipoxygenase [Fe(III) LOX] by octadeca-9,12-diynoic acid (ODYA), significant quantities of 11 oxooctadeca-9,12 diynoic acid (11-oxo-ODYA) are formed [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To elucidate the inactivation mechanism, a quantitative study into the relationship between the inactivation and 11-oxo-ODYA formation was carried out. The following observations were made (1) LOX (0.84 microM) was completely inactivated by 10 to 80 microM ODYA. However, at ODYA concentrations greater than 100 microM, LOX was only partially inactivated, and there was no inactivation at all at ODYA concentrations above 750 microM. The average number of turnovers in which 11-oxo-ODYA was formed increased from 1.2 to 12 when the ODYA concentration increased from 1 to 50 microM and then decreased again to 1.2 at 1000 microM ODYA. (2) The enzyme that was not irreversibly inactivated by ODYA was in the Fe(III) form at ODYA concentrations below 10 microM but in the Fe(II) form at ODYA concentrations greater than 100 microM. (3) In the presence of 750 microM ODYA and 25 microM 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid, all of the enzyme was inactivated. On the basis of these results, it is proposed that the dioxygenation product of ODYA is 11-hydroperoxyoctadeca-9,12-diynoic acid (11-HP-ODYA), which can convert Fe(II)-LOX into its Fe(III) form. However 11-HP-ODYA is converted into 11-oxo ODYA, which cannot perform the oxidation. It is proposed that the inactivating agent is either 11-HP-ODYA or the 11-peroxy-octadeca-9,12-diynoic acid radical (11-peroxy-ODYA radical), formed from the ODYA radical and O2. The oxidation of Fe(II)-LOX into its Fe(III) form as well as the inactivation of Fe(III)-LOX is competitively inhibited by ODYA PMID- 8639490 TI - High-resolution solution structure of the EGF-like domain of heregulin-alpha. AB - The solution structure of the 63-residue heregulin-alpha (HRG-alpha) epidermal growth factor (EGF)-like domain, corresponding to residues 177-239 of HRG-alpha, has been determined to high resolution using data from two-dimensional and three dimensional homo- and heteronuclear NMR spectroscopy. The structure is based on a total of 887 internuclear distance and dihedral restraints derived from data obtained using unlabeled and uniformly 15N-labeled protein samples, at pH 4.5, 20 degrees C. A total of 20 structures were calculated using a hybrid distance geometry-simulated annealing approach with the program DGII, followed by restrained molecular dynamics using the program DISCOVER. The average maximum violations are 0.12 +/- 0.01 angstroms and 1.4 +/- 0.3 degrees for distance and dihedral restraints, respectively. The backbone (N,C(alpha),C) atomic rms distribution about the mean coordinates for residues 3-23 and 31-49 is 0.29 +/- 0/07 angstroms. The N-and C-terminal residues (1-2 and 50-63) and 24-30 are disordered. Comparison of the HRG-alpha EGF-like domain structure with the previously determined structure of human EGF [Hommel et al. (1992) J. Mol. Biol. 227, 271-282] reveals a high degree of structural similarity; excluding the N terminal region (residues 1-13), the disordered phi-loop region (residues 24-30) that contains a three-residue insertion in HRG-alpha relative to hEGF, and the disordered C-terminal region (residues 50-63), the C(alpha) alignment between the HRG-alpha and hEGF minimized mean structures has a rms difference of approximately 1 angstrom. In HRG-alpha the N-terminal residues 2-6 form a well defined beta strand rather than being disordered as found for hEGF. This structural difference correlates with functional data which suggest that the N terminal region of the HRG-alpha EGF-like domain is responsible for the observed receptor specificity differences between HRG-alpha and EGF. PMID- 8639491 TI - Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin. AB - Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5 dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31 59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation. PMID- 8639492 TI - Standard free energies for uridylyl group transfer by hexose-1-P uridylyltransferase and UDP-hexose synthase and for the hydrolysis of uridine 5' phosphoimidazolate. AB - The reversible reaction of UDP-glucose with imidazole (Im) to produce uridine 5' phoshoimidazolate (UMPIm) and glucose-1-P is catalyzed by UDP-hexose synthase, which is the mutant H166G of hexose-1-P uridylyltransferase (EC 2.7.7.12) [Kim, J., Ruzicka, F.J., & Frey, P.A. (1990) Biochemistry 29, 10590-10593]. The availability of UDP-hexose synthase allows the equilibrium constant for the reaction UDP-glucose + Im = UMPIm + glucose-1-P to be measured, and it is found to be 2.2 x 10(-2) at pH 8.5 and 27 degrees C. At pH 7.0 and 27 degrees C the equilibrium constant is 6.4 x 10(-4). The equilibrium constant for the formation of the covalent uridylyl-enzyme intermediate of hexose-1-P uridylyltransferase (E His(166) + UDP-glucose = E-His(166)-UMP + glucose-1-P) is found to be 1.8 x 10( 4) at pH 7.0 and 25 degrees C, which is slightly less favorable than the formation of UMPIm from UDP-glucose and Im. These equilibrium constants, when considered in the light of other data in the literature, allow the standard free energy changes for the hydrolysis of UMPIm and the analogous covalent uridylyl enzyme intermediate to be calculated. The results show that delta G' degrees (delta G degrees (ph)(7.0)) for the hydrolyses of UMPIm and E-His(166)-UMP are 14.7 and -15.4 kcal mol(-1), respectively at pH 7.0. At pH 8.5, the corresponding values of delta G degrees (ph) (8.5) are -12.6 and -9.9 kcal mol(-1), respectively. It is concluded that noncovalent binding interactions between the active site and the UMP group of E-His(166)-UMP provide little or no stabilization in the formation of this species as an intermediate in the reaction of hexose-1-P uridylyltransferase. PMID- 8639493 TI - Structural and electronic properties of the heme cofactors in a multi-heme synthetic cytochrome. AB - Resonance Raman, absorption, and electron paramagnetic resonance spectra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome beta maquette described by Robertson et al. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is composed of 62 amino acid residues arranged in a di-alpha-helical unit which dimerizes in solution to form a four helix bundle. Each di-alpha-helical unit contains histidine residues at the 10,10' positions which serve as ligands to the hemes. When protoheme IX is incorporated, both hemes in the dimer are bis-ligated and low spin. The two hemes are inequivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studies were conducted on peptides reconstituted with protoheme IX (PHa) and several related variants. These hemes include 2-vinyldeuteroheme (2-VDH), 4-vinyldeuteroheme (4 VDH), protoheme III (PHs), and 1-methyl-2-oxomesoheme XIII (2-OMH). Collectively, the spectroscopic studies reveal the following: (1) 2-VDH, 4-VDH, and 2-OMH bind to the protein and form bis-ligated low-spin complexes similar to PHa. The structures of the two hemes in the dimers are identical as are the immediate protein environments around the bound cofactors. These results indicate that the redox inequivalence of the two hemes is due to heme-heme electronic interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arranged in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the vinyl group(s) are in the hydrophobic interface between the two units which comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs protein differs from the other synthetic proteins in that it contains one pentacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination site on the high-spin heme is inaccessible to exogenous imidazole but readily bind cyanide, suggesting that the alpha-helix containing the unbound histidine is nearby and partially shields the coordination site. The high-spin heme converts to low-spin at low-temperature, presumably via binding of the histidine residue on this nearby alpha-helix. It is suggested that the different behavior observed for the PHs protein is due to the fact that this heme is symmetric with respect to rotation about the alpha,gamma-axis of the macrocycle which bisects the meso carbons between the vinyl groups and propionic acid residues. This symmetry precludes rotational isomerism about the alpha,gamma-axis to establish an unhindered fit. In contrast, all the other hemes examined contain at least one substituent smaller than a vinyl group which together with the fact that two different alpha,gamma-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid the like-substituent--like-substituent heme- heme interactions of PHs. The propensity to avoid such interactions could explain the inequivalent binding properties of the two hemes in the dimer. For the PHs protein wherein these these interactions cannot be mitigated by rotation of the heme, other rearrangements of the protein must occur. These rearrangements could force the second-bound heme to assume a high-spin configuration. PMID- 8639494 TI - Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibration kinetics in carbonic anhydrase II. AB - Direct metal ligands to transition metals in metalloproteins exert a profound effect on protein-metal affinity and function. Indirect ligands, i.e., second shell residues that hydrogen bond to direct metal ligands, typically exert more subtle effects on the chemical properties of the protein-metal complex. However, E117 of human carbonic anhydrase II (CAII), which is part of the E117-119-Zn(2+) triad, is a notable exception: E117-substituted CAIIs exhibit dramatically increased kinetics of zinc complexation, and the E117Q variant exhibits enormously diminished catalytic activity and sulfonamide affinity. The three dimensional structures of zinc-bound and zinc-free E117Q CAII reveal no discrete structural changes in the active site that are responsible for enhanced zinc equilibration kinetics and decreased activity. Additionally, the structure of the acetazolamide complex is essentially identical to that of the wild-type enzyme despite the 10(4)-fold loss of enzyme-inhibitor affinity. We conclude, therefore, that the functional differences between E117Q and wild-type CAIIs arise from electrostatic and not structural differences in the active site. We propose that the E117Q substitution reverses the polarity of the residue 117-H119 hydrogen bond, thereby stabilizing H119 as a histidinate anion in the E117Q CAII holoenzyme. The additional negative charge in the first coordination sphere of the metal ion increases the pK(a) of the zinc-water ligand, destabilizes the transition state for CO(2) hydration, and facilitates the exchange of a zinc histidine ligand with an additional water molecule by decreasing the stability of the tetrahedral zinc complex. These novel properties engineered into E117Q CAII facilitate the exploitation of CAII as a rapid and sensitive Zn(2+) biosensor. PMID- 8639495 TI - Biosynthesis of methanopterin. AB - This paper establishes the pathway for the biosynthesis of methanopterin in Methanosarcina thermophila to proceed by the following series of reactions. First, 5-phospho-alpha-D-ribosyl diphosphate (PRPP) and 4-aminobenzoic acid condense together to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate, which then reacts with 6-hydroxymethyl-7,8-H2pterin pyrophosphate to produce 7,8 H2pterin-6-ylmethyl-4-(beta-D-ribofuranosyl)aminobenz ene 5'-phosphate (3'). Compound 3' is then reduced to 7,8-H2pterin-6-ylmethyl-l-(4-aminophenyl)-1-deoxy D-ribitol 5'-phosphate (4') in a reaction stimulated by the addition of FMN or factor F(420). Dephosphorylation of compound 4' leads to 7,8-H2pterin-6-ylmethyl 1-(4-aminophenyl)-1-deoxy-D-ribitol (5'). Compound 5' then condenses with another molecule of PRPP to form 7,8-H2pterin-6-ylmethyl-1-(4-aminophenyl)-1-deoxy-5-[1 alpha -D- ribofuranosyl 5-phosphate]-D-ribitol (9'). Compound 9', in the presence of ATP, then condenses with (S)-2-hydroxyglutaric acid to form demethylated H2methanopterin, a known precursor to methanopterin. The occurrence of this pathway was confirmed by (a) the chemical and/or biochemical synthesis of most of the proposed intermediates, (b) the detection of these intermediates in cell-free extracts, and (c) by the measurement of their conversion to demethylated methanopterin and/or other intermediates in the pathway. PMID- 8639496 TI - Inhibition of human steroid 5alpha reductases type I and II by 6-aza-steroids: structural determinants of one-step vs two-step mechanism. AB - We have discovered that 17beta-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5alpha-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5 alpha-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 degrees C, an initial finasteride-reductase complex is formed with a K(i)(app) of 11.9 +/- 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 +/- 0.01 s(-1). In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a K(i) of 6.8 +/- 0.2 nM. The reversible formation of a final complex, with an overall K(i) of 0.07 +/- 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 +/- 0.0001 s(-1) for the forward step and 0.00025 +/- 0.00006 s(-1) for the backward step. All rate constants for the two-step mechanism were obtained by using a general numerical integration method. The best fit values for the association and dissociation rate constants were 5.0 microM(-1) s(-1) and 0.033 +/- 0.008 s(-1), respectively, and the isomerization rate constants were 0.0035 +/- 0.007 s(-1) and 0.000076 +/- 0.000019 s(-1). These values correspond to an initial K(i) of 6.5 nM and an overall dissociation constant of 0.14 nM. The data presented here show that both finasteride and the 6-aza-steroid analogs are potent against type II 5alpha reductase, although their mechanisms of inhibition are different. PMID- 8639497 TI - Folding and functional complementation of engineered fragments from yeast phosphoglycerate kinase. AB - A set of protein fragments was produced by site-directed mutagenesis followed by chemical cleavage of phosphoglycerate kinase according to a previously described method [Pecorari et al. (1993) Protein Eng. 6, 313-325]. The cleavage positions were chosen in order to correspond to limits between structural subdomains. These isolated fragments were studied by circular dichroism, folding transitions, and cross-linking analyses. It appears that fragments corresponding to globular subdomains in the protein can recover the expected helix content. However, the cooperativity classically observed in the folding transitions of natural proteins is only observed for fragments larger than a domain. Previous studies have shown that the isolated C-terminal domain is an autonomous folding unit which displays a single cooperatine transition [Missiakas et al. (1990) Biochemistry 29, 8683 8689]. The results presented here show that the presence in a fragment of a sequence overpassing that of the C-terminal domain modifies its folding process. Reassociation experiments suggest that the efficiency of the complementation process is not related to the folding autonomy of the isolated fragments. PMID- 8639498 TI - Presence of a slow dimerization equilibrium on the thermal unfolding of the 205 316 thermolysin fragment at neutral pH. AB - Differential scanning calorimetry and size-exclusion chromatography have been used to characterize the dimerization and unfolding of the 205-316 C-terminal fragment of thermolysin at pH 7.5. We show that the folded fragment dimerizes at low temperature with a moderate affinity and undergoes thermal unfolding according to a N(2) <==> 2N <==> 2U model. This behavior has already been observed at acid pH, where a similar dissociation equilibrium has been found [Azuaga, A., Conejero-Lara, F., Rivas G., De Filippis, V., Fontana A., & Mateo, P. L. (1995) Biochim. Biophys. Acta 1252, 95-102]. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30 degrees C, with virtually no interconversion between the monomeric and the dimeric states of the fragment. We have studied the kinetics of interconversion between monomer and dimer by size exclusion chromatography experiments and have shown that a very high energy barrier (83.8 kJ/mol at 26.5 degrees C) exists between either state. A mathematical analysis of the DSC thermograms on the basis of the proposed model has allowed us to obtain the thermodynamic characterization of the dimerization and the unfolding processes of the fragment and confirms the kinetic parameters obtained in the chromatographic experiments. The thermodynamic functions for the unfolding of the fragment are compatible with some degree of disorder in the structures of both the monomer and the dimer. According to circular dichroism measurements, the dimerization of the fragment seems to be linked to some conformational change in the subunits, most probably due to a rearrangement of the existing secondary-structure elements. This fragment displays several features already observed in folding intermediates, such as the partial disorder of the polypeptidic chain, association processes, and kinetic barriers between different regions in the conformational space. PMID- 8639499 TI - Probing heme protein conformational equilibration rates with kinetic selection. AB - Double-pulse flash photolysis experiments on solutions of carbonmonoxymyoglobin (MbCO) are used to determine the time scale for protein conformational averaging. The interconversion times for transitions between the "open" and "closed" subpopulations of MbCO are found to be 10(-6)-10(-4)s, depending on solvent composition and temperature. In aqueous solution at 273 K, the interconversion rate is found to be 1.4 x 10(6)s. Since the interconversion rate is comparable to or slower than the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponential for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly than the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long-time tail of the relaxation of the protein (Tian et al., 1992). Bimolecular rebinding takes place at a statistically averaged rate, since the interconversion and relaxation rates are faster than the bimolecular kinetics. The geminate and bimolecular kinetics are analyzed quantitatively as a function of pH using this approach and the spectroscopically determined populations of the open and closed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be I(0)g = 32% and k(0)g = 1.3 x 10(7)s(-1) for the open state and I(1)g = 9.3% and k(1)g = 1.4 x 10(6)s(-1) for the closed state. In 75% glycerol solution at 264 K, the dominant component of the geminate rebinding is characterized by I(0)g1 = 89% and k(0)g1 = 3.1 x 10(6)s(-1) for the open state and I(1)g1 = 26% and k(1)g1 = 3.1 x 10(6)s(-1) for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solution is consistent with the idea that the open state provides an important pathway for ligand escape from (or entry to) the heme pocket (Tian et al., 1993). The increased viscosity of 75% glycerol solution delays the closed--> open interconversion until the end of the geminate phase, which forces the ligand to find alternative pathways to the solution. This observation, in conjunction with the near equivalence of the geminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protein-ligand dynamics. PMID- 8639500 TI - Importance of highly conserved anionic residues and electrostatic interactions in the activity and structure of the cardiotonic polypeptide anthopleurin B. AB - Several polypeptide toxins from sea anemones caused delayed inactivation of mammalian voltage-dependent sodium channels, resulting in a positive inotropic effect on the heart. Anthopleurin B (ApB), a toxin produced by the sea anemone Anthopleura xanthogrammica, is the most potent of all known anemone toxins. Previous studies in this laboratory have both defined and revealed an important role for the cationic cluster of Arg-12, Arg-14, and Lys-49 in the expression of ApB's biological activity. In the present investigation, we explore the role of all remaining charged residues by producing and characterizing mutants of ApB at Asp-7, Asp-9, Lys-37, His-39, and His-34. Recombinant toxins have been purified to homogeneity and their abilities to enhance veratridine-dependent sodium uptake in cell lines expressing either the neuronal or cardiac isoform of the sodium channel evaluated. Replacement of Asp-7 results in a product that fails to fold, while muteins H39A and H34A have activities very similar or identical to wild type ApB. In contrast, the D9N and K37A muteins are 7-12-fold less active that wild-type ApB, and truncation of the side chain in D9A results in a further decrease in activity, especially in the cardiac model. We conclude that although a negative charge per se is not essential at position 9, the presence of a hydrogen-bond forming side chain is critical both for appropriate folding and for interaction with the sodium channel. Because the K37A and H39A mutant toxins can fold normally, neither Lys-37 nor His-39 seem to participate in an intramolecular salt bridge, in contrast to suggestions arising from NMR studies of ApA and ApB. However, Lys-37 may play a role in channel interaction. PMID- 8639501 TI - Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule. AB - We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250 fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin. PMID- 8639504 TI - Sequence dependence and direct measurement of crossover isomer distribution in model Holliday junctions using NMR spectroscopy. AB - A 32-base-pair model of the Holliday junction (HJ) intermediate in genetic recombination has been prepared and analyzed in-depth by 2D and 3D (1)H NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permutation of the base pairs at the junction relative to a previously studied HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11459], designed to probe the effect of the sequence at the n - 1 position (where n is the residue directly at the branch point) on the stacking geometry. Observation of several interbase nuclear Overhauser effects (NOEs) clearly indicates a strong preference for the isomer opposite that observed for J2, confirming the dependence of stacking isomer preference on the sequence at the junction. As for other model HJs studied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single (15)N-labeled thymine residue at the branch point. 1D (15)n-filtered (1)H-detected experiments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more direct and accurate measure of the crossover isomer distribution. The comparative analysis of our immobile HJs and a model cruciform structure [Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the relevance of crossover isomer preference in vivo. PMID- 8639502 TI - Purification and properties of a Ca(2+)-independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes. AB - In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL( 1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis. PMID- 8639503 TI - Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases. AB - Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA. Single stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease. Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase. Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding. Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures. Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin. Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension. These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex. Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates. PMID- 8639505 TI - Origins of binding specificity of the A1 heterogeneous nuclear ribonucleoprotein. AB - The A1 heterogeneous nuclear ribonucleoprotein (hnRNP) is the best studied of the "core" hnRNP proteins that are tightly associated with heterogeneous nuclear RNA (hnRNA) within eukaryotic nuclei. Previous studies suggested that hnRNP A1 preferentially binds (under nonequilibrium conditions) to the pyrimidine-rich span of sequence at the 3'splice site of most introns [Swanson, M.S., & Dreyfuss, G. (1988) EMBO J. 11, 3519-3529; Buvoli et al. (1990) Nucleic Acids Res. 18, 6595 6600; Ishikawa et al. (1993) Mol. Cell. Biol. 13, 4301-4310]. Recently, Burd and Dreyfuss [(1994) EMBO J. 13, 1197-1204] used selection/amplification from pools of random sequence RNA to uncover an even higher-affinity A1 oligo that contained two copies of a high-affinity consensus sequence, UAGGGU/A. We have extended these studies by using a fluorescence assay to characterize the equilibrium binding properties of A1 to each of these oligonucleotides. By also characterizing the binding of A1 to sequence-randomized control oligonucleotides, we have been able to better evaluate the inherent "sequence-specific" binding properties of A1. Although these studies indicate that under equilibrium conditions A1 cannot specifically recognize the beta-globin, 3'-splice site DNA oligo analogue studied by Buvoli et al. (1990), they confirmed the high-affinity binding to the "winner" 20-mer RNA that was uncovered via selection/amplification and that has the sequence UAUGAUAGGGACUUAGGGUG (Burd & Dreyfuss, 1994). In 0.1 M NaCl, we found that A1 has approximately 100-fold higher affinity for this winner sequence sequence than it does for either a randomized version of this sequence or a 20-mer oligo corresponding to an unrelated beta-globin intron sequence. This winner RNA oligo aggregates in solution to form an apparent dimer that may represent a G-quartet resulting from dimerization of two Hoogsteen base-paired hairpins. On the basis of salt sensitivity studies carried out with various fragments of A1, the ability of A1 to discriminate the winner sequence from its randomized control results primarily from increased ionic interactions with the glycine-rich, COOH terminal domain of A1 that extends from residue 196 to 319. Nonetheless, most of the overall energy of binding for the A1 winner complex results from determinants that are resident within the first 195 residues of A1. The unique ability of the winner sequence (but not its sequence-randomized control) to form a higher-order aggregate, which may correspond to a G-tetrad, appears to facilitate the additional ionic interactions with the COOH terminal domain. Taken together, these data suggest the need to reevaluate possible and probable functions of A1 in vivo. PMID- 8639506 TI - Improved nearest-neighbor parameters for predicting DNA duplex stability. AB - Thermodynamic data were determined from UV absorbance vs temperature profiles of 23 oligonucleotides. These data were combined with data from the literature for 21 sequences to derive improved parameters for the 10 Watson-Crick nearest neighbors. The observed trend in nearest-neighbor stabilities at 37 degrees C is GC > CG > GG > GA approximately GT approximately CA > CT > AA > AT > TA (where only the top strand is shown for each nearest neighbor). This trend suggests that both sequence and base composition are important determinants of DNA duplex stability. On average, the improved parameters predict deltaG degrees(37), deltaH degrees, deltaS degrees, and T(m) within 4%, 7%, 8%, and 2 degrees C, respectively. The parameters are optimized for the prediction of oligonucleotides dissolved in 1 M NaC1. PMID- 8639507 TI - Differential proximity probing of two DNA binding sites in the Escherichia coli recA protein using photo-cross-linking methods. AB - The DNA strand-exchange reaction catalyzed by the Escherichia coli RecA protein occurs between the two DNA binding sites that are functionally distinct. Site I is the site to which a DNA molecule (normally single-stranded DNA) binds first; this first binding makes site II available for additional DNA-binding (normally double- stranded DNA). Photo-cross linking was employed to identify the amino acid residues located close to the bound DNA molecule(s). A ssDNA oligo containing multiple 5-iodouracil residues (IdU) was cross-linked to RecA by irradiation with a XeC1 pulse laser (308 nm), and the cross-linked peptides were purified and sequenced. To differentiate the two DNA binding sites, we used two protocols for making RecA-ssDNA complexes: (1) IdU-containing oligo was mixed with a stoichiometric excess of RecA, a condition which favors the binding of the oligo to site I, and (2) RecA was first allowed to bind to a nonphotoreactive oligo and then chased with the IdU-containing oligo, a condition which favors the binding of the IdU-oligo to site II. We observed that when RecA was in excess (site I probing), cross-linking occurred to Met-164 which is located in the disordered loop 1 of the RecA crystal structure [Story, R.M., Weber, I.T., & Steitz, T.A. (1992) Nature 355, 318-325]. When site II was probed, the majority of cross-linking occurred to Met-202 or Phe-203, located in loop 2. These results support the idea that, as predicted by Story and co-workers (1992), the disordered loops are involved in DNA binding. The results also suggest that the two sites are not only functionally but also physically distinct. PMID- 8639508 TI - Human DNA polymerase epsilon: enzymologic mechanism and gap-filling synthesis. AB - DNA polymerase epsilon (pol epsilon) was purified to apparent homogeneity from human placentas. The purified enzyme contains a single polypeptide of approximately 170 kDa (apparent mass) and has both DNA polymerase and 3'-5' exonuclease activities. Competitive inhibition studies indicate that like DNA polymerases alpha and delta (pol alpha and pol delta, respectively), free pol epsilon binds single-stranded but not double-stranded DNA. This conclusion was confirmed by sedimentation binding analysis. Also like pol alpha and pol beta, pol epsilon exhibits induced dNTP inhibition in the presence of template annealed to complementary primer containing a 2',3'-H (dideoxy)-terminus. Together, these data suggest that pol epsilon follows an ordered sequential ter-reactant mechanism of substrate recognition and binding; it binds template first followed by annealed primer and then template-specified dNTP. Enzymologic studies suggest that in contrast to both pol alpha and pol delta, pol epsilon functions more efficiently as gap size decreases. This observation is consistent with a specific role for pol epsilon in gap-filling in vivo. Gap-filling is essential for both replication and repair. PMID- 8639509 TI - Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin. AB - alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alphaB-crystallin are modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alphaB crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein. PMID- 8639510 TI - Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases. AB - Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 4-kinases is the first step in the synthesis of polyphosphoinositides, the lipid precursors of intracellular signaling molecules. We have recently identified a cytosolic PtdIns 4-kinase (cPI4K) in the bovine adrenal cortex that is distinguished from previously known PtdIns 4-kinases by its sensitivity to the PtdIns 3-kinase inhibitor wortmannin (WT). The present study has further characterized this soluble enzyme and compared its properties to those of the membrane-bound, type II PtdIns 4-kinase activity of the adrenal cortex and the type III enzyme of bovine brain. The enzymatic activity of adrenal cPI4K was inhibited not only by WT (IC50 approximately 50 nM) but also by LY-294002 (IC50 approximately 100 microM), another inhibitor of PtdIns 3-kinase, and neither compound affected type II PtdIns 4-kinase at concentrations that inhibited cPI4K. In contrast to the type II enzyme, cPI4K had a significantly higher Km for ATP, was relatively insensitive to inhibition by adenosine (Ki approximately 800 microM vs approximately 40 microM), had lower affinity for PtdIns, and was not inhibited by Ca2+ ions. These properties identify the WT-sensitive adrenal cPI4K as a type III PtdIns 4-kinase that is distinct from the tightly membrane-bound, Ca2+- and adenosine-sensitive, type II PtdIns 4-kinase. The type III PtdIns 4-kinase prepared from bovine brain exhibited similar kinetic parameters as the adrenal cPI4K, and was also inhibited by WT with an IC50 of 30-50 nM. Since WT inhibits the synthesis of agonist-regulated phosphoinositide pools in intact cells at micromolar concentrations, these findings indicated that type III rather than type II PtdIns 4-kinases are responsible for the maintenance of the precursor phospholipids required for intracellular signaling through the inositol phosphate/Ca2+ pathway. PMID- 8639511 TI - Kinetics and mechanism of the hydrolysis of depsipeptides catalyzed by the beta lactamase of Enterobacter cloacae P99. AB - The steady-state kinetics and mechanism of the hydrolysis and aminolysis of a series of acyclic depsipeptides, catalyzed by the class C beta-lactamase of Enterobacter cloacae P99, have been studied in order to more firmly establish the nature of the transition states involved. The class C beta-lactamase of Enterobacter cloacae P99 was employed. The depsipeptide substrates contained a constant acyl group, (phenylacetyl)glycyl, and chemically different leaving groups, m-carboxyphenoxide, m-carboxythiophenoxide, 3-carboxyl-4-nitrophenoxide, lactate, and thiolactate. Evaluation of the steady-state kinetic parameters and the effect of the alternative nucleophile methanol on these parameters and on the product distribution showed that deacylation was largely rate-determining to turnover of the aryl esters under conditions of substrate saturation, while acylation was rate-determining to the alkyl esters. The earlier conclusion [Govardhan & Pratt (1987) Biochemistry 26, 3385-3395] that acylation largely limited the turnover of the aryl esters was shown to be an artifact of phosphate buffer inhibition. The aminolysis of both the aryl the alkyl esters by D phenylalanine was influenced by binding of the substrate at a second binding site on the acyl-enzyme intermediate. A study of inhibiton of the hydrolysis of (phenylacetyl)-glycyl-D-thiolactate by the aminolysis product (phenylacetyl)glycyl-D-phenylalanine indicated that the second binding site is also available for ligands to bind the free enzyme and to the noncovalent Michaelis complex with this substrate. It is likely that penicillin-recognizing enzymes in general, both beta-lactamases and DD-peptidases, possess an extended substrate-binding site into which a variety of small ligands may bind at any point along the reaction coordinate and, to a greater or lesser extent depending on circumstances, affect catalysis. PMID- 8639512 TI - Beta-secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis. AB - Beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]-oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism. The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1,002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites. PMID- 8639513 TI - Cryptic initiation at the human D4 receptor reveals a functional role for the amino terminus. AB - It was found that deletion of the initiator methionine of the D4 receptor results in the use of a cryptic initiation site in the putative first transmembrane region. We made use of this observation to investigate the role of the amino terminus of the D4 receptor. In vitro transcription and translation of D4.4 and a D4.4 deleted for the initiation codon (D4.4 delta NH2) resulted in the formation of protein products with a molecular mass of about 44 and 40.5 kDa, respectively. The molecular mass of 40.5 kDa suggests initiation in the putative first transmembrane region. Transient expression of various deletion mutants indicated that this receptor form can be expressed at up to 70% of the D4.4 control levels and provided support for the existence for an alternative translation initiation site in the first transmembrane domain, most likely at nucleotide +112 (the initiator methionine codon is designated as +1). The D4.4 delta NH2 mutant was stably expressed in CHO cells. Pharmacological analysis demonstrated no major differences in antagonist binding with the regular D4.4 receptor, while dopamine and quinpirole binding affinities were about 5-fold decreased. The half-maximal level (EC50) for blocking forskolin-stimulated cAMP levels by dopamine was about 10-fold lower as compared to D4.4. Furthermore, the functional efficacy is decreased by about 40%. These data suggest that the amino-terminal domain is not essential for proper expression, but does interfere with the functional activity of the receptor, possibly through stabilization of the active state. To our knowledge this is the first demonstration that the amino terminus of a dopamine receptor is involved in signal transduction. PMID- 8639514 TI - Specificity of C-glycoside complexation by mannose/glucose specific lectins. AB - The binding of the mannose/glucose specific lectins from Canavalia ensiformis (concanavalin A) and Dioclea grandiflora to a series of C-glucosides were studied by titration microcalorimetry and fluorescence anisotropy titration. These closely related lectins share a specificity for the trimannoside methyl 3,6-di-O (alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, and are a useful model system for addressing the feasibility of differentiating between lectins with overlapping carbohydrate specificities. The ligands were designed to address two issues: (1) how the recognition properties of non-hydrolyzable C-glycoside analogues compare with those of the corresponding O-glycosides and (2) the effect of presentation of more than one saccharide recognition epitope on both affinity and specificity. Both lectins bind the C-glycosides with affinities comparable to those of the O-glycoside analogues; however, the ability of both lectins to differentiate between gluco and manno diastereomers was diminished in the C glycoside series. Bivalent norbornyl C-glycoside esters were bound by the lectin from Canavalia but only weakly by the lectin from Dioclea. In addition to binding the bivalent ligands, concanavalin A discriminated between C-2 epimers, with the manno configuration binding more tightly than the gluco. The stoichiometry of binding of the bivalent ligands to both di- and tetrameric lectin was two binding sites per ligand, rather than the expected 1:1 stoichiometry. Together, these results suggest that concanavalin A may possess more than one class of carbohydrate binding sites and that these additional sites show stereochemical discrimination similar to that of the previously identified monosaccharide binding site. The implications of these findings for possible in vivo roles of plant lectins and for the use of concanavalin A as a research tool are discussed. PMID- 8639515 TI - Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning. AB - The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins. PMID- 8639516 TI - Peptidyl-prolyl cis-trans isomerase of Bacillus subtilis: identification of residues involved in cyclosporin A affinity and catalytic efficiency. AB - The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%). Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP). Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B. subtilis and 3000 nM for E. coli. In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM( 1) s(-1) for the B. subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM( 1) s(-1) for E. coli, were detected using the common substrate suc-Ala-Ala-Pro Phe-pNA. Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values. Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E. coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM). In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E. coli protein. In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB. Replacement of V52 to M52, which is conserved in E. coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but it raises the catalytic efficiency by 12-fold to k(cat)/K(m) of 14 mM(-1) s( 1). In conclusion, our studies suggest that the unique histidine residues H90 and H109 in B. subtilis PPIase are, at least in part, responsible for its intermediate CsA affinity and that the v52 residue confers the low conversion rate. PMID- 8639517 TI - Conformation of the acylation site of palmitoylgramicidin in lipid bilayers of dimyristoylphosphatidylcholine. AB - Gramicidin A(gA) can be palmitoylated by means of an ester linkage to the OH group of the terminal ethanolamine that sits at the membrane-water interface in the functional gA channel. We have investigated palmitoyl-gA as a model transmembrane acylprotein. Ethanolamine-d(4) (NH(2)CD(2)CD(2)OH) was incorporated into gA by total synthesis, and a portion of the labeled gA was palmitoylated. Solid-state (2)H-NMR spectra of acyl- and nonacyl-gA in hydrated dimyristoylphosphatidylcholine (DMPC) bilayers were compared. The spectra for both oriented and nonoriented samples at 4 and at 40 degrees C indicate that the ethanolamine of gA is highly mobile prior to acylation, but essentially immobile after palmitoylation. The (2)H quadrupolar splittings allow the conformation of the ethanolamine group in acyl-gA to be determined. By combining our data with the previously determined quadrupolar splittings for deuterium labels on the palmitoyl chain [Vogt, T.C.B., Killian, J.A., & de Kruijff, B. (1994) Biochemistry 33, 2063-2070], we also propose a model for the acyl chain. The ethanolamine group rotates over Leu(10) and toward the outside of the gA channel's cylinder upon acylation, so that the attached acyl chain passes between the side chains of Trp(9) and Leu(10). To accommodate the acyl chain, the six membered portion of the indole ring of Trp(9) is displaced by about 0.9 angstroms, by means of 1-2 degree rotations in chi(1) and chi(2). PMID- 8639518 TI - Internal electron-transfer reactions in cytochrome c oxidase. PMID- 8639519 TI - Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. AB - To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1) 1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex cell interactions. PMID- 8639520 TI - Kinetics of halide release of haloalkane dehalogenase: evidence for a slow conformational change. AB - Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides. The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity, but the kcat values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s 1, respectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall kcat. Furthermore, at 5mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s-1 (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5mM 1,2-dibromoethane occurred at a rate of only 8 s-1. The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of kobs on the halide concentration suggested that there were two parallel routes for halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations. The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s-1 for bromide and chloride, respectively). We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion. A solvent kinetic isotope effect of 2H2O was found both on kcat and on the rate of halide release. 2H2O mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of 2H2O on the conformation of proteins. PMID- 8639521 TI - Structure and dynamics of a CheY-binding domain of the chemotaxis kinase CheA determined by nuclear magnetic resonance spectroscopy. AB - The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals. Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which binds to the downstream targets of phosphorylation, the response regulators CheY and CheB. This protein fragment contains the CheY-binding domain flanked on each side by regions that correspond to domain linkers in the intact protein. The structure of the domain was determined from 1429 restraints derived from heteronuclear multidimensional NMR experiments. Hybrid distance geometry--dynamical simulated annealing methods were used to calculate a family of structures that satisfy the experimental distance restraints and torsion angle restraints. The root mean square deviation of the 69 ordered residues in the domain is 0.52 A for the backbone heavy atoms and 0.99 A for all heavy atoms. The residues that have been implicated as important for CheY binding form a face consisting of several partially buried hydrophobic residues, framed by charged residues. The dynamic properties of this protein fragment were measured and analyzed using both isotropic and anisotropic models of molecular motion. The linker regions are very flexible and disordered, as evidenced by the very dynamics properties as compared to the CheY-binding domain. The CheY-binding domain of CheA is structurally similar to the histidine-containing phosphocarrier, HPr, which is a protein involved in the phosphoenolpyruvate:sugar phosphotransferase (PTS) pathway. This structural similarity suggests a possible evolutionary relationship of the PTS and chemotaxis pathways. PMID- 8639522 TI - Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5'-triphosphate. AB - Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis. PMID- 8639523 TI - An NMR spectroscopy and molecular mechanics study of the molecular basis for the supramolecular structure of lipopolysaccharides. AB - Lipopolysaccharides from Gram-negative bacteria interact with the mammalian immune system to trigger a cascade of physiological events leading to a shock syndrome which results in the death in over 70% of cases of severe shock. It is known that the supramolecular structures of lipopolysaccharide aggregates are critical contributors to their biological activities. Despite this, the molecular basis for the formation if the regular hexagonal plates and arrays observed in lipopolysaccharide films and suspensions is unknown. Since these structures are two dimensional, it is unlikely that X-ray crystallographic methods will shed much light on their detailed structure. Knowing this structure is important since it is becoming increasingly likely that the insertion of the lipopolysaccharide hydrocarbon chains in the target host cell membrane may be involved in triggering host responses. This work describes the three-dimensional structure of the lipopolysaccharide lipid A moiety. The structure was obtained by a combination of molecular mechanics calculations and nuclear magnetic resonance spectroscopy. This involved calculation of the dihedral angle between the two glucosamine residues of the lipid A molecule from coupling constants and measuring critical interresidue NOE values. The study also takes into account information from X-ray powder diffraction and electron microscopy studies. PMID- 8639524 TI - A crystallographic and spectroscopic study of the complex between d(CGCGAATTCGCG)2 and 2,5-bis(4-guanylphenyl)furan, an analogue of berenil. Structural origins of enhanced DNA-binding affinity. AB - 2,5-Bis(4-guanylphenyl)furan ("furamidine") is a dicationic minor groove binding drug that has been shown to be more effective than pentamidine against the Pneumocystis carinii pathogen in an immunosuppressed rat model. It has a close structural similarity to the antitrypanosomal drug berenil, differing only on the replacement of the central triazene unit with a furan moiety. we have determined the crystal structure of the complex between furamidine and the DNA dodecamer d(CGCGAATTCGCG)2 and compared it with the same DNA sequence by UV-visible, fluorescence, and CD spectroscopy. Furamidine shows tighter binding to this sequence (Keq = 6.7 x 10(6)) than berenil (Keq = 6.6 x 10(5)). The crystal structure reveals that, unlike berenil, furamidine makes direct hydrogen bond interactions with this DNA sequence through both amidinium groups to O2 atoms of thymine bases and is more isohelical with the minor groove. Molecular mechanics calculations support the hypothesis that these differences result in the improved interaction energy between the ligand and the DNA. PMID- 8639526 TI - Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant. AB - The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+ dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity. PMID- 8639525 TI - Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N mutant. AB - On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J.A., Gerlt, J.A., Kozarich, J.W., Koo, C.W., Shah, V.J., Kenyon, G.L., Neidhart, D.J., Fujita, J., & Petsko, G.A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R) and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., Kenyon, G.L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.R., Petsko, G.A., & Kenyon, G.L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S)-atrolactate, with the exception that the side chain of His 297 is tilted and displaced approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S) mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R) mandelate with solvent and the stereospecific elimination of bromide ion from (S) p-(bromomethyl)mandalate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pKa of His 297 in wild-type MR. PMID- 8639527 TI - Conserved C-terminus of the phosphatase CheZ is a binding domain for the chemotactic response regulator CheY. AB - CheZ is the phosphatase of the chemotactic response regulator, CheY. There are three conserved domains on CheZ. Here we determined the function of the C terminal domain (residues 202-214). A truncated form of CheZ, missing residues 202-214, hardly bound to the phosphorylated form of CheY. Conversely, a peptide composed of the last 19 amino acid residues of the CheZ (residues 196-214), generated by tryptic digestion, bound specifically to the phosphorylated form of CheY. This was demonstrated by both fluorescence depolarization of the peptide (labeled with fluorescein) and cross-linking. It is concluded that the conserved C-terminus of CheZ functions as a CheY-binding domain. PMID- 8639528 TI - Specificity for the exchange of phospholipids through polymyxin B mediated intermembrane molecular contacts. AB - Structural specificity for the direct vesicle-vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O.G., & Jain, M.K. (1996) Biochemistry 35, 299-308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange of covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that the stable PxB-mediated vesicle-vesicle contacts may be structurally and functionally distinct from "stalks", the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundation in interfacial equilibria and catalysis. PMID- 8639530 TI - Activation of epidermal growth factor receptor by epidermal growth factor. AB - The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) induces dimerization of the receptor and activation of its protein tyrosine kinase. Each of these three steps was followed as a function of the concentrations of EGF and of EGF receptor. Binding of EGF was followed by sedimentation of the complex between [3H]EGF and EGF receptor, dimerization was measured by quantitative cross-linking with glutaraldehyde, and the activation of the protein tyrosine kinase was monitored under the same conditions by following the initial velocity of the phosphorylation of peptides containing tyrosine. The binding of epidermal growth factor to its receptor was measured as a function of the concentration of epidermal growth factor, and the relationship was sigmoid with an average value of 1.7 for the Hill coefficient. Both dimerization and the activation of the tyrosine kinase displayed saturation as a function of the concentration of EGF. The ranges of the concentrations of EGF where dimerization and activation of the tyrosine kinase activity were half-maximal were 15-30 and 50-200 nM, respectively, but the value for the concentration of EGF at the half maximum for the activation of the tyrosine kinase was a complex function of the concentration of EGF receptor. The observed behavior of the binding of EGF, the dimerization of EGF receptor, and the activation of the tyrosine kinase were used as criteria against which to test mechanisms for the process of activation. Equations were derived for various reversible and irreversible mechanisms and used to calculate the theoretical behaviors of the three properties. In direct comparisons of the experimental and the theoretical data, several of the previously proposed reversible and irreversible mechanisms for the activation of EGF receptor were found to be inadequate, but a reasonable mechanism was formulated that was compatible with the experimental data. In this mechanism, dimeric EGF receptor must be occupied by two molecules of EGF for enzymatic activity to be expressed. PMID- 8639529 TI - Cholesterol-induced interfacial area condensations of galactosylceramides and sphingomyelins with identical acyl chains. AB - The interfacial interactions occurring between cholesterol and either galactosylceramides (GalCers) or sphingomyelins (SMs) with identical acyl chains have been investigated using Langmuir film balance techniques. Included among the synthesized GalCers and SMs were species containing palmitoyl (16:0), stearoyl (18:0), oleoyl [18:1 delta 9(c)], nervonoyl [24:1 delta 15(c)], or linoleoyl [18:2 delta 9,12(c)] acyl residues. The cholesterol-induced condensations in the average molecular areas of the monolayers were determined by classic mean molecular area vs composition plots as well as by expressing the changes in terms of sphingolipid cross-sectional area reduction over the surface pressure range from 1 to 40 mN/m (at 1 mN/m intervals). The results show that, at surface pressures approximating bilayer conditions (30 mN/m), acyl heterogeneity in naturally occurring SMs (bovine of egg SM) enhanced the area condensation induced by cholesterol compared with their predominant molecular species (e.g. 18:0 SM in bovine SM; 16:0 SM in egg SM). Nonetheless, cholesterol always had a greater condensing effect on SM compared to GalCer when these sphingolipids were acyl chain matched and in similar phase states (prior to mixing with cholesterol). Also, the cholesterol-induced area changes for a given sphingolipid type (e.g. SM or GalCer) were similar whether the acyl chains were saturated, cis-delta 9 monounsaturated, or cis-delta 9,12-diunsaturated if the sphingolipids were in similar phase states (prior to mixing with cholesterol) and compared at equivalent surface pressures. These results indicate that, under conditions were hydrocarbon structure is matched, the sphingolipid head group plays a dominant role in determining the extent to which cholesterol reduces sphingolipid cross sectional area. Despite the larger cholesterol-induced area condensations observed in SMs compared to those in GalCers, the molecular-packing densities showed that equimolar GalCer-cholesterol films were generally packed as tight as or slightly tighter than those of the SM-cholesterol films. The results are discussed in terms of a molecular model for sphingolipid-cholesterol interactions. Our findings also do only raise questions as to whether cholesterol induced condensation data provide a reliable measure of the affinity, i.e. interaction strength, between cholesterol and different lipids but also provide insight regarding the stability of sterol/sphingolipid 1-1 rich microdomains thought to exist in caveolae and other cell membrane regions. PMID- 8639531 TI - Cellular and in vitro transport of glutathione conjugates by MRP. AB - MRP is a recently identified ATP-binding cassette transporter. We previously established that MRP confers resistance to a spectrum of natural product cytotoxic drugs [Kruh, G.D., (1994) Cancer Res. 54, 1649-1652], that expression of MRP is associated with enhanced drug efflux [Breuninger, L.M., (1995) Cancer Res. 55, 5342-5347], and that MRP transcript is widely expressed in human tissues and solid tumor cell lines [Kruh, G.D., (1995) J. Natl. Cancer Inst. 87, 1256 1258]. In the present study the relationship between MRP and drug glutathione S conjugates was examined. We observed that MRP was labeled by azidophenacylglutathione (APA-SG), a photoaffinity analog of glutathione, and that inside-out membrane vesicles prepared from MRP-overexpressing HL60/ADR cells transported this compound. Transport into membrane vesicles was ATP-dependent, sensitive to osmolarity, and saturable with regard to APA-SG and ATP concentrations, with Km values of 15 and 61 microM, respectively. APA-SG transport was competitively inhibited by the natural product cytotoxic drugs daunorubicin, vincristine, and etoposide, with Ki values of 4.8, 3.8, and 5.5 microM, respectively. Oxidized glutathione, the drug-glutathione S-conjugate DNP SG, the LTD4 antagonist MK571 and arsenate were also competitive inhibitors, with Ki values of 9.0, 23.4, 1.1, and 15.0 microM, respectively. Analysis of the fate of monochlorobimane in MRP transfectants revealed reduced intracellular concentrations of drug-glutathione S-conjugates associated with enhanced efflux and altered intracellular distribution. These results indicate that MRP can transport glutathione conjugates in vitro and in living cells and suggest the possibility that the transporter may represent a link between cellular resistance to some classes of cytotoxic drugs and glutathione-mediated mechanisms of resistance. In addition, the observation that both mildly cationic or neutral natural product cytotoxic drugs and anionic compounds such as DNP-SG, MK571, and arsenate are competitive inhibitors of MRP action suggests that the substrate specificity of the transporter is quite broad. PMID- 8639532 TI - ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states. AB - The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements. PMID- 8639533 TI - Ligand-induced formation of triple helices with antiparallel third strands containing G and T. AB - We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I. PMID- 8639535 TI - Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8). AB - The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered. PMID- 8639534 TI - Carboranyl oligonucleotides. 3. Biochemical properties of oligonucleotides containing 5-(o-carboranyl-1-yl)-2'-deoxyuridine. AB - Boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology. The biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl) 2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied. 5-(o-Carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5' phosphodiesterases compared to the corresponding unmodified oligomer. They were substrates for T4 polynucleotide kinase and primers for Escherichia coli polymerase I and human immunodeficiency virus type 1 reverse transcriptase but not for human DNA polymerase alpha and beta. They also formed heteroduplexes that were substrates for E. coli RNase H, an essential property for antisense technology. These studies indicate that the carboranyl-containing oligonucleotides have desirable properties that need to be exploited further in the design of novel biopharmaceuticals. PMID- 8639536 TI - Cationic lipid binding to DNA: characterization of complex formation. AB - We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies. PMID- 8639537 TI - DNA replication machinery: functional characterization of a complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C suggests an asymmetric DNA polymerase dimer. AB - By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template. Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells. PMID- 8639538 TI - Modulation of DNA topoisomerase I activity by p53. AB - The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by most anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increase in catalytic activity occurs in parallel with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 h of drug treatment. Furthermore, topoisomerase I co immunoprecipitates with nuclear p53, suggesting that the activation of topoisomerase I may be a result of a direct interaction between the two proteins. In vitro experiments with purified recombinant proteins show that p53 increases the catalytic activities of topoisomerase I as measured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and phosphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoisomerase I sediments at a higher molecular weight in the presence of p53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, protecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in vivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I. PMID- 8639539 TI - In vivo topoisomerase II cleavage sites in the ribosomal DNA of Physarum polycephalum. AB - We have analyzed the topoisomerase II cleavage sites in the extrachromosomal ribosomal DNA of the lower eukaryote Physarum polycephalum using the topoisomerase II-specific inhibitor, 6,8-difluoro-7-(4-hydroxyphenyl)-1 cyclopropyl-4-quinolone-3-carboxylic acid. Most of the in vivo topoisomerase II cleavage sites were found either in the transcribed region of ribosomal DNA or in the palindromic region surrounded by the replication origins. Two classes of sites were identified: those which correlate with DNase I hypersensitive sites and corresponding to an open chromatin configuration (transcribed region) and internucleosomal cleavage sites (in the region of replication origins). Topoisomerase II drug-induced cleavage in the ribosomal DNA was considerably reduced upon Physarum differentiation to a dormant stage of life, the spherules. In contrast, the amount of drug-dependent cleavage was found to increase during the metaphase of mitosis, when rDNA transcription is shut off. These findings suggest a role for topoisomerase II in the ribosomal DNA minichromosomes segregation, in addition to its role in transcription. Finally, the similarity between in vivo sites and those observed following drug treatment of isolated nuclei indicates that no profound change occurs in rDNA chromatin conformation during nuclei isolation. By contrast, in vitro cleavage sites with purified topoisomerase II weakly correlate to in vivo, indicating a prominent role for chromatin structure in determining the interaction sites of topoisomerase II with DNA in vivo. PMID- 8639540 TI - Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing. AB - Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction. PMID- 8639541 TI - Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen. AB - Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa. PMID- 8639542 TI - Evidence of intramolecular cross-linked A alpha.gamma chain heterodimers in plasma fibrinogen. AB - A peptide band of approximately 105 kDa migrating near the gamma dimer position of disulfide bond reduced human plasma fibrinogen prepared from fresh single donor or outdated plasma was identified by SDS-PAGE. The band, amounting to approximately 2% of the total A alpha/gamma chain population, was thrombin and plasmin sensitive and reacted with antibodies to A alpha or gamma chains but not with antibodies to B beta chains, plasminogen, or factor XIII. Amino acid sequencing revealed a double sequence corresponding to that of A alpha and gamma chains, indicating that the band consists of covalently cross-linked A alpha.gamma chain heterodimers. A alpha.gamma heterodimers were identified as a component of monomeric fibrinogen by two-dimensional SDS-PAGE and by SDS-PAGE analysis of the monomer fraction isolated by gel sieving chromatography, thus indicating that A alpha.gamma heterodimers arise by intramolecular A alpha/gamma chain cross-linking. PMID- 8639543 TI - Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis. AB - Although the rapid thrombin-induced release of arachidonic acid in human platelets has been known for over 20 years, the amount of arachidonic acid mass mobilized and the source of the released arachidonic acid has remained a subject of intense controversy. Herein, we exploit the analytic power and sensitivity of electrospray ionization mass spectrometry to identify plasmenylethanolamines as the largest source of arachidonic acid mass released during thrombin stimulation and to demonstrate the presence of multiple novel molecular species of plasmenylethanolamines in human platelets. Specifically, 90 s after thrombin stimulation a total of 60.1 nmol of arachidonic acid-containing phospholipids/10(9) platelets was hydrolyzed which included the loss of 31.8 nmol/10(9) platelets from ethanolamine glycerophospholipids (hydrolysis of plasmenylethanolamines represented 63% of the mass lost from the ethanolamine glycerophospholipid pool) but only 10.9 nmol/10(9) platelets from choline glycerophospholipids. Human platelet phosphatidylserine and phosphatidylinositol pools contained similar amounts of arachidonic acid mass in resting platelets (approximately equal to 20 nmol/10(9) platelets), and each pool contributed 8.7 nmol/10(9) platelets after thrombin stimulation. From these results, a lower boundary for the rate of thrombin-induced arachidonic acid mobilization in human platelets can be set at > 60 nmol/10(9) platelets, thereby identifying specific kinetic characteristics and substrate selectivities of the phospholipase(s) activated during platelet stimulation. Collectively, these results underscore the importance of plasmenylethanolamines as the major storage depot of arachidonic acid in resting platelets and as the major source of arachidonic acid mobilized after thrombin stimulation of human platelets. PMID- 8639544 TI - Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure. AB - We have previously showed that factor X activator of Russell's viper venom (RVV X) contains six N-linked oligosaccharide chains: four in the heavy chain and one in each of the two light chains [Gowda, D.C., Jackson, C.M., Hensley, P., & Davidson, E.A. (1994) J. Biol. Chem. 269, 10644-10650]. In the present study, we have investigated the role of the carbohydrate moieties in the structure and functional activity of RVV-X. Sequential removal of sugar residues from the terminal ends by exoglycosidases, up to 50% of total carbohydrates, did not significantly alter the activity of RVV-X, demonstrating that the peripheral carbohydrate moieties are not involved in interactions with factor X. However, removal of whole oligosaccharide chains by N-glycanase caused an almost total loss of the ability of RVV-X to activate factor X to factor Xa. In parallel with these observations, circular dichroism spectroscopy showed that complete deglycosylation, but not the removal of peripheral sugars, caused a significant change in the secondary structure. Together, these data demonstrate that the oligosaccharide chains are necessary for the functional activity, and that the trimannosylchitobiose core residues are sufficient for the maintenance of the native polypeptide structure. PMID- 8639545 TI - Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation. AB - A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed. PMID- 8639547 TI - Introduction of a fifth carboxylate ligand heightens the affinity of the oncomodulin CD and EF sites for Ca2+. AB - The acid-pair hypothesis, proposed by Reid and Hodges [(1980) J. Theor. Biol. 84, 401-444], suggests that the affinity of an EF-hand motif for Ca2+ will be maximal with four acidic ligands, paired along the +x, -x and +z, -z axes. Addition of a fifth anionic ligand is predicted to reduce Ca(2+)-binding affinity, as a consequence of increased electrostatic repulsion. Interestingly, for oncomodulin, we observe that introduction of a fifth carboxylate residue at the +z position in the CD coordination sphere or at the -x position in the EF coordination sphere significantly increases the affinity of those sites for Ca2+. The variants resulting from replacement of serine-55 by aspartate (S55D), glycine-98 by aspartate (G98D), and the combined mutations (55/98) have been examined in Ca(2+) and Mg(2+)-binding studies, titration calorimetry, and differential scanning calorimetry. The KCa for the CD site is reduced from 800 to 67 nM by the S55D mutation, while KCa for the EF site is reduced from 45 to 4 nM by the G98D mutation. Both mutations destabilize the apo form of the protein and increase the thermal stability on the Ca(2+)-bound state. Interestingly, the S55D mutation also increases the affinity of the oncomodulin CD site for Mg2+, decreasing the dissociation constant from > 1 mM to approximately 30 microM. This increase in affinity is reflected in a substantially increased thermal stability of the Mg(2+)-bound form of the protein. In 0.15 M NaCl, 0.025 M Hepes (pH 7.4), and 0.01 M Mg2+, the wild-type protein denatures at 68.5 degrees C. By contrast, under identical conditions, the S55D mutations denatures at 79.0 degrees C. The increased metal ion-binding affinity displayed by the variant proteins may result in part from preferential destabilization of the apo-protein by the additional carboxylate. PMID- 8639546 TI - Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation. AB - Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle. PMID- 8639548 TI - A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: part of the F helix tilts in the M to N transition. AB - The N intermediate of the bacteriorhodopsin photocycle was trapped for electron diffraction studies in glucose-embedded specimens of the site-directed mutant Phe219 --> Leu. At neutral pH, the N-bR difference Fourier transform infrared spectrum of this mutant is indistinguishable from published difference spectra obtained for wild-type bacteriorhodopsin at alkaline pH. An electron diffraction difference map of the N intermediate in projection shows large differences near the F and the G helix, which are very similar to the features seen in the M intermediates of the Asp96 --> Gly mutant [Subramaniam et al. (1993) EMBO J. 12, 1-8]. This similarity was anticipated on the basis of Fourier transform infrared data, which have shown that the M intermediate trapped in Asp96 mutants already has the protein structure of the N intermediate [Sasaki et al. (1992) J. Biol. Chem. 267, 20782-20786]. A preliminary three-dimensional difference map of the N intermediate, calculated from electron diffraction data of samples tilted at 25 degrees, clearly shows that the change on the F helix consists of an outward movement of the cytoplasmic end of the helix. In addition, the cytoplasmic side of the G helix moves or becomes more ordered. Comparison with published difference maps of the M intermediate indicates that the F helix tilt occurs in the M to N transition, but the G helix change represents an earlier step in the photocycle. PMID- 8639550 TI - Analysis of substrate-induced electronic, catalytic, and structural changes in inducible NO synthase. AB - Inducible nitric oxide synthase (iNOS) catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine and O2. In addition to serving as substrate, L-arginine alters the enzyme's heme iron spin equilibrium, increases its NADPH oxidation, and promotes assembly of active dimeric iNOS from inactive monomers. To understand what structural aspects of L-arginine are important for causing these effects, we have studied the interactions of iNOS with several L-arginine and guanidine analogs. Very few analogs supported NO synthesis even when bound to iNOS at saturating or near-saturating levels. In contrast, almost all analogs shifted the heme iron spin equilibrium and either increased or decreased NADPH oxidation by iNOS. The guanidine analogs displayed the same pattern of effects as their amino acid counterparts but exhibited a lower affinity except for analogs containing S-alkylisothiourea or aminoguanidine groups. Most analogs also promoted iNOS dimerization, with hydroxyguanidine and S ethylisothiourea promoting more dimerization than L-arginine itself. Although the analog concentrations required to promote dimerization of monomers were somewhat higher than those required for binding to dimeric iNOS, they followed the same rank order. The degree of dimerization promoted by each analog did not correlate to its binding affinity, its causing a high- or low-spin shift in heme iron spin state, or to its increasing or decreasing NADPH oxidation. Together, we conclude that the enzyme's high degree of substrate specificity only applies to NO synthesis, in that a number of "inactive" structural analogs still bind to iNOS and affect its heme chemistry and structure in the absence of supporting NO synthesis. These latter affects are mediated through binding of the guanidinium portion of L-arginine and its analogs to a single site within iNOS and are relatively independent of the amino acid portion of the molecule. PMID- 8639549 TI - Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae. AB - The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2). To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis. The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity. The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined. Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains. The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2. PMID- 8639551 TI - Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL. AB - Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136 139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM. PMID- 8639552 TI - Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin. AB - The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a rate-limiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration. PMID- 8639553 TI - Charge-shielding and the "paradoxical" stimulation of tubulin polymerization by guanidine hydrochloride. AB - Low concentrations of guanidine hydrochloride (GuHCl) increase the rate (and to a lesser degree, the extent) of tubulin polymerization as assessed by light scattering. Maximum enhancement occurs at 120-160 mM GuHCl followed by decreases at higher GuHCl. The latent period is decreased, and there is a 3-4 fold reduction in the critical concentration of polymerization. Electronmicrographs reveal microtubules in the controls and an increasing fraction of total polymers present as aberrant microtubules as the GuHCl concentration is increased from 20 to 100 mM. The GuHCl effect is markedly reduced, but not abolished, in tubulin S (in which the anionic C termini of both monomers have been removed). The GuHCl induced polymerization has an absolute requirement for GTP and taxol or DMSO, is very sensitive to podophyllotoxin inhibition, and can overcome urea-mediated inhibition of polymerization. Guanidinium analogues mimic the GuHCl effect roughly as a function of the number of potential hydrogen bonds. The anions of the guanidine salts superimpose their inhibitory action on the guanidinium cation effect according to the lyotropic series. At higher GuHCl concentrations (peak effect 500-700 mM), a different polymer (type II) is formed that is GTP and taxol independent, but whose polymerization is retarded but not prevented by podophyllotoxin. Its structure resembles the fibrillar network seen in unfolding intermediates of other proteins. We conclude that both charge and hydrogen bonding ability are major contributors to the GuHCl-induced promotion of tubulin polymerization, and that charge-shielding is likely to be the basis for this effect. PMID- 8639554 TI - Primary structure of a new neuropeptide, cerebral peptide 2, purified from cerebral ganglia of Aplysia. AB - We report the purification and characterization of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed cerebral peptide 2 (CP2) because it was the larger of two peptides predominantly synthesized in the cerebral ganglia and transported to other regions of the central nervous system. The purification of CP2 from extracts of cerebral ganglia using three sequential modes of high-pressure liquid chromatography (HPLC) was followed using the [35S]methionine-labeled peptide obtained from transport experiments. The primary structure of CP2 was determined by automated Edman degradation of native CP2 and its proteolytic fragments in conjunction with mass spectrometry. CP2 is a 41 amino acid peptide with an amidated carboxyl terminal. A peptide with the proposed sequence of CP2 was synthetized and compared by HPLC with the native peptide. Chromatographic properties of the synthetic and native peptide labeled in vivo were found to be identical. CP2 does not appear to be a member of any previously identified peptide family. PMID- 8639555 TI - Three-dimensional structure of the reduced C77S mutant of the Chromatium vinosum high-potential iron-sulfur protein through nuclear magnetic resonance: comparison with the solution structure of the wild-type protein. AB - The full 1H NMR assignment of the reduced C77S mutant of Chromatium vinosum high potential iron-sulfur protein (HiPIP) was achieved by taking advantage of the assignment available for the wild-type protein. A total of 1565 nuclear Overhauser effect (NOE) spectroscopy cross peaks were integrated and converted into distance constraints, of which 497 were found to be irrelevant. An additional 24 dipolar constraints were obtained from one-dimensional NOE difference spectra by saturating hyperfine-shifted beta CH2 cysteine/serine protons. Forty-six 3JNH-H alpha coupling constants and eight hydrogen bonds provided further constraints. Through a distance geometry approach, a family of 15 structures was calculated, which was subsequently subjected to restrained energy minimization. The root mean square deviations of the minimized structures were 0.62 +/- 0.09 and 1.09 +/- 0.11 A for backbone and heavy atoms, respectively. The resulting solution structures are very similar to those of the reduced wild-type protein (WT). An analysis of the NOEs experienced by the protons of Ser-77 in both the reduced and oxidized forms reveals that they are very similar to those experienced by Cys-77 in WT. On the basis of the hyperfine shifts observed for the Ser-77 protons and of the present structural analysis, it is concluded that the serine O gamma atom is coordinated to the polymetallic center, thus confirming the strict analogy of the electronic structures of the polymetallic center in both proteins. Capillary electrophoresis experiments demonstrate coordination of Ser-77 as an anion. Serine versus cysteine coordination in iron-sulfur proteins is briefly discussed. PMID- 8639556 TI - Characterization of two transposon mutants from Haemophilus influenzae type b with altered lipooligosaccharide biosynthesis. AB - Two isogenic mutants of Haemophilus influenzae type b (Hib) strain A2 were prepared by random m-Tn3(Cm) insertions into the 7.4-kb lsg (lipooligosaccharide synthesis genes) region of Hib DNA, which consists of seven complete and one partial open reading frame (orfs). Compared to the parent A2 strain which produces a complex mixture of lipooligosaccharides (LOS), the mutant strains 281.25 and 276.4 produced only a few LOS species. The precise locations of transposon insertions into the lsg loci of these mutants were determined (base 3546 in orf 4 for strain 281.25 and base 4402 in orf 5 for strain 276.4), and the effects of these mutations on LOS biosynthesis and epitope expression were evaluated. When the O-deacylated LOS were analyzed by mass spectrometry, both strains contained major LOS species of M(r) 2601, 2439, and 2277, which consisted of a common heptose trisaccharide core structure [Hep3(PEA)Kdo(P)-lipid A, where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, and PEA is phosphoethanolamine] and four, three, or two hexoses, respectively. These species represent the smallest components of the wild-type LOS mixture. The major LOS oligosaccharide obtained from the strain 281.25 by mild acid hydrolysis was dephosphorylated and shown by composition analysis, methylation analysis, mass spectrometry, and 2D NMR studies to be a triantennary structure consisting of a heptose trisaccharide core with two glucose disaccharide branches: Hep alpha 1 - > (Glc beta 1 --> 4Glc alpha 1 --> 3) 2Hep alpha 1 --> (Glc beta 1 --> 4Glc beta 1 --> 4)3Hep alpha 1 --> anhydroKdo. Unlike the parent A2 strain, mutant strain 281.25 cannot add galactoses to the branches of this octasaccharide. Strain 276.4 is similarly deficient, except that it can still utilize a minor biosynthetic pathway leading to the addition of sialyl-N-acetyllactosamine. PMID- 8639557 TI - Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent. AB - Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process. In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of lambda repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (delta Cp). Denaturation curves based on aromatic 1H NMR spectra give identical denaturation curves, confirming purely twostate folding under all conditions studies. The denaturation surface can be fit with constant delta Cp and delta In KD/delta[urea] (KD is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic 1H NMR spectrum of the cold denatured state at 0 degree C in 3 M uea is essentially identical to the spectrum at 70 degree C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent. PMID- 8639558 TI - A symmetry-driven search for electrostatic interaction partners in charybdotoxin and a voltage-gated K+ channel. AB - A structural model of charybdotoxin bound to a Shaker K+ channel has emerged from mechanistic and mutagenic analysis of toxin-channel interactions. We test this model by predicting through-space electrostatic interactions between specific pairs of channel-toxin residues. Dissociation constants of channel-toxin variants, determined by radiolabeled toxin binding to Shaker-transfected COS membrane fragments, were used to identify pairs of residues located closely enough to interact electrostatically. The results further refine the structural model of the bound complex and produce a more detailed view of the vestibule of the Shaker channel. PMID- 8639559 TI - Three-dimensional solution structure of the N-terminal domain of DNA polymerase beta and mapping of the ssDNA interaction interface. AB - DNA polymerase beta (beta-Pol) consists of an N-terminal ssDNA binding domain with deoxyribose phosphodiesterase activity and a C-terminal domain with nucleotidyltransferase activity. The solution structure of the cloned N-terminal domain of beta-Pol has been determined by multidimensional heteronuclear NMR using experimental restraints that included 1030 distances based on analysis of NOE connectivities, 68 phi, chi 1, and chi 2 torsion angles based on analysis of couplings, and 22 hydrogen bonds. Hydrogen bonds were assessed only within helices by the absence of saturation transfer from water at pH 6.7, by NOEs and JNH alpha couplings indicative of well-structured helices, and by 13C alpha chemical shifts characteristic of helices. The root mean square deviation for heavy backbone atoms within the helices was 0.64 A in 55 structures. The solution structure of the N-terminal domain is formed from four helices packed as two antiparallel pairs crossing at 50 degrees in a V-like shape. The domain binds p(dT)8, a template analogue, as a 1:1 complex in 100 mM NaCl (KD = 10 microM). Analysis of the binding equilibria at increasing NaCl concentrations indicated that ionic contacts contribute to the complex. The binding interaction was mapped to one face of the domain by characterizing backbone 1H and 15N chemical shift changes. Assigned intermolecular NOEs from 2D NOESY support the assessment of the binding interface. The structure that forms the interaction surface includes an antiparallel helix-3-turn-helix-4 motif and residues adjacent to an omega-type loop connecting helix-1 and helix-2. Sites appropriate for nucleotide contact on the structure are described. The mapped interaction interface for a ssDNA template is the first described for a DNA polymerase. PMID- 8639560 TI - The three-dimensional solution structure of the SH2 domain from p55blk kinase. AB - Signal transduction in B cells is mediated, in part, by the interaction of the cytoplasmic components of the antigen receptor complex and various members of the src family tyrosine kinases. Key to this process appears to be the interaction of the tyrosine kinase SH2 domains with the tyrosine-phosphorylated cytoplasmic domain of Ig-alpha, a disulfide-bonded heterodimeric (with Ig-beta or Ig-gamma) transmembrane protein that noncovalently associates with the antigen receptor immunoglobin chains. In addition to binding to the phosphorylated cytoplasmic domains of Ig-alpha and Ig-beta, blk and fyn(T), two members of the src family kinases, have been shown to bind overlapping but distinct sets of phosphoproteins [Malek & Desiderio (1993) J. Biol. Chem. 268. 22557-22565]. A comparison of their three-dimensional structures may elucidate the apparently subtle differences required for phosphoprotein discrimination. To begin characterizing the blk/fyn/phosphosphoprotein interactions, we have determined the three-dimensional solution structure of the SH2 domain of blk kinase by nuclear magnetic resonance (NMR) spectroscopy. 1H, 13C, and 15N resonances of the SH2 domain of blk kinase were assigned by analysis of multidimensional, double- and triple-resonance NMR experiments. Twenty structures of the blk SH2 domain were refined with the program X-PLOR using a total of 2080 experimentally derived conformational restraints. The structures converged to a root-mean-squared (rms) distance deviation of 0.51 and 0.95 A for the backbone atoms and for the non-hydrogen atoms, respectively. The blk SH2 domain adopts the prototypical SH2 fold. Structurally, blk SH2 is most similar to the crystal structure of the v-src SH2 domain [Waksman et al. (1993) Nature 358.646-653] and superimposes on the crystal structure with an rmsd of 1.52 A for the backbone atoms. The largest deviations occur in the four loops interconnecting beta-strands A-E, which are the least well-defined regions in the NMR structure. Exclusion of these loops lowers this rmsd to 0.82 A. The conformation of the BC loop in the blk SH2 domain is similar to the open conformation in the apo lck SH2 domain, suggesting that, like the lck SH2 domain, the blk SH2 domain may have a gated phosphopeptide binding site. Finally, it is proposed that the amino acid substitution of Lys 88 (blk) for Glu [fyn(T)] is important for the observed differences in specificity between blk and fyn(T) SH2 domains. PMID- 8639561 TI - Adduction of the human N-ras codon 61 sequence with (-)-(7S,8R,9R,10S)-7,8 dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene: structural refinement of the intercalated SRSR(61,2) (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9,10- tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct from 1H NMR. AB - The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,910-tetrahydrobenzo [a]pyrenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'-5' d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R,10S)-7, 8-dihydroxy-9,10-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 369 NOEs from 1H NMR. This was named the SRSR(61,2) adduct, derived from the N-ras protooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. NOEs between C5, S.R.S.R A6, and A7 were disrupted, as were those between T17 and G18. NOEs between benzo[a]pyrene and DNA protons were localized on the two faces of the pyrenyl ring. The benzo[a]pyrene H3-H6 protons showed NOEs to T17 CH3, while H1, H2, and H3 showed NOEs to T17 deoxyribose; the latter protons and H4 showed NOEs to T17 H2', H2" and to T17 H6. Noes were observed between H11 and H12 and C5 H]',H2', H2". G18 N1H showed NOEs to both faces of benzo[a]pyrene. Upfield shifts of 2.6 ppm for T17 N3H and 1.8 ppm for G18 N1H. 1 ppm for T17 H6 and CH3, and 0.75 ppm for C5 H5, with a smaller shift for C5 H6, and a 1.5 ppm dispersion of the pyrenyl protons suggested that benzo[a]pyrene intercalated above the 5'-face of S.R.S.R A6. The precision of the refined structures was monitored by pairwise root mean square deviations. which were < 1.5 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 8.1 x 10(-2). Interstrand stacking between the pyrenyl ring and the T17 pyrimidine and G18 purine rings was enhanced by the bay ring. Changes of +30 degrees and -25 degrees in buckle for C5.G18 and S.R.S.R A6.T17, respectively, were calculated, as was a -40 degrees change in propeller twist for C5.G18. The rise between C5.G18 and S.R.S.R A6.T17 was calculated to be 7 A. The work extended the pattern for adenine N6 benzo[a]pyrene adducts, in which the R stereochemistry at C10 predicted 5'-intercalation of the pyrenyl moiety. PMID- 8639562 TI - Engineering stabilized ion channels: covalent dimers of alamethicin. AB - The peptide alamethicin forms channels with a variety of conductance states. Selective stabilization of a particular state should simplify the task of understanding conductance in terms of channel structure. We synthesized two different covalent dimers of alamethicin in which peptides were linked at their C terminal ends by flexible tethers. Both dimeric peptides formed channels with conductances that matched those of alamethicin channels. Particular conductance states were selectively stabilized, however, with lifetimes up to 170-fold longer than the same states observed with monomers. In addition, tethering appeared to limit the size of the structures formed so that, even at higher peptide concentrations, a single predominant conductance state was obtained. We suggest this state corresponds to a channel made from six alamethicin molecules (three dimers). PMID- 8639563 TI - NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump. AB - The Na+-activated NADH:ubiquinone oxidoreductase of Vibrio alginolyticus was extracted from the membranes with lauryldimethylamine-N-oxide and purified by two successive anion exchange columns. This preparation, yielding four major and several minor stained bands after SDS-PAGE, retained the NADH-dehydrogenase activity (with menadione as an artificial electron acceptor) and ubiquinone-1 (Q) reductase activity. On further fractionation of the enzyme, the Q-reductase activity essentially disappeared. Chemical analyses revealed the presence of FAD but not FMN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiquinone-8 in the purified Q-reductase preparation. The participation of an iron-sulfur cluster of the [2Fe-2S] type in the electron translocation was demonstrated by the appearance of a typical EPR signal for this prosthetic group after the reduction of Q-reductase with NADH. A strong EPR signal typical for a radical observed upon reduction of the enzyme might arise from the formation of quinone radicals. In the absence of Na+, the path of the electrons apparently ends with the reduction of ubiquinone-1 to the semiquinone derivative which in the presence of O2 becomes reoxidized with concomitant formation of superoxide radicals. In the presence of Na+, these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative. These results indicate that the Na+-dependent step in the electron transfer catalyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiquinone to ubiquinol. After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-1 was coupled to Na+ transport with an apparent stoichiometry of 0.5 Na+ per NADH oxidized. The transport was stimulated by valinomycin (+ K+) or by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The transport of Na+ is therefore a primary event and does not involve the intermediate formation of a proton gradient. PMID- 8639564 TI - Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems. AB - The "selective" cholesterol uptake pathway represents a bulk pathway by which many steroidogenic cells internalize lipoprotein-delivered cholesteryl esters. In the current report, we question whether cholesteryl esters entering cells via this pathway are capable of governing standard cholesterol end product feedback repression mechanisms. Cultured rat ovary granulosa cells which utilize both the "selective" and "endocytic" pathways to internalize lipoprotein-derived cholesteryl esters were used as a model system. ApoE-free hHDL3 was used to deliver cholesteryl esters to the cells exclusively by the selective pathway; hLDL was used as a control ligand which when internalized by the endocytic pathway releases cholesteryl esters which subsequently regulate the expression of the B/E (LDL)-receptor, HMG CoA reductase, and acyl-CoA:cholesterol acyltransferase (ACAT). Whereas trophic hormone (Bt2cAMP) stimulation by itself increased the activity, mRNA, and protein levels of both B/E-receptor and HMG CoA reductase, pretreatment with either lipoprotein (adjusted for equal cholesterol ester content) down-regulated this expression. Linked with these lipoprotein related changes was an increase in activity (though not gene expression) of ACAT. The level of change in mRNA levels, protein content, and activity for the examined regulatory proteins was essentially equivalent whether the lipoprotein provided to the cells was hLDL or hHDL3. Thus, similar signals appear to have been received by the cells despite differences in the uptake and processing of the ligand-derived cholesteryl esters, and these signals resulted in identical homeostatic responses by the cells. PMID- 8639565 TI - Blue and ultraviolet light-absorbing opsin from the retinal pigment epithelium. AB - The retinal pigment epithelium (RPE) contains an abundant opsin that is distinct from rhodopsin and cone visual pigments and is able to bind the retinaldehyde chromophore. The putative retinal G protein-coupled receptor (RGR) was isolated in digitonin solution from bovine RPE microsomes and copurified consistently with a minor 34-kDa protein. The absorption spectrum of RGR revealed endogenous pH sensitive absorbance in the blue and near-ultraviolet regions of light. Membrane bound RGR was incubated with exogenously added all-trans-retinal and formed two long-lived pH-dependent photopigments with absorption maxima of 469 +/- 2.4 and 370 +/- 7.3 nm. The effects of hydrogen ion concentration suggest that the blue and near-UV photopigments are tautomeric forms of RGR, in which an all-trans retinal Schiff base is protonated or unprotonated, respectively. The RPE pigment was also demonstrable by its reactivity to hydroxylamine in the dark. The retinaldehyde-RGR conjugate at neutral pH favors the near-UV pigment and is a novel light-absorbing opsin in the vertebrate eye. PMID- 8639566 TI - Structure around C6-C7 bond of the chromophore in bathorhodopsin: low-temperature spectroscopy of 6s-cis-locked bicyclic rhodopsin analogs. AB - To elucidate the structural changes near the beta-ionone ring region of the chromophore during the photobleaching process of rhodopsin, the photochemical and subsequent thermal reactions of rhodopsin analogs, whose retinylidene chromophores were fixed in a 6s-cis form with a five-membered ring (6,5 rhodopsin) and a seven-membered ring (6,7-rhodopsin), respectively, were investigated by low-temperature spectroscopy. Like rhodopsin, both the rhodopsin analogs convert to the respective batho-intermediates upon absorption of light at -190 degrees C. The extinction coefficient of batho-intermediate of 6,5-rhodopsin is similar to that of bathorhodopsin, while that of 6,7-rhodopsin is considerably smaller than that of bathorhodopsin. Like bathorhodopsin, the batho-intermediate of 6,5-rhodopsin directly converts to lumi-intermediate, while that of 6,7 rhodopsin first converts to a blue-shifted intermediate and then to lumi intermediate. These results strongly suggest that the structure around the beta ionone ring region of the bathorhodopsin chromophore resembles 6,5-retinal rather than 6,7-retinal. From the comparison of the structural features among retinal, 6,5-retinal, and 6,7-retinal, a possible conformation around C6-C7 bond of the bathorhodopsin chromophore is discussed. PMID- 8639567 TI - Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase. AB - In contrast to the membrane guanylate cyclases which are stimulated by extracellular ligands, rod outer segment membrane guanylate cyclase (ROS-GC) activity is modulated intracellularly by calcium in two ways: one, where it is inhibited, and the other, where it is stimulated. The former way is linked to the phototransduction, and physiology of the second is unknown. In both ways calcium modulation of the cyclase occurs through the calcium binding proteins: through guanylate cyclase activating proteins (GCAPs) in the case of phototransduction, and through the recently discovered calcium-dependent GCAP (CD-GCAP) in the case of the other way. The kinase-like domain of ROS-GC is critical for the phototransduction-linked process. The present study shows the expression of alpha and beta chains of S100A1-S100B protein in the bovine retina and demonstrates that this protein stimulates ROS-GC activity in a dose-dependent fashion, that the stimulation is calcium dependent with an EC50 of 17 microM, and that the kinase-like domain is not involved in the calcium-modulated cyclase activation. Instead the involved domain resides at the C-terminal segment, between amino acids 731 and 1054. Thus, this S100A1-S100B protein-mediated calcium-modulated signal transduction mechanism is novel. Furthermore, this study provides the molecular understanding of the two transduction processes mediated by the same ROS-GC, one linked to the low and the other to the high calcium levels. PMID- 8639568 TI - Identification of insulin-stimulated phosphorylation sites on calmodulin. AB - Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin. PMID- 8639569 TI - Mutational analysis of the catalytic subunit of muscle protein phosphatase-1. AB - A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group. PMID- 8639570 TI - In vitro inhibition of MAP kinase (ERK1/ERK2) activity by phosphorylated glia maturation factor (GMF). AB - We report that recombinant glia maturation factor (GMF), a 17-kDa brain protein, inhibits the activity of mitogen-activated protein (MAP) kinase in the test tube assay, in particular the ERK1/ERK2 isoforms. A preliminary phosphorylation of GMF by protein kinase A (PKA) dramatically increases its inhibitory effect by over 600-fold (Ki approximately 3 nM), making it the most potent MAP kinase inhibitor ever reported. Immunoprecipitation of GMF from cell extracts using its specific antibody coprecipitates ERK (and vice versa), suggesting the association of the two proteins in the cell. The inhibitory effect of PKA-phosphorylated GMF is specific, as it does not suppress the activity of cdc2 kinase, another proline directed kinase. Nor does it inhibit MAP kinase kinase (MEK) and MAP kinase activated protein (MAPKAP) kinase-2, the two enzymes immediately upstream and downstream, respectively, of ERK. Of the other three enzymes that can phosphorylate GMF, only p90 ribosomal S6 kinase (RSK) enhances the inhibitory function of GMF on ERK; protein kinase C (PKC) and casein kinase II (CKII) are without effect. The inhibition of ERK by PKA-phosphorylated GMF suggests that GMF could be one of the mediators of the suppressive effect of the PKA pathway on the MAP kinase pathway. On the other hand, that RSK-phosphorylated GMF also inhibits ERK implies a negative feedback loop in the regulation of MAP kinase activity. PMID- 8639571 TI - Kinetics of substrate reaction in the course of hydroperoxide-mediated inactivation of cytochrome P450 1A1. AB - A schematic kinetic model is proposed for the hydroperoxide-mediated substrate reaction and cytochrome P450 inactivation. From the model, the relationships between the product concentration at infinite time (P infinity), the apparent rate constant of P450 inactivation (A), and the substrate concentrations are predicted, and the predictions were experimentally examined. The reciprocal of P infinity is proportional to the reciprocal of the substrate concentration in both CuOOH- and H2O2-supported substrate reactions. The reciprocal of P infinity is proportional to cumene hydroperoxide (CuOOH) concentration, but P infinity is independent of H2O2 concentration, indicating different effects of CuOOH and H2O2 in P450 inactivation. The apparent rate constant (A) is proportional to the reciprocal of the substrate concentrations, suggesting substrate protection of P450 from hydroperoxide inactivation. The model also suggests that a second CuOOH molecule may compete with substrate for binding to the P450 substrate binding site. Simulated kinetics of production formation vs time are quite consistent with the experimental kinetics. PMID- 8639572 TI - A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene. AB - An open reading frame located upstream of the bacterioferritin gene in Escherichia coli encodes a hypothetical 64-residue protein [Andrews, S.C., Harrison, P.C., & Guest, J.R. (1989) J. Bacteriol. 171, 3940-3947)]. The spacing of the four cysteine residues in this hypothetical protein is identical to that in a region of NIFU, a [2Fe-2S] protein found in nitrogen-fixing bacteria [Fu, W., Jack, R.F., Morgan, T.V., Dean, D.R., & Johnson, M.K. (1994) Biochemistry 33, 13455-13463)]. The NIFU-like E. coli gene was cloned and overexpressed with a C terminal "His tag" in E. coli using the T7 RNA polymerase/promoter system, and the protein was purified by metal-chelate affinity chromatography. UV-vis absorption and EPR spectra together with iron and amino acid analyses conclusively established that this NIFU-like E. coli protein contains one [2Fe 2S] cluster which can exist in at least two oxidation levels: +2 for the as purified protein, and +1 for dithionite-reduced protein. Size-exclusion chromatography established that this His-tagged [2Fe-2S] protein is monomeric in solution. Affinity chromatography demonstrated specific complex formation between bacterioferritin (Bfr) and this NIFU-like [2Fe-2S] protein, which is dubbed Bfd. An open reading frame encoding a homologous Bfd is located near a Bfr gene in at least one other bacterium. Bfd may, therefore, constitute a general redox and/or regulatory component participating in the iron storage or mobilization functions of Bfr. PMID- 8639573 TI - Cold-sensitive assembly of a mutant manganese-stabilizing protein caused by a Val to Ala replacement. AB - Photosystem II (PSII) is a multisubunit transmembrane protein complex that oxidizes water and evolves O2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxidation. The 33-kDa water soluble PSII subunit, or manganese-stabilizing protein (MSP), stabilizes the O2 evolving manganese cluster and accelerates O2 evolution. Spinach PSII can be depleted of native MSP under conditions which retain a functional manganese cluster. Reconstition of MSP-depleted PSII with recombinant MSP was equally efficient at 4 and 22 degrees C. Replacement of Val235 (a conserved residue near the C-terminus of MSP) with Ala inhibited assembly of MSP at 4 degrees C, but not at 22 degrees C. Once assembled, [V235A]MSP remained bound to PSII even at 4 degrees C and in the presence of low concentrations of urea. Results from far-UV circular dichroism spectrometry indicated that [V235A]-MSP was destabilized by low temperature to a greater extent than the wild-type protein. However, the effect of temperature on the secondary structure of both the mutant and wild-type proteins was small compared to the temperature-independent destabilization of secondary structure induced by the mutation. These results demonstrate that the V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhibiting isomerization of one or more prolyl peptide bonds required for assembly. PMID- 8639574 TI - Effects of arginine-82 on the interactions of internal water molecules in bacteriorhodopsin. AB - Arg82, one of the residues near the protonated Schiff base of bacteriorhodopsin, facilitates proton release to the medium during the L-to-M reaction of the photocycle, but retards the rate of proton transfer from the Schiff base to Asp85. In order to understand the role of Arg82 in these processes, the structural changes upon formation of the M intermediate were studied by Fourier transform infrared spectroscopy of the hydrated films of Arg82 mutants at pH 9.5. The negative band at 1700 cm-1 in the BR --> M spectrum due to the deprotonation of Glu204 was absent when Arg82 was replaced with alanine (R82A), but present with small amplitude when residue 82 was a glutamine (R82Q), or a lysine (R82K), with a shift to 1696 cm-1. The O-H stretch of water at 3643 cm-1 is shifted toward a lower frequency in R82Q, R82K, and R82A in the unphotolyzed state. However, R82Q retains a fraction of the unshifted band. Another O-H stretch is prominent in R82Q around 3625 cm-1 but absent in R82A and probably in R82K. In parallel, R82Q retains a fraction of the slow component of the formation of the M intermediate, which is almost completely absent in R82K and R82A. These results, along with previous data for the mutants of Glu204, suggest that the guanidium group of Arg82 influences the H-bonding of water molecules located close to Asp85 and Arg82-Glu204 regions, and the rate of proton transfer from the Schiff base to Asp85. The amide group of Gln82 can substitute for it but weakly. PMID- 8639575 TI - Interactions and properties of smooth muscle myosin phosphatase. AB - Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c. PMID- 8639576 TI - Purification and characterization of two monomeric kinesin constructs. AB - Steady-state and pre-steady-state kinetic methods were used to analyze two shorter Drosophila kinesin constructs (K341 and K366) in comparison to K401. K341, K366, and K401 represent the kinesin motor domains containing the N terminal 341, 366, or 401 amino acids, respectively. K401 is dimeric (Kd = 37 +/- 17 nM) whereas both K366 and K341 are monomeric [Correia et al. (1995) Biochemistry 34, 4898-4907]. Like native kinesin and K401, K341 and K366 demonstrate low ATPase activity in the absence of microtubules (0.03 and 0.01 s 1, respectively), and ADP release is rate-limiting during steady-state turnover. Microtubules activate the steady-state ATPase to 84 s-1 for K341 (K(m),ATP = 100 microM; K0.5,MT = 3.2 microM tubulin) and 64 s-1 for K366 (K(m),ATP = 65 microM; K0.5,MT = 2.5 microM tubulin) in comparison to K401 at 20 s-1 (K(m)ATP = 60 microM; K0.5,MT = 1 microM tubulin). The rapid quench experiments for all three constructs show a burst of product formation during the first turnover, indicating the rate-limiting step for the microtubule-activated ATPase occurs after ATP hydrolysis. The interaction of K341 and K366 with the microtubule was analyzed by electron microscopy. The results show that K341 and K366, like K401, bind to the microtubule with an 8 nm axial periodicity. However, the addition of K366 to microtubules resulted in significant aggregation of microtubules. The pre steady-state kinetic results show that K341 retains the kinetic and structural properties necessary to compare directly the kinetic properties of monomeric and dimeric kinesins, although the microtubule-activated ATPase is significantly faster for the monomeric constructs, suggesting possible interactions in the dimer which inhibit ATP turnover as part of the coupling to force production. PMID- 8639578 TI - Calcium and gadolinium ions stimulate the GTPase activity of purified chicken brain tubulin through a conformational change. AB - Ca2+ and Gd3+ stimulated the GTPase activity of chicken brain tubulin 13- and 26 fold, respectively. Mg2+, Tb3+, and Na+ had no effect. This GTPase activity showed a saturation behavior with Ca2+ and Gd3+ with a maximal activity of 0.26 +/- 0.026 and 1.15 +/- 0.78 nmol min-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 +/- 0.18 and 0.011 +/- 0.007 mM, respectively. In the presence of Ca2+, the GTPase activity was proportional to tubulin concentration in the range 0.9-31.8 microM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca2+ were 0.71 +/- 0.1 and 0.049 +/- 0.043 mM, respectively. The similarity of the Ca2+ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not directly involved in the mechanism of inhibition of Ca2+ -dependent tubulin assembly. This inhibition could be better explained by the formation of a nonfunctional conformational state of tubulin induced by Ca2+ that is responsible for the GTPase activity. Quenching of the intrinsic fluorescence of tryptophan induced by Ca2+ showed an apparent dissociation constant of 0.14 +/- 0.005 mM, in the range of values determined through tubulin polymerization inhibition or through the induction of GTPase activity by Ca2+. Acrylamide-induced quenching of the intrinsic fluorescence showed values of the Stern-Volmer constants of 5.4 +/- 0.12 and 5.0 +/- 0.15 M-1 in the absence and presence of Ca2+, respectively. These results support the hypothesis that the inhibition of tubulin polymerization and the induction of the GTPase activity by Ca2+ is mediated by a conformational change. Ca2+ failed to induce depolymerization of GDP-AIF4 microtubules; this could be explained by a model in which Ca-tubulin is unable to assemble into microtubules and the rate of dissociation of GDP-Pi-tubulin from the microtubule ends is extremely slow compared with the rate of GDP-subunit dissociation, supporting the concept that the GTP- and GDP-Pi-tubulin cap at the ends of microtubules regulates their dynamic instability. PMID- 8639577 TI - Quinacrine noncompetitive inhibitor binding site localized on the Torpedo acetylcholine receptor in the open state. AB - Open-channel blockers of the nicotinic acetylcholine receptor (nAcChR) are widely thought to act sterically by entering and "plugging" the open channel of the nAcChR. However, quinacrine, a fluorescent open-channel blocker, has been recently shown to bind to the nAcChR at a site near the lipid bilayer while the receptor is in a closed, desensitized state, suggesting that at least one open channel blocker might act allosterically outside the channel [Valenzuela et al. (1992) J. Biol. Chem. 267, 8238]. To determine whether or not quinacrine also binds near the lipid bilayer when the receptor is in an open state, a short-range lipophilic quencher (5-doxylstearate, 5-SA) was used to assess the proximity of the nAcChR-bound quinacrine to the lipid bilayer while the receptor was transiently open by an agonist. Initial experiments using a stopped-flow instrument established the conditions required to monitor a portion of the changes in quinacrine fluorescence associated with its binding to the receptor in the open state. 5-SA (80 microM) reduced the amplitude of the rapid agonist induced change in quinacrine emission to 44% +/- 12% of the control value, indicating that the quinacrine was binding to a site proximal to the membrane partitioned 5-SA. Control experiments established that 5-SA had no effect on the ability of the receptor to undergo agonist-induced conformational changes, suggesting that little, if any, 5-SA distributed into the channel lumen and perturbed the functional activity of the receptor. Together, the results indicate that quinacrine binds to a site on the open receptor that is in contact with the lipid bilayer and not in the channel lumen. PMID- 8639579 TI - New insights into the catalytic cycle of flavocytochrome b2. AB - Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b2 heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1. Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [Ki = 30 mM; Lederer, F. (1978) Eur. J. Biochem, 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state. PMID- 8639580 TI - Interaction of cytochrome c with flavocytochrome b2. AB - Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1. Inhibition of the pre steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408 422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6345-6350]. PMID- 8639581 TI - Kinetic isotope effects as a probe of the beta-elimination reaction catalyzed by O-acetylserine sulfhydrylase. AB - Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction. PMID- 8639583 TI - Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. AB - The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays. Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. PMID- 8639582 TI - Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii. AB - We have expressed catalytically active Toxoplasma gondii dihydrofolate thymidylate synthase (DHFR-TS) and the individual TS and DHFR domains in Escherichia coli using the T7 promoter of pET-15b. DHFR-TS constituted approximately 10% of the total soluble cell protein and was purified using methotrexate-Sepharose chromatography to yield 10 mg of homogeneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to recover soluble, active enzyme. The TS domain was overexpressed as a soluble protein by growing the cells at 24 degrees C; this is the first report of the expression of an active TS domain from a bifunctional enzyme. The kcat and K(m) values for DHFR-TS are similar to those of other previously characterized protozoan DHFRs and TSs. The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional protein in accord with their effects in crude enzyme preparations and in vivo systems. Kinetic parameters and Ki values for TMP and Pyr with the isolated DHFR domain were identical to the values for DHFR in the bifunctional enzyme. Evidence of kinetic channeling of the dihydrofolate product of TS to the DHFR domain in the bifunctional enzyme was obtained by kinetic and inhibition studies. Properties such as yield, stability, and activities of the recombinant T. gondii DHFR-TS provide clear advantages over other bifunctional DHFR-TSs as a model for future studies. PMID- 8639585 TI - Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions. AB - By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g parallel = 2.0565 and g perpendicular = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (017-017)A perpendicular = 63 G and (017-016)A perpendicular = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket. PMID- 8639584 TI - The carbohydrate-recognition domain of E-selectin is sufficient for ligand binding under both static and flow conditions. AB - Selectins are a family of adhesion molecules with a well-defined domain structure comprised of a lectin or carbohydrate-recognition domain (CRD), an epidermal growth factor (EGF)-like motif, and a variable number of consensus repeats (CRs). While it is clear from various lines of evidence that the CRD plays a pivotal role in selectin-ligand interactions, little is known about the role of the non lectin selectin domains. We expressed a series of soluble chimeric proteins with various domains switched between E- and L-selectin and measured binding of the resulting chimeras to sialyl Lewis(a) and sulfatide, two carbohydrate structures which are specific for the E- and L-CRDs, respectively. Both CRDs bind to their respective ligands with the same affinity regardless of the origin of the other domains they are attached to. The domain-switched chimeras were assayed for their ability to support static binding and rolling of various cell lines which bind specifically to E-selectin. In these assays, the E-CRD was indispensable for both static binding and rolling under physiological flow conditions. The E-CRD alone, when substituted into L-selectin, supported rolling without the requirement for additional ligand-recognition elements. We conclude that the EGF domain or the CRs of E- and L-selectin have no influence on the CRD's specificity to carbohydrates. Furthermore, at least in the case of E-selectin, they do not contribute to the specificity of binding to cell surface ligands. PMID- 8639586 TI - Conformation of a beta-adrenoceptor-derived signal transducing peptide as inferred by circular dichroism and 1H NMR spectroscopy. AB - The peptide T345-359 representing the fourth intracellular loop of the avian beta adrenoceptor has been shown to strongly inhibit receptor-mediated adenylate cyclase activity [Munch, G., Dees, C., Hekman, M., & Palm, D. (1991) Eur. J. Biochem. 198, 357-364]. Circular dichroism and two-dimensional 1H NMR techniques were used to investigate the three-dimensional structure of the peptide in trifluoroethanol, phospholipid micelles, and small unilamellar phospholipid vesicles. The prepared vesicles were tested for size distribution and stability by using electron microscopy, photon correlation spectroscopy, and 31P NMR spectroscopy. The peptide T345-359 adopted a predominantly alpha-helical conformation in either trifluoroethanol or phospholipid micelles and vesicles. No structural differences were found for the conformation of the peptide in the presence of phospholipid micelles or vesicles, respectively, using 2D 1H NMR techniques, suggesting a unique conformation of T345-359 when associated with model membranes. A computer-aided model of the micelle-associated peptide was derived. The relevance of the 3D structure of the intracellular loops of receptors to communicate with the G protein in the signal transduction cascade is discussed. PMID- 8639587 TI - Nonrandom distribution of the one-disulfide intermediates in the regeneration of ribonuclease A. AB - The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A. PMID- 8639588 TI - Stabilization of the veratryl alcohol cation radical by lignin peroxidase. AB - Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of veratryl alcohol (VA) to veratryl aldehyde, with the enzyme-bound veratryl alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860 16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation. PMID- 8639589 TI - Domain closure in adenylate kinase. AB - The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK). A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively. The latter was subsequently labeled with either 5- or 4 acetamidosalicylic acid (the acceptor). From the comparative analysis of AK crystal structures [Schulz, G.E., Muller, C.W., & Diederichs, K. (1990) J. Mol. Biol. 213, 627-630] (apo-AK,AK.AMP complex and AK.AP5A [P1,P5-di(adenosine-5') pentaphosphate] complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure. The domain closure reduces interdomain distances in a two-step manner. Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E. coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex. Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex. The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively. Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A. The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E. coli AK in solution. PMID- 8639590 TI - Mechanism of proton transfer in the isomerization of 5-androstene-3, 17-dione by 3-oxo-delta 5-steroid isomerase and its D38E mutant. AB - The stereochemistry of proton transfer in the isomerization of [4 beta-2H]-5 androstene-3,17- dione (1d) to 4-androstene-3,17-dione (3) catalyzed by 3-oxo delta 5-steroid isomerase (KSI) has been reinvestigated. In H2O, approximately 65% of the label is retained in the product (3); of this, one-third is at C-4 and two-thirds at C-6 beta. When the same reaction is catalyzed by the D38E mutant of KSI, ca. 60% of the label is retained in the product, but almost all of it is at C-4. These reactions run in deuterium oxide result in 13% incorporation of a second deuterium with the wild type (WT) enzyme and 75% incorporation with the D38E mutant. When unlabeled 1 is isomerized in D2O, there is little incorporation of deuterium with WT (ca. 5 at. %) but substantial incorporation with D38E (130 at. %). These results are consistent with competitive abstraction of both the C-4 alpha and C-4 beta protons, as proposed by Viger et al. [(1981) J. Am. Chem. Soc. 103, 4151], and demonstrate that the KSI reaction is not completely stereospecific. A mechanism is proposed to account for these observations. PMID- 8639591 TI - Contributions of the ionizable amino acids to the stability of staphylococcal nuclease. AB - To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (delta GH2O) and sensitivity to changes in denaturant concentration [mGuHCl = d(delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (delta delta G) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge. PMID- 8639592 TI - Amino acid sequence and carbohydrate structure of a recombinant human tissue factor pathway inhibitor expressed in Chinese hamster ovary cells: one N-and two O-linked carbohydrate chains are located between Kunitz domains 2 and 3 and one N linked carbohydrate chain is in Kunitz domain 2. AB - Human tissue factor pathway inhibitor is a protease inhibitor with three tandem Kunitz-type inhibitory domains. The recombinant protein (r-hTFPI) was produced using Chinese hamster ovary cells, and its polypeptide and carbohydrate chain structures were analyzed. The complete amino acid sequence, composed of 276 residues, was determined using a protein sequencer after protease digestion and it was identical to that predicted from the cDNA sequence. Among three potential N-glycosylation sites, both Asn117 and Asn167 were fully N-glycosylated but Asn228 was not. Thr175 was also fully O-glycosylated, but Ser174 was partially O glycosylated. Carbohydrate composition and mass spectrometric analyses of the undecapeptide OG-11 (residues Leu 170approximately Leu180) showed that two O linked carbohydrate chains consisted of a type-1 core structure (Gal-GalNAc Ser/Thr) with 0-3 mol of N-acetylneuraminic acid(s). The N-linked carbohydrate chains were analyzed by two-dimensional carbohydrate mapping combined with sequential glycosidase digestion, after the reducing-ends of carbohydrate residues were tagged with 2-aminopyridine and non-reducing-end sialic acids were removed with sialidase. All the N-linked structures in r-hTFPI were complex-type carbohydrate chains with one fucose residue attached to the reducing-end GlcNAc and consisted of bi-, tri-, and tetraantennary carbohydrate chains in the ratio 1.9:1.3:1.0. Fucosylated tri- and tetraantennary carbohydrate chains with one or two N-acetyllactosaminyl repeats were also found (30% of carbohydrate chains determined). Thus, the region between Kunitz domains 2 and 3 encoded by exon 7 was highly glycosylated by two O-linked carbohydrate chains at Ser174 and Thr175 and one N-linked carbohydrate chain at Asn167. These results indicated that the region is occupied by a cluster of three bulky and acidic carbohydrate chains. PMID- 8639593 TI - Paraferritin: a protein complex with ferrireductase activity is associated with iron absorption in rats. AB - Recent studies reported that iron salts were absorbed in the duodenum utilizing a pathway involving membrane-associated integrin and a cytosolic protein named mobilferrin. In addition, a large molecular weight cytoplasmic complex was labeled with radioiron during mucosal uptake of iron in the duodenum. The molecular mass of this protein was 520 000 daltons, slightly larger than ferritin. On denaturing SDS-PAGE, the purified protein complex appeared to consist of at least four polypeptides, closely associated with each other. This complex was called paraferritin because its hydrodynamic volume resembled ferritin. In the present work, antibody studies demonstrate the presence of integrin, mobilferrin, and flavin monooxygenase in the water-soluble complex. Biochemical studies demonstrate the presence of a NADPH-dependent flavin monooxygenase ferrireductase activity that reduces Fe(III) to Fe(II). Antibodies against either integrin or mobilferrin inhibit monooxygenase activity. Inhibition of monooxygenase activity decreases radioiron uptake by tissue culture intestinal cells. Thus, we postulated that paraferritin plays a role in the mucosal uptake and transport of inorganic iron in small intestinal absorptive cells and is a mechanism for both the internalization of integrin from membranes to cellular cytosol and the delivery of iron to cellular constituents in an appropriate redox state. PMID- 8639594 TI - The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. AB - Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH dependent tertiary structural changes proximal to these chromophores. The pH dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well defined tertiary structure. PMID- 8639595 TI - Importance of specific adenosine N3-nitrogens for efficient cleavage by a hammerhead ribozyme. AB - Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazzaadenosine (c3A) base analogue for residues A6, A9, A13, A14, or A15.1. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/K(M), for the c3A6, c3A9, and c3A14 complexes were only marginally reduced (< or = 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c3A13 and c315.1 complexes were reduced by 9 fold and 55-fold, respectively. these reductions in cleavage efficiency are primarily a result of lower kcat values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N3-nitrogen of the A13 residue and particularly the A15.1 residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of these critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization. PMID- 8639596 TI - RNA conformation in the Tat-TAR complex determined by site-specific photo-cross linking. AB - Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. We have used a site-specific cross-linking method based on 4-thiouracil (4 thioU) photochemistry to determine the conformation of TAR RNA and its interaction with Tat protein under physiological conditions. Three different TAR RNA constructs with a single 4-thioU residue at position 23, 38, or 40 were synthesized. Upon UV irradiation, 4-thioU at all three positions formed interstrand covalent cross-links in TAR RNA. Determination of cross-link sites by RNA sequencing revealed that 4-thioU at position 23 makes a direct contact with U40, while a 4-thoU at position 40 cross-links to C24 and C25, and at position 38, 4-thioU contacts G26 in TAR RNA. The addition of arginine did not alter the yield or the site of RNA-RNA cross-link. However, in the presence of Tat(38-72), UV irradiation of RNA modified with 4-thioU at position 23 or 38 resulted in RNA- protein cross-links, but no RNA-RNA cross-links were observed. 4-thioU at position 40 formed both RNA-RNA and RNA-protein cross-links in the presence of Tat(38-72). An intriguing finding of our studies was that a cross-linked TAR RNA with 4-thioU at position 40 retained specific Tat-binding activity. Our results establish four important conclusions about Tat-TAR structure. (1) U23 of free TAR RNA is in close contact with U40. (2) U40 is in close proximity to C24 and C25 both in free TAR RNA and in a complex with Tat. (3) Tat protein directly contacts U23, U38, and U40 in the major groove of TAR RNA. (4) Tat protein can recognize a TAR RNA structure containing an interrupted bulge which is formed by a covalent link between U40 and two bulge residues, C24 and C25. These structural studies provide new insights into tertiary folding of TAR RNA and its interaction with Tat protein. PMID- 8639597 TI - Deletion of a B800-850 light-harvesting complex in Rhodospirillum molischianum DSM119 leads to "revertants" expressing a B800-820 complex: insights into pigment binding. AB - A B800-850 light-harvesting complex (also called LH2) deficient strain of Rhodospirillum molischianum was constructed by replacing a portion of the LH2 gene cluster by a kanamycin resistance gene cartridge. The LH2 deficient strain was characterized spectroscopically and by Southern blot analysis. Surprisingly, pseudorevertants were obtained which express a B800-820 complex which could not be observed in the wild type. This B800-820 complex was isolated and characterized. It consists of an alpha- and a beta subunit with 56 and 45 amino acid residues, respectively. The amino acid sequences of both subunits are extremely similar to those of the corresponding B800-850 complex. Resonance Raman spectroscopy shows that in the B800-820 complex the two 2-acetylcarbonyl groups of the bacteriochlo-rophyll a (BChl a) molecules absorbing at 820 nm are free from hydrogen bond interactions, whereas one of the two 2-acetylcarbonyl groups of the pair of BChl a molecules absorbing at 850 nm of the B800- 850 complex is involved in hydrogen bonds. These different protein- pigment interactions are due to the replacement of alpha Trp43 in the B800-850 complex by a Phe in the B800- 820 complex. Comparison of the amino acid sequences of the B800-850 and B800-820 complexes of Rs. molischianum and Rhodopseudomonas acidophila reveals a conserved motif comprised of three amino acid residues. Molecular modeling using the known LH2 structure of Rps. acidophila Ac 10050 indicates that this motif might be important for the precise structural arrangement of the native complex and fine tuning of its spectroscopic properties. PMID- 8639598 TI - Stopped-flow, classical, and dynamic light scattering analysis of matrix protein binding to nucleocapsids of vesicular stomatitis virus. AB - During the process of assembly of enveloped viruses, binding of the nucleoprotein core of the virus (nucleocapsid) to the host membrane is mediated by the viral matrix (M) protein. Light scattering properties of vesicular stomatitis virus (VSV) nucleocapsids and nucleocapsid-M protein (NCM) complexes assembled in vivo were determined following solubilizaton of the virion envelope with detergents at varying ionic strength to vary the extent of M protein binding. Three factors were found to contribute to the light scattering properties of VSV nucleocapsids: their conformation, extent of self-association, and amount of bound M protein. All three were affected by changes in ionic strength but could be distinguished by several parameters. Conformational changes in nucleocapsids and NCM complexes occurred rapidly (millisecond time scale) upon changing salt concentration and were reflected in changes in the angular dependence of light scattering intensity (i.e., changes in radius of gyration, RG). Changes in extent of self-association occurred relatively slowly (seconds to minutes time scale) and could be distinguished by the concentration dependence of the apparent molecular mass and diffusion coefficient of the NCM complex. Changes in M protein binding occurred on an intermediate time scale (t1/2 approximately one s) and reflected changes in both molecular mass and RG. The data presented here provide criteria for assessing binding of M protein to nucleocapsids under conditions of minimal perturbation of the NCM complex assembled in vivo and at low protein concentrations so that self-association of the NCM complex was minimal and reversible. PMID- 8639599 TI - Design challenges for hemoproteins: the solution structure of apocytochrome b5. AB - In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding. PMID- 8639600 TI - Engineered biosynthesis of novel polyketides: regiospecific methylation of an unnatural substrate by the tcmO O-methyltransferase. AB - TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6 methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates. PMID- 8639601 TI - NMR studies of the effects of the 5'-phosphate group on conformational properties of 5-methylaminomethyluridine found in the first position of the anticodon of Escherichia coli tRNA(Arg)4. AB - 5-Methylaminomethyluridine (mnm5U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA4Arg for codons AGA/AGG. In the present study, the temperature dependence of the ribose-puckering equilibrium of pmnm5U was analyzed by proton NMR spectroscopy. Thus, the enthalpy difference (delta H) between the C2'-endo and C3'-endo forms was obtained at 0.65 kcal.mol-1. By comparison of the delta H values of pU and pmnm5U, the 5-substitution was found to increase the relative stability of the C3'-endo form over the C2'-endo form significantly (by 0.56 kcal.mol-1). Furthermore, this conformational "rigidity" was concluded to depend on the 5'-phosphate group, because nucleoside U exhibits only a negligible change in the ribose-puckering equilibrium upon the 5 methylaminomethyl substitution. Further NMR analyses and molecular dynamics calculations revealed that interactions between the 5-methylaminomethyl and 5' phosphate groups of pmnm5U restrict the conformation about the glycosidic bond to a low anti form, enhancing steric repulsion between the 2-carbonyl and 2' hydroxyl groups in the C2'-endo form. This intrinsic conformational rigidity of the mnm5U residue in position 34 may contribute to the correct codon recognition. PMID- 8639602 TI - Structural features of a six-nucleotide RNA hairpin loop found in ribosomal RNA. AB - The hairpin loop GUAAUA occurs frequently in ribosomal RNA. Optical melting studies show that r(GGCGUAAUAGCC) folds into a hairpin containing this loop. The structural features of the r(GGCGUAAUAGCC) hairpin have been determined by NMR and molecular modeling. NOEs from G4-H1' to A9-H2 and from A9-H2 to G10-H1' show that G4 and A9 form a sheared base pair with two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3. One-dimensional NOE data show no NOEs between the imino protons of U5 and U8, but NOEs are observed between the U5-H1' and the U8-H6 and U8-H5, thus orienting the U8 imino proton away from U5. Thus U5 and U8 do not form an imino hydrogen-bonded U.U pair. The U5-H2' exhibits NOEs to both the A6 H8 and A7-H8, and the 3' phosphorus resonances of U5 and A6 are shifted downfield. This suggests that the helix turn is between the U5 and A6 nucleotides. The JH1'-H2 and JH3'-H4' coupling constants indicate that the loop is dynamic, particularly at 35 degrees C, well below the melting temperature of 63 degrees C. Structures were generated using 75 distance and 46 dihedral angle restraints. In these structures, the U5 base is stacked on the sheared base pair formed by G4 and A9 and can initiate a uridine turn similar to that observed in the anticodon loop of tRNA. The A6, A7, and U8 bases can stack on one another with their hydrogen-bonding surfaces exposed to the solvent, suggesting that they are available for tertiary interactions or protein recognition in rRNA. A range of loop structures are consistent with the data, however. The lack of formation of a U.U mismatch is consistent with a recent model that predicts the stability of hairpin loops of six nucleotides on the basis of the closing base pair and first mismatch in the loop [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289-14296]. PMID- 8639603 TI - Roles of the propeptide and metal ions in the folding and stability of the catalytic domain of stromelysin (matrix metalloproteinase 3). AB - Matrix metalloproteinases (MMPs) are an unique class of zinc metalloproteases in that 12 A from the catalytic zinc site is a second zinc site, the function of which has yet to be determined. In the pro form, the protease is inactive. Here we show that the heat-induced autocatalytic activation of pro to mature MMP3 is bimolecular. Further, the process is modulated by a low-affinity zinc. A mechanism is proposed by which the second zinc site may act as an enzymatic activator for the mature protease. A method for preparing completely metal-free protein is described. Surprisingly, there is a much more dramatic structural change between the apo and holo forms of the mature protein than there is between apo and holo proprotein. Apo mature MMP3 appears to form a native-like stable intermediate structure in which one or more of the tryptophan side chains is more solvent-exposed than in the holo form. Apo MMP3 is remarkably stable to thermal unfolding as monitored by CD; thus the metal ions do not appear to significantly stabilize the secondary structure of the catalytic domain. The apo mature MMP3 intermediate can be unfolded with heat, subsequently refolded, and reactivated by addition of zinc and calcium. Thus for MMP3, unlike subtilisin or alpha-lytic protease, the propeptide is not required for protein folding in a timely fashion and the role of intramolecular chaperone is not a universal one for the propeptides of proteases. PMID- 8639604 TI - A tRNA identity switch mediated by the binding interaction between a tRNA anticodon and the accessory domain of a class II aminoacyl-tRNA synthetase. AB - Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl , glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity. PMID- 8639605 TI - Heteronuclear (1H, 13C, 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer. AB - A full high-resolution three-dimensional solution structure of the monocyte chemoattractant protein-1 (MCP-1 or MCAF) homodimer has been determined by heteronuclear multidimensional NMR. MCP-1 is a member of a family of small proteins which play a crucial role in immune surveillance by orchestrating the recruitment of specific leukocytes to areas of immune challenge. The protein was uniformly isotopically enriched with 13C and 15N by expression in Escherichia coli, and complete sequence-specific resonance assignments were obtained by a combination of heteronuclear double- and triple-resonance experiments. The secondary structure was deduced from characteristic patterns of NOEs, 13 C alpha/beta chemical shifts, measurements of 3JHNH alpha scalar couplings, and patterns of slowly exchanging amide protons. Because MCP-1 forms symmetrical homodimers, additional experiments were carried out to unambiguously establish the quaternary contacts. NOEs from these novel experiments were merged with more traditional heteronuclear separated NOE measurements in an iterative strategy to partition the restraints between explicit inter/intrasubunit contacts and a class wherein both were retained as ambiguous. With more than 30 restraints per residue, the three-dimensional structure is well-defined with a backbone rmsd of 0.37 A to the mean over residues 5-69 of the dimer. We compare the structure with those recently reported for the related chemokines MIP-1 beta and RANTES and highlight the differences in terms of receptor specificity and function as well as interpret the known biological activity data of MCP-1 mutants. PMID- 8639606 TI - Protein kinase A-catalyzed phosphorylation and its effect on conformation in phytochrome A. AB - Phytochromes are ubiquitous red/far-red wavelength-sensitive photoreceptors in plants. Oat phytochrome A is a phosphoprotein. Phytochrome A (phyA) possesses two spatially different sites for phosphorylation with cAMP-dependent protein kinase (PKA) [McMichael & Lagarias (1990) Biochemistry 29, 3872-3878]. To assess the modulation of protein conformation by phosphorylation/dephosphorylation and its possible implication in phytochrome-mediated signal transduction, the conformations of phytochrome have been probed by PKA catalyzed phosphorylation. The phosphorylated species were purified and analyzed, along with untreated phytochrome, by limited proteolysis, circular dichroism (CD) and fluorescence quenching measurements. No significant changes in secondary structure of the phyA molecule after its phosphorylation were observed by CD. However, a subtle topographic and/or electrostatic effect of the phytochrome phosphorylation was detected by the time-resolved fluorescence quenching of Trp residues with Cs+ ions. N-Terminal phosphorylation at Ser17 was unique to the Pr form, but both Pr and Pfr phytochromes were phosphorylated at the hinge region to some extent. Phosphorylation at the hinge region resulted in noticeable changes in the proteolytic patterns, inhibiting cleavage near the phosphorylation site and favoring tryptic digestion of the Lys536-Asn537 peptide bond. Phosphorylation at the N-terminus did not cause observable changes in the helical structure of this region, but had an inhibitory effect on proteinase V8 accessibility at a site near the chromophore attachment. The functional relevance of protein phosphorylation of phyA is also discussed. PMID- 8639607 TI - Autoxidation of ubiquinol-6 is independent of superoxide dismutase. AB - Ubiquinone (Q) is an essential, lipid soluble, redox component of the mitochondrial respiratory chain. Much evidence suggests that ubiquinol (QH2) functions as an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids. It has been proposed that superoxide dismutase (SOD) may protect QH2 form autoxidation by acting either directly as a superoxide-semiquinone oxidoreductase or indirectly by scavenging superoxide. In this study, such an interaction between QH2 and SOD was tested by monitoring the fluorescence of cis-parinaric acid (cPN) incorporated phosphatidylcholine (PC) liposomes. Q6H2 was found to prevent both fluorescence decay and generation of lipid peroxides (LOOH) when peroxidation was initiated by the lipid-soluble azo initiator DAMP, dimethyl 2,2'-azobis (2-methylpropionate), while Q6 or SOD alone had no inhibitory effect. Addition of either SOD or catalase to Q6H2-containing liposomes had little effect on the rate of peroxidation even when incubated in 100% O2. Hence, the autoxidation of QH2 is a competing reaction that reduces the effectiveness of QH2 as an antioxidant and was not slowed by either SOD or catalase. The in vivo interaction of SOD and QH2 was also tested by employing yeast mutant strains harboring deletions in either CuZnSOD and/or MnSOD. The sod mutant yeast strains contained the same percent Q6H2 per cell as wild-type cells. These results indicate that the autoxidation of QH2 is independent of SOD. PMID- 8639608 TI - Proton transport by halorhodopsin. AB - In halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently deprotonates after photoexcitation with light > 530 nm, like in the light-driven proton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin, pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surface as described earlier for halorhodopsin from Halobacterium salinarium. This azide dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellular direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH change. We conclude that in halorhodopsin an azide bound to the extracellular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp 85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although halorhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a donor, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin through replacement of Asp-85 with a threonine, that protein became a chloride ion pump. PMID- 8639609 TI - Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides. AB - The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner. PMID- 8639610 TI - Contributions of asparagine at alpha 97 to the cooperative oxygenation process of hemoglobin. AB - According to the X-ray crystallographic results from human deoxyhemoglobin, beta 99Asp at the alpha 1 Beta 2 interface forms hydrogen bonds with alpha 42Tyr and alpha 97Asn. To clarify the structural and functional roles of the hydrogen bond between alpha 97Asn and beta 99Asp, we have engineered a recombinant hemoglobin in which alpha 97Asn is replaced by Ala, and have investigated its oxygen-binding properties, and have used proton nuclear magnetic resonance spectroscopy to determine the structural consequences of the mutation. Recombinant Hb (alpha 97Asn-->Ala) shows a milder alteration of functional properties compared to the severely impaired beta 99 mutants of the human abnormal hemoglobins. The addition of inositol hexaphosphate, an allosteric effector, causes recovery of the functional properties of recombinant Hb (alpha 97 Asn-->Ala) almost to the level of human normal adult hemoglobin without this allosteric effector. r Hb (alpha 97 Asn-->Ala) shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 interface compared to those of human normal adult hemoglobin. The proton nuclear magnetic resonance spectrum of the deoxy form of this recombinant hemoglobin shows the existence of an altered hydrogen bond which is believed to be between alpha 42Tyr and beta 99Asp at the alpha 1 beta 2 interface. Thus, the present results suggest that the intersubunit hydrogen bond between alpha 97 Asn and beta 99Asp at the alpha 1 beta 2 interface is not as crucial as the one between alpha 42Tyr and beta 99Asp in the deoxy quaternary structure. Preliminary molecular dynamics simulations have been used to calculate the contributions of specific interactions of several amino acid residues in r Hb (alpha 97Asn-->Ala) to the free energy of cooperativity of this recombinant hemoglobin. The results of these calculations are consistent with the experimental results. PMID- 8639611 TI - A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system. AB - The fluorescence properties of six different single Trp mutants of the mannitol specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan minus mutant (Robillard et al., 1996). The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I-. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation). The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991). When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30. PMID- 8639612 TI - D38 is an essential part of the proton translocation pathway in bacteriorhodopsin. AB - At present, almost no knowledge exists about the functional relevance of the amino acid residues at the cytoplasmic (CP) surface of the light-driven proton pump bacteriorhodopsin (BR) although a prerequisite for efficient vectorial proton translocation is the efficient capture of protons from the alkaline cytoplasm of the cell. To identify residues involved in the proton transfer reaction steps in the CP part of BR, the aspartic and glutamic amino acids D36, D38, D102, D104, and E161 were replaced by cysteine and arginine (i.e., a negatively charged residue by a neutral or positive one at the pH of investigation). The effect of these replacements on the photo- and transport cycle was examined by time-resolved visible and infrared spectroscopy, biochemical modification studies, and activity assays in intact cells. Of the five CP amino acids studied, only the replacement of D38 resulted in severe alterations of the reaction steps in BR during the second half of the photocycle. Our data show that D38, which seemed to be a freely accessible CP surface residue lacking functional importance, is an essential part of the CP proton uptake pathway connecting the membrane surface with the Schiff base of BR, probably as the first amino acid residue at the CP entrance. D38 influences the late steps in the functional cycle, such as the occurrence of the intermediates N and O, the modulation of the hydrogen-network, the conformational changes in the protein moiety, and the deprotonation/reprotonation of D96. Opposed to this function, the surface-exposed amino acids D36, D102, D104, and E161 seem to efficiently collect protons from the aqueous bulk phase and funnel them to the entrance of the CP proton pathway. PMID- 8639613 TI - Photoaccumulation in photosystem I does produce a phylloquinone (A1.-) radical. AB - Previous work has challenged the assignment of a photoaccumulated EPR signal to the phylloquinone electron acceptor in photosystem I, A1.-. Biosynthetic deuteration of the phylloquinone in the cyanobacterium Anabaena variabilis has been shown to narrow this photoaccumulated signal, demonstrating that the signal arises from A1.-. The ESP signal attributed to P700.+A1.- is also narrowed by this deuteration, showing that the photoaccumulated EPR signal and the ESP signal are monitoring the same redox component. Confirmation that the photoaccumulated EPR signal comes from deuterated phylloquinone was obtained by exchanging the deuterated for protonated phylloquinone, which broadened the photoaccumulated EPR signal. PMID- 8639614 TI - ENDOR and special triple resonance spectroscopy of A1.- of photosystem 1. AB - The photoaccumulated radical state of the photosystem 1 secondary electron acceptor A1, A1.-, has been studied in spinach and the cyanobacterium Anabaena variabilis strain Met27 using electron nuclear double resonance (ENDOR) and electron--nuclear--nuclear special triple (ST) resonance spectroscopies. Spectra of A1.- in both these species are very similar. ENDOR spectra of the phylloquinone anion radical in solvent glass were also obtained. Comparison of the spectra of the in vivo and in vitro radicals shows that A1.- is a phylloquinone anion radical with a distorted electron spin density distribution. Hyperfine couplings to the A1.- methyl group and to two protons hydrogen bonded to the quinone oxygens have been identified using biosynthetic deuteration in A. variabilis. Possible hyperfine coupling to a methylene proton of the phytyl side chain of the quinone has also been identified. These results are compared with those from previous studies of protein-bound semiquinones in the light of the unusual redox potential of A1. PMID- 8639615 TI - Bile salt stimulated cholesterol esterase increases uptake of high density lipoprotein-associated cholesteryl esters by HepG2 cells. AB - Bile salt stimulated cholesterol esterase is predominantly synthesized in the pancreas. However, this enzyme is also synthesized by the liver and was found to be present in plasma. The physiologic role of the systemic cholesterol esterase has not been clearly defined. In the current study, the human hepatoma cell line HepG2 was used as a model to determine the role of cholesterol esterase on hepatic uptake of high density lipoprotein (HDL)-associated cholesteryl esters. The results showed that hepatic uptake of the cholesteryl esters analog [3H]cholesteryl ether on reconstituted HDL was inhibited by anti-cholesterol esterase antibodies. The HDL-associated cholesteryl ester transported to HepG2 cells was also increased 2-fold in the presence of taurocholate, an activator of the cholesterol esterase. These results suggest that liver-derived cholesterol esterase may play an important role in cellular uptake of cholesteryl esters from HDL. This hypothesis was supported by demonstrating the ability of exogenously added cholesterol esterase to further enhance hepatic uptake of HDL-associated cholesteryl esters. The results of the current study also showed that cholesterol esterase increased free-to-esterified cholesterol ratio in the lipoprotein. Thus, alteration of HDL structure and composition contributes to the cholesterol esterase-induced cellular uptake of HDL-associated cholesteryl esters. On the basis of these observations, we propose that liver-derived cholesterol esterase may play an important role in lipoprotein metabolism. PMID- 8639616 TI - Cholesterol redistribution within human platelet plasma membrane: evidence for a stimulus-dependent event. AB - The fluorescent analog NBD-phosphatidylethanolamine and the analogs of cholesterol NBD-cholesterol and cholestatrienol were used to study the distribution of these lipids within the plasma membrane bilayer of human platelets. The probes were incorporated into platelets using phosphatidylcholine donor vesicles. The distribution of NBD lipid and of cholestatrienol in the platelet plasma membrane bilayer was followed by quenching with dithionite and TNBS, respectively. The t1/2 of cholestatrienol incorporation into platelet membranes was 39 min, and approximately 65% of the probe was quenched by addition of TNBS. When platelets were exposed to collagen or to ADP, a portion of the probe became inaccessible to quenching. Within 2 min of stimulation by collagen (10 micrograms/mL), the percentage of cholestatrienol fluorescence quenched by TNBS decreased to 45%. The fluorescent probe was not found to be associated either with the intracellular membranes or in the extracellular media after collagen stimulation. Similar data were obtained with NBD-cholesterol, but the decrease in accessibility of this probe to quenching was considerably slower. The redistribution of endogenous membrane cholesterol was also measured using cholesterol oxidase. Exposure of platelets to collagen decreased the accessibility of endogenous membrane cholesterol to enzymatic oxidation with cholesterol oxidase. Taken together, the foregoing observations are consistent with the stimulus-dependent translocation of cholesterol out of the outer monolayer. Coincident with the redistribution of cholesterol is the reciprocal movement of NBD-phosphatidylethanolamine into the outer monolayer. In the presence of the chaotropic agents urea and guanidine HCl, the movement of cholestatrienol upon collagen stimulation was prevented, but the redistribution of NBD-phosphatidylethanolamine was still detected. We propose that cholesterol translocates to the inner platelet monolayer following collagen stimulation, but the possibility that the sterol moves laterally within the outer membrane monolayer cannot be rigorously excluded. PMID- 8639617 TI - Study by infrared spectroscopy of the interdigitation of C26:0 cerebroside sulfate into phosphatidylcholine bilayers. AB - The insertion mode of the long fatty acid chain of the asymmetric glycosphingolipid C26:0-cerebroside sulfate (C26-CBS) in symmetric matrices of phosphatidylcholines of different acyl chain length has been investigated by transmission and attenuated total reflectance (ATR) infrared spectroscopy. The concentration of C26-CBS in myelin is increased in the demyelinating disease adrenoleukodystrophy. The conformational order and the orientation of the chains of the asymmetric glycosphingolipid have been evaluated for C26-CBS incorporated at 8 mol % in perdeuterated dimyristoylphosphatidylcholine (DMPC-d54) and perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The results, for the gel phase, are consistent with interdigitation of the C26-CBS long acyl chain across the bilayer center of an all-trans-DMPC bilayer in which DMPC is less tilted than in the absence of CBS. In contrast, in DPPC the results suggest that although the CBS long chain interdigitates across the center of the bilayer, it does not change the tilt angle of the DPPC molecules in the gel phase. Furthermore, in DPPC, C26-CBS is less well oriented than the host DPPC molecules and it increases the gauche content of the DPPC acyl chains. The observation of the amide spectral region indicates that exposure of the sphingosine amide moiety to buffer is greater in the longer chain length DPPC bilayer than in the shorter chain length DMPC bilayer. The thermotropic behavior of the lipid mixtures of C26-CBS at 8 mol % in DMPC or DPPC shows that the glycosphingolipid stabilizes the gel phase of the short chain length bilayer while it destabilizes the long chain length one. Our results further demonstrate that, at this concentration, C26-CBS is completely miscible in DMPC and DPPC in the gel and the liquid crystalline phases. The difference in behavior of C26-CBS in DMPC and DPPC is a consequence of the greater mismatch between the C26 chain length and the bilayer thickness of DPPC relative to DMPC. They may help to understand the deleterious effects of glycosphingolipids with very long chain fatty acids in adrenoleukodystrophy. PMID- 8639618 TI - Influence exercised by histidine-95 on chloride transport and the photocycle in halorhodopsin. AB - The anion pumping mechanism of halorhodopsin was studied using site-directed mutagenesis. Comparison of the amino acid sequence revealed that the B-C interhelix loop segment was highly homologous in all known halorhodopsins. Especially a basic residue, histidine-95, was conserved in all halorhodopsins. Using the expression-vector plasmid carrying the bop promoter, two His-95 mutants (H95R, H95A) were successfully expressed in Halobacterium salinarium. The expression levels of these halorhodopsin mutants were slightly lower than that for the wild-type halorhodopsin. In addition, these mutants were unstable under illumination compared with the wild-type. It suggested that His-95 is probably important for stabilizing the structure of halorhodopsin. The absorption maxima of these mutants are approximately 15 nm blue-shifted compared with the wild type, suggesting that His-95 interacts with the retinal Schiff base. At low chloride concentrations, the light-induced chloride pumping activity of these mutants was more than 20 times lower than that for the wild-type. Only under physiological conditions, the chloride pumping activity was detected. Even at a high chloride concentration (1 M NaCl), the HR520 intermediate could not be detected for these mutants. These results clearly indicate that His-95 has a crucial role in the chloride transport of halorhodopsin. PMID- 8639619 TI - Asp76 is the Schiff base counterion and proton acceptor in the proton translocating form of sensory rhodopsin I. AB - Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin. PMID- 8639620 TI - Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: substitutions at the gateway position. AB - The ion binding parameters of the EF-hand Ca2+ binding motif are carefully tuned for different biological applications. The present study examines the contribution of the ninth position of the Ca2+-coordinating EF-loop to the tuning of Ca2+ affinity and selectivity, using the model EF-loop of the Escherichia coli galactose binding protein. Eight side chains, representing the entire set of side chains commonly observed in natural EF-loop sequences, are tested at the ninth position of the model EF-loop to determine their effects on equilibrium ion binding parameters. Using the spherical metal ions of groups Ia, IIa, and IIIa and the lanthanides as probes, both the Ca2+ affinities and ionic selectivities of the engineered sites are quantitated. Neutral side chains of different size at the ninth EF-loop position [Gln (wild type), Asn, Thr, Ser, Ala, Gly] are observed to yield similar Ca2+ affinities and retain the native ability to exclude the physiological competing metal cations Na+, K+, and Mg2+. Acidic gateway side chains (Glu, Asp) are found to reduce Ca2+ affinity and shift the ionic charge selectivity as much as 10(3)-fold toward trivalent cations. Relative to the native Gln, all engineered side chains cause a partial loss of ionic size selectivity, stemming from enhanced affinities for nonphysiological large ions. Overall, the results have implications for the molecular mechanisms used by the EF-loop to control both (i) charge selectivity, which is proposed to stem from the electrostatic repulsion between the coordinating oxygens, and (ii) size selectivity, which is theorized to involve complex interactions between multiple coordinating side chains. Finally, it has recently been shown that the ninth EF loop position serves as a "gateway" to modulate the kinetics of Tb3+ binding and release without shifting the equilibrium affinity of this ion [Drake, S. K., & Falke, J. J. (1996) Biochemistry 35, 1753-1760]. The present results confirm that isoelectric substitutions at the gateway position have little effect on Ca2+ affinity, thereby supporting the hypothesis that the gateway side chain provides kinetic tuning of Ca2+ signaling proteins independently of their Ca2+ activation thresholds. PMID- 8639621 TI - Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization. AB - We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin. The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein. The protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (Cys, His, Asn) characteristic for cysteine proteases of the papain superfamily. Human bleomycin hydrolase has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. The 52 kDa recombinant protein forms a hexamer of 310 kDa and acts strictly as an aminopeptidase with a broad substrate specificity. The lack of a leader sequence and its pH optimum at 7.2 suggest a cytosolic/nuclear localization. Human bleomycin hydrolase was detected at low to moderate expression levels in most of the human organs tested. Significantly higher RNA levels have been observed in a variety of tumor cell lines. The human enzyme effectively degrades both forms of bleomycin (A2 and B2) in vitro and could indeed be responsible for the resistance of various tumors to this widely used anticancer drug. PMID- 8639622 TI - The role of Glu 57 in the mechanism of the Escherichia coli MutT enzyme by mutagenesis and heteronuclear NMR. AB - The role of the conserved residue Glu-57 in the mechanism of the MutT enzyme from Escherichia coli was investigated by mutagenesis and heteronuclear NMR methods. The enzymatic activity of the E57Q mutant is at least 10(5)-fold lower than that of the wild type enzyme. The solution structure of the E57Q mutant, based on comparisons of 1H-15N NOESY HSQC spectra and 1H-15N HSQC spectra to those of the wild type enzyme, differs in a region near Glu-57. The dissociation constants (KD) of the E-Mg2+ and E-Mn2+ complexes increased 3.3- and 3.6-fold, respectively, in the E57Q mutant, while the KD of E-dGTP is unaltered from that of the wild type enzyme. The enhanced paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons is halved in the E57Q mutant indicating an altered metal binding site. 1H-15N HSQC titrations of E57Q with MnCl2 show selective attenuation of the side chain NH signals of Gln-57 and the backbone NH signals of Gly-37, Gly-38, Lys-39, Glu-53, Glu-56, Gln-57, and Glu-98, indicating proximity of bound Nm2+ to these residues. The same resonances of the wild type and the E57Q mutant enzymes are attenuated by Mn2+, but significantly smaller paramagnetic effects (relative to the largest effect on Lys-39) are found on Gly 37, Gly-38, Val-58, and Glu-98 of the mutant, indicating an altered position of the bound divalent cation. Thus Glu-57 is probably a ligand to the enzyme-bound metal, and the profound loss of catalytic activity in the E57Q mutant results from structural and electronic changes at the site of the enzyme-bound divalent cation. 1H-15N HSQC titrations of the wild type enzyme with MgCl2 show changes in chemical shifts of 15N and NH resonances in regions closely overlapping those induced by the E57Q mutation itself, suggesting that the loss of the negative charge at Glu-57, either by mutation or by neutralization with Mg2+, induces a similar effect. In the E57Q mutant, the slow exchange of the side chain NH2 protons of Gln-57 and NOE's from the NH2 protons of Gln-57 to the beta and gamma protons of Glu-98 suggests hydrogen bonding of Gln-57 to Glu-98 in the free enzyme. 1H-15N HSQC titrations of both the wild type and mutant enzymes with dGTP show changes in 15N and NH chemicals shifts of residues in a cleft formed by beta strands A, C, and D on one side and loop I, the end of loop IV, and the beginning of helix II on the other side, suggesting this cleft to be the nucleotide binding site. These changes in chemical shift were smaller or absent in titrations of the wild type or mutant enzymes with AMPCPP or Mg2+-AMPCPP, in accord with the strong preference of the MutT enzyme for guanine over adenine nucleotide substrates. PMID- 8639623 TI - Mutational analysis of the oligosaccharide recognition site at the active site of Escherichia coli maltodextrin phosphorylase. AB - A mutagenesis approach was applied to identify specific amino acid residues that are tentatively involved in binding of the oligosaccharide substrate at the active site of Escherichia coli maltodextrin phosphorylase. From ten residues located within a proposed channel connecting the enzyme surface with the active site, nine displayed significant effects on the reaction with oligosaccharide substrates when exchanged by mutagenesis. While several mutant enzymes (N258A/D259A/N260A, N307A, E350A, and Y578F) exhibited moderate decreases in apparent binding (about 4-17-fold), two mutations, H536L and E67A, weakened apparent binding of oligosaccharide substrates by 2 orders of magnitude. Two further mutant enzymes (T346G and H310A) displayed a 10-fold increase in the apparent Km of the oligosaccharide in the degradation reaction, while binding in the synthesis direction seemed less affected, indicating partially differential binding modes of oligosaccharides in synthesis and degradation. Quite uniquely, the H310A mutant enzyme exhibits a more than 100-fold-lowered Ki for gluconolactone, indicating the existence of an inhibitor binding site similar to that expected for a carbonium ion-like transition state. This is further confirmed by the finding that glucose, which does not inhibit wild-type enzyme, became an inhibitor of the H310A mutant enzyme (Ki = 20 mM). Since mutation of D308 did reduce kcat about 10-100-fold while Km values remained unchanged, a participation of this residue in transition state binding is probable. The insight into substrate recognition derived from this mutagenesis study corroborates a binding model where maltopentaose fits into the phosphorylase b structure in a distorted form. PMID- 8639624 TI - Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues. AB - Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin. They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system. There are at least three mammalian isozymes which are tissue specific and developmentally regulated. To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (> 35%), and contain no disulfide bonds. Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C. These transitions are characterized by thermodynamic parameters typical of single domain globular proteins. Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (Kd = 0.5 microM), and the results are similar to the kinetically determined Km for ubiquitin ethyl ester (o.6 microM). The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme. PMID- 8639625 TI - Ligand effects on the fluorescence properties of tyrosine-9 in alpha 1-1 glutathione S-transferase. AB - A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H site, Tyr-9 is fully protonated, with little charge-transfer character. PMID- 8639627 TI - Low-temperature thermochromism marks a change in coordination for the metal ion in manganese superoxide dismutase. AB - We have observed thermochromism (temperature-dependent absorption) for anion complexes of manganese superoxide dismutase indicating a change in coordination number for the metal complex at low temperatures. The ligand field spectra for the Mn(III) ion, characteristic of five-coordination for the azide complex at 295 K, cleanly convert to spectra reflecting six-coordination at low temperature, with a midpoint for the transition near 200 K. The active site structure is temperature-dependent, a relatively rigid, distorted octahedral low-temperature Mn complex melting with dehydration (or displacement of one of the protein ligands) to form a five-coordinated complex under physiological conditions. Thermodynamic parameters for the transition estimated from van't Hoff analysis (delta HvH = 5 kcal/mol; delta SvH = 22 cal/mol K) are consistent with reduced chemical binding and increased fluxionality at room temperature. This thermochromism of MnSD demonstrates the existence of distinct isomeric forms of the active site metal complex, whose relative stability depends on the degree of vibrational excitation. The marginal destabilization of the six-coordinate anion complex under physiological conditions suggests that the enzyme may thermally control the stability of intermediates in a dissociative displacement mechanism for substrate binding and redox. PMID- 8639626 TI - Analysis of the substrate-recognition mode of aromatic amino acid aminotransferase by combined use of quasisubstrates and site-directed mutagenesis: systematic hydroxy-group addition/deletion studies to probe the enzyme-substrate interactions. AB - Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids. Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question. This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and [Tyr70-->Phe] mutant enzymes. Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate. When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates. From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms. This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta carboxylate group of substrates. The [Arg292-->Ala] or [Arg292-->Leu] mutation increased the Km values for aromatic amino acids 5-10 fold, and the [Arg292- >Lys] mutation increased these values 10-100-fold, without affecting the kcat values. This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT. PMID- 8639628 TI - Role of the prodomain in folding and secretion of rat pancreatic carboxypeptidase A1. AB - Pancreatic carboxypeptidase A1 (CPA1) is synthesized as an inactive precursor, proCPA1, which is processed to the active enzyme by the proteolytic removal of the 95-amino acid N-terminal prodomain. Purified rat proCPA1 is renatured in vitro after denaturation in guanidine or in guanidine plus reducing agents. In contrast, purified CPA1 is not renatured under any of the conditions tested. While proCPA1 is secreted in yeast when fused to the alpha-factor signal sequence in place of its endogenous signal sequence, mature CPA1 is not secreted and is trapped and degraded intracellularly. Thus, in addition to maintaining CPA1 in the inactive state, the prodomain promotes folding and secretion of the proenzyme. Neither of these functions can be restored by supplying the prodomain to CPA1 in trans. The three-dimensional structure of porcine proCPA reveals a number of extensive contacts made between the prodomain and the enzyme active site which account for its inhibitory properties [Guasch et al. (1992) J. Mol. Biol. 224, 141-157]. Among these contacts are salt bridges formed between Arg-71 and Asp-A36 and between Arg-124 and Asp-A89. Mutation of any of these four residues inhibits secretion of proCPA1 from yeast and results in its intracellular degradation. The effect of the mutations on secretion suggests that interactions which stabilize the binding of prodomain to the native enzyme active site may also be important for the successful folding of proCPA1. PMID- 8639629 TI - Initial rate and equilibrium isotope exchange studies on the ATP-dependent activity of polyphosphate Glucokinase from Propionibacterium shermanii. AB - Polyphosphate glucokinase [EC 2.7.1.63] catalyzes the phosphorylation of glucose using either inorganic polyphosphate [poly(P)] or ATP as the phosphoryl donor. Both activities purified from Propionibacterium shermanii are the functional properties of a single enzyme with separate binding sites for the two phosphoryl donor substrates. The enzyme was found to utilize poly(P) much more efficiently than it does ATP, with a kcat/Kpoly(P) to kcat/KATP ratio of 2800. The catalytic constant for poly(P) is about 2-fold higher than for ATP. Other nucleotides like GTP and dATP also served as substrates with good efficiencies. The ATP-dependent reaction was analyzed using steady-state kinetics and isotopic exchange kinetics at chemical equilibrium. Intersecting initial velocity patterns for both glucose and ATP indicate sequential addition of substrates. Product inhibition studies resulted in two competitive and two noncompetitive patterns, which is characteristic of a Theorell-Chance mechanism or a random mechanism with two dead end complexes. Results of isotope exchange experiments, however, rule out a Theorell-Chance mechanism, as well as a truly random mechanism. They are not consistent with a partially random mechanism (although a kinetically compulsory order of substrate binding is not excluded), where glucose is preferentially bound to free enzyme before ATP, and ADP is preferentially released as the first product, followed by glucose 6-phosphate. Dead-end inhibition analysis confirms this order of substrate binding. Competitive inhibition of ADP vs ATP is explained as resulting primarily from binding as a dead-end inhibitor (E.Glc.ADP) and not as a product. Another weaker abortive complex, E.ATP.G6P, is also formed. The chemical transformation or the release of ADP is the rate-limiting step in ATP utilization. PMID- 8639630 TI - Importance of two buried salt bridges in the stability and folding pathway of barnase. AB - The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition rate for unfolding, and a folding intermediate has been analyzed by protein engineering, kinetic, and thermodynamic studies. The aspartate residues in the bridges Arg69-Asp93 and Arg83-Asp75 were replaced by the isosteric analogue asparagine, while various replacements were probed for the positively charged arginine partners. The mutations are very destabilizing, lowering stability by up to 5.4 kcal/mol. A value of 3.0-3.5 kcal/mol was derived for the coupling energy between Arg and Asp from a double mutant cycle analysis. Despite the radical nature of these mutations, they do not appear to alter the pathway of folding. The interaction between Arg69 and Asp93, located in a relatively conserved region among ribonucleases, is predominantly formed in the major transition state along the folding pathway, as found previously from an analysis of more benign mutations; the value of phi(F) for all mutations at positions 69 and 93 are 0.8-0.9 in the major transition state for folding where phi(F) = 0 = fully unfolded and phi(F) = 1 = fully folded interaction energies). In contrast, the interaction between Arg83 and Asp75 in the active site of barnase is formed only in the native state of the protein. The analysis of folding pathways and the structure of folding intermediates by making kinetic and thermodynamic measurements on mutants appears even more robust than expected. PMID- 8639631 TI - New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase. AB - We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase. The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue. This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation. The semiburied salt bridge studied here is between Asp93 and Arg69. Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69. The Asp93 Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions. The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway. This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable. The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways. PMID- 8639632 TI - Interaction of vinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine. AB - Vinca alkaloids are antimitotic drugs that inhibit microtubule assembly and induce tubulin self-association into coiled spiral aggregates. Previous sedimentation velocity results with vinblastine have been interpreted by a mechanism involving isodesmic ligand-mediated or ligand-mediated plus ligand facilitated self-association [Lobert et al. (1995) Biochemistry 34, 8050-8060]. In this study, we compare the vincristine- or vinorelbine-induced self association of porcine brain tubulin with our prior vinblastine studies in the presence of 50 microM GDP or 50 microM GTP. Vincristine demonstrates the highest overall affinity for tubulin, K1K2, and vinorelbine the lowest (vincristine > vinblastine > vinorelbine). These and the first quantitative studies comparing the interaction of a new vinca alkaloid derivative, vinorelbine (Navelbine), with other vinca alkaloids. The relative binding affinities reported here correlate with the weekly drug doses used clinically in cancer chemotherapy, where vincristine is used at the lowest dosages and vinorelbine at the highest. Surprisingly, K1, the affinity of drug for tubulin heterodimers, is identical for all three drugs. When data are fit with the ligand-mediated model, the differences in overall affinity are due to effects on K2, the affinity of liganded heterodimers for spiral polymers. When data are fit with the ligand mediated plus-facilitated model, affinity differences are also reflected in K3, the binding of the drug to unliganded polymers. We find that GDP enhances self association in the presence of all three drugs 3-5-fold over GTP. The enhancement is manifested in K2 and K3 and amounts to an average of 0.90 +/= 0.17 kcal/mol. Thus, nucleotide enhancement is linked to the self-association step. Data collected at 5, 25, and 36 degrees C for all three drugs show increased maximum s 20,w values with increasing temperature and are consistent with an entropically driven reaction for the overall process. To investigate these results further, stopped-flow light scattering experiments have been conducted. Relaxation times are longest for the largest vincristine polymers and shortest for the smallest vinorelbine polymers, consistent with a cascade of events corresponding to successive dissociation events from spiral polymers, the larger the polymers the longer the relaxation time. Relaxation times for any single drug decrease with increasing tubulin concentration, consistent with the occurrence of oligomer annealing in addition to the association of liganded heterodimers to the ends of the growing spirals. Relaxation times were used to estimate on and off rates for liganded heterodimer association with spirals, and their ratio gives affinity constants (Kapp) that are independently consistent with K2 estimates from sedimentation velocity results for vinblastine and vinorelbine. For vincristine induced tubulin polymers, a two-step process is observed with a second relaxation time more than 20-fold longer than times observed for vinblastine or vinorelbine. Sedimentation velocity experiments at low speeds and electron microscopy are consistent with the presence of a small amount of larger polymers (> or = 40S) in the vincristine samples, possibly involving alignment of spirals. Under our experimental conditions, these larger polymers appear to have a minimal effect on the estimated energetics of the vincristine-induced self-association of tubulin. PMID- 8639633 TI - Variable conformation and dynamics of calmodulin complexed with peptides derived from the autoinhibitory domains of target proteins. AB - Calcium-saturated calmodulin (CaM) can bind and activate many target proteins through the direct association with the respective autoinhibitory domains. The CaM binding sequences within the autoinhibitory domains of these proteins have little sequence homology, and the mechanisms associated with CaM's ability to recognize and productively bind with these variable sequences is unclear. Common structural features of CaM bound to five peptides that are homologous to the autoinhibitory domains of smooth muscle myosin light chain kinase, CaM-dependent protein kinase II alpha, the plasma membrane Ca-ATPase, a MARCKS homolog, and glycogen phosphorylase kinase were assessed using frequency-domain fluorescence spectroscopy. In addition, the structural features of CaM complexed with the peptide melittin was also considered. We observe similar decreases in the average fluorescence lifetime and similar increases in the solvent accessibility of N-(1 pyrenyl)maleimide (PM) bound at Cys27 in calcium binding loop I in the amino terminal domain of CaM upon association with all six target peptides. Likewise, using fluorescence resonance energy transfer to measure the spatial separation between the opposing globular domains in CaM, we observe a similar spatial separation between the opposing globular domains of CaM bound to all six peptides. This indicates that CaM undergoes comparable structural changes upon association with all six target peptides. However, there are significant differences in the observed lifetime, solvent accessibility, correlation time associated with the segmented rotational motion of PM-CaM, and in the spatial separation between the opposing globular domains in CaM upon association with the individual target peptides, which indicates that CaM adopts a different tertiary structure that is dependent on the structural features of the bound target peptide. The correlation times associated with the overall hydrodynamic properties of CaM complexed with all six peptides are nearly identical (phi 2 approximately 10.6 +/- 0.4 ns) and are consistent with the known dimensions of CaM complexed to a peptide homologous to the CaM binding sequence of CaM dependent protein kinase II alpha. Therefore, while these results are consistent with a common binding mechanism between CaM and all six target peptides, they indicate that the binding domains of CaM adopt different tertiary structures that allow them to bind with the variable sequences found in the autoinhibitory domains of target proteins with high affinity. PMID- 8639634 TI - Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes. AB - Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K+ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of alpha-helical structures in membrane mimetic environments, the alpha-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10(4) and 10(5) M-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results are in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization. PMID- 8639635 TI - Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family. AB - Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins. PMID- 8639636 TI - Heparinase I from Flavobacterium heparinum. Identification of a critical histidine residue essential for catalysis as probed by chemical modification and site-directed mutagenesis. AB - We recently identified cysteine-135 as an important amino acid for heparinase I (EC 4.2.2.7) activity. In this study, we have identified a second residue critical for enzymatic activity. We observe concentration-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 microM DEPC results in a 85% loss of heparinase I activity in 15 min. Heparinase I activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase I by DEPC is specific for histidine residues. Chemical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and [14C]DEPC indicates that between one and two histidine residues are modified. Chemical modification of the surface-accessible histidines, in the presence and absence of heparin, suggests that the histidine(s) lie(s) in or near the active site of heparinase I. The wild-type heparinase I has four histidine residues; site-directed mutagenesis of H129A, H165A, and H339A did not affect enzyme activity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity. However, H203A inactivates heparinase I while a H203D mutant has residual activity, indicating a role of this residue in catalysis. We propose that histidine-203, contained in the heparin binding site, is immediately adjacent to cysteine-135, and these residues together form the catalytic domain of heparinase I. PMID- 8639637 TI - Solubilization of a complex of tryptic fragments of Na,K-ATPase containing occluded Rb ions and bound ouabain. AB - The nonionic detergent C12E10 (polyoxyethylene 10-laurylether) has been used to solubilize a complex of tryptic fragments of Na, K-ATPase containing occluded Rb ions and bound ouabain. The aim was to define which fragments are required to maintain Rb occlusion. The experiments utilize "19 kDa membranes" consisting of a 19 kDa and several smaller tryptic fragments (8-11.7 kDa) of the alpha subunit, which include trans-membrane segments M7/M10 and the pairs M1/M2, M3/M4, and M5/M6 [Capasso, J. M., et al (1992) J. Biol. Chem. 267, 1150-1158]. The beta subunit is partially split into a 16 kDa fragment and a glycosylated approximately 50 kDa fragment. Cation occlusion and ouabain binding are intact. After preincubation of "19 kDa membranes" with Rb (5 mM) and then ouabain (10 mM), 90-100% of occluded Rb was solubilized by C12E10 at 0 degrees C. All fragments of the alpha and beta subunits, and also the gamma subunit, were cosolubilized by C12E10, and were observed to sediment together on a sucrose density gradient as a complex containing occluded Rb ions. The soluble complex consists of a monomer containing one copy of each fragment, as indicated by size exclusion HPLC, as well as estimates of specific Rb occlusion (20.0 +/- 1.2 nmol/mg of protein). In the absence of Rb ions and ouabain, the complex was unstable. Whereas the 19 kDa fragment (M7-M10) and beta subunit remained associated, the smaller fragments, containing M5/M6 and M3/M4 and M1/M2, and the subunit dissociated. Observations on the thermal inactivation of Rb occlusion, and effect of pH and ionic strength, suggest that the soluble complex is stabilized by multiple interactions, both within the lipid bilayer and in hydrophilic domains (e.g., salt bridges). PMID- 8639638 TI - Inositol 1,4,5-trisphosphate-mediated Ca2+ release from platelet internal membranes is regulated by differential phosphorylation. AB - Platelets are activated by an increase in cytosolic Ca2+, and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from internal stores via the InsP3 receptor. Cytosolic cAMP inhibits platelet activation, and experiments were designed to determine if cAMP-dependent phosphorylation affects the rate of InsP3-mediated Ca2+ release. Western blotting of platelet internal membranes with anti-InsP3 receptor and anti-actin binding protein antibodies revealed that the platelet contains type 1 InsP3 receptor and that it is distinct from actin binding protein. The platelet InsP3 receptor was shown to be phosphorylated by endogenous, membrane-bound kinases as well as by exogenous protein kinase A. Prior phosphorylation of the insP3 receptor by endogenous kinases inhibited additional protein kinase A-dependent phosphorylation by 60%. Furthermore, endogenous phosphorylation resulted in a 2 fold increase in the InsP3-mediated Ca2+ release rate relative to dephosphorylated controls. Following endogenous phosphorylation, additional phosphorylation by protein kinase A returned the Ca2+ release rate to control values, while protein kinase A-dependent phosphorylation of dephosphorylated membranes did not affect the release rate. These results suggest that the InsP3 receptor within intact platelets is phosphorylated by endogenous kinases which results in a high InsP3-mediated Ca2+ release rate, and that increases in cAMP result in additional phosphorylation that inhibits Ca2+ release, thus contributing to inhibition of platelet activation. PMID- 8639639 TI - Immobilization of the type I receptor for IgE initiates signal transduction in mast cells. AB - Clustering of the type I receptor for IgE (Fc(epsilon) RI) on mast cells initiates a cascade of biochemical processes that results in the secretion of inflammatory mediators. We have studied this clustering process in order to obtain information about receptor density and mobility required for initiating that cascade. Specifically, we examined the role of new cluster formation in sustaining the secretory response and the minimal cluster density required for initiating secretion. The experimental protocol adopted for these studies employed photoactivatable antigens and antigen-carrying solid surfaces which enabled us to control the density and mobility of the Fc epsilon RI within the cluster. Our results show that recruitment of new Fc(epsilon) RI into clusters, either by antigen exchange among Fc(epsilon) RI-bound IgE molecules or by IgE bound Fc(epsilon) RI exchange with vacant receptors, is not required for sustaining the cellular secretory response. Furthermore, we find that the cell's secretory response is very sensitive to the density of immobilized Fc(epsilon) RIs, increasing steeply above a density of ca. 1000 immobilized molecules/microns 2. Taken together, these finding suggest that immobilization of a fraction of the randomly distributed Fc(epsilon) RIs that are in sufficient proximity on the surface of mucosal-type mast cells of the RBL-2H3 line initiates a degranulation signal, and that this is maintained as long as these receptors are kept within this distance. The above conclusions and the experimental protocol presented in this study are expected to have wider applications for the study and understanding of signaling by immuno (as well as other) receptors. PMID- 8639640 TI - Unesterified long chain fatty acids inhibit the binding of single chain urokinase to the urokinase receptor. AB - The interaction of single chain urokinase with its receptor accelerates plasminogen activator activity on cell surfaces and induces intracellular signalling in several cell types. To date, no physiologic inhibitor of this binding has been identified. We report that the binding of scuPA to its cellular receptor is inhibited by long chain fatty acids such as oleic acid (C18, delta 9) at physiological plasma concentrations. Inhibition of single chain urokinase binding to human trophoblastic cells by long chain fatty acids was dose-dependent and saturable. Fifty percent of the binding was inhibited at an oleic acid concentration of 27 microM, while inhibition was maximal (75%) at 150 microM oleic acid. The inhibitory potency of oleic acid was unaffected by fatty acid free albumin or human plasma. Inhibition of single chain urokinase binding by free fatty acid analogues was critically dependent on chain length (> C14 required for inhibition) and was proportional to the extent of unsaturation. Only the fraction of specific scuPA binding to trophoblasts that was dependent on uPAR was susceptible to inhibition by oleic acid, while binding of scuPA to vitronectin, thombospondin, and the alpha 2-macroglobulin receptor/low-density lipoprotein-related receptor was not. [3H]Oleic acid bound specifically to recombinant soluble uPAR in a 1:1 molar ratio in the presence or absence of plasma and totally blocked its specific binding to a cell line expressing glycosyl phosphatidylinositol-linked single chain urokinase. These results indicate that oleic acid and other unsaturated long chain free fatty acids may serve as physiologic regulators of proteolytic events and intracellular signalling that depend upon the interaction of urokinase with its receptor. PMID- 8639641 TI - Role of CAS, a human homologue to the yeast chromosome segregation gene CSE1, in toxin and tumor necrosis factor mediated apoptosis. AB - We have previously isolated by expression/selection cloning plasmids containing human cDNAs that rendered MCF-7 breast cancer cells resistant to immunotoxins, Pseudomonas exotoxin (PE), and diphtheria toxin (DT) [Brinkmann et al. (1995) Mol. Med. 1, 206-216]. Here we describe that one of these resistant plasmids, which contains an antisense cDNA fragment homologous to the yeast chromosome segregation gene CSE1 [CAS; Brinkmann et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10427-10431], reduces the intracellular content of the human CSE1 homologue CAS protein. CAS reduction confers resistance not only to the ADP-ribosylating toxins PE and DT, but also to tumor necrosis factor alpha and beta. The resistance was observed as reduced apoptosis. CAS antisense did not affect the cell death induced by staurosporine, cycloheximide, or etoposide. The observation that CAS antisense can interfere with apoptosis mediated by TNF and ADP ribosylating toxins suggests that CAS may play a role in selected pathways of apoptosis. PMID- 8639642 TI - Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site. AB - The (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein. In order to identify protein structural elements involved in cap methylation, we introduced eight alanine substitution mutations within two sequence motifs of D1(498-844)-(594)VLAIDFGNG(602) and (681)IHYSF(685)--that are conserved in the cap methyltransferase from yeast. The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and the recombinant D1(498-844)/D12 heterodimers were purified. Alanine substitutions at five positions--Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685--had little or no effect on methyltransferase activity. Mutations at three conserved residues were deleterious. Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wild-type protein. Substitutions at His-682 and Tyr-683 reduced activity to 4% and 0.05%, respectively. By further mutating Tyr-683 to Phe and Ser, we established that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable. Specific binding of the methyltransferase to the RNA cap was demonstrated by UV cross-linking to [32P]GMP labeled capped poly(A). Label transfer occurred exclusively to the D1(498-844) subunit and was competed by the cap analogs GpppA and m7GpppA. Cap-specific cross linking to m7GpppA(pA)n was stimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but stimulated by AdoMet. We suggest that occupancy of the methyl donor site either enhances the affinity for the cap guanosine or alters the protein interface so that a photoreactive moiety is brought closer to the cap structure. The catalytically defective H682A, Y683A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy. The catalytically defective G600A mutant did cross-link to the cap in the presence of AdoHcy, suggesting that this mutation affects the chemical step of transmethylation. PMID- 8639643 TI - Recognition of anionic porphyrins by DNA aptamers. AB - DNA sequences were isolated by in vitro selection for binding to N methylmesoporphyrin IX (NMM), a molecule that behaves as a stable transition state analogue for porphyrin chelatases. Clones approximately 280 and approximately 120 nucleotides long were obtained, which bound to NMM with sub micromolar affinity but bound mesoporphyrin IX (MPIX), as well as various metalloderivatives of MPIX, with lower affinity. Footprinting experiments with dimethyl sulfate, DNase I, and bound hemin molecules activated by superoxide identified a series of short guanine-rich motifs to be the binding sites for the various porphyrins. One clone, PS2, examined in depth, gave a methylation footprint with bound NMM but not with bound MPIX nor with a number of metalloporphyrins. The binding domain PS2, synthesized as a short oligonucleotide, itself showed high-affinity binding to NMM. The binding sequences from different clones were loosely homologous, and the footprinting data were consistent with their folding to form one or more guanine quartets in the presence of NMM. Ultraviolet--visible absorption and circular dichroism spectroscopy of the DNA--NMM complexes indicates, however, that the interaction is not primarily intercalative in nature. The preferential binding of NMM by these aptamers raises the possibility of their being able to catalyze the chelation of metal ions by the porphyrin MPIX. PMID- 8639645 TI - Synthesis and characterization of a novel spin-labeled affinity probe of human erythrocyte band 3: characteristics of the stilbenedisulfonate binding site. AB - A new spin-labeled maleimide derivative of the anion exchange inhibitor 4-4' diaminodihydrostilbene-2,2'-disulfonate (H2DADS) has been synthesized as a site specific molecular probe of the stilbenedisulfonate binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H2DADS maleimide, specifically and covalently labels the Mr 17 kDa integral membrane segment of band 3 with a 1:1 stoichiometry and inhibits essentially 100% of the band 3-mediated anion exchange. The linear V1 EPR spectrum of spin-labeled intact erythrocytes is indicative of a spatially isolated probe which is effectively immobilized on the submicrosecond time scale. Several independent lines of experimental evidence have shown that the nitroxide moiety of SL-H2DADS-maleimide labeled band 3 is sequestered in a highly protected protein environment. These results are consistent with the observation that the spin-label is rigidly linked to band 3 in a fixed orientation with respect to the membrane normal axis [Hustedt, E. J., & Beth, A. H., (1996) Biochemistry 35, 6944-6954]. The nitroxide moieties of the SL-H2DADS-maleimide-labeled band 3 dimer are greater than 20 A from each other and are also more than 20 A from a monomer-monomer contact surface defined by cross-linking with the spin-labeled reagent BSSDA [bis(sulfo-N succinimidyl)doxyl-2-spiro-5'-azelate]. These properties make SL-H2DADS-maleimide an extremely useful molecular probe for characterization of the physical properties of the band 3 stilbenedisulfonate binding site, determination of distances between the stilbenedisulfonate site and other segments of band 3, and investigation of the global rotational dynamics of human erythrocyte band 3. PMID- 8639644 TI - Inhibitory RNA ligand to reverse transcriptase from feline immunodeficiency virus. AB - High-affinity, high-specificity RNA ligands for reverse transcriptase from feline immunodeficiency virus (FIV) were isolated from an RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. The selected RNA ligands bound to FIV reverse transcriptase with dissociation constants in the nanomolar range. One of the ligands was a potent inhibitor of the RNA-dependent DNA polymerase activity of both the recombinant and the virion derived FIV reverse transcriptase. It also inhibited the reverse transcriptase from an FIV mutant that is resistant to 3'-azido-3'-deoxythymidine (AZT). The inhibition of FIV reverse transcriptase was competitive with respect to template primer and noncompetitive with respect to deoxyribonucleoside 5'-triphosphates. This ligand was specific for the FIV enzyme and did not inhibit other reverse transcriptases tested (avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus type 1). PMID- 8639647 TI - Controlling topology and native-like behavior of de novo-designed peptides: design and characterization of antiparallel four-stranded coiled coils. AB - The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3 Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins. PMID- 8639646 TI - Determination of the orientation of a band 3 affinity spin-label relative to the membrane normal axis of the human erythrocyte. AB - The orientation of the nitroxide moiety of an isotopically substituted spin labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5 nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal. PMID- 8639648 TI - Subcellular metabolite transport and carbon isotope kinetics in the intramyocardial glutamate pool. AB - The pathophysiological state of the cell must be translated into the mitochondria to meet the demands for oxidative energy production. Metabolite exchange across the mitochondrial membrane provides this communication and was observed with 13C NMR spectroscopy of hearts oxidizing [2-13C]-butyrate at normal or high cytosolic redox state. Previous NMR observations of 13C turnover within the glutamate pool of intact tissues have indicated its relationship with metabolic flux through the tricarboxylic acid (TCA) cycle, but the direct influence of isotope exchange between the TCA cycle intermediates in the mitochondria and the cytosolic glutamate pool has been much less considered. This current study was designed to determine whether the physical transport of metabolites across the mitochondrial membrane of intact heart tissues could be discerned as a rate determinant for isotope turnover in the NMR-detectable glutamate pool. 13C entry into glutamate provided measures of TCA cycle flux and the interconversion between mitochondrial intermediates and cytosolic glutamate. The influence of the malate-aspartate shuttle activity was examined by comparing two groups of hearts: one group oxidizing 2.5 mM [2-13C]-butyrate (n = 5) and the other oxidizing 2.5 mM [2 13C]butyrate in the presence of a lactate (2.5 mM)-induced elevation in the cytosolic redox to stimulate shuttle activity (n = 5). High redox state did not affect TCA cycle flux but increased the rate of interconversion between alpha ketoglutarate and glutamate from 3.1 +/- 0.2 mumol min-1 (g dry)-1 to 14.3 +/- 2.0. High resolution 13C NMR spectra of tissue extracts confirmed that the exogenous lactate did not contribute as a carbon source for the formation of either the TCA cycle intermediates or glutamate. In both groups, over 95% of the acetyl-CoA was derived from the short-chain fatty acid butyrate, irrespective of the presence of lactate. Additional hearts perfused with unlabeled butyrate and [3-13C]lactate showed no label entry into glutamate, but rather the formation of [3-13C]alanine, indicating the net reverse flux through lactate dehydrogenase to increase NADH production. Thus, the addition of lactate served only to augment cytosolic redox state to drive the malate-aspartate shuttle. The dynamic-mode acquisition of 13C NMR data from intact hearts, oxidizing [2-13C]-butyrate with or without additional lactate, demonstrated the influence of malate-aspartate shuttle activity on the 13C enrichment rates within glutamate. These data indicate metabolic communication between the mitochondria and cytosol in response to the physiological state of intact tissues. PMID- 8639649 TI - Random mutagenesis targeted to the active site of the EcoRV restriction endonuclease. AB - Two segments of the gene for the EcoRV restriction endonuclease, each encoding 10 amino acids at the active site, were subjected to random mutagenesis with degenerate oligonucleotides. Mutations that abolished the activity of the EcoRV endonuclease were selected by viability in a strain of Escherichia coli that lacks the EcoRV methyltransferase, under conditions where the gene for the wild type endonuclease is lethal to the cell. Sixty-five mutants were isolated and analyzed by DNA sequencing to identify the mutations. The collection of null mutants contained 49 with single amino acid substitutions, 15 with double substitutions, and one with a triple substitution. The single substitutions were located at many different positions within the two 10-amino acid segments, though several hot-spots gave rise to null mutants at high frequencies. Some hot-spots were readily explained by reference to the crystal structure of EcoRV since they were at the amino acids immediately adjacent to the scissile phosphodiester bond: for example, Asp90 and Lys92. These residues may be directly involved in the catalytic mechanism. Other hot-spots, such as Gln69, Tyr72, and Ala88, were at unexpected positions that appear to have no direct role in DNA binding or catalysis. At some of the unexpected hot-spots, the side chain of the amino acid lies distant from the DNA, yet the enzyme was still inactivated by conservative substitutions at these positions. The sensitivity of the EcoRV endonuclease to conservative substitutions may be due to its requirement to take up one particular conformation at the DNA-protein interface out of a large number of alternative conformations. PMID- 8639650 TI - An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese. AB - The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild type EcoRV. The Ile-->Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV. PMID- 8639651 TI - Local mobility within villin 14T probed via heteronuclear relaxation measurements and a reduced spectral density mapping. AB - Villin 14T, a representative domain from the actin severing and bundling protein villin, binds calcium ions and actin monomers. To begin to understand the contributions of mobility to the villin-calcium and villin-actin interactions, relaxation rates for magnetization involving the amide nitrogens and protons have been measured for 15N-labeled villin 14T in solution. Although we have measured the complete set of rates required for a full spectral density map, difficulties in the accurate measurement of relaxation rates for antiphase coherence and two spin order led us to consider a reduced mapping formalism. From the reduced spectral density map, a model-free analysis, or directly from the measured Nx,y relaxation rates, local variations in mobility along the backbone of villin 14T have been revealed. Fast motions are evident not only at the amino and carboxyl termini but also in the turn between strands beta 4 and beta 5 of the central beta-sheet and in the turn between helix alpha 3 and strand beta 7. Slower motions are suggested for the turn between strands beta 2 and beta 3. Motions on the microsecond to millisecond time scale have been probed directly by examining the dependence of the proton transverse relaxation rate on the spin-locking field strength. Leu11 shows a strong dependence on field strength, implying conformational exchange with a time constant of 125 +/- 69 microseconds. The backbone at the actin-binding interface appears to be rather rigid. PMID- 8639652 TI - Unambiguous structure characterization of a DNA-RNA triple helix by 15N- and 13C filtered NOESY spectroscopy. AB - DNA.DNA*RNA triple helices of the pyrimidine.purine*pyrimidine motif (where . indicates Watson-Crick pairing and * indicates Hoogsteen pairing) appear to be very stable, which has important implications for the development of novel antisense strategies. Here we present the first structural NMR studies on such a system, composed of a DNA hairpin with a homopurine-homopyrimidine stem sequence and a single-stranded RNA oligonucleotide containing exclusively pyrimidine residues. In these investigations an unlabeled DNA hairpin and a uniformly 13C/15N-enriched RNA oligonucleotide were utilized in combination with X-edited 1H NMR spectroscopy. Improved 15N (omega 2) filtered NOESY and 13C (omega 1) filtered NOESY are presented by which we were able to differentiate between intrastrand, i.e., DNA-DNA and RNA-RNA, and interstrand, i.e., DNA-RNA, NOE contacts. It is unambiguously established that the complex forms a right-handed triple helix, with the RNA strand situated in the major groove of the Watson Crick stem of the hairpin. The interaction is stabilized by the formation of Hoogsteen-type base pairs between the RNA strand and the purine strand of the DNA. These strands run parallel to each other. The characterization of the DNA RNA triple helix structure described here shows that this type of experiment forms a valuable instrument in the structure determination of bimolecular systems of nucleic acids. PMID- 8639653 TI - The high-resolution crystal structure of human annexin III shows subtle differences with annexin V. AB - The structure of recombinant human annexin III was solved to 1.8 A resolution. Though homologous to annexin I and V, the annexin III structure shows significant differences. The tryptophan in the calcium loop of the third domain is exposed to the solvent, as in the structure of annexin V crystallized in high calcium concentrations, although the annexin III crystals were prepared at low calcium concentrations. The position of domain III relative to the other domains is different from both annexin V and I, suggesting further flexibility of the molecule. The entire N-terminus of the protein is well-defined in the present structure. The side chain of tryptophan 5 interacts with the hinge region of the hydrophillic channel, which could have an effect on the potential mobility of this region, as well as on its possible calcium channel behavior. PMID- 8639654 TI - Mutagenesis of N-glycosylation sites in the human vasoactive intestinal peptide 1 receptor. Evidence that asparagine 58 or 69 is crucial for correct delivery of the receptor to plasma membrane. AB - The functional role of N-linked carbohydrates in the human vasoactive intestinal peptide (VIP) 1 receptor was investigated by site-directed mutagenesis (Asn- >Thr) of the four consensus N-glycosylation sites on Asn58, Asn69, Asn100 (N terminal extracellular domain) and Asn293 (second extracellular loop). Mutated receptors were investigated after transient expression in Cos-7 cells, by ligand binding assay, affinity cross-linking, western blotting, and confocal laser microscopy of epitope-tagged receptor proteins. Mutations of each consensus site revealed that Asn58, Asn69, and Asn100 were occupied by a 9-kDa N-linked carbohydrate whereas Asn293 was not used for glycosylation. Each mutated receptor was expressed (western blot) and delivered at the plasma membrane (confocal microscopy) of Cos-7 cells. They displayed a dissociation constant similar to that of the wild-type receptor, i.e., 0.5-1 nM. In contrast, no VIP binding to Cos-7 cells could be observed with the mutant devoid of consensus N-glycosylation sites due to a strict sequestration of this mutant in the perinuclear endoplasmic reticulum. However, when solubilized with a zwitterionic detergent, this mutant bound [125I]VIP specifically, indicating that it retained intrinsic binding activity. The construction of other mutants in which three out of four N glycosylation sites were altered, demonstrated that N-glycosylation at either Asn58 or Asn69 is necessary and sufficient to ensure correct delivery of the receptor to the plasma membrane. Further pharmacological studies involving incubation of Cos-7 cells with castanospermine or deoxymannojirimycin immediately after transfection of mutated cDNAs encoding receptors with a single glycosylation site at Asn58 or Asn69 suggested that carbohydrate at Asn58 was involved in a calnexin-dependent folding process of the receptor whereas carbohydrate at Asn69 was not. These studies highlight the functional importance of the N-glycosylation of the human VIP 1 receptor which belongs to a new subfamily of seven membrane-spanning receptors. PMID- 8639656 TI - In vivo phosphorylation of histone H1 variants during the cell cycle. AB - In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels. PMID- 8639655 TI - Kinetic tuning of the EF-hand calcium binding motif: the gateway residue independently adjusts (i) barrier height and (ii) equilibrium. AB - In EF-hand calcium binding sites of known structure, the side chain provided by the ninth EF-loop position lies at the entrance of the shortest pathway connecting the metal binding cavity to solvent. The location of this residue suggests that it could serve as a "gateway", providing steric and electrostatic control over the kinetics of Ca2+ binding and dissociation. To test this hypothesis, the present study has engineered the putative gateway side chain of a model EF-hand site and determined the resulting effects on metal ion affinity and dissociation kinetics. The model site chosen was that of the Escherichia coli galactose binding protein (GBP), in which the putative gateway is a Gln side chain. Nine engineered GBP molecules were generated and isolated, each exhibiting native-like activity and global conformation. Two control substitutions at the fourth EF-loop position, a noncoordinating surface residue, had no significant effect on either the equilibrium or the kinetics of the model site. The remaining seven proteins, which possessed unique substitutions at the ninth EF-loop position (Glu, Asn, Asp, Thr, Ser, Gly, Ala), in each case significantly altered the affinity or dissociation kinetics of the site for Tb3+, used as a probe metal ion. Neutral side chains at the gateway position yielded a 590-fold range of Tb3+ dissociation kinetics but only a 3-fold range of Tb3+ affinities, indicating that the size or polarity of these substitutions alters the transition state barrier for metal binding and release without substantially shifting the equilibrium. In contrast, acidic side chains yielded as much as a 34-fold decrease in the Tb3+ off-rate and a 33-fold increase in Tb3+ affinity, suggesting that a negative charge at the gateway position increases the equilibrium stability of the bound metal ion and thereby slows metal release. Thus, kinetic tuning by the gateway side chain exhibits both transition state and ground state tuning mechanisms. In natural EF-hand sequences, different gateway side chains are used by slow buffering sites and rapid signaling sites, providing evidence that the gateway position is an important physiological determinant of metal binding kinetics. PMID- 8639657 TI - 1,2-Dithiolan-3-one 1-oxides: a class of thiol-activated DNA-cleaving agents that are structurally related to the natural product leinamycin. AB - Leinamycin is a recently discovered, thiol-dependent DNA-cleaving natural product. The mechanism of DNA cleavage by leinamycin is unknown. Inspired by this intriguing natural product, we have investigated the DNA-cleaving properties of three 1,2-dithiolan-3-one 1-oxides (1-3) that are structurally related to the suspected DNA-cleaving "core" of leinamycin. It was found that, similar to leinamycin, these three 1,2-dithiolan-3-one 1-oxides are thiol-dependent DNA cleaving agents. At the concentrations of 1-3 used in these experiments (approximately 100 microM), efficient DNA cleavage is absolutely dependent on added thiol, with optimum cleavage occurring at 5-10 equiv (500 microM-1 mM) of added thiol. 2-Mercaptoethanol, glutathione, dithiothreitol, and thiophenol function with approximately equal efficiency as triggering agents for the cleavage reaction. DNA cleavage by 1-3 is not highly pH-dependent. Cleavage of DNA by these sulfur heterocycles is diminished by the removal of molecular oxygen from the reaction medium, by the radical scavengers methanol, ethanol, and mannitol, and by the enzyme catalase. Superoxide dismutase does not suppress DNA cleavage by these compounds. When diethylenetriaminepentaacetic acid is employed in these reactions as a chelator of adventitious trace metal ions, DNA cleavage is efficiently inhibited. The S-deoxy analog of 1 does not cleave DNA under conditions where 1 effects efficient thiol-mediated cleavage of DNA. These experiments indicate that, in concert with thiols, 1,2-dithiolan-3-one 1-oxides convert molecular oxygen to DNA-cleaving oxygen radicals. The marked effect of catalase further suggests that molecular oxygen is converted to hydrogen peroxide which ultimately cleaves DNA via a trace metal-dependent Fenton reaction. This work demonstrates that 1,2-dithiolan-3-one 1-oxides represent a general class of thiol-potentiated DNA-cleaving molecules. PMID- 8639658 TI - Purification and characterization of a DNA polymerase beta promoter initiator element-binding transcription factor from bovine testis. AB - A low-abundance DNA-binding protein for the DNA polymerase beta (beta-pol) promoter initiator element was purified from bovine testis. The transcriptional initiator element (Inr) of the mammalian beta-pol promoters characterized is highly conserved, and the bovine beta-pol promoter Inr has the sequence 11CAGAGGCGGCCATTGTT+6. The purified initiator element-binding protein (Inr-BP) binds with high affinity to an oligonucleotide corresponding to the beta-pol promoter Inr (Kd = 5 pM), and increasing ionic strength decreases stability of the protein-DNA complex. Mutational analysis of the Inr shows that the purified Inr-BP binds with sequence specificity to the sequence CCAT at -2 to +2 of the Inr, but that seven residues on the 5' side and three residues on the 3' side of the CCAT sequence are required also. Using an in vitro transcription assay with HeLa cell nuclear extract, we find that the endogenous Inr-BP is required for transcriptional activity of the beta-pol promoter; addition of purified Inr-BP restores activity to the nuclear extract depleted in Inr-BP by affinity chromatography. These results, based upon the sequence specificity for DNA binding, indicate that Inr-BP is a YY1-like protein and suggest that it is a required transcription factor in beta-pol gene expression. PMID- 8639659 TI - Evidence for interdomain interaction in the Escherichia coli repressor of biotin biosynthesis from studies of an N-terminal domain deletion mutant. AB - The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA binding protein. The protein is composed of three structural domains. Contact with the biotin operator (bioO) in the transcriptional repression complex is made by the N-terminal domain which contains a helix-turn helix structural module. The central domain is required for the catalytic functions of BirA including synthesis of biotinyl-5'-AMP from substrates ATP and transfer of biotin from the adenylate to a lysine residue of the biotin carboxyl carrier protein (BCCP) of acetyl CoA carboxylase. The adenylate serves not only as the activated intermediate in the biotin transfer reaction but also as the positive allosteric effector for site-specific DNA binding. Little interaction between the N-terminal and central domains is observed in the apo-repressor structure (Wilson et al., 1992). In this work, we have engineered an N-terminal deletion mutant of BirA, BirA65-321. Biochemical analysis of the purified truncated repressor indicates that, as expected, it does not bind to biotin operator DNA. BirA65-321 is, moreover, identical to intact BirA in catalysis of synthesis of bio-5'-AMP and in transfer of biotin from the adenylate to BCCP. Deletion of the DNA binding domain severely compromises the ability of BirA to bind to biotin or bio-5'-AMP. The affinity of BirA65-321 for biotin is decreased 100-fold while that for bio-5'-AMP is decreased 1000-fold, relative to intact BirA. The significant functional role of the DNA binding domain in tight binding of the two ligands to the central domain may be indicative of formation of extensive interdomain contacts in the holorepressor structure. PMID- 8639660 TI - Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis. AB - The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered. PMID- 8639661 TI - Direct determination of the membrane affinities of individual amino acids. AB - Amino acids have distinct lipid bilayer affinities which influence the insertion and topology of membrane-bound polypeptides and proteins. To measure membrane affinities, 14 uncharged amino acids were introduced individually at a guest site in a 25-residue peptide derived from the membrane-binding presequence of yeast cytochrome c oxidase, and the peptides were labeled with a nitroxide spin-label. The free energies of transfer from phospholipid bilayers to water (delta delta Gbilayer) were determined directly by examination of partitioning into phospholipid bilayers using electron paramagnetic resonance. The delta delta Gbilayer values are in agreement with hydrophobicities assessed from 1-octanol water partitioning of N-acetyl amino acid amides [Fauchere, J.-L., & Pliska, V. (1983) Eur. J. Med. Chem. 18, 369-375; Eisenberg, D., & McLachlan, A. (1986) Nature 319, 199-203] and quantitatively demonstrate the role of the hydrophobic effect in membrane-protein interactions. PMID- 8639662 TI - Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A 1H NMR study. AB - Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gln in stellacyanin. PMID- 8639663 TI - Interactions between diphenylcarbazide, zinc, cobalt, and manganese on the oxidizing side of photosystem II. AB - The inhibition of DPC-mediated DCIP photoreduction by exogenous MnCl2 in Tris treated photosystem II (PSII) membrane fragments has been used to probe for amino acids on the PSII reaction center proteins, including D1His337, that provide ligands for binding manganese [Preston, C., & Seibert, M. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. I, pp 925-928, Kluwer Academic Publishers, Dordrecht, The Netherlands; Preston, C., & Seibert, M. (1991) Biochemistry 30, 9615-9624 and 9625-9633]. At a concentration of 200 microM, DPC is photooxidized at both a high-affinity and a low-affinity site in PSII at approximately the same initial rate. Addition of 10 microM MnCl2 noncompetitively inhibits DPC photooxidation at the high-affinity site, with a Ki of 1.5 microM, causing a decrease of about 50% in the overall DCIP photoreduction rate. The high-affinity site for Mn binding was deconvoluted into four independent components. In earlier work, the inhibition was attributed to the tight association of either Mn2+ or Mn3+ with the PSII membrane. We report here that inhibition of DPC photooxidation may involve two different types of high affinity, Mn-binding components: (a) one that is specific for Mn, and (b) others that bind Mn, but may also bind additional divalent cations, such as Zn and Co, that are not photooxidized by PSII. These conclusions are based on the observations that (a) DPC photooxidation can be inhibited by Zn2+ and Co2+; (b) Zn2+ and Co2+ interact with Mn2+ in a nonmutually exclusive manner, suggesting that they may share some binding components with Mn2+; (c) high-affinity Mn2+ (but not Zn2+ or Co2+) inhibition of DPC photooxidation is accompanied by nondecaying fluorescence emission, following a single saturating flash, indicating efficient electron donation by Mn2+ to YZ+; (d) Mn2+ photooxidation in the presence of DPC is not inhibited by Zn2+ or Co2+; and (e) kinetic modeling of the interaction between high-affinity Mn2+ and DPC in PSII indicates inhibition of steady-state Mn2+ photooxidation by DPC, but allows for a single photooxidation of Mn2+. We conclude that Mn inhibition of DPC photooxidation can be used to identify Mn-binding sites of physiological importance, and suggest that the Mn-specific component of the high-affinity, Mn-binding site involves the ligand to the first Mn bound during photoactivation (i.e., Asp170 on D1, as found by other investigators). PMID- 8639664 TI - Properties of the chloride-depleted oxygen-evolving complex of photosystem II studied by electron paramagnetic resonance. AB - The effects of different Cl- depletion treatments in photosystem II (PS-II) enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy and by measurements of oxygen-evolving activity. The results indicated that the oxygen-evolving complex of PS-II exhibits two distinct Cl(-) dependent properties. (1) After Cl(-)-free washes at pH 6.3, a reversibly altered distribution of structural states of PS-II was observed, manifested as the appearance of a g = 4 EPR signal from the S2 state in a significant fraction of centers (20-40%) at the expense of the S2 multiline signal. In addition, small but significant changes in the shape of the S2 multiline EPR signal were observed. Reconstitution of Cl- to Cl(-)-free washed PS-II rapidly reversed the observed effects of the Cl(-)-free washing. The anions, SO4(2-) and F-, which are often used during Cl- depletion treatments, had no effect on the S2 EPR properties of PS-II under these conditions in the absence or presence of Cl-. Flash experiments and measurements of oxygen evolution versus light intensity indicated that the two structural states observed after the removal of Cl- at pH 6.3 originated from oxygen-evolving centers exhibiting a lowered quantum yield of water oxidation. (2) Depletion of Cl- in PS-II by pH 10 treatment reversibly inhibited the oxygen-evolving activity to approximately 15%. The pH 10 treatment depleted the Cl- from a site which is considered to be equivalent to that studied in most earlier work on Cl(-)-depleted PS-II. The S2 state in pH 10/Cl(-) depleted PS-II was reversibly modified to a state from which no S2 multiline EPR signal was generated and which exhibited an intense S2 g = 4 EPR signal corresponding to at least 40% of the centers but possibly to a much larger fraction of centers. The state responsible for the intense S2 g = 4 signal generated under these conditions is unlike that observed after removal of Cl- from PS-II at pH 6.3, in that this state was more stable in the dark, showing a half-decay time of approximately 1.5 h at 0 degrees C, and was unable to undergo further charge accumulation. Nevertheless, a fraction of centers, probably different from those exhibiting the S2 g = 4 signal, was able to advance to the formal S3 state, giving rise to a narrow EPR signal around g = 2. Addition of the anions SO4(2-) or F- to pH 10/Cl(-)-depleted PS-II affected the properties of PS II, resulting in EPR properties of the S2 state similar to those reported earlier following Cl- depletion treatment of PS-II in the presence of these anions. Surprisingly, after addition of F-, the g = 4 EPR signal showed a damped flash dependent oscillation. In addition, a narrow signal around g = 2, corresponding to the formal S3 state, also showed a damped flash-dependent oscillation pattern. The presence of oscillating EPR signals (albeit damped) in F(-)-treated pH 10/Cl( )-depleted PS-II indicates functional enzyme turnover. This was confirmed by measurements of the oxygen-evolving activity versus light intensity which indicated that in approximately 45% of oxygen-evolving centers the enzyme turnover was slowed by a factor of 2. The distinct Cl- depletion effects in PS-II observed under the two different Cl- depletion treatments are considered to reflect the presence of two distinct Cl(-)-binding sites in PS-II. PMID- 8639665 TI - Interactions of substrate and product with cytochrome P450 2B4. AB - Interactions of the substrate(s) benzphetamine and the product (P) desmethylbenzphetamine with cytochrome P450 2B4 were studied by difference spectrophotometry. A two-sites model in which site 1 binding, causing Type I transition (low- to high-spin) must precede site 2 binding, causing Type II transition (high- to low-spin), gave an acceptable fit to the spectral titration data. The equilibrium association constant of substrate for site 1 (K1) was greater than that for site 2 (K2), and the K2 for the product was greater than K1, indicating that the substrate binds preferentially to site 1 and the product prefers site 2. In addition, competition between P and a strong Type II ligand (1 benzylimidazole) and a noncompetitive type of interaction between S and the same Type II ligand was observed. This indicates that P binds to the same site as the Type II ligand and S binds to a different site. The observed high-spin maxima for both P (EP1HSmax) and S (ES1HSmax) were similar to those calculated using the K1 and K2 values obtained from the curve-fitting procedure, indicating that the equilibrium concentration of the high-spin species is controlled entirely by K1 and K2. Simultaneous presence of the substrate and product decreased K1 of the substrate and K2 of the product, indicating that there is interaction between the substrate-preferred and the product-preferred sites. A possible functional significance of the differences in the site preferences of the substrate and product is discussed. PMID- 8639666 TI - Modulation of arrestin release in the light-driven regeneration of Rh1 Drosophila rhodopsin. AB - We report studies of the in vitro regeneration of Rh1 Drosophila rhodopsin using immunochemical and spectroscopic probes for the release of arrestin (49 kDa). Upon illumination of metarhodopsin-containing membrane suspensions isolated from homogenized Drosophila heads, arrestin was released into the aqueous medium. In contrast, no release of arrestin was observed upon illumination of metarhodopsin in lipid/detergent micellar extracts. The spectroscopic changes associated with the transition from metarhodopsin to rhodopsin were, however, similar in membrane suspensions and in micellar extracts. The light-driven release of arrestin was restored in reconstituted liposomes formed by dialysis of detergent from the micellar extracts. We conclude that micellar solubilization of membranes decouples the light-driven release of arrestin from rhodopsin structural changes which are responsible for altering the lambda max of the chromophore. The finding that arrestin release from rhodopsin can be modulated by changes in the local membrane environment provides an opportunity to further characterize the nature of rhodopsin conformational changes during regeneration. PMID- 8639668 TI - Catalytic mechanism of glucoamylase probed by mutagenesis in conjunction with hydrolysis of alpha-D-glucopyranosyl fluoride and maltooligosaccharides. AB - The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using alpha-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116-->Ala, Trp120 ->Phe, Asp176-->Asn, and Glu400-->Gln. These replacements decreased the activity (kcat/KM) toward maltose by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GAs, showed wild-type and twice wild-type level activity, while Asp176-->Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild type GA complexed with GF, the pH-activity (kcat) profile shows a pKa of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis. PMID- 8639667 TI - Cell line and site specific comparative analysis of the N-linked oligosaccharides on human ICAM-1des454-532 by electrospray ionization mass spectrometry. AB - Sialylated oligosaccharide structures were determined by the technique of electrospray ionization mass spectroscopy at seven of eight N-linked glycosylation sites of recombinant human ICAM-1des454-532 [tICAM(453)] purified from the tissue culture fluid of Chinese hamster ovary, human embryonic kidney, and mouse myeloma cell lines. The number of structures at each site depended on the cell line and ranged from 8 to 34. N-Glycolyneuraminic acid, a human oncofetal antigen, was found at all sites of all three cell line derived forms of tICAM(453). Tetraantennary complex structures containing one and/or two galactose beta 1,4 N-acetylglucosamine repeats, characteristic of membrane bound proteins, were found on soluble tICAM(453) primarily at Asn-379. Asn-379, located between the D4 and D5 domains, is believed to be located close to the membrane surface in membrane bound ICAM-1. It has been proposed that the extent of N-linked glycosylation at Asn-240 and Asn-269 in the third domain of ICAM-1 may regulate the binding avidity of ICAM-1 to Mac-1 [Diamond, M. S., Staunton, D. E., Marlin, S. D., & Springer, T. A. (1991) Cell 65, 961-971]. In the present study the tICAM(453) Asn-269 site was found to contain predominantly one oligosaccharide structure that is conserved in all three cell lines. On the other hand, the Asn 240 site was found to contain cell line dependent oligosaccharide structural heterogeneity particularly in the degree of sialylation. PMID- 8639669 TI - Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L serine intermediates. AB - The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands. PMID- 8639670 TI - Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis. AB - The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity. ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans. The active-site aspartate 52 residue of ChEWL, which is postulated to stabilize the oxocarbenium ion intermediate, has no counterpart in GoEWL. The substrate specificity of the D52A ChEWL mutant was compared with those of wild-type ChEWL and GoEWL. D52A ChEWL retains approximately 4% of the wild-type catalytic activity in reactions with three different bacterial cell suspensions. Asp52 therefore is not essential to the catalytic mechanism, accounting for only a 2 kcal/mol decrease in delta G++. The function of Asp52 in D52A ChEWL- and GoEWL-catalyzed cleavage of (carboxymethyl)chitin may be partially fulfilled by an appropriately positioned carboxyl group on the substrate (substrate-assisted catalysis). D52A ChEWL and GoEWL, unlike wild-type ChEWL, exhibit biphasic kinetics in the clearing of Micrococcus luteus cell suspensions, suggesting preferences for subsets of the linkages in the M. luteus peptidoglycan. These subsets do not exist in the peptidoglycans of Escherichia coli or Sarcina lutea, since neither D52A ChEWL nor GoEWL exhibits initial bursts in reactions with suspensions of these bacteria. We propose that substrate-assisted catalysis occurs in reactions of D52A ChEWL and GoEWL with M. luteus peptidoglycans, with the glycine carboxyl group of un-cross linked peptides attached to N-acetylmuramic acid partially substituting the function of the missing Asp52. PMID- 8639671 TI - Synergistic contributions of asparagine 46 and aspartate 52 to the catalytic mechanism of chicken egg white lysozyme. AB - The X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and Asp52 with the D-subsite N-acetylmuramic acid residue help to distort that pyranose ring into the reactive half-chair conformation and that a hydrogen bond is formed between Asn46 and Asp52 [Strynadka, N. C. J., & James, M. N. G. (1991) J. Mol. Biol. 220, 401-424]. These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL. The Michaelis constants of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type KM; the corresponding kcat values are 25- and 50 fold lower, respectively, than the wild-type kcat. These results support the proposal of Strynadka and James. The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do not vary. The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues. The mutations do not change the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived substrates differ in conformation from complexes with bacterial peptidoglycans. PMID- 8639672 TI - Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity. AB - This report describes the effect on antigen binding of an isomerized aspartate residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies. PMID- 8639673 TI - Kinetics of human factor VII activation. AB - In this study the activation of human factor VII by a variety of potential activators in the presence and absence of mixed phospholipid vesicles [25% phosphatidylserine (PS), 75% phosphatidylcholine (PC)] is evaluated. At the plasma concentration of factor VII, 10 nM, the activation rate of the zymogen by 0.05 nM factor Xa is anionic phospholipid (PCPS) dependent and achieves a maximum value of 18 pM/s at 5-20 microM PCPS; further increases in the levels of PCPS decrease the activation rate of factor VII. The maximum activation rate of factor VII (10 nM) by the factor VIIa-tissue factor complex (0.1 nM), 0.76 pM/s, is achieved at 200 microM PCPS. No detectable activation of 10 nM factor VII is observed under similar conditions when either thrombin (0.1 nM) or factor IXa (0.1 nM) is used as an activator. Factor VIIa (10 nM) and factor XIa (1 nM) are not observed to activate factor VII at detectable rates. The observed Michaelis Menten constants (KM) for factor VII activation in the presence of PCPS at optimal concentrations vary from 1.2 microM for factor Xa to 3.2 microM for the factor VIIa-tissue factor complex. The highest catalytic constant (kcat) value (15.2 s-1) is observed for factor Xa-PCPS. The factor VIIa-tissue factor complex, factor IXa, and thrombin kcat values are 1.4, 0.32, and 0.061 s-1, respectively. Tissue factor does not increase the factor VII activation rate by factor Xa, factor IXa, or thrombin. Factor VIIIa in the presence of PCPS has no effect on factor VII activation by factor IXa. In contrast, factor Va decreases the factor VII activation rate by factor Xa, reaching saturation at concentrations consistent with complete prothrombinase complex formation. The formed prothrombinase complex activates factor VII at approximately 30% the rate of factor Xa bound to phospholipids. These data allow us to conclude that the predominant physiological factor VII activator is, most likely, membrane-bound factor Xa. PMID- 8639674 TI - Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase. AB - Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2. PMID- 8639675 TI - Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin antithrombin reaction. AB - In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000 fold and made it possible to demonstrate a two-step reaction mechanism for des PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed. PMID- 8639676 TI - Domain structure and conformation of histidine-proline-rich glycoprotein. AB - The complete primary structure of rabbit plasma histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, was determined by a combination of cDNA and peptide sequencing. Limited proteolysis with plasmin yielded three disulfide-linked fragments that were further purified. Reduction of the disulfide bonds with dithiothreitol under nondenaturing conditions releases the central, histidine-proline-rich domain, which contains 15 tandem repeats of the pentapeptide [H/P]-[H/P]PHG. The N-terminal fragment (295 amino acids), consisting of two cystatin-like modules, is bound to the proline-rich C-terminal fragment (105 amino acids) via a buried disulfide bond whose reduction requires prior denaturation. Far-UV circular dichroism spectra revealed beta-sheet with some alpha-helix, polyproline-II helix, and random coil in the secondary structure of the N-terminal, central, and C-terminal domains, respectively. The modular architecture of HPRG suggests that it may have several independent binding sites and that its biological role may be to bring two or more ligands together. The histidine-proline-rich domain, which contains 34 of the 53 histidine residues of HPRG, binds heparin and has an isoelectric point of 7.15 and a relatively high apparent pKa (7.0) of its histidine residues, and thus it probably mediates the interaction between HPRG and heparin, which is strikingly sensitive to pH in the range 7.0-7.4 [Peterson et al. (1987) J. Biol. Chem. 262, 7567-7574]. Solvent perturbation and second-derivative UV spectroscopy of HPRG revealed changes in the environment of tryptophan residues upon lowering the pH. This transition had a midpoint at pH 6.0 and required the disulfide bond bridging the histidine-proline-rich domain to the N/C fragment. The data are consistent with the mutual repulsion of protonated histidine residues in the histidine proline-rich region causing a conformational change transmitted to the rest of the molecule via the disulfide bond. PMID- 8639677 TI - Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin. AB - The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6 fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11). PMID- 8639678 TI - Formation and decay of the S3 EPR signal species in acetate-inhibited photosystem II. AB - A 230-G-wide EPR signal is induced in acetate-treated photosystem II by 30 s of illumination at 277 K followed by freezing under illumination to 77 K [MacLachlan, D. J., & Nugent, J. H. A. (1993) Biochemistry 32, 9772-9780]. This signal, referred to as the S3 EPR signal, has been interpreted to arise from an S2X+ species where X+ is an amino acid radical. Investigation of the factors responsible for the formation and decay of the S3 EPR signal reveals that the yield of the S3 EPR signal is strongly temperature-dependent and depends on the rate of oxidation of QA-. Quantitation of the number of centers contributing to the S3 EPR signal produced by the optimal continuous illumination times of 3 min at 250 K, 30 s at 273 K, and 5 s at 294 K gave values of 13, 38, and 49 +/- 3%, respectively. By using 5 s of illumination at 294 K to induce the S3 EPR signal, and then illumination at 200 K to reduce QA, both the S3 and QA(-)Fe EPR signals were induced in high yield. This result indicates that the S3 EPR signal does not arise from an acceptor-side species. When saturating laser flashes were used to induce the S3 EPR signal in a dark-prepared, dark-adapted, acetate-treated sample, the yield was small after one flash and close to maximal after two flashes. An EPR signal at g = 4.1 was observed to be formed at intermediate times during the decay of the S3 EPR signal in the dark; the rates of decay of the S3 EPR signal at 273 and 294 K corresponded to the rates of formation of the g = 4.1 EPR signal. These results, together with the flash results, indicate that two steps are involved in both the generation and decay of the S3 EPR signal. The rates of formation and decay of both the S3 and QA(-)Fe EPR signals were measured at 250, 273, and 294 K. A kinetic model is presented that accounts for these kinetic data and the yield of the S3 EPR signal. PMID- 8639679 TI - Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPII) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII. AB - This investigation is focused on the conformational behavior of the blood group Lewisa (Le(a)-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLNFPII. The latter two are more potent oligosaccharide ligands for the animal lectins, E- and L-selectin, and the natural killer cell receptor, NKR-P1, than are the shorter chain analogs based on the trisaccharide Le(a) domain. We report here that the three oligosaccharides based on the fucopentasaccharide have very similar average solution conformations as determined from NMR spectroscopical parameters, in particular 13C chemical shift differences. From restrained simulated annealing and restrained molecular dynamics (MD) simulations performed in order to determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that are in good agreement with experimental parameters, such as rotating-frame Overhauser effects (ROE's) and long-range 1H,13C coupling constants across the glycosidic linkages. In these model structures the Le(a) domain at the non-reducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The Gal beta 1-4Glc linkage at the reducing end is also rather rigid, showing only little more flexibility than the Le(a) domain. However, the NeuAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkage in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = -20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequence 3' sialyl Le(x), and (phi = -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of drug designs, it will be important to establish which conformational distribution is assumed for the GlcNAc beta 1-3Gal linkage in these longer chain oligosaccharides in the bound state. PMID- 8639680 TI - Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form. AB - The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure. The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible. PMID- 8639681 TI - Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine. AB - Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946 30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements. PMID- 8639683 TI - Mutagenic and thermodynamic analyses of residual structure in the alpha subunit of tryptophan synthase. AB - The alpha subunit of tryptophan synthase from Escherichia coli has been previously shown to contain residual structure at 5 M urea, conditions where the secondary structure is entirely disrupted and the tyrosine residues are exposed to solvent [Saab-Rincon, G., Froebe, C. L., & Matthews, C. R. (1993) Biochemistry 32, 13981-13990]. The residual structure can be monitored by one-dimensional NMR spectroscopy studies of histidine 92 whose C epsilon proton is sensitive to the slow exchange between this form and the unfolded protein. The temperature dependence of the cooperative urea-induced unfolding transition between intermediate and unfolded forms demonstrates that this process involves negative values for both the enthalpy and entropy changes at 25 degrees C. The effects of replacements of several nonpolar side chains adjacent to histidine 92 on the slopes and midpoints of the unfolding transition curve show that these side chains participate in the residual structure. A 15-residue peptide spanning histidine 92 and the mutated residues, however, is not sufficient to define this structure. These results demonstrate that the residual structure in the alpha subunit is stabilized by the hydrophobic effect and may involve side chains which are distant in sequence to histidine 92. PMID- 8639682 TI - Ferrous active site of isopenicillin N synthase: genetic and sequence analysis of the endogenous ligands. AB - Isopenicillin N synthase (IPNS) from Streptomyces jumonjinensis (M(r) 37,902) is a non-heme ferrous iron-containing enzyme that catalyzes the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies [reviewed in Cooper, R. D. (1993) Biomed. Chem. 1, 1-17] have led to a model for the coordination environment of the iron atom possessing three histidine and one aspartic acid endogenous ligands and a solvent molecule. A refinement of that model proposes that formation of the Fe(II) IPNS-ACV complex occurs with displacement of the H2O from the metal center and that one of the histidines is subsequently replaced by a solvent molecule on binding of dioxygen. Here we report genetic studies to determine the nature and location of the endogenous ligands in the S. jumonjinensis IPNS primary amino acid sequence that constitute the ferrous active site. Site-directed mutagenesis was used to exchange each of the seven histidines and the five aspartic acids that are conserved in bacterial and fungal IPNS proteins. Biochemical analysis of the alanine-substituted mutant proteins shows that two histidines, His212 and His268, and one aspartic acid, Asp214, are essential for enzyme activity. The other mutant enzymes have specific activities 5-68% that of wild type. Sequence analysis of 10 IPNS and 42 other non-heme ferrous iron-dependent dioxygenases reveal the presence of a common motif, HisXAsp(53-57)XHis, which in IPNS contains the identical two histidines and one aspartic acid essential for function. Accordingly, we have assigned residues His212, His268, and Asp214 as three of the four endogenous ligands postulated to form the IPNS ferrous active site. Compelling support for these conclusions comes from the recent crystal structure determination of the manganese form of a fungal IPNS [Roach et al. (1995) Nature 375, 700-704]. PMID- 8639685 TI - Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin. AB - The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2 methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite. PMID- 8639684 TI - Thermodynamic cycles as probes of structure in unfolded proteins. AB - The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone. PMID- 8639686 TI - Association between the amino- and carboxyl-terminal halves of lactose permease is specific and mediated by multiple transmembrane domains. AB - Lactose permease of Escherichia coli is a polytopic membrane transport protein containing 12 membrane-spanning segments. When the amino (N6)- and carboxy (C6) terminal halves are expressed as separate gene fragments, association of the first half (N6) of the permease with the second half (C6) is necessary for stable insertion of C6 [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329]. In this report we demonstrate that N6-C6 interaction is specific, since N6 fragments derived from the structurally related tetracycline or sucrose transporters are unable to stabilize insertion of C6 from lactose permease. Furthermore, this association appears to be mediated by multiple transmembrane domains, since co-expression of progressively truncated N-terminal fragments (N5, N4, N3, N2, N1) with C6 leads to markedly decreased, but detectable amounts of C6 in the membrane. The results indicate that the N- and C-terminal six transmembrane domains of lactose permease are integrated into the membrane as separate units, and insertion of the C-terminal half is directed by specific interactions with the N-terminal half of the protein. PMID- 8639687 TI - Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques. AB - The phosphorylation of a peptide substrate by the catalytic subunit of cAMP dependent protein kinase was monitored over short time periods (2-1000 ms) using a rapid quench flow mixing device and a radioactive assay. The production of phosphokemptide [LRRAS(P)LG] as a function of time is characterized by a rapid "burst" phase (250 s-1) followed by a slower, linear phase (L/[E]t = 21 s-1) at 100 microM Kemptide. The amplitude of this "burst" phase varies linearly with the enzyme concentration and represents approximately 100% of the total enzyme concentration, indicating that the "burst" phase is not due to product inhibition. The observed rate constants for the "burst" and linear phases and the "burst" amplitude vary hyperbolically with the substrate concentration. From these dependencies, a maximum "burst" rate constant of 500 +/- 60 s-1 and a Km and Kd for Kemptide of 4.9 +/- 1.4 and 200 +/- 60 microM were determined. The kcat and Km data extracted from the linear portion of the rapid quench flow transients are indistinguishable from those obtained by standard steady-state kinetic analyses using low catalytic subunit concentrations and a spectrophotometric, coupled enzyme assay. Both rate constants for the "burst" and linear phases decreased in the presence of Mn2+. The data imply that the phosphorylation of Kemptide by the catalytic subunit occurs by a mechanism in which the substrate is loosely bound, is rapidly phosphorylated at the active site, and is released at a steady-state rate that is likely controlled by the dissociation rate constant for ADP. The combined pre-steady-state kinetic data establish a comprehensive, kinetic mechanism that predicts all the steady-state kinetic and viscosometric data. This study represents the first chemical observation and characterization of phosphoryl transfer at the active site of a protein kinase and will be useful for further structure-function studies on this and other protein kinases. PMID- 8639689 TI - Thermodynamic studies of the core histones: pH and ionic strength effects on the stability of the (H3-H4)/(H3-H4)2 system. AB - The self-associative behavior and the thermal stability of the H3/H4 histone complex was studied in low-ionic strength conditions by several physicochemical techniques, including differential scanning calorimetry and circular dichroism spectroscopy. At neutrality, the major molecular species present in solution is the (H3-H4)2 tetramer. Its thermodynamic properties cannot be studied directly though, since its thermal denaturation is completely irreversible even at the lowest salt concentrations. However, a complete thermodynamic analysis can be performed at low ionic strength and pH 4.5, where the (H3-H4)2 tetramer is quantitatively dissociated into two H3-H4 dimers and where almost complete reversibility of the thermal transitions is attained. The unfolding transition temperature of the 26.5 kDa H3-H4 dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. The thermal denaturation of the H3-H4 dimer is characterized by the presence of a single calorimetric peak, centered at 58 degrees C, with a corresponding enthalpy change of 25 kcal/mol of a 13 kDa monomer unit and a change in heat capacity upon unfolding of about 0.6 kcal/(K mol of 13 kDa monomer unit). The complex between histones H3 and H4 (tetramer or dimer) is stable between pH 9.5 and 3.0. At pH 1.5, the system is almost completely unfolded at all temperatures. At low ionic strengths and pH values between 5.0 and 2.5, the H3-H4 dimer behaves as a highly cooperative system, melting as a single unit; i.e. individual H3 and H4 folded monomers are not detectable during the treatment. The two-state mechanism accounting for the unfolding of the H3-H4 dimer at pH 4.5 is the same as that described for the H2A-H2B dimer at neutrality. Just like for the H2A and H2B histones, the H3 and H4 polypeptides are properly folded only when assembled as H3-H4 dimers or in higher-order histone assemblies. Therefore, coupling along the interfaces of the two chains within the heterodimer is the major factor contributing to the stabilization of the secondary and tertiary structures of the chains as well as of the histone dimers. PMID- 8639688 TI - Synthesis and photochemical properties of a kainate precursor and activation of kainate and AMPA receptor channels on a microsecond time scale. AB - Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolylpropionate (AMPA) receptors are transmembrane proteins that can form ion channels upon binding a specific ligand. The receptors are located at major excitatory synapses in the mammalian central nervous system. Kainate and AMPA receptors participate in many physiological activities of the brain, including learning and memory, and are involved in many neurological disorders. Elucidation of the mechanisms of receptor transmembrane channel formation, inhibition, and regulation is important in understanding fundamental central nervous system function and in designing potential therapeutic agents. Kainate can activate both kainate and AMPA receptors, leading to channel opening in the microsecond to millisecond time region. A newly developed laser pulse photolysis technique, with a microsecond time resolution, has been successfully used to study the chemical reactions of receptor proteins in the microsecond to millisecond time region. To apply the technique to kainate and AMPA receptors, a photolabile kainate precursor in which a caging group, the alpha-carboxy-2-nitrobenzyl group, is attached to the gamma carboxyl group of kainic acid has been synthesized. The photolytic release of free kainate from the caged kainate on the microsecond time scale, initiated by a pulse of laser light at 308 nm, was measured. The quantum yield is 0.34 at pH 6.8 and room temperature. The half-life of the major component (approximately 86%) of the photolytic reaction is 45 microseconds, while that of the minor component (approximately 14%) is 0.7 ms. The effects of the caged kainate on kainate and AMPA receptors endogenously expressed in rat hippocampal neurons were also evaluated. Caged kainate (750 microM) did not activate the receptor channels, nor did it potentiate or inhibit the kainate response. Photolysis of the caged kainate by a pulse of 333-nm laser light resulted in a rapid rise (with a t1/2 of 0.4 ms) in the whole-cell current due to the opening of kainate-activated receptor channels. The results presented demonstrate that this kainate precursor is suitable for rapid chemical kinetic investigations of the kainate and AMPA receptors in the microsecond to millisecond time region. PMID- 8639690 TI - Critical pressures in multicomponent lipid monolayers. AB - Epifluorescence microscopy has been used previously to study coexisting liquid phases in lipid monolayers of dihydrocholesterol and dimyristoylphosphatidylcholine at the air/water interface. This binary mixture has a critical point at room temperature (22 degrees C), a monolayer pressure of approx. 10 mN/m, and a composition in the vicinity of 20-30 mol% dihydrocholesterol. It is reported here that this critical pressure can be lowered, raised, or maintained constant by systematically replacing molecules of this phosphatidylcholine with molecules of a phosphatidylethanolamine, or an unsaturated phosphatidylcholine, or mixtures of the two, while maintaining the dihydrocholesterol concentration at 20 mol%. Thus, even complex mixtures of lipids may be characterized by a single, well-defined second-order phase transition. In principle, such transitions might be found in biological membranes. PMID- 8639691 TI - Molecular cloning and tissue distribution of rat peptide transporter PEPT2. AB - A cDNA encoding rat H(+)- coupled peptide transporter PEPT2 was isolated. The cDNA encoded a protein of 729 amino acids with 48% amino acid identity to the rat PEPT1. The mRNA expression of rat PEPT2 was predominant in the kidney. When expressed in Xenopus oocytes, rat PEPT2 stimulated the uptake of bestatin, a dipeptide-like drug. PMID- 8639692 TI - Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells. AB - Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel. PMID- 8639693 TI - Is vitrification involved in depression of the phase transition temperature in dry phospholipids? AB - Recent literature has suggested that the depression of the phase transition temperature (Tm) in dry phospholipids by sugars may be ascribed to vitrification of the stabilizing solute, rather than by the direct interaction between sugar and phospholipid we have proposed. Koster et al. ((1994) Biochim. Biophys. Acta 1193, 143-150) claim that the only necessity is that the glass transition (Tg) for the sugar exceed Tm for the lipid. Evidence is presented in the present paper that this is not sufficient. Based on the vitrification hypothesis of Koster et al., the predicted order of effectiveness in depressing Tm in dry dipalmitoylphosphatidylcholine (DPPC) is dextran > or = hydroxyethyl starch > stachyose > raffinose > trehalose > sucrose > glucose. In fact, the opposite order was seen. The effect of raffinose, sucrose, or trehalose on Tm in dry DPPC depends on the thermal history of the sample, as we have reported previously. When DPPC dried with trehalose is heated for the first time, Tm is about 55 degrees C, but on the second and subsequent heating scans Tm falls to about 25 degrees C. Koster et al. suggest that this effect is due to heating the sample above Tg rather than to melting the hydrocarbon chains. We present evidence here that all that is required is for the chains to be melted. Further, we show that retention of residual water by DPPC dried with trehalose depends on the drying temperature, but is independent of drying temperature with glucose, a finding that is consistent with direct interaction. We conclude that vitrification is not in itself sufficient to depress Tm in dry phospholipids. PMID- 8639695 TI - A voltage-dependent chloride channel from Tetrahymena ciliary membrane incorporated into planar lipid bilayers. AB - Membrane vesicles from cilia of Tetrahymena thermophila were incorporated into a planar phospholipid bilayer membrane, and single-channel currents across the planar membrane were recorded under voltage-clamp conditions. A novel and reproducible chloride channel was observed when a mixture of phosphatidylethanolamine and phosphatidylcholine was used to form the planar lipid membrane but not when acidic phospholipid mixtures such as asolectin or a mixture containing phosphatidylserine. Using symmetrical 100 mM KCl solutions, the single-channel conductance of the fully open state (O1) was 73.1 pS, with sub level (O2) conductance of 9.0 pS. The permeability ratio Pc1/Pk was calculated as 3.7, according to the Goldman-Hodgkin-Katz current equation. This channel exhibited characteristic voltage-dependent burst activities. With an increase in membrane potential, the lifetimes of both the burst and interburst states decreased. In the burst state, the frequency of transition between the O1 and O2 states was also voltage-dependent, mainly due to the decrease in the lifetime of the O1 state, with an increase in membrane potential. In addition, channel activity was inhibited by indanyloxyacetic acid-94 (IAA-94), an inhibitor of epithelial chloride channels. PMID- 8639694 TI - Antitumor sulfonylurea-inhibited NADH oxidase of cultured HeLa cells shed into media. AB - Conditioned culture media of HeLa S cells contain a soluble NADH oxidase activity inhibited by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N' -(4 chlorophenyl)urea (LY181984) similar to that associated with the outer surface of the plasma membrane. This activity was absent from media in which cells had not been grown and was present in conditioned culture media from which cells had been removed by centrifugation both for serum-containing and serum-free media. The Km with respect to NADH and response to thiol reagents were similar to those of the corresponding activity of the plasma membrane of HeLa cells. The conditioned HeLa culture media bound [3H]LY181984 with high affinity. Both antitumor sulfonylurea inhibited and -resistant forms of the NADH oxidase were isolated by free-flow electrophoresis. The antitumor sulfonylurea-inhibited activity was purified to apparent homogeneity and was identified with a 33.5 kDa protein with an isoelectric point of about pH 4.5. The 33.5 kDa protein from conditioned HeLa culture medium both bound [3H]LY181984 and retained an LY181984-inhibited NADH oxidase activity. A polyclonal antisera was raised in rabbits to the purified 33.5 kDa constituent from conditioned HeLa culture medium. The antisera blocked the activity of the LY181984-inhibited NADH oxidase activity, immunoprecipitated the activity and reacted with a 33.5 kDa protein on Western blots while preimmune sera did not. Also inhibited and immunoprecipitated was NADH oxidase activity from HeLa plasma membranes. The findings are consistent with the 33.5 kDa drug inhibited NADH oxidase activity of the culture media being a shed form of the corresponding native 34 kDa antitumor sulfonylurea-inhibited NADH oxidase activity of the HeLa cell plasma membrane. PMID- 8639696 TI - Characterization of P2Z purinergic receptors on phagocytic cells of the thymic reticulum in culture. AB - The thymic microenvironment is under intrinsic and extrinsic control circuits by several elements including hormones, neuropeptides, lymphokines, innervation and cell contact. P2 purinergic receptors have been described in a number of cells including macrophages, thymocytes, and other cells of the immune-inflammatory system. Here, we use the whole-cell patch-clamp technique and dye permeabilization assays to investigate the presence of ionic channels and purinergic receptors in one microenvironmental thymic component, namely the phagocytic cell of the thymic reticulum. At holding potentials ranging from -30 to -60 mV, applications of extracellular ATP in the vicinity of the cell membrane induce a transient and fast-activating inward current followed in most cells by an outward current. The whole event lasts 5-20 s. The inward current has a reversal potential close to 0 mV and the outward current can be ascribed to a Ca2+ -dependent K+ conductance. Both currents are inhibited by Mg2+, suggesting that the phenomenon is mediated by ATP4-. ATP-gamma-S can also induce both inward and outward currents. Exposure of phagocytic cells of the thymic reticulum to 5 mM ATP for 10 min induced permeabilization to lucifer yellow but not to the larger dyes trypan blue and rhodamine-dextran, suggesting a molecular weight cut off smaller than 900. These observations lead us to conclude that phagocytic cells of the thymic reticulum express P2Z purinergic receptors that can mobilize Ca2+, induce the opening of ionic channels and permeabilize the cell membrane. PMID- 8639697 TI - Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals. AB - The efficiency and selectivity of photosensitized damage to membrane functions may be influenced strongly by the identity of the initial reactive species formed by the photosensitizer. To test this possibility, a photosensitizer, rose bengal (RB), was used that resides in the plasma membrane and which generates singlet molecular oxygen (1O2*) upon excitation with visible light, and radicals plus 1O2* upon excitation with UV radiation. With this approach, 1O2* and radicals are formed at the same locations in the plasma membrane. The response of three plasma membrane functions, namely, proline transport, membrane potential, and membrane impermeability to charged dye molecules, was assessed. The efficiencies of the responses in the presence and absence of oxygen were compared per photon absorbed by RB at two wavelengths, 355 nm (UV excitation) and 532 nm (visible excitation). The efficiency of oxygen removal before irradiation was assessed by measuring the RB triplet lifetime. The three membrane functions were inhibited more efficiently at 355 nm than at 532 nm in the presence of oxygen indicating that the radicals are more effective at initiating damage to membrane components than 1O2*. The ratio of photosensitized effects at the two wavelengths in the presence of oxygen was the same for two membrane functions but not for the third suggesting that 1O2* and radicals initiate a common mechanistic pathway for damage to some membrane functions but not to others. Removing oxygen reduced the efficiency of 355 nm-induced photosensitization by factors of 1.4 to 7. The sensitivity of the three membrane functions to 1O2*-initiated damage varied over a factor of 50 whereas radical initiated damage only varied by a factor of 15. In summary, these results indicate that radicals and 1O2* formed at the same locations in the plasma membrane vary in their efficiency and specificity for membrane damage but may, in some cases, operate by a common secondary damage mechanism in the presence of oxygen. PMID- 8639698 TI - Chloride accumulation in freshly isolated Ehrlich ascites tumor cells: the role of the Na/K/2Cl cotransporter. AB - When Ehrlich ascites tumor cells are removed from the peritoneal cavity and incubated in a saline solution, cells lose water, sodium, lactate and hydrogen ions and gain chloride. The gain of intracellular chloride exceeds that predicted from passive distribution. As chloride has been purported to play a role in volume regulation, it was of interest to identify factors responsible for controlling or maintaining intracellular chloride out of electrochemical equilibrium in Ehrlich cells. The results demonstrate that chloride accumulation in freshly isolated Ehrlich cells is sensitive to bumetanide, low extracellular K+ and low extracellular Na+, and is insensitive to DIDS. We conclude that chloride accumulation occurs due to the activity of the Na/K/2Cl cotransporter. PMID- 8639699 TI - Towards the localization of the essential arginine residues in the band 3 protein of human red blood cell membranes. AB - The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5 maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding. PMID- 8639701 TI - Cytoplasmic and mitochondrial forms of yeast adenylate kinase 2 are N-acetylated. AB - Yeast major adenylate kinase (Aky2p), encoded by a single gene, occurs in two subcellular compartments, mitochondria and cytoplasm. Only 6-8% of the protein which has no cleavable presequence is imported into the organelle (Bandlow et al. (1988) Eur. J. Biochem. 178, 451-457). In the wild type two AKY2-derived signals (a major and a minor one) were detected by a monospecific antibody after two dimensional gel electrophoresis and Western blotting. The signals reflected identical electrophoretic mobilities and were absent from an AKY2-disrupted strain suggesting that they were due to differently modified forms of Aky2p. Two similar signals were found in a mutant defective in protein N-acetylation, however, the pI values of both spots were shifted towards alkaline pH by one charge. This indicated that both forms of Aky2p were N-acetylated in the wild type and that their charge difference was not caused by incomplete N-acetylation. This observation furthermore suggested that, in the wild type, two different modifications exist one of which is N-acetylation. The second modification remains unidentified. We analysed the influence of protein N-acetylation on mitochondrial import. Both versions of Aky2p occurred in the cytoplasm and in mitochondria. Their proportion was unchanged in the N-acetylation mutant showing that neither modification affected the efficiency of import of adenylate kinase into mitochondria. It is discussed that N-acetylation occurs during or immediately after translation in the cytoplasm so that import of adenylate kinase may ensue co-translationally. PMID- 8639700 TI - Effects of intracellular pH on high pressure-induced hemolysis of anion transport inhibitor-treated erythrocytes. AB - Effects of anion transport inhibitors such as 4,4'-diisothiocyanostilbene-2,2' disulfonate (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate on hemolysis of human erythrocytes at 200 MPa were examined by changing intracellular pH (7.2-7.9). These inhibitors suppressed the hemolysis at neutral pH but enhanced it at alkaline pH. However, such an enhancement was suppressed by cross-linking of membrane proteins using diamide. From the near-UV CD spectra of band 3 and the relation between hemolysis and anion transport in intact or trypsin-treated erythrocytes, it was found that such hemolytic properties were characterized by the binding of inhibitors to band 3. In addition, spectrin detachment from the erythrocyte membrane by high pressure was considerably suppressed by DIDS treatment at neutral pH, but not by DIDS labeling at alkaline pH. These results suggest that the interaction of the cytoplasmic domain of band 3 with the cytoskeleton, which is induced by the binding of ligands to the exofacial domain of band 3, is dependent on the intracellular pH, i.e., the linking is tightened at neutral pH but relaxed at alkaline pH. PMID- 8639702 TI - Rate of Na+/Ca2+ exchange across the plasma membrane of synaptosomes measured using the fluorescence of chlorotetracycline. Implications to calcium homeostasis in synaptic terminals. AB - It is shown that the fluorescence of chlorotetracycline (CTC) can be used to continuously monitor Ca2+ fluxes mediated by the Na+/Ca2+-exchanger of the plasma membrane of synaptosomes. The kinetics of Ca2+ uptake can be followed from the kinetics of the increase of CTC fluorescence with external Ca2+ concentrations in the micromolar range. Since the fluorescence of CTC is not sensitive to Ca2+ concentration below 20 microM this avoids any significant contribution of Ca2+ flux through Ca2+ channels to CTC fluorescence. By replacing KCl by choline chloride in the buffer to avoid plasma membrane depolarization it is shown that the amplitude of the CTC fluorescence change is dependent upon the Na(+)-gradient preimposed across the plasma membrane, and the rate constant of the kinetic process is dependent upon the Ca2+ concentration. The rate constant of the Ca2+ influx measured with depolarized and non-depolarized synaptic plasma membrane vesicles at 37 degrees C and pH 7.4 were 0.55 +/- 0.10 and 0.25 +/- 0.02 min-1, respectively. The overall rate of Na+/Ca2+ exchange calculated under conditions close to physiological Na+ and Ca2+ gradients and membrane resting potential ranged from 15 to 25% of the activity of the plasma membrane Ca2+ pump under these experimental conditions. The results also point out that membrane depolarization increases approx. 2-fold the rate of Na+/Ca2+ exchange in synaptic plasma membrane vesicles. PMID- 8639703 TI - Asymmetric and functional reconstitution of band 3 into pre-formed phosphatidylcholine vesicles. AB - Human erythrocyte band 3 protein was purified in 0.1% Triton X-100 and reconstituted into pre-formed phosphatidylcholine vesicles by a Triton X-100 mediated procedure [1]. Band 3 (and its transmembrane domain) could be asymmetrically reconstituted into phosphatidylcholine vesicles with retention of sulfate transport activity which showed behaviour characteristic of red cell anion transport in response to pH, H2DIDS and temperature. Successful reconstitution was also possible using high mol ratios of band 3/phosphatidylcholine (1:200), which are not achieved by any other method. PMID- 8639704 TI - The WT1 Wilms' tumor suppressor gene: how much do we really know? PMID- 8639705 TI - Retroviral insertional mutagenesis as a strategy to identify cancer genes. PMID- 8639706 TI - Mechanisms of eukaryotic transcription: surfaces, complexes, and contexts. (Cold Spring Harbor Cancer Cells Meeting, August 30-September 3, 1995). AB - As with other biological processes, evolution has resulted in numerous, alternative pathways to achieve regulation of gene expression. In the elaborate series of steps required to transcribe a gene, necessitating assembly and subsequent disassembly of multiple complexes, subtle alterations in specific protein-protein interactions can lead to dramatically different transcriptional consequences. PMID- 8639707 TI - P53 and angiogenesis. PMID- 8639708 TI - Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5'-phosphate. AB - The alpha-amino group of Lys-1 and the epsilon-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5'-phosphate (PLP) separately. The effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decreased obviously during denaturation by low concentrations of GdnHCl indicating conformational change of the active site is a parallel event to the inactivation of the enzyme. Time-correlated fluorescence lifetime measurements revealed the existence of two conformational states of PLP modified RNase A and a shift of the conformation in favor of the slow-decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. PMID- 8639709 TI - Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation. AB - A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) [1]. But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability. PMID- 8639711 TI - Identification of a tyrosine residue in ovine placental lactogen as essential for its binding to receptors. AB - Nitration of ovine placental lactogen (oPL) with a 10-fold molar excess of tetranitromethane over protein content resulted in the modification of 0.8 tyrosine residue. No conformational changes were observed by either fourth derivative spectral analysis or circular dichroism. Nitration significantly decreased the binding capacity of the hormone to lactogenic and somatogenic rat liver receptors. This binding capacity was not restored by reduction of the nitro groups, thus indicating that the decrease was not due to the difference in pK between tyrosine and nitrotyrosine. The nitrotyrosine-containing peptide was isolated from a tryptic digest by HPLC and its modification extent was of 67%, which is consistent with the decrease in binding capacities (65% and 70%). Its amino acid sequence was determined and the modified tyrosine residue was identified as Tyr-46. These results provide the first evidence of the involvement of a tyrosine residue in the binding of oPL to both lactogenic and somatogenic receptors. This tyrosine appears to be a shared binding epitope between oPL and the prolactins. PMID- 8639710 TI - Interaction of a troponin I inhibitory peptide with both domains of troponin C. AB - Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC. PMID- 8639712 TI - Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine. AB - We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis. PMID- 8639713 TI - Retinoid binding to retinol-binding protein and the interference with the interaction with transthyretin. AB - The retinol carrier retinol-binding protein (RBP) forms a complex with the thyroid hormone binding protein transthyretin in the plasma of a number of vertebrate species. The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with transthyretin have been investigated by means of fluorescence anisotropy studies. The presence of two independent and equivalent RBP binding sites per transthyretin molecule has been established for proteins purified from species distant in evolution. Although the natural ligand retinol participates in the interaction between retinol-RBP and transthyretin, its binding to RBP is not a prerequisite for protein-protein interaction. The dissociation constants of human transthyretin binding liganded and unliganded forms of human RBP were determined to be: all-trans retinol-RBP, Kd approximately 0.2 microM; apoRBP, Kd approximately 1.2 microM; all-trans retinoic acid-RBP, Kd approximately 0.8 microM; all-trans retinyl methyl ether-RBP, Kd approximately 6 microM. The complex of RBP with the synthetic retinoid fenretinide, which bears the bulky hydroxyphenyl end group, exhibits negligible affinity for transthyretin. The replacement of RBP-bound retinol with synthetic retinoids affects RBP-transthyretin recognition to an extent that appears to be well correlated with the nature and steric hindrance of the groups substituting the retinol hydroxyl group, consistent with their location at the interface between the contact areas of RBP and transthyretin. PMID- 8639714 TI - A type I ovine interferon with limited similarity to IFN-alpha, IFN-omega and IFN tau: gene structure, biological properties and unusual species specificity. AB - A gene encoding a 195 amino-acid (a.a.) polypeptide with a putative 23 a.a. signal sequence that had about 60% a.a. sequence identity to ovine interferon omega (OvIFN-omega) and 55% or less identity to BoIFN-tau, OvIFN-tau and all known IFN-alpha and -beta has been identified from an ovine genomic DNA library. Surprisingly, it shared almost complete identity to genes for rabbit IFN-omega within its coding sequence and proximal promoter region, although the two were different in their 3'-ends. This IFN (tentatively termed ovine IFN-omega variant, OvIFN-omegav), purified in recombinant form from E. coli, had normal antiviral activity when tested on sheep fetal tongue and brain cells and rabbit kidney cells, but very low activity towards bovine, goat and human cells. It competed with 125I-labeled BoIFN-tau for binding to IFN receptors on ovine cells. Expression of OvIFN-omegav was not detected by reverse transcription-PCR either in ovine peripheral blood leukocytes infected with Sendai virus, or in any other tissues examined. OvIFN-omegav may represent a previously unrecognized, non virally inducible type I subtype distinct from IFN-alpha, -beta, -omega and -tau. The presence of a conserved gene in rabbit and sheep could reflect a recent interspecies transfer. PMID- 8639715 TI - Effects of salts of alkali earth metals and calcium chloride on the stability of cytochrome c and myoglobin. AB - This study suggests a procedure by which binding of denaturants could be detected without any additional information other than that provided in the denaturation profiles of proteins. Two predominantly alpha-proteins, namely ferricytochrome c and metmyoglobin were denatured by guanidine hydrochloride (GdnHCl) in the presence of low fixed concentrations of salts at 25 degrees C and transition between native and denatured states was followed by absorbance measurements in the visible region (500-350 nm). The raw data were converted into transition curves from which Cm, the midpoint of GdnHCl-induced transition, and deltaGapp, the free energy changes on denaturation, were calculated assuming a two-state mechanism, and values of deltaGapp at zero concentration of the denaturant were estimated. It has been observed (1) that chlorides of Na, K, Cs, and Rb do not affect the native conformation of proteins, (2) that GdnHCl-induced denaturations of proteins in presence and absence of sodium bromide, sodium perchlorate and salts of lithium and calcium are reversible, (3) that optical properties of the GdnHCl-denatured state of proteins remain unchanged in presence of the second denaturant, (4) Cm decreases with an increase in the denaturant concentration, and (5) that except for GdnHCl there exist one or more binding sites on the native proteins for the denaturants. PMID- 8639716 TI - Iron site structure of two irreversible hemichromes from human hemoglobin, untreated and oxidized to sulfoxide at MetD6(55)beta. AB - The Fe K-edge X-ray absorption near-edge structure (XANES) spectra of two irreversible human hemichromes, spontaneously formed from HbA and HbMetSO (a hemoglobin derivative, where MetD6(55)beta has been previously oxidized to sulfoxide by chloramine T) were determined. The results show that the hemichrome from HbMetSO is characterized by the distal histidyl imidazole moved within the bonding distance of the heme iron. Such structure is different from that of the hemichrome spontaneously produced from native human hemoglobin, which probably has a hydroxide group as sixth heme ligand. PMID- 8639718 TI - Conformation, stability and interactions of corneal keratan sulfate proteoglycan. AB - We have monitored the molecular conformation, stability, interaction and dynamics of keratan sulfate proteoglycan, the major structural protein component of the cornea, in solution, by studying the fluorescence spectral features of its tryptophan residues as component-specific intrinsic spectral probes (collagen, the other structural component of the cornea, has no tryptophans). Our study suggests that the Trp region of the molecule is in a motionally restricted environment as it exhibits a fluorescence red-edge effect and shows dipole relaxation. The extrinsic spectral probe 8-anilinonaphthalene 1-sulfonate reveals keratan sulfate proteoglycan to possess significant surface hydrophobicity. This dual character of keratan sulfate proteoglycan allows us to label it as an 'ambidextran' proteoglycan. The molecule is stable between pH 5-8 and has a Tm value of 72 degrees C. Disulfide bonds play a role in the stability of the molecule. KSPG is seen to interact with collagen and the model compound, poly(L proline). Interaction of the proteoglycan with unilamellar vesicles appears to be more interfacial than penetrative. This dual interaction displayed by KSPG with collagen and with lipid assemblages suggests that it plays the role of a 'filler' in the extracellular matrix of the cornea. PMID- 8639717 TI - High substrate specificity and induction characteristics of trimethylamine-N oxide reductase of Escherichia coli. AB - Using a wide variety of N- and S-oxide compounds we have shown by kinetic analysis that only two N-oxides, trimethylamine-N-oxide and 4-methylmorpholine-N oxide, can be considered good substrates for trimethylamine-N-oxide (TMAO) reductase on the basis of their kcat/Km ratio. This result demonstrates that TMAO reductase possesses a high substrate specificity. Induction of the torCAD operon using the same S- and N-oxide compounds was also analyzed. We demonstrate that there is no correlation between the ability for a compound to be reduced by TMAO reductase and to induce TMAO reductase synthesis. PMID- 8639719 TI - A calorimetric study of the binding of AMP to liver glycogen phosphorylase b. AB - The energetics of the interaction between liver glycogen phosphorylase b and the adenosine 5'-monophosphate (AMP) have been studied by equilibrium dialysis and isothermal titration calorimetry (ITC) at 25 degrees C. A concomitant net release of protons with AMP to phosphorylase binding was detected carrying out calorimetric experiments in three buffers having different heats of ionization at 25 degrees C. Four binding sites were found for AMP in the dimeric enzyme, which would correspond to the activator and the inhibitor sites identified in the muscle isozyme. The affinity of AMP for these four sites is similar. Thus, the binding of AMP to the activator sites seems to be non-cooperative and it does not perform the conformational change necessary to activate the enzyme. Moreover, the inhibitor sites are occupied almost in the same extension that the activator sites, which would impair any activation of the enzyme. PMID- 8639720 TI - Involvement of tyrosine residue in the inhibition of plant vacuolar H(+) pyrophosphatase by tetranitromethane. AB - Plant vacuolar vesicles contain a novel H(+)-translocating pyrophosphatase (H(+) PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H(+) PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H(+)-translocating pyrophosphatase activity. The half maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H(+)-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H(+)-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H(+)-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H(+)-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H(+)-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H(+)-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H(+)-PPase. Taken together, we suggest that vacuolar H(+) pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM. PMID- 8639721 TI - Five-year survival curves: how much data are enough for patient-physician decision making in general surgery? AB - OBJECTIVE: To find out whether patients' preferences for a particular type of treatment are changed by the way in which the information is presented (point estimates-year 0 and year 5 data points-or two five-year survival curves). DESIGN: Prospective study. SETTING: University-based Department of Veterans Affairs Medical Center, USA. SUBJECTS: 236 Consecutive patients seen in a general medical outpatient clinic. INTERVENTIONS: Patients were asked to choose between two treatments, one with a 10% immediate mortality and 37% five-year survival (treatment A) and one with no immediate mortality and 22% five-year survival (treatment B). They were randomised to be given the survival data either as point estimates or as two five-year survival curves. MAIN OUTCOME MEASURES: Number of patients who chose each treatment. RESULTS: The mean age was 67 years (range 40 84) and mean duration of education was 13 years (range 0-22). Significantly fewer patients chose the treatment with better immediate survival when they were given the data as survival curves (47/140, 34% compared with 93/140, 66% p < 0.0001) than when they were given data as point estimates (57/96, 59%, compared with 39/96, 41%). CONCLUSION: Patients are significantly more likely to make different decisions when given more data (in the form of five-year survival curves which show short, medium, and long term results) than when they are presented with only point estimates. PMID- 8639722 TI - Immune and nutritional effects of early enteral nutrition after major abdominal operations. AB - AIM: To investigate the effect of early postoperative enteral nutrition enriched with arginine, RNA and omega-3 fatty acids on immunological and nutritional variables after elective curative operations for gastric or pancreatic cancer. DESIGN: Randomised controlled trial. SETTING: University hospital, Italy. SUBJECTS: 78 Consecutive patients who were to undergo curative operations for gastric or pancreatic cancer, 60 of whom were suitable for the study. INTERVENTIONS: Patients were randomly allocated to three groups (n = 20 each) according to the type of postoperative nutritional support: standard enteral diet, the same diet enriched with arginine, RNA, and omega-3 fatty acids or total parenteral nutrition. The daily nutritional goal was 25 kcal (105 kJ)/kg and 0.25 g nitrogen/kg for all patients. MAIN OUTCOME MEASURES: Serum concentrations of immunoglobulins, albumin, transferrin, prealbumin, retinol binding protein (RBP); cholinesterase activity, weight loss, duration of operation, operative blood loss; blood transfusion; delayed hypersensitivity responses, number of lymphocyte subsets, phagocytic ability of monocytes, number of interleukin-2 (IL-2) plasma receptors, interleukin-6 (IL-6) plasma concentrations, postoperative infections and sepsis scores. RESULTS: All enterally fed patients but one completed the nutritional programme. There were significant postoperative reductions in both nutritional and immunological variables in all groups. On postoperative days 4 and 8 prealbumin concentration (p < 0.05), RBP concentration (p < 0.05), delayed hypersensitivity responses (p < 0.05), phagocytic ability of monocytes (p < 0.01) and concentration of IL-2 receptors (p < 0.009) had all recovered more in the group receiving the enriched solution. There was no difference in the postoperative infection rates among the three groups, but the infections were less severe in the enriched group (p < 0.005). CONCLUSION: Early enteral feeding was well tolerated. Patients who received the enriched solution recovered both their nutritional and immunological status quicker than those in the other two groups. PMID- 8639723 TI - Changes in permeability of sheep's lungs by oleic acid are dose dependent. AB - OBJECTIVE: To evaluate a two-isotope technique to detect graded changes in pulmonary microvascular permeability. DESIGN: Open experimental study. SETTING: University hospital, Sweden. MATERIAL: Fifty-seven sheep. INTERVENTIONS: Catherisation of one carotid artery, pulmonary artery and central vein. Control group 1 (n = 10), control group 2 (n = 12) had an additional pulmonary artery balloon catheter inserted, experimental group 3 (n = 9) was given oleic acid 0.005 mg kg-1 BW, experimental group 4 (n = 12) received oleic acid 0.02 mg kg-1 BW and experimental group 5 (n = 11) 0.05 mg kg-1 BW. Groups 3-5 had all PA catheters. All animals were intubated and ventilated artificially. Duration of experiments was 6 hours. OUTCOME MEASURES: Transferrin was labelled in vivo with 113mIn chloride and erythrocytes with 99mTc following injection of stannous chloride. External gamma counting was corrected for background, decay and scatter. Blood activity was used as reference. Normalised slope index (NSI) and transferrin leak index (TLI) were calculated as measures of pulmonary microvascular permeability. RESULTS: A graded response in both NSI and TLI was found. Insertion of the PA catheter (group 2) significantly increased NSI from (group 1) (1.4 (0.1)) 10(-4) min-1 to (11 (2)) 10(-4) min-1 (p < 0.05). TLI increased significantly from (9 (2)) 10(-4) min-1 to (72 (13)) 10(-4) min-1. Oleic acid increased NSI significantly to (13 (1)) 10(-4) min-1, (32 (2)) 10(-4) min-1 and (61 (5)) 10(-4) min-1 in groups 3-5, respectively. Corresponding values for TLI were (95 (13)) 10(-4) min-1, (162 (6)) 10(-4) min-1 and (228 (26)) 10-4 min-1, respectively. CONCLUSION: The double-isotope technique of external monitoring of permeability changes to protein in the lungs was sensitive to pick up graded increments in leakage, related in a dose-dependent way to lung injury. PMID- 8639724 TI - Prognosis in posttraumatic acute renal failure is adversely influenced by hypotension and hyperkalaemia. AB - OBJECTIVE: To see if it is possible to predict mortality in isolated post traumatic acute renal failure. DESIGN: Retrospective study 1984-1990 inclusive. SETTING: Teaching hospital, South Africa. SUBJECTS: Thirty-nine patients who developed isolated post-traumatic acute renal failure out of 106526 admissions for trauma. INTERVENTIONS: Standard aggressive management of traumatic injury and acute renal failure. MAIN OUTCOME MEASURE: Death. RESULTS: Fifteen of the 39 patients who developed post-traumatic acute renal failure died (39%). Blunt trauma from assaults was a major cause of acute renal failure (16/39, 41%). Hypotension and hyperkalaemia were the two main predictors of death having a mortality of 63% and 52%, respectively. CONCLUSION: Clinicians should be aware of the risks of hypotension and hyperkalaemia in injured patients. Preventive measures such as aggressive resuscitation and timely correction of serum electrolyte concentrations are essential in such patients. PMID- 8639725 TI - Incisional hernia after midline laparotomy: a prospective study. AB - OBJECTIVE: To study the healing of midline laparotomy wounds. DESIGN: Prospective clinical study. SETTING: County hospitals, Sweden and Iceland. SUBJECTS: 861 patients who underwent midline laparotomy between August 1989 and November 1992. 453 of whom were operated on during the first 20 months, and 408 of whom were operated on during the second 20 months after surgeons had been asked to adjust their technique to achieve a suture length: wound length ratio of more than 4. MAIN OUTCOME MEASURES: Wound dehiscence, wound infection, and incidence of incisional hernia at 12 months. RESULTS: 78/861 patients (9%) developed wound infection. This incidence correlated with previous midline laparotomy and degree of wound contamination. Mean (SD) suture length: wound length ratio increased from 3.6 (1.3) in the first period to 4.9 (1.6) during the second period (p < 0.01), as a result of a reduction in the stitch interval from 1.2 (0.2)-0.9 (0.2) cm (p < 0.01). All other recorded variables were comparable in both study periods. The rate of incisional hernia decreased from 19% (68 of 363) during the first period to 11% (35 of 320) during the second period (p < 0.01). Suture length: wound length ratio < 4, wound infection and age 60 years or more were significantly and independently associated with an increased incidence of incisional hernia. CONCLUSION: Suture technique is a major determinant of incisional hernia in continuously sutured midline laparotomies. Simple adjustments in technique can considerably improve late operative results. PMID- 8639726 TI - Operations in unselected patients with ulcerative colitis and indeterminate colitis. A long-term follow-up study. AB - OBJECTIVE: To assess the need for operative treatment for ulcerative colitis and indeterminate colitis. DESIGN: Retrospective study. SETTING: University Hospital, Sweden. SUBJECT: All patients diagnosed from 1958 to 1982, in Malmo, Sweden ulcerative colitis (n = 471) and indeterminate colitis (n = 100). MAIN OUTCOME MEASURES: Incidence of colectomy. RESULTS: The mean follow-up was 15 years. The incidence of colectomy was 7.51, 1.90 and 36.13/1000 person years for definite ulcerative colitis, probable ulcerative colitis and indeterminate colitis, respectively. The incidence of colectomy in patients with definite ulcerative colitis in the present study was low compared with other studies. In contrast to other reports, men had a threefold risk while extent of inflammation did not influence the incidence. Patients with indeterminate colitis were a high risk group with a high incidence of colectomy. The high incidence among patients with indeterminate colitis compared with that in patients with definite ulcerative colitis was also seen in subgroups such as patients with total colitis at diagnosis, in patients in remission after the first attack, and in patients with a severe attack. CONCLUSION: As patients with indeterminate colitis seem to be at increased risk of colectomy it could be important to distinguish them from those with ulcerative colitis. Today, however, it is not possible to identify all patients with indeterminate colitis early in the course of the disease. PMID- 8639727 TI - Surgical treatment of symptomatic rectosigmoid endometriosis. AB - OBJECTIVE: To assess the results of surgical treatment of symptomatic intestinal endometriosis. DESIGN: Retrospective study. SETTING: University hospital, The Netherlands. SUBJECTS: 14 patients in whom medical treatment had failed. INTERVENTIONS: Hysterectomy (n = 5), salpingo-oophorectomy (n = 8, bilateral in 5), posterior vaginal wall excision (n = 14), and partial resection (n = 6) or anterior wedge excision (n = 8) of the rectum. MAIN OUTCOME MEASURES: Blood loss, operating time, morbidity, and relief of symptoms. RESULTS: 9 patients became symptom free (64%), 5 of whom developed postmenopausal symptoms later on. 2 had unrelated vague abdominal symptoms and 3 more could not be assessed because they underwent in vitro fertilisation and developed stimulation-related abdominal symptoms. All were content with the operative results. CONCLUSIONS: Operations for rectosigmoid endometriosis are technically difficult with a long operating time and considerable blood loss, but they are successful in relieving symptoms when conservative treatment has failed. PMID- 8639728 TI - Simplified sutured sacral rectopexy for complete rectal prolapse in adults. AB - OBJECTIVE: To assess the results of simplified sutured sacral rectopexy for complete rectal prolapse. DESIGN: Prospective study. SETTING: University hospital, India. SUBJECT: 65 patients with complete rectal prolapse treated by modified sutured sacral rectopexy. MAIN OUTCOME MEASURES: Morbidity, mortality and recurrence. RESULTS: During a follow-up of 2-10 years (mean 5.4 years) none of the patients developed a recurrence of complete prolapse of the rectum but 3(5%) developed mucosal prolapse. The incontinence improved in 12 of 16 patients (75%) and constipation improved in 5 of 6 (83.3%). CONCLUSION: We conclude that the simplified sutured sacral rectopexy is a simple operation for treating complete rectal prolapse in adults, and gives good results. PMID- 8639729 TI - Chylothorax complicating Ivor Lewis oesophagectomy. PMID- 8639730 TI - Cysts of the tailgut. PMID- 8639731 TI - Primary perianal actinomycosis. PMID- 8639732 TI - Squamous metaplasia of the rectum: a surgical curiosity. PMID- 8639733 TI - Weight reduction after gastric bypass and horizontal gastroplasty for morbid obesity. PMID- 8639734 TI - Breast cancer. PMID- 8639735 TI - Present and future state of surgical management in breast cancer. PMID- 8639736 TI - Changes in the treatment of early breast cancer at the Mayo Clinic: 1988-1992. AB - OBJECTIVE: To investigate the temporal patterns in the surgical management of early (stages 0-2) breast cancer at Mayo Clinic between 1988 and 1992. DESIGN: Retrospective study. SETTING: Academic medical center, United States. SUBJECTS: 2551 patients with stage 0-2 primary breast cancer who underwent surgical treatment. MAIN OUTCOME MEASURE: Change in the number of patients who underwent breast conserving surgery during the five year period. RESULTS: The overall number of patients who underwent breast conserving surgery ranged from 73/498 (15%) in 1988 to 187/547 (34%) in 1992. When the patients who were ineligible to undergo breast conservation were subtracted from those who underwent mastectomy (leaving only those who chose mastectomy), the proportion nearly reached 50% in 1991, and breast conservation procedures exceeded mastectomies in 1992. CONCLUSIONS: The majority of women primarily treated for early breast cancer during the five year period 1988-1992 undergo mastectomy. The reasons appear to be multifactorial, but a significant percentage are not eligible for breast conservation according to our criteria. PMID- 8639737 TI - Epidemiology of breast cancer: the Nordic contribution. AB - In the Nordic countries, breast cancer, is the most common malignant disease among women and the incidence has been increasing steadily for the past 30 years. The age-specific incidence curve of female breast cancer suggests that the menopause has a protective effect. Nordic studies have provided evidence of an increased breast cancer risk in women of low parity, those who have their first child late, and those who have used oral contraceptives and hormone replacement therapy for a long time. PMID- 8639738 TI - Cloning and initial characterization of 14 myb-related cDNAs from tomato (Lycopersicon esculentum cv. Ailsa Craig). AB - myb-related transcription factors contain highly conserved DNA-binding domains. Using a mixture of degenerate oligonucleotides derived from the highly conserved region as probe, 14 myb-related clones were isolated from a cDNA library constructed using tomato hypocoyl mRNA. The expression of these clones was studied by northern blot analysis using poly(A)+ RNA from 7 tissue types (hypocotyl, leaf, root, green and red fruit, immature and mature flower). This study has revealed a wide range of expression patterns which include multiple and single transcripts, some of which show marked tissue specificity. Two clones showing different expression patterns have been fully sequenced. The DNA-binding domains of these two tomato myb clones are compared with myb genes from other plant species and organisms. Of the three clones analysed so far by Southern hybridization, two are single-copy genes and one has multiple genomic copies. PMID- 8639739 TI - Isolation and characterization of a Tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated. AB - Sequence analysis of the two cDNA clones 47/11 and 50A which were isolated by differential screening of an explant cDNA library obtained from the monocot Tritordeum (hexaploid hybrid of diploid wild barley and tetraploid wheat lines) reveals that both clones include the same open reading frame (ORF). The sequence of this ORF shows a high degree of similarity with dicot S-adenosylmethionine decarboxylase (SAMDC) gene sequences and contains regions highly conserved in all known SAMDC sequences. It is further shown that the sequence represented by the cDNA clones 47/11 and 50A is derived from the wild barley (Hordeum chilense) genome, where it is present as a single-copy gene. Northern analyses indicate the corresponding transcript to accumulate in response to wounding and the transcript level changes with a circadian rhythm, having a beak in the middle of the light period. The periodicity continues in constant light, but is changed in constant darkness. PMID- 8639740 TI - Isolation and characterization of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor. AB - A full-length cDNA encoding the 206 amino acid open reading frame of a trypsin/chymotrypsin inhibitor abundant in the corms of giant taro (Alocasia macrorrhiza) was isolated. An internal fragment was cloned using degenerate primers corresponding to a region of the mature protein sequence and the 'rapid amplification of cDNA ends' (RACE) method used to generate a composite cDNA sequence. The length of the cDNA was close to the predicted size of the corresponding transcript deduced from northern blot analysis of corm mRNA. The inhibitor was expressed strongly in the mature corm, at low levels in leaf blades and petioles but not in roots. Southern blot analysis of the giant taro DNA indicated that this inhibitor is encoded by a small multigene family and this was further supported by the isolation of two different sequence classes from corm cDNA using primers to the ends of the composite sequence. PMID- 8639741 TI - Cloning and characterization of an Arabidopsis thaliana cDNA clone encoding an organellar isoform of serine acetyltransferase. AB - We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15. The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent Km for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm3 respectively) on the cysE mutant. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence. Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences. PMID- 8639742 TI - Isolation by PCR of a cDNA clone from pea petals with similarity to petunia and wheat zinc finger proteins. AB - The C2H2 TFIIIA/Kruppel class of zinc finger proteins are an important group of regulatory nucleic acid binding factors and have been extensively studied in humans, Drosophila and yeast. We have employed 3' RACE PCR, using a highly degenerate oligonucleotide primer, for the facile isolation of a C2H2 zinc finger protein cDNA (Pszf1) from pea petals. The Pszf1 cDNA open reading frame potentially encodes a protein with two widely separated zinc fingers similar to zinc finger proteins from petunia and wheat. This class of two-fingered zinc finger proteins, possessing a wide and variable linker sequence, appears to be unique to plants. Three regions outside the zinc finger domains are also conserved between the members of the plant zinc finger protein family and one of these regions is a candidate nuclear localisation signal. The Pszf1 amino acid sequence is most similar to that of the petunia Epf1 protein, they possess an interfinger linker sequence of approximately the same length and they have a similar expression pattern with maximal transcript accumulation in mature petals, suggesting that Pszf1 may be the pea homologue of the petunia Epf1 zinc finger gene. PMID- 8639743 TI - Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings. AB - A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome. PMID- 8639744 TI - Comparison of the expression patterns of genes coding for wheat gluten proteins and proteins involved in the secretory pathway in developing caryopses of wheat. AB - The synthesis of gluten proteins in the developing caryopsis of wheat is highly coordinated, with mRNAs for the various groups being detected from 11 days after anthesis, and the proteins from about 14 days. In contrast, the levels of transcripts for BiP, PDI and PPI are highest at earlier stages of development. The levels of transcripts for two small GTP binding proteins involved in the secretory pathway (Rab1 and Rab5) are also highest early in development, which is consistent with the retention of most of the gluten proteins within the ER to form protein bodies. PMID- 8639745 TI - Seeing blue: the discovery of cryptochrome. PMID- 8639746 TI - Expression analysis of a sucrose synthase gene from sugar beet (Beta vulgaris L.). AB - To investigate the expression pattern of sucrose synthase, a cDNA from tap roots of sugar beet (Beta vulgaris L.) was isolated using a heterologous sucrose synthase cDNA from potato. The 2762 bp long cDNA clone designated SBSS 1 encodes for a 822 amino acid polypeptide of a predicted molecular mass of 93.7 kDa. The deduced amino acid sequence of sugar beet sucrose synthase has homologies of 65 70% when compared to predicted amino acid sequences of sucrose synthases from other species. RNA blot analysis shows that SBSS1 is expressed most predominant in tap root under normal growth conditions. Cold treatment and anaerobiosis lead to an increase in the steady-state levels of SBSS 1 mRNA in leaf and root tissue. In tap root slices, sugars in various concentrations had no influence on the SBSS 1 transcript level. On the other hand, wounding resulted in a decreased transcript level. PMID- 8639747 TI - Expression of protein disulfide isomerase is elevated in the endosperm of the maize floury-2 mutant. AB - A maize protein disulfide isomerase (PDI, EC 5.3.4.1) cDNA clone was isolated and characterized. The deduced amino acid sequence contains two regions characteristic of the active sites for PDI and a carboxyl-terminal endoplasmic reticulum (ER) retention sequence, Lys-Asp-Glu-Leu. Southern blot analysis indicated the maize PDI is encoded by a single gene that maps to the short arm of chromosome 4. When isolated from the cisternal and protein body ER, the PDI protein resolves into a fast and a slow form on SDS-PAGE. During endosperm development, the PDI RNA level increases between 10 and 14 days after pollination. In floury-2 (fl2) endosperm, which contains an abnormally processed alpha-zein protein, PDI expression is significantly increased, and the level of PDI protein and RNA is positively correlated with the dosage of fl2 alleles. The increase of PDI in fl2 occurs mainly in the cisternal ER fraction, whereas the most dramatic increase of binding protein (BiP) is in the protein body ER. We propose that the induction of PDI in the fl2 mutant reflects its role as a molecular chaperone, and that PDI functions in concert with BiP at different stages of zein processing and assembly into protein bodies. PMID- 8639748 TI - Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions. AB - Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N sufficient conditions. PMID- 8639750 TI - Isolation, characterization and expression of the maize Cat2 catalase gene. AB - The maize Cat2 gene was isolated by direct cloning and PCR. The clones were mapped and sequenced. The start site of transcription was determined by primer extension. Computer analysis of the 1.6 kb Cat2 promoter sequence has revealed an obvious TATA box, tow GC boxes, a putative GA response element, and several ACGT core sequences known to have diverse regulatory functions in plants. Several other protein binding motifs were also identified within 800 bp upstream from the transcriptional start site. Five introns were identified in the Cat2 coding region. All five Cat2 introns are located in exactly the same position as five of the six introns in Cat1. Two of the Cat2 introns are located in the same position as the two Cat3 introns. The identical positioning of these introns suggests an evolutionary link between all three maize catalase genes. The response of the CAt2 gene to plant growth regulators was examined. Results clearly showed that the response of CAt2 to several environmental factors are developmental stage dependent. Thus, complex regulatory mechanisms appear to be involved in the regulation of Cat2 expression in maize. PMID- 8639749 TI - An endochitinase gene expressed at high levels in the stylar transmitting tissue of tomatoes. AB - A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5'-flanking DNA from the Chi2;1 gene was shown to drive high level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles. PMID- 8639751 TI - Degradation of mistargeted OEE33 in the chloroplast stroma. AB - OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease. PMID- 8639752 TI - Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis). AB - Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level. PMID- 8639753 TI - A low-temperature-responsive gene from barley encodes a protein with single stranded nucleic acid-binding activity which is phosphorylated in vitro. AB - A low-temperature-responsive gene, blt 801, isolated from a winter barley (Hordeum vulgare L.) cDNA library prepared from leaf meristematic tissue, was sequenced. The deduced amino acid sequence predicts a glycine-rich RNA-binding protein (GR-RNP) which was homology to stress-responsive GR-RNPs from several other plant species. BLT 801 is a two-domain protein, the amino-terminal domain comprises a consensus RNA-binding domain similar to that found in many eukaryotic genes and the carboxy-terminal domain is extremely glycine-rich (68.5% glycine). Blt 801 mRNA also accumulates in response to the phytohormone abscisic acid. The protein encoded by blt 801 has been produced as a recombinant fusion protein using a bacterial expression vector. The fusion protein, a chimaera of glutathione S-transferase and BLT 801, has been used in studies to determine nucleic acid binding and other characteristics. Binding studies with single stranded nucleic acids show that BLT 801 has affinity for homoribopolymers G, A and U but not C, it also binds to single-stranded DNA and selects RNA molecules containing open loop structures enriched in adenine but low in cytosine. Blt 801 has a consensus motif for phosphorylation by cAMP protein kinase (PKA) at the junction between the two domains which can be phosphorylated by PKA in vitro and which, by analogy to animal studies, may have significance for controlling enzyme function. PMID- 8639754 TI - Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato. AB - Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whither the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. The increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts synergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions. PMID- 8639755 TI - Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings. AB - A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family. PMID- 8639756 TI - Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations. AB - The S3 allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S1 allele, preceded by an 18 amino acid signal sequence. Expression of the S3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S1 protein. In contrast to other S proteins identified to date, S3 protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of Western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S3 allele with antiserum raised to S3e, revealed a protein (S3s) which was indistinguishable in pI and Mr from that in the Shirley population. A cDNA encoding S3s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level. PMID- 8639757 TI - Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria. AB - A cDNA clone (RaRO47) encoding a sulfotransferase (ST) has been isolated from Arabidopsis cell suspensions. The deduced polypeptide of 302 amino acids is highly related to plant flavonol sulfotransferases (FSTs), characterized for the first time in Flaveria, and also to STs from animal tissue. The expression of the Arabidopsis ST gene(s) corresponding to RaR047 was examined during different developmental stages. It was found that, at the level of steady-state mRNA, expression of gene(s) encoding this ST was rapidly induced in the aerial parts of young seedlings, and during growth of Arabidopsis cell cultures. No expression could be detected in roots. Treatment of Arabidopsis seedlings with hormonal or stress-related compounds, showed that RaR047 mRNA accumulation was more particularly induced in response to salicylic acid and methyl jasmonate. Furthermore, in the leaves of mature plants or in cell suspensions, accumulation of RaR047 mRNA was observed upon infection with bacterial pathogens. This expression was observed preferentially in response to avirulent pathogens causing an hypersensitive reaction, as compared to virulent pathogens, which lead to disease. PMID- 8639758 TI - The transcriptional control of hematopoiesis. PMID- 8639759 TI - In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells. AB - Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG-2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome. PMID- 8639760 TI - Enhanced engraftment of hematopoietic progenitor cells in mice treated with granulocyte colony-stimulating factor before low-dose irradiation: implications for gene therapy. AB - Gene therapy for inherited disorders of blood cells will require both efficient methods for stable gene transfer and nonablative bone marrow conditioning regimens to allow engraftment of modified hematopoietic progenitor cells (HPCs). We have used a sensitive murine system for detecting HPC engraftment using congenic C57BL/6 mice that differ at the Ly5 locus, which encodes the leukocyte common antigen. The system relies on the ability of monoclonal antibodies with specificity for Ly5.1 and Ly5.2 (revised nomenclature: CD45.1 and CD45.2, respectively) to distinguish donor and recipient peripheral blood leukocytes after transplantation of purified Sca-1+ bone marrow-derived HPCs. No detectable engraftment occurred in nonirradiated recipient mice, even when as many as 2.0 x 10(6) SCa-1+HPCs were transplanted. However, in mice receiving total body irradiation (TBI), engraftment increased as a function of pretransplantation radiation dose, number of transplanted cells, and time after transplantation. Moreover, mice receiving either granulocyte colony-stimulating factor (G-CSF) or G-CSF+ stem cell factor before low-dose TBI (160 cGy) exhibited a marked increase in engraftment compared with mice receiving a vehicle control before low-dose TBI (18.9% and 20.6% v 5.6% at a 1 month, respectively; 29% and 35% v 15.1% at 4 months, respectively). Use of growth factor pretreatment even allowed TBI doses as low as 30, 70, or 120 cGy to achieve significant engraftment of donor progenitors (0.3%, 1.5%, and 6.8% at 1 month, respectively; 1.7%, 5.8%, and 13.9% at 4 months, respectively). All animals remained healthy during the observation periods. Thus, growth factor preconditioning of the recipient followed by low dose TBI may provide an optimal balance between safety and efficacy in achieving required levels of engraftment for gene therapy of blood disorders. PMID- 8639761 TI - Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells. AB - Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele. PMID- 8639762 TI - Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. AB - Thrombopoietin (TPO), the ligand for c-mpl, stimulates proliferation of committed megakaryocytic progenitors and induces maturation of megakaryocytes. To better understand factors regulating TPO levels, we measured blood levels of TPO in patients with impaired platelet production due to aplastic anemia (AA) and with platelet destructive disorders, including idiopathic thrombocytopenic purpura (ITP), posttransfusion purpura (PTP), drug purpura (DP), and X-linked thrombocytopenia (XLTP). The TPO receptor capture enzyme immunoassay (EIA) used had a detection limit of integral of approximately-150 to 200 pg/mL. TPO was undetectable in 88 of 89 normal individuals. Eighteen of 19 patients with AA and a mean platelet count (MPC) of 18,000/microliters (2,000 to 61,000/microliters) had markedly elevated TPO levels (mean, 1,467 pg/mL; range, 597 to 3,834 pg/mL). Eight AA patients who responded to immunosuppressive therapy with their MPC increasing to 140,000/microliters (92,000 to 175,000/microliters) had substantial decreases in TPO (mean, 440 pg/mL; range, 193 to 771 pg/mL). Initial TPO levels did not differ significantly between responders and nonresponders. In contrast, all 21 patients with ITP and an MPC of 16,000/microliters (1,000 to 51,000 /microliters) had undetectable TPO levels, as did 6 patients with acute PTP or DP and 2 patients with XLTP. Megakaryocyte mass, reflected in the rate of platelet production, appears to be the major determinant of TPO levels in thrombocytopenic patients rather than circulating platelet levels per se. Measurement of serum TPO may be useful in differentiating thrombocytopenias due to peripheral destruction from those due to thrombopoietic failure. PMID- 8639763 TI - Efficacy of interferon in treating chronic hepatitis C in children with a history of acute leukemia. AB - Interferon (IFN) is effective in treating adults as well as children with chronic hepatitis C. We investigated the efficacy of IFN therapy in 13 children with underlying acute leukemia who had chronic hepatitis C (age range, 5 to 17 years; mean age, 9.9 years). Natural IFN- alpha was administered at a dose of 0.1 mega unit (MU)/kg (maximum dose, 6.0 MU) daily for 2 weeks and then three times per week for an additional 22 weeks (total dose, 8 MU/kg). IFN treatment was initiated at least 2 years after the completion of treatment for acute leukemia. A complete response was obtained in 5 children (38%). The serum level of anti hepatitis C virus core antibody was closely related to the response to IFN. IFN therapy was well tolerated by all but 1 of the children, who developed mild transient heart failure 4 months after the initiation of therapy. IFN therapy for children with chronic hepatitis C who had underlying acute leukemia was beneficial. However, further trials are required to confirm the safety and improve the dosage schedule of IFN therapy. PMID- 8639764 TI - Maintenance treatment of the anemia of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor and erythropoietin: evidence for in vivo synergy. AB - Patients with myelodysplastic syndromes (MDS) have refractory cytopenias leading to transfusion requirements and infectious complications. In vitro marrow culture data have indicated that granulocyte colony stimulating factor (G-CSF) synergizes with erythropoietin (EPO) for the production of erythroid precursors. In an effort to treat the anemia and neutropenia in this disorder, MDS patients were treated with a combination of recombinant human EPO and recombinant human G-CSF. Fifty-five patients were enrolled in the study of which 53 (96%) had a neutrophil response. Forty-four patients were evaluable for an erythroid response of which 21 (48%) responded. An erythroid response was significantly more likely in those patients with relatively low serum EPO levels, higher absolute basal reticulocyte counts and normal cytogenetics at study entry. Seventeen (81%) of the patients who responded to combined G-CSF plus EPO therapy continued to respond during an 8 week maintenance phase. G-CSF was then discontinued and all patients' neutrophil responses were diminished, whereas 8 continued to have an erythroid response to EPO alone. In 7 of the remaining 9 patients, resumption of G-CSF was required for recurrent erythroid responses. The median duration of erythroid responses to these cytokines was 11 months, with 6 patients having relatively prolonged and durable responses for 15 to 36 months. Our results also indicate that approximately one half of responding patients require both G-CSF and EPO to maintain an effective erythroid response, suggesting that synergy between G-CSF and EPO exists in vivo for the production of red blood cells in MDS. PMID- 8639765 TI - Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells. AB - It has been hypothesized that distinct stromal cells from niches within the microenvironment that selectively regulate stem cell functions. To test this hypothesis, we derived a panel of matched stromal cell lines from murine fetal liver. The lines were immortalized with a retroviral vector encoding a temperature sensitive SV40 T antigen, to provide a snapshot of potential heterogeneity of the in vivo stroma compartment. All the stromal cell lines tested, supported the proliferation and differentiation of myeloid cells in Dexter type bone marrow cultures. Furthermore, RT-PCR analysis indicates that these lines are similar with respect to the production of an array of cytokines. However, the stromal cell lines differed markedly in their ability to maintain in vitro stem cells with in vivo repopulating capacity. Stem cell levels were measured in the competitive repopulation assay, following 3 weeks of coculture on individual stromal cell lines. Three classes of stromal cell lines were identified: (1) lines that did not support stem cells, (2) lines that sustained low levels of stem cells that often showed limited persistence in vivo, and (3) an infrequent line (1 out of 16 lines tested) that maintained high levels of primitive, long-term repopulating stem cells. This suggests that stromal cells that can support primitive stem cells are rare in the hematopoietic microenvironment. Taken together, these data substantiate the hypothesis that distinct stromal cells interact selectively with stem cells. PMID- 8639766 TI - Isolation and biological characterization of two classes of blast-cell colony forming cells from normal murine marrow. AB - In this study, a primitive progenitor cell, the blast-cell colony-forming cell (BC-CFC), which is thought by some to be equivalent to the hematopoietic stem cell (HSC), those cells capable of long-term marrow repopulation, has been isolated from normal murine marrow. The cell separation method we employed has, as its final step, the purification of marrow cells based on their ability to take up (bright) or exclude (dull) the mitochondrial dye, Rhodamine (Rho)-123. Rho-bright and Rho-dull cells are enriched for multipotential progenitor cells or for HSC, respectively. It was found that Rho-bright cells contain more BC-CFC than Rho-dull cells (310 +/- 50 v 120 +/- 40 per 10(5) purified cells, respectively). However, the BC-CFC that copurified with the Rho-bright and the Rho-dull cells were different in terms of replating efficiency and response to interleukin-3 (IL-3) and stem cell factor (SCF). In fact, on replating, the blast cell colonies cultured from the Rho-dull population give rise to many more secondary colonies and had a greater replating efficiency than those obtained from Rho-bright cells (replating efficiency: 29.0 +/- 6.3 v 19.5 +/- 3.7, respectively, P < .01). Furthermore, while the same numbers of blast-cell colonies were detected in culture of Rho-bright cells stimulated with IL-3 alone or in combination with SCF, blast-cell colonies were generated in cultures of Rho dull cells only in the presence of both IL-3 and SCF. After 5 days in suspension culture stimulated with IL-3 and SCF, Rho-dull cells generated BC-CFC whose replating potential was similar to the replating potential of BC-CFC contained in the Rho-bright population. These results indicate that BC-CFC contained in the Rho-bright and -dull populations are qualitatively different. Because the Rho dull population contains HSC, the results indicate that few, if any, BC-CFC are HSC. PMID- 8639767 TI - Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells. AB - We investigated the effects of stem cell factor (SCF) on the migration of murine bone marrow hematopoietic progenitor cells (HPC) in vitro using a modification of the checkerboard assay. Chemotactic and chemokinetic activities of SCF on HPC were evaluated by the numbers of HPC migrated on positive and negative gradients of SCF, respectively. On both positive and negative gradients of SCF, HPC began to migrate after 4 hours incubation, and their numbers then increased time dependently. These results indicated that SCF functions as a chemotactic and chemokinetic agent for HPC. Analysis of types of colonies derived from the migrated HPC showed that SCF had chemotactic and chemokinetic effects on all types of HPC. When migrating activities of other cytokines were examined, interleukin (IL)-3 and IL-11 also affected the migration of HPC, but the degrees of each effect were lower than that of SCF. The results of the present study demonstrated that SCF is one of the most potent chemotactic and chemokinetic factors for HPC and suggest that SCF may play an important role in the flow of HPC into bone marrow where stromal cells constitutively produce SCF. PMID- 8639768 TI - Early ontogeny of the human marrow from long bones: an immunohistochemical study of hematopoiesis and its microenvironment. AB - We examined long bones from 42 human embryos and fetuses whose gestational ages ranged from 6 to 28 weeks. Bone rudiment sections were stained using a panel of monoclonal antibodies directed at antigens expressed by hematopoietic cells, endothelial cells, smooth muscle cells, fibro-blasts, and stromal cells, to describe the events preceding and accompanying the onset of hematopoiesis in the diaphyseal region. Five distinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [gw]) was that of entirely cartilaginous rudiments: chondrocytes were dilated and capillaries with CD34+ endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68+ cells were observed, interspersed within the marrow cavity. Stage III (9 to 10.5 gw) was characterized by the development of the vascular bed in the absence of detectable hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells organized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34+ endothelial cells flanked on their abluminal side by alpha SM actin+ myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increased in size. Logettes filled with hematopoietic cells were immersed within large and almost empty vascular sinuses. Logettes were attached by a short pedicle to connective tissue adjacent to bone/cartilage remaining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found solely in the diaphyseal area. Most hematopoietic cells found inside logettes were CD15+ myelocytes; rarely seen were glycophorin A+ immature erythroblasts and CD34+ nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin+ myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long bones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient long bone hematopoiesis: 1) absence of CD34+ hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the emerging blood-forming marrow, 2) predominance of granulopoiesis, and 3) exclusive development in specific structures organized by vascular cells. This study also suggests that CD68+ cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid cells) may be the critical microenvironment at the onset of hematopoiesis. PMID- 8639769 TI - Induction of LFA-1 on pluripotent CD34+ bone marrow cells does not affect lineage commitment. AB - Leukocyte function associated antigen 1 (LFA-1) is an adhesion molecule indispensable in immune and inflammatory reactions, but its role in hematopoiesis remains obscure. Since LFA-1 is predominantly expressed by leukocytes, it is considered as a marker of late stage stem cell maturation when expressed on CD34+ bone marrow cells, and represents more mature hematopoietic progenitor cells. We observed that freshly isolated CD34+ bone marrow cells express LFA-1, and that the level of expression is highly variable. Interestingly, the expression of LFA 1 specific activation epitope L16 on these cells is low, even after culture. This demonstrates the LFA-1 is not activated, as was confirmed by low adhesion to ICAM 1. Culturing sorted CD34+ LFA-1+ cells in single cell per well assays in medium supplemented with SCF, Epo, IL-3, Il-6, GM-CSF, and G-CSF revealed that they gave rise to dispersed macrophage-like colonies, supporting the notion that CD34+LFA 1+ cells indeed consist of a mature committed cell population. In contrast, sorted CD34+LFA-1- cells had high proliferative potential and developed into large multilineage colonies within 14 days of culture. Unanticipated, in time course experiments we observed that these CD34+LFA-1- cells expressed LFA-1 within 24 hours upon culture. This induction was neither caused by the monoclonal antibody used to tag CD34 cells, nor dependent on growth factors present in the medium. These findings demonstrate that two populations of CD34+LFA-1+ cells can be discriminated: leukocyte lineage committed CD34+ cells in freshly isolated bone marrow cells, and multipotent CD34+ cells that acquired LFA-1 upon in vitro culture. These in vitro findings support the hypothesis that once contacts with bone marrow stroma are lost, LFA-1 is upregulated by default, due to the lack of negative regulating signals from stromal cells. This might also explain the widely variable expression of LFA-1 as a result of crowding of cells in the bone marrow with subsequent loss of contact with stroma and upregulation of LFA-1, providing those cells with adhesion receptors enabling migration in the periphery. PMID- 8639770 TI - Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony stimulating factor. AB - Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine-SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression. PMID- 8639771 TI - CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells. AB - Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population. PMID- 8639772 TI - Interleukin-1 beta induces production of granulocyte colony-stimulating factor in human hepatoma cells. AB - Interleukin-1 (IL-1) is a proinflammatory cytokine that participates in the activation of the acute-phase plasma protein genes in hepatic cells during infection and injury. In human hepatoma HepG2 and Hep3B cells, IL-1 beta induced production of the granulocyte colony-stimulating factor (G-CSF) in a dose dependent manner. Activation of G-CSF gene expression was an early and transient response. In HepG2 cells, G-CSF mRNA was strongly upregulated 2 hours after IL-1 beta treatment and returned to the pretreatment level by 6 hours. The secreted G CSF was biologically active, as shown by the induction of gene transcription through the G-CSF receptor. Maximal G-CSF activity released to culture medium occurred after 8 hours. Previous studies have shown that liver expression of G CSF was augmented in mice challenged by inflammatory stimuli. Our data suggest that IL-1 beta mediates, at least in part, this cytokine activation program in parenchymal cells and that liver-derived G-CSF may contribute to the regulation of hematopoiesis during the acute-phase response. PMID- 8639773 TI - Interferon-gamma constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition. AB - Clinical and laboratory studies have suggested involvement of interferon-gamma (IFN-gamma) in the pathophysiology of aplastic anemia. T cells from aplastic anemia (AA) patients secrete IFN-gamma in vitro, activated cytotoxic lymphocytes infiltrate aplastic bone marrow (BM), and IFN-gamma mRNA, not detected in normal BM, is present in BM from most AA patients. Many patients respond to immunosuppressive therapy with antithymocyte globulin and cyclosporine. Using long-term BM cultures (LTBMC) as a tissue culture model of hematopoiesis, we show that IFN-gamma is a potent inhibitor in the long-term culture-initiating cell (LTC-IC) assay, the best in vitro surrogate test for human hematopoietic stem cells, as well as of the output of committed progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]). In LTBMC, continuous addition of relatively high IFN-gamma concentrations (1,000 U/mL weekly or 200 U/mL every 2 days) was required for inhibition of secondary colony formation, a measure of LTC-IC number and clonogenicity. To mimick local production of IFN-gamma, human stromal cells were engineered by retroviral mediated gene transfer to express a transduced IFN-gamma gene. IFN-gamma secreted by stromal cells was far more potent than exogenous IFN-gamma in its effects in the LTC-IC assay. For purified CD34+ cells culture in the presence of IFN-gamma stroma dramatically reduced secondary colony numbers as well as production of CFU GM and BFU-E. Supernatants from these cultures contained only about 20 U/mL of IFN-gamma; this quantity of cytokine, when added to LTBMC, had little effect on hematopoiesis. The mechanism of hematopoietic suppression was related to the inhibition of cell cycle progression and induction of apoptosis of CD34+ cells. There was no apparent effect of local low-level IFN-gamma production on stromal cell function, as reflected in cell morphology, cell surface phenotype, or expression of hematopoietic growth factor genes. LTBMC with genetically altered stromal cells offers an in vitro model of immune suppression of hematopoiesis in AA and may be helpful in testing certain therapeutic modalities. We infer from our data that local production of low levels of inhibitory cytokine is sufficient to markedly inhibit hematopoiesis and to destroy stem cells and more mature progenitor cells. PMID- 8639774 TI - Relative reactivity of platelets from thrombopoietin- and interleukin-6-treated dogs. AB - Previous reports have shown that interleukin-6 (IL-6) enhances the responsiveness of platelets to thrombin stimulation and has modest thrombocytopoietic effects in vivo. Thrombopoietin (TPO; mpl ligand) has been shown to have dramatic thrombocytopoietic effect in vivo, but little is known of its capacity to alter platelet function. In this study, a direct comparison of the effects of IL-6 and TPO on platelet function in dogs has been performed, with modest doses of TPO (1 microgram/kg/d) chosen to match or moderately exceed the platelet counts achieved with IL-6 (40 micrograms/kg/d) for 10 days. Platelet responsiveness to thrombin stimulation was assessed in TPO-treated, IL-6-treated, and control dogs by flow cytometric measurement of P-selectin expression. On day 5, the dose of thrombin promoting half maximal stimulation (EC50) of platelets was not significantly changed in TPO-treated dogs, whereas in IL-6-treated dogs the EC50 decreased to 73.1% +/- 6.1% (mean +/- 1 SD; n = 5) of control values (P < 0.01). These experiments were performed on both gel-filtered platelets and washed whole blood, indicating that the observed changes in EC50 were caused by cytokine-mediated alteration of platelets rather than plasma components. Because it has been shown that thiazole orange specifically labels a subpopulation of dog platelets that is less than 24 hours old, the thrombin responsiveness of these young, newly synthesized platelets was determined. The EC50 of thiazole orange-positive platelets from IL-6-treated dogs decreased dramatically by day 5 to 46.5% +/- 13.1% (n = 4) of control values (P < 0.001), whereas TPO-treated dogs did not significantly change. When TPO was directly incubated with platelets ex vivo, no effects on either thrombin-mediated P-selectin expression or adenosine diphosphate-induced fibrinogen binding were observed. These data show that IL-6 alters platelet function, as measured by reactivity to thrombin, whereas TPO does not. This divergence in function is observed even though TPO is equally, or more, effective at promoting platelet production under these experimental conditions. PMID- 8639775 TI - Protein C Nagoya, an elongated mutant of protein C, is retained within the endoplasmic reticulum and is associated with GRP78 and GRP94. AB - Protein C Nagoya, an elongated variant of the human protein C, is retained and degraded within the cells in which it is produced (Yamamoto et al, J Clin Invest 90:2439, 1992). To determine the subcellular localization of the protein C Nagoya, the recombinant protein C bearing this mutation was expressed in Chinese hamster ovary (CHO) cells. The mutant protein C was not secreted from the cells and remained susceptible to endo-beta-N-acetylglucosaminidase H (endo H). Immunoelectron microscopy indicated that protein C Nagoya was retained in the endoplasmic reticulum (ER), whereas wild-type protein C was observed in both the ER and the Golgi apparatus. Metabolic radiolabeling with [35S] methionine in combination with chemical cross-linking showed that the protein C Nagoya existed in the ER as a complex with 78-kD glucose-regulated protein (GRP78) and 94-kD glucose-regulated protein (GRP94). Because both GRP78 and GRP94 associate to a far lesser degree with wild-type protein C than with protein C Nagoya, our data suggest that both stress proteins function as molecular chaperones and work in concert with the folding and assembly of protein C. These findings extend our understanding the molecular pathogenesis of protein C deficiency. PMID- 8639776 TI - Mouse P-selectin glycoprotein ligand-1: molecular cloning, chromosomal localization, and expression of a functional P-selectin receptor. AB - A mouse homolog of P-selectin glycoprotein ligand-1 (PSGL-1), a P-selectin receptor on myeloid cells, has been cloned using the human cDNA sequence to probe a cDNA library prepared from the mouse WEHI-3 monocytic cell line and a genomic DNA library prepared from 129/SvJ mouse tissue. The gene flanking the entire open reading frame of 397 amino acids is composed of a single exon. Mouse and human PSGL-1 show an overall similarity of 67% and an identity of 50% and contain a similar domain organization. However, there are 10 threonine/serine-rich decameric repeats in mouse PSGL-1 as compared with 15 threonine-rich repeats in human PSGL-1. When the mouse PSGL-1 cDNA is coexpressed with an alpha 1,3/1,4 fucosyltransferase cDNA in COS cells, a functional protein is expressed on the COS cell surface mediating binding to human P-selectin. The mouse PSGL-1 gene, Selpl, was mapped to a position on mouse chromosome 5 (Chr 5). Northern blot analyses of mouse tissues showed moderate expression of a PSGL-1 mRNA species in most tissues including heart, kidney, liver, muscle, ovary, and stomach and high levels of expression in blood, bone marrow, brain, adipose tissue, spleen, and thymus. Whereas certain mouse myeloid cell lines including PU5-1.8, WEHI-3B, and 32DC13 express high levels of PSGL-1 mRNA, only WEHI-3B and 32DC13 bind to P selectin; this interaction is blocked by anti-PSGL-1 antibody. WEHI-3B cells bind significantly better to P-selectin than to E-selectin. Although comparable P selectin binding is observed in 32DC13 cells, these cells bind better to E selectin. Binding of 32DC13 cells to E-selectin is not blocked by anti-PSGL-1 antibody. Treatment of WEHI-3B cells with trypsin or neuraminidase abolished their ability to interact with P-selectin. These results indicate that mouse PSGL 1 has structural and functional homology to human PSGL-1 but is characterized by differences in the composition and number of the decameric repeats. PSGL-1 on mouse myeloid cells is critical for high-affinity binding to P-selectin but not E selectin. PMID- 8639777 TI - Activation of factor VII during alimentary lipemia occurs in healthy adults and patients with congenital factor XII or factor XI deficiency, but not in patients with factor IX deficiency. AB - Factor VII activity (FVIIc), a risk marker for coronary heart disease, is increased during postprandial lipemia. Factor VII activation accompanies lipolysis of triglyceride-rich lipoproteins, but the nature of this association and whether it is causal remain uncertain. To explore this issue, four patients with homozygous factor XII deficiency, four with complete factor XI deficiency, six with factor IX deficiency, and their respective age- and sex-matched controls were given two isocaloric dietary regimens, one providing on average 136 g fat and the other 19 g fat. Blood was taken before breakfast, immediately before lunch at 195 minutes, and at completion of the study at 390 minutes. All samples for each subject and matched control were assayed as one batch for FVIIc, activated factor VII, and factor VII antigen (FVIIag). Activation of factor VII was observed with the high-fat regimen but not with the low-fat regimen in all controls, factor XII-deficient patients, and factor XI-deficient patients. No factor VII activation was observed during either regimen in factor IX-deficient patients, but a normal postprandial responsiveness of factor VII to dietary fat was restored in one patient who replicated the study after factor IX therapy. Plasma FVIIag was not altered postprandially in either regimen in any group of patients or controls. Factor IX apparently plays an obligatory role in the postprandial activation of factor VII, although the mechanism remains to be determined. PMID- 8639778 TI - A frame shift mutation in the fibrinogen A alpha chain gene in a kindred with renal amyloidosis. AB - A new American kindred with amyloidosis was found by single-strand conformation polymorphism analysis to have a mutation in the fibrinogen A alpha chain gene. Affected members in this kindred have autosomal dominant amyloid nephropathy. DNA sequencing showed a single nucleotide deletion at the third base of codon 524 of the fibrinogen A alpha chain genes (4904delG) that resulted in a frame shift and premature termination of the protein at codon 548. Antiserum was produced to a portion of the abnormal peptide predicted by the DNA sequence and amyloid deposits were immuno-histologically proven to contain this abnormal peptide. Two of the propositus' 4 children were positive for the mutant fibrinogen A alpha chain gene by restriction fragment length polymorphism analysis based on polymerase chain reaction. These two mutant gene carriers now in the second decade of life show no clinical symptoms of amyloidosis as yet but have lower plasma fibrinogen concentrations when compared with their normal siblings. This the first description of a kindred with renal amyloidosis and low plasma fibrinogen and also the first report of amyloidosis caused by a frame shift mutation. PMID- 8639779 TI - Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance. AB - Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator-induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance. PMID- 8639780 TI - Correction of the platelet adhesion defect in delta-storage pool deficiency at elevated hematocrit--possible role of adenosine diphosphate. AB - Previous studies on patients with storage pool deficiency (SPD) who are specifically deficient in platelet dense granules (delta-SPD) have suggested a role for dense granule substances, in all likelihood adenosine diphosphate (ADP), in mediating thrombus formation on subendothelium at high shear rates. The role of dense granule substances in mediating platelet adhesion appears to be more complicated Previous studies in delta-SPD suggested an adhesion defect that was strongly influenced by the patient's hematocrit (Hct) value. To explore further the possibility that red blood cells (RBCs) may influence the role that platelet storage granules play in mediating adhesion at high shear rates, we have measured adhesion (and thrombus formation) throughout a preselected range of Hct values (30% to 60%) in normal subjects and in patients with delta-SPD. The present studies confirm the defect in platelet adhesion in patients with delta-SPD, most significantly at Hct values of 30% to 40%. This defect (but not that of thrombus formation) can be completely corrected by the addition of RBCs. The correction of the platelet adhesion defect by RBCs was specific for delta-SPD; it was not observed in either von Willebrand's disease or thrombasthenia. Studies performed on normal blood under conditions that could be expected to block any effect of ADP on adhesion and an analysis of the type of adhesion defect in delta-SPD suggest that ADP may be involved in the process required for platelet spreading on the subendothelium. The corrective effect of RBCs on platelet adhesion in delta-SPD appears to be chemical rather than physical in nature, possibly due to shear-induced release of RBC ADP or to other recently described properties of RBCs that enhance collagen-induced platelet interactions. PMID- 8639781 TI - Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. AB - Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximately 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximately 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. vWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, of calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe CHN2. Activation by divalent metal ions was found to increase in the following order: Zn2+ approximately Cu2+ approximately CD2+ approximately Ni2+ approximately Co2+ 9.3 and 5.8 years, respectively). In terms of progression-free survival, MCL showed a behavior similar to the low-grade group, with frequent relapses. Follicle center cell lymphomas behaved as low-grade lymphomas as defined by the WF and diffuse large B-cell lymphomas as the WF intermediate grade group. Because several NHL entities have a clinical behavior of their own, their recognition by the REAL classification offers clinicians additional information that is not obtained when the WF is used. PMID- 8639797 TI - Activated abl oncogenes and apoptosis: differing responses of transformed myeloid progenitor cell lines. AB - Activation of the c-abl protooncogene occurs during the generation of both the Abelson murine leukemia virus and the bcrabl fusion gene. To further dissect the biological properties of these proteins, we studied their effect on apoptosis. Using dimethyl sulfoxide (DMSO) to induce apoptosis in the murine myeloid progenitor cell line 32Dcl3, we examined the effect of expression of both v-abl and bcrabl transgenes on apoptosis. v-abl expressing 32Dcl3 cells are sensitive to apoptosis induction, similar to parental 32Dcl3 cells. In contrast, bcrabl expression 32Dcl3 cells are protected from the apoptotic stimulus resulting from DMSO exposure. Analyzing the expression patterns for Bcl-2 and Bax, two proteins known to modulate the apoptotic response, we found a downregulation of Bcl-2 and enhanced expression of Bax in 32Dcl3 cells. In 32Dcl3/v-Abl cells, Bcl-2 expression remained constant while Bax was upregulated, whereas in 32Dcl3 cells expressing bcrabl, there was continuous expression of Bcl-2 at a level greater than observed in v-abl transformed cells. Taken together, our data demonstrate that although both activated abl gene products promote overlapping effects of some biological responses (i.e., factor-independent proliferation) they diverge in their effect on apoptotic signaling pathways. PMID- 8639798 TI - The p53 gene in pediatric therapy-related leukemia and myelodysplasia. AB - We investigated the frequency of p53 mutations in 19 pediatric cases of therapy related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2-basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting. PMID- 8639799 TI - Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion. AB - Several cytokines have been suggested to play a regulatory action on the neoplastic clone of patients with B-cell chronic lymphocytic leukemia (B-CLL) by interfering in the differentiation, proliferation, or death/survival pathways. Interleukin-8 (IL-8) is a chemoattractant protein constitutively expressed at the mRNA level and released by B-CLL cells. In view of the presence of the IL-8 receptor mRNA and of specific IL-8 binding, confirmed also by Scatchard analysis using 125I-IL-8, the study was extended to evaluate the possible regulatory role of this cytokine on B-CLL cells. IL-8 failed to show any in vitro proliferative effect on leukemic B-CLL cells. By contrast, the propidium iodide (PI) staining of the DNA content showed that IL-8 could prolong the survival of resting B-CLL cells in 11 of 16 cases studied. In the remaining 5 cases, 90.6% +/- 4.39% SD of the cells after culture remained viable and IL-8 could exert a significant death protection action after pretreatment with 10(-4) mol/L hydrocortisone, which reduced the percentage of viable B-CLL cells. The dose range of IL-8 capable of inducing the prolonging survival effect is comparable with the levels of IL-8 released constitutively by B-CLL cells, indicating that the death protection action is exerted at physiologic doses. The in vitro rescue from death induced by IL-8 is reflected by an increased expression of bcl-2 mRNA in B-CLL cases incubated in the presence of IL-8. These findings were further confirmed at the protein level, because in B-CLL cells that displayed a bimodal bcl-2 intracytoplasmatic protein expression IL-8 was capable of upmodulating the bcl 2high expression peak. The potential autocrine regulatory action exerted by IL-8 is supported by the evidence that exogenous IL-8 can upregulate IL-8 mRNA in B CLL cells. These results, together with the demonstration that antibody-mediated neutralization of endogenous IL-8 could induce a significant in vitro reduction in the number of living cells, further support the hypothesis that, in B-CLL, the physiologic doses of IL-8 released constitutively by the leukemic clone may play an autocrine role in the process of cell accumulation characteristic of this disease. PMID- 8639800 TI - Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation. AB - The BCL1 lymphoma in Balb/c mice can be successfully treated with bispecific (anti-CD3 x anti-idiotype) antibodies (BSABs). In these experiments, animals were injected intraperitoneally (IP) with 5 x 10(3) tumor cells (day 0) and treated with one single intravenous (IV) injection of 5 micrograms BSAB (day 9). Because cross-linking of the CD3 complex is not in itself sufficient to activate resting T cells, the therapeutic success was mainly based on the progressive retargeting of the relatively small cytotoxic T-lymphocyte effector cell pool already in existence in vivo. For this reason, the therapy lost its effectiveness at higher tumor loads. Two possibilities were explored to treat mice with a higher tumor load (10(5) tumor cells). First multiple injections of BSABs were used, but a dose-related monovalent anti-CD3 immunosuppression was induced. This approach was only beneficial when the immune system was able to recover from the previous injection of BSAB. As a second approach, CD28 costimulation together with BSABs were used in an attempt to activate resting T cells, ultimately enlarging the effector T-cell pool. However, we were repeatedly unsuccessful in attempts to improve our in vivo results using young, naive animals in which the majority of T cells are of the naive phenotype. Only when animals were primed to induce the memory T-cell type was a significantly better outcome observed, and then only with multiple injections of BSABs and anti-CD28 monoclonal antibodies (MoAbs), rather than with BSAB or anti-CD28 MoAb alone. These results suggest that this combination was able to activate memory and effector T cells and to focus them on the tumor, but was unable to activate naive T cells fully. The in vivo potency of the BSAB and CD28 costimulation was shown by the fact that one-tenth of the quantity of BSAB was required to cure animals with 20 times the tumor load. PMID- 8639801 TI - Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines. AB - Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All-trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage. PMID- 8639802 TI - Protein phosphorylation in neutrophils from patients with p67-phox-deficient chronic granulomatous disease. AB - Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma-32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma -32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox. PMID- 8639803 TI - Release of oxygen metabolites from chemoattractant-stimulated neutrophils is inhibited by resting platelets: role of extracellular adenosine and actin polymerization. AB - The effect of human platelets on chemoattractant-induced generation of oxygen metabolites in neutrophils was investigated, using luminol-enhanced chemiluminescence (CL). Resting platelets inhibited the extracellular, but not the intracellular, production of oxygen radicals in formyl-methionyl-leucyl phenylalanine (fMet-Leu-Phe)-stimulated neutrophils. Maximal effect was obtained at the physiological neutrophil/platelet ratio of 1/50. Similar results were acquired by adding supernatants of platelets, indicating a role for a soluble factor. Removal of extracellular adenosine by adenosine deaminase (ADA), or blocking of adenosine-receptors by theophylline, antagonized the inhibitory effects of platelets (or the equivalent supernatant) on the neutrophil respiratory burst. In contrast, accumulation of adenosine by apyrase enhanced the inhibition. Exogenous adenosine mimicked the effects of platelets on the fMet-Leu Phe-induced respiratory burst. To further assess the role of platelet-derived adenosine, the platelets were fixed with paraformaldehyde. We found that fixed platelets, as well as their supernatant, inhibited the fMet-Leu-Phe-induced CL response to the same extent as viable cells. These effects were also reversed by ADA and theophylline, respectively. A prior removal of adenosine in the platelet suspension by ADA, followed by treatment with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) to inactivate ADA, did not reverse the inhibitory action of platelets on the fMet-Leu-Phe-induced CL-response in neutrophils. However, if adenosine receptors of neutrophil at the same time were blocked with theophyline, the inhibition was significantly reduced. Platelets markedly increased the generation of adenosine in a neutrophil suspension. The effect was antagonized by S-(4-Nitrobenzyl)-6-thioguanosine (NBTG), but unaffected by alpha, beta-methyl eneadenosine5'diphosphate (AMP-CP), indicating that the platelet-dependent accumulation of adenosine is due to an increased release of endogenous adenosine from neutrophils and not to a degradation of extracellular AMP. In correlation, NBTG, but not AMP-CP, reversed the platelet-mediated inhibition of the fMet-Leu Phe-induced CL-response in neutrophils. Consequently, these data suggest that a platelet-derived factor increases the release of endogenously formed adenosine from neutrophils, terminating the production of oxygen radicals. The inhibition of oxidase activity was also associated with a platelet-induced polymerization of actin in the margin of the neutrophils. Treatment of neutrophils with cytochalasin B reversed the effects of platelets, both on F-actin content and CL response. In summary, resting platelets limit the release of oxygen radicals from chemoattractant-stimulated neutrophils, thus preventing excessive damage to host tissues in the vascular space. This effect is suggested to be associated with an increase generation of neutrophil-derived adenosine enhancing an autoregulatory inhibitory pathway, and a peripheral accumulation of actin filaments forming a barrier for extracellular release of reactive oxygen radicals. PMID- 8639804 TI - Clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated Fanconi anemia complementation group C polypeptide with partial activity. AB - Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital anomalies, aplastic anemia, and cancer susceptibility. Mutations within the FA complementation group C (FAC) gene account for approximately 14% of diagnosed FA cases. Two mutations, one in exon 1 (delG322) and one in exon 4 (IVS4 + 4 A to T), account for 90% of known FAC mutations. The delG322 mutation results in a mild FA phenotype, while the IVS4 + 4 A to T mutation results in severe FA phenotype. To determine the molecular basis for this clinical variability, we analyzed patient-derived cell lines for the expression of characteristic mutant FAC polypeptides. All cell lines with the delG322 mutation expressed a 50-kD FAC polypeptides, FRP-50 (FAC-related protein), shown to be an amino terminal truncated isoform of FAC reinitiated at methionine 55. All cell lines with the IVS4 + 4 A to T mutation lacked FRP-50. Overexpression of a cDNA encoding FRP-50 in an FA(C) cell line resulted in partial correction of mitomycin C sensitivity. In conclusion, expression of an amino terminal truncated FAC protein accounts, at least in part, for the clinical heterogeneity among FA(C) patients. PMID- 8639805 TI - The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum. AB - We studied 20 individuals from 17 unrelated families with congenital dyserythropoietic anemia (type II; CDAII). The clinical phenotype was mild to moderate. The inheritance pattern was invariably recessive. Coomassie blue stained gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) show that band 3 was thinner and migrated slightly faster than usual. In addition, staining showed two unknown minor bands (in the patients), but not in normal controls, the obligate carrier parents, or in patients with other anemic syndromes. These minor proteins were studied using partial digestion, amino acid sequencing, Western blotting, immunofluorescence, and immunogold electron microscopy. They were identified as the glucose-regulated protein GRP78 and calreticulin that are resident proteins of the endoplasmic reticulum (ER). Using specific antibody, we showed that protein disulfide isomerase (PDI), a third major protein of the ER, was also present on the SDS-PAGE of red blood cell (RBC) ghosts. Immunofluorescence colocalized PDI with the dense discontinuous ring decorating the RBC membrane. Immunogold electron microscopy showed that PDI was localized in the lumen of the cisternae, confirming that these originate from the smooth ER. From a practical point of view, screening the above minor proteins in RBC membranes appears to be a straightforward and reliable diagnostic test for CDAII. PMID- 8639806 TI - Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst. AB - The vasoocclusive process in patients with sickle cell disease (SCD) is complex and involves interactions among sickle erythrocytes (SS-RBC), vascular endothelium, and plasma and cellular components. The role of neutrophils (PMN) in vasoocclusion has not been examined. Patients with SCD appear to have chronically activated PMN. Because the first step in PMN activation is particle recognition, we explored whether normal PMN recognize SS-RBC and whether this recognition results in PMN monolayers, significantly more SS-RBC adhered to the PMN than did normal erythrocytes (AA-RBC; P < .001). Preincubation of erythrocytes with autologous plasma significantly increased the adherence of SS-RBC to PMN but had no effect on AA-RBC (P < .001). When adhesion of density fractionated SS-RBC was performed, dense SS-RBC showed greater adherence to the PMN monolayers than did light SS-RBC (P < .001). To determine mechanisms of this adhesion, IgG and Arg Gly-Asp-Ser (RGDS) receptor sites on PMN were saturated. IgG inhibited adherence of dense SS-RBC, whereas RGDS inhibited adherence in both fractions, although to a greater extent in the light fraction. We measured SS-RBC activation of PMN by incubating SS-RBC with 2', 7'-Dichloro-fluroescin Diacetate (DCF)-labeled PMN. Incubation of PMN with SS-RBC resulted in a significant increase in fluorescence compared to AA-RBC. We show here that PMN recognize SS-RBC through multiple mechanisms and that this recognition results in activation of PMN. These findings contribute to the understanding of vasoocclusive crisis in patients with SCD and may have therapeutic implications. PMID- 8639807 TI - Cysteine proteinase inhibitors block early steps in hemoglobin degradation by cultured malaria parasites. AB - Erythrocytic malaria parasites degrade hemoglobin as a source of amino acids for parasite protein synthesis. Cysteine proteinase inhibitors have been shown to block the hydrolysis of globin by cultured parasites, indicating that a malarial cysteine proteinase is required for this process. In the present study, we have evaluated the role of parasite proteinases in earlier steps of hemoglobin degradation, namely the disassociation of the hemoglobin tetramer and the separation of heme from globin. Hemoglobin did not spontaneously denature or release heme under the pH and reducing conditions of the malarial food vacuole, suggesting that parasite enzymatic activity is necessary for early steps in hemoglobin degradation. The incubation of cultured parasites with cysteine proteinase inhibitors inhibited the denaturation of hemoglobin and the release of heme from globin. These results suggest that, in addition to its role in globin hydrolysis, a malarial cysteine proteinase participates in the dissociation of the hemoglobin tetramer and the release of heme from globin. Thus, the malarial cysteine proteinase is a promising target for antimalarial chemotherapy. PMID- 8639808 TI - High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation. AB - As compared with related HLA-identical sibling donors, bone marrow transplantation (BMT) with phenotypically HLA ABDR-compatible unrelated donors is associated with increased mortality. This may be due to hidden HLA incompatibilities not detected by conventional typing. We have analyzed 44 unrelated patient-donor pairs who were matched for HLA-A, -B, and -DR by routine tissue typing. Our comprehensive HLA typing approach consisted of serology, cytotoxic T-cell precursor (CTLp) tests, T-cell cloning, oligotyping, and DNA sequencing. Using these techniques, we identified numerous HLA allele mismatches not detected by the previously applied routine typing. Twenty-four patient-donor pairs were highly matched and had a low CTLp frequency, whereas the remaining 20 pairs were allele-mismatched for HLA-A, -B, -C, -DR, -DQ antigens and/or had a positive result of the CTLp test. Patient and donor age, diagnosis, and treatment did not differ significantly between the matched and mismatched transplants. The probability for severe acute graft-versus-host disease grades III-IV was 21% in the matched and 47% in the mismatched patients (P = .0464). Transplant-related mortality was 21% and 57% (P = .0072) and actuarial patient survival rates at 3 years were 61% and 13% (P = .0005). We conclude that both HLA class I and class II allele mismatches between unrelated phenotypically ABDR-compatible patient donor pairs are frequent and associated with increased incidence of posttransplant complications. PMID- 8639811 TI - Numerical chromosome aberrations in CD30+ cells of Hodgkin's disease: truth or FICTION. PMID- 8639809 TI - Murine gamma/delta-expressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens. AB - T cells with antidonor specificities have been isolated from human recipients experiencing graft rejection after allogeneic bone marrow transplantation (BMT). Partial T-cell depletion of unrelated BM grafts with an anti- T-cell receptor (TCR) monoclonal antibody (MoAb) directed against the TCR alpha/beta heterodimer have shown that the incidence of graft-versus-host disease is low and that the incidence of durable engraftment is high. These studies suggest either that the number of residual TCR alpha/beta+ cells was sufficient to permit alloengraftment or that the preservation of cells other than TCR alpha/beta+ cells was beneficial for engraftment. With respect to the latter, one such candidate cell is the TCR gamma/delta+ T cell. Because no studies have specifically examined whether TCR gamma/delta+ cells might be capable of eliminating BM-derived hematopoietic cells, we established a new graft rejection model system in which transgenic (Tg) H-2d mice (termed G8), known to express gamma/delta heterodimers on high proportion of peripheral T cells, were used as BMT recipients. These Tg TCR gamma/delta+ cells respond vigorously to target cells that express the nonclassical major histocompatibility complex (MHC) class lb region gene products encoded in H-2T region of H-2T(b)+ strains. G8 Tg mice were used as recipients for C57BL/6 (B6: H-2(b); H-2T(b)) T-cell-depleted (TCD) donor BM. We show that G8 Tg (H-2(d), H-2T(d)) mice are potent mediators of B6 BM graft rejection and that the rejection process was inhibited by anti-TCR gamma/delta MoAbs. In contrast, BM from a B6 congenic strain that expresses the H-2T(a) allele, B6.A-Tl(a)/BoyEg, was readily accepted, suggesting that H-2T antigens on repopulating donor BM cells are the targets of host graft rejecting T cells that express the TCR gamma/delta heterodimer. PB chimerism studies were performed at > or = 1.5 months post-BMT using TCD BM from severe combined immunodeficient allogeneic donors, which is highly susceptible to rejection by the host. The addition of donor G8 TCR gamma/delta+ cells to TCD donor BM was shown to significantly increase alloengraftment in B6 recipients. These results show that (1) host TCR gamma/delta+ cells can reject repopulating donor cells, presumably by responding to nonclassical MHC class lb gene products expressed on BM-derived hematopoietic progenitor cells; and (2) donor TCR gamma/delta+ cells can facilitate the alloengraftment of rigorously TCD donor BM. PMID- 8639810 TI - Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. AB - We prospectively studied 98 chronic myeloid leukemia (CML) patients after bone marrow transplantation by competitive polymerase chain reaction to detect and quantify leukemia-specific BCR-ABL mRNA. Of 69 patients who had persistently undetectable, decreasing, or low BCR-ABL levels ( < 50 transcripts/microgram RNA) on sequential analysis, one (1%) subsequently relapsed. Of 29 patients who had increasing or persistently high BCR-ABL (> 50 transcripts/microgram RNA) on sequential analysis, 21 (72%) have relapsed (P < .00001). In 19 patients studied sequentially, a constant logarithmic increase in the number of BCR-ABL transcripts was found before relapse, indicating a constant BCR-ABL transcript doubling time. The doubling time for patients relapsing cytogenetically or into chronic phase (median, 24.7 days) was significantly longer than that of patients relapsing into advanced phases (median, 14.7 days; P = .005). Eight patients were treated for relapse by donor leukocyte transfusions. The doubling time of responders was significantly longer than that of nonresponders (P = .017). We conclude that quantification of BCR-ABL transcripts after bone marrow transplantation (BMT) is valuable in predicting relapse: a more rapid BCR-ABL transcript doubling time before relapse might indicate a more aggressive disease. PMID- 8639812 TI - Burst-forming units-erythroid from erythropoietic protoporphyria patients fluoresce under 405 nm light. PMID- 8639813 TI - Further evidence for in vivo isotype switching in B-cell chronic lymphocytic leukemia. PMID- 8639814 TI - Mantle cell lymphoma-- an entity comes of age. PMID- 8639815 TI - Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis. AB - Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation. PMID- 8639816 TI - The differentiation and maturation mediator for human myeloid leukemia cells shares homology with neuroleukin or phosphoglucose isomerase. AB - The identity of the maturation inducer capable of mediating the differentiation of human myeloid leukemic HL-60 calls to terminal monocytic cells was investigated. One of such inducers from T cells was purified as a 54.3-kD peptide. The amino acid sequence of a tryptic peptide and the enzyme cleavage sites revealed 100% homology to neuroleukin or phosphoglucose isomerase (PGI). Neuroleukin mediates differentiation of neurons and is homologous to PGI, which catalyzes the interconversion of glucose-6-phosphate and fructose-6-phosphate. The 54.3-kD inducer was shown to have PGI enzymatic activity. Separately purified PGI by substrate-elution exhibited identical specificity as the maturation inducer for HL-60 cell differentiation. They mediated a reduction of proliferating S and G2M cells, and the mature monocytic calls acquired complement receptors, phagocytic capacity, and adherence morphology. The magnitude of differentiation was dosage dependent on the inducer, with a bell-shaped curve. At the excess dose range cells did not undergo differentiation and remained in a proliferating cycle. Abnormally elevated PGI enzyme activities were detected in the plasma of acute myelogenous leukemia patients. Whether they represent an excess of the differentiation regulator in patients and are important in leukemogenesis remain to be investigated. PMID- 8639817 TI - Prenatal diagnosis of triosephosphate isomerase deficiency. AB - First-trimester prenatal diagnosis was undertaken by chorionic villus DNA analysis in two unrelated families with the inherited glycolytic disorder triosephosphate isomerase (TPI) deficiency. The propositus in each family was shown to be homozygous for a missense mutation (GAG --> GAC) at codon 104 of the TPI gene. In the first case the fetus was heterozygous for the codon 104 mutation and therefore clinically unaffected. Prenatal diagnosis in the second case showed the fetus to be homozygous for the codon 104 mutation and thus affected by TPI deficiency. This represents the first molecular diagnosis during early pregnancy of a human glycolytic enzyme disorder. PMID- 8639818 TI - Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells. AB - Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo. PMID- 8639820 TI - Association of lymphomatoid granulomatosis with Epstein-Barr viral infection of B lymphocytes and response to interferon-alpha 2b. AB - Lymphomatoid granulomatosis (LyG) is an angiodestructive lymphoproliferative disorder (LPD) often involving the lungs. Its etiology is uncertain, but a number of previous studies had suggested it is a T-cell LPD associated with Epstein-Barr virus (EBV). Because of the similarity between LYG and nasal angiocentric lymphoma, the term angiocentric immunoproliferative lesion was proposed for both entities. Optimal therapy is unknown, but chemotherapy is often used. We studied four patients with LYG over a 5-year period. Biopsy samples were analyzed by immunohistochemistry, EBV in situ hybridization, and for Ig heavy-chain (IgH) gene rearrangements, Clinically, we assessed EBV serology, lymphocyte subsets, and the efficacy of interferon-alpha2b (IFN-alpha2b), All biopsy samples showed an exuberant T-cell infiltrate with scattered atypical large B cells. Double labeling showed EBV in the B cells but not T cells. Clonal IgH gene rearrangements were detected in 2 of 3 patients studied, 1 of whom had three distinct clones, and light-chain restriction showed two clones in an additional patient. All patients had positive EBV serologies. and markedly abnormal lymphocyte subsets. With IFN, 3 patients are alive and disease free at 36, 43, and 60 months; 1 patient achieved a partial response for 16 months but discontinued therapy and died with lymphoma. These results indicate that LYG is a T-cell-rich EBV-associated B-cell LPD in which the infiltrating T cells are numerous but reactive. IgH gene rearrangements may be polyclonal, monoclonal, or oligoclonal. Its association with immune defects suggests it is related to posttransplant LPD. However, LYG and nasal angiocentric lymphoma are distinct entities and should no longer be included together under the term angiocentric immunoproliferative lesion. IFN is effective therapy and should be studied further. PMID- 8639819 TI - Dendritic cells and macrophages can mature independently from a human bone marrow derived, post-colony-forming unit intermediate. AB - CD34+ precursors in normal human bone marrow (BM) generate large numbers of dendritic cells alongside macrophages and granulocytic precursors when cultured for 12 to 14 days in c-kit ligand, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). This study reports an intermediate cell type that develops by day 6, and has the potential to differentiate into either macrophages or dendritic cells. When the d6 progeny are depleted of mature macrophages and residual CD34+ precursors, a discrete CD14+ HLA-DR+ population persists in addition to immunostimulatory CD14- HLA-DR() dendritic cells. Half of the CD14+ HLA-DR+ population is in cell cycle (Ki-67+), but colony-forming units (CFUs) are no longer detectable. The calls are c-fms+, but lack myeloperoxidase and nonspecific esterase. They also possess substantial phagocytic and allostimulatory activity. These post-CFU, CD14+ HLA-DR+ intermediates develop into typical macrophages when recultured in the absence of exogenous cytokines. M-CSF supports up to approximately 2.5-fold expansion of macrophage progeny. In contrast, the combination of GM-CSF and TNF-alpha supports quantitative differentiation into dendritic cells, lacking c-fms, CD14, and other macrophage properties, and expressing HLA-DR, CD1a, CD83, CD80, CD86, and potent allostimulatory activity. Therefore, normal CD34+ BM precursors can generate a post-CFU bipotential intermediate in the presence of c-kit ligand, GM-CSF, and TNF-alpha. This intermediate cell type will develop along the dendritic cell pathway when macrophages are removed and GM-CSF and TNF-alpha are provided. Alternatively, it can differentiate along a macrophage pathway when recultured with or without M-CSF. PMID- 8639821 TI - Wiskott-Aldrich syndrome: report of an autosomal dominant variant. AB - The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a clinical triad of thrombocytopenia with small platelets, eczema, and immunodeficiency. Impaired CD43 glycoprotein expression on lymphocytes is a typical hallmark of this disorder. The CD43 gene is located on chromosome 16, and the WAS gene, WASP, was recently isolated from the chromosome X p11.22-p11.23. This gene, mutated in WAS patients, encodes a protein that is likely to play a role in controlling the expression of CD43. However, the molecular mechanism(s) causing WAS are not yet known. Herein, we describe a three-generation family in which clinical and laboratory WAS features were expressed in six of nine subjects available for study. At variance with classic X-linked WAS, this disorder was characterized by the presence of thrombocytopenia with a broad spectrum of platelet size, including giant platelets, and was inherited as an autosomal dominant trait. This last finding led us to hypothesize a mutation of the CD43 gene. However, Southern blot analysis failed to detect structural abnormalities of this gene, and genotype analysis ruled out the possibility that a CD43 allele might be shared by the affected individuals. These findings indicate that an alteration(s) of an autosomal gene distinct from the CD43 gene is responsible for the disease. Thus, results from this family, providing the first observation of an autosomally transmitted WAS variant, indicate that genetic mechanism(s) leading to WAS are more complex than previously recognized. PMID- 8639822 TI - Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. AB - Recently, the ligand for the Mpl receptor (ML) was identified to be thrombopoietin, the principal regulator of megakaryocytopoiesis and thrombopoiesis. We examined the effects of ML, as a single factor or in combinations with early acting factors such as steel factor (SF), interleukin (IL)-3, IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF), on colony formation from primitive progenitors of mice. Cells enriched for cell cycle dormant primitive progenitors were isolated from bone marrow cells of 5 fluorouracil (5-FU)-treated mice by a combination of Nycodenz density gradient separation, immunomagnetic selection for lineage-negative cells, and fluorescence activated cell sorter (FACS) sorting for Ly-6A/E+Kit+ cells. ML, in the presence of erythropoietin, could support the formation of only a few megakaryocyte colonies. However, ML acted synergistically with SF or IL-3 to support the formation of multiple types of hematopoietic colonies including multilineage colonies. Effects of the combination of ML and SF on multipotential progenitors were not mediated through other cells, as demonstrated by micromanipulation of individual progenitors. In suspension culture, the combination of ML and SF increased the number of multipotential progenitors. ML also acted synergistically with IL-11, IL-6, or G-CSF to support colony formation in serum-containing, but not in serum-free, cultures. However, the multilineage colony formation seen in serum-containing culture was completely abrogated by addition of ACK2, a neutralizing antibody to Kit protein. Serial observation (mapping studies) of colony development from multipotential progenitors suggested that ML triggers the cell division of dormant progenitors. Based on these observations, we propose that ML can function as an early acting cytokine and stimulate the proliferation of cell cycle dormant progenitors by shortening their G0 period. PMID- 8639824 TI - Maintenance of murine long-term repopulating stem cells in ex vivo culture is affected by modulation of transforming growth factor-beta but not macrophage inflammatory protein-1 alpha activities. AB - Transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-l alpha (MIP-1 alpha) are both well-described inhibitors of committed and multipotential hematopoietic progenitors. The effect of these cytokines; on true stem cell activity in ex vivo culture systems as assayed by murine long-term repopulating activity (LTRA) has not been examined. We studied the stem cell effects of the addition of these cytokines to ex vivo cultures containing interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), using the murine competitive repopulation assay. We also tested the impact of adding an anti-TGF beta neutralizing antibody, to ask whether abrogation of autocrine/paracrine TGF beta may protect or enhance the survival of LTRA during ex vivo culture. TGF-beta 1 had significant suppressive effects on both short- and long-term repopulating activities, and anti-TGF-beta antibody had enhancing effects compared with control cultures containing IL-3, IL-6, and SCF alone. MIP-1 alpha had no significant effects on either short- or long-term repopulating ability. These data suggest that abrogation of TGF-beta during suspension culture may allow enhanced survival or even expansion of primitive cells ex vivo, with implications for many applications, including gene therapy. PMID- 8639823 TI - Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO. AB - UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT 7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO. PMID- 8639825 TI - Structural and functional differentiation of sinusoidal endothelial cells during liver organogenesis in humans. AB - During fetal life, human liver sinusoids, which differentiate between 4 and 12 weeks of gestation from capillaries of the septum transversum, must support an important hematopoietic function and acquire the structural and functional characteristics of adult sinusoids. To gain insight into their differentiation process, we studied the expression of (1) markers of continuous endothelia, absent from adult sinusoidal endothelial cells (PECAM-1, CD34, and 1F10); (2) functional markers of adult sinusoidal endothelial calls (CD4, 1CAM-1, CD32, and CD14); and (3) extracellular matrix components (laminin, tenascin, fibronectin, and thrombospondin) in 37 fetuses of different gestational ages. We identified two successive differentiation events. (1) An early structural differentiation, occurring from 5 to 12 weeks of gestation, was characterized by the loss of continuous endothelial cell markers and a reduction in the perisinusoidal amount of laminin and in the deposition of tenascin, fibronectin, and thrombospondin; at the end of this process, fetal liver sinusoids present structural characteristics comparable to those of the sinuses in adult hematopoietic bone marrow. (2) A later functional differentiation was characterized by the acquisition of the markers of adult sinusoidal endothelial cells, initiating at 10 weeks of gestation and completed by 20 weeks of gestation; this process likely contributes to adapt liver sinusoids to the specific functions of the adult hepatic tissue. PMID- 8639826 TI - Prophylactic pretreatment of mice with hematopoietic growth factors induces expansion of primitive cell compartments and results in protection against 5 fluorouracil-induced toxicity. AB - The aim of this study was to expand the primitive and committed hematopoietic cell compartments in vivo in order to confer resistance of the blood cell forming system against the cytotoxic, cell cycle specific drug 5-fluorouracil (5-FU). Possible chemoprotective effects of such a pretreatment could result from increased numbers of hematopoietic cells, present before 5-FU administration. In addition, we hypothesized that an enhanced number of primitive and progenitor calls would result in a reduced cycling activity, ie, 5-FU sensitivity, of these same cells, due to normal physiological feedback loops. Administration of stem cell factor (SCF) plus interleukin-11 (IL-11) to mice was shown to result in expansion of the various immature cell compartments in marrow and, in particular, spleen. The total body content of the primitive cobblestone area forming cells (CAFC)-day 28 was increased to 140%, whereas the more committed cells (CAFC-day 7, erythroid and granuloid progenitors) were increased to 500%. This in vivo expansion resulted in a decreased 5-FU sensitivity of the hematopoietic system. In particular, mice that had received 5-FU 24 hours after discontinuation of growth factor pretreatment showed significantly less toxicity of committed cell stages. Compared with mice not pretreated, it appeared that in pretreated mice, 24 hours after 5-FU administration, the absolute number, but also the fraction of surviving CAFC, was much higher in both marrow and spleen. This was caused by a decrease in the cycling activity of all primitive cell subsets. To explore the possible use of this finding in a chemotherapeutic setting, we determined the interval between two subsequent doses of 5-FU (160 mg/kg) that was required to prevent drug-induced mortality. When control mice received a second dose of 5-FU 7, 10, or 14 days after the first, respectively 0%, 20%, and 80% survived. In contrast, 40% and 100% of mice that received SCF + IL-11 before the first dose of 5-FU, survived a second dose of 5-FU given respectively after 7 or 10 days. To assess whether chemoprotection in this setting could be ascribed to protection of the hematopoietic system, we transplanted a high number of normal bone marrow cells (sufficient to compensate for any hematopoietic deficiency) to normal and pretreated mice after they had been administered 2 doses of 5-FU, given 7 days apart. Bone marrow transplantation (BMT) could only rescue 50% of mice not pretreated, showing that a significant part of the mortality was because of nonhematologic toxicity. However, a BMT given to growth factor pretreated mice saved all mice, indicating that in this setting SCF + IL-11 had additional protective effects on cell systems other than hematopoiesis. In conclusion, our study showed fundamental knowledge about the behavior of primitive cells in vivo and has shown that manipulation of these and other cell compartments with appropriate growth factors may confer resistance against cytotoxic drugs. PMID- 8639827 TI - Ex vivo expansion with stem cell factor and interleukin-11 augments both short term recovery posttransplant and the ability to serially transplant marrow. AB - The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11. PMID- 8639828 TI - Hematopoietic supportive functions of mouse bone marrow and fetal liver microenvironment: dissection of granulocyte, B-lymphocyte, and hematopoietic progenitor support at the stroma cell clone level. AB - We dissected the functions of the microenvironment of bone marrow (BM) and fetal liver (FL) at the cellular level by cloning individual stromal calls and characterizing their phenotypical and functional features. Stromal cell clones derived from FL are large in size (mean forward light scatter intensity [mFSC] of 450), express the surface antigen Thy-1 but not Sca-1 and 6 out of 6 are able to differentiate into fat accumulating adipocytes. BM derived stromal cell clones are either small (mFSC of 250) or large (mFSC of 450), express Sca-1 but not Thy 1 and only 2 out of 7 differentiate towards adipocytes. Heterogeneity in terms of vascular adhesion molecule-1, intracellular adhesion molecule-1 and heat stable antigen expression was found among the different cell clones. Functional assays using long- and short-term cocultures of stromal and hematopoietic calls revealed: (1) the capacity of 8 out of 12 stromal cell clones to support the expansion of primitive hematopoietic progenitors (colony forming unit spleen day 12) more than 10 weeks. Fat accumulation but not expression of stem cell factor by stromal cells did correlate with this supportive function. (2) Better support of granulocyte maturation and proliferation by BM- compared to FL-derived stromal cell clones. However, stromal cell clones from both organs expressed macrophage colony stimulating factor. (3) The ability of 4 out of 12 stromal cell clones (derived from both, FL and BM) to support the expansion of Interleukin-7 dependent pre-B cells from the BM. Pre-B cell growth stimulating factor was not restricted to supporters. (4) Mutual exclusiveness of myeloid and lymphoid support in that a given stromal cell clone supported either pre B-cell or granulocyte expansion. Experiments comparing the support of BM- and FL-derived hematopoietic progenitors showed identical responses of late (B220+/c-kit-) but strikingly different responses of early (B220+/c-kit+) pre-B cells, revealing different proliferation requirements for FL- versus BM- derived early pre-B cells in vitro. PMID- 8639829 TI - The proto-oncogene HLF and the related basic leucine zipper protein TEF display highly similar DNA-binding and transcriptional regulatory properties. AB - Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To address these questions, we cloned the human homologue of TEF/VBP, a bZIP protein closely related to HLF. Using a binding site selection assay, we found that TEF bound preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site. TEF and HLF activated transcription of consensus site-containing reporter genes in several different cell types with similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to a discrete approximate 40 amino acid region of TEF and HLF within which they share 72% amino acid identity and 85% similarity. The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs. The THAD contained most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogated transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF and HLF share indistinguishable DNA binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important. PMID- 8639830 TI - Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells. AB - Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture. PMID- 8639832 TI - Characterization of porcine platelet glycoproteins recognized by human natural "anti-gal" antibodies. AB - Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180 , 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain. PMID- 8639831 TI - Characterization of vasoactive intestinal peptide receptors on human megakaryocytes and platelets. AB - Vasoactive intestinal peptide receptor I (VIPRI) expression was examined in megakaryocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). VIPRI protein was characterized in platelet membranes using covalent crosslinking techniques. Human megakaryocytes were isolated from suspension cultures of cord blood and adult bone marrow mononuclear cells using a murine monoclonal antibody to human platelet glycoprotein IIB/IIIA (CD41) and immunomagnetic beads. RT-PCR primers were constructed for the VIP, VIPRI, and VIPRII genes as well as for megakaryocyte specific genes, c-mpl and platelet factor 4 (PF-4). VIP, VIPRI, c mpl, and PF-4 were coexpressed in megakaryocyte mRNA. Southern blot analysis confirmed the expression of VIPRI. 125I-VIP was covalently cross-linked to human platelet membranes using the homobifunctional reagent disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. A 125I-VIP protein complex of Mr = 50,000 was identified. Labeling of the Mr = 50,000 component was completely abolished by unlabeled VIP, but not by peptide histidine methionine or growth hormone releasing factor, indicating specific binding of VIP to the platelet membranes. Taken together, these results suggest that VIP may have direct effects on megakaryocytopoiesis and support our earlier observations of VIP modulation of platelet aggregation. PMID- 8639833 TI - Molecular basis of a hereditary type I protein S deficiency caused by a substitution of Cys for Arg474. AB - The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family. PMID- 8639834 TI - Platelet-derived microparticles associate with fibrin during thrombosis. AB - Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P-selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P-selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi. PMID- 8639835 TI - Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists. AB - Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin. PMID- 8639836 TI - Sustained expression of therapeutic levels of human factor VIII in mice. AB - Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression. PMID- 8639837 TI - Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes. AB - The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans-acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation. PMID- 8639838 TI - A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I. AB - A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation. PMID- 8639840 TI - Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. AB - The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-->Ala-substituted rAPC (rAPCgamma20A), and membrane bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S. PMID- 8639839 TI - Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay. AB - Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC. PMID- 8639841 TI - Lack of plasminogen activator inhibitor-1 effect in a transgenic mouse model of metastatic melanoma. AB - Tumor cell invasion and metastasis is a complex, multistep process that is postulated to require degradation of extracellular matrix at several steps. Urokinase-type plasminogen activator (uPA) is expressed on the cell surface of B16 murine melanoma cells and is thought to contribute to the pericellular proteolysis necessary for tumor cell migration. In vitro modification of B16 melanoma cell surface uPA activity has been shown to alter the invasive and metastatic potential of these murine melanoma cells in vivo. Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of both uPA and tissue-type plasminogen activator (tPA) is the major physiologic regulator of plasminogen activator activity. To test the role of host PAI-1 in the invasive and metastatic capacity of B16 melanoma cells we analyzed local tumor growth and pulmonary metastasis in transgenic mice engineered to overexpress murine PAI-1 in multiple tissues including lung, and in mice completely deficient in PAI-1. No significant difference in the number of pulmonary metastases was observed after intravenous inoculation of tumor cells into PAI-1-overexpressing and PAI-1-deficient mice when compared with wild-type controls. Similarly, in a spontaneous metastasis model, PAI-1-overexpressing and PAI-1-deficient mice demonstrated no difference in primary tumor size or overall survival. These data demonstrate that wide variations of host PAI-1 expression, from complete absence to marked overexpression, does not significantly influence the metastatic potential of B16 melanoma cells in a murine model. PMID- 8639842 TI - Engagement of the adhesion receptor CD22 triggers a potent stimulatory signal for B cells and blocking CD22/CD22L interactions impairs T-cell proliferation. AB - The B-lymphocyte-restricted adhesion protein CD22 mediates sialic acid-dependent cell-cell interactions. Engagement of CD22 on B lymphocytes with a CD22 monoclonal antibody (MoAb) HB22.7 that blocks the binding of CD22 to its ligand(s) directly stimulated B-cell proliferation. In addition, the HB22.7 MoAb costimulated B-cell proliferation with either anti-IgM, interleukin-2 (IL-2), IL 4, or CD40 and triggered predominantly B-cell IgG secretion with IL-2. Even more striking levels of B-cell proliferation occurred with HB22.7 MoAb under culture conditions that enhanced B-B-cell interactions. In contrast, a nonblocking CD22 MoAb (CD22.5) poorly costimulated in similar experiments. The functional differences between the two antibodies likely result from differing abilities to trigger downstream signaling events as significant differences in CD22 tyrosine phosphorylation and the recruitment of the tyrosine kinase p53/56lyn and the tyrosine phosphatase SH-PTP1C were found. Besides their role in B-cell stimulation, CD22/CD22L interactions may also assist in regulating T-cell proliferation because inhibition of CD22-CD22L engagement with the HB22.7 MoAb impaired T-cell proliferation in a costimulatory assay. Thus, CD22/CD22L interactions result in stimulatory signals for both B and T lymphocytes. PMID- 8639843 TI - Interferon-alpha stimulates production of interleukin-10 in activated CD4+ T cells and monocytes. AB - In the present study, we investigated the effect of interferon-alpha (IFN-alpha) on the expression of interleukin-10 (IL-10) mRNA and protein synthesis in human monocytes and CD4+ T cells. In mononuclear cells, IFN-alpha induced expression of IL-10 mRNA and further enhanced lipopolysaccharide (LPS)-stimulated IL-10 expression. In purified monocytes, a strong expression of IL-10 mRNA induced by LPS was not further enhanced by IFN-alpha. In highly purified CD4+ T cells, IFN alpha upregulated IL-10 mRNA upon activation with phytohemagglutinin and phorbol myristate acetate. In purified monocytes, an effect of IFN-alpha on IL-10 protein synthesis was dependent on costimulation with LPS. Maximal stimulation of IL-10 protein by IFN-alpha was seen after prolonged incubation periods of 48 to 96 hours, whereas IFN-gamma reduced IL-10 production in the early incubation period. Similar effects of IFN-alpha were observed in CD4+ T cells activated with CD3 and CD28 monoclonal antibodies. Addition of IFN-alpha caused an increase of IL-10 in culture supernatants of activated T-helper cells of more than 100% after 96 hours of incubation. In contrast, other cytokines, including IFN-gamma and IL-4, had no influence on IL-10 secretion stimulated by CD3 and CD28 in CD4+ T cells. In serum samples of IFN-alpha-treated individuals, we failed to detect an influence of cytokine treatment on IL-10 serum levels, confirming the requirement of additional activating signals for IFN-alpha-mediated effects on IL-10 synthesis. In conclusion, IFN-alpha enhances the late induction of IL-10, which physiologically occurs upon stimulation of monocytes and T cells. Biologically, this effect might enhance the negative-feedback mechanism ascribed to IL-10, which limits inflammatory reactions. PMID- 8639844 TI - A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1. AB - In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection. PMID- 8639845 TI - In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals. AB - Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3-mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N-acetyl-L cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death. PMID- 8639846 TI - Characterization of a hierarchy in human acute myeloid leukemia progenitor cells. AB - Despite the usual uniform and primitive appearance of cells derived from the leukemic clone in most patients with acute myeloid leukemia (AML), there is considerable heterogeneity among leukemic blasts, particularly with respect to their capacity to proliferate and/or self renew. We have assessed whether these differences in proliferative potential are correlated with the phenotypic changes that characterize normal hematopoiesis, which might suggest an analogous hierarchy of AML progenitors. We have used the ability of primitive AML cells to persist or produce blast colony forming cells (CFU-blast) detected after 2 to 8 weeks in the presence of growth factors in suspension cultures (SC) termed SC initiating cells (IC), or with stroma in long-term cultures (LTC-IC) as a quantitative assay for a cell that may have primitive characteristics. This SC assay is linear, cell concentration independent, and the frequency of SC-IC by limiting dilution analysis is lower than primary CFU-blast. The average output of CFU-blast after 2 to 8 weeks by individual SC-IC varied between 2 and more than 100 in individual patients. Leukemic blasts were sorted based on their expression of antigens previously found useful to characterize normal progenitor differentiation, and analyzed for the percentage of CFU-blast SC-IC, and leukemic LTC-IC within each fraction. All of these progenitor types were heterogeneous in their expression of CD45RA and CD33, but expressed uniformly low levels of CD15 and differed from normal primitive progenitors in their high expression of HLA DR. CFU-blast had a significantly higher expression of CD71 and CD38 as compared with SC-IC or leukemic LTC-IC. In patients with CD34+ blasts, the majority of their SC-IC at 4 weeks were CD34+/CD38-; however, patients with CD34- blasts had at least some CD34- progenitors. These results show that while heterogeneity exists between patients, it is possible to physically separate subpopulations of AML cells with different proliferative potentials. It also provides some support for the concept that quantitation of leukemic cells capable of producing CFU blast for 4 weeks or more in vitro measures a less frequent leukemic progenitor with higher proliferative potential that may be the only relevant cell for maintaining the leukemic clone in vivo. PMID- 8639847 TI - Quantifiable excess of bone resorption in monoclonal gammopathy is an early symptom of malignancy: a prospective study of 87 bone biopsies. AB - To determine if excessive osteoclastic-mediated bone resorption (BR) is an early tumor-induced event in multiple myeloma (MM), BR was assessed at first presentation on quantitative bone biopsy in 87 individuals evaluated for monoclonal gammopathy of undetermined significance (MGUS) and reinterpreted according to the presenting features and subsequent follow-up evaluation. As a reference population, 48 patients with previously untreated overt MM were evaluated under similar conditions. The median level of BR was significantly higher in 48 overt MM versus 87 MGUS patients (12.2% v 5.1% [normal level, <6%], P <.01). Actually, 93% of overt MM patients had an excessive BR versus 45% of MGUS patients at presentation (P <.01) According to simple presenting parameters (> or <5% plasma cells within the bone marrow, presence or absence of mild anemia/neutropenia), 31 individuals were classified as low-risk MGUS, 32 high risk MGUS, and 24 indolent MM. An excessive BR was observed in 16% of low-risk MGUS, 46% of high-risk MGUS (P <.01 v low-risk MGUS), 79% of indolent MM (P <.05 v high-risk MGUS), and 93% of overt MM patients. Of major interest, the level of BR in indolent MM (11.2%) was identical to that in overt MM (12.2%) but significantly higher than in both low-risk (4%, P <.01) and high-risk (5.6%, P <.01) MGUS. When considering the follow-up evaluation of MGUS patients, an excessive BR at presentation was observed in 52% of MGUS cases that turned out to be unstable or developed subsequent MM, but in only 4% of stable MGUS (P <.01). More precisely the level of BR of low-risk MGUS that either turned out to be unstable or that developed into MM was significantly higher at presentation than that of subsequent stable MGUS (4.4% v 2.9%, P <.05). The same difference was observed in both high-risk MGUS and indolent MM according to subsequent follow-up studies (8.1% v 3.4% and 11.7% v 6%, respectively, P <.05). Of major interest, the level of BR in 11 stable high-risk MGUS cases actually fulfilling the diagnostic criteria of smoldering MM was very low (3.4%) and similar to that in stable low-risk MGUS (2.9%). We conclude that a quantifiable excess of BR in MGUS is significantly associated with progression and thus is an early symptom of malignancy in these individuals. PMID- 8639848 TI - BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients. AB - We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS) selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC-IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation. PMID- 8639849 TI - Integrin function in chronic lymphocytic leukemia. AB - Integrins are central to many aspects of the tissue localization of normal and malignant lymphocytes. We examined how integrin function, rather than simple expression, might determine disease behavior in chronic lymphocyte leukemia (CLL). Using fluorescence-activated cell sorting (FACS) and immunoprecipitation, we first established the precise integrin heterodimer expression of a representative group of CLL patients (beta1 consistently present, with variable alpha 3, alpha 4, and alpha 5; alpha 4 beta 7 often expressed; alpha L beta 2 high; alpha V beta 3 absent). Regarding function, we initially examined the ability of CLL cells to interact with endothelium, because such interaction is the initial event determining the entry of CLL lymphocytes into tissues. The abnormal lymphocytes were shown to bind at low levels to unstimulated endothelium via beta 2/intercellular adhesion molecule (ICAM). However, when the endothelium was stimulated, markedly enhanced interaction with endothelium was observed in approximately half the cases; in these patients, the neoplastic population expressed alpha 4 beta 1, which conferred the ability to adhere strongly to stimulated endothelium via the alpha 4 beta 1 ligand, vascular cellular adhesion molecule-1 (VCAM-1). In relation to the migration of CLL cells within tissues, the abnormal lymphocytes showed differential binding to various adhesive proteins; they did not attach to basement membrane components, but displayed variable adhesion to fibronectin (FN). Finally, we examined the role of cell activation in these processes, and showed that activated CLL lymphocyte populations showed an increased capacity to adhere to both endothelium and matrix. Moreover, ex vivo CLL cells showed no capacity to migrate through endothelium/stroma, but were able to do so after cytokine stimulation. These studies show how the constitutive integrin expression/function, the intrinsic activation state of the cell, and the capacity of cytokines to modify integrin mediated function all combine to determine the different patterns of clinical disease observed in CLL. PMID- 8639850 TI - Persistence of multipotent progenitors expressing AML1/ETO transcripts in long term remission patients with t(8;21) acute myelogenous leukemia. AB - The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT-PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X-chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality. PMID- 8639851 TI - Interleukin-4 enhances the survival of severe combined immunodeficient mice engrafted with human B-cell precursor leukemia. AB - Human interleukin-4 (huIL-4) has been shown to inhibit the growth in vitro of cells from patients with acute lymphoblastic leukemia (ALL). With the aim of determining whether this cytokine might be useful in the treatment of patients with ALL, the effects of huIL-4 on human B-cell precursor ALL engrafted in severe combined immunodeficient (SCID) mice were examined. The inhibition of [3H] thymidine uptake of primary ALL cells by huIL-4 was maintained following engraftment and passage of leukemia in SCID mice. Five of seven xenograft leukemias showed significant inhibition in vitro by huIL-4 at concentrations as low as 0.5 ng/mL; furthermore, huIL-4 counteracted the proliferative effects of IL-7. When used to treat two human leukemias engrafted in SCID mice, huIL-4 200 microgram/kg/d, as a continuous 14-day subcutaneous infusion, suppressed the appearance of circulating lymphoblasts and extended survival of mice by 39% and 108%, respectively, the first demonstration of IL-4 activity against human leukemia in vivo. The mean steady-state huIL-4 level in mouse plasma during the infusion was 1.46 ng/mL (SEM +/- 0.14 ng/mL), which was similar to concentrations found to be effective in vitro. ALL cells obtained from mice relapsing after huIL 4 treatment continued to show inhibition by the cytokine in vitro. These data suggest that IL-4 may be useful in the treatment of patients with ALL. PMID- 8639852 TI - Molecular analysis of t(11;19) breakpoints in childhood acute leukemias. AB - MLL is fused to ENL or ELL in acute leukemias that contain t(ll;19)(q23;p13). Although ENL and ELL localize to chromosome 19, bands p13.3 and p13.1, respectively, these breakpoints are not always readily distinguished by standard cytogenetics. We therefore used reverse transcriptase-polymerase chain reaction (RT-PCR) assays to analyze 26 cases of childhood acute leukemia containing t(11;19) to determine the frequencies of ENL and ELL involvement. All 17 cases of acute lymphoblastic leukemia (ALL) had MLL/ENL fusion transcripts. By contrast, of the 9 cases of acute myeloid leukemia (AML) analyzed, 6 had MLL/ENL fusions, 2 had MLL/ELL fusions, and 1 case had no RT-PCR-detectable MLL fusion mRNA. These data suggest that the majority of 11;19 translocations involve ENL, whereas involvement of ELL is relatively uncommon in childhood acute leukemia and may be restricted to AML. PMID- 8639853 TI - Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity. AB - We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies. PMID- 8639854 TI - Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI. AB - Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI. PMID- 8639855 TI - Defective iron supply for erythropoiesis and adequate endogenous erythropoietin production in the anemia associated with systemic-onset juvenile chronic arthritis. AB - Systemic-onset juvenile chronic arthritis (SoJCA) is associated with high levels of circulating interleukin-6 (IL-6) and is frequently complicated by severe microcytic anemia whose pathogenesis is unclear. Therefore, we studied 20 consecutive SoJCA patients with hemoglobin (Hb) levels <12 g/dL, evaluating erythroid progenitor proliferation, endogenous erythropoietin production, body iron status, and iron supply for erythropoiesis. Hb concentrations ranged from 6.5 to 11.9 g/dL. Hb level was directly related to mean corpuscular volume (r = .82, P < .001) and inversely related to circulating transferrin receptor (r = .81, P < .001) suggesting that the severity of anemia was directly proportional to the degree of iron-deficient erythropoiesis. Serum ferritin ranged from 18 to 1,660 microgram/L and was unrelated to Hb level. Bone marrow iron stores wore markedly reduced in the three children investigated, and they also showed increased serum transferrin receptor and normal-to-high serum ferritin. All 20 patients had elevated IL-6 levels and normal in vitro growth of erythroid progenitors. Endogenous erythropoietin (epo) production was appropriate for the degree of anemia as judged by both the observed to predicted log (serum epo) ratio 10.95 +/- 0.12) and a comparison of the serum epo-Hb regression found in these subjects with that of thalassemia patients. Multiple regression analysis showed that serum transferrin receptor was the parameter most closely related to hemoglobin concentration: variation in circulating transferrin receptor explained 61% of the variation in Hb level (P < .001). In 10 severely anemic patients, amelioration of anemia following intravenous iron administration resulted in normalization of serum transferrin receptor. Defective iron supply to the erythron rather than blunted epo production is the major cause of the microcytic anemia associated with SoJCA. A true body-iron deficiency caused by decreased iron absorption likely complicates long-lasting inflammation in the most anemic children, and this can be recognized by high serum transferrin receptor levels. Although oral iron is of no benefit, intravenous iron saccharate is a safe and effective means for improving iron availability for erythropoiesis and correcting this anemia. Thus, while chronically high endogenous IL-6 levels do not appear to blunt epo production, they are probably responsible for the observed abnormalities in iron metabolism. Anemia of chronic disease encompasses a variety of anemic conditions whose peculiar features may specifically correlate with the type of cytokine(s) predominantly released. PMID- 8639856 TI - The pathophysiology of pure red cell aplasia: implications for therapy. AB - To determine the utility of marrow culture in defining the natural history and therapeutic response of pure red cell aplasia we have studied 37 patients. Patients were evaluated at the University of Washington before specific therapies (n = 21) or at the time of treatment failure in = 16). Evaluation included a medical and drug exposure history, a physical examination, a chest x-ray or computed tomography to rule out thymoma, lymphocyte immunophenotype studies, anti nuclear antibody and rheumatoid factor determinations, marrow cytogenetics, and marrow progenitor cell cultures. Retrospective Southern analyses to detect human parvovirus B19 was performed in the 27 patients for whom sera was stored. Clinical follow-up was obtained to document therapeutic responses. Normal burst forming unit-erythroid (BFU-E) growth (>30 bursts/10(5) marrow mononuclear cells [MMNC]) in culture proved an outstanding predictor of clinical response, as 27 of 29 individuals with normal frequencies of erythroid bursts in culture responded to immunomodulating therapies (sensitivity 96%, specificity 78%, predictive value 93%, P = .0001 with two-tailed chi square analysis). Overall, 28 patients responded to either immunomodulating therapies or drug withdrawal. Twenty-four patients obtained a normal hematocrit (complete response [CR] and 4 additional patients became transfusion independent (partial response). Although responding patients often required several therapies, 20 of 24 (83%) patients who obtained a CR have sustained a normal hematocrit without maintenance therapy at the time of last follow-up (median 5 years). In contrast, of 8 patients with poor in vitro BFU-E growth (<6 bursts/10(5) MMNC), 7 failed to respond to any therapy and all died (median survival time 17 months). Our data suggest that in individuals, from whom BFU-E mature appropriately in culture, immunosuppressive drugs should be used sequentially until a CR is obtained and a durable remission is the expected outcome. PMID- 8639857 TI - Human erythrocytes bearing electroinserted CD4 neutralize infection in vitro by primary isolates of human immunodeficiency virus type 1. AB - Human erythrocytes bearing electroinserted full-length CD4 (RBC-CD4) can bind and fuse with a laboratory strain of human immunodeficiency virus type 1 (HIV-1) or with T cells infected by HIV-1. Here we show that RBC-CD4 neutralize primary HIV 1 strains in an assay of cocultivation of peripheral blood mononuclear cells (PBMC) from HIV-1-infected persons with uninfected PBMC. RBC-CD4 inhibited viral p24 core antigen accumulation in these cocultures up to 10,000-fold compared with RBC alone. Viral p24 accumulation was inhibited equally well when measured in culture supernatants or in call extracts. The inhibition was dose-dependent and long-lived. Two types of recombinant CD4 tested in parallel were largely ineffective. The neutralization of primary HIV-1 by RBC-CD4 in vitro was demonstrated in PBMC cultures from 21 of a total of 23 patients tested at two independent sites. RBC-CD4 may offer a route to blocking HIV-1 infection in vivo. PMID- 8639858 TI - Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro. AB - Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-I beta (IL-1 beta) stimulated endothelial calls in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1 beta for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: alpha v beta 3 after 1 hour of IL-1 beta stimulation; E-selectin after 4 hours of IL-1 beta stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1 beta stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes. PMID- 8639859 TI - Molecular basis of the altered antigenic expression of RhD in weak D(Du) and RhC/e in RN phenotypes. AB - The RH blood group locus is composed of two sequence-related genes, RHD and RHCE, encoding the D, Cc, and Ee antigens in common Rh-positive phenotypes. In this report, we have analyzed the molecular basis of Rh antigens expression in weak D (Du) and RN donors, in whom there is a severe reduction of the D and C/e antigens, respectively. Genomic and transcript analysis of three unrelated low grade weak D (Du) variants indicated that the very low expression of the D antigen is not the result of rearrangement or mutation in the coding sequence of the RHO gone. Accordingly, weak D (Du) erythrocytes should carry a normal RhD polypeptide, which is in agreement with the observation that these variants never produce anti-D antibodies. Comparative polymerase chain reaction analysis showed a lower steady-state level of RhD transcripts in weak D (Du) reticulocytes, as compared with normal RhD-positive controls, thus providing direct evidence that the difference between the D antigen of D-positive and weak D (Du) red blood cells is quantitative only. Conversely, analysis of the molecular genetic basis of the RN phenotype Indicated that the severely decreased expression of the RhC and Rhe antigens in three variants is associated with a qualitative alteration identified as a segmental DNA exchange between the RHCE and RHD genes. These genomic rearrangements, which resulted in hybrid RhCe-D-Ce proteins expressing the low frequency Rh32 but not the high incidence Rh46 antigens, involved either axon 4 alone or both exons 3 and 4. These findings show that an identical phenotypical alteration of Rh antigens (reduced expression) may result either from a quantitative or a qualitative alteration of the RH genes expression. PMID- 8639860 TI - Glycoprotein IV-independent adhesion of sickle red blood cells to immobilized thrombospondin under flow conditions. AB - The abnormal adherence of red blood cells (RBC to the blood vessel wall is believed to contribute to the vascular occlusion observed in patients with sickle call anemia. The cell adhesion receptors GPIV (CD36) and integrin alpha 4 beta 1 (CD49d/CD29) were previously identified on circulating sickle reticulocytes, and shown to mediate sickle RBC adhesion to the endothelium. The presence of damaged endothelium in these patients suggests that exposed extracellular matrix proteins could provide a potential substrate for sickle RBC adhesion. To determine whether RBC adhesion receptors could mediate adhesion to extracellular matrix proteins, we tested their ability to adhere to a variety of immobilized, purified proteins under flow conditions. Neither sickle nor normal RBC adhered to fibronectin, vitronectin, fibrinogen, or collagen. In contrast, we observed substantial adhesion of sickle but not normal RBC to thrombospondin (TSP). The adhesion was not inhibited with known antagonists of the GPIV-TSP interaction, nor by inhibitors of several other known binding domains in TSP. Moreover, the adhesion was resistant to inhibition by soluble TSP, suggesting that immobilization of TSP exposes an adhesive site that is cryptic on TSP in solution. However, the glycosaminoglycans, chondroitin sulfate A, and dextran sulfate were potent inhibitors of this adhesion. These results suggest that a mechanism distinct from GPIV is responsible for sickle RBC adhesion to immobilized TSP under flow conditions. PMID- 8639861 TI - Differential cytotoxicity of iron chelators on malaria-infected cells versus mammalian cells. AB - Iron chelators of the hydroxamate class arrest in vitro proliferation of malaria parasites end of mammalian cells. The factors determining the biological activity of the chelators have classically been attributed to the chelators' capacity for binding iron and to their ability to traverse membranes as free chelators and as chelator-iron complexes. We show in this work that the nature of the chelatable pool of cell iron also contributes to the susceptibility of cells to iron chelators. A class of N-terminal (Nt derivatives of desferrioxamine (DFO), (Nt DFO), is shown here to differentially affect growth and replication of intraerythrocytic parasites (Plasmodium falciparum). Methyl-anthranilic DFO (MADFO), the relatively less hydrophilic member of the Nt-DFOs series, reduced parasite proliferation (48 hour test) with an IC50 of 4 +/- 1 micromol/L and mammalian cell (K562 and HepG2) proliferation with an IC50 > 100 micromol/L. On the other hand, the more hydrophilic Nt-free DFO, displayed IC50 values of 21 +/- 5 micromol/L for parasites and 7 +/- 1 micromol/L for mammalian cells. The selective antiparasitic activity of MA-DFO, as reflected in the speed of action and IC50 values on cell proliferation, is attributed primarily to membrane permeation and iron (III) binding properties of the drug. In contrast, the relatively low antiproliferative activity of the more permeant MA-DFO on mammalian cells, resulted from MA-DFO's reduced capacity for scavenging intracellular iron. This is apparent from MA-DFO reduced effects on: (1) the chelatable iron (II) pool that is associated with the cell cytosol; (2) the cell chelator-extractable iron, and (3) cell ferritin levels. The potent antimalarial efficacy and biological selectivity of MA-DFO relative to the parent DFO, is of importance for improved design of chemotherapeutic agents. PMID- 8639862 TI - Increased adhesion of erythrocytes to components of the extracellular matrix: isolation and characterization of a red blood cell lipid that binds thrombospondin and laminin. AB - Red blood cell (RBC) adhesion to the vascular endothelium is increased in several pathologic conditions, including sickle cell disease and malaria. However, RBC interactions with components of the subendothelial matrix are not well characterized. Under in vitro flow conditions of 1 dyne/cm2, washed RBCs bound to the purified adhesive molecules thrombospondin (TSP) and laminin. Sickle RBCs had the greatest adhesion of all tested RBCs. The adhesion of sickle RBCs to immobilized TSP was inhibited by the anionic polysaccharides high molecular weight (MW) dextran sulfate and chondroitin sulfate A, but not other anionic polysaccharides of similar structure and/or charge density. These data were consistent with the RBC adhesive molecule being a sulfated glycolipid. Therefore, TSP-binding lipids from normal and sickle RBCs were isolated and characterized. The TSP-binding lipid was purified by alkaline methanolysis, anion exchange chromatography and preparative thin layer chromatography (TLC). A homogeneous band on TLC was identified using a specific overlay TSP-binding assay. TSP binding to the purified lipid was stable to bass and neuraminidase treatment, labile to acid treatment, and was inhibited by high MW dextran sulfate, similar to that seen with intact RBCs binding to immobilized TSP under conditions of flow. In addition, soluble laminin bound to the purified RBC lipid. This acidic TSP- and laminin-binding lipid(s) isolated from both sickle and normal RBC membranes may contribute to erythrocyte interactions with the subendothelial matrix, hereby participating in the pathogenesis of vaso-occlusive diseases. PMID- 8639863 TI - Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: a method to ameliorate graft-versus-host disease and extend the donor pool. AB - Graft-versus-host disease (GVHD) is initiated by adoptively transferred donor T cells that recognize host alloantigens. Whereas the absence of donor T-cell proliferation to host alloantigens in a mixed-leukocyte reaction does not predict freedom from GVHD, the frequency of alloreactive precursor helper T lymphocytes (pHTL) is predictive. Complete blockade of 87 family-mediated costimulation, but not of major histocompatibility complex recognition or adhesion, induces host alloantigenic-specific energy by reducing cytokine production below threshold levels necessary for common gamma chain signaling. The associated reduction of alloreactive pHTL frequency below that predictive for GVHD, without depletion of either nonallospecific T cells or hematopoietic progenitors, has led us to embark upon human clinical trials of haplomismatched allogeneic bone marrow transplantation. PMID- 8639865 TI - Transplantation of allogeneic CD34+ blood cells. AB - Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA-identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD. PMID- 8639866 TI - Safety of filtered leukocyte-reduced blood products for prevention of transfusion associated cytomegalovirus infection. PMID- 8639864 TI - Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome. AB - Long-term survival and improved neuropsychological function have occurred in selected children with Hurler syndrome (MPS I H) after successful engraftment with genotypically matched sibling bone marrow transplantation (BMT). However, because few children have HLA-identical siblings, the feasibility of unrelated donor (URD) BMT as a vehicle for adoptive enzyme therapy was evaluated in this retrospective study. Forty consecutive children (median, 1.7 years; range, 0.9 to 3.2 years) with MPS I H received high-dose chemotherapy with or without radiation followed by BMT between January 27, 1989 and May 13, 1994. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients are alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute graft-versus-host disease (GVHD) was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD BMT because of graft rejection or failure. Of the 20 survivors, 13 children have complete donor engraftment, two children have mixed chimeric grafts, and five children have autologous marrow recovery. The BM cell dose was correlated with both donor engraftment and survival. Thirteen of 27 evaluable patients were engrafted at 1 year following URD BMT. Neither T-lymphocyte depletion (TLD) of the bone marrow nor irradiation appeared to influence the likelihood of engraftment. Ten of 16 patients alive at 1 year who received a BM cell dose greater than or equal to 3.5 x 10(8) cells/kg engrafted, and 62% are estimated to be alive at 3 years. In contrast, only 3 of 11 patients receiving less than 3.5 x 10(8) cells/kg engrafted, and 24% are estimated to be alive at 3 years (P = .05). The mental developmental index (MDI) was assessed before BMT. Both baseline and post-BMT neuropsychological data were available for 11 engrafted survivors. Eight children with a baseline MDI greater than 70 have undergone URD BMT (median age, 1.5 years; range, 1.0 to 2.4 years). Of these, two children have had BMT too recently for developmental follow-up. Of the remaining six, none has shown any decline in age equivalent scores. Four children are acquiring skills at a pace equal to or slightly below their same age peers; two children have shown a plateau in learning or extreme slowing in their learning process. For children with a baseline MDI less than 70 (median age, 2.5 years; range, 0.9 to 2.9 years), post BMT follow-up indicated that two children have shown deterioration in their developmental skills. The remaining three children are maintaining their skills and are adding to them at a highly variable rate. We conclude that MPS I H patients with a baseline MDI greater than 70 who are engrafted survivors following URD BMT can achieve a favorable long-term outcome and improved cognitive function. Future protocols must address the high risk of graft rejection or failure and the impact of GVHD in this patient population. PMID- 8639867 TI - Lack of constitutive activation of Janus kinases and signal transduction and activation of transcription factors in Philadelphia chromosome-positive acute lymphoblastic leukemia. PMID- 8639868 TI - Molecular basis for the hereditary hyperferritinemia-cataract syndrome. PMID- 8639869 TI - Purging of malignant cells from blood after short ex vivo incubation with NK-92 cells. PMID- 8639870 TI - Epstein-Barr virus-associated CD3- large granular lymphocyte leukemia presenting with polycranial nerve palsies. PMID- 8639871 TI - Dysmegakaryopoietic thrombocytopenia in patients with distal chromosome 11q deletion. PMID- 8639872 TI - Granulocyte colony-stimulating factor administration and peripheral blood progenitor cells collection in normal donors: analysis of leukapheresis-related side effects. PMID- 8639873 TI - Serum thrombopoietin levels in cyclic thrombocytopenia. PMID- 8639874 TI - CD40: a sensitive marker of Reed-Sternberg cells. PMID- 8639875 TI - Organization of the gene encoding the human Kell blood group protein. PMID- 8639876 TI - B-chronic lymphocytic leukemia: a malignancy of anti-self B cells. PMID- 8639877 TI - Defects in Wiskott-Aldrich syndrome blood cells. PMID- 8639878 TI - Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. AB - Human natural killer (NK) cells are bone marrow (BM)-derived CD2+CD16+CD56+ large granular lymphocytes (LGL) that lack CD3 yet contain the T-cell receptor zeta chain (zeta-TCR). NK cells provide requisite interferon-gamma (IFN-gamma) during the early stages of infection in several experimental animal models. A number of studies have shown that human CD3-CD56+ NK cells can be obtained from BM-derived CD34+ hematopoietic progenitor cells (HPCs) cultured in the presence of interleukin-2 (IL-2) and an allogeneic feeder cell layer, or IL-2 and other hematopoietic growth factors such as the c-kit ligand (KL). The failure to detect the IL-2 gene product within the BM stroma and the presence of NK cells in IL-2 deficient mice suggested that cytokines other than IL-2 may participate in NK cell differentiation from HPCs in vivo. IL-15 is a cytokine which, while lacking any sequence homology in IL-2, can activate cells via the IL-2 receptor. Here we show that human BM stromal cells express the IL-15 transcript, and supernatants from long-term BM stromal cell cultures contain IL-15 protein. In vitro, CD3 CD56+ NK cells can be obtained from 21-day cultures of CD34+ HPCs supplemented with IL-15 in the absence of IL-2, stromal cells, or other cytokines. The addition of the KL to these cultures had no effect on the differentiation of the CD3-CD56+ cytotoxic effector cells, but greatly enhanced their expansion. The majority of these cells lack CD2 and CD16, but do express zeta-TCR. Similar to NK cells found in peripheral blood, the CD2-CD16-CD56+ NK cells grown in the presence of IL-15 were found to be potent producers of IFN-gamma in response to monocyte-derived cytokines. Thus IL-15, like KL, appears to be produced by BM stromal cells. IL-15 can induce CD34+ HPCs to differentiate into CD3-CD56+ NK cells, and KL can amplify this. Therefore, IL-15 may be a physiologically relevant ligand for NK cell differentiation in vivo. PMID- 8639879 TI - Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation. AB - The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c-derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL 3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid-specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling. PMID- 8639881 TI - P2X1 receptor activation in HL60 cells. AB - Recent cloning of the human P2X1 receptor revealed high levels of its messenger RNA in differentiated promyelocytes (HL60 cells). We found expression of P2X1 receptor protein in HL60 cells by radioligand binding, by immunohistochemistry, using a receptor specific antibody, and by electrophysiology. The currents elicited by adenosine triphosphate (ATP) had the expected properties of P2X1 receptors (rapid desensitization, mimicked by alpha,beta-methylene-ATP). However, these currents were only observed in cells that were pretreated with apyrase, which destroys extracellular ATP, or with suramin, a P2X receptor antagonist. This implies that HL60 cells release ATP, which chronically desensitizes the receptor. ATP release was detected by direct measurement, using the luciferin luciferase assay. It is concluded that functional P2X1 receptors are present in the membrane of differentiated HL60 cells. PMID- 8639880 TI - Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors. AB - The MLL gene located at 11q23 is frequently disrupted by chromosomal translocation in a wide spectrum of newly diagnosed acute leukemias. Recently, it has become apparent that the MLL gene is very frequently disrupted by chromosomal translocations in patients with secondary leukemias associated with chemotherapeutic regimens incorporating topoisomerase II inhibitors. These secondary leukemias associated with topoisomerase II inhibitors (most commonly teniposide, etoposide, or doxorubicin) have distinct clinical and biologic features which have led to the speculation that they are induced by treatment with topoisomerase II inhibitors. We have identified a site within the MLL breakpoint cluster region (bcr) that is highly sensitive to double-strand DNA cleavage induced by topoisomerase II inhibitors. This finding is quite specific and highly reproducible. Although it was initially discovered in malignant lymphoblasts isolated from a patient receiving multiagent chemotherapy, this site specific double-strand DNA cleavage can be induced in tissue culture using malignant cell lines as well as peripheral blood from normal individuals. Site specific cleavage occurs in a significant fraction of cells using a variety of model systems, is both time and dose dependent, and can be induced with either doxorubicin or etoposide. This site-specific cleavage maps to the same region as a consensus topoisomerase II cleavage site within the MLL bcr. These results suggest that site specific cleavage within the MLL bcr induced by topoisomerase II inhibitors may be an early step leading to MLL translocations and secondary leukemia. PMID- 8639882 TI - Functional inactivation in mice of the gene for the interleukin-3 (IL-3)-specific receptor beta-chain: implications for IL-3 function and the mechanism of receptor transmodulation in hematopoietic cells. AB - The receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 and -5 (IL-3, IL-5) share a common signaling subunit (beta c). However, in the mouse, IL-3 can also use an alternative IL-3-specific receptor beta-chain (beta IL-3). To assess the relative contributions of beta c and beta IL-3 to IL-3 receptor formation and function, mice were generated in which the beta IL-3 gene was functionally inactivated by replacement of exons 9-13 with a neomycin resistance cassette. Bone marrow cells from these mice displayed a lower affinity IL-3 receptor than normal and were hyporesponsive to IL-3, but the mice displayed no obvious hematopoietic abnormalities. The data suggested that beta c and beta IL-3 are normally coexpressed on IL-3-responsive cells and have identical qualitative signaling capacities. Receptor transmodulation studies on bone marrow cells from wild-type, beta c -/-, and beta IL-3 -/- mice showed that the previously described hierarchical pattern of transmodulation was dependent on the relative numbers of both beta IL-3 and beta c receptor chains and also provided evidence for an unexpected interaction between beta c chains and G-CSF and M-CSF receptors. PMID- 8639883 TI - Recombinant human erythropoietin in transfusion-dependent anemic patients with multiple myeloma and non-Hodgkin's lymphoma--a randomized multicenter study. The European Study Group of Erythropoietin (Epoetin Beta) Treatment in Multiple Myeloma and Non-Hodgkin's Lymphoma. AB - One hundred twenty-one anemic, transfusion-dependent patients with multiple myeloma (MM) or low-grade non-Hodgkin's lymphoma (NHL) were randomly allocated to receive (a) recombinant human erythropoietin (rhEPO) 10,000 U/d subcutaneously 7 days a week (fixed dose group) (n = 38), or (b) rhEPO 2,000 U/d subcutaneously for 8 weeks followed by step-wise escalation of the rhEPO dose (titration group) (n = 44), or (c) no rhEPO therapy (control group) (n = 39). The total treatment period was 24 weeks. There were no differences between the three groups with regard to baseline clinical, demographic, or health status measures. The cumulative response frequency, defined as elimination of the transfusion need in combination with an increase in the hemoglobin concentration by >20 g/L, was 60% in both rhEPO treatment groups and 24% in the control group (P = .01 and .02, respectively, log rank test). For patients in the titration group the response rate on the first dose level (2,000 U/d) was only 14%. Cox's univariate regression analysis revealed that an inadequately low endogenous erythropoietin concentration in relation to the degree of anemia and a baseline platelet concentration > or = 100 x 10(9)/L were significant predictors for response to rhEPO therapy (P < .01). Multivariate regression analysis showed that relative erythropoietin concentration was the most important factor and the platelet count had no additional influence on response. Treatment with rhEPO was well tolerated. We conclude that treatment with rhEPO may be indicated in anemic MM and NHL patients with a relative erythropoietin deficiency. An initial dose of 5,000 U/d subcutaneously may be recommended. PMID- 8639884 TI - Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cell-independent mechanism involving apoptosis. AB - Loss of ovarian function leads to a significant increase in the number of bone resorbing osteoclasts. Estrogen replacement is known to manifest bone protective effects in the treatment of postmenopausal osteoporosis. In the present study, we used ovariectomized rats to examine the effects of estrogen loss at the osteoclast progenitor colony forming unit-granulocyte macrophage (CFU-GM) level. A significant increase in CFU-GM number was observed as early as 7 days following ovariectomy, and correlated directly with an increase in the number of osteoclast like cells generated in marrow cultures. The increase in CFU-GM following ovariectomy was abrogated in animals that received estrogen treatment in vivo. A similar suppressive effect was observed on CFU-GM number when ovariectomized rat marrow was treated with estrogen in vitro. This effect was blocked in the presence of the estrogen antihormone ICI 164,384. Thus, the data suggest the possibility that estrogen exerts a direct effect on osteoclast progenitors, and does so through the estrogen receptor-mediated mechanism. Ovariectomy also led to an increase in the early hematopoietic stem/progenitor cell population (Thy 1.1+ cells) as determined by FLOW cytometry methods. Morphological changes as well as terminal deoxynucleotidyl transferase assays revealed that estrogen treatment negated growth factor-induced proliferation of these early progenitors by promoting apoptosis. The cellular effects of estrogen in vitro together with the immunocytochemical detection of the estrogen receptor in these cells, strongly support the contention that in addition to osteoclast progenitors such as CFU-GM, earlier hematopoietic progenitors are also unique cellular targets for estrogen action. PMID- 8639885 TI - 1,25-dihydroxyvitamin D3 differentiates normal neutrophilic promyelocytes to monocytes/macrophages in vitro. AB - Although it is well established that the addition of 1,25-dihydroxyvitamin D3 (D3) to the culture of normal human granulocyte/macrophage progenitors induces monocyte/macrophage (Mo/M phi) colonies, the target cells of D3 in the Mo/M phi differentiation have not been identified. We examined whether neutrophilic promyelocytes are the target cells. As a source of the promyelocyte fraction, we used colonies after 5 days of culture (5-day colonies) of colony-forming unit granulocyte. The culture contained granulocyte colony-stimulating factor (G-CSF) as the growth factor and generated only neutrophilic colonies. The promyelocytic nature of the 5-day colonies was confirmed morphologically, cytochemically, and ultrastructurally. After morphological evaluation on part of the individual colonies, they were transferred into new semisolid cultures with or without D3 (10(-7) mol/L) in the presence of G-CSF, then incubated for the subsequent 7 days. With D3, the colonies were loose, and all the constituent cells were morphologically small macrophages, which were positive for alpha-naphthyl butyrate (alpha NB) esterase, strongly positive for CD14 antigen, and plastic adherent. While without D3, the colonies were rather compact, and all the constituent cells were morphologically mature neutrophils, which were positive for naphthol ASD-chloroacetate esterase and weakly positive for CD14 antigen. Secondary culture of the 8- or 10-day colonies with D3 induced a lower number of alpha NB-positive cells, in proportion to the percentage of promyelocytes at the time of transfer in each colony. Four days of secondary culture with D3 was sufficient to induce alpha NB-positive cells. G-CSF was not an essential factor to induce alpha NB-positive cells. These findings indicate that D3 differentiates normal human neutrophilic promyelocytes into the Mo/M phi lineage in vitro. PMID- 8639886 TI - Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure. AB - We have isolated and mapped the rHuEPO epitopes for three noncompeting anti-EPO monoclonal antibodies (MoAbs). The MoAb 9G8A recognizes a linear epitope that includes amino acids 13, 16, and 17. MoAb F12 recognizes a conformational epitope that includes amino acids 31 through 33, 86 through 91, and 138. MoAb D11 recognizes a conformational epitope that includes amino acids 64 through 78 and 99 through 110. MoAb D11 neutralizes rHuEPO activity which suggests that its epitope may contain the receptor binding domain. Analysis of the effect of mutations on folding allowed the identification of buried residues, alpha helical, and non alpha-helical regions. This data along with epitope mapping data of anti rHuEPO monoclonals was used to model rHuEPO protein structure. A model consistent with the data is a 4-helix bundle with short and long interconnecting loops. PMID- 8639887 TI - Isolation and characterization of conformation sensitive antierythropoietin monoclonal antibodies: effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure. AB - We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability. PMID- 8639888 TI - Gene transfer of multidrug resistance into a factor-dependent human hematopoietic progenitor cell line: in vivo model for genetically transferred chemoprotection. AB - To develop a rapid preclinical in vivo model to study gene transfer into human hematopoietic progenitor cells, MO-7e cells (CD-34+, c-kit+) were infected with multidrug resistance (MDR1)-containing retroviruses and then transplanted into nonobese diabetic severe combined immunodeficient mice (NOD SCID). MO-7e cells infected with a retrovirus encoding the human MDR1 cDNA showed integration, transcription, and expression of the transfered MDR1 gene. This resulted in a 20 fold increase in the resistance of MO-7e cells to paclitaxel in vitro. The expression of the MDR1 gene product was stable over a 6-month period in vitro without selection in colchicine. MO-7e and MDR1-infected MO-7e cells were transplanted into NOD SCID mice to determine whether MDR1 could confer drug resistance in vivo. A sensitive polymerase chain reaction method specific for human sequences was developed to quantitate the level of human cell engraftment in NOD SCID bone marrow (BM) cells. The percentage of human DNA in BM cells from MO-7e-transplanted mice was 10.9% and decreased to 0.7% in mice treated with paclitaxel. The percentage of human DNA in infected-MO-7e transplanted mice was 7.6% and that level was unchanged in mice treated with paclitaxel. These results show that expression of the MDR1 gene in human hematopoietic progenitor cells can confer functional drug resistance in an in vivo model. PMID- 8639889 TI - Potentiation of granulocyte colony-stimulating factor-induced mobilization of circulating progenitor cells by seven-day pretreatment with interleukin-3. AB - Granulocyte colony-stimulating factor (G-CSF) as a single agent is increasingly used for the mobilization of peripheral blood progenitor cells (PBPCs) for stem cell transplantation. In patients with perturbed hematopoiesis the mobilizing capacity of G-CSF alone may be inadequate. We have shown in rhesus monkeys that interleukin-3 (IL-3) pretreatment markedly potentiated the increase in PBPC numbers by subsequent administration of granulocyte/macrophage-CSF (GM-CSF). Here we studied the effect of IL-3 pretreatment on G-CSF-induced mobilization of PBPCs in 6 patients with Hodgkin's disease (n = 5) or non-Hodgkin's lymphoma (n = 1) who had low progenitor cell numbers because of previous chemotherapy. Patients were treated in cycle 1 with G-CSF at a dose of 5 microgram/kg/d for 5 days and, after a treatment-free interval, received cycle 2 consisting of 5 microgram/kg/d of IL-3 for 7 days followed by G-CSF again at a dose of 5 microgram/kg/d for 5 days. G-CSF alone increased the mean number of circulating colony-forming units GM (CFU-GM) by 21-fold, the number of burst-forming units-erythroid (BFU-E) by 9 fold, and the number of CFU-mix by 24-fold over pretreatment values. Treatment with 5 microgram/kg/d of IL-3 for 7 days did not mobilize by itself but significantly potentiated G-CSF-induced mobilization of all progenitor cell types leading to a 56-, 15-, and 46-fold increase over baseline of CFU-GM, BFU-E, and CFU-mix numbers, respectively. In 2 patients in whom leukapheresis was performed after G-CSF alone the target number of 2 x 10(6)/kg CD34+ cells was not reached. However, leukapheresis after the IL-3/G-CSF combination obtained > or =2 x 10(6)/kg CD34+ cells in 3 of 6 patients, including both patients who had inadequate collection after G-CSF alone. In one patient adequate function of mobilized progenitors could be shown by the demonstration of rapid trilineage engraftment after infusion of progenitors after myeloablative chemotherapy. Seven day pretreatment with IL-3 may be a useful mean to augment mobilization of circulating progenitors by G-CSF. The combination of IL-3 and G-CSF seems to allow the procurement of sufficient numbers of PBPCs in some patients who cannot be mobilized adequately by G-CSF alone. PMID- 8639890 TI - Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. AB - Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events. PMID- 8639891 TI - The Ly-6E.1 (Sca-1) gene requires a 3' chromatin-dependent region for high-level gamma-interferon-induced hematopoietic cell expression. AB - The Ly-6E.1/A.2 gene product recognized by the Sca-1 antibody has been found on murine hematopoietic stem cells and some hematopoietic precursors, T lymphocytes, and nonhematopoietic cell lineages, suggesting a complex array of gene regulatory elements. The ability to use the Ly6E.1/A.2 transcriptional regulatory elements to direct expression of heterologous genes will allow for the manipulation of these cells during development and in hematopoietic cell transplantations. To identify the elements necessary for high-level expression, we have made deletion constructs of Ly-6E.1 gene flanking regions containing DNase I hypersensitive sites, tested them for expression in hematopoietic cells, and have performed kinetic analyses to correlate the appearance of hypersensitive sites with gene transcription and protein expression. We show that a 3' region containing two DNase I hypersensitive sites at +8.7 and +8.9 kb is required for high-level, gamma-interferon (gamma-IFN)-induced expression of the Ly-6E.1 gene and that a consensus sequence for a gamma-IFN-responsive element localizes to the +8.7 site. We also provide a description of allele- and cell-specific DNase I hypersensitive site patterns of the Ly-6E.1 and Ly-6A.2 genes. Taken together, these data indicate that while both 5' and 3' hypersensitive sites are rapidly induced with gamma-IFN, the 3' most distal hypersensitive sites are involved in directing high levels of expression of Sca-1 in hematopoietic cells. PMID- 8639892 TI - Effects of recombinant human megakaryocyte growth and development factor on platelet activation. AB - The c-Mpl receptor for thrombopoietin and its recombinant related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF on platelet aggregation in platelet-rich plasma (PRP) and in whole blood. The results obtained indicate that MGDF by itself did not affect platelet aggregation. However, when added before other agonists such as adenosine diphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of MGDF was dependent on the dose and on the time of platelet preincubation, and it occurred both in PRP and in whole blood platelet aggregation. MGDF also "primed" the release of adenosine triphosphates and the production of thromboxane B2 by platelets stimulated with ADP, EPI, and THR. When added 15 seconds after the preincubation of platelets with subthreshold concentrations of ADP, EPI, and THR, MGDF exhibited a synergism with these agonists. Moreover, we observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential. PMID- 8639893 TI - Type I factor XIII deficiency is caused by a genetic defect of its b subunit: insertion of triplet AAC in exon III leads to premature termination in the second Sushi domain. AB - Factor XIII deficiency has been classified into two categories: type I deficiency, characterized by the lack of both the a and b subunits; and type II deficiency, characterized by the lack of the a subunit alone. To clarify the genetic bases of these diseases, previously reported cases of the type I deficiency were examined at the DNA level. DNA sequence analysis showed that a nucleotide triplet (AAC) was inserted within the codon for Tyr-80 in exon III of the gene for a female proband's b subunit, resulting in the creation of a stop codon. Restriction digestion of amplified DNAs confirmed that the proband and her sister were homozygotes, and their family members were heterozygotes of this mutant allele. A truncated protein composed of 79 amino acids could be synthesized by these homozygotes; however, such a protein would not be secreted or it would degrade quickly, because there were normal amounts of the mutant mRNA, but no b subunit was detected in these patients. The a subunit deficiency of these patients must be a secondary to the b subunit deficiency, as their gene for the a subunit was intact, and the a subunit in their platelets was present within normal levels. PMID- 8639894 TI - Evidence for a novel binding protein to urokinase-type plasminogen activator in platelet membranes. AB - Endogenous urokinase-type plasminogen activator (u-PA) has been identified in platelet membrane, and platelets have been shown to take up exogenous high molecular weight u-PA from the ambient medium. In this report, the mechanism of the association of u-PA with platelets was investigated using recombinant, single chain u-PA. When gel filtered human platelets were incubated with radiolabeled u PA, the u-PA was found to specifically and saturably bind to the resting platelets in a dose-dependent manner. Unlabeled u-PA and the amino terminal fragment of u-PA inhibited 125I-u-PA binding to platelets with a mean IC50 of 65 and 58 nmol/L, respectively. A single saturable binding site in intact resting platelets was found with a mean kd of 43 +/- 25 nmol/L and 2263 +/- 809 sites per platelet. In contrast to resting platelets, 125I-u-PA did not bind to thrombin induced platelets. Western blotting studies, using a monoclonal or a polyclonal antibody specific for the u-PA cell-surface receptor (u- PAR), failed to show evidence of u-PAR in resting platelets, whereas, u-PAR was found at approximately 54 and approximately 48 kD on U937 monocytes, which served as a positive control. Ligand blotting of platelet membrane and of U937 cell proteins with 125I-u-PA revealed a u-PA binding protein of approximately 70 kD in the platelets and one of approximately 54 kD in the U937 cells. Complexion of u-PA with a platelet membrane protein was also shown by gel filtration of a mixture of u-PA and platelet membrane proteins. A u-PA complex was further shown by enzyme-linked immunosorbent assay when microtiter plates were coated with platelet membrane proteins, and this complex formation was shown to be dose-dependent and saturable with an apparent kd of 17 nmol/L. It was concluded that platelet membrane contains a specific, high affinity u-PA-binding protein that is distinct from u PAR. PMID- 8639895 TI - Platelet glycoprotein (Gp) IX associates with GpIb alpha in the platelet membrane GpIb complex. AB - The platelet membrane glycoprotein (Gp) Ib complex consists of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta subunits; GpIX, tightly, but noncovalently associated with GpIb alpha-beta; and the more weakly associated GpV. It is not certain whether the association of GpIX to Gplb alpha-beta is via GpIB alpha, GpIb beta, or both subunits, although recently published evidence implicates an interaction with GpIb beta. We have investigated the interaction of GpIX with GpIb alpha-beta using polyclonal rabbit antibodies to GpIb alpha and GpIb beta raised by immunization with purified glycocalicin and with synthetic peptide sequences from GpIb beta, respectively, as well as monoclonal antibodies directed against GpIX (FMC-25) and against GpIb alpha (AP-1). We performed two types of experiments, using either purified GpIb complex or platelets. (1) When wells were coated with nonreduced GpIb complex, the binding of FMC-25 was inhibited 73% by GpIb alpha antibody, but only 30% by the GpIb beta antibody; when wells were coated with reduced complex, FMC-25 binding was inhibited by the same two antibodies by 86% and 13%, respectively. When wells were coated with polyclonal GpIb alpha or GpIb beta antibodies and then incubated with reduced GpIb complex, only wells coated with GpIb alpha antibodies captured GpIX reactivity. When wells were coated with FMC-25 and then incubated with nonreduced GpIb complex, both the GpIb alpha and GpIb beta polyclonal antibodies reacted strongly; in contrast, only GpIb alpha reactivity was retained when wells coated with FMC-25 were incubated with reduced GpIb complex. In the reciprocal experiment, AP-1-coated wells incubated with either nonreduced or reduced GpIb complex bound radiolabeled FMC-25. (2) The ability of polyclonal GpIb alpha and GpIb beta antibodies to inhibit binding of FMC-25 to platelets was studied by ELISA and by flow cytometry. In both systems, FMC-25 binding was inhibited by the GpIb alpha antibody, but not significantly by the GpIb beta antibody. We conclude that GpIX is strongly associated with GpIb alpha in the purified platelet GpIb complex and in the platelet membrane. PMID- 8639896 TI - Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics. AB - Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90-2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation. PMID- 8639897 TI - Evidence for oligoclonal diversification of the VH6-containing immunoglobulin repertoire during reconstitution after bone marrow transplantation. AB - Patients who have undergone bone marrow transplantation (BMT) remain immunodeficient for months to years posttransplantation. To evaluate the basic molecular events underlying reconstitution of the humoral immune response, we have performed a detailed nucleotide sequence analysis of VH6 containing rearrangements in circulating B cells from two BM donor/recipient pairs. Our results show that the third complementarity determining region (CDR3) diversity is much lower early after transplantation, compared with that of the donors, and that the clonal variability remains low for 3 months. Repertoire diversification follows an oligoclonal pattern where B lymphopoiesis appears to occur in waves up to 6 months posttransplantation. The repertoire among donated marrow cells is not reflected in peripheral blood lymphocytes from the transplanted patients. There is differential D gene utilization among both donor and patient samples, whereas JH gene usage is biased toward JH4, 5, and 6. One month after transplantation the vast majority of the sequenced clones are functional and contain a high frequency of replacement mutations in the CDRs of the VH6 gene. We conclude that Ig gene expression is very restricted early after BMT and that development of the B-cell repertoire appears to follow a wavelike pattern. PMID- 8639898 TI - Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14. AB - Human vascular endothelial cells (HUVECs), which do not display the lipopolysaccharide (LPS) receptor CD14, were examined for protein tyrosine phosphorylation after LPS stimulation in the presence and absence of soluble CD14 (sCD14). By phosphotyrosine Western blotting and immunocomplex kinase assays we show that LPS was capable of inducing in these cells rapid protein tyrosine phosphorylation and kinase activation of two members of the mitogen-activated protein kinase (MAPK) family erk-1 and the newly discovered p38, requiring the presence of sCD14. LPS-induced tyrosine phosphorylation of MAPK was associated with increased transcript- and surface protein expression of intracellular adhesion molecule-1 by HUVECs. MAPK phosphorylation and activation was induced by LPS in concentrations as little as 30 ng/mL and as early as 15 minutes after stimulation. Furthermore, tyrosine kinase inhibitors such as Genistein partially inhibited this effect. These results show that LPS triggers similar signaling events in both CD14+ myelo-monocytic cells and cells lacking the putative LPS receptor CD14, suggesting the presence of a common, yet unidentified element in LPS-signaling in both cell types. PMID- 8639899 TI - Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor. AB - Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti-CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF--cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo. PMID- 8639900 TI - Fas-mediated apoptosis in cultured human eosinophils. AB - Previous studies have shown that cytokine-dependent eosinophils undergo apoptosis, yet the mechanisms governing this phenomenon remain obscure. Fas antigen is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family. Cross-linking of Fas antigen in numerous cell types leads to apoptosis. In the present study, we examined the potential role of Fas antigen in the apoptosis of purified blood eosinophils from healthy donors. Cytokine deprived eosinophils exhibited a time-dependent loss in viability, accompanied by an increase in the number of apoptotic nuclei and in the expression of Fas antigen and its mRNA, as shown by flow cytometry and reverse transcriptase polymerase chain reaction, respectively. Cross-linking of Fas antigen with an agonistic anti-Fas monoclonal antibody (MoAb) induced a dose- and time-dependent increase in the number of apoptotic nuclei. Furthermore, using an in vitro coculture system, we showed engulfment of anti-Fas MoAb-treated eosinophils by monocyte-derived macrophages. Finally, incubation of eosinophils with the corticosteroid, dexamethasone, induced apoptosis and augmented that triggered by anti-Fas MoAb. Together, these observations suggest that Fas antigen expression and activation is involved in the apoptosis of human eosinophils and may contribute to the resolution of inflammatory allergic reactions in which eosinophil accumulation is a prominent feature. PMID- 8639901 TI - Phosphatase 2A participates in interferon-gamma's induced upregulation of C1 inhibitor mRNA expression. AB - C1 inhibitor (C1 INH) is the major inhibitor of the proteolytically active subcomponents of C1, kallikrein, activated forms of factor XII, and factor XIa in plasma. We determined the mechanism(s) how interferon-gamma (IFN-gamma) regulates C1 INH mRNA expression in HepG2 cells. Cycloheximide or anisomycin treatment alone did not increase C1 INH mRNA nor did it potentiate C1 INH mRNA expression after IFN-gamma stimulation. C1 INH mRNA levels on Northern blot from untreated and IFN-gamma-treated cells did not change for more than 20 hours after actinomycin D treatment. Actinomycin D and 5,6-dichloro-1-beta ribofuranosylbenzimidazole abolished IFN-gamma-induced C1 INH mRNA expression. Relatively more C1 INH mRNA precursor (heterogeneous nuclear RNA [hnRNA]) was detected in total RNA from IFN-gamma-treated HepG2 cells than unstimulated cells. Treatment of HepG2 cells with the phosphatase 1 and 2A inhibitors, okadaic acid (> or = 50 nmol/L) and calyculin (> or = 25 nmol/L), decreased IFN-gamma's ability to upregulated C1 INH mRNA. The phosphatase 2A inhibitor, cantharidin (> or = 10 micromol/L), also blocked the IFN-gamma induction of the C1 INH gene. In HepG2 cells total phosphatase 2A activity was significantly increased by C6 ceramide but not IFN-gamma. However, C6 ceramide itself did not increase C1 INH mRNA expression. These data indicate that phosphatase 2A is required to dephosphorylate a substrate in order for IFN-gamma to induce the transcriptional upregulation of C1 INH mRNA, but phosphatase 2A is not a direct stimulator of C1 INH gene expression. PMID- 8639902 TI - ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival. AB - ILA, a gene induced by lymphocyte activation, is a member of the human nerve growth factor tumor necrosis factor receptor family and the human homologue of murine 4-1BB. The present study analyzed the role of ILA in the regulation of human peripheral blood T-lymphocyte function. Antibodies raised against different fusion proteins recognized ILA on activated lymphocytes. These antibodies significantly increased anti-CD3--induced T-lymphocyte proliferation. When anti CD3--stimulated cells were incubated on ILA-expressing CHO cells, proliferation was inhibited. CHO cells transfected with a control construct and not expressing ILA did not reduce T-cell proliferation. A purified fusion protein containing the extracellular domain of ILA and the constant domain of human IgG (ILA-IgG) also inhibited lymphocyte proliferation. Results obtained by 3H-thymidine incorporation were confirmed by cell cycle analysis that showed a decrease in the number of lymphocytes in S phase. Lymphocyte morphology in cultures with ILA expressing CHO cells was suggestive of apoptosis. Flow cytometry on propidium iodide-stained cells showed a time-dependent increase in the number of hypodiploid apoptotic cells when lymphocytes were cultured on ILA-expressing CHO cells. Internucleosomal DNA cleavage was seen in these cultures, but not in the presence of ILA-negative CHO cells. Studies on the mechanism by which ILA regulates T-cell function showed that ILA-IgG inhibited anti-CD3-induced T-cell proliferation when presented in immobilized but not in soluble form. These results suggest that ILA may act by cross-linking its ligand and thereby inhibit T-cell proliferation. PMID- 8639903 TI - VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4-34) gene. AB - The immunoglobin heavy chain variable region (VH) gene usage in multiple myeloma (MM) has not been reported, although a few studies have incidentally identified the VH gene rearranged in small cohorts of MM patients. We used a reverse transcriptase-polymerase chain reaction based technique to analyze the VH gene usage in MM. The VH sequences were obtained after amplification of bone marrow cDNA using the seven VH family-specific and constant region primers. The VH sequences of 72 patients were successfully identified. The frequency of VH family usage in decreasing order was VH3>VH4>VH1>VH5>VH2>VH6>VH7 and corresponded to the functional germline complexity of the VH families. Individual VH genes (VH1-69, VH3-9, VH3-23, and VH3-30) were overrepresented in our cohort of MM patients; some VH genes [VH3-49, VH3-53, and VH4.21 (VH4-34)], which are rearranged with increased frequency in normal circulating B cells, autoimmune diseases, and other B-cell malignancies, were not detected in any MM patient. Compared with germline sequences, an average of 8.8% (range, 2.7% to 16.5%) of the nucleotides had evidence of mutation within each VH sequence. Based on these results, we conclude that (1) the VH gene usage in MM is unique compared with other malignant and nonmalignant B-cell populations, (2) the physiologic process of clonal deletion functions to remove clones that have rearranged VH genes (VH4.21) capable of expressing antibodies, which recognize self-antigens, and (3) the complete lack of VH4.21 gene rearrangement may help to partially explain the paucity of autoimmune phenomena in MM. PMID- 8639904 TI - Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease. AB - Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35 fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha. PMID- 8639905 TI - Expression of granulocyte colony-stimulating factor and granulocyte colony stimulating factor receptor genes in partially overlapping monoclonal B-cell populations from chronic lymphocytic leukemia patients. AB - B lymphocytes were purified from the peripheral blood of 30 B-cell chronic lymphocytic leukemia (B-CLL) patients and tested for the ability to produce granulocyte colony-stimulating factor (G-CSF) in vitro. Fifteen Staphylococcus aureus Cowan I (SAC)-stimulated, but not unstimulated, B-cell suspensions produced G-CSF in short-term cultures. Accordingly, G-CSF mRNA was detected only in SAC-stimulated B cells. Five CLL B-cell fractions that released G-CSF following exposure to SAC were also incubated with CD40 or anti-mu antibodies in the presence or absence of recombinant (r) interleukin-2 (IL-2) or IL-4. The 5 cell suspensions produced G-CSF only on culture with CD40 monoclonal antibody in combination with rIL-2 or rIL-4. CD5+ B lymphocytes, which represent the normal counterparts of most B-CLL proliferations, did not produce G-CSF under any of the above culture conditions. G-CSF produced by leukemic B lymphocytes was biologically active, because conditioned media of SAC-stimulated cells supported the in vitro growth of myeloid colonies from normal bone marrow progenitors. The colony stimulating activity of CLL B-cell supernatants was ascribed to both G-CSF and granulocyte-macrophage colony stimulating factor. G-CSF receptors (G-CSFRs) were detected on freshly isolated B lymphocytes from 7 of 11 B-CLL patients; 5 of these cell suspensions produced G-CSF in culture, whereas 2 did not. rG-CSF rescued 3 of the 7 G-CSFR+ cell fractions from spontaneous apoptosis but had no effect on their in vitro proliferation. PMID- 8639907 TI - Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis. AB - We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis. PMID- 8639906 TI - Rearrangement of the MLL gene confers a poor prognosis in childhood acute lymphoblastic leukemia, regardless of presenting age. AB - MLL gene rearrangements are associated with an extremely poor prognosis in infants with acute lymphoblastic leukemia (ALL), but little is known about their clinical significance in older children. Therefore, we studied 45 cases of childhood ALL with abnormalities of chromosome 11q23 for rearrangement of the MLL gene to determine if this feature confers a uniformly poor prognosis. MLL gene rearrangements were detected in all 18 cases with the common t(4;11), t(9;11) or t(11;19) translocations, whereas only 5 of 12 patients with either unbalanced or uncommon balanced translocations demonstrated a rearrangement. Abnormalities of the MLL gene were not detected in any of the 15 cases with a deletion or inversion of the chromosomes 11q23 region. The presence of an MLL rearrangement was significantly associated with age less than 1 year (P < .001), leukocyte count >50 x 10(9)/L (P = .003), and the absence of leukemic cell CD10 expression (P < .001). In a stratified statistical analysis adjusted for age and treatment protocol, MLL gene rearrangement was correlated with an inferior treatment outcome (P = .028). The 4-year event-free survival estimate (+/- SE) was 10% +/- 6.5% for cases with a rearranged MLL gene and 64% +/- 19.2% for other cases. When infants were excluded from the analysis, MLL rearrangement was still significantly associated with a poor outcome (P = .02), and remained so with the exclusion of t(4;11)-positive cases (P = .05). Thus, regardless of presenting age, MLL gene rearrangement identifies a high-risk subgroup of patients who are not likely to be cured with conventional treatment. PMID- 8639908 TI - Activation of erythroid-specific promoters during anthracycline-induced differentiation of K562 cells. AB - Anthracycline antitumor drugs such as aclacinomycin (ACM) and doxorubicin (DOX) used in subtoxic concentrations induce erythroid differentiation of the erythroleukemic cell line K562. To elucidate the possible role of erythroid genes of the erythropoietin receptor (EpoR) and the transcription factor GATA-1 in this effect, the regulatory regions of the above genes and human epsilon- and gamma globin and porphobilinogen deaminase (PBGD) genes were fused to the firefly luciferase gene. The resulting reporter constructs were tested in a transfection assay of the erythroleukemic cell line K562 stimulated to differentiate by treatment with the anthracycline drugs ACM and DOX or hemin (HEM). The results showed activation of the tested promoters after cell treatment with ACM, but not with DOX or HEM. In contrast to the mouse EpoR gene promoter, the activity of the human EpoR gene promoter (-659/-60) in the reporter construct was not modified by addition of the first intron sequence. In ACM-treated K562 cells, EpoR gene promoter activity completely correlated with EpoR and GATA-1 mRNA levels and the degree of erythroid maturation. In addition, ACM strongly activated the erythroid gene promoters that contain GATA binding sites. Nevertheless, less activation was also observed for the GATA-1 gene promoter (-312/-31) lacking any known GATA binding sites. Insertion of the GATA-1 gene enhancer with two canonic GATA binding sites, stimulated the ACM activation effect for EpoR and GATA-1 promoter containing constructs. Mutation of the enhancer GATA binding sites abolished this effect. All the regulatory regions tested (except gamma-globin promoter) were completely inactive in nonerythroid COS7 cells. These data indicate that (1) two structurally different anthracycline analogues, DOX and ACM, differ in their differentiation mechanisms, and (2) ACM switches on the erythroid program of K562 cells, at least in part because of interaction with a factor(s) that recognizes the GATA binding sites in the promoter region of erythroid genes leading to their activation. PMID- 8639909 TI - The 12;21 translocation involving TEL and deletion of the other TEL allele: two frequently associated alterations found in childhood acute lymphoblastic leukemia. AB - A recurrent t(12;21)(p13;q22) has recently been described in human acute lymphoblastic leukemias (ALLs). This translocation fuses TEL and AML1, two genes previously cloned from translocation breakpoints in myeloid leukemias. In addition, allelic loss of the TEL gene can be detected in 15% to 22% of childhood ALLs. In the present study, we have sought allelic deletions of TEL and the presence of the t(12;21) in 50 children with B-lineage ALL, using a combination of microsatellite typing, fluorescent in situ hybridization (FISH), and analysis of the fusion transcripts resulting from the TEL-AML1 gene fusion. Our results indicate that the association between the t(12;21) and the deletion of the nontranslocated allele of TEL is among the most frequent abnormalities observed in B-lineage ALLs. FISH analysis using several cosmid probes showed that, in one patient with a t(12;21) translocation involving TEL, the second allele had an intragenic deletion. This observation points to TEL as the actual target of 12p12 13 deletions in patients that associate a t(12;21) with a deletion. The TEL-AML1 fusion RNA was found in all patients with the t(12;21) whereas the reciprocal AML1-TEL transcript was only found in a subset of patients, suggesting that only the protein product encoded by TEL-AML1 is likely to play a role in leukemogenesis. The observation that, in two patients with the t(12;21), a deletion of TEL was only present in a subclone indicates that this deletion was a secondary event that occurred after the translocation. The frequent occurrence of TEL deletions in patients with t(12;21) suggests that the deletion of the normal TEL allele subsequent to the t(12;21) provides a further proliferative advantage to leukemic cells. PMID- 8639910 TI - Relation of autonomous and interleukin-2-responsive growth of leukemic cells to survival in adult T-cell leukemia. AB - We examined autonomous and interleukin-2 (IL-2)-responsive growth activities of leukemic cells derived from peripheral blood, as well as several clinical manifestations, including serum lactate dehydrogenase (LDH) level, of 35 patients with adult T-cell leukemia (ATL) to determine whether these properties were related to prognosis. Growth activities were measured by [3H]-thymidine incorporation of the cells after 24 hours' culture with or without exogenous IL 2. Both autonomous and IL-2-responsive growth activities were higher in the patients than in healthy controls and were significantly correlated with each other (P < .0001, r = .956). Both higher growth activities were significantly associated with shorter survival times (P = .0042, r = .472 and P = .0117, r = .421, respectively). An increased serum LDH value was also significantly associated with shorter survival times (P = .0011, r = .530), but corrected calcium level, sex, white blood cell count, or age were not. These results strongly suggest that both growth activities of primary tumor cells, in addition to the serum LDH value, are prognostic determinants in ATL. We propose a new prognostic classification combining LDH values and autonomous growth activity into three groups: (1) high growth activity and high LDH; (2) high growth activity and low LDH, or low growth activity and high LDH; and (3) low growth activity and low LDH, which showed a significant relationship to survival time (P = .0014; the median survival time for each group was 39, 94, and 340 days, respectively). PMID- 8639911 TI - CD30 (Ki-1)-positive malignant lymphomas: clinical, immunophenotypic, histologic, and genetic characteristics and differences with Hodgkin's disease. AB - This study compares the histologic and immunophenotypic features of 71 cases of primary CD30+ diffuse large-cell lymphomas (DLCL) and 128 cases of Hodgkin's disease (HD) and discusses the clinical features of 52 patients with CD30+ DLCL. It includes analysis of sites of involvement, staging, response to treatment, sites and treatment of recurrences, and disease-free and overall survival. Diagnostic immunophenotypic differences were found between CD30+ DLCL and HD. All cases of CD30+ DLCL were positive for one or more common or lineage-specific lymphocyte antigens or for EMA. In contrast, 96.9% of HD cases were negative for CD45, CD45-RO, CD43, and CD20. The four exceptions are discussed. All cases of HD were negative for EMA. In patients with CD30+ DLCL, a T-cell phenotype was found in 60%, a null-cell type in 22%, and a B-cell type in 18% of the cases. The median age of patients with T- and null-cell phenotype was 22 years (range, 4 to 72). Fifty-two percent of them had high-stage (III and IV) disease and 61% had extranodal involvement at presentation, including 25% with skin lesions. Lymph nodes draining the skin lesions became involved in seven of 11 patients. No patient had initial bone marrow involvement. Most patients were treated with chemotherapy, and 83% had a complete remission. Fifty-four percent remain free of disease with a median follow-up of 47 months. Thirteen patients (29%) had one or more recurrences and five of them remain free of disease after salvage therapy, with a median follow-up period of 79 months. The clinical stage did not affect survival, probably as a result of different therapy. The t(2;5) translocation was found in five of 15 patients who had cytogenetic abnormalities. Of the other 10 cases, the translocation was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in four of five cases studied. All nine cases were of T- or null-cell phenotype. The cases of B-cell CD30+ DLCL had a characteristic immunophenotype. All were negative for EMA. These patients were older and had frequent bone marrow involvement but no skin infiltration by lymphoma. All three patients who were human immunodeficiency virus-positive (HIV+) had lymphomas of B cell lineage. Detection of the t(2;5) translocation by molecular genetics is a useful and highly specific marker in the differential diagnosis between HD and CD30+ DLCL. PMID- 8639912 TI - Frequent expression of interleukin-10 by Epstein-Barr virus-harboring tumor cells of Hodgkin's disease. AB - Tumor cells of Epstein-Barr virus (EBV)-associated Hodgkin's disease (HD) express the viral protein, latent infection membrane protein-1 (LMP1), but evade cytotoxic responses normally directed at this antigen. We tested whether local production of the immunoregulatory interleukins (IL)-4 and -10 may have a role in this process. IL-4 RNA was not detectable in any of the HD cases. By contrast, isotopic in situ hybridization and correlation with the presence of EBV gene products showed significantly higher proportions of cases with IL-10 expressing tumor cells in LMP1-positive (17 of 26, 66%) as compared with LMP1-negative HD cases (six of 37, 16%). Absence of EBV BCRF1 RNA indicated that the transcripts originated from the cellular IL-10 gene. Similarly, an association between IL-10 expression and EBV-infection of tumor cells was found in AIDS-related malignant non-Hodgkin lymphomas (ARL). Very small proportions of EBV-infected cells, mainly blasts, expressed IL-10 in infectious mononucleosis tonsils. Thus, although not entirely exclusive to EBV-positive cases, IL-10 expression is frequently associated with EBV-infection in HD and ARL and appears to be upregulated by EBV, most likely through LMP1. In view of the established inhibitory effects of IL-10 on cell mediated immunity, it is suggested that IL-10 expression may contribute to evasion of LMP1-positive cells from cytotoxicity directed at viral antigens. PMID- 8639913 TI - Cure of disseminated xenografted human Hodgkin's tumors by bispecific monoclonal antibodies and human T cells: the role of human T-cell subsets in a preclinical model. AB - Cure of a single established human Hodgkin's tumor growing subcutaneously in severe combined immunodeficient (SCID) mice can be achieved with a complex protocol using two bispecific monoclonal antibodies (Bi-MoAb) directed against the Hodgkin's associated CD30 antigen and the T-cell triggering molecules CD3 and CD28, respectively, together with human T cells prestimulated in vitro with Bi MoAbs in the presence of CD30+ cells. To adapt this model to the clinical situation, disseminated tumors were established in SCID mice by intravenous injection of 2 x 10(7) cells of the Hodgkin's derived cell line L540CY. Treatment of SCID mice bearing disseminated CD30+ Hodgkin's tumors with the combination of CD3/CD30 and CD28/CD30 Bi-MoAbs and naive (ie, not in vitro prestimulated) human T cells resulted in the cure of all appropriately treated animals. T lymphocytes obtained from patients with advanced stage untreated Hodgkin's disease were as effective as lymphocytes from healthy controls. Treatment was effective even when delayed until 2 weeks after tumor inoculation, and application of Bi-MoAbs into SCID mice with circulating human T cells was as effective as injecting the Bi MoAbs before the lymphocytes. Treatment results with isolated CD4+ and CD8+ human T cells suggest that both subsets are necessary for the Bi-MoAb mediated cure of xenografted human tumors in vivo. The efficacy and practicability of this preclinical immunotherapy protocol support and form the basis for the clinical evaluation of this approach in patients with Hodgkin's disease resistant to standard therapy. PMID- 8639914 TI - Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML. AB - Recent studies have shown that tumor cells genetically modified by transduction of B7-1, a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, are rejected in syngeneic hosts. In these reports, transformed cell lines and drug-selected cells have been used for vaccinations. To determine the effectiveness of B7-1-transduced primary acute myelogenous leukemia (AML) cells on the induction of antitumor immunity, we have studied a murine AML model in which primary AML cells were retrovirally transduced with the murine B7-1 cDNA. A defective retroviral producer clone expressing B7-1 and secreting a high titer of virus was used for infection of AML cells. Unselected transduced AML cells, expressing a high level of B7-1, were used for in vivo vaccinations. Our results show that one intravenous (IV) injection of irradiated B7-1-positive (B7-1+) AML cells can provide long-lasting (5 to 6 months) systemic immunity against subsequent challenge with wild-type AML cells. Furthermore, one exposure to irradiated B7-1+ AML cells results in rejection of leukemia by leukemic mice when the vaccination occurs in the early stages of the disease. The antileukemia immunity is CD8+ T-cell-dependent and B7/CD28-mediated, since in vivo treatment of mice with anti-CD8 monoclonal antibody or CTLA-4 Ig leads to abrogation of the specific antileukemia immune response. These results emphasize that B7-1 vaccines may have therapeutic usefulness for patients with AML. PMID- 8639915 TI - 1 alpha,25-dihydroxyvitamin D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation. AB - Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL 60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3-induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3-induced inhibition of HL-60 cell proliferation. PMID- 8639916 TI - Generation of phenotypically aged phosphatidylserine-expressing erythrocytes by dilauroylphosphatidylcholine-induced vesiculation. AB - In vitro stored red blood cells (RBC) and RBC artificially induced to vesiculate by incubation with dilauroylphosphatidyl-choline were monitored for age- and vesiculation-dependent changes in cell density, membrane lipid asymmetry, and their ability to be recognized and cleared by reticuloendothelial cells. RBC demonstrated a progressive increase in density on self-forming Percoll gradients upon vesiculation and in vitro "aging." Uptake of vesiculated RBC by in vitro cultivated macrophages was increased threefold over non-vesiculated control RBC. The clearance rate of dense vesiculated RBC was biphasic and contained a rapid component and a slower second component consistent with the clearance rates of normal control populations. Determination of phosphatidylserine (PS) in the outer leaflet of RBC by the PS-dependent prothrombinase assay revealed that PS redistributed to the cell's outer leaflet upon in vitro storage and vesiculation. Inhibition of PS movement by oxidation of membrane sulfhydryls with pyridyldithioethylamine resulted in higher prothrombinase levels and enhanced clearance of vesiculated RBC. These experiments suggest that vesiculation contributes to alterations in membrane lipid asymmetry and cell density characteristic of the aged RBC phenotype. PMID- 8639918 TI - Expression of RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of Rh blood group antigens. AB - Retroviral-mediated gene transfer using cDNA transcripts of the RHD and RHCE genes resulted in the isolation of K562 clones expressing D and G or c and E antigens, respectively. These results represent the first direct demonstration that the RHD gene encodes the D and G antigens and the RHCE gene encodes the c and E antigens. Both c and E antigens were expressed after transduction of K562 cells with a single cDNA, indicating that the c antigen does not arise by alternative splicing (exon skipping) of the product of the RHCE gene, as has been suggested. PMID- 8639917 TI - Characterization of the gene encoding the human LW blood group protein in LW+ and LW- phenotypes. AB - The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig-like domain are encoded by the first exon. The 5'UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5'UT region, has been detected by sequencing polymerase chain reaction-amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes. PMID- 8639919 TI - Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme. AB - Human glucose 6-phosphate dehydrogenase (G6PD) has a particularly large number of variants resulting from point mutations; some 60 mutations have been sequenced to date. Many variants, some polymorphic, are associated with enzyme deficiency. Certain variants have severe clinical manifestations; for such variants, the mutant enzyme almost always displays a reduced thermal stability. A homology model of human G6PD has been built, based on the three-dimensional structure of the enzyme from Leuconostoc mesenteroides. The model has suggested structural reasons for the diminished enzyme stability and hence for deficiency. It has shown that a cluster of mutations in exon 10, resulting in severe clinical symptoms, occurs at or near the dimer interface of the enzyme, that the eight residue deletion in the variant Nara is at a surface loop, and that the two mutations in the A- variant are close together in the three-dimensional structure. PMID- 8639920 TI - Differential regulation of IRP1 and IRP2 by nitric oxide in rat hepatoma cells. AB - Iron-regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to stem-loop structures known as iron-responsive elements (IREs). IREs are located in the 5'- or 3'-untranslated regions (UTRs) of specific mRNAs that encode proteins involved in iron homeostasis. The binding of IRPs to 5' IREs represses translation of the mRNA, whereas the binding of IRPs to 3' IREs stabilizes the mRNA. IRP1 and IRP2 binding activities are regulated by intracellular iron levels. In addition, nitric oxide (NO.) increases the affinity of IRP1 for IREs. The role of NO. in the regulation of IRP1 and IRP2 in rat hepatoma cells was investigated by using the NO.-generating compound S-nitroso-N-acetylpenicillamine (SNAP), or by stimulating cells with multiple cytokines and lipopolysaccharide (LPS) to induce NO. production. Mitochondrial and IRP1 aconitase activities were decreased in cells producing NO(.). NO. increased IRE binding activity of IRP1, but had no effect on IRE binding activity of IRP2. The increase in IRE binding activity of IRP1 was coincident with the translational repression of ferritin synthesis. Transferrin receptor (TfR) mRNA levels were increased in cells treated with NO.-generating compounds, but not in cytokine- and LPS-treated cells. Our data indicate that IRP1 and IRP2 are differentially regulated by NO. in rat hepatoma cells, suggesting a role for IRP1 in the regulation of iron homeostasis in vivo during hepatic inflammation. PMID- 8639921 TI - A novel mutation found in the 3' domain of NADH-cytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia. AB - Congenital methemoglobinemia caused by an erythrocytic deficiency of cytochrome b5 reductase (b5R; type I) in African-American individuals was first reported by this laboratory. The rarity of this observation is possibly due to the difficulty detecting cyanosis that is masked by naturally occurring dark skin pigment. Since previous biochemical studies on the African-American family with variant enzyme b5R-Shreveport showed enzyme instability, we focused on molecular analysis of its transcript. The transcript size was the same as that of a normal control. The nucleotide sequence of both normal and variant transcripts were examined by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) product. The propositus was found to be homozygous for a G to A transition at codon 212 in exon 8, changing a glutamate to a lysine (E212K). In addition, a C to G transversion was found at codon 116 in exon 5, changing a threonine to a serine (T116S). Using allele-specific PCR, we determined that E212K was found only in the propositus and her heterozygous mother. Furthermore, E212K is predicted to disrupt an alpha-helix peptide structure of b5R, suggesting that this is likely the disease-causing mutation. In contrast, T116S was found to be a high-frequency polymorphism specific for the African-American population. The E212K mutation is uniquely present in the 3' end of the b5R gene (exon 8), which differs from those b5R mutations found among Japanese subjects (exons 3 and 5) and in an Italian subject (exon 4) and, thus, further contributes to our understanding of the structure/function relationship of this housekeeping enzyme. PMID- 8639922 TI - Pure red cell aplasia: association with large granular lymphocyte leukemia and the prognostic value of cytogenetic abnormalities. AB - From 1980 through 1994, we identified 47 adult patients with acquired pure red cell aplasia (median age, 64 years; range, 22 to 84 years). Associated clinical disorders included T-cell large granular lymphocytic (LGL) leukemia, thymoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Review of bone marrow findings in 40 patients showed absence of erythroid precursors in 14 patients and rare pronormoblasts in 26. None had morphologic evidence of myelodysplasia. T cell receptor gene rearrangement studies with Southern blot technique in 14 patients showed clonal rearrangements in nine. Karyotypic analyses performed in 28 patients showed clonal abnormalities in four. Overall, 28 of 47 patients (60%) responded to immunosuppressive therapy, but none were the patients with cytogenetic abnormalities. There was a trend toward superior response to immunosuppressive agents in the patients with T-cell LGL leukemia. Cyclophosphamide, with or without corticosteroids, was the most useful treatment agent. Cyclosporine A was effective for refractory disease. Neither the presence of an associated clinical disorder nor the existence of detectable erythroid precursors affected overall survival. We conclude that (1) T-cell LGL leukemia is the disorder most commonly associated with pure red cell aplasia, (2) the presence of clonal cytogenetic abnormality predicts poor response to immunosuppressive therapy, and (3) oral cyclophosphamide and cyclosporine A are effective treatment regimens. PMID- 8639923 TI - Investigation of the relative infectivity and pathogenicity of different hepatitis C virus genotypes in hemophiliacs. AB - To assess the relative infectivity and pathogenicity of variants of hepatitis C virus (HCV) genotypes, the distribution of genotypes in hemophilic patients who had been treated with nonvirally inactivated factor concentrates or cryoprecipitates prepared from local blood donors was compared with those found in the respective blood donor populations. Genotype frequencies differed markedly in the four countries investigated (Scotland, Hungary, South Africa, and Thailand) but in each, the HCV genotype distributions in hemophiliacs and blood donors were similar. In addition, HCV genotypes in recipients of commercially manufactured concentrates were similar to those found in the US general population. These findings provide no evidence that HCV genotypes differ significantly from each other in replication rate, transmissibility, or infectivity. PMID- 8639924 TI - Donor immunization with Haemophilus influenzae type b (HIB)-conjugate vaccine in allogeneic bone marrow transplantation. AB - Bone marrow transplant patients are at increased risk for infections with polysaccharide encapsulated organisms and respond poorly to polysaccharide vaccines. We evaluated the effect of donor immunization with Haemophilus influenzae type b (HIB) polysaccharide-conjugate vaccine on recipient antibody responses following allogeneic bone marrow transplantation. Thirty-two allogeneic transplant patients and their donors were immunized before transplantation with HIB-conjugate, tetanus toxoid and 23-valent pneumococcal vaccines. Following transplantation, patients received HIB-conjugate and tetanus toxoid vaccines at 3, 6, 12, and 24 months and 23-valent pneumococcal vaccine at 12 and 24 months. Thirty-three patients with unimmunized donors were immunized following transplantation in an identical manner. Patients whose donors were immunized had significantly higher total anti-HIB antibody concentrations at 3 months (P = .0001), 6 months (P = .0001), 12 months (P = .0001), and 24 months (P = .002) after transplant compared with patients whose donors were unimmunized. Higher antitetanus toxoid antibody concentrations were also noted in patients with immunized donors, whereas donor immunization with pneumococcal vaccine had no effect on antibody concentrations following transplantation. Donor immunization with HIB-conjugate vaccine resulted in higher antibody concentrations in patients as early as 3 months after allogeneic transplantation and may be an effective strategy to prevent HIB infections. PMID- 8639925 TI - Different T-cell receptor repertoires between lesions and peripheral blood in acute graft-versus-host disease after allogeneic bone marrow transplantation. AB - From the viewpoint of T-cell receptor (TCR) repertoire, we studied the role of T cells in acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) from an HLA-identical sibling. By means of inverse polymerase chain reaction method and DNA sequencing, we analyzed TCR-alpha and beta transcripts from GVHD lesions and peripheral blood (PB) in a patient with typical GVHD together with PB from donor. At the initial onset of GVHD, V alpha-7 and -19 subfamilies were oligoclonally expanded in the PB compared with those in the oral mucosal lesions. At the second onset, V alpha-2, and V beta-6 subfamilies were more frequently detected in the cutaneous lesion than in the PB. Some TCR transcripts were recurrently found either in the mucosal or cutaneous lesions (or in both) and not in the PB. Furthermore, some of recurrent TCR transcripts in the lesions shared V gene segments and common motifs of complementarity determining region-3. These findings suggested that T cells infiltrating the GVHD lesions recognized a limited kind of antigens presented by patient's tissues with GVHD, and that T-cell repertoire in the GVHD lesions was different from that in the PB. PMID- 8639926 TI - Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors. AB - Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL. PMID- 8639927 TI - Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions. AB - We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology. PMID- 8639929 TI - Active immunization of murine allogeneic bone marrow transplant donors with B cell tumor-derived idiotype: a strategy for enhancing the specific antitumor effect of marrow grafts. AB - Persistence of the underlying malignancy remains the major obstacle limiting the success of high-dose chemoradiotherapy with allogeneic bone marrow transplantation (BMT) for lymphomas and multiple myeloma. We used the C3H 38C13 murine B-cell lymphoma, which expresses and secretes clonally derived Ig, the idiotype of which can serve as a tumor-specific antigen, to test the principle of transfer of tumor idiotype-specific immunity with BM. BALB/c marrow donors were twice immunized with 38C13-derived Ig, or with an isotype-matched control Ig, conjugated to keyhole limpet hemocyanin. Lethally irradiated C3H recipients reconstituted with marrow from idiotype immune, but not nonspecifically immune, donors demonstrated protection against subsequent lethal tumor challenge. The immunoprotective effect of immune allogeneic marrow was abrogated by T-cell depletion of the marrow graft before infusion. Low levels of serum anti-idiotypic antibody remained unaltered in recipients of T-cell-depleted immune marrow, consistent with a primary role for T-cell immunity in the cellular mechanism of this phenomenon. A modest therapeutic effect of immune allogeneic marrow was observed against 10 day, 1 cm established subcutaneous tumors, but only in combination with a booster immunization of the recipient post-BMT. These results provide the rationale for a novel strategy for enhancing the specific antitumor effect of allogeneic marrow grafts. PMID- 8639928 TI - Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. AB - Patients successfully treated with a marrow transplant often have concerns about fertility and pregnancy. This study was performed to determine pregnancy outcome among patients who had received high-dose chemotherapy alone or with total-body irradiation (TBI) and marrow transplantation for aplastic anemia or hematologic malignancy. Records of 1,326 postpubertal and 196 prepubertal patients currently more than 12 years of age after marrow transplant in Seattle from August 1971 to January 1992 were reviewed to determine the patients with normal gonadal function and pregnancies. Among 708 postpubertal women, 110 recovered normal ovarian function and 32 became pregnant. In addition, nine formerly prepubertal girls with normal gonadal function became pregnant. Among 618 postpubertal men, 157 recovered testicular function and partners of 33 became pregnant. An additional two formerly prepubertal men had partners who became pregnant. Forty-one female patients and partners of 35 male patients had 146 pregnancies after transplant. All 76 patients responded to a questionnaire requesting pregnancy history, outcome, infant birth weight, and congenital anomalies information for all clinically recognized pregnancies. There were 115 live births among 146 (79%) pregnancies. Spontaneous abortion terminated four of 56 (7%) pregnancies for 28 female cyclophosphamide (CY) recipients and six of 16 (37%) pregnancies for 13 TBI recipients (P = .02). Partners of 28 male CY recipients had four of 62 (6.4%) pregnancies terminate with spontaneous abortion, but there were no spontaneous abortions among eight pregnancies of five TBI recipients' partners. Preterm delivery occurred for eight of 44 (18%) and five of eight (63%) live births for 24 CY and eight TBI female recipients (P = .01). This 25% incidence among all female patient pregnancies is higher than the expected incidence of 8% to 10% (P = .0001). The 13 preterm deliveries resulted in 10 low birth weight ([LBW] 1.8 to 2.24 kg) and three very low birth weight ([VLBW] < or = 1.36 kg) infants, for an overall incidence of 25%, which is higher than the expected incidence of 6.5% for the general population (P = .0001). Twelve of the 13 premature infants survive. Congenital anomalies were seen among two of 52 (3.8%) live-born infants of female and six of 63 (9.5%) live-born infants of male patients, which is not different from the 13% of single congenital anomalies reported for the general population. These data demonstrate that clinically recognized pregnancies among women who have received a marrow transplant incorporating TBI are likely to be accompanied by an increased risk of spontaneous abortion. Pregnancies among all women who received a marrow transplant are likely to be accompanied by preterm labor and delivery of LBW or VLBW babies who do not seem to be at an increased risk of congenital anomalies. However, determination of possible adverse effects of parental exposure to high-dose alkylating agents with or without TBI on children born posttransplant requires longer, additional follow-up. PMID- 8639930 TI - Desmopressin-induced thrombocytopenia in von Willebrand disease patients with the Arg611His mutation in the A1 domain of von Willebrand factor. PMID- 8639931 TI - Decreased stability of the O allele mRNA transcript of the ABO gene. PMID- 8639932 TI - High-dose melphalan and autologous bone marrow transplantation for systemic AL amyloidosis with cardiac involvement. PMID- 8639933 TI - Apoptosis in bone marrow of myelodysplastic syndrome patients. PMID- 8639934 TI - Thrombopoiesis and thrombopoietin: the significance of "non-Tpo" cytokines. PMID- 8639935 TI - Intron 22 of factor VIII gene--a hot spot for structural aberrations causing severe hemophilia A. PMID- 8639936 TI - In vivo induction of HLA-DR on human neutrophils in patients treated with interferon-gamma. PMID- 8639937 TI - The circadian rhythm of thermoregulation in Japanese quail: III. Effects of melatonin administration. AB - Recent studies indicate that the circadian pacemakers in the eyes of Japanese quail are coupled to the rest of the circadian system by both neural and hormonal outputs. The effects of exogenous melatonin administration on circadian body temperature and activity rhythms of quail were tested to determine whether melatonin could be the hormonal link involved. Continuous melatonin administration caused arrhythmicity or period changes in the body temperature and activity rhythms of pinealectomized and sham-pinealectomized birds held in constant darkness and also significantly decreased the amplitude of the body temperature rhythms of normal birds held on light:dark 12:12. Further, melatonin entrained the body temperature and activity rhythms of normal birds when administered daily via the drinking water. The results show that melatonin can affect the circadian system of quail and support the hypothesis that melatonin is importantly involved in linking pacemakers in the eyes to the rest of the circadian system. PMID- 8639938 TI - Two distinct retinal projections to the hamster suprachiasmatic nucleus. AB - Electrical stimulation of the intergeniculate leaflet (IGL) region in hamsters can induce expression of Fos-like immunoreactivity (lir) in a restricted portion of the dorsolateral suprachiasmatic nucleus (SCN). The authors investigated whether the mechanisms by which stimulation affects SCN cells involves orthodromic activation of IGL cells projecting to the SCN or antidromic activation of retinal ganglion cells that send bifurcating axonal projections to both the IGL and the SCN. Bilateral optic enucleation strongly reduced induction of Fos-lir in SCN cells in response to electrical stimulation of the IGL region, implicating antidromic activation of retinal ganglion cells as the mechanism. This result implies that a class of retinal ganglion cells that project to the IGL also project selectively to the dorsolateral SCN. Earlier pharmacological studies suggest that this anatomically distinct retinal projection to the SCN is also neurochemically different from that innervating the rest of the nucleus. PMID- 8639939 TI - Is a critical interval of the circadian pacemaker at dusk responsive to light and melatonin responsible for the timing of estrus in the Romney Marsh ewe? AB - Two experiments, using Romney Marsh ewes, tested for the existence and role of a critical interval of the circadian pacemaker located near dusk that may be integrally involved in the precise timing of the breeding season. Groups of Romney Marsh ewes (n = 6) were provided with exogenous melatonin by injection at dusk (Experiment 1) or by infusion at dawn or subjected to extended darkness at dawn (Experiment 2) from the winter to the summer solstice before being exposed to natural photoperiod at latitude 35 degrees S. Other than the experimental protocols, all animals were held in natural photoperiod. The onset of the breeding season (defined as cyclic ovarian activity as indicated by plasma progesterone monitoring) was normal in those animals treated with morning melatonin but was delayed in those animals treated with melatonin at dusk or extended darkness at dawn compared to controls in natural photoperiod (p < .01). Exogenous melatonin at dusk was associated with a phase advance of the onset of the circadian pacemaker (as measured by endogenous melatonin in acutely extended darkness); additional darkness at dawn was associated with a phase delay of both the onset and the offset of the circadian pacemaker. Exogenous morning melatonin did not change the phase of the circadian pacemaker relative to the controls. The results are consistent with an external coincidence model of seasonal breeding in which a critical interval of the circadian pacemaker requires exposure to light during spring/summer to time estrus correctly. The proposed critical interval appears to be located near dusk in this model and is phase locked to the circadian pacemaker. The effect of the exogenous melatonin on the timing of the breeding season is similar to darkness when administered at dusk but is not equivalent to darkness at dawn. The timing of anestrus was not affected by any of the experimental treatments and may reflect a common response to an environmental influence. PMID- 8639940 TI - Circadian aspect of photoperiodic time measurement in a female house sparrow, Passer domesticus. AB - Endogenous circadian rhythms are involved in various photoperiodic responses of birds. Investigations involving the mechanisms of photoperiodic time measurement in birds have been confined mainly to temperate zone species using males exclusively. Due to the paucity of experimental evidence on subtropical birds that have very wide distribution, the present study was performed using subtropical female house sparrows. Photoperiodic sparrows were subjected to various T cycles for 60 days in which a fixed 3-h photophase was combined with scotophase in cycles of 20-30 h duration. Simultaneously, two groups of birds were also exposed to short days (light:dark = 8L:16D) or long days (15L:9D) as controls. Significant ovarian growth was found only in cycles of 3L:17D, 3L:23D, 3L:25D, and 3L:27D as well as in 15L:9D, whereas no positive response was detected in 3L:19D, 3L:21D, and 8L:16D. Plasma levels of estradiol showed a close relation to ovarian growth. The results indicate that photoperiodic time measurement in house sparrows involves an endogenous circadian rhythm. PMID- 8639941 TI - Seasonal variation in human illumination exposure at two different latitudes. AB - The authors measured ambient illumination exposure in healthy volunteers in San Diego, California (latitude 32 degrees 43' N, n = 30), and Rochester, Minnesota (latitude 44 degrees 1' N, n = 24), during each of the four quarters of the year, which were centered on the solstices and equinoxes. Subjects wore photosensors on their wrists and lapels (or foreheads while in bed) 24 h per day for an average of 5-6 days per quarter. The maximum of the two illumination readings was stored each minute. Annual average time spent per day in outdoor illumination (> or = 1000 lux) was significantly higher in San Diego than it was in Rochester (p < .04). Daily durations of illumination at or exceeding thresholds of 1, 10, 100, 1000, and 10,000 lux were highly seasonal in the sample as a whole (p < .01 at 1 lux, p < .0001 at other thresholds). Seasonal variation in outdoor illumination was far more pronounced in Rochester than it was in San Diego (interaction p < .001) but remained significant in San Diego (p < or = .03). Seasonal variation in indoor illumination was generally similar in the two cities. The median Rochester subject experienced illumination > or = 1000 lux for 2 h 23 min per day during summer and 23 min per day during winter. The corresponding times in San Diego were 2 h 10 min and 1 h 20 min. Neither age nor gender predicted illumination duration at any level. Both season and geographic location strongly influenced human illumination exposure, and behavior (choice of indoor vs. outdoor environment) was the most important mediating factor. PMID- 8639942 TI - Is there an endogenous tidal foraging rhythm in marine iguanas? AB - As strictly herbivorous reptiles, Galapagos marine iguanas graze on algae in the intertidal areas during low tide. Daily foraging rhythms were observed on two islands during 3 years to determine the proximate factors underlying behavioral synchrony with the tides. Marine iguanas walked to their intertidal foraging grounds from far-off resting areas in anticipation of the time of low tide. Foraging activity was restricted to daytime, resulting in a complex bitidal rhythm including conspicuous switches from afternoon foraging to foraging during the subsequent morning when low tide occurred after dusk. The animals anticipated the daily low tide by a maximum of 4 h. The degree of anticipation depended on environmental parameters such as wave action and food supply. "Early foragers" survived in greater numbers than did animals arriving later at foraging sites, a result indicating selection pressure on the timing of anticipation. The timing of foraging trips was better predicted by the daily changes in tabulated low tide than it was by the daily changes in actual exposure of the intertidal foraging flats, suggesting an endogenous nature of the foraging rhythms. Endogenous rhythmicity would also explain why iguanas that had spontaneously fasted for several days nevertheless went foraging at the "right" time of day. A potential lunar component of the foraging rhythmicity of marine iguanas showed up in their assemblage on intertidal rocks during neap tide nights. This may indicate that iguanas possessed information on the semi-monthly rhythms in tide heights. Enclosure experiments showed that bitidal foraging rhythms of iguanas may free run in the absence of direct cues from the intertidal areas and operate independent of the light:dark cycle and social stimuli. Therefore, the existence of a circatidal oscillator in marine iguanas is proposed. The bitidal foraging pattern may result from an interaction of a circadian system with a circatidal system. Food intake or related stimuli may be used as tidal zeitgebers in synchronizing the foraging rhythms of these reptiles under natural conditions. PMID- 8639943 TI - Models of risk assessments for biologicals or related products in the European Union. AB - In the context of veterinary biologicals, environmental risk assessment means the evaluation of the risk to human health and the environment (which includes plants and animals) connected with the release of such products. The following categories or types of veterinary biologicals can be distinguished: non genetically modified organisms (non-GMOs) (inactivated/live) GMOs (inactivated/live) carrier products related products (e.g. non-specific "inducers'). Suitable models used in risk assessment for these products should aim to identify all possible adverse effects. A good working model should lead, at least, to a qualitative judgement on the environmental risk of the biological product (e.g. negligible, low, medium, severe, unacceptable). Quantifiable outcomes are rare; therefore, the producer of a biological product and the European control authorities should accept only models which are based on testable points and which are relevant to the type of product and its instructions for use. In view of animal welfare aspects, models working without animals should be preferred. In recent years, some of these methods have been integrated into safety tests described in European Union Directives and in monographs of the European Pharmacopoeia. By reviewing vaccine/registration problems (e.g. Aujeszky's disease live vaccine for pigs, and vaccinia-vectored rabies vaccine), several models used in risk assessment are demonstrated and discussed. PMID- 8639944 TI - Models used in Australia in risk assessments for veterinary biologicals. AB - In Australia, the development of models for risk assessment in the field of veterinary biologicals is based on the chapter on "Import risk analysis' in the Office International des Epizooties International Animal Health Code. The development process involves examination of the following: country factor (source of the product), commodity factor, nature of the product, potential to carry contaminating organisms, end-use, risk reduction features of the manufacture or nature of the product (e.g. inactivation or extraction techniques, dilution). Finally, the Australian risk model and quarantine process carefully examine the risk of domestic exposure. Consideration goes beyond potential in vivo or in vitro use of the product and encompasses the wider potential for access to animals (domestic and farm, native and feral) after arrival in Australia. Practice involves the limitation of certain agents to appropriate biocontainment facilities, or--where therapeutic or immunotherapeutic use is planned--continuous assessment of the production systems of the manufacturing company. These assessments are developed into templates or precedents which may be re-used to lend consistency and security to the manufacturing process. Australian practice and assessment recognise the wide variety of biologicals and the number of diseases which they can potentially introduce. The author examines several types of biologicals and emerging threats, including that presented by bovine spongiform encephalopathy. This paper complements the paper by J. Owusu in this issue of the Scientific and Technical Review, which addresses, in particular, the assessment of vaccines in Australia. The National Registration Authority plays a specific role which is complementary to that of the other quarantine authorities. Attitudes to risk assessment in Australia derive from the freedom of this country from many diseases, from the potential for inoculation of imported biologicals into animals with direct transmission of contaminants, and from the difficulty of identifying the original source country in many cases (due to the freedom of movement of many biologicals, particularly the substrates of many immunobiologicals). PMID- 8639946 TI - Risk analysis for the importation of veterinary biologicals into the United States of America. AB - International trade in veterinary biological products has been restricted by the following factors: a) concerns that contaminated products could result in the introduction of foreign animal disease agents into the importing country b) differences between countries in the technical requirements for product registration. The provisions of the North American Free Trade Agreement (NAFTA) and the General Agreement on Tariffs and Trade (GATT: now the World Trade Organisation [WTO]) require importation decisions to be science-based and transparent. This requires regulatory agencies to implement valid, credible, and science-based risk analysis models for decision-making. The Veterinary Biologics section of the United States Department of Agriculture, Animal and Plant Health Inspection Service, currently uses a formalized risk analysis model to evaluate the safety risks associated with proposals to field test and license new and biotechnology-derived veterinary biological products. This model for evaluating field tests has been modified to evaluate proposals to import veterinary biological products into the United States of America. The authors describe this risk analysis model, which was specifically designed to evaluate the risks of importing veterinary biological products potentially contaminated with foreign animal disease agents. PMID- 8639945 TI - Risk analysis for veterinary biologicals released into the environment. AB - All veterinary biologicals licensed in Canada must be shown to be pure, potent, safe and effective. A risk-based approach is used to evaluate the safety of all biologicals, whether produced by conventional methods or by molecular biological techniques. Traditionally, qualitative risk assessment methods have been used for this purpose. More recently, quantitative risk assessment has become available for complex issues. The quantitative risk assessment method uses "scenario tree analysis' to predict the likelihood of various outcomes and their respective impacts. The authors describe the quantitative risk assessment approach which is used within the broader context of risk analysis (i.e. risk assessment, risk management and risk communication) to develop recommendations for the field release of veterinary biologicals. The general regulatory framework for the licensing of veterinary biologicals in Canada is also presented. PMID- 8639948 TI - Risk analysis methods and techniques for veterinary biologicals used in Australia. AB - Advances in modern science and technology and the globalisation of the veterinary manufacturing industry, coupled with the relaxation of trade restrictions by the General Agreement on Tariffs and Trade treaty on sanitary and phytosanitary measures, call for an international approach to standards of acceptable risk and risk analysis methodology. In Australia, different elements of risk analysis are undertaken by different agencies. The agencies employ screening risk assessment, which uses simple worst-case scenarios and conservative data to set priorities and identify issues of limited risk. The approach is multi-factorial, assessing risk to public health, animals and the environment. The major components of the analysis process are risk assessment, risk management, and procedures for communicating and monitoring risk. The author advocates the possible use of quantitative risk assessment, based on acceptable international standards, in making international trade decisions. In the absence of acceptable international standards, it is proposed that countries adopt mutual recognition of comparable standards and specifications employed by national agencies. PMID- 8639947 TI - Biologicals: test procedures available to assess components and products, with limitations. AB - Contamination with a pathogen, residual live organisms in inactivated products, and a lack of safety of live products may be considered to constitute the greatest risks associated with the use and the international movement of biological products. Although tests are conducted to monitor the risks, the limitations of such tests must be recognised and steps should always be taken to minimise and, as far as possible, avoid the risk. Tests must be suitably sensitive and specific for their function, but they must also be practical and reproducible. Studies on the sensitivity of tests provide a measure of their limitations. The author describes points to consider for the assessment of the suitability of different test methods. Emphasis is placed on tests for parameters associated with risk, such as extraneous agent testing. Examples of such analyses are discussed. There is a need for an exchange of information and a scientific debate on such issues if international acceptance of test methods and harmonization of requirements are to be achieved. PMID- 8639949 TI - Quantitative risk analysis applied to innocuity and potency tests on the oil adjuvanted vaccine against foot and mouth disease in Argentina. AB - The authors describe the method used in Argentina for quantification of risk in controls of the potency and innocuity of foot and mouth disease vaccine. Quantitative risk analysis is a relatively new tool in the animal health field, and is in line with the principles of transparency and equivalency of the Sanitary and Phytosanitary Agreement of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT: now World Trade Organisation [WTO]). The risk assessment is presented through a description of the steps involved in manufacturing the vaccine, and the controls performed by the manufacturer and by the National Health Animal Service (Servicio Nacional de Sanidad Animal: SENASA). The adverse situation is considered as the lack of potency or innocuity of the vaccine, and the risk is estimated using a combination of the Monte Carlo simulation and the application of a Bayesian model. PMID- 8639950 TI - Registration of veterinary products in Argentina. AB - A scheme for registering pharmaceutical and biological products for veterinary use was introduced in Argentina in 1994, as part of a joint scheme for countries of the Common Market of the South (Mercado Comun del Sur: "Mercosur'). The authors describe the main features of these regulations, and the process which led to their development. PMID- 8639951 TI - Regulation of immunological veterinary medicinal products in the European Union. AB - Since 1990, immunological veterinary medicinal products have been covered by the pharmaceutical legislation of the European Union (EU) and, as a consequence, these products are now subject to the relevant general provisions regarding manufacturing and marketing authorization. For new veterinary immunologicals, the legislation entered into force on 1 April 1993. For products already on the market, a transitional period of five years was granted, during which Member States must proceed with the review of these products. Member States are coordinating this review and have set a calendar for the various species-specific products. Despite the extensive harmonization of requirements and criteria, and the existence of procedures for marketing authorization involving the Committee for Veterinary Medicinal Products (CVMP), the actual decision whether to authorize a product was still taken by individual Member States, thus leading to divergences at the very last stage of the process. Therefore, in 1993, the European Council adopted a Regulation and Directives; this legislation modified the current system, introduced two new procedures for veterinary medicinal products and established the European Medicines Evaluation Agency. Under the new system, innovatory medicinal products obtained through biotechnology will be authorized centrally and marketed throughout the EU. Conventional medicinal products will be subject to mutual recognition of authorizations, with binding arbitration by the Agency in case of disagreements between Member States. PMID- 8639952 TI - Present systems and future needs for risk assessment of biologicals in the United States of America: the perspective of the regulator. AB - The authority for regulating veterinary biologicals in the United States of America (USA) is provided in the Virus-Serum-Toxin Act, enacted in 1913 and amended in 1995. The Act authorizes the Secretary of Agriculture to prescribe regulations governing the preparation and marketing of veterinary biologicals shipped into, within or from the USA. The mandate of the United States Veterinary Biologics Program is to ensure that all veterinary biological products under Government jurisdiction in the USA are pure, safe, potent and effective. The Program is based on licensing, inspection and testing. Risk assessment techniques, and effective strategies for risk management and risk communication, are essential tools for regulatory officials charged with the responsibility of developing requirements for licensing veterinary biological products and production facilities. To accommodate scientific advances, heightened consumer awareness and the international harmonization of requirements, regulatory agencies must continually review and update programme requirements. The author discusses programme updates initiated to address future needs of the United States Veterinary Biologics Program. PMID- 8639953 TI - Present systems and future needs for risk assessment of veterinary biologicals in Australia: the perspective of the regulator. AB - The increasing range and complexity of biologicals, and the greater demand for these products, have resulted in a greater volume of trade in animal-based biological material. This has given rise, in turn, to many approaches to the regulation of importation of these materials, as countries seek protection against the introduction of disease. Harmonization of these regulatory approaches would contribute significantly to the availability of veterinary biologicals, to their manufacture and trade, and to disease security. Australia has developed systems for the categorisation and evaluation of biologicals, control by import permits, and specific procedures at point-of-entry and in institutions where these products are used. Computerised records and precedents assist in evaluation and in the issuing of permits. Recognition that some materials must be subject to further control has led to a system of registration of institutions based on levels of biosecurity, and approved use and disposal programmes. Institutions vary from high-security animal health laboratories to human in vitro fertilisation clinics, which use animal-derived media and materials. Such institutions are regulated through quality assurance contracts. Quarantine authorities have linkages with other agencies which have an interest in these products. These linkages reflect the administrative structures of government in Australia, and provide for management of all forms of risk. The author describes these systems and overviews their biological basis. PMID- 8639955 TI - Present systems and future needs for risk assessments of biologicals: the perspective of the regulator in the People's Republic of China. AB - Regulation of veterinary biologicals in the People's Republic of China is governed by the Animal Drug Administration Regulations issued by the State Council in 1987. These regulations authorize the Ministry of Agriculture to prescribe requirements governing the production and marketing of veterinary biologicals shipped into, within or from China. The goal of the veterinary biologicals programme is to ensure that all veterinary biological products are pure, safe, potent and effective. The programme is based on review, licensing, inspection and post-licensing testing. Risk assessment procedures and requirements for biotechnology-derived veterinary biologicals have been established. China needs international harmonization of trade in veterinary biologicals and will make a great contribution in this field. PMID- 8639954 TI - Regulatory framework and requirements for managing risks associated with veterinary biological products in Africa: present systems and future needs. AB - Veterinary vaccines are considered to be medicinal products. As such, they are subject to assessment for managing risks associated with their marketing and use. The current risk assessment procedures used in Africa are based on the quality testing methods standardised by the Pan-African Veterinary Vaccine Centre (PANVAC). The authors examine the risk assessment procedures related to the importation of products and to the release of live products into the environment. The lack of infrastructures, specialised personnel and financial resources prevents each individual country from establishing its own system for managing the risks associated with the importation of veterinary vaccines. Regional co operation between African countries is therefore recommended, and must be based on the existing PANVAC network for the quality testing of priority vaccines. This is justified by the results obtained by PANVAC in the standardisation of production technologies for vaccines against rinderpest and contagious bovine pleuropneumonia, and in other areas. The authors recommend that PANVAC be used to aid regional co-operation in Africa in the management of risks associated with the marketing and use of veterinary vaccines. PMID- 8639957 TI - Application of risk assessment to veterinary biologicals: the perspective of the animal health industry. AB - The authors review the perspective of the animal health industry on the changing regulatory climate for the registration and free circulation of veterinary vaccines. The industry supports the increased mutual acceptance of technical standards, the harmonization of these standards and the use of proper risk analysis for regulating the free movement of veterinary vaccines. The veterinary vaccine industry is relatively small, but a large number of products are manufactured and the regulation of these products is highly complex. This complexity results in divergent policies on the importation of vaccines, thus limiting the flexibility of the industry in deciding where to develop and manufacture products. The industry welcomes moves by governments to dispense with "zero risk' policies and to adopt consistent scientific risk analysis approaches, in line with the World Trade Organisation agreement on the application of sanitary and phytosanitary measures. The risks involved in the handling, development and manufacture of veterinary biological products are reviewed, and the factors to be taken into account in the assessment and in the management or control of these risks are presented. Dissection of the production process into its various phases enables identification of the critical points and application of specific risk control measures. An important aspect of the risk assessment is the evaluation of the existing testing methods. A specific programme intended to establish the equivalence or harmonization of various test methods is being proposed. In conclusion, the industry is willing to be actively involved in the harmonization process and expects an equally clear political and technical commitment from the governments of the major trading countries. PMID- 8639956 TI - The role of international and regional organisations in the regulation of veterinary biologicals. AB - The authors discuss the role played by international and regional organisations in the registration and testing of veterinary biological products. International organisations which contribute significantly to this field include the Office International des Epizooties (OIE)--through the work of the Standards Commission and the publication of the Manual of standards for diagnostic tests and vaccines- , the Food and Agriculture Organisation of the United Nations (FAO) and the World Health Organisation (WHO)--through the work of the Joint FAO/International Atomic Energy Agency (IAEA) Division of Nuclear Techniques in Food and Agriculture in standardising enzyme-linked immunosorbent assay (ELISA) techniques, as well as through WHO Expert Committees. In Europe, the most important regional organisations are the European Commission and the European Pharmacopoeia. In the Americas, the most significant contribution is made by the two specialised institutes of the Pan American Health Organisation (namely INPPAZ [Pan American Institute for Food Protection and Zoonoses] and PANAFTOSA [Pan American Foot and Mouth Disease Centre]), and by the Inter-American Institute for Cooperation in Agriculture. In Africa, PANVAC (the Pan-African Veterinary Vaccine Centre) continues to perform valuable work in testing veterinary vaccines. For the industrialised countries, the Organisation for Economic Co-operation and Development (OECD) is involved in the regulation of biotechnology products and in standardising "good laboratory practice' for vaccine manufacture. A table is presented which summarises and compares the respective roles of these organisations in the harmonization of licensing and testing procedures, the distribution of reference reagents, vaccine testing and the creation of vaccine banks. PMID- 8639958 TI - A European farmers' view of free trade and risk assessment for veterinary biologicals. AB - In Europe, farmers request that they be supplied with the right drug in the right place for the right animal at the right price: no more, no less. They are not concerned which country or company produces the veterinary biologicals used. Farmers are advised by the veterinary practitioner, but knowledge of neither the country of manufacture nor the producer determines their choice. This attitude is due to the quality of the pharmaceutical products available. European farmers support free trade and the implementation of scientific risk assessment. In the view of European farmers, the system of equivalent measurement should be adopted: countries may have different systems to check the quality of veterinary products, but the outcome should be the same, i.e. a guarantee of quality, safety and efficacy. PMID- 8639959 TI - Proceedings of the 1st International Symposium on Risk Assessment for Veterinary Biologicals: The Next Step in International Harmonisation. Arlington, Virginia, 5 7 December 1994. PMID- 8639960 TI - Limitations, definitions, principles and methods of risk analysis. AB - Decisions on veterinary biologicals involve large uncertainties, complexities which cut across many scientific and technical disciplines, and large potential adverse impacts on public health and on important sectors of the economy. How should risk assessment help to guide the decision process on veterinary biologicals? How can risk assessment practices be harmonized internationally, given the different regulatory traditions and institutions of different countries? A broad view of risk assessment is needed, that risk assessment is a framework for summarizing applicable scientific judgement in support of regulatory decision-making. Support for this view of risk assessment is found in the major reports which have defined risk assessment as currently practised by many regulatory agencies in the United States of America (USA). However, some interested and affected parties perceive risk assessment in the USA as overly quantitative and narrowly focused on regulatory standards for carcinogens. An example of risk assessment for microbial contamination indicates how quantitative methods can be used when data are sparse and decisions must be made in the face of great uncertainty. Such quantitative methods can be used to improve communication about risk, to promote consensus in support of controversial decisions, and to identify valuable opportunities for research to reduce the important sources of uncertainty. PMID- 8639961 TI - Risk analysis systems for veterinary biologicals: a regulator's tool box. AB - Recent advances in biology and technology have significantly improved our ability to produce veterinary biologicals of high purity, efficacy and safety, virtually anywhere in the world. At the same time, increasing trade and comprehensive trade agreements, such as the Uruguay Round of the General Agreement on Tariffs and Trade (GATT: now the World Trade Organisation [WTO]), have put pressure on governments to use scientific principles in the regulation of trade for a wide range of products, including veterinary biologicals. In many cases, however, nations have been reluctant to allow the movement of veterinary biologicals, due to the perceived threat of importing an exotic disease. This paper discusses the history of risk analysis as a decision support tool and provides examples of how this tool may be used in a science-based regulatory system for veterinary biologicals. A wide variety of tools are described, including qualitative, semi quantitative and quantitative methods, most with a long history of use in engineering and the health and environmental sciences. PMID- 8639962 TI - Risk analysis in the manufacture of veterinary biologicals. AB - "Primum non nocere' must be the first quality of veterinary immunological medicinal products. Throughout the manufacturing process, stage by stage, various ingredients and/or operations could be responsible for safety problems observed when the product is administered. During the preliminary stages, various ingredients--e.g. master seed strains (virus, bacteria or parasite), cell substrates (cell-lines or primary cells) and substances of animal origin--could be contaminated by extraneous agents. These represent the most important risks. During the process--i.e. preliminary stages, microorganism growth, downstream processing (harvest, purification, concentration, inactivation, etc.), formulation, filling, freeze-drying and finally packaging--environmental conditions (working areas, equipment, etc.) might also be defective and responsible for product contamination. The author examines all aspects of risks and their assessments through consideration of "good manufacturing practice' based on quality assurance and quality control systems. PMID- 8639963 TI - Risk of introducing exotic disease through importation of animals and animal products. AB - Between 1870 and 1929, nine separate outbreaks of foot and mouth disease (FMD) occurred in the United States of America (USA); additional outbreaks in North America include one in Mexico (1947) and two in Canada (1870 and 1952). In 1930, the United States Congress enacted a law prohibiting importation of live ruminants or swine or fresh meat from these species into the USA from countries affected with FMD or rinderpest. Although the effect of this prohibition may be debated, the USA has remained free of FMD since its enactment. A hidden benefit of this prohibition was probably the limitation on importing other disease agents from countries of the world where FMD was present. As many regions of the world make progress towards the control and eradication of FMD, North America must take greater cognizance of other disease agents with which it has not been concerned to date, as these existed only in regions of the world affected with FMD and/or rinderpest. One of the methods of dealing with these other diseases is by using risk assessment and risk management methodologies. For risk assessment to work, however, the available management technologies must be examined, and levels of risk assigned to match the available technology. The authors explore risk analysis options for the importation of animals and animal products in a manner which will continue to protect the livestock industry in the USA. They also examine the role of veterinary biologicals as a management tool to mitigate the attendant risks. PMID- 8639964 TI - Risks related to the introduction of exotic diseases: a European view. AB - The European Union (EU) has historically been free of a number of epidemic diseases of livestock and is approaching eradication of several others. This situation is put at risk by the importation of live animals and animal products. Animal products include products which are broadly described as veterinary biologicals. For the purposes of assessing and controlling risks, these fall into two groups: crude products (e.g. serum and other blood products) and medicinal products (e.g. vaccines and other immunological products). This second group is covered by another paper in this issue of the Review. Risk reduction methods may be applied to crude veterinary biologicals pre- or post-importation. Pre importation methods include product treatment, or ensuring that the source of the product is "risk-free'. Post-importation risk reduction involves product testing, or treatment achieved by channelling products to commercial operations which are covered by European medicines legislation. At present, EU Member States issue health certificates individually for crude veterinary biologicals, and conditions for entry may differ between States. This process will soon be harmonised (the provisions for marketing medicinal products have already been harmonised). PMID- 8639965 TI - Environmental safety assessment of vaccines derived from biotechnology. AB - Products derived from biotechnology are generally safer than previous products obtained by conventional, more empirical methods. Given the great variety of biotechnology-derived products and use situations, risk assessment procedures must be based on a case-by-case analysis. It is therefore difficult to propose a generic, well-defined risk assessment procedure. In the case of vectored vaccines, two main risks must clearly be assessed: the possible risk of tissue or species tropism modification, with special regard to non-target species, the risk of recombination with wild counterparts of the vector. This paper begins with a classification and description of the main biotechnology-derived products to be used in veterinary medicine, including deoxyribonucleic acid (DNA) vaccines, and then attempts to define general rules for the risk assessment of each category of products. PMID- 8639966 TI - Risk assessment related to veterinary biologicals: side-effects in target animals. AB - Despite established control measures, the large-scale use of veterinary biologicals may involve side-effects. The most common side-effects observed include the following: injection site reactions, systemic reactions, allergic reactions, effects on the immune system, residual pathogenicity, inadequate inactivation, genetic recombination, contamination. New technologies, new harmonised regulations, and commitment to quality will ensure a continuous supply of safe and innovative products. PMID- 8639967 TI - Risk assessment related to veterinary biologicals: specific issues in developing nations. AB - The author reviews both technical and socio-economic issues in developing nations, in relation to veterinary biologicals. Health risk assessment is a specific process to estimate the likelihood that animals, humans or ecological systems will be affected adversely by a chemical or physical agent, or biological product, under a specific set of conditions. Some technical issues (quality assurance, good manufacturing practice, education of end-users, field monitoring) apply equally well in developed, industrialised and in developing, pre industrialised nations. Many regions have documented unique diseases (trypanosomosis, tick diseases, theileriosis) or high disease prevalence which may influence risk assessment results. This emphasises the need for scientifically-valid risk assessment methodologies in developing nations. Developing nations also have various socio-economic concerns, which may not be based on scientific fact but, nonetheless, affect trade in, and use of, veterinary biologicals. These non-scientific but perceived problems and issues are briefly discussed, and possible solutions are presented. The way in which countries deal with such perceived problems and issues in a context of internationally harmonised norms for risk assessment impinges on livestock farmers in developing nations. Finally, the author presents possible ways to correct the potentially widening cost gap between conventional, proven veterinary biologicals and newly-developed products. The results of risk assessment of veterinary biologicals influence risk management in both developed and less developed nations. It is important to agree upon scientifically-based risk management guidelines which may be applied in all countries. The effect of the agreements of the Uruguay Round of the General Agreement on Tariffs and Trade on trade in veterinary biologicals in developing nations is reviewed. PMID- 8639968 TI - Major decrements in glycated hemoglobin levels between 1978 and 1989 in patients with insulin-dependent diabetes mellitus. AB - The Diabetes Control and Complications Trial has shown that intensive treatment can deter the development and progression of diabetic complications. Integral to intensive treatment is improved glycemic control. To describe the trend in glycemic control for subjects with insulin-dependent diabetes mellitus, we examined the medical records of 662 subjects seen between 1978 and 1989 at the Model Demonstration Unit of the Diabetes Research and Training Center (Washington University School of Medicine). Mean value of glycated hemoglobin showed steady decline from a peak of 11.5% in 1979 to 9.0% in 1989. This decline was observed both in subjects evaluated only once (annual rate of decline estimated from linear regression, -0.17 +/1 0.03; p = 0.0001) and in subjects evaluated more than once (annual rate of decline estimated from growth curves, -0.18 +/- 0.06; p = 0.0001). These results suggest that substantial lowering of glycated hemoglobin has occurred during the last decade. This reduction should result in a lowered risk of diabetic complications. PMID- 8639969 TI - Vascular responses by transcutaneous oximetry in adolescents with and without diabetes. AB - The vascular response of the skin was evaluated by transcutaneous oximetry (TcPO2) in the forearm in 119 adolescents with type I diabetes aged 10.4-19.8 (median 15.3) years, with a duration of diabetes 0.7 to 18.3 (median 7.8) years, and 49 nondiabetic adolescents aged 11.3-18.8 (median 15.5) years. Two different vascular stimuli were used: heating of the probe to 43 degrees C and 5 min of ischemia. Baseline TcPO2 after 13 min of equilibration at a probe temperature of 43 degrees C, postischemic maximum TcPO2, and the postischemic TcPO2 increase were significantly lower in the diabetic group compared to the control group (p = 0.0001, p < 0.0001, and p = 0.0001, respectively). In both the diabetic and the control groups, gender differences were found for baseline TcPO2 (p = 0.0001 and p = 0.0009, respectively) and postischemic maximum TcPO2 (p = 0.0001 and p = 0.005, respectively), the girls having consistently higher values. After controlling for gender by multiple linear regression analysis, duration of diabetes showed a significant effect on postischemic maximum TcPO2 (R2 = 22%, p = 0.02). The postischemic TcPO2 increase was not affected by gender. Lower values for the postischemic TcPO2 increase were related to higher GHb values (R2 = 4%, p = 0.03). Abnormal values for oximetry were associated only with some autonomic nerve function abnormalities. Differences in the vascular response to heat and ischemia as measured by transcutaneous oximetry can be demonstrated between adolescents with type I diabetes and nondiabetic controls, as well as between girls and boys. Lower values in diabetic subjects are weakly associated with diabetes duration and metabolic control, independent of gender. PMID- 8639970 TI - Diabetes mellitus and malignant external otitis: a case study. AB - Malignant external otitis (MEO) is an infection of the external auditory meatus, that affects elderly diabetic patients. As this disease results in a high percentage of deaths, especially if the diagnosis is delayed, we thought that it would be useful to cite a recent case study that was resolved in a positive way, in spite of the extent of the disease. PMID- 8639971 TI - Aminoguanidine does not inhibit aldose reductase activity in galactose-fed rats. AB - Aminoguanidine, nucleophilic hydrazine derivative, has been shown to inhibit diamine oxidase, the formation of advanced glycation endproducts, nitric oxide synthase, and catalase. Prompted by the reports that aminoguanidine also inhibits aldose reductase (AR), we have investigated the effect of aminoguanidine, 1,3 diaminoguanidine, and methylguanidine on AR activity in vitro, and in vivo. In vitro, we have measured the inhibition of AR isolated from bovine lenses; in vivo, we have examined the effect on the galactitol levels in the red blood cells, sciatic nerve, retina, and lens of rats administered the test compounds for 11 days in the drinking water and, for the last 4 days, given access to a 20% galactose diet. Two known, structurally distinct AR inhibitors, tolrestat and compound WAY-121,509, were used as reference. In vitro, at concentrations up to 1.0 mmol/L, none of the tested guanidine derivatives had any effect on AR. As a corollary, in vivo, at doses ranging from 201 to 349 mg/kg/day, none of the guanidine derivatives affected tissular galactitol levels. We conclude that, in short-term galactose-fed rats, at the doses tested, aminoguanidine, 1,3 diaminoguanidine, and methylguanidine do not inhibit AR. PMID- 8639972 TI - Molecular and physiological aspects of nephropathy in type I (insulin-dependent) diabetes mellitus. PMID- 8639973 TI - Correlation between myocardial dysfunction and changes in myosin isoenzymes in diabetic rat hearts. AB - We investigated the relationship between papillary muscle function and the myosin isoenzyme pattern, collagen content, and the type of myocardial collagen in diabetic rats to elucidate the mechanism of short-term myocardial dysfunction in diabetes. Diabetes was induced in 9-week-old male Wistar rats with a single intravenous injection of streptozotocin. One-half of the diabetic rats were treated with insulin. Age-matched control rats were also studied. The time to peak tension (TPT) of isometric papillary muscle contraction, time to 1/2 relaxation, and time from the peak tension to the peak decrease in tension (TPN) were significantly prolonged in diabetic rats at 4, 8, and 12 weeks. The peak increase and decrease in tension were slower in the diabetic rats compared with control rats. The level of the myocardial myosin isoenzyme V3 was greater in diabetic rats than in control rats at each interval. Findings in insulin-treated rats were similar to those in controls. The collagen content and the ratio of type I collagen to type III collagen were similar in all groups. The V3 level was significantly correlated with mechanical parameters (TPT versus %V3: r = 0.81, p < 0.01; TPN versus % V3: r = 0.78, p < 0.01). Our findings suggest that short term myocardial dysfunction in diabetic rats is related to changes in the myosin isoenzyme pattern. PMID- 8639974 TI - Reversal of defective peripheral nerve conduction velocity, nutritive endoneurial blood flow, and oxygenation by a novel aldose reductase inhibitor, WAY-121,509, in streptozotocin-induced diabetic rats. AB - The main aim was to investigate whether 1 month of aldose reductase inhibitor treatment could correct a deficit in sciatic nerve nutritive blood flow following 1 month of untreated streptozotocin-induced diabetes in rats. Treatment was with two doses of WAY-121,509, both of which completely blocked neuronal sorbitol accumulation. The high dose fully corrected a motor conduction velocity deficit, whereas the low dose caused 51.3% amelioration. Nutritive endoneurial blood flow, monitored by hydrogen clearance, was 43.4% reduced after 1 month of diabetes. This was completely corrected by the high dose of WAY-121,509. In addition, vascular conductance was supranormal and there was a decrease in arteriovenous shunt flow. Low dose treatment caused a 55.6% improvement of the nutritive endoneurial blood flow deficit, paralleling the conduction velocity effect. WAY 121,509 did not alter nerve perfusion in nondiabetic rats. Data from multiple sciatic nerve penetrations by oxygen sensitive microelectrodes revealed a 42.0% deficit in mean endoneurial oxygen tension with diabetes, whereas tensions were in the nondiabetic range for high dose WAY-121,509 treatment. Thus, the data highlight neurovascular actions of aldose reductase inhibition, and suggest that neuronal polyol pathway metabolite levels are a poor predictor of functional efficacy. PMID- 8639975 TI - Little association of lipid parameters and large sensory nerve fiber function in diabetes mellitus. AB - The natural history of diabetic neuropathy and its risk factors are not well understood. The potential association of various lipids [e.g., high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides], and lipoprotein(a) [Lp(a)] concentrations, with large sensory nerve fiber function as assessed by vibratory thresholds was examined in a group of 91 individuals with diabetes mellitus. In multivariate analyses, no independent relationships of any of the lipid or lipoprotein parameters measured in this study were found with vibratory thresholds (i.e., dependent variable). Independent associations of age, duration of diabetes, height, and medications that lower blood pressure with vibratory thresholds were shown and explained 51% of the overall variability of the model. In gender-specific models, age, height, and medications that lower blood pressure were statistically significant independent determinates (i.e., males R2 = 0.61, females R2 = 0.39). These cross sectional data suggest that lipid and lipoprotein parameters measured in this study have little association with large sensory nerve fiber dysfunction. The interesting association with the use of medications that lower blood pressure and vibratory thresholds warrants further investigation. PMID- 8639976 TI - Perioperative management with prolonged intravenous insulin infusion versus subcutaneous insulin in children with type I diabetes mellitus. AB - Our objective was to retrospectively evaluate glycemic excursion and insulin dosage in the perioperative period in children and adolescents with type I diabetes mellitus receiving prolonged intravenous insulin infusion for 2-3 days compared to conventional subcutaneous insulin treatment. A retrospective review of surgical admissions at the Children's Hospital of Los Angeles in patients with type I diabetes mellitus was conducted for the 3-year period from July 1989 to June 1992, to evaluate two treatment protocols used during that period. For the nine admissions in group 1, patients received 0.06-0.1 units regular insulin/kg/h beginning 2 h prior to surgery and lasting for 2-3 days postoperatively; while, for the ten admissions in group 2 subjects were given subcutaneous regular and intermediate-acting insulin as 2-4 injections daily, with the regular insulin dose prior to surgery decreased to 66-75% of usual. Blood glucose levels were determined at the bedside at hourly intervals and insulin dose adjustment done with the aim of achieving blood glucose levels between 5.5 and 8.3 mmol/L (100 150 mg/dL). The mean bedside blood glucose levels for group 2 were significantly higher 1 h prior to surgery and during the intraoperative period (p < 0.05). In the postoperative period, group 2 blood glucose levels were significantly higher at multiple times for up to 3 days with multiple levels greater than 11.1 mmol/L (200 mg/dL), which was not seen in group 1. The mean insulin dosage (units/kg) prior to admission was not different for the two groups. On the day of surgery and during postoperative days 1 and 2, patients in group 1 received a greater insulin dosage than group 2 subjects (p < 0.025). In group 1, insulin dosage was increased 23% and 15% over baseline for postoperative days 1 and 2, respectively, then, by day 3, was decreased back toward the baseline. In group 2 subjects, a 13.8% increase occurred on the day of surgery due to extra insulin given immediately following the procedures, followed by a 5.4, 44.2, and 66.6% increase over baseline for postoperative days 1 through 3, respectively. In conclusion, meticulous glycemic control was readily achieved in the perioperative period with a constant intravenous insulin infusion for up to 3 days in children and adolescents with type I diabetes. To achieve glycemic control, insulin dosage needs to be increased on the day of surgery and for approximately 2 postoperative days. PMID- 8639977 TI - Proposed federal legislation jeopardizes patient privacy. AB - In the last year there has been a move to enact federal legislation concerning private health-care information. This move has been fueled by a growing trend toward the computerization and electronic transmission of health-care information. These advances in technology call for appropriate new protections of patients' privacy. Unfortunately, the proposed legislation has not received adequate attention in the medical community. Physicians and patients in general are not aware of the legislation and have not been engaged in shaping its contents. In its current form, the legislation would seriously undermine traditional protections of confidentiality that are ensured by physicians. The flaws of the proposed legislation are examined in this article. PMID- 8639978 TI - Forensic and policy implications of the transracial adoption debate. AB - The adoption of black children by white families continues to garner significant attention from legislatures, the media, and scholars in many disciplines. Still, forensic psychiatrists have said little about this form of transracial adoption, and they seem willing to allow other disciplines to map out singlehandedly the public policy in this area. This policy is expected to affect an estimated 175,000 black children nationally who live in some form of out-of-home placement. Forensic psychiatrists should increase their understanding of and involvement in the debate over this special form of adoption. This article highlights several principles that must be better understood if forensic psychiatrists are to participate in the debate with clarity and understanding. PMID- 8639979 TI - Need for expansion of forensic psychiatrists' role in sexual harassment cases. AB - Apparently intimidated by the current climate of political correctness, forensic psychiatrists in sexual harassment cases often limit themselves to a determination of damages. Yet they are in a unique position to help in the resolution of more complex issues: they need not merely accept plaintiffs' allegations at face value, as treating psychiatrists generally do, but can assess credibility and identify psychodynamics that could be crucial in the clarification of legal questions such as "welcomeness." This article discusses the significance of pertinent data, such as a history of childhood sexual abuse, but emphasizes that such information does not necessarily invalidate the plaintiffs allegations. It also reviews the obstacles that can stand in the way of a complete psychiatric examination and thereby limit the forensic psychiatrist's ability to help the courts. Increased involvement by forensic psychiatrists could contribute to a more impartial evaluation of sexual harassment cases and help establish the distinction between valid claims and frivolous ones. PMID- 8639980 TI - A comparison of female versus male insanity acquittees in Colorado. AB - This study was undertaken to investigate the authors' clinical impression that there are significant differences between the male and female insanity acquittees in Colorado, and that these differences result in significantly different treatment needs. The study sample included 149 patients: 112 men and 37 women committed to the Colorado Mental Health Institute at Pueblo as not guilty by reason of insanity (NGRI). Data were collected from a computerized data system and from chart reviews. The study provides descriptive data regarding demographic, legal, and mental health parameters of these acquittees. Demographic items included prior history of incarceration, age at first arrest, type of NGRI crime committed, and severity of NGRI crime. Mental health variables included prior psychiatric hospitalization history of suicide attempts, substance abuse history, inpatient substance abuse treatment history, diagnoses, escape history and length of stay. Percentages of male and female subjects were calculated for those variables with discrete categories. Means and medians were calculated for continuous variables. Results indicate that women are significantly more likely to be given a diagnosis of mood disorder or borderline personality disorder, are significantly older than men at the time of commitment, and are statistically more likely to have committed a single violent crime than men. Men were found to have a significantly higher rate of prior and current substance abuse, a significantly higher rate of antisocial personality disorder, a significantly greater history of violent crime prior to the NGRI offense, and arrests beginning at a significantly younger age than women. Despite the higher severity of crime rating for women, their length of stay was significantly shorter than for men. The implications of the findings with regard to different treatment needs are discussed, and the findings are compared to four other studies addressing female versus male insanity acquittees in other states. PMID- 8639981 TI - Civil commitment viewed from three perspectives: professional, family, and police. AB - This study was designed to uncover differences in interpretation and implementation of civil commitment laws. Such problems in interpretation may contribute to mentally ill persons remaining untreated and potentially joining the thousands of homeless mentally ill persons in our communities or the incarcerated mentally ill persons in our prisons and jails. The study examines differences in the assessments of the severity of mental illness, and the appropriateness and judged feasibility for commitment in different commitment categories, made by emergency room admitting personnel, police officers, and families of mentally ill persons. The results demonstrate that police are significantly less likely than families or mental health professionals to perceive mental disability or a need for involuntary commitment on any grounds. Professional psychiatric staffs were much more likely than the other two groups to consider commitment in all three cases as legally feasible. Family ratings of appropriateness for commitment based on the presented symptomatology are similar to those of professionals. However, they are significantly less likely than professionals to judge the cases as legally feasible for commitment, and they interpret the laws similarly to the police raters in believing that commitment laws will not allow involuntary hospitalizations. Consequences and implications of these differences are discussed. PMID- 8639982 TI - The mental state of arsonists as determined by forensic psychiatric examinations. AB - In this study we evaluated whether arsonists (n = 98) differ from homicide offenders (n = 55) in regard to psychiatric disorders, suicidality, and criminal responsibility in the context of forensic psychiatric pretrial examinations. Arsonists were mainly male, poorly educated, unemployed, and living in rural areas. Eighty-four percent of the arsonists and 62 percent of the homicide offenders had an alcohol abuse problem. This difference was statistically significant (p = .002). The arsonists more commonly had suicidal thoughts and attempted suicides. Over one-third of the arsonists used fire-setting as a suicide attempt. In comparing the arsonists with the control group, there was a statistically significant difference in the variables that indicate suicidality. Arsonists more commonly had diagnosed psychiatric diseases (p = .008). The incidence of psychoses was fourfold, chronic or severe depression about threefold, and mental retardation twofold when compared with the homicide offenders. Eighty-five percent of the arsonists had received psychiatric care before the crime was committed. The arsonists were more often found to be not criminally responsible for the crime committed (p = .01). PMID- 8639983 TI - Incurable psychopaths? AB - Treatment, comprising pharmacotherapy and an educational program based on cognitive behavior therapy, of four psychopathic, criminal men fulfilling the criteria for borderline personality disorder and antisocial personality disorder is described. The diagnoses were made during a forensic psychiatric evaluation. An estimation of the capacity of the central serotonergic system was performed by analysing the platelet monoamine oxidase (MAO) activity. The pharmacotherapy was combined with an educational program involving strategies for developing better impulse control. All four men had earlier been regarded as resistant to conventional therapy. In the present cases, a combined psychosocial and biological approach seemed to be effective in developing an increased control of impulses, leading to improved coping strategies. Controlled studies are needed in order to clarify whether the described treatment program proves beneficial. PMID- 8639984 TI - Rape trauma syndrome in the military courts. AB - The military courts have developed a rich case law tradition in the area of rape trauma syndrome testimony. These cases are particularly important in the context of a military that is both increasingly female and increasingly sensitive to mixed gender relationships. This article reviews the military court's approach to rape trauma testimony over the past 15 years. The author notes that military courts have been accepting of this testimony within certain well defined limits. The author analyzes the approach to testimony at one military medical center and offers a testimony model for the forensic psychiatrist who testifies in a military setting. PMID- 8639985 TI - Screening prison inmates for mental disorder: an examination of the relationship between mental disorder and prison adjustment. AB - The rapidly expanding population of prison inmates has severely challenged the prison system's ability to effectively screen incoming inmates for mental disorders and mental health service needs. This study describes a comprehensive mental health screening of inmates at a maximum security prison, using a modified version of the Referral Decision Scale (RDS), a screening measure developed from the Diagnostic Interview Schedule. Modified items and adjusted cut-off scores were used in order to reduce the rate of false positives. Survey results indicate that this modified version of the RDS may be an effective screening measure for correctional settings. Subjects who were positive on the RDS were compared to negative subjects on a variety of offense and prison adjustment variables. Findings indicate that while subjects who were positive on the RDS experienced some initial adjustment problems within the prison, they were generally not found to be involved in an elevated rate of prison violence and were not more often remanded to disciplinary units than those subjects who were negative on the RDS. PMID- 8639986 TI - Experiencing a shame response as a precursor to violence. AB - The shame response is a primitive physiological response to a rejection of oneself by another. The discomfort of this response may vary from intense physical pain to one that is barely noticeable, if at all. When this pain is sufficient, it causes anger that may be directed outward against another or inward against oneself. The intensity of the shame response, hence the intensity of the pain and anger, is related to the significance of the other, the significance of witnesses to the rejection, one's vulnerability, whether or not the rejection is of oneself or an aspect of oneself, and if the rejection comes as a surprise. When most intense (i.e., most painful), the shame response may include a tightness of the throat, nausea, stomach pain, and a sense that the contents of one's chest and abdomen are collapsing, exploding, or imploding. In reviewing what preceded an act of violence, it is necessary to determine whether the assailant had experienced a shame response and how intense it was. Understanding that a shame response can lead to anger and violence allows for the prevention of violence. This requires that individuals do not experience rejections that are so painful as to lead to violence. PMID- 8639987 TI - Differential use of admission status in a psychiatric emergency room. AB - The goal of this study is to understand how different admission statuses of varying degrees of restrictiveness (informal, voluntary, emergency admission, and involuntary admission on medical certification) are used in the psychiatric emergency room. The study included 656 consecutively admitted patients from a psychiatric emergency room over 28 months. Data were analyzed univariately and using two discriminant function models. Only six (0.9%) patients were informal admissions. Voluntary admissions (24.9%, n = 163) tended to be for patients with affective disorders, those who were self-referred, suicidal risks, those who had a marital or family problem, and those who were over age 60. Nonvoluntary admissions (74.2%) tended to be for patients with schizophreniform symptoms and those referred by police or court. Involuntary admission on medical certification (53.2%, n = 349) tended to be for patients who were family referred, younger than 20 years old, had social interpersonal nonfamily stressors, were suicidal risks, were or had been married, had organic psychotic disorder, history of violence, and manic episode or schizophrenia. Emergency admission patients (21%, n = 138) were characterized by being between 40 to 50 years old, having a diagnosis of psychoactive substance abuse, having previous outpatient treatment, and having been referred by emergency service. The major difference between involuntary admissions and voluntary was that the former were more often actively psychotic or referred by police or court. The major difference between emergency admission and involuntary admission on medical certification seemed to be that patients with a more available support system, whose primary diagnoses was not substance abuse and who were suicidal, were preferred for involuntary admission on medical certification. PMID- 8639988 TI - Violence and schizophrenia: clozapine as a specific antiaggressive agent. AB - Pharmacological management of persistent aggression in patients with schizophrenia is a difficult clinical dilemma. Clozapine has been shown to be an effective agent in this regard. This study sought to compare the symptomatic response on the Brief Psychiatric Rating Scale (BPRS) between hostile schizophrenic patients and patients without aggression. While dramatic improvements were evident in aggression, both groups were indistinguishable with respect to BPRS response. These results suggest that clozapine may have a selective antiaggressive effect. PMID- 8639989 TI - Chronic fatigue syndrome associated with a psychotic state resulting in multiple murders. AB - A 28-year-old, ambitious, academically successful Asian man with a zeal for hard work develops infectious mononucleosis and its resultant lethargy and fatigue. He becomes depressed, then develops symptoms of mania before turning floridly psychotic. In his psychotic state he develops grandiose delusions about being the second son of God after Christ and takes it upon himself to rid the world of all evil by defeating the anti-Christ. He kills four people and seriously injures a fifth. He is arrested and found not guilty by reason of insanity. He remains a diagnostic puzzle for a long time before starting to respond to neuroleptic medication. PMID- 8639990 TI - [The applications of molecular biology in taxonomy and epidemiology]. PMID- 8639991 TI - [Basic in-vitro toxicity testing on cell cultures]. PMID- 8639992 TI - [Acute infectious diarrheal disease. The dimensions--the outlook]. PMID- 8639993 TI - [The epidemiology of transmissible diseases and the epidemiology of nontransmissible diseases--identity, diversity or incompatibility?]. PMID- 8639995 TI - [Diarrheal syndromes in hospitalized patients]. PMID- 8639996 TI - [The epidemiologic role of patients with "atypical" diarrheal diseases as outpatients in maintaining and spreading enteric infections]. PMID- 8639994 TI - [The epidemiological aspects of indeterminant intestinal infections]. PMID- 8639997 TI - [The epidemiologic potential of neonatology units in the occurrence of acute diarrheal diseases in outpatient newborns]. PMID- 8639998 TI - [Cholera in Neamt County 1994]. PMID- 8639999 TI - [An epidemiological assessment of acute diarrheal diseases in Iasi city and County (1985-1994)]. AB - Acute diarrhoea, with a remarkable etiologic, clinic and epidemiologic polymorphism represents, for various reasons, an important concern for health preservation both in industrialised countries and, mainly, in the developing ones. It is that, at world level, 1.2-1.5 milliards of diarrhoea episodes, of which 750-950 millions in children, causing death in over 4 millions aged 0-5 years, are recorded. In Romania, although the real data of morbidity by acute diarrhoea are not known, the reports show a significant decrease in the past 10 years. In 1993, 420.2 cases at 100,000 inhabitants were reported, the most commonly affected being the children age 0-4 years. In Moldavia (Romania), numerous extensive studies on the epidemiology of acute diarrhoea have been carried out. This paper present and aims rendering evident some epidemiological aspects of acute diarrhoea cases reported in the interval 1985-1994 in Iasi County and town. PMID- 8640001 TI - [The improvement of methods for epidemiological assessment in cardiovascular diseases via biological markers]. PMID- 8640000 TI - [An epidemiological assessment of viral hepatitis B in Neamt County in the period of 1985-1994]. PMID- 8640002 TI - [The recrudescence of sexually transmitted diseases--an alarm signal for an increase in the sexual transmission of HIV infection]. PMID- 8640003 TI - [Nosocomial infections in the case of viral hepatitis]. PMID- 8640004 TI - [The use of cefuroxime (Zinacef) in bacterial pneumonias in children]. PMID- 8640005 TI - [The comparative identification of Campylobacter strains by traditional enzymatic tests and the gene amplification reaction]. AB - 39 strains of Campylobacter isolated from 153 diarrhoeal children (0-3 years) were comparatively identified by the traditional enzymatic tests and by the Polymerase Chain reaction (PCR). The hippurate hydrolysis test appreciated 27 strains as Campylobacter jejuni (69.2%) and 12 strains as Campylobacter coli (30.8%). The P.C.R. realised in France has appreciated 29 strains as Campylobacter jejuni (74.4%) and 10 strains as Campylobacter coli (25.6%). The analysis of the results discrepancy permitted to reveal 4 strains appreciated by two methods as different ones. The supplementary examinations of dubious strains by API-Campy test systems permitted to confirm the PCR results and to explain their divergence in contrast to hippurate hydrolysis test results. Two results were appreciated as false ones for hippurate test (5.1%). Other two errors were due to two hippurate-negative Campylobacter jejuni strains. The PCR results were exact, without errors and not influenced by modified phenotypical characters of Campylobacter strains. Thus, the efficiency of the identification by the hippurate hydrolysis test was only 89.7% in comparison to 100% efficiency of PCR (p<0.05). The discrepant cases indicated the necessity of supplementary differentiation of hippurate-negative Campylobacter strains including genetical methods in order to define the species exactly and to prevent the grave consequences especially characteristic of Campylobacter jejuni. PMID- 8640006 TI - [The thermonuclease test used in detecting S. aureus multiplication in food]. PMID- 8640007 TI - [The E-test--an accessible method of determining minimal concentrations of inhibitors (MCI)]. PMID- 8640008 TI - [The plasmid profile, a useful marker in studying a focus of Salmonella enteritidis food poisoning]. PMID- 8640010 TI - [Infection with species of the genus Bordetella in adults, a factor in maintaining morbidity for whooping cough. Serological studies]. AB - A number of 1021 serological investigations were performed to identify agglutinating antibodies against B. pertussis and B. parapertussis in adults. The investigations were initiated to study the presence of agglutinins in serum, in response to a previous infection with Bordetella species. The serological tests revealed the presence of agglutinating antibodies at titres considered to be positive (> or = 1/320) in 136 (13.3%) sera. The results obtained suggest that pertussis is relatively frequent in adults, and must be considered as a factor of maintaining morbidity. PMID- 8640009 TI - [The measurement of plasma viral loads in HIV-infected children. Their possible role in parenteral transmission]. PMID- 8640011 TI - [A case of Lyme disease detected in Romania]. PMID- 8640012 TI - [False-positive ELISA reactions for the hepatitis C virus and the human immunodeficiency virus after anti-influenzal vaccination]. AB - Possible appearance of ELISA false-positive results for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) after vaccination against influenza was studied on 402 volunteers before, and 30 and 90 days after that. Our results prove that influenza vaccine can sometimes induce false-positive ELISA for HCV, but not for HIV. Their frequency is relatively small--0.96% in 10-20-years-old group and 3.1% in over 60 years age, while in 3-6 years age there were not registered this kind of reactions. The surveillance of individual ELISA-positive cases over time proves that cross-reacting antibodies HCV raised after vaccination against influenza are present only in second sera, that means 30 days after vaccination, but these antibodies disappeared 90 days later. PMID- 8640013 TI - [The relevance of phenotypic markers for progression in AIDS in HIV-1+ children]. PMID- 8640015 TI - [Lyme disease--human borreliosis redefined]. PMID- 8640014 TI - [The beneficial effect of immunological and antiviral preparations in herpetic and papillomatosis infections]. PMID- 8640016 TI - [Malaria resistant to chemotherapy]. PMID- 8640018 TI - Sacral nerve terminal motor latency after ileal J pouch-anal anastomosis for ulcerative colitis. AB - Using a new transcutaneous magnetic stimulation technique, sacral nerve terminal motor latencies (SNTML) were measured after ileal J pouch-anal anastomosis in eight patients with ulcerative colitis, and the results were compared with those obtained from 15 normal subjects. The conduction delay of the SNTML in patients with soiling was significantly longer than that of the continent group as well as that of normal subjects (P < 0.01). There were no significant differences in the conduction delay between the continent group and the control subjects. These findings therefore support the hypothesis that such soiling, which is sometimes seen after ileal J pouch-anal anastomosis, is partly due to damage to the sacral nerves. PMID- 8640017 TI - The influence of excess body weight on the surgical treatment of patients with gastric cancer. AB - Sixty-two overweight gastric cancer patients were compared with 201 normal-weight patients to clarify the influences of excessive weight on the surgical treatment of gastric cancer. The frequencies of hypertension and diabetes mellitus were significantly higher in the overweight group (P < 0.01), but no pathologic differences in the resected tumor were found between the two groups. The operative times were longer (P < 0.01) and the number of lymph nodes extirpated and examined was smaller (P < 0.01) in the overweight group. The incidence of postoperative complications was not higher in the overweight group. The postoperative survival rate of patients with nodal metastasis was statistically lower in the overweight group (P < 0.05). Regarding the causes of death in patients with nodal metastasis, 61.1% of overweight patients and 43.8% of normal weight patients died of recurrence of gastric cancer. In conclusion, surgical treatment of overweight patients with gastric cancer was found to be technically more difficult and the prognosis of such patients with nodal metastasis may thus be worse than that of their normal-weight counterparts. PMID- 8640019 TI - Venous invasion as a prognostic factor in colorectal cancer. AB - Venous invasion as a prognostic factor was evaluated in 124 patients with colorectal cancer. By classifying the patients as having either negative to mild invasion or moderate to marked invasion, a significant correlation was found between the degree of venous invasion and clinicopathological variables such as lymphatic invasion, lymph node metastasis, liver metastasis, and DNA ploidy. Significantly more favorable survival was seen in those with a lower degree of vascular invasion; however, of the six prognosticators analyzed by Cox's proportional hazard model, the only significant factors were depth of invasion and DNA ploidy. Although venous invasion showed no significance, it is still considered a valuable prognostic indicator that is easy and economical to perform. PMID- 8640020 TI - A high level of prostaglandin E2 (PGE2) in the portal vein suppresses liver associated immunity and promotes liver metastases. AB - Prostaglandin E2 (PGE2) is generally accepted to be an immunosuppressant produced by cancer cells and their surrounding macrophages. Although several investigators have reported detecting high concentrations of PGE2 in the portal veins of patients with colorectal cancer, the relationship between these high concentrations of PGE2 in the portal vein and liver-associated immunity remains unclear. In this study, we attempted to determine if the portal administration of PGE2 suppresses the immune function of the liver in a rat model. Donryu rats were administered PGE2 via the portal vein for 7 days, following which the cytotoxic activity of hepatic sinusoidal lymphocytes (HSL) against natural killer (NK) sensitive YAC-1 and rat syngeneic AH60C tumor cells was assessed. Purified HSL are spontaneously cytolytic; however, the continuous administration of PGE2 dramatically suppressed the cytotoxic activity of HSLs in a dose-dependent fashion. Flow cytometric analysis showed that the large granular lymphocyte (LGL) fraction, hepatic natural killer (pit) cells, and CD4-8+ killer/suppressor T cells were mainly reduced in number in the HSLs following PGE2 infusion. In this rat AH60C metastasis model, the continuous administration of PGE2 increased the number and size of metastatic tumor nodules in the liver, suggesting that high concentrations of PGE2 in the portal vein suppress liver-associated immunity and promote the formation of hepatic metastasis. PMID- 8640021 TI - Long-term survival of 5 years following initial surgery for gastric cancer and simultaneous disseminated peritoneal metastasis: report of a case. AB - We report herein the rare case of a patient who survived for 5 years and 10 months after commencing treatment for gastric cancer with simultaneous disseminated peritoneal metastasis. A 45-year-old man was diagnosed as having advanced gastric cancer following the discovery of numerous nodules in the peritoneal cavity at laparotomy. The patient was treated by palliative gastrectomy and continuous hyperthermic peritoneal perfusion (CHPP) immediately after surgery on November 11, 1987. Postoperatively, he underwent radiofrequency (RF) hyperthermia with intraperitoneal cisplatin a total of seven times. He continued on a combination of uracil and tegafur (UFT) administered orally with a protein-bound beta-D-glucan extracted from the mycelia of Cariolus versicolor (PSK). Long-term survival was achieved following the initial palliative gastrectomy despite simultaneous disseminated peritoneal metastasis. PMID- 8640022 TI - Venous hemangioma of the mesoappendix: report of a case and a brief review of the Japanese literature. AB - We present herein the rare case of a 48-year-old man in whom an abdominal mass, revealed by celiotomy to be a solid tumor of the mesoappendix, was histologically diagnosed as having a venous hemangioma. To our knowledge, only 18 cases of mesenteric hemangioma have been reported in Japan, including the present case. However, establishing a correct diagnosis preoperatively is extremely difficult despite advanced imaging techniques. In fact, a mesenteric mass was diagnosed preoperatively in only 3 of these 18 cases. Complete excision with or without bowel resection was performed in 16 cases. Interestingly, the histological diagnosis of all the previous cases was cavernous hemangioma, confirming that this report documents the first case of venous hemangioma of the mesentery in the Japanese literature. PMID- 8640023 TI - Retroperitoneal abscesses--rare causes and atypical manifestations: report of two cases. AB - Retroperitoneal space abscesses are a life-threatening illness which is difficult to diagnose and treat because of both their rarity and insidious clinical manifestations. The insidious development of this illness is a challenge for all medical and surgical subspecialists. The discovery of the real source of the infection is very important but not always possible, and knowledge of the anatomy and borders of the retroperitoneal space is helpful in understanding all such atypical cases and for establishing a definitive treatment. Two cases are reported that are both characterized by rare causes, uncommon development, and atypical manifestation. The normally undefined inferior borders of some parts of the retroperitoneal space or previous retroperitoneal surgery could have been the cause of the abnormal and misleading development of the abscesses. Chronically infected organs can be the true origin of this abscess, and their discovery in a nonemergency situation is mandatory for complete surgical treatment. These cases show that knowledge of the anatomy of the retroperitoneal space is important to establish the suspicion of an abscess, to understand its manifestations, which are sometimes caused by a rare pathophysiology, and to initiate appropriate treatment. PMID- 8640024 TI - Cloacogenic anal carcinoma presenting with humoral hypercalcemia: report of a case. AB - We present herein the case of a 60-year-old man found to have a rare type of cloacogenic anal carcinoma. The disease was advanced and aggressive with local invasion to the prostate as well as distant lymph node metastases to the neck and paraaortic region on presentation. Therefore, a palliative abdominoperineal resection was performed, 10 weeks following which the patient developed humoral hypercalcemia. Despite treatment with hydration, furosemide, steroids, and calcitonin, serum calcium continued to rise and the patient died on the 95th postoperative day. Laboratory findings revealed a decreased parathyroid hormone (PTH) level and marked elevation of parathyroid hormone-related protein (PTHRP). The detection of the PTHRP in the tumor extract and the positive immunohistochemical staining for this in the tumor cells suggested that the humoral hypercalcemia was definitely caused by its associated tumor production. Although hypercalcemia is not an uncommon complication of solid cancers in their late stage, only three other cases of rectal cancer with hypercalcemia have ever been reported. To our knowledge, this is the first documentation of cloacogenic anal carcinoma accompanied by PTHRP-induced severe humoral hypercalcemia. PMID- 8640025 TI - Successful resection of a large hepatoblastoma in a young adult: report of a case. AB - Hepatoblastoma (HB) rarely occurs in adults, and very few cases of successful resection have been documented. We report herein the unusual case of a 22-year old, otherwise healthy woman with no history of liver disease who presented with upper abdominal pain and hepatomegaly. Tests for hepatitis B virus (HBV) and hepatitis C virus (HCV) were negative, but the AFP was mildly elevated at 77 ng/ml, the normal being < 20. There was no evidence of liver cirrhosis on either the laboratory or histologic examinations. A well-demarcated solid mass of 14 cm in diameter, which was lobulated and partly necrotic, was detected in the liver by computed tomography (CT). The lesion was echogenic on ultrasound, slightly hypodense on CT, and mildly hypervascular on arteriogram. The entire tumor was resected by extensive hepatectomy preserving only the lateral segment and part of the posterior segment of the liver. Histologically, the neoplasm was diagnosed as a pure epithelial HB of the fetal type. Following the operation, the patient has been well and free of recurrence for 38 months, maintaining low alpha-fetoprotein (AFP) levels at around 5 ng/ml. To our knowledge, this is the longest reported survival of an adult following surgical resection of an epithelial HB. PMID- 8640026 TI - Aneurysm of the transverse cervical artery occurring in association with a cavernous hemangioma as a complication of Klippel-Trenaunay syndrome: report of a case. AB - We report herein the case of a 14-year-old girl with Klippel-Trenaunay syndrome who developed an aneurysm of the transverse cervical artery. Because it was continuing to increase in size, with an associated risk of rupture, an aneurysmectomy was performed. Pathological examination of the resected specimen revealed a cavernous hemangioma located near the aneurysm. To our knowledge no other case of an aneurysm occurring in association with a cavernous hemangioma as a complication of Klippel-Trenaunay syndrome has ever been reported. PMID- 8640027 TI - Left partial anomalous pulmonary venous connection found during a lobectomy for lung cancer: report of a case. AB - We report herein the case of a 68-year-old man in whom a partial anomalous pulmonary venous connection (PAPVC) was found during an operation for primary lung cancer. The preoperative clinical findings did not suggest a vascular shunt, and intraoperatively the anomalous vein was seen to drain only from the left upper lobe into the left innominate vein. The lower pulmonary vein connected normally, and there was no atrial septal defect nor any other anomalous condition. A left upper lobectomy with ligation of the anomalous connected vein was performed uneventfully. This type of PAPVC is extremely rare, and is especially noteworthy because there were no clinical signs. PMID- 8640028 TI - Lung metastasis from parathyroid carcinoma causing recurrent renal hyperparathyroidism in a hemodialysis patient: report of a case. AB - We report herein the case of a 46-year-old woman on hemodialysis (HD) who developed recurrent renal hyperparathyroidism induced by lung metastasis from parathyroid carcinoma. The patient had been commenced on HD for chronic renal failure about 20 years earlier and had undergone a parathyroidectomy for advanced renal hyperparathyroidism 8 years later. After the initial operation, further explorations of the neck were performed due to recurrence, despite which the hyperparathyroidism persisted and she was finally referred to our department. The appearance of multiple coinlike lesions on a chest X-ray and computed tomography led to the diagnosis of recurrent hyperparathyroidism induced by lung metastasis from parathyroid carcinoma. A pulmonary wedge resection was performed and the metastatic parathyroid nodules were removed. Of the several hypotheses about the etiology of parathyroid carcinoma in HD patients, it is most likely that the parathyroid hyperplasia induced by chronic renal failure develops into carcinoma. Even in renal hyperparathyroidism, we should bear in mind the possibility that metastatic parathyroid carcinoma is a possible source of excess parathyroid hormone secretion at recurrence. PMID- 8640029 TI - The effect of high-energy shock wave therapy combined with cisplatin on mouse hepatoma. AB - It is well documented that high-energy shock waves (HESW) can produce antitumor effects in vivo and in vitro. Furthermore, because HESW can be focused on a limited area, this therapy is considered applicable to the treatment of localized cancer. In this study, we investigated the effects of HESW therapy combined with cisplatin (CDDP) on MH134 hepatoma in a mouse model. Tumor growth was inhibited by 1 mg/kg CDDP treatment in combination with 2,000 HESW administration, but not by 1 mg/kg CDDP treatment only. Moreover, the CDDP concentration in the tumor increased after HESW administration. The active oxygen induced by HESW was then investigated by the electron spin resonance system, and it was found that HESW generated hydroxy-radicals. As oxygen radicals have been reported to change cell membrane potential, it is supposed that active oxygen induced by HESW changes cell membrane permeability, and that CDDP is concentrated in the tumor. Therefore, the combined therapy with HESW and CDDP showed synergistic inhibitory effects on tumor growth. PMID- 8640031 TI - Closure of the distal pancreatic stump with a seromuscular flap. AB - We describe herein our new method for transecting the pancreas and closing its stump in distal pancreatectomy, devised to decrease the risk of pancreatic fistula formation. With this technique, the pancreas is transected in such a way that a convex stump is left, whereby the pancreatic secretions from the parenchyma near the pancreatic stump are fully drained into the main pancreatic duct. A pedicled seromuscular flap of the stomach or jejunum is then used to cover the cut surface of the pancreas. This new technique provides tight closure of the pancreatic stump after distal pancreatectomy. PMID- 8640030 TI - Use of the double-stapling technique in esophageal surgery. AB - This study was conducted to examine the effectiveness of the double-stapling technique (DST) for performing esophageal anastomoses. A total of 17 patients, 10 with esophageal cancer and 7 with gastric cancer, underwent esophageal anastomosis employing this technique. Of the ten patients who underwent esophageal operations, eight received esophageal-gastric tube anastomosis, one esophageal-colon and colon-residual stomach anastomosis, and one pharyngeal gastric tube anastomosis, while all seven of the patients who underwent operations for gastric cancer received esophagojejunostomy. Thus, a total of 18 anastomoses were performed. No problems were encountered during the anastomoses, and no leakage occurred in any of the patients. Thus, we conclude that DST is a safe and easy technique for performing esophageal anastomosis, especially intrathoracic or intramediastinal anastomoses. PMID- 8640033 TI - Passive immunity against diarrhoea. AB - Passive immunity against a variety of respiratory and gastrointestinal pathogens has recently been increasingly used clinically, and oral administration of antibodies of both human and non-human origin has been tried both for prophylaxis and treatment of infections. Although the former type of therapy has been shown to be effective, data on the latter are still scarce. This commentary focuses on recent studies on successful oral therapeutic administration of bovine immunoglobulins. PMID- 8640034 TI - Psychomotor and mental development at four years of age: relation to psychosocial conditions and health. PMID- 8640032 TI - Intratracheal stent intubation under extracorporeal lung assist. AB - An artificial stent was intubated using extracorporeal lung assist (ECLA) in two patients with inoperable tracheal stenosis. In a patient with an endotracheal tumor, an airway obstruction due to a partial stent collapse was overcome by an immediate ECLA perfusion. In another patient with chronic inflammatory tracheal stenosis, a repetitive balloon dilation of the trachea could be safely performed utilizing ECLA perfusion. Preventive femoral cannulation, employing the assistance of an ECLA circuit, is thus considered to be a safe and effective procedure for the treatment of inoperable tracheal stenosis. PMID- 8640035 TI - Decreasing hospital admissions for childhood asthma in Sweden. PMID- 8640036 TI - The relationship between the intrauterine environment and blood pressure in 3 year-old Japanese children. AB - In 195 Japanese children the systolic pressure at 3 years of age was inversely correlated to the body weight at birth and positively correlated to the mothers' systolic pressure during pregnancy: the average systolic pressure in children whose body weight at birth exceeded 3510 g was 3.0 mmHg, which was lower than that of children whose body weight at birth was 2990 g or less. There was an increase of 0.12 mmHg in the children's systolic pressure with each increment of 1 mmHg in the systolic pressure of their mothers. The systolic pressure at 3 years in children of mothers who had had pretibial oedema during pregnancy (101.0 +/- 8.8 mmHg) was significantly higher compared with children whose mothers did not have oedema (96.6 +/- 9.6 mmHg). PMID- 8640037 TI - Red blood cell indices and iron status according to feeding practices in infants and young children. AB - With the electronic counters routinely used, it has become practical to determine the concentration of hemoglobin, red cell indices, and RDW concurrently in association with transferrin saturation and ferritin in accordance with feeding practices. The 1028 infants and children aged 6 to 24 months, who had been mainly admitted with acute infectious or inflammatory diseases, were divided into three groups, i.e., children who were exclusively breast-fed more than 6 months (group A), those who had been given iron-fortified formula milk since birth (group B), and those who had been given breast milk for 5-6 months and then switched to the iron-fortified formula (group C). Children with anemia comprised 34.8% (104/299) of group A, significantly more than 5.6% (34/608) of group B and 6.6% (8/121) of group C (p < 0.001, respectively). Children with MCV < 70 fl comprised 39.5% (118/299) of group A, significantly more than 7.1% (43/608) of group B and 13.2% (16/121) of group C. Out of the total 146 patients with anemia, 82.2% (n = 120) had laboratory evidence of iron deficiency, which was mostly suggested by a dietary history. The sensitivity of MCV values < 70 fl in IDA patients was 90.0%; specificity was 53.8%. The sensitivity of RDW values > or = 15% was 83.3%; specificity was 57.7%. The positive predictive value could be increased to 97.8% by combining MCV < 70 fl and RDW > or = 15%. The sensitivity of serum ferritin concentrations < 10 ng/ml was 62.4% and specificity was 100%. The sensitivity of transferrin saturation < 12% was 72.3% and specificity was 81.3%. By combining the hemoglobin with MCV and RDW in screening for iron deficiency, the diagnostic accuracy of IDA can be increased. We support the use of appropriately iron fortified weaning foods or the routine iron supplement starting at 6 months of age in exclusively breast-fed infants. PMID- 8640038 TI - The A985 to G mutation of the medium-chain acyl-CoA dehydrogenase gene and sudden infant death syndrome in Normandy. AB - Medium-chain acyl-CoA dehydrogenase deficiency is the most common genetic defect of hepatic fatty acid oxidation. Clinical signs are somnolence and lethargy potentially leading to coma. Death occurs during the first attack in about 20% of cases, suggesting sudden infant death syndrome. A point mutation (adenine to guanine at position 985) in exon 11 of the medium-chain acyl-CoA dehydrogenase gene accounts for 90% of medium-chain acyl-CoA dehydrogenase deficiency-causing alleles. Such a high prevalence of a single mutation makes it possible to estimate the incidence of medium-chain acyl-CoA dehydrogenase deficiency in the general population and in sudden infant death syndrome. The study was performed by polymerase chain reaction amplification from blood spots on filter paper in 2000 randomly selected newborns (group I) and in 225 infants dead from sudden infant death syndrome (group II). Among 2000 newborns, 17 were found to be heterozygote for the G985 mutation. In group II, one child was found with a single copy of the G985 mutation. So, the estimated frequency of the G985 mutation in the general population was 1/118 and the incidence of medium-chain acyl-CoA dehydrogenase deficiency was calculated as around 1/45,000 in Normandy. PMID- 8640039 TI - Increased diarrhoeal and respiratory morbidity in association with zinc deficiency--a preliminary report. AB - Plasma zinc levels, in a cohort of 116 children aged 12-59 months recently recovered from an episode of acute diarrhoea attending a community health clinic in an urban slum, were related to diarrhoeal and respiratory morbidity in the 3 month period following recovery. Children with low plasma zinc levels (< 60 micrograms dl-1) spent a significantly greater number of days with watery diarrhoea (rate ratio 1.69 (95% confidence intervals 1.03-2.78)), diarrhoea associated with fever (rate ratio 1.88 (95% confidence intervals 1.05-3.34)), and acute lower respiratory tract infections (rate ratio 2.69 (95% confidence intervals 1.64-4.38)). PMID- 8640040 TI - A field trial of wheat-based oral rehydration solution among Afghan refugee children. AB - A total of 326 Afghan children aged between 6 months and 5 years with uncomplicated nondysenteric diarrhea for the previous 24 h to 5 days were treated at home by their mothers with either wheat-salt solution (WSS) or World Health Organization recommended glucose-oral rehydration salts (G-ORS). For 7 consecutive days the children were examined in the household and the mothers interviewed to assess the progress, feeding practices, and perception of treatment efficacy. Children treated with WSS recovered significantly earlier; the mean duration on treatment was 4.0 days (SD 1.7 days) on WSS compared to 6.4 days (SD 1.7 days) on G-ORS. By the second day of treatment, significantly more mothers using WSS (56%) reported that their children had formed stools versus 11% of their G-ORS counterparts; the mean stool frequency after 2 days was also significantly reduced; 3 stools day-1 (SD 2.1) on WSS versus 5 (SD 2.9) on G-ORS. The cereal-based solution was not confused with normal food and led to better feeding patterns. By day 2, 74% of the mothers using WSS had resumed their normal feeding frequencies as opposed to 33% of G-ORS mothers. On recovery the WSS group had gained significantly more weight; the WSS group gained 169 g (SD 142 g) while the G-ORS group lost 150 g (SD 174g). This study suggests by subjective and objective measures that WSS could be considered as an effective home fluid for the first-line treatment of diarrhea. PMID- 8640041 TI - Hand eczema in children with atopic dermatitis: a low prevalence? PMID- 8640042 TI - Interleukin-1 alpha and interleukin-1 receptor antagonist in the urine of children with acute pyelonephritis and relation to renal scarring. AB - Urinary concentrations of interleukin-1 alpha (IL-1 alpha) and interleukin-1 receptor antagonist (IL-lra) standardized to urinary creatinine concentrations were studied. The median standardized IL-1 alpha creatinine quotient in children with first-time acute pyelonephritis was 3.6 pg/mumol, but was nondetectable in children with recurrent pyelonephritis, children with non-renal febrile conditions and children convalescent after acute pyelonephritis (p < 0.05-0.01). IL-lra levels were also significantly higher in children with acute first-time pyelonephritis (median of 239 pg/mumol) compared to these three groups of children (p < 0.01-0.001). The highest urinary IL-lra levels, however, were found in the healthy controls (median value 1.019; p < 0.001). Both cytokines were higher among children younger than one year compared to older children. The acute IL-1 alpha creatinine quotients were lowest in children with uptake defects on 99mTC-dimercaptosuccinic acid (DMSA) scintigraphy both during the acute infection (reflecting the acute inflammation) (p < 0.001) and 1 year after the acute infection (reflecting permanent kidney scarring) (p < 0.001). In conclusion, persisting high urinary levels of IL-1 alpha were associated with less renal inflammation and scarring. PMID- 8640043 TI - MxA protein in infants and children with respiratory tract infection. AB - MxA protein--a stable product of cells stimulated by type I interferons--was examined prospectively for its ability to discriminate between viral and bacterial respiratory tract infections (RTIs) in 182 infants and children. The nasopharyngeal secretions (NPSs) of all of them were tested for MxA using enzyme linked immunosorbent assay (ELISA), and the whole blood of 92. Seventy-three children undergoing elective surgery served as controls. These apparently healthy children had higher levels of serum MxA than adult controls. Using antigen detection and serology, a viral aetiology was diagnosed in 81/182 cases. The sensitivity and specificity of MxA ELISA were assessed at 92 and 76% for the blood test and at 40 and 91% for the NPS, respectively. The positive predictive value for a viral RTI was superior to a leucocyte count or C-reactive protein when determined only once. PMID- 8640045 TI - Decreasing admissions for childhood asthma to a Swedish county hospital. AB - Hospital admission rates for childhood asthma have increased in many countries. To study if this is also true for Norrkoping Hospital, paediatric admission rates for asthma every fifth year were examined for the period 1973 to 1993. Admission rates were found to have fallen over the last 10 years, especially in children of school age. Among the younger age groups (below 5 years of age) a fall in admission rates was also observed over the last 5 years. This fall occurred in spite of reported increases in the prevalence of childhood asthma. The relative risk for admission due to asthma thus decreased from 1 in 1973 to 0.09 in 1993. The readmission rate has been stable. The mean length of stay in hospital for asthma decreased significantly. The observed decreasing trend in hospital admissions for childhood asthma is contrary to that found in many other countries. Possible explanations are discussed. PMID- 8640044 TI - Fungal pneumonia: the predominant lung infection causing death in children undergoing bone marrow transplantation. AB - The study included 6 children (aged 4-14 years) receiving a conditioning regimen for bone marrow transplantation (BMT) and 14 children (aged 2 14 years) with bone marrow transplants (13 allogeneic, 1 autologous). The children underwent flexible fibre-optic bronchoscopy (FFB) with bronchoalveolar lavage during 6 and 17 episodes of pneumonia, respectively. The aim was to compare the results of the two groups with respect to bronchoscopy findings, pneumonia-causing agents and outcome. During the conditioning regimen, the aetiological agents were recovered by bronchoscopy in 1/6 (17%) episodes and revealed by autopsy in another episode. In three episodes where the aetiology was uncertain, bacterial pneumonia was suspected in two, and Candida pneumonia in one. In episodes after transplantation the aetiological agents were recovered from bronchoscopy material in 14/17 (82%) patients. Autopsy confirmed the premortal diagnosis in the four children who died. In three episodes, bacterial pneumonia was clinically suspected. Based on clinical manifestations, FFB and autopsy findings, bacterial and fungal pneumonia were the most common diagnoses both during conditioning and after BMT. Fungal pneumonia was the most common cause of death in both groups. PMID- 8640046 TI - Influence of breathing pattern on transcutaneous oxygen and carbon dioxide tension during histamine provocation in children with bronchial asthma. AB - The influence of the breathing pattern on transcutaneous blood gases was evaluated in 18 boys and 8 girls 7-18 years of age, with bronchial asthma, during bronchial provocation with histamine-HCl. Transcutaneous oxygen tension (tcPO2), carbon dioxide tension (tcPCO2) and the breathing pattern assessed by the transthoracic impedance technique were continuously monitored during the provocation. At reaction, when the fall in the forced expiratory volume in 1 s (FEV1) was 20% or more, the tcPO2 fell by 15% or more below the baseline in 22/26 and by 20% or more in 14/26 children. In some children, a marked fall in the tcPO2 was already noted after the saline inhalation and the first histamine dose steps without simultaneous changes in the FEV1. This early fall in the tcPO2 correlated to changes in the breathing pattern and was interpreted as a sign of compensatory hypoventilation secondary to the hyperventilation observed during the inhalations. We conclude that transcutaneous oxygen tension can be used as an indicator of a bronchial reaction during bronchial provocation tests in children only if one takes account of the fact that the breathing pattern during the inhalation of the challenge compound per se has an effect on the oxygen tension. PMID- 8640047 TI - Nebulized budesonide for the treatment of moderate to severe asthma in infants and toddlers. AB - Maintenance treatment with nebulized budesonide was studied in young children with asthma not controlled without steroids. In a blind parallel-group study for 18 weeks, 102 children, mean age 22 (5- 47) months, were randomized for treatment starting with 0.25 or 1 mg b.i.d. The patients were reviewed every 3 weeks, and if symptom control had been achieved the dose was reduced, otherwise it was kept. The clinical effect was very good with both dose regimens. The median time to 7 consecutive days without any asthma symptoms was about 1 month with both, highlighting the importance of the duration of therapy rather than the benefits of a high starting dose. In 18 of 24 children who attained the placebo stage, symptoms had reappeared at the last visit. Although an overall minimal effective maintenance dose could not be demonstrated, 47% achieved symptom control on 0.25 mg b.i.d., i.e. fulfilled criteria for further dose reduction. No significant side effects were seen. On average, 25% of the nominal dose reached the patients. PMID- 8640048 TI - Asthma treatment in schoolchildren: lung function in different therapeutic groups. AB - Dynamic spirometry and peak expiratory flow were measured in 297 school-aged children with asthma during their control visit at the outpatient clinic in 1993. Sixty (20%) children had no maintenance drugs, 169 (57%) used cromoglycate (n = 97) or nedocromil (n = 72), and 68 (23%) budesonide. The treatment of each child had been selected on clinical grounds according to the principles of the international consensus statement from 1989. The mean values of peak expiratory flow (PEF), forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) were over 95% of the height-related reference values in all treatment groups. The lower limits of the 95% confidence intervals were at the level of more than 90% of those predicted. The mean FEV1/FVC ratio (FEV%) was over 85%, and the mean maximal mid-expiratory flow (MMEF) over 75% of the reference values. Decreased PEF values ( < 75%) were present in 10%, decreased FVC, FEV1, or FEV% ( < 75%) values in 4.6%, and decreased MMEF ( < 65%) values in 18%. Only minor differences between the different therapeutic groups were observed. Our results show that the clinical selection of children between the three therapeutical groups was adequate. In our area up to 70% of children requiring maintenance therapy for asthma can be treated with cromones. PMID- 8640049 TI - Diagnosis of the Prader-Willi syndrome by proving the absence of the unmethylated PW71 DNA fragment. AB - The Prader-Willi syndrome (PWS) is a genetic disorder which is difficult to diagnose from clinical symptoms in newborns and young children. However, it is known that in PWS a fragment within the q11-13 region of the paternally derived chromosome 15 is deleted. Recently it has been observed that the D15S63 (PW71) locus in chromosome 15q11-13 is methylated on the maternally derived chromosome, but unmethylated on the paternally derived chromosome. Based on this observation a rapid diagnostic test (the PW71 methylation test) using methylation-sensitive restriction enzymes has been developed for patients presumed to have PWS. We have studied 56 patients; 30 patients with classical features of PWS and 26 patients with only psychomotor retardation and obesity, referred to us from different part of Sweden. Twenty-nine of the 30 classical PWS patients were found to have an absence of the unmethylated paternally derived PW71(D15S63) locus in chromosome 15q11-13. None of the patients with only obesity and psychomotor retardation had this "absence" pattern on chromosome 15q11-13. Using the PW71 methylation test on patients with PWS, a concordance of 96% was found. The PW71 methylation test is presently the method of choice for rapid diagnostic testing of patients suspected of having PWS. PMID- 8640050 TI - Going Dutch in nocturnal enuresis. AB - Based on several intervention programmes, a strategy for the treatment of nocturnal enuresis has recently been developed by an expert committee in the Netherlands. It consists of three parts. First, two structured interviews are given: one to differentiate between enuresis and incontinence and one to detect associated problems such as diurnal enuresis, constipation or behavioural problems. Secondly, a medical examination is made, confined to the inspection of the external genitalia and lower back, palpation of the abdomen and urine examination. Thirdly, the following guidelines for treatment at different age levels are applied: up to the age of 6 years no intervention is needed; between the ages of 6 and 8 years, lifting out of bed and/or the calendar method; between the ages of 8 and 12 years, enuresis alarm (if not successful, medication with desmopressin is prescribed for a restricted period of time), and ambulatory dry bed training in a group setting may follow; over 13 years of age, clinical dry bed training according to the Messer/Azrin method is advised. According to the expert committee, these guidelines offer sufficient possibilities to deal with the problem of nocturnal enuresis. PMID- 8640051 TI - Amphetamine addiction during pregnancy: 14-year follow-up of growth and school performance. AB - Sixty-five children born to women who all abused amphetamine during pregnancy have been followed prospectively since their birth in 1976-77. At the age of 14 15 years, information about growth and school achievement was collected from school records. For comparison of school achievements the means of schoolmates were used, and for growth a group of Stockholm children born in the same year. By the age of 14 years only 14 children (22%) had stayed with their biological mothers for the whole period since birth. In the eighth grade, 10 (15%) were one grade lower than indicated by their biological age. The norm for Sweden is less than 5%. The means of the points in mathematics, Swedish language and sports were statistically below those of their classmates. At the age of 10 years the girls were significantly shorter and lighter than their peers born in 1976. At the age of 14 years the boys were statistically taller and heavier than their peers. It can be concluded that maternal amphetamine abuse during pregnancy will influence children at lest up to the age of 14-15 years even though many of them have been living in foster homes since a young age. PMID- 8640052 TI - Effects of information on smoking behaviour in families with preschool children. AB - An information programme on measures to prevent passive smoking by children, designed for use during well-child visits, was tested. A total of 443 consecutive families with one or two smoking parents, attending mother and child health centres in Oslo, Norway, were randomly allocated to an intervention group (n = 221) and a control group (n = 222). Eighty families (18%) dropped out during the study period. For the intervention group, the communication between the health visitor and the family was prolonged at one well-child visit with a brief session on smoking, and the parents were given three brochures. The families in the control group received no information on smoking. Changes in practical measures to prevent passive smoking by the children (e.g. no smoking indoors) as well as changes in daily smoking and smoking quantity were assessed by parental reports. We found no significant differences between the groups with respect to change in smoking behaviour. PMID- 8640053 TI - Maternal smoking and body composition of the newborn. AB - The influence on neonatal anthropometry of maternal cigarette smoking in pregnancy was investigated in 933 parous women. Anthropometric growth parameters including skinfold measurements were studied in the newborns. After adjustment for maternal age, pre-pregnancy weight, height and pregnancy weight gain, smoking had a clear dose-dependent negative effect on all anthropometric characteristics in the infant. In contrast to the results obtained in other investigations, the reduced birth weight of the infants of smoking mothers was not found to be primarily due to a reduction in lean body mass; nor was fat deposition found to be reduced. Fetal anthropometry was also negatively affected in infants born to mothers who stopped smoking during pregnancy. PMID- 8640054 TI - Influence of plasma preparations and donor red blood cells on the antioxidant capacity of blood from newborn babies: an in vitro study. AB - We investigated in an in vitro transfusion model the early effects of plasma preparations and donor red blood cells on the antioxidant capacity of the cord blood from babies. Addition of pasteurized plasma protein solution to plasma from babies decreased the peroxyl radical trapping capacity (p < 0.02). In contrast, fresh frozen plasma did not lower this capacity. Addition of adult donor red blood cells to the babies' red blood cells did not significantly decrease the glutathione-recycling capacity of the blood. On the basis of these in vitro results we hypothesize that the use of resuscitation fluids with low antioxidant capacity may temporarily decrease the ability of the baby to catabolize reactive oxygen species. PMID- 8640055 TI - Ultrasonographic evaluation of breech presentation as a risk factor for hip dysplasia. AB - In order to gain more information of breech position as a risk factor for congenital hip dysplasia or dislocation, the hips of 408 newborns delivered in the breech position were examined by ultrasound. Clinical examination was performed by both experienced paediatricians and orthopaedic surgeons. The infants were re-examined by ultrasound at 2-3 months of age. Twenty-five newborns (6.1%) had neonatal hip instability. Breech presentation as a risk factor was confirmed, with first borns, breech position with extended knees, and high birthweight as special high-risk groups. Ultrasound showed subluxation in most of the unstable hips. The main benefit of using ultrasound was that direct visualization permitted more reliable evaluation, especially when the clinical findings were uncertain. Normal ultrasound findings in false positive and uncertain Ortolani tests reduced the frequency of unnecessary treatment. Because ultrasound was used in follow-up, the need of radiography was reduced. There were no late-detected cases of hip dysplasia or dislocation, indicating that routine follow-up is not necessary in breech infants with normal hips at birth, provided that the neonatal screening is optimal. PMID- 8640056 TI - Early diet of preterm infants and bone mineralization at age five years. AB - Bone disease with significantly reduced bone mineralization is common in preterm infants, and associated with later linear growth stunting at 18 months of age. Dietary insufficiency of calcium and phosphorus is thought to be the principal aetiological factor. We studied 54 children at mean age 5 years who were born preterm and had participated in a prospective multicentre study of effects of early diet on later growth and development. Diets compared were banked donor breast milk and preterm formula fed as a supplement to mother's own milk. Increasing human milk intake was strongly positively associated with later bone mineral content. Children fed predominantly human milk had greater bone mineral content than children of similar size born at term. These data suggest that the early nutritional environment of the preterm infant could play an important role in determining later skeletal growth and mineralization. PMID- 8640057 TI - Focal intestinal perforation in preterm infants is an emerging disease. AB - In order to elicit the pathogenesis of focal intestinal perforation in preterm infants we contrasted 8 infants who developed this disease with 16 gestation matched controls. The cases were found to have lower birthweights for gestation (median standard deviation score of -1.02 in cases versus -0.08 in controls), and more frequently had pre-existing patent ductus arteriosus and intraventricular haemorrhage (88 and 63% in cases versus 25 and 6% in controls, respectively). There were similar rates of other perinatal variables in the two groups, including indomethacin and umbilical arterial catheter use. Conditions associated with fetal or neonatal hypoxia are important antecedents for this emerging distinct clinical entity. PMID- 8640058 TI - Small bowel biopsy in Swedish paediatric clinics. AB - The capsule technique for small bowel biopsy performed at Swedish paediatric clinics was evaluated using two questionnaires in 1990 and 1993, respectively. Replies were received from all 45 centres which together perform approximately 2300 biopsies per year. Clotting function tests prior to biopsy were carried out in 42% of the centres. The biopsies were performed under intubation anaesthesia in 13% of the centres. The most striking difference between the answers to the two questionnaires was the mode of sedation. The use of intravenous sedatives increased from 40% of the centres in the first questionnaire to 59% in the second one. The use of the oral, rectal and intramuscular routes decreased correspondingly. The most frequently used drugs for intravenous sedation were benzodiazepines, in the first questionnaire diazepam and in the second one midazolam. The failure rate was approximately 5%. In the first questionnaire, no complication was encountered. In the second questionnaire, three cases of intramural duodenal haematoma were reported, one of which led to pancreatitis. We conclude that by focusing on questions of sedation these rather simple questionnaires may have resulted in more effective sedation of children undergoing small bowel biopsy. PMID- 8640059 TI - Physiological anticoagulants and activated protein C resistance in childhood stroke. AB - Immunological and functional protein S, protein C and antithrombin III levels and anticoagulant responses to activated protein C were measured in 24 patients with stroke in childhood. No hereditary deficiencies were found. The protein S levels in healthy controls of younger age did not differ from the adult levels. For optimal screening of protein S deficiency, measurements using functional as well as immunological assays are recommended. Appropriate criteria for the diagnosis of the deficiencies must be carefully applied if unnecessary anxiety and inappropriate treatment of children are to be avoided. PMID- 8640060 TI - Aortic thrombosis in a neonate with hereditary antithrombin III deficiency: successful outcome with thrombolytic and replacement treatment. AB - We report the case of a newborn male who presented an aortic thrombosis during the neonatal period and was subsequently diagnosed as having antithrombin III (AT III) hereditary deficiency type I. This hypercoagulable condition is known to predispose young adults to venous thrombosis, but in our patient the primary thrombotic incident affected the arterial vessels within the first few days of life. Combined treatment with thrombolytic agents and AT III concentrates recovered aortic permeability, suggesting that the use of AT III may be beneficial for the treatment of thrombotic complications during the first few days of life. PMID- 8640061 TI - Increased risk of leukaemia in patients with juvenile chronic arthritis treated with chlorambucil. AB - Two cases of acute leukaemia have developed in a group of 77 patients treated with chlorambucil (Chl) because of severe juvenile chronic arthritis. The total follow-up from the beginning of Chl treatment in these patients was 560 years, indicating a highly increased risk of leukaemia. Despite favourable results, especially in patients with secondary amyloidosis, Chl should only be used in selected cases. PMID- 8640063 TI - Familial hypercholesterolaemia with severe cardiac involvement in a boy: successful management and mid-term follow-up. AB - A 13-year-old boy with double heterozygosity for familial hypercholesterolaemia with a 90% left coronary artery main stem stenosis is reported. The patient's cholesterol levels were effectively controlled with weekly sessions of selective low-density lipoprotein cholesterol removal through immunoadsorption by use of an extracorporeal system. Left main coronary artery stenosis was successfully treated with percutaneous transluminal balloon dilation. At 30 months after the intervention and still under treatment with weekly sessions of low-density lipoprotein apheresis the patient is free of cardiac symptoms. He shows normal exercise capacity and normal myocardial perfusion. It is concluded that aggressive management is justified in such patients and may result in a near normal quality of life. PMID- 8640062 TI - Treatment of children with congenital heart disease and growth retardation with recombinant human growth hormone. AB - Seven prepubertal short children with congenital heart disease were treated with recombinant human growth hormone (GH). Although complete surgical correction was performed for their heart disease at least 2 years before the start of GH therapy, improvement in growth was less than expected in these children. They received 0.5 IU kg-1 week-1 of GH daily for 2 years or more. The growth rate increased from a mean of 4.3 cm year-1 before treatment to a mean of 7.8 cm year 1 in the first year and to a mean of 6.3 cm year-1 in the second year of treatment. Their mean standardized height improved from -3.41 +/- 0.78 to -2.54 +/- 0.62 after 2 years. The mean height age difference minus the bone age difference became positive in these children. We conclude that recombinant GH increases the growth rate in children with congenital heart disease and prepubertal growth retardation. PMID- 8640064 TI - Xanthogranulomatous pyelonephritis--an unusual renal disease in childhood. AB - We report on a 6-year-old boy with Xanthogranulomatous pyelonephritis. This unusual type of renal inflammation has a typical clinical picture that is important to bear in mind. The affected child is often seriously ill with a palpable flank mass leading to initial suspicion of malignancy. Modern imaging procedures help in obtaining a pre-operative diagnosis. PMID- 8640065 TI - New treatment options for patients with melanoma: review of melanoma-derived T cell epitope-based peptide vaccines. AB - Human melanoma represents the principal cause of death in patients with skin cancer in the United States and Europe. Tumour infiltrating lymphocytes recognizing melanoma have been used to identify the tumour antigens recognized by T-cells in the context of MHC class I or class II molecules. Such antigens include MAGE-1, MAGE-3, MART-1/Melan-A, gp100, tyrosinase, the tyrosinase-related antigen gp75, the antigen gp15 and the mutated CDK4 and beta-catenin gene products. The identification of these T-cell epitopes provides us with novel reagents for the development of state-of-the-art treatments and for the (immuno )monitoring of patients with melanoma. In order for treatments, including peptide based vaccines, to be successful, several conceptual criteria must be met: (1) The patient's tumour must present the relevant epitope(s) integrated into the vaccine, (2) the tumour should express the appropriate restricting major histocompatibility complex (MHC) molecule(s) required for patient cytotoxic T lymphocyte (CTL) reactivity, and (3) the patient's T-cell repertoire should be able to react productively against the melanoma antigens present in the vaccine. Clinical trials implementing peptide-based vaccines or whole protein therapies have been initiated in the United States and Europe. We suggest that such treatments should include the careful monitoring of anti-tumour T-cell responses. This should include examination of melanoma antigen and MHC class I allele expression in the individual patient's tumour, assessment of the status of the peptide transporter molecules TAP1/TAP2 and evaluation of T-cell mediated immune responses reactive against peptides and autologous melanoma. Evaluation of clinical parameters (such as disease-free survival) in conjunction with an examination of immunological parameters may facilitate our understanding of the immune responses against T-cell antigens that are shared among melanoma and normal melanocytes, and may ultimately help to identify the most effective immunotherapy for patients with melanoma. PMID- 8640066 TI - c-KIT receptor expression in cutaneous malignant melanoma and benign melanotic naevi. AB - To investigate the role of c-KIT receptor in melanocytic tumour development and progression, we analysed the expression and localization of c-KIT by immunohistochemistry and Western blotting. In contrast to the positive staining shown by melanocytes and naevus cells in the epidermis of common naevi (n=20), all dysplastic naevi (n=13) were negative, as were dermal melanocytic cells of blue naevi (n = 4) and common naevi (n = 26). Three out of four superficial spreading melanomas lost c-KIT expression both in the epidermal and dermal parts, while nodular melanomas showed no expression of c-KIT except in partially positive cells, and six out of seven metastatic melanomas were negative. In acral lentiginous melanomas (n = 8), in contrast to other types of melanoma, all cases with melanoma cells growing basally in the epidermis showed strong c-KIT positivity, but melanoma cells growing at the upper layers of the epidermis and vertically into the dermis lost c-KIT expression. Using the Western blot method on cultured pigment cells, human epidermal melanocytes, junctional naevus cells and one out of three metastatic melanoma cell lines showed 125 and 145 kDa bands corresponding to c-KIT, whereas dermal naevus cells did not. These results suggest that dysplastic naevi are distinct from ordinary naevi in terms of c-KIT expression and that basally growing cells in acral lentigenous melanomas could be at an initial stage of tumour progression, before c-KIT loss occurs. PMID- 8640067 TI - Expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma. AB - In cutaneous melanoma, the standard CD44 molecule is abundantly expressed, whereas the expression of certain splice variants is related to tumour progression and to the metastatic potential of the cell line. In the present study we have investigated the expression of CD44 and the pattern of CD44 alternative splicing in uveal melanoma in relation to the cell type, diameter and invasiveness of the tumour. All uveal melanomas strongly stained with antibodies to the standard portion of CD44. No expression of the CD44 variant (v) exon CD44v7 was found, whereas v5, v6 and v10 were expressed (in 2/12, 5/12 and 8/12 cases, respectively). No correlation was observed between expression of particular splice variants and cell type, tumour diameter or invasion of the sclera or Bruch's membrane. All three uveal melanoma cell lines tested were strongly CD44 positive and expressed low levels of v6-containing isoforms at the cell surface, but v5, v7 and v10 were absent. Our results show that CD44 is strongly expressed in uveal melanoma and that the pattern of CD44 alternative splicing is similar to that observed in cutaneous melanoma. However, in uveal melanoma this alternative splicing does not appear to be related to prognostic parameters. PMID- 8640068 TI - Diagnostic impact of nuclear DNA content and proliferative activity in benign and malignant melanocytic lesions. AB - Nuclear DNA content was assessed, using image cytometry, in adult melanocytes in normal skin and in 20 intradermal naevi, 60 junction naevi, 107 compound naevi, 61 dysplastic naevi, 17 melanomas in situ and 101 primary malignant melanomas. Proliferation was estimated using mitotic counting and immunohistochemical staining by anti-Ki-67 (clone MIB1) monoclonal antibodies. All normal adult melanocytes; and intradermal naevi (97% junction naevi, 98% compound naevi, 66% dysplastic naevi) were diploid. Thirty-four percent of the dysplastic naevi, 88% of the melanomas in situ and 96% of the malignant melanomas were clearly aneuploid. Proliferation, as assessed by mitotic counting and MI81 index, was significantly higher in aneuploid invasive malignant melanomas than in aneuploid dysplastic naevi (P<0.0001). The results indicate that histomorphologically defined entities of melanocytic lesions are characterized by typical DNA distribution patterns. Distinct aneuploidy combined with high proliferation rates generally seem to be well correlated to an increased malignancy potential of the lesion. In dysplastic naevi, the clonic expansion of aneuploid naevus cells may be limited by host defence mechanisms. Thus, DNA aneuploidy seems to indicate increased risk of malignant transformation, but has to be combined with other data, such as proliferation, in order to differentiate more precisely between different melanocytic lesions. PMID- 8640070 TI - The case for therapeutic vaccines. PMID- 8640069 TI - Detection and quantitation of melanoma cells in the circulation of patients. AB - A method for the quantitation of tyrosinase mRNA in the bloodstream of melanoma patients has been developed by using competitive polymerase chain reaction (PCR) with a heterologous DNA (PCR MIMIC) as an internal standard. The method was validated by demonstration of similar amplification efficiencies for both molecules and by accurate quantitation of an artificial fourfold difference in the level of tyrosinase mRNA. The ratio of amplified target to amplified standard (At/As ratio) was determined for a range of melanoma patients who had previously tested positive for tyrosinase. Sequential samples were also analysed to examine the level of tyrosinase in individual patients over time. When tyrosinase levels in melanoma cell lines were compared for a constant amount of total RNA, or for a constant number of cells, tyrosinase mRNA was found to vary more than a thousand fold between cell lines. Because of this, the At/As ratio of patients was compared with the At/As ratios obtained when 10-fold serial dilutions of cells from a control melanoma cell line (MM200) were added to 2 ml of packed blood. An 'MM200 equivalence' value was thus calculated, giving the equivalent number of MM200 cells in the bloodstream of melanoma patients. Prolonged follow-up will be needed to determine the prognostic significance of the detection and levels of tyrosinase mRNA in the bloodstream of melanoma patients. PMID- 8640071 TI - A sensitivity and specificity analysis of the surface microscopy features of invasive melanoma. AB - In vivo cutaneous surface microscopy, epiluminescence microscopy, dermoscopy, dermatoscopy and magnified oil immersion diascopy, are terms that describe the use of an incident light magnification system to examine cutaneous lesions, usually with immersion oil at the skin-microscope interface. The result is the visualization of a multitude of morphological features, not visible with the naked eye, that enhance the clinical diagnosis of nearly all pigmented lesions. Sixty-two invasive melanomas and 159 randomly selected non-melanoma pigmented lesions were used in the study. The non-melanomas, while randomly selected from a large data base, were all clinically atypical. Using the x 10 magnification of hand-held surface microscopes (Dermatoscope, Episcope), we present an analysis of 72 surface microscopic variables (constituting over 15,000 single observations) for the diagnosis of invasive melanoma. Forty of the 72 features studied were shown to differ significantly between invasive melanoma and non-melanoma pigmented lesions. Blue-white veil, multiple brown dots, radial streaming and pseudopods had a specificity greater than 95% for melanoma. Two features, symmetrically irregular pigment (non-uniform pigmentation with point and axial symmetry) and the presence of a single colour, had a sensitivity of 0%, i.e. were absent, in melanoma. The other significant features are presented, with their sensitivity and specificity for melanoma. PMID- 8640073 TI - Malignant melanoma co-existing with sarcoidosis: implications for prognosis and management. PMID- 8640072 TI - Biological activity and clinical efficacy of intravenous high-dose thymopentin in metastatic melanoma patients. AB - Eight patients with cutaneous metastatic melanoma were submitted to high-dose intravenous thymopentin (TP5) treatment for 5 weeks: three patients received 1 g three times a week, three received 1 g daily and two received 2 g daily. Four out of eight patients presented a partial response of cutaneous lesions lasting for 1 7 months, and six remain alive with evidence of disease after a follow-up of 2-7 months. A remarkable histologic observation is the presence of tumour necrosis, which was seen as both single cells and large confluent areas. The majority of lymphoid cells present in the tumour are CD45RO+ and CD4+. The CD4+ cells might play an important role in the anti-tumour immune local response by secreting cytokines and inducing apoptotic and necrotic cell death. This hypothesis seems to be confirmed by the presence of a high number of CD4+ cells around intratumoral vessels, while the presence of endovascular micro-thrombosis provides indirect evidence of cytokine activity. Cellular lysis may be produced by the activity of both CD8+ and CD4+ lymphoid cells. The role of TP5 may be an activation of CD4+ and CD8+ lymphoid cells. Clinical and pathological data indicate that TP5 is able to produce consistent clinical and immunological effects in melanoma patients with cutaneous metastases. PMID- 8640074 TI - [Cytoplasmic ribosomes of Plasmodium and prophylaxis of P. falciparum malaria by various antibiotics]. PMID- 8640075 TI - [Sensitivity in vivo and in vitro of Plasmodium falciparum to chloroquine and amodiaquine in Bangangte (west Cameroon)]. AB - A cross-sectional malaria survey was conducted during the rainy season in Bangangte (West-Cameroon), a town of 90,000 inhabitants located in the high tablelands zone of Cameroon. An in vivo study revealed that 50% of P. falciparum malaria patients were resistant to chloroquine (25 mg/kg over 3 days) and 26.3% to amodiaquine (35 mg/kg over 3 days) at a RII/RIII level. In addition, plasmodial indices indicated that malaria was mesoendemic in this area of Cameroon. PMID- 8640076 TI - [Prognostic value of lactate determination in cerebral malaria in the Cameroonian child]. AB - In this study, we investigated the disturbances of glucose metabolism in 61 Cameroonian children aged 1 to 15 years old, infected with cerebral malaria and severe malaria cases admitted in hospitals in Yaounde. Parasitaemia in the two groups was respectively 8.10 +/- 0.35% in cerebral malaria group and 4.47 +/- 0.84% in those admitted for severe malaria. The majority of our patients (n = 42) had normal glycaemia, while 15 cases were hyperglycemic and only 4 subjects all belonging to the cerebral malaria group were hypoglycemic. No case of hyperinsulinaemia was recorded. The level of lactate was higher in 74% of patients (19 over 26) admitted for cerebral malaria virsus 37% patients (13 over 35) admitted for severe cases. We found a positive correlation between the level of parasite and that of lactate (P < 0.01 r = 0.47). Whereas no positive correlation was found in one hand between the level of parasite and glycaemia, the level of parasites and insulin, and in the other hand between glycaemia and the level of lactate, glycaemia and insulin. PMID- 8640078 TI - [Epidemiological study of Paragonimus sp. in south Cameroon]. AB - A study was carried out in 1993 in south Cameroon in order to assess the changes in the incidence rate of human paragonimiasis, and to get information on the transmission pattern of Paragonimus sp. Two human cases have been recorded, and one dog was found infected in the Ntem Valley. The results suggest that a decrease of the incidence rate of paragonimiasis occurred in humans and animals. Paragonimus metacercariae were found in three species of freshwater crabs: Sudanonautes africanus, S. aubryi et S. granulatus, the latter being a new host. Prevalence of infection in crabs increased southerly, and significant seasonal changes were found. Two cats were infested with metacercariae from S. africanus, and studies are in progress to identify the parasite(s) at a specific level. PMID- 8640077 TI - Peripheral nervous system involvement in human and experimental chronic American trypanosomiasis. AB - An electrophysiological and histological study of the muscle and the peripheral nervous system (PNS) was carried out in chronic human American trypanosomiasis (Chagas' disease) and in an experimental Chagas' disease (Chd) mouse model. Altogether 995 patients with chronic Chd and 261 mice, experimentally infected with RA and CA-I parasite strains, were investigated. Results were compared with matched controls. Techniques employed in humans were: clinical assessment, conventional electromyography (EMG), estimated number of motor units, motor and sensory nerve conduction velocities, repetitive nerve stimulation and muscle and sural nerve biopsies. In mice conventional EMG, sciatic nerve conduction time, sciatic nerve action potential amplitude, in vitro miniature end-plate potentials (MEPPs) and end-plate potentials (EPPs) recordings, muscle, nerve and spinal cord histology and identification of cell phenotypes within the inflammatory infiltrates were the employed procedures. Out of 511 patients submitted to clinical examination, 52 disclosed signs and symptoms of mixed peripheral neuropathy. By employing electrophysiological techniques, it could be shown that about 30% of the investigated patients had one or more of the following features: diminished interference pattern, most of the remainder motor unit potentials being (MUPs) polyphasic; reduced number of functional motor units in the thenar, hypothenar, soleus and/or edb muscles; slow sensory and motor nerve conduction velocities; low sensory action potential amplitude and impairement of neuromuscular transmission. In mice, MUPs duration and amplitude were increased at later stages of the infection, nerve conduction was slow, nerve action potentials were of low amplitude, mepps were of low amplitude and double epps were frequently found. Muscle histology in humans with chronic Chd showed type I and type II grouping, atrophic angular fibers and targetoid muscle fibers. In mice perivascular mononuclear cells infiltrates, small round fibers, muscle fibers necrosis, atrophic angular fibers, type II muscle fibers grouping and grouped muscle fibers atrophy were found. Sural nerve samples showed segmental and paranodal demyelination and axonal loss. The same features were observed in mice nerves, also in this model mononuclear cells infiltrates at the nerve, dorsal root ganglia and meninges surrounding the spinal cord were observed. Muscle and nervous tissues infiltrates were mainly composed of T lymphocytes with predominance of CD8 or CD4 subsets according to the parasites strain employed for infecting the animals. These findings suggest that the skeletal muscle and the PNS may be involved in chronic American trypanosomiasis. PMID- 8640079 TI - [Apropos of 2 cases of severe malaria contracted in the port of Marseille]. AB - Acute Plasmodium falciparum malaria occurred during summer 1993 in two inhabitants living close to Marseille harbour. History of blood transfusion and travel outside France were excluded as was also discarded airport malaria. Entomological investigations confirmed the absence of Anopheles breeding sites in the port area. An hypothesis is a vectorial transmission following introduction of one or several anopheles arrived on a ship coming from tropical Africa. During this season, the weather conditions were favourable to the survival of anopheles and the completion of P. falciparum sporogonic cycle. Physicians were advised to take into consideration malaria in the differential diagnosis of fever from unknown origin in any patient working or living inside or around the harbour area regardless history of previous travel in malaria endemic region. PMID- 8640080 TI - [Prevalence of pulmonary tuberculosis and HIV infection in the district of Dire in Mali]. AB - In 1990, a study was made in the district of Dire, North Mali in West Africa, to determine the prevalence of pulmonary tuberculosis and HIV infections in general population. From 1,814 subjects tested, 1.1% (n = 20) have active pulmonary tuberculosis and 0.2% (n = 4) were seropositive with HIV1 or HIV1 + 2. No relation was made between the two diseases. PMID- 8640081 TI - [Current point of leishmaniasis epidemiology in Algeria]. AB - The authors describe in this study the current situation of cutaneous and visceral leishmaniasis in Algeria. After a review of the clinical forms and the epidemiological profile of the diseases in this country, the authors mention a resurgence of the number of cases, and the appearance of numerous new foci for both forms of the disease. PMID- 8640082 TI - [Presence of Borrelia burgdorferi sensu lato in Ixodes (Trichotoixodes) pari Leach, 1815 (Acari:Ixodidae), tick strictly specific for birds]. PMID- 8640083 TI - [Seasonal variations of female aggressiveness and population dynamics of Culex quinquefasciatus Say 1823 in Abidjan (Republic of Cote d'Ivoire)]. AB - Seasonal variations of Culex quinquefasciatus aggressiveness were studied in Abidjan where this mosquito is very harmful. Abidjan is crossed from East to West by the Ebrie lagoon, which is a vast expanse of brackish water (about 566 km2). In the North of this expanse of water lay continental plateaux and in the South lay low-plateaux. The city is composed of 10 districts, most of which are situated on the continental plateaux. Culex quinquefasciatus adult populations were caught monthly in 1989 on human bait traps in Abidjan 10 districts, one study place was located in each district. Captures took place outside the houses, in the dusk and during the first part of night, that is to say between 4 p.m. and 1 a.m. In Abidjan, Culex quinquefasciatus population density varies considerably within the city area. In the areas where the species is numerous, a high percentage of it seems to be developing all year through from breeding places which are, in most cases, permanent. Besides, the aggressiveness seasonal cycle which was registered in the above mentioned areas is particularly diversified. PMID- 8640084 TI - [Seroprevalence of Chlamydia trachomatis infection in STD consultants in Morocco]. AB - We have conducted a seroepidemiological survey of Chlamydia trachomatis infection among 400 STD consultants in comparison with 400 blood donors. The study was performed by using the indirect microimmunofluorescence technique with Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae as antigens. The overall seroprevalences were 60% and 46% for STD consultants and blood donors respectively. The seroprevalences of Chlamydia trachomatis alone were 12.5% for STD consultants and 7.5% for blood donors. No differences were observed according to age in the two groups and people of 20-29 and 30-39 years old, of both sexes were the most concerned. We conclude that Chlamydia trachomatis infection remains an important problem in Morocco. PMID- 8640085 TI - [Etiologic aspects and therapeutic problems of purulent pleurisy in Abidjan (Cote d'Ivoire)]. AB - We present the results of a retrospective study of 127 cases of empyema admitted to the pneumophtisiology department of the Centre hospitalier universitaire de Treichville (Abidjan), between January 1985 and December 1989. We present the pathogens identified in the pleural fluid and the course of the disease during treatment by repeat thoraco-centesis and systemic antibiotics. During the study period, pleural empyema represented 2.7% of all admissions to the pneumophtisiology department, and 20.5% of those presenting with pleural effusions. Bacteriological examination was recovered in 88 of the 127 patients, and was positive in 57 cases (64.7% of those examined). Of those with positive bacteriology, 50 (56.8%) had non-tuberculous bacterial infections, and 7 (7.9%) had tuberculous infection. Among the non-tuberculous bacterial infections, Gram negative bacilli were most common (72%), and Pseudomonas was the species most frequently identified (48%). The mean stay in hospital was 47 days (range 10-143) and in 82 patients (64.6%), the outcome was favourable. The presentation was complicated by encystment in 36 cases (28.4%) and 9 patients (7%) died in hospital. PMID- 8640086 TI - [Bacteriological aspects of cholera in Chad (1991 and 1994 epidemics)]. PMID- 8640087 TI - [Current situation of meningitis, infectious encephalitis and intracranial suppurations in African tropical zone]. PMID- 8640088 TI - [Dedication of the "Medical Inspector Alphonse Laveran" year, 1994]. PMID- 8640089 TI - Detection and sequence analysis of a new herpesvirus-like agent in AIDS and non AIDS Kaposi's sarcoma in Taiwan. AB - A new herpesvirus-like DNA sequence (KSHV) has been recently identified in Kaposi's sarcoma from acquired immunodeficiency syndrome (AIDS) and non-AIDS patients. In order to verify the role of this new viral agent in Taiwan, a series of Kaposi's sarcoma specimens obtained from AIDS and non-AIDS patients in three medical centers representing different geographic locations of Taiwan were surveyed. A total of seven specimens from AIDS patients and 22 specimens from non AIDS patients were tested for the presence of this herpesvirus-like agent by polymerase chain reaction (PCR) using the published Kaposi's sarcoma 330-233 primers. The tumor DNA was also tested for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpes-virus type 6 (HHV-6). The Kaposi's sarcoma specimens from AIDS and non-AIDS patients were positive for KSHV except in one case. Only occasional specimens were positive for CMV and EBV, with a slightly higher positive rate for HHV-6 in AIDS and non-AIDS patients. Cloning and sequence analysis of the PCR products from two cases of Kaposi's sarcoma (one each for AIDS and non-AIDS) revealed only minor differences in this DNA sequence, as compared with the published data from north America. These results further support the assumption that this new KSHV is strongly associated with Kaposi's sarcoma in AIDS and non-AIDS patients in Taiwan. PMID- 8640090 TI - Nosocomial candidemia in a university hospital in Taiwan. AB - The incidence of nosocomial candidemia increased 27-fold over the past 13 years at National Taiwan University Hospital. In order to investigate its predisposing factors, clinical manifestations and prognostic determinants, a prospective observational study of nosocomial candidemia was undertaken at the hospital. From 1 May 1994 to 30 April 1995, 118 consecutive adult patients with 120 Candida spp blood isolates were analyzed. Clinical presentations included fever (100%) with a median duration of 3 days, diarrhea within 7 days of candidemia (27.9%) and macronodular skin emboli (7.6%). Laboratory studies showed worsening azotemia (35.6%), elevation of aminotransferase (28.3%), leukocytosis (27.1%) and thrombocytopenia (23.3%). Use of multiple antibiotics, retained vascular catheters and parenteral nutritional support were the three most common predisposing factors for candidemia. C. albicans was the most common isolate (50%), followed by C. tropicalis (20%), C. glabrata (14%), C. parapsilosis (9.2%). C. guilliermondii (2.5%), and C. Krusei (1.7%). C. tropicalis was more frequently the cause of candidemia in patients with leukemia and neutropenia, while C. glabrata was more commonly seen in patients receiving fluconazole prophylaxis. A severity scoring system adopted from prospective bacteremia studies proved to be highly predictive of mortality in candidemic patients. The overall case fatality rate was 70/118 (59.3%), and 51/70 (72.9%) were attributable to candidemia. In a multivariate analysis, the prognostic factors adversely influencing outcome were: higher severity scores, no removal of the catheter, persistent candidemia and lack of antifungal therapy. PMID- 8640091 TI - Antimicrobial resistance and serotype distribution of Streptococcus pneumoniae strains isolated in southern Taiwan. AB - The antimicrobial susceptibilities and serotypes of 115 Streptococcus pneumonia strains isolated in southern Taiwan from January 1990 to December 1993 were determined. All isolates were susceptible to cephalothin, cefotaxime, trimethoprim/sulfamethoxazole and vancomycin, and 14 of the isolates were resistant to penicillin G. The oxacillin disk method for presumptive detection of resistance to penicillin had a sensitivity of 85.7% and specificity of 97.0%. Resistance rates were as follows: erythromycin 62.2%, tetracycline 71.3%, clindamycin 46.1% and chloramphenicol 19.1%. Eighty-four percent of the isolates were resistant to one or more of the antibiotic tested. Multiple resistance (to three or more classes of antibiotics) was identified in 40.9% of all the isolates and 100% of penicillin-resistant isolates. The predominant serotypes were: 14 (19.1%), 3 (17.4%), 23 (15.7%), 6 (10.4%), and 15 (6.1%). Serotypes 14 and 63 most commonly caused childhood infections, while serotypes 3 and 23 were frequently encountered in adults. The proportion of coverage in 23-valent pneumococcal polysaccharide vaccine was 92.2%, if vaccine-related serotypes were considered to be cross-protecting. Seven (58%) of 12 typable penicillin-resistant isolates belonged to serotype 23 and two (16.7%) to serotype 6. All isolates of serotype 14, 23, 15 and 19 were resistant to one or more antibiotics. Multiple drug resistance was frequently associated with serotype 23 (31.9%), 14 (23.4%) and 6 (17.0%). Sixty-five percent of isolates of serotype 3 were susceptible to all antibiotics tested. The high level of antimicrobial resistance in S. pneumoniae mandates the continuous surveillance of resistance and the strict control of antibiotic use in Taiwan. PMID- 8640092 TI - Seroepidemiology of measles in southern Taiwan: two years after implementation of the measles elimination program. AB - The seroprevalence of the measles antibody in southern Taiwan 2 years after the launch of the two-dose measles elimination program was studied using serological surveillance. Sera from 868 healthy inhabitants were collected from January to December 1993. Measles IgG antibody was detected by enzyme-linked immunosorbent assay. Measles antibody was found in 82% of infants under 3 months of age, 26% of those between 4 and 9 months and 54% of those between 10 and 15 months of age. The most common reason for postponement of vaccination was due to concomitant illness during the scheduled vaccination period. The presence of measles antibody rose sharply in children over 15 months of age, reached 90% among preschool-aged children and was 93% in school-aged children. These data suggest that a high immunization coverage rate was achieved in children over 15 months of age (92%) and that a third measles immunization at age 12 is not necessary. Future strategies for measles control should aim at increasing the immunization coverage for children between 9 and 15 months of age. PMID- 8640094 TI - Home parenteral nutrition in children. AB - From 1985 to 1994, home parenteral nutrition (HPN) was used as the method of feeding nine pediatric patients. Indications for HPN included congenital or acquired short bowel syndrome. Crohn's disease, chronic intractable diarrhea, chronic idiopathic intestinal pseudo-obstruction and Hirschsprung's disease. During the period, two patients died; one of sepsis and the other from hepatic failure. Three of the remaining patients have since moved on to oral feeding, but four patients continued on HPN. The majority of these patients have attained a normal weight and height for age while receiving HPN. All patients were fed via an implanted silicone catheter. Catheters were removed and replaced due to complications including nine episodes of infection, four episodes of occlusion, three episodes of breakage and two episodes of dislodgement. Catheter-related sepsis was the most common cause of morbidity and hospital readmission in patients receiving HPN, but was acceptably rare. Staphylococcus aureus was the most frequently cultured organism from either the catheter tip or the blood. HPN is a relatively safe feeding method for patients who would otherwise remain hospitalized for prolonged periods on parenteral nutrition for permanent or prolonged intestinal failure. PMID- 8640093 TI - Hyperglycemia induced activation of type-1 protein phosphatase activator (kinase FA) in perfused human placenta. AB - We report the identification of type-1 protein phosphatase activating factor (kinase FA), a unique biologic mediator for both insulin and epidermal growth factors in the human placenta. The activity of kinase F, was found to be extremely labile in the unperfused placenta. Fresh term placentas lost more than 50% of the total kinase FA activity within 6 hours when exposed to air of incubated in medium but not perfused. In contrast, the activity of kinase FA was stable when the human term placenta was dually perfused. This indicates that placental dual perfusion is a useful method for studying protein phosphorylation dephosphorylation involved in signal transduction. When fresh placentas were perfused with media containing glucose at 141 +/- 10, 242 +/- 12 and 436 +/- 20 mg/dL, kinase FA activity was stimulated several-fold in a glucose concentration dependent manner when compared with control levels at delivery. The results suggest that hyperglycemia-mediated activation may represent a previously unknown control mechanisms for the regulation of protein kinase FA. The results also suggest that human placental perfusion is a good in vitro system for studying signal transduction mechanisms involved in hormonal actions and metabolic regulation. PMID- 8640095 TI - Microbial threats in Taiwan. PMID- 8640096 TI - Transfusion-acquired AIDS in Taiwan. AB - Human immunodeficiency virus type 1 (HIV-1) can be transmitted through blood transfusion. The first transfusion-acquired immunodeficiency syndrome (AIDS) patient in Taiwan was a 46-year-old woman who received two units of whole blood during a hysterectomy at a provincial hospital in 1985. In 1991, she experienced a herpes zoster infection. In March 1993, she had extensive herpetic gingivostomatitis and another herpes zoster attack, and was treated at the same hospital. Two months later, she had oral candidiasis and was treated at a medical center. She was not tested for HIV-1 infection until she developed Pneumocystis carinii pneumonia in June 1993. In February 1994, and developed cytomegalovirus retinitis and died 6 months later. Donor blood given to the patients during the hysterectomy was HIV-1 positive. The donor's HIV infection was discovered in 1991 and he died of AIDS in 1993. As blood centers in Taiwan did not start screening for HIV-1 until January 1988, it is urgently recommended that any individual who received a blood transfusion between 1984 and 1987 in Taiwan and who currently experiences repeated episodes of opportunistic infections have an HIV-1 blood test. The receipt of a blood transfusion between 1984 and 1987 should be listed by the Department of Health as an indication for HIV-1 screening. PMID- 8640097 TI - Endogenous Escherichia coli endophthalmitis. AB - Metastatic bacterial endophthalmitis caused by Klebsiella pneumoniae is a unique but well-known phenomenon in Taiwan, where most cases occur in diabetic patients with pyogenic liver abscesses. However, endogenous Escherichia coli endophthalmitis is quite rare. The most common primary site of this infection is the urinary tract and the incidence of concurrent systemic infections, such as abscess formation and infective endocarditis, is high. This illness has a rapidly progressive clinical course associated with a poor prognosis for recovery of visual acuity. This is a report of a case of endogenous E. coli endophthalmitis originating from a renal abscess in a diabetic woman. Despite aggressive local and systemic treatment, evisceration was required to prevent the infection from spreading. PMID- 8640098 TI - Viral hepatitis in East Asia. AB - Viral hepatitis has a high prevalence in East Asia and is an important problem. Identification of the individual hepatitis viruses. A-E, has enabled researches to investigate the epidemiology and pathogenesis of viral hepatitis and its sequelae, and possible means of prevention. Because of improvement in hygiene in East Asia in recent decades, hepatitis A virus infection has decreased markedly. However, this has resulted in the younger population being susceptible to hepatitis A. Fortunately, effective active immunization for hepatitis A has become available. Hepatitis B is still rampant, especially in the southern part of East Asia where chronic infection is common. Patients who are chronic hepatitis B virus carriers are reservoirs for the virus and have a much higher risk of chronic liver disease and hepatocellular carcinoma (HGC). Currently, hepatitis B infection is being brought under control in East Asia through mass immunization. Serologic and molecular epidemiologic studies have also revealed that Hepatitis C is prevalent in the region. Hepatitis C virus also contributes to the development of cirrhosis and HCC. No effect immunization is currently available, and hepatitis C can only be controlled by preventative measures. The epidemiology and pathogenesis of viral hepatitis is discussed in this review, including new viral hepatitis agents possibly responsible for non-A-E hepatitis. PMID- 8640099 TI - Epidermolysis bullosa letalis with pyloric atresia in an infant. AB - Epidermolysis bullosa (EB) is a group of inherited diseases, that are characterized by vesiculobullous lesions that arise in response to minimal trauma or friction. The three major groups of EB differ according to the ultrastructural level of cleavage namely: simplex (epidermolytic), junctional and dystrophic (dermolytic). The combination of EB and pyloric atresia in rare and there is a definite association between them. We report a baby boy who died epidermolysis bullosa complications despite successful surgical correction of this pyloric atresia. PMID- 8640100 TI - Gastric actinomycosis. AB - A case of actinomycosis of the stomach in a 61-year-old woman is reported. The patient presented to the hospital with a history of epigastric pain, fatigue, poor appetite, constipation and mild fever of 20 days' duration. On physical examination, a tender mass was felt at the epigastrium. Computed tomography (CT) showed a heterogeneously enhanced mass at the posterior wall of the stomach. Upper gastrointestinal series revealed a submucosal mass at the gastric antrum. A malignant tumor was suspected and surgery was recommended. A 5 x 4 x 2 cm mass was found at the posterior wall of the distal gastric antrum. A Billroth II subtotal gastrectomy was performed. Pathologic examination revealed suppuration and sulfur granules in the indurated mass. The patient was subsequently treated with intravenous penicillin-V for 12 days and then with oral penicillin-G for 4 months. She remained well at her last follow-up appointment. Although the initial radiologic findings were nonspecific, CT was of importance in delineating the location and evaluating the extent of the lesion. PMID- 8640102 TI - Scalene lymph node metastases with the negative pelvic nodes in invasive cervical carcinoma. AB - Cervical carcinoma spreads predominantly by lymphatic routes and lymph node metastases may occur even in early stages of disease. Metastases usually first appear in pelvic lymph nodes, then disseminate along the efferent lymphatic chain to the extrapelvic lymph nodes. Cases of positive lymph node metastases with negative pelvic nodes in invasive cervical carcinoma are extremely rare. We report a 50-year-old woman with bulky stage IIA cervical carcinoma who had scalene lymph node metastases in the absence of pelvic lymph node metastases after radical hysterectomy and postoperative pelvic irradiation. This rare "skipping" nodal metastasis was probably via posterior trunk lymphatic drainage of the bulky cervical carcinoma which mainly invaded the posterior vaginal cuff. Neoadjuvant or adjuvant chemotherapy is recommended to prolong survival of patients in such cases. PMID- 8640101 TI - Prevention of false results from preferential PCR amplification or VNTR alleles. AB - In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework. PMID- 8640103 TI - Identification of malnutrition-related diabetes mellitus among hospitalized patients in Taiwan. AB - A retrospective review of 1,089 diabetes mellitus admitted to the metabolic ward of Chang Gung Memorial Hospital was performed to screen for malnutrition-related diabetes mellitus (MRDM). None of the patients fulfilled the MRDM criteria, although 20 patients with a body mass index < or = 15 kg/m2 were further analyzed. These patients presented with poor glycemic control and most had associated chronic diseases. C-peptide levels were < 0.8 ng/mL in 17 patients and undetectable in six patients. Two patients experienced diabetic ketoacidosis (DKA). Insulin treatment was required by 18 patients with a mean (+/- SD) dosage of 30 +/- 9 U/day. During follow-up of 14 of the patients for 9.8 +/- 4.3 months, HbA1c decreased significantly. Their mean body weight and body mass index increased significantly from 36.6 +/- 4.6 kg to 47.2 +/- 6.2 kg and 13.9 +/- 0.9 to 18.0 +/- 1.9 kg/m2, respectively. Careful attention should be paid to diabetic control and associated chronic illness in patients with severe undernutrition. PMID- 8640104 TI - Postoperative ambulation of patients with intraspinal neurilemomas and meningiomas. AB - Factors affecting the postoperative ambulatory state of patients with intraspinal neurilemomas and meningiomas were evaluated in 92 patients who underwent surgery at the Chang Gung Memoriam Hospital. The patients' records were reviewed retrospectively and leg power grading was noted (Medical Research Council of Great Britain grading system, zero to five). Of the 89 patients with a preoperative leg power of 1 or better, 87 could walk with or without aids shortly after surgery. The remaining three patients, with a preoperative leg power of zero, were all wheelchair-bound postoperatively. The presence of sensory deficits and sphincter incontinence did not correlates with a poor postoperative ambulatory state, provided the preoperative leg power was above zero. The average period between the onset of the earliest symptoms and the establishment of the diagnosis was 68 weeks for patients with intraspinal neurilemomas and 71 weeks for those with meningiomas. Surgical for patients with neurilemomas were as good as those with meningiomas. Patients with multiple spinal neurilemomas did not fare any worse after surgery. PMID- 8640105 TI - Characterization of the lactose transport system in the strain Bifidobacterium bifidum DSM 20082. AB - Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose 14C. Km and V(max) values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the beta-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-beta-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na+, Li+, or K+ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the stain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system. PMID- 8640106 TI - Effect of extracellular hydrogen on organic acid utilization by the ruminal bacterium Selenomonas ruminantium. AB - The objective of this study was to evaluate the effect of extracellular H2 on organic acid utilization by two lactate-utilizing strains of Selenomonas ruminantium (HD4, H18). Both strains were able to grow (optical density at 600 nm > or = after 9 h) on either aspartate, fumarate, or malate in the presence of 1 atmosphere (atm) of H2. Succinate was the major end product produced in these fermentations. When cells were incubated with lactate plus 1 atm H2, growth was minimal little lactate was fermented. The electron transport inhibitor, acriflavine, was strong inhibitor of growth when either strain was incubated in the presence of organic acid plus H2. Compared with glucose- or lactate-grown cells, cellular carbohydrate levels were lower for both strains in cells grown on either organic acid plus H2. These results suggest that electron transport plays a role in organic acid utilization by S. ruminantium. PMID- 8640107 TI - Recombination-induced variants of Clostridium acetobutylicum ATCC 824 with increased solvent production. AB - Three sporulation-specific genes (orfA, sigE, sigG) from clostridium acetobutylicum ATCC 824 are arranged in a cluster, encoding the putative sigma E processing enzyme, sigma E, and sigma sigma G respectively. When they were transformed into Clostridium acetobutylicum while on a plasmid functional in this organism, transformants did not survive. Three kinds of recombinations were then attempted with nonreplicative plasmids: duplication of orfA and sigE, replacement of all of the three genes, and inactivation of orfA. While the wild-type strain ceased to grow and produce solvents in batch cultures after approximately 24 h, mutant strains were isolated that showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol in test tube cultures. In addition, one of the derived strains showed a significantly higher growth rate. Features of the restriction maps of the recombinants did not correlate with expected maps, indicating possible complications occurring during the recombination events. PMID- 8640108 TI - [A cytogenetic study of hematopoietic cells in fish from the reservoirs of the Noril'sk-Pyasina water system (Taimyr)]. AB - The levels of chromosome mutations in hemopoietic cells of various fish from water bodies near the Noril'sk mining and smelting complex were estimated by the micronucleus test. In some cases very high frequencies of erythrocytes with micronuclei were observed in fish inhabiting highly polluted water bodies. On the whole, the number of cells with chromosome aberrations was within the limits of spontaneous mutagenesis. Marked species-specific, seasonal and local differences were found in the frequency of erythrocytes with micronuclei. The problems related to the influence of chemical pollutants on fish are discussed. PMID- 8640109 TI - [Alpha-fetoprotein suppresses keratinocyte proliferation in vitro]. AB - The influence of alpha-fetoprotein (AFP) on the proliferation of human epidermal keratinocytes in vitro has been studied. AFP (1p microgram/ ml) inhibits the proliferation of keratinocytes cultivated in a serum-free medium. The addition of fetal bovine serum significantly enhances the effect of AFP in this culture. A similar effect of AFP is observed in a medium with a low content of calcium ions. PMID- 8640110 TI - [The genetic links and species affiliation of the field mouse (Rodentia, Muridae, Sylvaemus) from the Pamir-Alai]. AB - Analysis of variation in 30 presumptive biochemical loci in Sylvaemus sylvaticus pallipes from Tadjikistan was carried out with reference to the geographically closely related species S. uralensis. The data obtained support the genetic identity of pallipes, on the one hand, and suggest a rather low level of genetic divergence (D'Nei = 0.154, Pfd = 13) from an ancestral taxon, on the other hand. Analysis of genetic variation of diagnostic morphological features suggests that pallipes is the most extreme form of geographic population series of the uralensis species group. Both genetic and morphometric data suggest that S. pallipes is an allospecies of the uralensis species complex. PMID- 8640111 TI - [The characteristics of the distribution of the great gerbil (Rhombomys opimus) on the territory of China]. AB - The distribution of the great gerbil (Rh. opimus) in China was analyzed on the basis of published data, museum collections, natural maps, and authors' field observations. A preliminary scheme was plotted for range regionalization, in which three parts have been isolated or, according to classification of Dubrovskii and Kucheruk (1971), three regional complexes of autonomous groups of populations: Dzungarian, Beishanian and Alashanian. West of the main area of the great gerbil distribution, an isolated colony is located in the Ili River valley, connected with the Kazakhstan portion of the range (Ili regional complex of autonomous groups of populations). Additional field observations are required for more detailed description of the great gerbil range. PMID- 8640113 TI - [The morphofunctional characteristics of the harderian gland in some mammalian species]. AB - The structure of harderian glands was studied using electron microscopy and histochemistry in six rodent species, including three Phodopus species. Based on the concept of cell morphotype it was shown that the harderian glands of the studied mammalian species are lipid-secreting and have secretory cells of the species specific morphotypes. The secretory cells are distinguished by the ultrastructural features not only between different genera, but also between different species of the same genus. The data obtained allow species identification according to the ultrastructure of secretory cells. The role of harderian glands in chemical communication was shown for the golden hamster (Mesocricetus auratus). Their secretory substance contains information about the sexual and species identity of animals. PMID- 8640112 TI - [The mutagenic and modifying properties of emoxipin studied by micronucleus assay in liver cells]. AB - Emoxypin is a medicinal drug from the group of 3-oxypyridines. We studied the capacity of emoxypin to affect the spontaneous level of micronuclear aberrations in the hepatocytes (to decrease or increase it by exerting a mutagenic effect) using the micronucleus test, as well as the capacity to modulate (enhance or weaken) the effects of nitrosomethylurea and X-irradiation. The results obtained do not suggest cytogenetic activity of emoxypin. The nature of "spontaneous" micronuclear aberrations in the liver are discussed, as well as the causes of their age-related increase and adequacy of this model to search for antimutagens. PMID- 8640114 TI - [Changes in the opsonizing capacity of the blood serum in rats after irradiation and after a combined radiation-thermal lesion]. AB - We studied changes in the blood serum opsonic capacity after irradiation, thermal burn, or combined radiation-thermal injuries. It was shown using chemiluminescence of macrophages, that phagocytized zymosan opsonized by the serum of control or experimental animals, and inhibition of complement-dependent reactions of opsonization was observed only after combined radiation-thermal injuries and was rather short-term. Incubation of normal macrophages with a serum obtained after the studied influences decreased the chemiluminescent response of macrophages to latex absorption. The most pronounced decrease in the intensity of luminescence was observed in the presence of the serum of rats subjected to combined radiation-thermal injuries and was due, within 24 h, to the burn component and, within 3 and 7 days, to the summed burn and radiation components of combined radiation-thermal injuries. We propose that the decreased opsonic capacity of the serum is due to the appearance of inhibitors of phagocytosis in the blood of animals after combined radiation-thermal injuries, rather than to deficiency of opsonizing factors. PMID- 8640115 TI - [Treatment of permeabilized cells by Bal31 nuclease leads to excision of DNA loops from the genome]. PMID- 8640116 TI - [Dependence of the binding of (3H)-dopamine by synaptosomes on the physico chemical state of the membranes under hyperbaric conditions]. PMID- 8640117 TI - [Functional transformation of cytotoxic T-lymphocytes into T-suppressors under the effect of major histocompatibility class I antigens and thymus hormone]. PMID- 8640118 TI - [Undeveloped territories in the center of Moscow as a habitat for mouse-like rodents]. PMID- 8640119 TI - [Dynamics of sizes of daily home ranges of female bank voles during the breeding period]. PMID- 8640120 TI - [Effect of dexamethasone on transplantation regeneration processes of rat skeletal muscles]. PMID- 8640122 TI - [Participation of the hypothalamo-hypophyseal part of the neuroendocrine system in development of immune response in rat fetuses]. PMID- 8640121 TI - [Effect of calcium ions on cell death by apoptosis, induced by cytotoxic proteins from LAK cells]. PMID- 8640123 TI - [Ultrastructure of the motor ends of the beta-axon on intra- and extrafusal muscle fibers in rat quadriceps muscle]. PMID- 8640124 TI - [Cytogenetic study of residents three years and more after the accident at the Chernobyl Atomic Power Plant]. PMID- 8640125 TI - [The human adenosine deaminase gene contains a p53-responsive element]. PMID- 8640126 TI - ["External" correction of DNA replication errors in rat cells]. PMID- 8640127 TI - [Effect of para-aminobenzoic acid on the fibrinolytic activity of blood]. PMID- 8640128 TI - [Physiological effects of dermorphin and its analog (dAlA4)-dermorphin]. PMID- 8640129 TI - [Inter-hemisphere asymmetry of cerebral lipid peroxidation in rats with various types of behavior as a prognostic indicator of their resistance to cerebral ischemia and effectiveness of the anti-ischemic action of substance P]. PMID- 8640130 TI - [Effect of a specific promotor sequence on nucleosome phasing]. PMID- 8640131 TI - [Mathematical modeling of infectious diseases]. PMID- 8640133 TI - [Population-based study of human platelet mitochondrial monoamineoxidase and an epigenetic model of its regulation]. PMID- 8640132 TI - [Mutant human gamma-interferon with increased antiviral activity]. PMID- 8640135 TI - [Effect of a hydrophilic cadmium complex with anabasine on energized rat liver mitochondria]. PMID- 8640134 TI - [Inhibition of the dopamine reuptake system by analogs of the neurotoxic metabolite MPP (1-methyl-4-phenylpyridine). The structure-activity relationship]. PMID- 8640136 TI - [The purine-pyrimidine tract activates transcription of the minimal retroviral promotor]. PMID- 8640137 TI - [Sex chromosomes: what are they for? (A new concept)]. PMID- 8640138 TI - [Features of biosynthesis of (14C)-arachidonic acid metabolites in metastasizing tumors]. PMID- 8640140 TI - [Role of post-tetanic potentiation in appearance of dominant properties of a defensive conditioned reflex]. PMID- 8640139 TI - [Activation of transcription factor p91 by an internalized epidermal growth factor receptor in A-431 cells]. PMID- 8640141 TI - [Characteristics of beta-specific multigene family gene expression in D. virilis. One "isoenzyme" can consist of two proteins]. PMID- 8640142 TI - [Arobiosis in the evolution of tapeworms]. PMID- 8640143 TI - [Biography and contributions to the development of Polish Allergy by Prof. zw hab. med. Sabina Chyrek-Borowska]. PMID- 8640144 TI - [An immunologic evaluation of Polish pollen extract Catalet "C" and Catalet "D" in patients with pollinosis]. AB - In a group of 41 hay fever and/or seasonal asthma patients, sensitive to weed or tree pollens the influence of specific immunotherapy (sIT) on the level of IgE, specific IgE and IgG4, and HR induced by anti-IgE and specific allergens was evaluated. It was shown that both allergenic extracts, Catalet C and Catalet D, are really immunogenic, which is manifested in a decrease in specific IgE and in a marked increase in specific IgG4, after 2 years of sIT. In comparison with the placebo, the sensitivity of blood basophils to both specific allergens and non specific factors significantly decreased. PMID- 8640145 TI - [Clinical evaluation of Polish allergen extracts Catalet C and Catalet D in patients with pollinosis]. AB - The aim of this paper was to evaluate the clinical efficacy, individual tolerance and side effects of allergen extracts, a weed pollen vaccine Catalet C and a tree pollen vaccine Catalet D. The study was carried out on a group of 41 patients aged from 15 to 46 years in comparison with a placebo group (36 patients). A good tolerance, no serious side effects and a high clinical efficacy, manifested in diminution of pollinosis symptoms in more than 80% of patients was noted. In the treated patients a significant reduction of antiallergic drug consumption in the pollen season was noted as compared with the placebo. The extracts Catalet C and Catalet D are usefull in the specific immunotherapy in weed and tree pollen allergic patients. PMID- 8640147 TI - [Specific immunotherapy in patients with bronchial asthma hypersensitive to housedust mites]. AB - An efficacy and effects of immunotherapy (IT) on skin and bronchial reactivity, and serum levels of some immunoglobulins in mite asthma patients were evaluated. A usefulness of some tests was assessed additionally. The studies were carried out on 37 asthmatics. Thirty of them were treated with Alutard SQ and 7 were observed as a control group for a period of 24 months. During the successive visits to the clinic the following examinations were carried out: physical examination, checking patient diary cards, skin prick tests, PS test and blood samples were taken for determination of IgE, sIgE Dpt, sIgG4 Dpt. Specific and nonspecific bronchial hyperactivity (BHR) were evaluated before, after 1 year and 2 years of IT. The obtained results evidenced a clinical improvement, a reduction of other antiasthmatic drugs consumption, decreases in serum IgE and sIgE Dpt levels and increases in serum sIgG4 Dpt levels. The changes in humoral response did not correlate with the clinical improvement. At the successive visits were needed higher concentrations of allergen to cause the positive allergic reaction in PS test. The reductions in specific and nonspecific DHR during IT were observed. The bronchial allergen challenge test seems to be a very valuable method in monitoring of IT in asthma patients. PMID- 8640146 TI - [Efficacy and tolerance of allergenic extracts Allergovit and Novo-Helisen depot in immunotherapy of allergic diseases]. AB - The aim of this study was to evaluate the efficacy, tolerance and immunogenity of allergenic extracts: Allergovit and Novo-Hellisen depot (produced by Allergopharma--Germany), used for sIT in pollinosis and house dust mite allergy. The treatment was carried out on 40 patients for three years. The evaluation included the results of a physical examination, the score of symptoms from self observations and the consumption of antiallergic medicines. In addition to the serum IgE and IgG4 the percentage of histamine, liberated by a-IgE and specific allergens, from peripheral blood basophils was determined. In some of patients treated with Novo-Helisen depot the specific and nonspecific bronchial challenge tests were made. It was shown that the allergenic extracts Allergovit and Novo Helisen depot are effective in treatment of pollinosis and house dust mite allergy, and are well tolerated by the patients. The changes in humoral response during sIT manifest their immunogenity. The sIT with Allergovit decreases the sensitivity of the peripheral blood basophils, whereas the treatment with Novo Helisen depot causes a diminution of bronchial specific and nonspecific reactivity and a negativisation of late allergic reaction. PMID- 8640148 TI - [CAP system methods versus FAST methods in immunologic monitoring of specific immunotherapy in pollen allergy]. AB - The aim was to study the value of the CAP system (Pharmacia) in comparison with the FAST method (Bio Whittaker) in determination of total IgE, specific IgE and IgG4 during specific immunotherapy (sIT). The studies were carried out on a group of 67 patients with pollinosis treated with various pollen allergen extracts. The immunological studies were performed before and after several courses of sIT. A higher reproducibility and sensitivity of the CAP System was shown. The values of specific IgE were somewhat higher on using the CAP system than using the FAST method. The CAP system seems to be of great value in diagnostics and monitoring sIT, especially in birch pollen allergy. PMID- 8640149 TI - [Behavior of various spirometry parameters during bronchial provocation tests with D. pteronyssinus in patients with asthma, sensitized to housedust mites]. AB - Changes in particular spirometry parameters during specific bronchial provocation test (BPT) with an allergen in asthmatics sensitized to house dust mites were evaluated. Studies were carried out on 130 patients. Specific BPT with D. pteronyssinus was performed according to the Ryan's method using a jet De Villbiss 646 nebuliser powered by compressed air and connected to the French Rosenthal dosimeter. The spirometry was measured using Pneumoscreen II (Jaeger - Germany). The provocation was terminated when a value of FEV1 fell at least 20% from baseline and then the spirometry parameters were measured hourly for a period 8 hours. Early and late asthmatic responses were founded. Results were expressed as a percentage of the maximum decrease from the baseline in particular parameters. To the statistical calculations only positive sBPT results founded in 96 patients were selected. The mean values of the decreases during of the EAR were as follow: FEV1 = 30.6 +/- 9.9%, FVC = 18.9 +/- 9.9%, FEV/FVC = 14.6 +/- 9.2%, FEV25-27 = 41.5 +/- 15.7%, PEF = 30.8 +/- 15.7%. The correlation coefficients were between FEV1 and FVC -0.57, FEV1 and FEV1/FVC -0.56, FEV1 and FEF25.75 -0.65, FEV1 and PEF -0.6. The results were statistically significant. Decreases in FEV1 and PEF were very similar each other. The greatest changes were observed in FEF25.75. The curves of the FEV1 and PEF averages observed during LAR were quite close to each other and almost parallel. The usefulness of the both parameters seems to be equal. PMID- 8640150 TI - [Production of selected cytokines by monocytes (IL-1 beta, IL-6) and lymphocytes (IL-2, IL-4) in peripheral blood of patients with nonallergic bronchial asthma treated with Broncho-Vaxom]. AB - In 16 patients with nonallergic bronchial asthma treated with Broncho-vaxom and 10 healthy persons the mononuclear peripheral blood cells ability for IL-1 beta, IL-2, IL-4 and IL-6 production were studied. Nonallergic asthmatics were characterised by increased levels of IL-1 beta and IL-6 produced by monocytes. After Broncho-vaxom therapy a decreased for IL-1 beta and IL-6, and an increased production of IL-2 were observed. These findings indicate that orally administered Broncho-vaxom affects on biological activity of mononuclear peripheral blood cells. PMID- 8640151 TI - [Effect of Ribomunyl therapy on production of selected cytokines by mononuclear peripheral blood cells in nonatopic bronchial asthma]. AB - In a group of 14 patients with nonatopic bronchial asthma, with recurrent infections of airways (approximately 5 - 10 episodes/year) and in 9 healthy persons the effect of Ribomunyl on IL-1 beta, IL-2, IL-4 and IL-6 production by mononuclear peripheral blood cells were assessed. After Ribomunyl treatment in asthmatics the significant decrease in IL-1 beta and IL-6 production by monocytes stimulated with lipopolysaccharides (LPS) and significant increase in IL-2 production by lymphocytes were observed. PMID- 8640152 TI - [Effect of flunisolide on exacerbation of clinical symptoms, spirometric parameters, consumption of B2-adrenergic drugs and levels of cortisol and ECP in serum of patients with bronchial asthma]. AB - The effects of flunisolide on a symptoms, spirometry, beta 2-agonist consumption, serum cortisol and ECP levels in 30 asthmatics were assessed. The patients qualified for the study after a two week run--in period, inhaled flunisolide from MDI--1000 micrograms daily (2 x 2 puffs) for a period of 4 weeks. Patients noted daily asthma symptoms, morning and evening PEF, the beta 2-agonists consumption and side effects in their diary cards. Each patient had 3 visits to the clinic, before and after the run-in period and at the end of the study. During the visits spirometry were performed. After the flunisolide therapy, statistically significant decreases in the daily and nightly asthma symptoms, diurnal variations of PEF, beta 2-agonist consumption, serum ECP levels and increases in morning and evening PEF and in serum cortisol levels. The patients tolerated the drug well. No serious side effects were observed during the study. PMID- 8640153 TI - [Introductory clinical evaluation of fluticasone propionate efficacy for treatment of allergic rhinitis in children]. AB - The investigations were carried out on 13 children with seasonal rhinitis, and in 10 children with perennial rhinitis. The fluticasone propionate (Flixonase-Glaxo) was introduced in acute state of disease: one dose to each nostril (100 micrograms) for the patient 7-11 years old, and in the patients under 11 years old--two doses to each nostril (200 micrograms) in the morning, during 3 weeks. The clinical effects were established using score-system in 0-3 points scale which included essential symptoms like: itching, sneezing, nasal blockade, rhinorrhoea, mucosal oedema and eyes irritation. Very good and good effects of the treatment in patients with seasonal rhinitis in 93% of children was observed. In the group of patients with perennial rhinitis, very good and good results, was observed in 80% of children. Easy dosage, high efficacy, very nice smell and no side-effects make this medicine very usefull in the treatment of allergic rhinitis in children. PMID- 8640154 TI - [Morphology of mast cells in experimental pulmonary fibrosis induced with bleomysin]. AB - Bleomycin induces local inflammatory process with subsequent pulmonary fibrosis. An unknown chemoattractant induces the mast cell (MC) migration to the injured lung tissue. Aim of this study was evaluation of the number, topography and ultrastructure (TEM) of the MC in rat lungs with bleomycin-induced fibrosis. The bleomycin was administered once intratracheally (3.6 mg/kg). The animals were sacrificed on 7th. 14th and 21st day of experiment. MC were stained with Csaba's method. An evident increase in MC number related to the phase of experiment and stage of lung fibrosis was observed: 520, 1200, 4745 per cm2 section on the 7th, 14th and 21st day respectively. In control the MC number was 163 per cm2 section (p < 0.001). On the 7th day the MC were rich in red granules (mature granules with preponderance of heparin). They were located mainly in pleura and around the blood vessels, as in control. On the 14th and 21st day the majority of MC was situated in places of active fibrosing. They contained exclusively blue granules (the young granules with preponderance of biogenic amines), mixture of increased secretory function of the MC in the fibrotic lungs. Some of the MC granules showed fusion and altered matrix contents, other were emptied in piecemeal manner. A net of microtubules connected the granules was observed. Degranulation of MC may release heparin and cytokines able to stimulate synthesis of extracellular matrix. It has been suggested that heparin contributes to the fibrotic process and angiogenesis, stimulating directly or indirectly collagen synthesis by binding, stabilization and activation of fibroblast growth factor (FGF) and cell adhesion glycoproteins. PMID- 8640155 TI - [Twenty-five years at the Allergy Clinic AM in Bialymstok]. PMID- 8640156 TI - [Progress in pathophysiology and therapy of bronchial asthma]. PMID- 8640157 TI - Is blood a better source of allogeneic hematopoietic stem cells for use after radiation accidents? AB - Peripheral blood and cord blood are increasingly used as sources of hematopoietic stem cells. They offer several advantages over bone marrow-derived stem cell grafts and are expected to replace bone marrow transplantation for treatment of malignant hematological disorders and solid tumors. Since the benefit of bone marrow transplantation for treatment of victims of radiation accidents has been seriously questioned, it is reasonable to examine if these alternative stem cell sources may change future strategies. Cord blood is attractive since it is an immediately available and fully characterized allogeneic stem cell source. However, experience with transplants in children suggests that recovery times are too long to benefit a victim of a radiation accident. Because of their potential to rapidly restore hematopoiesis, peripheral blood stem cells may currently be the best stem cell source for a patient who is expected to die from the consequences of prolonged pancytopenia (and not from damage of nonhematopoietic tissue). However, before getting accepted as the optimal stem cell source, the risk of acute and chronic GVHD must be confirmed to be low and unrelated donors must be shown to accept the potential risk of treatment with mobilizing agents. Future strategies should aim at developing methods of transiently rather than permanently engrafting allogeneic stem cells, since essentially all patients exposed to high doses of irradiation are expected to recover autologous marrow function. PMID- 8640158 TI - Hematopoietic precursor cell transplants in Europe: activity in 1994. Report from the European Group for Blood and Marrow Transplantation (EBMT). AB - This report, the fifth of a series, summarizes indications and donor source of hematopoietic precursor cell transplants performed in Europe in 1994 and illustrates the evolution since 1990. 306 teams in 30 participating countries performed a total of 10 066 transplants, 3502 allogeneic and 6564 autologous transplants. In 1990, there were only 143 teams and a total of 4234 transplants, 2137 allogeneic and 2097 autologous transplants. Main indications in 1994 were leukemias with 3881 transplants (39%), 2635 allogeneic (75%) and 1246 autologous (19%) transplants; lymphomas with 3760 transplants (37%), 245 allogeneic (7%) and 3515 autologous (54%) transplants; severe aplastic anemia with 250 transplants (2%), 248 allogeneic (7%) and 2 autologous transplants; solid tumors with 1800 transplants (18%), 13 allogeneic and 1787 autologous (27%) transplants and congenital disorders with 375 (4%) transplants, 361 allogeneic (11%) and 14 autologous transplants. The relative proportion of the main indication has stayed unchanged for allogeneic transplants. There was, in contrast, a marked increase in lymphomas and solid tumors, ie mainly for breast carcinoma, as an indication for autologous transplants since 1990. For the 3502 allogeneic transplants, the donor was an HLA-identical sibling in 2677 cases (76%), an HLA non-identical family member in 195 cases (6%), a syngeneic twin in 45 cases (1%) and an unrelated donor in 585 cases (17%). Donor source was bone marrow for 3322 allogeneic (95%) and 1869 autologous (29%) transplants, peripheral blood for 180 allogeneic (5%) and 4289 autologous (65%) transplants and a combination of bone marrow and peripheral blood for a few allogeneic and 406 autologous (6%) transplants. The relative proportion of peripheral blood as a source for autologous transplants has changed since 1991 from 15 to 72.1% in 1994. A similar trend for allogeneic transplants is just emerging. PMID- 8640159 TI - High-dose therapy with peripheral blood progenitor cell transplantation in low grade non-Hodgkin's lymphoma. AB - It was the objective of our study to evaluate the efficacy of a sequential high dose therapy with peripheral blood progenitor cell (PBPC) support in patients with low-grade non-Hodgkin's lymphoma (NHL). Since July 1991, 48 patients (23 male/25 female) with a median age of 43 years (range 26-55) were included in the study. At the time of entry, 28 patients were in first and seven in second or higher remission. Twelve patients had relapse of disease and one patient had tumor progression. PBPC were collected during granulocyte colony-stimulating factor (G-CSF)-enhanced leukocyte recovery following treatment with high-dose cytarabine and mitoxantrone (HAM). A median of two leukaphereses (range 2-7) resulted in 6.9 x 10(6) CD34+ cells/kg (median, range 2.1 x 10(6)-38.8 x 10(6)). A comparison was made between the harvests obtained from patients in first remission and those from patients in second remission, in relapse or progressive disease. Patients mobilized in first remission tended to have a greater collection efficiency for CD34+ cells comprising a significantly greater proportion of more primitive CD34+/Thy-1+ progenitor cells. Conversely, leukapheresis (LP) products collected during first remission contained a significantly smaller proportion of CD34+/CD45RA+ cells and CD34+/c-kit+ cells, subsets which reflect a more differentiated progenitor cell stage. Following high dose therapy and PBPC autografting, the median time to reach platelets > or = 20 x 10(9)/l and neutrophils > or = 0.5 x 10(9)/l and 12 and 13 days, respectively. Two patients died of treatment-related toxic organ failure. Thirty-nine patients are alive in remission after a median follow-up time of 15 months (range 1-31), while seven patients relapsed between 5 and 29 months post-transplantation. Except for one patient autografted in first remission, the patients with relapse had a history of previous relapse or progressive disease. Since the probability of disease-free survival appears to be related to the disease status at the time of autografting, PBPC-supported high-dose therapy including total body irradiation should be investigated further for patients with low-grade NHL while they are in first remission. PMID- 8640160 TI - Tandem high-dose chemotherapy supported by hematopoietic progenitor cells yields prolonged survival in stage IV breast cancer. AB - The aim of this phase II study was to determine the feasibility of using two (tandem) courses of high-dose alkylating agents with bone marrow or peripheral blood progenitor cell support in women with stage IV breast cancer. Women with stage IV breast cancer who had achieved a CR or PR during conventional chemotherapy were enrolled in a phase II trial of high-dose cyclophosphamide 7500 mg/m2 and thiotepa 675 mg/m2 (C+T) followed within 180 days by high-dose melphalan (M) 140 mg/m2. Bone marrow and/or GM-CSF mobilized peripheral blood hematopoietic progenitor cells were used to support high-dose C+T and high-dose M. Twenty-seven women were enrolled in this trial. The median age was 45 years (range 32-56). The median PS was 0 and all patients had achieved either a CR (4/27, 15%) or PR (23/27, 85%) to conventional chemotherapy. All 27 women underwent high dose C+T. The predominant toxicities were mucositis (81%), and diarrhea (81%); two patients (7%) died from infectious complications. Following C+T, the median time to hematologic recovery for neutrophils (ANC > 500 cells/mu 1) was 12 days and for platelets (>20 000 cell/mu 1), 23 days. Following C+T, 18 of 22 patients received high dose M; the predominant toxicities were nausea, vomiting (70%), and mucositis (91%). The median time to hematologic recovery for the ANC was 13 days and for platelets, 18 days. The overall response after high dose C+T and high dose M was 67% (CR, 15/27 patients (56%) and PR* (complete resolution of all measurable disease but persistent lytic disease or positive bone scan) 3/27 patients (11%). With median follow-up of 24 months, the actuarial freedom from relapse or treatment failure is 56% at 24 months. At 30 months 56% of patients are alive. For patients who achieve a CR or PR* the actuarial freedom from relapse or treatment failure at 24 months is 88%. In women with stage IV breast cancer who attain a CR or PR to conventional chemotherapy, tandem high dose chemotherapy with ABMT can lead to prolonged relapse-free survival. PMID- 8640161 TI - Changes in reticulocyte fractions during peripheral stem cell harvesting: role in monitoring stem cell collection. AB - We prospectively evaluated the changes in immature reticulocyte fractions during peripheral blood stem cell mobilization to determine any possible relationship with mobilization of stem cells into the peripheral blood. Circulating neutrophils, immature reticulocyte fractions (% of HFR + MFR) (HMFR) (measured by flow cytometry), circulating CD34+ cells (measured by flow cytometry) and CFU-GM (measured by semisolid media assay in the apheresis fluid) were closely monitored following priming with chemotherapy and colony-stimulating factors in 15 patients with hematological or solid tumors (group I). Day 0 was defined as the day on which the neutrophil count fell below 0.5 x 10(9)/l. In a second group of nine patients (group II) reticulocyte fractions and CD34+ cells were measured directly on the days on which they were predicted to increase using the data from group I. Reticulocyte counts and HMFR were also monitored in 18 patients who were mobilized with G-CSF alone. In group I, a significant rise in HMFR and CD34+ cells occurred on days 2, 4 and 6, and a linear correlation between HMFR on day 2 and CD34+ cells on day 4 was demonstrated (P = 0.0068, r = 0.74). In group II similar patterns of recovery were found. During mobilization with G-CSF alone HMFR significantly increased on days 2, 4 and 6 of treatment with respect to baseline values, and a multiplicative relationship between the increase of HMFR and neutrophils was observed (r = 0.707, P < 0.00001). Unfortunately, patients who did not mobilize CD34+ cells (one in groups I and II and three in the G-CSF group) showed similar HMFR kinetics to those who mobilized CD34+ cells. An increase in immature reticulocyte fractions precedes the presence of circulating CD34+ cells by about 2 days in patients mobilized with chemotherapy and growth factors, and it could thus serve as an indirect surrogate marker for monitoring the timing of stem cell harvesting. An unexpected increase of HMFR during treatment with G-CSF alone was found, indicating an effect of this factor on erythropoiesis in vivo. PMID- 8640162 TI - The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. AB - Our previous data obtained by flow cytometry and by clonogenic assay had consistently shown a lower cloning efficiency of hematopoietic progenitor cells in bone marrow (BM) compared to those in blood (PB) or in cord blood (CB). Also, recent clinical reports have described more rapid reconstitution after PB than after BM transplantation. We have applied two- or three-color flow cytometric analysis using monoclonal antibodies directed against the stem- and progenitor cell antigen CD34, in combination with other cell surface markers. We report significant differences in the composition of progenitor cells contained in BM (238 specimens from 53 healthy donors and from patients in remission), PB (301 samples from 92 patients with or without cytokine support) and CB (n = 37). Leukapheresis products (Pher, n = 69) were included in the study as well as positively selected CD34+ cells obtained from BM (BMsel, n = 2), PB (PBsel, n = 28) and CB (CBsel, n = 5). We used monoclonal antibodies directed against CD7, CD19, CD34, CD38, CD45RA and glycophorin A. The highest proportion of CD34+ cells (in % of the MNC) was found in BM (mean 5.37% +/- 4.5). In the other sources, the mean values were 1.79% +/- 2.46 (PB), 1.48% +/- 1.81 (Pher) and 1.1% +/- 1.69 (CB). However, BM was the only source in which a considerable proportion of the CD34+ cells coexpressed the B cell antigen CD19 (mean 30.1%, median 28, range 0 to 84%). The amount of earlier myeloid progenitors as determined by their non expression of the CD45RA antigen was lowest among BM CD34+ cells (26.7% +/- 16.6). In the other sources, the respective values were 57.5% +/- 17.9 (PB), 63.6% +/- 13.9 (Pher) and 70.4% +/- 16.1 (CB). These results were confirmed by subtype analyses of the CD34+ cells positively selected from the three sources. Enrichment showed minor CD34+ subpopulations to be identified. The mean proportions of B cell progenitors were 0.11% +/- 0.24 (PBsel) and 1.3% +/- 1.42 (CBsel) of the CD34+ cells. The CD34+ cells from all cell sources coexpressed GPA (median 0.15%, range 0 to 1.8%) and CD7 (median 0.25%, range 0 to 1.2%). The proportion of CD38- cells ranged from 0.7 to 4% of the CD34+ MNC. Thus, despite higher CD34 counts in BM, the relative proportions of myeloid progenitors are higher in PB and in CB. This suggests that, if timely reconstitution depends on the number of CD34+ cells transplanted, the mean number of "stem cells' (SC) required is 1.4-fold (for myeloid cells) or 2.2-fold (for earlier myeloid cells) higher for BM than for PB. PMID- 8640163 TI - Comparison of four cytokine regimens for mobilization of peripheral blood stem cells: IL-3 alone and combined with GM-CSF or G-CSF. AB - Improved methods for mobilization may reduce the number of aphereses required to collect adequate numbers of peripheral blood stem cells (PBSC) and hasten engraftment following high-dose therapy. Mobilization with cytokines alone enables engraftment after myeloablative therapy. The optimal cytokine regimen for mobilization has not been established. The study evaluated the effects of four interleukin-3-containing cytokine regimens administered during steady state hematopoiesis on PBSC mobilization in 30 patients with breast cancer or lymphoid malignancies. These regimens included IL-3 alone (Arm 1), sequential IL-3 --> G GSF (Arm 2), sequential IL-3 --> GM-CSF (Arm 3) and combined IL-3 + G-CSF (Arm 4). Consecutive days of apheresis were performed until a target of 4-6 x 10(8) mononuclear cells/kg were collected. All patients received intravenous GM-CSF after PBSC infusion. Median days to an ANC > or = 500/ microliters in Arm 3(22 days) was significantly slower than for patients in Arm 2 (13 days) but not significantly different from patients in Arm 1 or Arm 4. There was no significant difference in platelet engraftment or days of hospitalization between the study arms. Addition of GM-CSF to IL-3-containing mobilization regimens results in collection of PBSC that lead to delayed engraftment. Further development of Arms 1, 2, and 4 appear warranted. PMID- 8640164 TI - Serum concentration of the soluble interleukin-2 receptor for monitoring acute graft-versus-host disease. AB - Soluble interleukin-2 receptors (sIL-2R) are elevated in various disorders involving the activation of T cells. We measured serial serum concentrations of sIL-2R in 30 patients receiving allogeneic BMT to evaluate the usefulness of sIL 2R as a parameter for acute GVHD. In the 17 patients who developed acute GVHD, the sIL-2R concentration rose significantly on day 3 following transplantation, preceding the occurrence of acute GVHD. This change was not seen in the 13 patients without acute GVHD. The serum concentration of sIL-2R decreased as the acute GVHD subsided. The peak concentration of serum sIL-2R correlated with the severity of the acute GVHD. Simultaneous measurement of tumor necrosis factor alpha (TNF alpha) showed a significant rise in patients with acute GVHD, that became evident earlier than the sIL-2R elevation. TNF alpha concentrations also decreased following treatment of the acute GVHD. However, significant rise in TNF alpha were also seen in the early phase of allogeneic BMT in patients who did not develop acute GVHD. Our data suggest that the serum concentrations of sIL-2R as well as TNF alpha might reflect the severity of acute GVHD, and that the serum sIL-2R concentration might be a sensitive and practical indicator for acute GVHD. PMID- 8640165 TI - Allogeneic bone marrow transplantation vs aggressive post-remission chemotherapy for children with acute myeloid leukemia in first complete remission. A prospective study from the French Society of Pediatric Hematology and Immunology (SHIP). AB - The objective of this study was to compare allogeneic bone marrow transplantation (BMT) with high-dose cytarabine containing chemotherapy in children with acute myeloid leukemia (AML) in first complete remission (CR). One hundred and seventy one children were enrolled on the LAME89/91 protocol. Induction chemotherapy was a combination of cytarabine and mitoxantrone. After achieving CR, patients who had an HLA-identical sibling donor underwent allogeneic BMT. Children not eligible for BMT received post remission chemotherapy which included two consolidation courses, the second consolidation consisting of high-dose cytarabine with amsacrine and asparaginase. CR was achieved in 149 children (87%). Thirty-two had an HLA-identical sibling donor and were eligible for BMT. These 32 patients, as well as an additional child who had a one antigen HLA mismatched father, received BMT during first CR. Consequently, 33 patients were analyzed in the BMT group and 116 in the chemotherapy group. The 4-year probability of relapse was 26 +/- 15% in the BMT group and 47 +/- 10% in the chemotherapy group (P = 0.04). The risk of therapy-related death was 3% for BMT and 7.7% for chemotherapy. Disease-free survival (DFS) was 72 +/- 15% in the BMT group and 48 +/- 10% in the chemotherapy group (p = 0.02). We conclude that allogeneic BMT from a matched sibling donor is the treatment of choice for reducing the relapse risk and for increasing DFS in children with AML in first CR. PMID- 8640166 TI - Use of cryopreserved bone marrow in unrelated allogeneic transplantation. AB - Cryopreservation of donor bone marrow can facilitate scheduling allogeneic bone marrow transplantation (BMT) by affording independence of a preset time for donation. Previously, we and others have shown the feasibility of using cryopreserved related allogeneic bone marrow. Here, we report the results of the first 10 patients receiving cryopreserved unrelated bone marrow between 1992 and 1995. All evaluable patients (n = 9) engrafted. Time to reach an absolute neutrophil count (ANC) >0.2 x 10(9)/1 and ANC >0.5 x 10(9)/l was 21.4 +/- 9.1 days and 22.6 +/- 9.2 days, respectively. The incidence of acute GVHD > or = grade II and chronic GVHD was 75 and 20%, respectively. Five of nine evaluable patients were alive 100 days post-transplantation. We conclude that cryopreserved unrelated donor bone marrow may be used for allogeneic transplantation. PMID- 8640168 TI - Unexplained effusions: association with allogeneic bone marrow transplantation and acute or chronic graft-versus-host disease. AB - We evaluated patients presenting with large and recurrent sterile serosal effusions following bone marrow transplants. From a review of the Minnesota BMT Database from 1974 to 1993, seven patients with unexplained multiple effusions involving two or more of the pleural, pericardial or peritoneal cavities were identified. Patients with veno-occlusive disease (VOD), infections, cardiac insufficiency, tumor relapse and GM-CSF toxicity were excluded. All had onset following engraftment and six occurred before day 100. Unexplained multiple effusions were observed in recipients of allogeneic transplants but not autologous transplants and were found only in patients with acute and/or chronic GVHD. Five of seven patients also had cytomegalovirus (CMV) disease. Multiple effusions appear to be part of the presentation of severe acute or chronic GVHD, often in association with CMV disease in patients who receive allogeneic donor marrow. PMID- 8640167 TI - High-dose chemo-radiotherapy followed by autologous Philadelphia chromosome negative blood progenitor cell transplantation in patients with chronic myelogenous leukemia. AB - Twenty-three patients with chronic myelogenous leukemia in early chronic phase (ECP) and not previously treated with alpha-interferon (IFN-alpha) (10 patients), in ECP but pretreated with IFN-alpha (<12 months) (seven patients) and in late chronic phase (LCP) pretreated with IFN-alpha (>12 months) (six patients) underwent autografting with Philadelphia (Ph) chromosome-negative blood progenitor cells (BPCs) (20 patients), or partially/totally Ph-positive BPCs (three patients), previously mobilized during the early phase of recovery after aplasia induced by intensive chemotherapy. The conditioning regimen consisted of high-dose chemotherapy alone or followed by total body irradiation (TBI). Recombinant G-CSF was given after BPCs infusion on day +8. All patients in ECP not pretreated with IFN-alpha are alive and five of them are Ph-negative in the marrow after autografting. Six of seven patients autografted with Ph-negative BPCs in the group of ECP pretreated with IFN-alpha (<12 months) are alive and two of them are still Ph-negative in the marrow. In the same group, the only patient transplanted with partially Ph-positive BPCs, died of blastic transformation 2 months after reinfusion. Three patients (two patients autografted with Ph negative BPCs and one patient with Ph-positive BPC) in the group of LCP pretreated with IFN-alpha >12 months are alive but Ph-positive after autografting. The other three patients of the same group died of procedure related toxicity (two patients) and blastic transformation (one patient). Seventeen patients (10/10 ECP not pretreated with IFN-alpha; 5/7 ECP pretreated with IFN-alpha and 2/6 LCP pretreated with IFN-alpha) of 23 autografted patients were treated with IFN-alpha +/- IL-2. Toxicities after autografting were mostly related to myelosuppression, particularly thrombocytopenia. All patients of the two groups pretreated with IFN-alpha developed febrile episodes during the aplastic phase following BPCs reinfusion. No patient autografted in ECP and those not pretreated with IFN-alpha developed febrile episodes. This is also probably due to the use of i.v. antibiotic and antimicotic prophylaxis when neutrophils were < or = 1 x 10(9)/l after autografting. Greater toxicity was observed in patients pretreated with IFN-alpha, being lethal in two cases in LCF. In conclusion, the "in vivo' manipulation approach employed in our institution is a safe procedure and it results in a high collection of Ph-negative cells in the blood if the cells are harvested: (1) in early chronic phase; (2) in early phase of recovery after chemotherapy-inducing aplasia; (3) in patients not extensively pretreated with IFN-alpha. The data presented here have shown encouraging trends in chronic phase of CML and offer new perspective for patients without an HLA identical donor or for patients who do not respond to IFN-alpha. PMID- 8640169 TI - Outcome of autologous rescue after failed engraftment of allogeneic marrow. AB - Ten patients experiencing primary graft failure after allogeneic bone marrow transplantation received autologous marrow or blood cells as hematopoietic rescue 21-40 (median 22.5) days after the original transplant after which immunosuppression with cyclosporine was tapered off rapidly. The leukocyte count at the time of rescue was 0.1-0.3 x 10(9)/l with < 0.1 x 10(9)/l neutrophils. The clinical course was uneventful after rescue with hematologic recovery to 0.5 x 10(9)/l neutrophils occurring 9-61 days (median 20) later in nine patients, and to 50 x 10(9)/l platelets 17-595 days (median 40) later in eight patients. No patient had a life-threatening infection at the time of or after rescue. No patient experienced acute graft-versus-host disease. Four patients with acute leukemia had recurrent disease 46-117 days after autologous rescue, and died of relapse. Five patients with acute leukemia are alive in continuous remission at 270-3382 days, and one patient with chronic myeloid leukemia is alive in hematologic remission at 228 days. We conclude that infusion of back-up autologous cells can prevent the immediate infectious consequences of primary allogeneic graft failure through consistent myeloid recovery, and may result in long-term survival of patients with good-risk disease. PMID- 8640170 TI - Major salivary gland dysfunction in human acute and chronic graft-versus-host disease (GVHD). AB - Salivary gland dysfunction is frequently observed in patients suffering from acute (a) and chronic (c) GVHD. We studied the influence of GVHD on the function of major salivary glands in 20 patients with GVHD (cGVHD, 15; aGVHD, 5). A subjective evaluation of salivary function was performed, in which the score ranged from 0-4 where a higher score indicated more oral dryness. Patients with aGVHD scored 4.0 while patients with cGVHD scored 2.1 (P < 0.01). In addition to this subjective evaluation, patient's salivary flow rates were measured and a reduction of 90% and 60% in aGVHD and cGVHD patients respectively, was observed as compared to controls (P < 0.01; P < 0.05). No hyposalivation was observed in patients who underwent bone marrow transplantation but did not develop GVHD as compared to normal individuals. A direct correlation was observed between the degree of hyposalivation and the severity of the GVHD. Hyposalivation was also documented by scintigraphy of the major salivary glands in the GVHD patients. Furthermore, hyposalivation was associated with extensive mucosal atrophy, erythema, tongue surface depapillation, lichenoid lesions of the buccal and labial mucosa as well as lupus-like lesions. Routine assessment of these glands in patients with GVHD could play a role in monitoring response to therapy. PMID- 8640171 TI - Association of busulfan area under the curve with veno-occlusive disease following BMT. AB - Busulfan pharmacokinetics, specifically area under the concentration curve (AUC), have been correlated with the occurrence of veno-occlusive disease (VOD) following BMT. To evaluate the risk of VOD, we studied 66 patients who received pharmacotherapeutically monitored busulfan regimens in combination with CY, etoposide (VP16) and/or Ara-C in preparation for BMT. These patients received a total of 16 doses of busulfan dosed as 1 mg/kg/dose q 6 h beginning at 09.00 (n = 39), 18.00 (n = 2), 21.00 (n = 1) or 24.00 (n = 24) h. With the first dose, blood samples were obtained at baseline, every 15-30 min for 2 h, then every 1-2 h for 4 h. Blood was analyzed for busulfan concentration by high performance liquid chromatography and AUC calculated by the trapezoidal rule. Seventeen patients (25.8%) were not evaluable for AUC calculation due to slow absorption and/or elimination: 13 of 27 (48.1%) received the first dose between 18.00-24.00 vs four of 39 (10.2%) patients who received the first dose at 09.00 (P < 0.001). Eighteen of 51 (35.3%) evaluable patients had an AUC > 1500 mumol x min/l; 10 of whom received doses reduced proportionally to achieve an AUC = 1200 mumol x min/l starting with the 10th to 15th dose. Six of 18 (33.3%) patients with an initial AUC > 1500 mumol x min/l developed VOD vs one of 33 (3.0%) patients with an initial AUC < 1500 mumol x min/l (relative risk = 11.1; P = 0.0056). Other pharmacokinetic parameters, age, gender, type of BMT, previous therapy or pre transplant liver function tests were not predictive of VOD. A higher incidence of VOD occurred in patients receiving BUCY (4 of 10) compared to those receiving BUCYAra-C (1 of 18) or BUCYVP16 (7 of 38), which could not be attributed to increased busulfan exposure in the BUCY patients. Routine pharmacotherapeutic monitoring of busulfan is recommended with further study to evaluate the impact of earlier and greater overall dose reduction in patients with high initial busulfan exposures. PMID- 8640172 TI - FISH detection of mixed chimerism in 33 patients submitted to bone marrow transplantation. AB - Fluorescence in situ hybridization (FISH) and cytogenetic analysis were carried out in 33 transplanted patients suffering from different hematologic disease using probes for X and Y chromosomes and ABL and BCR genes. FISH showed that recipient cells were invariably present during post-transplant follow-up. Stable minimal residual disease was associated with clinical and hematologic remission, while a progressive increase of host cells was strictly related with disease relapse. Cytogenetic investigation on the same samples showed recipient cells only in few cases. It was concluded that FISH analysis is useful for: (1) characterizing cases in which standard cytogenetic analysis has failed; (2) detecting host cells in sex-mismatched transplanted patients; and (3) evaluating Ph-negative CML with the BCR/ABL rearrangement. The possibility of detecting chromosome rearrangements in interphase nuclei using FISH analysis improves diagnosis and prediction of disease evolution and prompts earlier therapeutic approaches. PMID- 8640173 TI - Salivary immunoglobulins in recipients of bone marrow grafts. III. A longitudinal follow-up of CMV specific antibodies. AB - Cytomegalovirus (CMV) infection is a major complication of BMT. The oral cavity is a common route for CMV infection, whose protection is provided by salivary anti-CMV antibodies. We developed an ELISA assay for the detection of CMV specific antibodies in parotid saliva. Saliva of patients receiving BMT from CMV positive donors was transiently reconstituted with IgG and IgA anti-CMV antibodies shortly after transplantation. The concentration of these antibodies gradually decreased during the 2 months after transplantation and increased again around day 80. A remarkable rise in the salivary concentrations of IgG and IgM anti-CMV was observed shortly after i.v. administration of Sandoglobulin. These results demonstrate, for the first time accurate monitoring of CMV-specific antibodies in saliva using a quantitative ELISA assay. The study suggests that secretion of CMV-specific antibodies in saliva of immunocompromised patients can be reconstituted by donor-derived B and plasma cells transferred with the BM or by i.v. administration of pooled Ig. PMID- 8640175 TI - Quality of life in long-term survivors of marrow transplantation: comparison with a matched group receiving maintenance chemotherapy. AB - A retrospective descriptive study was designed to assess the quality of life (QoL) and psychosocial adjustment in long-term BMT survivors compared with a group of patients with haematological malignancies receiving maintenance chemotherapy (MC), matched for age, post-treatment time, sociodemographic and disease characteristics. The sample consisted of 91 long-term BMT survivors and 73 MC patients from three teaching hospitals in the UK. The results indicated that most of the BMT subjects had a good to excellent quality of life and, in some domains, even better adjustment than the MC patients. However, 20% of the BMT subjects had failed to return to full-time employment at a mean post-BMT time of almost 40 months. A significant number of BMT subjects were also identified with symptoms of anxiety and depression. The physical symptomatology had an association with psychological status. Impotence-related difficulties, decreased sexual satisfaction and altered body image were the main characteristics of psychosexual dysfunction in the BMT group. Poorer quality of life was predicted by the presence of depressive symptoms, low affirmation, and impoverished social adjustment. PMID- 8640174 TI - Gamma delta T lymphocyte regeneration after T lymphocyte-depleted bone marrow transplantation from mismatched family members or matched unrelated donors. AB - The recovery of gamma delta T lymphocytes was studied in 31 recipients of T cell depleted allogeneic bone marrow (BMT) to determine if the dynamics of reconstitution could be related to graft-versus-host disease (GVHD) or other complications of marrow transplantation. Two distinct patterns of regeneration were apparent. In 12 patients, there was a progressive rise in both the percentage and the absolute number of peripheral blood gamma delta T cells over the first year post-transplantation, but these increases never breached levels found in 14 healthy donors. Each of the 19 remaining patients had abnormally high proportions and numbers of gamma delta T cells on at least two occasions following transplantation. The clinical factor that best explained these observations was the frequency of intercurrent infections. Of 19 patients with abnormally increased percentages and numbers of gamma delta T lymphocytes, 18 had one or more episodes of confirmed viral or fungal infection, contrasted with only two of 12 in the comparison group (P < 0.001). There was no significant association of gamma delta T cell recovery patterns with the presence of GVHD (P = 0.33). We conclude that the recovery of gamma delta T lymphocytes after marrow transplantation may vary. Supranormal levels of this T cell subset are associated with infection and may contribute significantly to cellular immune defenses against fungal or viral disease. PMID- 8640176 TI - Positive selection of CD34+ cells from cryopreserved peripheral blood stem cells after thawing: technical aspects and clinical use. AB - Autologous stem cell transplantation has become an important therapy in lymphoma, multiple myeloma and solid tumors. The rationale for the selection of CD34+ cells from peripheral blood or bone marrow progenitor cell collections is based on the observation that contaminating tumor cells can be depleted approximately 3 to 6 logs. This procedure may be limited because of lack of sufficient numbers of progenitor cells in the leukapheresis concentrates. The use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples makes it possible to pool two or even more stem cell harvests collected at different time points to increase the total number of CD34+ progenitor cells. We report in this work the feasibility of frozen/thawed peripheral blood CD34+ positive cell selection, using the large scale (Ceprate SC) and the lab-scale avidin-biotin immunoadsorption system (Ceprate LC). This procedure consists of a washing step and a positive selection step. Our results show that frozen/thawed CD34+ cells were obtained with a purity of 86.68 +/- 3.62%, a viability of 97.94 +/- 0.97% and a recovery of 91.85 +/- 10.84% (range 80 to 112%). The CFU-GM assays were performed in a methylcellulose based medium; 89.13 +/- 19.63 colonies were obtained for 10(3) cells plated. Two patients were grafted with peripheral blood CD34+ cells selected after freezing. Our clinical data show that these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy. PMID- 8640177 TI - PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents. AB - Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT. PMID- 8640178 TI - Improved single-step PCR assay for sex identification post-allogeneic sex mismatched BMT. AB - A method is presented which allows sex-identification simultaneously of both male and female cells in cell mixtures. The sensitivity of this PCR-based assay is one male cell in 10(6) female cells. The test can be used routinely to analyze bone marrow and peripheral blood lymphocytes post-allogeneic sex-mismatched bone marrow transplantation to detect early engraftment or rejection, as well as host cell regeneration. PMID- 8640180 TI - Paecilomyces varioti fungemia in a bone marrow transplant patient. AB - Paecilomyces varioti, a fungus resembling penicillium spp, has been described in conjunction with impaired host defence or foreign body implants. We report a case of Paecilomyces varioti catheter-related fungemia that occurred during neutropenia in an allogeneic BMT patient receiving antifungal prophylaxis with fluconazole. Successful treatment was achieved by removal of central venous catheter, intravenous amphotericin B and oral itraconazole. PMID- 8640179 TI - Endothelium and bone marrow transplantation. AB - Thrombotic complications may occur early after marrow transplantation and many data suggest that endothelial injury plays a pivotal role in their pathogenesis. Since plasma thrombomodulin and P-selectin are thought to be of value as markers of vascular endothelial cell membrane injury, we investigated their plasma concentration in bone marrow transplant patients aiming better to clarify the degree of endothelial involvement. Plasma thrombomodulin and P-selectin were monitored in 25 patients without thrombotic complications before transplant, on day 0 and weekly for 1 month thereafter, while in three patients who developed VOD monitoring continued until day +52. These proteins were in the normal range in all the uncomplicated patients and in two with reversible VOD, while they were always very high in the only patient who developed very severe and lethal VOD. In conclusion, we suggest that endothelial activation/damage occurs rarely in the course of BMT for hematological malignancies; we were able to document endothelial injury in only one patient with very severe thrombotic complication. PMID- 8640181 TI - Herpes simplex virus (HSV) colitis in a bone marrow transplant recipient. AB - Herpes simplex virus (HSV) infections are common in bone marrow transplantation patients. Unusual sites may be involved, however colonic disease with HSV is rare. We report a successfully treated case of colitis due to HSV, cytomegalovirus, Clostridium difficile and graft-versus-host disease in an allogeneic marrow recipient. PMID- 8640182 TI - High-dose cytosine arabinoside and fractionated total body irradiation as a preparative regimen for the treatment of children with acute lymphoblastic leukemia and Down syndrome by allogeneic bone marrow transplantation. AB - The early toxicity, incidence of graft-versus-host disease (GVHD) and long-term follow-up were evaluated in two children with Down syndrome (DS) treated for acute lymphoblastic leukemia (ALL) in second complete remission by HLA-matched sibling allogeneic bone marrow transplantation (BMT). Preparative conditioning therapy consisted of cytosine arabinoside (Ara-C) and fractionated total body irradiation (F-TBI) and GVHD prophylaxis of cyclosporin A. The conditioning regimen was well tolerated, the only acute complication being mild mucositis. Engraftment (polymorphonuclear cells >500/microliter) was documented by day +17 in both patients. One child remains in continuous complete remission, without medical problems, 60 months after BMT. The second patient died from complications associated with chronic GVHD 21 months following BMT. Ara-C and F-TBI is a well tolerated preparative regimen for children with DS undergoing allogeneic BMT. PMID- 8640183 TI - Thalidomide responsive chronic pulmonary GVHD. AB - A 29-year-old male underwent allogeneic bone marrow transplantation for progressive multiple myeloma. His post-transplant course was complicated by severe chronic pulmonary graft-versus-host disease (GVHD) resistant to cyclosporin A, corticosteroids and azathioprine. The introduction of thalidomide resulted in a dramatic improvement in his lung function which has been maintained even after cessation of thalidomide. He remains well 40 months after transplantation. PMID- 8640184 TI - Fulminant adenovirus hepatitis after allogeneic bone marrow transplantation. AB - Adenoviruses may cause severe infections in bone marrow transplant recipients. We report the case of a patient who developed fulminant hepatitis 5 months after bone marrow transplantation. Adenovirus type 2 was cultured from stool and blood samples. The patient died from liver failure. Histologic examination of post mortem liver samples showed extensive necrosis with nuclear inclusions. Adenovirus was identified in liver cells by electron microscopy and immunohistochemical staining using a monoclonal anti-adenovirus antibody. No other pathogen was identified. PMID- 8640185 TI - Use of vancomycin in central venous catheter-related infections. PMID- 8640186 TI - Composition of Pneumocystis carinii neutral lipids and identification of coenzyme Q10 as the major ubiquinone homolog. AB - The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ10 was the predominant homolog with lesser amounts of CoQ9 present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ9 and lesser amounts of CoQ8, but no detectable CoQ10. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ10 (if not both CoQ10 and CoQ9) in P. carinii was not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism. PMID- 8640187 TI - An arrayed bacteriophage P1 genomic library of Pneumocystis carinii. AB - We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of approximately 4800 independent clones with an average insert size of approximately 55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4-5 positive clones for each, consistent with a several-fold coverage of the approximately 10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library of P. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen. PMID- 8640188 TI - Microsporidian spore wall: ultrastructural findings on Encephalitozoon hellem exospore. AB - A study of the spore wall of Encephalitozoon hellem was performed on thin sections, freeze-fracture, and deep-etched samples to obtain information on spore wall organization and composition. Our observations demonstrate that the spore wall is formed by an inner 30-35 nm electron-lucent endospore and an outer 25-30 nm electron-dense exospore. The exospore is a complex of three layers: an outer spiny layer, an electron-lucent intermediate lamina and an inner fibrous layer. Freeze-fracture and deep-etching techniques reveal that the intermediate lamina and the inner fibrous layer result from the different spatial disposition of the same 4-nm thick fibrils. In thin sections the endospore reveals a scattered electron-dense material that appears in the form of trabecular structures when analyzed in deep-etched samples. The presence of chitin in the exospore is discussed. PMID- 8640189 TI - Evidence for heterotrimeric GTP-binding proteins in Toxoplasma gondii. AB - Toxoplasma gondii, an intracellular protozoan parasite, resides within a host derived vacuole that is rapidly modified by a parasite-secreted membranous tubular network. In this study we investigated the involvement of heterotrimeric G proteins in the secretory pathway of T. gondii. Aluminum fluoride (AlFn), a specific activator of heterotrimeric G proteins, induced secretion from isolated tachyzoites of T. gondii in vitro, as seen by light optics and electron microscopy. In Western blot analyses, antibodies to G protein alpha subunits reacted with 39-42 kDa proteins from T. gondii isolates. Antibodies to G(o) alpha and Gs alpha coupled to the fluorescent probe fluorescein isothiocyanate localized to the paranuclear region of T. gondii. Gi3 alpha immunoprobes were confined to the cytoplasmic matrix of T. gondii and also labeled the parasitophorous vesicle. Fluorescein isothiocyanate-conjugated GA/1, an antipeptide antisera directed toward the GTP binding site common to G protein alpha subunits, was confined to the lateral cytoplasmic domain of the parasites where secretion is most prominent. In time-sequence studies using the GA/1 probe, the immunoreactive material shifted position during invasion of target cell to areas of active secretion. PMID- 8640190 TI - An improved method to obtain high molecular weight DNA from purified micro- and macronuclei of Tetrahymena thermophila. AB - An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described. Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (>100 kb) DNA fragments. Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending. We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM). To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods. High molecular weight micro- and macronuclear DNA was obtained using the new protocol. PMID- 8640191 TI - Nucleotide sequences of DNA fragments of Encephalitozoon cuniculi amplified by polymerase chain reaction with primers regarded as specific for Echinococcus. AB - Encephalitozoon-like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi, which was shown to have En. cuniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus, were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus-specific primer sets could amplify not only Ec. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus-specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis. PMID- 8640194 TI - Lipid requirements and lipid uptake by Giardia lamblia trophozoites in culture. AB - To better understand the lipid requirements of Giardia lamblia trophozoites and the mechanisms of lipid uptake, we supplemented serum-free TYI-S-33 medium with lipids incorporated into different lipid carriers. We found that serum lipoproteins, beta-cyclodextrins, and bile salts are able to supply cholesterol and phospholipids to Giardia and to support the multiplication of the parasite in vitro. The growth rates obtained with different lipoproteins or bile salts and lipid mixtures were similar to that in standard culture medium containing serum. Pulse labelling experiments using fluorescent lipid analogs demonstrated that Giardia can take up lipids from lipoproteins, beta-cyclodextrins, or bile salt micelles, but with different kinetics, and that bile salts greatly facilitated lipid transfer from lipoproteins and cyclodextrins to the parasite surface. The binding of different radioiodinated lipoprotein classes to the trophozoite surface, inhibition of lipoprotein interiorization at 4 degrees C or by cytochalasin D, and incorporation studies using fluorescent LDL suggested that a small component of lipid uptake by trophozoites was likely due to endocytosis of lipoproteins. PMID- 8640192 TI - Insights into the evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences. AB - The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi, were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait. PMID- 8640193 TI - Cytoplasm rescues an arrhythmic mutant on the circadian rhythm of mating reactivity in Paramecium bursaria. AB - Cells of an unusual Paramecium bursaria stock (Sj2) expressed rhythmic mating reactivity in a light/dark cycle (LD) and under continuous illumination (LL). When placed in continuous darkness (DD), did not show rhythmicity but rather demonstrated a continuous high mating reactivity. However, mating reactivity was reduced following exposure to a 6-h light pulse interrupting the DD, and then recovered to its former condition. Genetic analysis showed the arrhythmicity in DD to be a dominant character inherited in a Mendelian ratio. On the other hand, a clone (MC1w) that did not show the rhythmicity in either DD or LL was isolated from the parent stock Sj2w following a 5-h treatment with 2 micrograms/ml nitrosoguanidine (MNNG). The MC1w cells expressed weak rhythmicity in LD, but were insensitive to a 6-h light pulse in DD. The arrhythmicity in LL was inherited cytoplasmically. In addition to this, rhythmicity in LL could be recovered by injection of cytoplasm from the wild-type cell when the recipient cell was homozygous for the wild-type nuclear gene (+/+). The cytoplasmic components or factors are assumed to control the functional circadian system and genetically determine the rhythmicity of mating reactivity. PMID- 8640196 TI - Academy for eating disorders. PMID- 8640195 TI - Gametocytogenesis of Leucocytozoon caulleryi in in vitro culture: effect of human red blood cells on the development of second-generation merozoites. AB - For investigating development of second-generation merozoites of Leucocytozoon caulleryi into mature gametocytes, infected erythrocytes from chickens at 15 d after sporozoites inoculation were cultured in RPMI-1640 modified medium supplemented with 10% horse serum and 0.5 ml of human erythrocytes (type O). When culture was carried out at 37 degrees C in a humidified atmosphere or 5% CO2 for 7 d, the very small number of second-generation merozoites developed into morphologically mature gametocytes. However, in the high carbon dioxide and low oxygen condition, mature gametocytes weren't observed in culture. The role of human erythrocytes added has not been clarified yet. PMID- 8640197 TI - Anorexia nervosa following gastroplasty in the male: two cases. AB - The first stage in the development of anorexia nervosa involves voluntary restriction of food intake, that is, diet. Marked weight loss, impairment in body image, and deterioration of health ensue. Anorexia nervosa is much more prevalent among women than men. Its etiology and presentation are mostly similar for both sexes. Certain features, such as greater premorbid obesity and sexual identity concerns, are thought to be more prevalent in male patients. We present 2 male patients who had undergone gastroplasty for morbid obesity and subsequently developed anorexia nervosa. Both evinced signs of identity confusion. Neither one of the patients underwent psychiatric evaluation before surgery. The cases described illustrate that anorexia nervosa may succeed acute and marked weight loss following gastroplasty. This emphasizes the need for a psychiatric assessment before bariatric surgery, and should alert clinicians to search for elements that may predispose vulnerable individuals to a risk of developing anorexia nervosa. PMID- 8640198 TI - Food preferences and desire to eat in anorexia and bulimia nervosa. AB - OBJECTIVE: To determine whether eating disordered patients and controls differ in visual analog scale (VAS) ratings of liking and desire to eat various foods and whether ratings differ according to caloric or macronutrient content of the foods. METHOD: Fifty-five female inpatients with eating disorders and 15 controls rated their liking of and desire to eat 50 common foods at admission and discharge using 100-mm VAS. RESULTS: All patient groups rated their desire to eat high-calorie foods significantly lower than their desire to eat low-calorie foods whereas controls rated their desire to eat high- and low-calorie foods equally. Patients also differed from controls more in ratings of desire to eat than in liking when foods were classified according to macronutrient content. In restricting anorexics (N = 25), ratings of liking and desire to eat for high fat/low-carbohydrate (CHO) and high-fat/high-CHO foods were not significantly correlated at admission. Disparity in correlations between restrictors, bulimics (N = 18), and controls was attenuated with treatment while anorexics with bulimic features (N = 12) became less like controls from admission to discharge. DISCUSSION: Differences in the way patients and controls perceive foods should be borne in mind during the treatment process. Furthermore, since patients had not completely normalized by discharge, treatment strategies should emphasize acceptance of foods varying in macronutrient and caloric content, as intake of a varied diet is of key importance in regaining and maintaining good health. PMID- 8640199 TI - Eating disturbances before and after vertical banded gastroplasty: a pilot study. AB - OBJECTIVE: We interviewed 24 female patients who had undergone vertical banded gastroplasty (VBG) to examine what effect eating disturbances have on weight loss outcome after VBG. METHOD: Subjects were recruited from 120 patients who had undergone VBG in the last 3 1/2 years. Subjects were asked questions relating to past and present weight status, weight loss methods, eating behaviors, and psychiatric status. RESULTS: Eating disturbances before VBG in the form of binge eating disorder (BED), bulimia nervosa (BN), night eating syndrome, and drinking large quantities of fluids were very common (75%), and they tended to persist after VBG. Although VBG led to varying weight loss, some patients exhibited a weight regain as postoperative time increased. Patients currently diagnosed with an eating disturbance were more likely to exhibit weight regain than people without these disturbances. VBG did not affect psychiatric status. DISCUSSION: VBG appears to be successful in producing weight loss in the morbidly obese, but less successful in altering eating behavior or psychiatric status. Future studies should aim to clarify the role of eating behavior in determining the outcome of bariatric surgery and examine whether treatments such as cognitive behavior therapy targeted specifically at the eating disturbance can improve surgical outcome. PMID- 8640200 TI - Seasonality of symptoms in anorexia and bulimia nervosa. AB - OBJECTIVE: Recent research has suggested that a large proportion of patients with bulimia nervosa have seasonal (winter) worsening of mood symptoms similar to seasonal affective disorder (SAD). The objectives of this study were to determine the specificity of this finding in anorexia and bulimia nervosa, and to further delineate the seasonal mood and eating patterns in bulimia nervosa. METHOD: A modified Seasonal Pattern Assessment Questionnaire (SPAQ) was administered to consecutive female patients assessed at an Eating Disorders Clinic with DSM-III-R diagnoses of bulimia nervosa (BN, N = 60) and anorexia nervosa (AN, N = 31), and to female nonclinical comparison subjects (NC, N = 50). RESULTS: The BN group had higher global seasonality scores and more presumptive diagnoses of SAD than the other two groups; the AN patients, whether they had the restricting or binge eating/purging subtype, did not differ from the NC subjects. Thirty-two percent of the identified seasonal BN patients did not have parallel worsening of mood and eating symptoms in the same season. DISCUSSION: These results suggest that seasonality of symptoms is specific to BN and that there may be separate mechanisms for the seasonality of mood and eating symptoms in some BN patients. PMID- 8640201 TI - Comorbidity of binge eating disorder and the partial binge eating syndrome with bipolar disorder. AB - OBJECTIVE: The authors examined the prevalence of binge eating disorder (BED), partial binge eating syndrome, and night binge eating syndrome in subjects with bipolar disorder (BD). METHOD: Sixty-one subjects in whom BD was established using DSM-III-R criteria received a semistructured clinical interview including a detailed description of binge eating behavior and of night binge eating. Frequencies were compared to prevalence estimates in community samples. RESULTS: Eight subjects (13%) met DSM-IV criteria for the diagnosis of BED. An additional 15 subjects (25%) exhibited a partial binge eating syndrome. These two otherwise identical groups of binge eaters were separated only by the DSM-IV frequency criterion. The rates found were higher than rates found in community samples. Ten subjects reported night binge eating in addition to their usual binge eating behavior. This occurred consistently between 2:00 and 4:00 a.m. CONCLUSIONS: Possible underlying mechanisms for the high frequency of binge eating among bipolar subjects are discussed including a model of serotonin-mediated self modulation of mood. The finding of two groups of binge eaters separated only by the frequency criterion raises questions as to whether the frequency criterion as presently defined in DSM-IV is valid or should be modified. PMID- 8640202 TI - Effects of weight cycling on the resting energy expenditure and body composition of obese women. AB - OBJECTIVE: Numerous reports have suggested that cycles of weight loss and regain (i.e., weight cycling) are associated with adverse health consequences, a concern that may lead some obese individuals to forgo weight control efforts. The present study examined whether weight cycling was associated with a reduction in resting energy expenditure (REE) and with increases in both total and upper body fat. METHOD: REE, body composition, and body fat distribution were measured before and after weight loss, and following full weight regain, in 12 women who before treatment had a mean (+/- SEM) age of 38.8 +/- 3.4 years and weight of 98.0 +/- 3.2 kg. RESULTS: At the end of treatment, patients lost 18.9 +/- 2.6 kg which was comprised of significant decreases in body fat and fat-free mass of 15.2 +/- 2.2 and 3.7 +/- 0.8 kg, respectively (both ps < .001). REE also fell during this time from 1,631 +/- 82 to 1,501 +/- 51 kcal/d (p < .03). All of these measures, however, returned to their baseline values when patients regained their lost weight. Body fat distribution was unchanged throughout the study. DISCUSSION: These results do not support claims that weight cycling adversely affects REE, body composition, or body fat distribution. PMID- 8640203 TI - Season of birth and eating disorders. AB - A statistically significant season of birth variation is found in an unselected nationwide sample of 1,939 eating disorders patients, with peak season of birth occurring in May. However, among younger patients (n = 882), peak season of birth is in March, which is statistically significantly different to that expected from the general population season of birth cycle. This finding may imply links between etiology of earlier-onset eating disorders and the psychoses; similar first quarter peak seasonal patterns of birth have been found in schizophrenic and affective psychoses--with birth peaks in January and February. In contrast, for the neuroses and personality disorders, birth peaks have been found to be in June and August, similar to the June birth peak found in this study for later onset eating disorders (n = 1,057), which was not statistically significantly different to season of birth peaks expected from general population data. PMID- 8640205 TI - Weight and shape overconcern and emotional eating in binge eating disorder. AB - OBJECTIVE: This study investigated two issues: the level of weight and shape concerns, and the self-reported tendency to eat in response to negative emotions among obese individuals with binge eating disorder (BED), eating disorder not otherwise specified (EDNOS), and no eating disorder (CONTROL). METHOD: On the basis of demographic and diagnostic surveys, 156 participants in a weight loss program were categorized on two dimensions, eating disorder category and weight (BED vs. EDNOS vs. CONTROL/low vs. high body mass index), yielding a 2 x 3 experimental design. RESULTS: Individuals with BED reported a greater tendency to eat in response to negative mood states than CONTROL subjects and low weight EDNOS subjects, but not high weight EDNOS subjects. Weight did not influence self reported weight and shape concerns. Individuals with BED expressed greater concern for weight and shape than non-eating disordered CONTROLs. DISCUSSION: The findings suggest that overconcern with weight and shape be further investigated as a diagnostic feature of BED and that emotional eating is associated with BED but not obesity per se. PMID- 8640206 TI - On the relationship between dieting and "obese" and bulimic eating patterns. AB - OBJECTIVE: Relationships were studied between emotional, external and restrained eating behavior, and bulimia, and also between these types of eating behavior and body dissatisfaction and drive for thinness. METHOD: The sample consisted of female adolescents. Eating behavior and body evaluation were measured with scales of the Dutch Eating Behaviour Questionnaire (DEBQ) and the Eating Disorders Inventory (EDI). RESULTS: Significant and high relationships were found between emotional and external and bulimic eating behavior. The same was true for restrained eating, and body dissatisfaction and drive for thinness. However, results from factor analyses suggest that the three types of overeating do not point to one and the same construct. Further, also restrained eating was found to point at a different construct than the construct associated with body dissatisfaction and drive for thinness. Finally, no high relationships were found between restrained eating and "obese" or bulimic eating patterns. DISCUSSION. The low relationship between restrained eating and various types of overeating is in line with earlier results with the DEBQ Restraint scale and offers further support for the contention that the excessive food intake found in subjects with high scores on the Herman and Polivy's Restraint Scale (RS) may be an artefact of the RS, as a result of its bias towards the selection of a counterregulating sample. PMID- 8640204 TI - Structure of disordered eating in a twin community sample. AB - OBJECTIVE: To describe the types of eating problems experienced by women in an Australian twin population. METHOD: Questions assessing preoccupation with weight or shape, use of various methods of weight control, difficulties with weight control, disordered eating, or binging, were administered to a group of 3,869 female twins. Exploratory and confirmatory factor analyses were used to summarize and describe problematic eating behaviors. RESULTS: For those women experiencing problems with eating, five groups could be identified. These were overweight women who were dissatisfied with their weight and shape, underweight women struggling with anorexic behaviors, women who were having problems with binging, women who were using more extreme methods of weight control such as vomiting, laxatives, and starvation, and overweight women who were using slimming and fluid tablets for weight control. Confirmatory factor analysis suggested that this factor model is an acceptable fit to the data and that the factor structure generalizes well across two groups viz, the first-born and second-born twins. DISCUSSION: It was concluded that future studies aiming to develop a general description of eating problems in the community should specifically assess the purging behaviors used by women. PMID- 8640207 TI - The eating attitudes test and the eating disorders inventory in four Bulgarian clinical and nonclinical samples. AB - OBJECTIVE: As part of the International Collaborative Study of Eating Disorders in Adolescence, with sites in West and East Berlin, Zurich, and Sofia, eating attitudes and disorders were studied in four Bulgarian samples. METHOD: The Eating Attitude Test (EAT) and the Eating Disorders Inventory (EDI) were used in this study. Besides a clinical group of anorectic patients, students attending either a special secondary school, a standard secondary school, or a sports school participated in this study. RESULTS: Findings revealed excellent discriminant validity of the EAT total score and the three subscales. This also applied to four out of eight subscales of the EDI. Discriminant validity was less satisfactory in three further scales and entirely missing for Maturity Fears. DISCUSSION: The findings are discussed in a transcultural perspective. PMID- 8640209 TI - Simple minds and complex traits. PMID- 8640208 TI - Treating bulimia nervosa in primary care: a pilot study. AB - OBJECTIVE: To develop and evaluate a simplified and condensed cognitive behavioral treatment for bulimia nervosa suitable for use in primary care. METHOD: Once developed, the treatment was tested on a consecutive series of 11 patients. RESULTS: Six patients did well, 3 were nonresponders, and in the other 2 external events interfered with progress. DISCUSSION: This form of treatment may benefit a significant proportion of those patients with bulimia nervosa who are seen in primary care. It can be administered by nonspecialist therapists. PMID- 8640210 TI - Drosophila inherit diseases. PMID- 8640211 TI - Changing tack on the map. PMID- 8640212 TI - Why mice drink. PMID- 8640213 TI - Tapping into tumours. PMID- 8640214 TI - PAX6 missense mutation in isolated foveal hypoplasia. PMID- 8640215 TI - Research in India. PMID- 8640216 TI - CNTF and psychiatric disorders. PMID- 8640217 TI - CNTF and psychiatric disorders. PMID- 8640218 TI - Biotinidase mutational "hotspot'. PMID- 8640219 TI - Identification of sex-specific quantitative trait loci controlling alcohol preference in C57BL/ 6 mice. AB - Mice from various inbred strains consume alcoholic beverages at highly reproducible and strain-specific levels. While most mice consume alcohol in moderate amounts, C57BL/6J animals exhibit sustained oral ingestion of high levels of alcohol in the presence of competing water and food. We now report a genetic investigation of this phenotype as one potential model for alcoholism. An intercross-backcross breeding protocol was used to identify two recessive alcohol preference quantitative trait loci (QTLs) that are both sex-restricted in expression. A comparison of our results with those of an earlier morphine preference study argues against the hypothesis of a single unified phenotype defined by a preference for all euphoria-producing drugs. PMID- 8640220 TI - Minisatellite diversity supports a recent African origin for modern humans. AB - In a study of human diversity at a highly variable locus, we have mapped the internal structures of tandem-repetitive alleles from different populations at the minisatellite MS205 (D16S309). The results give an unusually detailed view of the different allelic structures represented on modern human chromosomes, and of the ancestral relationships between them. There was a clear difference in allelic diversity between African and non-African populations. A restricted set of allele families was found in non-African populations, and formed a subset of the much greater diversity seen on African chromosomes. The data strongly support a recent African origin for modern human diversity at this locus. PMID- 8640221 TI - A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2. AB - Non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is a common disorder of middle-aged individuals characterized by high blood glucose levels which, if untreated, can cause serious medical complications and lead to early death. Genetic factors play an important role in determining susceptibility to this disorder. However, the number of genes involved, their chromosomal location and the magnitude of their effect on NIDDM susceptibility are unknown. We have screened the human genome for susceptibility genes for NIDDM using non-and quasi parametric linkage analysis methods in a group of Mexican American affected sib pairs. One marker, D2S125, showed significant evidence of linkage to NIDDM and appears to be a major factor affecting the development of diabetes mellitus in Mexican Americans. We propose that this locus be designated NIDDM1. PMID- 8640222 TI - Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching. AB - Cross-species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty-six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the "text string' option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification. PMID- 8640223 TI - A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. AB - X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man. PMID- 8640224 TI - Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. AB - Inherited hypokalaemic alkalosis with low blood pressure can be divided into two groups-Gitelman's syndrome, featuring hypocalciuria, hypomagnesaemia and milder clinical manifestations, and Bartter's syndrome, featuring hypercalciuria and early presentation with severe volume depletion. Mutations in the renal Na-Cl cotransporter have been shown to cause Gitelman's syndrome. We demonstrate linkage of Bartter's syndrome to the renal Na-K-2Cl cotransporter gene NKCC2, and identify frameshift or non-conservative missense mutations for this gene that co segregate with the disease. These findings demonstrate the molecular basis of Bartter's syndrome, provide the basis for molecular classification of patients with inherited hypokalaemic alkalosis, and suggest potential phenotypes in heterozygous carriers of NKCC2 mutations. PMID- 8640225 TI - Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. AB - Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair. PMID- 8640226 TI - Expanded polyglutamine in the Machado-Joseph disease protein induces cell death in vitro and in vivo. AB - Recently, we identified a novel gene, MJD1, which contains an expanded CAG triplet repeat in Machado-Joseph disease. Here we report the induction of apoptosis in cultured cells expressing a portion of the MJD1 gene that includes the expanded CAG repeats. Cell death occurs only when the CAG repeat is translated into polyglutamine residues, which apparently precipitate in large covalently modified forms. We also created ataxic transgenic mice by expressing the expanded polyglutamine stretch in Purkinje cells. Our results demonstrate the potential involvement of the expanded polyglutamine as the common aetiological agent for inherited neurodegenerative diseases with CAG expansions. PMID- 8640227 TI - Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse. AB - Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis. PMID- 8640228 TI - A functionally defective allele of TAP1 results in loss of MHC class I antigen presentation in a human lung cancer. AB - Tumours express a variety of novel epitopes which represent potential immune targets, and thus clinically evident tumours are thought to have effectively avoided immune recognition and elimination. Transporters associated with antigen presentation (TAP) are thought to be responsible for conveying intracellular peptides into the endoplasmic reticulum for complex formation with class I MHC and subsequent recognition by cytotoxic T lymphocytes. In this study, we evaluated 79 human solid tumours and cell lines for genetic abnormalities in TAP1 that might have led to an acquired loss of antigen presenting ability. A novel sequence (R659Q) was discovered near the ATP binding site in a human small cell lung cancer (SCLC) cell line, H1436. This cell line is heterozygous for this allele, but only the R659Q allele is transcribed into RNA. Even though the R659Q protein is expressed, these cells act as if they were TAP deficient by peptide binding and antigen presentation studies, which are restored after transfection of a functional TAP1 allele. This is the first evidence for a naturally occuring protein structural defect resulting in defective peptide transport in a human solid tumour. PMID- 8640229 TI - Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis. AB - Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect. PMID- 8640231 TI - Characterization of DRP2, a novel human dystrophin homologue. AB - The currently recognised dystrophin protein family comprises the archetype, dystrophin, its close relative, utrophin or dystrophin-related protein (DRP), and a distantly related protein known as the 87K tyrosine kinase substrate. During the course of a phylogenetic study of sequences encoding the characteristic C terminal domains of dystrophin-related proteins, we identified an unexpected novel class of vertebrate dystrophin-related sequences. We term this class dystrophin-related protein 2 (DRP2), and suggest that utrophin/DRP be renamed DRP1 to simplify future nomenclature. DRP2 is a relatively small protein, encoded in man by a 45 kb gene localized to Xq22. It is expressed principally in the brain and spinal cord, and is similar in overall structure to the Dp116 dystrophin isoform. The discovery of a novel relative of dystrophin substantially broadens the scope for study of this interesting group of proteins and their associated glycoprotein complexes. PMID- 8640230 TI - A murine model of Menkes disease reveals a physiological function of metallothionein. AB - Human Menkes disease and the murine Mottled phenotype are X-linked diseases that result from copper deficiency due to mutations in a copper-effluxing ATPase, designated ATP7A. Male mice with the Mottled-Brindled allele (Mo-brJ) accumulate copper in the intestine, fail to export copper to peripheral organs and die a few weeks after birth. Much of the intestinal copper is bound by metallothionein (MT). To determine the function of MT in the presence of Atp7a deficiency, we crossed Mo-brJ females with males that bear a targeted disruption of the Mt1 and Mt2 genes (Mt-/-). On an Mt -/- background, most Mo-brJ males as well as heterozygous Mo-brJ females die before embryonic day 11. The lethality in Mo-brJ females can be explained by preferential inactivation of the paternal X chromosome in extraembryonic tissues and resultant copper toxicity in the absence of MT. In support of this hypothesis, cell lines derived from Mt -/-, Mo-brJ embryos are very sensitive to copper toxicity. PMID- 8640232 TI - A synaptobrevin-like gene in the Xq28 pseudoautosomal region undergoes X inactivation. AB - The X and Y chromosomes that maintain human dimorphism are thought to have descended from a single progenitor, with the Y chromosome becoming largely depleted of genes. A number of genes, however, retain copies on both X and Y chromosomes and escape the inactivation that affects most X-linked genes in somatic cells. Many of those genes are present in two pseudoautosomal regions (PARs) at the termini of the short (p) and long (q) arms of the sex chromosomes. For both PARs, pairing facilitates the exchange of information, ensuring the homogenisation of X and Y chromosomal material in these regions. We report here a strikingly different regulation of expression of a gene in Xq PAR. Unlike all Xp PAR genes studied so far, a synaptobrevin-like gene, tentatively named SYBL1, undergoes X inactivation. In addition, it is also inactive on the Y chromosome, thereby maintaining dosage compensation in an unprecedented way. PMID- 8640233 TI - Sex reversal by loss of the C-terminal transactivation domain of human SOX9. AB - Haploinsufficiency for SOX9 has recently been identified as the cause for both campomelic dysplasia (CD), a human skeletal malformation syndrome, and the associated autosomal XY sex reversal. SOX9 contains a putative DNA-binding motif known as the high-mobility group (HMG) domain characterizing a whole class of transcription factors. We show in cell transfection experiments that SOX9 can transactivate transcription from a reporter plasmid through the motif AACAAAG, a sequence recognized by other HMG domain transcription factors. By fusing all or part of SOX9 to the DNA-binding domain of yeast GAL4, the transactivating function was mapped to a transcription activation (TA) domain at the C terminus of SOX9. This non-acidic TA domain is evolutionarily conserved and rich in proline, glutamine and serine. With one exception, all SOX9 nonsense and frame shift mutations described so far in CD/sex reversal patients lead to truncation of the TA domain, suggesting that impairment of gonadal and skeletal development in these cases results, at least in part, from loss of transactivation of genes downstream of SOX9. PMID- 8640234 TI - Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. AB - The longitudinal growth of the skeleton arises from the continuous process of endochondral ossification occurring at the ends of growing long bones. Dwarfism results when this process is disrupted, as in the autosomal dominant human skeletal diseases hypochondroplasia (HCH), achondroplasia (ACH) and thanatophoric dysplasia (TD). Interestingly, these disorders display a graded spectrum of phenotypic severity and are the result of distinct missense mutations in the fibroblast growth factor receptor 3 gene (FGFR3). TD, characterized by neonatal lethality and profound dwarfism, is the result of FGFR3 mutations, including an R248C substitution in the extracellular domain or a K650E substitution in the tyrosine kinase (TK) domain. ACH, which is non-lethal and presents less severe dwarfism, results almost exclusively from a G380R substitution in the transmembrane domain. Homozygous achondroplasia resembles the phenotype of TD. In this report the effect of the ACH and TD mutations on the activity and regulation of FGFR3 are analysed. We showed that each of the mutations constitutively activate the receptor, as evidenced by ligand-independent receptor tyrosine phosphorylation and cell proliferation. Moreover, the mutations that are responsible for TD were more strongly activating than the mutation causing ACH, providing a biochemical explanation for the observation that the phenotype of TD is more severe than that of ACH. PMID- 8640235 TI - BRCA2 mutations in primary breast and ovarian cancers. AB - The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1. PMID- 8640236 TI - Low incidence of BRCA2 mutations in breast carcinoma and other cancers. AB - Inherited mutant alleles of familial tumour suppressor genes predispose individuals to particular types of cancer. In addition to an involvement in inherited susceptibility to cancer, these tumour suppressor genes are targets for somatic mutations in sporadic cancers of the same type found in the familial forms. An exception is BRCA1, which contributes to a significant fraction of familial breast and ovarian cancer, but undergoes mutation at very low rates in sporadic breast and ovarian cancers. This finding suggests that other genes may be the principal targets for somatic mutation in breast carcinoma. A second, recently identified familial breast cancer gene, BRCA2 (refs 5-8), accounts for a proportion of breast cancer roughly equal to BRCA1. Like BRCA1, BRCA2 behaves as a dominantly inherited tumour suppressor gene. Individuals who inherit one mutant allele are at increased risk for breast cancer, and the tumours they develop lose the wild-type allele by heterozygous deletion. The BRCA2 coding sequence is huge, composed of 26 exons that span 10,443 bp. Here we investigate the rate of BRCA2 mutation in sporadic breast cancers and in a set of cell lines that represent twelve other tumour types. Surprisingly, mutations in BRCA2 are infrequent in cancers including breast carcinoma. However, a probable germline mutation in a pancreatic tumour cell line suggests a role for BRCA2 in susceptibility to pancreatic cancer. PMID- 8640237 TI - Mutation analysis in the BRCA2 gene in primary breast cancers. AB - Breast cancer, one of the most common and deleterious of all diseases affecting women, occurs in hereditary and sporadic forms. Hereditary breast cancers are genetically heterogeneous; susceptibility is variously attributable to germline mutations in the BRCA1 (ref. 1), BRCA2 (ref. 2), TP53 (ref. 3) or ataxia telangiectasia (ATM) genes, each of which is considered to be a tumour suppressor. Recently a number of germline mutations in the BRCA2 gene have been identified in families prone to breast cancer. We screened 100 primary breast cancers from Japanese patients for BRCA2 mutations, using PCR-SSCP. We found two germline mutations and one somatic mutation in our patient group. One of the germline mutations was an insertion of an Alu element into exon 22, which resulted in alternative splicing that skipped exon 22. The presence of a 64-bp polyadenylate tract and evidence for an 8-bp target-site duplication of the inserted DNA implied that the retrotransposal insertion of a transcriptionally active Alu element caused this event. Our results indicate that somatic BRCA2 mutations, like somatic mutations in the BRCA1 gene, are very rare in primary breast cancers. PMID- 8640238 TI - A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families. AB - Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is an uncommon inherited disorder characterized by salt-wasting and end-organ unresponsiveness to mineralocorticoids. A complete genome search using homozygosity mapping in eleven consanguineous families with PHA1 provided conclusive evidence of linkage with heterogeneity. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. These two chromosomal regions harbour the genes encoding the three subunits of the human amiloride sensitive epithelial sodium channel (hENaC): SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Our linkage results have been further supported by the recent report of mutations in the alpha and beta subunit genes in PHA1 patients. We now report the identification of a 3' splice site mutation in SCNN1G (318-1 G-->A) in three families showing linkage to 16p. Abnormal splicing results with the production of two messenger RNAs, one arising from activation of an adjacent cryptic splice site and the other from skipping of the downstream exon. The two corresponding mutant gamma hENaC subunits are predicted to have three highly conserved amino acids in the extracellular domain replaced by a novel amino acid (KYS106-108-->N) and truncation from 649 to 134 amino acids respectively. These three families all originate from the Indian sub-continent and the probands have severe generalized PHA. They share a common haplotype which suggests the presence of a founder mutation in this sub-population. PMID- 8640239 TI - Biochemical bases of the pharmacological action of the flavonoid silymarin and of its structural isomer silibinin. AB - The flavonoid silymarin and one its structural components, silibinin, have been well characterized as hepato-protective substances. However, little is known about the biochemical mechanisms of action of these substances. This review deals with recent investigations to elucidate the molecular action of the flavonoid. Three levels of action have been proposed for silymarin in experimental animals: a) as an antioxidant, by scavenging prooxidant free radicals and by increasing the intracellular concentration of the tripeptide glutathione; b) regulatory action of the cellular membrane permeability and increase of its stability against xenobiotic injury; c) at the nuclear expression, by increasing the synthesis of ribosomal RNA by stimulating DNA polymerase I and by exerting a steroid-like regulatory action on DNA transcription. The specific hepatoprotective action of silibinin against the toxicity of ethanol, phenylhydrazine and acetaminophen is also discussed. It is suggested that the biochemical effects observed for the flavonoid in experimental models may settle the basis for understanding the pharmacological action of silymarin and silibinin. PMID- 8640240 TI - Comparison of the effect of hypothalamic neuropeptides upon luteinizing hormone secretion by cultured rat anterior pituitary cells. AB - Bovine median eminence contains a factor difference from gonadotropin-releasing hormone (GnRH) than increases basal luteinizing hormone (LH) secretion and potentiates GnRH-stimulated LH release. We compared the effect of hypothalamic neuropeptides on basal and GnRH-stimulated LH secretion using rat pituitary cells under static incubation conditions to determine if any of them mimics the LH releasing activity no attributable to GnRH present in bovine median eminence extracts. Both, galanin and neurotensin (10(-9)-10(-5)) stimulated basal LH secretion in a dose-response manner. Galanin increased 3-4 fold and neurotensin doubled the basal LH secretion. The GnRH antagonist Nal-Glu 10(-6) M abolished the effect of 10(-7) M GnRH and 10(-5)M neurotensin, but did not block the LH releasing activity of galanin. Leucin-enkephalin, beta-endorphin, substance P and neuropeptide Y (NPY) did not alter basal LH secretion. Neuropeptides produced three types of response on GnRH-stimulated LH release. First, leucine-enkephalin and beta-endorphin (10(-9)-10(-5) M) showed a dose-dependent inhibition of GnRH stimulated LH release. At 10(-5) M the inhibition was complete with leucine enkephalin and only 30% with beta-endorphin. Both were blocked by naloxone. Second, substance P showed an inverted U type response on GnRH-stimulated LH secretion. At 10(-9) M this peptide potentiated the action of GnRH. This effect decreased when the dose of substance P was increased to 10(-7) M and turned inhibitory at 10(-5) M when 10(-7) M GnRH was used. Third, galanin and NPY potentiated the effect of GnRH on LH secretion. Neurotensin had no effect on GnRH stimulated LH release. In conclusion, rat gonadotrophs present diverse responses to neuropeptides at physiological concentrations, and -apart from GnRH-galanin is most likely the other factor present in bovine median eminence extracts that stimulates LH secretion. The data lend further support to a role of galanin in the control of LH secretion. PMID- 8640241 TI - Number, size and distribution of ganglion neurons in urinary bladder of rodents. AB - Whole-mount preparations of urinary bladders stained with a modified Giemsa technique were obtained from three rodent species (Guinea-pig, Calomys callosus and the C57/BLJ isogenic mouse) to identify and estimate the relative number and size of ganglionic neurons within the wall of the organ. The distribution of the ganglia was not uniform among the three species: ganglia were concentrated around the ureteral orifices in the Guinea-pig, they were scattered throughout the organ in Calomys callosus, and they were concentrated near the internal urethral orifice in the C57/BLJ mouse. In the Guinea-pig, the size of approximately 50% of the neurons lie in the range of 200 to 300 microns2. In Calomys callosus, 40% of the neurons lie in the range of 200 to 250 microns2, with 28% in the range of 50 to 100 microns2. For the C57/BLJ mouse, approximately 60% of the neurons have an area of 250 to 400 microns2. PMID- 8640242 TI - Stress-induced hyperglycemia and hypoinsulinemia are suppressed by sulfonylurea. Predominant role of insulin. AB - Based on the in vitro blockade of adrenal catecholamines release by sulfonylurea, we searched for an anti-stress activity of this drug. Stress-induced hyperglycemia and insulin inhibition were employed as adrenergic stress indicators. A standard dose of the oral sulfonylurea glipizide (200 micrograms/100 g), administered 15 min before a 1-h restraint stress to intact or 80% pancreatectomized rats, produced total suppression of the stress-induced hyperglycemia-hypoinsulinemia, an effect followed by a significant post-stress hypoglycemia of 1 h duration. The latter effect was elicited by all the sulfonylureas assayed. In the 80% pancreatectomized rats, glipizide nearly halved the increases in plasma catecholamines at 30 min of stress, but did not modify those attained at 60 min, when glycemia was decreasing and insulinemia was still increasing. Moreover, behavioral experiments in intact stressed rats showed that the adrenergic overt behavior inhibition caused by propranolol was not produced either by glipizide or insulin, reinforcing that glipizide affect was not mediated by catecholamine inhibition. These findings suggest a blockade of catecholamines hepatic receptors by the anticipated insulin release induced by sulfonylurea. Thus, insulin fully dominated when insulin and catecholamines were administered in a stress-like sequence. A confirmation of these findings in diabetic patients subjected to surgical stress would allow a new therapeutic application of sulfonylurea. It is concluded that an anticipated insulin release plus an insulin dominant role over catecholamines activity might explain the anti stress effect of sulfonylurea. PMID- 8640243 TI - Phrenic nerve activity during artificial ventilation at different body temperatures and its relationships with carotid chemosensory activity. AB - While the chemoreceptor discharges of carotid bodies in vitro are highly dependent on temperature, these chemoreceptors in situ contribute only moderately to the ventilatory adjustment to changing body temperature (Tb), probably because of the concomitant and reverse changes in natural chemoreceptor stimuli in closed loop preparations. Accordingly, we studied the frequency of carotid chemosensory discharge (fx) and the phrenic integrated electroneurogram (IENGph) in pentobarbitone anesthetized cats, paralyzed with alcuronium and artificially ventilated, at three steady-state levels of Tb (35.5, 37.5 and 40.2 degrees C), modifying the frequency and volume of the ventilator to maintain PETCO2 within normal range. While fx increases along with Tb when PETCO2 is allowed to fluctuate freely, its mean basal value was not consistently different at the three Tb's studied under controlled conditions. The amplitude of IENGph was reduced and the frequency of phrenic inspiratory cycles was increased as Tb was raised from 35.5 to 37.5 degrees C and then to 40.2 degrees C. Brief 100% O2 inhalations and i.v. injections of dopamine produced minimal depressions of IENGph amplitude in hypothermia, but pronounced although similar depressions in normothermia and hyperthermia. i.v. injections of NaCN augmented fx and IENGph in dose related manner, and the relationships between both variables showed larger changes in IENGph at the hypothermic and normothermic conditions when expressed in absolute terms, but not when expressed in relative terms. Thus, the chemosensory input is not consistently modified by thermal levels under controlled ventilatory conditions, but the chemosensory drive of the ventilatory output is less pronounced in hypothermia. The chemosensory input is similarly affected by varying degrees of cytotoxic hypoxia at different Tb's, but the ventilatory output is less vigorously increased in hyperthermia, pointing to a decreased reflex gain in that condition. PMID- 8640244 TI - Alexander Lipschutz (1883-1980). A biographical synopsis. AB - Alexander Lipschutz was born in Riga, Latvia, in 1883. He obtained his M.D. from the University of Gottingen, Germany, in 1901. He conducted research at the Universities of Zurich, Bonn, Gottingen, Bern and Vienna. He was full Professor of Physiology at the Universities of Dorpart (1919-1926), in Tartu, Estonia, and Concepcion (1927-1936), in Chile. Later, he became the first Director of the Institute of Experimental Medicine, of the Chilean National Health Service. He authored 22 books and a large number of scientific papers, mostly on Endocrinology and Oncology. He directed 16 medical theses at the University of Concepcion and 81 at the University of Chile. He was awarded with the first Chilean National Prize in Science (1969). He died in Santiago, Chile, in 1980. PMID- 8640245 TI - Epistemic restrictions in population biology. AB - Biologists have believed that the application of statistical or mathematical models to population biology has always been a correct and helpful tool to acquire knowledge. The present article demonstrates that the standard interpretation of statistical results yielded by the application of mathematical models to some populational processes, not only hides knowledge, but may lead to wrong knowledge. These epistemic restrictions (type I and II errors). A new more versatile conditional interpretation of statistical results is proposed. PMID- 8640246 TI - Biological sciences in Chile and South America, 1981-1991: a citationist perspective. Output data and specialty area impact trends. AB - The purpose of this report is to examine the biological sciences in Chile and South America in bibliographic terms -the number of papers each nation published from 1981-1991 and the number of citations to them in the international research literature. The database consists of 34,600 biological science papers from Argentina, Brazil, Chile, and Venezuela in the 1981-1991 Science Citation Index files of the Institute for Scientific Information. Twelve specialty areas were selected to represent the biological sciences of special interest to Chile: animal sciences, biochemistry/biophysics, environmental sciences, experimental biology/medicine, immunology, microbiology/cell biology, molecular biology/genetics, neurosciences, pharmacology, physiology, plant sciences, and reproductive sciences. Data are reported on the number of papers in these fields, combined, by authors based in Chile and other South American nations. In addition, time-series trends in the impact (average citations per paper) of Chilean research relative to South America as a whole, overall and in each specialty, are presented and discussed. PMID- 8640247 TI - Finance ... U.S. acute care hospitals spent less in indigent care in 1994. PMID- 8640248 TI - Managed care. Currying AARP's favor. PMID- 8640250 TI - On the Internet. Security alert! PMID- 8640251 TI - Information systems ... health information networks could soon be linked. PMID- 8640249 TI - Payment ... Medicare reimbursement comes up short in covering the cost of total hip replacements. PMID- 8640252 TI - Public hospitals ... New York City's public hospital system is headed for a "near meltdown". PMID- 8640254 TI - Build it, and they might come. PMID- 8640253 TI - Buyouts & ventures. Selling ... or selling out. AB - Charity begins at home, yet across the country hometown not-for-profit hospitals are forming joint partnerships with investor-owned systems. In some of these cases no foundation is created to continue the hospital's good deeds. In this special report, Hospitals & Health Networks looks at the issues and asks, "Do these joint ventures need more public scrutiny?" PMID- 8640255 TI - Managed care. A capital way to put doctors in charge. PMID- 8640256 TI - In the OR. The humane touch. PMID- 8640258 TI - Virtual training. A high-tech dress rehearsal. PMID- 8640259 TI - HospitalPulse ... January 1996. PMID- 8640257 TI - Medicine & law. Tightening the chain of evidence. PMID- 8640260 TI - Fixing the system. Round two in Kentucky's reform fight. PMID- 8640261 TI - Tuning consumers in to healthy programming. PMID- 8640264 TI - Utilization ... Blue Cross/Blue Shield Association. PMID- 8640262 TI - Voters concerned about health care. PMID- 8640263 TI - Payment ... Operation Restore Trust. PMID- 8640265 TI - Home health's growth spurt. PMID- 8640266 TI - There's no business like big business. PMID- 8640267 TI - Leaps of faith. PMID- 8640269 TI - Betting on health care. PMID- 8640268 TI - Betting against health care. AB - Health care firms of all types helped fuel the biggest short-selling frenzy in the New York Stock Exchange's history, recently hitting a record 2.2 billion shares. While some analysts say this means nothing, the fact is that many investors are "shorting" the stock; in other words, they're betting against it. What appears as a lack of confidence may be nothing more than a simple quirk of Wall Street. Good, bad or indifferent, selling short is no tall tale. PMID- 8640271 TI - Ready. Set. Wait! PMID- 8640270 TI - The storm before the qualm. AB - More than half of all changes in organizations fail. But there is hope, suggests Rick Maurer, author of ?Beyond the Wall of Resistance.? He says that it isn't resistance itself--since resistance is nothing more than people's need to protect themselves from harm--but how you handle it that creates the problem. Here in this exclusive excerpt, he argues that you shouldn't try to overcome resistance, but embrace it. PMID- 8640272 TI - Partnerships. Synergy in Seattle. PMID- 8640273 TI - Ethics. The moralsmobile rolls in. PMID- 8640274 TI - Policy. A lean, mean academic medicine machine. PMID- 8640275 TI - Executive chartbook. 8 steps to making capitation work. PMID- 8640276 TI - Information systems. California's doing more than dreaming. PMID- 8640277 TI - Physicians. Help wanted. PMID- 8640278 TI - State reform. Will ERISA spell relief for small businesses? PMID- 8640279 TI - Outreach. How race colors breast cancer. PMID- 8640280 TI - Influenza and respiratory syncytial virus surveillance. PMID- 8640281 TI - The future of endocrinology in Europe--a view from across La Manche on connection to the mainland. PMID- 8640282 TI - Birth defects in diabetic pregnancies: where do we go from here? PMID- 8640283 TI - Aminoguanidine and diabetic neuropathy. PMID- 8640284 TI - Measurement of activin in physiological fluids. PMID- 8640286 TI - Functional aspects of the pineal hormone melatonin in combating cell and tissue damage induced by free radicals. PMID- 8640287 TI - Knockout mice lacking GLUT4 glucose transporters. PMID- 8640285 TI - Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements. AB - Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate. PMID- 8640288 TI - Dichlorodiphenyltrichloroethane and androgen receptor. PMID- 8640289 TI - Endocrinology meets immunology: T lymphocytes as novel targets for melatonin. PMID- 8640290 TI - Intercellular chatter: osteoblasts, osteoclasts and interleukin 6. PMID- 8640291 TI - Insulin-like growth factor binding protein 3 (IGFBP-3): a novel target of the tumor suppressor p53 inhibiting cell growth. PMID- 8640292 TI - New mechanisms of insulin resistance at the nuclear level. PMID- 8640293 TI - Gonadal function after multimodality treatment in men with testicular germ cell cancer. AB - We evaluated gonadal function in 63 patients with testicular cancer both within 1 month of unilateral orchiectomy before further treatment (pretreatment) and 3 years after treatment discontinuation (post-treatment). Sixteen patients underwent orchiectomy alone (group 1), nine patients underwent infradiaphragmatic radiotherapy (group 2) and 28 patients received four cycles (group 3) and 10 patients received six cycles (group 4) of cisplatin-based chemotherapy (cisplatin, vinblastine and bleomycin-PVB, or cisplatin, etoposide and bleomycin PEB). Pretreatment semen analyses showed reduced sperm cell density, motility and impaired morphology of spermatozoa in all four groups (p > 0.05). At the same time elevated estradiol and decreased serum follicle-stimulating hormone (FSH) levels in 28.5% of subjects were correlated with high serum beta human chorionic gonadotropin concentrations. Semen analyses revealed the lowest values for all parameters after infradiaphragmatic radiotherapy. Sperm cell count, motility and morphology were significantly better in patients treated with orchiectomy alone or with a conventional dose of chemotherapy than in the groups that received radiotherapy or high doses of chemotherapy (p < 0.05). We also observed a correlation between serum FSH values and sperm cell density for both pretreatment and post-treatment in every group of patients (p < 0.05). Persistent subclinical Leydig cell dysfunction in groups treated with radiotherapy or high doses of chemotherapy was expressed by increased basal luteinizing hormone levels (78% of patients in group 2 vs 60% of patients in group 4) (p < 0.05) and by normal testosterone serum values (89% of patients in group 2 vs 80% of patients in group 4). Spermatogenesis and Leydig cell function are, therefore, persistently impaired in the majority of testicular cancer patients treated with radiotherapy or with more intensive chemotherapy. PMID- 8640294 TI - Circulating levels of immunoreactive parathyroid hormone-related protein and intact parathyroid hormone in human fetuses and newborns. AB - Undetectable or extremely low levels of circulating immunoreactive parathyroid hormone (PTH) have been reported in human newborns while PTH bioactivity was high. This prompted the hypothesis that the fetal calcemic hormone might be PTH related protein. The purpose of this study was to measure circulating immunoreactive PTH-related protein in human fetuses and newborns in order to investigate this hypothesis. Parathyroid hormone-related protein (PTHrP(1-86) and intact PTH were measured using two-site immunoradiometric assays in plasma obtained by cordocentesis from 23 fetuses (19-33 weeks of gestation), from 17 newborns at term (38-41 weeks), from their mothers and from 22 normal women of reproductive age. Plasma PTHrP was detectable in all but one of the fetuses and newborns and in all the mothers and the controls. The mean level was similar among fetuses (19-33 weeks) (0.43 +/- 0.18 pmol/l), newborns (0.48 +/- 0.12), mothers (0.48 +/- 0.14) and normal controls (0.46 +/- 0.09). Plasma PTH was found to be significantly higher in fetuses at midgestation (1.0 +/- 0.99 pmol/l) than in the newborns (0.22 +/- 0.21) (p < 0.0025); maternal PTH was significantly higher compared to fetal level at mid-gestation (2.1 +/- 1.0, p < 0.01) as well as at term (2.69 +/- 1.40, p < 0.001). In the control women PTH was 3.07 +/- 1.25 pmol/l. These results showed that plasma amino-terminal PTHrP-(1-86) is detectable during the second half of human fetal life and its level remains unchanged during this period of time, in contrast to changing levels of fetal plasma PTH. The relatively low PTHrP-(1-86) level that we found in the newborns is not responsible for the high PTH-like bioactivity found by some investigators in cord blood at term. PMID- 8640295 TI - Earliest prevention of endemic goiter by iodine supplementation during pregnancy. AB - During pregnancy complex changes of maternal thyroid function occur and they are influenced by the maternal iodine supply. It has been demonstrated that with decreasing iodine supply maternal goiter and hypothyroxinemia as well as fetal and neonatal hypothyroidism become more prevalent. Therefore iodine supplementation during pregnancy is now strongly recommended also in areas of moderate iodine deficiency. To monitor the success of iodine supplementation and its theoretical risk of increasing the frequency of thyroid autoantibodies, we have investigated the thyroid volume, thyroid function, urinary iodine excretion and antibodies to thyroid peroxidase at 10-12 weeks of gestation and postpartum in 38 mothers receiving 300 micrograms potassium iodide/day and in 70 mothers without iodine supplementation. In all of their newborns thyroid volume was determined by ultrasound. The thyrotropin (TSH) levels and antibodies to thyroid peroxidase (TPO-ab) in the neonates were measured in dried blood spots on filter paper from their newborn screening. Urinary iodine excretion was increased significantly after iodine supplementation in mothers (p < 0.001) and their newborns (< 0.05). No hypo- or hyperthyroidism was observed in the mothers or newborns. Interestingly, no difference of maternal thyroid volumes was observed between the two groups after pregnancy, but the volumes of the thyroid glands in newborns of mothers who received iodine were significantly (p < 0.004) lower (0.7 +/- 0.4 ml) than in the control group (1.5 +/- 1.1 ml). There was no change in the frequency of TPO-ab in either group after pregnancy. In four mothers transplacental passage of these antibodies was documented by positive measurement in the blood sample of the newborn. This study documents that iodine supplementation during pregnancy in an area of moderate iodine deficiency results in a lower size of neonatal thyroid volume and that this supplementation was not accompanied by an increase in the frequency of TPO-ab. PMID- 8640296 TI - Major histocompatibility complex class II and complement polymorphisms in postpartum thyroiditis. AB - The objective was to re-evaluate the association between class II HLA-DR and DQ MIIC antigens and postpartum thyroiditis (PPT) and to determine the prevalence of the class III complement allotypes of Properdin factor B (Bf), C4A and C4B in this condition. Two hundred and sixty-five (of 2897) pregnant women screened positive for thyroid autoantibody activity took part. Further blood samples were obtained for HLA class II (185) and complement (193) typing. The severity of the ensuing PPT was assessed by measuring thyroid function during the postpartum year. The HLA-DR and DQ phenotypes were assigned from restriction fragment length polymorphism analysis, and Bf, C4A and C4B allotypes were determined by immunofixation with anti-Bf or anti-C4 antibodies after electrophoresis. A weak association between the HLA class II antigens and PPT, as indicated by a reduced frequency of DR15 and DQ6 together with an increased frequency of-DR5 and DQ7, was confirmed. However, only the change in DR5 frequency remained significant after correction (corrected p < 0.05). Postpartum thyroiditis was also associated with frequency disturbances in BI and C4A allotypes but not C4B allotypes. Whilst this study has not provided evidence of a strong marker gene for PPT, it does not preclude the involvement of the MIIC in this condition. These data show disturbances in complement allotype frequencies, suggesting that the class III region may provide a useful focus for further study of this pathology. PMID- 8640297 TI - Effects of the dopamine agonist cabergoline in patients with prolactinoma intolerant or resistant to bromocriptine. AB - Cabergoline is a new long-acting crgoline derivative used to treat hyperprolactinaemia. Its effect was assessed in 10 patients (eight women and two men) with prolactinoma who were intolerant (group I: N = 7) or resistant (group II: N = 3) to bromocriptine. In group I, no side effect was observed on cabergoline therapy; two patients became pregnant and normoprolactinaemia was achieved in the live others. In group II, cabergoline was active and well tolerated in two out of the three patients: one woman had three consecutive pregnancies: in another patient normoprolactinaemia was restored and the tumour shrank by 60%; in the third patient cabergoline was discontinued because of side effects and inefficacy. Thus, cabergoline appears to be an alternative of choice as treatment of hyperprolactinaemic patients who are intolerant or resistant to bromocriptine. PMID- 8640298 TI - Insulin treatment fails to abolish the teratogenic potential of serum from diabetic rats. AB - Maternal diabetes during pregnancy constitutes an increased risk for congenital malformations in the offspring. Previous studies have identified several serum components with teratogenic activity, e.g. glucose and beta-hydroxybutyrate, but have also suggested that the teratogenic influence of the diabetic environment on the developing embryo is multifactorial and may depend upon changed concentrations of several maternal metabolites. In the present investigation we aimed to assess the teratological impact of small, concomitant alterations in a series of metabolites, particularly those not previously identified as teratogens. We thus investigated the influence of a mild diabetic environment by culturing gestational day-9 rat embryos in serum from insulin-treated diabetic rats for 48 h in vitro, and compared the embryonic outcome with that obtained after culture in normal serum and in serum from manifestly diabetic rats without insulin treatment. The glucose concentration was adjusted to 10 or 30 mmol/l in the cultures, and the embryos were evaluated with respect to crown-rump length, protein and DNA content, number of somites and malformation score (comparing major, minor or no malformations). We found that increased glucose levels caused embryonic maldevelopment in both normal and diabetic serum, and that despite normalization of the diabetic state, the serum from the insulin-treated diabetic rats caused more growth retardation than the nondiabetic control serum. The normalized diabetic serum was also more teratogenic than the normal serum at the low glucose concentration, whereas the serum from the manifestly diabetic rats tended to cause more dysmorphogenesis at 30 mmol/l glucose than both the normal and normalized diabetic serum. The results suggest that the teratogenicity of maternal serum in diabetic pregnancy is not mediated exclusively by increased concentrations of glucose and ketone bodies. The efforts to diminish the teratogenic effects of a diabetic environment should therefore include normalization of a multitude of serum factors, including glucose and ketone bodies. PMID- 8640299 TI - Slowing of peripheral motor nerve conduction was ameliorated by aminoguanidine in streptozocin-induced diabetic rats. AB - The aims of this study were to investigate the effect of aminoguanidine (AG) on slowing of motor nerve conduction velocity (MNCV) of the sciatic nerve in streptozocin-induced diabetic rats and to assess its mechanism of action. The MNCV of the sciatic nerve was measured electrophysiologically in diabetic rats treated with and without AG for 16 weeks. To elucidate the action of AG, morphological lesion and abnormality of polyol pathway metabolism in the nerve were examined and tissue levels of advanced glycosylation end-products (AGE) were determined as an indicator of AGE accumulation in tissue. Diabetic rats were treated with AG at three doses of 10, 25 and 50 mg/kg for 16 weeks. Myelinated fiber morphometry and nerve Na+,K(-)-ATPase activity were determined. The AGE levels in renal cortex were measured by a specific ELISA. Aminoguanidine dose dependently ameliorated slowing of MNCV 16 weeks after the treatment without changing body weight or blood glucose levels. No difference in myelinated fiber morphometry or Na+,K(+)-ATPase activity with or without AG treatment was detected in diabetic rats. Diabetes increased the AGE level in the renal cortex by six times compared to non-diabetic rats, and AG reduced the rise in the AGE level by 40%. The MNCV was inversely correlated with the AGE levels. We conclude that improvement of conduction slowing by AG in experimental diabetes may be through decreasing the AGE level in the peripheral tissues. Aminoguanidine may have a therapeutic potential in controlling diabetic peripheral neuropathy. PMID- 8640300 TI - Modulation by retinoic acid of insulin-like growth factor (IGF) and IGF binding protein expression in human SK-N-SH neuroblastoma cells. AB - Growth in neuroblastoma cells is regulated by insulin-like growth factors (IGFs) whose action is modulated by IGF binding proteins (IGFBPs). In this study, SK-N SH neuroblastoma cells were shown to produce IGF-II, IGFBP-2, IGFBP-4 and small quantities of IGFBP-6. We have studied the effects of a natural morphogen, retinoic acid (RA), on growth and IGFBP expression in these cells. In all experiments, cells were cultured in serum-free medium and treated with 1 mumol/l RA for 12 h. Cell number increased by almost 50% during the first 24 h after the beginning of treatment. This stimulation was inhibited by 80% or more in the presence of the anti-type 1 IGF receptor antibody alpha-IR3 and anti-IGF-II antibody. The IGF-II concentrations in the culture media, measured after acidic gel filtration, increased about 1.5-fold and Northern blotting showed a concomitant increase in IGF-II mRNA levels. The mitogenic effect of RA therefore reflects its stimulation of IGF-II production. The availability of IGF-II to the cells may also be enhanced because of the proteolysis of IGFBP-2 to which it is bound. After this initial phase, proliferation ceased despite continued IGF-II production between 24 and 72 h. Both IGFBP-2 and IGFBP-4 production decreased, whereas that of IGFBP-6 increased. These changes appeared both in the protein quantities and in their mRNAs. Insulin-like growth factor binding protein 6 has a strong affinity for IGF-II, 5-10 times that of IGFBP-2 and at least 10 times that of the type I IGF receptor, and the arrested proliferation may result, at least in part, from sequestration by IGFBP-6 of the IGF-II secreted. PMID- 8640301 TI - Measurement of activin in biological fluids by radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin. AB - Activin, a dimer of the beta-subunits of inhibin, is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors and has a widespread range of actions in a variety of tissues. The investigation of the physiology of activin action has been facilitated in recent years by the availability of immunoassays in addition to bioassays. Follistatin has been shown to bind to activin with a high affinity and therefore interferes in both radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). In this study we examined the effect of various surfactants and 1.4-dioxane on the measurement of activin in the presence of follistatin by radioimmunoassay. The addition of a combination of sodium deoxycholate. Tween 20 and sodium dodecyl sulphate removed the interference of follistatin in the radioimmunoassay. The measured content of activin in male rat serum, human male serum, human female serum and bovine follicular fluid rose from 3.29 to 4.15, < 0.48 to 2.87, 2.42 to 4.17 and 30.9 to 85.6 ng/ml, respectively, when assayed in the presence of the dissociating reagents. It was unclear whether the altered potencies were due to a dissociation of the follistatin/activin complex rather than the exposure of the epitope on activin recognized by the antiserum. Serum concentrations of activin were lower than those found in testicular cytosols, and after castration no change in serum activin levels was observed, suggesting that the testis does not contribute significantly to circulating activin levels. The use of the dissociating reagents in the radioimmunoassay will enable studies to be carried out that more accurately measure the activin content of various biological fluids, and thus lead to a greater understanding of the physiology of this growth factor. PMID- 8640302 TI - Expression of endothelin precursor genes in human trophoblast in culture. AB - We have shown previously the presence of immunoreactive endothelin in cultured trophoblastic cells from human term placenta as well as in the trophoblast conditioned medium. To confirm whether or not the differentiated syncytiotrophoblast is a site for endothelin synthesis, we investigated, by reverse transcription and polymerase chain reaction, the expression of the three preproendothelin genes in 3-day cultured trophoblast. While no endothelin-2 precursor mRNA was detected, preproendothelin-1 mRNA was found to be expressed by the trophoblast. The endothelin-3 precursor gene was also expressed, but at low level and it was detected only after Southern blotting and oligonucleotide hybridization. The ability of trophoblast in culture to express the endothelin precursor genes supports the idea that, in human term placenta, villous syncytiotrophoblast that lines the intervillous space containing maternal blood acts as an endothelial layer. PMID- 8640303 TI - Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin. AB - Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows. The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG. Granulosa cells (2-3 x 10(5) viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days. Whilst progesterone production (ng/micrograms DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro. The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations. PMID- 8640304 TI - Expression of sterol carrier protein 2 (SCP2) in human adrenocortical tissue. AB - Sterol carrier protein 2 (SCP2) has been implicated in adrenal steroidogenesis by in vitro studies. In order to clarify the clinical significance of SCP2 in human steroidogenesis, we investigated the expression of SCP2 mRNA in various types of adrenocortical tissue and one testis and examined the correlation between the amounts of SCP2 and other values such as the free cholesterol content and the cholesterol side-chain cleavage (SCC) activity in the tissue mitochondria. The types of adrenocortical tissue examined included adrenocortical carcinomas (N = 3), adrenocortical adenomas from patients with Conn's syndrome (N = 3) and from patients with Cushing's syndrome (N = 3), non-functioning adrenocortical adenomas (N = 2) and normal adrenal glands (N = 2). Northern blot hybridization predominantly revealed a 1.8-kb SCP2 mRNA in all tissue specimens examined. The mRNA concentrations of SCP2 in two out of three adrenocortical carcinomas were relatively lower than those in other types of tissue. No other special tendency was observed regarding the mRNA expression levels in various tissue specimens. The mRNA concentrations of SCP2 correlated significantly with mitochondrial contents of free cholesterol (r = 0.67, p < 0.01), but was not correlated with the SCC activities in mitochondria measured by an in vitro enzyme assay. The mitochondrial SCC activities, however, were correlated significantly with the protein levels of mitochondrial P-450 scc determined by a Western blot analysis (r = 0.79, p < 0.01). The significant positive correlation between mRNA concentrations of SCP2 and the mitochondrial content of free cholesterol suggests that the central role of SCP2 in human steroidogenic tissues may be in part a translocation of cytoplasmic free cholesterol to the mitochondria, as demonstrated previously by in vitro studies. PMID- 8640305 TI - Progesterone does not alter sympathetic activity in tissues involved in energy balance. AB - Female rats acclimated to thermoneutrality to avoid cold influences received progesterone by means of subcutaneous implants. They increased their food intake and body weight above the values recorded in control animals. None the less, despite the enhanced food intake, no sign of activation of the sympathetic nervous system was observed, as judged by the unaltered noradrenaline content, half-life and turnover rate in brown adipose tissue, pancreas and heart. This indicates that progesterone increases food intake but prevents non-energy conservation processes regulated by the sympathetic nervous system from taking place. Thus, it facilitates in two different ways the building up of energy stores. Because overfeeding induced by palatable diets increases the sympathetic tone to the organs studied, it is suggested that the central mechanisms regulating energy balance are probably influenced in a different manner by progesterone than by the sensory properties of palatable diets. PMID- 8640306 TI - Selective macrophage depletion in the liver does not prevent the development of the sick euthyroid syndrome in the mouse. AB - A decreased serum triiodothyronine (T3) level is one of the main characteristics of the sick euthyroid syndrome, caused mainly by a decreased 5'-deiodination of thyroxine (T4) in the liver. Cytokines have been implicated in the pathogenesis of the changes in thyroid hormone metabolism during illness. We therefore investigated the role of cytokines produced by the liver macrophages (Kupffer cells) in the development of the sick euthyroid syndrome, which was induced in mice by a single injection of bacterial endotoxin (lipopolysaccharide) or by 24-h starvation. Experiments were carried out with or without previous selective depletion of liver macrophages by intravenous administration of liposome encapsulated dichloromethylene diphosphonate. Relative to saline-injected pair fed controls, the administration of lipopolysaccharide caused a decrease of serum T3 and T4 and liver 5'-deiodinase mRNA. Selective depletion of liver macrophages, did not affect these changes. Starvation for 24h decreased serum T3 and T4, associated with a slight decrease of liver 5'-deiodinase mRNA. There were no differences between macrophage-depleted and non-depleted animals in this respect. In summary, selective depletion of liver macrophages did not affect the decrease in serum T3, T4 or liver 5'-deiodinase mRNA induced by lipopolysaccharide or 24-h starvation in mice. We conclude that cytokines produced by Kupffer cells are not involved in the pathogenesis of the sick euthyroid syndrome in this experimental model. PMID- 8640307 TI - Serum iodothyronine concentrations in intestinally decontaminated rats treated with a 5'-deiodinase type I inhibitor 6-anilino-2-thiouracil. AB - Enteric bacteria have been postulated to have a role in thyroid economy by promoting the hydrolysis of thyroid hormone conjugates of biliary origin, thus permitting the absorption and recycling of thyroxine (T4) and triiodothyronine (T3). An enterohepatic circulation of T3 might be more pronounced under conditions in which type I iodothyronine deiodinase activity (5'D-I) is inhibited, because this augments the accumulation of T3 sulfate conjugates in bile. This potential of increased gut reabsorption of T3 might explain, at least in part, the failure of serum T3 values to decrease appreciably when marked reductions in peripheral 5'D-I activity are induced by selenium deficiency or 6 anilino-2-thiouracil (ATU) administration. Thus, studies were performed to determine the effect of intestinal decontamination, in the absence and in the presence of 5'D-I inhibition, on plasma T4 and T3 concentrations. Groups of adult male rats received either enteric antibiotics or no antibiotics for 12 days and then, in half of the rats in each group, treatment for 10 days with ATU, a 5'D-I inhibitor that does not affect thyroid hormone synthesis. The activity of intestinal arylsulfatase and arylsulfotransferase, enzymes that catalyze hydrolysis of thyroid hormone conjugates, was reduced markedly by approximately 87% in rats that received antibiotics, regardless of whether or not they also received ATU. The ATU treatment markedly inhibited liver 5'D-I activity in antibiotic-treated as well as in non-antibiotic-treated rats (control = 399 +/- 32 U/mg protein (mean +/- SEM); ATU = 152 +/- 17: antibiotics = 351 +/- 29; antibiotics + ATU = 130 +/- 10; p < 0.01) and significantly increased plasma T4 and T3 sulfate (T4S, T3S) concentrations (control: T4S = 2.8 +/- 0.4 and T3S = 6.7 +/- 1.3 ng/dl; ATU: T4S = 6.2 +/- 1.4 and T3S = 10.6 +/- 2.1 ng/dl; antibiotics: T4S = 1.8 +/- 0.2 and T3S = 3.6 +/- 1.0 ng/dl; antibiotics + ATU: T4S = 6.8 +/- 0.7 and T3S = 9.7 +/- 1.8 ng/dl; p < 0.05). The ATU treatment was associated with a significant increase in plasma T4 and rT3 concentrations but did not affect plasma T3 concentrations, and intestinal decontamination did not alter these ATU-associated effects on circulating thyroid hormones. These results suggest that anaerobic enteric bacteria in the rat do not have an important role in recycling of thyroid hormones, either under normal conditions or in circumstances where 5'D-I activity is markedly reduced, and that increased gut absorption of T3 from T3S cannot explain the near-normal serum T3 values found when peripheral 5'D-I activity is markedly decreased. PMID- 8640308 TI - Perinatal ontogeny of porcine growth hormone receptor gene expression is modulated by thyroid status. AB - The ontogeny of growth hormone receptors (GHR) represents a critical stage in growth and metabolism. We have investigated the perinatal ontogeny of hepatic and skeletal muscle GHR gene expression in piglets, and its modulation by GH and thyroid hormones. Test piglets were rendered hypothyroid in late gestation by feeding the sow a high-glucosinolate rapeseed meal. Plasma and tissue samples were obtained from test and control piglets at various ages between 80 days of fetal life (80F) and 2 days postnatally. Plasma hormone levels were determined by radioimmunoassay and GHR mRNA by RNase protection assays. In controls, plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels increased between 80F and birth and the early postnatal period was characterized by a marked surge in plasma T3. Test piglets were hypothyroid at 110F with total T4, total T3 and free T3 levels being reduced by 28, 53 and 33% respectively. By contrast, the postnatal increase in T3 was more marked in test than in control animals. Plasma GH levels decreased over the perinatal period and there was no effect of treatment. Hepatic GHR mRNA was at the lower limit of detection at 80F but by 110F was expressed in both groups of animals. However, fetal hypothyroidism at 110F resulted in a marked 70% decrease in hepatic GHR mRNA (p < 0.01). The higher postnatal rise in T3 in test piglets was accompanied by a recovery of hepatic GHR mRNA levels. By contrast with liver, skeletal muscle (longissimus dorsi) expressed high levels of GHR mRNA at 80F and hypothyroidism induced a 68% increase in GHR mRNA (p < 0.001). The present results suggest that thyroid hormones may modulate the perinatal ontogeny of GHR gene expression, in addition to other hormonal factors, and that this modulation is tissue-specific. PMID- 8640310 TI - [New hepatitis viruses "E, GB, etc."]. AB - Some cases of hepatitis were non A, non B, non C, non D. Hepatitis E virus (Calicivirus) transmission is fecal-oral similar to that of the hepatitis A virus. Viral hepatitis E is endemic and frequently epidemic in many developing countries, but exceptionally observed in France. A high mortality rate was observed in pregnant women. Recently, ELISA assays for IgM anti-HCV are available. Many hepatitis cases (acute and chronic) are not caused by known viruses (non A-non E). Four Flavivirus like have recently been cloned from infectious tamarin serum, derived from human viral hepatitis. Three viruses GB Virus A (GBV-A), GB Virus B (GBV-B) and GB Virus C (GBV-C) were identified. The GB viruses and HG Virus are not genotypes of hepatitis C Virus (HCV) and perhaps that GBV-A/GBV-C and HGV are closely related. GBV-A could be an indigenous tamarin virus. The new hepatitis (non A-non E) viruses were associated with blood transmission and with chronic diseases. The development of specific diagnostic reagents using polymerase chain reaction (PCR) and ELISA assays are essential to answer many questions on the epidemiology (and the incidence of post-transfusion hepatitis), and the pathogenesis of GBV and HGV viruses. PMID- 8640309 TI - [Estimation of risk of virus transmission in hepatitis B and C and human retrovirus via transfusion of labile blood derivatives]. AB - This study estimates the risk of transmitting human immunodeficiency virus (HIV), human T lymphotropic virus (HTLV), hepatitis C virus (HCV) and hepatitis B virus (HBV) from blood units using a seroconversion incidence model. Data from 13 blood transfusion centers collecting about 1 million donations per year and belonging to the Retrovirus and the Viral Hepatitis study groups were analyzed during the 3 year study period (1992-1994) for HIV, HTLV, and HBV and a 2 year study period for HCV (1993-1994). Seroconversion incidence rates were calculated and multiplied by estimates of the serological window period for each agent to obtain residual risk. The risk that an infectious donation was made during the window period was estimated to be 1 in 2 millions (95% CI: 1/10(7)-1/450000) for HTLV, 1 in 588000 (1/3 300000-1/227000) for HIV, 1 in 217000 (1/714000-1/83000) for HCV and 1 in 112000 (1/333000-1/43500) for HBV. This risk was estimated for the totality of donations collected in France for HIV and HTLV. For HIV it was the same as above (1 in 588000) and for HTLV it was much lower (1 in 7 millions). PMID- 8640311 TI - [Quality assurance and prevention of immuno-hemolytic incidents in blood transfusion]. AB - Direct and indirect measures of the reliability of the transfusion process are described. These measures can be used to assess the improvement of the transfusion process with a view to preventing hemolytic incidents. Quality assurance arrangements required by their use are made clear. The stress is put upon four points: processes must be formalized and standardised; quality audits must become a routine part of the transfusion process; the system of error reporting must be extended to include all failures; anonymity must be insured to improve reporting. PMID- 8640312 TI - [Quality assurance of bacterial incidents associated with blood transfusion]. AB - Transfusion associated bacterial sepsis, TABS, may be caused by-donor bacteremia, bacteria from the donors skin,-bacteria brought by one of the persons handling the donation or the blood product, from collection and components preparation to the actual transfusion,-or bacteria introduced by a defect in one of the disposables used. Quality Assurance relies on the Good Transfusion Practices of the French Blood Agency all along the transfusion chain from the donor to the recipient. Active involvement of personnel, standardisation and controls as well as improvement of technics particularly as regarding donor selection, skin disinfection technics and collection are required. The ultimate control is hemovigilance with proper identification and investigation of all cases of TABS even with mild symptoms. Centralisation of data in the Haemovigilance scheme is a further step toward a better understanding and improvement of blood products bacterial security. PMID- 8640313 TI - [Bacterial complications of blood transfusions]. PMID- 8640314 TI - [Human herpesvirus 8]. AB - Human herpesvirus 8 (HHV-8, KSHV) was discovered in 1994 by means of a molecular biology approach which permitted to characterize fragments of its genomic sequence. The partial analysis of nucleotide sequence showed that HHV-8 was closely related to herpesvirus saimiri and Epstein-Barr virus, two members of Gammaherpesvirinae sub-family. So far, the virus has been neither isolated nor observed using electron microscopy. It is only detected by means of polymerase chain reaction (PCR) and molecular hybridization. HHV-8 detection in human tissues is strongly associated with three diseases: Kaposi's sarcoma, body-cavity based lymphomas and Castleman's disease. This association raises the question of the causative role of HHV-8 in the occurrence of these three disease. PMID- 8640315 TI - [Why remove leukocytes from labile blood products in 1995?]. AB - During the last 15 years, the techniques to prepare leukocyte-poor cellular blood components greatly improved, as well as our knowledge about the role of leukocytes in many adverse effects of transfusion. These two facts favor the extension of indication of leukocyte-poor blood components. Leukocytes in blood components may be detrimental to their storage, due to their metabolic needs and to their progressive lysis, leading to the release of cytokines. Leukocytes are the exclusive vector in blood of CMV and HTLV viruses. Leukocytes are a key element of the immune modifications induced by transfusion. HLA alloimmunization is favored by the transfusion of large quantities of leukocytes HLA different from the recipient whose immune functions are intact. Conversely, the risk of transfusion associated graft versus host disease is dependent of the transfusion of mature T lymphocytes sharing a partial identity with the recipient, and/or an immune deficient status of the recipient. Between these two extremes, many other effects related to the presence of leukocytes in cellular blood components, as are the transfusion effect observed in transplant recipients, the increased risk for bacterial infection after transfusion, the increased risk for tumor recurrence or the reactivation of virus infections, remain to be fully understood. Despite recent significant improvements, further studies, experimental as well as clinical, will be needed to expand the indications of leukocyte-poor blood components. PMID- 8640316 TI - [Residual risks of viral transmission by transfusions and projected yields of additional screening tests. Retrovirus Epidemiology Donors Study (REDS)]. AB - The residual risk of virus transmission associated with infectious seroconversion, window period donations can be estimated by combining the incidence rate of infection among repeat donors with the duration of the pre seroconversion window period, thus deriving the rate of window phase donations missed by current screening. The residual risks are for HIV 2.0/10(6); HTLV 1.6/10(6); HCV 9.7/10(6); HBV 15.0/10(6). Similarly the net yield and incremental reduction in risk of routine implementation of new tests can be estimated, demonstrating the potential interest of nucleic acid detection. PMID- 8640317 TI - Viral inactivation of blood components: recent advances. PMID- 8640318 TI - Hypermagnesemia: elderly over-the-counter drug users at risk. PMID- 8640319 TI - Hypermagnesemia: elderly over-the-counter drug users at risk. PMID- 8640320 TI - Calculating a clinic's childhood immunization rate: costs and returns. PMID- 8640321 TI - Caring for Simona. PMID- 8640322 TI - The health care experience of patients with low literacy. AB - OBJECTIVES: To understand the difficulties that patients with poor reading ability have interacting with the health care system and to identify the coping mechanisms they use to deal with these problems. DESIGN: Focus groups and individual interviews with patients who are illiterate and patients with low literacy. SETTING: Two large, urban public hospitals. PARTICIPANTS: Sixty patients with marginal to poor reading abilities as measured by the Rapid Estimate of Adult Literacy in Medicine were interviewed in focus groups or individual interviews. MEASUREMENTS AND MAIN RESULTS: Patients with low literacy harbor a deep sense of shame, which is reinforced by hospital staff who become frustrated or angry when someone cannot complete a form or read instructions. Seeking medical care is intimidating for patients with low literacy because they cannot understand signs and registration forms. Many patients recounted serious medication errors resulting from their inability to read labels. To cope with these problems, the patients with low literacy rely heavily on oral explanations, visual clues, and demonstrations of tasks to learn new material. Most also use a friend or family member as a surrogate reader. CONCLUSIONS: Patients with poor reading ability have important problems accessing the health care system, understanding recommended treatments, and following the instructions of providers. Because of their shame, patients with low literacy may be unwilling to disclose their problem to health care providers, and screening tests of reading ability may be necessary to identify those who need special assistance. Patients' coping mechanisms give insight into possible interventions that may improve their interactions with the health care system. PMID- 8640325 TI - Hormone replacement therapy and breast cancer. PMID- 8640324 TI - Hormone replacement therapy and breast cancer risk. AB - The role of estrogen therapy in the risk of breast cancer has been a concern for both physicians and patients. There is some evidence that women taking estrogen who develop breast cancer have a better prognosis. During 8 to 18 years of follow up of 256 postmenopausal women with breast cancer from our hospital, median survival time was 84 months for those who never used estrogen, 80 months for past users, and 143 months for current users. More than 50 studies have shown that there is no increased risk of breast cancer even with long-term estrogen use, while some studies suggest an increased risk. Several studies indicate that when progestogens are added to estrogen therapy, there is a significant reduction in the risk of breast carcinoma. Indirect evidence is accumulating to show why added progestogen should decrease the risk of breast cancer. Preliminary studies further indicate that estrogen therapy, which has been contraindicated in breast cancer survivors in the past, may be safe, and added progestogens may decrease recurrences and deaths. Some medical oncologists and surgeons now advocate estrogen use in women with previous carcinoma of the breast. PMID- 8640323 TI - Educating patients about cystic fibrosis carrier screening in a primary care setting. AB - OBJECTIVE: To assess the effectiveness of education about cystic fibrosis carrier screening in a primary care setting. DESIGN: Participants were asked to read a brochure, and were offered cystic fibrosis carrier screening. They were assessed for knowledge after reading the brochure and again after having an opportunity to ask questions and reread the brochure at home, at which time consent for testing was obtained. SETTING: Two sites of a health maintenance organization in the Baltimore, Md, area. PARTICIPANTS: Enrollees in a health maintenance organization aged 18 to 44 years. Of 608 enrollees approached, 477 completed an initial knowledge questionnaire, and 143 consented to testing. MAIN OUTCOME MEASURE: Change in knowledge score. RESULTS: Knowledge scores improved from a mean of 69% correct initially to 75% at the time of consent (P < 0.1, Student's paired t test). When participants were stratified by educational attainment, significant improvement was observed only for participants with no more than a high school education. However, their final knowledge score was significantly lower than that of college graduates. CONCLUSIONS: For people with more formal education, printed materials augmented by a chance to ask questions may be sufficient to ensure informed consent. For less well-educated persons, additional education may be necessary to ensure understanding of difficult concepts. PMID- 8640327 TI - The association of urinary tract infection with a recent pelvic examination in women. AB - We tried to determine whether women with a urinary tract infection (UTI) were more likely to have had a recent pelvic examination than were women seen for other reasons. We compared 56 women who were diagnosed as having a UTI with 49 controls who had an unrelated complaint (sinusitis). Significantly more women with UTIs had received a pelvic examination within the preceding 2 months (43% vs 16%, P = .01). We conclude that having a pelvic examination is associated with an increased risk of a UTI developing within the following 2 months. This may be due to physical factors related to the examination or to risk factors related to the patients' reasons for obtaining a pelvic examination. Further study is needed to determine if the pelvic examination is an independent risk factor. If so, established preventive measures could reduce this risk. PMID- 8640328 TI - The association of urinary tract infection with a recent pelvic examination in women. PMID- 8640329 TI - Gentamycin reduces bacteremia and mortality rates associated with the treatment of experimental peritonitis. PMID- 8640326 TI - Angiotensin II receptor blockers. A new class of antihypertensive drugs. AB - Angiotensin II (AII) receptor antagonists, a new class of antihypertensive agents, recently became available for the treatment of clinical hypertension. These agents have a unique mechanism of action: they selectively block the AII receptor type I thereby blocking all known physiological actions of AII that are relevant to hypertension. Controlled clinical trials have shown losartan potassium, the first of the AII receptor type I antagonists approved for clinical use, to be effective, providing long-term control of blood pressure in once daily oral doses in patients who have mild to moderate essential hypertension. Losartan is as efficacious as enalapril maleate and atenolol in these patients. Its antihypertensive effect is enhanced when used in combination with a thiazide diuretic. Losartan is well tolerated and generally free of the side effects that are commonly associated with angiotensin converting enzyme inhibitors and other currently available antihypertensive drugs. Thus, AII receptor blockers represent an important therapeutic advance in treating hypertension and provide a targeted treatment approach to block activation of the renin-angiotensin system. PMID- 8640330 TI - Noradrenaline-induced relaxation of rat oesophageal muscularis mucosae: mediation solely by innervated beta 3-adrenoceptors. AB - We investigated the effects of cocaine and corticosterone on the noradrenaline induced relaxation of rat oesophageal smooth muscle in the absence and presence of the selective beta2-antagonist, ICI 111,551. It was found that the concentration-response curve (CRC) of noradrenaline was not shifted by ICI 118,551 at 1 microM, whereas a clear shift to the right was observed at 100 microM of the antagonist. In the presence of corticosterone (10 microM), CRC's were clearly shifted to the left; with cocaine (10 microM) additionally present, a further leftward shift was observed, indicating the involvement of both neuronal and extraneuronal uptake sites. It was concluded that the relaxation of rat oesophageal muscularis mucosae by noradrenaline is solely mediated by beta3 adrenoceptors which are sympathetically innervated. PMID- 8640332 TI - Deoxyribose analogues of N6-cyclopentyladenosine (CPA): partial agonists at the adenosine A1 receptor in vivo. AB - 1. The purpose of the present study was to quantify the cardiovascular effects of the 2'-, 3'-, 5'-deoxyribose analogues of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) in vivo. The blood concentration-effect relationships of the compounds were assessed in individual rats and correlated to their receptor binding characteristics. 2. The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous infusion of 0.80 mg kg (-1) (63 micromol kg(-1)of 2' dCPA. The heart rate (HR) and mean arterial blood pressure (MAP) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. 3. The relationship between blood concentrations and the reductions in both heart rate and blood pressure were described according to the sigmoidal Emax model. For the bradycardiac effect, the potencies based on free drug concentrations (EC50,u) of 5'dCPA, 3'dCPA, 2'dCPAin blood were 5.9 +/- 1.7, 18 +/- 4 and 260 +/- 70 ng ml (-1) (19 +/- 6, 56 +/- 11 and 830 +/- 210 nM), respectively, and correlated well with the adenosine A1 receptor affinity in vitro. The Emax value of 2'dCPA was significantly less than those of the other compounds, suggesting that this compound may be regarded as a partial agonist when compared to the other analogues. The rank order of the maximal reduction in heart rate of the compounds corresponded well with the order of the GTP-shifts, as determined in vitro. 4. It is concluded that deoxyribose derivatives of CPA may be partial agonists for the adenosine A1 receptor and may serve as tools for further investigation of adenosine receptor partial agonism in vivo. PMID- 8640331 TI - Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the beta adrenoceptor and adenylate cyclase in rat heart. AB - 1. The effects of SR 33589 and amiodarone on the cardiac beta-adrenoceptor were studied in vitro and after chronic treatment by means of [125I]-(-) iodocyanopindolol ([125I]-(-)-CYP) binding and measurement of adenylate cyclase activity. 2. Binding of [125I]-(-)-CYP was inhibited in a dose-dependent manner by SR 33589 (IC50=1.8 +/- 0.4 microM, nH=0.93 +/- 0.06) and amiodarone (IC50=8.7 +/- 2.0 microM, nH=9.2 +/- 0.03). Saturation binding experiments indicated a non competitive interaction such that SR 33589 (1 and 3 microM) and amiodarone (5 and 10 microM) reduced the Bmax of [125I]-(-)-CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]-(-)-CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 microM) and amiodarone (30 microM).3. Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose-dependent, but non-competitive manner, by SR 33589 (isoprenaline-, glucagon- and secretin stimulated enzyme inhibited 50% at 6.8 +/- 0.6 microM, 31 +/- 10 microM and 12 +/ 3 microM, respectively) while the basal, GTP- and GPP(NH)p-stimulated enzyme was inhibited by 5-10% and the NaF and forskolin-stimulated enzyme by 50% at 500 microM. Amiodarone exhibited a similar pattern of inhibition. 4. After chronic oral treatment (50, 100, 150 mg kg(-1) per day, 14 days), both SR 33589 and amiodarone produced a dose-dependent decrease in Bmax without any effect on KD as determined from [125I]-(-)-CYP saturation experiments and a decrease of the isoprenaline- and glucagon-stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units. 5. Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4, and rT3 were changed after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and the level of rT3 were dose-dependently increased by amiodarone treatment. 6. In vitro, SR 33589 and amiodarone were characterized as non competitive beta-adrenoceptor antagonists. Chronic treatment led to a down regulation of the beta-adrenoceptor; the down-regulation cannot be attributed to an indirect effect mediated by the thyroid hormones. To reconcile these opposing observations, we propose that SR 33589 and amiodarone interact with the beta adrenoceptor at a site close to the intracellular loops which are involved in the coupling with Gs and contain the phosphorylable sites. PMID- 8640333 TI - Alterations of insulin response to different beta cell secretagogues and pancreatic vascular resistance induced by N omega-nitro-L-arginine methyl ester. AB - 1. We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different beta cell secretagogues and also on pancreatic vascular bed resistance. 2. This study was performed in the isolated perfused pancreas of the rat. Blockage of NO synthase was achieved with Nw-nitro-L arginine methyl ester (L-NAME); The specificity of the antagonist was checked by using its D-enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose-induced insulin secretion. 3. Arginine (5 mM) induced a monophasic response which was, in the presence of L-NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D-NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4. The small biphasic insulin secretion induced by L-leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L-NAME (5 mM) vs 2 times in the presence of D-NAME (5 mM). 5. beta cell responses to KCl (5 mM) and tolbutamide (0.185 mM) were only slight increased by L-NAME (5 mM) to values not far from the sum of the effects of L-NAME and of the two drugs alone. D-NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6. L-NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1-0.5 mM) to blunt the classical biphasic pattern of beta cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D-NAME (5 mM) was unable to induce similar effects. 7. SNP alone at 3 microM was ineffective but at 30 microM substantially reduced to second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L-NAME (5 mM) and restored a biphasic response. PMID- 8640334 TI - YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase. AB - 1. Our previous study demonstrated that YC-1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC-1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2. In human washed platelets, YC-1 inhibited platelet aggregation and ATP released induced by U46619 (2 microM), collagen (10 micro ml(-1)) and thrombin (0.1 u ml(-1)) in a concentration-dependent manner with IC50 values of (microM) 2.1 +/- 0.03, 11.7 +/ 2.1 and 59.3 +/- 7.1, respectively. 3. In a 30,000 g supernatant fraction from human platelet homogenate, YC-1 (5-100 microM) increased sGC activity in a concentration-dependent manner. At the same concentration-range, YC-1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4. MY-5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC-1, and shifted the concentration-anti-aggregation curve of YC-1 to the left. In contrast, HL-725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti-aggregatory effect caused by YC-1. 5. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC-1, and attenuated markedly the anti-aggregatory effect of YC-1. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA) did not affect YC-1-induced inhibition of platelet aggregation. 6. Haemoglobin, which binds NO, prevented the activation of sGC and anti-aggregatory effect caused by sodium nitroprusside, but did not affect YC-1 response. 7. These results would suggest that YC-1 activates sGC of human platelets by a NO-dependent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway. PMID- 8640335 TI - Actions of ADP, but not ATP, on cytosolic free Ca2+ in single rat hepatocytes mimicked by 2-methylthioATP. AB - 1. Aequorin-injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin-injected hepatocytes to ADP and ATP. 2. The effects of the phosphorothioate analogue of ATP, 2-methylthioATP (2-meSATP), have been examined on single rat hepatocytes. This analogue is belived to be the most potent agonist at the P2Y1 subclass of purinoceptor. 3. The [Ca2+]i transients induced by 2 meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4. At hig concentrations, 2-meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. 5. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2-meSATP- and ADP-induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP-induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. 6. ATP-induced transients were specifically potentiated by the co-addition of alpha, beta-methyleneATP, whereas 2-meSATP- and ADP-induced transients were unaffected by this treatment. 7. We conclude that 2 meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from teh receptor(s) mediating the effects of ATP. PMID- 8640337 TI - Mucosa-dependent muscarinic liberation of prostaglandins from rat isolated trachea. AB - 1. The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid-derived mediators from rat isolate tracheae. 2. Tracheae were preincubated with [3H]-arachidonic acid and the outflow of 3H compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor-preferring antagonist rho-fluoro-hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine. 3. High performance liquid chromatography analysis (methanol gradient) of the released 3H-compounds showed that one peak, co-eluting with [14C]-prostaglandin E2([14C]-PGE2) and [3H]-PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]-arachidonic acid remained unaffected. 4. Using an acetonitril gradient separation it was shown that [3H]-PGE2, [3H]-PGD2 and [3H]-PGF2alpha are released spontaneously, but [3H]-PGE2 represented the major fraction of 3H-prostaglandins. Acetylcholine enhanced the release of all three 3H-prostaglandins, but the effect on PGE2 was most pronounced and most consistent. 5. After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H prostaglandins ([3H]-PGE2/PGD2 peak) was abolished. 6. Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa dependent manner. 7. After inhibition of cyclo-oxygenase by 3 microM indomethacin, the outflow of 3H-prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release [3H]-arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 microM) also blocked the effect of acetylcholine on the outflow of 3H-prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]-arachidonic acid. 8. In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa. PMID- 8640336 TI - Prevention by alpha-tocopherol and rutin of glutathione and ATP depletion induced by oxidized LDL in cultured endothelial cells. AB - 1. Oxidized low density lipoproteins (LDL) are thought to play an important role in atherogenesis. Mildly oxidized LDL are cytotoxic to cultured endothelial cells. Toxic doses of oxidized LDL promote the peroxidation of cellular lipids (beginning at 6 h and being maximal after 12 h of pulse with oxidized LDL) and glutathione and ATP depletion (beginning after 15 h of pulse and evolving concurrently with the cytotoxicity). 2. Antioxidants from 3 different classes (rutin, ascorbic acid and alpha-tocopherol) were compared as to their ability to inhibit the cytotoxic effect of oxidized LDL to endothelial cells. 3. Effective concentrations of alpha-tocopherol inhibited cellular lipid peroxidation, glutathione and ATP depletion and the cytotoxic effect. 4. Ascorbic acid was less effective than alpha-tocopherol and rutin, and exhibited a dose-dependent biphasic effect in the presence of oxidized LDL. 5. Effective concentrations of rutin inhibited glutathione and ATP depletion as well as cytotoxicity, but did not block cellular lipid peroxidation. This suggests that the glutathione and ATP depletion is directly correlated to the cytotoxicity of oxidized LDL, whereas cellular lipid peroxidation is probably not directly the cause of cellular damage leading to cell death. 6. The association of antioxidants of 3 different classes allowed the suppression of the biphasic effect of ascorbic acid and increased the efficacy of the protective effect. The potential consequences for prevention of the pathogenic role of oxidized LDL in endothelial injury are discussed. PMID- 8640338 TI - The formation of nitric oxide donors from peroxynitrite. AB - 1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo. PMID- 8640339 TI - Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats. AB - 1. A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically-instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 micro g kg(-1) h(-1)) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4-8 after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well maintained over the following 8 h of LPS infusion. 2. By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta. (43.3 +/- 7.8, 28.8 +/- 3.3, 50.8 +/- 7.2, 3.04 +/- 0.29, 3.76 +/- 0.94 pmol min(-1) mg(-1) protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 +/- 1.23 to 29.44 +/- 7.08 micromol l(-1)), and further by 6 h (228.10 +/- 29.20 micromol l(-1)), but were less 24 h after onset of LPS infusion (74.96 +/- 11.34 micromol l(-1)). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8-24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3. Pretreatment with NG-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1) bolus, 30 mg kg(-1) h(-1) infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6-8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation. PMID- 8640341 TI - Long-term carbachol treatment-induced down-regulation of muscarinic M2-receptors but not m2 receptor mRNA in a human lung cell line. AB - 1. The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2. Saturation studies performed with the non-selective hydrophilic ([3H]-N-methyl-scopolamine, [3H] NMS) and lipophilic (3H]-quinuclidinyl benzilate, [3H]-QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3. Carbachol (1 mM) induced a rapid down-regulation of [3H]-NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4. The loss of [3h]-QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]-NMS binding sites as a result of receptor sequestration. 5. Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin-induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6. The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol had no effect on the stability of m2 mRNA. 7. The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run-on assay was unaltered by carbachol stimulation. 8. These results suggest that homologous sequestration, desensitization, and down-regulation of M2 modifications of m2 muscarinic receptor mRNAs. PMID- 8640340 TI - Study of NO and VIP as non-adrenergic non-cholinergic neurotransmitters in the pig gastric fundus. AB - 1. The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non-adrenergic non-cholinergic (NANC) relaxations in the pig gastric fundus was investigated. 2. Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5-hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced responses were abolished by tetrodotoxin. 3. The short lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG-nitro-L-arginine methyl ester (L-NAME). Pretreatment with L-arginine but not D-arginine, prevented the inhibitory effect of L-NAME except for sustained relaxations in the longitudinal muscle strips. 4. Sodium nitroprusside, forskolin, zaprinast and 3-isobutyl-l-methylxanthine induced concentration-dependent relaxations. Exogenous NO mimicked the short-lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L-NAME. 5. alpha-Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips. 6. The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation. 7. Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double-labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase-histochemistry showed the same pattern of nitrergic neurones and nerves as NOS-immunocytochemistry. 8. It is concluded that a cyclic GMP- and a cyclic AMP-dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short-lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation. PMID- 8640342 TI - Characterization of the pharmacology and regional distribution of (S)-[3H]-5 fluorowillardiine binding in rat brain. AB - 1. This study examined the binding of the new radioligand (S)-[3H]-5 fluorowillardiine to rat brain synaptic membranes. Specific binding represented greater than 80% of the total binding and was increased by 10% in the presence of 100mM potassium thiocyanate (KSCN). 2. In the absence of KSCN, (S)-[3H]-5 fluorowillardiine identified two binding sites with KD1=22.5 nM, Bmax1=1.4 pmol mg(-1) protein and KD2=1.5 microM, Bmax2=10.8 pmol mg(-1) protein. In the presence of 100 mM KSCN the affinities of both the binding sites were increased, yielding values of KD1=6.9 nM and KD2=0.4 microM KSCN was without effect on the Bmax values. 3. (S)-[3H]-5-fluorowillardiine binding was displaced by non-NMDA receptor ligands with the rank order of potency: 2,3-dihydroxy-6-nitro-7 sulphamoyl-benzo(F)quinoxaline (NBQX) > domoate > (S)-alpha-amino-3-hydroxy-5 methyl-4-isoxazolepropionice acid (AMPA) = L-glutamate > 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX) > kainate >> (R)-5-fluorowellardiine. In contrast, both N-methyl-D-aspartate (NMDA) and the metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were inactive. 4. By use of quantitative autoradiography the regional distribution of (S)-[3H]-5-fluorowillardiine binding in rat brain was assessed. The highest levels of binding were in the dentate gyrus and the CA1 region of the hippocampus. Lower levels of binding were detected in the cerebral cortex, olfactory system, lateral septum, caudate putamen and nucleus accumbens. 5. We conclude that the pharmacological profile and regional distribution of (S)-[3H]-5 fluorowillardiine binding is consistent with its specific interaction with AMPA receptors. PMID- 8640343 TI - Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig. AB - 1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells. PMID- 8640344 TI - Reflex circulatory collapse following intrapulmonary entrapment of activated platelets: mediation via 5-HT3 receptor stimulation. AB - 1. The role of 5-HT2 and 5-HT3 receptors in the mediation of direct and reflex vascular responses to intrapulmonary platelet activation was investigated. 2. Anaesthetized rabbits were challenged intravenously with an emulsion of autologous bone marrow that produced a sharp increase in pulmonary blood pressure, a fall in systemic blood pressure, platelet consumption and death. 3. Platelet depletion before the challenge nearly abolished all the cardiovascular effects and prevented death. Bilateral vagotomy prevented the fall in systemic blood pressure and death but did no prevent the increase in pulmonary pressure. The intravenous administration of the 5-HT2 antagonist, ketanserin, only reduced the increase in pulmonary pressure without affecting the systemic response or mortality. 4. The effects of intravenous 5-HT and of electrical stimulation of the cephalic ends of the cut vagi nerves were also explored. 5-HT injection increased the pulmonary vascular pressure but its effects on systemic blood pressure were variable. These response were modified by the 5-HT antagonists in a manner that resembles their effects on bone marrow embolism. Afferent vagal stimulation produced a fall in systemic blood pressure that was not prevented by MDL-7222. 5. This study indicates that a centrally mediated reduction of peripheral vascular tone is the cause of the potentially lethal circulatory collapse that follows the intrapulmonary entrapment of activated platelets. This reflex is initiated by the action of 5-HT on 5-HT3 receptors in the lung. PMID- 8640345 TI - Electrophysiological actions of felbamate on rat striatal neurones. AB - 1. We have investigated the effects of the anticonvulsant drug, felbamate (FBM), on striatal neurones, recorded in vitro by using both intracellular and extracellular conventional recordings in slices and whole-cell recordings in acutely isolated neurones. 2. FBM, at therapeutically relevant concentrations (30 300 microM) showed multiple mechanisms of action. Like other antiepileptic drugs, FBM (30-300 microM) showed a direct inhibitory action on current-evoked firing discharge of striatal neurones. A patch-clamp analysis of this effect revealed a dose-related reduction of voltage-dependent sodium (Na+) currents (10-100 microM), with a half inhibiton dose (IC50) value of 28 microM. 3. We also tested whether FBM affected corticostriatal glutamate transmission. In control medium (1.2 mM external magnesium), both extracellularly recorded field potentials and intracellularly recorded excitatory postsynaptic potentials (e.p.s.ps) evoked by cortical stimulation were no affected by bath application of 30-300 microM FBM. 4. When magnesium was removed from the perfusing solution, a procedure which reveals a N-methyl-D-aspartate (NMDA)-mediated component in the corticostriatal synaptic potential, FBM (30-300 microM) produced a dose-dependent reduction of the amplitude of both the field potential and the e.p.s.p. 5. FBM reduced the inward currents produced either by bath or by focal applications of 30 microM NMDA, finding consistent with the hypothesis that the observed reduction of the NMDA-mediated component of the synaptic potentials may be caused at postsynaptic level. 6. The reduction of the NMDA-mediated component of the synaptic transmission by FBM and its depressant effect on the voltage-dependent Na+ channels, may account for the antiepileptic action of this drug. Moreover, the pharmacological properties of FBM might render this drug interesting as a neuroprotectant agent. PMID- 8640346 TI - Determination of KA values by controlled receptor expression in Xenopus oocytes. AB - 1. In the present study we estimated the KA value of endothelin-1 (ET-1) for ETA receptors by a new method in which the level of expression of ETA-receptors in Xenopus oocytes was altered in a controlled way. 2. Kvl.2 (a delayed rectifier type K channel) c RNA at the fixed concentration of 0.2 micro g micro l(-1) was mixed with ETA-receptor cRNA at various concentration ratios (10(-3)-3). Oocytes were examined 2-4 days after the injection of the cRNA mixtures. 3. In these oocytes, ET-1 suppressed the amplitude of Kvl.2 current in a dose-dependent manner in the range of 0.1-100 nM; the maximum inhibition produced by ET-1 was larger and the EC50 value for the inhibition by ET-1 was smaller as the mixture ratio was increased. Double-reciprocal plots of equiactive concentrations of ET-1 in 1/1- and 1/30-injected oocytes yielded a KA for ET-1 of 7.4 nM. The number of ETA-receptors in 1/30-injected oocytes was 13% of that in 1/1-injected oocytes, whereas the inhibition of the current in 1/30-injected oocytes was about 60% of that in 1/1-injected oocytes. This suggests the presence of spare receptors of ETA in the latter. 4. A saturation binding experiment estimated a KD value of 0.1 nM for ET-1 at ETA-receptors and the number of ETA-receptors in 1/30-injected oocytes was 23% of that in 1/1-injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and KD, which could be due to factors unique to the expression system employed in the present study. PMID- 8640347 TI - Differential effects of fibrates on the acyl composition of microsomal phospholipids in rats. AB - 1. The time-course and comparative effects of treatment with clofibrate (CFB), bezafibrate (BFB), and gemfibrozil (GFB) on the acyl composition of the main microsomal phospholipids, i.e. phosphatidylcholine and phosphatidylethanolamine, have been studied in male Sprague-Dawley rats. 2. The administration of the three fibrates caused a strong peroxisomal induction and a hypolipidaemic effect. Concerning the changes in acyl composition, CFB and BFB behaved in a similar way, with differences which could be attributed to their different potency as peroxisome inducers, whereas GFB showed a somewhat distinct profile. 3. The three drugs increased the relative content of palmitic, palmitoleic and oleic acids, whereas the levels of stearic acid and also those of long chain, highly unsaturated fatty acids docosatetraenoic, docosapentaenoic and docosahexaenoic acids were reduced. In general, these effects appeared from the first day of treatment and were highly correlated with peroxisomal proliferation. In addition, they were more evident in the phosphatidylcholine than in the phosphatidylethanolamine fraction. 4. Fibrates increased total monounsaturated fatty acids, whereas a decrease in total polyunsaturated fatty acids in the phosphatidylcholine fraction was observed in CFB- and BFB-, but not in GFB treated rats. Clear differences appeared between CFB and BFB on the one hand, and GFB on the other when the influence of fibrate treatment on the molar percentages of linoleic, eicosatrienoic, arachidonic and mead acids was analyzed. 5. GFB increased linoleic acid content in phosphatidylethanolamine, whereas CFB and BFB decreased its level in both phospholipid fractions. In contrast, CFB and BFB enhanced eicosatrienoic and mead acids in both fractions and arachidonic acid in phosphatidylethanolamine, whereas GFB had practically no effect. PMID- 8640348 TI - New high affinity peptide antagonists to the spinal galanin receptor. AB - 1. The role of endogenous galanin in somatosensory processing has been studied with galanin receptor antagonists. The new galanin receptor ligands C7, M32, M38 and M40 bind with high affinity (Kd in nanomolar range) to spinal cord galanin receptors and possess oxidative stability as compared to earlier generations of peptide ligands. These peptides have been examined in the spinal flexor reflex model where exogenous galanin exhibited biphasic excitatory and inhibitory effects. 2. Intrathecal administration of C7 [galanin(1-13)-spantide] and M32 [galanin (1-13)-neuropeptide Y(25-36) amide] blocked facilitation of the nociceptive flexor reflex induced by 30 pmol intrathecal galanin in decerebrate, spinalized rats in a dose-dependent manner, thus behaving as antagonists of the galanin receptor. In contrast, M38[galanin(1-13)-(Ala-Leu)3-Ala amide] and M40 [galanin(1-13)-Pro-Pro-(Ala-Leu)2-Ala amide], exhibited only weak antagonism at high doses in this model. Moreover, lower doses of M40 potentiated galanin induced reflex facilitation. C7 was neurotoxic at high doses in the rat spinal cord. 3. M32 and C7 were potent antagonists of galanin receptors in rat spinal cord, in correlation with their in vitro binding characteristics. In contrast, M38 and M40, despite their high in vitro affinity, exhibited only very weak antagonism. Moreover, M40 may also behave as a partial agonist. 4. Previous studies have shown that the galanin receptor may be heterogeneous. The discrepancy between in vitro binding and in vivo antagonistic potency of M38 and M40 may also suggest the presence of different galanin receptor subtypes within the rat spinal cord. However, other explanations for the discrepancy, such as differences in metabolic stability, diffusion rates and penetration to the site of action are also possible. PMID- 8640349 TI - A re-evaluation of the role of alpha 2-adrenoceptors in the anxiogenic effects of yohimbine, using the selective antagonist delequamine in the rat. AB - 1. The acute behavioural effects of the alpha2-adrenoceptor antagonists, yohimbine, idazoxan and delequamine (RS-15385-197) were compared in two tests of exploratory behaviour in the rat, operated in tandem. These were the elevated X maze test (5 min) and a modified holeboard test (12 min), which comprised a holeboard arena with a small roof in one corner as a 'refuge'. Rats were first placed into this corner, thus enabling measurements of initial emergence latency and the number of forays. The experiments were always done with a concomitant vehicle control group, with 10-12 rats per group, and with the treatment blinded. 2. In order to validate the tests, the effects of representatives of four classes of psychoactive agents were examined, viz. picrotoxin (anxiogenic), chlordiazepoxide (anxiolytic), (+)-amphetamine (stimulant) and diphenhydramine (sedative). The modified holeboard tended to be more sensitive than the measurement of total arm entries in the elevated X-maze at detecting drug effects on exploratory behaviour, but unlike the X-maze it could not clearly identify each class of agent. Thus, picrotoxin (5 mg kg(-1), i.p.) reduced total arm entries and open arm exploration in the X-maze (P<0.02) and suppressed most measures of activity in the holeboard (P<0.05); chlordiazepoxide (7.5 mg kg(-1), i.p.) increased total arm entries and open arm exploration (P<0.02) in the X maze, without clear-cut effects in the holeboard; (+)-amphetamine (1 mg kg(-1), i.p.) had no significant effects in the X-maze, but increased most holeboard activities (P<0.05), and diphenhydramine (30 mg kg(-1), i.p.) reduced total arm entries in the X-maze (P<0.002) and hole exploration in the holeboard (P<0.05). PMID- 8640350 TI - Mastoparan-induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells. AB - 1. The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 microM) caused an accumulation of diacylglycerol (DG) and phosphatidic acd (PA) accompanied by choline release in a concentration- and time-dependent manner. 2. In the presence of 2% n butanol, mastoparan (3-100 microM) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 microM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3. Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4. A protein kinase C (PKC) inhibitor, calphostin C (1 microM), did not inhibit mastoparan-induce PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5. PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6. Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7. These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins. PMID- 8640351 TI - Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats. AB - 1. Isoarecolone was approximately 250 times less potent than nicotine as an inhibitor of [3H]-nicotine binding to rat brain membranes. Isoarecolone failed to inhibit the binding of the nicotinic ligand [125I]-alpha-bungarotoxin or of the muscarinic ligand [3H]-QNB. 2. Nicotine (0.01-30 microM) evoked the release of [3H]-dopamine from striatal and frontal cortex synaptosomes, with EC50 values of approximately 0.5 microM in each case. This release was largely mecamylamine sensitive. 3. Isoarecolone (1-200 microM) evoked predominantly mecamylamine sensitive dopamine release from both striatal and cortical synaptosomes, with a potency at least 20 times less than that of nicotine. The maximum effect of isoarecolone was less than that of nicotine, particularly in the frontal cortex preparation. 4. In control rats treated chronically with saline, neither nicotine nor isoarecolone had clear effects on locomotor activity at the doses tested. Chronic treatment with nicotine clearly sensitized rats to the locomotor activating effect of isoarecolone was seen at a dose about 40 times larger than that of nicotine. 5. The low potency and efficacy of isoarecolone in facilitating sensitized locomotor activity resembled its lower potency and efficacy, compared with nicotine, in evoking dopamine release in vitro. The agonist profile of the nicotinic receptor population mediating dopamine release may determine the pharmacological characteristics of consequent locomotor behaviour. PMID- 8640352 TI - Pharmacological evidence for a single bradykinin B2 receptor in the guinea-pig. AB - 1. The present study addresses the possibility of the existence of different kinin B2 receptor subtypes in the guinea-pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence o indomethacin (3 microM), atropine (10 microM) and captopril (10 microM). 2. BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50 of 13.2 +/- 1.4 nM (n=27), 11.2 +/- 2.1 (n=26), 23.6 +/- 6.3 (n=26), and 33.0 +/- 6.5 (n=27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.34.11) inhibitor and MERGETPA (DL-2-mercaptomethyl-3 guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK-induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments. 3. The peptide B2 receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. IN order to compare the inhibitory potency of these compounds between tissues, pKB values were determined. Mean values of pKB for Hoe 140 were 8.05 +/- 0.07, 8.43 +/- 0.11, 8.13 +/- 0.18, and 8.52 +/- 0. 25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKB values of 6.89 +/- 0.10, 7.57 +/- 0.12, 7.36 +/- 0.12 adn 7.51 +/- 0.28 in the JV, GPI, LP and GPT, respectively. 4. D-Arg [Hyp3, D-Phe7, Leu8]BK and D-Arg[Hyp3, D-Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration-response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D-Arg [Hyp3, D-Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567. PMID- 8640353 TI - Action of adenosine receptor antagonists on hypoxia-induced effects in the rat hippocampus in vitro. AB - 1. We have studied three hypoxia-induced phenomena in the CA1 stratum pyramidale of the rat hippocampal slice: (a) the increase in extracellular potassium ion concentration ([K+]e) measured with ion-sensitive microelectrodes, (b) the intracellularly-recorded pyramidal cell hyperpolarization and (c) the extracellularly-recorded depression of the synaptically-evoked field potential recorded in stratum pyramidale. 2. The extracellular potassium ion concentration ([K+]e) rose from 3 mM to 4.1-4.4 mM at a time when the pyramidal cells hyperpolarized by about 6 mV and neurotransmission was virtually abolished. 3. Presumed glial cells depolarized in response to hypoxia. The shape and time course of this response was remarkably similar to the rise in [K+]e so induced. This is consistent with findings that glial cell membrane potential is dependent on transmembrane K+ gradient. 4. We investigated the effects of theophylline (100 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM) on these effects. We have found that these compounds attenuated by about half the hypoxia induced increase in [K+]e; however, they did not reduce the hypoxia-induced hyperpolarization. We have confirmed that they dramatically reduced the suppression of excitatory transmission caused by the hypoxia. We conclude that adenosine A1 receptors may be involved in the alteration of K+ homeostasis in the hippocampal slice during hypoxia. PMID- 8640356 TI - Voltage-dependence of Ca2+ agonist effect of YC-170 on cardiac L-type Ca2+ channels. AB - 1. We investigated the voltage-dependence of the agonist actions of YC-170, a dihydropyridine (DHP) derivative, on cardiac L-type Ca2+ channels in rabbit ventricular cells, using the patch clamp technique. The characteristics of YC-170 were compared with those of other DHP Ca2+ agonists (Bay K 8644, CGP 28392). Ca2+ channel activities were elicited by depolarizing pulses to 0 mV from a holding potential (HP) of either -80 mV or -40 mV. 2. YC-170 (10 microM) increased Ca2+ channel activities when HP was set at -80 mV. However, decreasing HP to -40 mV abolished the agonist action. The agonist effect of Bay K 8644 (1 microM) on Ca2+ channels was elicited to the same extent at either HP. CGP 28392 (10 microM) also increased Ca2+ channel activities at both HPs, but its agonist effect was significantly larger at an HP of -80 mV than at -40 mV. 3. All of the three DHP Ca2+agonists prolonged open times of Ca2+ channels, but the prolongation did not correspond to the voltage-dependence of Ca2+ agonist effects of the three DHPs. 4. YC-170 markedly reduced the closed time of the Ca2+ channel when the HP was at -80 mV, but prolonged it at HP of -40 mV. Bay K 8644 reduced closed times at an HP of -80 mV. At an HP of -40 mV, Bay K 8644 slightly reduced closed times. CGP 28392 reduced closed times at an HP of -80 mV and prolonged those at an HP of -40 mV. Thus the voltage-dependence of the agonist effects of these agents was in good agreement with the voltage-dependence of changes in closed times of Ca2+ channel. 5. Two mechanisms may be involved in the agonist action of YC-170; a prolongation of open times, and a reduction of closed times of Ca2+ channels, i.e. an increase in reopening. The former mechanism is not dependent on Hp and the latter mechanism is highly dependent on HP. Thus, the voltage-dependence of the agonist action may be attributed to the voltage-dependence of their enhancing effect on reopening of Ca2+ channels. PMID- 8640355 TI - Anxiolytic activity of adenosine receptor activation in mice. AB - 1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8 cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5 dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID- 8640354 TI - Pharmacological characterization of behavioural responses to SK&F 83959 in relation to 'D1-like' dopamine receptors not linked to adenylyl cyclase. AB - 1. Behavioural responses to the new benzazepine derivative, SK&F 83959, a compound that both fails to stimulate adenylyl cyclase and inhibits the stimulation of adenylyl cyclase induced by dopamine, were characterized in detail. 2. In rat striatal membrane preparations, radioligand binding studies with [3H]-SCH 23390 and [3H]-spiperone indicated SK&F 83959 had a high affinity and >250 fold selectivity for D1 over D2 receptors. 3. Using a rapid time sampling behavioural check list technique, SK&F 83959 (0.01-1.25 mg kg(-1)) induced grooming in the manner of all known D1 receptor agonists, together with some vacuous chewing, which declined at higher doses with the emergence of directed chewing and rearing as an adjunct to prominent sniffing; no stereotyped behavioural was evident. 4. Grooming to SK&F 83959 (0.05 mg kg(-1)) was blocked by the selective D1 receptor antagonists, SCH 23390 (0.01-1.0 mg kg(-1)) and BW 737C (0.04-5.0 mg kg(-1)) and was attenuated by the selective D2 receptor antagonist, YM 09151-2 (0.005-0.5 mg kg(-1)); vacuous chewing to SK&F 83959 was not influenced by either SCH 23390 or BW 737C and was enhanced by YM 09151-2. 5. The paradoxical induction of typical D1 receptor agonist-induced grooming by SK&F 83959, an agent satisfying criteria for a D1 receptor antagonist as classically defined, together with its blockade by typical D1 antagonists, strongly suggests mediation via a 'D1-like' site that appears to respond similarly to agents independent of whether they exert agonist or antagonist actions at the classical adenylyl cyclase-coupled D1 receptor. This direct functional evidence for a 'D1 like' site that is not linked to adenylyl cyclase readily complements neurochemical data suggesting the existence of a cyclase-independent 'D1-like' receptor that may be coupled to phosphoinositide hydrolysis. PMID- 8640359 TI - Quantitative recording of vitality patterns in living multicellular spheroids by confocal microscopy. AB - Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or intravascular microregions of large tumors. With progressing growth three distinct concentric annular shells develop. A rim of proliferating cells in the periphery is followed towards the center by layers of quiescent cells and at a defined spheroid diameter cell death occurs in the central core. Fluorescein diacetate (FDA) and Calcein/AM were used as vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for dead cells. For loading multicellular spheroids with the esterase substrate dyes we used a two step cold incubation technique to avoid dye accumulation in the most peripheral cell layers. Homogenously stained tissue allowed to describe the fluorescence attenuation in depth as a monoexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 x 10(-3) in vital stained tissue and 17.9 x 10(-3) in lethal stained tissue. Using the respective attenuation coefficient the raw data were corrected for light absorption and scattering in depth. In radial recordings of the vitality status of multicellular glioma spheroids using CLSM-technique we showed that spheroids up to a diameter of 250 microns were homogenously stained with Calcein/AM and FDA. Spheroids larger than 250 microns consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 microns. The thickness of the rim of vital stained cells decreased with increasing diameter of the spheroids to 64 +/- 7 microns in spheroids of a diameter of 550 +/- 25 microns. Thereafter the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93 +/- 9 microns in spheroids of 700 microns in diameter. The first signs of dead cell staining in the central core occurred at a diameter of 400 +/- 25 microns. The radius of the core increased in an exponential way. The cell layer which was stained neither by vital nor by lethal dyes showed a thickness of 150 microns in spheroids of 550 +/- 25 microns in diameter. Our staining technique and the radial recording of mean field fluorescence signals in living multicellular spheroids will be a valuable tool for experimental cancer research providing a non invasive quantification of cell vitality in living multicellular spheroids. PMID- 8640358 TI - Action of angiotensin II, 5-hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit. AB - 1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions. PMID- 8640357 TI - Enhancement by cyclo-oxygenase inhibitors of platelet-activating factor production in thapsigargin-stimulated macrophages. AB - 1. Thapsigargin stimulated the accumulation of cell-associated platelet activating factor (PAF) and extracellular prostaglandin E2 (PGE2) in rat peritoneal macrophages. PAF in the conditioned medium was less than the detectable amount. To obtain further insight into the mechanism of PAF accumulation, the role of PGE2 in PAF accumulation was investigated. 2. When macrophages were incubated in medium containing thapsigargin (30 ng ml(-1), 46.1 nM) and cyclo-oxygenase inhibitors such as indomethacin, naproxen or ibuprofen, the PAF content of the cells at 10 min was increased in a concentration-dependent manner in accordance with inhibition of PGE2 production. The stimulation by thapsigargin, cyclo-oxygenase inhibitors did not increase PAF accumulation. 3. In thapsigargin-stimulated macrophages, when PGE2(10(-7) M) was added to the medium, the cyclo-oxygenase inhibitor-induced stimulation of PAF accumulation at 10 min was markedly inhibited. 4. The accumulation of PAF induced by thapsigargin alone at 10 min was inhibited by exogenous PGE2 (10(-8) and 10(-7) M), or arachidonic acid (10(-6) and 10(-5) M) in accordance with the increase in PGE2 production. 5. The accumulation of PAF induced by thapsigargin alone or by thapsigargin and indomethacin (10(-6) M) was inhibited by dibutyryl cyclic AMP. 6. These results indicate that the concurrently produced PGE2 in thapsigargin-stimulated macrophages down-regulates PAF accumulation by increasing intracellular cyclic AMP levels, and that cyclo-oxygenase inhibitors increase PAF accumulation by inhibiting PGE2 production. PMID- 8640360 TI - Structural organization of the cell surface of pathogenic protozoa. AB - The surface of parasitic protozoa plays an important role in the process of their interaction with cells from the host. The present review analyzes the structural organization of the surface of sporozoa, trypanosomatids, Entamoeba and Trichomonas, as evaluated by conventional transmission electron microscopy, cytochemical techniques and freeze-fracture. In most protozoa, no special region of surface membrane is detected. In others, however, special membrane domains have been described. As examples, we can mention the cytostome found in epimastigote forms of Trypanosoma cruzi, the region of attachment of the flagellum to the protozoon body in Trypanosomatidae and Trichomonadidae, and the inner membrane complex of Apicomplexa. PMID- 8640361 TI - Nucleoid proteins. AB - This article examines the published evidence in support of the classification of organisms into three groups (Bacteria, Archae, and Eukarya) instead of two groups (prokaryotes and eukaryotes) and summarizes the comparative biochemistry of each of the known histone-like, nucleoid DNA-binding proteins. The molecular structures and amino acid sequences of Archae are more similar to those of Eukarya than of Bacteria, with a few exceptions. Cytochemical methodology employed for localizing these proteins in archaeal and bacterial cells has also been reviewed. It is becoming increasingly apparent that these proteins participate both in the organization of DNA and in the control of gene expression. Evidence obtained from biochemical properties, structural and functional differences, and the ultrastructural location of these proteins, as well as from gene mutations clearly justifies the division of prokaryotes into bacterial and archaeal groups. Indeed, chromosomes, whether they be nuclear, prokaryotic, or organellar, are invariably complexed with abundant, small, basic proteins that bind to DNA with low sequence specificity. These proteins include the histones, histone-like proteins, and nonhistone high mobility group (HMG) proteins. PMID- 8640362 TI - Correlation of mono and combined amitriptyline/lithium therapy with therapeutic and side effects. AB - The role of lithium in combination with tricyclic antidepressants (TCA) used in the treatment of unipolar depression has been much less studied than in the case of bipolar disorders. The aim of this study was to compare the therapeutic and side effects with doses and plasma concentrations in the patients with major (unipolar) depression (DSM-III-R criteria) treated by amitriptyline combine (At+Li) and mono-therapy. All three, of these regimens, were therapeutically effective, but after 6 weeks combined (At+Li) therapy showed the absence of the increased number of side effects. PMID- 8640363 TI - The psychoanalytical concept from a neurophysiological perspective. AB - Many neurobiologists remain reluctant concerning the perspectives of a fruitful dialogue between psychoanalysis and neurosciences. Our attempt aims to underline the impact of recent neurophysiological and psychophysiological data on a scientific foundation of the three basic components of psychoanalysis: unconsciousness, sexuality and dreams. The psychoanalytical and the neuroscientific theory and practice of the posttraumatic stress disorders and of the stress concept are also discussed. PMID- 8640364 TI - Questions of the end of the century and millennium. Ecologic pharmacology. AB - The author starts from the idea that drugs are factors of the external environment, which can produce the pollution of the organism. This raised the problem of the necessity to practice an ecologic pharmacology. Referring to the drugs as part of the material, the author underlines that they represent a unit, substance-energy-information. He considers that the pharmacologic effects are the result mainly of information transmission, while the pharmacotoxicologic effects are produced by substance. There is a single way to reduce the toxic effects, by diminution of the dosage, which is achieved by homeopathy. The author proposes that pharmacology should include two branches, allopathy and homeopathy, the only possibility to perform a pharmacology with a dominant, defining ecologic character. PMID- 8640365 TI - Integrative cybernetic model of oxygen homeostasis. AB - The paper presents an integrative cybernetic model of the complex physiological process generically called "oxygen homeostasis". The model has been designed in such a manner as to enable a mathematical approach, in quantitative terms, to the various functions involved in the generation and uninterrupted functioning of the mentioned process. Those functions are: pulmonary and tissular respiratory function, blood circulation, transport, erythropoiesis, as well as other functions involved in the blood pH regulation at renal and hepatic level (the resorption of Na+ ions from the glomerular filtrate and the ammonio-genetic functions of the kidneys and, respectively, the glyco-genetic, ureo-genetic and metabolization functions of the liver). All of these functions have been assumed as regulated and controlled within a unitary cybernetic system, interpreted as an open, dynamic system. That system consists of seven multicompartment subsystems which are open and interconnected by means of exchange relations, biophysical and biochemical processes involved in the performance of the respective function. Special attention was given to the mathematical modelling of the exchange processes going on in the erythrocytic membrane as part of the transport function of the circulating blood. The mathematical description of the entire homeostatic system operation was done by means of a set of differential and algebraic equations. Some of those equations (those describing the continuity of several chemical species in a compartment of the circulatory system and those characterizing the state of thermodynamic balance of the blood subsystem within the respective compartment) enable one to determine the value of the state parameters at a given moment, as a function of the external and internal conditions and as a function of the intensity of processes specific to the subsystems considered. There are other equations (equations of regulation and control devices) that enable one to determine the variations in intensity of the processes mentioned that are necessary in order to restore the system to its "normal" state, in case of possible deregulations caused by disturbing factors. PMID- 8640366 TI - [Current information on nitric oxide as a neuronal messenger]. PMID- 8640367 TI - Hypobaric hypoxia: dual sympathetic control in the light of RR and QT spectra. AB - ECGs of 21 candidate-pilots and 19 pilots were recorded during: 1) exposure to 5500 m hypobaric hypoxia (HH) while sitting, in the sequence: 1a) initial 7 min of adaptation (A); 1b) later 7 min of recovery (R) after short but intense tread mill effort; and 2) final 7 min baseline (B), while sitting and requested to relax, at "0 m altitude" in the hypobaric room. RR and QT short-term variability were studied using spectral powers within Traube-Hering-Mayer (THM: 0.05-0.15 Hz) and respiratory (RESP: 0.2-0.4 Hz) bands. Mean RR proved highest capability to aggregate individual response-profiles: 15 pilots and 9 candidates entered the main (normal) cluster, featured by a comparison "triangle" set as expected: A > R < B > A. QT-THM power closely followed: 10 subjects (ss) in very normal cluster, defined as: A < R > B < A, while secondary clusters in candidates and pilots were interpreted by not-successful relaxation and exaggerated start-effects, respectively. Subjects with QT-THM normal clusters (A < R > B < A has group averages, p < = 0.05), also showed a quasi-normal "triangle" for mean RR, (A = R < B > A). During adaptation to hypoxia, pilots' QT-THM was higher than candidates' one (p < 0.02, Wilcoxon test). Study supports the emerging capability of QT-THM spectral power to index ventricular sympathetic control. Exposure to hypobaric hypoxia proved to be in these subjects a psycho-physiologic rather than a purely physiologic test. PMID- 8640368 TI - Attempts to identify the active factor of reticulin-M by high voltage electrophoresis and by acetone precipitation. AB - The reticulin-M forte (R forte), an antianaphylactic peptide, extracted from organs rich in RES, previously stimulated with India ink, was analysed after acetone precipitation by paper high voltage electrophoresis. Finally the biological activity remains concentrated in the second, arbitrary group of basic peptides, fractions 1 and 3. PMID- 8640369 TI - The effect of reduced glutathione (TAD-600) on the behavior of rats neonatally intoxicated with ethanol. AB - The study was performed on three groups of male rats of the Wistar strain at the age of three months: group 1 (n = 10), the control group; group 2 (n = 10), consisting of animals treated neonatally (from day 4 through day 10) with ethanol 20%, administered by gavage to the mothers, at the dose of 5 g/kg body/day; group 3 (n = 10) of animals exposed to ethanol under similar conditions, but whose mothers have been injected a reduced glutathione (TAD-600) solution in a dose of 2 mg/100 g body weight of the mother. All the animals were weight ed before the test of behavior. The neonatal administration of ethanol caused ponderal weight retardation, which was not influenced by TAD-600 administrations. Also, a decrease in the scores of emotivity and spontaneous motility in the open field was noticed, as well as difficulties in the acquisition of active avoidance conditioned reflexes in the shuttle-box. Concomitant glutathione administration during the neonatal period significantly improves the learning ability at the age of three months, as well as the score of emotivity, without improving the spontaneous motility score. PMID- 8640370 TI - Folic acid age correlation established on folates determinations in cell homogenates. AB - From the available data, there has resulted that folic acid quantity in homogenate, coming from various organs, collected from young animals is much larger than for the other age categories. These results may be correlated with intense cell proliferation and development processes that take place in the young tissues as compared to rather low values established from grown-ups and old ranges. When comparing the folates quantity found in homogenate resulting from various organs we may ascertain that, if the folates are related to grams of tissue under the test, they are more in spleen than in kidneys, liver and brains respectively. If the folates are reported to tissue protein milligrams it may be ascertained that a larger quantity of folates is found in the brains than in the other studied organs that may be due to big dilution of the proteins in the brains. PMID- 8640372 TI - [Experimental studies of the nootropic-type central nervous effects of nifedipine and nimodipine]. PMID- 8640371 TI - The evolution of the painful sensitivity in acute and chronic stress. AB - The clinical research was made on two groups of young volunteer students. We considered stress consisting in chronic informational overexposure during the examination session and the acute stress from their emotions before a hard examination. The painful sensitivity was analysed by measuring the retraction time of the finger from water at 55 degrees C. The experimental research was made on a group of 100 male mice. The acute stress was performed by subjecting each mouse to swim (behavioral despair test). Painful sensitivity was determined by the test of the hot plate heated at 50 degrees C. Individuals with hyper (H) and hypo (h) painful sensitivity were selected for the tests. In chronic stress, the results proved increased painful sensitivity (hyperalgia) more important at "h" compared to "H" (p < 0.05). In acute stress decreased painful sensitivity (stress analgesia) was noticed more significant at "H" compared to h" (p < 0.05). All these results suggested that the extreme "H" and "h" are two different stress behaviors with opposite mechanisms involved in stress analgesia. This hypothesis is related with studies which demonstrate the involvement in stress analgesia of non-opioid monoaminergic mechanisms together with the opioid mechanisms (Lewis, 1980). PMID- 8640373 TI - Lithium ion influence on N-acetyl-beta-glucosaminidase isoenzymes serum level. AB - N-acetyl-beta-glucosaminidase (NAG) is a lysosomal enzyme found in most human cells, including those from the nervous system and kidneys. There are several different NAG forms in human tissues and fluids, but isoenzymes A and B are the most abundant, presenting about 98% of the whole NAG activity in normal serum. The paper is dealing with the total NAG serum level, main isoenzymes (A and B) activity, their ratio and possible lithium ion influence in affectively disordered patients. The sample consisted of thirteen bipolar-euthymic patients who had been receiving lithium for more than two years. The results pointed out reduction in total serum NAG activity, the lithium ion influence absence on total serum NAG activity, but at the same time the important modifying effects on isoenzyme NAG activities. PMID- 8640374 TI - Administration of lithium prophylaxis. AB - Successful prophylaxis of manic-depressive disorder requires more than the prescription of lithium carbonate. The administrative arrangements in an area of Scotland were accompanied by a 300% increase in the frequency of admissions for mania, whereas in an area of the West Midlands, a large decrease was achieved. PMID- 8640375 TI - KIM127, an antibody that promotes adhesion, maps to a region of CD18 that includes cysteine-rich repeats. AB - A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18 dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats. PMID- 8640377 TI - Reversible inactivation of purified leukocyte integrin CR3 (CD11b/CD18, alpha m beta 2) by removal of divalent cations from a cryptic site. AB - Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, alpha m beta 2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that "inactive" CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 +/- 4.5 nM) similar to untreated, active receptor (12.5 +/- 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the alpha chain of CR3. We found that monomeric C3bi binds directly to recombinant I domain in a Mg(2+)-dependent fashion with an affinity of 300 +/- 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3. PMID- 8640376 TI - A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins. AB - Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-beta 1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays. PMID- 8640378 TI - Plasmodium falciparum: the adherence of erythrocytes infected with human malaria can be mimicked using pfalhesin-coated microspheres. AB - Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542-555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by immunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells transfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin) were produced. The pfalhesin coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546-553 had little binding capacity for melanoma cells. These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel 'artificial' P. falciparum-infected erythrocytes for use in in vitro and in vivo studies. PMID- 8640379 TI - Contribution of cadherins to directional cell migration and histogenesis in Xenopus embryos. AB - Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the beta-catenin. PMID- 8640380 TI - The scapula: coracoid, acromial, and avulsion fractures. AB - Fractures of the coracoid and acromial processes and avulsion fractures of the scapula are uncommon with the vast majority being managed quite successfully nonoperatively. On occasion, however, these injuries may be significantly displaced and of functional importance, thus making surgical management a consideration. Diagnosis is radiographic. Due to the complex anatomy in the area, CT scanning is often necessary to detect and accurately define these injuries. The various fracture patterns that occur as well as mechanisms of injury are described. Surgical indications are detailed as are the various fixation techniques that may be employed or required. The postoperative rehabilitation program is as important as the surgical procedure. PMID- 8640381 TI - Rupture of the extensor pollicis longus tendon. AB - Rupture of the extensor pollicis longus (EPL) tendon after nondisplaced fracture of the distal radius is a known complication, but can also occur in nonrheumatoid patients without a history of fracture. This study is a retrospective analysis of seven patients treated between 1985 and 1992. Five EPL ruptures occurred with nondisplaced Colles' fractures. Two involved a chronic tenosynovitis of the second dorsal extensor compartment of the wrist. The age of the patients ranged from 29 years to 68 years (mean, 42 years). Length of follow-up ranged from 6 weeks to 62 months (mean, 30 months). Rupture occurred at a median of 7 weeks (range, 2 weeks to 11 months) from the time of the distal radius fracture. Three extensor indicis proprius tendons and four free palmaris longus tendon grafts were performed. At final follow-up, six patients had an extensor lag < 10 degrees or normal thumb interphalangeal joint motion. One patient had a 25 degrees extensor lag. The causes of EPL rupture include mechanical irritation, attrition, and vascular impairment leading to delayed rupture. Synovitis of the extensor carpi radialis due to repetitive use may invade the EPL tendon and lead to rupture. Extensor indicis proprius transfer or free palmaris longus grafts yield good results after EPL rupture. PMID- 8640382 TI - Prophylaxis of deep venous thrombosis after total hip arthroplasty by using intermittent compression of the plantar venous plexus. AB - A randomized, prospective, blinded study comparing the efficacy of prophylaxis of deep venous thrombosis by using (A) heparin-aspirin therapy, (B) intermittent pulsatile pneumatic-pump compression of the plantar venous plexus, or (C) both methods, was conducted in patients undergoing elective total hip replacement arthroplasty. Duplex ultrasonography was obtained in all 75 patients before surgery, at 1 week, and 2 weeks after surgery, to detect the presence or absence of deep venous thrombosis, with venograms confirming all positive results. Five of 25 patients in group A (heparin-aspirin) developed deep vein thrombosis. No deep venous thrombi were detected in groups B or C. One pulmonary embolus was detected in group A. The reduction in detectable deep venous thrombosis by the use of intermittent compression of the plantar venous plexus was significant. Wound drainage was decreased by 2 to 3 days (P < 0.05) in group B. It is concluded that, in this group of 75 consecutive patients, intermittent pulsatile compression of the plantar venous plexus was superior to heparin/aspirin pharmacologic prophylaxis for the prevention of deep venous thrombosis proximal to the calf. PMID- 8640383 TI - Tuberculous arthritis of the knee presenting as a meniscal tear. AB - Nonspinal skeletal tuberculosis is a rare, indolent disease that is often difficult to diagnose. The incidence in the United States has recently increased. Pain and swelling are common symptoms. Radiographs may reveal normal findings, or in more advanced cases, demonstrate osteopenia, marginal erosions, and eventually, joint space narrowing and destruction. Treatment depends on the extent of the disease. Prolonged therapy with antitubercular agents is the mainstay of treatment. Synovectomy, osseous debridement, and arthrodesis also have a role in the treatment of this infection. PMID- 8640384 TI - Femoral nerve palsy after arthroscopic surgery with an infusion pump irrigation system. A report of three cases. AB - One patient developed complete, and two patients, partial, femoral nerve palsy after arthroscopic surgery in which an infusion pump was used to operate an irrigation system. In one case, hip flexor and quadricep function was completely lost after the patient underwent arthroscopic partial medial meniscectomy without the use of a tourniquet. A CT scan of the pelvis demonstrated considerable fluid accumulation in the thigh and inguinal regions. The remaining two patients developed quadriceps weakness, but not complete femoral nerve palsy, after arthroscopic-assisted anterior cruciate ligament reconstructions. Although tourniquets were used in these latter two procedures, the pressures were low (300 to 325 mm Hg) and the tourniquet times not excessive, suggesting that femoral nerve palsy in these two patients resulted from fluid extravasation. In all three cases, muscle function returned within 6 to 7 months, but sensory nerve deficits were still present at that time. PMID- 8640386 TI - Thoracolumbar burst fractures treated with combined anterior and posterior surgery. AB - Between 1985 and 1992, 84 burst fractures were surgically managed with anterior decompression, autologous iliac crest strut graft, and posterior instrumentation and fusion. Fifteen were lost to follow-up, leaving 69 patients for detailed review. Of the 22 patients with complete or partial neurologic injury, 12 patients either totally or partially recovered function following surgery. Of these 12 patients, 6 improved 1 Frankel grade; 5 improved 2 Frankel grades, and 1 improved 3 Frankel grades. Follow-up was 12 to 91 months (average 41 months). For all of the fractures in this series, the mean operative correction in sagittal kyphosis was 14 degrees, but this decreased to 7 degrees at final review. No patient had significant scoliosis, and 66 patients achieved solid arthrodesis with 3 pseudoarthroses (4%). Mean operative time for 2-in-1 procedures was 5 hours 42 minutes, with an estimated blood loss of 1,455 mL. Of 62 patients available for follow-up telephone interview, 42 (68%) had minimal or no pain; 11, mild pain; 8, moderate pain; and 1, severe pain. Function in daily activities was assessed as normal or minimally impaired in 43 of 51 patients (84%) with normal neurologic function by physical examination. We conclude that anterior decompression, strut autografting, and posterior instrumented autogenous fusion, either as a combined or staged procedure, is a safe surgical option for thoracolumbar burst fractures. PMID- 8640387 TI - A 40-year-old man with "stabbing" right-side chest pain. AB - The following case is presented to illustrate the roentgenographic and clinical findings of a condition of interest to orthopedic surgeons. The initial history, physical findings, and roentgenographic examination are found on the first page. The final clinical and roentgenographic diagnosis is presented on the following pages. PMID- 8640385 TI - Recurrent dislocation of the pisiform bone. AB - A 17-year-old man had chronic pain and recurrent radial dislocation of the pisiform bone following a fall on his outstretched arm. The bone was excised, along with a 3 x 4-mm osteocartilaginous fragment from the distal triquetrum. Pisotriquetral joint instability should be considered in the differential diagnosis of palmar-ulnar wrist pain. PMID- 8640388 TI - Imaging barium-free bone cement. AB - Prior to 1972, polymethylmethacrylate, or PMMA, did not contain barium sulfate and was, therefore, radiolucent. We describe a technique using magnetic resonance imaging that is very useful in imaging barium-free PMMA. PMID- 8640390 TI - Cutting the uncuttables. PMID- 8640389 TI - Mediocrates already has the money. PMID- 8640391 TI - Total hip arthroplasty after acute displaced femoral neck fractures. AB - Displaced femoral neck fractures have three treatment options; reduction and fixation, hemiarthroplasty, and total hip arthroplasty. Total hip arthroplasty is often avoided; this is commonly owing to its assumed increased risk of dislocation. A careful and critical review of the literature reveals that it does offer certain advantages in selected patients. If acute dislocations (less than 4 months) are prevented, the dislocation rates after a fracture appear to be the same, regardless of whether a hemiarthroplasty or total hip arthroplasty is used. PMID- 8640392 TI - Surgical treatment for osteoarthritis at the base of the thumb. AB - Osteoarthritis of the trapeziometacarpal joint can cause pain and disability. Surgical procedures for this problem include extra-articular ligamentous reconstruction, arthrodesis of the trapeziometacarpal joint, osteotomy of the thumb metacarpal, total joint arthroplasty, silicone interposition arthroplasty, and partial or complete resection of the trapezium. None of the operations results in a painless, stable thumb with good strength and motion in all cases. Disorders of the thumb metacarpophalangeal joint and the scaphotrapezio-trapezoid joints may accompany osteoarthritis of the trapeziometacarpal joint. PMID- 8640393 TI - New Zealand population data at five VNTR loci: validation as databases for forensic identity testing. AB - Databases were developed for three New Zealand ethnic groups (Caucasian, Maori and Polynesian), at five VNTR loci (D1S7, D2S44, D4S139, D5S110 and D12S11), and validated for interpretation of forensic identity tests. A +/-2.8% sliding window was used to define the alleles at each locus and allelic frequency distributions were obtained for each locus. The conservative nature of the sliding window approach for forensic casework was demonstrated. Tests for independence of alleles within and between loci showed good agreement with the expectation of independence. Although Polynesians are known to have reduced genetic diversity at some VNTR loci, this was found not to be a concern for the present methodology. Procedures for the analysis and reporting of DNA profile results used by New Zealand forensic scientists are therefore appropriate. PMID- 8640394 TI - Drug addict deaths in north and west London and prevalence of HIV and hepatitis B infection. AB - In a survey of 176 drug addict deaths occurring from 1987 to 1992 inclusive, in North and West London, nine were HIV positive, eight were hepatitis B positive, and two were positive for both infections; 63% were injectors. Of the 176 deaths, 115 were directly related to drug poisoning, 38 were due to complications of drug ingestion and 23 were from unrelated causes. No increase in the incidence of suicide was observed in addicts who knew that they were HIV positive. PMID- 8640395 TI - Mental disease in a forensic autopsy population of alcoholics. AB - A retrospective examination of fatalities was carried out during a five-year period among alcoholics in Copenhagen who had undergone forensic examination. The frequency of mental disease was compared with that among a non-alcoholic control group. In an examination of 2298 deaths, it was found that alcoholics had a higher frequency of unipolar affective disorder and of general "nervousness'. Previous suicide attempts were more frequent in alcoholics, and both alcoholics and controls had a higher frequency of previous suicide attempts among the depressed and those who later committed suicide than among those who did not. PMID- 8640396 TI - Human sex determination using multiplex polymerase chain reaction (PCR). AB - A sex determination method has been developed using the polymerase chain reaction (PCR) which involved the amplification of the sex-determining region Y (SRY) gene and the amplification of the HLA-DQa gene as an internal control. This method, which can be applied to old and degraded DNA samples, allowed confident sexing of biological samples producing readily identifiable PCR products using two sets of primers in the one PCR reaction. The PCR products were short DNA sequences of 139bp and 239/242bp for the SRY and HLA-DQa regions respectively. The application of this method in forensic science will allow determination of the gender of perpetrators of crimes involving biological materials. PMID- 8640397 TI - Apolipoprotein(a) kringle IV repeat number predicts risk for coronary heart disease. AB - A high plasma concentration of lipoprotein(a) [Lp(a)] has been suggested as a risk factor for coronary heart disease (CHD), but some recent prospective studies have questioned the significance of Lp(a). Lp(a) concentrations are determined to a large extent by the hypervariable apo(a) gene locus on chromosome 6q2.7, which contains a variable number of identical tandemly arranged transcribed kringle IV type 2 repeats. The number of these repeats correlates inversely with plasma Lp(a) concentration. We analyzed whether apo(a) gene variation (kringle IV repeat number) is associated with CHD. Apo(a) genotypes were determined by pulsed-field gel electrophoresis/genomic blotting in CHD patients who had undergone angiography (n = 69) and control subjects matched for age, sex, and ethnicity (n = 69) and were related to Lp(a) concentration, apo(a) isoform in plasma, and disease status. Apo(a) alleles with a low kringle IV copy number ( < 22) and high Lp(a) concentration were significantly more frequent in the CHD group (P < .001), whereas large nonexpressed alleles were more frequent in control subjects. The odds ratio for CHD increased continuously with a decreasing number of kringle IV repeats and ranged from 0.3 in individuals with > 25 kringle IV repeats on both alleles to 4.6 in those with < 20 repeats on at least one allele. This provides direct genetic evidence that variation at the apo(a) gene locus, which determines Lp(a) levels, is also a determinant of CHD risk. PMID- 8640398 TI - Coronary artery disease in IDDM. Gender differences in risk factors but not risk. AB - Insulin-dependent diabetes mellitus (IDDM) increases the risk of developing coronary artery disease (CAD) compared with that seen in the general population, while the sex differential in rates of CAD is considerably reduced in IDDM populations. To further our understanding of these observations, the effects of gender on baseline risk factors for CAD incidence were examined. Participants in the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study were recruited from the Children's Hospital of Pittsburgh IDDM registry and had been diagnosed between 1950 and 1980. Subjects completed a series of questionnaires and were given a full clinical examination at baseline (1986 through 1988) and every subsequent 2 years. This report is based on the first 4 years of follow-up. Similar incidence rates of new CAD events were observed in men and women. In neither sex was glycemic control a predictor of later CAD. Sex-specific Cox proportional hazards models showed that for men, duration of IDDM, HDL cholesterol, fibrinogen, hypertension, and smoking were all significantly associated with the onset of CAD. Hypertension, fibrinogen, and smoking were all replaced by nephropathy when this latter variable was added to the model. For women, duration, hypertension, waist-hip ratio, physical activity, and depressive symptomatology were all significant independent predictors of CAD. Nephropathy status did not enter the model for women. While 4-year incidence of CAD in IDDM varies little by sex in this population, the predictive risk factors vary considerably. In particular, the effect of renal disease was stronger in men, while the cluster of physical activity, waist-to-hip ratio, and depressive symptomatology were more important in women. These results may help explain the relatively greater impact IDDM has on CAD risk for women and suggest new potential preventive approaches. PMID- 8640399 TI - Low whole-blood S-adenosylmethionine and correlation between 5 methyltetrahydrofolate and homocysteine in coronary artery disease. AB - Mild elevation of plasma homocysteine is an independent risk factor for vascular disease. We studied the role of 5-methyltetrahydrofolate (5-MTHF), the folate form directly involved in homocysteine metabolism, in contrast to previous studies, which used total folate measurements, in 70 coronary artery disease (CAD) patients and control subjects. We also measured S-adenosylmethionine (SAM), which controls the activity of critical enzymes of homocysteine metabolism. Fasting plasma total homocysteine was elevated (> 12.4 mumol/L for women, > 13.3 mumol/L for men) in 17% of patients, in accordance with earlier studies. These patients showed lower 5-MTHF (12.4 +/- 1.0 mumol/L, mean +/- SD) than control subjects (24.2 +/- 15.0, P < .001), and there was a clear correlation (multiple linear regression analysis: P = .002) of this relevant form of folate with homocysteine. However, 37% of the normohomocysteinemic patients also revealed similarly low 5-MTHF levels, suggesting that a decrease of 5-MTHF does not necessarily cause hyperhomocysteinemia. SAM was significantly decreased in patients (1.4 +/- 0.4 mumol/L) compared with control subjects (1.8 +/- 0.3, P < .001) but was not correlated to homocysteine or 5-MTHF. The correlation between homocysteine and 5-MTHF that was found in CAD patients but not in control subjects confirms the direct relationship between these compounds in vivo. The new finding of low SAM in patients demands further studies, since it might indicate that low levels pose risk and that SAM might be a protective factor against the development of CAD. PMID- 8640401 TI - Thrombotic risk in hereditary antithrombin III, protein C, or protein S deficiency. A cooperative, retrospective study. Gesellschaft fur Thrombose- und Hamostaseforschung (GTH) Study Group on Natural Inhibitors. AB - A cooperative, retrospective study was performed on data from 8 coagulation laboratories and thrombosis units in Austria, Germany, and Switzerland to assess the risk for thrombosis in patients with hereditary antithrombin III (AT-III), protein C (PC), and protein S (PS) deficiencies; to compare the clinical manifestations of these 3 deficiency states; and to estimate the risk for development of thrombosis in high-risk situations. Two hundred thirty patients from 71 families with a documented hereditary deficiency of a natural coagulation inhibitor were included in the study. The patient group comprised 69 patients from 25 families with AT-III deficiency, 86 patients from 27 families with PC deficiency, and 75 patients from 19 families with PS deficiency. Diagnosis of the deficiency state was made at each participating center. Clinical data were documented in a questionnaire that was completed during each patient's visit to the participating center. The questionnaires were sent to the coordinating center (Vienna) and analyzed centrally. The probability of developing thrombosis was 80% to 90% for all deficiency states by 50 to 60 years of age, and this figure did not change considerably after data from the propositi were excluded. AT-III deficient females developed thrombosis earlier in life compared with PC- and PS deficient females due to a high thrombotic risk during pregnancy (40% in patients with AT-III deficiency) and oral contraceptive use. The clinical features of thromboembolism were similar in the three deficiency states except for a higher frequency of superficial thrombophlebitis in patients with PC and PS deficiencies. Mesenteric vein thromboses occurred in 4% to 10% of patients and in 2 of 8 patients as a recurrent event. The recurrence rate was 63% (60% of recurrent events occurred spontaneously) and did not differ significantly among the three deficiency states. Before 14 years of age only 1 of 80 surgical procedures and 0 of 21 leg injuries were followed by thrombosis. After 14 years of age thromboembolic events occurred after abdominal surgery or leg injury in one third of patients. Between 40% and 50% of symptomatic patients reported that they felt handicapped by a postthrombotic syndrome. We conclude that diagnosis of a coagulation inhibitor deficiency state should be made before 14 years of age. During childhood thrombosis prophylaxis cannot be regularly recommended but should be instituted after 13 years of age during/after abdominal surgery, including appendectomy, and after leg injury in AT-III-, PC-, and PS-deficient patients. The high recurrence rate (60% spontaneous recurrence) and the relatively high frequency of mesenteric vein thrombosis as a recurrent event favor introduction of long-term oral anticoagulant treatment after the first thrombotic event in patients with a documented hereditary deficiency of AT-III, PC, or PS. PMID- 8640400 TI - 'Silent' cerebral infarction is associated with hypercoagulability, endothelial cell damage, and high Lp(a) levels in elderly Japanese. AB - "Silent" lacunar stroke, often found in the elderly, has been proposed as a predisposing condition for clinically overt stroke. However, the risk factors related to this condition have not been studied thoroughly. We conducted brain magnetic resonance imaging and measured the levels of fibrinogen, molecular markers of coagulation activation [prothrombin fragment 1 + 2 (F1 + 2)] and endothelial cell damage [von Willebrand factor (vWF) and thrombomodulin], and lipid profiles including lipoprotein (a) [Lp(a)] in 178 asymptomatic, high-risk, Japanese subjects aged 44 to 93 years. We also studied 32 symptomatic patients with lacunar stroke (symptomatic lacunar group). The prevalence of silent lacunar stroke increased with age up to 85 years but decreased with age in those 85 years old and older. Of the 160 elderly subjects ( > or = 60 years) 84 (53%) had > or = 1 lacunar infarcts (silent lacunar group) and the remaining 76 were considered as the nonlacunar group. Fibrinogen and F1 + 2 levels in the silent lacunar group were significantly higher than those in the nonlacunar group (P < .01). Mean Lp(a) levels and the prevalence of subjects with an Lp(a) level > 30 mg/dL were significantly higher in the symptomatic lacunar group than the nonlacunar group (P < .05), whereas these levels in the silent lacunar group were intermediate to those of the other two groups. When we further classified the silent lacunar group into three subgroups based on the number of lacunes (few lacunes, 1 or 2; moderate number of lacunes, 3 or 4; and numerous lacunes, > or = 5), levels of Lp(a), F1 + 2, vWF, and thrombomodulin were significantly higher and Lp(a) levels > 30 mg/dL more common in the numerous-lacune than in the few-lacune subgroup. We conclude that silent lacunar stroke is often found in asymptomatic, high-risk, elderly Japanese patients and that silent multiple lacunar stroke is associated with hypercoagulability, endothelial cell damage, and high Lp(a) levels. PMID- 8640402 TI - Decreased serum testosterone in men with acute ischemic stroke. AB - Serum levels of total and free testosterone and 17 beta-estradiol were determined in 144 men with acute ischemic stroke and 47 healthy male control subjects. Blood samples from patients were drawn a mean of 3 days after stroke onset and also 6 months after admission in a subgroup of 45 patients. Initial stroke severity was assessed on the Scandinavian Stroke Scale and infarct size by computed tomographic scan. Mean total serum testosterone was 13.8 +/- 0.5 nmol/L in stroke patients and 16.5 +/- 0.7 nmol/L in control subjects (P = .002); the respective values for free serum testosterone were 40.8 +/- 1.3 and 51.0 +/- 2.2 pmol/L (P = .0001). Both total and free testosterone were significantly inversely associated with stroke severity and 6-month mortality, and total testosterone was significantly inversely associated with infarct size. The differences in total and free testosterone levels between patients and control subjects could not be explained by 10 putative risk factors for stroke, including age, blood pressure, diabetes, ischemic heart disease, smoking, and atrial fibrillation. Total and free testosterone levels tended to normalize 6 months after the stroke. There was no difference between patients and control subjects in serum 17 beta-estradiol levels. These results support the idea that testosterone affects the pathogenesis of ischemic stroke in men. PMID- 8640403 TI - Apolipoprotein A-II influences the substrate properties of human HDL2 and HDL3 for hepatic lipase. AB - Hepatic lipase has a demonstrated dual role in plasma lipid transport in that it participates in the removal of remnants of triglyceride-rich lipoproteins from the circulation and in the metabolism of plasma HDL. The study presented here investigated the substrate properties for hepatic lipase of HDL differing in density and apolipoprotein (apo) composition. Rates of fatty acid liberation were twofold higher in HDL2 compared with the respective HDL3 subspecies. Within each density class, enzyme-catalyzed fatty acid release was nearly twofold higher from HDL containing apoA-II compared with HDL devoid of apoA-II. When native HDL3 devoid of apoA-II was reconstituted with dimeric apoA-II in vitro, rates of fatty acid liberation in reconstituted particles were similar to those in native HDL3 containing apoA-II. HDL containing apoA-II competed more effectively with small VLDL for binding of hepatic lipase than HDL devoid of apoA-II. HDL3, particularly apoA-II-containing HDL3, reduced lipolysis of triglyceride and total fatty acid liberation in small VLDL. We conclude that the substrate properties of HDLs for hepatic lipase are influenced by both their size and apoA-II content. Moreover, size as well as apoA-II content may indirectly affect remnant clearance. PMID- 8640404 TI - Fenofibrate reduces plasma cholesteryl ester transfer from HDL to VLDL and normalizes the atherogenic, dense LDL profile in combined hyperlipidemia. AB - The effect of fenofibrate on plasma cholesteryl ester transfer protein (CETP) activity in relation to the quantitative and qualitative features of apoB- and apoA-I-containing lipoprotein subspecies was investigated in nine patients presenting with combined hyperlipidemia. Fenofibrate (200 mg/d for 8 weeks) induced significant reductions in plasma cholesterol (-16%; P < .01), triglyceride (-44%; P < .007), VLDL cholesterol (-52%; P = .01), LDL cholesterol (-14%; P < .001), and apoB (-15%; P < .009) levels and increased HDL cholesterol (19%; P = .0001) and apoA-I (12%; P = .003) levels. An exogenous cholesteryl ester transfer (CET) assay revealed a marked decrease (-26%; P < .002) in total plasma CETP-dependent CET activity after fenofibrate treatment. Concomitant with the pronounced reduction in VLDL levels (37%; P < .005), the rate of CET from HDL to VLDL was significantly reduced by 38% (P = .0001), whereas no modification in the rate of cholesteryl ester exchange between HDL and LDL occurred after fenofibrate therapy. Combined hyperlipidemia is characterized by an asymmetrical LDL profile in which small, dense LDL subspecies (LDL-4 and LDL-5, d = 1.039 to 1.063 g/mL) predominate. Fenofibrate quantitatively normalized the atherogenic LDL profile by reducing levels of dense LDL subspecies (-21%) and by inducing an elevation (26%; P < .05) in LDL subspecies of intermediate density (LDL-3, d = 1.029 to 1.039 g/mL), which possess optimal binding affinity for the cellular LDL receptor. However, no marked qualitative modifications in the chemical composition or size of LDL particles were observed after drug treatment. Interestingly, the HDL cholesterol concentration was increased by fenofibrate therapy, whereas no significant change was detected in total plasma HDL mass. In contrast, the HDL subspecies pattern was modified as the result of an increase in the total mass (11.7%) of HDL2a, HDL3a, and HDL3b (d = 1.091 to 1.156 g/mL) at the expense of reductions in the total mass (-23%) of HDL2b (d = 1.063 to 1.091 g/mL) and HDL3c (d = 1.156 to 1.179 g/mL). Such changes are consistent with a drug-induced reduction in CETP activity. In conclusion, the overall mechanism involved in the fenofibrate-induced modulation of the atherogenic dense LDL profile in combined hyperlipidemia primarily involves reduction in CET from HDL to VLDL together with normalization of the intravascular transformation of VLDL precursors to receptor-active LDLs of intermediate density. PMID- 8640405 TI - A cytotoxic electronegative LDL subfraction is present in human plasma. AB - By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium. PMID- 8640406 TI - Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations. AB - Oxidative modification of LDL is thought to be a radical-mediated process involving lipid peroxides. The small dense LDL subpopulations are particularly susceptible to oxidation, and individuals with high proportions of dense LDL are at a greater risk for atherosclerosis. An oxidatively modified plasma LDL, referred to as LDL-, is found largely among the dense LDL fractions. LDL- and dense LDL particles also contain much greater amounts of lipid peroxides compared with total LDL or the more buoyant LDL fractions. The content of LDL- in dense LDL particles appears to be related to copper- or heme-induced oxidative susceptibility, which may be attributable to peroxide levels. The rate of lipid peroxidation during the antioxidant-protected phase (lag period) and the length of the antioxidant-protected phase (lag time) are correlated with the LDL- content of total LDL. Once LDL oxidation enters the propagation phase, there is no relationship to the initial LDL- content or total LDL lipid peroxide or vitamin E levels. Beyond a threshold LDL- content of approximately 2%, there is a significant increase in the oxidative susceptibility of nLDL particles (ie, purified LDL that is free of LDL-), and this susceptibility becomes more pronounced as the LDL- content increases. nLDL is resistant to copper- or heme induced oxidation. The oxidative susceptibility is not influenced by vitamin E content in LDL but is strongly inhibited by ascorbic acid in the medium. Involvement of LDL(-)-associated peroxides during the stimulated oxidation of LDL is suggested by the inhibition of nLDL oxidation when LDL- is treated with ebselen prior to its addition to nLDL. Populations of LDL enriched with LDL- appear to contain peroxides at levels approaching the threshold required for progressive radical propagation reactions. We postulate that elevated LDL- may constitute a pro-oxidant state that facilitates oxidative reactions in vascular components. PMID- 8640407 TI - Differences in receptor binding of LDL subfractions. AB - Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d = 1.020 to 1.050 g/mL) from subjects with a predominance of large (> or = 272 A), medium (259 to 271 A), and small (< or = 257 A) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels < 160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C > or = 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d = 1.026 to 1.032 g/mL; II, d = 1.032 to 1.038 g/mL; and III, d = 1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (Kd) of LDL-II was similar for patterns A and B (9.2 +/- 1.4 and 9.4 +/- 0.7, respectively) and 30% lower in LDL-III from both groups (P < .05). The binding affinity of LDL-I in pattern A (12.6 +/- 1.5 micrograms/mg) was lower (P < .05) than that in LDL-II and LDL-I from pattern B (8.0 +/- 2.4 micrograms/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (Kd = 20.0 and 19.2 micrograms/mg) than in pattern A (Kd = 13.2 and 14.2 micrograms/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P < .001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo. PMID- 8640408 TI - Measurement of the free form of TFPI antigen in hyperlipidemia. Relationship between free and endothelial cell-associated forms of TFPI. AB - Tissue factor pathway inhibitor (TFPI), a protease with three tandem Kunitz-type (K1, K2, and K3) domains, inhibits the initial reaction of the TF-mediated coagulation pathway. TFPI occurs in a free and a lipoprotein-associated form in plasma as well as an endothelial cell-associated form on vascular walls. In a previous study we had demonstrated that free-form TFPI activity was lower in hyperlipidemic patients. In the present study we established a new enzyme immunoassay method for measuring free-form TFPI antigen; this new method uses a monoclonal antibody that recognizes the K3 domain of free-form TFPI but not lipoprotein-associated TFPI. Free-form TFPI antigen was significantly lower in hyperlipidemic patients compared with those in normolipidemic individuals. We applied this new method to measure the amount of endothelial cell-associated TFPI, which can be released by heparin injection, as "free-form TFPI." We found that free-form TFPI antigen in plasma was positively correlated with the endothelial cell-associated form. These results indicate that both of these forms of TFPI are in equilibrium in vivo and that our new method can be used for assessing changes in the levels of endothelial cell-associated TFPI antigen and, hence, for assessing thrombotic tendencies in various disease states. PMID- 8640409 TI - Gene expression of acyl-coenzyme-A:cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation. AB - The gene expression and enzyme kinetics of acyl coenzyme A:cholesterol acyltransferase (ACAT) were investigated in human monocytes, macrophages, and foam cells. Northern blot analysis using a 1.65-kb coding region of human ACAT cDNA as the probe showed that each of the cell types exhibited four mRNA transcripts. The levels of the 4.2- and 3.7-kb ACAT transcripts were three- and sixfold higher, respectively, in macrophages than monocytes. These transcripts were expressed at the same high levels after conversion of macrophages to foam cells. In contrast, the 6.3- and 4.4-kb transcripts for ACAT were expressed at a relatively constant level in all three cell types. The expression of mRNA for glyceraldehyde phosphate dehydrogenase, the control gene in this study, was also expressed at a constant level in each of the cell types. The increase in ACAT mRNA was accompanied by changes in the kinetic properties of the enzyme. Specifically, there was a 14-fold increase in Vmax and a 71% decrease in Km with respect to oleoyl coenzyme A. Although not definitive, the concomitant changes in mRNA and Vmax strongly suggest that the amount of ACAT protein increases upon conversion of monocytes to macrophages. The data show that ACAT in monocytes can be regulated by both substrate and gene expression. PMID- 8640410 TI - Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. AB - It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro. PMID- 8640411 TI - Distinct rat aortic smooth muscle cells differ in versican/PG-M expression. AB - Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call pi SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether pi and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [35S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 Kav on Sepharose CL-2B) that was not detectable in a pi SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial-type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat pi SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types. PMID- 8640412 TI - Magnetic resonance imaging in potential postsurgical recurrence of breast cancer: pitfalls and limitations. AB - OBJECTIVE: To determine the sensitivity and specificity of magnetic resonance imaging (MRI) of the breast for detecting recurrent carcinoma. PATIENTS AND METHODS: Thirteen patients ranging in age from 47 to 77 years who had undergone lumpectomy 5 months to 8 years earlier and who had mammographic findings suggestive of recurrence underwent contrast-enhanced dynamic MRI. Histologic confirmation was obtained in all cases. RESULTS: Of the eight lesions (in seven patients) for which biopsy proved recurrence, MRI correctly identified six; there were two false negative results. Of the six benign lesions, four were correctly identified by MRI. The two false positive results involved fat necrosis and a foreign-body reaction respectively. CONCLUSION: These results confirm previous reports of the poor specificity of MRI of focal breast lesions. The authors therefore recommend caution in the use of breast MRI in the assessment and management of suspected recurrent carcinoma. PMID- 8640413 TI - Squamous cell carcinoma of the breast. AB - OBJECTIVE: To review the mammographic and ultrasonographic features of primary breast cancer containing squamous cell carcinoma. PATIENTS AND METHODS: From medical records for breast cancer patients seen over a 37-year period the authors identified 19 patients with squamous cell carcinoma, for 5 of whom histologic sections and imaging studies were available. The tumours were classified on the basis of histologic findings as pure (in two patients) or predominantly (in three patients) squamous cell carcinoma. Mammograms were available for four of the patients, and the mammographic report only was available for analysis for the fifth. Ultrasonography had been performed for four of the patients; the images were available for two of the patients and the reports only for two. RESULTS: The median age of the patients was 55 years. The mean size of the tumours, all of which were palpable, was 5.0 cm. None of the tumours was connected to the skin, arose in the nipple-areolar complex or was metastatic. On mammography, the margins of all five masses, which were oval in shape, were indistinct and partly well-circumscribed; in three cases, the tumour margin was also partly spiculated. No malignant microcalcifications were seen. The two "pure" squamous cell carcinoma tumours appeared on ultrasonography as solid hypoechoic masses, and two of the predominantly squamous cell carcinoma tumours had both cystic and solid components. At gross pathological examination, four of the tumours (two "pure" and two predominantly squamous cell carcinoma) were cystic, which reflected areas of necrosis and cyst formation. CONCLUSION: Although "pure" or predominantly squamous cell carcinoma is a rare histologic variant of breast cancer that lacks any typical mammographic features, this tumour can be added to the differential diagnosis of cystic breast masses seen on ultrasonography. PMID- 8640414 TI - Computed tomography appearance of malignant schwannoma of the liver. AB - Malignant schwannoma of the liver without associated neurofibromatosis is rare. The authors present one such case in a 35-year-old woman. Computed tomography demonstrated large liver masses with contiguous involvement of adjacent stomach tissue, consistent with local invasion. PMID- 8640415 TI - Colouterine fistula: computed tomography and vaginography findings. AB - Colouterine fistulas are difficult to demonstrate radiologically. The authors present a case in which such a fistula was observed with computed tomography and confirmed by vaginography. PMID- 8640416 TI - Follow-up after insertion of Bird's Nest inferior vena caval filters. AB - OBJECTIVE: To determine the long-term clinical and radiographic outcome of patients who undergo insertion of a Gianturco-Roehm Bird's Nest vena caval filter (Cook Inc., Bloomington, Ind.). PATIENTS AND METHOD: The medical records of 40 patients who, over a 34-month period, underwent insertion of a Bird's Nest filter were reviewed and the reasons for filter insertion determined. The causes of any subsequent deaths were noted, and the autopsy findings, when available, were reviewed. Surviving patients were contacted, and 12 were willing to return for follow-up imaging, which consisted of plain radiography, real-time ultrasonography, colour Doppler imaging and contrast-enhanced computed tomography. Changes in filter position, the presence of thrombus and perforation of the vessel wall by the filter struts were documented. RESULTS: All 10 patients who underwent filter insertion while in an intensive care unit (ICU) died, an average of 22 days after the procedure. Eleven other patients also died. Recurrent pulmonary embolism was not suspected in any of these patients, and five autopsies revealed no caval thrombosis. Imaging studies in 12 of the surviving patients revealed no occlusion of the inferior vena cava and no filter migration; however, the vena caval wall was perforated in all of these patients. Nonocclusive intrafilter thrombus was detected by colour Doppler imaging in three patients. CONCLUSIONS: In this small group of patients the Bird's Nest filter was effective in preventing recurrent pulmonary embolism and caused less caval thrombosis than has previously been suspected, although intrafilter clot was found in 25% of the patients who underwent follow-up imaging. Colour Doppler imaging is the method of choice for detecting nonocclusive thrombus with this type of filter. Perforation of the caval wall was universal but not clinically symptomatic. Finally, guidelines should be established to ensure the cost effective use of inferior vena caval filters in ICU patients. PMID- 8640417 TI - Ultrasound monitoring in cannulation of the internal jugular vein: anatomic and technical considerations. AB - OBJECTIVE: To examine the effect of variations in anatomic features and operator experience on the success and complication rates of sonographically monitored cannulation of the internal jugular vein. PATIENTS AND METHODS: The authors prospectively collected data for ultrasound-monitored cannulation of the internal jugular vein in 150 patients. In all cases the radiologist recorded the side of puncture, the number of passes needed, the number of vein punctures (one or two), whether the walls were opposed during puncture and any complications. For the last 80 patients the following information was also recorded: the distance from the skin to the internal jugular vein, the diameter of the vein with the Valsalva manoeuvre and the location of the vein relative to the carotid artery. All but three of the cannulations were performed by one of three radiologists, all of whom had at least 5 years of experience. RESULTS: Cannulation was successful in all of the patients, and the first pass was successful in 133 (88.7%). These results are better than those of blind placement techniques reported in the literature. The only complications were hematoma and carotid puncture, which both occurred in the same two patients (1.3%). There was no significant difference among the radiologists in the number of passes needed (one-way analysis of variance, p > 0.05). The number of passes was independent of anatomic factors, including depth from skin, vein diameter or relative location. However, significantly more passes were needed for left-side punctures than for right-side punctures (Student t-test, p < 0.05). CONCLUSIONS: Real-time ultrasound monitoring is superior to blind techniques in cannulation of the internal jugular vein because of its ease, accuracy and safety. Sonographic real-time monitoring minimizes the impact of anatomic factors on success and complication rates. It is a safe and efficacious approach that should be preferred in the placement of central lines. PMID- 8640418 TI - The snapping hip: clinical and imaging findings in transient subluxation of the iliopsoas tendon. AB - OBJECTIVE: To define the clinical, ultrasonographic and magnetic resonance imaging (MRI) findings in patients with painful snapping of the hip secondary to transient subluxation of the iliopsoas tendon. PATIENTS AND METHODS: Seven patients, ranging in age from 17 to 30 years, with a total of eight painful snapping hips were examined with static and dynamic ultrasonography and MRI during hip motion producing the painful snapping. The duration of symptoms, the level of disability and the response to therapy were recorded. RESULTS: Static ultrasonography showed thickening of the iliopsoas tendon (tendinitis) in two cases and a peritendinous fluid collection in two cases. In all cases dynamic ultrasonography of the iliopsoas tendon during hip motion showed distinct abnormal motion of the tendon corresponding temporally to the painful palpable and audible sensation. MRI showed normal intra-articular structures in all cases, tendinitis in two cases and iliopsoas bursitis in one case. Clinically, subluxation of the iliopsoas tendon is a chronic (mean duration of symptoms in this series, 23 months) disabling condition that may be relieved by surgical tendon release. CONCLUSIONS: Dynamic ultrasonography is useful for detecting transient subluxation of the iliopsoas tendon in patients with a painful snapping hip. MRI is useful for excluding intra-articular abnormalities in patients with this condition. PMID- 8640419 TI - Radiation-associated gliosarcoma. AB - The authors report a case of radiation-associated intracerebral gliosarcoma with fibrosarcomatous predominance, arising at the site of a low-grade glioma treated 10 years previously. The features of this case conform to the accepted criteria for radiation-induced tumour, in that the tumour developed within the radiation field, differed dramatically in histologic features from the original tumour and did not develop until 10 years after treatment. Although such tumours are most uncommon, this case suggests that radiation-induced gliosarcoma should be considered in the differential diagnosis of recurrent mass at the site of a treated intracranial neoplasm. PMID- 8640420 TI - A prospective randomized clinical trial comparing two film-screen systems for chest radiography. AB - OBJECTIVE: To compare conventional and asymmetric film-screen chest radiography systems in a prospective, randomized trial. PATIENTS AND METHODS: Posteroanterior and lateral films were obtained with each system for one healthy volunteer and 49 consenting patients referred from pulmonary clinics and wards (for a total of 27 male and 23 female subjects ranging in age from 16 to 82 [mean 58] years). The radiographs, obtained and presented in random order, were reviewed and rated independently by two experienced radiologists and one resident in radiology; all observers were blinded to patient identification and film type. The Wilcoxon signed rank sum nonparametric test for paired samples was used to test for significant differences between the two film-screen systems. A second evaluation involving direct (blinded) comparison of the two types of films was then performed for each of the 25 patients in whom abnormality was noted during the first evaluation. RESULTS: For the posteroanterior radiographs, the asymmetric film-screen system was significantly better for assessing the trachea and mainstem bronchi, the descending thoracic-aortic edge, the left paraspinal line, the thoracic vertebral body interspace and the azygo-esophageal line (p < 0.05), whereas the conventional system had superior conspicuity in the lateral subpleural zones (p < 0.05). For the lateral radiographs, the asymmetric system was superior for assessing retrosternal lung markings (p < 0.05) but inferior for assessing fissures (p < 0.05). CONCLUSIONS: In general, the asymmetric system was superior for assessing mediastinal features and inferior for assessing the lateral subpleural zones in the posteroanterior radiographs. The asymmetric system was superior for assessing retrosternal lung markings and inferior for assessing fissures in the lateral radiographs. The results for the posteroanterior radiographs were consistent with the results of nonblinded studies reported elsewhere. PMID- 8640421 TI - Residents' corner: Answer to case of the month #37. Hepatic ascariasis. PMID- 8640422 TI - Contrast-enhanced imaging studies contraindicated in patients receiving Glucophage. PMID- 8640423 TI - Clinical monitoring of treatment course in child physical abuse: psychometric characteristics and treatment comparisons. AB - Weekly reports of high-risk indicators designed to monitor the course of treatment were obtained from physically abused, school-aged children and their parents/guardians who were randomly assigned to Individual Child and Parent Cognitive-Behavioral Treatment (CBT) or Family Therapy (FT). Measures of parental anger and physical discipline/force, and family problems were obtained each session. The measures showed moderate stability and parent-child correspondence. Between 20% and 23% of the two informant's reports acknowledged high levels of physical discipline/force during the early and late phases of treatment, respectively, and an even higher percentage of cases reported heightened parental anger and family problems. Early treatment reports from both informants predicted late period reports, but only parent reports were related to validity measures. The overall levels of parental anger and physical discipline/force were lower in CBT than FT families, though each group showed a reduction on these items from the early to late treatment sessions. The importance of routine monitoring of clinical course during intervention, especially in the identification of cases at risk of reabuse, is discussed. PMID- 8640424 TI - It's time to have another look at the medical model. PMID- 8640425 TI - Trauma history and personal narratives: some clues to coping among survivors of child abuse. AB - Narrative features of the life stories of child abuse survivors and nonvictimized respondents were compared. Particular emphasis was placed on relatively "objective" features, given that the content of the narrative typically precluded blind coding. The research focused on both the relative emphasis on the past versus present and future and on the self versus others in respondents' stories. The narratives of child abuse survivors differed from the comparison group on both of these features; their stories focused more on the past and de-emphasized the central role of the self. Greater emphasis on others was the best predictor of poor present coping among child abuse survivors. The narratives of a second sample of respondents who reported having experienced traumatic parental divorce were studied for comparison purposes. Despite some similarities in narrative construction, the increased emphasis on others, with its maladaptive associations, was unique to child abuse survivors. PMID- 8640426 TI - [The pediatrician, sexual abuse and anogenital warts in prepuberty]. AB - The increase of incidence in human papillomavirus infection in population has favored the more frequent appearance of anogenital warts in children. Types 6 and 11 are the most frequently associated to this pathology. Though sexual transmission is common, with a very lengthy incubation period, it is not the only way. Sexual abuse is always a possible source of infection and should be excluded in all cases. An actuation protocol has to be established for the pediatrician depending on the clinical, epidemiological and the social characteristics. PMID- 8640427 TI - Factors associated with entry into therapy in children evaluated for sexual abuse. AB - It is widely accepted that therapeutic intervention is an important and effective component in the treatment of the sexually abused child. The goal of this study was to identify children who are at risk for not receiving mental health services following sexual victimization. Nine-hundred and seventy-two children were followed for 6 months, and intervention by the Department of Children's Services (DCS), law enforcement agencies, and mental health professionals was monitored. Those children who were most likely to enter therapy were Caucasian, between the ages of 7 and 13, had cases in which DCS or law enforcement were involved, were placed outside the home, and experienced abuse of greater frequency. Implications of this study for intervention with child sexual abuse victims are discussed. Recommendations are made for future research. PMID- 8640428 TI - Interviewing children about past events: the influence of peer support and misleading questions. AB - The present experiment investigated the influence of peer support and leading and misleading questions on children's reports of a neutral event. Twenty-four children aged between 5 and 7 years and 24 children aged between 8 and 10 years took part in an event which focused on the parts and functions of the human body. Three days later they were interviewed about the event either alone or with a same-sex peer. The younger children recalled less information than the older children during prompted recall, and both age groups made very few errors. For questions, younger children made significantly more errors in response to directly misleading questions that to indirectly misleading questions. Both age groups were very accurate in response to directly and indirectly leading questions. Peer support did not influence children's prompted recall reports about the event or their responses to questions. These results are discussed in the context of their implications for interviewing children about past events. PMID- 8640429 TI - The long-term impact of the physical, emotional, and sexual abuse of children: a community study. AB - The associations between giving a history of physical, emotional, and sexual abuse in children and a range of mental health, interpersonal, and sexual problems in adult life were examined in a community sample of women. Abuse was defined to establish groups giving histories of unequivocal victimization. A history of any form of abuse was associated with increased rates of psychopathology, sexual difficulties, decreased self-esteem, and interpersonal problems. The similarities between the three forms of abuse in terms of their association with negative adult outcomes was more apparent than any differences, though there was a trend for sexual abuse to be particularly associated to sexual problems, emotional abuse to low self-esteem, and physical abuse to marital breakdown. Abuse of all types was more frequent in those from disturbed and disrupted family backgrounds. The background factors associated with reports of abuse were themselves often associated to the same range of negative adult outcomes as for abuse. Logistic regressions indicated that some, though not all, of the apparent associations between abuse and adult problems was accounted for by this matrix of childhood disadvantage from which abuse so often emerged. PMID- 8640431 TI - Child sexual abuse continues to present many dilemmas for the practicing pediatrician. PMID- 8640430 TI - Semistructured child sexual abuse interviews: interview and child characteristics related to credibility of disclosure. AB - This study provided the first empirical description of child and interviewer behaviors occurring within semistructured assessment interviews with children suspected of being victims of sexual abuse. Specifically, relationships between child and interviewer characteristics and interview credibility were examined. Using the Child Abuse Interview Interaction Coding System (CAIICS, Wood, 1990), 55 videotaped interviews of high-risk sex abuse cases seen at a multidisciplinary assessment center were behaviorally coded. Support was found for the interrater reliability and criterion related validity of the CAIICS was found. Results also revealed that children were initially rated as relaxed and displayed few emotional behaviors. Thus, the assumption that a credible disclosure of abuse must necessarily include the display of emotion by the child was not supported. Second, several behavioral differences between preschool and school-aged children were identified: however, no meaningful gender differences were found. Third, supporting evidence was found for both age and gender effects in judgments of interview credibility, with girls and school-aged children judged as more credible. Fourth, while the interviewer did engage in so called leading behaviors, these behaviors were not found to be related to rating of interview credibility. However, interviewer behaviors may have affected interview credibility through an intervening variable. Finally, implications, for further use of the CAIICS for examining interviewer-child interactions, evaluating standards of practice, and assisting with interviewer training are discussed. PMID- 8640432 TI - Limbal sub-Tenon's administration of retrobulbar anesthesia using a blunt irrigating cannula. AB - BACKGROUND AND OBJECTIVE: To eliminate sharp needles when administering local anesthesia to the eye in order to reduce serious complications caused by needle perforation. PATIENTS AND METHODS: After topical anesthesia, limbal conjunctival incision, and sub-Tenon's dissection, a retrobulbar irrigation of an equal mixture of bupivacaine (5 mg/ml) and lidocaine (20 mg/ml) was given using a blunt cannula. RESULTS: The technique was used in both vitreoretinal surgery (n = 70) and anterior segment surgery (n = 235) with good analgesic and akinetic effects. No serious adverse were noted. CONCLUSION: This proved to be a safe and efficient technique that abandoned the use of sharp needles. PMID- 8640433 TI - Accommodation and iridotomy in the pigment dispersion syndrome. AB - BACKGROUND AND OBJECTIVE: Iris concavity has been noted in pigment dispersion syndrome, and could have a role in producing iris-zonule contact. Iris concavity is most likely caused by a relative increase in anterior chamber pressure. The method by which this occurs remains speculative. The authors used ultrasound biomicroscopy to examine the role of accommodation in producing iris concavity and to document changes that occur following iridotomy. PATIENTS AND METHODS: Thirteen patients with clinically diagnosed pigmentary dispersion and pigmentary glaucoma underwent accommodation studies while being continuously imaged with ultrasound biomicroscopy. Anterior chamber depths were measured and iris configuration noted on distance and near fixation. These studies were repeated in 6 patients following laser iridotomy. RESULTS: All patients showed a decrease in anterior chamber depth with accommodation. Ten patients had a planar iris configuration on distance fixation and 3 concave. Eleven of 13 patients showed increased concavity of the iris on near fixation as compared with distance fixation. Following iridotomy in 6 patients, the iris showed a planar configuration that remained unchanged on near fixation. CONCLUSION: Accommodation increases iris concavity in some patients with pigment dispersion syndrome. The most likely explanation is an accommodation-induced relative increase in anterior chamber pressure secondary to anterior movement of the lens surface. Iridotomy prevents change in the iris profile with accommodation. PMID- 8640435 TI - The Castroviejo square graft. AB - The histopathologic features of three keratoplasty specimens from three cases involving square graft procedures performed by Dr. Castroviejo are reported. Light and electron microscopy were performed. In two of the cases, repeat keratoplasties were performed because of recurrent corneal dystrophies. In the third case, a repeat keratoplasty was performed because of graft failure. PMID- 8640434 TI - Anterior chamber fluid cultures following phacoemulsification and posterior chamber lens implantation. AB - BACKGROUND AND OBJECTIVE: A randomized, prospective study was undertaken to evaluate the incidence of positive cultures in the anterior chamber following cataract surgery when using intracameral gentamicin compared with no antibiotic. PATIENTS AND METHODS: A total of 97 patients were enrolled-- 48 receiving gentamicin in the irrigating solution and 49 receiving no intracameral antibiotics (control group). Each cataract extraction was accomplished by phacoemulsification followed by intraocular lens implantation. At the end of surgery, o.1 ml of anterior chamber fluid from each case was collected and cultured. RESULTS: All cultures were negative in the group receiving gentamicin. However, one positive culture occurred in the control group. There was no significant difference in the positive culture rate between the two groups (P = .52). CONCLUSION: This study suggests that cataract extraction with phacoemulsification and intraocular lens implantation has greatly reduced the incidence of bacterial contamination of anterior chamber fluid, with or without the use of intracameral antibiotic. PMID- 8640436 TI - Role of dacryocystectomy in the management of failed dacryocystorhinostomy associated with chronic dacryocystitis. AB - Three patients had recurrent chronic dacryocystitis but no epiphora associated with failed dacryocystorhinostomies. Dacryocystectomy was performed on all three patients. These patients were cured of dacryocystitis following dacryocystectomy and none had epiphora postoperatively. There may be a group of patients with dry eyes and chronic dacryocystitis in whom dacryocystorhinostomy is likely to fail. Such patients may respond favorably to dacryocystectomy. PMID- 8640437 TI - Vitrectomy and juvenile epiretinal membrane. AB - The cases of two children who had idiopathic epiretinal membranes are reported. Causes for juvenile epiretinal membranes, including trauma, pars planitis, toxocariasis, Coats' disease, or combined hamartomas, were not present. Both patients previously had documented normal vision in the affected eye. Observation revealed deterioration of vision, and a pars plana vitrectomy and a membranectomy were performed. The 5-year -old girl's vision improved from counting fingers at 5 feet to 20/80. The 12-year-old boy's vision improved from 20/200 to 20/80. Selected cases of juvenile epiretinal membranes may benefit from surgical excision. PMID- 8640438 TI - Benign orbital fibrous histiocytoma simulating a lacrimal gland tumor. AB - The case of a patient with a lacrimal fossa mass that was believed to be a primary lacrimal gland tumor is reported. However, at lateral orbitotomy the tumor was found within the lacrimal fossa, but distinct from the lacrimal gland. Histopathology revealed a benign fibrous histiocytoma. The tumor was totally excised, has not recurred over a 3-year follow-up period. This mesenchymal tumor should be included in the differential diagnosis of lacrimal fossa mass in adults. PMID- 8640440 TI - Retinal hemorrhages associated with ocular decompression after glaucoma surgery. AB - Two cases of retinal hemorrhages in glaucomatous eyes associated with ocular decompression following glaucoma filtration surgery are presented. Both patients were young, healthy men with markedly elevated preoperative intraocular pressures (IOPs). The hemorrhages were blot shaped and scattered throughout the macula (including the fovea) in each eye that underwent surgery. One eye had hemorrhages with distinct white centers. Each eye that underwent surgery experienced a permanent decrease in visual acuity after resolution of the hemorrhages. A gradual decrease of IOP preoperatively and intraoperatively is recommended in order to avoid this complication. PMID- 8640439 TI - Cluster headache presenting with orbital inflammation. AB - Acute, noninfectious orbital inflammations often defy a specific diagnosis despite a thorough medical evaluation and are grouped in the nonspecific diagnostic category of idiopathic inflammation of the orbit (pseudotumor). An atypical case of cluster headache presenting with intermittent migratory facial swelling and orbital inflammation, and 3 mm of exophthalmos simulating idiopathic orbital inflammation is presented. Although periobital pain is a frequent finding in patients suffering from cluster headaches, to the authors' knowledge this is the first reported case of orbital inflammation with cluster headache. After unsuccessful treatment directed at potential infectious and inflammatory causes, the patient responded well to methysergide, an established treatment for cluster headaches. A review of the known mechanism for this disorder, peripheral and central nervous system mechanisms of pain, and migratory angioedema reveals overlapping pathophysiology, clinical findings, and associated symptoms. PMID- 8640441 TI - Postoperative manipulation of the Krupin valve. AB - Five patients were noted to have elevated intraocular pressure following implantation of the Krupin glaucoma tube-to-plate device. In each, conjunctiva was draped flat over the plate. A muscle hook was used to manipulate conjunctiva over the tube-plate junction at the site of the valve, producing immediate increased flow through the tube, reduction of intraocular pressure, and bleb formation. In all five cases, intraocular pressure has been controlled at longest follow-up. One cause of elevated intraocular pressure following implantation of the Krupin tube-to-plate device is excessive resistance at the valve site, which can be relieved by manipulation with a muscle hook. PMID- 8640443 TI - Hinge keratoplasty. AB - A simple trephination technique is described that allows the surgeon to be prepared to handle an expulsive hemorrhage before it occurs. After trephination is performed, the host button is excised except for a 1 clock hour width of tissue. This hinge allows the attached button to fold over the corneal rim away from the surgical field. There is no interference with subsequent intraocular procedures. Should expulsive hemorrhage develop, the attached button can be immediately flipped into place and the wound quickly sewn closed. PMID- 8640442 TI - Relaxing retinotomy under perfluorocarbon liquid. AB - A technique of performing a relaxing retinotomy under the perfluorocarbon liquid perfluoroperhydropenanthrene (Vitreon, Vitrophage, Inc., Lyons, IL) is described. This procedure allows perfluorocarbon liquid to be used to maintain the retina in the desired position and to dissect proliferative membranes prior to the retinotomy without having to remove the Vitreon to perform the retinotomy. PMID- 8640444 TI - Diagnosis of traumatic cyclodialysis by ultrasound biomicroscopy. AB - BACKGROUND AND OBJECTIVE: To evaluate the ability of high-frequency ultrasound biomicroscopy to diagnose traumatic cyclodialyses not evident on clinical examination. PATIENTS AND METHODS: Six eyes to six patients with posttraumatic hypotony and/or shallow anterior chamber and suspected cyclodialysis clefts were examined with slit-lamp biomicroscopy, gonioscopy, B-scan ultrasonography, and ultrasound biomicroscopy. Ultrasound biomicroscopy provided high resolution of cross-sectional images of the anterior chamber angle, posterior chamber, and anterior uveal tissue. RESULTS: Ultrasound biomicroscopy confirmed the disinsertion of the ciliary body from the scleral spur and associated ciliary body detachment in all eyes. Gonioscopy failed to demonstrate a cyclodialysis cleft in five eyes because of hyphema (two eyes) and abnormal iris architecture (related to trauma) precluding visualization of the angle recess (three eyes). Using information from ultrasound biomicroscopy imagining, one patient underwent a ciliary body reattachment procedure and repair of the cyclodialysis cleft. CONCLUSION: Ultrasound biomicroscopy is a noninvasive method that can accurately diagnose the presence of traumatic cyclodialyses and can aid in surgical management. It is particularly useful in the presence of hazy media, hypotony, and/or abnormal anterior segment anatomy. PMID- 8640445 TI - Cell surface binding characteristics correlate with consensus type I interferon enhanced activity. AB - The binding characteristics of a genetically engineered consensus interferon with unusually high biologic activity were compared to the characteristics of recombinant interferon-alpha 2. Both interferon-alpha 2 and the consensus interferon produced typical biphasic Scatchard plots, indicating multiple independent binding sites. The consensus interferon, which exhibited a biologic potency more than 10-fold greater than all other type I interferons, also exhibited binding site affinities greater than those for IFN-alpha 2b. In addition, a larger number of high, and low-affinity cell surface sites were recognized by the consensus interferon, resulting in equivalent numbers of sites at reduced molar concentrations compared to IFN-a2b. Thus, at any given biologic activity, similar numbers of sites were bound by the consensus interferon and IFN alpha 2, despite differences in their molar concentrations. No differences in internalization kinetics were identified between the two interferons, indicating that the differences in cell surface binding may be sufficient to produce the differences in biologic activity. PMID- 8640446 TI - Potentiation by thyroxine of interferon-gamma-induced HLA-DR expression is protein kinase A- and C-dependent. AB - L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) potentiate the antiviral state induced by interferon-gamma(IFN-gamma) in homologous cells by a mechanism that is dependent upon calcium/phospholipid-dependent protein kinase (PKC). L-T4 and T3 also potentiate induction by IFN-gamma of MHC class II HLA-DR antigen expression in HeLa cells. In the present studies of HLA-DR expression, the PKC inhibitor staurosporine (0.1-1 nM) enhanced the expression of HLA-DR when the inhibitor was added simultaneously with IFN-gamma, 100 IU/ml. In the presence of IFN-gamma and 10(-7) M T4, the same concentrations of staurosporine inhibited potentiation of HLA-DR expression by thyroid hormone. A more specific PKC inhibitor, CGP41251 (0.5-5 nM), similarly enhanced HLA-DR expression in the presence of IFN-gamma but inhibited thyroid hormone potentiation of antigen expression. Both actions of CGP41251 were suppressed when cells were also treated with phorbol 12-myristate 13-acetate (PMA). A phospholipase C inhibitor, U73122 (1-1000 nM), did not alter the potentiating ability of T4, although it inhibited in a concentration-dependent manner the expression of HLA-DR induced by IFN gamma. The potentiating effect of T4 was much more sensitive to a cyclic AMP dependent protein kinase (PKA) inhibitor,KT5720 (1-1000nM), than was the induction of HLA-DR by IFN-gamma. The inhibitory effects of KT5720 were reversed by concurrent 8-bromo-cAMP treatment. The calmodulin antagonist W-7 (5-50 microM) did not alter IFN-gamma induction of HLA-DR in either the presence or absence of T4. HLA-DR expression in HeLa cells appears to be under PKC-associated inhibition; IFN-gamma reverses this inhibition to promote the appearance of the DR antigen. In contrast, potentiation by T4 of induction of HLA-DR by IFN-gamma requires activation of PKC. PKA is involved both in DR induction by IFN-gamma and in potentiation of the latter by T4. Thus, PKA and PKC have discrete roles in IFN gamma-induced MHC class II antigen expression and its modulation by thyroid hormone. PMID- 8640447 TI - Cloning and biologic activities of a bovine interferon-alpha isolated from the epithelium of a rotavirus-infected calf. AB - A cDNA encoding a distinct bovine (Bo) interferon (IFN) alpha, designated BoIFN alpha E, was generated from gut epithelial cells isolated from a rotavirus infected calf. The BoIFN-alpha E cDNA sequence shared a greater than 90% identity with the other BoIFN-alpha subtypes. The cDNA encoding BoIFN-alpha E has been expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Insect cells infected with recombinant virus secreted a protein with a relative molecular mass of 19,500 into the culture medium not observed in cells infected with wild-type AcMNPV. Supernatants harvested from cultures of insect cells infected with the recombinant AcMNPV encoding IFN-alpha E inhibited the replication of Semliki Forest virus in a bovine cell line and typically showed 10(6) dilution units/ml of antiviral activity. However, differences were observed between the activities of recombinant BoIFN-alpha E and BoIFN-alpha 1 1 on the proliferation of WC1+ gamma/delta T cells. Purified ( > 99%) WC1+ gamma/delta T cells failed to proliferate to IFN-alpha 1 1 or concanavalin A and IFN-alpha E acted as a weak proliferative signal to these cells, demonstrating a functional difference between two closely related BoIFN-alpha subtypes. PMID- 8640448 TI - MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt. AB - MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed. PMID- 8640449 TI - Early cytokine synthesis in the excised mouse cornea. AB - Corneas excised from normal BALB/c mice and incubated in vitro were analyzed for the production of "early-warning" cytokines via reverse transcription-polymerase chain reaction and ELISA. It was found that the trauma of excision stimulated rapid IL-1 alpha synthesis, with peak protein accumulation occurring at 6 h, whereas IL-6 synthesis was maximal at 18 h. Neither IL-1 beta protein nor message was detected at any point, and TNF-alpha synthesis never increased above constituted levels. Antibody neutralization of endogenous IL-1 alpha blocked IL-6 synthesis. Addition of exogenous IL-1 alpha induced IL-1 alpha and IL-6 synthesis in vitro. Inoculation of IL-1 alpha into the cornea induced IL-6 synthesis in vivo. Addition of IL-1 alpha could stimulate IL-1R, IL-1 alpha, and IL-6 mRNA synthesis in the epithelial, stromal, and endothelial components of the cornea. However, protein production was readily detected only in the epithelial layer. We concluded that mechanical trauma to the mouse cornea triggers the enhanced synthesis of IL-1 alpha and IL-1R, which in turn results in the production of IL 6 and more IL-1 alpha. That corneal excision did not stimulate the synthesis of IL-1 beta or TNF-alpha indicates that there is a selective induction of early cytokine expression in this specialized tissue. PMID- 8640450 TI - Induction of a cytokine network by superantigens with parallel TH1 and TH2 stimulation. AB - Superantigens cross-link the MHC class II molecule on accessory cells with the V beta region of the TCR outside the antigen binding sites. In this study, we compared the capacity of the staphylococcal entertoxins (SE) A, B, C1, C2, C3, D, and E, the toxic shock syndrome toxin (TSST) 1, the exfoliative toxin (ExFT) A, and the Streptococcus pyogenes erythrogenic exotoxins (SPE) B and C to induce cytokine release in human peripheral blood mononuclear cells. We showed that all toxins tested induced IL-1 alpha and beta, IL-2, IL-4, IL-6, IFN-gamma, and TNF alpha, but not IFN-alpha. However, we found that SPEB differed from all other toxins tested, because its cytokine induction was significantly lower than that of the other toxins. This was not true of IL-6 and IL-10 induction, in which SPEB showed similar amounts of IL-6 compared with all other toxins and of IL-10 in comparison to SEC2. SPEB showed a specificity for TH2 cells, whereas the other toxins stimulated TH1 as well as TH2 cells very strongly. As a result, superantigens appear to be able to uncouple the TH1/TH2 antagonism. Collectively, our results indicate that SPEB seems not to be a superantigen or represents a different group of microbial superantigens. Furthermore, superantigens stimulate TH1 as well as TH2 cells without any preference and therefore they are able to induce humoral as well as cellular immunity. This could be one reason for the existence of autoantibodies and autoreactive T cells in autoimmune diseases and one major step in the beginning of the induction of autoreactivity. PMID- 8640451 TI - Poly ICLC inhibits Plasmodium cynomolgi B malaria infection in rhesus monkeys. AB - Prophylatic treatment with a single dose of 1.0 or 2.0 mg/kg (body weight) of polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (Poly ICLC), a potent interferon (IFN) inducer and immune enhancer, 18 h before intravenous inoculation of sporozoites (1.04 x 10(5)-0.70 x 10(6) sporozoites) of Plasmodium cynomolgi B in the rhesus monkey, completely abolished the infectivity of sporozoites. The inhibitory effect of Poly ICLC is dose dependent in monkeys infected with P. cynomolgi B sporozoites. Treatment with lower doses of Poly ICLC (0.5 mg/kg) provided significant protection, but the lowest dose of Poly ICLC used (0.1 mg/kg) failed to provide any protection. Prophylactic treatment with Poly ICLC, however, had no protective effect against trophozoite-induced infection. PMID- 8640452 TI - Interleukin-8 inhibits non-small cell lung cancer proliferation: a possible role for regulation of tumor growth by autocrine and paracrine pathways. AB - Interleukin-8 (IL-8) is an 8 kD chemokine and angiogenic factor produced by alveolar macrophages, endothelial cells, monocytes, fibroblasts, T lymphocytes, and epithelial cells in response to a variety of stimuli, including LPS, TNF alpha, IL-1, IL-7, and hypoxia. Pulmonary tumors produce a variety of growth factors and cytokines that may act in both autocrine and paracrine fashion. A549, a well-characterized human lung adenocarcinoma line, was cloned for different levels of IL-8 production by limiting dilution. Clone 3B4 produced 361 +/- 73 pg/ml, and clone 2B2 produced 7818 +/- 614 pg/ml of IL-8 (p = 0.003). Clone 3B4 proliferated at 1.7 times the rate of 2B2. Anti-IL-8 reversed the decrement in proliferation of clone 2B2 by 50%, but recombinant IL-8 decreased the proliferation of 3B4 by 40-55% compared with control. In addition to A549, three other non-small cell lung cancer (NSCLC) lines showed significantly decreased proliferation in response to exogenous recombinant IL-8 (5-30 ng/ml; p < 0.05). These findings suggest that in addition to its chemotactic and angiogenic activities, IL-8 may inhibit lung tumor proliferation by both autocrine and paracrine pathways. PMID- 8640454 TI - Murine cardiotrophin-1 stimulates the acute-phase response in rat hepatocytes and H35 hepatoma cells. AB - Mouse cardiotrophin-1 (CT-1) is a hypertrophy-inducing factor for cardiac myocytes and interacts with cell surface receptors that incorporate the signaling molecule gp130. Because other cytokines utilizing this receptor subunit stimulate acute-phase protein synthesis, we tested cardiotrophin-1 in in vitro assays of protein synthesis by primary rat hepatocytes, rat hepatoma cells (H35), and human hepatoma cells (HepG2). CT-1 showed a dose-dependent induction of protein synthesis by primary rat hepatocytes, with effective concentrations ranging from 0.1 to 100 ng/ml. Production of a number of acute-phase proteins, including alpha 1-cysteine proteinase inhibitor ( alpha 1-CPI), alpha 1-proteinase inhibitor (alpha 1-Pi), alpha 2-macroglobulin, and alpha 1-acid glycoprotein, was markedly increased at 48 and 72 h of cytokine stimulation. In rat H35 cells, CT-1 stimulated alpha 1-Pi and alpha 1-CPI protein production and upregulated alpha 1 CPI mRNA levels with similar potency. Compared with other IL-6-type human cytokines at optimal concentrations in parallel assays, CT-1 induced similar levels of acute-phase proteins as human oncostatin M (OM) and leukemia inhibitory factor (LIF), whereas human IL-6 induced the greatest levels of alpha 1-CPI or alpha 1-Pi production by H35 cells. When tested on human HepG2 cells, murine CT-1 was far less effective, in that it stimulated alpha 1-antichymotrypsin production only at very high concentrations (100 ng/ml) but did not alter haptoglobin or alpha 1-Pi. Human OM and IL-6 were effective at lower concentrations and induced much higher levels of acute-phase protein synthesis, whereas LIF activity was similar to that to CT-1. These results show that murine CT-1 is a strong acute phase mediator for rat hepatocytes in vitro and its activity is similar to LIF on rat hepatocytes, H35 cells, and HepG2 cells. PMID- 8640453 TI - Antisense manganese superoxide dismutase mRNA inhibits the antiviral action of interferon-gamma and interferon-alpha. AB - Manganese superoxide dismutase (MnSOD) is induced by interferon-gamma (IFN-gamma) in various cell lines. To determine whether MnSOD plays a role in the antiviral action of IFN-gamma, we employed an antisense strategy to inhibit the expression of MnSOD in the human melanoma cell line, A375. Three antisense-containing clones that exhibited reduced induction of MnSOD were investigated with respect to their response to the antiviral protective effects of IFN-gamma and IFN-alpha. We observed a striking decrease in the ability of IFN-gamma to protect antisense clones from vesicular stomatitis virus infection (VSV). The IFN-alpha induced antiviral state was also impaired, but to a lesser degree than was observed with IFN-gamma. We excluded the possibility that these effects were caused by a higher sensitivity of the antisense cells to VSV itself and found that the antisense clones actually were less sensitive to VSV. Therefore, we conclude that MnSOD is involved in the establishment of the IFN-gamma-induced antiviral state and to a lesser degree in the antiviral actions of IFN-gamma. PMID- 8640455 TI - Stimulation of natural interferon-alpha/beta-producing cells by Staphylococcus aureus. AB - Human peripheral blood mononuclear cells (PBMCs) produced high levels of antiviral activity, as determined by bioassay, when stimulated by Staphylococcus aureus Cowan I (SAC) and E. coli. Specific immunoassays demonstrated the presence of both IFN-alpha and gamma and, for SAC, also low levels of IFN-beta. The frequencies of SAC-induced IF N-alpha-producing cells (IPCs) were up to 1-2 per 10(3) PBMCs. These IPCs expressed the HLA-DR and CD4 antigens but not CD3, CD14, or CD19, thus resembling the natural IFN-alpha-producing cells (NIPC). The SAC was more efficient as IFN inducer when heat killed than when streptomycin inhibited. The SAC was inhibitory to virally induced IFN-alpha responses, in particular when streptomycin inhibited. Both pronase treatment and mechanical disruption of SAC cells abolished their capacity to induce IFN-alpha production. Staphylococcal strains lacking or expressing low levels of protein A (SpA) showed a decreased ability to induce IFN-alpha production. However, purified SpA did not itself induce IFN-alpha. Possibly, SpA together with other bacterial surface proteins is important for the capacity of SAC to induce IFN-alpha production in NIPC. PMID- 8640456 TI - Calcium signals and protein tyrosine kinases are required for the induction of c jun in Jurkat cells stimulated by the T cell-receptor complex and oxidative signals. AB - The regulation of c-jun plays an important role in T cell activation, proliferation, and expression of interleukin-2. In the present study, we determined whether Ca2+ signals and the activity of protein tyrosine kinases (PTKs) were required for the induction of c-jun in Jurkat cells stimulated with cross-linked anti-T cell receptor/CD3 antibodies or exposed to oxidative stress in the form of micromolar concentrations of H2O2. Jurkat cells exhibited rapid elevations in intracellular calcium [Ca2+]i levels in response to H2O2 and cross linked anti-CD3 antibodies that mainly reflected the influx of extracellular Ca2+. The Ca2+ flux in response to oxidative signals was distinguished by an exquisite sensitivity to inhibition with Ni2+, suggesting the involvement of cation channels. PTK activity was needed for [Ca2+]i elevations in response to both oxidative and anti-CD3 signals, although H2O2 induction of [Ca2+]i increases was more resistant to inhibition by genistein than anti-CD3 [Ca2+]i responses. Both oxidative signals and anti-CD3 stimulation induced increased levels of c-jun and c-fos mRNA. The increased expression of c-jun with H2O2 was preceded by [Ca2+]i increases and accompanied by activation of c-Jun aminoterminal kinases (JNKs), as well as increased AP-1 binding activity. Induction of c-jun with oxidative signals and anti-CD3 was also shown to be crucially dependent on [Ca2+]i elevations because the chelation of [Ca2+]i with BAPTA resulted in a dose dependent inhibition of c-jun expression. Furthermore, inhibition studies demonstrated that the optimal induction of c-jun mRNA in response to oxidative signals required PTK as well as protein kinase C (PKC). Thus, these findings suggest that both [Ca2+]i signals and the activity of PTKs are essential for the optimal expression of c-jun in response to TCR/CD3 signals and changes in redox potentials. PMID- 8640457 TI - [Mammalian small stress proteins and responses to stress]. AB - When cells are exposed to heat stress or chemical stress, expression of genes for heat shock proteins or stress proteins (HSPs) is enhanced and the proteins are accumulated in cells. The cells with increased HSPs exhibit tolerance against the additional stress. HSPs are expressed also in unstressed tissues or cells for essential biochemical cellular processes including growth and differentiation. Since the responses of HSPs in tissues to stress loaded to a whole living body are much more sensitive compared to those in cultured cells, it is suggested that endogenous factors modulate the stress-induced expression of HSPs. Here we summarize the responses of small HSPs (alpha crystallins, HSP27 and p20) to stress and their modifications by various factors. PMID- 8640459 TI - Permanent occlusion of bilateral internal carotid arteries produces cognitive deficits in two learning behavior tasks. AB - We investigated the effects of permanent bilateral occlusion of the internal carotid arteries (2ICAO) on the learning and memory performances in rats to evaluate the permanent 2ICAO rats as a model for vascular dementia. The learning and memory performance was tested by a step-through passive avoidance task and an 8-arm radial maze task. Permanent 2ICAO decreased cerebral blood flows in the cortex and hippocampus by 46.3 +/- 3.3 and 21.1 +/- 4.6%, respectively, when measured at 15 min after occlusion. In the passive avoidance task, the 2ICAO rats showed no impairment of learning or of memory retention when tested 1 h after learning trial, while they showed a shorter latency than sham-operated rats when tested 24 h after learning trial. In the radial maze learning task, the non pretrained 2ICAO rats showed impairment. The pretrained 2ICAO rats had no deficit in the radial maze retention task but they showed impaired performance when a 3 min delay was interposed in the task. These results suggest that permanent 2ICAO is a useful animal model for studying vascular dementia. PMID- 8640458 TI - Modification by MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, of morphine sensitization: evaluation by ambulation in mice. AB - The effects of five administrations (3- to 4-day intervals) of morphine (MOR: 10 and 20 mg/kg, s.c.) alone, MK-801 (dizocilpine: 0.03, 0.1, 0.3 and 1 mg/kg, i.p.) alone, and combinations of MOR with MK-801 on the ambulation in mice were investigated. MK-801 at 0.3 and 1 mg/kg, but not at 0.03 and 0.1 mg/kg, significantly increased the ambulation of mice. Although the mice given repeated administrations of MK-801 (0.3 and 1 mg/kg) exhibited enhancement and reduction, respectively, in the ambulation-increasing effect of the individual doses, they showed significantly higher sensitivity than the saline-treated mice to the challenge with MOR (10 mg/kg). The repeated administrations of MOR (10 and 20 mg/kg) induced a progressive enhancement of the ambulation-increasing effect. The mice repeatedly given MOR (10 mg/kg) exhibited significant increase in the sensitivity to MK-801 (0.03-0.3 mg/kg). The coadministrations of MOR with MK-801 intensified the ambulation-increasing effect, and repeated coadministrations induced progressive enhancement of the effect, except for the combinations of MOR (10 or 20 mg/kg) with MK-801 (1 mg/kg). However, the induction of MOR sensitization was not modified by any doses of MK-801, except for the case of combination of MOR (20 mg/kg) with MK-801 (1 mg/kg) which was highly toxic (i.e., eliciting death or a moribund condition). On the other hand, simultaneous treatment with SCH 23390 (0.05 mg/kg, s.c.) or nemonapride (0.05 mg/kg, s.c.), or 4-hr pretreatment with reserpine (1 mg/kg, s.c.) strongly, and 4-hr pretreatment with alpha-methyl-p-tyrosine (200 mg/kg, i.p.) partially reduced the ambulation increasing effect of both MOR (10 mg/kg) and MK-801 (0.3 mg/kg). Simultaneous treatment with naloxone (1 mg/kg, sc) selectively reduced the effect of MOR. However, simultaneous treatment with apomorphine (0.1 mg/kg, s.c.) did not modify the effects of either drug. These results suggest that the characteristics of the ambulation-increasing effects of MOR and MK-801 are similar to each other, and that the repeated treatments with MK-801 induce a cross-sensitization to MOR and vice versa. PMID- 8640460 TI - Long-lasting inhibition of 5-HT uptake of platelets in subjects treated by duloxetine, a potential antidepressant. AB - Duloxetine is an inhibitor of serotonin and norepinephrine uptake, and is being developed as a new antidepressant. In the present study, using healthy volunteers who took 20 mg of duloxetine for 7 days, the plasma concentrations of duloxetine and the ex vivo serotonin uptake rate in the platelets were simultaneously monitored during and after administration. Furthermore, a comparison was made by measuring parameters for serotonin uptake in vitro and [3H]paroxetine binding before and after administration. Actual values of the uptake inhibition rate ex vivo were stronger than those expected in spite of the dilution of plasma in the experiment, and the inhibitory effect was seen even after the drug was no longer detected in plasma. No significant changes were observed in Vmax, Km, Bmax or Kd. Thereafter, the effect of the washing procedure was examined in platelets treated with different antidepressants in vitro. The minimum effect was seen in platelets treated by duloxetine or paroxetine, while desipramine-treated platelets showed susceptibility to the procedure. These results suggest that duloxetine was hardly dissociated from the serotonin uptake site, which was responsible for the strong and long-lasting effect of plasma. PMID- 8640461 TI - [Clinical effects of cortisol synthesis inhibition on treatment-resistant depression]. AB - Clinical trial with a steroid suppressive agent, metyrapone, was carried out in 6 patients with treatment-resistant depression (3 patients with major depression and 3 with bipolar disorder). Up to 2,000 mg/day of metyrapone was administered for 4 weeks, and 10 trials of the therapy were done in these patients. Most patients completed the therapy without remarkable side effects. As a result, three patients (6 trials) showed remission within 4 weeks and one patient (one trial) showed a partial response. In the remitted patients, plasma cortisol levels were suppressed below 10 micrograms/dl during the therapy and plasma ACTH levels were elevated. These results indicate that "hypercortisolemia-induced depression" similar to Cushing's disease may be present in patients with treatment-resistant depression. PMID- 8640462 TI - Prediction of first-trimester miscarriage from embryonic bradycardia and embryonic growth delay. AB - OBJECTIVE: The purpose of this study was to determine whether subsequent and simultaneous measurements of embryonic heart rate (EHR) and crown-rump length (CRL) are useful in predicting miscarriage. STUDY DESIGN: A prospective cross sectional study was performed on miscarriages with detectable embryonic heart rate (n = 33). Chromosomal tests were carried out in 9 of 33 cases. RESULT: In cases with embryonic bradycardia and/or embryonic growth delay, the mean CRL was 6.6 mm (n = 16), and embryonic death occurred at CRL values under 20 mm. In cases where neither bradycardia nor growth delay was detected, the CRL was 23.9 mm (n = 17) at embryonic death. Chromosomal abnormalities were present as triploidy (n = 5) and trisomy-16 (n = 2) in both and embryonic-growth-delay cases. CONCLUSION: Subsequent and simultaneous measurements of EHR and CRL are useful in predicting miscarriage in the first trimester, especially when the CRL is under 20 mm. PMID- 8640463 TI - Enhanced magnetic resonance imaging in monitoring of conservative treatment of cervical pregnancy. AB - We report on case of cervical pregnancy successfully treated by two 5-day courses of etoposide, embryocide with the injection of KCl, the injection of methotrexate into the placenta, and cervical curettage. We evaluated the effects of these treatments by measuring urinary human chorionic gonadotropin (hCG) titers and serial hCG beta-carboxy-terminal peptide (CTP) levels. Moreover, we used ultrasonography and magnetic resonance imaging (MRI) enhanced by gadolinium diethylenetriamine-pentaacetic acid (Gd-DTPA) to diagnose and to evaluate the blood supply to the trophoblast. The beneficial outcomes suggest that Gd-DTPA enhanced MRI is useful in detecting the blood supply to the trophoblast, in order to avoid the massive bleeding that results from conservation treatments. This is the first report of the usefulness of Gd-DTPA-enhanced MRI for the successful conservative treatment of cervical pregnancy. PMID- 8640464 TI - Detection of lymph node metastasis in ovarian carcinoma and uterine corpus carcinoma by preoperative computerized tomography or magnetic resonance imaging. AB - OBJECTIVE: Preoperative CT or MRI findings were compared with the results of staging the laparotomy to evaluate the accuracy of CT or MRI for detecting pelvic and para-aortic lymph-node metastases. METHODS: In evaluating CTs in 95 ovarian carcinomas, we examined plain and contrast images made in 1- to 1.5-cm-thick slices from the pubis to the xiphoid process. Lymph nodes 1.5 cm or larger were considered to be positive. MRIs of 60 uterine corpus carcinomas utilized T1 weighted contrast, T2-weighted, and short-inversion time inversion-recovery (STIR) images. RESULTS: CT had a sensitivity of 60.9% and a specificity of 93.1%. The positive and negative predictive values were 73.7% and 88.2%, respectively. The diagnostic accuracy of CT for detecting para-aortic lymph-node metastases exceeded that for pelvic node metastases. The results of MRI indicated that the T1-weighted image and STIR image were the most accurate in identifying metastatic nodes. CONCLUSIONS: These results indicate that the most practical approach might be to search for enlarged lymph nodes by CT, and to follow-up with MRI when CT scans are questionable. PMID- 8640466 TI - Early oral hydration: a novel regimen for management after elective cesarean section. AB - OBJECTIVE: To compare early oral hydration versus the conventional intravenous fluid replacement after elective cesarean section. METHODS: Two hundred women performed elective cesarean section were selected and randomized to: Regimen I (n = 100): early oral hydration (sips of fruit juices sweetened with honey once the women felt thirst immediately after the operation, then solid food 24 hours later, the amounts of juice or food were determined by the patient herself, i.e. she would regulate her physiological needs) versus regimen II (n = 100): conventional intravenous hydration (2-3 L of dextrose saline/24 hours and solid food thereafter). Biochemical, metabolic values, postoperative nausea and vomiting, propulsive bowel movements, tolerance of solid food, hospital stay and successful continuation of breast feeding were compared in both groups. RESULTS: The two regimens were equally effective in maintaining fluid balance and normal plasma and urinary electrolytes without any observed differences in biochemical or metabolic values. The return of bowel sounds, first occurrence of flatus and tolerance of solid food were achieved in regimen I at time periods significantly shorter than those attained with regimen II (p < 0.0001). The incidence of nausea and vomiting, ileus and deep vein thrombosis was higher in regimen II than in regimen I but the difference is statistically insignificant (p > 0.05). Patients in regimen II were hospitalized for significantly longer times than those on regimen I (p < 0.0001). Successful continuation of breast feeding was significantly more in regimen I than in regimen II (p < 0.01). CONCLUSION: Early oral hydration after elective cesarean section effectively maintained fluid balance and it was associated with rapid return of propulsive bowel movements, successful breast feeding, less side effects and shorter hospital stay than the conventional intravenous hydration. So, it should be used in most cases after elective cesarean section. PMID- 8640465 TI - Prevalence, genotypes and antibody titer of hepatitis C virus in pregnant women in Taiwan. AB - OBJECTIVES: To investigate the prevalence and genotypes of hepatitis C virus (HCV) in pregnant women in Taiwan, as well as to examine whether any correlation occurs between HCV genotype and anti-HCV titer. METHODS: Forty-three pregnant women with positive anti-HCV and HCV-RNA were selected among 3,400 cases screened from January 1992 to March 1994. Each blood specimen was assayed for HCV genotypes by PCR method to detect HCV I, II, III and IV. Anti-HCV titer was determined by a second-generation EIA kit with serial dilutions. RESULTS: Twenty eight cases (65%) belonged to HCV II, 11 (26%) HCV III, 3 (7%) HCV IV and 1 (2%) HCV II+III. Besides, the anti-HCV titers in HCV II and HCV III groups were similar, ranging from 8x to 40,000x and revealing no statistical significance (p = 0.75). CONCLUSIONS: Our data have verified that the prevalence rate of HCV infection in pregnant women is around 1.3% and no significant difference of anti HCV titer occurs between HCV II and HCV III pregnant women. PMID- 8640467 TI - Clinical trial of daily low-dose oral etoposide for patients with residual or recurrent cancer of the ovary or uterus. AB - OBJECTIVE: To determine the efficacy of long-term therapy of oral etoposide in patients with residual or recurrent gynecological malignancies. METHODS: Twenty five Japanese patients with resistant or recurrent carcinoma of the uterus or ovary were treated with oral etoposide at a dose of 25 mg/day/body for 21 consecutive days, and cycles were repeated every 4 weeks. The residual or recurrent lesion could be objectively evaluated in all patients by measuring it directly. RESULTS: The response rate after 6 cycles of therapy was 40% for the group of all patients, and 42.8%, 28.6% and 50% for those with ovarian carcinoma, cervical carcinoma, and endometrial carcinoma, respectively. Side effects of etoposide treatment included gastrointestinal discomfort in 14 patients and leukopenia of grade 3 or higher in 2 patients. However, these side effects were mild, and all patients could continue treatment. CONCLUSION: These findings indicate that long-term, low-dose oral etoposide was effective for and well tolerated by patients with refractory or recurrent carcinoma of the ovary or uterus. PMID- 8640468 TI - Anaphylactic reaction to cefazolin in pregnancy. AB - Anaphylaxis is a rare event in pregnancy, but is one that can have serious implications for both the mother and fetus. We report a case of severe anaphylactic reaction to intravenous (i.v.) cefazolin (CEZ) given during labor with premature rupture of membranes. Maternal shock and prolonged fetal bradycardia occurred. Both the maternal and fetal outcomes were good following treatment with ephedrine, glucocorticoids, and emergency caesarean section. PMID- 8640469 TI - Effect on neonatal thyroid function of povidone-iodine used on mothers during perinatal period. AB - OBJECTIVE: Our objective was to evaluate the effect on the neonatal thyroid function of povidone-iodine (PVP-I) used on mothers during the perinatal period. METHODS: Eight consecutive-term pregnant women were randomly separated into 4 groups. Either PVP-1 or benzethonium chloride (BC) was used to disinfect both maternal skin during labor and vaginal lacerations after delivery in Group I and IV; and PVP-1 or BC was supplied to either skin or lacerations in Groups II and III. On day 5, we measured the iodide concentration in breast milk and random urine of neonates, and the levels of neonatal thyroid-stimulating hormone (TSH) and free thyroxine. RESULTS: The TSH levels in Group I and the breast-milk iodide concentrations in Groups I and II were significantly higher than those in Group IV (p < 0.05). Dietary iodide intake from breast milk correlated significantly with urinary iodide concentrations (r = 0.52, p < 0.001). In our clinic, the recall rate at the time of screening for congenital hypothyroidism was significantly decreased by discontinuing the use of PVP-I (4.47% vs 0.74%, p < 0.001). CONCLUSIONS: These findings suggest that the use of PVP-I on mothers is associated with transient neonatal hyperthyrotropinemia, in which breast milk might play an important role. Therefore, we recommend that the use of PVP-I be avoided during labor. PMID- 8640470 TI - A randomized study comparing suture with non-suture cold-knife conization. AB - OBJECTIVE: To compare the short-term outcome of cold-knife conization between suture and non-suture (with Monsel's solution pack) methods. METHODS: Between February, 1990 and March, 1992, 142 patients underwent cold-knife conization were randomly allocated into 2 groups. The hemostasis was done by suture in one group and by Monsel's solution pack without suture in the other group. The patients' and cone specimens' characteristics as well as short-term outcome were recorded and compared using Chi-square test, Fisher exact test and t-test where appropriate. RESULTS: The patients' characteristics, cone size, and histologic diagnosis of both groups were comparable. The non-suture group had shorter operative time (10.69 +/- 7.86 vs. 20.04 +/- 6.99 minutes) and lesser late hemorrhage (2.9 vs. 15.9%) than the suture group (p < 0.05). There was no statistical difference in the operative blood loss. CONCLUSION: The non-suture method of conization should replace the traditional suture method. PMID- 8640471 TI - Immunohistochemical detection of p53 overexpression in formalin-fixed, paraffin embedded sections of endometrial carcinoma. AB - OBJECTIVE: In the past, immunohistochemical detection of p53 has been possible only in the case of fresh-frozen tissues. However using CM1 and an enhanced method based on microwave heating and protease digestion enabled the immunohistochemical detection of p53 overexpression in formalin-fixed, paraffin embedded sections of endometrial carcinoma, although very few positive stainings were obtained without such an enhanced treatment. To evaluate the enhancement method, a comparative study was performed on frozen materials and paraffin embedded samples. METHODS: Fifty-three frozen materials, including normal endometrial glands, endometrial hyperplasias and carcinomas, and paraffin embedded samples from the same patients were used in this study. PAb1801 (mouse monoclonal antibody) was used to analyse p53 overexpression in the frozen sections, while CM1 (rabbit polyclonal anti-p53 antibody) was used for the paraffin sections. RESULTS: PAb1801 and CM1 staining on normal endometriums and endometrial lesions showed a significant degree of concordance between frozen and paraffin-embedded materials. CONCLUSION: Enhanced immunodetection of p53 in paraffin-embedded tissues will provide a useful alternative to the usual fresh tissue assay. PMID- 8640472 TI - The chromosome complement of human uncleaved oocytes. AB - OBJECTIVE: Investigation of human oocyte chromosomes that fail to fertilize may provide data essential to the assessment of reproductive failure. In view of this, 121 uncleaved human oocytes were analyzed cytogenetically to assess the type and frequency of chromosomal abnormality. METHODS: Oocyte recovery was done from patients undergoing in vitro fertilization (IVE) procedures. Then, these oocytes were preincubated and inseminated in vitro. Karyotyping was attempted in 121 oocytes lacking signs fertilization 50 h after insemination. RESULTS: Sixty nine oocytes were adequately karyotyped. The overall frequency of chromosomal aberration was 47.6%. Amongst these, 34.8% were aneuploidy, the frequency of which was significantly higher (p < 0.05) in patients > 35 years of age. Diploidy and hyperploidy was noted in 7.2% and 2.8% respectively. Translocation were noted in 2.8% and in 18.8% of human oocytes sperm chromosome condensation appeared prematurely in the G1 phase. No correlation was a found between specific chromosomal aberrations and type of fertility, stimulation treatment or gonadotropin levels. CONCLUSION: The high rate of chromosomal disorders (47.6%) in human oocytes may contribute significantly towards their failure to fertilize in vitro. PMID- 8640474 TI - Allelic deletion mapping of putative tumor suppressor genes on 17q in sporadic ovarian cancer. AB - OBJECTIVES: 1) To study whether the same or different chromosome 17q genes may be involved in the oncogenesis of familial and sporadic ovarian malignancies. 2) To localize the candidate gene in the sporadic ovarian cancers. METHODS (STUDY DESIGN): Using DNA extracted from ovarian tumors and corresponding peripheral leukocyte, we examined the status of loss of heterozygosity (LOH) at 12 loci spanning chromosome 17q12-q25 by Southern hybridization and polymerase chain reaction. RESULTS: Comparison of the extent of LOH among 25 epithelial ovarian tumors showing allele loss at one or more loci on 17q, the smallest overlapping region of allelic deletion is between D17S579 and GIP, with a genetic distance of approximate 2 cM. CONCLUSIONS: From our allelic deletion analysis of chromosome 17q loci, it indicates that there are more than one ovarian cancer candidate genes on chromosome 17q. PMID- 8640473 TI - Antibodies prevalence against HPV-6b and -16 recombinant fusion proteins in Korean patients with cervical neoplasia. AB - OBJECTIVES: This study was to investigate the adjunctive diagnostic utility of HPV fusion proteins from the sera in the patients with cervical neoplasia. Immunologic researches on host factors in HPV infection could contribute to better understanding of patho-physiologic mechanisms of cervical carcinogenesis, and provide valuable information for HPV vaccine development. METHODS: Prevalence of antibodies against human papillomavirus (HPV) in sera of the patients with carcinoma (n = 81), precancers (n = 25) of uterine cervix and normal controls (n = 40) in Korean women were investigated by Western blot immunoassay using partially purified TrpE fusion recombinant proteins for HPV-6b and HPV-16. RESULTS: In 81 patients with cervical cancer, 15 (19%) and 54 (67%) patients were positive for antibodies to at least one of the tested recombinant proteins related to HPV-6b or HPV-16, respectively. And in 25 patients with HPV-related squamous intraepithelial lesion (SIL) of cervix, 10 were positive for at least one of the tested antibodies for HPV-6b gene products (40%) and 11 were positive for HPV-16 related antibodies (44%). The prevalence rates of antibodies for HPV 6b in the sera of SIL patients were significantly higher than those from the sera of cervical cancer patients (p < 0.05). There was an inverse correlation between increasing the positivity against HPV-16 E6 protein and progression of cervical lesions, but there was no significant correlation between the positivities of the other antibodies to HPV-16 and the severity of disease. CONCLUSION: These results suggest that the recombinant proteins related to HPV-6b and HPV-16 could be useful in evaluation of the patients with HPV-related cervical lesions and these might play an adjunctive role in diagnosis and management of cervical neoplasia. PMID- 8640475 TI - Filtration of human seminal specimens using LeukoSorb leukocyte-retention medium. AB - OBJECTIVE: To compare LeukoSorb filtration with Percoll centrifugation in preparing sperm. METHODS: Each of 26 human seminal specimens were analyzed before and after two preparation methods. RESULTS: In normospermic specimens (n = 11), LeukoSorb produced higher results than Percoll did with regard to mean percentage of motility (72.7% vs 59.2%), total swollen sperm in a hypo-osmotic swelling test (84.8% vs 59.0%), (g)-type swollen sperm (37.8% vs 23.4%) and tail abnormality (20.4% vs 10.1%). In asthenospermic specimens (n = 15), LeukoSorb produced higher results than Percoll did with regard to mean percentage of motile sperm recovery (22.2% vs 10.1%), motility (46.9% vs 23.2%), viability (75.6% vs 54.7%), total swollen sperm (84.8% vs 59.0%) and tail abnormality (42.2% vs 24.3%), although the mean percentage of head abnormality was lower with LeukoSorb than with Percoll (30.6% vs 40.2%) vs 40.2%). In pyospermic specimens (n = 10), the mean concentration of round cells after LeukoSorb decreased significantly more than with Percoll (p = 0.029). The filtration time correlated with the total sperm concentration. CONCLUSION: LeukoSorb filtration is useful for preparing asthenospermic and pyospermic seminal specimens. PMID- 8640476 TI - Purification and characterization of 1,25(OH)2D3 receptor from human placenta. AB - OBJECTIVE: To characterize the receptor for 1,25-dihydroxycholecalciferol [1,25 (OH)2D3], we purified it from nuclear fractions of human placentae. METHODS: Human placental fractions were concentrated with ammonium sulfate, extracted from hydroxylapatite, and then chromatographed on Sepharcryl S-200 and DEAE-cellulose. RESULTS: The receptor for [1,25(OH)2D3] was purified approximately 1,500-fold. The molecular weight of the receptor was estimated to be 55 K dalton by gel filtration. The receptor fractions showed a dissociation constant (Kd) of 3.0 x 10(-10) mol/l, and adsorbed to the DNA cellulose column. D3 analogs, estradiol, and progesterone had almost no effect on 1,25(OH)2D3 binding. CONCLUSION: These properties of the 1,25(OH)2D3 receptor in human placenta are similar to those of the chicken intestinal 1,25(OH)2D3 receptor. PMID- 8640477 TI - Cytogenetic effects of cryopreservation on human sperm: assessment using an improved method for analyzing human sperm chromosomes. AB - OBJECTIVE: To evaluate any cytogenetic effects of cryopreservation on human sperm by comparing the frequencies of sperm chromosome anomalies and sex ratios before and after freezing. METHODS: Using in vitro fertilization of zona-free hamster oocytes, analysis of sperm chromosomes was first performed on portions of fresh human semen samples. The residual semen was then analyzed for sperm chromosomes after cryopreservation for several weeks. Sperm donors were 5 healthy men aged 26 38 years. RESULTS: A total of 166 sperm karyotypes were analyzed, 94 before freezing and 72 after freezing. The results indicated no significant differences between fresh and frozen sperm in either frequencies of aneuploidy (fresh: 0%, frozen: 2.8%) or structural anomalies (fresh: 7.5%, frozen: 9.7%). The sex ratios did not differ from the expected 1:1 ratio under either condition. CONCLUSIONS: The results of these studies indicate that cryopreservation does not exert any cytogenetic mutagenicity on human spermatozoa or alter X/Y ratio of human sperm. PMID- 8640479 TI - [Interaction effects in the lateral visual field: an extension of Bouma's (1970) approach]. AB - The recognizability of a letter is impaired by the presence of additional letters. This phenomenon is called lateral masking. Proceeding on the assumption of Bouma (1970) lateral masking can be described in terms of retinal eccentricity of the target letter and the distance between target and flanking stimuli. In this paper, we will address to the question if these kinds of parameters are adequate for describing lateral masking effects. For this purpose, characteristics of the string like its length and its homogenity are varied. The subject's task is to identify the central letter of a tachistoscopically presented string. The targets in the string are flanked both by only x and by different randomly selected letters. Additionally, we varied the length of the string. The data show that these variations influence the strength of the masking effect. It is concluded that a pure sensorical approach is not sufficient for an explanation of lateral masking effects. PMID- 8640478 TI - [Intuitive evaluation of guilt of violent offenders in relation to offender personality]. AB - The distinction between overcontrolled and undercontrolled offenders in criminal psychology is applied to the forensic issues related to crimes of passion under German penal law. The effects of descriptions of the offenders' personality on the attribution of responsibility were studied in a judgment experiment using fictional cases. N = 193 students of law and of psychology, respectively, gave judgments on an offender's legal responsibility and related criteria. Vignettes with four different homicide case descriptions were used as stimuli. Offender personality (overcontrolled vs undercontrolled) was varied as a second stimulus factor. Higher proportions of overcontrolled offenders were judged to be not responsible for their deeds. Law students were more lenient in their judgments than students of psychology. A factor analysis of subjects' ratings of 15 diagnostic criteria yielded four dimensions: intensity of affective excitement, incomprehensibility of the offense, strangeness to the offender's personality, and inevitability. PMID- 8640480 TI - [Modern development of child psychiatry and Hartmann's theory of biopsychosocial integration]. AB - The author gives an account of the views of L. Hartmann, president of the American Psychiatric Association, professor of child psychiatry, specially of his theory of biopsychosocial integration. It is a selective abbreviated explanation of Hartman's concept which seems to be a good baseline of contemporary child psychiatry. PMID- 8640481 TI - [A case of pathologic (F63.0) or dangerous (Z72.6) gambling?]. AB - The authors recapitulate the term of pathological gambling in different classifications incl. the 10th revision of the ICD. It is a new concept which involves certain obscure points also from the linguistic aspect. They demonstrate on a forensic expert opinion elaborated by the Faculty why the statement of the accused that he committed extensive property offenses as a result of pathological gambling was not accepted by experts. PMID- 8640482 TI - [The establishment of mandatory confidentiality in psychiatric practice. I. Survey of legal regulations]. AB - The problem to keep medical secret has its moral, legal and psychological aspects. Although in the submitted paper the importance of moral and psychological aspects of the legal component of the problem, described in this country usually as "mandatory medical secret". The paper analyzes on a historical background the state of legislative norms of medical secret from the last century to the present time. It emphasizes the continuity of legislative solutions which reflect to a certain extent social changes during the period of investigation and demonstrates the present trend of gradual minimalization of state interference and accentuation of personal responsibility, consistent with the List of basic rights and freedom. The author compares also the principles of solution of the problem with selected experience from abroad. In the conclusion he informs on the present state of legal norms which are an amendment of the penal code valid since 1994 and defines the basis of unlawful use of personal data, incl. defined sanctions. Thus strengthening of the legal support of the investigated problem of medical secret was achieved. PMID- 8640483 TI - [The effect of violence in the media on children and adolescents from the viewpoint adolescent psychiatry]. PMID- 8640484 TI - [Types of sexual arousal in women during heterosexual activity]. AB - Drawing of typical personal curve of the level of excitement before, during and after intercourse was used as a part of a questionnaire for the investigation of female sexuality. The respondents were encouraged by the instructions to draw also a second, alternative curve, if they wished. Drawings obtained from 200 female neurotics and 100 female health professionals and counselors were analyzed. They showed a large variability. We divided them into six types. Curves with gradual increase of the excitement up to one climax with following gradual decrease were most frequent in both samples (in 56% neurotics and 48% of health professionals). Curves with more than one climax (multiorgasmic) were on th second place in frequency in health professionals (19% typical plus 8% alternative), while curves without climax (anorgasmic) were more frequent in neurotic females (19% plus 9%). Curves with a pronounced plateau phase, curves with some oscillations of the excitement, and curves with noncoital climax (before or and after intercourse) followed. PMID- 8640485 TI - [Neuropsychology of traumatic brain damage in children]. AB - The author deals with the neuropsychology of traumatic brain damage in children. He mentions briefly the development of neuropsychology with regard to problems encountered in children. Based on an analysis of the literature he assesses four basic factors when establishing the diagnosis. These factors are: age, type and extent of damage, duration of coma and posttraumatic amnesia and premorbid personality. The author finds discrepancies between assessment of coma in different experimental publications and emphasizes the practical aspect of these basic factors in clinical reflections. He presents results of studies concerning mild, medium and severe cerebral affections in children. He emphasizes the importance of psychotherapy, neuropsychological rehabilitation and crisis intervention. PMID- 8640486 TI - [Psychodynamic and psychotherapeutic outcomes in anorexia nervosa during adolescence]. AB - The authors discuss problems of psychogenic disorders of food intake and their basic variants--anorexia nervosa and bulimia. They try to define socio-cultural, familial and psychological characteristics of these diseases and find their relationship to adolescence as a risk period for the development of anorexia and bulimia. They mention specific personality characteristics and family structures in conjunction with the restrictive and bulemic variant of the disease and seek solutions of optimal psychotherapeutic intervention. PMID- 8640487 TI - [150th anniversary of the birth of Dr. Benjamin Cumpelik]. PMID- 8640488 TI - [Use of behavioral scales in the diagnosis of dementia in the aged]. AB - The authors present their own experience with the use of the behavioural scale elaborated by Blessed, Tomlinson and Roth usually called Blessed Dementia Scale BS. They examined 76 patients (59 dementia, 17 with major depression, aged 57 - 87 years (mean age 70.3 +/- 7.2 years). The results obtained by BS were compared with WAIS-R, Wechsler's memory scale (WMS) and the MSE cognitive scale (Knopman et al., 1985). They found that BS differentiates satisfactorily patients with dementia and major depression (dementia X = 8.16 +/- 5.4, depression X = 4.41 +/- 2.41, t = 4.07, p < 0,001). The results of examinations by means of BS correlate significantly inversely with the results of examinations of cognitive functions. The closest correlation is between the total BS score and MSE (r = -0.705, p < 0,001). It was confirmed that BS can be used in the diagnosis of dementia for the differential diagnosis of dementia and depressions. Evaluation of partial BS scores (associated with instrumental ADL, basic ADL and personality changes, drives and interests) provides information important for evaluation of the functional capacity of the patient and for the procedure after termination of in patient treatment. It is important that the total BS score and the partial BS score is not influenced by the patients' age. PMID- 8640490 TI - [Results of gastric resection using laser technique]. PMID- 8640489 TI - [Prevention of complications in laparoscopic cholecystectomy]. PMID- 8640491 TI - [Features of resection of perforating pyloric-duodenal ulcer in patients with diffuse peritonitis]. PMID- 8640492 TI - [Differentiated approach to repeated practicable cleansing of the abdominal cavity in patients with acute diffuse peritonitis]. PMID- 8640493 TI - [Open method of the treatment in acute diffuse suppurative peritonitis]. PMID- 8640494 TI - [Diagnosis and treatment of acute postoperative intestinal obstruction]. PMID- 8640495 TI - [Ways of improving results of the treatment of patients with acute intestinal obstruction]. PMID- 8640496 TI - [Correction of surgical stress during different stages of the treatment of children with acute diseases of abdominal organs]. PMID- 8640497 TI - [Characteristics of new pathogenetic criteria in extreme pathology]. PMID- 8640498 TI - [Principal approach to medical assistance to victims with multiple trauma during the prehospital stage]. PMID- 8640499 TI - [Prevention and treatment of acute suppurative infection in emergency abdominal surgery]. PMID- 8640500 TI - [Morphological changes in the gastric mucosa in peptic ulcer hemorrhage]. PMID- 8640501 TI - [Morphological analysis of reaction of the neuroendocrine apparatus of the appendix in different forms of appendicitis]. PMID- 8640502 TI - [Supraduodenal choledochoduodenostomy]. PMID- 8640503 TI - [Role of metabolic arachidonic acid cascade and prostaglandin E2 in the pathogenesis of immunodeficiency in traumatic shock and acute hemorrhage]. PMID- 8640505 TI - [Diagnostic and tactical error in the treatment of closed abdominal trauma]. PMID- 8640504 TI - [Endoscopic sclerotherapy of esophageal and gastric varicose veins in liver cirrhosis with portal hypertension]. PMID- 8640506 TI - [A method of laparotomy in the treatment of peritonitis]. PMID- 8640507 TI - [Method of treating patients with burn-induced esophageal stenosis with vibration massage bougies]. PMID- 8640509 TI - [Fixation of pathologically movable right kidney with a graft]. PMID- 8640510 TI - [Features of the clinical course of anaerobic peritonitis]. PMID- 8640511 TI - [Intra-abdominal hemorrhage from epigastric vessels]. PMID- 8640508 TI - [A method of cleansing the pleural cavity in patients with pleural empyema]. PMID- 8640512 TI - [Relationship of blood calcium level and manifestations of clinical symptoms of perforated gastroduodenal ulcer]. PMID- 8640513 TI - [Transverse rupture of the pancreas in blunt abdominal trauma]. PMID- 8640515 TI - [A rare complication of destructive pancreatitis]. PMID- 8640514 TI - [Angioleiomyoma of the small intestine as a cause of recurrence of hemorrhage]. PMID- 8640516 TI - [A method of removal of bronchial foreign bodies]. PMID- 8640517 TI - [Pelvic trauma with retroperitoneal hematoma as a cause of the etiology of mechanical obstruction of the large intestine]. PMID- 8640518 TI - [Isolated rupture of the thoracic duct in closed trauma of the thorax]. PMID- 8640519 TI - [Megaduodenum]. PMID- 8640520 TI - [Acute appendicitis and necrosis of the ileum in strangulated femoral hernia in an aged patient]. PMID- 8640521 TI - [One-stage surgical treatment of cholelithiasis and urolithiasis, renal cysts and ovarian cysts]. PMID- 8640522 TI - [Assessment of regional blood flow of the stomach in patients with peptic ulcer stenosis before and after surgery]. PMID- 8640523 TI - [Possibilities of laparoscopic cholecystectomy in acute cholecystitis]. PMID- 8640524 TI - Effect of metal ions on the hydrolytic reactions of nucleosides and their phosphoesters. PMID- 8640525 TI - Mechanistic insight from kinetic studies on the interaction of model palladium (II) complexes with nucleic acid components. PMID- 8640526 TI - NMR studies of oligonucleotide-metal ion interactions. PMID- 8640527 TI - Metal ion interactions with DNA: considerations on structure, stability, and effects from metal ion binding. PMID- 8640528 TI - Electron transfer reactions through the DNA double helix. PMID- 8640529 TI - Role of metal ions in ribozymes. PMID- 8640530 TI - Ternary metal ion-nucleic acid base-protein complexes. PMID- 8640531 TI - Metal-responsive gene regulation and the zinc metalloregulatory model. PMID- 8640532 TI - Role of iron-sulfur proteins in gene regulation. PMID- 8640533 TI - Current status of structure-activity relationships of platinum anticancer drugs: activation of the trans geometry. PMID- 8640534 TI - Cisplatin and derived anticancer drugs: mechanism and current status of DNA binding. PMID- 8640535 TI - Proteins that bind to and mediate the biological activity of platinum anticancer drug-DNA adducts. PMID- 8640537 TI - More on death and dying. PMID- 8640538 TI - The world of biomedical research. PMID- 8640536 TI - Interactions of metallopharmaceuticals with DNA. PMID- 8640539 TI - Progress on polio in India. PMID- 8640540 TI - Nonstop treatment of cystic fibrosis. PMID- 8640541 TI - A role for GAPDH in apoptosis and neurodegeneration. PMID- 8640542 TI - Spectacles and young eyes. PMID- 8640543 TI - Japan's anti-AIDS drug arsenal slowly expands. PMID- 8640544 TI - The advent of the "unpatients'. AB - Predictive diagnosis by molecular methods will change the scientific basis of prognostics. At the same time, it will change the ethical dimensions of the relation among patients, their doctors and other providers of care. PMID- 8640545 TI - HIV viral load markers in clinical practice. AB - Plasma HIV RNA determinations are an important prognostic marker of disease progression and, when used appropriately, provide a valuable tool for the management of individual patients. But what constitutes appropriate use? PMID- 8640546 TI - Microsatellite instability: the mutator that mutates the other mutator. PMID- 8640547 TI - Serine protease inhibitors (SERPINS): where mechanism meets medicine. PMID- 8640548 TI - Selective gene therapy for proliferative disorders: sense and antisense. PMID- 8640550 TI - Tissue sealants: current status, future potential. PMID- 8640549 TI - Pyrazinamide--on the antituberculosis drug frontline. PMID- 8640551 TI - Banking on spermatogonial stem cells: frozen assets and foreign investments. PMID- 8640552 TI - Fusin--a place for HIV-1 and T4 cells to meet. PMID- 8640553 TI - Ribozyme trans-splicing and RNA tagging: following the messenger. PMID- 8640554 TI - Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. AB - In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases. PMID- 8640555 TI - Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene therapy. AB - Mutations in the beta subunit of the cGMP phosphodiesterase gene (beta PDE) can cause a recessively inherited retinal degeneration in several species, including mice, dogs and humans. We tested the possibility of altering the course of retinal degeneration in the rd mouse through subretinal injection of a recombinant replication-defective adenovirus that contains the murine cDNA for wild-type (beta PDE, Ad.CMV beta PDE. Subretinal injection of Ad.CMV beta PDE results in beta PDE transcripts and increased PDE activity and delays photoreceptor cell death by six weeks. The findings demonstrate cell rescue by in vivo gene transfer, thus supporting the feasibility of treating an inherited retinal degeneration by somatic gene therapy. PMID- 8640556 TI - Vacuolar myelopathy in transgenic mice expressing human immunodeficiency virus type 1 proteins under the regulation of the myelin basic protein gene promoter. AB - Vacuolar myelopathy is a common neurological complication in AIDS patients. The pathogenesis of this spinal cord white matter disease remains unclear and it is still debated whether infection of spinal cord with the human immunodeficiency virus type 1 (HIV-1) is causing the disease. We have generated transgenic mice expressing the entire HIV-1 genome under the regulation of an oligodendrocyte specific promoter. These mice develop spinal cord vacuolar lesions similar to those found in AIDS patients. This animal model provides in vivo evidence linking the expression of HIV-1 proteins in oligodendrocytes to the spinal cord damage found in vacuolar myelopathy. PMID- 8640557 TI - Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. AB - Naturally pyrazinamide (PZA)-resistant Mycobacterium bovis and acquired PZA resistant M. tuberculosis strains lose pyrazinamidase (PZase). To investigate the molecular mechanism of PZA resistance, we have cloned the gene (pncA) encoding M. tuberculosis PZase. Mutations in pncA were identified in both types of PZA resistant strains, and transformation of these strains with a functional pncA gene restored PZase activity and PZA susceptibility. These findings, besides providing the basis for understanding how PZA works, should have implications for rapid detection of PZA-resistant clinical isolates of M. tuberculosis and also for rapid differentiation of M. bovis from M. tuberculosis strains. PMID- 8640558 TI - Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. AB - Substantial evidence exists supporting a direct role for raf kinases in the development and maintenance of certain human malignancies. Here we test the potential of phosphorothioate antisense oligodeoxynucleotides targeted against human C-raf-1 kinase to specifically inhibit C-raf-1 kinase gene expression and tumor progression in cell culture and in vivo, using human tumor xenograft mouse models. Treatment of human tumor cells with appropriate phosphorothioate antisense oligodeoxynucleotides led to specific inhibition of C-raf kinase gene expression in cell culture and in vivo at well-tolerated doses. Moreover, oligodeoxynucleotide treatment resulted in potent antiproliferative effects in cell culture and potent antitumor effects in vivo against a variety of tumor types that were highly consistent with an antisense mechanism of action for these compounds. These studies strongly suggest that antisense inhibitors targeted against C-raf-1 kinase may be of considerable value as antineoplastic agents that display activity against a wide spectrum of tumor types at well-tolerated doses. PMID- 8640559 TI - Somatic microsatellite mutations as molecular tumor clocks. AB - Microsatellite (MS) mutations can potentially unravel the past of mutator phenotype tumors, with greater genetic diversity expected in older regions. Rapid clonal expansions of xenografts were characterized by relatively homogenous MS alleles, whereas greater diversity was observed in a colorectal cancer with the greatest variation in its adjacent adenoma. A subcutaneous lung cancer metastasis demonstrated diversity consistent with its one-month clinical duration and evidence of active mitosis during dormancy. The genetic legacy inherent to multistep tumorigenesis provides direct estimates of tumor ages, with up to thousands of cell divisions and high death rates necessary to yield the observed diversities. MS molecular tumor clocks have the unique potential to systematically reconstruct the early and occult evolution of individual human mutator phenotype tumors. PMID- 8640560 TI - Frequent microsatellite alterations at chromosomes 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment. AB - To better understand genetic alterations in oral premalignant lesions, we examined 84 oral leukoplakia samples from 37 patients who had been enrolled in a chemoprevention trial. The samples were analyzed for two microsatellite markers located at chromosomes 9p21 and 3p14. Loss of heterozygosity (LOH) at either or both loci was identified in 19 of the 37 (51%) patients. Of these 19 patients, seven (37%) have developed head and neck squamous cell carcinoma (HNSCC) while only one of 18 (6%) of patients without LOH developed HNSCC. Our data suggest that clonal genetic alterations are common in oral premalignant lesions; that multiple genetic alterations have already occurred in oral premalignant lesions, allowing at least a focal clonal expansion; and that losses of the 9p21 and 3p14 regions may be related to early processes of tumorigenesis in HNSCC. These genetic alterations in premalignant tissues may serve as markers for cancer risk assessment. PMID- 8640561 TI - Growth arrest of solid human neuroblastoma xenografts in nude rats by natural IgM from healthy humans. AB - Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and is associated with very poor prognosis in patients with advanced disease. Current therapeutic regimens of advanced NB which combine surgical resection with radiation therapy and/or chemotherapy brought some improvements, but in a significant number of patients, a cure remains elusive. Normal human serum of healthy adults contains natural IgM antibodies that are cytotoxic for human NB cells. In this study, we evaluated the anti-NB activity of these natural IgM antibodies in nude rats bearing solid human NB tumors. A single intravenous (i.v.) injection of purified cytotoxic IgM led to uptake of IgM into the tumors with massive perivascular complement activation and accumulation of neutrophil granulocytes after 24 hours. Five consecutive i.v. injections of purified cytotoxic IgM into NB-bearing animals resulted in complete growth arrest of even large and established solid tumors which lasted for several weeks after discontinuation of the injections, whereas tumors of control animals continued to grow exponentially during the observation period. These studies suggest that natural anti-NB IgM may have a potential as a novel therapeutic modality in the treatment of human NB. PMID- 8640562 TI - Angiostatin induces and sustains dormancy of human primary tumors in mice. AB - There is now considerable direct evidence that tumor growth is angiogenesis dependent. The most compelling evidence is based on the discovery of angiostatin, an angiogenesis inhibitor that selectively instructs endothelium to become refractory to angiogenic stimuli. Angiostatin, which specifically inhibits endothelial proliferation, induced dormancy of metastases defined by a balance of apoptosis and proliferation. We now show that systemic administration of human angiostatin potently inhibits the growth of three human and three murine primary carcinomas in mice. An almost complete inhibition of tumor growth was observed without detectable toxicity or resistance. The human carcinomas regressed to microscopic dormant foci in which tumor cell proliferation was balanced by apoptosis in the presence of blocked angiogenesis. This regression of primary tumors without toxicity has not been previously described. This is also the first demonstration of dormancy therapy, a novel anticancer strategy in which malignant tumors are regressed by prolonged blockade of angiogenesis. PMID- 8640563 TI - Reconstitution of spermatogenesis from frozen spermatogonial stem cells. AB - Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at -196 degrees C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal. PMID- 8640564 TI - Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients. AB - Neuronal growth factors hold promise for providing therapeutic benefits in various neurological disorders. As a means of ensuring adequate central nervous system delivery of growth factors and minimizing significant adverse side effects associated with systemic delivery methods, we have developed an ex vivo gene therapy approach for protein delivery using encapsulated genetically modified xenogeneic cells. Ciliary neurotrophic factor (CNTF) has been shown in various rodent models to reduce the motor neuron cell death similar to that seen in amyotrophic lateral sclerosis (ALS). The initial trials focusing on the systemic administration of CNTF for ALS have been discontinued as a result of major side effects, thus preventing determination of the potential efficacy of the molecule. In order to deliver CNTF directly to the nervous system, we conducted a phase I study in which six ALS patients were implanted with polymer capsules containing genetically engineered baby hamster kidney cells releasing approximately 0.5 microgram of human CNTF per day in vitro. The CNTF-releasing implants were surgically placed within the lumbar intrathecal space. Nanogram levels of CNTF were measured within the patients' cerebrospinal fluid (CSF) for at least 17 weeks post-transplantation, whereas it was undetectable before implantation. Intrathecal delivery of CNTF was not associated with the limiting side effects observed with systemic delivery. These results demonstrate that neurotrophic factors can be continuously delivered within the CSF of humans by an ex vivo gene therapy approach, opening new avenues for the treatment of neurological diseases. PMID- 8640566 TI - The role of endogenous nerve growth factor in human diabetic neuropathy. AB - Nerve growth factor (NGF) is trophic to sensory and sympathetic fibers. In animal models, NGF is depleted in diabetic nerves and NGF deprivation produces hypoalgesia. Exogenous NGF can reverse some of the pathological changes in diabetic nerves and NGF excess leads to hyperalgesia. We have quantified sensory and autonomic function in early diabetic polyneuropathy and correlated changes with levels of NGF and neuropeptides in affected skin. We describe an early length-dependent dysfunction of sensory small-diameter fibers, prior to dysfunction of sympathetic fibers, with depletion of skin NGF and the sensory neuropeptide substance P. We describe a significant correlation between NGF depletion and decreased skin axon-reflex vasodilation, mediated by small sensory fibers partly via substance P release. Immunostaining shows depletion of NGF in keratinocytes in diabetic skin. We propose that a decrease in endogenous skin derived NGF influences the presentation of diabetic polyneuropathy, although metabolic or vascular abnormalities may be the cause of the neuropathy. As loss of nociception and axon-reflex vasodilation contribute to diabetic foot ulceration, early and prolonged NGF treatment at an appropriate dose may provide rational prophylaxis for this condition. PMID- 8640567 TI - Stereotactic radiosurgery. PMID- 8640565 TI - Striatal dopamine nerve terminal markers in human, chronic methamphetamine users. AB - Methamphetamine is a drug that is significantly abused worldwide, Although long lasting depletion of dopamine and other dopamine nerve terminal markers has been reported in striatum of nonhuman primates receiving very high doses of the psychostimulant, no information is available for humans. We found reduced levels of three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase and the dopamine transporter) in post-mortem striatum (nucleus accumbens, caudate, putamen) of chronic methamphetamine users. However, levels of DOPA decarboxylase and the vesicular monoamine transporter, known to be reduced in Parkinson's disease, were normal. This suggests that chronic exposure to methamphetamine does not cause permanent degeneration of striatal dopamine nerve terminals at the doses used by the young subjects in our study. However, the dopamine reduction might explain some of the dysphoric effects of the drug, whereas the decreased dopamine transporter could provide the basis for dose escalation occurring in some methamphetamine users. PMID- 8640568 TI - Second-generation adenovirus vectors. PMID- 8640571 TI - Denturists. PMID- 8640570 TI - Denturists. PMID- 8640569 TI - Denturists. PMID- 8640573 TI - Ancillary dental personnel. PMID- 8640572 TI - Denturists. PMID- 8640574 TI - Promotional flyers. PMID- 8640575 TI - The dental interview. PMID- 8640576 TI - Oralife managed care plan is a no go. PMID- 8640577 TI - Orthodontic retention. AB - This article briefly describes several factors that should be considered during orthodontic retention. These factors may influence occlusal stability following the completion of the active phase of orthodontic therapy. Orthodontic retention is an important part of orthodontic treatment. It should always be considered and included in the initial stages of treatment planning, rather than added on at the end of treatment as an after-thought. PMID- 8640578 TI - Orthodontic treatment in the mixed dentition. AB - In the mixed dentition, the goal of orthodontic treatment is to maintain or improve arch integrity for the eruption of the permanent teeth, and to prevent the development of a more complicated malocclusion. The importance of correct diagnosis prior to the treatment of mixed-dentition patients cannot be over emphasized. An understanding of the mixed dentition's normal development allows clinicians to determine if the occlusion is developing outside of the normal range. Appropriate treatment can only be planned after this determination has been made. Once it is concluded that orthodontic treatment is required, decisions must be made regarding the timing of treatment, the specific type of treatment needed, and the provider of treatment. By employing a systematic approach to examination and treatment planning, the general dental practitioner can provide an important service to patients with a mixed dentition. PMID- 8640579 TI - Adjunctive orthodontic treatment for adult patients. AB - An increasing number of adult patients are becoming more aware of their orthodontic needs, and more willing to pursue treatment. This article identifies some of the major differences in the orthodontic treatment of children and adults, and summarizes important aspects of adjunctive orthodontic treatment for adults that may facilitate other dental treatment. In addition, a number of clinical scenarios in which adjunctive orthodontics may enhance the provision of restorative treatment in adult patients are identified. PMID- 8640580 TI - What is the INR? AB - The World Health Organization (WHO), the International Committee for Thrombosis and Hemostasis, and the International Committee for Standardization in Hematology, have strongly suggested that INR (International Normalized Ratio) values should be used to report a patient's level of coagulation. This paper discusses the use of the INR to monitor a patient's coagulation level, and reviews the recommendations on the INR levels at which dental extractions and similar oral surgical procedures may be performed safely. Studies are needed to determine whether new local hemostatic agents are sufficient for the safe management of patients at higher INR levels, thereby avoiding the need for hospitalization and heparin therapy. PMID- 8640581 TI - [Oro-dental manifestations of leukemia in children]. AB - Every year in Canada, approximately one thousand cancer cases are reported in children from birth to age 14. Leukemia accounts for approximately 30 per cent of these cases. Leukemia and its treatment are likely to cause to children more oral complications than all other types of cancer. Both the leukemic condition itself and the therapy cause oral signs and symptoms with significant morbidity. Since life expectancy for a patient with leukemia has been greatly improved, dentists have an increasing role to play before, during and after a treatment against leukemia. A review of the oral manifestations related to leukemia and its treatment is presented. PMID- 8640582 TI - Descriptors and techniques for quantitative structure-biodegradability studies. AB - Biodegradation occurs mainly through microbial enzyme attack, and enzyme catalysed reactions are known to depend on hydrophobic, electronic and steric effects. However, most QSBR studies involve correlation with a single parameter, but there is no consistency regarding the class of parameter. This suggests that biodegradation occurs through a range of different mechanisms. A few QSBR studies have reported the need to include more than one class of parameter in correlations. As well as linear regression analysis, other correlation methods have been used in QSBR investigations. These include discriminant analysis, neural networks and comparative molecular field analysis (CoMFA). PMID- 8640583 TI - A mechanistic approach to deriving quantitative structure-activity relationship models for microbial degradation of organic compounds. AB - Quantitative Structure-Biodegradability Relationships (QSBRs), relate the molecular structure of an organic chemical to its biological degradability. The high complexity of the microbial degradation process as well as great variety of the interactions between organism, organic pollutant and the environment make it difficult to interpret the results from biodegradability experiments. Literature concerning the various approaches applied in Structure-Biodegradability Relationship modeling is reviewed and the reliability and applicability of the results obtained from different biodegradation tests is discussed. A mechanistic approach, based on comparison of the data measured at different organization levels, will hopefully contribute to a better understanding of mechanisms taking place during the biodegradation. Their description and quantification may lead to improvement of existing QSBR models. PMID- 8640584 TI - Impact of biodegradation test methods on the development and applicability of biodegradation QSARs. AB - The biodegradability of a substance depends on the structure and physical form of the substance, the time that has been available for acclimation, and the environmental conditions. Importantly, these later factors can be just as important as structure in determining the outcome of a biodegradation test. The development of appropriate QSARs for biodegradation and the ultimate value of the final QSAR depends on understanding these factors. This paper will describe what is known about the effect of test conditions on the results of biodegradation tests. The ability of these tests to reflect real environmental conditions will also be examined. Finally, we will discuss what we believe, in the light of this information, should be the goal of biodegradation QSARs and how these QSARs can be most appropriately used in fate assessments. PMID- 8640585 TI - Qsar study of the toxicity of benzothiazolium salts against Euglena gracilis: the Free-Wilson approach. AB - The Free-Wilson QSAR model in the Fujita-Ban modification was used to calculate the quantitative substituent activity contributions to the toxicity against Euglena gracilis in a series of 91 benzothiazolium salts. An important increase of activity was found for R1 = styryl or SCH2C6H5 group and for R2 = propargyl or allyl group. The substituents on benzene ring also enhance the activity. The possible relations between structure of active groups and their electronic or physicochemical properties are discussed. By using the calculated values it is possible to predict toxicity for 1300 compounds. New compounds with assumed high or low toxicity are predicted. PMID- 8640586 TI - Physician resource planning--you can never plan the future by the past (Edmund Burke). PMID- 8640588 TI - Temporary axillofemoral bypass: its use in high risk cardiac patients requiring urgent abdominal aneurysm repair. AB - The creation of a temporary axillofemoral bypass in a patient undergoing surgery for an 8.1 cm symptomatic infrarenal aortic aneurysm is described. This adjunctive procedure, rarely used in abdominal aortic surgery, may have contributed to minimizing variations in afterload in a patient with a 20% ejection fraction and severe mitral regurgitation. PMID- 8640587 TI - A symptomatic atypical mycobacterial infection masquerading as a vascular ring on chest x-ray. AB - Atypical mycobacterium is rare in children. There are few case reports of childhood pulmonary diseases caused by atypical mycobacterium. The case of a 20 month-old boy who had stridor and respiratory distress is presented. Chest x-ray showed right-sided narrowing of the trachea and left-sided aortic knob. Ziehl Neelsen stain showed acid-fast bacilli. The culture showed Mycobacterium malmoense. PMID- 8640589 TI - Standards for training in adult cardiac catheterization and angiography. Canadian Cardiovascular Society Committee. PMID- 8640590 TI - Standards for coronary angioplasty training. Canadian Cardiovascular Society Committee. PMID- 8640592 TI - Standards for training in adult clinical cardiac electrophysiology. Canadian Cardiovascular Society Committee. PMID- 8640593 TI - Standards for adult nuclear cardiology training. Canadian Cardiovascular Committee. PMID- 8640591 TI - Standards for adult echocardiography training. Canadian Cardiovascular Society Committee. PMID- 8640594 TI - Right heart failure due to ventricular adiposity: 'adipositas cordis'--an old diagnosis revisited. AB - Adiposity of the heart is characterized by an increase in the amount of epicardial and other adipose tissue. The most pronounced changes involve the right ventricle. The adipocytes may be interposed between myocytes, and in severe cases the normal mechanics and function of the ventricle are impaired. Adiposity of the heart is usually an incidental finding at autopsy, and only rarely is it of clinical significance. This report describes a 46-year-old female with multiorgan failure secondary to bronchopneumonia, purulent pericarditis, tamponade and sepsis, whose clinical course was altered due to severe adiposity of the heart, so-called 'adipositas cordis'. PMID- 8640595 TI - Spontaneous iliopsoas hematoma in patients with unstable coronary syndromes receiving intravenous heparin in therapeutic doses. AB - OBJECTIVE: To identify the relationship between the use of anticoagulants, specifically heparin, and the development of iliacus and psoas muscle hematoma. Three patients with unstable angina who developed groin pain while on heparin anticoagulation are presented. Patients who are anticoagulated with heparin are at increased risk of developing iliacus or psoas hematoma, manifesting a wide range of symptoms from groin pain to massive bleeding and shock. Identification of these patients is crucial in cardiology practice. DATA SOURCES: MEDLINE searches under "iliacus', "psoas' and "iliopsoas hematoma' were conducted and cross-referenced with patients on anticoagulant therapy. Only English language articles were included. STUDY SELECTION: The search covered January 1966 to February 1995. Fifty-one articles were studied. DATA SYNTHESIS: The current literature suggests that anticoagulation can cause iliacus or psoas muscle hematoma and usually presents as femoral neuropathy. However, the presented case reports provide evidence that an earlier manifestation of this entity is the development of groin pain, and that early identification is crucial to improving patient morbidity and mortality. CONCLUSIONS: Patients who are on heparin anticoagulation should be carefully monitored for development of groin pain or leg weakness. In such cases, early recognition of possible iliacus or psoas hematoma should be by abdominal ultrasound or computed tomography, and heparin anticoagulation should be modified according to its clinical requirement. PMID- 8640596 TI - False-positive 201thallium study in Wolff-Parkinson-White syndrome. AB - Wolff-Parkinson-White syndrome (WPW) is known to cause abnormal rest electrocardiogram and stress tests. Consequently, the diagnosis of coronary artery disease (CAD) may be difficult in patients with WPW. Previous reports have described false positive stress 201thallium studies in WPW, but the absence of CAD has rarely been confirmed by coronary angiography. A case of false positive stress 201thallium scintigraphy confirmed by angiography is presented. Left ventricular contractile asynchrony can explain the defects observed in myocardial perfusion scintigraphy. The importance of the defects relates to the length of the delta wave. PMID- 8640598 TI - Left ventricular hypertrophy regression as a process with variable biological implications. AB - OBJECTIVE: To determine whether myocardial hypertrophy regression, similarly to hypertrophy itself, is a process of variable nature with different biological implications. DATA SOURCES: Current Contents and MEDLINE searches under myocardial hypertrophy and regression of hypertrophy-related headings were conducted. DATA EXTRACTION: The search covered the period from 1969 to 1994, and 89 articles dealing with both human and animal studies were used. DATA SYNTHESIS: The positive adaptive effect of left ventricular myocardial hypertrophy may be counterbalanced by increased incidence of heart failure, myocardial infarction or sudden death. The risk of undesirable cardiovascular events varies according to the character of hypertrophic growth. Reduction of cardiac mass to that of a normal heart need not always mean that the ventricle is normal in all aspects. Several forms of left ventricular hypertrophy regression may be distinguished. The hypertrophy regression of the athletic heart is of a physiological nature. The spontaneous regression of left ventricular hypertrophy as seen in the rabbit model of aortic insufficiency has a pathological aspect resulting in heart failure. The nature of therapeutically induced regression of hypertrophy may vary according to fibrotic tissue concentration, energetical state and the function of the regressed heart. CONCLUSION: The biological implication of hypertrophy regression varies especially with respect to the nature of hypertrophy and the mode of achievement of hypertrophied mass reversal. Only long term prospective studies can clarify the question as to which types of hypertrophy regression result in decreased cardiovascular risk. PMID- 8640597 TI - Cardiac manifestations of Lyme disease: a review. AB - OBJECTIVE: To describe the clinical features of cardiac manifestations of Lyme disease, the most common vector-borne illness in North America, which occasionally results in cardiac involvement. DATA SOURCES: A review of the English-language clinical literature pertaining to Lyme disease and Lyme carditis indexed in MEDLINE from 1975 to 1995. DATA EXTRACTION: Studies describing diagnosis, clinical features, treatment or outcome were reviewed. DATA SYNTHESIS: Cardiac complications of Lyme disease may occur in up to 8% of patients. Cardiac manifestations occur in the early phase of the illness, at a median of 21 days from the onset of erythema migrans. Manifestations of Lyme carditis include atrioventricular block, myopericarditis, intraventricular conduction disturbances, bundle branch block and congestive heart failure. Temporary cardiac pacing may be required in up to a third of cases and complete recovery occurs in most (greater than 90%) patients. The overall prognosis of Lyme carditis is very good, although recovery may be delayed and late complications such as dilated cardiomyopathy may occur. CONCLUSION: Lyme disease is a tick-borne spirochetal infection caused by Borrelia burgdorferi. Cardiac complications of Lyme disease generally occur in the early phase and include conduction system disturbances, myopericarditis and congestive heart failure. PMID- 8640600 TI - Improvement of ventricular function after atrioventricular junction ablation in a patient with atrial flutter after Mustard's operation. AB - Arrhythmias are common problems following Mustard's operation for D-transposition of the great arteries. A 19-year-old male is presented who was diagnosed at birth with D-transposition of the great arteries and a right aortic arch and underwent a Mustard procedure at 12 months of age. He developed sinus node dysfunction and atrial flutter unresponsive to antiarrhythmic drugs and dual chamber pacing. Following complete heart block with radiofrequency catheter ablation of the atrioventricular junction, the patient's heart was paced in VVIR mode. Ventricular function improved after the ablation and persisted in two years' follow-up. PMID- 8640599 TI - An irreversible A1-selective adenosine agonist preconditions rabbit heart. AB - This study tested whether an irreversible agonist of the A1 adenosine receptor, m DITC ADAC, can mimic the protective effect of ischemic preconditioning in the rabbit heart. Isolated Krebs buffer-perfused rabbit hearts experienced 30 mins of regional ischemia and 120 mins of reperfusion. Infarct size was measured with tetrazolium staining. In untreated hearts 32 +/- 2% of the risk zone infarcted while only 9 +/- 2% infarction was seen in hearts that were preconditioned with 5 mins of global ischemia followed by 10 mins of reperfusion (P < 0.05 versus control). Exposure to 200 nM of the A1-selective agonist 2-chloro-N6 cyclopentyladenosine (CCPA) for 5 mins followed by 10 mins of washout protected the hearts as well as preconditioning with 13 +/- 7% infarction (P < 0.05 versus control). Protection from CCPA was completely blocked by 200 nM DPCPX (8 cyclopentyl-1,3-dipropylxanthine) with 34 +/- 7% infarction (P < 0.05 versus CCPA) confirming that protection was via the A1 adenosine receptor. m-DITC ADAC, which irreversibly stimulates the A1 adenosine receptor, also protected the hearts with only 15 +/- 4% infarction (P < 0.05 versus control). It was concluded that m-DITC ADAC does mimic ischemic preconditioning and that an irreversible agonist might be a novel way to provide an extended window of protection to the heart from a single intracoronary injection. PMID- 8640602 TI - Proximal narrowing of anomalous right coronary artery from the left coronary sinus: delineation by omniplane transesophageal echocardiogram. AB - Anomalous origin of the right coronary artery from the left sinus of Valsalva is a serious and potentially fatal, albeit rare, congenital abnormality. It can be associated with marked functional impairment and even sudden death. Transesophageal echocardiography can identify and confirm the course of aberrant coronary arteries and their relationship to the great vessels, as is demonstrated in this report of a 51-year-old female presenting with anomalous right coronary artery with narrowing of its proximal portion. PMID- 8640601 TI - Congenitally corrected transposition of the great arteries and exercise-induced ventricular tachycardia. AB - Congenitally corrected transposition of the great arteries (CCTGA) is an uncommon condition. Few patients survive past 50 years of age, because of associated congenital defects, systemic (morphological right) ventricular dysfunction, atrioventricular valvular insufficiency or complete heart block. A unique case is presented of exercise-induced ventricular tachycardia, which led to the diagnosis of CCTGA, without atrioventricular block or other cardiac anomalies. PMID- 8640603 TI - Isolation and characterization of resin acid degrading bacteria found in effluent from a bleached kraft pulp mill. AB - Thirteen resin acid degrading bacteria enriched on abietic or dehydroabietic acids were isolated from waste water from the aerated stabilization basin of a bleached kraft pulp mill. Standard biochemical tests were used to characterize each isolate. Each isolate was tested for its ability to degrade six abietane- and pimarane-type resin acids. Resin acid concentrations were determined by high pressure liquid chromatography and UV absorbance. Cluster analysis based on phenotypic characteristics identified two distinct clusters of degraders that differed in their ability to utilize carbohydrates as carbon sources. Fatty acid methyl ester analysis of representative isolates from each cluster identified A19 6a and D11-13 as Comamonas and Alcaligenes species, respectively. To determine genotypic relatedness, enterobacterial repetitive intergenic consensus sequences were used to amplify genomic DNA fragments from 10 isolates. These results supported the phenotypic analysis for all isolates tested except A19-5 and A19 6b. These two organisms were clustered closely together based on phenotype but had distinctly different banding patterns, suggesting that they are not related genotypically. All isolates degraded a subset of the six resin acid congeners. Isolates A19-3, A19-6a, A19-6b, and D11-37 were the most effective at degrading all six congeners. PMID- 8640604 TI - Production of beta-glucosidase and diauxic usage of sugar mixtures by Candida molischiana. AB - The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol). PMID- 8640605 TI - Use of continuous culture to screen for lipase-producing microorganisms and interesterification of butter fat by lipase isolates. AB - The continuous cultivation technique was used to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes. Using this technique, three strains of lipase producing bacteria were isolated and identified: Pantoea agglomerans (BB96CC1, BB168CC2) and Pseudomonas fluorescens (BW96CC1). In addition, butter fat induced more lipase production when present in the growth medium. Interesterification of butter fat triacylglycerols by enzymatic extracts of the isolated strains of microorganisms resulted in an appreciable interesterification yield, implying that hydrolysis was suppressed and interesterification of butter fat triacylglycerols was maximized in a microemulsion free-cosurfactant system. PMID- 8640606 TI - The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis. AB - The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis. PMID- 8640607 TI - Anti-adhesin antibodies that recognize a receptor-binding motif (adhesintope) inhibit pilus/fimbrial-mediated adherence of Pseudomonas aeruginosa and Candida albicans to asialo-GM1 receptors and human buccal epithelial cell surface receptors. AB - Pseudomonas aeruginosa and Candida albicans were reported to adhere to the glycosphingolipid asialo-GM1 by means of pili and fimbriae, respectively. These diverse adhesins have been previously reported to have an immunologically conserved antigenic epitope and the role of this cross-reactive epitope in adherence to asialo-GM1 was investigated in this study. Both the unbiotinylated PAK pilus and fimbrial adhesins inhibited biotinylated pili from P. aeruginosa PAK and biotinylated C. albicans fimbriae binding to asialo-GM1 and receptors present on human buccal epithelial cells (BECs), which suggested that the same receptor sites were recognized by the two adhesins. Monoclonal antibodies PK99H and Fm16 raised against the P. aeruginosa PAK pili and C. albicans fimbriae, respectively, recognized a conserved epitope present on the two adhesins. Both Fm16 and PK99H blocked fimbriae binding to asialo-GM1 and BEC receptors and also inhibited P. aeruginosa and C. albicans whole cell binding to BECs. These data suggested that the conserved epitope confers receptor-binding properties to the adhesins, demonstrated that (i) asialo-GM1-like receptors present on epithelial cell surfaces are utilized by the pilus and fimbrial adhesins and (ii) the binding to these glycoreceptors is mediated by a conserved epitope that has receptor-binding properties. PMID- 8640608 TI - Isolation and characterization of Azospirillum lipoferum locus that complements Rhizobium meliloti dctA and dctB mutations. AB - A DNA probe containing the structural gene for dicarboxylate transport (dct A) of Rhizobium meliloti hybridized strongly with the fragments of Azospirillum lipoferum genomic DNA. A genomic library of A. lipoferum was screened for the dct A gene by complementation of a dct A mutant of Rhizobium meliloti. A recombinant cosmid, p37D, capable of restoring growth of the dct A mutant on dicarboxylates was isolated and found to hybridize to the dctA probe. The ability of p37D to complement the dct B mutant of R. meloliti indicated that dct A and dct B genes in A. lipoferum may be organized adjacent to each other. PMID- 8640610 TI - Cyanide degradation by an Escherichia coli strain. AB - Chemical formation of a glucose-cyanide complex was necessary for metabolic degradation of cyanide at concentrations up to 50.0 mg/L by a strain of Escherichia coli isolated from gold extraction circuit liquids. Ammonia accumulating during the culture log phase as the sole nitrogen by-product was further utilized for bacterial growth. Washed (intact) cells, harvested at different periods of bacterial growth on cyanide, consumed oxygen in presence of cyanide. These findings suggest that metabolism of cyanide involved a dioxygenase enzyme that converted cyanide directly to ammonia, without the formation of cyanate. PMID- 8640609 TI - Negative chemotaxis in Cytophaga johnsonae. AB - Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria. The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl-, and N-chlorotaurine, as well as by low pH. Its response to H2O2 was eliminated by catalase. Nalidixic acid at 200 microM, which inhibits the growth but not the motility of C. johnsonae, did not interfere with its negative chemotactic response to H2O2, whereas sodium phosphate at 10 mM, which inhibits motility, did so. Cytophaga johnsonae was not repelled by taurine, n-octanol, phenol, L-valine, or high pH. Chemotaxis can be conveniently studied in gliding bacteria such as C. johnsonae. PMID- 8640611 TI - The review process. PMID- 8640612 TI - The response of critically ill patients to nutritional support. PMID- 8640613 TI - Canadian Association of General Surgeons' position statement on ambulatory care. PMID- 8640614 TI - Case 9. Presentation. Osteochondritis dissecans. PMID- 8640615 TI - Thymoma found during routine cardiac imaging. PMID- 8640616 TI - Presidential address, 1995. Surgery 2000: a look back to the future. AB - The present demoralized state of Canadian surgery is due to a number of short term influences. They include financial restraints, the desire of government agencies to off-load blame for unpopular decisions onto doctors and altered public expectations. The major long-term challenge will be a shortage of physicians and a severe shortage of general surgeons because of the superimposition of longer-term trends in medical demographics on short-term political reactions to a perceived oversupply of doctors. General surgeons need to identify the significant, long-term threats and challenges. If they can do this and plan their responses knowledgably, with some measure of altruism, the future in general surgery will be a bright one for present and future residents and medical students. PMID- 8640617 TI - An animal model of benign bile-duct stricture, sclerosing cholangitis and cholangiocarcinoma and the role of epidermal growth factor receptor in ductal proliferation. AB - OBJECTIVE: To adapt an animal model of benign bile-duct stricture, sclerosing cholangitis and cholangiocarcinoma in order to determine if the expression of epidermal growth factor receptor (EGFr) could be used to differentiate these lesions. DESIGN: A prospective control study with blinded interpretation of liver biopsy histology and immunohistochemical staining as the criterion standards. SETTING: A university-affiliated research centre. SUBJECTS: Male Syrian Golden hamsters (40 for benign duct stricture, 29 for sclerosing cholangitis and 27 for cholangiocarcinoma). INTERVENTIONS: Ligation of the common bile duct with 6-0 catgut for benign duct stricture; injection of the biliary tree with 0.15 mL of formalin for sclerosing cholangitis; and weekly subcutaneous injections of 500 mg/kg of di-isopropanolnitrosamine for 10 weeks followed by ligation of the common bile duct with 6-0 catgut for cholangiocarcinoma. Routine histologic preparation of liver biopsies obtained at autopsy 10 weeks postoperatively then immunohistochemical staining of specimens for EGFr. MAIN OUTCOME MEASURES: The development of benign or atypical biliary ductal proliferation, including adenoma and carcinoma formation. The presence or absence of immunohistochemical staining for EGFr. RESULTS: Benign ductal proliferation without atypia was seen in 15 of 21 animals in the bile-duct-stricture group that were sacrificed, in 15 of 24 animals in the sclerosing cholangitis group and in 17 of 18 animals in the cholangiocarcinoma group. Atypical proliferation was seen in 13 of 18 animals with cholangiocarcinoma but not in the other two groups. The differential occurrence of atypical ductal proliferation was statistically significant (p < 0.00001) for both groups. No evidence of EGFr expression was found in any group. CONCLUSION: Although the animal model was valid histologically for comparing benign and malignant biliary disease, EGFr does not play a role in biliary ductal proliferation and so cannot be used to differentiate between benign and malignant lesions. PMID- 8640618 TI - Small-bowel resection for metastatic melanoma. AB - OBJECTIVE: To determine whether complete resection of small-bowel metastases from melanoma improves patient survival. DESIGN: A computer-aided chart review. SETTING: Hospitals associated with McGill University. PATIENTS: Twenty patients (17 men, 3 women), identified from 1524 patients with melanoma, who underwent surgery to the small bowel for metastases. Patient age and clinical presentation, tumour site and stage were recorded. INTERVENTION: Exploratory laparotomy with complete or partial resection of involved small bowel. MAIN OUTCOME MEASURES: Operative morbidity, mortality and length of survival related to the extent of small-bowel resection. RESULTS: Eleven patients had complete resection, 8 patients had partial resection and 1 patient had a palliative bypass only. Long term survival (ranging from 2 to 10 years) was 36% in those who had complete resection and 0% in those who had partial resection; operative morbidity and mortality were 20% and 15% respectively. CONCLUSION: Complete resection of small bowel metastases in patients with metastatic melanoma can result in long-term survival. PMID- 8640619 TI - Chemical burns. AB - OBJECTIVES: To report a burn unit's experience with chemical burns and to discuss the fundamental principles in managing chemical burns. DESIGN: A chart review. SETTING: A burn centre at a major university-affiliated hospital. PATIENTS: Twenty-four patients with chemical burns, representing 2.6% of all burn admissions over an 8-year period at the Ross Tilley Regional Adult Burn Centre. Seventy-five percent of the burn injuries were work-related accidents. Chemicals involved included hydrofluoric acid, sulfuric acid, black liquor, various lyes, potassium permanganate and phenol. RESULTS: Fourteen patients required excision and skin grafting. Complications were frequent and included ocular chemical contacts, wound infections, tendon exposures, toe amputation and systemic reactions from absorption of chemical. One patient died from a chemical scald burn to 98% of the body surface area. CONCLUSIONS: The key principles in the management of chemical burns include removal of the chemical, copious irrigation, limited use of antidotes, correct estimation of the extent of injury, identification of systemic toxicity, treatment of ocular contacts and management of chemical inhalation injury. Individualized treatment is emphasized. PMID- 8640620 TI - Effect of nutritional support on routine nutrition assessment parameters and body composition in intensive care unit patients. AB - OBJECTIVES: To determine whether routine nutrition assessment parameters and body composition change after nutritional support in intensive care unit (ICU) patients and whether the changes, if any, are related to cumulative energy and fluid balances. DESIGN: A prospective study. SETTING: A university teaching hospital. PATIENTS: Forty-five mechanically ventilated medical and surgical patients admitted to the ICU who received nutritional support for 7 days (group 1) and 9 patients of this group who received nutritional support for 3 weeks or longer (group 2). INTERVENTIONS: Enteral and parenteral nutritional support prescribed on the basis of metabolic cart measurements of energy expenditure. OUTCOME MEASURES: Routine nutrition assessment, including determinations of weight, serum albumin and prealbumin, and lymphocyte count and body composition, including measurements of body cell mass, extracellular fluid and body fat, determined from bioelectric impedance analysis. RESULTS: In group 1 patients, weight, albumin and prealbumin levels, and extracellular mass changed, but there was no change in lymphocyte count, body cell mass or body fat. Changes in weight and extracellular mass were slightly related to cumulative fluid balance; changes in albumin and prealbumin levels were not related to cumulative energy or fluid balance. The findings were similar for group 2 patients. CONCLUSIONS: Changes in routine nutrition assessment parameters and body composition are slightly affected by fluid balance but not by energy balance; thus, they are not specific indicators of the adequacy of nutritional support in ICU patients. Improved nutrition assessment parameters are required to better monitor the response to nutritional support in critically ill patients. PMID- 8640621 TI - The efficacy of a computer-assisted preoperative tutorial for clinical clerks. AB - OBJECTIVE: To assess the effectiveness of computer-assisted preoperative tutorials on human anatomy in improving the operating-room learning experience for clinical clerks. DESIGN: Crossover trials with immediate assessment and approximately 1-week delay between trials. SETTING: General surgery operating room of a university teaching hospital. SUBJECTS: Eight 4th-year clinical clerks on a single 8-week surgical rotation. MAIN OUTCOME MEASURES: The senior surgeon's assessment of the clerk's knowledge and understanding of the operation, according to a six-item questionnaire, and the clerk's own assessment of the experience in the operating room, according to an eight-item questionnaire. RESULTS: Surgeons rated the clerks' performances in the operating room as better when they had received the preoperative tutorial (mean [and standard deviation], 3.7 [0.4]) than when they had not received the preoperative tutorial (3.0 [0.3]). The difference (0.7 [0.6]) was statistically significant (t7 = 3.3, p < 0.01). Similarly, clerks rated their own experience more positively when they had received the tutorial (4.0 [0.2]) than when they had not (3.1 [0.3]), with the difference (0.9 [0.5]) being statistically significant (t7 = 4.9, p < 0.001). CONCLUSIONS: Short, preoperative, computer-assisted tutorials on human anatomy can have a positive impact on the clerk's level of knowledge and confidence in the operating room. Further research is warranted into the extent to which students spontaneously make use of these tutorials. PMID- 8640622 TI - Role of endoscopic sphincterotomy alone in patients with choledocholithiasis and cholelithiasis. AB - OBJECTIVE: To study the long-term effects of endoscopic sphincterotomy alone in elderly patients with choledocholithiasis and cholelithiasis. DESIGN: A chart review. SETTING: A university-affiliated hospital. PATIENTS: Twenty-one patients over 60 years of age, who presented with cholecystitis, jaundice or cholangitis. The follow-up ranged from 1 to 5 years (mean 3.51 years). INTERVENTION: Endoscopic sphincterotomy. MAIN OUTCOME MEASURES: The occurrence of postsphincterotomy pancreatitis, cholangitis and cholecystitis. RESULTS: In the early postsphincterotomy period, 3 of the 21 patients had pancreatitis, which was treated conservatively. One patient had cholangitis. In this patient the initial sphincterotomy did not clear the common bile duct of all stones, but a repeat procedure was successful and the cholangitis resolved. One patient had mild cholecystitis that responded to conservative therapy. In the long term, 9 of the 21 patients had cholecystitis. This was treated conservatively in six patients, but three required cholecystectomy. One patient had choledocholithiasis requiring exploration of the common bile duct. CONCLUSIONS: In elderly debilitated patients with cholelithiasis and choledocholithiasis, endoscopic sphincterotomy alone may be adequate therapy. However, patients presenting with cholangitis and cholelithiasis may eventually require cholecystectomy. PMID- 8640623 TI - Laparoscopic inguinal herniorrhaphy: appraisal of a cohort study. AB - OBJECTIVES: To assess the safety and effectiveness of individualized laparoscopic herniorrhaphy and to compare its intraoperative cost to that of the standard Bassini operation. DESIGN: An analytic cohort study. SETTING: A university teaching hospital. PATIENTS: One group of 158 patients underwent 167 laparoscopic herniorrhaphies for symptomatic groin hernias. The approach was transabdominal preperitoneal for the first 124 patients and totally preperitoneal for the last 34 patients. A second group of 50 patients underwent a conventional Bassini operation. INTERVENTION: Individualized laparoscopic inguinal herniorrhaphy or Bassini herniorrhaphy. MAIN OUTCOME MEASURES: Complications and recurrences encountered in the laparoscopic group. Total operative time and intraoperative cost involved in both procedures. Analgesia required in each group during the first 2 postoperative days. RESULTS: Intra- and postoperative complications of the laparoscopic approach were not life threatening. The recurrence rate at a mean follow-up of 16.8 months was 1.2%. Total operative time was significantly (p < 0.001) longer in the laparoscopy group than in the Bassini group. Patients in the Bassini group took more parenteral analgesics than those in the laparoscopy group (p = 0.02), but there was no difference with respect to the number of times enteral analgesics were required (p = 0.32). Use of mesh and staples was more expensive than sutures alone inserted laparoscopically. The Bassini procedure was a less expensive procedure than laparoscopic herniorrhaphy. CONCLUSIONS: The laparoscopic treatment of groin hernias is safe. The recurrence rate is low. Primary unilateral inguinal hernias could be adequately treated at a lesser cost by a standard approach. Bilateral, recurrent and femoral hernias could benefit from a laparoscopic approach. PMID- 8640624 TI - Lower extremity free flaps: a review. AB - OBJECTIVE: To identify factors related to free-flap coverage of lower extremity fractures that are linked to a negative outcome. DESIGN: A chart review. SETTING: A large microsurgical referral centre. PATIENTS: From 1981 to 1989, the records of all patients who underwent free-tissue transfer to the lower extremity with more than 1 year of follow-up were selected. From this was drawn a subgroup of 49 patients (mean age, 36 years) who had tibial fractures (55% were motor vehicle injuries) and in almost all cases established soft-tissue or bony defects. They formed the study group. INTERVENTION: Free-flap transfer. OUTCOME MEASURES: Factors that might be associated with free-flap failure: mechanism of injury, grade of tibial fracture, history of smoking, diabetes, peripheral vascular disease, ischemic heart disease, vascular compromise in the leg preoperatively, recipient artery used, type of anastomosis, and hypertension or hypotension intraoperatively. RESULTS: Type IIIB tibial fractures were the most frequent (67%) and carried a significantly (p = 0.02) higher risk of free-flap failure than other types of fracture. Patients underwent a mean of four procedures before referral for free-tissue transfer. The mean time from injury to flap coverage was 1006 days. Stable, long-term coverage of the free flaps was achieved in 78% of patients. Wound breakdown was most often caused by recurrent osteomyelitis (65%). Seventy-four percent of the fractures healed. The amputation rate was 10%. Four patients required repeat free-flap transfer for limb salvage. CONCLUSIONS: Only the grade of tibial fracture could be significantly related to postoperative free flap failure. PMID- 8640625 TI - Hemobilia complicating elective laparoscopic cholecystectomy: a case report. AB - Iatrogenic injury to the hepatic or cystic arteries can occur during laparoscopic cholecystectomy and can be seen in isolation or in association with bile-duct injury. The most common manifestation of arterial injury is intraoperative hemorrhage; also, interruption of the right hepatic artery can occur without hemorrhage, and this can be clinically insignificant or associated with hepatic ischemia. A less common manifestation of arterial injury during laparoscopic cholecystectomy is presented. A 48-year-old woman had a pseudoaneurysm of the major anterior branch of the right hepatic artery in association with an injury to the common hepatic duct. This complication presented as massive hemobilia after she had been discharged from the hospital. Definitive repair of the pseudoaneurysm was carried out at the time of Roux-en-Y hepaticojejunostomy for correction of the associated duct injury. This unusual vascular complication should be considered in patients after laparoscopic cholecystectomy who demonstrate evidence of late occult or obvious hemorrhage. PMID- 8640626 TI - Splenorenal arterial bypass in a child with Takayasu's disease: a case report. AB - In adults splenorenal arterial bypass is a highly effective treatment for renovascular hypertension, but in children the procedure has been less successful because of the small size of the splenic artery. However, with the improvement in microvascular techniques the procedure is now possible in children. A 2-year-old child with Takayasu's arteritis, previous right nephrectomy and severe renovascular hypertension required revascularization to salvage his remaining left kidney. A splenorenal arterial bypass was performed through a left retroperitoneal flank incision. Postoperatively his creatinine level returned to normal and his requirements for antihypertensive medication were markedly diminished. The advantages of a splenorenal arterial bypass for left renal revascularization in a developing child are discussed. PMID- 8640627 TI - Chemotherapy for desmoid tumours in association with familial adenomatous polyposis: a report of three cases. AB - OBJECTIVE: To determine the efficacy of chemotherapy for inoperable desmoid tumours associated with familial adenomatous polyposis. DESIGN: A review of three cases of unresectable desmoid tumours and of the literature on the subject. SETTING: The Steven Atanas Stavro Polyposis Registry at Mount Sinai Hospital in Toronto. PATIENTS: Three patients with symptomatic, unresectable desmoid tumours associated with familial adenomatous polyposis and unresponsive to conventional hormone therapy. INTERVENTION: A chemotherapy regimen of seven cycles of doxorubicin (dose ranging from 60 to 90 mg/m2) and dacarbazine (1000 mg/m2), followed by carboplatin (400 mg/m2) and dacarbazine. OUTCOME MEASURES: Clinical improvement and tumour regression demonstrated by computed tomography. RESULTS: In each of the three cases significant tumour regression was seen clinically and radiologically. CONCLUSIONS: Cytotoxic chemotherapy is an effective treatment for desmoid tumours associated with familial adenomatous polyposis. The chemotherapy should be started early in cases of symptomatic desmoid tumour unresponsive to conventional medical therapy. PMID- 8640628 TI - Tuberculosis of the parotid gland. PMID- 8640629 TI - Renal autotransplantation. PMID- 8640630 TI - Open versus arthroscopic acromioplasty. PMID- 8640632 TI - An ethicist's commentary on whether a veterinarian should inform a client that a previous practitioner had left a sponge in a dog. PMID- 8640631 TI - Bovine somatotropin and the regulatory process for veterinary drug approval. PMID- 8640633 TI - [Intravenous anesthesia in the horse: comparison of xylazine-ketamine and xylazine-tiletamine-zolazepam combinations]. AB - Intravenous anesthesia in the horse: Comparison of xylazine-ketamine and xylaxine tiletamine-zolazepam combinations. Six healthy adult horses were anesthetized twice at random with following intravenous combinations: 1.1 mg/kg of body weight (BW) of xylazine followed by 2.2 mg/kg BW of ketamine (X-K) and 1.1 mg/kg BW of xylazine followed by 1.65 mg/kg BW of tiletamine-zolazepam (X-TZ). The modifications of some cardiorespiratory parameters and the duration of anesthesia were evaluated and compared for the 2 protocols used. Few significant differences were observed between the 2 protocols in regard to the cardiorespiratory parameters measured. The respiratory rate was lower (7 breaths per minute) and the heart rate was higher (34 beats per minute) with the X-TZ combination. The duration of anesthesia with this technique was 33 +/- 3 minutes (X +/- Sx) and longer than with X-K (18 +/- minutes (X +/- Sx)). Superficial analgesia lasted 14,5 +/- 3 minutes with the X-K combination and 31,7 +/- 3,2 minutes for the X-TZ combination. The 2 protocols are associated with a reduction of PaO2. PMID- 8640635 TI - Neospora abortions in eastern Ontario dairy herds. PMID- 8640634 TI - Detection of Staphylococcus aureus in bulk tank milk using modified Baird-Parker culture media. AB - The purpose of this project was to evaluate the use of 2 selective/differential culture media for detecting Staphylococcus aureus in bulk tank milk. One medium was Baird-Parker agar base supplemented with egg york tellurite emulsion and acriflavine. The other medium was Baird-Parker agar base supplemented with rabbit plasma/bovine fibrinogen and acriflavine. An increased inoculum of bulk tank milk (0.3 mL) was used to enhance the detection of S. aureus in samples containing low numbers of organisms. The sensitivity and specificity for detecting S. aureus in bulk tank milk were 94.8% and 100%, respectively, using Baird-Parker agar base supplemented with egg yolk tellurite emulsion and acriflavine, and 89.7% and 100%, respectively, using Baird-Parker agar base supplemented with rabbit plasma/bovine fibrinogen and acriflavine. Both media are practical for detecting S. aureus in bulk tank milk and monitoring its spread in lactating dairy herds in Alberta. PMID- 8640636 TI - Use of cisapride in the resolution of pelvic flexure impaction in a horse. PMID- 8640637 TI - Clostridium botulinum type C intoxication in feedlot steers being fed ensiled poultry litter. PMID- 8640638 TI - Phaeohyphomycosis in a cat. PMID- 8640639 TI - Microsporidian encephalitis of farmed Atlantic salmon (Salmo salar) in British Columbia. PMID- 8640640 TI - Copper penny ingestion in a cat. PMID- 8640641 TI - Emergency rescue treatment. PMID- 8640642 TI - Issues to consider when buying or selling a veterinary practice. PMID- 8640643 TI - Treating moderate and severe pain in small animals. PMID- 8640644 TI - Diagnostic ophthalmology. Periocular discharge and swelling around the eye in a dog. PMID- 8640645 TI - Induction of parturition in ewes with dexamethasone or dexamethasone and cloprostenol. PMID- 8640646 TI - Fatal pneumonia in adult dairy cattle associated with active infection with bovine respiratory syncytial virus. PMID- 8640647 TI - A monoclonal gammopathy-induced canine renal amyloidosis. PMID- 8640648 TI - Epidural abscess and discospondylitis in a dog after administration of a lumbosacral epidural analgesic. PMID- 8640649 TI - Inherited myopathy in a litter of Labrador retrievers. PMID- 8640650 TI - Anesthesia for canine cesarian section. PMID- 8640651 TI - Understanding your client's decision-making process. PMID- 8640652 TI - Mirror of the past provides image for future. PMID- 8640653 TI - Dry sow stalls--an animal welfare issue. PMID- 8640654 TI - An ethicist's commentary on how the veterinarian should deal with an injured, unowned dog. PMID- 8640656 TI - Nonsurgical videolaparoscopy for determination of reproductive status of the Arctic charr. AB - The efficient commercial production of Arctic charr is hampered by the limited and subtle sexual dimorphism shown by this species. This project examined the feasibility of low-cost videoendoscopy through the genital pore to determine sex, stage of maturity, and safety for 80, randomly selected, anesthetized Arctic charr. The sensitivity and specificity for correct assignment of male sex were 90% and 100%, respectively, among 3 observers in a single-blind design. Sensitivity and specificity for relative sexual maturity in the same fish were 96% and 54%, respectively. The observed mortality rate of 7% at the end of 3 weeks was confounded by the presence of unrelated disease. We conclude that the technique holds promise for application by producers to improve production efficiency. PMID- 8640655 TI - Risk factors associated with the incidence of foal mortality in an extensively managed mare herd. AB - The purpose of this study was to determine the incidence of neonatal mortality in a large, extensively managed mare herd and what risk factors were involved in foal mortality. For a 6 wk period between April 18, 1994, and May 31, 1994, 334 foals were born, of which 74 died before reaching 10 d of age, giving an overall mortality of 22% for this period. Seventy four percent of the foal deaths occurred within 48 h of parturition. The major causes of foal mortality included starvation/exposure 27%, septicemia 26%, and dystocia 20%. Weekly incidences varied significantly, ranging from 67% for week 1 to 14% for week 5 (P < 0.01). Other risk factors that were associated with foal death included failure of passive transfer (P < 0.0001), poor mothering ability (P < 0.0001), the presence of dystocia (P < 0.0001), low birth weight (p < 0.05), lack of rainfall (P < 0.01), and low temperatures (P < 0.1). The effect of sire, mare age, mare body condition, and foal sex were not significant risk factors for foal survival (P > 0.1). Further studies are required to determine if changing management procedures will be effective in reducing the incidence of neonatal foal mortality in this extensively managed herd. PMID- 8640657 TI - Unusual endocrine presentations of nasopharyngeal carcinoma. AB - BACKGROUND: Nasopharyngeal carcinoma is endemic in Southern China and the majority of patients present with local symptoms due to the tumor. METHODS: This report describes two unusual cases of occult nasopharyngeal carcinoma in which the patients initially presented with endocrine manifestations. RESULTS: The first patient presented with Cushing's syndrome secondary to ectopic adrenocorticotropic hormone (ACTH) production. Nasolaryngoscopy showed a growth in the left nasal fossa and biopsy revealed a poorly differentiated nasopharyngeal carcinoma that exhibited positive immunostaining for ACTH. The second patient presented with a 10-month history of bone pain over both lower limbs. She was normocalcemic but her serum alkaline phosphatase was markedly elevated. A bone biopsy showed both osteoclastic and osteoblastic activity with widespread fibrosis suggestive of Paget's disease. Three months later, she developed third cranial nerve palsy. Computed tomography investigation revealed a soft tissue mass filling the sphenoid and ethmoid sinuses. Biopsy showed a poorly differentiated nasopharyngeal carcinoma. The bone biopsy was reviewed and immunohistochemistry demonstrated the presence of cells positive for the epithelial marker AE1/3 within the fibrous stroma. Radio-labeled in situ hybridization showed that Epstein-Barr virus early RNA was present in these tumor cells and the bone lesions were in fact metastases. CONCLUSIONS: Nasopharyngeal carcinoma can present with rather atypical symptoms that may lead to a delay in diagnosis. Therefore, in high risk populations, it is important to consider nasopharyngeal carcinoma as a possible primary tumor in patients with occult carcinomas. PMID- 8640658 TI - Intensive short cause chemotherapy followed by radiotherapy of locally advanced nasopharyngeal carcinoma. AB - BACKGROUND: We designed a protocol with the goal of improving the disease free and overall survival of patients with previously untreated Stage IV nasopharyngeal carcinoma (NP(C)). The regimen consisted of intensive induction chemotherapy followed shortly thereafter by radiation therapy. METHODS: Between March 1986 and March 1992, 27 patients with T3-4, N2-3, M0 squamous cell carcinoma of the nasopharynx were treated with 2 cycles of chemotherapy, using cisplatin, 100 mg/m2 intravenously, on Day 1, and 5-fluorouracil (5-FU), 1000 mg/m2 per day continuous infusion, on Days 2-5. The second cycle was given on Day 16 and was followed by radiotherapy (RT), 70 Gray, given on Day 31. RESULTS: The objective response rate to chemotherapy was 93%, with a 37% complete response (CR) rate and a 56% partial response (PR) rate. The overall CR rate after RT was 85%. With a median follow-up of 60 months, the overall actuarial survival rate was 66%. Patients who had a CR after chemotherapy had a superior survival probability (100%). Toxicity was tolerable, without lethal complications. CONCLUSIONS: This study demonstrates that cisplatin/5-FU chemotherapy given in an intensive schedule and followed shortly thereafter by radical RT can improve the CR rate and survival of patients with locally advanced NPC, with tolerable toxicity. PMID- 8640659 TI - Epirubicin, cisplatin, and protracted venous infusion of 5-fluorouracil for esophagogastric adenocarcinoma: response, toxicity, quality of life, and survival. AB - BACKGROUND: The results of chemotherapy for patients with esophagogastric carcinoma have generally been modest but regimens developed more recently have produced higher response rates, and rekindled interest in neoadjuvant chemotherapy. One such regimen is epirubicin, cisplatin, and 5-fluorouracil (ECF). This study evaluates its efficacy, toxicity, impact on quality of life (QL), and impact on survival in a large consecutive series of patients with metastatic and locally advanced disease (LAD). METHODS: Patients with histologically confirmed esophagogastric carcinoma were treated with ECF (epirubicin 50 mg/m2 and cisplatin 60 mg/m2 every 3 weeks with continuous infusion of 5-fluorouracil (5-FU) 200 mg/m2/d). Responses were evaluated with computed tomography (CT) scan and endoscopy. QL was assessed using the European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire. RESULTS: A total of 235 patients were treated, 173 with metastatic disease and 62 with LAD. The mean number of cycles delivered was 6 (range: 1-11) and patients were followed-up for a median of 8 months. Response was observed in 135 of 220 (61%) evaluable patients, with a complete response (C(R)), 11% of the patients and a partial response in 50% of the patients. Patients with moderately differentiated adenocarcinomas and LAD responded most favorably. Symptomatic improvement was achieved in the majority of cases (63-78% depending on the symptom). Toxicity was generally only mild to moderate, with severe non hematologic toxicity in less than 12% of the patients and only 6 (2.5%) treatment related deaths. QL assessment showed no significant negative impact on emotional functioning and good symptomatic control. Surgery following response to ECF was performed in 29 of the LAD patients, and in 19 cases (66%) a potentially curative resection was possible, with histologic CR in 32% of the patients. CONCLUSIONS: ECF is a highly active regimen with acceptable toxicity in patients with esophagogastric adenocarcinoma. In a proportion of patients with LAD, chemotherapy enabled potentially curative surgery to be performed. The results justify further investigation of this regimen in a neoadjuvant setting. PMID- 8640660 TI - Multiple primary esophageal and concurrent upper aerodigestive tract cancer and the aldehyde dehydrogenase-2 genotype of Japanese alcoholics. AB - BACKGROUND: Multiple intraesophageal primary cancer and upper aerodigestive tract (UADT) cancer associated with esophageal cancer are common diseases, especially in heavy drinkers. They are often explained by the concept of field cancerization, which suggests a similar etiology. However, little is known about the nature of the hypothesized etiology. METHODS: Among 901 Japanese male alcoholics systematically screened by upper gastrointestinal endoscopy (with esophageal iodine staining), 33 had squamous cell carcinoma of the esophagus. The multiplicity of their esophageal carcinoma and their concurrent UADT cancer was compared with their genotype for aldehyde dehydrogenase-2 (ALDH2), the major determinant of blood acetaldehyde concentration after drinking. RESULTS: Of 17 patients with inactive ALDH2, 13 (76.5%) had multiple primary carcinoma of the esophagus, whereas 5 of 16 (31.3%) with active ALDH2 had multiple carcinomas (P < 0.01). The prevalence of concurrent UADT cancer was 29.4% in those patients with inactive ALDH2, compared with 6.3% in those patients with active ALDH2. CONCLUSIONS: Inactive ALDH2 is a risk factor for multiple carcinoma of the esophagus in alcoholics. Acetaldehyde, a recognized animal carcinogen, appears to play a critical role in field cancerization. PMID- 8640661 TI - Marked elevation of plasma carcinoembryonic antigen and stomach carcinoma. AB - BACKGROUND: This study was undertaken to clarify the relationship between marked elevation of plasma carcinoembryonic antigen (CEA) and signet ring cell carcinoma of the stomach. METHODS: To elucidate the contributing factor of extreme elevation of plasma CEA value, the histologic and biochemical records for 310 cases of stomach carcinoma, including 202 advanced and 108 early, collected between 1980 to 1994 from the San-in Rosai Hospital and Tottori University Hospital were studied. Immunohistochemical localization of CEA in the stomach was performed using a peroxidase antiperoxidase (PAP) staining technique. RESULTS: Among 310 cases of gastric carcinoma, 44 (14%) had abnormal plasma CEA values. The positivity rates of early and advanced gastric carcinoma were 3.7% (4/18) and 19% (40/202), respectively. Concerning advanced gastric carcinoma, 20 cases had more than 51 ng/mL, and 20 cases had between 5 ng/mL and 50 ng/mL. Four cases with plasma CEA values of more than 1,000 ng/mL had histological signet ring cell carcinoma (one case), and poorly differentiated adenocarcinoma with signet ring cell carcinoma (2 cases). Three cases of signet ring cell carcinoma or poorly differentiated adenocarcinoma of the stomach were massive local infiltration rather than hepatic metastasis. Among 40 cases with elevated plasma CEA, a multivariate regression analysis showed that only one variable (lymph node metastasis) was an independent factor (P < 0.05). Significantly higher rates of peritoneal metastasis (P < 0.0001) and lymph node metastasis (P < 0.05) were observed in patients with marked elevations of plasma CEA than in patients with moderate elevations of plasma CEA. No correlation was obtained between plasma CEA value and several biochemical tests. CONCLUSIONS: Marked elevation of plasma CEA may be found in the absence of liver metastasis from signet ring or poorly differentiated gastric carcinoma. Patients with marked elevations of CEA also had lymphatic and peritoneal dissemination. PMID- 8640662 TI - Characteristics of Epstein-Barr virus-associated gastric carcinoma with lymphoid stroma in Japan. AB - BACKGROUND: Gastric carcinoma with lymphoid stroma (GCLS), known to have a more favorable prognosis than ordinary gastric carcinoma, has been suggested to be closely associated with the Epstein-Barr virus (EBV). However, there are many clinicopathologic problems that remain unsolved. METHODS: In 21 patients, 26 GCLS lesions and 4 non-GCLS intramucosal adenocarcinomas that developed synchronously or metachronously with GCLS were examined for EBV involvement by in situ hybridization (ISH) and were analyzed clinicopathologically. In addition, nine patients who had advanced gastric carcinoma with massive liver metastases, who showed good response to chemotherapy and had prolonged survival, were examined for the presence or absence of EBV-associated GCLS. RESULTS: On ISH with EBV encoded small RNAs, diffuse hybridization signals were noted in 22 (84.6%) of 26 GCLS. Hybridization signals were also noted in all four non-GCLS adenocarcinomas accompanying GCLS. As a result, hybridization signals were noted in nine of ten cancerous lesions in four cases of synchronous multiple cancers and in all five cancerous lesions in two cases of metachronous multiple cancers. Long term survivors with liver metastases included two patients with EBV-associated GCLS. CONCLUSION: Approximately 84.6% of GCLS were related to EBV. EBV-associated GCLS constitutes one-half of the EBV-infected stomach cancers in our institution. The complete response and long term survival after conventional chemotherapy of two patients with Stage IV GCLS suggests that this form of gastric carcinoma may be especially sensitive to this treatment. The identification of EBV-associated synchronous multicentric cancers of both GCLS and non-GCLS type suggests that EBV infection may be an early event in the induction process of these tumors. PMID- 8640663 TI - The clinical significance of p53 accumulation in gastric carcinoma. AB - BACKGROUND: Alterations in the expression of p53 tumor suppressor protein is a frequent event in human cancer but the practical implications of this phenomenon are yet to be fully exploited. The objective of this study was to determine the value of p53 accumulation as a marker of tumor progression and prognosis of gastric carcinoma patients and to evaluate whether this parameter can be properly assessed prior to surgery. METHODS: The expression of p53 was studied immunohistochemically in 200 gastric carcinomas using paraffin embedded surgical specimens and endoscopic biopsies. The correlation between p53 expression in tumor tissue, selected clinicopathologic variables, and the course of the patients' disease were analyzed. RESULTS: Results showed that 42.5% of the gastric carcinomas expressed elevated levels of p53 protein. P53 accumulation positivity correlated with increasing tumor stage and size (P < 0.001 and P = 0.025, respectively). P53 positive tumors had a higher propensity for lymph node and distant metastases (P < 0.001). P53 accumulation was also more frequently detected in carcinoma from proximal rather than distal stomach (P = 0.027). In patients receiving potentially curative resection for advanced cancer, p53 accumulation was an independent parameter and the strongest for poor prognosis (RR = 3.7, P < 0.001). There was complete concordance between immunohistochemical detection of p53 in endoscopic and surgical material. CONCLUSIONS: A preoperative assessment of p53 expression in gastric carcinoma can be helpful to identify patients at high risk of metastatic spread to regional lymph nodes and independently to identify those with especially poor prognosis. When combined with routine procedures, this simple and inexpensive test might allow appropriate planning of better treatment strategies. PMID- 8640664 TI - Frequency and clinical features of multiple tumors of the large bowel in the general population and in patients with hereditary colorectal carcinoma. AB - BACKGROUND: Reports on the frequency of multiple carcinomas of the colon and rectum have varied from 3-4% to more than 10% of all tumors of the large bowel. METHODS: We reviewed the files of a specialized colorectal cancer registry with the following objectives: a) to determine the frequency of multiple tumors (synchronous or metachronous) in the general population; b) to compare these values with those observed in patients with hereditary nonpolyposis colorectal carcinoma (HNPCC); and c) to evaluate the clinical outcome of patients with multiple tumors and the role of other clinical parameters in the development of these neoplasms. RESULTS: From 1984 to 1992, 53 patients with multiple tumors (of 1298 registered patients, 4%) had large bowel carcinoma; 33 (2.5%) were synchronous and 20 (1.5%) metachronous. The total number of multiple colorectal carcinomas was 95, which was 7% of all registered colorectal carcinomas (1337 carcinomas in 1298 patients). Multiple tumors occurred significantly more often in patients with HNPCC than in those with sporadic carcinomas (P < 0.001); this increased prevalence was more marked for metachronous lesions, which occurred almost 4 times more often in patients with HNPCC (5.8% vs. 1.3%; P < 0.001). The average interval of time between the first and the second malignancy was 8.7 years; there was no significant difference between hereditary and sporadic tumors. Patients with synchronous tumors did not show appreciable differences in survival when compared with individuals who had single neoplasms. In contrast, a poor clinical outcome was observed in patients with metachronous tumors after the development of the second carcinoma. Finally, polypoid adenomas of the large bowel were found significantly more often in patients with multiple primary tumors than in those with a single tumor. CONCLUSIONS: These results emphasize the importance of preoperative pancolonoscopy for the identification of possible synchronous tumors (both benign and malignant) and long-lasting endoscopic follow up for the detection of recurrent or metachronous lesions. The conclusions are even more pertinent for patients with HNPCC, whose risk of metachronous tumors is significantly higher than that of patients with sporadic carcinoma. PMID- 8640665 TI - Factors correlated with portal venous invasion by hepatocellular carcinoma: univariate and multivariate analyses of 232 resected cases without preoperative treatments. AB - BACKGROUND: The postoperative intrahepatic recurrence of hepatocellular carcinoma (HCC) is high. It is difficult to distinguish whether the recurrence is metastatic or new primary lesion. To determine the malignant potential of HCC itself, we analyzed the risk factors associated with portal venous invasion since this is direct evidence of tumor invasiveness. METHODS: Two hundred and thirty two patients who underwent curative hepatectomy for HCC without preoperative treatments were included in this study, because preoperative treatment caused the tumor to undergo a variety of histologic change. We analyzed the risk factors linked to portal venous invasion by both univariate and multivariate analyses. RESULTS: In an univariate analysis, tumors larger than 3 cm, high histologic grade (III or IV), the presence of fibrous capsule, necrosis, mitotic rate of more than 4/10 high power fields, peliotic change, presence of tumor giant cells, high platelet count, low level of indocyanine green retention rate at 15 minutes, and the absence of cirrhosis were significantly correlated with portal venous invasion. In multiple stepwise logistic regression analysis, tumors larger than 3 cm, high histologic grades, and the presence of fibrous capsule were strong predictors of portal venous invasion by HCC. CONCLUSIONS: Because the blood vessels of the fibrous capsule were frequently invaded by cancer cells, it may have been possible to prevent postoperative metastatic recurrence if HCC were resected before becoming large enough to have a fibrous capsule. PMID- 8640666 TI - Treatment of small cell lung carcinoma in the elderly. AB - BACKGROUND: The number of elderly people with small cell lung carcinoma (SCLC) is increasing and currently nearly 25% are older than 70 years. Elderly patients may not tolerate intensive therapy and, therefore, often do not receive such treatment. Additionally, age may be an independent predictor for response and survival. We compared the investigation, staging procedure, and management of patients less than 60 years, 60 to 69, and older than 70 years who were diagnosed with SCLC between 1985 and 1991. We hypothesized that elderly patients were investigated and treated less aggressively, and that their outcome was poorer than that of younger patients with SCLC. METHODS: Information on weight loss, performance status, coexisting disease, staging investigations, and treatment was recorded. Treatment was categorized as optimal or suboptimal using predetermined criteria, and correlated with patient age. Toxicity grade, response to treatment, and survival were noted. RESULTS: There were no differences among the 3 age groups with respect to disease stage, and weight loss, although poorer performance status and comorbidity were more common in those patients older than 70 years. Elderly patients were investigated and treated less aggressively than the 2 younger patient groups. The oldest group received smaller chemotherapy dosage, fewer cycles, and had more dose reductions compared to the younger patients. Only 1 of 81 elderly patients was enrolled on an experimental protocol as compared with 19% and 28% of the younger patient groups. Furthermore, elderly patients had the highest frequency of supportive care alone. There was a significant relationship between advanced age and suboptimal treatment, with those older than 70 years having an odds ratio (OR) of 0.30 (95% confidence interval (CI) 0.15-0.61), for having received optimal treatment. Despite this, survival was similar for younger and older groups of patients (OR 0.89, CI 0.6 1.3). CONCLUSIONS: Elderly patients had poorer pre-treatment performance status, greater comorbidity, were more likely to have suboptimal therapy and were almost never entered into clinical trials. Despite this their survival did not differ from that of younger patients with SCLC. Randomized trials of treatment, with assessment of quality of life, are necessary to determine the effect of modified regimens for elderly patients with SCLC. PMID- 8640668 TI - Proliferation index and vascular density of giant cell tumors of bone: are they prognostic markers? AB - BACKGROUND: The proliferation index (PI) and vascular density (VD) in giant cell tumors (GCT) of bone were studied to assess then value as prognostic markers. METHODS: Formalin fixed, paraffin embedded tissue from 7 nonrecurrent (NR-GCT) and 13 recurrent (R-GCT) tumors were stained with MIB-1, a monoclonal antibody against Ki-67 (nuclear proliferation antigen), and QBEND-10, a monoclonal antibody against CD34 (endothelial antigen). A minimum of ten fields/case were analyzed on the SAMBA 4000 Image Analyzer. Only mononuclear cell nuclei were analyzed for MIB-1 staining. RESULTS: Mean values +/- 1 Standard deviation (SD) for PI (stained nuclear area/total nuclear area) were: NR-GCT, 10.98 +/- 3.70; R GCT (initial presentation), 8.94 +/- 3.57; and R-GCT (first recurrence), 9.25 +/- 3.52. In addition, the mean PI +/- 1 SD for mononuclear round-ovoid cells was 8.71 +/- 3.47 and was 9.16 +/- 10 for mononuclear spindle cells. The mean values +/- 1 SD for VD (stained area/total area of structures) were: NR-GCT, 10.61 +/- 6.37; R-GCT (initial curettage), 9.40 +/- 4.94; and R-GCT, (first recurrence), 12.56 +/- 5.88. Comparing the means for PI and VD for both groups of tumors using Student's t test showed no statistically significant differences (P = 0.34). In one case of histologically benign GCT that metastasized to the lungs, the PI of the metastatic tissue was not significantly different from the primary tumor. CONCLUSIONS: This study presents evidence that the degree of tumor cell proliferation and vascularity are not useful parameters to predict the recurrence of GCT of bone and that there are no significant differences between the PI of mononuclear round-ovoid cells and mononuclear spindle cells. PMID- 8640669 TI - Amelanotic lentigo maligna melanoma: a diagnostic conundrum-- presentation of four new cases. AB - BACKGROUND: The clinical appearance of amelanotic lentigo maligna melanoma (ALMM) is quite confusing and usually is not diagnosed prior to histopathologic examination. METHODS: We have studied four new patients with ALMM whose correct diagnosis was not obtained from any one clinical finding. We arrived at the final diagnosis in an unexpected way, having had biopsied the lesions for diagnoses other than malignant melanoma. RESULTS: ALMM presents as a nonspecific skin lesion with no single indicative characteristic. A search of the literature confirmed our difficulty in making the diagnosis. CONCLUSIONS: To diagnose ALMM, one has to be cognizant of this condition and has to consider a constellation of findings that are unusual with melanocytic lesions. Questionable lesions must be biopsied for definitive histopathologic diagnosis. PMID- 8640670 TI - Endometrial carcinoma associated with breast carcinoma: low incidence with tamoxifen use. AB - BACKGROUND: Women treated with tamoxifen for breast cancer are at increased risk of endometrial cancer. This study examines the experience at Roswell Park Cancer Institute (RPCI) with women diagnosed with both endometrial carcinoma (EC) and breast carcinoma (BC) to determine the risk and stage of endometrial carcinoma among women treated with tamoxifen. METHODS: The tumor registry was searched for women with diagnoses of both BC and EC between 1980 and 1993. Systemic therapy was classified for all analytic cases of breast carcinoma (women who received primary BC treatment at RPCI). Medical records of all women with both BC and EC were reviewed, including all analytic and nonanalytic cases. RESULTS: There were 1947 analytic and 1534 nonanalytic BC cases and 877 analytic and 239 nonanalytic EC cases. Thirty-six women in the nonanalytic breast cancer group also had endometrial carcinoma. Fifteen had endometrial carcinoma before breast carcinoma, and 20 of 21 women with breast cancer first had no record of tamoxifen use. Thirty-seven women in the analytic breast carcinoma group had endometrial carcinoma. Endometrial carcinoma preceded breast carcinoma in 29 women. Breast carcinoma preceded endometrial carcinoma in eight women, and two of these developed endometrial carcinoma during or after tamoxifen therapy. Therefore, a total of three women developed endometrial cancer during or after tamoxifen therapy (two analytic and one nonanalytic). The EC was classified as International Federation of Gynecology and Obstetrics (FIGO) Stage IA (1 patient) and IB (2 patients) with one patient each with histologic Grade I, II, and III after 1, 2, and 5 years of tamoxifen therapy, respectively. No patients had recurrence or died from endometrial carcinoma. The risk of endometrial carcinoma with tamoxifen was determined from the number of women in the breast cancer analytic group receiving tamoxifen. Hormonal therapy was coded as part of systemic treatment in 652 of 1947 analytic patients (33%; 510 as adjuvant therapy and 142 for metastatic cancer). Of these patients, 172 of 652 women (26%) had undergone hysterectomy prior to breast cancer diagnosis, and another 71 women (11%) received nontamoxifen hormone therapy (e.g., prednisone). Tamoxifen therapy was documented in 402 women in the analytic group. (The median age of these women at BC diagnosis was 63 years). Therefore, the maximum estimate of endometrial carcinoma risk is 2 of 402 cases (0.5%). CONCLUSIONS: The risk of endometrial carcinoma with tamoxifen use is low. The value of routine invasive screening for endometrial carcinoma for women receiving tamoxifen should be determined by prospective study. PMID- 8640667 TI - High neuron specific enolase levels in bronchoalveolar lavage fluid of patients with lung carcinoma: diagnostic value, relation to serum neuron specific enolase, and staging. AB - BACKGROUND: High levels of neuron specific enolase (NSE) have recently been described in the bronchoalveolar lavage (BAL) fluid of patients with lung carcinoma. Although its value in serum has been extensively studied, its diagnostic value in BAL fluid in terms of sensitivity, specificity, and predictive value have not been evaluated. In addition, its value in staging and relation to serum NSE are yet unknown. METHODS: NSE levels were determined on the same day in the BAL fluid and the sera of two groups of patients: those with newly diagnosed lung carcinoma and those with smoking related chronic obstructive pulmonary disease (COPD). Clinical TNM staging was also performed. Levels of NSE in BAL fluid were expressed as nanograms per 100 international units of lactate dehydrogenase. BAL fluid NSE levels of the two groups were compared with staging and serum NSE. RESULTS: A highly significant difference exists in BAL NSE in the two groups. For diagnostic purposes, the simultaneous measurements of serum NSE increases its sensitivity, but specificity remains unchanged. No correlation exists between BAL NSE and serum NSE, tumor size, nodal status, or the presence of metastases. BAL NSE is a better predictor of malignancy than serum NSE. CONCLUSION: BAL fluid measurements of NSE may have diagnostic value, specially if it is simultaneously measured in the serum. However, our study does not show any value for this technique in staging of lung carcinoma. Also it has no correlation with serum NSE. Studies will have to be performed to determine if BAL NSE can predict chemotherapeutic sensitivity. PMID- 8640671 TI - Analysis of numerical aberrations of specific chromosomes by fluorescent in situ hybridization as a diagnostic tool in breast cancer. AB - BACKGROUND: Biopsy by fine-needle, aspiration has become a routine technique for the diagnosis of a dominant breast mass. In this study, fluorescent in situ, hybridization (FISH) analysis of interphase nuclei allowed the authors to detect genetic aberrations that are difficult to identify by conventional cytology. METHODS: To investigate ways of minimizing misdiagnosis of the cytology of breast tumors, and detecting genetic aberrations preoperatively, the authors performed FISH with specimens obtained by fine-needle aspiration biopsies of 106 primary breast tumors (78 primary breast cancers, 2 phyllodes tumors, and 26 benign breast tumors). Numerical chromosome aberrations were investigated using 3-color FISH performed with (peri)centromere-specific probes for chromosomes 1, 11, and 17. RESULTS: Sufficient materials for FISH analysis were obtained with aspiration biopsy from 98 of the 106 breast tumors (93.4%). None of the benign tumors nor phyllodes tumors showed evidence of aneusomy for any of the 3 chromosomes. However, 71 of the 74 breast cancers (95.9%) for which sufficient material was available demonstrated aneusomy of at least 1 of the 3 chromosomes tested. The FISH analysis also suggested a possible correlation between aneusomy of chromosome 17 and metastasis to regional lymph nodes (chi-square test = 7.78; P < 0.05). CONCLUSIONS: FISH analysis of fine-needle aspiration biopsies can be a practical and useful method for the preoperative diagnosis of breast carcinoma. PMID- 8640672 TI - Evaluation of factors potentially associated with inadequate follow-up of mammographic abnormalities. AB - BACKGROUND: To increase the proportion of women who receive the recommended follow-up for mammographic abnormalities, factors which inhibit follow-up must be identified. Patient and health care delivery related factors were evaluated, stratified by type of follow-up recommendation, to determine reasons for inadequate follow-up. METHODS: All Caucasian and African American women at the Henry Ford Medical Group, in southeastern Michigan, with an abnormal screening mammogram result between January 1, 1992 and July 31, 1992 were identified. All women with inadequate follow-up, and a random sample of women with adequate follow-up, were invited to participate in a telephone interview that assessed three major dimensions of the Health Belief Model (susceptibility, benefits, and barriers), general health and health behaviors, and related characteristics. The relationship between these factors and inadequate follow-up was evaluated separately for women with immediate and 6-month follow-up recommendations, using univariate and multivariate analyses. RESULTS: A total of 555 women were invited to participate in the study (219 with inadequate follow-up and 336 with adequate follow-up). Interviews were completed for 418 women (75.3%). Women who were not notified of their mammographic abnormality were excluded from this study, leaving 399 women available for analysis. Among the women who had the recommended immediate follow-up, those who reported difficulty in obtaining medical appointments were 4 times more likely to have inadequate follow-up (95% confidence interval [CI] 1.5, 11.3), after adjusting for other variables. Among the women with six-month follow-up recommended, those who received fewer mammograms in the past 5 years were more likely to have inadequate follow-up (odds ratio [OR] = 4.0; 95% CI 1.6, 10.4). In this group, sociodemographic characteristics were not associated with inadequate follow-up, although women with transportation problems were at a higher risk (crude OR = 5.2; 95% CI 1.6, 16.7; adjusted OR = 3. 1; 95% CI 0.5, 18.3). Among women with 6-month follow-up recommended, those who perceived their health as poor or fair (crude OR = 2.4; 95% CI 1.2, 5.1; adjusted OR = 2.3; 95% CI 0.8, 6.8) and those who did not examine their own breasts frequently (crude OR = 2.3; 95% CI 1.0, 5.4; adjusted OR = 2.7; 95% CI 0.5, 18.3) were also more likely to have inadequate follow-up. CONCLUSIONS: Results from this study demonstrate that the relative importance of factors associated with inadequate follow-up of abnormal mammograms differs according to the type of follow-up recommended. For both types of recommendations, the factors identified are noteworthy because they are amenable to intervention. Efforts should be made to facilitate appointment scheduling for follow-up of abnormal mammograms, and information about previous mammography screening should be obtained to identify women who are at a high risk for inadequate follow-up. PMID- 8640673 TI - Endothelial area as a prognostic indicator for invasive breast carcinoma. AB - BACKGROUND: Vascular enumeration using antibodies to Factor VIII has been reported to be an independent prognostic indicator of invasive breast carcinoma. METHODS: To eliminate potential subjectivity in distinguishing between individual vessels, especially in areas of tangled capillaries, total endothelial area (EA) was assessed using a Samba 4000 image analyzer. One hundred seventy-eight invasive breast carcinomas (Stage 1 and 2, mean follow-up: 71 months) were immumostained for the presence of CD34, the human hematopoietic progenitor cell antigen also present in endothelium, and EA was quantitated within 5 adjacent 20X fields (0.74 mm2). Additionally, these same vessels were manually counted from the image analyzer. Manual counts were also made from a photomicrograph representative of a single 10X field (1.06 mm2). RESULTS: High grade carcinomas contained greater endothelial area than low grade carcinomas (P = 0.0001). Endothelial area was prognostically significant (P = 0.004) in univariate analysis of disease-free survival (DFS) and overall survival (OS), as were stage of disease, tumor size, and combined histologic grade (P < or = 0.024). Manual vessel counts from the monitor were significant for OS only. Manual vessel counts from photomicrographs showed no statistically significant association with DFS or OS. In multivariate analysis, EA, but not vessel enumeration, remained as an independent predictor for OS (lymph node negative patients only, n = 87) and for DFS (lymph node positive patients only, n = 91). For the entire group of patients (lymph node negative and lymph node positive) independent predictors of DFS and OS were tumor grade and size (P < or = 0.006). CONCLUSIONS: Of the three methods used to evaluate tumor angiogenesis, total endothelial area, as objectively evaluated by image analysis, was the only independent prognostic indicator for OS for patients with lymph node negative invasive breast carcinoma. PMID- 8640674 TI - Is paclitaxel and cisplatin a cost-effective first-line therapy for advance ovarian carcinoma? AB - BACKGROUND: Paclitaxel and cisplatin use for the treatment of advanced ovarian carcinoma (AOC) has been shown to increase median survival duration. An evaluation was performed on the economic consequences of treating AOC patients with combined paclitaxel and cisplatin chemotherapy compared with current usual care, i.e., combined cyclophosphamide and cisplatin chemotherapy. METHODS: Linear modeling techniques combined with retrospective chart analysis were used to predict the clinical progression and treatment of AOC patients until death. Cost effectiveness analysis comparing paclitaxel and cisplatin and usual care was performed from a simplified Ministry of Health perspective. RESULTS: Assuming a 50% increase in survival for paclitaxel and cisplatin patients, an assumption supported by recent clinical trial data, this treatment showed an average lifetime cost per patient of $50,054 Cdn compared with a cost of $36,837 Cdn for usual care. The incremental cost of the paclitaxel and cisplatin treatment over the usual treatment was $20,355 Cdn per life year gained. These results withstood extensive sensitivity analyses. CONCLUSIONS: Paclitaxel, in combination with cisplatin, appears to be a cost-effective first-line treatment for AOC. A moderate increase in incremental cost compares favorably with other life-saving strategies currently in use. As more data become available for the use of paclitaxel, this pilot study will provide a basis for more extensive economic evaluation of paclitaxel. PMID- 8640675 TI - Expression of cell regulatory proteins in ovarian borderline tumors. AB - BACKGROUND: Tumors of borderline malignancy are still a controversial subgroup of ovarian neoplasms. The expression of several cell regulatory proteins was studied to characterize the molecular phenotype of these tumors, and to compare them with their benign and malignant counterparts. METHODS: Specimens from 22 patients with tumors of borderline malignancy (11 serous and 11 mucinous tumors), 12 patients with benign tumors, and 16 patients with invasive ovarian carcinomas were evaluated for expression of epidermal growth factor receptor (EGFR), HER-2/neu, PTP1B, and p53 by immunohistochemical techniques. RESULTS: One or both of the tyrosine kinase growth factor receptors EGFR and HER-2/neu was expressed by 42% of benign, 59% of borderline, and 81% of malignant ovarian tumors. EGFR was expressed in a significantly greater fraction of malignant lesions (69%) than borderline lesions (18%) (P< 0.004). EGFR expression was not observed among the 11 mucinous borderline tumors. HER-2/neu was expressed by 50% of borderline tumors and was not a marker for malignancy. The tyrosine phosphatase PTP1B was expressed by a similar fraction of benign (17%), borderline (27%), and malignant (19%) tumors. The number of cases studied precluded correlation of kinase and phosphatase activity. However, among 12 tumors with PTP1B expression, 9 also expressed EGFR or HER-2/neu. Overexpression of p53 was observed only in malignant serous tumors and was not found in malignant mucinous, borderline, or benign lesions. CONCLUSIONS: Either EGFR or HER-2/neu was detected in a majority of borderline cancers. PTP1B was present only in a minority of these cancers. Frankly malignant serous lesions differed from borderline and benign tumors with regard to expression of EGFR and overexpression of p53. PMID- 8640676 TI - Unreliability of modified inguinal lymphadenectomy for clinical staging of penile carcinoma. AB - BACKGROUND: In 1988, Catalona proposed a modified bilateral inguinal lymphadenectomy for staging of lymph node metastasis from penile carcinoma. All three patients with penile carcinoma submitted to this procedure and without histologically confirmed metastases were free of disease within a mean follow-up time of 14.6 months. METHODS: In a prospective study, the authors evaluated thirteen patients staged by the TNM system and submitted to modified bilateral inguinal lymphadenectomy. RESULTS: None of the patients had histologic metastases in the medial quadrant lymph nodes. Two of these patients developed regional lymph node metastases within 13.2 months (mean follow-up time). CONCLUSIONS: Catalona's procedure was not reliable. We therefore recommend standard inguinal lymphadenectomy as the minimal treatment for patients with infiltrating carcinoma of the penis. PMID- 8640677 TI - Do prostate specific antigen and prostate specific antigen density enhance the detection of prostate carcinoma after initial diagnosis of prostatic intraepithelial neoplasia without concurrent carcinoma? AB - BACKGROUND: Prostatic intraepithelial neoplasia (PIN) is considered to be a precursor of prostate carcinoma in which serum levels of prostate specific antigen (PSA) have been correlated with PIN grades. The aim of this study was to determine whether PSA and prostate specific antigen density (PSAD), obtained at the time of initial diagnosis of PIN without concurrent carcinoma, can be used as predictive factors to discriminate patients with subsequent cancer on repeat biopsy. METHODS: We studied, retrospectively, the records of 93 patients with PIN (low and high grade) without concurrent carcinoma at the time of their first needle biopsy. We assessed the relationship between initial PIN grade, PSA, and PSAD with later detection of carcinoma on repeat biopsy. Patients were divided into 3 subgroups for analysis according to their initial PSA level (0-4, 4.1-10, >10 ng/mL). RESULTS: Carcinoma detection rate on repeat biopsy was 13.3% for patients with low grade PIN and 47.7% for patients with high grade PIN (P < 0.006). High grade PIN was frequently associated with subsequent carcinoma whatever the PSA level (33.3-61.9%). Low grade PIN was associated with subsequent carcinoma in 42.8% of the cases when PSA was greater than 10 ng/mL. When PSA was between 4 and 10 ng/mL, low grade PIN carcinoma was found on repeat biopsies in only 10.7% of the cases (P = 0.05). In none of the PSA subgroups did PSAD enhance later cancer detection. CONCLUSIONS: For patients with high grade PIN, the incidence of subsequent carcinoma is high, whatever the PSA values. For these cases repeat biopsies should be recommended. Patients with low grade PIN and PSA greater than 10 ng/mL should have repeat biopsies because the incidence of subsequent carcinoma is high and comparable to high grade PIN. PSAD did not provide additional information. PMID- 8640678 TI - Testicular germ cell tumors in patients with human immunodeficiency virus infection. AB - BACKGROUND: There has been evidence of a higher incidence of testicular germ cell tumors (GCT) in human immunodeficiency virus (HIV)-seropositive men than in the non HIV-infected male population. Most authors recommend standard therapy for HIV positive patients with GCT but the immumosuppressive effects of chemotherapy and/or radiotherapy must be considered. METHODS: The records of all patients in whom testicular cancer was diagnosed and/or treated at a single institution between January 1986 and July 1995 were reviewed with regard to HIV seropositivity. Tumor histology, initial staging, treatment, and the patients' outcomes were analyzed in connection with a review of the literature. RESULTS: Six patients with GCT and documented HIV seropositivity at the time of tumor diagnosis (four homosexuals, one bisexual, and one heterosexual former intravenous drug abuser) of 192 documented cases of testicular cancer are reported. In addition, 1 patient proved to be HIV seropositive 34 months after completing chemotherapy (vinblastine, ifosfamide, and cisplatin) for Stage IIB (minimal disease) seminoma. Intensified platinum-based chemotherapy was administered to two patients with clinical Stage IIIC (advanced disease) nonseminomatous germ cell tumors (NSGCT). Both patients achieved a transient partial response but suffered from progressive HIV disease and died 24 and 7 months, respectively, after orchiectomy. One patient with Stage IIIA (moderate disease) seminoma received four courses of chemotherapy (etoposide, ifosfamide, and cisplatin) and has remained in complete remission for 40 months. One patient with bilateral Stage I seminoma underwent adjuvant radiotherapy but was lost to follow-up. One patient with clinical Stage IIA (minimal disease) NSGCT refused any further treatment after hemiorchiectomy, but four courses of chemotherapy (cisplatin, etoposide and bleomycin) had to be given 32 months later because of symptomatic abdominal disease. A partial remission was obtained and there was no evidence of active tumor 16 months after the completion of chemotherapy. A retroperitoneal lymph node dissection was performed in 1 patient with Stage I NSGCT who was free of disease 111 months after diagnosis. The Centers for Disease Control classification for HIV infection and acquired immune deficiency syndrome (AIDS) did not change after therapy in two patients, whereas three patients suffered from progressive HIV disease. CONCLUSIONS: HIV infection should be considered in patients with testicular cancer who belong to an urban population. Oncologic therapy based on a patient's individual situation is recommended. PMID- 8640679 TI - Combination chemotherapy with doxorubicin, bleomycin, and vindesine for AIDS related Kaposi's sarcoma. AB - BACKGROUND: Kaposi's sarcoma is the most common neoplasm in patients with human immunodeficiency virus (HIV) infection. Although the best therapeutic approach is still unclear, patients with advanced KS are usually treated with systemic chemotherapy. METHODS: A prospective multiinstitutional Italian study evaluated the efficacy and toxicity of combination chemotherapy with doxorubicin, bleomycin, and vindesine (ABVi) in patients with progressive and extensive HIV related KS. Patients were given doxorubicin, 20 mg/m2 on Day 1; bleomycin, 15 mg on Day 1, and vindesine, 4 mg on Day 1 biweekly +/- granulocyte-colony stimulating factor. RESULTS: Overall, 21 of 38 evaluable patients (55%) achieved an objective response (OR): there was 1 complete response and 20 partial responses. The most important bone marrow toxicity was granulocytopenia in 61% of the evaluable patients; 34% had Grades 3-4 toxicity, according to the World Health Organization Classification. The majority of patients (64%) developed some type of opportunistic infection (OI) during chemotherapy or the follow-up, with cytomegalovirus infection being the most frequent OI observed. The median duration of survival from KS diagnosis and from the start of ABVi therapy was 19 months (range, 3.4-88.5 months) and 9.9 months (range, 0.1-42.4 months), respectively. CONCLUSIONS: The high rate of OI during ABVi chemotherapy and the follow-up is of concern, although these infections possibly could be due to our patients' low CD4+ lymphocyte counts. However, no toxic death was observed in our patients, suggesting that ABVi could be used in patients with aggressive disease, especially those who were previously untreated. PMID- 8640680 TI - Anaphylactoid reactions to methotrexate. AB - BACKGROUND: Methotrexate is a chemotherapeutic agent frequently utilized for the treatment of malignant, rheumatic, and pulmonary diseases. Although this agent has been extensively used for more than 45 years, there are few reports of immediate systemic hypersensitivity reactions. The reported immediate reactions include anaphylaxis, urticaria, and hepatitis. However, these reactions have been reported to occur only after some prior exposure to methotrexate. No immediate hypersensitivity reactions to methotrexate have been reported during the initial exposure. We describe two patients who developed immediate systemic hypersensitivity reactions during the initial administration of methotrexate. METHODS: The clinical outcomes of two patients treated by the Hematology/Oncology department at a tertiary care military medical center are described. The National Library of Medicine in Bethesda, Maryland, was electronically searched for the literature review. RESULTS: Patient 1, a 30-year-old male with localized high grade osteosarcoma of the left distal femur, developed generalized pruritus, urticaria, angioedema, and pharyngeal edema within 10 minutes of receiving the initial administration of intravenous high-dose methotrexate. No other pharmaceutical agents, such as antiemetics, were found to cause symptoms on rechallenge. The severity of this reaction precluded continuation of methotrexate therapy. Patient 2, a 23-year-old male with localized high grade osteosarcoma of the right distal tibia, developed pruritus and urticaria within 30 minutes of receiving the initial administration of intravenous high dose methotrexate. This patient, like most patients with immediate hypersensitivity reactions to methotrexate, developed recurrent symptoms during rechallenge of this agent despite prophylactic premedication. CONCLUSIONS: Unlike prior reports in our literature, our cases demonstrate that anaphylactoid reactions can occur during the initial exposure to methotrexate. Clinicians must be prepared to treat potentially life-threatening reactions with both the initial and subsequent doses of methotrexate. PMID- 8640681 TI - Second-line chemotherapy in human immunodeficiency virus-related non-Hodgkin's lymphoma: evidence of activity of a combination of etoposide, mitoxantrone, and prednimustine in relapsed patients. AB - BACKGROUND: There is very little experience reported in the literature on the treatment of patients with relapsed or resistant human immunodeficiency virus related non-Hodgkin's lymphoma (HIV-NHL). We performed a prospective study to evaluate the feasibility and activity of a second-line chemotherapy regimen consisting of etoposide, mitoxantrone, and prednimustine (VMP) in this setting. METHODS: Twenty-one patients were consecutively treated. Thirteen patients were resistant to primary chemotherapy and 8 patients had relapsed after their first complete remission (CR). Etoposide and prednimustine were both given orally at doses of 80 mg/m2 daily for 5 days, and mitoxantrone was given intravenously at a dose of 10 mg/m2 on Day 1; the cycles were repeated every 3 weeks. RESULTS: Nineteen of 21 patients were evaluable for response. The median number of cycles administered was 2 (range, 1-5). A CR occurred in 5 of 19 patients (26%; exact 95% confidence interval; 9-51%). Four of these CRs were observed in the 7 evaluable relapsed patients. Of 45 cycles evaluable for toxicity, severe neutropenia (< 500/microL) occurred in 19 (42%) cycles and severe thrombocytopenia (< 25,000/microL) in 6 (13%) cycles. One toxic death occurred due to sepsis during neutropenia. The overall median survival was 2 months (range, < 1-13 months); the median survival time for the 5 patients with CR (13 months; range, 6-13 months) was statistically significantly longer than that observed in patients without CR (2 months; range, < 1-7 months). CONCLUSIONS: Although the overall prognosis of patients with resistant or relapsed HIV-NHL is very poor, palliative therapy with VMP can be effective and relatively safe in the latter group. Prolonged survival has been observed in some patients who had relapsed after initial chemotherapy. PMID- 8640682 TI - Herpesvirus-like DNA sequences and Kaposi's sarcoma: relationship with epidemiology, clinical spectrum, and histologic features. AB - BACKGROUND: The evidence of an infectious agent other than human immunodeficiency virus (HIV) acting as a possible etiologic cause of Kaposi's sarcoma (KS) has received considerable attention in the last years. Recently, DNA sequences from a new herpesvirus (HHV-8) have been observed in several cases of KS. The discovery suggests that this virus may play a role in the pathogenesis of KS. To evaluate these results, we determined the frequency of HHV-8 DNA sequences in 78 specimens of KS according to different epidemiologic origins (sporadic KS: 6, immunosuppressive drug-associated: 11, and AIDS-associated: 61), clinical forms (cutaneous: 69, mucocutaneous: 4 and visceral; 5) and histologic variants (early patch: 40, late-plaque or nodular: 38). METHODS: We used the hot start polymerase chain reaction amplification method with KS330 primers specific for HHV-8 DNA. Tumoral or nontumoral skins and visceral specimens free of KS, originating from patients with KS positive for HHV-8 DNA sequences or from immunosuppressed patients without KS, served as controls. Normal skin from healthy HIV seronegative patients was also included. RESULTS: HHV-8 DNA sequences were found in 3 of the 6 sporadic KS (50%), in 5 of the 11 immunosuppressive drug-associated KS (45%), in 41 of the 61 AIDS-associated KS (67%), in 32 of the 69 cutaneous KS (46%), in 3 of the 4 mucocutaneous KS (75%) in 2 of the 5 systemic KS (40%), in 23 of the 40 early or patchstage KS (58%), in 30 of the 38 late plaque or nodular stages KS (79%). These sequences were also demonstrated in one sample of skin with scabies and in a glomerulonephritis lesion from two immunosuppressed patients with KS who were also positive for herpesvirus-like in their KS lesions. None of the other skin or visceral specimens, originating from healthy, AIDS, or transplanted patients without KS, were positive. CONCLUSIONS: Our results reinforce the hypothesis that HHV-8 is incriminated in different epidemiologic, clinical and histologic stages of KS and could contribute to the pathogenesis of this tumor. However, the presence of HHV-8 DNA sequences in skin and visceral samples free of KS from KS indicates that the virus is not restricted to the tumor tissue, and thus is able to disseminate in many organs of the target individual. Absence of the virus from healthy, AIDS, or transplanted patients without KS suggests that the viral Sequences either do not spread easily or do not easily maintain themselves in the human population. PMID- 8640683 TI - Nasal T-cell lymphoma causally associated with Epstein-Barr virus: clinicopathologic, phenotypic, and genotypic studies. AB - BACKGROUND: The authors have previously demonstrated nasal T-cell lymphoma (NTL) associated with Epstein-Barr virus (EBV). The detailed clinical, phenotypic, and genotypic features and the role of EBV in lymphomagenesis remain to be clarified. METHODS: The study group consisted of 18 patients with NTL. The phenotype was determined by immunoperoxidase staining with various monoclonal antibodies. Genotypic study was done using Southern blot hybridization. The presence of EBV encoded small nuclear early region (EBER) RNA and EBV DNA were determined by in situ hybridization. The expression of EBV-encoded nuclear antigen (EBNA) and latent membrane protein (LMP1) were identified by immunohistologic methods. Clonotypic analysis of EBV genomes was performed by Southern blot hybridization with EBV termini fragment probe. RESULTS: The clinical features of NTL were characterized as prolonged fever (16 patients), widespread dissemination into distant sites (13 patients), and poor prognosis with a median survival of only 6 months. EBER transcripts were identified in 16 of 18 patients. Monoclonal EBV genomes EBNA1 and LMP1 were also detected in all EBER-positive cases tested. All 18 patients expressed pan-T antigens such as MT1, CD45RO, and/or CD2. The rearrangements of T-cell receptor (TCR)-beta, -gamma, and/or -delta genes were shown in all 11 patients tested. The natural killer (NK) cell phenotype CD56 was expressed in all EBV-positive cases tested, and was not detected in EBV-negative cases. Seven EBV-positive cases expressed a TCR-delta chain with rearranged TCR gamma or -delta genes whereas both EBV-negative cases corresponded to alpha beta T-cell lymphoma, which expressed a TCR-beta chain with a rearranged TCR-beta gene. CONCLUSIONS: These data suggest that EBV-positive NTL may be derived from the lineage of NK-like T-cells or gamma delta T-cells, and that EBV may play a role in lymphomagenesis. Therefore, we propose that NTL which has peculiar clinical and histologic features could be classified as a new lymphoma entity. PMID- 8640685 TI - Risk factors for essential thrombocythemia: A case-control study. Italian Leukemia Study Group. AB - BACKGROUND: Very little information is presently available regarding risk factors for essential thrombocythemia (ET). METHODS: A case-control study was performed to study the possible association between ET and selected behavioral, occupational, and environmental exposures. RESULTS: Thirty-nine patients aged 20 years or older and 156 controls were enrolled in 2 Italian Hematology Departments located in Rome and Pavia. Controls were recruited among outpatients seen in the same hospitals and matched 4:1 to the patients after stratification by age and sex. Odds ratio (OR) estimates suggest an association between ET and hair dye use (in particular the use of dark hair dye for periods longer than 10 years: OR - 5.3; 95% confidence interval [CI], 1.4-19.9), living in houses built with tuff (a material with a high concentration of gamma-emitting radionuclides and radon) for longer than 9 years (OR = 5.1; 95% CI, 1.2-22.1), and selected occupations (electrical worker and shoemaker, OR +infinity and 2.7; 95% CI, 0.5-16 respectively). CONCLUSION: Behavioral exposures such as hair dyes, living in a tuff house, and working as an electrician are significantly associated with ET development. The data are consistent with those observed in acute leukemias. PMID- 8640684 TI - Treatment of children with newly diagnosed brain stem gliomas with intravenous recombinant beta-interferon and hyperfractionated radiation therapy: a childrens cancer group phase I/II study. AB - BACKGROUND: Prognosis for the majority of children with brain stem gliomas is dismal. In previous studies, recombinant beta-interferon (r beta IF) has been shown to be effective for children with recurrent brain stem gliomas and may also act synergistically with radiotherapy (RT). METHODS: Thirty-two children with diffuse intrinsic brain stem gliomas were treated with (r beta IF) and 7200 centigray (cGy) of hyperfractionated RT (100 cGy twice-daily fractions) to determine the toxicity of treatment and the tolerance of the brain stem to this regimen, as well as to assess survival. Patients were treated with r beta IF 3 times per week during RT and then for 8 weeks following RT. Initially, a dose escalation trial was performed. RESULTS: Interferon was initially begun at 12.5 x 10(6) IU/m2 and escalated up to 400 x 10(6) IU/m2. The safe starting dose was determined to be 100 x 10 (6) IU/m2. Due to unacceptable toxicity, the maintenance dose was reduced to 200 x 10 (6) IU/m2. Therapy was relatively well tolerated, although 13 of the patients required dose modifications due to hepatic or hematologic toxicity. Four of the patients had to discontinue treatment due to this toxicity. One patient died while receiving maintenance IF of encephalopathy, seizures, and brain stem dysfunction; believed possibly due to the r beta IF. Thirty of the 32 patients have developed progressive disease. The median time to progression from study entry was five months and the median time to death was 9 months. CONCLUSIONS: We conclude that r beta IF plus hyperfractionated therapy can be tolerated by children with newly diagnosed brain stem gliomas, although there is occasional dose-limiting hepatic, blood, and central nervous system toxicity. This therapy did not result in a higher rate of disease control. PMID- 8640686 TI - The National Cancer Data Base report on longitudinal observations on prostate cancer. AB - BACKGROUND: Data on 349,154 prostate cancer cases diagnosed since 1986 have been entered to the American College of Surgeons National Cancer Data Base (NCDB). Previous annual reports have examined subsets of these data. The present report highlights major trends in the presentation and treatment of prostate cancer in the United States evident from longitudinal analyses of the entire data. METHODS: NCDB data are collected following a computerized, standard format. Hospital participation is voluntary. RESULTS: Since the first year of data collection, the number of participating hospitals has increased from 496 to 996 and the number of prostate cancer patients reported to the NCDB increased from 19,531 to 84,408. The proportion of men diagnosed at ages younger than 70 years increased from 37.8% in 1986 to 46.9% in 1993. Completeness of reporting stage of disease and tumor grade has improved. The proportions of both the earliest (American joint Committee an Cancer [AJCC] Stage Groups 0 and I) and the most advanced (AJCC++ Stage Group IV) stages declined. The proportion of Grade 2 (moderately differentiated) tumors increased from 38.6% in 1986 to 57.5% in 1993. The proportion of AJCC Stage II prostate cancer increased from 19% in 1986 to 48.4% in 1993. The proportion of patients treated by prostatectomy increased from 9.9% in 1986 to 29.2% in 1993. The proportion of patients receiving no cancer directed treatment declined from 41.8% in 1986 to 21.6% in 1993. Less change was observed in the use of radiation and hormonal treatments. CONCLUSIONS: These data show that the clinical patterns of prostate cancer have changed markedly in recent years. PMID- 8640687 TI - Paraffin section immunocytochemistry for estrogen receptor: the time has come. PMID- 8640688 TI - Prognosticators determining survival subsequent to distant metastasis from nasopharyngeal carcinoma. AB - BACKGROUND: Distant metastases are common in patients with nasopharyngeal carcinoma (NPC), and their presence is the most important factor in limiting survival. We aimed to study the prognosticators determining survival subsequent to distant metastasis from NPC. METHODS: A study by both mono- and multivariate analyses was carried out in 945 patients presenting between 1984 and 1989. Forty two presented with metastases and 247 developed metastasis after primary radiotherapy. RESULTS: Patients who presented with distant metastasis (M1 classification) had a significantly shorter survival than those who developed metastases after primary radiotherapy. The presence of hepatic metastases, short metastasis free interval, and older age at presentation significantly predicted short survival after the diagnosis of distant metastasis. Patients with metastases preceded by, and/or accompanied with, locoregional recurrence had comparable survival to those without, despite their association with a significantly longer metastasis free interval. A history of locoregional recurrence was however not compatible with long term, disease free survival, and, in its presence, advanced T-classification on presentation predicted poor survival subsequent to metastasis. Long term disease free survival (64-117 months) was attained in 4 young patients (age < 40 years) with isolated intrathoracic metastases in the absence of locoregional recurrence after achieving a complete response to aggressive treatment, with chemotherapy, radiotherapy, and/or surgery, usually multimodal. CONCLUSIONS: Some of the clinical prognosticators have been identified and an attempt was made to subclassify distant metastases according to possible differences in prognosis. A subset of metastatic NPC was identified which is compatible with long term, disease free survival. Investigations during follow-up should be directed toward the early detection of such potentially salvageable cases. PMID- 8640689 TI - Phase II trial of 5-fluorouracil, leucovorin, interferon-alpha-2a, and cisplatin as neoadjuvant chemotherapy for locally advanced esophageal carcinoma. AB - BACKGROUND: Most patients with esophageal carcinoma present with locally advanced disease and a poor prognosis. Surgery or radiation provides palliation for locally advanced esophageal carcinoma. The role of neoadjuvant therapy remains to be defined. We administered neoadjuvant chemotherapy consisting of 5-fluorouracil (5-FU), leucovorin, interferon-alpha, and cisplatin to 11 patients with locally advanced disease. METHODS: Eleven patients with squamous cell or adenocarcinoma of the esophagus were treated peroperatively with two to three cycles of combination chemotherapy. Nine patients underwent resection with curative intent. RESULTS: Six patients received three cycles of chemotherapy, and five received two. Dose reduction was necessary for two patients. One patient achieved a pathologic complete response, histologically confirmed. Of the eleven patients, two did not undergo surgery because of progressive disease during chemotherapy. Seven of the 9 patients relapsed after surgery and 2 have been disease free for 27 months. CONCLUSIONS: The combination 5-FU leucovorin, interferon-alpha-2a, and cisplatin administered in a neoadjuvant setting resulted in a median survival of 11.8 months with a median time to relapse of 7 months. PMID- 8640690 TI - Type 73 human papillomavirus in esophageal squamous cell carcinoma: a novel association. AB - BACKGROUND: Human papillomaviruses (HPVs) commonly cause proliferative lesions of squamous epithelia, and infection with certain HPV types carries a high risk of malignant transformation, especially in the uterine cervix but also at other sites, including the esophagus. We used molecular techniques to detect and type HPV in an in situ squamous cell carcinoma in the esophagus of a 39-year old woman. METHODS: DNA was extracted from paraffin sections of the esophageal lesion and of the uterine cervix (which was removed several years earlier), and analyzed for HPV by polymerase chain reaction (PCR). Primers complementary to highly conserved regions of the open reading frame of most genital HPVs were used to amplify a approximately 450 base pair product that contained both conserved and divergent regions. The PCR products were hybridized with probes specific for HPV 6, HPV-11, HPV-16, and HPV-18, and with a consensus probe. A conspicuous band in the esophageal sample failed to hybridize with any of the probes. The amplimer was subcloned and sequenced. The sequence was compared with other known HPVs. RESULTS: The intraepithelial neoplasia in the patient's cervical cone biopsy contained HPV-16. The esophageal lesion contained HPV that did not hybridize with probes for types 6, 11, 16, or 18, but exhibited 98.3% homology with HPV-73. CONCLUSIONS: Squamous cell carcinoma in situ of the esophagus may be associated with infection by HPV-73. PMID- 8640691 TI - Prediction of early and late recurrence after curative resection for gastric carcinoma. AB - BACKGROUND: Although several prognostic factors for patients with gastric carcinoma have been demonstrated, those predictive of early and late recurrences after surgery are still unclear. METHODS: Of 479 patients who underwent curative resection for gastric carcinoma from 1977 to 1987, 115 patients who died of recurrence were studied. RESULTS: The patients were divided into 2 groups: an early recurrence group that included 80 patients who died within 3 years after surgery, and a late recurrence group, that included 35 patients who died more than 3 years after surgery. Early recurrence was characterized by a large tumor (8.1 cm vs. 6.7 cm; P<0.05) and many involved lymph nodes (7.7 vs 4.4; P<0.01). However, the depth of invasion, as well as the level of positive lymph nodes were not different between the two groups. Multivariate analysis showed that tumor size and the number of positive lymph nodes independently influenced the survival period. CONCLUSIONS: Tumor size and the number of positive lymph nodes are the most important factors predicting the timing of recurrence after curative resection for gastric carcinoma. PMID- 8640692 TI - The influence of cigarette smoking, alcohol, and green tea consumption on the risk of carcinoma of the cardia and distal stomach in Shanghai, China. AB - BACKGROUND: The divergent incidence patterns of gastric cardia and distal stomach cancer may suggest different etiologies. This study examined the role of cigarette smoking, alcohol drinking, and green tea consumption as risk factors for carcinoma by anatomic subsite of stomach. METHODS: Newly-diagnosed stomach carcinoma patients (n = 1124) and frequency-matched population controls (n = 1451) were interviewed in person. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression models. RESULTS: Excess risks associated with cigarette smoking and alcohol consumption were observed largely among men. The adjusted ORs for all stomach cancer combined were 1.35 (CI: 1.06-1.71) for current smokers, and 1.26 (CI: 0.86-1.84) for ex-smokers. For tumors of the distal stomach, statistically significant positive dose-response trends were found for the number of cigarettes smoked per day, the duration and pack-years of smoking, and inverse trends for years of stopped smoking. For tumors of the gastric cardia, however, a monotonic association was found only for the number of cigarettes smoked per day (P=0.06). Alcohol consumption was not related to the risk of cardia cancer, while a moderate excess risk of distal stomach cancer (OR: 1.55; CI: 1.07-2.26) was observed among heavy alcohol drinkers. Green tea drinking was inversely associated with risk of stomach cancer arising from either subsite, with ORs of 0.77 (CI: 0.52-1.13) among female heavy drinkers, and 0.76 (CI: 0.55-1.27) among male heavy drinkers. CONCLUSIONS: Our findings provide further evidence that cigarette smoking and, possibly, alcohol consumption increase the risk of stomach carcinoma, notably of the distal segment. An inverse association with green tea drinking was also observed. PMID- 8640694 TI - United States lung carcinoma incidence trends: declining for most histologic types among males, increasing among females. AB - BACKGROUND: Lung carcinoma is now the most frequently diagnosed major cancer in the world and is also the most common cause of cancer deaths in males and females in the United States and worldwide. Based on trends in cigarette smoking and on analysis of lung cancer rates by birth cohort, it was predicted that a decline would occur in age-adjusted lung cancer rates, initially in males, and approximately 10 years later in females. We evaluated age-adjusted lung cancer incidence rates for changes in trends by race, sex and histologic type to determine if and when rates may have started declining. METHODS: We analyzed population-based incidence data from the National Cancer Institute's Third National Cancer Survey conducted between 1969 and 1971 and from the Surveillance, Epidemiology and End-results (SEER) program conducted between 1974 and 1991. Age adjusted rates were plotted by time period using a logarithmic scale for the ordinate. We used regression methods for grouped time-to-response data to fit a model to the disease rate for age, and calendar year to estimate the calendar year of maximum disease rate. RESULTS: During this period, the overall age adjusted lung cancer incidence rate rose from 37.8 to 68.2 per 100,000. Lung cancer rates in both white and black males climaxed around 1984 and declined subsequently. Furthermore, among white and black males, the rates of squamous cell carcinoma, small cell carcinoma, and large cell carcinoma declined after peaks in 1981 and 1982, 1986 and 1987, and 1986 and 1988, respectively. The rates for adenocarcinoma in black males peaked in 1987 whereas the rates in white males appeared to have plateaued between 1989 and 1991. Total lung cancer rates in males exceeded those in females, with rates in black males exceeding rates in white males. Age-adjusted lung carcinoma rates among white and black females continued to increase for all histologic types with the exception of large cell carcinoma among whites, bronchioloalveolar carcinoma among whites and blacks, and adenosquamous carcinoma among blacks. CONCLUSIONS: The cumulative effect of these trends has resulted in a plateau of total lung carcinoma incidence in all persons combined, and a decline might be expected soon, as has already been observed among males. Most of these changes reflect past cigarette smoking patterns. Demonstration of declines and tapering increases among several population subgroups suggests impending reductions in the incidence and mortality rate for this highly fatal cancer. PMID- 8640693 TI - Paclitaxel, carboplatin, and extended schedule etoposide in the treatment of small cell lung carcinoma. AB - BACKGROUND: Paclitaxel is an active agent in the initial treatment of patients with small cell lung carcinoma. The authors evaluated the toxicity and efficacy of paclitaxel (1-hour infusion) added to a standard combination regimen of carboplatin and etoposide in a Phase II trial for the treatment of patients with small cell lung carcinoma. METHODS: Thirty-eight patients with previously untreated small cell lung carcinoma were treated with a combination regimen including paclitaxel, 135 mg/m2 by 1-hour intravenous (i.v.) infusion, on Day 1; carboplatin at AUC 5, on Day 1; and oral etoposide, 100 mg alternated with 50 mg, on days 1-10. Prior to availability of reimbursement for oral etoposide, 13 patients received etoposide, 25 mg/m2 i.v. on Days 1-5 and 8-12. Treatment courses were repeated every 21 days for a total of 4 courses. Patients with limited stage disease received radiation therapy (4500 centrigray in 25 fractions) concurrently with the last 2 courses of chemotherapy. RESULTS: This combination chemotherapy regimen was easily tolerated. Eleven episodes of Grade 3 or 4 leukopenia occurred in 9 patients (8% of courses); Grade 3 and 4 thrombocytopenia and anemia were also infrequent. Fifteen patients were hospitalized for treatment of fever associated with leukopenia. Concurrent treatment with chemotherapy and radiation therapy was also tolerable, but was more toxic; 6 of 15 patients (40%) developed esophagitis (Grade 3 in 5 patients, Grade 4 in 1 patient), and 45% of all episodes of Grade 3/4 leukopenia occurred during concurrent therapy. Other nonhematologic toxicity was uncommon. Twenty nine of 38 patients (76%) achieved a partial or complete response to treatment (limited stage, 14 of 15 patients, 93%; extensive stage, 15 of 23 patients, 65%). The complete response rate was 26% (limited stage disease, 40% versus extensive stage disease, 17%). Median actuarial overall survival was 7 months for patients with extensive stage disease, and 17 months for patients with limited stage disease. Prophylactic whole brain irradiation was not used, and seven patients developed brain metastases as their initial site of relapse. CONCLUSIONS: The combination of paclitaxel, administered by 1-hour infusion, carboplatin and extended schedule etoposide is feasible and well tolerated in the doses administered in this Phase II trail. This regimen was highly active with treatment results comparable to other standard regimens. Increased doses of both paclitaxel and carboplatin could probably be tolerated and are currently being evaluated. Precise definition of the role of paclitaxel in the treatment of small cell lung carcinoma awaits the results of randomized studies. PMID- 8640695 TI - Primary malignant melanoma (clear cell sarcoma) of bone: report of a case arising in the ulna. AB - BACKGROUND: To the authors' knowledge, there has been no previous report of primary malignant melanoma of bone. METHODS: A 33-year-old woman presented with a tumorous lesion in the olecranon of the right ulna. The histologic diagnosis was malignant melanoma with close similarity to clear cell sarcoma. To exclude the possibility of malignant melanoma metastatic to the bone, clinical investigations including gallium 67-citrate scintigraphy, brain, chest, and abdominal computed tomography, and upper and lower gastrointestinal endoscopic examinations were performed. Conventional histopathologic, immunohistochemical, and electron microscopic studies were also performed. RESULTS: Clinical investigations showed no lesion suggestive of a primary melanoma other than that in the right ulna. Histologically, the tumor was comprised of polygonal or fusiform cells with clear or granular cytoplasm and vesicular nuclei containing one or two prominent nucleoli. The features were similar to those of clear cell sarcoma (malignant melanoma of soft parts). Fontana preparations and immunohistochemical staining for S-100 protein and HMB-45 (melanoma specific antigen) also revealed that the tumor cells had the characteristics of malignant melanoma. The patient has remained alive and well for more than 5 years after the initial treatment. CONCLUSIONS: The clinicopathologic findings in this case strongly suggested that the lesion was a primary malignant melanoma of bone. Therefore, this is the first report to indicate that malignant melanoma and related diseases can occur even in bone tissue. PMID- 8640696 TI - Intensive treatment of patients age 60 years and older with de novo acute myeloid leukemia: analysis of prognostic factors. AB - BACKGROUND: This study aimed to define pre-treatment parameters with prognostic significance in elderly patients with de novo acute myeloid leukemia (AML) who were treated with aggressive regimens. METHODS: We analyzed, retrospectively, the clinical and laboratory features of 159 consecutive patients age >60 years with AML. Ninety-two patients presenting as de novo AML were considered suitable for aggressive chemotherapy according to inclusion criteria not different from those commonly used for younger adults. They belonged to all of the French-American British classification types except M3, and their median age was 67 years (range: 60-79). Antileukemic treatment consisted of 1 of 3 sequential protocols adopted at the S. Eugenio University Hospital of Rome between 1987 and 1993. The three therapeutic groups were similar in number and presenting characteristics. In addition to arabinosylcytosine, induction schedules included mitoxantrone (Groups I and II) or daunorubicin (Group III), and etoposide (Groups I and III). Once in complete remission (CR), patients were consolidated with two other courses of chemotherapy using reduced dosages of the same drugs given during induction. RESULTS: Induction treatment achieved a 52.2% CR rate, with median remission duration and event free survival (EFS) of 35 and 27 weeks, respectively. Because no significant differences between the results of the three therapeutic groups were observed, all cases were pooled to evaluate the prognostic factors. In univariate analysis, the only presenting characteristic significantly associated with failure of induction treatment was age >67 years (P=0.007). Factors associated with an increased likelihood of shorter remission duration were CD7 expression on leukemic cells (P=0.007) and an abnormal karyotype (P=0.010; those predicting shorter EFS were a chromosomal status other than normal (P=0.002) and detection of CD14 antigen (P=0.008). Logistic regression results identified age and CD14 expression as the variables with independent prognostic impact on CR achievement. In a stepwise proportional hazards general linear model, CD7 and karyotype retained their predictive value regarding remission duration, whereas the karyotypic pattern at diagnosis and CD14 antigen expression were the most important determinants of EFS, with age showing a borderline statistical value. A simple "risk factor score" was developed that would allow for stratification of patients into prognostic groups. CONCLUSIONS: Cytogenetic analysis and immunophenotyping might help to select elderly patients with AML who have little benefit from current therapeutic strategies and with whom new approaches might be experimented. PMID- 8640697 TI - Nonmelanoma skin carcinoma in Albuquerque, New Mexico: experience of a major health care provider. AB - BACKGROUND: Although nonmelanoma skin carcinomas (NMSC) are increasing nationwide, rates are difficult to measure precisely, because few registries include types of carcinoma. Albuquerque, New Mexico is a high risk site for both melanoma and NMSC. In a National Cancer Institute (NCI) survey conducted between 1977 and 1978, NMSC incidence rates among non-Hispanic white males were the highest of 8 national study sites. METHODS: Experience with NMSC over 28.5 years at the Lovelace Health Systems in Albuquerque, New Mexico is described. The impact of multiple tumors was emphasized, recent annual incidence rates in members of the Lovelace Health Plan (LHP) were calculated, and trends in incidence rates since the NCI survey were estimated. RESULTS: There were 10,760 tumors among 4958 people between 1964 and mid-1992, with an average of 2.2 lesions per person (range: 1-92 lesions). Basal cell carcinoma (BCC) was 6.6 times more common than squamous cell carcinoma (SCC), and more likely to be multiple. There was an excess of males among persons with each tumor type and among subjects with multiple tumors. Only 46.1% of NMSC were first tumors; multiple or subsequent tumors comprised 53.7%. More than half of the concomitant or subsequent tumors were diagnosed within 1 year of the first, but new tumors were still appearing in the same subjects more than 10 years later. The incidence of NMSC between 1989 and 1991 among LHP members increased with age, and rates were higher for non-Hispanic males than females. Hispanics had much lower rates than non-Hispanic whites (NHW), with no sex differential. Incidence rates of BCC have increased markedly since 1977 and 1978 in NHW males, and to a lesser extent in other groups. Rates of SCC have not changed. The ratio of NMSC to other carcinomas at Lovelace has doubled over the same interval and has increased 10 fold over the last 2 decades. CONCLUSIONS: These data confirm the marked effect of age, ethnicity, and sex on NMSC rates. Differences in trends in BCC and SCC highlight their somewhat different etiologies. The increasing rate of BCC since the late 1970s and the large numbers of multiple tumors make a powerful case for intensified efforts at both primary and secondary prevention. PMID- 8640698 TI - Radiation-associated angiosarcoma: diagnostic and therapeutic implications--two case reports and a review of the literature. AB - BACKGROUND: Angiosarcoma (AS) accounts for 1 to 2% of all soft tissue sarcoma. Both primary and secondary AS may occur, the latter being reported in the upper extremity with lymphedema after extended radical mastectomy for breast cancer (postmastectomy AS) or following radiotherapy of the breast, the thoracic wall, or other sites (radiation-associated AS). The authors report two cases of cutaneous radiation-associated AS and review literature regarding treatment planning and follow-up data to define the most appropriate therapy for cutaneous and noncutaneous radiation-associated AS. METHODS: The clinical records of two patients with radiation-associated AS were analyzed and previously reported cases were reviewed. RESULTS: Case 1: a female age 67 years developed cutaneous AS in the residual breast 27 months after breast-conserving therapy and conventional external beam radiotherapy (EBR). She underwent chemotherapy followed by simple mastectomy and chemotherapy with the same regimen but developed early recurrence that was treated with hyperthermia and EBR, wide excision, and second-line chemotherapy. She died 30 months after primary diagnosis of AS with multiple metastases. Case 2: a male age 59 years developed cutaneous AS in the left groin, 10 years after conservative surgery and EBR for a penile carcinoma. Early recurrence following wide excision was treated with chemotherapy, re-excision, and immunochemotherapy but the patient died 24 months after the primary diagnosis of cutaneous AS with local progression and distant metastases. CONCLUSIONS: The prognosis of radiation-associated AS is dismal, due mostly to its poor differentiation and frequent diagnostic delay. Simple mastectomy is advised for patients with cutaneous AS after breast-conserving surgery with wide tumor-free margins. If primary surgery fails, survival is seriously compromised because adjuvant or palliative treatments are not effective. PMID- 8640699 TI - Fadrozole HCL (CGS-16949A) versus megestrol acetate treatment of postmenopausal patients with metastatic breast carcinoma: results of two randomized double blind controlled multiinstitutional trials. AB - BACKGROUND: Breast cancer patients with prior response to endocrine therapy achieve subsequent benefit from additional endocrine therapies. The efficacy and safety of an aromatase inhibitor, fadrozole HCL, were compared with megestrol acetate in post menopausal patients who had disease progression after receiving antiestrogen therapy either for metastatic disease or as adjuvant therapy. METHODS: In 2 multiinstitutional prospective trials, 683 postmenopausal patients were randomized to receive either fadrozole HCL, 1 mg twice daily, or megestrol acetate, 40 mg 4 times daily, in a double blind fashion after progression on first-line hormonal therapy. Objective response rates, time to progression, survival and safety of the two regimens were compared. RESULTS: Results of intent to-treat analyses are presented in this study. No significant differences were detected between the two treatment groups in time to progression, objective response rates, duration of response, and survival in either trial. There were no clinically meaningful differences between the treatment groups in the incidence and severity of adverse experiences, except that weight gain, fluid retention, and dyspnea were observed in more patients in the megestrol acetate group compared with those receiving fadrozole HCL, whereas nausea and vomiting were observed in more patients in the fadrozole HCL group compared with those receiving megestrol acetate. CONCLUSIONS: Fadrozole HCL was as efficacious as megestrol acetate in postmenopausal patients with metastatic breast carcinoma after one hormonal therapy. Adverse experiences were mild with both therapies, but megestrol acetate was associated wiht a higher frequency of weight gain, fluid retention and dyspnea, whereas fadrozole HCL was associated with a higher frequency of nausea and vomiting. PMID- 8640700 TI - Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays. AB - BACKGROUND: Historically, estrogen receptor (ER) determinations have been made by the ligand-binding assay of tumor homogenates, primarily by the dextran-coated charcoal method (DCC). Immunocytochemical assays (ICA) for ER are more recent and have been executed mostly on frozen sections with the monoclonal antibody H222Sp gamma (H222). Lately, new monoclonal antibodies derived by recombinant ER technology have been developed that work well on paraffin embedded, formalin fixed tissue sections. However, there is little information as to whether such assays prognosticate endocrine response. METHODS: Using antigen retrieval, the immunoglobulin G1 monoclonal antibody ER1D5, and the streptavidin-biotin detection system, 74 patients with breast cancer in whom endocrine response was known were assayed and the results compared with ER by DCC and ER by ICA in frozen section with H222. RESULTS: ER1D5 in paraffin provided the highest correlation with endocrine response (Kendall's tau [r] = 0.57; P<0.001) whereas ER by DCC failed to correlate (r= -0.002; P<0.99). ER1D5 in paraffin correlated weakly though significantly with DCC (Kappa Statistic [K] = 0.204; P<0.02). H222 in frozen sections also correlated moderately with endocrine response (r = 0.34; P<0.001). CONCLUSIONS: ER can be detected in routine tissue sections processed with antigen retrieval and ER1D5, and can be relied upon to provide accurate prognostic information regarding response to endocrine therapies in breast cancer patients. PMID- 8640701 TI - A pilot evaluation of alternating preoperative chemotherapy in the management of patients with locoregionally advanced breast carcinoma. AB - BACKGROUND: This prospective trial was conducted to evaluate the outcome of patients treated with preoperative and post operative chemotherapy, mastectomy, and irradiation for locoregionally advanced breast carcinoma. METHODS: Between June 1986 and September 1990, 71 patients received 2 cycles of doxorubicin that alternated with 2 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil prior to mastectomy; irradiation was administered when the tumor was not amenable to surgical resection. Additional chemotherapy and tamoxifen, in hormone receptor positive tumors, was used after mastectomy. Post-operative irradiation was given on a selective basis for patients at high risk for locoregional disease recurrence. RESULTS: Although 5 patients (7%) had disease progression, clinical partial or complete tumor response to preoperative chemotherapy was noted in 46 patients (65%). Sixty-eight patients (96%) underwent mastectomy. With a median follow-up of 52 months, the relapse-free and overall survival rates at 5 years were 42% and 57% respectively. Locoregional tumor recurrence occurred in 14 patients (20%), and 28 patients (39%) developed metastatic disease. Menopausal status, clinical presentation (noninflammatory vs. inflammatory), and American Joint Committee on Cancer clinical stage were independent covariates associated with patient outcome. CONCLUSIONS: Preoperative alternating chemotherapy, with the selective use of irradiation, resulted in significant locoregional disease regression and the successful integration of mastectomy into the therapeutic strategy. Locoregional tumor control and relapse-free and overall survival estimates for the approach described herein compared favorably with other comtemporary reports for this condition. PMID- 8640702 TI - Atypical apocrine adenosis of the breast: a clinicopathologic study of 37 patients with 8.7-year follow-up. AB - BACKGROUND: Apocrine metaplasia is occasionally superimposed on sclerosing adenosis (apocrine adenosis) in breast biopsies, and cytologic atypia is sometimes present (atypical apocrine adenosis). The long term risk of patients developing breast carcinoma subsequent to the diagnosis of this lesion is unknown. METHODS: Atypical apocrine adenosis was defined as apocrine adenosis with enlarged nucleoli and a greater than threefold variation in nuclear area. Lesions with recognizable cytoarchitectural patterns of intraductal carcinoma were excluded. Surveillance, Epidemiology and End Results (SEER) data were used as the reference population for calculations of relative risk. RESULTS: Thirty seven women with atypical apocrine adenosis had a mean follow-up of 8.7 years. Four patients developed invasive ductal carcinoma of the breast (3 ipsilateral, 1 contralateral) after a mean of 5.6 years. The relative risk of developing carcinoma was 5.5 (95% confidence interval [CI], 1.9-16). All patients who developed carcinoma were older than age 60 at the time of breast biopsy showing atypical apocrine adenosis, and carcinoma developed at a mean age of 70 years. In the older than 60 years age group (11 patients), the relative risk of developing carcinoma was 14 (95% CI, 4.1-48). CONCLUSIONS: Atypical apocrine adenosis confers an increased risk of developing breast carcinoma in women older than age 60, and the risk in younger women is probably low. Some cases of atypical apocrine adenosis may represent in situ apocrine carcinomas that are difficult to diagnose because of the absence of the usual architectural features of intraductal carcinoma. PMID- 8640703 TI - Human papillomavirus DNA after treatment of cervical dysplasia: low prevalence in normal cytologic smears. AB - BACKGROUND: The presence of human papillomavirus (HPV) DNA in relation to cervical cytology was evaluated after treatment of cervical dysplasia. METHODS: Forty patients, 22 with normal and 18 with abnormal cytology (mild or moderate dyskaryosis), with a history of cervical dysplasia were selected. Only patients with HPV DNA positive biopsies obtained before treatment were included. The presence of HPV was assessed in cervical smears at least 1 year after treatment of cervical dysplasia by using a polymerase chain reaction (PCR) with consensus primers (CPI/IIG). HPV typing was done by direct sequence analysis of the CPI/IIG PCR generated amplimers. RESULTS: Smears from 3 of the 22 patients with normal cytology after treatment were positive for HPV DNA (14%). HPV DNA positive smears were found in 13 of the 18 patients with abnormal cytology after treatment (72%) (relative risk: 5.3; 95% confidence interval: 1.78-15.75). In 11 of the 16 HPV DNA positive smears (69%), the HPV type was different from that before treatment. In 35 of 40 patients, the HPV type before treatment could not be detected after treatment (88%). CONCLUSIONS: A minority of the patients with normal cytology after treatment of cervical dysplasia had detectable HPV DNA. In contrast, a high prevalence of HPV DNA was found in cervical smears of patients with abnormal cytology after treatment of cervical dysplasia. After treatment, none of the patients with abnormal cytology but HPV DNA negative smears had recurrence of cervical intraepithelial neoplasia. This suggests the value of supplementary HPV DNA testing during follow-up of patients treated for cervical dysplasia. PMID- 8640704 TI - Trousseau's syndrome in association with ovarian carcinoma. AB - BACKGROUND: The association of neoplastic disease and thromboembolic disorders was first recognized by Trousseau in 1865. Abnormalities of blood coagulation tests have been reported in the majority of patients with cancer, including ovarian carcinoma. However, Trousseau's syndrome has rarely been reported in women with ovarian carcinoma. METHODS: A literature search for cases of Trousseau's syndrome in association with ovarian carcinoma was performed using the MEDLINE database. Case notes of patients with ovarian carcinoma treated at St. George's Hospital were reviewed and cases of Trousseau's syndrome identified. RESULTS: We report a series of 4 cases of Trousseau's syndrome in association with ovarian carcinoma occurring over a 3- to 4-year period, and highlight the particular difficulties associated with the management of these thromboembolic effects. CONCLUSIONS: Trousseau's syndrome can impair quality of life in patients with advanced ovarian carcinoma and enhanced anticoagulation regimens may be required for effective palliation. PMID- 8640705 TI - Tandem high-dose chemotherapy with ifosfamide, carboplatin, and teniposide with autologous bone marrow transplantation for the treatment of poor prognosis common epithelial ovarian carcinoma. AB - BACKGROUND: A phase I or II trial was conducted to assess the toxicity and the efficacy of a tandem high dose chemotherapy combining ifosfamide, carboplatin, and teniposide in patients with poor prognosis ovarian carcinoma. METHODS: Thirty seven patients were scheduled to receive tandem high dose therapy combining ifosfamide 7500 to 11250 mg/m2, carboplatin 875 ot 1000 mg/m2 and teniposide 750 to 1000 mg/m2, followed by autologous bone marrow transplantation (ABMT). Eight patients were refractory to the platin-based regimen, 7 were treated in chemosensitive relapse, and 22 in partial or complete response (PR/CR) were treated. Sixty-six cycles were administered. Sixteen patients were evaluated for response. RESULTS: The overall response rate was 56% (CR rate: 12%). Toxic effects consisted of mainly renal toxicity, esophagitis, and enterocolitis. Three patients died of therapy-related complications. Since the time of ABMT, the median overall survival (OS) duration of the whole population was 18 months and the survival rate was 14% at 60 months. For the 22 patients treated after PR or CR, the median OS duration was 24 months and the survival rate was 32% at 60 months. Tandem high dose therapy with ABMT was unable to circumvent resistance to conventional chemotherapy or to prolong the duration of survival for patients treated in chemosensitive relapse. For patients treated after CR or PR, the survival results were similar to that achieved with conventional therapy. CONCLUSIONS: Prospective, randomized studies, including patients only after CR or with minimal residual disease, are urgently required to evaluate the activity of high dose therapy in the treatment of advanced ovarian carcinoma. PMID- 8640706 TI - Adjuvant immunotherapy treatment of renal carcinoma patients with autologous tumor cells and bacillus Calmette-Guerin: five-year results of a prospective randomized study. AB - BACKGROUND: Active specific immunotherapy (ASI) is a strategy that attempts to boost the host's immune response specifically against its own tumor. The purpose of this study was to investigate the effect of adjuvant ASI in patients with renal carcinoma. METHODS: Of 120 consecutive patients, 60 were randomized to a control group and 60 to receive ASI comprised of 3 intradermal injections of 10(7) autologous irradiated tumor cells mixed with 10(7) Bacillus Calmette-Guerin (in the first 2 vaccinations) or alone. At randomization and 1, 6, and 12 months after completing immunotherapy, the treated patients were evaluated for the development of delayed type cutaneous hypersensitivity (DTCH) response to autologous tumor and autologous normal renal cells. RESULTS: The baseline DTCH responses were negative in all patients. One month after completing ASI, 38 of 54 immunized patients showed a significant (P<0.01) DTCH response to autologous tumor but not to autologous normal renal cells. The response was persistent at 6 months in 25 of 44 patients and at 12 months in 16 of 28 patients. DTCH response remained negative in the nonimmunized control patients. There was no systemic toxicity but local ulcerations that healed in 2 months were observed. After a median follow-up of 61 months, the probability of 5-year disease free survival (DFS) was 63% for treated patients and 72% for control patients. The corresponding probability of 5-year overall survival (OS) was 69% and 78%, respectively. These differences were not statistically significant. CONCLUSIONS: This is the first prospective randomized study of ASI in a large population of patients with renal carcinoma after radical nephrectomy. Our data clearly indicate that ASI can increase the reactivity to autologous tumor, as measured by the DTCH test, but it appears unable to affect DFS and OS of patients. PMID- 8640707 TI - Cytogenetic analysis of aggressive meningiomas: possible diagnostic and prognostic implications. AB - BACKGROUND: Published karyotypes from aggressive (atypical and malignant) meningiomas are few, but suggest clonal evolution from benign tumors with monosomy 22 to aggressive forms with additional abnormalities. The goal of this study was to identify the most frequent karyotypic abnormalities associated with aggressive histopathology and biologic behavior. METHODS: Eight intracranial meningiomas exhibiting histologically atypical features at the time of intraoperative diagnosis were chosen for cytogenetic analysis. The study set was comprised entirely of histologically atypical meningiomas. Four were considered malignant; three on the basis of brain invasion and one due to extracranial metastases. None was histologically anaplastic. RESULTS: Chromosomal abnormalities were demonstrated in 6 cases (75%), 5 of which were complex (63%). Loss of chromosome 22 was identified in two cases, both of which were associated with additional aberrations. Abnormalities most frequently involved chromosomes 1 (63%), 3 (50%), and 6 (63%). Four cases (50%) had dicentric or ring chromosomes. An additional 47 previously reported karyotypes from atypical and malignant meningiomas were reviewed. Comparison with published karyotypes of 200 histologically benign meningiomas served to underscore the increased frequency of complex karyotypes, chromosome 1, 3, and 6 abnormalities, and telomeric associations in the aggressive tumors. Apparently normal karyotypes as well as monosomy 22 alone were more frequently associated with benign, nonatypical histopathology. CONCLUSIONS: These findings suggest a possible role for cytogenetic analysis in determining the prognosis and perhaps in refining the diagnosis of atypical or aggressive meningiomas. Further studies are necessary to determine the significance of complex karyotypes, chromosome 1, 3, and 6 abnormalities, and telomeric associations, particularly whether they portend a more aggressive clinical course in meningiomas lacking features of histologic atypia. PMID- 8640708 TI - Prospective randomized clinical trial to evaluate the optimal dose of 131 I for remnant ablation in patients with differentiated thyroid carcinoma. AB - BACKGROUND: Radioiodine has been used for more than a half-century to ablate thyroid remnants following thyroid surgery, but a single optimal dose has not been established. We designed a prospective randomized trial to determine the optimal dose of 131 I for remnant ablation. METHODS: Using a simple randomization technique, 149 patients with remnant thyroid were incorporated into 4 treatment groups. Twenty-seven of these patients were administered 25 to 34 millicurie (mCi) of 131 I (30 +/- 1.5), 54 received 35 to 64 mCi (50.6 +/- 5.4), 38 received 65 to 119 mCi (88.6 +/- 14) and 30 patients received 120 to 200 mCi (155 +/- 28.7). Six months to 1 year after treatment, all subjects were reassessed after withdrawing L-thyroxine for 4 to 6 weeks. A successful ablation was defined as the absence of thyroid bed activity in 5 mCi 131 I neck scan at 48 hours along with 2 adjunctive criteria which were the neck uptake of <0.2% of the administered activity and the thyroglobulin (Tg) value of <10 ng/mL. RESULTS: Applying the above criteria, we observed complete ablation of 17 of 27 thyroid gland remnants (63%) in the 30 mCi group, 42 of 54 (77.8%) in the 50 mCi group, 28 of 38 (73.7%) in the 90 mCi group and 23 of 30 (76.7%) in the 155 mCi group. When the radiation-absorbed dose was calculated, a 30 mCi dose delivered approximately 20,000 centigray (cGy), a 50 mCi dose about 30,000 cGy, a 90 mCi dose about 50,000 cGy, and a 155 mCi dose about 130,000 cGy. CONCLUSIONS: Increasing the empirical 131 I initial dose to more than 50 mCi results in plateauing of the dose-response curve and thus, conventional high dose remnant ablation needs critical evaluation. Based on dosimetry results, one should aim to deliver about 30,000 cGy to the thyroid remnant, as higher doses do not appear to yield a higher ablation rate. PMID- 8640709 TI - Oncogene expression in carotid body tumors. AB - BACKGROUND: The genetic etiology of carotid body tumors is suggested by the familial occurrence of the neoplasm. Environmental influences are also implied by the fact that the tumor is more common in those living at high altitudes. However, the development of sporadic tumors occurring at sea level, which account for the majority of cases, remains unknown. METHODS: The clinical and pathologic features of 13 carotid body tumors excised in 13 patients were reviewed. Two patients had bilateral tumors, one with a strong family history, and two patients had recurrent carotid body tumors. All tumors were benign except for one that had local lymph gland metastases. All patients were followed up for a period ranging from 1 to 17 years. Each tumor was examined for the oncoproteins c-myc, bcl-2 c erbB-2, c-erbB-3 and c-jun and for the proliferating cell nuclear antigen (PCNA) by immunohistochemistry. RESULTS: c-myc immunoreactivity was observed in all tumors and, in 12 of 13 cases, was present in more than 10% of tumor cells, bcl-2 immunoreactivity was found in 11 cases with 6 tumors exhibiting more than 10% immunopositive cells, c-jun expression was found in 5 cases with 3 tumors containing more than 10% immunopositive cells. Only two tumors were positive for c-erb-B2 immunoreactivity with a cytoplasmic staining pattern. One tumor was positive for c-erb-B3. CONCLUSIONS: The oncogenes c-myc, bcl-2 and c-jun, are abnormally expressed in some carotid body tumors. Their expression may contribute to the genesis of carotid body tumors. PMID- 8640710 TI - Acute pancreatitis associated with continuous infusion cytarabine therapy: a case report. AB - BACKGROUND: While acute pancreatitis is a recognized complication of numerous drugs, cytarabine's role in causing this complication is controversial. Approximately 15 cases have been reported to the Food and Drug Administration linking cytarabine with pancreas-related toxicities. Previous case reports have been complicated by comorbid illnesses and the coadministration of other drugs associated with acute pancreatitis. METHODS: This report describes the clinical course of a patient with acute myelogenous leukemia (AML) who developed recurrent pancreatitis associated with cytarabine therapy. RESULTS: A male age 36 years with French-American-British M5B acute myelogenous leukemia received induction cytarabine (200 mg/m2/day) by continuous infusion for 7 days, and subsequently developed acute pancreatitis. The patient was rechallenged with intermittent, bolus, high dose cytarabine (HDAC) (3 g/m2bid administered over 3 hours) during the following intensification treatment, but did not develop clinical acute pancreatitis. Retreatment with continuous infusion cytarabine at a later time resulted in recurrence of acute pancreatitis. CONCLUSIONS: This case illustrates that cytarabine treatment may cause acute pancreatitis, and that this toxicity may be schedule dependent. In those with known sensitivity to cytarabine, altering the administration technique may avoid this complication. PMID- 8640711 TI - Thymic lymphoblastic lymphoma of committed natural killer cell precursor origin: a case report. AB - BACKGROUND: Recently, it was demonstrated that the human fetal thymocyte contains a bipotential progenitor capable of both T lymphocyte and natural killer (NK) cell differentiation. However, prior to this report a malignant neoplasm arising from these cells had not been documented. METHODS: A Japanese female age 38 years was examined by morphology of light and electron microscopy, immunohistochemistry, 3-color flow cytometry, cytotoxic assay, and Southern blotting. RESULTS: The patient presented with a mediastinal mass and pleural effusion. Leukemic progression was identified following chemotherapy and complete clinical remission. Immunophenotyping of lymphoma revealed CD45++, c-kit dim+, terminal deoxynucleotidyl transferase (TdT)-<+, CD38++, CD34+<++, CD33+<-, CD13dim+approximately+, HLA-DR+, CD7+, cytoplasmic CD3 (cCD3)+, surface CD3 (sCD3)-, CD2dim+, CD56+, CD16-, CD11b+, CD57-, CD1a-, CD5-, TCR alpha beta-, TCR gamma delta-, CD4-, CD8-, CD28-, CD10-, CD19-, CD20-, CD22-, surface immunoglobulins-, and CD14-. Functional NK activity of the lymphoma cells was extremely low. DNA analysis revealed no gene rearrangement in TCR beta, gamma, and delta or immunoglobulin heavy and light chain genes. CONCLUSIONS: Lymphoma cells of this case were derived from a distinct subtype of lymphocyte that originate from a thymic precursor committed to NK cell differentiation. This category is different from those of thymic T or precursor B cell pheno-/genotype. PMID- 8640712 TI - Regression of monoclonal B-cell expansion in patients affected by mixed cryoglobulinemia responsive to alpha-interferon therapy. AB - BACKGROUND: Several authors have reported on the effectiveness of alpha interferon (IFN-alpha) in the treatment of patients with mixed cryoglobulinemia. This prompted the authors to investigate the long term effects of this drug on clinical, hematologic, and virologic parameters in a group of 20 patients (13 women and 7 men) affected by mixed cryoglobulinemia. METHODS: In all patients, bone marrow biopsy, phenotyping of marrow cells, and polymerase chain reaction (PCR) immunoglobulin gene rearrangement in peripheral blood lymphocytes were performed before therapy and at the end of the follow-up. A liver biopsy was obtained in patients with biochemical signs of chronic liver disease. The presence of hepatitis C virus (HCV) RNA in serum was assessed by detection of anti-HCV antibodies, and by PCR amplification of the 5' untranslated region of HCV. The HCV genotype was also determined by PCR amplification of the core region of the virus with type-specific primers. The treatment schedule followed by all patients was 3 million units of recombinant IFN-alpha 2b 3 times weekly for 1 year. RESULTS: In 6 patients, the marrow histology before therapy showed a massive (more than 50%) monomorphous infiltration by plasmacytoid lymphocytes, indicating the presence of low grade non-Hodgkin's lymphoma. Anti-HCV antibodies were present in 19 (95%) subjects, and HCV-RNA was detectable in all patients. In addition, all patients affected by Type II mixed cryoglobulinemia showed a monoclonal B-cell expansion in peripheral blood mononuclear cells (PBMC). With therapy, 5 patients (25%) achieved a complete response and 11 patients (55%) a partial response, whereas minor responses were observed in the remaining 4 patients (20%). One of the complete responders and all patients showing partial responses relapsed a few months after therapy withdrawal. At the end of the follow-up, four patients had obtained a complete remission. Bone marrow examination showed that B-lymphocytic monoclonal infiltrate disappeared in three patients. Moreover, these three patients had become negative for B-cell expansion in PBMC. Lack of response, or relapse, was associated with the presence of Type II HCV. CONCLUSIONS: HCV may be the cause of mixed cryoglobulinemia. The disease is associated with a high prevalence of bone marrow B-cell lymphomas. IFN-alpha appears to be an effective agent for the treatment of mixed cryoglobulinemia. It also seems able to determine regression of the lymphoproliferative disorder. The HCV genotype appears to be the most important predictive factor for the response to antiviral therapy. PMID- 8640713 TI - Control of neuroblastoma cell proliferation and differentiation by human bone marrow. AB - BACKGROUND: Neuroblastoma (NB) is one of the few tumors known to undergo spontaneous regression. Its progression, however, often leads to bone marrow (BM) metastasis. Proliferation and differentiation of human NB cells may be regulated in vitro by a variety of biologic agents, some of which are released by low density BM and peripheral blood (PB) cells. Little is known regarding BM cell derived control of NB cell growth and differentiation. METHODS: The proliferative and differentiative responses of NB cells, to BM cell-, and to PB cell-derived conditioned medium (CM) were evaluated in comparison to cytokine-induced responses. RESULTS: CM from unstimulated cultures of low density BM and PB cells, from healthy donors, from newborn infants, and from NB patients, significantly and reproducibly stimulate NB cell growth in vitro. The intensity of CM-induced stimulation was not attained by recombinant human tumor necrosis factor (rhTNF), interferon (rhIFN), or granulocyte-monocyte colony stimulating factor (rhGM-CSF); and although epidermal growth factor (rhEGF) and transforming growth factor alpha (rhTGF alpha) were strongly stimulatory, neutralizing antibodies against each of these agents did not affect CM-derived activity. In contrast to growth stimulation, differentiation of CM-treated NB cells, was reproducibly suppressed, as reflected in abrogation of neuronal cell morphology as well as of neurofilament and neuron specific enolase expression. CONCLUSIONS: Spontaneous regression of NB tumors, on one hand and BM metastasis on the other may be associated with the extent and nature of the NB cell response to regulatory activity released by BM and PB cells. PMID- 8640714 TI - Analysis of morbidity and mortality in 60 patients with peritoneal carcinomatosis treated by cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy. AB - BACKGROUND: Peritoneal carcinoma has been regarded as a uniformly lethal clinical entity. A treatment plan combining cytoreductive surgery and heated intraoperative intraperitoneal chemotherapy (HIIC) was devised and tested to treat such patients. The purpose of this study was to evaluate the morbidity and mortality associated with this treatment approach. METHODS: Sixty patients with peritoneal carcinomatosis from adenocarcinoma of the colon or appendix were included in the study. Extensive cytoreductive surgery was combined with heated intraperitoneal mitomycin in an intraoperative lavage technique followed by one cycle of early postoperative intraperitoneal 5-fluorouracil. Eleven clinical variables were selected and statistically correlated with morbidity and mortality. RESULTS: Twenty-five complications occurred in 21 patients (morbidity = 35%). Morbidity related to gastrointestinal function included anastomotic leak (n=6), bowel perforations (n=5), bile leak (n=3), and pancreatitis (n=2). Four patients presented with severe hematologic toxicity (Grade 3 or 4). There were three cases of postoperative bleeding, one case of abdominal wound dehiscence, and one case of pulmonary embolism. Morbidity was significantly associated with three clinical factors: male sex, high intraabdominal temperature during HIIC, and duration of the surgical procedure. Enteral complications (bowel fistula and anastomotic leak) occurred in patients with a significantly higher number of peritonectomy procedures and a significantly longer operation. Three patients died within 8 weeks after the procedure (mortality = 5%). Mortality was significantly associated with age and intraabdominal temperature. CONCLUSIONS: Cytoreductive surgery combined with HIIC is associated with a 35% morbidity rate and a 5% mortality rate. Extensive surgery (duration and number of peritonectomy procedures) and high intraabdominal temperature represent the major risk factors for postoperative morbidity and mortality of patients treated with this new therapeutic approach. PMID- 8640715 TI - When the diagnosis is cancer: patient communication experiences and preferences. AB - BACKGROUND: Discrepancies exist between reported experiences of patients when they have been given a diagnosis of cancer, published guidelines for telling a diagnosis, and patterns of communication patients rate as favorable. Several studies have identified what happened and what is important to cancer patients when told their diagnosis, but no studies have addressed subsequent communications concerning the implications of the diagnosis and treatment choices. This study extended previous research by investigating the experiences and preferences for communication about diagnosis, prognosis, and treatment of patients diagnosed with breast cancer or melanoma. METHODS: A self-report questionnaire was designed for this study based on previous research and qualitative data generated from focus groups. Patients with breast cancer or melanoma answered questions about their experiences with communication at the time of diagnosis and concerning prognosis, treatment and related issues. Comparisons were made between patient experiences, preferences and published guidelines. Differences between the experiences of breast cancer and melanoma patients were tested and the relationship between communication and subsequent psychological adjustment to cancer was assessed. RESULTS: Patient preferences for communication during diagnostic consultation were not always consistent with published guidelines. Type of cancer did not significantly affect patient preferences. Psychological adjustment was related to patient ratings of the quality of doctor discussion about treatment options, but not about the diagnosis of cancer and its implications. Patients who wanted more emotional support at the time of diagnosis subsequently experienced poorer psychological adjustment. CONCLUSIONS: The differences in patient preferences show that a list of prescriptions for how to disclose a cancer diagnosis is too simplistic. Guidelines for clinicians should be derived from patient-based data rather than be limited only to clinical opinion. Guidelines concerning communication at the time of diagnosis also need to address discussions concerning the implications of the diagnosis and making treatment decisions. PMID- 8640716 TI - Laparoscopic unilateral ovarian transposition prior to irradiation: prospective study of 20 cases. AB - BACKGROUND: The objective of this study was to evaluate the feasibility, morbidity, and efficacy of unilateral laparoscopic ovarian transposition on the preservation of hormonal function in premenopausal patients requiring pelvic irradiation (external and/or intracavity by brachytherapy). METHODS: This prospective study was based on 20 patients: 17 presenting with cervical cancer, 2 with Hodgkin's disease, and 1 with ependymoma of the cauda equina. The operative technique consisted of releasing the right ovary from its pelvic attachments, and placing it as high and as laterally as possible in the right paracolic gutter, after creating a pedicle on the infundibulopelvic ligament. The follow-up of ovarian function was more than 1 year for 14 patients. RESULTS: The therapeutic protocol was not modified as a result of ovarian transposition. No intraoperative or postoperative complications were observed. The mean dose of irradiation received by the transposed ovary was 1.75 gray (Gy) (range 0.4-3.7). Mean follow up was 2 years. Two cases of menopause (14.7%), in the only 2 patients older than 40 years, were observed among the 14 patients followed for more than 1 year. The success rate was 100% for patients younger than age 40 years. CONCLUSIONS: Laparoscopic ovarian transposition is a simple and reliable method, which does not complicate subsequent therapeutic protocol. Its short term efficiency is comparable to results obtained by laparotomy, with a lesser morbidity. Although long term evaluation is necessary, laparoscopic surgery should be considered as an alternative to laparotomy for ovarian transposition. PMID- 8640717 TI - MLL/ENL fusion in congenital acute lymphoblastic leukemia with a unique t(11;18;19). AB - To elucidate the events leading to a unique complex translocation involving chromosomes 11, 18, and 19 in a congenital progenitor B-cell acute lymphoblastic leukemia, we have performed comprehensive cytogenetic and fluorescence in situ hybridization (FISH) analyses as well as molecular genetic studies on the DNA and RNA level. We were able to confirm the cytogenetic interpretation of this complex t(11;18;19)(q23;q22;p13.3) by chromosome painting. Involvement of the MLL gene on 11q23 became evident by Southern blot analysis as well as by FISH with a YAC clone containing the respective gene. Despite the fact that the additional signals of the split YAC clone were observed on the abnormal chromosome 18, reverse transcription polymerase chain reaction (RT-PCR) revealed a MLL/ENL hybrid mRNA, which is specific for a t(11;19)(q23;p13.3). This gene fusion most probably represents the critical part of this rearrangement. The transfer of the translocated part of the split YAC clone onto chromosome 18 indicates that the second break must have occurred in the vicinity of the first one, at a distance too close to be resolved by FISH. Whether this break took place within chromosome 11 or 19 sequences, up- or downstream of the MLL/ENL fusion, and whether this translocation results from a concerted simultaneous exchange of material or from two separate sequential events in consecutive cell generations remains open. PMID- 8640718 TI - Long-term follow-up of minimal residual disease in childhood acute lymphoblastic leukemia patients by polymerase chain reaction analysis of multiple clone specific or malignancy-specific gene markers. AB - Two types of markers, namely the clone-specific markers including T-cell receptor (TCR) gamma, TCR delta, and Ig heavy-chain (IgH) gene rearrangements, and malignancy-specific fusion gene mRNA such as SIL-TAL-1, BCR-ABL, and HRX-partner genes, were investigated by molecular biology techniques in 65 Chinese patients with acute lymphoblastic leukemia (ALL). In combination, these markers were informative among 96% of patients. Minimal residual disease (MRD) was followed up in 23 of these patients with available materials over a period varying from 8 to 54 months with at least one leukemia-specific probe. In most children, MRD was decreased continuously to an ultimately undetectable level within 6 to 12 months after remission induction therapy. One patient exhibited low-level residual leukemic cells for 4 years before the MRD turned negative. Another patient remained in complete remission for 45 months, although a positive signal was detected at 34 months using TCR delta probe, but was negative with a TCR gamma marker which was positive at presentation. In three patients who relapsed, MRD either persisted through the clinical course or became positive and eventually increased 3-11 months before clinical relapse. These data suggested that the combined use of multiple gene markers is a valuable tool for the PCR-based MRD detection, since it can cover most ALL patients. Furthermore, long-term follow-up of MRD is helpful for determining the dosage as well as the period of maintenance chemotherapy and for predicting impending relapse. PMID- 8640719 TI - Localization of anti-CENP antibodies and alphoid sequences in acentric heterochromatin in a breast cancer cell line. AB - Karyotype alterations are a hallmark of cancer cells. Of particular interest to our laboratory are the inactive centromeres and blocks of heterochromatin devoid of the accompanying centromere. When purified or monospecies anticentromere proteins (CENP) antibodies or the whole serum from scleroderma (crest) patients are applied to human chromosomes, the centromere region exhibits the label. When we treated MDA 435 cells with the anti-CENP-A, anti-CENP-B, or the whole serum, the label was apparent on heterochromatin pericentric to the active and inactive centromeres. Moreover, blocks of heterochromatin not associated with any centromere also exhibited the label. Anti-CENP-C, however, is more strictly confined to the centromere in discrete dots and is not detected on any region except the sites of active centromeres. Distribution of alpha sequences also shows a pattern compatible with its distribution in the heterochromatin. Apparently, the use of anti-CENP-A and anti-CENP-B antibodies or alphoid DNA may not detect either the centromere (primary constriction) or the kinetochore; CENP C may be an exception. PMID- 8640720 TI - Cytogenetic abnormalities are frequent in uncultured prostate cancer cells. AB - Despite attempts by several laboratories to identify consistent chromosome abnormalities in cancer of the prostate, relatively few clonal changes have been found. We compared analysis of metaphases from uncultured specimens of primary prostate cancer (direct preparations) with those obtained from short-term culture using various media. While the number of metaphases in uncultured specimens was low, and chromosome morphology fair to poor, structural chromosome changes could be identified as clonal in 5 of 14 specimens (36%). In contrast, while clonal abnormalities were found in 20 of 61 (33%) specimens analyzed after short-term culture, these abnormalities were predominantly numerical and simple structural changes. Two tumors metastatic to lymph nodes were studied using direct preparations; both were near tetraploid, with multiple structural abnormalities, including isochromosome 8q in both and del(8)(p21) in one. Cytogenetic analyses of metastatic prostate tumors have been very limited, and these data suggest that formation of an i(8q) may be the mechanism by which loss of heterozygosity of 8p, reported frequently in molecular analyses, occurs. Our findings indicate that prostate cancers, like most solid tumors, do have clonal chromosome abnormalities that are frequently complex, but the method that reproducibly yields representative karyotypes from this particular tumor remains to be identified. PMID- 8640721 TI - No FISH evidence for trisomy 7 in normal or leukemic bone marrow. AB - Trisomy 7 has been found as the sole chromosomal anomaly in both benign and malignant tumors, as well as in nonneoplastic lesions. It has been reported that +7 may occur in tumor-infiltrating as well as in peripheral T cell and in the thymus. The precursor cells, which mature in the thymus to T lymphocytes, originate in the bone marrow and the present study was undertaken to investigate whether cells carrying an extra chromosome 7 can be detected there. Bone marrow samples from five hematologically normal individuals and 12 children with acute lymphoblastic leukemia (ALL) were analyzed by interphase fluorescence in situ hybridization (FISH) using a chromosome 7 centromere-specific probe. Half of the ALLs were karyotypically normal, whereas the other half displayed clonal abnormalities (one pseudodiploid and five hyperdiploid karyotypes, none of which had +7). The FISH analysis showed no evidence of cells with trisomy 7 in the bone marrow samples from the controls or ALLs. This suggests that the gain of a chromosome 7 by T lymphocytes does not occur before the bone marrow precursor cells are conditioned in the thymus. PMID- 8640722 TI - The BAX gene maps to the glioma candidate region at 19q13.3, but is not altered in human gliomas. AB - The bax protein regulates apoptosis in a cellular pathway that involves both bcl 2 and p53, two molecules associated with human glioma tumorigenesis. We therefore evaluated the possibility that BAX functions as a glioma tumor suppressor gene. Somatic cell hybrid panels, fluorescence in situ hybridization and cosmid mapping localized the BAX gene to 19q13.3, approximately 300 kb centromeric to HRC. Thus BAX maps to the region of chromosome 19 most frequently deleted in gliomas. Routine and pulsed-field gel electrophoresis/Southern blotting studies, however, failed to reveal large-scale deletions or rearrangements of the BAX gene in gliomas. In addition, single strand conformation polymorphism analysis of all six BAX exons and flanking intronic sequences did not disclose mutations in 20 gliomas with allelic loss of the other copy of 19q. A C/T polymorphism was detected in intron 3 and was common in the general population. Therefore, although BAX maps to the glioma candidate region on the long arm of chromosome 19, BAX is probably not the 19q glioma tumor suppressor gene. PMID- 8640723 TI - Multiple unrelated clones in myelodysplastic syndrome and in acute myeloid leukemia. AB - We identified cytogenetically unrelated clones in the bone marrow of 12 of 240 patients with myelodysplastic syndrome (MDS) and in 3 of 232 patients with acute myeloid leukemia (AML). In addition, unrelated single-cell abnormalities were found in six MDS and two AML patients. The most commonly encountered abnormalities present in the unrelated clones in patients with refractory anemia (RA) were del(5q), +8, and -7. In blastic types of MDS and AML trisomy 8 was found in two of eight patients while in the remaining cases the chromosome abnormalities were diverse and nonspecific. The presence of the chromosomally unrelated clones, together with recent data on the early appearance of monoclonality provided by molecular biology studies, support the interpretation that aberrations such as +8 and del(5q) are actually secondary abnormalities that develop during tumor progression in a cell with a primary submicroscopic genomic rearrangement. PMID- 8640725 TI - Persistence of AML1 rearrangement in peripheral blood cells in t(8;21). AB - Translocation (8;21)(q22;q22) involves fusion of the AML1 gene with the ETO gene, generating an AML1/ETO fusion transcript that can be detected by the polymerase chain reaction (PCR). Persistence of the AML1/ETO transcript has been demonstrated by PCR in patients with t(8;21) in long-term remission, but the rearranged AML1 gene could not be detected by Southern analysis, showing that the t(8;21) clone existed as minimal residual disease (MRD). In one patient with t(8;21), AML1/ETO could be detected serially in the peripheral blood. However, rearrangement of the AML1 gene was also found to persist. Furthermore, the hybridization intensities of the rearrangement bands showed that some of the mature myeloid cells also possessed the AML1 rearrangement. Thus, the presence of AML1/ETO in this case appeared to be due to persistence of the mutated clone as mature myeloid cells instead of MRD, implying that the t(8;21) had occurred in a preleukemic myeloid progenitor cell capable of differentiation. PMID- 8640724 TI - Assignment of the TP53 orthologue to a new linkage group (LG XIV) in fish of the genus Xiphophorus (Teleostei: Poeciliidae). AB - Using a p53 encoding cDNA fragment of rainbow trout (Oncorhynchus mykiss) as probe, a lambda clone from a platyfish (Xiphophorus maculatus) genomic library was isolated. DNA sequencing of the insert from this clone revealed that it contained the highly conserved domains IV and V of the p53 polypeptide. To map the Xiphophorus p53 gene, joint segregation analysis of the inheritance of a PstI generated DNA restriction fragment length polymorphism (RFLP) and the inheritance of 36 polymorphic protein and DNA markers was performed in backcross hybrids of X. clemenciae x (X. clemenciae x X. milleri) and X. helleri x X. (helleri x X. maculatus Jp 163 B) using Oncorhynchus cDNA and Xiphophorus genomic p53 probes, respectively. The p53-hybridizing sequence (TP53) was linked to the ACO1 (cytosolic aconitase) locus in both crosses, and defines a new Xiphophorus linkage group, designated LG XIV. This is the first mapping assignment of a known human tumor suppressor gene in fish. Since ACO1 is not linked with melanoma severity in X. helleri x X. maculatus Jp 163 A backcross hybrids, these data indicate that homozygosity for the X. helleri TP53 genotype in backcross hybrids of the cross type is not associated with genetically regulated malignant melanoma formation in the Gordon-Kosswig hybrid melanoma model. PMID- 8640726 TI - FISH studies on the Y chromosome in male urinary cells. AB - To ascertain the meaning of the loss of the Y (-Y) in bladder cancer, we addressed the incidence of -Y in urinary cells in relation to age in 35 men without bladder cancer. Aside from the bone marrow and blood, -Y has not been examined critically in other tissues and organs in a large series of men. The present study clearly demonstrated that -Y in the group studied was an infrequent finding and not related to age. PMID- 8640728 TI - Chromosomal anomalies exclusive of telomeric associations in giant cell tumor of bone. AB - The occurrence of telomeric associations in giant cell tumor of bone is well recognized. However, there has been little emphasis on clonal structural abnormalities that may be seen in addition to or in the absence of telomeric associations. Two cases of giant cell tumor of bone are presented in this study demonstrating clonal karyotypic anomalies other than telomeric association with a review of previously reported cases showing similar findings. PMID- 8640727 TI - Chromosome changes in nonneoplastic tissue. Numerical and structural abnormalities in nasal polyps with atypical stromal cells. AB - Cytogenetic investigation on short-term cultures of 13 nasal polyps disclosed the presence of chromosome aberrations in three cases: one (a recurrence) showed numerical changes; the other two had structural abnormalities, an inv(12)(q15q22) in one case, a der(6)t(6;12)(q22;q15) in the other. The three cases were characterized histologically by the presence of frequent atypical stromal cells, and were positive for vimentin and smooth muscle actin. Of the remaining 10 cases, three were not analyzable, and seven had normal karyotypes, although random structural changes were seen in two of them. PMID- 8640729 TI - A new translocation, t(5;21)(q13;q22) in acute myelogenous leukemia. AB - We report a case of acute myelogenous leukemia with a variant translocation involving chromosomes 5 and 21 with breakpoints on 5q13 and 21q22 as revealed by various techniques including fluorescence in situ hybridization. The unusual presentation of t(5;21)(q13;q22), as the sole abnormality in acute myelogenous leukemia, is atypical. PMID- 8640730 TI - Report of a complex karyotype in recurrent metastatic fibrolamellar hepatocellular carcinoma and a review of hepatocellular carcinoma cytogenetics. AB - Metastatic fibrolamellar hepatocellular carcinoma (HCC) was detected in the abdominal lymph nodes of an adolescent male after resection of the primary tumor. No dividing cells were isolated from attempted cytogenetic studies of the primary tumor. However, cytogenetic analysis of lymph node metastases detected 9 and 12 months after partial hepatectomy revealed abnormal hypertriploid karyotypes, with a suggestion of clonal evolution: 62-92 < 3n >,XX, -Y, +3, +6, +6, +7, +7, +8, +10, +13, +15, +16, +20, -21, -22, +mar1 x 2, +mar[cp6]/46,XY[8] and 78 < 3n >,XX, -Y,der(1)t(1;1)(p36.1;q21), +4, +6, +6, +7, +7,i(8)(q10), +10, +15, +20, 21, -22, +mar1 x 2, +mar2[3]/46, XY[17], respectively. Karyotypes of this variant of HCC have not been reported previously. The cytogenetics of HCC are reviewed. PMID- 8640731 TI - Incidence of human papilloma virus in patients with invasive cervical carcinoma. AB - The dot-blot hybridization of biotin-dUTP-labeled HPV-16 DNA with genomic DNA extracted from biopsies taken from patients with invasive carcinoma and abnormal cytology showed the presence of HPV-DNA in 88% and 80% cases under relaxed conditions and 40% and 20% cases under stringent conditions of hybridization, respectively. Southern blot revealed the HPV-DNA in randomly integrated form in two cases and in episomal form in the other two. PMID- 8640732 TI - Bilateral adrenocortical carcinoma showing loss of heterozygosity at the p53 and RB gene loci. AB - Presented is a rare case of nonfamilial, hormonally nonfunctional adrenocortical carcinoma with synchronous bilateral adrenal involvement. We investigated adrenal and metastatic tumors for loss of heterozygosity affecting four genetic loci containing the tumor suppressor genes p53, RB, DCC, and APC, using polymerase chain reaction and restriction fragment length polymorphism assay. Allelic losses at the p53 and RB loci were detected in all tumor samples, suggesting that the p53 and RB genes are involved in the tumorigenesis of adrenocortical carcinoma. PMID- 8640733 TI - Common and rare fragile sites on human chromosomes. The cytogenetic expression of active and inactive genes? PMID- 8640735 TI - Increased interleukin-10 serum levels in patients with solid tumours. AB - In 40 out of 99 patients (40.4%) with solid tumours of different tissue, but the same stage (IV), elevated serum levels of interleukin-10 were observed. The mean levels of the cytokine in patients with malignant melanoma (24.3 ng/ml), pancreatic (6.8 ng/ml) or gastric (6.3 ng/ml) adenocarcinoma were significantly higher than in healthy subjects (3.4 ng/ml) or in patients with uterine fibroma (1.7 ng/ml). Patients with colon (6.8 ng/ml) and renal (5.7 ng/ml) carcinoma had similar values of interleukin-10 but did not significantly differ from controls. Interleukin-10 is known to suppress the functions of both T lymphocytes and macrophages, working as a general dampener of the immune and inflammatory responses. The observation of increased circulating levels of interleukin-10 in cancer patients may have important implications for future investigations, immunological monitoring and therapeutic intervention on neoplastic patients, and suggests a mechanism for tumour cells escaping from immune surveillance. PMID- 8640734 TI - Study on aneuploidy and p53 mutations in astrocytomas. AB - To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed. PMID- 8640736 TI - p53 gene mutation in cerebral primitive neuroectodermal tumor in Taiwan. AB - p53 mutation has been rarely reported in cerebral primitive neuroectodermal tumors (PNET). To determine the significance of p53 mutations in the development of cerebral PNET, we studied cerebral PNET samples from 14 patients, 8 females and 6 males with a mean age of 38 years (range 10 months to 77 years) who had total or subtotal surgical resection. Histological typing of PNET with neuronal (N) and non-neuronal (NN) differentiation groups revealed 8 and 6 cases, respectively. Six (43%) of the 14 patients had p53 mutation. The p53(+) and p53( ) groups had an age range of 19-77 with a mean of 49 years and 10 months to 57 years with a mean of 30 years, respectively. p53 expression between the PNET-N and PNET-NN groups was 5 of 8 (62.5%) and 1 of 6 (16.7%), respectively. The mutations contained 3 transitions, 2 transversions and 1 frameshift; none of them occurred at the site of 'hot-spot' residues (codons 175, 248, 273). The results suggest that: (1) p53 mutation in cerebral PNET tends to show a higher incidence of neuronal differentiation and occurs in the older age group in Taiwan, (2) there was no difference in survival time between the PNET-N and PNET-NN groups (7 months and 6 months) (P = 0.54), and between p53(+) and p53(-) groups (6 months and 7 months) (P = 0.57), and (3) PNET may be an entity of a heterogenous group of tumors with different genetic mechanisms controlling their trends of differential lineage. Further studies are needed to determine the significance of p53 mutations in PNET development, especially the role of carcinogens in the genesis of PNET in Taiwan. PMID- 8640737 TI - Chronological observation of mouse endometrial carcinogenesis induced by N-methyl N-nitrosourea and 17 beta-estradiol. AB - Chronological observation of the neoplastic endometrial lesions induced by N methyl-N-nitrosourea (MNU) and 17 beta-estradiol (E2) were studied. E2 induced cystic glandular hyperplasia, but adenomatous hyperplasia was induced predominantly by MNU. Atypical hyperplasia and adenocarcinoma were induced cooperatively by E2 and MNU, and first found at week 12; the incidence of adenocarcinoma increased in accordance with the week-course. Some atypical hyperplasia might be changed to adenocarcinoma. Mean AgNORs numbers in (pre)neoplastic lesions increased in accordance with neoplastic changes. This model was useful for clarifying histogenesis of human endometrial carcinogenesis. PMID- 8640738 TI - Expression and the role of cathepsin H in human glioma progression and invasion. AB - Proteinases and their inhibitors may play a role in the development and progression of many cancers. Several studies suggested that lysosomal proteinases cathepsin B, L, and D may be involved in the malignant progression of some human neoplastic diseases. In this study, we determined the levels of cathepsin H in human glioma progression and the significance of cathepsin H in glioma cell invasion. Levels of cathepsin H antigen were found to be significantly higher in glioblastomas and anaplastic astrocytoma when compared with normal brain tissue and low-grade gliomas. Western blotting confirmed the presence of authentic cathepsin H with a doublet at 27 and 25 kDa in normal brain tissue and tumor samples. However, the intensity of the band increased significantly in glioblastoma samples. Cathepsin H antibody inhibited the invasion of glioblastoma cell lines through Matrigel invasion assay. These data suggest that the tumor specific increase in antigen may be a useful independent marker of tumor progression in central nervous system neoplasms. PMID- 8640739 TI - Promotion potential of tamoxifen on hepatocarcinogenesis in female SD or F344 rats initiated with diethylnitrosamine. AB - The liver promotion potential of tamoxifen (TAM), which has been widely used in the treatment of hormone-dependent breast cancers, was investigated using female SD or F344 rat initiated with diethylnitrosamine (DEN). In Experiment 1, 45 newborn female SD rats were administered DEN (100 mg/kg, i.p.) (Groups 1 and 2) or saline (Group 3) 24 h after birth. After weaning at week 3, Groups 1 and 3 were subcutaneously injected with TAM citrate (1 mg/rat per day), suspended in corn oil, in the subscapular region, while Group 2 was given the vehicle alone (s.c.) daily for 9 weeks, and killed at week 12. In Experiment 2, 70 female F344 rats at 7 weeks of age were divided into five groups. All animals were initially given DEN (200 mg/kg i.p.) for initiation. Two weeks later Groups 1-4 were given diets containing 100, 250, 500 ppm TAM, or 500 ppm PB for 6 weeks, respectively, while Group 5 was administered basal diet as a control for the same period. The rats were subjected to two-thirds partial hepatectomy (PH) at week 3 and were killed at week 8. The enhanced development of glutathione S-transferase-placental form (GST-P)-positive liver cell foci after DEN exposure in both newborn SD and adult F344 rat medium-term liver bioassay models (Experiments 1 and 2). This suggests that TAM exerts promotion potential for hepatocarcinogenesis in female rats. PMID- 8640740 TI - Low expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci induced in rat liver by diethylnitrosamine or phenobarbital. AB - The expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci (EAF) induced by diethylnitrosamine (DEN) and phenobarbital (PB) was examined. p21 expression in the nucleus of hepatocytes in EAF was decreased compared to surrounding tissue. Fifty-eight percent of all GST-P-positive EAF induced by DEN and 79% of the EAF induced by PB were p21-negative. The proportion of p21 negative EAF increased with the size of the foci and more than 90% of the largest EAF were p21-negative. p21 is a mediator of p53 signals leading to block of the cell cycle. In conjunction with previous data indicating that p53 is not induced in GST-P-positive hepatocytes isolated from EAF-bearing rats, the results of this study suggest a role for altered signaling in the G1-S check point in rat hepatocarcinogenesis. PMID- 8640741 TI - Gestational choriocarcinoma of the ovary diagnosed by analysis of tumour DNA. AB - Gestational choriocarcinoma of the ovary is a rare form of malignancy which can be difficult to distinguish from primary ovarian choriocarcinoma. The ability to make such a diagnosis could, however, have important implications for therapy. We report here a case of choriocarcinoma whose origins were difficult to determine and which behaved clinically more like a primary rather than a gestational choriocarcinoma. We have analysed DNA from this tumour by using polymerase chain reaction (PCR) amplification of a range of polymorphic alleles and have demonstrated that the tumour was in fact gestational. Furthermore, the lack of chromosome Y sequences and the presence of heterozygosity of the spouse's alleles, indicated that this tumour arose as a result of dispermic fertilisation of an empty ovum by sperm carrying the X chromosome. PMID- 8640742 TI - Anti-tumor activity of the crude saponins obtained from asparagus. AB - The crude saponins from the shoots (edible part of asparagus) of asparagus (asparagus crude saponins; ACS) were found to have antitumor activity. The ACS inhibited the growth of human leukemia HL-60 cells in culture and macromolecular synthesis in a dose and time dependent manner. The ACS at 75-100 micrograms/ml range was cytostatic. ACS concentrations greater than 200 micrograms/ml were cytocidal to HL-60 cells. The ACS at 6 and 50 micrograms/ml inhibited the synthesis of DNA, RNA and protein in HL-60 cells by 41, 5, and 4, respectively, or by 84, 68 and 59%, respectively. The inhibitory effect of ACS on DNA synthesis was irreversible. PMID- 8640743 TI - Low susceptibility of the spontaneously hypertensive rat (SHR) to quinoline induction of hepatic hemangioendothelial sarcomas. AB - Effects of quinoline and captafol, both of which are hemangiocarcinogenic agents, were investigated in spontaneously hypertensive rats (SHR). Male SHR and Wistar Kyoto rats (WKY), the parent strain of SHR, were administered quinoline (0.2%) or captafol (0.15%) supplemented in the diet for 32 weeks. Resultant incidences of hepatic hemangioendothelial sarcomas were in animals receiving quinoline 93% for WKY and only 7% for SHR. A few hepatocellular nodules were also induced in both strains. No histopathological lesions were observed in the other organs. Thus, the SHR proved unexpectedly less susceptible to vascular carcinogenicity than its WKY counterpart. PMID- 8640744 TI - Inhibition by rosemary and carnosol of 7,12-dimethylbenz[a]anthracene (DMBA) induced rat mammary tumorigenesis and in vivo DMBA-DNA adduct formation. AB - Extracts of the spice Rosemary officinalis L. have been reported to inhibit experimental carcinogenesis. Two rosemary components, carnosol and ursolic acid, appear to be partly responsible for the antitumorigenic activity of rosemary. The present studies were conducted in order to evaluate the activity of rosemary extract, carnosol and ursolic acid in inhibiting the in vivo formation of mammary 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts and the initiation of DMBA induced mammary tumorigenesis in female rats. Supplementation of diets for 2 weeks with rosemary extract (0.5% by wt) but not carnosol (1.0%) or ursolic acid (0.5%) resulted in a significant decrease in the in vivo formation of rat mammary DMBA-DNA adducts, compared to controls. When injected intraperitoneally (i.p.) for 5 days at 200 mg/kg body wt, rosemary and carnosol, but not ursolic acid, significantly inhibited mammary adduct formation by 44% and 40%, respectively, compared to controls. Injection of this dose of rosemary and carnosol was associated with a significant 74% and 65% decrease, respectively, in the number of DMBA-induced mammary adenocarcinomas per rat, compared to controls. Ursolic acid injection had no effect on mammary tumorigenesis. Therefore, carnosol is one rosemary constituent that can prevent DMBA-induced DNA damage and tumor formation in the rat mammary gland, and, thus, has potential for use as a breast cancer chemopreventative agent. PMID- 8640745 TI - Inhibition of the hepatocarcinogenicity of aflatoxin B1 in rats by low levels of the phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene. AB - The phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were studied for inhibition of aflatoxin B1 (AFB1) hepatocarcinogenesis in male Fischer 344 rats. The antioxidants were administered at 5, 25, or 125 ppm in AIN-76A diet for 42 weeks. Beginning with week 2, 5 micrograms/kg of AFB1 was given by intragastric instillation three times a week for 40 weeks either alone or concurrently with BHA or BHT feeding. The development of hepatocellular altered foci (HAF) induced by AFB1, as indicators of hepatocarcinogenesis, was monitored using immunohistochemical staining for the placental form of glutathione S-transferase. By 16 weeks the multiplicity of foci was 1.97/cm2 of liver area in rats given only AFB1, and this increased to 4.11/cm2 at 24 weeks and to 10.60/cm2 at 32 weeks. At the final sacrifice at 42 weeks, the multiplicity of foci was 12.90/cm2 compared to 0.75/cm2 in untreated controls. In rats given antioxidants in addition to AFB1, the high dose of BHA reduced the multiplicity to 7.72/cm2 and the high dose of BHT reduced the multiplicity to 9.35/cm2. Lower levels did not reduce foci induction. Thus, in male rats under the conditions of this experiment, the level of 125 ppm of either BHA or BHT inhibited the initiation of hepatocarcinogenesis by AFB1. The BHA effect was slightly greater than that of BHT.